Solid State Fermentation and Its Applications[1]

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Chapter 18. Solid State Fermentation and Its Applications

Liping Wang and Shang-Tian Yang

Department of Chemical and Biomolecular Engineering, The Ohio State University, 140 West 19th Avenue, Columbus, Ohio 43210, USA

1. INTRODUCTION

Solid-state fermentation has long been applied to the food industry. SSFs are processes carried out with microbes growing on nutrient impregnated solid substrate with little or no free water. The growth of koji, an enzyme-rich mold grown on shallow trays of steamed rice, is a classical example of SSF. Solid state fermentation (SSF) can be directly carried out with abundant low-cost biomaterials (starch, cellulose, lignin, hemicellulose, chitin, etc.) with minimal or no pretreatment, and thus is relatively simple, uses less energy than submerged fermentation (SmF), and can provide unique microenvironments conducive to microbial growth and metabolic activities. Currently, SSF is undergoing a renewed surge of interest, primarily because of the opportunities that SSF affords for increased productivity and product concentration as compared to SmF [1, 2]; new product possibilities, cheaper product recovery, and the prospect of using a wide range of agri-industry commodities and waste streams as substrates. Large amounts of excess plant biomass are produced by the agri-industry. It is desirable to use this as a renewable resource for sustainable chemical production via microbial cultivation. If not used to generate a value-added product, the biomass would remain in the waste stream and require expensive disposal or treatments. The major reason that Western industry is reluctant to use SSF is a lack of knowledge and of scalable bioreactor technologies. There are very few data on growth and product formation kinetics, reactor design, or process control in SSF available in the literature. However, with the increased interest in SSF with the goal of developing industrially applicable SSF systems, progress is being made. This chapter will review the recent advances in SSF system research and development.

2. PRODUCTS FROM SOLID STATE FERMENTATION (SSF)

SSF has been applied in the preparation of traditional foods since ancient times, especially in the Orient. Koji processing is a well-known example. In the koji process, steamed rice is inoculated with the spores of Aspergillus oryzae or Aspergillus sojae and kept in a temperature and humidity controlled room for a number of days. The mold germinates and produces hydrolytic enzymes which act on the rice starch, and when the fermentation is

Bioprocessing for Value-Added Products from Renewable ResourcesShang-Tian Yang (Editor)© 2007 Elsevier B.V. All rights reserved.

L. Wang and S.T. Yang

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finished, the koji is used for the enzyme treatment of other fermentation substrates or is itself used as a substrate for a subsequent fermentation. Soy sauce production, for example, begins with koji fermentation. With the development of modern biotechnology, solid state fermentation can be applied to produce various products, including enzymes, organic acid, secondary metabolites (e.g., antibiotics), biofuels (e.g., ethanol), aroma compounds, and even many bioactive products like mycotoxins, plant growth factors, immuno-suppressive drugs, etc. [3−5]. Enzymes produced by SSF have many industrial applications, such as enzyme assisted ensiling, bioprocessing of crops and crop residues, fiber processing, feed supplement, biopulping, soil bioremediation, biopesticide, etc. [6]. Tables 1−3 list some important products from SSF, both on a laboratory and an industrial scale.

2.1. Organic acids from SSF Some organic acids, such as citric acid, have long been produced from SSF. Others, lactic

acid, fumaric acid, oxalic acid, gluconic acid, etc., were reported to be produced by SSF only in recent years (Table 1).

2.1.1. Citric acid There is a large and still growing market for citric acid in the food, pharmaceutical, and

other industries all over the world. It is one of the world’s largest fermentation products with an estimated annual production of 1,000,000 tons [7]. Currently, it is produced mainly by SmF using Aspergillus niger or Candida sp. from different sources of carbohydrates, such as molasses and starch based media [8].

With the renewed interest in SSF, production of citric acid in SSF with low-cost agricultural products or residues has attracted attention (Table 1). Cost reduction can be expected when using these less expensive substrates, such as cassava bagasse [9], sugar cane bagasse [10, 11], fruits waste [12], sugar beet pulp [13, 14], apple pomace [15], coffee husk, and cassava bagasse [10].

In solid state fermentation, citric acid production is affected by several critical factors including carbon, phosphorous and nitrogen sources, trace elements, alcohols, pH value, oxygen availability, and CO2 accumulation [9, 16]. The fermentation yield of citric acid (g/kg dry substrate) varies depending on the substrates added; e.g., it was higher with tapioca and cane molasses than with wheat bran or potato waste pulp [17]. Several researchers have reported that the addition of methanol (3−6% w/w) into the solid substrates stimulates the production of citric acid [18–20]. It was also found that the stimulation of citric acid production by methanol was affected by moisture content of the substrates and of the strains. Addition of methanol increased production of citric acid only when using low-active strains of A. niger or highly moist substrate. However, the addition of methanol always eliminated the sporulation of A. niger strains [12, 13].

With A. niger, citric acid production is aided by a low aeration rate and a high concentration of CO2 because a low-oxygen environment limits the respiration activity of the cells, which consequently turn to citric acid synthesis [9, 21]. Various kinds of SSF reactors for citric acid production have been tested. Packed-bed reactors were found to be superior to flasks, trays and drum reactors [3, 9, 22]. Improved heat and mass transfer are thought to be

Solid state fermentation and its applications

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the reason. However, reactor design and scale up are complicated tasks. A detailed study will be necessary to test all the factors that may affect the fermentation process and to evaluate the efficiency of various kinds of reactors for the production of citric acid.

Table 1 Some organic acids produced in solid state fermentations

Product Organism Substrate Reference

Citric acid Aspergillus niger

Kumara (starch) Sweet potato Cassava bagasse Sugarcane bagasse

[23] [24] [9] [11, 12]

Lactic acid Lactobacillus amylophilus Lactobacillus paracasei Lactobacillus casei Rhizopus sp. and Acremonium thermophilus Rhizopus oryzae and Aspergillus niger Rhizopus oryzae

Wheat bran (starch) Sweet sorghum Sugar-cane press mud Corncob (cellulose) Carrot processing waste Sugarcane bagasse

[25, 26] [27] [28] [29] [30] [31, 32]

Oxalic acid Aspergillus niger White rot fungi

Sweet potato Spruce sapwood chips

[24] [33]

Fumaric acid Rhizopus arrhizus Rhizopus sp.

