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Microbial utilization of mono- and di-saccharide residues
Microbial conversion of starchy residues
Microbial conversion of complex mixtures of compounds (Polysaccharides, Proteins, Lipids, etc.)
Microbial utilization of cellulose and ligno-cellulose residues
Algal culture as a source of biomass
Microbial utilization of silviculture biomass
Micro-organisms and marine and freshwater biomass
International studies on processing organic residues
Department of Microbiology University of Iowa, Iowa City, Iowa, USA
Micro-organisms have been closely associated with transforming or cycling organic matter in nature for as long as such material has existed. But it has only been within the past 100 years that certain of these associations have become known, or that advantage has been taken of helpful microbes in rural agricultural practices. Predictions are, however, that greater use will have to be made of beneficial micro-organisms.
I need not discuss the important part micro-organisms play in the production of humus, nor how they help cycle all elements or substances in the soil and thereby provide the nutrients necessary for healthy crops. These topics are beyond the scope of this paper. Neither will I discuss in depth how microbes fix an estimated 150 to 175 million tons of atmospheric nitrogen per year, which is several times more than the total commercial production of nitrogen fertilizer in 1977; nor how micro-organisms may be degrading over 1,500 million tons of pesticides and large quantities of other complex synthetic substances that find their way into the environment each year.
The concept of utilizing excess biomass or waste from agricultural and agro-industrial residues to produce energy, feeds or foods, and other useful products is not necessarily new. For centuries agricultural residues and wood have been used as sources of fuel, food, construction materials, and paper-making, as well as for other purposes. Recently, fermentation of biomass has gained considerable attention because of the forthcoming scarcity of fossil fuels, and because it is necessary to increase the world food and feed supplies - especially those high in protein.
Most attention today is being given to the possible use of micro-organisms to convert relatively high-quality biomass (corn and grains, sugar-cane juice, etc.) to fuel. Although this topic will be discussed later, certain technical and economic restrictions exist that must be removed if significant fuel production is to result from fermentation of such high-quality biomass, because these substrates have other important possible uses. This does not mean, however, that residues from farm crops, livestock feedlots, agro-industries, forest operations, and other similar practices should be excluded. This is especially so in circumstances where their removal does not eventually reduce the quality of the land, permit soil erosion, or produce other harmful effects on crops.
A logical classification of agricultural and agro-industrial materials has recently been published by Rolz (1) His data (Table 1) illustrate the variation in the structure of the substances, and the nature of the by-products that may be available for utilization by micro-organisms
TABLE 1. Classification of Agricultural and Agro-Industrial By-products
|I||High proportion of di- and mono-saccharides||Sugar-cane growing and processing||Molasses|
|Pulp elaboration||Sulphite liquors|
|II||Di- and mono-saccharides with some structural polysaccharides||Fresh fruit collection centres||Rejected or damaged fruit|
|Rum and liquor making||Wash waters|
|III||Mixture of soluble organic
compounds, including starch,
sugars, proteins, pectin,
|Fruit and vegetable processing||Wastes from washing, peeling, and blanching|
|Tuber and grain processing||Wastes from sorting and washing|
|Coffee processing||Washing and pulping waters|
|Meat processing (beef, pork, poultry)||Washing and scalding waters|
|IV||Complex mixtures of structural
polysaccharides and other
compounds such as proteins,
|Fruit and vegetable processing||Peels, insoluble solids from pulp and seeds|
|Animal and poultry production||Manure|
|Animal slaughtering and meat processing||Suspended solids|
|Sugar-cane and oil palm processing||Residual solids|
|Alcohol and alcoholic beverage processing||Residual solids|
|V||Structural cellulose and lignin in high proportion||Cereal, sugar-cane, and rice growing and processing||Straw, husks, bagasse|
|Corn growing and processing||Stocks and cobs|
|Citronella and lemon grass processing||Bagasse|
|Coffee and cacao processing||Husks|
|Cotton seed processing||Hulls, linters|
|Forest processing||Bark, sawdust, wastes|
Source: Rolz (1).
