New developments in solid state fermentation: I-bioprocesses and products

Process Biochemistry 35 (2000) 1153 – 1169 www.elsevier.com/locate/procbio

New developments in solid state fermentation: I-bioprocesses and products Ashok Pandey a,*, Carlos R. Soccol a, David Mitchell b a

Process Biotechnology Laboratory, Department of Chemical Engineering, Federal Uni6ersity of Parana (UFPR), CEP 81531 -970, Curitiba-PR, Brazil b Department of Soil, Sector of Agricultural Sciences, UFPR, CEP 80035 -050, Curitiba-PR, Brazil Received 29 November 1999; received in revised form 2 February 2000; accepted 12 February 2000

Abstract The last decade has witnessed an unprecedented increase in interest in solid state fermentation (SSF) for the development of bioprocesses, such as bioremediation and biodegradation of hazardous compounds, biological detoxification of agro-industrial residues, biotransformation of crops and crop-residues for nutritional enrichment, biopulping, and production of value-added products, such as biologically active secondary metabolites, including antibiotics, alkaloids, plant growth factors, etc. enzymes, organic acids, biopesticides, including mycopesticides and bioherbicides, biosurfactants, biofuel, aroma compounds, etc. SSF systems, which during the previous two decades were termed as a ‘low-technology’ systems, appear to be a promising one for the production of value-added ‘low volume-high cost’ products such as biopharmaceuticals. SSF processes offer potential advantages in bioremediation and biological detoxification of hazardous and toxic compounds. With the advent of biotechnological innovations, mainly in the area of enzyme and fermentation technology, many new avenues have opened for the application of SSF. This review discusses more recent developments in the area of SSF leading to the developments of bioprocesses and products. © 2000 Elsevier Science Ltd. All rights reserved. Keywords: Solid state (substrate) fermentation; Bioremediation; Biodegradation; Biological detoxification; Crop residues; Biotransformation; Biopulping; Enzymes; Biologically active secondary metabolites; Organic acids; Ethanol; Biopesticides; Aroma compounds; Biosurfactants

1. Introduction In recent years, solid state fermentation (SSF) has shown much promise in the development of several bioprocesses and products. It seems that two terms, solid state fermentation and solid substrate fermentation have often been ambiguously used. As has been pointed out by Pandey et al. [1], it would be logical to distinguish these two terms only. Solid substrate fermentation should be used to define only those processes in which the substrate itself acts as carbon/energy source, occurring in the absence or near-absence of free water: Solid state fermentation should define any fermentation process occurring in the absence or near-ab* Corresponding author. Current address: Biotechnology Division, Regional Research Laboratory, CSIR, Trivandsum-695019, India. Tel.: + 55-41-3613196; fax: +91-471-491712. E-mail address: [email protected] (A. Pandey)

sence of free water, employing a natural substrate as above, or an inert substrate used as solid support [1]. During 1991–1992, two reviews were published on SSF describing the general features of SSF and aspects of fermenter design in SSF [2,3]. These reviews traced the history of SSF and discussed various developments since historical time. Since then, there has been a vast change in the scenario of SSF research all over the world. Tracing the history from the period since these were published revealed significant developments in SSF. During this period (1991–1999), more than 1000 publications have appeared in various journals, proceedings and books, apart from four important publications in book form [4–7]. A few reviews have also been presented discussing some particular features of SSF from time to time [8–13]. The latest in this has been a special one by CW Hesseltine [14] as the Thom Award Address, which is reprinted from his work in 1981. This very well signifies the biotechnological potential of SSF

0032-9592/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 0 0 ) 0 0 1 5 2 - 7

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globally. Significantly, it has been C.W. Hesseltine who first consolidated the scientific information on SSF in 1977. This article has the objective of reviewing the more recent developments in the important and fast growing area of SSF. The scope of the review would be limited to various bioprocesses and products developed during the present decade and no coverage has been made on engineering and modelling aspect as these will be dealt with separately.

2. Bioprocesses based on SSF

2.1. General considerations There are several important factors, which affect SSF processes. Among these, selection of a suitable strain and substrate and selection of process parameters (physical, chemical and biochemical) are crucial. While

efforts largely continued to exploit filamentous fungi and yeast for the production of various products, attempts also have been made to explore the possibilities of using bacterial strains in SSF systems [15–22]. Enzyme production has been an area in which several bacterial strains have been used and successes have been achieved [15,20]. A novel success in this regard was reported on the production of inulinase enzyme using strains of Staphylococcus sp. [20,22–26] and another for the production of L-glutamic acid using a strain of Bre6ibacterium sp., which was grown on sugarcane bagasse under solid state conditions [27]. Among the filamentous fungi, Phycomycetes (Mucor and Rhizopus), Ascomycetes (Aspergillus and Penicillium) and Basidiomycetes (white-rot fungi) continue to be the most preferred choice (see Tables 1–4). The selection of a substrate for SSF process depends upon several factors mainly related with cost and availability and thus may involve screening of several agro-industrial residues. In the SSF process, the solid

Table 1 Production of bioactive compounds in SSF Compound

Source

Substrate

Function

Reference

Aflatoxin Ochratoxin

A. oryzae, A. panasitus A. ochraceus, P. 6iridicatum, A. carbonarius Bacillus thuringiensis

Wheat, Oat, Rice, maize, peanuts Wheat, rice, corn, corn kernels

Mycotoxin Mycotoxin

[21,88] [21,89]

Coconut waste

Insecticide

[21]

Gibberella fujikuroi, Fusarium moniliforme Fusarium moniliforme Cla6iceps purpurea, C. fusiformis Penicillium chrysogenum Cephalosporium armonium Streptomyces cla6uligerus

Plant growth hormone

[35,90–94]

Growth promoter Disease treatment Antibiotic Antibiotic Antibiotic

[21] [95,96] [21,36] [21] [97,98]

S. 6iridifaciens

Wheat bran, corn cob, cassava flour, sugarcane bagassea Corn Sugarcane bagassea Sugarcane bagassea Barley Wheat rawa with cottonseed cake and sunflower cake Sweet potato residue

Antibiotic

[21]

S. rimosus Bacillus subtilis S. coelicolor

Corn cob Okara, wheat bran Agar mediuma

Antibiotic Antibiotic Antibiotic

[99,100] [32,101–104] [21]

B. subtilis Humicola fuscoatra Tolypocladium inflautum

Soybean residue Okara Agar mediuma Wheat bran

[21,101,102,105] [106] [107–109]

Ustiloxins

Ustilaginoidea 6irens

rice panicles

Antifungal volatiles Destrucxins A and B Clavulanic acid

B. subtilis

Impregnated loam based compost

Antibiotic Antibiotic Immuno suppressive drug Antimitotic cyclic peptide Antifungal compounds

Metarhizium anisopliae

Rice, rice bran, rice husk

Cyclodepsipeptides

[33]

S. cla6ulingerus P. bre6icompactum

b-Lactamase inhibitor, antibacterial –

[110]

Mycophenolic

Wheat rawa with soy-flour and sunflower cake Wheat bran

Bacterial endotoxins Gibberellic acid Zearalenone Ergot alkaloids Penicillin Cephalosporin Cephamycin C Tetracycline chlorotetracycline Oxytetracycline Iturin Actinorhodin, Methylenomycin Surfactin Monorden Cyclosporin A

a

As inert solid support.