Orange peels Raw cassava starch

[34] [31, 32]

Gluconic acid Aspergillus niger Fig extract Glucose Fig

[35] [36] [37]

Gallic acid Aspergillus foetidus and Rhizopus oryzae

Mixed tannin-rich agro-products

[38]

2.1.2. Lactic acid Lactic acid can be produced by the fermentation of bacterial or fungal strains. Lactobacillus

sp. (bacteria) and Rhizopus sp. (fungi) are the commonly used strains. Although industrial lactic acid fermentation is currently mainly carried out with homolactic acid bacteria in SmF, there has been increasing interest in fungal fermentation with Rhizopus oryzae for lactic acid production because of its unique ability to produce optically pure L(+)-lactic acid from glucose, xylose, and starch. However, controlling the fungal mycelial morphology and broth rheology is important to fungal fermentation. The highly branched fungal mycelia usually cause complex (viscous) broth rheology and difficulty in mixing and aeration in SmF with conventional agitated tank fermenters. Various cell immobilization methods to control the cell morphology and to achieve high cell density and high reaction rate have been studied. Tay


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and Yang [39] have developed a rotating fibrous bed bioreactor for immobilizing fungal cells and achieved excellent lactic acid production from glucose and soluble starch, with high lactic acid yield (>90% from glucose) and productivity (2.5 g/L⋅h or 467 g/h⋅m2). However, their good fermentation results were only obtained with increased oxygen tension (90%), and the results were not as good at a lower oxygen tension (25%−50%), indicating that oxygen transfer limitation was severe in the immobilized cell culture. This disadvantage has also greatly limited the industrial application of immobilized fungal cell fermentation. Furthermore, the reactor productivity is limited by the available surface area for cell attachment, and such a surface-dependent immobilized cell bioreactor is difficult to scale up. There is another disadvantage with SmF when starch is used as the substrate: starch has a relatively low solubility in water, and insoluble starch granules are difficult to handle (e.g., pumping and mixing) in conventional SmF. It is very difficult to use crude (insoluble) starch directly in SmF, especially with immobilized cells, as there would be increased mass transfer barriers. Consequently, the fermentation is much slower. In recent years, production of lactic acid from SSF has been reported [3]. Different crops or crop residues, such as sweet sorghum, corncob, sugarcane press mud, and carrot-processing waste, were used as substrates (Table 1). Soccol et al. [31, 32] evaluated lactic acid production in SmF and SSF (with inert support) by using a strain of R. oryzae, and found that both production level and productivity were higher in SSF. This indicates that SSF also has the good potential to economically produce lactic acid and other organic acids and alcohols from starch present in solid substrates.

2.2. Enzymes from SSF Historically, enzymes have long been produced from SSF. Several reviews of the

production of enzymes from SSF have been published in recent years [3, 5, 40]. Recordings of SSF can be found in Asia starting from thousands of years ago, e.g., the Koji process mentioned above. Evidently, the SSF process originated from food fermentation and production of enzymes. In theory, all the enzymes that are presently known and produced by any means are able to be produced under SSF. Some examples are listed in Table 2. The microorganisms involved can be filamentous fungi, yeasts, or bacteria.

2.2.1. Amylases Amylases are among the most important industrial enzymes in commercial biotechnology

[3]. Generally, hydrolytic enzymes and amylolytic enzymes are commonly produced by filamentous fungi; the preferred strains include Trichoderma spp., Aspergillus spp., and Rhizopus spp. [2]. Four of the amylases are of special interests: α-amylase, β-amylase, glucoamylase, and pullulanase. Each of them attacks the starch molecule at different sites, resulting in products of various chain lengths. Amylase production has been reported in the species of the genera Aspergillus, Penicillium, Cephalosporium, Mucor, Candida, Neurospora, Rhizopus, etc. [40, 41].

In industry amylases are mainly produced by fungi and bacteria through SmF. In recent years, the feasibility of applying SSF to the production of amylases has been intensively investigated. All kinds of agro-products or residues can be used as substrates in SSF, such as oil cakes (coconut oil cake, sesame oil cake, groundnut oil cake, palm kernel cake, olive oil


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cake, etc.), wheat bran, rice husks, spent brewing grains, banana peels, potato peels, etc. [42–48]. The most attractive feature of SSF for the production of enzymes is that it is much more economical than SmF. It is generally recognized that production of many industrial enzymes by SSF is much less expensive than by submerged fermentation [2]. In submerged fermentations, the product enzyme is often produced at a low concentration and with a low yield and productivity. Recovery of the enzyme product from the dilute fermentation broth is also costly. Current methods for using either starch or cellulose as fermentation feedstock usually include a hydrolysis step followed by fermentation. The hydrolysis of these polymeric biomaterials for fermentation use is, however, costly. Of special interest to industry are processes which combine microbial cultivation simultaneously with the hydrolysis of the biomass, as found in many SSF systems. Simultaneous saccharification and fermentation eliminates the hydrolysis step, thereby reducing production costs. Furthermore, many fungal enzymes can only be produced at high quantities under SSF conditions.

