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Microbial conversion of complex mixtures of compounds (Polysaccharides, Proteins, Lipids, etc.)

The residues in this group (Table 1) consist of mixtures of various complex compounds resulting from several agro-industrial activities. Some of the compounds are soluble, others are colloidal or solid (1). In certain cases the residues may be fairly uniform in character (peels from potatoes or apples), whereas in other instances the material may be of varied composition (manure).

Under natural conditions, rarely are substances in this group transformed by a single microbial species. Rather, a mixed flora is usually responsible for the conversions that occur. Thus, it is generally impossible to single out separate species as being responsible for any transformations that take place. Some of the residues are such, however, that they could be utilized for alcohol fermentation or SCP production by yeast, or they could serve as a substrate in rural areas for biogas production, and for algal culture. Because these processes are currently receiving considerable attention, and it is not easy to single out other unique processes where pure cultures can be employed, the reader is referred to reviews by Rolz (1) and DaSilva et a). (2), and the treatise on methane generation from human, animal, and agricultural wastes (19).

Microbial utilization of cellulose and ligno-cellulose residues

The most abundant renewable biomass on earth consists of cellulose, with between 5 and 15 tons per person being synthesized annually by photosynthesis. Much of the cellulose in nature is bound physico-chemically with lignin.

Because lignin is highly resistant, it protects cellulose against attack by most microbes, and it must be degraded by chemical or biological means before the cellulose can be utilized. Some higher fungi such as the basidiomycetes (Planerocheate chrysosporium) can degrade lignin, and mush rooms (Lentinus, Volveriella, and Pleurotus species) convert ligno-cellulose directly into fungal protein suitable for human consumption.

Table 4 lists some cellulose-utilizing organisms together with the general products they form, and their current status of development as useful agents. Brief mention may be made of each of these groups.

TABLE 4. Products of Some Cellulose-Utilizing Organisms

Volvariella Species

Mushrooms of the genus Volvariella (V. volvacea, V. esculenta, and V. diplasia) are cultivated mainly on rice straw and similar cellulosic materials by individual families in Asia and Africa Commercially, mushrooms in this genus account for about 4 per cent of the world production of some 916,000 tons. They have promise of expanded use in regions of the tropics where the grain is grown Production usually involves simply inoculating pre-soaked straw in flat stacks with spores (spawn), maintaining optimal moistures, and harvesting several crops of mushrooms. The spent straw is used to inoculate new straw stacks, and is a rich animal feed (20).

Lentinus edodes

The mushroom Lentinus edodes has been cultivated for centuries in China and Japan, where it is commercially produced in a multi-million-dollar industry; it accounts for about 15 per cent of world production. (Both in Asia and more especially in western countries, Agaricus bispora is the most important mushroom species and accounts for about 75 per cent of world production [20].)

L. edodes has potential for bioconversion of lignified residues and low-quality wood into fungal protein. Such protein is easily digested by ruminants, but its use as a feed supplement has received little attention.

Pleurotus Species

Mushrooms of the genus Pleurotus (P. ostreatus, P. sajorcaju, P. florida, P. cornucopiae, etc.) are called "White-rot" fungi, and they decompose lignin and polysaccharides in wood. They have potential in the conversion of waste and low-grade wood into protein-rich food for human consumption. P. cornucopiae is grown commercially in Japan, but none of the species is grown in western countries. P. ostreatus and P. florida have temperature optima near 30 C, making them promising for processing organic residues in the tropics. All can be cultivated on mixtures of sawdust and grain, manure, and food processing wastes (20 - 22).

Basidiomycetes

Wood-decaying fungi, such as Phanerochaeta chrysosporium, are widely distributed in northern countries where they are commonly called "white-rot" fungi. P. chrysosporium decomposes both the lignin and cellulose in wood; it is unique in that (i) it produces copious quantities of spores, making it easy to transfer; (ii) it is thermotolerant, growing rapidly at 35 to 40 C, but also well at 25 C; and (iii) it has simple nutritional requirements. This fungus has been fed to fish and rats as a source of protein, but it has not been studied extensively as a nutrient for other animals. It should be considered as a means of converting wood processing residues and other lignified wastes into partially de-lignified products for feed or fibre use, or for further conversions (23).

