'fnstltut Français de Recherche Scientifique pour le Développement en Coopération "ME-Délégation Régionale Cone Sud Programme "Technologie Agroallmentalre et Agromdustriahsation" 1n ternational Training (ourse on Sulid State Fermentation Maurice RAIMBAUL Carlos RIcardo SOCCOL Gérard CIIU S97 6-10 OctolJer 1 Curi lïba-(Parami) BRnSIL CNPq rio Parana Conselho Nacional de Pesquisa e Desenvolvimento Cientifico e Tecnologico Document ORSTOM Montpellier, 1998 ; n° 1 Maurice RAIMBAULT Carlos Ricardo SOCCOL Gérard CHUZEL INTERNATIONAL TRAINING COURSE ON SaUD STATE FERMENTATION 1 8 JL11 N 1998 ~\2 OtltJ l v ~/:'3 -'/ ~ It-c; y ~'l( C Montpellier üRSTOM, 1998 ~ F j (0 \ ),);" ' DASTOM Oocument8lion 11111111 ~I I I I ~ 1~I I I I I 010055116 1111 «( Les opinions exprimées dans ce document n'engagent que la responsabilité de leurs auteurs» Proceedings of the International Training Course on Solid State Fermentation FMS 97 - Curitiba 6-10 Oct. 1997 Contact Address: Laboratoire de Biotechnologie Microbienne Tropicale 911, Avenue Agropolis; BP: 5045 34032 Montpellier Cedex, France Tel: (33) +467416281; Fax (33) +467416283 E-mail: MauriceRaimbault@mpl.orstom.fr © Orstom, 1998, Centre de Montpellier No part of the material protected by this copyright may be reproduced or utilised in any form without written permission from the autors. Printed in France. ORGANIZER COMMITEE - Dr. Carlos R. SOCCOL, Professor UFPR - Dr. Maurice RAIMBAULT ,Researcher ORSTOM - Dr. Gérard CHUZEL, French Expert, MAE-Cône Sud SPONSORS UFPR - Universidade Feredal do Parana, Curitiba (Brasil) ORSTOM (MAA, SRE, DIST) - lnstirut Français de Recherche Scientifique pour le Développement en Coopération (Paris -France) MAE - Délégation Régionale Cône Sud; Progarnme « Technologie Agroalimentaire et AgroindustrieIJe» CNPq - Conselho Nacional de Pesquisa e Desenvolvimento Cientifico e Tecnologico (Brasilia -Brasil) INVITED SPEAKERS Dr. Dr. Dr. Dr. Dr. Dr. Dr. Dr. Christopher AUGUR (ORSTOM-Mexico) Pierre CHRlSTEN (ORSTOM-Montpellier) Ricardo PEREZ-CORREA (PUC-Santiago) Maurice RAIMBAULT (ORSTOM-Monrpellier) José RODRIGUEZ LEON (IClOCA - La Havana) Sebastianos ROUSSOS (OR~TOM- Montpellier) Gerardo SAUCEDO CASTANEDA (UAM-Mexico) Carlos Ricardo SOCCOL (UFPR- Curitiba) The group of speakers during the course FMS97 in Curitiba. From the lep: Gérard Chuzcl, Ricardo Pèrez, Carlos Soccol, Christopher Augur, Maurice Raimbault, José Rodriguez Leon, Sebastianos Roussos, Gerardo Saucedo, and Pierre Christen Cnot on the photo). CONTENTS Foreword 1. General aspects ofSSF - General and microbiological aspects ofSSF.MRaimbault. - Continuous enzymes and fungal metabolites production in Solid state fermentation . using a counter-current reactor. S. Roussos & D.L. Pyle 21 - Production of enzymes by SSF: Relation SubstratelEnzyme and Induction/Catabolic Repression. G. Viniegra & CAugur 37 - Fruity aromas production in SSF by the fungus Ceratocystis fimbriata.P. Christen .43 Il. Biological aspects of fungi in SSF: - Fungal biomass estimation in batch solid substrate cultivation using asymptotic observation. R. Pérez-Correa, A. Ebner, 1. Solar, G. Acuna & E. Agosin .... ... 51 - MUlagenesis and adapted strains to the growth in liquid or solid substrates. G. Viniegra & C Augur..... . 63 - Fungal genetics, case of Aspergillus. C Augur..................................................... 69 - Growth and production of immobilised lipase From Rhizopus de!emar cultivated in SSF on a synthetic resin (Amberlite). P. Christen . 77 lIl. Kinetics aspects in SSF processes: - Relation BiomasslRespiration: Theoretical and practical aspects. M. Raimbault, CR. Soccol, S. C Stertz, L. Porto de Souza ... - Kinetics of the solid state fermentation of raw Cassava Oour by Rhizopus formosa 28422. J Rodriguez-Leon, S. Stertz. CR. Soccol & M. Raimbault .87 103 IV Applications in SSF - Valorization ofagro-industrial residues by Solid State Fermentation in Brazil. CR. Soccol . III - Citric acid and glucoamylase production From Cassava by-products in Solid State Fermentation CR. Sacco!. . . 125 - Yeasts cultivation in SSF : control ofmetabolism of Schwaniomyces casle/ii during solid cultivation on starch. G Saucedo 137 - Sugarcane bagasse used in Solid state fermentation for cellu1ases production. S. Roussos 139 - Biotechnological management of coffee pulp. S. Roussos, 1. Perraud-Gaime & S. Denis 151 - Molecular techniques applied to fungal strain upgradation capability related to SSf cultures. C Augur 163 V Models and slralegies [or scaling-up - Theory and strategy for scale-up: Development of scale-up criteria for bio-reactors for solid state fermentation (SSf). G Saucedo 169 - Multivariable model predictive control of a solid substrate pilot bioreactor: a simulation study. R. Pérez-Correa, H Paran , 1. Solar & E. Agosin 171 - An aseptic pilot bioreactor for solid substrate cultivation processes. E. Agosin, R. Père::185 Correa, 1. Solar, M. Fernandez & L. Chiang ..... Annexes: - List of papers published by the group of invited speakers on SSF - Programme of the course in Curitiba - List of participants 195 FOREWORD Solid Substrate Fennentation (SSF) is a microbial processes of grow1h on solid materials which contain water and soluble nutrients. Generally, sol id substrates or suppons are biodegradable heterogenous plant material. Starch, ligno-cellulose. narural fibers. inen polyurethane, matrices and chemical polymers are the most common materiaJ used for cultivating fungi and other microorganisms in SSF. Researches in this field were recently developed which indicated that does exist an alternative for new processes generating less effluents, recycling wastes or by-products in tropicaJ agro-industry. This new technology represents a promising alternative for Latin American. Par1icularly, respirometry is of high interest for the on line control of the SSF and groW1h of mycelia biomass. The general objectives of this courses consist in training professional or high level students in the area of agro-industry and fennentation. Theoretical and practical aspects of SSF, on solid substrates or impregnated supports, were studied with special attention to the practice of the technique of respiration measurement. Creation of a network of professional and academic researchers involved in Research and Development on SSf projects are of interest for Brazil and Latin American Countries. Specific Objectives consisted in the presentation and discussion of direct experiences from Researchers and Professors on related topics to Microbiology, Biochemistry. Physiology and Scale-up of filamentous fungi cultivation by SSf. Revision of the Biological, Biochemical and Molecular Biology concepts involved in the bioconversion and respirometry aspects of the SSf was also studied. finally, laboratory training and practice of SSf and respirometry analysis of fungal metabolism of A~pergillus and Rhizopus cultivated on Cassava by-product was perfonned. This course was devoted to 30 professionals of the industrial sector, academic researchers, professors or students of Brazil (1/2) and from other countries of Latin American countries (1/2): Argentina, Mexico, Clule, Uruguay, Colombia and Cuba. The Organizer Commitee thanks sponsors Institutions: UfPR, ORSTOM, CNPq and French Cooperation for lheir financial support, and all inviled speakers for their active par1icipation in the course. Maurice Raimbault, Carlos Ricardo Soccol, Gérard Chuzel GENERAL AND MICROBIOLOGICAL ASPECTS OF SOLID SUBSTRATE FERMENTAnON Maurice Raimbault Laboratoire de Biotechnologie Microbienne Tropicale, Centre ORSTOM-LBMT 9ll av. Agropoli.s - B.P.:5045 - 34032 Montpellier (France) Summary We present at first some generaJ considerations about specificity and characteristics of SSF, their advantages and disadvantages compared to LSF. We speak about micro-organisms involved in SSF processes, considering the better performances of fi lamentous fungi. The solid substrates and their basic macromolecular compounds are detailed in relation to this complex and heterogeneous systems. Biomass measurements are examined in detail, as so as envirorunental factors, both essential for studying and optimising SSF processes. 1. General considerations. Aerobic microbiaJ transformation of solid materials or "Solid Substrate Fermentation" (SSF) can be defined in terms of the following properties of the substrate to be transformed: - A solid porous matrix which can be biodegradable or not but with a large surface area per unit volume, in the range of 10 3 to 10 6 m 2/1 for a ready microbial growth on the solid/gas interface. - The matrix should absorb water once or several times its dry weight with a relatively high water activity on the solid/gas interface in order to allow fast rates of biochemical processes. - Air mixture of oxygen with other gases and aerosols should flow under a relatively low pressure and mixing the fermenting mash. - The solid/gas interface should be a good habitat for the fast development of specifie cultures of moulds, yeasts or bacteria, either by isolated or mixtures of species. - The mechanical properties of the solid matri"X should stand compression or gentle stirring as required for a given fermentation process. This requires smaJI granular or librous particles which do not tend to break or stick to each other. - The solid matrix should not be contarninated by inhibitors of microbial activities and should be able to absorb or contain available microbial foodstuffs such as carbohydrates (cellulose. starch, sugars) nitrogen sources (arrunonia, urea, peptides) and mineraI salts. Typical examples of SSF are traditional fermentations such as: - Japanese "koji" which uses steamed rice as solid substrate inocuJated with solid strains of the mould Aspergillus oryzae. - Indonesian "tempeh" or Indian "ragi" which use steamed and cracked legurne seeds as solid substrate and a variety of non toxic mou Ids as microbial seed. - French "blue cheese" which uses perforated fresh cheese as substrate and selected moulds, such as Penicillium roque/orlii as inoculum. - Composting of lignocellulosic fibres, nalUIally contaminated by a large variety of organisms including cellulolytic bacteria, moulds and Streptomyces sp. - ln addition ID traditional fennentations new versions of SSF have been invented. For example, it is estimated that nearly a third of industrial enzyme production in Japan which is made by SSF process and koji fennentation has been modemised for large scale production of citric and itaconic acids. Furthennore, new applications of SSF have been suggested for the production of antibiotics (Barrios & al., 1988) or enriched foodsruffs (Senez et al., 1979). Presently SSF has been applied to large scale industrial processes mainly in Japan. Traditional koji, manufaclUIed in small wooden and bamboo trays, has changed gradually to more sophisticated processes: fixed bed room fennentations, rotating d.rurn processes and automated stainless steel chambers or trays with microprocessors, electronics sensors and servomechanical stirring, loading and discharging. The usual scale in sake or misa factories is around 1 or 2 metric tons per batch but reactors can be made and delivered by engineering finns to a capacity as large as 20 tons (Fujiwara, Ltd.). Outside Japan, Kumar (1987) has reported medium scale production of enzymes, such as pectinases, in India. Koji type processes are widely used in small factories of the Far East (Hesseltine, 1972) and koji fennentation as been adapted to local conditions of United States and other Western countries, including Cuba (Ill A). In France, a new finn (Lyven SA) was recentIy created to commercialise a process for pectinase production From sugarbeet pulp. Blue cheese production in France is being modemised with improvements on the mechanical conditioning of cheeses, production of mould spores and control of envirorunent conditions. Composting which was produced for small scale production of mush rooms has been modemised and scaled up in Europe and United States. Aiso , various finns in Europe and USA produce mushroom spawn by cultivating aseptically Agaricus, Pleurotus or Shii-Take on steri le grains in static conditions. New versions for SSF reactors have been developed in France (Durand et al., 1988), Cuba (Cabello, 1985; Enriquez, 1983 and Rodriguez, 1984) and fundamental srudies on process engineering are being conducted in Mexico (Saucedo, 1987). SSF is usually a batch process using heterogeneous materials with various ages, (Raimbault, 1980 and Tengerdy, 1985), giberellic acid (Agosin et al, 1987), pectinases (Kumar, 1987; Oriol, 1988), cellulases (Roussos, 1985), spores as biopesticides, flavours and frangancies and feed detoxification. Ali that points will be discussed during the course. Generally, most of the recent research activity on SSF is being done in developing nations as a possible alternative for conventional submerged cultures which are the main process for pharmaceutical and food industries in industrialised nations. SSF seems to have theoretical advantages over LSF. Nevertheless, SSF has several important limitations. Table 1 shows advantages and disadvantages of SSF compared to LSF. Table 2 presents a list of SSf process in econornical sectors of agro-industry, agriculture and lndustrial fennentation. Most of the processes are commercialised in South-East Asian, African, and Latin American countries. Nevertheless, a resurgence of interest has occurred in Western and European countries over last 10 years. The future potentials and applications of SSF for specifie processes are discussed in other cessions. But briefly, we can say: 2 TABLE 1. Comparison between Liquid and Solid Substrate Fermentations. Solid Substrate Fermentation Polymer Insoluble Substrates Starch Cellulose Pectines ~ubstrates lignin Heat sterilisation and aseptic Vapor treatment. non sterile Aseptic conditions conditions conlrol High volumes or water consu- Limited Consumption of wa~ater ter; low Aw. No effluent med and effluents discarded Metabolic Heating Low heat transfer caDacity Easv contTol of temoerature Limitation oby soluble oxygen Easy aeration and high surfal<\eration (02) ce exchange airlsubstrate High level of air reauired hiH control Buffered solid substrates Easv oH control lNlecanical alritation Good homogeneization Static conditions orefered Industrial equipments Need for Engineering & Iscale up New design Equipment Available Easy inoculation, continuous spore inoculation, batch Unoculation lorocess Risks of contamination for Risk of contamination for low k::ontamination single strain bacteria rate growth fungi IEncrgetic consideration High energy consuming Low ener!2V consuming High volumes and high cost Low volumes & low costs of Ivolume of Equipment technology equioments High volumes of polluting No effluents, less pollution IEmuent & pollution effluents Concentration S IProducts 30-80 12/1 100/3000:1 FACTOR Liquid Substrate Fermentation Soluble Substrates (sugars) - PotentialJy many high value products as enzymes, metabolites, antibiotics. could be produced in SSF. But improvements in engineering and socio-economic aspects are required because processes must use cheap substrate locally available, low technology applicable in rural region, and processes must be simplified. - Potential exists in developed countries, but require close cooperation and exchanges between developing and industrialised countries for funher application of SSF. - The greatest socio-economical potential of SSF is the raising of living standards through the production of protein rich foods for human consumption. Protein deficiency is a major cause of malnutrition and the problem will become worse with further increases in the world population. Two ways can be explored for that: - Production ofprotein-enriched fermented foods for direct human consumption. This alternative involve staIchy substrates for its initial nutritional calorific value. Successful production of such food will require demonstration of econornical feasibility, safety, significant nutritional improvement, and cultural acceprability. - The second alternative consists to produce fermcnted products for animal feeding. Srarchy fermented substrates with protein enrichment could be fed to monogastric animais or poultry. Fermented lignocellulosic substrates by increasing in the fibre digestibility couId be fed to tu- 3 minants. In this case, the economical feasibility should he decisive in comparison to the common model using protein of soybean cake, a by-product of soybean oil. Table 2. Main applications of SSF processes in various economical sectors Economical Sector Agro-Food Industry Examples Application KOJI, ! empeh, Kagl, Attleke, Fennented cheeses Mushroom Production & spawn Agancus, Pleurotus. ~hll-lake [Sugar cane Bagasse Coffee pulp, Silage Bioconversion By-products ComooslinQ. Delo"icalion Flavours, DyeslulJs, EssentIal Food Additives Fat and organic acids ,tleauvena, MelarliùJUm.lnchoBiocontrol , Bioinsecticide denna 1 Gibberelhns, Rhizobium, T nPlant Growth, Honnones chodenna Mycorrhization, Wild Mushroom [Plant InoculatIOn, Amylases, Cellulases, Proleases. Enzymes production Pectinases, Xylanases .. .. Traditional Food Fennentarions 1 Agriculture n"ustna Fennentation 1 Antibiotic production Organic acid Production Ethanol Production Fungal Melllbolites [PeneclllIn, teed & Probiotics [C1tnc aCld Fumaric acid Gallic acid Lactic acid ISchwanOlomyces sp. Starch Maltin o and Brewin o Honnones, Alcaloides. Since 15 years, the Orstom group worked on solid fennenllltion process for improving protein content of cassava and other tropical starchy substrales using fungi, specially from Aspergillus group in order 10 transfonn starch and minerai salts into fungal proteins (Raimbault, 1981). More recently, C. Soccol working al our Orstom laboratory in Montpellier. obtained good results with fungi of the Rhizopus group, of special interest in human traditional fennented foods (Soccol, 1993).These works are now continued in the view of increasing knowledge about specificity of strains of Rhizopus able to degrade the crude granules of starch, what could be simplify drastically the process of SSF. In another hand, the ORSTOM group is collaborating since 1981 with the Mexican UA M group on the following aspects : - Protein enriclunent of Cassava and starchy substrates - Production of organic acids or ethanol by SSF from starchy substrate and Cassava - Digestibility of fibres and lignocellulosic materials for animal feeding 4 - Degradation of caffeine in coffee pulp and ensiling for conservation and detoxification - Enzymes and fungal metabolites production by SSF using sugarcane bagasse Main results will be discussed further in this course by the respective speakers. We are hoping that in the future, an extended collaborative prograrn could be fined for a best interconnection first with ail other Latin-American groups of research involved in SSF, th en tentativeIy, create an international network including American, Asian, European and Australian groups of research. 2. Micro-organisIDs Bacteria, yeasts and fungi can grow on solid substrates, and find application in SSF processes. Filamentous fungi are the best adapted for SSF and dominate in research works. The Table 3 reports sorne examples of SSF processes for each category of micro-organisms involved. Bacteria are mainly invoJved in composting, ensiling and sorne food processes (Ooelle et al.. J 992). Yeasts can be used for ethanol and food or feed production (Saucedo et al., 199 J, 1992). But filamentous fungi are the most important group of micro-organisms used in SSF process owing to their physiological, enzymological and biochemical properties. The hyphal mode of fungaJ gro\Vth and their good tolerance for low Aw and high osmotic pressure conditions make fungi efficient and competitive in natural microflora for bioconversion of solid substrates. Koji and Tempeh are the two most important applications of SSF with filamentous fungi. Aspergillus oryzae is grown on wheat bran and soybean for Koji production, which is the first step of soy sauce or citric acid fermentation. Koji is a concentrated hydrolytic enzymes required in further steps of the fermentation process. Tempeh is an Indonesian fermented food produced by the growth of Rhizopus oligosporus on soybeans. The fermented product is consumed by people after cooking or toasting. The fungal fermentation allows beller nutritive quality and degrades sorne antinutritional compounds contained in the crude soybean. The hyphal mode of gro\vth gives to filamentous fungi a major advantage over uniceUular micro-organisms in the colonisation of solid substrates and for the utilisation of available nutrients. The basic mode of fungal gro\Vth is a combination of apical extension of hyphal tips, plus the generation of new hyphal tips through branching. An important feature is that although extension occurs only at the tip at a linear and constant rate, the frequency of branching make the gro\Vth of the total biomass at exponential kinetic pattern, mainly in the first steps of the vegetative stage. That point is of importance for the modelling of the gro\Vth, and we wiU be discussed further. The hyphal mode of growth gives also the filamentous fungi the power to enter into the solid substrates. The cell wall structure attached to the tip and the branching of the mycelium ensure fmn and sol id structure. The hydrolytic enzymes are excreted at the hyphal tip, without large dilution like in the case of LSF, that makes very efficient the action of hydrolytic enzy- 5 mes and allows penetration into most solid substrates. Penetration increases the accessibility of a1l available nutrients within particles. Table 3 Main groups of micro-organisms involved in SSF processes. Microf1ora ISSfi Process Bacteria Bacillus Sb, Pseudomonas sp. Serraria so Stref)foccus sp. Lactobacillus SP. Clostidrium sp. Composting, Natto, amylase Composting Composting Composting Ensiling, Food Ensiling, Food Yeast Endomycopsis burronii Saccharomyces cerevisiae Schwanniomvces castellii Tape, cassava, rice Food. Ethanol Ethanol. Amylase Fungi Altemaria sp. [Aspen?illus sp. Fusarium sp. Monilia SP. Mucor so. Rhizopus sp. Phanerochaete chrysosporium Trichoderma sp. Beauveria sp., Merharizium so. [Amylomyces rO/.I.Xii IAsoerJ!illus oryzae Rhizoous olif!osoorus AsperRillus niRer Pleurorus oesrrearus. sa;or-caju Lenrinus edodes Penicilium norarum, roqueforrii Composting Composting, Industrial, Food Composting, gibberellins Composting Composting, Food, enzyme Composting, Food, enZYmes, organic acids Composting, lignin degradation Composting, BiologicaJ control, Bioinsecticide Biological control, Bioinsecticide Tape cassava, rice Koji, Food, citric acid Tempeh, soybean, amYlase, liPase Feed, Proteins, Amylase, citric acid Mushroom Shii-(ake mushroom Penicillin, Cheese Fungi can not transport the macromolecular substrate, but the hyphal grovvth allows a close contact between hyphae and substrate surface. The fungaJ mycelium synthesise and excrete high quantities of hydroiytic exoenzymes. The resulting contact catalysis is very efficient and the simple products are in close contact to the mycelium where they can enter across the œil membrane for biosynthesis and fungal metabolic activities. This contact catalysis by enzymes can expIain the logistic model of fungaJ growth commonly observed (Raimbault, 1981). Aiso that point will be discussed further. 6 3. Substrates All solid substrates have a common feature: their basic macromolecular structure. In generaL substrates for SSF are composite and heterogeneous products From agriculture or by-products of agro-industry. This basic macromolecular structure (e.g. cellulose. starch, pectin, lignoceJlulose, fibres etc ..) confers the properties of a solid to the substrate. The structural macromolecule may sim ply provide an inert matrix within which the carbon and energy source (sugars. lipids, organic acids) are adsorbed (sugarcane bagasse, inert fibres, resins). But generally the macromolecular matrix represents the substrate and provide also the carbon and energy source. Preparation and pre-treatment represents the necessary steps to convert the raw substrate into a form suitabJe for use, that include: -size reduction by grinding, rasping or chopping - physical chemical or enzymatic hydrolysis of polymers to increase substrate availabiliry by the fungus. - supplementation with nutrients (phosphorus. nitrogen, salts) and adequation to pH and moisture content, through a minerai solution - Cooking or vapour treatment for macromolecular structure pre-degradation and elimination of major contaminants. Pre-treatments will be discussed under individual applications. The most significant problem of SSF is the high heterogeneiry which makes difficult to focus one category of hydrolytic processes, and leads to poor trials of modelling. This heterogeneity is of different nature: - non-uniform substrate structure (mixture of starch, lignocellulose, pectin) - Variability between batches of substrates limiting the reproducibility - Difficulty of mixing solid mass in fermentation, in order to avoid compactation, which causes non Wliform cultivation, gradients of temperature, pH and moisture with virtual impossibility to obtain a representative sam pie. Each macromolecular type of substrate presents different kind of heterogeneity: LignocelluIose occurs within plant cell walls which consists of cellulose microfibrils embedded in lignin, hemicellulose and pectin. Each category of plant material contain variable proportion of each chemical compounds. Two major problems can iimit lignocellulose breakdown: - cellulose exists in four recognised crystal structures known as celluloses UI,I11 and IV. Various chemical or thermaltreatment can change the amorphous form of cristalinity. - different enzymes are necessary in order degrade cellulose, e.g. endo and exo-celluJases plus cellobiase Pectins are polymers of galacturonic acid with different ratio of methylation and branching. Exo-and endo pectinases and demethylases hydrolyse pectin in galacturonic acid and metha- 7 nol. HemicelJulases are divided in major three groups: xylans, mannans and galaclans. Most of hemicellulases are heteropolymers containing two to four different types of sugar residue. Lignin represents between 26 to 29% of 1ignocelJulose, and is strongly bounded to cellulose and hemicelJulose, ruding them and protecting them from the hydrolase attack. Lignin peroxydase is the major enzyme involved in lignin degradation. Phanerochaele chrysosporium is the most recognised fungi for 1ignin degradation. So the lignocellulose hydrolysis is a very complex process. Effective cellulose hydrolysis requires the synergetic action of several cellulases, hemicellulases and Iignin peroxydases. But lignocellulose is a very abundant and cheap, natural, renewable material. so a lot of works were dedicated to micro-organisms breakdown, specially fungal species. Starch is another very imponant and abundant natural solid substrate. Many microorganisms are ca a I l hydrol se starch, but generally tile efficient hydr I:sis requires previous gelatinization. Some recent works concem the raw (crude or native) starch like it occurs naturally. The chemical structure of starch is relatively simple compared to lignocellulose substrates. Essentially starch is composed of two related polymers in different proponion folJowing plant material: amylose (16-30%) and amylopeclin (65-85%). Amylose is a polymer of glucose Iinked in a -1,4 bonds mainJy in linear chains. AmyJopectin is a large higWy branched polymer of glucose including a1so a -1,6 bonds al the branch points. Within the plant, cell starch is stored in the form of granules located in amyloplasts. intracellular organelles surrounded by a Iipoprotein membrane. Starch granules are highly variable in size and shape depending on the plant material. Granules contain both amorphous and crystalline internai regions in respective proponions of about 30170 . During the process of gelatinization, starch granules swell when heated in the presence of water, wruch involves the breaking of hydrogen bonds, especially in the crystalline regions. Many micro-organisms can hydrolyse starch , specially fungi wruch are sui table for SSF application involving starchy substrates. GJucoamylase, a-amylase, b-amylase, puliulanase and isoamylase are involved in the processes of starch degradation. Mainly a-amylase and glucoamyiase are of importance for SSF. a-amylase is an endo-amylase attacking a-l,4 bonds in random fashion which rapidly reduce molecular size of slarch and consequently its viscosity and liquefaction. GJucoamylase occurs almost exclusively in fungi including Aspergillus and Rhizopus groups. This exo amylase produces glucose units from amylose and amylopectin chains. Micro-organisms generaJly prefer gelatinised starch. But large quantity of energy is required for gelatinization, and it would he anractive to use organisms growing weil on raw (ungelatinised) starch. Different works are dedicate to isolale fungi producing enzymes able to degrade raw stareh, as has been done by Soccol el al (1991). Bergmann et al. (1988) and Abe et al. (1988). 8 In our lab we developed many studies conceming SSF of cassava, a very common tropical starchy crop, in the view of upgrading protein content, both for animal feeding using Aspergillus sp. or better for direct human consurnption, using Rhizopus. Table 4 indicates the protein enrichment with different fungi. Table 4. Protein enrichment of Cassava by various selected strains of fungi. (Raimbault et aL, 1985) - 1-..- .......- ._ . . _1. _ 12 ....... _ _ WI40 ~ ..- .... _7 ~~~ ~ .. _TI ~"'_SI ~ .. _14 ~_ ~ .. ~ ~ ... IU _WIOI .. _72 _ _ I' ..-. ~ . . _WI47 ............. _17 ~ ()) e.-a 2S Xf/l III ~("'cIoy-) Pt-.. ~ .. _WI:I T.... _ ILS 111.' n.e n.1 lAjl 1!o6 -.. -.. I!.I 21.5 S2.J JllS 30... .. 1 Jt.o Jt.S ..... •a ...... .,.... •• ..., e-.. • .,.... _~_27 ~ - no.. 0...... lAjl 10 '4.' 14.1 14.7 III <:-. III III JIl • ,... .,.... fi - Il 10 Il ~ III Ilaw_ loilIoI _ _ 50%. _ _ 14.' 14.2 14.1 .4.. 1S.1 IS.O 11.7 IS.. 11.1 10.' 2.30 ",-arc. P.' «1.1 1l.4 :&2 ... n4 f!.2 ... 4Cl.0 to.oo Recently good results were obtain by Soccol for the protein enrichment of cassava and cassava bagasse using selected strains of Rhizopus, producing biotransfonned starchy flours containing 10-12% of good protein, comparable to cereal. Such biotransfonned Cassa va flour can be used as cereal substitute for breadmak..ing until 20% without sensible change for the consurner . 4. Biomass Measurement Biomass is a fundamental parameter in the characterisation of rnicrobial growth. 1ts measurement is essential for k.inetic studies on SSF. Direct detennination of biomass in SSF is very difficult due to problems of separation of the microbial biomass from the substrate. This is especially true for SSF processes involving fungi, because the fungal hyphae penetrate into and bind the mycelium tightly to the substrate. On the other hand, for the calculation of growth rates and yields it is the absolute amount of biomass which is important. Methods that have been used for biomass estimation in SSF belong to one of the following categories. Direct evaluation of biomass Complete recovery of fungal biomass is possible only under artiticial circumstances in membrane tiller culture, because the membrane tilter prevents the penetration of the fungal 9 hyphae into the substrate (Mitchell et al, 1992). The whole of the fungal myceliwn can be recovered simply by peeling it off the membrane and weighing it directly or after drying. This method obviously canoot be used in actual SSF. However, it could find application in the calibration of indirect methods of biomass determination. Indirect biomass estimation methods should be calibrated under conditions as similar as possible to the actual situation in SSF. The global myceliwn composition could be appreciate through analysis of the myceliwn cultivated in LSF in conditions as close as possible than SSF cultivation. Microscopic observations can also represent good way to appreciate fungal growth in SSF. Naturally, optic examination is not possible at high magnitude but only at stereo microscope. Scanning Electron Microscope (SEM) is an useful manner to observe the mode of grov.·1h in SSF. New approach and researches are developed for image analysis by computing software in order to evaluate the totallength or volwne of mycelium on SEM photography. Another nel,',' approach very promising is the ConfocaJ Microscopy based on specific reaction of fungal biomass with specific fluorochrome probes. Resulting 3D images of biomass can open new way to appreciate and may be measure biomass in situ in a near future. Anyway, direct measurement of exact biomass in SSF is a very difficult question, then other approaches were preferred by workers. For that we can consider the global stoechiometric equation of the microbial growth: Carbon source + Water + Oxygen + Phosphorus + Nitrogen ! Biomass + C02 + Metabolites + Heat Each component is under strict variation of others when ail coefficients are maintained constants. For that measuring one ofthem can indicate the evolution of the others. Metabolie measurement of tire biomass - Respiratory metabolism Oxygen consumption and carbon dioxide release result from the respiration, the metabolic process by which aerobic micro-organisms derive most of their energy for growth. These metabolic activities are therefore growth associated and can be used for the estimation of biomass biosynthesis. As carbon compounds within the substrate are metabolised, they are converted into biomass and carbon dioxide. Production of carbon dioxide causes the weight of fermenting substrate to decrease during growth, and the amount of weight lost can be correlated to the amount of growth that has occurred. Growth estimation based on carbon dioxide release or oxygen consumption asswnes that the metabolism of these compounds is completely growth associated, which means that the amount of biomass produced per unit of gas metabolised must be constant. Sugarna and Okazaki (1979) reported that the ratio of mg C02 evolved to mg dry mycelia formed by Aspergillus oryzae on rice ranged from 0.91 to 1.26 mg C02 per mg dry mycelium. A graduai 10 increase in the ratio was observed late in growth due to endogenous respiration. Drastic changes can be observed for the respiratory quotient which conunonly changes with the 21 0.00 <lOQ 00 00000 18 ocP 0,\\ Cf ~' ,cPo' 15 )c~1 cf <}? N 12 0 ~ N 0 1 oocP°''\, 9 u 6 3 0 ooa 0 .0 0000000 l Jl" 1 1 12 24 cf \ '\00" '00, 0'0 48 36 60 72 000 ~o 84 Tempo (h) Fig. 1.- Kinetic evolution of C02 and 02 in air flow during fermentation of Rllizopus on cassava. groWlh phase, i.e: germination, rapid and vegetative groWlh, secondary metabolism. conidiation and degeneration of the myceliwn. The measurement of either carbon dioxide evolution or oxygen conswnption is most powerful when coupled with the use of a correlation mode!. The terrn correlation model is used here to denote a model which correlates biomass with a measurable parameter. Correlation models are not groWlh models as such since they make no predictions as to how the measurable parameter changes with time. The usefulness of correlation models is that by following the profile for the change in the parameter during growth, a biomass profile Can be constructed. Application ofthese correlation models involving prediction ofgroWlh from oxygen uptake rates or carbon dioxide evolution rates requires the use of numericaJ techniques to solve the differential equations. A computer and appropriate software is therefore essentia!. If both the monitoring and computational equipment is available then these correlation models provide a powerful meanS of biomass estimation since continuous on-fine measurements can be made. Other advantages of monitoring effluent gas concentrations with paramagnetic and infrared analysers include the ability to monitor the respiratory quotient to ensure optimal substrate oxidation, the ability to incorporate automated feedback control over the aeration rate, and the non-destructi ve nature of the measurement procedure. The metabolic activity in SSF is so important that we have dedicated a special lecture to study ail theoretical and practical aspects of respirometric measurement of fungal biomass cultivated II in SSF. Other speaker also will present a lot of data conceming lab and scale up experiences of respirometric measurement for several applications. More, during the practicaJ training on the aftemoons, you shall practice the laboratory methodology that we have specially design to study fungal growth on SSF based on the gas chromatography analysis of the effluent gas. - Production of extracellular enzymes or primarv metabolites Another metabolic activity which may be growth associated is extracellular enzyme production. Okazaki and co-workers (1980) claim that the a-amylase acti vity was directl)' proportional to mycelial weight for Aspergillus oryzae grown in SSF on steamed rice. For growth of Agaricus bisporus on mushroom compost, mycelial mass was directly proportional to the extracelluJar laccase activity for 70 days (Wood, 1979). In our works we observed generally a good adequation between growth and hydrolytic enzymes as amylases, cellulases or pectinases (see annexed list). In another hand, we observed frequently a good correlation between mycelial growth and organic acid production, which can be measured by th pH me m n or a posreriori orrelted by HPLC analysis on extracts. In the case of Rhizopus, Sacco1 demonstrated a close correlation between fungal protein (Biomass) and organic acids (citric. fumaric, lactic or acetic). Biomass Components The biomass can also be estimated from measurements of a specific component. until the composition of the biomass is constant and stable and the fraction of the component be representative. Protein content: The most readily measured biomass component is protein. We used the protein content (as deterrnined by the Lowry method) to measure the growth rate of Aspergillus niger on cassava meal ( Raimbault and Alazard, 1980). For growth of Chaelomium cellulolylicum on wheat straw the TCA insoluble nitrogen was deterrnined using the Kjeldahl method (Laukevics et al. 1984), biomass protein was then calculated as 6,25 times this value. In all cases the protein content of the biomass was assumed to be constant. Biomass protein contents measured b y the biuret method were consistent with those measured by the Kjeldahl method. But unfortunately the biuret method was not suitable for application to SSF itself because of non-specific interference by the starch from the substrate. The Folin method is more sensitive and allowed a greater dilution of the sarnple which avoided interference from the starch in the substrate. For that we choose the Folin technique to measure protein enrichrnent in starchy substrates . Nucleic acids DNA production has been used to estimate the biomass of Aspergillus oryzae on rice (Bajracharya & Mudgen. 1980). The method was calibrated using the DNA contents of fungaJ mycelia obtained in submerged culture. DNA contents were higher during early growth and then decreased. levelling off as stationary phase was approached. The method was corrected for the DNA content of the rice, which did not change since Aspergillus oryzae did not produce extracellular DNases. DNA or RNA methods are reliable only ifthere is linle nucleic acid in the substrate. and if no interfering chemicals are present. 12 Glucosamine A useful method for the estimation of fungal biomass in SSF is the glucosamine method. This method takes advantage of the presence of chitin in the cell walls of many fungi. Chitin is a poly-Nacetylglucosamine. Interference with this method may occur with growth on complex agricultural substrates containing gJucosamine in glucoproteins (Aidoo et al, 1981). The accuracy of the glucosamine method for determination of fungal biomass depends on establishing a reliable conversion factor relating glucosamine to mycelial dry weight (Sharma et al, 1977). However, the proportion of chitin in the mycelium will vary with age and the environmental conditions. Mycelial glucosamine contents ranged from 67 to 126 mg per g dry mycelium. Another disadvantage of the glucosamine method is the tedious extraction procedures and processing times of over 24 hours which make it inconvenient to perform. Ergosterol Ergosterol is the predominant sterol in fungi. Glucosamine estimation was therefore compared with the estimation of ergosterol for determination of the growth of Agaricus bisporus (Matcham et al, 1985). In solid cultures directly proportional relationships for glucosamine and ergosterol against linear extension of the mycelium were obtained. Determination of ergosterol was claimed to be more convenient than glucosarnine. Il couId be recovered and separated by HPLC and quantified simply by spectrophotometer, providing a sensitive index of biomass at low levels of growth. HPLC was necessary to separa te the ergosterol from sterols endogenous to the sol id substrate. However, Nout et al. (1987) showed that the ergosterol content of Rhizopus oligosporus varied from 2 to 24 micrograms per mg dry biomass, depending on the culture conditions. Ergosterol content was influenced by aeration. phase of growth and substrate composition. They concluded that it was an unreliable method for following growth of Rhizopus otigosporus in SSF. Physical measurement of biomass Peiialoza (1990) used another physical parameter to evaluate mycelial growth, based on the difference in the electric conductivity between biomass versus the substrate. Good correlation with biomass was obtained and a model was proposed. Recently Auria et al.( 1990) monitored the pressure drop in a packed bed du ring SSF of Aspergillus niger on a model solid substrate consisting of ion exchange resin beads. Pressure drop was closely correlated with protein production. Pressure drop is a parameter which is simple to measure and can be measured on-line. Further studies are required to determine whether the use of pressure drop in monitoring grow1h in forcefully aerated SSF bioreactors is generally applicable. An interesting point of this physical technique resides in the fact that it is sensible to the conidiation: early conidiophore stage make the pressure drop drastically and a breaking point can be easily observed. ln conclusion, the measurement of biomass in SSF is important to follow the kinetics of growth in relation to the metabolic activity. Measurement of metabolic activity by carbon 13 dioxide evolution or oxygen consumption can be generally applied, whereas extracellular enzyme production will only be useful when enzyme production is reasonably growth-associaled. Vital staining with fluorescein diacetate has potential in providing basic infonnation as to the mode of growth of fungi on complex solid surfaces as this method can show the distribution of metabolic activity within the mycelium. But it can not be measured on line. On the other hand, in the production of protein enriched feeds, the protein content itself is of greater importance than the actual biomass concentration, and the variation in biomass protein content during growth becomes less relevant. Overall, oxygen uptake and carbon dioxide evolution methods are probably the most promising techniques for biomass estimation in aerobic SSF as they provide on-line infonnation. The monitoring and computing equipment is relatively expensive and will not be suitable for low technology or rural applications. None method is ideally suiled 10 all siluations so the method most appropriate ta the particular SSF application must be chosen on the basis of simplicity, cost and accuracy. The best choice could be to cross two or three, or more, techniques for measurement of various parameters, and the total balance could be highly correlated to the actual biomass. 5. Environmental Factors Envirorunental factors such as temperature, pH, water activity, oxygen levels and concentrations of nUlrients and products significanlly affect rnicrobial growth and producl fonnation. ln submerged stirred cultures environrnental control is relatively simple because of the homogeneity of the suspension of rnicrobial cells and of the solution of nutrients and products in the Iiquid phase. The low moisture content of SSF enables a smaller reactor volume per substrate mass to be used for microbiaJ cultivation than with submerged cultures and also simplifies recovery of the product (Moo- Young et al., 1983). Serious problems, however, are encountered in respect of rnixing, heat exchange, oxygen transfer, moisture control and the localisation of pH gradients and nutrient and product leveJs as a consequence of the heterogeneity of the culture. The laner characteristic of SSF renders the measurement and control of the above mentioned parameters difficult, laborious and often inaccurale. thereby limiting the induslrial pOlential of this technology (Kim et al., 1985). Due to these problems, the micro-organisms that have been selected for SSF are more tolerant to a wide range of cultivation conditions (Mudgett, 1986). Moisture content and Water activity (Aw) SSF process can be defmed as microbial growth on solid particles without presence of free water. The water present in SSF systems exists in a complexed form within the solid matrix or as a thin layer either absorbed to the surface of the particles or Jess tightly bound within the capillary regions of the solid. Free water will only occur once the saturation capacity of the solid matrix is exceeded. The moisture level al which free moisture becomes apparent varies 14 considerably between substrates, however, and is de pendant upon their water binding characteristics. For example, free water is observed when the moisture content of solid substrates such as maple bark exceeds 40% and when it exceeds 50-55% in rice and cassava (Oriol et al, 1988). With most lignocellulosic substrates free water becomes apparent before the 80% moi sture level is reached (Moo- Young et al, 1983). The moisture levels in SSCF processes which vary between 30 and 85% has a marked effect on the growth kinetics, as shown on Figure 1 (Oriol et al, 1988). The optimum moi sture level for the cultivation of Aspergillus niger on rice was 40%, whereas on colTee pulp the level \Vas 80%. which illustrates the unreliability of moisture level as a parameter for predicting the growth of a micro-organism. Il is now generally accepted that the water requirements of microorganisms should be defined in lenns of the waler activity (Aw) of the environment rather than the water content of the solid substrate. This parameter is defined by lhe ralio of the vapour pressure of the water in the substrate (p) to the vapour pressure of pure water (Po) at the same temperature, i.e Aw = p/po. This concept is related to other parameters such as relative humidity (%RH = 100 x Aw) and water potentiaJ (psi = RTN. ln Aw; where R is the ideal gas constant, T is the absolute temperature and V is the mol volume of waler) (Griffin, 1981). .45 - ..'.c. 1C1D 4I E 1pO aw FIg. 2 ~vol,:,tiOr1. of the specifie growth rate ( - . - ) and of the germination Ume (-0 -) as a funetion of the initial water aetivity of the medium The reduction of Aw has a marked effect on microbial growth. Typically, a reduction in A w extends the lag phase, decrease the specific growth rate, and results in low amount of biomass 15 produced (Oriol et al. 1988) as it is shown in fig.2. In general, bacteria require higher values of Aw for growth than fungi, thereby enabling fungi to compete more successful1y at the A w values encountered in SSC processes. With the exception of halophilic bacteria., few bacteria grow at Aw values below 0.9 and most bacteria investigated show considerably higher nùnimum Aw values for growth. Sorne fungi. on the other hand, only stop growing at A w values as low as 0.62 and a number of fungi used in SSC processes have minimum growth A w values between 0.8 and 0.9 . The optimum moi sture content for growth and substrate utilisation is between 40 and 70% but depended upon the organism and the substrate used for cultivation. For example. cultivation of Aspergillus niger on stafchy substrates such as cassava (Raimbault & Alazard, 1980) and wheat bran (Nishio et al. 1979) was optimal at moisture levels considerably lower than on coffee pulp (Penaloza et al. 1985) or sugarcane bagasse (Roussos et al., 1989), possibly because of the greater water holding capacity of the latter substrate (Oriol et al. 1988). The optimum Aw for growth of a limited number of fungi used in SSF processes was at least 0.96 whereas the nùnimum growth Aw was generally greater than 0 9. This suggests that fungi used in F processes are not especiall xerophi1ic. The optimum Aw values for sporulation by Trichoderma viride and Penicillium roqueforti were lower than those for growth (Gervais et al.. 1988). Maintenance of the Aw at the growth optimum would pennit fungal biomass to be produced without sporulation. Tempera/ure and Real Transfer Stoechiometric global equation of respiration is highly exothennic and heat generation by high levels of funga1 activity within the solids lead to thennal gradients because of the limited heat transfer capacity of solid substrates. In aerobic processes, heat generation may he approximated from the rate or C02 evolution or 02 consumption. Each mole of C02 produced during the oxidation of carbohydrates released 673 Kca! . That is for why it is of high interest to measure C02 evolution during a SSF process, because it is directly relied to the risk of elevation of temperature. Detailed calculation of the relation between respiration, metaboJic heat and temperature were discussed in early works on SSF with Aspergillus niger growing on cassava or potato starch (Raimbault, 1981). The overall rate or heat transfer may be limited by the rates of intra- and inter-partic le heat transfer, by the rate at which heat is transferred from the particles surface to the gas phase. The thennal characteristics of organic material and the low moisture content in SSF are special difficult conditions for heat transfer. SaucedoCastaneda and co-workers ( 1990), developed a mathematical model for evaJuating the fundamental heat transfer mechanism in static SSF and more specifical1y to assess the importance of convection and conduction in heat dissipation. Saucedo will explain in his lecture how this model could be used as a basis for automatic control of static bioreactors. Heat removal is probably the most crucial factor in large scale SSF processes , and conventional convection or conductive cooling devices are inadequate for dissipating metabolic hem due to the poor thennal conductivity of mosl solid subslrales and result in non acceptable temperalure gradients. Only evaporative cooling devices provide sufficient heat elimination. Although the primary function of aeration during aerobic solid stale cultivations was to supply oxygen for cel! growth and to flush out the produced carbon dioxide, it also serves a critical function in heat and moisture transfer between the solids and the gas phase. The most 16 efficient processes for tempe rature control consists in evaporating water, what needs in return to complete the 10ss to avoid desiccation. Maintaining a constant temperature and moisture content in large scale solid substrate cultures is generaJly difficult , but as you will realise sorne alternative equipment begin to fit that function, and al! that will be discussed by Perez and Saucedo. The reactor type can have a large influence on the quaJity oftemperature control achieved. It depends highly of the type of SSF: static on clay or vertical exchangers, drums or mechanicaJly agitated with parameters controls, ail that aspect will be discussed in cessions about Engineering aspects of SSF. pH control and risks of contamination. The pH of a culture may change in response to nUcrobiaJ metabolic acllVllles. The most obvious reason is the secretion of organic acids such as citric, acetic or lac tic acids, which will cause the pH to decrease, in the sarne way than anunonium salts consumption. On the other hand, the assimilation of organic acids which may be present in certain media will lead to an increase in pH, and urea hydrolysis result in an alcalinisation. The changes in pH kinetics depends also higWy on the micro-organism. With Aspergillus sp., Penicillium sp. , and Rhizoplls sp. the pH can drop very quickly untilless than 3.0; for another type of fungi, Iike Trichoderma, Sporotrichum. Pleurotus sp. the pH is more stable berween 4 and 5. Besides, the nature of the subsrrate influence higWy pH kinetics, due to the buffering effect of lignocellulosic materials. In our case we used a mixture of anunonium salt and urea to regulate the pH decrease during A. niger growth on starchy substrates (Raimbault, 1980). A degree of pH control may be obtained by using different ratios of anunonium salts and urea in the substrate. Hydrolysis of urea Iiberates anunonia, which counteracts the rapid acidification resulting from uptake of the anunonium ion (Raimbault & Alazard. 