[21] [21]

[111]

Micro-organisms

Enzymes

Strains of Candida sp., Aspergillus sp., Rhizopus sp., Neurospora sitophila, P. candidum, Mucor sp. A. niger, A. oryzae, A. fonscaeus, Rhizomucor, Kluy6eromyces Wheat bran, soybean cake residue lactis Strains of Aspergillus sp., Rhizopus sp., Mucor sp., Bacillus Wheat bran, rice bran, rice husk, coconut cake, tea waste, sp., Saccharomyces sp. cassava, cassava bagasse, sugarcane bagasse. Banana waste, corn flour, saw-dust, soybean meal, sweet potato, potato, rice hull, sugar beet pulp Vibrio costicola Wheat bran, rice husk, saw dust, coconut oil cake Staphylococcus sp., Kluy6eromyces marxianus Wheat bran, chicory roots A. ficuum, A. carbonarius Canola meal R. oryzae Wheat bran+tannic acid Penicillium pinophilum Wheat straw

a

Source, Refs. [16–20,23,118–125].

Glutaminase Inulinase Phytase Tannase Feruloyl para-coumaroyl esterase

a-Amylase, b-amylase, glucoamylase

a-Galactosidase, b-galactosidase

Wheat bran, sunflower flour, coffee husk, soybean meal, rice bran, corn bran, rice hull, aspen wood, sweet potato residue, waste hair Wheat bran, peanut press cake, coconut cake, rice bran

Bagasse, wheat bran, wheat straw, saw-dust, cotton stalk, kraft lignin, cellulose powder, wood chips

Lipases

Proteases (acidic, neutral and alkaline)

Laccase, Li-peroxidase, Mn-peroxidase, aryl-alcohol oxidase, catalase, phenol oxidase

Strains of Aspergillus sp., Trichoderma sp., Penicillium sp., Phlebia radiata, P. eryngii, Melanocarpus albomyces, P. sanguineous, Thermomyces lanuginosa, Humicola lanuginosa, Thermascus aurantiacus, Talaromyces emersonii, Thermomonospora sp. Strains of Penicillium sp., Pleurotus sp., Phlebia radiata, Trametes 6ersicolor, Flammulina 6elutipes, Polyporus sp., Panus tigrinus, Trichoderma 6ersicolor Strains of Aspergillus sp., Penicillium sp., Rhizopus sp., Bacillus sp., Trichoderma sp.

Bagasse, coconut coir pith, rice husk, rice straw, wheat bran, wheat straw, tea waste, sweet sorghum, silage, sugar beet pulp, saw-dust, grape-wine cutting waste, palm oil mill waste, sago hampas, cassava waste, sweet sorghum, soy-hull, paddy straw, etc. Rice straw, corn hull, corncobs, wheat bran, wheat straw, bagasse, rice straw, cotton stalks, soy-hull, kraft pulp, sugar beet pulp, rice husk, apple pomace,corn cobs, coffee processing waste, barley straw, oat straw

Substrates

Xylanases, b-xylosidase a-arabinofuranosidase, acetoesterase, catechol-oxidase

Strains of Aspergillus sp., Trichoderma sp., Lentinula sp., Penicillium sp., Pleurotus sp., Neurospora sp., Sporotrichum sp., pul6erulentum, Cerrena sp., Botritis sp., Gliocladium sp., Phanerochaete sp., etc

Fungal cellulases and hemicellulases Fungal cellulases and hemicellulases Fungal pectinases cellulases, hemicellulases Amylases, proteases, lipases, cellulases, hemicellulases Xylanases Hydrolytic enzymes Laccases, ligninases Trichoderma harzianum cellulases T. harzianum cellulases for helper function

(A) Industrial applications Enzyme assisted ensiling Bioprocessing of crops and crop residues Fibre processing (retting) Feed supplement Biopulping Directed composting Soil bioremediation Post harvest residue decomposition Biopesticide

(B) Production Cellulase, b-glucosidase, CMCase, laccase, xylanase, polygalacturonase, ligninase

Enzymes

Process

Table 2 Industrial applications of enzymes produced under SSF [116] and production of enzymes by SSFa

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1156 Table 3 Production of organic acids in SSF Micro-organism Citric acid Aspergillus niger A. foetidus A. niger A. niger A. A. A. A. A. A. A. A. A. A.

niger niger niger niger niger niger niger niger niger niger

Fumaric acid Rhizopus sp. Lactic acid Rhizopus sp. R. oryzae R. oryzae Lactobacillus casei L. hel6eticus L. paracasei Streptococcus thermophilus Oxalic acid Aspergillus niger Gallic acid Rhizopus oryzae

a

Substrate

Reference

Sweet potato Pineapple waste Pineapple waste Kumara (starch containing root) Carrot-processing waste Okara (soy-residues) Carob pod Corncobs Polyurethanea Cassava Cassava bagasse Amberlitea Sugarcane press-mud Coffee husk

[127] [128] [128,129] [130,131] [132] [133] [134] [135] [136] [70] [17,18,137,142] [138,139] [140] [141]

Cassava

[70]

Cassava Carrot-processing waste Sugarcane bagassea Sugarcane press-mud Sugarcane press-mud Sweet sorghum Sugarcane press-mud

[68,142] [132] [143] [144] [144] [145] [144]

Sweet potato

[127]

Gallo seeds cover powder

[46]

As inert solid support.

