Solid State Fermentation-An

Overview

C. W. Hesseltine
Northern Regional Research Center, Agricultural Research Service, US Department of Agriculture, Peoria, Illinois 61604, USA 17 February 1987; accepted 24 February 1987)

(Received

ABSTRACT

Since ancient times many solid state fermentations have utilized fungi and bacteria, almost always in mixed culture. The discovery and development of penicillin led to extensive use of liquidfennentations using actinomycetes and Jitngi and to subsequent neglect of research on solid statefermentations and the use of mixed cultures. This paper reviews the types of solid state fermentations, equipment used, products made, as well as the advantages and disadvantages of solid state fermentations. Products resulting from old and new solid and liquid substrate fermentations are enumerated.

TYPES OF SOLID STATE FERMENTATIONS Solid state fermentations are those processes in which all or parts of the substrate are in a solid state (Hesseltine, 1972). They are more natural than liquid fermentations because they approach the conditions under which most microorganisms grow in the wild. While some groups of microorganisms, such as some bacteria and yeasts, grow in liquids, a large number of other microorganisms grow attached to solid substrates. The major groups of microorganisms used in fermentation are bacteria, actinomycetes, yeasts, and fungi. Bacteria grow on a solid matrix in the soil, on the roots of plants, inside plants, and on leaf surfaces. Even human and animal pathogens grow on surfaces. Streptococcus produces illness because this bacterium grows on the surface of cells of the throat. The bacterial flora covering the surface
79 0 US Government-1987.

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of mineral fragments or other packing in a sewage trickling filter is an example of a continuous fermentation reactor. The effluent biological oxygen demand is reduced as sewage passes over the film of bacteria on the packing. A compost pile is a good example of a multiple culture solid state fermentation. Bacterial cells are surrounded by exopolysaccharides, which attach the cell to solid objects. Although most bacteria in nature are growing in and on a solid state matrix, some are free of attachment as they grow in water, blood, and milk. Many species and genera of actinomycetes grow in soil and attack the more microbiologically resistant compounds. Although some actinomycetes live and multiply in water, most inhabit the soil. Fungi certainly are organisms that grow mostly on or in solid substrate material. Aspergillus species typically grow on and in cereals, concentrated media such as jams, heating compost, and decaying plant material in soil. However, some fungi are unattached in water. Yeasts grow and reproduce freely in fruit juices and other liquids containing simple carbohydrates but also may grow on solid substrates. Therefore, it is not surprising that solid state fermentations have been used and are still used widely because the growth conditions are similar to those under which many organisms have grown for thousands of years. Solid substrate fermentations may take on a number of different forms: (A) All the fermented material is solid. (1) Solid substrate is undisturbed. For example the shiittake mushroom (Lentinus edodes (Berk.) Sing.) is inoculated into oak logs and the mushroom mycelium grows in the wood, eventually producing sporocarps. (2) Only solid material is used, but the substrate is occasionally stirred and mixed thoroughly, either manually or mechanically. Koji is typically prepared by combining rice or roasted cracked wheat with flaked soyabeans, which mixture, after inoculation with Aspergillus oryzae, is stirred occasionally to maintain uniform moisture conditions, provide aeration, and break up the koji cake. (3) Solid material is continuously agitated on a shaker. The method developed at NRRC to produce gram quantities of aflatoxin, a mycotoxin produced by Aspergillus jlavus, used moist whole grain rice in flasks, which was sterilized with steam, then cooled and inoculated. The flasks were placed on a shaker and continuously shaken or stirred for 5 days, at which time the rice contained over a gram of aflatoxin per kilogram of rice. This method works especially well when small amounts of a compound such as aflatoxin are required.

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(B) Solid material is suspended in a liquid. (1) The solid material is placed in columns and the liquid medium circulated through it, as occurs in the Ex-Ferm process developed as a method to convert sugar cane into ethanol. (2) The solid material may be suspended in a liquid medium, which is either left stationary or agitated. Kaffir beer is a liquid but contains suspended particles of corn or sorghum or both in the fermentation, and the solid particles remain in the final product. These various methods contrast to fermentations based on the use of liquid media. The use of liquid media for fungal fermentations, aside from alcoholic beverages such as wine, beer and fermented milk, came about in the pre-penicillin days. As far as can be determined, Kluyver and Perquin (1933) were the first to publish and illustrate a method of using liquid medium with agitation to produce a fungal product. They illustrated their paper with a photograph of a flask of liquid medium containing balls of Aspergillus flaws mycelium from a kojic acid fermentation. The objective of Kluyver and Perquins research was to produce mycelium that was homogeneous; this cannot be done if one grows fungus colonies on the surface of liquid or solid medium. Aerial mycelium would be different in its enzyme content from submerged mycelium. Ward (1970) in his review of the contributions of USDA fermentation research, notes that in the gluconic acid fermentation Shake flask techniques were not yet developed, and the first submerged studies were made by aerating inoculated fermentation medium in sintered glass false-bottom gas washing bottles mounted in an autoclave which was placed under air pressure; the air flow through each bottle was controlled with a needle valve (May et al., 1934). It is apparent that, at that date, shake flasks were unknown for growing tilamentous microorganisms, although rotary drums were used (Herrick et al., 1935). The use of shaken liquid substrates in flasks was adopted for most mold fermentations and was used very early in the development of penicillin. All sorts of studies of nutrients, fermentation time, improved strains, amounts of agitation, and product recovery were made in agitated flasks on a variety of shaking machines, typically homemade. The first commercial penicillin was made by growing Penicillium on the surface of stationary liquid medium. This technique was shortly replaced by fermentations in which thousands of gallons of liquid media were vigorously agitated in tanks. This established the practice for the funguslike Streptomyces antibiotic fermentations. Streptomyces likewise produce typically fungus-like pellets in shake flasks and in large commercial fermentors.