1980). ln this manner, we obtained optimal growth of Aspergillus niger on granulated cassava meaJ when using a 3:2 ratio (on a nitrogen basis) of anunonium to urea. We observed that during the first stage of the cultivation the pH increased as the urea was hydrolysed. During the subsequent rapid growth arrunonium assimilation exceeded the rate of urea hydrolysis and the pH decreased, but increased again in the stationary phase. During the cultivation the pH remained between the limits of about pH 5 to pH 6.2, whereas a lower urea concentration resulted in a rapid decrease in pH. ln a sarne way, pH adjustment during the cultivation of Trichoderma viride on sugar-beet pulp by spraying with urea solutions was effective due to the urease activity of the microorganism causing an increase in pH at pilot plant level experimentation (Durand et al. 1988). Finally, in a process of cultivation of filarnentous fungi or yeasts, bacterial contamination may be minimised or prevented by employing a suitably low pH. Aeration Aeration fulfils four main functions in sol id state processes, namely (i) to maintain aerobic conditions, (ii) for carbon dioxide desorption, (iii) to regulate the substrate temperature and (iv) to regulate the moisture level . The gas environment may significantly affect the relative leveJs of biomass and enzyme production. ln aerobic submerged cultures oxygen supply is 17 often the growth limiting factor due to the low solubility of oxygen in water. ln contrast, a solid state process allows free access of atmospheric oxygen to the substrate, aeration may he easier than in submerged cultivations because of the rapid rate of oxygen diffusion into the water film surrounding the insoluble substrate particles and also the very high surface of contact between gas phase, substrate and aerial mycelial. The control of the gas phase and air flow is a simple and practical mean to regulate gas transfer and generally no oxygen limitation are observed in SSF processes. when the solid substrate is particular. Il is important to maintain a good balance between the three phases gas, liquid and gas in SSF processes (Auria, 1989; Saucedo et al. 1984). Modelling mass transfer in SSF is a key to keep good conditions for the development of the mycelium. By this very simple aeration process, it is also possible to induce metabolic reaction, either by water stress, heat stress or temperature changes, al! processes that can be drastically change biochemical, physiological or metabolic behaviour. 6. Conclusion SSF is a well adapted process for cultivation of fungi on natural vegetal materials which are breakdown by excreted hydrolytic enzymes. In contrast with LSF, in SSF processes. water related to the water activity is a limiting factor, both parameters no involved in LSF where water is in large excess. On the other hand, oxygen is a limiting factor in LSF but not in SSF where aeration is facilitated by the porous and particular structure and high surface contact area which facilitate transfers between gas and liquid phases. SSF are aerobic processes where respiration is a predominant processes for energy supply to the mycelium; but it can cause severe limitation of the growth when heat transfer is not efficient enough causing rapid elevation of the temperature. Is the reason why it is so important to study and control respirometry in SSF . We developed a laboratory technique to measure C02 and 02 on fine in SSF. A special lecture will be dedicated to the theory, modelling and basic concept of respirometry. Also it will be organise training cessions at the lab, to practice respirometric measurement and kinetics analysis. References - Alazard, D. and Raimbault, M. 1981. Comparative study of amylolytic enzymes production by Aspergillus niger in liquid and solid state cultivation Eur. J Appt. Microbiol. Biolechnof. 12:113-117. - Auria, R., Hemandez, S., Raimbault, M. and Revah, S. 1990. Ion exchange resin: a model support for solid state growth fermentation of Aspergillus niger. Biolechnof. Techniques. 4: 391-396. - Deschamps, F., Raimbault, M. and Senez, J.c. 1982. Solid state fermentation in the development of agro-food by-products. lndusry & Environ. 5 (2): 27-30. - Doelle H. W., Mitchell DA & Rolz C.E. (1992). Solid Substrate Cultivation. Eisiever Sci. Pub!. Itd;London & New York; 466 p. - Durand A. & Chereau D. 1988. A new pilot reactor for solid state fermentation: application to the protein enrichment of sugar beet pulp. Biotechnol. Bioeng. 31: 476-486. 18 - Durand, A., Renaud, R., Maratray, J., Almanza, S. 1997. The INRA-Dijon Reactors: Designs and applications. In Roussos, S., Lonsane, B.K., Raimbault, M. and ViniegrazGonzalez, G. (Eds.), Advances in solid slale fermenlalion, Kluwer Acad. Pub!.. Dordrecht. chapter 7 pp. 71-92. - Moo- Young M., Moriera A.R. & Tengerdy R.P. 1983. Principles of solid state fermentation. In The filamentous fungi, Vol. 4, Fungal Biotechnology. Smith lE, Berry D.R & Kristiansen B. Eds., Edward Arnold Publishers, London, pp. 117-144. - Oriol, E., Schettino, B., ViIÙegra-Gonzalez, G. and Raimbault, M. 1988a. Solid- state culture of Aspergillus niger on support. J Fermenl. Technol. 66: 57-62. - Oriol, E., Raimbault, M., Roussos, S. and ViIÙegra-GonzaJes, G. 1988b. Water and water activity in the solid state fermentation of cassava starch by Aspergillus niger. Appl. Microbiol. Biolechnol., 27: 498-503. - Raimbault M. - (1981). "Fermentation en milieu solide: croissance de champignons filamenteux sur substrats amylacés". Ediled by: ORSTOM-Paris; Série Travaux et Documents n° 127; 291 p. - Raimbault, M. and Alazard, D. 1980. Culture method to study fungal groWlh in solid fermentation. Eur. J Appl. Microbiol. Biolechnol. 9: 199-209. - Raimbault, M., Revah, S., Pina, F. and Villalobos P. 1985. Protein enrichment of cassava by solid state fermentation using molds isolated From traditional foods. J Ferment. Technol. 63: 395-399. - Roussos, S., Olmos, A., Raimbault, M., Saucedo-Castaiieda, G. and Lonsane, B.K. 1991. Strategies for large scale inocuJum development for solid state fermentation system : Conidiospores of Trichoderma harzianum.. Biolechnol. Tech. 5: 415-420 - Roussos, S., Raimbault, M., ViIÙegra-GonzaJez, G., Saucedo-Castafieda. G. and Lonsane. B.K. 1991. Scale-up of cellulases production by Trichoderma harzianum on a mixture of sugar cane bagasse and wheat bran in solid state fermentation system Micol. Neolrop. Apl. 4 : 83-98. - Roussos, S., Raimbault, M., Prebois, J-P. and Lonsane, B.K. 1993. Zymotis, A large scale solid state fermenter: Design and evaluation Applied Biochem. Biolechnol. 42: 37-52. - Saucedo-Castaiieda, G., Gutierrez-Rojas, M., Bacquet, G., Raimbault, M. and ViniegraGonzalez. G. 1990. Heat transfert simulation in solid substrate fermentation. Biolechnol. Bioeng. 35: 802-808. - Saucedo-Castaiieda, G., Lonsane, B.K., Navarro, J.M., Roussos, S. and Raimbault, M. 1992a. Potential of using a simple fermenter for biomass built up, starch hydrolysis and ethanol production: Sol id state fermentation system involving Schwanniomyces caslellii , Appl. Biochem. Biolechnol. 36: 47-61. - Saucedo-Castaneda, G., Lonsane. B.K., Krishnaiah, M.M., Navarro, J.M., Roussos, S. and Raimbault, M. 1992b. Maintenance of heat and water balances as a scale-up criterion for the production of ethanol by Schwanniomyces caslel/ii in a solid state fermentation system. Process Biochem. 27: 97-107 - Senez, J.c., Raimbault, M. and Deschamps, F. 1980. Protein enrichment of starchy substrates for animal feeds by solid state fermentation. World Animal Rel'. 35: 36-40. - Saccol, c., Marin, B., Raimbault, M. and Lebeault, lM. 1994. Potential of solid state fermentation for production of L( +) lactic acid by Rhizopus oryzae. Appl. /vlicrobiol. Biolechnol. 41: 286-290. 19 - Trejo-Hemandez, M.R., Raimbault, M., Roussos, S. and Lonsane, B.K. 1992. Potencial of solid state fennentation for production of ergot alka1oids. Let. Appl. Microbiol. 15: 156-159. - Trejo-Hemandez, M.R., Lonsane. B.K., Raimbault, M. and Roussos, S. 1993. Spectra of ergot a1kaloids produced by C/aviceps purpurea 1029c in solid state fennentation system: Influence of the composition of liquid medium used for impregnating sugar cane pith bagasse. Process Biochem. 28: 23-27. 20 CONTINUOUS ENZYMES AND FUNGAL METABOLITES PRODUCTION IN SOLID STATE FERMENTAnON USING A COUNTER-CURRENT REACTOR S. Roussos 1 and D.L. Pyle 2 Laboratoire de Biotechnologie, Centre ORSTOM, BP 5045; 34032 Montpellier Cedex 1, France Biotechnology and Biochemical Engineering Group Department of Food Science and Technology, University of Reading, Whiteknights PO BOX 226, Reading RG6 6AP, UK. 1 2 Abstract: This work presents the continuo us production of fungal biomass and enzymes by so!id state fermentation (SSF) in a counter-CUITent reactor adapted for this purpose. Pre-gemlinated conidia of Aspergillus niger were used as an inoculum and sugarcane bagasse. embedded \Vith a nutritive solution, was the solid support. The Solids residence time distribution (RTD) was carried out by feeding one impulse of blue-coloured hurnidified bagasse and this RTD was fixed at 20 hours. This study demonstrated that the values of the measured parameters (pH, moisture, biomass, glucoamylases production) were sinlilar to those reported for batch SSF using the same solid support and nlicro-organism. A marked increase in biomass occurred from the progressive comparnnent (from comparnnent no.l to no.9) into the reactor and the enzyme production was important (40 IU/g dry exit solids). No mycelium damage or sporulation was observed. The above results confirmed that the continuous production of enzymes by SSF under no sterile conditions was successful. Inoculation with pre-germinated conidia shortened the processing time and allowed control of the age of the mycelium in each compartmenl. Aeration was accomplished by natural convection and moisture content had to be controlled. This process can be applied to the continuous production of fungal biomass and metabolites in SSF with industrial applications using environmentally friendly biotechnology. Key words: Solid State fermentation, Continuous enzyme production, glucoamylases, A.lliger, pre-germinated conidia, counter-current reaClor, fungal metabolite. Definition of SSF: Solid State Fermentation (SSF) is a microbial process occurring mostly on the surface of solid materials that have the property to absorb or contain water, with or without soluble nutrients (Viniegra Gonzalez 1997). The solid materials could other be biodegradable or nol. For example, starch and cellulose are solid materiaJs of the first type, whereas, amberlite or polyurethane belong to the second type (Alazard and Raimbault 1981; Moo-Young el al. 1983, Barrios Gonzalez el al. 1988, Oriol el al. 1988a. Auria el al 1990, GonzaJez-Blanco el al. 1990, Roussos el al 1991). Advaotages and disadvantages of SSF: Solid state fermentation offers various advantages in comparison with submerged ones (Aido el al. 1982, Lonsane el al. 1992). Aeration is facilitated through the spaces between the substrate (Lambraki el al 1994, Soccol el al 1994). Substrate agitation, when necessary, is discominued (Senez el al J980, Deschamps el al 1982). The absence of a liquid phase and a low water content permit a) reduction of fermentor volume of liquid effluents from the process, b) reagents saving during metabolites recovery, c) 21 reduction of bacteria contamination and d) use of no sterile solid substrate in sorne cases. Culture media are simple mainly composed of agro-indusoial residues (Lonsane et al. 1985. Roussos et al. 1991). Culture growth conditions are close to those in me natural environrnent (Roussos et al. 1997). Its main disadvantages are me following: a) risks of high temperature rise (Saucedo-Castai1eda et al. 1990, Rodriguez et al. 1991, Saucedo et al. 1992a), b) difficult)' in parameter regulation (Durand et Cherau 1988), c) need of pre-treatment of solid material (Raimbault et al. 1985) d) high 10ss of hurnidity in fennentations lasting of long, e) necessity for high inoculation when natura1 microflora is not used (Roussos et al. 1991), and f) critical role of water and water activity (Oriol et al. 1988b, Gervais and Bensoussan 1995). Metabolites production in SSF: There has been a considerable amount of attention given to the physiology of me micro-organisms invo1ved and me characteristics of the metabolites produced (Trejo -Hernandez et al. 1993, Gutierrez-Rojas et al. 1995). Culture of filamentous fungi on solid supports has been applied to the production of enzymes, primary and secondary metabolites (Oriol et al. 1988b; Saucedo-Castai1eda et al. 1992b; Trejo-Hernandez et al. 1992; Christen et al. 1995). It has also been used for me detoxification of a wide variety of materials (Aquiahuat1 et al. 1988). Bioreactors: Considering to all these aspects mentioned above, bioreactors have been developed traditionally for different purposes and SSf has been carried out as a batch process in laboratory sca1e (Raimbault and Alazard 1980; Lepilleur et al. 1997), in pilot plant scale (Deschamps et al. 1985; Lonsane et al. 1984; Durand et 011. 1985; Pandey 1991, Roussos et al. 1993) and in industrial scale (Deschamps et al. 1982; Lonsane et al. 1992; Bandoor et al. 1997; Durand et al. 1997). However, continuous production of biomass and metabolites in SSf has not been reported yet. What is a CCR? In the early 1980s, a new CCR has been deveJoped by the Commonwealth Scientific and Industrial Research Organization (CSIRO) of Austra1ia in cooperation with Bioquip Australia Pty Limited (Casimir, 1983). Il has been demonstrated that this unit has a potential for high yields of soluble soLids, flavours and colours. Leach (1993) assessed the effect of processing variables on the perfonnance of this reactor in the extraction of apple juice. The perfonnance was strongly influenced by temperature. The effects of draft ratio, screw speed and addition of pectinase enzyme were also investigated. More recently, this extractor has been successfully studied by Gutierrez-Lopez et al. (1996) based on a chemical reaction engineering the ory, where the extractor was divided into three different zones, according to the flow patterns present. The counter-current reactor has also been used as a solid-Iiquid extractor for processing fennented products such as enzymes, organic acids, antibiotics, phytohonnones and salts (Greve and Kula 1991; Johansson et al. 1985; Klyueva and Zakharevich 1985; Kurnar and Lonsane 1987; Likidis et al. 1989; Schwarlzberg 1980; Srikanta et al. 1987). Objective: The objective of this work is to describe the continuous production of glucoamylases by Aspergillus niger, a Gras fungus (Samson et al. 1997) grown in SSf using a CCR adapted for this purpose because there are not any bioreactors reported in the literature, capable of working in a continuous process for the production of fungal biomass or enzymes in SSf. Before the onset of fennentations it was necessary to study the flow of the sol id 22 support (sugarcane pith bagasse) in the extractor in conditions similar to those used later during the fungaJ growth in SSf. Il was important to know precisely the dependence of the mean residence time of the solid on the different possible programs of the extractor. Il was also necessary to know the degree of mixing in different experimental conditions. 1t was desirable that during the fermentation two solid particles fed atthe same time came out of the extractor approximately at the same time too. Otherwise, in each point of the bioreactor there wou Id co-exist micro-orgarusms at different stages of development. Residence time distribution The counter-current extractor consists of a stainless steel ribbon flighted screw situated in a U-shaped stainless steeltrough. The screw transports the solid material in both forward and backward directions. Il is driven by an extemaJ molor which can work inlerminently in both directions. In lhis way, the screw reverses its direction of rotation and that is the distinctive characteristic of the reactor. This reversing movement results in a very efficient solid-solid contact because the solid is constantly in movement between both sides of the CCR. The reversing movement is controlled by four programmable timer switches located on the control panel of the extractor. One timer selects the desired time of forward movement and another timer, the time of backward movement. The other two timers control the stopping interval between the forward and backward motions. The screw is operated with a much more forward movement a backward. The solids are thereby given a net forward movement. The screw speed of rotation is also selected in the control panel. Obviously the combination of a variable screw speed and variable forwardlbackward cycles enable a considerable flexibility in the control of the solid phase residence times l and mixing parameters. The ideaJ unmixed flow panern of the solid particles in a CCR is known as plug-flow. In this ideaJ flow no solid particle overtakes any other particle ahead or behind. Thus, ail the particles take exactly the same time to go through the extractor. However, reaJ reactors never fully follow this flow pattern. Usually each particle may take differenl roUles lhrough the reactor and, as a result, different lengths of time are required 10 reach the exit. ln sorne cases, the devialion From ideality is considerable. This deviation always lowers the performance of the unit. To determine the extent of deviation From the ideaJ flow we usually just need to know how long the individual particles stayed in the equipment or, more precisely, the residence time distribution (RTD) of the flo\oVing stream. The RTD can be obtained experimentally using a stimulus-response technique. The system in study is disturbed somehow and the way the system responds to this stimulus gives us the desired information. The nominal mean solids residence time is given by equation 1. N t F +t s +2.t s T=-X (1) n t F -t s where r = nominal mean residence time (in minutes) N = number of flights of the screw n = screw speed (in revolutions per minute. rpm) IF = time for which the screw is set to move forward ta = time for which the screw is setto move backward 1 The residence lime is the lime necessary for the feed to traveJ from the feed-end 10 Ihe discharge- end. 23 ts = time for which the screw is set to be stationary. In each fermentation the CCR was initially filled with the amount of non-inoculated bagasse corresponding to the hold-up. This bagasse was uniforrnly distributed throughout the comparnnents. Then, the screw movement was staned at 1 rpm under the conditions presented in Table 1. Table 1. Screw movement program for the continuo us glucoamylases production in SSF using a CCR. Screw speed = 1 rpm. Screw Movement Movement Forward Stopping Backward Stopping Time (sec) 36.0 870.3 23.4 870.3 We knew from the results of the preliminary experiment that the dispersion should he low if there were 1.2 Kg per compartment. This corresponds approximately to a feeding rate of 0.5 Kg / h and that is the rate used in the flrst run. In the other IWO runs higher rates were tested. The residence time distributions obtained in these 3 runs are represented in Figures 1,2 and 3. In this ex periment, 3 nulS with tracer were carried out in which we tested the effect of the feecling rate on the dispersion using the program of 36.0 seconds for,"vard / 23.4 seconds backward 2. The lime needed to recover 90 % of the tracer fed gave us an idea of the dispersion. This value was calculated for each one of the runs and is given in Table 2. The variances of the residence time distributions were also calculated for each one of the runs and are presented in the same 00,-- --, ., 10 O+-15 ..... "--------r----'-_~_---...., J) 25 Time (h) Figure 1.: RTD of the tracer in the first run. Feeding rate = 0.50 Kg / h. , The objective was to work with exaclly the same program used laler in Ihe fermentations. For practical reasons we were interested in having cycles of exactly half an hour during the fermentation. For this reason, it was necessary. to use a program in which the backward movemenl lasted 23.4 second in order to have a mean residence time of 20 h. 24 "'r------------------, ., 10 ., - ., 10 o ...._ _L ........--~~~ ..............- - - - - - J )) 25 Time (h) 15 Figure 2 : RTD of the tracer in the second run. Feeding rate = 0.75 Kg / h. "',-------------------, II) )) - ., '"OM........- - . - - - - _ - -......... ~.._"-~_ 25 15 Time (h) Figure 3 : RTD of the tracer in the tbird run. Feeding rate = 1.00 Kg / h. table. These values were obtained using Equation 3. The experimental hold-up is also presented. as weil as the hold·up calcuJated by multiplying the feeding rate by the residence time (Equation 1) . Table 2. Time necessary to recover 90 % of the tracer, variance of the RTD and experimental and calculated bold·ups. Hold-up (Kg) t90% (h) d experimental calculated 3 15 \0.7 10 2 45 2.6 15.1 15 3 12 16 21.8 20 Run Il can be seen From Figures 1,2 and 3 and from the values of t90% and cr' in Table 2 that, the dispersion increases proportionally with the feeding rate. This was expected and as explained before, this happened because as the feeding rate increased, the hold-up increased too and 25 hence so, the accwnuJation of bagasse on the ribbon was higher and it was easier for the bagasse to go over the ribbon, pass over the axis of the screw and finally fall in another compartment. Comparing the experirnentaJ with the calcuJated hold-ups a similarity was observed. Therefore, it was confirmed that the mean residence time was 20 h. Il is important to note that, although the hold-up and the feeding rate were expressed in units of mass, it was not the weight of the sol id inside the equipment that influenced the dispersion. The dispersion occurred when the vo)wne of the solids was so high that the solid passed over the axis to another compartment. In our case, as all the experiments were made with the same solid, we could aJways workjust with the weight. However, if, for instance, the water content of the bagasse was changed, its density would change too and the results would be different. Il would be interesting to carry out sinùlar experiments \\'Îth different solids and observe if experirnents done with similar volwnetric flow rates but different solids gave similar results in terms of dispersion. Continuous glucoamylase production in SSF Previously sterilised solid material was inoculated with 2xl0 7 conidia of A. niger per gram of dry bagasse and ail this material was incubated at 37 oC for 8 hours. Follo\\'Îng that. this pregerminated material was stored at 4°C for a few hours. At two hours intervals, one plastic bag was taken from the cold room to the incubator at 37°C and Jeft for an hour before feed of the CCR. 28 feedings (0.5 Kg each) of the pre-germinated material were carried out each hour. Non-inoculated solid material was fed in the subsequent ten feedings. The CCR screw movement was monitored as shown in Table 1. In this way the mean residence time of the solids in the reactor was 20 hours. During the fermentation no sterile conditions was followed. No special precautions have been taken to avoid the exchange of micro-organisms with the exterior of the reactor. Despite the metaJlic covers being used, there was contact at the discharge-end and at the feeder. No forced aeration has been used. The natural movement of the air would probably be sufficient enough to supply the necessary oxygen and remove the carbon dioxide produced during fermentation. Samples from ail the compartments (1 to 9) of CCR were collected at 16, 20, 24, 30 and 38 hours of fermentation. At the end of SSF in the CCR, analysis of the samples was carried out to observe the evolution of the main fermentation parameters (water content, pH, biomass, reducing sugars and glucoamylases). The results ofthese analysis are presented in Figure 4. Water content: Kinetics of water content in the fermented solid material for each compartment of the CCR show that the humidity of the materia! decreased from 75 to 65 % from compartments 1 to 9. Ali kinetics present sirnilar patterns (Figure 4.A). Only at 20 (companment 9) and 38 hours (compartments 2 and 3) kinetics were different. pH: The pH evolution was the same for ail kinetics. At the beginning (compartments 1 and 2) the pH was stable and around 5.5. In companments 3-6 it decreased and a value of 3 was reached. This acidic conditions were maintained in compartments 7·9 (Figure 4.8). Only at 38 a different pattern was shown. In companments 1-6 the pH was al ways higher than the other kinetics. From compartment 7 onwards the pH changes were similar to the other kinetics. 26 Biomass: Biomass has only been analysed at 20, 24 and 30 hours of fennentation. Kinetics at 20 and 30 ho urs presented a similar panem. At 24 hours kinetic was differenl. In this study, the change in biomass concentration panern 20 and 30 hours only is described. At compartments \-3 the biomass concentration was very low. Compartments 3-7 showed an exponential growth. The growth slowed down after compartment 7 and there was even a decrease in biomass (Figure 4.C) The biomass change ranged between approximately 1 mg! g of dry maner at the first compartment and 40 mg!g of dry maner at the 9th compartment, reaching a maximum of 55 mg/g of dry maner at compartrnents 7 and 8. Reducing Sugars: The change in reducing sugars is heterogeneous for the different kinetics. However, in general an initial increase in their concentration could be observed, a maximum of 70 mg/g of dry maner being reached around the 4th compartmenl. Following that, a decrease occurred until compartment nO.7 (Figure 4.D). Only at 38 hours the kinetics was c1early differenl. Glucoamylases: Glucoamylases production was only analysed at 20 hours fennentation in the CCR. Compartments 1-4 presented a very low enzymatic activiry (approx. 10 IU/g of dry maner). Then an increase in activiry was predominant (compartrnents 5-9) until a maximum of 40 IU/g of dry maner was reached in the last compartment (Figure 4.E). Fungal aspect during a continuous SSF process: Microscopically observations have also been carried out with samples at 20 and 30 hours. From each sample a very smalt particle of fennented solid material was taken and stained with a "blue Co!1on" dye. This preparation was observed under 100x, 200x or 400x magnification. At the 1 st compartment the conielia had already germinated and the germinative tube length was approx. 3 times the diameter of the conidia. At the 2nd compartment the genninative tubes were 5-10 times longer than the conidia diameter and at the 3rd compartment around 100 times. From compartment no.3 onwards the mycelium was vigorous, thick and presented ramifications. At compartment no. 4 the mycelium was very weil grown. There was a strong ramification of the hyphae and the surface of the soJid material had been invaded. Compartments no. 4 showed a strong evolution. The surface of the solid material had been completel)' covered and the starch particles were degraded. At compartment no.6 the mycelium replaced the hydrolysed starchy solid and offered a solid structure for the fermented substrate. Compartments no.7, 8 and 9 presented similar change to that of compartment nO.6. The only difference was that in compartments no.8 and 9 the formation of a few asexual reproductive forms was detected. Phialides were formed however, these forms were still very young and no conidia were produced. This first A. niger solid state fermentation in the CCR, a good fungal growth was present and a reasonable amount of glucoamylases was produced. The water content decreased significantly from the first to the last compartmenl. Hence, a second SSF was carried out (results not shown in this paper) with a higher initial water content and with no use of the heating jacket of the CCR. The feeding rate increased to 0.8 Kg/h because, with the feeding rate of 0.5 Kg!h used in the first fermentation, just a small part of the capaciry of the reactor was in use. 27 The mycelium in each compartment was at a similar physiologica! state. At the first compartment, oruy germinated conidia were present. Successive compartments showed an increase in the colonisation of the substrate and at compartment nO.5 the substrate was covered in dense mycelium. A marked increase in biomass occurred from compartment no.5 onwards. No mycelium damage or sporulation was observed. In Table l, the change of the measured parameters is shown for each compartment of the CCR. These values were sirnilar to those reponed for batch SSF using the same substrate and rnicro-organism (Oriol el al. 1987). However, moisture content dropped from about 74% in companment no.l to 64% in companment no.9 due to the water evaporation efTect caused by the high tempe rature of the fermented material. In solid state fermentations severa! kinetic parameters have been analysed and. in general, ail of them presented a similar behaviour. To facilitate the discussion of the results a representative kinetics has been ca!culated, from different values of the fermentation (Table 3). However, reference to the other kinetics was also made whenever necessary. Parameters evolutioo io each CCR compartmeot duriog SSF ln this fermentation, 20 hours was selected as the most representative one. In Table 3. changes in water content, pH, biomass, reducing sugars and glucoamylases are shown at 20 hours fermentation. Table 3: Parameters changes in each CCR compartment over 22 hours fermentation. Sugarcane bagasse moistened with a nutritive solution and contaioing 10 hours old pre-germinated conidia of A. niger was used for feeding in the contiouous glucoamylases production under non-aseptic conditions du ring 38 hour SSF. Compartment Parameter 2 3 4 5 6 7 8 9 30 35 37 39 40 38 39 39 36 Water content (% w.b.) 74 74 73 72 73 72 71 66 64 4.9 4.4 3.1 3.0 2.9 2.8 2.9 45 47 55 42 Temperature (oC) pH 5.1 5.3 Biomass (mg/g) 0.4 2.4 Il 22 41 Reducing sugars (mg/g) 8.0 22 45 63 38 36 39 47 32 Glucoamylases (IU/g) 5.0 9.0 12 9.0 22 23 25 39 40 28 90 as _ _ 16h 00 -<>-20h 75 7IJ 24h _ _ 30h 66 -<>--38h 00 55 50 7.5 65 -.-16h -Q-2Oh 55 i: 45 24h _ _ 30h 3.5 -o-38h 25 00 50 C 40 -'1-2Oh 30 24h -w.-30h 100 . . . - - - - - - - - - - - - - - - - - - - - - - . . . , D --.16h -<>-20h 24 h ~30h -<>--38h 50...- -, E 40 ---2Oh 20 10 O+-----i---t---f----t---+---+---+--6 Compartment numt)er Figure 4 : Water content (A), pH (8), biomass (C), reducing sugars (D) and glucoamylases (E) evolution in each compartment of the CCR during the fermentation. Sugarcane bagasse media containing pre-germinated A. niger conidia was continuously fed. Residence time = 20 bours. 29 Water Content Evolution Before culture solid medium sterilisation, the inert solid support was moistened to 50% with a nutritive solution.Then, the water content was adjusted to 74% with the spores suspension. The change in water content for the 40 hour SSF in a CCR is represented in Table 3. A decrease from 74% in compai1ment no.l to 64 % in compartment no.9, however is evident this amount ofwater is enough to ensure good conidia germination and mycelium groWlh. The last compartments were characterised by a low water content, but it is believed that it did not affect the enzymes production because other studies confirmed the production of glucoamylases in SSF with water content lower than 60 % (Raimbault and Alazard 1980), using starch material at 50% water content and had good glucoamylases production). As long as it does not affect the fungal metabolism, a low water content is good because it avoids bacteria! contaminations. On the other hand, when there is need to implement the process at an industrial scale, a high water content in the final fermented product could he helpful in the recovery of metabolites. For example, if this product had a water content of 75% ,80% of the fermented juice could he recovered by just hydraulically pressing the fermented solid material (Roussos el al., 1992). Thesis one of the simplest and cheapest methods available to recover fungal metabolites. Glucoamylases Continuous Production lnitially at 20 hours there was a slow increase in the concentration of glucoamylases between compartments nO.1 and noJ. From compai1ment no.3 to noA a small decrease in concentration was observed but after the 4th compartment a share increase was evident. We think that the glucoamylases present at the first four compartments are the glucoamylases that were contained inside the conidia and were liberated during germination. These glucoamylases were responsible for the initial increase in the reducing sugars concentration described above. The biosynthesis of new glucoamylases started immediately after the 4th compartment and was clearly associated with the growth of mycelium. During this phase, a decrease in the reducing sugars concentration was observed because, although the glucoamylases production was high, it was not sufficient to compensate the reducing sugars consumption by the microorganism. The micro-organism was on the exponential phase of groWlh and, as soon as the reducing sugars were produced, they were consumed by the mould. [t is very interesting to note that, although the reducing sugars concentration was high at co'mpartments noA-8, the biosynthesis of glucoamylases was not repressed. On the contrary, in submerged fermentations a strong catabolic repression in the biosynthesis of glucoamylases was observed when the reducing sugars concentration was high. This fact could be explained thinking of what happened in the surroundings of the mycelium in the solid state fermentation. The mycelium was surrounded by a thin layer of water. The glucoamylases produced by A. niger in SSf had to move toward this layer in order to reach the starch material. When the reducing sugars were liberated fTom the starch, they had to diffuse back through the water layer until they reach the micro-organism. As this diffusion process was 30 low, as soon as the sugars reached the micro-organism they were consumed by il. In this way. the concentration of sugars close to the micro-organism was very Jow and the micro-organism continued the production of glucoamylases in SSF, even if this concentration away from the mould was high. In submerged fennentations, a layer of stagnant solution is also found around the microorganism. However, due to the agitation of the fennentation broth, its thickness is much smaller and the micro-organism is belter infonned about the concentrations away from it (Favelia-Torres et al. 1997). Spore Germination and Mycelium DeveIopment. Microscopy observations showed thatthe conidia were unifonnly distributed throughout the 7 solid material and the inoculation had been homogeneous. The amount of conidia used (2x 10 conictialg of dry bagasse) was enough to invade sugarcane bagasse during the mycelium growth, without being excessive. A high percentage of conidia was genninated. However, when the pre-genninated solid material was fed to the CCR, the spores were still at the beginning of germination. Probably the temperarure and time of incubation were not the most adequate. At compartment no.3 the ramification of the mycelium was evidenl. At this phase, the exponential growth started (Figure 4.C). There was a clear association between the exponentiaJ increase of the biomass and the ramification and strong growth of the mycelium. After compartment no.8 the fonnation of sorne reproductive fonns was observed and this corresponded to the slight decrease observed in biomass from compartment no.8 to no.9. Conclusions The continuous production of enzymes by SSF has been demonstrated. Inoculation with pregenninated conictia shortened the processing time and allowed control of the age of the mycelium in each compartmenl. Aeration was accomplished by natural convection and moi sture content was controlled. The fungal growth occurred in good conditions, and the movement of the screw no had caused any damage for the myceliurn. The sugarcane bagasse was an excellent solid support used in this equipment. SimiJarities between batch fermentation and fennentation in CCR were observed. 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Extraction of gibberellic acid From dry mouldy bran produced under solid state fermentation. Process Biochem. 22: 139-143. Lambraki, M., Marakis, S. and Roussos, S. 1994. Effect of temperature and aeration flow on carob tannin degradation by Aspergillus carbonarius in soJid state fermentation system. Micol. Neolrop. Apl. 7: 23-34. Leach, G. C. 1993. Studies on the Counter Current Diffusional Extraction of Apple Juice, Ph. D. Thesis, University of Reading, U.K. Lepilleur, c., De Araujo, A. A., Delcourt, S., Colavitti, P. and Roussos S. 1996. Laboratory scaJe bioreactors for study fungaJ physiology and metabolism in solid state fermentation system. In Roussos, S., Lonsane, B.K., Rairnbault, M. and Viniegraz-Gonzalez, G. (Eds.), Advances in solid state fermentation, Kluwer Acad. Publ., Dordrecht, p.93-1 11. Likidis Z., Schlichting E., BischoffL. & Schuegerl K. 1989. Relative extraction of penicillin G from mycel-containing broth in a countercurrent extraction decanter. Biotechnol. 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Biotechnol., 27: 498-503. Pandey, A. 1991. Aspects of fermenter design for solid-state fermentations. Biochem. 26: 355-361. Process Raimbault, M. and Alazard, D. 1980. Culture method to study fungaJ growth fermentation. Eur. J. Appl. Microbiol. Biotechnol. 9: 199-209. ln 33 sol id Raimbault, M., Revah, S., Pina, F. and Villalobos P. 1985. Protein enriclunent of cassava by solid state fermentation using molds isolated from traditional foods. J Ferment. Technol. 63: 395-399. Rodriguez, J.A., Torres, A., Echevarria, J. and Saura, G. 1991. Energy balance in solid state fermentation processes. Ac/a Bio/echnol. II: 9-14. Roussos, S., Olmos, A., Raimbault, M., Saucedo-Castaiieda, G. and Lonsane, B.K. 1991. Strategies for large scale inoculum development for solid state fermentation system : Conidiospores of Trichoderma harzianum.. Bio/echnol. Tech. 5: 415-420 Roussos, S., Raimbault, M., Viniegra-Gonzalez, G., Saucedo-Castaileda, G. and Lonsane, B.K. 1991. Scale-up of cellulases production by Trichoderma harzianum on a mixrure of sugar cane bagasse and wheat bran in solid state fermentation system. Micol. Neo/r0p. Apl. 4 : 83-98. Roussos, S., Raimbault, M., Prebois, J-P. and Lonsane, B.K. 1993. Zymotis, A large scaJe solid state fermenter: Design and evaluation Applied Biochem. Bio/echnol 42: 37-52. Roussos, S., Bresson, E., Saucedo-Castaiieda, G., Martinez, P., Guymberteau, l and Olivier. J-M. 1997. Production of mycelial cells of Pleuro/us opun/iae on natural support in solid state fermentation. In Roussos, S., Lonsane, B.K., Raimbault, M and Viniegra-Gonzalez, G. (Eds.), Advances in Solid S/a/ejermenta/ion. Kluwer Acad. Pub!., Dordrecht, chapter 40: pp. 481-498. Samson, R.... 1996. Mycol. Research. Saucedo-Castaiieda, G., Gutierrez-Rojas, M., Bacquet, G., Raimbault, M. and ViniegraGonzalez, G. 1990. Heat transfert simulation in solid substrate fermentation. Bio/echnol. Bioeng. 35: 802-808. Saucedo-Castaiieda, G., Lonsane, B.K., Navarro, lM., Roussos, S. and Raimbault, M. 1992a. Potential of using a simple fermenter for biomass built up, starch hydrolysis and ethanol production: Solid state fermentation system involving Schwanniomyces cas/el/ii , Appl. Biochem. Bio/echnol. 36: 47-61. Saucedo-Castaiieda, G., Lonsane, B.K., KrislU1aiah, M.M., Navarro, J.M .. Roussos, S. and Raimbault. M. 1992b. Maintenance of heat and water balances as a scaJe-up criterion for the production of ethanol by Schwanniomyces cas/el/ii in a solid state fermentation system. Process Biochem. 27: 97-107. Schwartzberg, H. G. (1980) Continuous Counter-Current Extraction in the Food Industry. Chem. Engineer. Progress. 76: 67-85. Senez, le. 1979. Solid fermentation of starchy substrates. Food NII/r.Bull. 1: 18-20. Senez, lC., Raimbault, M. and Deschamps, F. 1980. Protein enriclunent of starchy substrates for animal feeds by solid state fermentation. World Animal Rev. 35: 36-40. Srikanta S., Jaleel S.A., Ghildyal N.P., Lonsane B.K. & Karanth N.G. 1987. Novel technique for saccharification of cassa va fibrous waste for alcool production. Starch 39: 234-237. 34 Soccol, c., Marin, B., Raimbault, M. and Lebeault, J.M. 1994. Potential of solid state fermentation for production of L(+) lactic acid by Rhizopus oryzae. Appl. Microbiol. Biolechnol. 41: 286-290. Trejo-Hemandez, M.R., Raimbault, M., Roussos, S. and Lonsane, B.K. 1992. Potencial of solid state fermentation for production of ergot alkaloids. LeI. Appl. Microbiol. 15: 156159. Trejo-Hemandez, M.R., Lonsane, B.K., Raimbault, M. and Roussos, S. 1993. Spectra of ergot alkaloids produced by C/aviceps purpurea 1029c in solid state fermentation system: Influence of the composition of liquid medium used for impregnating sugar cane pith bagasse. Process Biochem. 28: 23-27. Viniegra-Gonzalez, G. 1997. Solid state fermentation: Definition, characteristics, limitations and monitoring. In Roussos, S., Lonsane, B.K., Raimbault, M. and Viniegraz-Gonzalez. G. (Eds.), Advances in so/id slale jermenlalion, KIuwer Acad. Pub!., Dordrecht. chapter 2: pp. 5-22. 35 PRODUCTION OF ENZYMES BY SOLID SUBSTRATE FERMENTATION: RELATION SUBSTRATEfENZYME AND INDUCTION/CATABOLIC REPRESSION G. Viniegra-Gonzalez, Christopher Augur Universidad Autonoma Metropolitana, Unidad Iztapalapa, Departamento de Biotecnologia Av. Michoacan y Purisima, Col. Vicentina, Apdo. Postal 55-535. Mexico. D.F., c.P. 09340 Summary Solid substrate cultures of moulds behave in quite a difTerent way to conventional submerged cultures. Catabolic repression often observed in submerged fermentation can easily be overcome by using solid state fermentation. A. niger strains can be grown by SSF technique with the following advantages: enzyme titres and productivity are usually higher; there is a tendency to early enzyme excretion as compared to SmF technique. Conventional enzyme production in a Stirred Tank Reactor (STR) by microbial organisms requires, an inexpensive and ready to use carbon source (sugar or starch derivative). a specific enzyme inducer, and a mixture of minerai salts and other organic compounds. Unfortunately, the presence of high levels of the carbon source often inhibit the production of many enzymes. For exampie, it has been reported that the addition of 100 much of sugar or even pectin to a STR inoculated with Aspergillus niger was detrimental to pectinase production [2, 3]. But continuous and controlled addition of small amounts of sugar (fedbatch reactor) increased four or five times pectinase productivity [2]. The explanation of this phenomenon was related to a dynamic balance between supply and demand of sugars in the SmF process leaving the sugar concentration at a low level, although the total supply of sugars was very high [2]. But, Ramesh and Lonsane [4 ] using Bacillus sublilis grown by SSF in fixed bed reactors and adding starch as inducer did nol find the catabolic repression phenomenon commonly observed by the addition of high levels of carbohydrates. Solis-Pereira el al. [5) confirmed that in shake flasks (SmF) inoculated with a strain of Aspergillus niger, called CH4 , glucose levels of 30 g!L were inhibitory to pectinase production in the presence of 30 g!L of peclin (Table 1). BUI in SSF (packed bed reaclorS wilh bagasse) using the same strain and inducer, the addition of glucose enhanced pectinase production although the sugar level was 100 g!L in the absorbed broth (see Table 1). 37 TABLE 1. Effect of glucose addition to pectinase levels produced by SmF and SSF techniques 15 1. Substrates Smf (U/mL) SSF (U/g) 30 g!L Pectin A + 30 gIL glucose A + 100 g!L glucose 0.75 0.00 5.00 11.0 13.0 N.D. This observation has been confmned in our laboratory (unpublished data) using different A. niger strains and inducers ( suc rose, tannic acid) and assaying for the corresponding enzyme activities (invertase, tannase) as illustrated in figure 1. Invertase secreted by A niger C28B25 during solid and Iiquid fermentation 100 . . . - - - - - - - - - - - - - - - - - - - - - - - - , 80 60 40 20 o G SSf S SSf S+G SSF G SmF S SmF S+G SmF Figure 1. Invertase secreted by A. niger C28B25 during SSF and SmF. (G: Glucose, S: Saccharose). Catabolic repression is observed in SmF at 40g/L of glucose (Fig. 1). The repression at the same concentration of glucose is much reduced in SSF. Glucose added to saccharose in SSf does not affect invertase production. Production of other enzymes has also been tested in packed bed reactors with bagasse as solid support and in fixed bed reactors with polyurethane as solid matrix. [n ail those cases, enzyme production was increased using rugh levels of glucose or sucrose without much evidence of catabolic repression. 38 A consequence of such physiological behaviour is the increase of enzyme productivity by SSF. This was observed to a certain level. Garcia-Pefia (11) studied the effect of adding increasing amounts oftannic acid as an inducer during an SSF process and observed that over and above 10% tannic acid, tannase production decreased drarnatically. as shown in figure 2. Effects of' tannic acid concentratiol during grovvt:h on extracellular enzyrnatic activity 0.7 ____ 1 0.6 0.5 /0 - - - - - - 2% 4% 0.4 (U/n 0 0.3 ~100/0 0.2 _ 2 0 0/0 0.1 o o 72 48 96 Timo (h) Figure 2. Effects of tannic acid concentration during growth on extracellular tannase activity Another interesting observation is the fact that tannase has been reported to be bound to the hyphal biomass of A. niger when it is produced by SmF. Lekha and Lonsane (10) and Garcia-Pefia (11) have found !hat trus enzyme is mostly excreted to the culture medium of A. niger when produced by SSF (figure 3.). These intriguing differences between SSF and SmF techniques in the excretion of enzymes, seem to indicate !hat mou Ids can modulate the way to use enzymes, depending on the culture medium. Apparently, those organisms have "sensors" that pick up environmental signaIs and have also complex transducing systems that modulate !heir biochemical behaviour in order to adapt to a particular set of environmental variables (low or high a w , good or bad mixing of substrates, low or high temperature, etc). This adaptation gives plasticity to those organisms in order to survive in changing culture conditions. An unexplored solid state fermentation method using polyurethane foam (PUF) as inert carrier impregnated with a synthetic Iiquid medium was developed simulating the nutritional composition and culnue conditions of solid state fermentation on sugar cane bagasse. with this system. biomass, the important parameter involved in SSF process, could be measured directly. Tannase production of various previously selected overproducing A. niger strains was tested (Figure 4.). The three strains produce maximum activity around 48 hours of incubation. The specific activities are similar ta those obtained with bagasse [12]. However, maximum tannase production is observed much later (around 100 hrs) when sugar cane bagasse is used as solid support. 39 0.7 0.6 0.5 0.4 -.--Extra 0.3 - - - - Mye. ass. 0.2 0.1 o o 24 48 144 120 72. Time (h) Figure 3. Comparison of tannase extracellular activity witb activity associated with tbe mycelium. Specifie Activity of A. niger strains groW'n on PUF 14 12 10 8 pro 6 4 2 o 24 48 72 Tlrne (h) 96 120 Figure 4. Specifie activity of tannase from tbree A. niger strains during growth on PUF. TABLE 2. Comparison of pectinase productivity by SmF and SSF cultures of A. niger CH41IJ. Activity Endo-pectinase Exo-pectinase Pectin-Iyase Ratio SSF Smf (SSF/Smf) (SSF/Smf) 0.006 0.14 0.008 0001 0.002 0.0002 6.0 51.5 29.2 40 Acuna-Argüelles el al. [1] found that A. niger CH4 had a much higher pectinase productivity when cultured by SSF as shown in Table 2. Clearly, SSF cultures were more productive for ail pectinase activities assayed. such as. endo-pectinase by viscometry, exo-pectinase by the production of reducing compounds and pectin lyase by the changes in UV absorbing material [1]. Invertase production byA. niger C28B25 ln SmF Invertase production byA. niger C28B25 in SSF 100 140 ..El ... ,! ::: :o=- =.: ~ 'i '" 4.5 120 4 100 3.5 1 3 00 2 ~ j: i 2.5 ë 00 :. ::: 1S! ~ 1 • 40 20 25 140 : :: EoÔ .! ::: :;:. ~~ ~ '; ~~ 120 100 1.5 ~: ,:- =: 00 00 05 40 ! 20 0.5 0 "'-'~----l--+---+--+O 12 00 24 48 0 O_-==f'----+--+---+---+ 12 o 48 nme(I'll nm.(h) Figure 5. Invertase production of A. niger C28B25 in SmF and SSF. Squares represent extracellular invertase production and diamond represent intracellular invertase. [Note the difference in scales] In SrnF, intracellular accumulation is over twice that of SSF. Invertase is secreted earlier (maximum at 24 hrs) in SSF than in SrnF (maximum at 36 hrs). UnJike tannase which is mycelium associated in SrnF, invertase seems to be readily excreted in bath SmF and SSF. Such increases of productivity could have important economic consequences in the cost of enzyme production . Thus, Ghildyal el al. [6] have made an economic pro forma analysis of amylo-glucosidase production by SrnF and SSF. Their calculations indicate that due to a higher yield of SSF using A. niger CFTRI 1105, which produced 10 times higher titres than by SrnF, the ove rail economic picture was much better for SSF process. ln fact there are reports of successful large scale production of pectolytic enzymes by SSF in India [7] and also of fungal amylase scale-up by SSF [8]. Pandey [9] has reviewed the reports of enzyme production by SSF including, cellulases, amylase, glucoamylase, beta glucosidase, pectinases, catalase and proteases with a list of 28 microbial species in which Baeillus and Aspergillus are the most frequently used genus. 