substrate not only supplies the nutrients to the microbial culture growing in it, but also serves as an anchorage for the cells. The substrate that provides all the needed nutrients to the micro-organisms growing in it should be considered as the ideal substrate. However, some of the nutrients may be available in sub-optimal concentrations, or even not present in the substrates. In such cases, it would be necessary to supplement them externally. It also has been a practice to pre-treat (chemically or mechanically) some substrates before use in SSF processes (e.g. ligno-cellulosics) which makes them more easily accessible for microbial growth. Among the several factors, which are important for microbial growth and activity in a particular substrate, particle size and moisture level/water activity are the most critical [28–46]. Generally, smaller substrate particles would provide larger surface area for microbial attack and thus should be considered as a desirable factor. However, too small substrate particles may result in substrate aggomulation in most of the cases,

which may interfere with microbial respiration/aeration, and thus may result in poor growth. At the same time, larger particles provide better respiration/aeration efficiency (due to increased inter-particle space) but provide limited surface for microbial attack. Thus, it would be necessary to arrive at a compromised particle size for a particular process. Research on the selection of a suitable substrate has mainly centred around tropical agro-industrial crops and residues. These include crops such as cassava, soybean, sugar beet, sweet potato, potato, and sweet sorghum, crop residues such as bran and straw of wheat and rice, hull of soy, corn and rice, bagasse of sugarcane and cassava, residues of the coffee processing industry such as coffee pulp, coffee husk, coffee spentground, residues of fruit-processing industries such as pomace of apple and grape, wastes of pine-apple and carrot processing, banana waste, waste of oil-processing mills such as coconut cake, soybean cake, peanut cake, canola meal and palm oil mill waste, and others such as saw-dust, corn cobs, carob pods, tea waste, chicory roots, etc. (Tables 1–4). Wheat bran has been the prime among all. Recently, some reviews have been presented on biotechnological potential of several agro-industrial residues for value-addition in SSF [16–19]. Many processes have been developed that utilise these as raw material for the production of bulk chemicals and value-added fine products such as ethanol, single-cell protein (SCP), mushrooms, enzymes, organic acids, biologically active secondary metabolites, etc. It was highlighted that advances in industrial biotechnology offer potential opportunities for economic utilisation of agro-industrial residues such as cassava bagasse, sugarcane bagasse, coffee pulp and coffee husk. Due to their rich organic nature, these can serve as ideal substrates for microbial processes for the production of valueadded products. Solid state fermentation has been mostly employed for bioconversion processes. Application of agro-industrial residues in bioprocesses, on the one hand, provides alternative substrates, and on other, helps in solving pollution problems, which otherwise may cause their disposal. In relation to SSF processes using inert substrates, two approaches have been adopted; one in which synthetic materials such as amberlite or polyurethane are used, and other which utilises natural materials such as sugarcane bagasse as the inert solid support. Since natural substrates create problems in the fermentation kinetics studies (due to their heterogeneous nature), such studies could better be performed using synthetic inert solid substrates.

2.1.1. Concept of sterility Sterility is very often required in any SmF process because many contaminants could outcompete the process organism under the conditions of high water

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availability provided. Often SSF processes involve an organism which grows quite rapidly under the low water conditions, and, if an active inoculum is added to a (cooked) substrate, the process organism is able to outcompete the contaminating organisms, meaning that strict aseptic operation of the bioreactor may not be essential in SSF, although, of course, operation should be carried out in as clean a manner as possible. The less stringent design requirements for such bioreactors, and correspondingly lower costs, could be considered as a favourable point for the SSF process, providing an economic advantage over the SmF process. However, there are a number of products for which SSF has good potential but for which the process organisms grow generally relatively slowly, which could be overtaken by contaminants. For example, this is the case in the production of gibberellic acid by Giberella sp. In such cases, it would be essential to use a bioreactor, which can be operated under aseptic conditions. In such a case, the bioreactor costs would be expected to be similar to those for SmF bioreactors, and SSF would only be chosen if it could provide a specific advantage, such as higher product titres, or lower downstream processing costs, which finally could lead to better economic performance.

2.1.2. Academic research 6ersus industrial applications In order to understand this, some fundamental questions must be considered. There is a large volume current research into SSF — why? There are relatively few commercial applications (at least compared to SmF) — why? A general opinion about the choice of fermentation method for the production of any micro-

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bial product would normally be SmF, unless there appears a particular reason why SSF should be chosen? There is no question that SmF is intrinsically less problematic — heat transfer is better and homogeneity is much-much better. SSF would be chosen if (a) particular economic conditions favoured it (therefore, in some parts of the world some enzymes are produced by SSF, whereas in other parts, the same enzymes are produced by SmF); (b) the product is only produced in SSF — or if produced in both systems, the SSF product is far superior (for example, fungal spores for use as biopesticides tend to be much more robust when they are produced in SSF compared with when they are produced in SmF, and some fungi simply do not sporulate well in SmF); (c) use of solids becomes an imperative (government regulations in response to environmental pressures caused by dumping of organic solids). Thus, although SSF has potential mainly in specific areas, under the conditions that it is ‘off the main track’, it has received relatively little attention. Its potential to operate reliably at a large scale simply has not been investigated to the same degree as the SmF method. It is possible that SSF processes could be routinely operated at large scale, following rational design rules — but we currently do not know enough to be sure if this is the case or not. Certainly there are a few successful SSF processes — such as enzyme production processes, various biopesticide production processes, etc., even if many of these are at relatively small scales. There is also the koji industry. SSF should not be seen as a technology, which can simply replace SmF. In fact, SmF has many features,

Table 4 Other products by SSF Product

Micro-organism

Substrate

References

L-glutamic

acid Pigments Carotenoid Xanthan gum

Bre6ibacterium sp. Monascus purfureus Penicillium sp. Xanthomonas campestris

[27] [175] [176] [177,178]

Succinoglycan

Agrobacterium tumefaciens Rhizobium hedysari Saccharomyces cere6isiae, Schwanniomyces castellii, Zymomonas mobilis, Candida utilis, Torula utilis Rhizopus oryzae, Ceratocystis fimbriata, B. subtilis Citrobacter freundii, Klebsiella pneumoniae, Rhizopus oligosporus, R. arrhizus, R. stolonifer Cunninghamella japonica, Rhizopus sp. Bacillus subtilis Entomopathogenic and mycoparasitic fungi

Sugarcane bagasse Sugarcane bagasse Corn meal Apple pomace, grape pomace, citrus peels, spent malt grains etc agar medium, spent malt grains, ivory nut shavings and grated carrotsa Apple pomace, sorghum carob pods, sugar beet, sweet sorghum, sweet potato, wheat flour, rice starch Sugarcane bagasse, cassava bagasse, coffee husk, soybean Soybean tempeh

Ethanol

Aroma compounds Vitamins B12, B6, riboflavin, thiamine, nicotinic acid, nicotinamide Gamma-linolenic acid Biosurfactants Biopesticides/bioherbicide

a

As inert solid support.

Cereals, soybean Agro-industrial residues, molasses Sweet sorgum, rice flour, perlite-corn meal agar

[178,179] [17,18,151–161]

[17,18,166–174] [180,181]

[182,183] [184,185] [147–150,186]

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which would make it the preferred method in a case where SmF and SSF had similar economic performances. The greater homogeneity in a SmF system simply makes the process less problematic. The relatively few commercial SSF processes compared with the range of products, which have been investigated in the laboratory, could probably be a confirmation of this. However, there are a number of products for which SSF is the superior production technology, and a number of large-scale SSF processes have begun operating commercially. There is a continued need to develop SSF technology to allow such processes to operate to their full potential.