Table 2 Some enzymes produced in solid state fermentations


Amylases Aspergillus oryzae Brown rice [49] Glucoamylase A. niger Tea waste [50] Lipase A. niger

Penicillium simplicissimum Gingelly oil cake Soy cake

[51] [52]

Acid protease A. niger Wheat bran [53] Cellulase, Gluco-sidase

A. ellipticus, A. fumigatus; Fusarium oxysporum

Lignocellulosic waste Corn stover

[54] [55]

Cellobiase A. niger Waste pulp [56] Xylanase A. niger

Fusarium oxysporum Trichoderma longibrachiatum

Wheat bran Corn stover Wheat bran-malt sprouts mixture

[57] [55] [58]

Hemicellulase Thermomonospora strain 29 Coffee waste [59] Phytase A. niger

A. ficuum, Mucor racemosus, Rhizopus oligosporus

Wheat bran Canola, soybean meal, cracked corn, wheat bran

[60] [61, 62]

Mannanase A. ochraceus Coeffee waste [63] Pectinase A. niger Soy and wheat bran [64] Pectin lyase A. niger Misc. [65] Chitinase Trichoderma harzianum Wheat bran + chitin [66] Tannase A. niger; A. foetidus, R.. oryzus Tannin-rich powdered fruits [39]


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2.2.2. Phytase Phytase is an enzyme that makes the phosphorus from phytin available for animal

digestion. Up to now, phytase has been mainly used as a dietary supplement for swine and poultry. As phytase is increasingly used in animal feeds, science and technology related to this enzyme are rapidly evolved. Several detailed reviews have been published in recent years [67, 68].

The benefits of phytase are its double effects on reducing the use of expensive inorganic phosphorus in animal diets and the environment pollution from excessive manure phosphorus runoff [68]. In grains used for animal feed, about 60−75% of phosphorus is in an organically bound form known as phytate, which cannot be utilized by monogastric animals. Thus, the majority of this phytin phosphorus will pass directly through the digestive track of the animals and winds up in manure and liquid effluent. In the U.S., large-scale animal production (pig and poultry farms and cattle feedlots) generates enormous quantities of potentially hazardous wastes, with phosphorus being a major pollutant. It has been shown that adding 500 to 1000 units of phytase to monogastric animal feeds can replace approximately 1 gram inorganic phosphorus supplementation and reduce phosphorus in the manure by about 30–50% [68]. The rate of phytase inclusion in animal diets is depending on both the desired degree of phytin reduction and economical considerations. Phytase currently produced by submerged fermentation is relatively expensive, and may add US $2–3 per metric ton to the feed cost [61]. SSF would be an economical alternative for the production of the enzyme. Phytase produced in SSF can be easily extracted with water and the production cost is expected to be much lower than that in submerged fermentation because of higher enzyme concentrations and activities. Phytase produced by filamentous fungi on selected feed ingredients contains also accessory enzymes, fungal protein and organic acids that increase feed digestibility and access to phytin in plant cells [61]. The product can be directly mixed in feed rations as a value-added supplement, further reducing the cost for use in animal feed.

2.2.3. Chitinases Chitinases are hydrolytic enzymes responsible for the degradation of chitin, a high

molecular weight linear polymer of N-acetyl-D-glucosamine units. As chitin degrading enzymes, chitinases have received increasing attentions in applications in the biocontrol of fungal pathogens, because chitin is the major structural component of fungal cell walls. Biological control is an attractive alternative for fungal pathogen control, since it neither causes environmental pollution nor induces pathogen resistance, which is usually a side effect of synthetic antifungal agents [69, 70]. Chitinases can be found in a wide range of organisms, including fungi, viruses, bacteria, insects, plant, and animal. However, chitinases of filamentous fungi have been shown to have higher activity levels and a wider antifungal spectrum than those of plants and bacteria [71]. Li [72] has reviewed fungal chitinases in details. The potential of industrial production of chitinases by either bacteria or fungi has been studied in both submerged and solid state fermentations [66, 71, 73−79]. Trichoderma harzianum is one of the most widely studied microorganisms for the production of chitinases. Mass production of Trichoderma spores for biocontrol purposes is of a high interest. Unfortunately, large-scale production of chitinases at present is still too expensive and


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uneconomical to make this enzyme available in sufficient quantities. SSF production of chitinase and Trichoderma spores could greatly reduce the production costs and thus should have a good potential for commercial applications.

In spite of the high productivity and low cost of SSF process, currently the application of SSF in the production of industrial enzymes is still mostly limited to lab scales. Most of the research is focusing on strain screening, process parameter optimization, small scale reactor design, etc. The main problem is that the development of a simple, practical, and automated SSF fermenter has not yet been achieved.

2.3. Biological control agents (BCA) from SSF Chemical pesticides have been extensively used for many years in agriculture all over the

world. It is a general concern that the use of these chemical pesticides poses adverse effects on human health and the environment. For this reason, there has been interest in researching biological control agents (BCA) since the 1960s [90] (Table 3). A biocontrol agent may be a living microorganism, a natural product of microbial origin, or a chemically modified natural product of microbial origin. Several steps are required to develop a microbial pesticide, including isolation of the microorganism, identification and characterization, and pilot trials under real conditions [91]. Both submerged and solid state fermentation processes have been used for BCA production. In this part, we will discuss only the production of BCAs by solid state fermentation.

Table 3 Some biocontrol agents and other compounds produced in solid state fermentations


Biocontrol agents (aerial spores or conidia)

Trichoderma harzianum

Epicoccum nigrum

Coniothyrium minitans

Solid substrates;

Peat/vermiculite/lentile meal

[80]

[81]

[82]

Biofungicide Collectotrichum truncatum Perlite + corn meal [83]

Bioinsecticide Beauveria bassioana Clay granules + liquid medium [84]

Neomycin Streptomyces marinensis Various solid substrates [85]

Penicillin Penicillium chrysogenum Sugar cane bagasse [86]

Griseofulvin Penicillium griseofulvum Rice bran [87]

Red pigment Monascus sp. EBF1 Barley [88]

Biopulping Phanerochaete chrysosporium Ceriporiopsis subvermispora

Wood chips [89]

In the development of BCAs, it is important to develop methods for mass production.

