ArticlePDF Available

Utilization of agricultural and food waste and by-products by biotechnology

May 2001

May 2001
12(3):26-29

Authors:

Poonam Nigam

Ulster University

Ashok Pandey

Indian Institute of Toxicology Research

Biotechnology as "microbial technology" is one of the most applied technologies. This technology employing microbiological systems and their products, can be explored for the utilization of various wastes and by-products originating from agriculture, food and related industries. Biotechnology has established various applications In environment, agriculture, food, dairy and meat Industries, Fermentation technology such as solid-state cultivation process can be applied in more effective way in the biotransformation and biological upgrading of crop, agricultural and food industry by-products for improved or upgraded qualities. This article discusses the potential utilisation of various wastes for pollution control, safe disposal and recycling through biotechnology. story': Genistein and Saponins.

.1 Diverse range of agro-residues utilization in SSF technology

…

.3 Agro-residues used in SSF for enzyme production

…

.4 Agro-residues used for microbial growth studies

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UNCORRECTED PROOF

February 20, 2009 Time: 07:44pm t1-v1.4

Chapter 10

Solid-State Fermentation Technology

for Bioconversion of Biomass

and Agricultural Residues

Poonam Singh nee’ Nigam and Ashok Pandey

Contents

10.1 Agro-Residue Bioconversion inSSF ........ .................................. 198

10.1.1 NatureofSubstrates............................................... 200

10.2 A Bio-Technology Solid State Fermentation. . . . . . . . . . . . . . . . . . . . . . . ............. 201

10.3 AdvantagesofSSFOverConventional LiquidFermentation ...................... 202

10.4 Performance ControlofSSFProcess.......................................... 204

10.4.1 Performance ControlbyParticleSizeofAgroResidues ................. 205

10.4.2 Performance Control by Medium Preparation of Agro-Residues . . . . . . . . . . 206

10.4.3 Performance Control by Moisture Content Of Agro Residues . . . . . . . . . . . . 207

10.5 MicroorganismsUsedForAgro-ResiduesBioconversion......................... 209

10.6 DesigningAndTypesofSSF ................................................ 211

10.6.1 FermenterDesignforSSF.......................................... 211

10.6.2 TypesofSSFSystems ............................................. 211

10.6.3 SSF Bioreactors . . . ............................................... 212

10.7 Scale-UpStagesofSSF..................................................... 213

10.7.1 FlaskLevel ...................................................... 213

10.7.2 LaboratoryFermenterLevel ........................................ 213

10.7.3 PilotFermenterLevel.............................................. 213

10.7.4 Production Fermenter Level . . . . . . . . . . . . . . . . . . . . . . . . . . . ............. 214

10.8 Factors AffectingSSF ...................................... ................ 214

10.8.1 SigniﬁcanceofAerationandMixinginSSF........................... 214

10.8.2 SigniﬁcanceofControlofTemperatureandpHinSSF .................. 215

10.9 Processes and Products of SSF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............. 216

References ............................................................... 216

Abstract Solid-state fermentations (SSF) have attracted a renewed interest and

attention from researchers due to recent developments in the ﬁeld of microbial-

biotechnology. Hence, for the practical, economical and environmentally-friendly

P. Singh nee’ Nigam (B)

Faculty of Life and Health Sciences∗University of Ulster, Coleraine BT52 1SA,

Northern Ireland, UK

e-mail: P.Singh@ulster.ac.uk

P. Singh nee’ Nigam, A. Pandey (eds.), Biotechnology for Agro-Industrial Residues

Utilisation, DOI 10.1007/978-1-4020-9942-7 10,

Springer Science+Business Media B.V. 2009

197

UNCORRECTED PROOF

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198 P. Singh nee’ Nigam and A. Pandey

bioconversion of agro-industrial wastes, solid state or substrate fermentation has

been researched globally and proved to be the ideal technology for this purpose.

In this chapter some important aspects of solid-state cultivation system have been

discussed, including the variety of substrates and microorganisms used in SSF for

the production of various end products; and the performance control of system by

regulation of important factors.

Keywords Solid substrates ·Agricultural residues ·Solid state fermentation ·Wate r

activity ·Moisture ·Bioreactor

10.1 Agro-Residue Bioconversion in SSF

Commonly used substrates in SSF are natural agricultural products, as well as

agro-industrial waste residues and by-products serve as a source of carbon in SSF

(Table 10.1). Lignocellulosic materials of agriculture origin compose more than

60% of plant biomass produced annually through the process of photosynthesis.

This vast resource is the potential and renewable source of biofuels, biofertilizers,

animal feed and chemical feedstocks. Lignocellulose may be a substrate for the

production of value-added products (Table 10.2), such as biofuels, biochemicals,

biopesticides, biopromoters,or may even be a product itself after biotransformation

(e.g. compost, biopulp).

AQ1

AQ2 In all applications the primary requirement is the hydrolysis of lignocellulose

into fermentable sugars by lignocellulolytic enzymes, or appropriate modiﬁcation of

Table 10.1 Diverse range of agro-residues utilization in SSF technology

Substrates for SSF Microorganisms used in SSF Reference

Starchy raw materials Aspergillus spp Czajkowska and Ilnicka 1988

Bannana waste A. niger Baldensperger et al. 1985

Barley Husk Bjkendra adusta Robinson and Nigam 2008

Corn cob A. niger Singh et al. 1989

Citrus peel A. niger Rodriquez et al. 1985

Sugarcan by-products A. terreus Blanko et al. 1990

Cassava Rhizopus oryzae Daubresse et al. 1987

Sugarbeet pulp Trichoderma viride Durand 1998

Cassava T. resei & yeast Opoku and Adoga 1980

Wheat straw T. reesei & Endomycopsis ﬁbuleger Laukevics et al. 1984

Wheat straw T. reesei, Chaeotominum Abdullah et al. 1985

Sugarbeet pulp T. reesei and Fusarium oxysporum Nigam and Vogel 1988, 1990

Sugarcane bagasse Polyporus spp Nigam 1990

Saccharum munja- Pleurotus spp. Gujral et al. 1987

Residues Wheat straw Coprinus spp. Yadav 1989, 1988

Cassava Sporotrichum pulverulentum Smith et al. 1986

Straw Candida utilis Han 1987

Sweet potato Pichia bartonii Yang 1988

Fodder beets Saccharomyces cerevisiae Gibbon et al. 1984

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10 Solid-State Fermentation Technology 199

Table 10.2 Agro-industrial residues used for addcd-value products

Substrate Microorganisms Product Reference

Oat cereal Rhizopus oryzae Lactic acid; various

value added

products

Koutinas et al. (2007)

Agro-residues Aspergillus niger Citric acid Prado et al. (2004)

Distillers grain Aspergillus niger Citric acid Xie and West (2006)

Wheat bran Mucor meihei Rennet Thakur et al. 1990

Wheat bran Rhizopus oligosporus,

Mucor meihei

Rennet Karanth (1988)

Agar Trichoderma viride Cheese aroma Gervais (1988)

Agro-residues Fungal cultures Cheese ﬂavour Revah and Lebeault (1988)

Polished rice Neurospora spp Aroma Yamauchi et al. (1989)

Barley Strptomyces

Cephcdosportum

aermonium

Cephalosporin Jermini and Demain (1989)

Sweet potato Aspergillus Tetracyclines Yang and Ling (1984)

Rice grains Streptomyces Cephalosporin Wang et al. (1984)

Bagasse Pencillium chrysogenum Penicillin Barrios-G et al. (1990)

Cassava Aspergillus niger Aﬂatoxins Barrios-G et al. (1990)

Corn A. ﬂavus Mycotoxin Hesseltine (1972)

Soya Various moulds Mycotoxin Bhumiratna et al. 1980

Oat straw Pofyporovs spp Lignin degradation Bone and Munoz (1984)