3. Development of bioprocesses

3.1. Bioremediation and biodegradation of hazardous compounds SSF appears to be useful tool for bioremediation and biodegradation of hazardous compounds. Masaphy et al. [47] proposed a system for bioremediation by biodegradation of atrazine added to a mixture of cotton and wheat straw and inoculated with the white-rot fungus Pleurotus pulmonarius. Application of Pleurotus sp. was also proposed by Fan et al. [48] for bioremediation of caffeinated residues. Earlier, Berry et al. [49] also proposed the use of SSF technique to dispose off the pesticide waste. They compared several methods to dispose off atrazine and found that SSF resulted in significant reduction in leachability and bioavailability of pesticide. Kastanek et al. [50] studied biodegradation of polychlorinated biphenyls (PCBs) and volatile chlorinated ethenes (CIUs) in contaminated soils and ground water in field condition to evaluate the natural attenuation process. SSF reactors (15-m3 volume) were found to enhance reductive dehalogenation. A study was carried out to evaluate the technological feasibility of SSF to dispose carbofuran. Results were claimed to provide the basis for the development of an effective and economical method for the biodegradation or containment of waste pesticide residue [51]. Wiesche et al. [52] studied two-step biodegradation of pyrene by white-rot fungi and soil micro-organisms in SSF using two strains, Dichomitus squalens and Pleurotus sp. The fungi were incubated on wheat straw contaminated with C-14 pyrene. Results proved the technological feasibility of SSF for pyrene degradation by the fungal strains individually and in combination with soil natural microflora.

3.2. Biological detoxification of agro-industrial residues Certain agro-industrial residues contain toxic (antiphysiological and anti-nutritional) compounds such as

hydrogen cyanide, caffeine, tannins, polyphenols, etc. and pose difficulties in their effective utilisation. Their disposal is a problem for the processing industries as it leads to serious environmental concerns. SSF has been recently applied as a tool to detoxify such residues e.g. cassava peels, rapeseed meal, canola meal, coffee husk, coffee pulp, etc. and some successes have been achieved, Ofuya and Obilor [53] studied the effects of SSF on the toxic components of cassava peels. Fermentation for 96 h caused a drastic reduction ( 95%) in the HCN levels of the peels and about 42% reduction in soluble tannins. Essers et al. [54] also studied the effect of six individual strains of the dominant microflora in solid substrate fermenting cassava on cyanogen levels, which included Geotrichum candidum, Mucor racemosus, Neurospora sitophila, Rhizopus oryzae, Rhizopus stolonifer and Bacillus sp. It was concluded that both incubation and microbial activity are instrumental in reducing the potential toxicity of cassava during the SSF and that effectiveness varies considerably between the species of micro-organisms applied. Bau et al. [55] studied the effect of SSF using Rhizopus oligosporus on elimination of anti nutritional substances of de-fatted rapeseed meal. A 24 h fermentation induced a degradation of about 58% aliphatic glucosinolates and 97% indo glucosinolates. 3-N-oxalyl-L-2,3-diaminopropanoic acid (ODAP), a neurotoxic amino acid, present in the seeds of grass pea (Lathyrus sati6us), causes irreversible spastic paraparesis (neurolathyrism) when over-consumed. This limits its food value, which otherwise is considered a tasty, nutritious and easily cultivatable food crop. Kuo et al. [56] attempted to detoxify the seeds by SSF using Aspergillus niger for 48 h, followed by SSF for further 48 h using R. oligosporus. This resulted in 90% reduction in ODAP, apart from improving other nutritional qualities of the fermented seed meal. Coffee pulp and coffee husk, which are generated during the industrial processing of coffee cherries, possess anti-physiological components such as caffeine, tannins and polyphenols. Several solutions and alternative uses of the coffee pulp and husk have been attempted. These include uses as fertilisers, livestock feed, compost, etc. However, these applications utilise only a fraction of the available quantity and are not technically very efficient. Attempts have been made to detoxify it for improved application as feed, and to produce several products such as enzymes, organic acids, flavour and aroma compounds, and mushrooms, etc. [19,57,58]. Caffeine is an active compound, one of the nature’s most powerful and addictive stimulants. It is the principal substance causing the mild stimulation effect of coffee. It is also present in coffee pulp and husk at about 1.3% concentration on dry weight basis. Tannins are generally thought to be anti-nutritional factors and prevent coffee pulp from being used at greater than

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10% in animal feed. The anti nutritional effects of tannins in animal feed have been discussed by Alzueta et al. [59] and Terrill et al. [60]. SSF has been frequently used for the biological detoxification of coffee husk using fungal strains [61– 63]. By the selective screening on an agar medium coffee husk, three strains of Rhizopus sp., which showed good growth (radial growth and biomass production), were compared with two strains belonging to basidiomycetes, viz. Phanerochaete chrysosporium for the degradation of caffeine and tannins in coffee husk [62]. Both the cultures, i.e. Rhizopus sp. as well as P. chrysosporium grew well on coffee husk. Rhizopus sp., however, appeared superior to P. chrysosporium as it resulted in higher caffeine and tannin degradation in relatively shorter period. Under the optimised conditions of substrate pH, initial moisture, inoculum size, temperature and aeration, Rhizopus sp. degraded 87 and 65% caffeine and tannins in comparison to 70 and 60%, respectively, by P. chrysosporium. Leifa et al. [63] studied degradation of caffeine in coffee husk using a strain of P. ostreatus LPB 09. Media supplemented with caffeine showed caffeine tolerance by the strain. Mycelial growth rate and biomass were 16 and 14.5%, and 65.6 and 7.6% less with 100 and 1000 g/l caffeine concentration. At 2500 g/l concentration, there was no fungal growth at all. Mycelial analysis revealed the presence of caffeine in it, showing that P. ostreatus actually did not degrade caffeine but accumulated in it (0.575% caffeine in dry mycelia). In SSF, caffeine concentration in the residue after frutification diminished by 85.4%, however, the fruitbody contained 0, 157% caffeine. It was claimed that P. ostreatus LPB 09 could be used for bioremediation of residues containing caffeine. Some bacterial and fungal strains such as Bacillus coagulans, Pseudomonas aeruginosa, P. putida, Penicillium rouquifortii, P. curtosum, and Pleurotus sp. have been stated to have the capacity of degrading caffeine [63,64]. Roussos et al. [64] studied caffeine degradation by P. 6errucosum in SSF of coffee pulp with and without external nitrogen supplementation. Results indicated that in spite of the limited growth of the culture without any external nitrogen, caffeine degradation was almost complete. Addition of nitrogenous compounds rather inhibited caffeine degradation. Hakil et al. [65] achieved considerable degradation of caffeine using several strains of filamentous fungi. Porres et al. [66] studied the degradation of caffeine and polyphenols in coffee pulp through silage. Under different processing conditions, a reduction of 13 – 63, 28 – 70 and 51 –81% in caffeine, total polyphenols and condensed tannins, respectively, was achieved. It was concluded that silage presented and ideal method to reduce the anti-physiological compounds contents in coffee pulp.