Thermoactinomycos Species

The thermophilic, cellulolytic, and starch-utilizing actinomyces, such as Thermoactinomyces sp., may provide an opportunity to produce single-cell protein for feed supplements in tropical climates. The thermoactinomyces do not utilize ligno-cellulose directly, so treatment of such complex materials would be necessary. However, they grow rapidly at 55 to 65°C under aerobic conditions on a variety of cellulosic and starchy materials plus other simple nutrients. According to preliminary results of Humphrey and associates (24), cell yields of 0.45 9 cell/g cellulose utilized can be obtained in 20 to 24 hours; apparently four cell-bound enzymes are involved in the degradation process.

Trichodorma viride

Although a number of fungi are cellulolytic, only a few produce cell-free cellulose in sufficient quantity to be useful for large-scale development. Trichoderma viride and a number of its mutants do produce a stable cellulose that is capable of degrading cellulose (Figure 3). The fungus grows rapidly on simple media in the pH range of 5.0 to 2.5, thus reducing to a minimum contamination from other microbes. The broth containing the enzyme is then mixed with pure cellulose, or with treated lignocellulose to remove the lignin, and a glucose syrup results.

Figure. 3. Enzymatic Conversion of Waste Cellulose to Glucose Sugar

The Japanese are producing cellulose commercially from J. viride on a limited scale using the Koji process, and considerable research is being done in several laboratories to obtain mutants that yield more enzyme. The use of cellulose from T. viride holds great promise as a tool for processing cellulose residues (25 - 27).

Clostridium thermocellum

The only known thermophilic, anaerobic bacterium that degrades cellulose is Clostridium thermocellum. The organism has simple nutritional requirements and grows at higher temperatures (50 C) than do most bacteria, which has the advantage in a fermentation process of being less prone to contamination by other organisms. In pure culture, the chief products from cellulose (or treated ligno-cellulose) are cell mass (protein), acetate, ethanol, lactate, H2, and CO2. In a mixed culture, C. thermocellum and Methanobacterium thermosutotrophicum, yield from cellulose are cell mass, methane, and acetate (27, 28). This is not a process that could be easily adapted to rural areas unless proper equipment were available but it could be used to produce either ethanol or biogas (methane) from cellulosic wastes.

Pseudomonas flvorescens var. cellulosee, Cellumonas Species, Cellvibrio Species, and Other Cellulose-Degrading Organisms

Species in the genera Pseudomonas, Cellulomonas, and Celivibrio utilize cellulose, but they apparently are unable to degrade ligno-cellulose. Co-fermentative studies on cellulose have been conducted using P. fluorescens and Candida utilis, Cellulomonas species, and Alcaligenes faecalis, and with Cellulomonas flavigena and Xanthomonas campestris Supposedly, cofermentation of cellulose with a non-cellulolytic organism increases the rate of utilization of soluble sugars produced in the process and thereby hastens the reaction (29). In fact, Casas-Campillo and colleagues (personal communication, 1978) have found that C. flavigena and X. campestris together are much more active against cellulose than are T. viride or combinations of other organisms.

Thermophilic Sporocytophaga

The Sporocytophaga (Sporocytophaga myxococcoides, etc.) digest cellulose and other components of cell walls, but not ligno-cellulose. A thermophilic strain that grows at 55 to 65 C has been found. This organism might be useful for the production of cell mass, ethanol, acetate, and lactate from cellulose.

Algal culture as a source of biomass

For many years algae have been used by the shoreside populations of Lake Chad and Lake Texcoco (Mexico) as a source of food, and one algal species (Spirulina sp.) is now produced commercially in Mexico at the rate of several thousand tons per year.

Certain big-engineers and microbiologists believe that carefully selected genetic strains of algae (Scenedesmus acutus, Spirulina maxima, Cosmarium turpinii), or photosynthetic bacteria (Rhodospirillum sp. or Rhodopseudomonas sp.) are the organisms of choice for the production of single-cell protein (30, 31). Such microbes contain about 65 per cent crude protein of moderately high biological value. The protein appears to be well utilized by animals. In addition, most species are as good a source of the B vitamins as yeast, and they contain ascorbic acid.

In addition to algal biomass having many uses, the process can be used to help purify sewage, livestock manure, and other agricultural wastes. A unique feature proposed by Colombo (31) is the cultivation of the algae in plastic tubes that can be extended for considerable distances in arid regions over land that is not useful for cultivating crops. Such a system takes advantage of solar energy, saves water, requires little capital and labour for development, and can be used either in the rural areas of less developed countries or on a larger industrial scale.

