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S. BHARGAV et al., Solid-state Fermentation: An Overview, Chem. Biochem. Eng. Q. 22 (1) 49–70 (2008) 49Solid-state Fermentation: An OverviewS. Bhargav, a B. P. Panda, a,* M. Ali, a and S. Javed baPharmaceutical Biotechnology Laboratory, Faculty of Pharmacy,Jamia Hamdard (Hamdard University), Hamdard Nagar,New Delhi, India – 110062bMolecular Biology and Biotechnology Laboratory, Faculty of Science,Jamia Hamdard (Hamdard University), Hamdard Nagar,New Delhi, India – 110062ReviewReceived: November 14, 2006Accepted: September 15, 2007Solid-state fermentation (SSF) is defined as the growth of microbes without freeflowing aqueous phase. The SSF is alternative to submerged fermentation for productionof value added products like antibiotics, single cell protein, Poly unsaturated fatty acids,enzymes, organic acids, biopesticides, biofuel and aroma production. However, the advantagesof SSF in various processes are found to be greater than in submerged fermentation.This paper reviews the advantages of solid-state fermentation over submerged in productionof different value added products, important features of various bioreactor designs, recentdevelopments in utilization of various agro-industrial residues as substrates and theimportance of mathematical modeling. With advances through modeling and optimizationtechniques, production-using SSF is advantageous and appropriate for production of manyvalue added products like enzymes, antibiotics, and organic acids. This technique not onlydecreases the cost of the process but also makes product cheaper for consumers.Key words:Solid-state fermentation, submerged, aroma, biocontrol, biofuel, antibiotics, aroma, bioreactors,substrates, enzymes, acids, biochemical engineering, modeling, PUFA, exopolysaccharidesIntroductionSolid-state fermentation (SSF) is defined as thefermentation process in which microorganismsgrow on solid materials without the presence of freeliquid. 1 The concept of using solid substrates isprobably the oldest method used by man to makemicroorganisms work for him. In recent years, SSFhas shown much promise in development of severalbioprocesses and products. However, SSF has alsosome disadvantages. There are some processes inwhich solid-state fermentation cannot be used as inbacterial fermentation.Solid-state offers greatest possibilities whenfungi are used. Unlike other microorganisms, fungitypically grow in nature on solid substrates such aspieces of wood, seeds, stems, roots and dried parts ofanimals such as skin, bones and fecal matter i.e. lowin moisture. 2 In SSF, the moisture necessary for microbialgrowth exists in an absorbed state or in complexwith solid matrix. However, SSF differs fromsolid substrate fermentation. In solid substrate fermentation,the substrate itself acts as a carbon sourceand occurs in absence or near absence of free water.* Corresponding author: Bibhu Prasad Panda,E-mail: bibhu_panda31@rediffmail.com;Telephone no.: +91-11-26059688 Ext. 5627;+91-11-65631138However, in solid-state fermentation, the process occursin absence or near absence of free water by employinga natural substrate or inert substrate as solidsupport. 3 The aim of SSF is to bring cultivated fungior bacteria in tight contact with the insoluble substrateand to achieve the highest nutrient concentrationfrom the substrate for fermentation. This technologyso far is run only on a small scale, but has anadvantage over submerged fermentation.Two types of SSF systems have been distinguisheddepending on the type of solid phase used.The most commonly used system involves cultivationon a natural material and less frequently on an inertsupport impregnated with liquid medium. 4 Solid-statefermentation processes can also be classified based onwhether the seed culture for fermentation is pure ormixed. In pure culture SSF, individual strains are usedfor substrate utilization and with mixed culture, differentmicroorganisms are utilized for the bioconversionof agro-industrial residues simultaneously.Biochemical engineering aspectsof solid-state fermentationA considerable amount of work has been donein recent years to understand the engineering aspectsof solid-state fermentation.

50 S. BHARGAV et al., Solid-state Fermentation: An Overview, Chem. Biochem. Eng. Q. 22 (1) 49–70 (2008)Moisture and water activity in SSFThe concept of water availability in substratebecomes important. Water activity, A w is defined asthe ratio of vapor pressure of an aqueous solution tothat of pure water at the same temperature. 5 Scott in1953 introduced to microbiology the concept ofwater activity (A w ), which is a different way of describingthe water relations in medium from theterm “water content”. In solid-state, the low moisturewithin the substrate limits the growth and metabolismof microorganisms when compared to submergedfermentation. A w is a very useful parameterfor measuring water potential, characterizing theenergetic state of water. 6Water relations in SSF have been studied underquantitative aspects. 7,8,9 Water activity of substrateshas a strong influence on microbial activity. A w determinesthe type of organisms, that can grow inSSF. A w of the medium has been attributed as a fundamentalparameter for mass transfer of wateracross the microbial cells. The control of this parametercould be used to modify microbial metabolicproduction and its excretion. 10Grevais and Molin studied the effect of waterin solid culture medium on fungal physiology suchas cellular mechanisms, radial growth rate and orientationof fungus. They observed that the radialextension rate of mycelia related to the water activityvalue. The optimum water activity was found tobe 0.99 for T. viride, and below 0.90 no fungal developmentoccurred, for Penicillum roquefortii theoptimal water activity was 0.97. 11 Molin et al. describedthe effect of water activity on hyphal andradial growth. 12 Moreover, hyphal growth rate wasalways greater than radial growth rate, showing thathyphae do not simply extend in radial directions.Fungal spore production is also influenced bywater activity. Maximum sporulation value ofPenicillum roquefortii was obtained at water activityof 0.96 i.e. at lower water activity maximumsporulation occurs. The influence of low water activityvalue on fungal fermentation is not well understood;at low water activity there is loss of essentialnutrients necessary for fungal growth. 13,14Production of secondary metabolites also dependson water activity. It was found that there is adirect relation between the amounts of enzyme producedvs. A w . In a solid culture medium of T. viridebiosynthesis of polygalactouronase, D-xylanase and- galactosidase is affected by water activity of substrate.It was found that maximum polygalactouronaseand D-xylanase production occur at A w of 0.99.However, -galactosidase formation was optimumbetween A w of 0.96 and 0.98. 15 Lipase productionby Candida rugosa was enhanced by mixed solidsubstrate with initial A w value of 0.92. 16 A w also effectsthe fungal aroma production, bioconversion of2-heptanone from octanoic acid by T. viride wasoptimum at water activity value of 0.96. 11Moreover, higher A w also enhanced substrateconversion to fungal biomass as reduced water activitycauses lower mass transfer and little wateravailability for microorganisms. This results inspore production. Increase in A w of substrate increasesspecific growth rate and spore germinationtime of the fungus. 5Grajek and Grevais reported that decrease ofA w by 0.01 (equivalent to 1 % of relative air humidity)causes reduction in biomass production andprotein content of culture medium. 17 Naraharafound that optimum A w for Aspergillus spp. was between0.970 and 0.990. 18 Fungus was unable togrow below A w of 0.97. These data prove that fungalgrowth and their secondary metabolite productionduring SSF are strongly affected by A w of substrate.11,17,18Temperature and heat transferFungal growth and secondary metabolite productionin SSF are greatly influenced by temperatureand heat transfer processes in the substrate bed.During SSF a large amount of heat is generated,which is proportional to the metabolic activities ofthe microorganism. However, fungus can grow overa wide range of temperatures from range of =20°Cto 55 °C. Nevertheless, optimum temperature forfungal growth could be different from that requiredfor product formation. 19 The substrates used forSSF have low thermal conductivities that decreaseheat removal and increase its accumulation. Therefore,the key issues in SSF are heat removal, andhence most studies are focused on maximizing heatremoval.The heat transfer in or out of an SSF system isclosely associated with microbial metabolic activityand aeration of the fermentation system. High temperatureeffects fungal germination, metabolitesformation and sporulation. 20 The effect of temperatureon kinetic parameters of T. ressei QM9414have been estimated by measuring radial growth offungus, its glucosamine content and specific respirationactivity using wheat bran as substrate. Thefungal activity declined exponentially when optimumtemperature for growth reached above maximum.21Mitchell and Meien used modeling experimentsto study the potential of Zymotis packed bedbioreactor. The rate of cooling was higher at the topof the bed as compared to the bottom. Vertical temperaturegradients were decreased. Bioreactor performancewas estimated by determining the time requiredfor volume average biomass concentration to

S. BHARGAV et al., Solid-state Fermentation: An Overview, Chem. Biochem. Eng. Q. 22 (1) 49–70 (2008) 51reach 90 % of maximum biomass concentration.The highest biomass concentration was above thebottom of the bioreactor where inlet air temperatureis lower than optimum temperature. The biomassnear the top was less than maximum biomass indicatingunfavorable temperature. 22Coupled control of temperature and moisture isalso an important issue for consideration. The lowmoisture and poor thermal conductivity of the substratemakes heat transfer and temperature controldifficult in SSF. Minimizing the substrate bedheight can solve heat transfer problems, but it ispossible only in small-scale solid-state fermentation.Good mixing of substrate with sparged oxygencan also solve heat transfer problems. Moreovermixing not only aids in the homogeneity of the bedbut also ensures an effective heat and mass transfer.Continuous mixing along with addition of water isadvantageous for simultaneous control of temperatureand moisture in large-scale SSF. 20 Adjustingthe aeration rate controls temperature during evaporativecooling in SSF. If the temperature is too low,the aeration rate decreases, which increases temperaturein the bed due to respiration of the microorganism.But if temperature is too high, increasingthe aeration rate promotes cooling of the substrate.23,24,25 However, with larger size particles orby forming aggregates of available agro-industrialresidues, such as in the case of wheat bran, heat removalcan be achieved with forced cold aeration, aslarger particles have greater space between them.Biomass and growth kineticsBiomass is a fundamental parameter for characterizingthe fungal growth and is essential formeasuring growth kinetics in SSF. However, culturingfungus over membrane filter can perform directestimation of biomass. A disadvantage of thismethod is that the membrane filter prevents penetrationof fungal hyphae within the substrate. 26 Fungalbiomass in an SSF system can be estimated bydifferent techniques, like scanning electron microscope,reacting fungal biomass with specific flourochromeprobes, respiration rate of organism, andreflectance infrared (IR) spectroscopy. 27,28,29Determination of fungal biomass in SSF canalso be done through some biochemical methods.Changes in three fungal constituents such as Glucosamine,Ergosterol and total sugar representsthe effect on fungal biomass. Among these, Glucosamineis considered a good biomass indicator ifthe fermentation media has the same constituentsbut not the same carbon-to-nitrogen ratio. Theamount of Ergosterol varies throughout developmentof fungus with different medium compositionand appears only when the fungi sporulate. Hence,it does not allow estimation of mycelial growth butit is a good indicator for sporulation. In conclusion,Glucosamine is the most reliable measure formycelial biomass determination but it can only beused for optimization studies of nutrient concentration.Ergosterol is, however, an unreliable measureof biomass but when determined along with Glucosaminegives useful information regarding sporeamount and fungal sporulation ability. Estimationof total sugar is not a good biomass indicator becauseit depends on fungus age and medium composition.The disadvantage of these manual biochemicalmethods are that they are time consuming,and require lengthy analytical procedures. 29Direct methods such as measurement of CO 2 orO 2 consumed are most powerful when coupled withthe use of a correlation model, which correlates biomasswith a measurable parameter. 30 Desgranges etal. estimated the biomass in solid-state fermentationby Infrared measurement of cell components suchas Glucosamine, Ergosterol, medium residues (sucrose,nitrogen) and CO 2 evolution rate. Glucosamineestimation is not used when medium containsinsoluble nitrogen. When manual methods forbiochemical estimations are not possible, the CO 2production rate and IR measurement are more beneficial.In SSF system CO 2 production correlateswell with other parameters. Moreover, it can indicateeven low physiological activity since themethod is very sensitive. 31Mass transferIn SSF the fungal hyphae forms a mat on thesubstrate surface and penetrates by secreting secondarymetabolites and enzymes. 32,33 Interparticle concentrationgradients due to nutrient consumption incombination with mass transfer limitations can havea strong effect on the rate and efficiency of the process.34,35,36 Mass transfer in SSF involves micro-scaleand macro-scale phenomenon. Micro-scale masstransfer depends on the growth of microorganismswhich depends on inter and intra particle O 2 and CO 2diffusion, enzyme, nutrient absorption and metabolitesformation. Macro-scale mass transfer includesairflow into and out of the SSF system, types of substrate,mixing of substrate, bioreactor design, spacebetween particles, variation in particle size and microorganismswithin the SSF system. 20,37,38Micro-scale mass transferLimitation of O 2 affects the aerobic SSF performance.In SSF fungal mycelia develops on asolid surface and in a void area within the substrate.Fungal mycelia are filled with water and oxygen inthe space between substrate. 39 Oxygen consumptiontakes place at interface between the substrate parti-