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3.3. Biotransformation of crops and crop residues Biotransformation (biological upgradation) of crop and crop-residues for improved nutritional qualities has been among the most important area where SSF has been of potential application, offering techno-economical feasibility. White-rot fungi have commonly been employed for this purpose. Cassava, which plays a very significant role, as food for millions of people in Africa, Asia and South America, is not a balanced food due to its low protein, vitamin and mineral contents. It also lacks in sulphur-containing amino acids. Several attempts have been made to improve the nutritional quality of cassava using SSF [66–77]. Soccol et al. carried out extensive studies on biotransformation of cassava [69,70] and cassava wastes [73–76] for nutritional improvement. Several strains belonging to Rhizopus sp. were screened to select the suitable cultures capable of growing on raw and treated cassava. A strain of R. formosa (edible fungus) was found most suitable for improving the nutritional quality of raw cassava flour [71]. Several studies also have been made to cultivate edible mushrooms. Mushrooms represent a highly specialised group of edible fungi. They possess the ability to biodegrade and bioconvert a spectrum of inedible plant residues into useful form of food, aptly considered as ‘food delicacy’. About 2000 species of mushrooms are edible, of which about 80 have been grown experimentally and around 20 commercially cultivated using SSF [78]. Ligno-cellulosic crop residues represent a potential source of dietary energy to ruminants. These residues are characterised by high percentages of cellulose and hemicellulose (in addition to some lignin), but are poor in protein content. This limits their utilisation as an ideal animal feed. In addition, some times they pose poor digestibility and poor palatability. In order to improve their utilisation, it is necessary to improve their nutritional quality. This could be achieved using several physical, chemical and microbial methods [79]. Since physical and chemical methods are energy intensive and are expensive, focus has been made on developing microbial methods. SSF has been termed potential for this [67,78–81]. Zadrazil et al. [81] reviewed bioconversion of lignocellulose into protein enriched ruminant feed with white-rot fungi. Several fungal strains belonging to some species of Pleurotus, Ganoderma, Stropharia, Polyporus, Lentinus, Dichomitus, Sporotrichum and Trametes have been reported to be useful [81]. Fan et al. [57,58], using coffee industry residues, also reported strains belonging to P. ostreatus and L. edodes useful for mushroom production in SSF. Several crop residues have been used for protein enrichment and single cell protein (SCP) production such as citrus fruit peel by Penicillium camemberti and P. roquefortii [82], rye with Fusarium sp. [83], carob

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pods by A. niger [34], mango and date industry wastes by Pleurotus ostreatus [84], sugar beet pulp by filamentous fungi [85], wheat straw by Phanerochaete chrysosporium, Pleurotus ostreatus, Phlebia radiata, and Ceriporiopsis sub6ermispora, using individual cultures, or by co-culturing such as apple pomace by A. niger and Candida utilis [86]. Fermented products showed improved protein contents and better digestibility for the cattle.

3.4. Biopulping Biopulping has been defined as the SSF of wood chips. Generally biopulping is carried out to improve the mechanical pulping process. Wall et al. [87] reviewed the design and kinetics of biopulping process. White-rot fungi are of special interest in this area and among these two species, Phanerochaete chrysosporium and Ceriporiopsis sub6ermispora are most important. The former has been shown to successfully biopulp wood chips without the need of autoclaving or nutritional enrichment. The process has shown techno-economical feasibility [87].

4. Products based on SSF

4.1. Bioacti6e products Many practical advantages have been attributed to the production of biologically active secondary metabolites through the SSF route. Balakrishnan and Pandey reviewed the production of biologically active secondary metabolites in SSF [21]. Their paper discussed different strategies and processes that could be helpful in utilising SSF technology for the production of biopharmaceuticals. In spite of the realisation of the potential application of SSF systems to produce high value bioactive secondary metabolites, much remains to be done in this area to achieve commercial production. Mycotoxins, bacterial endotoxins, plant growth factors, antibiotics, immuno-suppressive drugs, alkaloids, etc. are among the important group of bioactive compounds, which have been produced through SSF (Table 1) [88–111]. SSF has been potentially used for the production of mycotoxins, which are potent carcinogenic agents. These are generally produced on a wide range of food grains and seeds such as wheat, oat, rice, maize, etc. [21,88]. Strains of Aspergillus sp. have been used for their production. Mycotoxins are required to study the safety levels in food grains and to determine their quality and for other toxicity studies. Wicklow et al. [89] reported the production of Ochratoxin A, a known mycotoxin with demonstrated toxicity to insects, from the sclerotia of the fungus Aspergillus carbonarius

NRRL 369. The sclerotia were harvested from SSF of corn kernels. Ochratoxin A accounted for the activity of the methanol extract against larvae of the detritivorous beetle Carpophilus hemipterus (Nitidulidae) (75% reduction in feeding rate) and corn ear worm Helico6erpa tea (50% mortality with 99% reduction in weight gain among surviving larvae) when incorporated into a pinto bean diet at levels less than those occurring naturally in the sclerotia. Gibberellins (GAs), which are a large family of isoprenoid plant hormones and some of which are bioactive growth regulators, controlling seed germination, stem elongation and flowering have been reported to be produced by SSF using the rice pathogen Gibberella fujikuroi (especially the bioactive compounds gibberellic acid (GA3). Recently, Tudzynski [112] reviewed the biomolecular aspects of biosynthesis of gibberellins in Gibberella fujikuroi. Durand et al. [90] and Tomasini et al. [91] compared SSF and SmF for gibberellic acid production. G. fujikuroi produced 23 mg of gibberellin per ml in 120 h of liquid fermentation. SSF using cassava flour showed high production of gibberellic acid (250 mg/kg dry solid matter) in a very short time (36 h). The use of polyurethane as inert solid support resulted in very poor growth of the culture [91]. SSF using wheat bran resulted in 3 g GA3 per kg dry substrate in 11 days [92]. While most of the studies reported above were on a laboratory scale, Bandelier et al. [92] performed SSF at pilot scale. Hernandez et al. [95,96] studied the production of ergot alkaloids in SSF using sugarcane bagasse as inert solid support. A total of 16 different combinations of the liquid nutrient medium were used for impregnating sugarcane pith bagasse for the production of total alkaloids by Cla6iceps purpurea 1029 C. The data indicated large differences in the alkaloids’ spectra. It was claimed that there was the possibility of achieving tailor-made spectra of ergot alkaloids by changing the liquid nutrient media composition. The production of total ergot alkaloids by Cla6iceps fusiformis in SSF was 3.9 times higher than that in SmF [95]. Several studies have been carried out on the production of various antibiotics in SSF. These include penicillin, cephalosprin, tetracyclines, chlorotetracyclines, oxytetracyclines, iturin, surfcatin, actinorhodin, methylenomycin and monorden (see Table 1). Most of the studies involved the application of agro-industrial residues as substrate, although some used inert substrates such as sugarcane bagasse or agar. While iturin and surfactin were produced using bacterial strains, others came from fungal strains. Iturin is a potent antifungal peptide antibiotic, which is effective in suppressing phytopathogens. It was produced on okara (soybean curd reside) and wheat bran [21]. The SSF process was six to eight times more efficient with respect to productivity than SmF. Yang and Swei [99]