41 Bibliography 1.- Acuiia-Argüelles, M., Gutiérrez-Rojas, M., Viniegra-Gonzâlez, G., Favela-Torres, E.: Production and properties of three pectinoJytic activities produced by Aspergillus niger in submerged and solid state fermentation. Appl. Microbiol. Biolechnol. 43 (1995), 1-6. 2.- AguiJar, G., and Huitr6n, c.: Application of fed-batch cultures in the production of extracellular pectinases by Aspergillus sp. Enzyne Microb. Technol. 9 (1986), 541-545. 3.- Maldonado, M. c., Stresser A., and CaJlieri, D.: Regulatory aspects of the synthesis of polygalacturonases and pectinesterases by Aspergillus niger sp. Sciences des aliments 9 (1989), 101-110. 4.- Ramesh, M.V., and Lonsane, B.K.: Regulation of alpha-amylase production in Bacillus /icheniformis M27 by enzyme end-products in submerged fermentation and its overcoming in solid state fermentation system. Bio/echno/. LeN. 13 (l99J), 355-360. 5.- Solis-Pereira, S., Favela-Torres, E., Viniegra-GorwiJez, G., and Gutiérrez-Rojas. M.: Effects of different carbon sources on the synthesis of pectinase by Aspergillus nige,. in submerged and solid state fermentation. Appl. Microbiol. Bio/echnol. 39 (1993), 36-41. 6- Ghildyal, N.P., Lonsane, B.K:, Sreekantiah, K.R., Sreenivasa-Murthy. V.: Economics of submerged and solid state fermentations for the production of amyloglucosidase. J Food Sci Techno/. 22 (1985),171-176 7.- Ghildyal, N.P., Ramakrishna, S.V., Nirmaia Devi, P., Lonsane, B.K.. Ashlana, H.N.: Large scale production of pectolytic enzyme by solid state fermentation. J Food Sci. Techno/ 18 (1985),244-257. 8.- Jaleel, S.A., Ghildyal, N.P., Sreekantiah, K.R., Sreenivasa Murthy, V. Production and scale-up of fungal amylase by solid statefermentation. Paper presenled a/ /9'h Anl1l1a/ Conference, Associa/ion ofMicrobi%gis/s, India, Baroda (as cited in 17) (1978). 9.- Pandey, A.: Recent process developments in solid state fermentation. Process Biochemis/ry. 27 (1992), 109-117. 10.- Lekha, PX., and Lonsane, B.K.: Comparative titres, location and properties of tannin acyl hydrolase produced by Aspergillus niger PKL 104 in solid state, liquid surface and submerged fermentations. Proc. Biochem. 29 (1994), 497-503. 1 1.- Garcia-Pena, E.l.: Produccion. purificaciàn y caraclerizacion de /anasa producida por Aspergillus niger, en fermen/acion en media so/ido M. Sc. thesis (Biotechnology). Universidad Aul6noma Metropolitana. Iztapalapa, D.F. (Méx.) (1995). 12.- Ramirez, A. Se/eccion de cepas produc/oras de lanasa por fermenta/ion en medio solido. Lic. thesis (Biotechnology) Universidad Aut6noma Metropolitana. Iztapalapa, D.F. (Méx.) ( 1996). 42 FRUITY AROMAS PRODUCTION IN SOLID STATE FERMENTATION BY THE FUNGUS Ceratocystisfimbriata Pierre Christen' & Sergio Revah l Laboratoire de Biotechnologie, Centre ORSTOM, BP 5045; 34032 Montpellier Cedex l, France l Universidad Autonoma Metropolitana, Unidad Iztapalapa, Departamento de Ingenieria, Av. Michoacan y Purisima, Col. Vicentina, Apdo. Postal 55-535. Mexico, D.F., C.P. 09340 1 Abstract Solid state fermentation (SSf) has been studied for enzymes, antibiotics, alcohol production or for prote in enrichment, but few papers report the production of aromas by such a process. In this work, the study of the production of fruit)' aromas in SSF by the fungus Ceralocyslis fimbriala is presented, with special interest in the nature of the supportJsubstrate, the importance of added precursors in the medium and the aeration. The aromas were characterised by "sniffing" technique and Ge headspace analysis; growth was followed by respirometry. It was shown !hat wheat bran, cassava and sugarcane bagasse were adequate supports for growth and a detectable aroma production. Among the nutritive media tested, the synthetic medium previously defined, used with a higher glucose concentration (200 gll) gave a strong apple aroma while those containing aminoacids precursors such as leucine and valine gave strong banana aroma. It was found that aroma production was growth dependent and the maximum aroma intensity was detected a few hours before or after the maximum respirometric activity, this varying belWeen 0.8 and 1.6 ml C02 / h. g dry matter, after 24 hours). Experiments made under various aeration rates (0.05 and 0.005 ml/h. g dry matter) showed that this parameter was not limiting for growth even if the exit gas was very poor in oxygen at the lower aeration rate giving in this case the most intense aroma. For experiments made without forced aeration, the same aromas were also found \Vith higher intensity. Fourteen compounds have been separated by Ge headspace and Il of them such as acetaldehyde, ethanol, ethyl acetate, isoamyl acetate and isoamyl alcohol were ident ified. Introduction Micro-organisms play an important role in the generation of natural flavowing compounds particularly in the field of food aromas. One can refer to the extensive reviews deaJing with flavour generation by micro-organisms in the past few years (Latrasse el al., 1985; Welsh el al., 1989; Janssens el al., 1992). As pointed out recently by Bigelis (1992) and Christen (1995), filamentous fungi are especially interesting because they are able to produce flavouring compounds or flavours-related enzymes. Sorne recent papers have reported the production of aromas in SSf: Yamauchi el al. (1989) obtained a fruiry flavour growing a Neurospora strain on pregelatinized rice; Gervais and Sarrette (1990) studied the production of coconut aroma by Trichoderma viride on agar and Hwnphrey el al. (1990) patented a process where an Aspergillus strain grown on cellulose fibres produced methyl ketones from cocon ut oil. Moreover, the capacity of sorne moulds from the genus Ceralocyslis to produce fruit- 43 like aromas has already been demonstrated (Hanssen and Sprecher, 1981; Senemaud 1988, Christen el al., 1994). In this work, the ability of Ceralocyslis jimbriala to produce aromas in SSF was explored. It involved the study of the influences of the substrates/supports used, the aeration flow rates and the presence of precursors, on both gro\\1h and aroma production. Organism and culture media. Ceralocyslis jimbriala CSS 374-83 was used. Il was periodically transferred onto Potato Dextrose Agar (PDA) slants and stored at 4°C. Four substrates / supports were used: wheat bran, sugarcane bagasse, cassava (donated by Pr. C. Soccol, UFPR, Sranl) and an anionic resin (Amberlite lRA-900, Rohrn & Haas). There were prepared according to Christen el al. (1993). When forced aeration (packed bed) was used, the cultures were carried out in small colurnns placed in temperature controlled bath. For experiments without forced aeration (surface culture), they were made in 500 ml Erlenrneyer flasks covered with gauze or tight-sealed. They were filled with 7.5 g Initial Dry Matter ([DM) for wheat bran and Amberlite, and 5.25 g !DM for bagasse and cassava. For all experirnents, initial conditions were: temperature, 30°C; pH, 6; inocuJurn size, Ixl0 7 spores/g !DM. Aeration rates were 0.05 or 0.005 Vh.g IDM. Initial water content was caJculated according to the maximum adsorption capacity of each support (wheat bran, 50%; Amberlite, 58%; sugarcane bagasse, 63% and cassava, 65%). Culture conditions are given in tables 1 and 2. SM (See table 1 and 2) refers to the synthetic medium optimised by Christen and Raimbault (1991). Il was used with 200 gI1 of glucose. For ail cases. an oligoelement solution previously used by these authors was added. Urea, leucine and valine (167 mmolll) were used as nitrogen source and/or precursor of the aroma. T a bei l e u lt ure con d'f 1 IOns WI'th f orce d aera Ion. *SM : syn th e f IC me d'mm Run Su bstrates/Supports Nutritive media Aeration rate (l/h.g) 1 Amberlite Potato broth 005 2 Amberlite Potato broth 0.005 3 SM* Amberlite 0.005 4 Wheat bran Urea 0.05 5 Wheat bran 0.005 6 Wheat bran Urea 0.005 7 Wheat bran Leucine 0.005 8 Wheat bran Valine 0.005 9 Sugarcane bagasse SM* 0.005 la Sugarcane bagasse Potato broth + glucose 0.005 Analytical procedures. The odors of the cultures were deterrnined by sensorial evaluation with a nontrained panel consisting of six members. with no restriction in descriptive terrns. Growth was characterised by respirometry measured by gas chromatography. For packed colurnn experiments, this allowed the caJculation of the carbon dioxide production rate (CDPR), the oxygen uptake rate (OUR) and the respiratory quotient (RQ) (Christen el al., 1993). For surface cultures, 02 and C02 concentrations evolu- 44 tion was followed. Water activity and pH were also determined at the end of the fer1 mentation. Table 2: Culture conditions witbout forced aeration. Each experiment was made with both gauze and tight-sealed. Run II 12 13 14 15 16 17 18 19 Nutritive media Su bslrates/Supports Wheat bran Wheat bran Wheat bran Wheal bran Cassava Cassava Cassava Cassava Sugarcane bagasse SM Leucine Urea SM Leucine Urea SM + Leucine Volatiles produced during the fermentation were characterised by gas chromalography (Hewlen-Packard 5890 equipped with a Megabore HP-I column (Iength, 5m) and Wilh a f1ame ionisation detector) of headspace vapour from the cultures (only for experimems withoUI forced aeration). Conditions were: Temperatures, injector and detector: 210°C, oyen he Id al 40°C during 2 minutes and then programmed al 10°C/min to 150°C. The nitrogen gas f10w rate was 1.5 ml/min and splil ratio 1:32. Table 3: Results of aroma production in packed cultures and forced aeration. • - none, + weak, ++ medium, +++ strong. # trnax: lime of maximum aroma perception. § in mllh.g IDM. Run 1 2 3 4 5 6 7 8 9 10 Aroma & Intensity • trnax (h)# CDPRmax~ - - banana +, lhen apple/pear + banana + pear/apple + pear/apple ++ pear/apple ++ banana +++ banana +++ pear/apple +. then peach +++ pear/apple ++. then peach ++ 67/91 2.58 0.95 Aw final 0.996 0973 pH final 6.76 3.05 2.65 8.76 9.06 8.95 9.03 9.06 2.71 6.24 91 42 17 39 17 17 20/91 0.89 2.45 1.04 0.72 0.79 0.84 0.990 0.983 0.988 0.985 0.989 0.990 0.993 68/116 070 0.998 !.JO 45 ResuJts are presented in two parts according to the modes of culture and aeration used. Packed cultures experiments with forced aeration. From the results presented in table 3, it can be seen that at the higher aeration rate (0.05 l/h.g !DM in runs 1 and 4), no or poor aroma was detected. It can he assumed that this rate swept away the volatiles produced and/or oxygenated conditions reduce the synthesis of such molecules. It is why a very low aeration was then used (0.005 l/h.g !DM). In that case, the overall aromas detected (peariapple and banana) were stronger. In particular, it was clearly shown that leucine and valine, when added to the medium, played a precursor role for the development of the banana aroma (runs 7 and 8). This fruity aroma appeared very rapidly (before the first 24 hours) in these cases. When no precursor was used, pear/apple aroma was also detected (runs 4, 5. 6, 9 and 10). It must be pointed the observations made in runs 9 and 10 when 2 successive kinds of aromas were detected, first the pear/apple one and then at 5 days a strong peach one. With this aeration rate, lower CDPR max were observed (Iess than 1.3 ml/h.g !DM) which indicates maybe that, in poorly aerated media, growth was limited and volatile metabolites production favoured. In terms of support evaluation, wheat bran (supplemented or not) and suppJemented bagasse gave better result than Amberlite. In all cases, water activity was maintained at a satisfactory level, but pH sometimes at the end of the fermentation were alkaline (for wheat bran) or acid (for bagasse). In all cases, no compounds were detected in headspace analysis of the cultures. As a conclusion, wheat bran and bagasse are adequate substrates and supports, very low aeration is recommended (0.005 l/h.g IDM), precursors like leucine or valine can shift fermentation to a particular aroma, although fruit), aroma was also produced without them. From table 3 and figures 1 and 2. it can be seen that in both cases, the maximum aroma was detected just after the maximum in CDPR was attained. Seemingly, aroma production was found to be growth related. Surface cultures experiments without forced aeration In this case 9 combinat ions were tested, and in each case, experiments were made with gauze cover (static aerated culture) and tightly-sealed (without aeration). Sensorial evaluation was only possible in the case of aerated cultures. In the second case, respiration was characterised by 02 (%) consumption and C02 (%) accumulation in the flask. Results are presented in Table 4. Ali of the three substrates/support were found to allow growth and aroma production in aerated conditions. Aroma detection was more important than in experiments made with forced aeration. The strongest aroma detected (banana) corresponded to the media in which leucine was added (runs 13, 17, 19) while pear/apple aroma, with a lower intensit)l, was obtained with wheat bran completed with synthetic medium and urea (runs 12 and 14). These aromas were detected with major intensity between the first and the second day. 46 Table 4: Results of aroma production in surface cultures without forced aeration. * - none, + weak, ++ medium, +++ strong. # tmax: time of maximum perception of the aroma. § in mllh.g !DM. 1 refers to aerated cultures and 2, to tight-sealed !lask cultures. Run Il 12 13 14 15 16 17 18 19 Aroma & Inlensity *1 tmax (h)#\ - - apple/pear ++ banana +++ apple/pear ++ banana ++ 44.3 35.8 41.3 40 - - banana +++ 40 - - banana +++ 39.3 CDPRmax §! cm max (%)2 1. 15 0.06 0.16 1.23 0.20 0.90 0.20 0.45 0.08 81.3 31.5 22.3 36.7 62.5 29.6 7.3 46.9 lU For tight-sealed !lask cultures, growth was also observed. As growth and substrate fermentation evolved, C02 was produced and the internai pressure increased. This pressure was released during sampling which provoked the increase in C02 concentration up 10 81 %. These values of C02 were coupled with low 02 (Iess than 2 % of residual oxygen) which channelled the metabolism toward the fermentative route. No sensorial evaluation was made for tight-sealed fiask cultures but it can be seen in figure 3 thallarge amounlS of volatile were also produced. Separation and identification of GC detected compounds For headspace chromatograms of aerated and tight-sealed cultures of run 19, fourteen compounds were detecled. Eleven compounds were identified lhrough relention lime comparison with a standard and can be classified according to their relative quantity (peak area): ethanol, ethyl acetate, ethyl propionate and isoamy 1 acetate are important; acetaldehyde, isoamyl alcohol and isobutyJ acetate are intermediate and 1propanol, 2-propanol, J -butanol and amyl acetate are in small amounts. Arnong them, isoamyl acetate and isoamyl alcohol are knOWI1 to be important compounds in the aroma of banana. while acetaldehyde, ethanol and ethyl acetate are al ways present in fruit aromas. Other minor compounds like ethy\ propionate and isobutyl ace ta te are also reported to participate in fruit aromas. Sorne difTerences can be observed berween aerated and tight-sealed culrures. Acetaldehyde peak is bigger. ethyl propionate peak is smaller and unknown peak # 13 is absent in the second case. Unfortunately, it is not possible to evaluate directly the impact of these differences on the aroma. 47 4 banana apple :E C Cl oC 0.8 3 3 0.6 QI -. E a: a. c () » ... 0 2 :::s Cl) 0.4 [ :::s Vl 0.2 0 '< 1 0 20 40 60 80 100 120 0 140 Time (h) Figure 1. Evolution of Carbon Dioxide Production Rate (CDPR) during production of banana/apple aroma. 48 banana peach -c 0.8 Cl 0.6 :?: ~ :::: E -a:: 4 3 3 Dl 2 0.4 C ;:, Cl) ;:, c. () » ... 0 CIl O.2~ o Œ1 0 '< 1 20 40 60 80 1 0 100 120 140 lime (h) Figure 2. Evolution of Carbon Oioxide Production Rate (CDPR) du ring production of banana/peach aroma. 49 Conclusion Wheat bran, cassava and sugarcane bagasse were found to be adequate substrates/suppon for aroma production by C. fimbria/a. Amino acids like valine or leucine seemed to be direct precursors ofbanana-like aroma. Other aromas (peach, apple) were also detected without adding any precursor. The corresponding compounds of banana aroma (isoamyl alcohol and isoamyl acetate) were detected in the headspace of the culture at relatively important amounts. A IOta1 of 14 compounds were separated by GC and among them II were identified (1 aldehyde,5 alcohols and 5 esters). Work is currently continued to identify the unknown peaks and to quantify the identified compounds. Very low aeration (0.005 l/h.g !DM or passive diffusion) favoured the detection of strong aromas. Results were highJy improved in the se conditions in comparison with those obtained by Christen e/ al. (1994) at higher aeration rates. In the tightsealed experiments, it was shown that the fungus was able to ferment the carbohydrates present in the mediwn (glucose in the case of bagasse, derivatives of starch in the case of wheat bran and cassava). The fact that very low or no aeration is required opens interesting technological perspectives for the production of fruity aromas by C. fimbria/a. References Bigelis, R. 1992. Flavor metabolites and enzymes from filamentous fungi. Food Technol 46 ( 10) : 151-161. Christen, P., RaimbauJt, M. 1991. Optimization of culture mediwn for aroma production by Cera/ocys/is fimbria/a. Bio/ech. Le". 13 (7) : 521-526. Christen, P., Auria, R., Vega, c., Villegas, E., Revah, S. 1993. Growth of Candida u/ilis in solid state fermentation. Bio/ech. Adv. II : 549-557. Christen, P., Villegas, E., Revah, S. 1994. Growth and aroma production by Ceralocys/is fimbria/a in various fermentation media. Bio/ech. Le". 16 (II) : 1183-1188. Christen, P. 1995. Producci6n de aromas en fermentaci6n s61ida. Topicos de lnves/igacion y Docencia. Submined. Gervais, P., Sarrene, M. 1990. Influence of age of mycelium and water activity on aroma production by Trichoderma viride. 1. Fermen/. Bioeng. 69 (1) : 46-50. Hanssen, H.P., Sprecher, E. 1981. In: Schreier P. (ed.), Flavour'81, 547-556, W. de Gruyler, West Germany. Humphrey, M., Pearce, S., Skill, B. 1990. Biotransformation of coconut fat to methyl ketones. A commercial scale solid-state fermentation. In: Abu Symp. Bioforma/ion of Flavours. London. Janssens, L., de Poo ter, H.L.. Schamp, N.M., Vandamme, EJ. 1992. Production of flavours by microorganisms. Process Biochem. 27 : 195-215. Latrasse, A., Degorce-Dwnas, J.R., Leveau, J.Y. 1985. Production d'arômes par les microorganismes. Sci Alim. 5 : 1-26. Senemaud, C. 1988. Les champignons filamenteux producteurs d'arômes fruités. Etudes de faisabilité sur substrats agro-industriels. Ph. D. Thesis, Université de Bourgogne (France), 170p. Welsh, F.W., Murray, W.D., Williams, R.E. 1989. Microbiological and enzymalic production of flavor and fragrance chemicals. Cri!. Rev. Bio/ech. 9 (2) : 105-169. Yamauchi, H., Akita, O., Obata, 1., Amachi, T., Ham, S., Yoshizawa, K. 1989. Production and application of a fruity odor in a solid-state culture of Neurospora sp. using pregelatinized polish rice. Agric. Biol Chem. 53 (11) : 2881-2886. 50 FUNGAL BIOMASS ESTIMATION IN BATCH SOLID SUBSTRATE CULTIVATION USING ASYMPTOTIC OBSERVATION 1 Armin Ebner 1, Ivàn Solar 1 , Gonzalo Acuna 2 , J. Ricardo Pèrez-Correa , and Eduardo Agosin 1. IOepto. de Ingenieria Quimica y Bioprocesos, Pontificia Universidad Catûlica de Chile. Casilla 306. 22, Chile. Universidad de Santiago de Chi le, CECTA, Casilla 33074, Santiago 33, Chile. ~antiago Abstract Asymptotic observers for biomass estimation have been generalized for kinetic models that include monality and maintenance coefftcients, making them applicable to fermentation batch processes. The observer, which use C02 measurements and a non-linear model of the process, was applied 10 laboratory experiments with the fungus Gibberella fujikuroi. Model parameters were calibrated with laboratory data, using a non-degradable support to simplify biomass measurements. Convergence of the estimator is assured as long as the maintenance or mortality coefficients are non zero, and the sugars concentration is kept at a high level. However. the speed of convergence cannot be modified. Introduction The Jack of on-line measurements of key variables is the main obstacle that hinders the development of proper control and optimization systems for fermentation processes. This is specially true for solid substrate cultivation (SSC), that lags behind submerged cultivation. To partially overcome this limitation of SSC processes, on-line observers of relevant variables such as biomass, secondary metabolites and nutrients concentration, can be developed. ln fact, the high cost and scarcity of reliable sensors adapted to SSC systems, forces the development of softsensors (software + sensors) [1]. A large number of applications of softsensors has been reported in the literature. For example, the extended Kalman filter has been used successfuJly in the observation of variables in processes such as the effluent processing [2], foods drying [3] and vegetable cells cultivation [4]. The adaptive Kalman filter has been also used to determine the process states and. at the same time, certain time variant kinetic parameters [5, 6]. However these filters, given the non-Iinearity of the represented processes, loose the optimality character that has the linear Kalman filter. To overcome this problem, Lj ung and Süderstrom [7] and then Goodwin and Sin [8] proposed an 51 algorithm called recursive predictive error. This algoritlun was successfully used by Chattaway and Stephanopoulos [9] to detect contamination of bioreactors. However, these kind of estimators are difficult to implement, are sensitive to model parameters variations and are not generally applicable. The observability of the system model must be guaranteed throughout the whole process. These conditions are rarely met in batch fermentation processes, panicularly in SSc. To overcome the limitations of these Kalman based observers, Bastin and Dochain [10] proposed asymptotic estimators (without arbitrary convergence speed) especially adapted for fermentation processes. These estimators are useful in processes whose observability is not guaranteed, as are many batch fermentation non-linear processes. They have been successfully applied. given their implementation simplicity, in many biotechnology processes [11-15]. The observer developed in the present work, is heavily based on this kind of estimators. First, the model of the SSC system is presented. Then, the observer is developed and conditions for convergence are analyzed. The application of the observer to laboratory data, with uncertainty in initial conditions, is discussed next. The work is concluded with some final remarks. Model The growth dynamics of Gibberella fujikuroi in a batch SSC process, can be represented by the next equation: dX dl p. X - Kd · X (1) The variable X represents biomass concentration as gr. of dry biomass / gr. of dry matter (inert support). The terrn Il represents the specific growth rate and it is usually considered a function of temperarure, pH, waler activity and Iimiting substrate concentrations. The present model assumes that Il depends only on nutrients concentration, since the other variables are kept relatively constant, then: G g·N P= LK,"X + N] . (2) IKe; + G] where N represents the substrate concentration in terrn of gr. of nitrogen / gr. of dry matter, G represents the hydrolyzed and non-hydrolyzed sugars concentration in gr. of sugars / gr. of dry matter, Ilm is the maximum specific growth rate, and KN and KG are kinetic parameters. It is assumed that the nitrogen consumption follows the Contois mode l, while sugars consumption folJows a Monod mode!. This is juslified since nitrogen is less available than sugars and its consumplion is Iimited by diffusion. 52 Assuming that nitrogen consumption for maintenance and production is negligibJe. only the nitrogen used for biomass growth is taken into account: dN di" (3) = -1l· X1Yq,v where YX/N is the biomass/nitrogen yield coefficient. Other authors [16, 17] used a different model that directly relates nutrients consumption with biomass growth kinetics: dIV", dl (4) Then, introducing eg. (1), yields: (5) This expression, which is different From eg. (3), is incorrectly used in the literature [18-20]. Then. eg. (4) is onJy applicable to the special case where autolysis is considered negligible. The model used here for sugars consumption. follows the development of Acevedo [17]: (6) where YXJG corresponds to the biomass/sugars yield coefficient, and mG to the maintenance coefficient. The dynamics of C02 production and 02 consumption rates are given by the following eguations: C dCO, - d l- = Il . X 1 YI' lCO, + "t-o, .- - - X K(;+C C dO, dl - =-11· X 1 YI'fO, -mOl -- - - X Kc +C (7) (8) Here. C02 and 02 are the accumulation of carbon dioxide formed and oxygen consumed, expressed in gr C02 1 (gr dry maner) and gr 02 1 (gr dry matter) respectively. Y X/C02 and Y XJ02 53 are the biomass/carbon dioxide and biomass/oxygen yield coefficients respectively, and finally. mC02 and m02 are the maintenance coefficients for carbon dioxide and oxygen respectively. ln the above equations, X, N, G and C02 and 02 are the state variables. where only carbon dioxide and oxygen outlet gas concentration are usually measured in SSC processes. Table 1 sununarizes the model parameters estimated from laboratory data. Parameter C0 20 Kr! KG KN Go m r()') ml. No mm Xo YYlrn7 y y Ir; YX/N Table l' Model Parameters value units [gr C0 20 .lgr d.m.l 0.0000 [I/hr] 0.0012 [gr sugars/gr d.m.l 0.0800 [gr nitrog.lgr biomass] 0.0500 [gr sugars.lgr d.m.l 0.5000 [gr C02 ace/gr biomass hrl 0.0231 [gr sugars/gr biomass hrl 0.0420 [gr nitrog.lgr d.m.1 0.0095 [1 Ihrl 0.1650 [gr biomass/gr d.m.] 0.0060 [gr biomass/gr C02 acc.l 1.6700 [gr biomass/gr d.m.] 0.5500 [gr biomass/gr nitrog.] 13.7000 Observer Development The aim of the observer developed in this work, is to estimate biomass concentration based on carbon dioxide measurements. The use of exponential observers, as the Extended Kalman filter. emerges as the first alternative to be analyzed. However, the structure of the system. with state variables strongly interactive due to Gand N influences, makes the observability and convergence analysis very curnbersome [21]. A much simpler and interesting alternative to exponential filters, are the asymptotic observers. as proposed in [10]. Unfortunately, the maintenance parameters in the model above, which depend on G, do not allow direct application of this method. ln the above mode!, there is no auxiliary variable Z, which is only function of observed states. In fact, the model equations, excluding oxygen since it is not measured, wrinen in vector forrn are: dx dï= K cI>(x)+f(x) (9) with x the state vector, 54 X T = [X,N ,cq,G] (10) K a constants vector, (1 1) with <1J(x) and r(x), non-linear vector functions of the states, defined as. <1J(X) = f1(N ,G)· X (12) (13 ) This state representation can be split into rwo partitions: dxa / di" = Ka . <1J(x) + ra(x) ( 14) (15) Since rank(K) = 1, these states can be chosen in the following way: Xa = X (16) x; = [N ,cq,G] ( 17) inducing the next partitions in K and G: Ka =1 (18) ( 19) fa(x) = -KJ X r:(x) = [o,mCOl (20) K0~G 55 X,-m(; K(;~GX] (21 ) According to the methodology proposed by Bastin and Dochain [la], this system can be transfonned into another that do not include the specifie kinetic vector, F(x). For this purpose. an auxiliary variable, Z, can be defined: (22) whose dynamics is given by: a (23) Finally, considering that the only observed variable is the C02 concentration, the vector Z can be reordered in the next form: [0] Z11 l/Yx/v (24) Z= ZZ: =Am-Xm+An-Xn= al -C02 + -IIY\,/co, [ ~ l/Yx/G from where it is easy to obtain the non-observed state, xn ' in terms of the observed state, x m . and the auxiliary variable, Z, yielding: X] N [G = [01 YI- -/ Yx /C02 YI 1y\, 0] [ZI cOli y\, / NO- Z) - co) a ! C02 ! G 1 ] (25) Z3 Using the above matrix equations, the following asymptotic observer can be established: 56 o (26) G A mG - - - , X K(j+G with - Y'Fo, YrFo,IY,!" (27) Y'/CO,/YXp A using the following initial conditions for Z: ~o ~] [~~~~] 1 where A (28) G(O) means estimated variable. Observer analysis A Here, the dynarnics of Z (eq. 26) is not independent of the non-observed states. Therefore. the above observer does not corresponds to the asymptotic observer as it was outlined in (10). Then, il is necessary to analyze carefully the stabiliry conditions of this new estimator. To do this. il is usefulto consider the error eSlimate vector dynamics, given by: (29) with 57 (30) and the functions Ci> C2 and C J , given by: Cl] [ c; ~ = [Kd' Yx {C021 Yx 1 N ] (Kd 1 y,. {C02 + mC02 . G 1( 1(; + G)) . }\ { co, (Kd 1 Yx 1 e + me . G 1(Kc + G)) . Yx (31) {CO 2 From the characteristic polynomial of eq. (29), and assuming that: G G (32) the eigenvalues of the error dynamics are: 1..1 = ÀJ = 0 and 1.. 2 = - C2. The above asswnption is valid only for sugars concentration values larger than 10'KG, that is, G > 0.7 (gr. 1 gr. d.m.), during ail the course of the cultivation. Therefore, the estimation error will disappear asymptotically for En, as long as C2 > O. ln the case of EZI and En, any initial error will not be drove to zero. In terms of the state variables, eq. (27) indicates that only X could be estimated asymptotically without error, since ils estimation depends only on Z 2' On the other hand, the estimation ofN and G will be always affected by the initial estimation errors. Results and Discussion Figure. 1 illustrates the above statements. In this simulated example, high sugars concentrations have been used. Different initial error estimates of N, G and X, have been considered; 10%, 10% and 500% respectively. As it was expected, the initial error remains in the estimation ofN and G. On the other hand, the estimation of X converges to its true value. 58 0.14,-------------------, 0.10 ooa 006 o."" 0.02 " 0.00 +--+--+--1--+----1----1----1---1 160 140 40 80 100 120 o 60 20 0.000 0 20 40 60 ao 100 lIme (hOtlrsl lime (hours) '20 ..0 160 3.0 (c) 2.5 20 1.5 1.0 -- - Simulated 1 0.5 E,ti mal "" 0.0 a 20 40 60 100 80 Tlme <"ours) 120 140 180 Figure 1: Estimation of biomass (a), nitrogen (b) and sugars (c). Initial estimation error of 10% for sugars and nitrogen concentrations and 500% for biomass. High initial sugars concentration. G(0)=2.5 (gr. / gr. d.m.). Symbols and discontinuous lines represent experimental measurements. The instability of the estimator for low sugars concentrations (G < 0.7 gr. / gr. d.m.) can be appreciated in Figure 2. In this case, ail the estimation errors growth without limit, even though the same initial estÏmate errors used in the previous figure were considered in this simulation. Il is wonh nOling thal Ihe developed observer for SSC processes converges in spile of being a batch process. Bastin and Dochain [10] have shown that the asymptotic estimaLOr converges in continuous and fed-batch fermentation, as long as the dilution rate, D, is different from zero for Jong periods. Since in batch processes D = 0, the asymptotic estimator does nol generally converges. However, the observer developed in the present work converges because the parameters Kd and mC02, which define the value of C 2 in eq. (31). are not zero. Finally, it must be pointed outthat with asymptotic observers the speed of convergenœ cannot be modified. This is an even more serious limitation in batch processes, since in continuous and fedbatch processes Ihe dilution rate can be used 10 speed up Ihe convergence. Unforlunately. parameters as Kd and mC02 are intrinsic of the process and Ihey cannOI be modified to acce\erale the estimation convergence. 59 In the example shown in Fig. l, it is seen that such convergence is reached, for biomass. approximately after 100 hours. This corresponds to approximately 60% of the total time of the cultivation process, and once the exponential gro\Vth phase has already ended. This is not very good from the process control point of view, since it is in such phase where coherent actions must be exercises to attain the desired biomass concentration level. The use of exponential or predictive observers, which provide faster convergence. stays as an alternative that will be dealt in future studies. To apply the se observers to the model described in the present work, high values of sugars concentration are also required. Figure 2: Estimation of biomass (a), nitrogen (b) and sugars (c). Initial estimation error of 10% for sugars and nitrogen concentrations and 500% for biomass. Low initial sugars concentration, G(0)=0.5 (gr. / gr. d.m.). Symbols and discontinuous lines represent experimental measurements. 60 Conclusions The observers development method proposed by Bastin and Dochain [10], has been generalized for kinetic models that include mortaliry and maintenance coefficients, and applied to a SSC process. In addition, an anaJysis of the estimator concluded that it will converge for the main variable (biomass concentration), provided that the carbon source concentration is high enough and the mortaliry or maintenance coefficients are different from zero. Acknowledgments This work has been supported by projects FONDEF 2-50, FONDECYT 1960360, D1CYT 9595AL and FONDECYT 1961299. References 1 Royce, P" 1993, A discussion of recenl developments in fermentation monitoring and control from a practical perspective, Cril. Rev. Bioteehnol., 13: 117-149. 2 Ayesa, E., Florez, 1., Garcia-Heras, J.L., Larrea. L:, 1991, State and coefficients estimation for the activated sludge process using a modified Kalman filter algorithm, Wal. Sei. Tech., 24:235-247. 3 Kiranoudis, C.T., Dimitratos. 1., Maroulis, Z.B., Marino-Kouris. D., 1993, State estimation in the batch drying of foods. Drying Teehnol., Il: 1053-1069. 4 AJbiol, 1., Robuste, J., Casas, c., Poch. M., 1993, Biomass estimation in plant cell cultures using and extended Kalman filter, Bioreehnol. Prog. 9: 174-178. 5 StephanopouJos G., San, K.Y.. 1984, Studies on on-Iine bioreactor identification 1: Theory, Biorechnol. Bioeng., 26: 1176-1188. 6 Shimizu, H.. Takamatsu. T., 1989, An algorithmic approach to constructing the on-line estimation system for the specific growth rate. Bioteehnol. Bioeng., 33:354-364. 7 Ljung, L., Sbderstrom T .. 1983, Theory and practice of recursive identification. MIT Press. 8 Goodwin. G.c., Sin, K.S., 1984, Adaptive filtering, prediction and control. Prentice-Hall, Englewood Cliffs. New Jersey. 61 9 Chattaway, T., Stephanopoulos, G.N., 1989, An adaptive state estimator for detecting contaminants in bioreactors, Biolechnol. Bioeng., 34:647-659. 10 Bastin, G., Dochain, D., 1990, On-line estimation and adaptive control of bioreactors, Elsevier Science Publishing Co., Amsterdam. II Acuiia, G., Latrille, E., Béai, c., Corrieu, G., Chéruy, A., 1994, On-line estimation of biological variables during pH controlled lactic acid fermentations, Biolechnol. Bioeng., 44:1168-1176. 12 Dochain, D., Pauss, A., 1988, On-line estimation of microbial specifie gro\vth-rates: an iJlustrative case study, Can. 1. Chem Eng.,66:626-630. 13 Flaus, J.M., Chéruy, A., Pons, M.N., Engasser, 1.M., 1989, Estimation de l'état physiologique des microorganismes dans un bioprocédé, RAIRO-APII, 23 :211-220. 14 Lubenova, V., Ignatova, M., Tsonkov, S., 1993, On-line estimation of specifie growth rate for a class of aerobic batch processes, Chem. Biochem. Eng. Q. 7: 10 1-1 06. 15 Pomerleau, Y., Perrier, M., 1990, Estimation of a multiple specifie growth rates in bioprocesses, AICheE 1. 36:207-215. 16 Raimbault M. 1981. Fermentation en Milieu Solide, Croissance de Champignons Filamenteaux sur Substrat Amylacé. Service des Publications de L' O.R.S.T.O.M. Paris, France, p 132- 132,149-153,166-167,176,222-223. 17 Acevedo F. 1988. Cinética de Fermentaciones. Tercer Curso Latinoamericano de Biotecnologia, Octavo Curso Internacional de Ingenieria Bioquimica. Universidad de Valparaiso, Escuela de Ingenieria Bioquimica, Organizaci6n de los Estados Cat6lica Americanos, Agosto 7-19 de 1988. 18 Menezes 1. C. , Alves S. S. 1994 Mathematical Modelling of Industrial Pilot Plant PenicillinG ; Fedbatch Fermentations. J. Chem. Tech. Biotechnol. 61, p 123-138. 19 Demain A. y Salomon N. 1988 Manual of Industrial Microbiology and Biotechnolgy. American Society for microbiology, Washinton D. C. , p 138. 20 Huang S. y. Chow M. S. 1990 Jinetic Model for Microbial Uptake of Insoluble Solid-State Substrate. Biotech. and Bioeng. 35, p 547-558. 21 Ljung, L., 1979, Asymptotic behaviour of the Extended Kalman filter as a parameter estimator for linear systems. IEEE Trans. aU/omo Control 24, 36-50. 62 MUTAGENESIS AND ADAPTED STRAINS TO THE GROWTH IN LIQUID OR SOLID SUBSTRATES Christopher Augur and G. Viniegra-Gonzalez Universidad Autonoma Metropolitana, Unidad Iztapalapa,IOepartamento de Biotecnologia Av. Michoacan y Purisima, Col. Vicentina, Apdo. Postal 55-535. Mexico, O.F .. c.P. 09340 Summary Mutation by UV Iight resulted in the isolation of A. niger strains geared lOwards high pectinase production in either SSF or SFM but never in both. Water activiry plays an essential role on the levels of activity of specific enzymes. Mutated strains showed growth and sporulation patterns distinguishable by morphometric techniques. when cuhured on defined media. Many enzymes which break down complex polymers are produced commercially or are being developed for such use. Biosynthesis of these enzymes is often controlled by carbon catabolic repression. Selection for mutants resistant to analogous of glucose has been used to isolate mutants which are deficient in carbon catabolic repression of these enzymes. For example, resistance to 2-deoxyglucose has been used to isolate deregulated mutants of Trichoderma, which overproduce cellulase [17] or as we shall see below. to select for mutants overproducing pectinases in either SSF or Smf. Shankaranand el al. [2] suggested that rnicrobial strains selected for SSF processes should be different to those selected for SmF processes. They found, for example. that in a collection of nearly 30 bacterial strains, the majoriry of them were good enzyme producers either in SSF or Smf but seldom in both. Antier el al. [1.3] isolated UV mutants of wild strain A. niger C28B25 [4]. The selection phenotype was 2-deoxy-glucose (DG) resistance (DG R) but in culture media with two different water activities (a" = 0.99 or 0.96) such phenotypes were labelled to belong to classes: a) A W99 and b) A W96 (when a" = 0.96 after adding 15% ethylene glycol). Mutants A W99 had an inverse correlation between their abiliry to produce pectinase by SSF on coffee pulp (a w = 0.96) as shown by solid bars in figure 1 with respect 10 the production of pectinase by Smf shown as c1ear bars in the same figure. Apparently there was a trade off relation between each kind of those phenorypes. Strain WT in that figure corresponds to wild type (C28B25) isolated by Boccas el al. [4]. Therefore. strain AW99-iii had seven times less potency for SSF pectinase production and more than lhree limes more potency for Smf as compared to WT (see figure 1). 63 19) ~------------------==-....., BIC WT AW99-b AW99·ii AW99-i AW99-c AW99-a AW99-iii STRAINS Figure 1. Comparison of pectinase activities of DG R mutants of A. niger C28B25 isolated at bigb wat ra tivity (a w = 0.99) and ultured in sbake flasks ( mF) or coffee pulp packed bed columns (SSF) according to Antier et al. [1]. U PEe = arbitrary enzyme viscomelTy units expressed by g of solid subslTate (SSF) or dry biomass (SmF). ln Figure 2 the same kind of results are shown for A W96 mutants. Here the inverse correlation of pectinase production by SSF and Smf is not as evident as in Fig. 1. Strain WT is the same wild type shown in the previous figure. Ail A W96 mutants had equal or higher potency in Smf than the wild type but some of them (strains A W96-1 and 3 in Fig. 2) increased their potency in SSF by nearly 40% over WT. Antier el al. [3] showed that DO R phenotype was independent from A W96 and A W99 phenotypes. Since a DO R reverting strain (A W96-3 became DOS) was found to retain high pectinase productivity in low water activity but became highly sensitive to DO as the \vild type [3]. ~50 ,--------------------------, WO 150 100 50 a WT AW96-4 AW96·2 AW96-1 AW96·3 5TRAIN5 Figure 2. Comparison of pectinase activities of DG R mutants of A. niger C28B25 (WT) isolated at low water activity (a w = 0.96) and cultured in shake flasks (SmF ) and coffee pulp packed bed columns (SSF) according to Antier et al. 111. U PEe are arbitrary enzyme units by viscometry expressed by g of solid substrate (SSF) or dry biomass (SmF). 64 Recent work in our laboratory (Romero el al., 1996 and 1997, unpublished data) has shown that strains AW96 and AW99 are derepressed for the production of pectinase. invertase and amylase, although they were selected on the basis of pectinase over production [1, 3]. Such observations support the existence of general regulatory mechanisms invo\ved in the adaptation of moulds to liquid or solid environments and controlling the yield and quality of enzymes best suited for each kind of culture medium. As a consequence, the selection of strains for the production of enzymes by SSF technique requires the use of specific protocols including the survivaJ to metabolic stress factors, namely, the presence of antimetabolites such as DG [1] or dinitro phenol [3] and also low levels of water activity. Concerning the importance of water activity for the production of enzyme by SSF technique, Narahara el al. [13] proposed the use of mixtures of steamed rice particles with lignocellulosic particles in order to increase the moisture content of koji SSF fermentation for the production of amylases. Oriol el al. [II] used mixtures of steam cooked cassava granules with sieved fibres of sugar cane bagasse. This was based on the fact that bagasse can absorb four-fold its dry weight in water whereas cassa va granules can only absorb one-fold its dry weight in water. The overall moisture content of those mixtures was a weighed average of the corresponding fractions of each materia1. Thus, initial moistures couId be adj usted in the range from 42% up to 70%. A summary of their results is shown in Table 3. Sirnilar results were obtained using different levels of glucose absorbed in bagasse, from lOto 300 gIL [Il]. The value of the specific groWlh rate was also an increasing function of a". In this system there was an optimum value for enzyme productivity (not shown in Table 2) because bagasse fibres did not contain starch nor were inoculated with mould spores. they were only used as a water reservoir. This experiment showed the presence of inter-particle mass transfer of moisture and suggested the use of mixed materials in order 10 correct for low moisture content in amylaceous substrates. Table 3. Effect of initial water activity (a....) on the maximal amount of biomass (X mu ) and substrate conversion of Aspergillus niger No. 10 rIll. initial SC Xma., glg Initial Dry glg Initial Dry aw · Weight Weight 0.933 0123 0.233 0.940 0.172 0.344 0.958 0.251 0.417 0.957 0.324 0.477 0.980 0.339 0558 Pandey [12] in his review of SSF processes comments "the types of micro-organisms that can grow in SSF systems are delermined by the water activity factor a".... defined as the relative humidity of the gaseous atmosphere in equilibrium with the substrate" ... "The microorganisms, which can grow and are capable of carrying out their metabolic activities at lower 65 a w values are sui table for SSF processes". Sarrete el al. [14] showed that addition of glycerolto a tempeh SSF fermentation by Rhizopus oligosporus changed the yield of various polysaccharidases having different optimal conditions, for example, polygalacturonases and xylanases were maximal for a", values between 1.00 and 0.99 but endocellulase production ,vas maximal when aw = 0.98. Acuiia-Argüelles el al. [15] used ethylene glycol as water depressor of SSF cultures of A. niger grown on bagasse as support. They found that decreasing values of a w from 0.98 to 0.90 resulted in decreasing activities of exopectinase, as shown in Table 4. Those results are in agreement with earlier observations [16] showing that the production of polygalacturonase and cellulase by Geolrichum candidum was very sensitive to a w reduction, from 1.00 to 0.98, by the addition of KCI, mannitol or polyethylene glycol. Also, Grajek and Gervais [10] found that the production of polygalacturonase, D-xylanase and ~-galactosidase by SSF cultures of Tichoderma viridae TS was influenced by water activity. Table 4. Erreet ofwater aetivity on the level of exopeetinase produeed by A. niger CH4, [151· Water activity 0.98 0.97 0.94 0.90 Exopectinase activity (end point mg of reducing compounds per ml) 20 18 15 5 Minjares-Carranco el al., [8] carried out physiological comparisons between pectinaseproducing mutants of A. niger adapted either to SSF or SmF through morphometric analyses. Parent and mutant strains were gro,vn on a specific medium and morphological measurements were performed with a digital image analyser. For the characterisation of each strain, two indexes were used lb (growth rate of a colony on a given medium relative to maximun growth rate on medium DEX) and la (sporulated area over total area of colony after 72 hrs of gro\.\>1h). Each strain was then represented by a plot on the la versus lb plane. Figure J shows the « mapping » of the wild type strain and the mutant strains. They map in three distinct regions. Although the mutants described above were selected for their resistance to 2deoxyglucose, the resuJting pectinase-producing strains from bath classes (A W99 and A W96) show distinct physiological and phenotypic patterns. The deoxyglucose resistance phenotype may not be directly involved with the complex patterns of physiological derepression and enzyme production. 66 la 0.6 A W type 0.4 0.2 •• • • AIN96 AVI/99 o -I---+.s:...I.~f---+--~f----i 0.8 0.4 06 0.2 o Figure 3.- la and lb values obtained for wild t)'pe (triangles) and mutant strains of series AW96 (square) and AW99 (circle). Each plot represents the average of triplicate measurements on a given strain. Bibliography 1. Antier, P., A., Minjares and G. Viniegra: Pectinase-hyperproducing mutants of Aspergillus niger C28B25 for solid state fermentation on colfee pulp. Enzyme Microb. Technol. 15 (1993a),254-260. 2. Shankaranand, V., Rarnesh, M.V. and Lonsane, B.K.: Idiosyncrasies ofsolid state fermentation systems in the biosynthesis of metabolites by sorne bacterial and fungaJ cultures. Process Biochem. 25 (1992), 33-36 3. Antier, Ph., Minjares, A., Roussos, S.. and Viniegra-Gonzalez, G.: New approach for selecting pectinase producing mutants of Aspergillus niger weil adapted to solid state fermentation. Bio/echnol. Adv. Il (1993),429-440. 4. Boccas, F., Roussos, S., Gutiérrez-Rojas, M., and Viniegra-Gonzalez, G.: Production of pectinase from coifee pulp in solid state fermentation system: selection of wild fungal isolales of high potency by a simple three step screening technique. J Food Sci. Technol. 31( 1) (1994), 22-26. 5. Loera-CorraJ, O.: Es/udios gené/icos de las mu/an/es de Aspergillus niger C28B25 sobre produclOras de peclinasas en suslralos liquidos y so/idos. M. Sc. thesis in Biotechnology. Universidad Aut6noma Metropolitana, Iztapalapa, D.F. (Méx.) (1994). 6. Allen. K.E., McNally, MT, Lowendorf. H.S., Slayman, C.W., and Free, S.J.: Deoxyglucoseresistant mutants of Neurospora crassa: isolation, mapping, and biochemical characterization .. J Bac/eriol. 171 (1989), 53-58 7. Alazard, D., Raimbault, M.: Comparative study of amylolytic enzyme production by Aspergillus niger in liquid and solid state cultivation. J Appl. Microbiol. Bio/echnol. 12 (1981), 113-117. 8. Minjares-Carranco, A., Trejo-Aguilar, B.A., Aguilar, G., and and Viniegra-Gonzalez, G.: Physiological comparisons between pectinase-producing mutants of Aspergillus niger adapted 67 either to solid-state fennentation or submerged fennentalion. Enzyme Mycrob. Technol. 21 (1997), 25-31. 9. AcUJ1a-Argüelles, M., Gutiérrez-Rojas, M., Viniegra-Gonzalez, G., Favela-Torres, E.: Production and properties of three pectinolytic activities produced by Aspergillus niger in submerged and solid state fennentation. Appl. Microbiol. Biorechnol. 43 (1995), 1-6. 10.-Grajek, W., and Gervais, P.: Influence of water activity on the enzyme biosinthesis and enzyme activities produced by Trichoderma viride TS in soIid state fennentation. Enzyme Microbiol. Technol. 9 (1987), 658-662. 11- Oriol, E., Raimbauit M., Roussos S., and Viniegra-Gonzalez G.: Water and water activity in the solid state fennentation of cassava starch by Aspergillus niger. Appl. Microbiol. Biorechnol. 27. (1988),498-503. 12.- Pandey, A.: Recent process developments in solid state fennentation. Process Biochemisrry. 27 (1992), 109-117. 13.- Narahara, H., Koyama, Y., Yoshida, T., Atthasampunna. P., Taguchi. H.: Control of water content in a solid-state culture of Aspergillus oryzae. J Ferment Technol. 62 (1984), 453-459. 14.- Sarrette, M., Nout, M.J.R., Gervais, P., and Rombouts, F.M.: Effect of water activity on the production and activity of Rhizopus oligosporus polysaccharidases. Appl. Microbiol. Biorechnol. 37 (1992), 420-425. 15.- Acuila-Argüelles, M., Gutiérrez-Rojas, M., Viniegra-Gonzalez, G., Favela-Torres. E.: Effect of water activity on exo-pectinase production by Aspergillus niger CH4 on solid state fennentation. Biorechnol. Leu. 16 (1) (1994), 23-28. 16.- Davis, L.L., and Baudoin B.A.M.: Erfect of osmotic potential on synthesis and secretion of polygalacturonase and cellulase by Geotrichum candidum. Can. J Microbiol. 33 (1987), 138-141. 17.