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Chapter 1 Raw Materials for Microbial Processes Microorganisms, like all other forms of life, require water and nutrients for growth, reproduction, and maintenance. In addition to suitable sources of utilizable carbon, nitrogen, and sulfur, microbes generally require sodium, potassium, phosphorus, iron, and other minerals. The major factor in select- ing raw materials for microbial processes is the source of carbon. Microbial processes have long been harnessed for the benefit of man in the production of foods, medicines, and alcoholic beverages. Nature employs microbes on a much grander scale to establish and maintain a balance among the diverse forms of life on this planet. The underlying agents responsible for the myriad syntheses, transformations, and other reactions caused by mi- crobes are the enzymesbiological catalysts of high specificity and efficiency. One important aim of science and technology has been to domesticate beneficial microbes, especially for the transformation of raw materials to worthwhile end products. In general, most raw materials are naturally occurring substances from which more useful materials can be produced. In this sense, microbes them- selves may be considered raw materials suitable for further processing. The use of microbes as single-cell protein (SCP) is an example (see Chapter 2~. In this report the discussion will be limited to major carbon sources found in nature, formed mostly by plants through photosynthesis, which can be used either for producing additional biomass (e.g., SCP) or for further transforma- tions (e.g., alcohol). In theory, any abundant carbon source might be employed for microbial processes, including coal, petroleum, lignocellulose, starch, sugar, organic acids, and even carbon dioxide. Some of these sources are currently used; others, such as coal and carbon dioxide, present considerable technological barriers. Coal would have to be converted first to a readily usable carbon base (perhaps paraffin or methanol) because it is biologically inert and may con- tain compounds potentially inhibitory or toxic to microbes. However, plant biomass and, to a lesser extent, animal biomass, represent utilizable sources of carbon for microbial processes. Well-known examples of microbial processes based on these sources are the production of alcohol from grain and cheese from milk. 10

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RAW MATERIALS FOR MICROBIAL PROCESSES 11 Although carbon dioxide is a form of carbon that can be assimilated by some microorganisms, this raw material is utilized mainly by plants through the mechanism of photosynthesis. Primary photosynthetic productivity (growth of plants using solar energy) of the earth has been estimated to be 155 x 109 t* of material per year on a dry weight basis. The distribution of plant biomass produced by photosynthesis is shown in Table 1.1. Land-based plants account for 65 percent of the weight of the biomass produced annually, even though they occupy only about 29 percent of the area. The dominant yearly production of land-based biomass, approxi- mately 42 percent, is produced as forest. Although agricultural crops account for only 6 percent of the primary photosynthetic productivity, they provide not only a vital portion of food for man and animals, but other essentials such as structural materials, textiles, and paper products as well. Agricultural raw materials are the most important source of carbon for microbial conversion processes. The historical multi- purpose use of agricultural crops has maintained a continued heavy depend- ence on this source. Most agricultural crops and residues are relatively free from toxic materials and this, in addition to their availability, may have stimulated their use as a raw material for microbial processes. Because of these advantages, along with the real technological barriers to using other carbon sources, agricultural crops and residues can be expected to retain their dominance as the carbon source for microbial processes. But this must not preclude the further explor- ation and exploitation of other sources, especially those indigenous to the less-developed nations. In addition, based on their unique environments and requirements, certain countries may have an opportunity to establish new plants and practices for microbial processes. Typical Raw Materials Some typical raw materials and fermentation products used in developed countries are listed in Table 1.2. Selected combinations of other materials are used as substrates for various products. These raw materials provide carbon, nitrogen, salts, trace elements, vitamins, and other requirements for the processes; hey are few in number because the conditions for large-scale mi- crobial processes impose limitations on the materials that may serve as sub- strates. In general, the raw materials mentioned in Table 1.2 are traditionally used for microbial processes because of their suitability for specific processes. But *In this report t represents metric ton.