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and Yang [100] reported the production of tetracycline from cellulosic substrates in SSF. Each gram of substrate produced 10– 11 mg of oxytetracycline in eight days. Surfactin, a lipopeptide antibiotic, which is inhibitor of fibrin clotting, was produced using okara [102,105]. Production in SSF was four to five times more efficient than in SmF. Ohno et al. applied SSF for the production of iturin (anti-fungal lipopeptide antibiotic) using okara [103] and wheat bran [104]. Joshi et al. [113] reported new verticillin and glisoprenin analogues from Gliocladium catenulatum, a mycoparasite of Aspergillus fla6us sclerotia by SSF cultures of the sclerotial mycoparasite. Antifungal and antibacterial metabolites from a sclerotium-colonizing isolate of Mortierella 6inacea grown in SSF has also been reported by Soman et al. [114]. One important tool in the discovery of new bioactive compounds could be the exploitation of marine microorganisms which are known to produce a diverse spectra of novel secondary metabolites. Exploitation of marine micro-organisms employing SSF with fishery wastes in place of conventional substrates could revolutionise industrial biotechnology [115]. Marine micro-organisms are unique in nature and differ widely in many aspects with their terrestrial counter parts. They are not fully understood with respect to their biology, which perhaps is the major reason for the lack of adequate recognition to their potential.

4.2. Enzymes SSF holds tremendous potential for the production of enzymes [20]. It can be of special interest in those processes where the crude fermented product may be used directly as enzyme source [116]. In addition to well-established applications in the food and fermentation industries, microbial enzymes have attained a significant role in biotransformations involving organic solvent media, mainly for bioactive compounds. Table 2 lists some of the possible applications of enzymes produced in SSF and Table 2B lists the spectrum of microbial cultures employed for enzyme production in SSF. The selection of a particular strain, however, remains a tedious task, especially when commercially significant enzyme yields are to be achieved. Agro-industrial residues are generally considered the best substrates for the SSF processes, and enzyme production in SSF is not an exception to that. A number of such substrates employed for the cultivation of micro-organisms to produce host of enzymes are shown in Table 2B. As is apparent, wheat bran, however, holds the key and has been used commonly in various processes. Currently, industrial demand of most of the enzymes is met by production using submerged fermentation (SmF), generally employing genetically modified strains. The cost of production in SmF is high and it is

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uneconomical to use many enzymes in several processes. This necessitates reduction in production cost by alternative methods. SSF should be considered an attractive alternative. Tengerdy [116] advocated that SSF was particularly suitable for ligno-cellulosic enzymes production for various agro-biotechnological applications. To illustrate this, a comparison was made for cellulase production in SmF and SSF [116,117]. In SmF, cellulase yields are generally about 10 g/l, and the average fermentation cost in a stirred tank bioreactor is about $200/m3. Thus, the production cost in the crude fermentation by SmF is about $20/kg. In SSF, the average production level is about 10 mg/g substrate and the average fermentation cost is only about $25/mt. Thus, the unit cost of SSF cellulase is just about $0.2/kg [116]. Much published information is available on the production of enzymes of industrial importance, such as protease’s, cellulase, ligninases, xylanase, pectinase, amylase, glucoamylase, etc. Attempts are also being made to study SSF processes for the production of inulinases, phytases, tannase, phenolic acid esterase, microbial rennet, aryl-alcohol oxidase, oligo-saccharide oxidase, tannin acyl hydrolase, a-L arabinofuranosidase, etc. using SSF systems (Table 2B) [20,23,118– 125]. Most of the results, however, are of laboratory, or semi-pilot-scale experiments. Recently, Alltech (Nicholasville, KY, USA) has established a large-scale enzyme production facility, which is claimed to be first in enzyme technology based on SSF [126]. This new facility is located in Serdan (Mexico). Alltech’s European Bioscience Centre, in cooperation with INRA, the French government agricultural research establishment, have been involved in growth conditions and fermenter design. The plant is meant for the production of phytase.

4.3. Organic acids The production of organic acids by SSF is associated with the historical development of SSF. Citric acid has been known produced in SSF for many years while lactic acid, fumaric acid and oxalic acid have been reported to be produced only in the present decade (Table 3) [127–145]. Citric acid is the most important organic acid produced in tonnage and is extensively used in food and pharmaceutical industries. It is produced mainly by submerged fermentation using Aspergillus niger or Candida sp. from different sources of carbohydrates, such as molasses and starch based media. However, SSF using alternative sources of carbon such as agro-industrial residues has great potential. Vandenberghe et al. [146] have reviewed recent development in citric acid production by presenting a brief summary of the subject, describing micro-organisms, production tech-

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niques, and substrates, etc. In SSF, almost the entire production has been obtained using crops and cropresidues as substrates and A. niger as production strain (Table 3). It has been generally found that addition of methanol increased citric acid production in SSF [129,134,135,137,146]. Leangon et al. [127] studied the influence of glycolytic rate on production of citric acid and oxalic acid. They proposed that over-production of citric acid was related to an increased glucose flux through glycolysis. At low glucose fluxes, oxalic acid was accumulated. Tran et al. [128] compared citric production from pineapple waste in different bioreactors (fermenters). They reported best production in flasks and lower yields in tray and rotating drum bioreactors. Lu et al. [130,131] compared citric acid production in packed bed bioreactors. A multi-layer packed bioreactor improved mass transfer considerably compared with a single-layer packed-bed operated under similar conditions. Packed-bed bioreactors showed superior production of citric acid than flask culture. Vandenberghe et al. (Vandenberghe LPS, Soccol CR, Pandey A, Lebeault JM, unpublished results) also obtained higher citric acid yields in packed-bed column bioreactors using cassava bagasse as substrate than in flasks. Improved aeration and heat and mass transfer effects were thought to be the reasons for this. Gutierrez-Rozas et al. [138] compared different mechanisms of heat removal (conductive, convective and evaporative) from packed-bed bioreactors in SSF for citric acid production with an inert support. Results showed that the conductive heat transfer was the least efficient mechanism (8.65%) when compared with convective (26.65%) and evaporative (64.7%). Lactic acid production using SSF has been carried out using fungal as well as bacterial strains. Strains of Rhizopus sp. have been common among the former, and that of Lactobacillus sp. for the latter. Different crops such as cassava and sweet sorghum and cropresidues such as sugarcane bagasse, sugarcane pressmud and carrot-processing waste were used as substrate in these processes (Table 3). Garg and Hang [132] compared lactic acid and citric acid production from carrot-processing waste by fungal strains. Based on sugars consumed, the yield of lactic acid was almost 50% more than citric acid. A strain of R. oryzae was used by Soccol et al. [143] to evaluate L-(+) lactic acid production in SmF and SSF. For SSF, an inert solid support (sugarcane bagasse impregnated with a nutrient solution) was used. Both production level and productivity were higher in SSF, although fermentation yields were 77% whatever was the medium. L-(+)-lactic acid production was of 93.8 and 137.0 g/l in SmF and SSF, respectively. The productivity was 1.38 g/l per h in liquid medium and 1.43 g/l per h in solid medium. However, the fermentation yield was about 77% whatever the medium. Richter and Trager [145] also com-