- Labudova, 1., and Farkas, V. Enriclunent technique for the selection of cataboliterepression -resistant mutants of Trichoderma as producers of cellulase. FEMS Microbiol Leuers 20 (1983) -211-215 68 FUNGAL GENETICS, CASE OF ASPERGILLUS Christopher Augur Universidad Autonoma Metropolitana, Unidad Iztapalapa, Departamento de Biotecnologia Av. Michoacan y Purisima, Col. Vicentina, Apdo. Postal 55-535. Mexico, D.F .. c.P. 09340 Summary Classical genetic techniques enabled the selection of an arginine minus auxotroph from each of the mutant strains AW99 and AW96. When crossed. these [wo deoxyglucose resistanl strains yielded a deoxyglucose sensitive diploid. Foreign DNA (via plasmids) can be introduced into the A W99 strain in a stable manner, by electroporation or by polyethylene glycol in the presence of protoplasts. Genes can be directed 10 a specific site of the genome by Random Enzyme Mediated Integration (REMI). Pectinases play an important role in industry in the processing of fruit and vegetable juices as they can alter the viscosity and facilitate extraction, fdtration and clarification processes [1 J. A favourite organism for the production of pectinases is A. niger which has been modifled by mutation or c1assical genetics [7] and through molecular genetics [6]. In both cases it was possible to increment production of speciflc pectinases. Antier el al. [3] devised a strategy to increase pectinase production using SSF (coffee puJp) to which the parent strain C28B25 was added. Following UV-induced mutagenesis, mutant srrains were derived from A. niger C28B25 belonging either to series AW99 [producing maximum pectinase levels in submerged fermentations (SmF)] or series A W96 [producing maximum pectinase level in sold state fennentation (SSF)]. Mutants selected by Antier el al were resistant to catabolic repression by saccharose whereas the parent strain was sensitive.. In order to genetically characterise these strains. a master strain was required. Due to incompatibility with known master strains, Loera el 01.[4] decided to devise their own. Il was decided to induce a mutation in the set of DG R strains in the form of an auxotrophy to arginine. FUrlhennore, DNA could then be introduced by transfonnation into the auxotrophic strains ofa plasmid pDHG25. This plasmid contained the argB gene which could complement a specific arginine deficient strain. For this study IWO pectinase hyperproducing strains were selected: AW99 and AW96 (Figure 2). These strains were mutagenized by irradiation with UV light. This step was folJowed by an enrichrnent step during which genninating spores on minimal medium were eliminated with the aim of picking up non gerrninated spores with a higher probability of being auxotrophic [5]. One arginine mutanl was oblained from each of Ihe Iwo parent strains. resulting in the following strains : AW99arg - and AW96arg -. These two strains were then crossed. Loera-Corral [4] obtained a dikaryon of the two DG R strains (A W99arg - x AW96arg -.). 69 20G Resistance of Haploid and Oiploid Strains of A. niger ---'-A\\' 96 --D~ 3 (A\\' 96 X AW 99) - A \ \ ' 99 2.5 2 1.5 1 0.5 o o 0.2 0.1 0.3 0.5 0.4 20G (gIl) Figure 1. Oeoxyglu 0 r istance of baploid and diploid strains of A. niger The 04 dikaryon shown above in Figure 1 became deoxyglucose sensitive (OG s ); ail other phenotypic characterislÏcs were that of strain AW99 [4]. Phenotypes OG R were complementary to each other (as seen when diploid 04 was obtained) but phenotype AW99 was dominant over A W96. The way in which the phenotype OG R is associated to the selection of A W96 and AW99 phenotypes is still unknown. Allen el al., [2] have found pleiotropic mutations of Nellrospora crassa which are associated to OG R phenotype, mapping as OG R point mutants in four different loci, and at the same time, show derepressed and modified patterns of invertase and amylase production. DSmF 150 , . . . . - - - - - - - - - - - - - - - - - - - - - - - - - - - - , .SSF 100 150 100 50 o WT AW9&-4 AW96-2 AW96·1 AW96·J STRAINS Figure 2. Comparison of pectinase activities of OG R mutants of A. niger C28B25 (WT) isolated at low water activity (a w = 0.96) and cultured in sbake flasks (SmF ) and coffee pulp packed bed columns (SSF) according to Antier et al. [3J. U PEC are arbitrary enzyme units by viscometry expressed by g of solid substrate (SSF) or dry biomass (SmF). 70 Recent work in our laboratory (Romero el al., 1996 and 1997, unpublished data) has shown that strains AW96 and AW99 are derepressed for the production of pectinase, invertase and amylase, although they were selected on the basis ofpectinase over production [3]. Such observations support the existence of general regulatory mechanisms involved in the adaptation of moulds to liquid or solid environments and controlling the yield and quality of enzymes best suited for each kind of culture medium. As a consequence, the selection of strains for the production of enzymes by SSF technique requires the use of specific protocols including the survivalto metabolic stress factors, namely, the presence of antimetabolites such as DG or dinitro phenol and also low levels of water activity. After development through classicaJ genetics of strain AW99argB-, we used molecular genetic methods to test for the introduction of foreign DNA (via plasmids) into A W99arg-. The introduction of foreign genes into fungi has required the sening up of methods adapted from yeasts and bacteria. The arginine auxotroph AW99argB- was used as a host to study the introduction of two plasmids, pDHG25 (an autonomously replicating plasmid; see description in Armex) and pDC 1 (an integrative plasmid, see description in Armex). Both plasmids were introduced into the host either by electroporation (see conditions in Armex) or by polyethylene glycol mediated intoduction in protoplasts. A scheme of the laner technique is presented in armex. Additionally, the REMI (random enzyme mediated integration) technique was tested. This technique consists of adding a restriction enzyme (BamH 1) along with the DN A to be introduced into the fungal ce Ils. If the fungal genome contains within its DNA, the restiction site. the introduced DNA integrates within that site in the genome. A. niger is known to have only one BamH 1 site in ils genome. Results of these experiments are presented in Armex. The first conclusion to be made is that it is possible to insert foreign DNA into the AW99arg- strain either by electroporation or by polyethylene glycol (PEG) via protoplasts. ln both cases, the nurnber of transformants depends greatly on the type of DNA introduced (Iinear, circuJar). The integrative plasmid (pDC)) yields a greater number of transformants, irrespective of the method employed. REMI via electroporation of the integrative plasmid pOC l, seems to be the method of choice. Introduction of the linear pDC 1 plasmid in the BamH 1 site of the genome induced a phenotypic mutation known as fluft), (data not shown) where no sporulation occurs. The integration of the plasmid therefore disrupted an essential gene involved in conidiation. Foreign genes can therefore be introduced into the laboratory strains for study. The introduced genes are stable and can either replicate autonomously (if on the pDHG25 plasmid) or integrate into the genome either randomly or specifically (via REMI). 71 TRANSFORMATION BY PROTOPLAST FUSION 00 0°0°0°0 ° 00°0 00 SPORES ~ o 0 YOUNG MYCELIUM (14 hrs) - .. , ------I.~ NOVOZYME 234 CL YTIC ENZYMES . , - , -. , , .. , .. .. Trichoderma harzianum) DNA ...... : .", ... .... . ... 1""----. -.', ' \ 72 PEG 0 1' - ................ RESULTS FROM ELECTROPORATlON STRAIN:A. nigerdgrAW99arg PLASMID • ~~I SPORE CONCENTRATION = 10' No. of TRANSFORMANTS //lg ONA Negative control pOHG25 (circuLar) pDHG25 linearlBarnHI pOC 1 (circular) À PULSE (ms) - 6.7 4 32 5.5 5.6 6.5 16 40 pOCl linearlBarnHI pOCllinear+ 1 U BarnHI pDC 1 linear + 5 U BamHI pOC 1 linear + 10U BarnHI 6.3 6.5 68 12 5.9 6.3 3 .. CondItIOns used for transforma lion' Voltage (kv) Resistance (Q) Concentration of ONA (/lg) Recuperation time (hr) 1.0 400 0.25 2.5 RESULTS FROM PROTOPLAST TRANSFORMATION STRAIN:A. nigerdgrAW99arg SPORE CONCENTRATION = 10' TYPE OF ONA No. TRANSFORMANTS/ Ilg ONA Negative control Positive control pOHG25 linear BarnHI TREATEO AT 65°C 1 pOHG25 (circular) IpOCl circular pOC 1 linear /BamHI pOCI linear /BamHI + 1 unit restriction enzyme 2-3 75 24 120 18 1 12 IpOCI linear/BamHI + 5 unit 6 73 restriction enzyme pDC 1 linear lBarnHI + 10 units restriction enzyme 2 ------------- 1 STABILITY RESULTS Strain: A. niger dgrAW99arg transformation method: Electroporation Percent stability 60% 68 % 75 % 70% 71 % 74 % 67 % Type of plasmid pDHG25 circular pDHG25 linearized pDC 1 circular pDC 1 linearized pDC 1 !inearized + 1 U BarnHI pDC 1 linearized + 5 U BarnHI pDC 1 linearized + 10 U BarnHI Strain: A. niger dgrA W99arg Transformation method: Protoplasts 1 Type of plasmid pDHG25 circular pDHG25 linearized pDC 1 circular pDC 1 !inearized pDC 1 !inearized + 1 U BarnHI pDC 1 linearized + 5 U BarnHI pDC 1 linearized + 10 U BamHI Percent stability 70% 65 % 79% 83 % 83 % 81 % 79% 74 Bibliography \.- Ward, O. P. (1985). Hidrolytic Enzymes. In 8lanch, H. W.; Drew, S.; Wang, D. 1. (Des) Comprehensive Biotechnology. Pergarnon Press. New York. 3: 819-835. 2.- Allen, K. E.; MacNally, M. T.; Lowendorf, H. S.; Slayman, C. W.: Free, S. J. (1989) Deoxiglucose-Resistance Mutants of Neurospora crassa: Isolation, Mapping and 8iochemical Characterisation. J Bacteriol. 171 (1): 53-58. 3.- Antier, P.; Minjares, A.; Viniegra, G. (1993). Pectinase-Hyperproducing Mutants of Aspergillus niger C28825 for Solid State Fermentation on Coffee PuJp. Enzyme Microb. Technol. 15:254260. 4.- Loera-Corral, O. (1994). Caracterizaci6n de Mutantes de Aspergillus niger Sobreproductores de Pectinasas. Tésis de Maestria. Universidad Aut6noma Metropolitana. México, D. F. 5.- 80S. C. 1.; Debets 1. M.; Nachtegaal, H.; Slakhorst, S. M.; Swart, K. Isolation of Auxotrophic Mutants of Aspergillus niger by Filtration Enrichrnent and Lytic Enzymes. Curr. Genet (1992) 21: 1.17-120. 6.- 8ussink, HJD Van den Homberg, JPTW, Van den Ijssel and Visser, J. Characterization of polygalacturonase-overproducing Aspergillus niger transformants. Applied Microbial Biotech. (1992) 37: 324-329 7.- Leuchtenberger, A and Mayer, G. Changed pectinase synthesis by aggregated mycelium of sorne Aspergillus niger mutants. Enzyme microbialtechnol. (1992) 14: 18-22 75 GROWTH AND PRODUCTION OF IMMOBILISED LIPASE FROM Rhizopus delemar CULTIVATED IN SSF ON A SYNTHETIC RESIN (AMBERLITE) Pierre Christen' & Sergio Revah 2 1 Laboratoire de Biotechnologie, Centre ORSTOM, BP 5045; 34032 Montpellier Cedex 1, France 2 Universidad Autonoma Metropolitana, Unidad Iztapalapa, Departamento de !ngenieria, Av. Michoacan y Purisima, Col. Vicentina, Apdo. Postal 55·535. Mexico, D.F., C.P. 09340 Abstract ln this work, a study of lipase production by Rhizopus delemar grown on a polymeric resin (Amberlite IRA 900) added with a medium previously optimised is presented. ln the first part, the activity of several commercial preparations and the R. delemar lipase produced in liquid was compared. Il also appeared th a! the resin can adsorb more than 24% of the lipase produced per g Amberlite. Desorption realised using 5g NaCI 1 g Amberlite at pH 5 allowed to recover 35 % of the adsorbed lipase. It was also shown that the resin displayed a thermo-protective effect since no loss in activity was observed when the adsorbed enzyme was heated at 80 oC for 24 hours. ln solid state fermentation (SSF), the fun gus was shown to produce high amounts of enzyme (93 U 1 g dry Amberlite against only 14 U Iml in submerged culture) when dextrin was used as carbon source after only 24 hours (against 48 hours in liquid culture). Significative activity was also detected with maltose and more surprisingly with glucose (68 and 57 U 1 g dry Amberlite respectively). The strong inhibitory effect of glucose observed in liquid culture was reduced in SSF. Introduction Lipases are wide!y used enzymes that can be obtained from animaIs, plants and micro-organisms. Microbial lipases have been used in the food industry, mainly in dairy products, and are also important in detergents, pharmaceutical, cosmetics and leather processing (Seitz, 1973). The enzyme modified cheeses (EMC) are also an interesting application involving lipases (Revah and Lebeault, 1989). New trends in that field are directed toward the use of irrunobilised lipases in organic solvent for ester synthesis, triglycerides hydrolysis, flavouring compounds synthesis (Christen and L6pez-Munguia, 1994). Solid state fermentation can be a sui table method for producing enzymes such as pectinases, amylases, or eellulases (Lonsane and Ghildyal, 1992), but few papers have dealt with lipases. Nevertheless, Yamada ( 1977) reported that, in Japan, most of the microbiaJ lipases cornes from Aspergillus strain cultivated in liquid culture (Le) and SSF. More reeently, Rivera Muî10z el al (1991), using Penicillium candidum grown on wheat bran, found that SSF has many advantages against Le for lipase production. To understand belter fungal growth, inert supports impregnated with a nutritive solution have been reported. The aim of this work was to study the growth and 77 the lipase production by Rhizopus delemar on Amberlite, a weil studied anionic resin (Auria el al., 1990; Chrîslen el al., 1994). This inc1ude, characlerisation of!he lipolYlic activity in LC, SSF experimenls and interaction support/enzyme. Micro-organisms and culture media Two Rhizopus delemar strains were tested: COSS H313 (CINVESTAV-MEXICO) and NRRL 1472. They were periodically transferred on Potato Dextrose Agar (PDA) slants and stored at 4°C. Spores were produced in Erleruneyer l1asks on PDA at 29°C during 6 days. The nutritive medium previously optimised by Maninez Cruz el al. (1993) was used bo!h in LC and SSF. LC were made in 250 rnJ Erleruneyer l1asks placed on a rotatory shaker. Initial conditions were : temperature, 29°C; pH, 6: inoculum size, Ixl0 7 spores/ml; agitation speed, 180 rpm. In solid state cultures. an anionic resin (Amberlite IRA-900, Rohm & Haas) was used and prepared according to Christen el al. (1993). Nutritive medium was added to the dried support to complete 58% final water conlent. the maximum ab rption capacity of !he re in. The cultures were carried out in smaJl columns placed in temperature controlled bath. Initial conditions were: temperature, 2~C; pH, 6; inoculum size, Ixl0 7 spores/g Initial Dry Matter (!DM) and aeration rate, 0.5 l/h.g !DM. Analytical procedures ln LC, growth was followed by Ihe evolution in dry weighl. In SSF, respirometry was used which allowed to calculate C02 production rate (CDPR) as previously described (Christen el al., 1993).Water activity, moisture content and pH were also delerrnined al !he end of the fermentation. Lipolytic activiry was assayed with the method used by Nahas (1988) with sorne modification. The substrate was a 5 % tributyrin emulsion prepared in a 1 % Tween Solulion in 2.5 M tris-maleate buffer (pH = 6) by homogenising wilh an Ultraturax apparalus (8000 rpm during 2 mn). The reaction mixture contained 18 rnJ subst raIe and 12 ml extract solution. ln the case of adsorbed lipase, one gram of Amberlite was added 10 the reaction medium. The delerminalion was achieved wi!h a Mellier DL 21 pH sIal, at 37°C and pH adjusted 10 6. The butyric acid released was litrated with 5 mM NaOH solution during 5 min. One unil (U) was defined as the amount of enzyme releasing one mmol of free fait)' acid per minute. Adsorption/desorption study R. delemar lipase adsonion study was achieved using entire. (average diameter : 0.53 mm), or ground Amberlite (average diameter: 0.10 mm). Amounts varying From 0.5 g to 6 g of Amberlite were contacted wi!h 50 ml of enzymatic extract in 250 ml Erleruneyer l1asks placed in a rotatory shaker (150 rpm) during 24 h. Temperature was 29°C and pH adjusted to 6. Desorption study was achieved using 2g of Amberlite in 50rnJ of chloride sodium concentrations ranging from lOto 120 g NaCI Il and at different pH. Conditions were similar to those of the adsorption studies. Results are reponed as : . Uads is defined as UO-Ures, where UO was !he initial and Ures the residual ac- 78 tivity of the extract after the adsorption experimenl. · Uexp which is measured and represents the activity shown by the Amberlite after the adsorption experimenl. · Udes tot : 100 x (UdeslUads) where Udes represents the desorbed lipase in the medium and is assayed as described previously. Il is expressed as percentage of total adsorbed lipase. · Udes rel: 100 x (UdeslUexp). It is expressed as percentage of total ex-pressed lipase. Results and Discussion Evaluation ofthe R. delemar strains Lipolytic activity of the 2 strains was evaluated according to Corzo (1993) by growing the mould in Petri dishes on PDA added with emulsified tributyrin (1 %). The diameter of the halo around the colony, corresponding to the hydrolysis of the substrate was measured after 3 days. The CDBB H313 strain gave an average diameter. calculated from one hundred colonies, of 2.75 mm against 2.28 mm for the NRRL 1472 strain. The former was then used in ail further experiments. Growth and lipase productÜJn kinetics in Le LC was used to produce the enzymatic extract needed to the adsorption/desorption experiments. Results are plotted in figure 1. It can be seen that maximum activity (14.07 U/ml) corresponds to maximum growth (12.3 mg/ml). These values were reached within 2 days and correspond to those obtained by Martinez Cruz el al. (1993). 11 was shown that a centrifugation al 5000 rpm during 5 llU1 was not recommended because a loss of more than 50% in 'lipo]ytic activity was observed, probably due to celJ bound protein. The important decrease in lipolytic activity observed after 2 days may be due to proteolytic activity and/or denaturation of the protein. This was not observed when Amberlite (2g/50ml) was present in the medium (Angeles, 1995). ln figure 2, it can be observed thatthe extract was more stable at pH 5 while the optimum activity was obtained at 6.5. Growth and lipase production in SSF Glucose is known to be a repressor of lipase production in LC Haas and Bailey (1993). One of the particular goal of the experiments in SSF was to see ifthis catabolic repression could be partially or totally overcome. Three carbon sources (20 gIl) were used in the SSF experiments: glucose, maltose and dextrin. Results are presented in figures 3 and 4. The growth, followed by CDPR. did not display significative differences between the 3 substrates. Maxima of this parameter were between 15 and 20 hours - a very short time in SSF - and reached values of 3.5 mllh.g !DM .. Maximum lipase production was found after 24 hours, just after the maximum of CDPR, corresponding to the lower pH in the medium. Best production was found for dextrin (95.6 U/g !DM) against68.2 U/g!DM for maltose and 57.7 U/g !DM for glucose (See Figure 9). The equivalence in U/ml reactor is given in table 1 and 79 it is shown that SSF gave a higher productivity with the same substrate (dextrin) than Le. This activity decrease after this maxima for the 3 cases. SSF Fermentation LC Maltose Glucose Dextrin Carbon source 18 18 48 Time max. production (h) 2.8 3.5 CDPR max (ml/h.g !DM) 1-1.3 1-1.3 R.Q. (Range) 4.9 5.7 pH max. production Aw 0.998 0.999 57.7 Lipolytic Act. (U/g 68.2 IDM) Lipolvtic Act. (Ulml) 14.08 10.21 11.33 Table 1. Comparative data of LC and SSF lipase production. Dextrin 15 3.48 1-1.4 5.6 0.994 95.6 15.66 In table 1 are summarised the most impOt1ani data about LC and SSf for lipase production by R. delemar. lt can be seen that the enzyme is produced faster and with a better productivity in SSF. Moreover, Amberlite is an adequate support for this purpose: it provided a good stability for pH, moisture content (which remained stable) and Aw, all three being key parameters in SSf. The best carbon source was dextrin as already observed in LC (Marti nez Cruz el al., 1993), but in SSf. the catabolic repression due to glucose was not as important as expected (onl)' a decrease of 40% against dextrin). R.Q. observed are typical of the oxidative use of the carbon sources. R. delemar lipase adsorptionldesorption study . Adsorption study Results for lipase adsorption on entire and ground Amberlite are represented in figures 5 and 6. It can be seen on figure 3 that the arnount of lipase adsorbed per g of Amberlite decreased for higher amounts of resin, while the contrary trend is observed for the residual activity. Only 3% of the adsorbed enzyme was active when 0.5 g of resin where used, but this increased to 26% for 6 g. The important losses observed in expressed activity may be due to partial denaturation of the protein. inactivation of the active sites due to the anionic properties of the support or partial diffusion of the protein inside the resin. ln the case of ground Amberlite (Cf figure 6), as low as 0.5 g were sufficient to adsorb ail the lipase present in the reaction medium. In the sarne way than for entire Amberlite, the relation between expressed and adsorbed lipase increased from 12.4% ta 34% with the increase of amount of resin in the medium. Nevertheless, the values for adsorbed and expressed activities are higher than for entire Amberlite, probably due to the increase in the contact area in that case. It may also be explained by a decrease in the limitation of diffusion. 80 t Figure 1 ; R. delemar IIplse production klnetlc ln Ellect of cenlrUugatlon on Ilpolytlc activlly 6 - -6- - Not centrifugated exlract ------ Biomass - -G- Centrifugaled extracl Le J" A _". Ë -12 :; 12 CD 0- . 3 on on u ,.. o .... .~ ... " -:.0 '" (h) lime Figure 2 : •• 12 4 • 2 4 Extract 3 - --0 stabllity at diU.rent pH 20- Ë :; .. ,, ,.. -; u ~ . --0-30 min - -e- 24 hour ,or • u ,.. -0 Q. 3 0 3 , 0 1 1 1 pH Figure 3 Respirometric actlvity 01 R, delemar grown in SSF wlth 3 different carbon sources i' e -. ------b- 3 Oe)(ltin -Maltose --0 -Glucose '" ~ l a: Q. 0 U 1 0 20 30 4 0 lime (h) 81 , 0 .0 10 100- Figure 4 : Llpolytlc Ictlvlly tor R. delemlr grown ln SSF wlth 3 dltterent clrbon source. i' ~ ~'" ~ .. 0· Glucose - • . Maltose • 0 • 6 0- ~ u œ u ., .0" t. t. •0 -'--"'E) .. : . , .... 0 ,, ;;, "0 a. ::; 2 0 0 10 0 2 0 Time 35 • 60 7 0 (hl R. delemar Iipaae adsorption on Amberlile Figure f or .0 • 0 J 0 Dextrin -0-- e ;::I '~2J :~ . -e· Uads 1 50 -e-Uexp ... -e_ ..... u ;;, "0 1 00 a. ::; -·e • 0 13 0 J 0 , Amberlite Figure 6 (9) : R delemar Iipaae ad sor pilon on ground i:::! ~ - -e-· Uads -e-Usxp - , 20 ~ .~ 90j ~ 60 ): ~ o a. :J ! JO e. -'-. -e· .- '-" e OO!-----;:--=~==~J~=~::=~.==~-----'. Amberlile 82 (9) Arnberllte Figure 7 : R. delemar lipase adsorption kinetlc on ground and enlire Amberlite 100-. ~/~-.--.--.-- ai ,.. . ~ u .-------- ... ------- .... ----. 80 ··.··ground .0 --e-entire .!! >- 4 0 1 Ci C. ::; 20~ : o ~----L8---,L2---,.LI.-_-;LO----'2 4 Time Figure 8 lipase 40 ,.. ~• ,.. Influence of (NeCI] on de.orp'ion (pH,6). - . Udes 101 , 5 ~ (h) --e-- Udes rel '0 2 5 20 ! , 5 :JCi ' : . • __ -. o -' o , , , 4 9 NeClig IDM Figure 9 Influence of pH on lipase desorption «(NaClj,2glg Amberli'e). '5 ~ '0 ~ 25 - -e-· Udes 101 -e-Udes rel .. i ~ 20 ,.. 15 Ci ,.' :3" ' 0 o r----.,---~----'---~-----' pH 83 ln figure 7, it can be seen that all the lipase present in the medium is adsorbed on ground Amberlite within about 2 hours while, entire Amberlite was sarurated after 8 hours, with only a Iittle more than 20% of the lipase present in the reactive medium. These experiments showed that ail the lipase was adsorbed on Amberlite in maximum 8 hours. The amount of adsorbed lipase and the adsorption dynamics de pend strongly on the size of the resin. This confirmed the hypothesis presented above. Moreover, the adsorption on Amberlite displayed a thermo protective effect since no loss in activity was observed after that a sample of adsorbed lipase stayed 24 hours at 80°C (Angeles, 1995). The adsorption of the produced enzyme on Amberlite during growth on SSF may serve as a method to concentrate it simultaneously . . Desorption study To study the recovery of the adsorbed R. delemar lipase on Amber!ite, the entire particle was used. The influence ofNaCI concentration and pH were explored (See figures 8 and 9). These experiments were realised at 29°C; agitation, 150 rpm during 24 hours. Addition of NaCl, previously used by Corzo (1993), for lipase desorption allowed a 38 % of desorption at 100 g NaCl!\ at an optimum pH of 5. Conclusions ln this study: · R delemar CDBB H313 strain was selected for its better lipase production. · In LC, the negative effect of centrifugation on lipase recovery. was demonstrated. 1t was established that the enzyme was more time stable at pH 5, while its optimum activity was at pH 6. · In SSF, the mould showed a good capacity to grow on Amberlite with various carbon sources (dextrin, maltose and glucose). Bestlipase production was found with dextrin (as in LC) white lower glucose repression was observed than in LC. · In sorption/desorption experiences, it was evidenced that entire Amberlite was saturated with 24 % of the lipase while ground Amberlite was able to adsorb ail the lipase present in the medium (about 700 U). There is an important difference between the "adsorbed" Amberlite (defined as initial rested from residual activity) and acrual active lipase on Amberlite (only 26% and 34% for entire and ground support). The desorption experiments showed thatthe recovery of the adsorbed enzyme was uneasy (only 38% with 2 gNacl/g !DM and pH 5). lt will be preferable to use the enzyme adsorbed on Amberlite than to try to desorb il. Furthermore, such lipase displayed a good thermostability. The use of Amberlite as a support opens interesting possibilities to study simultaneous enzyme production and separation in SSF. 84 References Angeles, N. 1995. Producci6n de lipasa por Rhizopus delemar sobre una resina sintética (Amberlita IRA 900). Licence thesis, UNAM, México. In press. Auria, R., Hemandez, S., Raimbault, M., Revah, S. 1990. Ion exchange resin: a model support for solid state growth fermentation of Aspergillus niger. Biolechnol. Techniques, 4(6): 391-396. Christen, P., Auria, R., Vega, c., Villegas, E., Revah, S. 1993. Growth of Candida Ulilis in solid state fermentation. Biolech Adv. 11 : 549-557. Christen, P., Auria, R., Marcos, R., Villegas, E., Revah, S. 1994. Growth of Candida ulilis on Amberlite with glucose and ethanol as sole carbon sources. Ad\'. Bioprocess Eng., E. Galindo and Q.T. Rarnirez (Eds), KJuwer Acad. Pub. : 87-93. Christen, P., Lopez Munguia, A. 1994. Enzymes and food flavor - A review. Food Biolechnol., 8(2&3): 167-190. Corzo. G. 1993. Estudio comparativo de la producci6n de lipasas por Yarrowia lipoIylica en sistemas liquido y s6lido. Master thesis, UNAM, México, 94 Pp. Haas, M.J., Bailey, D.G. 1993. Glycerol as carbon source for lipase production by the fungus Rhizopus delemar. Food BiOiechnol., 7( J) : 49-73. Lonsane, B.K., Ghildyal, N.P. 1992. Exoenzymes. ln: Solid substrate cultivation, H.W. Doelle, DA Mitchell, C.E. Rolz (Eds), 191-209, Elsevier Appl. Sci., London. Martinez Cruz, P., Christen, P., faITes, A. 1993. Medium optimization by a fractional factorial design for lipase production by Rhizopus delemar. J. Ferment Bioeng. 76(2) : 94-97. Nahas, E. 1988. Control of lipase production by Rhizopus oligosporus under various growth conditions. J. Gen Microbiol. 134: 227-233. Revah, S., Lebeau!t. J.M. !989. Accelerated production of blue cheese flavors by fermentation on granular curds with lipase addition. Lail, 69: 281-289. Rivera-Muftoz, G., Tinoco- Valencia, J.R., Sanchez, S., farres. A. 1991. Production of microbiallipases in a sol id state fermentation system. Biolechnol. Leif., 13(4) : 277280. Seitz, E. W. 1973. Industrial applications of microbial lipases: A review. J. Am. Qil Chem. Soc., 51: 12-16. Yamada. K. 1977. Recent advances in industrial fermentation in Japan. Biolechnol. Bioeng., 19: 1563-1621. 85 RELATION BIOMASS/RESPIRATION: THEORETICAL AND PRACTICAL ASPECTS M. Raimbault\ c.R. Soccol 2 , S.c. Stertz2 , L. Porto de Souza 2 iLaboratoire de Biotechnologie MicrobielUle Tropicale, Centre üRSTüM-LBMT 911 av. Agropoli.s - B.P.:5045 - 34032 Montpellier (France) 2Laborat6rio de Processos Biotecno16gicos; UFPR / Centro Politécnico / Jardim das Américas, Caixa Postal 190 Il - 081531-970 - Curitiba - PR, Brasil Summary We present here sorne theoretical consideration on the correlation model belWeen Biomass and respiration. The case of me growth of Aspergillus niger on starchy substrate is discussed as an example of the application of the calculation of fermentation parameters . lt is specially possible to calculate from the respiratory metabolism the specifie growth rate, the maintenance, yields in biomass, metabolic products, and also heat evolved during the fermentation on the basis 0 f Oxygen Uptake Rate, C02 evolved and the consumed substrate. ln a second part we detail the equipment and methodology of the lab standardised method we developed for ail physiological studies and optimisation of the culture conditions for SSF of fungi. Finally we illustrate the technique by the case of A. niger and Rhizopus oryzae cultivated on starchy SSF for showing the software developed for automatic calculation from the on line data obtained byepG. I. Theoretical Aspects 1. General aspect of tbe fungal growtb !Oneties: Exponential Model: Various models were proposed to fit up with the kinetics growth of micro-organisms. First of them were proposed by Monod for the unicellular grow1.h of bacteria and can be written in the exponential equation: dX/dt = Il ,x where X is Biomass; t is Time and Il the specific groWlh rate Il represents the biomass produce per hoUT and by g of biomass: it means that the groWlh rate is proportional to the actuaJ biomass. In the integrated form: X = ~,e~.t and the logarithmic form: ln X = )l.t + ln ~ Then you can calculate the specific groWlh rate (Il = In2/td) plotting the biomass on a log scaJe versus time. This type of exponential model is generally applied for lU]icellular bacteria or yeast when the nurnber of cells groWlh exponentially . Vegelalive growlh of mycelium: ln the case of mycelium, the groWlh is of different mode without cellular division. The exponential form could be the result of a linear groWlh at the apex of the hyphae combined 10 the frequency of the branching point which increase the 87 number of hyphaJ apex. Trinci caJcuJated that for A. nidulans, the apex grown exponentially until 120~m then the growth became linear and a new branching point appeared. Considering the biomass proportional to the totallength of the mycelium, he global equations become : X=k.L dUdt = kt. n n= k2.L with where L= length of mycelium; kl and k2 specific constants and n frequency of branching , which can be written : dL/dt = kl.k2 (L) dXldt = k. dL/dt = (kl.k2). k.L = kl.k2. X dXldt = ~. X (with ~= k 1.k2) Thus, it is possible to explain the fungal growth as an exponential equation. This model fit weil onJy in the first stage of the growth because rapidly, not ail the total mycelium can grows without limitation, and after 3 to 5 generation-time, a part of the mycelial biomass can not participate to the growth rate. Growth limitation: The common cause of the growth limitation is the decrease of the substrate concentration. Monod (1942) proposed a relation between growth rate and subslIate concentration: dX = Ilmax' dt S .X (kS + S) where the Ilmax is Il in the optimal condition, kS the saturation constant of the substrate en S the substrate concentration. Cubic Model o[growth: Pirt (1966) proposed a cubic root model to explain the fungal growth in pellet form: X' 3 = k. t + X o' 3 But this model fit up onJy in LSF and when the mycelium grow in pellet form and it do not fit weil for SSF. Logistic Model: The mycelium is not homogeneous, and can depend of the distance to the apex, with vacuoles in oldest parts. The concept is based on the fact that the medium composition can produce a 88 defmed maximwn of biomass, when the biomass increase, the rate slow down in reason of appearing limiting factors. the general equation of logistic is as follows: dX = Il. X (l·--X dt Xm a x This type of model generally is weil correlated with fungal growth (Edwards & Wilke, 1968), panicularly with batch cultures. Mainlenance cancepl: \\Then the growth stops and the biomass remains constant, the biomass needs to consume energy and substrate to maintain its viability and to realise its basic metabolic activities like respiration, secondary metabolisms., turnover of proteins and active transport (Pirt, 1965). The general equation is: Q.S- ...l... 4X...- + m. X dt Ys dt 2. Stoechiometrie equations of respiration and Biomass biosynthesis General equalians: ln the following, we suppose the growth of myceliwn in the exponential form, and for constants coefficients, we used the data established for Aspergillus niger cultivated on starch substrate (Raimbault, 1981). The global equation for the biomass is the result of starch hydrolysis, respiration and biosynthesis: Hydrolysis . lin (C6HI005)n + H20 ----> C6H1206 Respiration.. (a) C6HI206 + 602 -----> 6 C02 + 6 H20 (- 673 Kcal/mole) Biomass .. (b) C6H1206 + 0.84 NH40H + 30.4 H20---> 6(CH1.6200.62NO.14; 5.6 H20) Balance .... C6H1206+ 2.1 02+ 0.54 NH40H+17.6 H20 ------> 3.9 (CH1.6200.63NO.14j 5.6 H20 ) + 2.1 89 cm In the present case of A. ruger on starch, the proportion of glucose consumed for respiration (a) is 35% and 65% for biosynthesis. The mycelium composition in CHON was determined on the basis of the compositIOn of mycelium cultivated on liquid medium on starch substrate. The coefficients a and b were calcLÙated on the basis of total glucose consumed and the oxygen uptake folJowing the equation of respiration. where S is the substrate , Ys the cellLÙar yield, and m the maintenance coefficient. Starting from the global equation, it is possible to calculate the metabolic heat production considering the exothermic reaction of respiration: !!.9- = K.~ dOt dt dt dt ~ 0t%' F V 0t mol Q.Q . 673 Kcal dt k dT dt Kinetics o[Biomass: Conside ring the direct relation between C02 and 02, it is possible to get on line the evolution of the biomass, capturing data of Oxygen Uptake Rate or C02 evolution: _1_ Y02 + . dX dt m 02 ' X dCO Z dt _1_ y C02 ~ + mC02 ' X dt From trus equation we can write considering dXJdt = Il.X: d02 = (JL + m02 dt YOl Considering that d 02 ~ 02% = ). X OUR and F 02%.F = Y02 • Air Flow X dt The logarilhmic form ofthis equalion is: Ln (02%) = Ln X + constant , or Ln (02%) = Il . t + constant Similarly we can write the same equation for the C02 evolution (C02%) Ln (C02%) = Il. t + constant From the last two equation, it is thus possible to calcuJate the specifie growth rate of the mycelium using on line data of gas composition evolving from the incubator, without 90 destroying the sample, observing the kinelic evolution of lhe same sample all the time of the fennentalion. Thal represents a greal advantage of the SSF. 3. Equipment & Methodology In order to measure kinetics evolulion of fungal biomass cultivated on SSF, we have developed a simple column incubator device which allows to control air f10w and analysis on-line of the gas composition at the exit of the reactor (Raimbault, 1980; Alazard & Raimbault. 198 J). The figure 1 shows the laboratory device designed to realise lab experimentation with 24 incubator with temperature and air f10w control (Trejo-Hemandez, 1986; Oriol, 1987 ; Dufour. 1990 : Saucedo-Castaiieda, 1991 ; Soccol, 1992). Glass column reactors (5) of 2 or 4 cm diameter and 20 cm length are filled with the inocuJated and moistened solid substrate (100-150 g of WM). Jncubators are put on a humidificator (3) and installed in controlled water bath (2 & 4). The air 1l0w, pre-saturated in water, is controlled by oùcrovalves (6). So, the air f10w can be controUed for each column. Figure 1 : Incubation device for aerobic SSF. (1): air input: (2) Thermoregulaled water bath; (3) Humidficador; (4): Control heater; (5): Column incubator; 6: Microwalves. To analyse gas composition, different techniques were developed including trapping C02 in alkaline solution. paramagnetic analysis for oxygen, Infra Red analysis for C02. We describe here (figure 2) the Gas Chromatography technique that we used with success during various years at the Jaboratory scaJe. Other alternative are also presented by other speakers during the course. 91 The equipment is composed by a gas chromatograph (CPG) equipped with thermal conductivity detector and Alltech column CTR 1 (double concentric column : extern molecular screen 5 A° and internai Porapak as stationary phase. Figure 2: OD tiDe aDalysis equipment for gas measurement using CPG. 1 Air input; 2: Thermostatic; 3: Column incubator; 4 Silicagel ; 5: Sarnpler; 6 Interface for automatic injection. The conditions of chromatograph are as follows: Detector: Thermal conductivity Detector temperature: 60°C Column temperature: 60°C Gas phase: Helium Gas phase tlow: 40 ml.min-I Catharometer current: 120 mA Helium pressure: 1 bar Loop injection Volume: 2001..t1 Gas for calibration: Air:, C02 (0.0) / 02 (21.0 ) / N (79.0 ) Mixture 1: C02 (50) / 02 (5.0) / N (90.0 ) Mixture 2: C02 (10.0) / 02 ( 15.) / N (75.0 ) 92 I. Experimental Aspects The experimental part of the course was performed at the Laboratory of Biotechnological Processes of the Chemical Technology Department of the UFPR. So, the training part concemed practice cultivation of two fil amen tous fungi (Aspergillus oryzae and Rhizopus. formosa) on Cassava Bagasse, a Brazilian by-product of the industrial extraction of Cassava Starch and flour production for human consumption. Inoculum preparation: The two strains of fungi were cultivated in erlen flask on the surface of PDA medium during a week at 35°C. Then, conidia were cropped with a platinum loop in a laminar flux cabinet, in sterile tubes containing 10 ml of water, 1% of Tween and glass balls. The suspension is homogenised 15 min and successively diluted as necessary (10- 1/10. 7) for direct microscopic count in a Neubauer cell. The number of conidia in the cell, allows calculate the nurnber of conidia in the original suspension, using the formula: N (conidia/ml) = D (Dumber of cODidia iD couDtiDg cell ). (lfDilutioD factor). (25. 10 4 ) The adequate dilution is then prepared in order to get a good concentration of conidia a110wing final inoculation of Ixl0 7 conodialg of DM substrate. This suspension is kept al 4°C under constant agitation until utilisation (Soccol, 1991). Subslra/e prepara/ion: ln this experience, we used Cassava Bagasse, an indus trial by-product obtained from the Lorenz Company (Quatro Pontes, SC, Brazil). The raw materiai was grounded in order to get a granulometry of 0,8-2,0 mm diameter particles. The material was dried at 55-60 oC in oyen with circulating air during 12 hours. This materiai was analysed in agreement to the recommended methods described in Analytical Normas of the Adolfo Lutz Institute (Sao Paulo, 1985). The starch was determined by the NS-00396/85 method (National Starch Chemical Corporation, 1985). using the Thermamyl commercial a-amylase (Thermamyl). Prolein content was determined by the Stutzer method (Vervack, 1973). The following table shows the composition of the byproduct: 93 Table 1. Pbysico-cbemical composition of the Cassava Bagasse (Stertz, 1997) Compound Cassava Bagasse composition (gll OOg DM) Moisture Content Proteins Carbohydrates Lipids Fibres Ashes 10.70 1,60 63.40 0,53 22.20 1.50 Preparation ofthe mediumfor Solid Substrate Fermentation: The saline solution used to humidify flour contained: - (NH4 ) 2 504 -KH 2P0 4 - Urea - Water 4,34 g 1,7 g 0, 83 g 233 ml The pH of the solution was adjusted to 5,8 with Na2C03 (3N) The volume of saline solution necessary to moisten the flour of cassava bagasse is calculated by the formula: M% x Mass of substrate (g) Mass of Water (g) = 100 - M% ) For example, for 100 g of Cassava Bagasse substrate, and for a Moisture content of 65%, it would be used 185 ml of saline solution. AnalYlical procedure: In order to characterise kinetics biotransformation of the material by the fungaJ strains. sampies are picked up at regular duration of the fermentation and physical-chemical determinations are performed. For that purpose, samples are treated following the flow sheet showed on the figure 3. Ail analysis (pH, Moisture content, Ashes, Lipid, Fibres, Total acidity and sugars) were performed as recommended by ormas Analiticas do Intituto of Adolfo Lutz (Sao Paulo, 1985). 94 pH: pH was determined under agitation eleclronically with pH meter, after homogenisation of the suspension of 1g in 10 ml of distillated water. Moislure conlen/: Moisture content was determined from 5 gm of moistened sample by drying in a controlled oven during 24 h at 105°C. The Moisture Content is calculated by the formula: M% = (P2-Pl). 100 P2. with: M% = moisture content P2 Weight of the pre-treated sample (5 g) ; PI Weight after desiccation of the sample. Carbohydrales and primary melaboliles: Sugar, organic acids, ethanol, were determinated by H.P.L.e. The Slarch was determined by the NS-00396/85 method (National Slarch Chemical Corporation. 1985), using the NOVO Nordisk « Thermamyl 120L)} (liquid commerciaJ aamylase (Thermamyl). 4 grams ofsample are added in 100 ml of water: autoclaved at room pressure during 1 h , and adjusted to pH 6,0-6,5 with a solution of NaOH (1 N). \Vhen temperature is 95°C, 60 - 70 ppm of CacCI2 and 1 ml of Thermamyl Novo were added , and kept 15 min at this temperature then filtered on Whatman paper, washed and centrifuged ; finally the residuaJ materia! is dried at 105-110 oC during 1 h 30 and residual weight is determined. Starch % of residual dry matter was calculated by the fonnula: Starch % were PT = = (PT - PR ) . 100 PT Total mass and PR = Residual mass For true Protein content the Stutzer method was used (Vervack, J 973). Solid Slale jermen/alion cu!livalion in column. The device showed in figures 1 and 2 was used for incubation and respirometric analysis. under following conditions: Temperature of 35 oC Flow rate of air Oux : 100 ml 1 min The composition of air can be observed directly on the computer screen . After 24 hours and 48 hours samples columns are pick up for analysis. 95 Treatment of Samples in SSF ANALYSIS J Solid State Fennentation 1 1 C02 and 02 On ]ine CPG 1 1 ~ 1 Samples from Column & Graphies: QR 1 moisture Content 1 1 l Loss of Dry Matter 1 Dilution 1 1 1 1 Fungal Microflora & Contaminant Control 1 Homogeneisation Ultra Turrax .1 1 pH 1 : microscopie Observation 1 Centrifugation 30 min at 5 000 g -> Liquid Fraction 1 Biochemica! Analysis (proteins. sugars, enzymes) 1 1 1 Filtration 0.45 m Figure 3. HPLC Analysis (sugars, organic acids. alcohol) Flow sheet of samples treatment s for analysis in SSF 96 1 Typically Ù1e Table 1 represents resuJts obtained from SSF cultivation of Rhizopus ory=ae on Cassava. Figure 3 represent graphs obtained from on fine data captured by the computer. It can be calculate directly the oxygen consumption and C02 evolution, and also the specifie growth rate. In addition, biochemical analysis allowed to correlate aIl fennentation parameters. and caIcuIate ail Ù1e balance of Ù1e biotransfonnation process. AlI that infonnation is of importance for pilot and scale up further applications. Table 1.- Data of Solid Substrate Fermentation of Rhizopus oryrae MUCL 28168, cultivated on Cassava flour substrate. (Raimbault et al, 1995). INITIAL 142,20 60,4 5 85.00 Total MassofMateriai Dry Matter Content Air Flux ofaeration Total Dry Matter Duration of the gerrninating Timefor Maximum Rate ( Maximum 02 Uptake Rate(ml/H/g VMaximum C02 Evolved Rat e (ml/Hlg Mean 0 f Resoiratorv Total02 uotake (g / g Total C02 evolved (g / g Duration oft he exponential Soec ific Growth Ra te ( u) 85,96 7h 16 - 30 4.65 4.91 1 05 0.19 0.28 9h 0.263 Protein (%DM Total S1.JQar (% DM Loss in Dry Matter Yield Protein / &1gar (Y Yield 9.96 46.54 31,51 0.105 077 97 FINAL 128,40 45.85 <-- ---- --- --- ---- --- 58.87 <-- --- ---- --- --- --<-- ---- --- --- -----<-- --- ---- --- --- --<-- ---- --- --- ---- --<----- ---- --- --- --<-- --- --- ---- ----<-- --- --- ---- ----<-- --- --- ---- ----<-- --- --- ---- ----- 20.08 15.05 <-- --- --- ---- ----<-- --- --- ---- ----<-- --- --- ---- ----- -0- d02 1-<>- dC02 ~ 5 Cl :\~ 00 S4 : ~!JJ1Il DcD [c D cD N a 1 c QR 3 ('lr<1-=~~dC~ 0f~Q/tJ>~ c ~ D D~ ès> ~trM \,aPf ~ajl~ ! o~~~rsP' lf "0 c " N 82 1.5 <U\:o '" Ô c 9Y ë ;;" ~ 0 0 .~~L 10 15 0.5 0 20 25 30 40 35 45 50 Time (h) 1-0- JC02 -0- J02 c LN COli 250 ce ~c!tl1llCa 1lII11ll:f1dIltJ!Dlbltac 1.5 200 [~r. I5frr c c lins ::;: c c c c c c c c c !!! ISO E N 0 "0 § 100 N c c c c c 50 0 0 10 ~~ /~'" ":J Z -0.5 lll~~!Zl lt tdo 15 U .J ffé-rf C C 0 U N 0 0.5 -1 20 25 JO 35 40 45 50 Time (hl Figure 3. Kinetic of respiration characterislic duriog growlh of Rhizopus oryzae on crude Cassava flour. 98 References : - Alazard, D. and Raimbault, M. 198). Comparative study of amylolytic enzymes production by Aspergillus niger in 1iquid and solid sUlle cultivation. Eur. 1. App/. Microbio/. Biotechnol. 12: 113-117. - Auria, R., Hemandez, S., Raimbault, M. and Revah, S. 1990. Ion exchange resin: a model support for solid state growth fermentation of Aspergillus niger. Biotechnol. Techniques. 4: 39l-396. - Deschamps, F., Raimbault, M. and Senez, 1.c. 1982. Solid state fermentation in the deveJopment of agro-food by-products. lndusry & Environ. 5 (2): 27-30. - Doelle H.W., Mitchell D.A. & Rolz C.E. (1992). Solid Substrate Cultivation. Eisiever Sci. Publ. Itd;London & New York; 466 p. - Moo-Young M., Moriera A.R. & Tengerdy R.P. 1983. Principles of solid state fermentation. In The fiJamentous fungi, Vol. 4, Fungal Biotechnology. Smith 1.E, Berry d.R & Kristiansen B. Eds., Edward Arnold Publishers, London, pp. 117-144. - Lonsane, B.K., Saucedo-Castaii.eda, G., Raimbault, M., Roussos, S., Viniegra-Gon.zalez. G., Gildyal, N.P., Rarnakrishna, M. and Krishnaiah, M.M. 1992. Scale-up strategies for solid state fermentation systems. Process Biochem. 27: 259-273. - Oriol, E., Schenino, B., Viniegra-Gon.zalez, G. and Raimbaull. M. 1988a. Sol id- state culture of Aspergillus niger on support. 1. Ferment. Techno/. 66: 57-62. - Prebois, 1.P., Raimbault, M. and Roussos, S. 1985. Biofermenteur statique pour la culture de champignons filamenteux en milieu solide. Brevet Français N° 85.17.934. - Raimbault M. - (1981). "Fermentation en milieu solide: croissance de champignons filamenteux sur substrats amylacés". Edited by: ORS TOM-Paris; Série Travaux et Documents n° 127; 291 p. - Raimbault, M. and Alazard, D. 1980. Culture method to study fungal groWlh fermentation. Eur. 1. App/. Microbio/. Biotechnol. 9: 199-209. III solid - Raimbault, M, ; Ramirez Toro, C, : Giraud, E. : Soccol. C.R. ; Saucedo, G. Fermentation in cassava bioconversion. ln ; Dufour, D. ; O'brien, G.M. ; Best, R. (Eds) Cassava Flour and Starch ; Progress in Research and Devellopment - Session 4 - Bioconversion and Byoproduct us. ClAT Pub. N. 271,1996 p187-196. - Raimbault. M., Revah, S.. Pina. F. and Villalobos P. 1985. Protein enrichment of cassa va by solid state fermentation using molds isolated from traditional foods. 1. Ferment. Techno/. 63: 395-399. 99 - Raimbault, M., Roussos, S., Oriol, E., Viniegra, G., Gutierrez, M., Barrios-Gonzalez, J. 1989. Procédé de culture de microorganismes sur milieu solide constitué d'un support solide, absorbant, compressible et non fermentable. Brevet Français N° 89. 06558. - Raimbault, M. and Viniegra-Gonzalez, G. 1991. Modem and traditional aspects of solid state fermentation ln. Chahal, O.S. (Ed.), Food, feed and .fuel from biomass. Oxford & IBH Publis. Co. Pvn. Lld. New Delhi, p. 153-163. - Roussos, S., Oimos, A., Raimbault, M., Saucedo-Castaneda, G. and Lonsane. B.K. 1991. Strategies for large scale inOClÙum development for solid state fermentation system Conidiospores of Trichoderma harzianum.. Biotechnol Tech. 5: 415-420 - Rodriguez Leon, lA., Sastre, L., Echevarria, l, Delgado, G. & Bechstedt, W. (1988). A mathematical approach for the estimation of biomass production rate in solid state fermentation. Acta Biotechnol. 8, 307-310. - Sào Paulo (1985) Instituto Adolfo Lutz. Normas Analiticas do Instituto Adolfo Lutz, Sào Paulo, Brazil, 523p - Sato, K., Nagatani, M., Nakamuri, K. 1. & Sato, S. (1983). GroWlh estimation of Candida lipolyrica from oxygen uptake rate in a solid state culture with forced aeration. J. Ferment. Technol. 61, 623-629 - Saucedo-Castaneda, G., Gutierrez-Rojas, M., Bacquet. G.. Raimbault. M. and ViniegraGonzalez, G. 1990. Heat transfert simulation in solid substrate fermentation. Biotechnol Bioeng. 35: 802-808. - Saucedo-Castaneda, G., Gutierrez-Rojas, M., Bacquet, G., Raimbault, M. and ViniegraGonzalez, G. 1990. Heat transfert simulation in solid substrate fermentation. Biotechnol Bioeng. 35: 802-808. - Saucedo-Castafieda, G., Lonsane, B.K., Navarro, lM., Roussos, S. and Raimbault, M. 1992. Control of carbon dioxide in exhaust air as a method for equal biomass yields at different bed heights in colunm fermentor. ApplMicrobiolBiotechnol. 37: 580-582. - Saucedo-Castafieda, G., Trejo-Hemandez. M.R .. Lonsane, B.K., Navarro, J.M., Roussos, S.. Dufour, D. and Raimbault, M. 1993. On-line monitoring and control system for CO 2 and O 2 concentrations in aerobic and anaerobic solid state fermentations. Process Biochem. 29: 13-24. - Soccol, C.R. 1992. Physiologie et métabolisme de Rhizopus en culture solide et submergée en relation avec la dégradation d'amidon cru et la production d'acide L(+) lactique. Thèse de Doctorat, Université de Technologie de Compiègne, France, 218 p. - Socco1, C. R. Biotechnology products from cassava root by solid state fermentation. Journal 0 fScientific and Industrial Research, Vol. 55, may-june, 1996, pp 358-364. 100 - Soccol, C.R., Rodriguez-Leon, 1., Marin, B., Roussos, S., Raimbault, M. 1993. Growth kinetics of Rhizopus arrhizus in solid state fermentation of treated cassa va Biolechnol. Techniques, 7: 563-568. - Soccol, c., Marin, B., Raimbault, M. and Lebeault, 1.M. 1994. Breeding and growth of Rhizopus in raw cassa va by solid state fermentation Appl. Mierobiol. Bioleehnol. 41: 330-336. - Soccol, c., Marin, B., Raimbault, M. and Lebeault, J.M. 1994. Potential of solid state fermentation for production of L(+) lactic acid by Rhizopus oryzae. Appl. Microbiol. Bioleehnol. 41: 286-290. - Soccol, C.R., I1oki, 1., Marin, B., Roussos, S. and Raimbault, M. 1994. Comparative production of amylases and protein enrichement of raw and cooked cassava by Rhi;:opus strains in submerged and sol id state fermentation J. Food. Sei. Technol. 31: 320-323. - Soccol, C. R.; Raimbault. M.; Pinheiro, L. 1. Effect of C02 concentration on the micelium growth of Rhizopus species. Arq. Biol. Tecnol., 37( 1) : 203-210. 1994. - Soccol, C. R.: Stertz, S. C.; Raimbault, M.; Pinheiro, L. 1. Biotransformation of solid waste from cassava starch production by Rhizopus in solid state fermentation. Part 1 - Screening ofstrains. Arq. Biol. Tecnol., 38(4): 1303-1310, 1995. - Soccol, C. R.: Stertz, S. c.; Raimbault. M.; Pinheiro, L. 1. Biotransformation of solid waste From cassava starch production by Rhizopus in solid state fermentation. Part Il Optimization of the culture condition s and growth kinetics. Arq. Biol. Tecnol., 38(4) : 1311-1318,1995. - Soccol, C. R.: Stertz, S. c.; Raimbalt, M.; Pinheiro, L. 1. Biotransformation of solid waste from cassava starch production by Rhizopus in solid state fermentation. Part III - Scale-up sludies in different bioreactors. Arq. Biol. Tecnol., 38(4) : 1319-1326. 1995. - Soccol, C. R.; Stonoga, V.!.; Raimbault, M. Production of L(+) latic acid by Rhizopus species. World J. Microbiol. Biolechnol., 10(4) : 433-435, 1994. - SoccoI. C. R.; lloki. 1.; Marin. B.; Roussos, S.; Raimbault, M. Comparative production of amylase and protein enrichement of raw and cooked cassava by Rhizoplls strains in submerged and solid state fermentation. J. Food Sei. Teehnol.. 3: 320-323, 1994. - Soccol, C. R.; Marin, B.; Raimbault, M.; Lebeault, 1. M. fermentation for production of L( +) lactic acid by Rhizopus Bioleehnol. 