Usually, bacteria and yeast are produced in submerged fermentation, while many fungi are fermented in solid state fermentation. It was found that SSF has some advantages over SmF in


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producing high quality BCAs inocula. SSF can produce abundant, robust, and healthy conidia because SSF is more like fungal natural environmental conditions and because habitat aeration is better, while many filamentous fungi sporulate poorly in SmF. Coniothyrium minitans is a promising fungal BCA of the plant pathogen Sclerotinia sclerotiorum. It sporulates relatively poorly in liquid media, but sporulates well on solid substrates [82, 92–94]. C. minitans spores produced by SSF are of better quality, with greater resistance to UV-irradiation and desiccation during recovery procedures, and are more viable after storage [90]. De Cal et al. [95] reported that the conidial production of Penicillium frequentans was higher in SSF than in SmF. Fresh conidia produced by solid-state fermentation reduced the incidence of brown rot on plums by 75%. Agosin et al. [80] compared the aerial spores of T. harzianum P1, a potential biocontrol agent, produced in SSF against those produced in SmF. They found that spores from SSF had higher productivity, greater UV-resistance, and a longer shelf life. The SSF spores had a thicker outer wall and fewer organelles. The SmF spores, however, were usually mostly collapsed, inhibited many cytoplasmic organelles, and had a much thinner outer wall. Even some bacteria-originated BCAs had higher productivities in SSF than in SmF. Bacillus subtilis isolated by Shoda [96] exhibited a broad suppression spectrum to various plant pathogens mainly by producing lipopeptide antibiotic, iturin A. When this bacterium was grown on soybean curd residue in SSF, the productivity of iturin A was about 10 times higher than when grown in SmF [96].

However, the commercialization of these microorganisms as BCAs has been hampered by the lack of a cost-effective way of producing sufficient amounts of biomass for use. Design and scale-up of an appropriate SSF reactor with control and automation is a complicated task and a breakthrough has not yet been achieved.

3. ADVANTAGES AND UNSOLVED PROBLEMS

As listed in Table 4, SSF has many unique process characteristics that differ from and often provide advantages over SmF [1], especially for filamentous fungi that are usually more difficult to operate in SmF than bacteria and yeasts. Filamentous fungi have long been employed in the fermentation industry and continue to be the principal source of antibiotics and enzymes. As saprophytes, filamentous fungi naturally secrete large amounts of enzymes and metabolic products. As an additional advantage, secretion assures simplified and inexpensive product recovery because the product is purified from the spent medium rather than from the host biomass [97]. Currently, about 40% of industrial enzymes are produced by filamentous fungi [98], mainly by SmF processes. Compared to SmF, SSF is a process with low cost and high productivity. Many industrial enzymes have been shown to be better produced by SSF than by SmF [65]. It has been reported that the production cost of cellulase in SmF is ~$20/kg; while the cost is only ~$0.2/kg in SSF [3]. The production of pectin lyase by A. niger was 3 times higher in SSF than in SmF, and the produced enzyme in SSF constituted 65% of total extracellular proteins produced by the fungus [65]. Maldonado and De Saad [99] reported that enzyme production was four to six times higher in SSF than in SmF. In general, SSF is more suitable than SmF for the growth of filamentous fungi under conditions where catabolite repression applies [100].


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Table 4 Some unique characteristics and advantages of solid state fermentation

Characteristics Advantages/Comments

Low moisture content lower reactor volume required for a given productivity lower purification costs because of higher product concentrations lower costs for treatment of liquid effluent inhibition of contaminants

High interfacial surface area to liquid volume ratio

aeration is easily achieved generally has lower energy requirements

Simulates the natural environment for microbial growth

allows more complete genetic expression in the microbe some products are produced at higher rates in SSF some products are produced only in SSF yields are reliable and reproducible

Simple media often no more than unprocessed grains with minimal mineral supplementation or no supplementation at all may consist of agri-industry wastes such as corn fiber and bagasse

Substrate availability may increase during fermentation (or decrease or remain constant, as well) rather than always decrease as it does in SmF

In addition to the higher production titers and yields, phytase produced in SSF showed

better heat resistance and retained most of its activity after agglomeration and palletizing in the process of producing animal feeds [60]. Also, fungal spores or conidia produced in SSF had a thicker outer wall and showed greater resistance to UV light and heat, and also had a longer shelf-life when used as a biocontrol agent [80].

Recent advances in the molecular genetics of filamentous fungi have also allowed development of commercially promising recombinant fungal strains for production of enzymes, heterologous proteins, and other biochemicals [98, 101]. However, most filamentous fungi, including Aspergillus, secrete a diversity of extracellular proteases [102] which may cause a major problem for heterologous protein production because these extracellular proteases degrade heterologous proteins. The construction and use of protease-deficient host strains may alleviate this problem, but often at the expense of reduced expression of the protein product. Development of an optimal production medium and classical strain improvement programs (random mutagenesis and selection) have also been studied with limited success in improving protein yields. Although gene expression dynamics of filamentous fungi under SSF conditions remain largely unknown, there is a possibility that the attack of fungal proteases on heterologous proteins may be reduced in SSF, thus allowing filamentous fungi to be used as a more efficient producer for secreted recombinant protein (enzyme) production.

However, there are several major problems in the development of SSF on an industrial scale, including the mass and heat transfer limitations and difficulty in handling solids in existing reactors and the lack of kinetic and design data on various fermentations [103, 104].


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Heat and mass transfer are particularly critical issues for scaling-up SSF processes. Several workers have suggested forced air convection in packed bed reactors as at least a partial solution to the mass and heat transfer difficulties [105–109] noted in even shallow tray fermenters [110]. Although packed bed reactors successfully increase protein productivity, a temperature gradient exists in the reactor, because the lack of free-flowing water causes inefficient heat removal [111]. It is estimated that even with an airflow as high as one volume per reactor volume, up to 85% of the enzyme produced by the microorganism can be denatured because of the high heat accumulation by the end of the fermentation [112]. It also has been shown that the growth of biomass in the void space of packed beds is responsible for much of the decrease in effective diffusion coefficients observed in these reactors and is therefore much of the cause of the formation of gaseous concentration gradients [113]. Furthermore, it has been suggested that steric hindrance in packed beds prevents the continued growth of biomass [114]. Another common problem in SSF is bed caking caused by substrate matrix change during the fermentation process, which in turn causes difficulties in process control and downstream processing. Difficult solid handling in the process as run in existing reactors also presents a major problem in the development of SSF on an industrial scale [107]. In general, packed bed bioreactors are difficult to scale up and pose problems in solid handling [107]. They are also difficult to operate as continuous reactors.