Birch lignin Phanerochaete

chrysosporhun

Lignin conversion Mudagett and Paradis (1985)

Maple Wood Polyporus anceps Lignin conversion Matteau and Bone (1980)

Bagasse 2 Polyporous spp Lignin conversion Nigam (1990)

Aspen Wood Phubia tremelloasa Deligniﬁcation Reid (1989)

Wheat bran Fusarium monoliforme,

Gibberrela fujikuroi

Gibberellic acid Kumar and Lonsane

(1987a, b, c)

Wheat bran Fusarium monoliforme,

Gibberrela fujikuroi

Gibberellic acid Prema et al. (1988)

Wheat straw Poms tignium Hydroenperoxi de Maltseva et al. (1989)

Soya bean Cassava R. oligsoporus Tempeh and Koji Hesseltine (1972)

Koji-type SSF Filamentous fungi Fungal spores Vezina and Singh (1975)

Soya bean Filamentous fungi Fungal spores Lotong and Suwarnarit (1983)

AQ3

AQ4

the structure of lignocellulose. Economical and effective lignocellulolytic enzyme

complexes, containing cellulases, hemicellulases, pectinases and ligninases may be

prepared by SSF (Table 10.3). Lignocellulose is also the raw material of the paper

industry. To fully utilize the potential of lignocellulose, it has to be converted by

chemical and/or biological processes. Solid substrate fermentation (SSF) plays an

important role, and has a great perspective for the bioconversion of plant biomass.

Lignocellulose may be a good feedstock for the production of biofuels, enzymes

and other biochemical products by SSF. Crop residues (straw, corn by-products,

bagasse, etc.) are particularly suitable for this purpose, since they are available in

large quantities in processing facilities (Pandey et al. 2001).

Lignocellulose in wood may be transformed into good quality paper products

with the help of SSF biopulping and biobleaching. Agricultural residues may be

converted into animal feed enriched with microbial biomass, enzymes, biopromot-

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200 P. Singh nee’ Nigam and A. Pandey

Table 10.3 Agro-residues used in SSF for enzyme production

Substrates Microorganisms Enzymes Reference

Bagasse,

sawdust,corn

cobs

A. niger cellulose, beta

glucosidase

Madamwar et al. (1989)

Corn cobs A. niger cellulose Singh et al. (1989)

Wheat bran A. niger glucoamylase Pandey (1990)

Wheat bran A. niger glucoamylase Ramakrishnana et al. (1982)

Sugarbeet pulp A. phoenicis beta glucosidase Deschamps and Huet (1984)

Wheat bran A. ﬂavus protease Malathi and

Chakrabarty (1991)

Wheat bran A. carbonarius pectinase Karanth (1988)

Wheat bran A. niveus catalase Karanth (1988)

Sugarbeet pulp T. viride and A. niger cellulase and

amylase

Desgrenges and

Durand (1990)

Wheat bran and

rice straw

Trichoderma spp.

A. ustus, Botritis

spp.,

S. pulverulentum

Cellulose, beta-

glucosidase,

Xylanse

Shamala and

Sreekantiah (1986)

Wheat bran Pencillium spp.

Geotrichwn

Candidum, Mucor

meihei & 2

Rhizopus spp.

lipase Munoz et al. (1991)

Sugarbeet pulp P. capsulatum enzymes Considine et al. (1988)

Citrus pulp-pellets P. charlesii,

Talaromyces

ﬂavus,

Tubercularia

vulgaris

Pectic enzymes Siessere and Said (1989)

Citrus pulp T. vulgaris pectic enzymes Vieira et al. (1991)

Bagasse Polyporous spp. Cellulase &

ligninase

Nigam et al. (1987)

Lignocellulo sis Lentinula edodus enzymes Mishra and Leatham (1990)

Wheat bran Bacillus

licheniformis

alpha amylase Ramesh and Lonsane

(1987a, b, 1990)

Wheat bran Bacillus subtilis protease Jermini and Demain (1989)

Straw Neurospora crasse Carboymethyl

cellulase, beta

glucosidase

Macris et al. (1987)

ers, and made more digestible by SSF. Lignocellulosic waste may be composted to

targeted biofertilizer, biopesticide and biopromoter products. Post-harvest residue

may be decomposed on site by ﬁlamentous fungi and recycled to the soil with im-

proved biofertilizer and bioprotective properties.

AQ5

10.1.1 Nature of Substrates

The major organic material available in nature are polymeric in nature e.g. polysac-

charides (cellulose, hemicellulose, pectins, and starch etc.), lignin and protein,

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10 Solid-State Fermentation Technology 201

Table 10.4 Agro-residues used for microbial growth studies

Substrate Microorganism Reference

Cassava A. niger Raimbault and Alazard, 1980,

Casteada et al., 1990,

Oriol 1988

Barley Husk and Barley straw Bjkendra adusta Robinson and Nigam 2008

Citrus peel A. niger Rodriguez et al. 1985,

Cassva Rhizosporus oligosporus Mitchell et al. (1988, 1990)

Soya bean R. oligosporus Rathbun and Shuler 1983

Sugarbeet pulp T.viride,A.niger Desgrenges and Durand 1990

Sugarrbeet pulp T. viride, Sporotrichum

Pulverulentum and

Thermoascus auranticus

Grajek 1988

Buckwheat seeds Penicillium roqueforti Desfarges et al. 1987

Wheat straw Corinus ﬁmetarius Singh et al. 1990

which can be metabolized by different microorganisms as a source of energy. These

substrates that are insoluble in water, absorb water onto their matrix, which provides

required moisture in SSF system for the growth and metabolic activities of microor-

ganisms. Bacterial and yeast cultures grow on the surface of substrate ﬁbrils and

particles while fungal mycelia penetrate into the particles of substrate for nutrition.

The solid phase in SSF provides a rich and complex source of nutrients that may

AQ6

be sufﬁcient or sometimes insufﬁcient and incomplete with respect to the overall nu-

tritional requirements of that particular microorganismthat is cultivated on that sub-

strate. The constituents in the agricultural solids are approximately analysed in terms

of total carbohydrates, proteins, lipids, various elements and ash content. The solid

substrates generally contain some small carbon compoundswhereas the bulk of total

dry weight is a complex polymer. The polymeric forms require enzymatic hydrol-

ysis for their mineralisation as carbon-energy sources in microbialmetabolism. In

comparison with liquid-state fermentation, which generally use less complex carbon

energy sources, solid insoluble substrates provide mixed ingredients of high molec-

ular weight carbon compounds. Such complex carbon compounds may contribute

inhibition, induction, or repression mechanism in microbial metabolism during solid

state cultivation.

10.2 A Bio-Technology Solid State Fermentation

Solid substrate systems have been deﬁned in several ways:

1. Solid substrate fermentation (SSF) is the microbial transformation of biological

materials in their natural state, in contrast with liquid or submerged fermentation

that is carried out in dilute solutions or slurries (Pandey et al. 2001, 2004).

2. Solid substrate fermentation is generally deﬁned as the growth of microorgan-

isms on solid substrates or sometimes referred to as solid-state fermentation

since the process taking place is in the absence or near-absence of free water

in the system (Nigam and Singh 1994). The substrate however, must contain

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202 P. Singh nee’ Nigam and A. Pandey

enough moisture, which exists in absorbed form within the solid substrate matrix

and simulates the fermentation reaction occurring in nature. These moist solid-

substrates are insoluble in water and polymeric in nature, are a source of carbon

and energy, vitamins, minerals, nutrients and also provide their absorbed water

for microbial growth as well as anchorage.

3. Solid-state or solid-substrate fermentationmeans that the substrate is moistened,

often with a thin layer of water on the surface of the particles, but there is not

enough water present to make ﬂuid mixture. Weight ratios of water to substrate

in SSF are usually between 1:1 and 1:10.