52 S. BHARGAV et al., Solid-state Fermentation: An Overview, Chem. Biochem. Eng. Q. 22 (1) 49–70 (2008)cles and fungal hyphae. Several researchers havemodeled O 2 transport between the water and fungalhyphae by considering fungal hyphae as a biofilmof unicellular organisms. Mitchell et al. studiedintra-particle oxygen transfer of Rhizopus oligosporusin SSF conditions on a nutritionally definedmedium and reported that O 2 transfer depends onthe interfacial gas-liquid surface area and thicknessof the wet fungal layer. These two factors play animportant role for O 2 transfer in the SSF system.Similar to O 2 transfer, CO 2 diffusion takes placefrom fungal hyphae through the void area of thesolid substrate. 32Apart from O 2 ,CO 2 diffusion micro-scale masstransfer includes transfer of nutrients, enzymes, secondarymetabolites and different metabolic products.Enzymes secreted from the fungal hyphae actupon the complex organic substrate converting itinto simplified carbon sources, which are again utilizedby fungus. Therefore, in SSF there is masstransfer of enzymes from fungus to substrate andtransfer of nutrients from substrate to fungus. WhenRhizopus oryzae is cultured on a complex tanninsubstrate, the secreted tannase breaks the substrateinto glucose and gallic acid, and the glucose is utilizedby the fungus as a carbon source. 37Macro-scale mass transferMacro-scale mass transfer is directly influencedby type and design of the bioreactors used inSSF: transfer rate of O 2 ,CO 2 into and out of thebioreactor, transfer of water in the bioreactor, mixingspeed of solid substrate, water and microorganisms,thickness of bioreactor wall, and temperatureof the cooling agent in the bioreactor. The optimumdesign and suitable selection of a bioreactor is requiredfor maximum mass transfer process. 20 However,a particle simulation model for optimizingbetter design of rotating drum bioreactor was developedby Schutyser et al. and particle simulationmodels provide better details about the transportprocess and optimizes high mass transfer. 40ModelingMathematical modeling in the SSF system is animportant tool for optimum design and operation ofbioreactors. The cost procured in predicting fermentationconditions at larger scale eases with modeling.Models in bioreactor can be of two types: Kineticmodels and Transport models. A kineticmodel describes how the microorganisms are influencedby different process parameters, while thetransport model describes the mass and heat transferwithin the bioreactor SSF systems. Kinetic modelingdepends on particle size, packing density, respirationrate, pore size of substrate particles, depthof fungal mycelium penetration within the substrate,and water content of the substrate bed. Transportmodeling depends on the growth rate of fungus,rate of airflow through the bed, width of bioreactorwalls, height of the bed, and rate of the heat removalprocess. Moreover, mathematical modelshelp in finding the relationship between O 2 and thesubstrate consumption rate, substrate consumptionrate and biomass synthesis rate, oxygen uptake rateand biomass synthesis rate, heat evolved and its relationwith the biomass synthesis rate.Modeling and simulation of biological processesprovide a basis for economic and ecologicalevaluation, which enables integrated optimizationof the processes. This helps in prediction of the processat larger scale. Ecological assessments arebased on material and energy balance of the processto identify the most relevant materials and processsteps. 41Microbial growth kinetics along with operatingvariables such as moisture, temperature, product accumulation,heat and wastes accumulation play animportant role in continuous SSF processes. Lagemaatand Pyle, attempted to develop an unstructuredgrowth model, to form a basis of continuoustannase production. The model described the uptakeand growth kinetics of Penicillum glabrum on inertimpregnated polyurethane foam. The time delay betweenbiomass production and tannase and sporeformation was described using logistic kinetics,since tannase is not produced after stationaryphases. 42Determination of biomass production throughfungal indicators such as Glucosamine, Ergosterolis time-consuming but using sensitive methods likedetermining respiration saves time and improves accuracyof the process. A kinetic model for monitoringbed conditions in SSF was developed using respiratorygases such as O 2 ,CO 2 . The model wascalibrated using Gibberella fujikoroi on wheat branstarch in an agitated aseptic SSF bioreactor. Thevarious assumptions were divided into 4 groups: kinetic,evaporation, energy balance, and water balancemodules. The CO 2 production rate is a directindicator for biomass growth. First CO 2 productionrate, water content and bed temperature were obtainedthrough water and energy balance models,but due to the complexity of the process these estimationswere considered to result in significanterrors. Determination of the CO 2 production ratethrough kinetic models reduced the chances oferrors and noise production. Simulations in singlestep were enabled with help of kinetic models. 43A mathematical growth model for convertingthe absolute mass ratio to relative mass ratio was

54 S. BHARGAV et al., Solid-state Fermentation: An Overview, Chem. Biochem. Eng. Q. 22 (1) 49–70 (2008)and unloading of the reactor. The aim of Zymotis isto minimize vertical temperature gradients andmaximize the heat transfer through cooling water. 22In conclusion, most researchers for bioreactordevelopment have used mathematical modeling atmicroscopic scale using simple logistic kinetics.Logistic equations are simpler than complex partialdifferential equations and represent whole datawithout complexity. Modeling at microscopic leveldirectly affects heat and mass transfer in bioreactors,which affects scale-up of the bioprocess.Continuous operating bioreactors based on agro-industrialresidues are to be modeled. These bioreactorsshould have arrangements for substrate addition,water replacement and air inlet. However,validation of artificial results at smaller scale is necessaryin reality at a larger scale for success of themathematical modeling.BioreactorsIn fermentation processes, bioreactor systemsprovide the environment for growth, cultivation ofmicrobes. However, some of the factors affectingthe growth of the product in SSF bioreactors aretemperature, humidity of substrate bed, type of substrateused, size of the bioreactor, aeration, coolingrate, height of bed and fungal morphology. Whencompared to submerged fermentation, SSF is carriedout in simple bioreactor systems; SSF bioreactorsare fitted with a humidifier and with orwithout an agitator unit. Poor thermal conductivityof the substrate bed presents a great challenge tobioreactor design, but composition, particle size,porosity and water-holding capacity of the substrateused also affects the bioreactor.Various researchers have classified the SSFbioreactors broadly 20,50,52,53 but most bioreactors canbe distinguished by a factor whether they are usedat small scale and large scale.Small-scale bioreactorsSolid-state fermentation at laboratory scale iscarried out in Petri dishes, jars, wide mouthed Erlenmeyerflasks, roux bottles, and roller bottles. Thesesystems are simple and experiments are carriedout easily. 53 There are several forms of small-scalebioreactors such as column bioreactors. MaurisRaimbault and J. C. Germon in ORSTOM (InstitutFrançais de Recherche Scientifique pour le Developpmenten Cooperation, France) laboratory ofsoil microbiology designed it in 1980, for growingAspergillus niger on cassava meal in solid-state.Column bioreactors consist of small columns (diameter:D = 22 mm, height: H = 210 mm) whichcan hold 20 g of pre-inoculated solid material. Approximately24 such columns were put together inthermo-regulated water baths and water-saturatedair was passed through each column at a flow rateof Q = 4–6 Lh –1 . Column bioreactors are usefulfor optimization of the medium, and constitute animportant part of research. However, difficulty liesin obtaining the product and poor heat removal.Pandey et al. 54 determined the performance of thecolumn bioreactor for glucoamylase production.Columns of different diameters were used and arrangedwith different substrate bed heights of h =4.5, 9, 18, 22.5 cm. Enhancement in substrate bedheight increased the enzyme synthesis. The columnwith 18 cm of bed height produced maximum enzymein 48 h with an aeration rate of Q a = 1 to 1.5LL –1 min –1 . However, with further increase in bedheight, enzyme synthesis decreased. The decreasemay be due to the reduced aeration rate with increasedheight of the substrate bed.A few years later the INRA (Institut Nationalde la Recherche Agronomique, Dijon, France) teamdeveloped a reactor with one liter working volumefitted with a relative humidity probe, a cooling coilin heating circuit, and a cover for the reactor vessel.These reactors were filled with pre-inoculated solidmaterial, and a computer controlled all parametersof the reactor. Sampling for analysis was easier inINRA bioreactors. This was the advantage overORSTOM bioreactors. The automatic control ofrelative humidity and temperature makes these reactorsuseful in scale-up studies.Apart from non-agitated column reactors, thereare bioreactor systems with agitator systems such asperforated-drum reactor and horizontal paddlemixer (with or without water jacket). Drum bioreactorsare designed to mix the solid substrate byrotating horizontal rotation vessels (which are withor without baffles). The rotation or agitation createstumbling in the matrix of the solid medium by minimizingthe heat produced. However, mixing ofinoculum with substrate is uniform in drum bioreactorsbut fungi morphology is readily damagedby high shear, stress is not used in such bioreactors.However, an intermittent mixing strategy is involvedin these bioreactors, which gives balancebetween positive effects of increasing temperatureon deleterious effects on fungal hyphae. 52,55 A paddlemixer was developed by Wageningen Universityof Agriculture. It consists of a number ofblades making mixing more efficient than rotatingdrum. 52Various advancements have been made throughrotating drum bioreactors. Kalogeris et al. have developeda laboratory scale intermittent agitation rotatingdrum type bioreactor for SSF of thermophillicorganisms. The main parts of the apparatusare: perforated (pore size, S =1mm 2 ) cylindrical