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12 MICROBIAL PROCESSES in another geographical area, or for new or different processes, one need not be limited to what has been used in the past. Table 1.3 lists a variety of important food crops grown in developing countries. These crops or their residues may also be considered as raw materials for microbial processes. A variety of other common waste materials derived from agricultural, forest, and urban sources, may serve as substrates for microbial processes (Table 1.4~. TABLE 1.1 Estimated Primary Photosynthetic Productivity of the Earth Area (total = 5 10 million km2 ) Net Productivity (total = 155.2 billion tons dry wt/yr) Total Earth 100~o 100% Continents 29.2 64.6 Forests 9.8 41.6 Tropical Rain 3.3 21.9 Raingreen 1.5 7.3 Summer Green 1.4 4.5 Chaparral 0.3 0.7 Warm Temperate Mixed 1.0 3.2 Boreal (Northern) 2.4 3.9 Woodland 1.4 2.7 Dwarf and Scrub 5.1 1.5 Tundra 1.6 0.7 Desert Scrub 3.5 0.8 Grasslands 4.7 9.7 Tropical 2.9 6.8 Temperate 1.8 2.9 Desert (Extreme) 4.7 0 Dry 1.7 0 Ice 3.0 0 Cultivated Land 2.7 s.9 F; reshwater 0.8 3.2 Swamp& Marsh 0.4 2.6 Lake & Stream 0.4 0.6 Oceans 70.8 35.4 Reefs& Estuaries 0.4 2.6 Continental Shelf 5.1 6.0 Open Ocean 65.1 26.7 Upwelling Zones 0.08 0.1 Source: James A. Bassham. 1975. Cellulose as a chemical and energy resource. In Cellu- lose as a chemical and energy resource. New York: John Wiley and Sons.

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RAW MATERIALS FOR MICROBIAL PROCESSES TABLE 1.2 Typical Raw Materials and Products in Industrialized Countries Raw Materials Products Sulfite Waste Liquor Ethanol Methanol Whey Paraffins Molasses Single-Cell Protein (SCP) SCP Acetic Acid SCP SCP Lactic Acid SCP Citric Acid Amino Acids Ethanol Glutamic Acid TABLE 1.3 Estimated Production of Major Food Crops in Developing (countries* Metric Tons (in Crop thousands! Percen t Paddy 186230 21.36 Cassava 103486 1 1.87 Wheat 95048 10.90 Maize 7 33 28 8.41 Banana/Plaintain 55199 6.33 Coconuts 32664 3 75 Sorghum 3 1 173 3.57 Yams, Taro, etc. 28777 3.30 Potatoes 26909 3.09 (Pulses)** 25997 (2.98) Citrus 22040 2.53 Millet 2145 2 2.46 Barley 20775 2.38 Sweet Potatoes 17630 2.02 Soybeans 13842 1.59 Groundnuts 13502 1.55 Tomatoes 1275 5 1.46 Grapes 12720 1.46 Mangoes 12556 1.44 Watermelon 10436 1.20 Dry Beans 8537 0.98 Onions 6474 0.74 Percentage of Total Developing Country l God Crop Production 94.39 *Developing market economies as defined in the FAO Production Year- book (1977). **Pulsestotal legumes except soybeans and groundnuts. Sourc e: PA O Produc tion Yearbook. New Y lark: U N I PU B. ~ 9 7 7. 13

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14 MICROBIAL PROCESSES TABLE 1.4 Typical By-product Substrates for Use in Microbial Processes in Developing Countries Agricultural Other Molasses Maize Stover Straw Bran Coffee Hulls Cocoa Hulls Coconut Hulls Fruit Peels Fruit Leaves Bagasse Oilseed Cakes Cotton Wastes Tea Wastes Bark Sawdust Animal Manures Sewage Municipal Garbage Paper Mill Effluent Cannery Effluent Fishery Effluent Slaughterhouse Effluent Milk-Processirlg Effluent Of the lists of materials in Tables 1.2, 1.3, and 1.4, a few may hold special promise for use in the microbial processes that occupy the bulk of this report. The criteria for selecting these raw materials for research are Weir almost year-round availability in large volume in many developing areas and their ease of assimilation by microorganisms. Among raw materials commonly used for microbial processes (Table 1.2), molasses is probably the one most readily available for use as a substrate in developing countries. Because it contains both easily assimilable sugars and necessary micronutrients, it is a very useful substrate that is readily utilized by a variety of microorganisms. However, it contains very little nitrogen. Starch (or syrups produced from starch) is also a good substrate, and many potential sources are available. These include the cereal crops (maize, rice, wheat, etc.) and starchy tubers such as potato and cassava. In addition to the crops that may be good sources of starch, a few of the plentiful products and waste products listed in Tables 1.3 and 1.4 may merit particular consideration for some of the microbial processes. Cassava, for instance, may be a good choice as a substrate to produce ethanol, SCP, and other economically valuable substances. This milt be a better disposition of the crop than its present widespread use for food, since its low protein-to- calorie ratio makes it less than ideal nutritionally. Another promising raw material is coffee-processing waste, which is produced in large amounts (4.5 t of by-product for each t of dehulled coffee). It appears to be a good substrate for the growth of various fungi and yeasts. Taro (Colocasia esculenta), Cough a well-known staple food, has a more limited distribution than some of the agricultural products mentioned above, existing as a commercial crop only in Egypt, West Africa, Southeast Asia, and some Pacific and Caribbean Islands.