Microbial utilization of silviculture biomass

Several excellent reviews have been published on biomass production from forest ecosystems (32, 33), and data (Table 3) indicate that forestry remains and processing wastes are not fully utilized today. Chemical products that can be obtained from forestry biomass are ammonia, methanol, ethanol, fuel gas and oil, and charcoal. Ethanol fermentation by yeast from wood hydrolysis could become competitive within 10 to 15 years (32). But, as with methanol production from wood, the demand for ethanol will be satisfied for the near future by existing synthetic sources, unless it becomes necessary to use it in gasoline blends.

Micro-organisms and marine and freshwater biomass

Other sources of biomass deserving mention are marine plants, such as giant seaweeds (kelp), from the waters of the tropical and temperate oceans, and the so-called weeds, such as the water hyacinth.

Although the technology has not been proven, ocean-based kelp farming has some attraction for two reasons: (i) kelp is fairly efficient in converting sunlight into stored energy (2 per cent), and (ii) land and terrestrial waters are not constraints. Experimental data on marine plants have been collected by Wilcox of the US Navy's Ocean Food and Energy Farm Project (34). All aspects of this programme are interesting, but the only data involving microbiology are those concerned with the production of methane as a source of energy from kelp by anaerobic microbial digestion. In addition to methane, certain by-products remain in the sludge and liquid of the digester, which can be used for nitrogen fertilizer or animal feed supplements

There are no major nutritional deficiencies in kelp for mesophilic, anaerobic, methaneproducing microorganisms, so they grow readily on a slurry of the material. One precaution is that the salt concentration of the raw slurry must be reduced. An interesting research project would be to develop strains of methane-producing micro-organisms that are more salt-tolerant.

Economic studies indicate that the cost of methane production from kelp fermentation may range from US$2 to US$7 per GJ (per million BTU), depending on credit values received from feeds and other by-products. Thus, entry to the fuels market for kelp-derived methane will require research to provide a cheaper product, and capitalization. If preparatory methods for handling the kelp, and the fermentation, could be carried out in the open ocean where wind, wave, and solar energy could be used, the cost of the methane could be reduced below current land based fermentation processes.

One of the most prolific plant colonizers of rivers and lakes is the water hyacinth, which has spread in recent years from its natural habitat in South America to at least 50 tropical and sub-tropical countries around the globe. A few plants can multiply and spread over an area of 120 yd(2) of water in several months, depending on the nutrients in, and temperature of, the water, and the plant mass may represent many hundred tons of hyacinth.

Such water weeds are an environmental disaster in some countries because they interfere with water transportation and fishing, and they can be a health hazard as well by providing a suitable breeding site for the malarial mosquito. Ironically, the water hyacinth may be a promising candidate for solving needs of animal feed, energy, and control of water pollution, and in this regard, micro-organisms can play an important part.

Water hyacinths contain most of the essential nutrients for animal growth, but making a palatable feed from them is not easy because of the high moisture content of the plants. Research indicates, however, that the plants can be converted into silage by placing chopped plants in a closed container and allowing them to undergo microbial fermentation for about a month. Such silage has been shown to be highly palatable to sheep and other animals.

Potentially, the water hyacinth may be used as a source of energy, and for the purification of sewage. Considerable research has been done on these subjects, especially by Wolverton and McDonald at the NASA Space Technology Laboratories in Bay St. Louis, Mississippi (35). Biogas or methane production from the microbial anaerobic decomposition of water hyacinths has been investigated only on a laboratory scale. Many factors, such as carbon to nitrogen (C/N) ratios and temperature, affect the amount of gas and residue produced from the microbial digestion of the plant material. Based on research, it has been calculated that one hectare of water hyacinths can produce enough biomass each day to generate between 90 and 180 m³ of methane gas, and at the same time 0.5 ton of residue useful as a fertilizer. Further research is needed on the use of water weeds as a substrate for microorganisms.

International studies on processing organic residues

Considerable work is being done in several countries on the microbial production of food, energy, enzymes, and other useful substances from natural and agro-industrial wastes.

Some of these processes are, or could be, adapted to rural areas where the residues originate. A few examples are listed in Table 5, from the paper by Olembo (36) in the monograph on the Global Impacts of Applied Microbiology and Its Relevance to Developing Countries (37).