4. SSF can be deﬁned as a system with solid matrix particles, a liquid phase bound

to them and a gaseous phase entrapped within the particles. The physical prop-

erties of this system such as the water potential and water holding capacity, (can

be used as an index of aeration) and bulk density (which predicates the volume

of pore space) help to deﬁne the conditions of solid-state fermentation.

10.3 Advantages of SSF Over Conventional

Liquid Fermentation

Traditional SSF came about for two primary reasons:

1. The desire for more tasty food, as with Oriental fermented foods and mould-

ripened cheese; and

2. The need to dispose of agricultural and farm waste materials (as in composting).

A closer examination of SSF processes in recent years in several research cen-

tres throughout the world has led to the realisation of its numerous economical

and practical advantages (Lonsane et al. 1985; Steinkraus 1984). The attraction

of SSF comes from its simplicity and its closeness to the natural way of life for

many microorganisms. Since large amount of water are not added to the biolog-

ical systems, fermenter volumes remain small, necessary manipulations become

less expensive and the cost of water removal at the end of fermentation in min-

imised. This type of fermentation is especially suitable for growing mixed cul-

tures of microorganisms where symbiosis stimulates better growth and productivity

(Bushell and Slater 1981). Solid-state fermentations are clearly distinguished from

submerged cultures by the fact that microbial colonisaton occur at or near the sur-

faces of solid substrate, or in few cases the soluble substrate supported on the solid

insoluble-matrix in the environment of low-moisture contents. In contrast to liquid

fermentation, the substrates traditionally fermented in the solid-state are renewable

agricultural products, such as wheat, rice, millet, barley, corn and soybeans. The

non-traditional substrates, which can be used in industrial process development,

include an abundant availability of agricultural, forest and food-processing wastes.

¿From an engineering point of view, SSF offers many attractive features in com-

parison to conventionalstirred tank reactors or aerated liquid medium fermentations

because no free water is present, this leads to many beneﬁts.

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10 Solid-State Fermentation Technology 203

Solid-state fermentations can be used to provide low-shear environments for the

cultivation of shear-sensitive mycelial organisms. Solid state cultivationscan be and

have been used for mass productionof spores, which can than be used for the trans-

formation of organic compounds such as steroids, antibiotics, fatty acids, and car-

bohydrates. Fungal spores have applications in the production of food-ﬂavours and

insecticides. The advantage of solid state fermentation includes simplicity, yields

and the homogeneity of spore preparations. The expected advantages of SSF over

submerged fermentations are:

a. Smaller fermenter volume, relative to the yield of the product, as there is no

excess water taking space in the fermenter,

b. Lower sterilisation energy costs, as less volume of water needs to be heated,

c. Seed tanks are not necessary in all cases, as the spore inocula can be successfully

used to inoculate the solid medium.

d. Easier aeration, as air can circulate easily and freely between the substrate par-

ticles, and also because the liquid ﬁlm covering the substrate has a large surface

area compared to its volume. Aeration is facilitated by spaces between substrate

particles and particle mixing.

e. Reduced or eliminated capital and operating costs for stirring, since occasional

stirring is sufﬁcient.

f. Lower costs of productrecovery and drying; inmany cases the product is concen-

trated in the substrate and can be used directly e.g. Oriental foods and cheeses,

or the products can be directly incorporated into animal feeds.

g. If the product is to be extracted from the substrate e.g. enzymes andother metabo-

lites, then much less solvent is needed. The fermented solids may be extracted

immediately by direct addition of solvents or maintained in frozen storage before

extraction.

h. Reduced or eliminated capital and operating costs for efﬂuent treatment due to

lower water content in the system.

The other beneﬁts are:

1. The media are relatively simple; a natural, as opposed to a synthetic, medium is

used;

2. A more natural environment for microorganisms, e.g. agricultural wastes degrad-

ing organisms: many of these fungi grow and perform better under SSF than

submerged conditions;

3. A less favourable environment for many bacteria, which require a high mois-

ture level to survive, lowering the risk of contamination, therefore many SSF

processes need no sterilisation;

4. SSF is adaptable to either continuous or batch process and the complexity of

equipment is no greater than that required for submerged reactors.

Above described advantages are so attractive for the biological processing of

agricultural by products that most of the work has used SSF process. These

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204 P. Singh nee’ Nigam and A. Pandey

advantages can outweigh the disadvantages of SSF, which are the slowness of fer-

mentation and the difﬁculty of controlling the process precisely.

10.4 Performance Control of SSF Process

The difference in process control between SSF and SmF is mainly due to the use of

solid substrates with a very low moisture content in system. The disadvantages of

large-scale solid cultures are due to the problems of process-control, process scale-

up and the major problem of heat build-up. Despite these drawbacks, large-scale

SSF processes have been developed successfully in Japan for the manufacture of a

variety of products, including fermented foods and food-products,enzymes, and or-

ganic acids. The drawbacks have been overcome by carrying these fermentations in

stationary and rotary tray processes, where the temperatureand humidity-controlled

air is circulated through the stacked beds of fermenting solid substrate particles.

These tray methods of cultivation have been used for centuries in the manufacture

of traditional food products and the cultures experience the shear-sensitivity in some

of these processes. These are main reasons of less frequent use of rotary drum-type

fermenters.

Little information is available in the West on the details of modern control sys-

tems in large-scale solid-state cultivations. The control of temperature and humidity

within practical limits is exercised through water temperatures, which is used to

humidify the circulating air. The humidiﬁed air is circulated at ﬂow-rates to meet

the requirements of heat and mass transfer. The gas environment has been found to

signiﬁcantly affect the rate and extent of culture colonisation and product forma-

tion in SSF. In the commercial production of amylase using rice substrate in SSF,

oxygen pressures above atmospheric have been found to signiﬁcantly stimulate the

enzyme productivity, suggesting oxygen limitation at normal atmospheric pressure.

The DNA measurements revealed that this only caused a little effect on biomass

formation, but the carbon dioxide pressures above 0.01 atm severely affected the

process through the inhibition in amylase productivity.

In a protein production process by Aspergillus species using alfalfa residues,

cellulase and pectinase activities have been found stimulated by oxygen and car-

bon dioxide pressures above atmospheric levels, and with no effect on biomass

formation. These studies have been conducted in controlled gas environments at

constant partial pressures, which is maintained by admitting pure oxygen on demand

at pressures below a set point and purging carbon dioxide in 30% KOH at pressures

above a set point in a closed aeration system. In another type of SSF performed for

the degradation of natural birch lignin employing Phanerochaete chrysosporium,

high oxygen pressures have been found to be stimulating, whereas the high carbon

dioxide pressures have been found inhibiting the process. The stimulatory effect of

oxygen on breakdown of lignins has been conﬁrmed in laboratory studies by using

labeled synthetic lignins and natural wood lignins.

Given the present state of the art, the most promising approach in solid state

fermentation processes development happens to be the measurements and control of

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10 Solid-State Fermentation Technology 205

various parameters and process variables, similarly as in any liquid fermentation.In

SSF processes, various methods are selected to analyse the temperature, pH, humid-

ity, oxygen and carbon dioxide concentrations in gas phases, biochemical analysis

of fermented and unfermented solids and their extracts. The manufacturing produc-

tivities of some industrial scale submerged liquid fermentations have increased sig-

niﬁcantly over years, e.g. antibiotic production. This development has been possible

due to applied and basic research in microbial-biochemistry, microbial-physiology,

and genetics. To some extent the contribution also goes to engineering research

based on concepts of stoichiometry, kinetics, thermodynamics, and heat and mass

transfer in control of the microbial fermentation process and its environment.