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RAW MATERIALS FOR MICROBIAL PROCESSES However, it, too, has potential as a substrate. 15 Domestic sewage or industrial wastes offer possible fe~entation sub- strates for algae or bacteria. Unless the supply is properly planned, however, it cannot always be counted on to meet the demands of a large microbial process. The single most abundant potential source of carbonin developing coun- tries and elsewhereis cellulose (see Chapter 8~. This is a constituent of many foods,< fiber crops, agricultural residues, and wood and forest residues, some of which are mentioned in Table 1.4. Most of these selected substrates are rich in carbohydrates, but for certain microbes they may have to be supplemented with sources of nitrogen, salts, trace metals, and other requirements. Possible sources for some of these supplements in developing countries may be whole yeast or distiller's dried solubles from local alcoholic fermentations. In some countries meat or fish by-products such as slaughterhouse wastes or gutting and canning residues would be excellent supplements. Combinations of plants might also be used to meet the nutritional requirements of producing organisms. For example, cassava (carbohydrate) could be combined with soybeans (high nitrogen). Underutilized Raw Materials The materials so far discussed as possible substrates for microbial processes are widely cultivated and available. But there are many less-known plants, or plants that may be used only locally, that may be excellent candidates for this purpose. An example is the winged bean (Psophocarpus tetragonolobus), now becoming more popular as a food in Southeast Asia and West Africa because of its unique combination of protein-rich and edible seeds, tubers, and leaves. Certain tropical plants, such as basella and amaranths, have not received much attention as food sources, but they may give a greater yield than many crops in extensive use and may also be useful as substrates. However, under- developed raw materials selected for large-scale microbial processes will probably have to meet Me requirements discussed in the Introduction. Plants can also be grown specifically for biomass as a fermentation sub- strate. Plants selected for such use should grow and reproduce rapidly, con- tain a low crystallinity cellulose and a low lignin content, and be easily harvested and transported. Another desirable characteristic of plants for biomass would be an ability to grow in ecological niches in which they will not compete with or eclipse regular crops. For instance, the buffalo gourd (Cucurbita foetidissima) does well in arid conditions, and the salt bushes (A triplex spp.) and tamarugo (fro sopis tamarz~go) are salt tolerant and might be introduced into countries with arid

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16 MICROBIAL PROCESSES and saline areas. Aquatic plants such as the reed (Phragmites communist, cattails (Typha spp.), the papyrus reeds (Cypenus spp.), mat rum (Juncus effuses), textile screw pine (Pandanus tectonics), and eel grass (Zostera ma- rina) are examples of plants that grow in a saline aquatic environment. The use of the water hyacinth (Eichornia crassipes) to treat domestic sewage with large quantities of plant biomass produced during the process is receiving increased attention because of the tremendous growth rate of this plantas much as 850 kg/ha/day of dry plant material has been reported. This prolific growth has made the water hyacinth a troublesome weed in certain tropical and semitropical areas, particularly the Nile Basin and south- ern United States. Biomass from domestic waste treatment can be used to produce biogas and fertilizer, feed, or a protein concentrate. At present, domestic wastewater treatment using the water hyacinth is being demon- strated in the United States. Kelps and seaweeds are sources of carbohydrates other than cellulose. These plants have the drawback of high water and salt content. Farming such plants and harvesting them economically may also present special problems. Countries with a limited supply of oil and natural gas are not likely to consider petroleum hydrocarbons as a microbial substrate. But countries with large oil and gas deposits also have a supply of methane, which may be used as a carbon and hydrogen substrate for the grown of organisms for single-cell protein. Research Needs In many developing countries there is need to establish basic information about potential substrates for microbial processes by: Systematic identification of resources; Analysis of constituents and properties of promising individual sources; Identification of scientific, technological, and institutional resources and constraints; and Determination of optimal process based on best use of substrate, eco- nomic justification, available technical support, and unique area needs. References and Suggested Reading Bassham, J. A. 1975. Cellulose as a chemical and energy resource. In Cellulose as a chemical and energy resource: Cellulose Conference Proceedings, held under the auspices of the National Science Foundation, at the University of California, Berke- ley, June 25-27. 1974, C. R. Wilke, ea.' pp. 9-19. New York: John Wiley and Sons. Birch, G. G.; Parker, K. J.; and Worgan, J. T., eds. 1976. Food from waste. London: Applied Science Publishers Ltd.