S. BHARGAV et al., Solid-state Fermentation: An Overview, Chem. Biochem. Eng. Q. 22 (1) 49–70 (2008) 55drum (diameter, D = 0.15 m; length, L = 0.59m; capacity,V = 10 L), vessel with water jackets, motorconnected to controller, heat exchanger, condenserat air outlet, and humidifier. 56 Kalogeris et al. determinedthe efficiency of the bioreactor by producinghemicellulases and cellulases with a thermophillicfungus, Thermoascus aurantiacus, usingwheat straw as substrate. He examined the effectsof temperature, moisture and aeration. Among thevarious kinetic models tested, Le Duy model describedthe relationship between T. auranticusgrowth and enzyme production. However, increasein aeration rate and high moisture levels favored theenzyme and enzyme production. The bioreactor canbe used for production of hydrolases from thermophillicorganisms due to their superior thermo-stability,optimum activity at elevated temperatures,and high rates of substrate hydrolysis. 57A Zymotis packed bed reactor was developed atlaboratory scale by the ORSTOM team. Zymotisbioreactors are a modification of packed bed bioreactorswith internal plates in which cold water circulatesat an optimum temperature. Variation in optimumtemperature retards the initial growth of theculture used. In packed bed bioreactor, large axialtemperature gradients arise leading to poor microbialgrowth at the end of the base near the outlet,with an increase in vertical temperature gradients. 22Another bioreactor (Growtek) was developedby a team of scientists at IIT (Indian Institute ofTechnology) Kharagpur, India, for plant tissue culturebut later it was used for solid-state fermentation.This bioreactor has a cylindrical vessel (widthD = 11.3 cm, height H = 16 cm) with a spout at thebase, inclined at an angle of = 15° having a diameterof d = 2.6 cm and length of l = 8.5 cm. Bothvessel and spout have lids. A float of area A =72cm 2 is provided inside the vessel, which consists ofa perforated base. Fermentation of solid substrate iscarried out on a float to which the seed culture isadded, while liquid salt solution is kept below thefloat. An advantage of the Growtek bioreactor isthat the inoculated solid comes into direct contactwith the liquid medium. The products formed duringthe fermentation process leach in liquid medium.This type of bioreactor was used for productionof gallic acid from tannin-rich solid material. 37Ahamad et al. used a Growtek bioreactor for productionof mevastatin by Penicillium citrinum fromwheat bran. 58Large scale bioreactorsThe successful operation of large-scale bioreactorsdepends on design features obtained aftermathematical modeling, susceptibility of the substrateand fungal morphology to increase in temperature,effect of particle size and voids betweenthem, quantity of the substrate used, and height ofthe substrate bed. There are several forms oflarge-scale bioreactors used in SSF.Koji types of bioreactors are the simplest andwithout forced aeration. These types of bioreactorsare also known as tray bioreactors. The trays aremade of wood, plastic, metal. It is not necessarythat trays should be perforated. Trays are arrangedone above the other with suitable gaps betweenthem and placed in climatic controlled chamber undercirculating air, which maintain uniform temperature.An advantage of this technology is that by increasingthe number of trays, the scale-up is easier.However, the requirement of sterility, large spaceand labor makes the process difficult. Rodriguez etal. reported the tray bioreactor to be appropriate forproducing laccase by T. versicolor in solid-stateconditions operating with lignocellulosic supportsbesides their several disadvantages. 59 Rodriguez etal. also proved superiority of grape seeds over nylonsponge supports using tray configuration duringlaccase production (with Trametes hirsuta underSSF conditions). 60 A Shallow tray fermentor wasused at laboratory scale for cellulase productionwith Trichoderma reesei ZU02 strain on corncobresidues. 61However, BIOCON India has used this technologyfor large-scale production of immuno-suppressants.They have simplified problems of sterilityby keeping the trays in HEPA (High EfficiencyParticulate Air) filtered air, using automated machinesfor layering the substrate in trays.Packed bed bioreactors are modifications oflaboratory-scale column reactors. A sieve present atthe bottom is used for holding the substrate. Passingforced air through the static bed provides aeration.Heat produced during fermentation within the substratebed is a major limitation of these reactors.However, reduction in water content within the substratebed increases its hardening. This decreasesfungal penetration within the bed, causing reductionin product formation. Hence, it is advantageous touse thermo-stable fungus for producing a thermo--stable product (especially enzymes) in packed bedreactors. Heat produced in the packed column canbe reduced by increasing the moisture of air insideit, which is achieved by passing cold saturated airor using heat exchangers as in Zymotis. Pressuredrop is an important feature of the packed reactor.High-pressure drop reduces airflow from the substratefavoring channeling within the bed. 53 Thechanneling decreases optimum temperature requiredfor growth of organisms in the substrate bed.Some large-scale bioreactors like PLAFRACTOR 62use heat exchangers for controlling temperatureduring fermentation. However, enhancing agitation

56 S. BHARGAV et al., Solid-state Fermentation: An Overview, Chem. Biochem. Eng. Q. 22 (1) 49–70 (2008)in the unmixed static bed solves the problem of heatremoval and scale-up. Oostra et al. 50 using themodel of a scraped drum reactor have proved this.Another bioreactor used at industrial scale isthe rotating drum bioreactor (RDB). The RDBmixes the substrate with the cultivating organism,while rotation can be continuous or intermittent dependingon the bed height and speed of rotation.Some critical factors affecting RDB are type of substrate,height of bed, fungal physiology, and size ofsubstrate particles. Incorporation of baffles may improvethe operation of RDB. Various researchershave studied the impact of baffles within RDB.Schutyser et al. have used discrete particle simulatorsto predict that curved baffles have better radialmixing characters than other designs. Curved bafflesare independent of particle rotation, and continuousrotation of drum is possible with them. 40 RDBare characterized as mixed bioreactors. However,with higher shear rates and rotational speed, thefungal physiology may be destroyed. 20,50,63In fluidized bed reactors, the solid substrate isfluidized by upward airflow. The bioreactor operateson the flow of air and velocity with which thesubstrate moves upwards. Sufficient velocity of airis supplied to fluidize the solid substrate. The columnof the bioreactor is high enough for bed expansion.Widening of the column near the top allowsdisengagement of solids from the gas stream. Maintenanceof uniform conditions throughout the substrateand increase in surface area is an advantageof this bioreactor.After many advances in SSF reactors, heat controland scale-up still require consideration throughmodeling.Substrates used in solid-statefermentationFor SSF processes, different agro-industrialwastes are used as solid substrates. Selection ofagro-industrial residues for utilization in SSF dependson some physical parameters such as particlesize, moisture level, intra-particle spacing and nutrientcomposition within the substrate. In recentyears, some important agro-industrial residues suchas cassava bagasse, sugarcane bagasse, sugar beatpulp/husk, orange bagasse, oil cakes, apple pomace,grape juice, grape seed, coffee husk, wheat bran,coir pith etc. have been used as substrates forsolid-state fermentation.Cassava bagasse and sugarcane bagasse offeran advantage over other substrates such as ricestraw, wheat straw, because of their slow ash content.64 One of the factors making cassava an optimumsubstrate for SSF is its high water retentioncapacity. 65 Cassava bagasse is highly used for productionof citric acid, flavors, mushrooms, and differentbiotransformation processes. However, incomparison to sugarcane bagasse, cassava offers anadvantage as it does not require pretreatment, andcan probably be decomposed by most organisms forvarious purposes. 64 Some experiments have provedthat the protein mass fraction of cassava may be improvedfrom w = 1.67 % to 12 % by carrying out itsbiotransformation with tray bioreactors. 54 However,sugarcane bagasse and wheat bran are used forcommercial production of most compounds usingSSF, but their potential is still to be explored completely.Sugarcane bagasse has many efficient andeconomical applications, such as production of protein-enrichedcattle feed and protein. 64 Pretreatmentof sugarcane bagasse is an important aspect sinceassimilation and accessibility of microorganisms tosubstrate becomes easy, thus resulting in decompositionof hemi cellulose and lignin. 66Another upcoming agro-industrial substrate iscoffee pulp/husk due to its rich organic nature andhigh nutritive value. Obtaining coffee pulp or coffeehusk usually depends on the method employedfor refining the coffee cherries, i.e. wet or drymethod. However, fungi Basidiomycetes are foundmostly in husk rather than in pulp. 67Okara (soybean curd residue), rich in water-insolubleingredients, is a useful substrate for microbialfermentation. The main disadvantage of okarais natural spoilage when it is not refrigerated. Dehydrationof the soybean curd residue has been reportedto improve its utilization. Ohno et al. successfullyproduced an antibiotic iturin from okara. 68Substrates such as agro-industrial residues areproved by many researchers to be better for filamentousfungi. The morphology of filamentousfungi supports them to penetrate the hardest surfacedue to the presence of turgid pressure at the tip oftheir mycelium. Hence, the raw materials consideredas waste are used for production of valueadded fine products and reducing pollution problems.69Applications of solid-state fermentationProduction of enzymes by SSFEnzyme production is one of the most importantapplications of SSF. SSF has advantages oversubmerged fermentation such as high volumetricproductivity, low cost of equipment involved, betteryield of product, lesser waste generation and lessertime consuming processes etc.The type of strain, culture conditions, nature ofthe substrate and availability of nutrients are the