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As a consequence of this technique, research on solid state fermentations and their application fell into disuse in the West. However, this was not true in the Orient where research on solid state fermentations was pushed, especially as applicable to processes that required the production of enzymes in koji for a number of fermentations.

MODERN

VERSUS ANCIENT

FERMENTATIONS

Using microorganisms to carry out certain processes and fermentations goes back to antiquity. Table 1 lists a number of these processes that occur on solid substrate and on or in liquid media. These fermentations were already conducted before a written record occurred. Obviously a large number of useful microbial processes were employed on solid substrate, many more than in purely liquid media. Costerton (1985) points out that many of these solid substrate fermentations are still used today. On a volume and perhaps dollar basis, more traditional fermentation products are still made by solid state fermentation than by liquid fermentation. However, many more new fermentation products are made by liquid fermentation than by solid state fermentation (Table 2).
TABLE 1

Ancient
Solid state

Fermentation

Products
Liquid

Sauerkraut Koji (for rice wine, miso, & shoyu) Cheese Bread Mushrooms Shoyu Miso Natto Tempeh Ragi and related starters Fermented fish Fermented vegetables Sausages Silage Tea, coffee, vanilla, and cocoa Compost Kaffir beer Decomposition of plant and animal

Wine Acid milks Beer Vinegar Yogurt

material

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83

Modem Solid state Enzymes Mycotoxins Clean water from trickling

Products Liquid Penicillin Other antibiotics Citric acid Gluconic acid Torula and bakers yeast Enzymes Steroid transformations Amino acids Xanthan gum Vitamin B,z Riboflavin Lactic acid

filters

METHODS

AND EQUIPMENT USED IN SOLID STATE FERMENTATIONS

The revival of interest in solid substrate fermentations, at least in our laboratory, came about with the awareness of toxins produced by fungi. At the beginning of the research on aflatoxin, a method for producing quantities of aflatoxin was needed because much of the USDA program on aflatoxin depended upon the availability of grams of material. Aflatoxins of high purity were required for making standards and, more importantly, large quantities of material were needed for animal studies, especially for long-term feeding experiments in swine, poultry, beef, and dairy cattle. In attempting to produce these quantities, many methods were tried, beginning with agitated liquid media. The use of solid media in standing flasks was tried, but it was soon apparent that yield was not uniform unless the fermenting material was shaken and turned. This led to the inoculation of rice dry enough for the kernels not to stick together. The best yields were obtained by continuous agitation on a rotary shaker (Shotwell et al. 1966). Almost all the aflatoxin in the rice could be removed in a relatively pure form with three extractions. This method yielded gram quantities of aflatoxin in five days. This technique has since been used for producing mycotoxins from other fungi and has been scaled up in rotating drums equipped with baffles, which constantly mix the moist inoculated grain.

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A method widely used even today is to use rice, wheat, bran, or other cereals as the substrate, which is sterilized in open trays. The trays are typically made of wood because the wood can be soaked with water, thus keeping the fermenting grain next to the side and bottom of the tray moist, insuring a uniform moist substrate. This method is often used to produce fungus spores, or is scaled up into huge tray-like tanks in which tons of fermenting substrate are used to produce koji as the source of enzymes in a variety of foods and beverages. A modern development of the tray method uses a circular tank, which slowly rotates and is supplied with devices that break the mycelium at a certain age, improve aeration, and lifts the fermenting grain from the bottom to the top. Aeration is supplied both from above and from below. Such circular tanks may be harvested all at once or continuously, with harvesting going on at the end of the fermentation cycle while newly inoculated substrate is added just beyond the harvesting location. In an entirely different fermentation method the solid, moist substrate is placed in plastic bags or tubes. This moist inoculated material ferments in the plastic bag, and the bags with contents are sold to the consumer. Our laboratory developed this method for tempeh making, and this packaged product now is sold in Indonesia. Perhaps the simplest fermentation method occurs when a host of microorganisms breaks down organic material, such as a compost pile, and generates numbers of new compounds. A slightly more sophisticated process is the controlled composting and pasteurization of manure and straw used as the substrate for growing commercial mushrooms. All of the above fermentations are based upon solid substrate in which there is no free liquid. In one solid/liquid fermentation method the liquid media to be converted trickles through a cylinder, which houses microorganisms attached to a solid matrix. The solid material may be plant pieces, such as wood shavings in the acetic acid fermentation, or stones as in trickling filter beds. Another solid/liquid fermentation method is used to make kaffir beer, where solid particles of corn or sorghum are suspended in a liquid medium and the whole is agitated in a fermentor much like regular deep tanks used in purely liquid fermentations.