4. SSF REACTORS

To commercialize SSF processes for the production of enzymes, chemicals, and biologically active compounds (including enzymes and therapeutic proteins) from polymeric biomaterials produced in large quantities as low-value commodities or byproducts in the agricultural industry, it is necessary to develop efficient bioreactor systems [115]. The most commonly used SSF bioreactors are tray reactors, drum reactors, and packed bed reactors [3, 116]; they vary in their agitation and aeration conditions. It is recognized that continuously-mixed beds with forced aeration have the potential to perform better than other bioreactors due to their good heat and mass transfer characteristics. In recent years, many mixed SSF reactors have also been studied [3], such as rotating drum reactors and fluidized beds reactors. Durand [117] gave a detailed review of SSF reactor designs. Table 5 lists and compares some important SSF reactors.

4.1. Tray reactors Tray reactors, based on traditional Koji fermentation, are the simplest SSF reactors. They

are typically stacks of shallow trays loaded with fermentation substrates, which are inoculated with microorganisms, in an aerated and controlled environment room (Fig. 1). The top surface of trays is exposed to the air. The bottom surface is usually perforated but without forced air circulation through the tray. The temperature is controlled by controlling the temperature of the gas stream into the room. Moisture saturation is maintained by spraying sterilized water into the room, thus keeping an optimal moisture content level for the fermentation. The current industrial applications of SSF usually use this kind of reactor, as it is the most traditional form of SSF reactor and a lot of knowledge and experience have been


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accumulated. However, there are unsolved problems, among which heat removal is the most important one. The heat removal in trays depends mainly on conduction through the tray walls to air. In general, conductive cooling is insufficient to remove the metabolic heat for a large scale SSF system, even if it is continuously mixed [118]. Other problems include a high contamination risk and low substrate utilization efficiency resulting from low heat and mass transfer rates.

Table 5 Features and problems of commonly used SSF reactors

Reactor type Features / problems References

Tray reactors Simple structure, easy to operate Non-aeration, non-mixed Heat accumulation Temperature and moisture gradients generated Bed caking

[110, 118, 119]

Packed-bed reactors Forced-aeration, non-mixed Axial temperature and gas concentration gradients exist Difficult to scale-up Bed caking

[103, 107, 112, 120, 121]

Rotating-drum reactors Forced aeration, continuously or intermittently mixed Improved mass and heat transfer Shear effect may cause damage to organisms Slumping flow may cause little mixing Complicated reactor construction Difficult operation

[122, 123 – 129]

Fluidized-bed reactors Continuously mixed High mass and heat transfer rate No bed caking High power requirements High shear damage to microorganisms Difficult to fluidize large, coarse and sticky particles

[130 – 134]

Spouted-bed reactors Continuously or intermittently mixed High mass and heat transfer rate Lower power requirements than fluidized-beds Good in handling large, coarse, non-uniformly sized,

and sticky particles usually used in SSF Need further investigations on characterization and

scale-up

[49, 135– 141]

When heat is not sufficiently removed, temperature and moisture gradients will develop in

the matrix. Rathbun and Shuler [110] have experimentally studied temperature and gas concentration gradients in tray fermenters. They established a temperature gradient in a tray


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containing various depths of medium, and then measured temperature and concentration as cultivation proceeded over time. The vertical temperature gradient was larger than the lateral gradient as a result of the heating. When thin (~0.5 cm) beds were compared to relatively thick beds (~3.2 cm), little or no transport resistance was found for the shallow beds, allowing intrinsic growth parameters to be determined. The thicker beds had noticeable transport limitation effects, developing considerable gradients in both temperature and oxygen concentration.

Fig. 1. Schematic of a tray reactor.

Fig. 2. Schematic of a PlaFractor™ stacks fermenter (adapted from [119]).

The evaporating of water from the matrix removes a little of the metabolic heat, but still

cannot solve the problem because of the poor mass and heat transfer inside the fermentation matrix. Thus, the limitation is that only a thin layer in the tray can be effectively fermented. Bed caking, which is caused by the agglomeration of mycelium, makes the problem even worse. The caking can be a major resistance to the air flow. Some of these systems incorporate a “mixing” arrangement. However, mixing can cause shear damage to the mycelium and loss of productivity, so this has to be balanced against the productivity loss

Air Exhaust

Water spray


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resulting from improper temperature control or bed drying. In many cases, the productivity of unmixed tray culture systems is better than that of mixed systems [119].

There have been many efforts in improving this traditional fermentation process, e.g., Biocon India Ltd. developed a technology called PlaFractor™ in 2003 (Fig. 2) [119]. A PlaFractor™ stack is made of several tray modules arranged upon each other and sealed by gaskets in between. Each tray module has a mixing arm with blades, which rotate around the axis formed by the arms. It seems to be featured of contained environment, superior temperature control, automation and in-situ operations, and space and energy saving.

4.2. Packed bed reactors A packed bed bioreactor for SSF is a column with a perforated base for forced aeration. It

may have a jacket for water circulation to control the temperature during fermentation. Packed bed reactors allow forced convective mass transfer as air is pumped through the bed. Smaller gradients than those found in trays are found in packed bed reactors, but reduction in bed porosity with time can still be a problem [103]. Axial temperature and gas concentration gradients have been documented in packed columns [120], although they are much smaller than those observed in tray reactors [121]. Compared to tray reactors, packed bed reactors successfully increased the enzyme productivities. However, they are difficult to scale up, and with bed caking in the columns, solid handling becomes difficult [107]. They are also difficult to operate as continuous reactors. Despite these problems, efforts have been made in order to improve the performance of packed beds and to scale up it.