Direct economic comparisons of solid-state and liquid-state fermentations are not

possible, it is apparent that the large-scale solid-state fermentations (known as Koji

in Orient) have been developed in Japan on an economic basis. Potential economic

advantages ofsuch processes to employsuitable microbe-substratesystem include:

1. reduced thermal processing requirements, since many processes are not aseptic;

2. reduced energy requirements for agitation, since surface-to-volume ratios for gas

transfer are high and many processes do not require agitation due to their shear-

sensitivity;

3. high extracellular product concentrations, that can be efﬁciently recovered by

superﬁcial-extraction or leaching methods.

10.4.1 Performance Control by Particle Size of Agro Residues

SSF processes performance can be varied and controlled by changing physical and

chemical factors. It has been reported that substrates with ﬁner particles showed im-

proved degradation due to an increase in surface area for enzymatic action (Moloney

et al. 1984). The greater growth of fungal cultures has been found stimulated

by smaller particle size substrates. Higher enzyme productivity in SSF has been

achieved with substrates, which contained particles of mixed sizes from 180 ␮mto

1.4 mm.

Particles and kernels of grain must be of suitable size, but not be too small in

order to avoid particle agglomeration. The particle size must be in a limited size

range to be maintained at relatively low moisture content to prevent contamination.

The smaller particle size provides a larger surface area which facilitates heat transfer

and gas exchange. Smaller particle sizes also distribute equivalent moisture concen-

trations in thinner ﬁlms on external surfaces exposed to the gas environments, given

the same void volume fraction (porosity) and pore size distribution. Internal pores

maintain the same surface-to-volume ratios with respect to solid surfaces, based on

geometric considerations of spherical particles. This results in higher surface nutri-

ent concentrations and the diffusion of nutrients takes place via shorter pathways at

the surfaces as well as in the pores of those substrates which have same tortuosity.

Too small a particle size may result in closer packing densities of the substrates

and the void space between particles becomes considerable reduced. The reduced

space between particles tends to reduce the available area for heat transfer and

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206 P. Singh nee’ Nigam and A. Pandey

gas-exchange with the surrounding environment. If such condition arises, densely

packed particles in a cultivation system have to be sufﬁciently agitated to provide a

better separation of particles for the exchanges of gases and heat transfer. There may

be a lower limit in particle size at which the heat transfer or gas exchange becomes

rate limiting and there may be an upper limit at which the nutrient transfer becomes

limiting. Conclusively under any condition, the particle size of the substrate to be

used is one of the major variables in the SSF-process development. Various meth-

ods are available to obtain particle sizes such as milling, grinding, chopping and

sieving to obtain substrates of particular particle-sizes. In the case of lignocellulosic

substrates, smaller particle size substrate is usually obtained through ball-milling.

10.4.2 Performance Control by Medium Preparation

of Agro-Residues

Some SSF systems do not require any nutritional supplements as do most of

the traditional food fermentations. Medium supplementation is necessary in non-

traditional SSF fermentations, as it induces enzyme-synthesis, provides balanced

growth conditions for mycelial-colonisation and biomass formation, as well as pro-

longing the production of secondary metabolites. SSF employing brown-rot fungi,

require an additional carbon source for the induction of enzymes for the cellulose-

utilisation. Certain fungi including Lentinus lapidus, Poria monticola,andLezites

trabea can be cultivated on lignin-containing natural wood substrates from aspen,

pine and spruce, when the SSF medium is supplemented with glucose or cellobiose

in smaller quantities of 0.5%, w/v, and an even smaller amount of peptone, as-

paragine and yeast extract. In unsupplemented media, growth of these fungi was

very slow as negligible. A co-metabolite, such as glucose or cellulose, stimulates the

lignin-degrading system in white-rot fungi such as Phaenerochaete chrysosporium

and Coriolus versicolor when these organismsare cultivated on spruce lignin. Other

supplementations of cellobiose, mannose, xylose, glycerol or succinate have been

found less effective.

Studies for the nutritional requirements for a developmental microbe-substrate

system to be used on a large-scale SSF, can be done in preliminary experiments

in small-scale liquid or SSF on laboratory scale. There is a procedure for evaluat-

ing the effects of nutritional supplements on culture-growth and product formation,

in which microbial-cultures and the solid substrate are contained in separate com-

partments divided by a membrane with a molecular-weight-cut-off.The membrane

permits the passage of enzymes and small molecular weight compounds but restricts

microbial and substrate solids. One of the major difﬁculties in the development of

solid state fermentations has been the problem in separating microbial biomass from

the solid substrate particles after the mycelial growth has covered the substrate sur-

faces. In solid culture cultivation the microorganism and substrate are intimately

associated making the analytical methods of limited value in stoichiometric analy-

sis of SSF. The analysis of biomass yield and growth rate by the measurement of

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10 Solid-State Fermentation Technology 207

glucosamine, protein, RNA, DNA, oxygen consumption, and carbon dioxideor heat

evolution, can not be accurately used in samples of SSF.

Solid cultures for the production of secondary metabolites may have another

problem in that the nutrient, whose deﬁciency triggers the pathway leading to forma-

tion of secondary metabolite, may be available in excess when the microbial growth

becomes limited by other nutrient. Therefore, the selection of a solid substrate and

required-supplements is more critical for a SSF process for antibiotic production

that for a SSF designed for enzyme and organic acid biosynthesis.

10.4.3 Performance Control by Moisture Content

of Agro Residues

Solid-state or solid-substrate fermentation means that the substrate is moistened,

often with a thin layer of water on the surface of the particles, although there is not

enough water present to make a ﬂuid mixture. Weight ratios of water to substrate

in SSF are usually between 1:1 and 1:10 (Reid 1989a, b). Since biological activity

ceases below a moisture content of about 12%, this establishes the lower limit at

which SSF can take place. The upper limit is a function of absorbency and hence,

moisture content varies with the substrate material type.

Solid substrates may be viewed as gas-liquid-solid mixtures. The aqueous phase

in such mixtures is intimately associated with solid surfaces in various states of

sorption. The aqueous phase in a cultivation system is in contact with the gas phase

continuous with the external gas environment. Different types of solid substrates

can absorb different amounts of water. Depending on the moisture content of the

solid; some of the water is tightly bound to solid surfaces, some amount of water is

less tightly bound and remaining water may exist in a free state inside the capillary

regions of the solid substrates. The gas-liquid interface provides a boundary for

gaseous exchange between carbon dioxide and oxygen as well as for heat exchanges.

Water in biological materials exists in three states. The moisture isotherm mea-

surements determines that the solids sorb or desorb water vapour in equilibrium

with relative humidities in a gas phase (water activities), which can be maintained

by saturated salt solutions at a constant temperature. Water is tightly bound to solid

surfaces at the surface in a monolayer region. In case of agriculturalresidues, mono-

layer binding is generally 5 to 10g per 100 g of dry solids. Beyond the surface

monolayer in a multilayer region, water is less tightly bound in additional layers

at progressively decreasing energy levels. Then beyond the multilayer region, free

water exists in a region of capillary condensation. In terms of relationships between

water activity and moisture content, the distinction between the multilayer and cap-

illary regions is ambiguous. The electric measurements of an agricultural residue

containing high starch content has been used to determine the dividing line between

multilayer and capillary regions. The dividing line was deﬁned by a moisture content

of about 25 to 30% by weight at a water activity of 80 to 85%, which is the lower

limit for microbial growth except for some halophilic or osmophilic microbes.

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208 P. Singh nee’ Nigam and A. Pandey

The sorption isotherm may vary from one type of product to another, the hys-

teresis is seen in sorption and desorption isotherms. Water may exist in free state

at moisture levels of interest in solid state fermentation, which is in contrast with

general perception about SSF that the free water does not exist in such systems.