INOCULUM

FOR SOLID STATE FERMENTATION

For many solid state fermentations, inoculum can be prepared and used in the same fashion as in liquid media. Either preformed inoculum or dry spores in a carrier may be used to inoculate the substrate. However, in

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the case of fungi used commercially that do not sporulate in culture, such as Aguricus, Lentinus, Pleurotus, and Volvariellu, the inoculum has to be prepared as spawn. In this procedure, the fungus is grown on solid particles such as rye kernels, and the infected kernels are dispersed evenly throughout the substrate. Infected cereal kernel inoculum is never used in liquid fermentations because insufficient growth sites would make the fermentation time too long. Often in solid state fermentations, mixed cultures with two or more microorganisms are used. In starters used to degrade waste material, a diverse mixture of microorganisms may be added. Thus, certain species will attack one compound, and others will either further degrade the first products or attack separate compounds. In koji, several strains of Aspergillus oryzae are utilized to produce different enzymes to attack starch, protein, and lipid material. A different set of strains is used for each kind of koji. Using mixed cultures goes back centuries and offers many advantages (Hesseltine, 1981).

ADVANTAGES

OF SOLID STATE FERMENTATIONS

(1) The substrate may be very simple, often using only one ingredient plus water. For example, in the production of aflatoxin, only moistened polished rice is used. In shoyu koji, only cracked roasted wheat and soybean flakes are fermented. In some instances, to enhance growth, supplementary nutrients can be added directly to the water used to moisten the substrate. (2) Bacterial contamination rarely occurs with fungus fermentations in solid substrates because the moisture level at the surface of the substrate is too low to support bacterial growth. For this reason, many solid state fermentations can be carried out in open tanks, even though bacterial contamination will always be present in the air. Bacteria either cannot grow or, if they do, growth will be so slow and diluted that the fermentation will be unaffected. Therefore, fermentation time may be greatly extended to improve the yield of the product without forming undesirable products. (3) Since less substrate and water are used to produce the product, pollution problems are greatly reduced. Because there is no liquid fraction to be disposed of, the residual substrate can be dried easily or shipped wet as a feed. For example, in the Kaoliang (Taiwan) alcohol process for ethanol production, the moist residue of spent sorghum and yeast cells can be sold as a pig feed; there is no liquid waste to dispose of (Hesseltine, 1981).

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(4) When a product such as an enzyme or antibiotic

(5)

(6)

(7)

(8)

(9)

(10)

has to be extracted from the fermentation, less solvent is required for recovery. The recovery from the substrate may often be carried out directly in the fermentation vessel. In the Kaoliang process, alcohol isstripped from the solid sorghum substrate in the fermentation vessel. The fermentation vessel does not need to be as large as the one required for a liquid fermentation process. For example, with aflatoxin production one can obtain regularly over 1 g per kg whereas to produce an equal quantity in liquid media would require a much larger volume of fermentation liquor, and recovery would not be as good or as easy. The solid state fermentation process can be scaled up to either a large batch or a continuous fermentation. One of the biggest problems in continuous fermentations is the mutation or change in the mix of the organisms, resulting in reduced yield or changes in the chemical structure being produced. This may be avoided in continuous fermentation by constantly adding new stable inoculum as the old is being removed with the product. The older completed fermentation material does not get mixed with new substrate because there is no mixing of the two. as in a liquid process. In a continuous liquid fermentation, broth is being removed at the same time fresh medium is being added and, consequently, old cells are being mixed with the new. The conditions under which microorganisms, especially fungi, grow are similar to those in nature. All mushrooms grow on wood or on other solid plant material. The same can be said of genera commonly used in industrial processes, such as Penicillium, Aspergillus, and Rhizopus, which typically grow on substrates containing simple carbohydrates. In some fermentations, the indigenous microbial flora of the substrate may serve as the inoculum. In a fermentation process developed at our Center, liquid cattle waste and cracked corn were combined in a continuous solid state fermentation. The inoculum came from these two substrates, neither of which were sterilized or inoculated. When the solid material is not suspended in liquid, aeration is easier because of the large void spaces between the solid particles. Aeration is even better when the solid fermentation material is subjected to continuous agitation. Product yields are quite reproducible. For example, Hesseltine (1977) reported on four fermentations using Aspergillus sulphureus growing on wheat to produce ochratoxin. The separate fermentations gave ochratoxin yields of 180, 196, 199, 183 ,ug g- of substrate.