S. BHARGAV et al., Solid-state Fermentation: An Overview, Chem. Biochem. Eng. Q. 22 (1) 49–70 (2008) 57other important factors affecting yield of enzymeproduction. 70 It is crucial to provide optimized watercontent and control the water activity for goodenzyme production. Agro-industrial substrates areconsidered best for enzyme production in SSF. Thecost of enzyme production by submerged fermentationis higher compared to SSF. Tangerdy et al.have also proved this by comparing cellulase productioncosts in SSF and SMF. 71ProteaseVarious attempts for producing acid, neutraland alkaline protease have been done using agro-industrialresidues with SSF, depending on their catalyticactivity and optimum pH. Proteases are producedby fermentation of agro-industrial substratessuch as rice bran, wheat bran, soy meal, green gramhusk, rice husk, spent brewing grain, coconut oilcake, palm kernel cake, sesame oil cake, jackfruitseed powder, olive oil cake, lentil husk etc.Ikasari and Mitchell 72 used rice bran for producingacid proteases with Rhizopus oligosporus, asno toxin production occurred during SSF. Acid proteaseproduction has also been obtained using bacteriaMucor bacilliformis in SSF. 73 Tunga et al.produced extra-cellular alkaline serine proteaseusing Aspergillus parasiticus and Rhizopus oryzaeNRRL-21498 with wheat bran as substrate. 74Prakasham et al. obtained maximum alkaline proteaseproduction from green gram husk. 75 The alkalineproteases obtained using SSF of Aspergillusparasiticus was thermo-stable at = 50 to 60 °C,high pH, in the presence of surfactants, metals andoxidizing agents. 74 However, most researchers haveused wheat bran for production of alkaline protease.Protease production in SSF is higher than withsubmerged fermentation. Down streaming and extractionof the product is cheaper in SSF due tolesser water content of the substrate. Bacillussubtillis obtained from excreta of larvae Bruckdaolepachymerusnucleorum was also used for proteaseproduction using soy cake as substrate. Maximumprotease productivity was P = 15.4 U g –1 h –1 forSSF but it was P = 1.3 U g –1 h –1 . The enzyme productivitywas 45 % higher in SSF. 76 Some criticalfermentation parameters for obtaining maximumprotease production are accumulation density ofmicrobe within the substrate and height of the substratebed. Sandhya et al. studied the effect of parameterssuch as incubation temperature, pH,inoculum size and incubation time on neutral proteaseproduction through submerged fermentationand SSF. In solid-state, the total enzyme yield was31.2 U enzyme per gram of fermented substrate, butin submerged fermentation the yield was 8.7 U enzymeper gram of fermented substrate. The yield inSSF was 3.5 times higher than in SMF. 77 Similarly,George et al. compared the production of proteasesby Bacillus amyloliquefaciens in solid-state andsubmerged fermentation conditions. In submergedstate, 8 · 10 6 units of enzyme were obtained ina V = 20 L fermentor under optimal conditions.However, in solid-state the enzyme production was25 · 10 4 units g –1 . They reported SSF to be simplerin operation than submerged fermentation. 78LipaseIn recent years, considerable increase in lipaseproduction from microbes and agro-industrialwastes using SSF has gained importance. However,fungi are best lipase producers. 79 Most researchershave used wheat bran for maximum lipase production.Kamini et al. used gingelly oil cake for theproduction of lipase by Aspergillus niger. However,supplementation of various nitrogen sources, carbohydratesand inducers to substrates was ineffective.80 In contrast, SSF of babassu oil cake withPenicillum restrictum proved that lipase activity dependson the type of supplementation. Enrichmentof high carbon containing substances such aspeptone, olive oil, resulted in high lipase activity. 81Enhancement in lipase activity by olive oilsupplementation has also been proved by Cordovaet al. 82 The fungal spp. Rhizopus oligosporus hasbeen found to be the best mould among 10 differentfungal cultures for production of lipases without theaddition of supplements. 83Castilho performed the economic analysis ofsolid-state and submerged fermentation for productionof lipase using Penicillum restrictum. He reportedthat, for the production scale of 100 m 3lipase concentrate per year, a total capital investmentneeded for submerged process was 78 %higher than that for SSF. The unitary product costin submerged processes was 68 % higher than theproduct selling price. 84Mahadik et al. compared lipase production insubmerged and solid-state fermentation. In SMF asynthetic oil-based medium was used, but SSF usedwheat bran supplemented with olive oil, which resultedin increased lipase production. 79 Mateos Diazet al. reported that lipase from solid-state to bemore thermo-tolerant than from submerged fermentation(thermal stability, half life at = 50 °C wast = 0.44 h with SMF, and 0.72 h with SSF). Thetemperature at which maximum activity of lipasesoccurs was = 30 °C with SMF, and =40 °Cwith SSF. The lipase produced was from fungusRhizopus homothallicus. Process development requiresproper utilization of enzymes. Hence, theyshould show high functional stability. The enzymesobtained from thermophillic fungus have high ther-

58 S. BHARGAV et al., Solid-state Fermentation: An Overview, Chem. Biochem. Eng. Q. 22 (1) 49–70 (2008)mal stability, and activity at mesophillic temperaturesare generally utilized in chemical processes. 85Some of the agro-industrial wastes used in SSFfor lipase production are babassu oil cake, wheatbran, rice bran, gingelly oil cake, almond meal,mustard meal, coconut meal, rice husk, sugarcanebagasse, cassava bagasse, coconut oil cake, olive oilcake, etc.CellulaseCellulase is an enzyme complex used for theconversion of lignocellulosic residues and used forproduction of ethanol, single-cell protein, bleachingof pulp, for treatment of waste papers and for fruitjuice extraction. In SSF, using lignocellulosicwastes as substrates can reduce the cost of cellulaseproduction. 61 Lignocellulosic materials are cheaperand pretreatment is required to improve their utilization.Pretreatment of lignocellulosic matrix increasesthe potential of cellulases to act oncellulose, hemicelluloses.The concerted action of enzymes like endoglucanases,exoglucanases and - glucosidase isused for hydrolyzing cellulose. The rate-limitingstep is the ability of endoglucanases to reach amorphousregions within the crystalline matrix and createnew ends with which endoglucanases can act. 86Ethanol production from lignocellulose biomass requireshydrolysis by cellulase and hemicellulase forconverting lignocellulosic biomass to biofuel. 87Some factors like moisture content, particlesize, pH, incubation temperature, inoculum size, incubationperiod and enrichment of medium withcarbon and nitrogen were considered optimum forcellulase production by bacterial strain Bacillussubtilis on banana fruit stalk wastes. However, totalenzyme production was 12 times higher in SSFthan in submerged fermentation under similar experimentalcondition. 88 It is necessary to considerthese factors for cellulase production from lignocellulosicwastes as their nutrients are already depleted.Water content of substrate and aeration rate arecritical factors in cellulase production using SSF.Corncob residue was used for cellulase productionwith Trichoderma reesei ZU02 in shallow tray fermentors.Xia and Cen used a deep trough fermentorwith forced aeration for cellulase production.Forced aeration enhanced the mass transfer to agreater extent, which increased cellulase activity to305 IU per g of cellulose. 61 It has been reported byFujian et al. that substrates in solid-state with continuouscirculation of air and convective diffusionwith pressure are better for fungal propagation thanstatic cultures. This periodic air circulation increasesthe looseness of substrates and enhancescellulase activity. The filter paper activity of cellulaseenzyme increased to 20.4 IU g –1 at a bed heightof h = 9cm in t = 60 h, while maximum filter paperenzyme activity was 10.8 IU g –1 in 84 h withinstatic cultures. The work was performed usingsteam-exploded wheat straw as carrier with Penicillumdecumbens in SSF. 89 However, changes inthe amplitude of air pressure increased the oxygenavailability to the cultures used and heat removal.The variations enhanced the cellulase production byTrichoderma viride in SSF. 90Co culturing of two fungi in SSF enhances theenzyme production. Co culturing of Trichodermareesei mutants with Aspergillus spp. increased thecellulase production by 50 % and improved thecellulase glucosidase ratio, by partially removingproduct inhibition and its hydrolysis. 91 Co culturingAspergillus ellipticus and Aspergillus fumigatus resultedin improved hydrolytic and -glucosidaes activity.61,71 However, some newly developed agro-industrialwastes used for cellulase production arebanana wastes, rice straw, corn cob residue, ricehusk, wheat straw, banana fruit stalk, and coconutcoir pith.PectinasePectinases are constitutive or inducible enzymesproduced by microbes for breaking pectin.Different substrates used for production of pectinaseare wheat bran, soy bran, apple pomace, cranberrypomace, strawberry pomace, beet pulp, coffeepulp & husk, cocoa, lemon & orange peel, combinationof sugarcane bagasse and orange bagasse,wheat bran etc.Production of polygalactouronase (PG) andpectinesterase (PE) was 6.4 times higher in SSFcompared to submerged fermentation. PE and PGactivity was measured to be 500 U L –1 and 350UL –1 at 24 h of incubation with pectin as the solecarbon source with SSF, but in submerged fermentationenzyme production was 127 U L –1 and 55UL –1 at t = 48 h of incubation. Supplementation ofglucose decreased the production of enzymes due tocatabolite repression in submerged fermentation.However, PE and PG enzymes increased by 30 %and 33 % respectively with addition of glucose. 92Similarly, exopectinase activity increased from 623to 7150 (IU L –1 ) in SSF, but decreased from 1714 to355 (IU L –1 ) in submerged fermentation in the presenceof sucrose at A w of 0.995. Similar results wereat A w of 0.96. Increase in water activity increasedpectinase activity in SSF. 93 Production usingdeseeded sunflower head as substrate resulted instep-up of exopectinase and endopectinase enzymeusing SSF. In SMF the endopectinase was 18.9UmL –1 , which increased to 19.8 U mL –1 in SSF.