Moisture is a critical factor in SSF of aﬂatoxin production on rice; the yields of

aﬂatoxins have been found decreasing rapidly at moistures above 40%. The rice

particles become sticky at moistures above 30 to 35%. Moisture content plays an

important role on the growth of lactic acid bacteria on feedlot wastes liquids mixed

with cracked corn; growth and acid production was limited at moisture level less

than 35%, whereas the higher level above 42% in SSF-mixtures caused the contents

to become gummy and aggregate. One of the secrets of a successful SSF-process

is to keep the fermenting substrate moist enough for fungal-growth and coloni-

sation and to avoid higher moisture level not to promote the unwanted bacterial

growth. Therefore, the optimum moisture content for a particular type of SSF for

its microbe-substrate system should be determined for a particular end-product and

cultivation conditions of that SSF.

The level of moisture content affects the process productivity signiﬁcantly in

any SSF system, when available in lower or higher quantities than the optimum

value (Lonsane et al. 1985). Hence, it should be in limited and required amounts in

system. The presence of an optimum moisture content in SSF medium has been em-

phasised also for the cultivation of bacterial cultures (Ramesh and Lonsane 1990).

The process productivities are affected by water content because the physiochemical

properties of the solids depend and vary with moisture available to them. Therefore,

the major key factors determining the outcome of the SSF-process are the moisture

content and the relative humidity levels (Lonsane et al. 1985).

Heat removal during fermentation is mostly achieved by evaporative cooling.

This leads to an uneven distribution of water in system due to large quantities of

water evaporation. Workers have practised various ways to maintain the moisture

content of the solids (Lonsane et al. 1985; Ahmed et al. 1987).

10.4.3.1 Control of Water Activity Factor in SSF

Water activity of the substrate has been proposed as the condition of growth and

viability of the microbes and hence, the importance of awin SSF has widely been

studied (Nishio et al. 1979; Raimbault and Alazard 1980; Kim et al. 1985). Water

activity is deﬁned as the relative humidity of the gaseous atmosphere in equilibrium

with the substrate and the water activity factor, awof the substrate quantitatively

expresses the water requirement for microbial activity (Smith et al. 1985).

aw=−Vm ␾/55.5where,

V=number of ions formed,

m=Molar concentration of solute

␾=Molar osmotic coefﬁcient, and

55.5=molar concentration of a solution of pure water.

Pure water has an aw=1.00 and it will decrease with the presence of solutes.

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10 Solid-State Fermentation Technology 209

The types of the microorganisms that can grow in SSF systems are determined

by the water activity factor, aw. Bacteria mainly grow at higher awvalues while

ﬁlamentous fungi and some yeasts can grow at lower awvalues (0.6–0.7). The mi-

croorganisms capable of carrying out their metabolic activities at lower awvalues

are suitable for SSF processes. High awfavours sporulation in the course of growth

in SSF, but low awfavours spore germination and mycelial growth.

Numerous experiments have demonstrated the inﬂuence of awon microbial-

metabolism (Gervais and Buttut 1988), such as, on growth rate and sporogenesis AQ7

of ﬁlamentous fungi (Gervais et al. 1988), on enzyme biosynthesis by fungi (Grajek

and Gervais 1987), and on cheese aroma production (Gervais et al. 1988). AQ8

The awof the medium is a fundamental parameter for mass transfer of the water

and solutes across the cell membrane (Gervais and Sarrette 1990). The control of

this parameter could be used to modify the metabolic production or excretion of a

microorganism (Gervais 1989, 1990). A theoretical calculation based on the Ross

equation showed that awdecreased towards the end of the SSF-culture (Oriol 1988). AQ9

A kinetic model which relates the rate constant of the death of the microbial cells to

awand temperature has been proposed by Moser (1988), using the equation

k=k␣awexp −EAaw/RT

Constants k␣and EAare calculated from the experimental value of aw.Reg-

ulation of the awcan be controlled by the relative humidity of the air. Gervais and

Bazelin (1986) reported a SSF process allowing the control of awand Gervais (1989)

developed a new sensor for the continuous awmeasurement in SSF.

10.5 Microorganisms Used For Agro-Residues Bioconversion

Selection of a suitable microorganism is one of the most important criteria in SSF.

The vast majority of wild type microorganisms are incapable of producing com-

mercially acceptable yields of the desired products. The unique characteristics of

solid-state cultivations are their ability to provide a selective environment at lower

concentrations of moisture ideal for mycelial organisms. The mycelial organisms are

capable of producing a range of extracellular enzymes required for the hydrolysis

of complex, polymeric solid substrates. Such microorganisms are able to colonise

at high nutrient concentrations near solid surfaces. The mycelial organisms include

a large number of ﬁlamentous fungi and a few bacteria of actinomycetes. The im-

portance of microorganisms can be seen from the fact that a culture of Aspergillus

niger can produce as many as 19 types of enzymes, while enzyme alpha amylase

can be produced by some 28 different types of cultures (Fogarty and Kelly 1979;

Pandey 1992). SSF processes can be placed in two main classes based on the type

of microorganism involved:

1. Natural (Indigenous) SSF: Ensiling and composting are SSF processes, that

utilise natural microﬂora. In nature, SSF is often carried out by mixed cultures

in which several microorganisms show symbiotic cooperation.

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210 P. Singh nee’ Nigam and A. Pandey

2. Pure culture SSF: Known puriﬁed microorganisms are used in such processes

either singly or in mixed culture. SSF using a pure culture is known since antiq-

uity e.g. the Koji process with Aspergillus oryzae. A pure culture is necessary in

industrial SSF process for improved rate of substrate utilisation and controlled

product formation. A typical example of pure mixed culture SSF is the bio-

conversion of agricultural residues to fungal biomass (protein) using two pure

cultures of Chaetomium cellulolyticum and Candida utilis.

Several microorganisms have been employed in a wide range of SSF processes

for various objectives. The cultivation of ﬁlamentous fungi on solid substrates has

been widely used for different purposes at laboratory scale e.g. for Koji fermenta-

tion, for lignocellulose fermentation (Matteau and Bone 1980), for fungal spores

(Lotong and Suwarnarit 1983), and for mycotoxin production (Hesseltine 1972;

Bhumiratna et al. 1980). For various purposes, among the ﬁlamentous fungi three

classes, viz. Phycomycetes (Mucor and Rhizopus), Ascomycetes (Aspergillus and

Penicillium) and Basidiomycetes (Nigam and Prabhu 1985), have been most

widely used.

SSF has been most commonly used mploying Aspergillus niger for protein en-

richment (Rodriquez et al. 1985; Baldensperger et al. 1985; Czajkowska and Il-

nicka 1988) as well as forenzymes production, such as, cellulase (Singh et al. 1989;

Madamwar et al. 1989), amylase, glucoamylase (Ramakrishna et al. 1982; Pandey

1990), beta glucosidase, and protease (Malathi and Chakrabarty 1991). Production

of alcohols, ketones and aldehyde in rice fermentation was achieved by the use of

A. oryzae (Ito et al. 1990). For protein enrichment and kinetic studies related to

SSF process Rhizopus oligosporus has been employed (Rathbun and Shuler 1983;

Mitchell et al. 1988, 1990).

Fungal rennet has been produced by R. oligosporus and Mucor meihei (Karanth

1988). For enzyme production and protein enrichment cultures of Trichoderma

spp. have been employed in pure, single and mixed SSF (Daubresses et al. 1987;

Grajek 1988). Lipase enzyme production has been reported (Munoz et al. 1991)

using six species of Penicillium, two species of Rhizopus, Geotrichum candidum

and Mucor meihei, whereas the maximum lipase activity was obtained with P. can-

didum, P. camembertii and M. meihei. For the production of several other enzymes

e.g. hydrolases and pectic enzymes (Siesser and Said 1989) several other species of

Penicillium have been employed in SSF.

Production of the antibiotic penicillin was achieved in a non-sterile SSF pro-

cess on sugar cane bagasse impregnated with culture medium using Penicillium

chrysogenum. Protein enrichment of lignocellulosic substrates for animal feed pro-

duction (Nigam 1990; Nigam and Vogel 1990a, b), lignin degradation (Bone and

Munoz 1984), and cellulase and ligninase enzyme production (Nigam et al. 1987a,

b) have been obtained by white-rot cultures in SSF.