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(11) With constant agitation of the fermenting substrate, sporulation is almost completely blocked because the grinding action of the solid particles wears off sporophores. Because of this, spores do not exist, and the contamination of the fermentation work area and the hazard of spore inhalation by workers are reduced.

DISADVANTAGES

OF SOLID STATE FERMENTATIONS

(1) Probably the most important disadvantage is the generation of heat which occurs even in small fermentations of only 1 kg. Excess heat must be controlled, otherwise the temperature becomes so high that it destroys the desired microbial product or stops growth and fermentation completely. This problem is one of engineering: how to reduce heat and, if possible, how to use the generated heat productively in large operations. (2) If existing equipment does not suffice, it is necessary to develop devices to monitor temperature and to measure pH, O2 and COZ levels, moisture content, and the concentration of desired product. (3) Sometimes the substrate must be treated to make it more easily infected by the inoculum and to increase the surface area. For example, when wheat is used in the shoyu koji process, it is usually crushed and sometimes roasted. We also found it necessary to abrade the surface before the wheat was moistened and sterilized. The abraded surface allowed the quick entry of spore germ tubes into the kernel. (4) During the early stages of fermentation (48-72 h), the substrate sometimes dries to the point where mold growth stops. Consequently, care must be taken to add sterile water. However, this has to be done cautiously, otherwise excess moisture reduces growth and promotes contamination by organisms that require more moisture. Of course, this is not a problem when the substrate is in a solid/liquid fermentation. (5) With the concentrated medium, the initial amount of inoculum required may be quite high. It is even higher in a continuous fermentation because inoculum has to be added continuously to the fermentation. (6) For large fermentations requiring agitation, the energy costs may be high. Although the constant agitation of solid material would become quite expensive, it should be recognized that constant agitation might not be the best way to produce a given product. However, when small amounts of product are desired, this may be the fastest and most productive way to go.

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(7) Because microorganisms used may require different moisture levels in strictly solid media, moisture is a critical and limiting factor in many fermentations. For example, Aspergi1ZusJlavu.s requires a low moisture range to optimally produce aflatoxin, while Rhizopus oligosporus requires a much higher moisture level to produce its proteolytic enzymes. (8) In some instances, just as in liquid fermentations, one cannot obtain the desired product by solid substrate fermentation, or it is produced in low yields or in mixtures with other, undesirable, products.

CONCLUSION Solid state fermentations merit more study to develop new or improved processes, to improve methods for measuring and determining the optimum conditions for growth and for controlling this growth. Often microorganisms have optimum growth conditions which may be different from the optimum conditions for product formation. For example, afterFusatium grows for a few days, the temperature needs to be reduced in order to get maximum toxin production. For this reason, natural outbreaks oft;usarium mycotoxicosis are usually reported in late winter or early spring. Most traditional solid state fermentations need further study to improve the efficiency of these fermentations by reducing handling costs, shortening fermentation time, improving strains genetically, making the process continuous and using mixed cultures.

REFERENCES
Costerton, J. W. (1985). The role of bacterial exopolysaccharides in nature and

disease. Developments in Industrial Microbiology 26, 249-61. Herrick, H. T.; Hellbach, R.; May, 0. E. (1935).Apparatus for the application of submerged mold fermentation under pressure. Industrial and Engineering
Chemistry 27, 681-3.

Hesseltine, C. W. (1972). Solid state fermentations. Biotechnology and Bioengineeting 14, 5 17-32.

Hesseltine, C. W. (1977). Solid state fermentation-Part

12, 24-7.

1. Process Biochemistv
Developments in

Hesseltine,

C. W. (1981). A microbes view of fermentation.

Industrial Microbiology 22, l- 18.

Kluyver, A. J.; Perquin, L. H. C. (1933). Zur Methodik der Schimmelstoffwechseluntersuchung. Biochemische Zeitschrift 266, 68-81.

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May, 0. E.,; Herrick, H. T.; Moyer, A. J.; Wells, P. A. (1934). Gluconic acid production by submerged mold growths under increased air pressure.
Ind. Erg. Chem. 26, 575-8.

Shotwell, 0. L.; Hesseltine, C. W.; Stubblefield, R. D.; Sorenson, W. G. (1966). Production of aflatoxin on rice. Applied Microbiology 14, 425-8. Ward, G. E. (1970). Some contributions of the US Department of Agriculture to the fermentation industry. Advances in Applied Microbiology 13, 363-82.