Several calculations have been made of the quantities of biomass produced annually in the world by photosynthesis, and the resulting agro-industrial wastes One estimate is that 1.7 x 10(11) tons of biomass are produced annually, and that 98 per cent of this amount is not used in an economically sound manner DaSilva, Olembo, and Burgers (2) present data on some agricultural residues in six European countries (Table 2); these constitute about 98 million tons each year. For other countries, the three authors estimate the following: Malaysian oil palm and rice mill wastes in 1974 were 3 million and 250,000 tons, respectively. In Egypt, 600,000 tons of maize cobs, 1.5 million tons of dry rice straw, and 40,000 tons of sugar pith residues accumulate annually. About 100,000 tons of sugar-cane bagasse are burned in Bangladesh each year. In Western Australia, 10 million tons of wheat and barley straw and chaff are produced annually. Recent similar estimates for the United States by Pimentel and associates (3) are presented in Table 3.
TABLE 2. Agricultural Wastes in Certain European Countries
|Amounts in tons|
|Country||Cereal straw*||Corn stover||Beet pulp by-product|
Data from Battelle Document 75712, Courtesy DaSilva, Olembo, Burgers (2).
* Amount varied in different countries from 1.5 to 5.0 tons/ hectare.
** Yield 1.8 tons/hectare.
TABLE 3. Sources of Biomass Available Annually in the United States
kcal x 1012
|Food-processing wastes(20 - 70% moisture)||4||4||18|
|Food-processing wastes(70 - 90% moisture)||14||14||10|
|Municipal sewage||13||2||1 3|
Source: Pimentel et al. (3).
Before any organic residue or high-quality biomass material is considered for microbial conversion to other substances, a number of factors must be taken into consideration. For instance: (i) Is there a ready and continuous supply of the raw product to be converted? (ii) If the material is removed from cropland or forests, will this contribute to soil erosion and depletion of plant nutrients? (iii) Are expensive equipment and large amounts of capital necessary for the processing? and (iv) Are such things as an external energy supply and large amounts of water necessary?
After considering the above factors, the following question may be asked: Which microorganism or microorganisms possess potentials for the bioconversion of the organic material under consideration? First, we must keep in mind that in natural conditions the indigenous microbial flora is only one component of a complex, dynamic biomass undergoing interaction in the transformation of organic matter. Only in a few cases can any one species or genus be given sole credit for natural bioconversions. For example, in the transformation of green fodder or forage crops into silage, the complex fermentation process involves plant enzymes as well as several groups of microorganisms present in the fodder and in the environment. Likewise, in the production of biogas from organic wastes, methane bacteria may be responsible for the gases produced, but this is not the only biological process taking place in the digester.
Even though mixed cultures of micro-organisms are usually involved in the transformation of organic residues, there are cases where pure cultures of bacteria, yeasts, moulds, or enzyme preparations can be used for processing such materials; these will be discussed later
Because other papers in these proceedings are also devoted to topics that can be included under the broad title of this paper, the following discussion will be restricted to a few possible microbial processes involved in the transformation of by-products or residues listed in Tables 1-3. Some of these processes have been, or can be, adapted on a small scale to rural regions, but others currently require fairly sophisticated knowledge or equipment for operation.
One of the major problems facing us today is how to adapt technical skills to various regions where people differ in their cultural or social customs, where natural resources vary, where the economy is dissimilar, or where environmental conditions may limit certain processes. More careful thought must be given in future developments as to whether the so-called "high technology" will be the best choice for people in every nation, or whether more attention needs to be given to what Norman refers to as "soft technologies" (4); Hedén speaks of as "self-reliance in an equilibrium society" (5); or what DaSilva, Olembo, and Burgers consider "low capital" vs. "high capital" technologies (21. The results presented in this Symposium can help point the way for leaders in various countries to make certain important decisions for future development.
An extensive discussion of even the major organic residues that can be utilized by microorganisms in a rural environment cannot be covered in one article. So I have selected only a few substances from the groups used for classification in Table 1.
The by-products (molasses, sulphite liquor, whey) listed in group 1, Table 1, are rich in fermentable sugars, and they serve as a major source of carbon for a great variety of micro-organisms. At least 5,000 microbial metabolic products have been isolated from solutions in which the simpler sugars have served as the main source of carbon for metabolism by micro-organisms. These metabolites include not only simple alcohols, organic acids, gases, antibiotics, vitamins, enzymes, toxins, etc. but also some unique compounds whose use or function remains unknown. Great opportunities exist for finding uses for some of these substances, or for developing technologies that may be applicable to rural processing of such materials.