S. BHARGAV et al., Solid-state Fermentation: An Overview, Chem. Biochem. Eng. Q. 22 (1) 49–70 (2008) 59Similarly, exopectinase increased from 30.3 U mL –1in SMF to 45.9U mL –1 in SSF, at optimummoisture of s = 65 % and particle size of d = 500m. 94,95 Polyurethane, when used as inert supportfor exopectinase production using Aspergillus nigerin Erlenmeyer flasks, resulted in direct measurementof biomass production, substrate uptake andenzyme activity in culture medium. 93 Botella et al.used grape pomace for exopectinase productionwith Aspergillus awamori in SSF. Initial substratemoisture content of 65 % and supplementation of6 % glucose as carbon source enhanced enzyme activity.However, particle size did not influence theincreased enzyme production, which is contradictoryto earlier reports. 96,97Besides moisture, the production of pectinaseenzymes also depends on physical parameters ofthe bioreactor used, amount of substrate used, typeand pH of substrate and supplements added duringfermentation. A mixture of orange bagasse, wheatbran (1:1) was used for production of endo-polygalactouronase,exo-polygalactouronase and pectinlyaseby Penicillum viridicatum in Erlenmeyerflasks and polypropylene packs at 2 different moistures = 70 %, 80 %. However higher production ofall three enzymes was obtained in polypropylenepacks at s = 70 % moisture, but lower moisture contentwas highly significant for Pectinlyase production.Increase in pectinase enzymes using polypropylenepacks favored it for scale-up. 98Martins et al. used the Thermoascus auranticusfor pectinlyase and polygalactouronase production.The quantity of pectinlyase and polygalactouronasewas found to be higher when compared with otherpectinolytic strains such as Aspergillus niger,Penicillumitalicum,Aspergillus foetidus. However, additionof fibrous material such as sugarcane bagasseto the mixture of wheat bran, orange bagasse, resultedin intra-particle spacing, causing increase inaeration, nutrient and enzyme diffusion, which supportedpectinlyase production. These results provedthat media composition did not affect polygalactouronaseproduction. 99 Pectinase and polygalactouronaseusing Moniellia,Penicillum spp. wereproduced using orange bagasse, sugarcane bagasseand wheat bran as substrate. Addition of fibrousmaterial such as sugarcane bagasse caused interparticlespacing resulting in increase of aeration andnutrients supply. However, sugarcane bagasse asthe sole carbon source, did not allow the growth offungi, indicating that microorganisms are unable tohydrolyze cellulose, hemicelluloses fibers but theycan support mycelia formation. The sugarcane bagassewas used as the inert support for growth &pectinase production by Aspergillus niger,Penicillumviridicatum,Thermoascus auranticus. 100Solid substrates resulted in higher pectinaseproduction as they supply nutrients to microbialcultures along with anchorage. Nutrients, which areunavailable or are in sub-optimal concentration, areusually supplemented externally. A Bacillus sp.DT 7 was used for production of pectinase usingwheat bran, rice bran, apple pomace as substrates inErlenmeyer flasks. Maximal enzyme productionwas obtained from wheat bran supplemented withpolygalactouronic acid and neurobion. Supplementationof 27 L of neurobion enhanced pectinaseproduction by 65.8 %. 101PhytaseSeveral strains of bacteria, yeasts, fungi suchas Bacillus subtillis,E. coli,Bacillus amyloliquefaciens,Schwanniomycescastellii,Schwanniomycesoccidentalis,Hansenula polymorpha,Aspergillusflavipes,Aspergillus fumigatus,Aspergillus oryzaeand Aspergillus ficcum are employed for phytaseproduction in SSF systems. Some of the substratesgenerally used for phytase production are canolameal, coconut oil cake, wheat bran, black beanflour, cowpea meal, mustard cake, cotton cake,palm kernel cake, sesame oil cake, rapeseed meal,olive oil cake, groundnut oil cake etc.Phytase production increases with fermentationof mixed substrates. The increase may be due to theavailability of different nutrients from different substratesin the same reactor simultaneously. This maystep-up phytase production. Roopesh et al. usedSSF for phytase production from various substratecombinations of wheat bran and various oil cakesusing Mucor racemosus under SSF. Combination ofwheat bran and oil cake yielded greater Phytasewhen compared to their individual production. Optimizationof conditions resulted in 44.5 U g –1 ofphytase with the combination of wheat bran and oilcake, which were 1.5 times and 4 times higher, respectively,when the oil cake and wheat bran wereused as substrates. The type of carbon and nitrogensource used is an important factor for considerationin any fermentation process. 102 However, alongwith imbalance in the carbon-to-nitrogen ratio,many parameters affect phytase production such asincubation time, initial moisture content and incubationtemperature. Similarly, Ramachandran et al.used Rhizopus spp. with coconut oil cake, sesameoil cake, palm kernel cake, groundnut oil cake, cottonseedoil cake and olive oil cake for phytase production.Cottonseed oil cake and olive oil cakepoorly supported the phytase production individually,but mixed substrate fermentation of bothincreased enzyme production to 35 U/gds.Supplementation of glucose further enhancedphytase activity to 52 U/gds. Enhancement was re-

60 S. BHARGAV et al., Solid-state Fermentation: An Overview, Chem. Biochem. Eng. Q. 22 (1) 49–70 (2008)ported due to the combined effects of nutrients inthem. 103Various other substrates like wheat bran, oilcakes such as groundnut oil cake, canola meal,rapeseed meal and soybean meal when supplementedwith surfactants resulted in higher productionof phytase. Aspergillus ficcum was used forphytase production on canola meal using SSF. 104Extra cellular phytase produced using thermo-tolerantAspergillus niger was maximum with cowpeameal. Increase in membrane permeability of fungushas been reported to be the optimum cause forphytase enhancement in SSF. 105L-glutaminaseL-glutaminase is used as antileukaemic and flavorenhancing agent. Solid-state fermentation wasfound to be more suitable than submerged fermentationfor biosynthesis of L-glutaminase using Pseudomonasflourescens, as 25-fold enhancement wasobtained. 106 The moisture of substrate highly effectsenzyme production. L-glutaminase was produced byyeast Zygosacharomyces rouxii using wheat branand sesame oil as substrate with s = 64.2 % moisture,t = 48 h old inoculum and = 30 °C incubationtemperature. Addition of 10 % NaCl and seawaterto wheat bran and sesame oil enhanced enzymeproduction. The enzyme produced fromwheat bran and sesame oil cake in dry state was 2.2and 2.17 U/gds but with supplementation, the concentrationincreased to 7.5 and 11.61 U/gds respectively.107 Beauveria spp. an alkalophilic and salt-tolerantfungus isolated from marine sediment, wasused for L-glutaminase production using seawater-basedmedium supplemented with L-glutamine(w = 0.25 %) as substrate. 108 Seawater being a naturalreserve for marine organisms can provide themsufficient nutrients when used as supplement in SSFfor production of industrially important enzymes.AmylaseAmylase has potential application in a numberof industrial processes such as food, fermentation,textiles and paper industries. The two major classesof starch degrading amylases are glucoamylasesand -amylase. Solid-state fermentation holds tremendouspotential for production of these enzymesindustrially on a large scale.Production of -amylase is not limited to fungalcultures but it is also done by bacterial culturesof genus Bacillus. Bacterial cultures reduce the timerequired for fermentation, reducing a lot of expenditureinvolved. Wheat bran has been reported to bethe best substrate even for bacterial cultures. Babuand Satyanarayana produced -amylase with Bacilluscoagulans in SSF using wheat bran as substrateand supplemented with tap water as moisteningagent. They compared the production of enzyme indifferent vessels. 109 Ramesh and Lonsane produced-amylase with Bacillus licheniformis M27 underSSF conditions, using wheat bran as substrate. 38An advantage of SSF over submerged fermentationis the inhibition of catabolic repression byregulation of end product synthesis from the productformed. -amylase production by Bacilluslicheniformis decreased from 420 to 30 units mL –1with increase in fraction of starch from 0.2 to 1 %in SMF. However, in SSF the enzyme activity enhanced29-fold with a 42 times increase in concentrationof starch. Hence, inhibition of enzyme byproduct is overruled in solid-state fermentation. 110Ramachandran et al. used coconut oil cake, a dryproduct obtained from copra for production of-amylase with Aspergillus niger in SSF.Supplementation with starch, peptone and glucoseincreased the enzyme synthesis. 111Glucoamylase is still produced by large-scalesubmerged fermentation, but the production cost remainshigh in all respects. There are less reports ofglucoamylase production in SSF. However, cost ofproduction is lower in SSF. Cost reduction can beachieved by using cheaper substrates such as agro--industrial residues. Selection of the proper substrateis an important parameter for maximum enzyme activity.Ellaiah et al. optimized different substrateslike wheat bran, green gram husk, black gram bean,barley flour, jowar flour, maize bran, rice bran, andwheat rawa for maximum glucoamylase productionwith Aspergillus spp. under SSF. However, wheatbran showed maximum enzyme activity to be 247Ug –1 . Supplementation of w = 1 % fructose andw = 1 % urea enhanced enzyme activity. A veryhigh moisture content of 80 % favored maximumenzyme production. 112 Tea waste was used as substratefor maximum glucoamylase production usingAspergillus niger. Supplementation of tea wastewith mineral solution containing salts such as K,Mn, Mg, Zn and Ca resulted in 198.4 IU/gds ofenzyme. The results were at t = 96 h with 4 %inoculum, 60 % moisture, and pH of 4.5. Supplementationwith w = 1 % sucrose enhanced enzymeactivity to 212.6 IU/gds at 96 h. Further addition ofw = 1 % malt extract increased enzyme activity to226 IU/gds at 72 h after inoculation. 113Anto et al. produced glucoamylase using riceflakes (categorized as coarse, fine and medium),wheat bran and rice powder as substrates withAspergillus spp. HA-2. Supplementation of sucroseas carbon source to wheat bran and glucose tocoarse, medium waste enhanced enzyme production.Addition of yeast extract and peptone to thesubstrates also enhanced enzyme production. Maximumenzyme production was obtained in wheat

S. BHARGAV et al., Solid-state Fermentation: An Overview, Chem. Biochem. Eng. Q. 22 (1) 49–70 (2008) 61bran (264 0.64 U/gds) but glucoamylase productionin different categories of rice flakes varied.Maximum enzyme was produced in coarse (211.5 1.44 U/gds) and medium wastes (192.1 1.15U/gds). The factors affecting enzyme activity withcombination of wastes were attributed to the increasein agglomeration of particles, which resultedin reduced aeration and penetration by fungalmycelia. 114Nutrient supplementation from organic sourcesincreases enzyme production to a greater extentthan inorganic sources. It has also been found thatmost researchers used wheat bran as substrate for-amylase production because it contains sufficientnutrients, it remains loose even in moist conditions,and has a larger surface area. Due to these factors,aeration and mycelial penetration are easier inwheat bran. 109LigninaseLignin is a biopolymer with complex phenylpropanoidstructure and contributes to environmentalpollution. The most common organisms forlignin degradation are white rot fungi. However, activityof Phanerochaete chrysosporium is found tobe most suitable for efficient lignin degradation.Enzymes secreted by white rot fungi are ligninases.They are Lignin Peroxidase (LiP), ManganesePeroxidase (MnP) and H 2 O 2 generating enzymes.Secondary metabolism of these enzymes is triggeredby C, N, S depletion. 115,116The respiration rate of an organism is indicatorof the organism’s activity. Cordova et al. determinedthe relation between the CO 2 evolution rateand enzymatic activity of fungus when grown onsugarcane bagasse pith. There was an increase inCO 2 evolution rate with increase in metabolism. Activityof manganese peroxidase (MnP) was expressedin the idiophase where the residual glucoselevel was the least in the medium. The MnP activityenhanced and then decreased rapidly due to simultaneousprotease activity. Lignin peroxidase activitywas reported during the exponential phase of theorganism’s growth. However, it decreased after fungalsecondary metabolism. This indicates that LiP isless sensitive to MnP for proteolytic action. 117 CO 2generation took place after uptake of O 2 by fungus.However, LiP and MnP activities cannot be studiedin an anaerobic media.Fujian et al. compared lignolytic enzyme activitiesin submerged and solid-state fermentation usingsteam-exploded wheat straw as substrate. LiP,MnP activities in optimum conditions were 61.67UL –1 and 27.12 U L –1 in submerged fermentation,but in solid-state the activities were 365.12 U L –1and 265 U L –1 , respectively. However, in SSF themaximum enzymatic activities reached 2600 U L –1(LiP) and 1375 U L –1 (MnP). 118 This proves the feasibilityof solid-state over submerged fermentation.Developing semi solid-state conditions withinthe bioreactor also enhances lignin enzymes.Dominguez et al. developed a bioreactor based onrotating drums for producing lignolytic enzymes.The enzyme activities at 1 L L –1 min –1 of air werefound to be 1350 U L –1 (LiP) and 364 U L –1 (MnP)respectively. Semisolid-state conditions were maintainedby using nylon sponges for providing inertsupport, which is rotated by regularly passingthrough the nutrient medium. Nylon sponges wereused due to its hydrophobicity, porous nature, affinityfor fungus and moisture retaining capacity.Higher aeration rate avoided the support cloggingand made nutrient availability to organism easier. 116Similarly, Couto et al. used polypropylene spongesas inert support for lignolytic enzyme production. 115Lignin-degrading enzymes were obtained frommushroom Bjerkandera adusta by immobilizing iton polyurethane foams. 119Degradation by lignin enzymes is a non-specificreaction on the basis of free radicals resultingin destabilization of bonds and finally breaking ofmacromolecule. This characteristic increases thepotential for chemical industries; environmental industries;coal industries and extracting importantmetabolites from natural sources.XylanaseDifferent food processing with products suchas straws and brans of different cereals, corn, hulland cobs, sugarcane and cassava bagasse, varioussaw dusts and different fruit processing and oil processingresidues have been used for producingxylanase. Xylanase production requires substratesin very high concentration, with prominent waterabsorbance capacity. Xylanase are produced mainlyby Aspergillus and Trichoderma spp. 120,121 Xylanaseproduction was achieved successfully by Aspergillusfischeri,Aspergillus niger using wheat branand wheat straw as main substrates. 122,123Addition of nitrogen source as supplement isan important step for xylanase production. 123 Sodiumnitrite played a significant role in productionof alkali stable cellulose free xylanase by Aspergillusfischeri. Xylanase in conjunction withcellulolytic enzymes is used for bioconversion oflignocellulosic material to produce fuels and otherchemicals. 122The Koji process was used for productionof xylanase by Aspergillus sulphureus using drykoji. 124 Ghanem et al. produced xylanase usingAspergillus terreus on wheat straw medium. 55Topakas et al. used the Sporotrichum thermophile