Production of gibberellic acid has been reported using Fusarium monoliforme

and Gibberella fugikuroi (Kumar and Lonsane 1987a, b). Bacterial alpha amylase

production is reported using Bacillus licheniformis in SSF (Ramesh and Lonsane

1987, 1990). Several yeasts have been used for protein enrichment and ethanol

AQ10

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10 Solid-State Fermentation Technology 211

fermentation in SSF. For protein enrichment of straw (Han 1987) Candida utilis

was used whereas Saccharomyces cerevisiae has most commonly been employed

for ethanol production (Gibbons et al. 1984; Kargi et al. 1985).

10.6 Designing And Types of SSF

10.6.1 Fermenter Design for SSF

Several miscellaneous types of fermenters have been used in batch or continu-

ous mode in SSF processes (Hardin 2004). Process parameters are very impor-

tant factors and they have to be considered in a bioreactor design for any SSF.

Design considerations in types of SS-fermenters used by various researchers are

described by Aidoo et al. (1982). The engineering aspects, with major types of fer-

menters describing their advantages and drawbacks has been reviewed by Fernandez

et al. (2004). Solid state cultivations are not as well characterised on a fundamental

scientiﬁc or engineering basis, as are the liquid fermentation systems that are used

in the West for the industrial production of microbial-metabolites. Solid-state fer-

mentations are, however,widely used in the Orient and therefore, the old traditional

methods of cultivation systems which have been used in food-processing for more

than 2,000 years, have now been modernised and well characterised for their ex-

tended application to non-traditional products. Mitchell et al. (2004) have described

in detail the modelling aspects of SSF.

The physical state of the substrate and the products to be produced in the system

characterise the design-type of solid state cultivation process:

a. Low-moisture solids are fermented

1. without any agitation for the production of Tempeh and Natto;

2. by occasional stirring for the production of Miso and Soy sauce;

3. with continuous stirring for the production of Aﬂatoxin.

b. Suspended solids are fermented in packed bed columns

1. through which the liquid is circulated, as for the production of rice-wine;

2. which contain stationary or agitated liquid media, for the production of Kafﬁr

beer.

10.6.2 Types of SSF Systems

There are two types based on process design:

Type one- Fermentation in static reactor

e.g. Tray fermentations (Lonsane et al. 1985; Viesturs et al. 1987)

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212 P. Singh nee’ Nigam and A. Pandey

Type two- Fermentation with occasional or continuous agitation

e.g. Production of aﬂatoxin, ochtratoxin and enzymes (Lindenfelser and Ciegler

1975; Hesseltine 1977; Silman 1980).

Type two has 4 variations according to the need of process:

1. Occasional agitation, without forced aeration

2. Slow continuous agitation, without forced aeration

3. Occasional agitation with forced aeration

4. Continuous agitation with forced aeration.

10.6.3 SSF Bioreactors

Three basic groups of reactor exist for SSF, and these may be distinguished by the

type of mixing and aeration used. In laboratory scale, SSF occurs mainly in ﬂasks

whereas following reactors are used for large-scale product-formation.

10.6.3.1 Tray Bioreactors

Tray bioreactors tend to be very simple in design, with no forced aeration or mixing

of the solid substrate. Such reactors are restrictive in the amount of substrate that

can be fermented, as only thin layers can be used, so as to avoid overheating and

maintain aerobic conditions. Tray undersides are perforated to allow aeration of

the solid substrate, each arranged above each other. In such reactors, temperature

and relative humidity are the only controllable external parameters (Durand 1998).

Wooden trays were initially used for soy sauce production in Koji fermentations by

Aspergillus oryzae. The use of tray fermenters in large-scale production is limited

as they require a large operational area and tend to be labour intensive. The lack

of adaptability of this type of fermenter makes it an unattractive design for any

large-scale production.

10.6.3.2 Drum Bioreactors

Drum bioreactors are designed to allow adequate aeration and mixing of the solid,

whilst limiting the damage to the inoculum or product. As previously mentioned,

mixing and aeration of the medium has been explored in two ways: by rotating

the entire vessel or through the use of various agitation devices. Rotation or the

use of agitation can be carried out on a continuous or periodic basis. In contrast

to tray reactors, growth of the inoculum in drum bioreactors is considered to be

better and more uniform. Increased sheer forces through mixing, can however, have

a detrimental affect on the ultimate product yield.

Although the mass heat transfer, aeration and mixing of the substrate is increased,

damage to inoculum and heat build up through sheer forces may affect the ﬁnal

product yield. Application of drum reactors for large-scale fermentations also poses

handling difﬁculties.

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10 Solid-State Fermentation Technology 213

10.6.3.3 Packed Bed Bioreactors

Columns are usually constructed from glass or plastic with the solid substrate sup-

ported on a perforated base through which forced aeration is applied. They have

been successfully used for the production of enzymes, organic acids and secondary

metabolites. Forced aeration is generally applied at the bottom of the column, with

the humidity of the air kepthigh to avoid desiccation of the substrate. Disadvantages

associated with packed bed column bioreactors for SSF include difﬁculties in re-

trieving the product, non-uniform growth,poor heat removal and scale-up problems.

10.7 Scale-Up Stages of SSF

Scale-up of SSF has been deﬁned in many ways. There are mainly four stages:

10.7.1 Flask Level

This is smallest scale using 50–1000g substrate working capacity, and used for the

selection of the organism, optimisation of the process and experimental variables

in a short time and at low cost. The vessels used are conical ﬂasks and beakers

(Mitchell et al. 1986; Nigam et al. 1987a, b), Roux bottles (Gervais et al. 1988;

Nigam 1990), jars (Hang et al. 1986), and glass tubes (Raimbault and Alazard 1980).

10.7.2 Laboratory Fermenter Level

This is next to ﬂask scale using a 5–20kg substrate working capacity. It is used

for a selection of procedures such as, inoculum development, medium sterilisation,

aeration, agitation and downstream processing. Standardisation of various param-

eters, selection of control strategies and instruments, evaluation of economics of

the process and its commercial feasibility are also examined at this level. The fer-

menters used are glass incubators (Deschamps and Huet 1984; Oriol et al. 1988;

Smith et al. 1986), column fermenters (Oriol et al. 1988); polypropylene bags

(Yadav 1988), and miscellaneous types of fermenters (Raimbault and Alazard 1980;

Viesturs et al. 1981).

10.7.3 Pilot Fermenter Level

This scale is a stage before the commercial scale using 50–5000 kg of substrate.

This level is necessary for the conﬁrmation of laboratory data and selection of

optimised procedures. It facilitates market trials of the product, physicochem-

ical characterisation and determination of viability of the process. Most large

scale SSFs employ tray type fermenters as in the oldest soy sauce Koji process

(Daubresse et al. 1987), rotating drum type (Lindenfelser and Ciegler 1975; Han

and Anderson 1975; Hesseltine 1977), horizontal paddle fermenters and mixed layer

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214 P. Singh nee’ Nigam and A. Pandey

pilot plant fermenters (Laukevics et al. 1984). Durand and Chereau (1988) reported

the use of a pilot reactor having a one ton working capacity.

10.7.4 Production Fermenter Level

The commercial scale fermenter utilises 25–1000 tonnes of substrate and is per-

formed for streamlining of the developed process. Yokotsuka (1985) described deep

trough methods and mechanical continuous equipment for Koji production generat-

ing 50–100 tonnes of Koji per day.