In order to scale up the reactor, a mathematical model predicting temperature, moisture, biomass, and substrate profile is a valuable tool. Several models have been published. Earlier models usually concerned only energy balance [122, 142–146], which gave information on radial and axial temperature gradients, because heat removal is the main problem for large scale SSF reactors. The axial temperature gradients promote evaporation even if saturated air is used to aerate the column because the water carrying capacity of the air increases as it heats up. This evaporation not only removes 65% of the waste metabolic heat [147] but also dries the substrate, thus inhibiting cell growth. Water balance is therefore also very important. Oxygen supply has been reasonably ignored in most models if the bed is forced aerated. In reactors with forced aeration, the supply of oxygen to particle surfaces is not a limiting factor [120]. Several recent models have included the water balance. Weber et al. [142] proposed a model with enthalpy and water balances.

Enthalpy balance:

aai j

jjii hz

FHrhChCt ∂

∂⋅−∆⋅−=

⋅⋅+⋅⋅−

∂∂ ∑ ∑ ''

0'''

0)()()1( εε

Ci and Cj indicate concentrations of all components in the moist solid matrix and the gas phase, respectively.

Water balance:

[ ] gawwgxwxss zFrCCxC

tϕεϕε

∂∂

⋅−=⋅+⋅+⋅⋅−∂∂ ''''')()1(


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These equations can be simplified based on the following assumptions [142]: first, in the accumulation terms of the balances, all contributions of gases and all mass accumulation terms are negligible; second, there is a pseudo-steady state with respect to temperature and oxygen consumption rate; third, the air is at equilibrium with the solid matrix at any point in the bed; and last, the oxygen consumption rate is independent of temperature, provided that the air inlet and outlet temperatures are chosen within the optimal range found by McQuilken et al. [148]. The simplified equations are:

)(0 ''0

'''0 aa h

dzdFHr ⋅−∆⋅−=

[ ] gawxwxss zFYrCxC

tϕϕε

∂∂

⋅−⋅−=⋅+⋅⋅−∂∂ ''

0'''

0)()1(

The model was validated with the fermentation of the fungi Coniothyrium minitans and Aspergillus oryzae. The model gave accurate temperature predictions when online oxygen measurement was used as input.

Von Meien and Mitchell [149] developed a two-phase model for water and heat transfer within an intermittently-mixed solid-state fermentation bioreactor with forced aeration.

Water balance in gas phase:

)(' *ss

ggg aK

zG

tϕϕ

ϕϕρε −=

∂∂

+∂∂

⋅

Water balance in solid phase:

)()(')( *

tSC

tbSYaK

tS

xWBsss

∂∂

+∂∂

+−−=∂⋅∂ ϕϕϕ

Energy balance in gas phase:

)()()( sgg

PgPgg

PgPgg TThaz

TGCC

tT

CC −−=∂∂

⋅⋅++∂∂

⋅+⋅ νν ϕϕρε

Energy balance in solid phase:

∂∂

+∂∂

+−⋅−−=∂∂

⋅+⋅tSC

tbSYaKTTha

tTCCS xQsssg

sPwsPs )(')()( *ϕϕλϕ

This model predicts that it is impossible to prevent the bed from drying out when it is forced aerated unless the bed is intermittently mixed.

Even with heat removal by evaporation, some extra care may still be needed when scaling-up solid-state fermentation processes for the production of thermo-labile products. Muller dos Santos et al. [112] studied thermal denaturation in packed beds. Based on the mathematical model of a well-mixed bioreactor, it is suggested that even with airflows as high as one vvm, up to 85% of the enzyme produced by the microorganism can be denatured by the end of the fermentation.


479

4.3. Rotating drum reactors In a rotating drum reactor, the substrate bed is held within a horizontal or near horizontal

drum, which can be continuously or intermittently mixed (Fig. 3). The effect of operating conditions, particularly the rotating speed, have been studied by many researchers. Stuart et al. [123] revealed that the extent of the effect of shear caused by drum rotation is controversial. In some studies, high rotating speed reduced productivity, presumably because of the deleterious effects of shear. However, in some other studies, highest productivity was obtained at the highest speed. Based on their own experiments, Stuart et al. [123] concluded that the effects of operational variables on the performance of the reactors were mediated by their effects on transport phenomena, such as mixing, heat and mass transfer, and shear within the fermentation bed.

Another critical issue concerned with the performance of the rotating drum reactors is the slumping flow of the fermentation bed. At the low rotational speeds (a few rpm) typically used for rotating drum bioreactors, the bed in an unbaffled drum undergoes a slumping flow in which the bed as a whole slides down the internal surface of the drum, causing relatively

little mixing within the substrate bed. Under such conditions, the rotating drum does not perform any better than a tray bioreactor. In order to avoid the slumping flow, two strategies have been considered: high rotating speed (10-50 rpm) [123] and the addition of baffles inside the drums [124–126]. However, high rotation rates can adversely affect growth due to shear damage [123]. Thus, the use of baffles is preferred. Schutyser et al. [127] studied straight baffles and curved baffles in rotating drums. It was found that, in a drum with curved baffles, complete mixing in the radial and axial direction was achieved much faster than in the other designs. Problems arise in that as construction becomes more complicated, manual tasks become more difficult, and loading and unloading of the solid may also become more difficult.

The gas-flow pattern in rotating drum reactors is another issue for concern. The gas flow in

the headspace associated with the end-to-end aeration typically follows the pattern of plug-flow with axial dispersion [128], which causes significant axial temperature gradients in the substrate bed [123, 129]. This suggests that axial mixing, such as the use of angled lifters, is necessary [129]. However, this, possibly together with the replacing the end-to-end air flow with introduction and removal air at several points along the reactor axis, will greatly increase the complexity of the reactor construction. The scale up of rotating drum reactors is also limited by vessel size and mechanical gear design.

Fig. 3. Schematic of a rotating drum reactor. The reactor can be continuously or intermittently rotated. (adapted from [120]).