62 S. BHARGAV et al., Solid-state Fermentation: An Overview, Chem. Biochem. Eng. Q. 22 (1) 49–70 (2008)for xylanase production in a horizontal bioreactorwith wheat straw and destarched wheat bran as substrate.125Pretreatment of lignocellulosic substrates wasfound to be advantageous in many cases, howeverin some it proved disadvantageous. 26 Pretreatmentfor xylanase production using wheat straw byAspergillus terreus resulted in reduction of enzymeactivity. 123 Some other molds that can be used forproduction of xylanase using SSF are Melanocarpusalbomyces, 126 Thermomyces lanuginosus, 127Humicola insolens 128 and Aureobusidium pullulans.129Production of organic acids under SSFFermentation plays a key role in the productionof organic acids. The production of organic acidsprogressed with development of SSF. However,biotechnological processes for large-scale productionof organic acids are still in early phases of development.Organic acids are the most common ingredientsof food and beverages because of theirthree main properties: solubility, hygroscopic quality,and their ability to chelate. 130 Some of the acidsproduced using SSF are citric acid, lactic acid, gallicacid, fumaric acid, -linoleic acid, and kojicacid.Citric acidVarious agro-industrial residues such as sugarcanepress mud, coffee husk, wheat bran, cassavafibrous residue, de-oiled rice bran, carob pods, applepomace, grape pomace, kiwi fruit peels, okara,pineapple wastes, mausami wastes, kumara etc. aremost potential substrates for production of citricacid in SSF.Solid-state fermentation has been proved advantageousover SMF in many respects. Metal ionssuch as Fe +2 ,Mn +2 and Zn +2 repress biosynthesis ofcitric acid by Aspergillus niger in submerged fermentation,but this does not happen in SSF. It is reportedby Kumar et al. that addition of these metalions enhances production of citric acid by 1.4 – 1.9times in SSF. 3 Kumar et al. compared wheat branand sugarcane bagasse for maximum citric acidproduction with Aspergillus niger DS-1 strain. Sugarcanebagasse proved to be a better carrier whencompared with wheat bran. Agglomeration in wheatbran caused non-uniform mixing of the substrate,affected heat and mass transfer along with growthand product formation. Bagasse did not show agglomerationeven when moisture level was increasedfrom 65 % to 85 %. 3 Supplementation ofmethanol to agro-industrial residues enhanced citricacid production. Kumar et al. utilized pineapple,mixed fruit and mausami residues for producing citricacid using Aspergillus niger DS-1 in SSF.Supplementation of methanol to substrates wasdone at different moisture levels. Methanol being adehydrating agent enhanced the moisture requirementof substrates. In the presence of methanolsugar consumption decreased but citric acid productionincreased. The maximum citric acid yieldwas Y = 51.4, 46.5 and 50 % (based on sugar consumed)from pineapple, mixed fruit and mausamiresidues, respectively. 131 Similar reports with carobpods and corncobs have been published by Roukasand Hang et al., respectively. Addition of methanolto the substrate increased citric acid production.132,133 Figs were used for production of citricacid due to their high carbohydrate content andyielded 8 % citric acid. However, the addition ofmethanol increased citric acid production from64 – 490 g kg –1 dry fig. 134 Enhancement in citricacid also depends on other additives such as carbonand nitrogen sources and metal ions.Researchers have used different bioreactors forproduction of citric acid. Flasks proved their superiorityover tray and rotating drum bioreactors whencompared among different bioreactors using pineapplewaste as substrate for citric acid production,with strains of Aspergillus. 135 Packed bed reactorsproved their superiority when compared to flaskcultures in production of citric acid. Kumara,starch-containing substrate was used for citric acidproduction using Aspergillus niger with a packedbed bioreactor. Kinetic analysis of the packed bedbioreactor showed an overall reactor productivity ofP = 0.82 g h –1 citrate per kg wet mass kumara on 5 thday, which in the flask was 0.42 g h –1 citrate per kgwet mass kumara on 8 th day. Some adverse effectsof packed bed such as reactor blockage due to fungalbiomass, forced aeration and limited space,were dominated by optimizing particle size of substrate(d p = 4 – 8 mm) and airflow rate (Q =0.5–1.5Lmin –1 ). Higher bed height and larger particle sizeretard the citric acid production. Larger particle sizepromotes aeration at lower bed height in the reactor,which can destroy filamentous fungi. The high productivityin packed bed was due to initial utilizationof starch in the substrates for citric acid productionand occurrence of CO 2 throughout the process. 136However, some of the results above were contraindicatedwhen apple pomace was used as substratefor production of citric acid using Aspergillus nigerin packed bed bioreactor. Adjustment of the aerationrate provided sufficient O 2 to filamentousfungi, increase in bed height promoted physical stabilityand better air distribution in the reactor, optimummoisture content provided proper diffusion tofungal cells, and increase in particle size preventedcompaction of the substrate promoting aerationin the reactor. Hence, 124 g citric acid was pro-

S. BHARGAV et al., Solid-state Fermentation: An Overview, Chem. Biochem. Eng. Q. 22 (1) 49–70 (2008) 63duced from 1 kg dry apple pomace with a yield ofY = 80 % based on total sugar. 137Prado et al. related the metabolic activity offungus Aspergillus niger to citric acid productionon cassava bagasse. The cassava bagasse containingdifferent percentages of gelatinized starch was usedduring the experiment. Gelatinization of starch incassava bagasse made it more susceptible to fungalaction. In horizontal drum bioreactor and traybioreactor, it was observed that the lower respirationrate i.e. high O 2 uptake and low CO 2 production,favored the citric acid production in horizontaldrum, but the presence of high partial pressure ofCO 2 within the cassava bed also favored citric aciddue to entrapment of fungal spores. Maximum citricacid production in tray bioreactor was with 80 %gelatinized starch. But in horizontal drum bioreactors,the maximum results were obtained with100 % gelatinized starch. Increasing the bed heightin tray bioreactors enhanced citric acid production.138 When efficiency of different substrates suchas cassava bagasse, sugarcane bagasse and coffeehusk were compared for citric acid production withAspergillus niger, cassava bagasse proved to bebetter since it increased the protein content of fermentedmatter. The presence of calcium, phosphorus,vitamin B 2 , thiamine and niacin in cassava mayhave enhanced the yield of citric acid. 139Aspergillus niger has been found to be themost suitable strain for citric acid production. Moststrains are unable to produce citric acid in acceptableyields since it is a metabolite of energy metabolism.Its accumulation rises in appreciableamounts in drastic conditions. The main advantagesof using Aspergillus niger are its easy handling andits ability to ferment a wide variety of cheap rawmaterials. Enhancement in citric acid depends onthe selection of proper nutrient supplements, organismand substrates to prevent drastic changes inpH. 140 However, significant optimization may makeproduction cheaper in SSF.Lactic acidSome of the substrates used for production oflactic acid are wheat bran, wheat straw corncob,cassava, sweet sorghum, sugarcane bagasse, sugarcanepress mud and carrot processing wastes. Aerationof moistened medium is an important factor forthe SSF. It provides humidity to solid support andoxygen for growth. Soccol et al. used Rhizopusoryzae NRRL 395 on sugarcane bagasse impregnatedwith glucose and CaCO 3 to produce lacticacid. Lactic acid production in SSF and SMF was = 137.0 and 93.8 g L –1 respectively. Thus, productivitywas = 1.38 g L –1 per hour in liquid mediumand = 1.43 g L –1 per hour in solid medium, whichmakes SSF suitable for higher production of lacticacid. 141 Miura et al. utilized corncobs for L-lacticacid production using Acremonium thermophilusand Rhizopus in an airlift bioreactor. 142Inert support when used should provide goodconditions for fermentation along with the purity ofthe product. Sugarcane bagasse impregnated withthe sugar solution from gelatinized cassava bagassewas used as an inert support for production of lacticacid using Lactobacillus delbruecki. 143Naveena et al. used statistical analysis to optimizemedium for lactic acid production from wheatbran using Lactobacillus amylophilus GV6 in SSF.Wheat bran not only makes the process economicalbut also brings the organism closer to its naturalhabitat. Lactobacillus amylophilus has been foundto be efficient in direct fermentation of starch tolactic acid, avoiding the multi-step processes of simultaneoussaccharification and fermentation. 144Optimization of nutrients by response surface methodologyresulted in production of 36 g of lactic acidper 100 g of wheat bran having 54 g of starch, withthe Lactobacillus amylophilus strain. The increasein lactic acid production was 100 % (from 0.18 to0.36). The conversion from rough surface with extensivecomplex mesh to smooth surface particlesafter inoculation confirmed alteration of raw starchto glucose, which then converted to lactic acid. 145Some parameters to be considered for lactic acidproduction are aeration rate, substrate selection andnutritional supplementation.However, lactic acid production from cheapersubstrates is still a challenge in SSF, which has tobe overcome by development of low cost mediums.Gallic acidGallic acid is a phenolic compound. Tannaseenzyme is used for converting tannin to gallic acidusing Rhizopus oryzae on tannin-rich substrate ina Growtek bioreactor. 37 In the Growtek bioreactor,the solid substrate comes into direct contact withthe liquid medium, and thus heat removal is easy.This reactor was used for producing gallic acidwith mixed cultures of filamentous fungi such asRhizopus oryzae and Aspergillus foetidus. Co-culturingof two organisms has advantage of providinginternal regulation and product formation. 146Secondary metabolites productionunder SSF conditionSolid-state fermentation can be used for productionof secondary metabolites. Most of these areaccumulated in later stages of fermentation (idiophase).However, product formation has been foundsuperior in solid-state processes. Problems associatedwith secondary metabolite production in liquid