10.8 Factors Affecting SSF

Each microbe-substrate system is unique and the process variables must be con-

sidered in terms of the physical properties and chemical composition of its sub-

strate, growth characteristics and physiological properties of the microorganisms

to be cultivated in SSF. The nature of the product, if the process involves the

synthesis of primary or secondary metabolite may be based on the synthesis of

extracellular enzymes in growth-associated metabolism. The process variables af-

fecting a solid state cultivation include, pretreatment of substrates, particle-size of

substrates, medium-ingredients, supplementation of growth medium, sterilisation of

SSF-medium, moisture-content, inoculum-density, temperature, pH, agitation and

aeration. These variables should be considered in process-development of a SSF to

be carried out for different purposes. Some of these variables have been discussed

in some sections as above, the rest are discussed below.

10.8.1 Signiﬁcance of Aeration and Mixing in SSF

In any SSF-process an adequate supply of oxygen is required to maintain the

aerobic conditions and for the transfer of excess carbon dioxide produced during

metabolism. This requirement can be achieved through the process of aeration and

mixing of the fermenting solids. In certain cases, the mixture can not be agitated

vigorously or in some cases, at all, if the microorganism used in SSF is shear sen-

sitive. The shear sensitivity is attributed to disruption of mycelial-substrate contact;

this is particularly concerned to those organisms which possess mycelial-bound en-

zymes required for the hydrolysis of solid substrate-polymers. Most Koji processes

in Japan performed for the commercial production of enzymes do not involve great

agitation. The fermenting substrate is gently turned periodically just to bring the

bottom of Koji to the top. These processes have been developed in highly controlled

environments, using automated systems for inoculum mixing, and turning of the

fermenting substrate.

Most of the traditional food-fermentation in Japan use the rotary-tray method

for SSF with the circulation of humidiﬁed air to create the conditions suitable for

gas-exchange and heat-transfer. In the SSF for the production of certain secondary

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10 Solid-State Fermentation Technology 215

metabolites such as aﬂatoxin and ochratoxin, and in some processes for the enzyme

production, mixing and particle separation are achievedby agitation on shakers or in

rotating vessels with circulating conditioned air. Maximum rotation rates generally

decrease with the size of the fermentation-vessel. Therefore, solid-state fermenta-

tions are ideal for the cultivation of those microorganisms that are extremely sensi-

tive to the shear rates of the impeller speeds required for stringent oxygen demand

rates in liquid fermentaton. Such microorganisms colonise the solid substrates by

microbe-substrate attachment and there is no pellet formation in solid-state cultiva-

tion, which is added advantage to SSF.

Aeration plays an important role in solid state fermentations as compared to liq-

uid fermentation where it only helps in gas transfer. Aeration facilitates in heat,

gas and moisture transfer between the fermenting solid particles and the gas envi-

ronment of the system. The temperature of the gas phase serves by supplying or

removing heat, in maintaining the relative humidity in equilibrium with the liquid

phase. In liquid fermentations the substrates are dissolved in at low substrate con-

centrations in large volumes of ﬂuid, but in solid cultures with respect to moisture

transfer, the loss or gain of moisture during SSF is extremely sensitive to the water

activity of the gas-phase. Therefore, small changes in the relative humidity of the

gas phase in equilibrium with the solids may cause the large changes of moisture

content in the solid state, dependingon the sorption-desorptioncharacteristics of the

solid substrate.

There are two main functions of the gas phase in SSF, the primary function is

to supply oxygen and remove the carbon dioxide from the system. The secondary

function of aeration is in heat and moisture transfer that is more important, when

the rates of oxygen and carbon dioxide are not limiting. The gas phase can facil-

itate in the ontrol of solid cultures, due to the fact that direct measurements can

not be performed to estimate dissolved oxygen or carbon dioxide concentrations in

low-moisture solids during the course of the fermentation on either a continuous or

sampling basis. The methods of aeration may cause the conditions of gas transfer be-

ing relatively stagnant. This condition may be responsible for the oxygen limitation

at small penetration depths or may lead to inhibitory carbon dioxide concentrations

in normal atmospheric environments. The gas phase in the SSF during the course

of microbial metabolism, can be analysed for oxygen, and carbon dioxide pressures

using analysers which function on thermal-conductivity, paramagnetism, or infrared

absorption. The technique of gas chromatography can also be used for gas-analysis

of the gas phase of a SSF.

10.8.2 Signiﬁcance of Control of Temperature and pH in SSF

Two signiﬁcant variables affecting any SSF are the incubation temperature and the

pH of SSF-medium. Both variables are speciﬁc for each SSF process depending on

the microoganisms to be cultivated and the product to be formed. Unlike submerged

fermentation, these factors are difﬁcult to control in SSF. These variables can not

be directly measured in the liquid phase, as these are associated with the solids at

UNCORRECTED PROOF

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216 P. Singh nee’ Nigam and A. Pandey

lower moisture content without any free liquid in the fermenting medium. The other

difﬁcult situation arises when the growth temperature of cultivated microorganism

is different than the optimal temperature for the product formation. Such systems

require a possible need for temperature proﬁling or shift in the later stages of fer-

mentation. The thermal gradients may be induced within SSF-mixture due to the

rate of heat generation in SSF-system at high levels of biological activity. This

gradient may limit the heat transfer and may lead to sub-optimal conditions for

microbial-biomass and product formation.

The local pH levels at solid surfaces near which the biological activity occurs,

may be considerable different than the bulk pH of the liquid phase. This difference

in pH levels happens due to surface charge effects and ionic equilibria modiﬁed

by solute transport effects. There is no suitable method to measure the precise pH

of fermenting solid residues in SSF. A general method used for measuring pH of

solid agricultural residues involves mixing one part of fermented solids (dry weight)

and three parts of freshly boiled and cooled water, and measuring the pH of the

resultant liquid after ﬁve minutes using a glass electrode. This procedure can be

used to monitor pH changes during fermentation on intervals using minimum one

gram of the SSF-mixture.

It is easier to measure temperature of the fermenting SSF-mixture, in compari-

son to pH measurement. Temperature can be measured using thermistor or thermo-

couple probes at various depths of the SSF-mixture below the medium-surface. In

various SSF-processes for the production of enzymes, mycelial-biomass or organic

acids, total heat generation of up to 600kcal per kilogram of fermenting solids has

been observed. A study of composting of animal wastes and agricultural residue has

revealed that such heat generations may lead to rapid temperature rise of the fer-

menting mass in the system limited by heat transfer. The study also revealed that the

biological activity was considerably highernear the surface of the compost pile than

in the depth of pile that was at lower oxygen pressure. This phenomenon happens

due to a decrease in interior oxygen concentrations inside the SSF-mixture pile of

compost. Thus the heat generation in such fermentations is coupled to conditions

for heat as well as mass transfer.

10.9 Processes and Products of SSF

Various processes and products from bioconversion of agro-residues of industrial,

pharmaceutical, and environmental importance have been discussed in detail in fur-

ther chapters 11–24 under sections II, III.

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Chapter-10

Query No. Page No. Line No. Query

AQ1 198 31 “Robinson and Nigam 2008” is not listed in the refer-

ence list. please provide.

AQ2 198 34 “Blanko et al. 1990” is not listed in the reference list.

please provide.

AQ3 199 06 “Prado et al. 2004” is not listed in the reference list.

please provide.

AQ4 199 26 Please specify whether “Reid 1989” is “1989a” or

“1989b”

AQ5 200 27 Please specify whether “Nigam et al. 1987” is “1987a”

ar “1987b”.

AQ6 201 01 Please provide citation for table 10.4

AQ7 209 8 “Gervais and Buttut 1988” is not listed in the reference

list. Please provide.

AQ8 209 10 “Grajek and Gervais 1987” is not listed in the reference

list. Please provide.

AQ9 209 15 “Orial 1988, Gervais and Bazelin 1986” are not listed

in the reference list. Please provide.

AQ10 210 44 Please specify whether “Ramesh and Lonsane 1987” is

“1987a” or “1987b”.