480

4.4. Fluidized bed reactors Gas-solid fluidized beds have also previously been used to improve mass and heat transfer

in SSF [130, 131]. Yeasts have been successfully grown in fluidized beds [132]. The fluidized beds were intensively studied in the 1980s, but have attracted relatively little attention in recent years [3]. In a fluidized bed, the gas-flow rate required for fluidization is high enough to confirm a very good rate of heat and mass transfer. However, as a bioreactor for SSF, its shortcoming is obvious. Fluidization requires the use of fine particles and the minimum air velocity for fluidization is high [133], resulting in high power requirements; also, the high air flow rate causes high shear damage to the microorganisms, especially filamentous fungi which grow in the form of delicate hyphae. Large, coarse, and sometimes sticky particles, which usually appear in SSF, are difficult to fluidize. A spouted bed may provide good heat and mass transfer as well, while requiring a lower gas-flow rate and can easily handle the coarse particles. In the late 1980s, a spouted bed for the production of cellulase using immobilized Trichoderma viride cells was reported [134]; however it was a gas-solid-liquid reactor used for submerged fermentation.

4.5. Spouted Bed Bioreactor (SBB) Recently, a novel gas-solid spouted bed bioreactor has been proposed to overcome the

difficulties in SSF [49]. Gas-solid spouting, a variant of fluidization initially developed in the early 1950 for grain drying, permits good gas-solid contact for solid materials that are too coarse or dense for stable fluidization [135]. Spouted beds now have many applications in granulations, combustion, drying, and coating. Spouted bed reactors have been found to have many advantages over conventional fluidized beds [136, 137]. They are used when homogeneous and stable flow region is not attained in the fluidized bed, as in the cases of non-spherical particles and polydisperse and fine disperse systems [138]. Spouting is particularly appropriate for handling the large, coarse, non-uniformly sized, and sticky particles that are often used as SSF substrates. In a spouted bed, heat and mass transfer rates within the reactor are high, and solids are well mixed [139, 140]. The minimum air velocity required for spouting is lower than that required for fluidization. Gas-solid spouting in SSF also can prevent mycelial caking during the fermentation [49]. Because the particles in a spouted bed do not become packed together, problems associated with packed bed reactors will not arise in a spouted bed bioreactor.

In a gas-solid spouted bed (see Fig. 4), a stream of high velocity gas is injected into a bed of coarse particles; it jets through the middle of the bed, carrying a stream of particles with it, which then rain back onto the bed [136]. The rise of the particles in the middle of the bed is accompanied by the sinking of particles in the annular region. Agglomerations of particles are broken apart by high velocity impacts in the core region. Large particles, which can not be fluidized because they are too large, can often be spouted. Somewhat finer particles, which can be fluidized but only at high gas velocities, can be spouted at lower gas velocities. Therefore, the use of spouting rather than fluidization has the advantages of being applicable to larger substrate particles (such as grains) without requiring grinding and requiring less energy for air compression during fermentation.


481

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

0 5 10 15 20 25 30 35

Fermentation Time (h)

Prot

ein

(mg/

g ric

e)

0

10

20

30

40

50

η-, η-

, and

Glu

coam

ylas

e

Protein

β -Amylase (1000 U/g rice)

Glucoamylase (100 U/g rice)

α -Amylase (U/g rice)

SBF - 4 hr

0

0.4

0.8

1.2

1.6

Enz

yme

Pro

duct

ion

Rat

e

Static SSF1 hr Interval SBF

4 hr Interval SBFPacked Bed

α-Amylase β-Amylase Glucoamylase(U/g rice/h) (1000 U/g rice/h) (100 U/g rice/h)

A B Fig. 5. A. Kinetics of enzyme production from brown rice in a spouted bed bioreactor. B. Comparison of enzyme productivities in SBB and other types of reactors (adapted from [49]).

SSF have been studied for the production of α-amylase, β-amylase, and glucoamylase from

rice by Aspergillus oryzae in spouted bed, packed bed, and tray reactors [49]. The results showed that tray reactors with surface aeration had poor mass and heat transfer. The packed bed reactor with continuous aeration through the rice bed produced high protein and enzymes, but the fermented rice was difficult to remove and process due to the formation of large chunks of rice aggregates knitted together with fungal mycelia. Also, the fermentation in the packed bed was not uniform. The spouted bed bioreactor with intermittent air spouting achieved high production levels in both total protein and enzymes that were comparable to those found in the packed bed bioreactor, but without the non-uniformity and solids handling

Bed height

Fountain height

Air in

Air out

60°

Dc

Di

Hc

A B C

Fig. 4. A gas-solid spouted bed reactor – Gas flow upward while particles flow downward except in the central jet area where both gas and particles flow upward. A. Dimensions of the bioreactor. B. Spouted-bed structure. Arrows represent the grain flow direction. C. Spouting of various grains: brown rice, lentils, cracked corn, and soy bean (from left to right).


482

problems. Fig. 5A shows typical protein and amylases production kinetics in SSF with rice as the substrate. Fig. 5B shows that SSF with intermittent spouting had high enzyme production comparable to that of packed bed bioreactors with forced aeration and much higher than that of the static tray reactor. Fig. 6 shows the mycelium growth on grain particles in SBB.

Fig. 6. Morphology of filamentous fungi in SSF. Fungal mycelia with spores grown on solid substrate with starch granules. The right photo shows that the fungal mycelia penetrate through the grain’s skin and reach starch granules.

As a SSF bioreactor, SBB demonstrates high potential in obtaining better mass and heat transfer, and thus a higher productivity of enzymes and other products. Further investigation will be needed in order to better characterize and scale up the spouted bed bioreactor for solid state fermentation. In order to scale up the spouted-bed bioreactor, an understanding of reactor hydrodynamics is necessary. We have studied the minimum spouting velocity (Ums), particle mixing time, and the effects of column diameter (Dc), inlet diameter (Di), bed height (H), particle density (ρp) and fluid density (ρf) [141]. In general, the Ums increases when the static bed height, cone angle, or gas inlet diameter are increased. Numerous correlations have been suggested for the prediction of minimum spouting velocity, a critical design parameter in reactor scale-ups [141, 150, 151]. Many of these are empirical in nature, though a few have a dimensional analysis or theoretical basis. One of the earliest proposed, and still one of the most popular, is that of Mathur and Gishler [152]. Several improved or situation-specific correlations have since been proposed, although most of this work was done in the early 1970's. Table 6 lists several of these correlations.