pared SSF and SmF for lactic acid production using a bacterial strain of L. paracasei. Lactate concentrations and yields were 88–106 g/l and 91–95% for SmF, and 90 g/kg and 91–95% for SSF, respectively. The time required for SSF was 120–200 h in comparison of 24–32 h in SmF. Xavier and Lonsane [144] compared three bacterial strains for lactic acid production from sugarcane press-mud. A strain of L. casei produced a higher concentration of lactic acid in comparison to L. hel6eticus and Streptococcus thermophilus. Gallic acid (3,4,5-trihydroxybenzoic acid) is a phenolic acid and it finds application in various fields such as in manufacture of trimethoprim (used as antibacterial agent), preparation of gallic acid esters (antioxidants), etc. A process was developed to produce gallic acid by biotransformation of tannic acid in SSF using powder of gallo seed cover with Rhizopus oryzae [46].

4.4. Biopesticides It is largely considered that the challenge posed by insects and pests to agriculture industry can be effectively met by biopesticide which are considered environmental friendly. Recently, the use of entomopathogenic and mycoparasitic fungi for biological control of insects and pests has received increasing attention. It is believed that either fungal cells themselves or cell-free components would be equally effective. Deshpande [147] reviewed the production of biopesticides (mycopesticides) by SSF and SmF. The identification of a fungal strain with pesticide activity is the first step in developing infective propagules such as conidia, blastospores, chlamydospores, oospores, and zygospores. It was commented that the understanding of the molecular aspects of fungus–fungus and fungus–insect interactions, the role of hydrolytic enzymes, especially chitinases in killing process, and the possible use of chitin synthesis inhibitors would be the prime areas of research aimed at making fungi more effective either singly as in combination as mycopesticides. Attempts have been made to produce entomopathogenic fungi in SSF. Soccol et al. [148] developed a SSF based process, which investigated several agro-industrial substrates to produce spores from Beau6eria bassiana for use in the biological control of pests of banana, sugarcane, soybean and coffee. Desgranges et al. [149] also used SSF to produce B. bassiana for use against European corn borer. They used clay microgranules humidified with optimum nutrients. The bioinsecticide was produced in a pilot reactor of 1600-l capacity. The bioproduct showed a field efficiency of 80% Colletotrichum truncatum, is another fungal plant pathogen, which shows promise as a bioherbicide against the difficult weed Sesbania exaltata. C. truncatum spores were produced in SSF using solid vermiculite and solid perlite-corn meal-agar [150].

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4.5. Biofuel SSF has been explored as an alternate technology for the production of ethanol from agro-industrial residues. Several advantages such as elimination of the sugar extraction process (and thus savings in the cost), reduction in fermenter volume (due to elimination of water addition), reduction in distillation plant and energy costs, etc. have been attributed to this. Most of the studies involved yeast cultures [151 – 158], although attempts also have been made with bacterial strains. Zymomonas mobilis has been considered as an efficient culture [159]. Apple pomace is a useful substrate for ethanol production in SSF [151 – 153,156]. Sandhu and Joshi [151], Joshi and Sandhu [152] and Joshi et al. [153] studied various aspects of ethanol production from apple pomace in SSF. Natural fermentation of apple pomace was inferior to yeast inoculated fermentation. Highest yields were obtained from Saccharomyces cere6isiae. The performance of Torula utilis and Candida utilis was comparable. Henk and Linden [160] used sorghum as substrate for SSF. They reported that the addition of cellulase enzyme in the substrate improved ethanol yields. Roukas [154] used carob pods as substrate and reported that sterilisation of substrate was not necessary as ethanol yields from sterilised or non-sterilised substrates were the same. Attempts also have been made to use starchy substrates for ethanol fermentation in SSF. Saucedo-Castaneda et al. [155] described the potential of using a single fermenter for biomass built-up, starch hydrolysis and ethanol production in SSF using Schwanniomyces castellii. Results indicated effective utilisation of starch by the yeast culture to produce ethanol. Kiransree et al. [158,161] used several starchy substrates such as sweet sorghum, sweet potato, wheat flour, rice starch, soluble starch and potato starch for ethanol production using a thermotolerant yeast strain of S. cere6isiae. Rice starch and sweet sorghum gave highest ethanol yields.

4.6. Aroma compounds Most of the flavouring compounds are presently produced via chemical synthesis or extraction from natural materials. However, recent market surveys have demonstrated that consumers prefer foodstuff that can be labelled as natural. Plants have been major sources of essential oils and flavours but their use depends on natural factors difficult to control, such as weather conditions and plant diseases. An alternative route for flavour synthesis is based on microbial biosynthesis or bioconversion [162]. Several micro-organisms including bacteria and fungi, are currently known for their ability to synthesise different aroma compounds. Attempts to use these micro-organisms in SmF resulted in low pro-