41: 286-290. 1994. Potential of solid state oryzae. App. Mierobiol. - Soccol, C. R.: Leon. J. R.: Marin, B.: Roussos, S.: Raimbault, M. Growth kinelics of Rhizopus ln solid stale fennentation of lreated cassava. Sei. Teehnol. Lellers, 7(8): 563-568, 1993. - Vervack W (1973) Analysis des aliments, méthodes courantes d'analyses. Laboratoire de Biochimie de la Nutrition, UCL. Louvain-la-Neuve lOI KINETICS OF THE SOLID STATE FERMENTATION OF RAW CASSAVA FLOUR BY Rh izopus formosa 28422. J.A. Rodriguez-Leon l , S.c. Stertz 2, c.R. Soccol 2 and M. Raimbaulf IICIOCA. (Instituto Cubano de Investigaci6n de los Derivados de la Cana de Azucar) P.O. Box 4026, La Habana, Cuba. 2Laborat6rio de Processos Biotecnol6gicos; UFPR 1 Centro Politécnico 1 Jardim das Américas , Caixa Postal 190 Il - 081531-970 - Curitiba - PR, Brasil JORSTO M , Centre Montpellier, 911 Av. Agropolis, BP 5045,34032 - Montpellier - France The strain Rhizopus formosa 28422 was selected from a stock of ten stains from genera Rhizopus. for their capacity to anack raw cassava starch by solid substrate fermentation and showed the highest growth in this substrate. The optimal substrate composition, estimated by surface response design experiments, was 10 % cassava bagasse, 10 % soybean flour and 80 % cassava flour. Optimal fermentation conditions were temperature, 32 oC, moisture, 64 %, initial pH, 6.5 and inoculum rate, 10 6 spores/g DM. These conditions were employed for studying the kinetics of the biotransformation of cassava flour considering the Oxygen Uptake Rate (OUR) and the CO 2 evolved during the process. The respiratory quotient was nearly l, corresponding to an aerobic process in the first 24 h. Sporulation appeared after 26 hours of fermentation in which the respiralory quotient showed a trend to increase up to nearly 6. Biomass was estimated solving the OUR balance for a yield based on oxygen consumption (Y x/o) of 0.531 g biomass Ig consumed O 2 and a value for the maintenance coefficient (m) of the order of 0.068 g consumed 0ig biomass· 1 h· l • The corresponding value for the growth specific velocity at the exponential phase (~) was 0.16 h· l . Keywords : Solid Substrate Fermentation, Cassava, Rhizopus, Protein Enrichmenl, Kinetics Introduction Cassa va flour is a basic ingredient of Brazilian people diet. However, its protein content is low (2.1 % w/w, on a dry maner basis) and of poor nutritional quality (EI-Dash. 1994). A biotechnologicaJ alternative for improving content and quality of cassava flour proteins is the fermentation with filamentous fungi (Vanneste, 1982). Solid State Fermentation (SSF) of cassava is a relative simple procedure, which increases by four to five folds its protein content. On anoÙ1er hand, the protein quality of the final product is very acceptable when compared with FAO standards (Varmesle, 1982). Rhizopus are edible filamentous fungi. employed since thousand of years, mainly in Oriental Countries like Chine, Korea, lapan, Indonesia. Malaysian and others, for preparing fermented foods (Hesseltine, 1965; Raimbault & Alazard, ) 98). Rhizopus fermentation Jeads 10 protein enrichment and digeslibility improvement of foods (Soccol et al 1992, 1993, 1994). Aiso Rhizopus fermentation can restrain toxic products formation. like aflatoxins (Ko, 1974; Zhu el al.. 1989), produce active biocides against bacteria (Wang el al., 1969) and detoxify cyanogenic glycosides of cassava (Iinamarin) (Padmaja & Balagopal, 1985). As cassava has a naturally high slarch content, it would be inleresling 10 identify mould strains able 10 utilise this carbon source in ilS native forms, i. e., without lhe energy consuming for 103 gelatinization step. Thus, this research aims to develop a biotransfonned flour by solid state fennentation, in order to obtain a proper protein content, employing various strains of Rhizopus. able to altack native cassava starch and estimate the kinetics that describe such process. Substrate preparation: Cassava flour was prepared in the laboratory from fresh commercial roots. Roots were cleaned and handy ground. The fraction passed through 2.0 mm sieve was dried at 55-60 oC and the new fraction retained in 0.8 mm sieve was employed for fennentation studies. Cassa va bagasse was purchased from Yamakawa Industries (Paranavai. PR - Brazil). ft was further ground in a disc miU (Alpha) and the fraction 0.8 - 2.0 mm was retained for assays. Soybean flour was also prepared in the laboratory, from fresh commercial beans. Seeds were toasted (10 min/250 oC), dehulled and ground. The fraction retained in 0.8 - 2.0 nun sieves was selected and employed for fennentation studies. The solid substrate was initially a mixture of 80 % cassava flour, 10 % cassa va bagasse and 10% of soybean flour. Strain: The Rhizopusjormosa MUCl 28422 was employed due to their ability to grovith in raw cassa va. ft was replicated in potato-dextrose agar medium, incubated during 10 days al 28-32 oC and then kept at 4 oC during six months maximum. Inoculum preparation : Spores were first inoculated in Petri dishes containing cassava-agar medium and incubated at 28-32 oC for 10 days. Therefore, spores were collected with a platinum Joop under laminar flow and diluted in test tubes with 10 mL of 1 % (v/v) Tween 80 in distillated water, previously sterilised. Spore suspension was diluted in distillated water and spores counted in a Malassez cell counter, before keeping at 4a C. Cassava-agar medium preparation: 30 g of cassava Oour were diluled in 1 L dislillated waler and cooked during 1 hour in autoclave. Resulting solution was then filtered and mixed with 2.93 g (NH4hS04, 1.5 g KH 2P0 4, 0.72 g urea and 15 g agar. After dissolution by heating, pH was adjusted to 5.5 with Na2C03 (3N) and the final solution sterilised at 121°C during 20 minutes. Solid substrate fermentation conditions: The initial inoculum rate was 106 with an initial pH of 6.5 and an exit flow rate of 0.13 1/ h g dried matter. The inlet air was saturated. The running fennentation time was 36 h at a temperature of 32 oc. The reactor was a column type with 3.5 inner diameter submerged in a water bath. Analytical metbods : Protein content was detennined by the Stutzer method (Vervack 1973). Residual starch was measured by the NS-00396/85 method, employing commercial a-amylase (Thennarnyl). Other pararneters, like pH and moisture, ash. lipid, protein, fibber and carbohydrate contents and were detennined by the Institute Adolfo Lutz Reconunended Analytical Procedures (Sào Paulo 1985). 104 Results The kinetics of the solid fermentation was determined by measuring the Oxygen Uptake Rate (OUR), the CO 2 evolved and the respiration quotient (RQ) during the process. A balance was made for the estimation of the OUR and the CO 2 evolved in terms of volumetric flow (1Jh), considering an initial weight of 27 g of dry matter, the O2 and CO] percentage composition of the exhausted air flow (FouI) which was 0.\3 II h g initial dried weight and the inlet air flow (Fin) to the fermentor. The following equations were considered: v020UI = (% 020ul Il 00) FouI V C020UI = (% C0 20u / 100) FOuI VN20UI = (100 - % 020ul - % C0 20ul )/1 00) FouI and from the balance of O2 and N 2 is obtained that: V02uplake = (20.911 00) Fin - (% 02s 1100) FouI Relating the several equations considered, the following relationship for the irùet and outlet air flow is obtained: Fm (100 -%02- %CO,)Foul = ----------79.1 For the estimation of the OUR and the Cm evolved in mass flow units (mmoleslh), it was considered that the air is an ideal gas, the respective volumetric flows (V02uplake and VC020u,) and the proper corrections for temperature conditions, considering a temperature value of 32 oc. Table and Figure 1 show these results and the pattern of OUR and CO 2 evolved during the solid fermentation. From Table 1 is observed that the process showed the characteristics of an aerobic system in the tirst 26 h with an acceptable RQ which has a mean value of 0.94 for this time interval. Aftcr 26 h this pattern holds no more and it was observed a sustained increase in the CO 2 evolved in relation to the OUR and therefor an increase in the RQ as is shown in Figure 2. As is it observed from Table 2 there are not practically signiticant growth after the tirst 24 h with a very short lag phase of the order of only 1 h. 105 Table 1. OUR, CO 2 evolved and respiration quotient (RQ) pattern during tbe solid state fermentation of raw cassava flour by tbe strain Rhizopus for mosa 28422 at 32 oc. RQ CO 2 %0 2 %C0 2 OUR Time evolved in Fout in FouI (mmoleslh (mmoleslh (h) ) ) 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 20.90 20.67 20.36 20.08 19.62 19.21 18.29 17.32 16.77 16.04 14.42 13.51 14.04 16.59 17.65 1869 19.22 19.17 18.96 0.00 0.30 0.55 1.02 1.36 1.64 1.72 2.09 4.46 8.11 6.82 4.33 359 4.43 4.71 4.53 4.89 5.01 5.04 0.000 0.453 1.150 1.641 2.694 3.644 6.088 8.502 8.650 8.561 13.673 17.542 16.527 9.154 6.129 3.417 1.780 1.847 2.398 0.000 0.642 1.177 2.182 2.910 3.509 3.680 4.472 9.543 17.353 14.593 9.265 7.681 9.479 10.078 9.693 10.463 10.720 10.784 1.4 1.0 1.3 l.l 1.0 0.6 0.5 1.1 2.0 1.1 0.5 0.5 1.0 1.6 2.8 5.9 5.8 4.5 Table 2. Substrate and biomass cbaracteristics during tbe solid state fermentation of ra\\' cassava flour by Rhizopus formosa 28422 at 32 oc. Fermentation time (h) 24 h Oh 36 h Humid substrate weight (g) 75.00 70.02 69.25 Humidity (%) 64.02 64.11 66.91 initial protein due the inoculum (%) 0.12 biomass prote in content (%) (d.b.) 50.16 substrate protein content (%) (d.b.) 10.13 5.58 10.68 Total biomass (g) 0.065 1.809 1.881 Based in these results it was decided to proceed with the estimation of the biotechnological parameters considering the balance of the OUR. From this balance the following equation is obtained (Sato et. al., 1983): 106 11 a) d O ! ) ,.n-' dÜ2 ) ( i=n-l.) / + I,(_)t=i + 12 Xo-aI,x, dt ,., dt ;., dO! dt Xn= ( YX~I\_((_~=o+(_~.n 2 ( a) 1+2" where: a = m (Y x10) ôt 16.0 . . . - - - - - - - - - - - - - - - - - - , 14.0 12.0 10.0 8.0 6.0 4.0 2.0 0.0 ~!F-+-t_1~__t_+_+_+_t_+_+_+_t_1~_+--l 12 16 h:a:J 24 28 32 )S Figure 1. Kinetic pattern of tbe OUR and COz evolved during tbe solid state fermentation of raw cassava flour by the strain Rhizopus formosa 28422. Ra 7.0.,----------------., 6,0 5,0 4.0 3.0 2,0 1,0 2. 4 6 8 10 12 14 16 18 :a:J 22 24 28 28 :ll 32 34 )S Figure 2. Respiration quotient pattern (RQ) during the solid state fermentation of raw cassava flour by the strain Rhizopusformosa 28422. The procedure to estimate the biomass content in a particular moment (X n) consisl in make a trial and error estimation, assuming values for the biomass yield based in oxygen consumption (Y x10) 107 and for the maintenance coefficient (m), (Rodriguez Leon et al., 1988), considering in our case the biomass values analytically deterrnined at 24 and 36 h until the values predicted by the equation here considered agree with those deterrnined analytically. Using the data reported at Table 1 and Table 2 the system was solved for a value of 0.531 g biomass /g consumed O 2 for the biomass 1 1 yield based in oxygen consumption (Y xie) and 0.068 g consumed O 2 / g biomass· h· for the maintenance coefficient (m). ln Table 3 are reported the biomass estimation for the different times considered, calculated via the equation for (X n) reported before. Table 3. Biomass estimated from the OUR during the solid state fermentation of ra\\' cassava f10ur by the strain RilizoDus (ormosa 28422. relative error biomass time biomass (%) measured (h) estimated 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 (g) (g) 0.065 0.067 0.083 0.115 0.164 0.237 0.350 0.520 0.712 0.891 1.125 1.462 1.813 2.027 2.089 2.07 1.996 1.906 1.830 0.065 0 1.809 0.2 1.881 2.4 Table 3 report too the comparison between the values estimates for 24 and 36 h with the values determined analytically. As it can be seen the error in the biomass estimation are lower than 2.5%. From the data of the biomass estimation it was calculated the specifie growth velocity for the log phase (llmax) by a regression of ln X n vs l. The value obtained was 0.16 h- I with a regression coefficient of 0.997 considering the values between 2 and 24 h. 108 Discussion: The behaviour of the system after 26 h could be related to a improper air flow distribution due to air flow canalisation after the mycelium biomass was fully developed. At the same time this is a point were initial sporulation start, therefor this pattern indicate that the mycelial growth lasted until 26 h. This fact is corroborated by the results reported in Table 2 where is shown the data that characterise the subsrrate and biomass synthesis during the fenmentation. The value of Yx10 considered for solving the OUR balance seems to be relatively low and at the same time the value for the maintenance coefficient (m) seems high. This could be due to the characteristics of the substrate employed, raw cassava flour, and the necessity of synthesis by the micro-orgarusm of the proper amylases that allow the flour hydrolysis since the beginning of the process, considering that there is not lag phase in this system, as is shown by the kinetic pattern. ln this sense is notable that the pattern of enzyme synthesis seems more a constitutive and not an inductive one taking into account that the lag phase is practically nul!. ln other words. if the amylases synthesis is inductive as it could be expected, the process in this case, with raw cassava flour and srrain of Rhizopusformosa 28422, is really fast, provoking, a high maintenance coefficient and therefor a Iow biomass yield based in oxygen conswnption. The results here discussed corroborate that the process in which raw cassava flour is fenmented by a solid state fenmentation process with the slrain Rhizopus formosa 28422 is quite feasible and is developed in 24 h allowing the use of raw cassava flour without the necessity of previous pretreatment as nonmally is done and attaching a level of protein of the order of 10% dried basis. Acknowledgements We thank CNPq (Conselho Nacional de Oesenvolvimento Cientifico e Tecno!ogico), CAPES (Coordenaçao de Aperfeiçoamento de Pessoal de Nivel Superior). IClOCA (lnstituto Cubano de lnvestigcion de los Oerivados de la Cana de Azûcar) and European Union (project CEE/ST03 N° TS3-CT92-0 110) for its financial support. References - EI-Oash A, Mazzari MR, Germani R (1994) Tecnologia de Farinhas Mistas - Uso de farinha mista de trigo e mandioca na produçào de pàes. vol 1 Embrapa/Spipp, Brasilia. pp 17-18 - Hesseltine C\V (1965) A millenium of fungi food and fermentation. Mycologia 57: 149-197 - Ko SO (1974) Self protection of fermented foods against aflatoxin. Proceedings of the TV International Congress of Food Science and Technology v3 pp 244-253 - National Starch Quimical Corporation. NS - 00396185. (1985). - Padmaja G; Balagopal (1985) Cyanide degradation by Rhizopus. Can J Microbiol 35(8):663-69 - Phan-Tha-Luu R, Feneuille O. Mathieu O. (1983) Methodologie de la Recherche Experimentale. 4 volumes. LPRAIlYT. Marseille, France, - Raimbault M & Alazard 0 (1980) Culture method to study fungal groWlh in solid fermentation. Critical Reviews in Biotechnology 4: 199-209 109 - Rodriguez Le6n, J. A., Sastre, L., Echevania, 1., Delgado, G. & Bechstedt, W. (1988). A mathematical approach for the estimation of biomass production rate in solid state fermentation. Acta Biotechnol. 8,307-310. - Sao Paulo (1985) Instituto Adolfo Lutz. Normas Analiticas do Instituto Adolfo Lutz, Sào Paulo, Brazil, 523p - Sato, K., Nagatani, M., Nakamuri, K. 1. & Sato, S. (1983). Growth estimation of Candida lipolytica from oxygen uptake rate in a solid state culture with forced aeration. J. Ferment. Technol. 61, 623-629. - Saccol C R (1992) Physiologie et métobolisme de Rhizopus en culture soJid et submergée en relation avec la dégradation d'amidon cru et la production d'acide L(+)lactique. PhD Thesis. Université de Technologie de Compiègne, Compiègne, France, 219 p. - Soccol CR, Leon .JR Marin B, Roussos S, Raimbault M (1993) Growth kinetics of Rhizopus in solid state fennentation of treated cassava. Sci Technol Leners 7:563-568 - Saccol CR, Marin B, Raimbault M, Lebeault JM (1994) Breeding and growth of Rhizopus in raw cassava by solid state fermentation App Microbiol Biotechnol41 :330-336 - Soccol CR, Marin B, Roussos S, Raimbault M (1993) Scanning electron microscopy of the development of Rhizopus arrhizus on raw cassava by solid state fermentation. Micologia NeotTopical Aplicada 6:27-39 - Saccol C R (1994) Contribuiçao ao estudo da fermentaçao no estado s6lido em relaçào corn a produçào de àcido fumàrico biotransfonnaçào de residuo s6lido de mandioca por Rhizopus e basidiomacromicetos do gênero Pleuro/us. Tirular Professor Thesis, Parana Federal University, Curitiba, Brazil - Vanneste G (1982) Enrichissement proteique du manioc par fermentation fongique. Rev Ferm Ind Aliment 37: 19-24 - Vervack W (1973) Analysis des aliments, méthodes courantes d'analyses. Laboratoire de Biochimie de la Nutrition, UCL, Louvain-la-Neuve - Wang HL, Runle DI, Hesseltine CW (1969) Antibacterial compound From a soybean product fermented by Rhizopus oligosporus. Proc Soc Exper Biol Med 131 :579-583 - Zhu C R; Du MJ; Lei DN; Wan LQ A study on the inhibition of aflatoxin b induced l hepatocarcino-genesis by the Rhizopus delemar Mater Med Pol 21 (2) : 87-91 1989 110 V ALORIZATION OF AGRO-INDUSTRIAL RESIDUES BY SOLID STATE FERMENTATION IN BRAZIL Carlos Ricardo Soccol UFPR, Laborat6rio de Processos Biotecnol6gicos, Departamento de Engenharia Quimica.. Caixa Postal 19 0 Il; 81 531-970 - Curitiba ( PR) - Brasil AbstTact ln this work sorne experiments for the using agro-industrial residues as substrates in sol id state fermentation is presented and discussed. Since Brazilian industry of cassava (Manhioc escu/en/a. Crantz) cultures produce a large amount of solid wastes, these residues were employed as model systems. There were developed different processes such as protein enrichment for human and animal nutrition, production of mushroom and metabolites such as enzymes. organic acids \Vith successful perspectives. J- 8iotransformation of residues from Cassava Starch production by Rhizopus genus Parana State is nowadays one of the biggest Brazilian cassava producer, its annual production being about 3.2 ITÙlIions tons of roots. The Northwest region concentrates a significant number of starch industries because of this high production, sorne of them processing about 500 tons/day of roots. Although these industries grant econoITÙcal development ta the Northwest region. the produced wastes, being hundreds of tons of solid and liquids wastes every day. heavily polluted the environment, namely rivers and streams. Figure 1. shows a basic unitary operations used by one of these industries to produce cassava starch. This industry, which processing capacity is about 100 ton/day of roots, produces 0.47 tons of peel. 112 tons of solid waste (cassava bagasse) with 85% ofmoisture and 1060 m] ofliquid waste (manipueira). TItrough these data we can evaluate the pollulant charge and the damages against environment by each industrial unit. We believe Ihat research efforts in biotechnological processes 10 take advantage of these wastes as substrates could interest the industries, in order to reduce the impact against the environment. Table 1 shows the results of physicochemical determinations on dried cassava bagasse from Paranâ State starch industry. Residual starch is about 52.45% and total reducing sugars 59.63%. These results show slarch extraction processes deficiencies in these industries, producing high raw materiallosses, and therefore, reducing the process efficiency. The level of residual starch may be still higher than this: Cereda ([994), in samples from Paranâ. Sm Paulo and Minas Gerais States reported levels of 60-70 % of residual starch. We believe that cassava bagasse residual starch different contents found by other authors could be explained by variance on each industrial process conditions. Protein, lipids, fibbers and ashes percentages are respectively: 1.67; 0.53; 22.19 and 1.10%. III The main objective ofthis work was to select Rhizopus strains capable to attack raw cassava bagasse (WlgelatiIÙsed starch) for the attainment of a high level protein flour to he used for human or aIÙmal feeding. Cassava tOOI 100lon/day Walet Srarch extraction water - i Peels 0.47 Ion Washing and peeling i i i Washing watet Cunet Brushers Screen Solid Wasle (Cassava bagasse) 112 ton/day with 85% of moisrute - Slarch mil!< J. Centnfugallon _ i i i Liquid Waste (manipueita) 1060 m3 wilh 5% solids Slarch Drying Dtied Srarch (250 lon/dia) Figure 1 - Basic unitary operations utilised in cassava starch process in Parana State, BraziI. Table 1 - Physicochemical composition of cassava bagasse Composition Content (%)' Moislure \0.73 Proteins 1.67 Lipids 0.53 Starch 52.45 Total Sugar 59.63 Fibbets 22.19 Ashes 1.10 a) Dry basis 112 Table 2 shows the results of strain selection for different Rhizopus species able to anack and biotrasfonn raw cassava bagasse. Considering the 19 srudied strains, only 3 presented a significant development in raw cassava bagasse (R. oryzae 28627, R. delemar 34612 and Rhizopus oryzae 28168). Some of them presented a regular growth in this residue (R. sp. 25975 ; R jormosa 28422 ; R. slolonifer 28169 ; R. oryzae 22580 ; R ofigosporus 6203 ; R. microsporus 46436); and some showed a weak development (R. arrhizus 16179; R. ory::ae 25976; R. arrhizus 1526; R. oryzae 395 ; R. slolonifer 28181 ; R. arrhizus 2582 ; R. arrhi:lIs 28425). There were also those that did not grow on this substrate (R ofigosporllS 2710 ; R. circicans 1475 and. R. delemar 1472). Table 2 - Screeoing for Rhizopus strains able to bio-transform cassava bagasse straio Growth Residual starch Proteins g/100 g DM g/100 g DM + R. arrhizus MUCL 16179 44.4 2.45 R. oligosporus NRRL 2710 52.3 1.8 ++ R. sp. NRRL 25975 43 6.9 + R. oryzae NRRL 25976 46 3.3 R. circicansNRRL 1475 50 18 R. delemar NRRL 1472 49 1.9 + R. arrhizus NRRL 1526 46 3.8 ++ R.jormosa MUCL 28422 42 7.4 +++ R oryzae MUCL 28168 32 10 + R. oryzae NRRL 395 47 3.6 +++ R. oryzae MUCL 28627 30 10.5 ++ R. slOlonifer MUCL 28169 42 7.7 ++ R.oryzae ATCC 22580 45 5.6 ++ R. oligosporus ATCC 6203 43 5.9 ++ R.microsporusATCC46436 42 6.3 + R. slOlonifer MUCL 28181 47 3.5 +++ R. delemar ATCC 34612 31 9.9 + Rarrhizus NRRL 2582 48 3.5 + R. arrhizus MU CL 28425 49 2.8 Raw cassava bagasse 53 1.7 ATCC = American Culture Collection (Rocckeville, Maryland, USA) ; MUCl = Catholic Universiry of Leuven. Belgium) : NRRL = Nonhern Regional Research laboralOry (U.S Depanment of Agriculture, Peoria, lIIinois, USA) - no growth, + weak growth, ++ regular groWlh, +++ excellent groWlh Ideal growth conditions for Rhizopus oryzae 28627 in solid state fennentation using cassava bagasse were determined. These conditions were : temperature, 28-32 oC ; inoculation rate, 5 10 spores/g dry bagasse; initial moisture, 70%; CIN, 4.7-14; initial pH. 5.7-6.4. 113 Mould growth was evaluated through real synthesised protein determination. The results showed that its content varied from 1.67 gllOO g dry bagasse, in the beginning of the fermentation, to 12 gllOO g dry bagasse after 24 h of culture. Yield coefficient between produced protein and consumed starch and was about 0.5. Kinetic of Growth Figure 2A shows kinetic evolution of consumed starch and R. oryzae growlh in cassava bagasse evaluated through synthesised protein in optimised conditions. During fermentation, about 42.23% of starch present in cassava was consumed. After 30h of fermentation the proteic level was 12.8 gllOOg of dry cassava bagasse. Il is 7.7 fold of the initial value, in only 30 h of fermentation. 50% yield was obtained between consumed starch and synthesised protein, without considering that part of this starch was hydrolysed to reducing sugar equivalent in glucose (6.2 g/IOOg DM) and remained in cassava bagasse without use by the mould (Fig. 2B). Il was observed a slight pH increase in the first 12 h of fermentation and an important decrease during the following hours, rea hîng pH 4.6 at the end of fermentation. This reduction would induce undesirable bacteria development. Figure 2 C shows glucoamylase evolution during raw cassa va bagasse bio-transformation. 1t was verified an important glucoamylase production increase after 12 h of fermentation, reaching its maximum after 30 h of fermentation (108 UI g dry bagasse). These numbers are sirnilar to those obtained by SOCCOL (1992) and SOCCOL el al. (1994) working with raw cassava (ungelatinised) pellets. ln the same figure, it can be observed a moisture increase from 69.14% at initial stage of fermentation to 72.77% after 30 h of culture. This moisture increase during fermentation is a good indicator of micro-organism groWlh. Scale-up in Different Bio-Reactors The global balance of the biotransformation of the raw cassava bagasse in different bioreactors it is presented in Table 3. We saw that it was possible to repeat and even to produce a significant increase in the prote in richness, glucoamylase synthesis, as weil as in starch consumption, in relation to the initial values on Petri plates. The bioreactors tray type showed an excellent performance in the transformation of cassava bagasse. The use of screens on the botlom of this type of bioreactor allowed a betler aeration, as weil as a more uniform growth of the fungi in the whole bagasse mass. 114 56 +--~-~-..,...-----'---.....,...---.----r----.---, 14 ,2 10 PrO'en (gI'OOg DM) 35 30 RedlJ:irg sugars (9'10~ DM) Glucoamylase (U/g '20 " .00 60 71 " 70 20 Figure 2. Kinetics of Cassava bagasse bio-transformation by Rhizopus oryzae 28.627. Evolution of A) Consumed starch and synthesised protein; B) pH and reducing sugars and C) Moisture and glucoamylase. Ils Table 3 - Global Comparison of Scale-Up Studies in Different Bioreactors Bioreator s type Residual Starch gllOOb Proteins gllOOg DM Glucoamyl ase U/gDM Reducing Sugar gllOOg Final pH Final Moisture YP/S 0/0 DM DM Petri plate Perforated smalltray Perforated big tray 30.3 29.6 12.8 13.44 108 94 5.7 4.7 4.6 4.4 73 71 0.5 0.52 28.7 13.5 103 5.2 4.3 0.5 0.5 SmaIJ 27.9 11 80.5 4.2 4.8 74.5 0.38 31 9.4 74 3.54 49 77 0.36 column with forced aeration Big coJumn with forced aeration DM : Dry maner ; P : Protein ; S: Starch ; Time of fermentation : 24 h : Initial protein in bagasse 1.67; Initial starch in bagasse; 52.45 ; YP/S : Yield coefficient ln such types of bio-reactors the protein synthesis was 44% superior to the values obtained in column bio-reactors. Even with forced aeration of 18 litres/hour used in the colurnn type (big), the final concentration in reaJ protein in the transformed bagasse was 9.4 g/IOOg of dried matter. The evolution of temperature during the bio-transformation of cassa va bagasse by the fungus in column type bio-reactor with forced aeration is shown in Figure 3. The temperature rises at exponential rate after the first 10 hours of fermentation and reaches the maximum of 46 oC after 18 ho urs of fermentation. Physiologic studies showed that fungal growth can he affected when the temperature rises above 36°C. According 10 Raimbault (1980) and Crajek (1988) sorne fermentation can reach temperatures from 60-65°C due to the heat liberated by the micro-organism during its growth. This raising of the temperalure could destroy the microorganism and stop the fermentation, if not controlled. In our study the temperature did not affect the growth, although it was possible to notice a certain reduction in the development of the fungi. We believe that this problem could be minimised by a cooling coyer around the column. Moisture level is superior at the end of bio-transformation in colurnn type fermenters when compared to the tray type (Table 3). This air f10w rate allows a more intense respiratory activiry by the fungi, inducing an increasing in CO 2 and H 20 liberation. It was obtained superior values in glucoamylase activity in tray type bioreactors when compared to the column type. 116 47.5 __ -Bio-reaclOr tempcrsnJre -+--Chamber temperature 45.0 Bioreactor Tempe rai ure oC 42.5 40.0 375 35.0 32.5 30.0 27.5 0 10 15 20 25 30 Time (h) Figure 3. Evolution of the temperature in the core of the column t)'pe bio-reactor during the bio-transformation of cassava bagasse by Rhizopus oryzae 28.627. Reducing sugars from Ihe aClion of glucoamylase on Slarch cassava bagasse were found in every type of studied bio-reactors and represent an increase of nUlritional value of the biotransformed bagasse. Fermentation yield of 51% and 50% obtained in small and big tray are considered excellent when compared to the results obtained by Soccol (1992), working with pellels of raw cassava (38% yield). We believe Ihal Ihis high yield achieved wilh R. oryzae 28627 is due mainly to structural characteristic of the bagasse, though its less density and high fibber concentration could favour Ihe aeration, as weil microbial growth. Thickness Effect oftire Fermentation Red in Rio-Reactor Tray Type The effect of the thickness of the fermentation bed on the evolution of the temperalUre dwing Ihe bio-transformalion of cassava bagasse are shown in Figure 4. Il was verified a raise of Ihe fun gus metabolism after 14 hours of fermentation. This activity reached its maximum between 22 and 24 hours of culture. The evolution of Ihe temperalUre was proportional to the thickness of the fermentation bed in the bio-reactor. For a thickness of 8 cm, the inside lemperature reached values up to 44 oC bel ween 18 and 20 hours of fermenlation. When the layer was reduced to 6 cm, the maximum temperature was 42.5 oC; wilh 4 cm, it was 37.5 oC oc. and with 2 cm of thickness, it dropped to 31.5 The values of the microbial growth, measured by synthesized protein, were superior for Ihe Ihickness of 1.2 and 4.0 cm. These results showed Ihal a rise in Ihe temperature on the layers of 6.0 and 8.0 cm did nol affect significantly Ihe growth of the fungus. Yields in terms of synthesized proleins and consumed starch dwing the bio-transformation maintain Ihemselves equally high to the differenl sludied Ihickness (Table 4). We can see thal the proie in synlhesis did nol present a significant difference for ail Ihe sludied thickness (12.0-13.7 gllOOg DM). These results show the possibility of using almosl the total practical volume of the bio-reactors tray type without any important losses in Ihe protein synthesis. 117 46 44 42 40 U 0 ~ ë 38 36 1S.. 34 '" 32 E f- 30 28 26 24 0 5 15 10 20 30 25 Time (h) Figure 4. Effect of thickness on the ternperature of the fermentation bed during the biotransformation of cassava bagasse by Rhizopus oryzae 28.627 in tray type bio-reactor. Table 4 - Effeet of fermentation bed thickness on the bio-transformation of the eassava bagasse by Rhizopus oryzae 28627 in tray t)'pe bioreaetor. Fermentation Residual Bed Starch gllOO g Thiekness DM 1 29.3 28.8 2 4 30.0 6 31.0 8 30.2 Protein gllOOg DM 13.2 13.7 13.5 12.1 12.0 Reducing sugar gllOO g DM 4.22 4.\0 4.50 3.50 3.70 Bio-transformation conditions: Tirne : 30 hours Initial protein content in cassava bagasse: 1.67 gll OOg DM Initial starch in cassava bagasse: 53 gltOO g DM YP/S : Yield coefficient (synthesised proteinlconsumed starch) DM : Dried maner 118 pH YP/S 4.2 4.0 3.9 4.3 3.4 0.50 0.51 0.52 0.48 0.46 Microbiological evalualion oflhe biO-Iransformed cassava bagasse The results of the microbiological evaluation presented on Table 5 show that the biotransformation of cassava bagasse allows the elimination almost completely of the microorganisms present in cassava bagasse before the bio-transformation (sample 1 and 2). The biotransformed bagasse nour obtained showed an excellent sanitary condition (sample 2) and are perfectly inside the standards of the sanitary legislation, considering that the cassava bagasse was bio-transformed without any thermal process of sterilisation. being only dehydrated at 60 oC for about 24 h after fermentation. Il can also be observed that the exposure of the nour under the ultraviolet rays (sample 3 and 4) for 5 and 10 min. eliminate completely the small nurnber of yeast and moulds present at the sample 2. They reduced equally the accoum of mesophila bacteria, although did not eliminate it completely. The results showed that were no growth of the undesirable bacteria such as Staphylococcus aureus , Bacillus cereus. Salmonella and faecal coliforms. Table 5 - Microbiological Evaluation of tbe Bio-Transformed Cassava Bagasse Flour Microbiological examinations Mesophiles bacteria (NMP/g) Count total coliforms (NMP/g) Moulds and Yeasts (CFU/g) Slaphylococcus aureus (CFU/g) Bacillus cereus (CFU/g) Salmonella ( in 25g) 4 5 3.3 x 102 Samples 3 2.3 x 102 2.1 x 102 4.2x 102 64 Negative Negative Negative Negative 8 x 103 2 x 102 Negative Negative 4 x 102 Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative 1 2 2 x 105 Sample 1 - Cassava bagasse flour not bio-transformed Sample 2 - Cassava bagasse flour bio-transformed Sample 3 - Cassava bagasse bio-transformed and treated 5 min with UV Sample 4 - Cassava bagasse bio-transformed and treated 10 min with UV Sample 5 - Cassa va flour selling in Curitiba City CFU- Colony-forming units per g MPN - Most probable number 119 The microbiological analysis of cassava bio-transfonned bagasse flour and traditional cassava flour showed that the first one presented superior sanitary conditions. That is due probably to a bactericide action of R. oryzae 28627, associated to a reduction of pH that did not allow the development of a great number of bacteria, yeast and fungi. This results confinn those obtained by Wang et al. (1969), describing the capacity of certain strains of the genus Rhizopus 10 inhibit the growth of a large number of Gram (+) bacteria. This study shows the high potenlial of sorne fungi slrains like Rhizopus in bio-transfonning solid amylaceous residues such as cassava bagasse. The perfonnance of these strains was remarkable, considering that in only 24 hours of fennentation the protein content of the bagasse couId be increased almost 8 times. The efficiency in the synthesis of proteins together with the capacity of anacking the raw starch (not gelatinized) confer ta these strains important characteristics, which could contribute significantly in the simplification of a large number of biotechnological processes that aim to the production of different metabolites. 120 D. EDmLE MUSHROOMS Pleurotus sajor-caju PRODUCTION FROM CASSA V A BAGASSE This work shows the feasibi1ity of using cassava bagasse for production of edible mushrooms of the genus Pleurolus. Se le clion oflhe slrain Table 6 shows the results of the five different strains of Pleurolus Spp. grew on the gelled mediwn prepared trom cassava bagasse and agar. The strains PleurOlus sajor-caju CCB 19 and 20 showed the highest radial growth (13.3 and 11.3 mm/day, respectively) and also the highest biomass production (69.8 and 67.7 mg, respectively). These results led to the selection of stTain P. sajor-caju CCB 19 to further experiments. Table 6 - Radial growtb and biomass production of live Pleurotus spp. strain cultivated in Petri disbes (8.5 cm) on agar-cassava librous residue medium (15g11 agar , 40 gli cassllva). Conditions: 6 days of culture, 25 oC and pH 7.0. Strain Pleurolus OSlrealUS CCB 12 Pleural us OSlrealuS LPB 22 Pleurolus OSlrealus CCB 115 Pleurolus sajor-caju CCB 19 PleurOlus sajor-caju CCB 20 Radial growtb (mm/day) Biomass (mg/plate) 7.2 ± 1.27 27.3 ± 2.69 9.5 ± 0.92 53.6 ± 452 o. ± 0.64 7.4 ± 0.28 9.8 13.3 ± 0.0 69.8 ± 3.24 11.3± 0.71 67.7+3.18 Fruit body produclion ln order to verify the feasibility of using agro-industrial residues for Pleurolus sajar-caju CCB 19 development, severaJ experiments with different ratios cassava fibrous waste sugarcane bagasse were studied. as shows Table 7. BiologicaJ efficiency (BE) \Vas utilized to measure to growth of the mushroom on the solid residues. The results showed that the best value (30.7%) was obtained for isolated cassava residue; the addition of sugarcane bagasse to cassava fibrous waste did not improve EB. Cassava residue had about 60% of carbohydrate (Barbosa, 1996). 121 Table 7 - Biological efficiency obtained with different cassava fibrous waste : sugarcanf bagasse ratios for the fruit body production by Pleuro/us sajor-caju CCB19. Conditions spawn lOg, 55 days of culture (polyethylene bags) j 24-28 oC, 70% relative humidity Sugarcane bagasse Experiment Cassava bagasse (gO 80 1 20 60 2 40 40 3 60 20 4 80 5 100 6 100 BE - Biological efficiency : fresh mushroom weight (g)/ substrate dry weight BE(%) 18.2 23.8 24.5 26.6 30.7 26.4 The BE was obtained considering two flushes of fruit body production: the first at 45 days of culture (60% of the total yield) and the second at 55 days of culture This behavior was also observed by Chang et al (1981) and Nair (1989). Effec/ of spawn concentra/ion offruit body production Rajarathman and Bano (1987a) suggest 20% as the best value for seed-inoculum for Pleuro/us spp. cultivation. However, Figure 5 shows that the best BE 38% obtained for Pleuro/us salorcaju 10% of spawn inoculated to the medium ~ 35 >. u .. c: 'ü if w 30 ;; .!:! 0> 0 "0 25 ID 20 5 10 15 20 25 Spawn concentration (%) Figure 5. Effect of spawn concentration on biological efficiency (BE) obt.a.ined for tbe fruit bod~' production b~' PleurOlus sajor-caju CCB19. Conditions: 55 days of culture (polyetbylene bags) ; 24-28 ·C, 70 % relative bumidi~·. 122 Effecl ofsoybean addilion onfruil body produClion The addition of ground soybean to cassava fibrous waste produced an improvement of Pleuralus sajar-caju development (maximum BE 69%) (Table 8), as reported by many authors (Rajarathnam el al., 1987, Royse el al., 1991, Royse, 1992, Belinski el al. . 1994). This result couJd be explained by the reduction of carbon:nitrogen ratio caused by soybean addition. Table 8 - Biological efficiency obtained with diITerent levels of soybean addition to cassava fibrous waste for the fruit production by Pleurotus sajor ---caju CCB19. Conditions: spawn 10 g, 55 days of culture (polyethylene bags) ; 24-28 oC Soybean (%) Cassava bagasse (g) BE(%) 0.0 100 29.80 ± 0.58 5.0 95 32.77 ± 1.80 100 90 37.10±0.56 15.0 85 45.97 ± 3.37 20.0 80 69.00 ± 4.89 25.0 75 47.22 ± 2.46 BE - Biological efficiency : fresh mushroom weight (g)/substrate dry weight (g) Scale -up afIhe fruil body praduclian Using the culture conditions established previously, the scale-up of the fruit body production by Pleuralus sajar-caju was carried out raising the quantities of the experiments 10 and 100 times higher. Table 9 shows that the EB value obtained for small scale was comparable to the results obtained for large scale, confirming the feasibility of mushroom cultivation in a larger scale. Table 9 - Scale-up (1:10; 1:100) of the cultivation Pleurotus sajor-caju CCB19 on cassava bagasse. Soybean Cassava bagasse (g) BE(%) 20 80 69.00 ± 4.89 200 800 63.43 ± 2.55 2000 8000 65.27 ± 5.21 B.E - Biological efficiency : fresh mushroom weight (g)/substrate dry weight (g) 123 Conclusions Pleuro/us sajor-caju CCB 19 cultivated on cassava bagasse subslrate showed a biological efficiency (BE) of 30% The addition of soybean flour 10 Ù1e cassava bagasse raised the BE from 30 10 69% and Ù1e scale-up of process was successfully carried ouI. These results show that agro-industrial residues as cassava bagasse can effectively be used for commercial production of Ù1e edible mushroom by solid state fermentation. SimiJar advantageous results were also obtained for Ù1e production of Lentinula edodes with cassava bagasse. Acknowledgements: The author wishes to thank the financial support from the Brazilian Agency CNPq REFERENCES - Barbosa, M.C.S. Tese de mestrado . Setor de Teclmologia, Universidade Federal do Paran~, Curitiba, 1996, 117p. - Belinski, P. A . , Masaphy, S. , Levanon, O., Hadar, Y. and Dosoretz, c.G. Appl. MierobioL Bioteehnol., 40,629-633, 1994 - Cereda M . Res'duos da industrializaçào da mandioea no Brasil. S<o Paulo- Brasil, Editora Paulic_ia, 1994. P. 27. - Chang S.T., Lau, 0 W and Cho, K.Y. EuropeanJ. App/. Microbio/. Bio/eehnol., 12,58-62. 1981. - Grajek W. Cooling aspects of solid state cultures of mesophilic and thermophilic fungi . .J. ferment. Teehnol., 1988,66: 675-79. - Nair, N.G. Ann. App. Biol., 114, 167-176, 1989 - Raimbault M. Fermentation en milieu solide: croissance de champignons filamenteux sur substrat amylacé. Thèse de Doctorat es science, Univ. Paul Sabatier; Toulouse, 1980; 291 p. - Rajaratlmam, S. and Bano, Z.. CRe. Critieal Rev. Food Sei. Nutrition, 26. 157-223. 1987a. - Rajaratlmam, S. and Bano, Z .. CRe. Critieal Rev. Food Sei. Nutrition. 26, 243-311, 1987b. - Royse, D.J., Fales, S.L. and Karunanandaa, K. Appl. Mierobiol. Bioleeltnol., 36,425-429, 1991. - Royse, D.J. Appl. Mierobiol. Bioleellnol., 38,179-182, 1992. - Saccol, C. R., Raimbault, M. and Pinheiro, L. 1. Arq. Biol. Tecnol., 37, 203-210, 1994. - Soccol C.R . Physiologie et métabolisme de Rhizopus en culture solid et submerg_e en relation avec la degradation d'amidon cru et la production d'acide L(+) lactique. These de Doctorat. Mention/Genie enzymatique, Bioconversion et microbiologie, Universite de Teclmologie de Compiegne, Compiègne- France, 218p. 1992. - Wang H.L., Ruttle 0.1., Hesseltine C. W.. Antibacterial compound from a soybean product fermented by Rhizopus oligosporus. Proc. Soc. Exper. Biol Med., 1969; 131 : 579-583. 124 CITRIC ACID AND GLUCOAMYLASE PRODUCTION FROM CASSAVA BY-PRODUCTS IN SOLID STATE FERMENTATION Carlos Ricardo Soccol UFPR, Laborat6rio de Processos Biotecnol6gicos, Departamento de Engenharia Quimica,. Caixa Postal 19 0 II; 81 531-970 - Curitiba ( PR) - Brasil 1 - Ci tric acid production from Cassava Bagasse Six strains of Aspergillus niger were screened in liquid mediwn and strain LPB 21 was selecled for funher studies on 3 different agTo-industrial residues for production of citric acid by solid state fermentation. Cassava bagasse was found to he a better substrate than vegetal sponge and sugar cane bagasse. Citric acid production and yields were respectively 13.64 g/100g dTied substrate and 41.78 % of acid citric produced by starch conswned. In the studies under optimized conditions, the production of citric acid was 280 gIKg of dry bagasse at 120 h, which corresponds to a yield 70%, based on starch conswned. Kinetics studies on of pH changes, moisture level, 10ss of weight of the substrate, starch utilization and a-amylase production allowed an insight in the process. E./fec/ ofinitial mois/ure content ofcassava bagasse The water content of solid support has been reported as an important limiting factor for solid state fermentation (Pandey, 1992, Lonsane e/ al., 1992). Consequently, the effect of moi sture present in cassava bagasse was investigated in the range 45-65% initial moisture. Results showed that the production of citnc acid at 8 days increased with an increase in initial moisture content of the medium up to 50% (Table 1). Table 1. Effeet of Initial Moisture of Cassava Bagasse on Citric Aspergillus Niger LPB 21 Moisture 0/0 Citrie acid produced (g/100 g dried bagasse) Sugar consumed (g/IOOg dried bagasse) Acid Production by Yield 0/0 45 16.35 3692 44.28 67.96 50 27.25 40.1 55 25.32 36.83 68.75 2215 39.63 55.89 60 1939 37.19 52.14 65 Initial sugar : 46.5 g/lOO g dried support, Yield : g produced citric acid 1 g consumed sugar 125 A further increase in the initial moisture of cassava bagasse of 10% had an adverse effect on the synthesis of citric acid. Thus, the initial moisture content of 50% could be considered as ideal to produce high quantity of citric acid (272gfKg cassava bagasse). In this case, fermentation yield was approximately 70%. EfJect ofInitial pH Table 2 presents the results of production of citric acid by SSF using different initial pH values. Il can be seen that very low values (1.0) reduced the production of the metabolite: higher values favored the production and the maximum was obtained at pH 2.0: in this case. the concentration of produced citric acid was 246 g!Kg dried cassava bagasse with a yield of 71.45 %. These results confum those reported by many authors, that the initial pH of the medium for citric acid production is in the range of 1.6 to 3.5 (Kolicheski, 1995, Meers el al. 1991 ; Miller, 1981). Table 2- EfTect of Initial pH of the Medium on Citric Acid Production by Aspergillus Nige LPB 21 on Cassava Bagasse by Solid State Fermentation pH 1 2 3 Citric acid production (g/100 g dried bagasse) Consumed Sugar (g/100 g dried bagasse) Yield 18.12 24.64 21.93 28.32 34.54 34.19 63.98 7145 6414 (%) Initial sugar : 46.5 g/l 00 g dried support, Yield : g produced citric acid / g consumed sugar Aeration EfJecl Aeration is an important factor for citric acid production in colunm type bio-reaclors, since the air f10w helps to dissipate metabolic heat and also provide necessary oxygen for the growth of the microorganism. Table 3 shows that the optimal aeration flow was between 50 and 60 ml/min. Aeration rates above or below these values lead to a decrease in citric acid production. TemperalUre EfJecl The optimaltemperature for fermentation varies according to the microorganism and is usual1y between 25 and 35°C (Prescon and Ounn, 1959). In this work the highest production of citric acid occurred at 26 oC (Table 4), confirming those reported above. 126 Table 3 - EfTect of Aeration Rate on Citric Acid Production by Aspergillus Niger LPB 21 Grown on Cassava Bagasse in Solid State Fermentation. Aeration (mUmin/column) Citric acid produced (gllOO g dried support) Sugar consumed (gllOO g dried support) Yield 40 50 60 70 80 13.6 25.42 27.12 19.45 17.32 38.36 36.13 37.54 37.51 32.61 35.45 73.36 72.24 51.85 53.11 (%) Initial sugar : 46.5 g/l 00 g dried support, Yield : g produced ciuic acid / g consumed sugar Table 4. EfTect of Temperature on Citric Acid Production by Aspergillus niger LPB 21 Grown on Cassava Bagasse in Solid State Fermentation. Temperatures Citric acid Consumed sugar Yield oC production (gllOO g dried (gllOO g dried (%) bagasse) bagasse) 24 15.64 37.81 26 28.45 37.66 28 23.91 36.88 30 20.80 35.97 32 19.73 37.67 Initial sugar : 46.5 g/I 00 g dried support. Yield : g produced citric acid / g consumed sugar 41.36 75.54 64.83 57.83 51.96 Kinelics ofCitric Acid Produclion Figure lA shows the evoiution of consumption of starch and citric acid production during the fermentation. The production of citric acid started in the initial period of fermentation although its concentration reached 74 gJkg dried cassava bagasse at 48 h of culture. Between 48 and 72 h, ciuic acid productivity achieved its highest point (201.34 gIKg dried cassava bagasse) and From 72 ID 120 h, the final concentration was 280 gJKg dried cassava bagasse. which corresponds to a yield of 70% in relation with the consumed starch. These results are higher than those reported by sorne authors working with different substrates (Hang el al., 1987 ; Omar el al., 1992 ; Shakaranand and Lonsane, 1993). Production of a-amylase was higher during the first 72 h fermentation (192 IU/g dried cassava bagasse) (Figure 1B) and is associated with the higher consumption of starch in this period (Figure 1A). pH is gradually reduced as citric acid is accumulated in the medium and reaches its lowest value (pH 0.5) after 96 h fermentation (Figure 1B). Figure 1C shows the increase in medium moi sture and the support 1055 of weight of during the culture. An increase of moisture from 60.32 to 64.18 % is most likely due to fungal metabo1ism (Soccol, 1992 and 1994) and the 10ss of weight of the support is due to utilization of starch by micro-organism. 127 " " '" :xl JO Cime 1. 19 g DM] .",d SI wcll (gIg DM) 20 " 10 L~~~C-~""40~~""60~~~;::::::~'OO~::;::::;tIO~·~...,J'400 Titre (h) Figure lA - Kinetics of citric acid production by Aspergillus niger. Evolution of starch consuption and citric acid production AlphaAmylase actJ.VIl)' (lU/8 DM) Timc (hl Figure lB - Kinetics of citric acid production by Aspergillus f1iger. Evolution of alpha-amylase and pH during the fermentation Loss of \\'cight MOl.Slure t>(%) (%) --...-- Loss of Weil!h( '" (%) 1 Time (h) Figure 1 C. Kinetics of citric acid production by Aspergillus f1iger. Evolution of moisture and loss of weight during the fermentation 128 II - Glucoamylase production from Cassava Bagasse by Solid State Two strains of Rhizopus oryzae (MUCL 28627 and MUCL 28168) and one of Rhizopus delemar (ATCC 34612) were investigated for their ability to produce glucoamylase able to attack granular starch. Cassava (Manhioc esculenta, Crantz) bagasse was employed as substrate in solid state fermentation (SSF). Scanning electron microscopy was used to follow granular starch degradation by the fungal enzyme. An experimental design was used to optimize the fermentation time and the most suitable pH condition to extract the enzyme from SSF medium. Enzyme yield was evaluated as specific activity. R. oryzae MUCL 28627 presented most important specific activity after fermentation of crude bagasse. Higher specific activity values (> 10 katlkg) were obtained after 72 hours fermentation at 32 oC and extraction of the enzyme activity with pH 4.5 200 mM acetate buffer from the whoJe fermented biomass. Screening ofstrain Previous screening work realized by SOCCOL (1992) showed that three strain of Rhizopus were able to anack granular crude starch: Rhizopus oryzae MUCL 28627 and MUCl 28168 and Rhizopus delemar ATCC 34612. In this work, these selected strains were assayed to growth in cassava bagasse, an agroindustrial waste still having starch as the main component (50 %, dry basis), by solid state fermentation. Actually, the tluee strains presented a marked development in the substrate, characlerized by the colonization of the enlire surface of the Petri dish by mold mycelium, after 32 hours fermentation (results not shown). The specific activity recovery is low in the first 12 hours of fermentation (Figure 2). In fact, the initial 8-12 ho urs correspond ta the fmal of the latency phase and the star! of spore germination. Later the specific amylolytic activity enhances as a function of fermentation time until 32 hours for ail strains investigated (Figure 2). However, the activities of Rhizopus delemar ATCC 34612 and Rhizopus oryzae MUCl 28168 remain almost constant after this time period whereas the aClivity of Rhizopus oryzae MUCL 28627 shows a continuous increase in the studied period of 48 hours. On another hand, the absolute values observed for specific activity of R oryzae 28627 were always superior to those observed for the two other strains. As specific activity of glucoamylase From R. oryzae MUCL 28627 seemed to increase as a function of time (Figure 2), an experimental design with su perior time periods was employed to follow the activity yield enhancement (Table 5). Other factors, like bagasse initial moisture content and extraction pH were also invesligated. In fact, a high humidity content may affect enzyme production by favoring contaminant development or altering enzyme and oxygen diffusion. The correct choice of the pH for enzyme activity extraction, on the other hand, may improve further purification procedures. The experimental design was run twice, totaling 34 assays. The protein content and the activilies of glucoamylase and a-amylase were measured (Table 5). In experimental conditions of glucoamylase determination (60 jC temperature, pH 4.5 and 2-5 min. reaction lime) no significant activity of a-amylase was detected, since the amount of reducing sugars determined 129 8 Cl E ~ III 6 .:.:: >- ":0=> R. oryzae 28168 -0- R. oryzae 28627 ..... ::::J - -D- R. delemar 34612 4 u <C u ~ u CIl 2 a. l/) 0 0 12 36 24 48 Time (hours) Figure 2. Glucoamylase specific activity as a fUDction of tbe selected Rhizopus strain and tbe time of solid state fermentation. by Somogyi-Nelson method were coincident with the content of a-D-glucose measured enzymatically by glucose-oxidase/peroxidase method. As specifie activity of glucoamylase from R. oryzae MUCL 28627 seemed to increase as a function of time (Figure 2), an experimentai design with superior time periods was employed to fol1ow the activity yield enhancement (Table 5). Other factors, like bagasse initial moisture content and extraction pH were also investigated. In fact, a high humidity content may affect enzyme production by favoring contaminant development or altering enzyme and oxygen diffusion. The correct choice of the pH for enzyme activity extraction, on the other hand, may improve further purification procedures. The experimental design was run twice, totaling 34 assays. The protein content and the activities of glucoamylase and a-amylase were measured (Table 5). In experimental conditions of glucoamylase detennination (60 iC temperature. pH 4.5 and 2-5 min. reaction time) no significant activity of a-amylase was detected, since the amount of reducing sugars detennined by Somogyi-Nelson method were coincident with the content of a-D-glucose measured enzymatically by glucose-oxidase/peroxidase method. Experimental results of soluble protein content and specifie amylolytic actlvlty were submitted to ANOVA (Table 6 and 7). For soluble proteins, the correlation coefficient (R 2 = 0.938562) and the very low p-value for the lack-of-fit (p = 0.0019) indicate that the mathematicaJ model explains the experimental variation observed (Table 6). Fermentation time and extraction pH caused significant effects (p < 0.0005) on the response content of proteins/mL of crude extract, but not the initial moisture content. Indeed, the Jevels of added 130 Table S. Experimental design employed to optimize tbe extraction of glucoamylase activity from cassava bagasse fermented by Rhizopus oryzoe 28627 and the respective experimen tal responses. Run Time (hours) 1 2 3 4 5 6 7 8 9 10 53 34 72 34 72 34 72 34 53 72 II 27 79 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 53 53 53 53 53 53 34 72 34 72 34 72 34 53 72 27 79 53 53 53 53 53 Extraction Initial moisture (mL H 20/g dry pH bagasse.) 