Of the existing correlations, most were developed for roughly spherical particles. Many SSF substrates are not spherical, for example, rice grains have a length to width ratio of 3 or more. Furthermore, although grain in general and rice in particular have been studied in spouting beds before, these studies were all concerned with drying rice to fairly low moisture contents (generally less than 30%) [153], not with the somewhat higher moisture contents (30% to 45%) usually used in solid state fermentation. The higher moisture content can cause significant changes in particle size, density, and surface characteristics which can greatly affect the spoutability of the solid particles and hydrodynamics in the spouted bed reactor. A critical question that must be answered is whether the solid substrates suitable for SSF can be properly spouted in a spouted bed bioreactor. A comparison of experimental results with


483

existing correlations is also necessary in order to determine the correlations best suited to the design of a spouted bed bioreactor for SSF.

Table 6 Correlations for the minimum spouting velocity [141]

Correlation equation Reference

( ) 21

31

2

−

=

f

fp

c

i

c

pms Hg

DD

Dd

Uρρρ

SI units

[154]

U k V d Dms p p i= 0 6 0 6 0 2. . .ρ

CGS units

[155]

( )U d

g

DDD

HDms p

p f

f c

i

c c

DD

ic

=−

+

−ρ ρ

ρ

1 22 0 5 1 76

0 64 268. .. .

SI units

[156]

( )U d H gms p

p f

f=

−

0 0143 20 741 0 592

12

. . .ρ ρ

ρ

CGS units

[157]

( ) 324.0274.0615.0

2977.0

−

=

f

fp

c

i

c

pms Hg

DD

Dd

Uρρρ

SI units

[158]

530.0

677.0697.0714.0 )(

2

−

=

f

fp

c

i

c

pms gH

DD

Dd

aUρ

ρρ

SI units; a = 0.832 for Reactor I and 0.493 for Reactor II

[141]

5. CONCLUSIONS

Solid state fermentation has been applied to food production for ages. With a new surge of interest in SSF since the 1980s, its applications are much expanded across many fields of science and industry. Organic acids and industrial enzymes are the two most common categories of products from SSF. With little or no free water in the fermentation bed, SSF generates higher volumetric productivity and less waste water. The extraction of products from the fermentation bed can provide more concentrated solutions than submerged fermentation for the downstream processes. All the advantages of SSF suggest that SSF has a high potential as a much more economical industrial process. However, its industrial applications are very limited, especially in western countries. The main problem is that the development of a simple and practical automated fermenter for SSF processes has not yet been achieved. Traditionally, SSF is carried out in trays or packed-bed bioreactors. These


484

conventional reactors are simple to construct and widely used, but cannot provide enough mass and heat transfer, which are very important for fermentation processes. In recent years, many new designs of SSF reactors have been proposed in laboratories: rotating drums, fluidized beds, etc. However, each has its shortcomings, and is not yet feasible for scale-up. Recently, a novel gas-solid spouted bed bioreactor has been proposed to overcome the difficulties in SSF [49]. This reactor can provide good mass and heat transfer, easier solid handling, and experiments showed that higher enzyme production was obtained compared to static and packed bed reactors. It is demonstrated to have a high potential for industrial application, but further investigations are needed to better characterize it and to scale it up.

ACKNOWLEDGEMENTS

This work was supported in part by the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service (grant number 00-01797) and the Consortium for Plant Biotechnology Research, Inc. (grant number GO12026-229).

NOMENCLATURE

a, b, c, d, and e constants Cs concentration of support in bed (kg dry support m−3 support) CPg dry gas heat capacity (J kg−1 K−1) CPs heat capacity of dry solid (J kg−1 K−1) CPv water vapor heat capacity (J kg−1 K−1) CPw heat capacity of liquid water (J kg−1 K−1) Cwg concentration of water in air (kg water m−3 air) Cx concentration of biomass on support (kg dry biomass m−3 support) dp particle diameter; m dave particle average diameter; m deqv particle equivalent diameter; m Dc reactor body diameter; m Di air inlet diameter; m F″a superficial aeration rate (kg dry air m−2 s−1) ha enthalpy of (moist) air (J [kg dry air]−1) ha heat transfer coefficient for heat transfer between the solid and gas phases (J s−1 m−3 K) H Bed height; m ∆Ho reaction enthalpy (J [kg O2]−1) k constant in Charlton correlation K′a mass transfer coefficient for water transfer between the solid and gas phases (kg s−1 m−3) r′″o oxygen production biomass (kg O2 m−3 reactor s−1) r′″w water production biomass (kg H2O m−3 reactor s−1) S volumetric concentration of total dry solid (kg m−3) t time (s) Tg temperature of gas phase (K) Ts temperature of solid phase (K) Ums reactor superficial minimum spouting velocity; m/s V volume of spouting bed in Charlton correlation; cm3


485

xwx water content of biomass (kg water [kg dry biomass]−1) YQ heat yield from growth (J kg−1) YWB stoichiometric coefficient relating water production to growth (kg kg−1) z axial position in bed (m) ρg gas phase density (kg m−3) ρp particle density; kg/m3 ρf spouting gas density; kg/m3 ϕg water content of gas phase (kg kg−1 dry air) ϕs water content of solid phase (kg kg−1 dry solid) ϕs

* solid phase Water content for equilibrium with the gas phase at Tg (kg kg−1)

λ enthalpy of evaporation of water (J kg−1) ε void fraction (m3 air m−3 reactor)

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Solid State Fermentation and Its Applications[1]

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