64 S. BHARGAV et al., Solid-state Fermentation: An Overview, Chem. Biochem. Eng. Q. 22 (1) 49–70 (2008)fermentation are shear forces, increase in viscositydue to metabolite secretion, fungal morphology,and reduction in metabolite stability.Gibberellic acidsGibberellic acid is a fungal secondary metaboliteproduced in its stationary phase. The SSF systemnot only minimizes production and extractioncosts, but also increases the yield of gibberellic acids.Accumulation of gibberellic acid was 1.626times higher in SSF than SMF using wheat bran assubstrate with Gibberella fujikuroi P3. 147 Limitationin nitrogen sources stops the exponential growth offungus triggering production of secondary metabolitessuch as gibberellins. 148Various bioreactors using different substrateshave been used for production of gibberellic acid inSSF. Bendelier et al. developed an aseptic pilotscale reactor for production of gibberellic acid usingGibberella fujikoroi in fed batch SSF. 149 Gelmiet al. used amberlite as inert solid support in glasscolumn reactors under different conditions of temperatureand water activity for gibberellic acid production.148 A model using maize cob particlessoaked in an amylaceous effluent was developedfor studying factors affecting gibberellic acid productionin SSF. The factors were particle diameter,volume of liquid phase and substrate concentration.150 Tomasini et al. produced gibberellic acid ondifferent SSF systems such as cassava flour, sugarcanebagasse, and polyurethane foam. With SSF250 mg of gibberellin per kg of dry solid mediumwas produced in 36 h on cassava, but 23 mg ofgibberellin per L of the medium was produced insubmerged fermentation. 151 Scaling up and optimizingthe operation of solid-state cultivation bioreactorswill be simplified if there is availability ofaccurate process models, but problems may still existdue to absence of online measuring devices. 47Aroma productionMicroorganisms play an important role in generationof natural compounds, such as fruity aroma.Although bacteria, yeast and fungi produce aromacompounds only a few spp. of yeast and fungi havebeen preferred due to their GRSA (Generally Regardedas Safe) status. Solid-state fermentation hasbeen used for production of aromas by cultivatingNeurospora spp, Zygosaccharomyces rouxii, Aspergillusspp. and Trichoderma viride using pre-gelatinizedrice, miso, cellulose fibers, and agar.Medeiros et al. usedKluyveromyces marxianuson cassava bagasse, as substrate in packed bed reactorwith forced aeration at two different flow rates ofQ = 0.06 and 0.12 L h –1 g –1 . However, with loweraeration, the total volatile content increased at t =24 hbut with higher aeration the total volatile content wasless, and the rate of production decreased. Acetaldehyde,ethyl acetate and ethanol were the three majorvolatile compounds. Production of acetaldehydeand ethyl acetate decreased with increase in aerationrate. This has been attributed to the decrease in vaporpressure within the fermented medium with the increaseof the aeration rate. 152 Strain of Kluyveromycesmarxiamus was cultivated on five differentagro-industrial residues such as cassava bagasse, giantpalm bran, apple pomace, sugarcane bagasse, andsunflower seeds. Palm bran and cassava bagasseproved feasible for aroma production. Ethanol andethyl acetate were in highest concentration withpalm bran and cassava bagasse respectively in t =72h. Difference in composition of fermentation mediumhighly affected the aroma production. 153Ceratocystis fimbriata was used for producingvolatile compounds in column and horizontal drumbioreactors over coffee husk as substrate. However,production was higher in the horizontal drumbioreactor. The dominant compounds in the processwere ethyl acetate, acetaldehyde and ethanol. Thesewere extracted by using porous absorbents. Resinslike (Tenax and Amberlite) showed the best resultsfor volatile compounds. In the case of Tenax,acetaldehyde was recovered in significant amounts(n = 649.7 mol). The type of reactor used and absorbentinfluences the production. 154 Ceratocystisfimbriata when grown on different agro-industrialresidues medium containing cassava bagasse, applepomace, amaranth and soybean resulted in aromaproduction. The medium containing amaranth producedpineapple aroma. However, the medium containingcassava bagasse, apple pomace and soybeanresulted in fruity aroma. The aroma produced wasfound to depend on the growth rate of fungus andits respiratory rate. The greater the respiratory activityof fungus the greater is its growth. 155 Hot watertreated coffee husk was used for production ofaroma production by Ceratocystis fimbriata in SSF.Supplementation of glucose, leucine increased thearoma production but an opposite effect was seenby addition of saline solution. 156Ferron et al. reviewed the microbial prospectsof aroma production using SSF. 157 Longo and Sanromanreviewed the production of aroma compoundsfor the food processing industry by microbial cultures.Production form microorganisms has great advantagesover traditional methods, such as decreasein production costs, ease of downstreaming processes,and use of cheaper agro-industrial substrates. 158A cheap alternative to agro-industrial residuesfor aroma production is cereal grain. Various spp.of Aspergillus,Penicillum,Rhizopus etc, can becultivated on it for production of various aromacompounds such as esters, aldehydes, alcohols etc.

S. BHARGAV et al., Solid-state Fermentation: An Overview, Chem. Biochem. Eng. Q. 22 (1) 49–70 (2008) 65The development of novel, cheap production processessuch as solid-state fermentation may solvesome current limitations of microbial flavor production,as well as widen the spectrum of some biotechnologicallyaccessible compounds.AntibioticsMany antibiotics such as penicillin, cephamycinC, neomycin, iturin, cyclosporin A, cephalosporinsare produced by SSF.Penicillin was produced by using Penicillumchrysogenum with substrates such as wheat bran ofhigh moisture content (s = 70 %) and sugarcanebagasse. 159,160,161 Cephamycin C is produced by avariety of microorganisms including Streptomycescattleya,Streptomyces clavuligerus and Nocardialactamdurans. Wheat raw supplemented with cottonseed-de-oiledcake and sunflower cake was usedfor production of cephamycin C using SSF. 162Wheat raw supplemented with raspberry proved tobe optimum for production of neomycin by SSF.Some critical parameters considered to be optimumfor production of neomycin are particle size of substrate,initial moisture content, inoculum volume,and incubation temperature. 163Iturin, an anti-fungal antibiotic, produced bySSF had stronger antibiotic activity due to itslonger side-chain. 164 Dehydrated okara (containings = 82 % moisture) mixed with wheat bran, whentreated with Bacillus subtilis produced Iturin. 165,68The amount of iturin produced per unit mass of wetsubstrate in SSF was found to be 5–6 times higherthan in submerged fermentation when wheat branwas treated with Bacillus subtilis NB22. 164 Severalparameters such as perforation in SSF trays, solidsubstrate thickness, type and size of inoculum andeffect of relative humidity have been optimized forcyclosporin A production with wheat bran byTolypocladium inflatum in SSF. 166,167Adinarayana et al. optimized the additives andparameters such as incubation temperature, moisturecontent, and inoculum level etc. for maximumcephalosporin C production using various substrateswith Acremonium chrysogenum. However, wheatraw was found to be the best substrate. 168 Strains ofStreptomyces were assessed for tetracycline productionusing various agro-industrial residues as substrates.All the strains produced maximum tetracyclinewith peanut as carbohydrate source. 169Production of poly unsaturated fatty acids(PUFA) under SSFPoly unsaturated fatty acids (PUFA) have to besupplied in diet, as they are not produced in thebody. Submerged and solid-state fermentation canbe used for PUFA production. Fungus involved inPUFA production decreases anti-nutrient substances,such as phytic acid, within the substrate, and partiallyhydrolyzes the biopolymers in it making themsuitable for food and feed supplement. An importantparameter to be considered in SSF is masstransfer during fermentation. Problems involved inscale-up also require solving. 170Gamma linoleic acid (GLA) is the most extensivelystudied PUFA by SSF. In addition, SSFprocesses have also been used for production ofarachidonic acid, eicosapentanoic acid-rich byproducts.170 Cunninghamella japonica when grown onvarious cereal substrates such as unhurled barley,pearl barley, peeled barley, hulled wheat, hulledmillet, and polished rice in SSF produced GLA andlipids. The highest amount of lipid, GLA was obtainedduring SSF of rice and millet. 171 Similarly,optimization and screening of cereals was done inroller bottles and cultivation bags for maximumGLA production. Pearled barley resulted in highestGLA production. Supplementation of peanut oilfurther enhanced the production. 172 Among variousfungal strains, the highest GLA was obtained fromThamnidium elegans in a roller bottle with applepomace and spent malt grains as substrate. 173 Commercially,GLA is being produced in Japan usingthe genus Mortierella and Mucorales. 171,173Production of poly gamma glutamate (PGG)under SSFPoly gamma glutamate (PGG) is an anionic, water-soluble,and highly viscous polypeptide. PGG isused as thickener, humectant, drug carrier, heavymetal absorber and feed additive. Uncontrollablefoaming, limitation of O 2 and mass transfer decreasesPGG production in submerged fermentation, butcontrol over foaming and cost of substrates isachieved in SSF. A high protein-containing materialis a good substrate for PGG production using Bacillussubtilis. Hence, wheat bran supplemented withsoybean cake powder and additives such as glutamate,citric acid as substrate resulted in maximumPGG production (83.61 g per kg of dry substrate) ats = 60–65 % moisture level. However, with optimizationa four-fold enhancement was observed. 174Production of poly hydroxy alkanoates (PHA)under SSFPoly hydroxy alkanoates are microbial polyestersaccumulated by microbes as energy reserve.Production using SSF decreases environmentalproblems and costs involved in the process. Substratessuch as olive oil, babassu oil cake treatedwith Ralstonia eutropha can be used for PHA productionby SSF. Addition of sugarcane molasses tosoy cake increases PHA production. 175