6.5 4.5 4.5 8.5 8.5 4.5 4.5 8.5 6.5 8.5 6.5 6.5 3.8 9.2 6.5 6.5 6.5 6.5 4.5 4.5 8.5 8.5 4.5 4.5 8.5 6.5 8.5 6.5 65 3.8 9.2 6.5 6.5 65 2.8 1.9 1.9 1.9 1.9 3.7 3.7 3.7 2.8 3.7 2.8 2.8 2.8 2.8 I.S 4.1 2.8 2.8 1.9 1.9 1.9 1.9 3.7 3.7 3.7 2.8 3.7 2.8 2.8 2.8 2.8 I.S 4.1 2.8 131 Soluble proteins (mg/mL) Activil)' (nkat/mL) Specifie activity (mkat/mg) 177 163 50 372 296 182 37 354 164 316 181 104 53 321 189 157 186 204 175 41 390 282 194 53 367 109 280 198 74 59 296 221 171 193 281 386 462 168 273 431 461 142 291 301 175 288 256 125 244 367 285 259 418 481 161 305 395 495 124 301 304 190 324 290 148 258 310 306 1.59 2.39 9.25 0.45 0.92 2.37 12.46 0.40 1.77 0.95 0.97 2.77 4.83 0.39 1.29 234 153 1.27 2.39 11.74 0.41 1.08 2.04 9.34 0.34 2.76 1.09 0.96 438 492 0.50 1.17 1.81 1.59 water presently investigated are correspondent to 65 up to 79% of initial moisture. Saccol (1994) verified that this is the optimum range for protein production by R. oryzae 28627 by SSF, which explains why its ef'fect does not significant1y affects protein responses. In the same way, interaction effects of initial moisnue with other variables and quadratic effects of time variable were not significant (p > 0.10). They were eliminated of the analysis and the resulting regression equation was employed to plot Figure 3. The maximal response zone is located in the direction of minor fermentation time periods and higher pH values of the extraction buffer (Figure 3 ). The decrease on protein content as a function of time is supposed to be related to secretion of protease during fermentation and it is presently under investigation in our laboratory. The effect of the pH of extraction buffer is outstanding: the basic buffer (pH 8.5) extract 2 up to 6 fold more protein than the acidic one (pH 4.5), depending on fermentation time course. In fact, a great part of biosynthesized proteins present pl near to pH 4.5 and they are very soluble at basic pH (pH> 8) what could contribute to the observed increase of the extraction yield. Conversely, acidic pH acts as a selective extraction factor, since only proteins presenting net charge in acidic pH will be solublized (Chiarello et al. 1996). Table 6. ANOVA for experimental responses of soluble protein content (mg/ml of crude extract) Effeet A: Time B: pH C: Initial Moisture AB AC BC AA BB CC Laek-of-fit Pure error TOTAL SUIJl of Squares 5320519 256374.64 403.05 313600 25.00 225.00 108.28 9294.00 10181.90 13236.23 8558.83 354748.03 Degrees of Freedorn 1 1 1 1 1 1 1 1 1 5 19 33 Mean Square 53205.19 25637464 403.05 3136.00 25.00 22500 \08.28 9294.00 10181.89 2647.25 450.46 F. ratio p value 118.11 569.13 0.89 6.96 0.06 0.50 0.24 20.63 22.60 5.88 0.0000 0.0000 0.3661 0.0162 0.8188 0.4958 0.6347 0.0002 0.0001 0.0019 R' = 0.938562 Table 7. ANOVA for experimental responses of specifie glueoamylase aetivity (mkat/mg of soluble proteins). Effeet Sum of Squares p value Degrees of Mean F. ratio Freedom Square A: Time 81.44 1 81.44 157.32 0.0000 B: pH 134.88 1 134.88 260.55 0.0000 C: Initial Moisture 0.26 1 0.26 0.50 0.4939 AB 58.53 1 113.05 5853 0.0000 AC 1 1.02 1.02 1.97 0.1765 BC 0.01 1 0.01 0.01 0.9407 AA 1 83.05 83.05 Il.69 0.0029 BB 10.49 1 10.49 20.27 0.0002 CC 1.50 1 1.50 2.90 0.1051 Lack-of-fit 1906 5 3.81 7.36 0.0005 Pure error 9.84 19 0.52 TOTAL 323.07 33 R' = 0.910551 132 Proteins (~g/mL) 400 300 200 8.5 100 Extraction pH 62.5 72 Fermentation Tlme (hours) Figure 3. Response surface to soluble proteins as a function of fermentation time and extraction pH. Soluble proteins (mg/mL) = 157.42 -47.76A + 104.84B - 4.16C + 14AB + 26.32B 2 + 27.56C 2, where A is coded units of time, B, coded units of extraction pH and C coded units of initial moisture. R2 = 0.937552 Specifie ACllvity (~k.tJmg) 10 8.5 o 62.5 72 Fermentation Time (hours) Figure 4. Response surface to specifie glucoamylase actlvlty as a function of fermentation time and extraction pH. Specific activity (mkatlmg) = 1.67 + 1.87A - 2.41 B + 0.11 C-1.91 AB + 0.67 A2 + 0.88B 2, where A is coded units of time, B coded units of extraction pH and C coded units of initial moisture. R2 = 0.902743. 133 As observed for soluble protein responses, only the fermentation time and the extraction pH showed a significant effect on the glucoamylase specific activiry responses (Table 7). 2 Statistical analysis confrrmed that the mathematical model fits weil to experimental data (R = 0.910551and p value < 0.0005 for lack-of-fit). Since the initial moisture variable, its interactions and quadratic effects were also not significanl they were eliminated of the analysis and the resulting regression equation was employed to plot Figure 4. The best yield of specific activity was obtained with fermentation time of 72 hours and extraction pH of 4.5 (Figure 4). The acidic pH is supposed to protect enzyme against aspartic proteinases attack during a long period fermentation, since the pl of these fungal hydrolases is bellow pH 5.1 (Campos & Felix, 1995). On another hand, the low solubiliry of non enzymatic proteins at pH 4.5 seemed to contribute to the selective extraction of glucoamylase. ln fact, glucoamylase is a widely spread enzyme, found mainly in fungi and less often in bacteria and generally in more than one isoforrn in the same species (Ali et al. 1994 ; Fitatusuji et al. 1993). Depending on the enzyme source, the pl is also variable. Mold glucoamylases can display pl from 4.1 up to 8.4 (Speck et al. 1991 ; Yamasaki, 1978) and, probably, the glucoamylase !Tom R. oryzae 28627, able to attack granular starch , has a pl located on neutral or aJkaline zone, what ex plains its selective extraction at pH 4.5. Conclusion Glucoamylases of Rhizopus oryzae 28627 were able to attack and perforate granular cassava starch as observed by scanning electron microscopy. Higher yields of specific activity were obtained after 72 hours of solid state fermentation of cassava bagasse provided an extraction of the enzymatic protein with 200 mM pH 4.5 acetate buffer. Ongoing studies in this field are in progress to purify glucoamylases and to elucidale the action of proteases on glucoamylolytic activity losses. Acknowledgements: The author wishes to thank the financial support !Tom the Brazilian Agency CNPq REFERENCES - Ali, S.; Malek, ; Hossain, Z. (1994) Purification and characterization of a thermostable glucoamylase from a MyrOlhecium isolate. J App. Bacleriol., 76:210-215. - Barret, A. 1. (1986). An introduction to the proteinases. In: Barret. A. J. & Salvesen. PrOleinase lnhibilors. Amsterdam, Elsevier, 1986. p. 1-21.28. - Campos & Feliz, C.R. (1995). Purification and characterization of a glucoamylase from Humicola grisea Appt. Environ. Microbiot., 61(6):2436-2438. - Chiarelo, M. D.; Larre, c.; Kedzior. M.; Gueguen, 1. (1996). Pea seedling extract catalysis amine-binding and cross-linking. 1. Evidences for the role of a diamine oxidase. 1. Agric. Food Chem., 44(11<):3717-3722. 134 - Fitatsuji, M.; Ogawa, T.; FukudaA, H. (1993). Purification and properties of two forms of glucoamylase from Saccharomyces jibuligera. J Ferm. Bioeng., 76(6):. 521-523. - Raimbault M & Alazard D. Culture method to study fungal growth in solid fermentation. European J Appt. Microbiol., 1981. 9 : 199-209. - Soccol, C. R. Tese, Professor Titular em Biotecnologia e Tecnologia de Alimentos - Setor de Tecnologia, UlÙversidade Federal do Parant., Curitiba, 1994,228 p. - Soccol C.R . Physiologie et métabolisme de Rhizopus en culture solid et submerg_e en relation avec la degradation d'amidon cru et la production d'acide L(+) lactique. These de Doctorat. Mention/Genie enzymatique, Bioconversion et microbiologie, Universite de Technologie de Compiegne, Compiègne- France, 218p, 1992. - Specka, U.; Mayer, F.; Antranikian, G. (1991). Purification and properties ofa thermoactive Appl. Environ. Microbiol.. glucoamylase from Clostridium thermosaccharolyticum. 57(8):2317-2323. - Yamasaki, Y. & Suzukï. Y. (1978). Purification and properties of a-glucosidase and glucoamylase from Lentinus edodes (Berk) Sing. Agric. Biol. Chem., 42(5):917- 980. - Ayala, L. A. C.; Socco!, C. R.; Santos, H. R.; Todeschinil, M. L. Produçào de esporos de fungo entomopatogêlÙco (Beauveria bassiana) por fermentaçào no estado s6lido utilizando coma substrato refugo de batata. Anais do XI SINAFERM, Sociedade BrasiJeira de Microbiologia, Vol 2, pp. 574-579, 1996. - Barbosa, M.C.S. ; Saccol, C.R. ; Todeschini, M.L. ; Tonial, T. ; Flores, V. Cultivation of Pleurotus sajor-caju in cassava waste. ln: G.M. Zanin & F.F. Moraes (Eds). Anais W Seminario de Hidrolise Enzimaica de Biomassas. Editora Sthampa, 1994, p 179-181. - Barbosa, M.C.S ; Socco!. C.R ; Krieger, N ; ChiarelIo, M. D. Pleurotus sajour caju production from cassava waste by solid state fermentation. Fifth Brazilian Symposium on The Chemistry of Lignins and Other Wood Components. Curitiba (Pr) Brasil, 3118 to 05/09, 1997, P 557-569. - Beux. M.R. ; Soccol, C.R. ; Raimbault, M. Use cassava bagasse on cultivation edible fungus Lentinus edodes by solid state fermentation. ln: G.M. Zanin & F.F. Moraes (Eds). Anais IV Seminario de Hidr61ise Enzimatica de Biomassas. Editora Sthampa, 1994, p 190- 198. - Beux, M. R. & Soccol, C. R. - Cultivo do fungo comeslvel Lentinula edodes em residuos agroindustriais do Parana através do uso de fermentaçào no estado s6lido. Boletim do CEPPA, vo!. 14 (1) p. 11-21,1996. - Beux, M. R.; Soccol, C. R.; Marin, B.; Tonia!. T.; Roussos, S. Cultivation of Lentinus edodes on mixture of cassava and sugar cane bagasse. In: Roussos, S.Lonsane, B. K.; Raimbault, M.; Viniegra-Gonzalez. G. (Eds) Advances in Solid State Fermentation - Edible mushrooms/ fungi. Dordrecht, Kluwer Acad. Pub .. c. 41, 1997. p501-514. - Kolicheski, M. B.; Socco!. C. R.; Marin, B.; Medeiros, E.; Raimbault, M.citric acid production on three cellulosic supports in solid state fermentation. In: Roussos, S.Lonsane, B. K.; Raimbault, M.; VilÙegra-Gonzalez, G. (Eds) Advances in Solid State Fermentation Secondary metabolits. aroma. pigment and biopesticides. Dordrecht, Kluwer Acad. Pub., c. 36, 1997. p449-462. - Kolicheski. M. B. & Socco!. C. R. Produçào de acido citrico por fermentaçào estado s6lido (FES) utilizando coma substrato bagaço de mandioca. Anais do Xl SINAFERM, Sociedade Brasileira de Microbiologia, Vol!. pp 369-375. 1996. - Raimbault, M; Giraud, E.; Soccol, C.R. ; Saucedo, G. Fermentation in cassava bioconversion. ln ; Dufour, D. ; O'brien, G.M. ; Best, R. (Eds) Cassava Flour and Starch ; Progress in 135 Research and Devellopmenl - Session 4 - Bioconversion and By-products. ClAT Pub. N. 271,1996. pI87-196.. - Soccol, C. R. Agroindustrial Aplications of Solid State Fennentation Processes. In: E. Galindo (Ed.), Fronteras en Biotecnologia y Bioingeneria., pp. 349-358. Soeiedade Mexieana de Bio/een%gia y Biengeneria, 1996. - Soccol, C. R. Aplicaçôes da fennentaçào no estado s6lido na valorizaçào de residuos agroindustriais. França -F/ash Agrieu//ura, 4(2): 3-4, 1995. - Soccol, C. R. Contribuiçào ao estudo da fermentaçào no estado s61ido em relaçào com a produçào de âcido fumârico e biotransfonnaçào de residuo s6lido de mandioca por Rhizopus e Basidiomacromicelos do gênero P/euro/us. Tese de Professor Tilular. UFPR, Curitiba. 1994. 228p. - Soccol, C. R.; Marin, B.; Raimbault, M.; Lebeau!!, J. M. Potential of solid state fennentation for production of L(+) lactic acid by Rhizopus oryzae. App. Mierobiol Bio/eehno/. 41: 286-290, 1994. - Soccol, C.R. ; Cabrero, M.A. ; Roussos, S ; Raimbault, M . Selecion de cepas de Rhizoplls em base a su capacidade de crescimienlo en yuca. Soeiedade Mexieana de Bio/een%gia e Bioengennieria, V.3 ; N 1-2, p. 61-64,1993. 136 YEAST CULTIVATION IN SSF: CONTROL OF METABOLISM OF Schwanniomyces castellii DURING SOLID CULTIVATION ONSTARCH Gerardo Saucedo-Castaiieda Universidad Autonoma Metropolitana, Unidad Iztapalapa, Departamento de Biotecnologia Av. Michoacan y Purisima, Col. Vicentina, Apdo. Postal 55-535. Mexico, D.f., c.P. 09340, Mexico Abstract" Cu!tivation reports of yeasts in SSF are scarce, there are sorne work done on ethanol production from sweet sorghum and few papers on protein enrichmenl. Filamentous fungi are the most used microorganism in SSF. Nevertheless, yeasts systems pro vide an interesting experimentaJ model for studies concerning control of metabolism in SSF systems. Other advantage of yeasts is that cells can be easily separaled from support, then biomass detennination can be carried out using standard methods of submerged fennentation. SSF by Schw. castellii was selected as experimental modelto study a fennentation with two phases: an aerobic stage required for biomass buiJd up, amylase synthesis and an anaerobic phase for the transfonnation of hydrolyzed starch into ethanol. Switching of phases can be easily done by modifying the gas phase in the fennentor. Data on conversion of starch into biomass and ethanol indicated that the overall ethanol conversion was of 57 % of the theoretical values, nevertheless, the a1cohoJ yield in the anaerobic phase was 94 % of the theoretical value. The biomass fonnation is confined to aerobic stage. Solid state cu!tivation of yeasls was used also to develop a automated monitoring and control system for SSF. It pro vides on line data of carbon dioxide and oxygen in real time without disturbing the fennentation. In this sense, the estimation of specific growth rate by direct biomass detennination and gas analysis were similar. ln the recent years, our research team have supported development on these equipments using specific transducers and gas chromatography. To our knowledge this kind of in line automated systems appears to be a very useful feature for SSF systems. * The content of the lecture was publ ished in: Applied Biochemis/ry and Bio/echn%gy, vol. 36 (1992); pp. 47-61 137 SUGARCANE USED IN SOLID STATE FERMENTATION FOR CELLULASESPRODUCTION Sebastianos Roussos Laboratoire de Biotechnologie, Centre ORSTOM, BP 5045; 34032 Montpellier Cedex l, France Cellulases production and applications Cellulases, the enzymes capable of hydrolyzing cellulosic compounds, find extensive use in extraction of green-tea components, modification of food tissues, removaJ of soybean seed coat, improving canle feed quality, recovering juice as weil as other products from plant tissues and as component of digestive aid (Toyama, 1969; Lonsane and GhildyaJ.l 991). Cellulases can be produced by submerged or solid state fermentations. The laner technique is generaJly preferred as it offers many advantages such as two-three times higher enzyme production as weIl as protein rate, higher concentration of the product in the medium, direct use of air-dried fermented solids as source of enzyme which lead to eJimination of expenses on downstream processing, employment of natural celJuJosic wastes as substrate in contrast to the necessity of using pure cellulose in submerged fermentation (SmF) and the posibility of carrying out fermentation in nonaseptic conditions (Chahal 1983: Toyama and Ogawa, 1978; Pamment el al., 1978; Deschamps el al., 1985; Allen, 1983: Sternberg, 1976). The biosynthesis of cellulases in SmF process is strongly affected by catabolic and end product repressions (Gallo el al., 1978; Ryu and Mandels, 1980) and the recent reports on the overcoming of these repressions to significant extent in solid state fermentation (SSF) system (Ramesh and Lonsane, 1991 a, b), therefore, are of economic importance. The amenability of SSF technique to use upto 20-30% substrate, in contrast to the maximum of 5% in SmF process, has been documented (Pamment el a/~ 1978). Il is. therefore, not surprising that cellulases to the extent of 45 IOnnes/annum and worth about 170 xl 0 6 yen were produced by SSF in Japan as early as in 1975-76 (Yamada,1977). About 7.2 tonnes of cellulases were exported to West Germany and Australia from Japan in 1967-68 (Toyama,1969), thereby indicating its leading status in cellulase production by SSF system. An important potential application of cellulases is in the production of glucose. ethanol. high fructose syrup and other feedstocks From agro-industrial cellulosic residues and wastes (Emerts and Katzen, 1980; Lonsane and Ramakrishna, 1989). Extensive R&D efforts have been put up in last 20 years to produce the enzymes by microbial fermentations and thousands of publications as weil as patents are avaiJable on the production and application of the cellulases (Ryu and Mandels, 1980; Mandels, 1982; Frost and Moss, 1987). However. no commercial exploitation has emanated from these efforts because of high cost of cellulases even when these are produced by SSF system. For example, the cost of the enzyme was shown to comprise nearly 50% of the outlay required to produce sugar from corn SlOver (Perez el al. 1980). 139 Lignocellulosic residues/wastes solid substrat The agro-industriallignocellulosic residues/wastes form a most important renewable reservoir of carbon for a variety of vitally important chemica1 feedstocks and fuel in the O\'erall economy of any country. Their unlimited availability and environmental pollution potential. if not disposed-off properly, dictate renewed efforts for their efficient and economic uti lization. It is well known that the selection of appropria te and highly potent microorganism. use of cheaper and efficient substrate, selection of bioreactor, employment of standardized process parameters, characteristics of the enzyme produced, extent of downstream processing as weil as waste treatment, inocuJum development technique, degree of colonization of the substrate. and efficiency of each unit operations of the process are of vital importance in determining the economics of the process (Kumar and Lonsane, 1989; Ramesh and Lonsane. 1990; Mitchell and Lonsane, 1991; Roussos el_al. 1991 a,b,c; Lonsane and Krishnaiah, 1991). Criteria for celluJolytic microorganisms selection Efforts were, therefore, initiated for screening of potent and most appropriate microorganisms for cellulase production in SSF system. A large number of cultures from various fungal genera and species were screened and Trichoderma harzianum CCM F-470 was selected based on four different criteria, i.e., rapid apical growth which leads to higher degree of colonization of the substrate, good sporulation capability which is vital for uniform distribution of the culture in the moist solid medium during inoculation, rapid groWlh of the culture which facilitates fermentation in non-aseptic conditions and higher enzyme production ability (Roussos and Raimbault, 1982). The kinetics and the ratios of two different cellulosic enzyme activities on various solid substrates in colurnn fermenter were investigated in the continuation of these renewed efforts. These results are reported in the present communication due to their importance in effecting economy in the enzyme production cost. The data also allow the production of tailor-made activities of different celluJosic enzymes which may prove useful in efficient hydrolysis of different lignocellulosic materials. Natural MicroOora of sugarcane bagasse The microbial loads on fresh bagasse and after storage for 15 days in the normal conditions of storage in the sugar mill yard are presented in table 1. The data indicate tremendous increase in the microbial population in the bagasse stored for 15 days. For example, the total bacterial, total fungal and cellulolytic fungal counts were about 715, 917 and 2218 times over those of the fresh bagasse. Such high microbial counts will be disastrous in the fermentation process without sterilization. lt is, therefore, essential to sterilize the substrate before use in the fermentation process. The pretreatment of the substrate also leads to many advantages. For example, it is found to be efficient in killing a Jarger microflora present naturally on the substrate. No contamination of the medium by bacteria, yeasts and other fungi was observed during the entire course of fermentation in the cases when the medium is based on pretreated substrate. in spite of the use of non-aseptic conditions beyond the moist medium autoclaving stage. The contamination control was probably aided by the use of large inoculum (3 x 10 7 spores / g SOM) which probably allowed preferrential groWlh of T. harzianum and imparted it the status 140 of dominance. Other beneficiaJ changes due to pretreatment of the substrate are: 1) reduction in crvstallinitv of the cellulose due to formation of amorphous celluloses. 2) gelatinization of s~ch pres~nt in the substrate, 3) swelling of the substrate, 4) hydration of the substrate. 5) homogeneous distribution of mineral-salt media and a horde of other benefits (Tanaka and Matsuno, 1985). Table 1. Natural microflora of sugarcane bagasse. Sarnple Microbial load 1 g material Moisture % Total Total Cellulolytic bacteria fungi fungi Immêdlately as It eXit l'rom sugar uùll 44.4 2.63 X 106 \.08 X Hf 2.66 X 10> After 15 days storage in sugar uùll yard 59.6 1.88 x 109 9.90 x 1<J6 5.90 x 106 Untreated and pretreated substrates The necessity for pretreating the lignocellulosic residues and wastes to improve the accessibility of ceIJulose ta microbial attack has been weil established (Tanaka and Matsuno, 1985). A number of different physical and cheuùcal pretreatments. either individually or in combination, have been developed and include bail uùlling, compression uùlling, grinding, cryomiling, gamma ray dosage, microwave irradiation, steam explosion. rapid depressurization and autahydrolysis by vanous chemicals such as acids, alkalies. solvents, gaseous ozone etc .. (Tanaka and Matsuno, 1985). Most of these pretreatment methods are impractical at larger scaJe and highJy cost-intensive due to various reasons such as longer pretreatment lime. high energy requirement. need for using specific equipments or machinery and occurance of undesirable side reactions. The possibility of combining sterilization of the substrate and its pretreatment was, therefore, conceived in the present studies. The moiSI solids with 50% moisture were transferred in 100 g moist weight quantity in beaker for autoclaving at J21°C for 20 min. The results of the comparative enzyme production on untreated and pretreated substrates in column fermenters are depicted in table 2. The CMCase and FPA fractions were present right From 0-21 h in the medium without any pretreatment probably due to their presence in the substrate used for fermentation. These might have been formed during the storage of sugarcane bagasse in the sugar mill and institute premises before employing it for fermentation it the present studies. These enzymes. in contrast, were totally absent upto 21 h in the medium based on pretreated substrate. Obviously. the enzymes initially present on the substrate were destroyed during autaclaving of the moist medium. The enzyme production beyond 21 h was, however, at l'aster rate in both the media. The peaks in enzyme production were achieved at 30 h in untreated medium as compared to lhose al 48 h in the pretreated medium. However, the peak values of the enzymes in case of pretreated substrate were higher by about 2.7 and 141 Table 2. Comparative production of ceDulases in column fermenter by T. hanianum on treated and untreated substrates Fermentation time,h Medium pH A B CMCase production A B FPA production B A 0 6.2 5.4 1.1 0 1.1 0 6 6.2 5.4 0.7 0 1.1 0 21 4.8 53 0.1 0 2.8 0 30 5.9 4.2 45.9 31.6 7.1 3.4 44 7.4 5.1 35.4 124.6 5.4 10.9 48 7.7 5.1 14.1 125.8 3.5 12.8 67 83 5.5 7.6 12.4 2.8 11.2 A: Untreated substrate, B : treated substrate. The enzyme titres are expressed as IU 1 g SDM. Table 3. A typical fermentation data on the production of ceUnJases by T. harzianum at laboratory scale in colnmn fermenter under standardized parameters Attribute Unit Value Peak: value in CMCase IU/gSDM 204.4 Peak: value in FPA IU/gSDM 16.1 CMCase : FPA at peak: level Ratio 1: 0.08 Peak: enzyme production time h 48 Range of moisture content of the medium during fermentation % 68.3-73.9 Lowest pH in growth phase (at 28 h) 4.5 Highest pH during enzyme synthesis and Iiberation (at 48 h) 6.3 Contamination during fermentation Absent 142 \.8 times as compared to those on the untreated substrate. These values at 48 h fermentation were higher by about 8.9 and 3.7 times in case of the pretreated substrate. The microscopic examination of the sampies From both the fermentations. involving untreated and pretreated substrates, revealed extensive contamination by bacteria and yeast in the former case right From the begining of the fermentation. The groWlh rate of sorne of these contaminants, especiaJly the bacterial cultures, was much faster than that of T harzianum as the sampIes from laner phases of fermentation showed many bacterial cells and very few fungal mycelia or spores. Moreover, the mycelial cells of Tharzianum were noticed to be lysed probably by the contamÎnants or their products. The contamination of the order of 10 \\ cells of bacteria and yeast was a1so recorded by Pepe (1984) when sugar beet cosset was used without any autoclaving in large fermenter for prote in upgradation. The changes in the pH of the media, based on the use of untreated and pretreated substrates, showed interesting panem (Table 2). The pH dropped to 5.9 by 30 h and then increased to 8.3 by 67 h fermentation in case of untreated substrate. The general trend in pH drop and rise was similar in the medium based on pretreated substrate but the values were 4.2 and 5.5 at 30 and 67 h, respectively. It is interesting to note that the initial pH was much higher in case of the medium involving untreated substrate as compared to that of the medium based on pretreated substrate. Many times higher production of the enzymes by the culture in the medium based on pretreated substrate indicates that the heat treatment of the substrate in moist condition modifies it physically for imparting bener accessibility of the cellulose to microbial allack. Consequently, the substrate becomes more amenable to microbial growth and leads to improved production of the enzymes. The particle size reduction due to chopping of the bagasse might also have resulted in exposing larger surface area of the substrate to heat action and thus is partially responsible in making the substrate more accessible to the microorganism. Sugarcane bagasse alone as subslrale for T.harZ;anum growlh The manufacture of suc rose From cane sugar in the tropical countries results in the generation of large quantity of bagasse which are generally used as fuel in the sugar mil!. In another usage. the bagasses are depithed are the fibres thus obtained are used in the manufacture of paper. The sugarcane bagasse forms an excellent substrate in SSf processes. The results of the groWlh and metabolism of Tharzianum on bagasse in colmnn fermenter for 64 h under SSf system revealed that the conidiospores started germinating at about 10 h and the spore germination was \ 00 % by 20 h. The mycelial cells enveloped the substrate particles more or less fully by about 30 h. The moisture content of the medium during the course of fermentation was quite stable and ranged between 70.5 - 72.9 % (Fig. 3). Similar was the case for the kinetics of pH changes which were in the range of 5.9 - 6.3 during [mt 52 h fermentation. This is the fermentation period which led to maximum enzyme titres. Further continuation of fermentation beyond 52 h resulted in increasing the pH of the medium to 6.9 at 58 and 64 h fermentation. The data on the production of CMCase and FPA fractions indicated that no CMCase was produced upto 24 h, in contrast to the production of FPA at slower rate right from the start of fermentation (fig. 4). The rates of production of these enzymes were, however, faster between 24 to 44 h and about 80 % of the total enzyme was formed during this period. The peaks in enzyme titres were achieved ar 52 h for both the enzyme and their levels decreased if 143 Table 4. Large scale production of ceUuJases by T.harzUmum in Zymotis and laboratory scale colnmn fermenter run in paraDel Fermentation Moisture content of pH of the medium CMCase production FPA productim the medium, % time, h A B A B A B A B 0 71.0 713 5.5 5.6 0 0 0 0 10 72.6 70.5 5.6 5.5 0 0 0 0 22 71.5 70.4 4.8 53 0 0 0 0 26 71.4 71.5 4.2 4.7 0 0 0 0 30 72.4 71.6 4.5 4.4 93 3.1 1.2 0.8 34 72.6 72.1 5.0 4.7 29.9 15.1 3.4 3.2 46 733 73.1 5.7 5.6 74.1 62.2 5.6 4.4 48 72.7 76.6 5.8 5.8 74.2 71.8 8.1 5.5 A : Zymotis, B : laboratory scale column fermenter. The enzyme titres are expressed as IU/g SDM 100 O~~:::::::~:=:::::::==~==:=;::===::;:::==:::::==~O 40 GO Time d~~urs) o 20 Figure 4. CMCase and FPA cellulase production 144 by T. harzianum the fennentation was continued further (Fig. 4). The ratios of CMCase and FPA ranged between 8.3-10.0 during 44-52 h fennentation. Combination of bagasse and wbeat bran as substrate The use of sugarcane bagasse and wheat bran at the ratio of 80 : 20 in the column fennenter and under the fennentation parameters such as 20 g mediwn in the column, 72 % initial moisture content, 28 ± 1°C incubation temperature and aeration at 5 1air 1 h 1 column in the production of cellulolytic enzymes by T harzianum showed entirely different pal1erns in the enzyme synthesis during initial period of fennentation. The CMCase and FPA fractions were not fonned upto 20 h fennentation. Subsequently, the rate of production of these enzyme activities was at faster rate between 28-48 h (Fig. 6). The peak in enzyme titres was allained at 48 h in both the cases. The ratios of the se activities were about 6 upto 28 h but these changed to 11.0-12.3 in the subsequent period. The kinetics of moisture content of the medium showed that it increased graduaJly during the entire course of fennentation and was 4-5 % higher at the end of fennentation as compared to the initia! value at 0 h (Fig. 7 ). The kinetics of the pH changes are interesting. The pH decreased sharply from 5.8 to 4.8 between 12-28 h, the period which involves active groW'lh of the culture. The bagasse appears to have strong buffering action as the pH was not reduced to a value less than 4.8. Subsequently, the pH increased to 6.3 between 28-65 h fennentation, the period which corresponds to enzyme biosynthesis and its release in the medium. In contrast, the pH was nearly stable at the initial value of 5.8 during 0-12 h, the period which involves the gennination of the conidiospores. The data indicate that there is no need for pH control during the fermentation as the above kinetic changes in the pH seems to be helpful. Typical fermentation data at laboratory scale The production of cellulases by T harzianum in pretreated moist medium in laboratory scale column fennenter of 18 g working capacity under standardized parameters indicated that the maximum enzyme production was achieved at 48 h (Table 3). The peak values of enzymes were 204.4 and 16.1 lUI g SOM of CMCase and FPA fractions, respectively, thereby leading to the ratio of 1 : 0.08 at 48 h. These values are much higher than those in the pretreated moist medium under non-standardized parameters (Table 2), thereby indicating the efficacy of parameter standardization performed in this process (Roussos, 1987). The continuation of the fennentation under standardized parameters beyond 48 h. however, resulted in reduction in the titres of the enzymes, which was more drastic in case of FPA fraction. The moisture content of the mediwn during the course of fennentation ranged between 68.3 - 73.9% (Table 3). The pH of the medium decreased gradually in the initial 28 h fennentation from the initial value of 5.8 to 4.5. In the subsequent tennentation period, it started increasing and reached the value of 6.3 at 48 h. This confinns the trend of pH changes during groWlh and enzyme production phases and its utility in monitoring the fennentation as stressed earJier (Roussos el al., 1991 a). The microscopic examination of the fennenting solids at different intervals during the entire fermentation period has not revealed any contamination by bacteria, yeast and fungi other than T harzianum. The groWlh of the culture was found to be uniform throughout the solid mass in the fennenter. 145 The higher production of the enzymes at 48 h without the need for mainlaining aseptic conditions during fermentation, the ratio ofCMCase : FPA fractions ar 1 : 0.08, an absence of any contamination during the fermentation due to combinalion of substrate pretreatment \Vith autoclaving of the medium, the cheapness of the substrate and the homogeneous growth of the culture in the medium probably due to uniform distribution of the spore inoculum during inoculation collectively indicate the high potenliaJ of the system for economic exploitation at industrial scale. Hence, the scale-up trials were undertaken. Scale-up in Zymotis The data on the production of cellulases in Zymotis charged with 41.4 kg moist medium and in laboratory scale column fermenter run in parai leI, aJong with the changes in pH as weil as % moisture of the media are presented in Table 4. The titres ofCMCase and FPA fractions were at peak values at 48 h in both the fermenters except for that of CMCase in Zymotis at 46 h. The production of both these components of the cellulolytic enzyme were initiated ar 30 h and their accumulation increased steadily till the peak values were attained. The ratios of CMCase and FPA fractions at peak levels were 1 : 0.075 and 1 : 0.077 in Zymotis and parallel column fermenter, respectively. The ratio increased to 1 : 0.11 at48 h in Zymotis due to increase in FPA production between 46-48 h but no change in CMCase titre. The production of both of these enzymic fractions was higher and also at a faster rate in Zymotis as compared to those in the parallel column fermenter (Table 4), thereby indicating that the conditions were more favorable at the large scaJe than those at smaller scaJe fermentation. Sirnilar results were also reported earlier for other products in SSF system (Lonsane el al., 1991 ; Saucedo-Castaneda el al., 1991). The moisture content of the medium during entire period of fermentation was similar in both the fermenters and ranged between 71.0 - 73.3 and 70.4 - 73.] % in Zymotis and parallel column fermenter, respectively (Table 4). The pH of the medium at the start and also at the end of fermentation was same in both these fermenters. However, the drop in pH during the initial growth phase and the increase in pH during the subsequent enzyme production phase were faster in Zymotis (Table 4). This probably explains the faster rate of enzyme production as weil as its accumulation in Zymotis as compared to those in parallel column fermenter. Comparison of enzyme production at laboratory and large scares The production of CMCase and FPA fractions atlarger scale in Zymotis was 36.28 and 50.03 % of those produced by the culture in column fermenter at laboratory scale under standardized parameters (Tables 3 and 4). The production of such low enzyme at larger seale in Zymotis indicates sorne lacunae or deficiency at larger scale fermemation. The close similarity in the profiles of fermentation parameters, such as moisture content and pH of the medium, in the laboratory scale column fermentation under standardized parame ter and Zymotis rules out the possibility of any role played by fermentation paramelers in giving lower yields atlarger scale. The temperature, medium compositions, inoculum quality, inoculum ratio are also similar in both the cases. The deficiency in the performance of Zymotis or its design features in obtaining lower enzymes at Jarger scale is also ruled out as 146 the production of the enzyme in Zymotis and column fennenter, which was run in parallel to Zymotis, were also 35.15 and 34.27 % as compared to those in the laboratory scale column fennentation under standardized parameters (Table 3 and 4). In addition, the enzyme titres in Zymotis and the parallel column fennenter were also simiJar (Table 4). The analysis of the who le process and process methodology indicate that the only difference between laboratory scale column fennentation in Zymotis and the parallel fennentation in coJumn is the change in the substrate pretreatment method. The substrate pretreatment was carried out by charging the moist medium in 100 g quantity in a beaker for autoclaving at 121°C for 20 min in case of laboratory scale column fennentation under standardized parameters, in contrast to the use of 6 kg moist solid medium in cylindricaJ alum.i.n.ium vessel at 121°C for 60 min. The depth of the medium in the aluminium vessel during autoclaving was much higher (60 cm) as compared to that in the beaker (10 cm) with the use of 100 g moist medium in the laboratory scale process. In fact, it is for this reason that the autoc1aving time was extended from 20 to 60 min in the larger scale process for giving more time for heat transfer. The results, however, indicate that the heat transfer during autoclaving al larger scale is less than that achived at 1aboratory scale. Probably the temperature achieved al the centre of the moist medium held in the cloth sac during autoclaving at large scaJe was Jess than 121°C or the heating of each particle of the medium was not for 20 min at 121°C. The autoclaving of the medium has also been specified as problematic unit operation during scale-up of submerged fennentation processes (Bank., 1984). It is feh that the same productivity would be possible to achieve at larger scale either by increasing the autoclaving temperature or time. Both ofthese approaches are, however. energy and cost intensive. Use of perforated aluminium trays for substrate pretreatment-cumautoc1aving and the bed depth of about 10 cm, as generally employed in tray fennentation processes (Lonsane el al., 1985; Ghildyal el al., 1981), may provide a simple and economic approach 10 overcome the problem. Bibliography ACEBAL, c., M.P. CASTILLON, P. ESTRADA. 1. MATA, E. COSTA. J. AGUAOO, O. RaMERa and F. JIMENEZ, 1986. Enhanced cellulase production from Trichodermo reesei QM 9414 on physically treated wheat slraw. Appl. Microbiol. Biotechnol. 24 : 218-223. ACEBAL, c., M.P. CASTILLON, P. ESTRADA, 1. MATA, J. AGUAOO and O. RaMERa, 1988. Production of cellulases by Trichodermo reesei QM 9414 in batch and fed-batch cultures on wheat straw. Acta Biotechnol. 6: 48i-494. ALLEN, AL. 1983. Enzymic hydrolysis of cellulose to fermentable sugars. In: Wise. O.L. (Ed). Liquid fuel developments. pp. 49-64. CRC Press, Boca Raton, Florida. BANK, G.T., 1984. Scale-up of fermentation processes. In A. Wiseman (Ed.). 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SAUCEDO-CASTANEDA, G., B.K. LONSANE, M.M. KRlSHNAIAH, lM NAVARRO, S. ROUSSOS and M. RAIMBAULT, 1991. Maintenance of heat and water balances as a scale-up criterion for the production of ethanol by Schwanniomyces castel/ii in a solid state fermentation system. Process Biochem. 27: 97-107. STERNBERG, D., 1976. Production of cellulases by Trichoderma. Biotechnol. Bioeng. Symp. 6 35-53. 149 TANAKA, M. and R. MATSUMO, 1985. Conversion of lignocellulosic materials to single·cell prote in (SCP) : Recent developments and problems. Enzyme Microb. Technol. 7 : 196-206. TOY AMA, N., 1969. Application of cellulases in Japan. [n : Gould, R.E. (Ed.), Cellulases and their applications. Advances in Chemical Series, Vol. 95, pp. 259·390. American Chemical Society Publications, Washington, D.C. TOYAMA, N., 1976. Feasibility of sugar production from agricultural and urban cellulosic wastes with Trichoderma viride cellulase. Biotechnol. Bioeng. Symp. 6 : 207·219. TOY AMA, N. and K. OGA WA, 1978. Cellulase production of Trichoderma viride in solid and submerged culture methods. ln : Ghose, T.K. (Ed.), Bioconversions of cellulosic substrates for energy, chemicals and microbial prote in • Symp. Proc. (1 st), )977, Indian Institute of Technology, New Delhi. VIDAUD, c., S. ROUSSOS, M. RAIMBAULT and F. DESCHAMPS. 1982. Effet de divers prétraitements sur l'accessibilité de la cellulose de la paille de blé aux cellulases de Trichoderllla harzianum. Cah. ORSTOM sér. Biol. 45: 17·23. YAMADA, K., 1977. Bioengineering repon . Recent advances in industrial fermentations in Japan. Biotechnol. Bioeng. 19 : 1563·1621 150 BIOTECHNOLOGICAL MANAGEMENT OF COFFEE PULP Sebastianos Roussos, Isabelle Perraud-Gaime & Sylvain Denis Laboratoire de Biotechnologie, Centre üRSTüM, BP 5045; 34032 Monrpellier Cedex 1. France General inroduclion Agro-industrial residues/wastes are generated in large quantities throughout the world. Their non-utilization resuhs in loss of valuable nutrients and envirorunental pollution (Zuluaga. 1989). Their bener utilization by biotechnological means assumes social, economic and industrial importance. Considering these facts, üRSTüM parlicipated into a scientific collaboration with Universidad Autonoma Metropolitana (UAM), Mexico. for the development of biotechnological processes for bener utilization of agro-industrial byproducts/wastes, especially the coffee pulp (Viniegra el al, 1991). CoITee pulp. generated to the extent of 40% in the fermentation of coffee berries (Zuluaga, 1989), poses many problems in the coffee producing tropical countries. Its disposai in nature, without any trealment, causes severe envirorunental pollution, due to putrefaction of organic maner (Zuluaga, 1989). Hence, the possibility of utilizing coffee pulp in the biotechnological processes for production of different metabolites was investigated thorought by Roussos el al (1993). Corree pulp is the main byproduct on coffee explotation industr)'. Two tons of green coITee produces one ton of coffee pulp (dry matter). Ils production on world scale rised 2.400.000 tons (two million four hundred thausand tons) in the harvest cycle from 1986 to 1987. Coffee pulp is essentially composed of carbohydrates, proteins, aminoacids, mineraI salts. tannins, poly phenols and caffeine. The last {WO compounds are reported to he antiphysiological factors on animal feed. Hence. coffee pulp has to follow a preliminary treatment before is used. Moreover, this byproduct can occurs in the nature and spoiling hardly the environmenl. Researcb on this field is been canied out to study its further utilization in biotechnological processes. Several alternatives were founded and developed these decades to avoid and minimized the environmental impact due to coffee pulp. Previous assays of yeast culture on coffee pulp were realized by The Colombian Research Center CENICAFE in 195\. conducted by Dr. CaJie. Later. INCAP and ICAITI in Guatemala also reported research on this malter. However. the most important contribution was the happening of the First International Conference on CoITee Byproducts Utilization for Animal Feed and its furtherlndustrial Applications, wellcomed by the Costarican Center CATIE in 1974. More recently. in 1989 rNMECAFE in Veracruz. Mexico and in 199J CENICAFE, Manizales. Colombia. there are organized an International Symposium on Biotechnology of Agroindusty of Coffee. called SIBAC; where sessions and contributions presented original research on the potential of coffee pulp byproducts (aquaculture and feed). Aquaculture: Fish farming could be a way to produce animal protein using locally available feedstuffs but taking into account present market limitations (Ramos-Henao. 1988). Sorne 151 work has been do ne on feeding coffee pulp to Tilapia (Garcia and Baynes, 1974). carp and catfish (C1arius mossenbicus) as indicated by Christensen (1981). The level of coffee pulp used in the experirnentaJ diets was c10sed to 33% without negative effects on the growth rate and yields of the fish. Lagooning may be also used as a secondary water treatment process, after anaerobic primary treatrnent of spent waters in the coffee mill. Ruminant nutrition: Proximate composition of coffee pulp shows a relative low nutritive value due to high level ofwall materials, lignin and also due to the presence of cafeine, tanins and chorogenic acid. Therefore, the use of raw coffee pulp is been suggested to be lower than 20% in ruminant diets (Ruiz and Ruiz, 1977; Vargas et al. 1982; Abate and Pfeffer, 1986). High raw coffee pulp intake has been associated to negative nitrogen balance because of the cafeine diuretic effect (Cabezas, et al. 1974). Coffee pulp silage seems to correct this problem probably because of cafein leaching in the silage liquor (Cabezas et al. 1976). On the other hand, solid-state culture of fungal organisms such as Penicillium roque/orrii or Aspergillus niger may reduce to less than 10% the level of cafeine in coffee pulp, leaving a probiotic activity in the ftmgal biomass as indicated above (Tapia et al. 1989; Campos-Monliel, 1995). Therefore, despite the nutrilional limitations of raw coffee pulp, solid-state fungaJ culture and ensiling (the two step fermentation process discussed above) may increase the ruminant nutritional and market value of this material. This is an interesting feature which remains to tested in vivo. Recent work done int the Biotechnology Laboratory of Centre ORSTOM, Montpellier (France) and UAM (Mexico) has shown that it is possible to keep and improve the biochemical quality of coffee pulp by using a mixture of selected strains of lactic bacteria and filamentous fungi. Solid-state fermentation of this material yields a decafeinated product which can be dried or rensiled (Roussos et al., 1989; Perraud-Gaime. 1995) Sorne HPLC measurements suggest that a major fraction of phenolic compounds is broken down (PerraudGaime, 1995). On the other hand, work by Antier et al. (1993 a,b), has shown that coffee pulp is en excellent substrate for pectinase production by selected strains of Aspergillus niger (Boccas et al. 1994). The solid residue after such fermentation is done has been found to have probiotic effect when assayed in vitro by Tapia et al. (1988). This probiotic effect is apparently linked to a water soluble enhancement growth factor present in fungal biomass and acting on rumen cellulolytic bacteria (Campos-Montiel and Viniegra-Gonzalez, 1995, Islas et al. 1995). ln the first transperency the New alternative for the Biotechnological Upgradation of Coffee Pulp is presented. Fresh Coffee Pulp is subjected 10 Lactic Acid Fermentation and Coffee Pulp Silage thus obtained can be used as substrate for solid State Fermentation system for the production of Probiotics, enzymes, animal feeds and phytohormona. Content: During these presentation 1 will be covering mainly about production of coffee pulp, its biochemical composition, natural microflora, oriented silage, caffeine degradation and pectinases production by filamentous in SSF system by filamentous fungi like Aspergillus and Penicillium species. Word production: The data on the Worl Green colTee and Coffee Pulp Production for the period 1989-90 are presented in Table 1. The World ColTee Production is five and a half 152 million Ions. For every Ion of colfee produced half a ton of colfee pulp is generated in humid process. This is applicable only when the colfee is processed by humid process. In India the total production of coffee during this period was one hundred thirty thousand (130,000) tons. Ihe exacl quanliry of colfee pulp produced is not definitely known because both humid and dry process are used. Coffee pulp chemical composition: The chemical composition of coffee pulp is presented in Table 2. CoITee pulp is essentially composed of carbohydrates, protein. amino acids. minerai salts, tannins, and caffeine (Zuluaga 1989). The last two compounds are reported to be antiphysioJogical factors for animal consumption (Bressani et al. 1972).. The composition of soluble sugars present in colfee pulp are presented in Table 3. These represents about 23% of total solids by dry weight (Zuluaga, 1981). The presence of protein, sugars, minerais and water in colfee pulp obtained by humid process. offers itself as an excellent substrate for the growth of microorganisms. If it is not utilized immediately it causes environmental pollution particularly in the ri vers surrolU1ding the factory processing area. ln order to conserve the nutritional factors present in the colfee pulp and to maintain its qualiry throughout the year we have used sillage process (Perraud-Gaime and Roussos. 