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ductivity of aroma compounds [163], which hampered industrial application of these processes. SSF could be of high potential for this purpose [164]. One approach in this regard could be to use tropical agro-industrial residues such as cassava bagasse, sugarcane bagasse, coffee husk, coffee pulp, etc. [16–20]. Ferron et al. [165] reviewed the prospects of microbial production of food flavours. They also advocated use of SSF processes for their production. Production of aroma compounds in SSF using naturally occurring substrates could offer potential benefits in production of food and fruity aroma compounds for human consumption at low cost. One major difficulty in this regard, however, remains the isolation/recovery of compounds produced, especially if the compounds have low boiling points. A few attempts have been made in this regard by trapping such compounds in suitable inert materials such as resins by adsorption (Medeiros ABP, Pandey A, Christen P, Soccol CR, unpublished results). However, much remains to be done in this area. Fungi from the genus Ceratocystis produce a large range of fruit-like or flower-like aromas (peach, pineapple, banana, citrus and rose) depending on the strain and the culture conditions [166–168]. Among the genus, C. fimbriata has a great potential for ester synthesis. It grows rapidly, has a good ability to sporulate and produces a wide variety of aromas. Bramorski et al. [169] evaluated the potential of several agro-industrial residues such as cassava bagasse, apple pomace, amaranth and soybean using a strain of C. fimbriata. All media supported fungal growth. While amaranth medium produced pineapple aroma, media with other substrates produced strong fruity aroma. Aroma production was growth dependent and maximum intensity was detected a few hours before or after the maximum respirometric activity. Production of strong pineapple aroma was also reported by Soares et al. [170] when SSF was carried out using coffee husk as substrate by this strain. Medeiros et al. [171] cultivated a strain of Kluy6eromyces marxianus in SSF using different solid substrates such as cassava bagasse, giant palm bran, apple pomace, sugarcane bagasse and sunflower seeds. The feasibility of using cassava bagasse and giant palm bran as substrates to produce fruity aroma was confirmed, although the former proved to be superior. Esters are the source of the aromas. Pyrazines, especially alkylpyrazines are heterocyclic compounds found in a wide variety of foods, which possess nutty and roasty flavour. These compounds are used as food additive for flavouring [172]. Besson et al. [173] and Larroche et al. [174] studied the production of 2,5-dimethylpyrazine (2,5-DMP) and tetramethylprazine (TTMP) using B. natto and B. subtilis, respectively, on soybeans in SSF. Results demonstrated the suitability of SSF for production of these compounds.

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4.7. Miscellaneous compounds There are reports describing the application of SSF systems for the production of various other products such as L-glutamic acid, pigments, carotenoid, xanthan gum, vitamins, biosurfactants, etc. (Table 4) [17,18,27,147–161,166 – 186]. A novel SSF system was developed by Nampoothiri and Pandey [27] to produce L-glutamic acid by cultivating a bacterial strain of Bre6ibacterium sp. on sugarcane bagasse impregnated with glucose, urea, mineral salts and vitamins. Yields as high as 80 mg glutamic acid per g dry fermented matter were obtained. Sugarcane bagasse was also used for the production of pigments in SSF by a strain of Monascus purfureus [175]. Rotary cultures gave higher yields of crude red and yellow pigments than stationary cultures. Exopolysaccharide such as xanthan, succinoglycan, etc. are the future products of SSF. In a recent work, Stredansky and Conti [177] described an SSF-based process for the production of xanthan gum using a culture of Xanthomonas campestris. The exopolysaccharide was produced on a number of agro-industrial residues or by-products such as spent malt grains, apple pomace, grape pomace, and citrus peels. With most of the substrates, the gum production was comparable to those obtained with SmF. These authors also studied succinoglycan production by SSF with Agrobacterium tumefaciens [178] on various solid substrates, including agar medium, spent malt grains, ivory nut shavings and grated carrots, impregnated with a nutrient solution. Fermentations were performed on a laboratory scale, both under static conditions and with agitation, using bottles and a prototype horizontal bioreactor. Several fermentation parameters were examined and optimised, including carbon and nitrogen composition, water content and layer thickness of the substrate. The yields and rheological properties of the polymers obtained under different fermentation conditions were compared. The highest succinoglycan yield was achieved in static cultivation, reaching 42 g/l of impregnating solution, corresponding to 30 g/kg of wet substrate. Polymer production in the horizontal bioreactor was faster, but the final yield was lower (29 g/l of impregnating solution). A comparison of SmF and SSF for the production of bacterial exopolysaccharides (EPS) showed that the latter technique yielded two to four times more polymer than the former, on the laboratory scale. SSF was performed using inert solid particles (spent malt grains) impregnated with a liquid medium. The polymer yields obtained from SSFs, as referred to the impregnating liquid volumes, were as follows: 38.8 g/l xanthan from Xanthomonas campestris, 21.8 g/l succinoglycan from Rhizobium hedysari and 20.3 g/l succinoglycan from Agrobacterium tumefaciens PT45. The authors claimed that these results made this technique promising for use on the industrial scale. A further advantage

with this fermentation process could be in the availability and low cost of substrates, which are obtained as by-products or wastes from the agricultural or food industry [179]. Keuth and Bisping [180,181] described the formation of water soluble vitamins (vitamin B12, vitamin B6, riboflavin, thiamine, nicotinic acid and nicotinamide) during the tempeh SSF. The role of several strains of Rhizopus oligosporus, R. arrhizus, and R. stolonifer and the role of several bacteria in the vitamin formation process were checked. The Rhizopus strains formed riboflavin, nicotinic acid, nicotinamide and vitamin B6. The final concentrations of these substances depended on the different strains involved and on the fermentation time. Isolates of R. oligosporus were generally the best vitamin formers. The moulds did not produce physiologically active vitamin B12. The addition of bacteria, which had been selected in a screening for vitamin B12 production, resulted in an increase of physiologically active vitamin B12. Citrobacter freundii and Klebsiella pneumoniae showed the best formation capabilities. Biosurfactants, which have drawn attention in recent years due to their low toxicity, biodegradability and ecological acceptability, are another group of compounds successfully produced by SSF. Traditionally, biosurfactants have been produced from hydrocarbons, which, however, makes them unattractive due to high cost when compared with chemical surfactants. Application of non-conventional substrates such as agro-industrial residues and by-products could be an attractive possibility [184,185]. Biosurfactants could find potential application in microbial enhanced oil recovery.

5. Conclusions Critical analysis of current literature shows that SSF offers several potential advantages for bioprocessing and production of various value-added products. It is well established that in many cases such as enzymes, bioactive compounds, etc. the product titres produced in SSF are many-fold higher than SmF. Although the reasons of this are not fully understood, efforts are being made to realise this fact. There has been much development on application of SSF in various areas such as bioremediation and biodegradation of hazardous compounds, biological detoxification of toxic agro-industrial residues, biotransformation of crops and crop residues for nutritional enrichment, biopulping, etc., and product-developments such as biologically active secondary metabolites, including antibiotics and other drugs, enzymes, organic acids, biopesticides, including mycopesticides and bioheribicides, biofuel (ethanol and other organic solvents), biosurfactants, food flavour compounds, etc. based on SSF system will

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be the technologies of the future. Considering the difficulties in handling the large volume bioreactors related to the production of bulk chemicals and products in SSF, it could be more practical to use SSF for the production of ‘low volume-high cost’ products such as biopharmaceuticals, including antibiotics and other drugs, some enzymes and organic acids, etc.

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