AQ11 216 32 “Alazard and Raimbault 1981, Bajracharya and Mudget

1979, Barnard and Hall 1983, Barnes et al. 1972,

Barries-Gonzalez et al. 1988a, b, Biddlestone and

Gray 1991, B´ulock 1979, Cannel and Moo-Young

1980a, b, Carrizalez et al. 1981, Cochet et al. 1988,

Corpe 1980, Georgiou and Shuler 1986, Ghildyal

et al. 1985, Gibbon et al. 1986, Haﬁz et al. 1990, Han

and Steinberg 1987, Hang et al. 1982, Hesseltine

1983, Huang et al. 1985, Illanes and Schaffeld 1981,

Joleel et al. 1988, Knapp and Howell 1980, Kumar

and Lonsane 1988, Larroche and Gras 1986, Lar-

roche et al. 1988, Ladish and Tsoa 1986, Lonsane

et al. 1982, Lonsane and Karanth 1990, Lonsane and

Ramesh 1990, Massiat et al. 1989, Moo-Young et al.

1979, 1983, Narahara 1977, Narahara et al. 1982,

Nigam and Singh 1996a, b, Nigam 1988, 1989a, b,

Nishio et al. 1981, Okazaki et al. 1980, Pamment

et al. 1978, Pandey 1991, Raimbault et al. 1991, Ro-

driquez et al. 1986, Sato et al. 1991, Schaffeld and

Illanes 1986, Shah et al. 1991, Senez et al. 1980,

Sorulsky and Coxwarth 1988, Srikanta et al. 1992,

Streeter et al. 1981, Tautorus and Chalmers 1984,

Tengerdy 1985, Thomas and Turner 1981, Ulmer

et al. 1981, Vaccarince et al. 1989, Wei et al. 1981,

Yu et al. 1976, Zadrazil and Brunnet 1982, Zadrazil

UNCORRECTED PROOF

February 20, 2009 Time: 07:44pm t1-v1.4

Query No. Page No. Line No. Query

and Grabbe 1983, Zyta 1992” are not cited in the text

part. Please provide.

UNCORRECTED PROOF

February 20, 2009 Time: 07:44pm t1-v1.4

Optimal an extracellular alkaline protease production condition for Pseudomonas aeruginosa

Article

Apr 2013
AFR J MICROBIOL RES

Pseudomonas aeruginosa JM07 which produces an extracellular alkaline protease was isolated from the Yellow Sea in China. The cultural conditions were optimized for maximum enzyme production. Maximum enzyme activity was achieved when the bacterium was grown in soybean meal (3.5%, w/v), corn steep meal (1.5%, w/v), Na2HPO4 (0.4%, w/v), KH2PO4 (0.03%, w/v), Na2CO3 (0.02%, w/v), MgSO4 (0.02%, w/v), CaCl2 (0.2, w/v), at pH 7.0 and 20°C over 12 h incubation period. The enzyme had an optimum pH of around 10 and maintained its stability over a broad pH range between 7 and 12. Its optimum temperature is around 30°C, and exhibited a stability of up to 40°C. The enzyme activity was strongly inhibited by DFP, suggesting that it belongs to the family of serine proteases.

Use of Response Surface Methodology for Optimizing Process Parameters for the Production of ??-Amylase by Aspergillus oryzae

Article

Aug 2003
BIOCHEM ENG J

Optimization of three parameters (incubation temperature, initial substrate moisture and inoculum size) was attempted by using a Box–Behnken design under the response surface methodology for the optimal production of α-amylase by Aspergillus oryzae NRRL 6270 in solid-state fermentation (SSF). Spent brewing grains (SBG) was used as sole carbon source. The experimental data was fitted into a polynomial model for the yield of enzyme and an optimum level was arrived at which nutrient supplements were optimized. A Plackett–Burman design was employed to screen nineteen nutrient components for their influence on α-amylase production by the fungal culture. Three components (soybean meal, calcium chloride and magnesium sulphate) were selected based on their positive influence on enzyme formation. A Box–Behnken design was employed to optimize their composition, which showed that an incubation temperature of 30 °C, an initial moisture of 70% and an inoculum rate of 1×107 spores/g dry substrate were the best conditions to produce α-amylase with A. oryzae NRRL 6270 on SBG. Under optimized conditions of SSF, about 20% increase in enzyme yield was observed.

SCREENING OF POTENTIAL ISOLATE FOR THE PRODUCTION OF AMYLASE WITH DIFFERENT AGRO WASTES AS SUBSTRATE

Research

Full-text available

Apr 2018

Amylolytic bacteria were isolated from decayed pomegranate. The bacteria were screened for amylase activity using starch agar plate assay. Five different substrates were used namely banana peel, Gossypium oil cake, groundnut oil cake, cassava, and wheat bran; for microbial production of amylase by Submerged fermentation (SmF) and it was found that maximum amylase activity (2.005 U/ml) specific activity (0.520U/mg) was produced by bacterial isolate 8 using cassava as a substrate.

Fruit Processing Waste Management

Chapter

Dec 2007

Judit Monspart‐Sényi

This chapter is a review of the environmental impact of waste and by-products of fruit processing. Fruit processing waste characteristics and treatment options with regard to the economic feasibility are mentioned. Also included are the current utilization opportunities of fruit processing waste and related technologies. For example, converting fruit waste and by-products into valuable new products (pectin derivatives, polygalacturonase, citric acid, alcoholic beverages, etc.) or additives in other products (e.g., utilization of fruit waste fiber). Attention is drawn to present-day techniques of waste management in apple, citrus, peach, and banana processing technologies. New research areas for fruit waste utilization are also mentioned. The main environmental regulations are summarized. Finally, it is essential to consider the food safety aspects with a conscious effort to control pollutants that enter the environment as a result of food processing.

Solid-state fermentation for biological delignification

Article

Jan 1989

I.D. Reid

Principles of solid substrate fermentation the filamentous fungi

Article

Jan 1983

Degradation of carrot fibres with cell-wall polysaccharides-degrading enzymes

Article

Jan 1990

Carrot fibres were degraded with two enzymes preparations, SP249 from Aspergillus aculeatus and cellulast from Trichodermareesei. Enzymic activities of these complexes indicate that SP249 was particularly active on pectic polymers and Cellulast could degrade amorphous and crystalline cellulose. The combination of both preparation degraded carrot fibres with synergistic effect and led to the solubilisation of 95% of the cell-wall polysaccharides.

Fermented protein foods in the Orient: Shoyu and miso in Japan

Chapter

Jan 1998

From ancient times, nations all over the world have inherited their own alcoholic beverages, which are prepared principally by converting the sugars involved in raw materials into alcohol by the action of yeasts. At the same time, vinegars have been made from almost all of these alcoholic beverages, converting alcohol into acetic acid by the action of acetic acid bacteria. Thus, fruit wines have been made from sweet fruits such as grapes, apples, oranges, and so on. However, in the preparation of wines from a starchy raw material such as wheat, barley, rice, or corn, the raw material must be degraded into sugars, mainly glucose, in order to be fermented by yeasts. It must be noted that there is a major difference between the saccharification process of Western countries and that of the Orient. The amylolytic enzymes used for the saccharification in Western countries have been derived from malt, while in the Orient, Aspergillus or Rhizopus moulds have been utilized as the source of amylolytic enzymes. Accordingly, beer is prepared by first saccharifying the starch of barley by the use of malt, while in the preparation of alcoholic beverages from rice or wheat in the Orient, Aspergillus or Rhizopus moulds are cultured on part of these raw materials to produce amylolytic enzymes. These mould-cultured materials are called ‘koji’ in Japan, and this koji is mixed with the other remaining parts of rice or wheat and water to make mash, which is then concurrently subjected to enzymatic saccharification, lactic fermentation, and yeast fermentation.

Solid state fermentation

Article