1997). NaturaJ Microflora of coffee pulp: ln the first instance the natural microf1ora of colfee pulp was evaluated and the data is shown in Table 4. Bacteria represents nearly 95% of the rnicrof1ora whereas filamentous fungi and yeast population was only about 5% (GaimePerraud et al. 1993). Figure 1 gives an idea of groups of microorganisms (bacteria, yeast molds) present in Mexican and Columbian colfee pulp with its nutritional capacities such as amylolytic, cellulolytic, pectinolytic and lactic acid bacterial population. LONG TIME CONSERVATION OF COFFEE PULP 1- Preservation of coffee pulp by ensilage: Influence of bioJogicaJ additives CoITee pulp, as it is generated, contains 80-85% moisture (Bressani el al, 1972), in addition to appreciable quantities ofsucrose, proteins. amino acids and other nutrients. Ail these factors and nutrients allow various microf1ora to develop quickly on the coITee pulp and the development of the microorganisms cause the putrefaction of coffee pulp (Gaime-Perraud et al, 1993 ; Roussos et al, 1995). It is also not practicable to uti 1ize the coITee pu 1p immediately, after its generation during coffee berry treatment mainly because the season of coITee berry processing lasts for 3-5 months. During this season, the industry cannot divert attention to this waste, as its priority is focused on the quality of coITee seeds during the entire season. Moreover, quick dehydration of the colfee pulp is impracticable. conside ring the huge quantity of the waste, high energy requirement, larger capaciry of machinery needed and heavy investment on space and building, not only for dehydration, but also for stocking of the dehydrated pulp. till its utilization. Ensilage of colfee pulp, for its preservation and improvement of feed value, is one of the avenues for value-added utilization of coITee pulp. Ensilage, a quick anaerobic process involving !actic acid bacteria, has been extensively used for preservation of forage in the 153 temperate regions. Il allows the prevention of putrefaction of the forage with nurumum degradation of organic maner. The process is quicker and it also improves the nutritive quality of the forage (Mc Donald et al, 1991). Ensilage factors: A number of factors are of vital importance in obtaining a good silage. The substrate to be ensiled should have 30-40% dry maner, should be compactable to the desired level, amenable for anaerobiosis and contain utilizable sugars in sufficient quantities (Bertin, 1986). Il must also have the colour, which is most nearer to the raw material, the fruity aroma and slightly acidic taste. In terms of chemical characteristics and achievement of the organic maner stability, the ensilage should involve a minimum loss of dry maner and the resulting sil age should have a pH value lower than 4.5, higher than 3% lactic acid, but less than 0.5 and 0.3% acetic and butyric acid, respectively (Mc Donald et al, 1991). A nurnber of chemical and biological additives are mixed with the substrate for improving silage or reducing fermentation time. [n the case of biological additives, a lactic acid bacterial inoculurn is added, as a minimum of J0 5 lactic acid bacteria per g dry maner is required (Gouet, 1994) to convert the carbohydrates into [actic acid, but not into butyric acid. Enzymes are also added, when the rate of assimilation of sucrose by the endogenous laclic acid bacteria is slower (Bertin, 1986). Ensilage is also practiced in tropical countries, despite the problems in terms of temperalUre. humidity and rains. Consequently, the rate of ensilage is slower, putrefaction is common and there is need to use a nurnber of additives. A nurnber of reports have been produced on ensilage of coffee pulp (Bohkenfor and Fonseca, 1974; Murillo, [978; Carrizalezand Gonzalez, 1984). But, most ofthese are associated with the development of the ensilage tecl:mique or the effect of chemical additives on the process. For example, Murillo (1974) compared the silage of coffee putp, obtained by natural microf1ora based fermentation, with that involving the use of molasses or organic acids as additives. After 90 days of ensilage, the loss of dry maner was as high as 26.8%, in the case of the use of organic acids as additive, though it allowed to anain a pH of less than 4.0. Caffeine content of the drained water was reported to increase significantly, in the case of the use of organic acids, probably because it became more soluble in acidic pH. Table 1. Comparative physico-chemica[ characteristics of the coffee pulp ensiled using natural microfiora and biological additives (Perraud-Gaime. 1995). Ensilage of coffee pulp (28 days) Parameters Initial Without Natural L. pian/arum Commercial pulp additives microf1ora A6 inoculum Moisture 62,55 61,63 66,78 66.78 66,19 pH 4,44 3,90 3,91 3,92 4,1 J DM losses (%) 1,41 0,80 1,73 0,38 Lactic acid (%DM) 0,00 2,39 3,35 2,14 0,08 Acetic acid (%DM) 0,00 0,29 0,68 0,48 0.05 Reducing sugars (%DM) 4,72 4,85 4,56 3,67 8,32 Caffeine (%DM) 1.04 0,95 1,02 0.93 0.90 154 The ensilage of coffee pulp was investigated by Perraud-Gaime (1995) with respect to the microbiology and biochemistT)' of the process, aJong with the evaJuation of biologicaJ additives, for improving the process and also the quality of the silage. Accordingly, the studies involved a) aJlowing the endogenous lactic microt1ora to grow on coffee pulp for using the fenuented mass as inoculum for the next batch, b) use of monoculture of Lactobacillus plantarum A6 as a biologicaJ additive (Giraud et aJ. 1991) and c)the use of commerciaJ inoculum as yet another biological additive. The latter contained two lactic bacteria and an enzyme complex. Data on the influence of three biological additives on the ensilage of coffee pulp for its preservation show that the endogenous microt1ora of the coffee pulp is efficient enough to produce good quality silage, with acceptable levels of organic acid, dry matter loss and fmaJ pH. The use of inoculants, as biological additives, showed the efficiency of natural microt1ora grown on coffee pulp and the monoculture of Lactobacillus plantarum A6 in improving the physico-chemical characteristics of the silage, though commerciaJ inoculum was not efficient, due to several reasons (Perraud-Gaime, 1995). Degradation of caffeine was absent in aJl the cases. Cellulases as a biologicaJ additive showed increased sugar production during ensilage. The results on the kinetics of different microflora developmenl and physico-chemical characteristics during ensilage provide the insight into the microbiology and physiology of the process and point out a number of possibilities for improving the ensilage process as weil as the quality of the silage (Perraud-Gaime & Roussos, 1997). Data allow us to conclude that ensilage is a good technique for preservation of wet coffee pulp. The endogenous lactic acid flora of dry coffee pulp is sufficient enough to produce a good quality of silage (Table 5). However, addition of biologicaJ additives, such as lactic acid bacterial inoculants and enzymes, allows the improvement of the quality of the silage, in tenus of increesing of lactic acid production, without concomitant production of volatile organic acids and ethanol. Caffeine is nOI degraded during Ihe silage and hence il is necessary to decaffeinate the coffee pulp with appropriate fungi by solid state fenuentation (PerraudGaUne and Roussos, 1997), if the ensiled coffee pulp is to be used for animal feeding, as caffeine has antiphysiological effects (Bressani et al, '972). 2,- Selection of filamentous fungi for coffee pulp decaffeination in SSF It is of economic and industrial importance to note that only 5.8% of the solids of the coffee berry result in the ultimate coffee drink and the remaining 94.2% fonus water and various byproducts (Zuluaga. 1989). Among the latter, the coffee pulp is the maximum and represents 40% of the coffee berry in wet form (Tauk, 1986), corresponding to 29% of dry matter (Bressani et al, 1972). This large quanlity of the coffee pulp poses problems of disposai 10 coffee berry producers, due to putrefaction and causes environmental pollution if nol disposed after appropriate lreatment (Zuluaga, 1989). Due to ilS high organic matter content, coffee pulp can be utilized for beneficial purposes and intensive research on this lopic has been carried out al üRSTüM (Roussos el al, 1995) and also in collaboration with Universidad AUlonoma Metropolitana (UAM-I), Mexico (Viniegra-Gonzalez et al, 1991). 155 Direct use of colfee pulp in animal feeding poses problems, due to its chemicaJ composition (Viniegra-GonzaJez el al, 1991). For example, the colfee pulp of Co./fea arabica contains approximatelly 1% caffeine and has antiphysiological effects on the animaIs (Braham el al. 1973; Cabezas el al, 1974, 1976; Vargas el al, 1982). il is, therefore, necessary to decaffeinate the coffee pulp, before its use as animal feed. Moreover, the colfee pulp gets putrified. because of its high content of water and, hence, needs preservation by appropriate economic technique. At ORSTOM, Montpellier, the techniques of ensilage and fimgal degradation of caffein by solid state fermentation (SSF) have been selected for preservation and decaffeination of the colfee pulp, respectively, because of their economic character. If these two techniques are applied in succession, it is of vital importance that the decaffeination by fungi is achieved before the formation of conidiospores. In the case of conidiospore formation, it will be essentialto sterilize the decaffeinated coffee pulp, before ensiling. However, mycelial ceIls of fimgi can be elirninated during ensiling and hence sterilization step can be avoided to achieve economy (Perraud-Gaime, 1995). Isolation of new fungi strains: Isolation, purification and conservation of filamentous fimgi capable of degrading caffeine was carried out as shown in figure 2. A total of 350 fW1gi have been isolated From colfee domains (colfee plants, soils of coffee plantation, colfee byproducts, fermenting colfee berries, etc.) during the research at ORSTOM and UAM (Aquiahuatl el al, 1988; Viniegra-Gonzalez el al. 1991; Roussos el al, 1995). From this collection, a total of 8 filamentous fimgi, representing two strains of Penicillium and 6 strains of Aspergillus, were selected for use in the present studies, based on their higher capacity to degrade caffeine to the extent of90 to 100% in liquid culture (Roussos el al, 1989). One of the Penicillium strains selected (V33A25) showed negative effect on caffeine degradation, upon the addition of inorganic nitrogen to the medium in SSF process (Roussos el al, 1994). Selection of filamentous fungi to Caffeine degradation: The objective of this study was to select one or more of the filamentous fimgi to grow in SSF and to degrade caffeine to the extent of 80%, before the initiation of conidia formation. Work was also carried out to develop a simple criterion, to correlate grovith of the fimgi, degradation of caffeine and sporulation time, so that it can be used to stop fermentation at the most appropriate stage. The ability of seven fungal isolates which can degrade calfein totally is shown in table 6. They belong to the genus of Aspegillus and Penicillium. Of these, only two strains are belonging to the genus of Aspergillus species (VI2A25) and Penicillium species (V33A25) was selected to study the kinetic and biochemical pathway of caffeine degradation. Decaffeination of coffee pulp in Solid State Fermentation, to eliminate its antiphysiological effects on animais, was studied by aerobic fungal solid state fermentation, prior to the stage of initiation of conidiospore formation (Perraud-Gaime, 1995). Comparative data on performance of two strains of Penicillium and six strains of Aspergillus spp., selected for their high ability to degrade, indicated the potential of Penicillium sp V33A25 for caffeine degradation in aerobic solid state fermentation, before the initiation of sporulation by the culture. Kinetic studies pointed out that the evolution of CO 2 is the reliable criterion for the determination of the phase of fermentation, caffeine degradation, increase in medium pH and initiation of sporulation, without taking sample and subjecting it to analyses or disturbing the 156 fermentation. These advantages are not available, if rise in pH the mediwn is selected as a criterion. Arnongst 7 different factors, the fermentation temperature, level of CaC!" in the mediwn and autoclaving or non-autoclaving of the mediwn exhibited srrong effects on the initial time of sporulation, extent of CO 2 evolution, pH of the mediwn and caffelne degradation (Figure 3). The data allow to envisage the use of mixed culture of lactic acid bacteria and filamentous fungi for decaffeination and ensilage of the coffee pulp, or in two stage fermentation, involving any of the simpler order. Table Il. Comparative data on growth and metabolism of tbe filamentous fungal cultures in column fermenters under solid state fermentation (Perraud-Gaime. 1995). Time of initiation Respirometr Caffeine Lag CO 2 Strains phase h production y coefficient degradation of sporulation h') at 30 h. % ml/g MSI 0,34 Penicillium sp. 12,5 115 91 32 h V26A25 Penicillium sp. 11,5 0,34 94 95 30 h V33A25 Aspergillus sp. 13,0 0,30 32 h 100 80 CI6A25 Aspergillus 130 0,29 82 28 h sp. 10.5 VI2A25 Aspergillus 0,34 65 87 32 h sp. 17,0 C17825 Aspergillus sp. 20,0 65 0.26 12 42 h CI1825 Aspergillus sp. 11,0 0,30 94 100 32 h C28825 Aspergillus sp. Il,0 85 0.34 79 30 h C23825 Il cao be concluded that it is possible to decaffeinate the coffee pulp in 30 h under aerobic conditions by using selected fungal culture, i.e.. Penicillium sp. V33A25 in solid state fermentation, before initiation of the sporulation by the strain. It is also not necessary to sterilize the substrate. lt is, therefore. possible to envisage the inoculation of the coffee pulp with mixed culture of lactic acid bacteria, for the ensilage preservation of coffee pulp. along with the selected filamentous fungi, for degradation of the caffeine. ft can lead to decaffeinated and stabilized coffee pulp, which is suitable for animal feeding (Perraud-Gaime. 1995). ft is also possible that the stages of the fermentation cao be observed visually on the computer, through respirometric parameters, without removing the sample and subjecting it to analyses and also wÎthout disturbing the culture mediwn. This factor of CO 2 evolution permits to reliably estimate different phases of the development of Penicillium sp. V33A25. in terms of degradation of caffeine and time of the sporu1ation of the filamentous fungi. 157 Biochemical pathway (or caffeine degradation by filamentous (ungi: ln order to understand the biochemical pathway for caffeine degradation by filamentous fungi, synthetic caffeine was used under submerged fermentation system with defmed synthetic medium. Metabolic pathway of caffeine degradation by Pseudomonas pulida is shown in figure 4. The caffeine is degraded to urea as indicated in this figure. The same pathway may not hold good for filamentous fungi. The kinetic of caffeine degradation by Penicillium and Aspergillus species is presented in figure 5. In both cases the degradation of caffeine is total in 50 hours, but the intermediate metabolites produced are different (Denis. 1996). In the case of Penicillium onJy theophyline appeared, whereas with Aspergillus species, Theobromine, paraxanthine and 3-methyl xanthine appeared as intennediates. The proposed pathway for the degradation of caffeine by filamentous fungi is shown in Figure 6. Pectinases production (rom coffee pulp in SSF: Another utilization of coffee pulp is for the production of enzymes (Antier et al. 1993). Coffee pulp is an excellent substrate for pectinase production (Boccas el al. 1994, Augur el a/. 1997). The ability of four wild fungal isolates capable of producing pectinase in solid state fermentation system using coffee pulp is shown in table 9. Cafeine degradation by Aspergillus oryzae and Penicillium roqueforlii was also studied (Denis, (996). Conclusion ln conclusion it may be said that by selecting proper filamentous fungi it is possible 10 detoxify coffee pulp (caffein degradation) and upgrade the coffee pulp for animal feed. The sillage of coffee pulp permit to conserve the good potentiallities of the pulp for various uses as indicated in the trophic chain (figure 9). The silage of coffee pulp under anaerobic conditions inhibits polyphenol oxidation. Uner aerobic conditions the caffeic acid, chlorogenic acid and tarmic acid forms polyphenols which can be further oxidized in presence of air to form quinones (fig. 10). These quinones in presence of proteins and free ami no acids form a black water insoluble product (fig. Il). ln order to overcome this anaerobic fermentation of coffee pulp with selected lactic acid bacteria is most efficient for the total detoxification of coffee pulp, polyphenols. Bibliography Antier, P., Minjares, A.. Roussos, S., Raimbault, M. and Viniegra-Gonzalez G. 1993. Pecpinases hyperproducing mutants of Aspergillus niger C28B25 for solid state fermentation of coffee pulp. Enzyme Microbiol. Technol. 15: 254-260. Aquiahuatl. M.A.. Raimbault, M.. Roussos, S. and Trejo, M.R. 1988. Coffee pul p detoxification by solid state fermentation: Isolation, identification and physiological studies. ln , Raimbault, M. (Ed), Proceedings of Ihe seminar on solid Siale fermentalion in bioconversion ofagroinduslrial raw malerials, üRSTüM, Montpellier. France, pp 13-26. Augur, C., Minjares, A., Viniegra-Gonzalez. 1997. Comparatives studies of pectinase production by Aspergillus niger in solid state and subrnrged fermentation. In Roussos, S.. Lonsane, B.K .. Raimbault M., Viniegra·Gonzalez, G. (Eds), Proceeding of Advances in Solid Slale Fermenrarion, Kluwer, Dordrecht. pp. 347- 353. 158 Bertin, G. 1986. Utilisation des enzymes polysaccharolytiques dans les milieux d'ensilage. Moyen de sélection et résultats pratiques. Thèse d'Etat en Sciences. Université Paul Sabatier. Toulouse, France, 209 p. Boccas, F., Roussos, S., Gutierrez. M., Serrano. L., Viniegra, G. 1994. Fungal strain selection for pectinases production l'rom coffee pulp in solid state fermentation system. 1. Food Science Technol. 31: 22-26. Bohkenfor, B. and Fonseca, H. 1974. Calidad de ensilado con pulpa de café conteniendo diferentes nive les de humedad y varios aditivos. ln Primera Reuni6n Intemacional sobre la Utilizaci6n de Subproductos deI Café en la Alirnentaci6n Animal y otras Aplicaciones Agricolas e Industriales, CATIE, Turrialba, Costa Rica, 41 p. Braham, 1.E., Jarquin, R., Gonzalez, 1.M. and Bressani, R. 1973. Pulpa y pergamino de café. 3. Utilizaci6n de la pulpa de café en la alirnentaci6n de rurniantes. Turrialba 23: 41-47. Bressani, R., Estrada, E. and Jarquin, R. 1972. Pulpa y pergamino de café.!. Composici6n quimica y contenido de aminoacidos de la proteina de la pulpa. Turrialba 22: 299-304. Cabezas, MT., Gonzalez, J.M. and Bressani, R. 1974. Pu\pa y pergamino de café. 5. Absorci6n y retenci6n de nitr6geno en temeros alimentados con raciones elaboradas con pulpa de café. Turrialba 24: 90-94. Bressani, R., Estrada, E. and Jarquin, R. 1972. Pulpa y pergamino de café. 1. Composici6n quimica y contenido de aminoacidos de la proteina de la pulpa. Turrialba 22: 299-304. Cabezas, M.T., Estrada, E., Murillo, B., Gonzalez, J.M. and Bressani, R. 1976. Pulpa y pergamino de café. 12. Efecto dei almacenamiento sobre el valor nutritivo de la pulpa de café para temeros. Archivos Latinoamericanos de Nutricion 26: 203-215. Campos, R., Viniegra-Gonzalez, G. 1995. Utilization of a consortium of celluolytic bacteria grown on a continuous anaerobic digester as a bioassay of probiotic extracts l'rom fungal origin. Biotechnol. Tech. 9: 65-68. Carrizales, V. and Gonzalez, 1. 1984. Aprovechamiento de la pulpa de café. Estudio Experimental, Reporte final. Fundaci6n CIEPE, San Felipe, Venezuela. Christensen, M.S. 1981. Preliminary tests on the suitability of coffee pulp in the diets of Common Carp (Cyprius carpio L) and catfisf (C/arias mossambicus peters). Aquaculture, 25, 235-242. Denis, S. 1992. La dégradation de la caféine par dellx champignons filamentellx : Aspergillus oryzae et Penicillium roque/orti. Rapport de DEA, Sciences des Aliments. Université de Montpellier Il, France, 30 p. Denis S. 1996. Dégradation de la cafféine par Aspergillus sp. et Penicillium sp. Etude physiologique et Biochimique. Thèse de Doctorat, Université de Montpellier II. France.194 p. Gaime-Perraud. 1.. Roussos. S. and Martinez-Carrera. D. 1993. Natural microorganisms of the fresh coffee pulp. Mical. Neotrop. Apl. 6: 95-103. Garcia, R.C., Baynes, D.R. 1974. Cultivo de Ti/apia aurea (Staindachner) en corrales de 100m2. alimentada artificialmente con gallinazas y un alimento preparado con 30 % de pulpa de café. Ministerio de Agricultura y Ganaderia, Dir. Gen. de Pecursos Nat. Ren.. El Salvador, 23p. Giraud, E., Brauman. A.. Keleke, S., Lelong, B. and Raimbault. M. 1991. Isolation and physiological study of amylolytic strain of Lactobacillus plan/arum. Appl. Microbiol. Biotechnol. 36: 379-383. Gouet, P. 1994. Bactériologie des ensilages. ln De Roissart, H. and Luquet, F.M. (Eds.), Bactéries lactiques. vol. 2, Lorica, Grenoble, France, pp 257-270. 159 Luis, L. and Ramirez, M. 1985. Estudio de los principales grupos de microorganismos presentes en los ensilados de pastos de estrella de jamaicano (Cynodon n/emfuensis). Silos y su relaci6n con paràmetros bioquimicos. Pastos y Forrajes 8: 141-155. McDonald, P., Henderson. A.R. and Heron, SJ.E. 1991. The biochemistry of si/age. 2nd ed., Chalcombe Publications, Marlow, England. 340 pp. Murillo, B 1974. Composici6n quimica y fraccionamiento de los componentes celuJares de la pulpa de café ensilada con aditivos. In Reuni6n Intemacional sobre la Utilizaci6n de Subproductos dei Café en la Alimentaci6n Animal y otras Aplicaciones Agricolas e Industriales, CA TIE, Turrialba, Costa Rica, 41 p. Murillo, B. 1978. Ensilaje de pulpa de café. In Braham, JE. and Bressani. R. (Eds.), Pu/pa de café: Composicion, tecn%gia y uti/izacion, INCAP, Guatemala, pp 97-\\ O. Perraud-Gaime, 1. 1995. Cultures mixtes en rrùlieu solide de bactéries lactiques et de champignons filamenteux pour la conservation et la détoxication de la pulpe de café. Thèse de Doctorat, Université de Montpellier II, France, 209 p. Perraud-Gaime,1. and Roussos, S. 1995. Preservation of coffee pulp by ensiling : Influence of biological additives. In Roussos, S., Lonsane, B.K., Raimbault M., Viniegra-Gonzalez. G. (Eds), Proceeding ofAdvances in So/id State Fermentation, KJuwer, Dordrecht 193-207. Roussos, S., Hannibal, L., Aquiahuatl, M.A., Trejo, M. and Marakis, S. 1994. Caffeine degradation by Penicillium verrucosum in solid state fermentation of coffee pulp : Critical effect of additional inorganic and organic nitrogen sources. J. Food Sci. Technol. 31: 316-319. Roussos, S., Aquiahuatl, M.A., Cassaigne, J, Favela, E., Gutierrez, M., Hannibal, L., Huerta, S., Nava, G., Raimbault, M., Rodriguez, W., Salas, J.A., Sanchez, R.. Trejo, M., VirùegraGonzalez, G. 1989. Detoxificacion de la pulpa de café por fermentation solida. In Roussos, S.. Licona, F.R. and Gutierrez, R.M. (Eds.), 1 Seminario Internaciona/ de Biotecn%gia en /a Indus tria Cafeta/era., Memorias 1 SIBAC, Xalapa, Veracruz. p. 121-143.Roussos, S., Aquiahuatl, M.A., Trejo-Hemandez, M.R., Gaime-Perraud, 1., Favela, E., Ramakrishna, M" Raimbault, M. and Viniegra-Gonzalez, G. 1995. Biotechnological management of coffee pulp: Isolation, screening, characterization, selection of caffeine-degrading fungi and natural microf1ora present in coffee pulp and husk. Appl. Microbio/. Biotechnol. 42: 756-762. Tapia, I.M., Herrera-Saldafia, R., Viniegra-Gonzalez. G., Gutierrez-Rojas, M. and Roussos. S. 1989. Pulpa de café fermentada: su uso coma aditivo en la fermentacion de rumiantes. In Roussos, S., Licona, F.R. and Gutierrez, R.M. (Eds.), Seminario Internaciona/ de Biotecn%gia en /a Industria Cafeta/era. Memorias, Jalapa, Veracruz, Mexico, p. 153-175. Tauk, S.M. 1986. Estudo da decomposiçao da polpa de café a 45°C através do uso de microorganismos isolados da polpa. Turria/ba 36: 27\-280. Trejo-Hernandez, M.R. 1992. Physiologie de croissance de souches de C/aviceps: Production d'alcaloides par fermentation en milieu solide. Thèse de Doctorat, Université de Provence. AixMarseille L France, 161 p. Vargas, E., Cabezas, MT, Murillo, B .. Braham, J.E. and Bressani, R. 1982. Efecto de altos niveles de pulpa de cafe deshidratada sobre el crecimiento y adaptaci6n de novillos j6venes. Archivos Latinoamericanos de Nutricion 32: 973-989. Viniegra-Gonzalez, G .. Roussos, S. and Raimbault, M. 1991. Fermentations en rrùlieu solide corrune moyen de valorisation des produits agricoles tropicaux au Mexique.ORSTOM Actua/ités 34: 23-25. 160 Vitzthwn, O.G., Barthel, M. and Kwasny, H. 1974. Détermination rapide de la caféine dans le café décaféiné ou non par chromatographie en phase gazeuse avec détecteur d'azote. Zeilschr f Lebensm. Un/ers. u. Forsch (Munich) 154: 135-140. Weinberg, Z.G., Ashbell, G., Azrieli, A. and Brukental, 1. 1993. Ensilage peas. ryegrass and wheat with additives of lactic acid bacteria (LAB) and cell wall degrading enzymes. Grass Forage Sei. 48: 70-78. Zuluaga, J. 1981. Contribution à l'étude de la composition chimique de la pulpe de café (Coffea arabica L.). Thèse de doctorat es Sciences. Faculté des sciences. université de Neuchatel-Suisse Zuluaga.1. 1989. Utilizaci6n integral de los subproductos dei café. in Roussos S.. Licona R. y Gutierrez M. (Eds), Memorias 1 Sem. ln/ern. Biolecnol. Agroindusi. Café (1 SIBAC). Xalapa. Mexico, pp 63-76. 161 MOLECULAR TECHNIQUES APPLIED TO FUNGAL STRAIN UPGRADATION CAPABILITY RELATED TO SSF CULTURES Christopher Augur, Gustavo Viniegra-Gonzalez Universidad Autonoma Metropolitana, Unidad Iztapalapa, Departamento de Biotecnologia Av. Michoacan y Purisima, Col. Vicentina, Apdo. Postal 55-535. Mexico, D.F., c.P. 09340 Summary Mutant strains derived from A. niger C28B25 and belonging to series AW99 [producing maximum pectinase level in submerged fermentation (SmF)] and series AW96 [producing maximum pectinase level in solid state fermentation (SSF)]were compared conceming pectinase properties and thennal stability. Pectinases produced by AW96 exhibited increased thennal stability compared to AW99. Results support the idea that not only the quantity but also the quality of the enzymes can be modified when SSF or SmF techniques are used. The use of differential display polymerase chain reaction may enable the isolation of solid state fennentation-specific genes. Our group is trying to use molecular methods in order to study a fundamental problem of fungal fermentation, that is, the way fungi adapt to grow and reproduce in liquid or solid media. With ever growing new uses for enzyme production in SSF, there is a need to improve the basic understanding of the physiology of molds used to produce enzymes by SSF. This way, over fifteen years of SSF srudies can help to give a qualitative jump on the physiology and biochemisrry of filamentous fungi with specific reference to their interaction with a specific solid substrate. In fact, filamentous fungi in general (whether perfect or imperfecl. but growing as fiJamentous structures or mycelia) have a remarkable adaptability to the specific type of substrate. Such kind of adaptations are most surely controlled by the transcription and expression of different sets of genes. Identification of the «trigger)} genes controlling those fungal adaptations will help to use modem techniques of genetic engineering to clone, transfer and express new genes from any biological source coding for enzymes and other use fuI products to be 'produced by different fermentation techniques. Previous work in our laboratory [1] with a wild-type strain of A.niger called C28825 led to the isolation of two different kinds of mutants resistant to 2-deoxyglucose (2-DG), namely the DG R A W96 class of strains and DG R A W99 class of strains. Interestingly, these mutant strains behave in an opposite manner to each other that is, DG R A W96 proved to he endopectinase hyperproducers when cu1tivated by SSF but not as much when cultivated by Smf whereas DG R A W99 hyperproduced endopectinase when cultivated by Smf but were poor pectinase producers when cultivated by SSF [3]. Thus it can be considered that DG R A W96 strains are « adapted )} to produce pectinase on SSF and DG R A W99 are « adapted » to SmF production. Alazard and Raimbault [6] found that amylase activity produced by A. niger using SSF technique was more heat tolerant than the one produced by Smf technique. Acuna-Argüelles 163 and coworkers [2] confumed such observation by measuring pectinase activities produced by SSF and Smf and using an A. niger CH4 strain ln an effort to approach at a molecuJar level, the nature of the differences observed. zymographic patterns of pectin hydrolases and pectin esterases produced by a wild strain A. niger C28B25 and mutants AW96 and AW99 were obtained. For the detection of in silu pectinolytic activity, and to study the effect of heaL extracts from the growth of each strain in either SSf or Smf were obtained and divided into rwo samples. Only one sample was heated at 90° C for 60 s. The samples were electrophoresed and pectinolytic activity was detected in situ. AW99 lacked a pectin esterase band at 70 Kd when compared to the other strains. Neither the AW96 nor the wild-type strains hydrolytic activities could be detected before heating and only the A W99 mutant ex.hibited a slight hydroJytic activity band. Nevertheless, heating of the SSf extracts resulted in an apparent activation of an additional hydrolytic activity. Only two low molecuJar weight pectin esterase activities could be detected in SSF extracts but none showed the 70kDa activity that had previously been observed in Smf extracts. Ail esterase activities were lost by heating the extracts. The SSf culture technique, thus produced pectin hydrolases that had the remarkable property of needing a brief themla1 treatrnent in order to show catalytic activity and were different from the ones produced by the SmF culture technique which led to thermal-sensitive pectin hydrolases. Apparently, thermal sensitivity of this latter activity was greater than for those of the wild type or AW96 srrains which provides additional evidence for the presence of discreet differences at the molecular level. The exact nature of the differences, whether resulting from modifications of the polypeptide chain or differences in glycosylation patterns. has yet to be elucidated. Furthermore, thermal stability of in virro enzyme activity was studied by viscometry [mainly endopolygalacturonase (endoPG) activity]. EndoPG activities from AW96 strain produced by either SmF or SSf techniques declined slowly with thermal treatment whether in the presence or absence of substrate (figure 1.) 1000 . , - - - - - - - - - - - - - - - , 100 ~~~::::;:=======_1 10 +----+---t----+----+--.::.t ., •. 0.1 100 r,""------------, 10 0.1 o heating lime (min) ~ +---+--+----+---t---=-....' heatlng lime (min) Figure 1. Thermal stahility of in vitro endopectinase activity of SmF extracts (A) and SSF extracts (8) in the presence (c1osed symhols) or absence (open symbols) of pectin (SglL). A W96=square symbols. AW99=round symbols. T=92'C. NOle the logarithmic scale on the axis. Activity from AW99 produced by Smf showed a trend without any significant difference from that of AW96 extracts when heated in buffer. In contrast, the same extract 164 exhibited a much more pronounced inactivation slope when heated in the presence of substrate (Figure 1A). Samples from SSF exhibited a more pronounced inactivation slope especially in the case of AW99 (Figure 1B). In this case, no significant differences were found in the presence or absence of substrate. In ail cases, endoPG activity produced by AW96 strain was more thermostable than that produced by AW99 strain. Exopectinase activities (Figure 2) produced by both types of mutants in Smf had similar trends when heated in the presence or absence of substrate (Figure 2A). '00 100 -...:.::: 10 10 , , ,. ~ 0.1 0 1 2 3 4 5 ,- ~ healing lime (min) ~ 0 1 2 3 4 5 heallng lime (min) Figure 2. Thermal stability of in vitTO exopectinase activity of SmF extracts (A) and SSF extracts (B). in the presence (c1osed symbols) or absence (open symbols) of pectin (5g1L). AW96=square symbols. AW99=round symbols. T=92°C. Nole the logarilhmic scale on the ax:is. Likewise, exopectinase activities had similar trends in extracts obtained from both strains by SSF when heated in buffer (Figure 2B) however, extracts from the AW96 strain showed and increased thermal sensitivity when heated in the presence of substrate. Results therefore indicated that pectinolytic enzymatic complexes produced by each type of mutant slrain were different when produced by SmF or SSF. Peclin hydrolases produced by SSF technique were more resistant to heat denaturation than those produced by Smf technique. Pectin esterases were, instead heat labile in a similar way when produced either by SSF and Smf techniques. Pectinase activity produced by AW96 mutants was more heat tolerant than that produced by AW99 [2]. Thermal tolerence of pectin hydrolases is an interesting property when analyzed by elecrophoretic zymography. Zymographic differences were related to the use of SSF or Smf techniques and the nature of each given strain. For example. the SSF technique produced a pectin hydrolase band requiring previous heating in order to have activity in the gel. The nature of the heat tolerence of the protein produced by SSF requires further basic work. Perhaps this could be related to the activation of a zymogen or the inactivation of a thermolabile inhibitor associated with the native protein. ln relation to pectin esterase activites, Smf produced a distinct band at 70 kDa which was absent in cultures obtained by SSF. These results give further support to the idea that each given fermentation technique is responsible for the production of different pectinase patterns [2, 7]. On the other hand. pectinase activities measured in crude extracts by viscometry showed very important differences between culture techniques and strains. For example, pectinase activites produced by the AW96 strain were more thermostable than those 165 produced by the AW99 strain. This could be related to earlier reports which suggested that enzymes produced by SSf are more thennostable than those produced by Smf [2,6,7]. The efTect of heating on pectinase activities measured by viscometry did not seem to be the same as that revealed by the zymograrns, but this may be related to the interaction in the fonner arnong severaJ enzymes and soluble materials present in the reaction mixture; nevertheless, thennal stability analysis of enzyme extracts helps to distinguish arnong different enzymatic phenotypes produced by difTerent strains and culture techniques. The molecuJar basis of those differences would require purification and detailed biochemical characterization of each given enzyme but opens sorne interesting questions on the way the molds adapt to solid and Iiquid culture techniques using perhaps a difTerent set of genes or modifying their expression in a differential way. In brief, a1though both types of mutant strains (A W96 and AW99) were selected for their resistance to 2-deoxyglucose (DO R), the results showed that both classes are in fact different and a1so that the DO R phenotype may not be directly involved with the complex patterns of physiotogical derepression and enzyme production which is in agreement with the hypothesis of having pleiotropic mutations associated with the DO R phenotype. As stressed in the introductory comments, identification of the « trigger)} genes controlling fungal adaptations to SSF would greatly help in the understanding of the mechanisms that control the adaptability of molds. We intend to test in the near future, a relatively new technique known as differential display polymerase chain reaction (DD-PCR) in order to detennine whether it is possible to identify genes that are specific for solid state fennentation. The technique has been successfully used in our Jaboratory to identify genes that regulate caffeine degradation in A. niger. Differentiai display is a powerful screening technique used for the detection and identification of differentially expressed genes [4] related to solving developmental, environmental [5] and honnonal problems. ln this method, two or more RNA's (from SSF and Smf cultures) are used as templates to generate cDNA. Subsequently the cDNA fragments are arnplified by PCR using an arbitrary primer in the presence of a radiolabeled nucleotide (dNTP). After separation by denaturing polyacry lamide gel electrophoresis the gels are flXed and dried. DifTerentially arnplified cDNA's are identified by autoradiography. To produce sufficient DNA for further analysis, the difTerentially expressed DNA is eJuted from the gel and rearnplified and then cloned. Northern analysis then confinns that a given clone is SSF-specific. The above mentioned results seem to support the idea that not only the quantity but the quality of enzymes can be modified when SSF or Smf techniques are used. This may be accomplished either by ruming on and off different sets of genes coding for different polypeptides, by controlling the edition of the sarne polypeptides (i.e., difTerences in glycolsyJation) or by a combination of both kinds of mechanisms. Distinction between such hypothese the use of techniques such as DD-PCR which is part of the present research prograrn in our laboratory. 166 Bibliography 1.- Antier, P., A., Minjares and G. Viniegra: Pectinase-hyperproducing mutants of Aspergillus niger C28B25 for solid state fermentation on coffee pulp. Enzyme Microb. Technol. 15 (1993),254-260. 2.- Acufia-Argüelles, M., Gutiérrez-Rojas, M., Viniegra-GonzâJez, G., Favela-Torres. E.: Production and properties of three pectinolytic activities produced by Aspergillus niger in submerged and solid state fermentation. Appl. Microbiol. Biotechnol. 43 (1995), 1-6. 3.- Minjares-Carranco (1992) Obtenci6n de mutantes de Aspergillus niger C28B25 hiperproductoras de pectinasas por fermentation en medio solido de la pulpa de cafe. Tésis de maestria. Universidad 1beroamericana. Mexico D.F. 4.- Liang, P., and Pardee, A.B. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257, (1992) 967-971. 5.- Sharrna, Y.K. and Davis, K. R. Isolation of a novel Arabidopsis ozone-induced cDNA by differential display Plant Mol. Biol. 29: (1995) 91-98. 6.- Alazard, D., Raimbault, M.: Comparative study of amylolytic enzyme production by Aspergillus niger in liquid and solid state cultivation. 1. Appl. Microbiol. Biotechnol. l2 (1981). 113-117. 7.- Solis-Pereira, S., Favela-Torres. E., Viniegra-Gonzalez, G., and Gutiérrez-Rojas. M.: Effects of different carbon sources on the synthesis of pectinase by Aspergillus niger in submerged and solid state fermentation. Appl. Microbiol. Biolechnol. 39 (1993),36-41. 167 THEORY AND STRATEGY FOR SCALE UP: DEVELOPMENT OF SCALE-UP CRITERIA FOR BIO-REACTORS OF SOLID STATE FERMENTATION (SSF) Gerardo Saucedo-Castaneda Universidad Autonoma Metropolitana, Unidad lztapalapa, Departamento de Biotecnologia Av. Michoacan y Purisima, Col. Vicentina, Apdo. Postal 55-535. Mexico. D.F., c.P. 09340, Mexico. Abstract* Scale up is the cruciallink in transferring a laboralory scale process to a pilot plant and then tO a commercial production scale. During this exercise multidisplinary groups involving specialislS from biologicaJ and engineering sciences, are required. In this paper biologicaJ and engineering aspects concerning scale-up of bioreactor of solid state fermentation are discussed. The problems associated with scale up of bioreactor of solid state fermentation are not necessary the same than those found in submerged fermentation. in sorne cases particular, those can be listed as follows: Variation in the biomass due to an excessive subculturing. medium sterilization, inoculum production, heat removal and moi sture maintenance are particularly important because of the heterogeneiry of materials in solid cultivation. The general methodology used to develop criteria for scale up involves principles of geometric. thermal and biochemical sirnilariry as weil as heat and mass balances. In the sense sorne cases can be described. In SSF, inoculum is used at a very high ratio, consequently inoculum production is a particular unit operation in large scale SSF fermentors. In this sense. the production of conidiospores of T harz:ianum has been reponed before indicating a large production Level in a pilot plant reactor . ln another experience. the combinat ion of appropriate sterilization and trealment of substrate resulted in significant increase of cellulase aClivities. Probably the most important problem during SSF processes is heat removal and moisture control. To overcome this problem several strategies have been proposed including maintenance of heat and water balances and utilization of evaporative cooling as criteria for scaling - up. In our research team we believe that fundamentals of mathematical modeling are an alternative for scaling-up of bioreactors. Coming from energy and mass balances dimensionless numbers could be found to be useful in scale-up processes. The application of dimensionless numbers (Biot and Peclet) were first proposed for solid state fermentation for the case of cultivation of A. niger on cassava, since in those ratio are described the operating conditions, geometry and size of bioreactor, leading to improve solid state reactors systems. • The content of the lecture was published in : Procen Biochemislry , vol. 27 (1992); pp. 259-273 l69 MULllVARlABLE MODEL PREDICTIVE CONTROL OF A SOLID SUBSTRATE PILOT BIOREACTOR: A Simulation Study Harold Pajan, Ricardo Pérez-Correa, Ivan Solar, Eduardo Agosin. Depto. de Ing. Quîmica y Bioprocesos, Pontiticia Universidad Cat61ica de Chile, Casîlla 306, Santiago 22. Chile. Abstract Th is work deals with the modelling and control of a Solrl SuIHrate Cultivat ion pilot bioreactor (50 kg of capa;:ity) for gibœrellic acid prodJction with Gibœrella fujikuroi. A simplified physical lurnped parameters model W<lS developed. The model cao reproduce the observed quatitarive behaviour of the main process variables, such as bedtemperature and bed "ater content. In addition, the performances of several control strategies using classic algorithms (Proport ional/lntegraltDerivative. PID) and a prelictive algorithm (Dynamic Matrix Control, DMC) \\ere anab'zed by simulation. In thege control strategies, temperatureandrelative hurnrlity oftheinpl1 air, and also fre~ W<lter and nitrogen additions \\ere derUled as manipulated variables. On the other hand, bed lemperature, bed W<lter content and biomass con cent rat on were derlled as controlled variables. lt was verified that the employment of a DMC algorithm simpliiies the design and start up of the control system. DMC possesses a flexible structure that can be adapted to the process facilities and limitations. In this appncation, the effort devoted to tuning the temperature control loop was significantly reduced and a beller closed loop response was ach~ved with a DM C algorithm compared with a PID. Ke)"'IUrds: DM C, temperature control, water content control, soli:! substrate fenrentation. Introduction M oddling and control in soli:! substrate cultivation sy stems (SSC) are critical aspects t hat must be solved before scahng up theprocess to industriallevd. SSC systems are difficult to understand due to the strong interaction among soli:!, liquid, gaseous and biotic phases. In addition, the lack of adequate sensors limits the idem ification of the p rocess states. Cultivation in soli:! substrate has been traditionally used by humalkind for several cenruries. In this process, themicrobial groWlh and the formation of products occur in the interior of a soli:! matrix, under low water content condit ions. High Yiekls, concent rated products and low operaI ion cosls are usually ach~ved with this kind of cultivation However, despite ils adva1tages. SSC has a ser~s of drawbacks [1) that make it difficult to scal: up processes that are promising at laboratory sca~. Many of the difficulties are relaed wilh the regulation of the temperature and water content of the solK:! bed,and the control of the biomass concentratxJn. 171 In flXfd bed bioreactors with perndic agitation, water content control is particularly complex. Pan ofthewater is strongly bouro to the soIn, whie therest can exist in free form in the capillary areas of the material. Care must be taken to avon saturation of the soin matrix capacity [2]. On the other hand, there is a lack of appropriate and une>qJensive sensors for on line water content monitoring. Theil, the water content regulation is difficult, slow and not precise. For temperature control, the difficulty is mainly fouro in the limited dissipation capacity of the metooolic heat generated during the cultivation This heat canrot be removed by vigorous agitation. since this affects negi'iively the growth of the microor!!fllllim. The Jack of general and reliable biokinetic models for SSC processes is the main problem for the control of the biormss concentraton in the bioreactor. Fung grow prefèrably in the interpanicular voicls and in the soin surfuce that is used as support. Theil, a heterogeneous substrate is generated that changes throughout the cultivation cyce [1], inhibiting direct biomass measurements [3, 4. 5). On the other hand, substrate degradation, heat builiup and water content varat ions, are direct!y relaed with the microorl?flIlism activity [6]. Discrete PlO and on/off control algorithrns are currently used in pilot and industrial SSC bioreactors. Given the process difficulties mentioned above, these controUers are difficult to tune and demand human supervision during the whole cultivation process to opernte it effectively [7]. In the present work, theperformanceofan advaJced mode! predictive control algorithm is assessed and compared with a classic PlO control. The study is base;! on simulations with a phy sical mode! of a pilot SSC bioreactor for gibbereUicacid (GA)) production using Gibberella fujikuroi. MODELLING ln figure l, the SSC pilot bioreactor used for GA) production is described. This bioreactor has a nominal capacity of 50 kg and has been designed for an aseptic operntion in batch or fed -batch mode. The bed agitation system is mechanical and operntes like a plough, where the basket, which contains the sold bed, rotaes and the blades are static. Sterile water and specific nutrients can be adde;! sp o rad ically . The input air is previously filter-sterilized and then conclitioned before entering the bioreactor. The bioreactor operntes with a semi-automal ic straegy for bed temperat ure regulat ion and water content control. Details of the instrumental ion and control straegy can be seen in [7]. A simplified SSC process can be represented as a two phase's system. These are the sold bed that contains the substrate. the inert support. the liquids (water for the most pan) and the microorl?flIlism; and the l?flS that flows t hrough the bed. 172 FRESH WATER NUTRJENTS OUTLET " AIR AIR CONDITIONING Figure 1: Diagram of the solid substrate cultivatioD pilot reactor The main mode! assumplions are: a) homogeneous temperat ure and componenl's concentraton within the bioreactor b) biomass growth limited by nitrogen only c) the out et gas is saturated d) negligible accumulation of the gas phase in the reactor The folbwing set of equations can be derived through mass and energy balances in the solid and gaseous phases. These equations describe the evoh.1l ion of the main bioreactor variables. First, the funl}Js growth kinelics can be represented by: dx di" (1) = (J.1- Ko)x 173 where x is the bioIUlSs conœntratiln, K o is a death constant, and m is the specific growth rate estimated from the Contois mode!. This stru;;ture was used becaJse it has been argœd that this mode! behaves weil both al rugh and low bioIUlSs conœntratilns. In addition, it considers diffusional limitations of nutrients [8]. (2) The valœ of mM, maximum specific growth rate, is considered constant in the above equalion. sinœ temperature and water activity of the soli! bed are kept regulated. ln equation 2, N is the nitrogen conœntratiln. From trus kinctic model, the consumption rates of oxy~n and nutrients, and the production rate of caroon diollide can be derived, which aredescribed below: dN J.lx dl YI (3) Jo! Here, YxJN is the bioIUlSs/nitrogen y iekl coefficient. dG (4) dl where Yx/G is the bioIUlSs/glœose y iekl coefficient, and l1lG the mairuenance coefficient. dO; J.l - (--'--t- mo.)x -- Yx dl dCO) --= dl 0: (5) . J.l (~fI1cu)-X x (6) co: ln equalions 5 and 6, Yx/02 and Yx/cm are the biomass y iekl coefficients for O 2 and CO 2 respect ively and mo2 and Incü2 the mailllenancecoefficients; d0 2/dt and dC0 2/dt the production rates. for oxy g:n and caroon diollide. respectively. Ali these rates (eqs. J to 6) are referred to the total drioo mass within the bed, whose evohllion can be estimated from the CO 2 production rate according to, dM., ----;JI = -k e ·( (12/44) dCO, ON JMs~ (7) 174 where, Ms is the dried soli! mass and ~ is a constant that accexmts for the wei~t loss caused by the product ion of carbon-containing compounds diffèrent t han CO 2. The factor (12/44) rep resent s the ratD between the molecular wei[jlts of Carbon and CO 2, whie 0.44 is the elerrental carbon composition in the orgmic soli:! mixture. To estimate the bed watcr content evolution, appropriate mass balll1ces were defIl1ed, yiekling aftcr rearrangemel1t: [F}4- z_w_X dX dl dMs dl ] (8) J'v! s where X is the soli:! wate: content and F2 the fresh watcr addition rate; w is the watcr evaporation rate, estimated from a watcr balooce in the gas phase. On the othcr hand z represents the watcr production rate, which is eSlimated from the sloi::hiometric equaions that describe the aerobic metooolism during the growth phase of the microorgilIll>m [9], z 18· Ms dCO I -_._- = 44 dl where 44 and 18 are the molecular wei[jlts of the carbon dioxide and watcr respectively. (9) The evolut ion of the bed tempera! ure is obtained from an eneIgY balance, y ielding: dM, C dx [q'" ( FI· HI - F 3 HJ)-CPsT .s -dl- - M,· Tc PH . o--dIJ .. dT, dl (10) Ts is the bed temperat ure. FI and F J are the inlel and out et gas fhNTates, and HI and H J the inlel and out let gas entrnJpies. The speciflc heat of the wet soli:! (Cps) can be expressed in terms of the specific heats of the dried soli:! and the watcr (CPH2ü) [l, 10). The metooolic heat gencration, q, is estimated using themethodology proposed in [II]. Then: q dQ M Sdï = (11) where the specific heat gencralion rate. dQ/dt. is estimated through the next equaion. dQ - dl J.l = (12) -(-m(})x Y, (j rno is the mairnenance coefficienL and YxJQ is the biomass/heaI coefficient, obtained expcrirnentally from the heats of combustion of the substrate and of the biomass [Il]. 