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RAW MATERIALS FOR MICROBIAL PROCESSES 17 Conference or Capturing the Sun through Biocon version, Proceedings, sponsored by the United States Energy Research Development Administration and others, Washington, D.C., March 10-12, 1976. Washington, D.C.: Washington Center of Metropolitan Studies. Food and agriculture: readings from Scientific American. 1976. SanFrancisco:W.H. Freeman and Company. National Academy of Sciences. 1975. Underexploited tropical plants with promising economic value. Report of an Ad Hoc Panel of the Advisory Committee on Tech- nology Innovation, Board on Science and Technology for International Develop- ment, Commission on International Relations. Washington, D.C.: National Academy of Sciences. 1976. Making aquatic weeds useful: some perspectives for developing coun- tries. Report of an Ad Hoc Panel of the Advisory Committee on Technology Innova- tion, Board on Science and Technology for International Development, Commission on International Relations. Washington, D.C.: National Academy of Sciences. 1976. Renewable resources for industrial materials. Report of the Committee on Renewable Resources for Industrial Materials, Board on Agriculture and Renew- able Resources, Commission on Natural Resources. Washington, D.C.: National Acad- emy of Sciences. 1977. Methanegeneration from human, animal, and agricultural wastes. Report of an Ad Hoc Panel of the Advisory Committee on Technology Innovation, Board on Science and Technology for International Development, Commission on Inter- national Relations. Washington, D.C.: National Academy of Sciences. Perlman, D. 1977. Fermentation industries, quo wadis? Chemical Technology 7:434-443. Schlegel, H. G., and Barnea, J., eds. 1977. Mircrobial energy conversion. Oxford: Perga- mon Press. White, J. W., and McGrew, W., eds. 1977. Clean fuels from biomass and wastes. Proceed- ings of the symposium held on January 25-28, 1977, at Orlando, Florida, sponsored by the Institute of Gas Technology. Chicago: Institute of Gas Technology. Wilke, C. R., ed. 1975. Cellulose as a chemical and energy resource: Cellulose Conference Proceedings, held under the auspices of the National Science Foundation, at the University of California, Berkeley, June 25-27, 1974. New York: John Wiley and Sons. Research Contacts Carl-Goran Heden, Karolinska Institutet, Solnavagen 1, S-10401 Stockholm 60, Sweden. Arthur E. Humphrey, School of Engineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, U.S.A. F. K. E. Imrie, Tate and Lyle Ltd., Philip Lyle Memorial Research Laboratory, Univer- sity of Reading, P. O. Box 68, Reading RG6 2BX, England David Perlman, School of Pharmacy, University of Wisconsin, Madison, Wisconsin 53706, U.S.A. Steven R. Tannenbaum, Department of Nutrition and Food Science, Massachusetts Insti- tute of Technology, Cambridge, Massachusetts 02139, U.S.A. Noel D. Vietmeyer, National Academy of Sciences, 2101 Constitution Avenue, N.W., Washington, D.C. 20418, U.S.A. Daniel Wang, Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, U.S.A. J. T. Worgan, National College of Food Technology, University of Reading, St. George's Avenue, Weybridge, Surrey KT13 ODE, England.