TABLE 5. Products Obtained in Various Countries from Residues Using Micro-organisms

Source: Olembo (36); data from UNEP/U/ICRO Training Courses.

The main objectives of the studies reported range from the need for an increase in protein food production to pollution abatement, and from industrial expansion to innovative research on the use of beneficial micro-organisms to improve the environment and welfare of human beings throughout the world.

References

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14. Y.D. Hang, D.F. Splittstoesser, and E.E, Woodams, "Utilization of Brewery Spent Grains Liquor by Aspergillus niger," Appl. Microbiol. 30:879 (1975).

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19. National Academy of Sciences-National Research Council, Methane Generation from Human, Animal, and Agricultural Wastes, NAS, Washington, D.C., 1977.

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22. F. Zadrazel, "The Ecology and Industrial Production of Pleurotus ostreatus, P. florida, P. cornucopiae, and P. eryagii," Mushroom Sci. 9 (1): 621 (1976).

23. B.V. Hofsten and A.V. Hofsten, "Ultrastructure of the Thermotolerant Basidiomycete Possibly Suitable for Production of Food Protein," Appl. Microbiology 27: 1142 (1974).

24. A.E. Humphrey, A. Moreira, W. Armiger, and D. Zabriskie "Production of Single-Cell Protein from Cellulose Wastes,' Biotechnol. Bioeng. Symposium 7: 45 (1975).

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26. L.A. Spano, J. Medeiros, and M. Mandels, "Enzymatic Hydrolysis of Cellulosic Wastes to Glucose for the Production of Food, Fuel, and Chemicals. Trichoderma viride, Fungal Fermentative Agent," J. Wash. Acad. Sci. 66: 279 (1976).

27. C.L. Cooney and D.L. Wise, "Thermophilic Anaerobic Digestion of Solid Waste for Fuel Gas Production," Biotech. Bioeng. 17: 1119 (1975).

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29. P. Beguin, H. Eisen, and A. Roupas, "Free and Cellulose-bound Cellulases in a Cellulomonas Species," J. Gen. Microbiol. 101: 191 (1977).

30. R. H. Shipman, L.T. Fan, and I.C. Kao, "Single-Cell Protein Production by Photosynthetic Bacteria,"Adv. Appl. Microbiology 21: 161 11977).

31. U. Colombo, "A Contribution Towards Solving the Protein Deficient Problem in the Developing Countries," paper presented at the Asian Regional Seminar on the Contributions of Science and Technology to National Development, New Delhi, 4 - 6 October 1978.

32. R. E. Inman, "Silvicultural Biomass Farms," Mitre Tech. Report 7347 11): 1 - 62 (1977).

33. H.G. Schegel and J. Barnea, Microbial Energy Conversion, Pergamon Press, New York, 1977.

34. H.A. Wilcox, in N.T. Monney (ed.), Ocean Energy Resources, pp. 83 - 104, American Society of Mechanical Engineers, New York, 1 977.

35. B.C. Wolverton, R.C. McDonald, and J. Gordon, "Bioconversion of Water Hyacinths into Methane Gas: Part I," NASA Tech. Report TM-X-72715, Bay St. Louis, Mississippi, 1975.

36. R.J. Olembo, in W.R. Stanton and E.J. DaSilva (eds.), GIAM V. Global Impacts of Applied Microbiology. State of the Art: GIAM and its Relevance to Developing Countries, pp. 27 - 34, University of Malaya Press, Kuala Lumpur, 1978.

37. W.R. Stanton and DaSilva (eds.), GIAM V. Global Impacts of Applied Microbiology. State of the Art: GIAM and its Relevance to Developing Countries, University of Malaya Press, Kuala Lumpur, 1978.

John Roger Porter, a much appreciated participant at the Guatemala conference and contributor to these Proceedings, died suddenly in May 1979. A highly productive and renowned microbiologist and former Head of the Department of Microbiology at the University of Iowa, Dr. Porter was a member of the Science Information Council of the National Science Foundation and a member of the National Board of Medical Examiners in Microbiology. He served as Chairman of the Advisory Committee on Scientific Publications of the National Institutes of Health, and was the recipient of the Pasteur Award in 1961. As Editor-in-Chief of the Journal of Bacteriology for ten years, he compiled and edited the 50-volume index for the Journal. His text Bacterial Chemistry and Physiology was widely used. A man of great warmth and personal charm, Dr. Porter will be greatly missed by his friends and colleagues.

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