175 Constants, parameters and Initial Condtions The values of the coefficients and panrneters modd are shown in tab~ ). Yiekl and maintenance coefficients for Gibberella fujikuroi were experimentalJy measured in the laboratory, using urea and starch as nutrients, and vermiculite (mean particle size mesh 16) as inen sup port. These values were Table 1: mo de 1 parameters Uni~ Value Parameter l.l 000 kc [h'1 0.0012 KD [kg 1kgd.m. 0.2500 KN [kg 1 kgd.m. h 0.0 Il 0 rnc. 5 rJI kgdm. h 3.3'10 mo [kg 1kg d.m. h 0.0130 ffin, [kg 1 kg d.m. h 0.0140 1'J1c:o' 6.510,8 [kgd.mJ J Y"''O r- y x/G Yx/N IVx/d' IVx/CO' mM [kg 1 kg [kg 1 kg [kg 1 kg [kg 1 kg 0.5500 14.3970 0.9510 06250 0.2200 [h'1 then rea!ljusted with data obtained in the pilot bioreactor, using sterilized wheat bran as substrate and inert support. The values of K D and mM were estimated from the biormss growth curves. The constants K N and kc were estimated by least square error minimization, comparing the biorms growth and wei~t 10ss experimental curves with the modd. The specific heat of the driro solrl \Vas measured at 28"C, empbying a diflèrential scanning caJorimeter (OSC) Perkin Elmer, Series 1020, OSC7. Equations (1) to (12) were integrated to simulate a typical cultivation run of 150 hours. The initial conditions empbyed in the simulation were: Ms = 25.19 [kg); N = 0.0166 [kg4<.g d.m.); G = 0.6595 [kg4<.g d.m.); X = 1.5 [kg4<.g d.m.); T s = 27 ["C) and x = 0.005 [kgtkg d.m.). The initial driro soli:J is composed of 20 kg of sterilized wheat bran, 5 kg of starch, 0.068 kg of urea and 0.1253 kg of driro biormss. Nitrogen and total glucose in this substrate are 0.4161 and 16.6160 kg;, respectively. CONTROL The performance of classic and OM C controllers was analyzed basro on MA TLAB simulations using the above physicaJ mode!. Bed temperature (T s ), bed water content (X) and bed biormss concentratvn (x) were defmed as controlled variables, whie inlet air temperature (TI) and relative hllITli:Jity (R H1 ), and also fresh water (F 2 ) and nitrogen addition (N), were defmed as manipulated 176 variables. Transfer functions for bed temperature, water content and biormss concentraton were obtained from step input responses [12], and are computed as the differences between a defmed nominaltrajectory and the disturbed trajectory. Bed temperature response is flIst order for inlel air temperature and relàive hurn.i:lity disturbances. On the other hand. bed water content and biormss concentraton respond as pure integrators for fresh water and rutrogen addition respectively. Other transfer functions relating these varilbles arenegligible. Recent studies have defmed the optimum opernting conditions for acheving high productivity of gibberellic acid (GA 3), when GibbereJia fujikuroi is used in an SSC bioreactor. Temperat ure must be kept at 28"C throughout ail the cultivation cyck:. On the other hand. water content should be increased at a constant rateduring the groWlh phase and then maintained at about 70% w/w for the rest of the cultivation cyde [13]. Finally, biormss concentratKJn must be regulated by the nitrogen sources to optimize GA 3 production [14]. Based on these studies, in our simulations bed lemperature set point is kept constant at 28"C. In addition, bed water content set point was progarnmed to foIbw a linear trajectory from an initial condition of 1.5 [kg.1<g d.m.] up to 2.5 [kg.1<g d.m.], in 40 hotm, then the set point is kept constant; this is the way the SSC pilOl reaClor is currently operated [7]. FinaIJy, for bed biormss concentraton, the set point is kept constant at a high previously defmed value. Classic Control The bed temperature was controlled with the inle! air relalive hurn.i:lity (as in the pilOl reaClor) using a discrete PI, since the transfer function is flIst order. The controller pulse transfer funct ion is given by: (13) where z is the discrete transformed variable, and qo and q) are related with the standard PI tuning paraneters (proponional gain, Kc, and rese! time, t[ ) through: qo = T Kc [1 + ri J (14) (15) where T is thesampling time. Control of water content and biormss concentratKJn was acheved with a proponional algorithm. since bothrelevanttransfer funClions werepure integrators. Here. the pulse transfer funClion of the controller is simply: 177 (16) DMCControl The Dy mrnic M atrix Control algorithm, DM C, has been presented and disOlssed wideJy in stamard process control teXlS [15], then on1y a basi: description will be given here. This discrete controller uses a mulùvariable step response modeJ (Dy narnic M atrix) to compute fut ure manip ulated variables moves that woukl minimize a given quadratic cost funùion. This prediction is ach~ved using CUITent output values, accumulated past errors and two matrices in the cost funùion that wei~t independentJy theoutput errors and the control moves. If input and output restrictions are included in the minimization problem, the algorithm is usually known as QOM C (Qua:lratic Dynamic Mat rix Control). The cost funùion, J, of the DM C algorithm is given by the product of the weil:tlt mairix, t, and the square of the nonmlized error vector, e, plus the product of the weil:tlt matrix, 1, and the square of the nonmlized future control moves, Dm. Here, it is considered ail future vector errors up to time hp (prediction horizon), and ail future moves up to time hc (control horizon). Then, J can be represented by: hp J = he 2,e,re',+ 2,ôm"À~', 1 i (17) '"/ In this algorithm, the matrices t and 1, and the horizons hp and hc, are tuning parameters. Usually, hp is defIned to include 90% of the step response and hc is set to hp/2. The wei~ting matrices are diagmal, and their elerrents can be set to get tighter control in certain outputs and to move more somedesired inputs. Results and Discussion ln ail simulations. the same sampling time of 90 s \Vas used for the three output variables. This is t he value used in the temperat ure control loop in the p ilOl reaàor [7]. Figure 2 show> the results for bed temperature control with PI and DMC. Here, the system was subjected to intel air llo\.vrate and temperat ure (Pl only) dislUrbances. When a Pl controller is used the inlel air humility is manipulated, while with DMC, inlel air temperature and relative humidity are manipulated simultaneously. The PI controller was t uned by trial and error, where the values of Kc = 20 and t[ = 10 gavethe best overall perfonnance. The values of the tuning parameters of the 178 DMCwereset to hp = 40,hc = 20,\i = l and t, = 1 (forai] i). As seenin the figure, when the classic algorithm PI is used, the inlel flowrate and temperature disturbances strongly affect the control perfurmance. In this cast; response is oscillatory, slow (20 hours to reach 28"C). presents lar~ deviations and even fail when an inJel air temperalure disturbance is apptied at time 90 hr. On the other hand, the DM C seerns a more adequate algxithm for this process. A fast response can be achXved (2 hours), with praeticaJly no oscillations and with a simple tuning procedure. M oreover, with the DM C algorithrn, the bed temperature can be controlled witoout requiring eX'lremely low values of the manipulated variables, which is difficult and expensive to allain in a real sy stem. 30 r - - - - - - r - - - . . , - - - - - - - - - , , - - - - - , - - - - . . , - - - - - - - - . 29 ~ ~ 27 ~ ~ , , ------;----------;-------, ,, 26 _ 25 ~ , , _ -----~--------- , ,, , , -------~----------~---------..:------ .. ---_.--- .. ---------- .. --------- . .., ---------- ,.. --------- ...--- ------ 24 . L - -_ _---::'-:-_ _---:~-----'----.L-----L------I o 25 50 75 100 125 150 Time [hl Figure 2: Bed temperature c10sed loop response "ith PI and DMC control under inlet air nO"rate and temperature disturbances. The control of the bed water content and biomass concentration was prao ically the same \Vith both algorithrns. Here. a perfèct trac:king was obtained (Fig;. 3 and 4) and the manipulated variables behave similarly (not shown). 179 2.6 2.4 7 2.2 JJ JJ - 2.0 ~ 1.8 od 1.6 1.4 25 0 75 50 100 125 150 T1me (hl Figure 3: Bedwater content tracking with PI and DMC control. 0.25 ~ ~ ~ DMC;"":~ ~ 010 0.05 0.00 ~ ~ ~_~~~~,------o-_ _-I ~:~: --::::J~~--: ~ ~ ~---;------ :-:- - : _ V--- - - - - - - - - - - - - - - ----::-'=- L...-_ _ o Figure 4: 25 -'---50 -'---7':> Time rh] Bed biomass concentration 180 ....i..... ....i..... 100 125 _ 150 tracking with PI and DMC control. Conclusions A phy sica! modeJ of an SSC pilOl bioreactor was deveJoped. Simulations with this modeJ showed the same qualitative behavior as experirnental results in the reacror. Based on simulations with this modeJ, it wasconduded that a DMCcontrolierbehaves better thana simple Pl in the control of the bed temperature, a critical opemting vamble in trus kind of reactors. Furthermore, PI controller is difficult to tune anddemalds more control effort; these are severe limitations in SSC prooesses. The fact that the DM C used in trus app tication includes more manipulated than observed variables, explains the good performance achieved. Although PI and DM C gave the same perfurmance forwater content and biomass concentration. the P controllers were more difficult to tune, Moreover, in the simulat ions shown here, it was considered that fresh water andJÙtrogen addilion werecontinuously added to the reaetor. ln a real process this addilion must be do ne by pulses. since the reactor is agitated only periodicaJly. Then, it woukl be easer to adapt a DM C than a PI to this kind of manipulation. Acko~edgements This work was supported by projects FONDEF N° 2-50 and FONDECYT WI960360. References 1 Sargmtanis, J., Karim, N., Murphy, V. and Ryoo, D. 1993. Effeet ofoperatingconditionson solK! substrate fenrentation. BiotechnoJogy and Bioengineering, 42: 149-158. 2 Mudg:tt, R. E. 1986. Soln-state fermentations. In: Demain, A. and Solomon, N, Manual of IndustriaJ M icrobioloW and Biotechnology. American Society for M icrobioloW, Washington D.C. 3 Mitchell, D., Duong, D., Greenfield. P. and Doelle, H. 1991. A semimechanistic mathematical modeJ for groWlh of Rhizopus oliwsporus in a modeJ solK!-state fermentation system, Biotechnology and Bioengineering, 38:353-362. 4 Auria, R.. Ortiz, 1.. Vilk:gas, E. and Revro. S. 1995. Influence of GroWlh and high moukl conoent rat ion on the presure drop in solK! state fenrentations. Process Biochemistl)'. 30:8:751-756. 5 Colin. R.T. 1992. Imag: anat'sis: putting filanentous microorganisms in the picrure. Tibtech October (Vol 1OO~ 343-348. 6 Saucedo-Ca>taiieda G., GU! ierrez-Rojas, M., Backet and G.. Raimbault. M. 1990. Heat transfer simulation in soin substrate fermentation. Biotechnology and Bioengineering. 35:802-808. 181 7 Fernandez, M., Pérez-Correa, R., Solar, 1., and A go sin, E. 1996. Autcrnation of a Solû Substrate CuJtivation Pilot Bioreactor, Bioprocess Engineering 16: 1-4. 8 Menezes, J. and Alvez, S. 1994. Matœmatical modclling of industriaJ pilot-plant PenX:illing-G fed batch fenrentatioos. J. Chem Tech Biotechnol, 61:123-138. 9 Raimbault, M. 1981. Fenrentatioo en miœu solile, croissance de charrpignons filamenteux sur substrat amy hcé. Service des publicalons de L'O.RS.T.OM., France. 10 SWeal, V. 1989. Theonal properties of foods. Agri:u1tural Engineering Department, Texas A & M University Co1.k:ge satKm, Texas, 75-79. II Baiey, J. and Ollis, D. 1986. Biochemical Engineering Fundamentals. M cGraw-Hill Publishing Company. 12 Pajan, H. 1996. M odclaci6n, Simulaci6n y Control de un Bioreactor Pilot 0 de Cultivo de Sustrato S6li1o. M Sc thesis, Chem and Biopro. Eng. Dept., Pontificia Universidad Cat61ica de Chie. (in spanish) 13 Corona, A. 1995. Actividad de agua: un panimetro fundamentaJ en cultivo sobre sustrato s6lioo. M Sc thesis, Chem and Biopro. Eng. Dept., Pontificia Universidad Cat6Jica de Chile. (in spanish) 14 Pastrana. L., Gonz.àJez, M., Torrado, A. and Mur<rlo. M. 1995. A fed-batch culrure mode! for improved production of gibbereUicacid from a waste medium. Biotechnology Letters, 17:263-268. 15 Luyben, W. 1990. Process Modelling, Simulation and Control for ChemicaJ Engineers. M cGrawHill PublishingCompany, 281-288. 182 NOMENCLATURE Cps CPH20 CO 2 e FI F2 FJ G hc hp HI HJ RH' J kc Kc KD KN lI1G rnq ma, mco, Ms N O2 q qo, q, Q t T Ts T, w X x y y Y><IN Yx/G [llkgx"C] heat capa::ity, wet solils [11kg\."'C heat capa::ity, liquid water [kg! kg d.s.] ] concentratvn, caroon dioxide erroc veclor [kg! h] mass flowrale, inlel dry air [kg! h] mass flowrate, inlel fresh water [kg! h] mass flowrate, out~t dry air [kg! kg d.s.] concentratvn, glucose control horizon predict ion horizon [l!kg d.a] entœJpy, inJel air [l!kg d.a.] entœJpy, out let air [%] rel<live hUJTIijity, inlel air cost funetion, DMC weiltJt loss factor proport ional gain [ho'] deaÙJ factor [kg! kg d.s.] Contois constant mairnenance coefficient, nitrogen [kg! kg d.s. h] mairnenance coefficient, metabolic heat [l!kg d.s. h] mairnenance coefficient, oll.y ~n [kg! kg d.s. h] mairn enance coefficient, caroon diollide [kgC0 2Ikgd.s. h] dry solil mass [kgd.s.] concentratvn, nitrogen [kg! kg d.s.] [kg! kg d.s.] concentration. oxy g:n metabolic heat, rate [llh] constants, PI pulse trarnfer funetion metabolic heat, generation [l!kg d.s.] time [hl sample time [hl temperat ure. solid bed ["C] temperat ure. inlel air ["C] evaporation rate [kg! hJ bed water content [kg! kg d.s.J concentratvn. dry biormss [kg! kg d.s] controlled variable set point y ie\j coefficient, biormss-nitrogen [kg! kg] Yiekl coefficient, biormss-glucose [kg! kg] 183 YxJ01 YxK) YxJCO Z Z·I y ieki coefficient, biorrass-oxygen y ieki coefficient, biorrass-metabolic heat yieki coefficient, biorrass-carbon dioxide water generation shift operator GREE]( SYMBO LS m specific growth rate maximum specific growth rate outputs wei~t matrix, DMC inputs wei~t matrix, DMC PI integraJ time inp ut increments, DM C 184 [kgf kg] [kgf J] [kgf kg] [kgf h] AN ASEPTIC PILOT BIOREACTOR FOR SOLID SUBSTRATE CULTIVATION PROCESSES Eduardo Agosin'!, Ricardo Pérez-Correa 1, Mario Fernandez 2, Ivan Solar l , and Luciano Chiang3 • 1 Depto, de lng. Quimica y Bioprocesos, Pontificia Universidad Cat61ica de Chile, Casilla 306, Santiago 22, Chile. 20epto. de lng. Eléctrica, Fac. de Ciencias Fisicas y Matemâticas, Universidad de Chile, Casilla 412-3. Santiago, Chile. JOepto . de lng. Mecânica y Metalurgia, Pontificia Universidad Cat61ica de Chi le. Casilla 306. Santiago 22, Chile. Abstract An SSC general purpose aseptic pilot reactor has been developed and evaluated for the production of gibberellic acid (GA J ), a secondary metabolite. This anicle describes the reactor, its instrumentation and control system, and its main operation steps. lt is shown that the reactor can be used to obtain high yields of gibberellic acid in a reproducible way. Keywords: gibberellic acid, control, monitoring. Introduction Solid Substrate Cultivation processes (SSC) are characterized by the grow1h of microorganisms within a porous support without free wateT. This condition favors the deveJopment of filamentous fungi, given their unique capacity to colonize the interparticular spaces of solid matrices. In addition, the risk of bacterial contamination is reduced due to the low water activity (Iower than 0.98) of the solid medium. Some interesting advantages of SSC processes compared with submerged cultivation are: higher productivity, lower operation costs and higher products concentration [1]. Most of the research and development work on SSC has been carried out at laboralory scale, and only few processes have been scaled up to industrial level. Then. a generaJ purpose SSC pilot reactor can simplify the scaling up of laboratory studies. This reactor must operate aseptically to deaJ with different kind of processes. Currently. industrial, semi-industriaJ or pilot reactors. reponed so far, do not operate aseptically [2, 3]. However. aseptic operation has been obtained al laboratory scale, on a rotary reactor of 2 kg capacity [4]. ln this work, it is described a new pilot SSC bioreactor of 50 kg nominal capacity. which has been operated successfully for the production of gibberellic acid by the filamentous fungus Gibberella fujikuroi [5.6]. 185 REACTOR DESCRIPTION General features The structUJ;e of the stainless steel reactor is shown in Fig. 1, where the foHowing parts can be identified: a) A raisable lid that allows a hennetic close wough a rubber seaJ and a handle as in an autoclave. Il has perforations for air sampling and nutrients feecting, and observation windows. The agitation system (motor, double ann and blades) and the feeding system are mOlUlted on the lid. b) The principal body has a double jacket system for sterilization. c) An air chamber, located in the lower part of the reactor, is used for inlet air homogenization. Here, air temperature and relative humidity are measured. d) A rotating basket, of 1.15 m of diameter and 0.28 m height, which has a nominal capacity of 50 kg of wet solid. The basket base contains perforations of 2 mm of diameter with a separation of 3.5 mm. Air Conditioning As shown in Fig. 2, the air is forced into the reactor by a fan (a) with a prefilter (b). The speed of the air can be regulated manually between 0.25 and 7 mis wough a purge system (c). An absolute filter (d) insures the purity of the air. Il is able to retain particJes larger than 0.3 llm with a mirùmaJ efficiency of 99.99%. A heating system (e), made of electric resistances with 6 kW maximum capacity, can heat the air for reactor steriiization and process needs. In addition, the air can be cooled up to 0 oC with a fins cooler (f) and a refrigeration system (g). The relative humidity is controlled by vapor addition through a solenoid valve (h) connected to a steam boiler (i) with a worlùng pressure of 5-10 psi. Agitation The double arm bed agitation system is shown in detail in Fig. 3. One arm contains blades (a) that operate like a plough. The other arm has curved paddles (b) that scrape the basket base. 80th anns can be manuaJly regulated with cranks (c and dl, according to process needs. The basket movement is provided by a reducing motor of 1.5 kW. The motor shaft is connected to the basket shaft (e). An inverter driver, CDS 150, contrais the rotation speed, achieving a minimum of 5 rpm with a torque of 5500 Nm. An aseptic water seal (f) insures that ail the inlet air pass through the solid bed. The basket is supported on roller bearings (g) to get a unifonn and soft movement. Water and nutrient feediog A set of 3 sprinklers (not seen in the figures) are mounted on the Iid. They are used to add water and dissolved nutrients periodically. according to process needs. The solution is forced into the reactor with a peristaltic pump with a maximum Oow of JO l/min. To distribute the solution all over the solid bed, the agitation system is tumed on each time the solution is fed. 186 Figure 1:General view orthe pilot reactor for aseJtic SSC processes, (a) Lid; (b) Main body; © Air chamber; (d) Basket Figure 2: Air comitioning system. (a) Fan; (b) Prefilter; © Air purg::; (d) Absolute fùter; (e) Electric heaters; (t) Fins cooer; (g) Cooling system; (h) Solenoid valve; (i) Boier; U) PC ; (k) lnstrumentaion cabinet 187 . reactor Table 1: Main features of the SSC pilot VALUE UNITS CHARACTERISTICS EXl eTIa1 heigh t EXl eTIa1 diarnet er 2150 1440 mm mm Basket heiltJt Basket diarreter 279 1150 Basket wei&f1t 93 mm mm kg Maximum 1000 25 kg(d.m.) Useful volume 0.65 01 Nol1lllJ rotaion speed 5 rpm Maximum torque 5500 NOl 3 Agitation power 2 HP Air flow 0.25-7.1 mis Air Filter Efficiency (0.3mm) 99.99 % Flow area through basket base 51.3 % Air chamber height 550 mm Air chamber diameter 1000 mm Maximum heaingpower (operation) 3 Maximum cooling power (operat ion) 6.6 Kw Kw Process Monitoring A Programmable Logic Control 1er (PLC), Hitachi series EM-II, is used for on-line data acquisition. The PLC is provided with modules for input/output analog and digital signais and an interface with a PC IBM compatible U in Fig. 2). The process is observed through the following measuring devices (as shown in the Process and Instrumentation Diagram, P&I D, in Fig. 4.): a) Temperature (TT in Fig. 4) is measured in three different points in the solid bed: close to the rotation shaft, in the middle and close to the basket wall. ln addition, temperatures of inlel. out let and ambient air are also measured. K type thennocouples are used as sensing devices and a 45C-THM PLC module is used for signal conditioning. b) A Vaissala sensor!transmiller provides continuous monitoring of inlet air relative humidity (HT in Fig. 4) in the range 0-100%. The output signal (4-20 mA) from the Vaissala device is 188 Figure 3: Agitation system. (a) Blades; (b) Curved pades: (c) Crank for curved paddles ann regulation; (d) Crank for blades arm regulat ion; (e) Basket shaft; (f) Water sea~ (g) Roler bearing; (h) Basket base; (i) Lid. 189 connected to an indicator in the instrumentation cabinet (k in Fig. 2) and to the analog module of the PLC (ACTANA-S2). c) Outlet air CO 2 concentration (C02T in Fig. 4) is measured by infrared detection with a Horiba PIR 2000 device, which gives a 0-1 V output signal that is connected to the analog module. This measurement technique requires a constant air flowrate, which is controlled manually. d) The air pressure drop (DPT in Fig. 4) is measured with a differential pressure transmitter Modus Instr. T30, which provides an output signal of 4-20 mA connected to the analog module. Process Control The main objectives of the control system are the regulation of temperature and water activity of the solid bed. The bed temperature is regulated by manipulating the set point of the inlet air relative humidiry (RH); a PlO discrete algorithm is used to compute the control action. To control the inlet air RH, an ON/OFF regulation algorithm drives a solenoid valve that introduces vapor into the air stream. Inlet air temperature is kept constant through the cultivation process, manipuJating simultaneously the cooling and healing system. The water activiry is controlled by periodic addition of sterile fresh water. Water demand is computed from a mass balance based on off-line bed hurnidiry measurements. A special purpose control sofrware was developed. Il allows, through a graphic interface, direct intervention in the process (similar to a command console) and visualization of trend curves of process variables. Using this interface, the reactor can he operated in manual or automatic form. The control algorithms are executed in the PC, according to options and parameters selected from the keyboard. Different ON/Off and PlO algorithms have heen implemented. Details of the design and performance of the controlloops have been published elsewhere [5, 6]. Reactor Operation During the reactor operation for gibberellin production, the following main steps can he distinguished: a) sterilization, b) substrate loading, c) inoculation, d) cullivation. e) substrate unloading, and f) cleaning. Ail the se steps. except cultivation, are carried out manually. a) The sterilization process. performed with steam and forced hot air. lasts two hours. The minimum temperature inside the reactor during this process is 100 oc. Then, the reactor is cooled with forced chili air until the reactor reaches 28 oC; this Jasts another two hours. b) The humidified wheat bran, previously sterilized in an autoclave. is loaded manually in few minutes into the reactor. During substrate loading, the agitation system is turned on, to achieve an even substrate distribution in the basket. To keep a positive pressure, mjnimizing the risk of contamination, the air fan is also tumed on. 190 <r------' Air Inlet Figure 4: Process and Instrumentation Diagram, P&ID. (a) TT: temperature measuremeJ1l: (b) DPT: differeJ1lial pressure measurement; (c) HT: relative hwnidity rreasurement; (d) C02T: Carbon dioNde air concentration measurement; (e) TC: temperature controller;(f) HC: humidity controller; (h) HI: hurn.rlity iroicatŒ; (i) fIC: Rotation speed controUer. 9 8 ··.w················ o ......---~=~~=---'--------'--------'---~-_-J o 40 20 60 80 \00 120 140 Time (h) -.- Exp.l - Exp. 2 -Exp. 3 ---- Exp. 4 1 Figure 5: }(jnetics ofgibberellicacidproduction in the pilot SSC bioreactor, from several cultivatioos. (FDM = Final Dry Matter; Exp.= Experiment). 191 c) With the agitation system on, the inoculation is carried out through the sprinklers. For this purpose, vegetative mycelium of a non-sporulating strain of Gibberella fujikuroi propagated in submerged culture was added at a level of 0.2% (w/w). Then, the solution of nutrients is fed. Finally, the water content of the solid bed is measured to adjust it to the optimum initial content. d) Cultivation time is 5 to 6 days. During this period, the reactor is operated semi-automatically. Periods of agitation are of approximately 2 hours, since more frequent agitation seriously injures the mycelium. This situation favors the appearance of agglomerations of bran and mycelium. which are difficult to break by agitation without damaging too much the rest of the mycelium. e) At the end of cultivation, the substrate is cooled to 15°C forcing cool and dry air through the fermented bed. Then, the subslIate is manually harvested from the reactor and stored at 4°C (or 20°C) before GA 3 extraction. f) Reactor c1eaning is carried out with hot water containing a quatemary ammonium compound (cationic detergent) for equipment disinfection. The above procedure was used to cultivate Gibberella fujikuroi on solid substrate for gibberellic acid (GA)) production. The process is quite reproducible as shown in Fig. 5. which illustrates the evolution of GA) concentration in different cultivation nms. Yields obtained at pilot scale do not differ much from those obtained at laboratory scaJe (data not shown). Conclusions Although the reactor is not completely automatic, a semi-automatic control system for the cultivation step has been developed. This step requires just one operator with a low level of dedication, due to the alann's system and the process visualization in the graphic interface. The reactor cannot operate with frequent agitation since the mycelium can be damaged. In addition, solid agglomeration reduces the overall yield. A new 200 kg pilot reactor is now operating with an improved agitation system, which will minimize these difficulties. Acknowledgements This work was supported by project FONDEF N° 2-50. References 1 Doelle, H.W.; Mitchell. DA and Rolz, C.E., 1992. Solid Substrate Cultivation. Elsevier Applied Science, London, 466 pp. 2 Deschamps F.; Meyer F. and Prebois J.P., 198b. Mise au Point d une Unite Pilote de Fermentation Aerobie en Milieux Solides. Colloque Soc. Fr. Microbiol., Toulouse, pp. 135- 147. 192 3 Durand A. and Chereau D., 1988. A New Pilot Reactor for Solid-Slale Fennentalion: Application to the Protein Enrichrnent of Sugar Beet Pulp. Biolechnol. Bioeng.. 31: 476-486. 4 Ryoo D. Murphy V.G., Karim M.N. and Tengerdy R.P., 1991. Evaporalive Cooling and Moislure Control in a Rocking Reactor for Solid Substrate Fennentation. Biotechnol. Techniques, 5: 19-24. 5 Fernandez M.; Ananias 1., Solar, 1.; Pérez. R.; Chiang, L. and Agosin, E., 1997. Advances in the Development of a Control System for a Solid Substrate Pilot Bioreactor. In: Advances in Solid Substrate Fennenlation, Roussos, Lonsane, Raimboult & Viniegra-Gonzalez (Editors), Kluwer Academie Publish., Dordrecht, pp. 155-168. 6 Fernandez. M .. 1.R. Pérez-Correa, 1. Solar, E. Agosin, 1996. Automation of a Solid Substrate Cultivation Pilot Reactor. Bioprocess Engineering, 16: 1-4 193 ANNEXES List of papers published bv the group of invited speakers on SSF - Alazard, D. and Raimbault, M. 1981. Comparative study of amylolytic enzymes production by Aspergillus niger in liquid and solid state cultivation Eur. J. Appl. Microbio/. Biolechno/. 12: 113-117. - Alazard, D. and Baldensperger, 1. 1982. Amylolytic enzymesfrom Aspergillus henneberguii (Aspergillus niger group): Purification and characterization of amylases from solid and liquid cultures. Carbohyd. Res. 107: 231-241. - Antier. P., Minjares, A.. Roussos, S., Raimbault, M. and Viniegra-Gonzalez G. 1993. Pectinases hyperproducing mutants of Aspergillus niger C28B25 for solid state fermentation of coITee pulp. Enzyme Microbiol. Techno/. 15: 254-260. - Antier, P., Minjares, A., Roussos, S. and Viniegra-Gonzalez G. 1993. New approach for selecting pectinase producing mutants of Aspergillus niger weil adapted to solid state fermentation. Adv. Biolechno/. Il: 429-440. - Aquiahuatl, M.A. 1992. Detoxificaciôn de la pulpa de café: morfologia. fisiologia y bioquimica de hongos filamentosos que degradan la cafeina. Tesis de Maeslria en Bi%gia. UNAM, Mexico, 72 p. - Aquiahuatl, M.A., Raimbault, M., Roussos, S. and Trejo, M.R. 1989. Coffee pulp detoxification by solid state fermentation: isolation, identification and physiological studies of filamentous fungi. In Raimbault, M. (Ed.), Proceedings of Ihe seminar Solid Srale Fermenralion in Bioconversion of Agroindustria/ Raw Maleria/s. Montpellier 24-28 July 1988. ORSTOM, France, pp. 13-26 - Aufeuvre, A.M. and Raimbault, M. 1982. Etude au microscope électronique à balayage du développement d'Aspergillus niger Van Tieghem sur milieu solide. CR Acad Sc. Paris T 294: 949-956. - Augur, C. and Viniegra-Gonzalez, G. 1996. Comparatives studies of pectinases production by Aspergillus niger in solid state and submerged fermentations. In Roussos, S., Lonsane, B.K .. Raimbault, M. and Viniegraz-Gonzalez, G. (Eds.), Advances in solid slale fermenlalion, Kluwer Acad. Pub!., Dordrecht, p.347-353. - Auria, R.. Hernandez, S., Raimbault, M. and Revah, S. 1990. Ion exchange resin: a model support for solid state growth fermentation of Aspergillus niger. Biolechnol. Techniques. 4: 391-396. - Auria, R.. Palacios, 1. and Revah. S. 1992. Determination of the interparticular effective diffusion coefficient for COI and O~ in solid state femlentation. Biolechnol. Bioeng. 39: 898902. - Auria. R.. Morales. M., Villegas. E.. Revah. S. 1993. Influence of mold groWlh on the presure drop in aerated solid slale femlentors. Biolechno/. Bioeng. 41: 1007-1013. - Auria. R. and Revah. S. J 994. Pressure drop as a method to evaluate mold growth in solid Galido E. and Ramirez, 0.1. (Eds.), Advances in Bioprocess state fermentors. In: Engineering Kluwer Acad. Pub!.. Dordrecht, p. 289-294. - Auria, R., Ortiz. 1., Villegas, E. and Revah, S. 1995. Influence of groWlh and high mold concentration on the pressure drop in solid state fermentors. Process Biochem. 30: 75 J-756. 195 - Ayala, L. A. c.; Soccol, C. R.; Santos, H. R.; Todeschinil, M. L. Produçào de esporos de fungo entomopatogênico (Beauveria bassiana) por fermentaçào no estado s6lido utilizando coma substrato refugo de batata. Anais do XI SINAFERM, Sociedade Brasileira de Microbiologia, Vol 2, pp. 574-579, 1996. - Ayala, L. A. c.; Soccol, C.R. ; Santos, H. R. ; Viscarra, R.V. BiotechnologicaJ improvemente of potato ( SoJanum tuberosum) refuses to produce enthomopathogenic fungus Beauveria bassiana by solid state fermentation. ln: G.M. Zanin & F.F. Moraes (Eds). Anais IV Seminario de Hidro/ise Enzimalica de Biomassas. Editora Sthampa, 1994, p 182-189. - Baldensperger, J., Le Mer J., Hannibal, L. and Quinto, P.J. 1985. Solid state fermentation of banana wastes. Biolechnol. Lell. 7: 743-748. - Barbosa, M.C.S. ; Soccol, C.R. ; Todeschini, M.L. ; Tonial, T. ; Flores, V. Cultivation of Pleurolus sajor-caju in cassava waste. ln: G.M. Zanin & F.F. Moraes (Eds). Anais IV Seminario de Hidrolise Enzimaica de Biomassas. Editora Sthampa, 1994, p 179-181. - Barbosa, M.C.S ; SoccoJ, C.R ; Krieger, N ; Chiarello, M. D. Pleurolus sajour caju production from cassava waste by solid state fermentation. Fifih Brazi/ian Symposium on The Chemistry of Lignins and Olher Wood Componenrs.Curitiba- Pr- Brazil, 31/8 to 05/09, 1997, P 557-569. - Barbosa, M. C. S.; Soccol, C. R.; Marin, B.; Todeschini, M. L.; Tonial, T.; Flores, V. Prospecta production of PleurOlUS sajor-caju from cassava fibrous waste. ln: Roussos, S.Lonsane, B. K.; Raimbault, M.; Viniegra-Gonzalez, G. (Eds) Advances in Solid State Fermentation - Edible mushrooms/ fungi. Dordrecht, Kluwer Acad. Pub., 41, 1997 p 515-528. - Barrios-Gonzalez, J. and Anaya, S. 1987. Desarrollo de un sistema para el estudio de la germinaci6n de esporas de Aspergillus niger. Rev. Mex. Mie 3: 9-18. - Barrios-Gonzalez, J., Tomasini, A., Viniegra-Gonzalez, G. and Lopez, L. 1988. Penicillin production by solid state fementation. Biolechnol. Lell. 10: 793-798. - Barrios-Gonzalez, J., Martinez, c., Aguilera, A. and Raimbault, M. 1989. Germination of concentrated suspentions of sporesfrom Aspergillus niger. Biolechnol. Lell. Il: 551-554. - Barrios-Gonzalez, J., Rodriguez, G.M. and Tomasini, A. 1990. Envirorunental and nutritional factors controling at1atoxin production in cassava solid state fermentation. J Fermenr. Bioeng. 70: 329-333. - Bensoussan, M., Tisserand, E., Kabbaj, W. and Roussos, S. 1995. Partial characterization of aroma produced by submerged culture of morel mushroom. Cryplog. Mycol. 16: 65-75. - Beux, M.R. ; Soccol, C.R. ; Raimbault, M. Use cassava bagasse on cuitivation edible fungus Lenrinus edodes by solid state fermentation. ln: G.M. Zanin & F.F. Moraes (Eds). Anais IV Seminario de Hidro/ise Enzimalica de Biomassas. Edilora Slhampa. 1994. p 190- 198. - Beux, M. R. & Socco!, C. R. - Cultivo do fungo comesivel Lenlinula edodes em residuos agroindustriais do Paranâ através do uso de fermentaçào no estado s6lido. Bolelim do CEPPA, vol. 14 (1) p. 11-21.1996. - Beux, M. R.; Soccol, C. R.; Marin, B.: Tonial, T.; Roussos, S. Cultivation of Lenrinus edodes on mixture of cassava and sugar cane bagasse. ln: Roussos, S.Lonsane, B. K.; Raimbault, M.; Viniegra-GonzaJez, G. (Eds) Advances in Solid State Fermentation - Edible mushrooms/ fungi. Dordrecht, Kluwer Acad. Pub., c. 41, 1997. p501-514. 196 - Boccas, F., Roussos, S., Gutierrez, M., Serrano, L., Viniegra, G. 1994. Fungal strain selection for pectinases production from coffee pulp in solid state fermentation system. 1. 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Degradation de la cafféine par Aspergillus sp. et Penicillium sp. : Etude physiologique et biochimique. Thèse de Docloral. Université Montpellier Il, Frane. 200 p. - Deschamps, F., Raimbault. M. and Senez, 1.e. 1982. Solid state fermentation in the development of agro-food by-producls. Indusry & Environ. 5 (2): 27-30. - Deschamps, F., Giuliano, e., Asther. M., Huet, M.e. and Roussos, S. 1985. Cellulase production by Trichoderma harzianum in static and mixed solid state fermentation reactors under non-aseptic conditions. Biolechnol. Bioeng, 27: 1385-1388. - Favela. E., Huerta, S., Roussos, S., Olivares, G., Nava, G., Viniegra. G. and Gutierrez-Rojas. M. 1989. Producci6n de enzimas a partir de la pulpa de café y su aplicaci6n a la indùstria cafetalera ln Roussos, S.. Licona.. F.R. and Gutierrez, R.M. (Eds.), 1 Seminario Internacional de Biolecnologia en la Indusrria Cafeialera. Memorias, XaJapa. Veracruz, p. 145-151. - Gaime-Perraud, 1.. Hannibal. L., Trejo, H.M., Raimbault. 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Thèse de DoC/oral, Université de Technologie de Compiègne, France, 218 p. - Soccol, C.R., Rodriguez-Leon, 1., Marin, B., Roussos, S., Raimbault, M. 1993. Growth kinetics of Rhizopus arrhizllS in solid state fermentation of treated cassava Biolechnol. Techniques. 7: 563-568. - Soccol, C. R.; Marin, B.; Roussos, S.; Raimbault. M. Scaruùng electron microscopie of the development of Rhizopus arrhizus on raw cassava by solid state fermentation. Mic%gia Neo/ropica/ Aplicada, 6: 27-39. 1993. - Soccol. C.R. ; Cabrero, M.A. ; Roussos. S ; Raimbault, M . Selecion de cepas de Rhizopus em base a su capacidade de crescimiento en yuca. Sociedade Mexicana de Biolecn%gia e Bioengennieria, V.3 ; N 1-2, p. 61-64,1993. - Soccol, c., Marin. B., Raimbault, M. and Lebeault. 1.M. 1994. Breeding and growlh of Rhizopus in raw cassava by solid state fermentation. App/. Microbio/. Biolechno/. 41: 330-336. - Soccol, C. R.; Raimbault. M.: Pinheiro. L. 1. 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(Ed.), Proceedings of the seminar Solid State Fermentation in Bioconversion of AgroindustriaJ Raw Materials, Montpellier 24-28 July 1988. ORSTOM, France, pp. 67-72. - Viniegra-Gonzalez G., Roussos S. and Raimbault M. 1991. Fermentations en milieu solide comme moyen de Valorisation des Produits Agricoles Tropicaux au Mexique. ORSTOM Aclualifés, 34: 23-25. - Viniegra-Gonzalez, G. 1996. Solid state fermentation: Definition, characteristics, limitations and monitoring. In Roussos, S., Lonsane. B.K., Raimbaull. M. and Viniegraz-Gonzalez, G. (Eds.), Advances in solid slale jermenralion, Kluwer Acad. Pub!., Dordrecht, p.5-22 204 Prograrn FMS-97 Monday 6th of October: 9:00-09:30 Opening ceremony I. General aspects of SSF (9:30-12:00) Chairman: C. Augur . 09:30-10:00 General and microbiological aspects of 55F (M. Ralmbault) 10:00-10:30 Continuous enzymes and fungaJ metabolites production in 55F using a counter -current reactor (5. Roussos) 10:30-10:45 Coffee Break 10:45-11:30 Probiotics from Solid State Fermentation (G. Saucedo-Castaiieda) 11:30-12:00 Fruity aromas production in SSF by Ceratocystis fimbriata (P. Christen) 12:00-14:00 Lunch 14:00-18:00 Practice in the \aboratory Thuesday 7th of October: Il BiologicaJ Aspects of fungal growth in SSF Chainnan: S. Roussos 9:00-9:30 Fungal biomass estimation in batch solid substrate cultivation using asymptotic observation (R. Perez) 09:30-10:00 Mutagenesis and adapted strains 10 the growth in Iiquid or sol id substrates (c. Augur) 1000-10:30 Coffee Break 10:30-11 :15 Fungal genetics , case of Aspergillus niger (c. Augur) II: 15-12:00 Growth and production of immobilized lipase from Rhizopus delemar cultivated in SSF on Amberlite (P. Christen) 12:00-14:00 Lunch 14 :00-18 :00 Praclice in the laboralory Wednesday 8th of October: III Kinetics aspects in SSF processes Chairman: G. Saucedo-Castaiieda 9:00-9:30 Relation between Biomass and Respiration: Theoretical aspects (M. Raimbault) 09:30-10:00 Kinetics of the solid state fermentation of raw cassava llour by Rhizopus furmosa 28422 (J. Rodriguez-Leon) 10:00-10:30 Coffee Break 10:30-11: 15 Biotransformation of by-products and agro-industrial wastes by 55F: Pan 1: Biotransformation of solid wast es (c. Soccol) II: 15-12:00 Pan Il: Production of industrial metabolites (c. Soccol) J 2:00-14:00 Lunch 14:00-18:00 Praclice in the laboralOry Thuesday 9th of October: IV Applications in SSF Chairman: R. Perez-Conea 9:00-9:30 Yeasts cultivation in 55F: control of metabolism of Schwanniomyces castelii during S5F on starch (G. Saucedo-Castaneda) 09:30-10: 15 Mushroom mycelium cultivation and Pleurotus aroma production on sugarcane bagasse (5. Roussos and W. Kabbaj) 10:15-10:30 Coffee Break 10: 30- 1 l: 15 BiOlechnological management of coffee pulp (S.Roussos and 1. Gaime) Il: 15-12:00 Molecular techniques applied to fungal main upgradation capacity related to 55F cultures, (c. Augur) 12:00-14:00 Lunch 14:00-18:00 Practice in the laboratory Friday 10th of October: V Models and strategies for scale-up Chairman: J. Rodriguez-Leon 9:00-9:30 Theory and strategy for scale-up in 5SF. (G. Saucedo-Castaiieda) 09:30-10:00 Multivariable model predictive control of a solid substrate pilot bioreactor: a Simulation slUdy (R. Perez-Correa) 1000-10:30 Coffee Break 1030-1 l: 15 An aseptic pilot bioreaclor for S5F processes (R. Perez-Correa) Il: 15-12:00 Inoculum produclion for solid substrates 1200-14:00 Lunch 14:00-18:00 Practice in the laboratory Participants of the International Training on Solid State Fermentation 6 - 10 Detober 1997 Curitiba - Parana - Brasil Name: Christopher Augur Instilution : ORSTOM Address : Ciceron 609 Los Morales 11530 Mexico D.F. Mexico E-mail: augur@xanum.uam.mx Tel: 52 5 2820800 Fax: 1 52 5 5825866 AClual Position: Resercher al ORSTOM Graduation, Tille, Speciality: PhO in Biochemistry and Molecular Biology Name : Pierre Christen Inslilution : ORSTOM Address: Laboratoire de Biotechnologie BP 5045 34032 Montpellier France E-mail: christen@mpl.orstom.fr 003346741 6201 Tel: Fax: 00 33 4 67 41 62 83 Aclual Position: Investigador (Researcher) Graùuation, Tille, Speciality: Phd. In Biotechnology - Solid State Fermentation - Biofiltration Name : Gerard Chuzel Institution: DRCS Address : CERATIUNESP BP 237 18603-970 Botucatu Brasil E-mail: gchuzel@sti.com.br Tel: 0055148213883 Fax: 0055 14821 3438 Aclual Position: PeritoEmbaixada França Gradualion, Title, Speciality: Doutor em Ciências dos Alimentos Name : Ricardo Perez Institulion : PUC -Chi le Aùdress : Depto. de Ingeniera Quimica y Bioprocessos E-mail: perez@ing.puc.cl Tel: 562 686 5803 Fax: Aclual Position: pue Vicufia Mackenna 486 Gradualion, Tille, Speciality: Name : Geraldo Saucedo-Catafieda Institution: UAM Address : Mexico E-mail: saucedo@xanum.uam.mx Tel: 00 52 57 24 49 99 Fax: 00 52 57244712 Actual Position: Profesor/lnvestigador Titular C Graduation, Title, Speciality: Dr., SSF, Modeling, Probiotics. Bioreactor, Instrumentation Name : Carlos Ricardo Soccol Institution: UFPR Address: Centro Politécnico Caixa P tal19011 81531-970 Curitiba·PR Brasil E-mail: soccol@engquim.ufpr.br Tel: 00 55 41 36623 23 p3285 Fax: 005541 26602 22 A tu 1 Position: Professor Tirular em Biotecnologia Graduation, Title, Speciality: PhD in Biotechnology Name : Maurice Raimbault Institution: ORS TOM Address : Laboratoire de Biotechnologie BP 5045 34032 Montpellier France E-mail: maurice.raimbault@mpl. orstom.fr Tel: 00 33 4 6741 62 81 Fax: 003346741 62 83 Actual Position: Graduation, Title, Speciality: Dr. Biotechnology/ Université de Toulouse Name: Sevastianos Roussos Institution: ORS TOM Address: Laboratoire de Biotechnologie BP 5045 34032 Montpellier France E-mail: roussos@mpl.orstom.fr Tel: 0033467416231 Fax: 003346741 62 83 Actual Position: Directeur de Recherche Graduation, Title, Speciality: Docteur d'état en microbiologie Name: Gustavo Viniegra Institution: UAM Address : Mexico E-mail: Tel: Fax: Actual Position: Graduation, Title, Speciality: Name : Robert Tanner Institution: Address : Tennessee, USA E-mail: Tel: Fax: Actual Position: Graduation, Title, Speciality: Name : José Rodriguez Le6n Institution: ICfDCA Address: P.O. Box 4026 La Habana Cuba E-mail: Tel: icidca@ceniai.inf.cu 00537983003 Fa~ 00537338236 Actual Position: Senior Researcher Graduation, Title, Speciality: M Sc en Biotechnology Dr. en Ciencias Name : Raul Jorge Heman Castro G6mez Institution: Univ. Est. de Londrina Address : Campus Universitario Caixa Postal. 6001 Coord. de Pesquisa e P6sGraduaçào 86051 - 930 Londrina Brasil E-mail: rgomez@npd.uel.br Tel: 0055433714503 Fax: 00 55 43 3284320 Actual Position: Diretor de Pesquisa Graduation, Title, Speciality: Engenharia de Alimentos M Sc. Applied Microbiology Dr. Ciência dos Alimentos Microbiologia Aplicada Name : Mauricio Gonzâlez Sepulveda Institution: PUC-Chile Address: Vicena Mackenna 4860 - Macul Santiago Chile E-mail: mugonzal@ing.puc.cl Tel: 00 56 2 6864255 Fax: 00 56 2 6865803 Actual Position: PhD Student in Engineering Science Graduation, Title, Speciality: Biochemistry Name : Francisco Perez Institution: Universtidad de Chile Address : Las Palmeras 3425 Casilla 653 Santiaga Clule E-mail: frperz@abello.dic.uchile Tel: 005626787325 Fax: Actual Position: Assistant Professor Graduation, Title, Speciality: PhO Ooctor Plant Biochemistry Name : Viviana M.Taragano Institution: FCEyN-UBA Address: Ciudad Universitiria '428 Cap. Fed Argentina E-mail: vtaragano@di.fcen.uba.ar Tel: 00 54 1 788 8964 int 12 Fax: 00 54 1 781 5021/9 int 222 Actual Position: PhO Student Graduation, Title, Speciality: Licenciada en Ciencias Biol6gicas Name : Christina Ramirez Institution: Universidad dei Valle Address: Ciudad Universitaria Melendez 25360 Cali Colombia E-mail: crisrami@mafalda.univalle.edu.co Tel: 330 72 85 Fax: 3334907 Actual Position: Profmicrobiology lnd. Investigation Graduation, Title, Speciality: Bi61oga, M.Sc. Tecnologia Quimica Name : Magali Leonel Institution: UNESP/CERAT Address: Fazenda Experimental Lageado Caixa Postal 237 18600-000 BOlucatu-SP Brasil E-mail: Tel: 8213883pl58 Fax: Actual Position: PhO SlUdent Graduation, Title, Specialit)': Bi61oga, Mestre em Ciência dos Alimentos. Àrea de Pesquisa: Aproveitamento de Residuos Agroindustriais e Fermentaçào Name : Clàudio Leandro M. Kuboski Institution: UFPR Address: Rua Luis Leào, 0\ Curitiba-PR Brasil E-mail: kuboski@engquim.ufpr.br Tel: 0055412224911 Fax: 00 55 41 2662042 Actual Position: Estudante de Graduaçào Graduation, Title, Speciality: Name : Eliane Endres Institution: UNIMES Address: Rua Panicular Miguel Cirilo, 15 Apl. 102 11030-070 Santos Brasil E-mail: fect@unimes.com.br Tel: 00 55 13 238 1707 Fax: ActuaI Position: Professora Graduation, Title, Speciality: Médica Veterinària Mestre em Microbiologia Agricola e do Meio Ambiente Name : Leda dos Reis Castilho Institution: UFRJ Address: Rua Capitào Salomâo, 14 204 22271-040 Rio de Janeiro-RJ Brasil E-mail: leda@peq.coppe.ufrj.br Tel: 0055 21 2460804 Fax: 0055212597611 Actual Position: PhO Student Graduation, Title, SpeciaIity: Chemical Engineering M.Sc. Solid State Fermentation Oownstream Processing Name: Vanildo Luiz dei Bianchi Institution: UNESP Addrcss: Rua Cristovâo Colombo, 2265 15054-000 Sâo José do Rio Preto-SP Brasil E-mail: vanildo@eta.ibilce.unesp.br Tel: 00 55 17 224 4966 Fax: 0055 17 224 8692 Actual Position: Professor Assistant Graduation, Title, Speciality: M.Sc. Eng. Alimentos PhO Agronomia Name : Claudia Lareo Institution: Faculdad de Ingenieria Address: 1. Herrera y Reissig 565 11300 Montevideo Uruguay E-mail: clareo@fing.edu.uy Tel: 005982710871 Fax: 005982 71 5446 Actual Position: Professor Graduation, Title, Speciality: Ingeniera Quimica PhO en Ing. Quimica Name: Angela Elena Machuca Herrera Institution: Faculdade de Engenharia Quimica de Lorena Address : Caixa Postal 116 12600-000 Lorena Brasil E-mail: aemh@sbq.org.br Tel: 0055 21 553 3422 Fax: 0055215533165 Actual Position: Pesquisador visitante Graduation, Title, Speciality: Bioquimico Doutor en Cièncias Name : Jorge Alberto Vieira Costa Institution: Universidade do Rio Grande Address: Rua Alfredo Huch, 475 ai a Postal 474 96201-900 Rio Grande-RS Brasil E-mail: dqmjorge@super.furg.br Tel: 00 55 532 311 900 P170 Fax: 0055532 323 3659 Actual Position: Professor Graduation, Tille, Speciality: PhD Engenharia de Alimentos Name: Virginia Sanchez Institution: Universidad de Buenos Aeres Address : Bynon 2428 1846 Jose Marmol Argentina E-mail: vsanchez@overnet.com.ar Tel: 2941685 Fax: Actual Position: Docente Graduation, Title, Speciality: Lic. Ciências Biol6gicas Name: Sergio de Jesus Romero G6mez Institution: UAM Address: Av. Michoacan y la Purisima Col. Vicentina Izzapalapa, Mexico, D.F. Mexico E-mail: stagers@xanum.uam.mx Tel: 57244719 Fax: 57244712 Actual Position: PhD Student Graduation, Tille, Speciality: M.Sc. en BiolecnoJogia Name : Rodrigo de Oliveira Moraes Institution: UNICAMP Address: DPB/FEQ!UNICAMP Caixa Postal 6066 13083-9701 Campinas -SP Brasil E-mail: alcahol@feq.unicamp.br Tel: 00 55 19788 78 40 Fax: 0055192394717 Actual Position: M Sc Student Graduation, Title, Speciality: Engenheiro de Alimentos Name : Maria Helena Andrade Santana Institution: UNICAMP Address : DPBIFEAIUNICAMP Caixa Postal 6066 13083-970 Campinas-SP Brasil E-mail: lena@feq.unicamp.br Tel: 0055 19 788 78 40 Fax: 0055 1923947 17 Actual Position: Professor Docente Graduation, Title, Speciality: Engenheira Quimica Naroe : Fidel Domenech L6pez Institution: ICIOCA Address : Via Blanca, 804 Esq.C.CentraIS.M. P Ciudad Habana Cuba E-mail: icdca@ceniai.inf.cu Tel: 00537983003 Fax: 00537338236 Actual Position: Investigadpr Agregado Graduation, Tille, Speciality: Eng. Quimico Name: Sônia Cachoeira Stertz Institution: UFPR Address : Centro Politécnico- LPB Caixa Postal 190 II 81531-970 - Curitiba-PR Brasil E-mail: soccol@engquim.ufpr.br Tel: 0055 41 36623 23 p.3285 Fax: 00 55 41 266 02 22 Actual Position: Quimica!PesquisadoralUFPRlLPB PhD Student in Biotechnology Graduation, Title, Specialil)': M Sc Tecnologia QuimicaiAlimentos Name : Luciana Porto de Souza Vandenberghe Institution: UFPR Address: Centro Politécnico- LPB Caixa Postal 1901 1 81531-970 - Curitiba-PR Brasil E-mail: Ivandenb@engquim.ufpr.br Tel: 0055 41 36623 23 p3285 Fax: 005541 2660222 Actual Position: PhD Student in Biotechnology Graduation, Title, Speciality: M.Sc. Tecnologia QuimicaiAlimentos Engenheira Quimica Name : Fabiola Stencel Carta Institution: UFPR Address : Centra Politécnico- LPB Caix a Postal 19011 81531-970 - Curitiba-PR Brasil E-mail: cartacwb@per.com.br Tel: 0055 41 36623 23 p3285 Fax: 005541 266 02 22 Actual Position: MSc. Student Graduation, Title, Speciality: Eng. Qui. Name : Débora Brand Institution: UFPR Address : Centro Politécruco- LPB Caixa Postal 190 II 81531-970 - Curitiba-PR Brasil E-mail: brand@per.com.br Tel: 00 55 413662323 pJ285 Fax: 00 55 41 266 02 22 Actual Position: M Sc Student Graduation, Title, Specialit)': Bioquimica Fannacêutica Industrial Name : Adriane B. Pedroni Medeiros Institution: UFPR Address : Centro Politécnico- LPB Caixa Postal 190 Il 81531·970 - Curitiba-PR Brasil E-mail: apedroni@engquim.ufpr.br Tel: 00 55 4 \ 3662323 p.3285 Fax: 00 55 41266 02 22 Actual Position: M Sc Student Graduation, Title, Speciality: Eng. Quimica Name : Adenise L. Woiciechowski Institution: UFPR Address : Centro Poiitécnico- LPB Caixa Postal 190 II 81531-970 - Curitiba-PR Brasil E-mail: soccol@engquim.ufpr.br Tel: 00 55 413662323 pJ285 Fax: 00 55 41 266 02 22 Actual Position: PhD Studant Graduation, Title, Speciality: M Sc Tecnolgia QuimicaiAlimentos Name : Mariene Soares Institution: UFPR Address : Centro Politécnico- LPB Caixa Postal 190 11 81531-970 - Curitiba-PR Brasil E-mail: msoares@engquim.ufpr.br Tel: 00 55 41 3662323 pJ285 Fax: 00 55 41 266 02 22 Actual Position: M Sc Student Graduation, Title, Speciality: Engenheira de Alimentos Name : Sandro Gennano Institution: UFPR Address : Centro Politécnico- LPB Caixa Postal 190 II 81531-970 - Curitiba-PR Brasil E-mail: soccol@engquim.ufpr.br Tel: 00 55 41 3662323 p.3285 Fax: 00 55 41 266 0222 Actual Position: Ph D Student Graduation, Tille, Speciality: Farmacêutico Bioquimico M Sc Bioquimica Name : Renata E. Freitas de Macedo Institution: UFPR Address : Centro Politécnico- LPB Caixa Postal 1901 1 81531-970 - Curitiba-PR Brasil E-mail: m.macedo@com.br Tel: 00 55 41 36623 23 p.3285 Fax: 00 55 41 26602 22 Actual Position: M Sc Student Graduation, TitIe, Speciality: Médica Veterimiria Name : Ana Maria Rodrigues Institution: Universidade de Blumenau Address: Rua Antonio da Veiga, 140 Caixa Postal 1507 89010-970 - Blumenau-SC Brasil E-mail: ana@furb.rct.sc.br Tel: 0055473237200p.155 Fax: 00 55 47 323 72 00 Actual Position: PesquisadoraiPh 0 Student Graduation, Title, Speciality: M Sc Ciência dos Alimentos Name : Isis Kaminski Caetano Institution: UNICENTRü Address : Rua Presidente Zacarias, 875 Guarapuava- PR Brasil E-mail: Tel: 005542 723 1869 p.270 Fax: 00 55 42 723 8644 Actual Position: ProfessoralPhD Student Graduation, Title, Specialil)': M Sc Engenharia Quimica Name : José Francisco Warth Institution: UFPR Address: Centro PolitécnicoDept. Patologia Basica Caixa Postal 19031 81531-970 - Curitiba-PR Brasil E-mail: j fgw@garoupa.bio.ufpr.br Tel: 005541 2574509 Fax: 0055 41 2662042 Actual Position: Professor Graduation, Title, Specialil)': Med. Veterinarial M Sc Microbiologia Name : Crislina L. Bastos Monteiro Institution: UFPR Address : Rua Jûl ia da Costa. 1151 apI. 60 J 80430-010 Curitiba-PR Brasil E-mail: Tel: 005541 3363282 Fax: 0055 41 2662042 Actual Position: Professor Assistente/Ph 0 Student Graduation, Title, Speciality: Bi610gal M Sc Tecnol. Quimica A group of trainees in the FMS97 course on Soliù State Fermentation in Curitiba View publication stats