66 S. BHARGAV et al., Solid-state Fermentation: An Overview, Chem. Biochem. Eng. Q. 22 (1) 49–70 (2008)Exo-polysaccharides production under SSFExo-polysaccharides such as succinoglycan,xanthan are the future products of SSF. 65 Agrobacteriumspp. has been used for succinoglycan productionon an industrial scale as they are non-pathogenicand produce good yield of polysaccharide.Succinoglycan was produced using Agrobacteriumtumaefaeciens on agar medium, spent malt grains,ivory nut and grated carrot in a horizontal bioreactor.176Similarly, Xanthomonas compestris was grownon a variety of solid substrates such as spent maltgrains, apple pomace and citrus peels for xanthangum production. Fermentation was done in rotatingculture bottles. 177Production of biocontrol agents under SSFDevelopment of sustainable agriculture througheco-friendly pesticides is needed due to their stabilityand reliability. They are used as an alternative totoxic residues. Among the various microbial agentsas biocontrol agents, fungal agents are found tohave greater potential because of their differentmodes of action. Liagenidium giganteum, a fungalagent used for control of mosquitoes, act by encystingon their larvae. It uses the larvae as a substratefor growing by entering inside it. This fungus actsby producing motile zoospores. 30The recognition of the fungal strain with pesticideactivity is significant for development of infectivepropagules such as conidiospores, blastospores,chlamydospores, oospores and zygospores, 65 alongwith assessment of its biocontrol activity. Similarly,the mechanism of infection is also an important parameterto be considered for large-scale productionstrategy. 3 Retention of moisture is an important aspectfor development of biopesticide formulation.Vrije et al. 179 illustrated the various mode of actionand requirements of down stream processing for thebiocontrol agents.Spores produced in SSF are more heat resistantand are more stable through SSF. 70 Heat evolutionand retention are the big problem in SSF system,but using lignocellulosic substrates, which increasethe water retention capacity and forced aeration, 178can prevent it.E. nigrum produced on peat, vermiculite andlentil meal is an antagonist of Monilinia laxa, responsiblefor loss in stone fruits. Production usingSSF is done in plastic bags because increasing thenumber of bags can do scale-up. 180 A glass columnreactor was used with forced aeration with differentsubstrate bed height and diameter for the productionof spores of Metarhizium anisophilae. Forcedaeration reduces heat transfer. 128 Similarly, a packedcolumn bed reactor has been used to study interactionsbetween Fusarium culmorum and its potentialbiocontrol agent, Trichoderma harzianum.Solid-state fermentation of broiler litter has beendone for the production of spores of B. thuringenesisand P. flourescens. 181 Oostra et al. used mixedbioreactors for large-scale production of spores forbiocontrol agent Coniothyrium minitans and evaluatedthe heat produced during the process. C.minitans has the ability to grow and sporulate in awide temperature range and is responsible for controllingSclerotinia sclerotinium infections. 50Production of biofuel by SSFEthanol is the most widely used biofuel today.Although it is easier to produce ethanol using submergedfermentation, SSF is preferred due to itslower water requirement, smaller volumes of fermentationmash, prevention of end product inhibition,and disposal of less liquid water, which decreasespollution problems.Cellulosic materials are receiving major attentionfor ethanol production because of their abundantavailability. 182 Solid-state fermentation of applepomace supplemented with ammonium sulfate andcontrolled fermentation with Saccharomyces cerevisiaehas been reported to produce ethanol. 183 Kargi etal. used the rotating drum fermentor to estimate theinfluence of rotational speed on the rate of ethanolformation. The rate of ethanol formation decreasedwith the increase in rotational speed. 184 Various spp.of yeasts that can be used for ethanol production bySSF are Saccharomyces cerevisiae,Kloeckera apiculate,Candidastellata,Candida pulcherrina andHensenula anomala. Bacteria like Zymomonas mobilisand fungus like Fusarium oxysporum also havethe ability to produce ethanol. Ethanol productionusing thermotolerant yeasts has been supported becauseof lower cooling costs required and faster determinationrates. 149,185 Various substrates that can beused for alcohol production are sweet sorghum,sweet potato, wheat flour, rice starch, soluble starchandpotatostarchusingSaccharomyces cerevisiae.However, maximum ethanol production is achievedby using mixed substrates. 186,187ConclusionsThere is a continuous development in SSFtechnology over the last two decades. The advantagesof SSF processes overweigh the obstacles dueto engineering problems involved in fermentationprocesses. Presently, in most SSF systems fungi aremore suitable than bacterial strains and yeasts, butgenetically improved or genetically modified bacterialand yeast strains may be made to suite SSF processes.Bacterial cultures decrease the time required

S. BHARGAV et al., Solid-state Fermentation: An Overview, Chem. Biochem. Eng. Q. 22 (1) 49–70 (2008) 67for fermentation and hence reduce the capital involved.Many difficulties are involved in SSF, thatrequire extensive attention, such as: difficulty inscale-up, requirement for controlling process variableslike heat generation, unavailability of directanalytical procedures to determine the biomass directlyin the substrate bed, and heterogeneous fermentationconditions. It has been noted that the useof inert support conditions provides good conditionsfor fermentation along with the purity of theproduct. 93,143 Improvement in bioreactors, processcontrol for continuous SSF is required in the biotechnologyindustry for producing most valueadded products. Analysis of existing literature hasproved that most value added products could beproduced in higher amounts by SSF than by submergedfermentation. Optimization of the propersubstrate and additives are an important part of theprocess. Recent developments made by various researchers,show that control of heat transfer,scale-up in SSF should be solved through prior laboratory-scalemathematical modeling.List of symbolsA – float of area, cm 2A w – water activityD – diameter of column, mm, md – diameter, mmd p – particle diameter, mmH – height of column, mm, mh – height of substrate bed, cml – length, mn – amount of substance, moln S– stirring speed, min –1P – productivity, U g –1 h –1Q – volume flow rate, L h –1s – moisture, %S – hole surface, mm 2t – time, hV – volume, Lw – mass fraction, %Y – yield, % – inclination angle, ° – mass concentration, g L –1 – temperature, °CList of abbreviationsSSF – solid-state fermentationSMF – submerged fermentationGDS – gram of dried substrateIU/gds – International Unit per gram of dried substrateReferences1. Cannel,E.,Moo-Young,M., Process Biochem. 4 (1980) 2.2. Hesseltine,C. W., Process Biochem. July/August 1977 24.3. Kumar,D.,Jain,V. K.,Shanker,G.,Srivastava,A., ProcessBiochem. 38 (2003) 1731.4. Oojikaas,L. P.,Weber,F. J.,Buitelaar,R. M.,Tramper,J.,Rinzema,A., Trends Biotechnol. 18 (2000) 356.5. Oriol,E.,Raimbault,M.,Roussos,S.,Gonzales,G. V.,Appl. Microbiol. Biotechnol. 27 (1988) 498.6. Scott,W. J., Aust. J. Biol. Sci. 6 (1953) 549.7. Nishio,N.,Tai,K.,Nagai,S., Eur. J. Appl. Microbiol.Biotechnol. 1979.8. Raimbault,M.,Alazard,D., Eur. J. Appl. Microbiol. 9(1980) 199.9. Kim,J. H.,Hosobuchi,M.,Kishimoto,M.,Seki,T.,Yoshida,T.,Taguchi,H.,Ryu,D. D. Y., Biotechnol.Bioeng. 27 (1985) 1445.10. Pandey,A.,Selvakumar,P,Soccol,C. R.,Nigam,P., Curr.Sci. 77 (1999) 149.11. Grevais,P.,Molin,P., Biochem. Eng. J. 13 (2003) 85.12. Molin,P.,Grevais,P.,Lemiere,J. P.,Davet,T., Res.Microbiol. 143 (1992) 777.13. Charlang,G. W.,Horowitz,N. H, Proc. Natl. Acad. Sci. 68(1971) 260.14. Charlang,G. W.,Horowitz,N. H., J. Bacteriol. 117 (1974)261.15. Grajek,W.,Grevais,P., Enzyme Microb. Technol. 9(1987) 658.16. Benjamin,S.,Pandey,A., Acta Biotechnol. 18 (1988) 315.17. Grajek,W.,Grevais,P., Appl. Microbiol. Biotechnol. 26(1987) 537.18. Narahara,H., J. Fermentation Technol. 55 (1977) 254.19. Yadav,J., Biotechnol. Bioeng. 31 (1988) 414.20. Raghavarao,K. S. M. S.,Ranganathan,T. V.,Karanth,N.G., Biochem. Eng. J. 13 (2003) 127.21. Smits,J. P.,Rinzema,A.,Tramper,J.,Van Sonsbeek,H. M.,Hage,J. C.,Kayank,A.,Knol,W., Enzyme Microb.Technol. 22 (1998) 50.22. Mitchell,D. A.,Meien,O. F., Biotechnol. Bioeng. 68(2000) 127.23. Sargantanis,J.,Karim,M. N,Murphy,V. G.,Ryoo,D.,Biotechnol. Bioeng. 42 (1993) 149.24. Nagel,F. J. I.,Tramper,J.,Bakker,M. S. N.,Rinzema,A.,Biotechnol. Bioeng. 71 (2000) 219.25. Nagel,F. J. I.,Tramper,J.,Bakker,M. S. N.,Rinzema,A.,Biotechnol. Bioeng. 72 (2001) 231.26. Jain,A., Process Biochem. 30 (1995) 705.27. Auria,R.,Morales,M.,Villegas,E.,Revah,S., Biotechnol.Bioeng. 41 (1993) 486.28. Raimbault,M., Electron. J. Biotechnol. 1 (1998) 1.29. Desgranges,C.,Vergoignan,C.,Georges,M.,Durand,A.,Appl. Microbiol. Biotechnol. 35 (1991) 200.30. Krishna,C., Crit. Rev. Biotechnol. 25 (2005) 1.31. Desgranges,C.,Vergoignan,C.,Georges,M.,Durand,A.,Appl. Microbiol. Biotechnol. 35 (1991) 206.32. Mitchell,D. A.,Greenfield,P. F.,Doelle,H. W., World J.Microbiol. Biotechnol. 6 (1990) 201.33. Mudgett,R. E., Solid-state fermentations, In: Manual ofIndustrial Microbiology and Biotechnology, American Societyfor Microbiology, Washington DC, 1986, pp 66-83.34. Georgiou,G.,Shuler,M. L., Biotechnol. Bioeng. 28 (1986)405.35. Moo-Young,M.,Moreira,A. R.,Tengerdy,R. P., Principlesof solid-substrate fermentation, In: Fungal Biotechnology

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