Large quantities of molasses are produced in countries where sugar-cane is grown and processed. Rolz, for example, estimates that over 6.3 million tons are available annually in the major sugar-cane-growing countries of Latin America (1).
The sugar in molasses can be metabolized by many micro-organisms and by several known pathways. The particular pathway followed, and the end-products produced, depend not only on the particular microbe, but also on a variety of environmental factors.
Special strains of Saccharomyces cerevisiae, S. fragilis, and Candida utils are used in the baking industry, as feed and food supplements, and for other purposes. World production of such yeast is over 300,000 tons per year. The raw materials for cultivation of such yeasts are generally a mixture of molasses, ammonium salts, and other essential inorganic salts.
In recent years the production of filamentous fungi as a source of protein has been emphasized. Espinosa et al., for example, have shown that the growth of Verticillium sp. on cane blackstrap molasses and coffee-waste water is technically feasible (6).
Mushroom mycelium has also been grown in molasses, as well as in vinasse, a waste product from the distillation of fermented sugar-cane juice.
Perhaps the greatest potential use of molasses, other than as a sweetener in foods for human consumption, and as a livestock feed supplement, is for the production of ethanol by fermentation, or as a feedstock for the manufacture of other useful products. The fermentation of molasses to ethanol by yeast is not an especially complex process, and it can be easily adapted on a small scale to rural areas. In Brazil, however, the production of ethyl alcohol from sugar-cane, manioc, and other tropical plants has become a major project of the government to reduce petroleum imports (Figure 1). Approval was given by Brazil's National Alcohol Commission for government financing in the amount of US$800 million in 1977 for over 30 of the 170 proposed distilleries. The plan calls for increasing alcohol production to over 3,800 million I by 1982. As fossil fuels become scarcer, many nations may need to turn to the ethanol fermentation of waste saccharide materials as a source of energy (7).
Figure. 1. Fermentation of Biomass to Ethanol or Other Organic Chemicals, and Other Organic Chemicals (From Altepohl )
Sulphite Waste Liquor
Several million tons of sugar occur in the sulphite liquor that results from the production of paper products; most is discarded in the United States (Table 3), and similar amounts are probably considered waste in other countries. Apart from the fact that sulphite liquor from the paper mills causes a disposal problem, it is also an economic loss because it can be converted into single-cell protein (SCP), ethanol, or D-lactic acid.
Candida utilis has been used for alcohol and feed yeast production from paper mill waste because it has a high tolerance for sulphite and can convert both hexoses and pentoses into yeast protein. A commercial operation called the Pekilo Process has been developed in Finland for the production of single-cell animal feed. Spent liquor from sulphite pulp mills is used as the substrate, and the fungus Paecilomyces variotil, which consists of 55 to 60 per cent protein, is used in the fermentation process. The first Pekilo plant built produces about 10,000 tons of single-cell protein annually.
Lactobacillus pentosus seems superior to other bacteria for producing D-lactic acid from sulphite waste liquors. Estimates for a mill producing 100 tons of pulp daily are that over 3 million kg of lactic acid can be manufactured annually.
Mushroom mycelium has been grown in sulphite waste liquor, and the process has been granted a patent.
In countries where cheese-making is important, large volumes of whey accumulate and must be disposed of as a waste, as profitable uses have not been found for the material. Development of new uses for whey would do much to reduce the waste and avoid the loss of milk nutrients. The possibilities for such developments offer some of the most interesting challenges in applied science.
Whey has some limitations as a substrate for attack by micro-organisms because fewer microbes utilize lactose than other sugars such as glucose. The best suited organisms for fermentation of whey are lactobacilli and certain yeasts.
Lactobacillus bulgaricus is capable of converting over 90 per cent of the lactose in whey to DL-lactic acid, and the organism is now used commercially for this purpose. Various lactosefermenting yeasts (Saccharomyces fragilis, Candida pseudo-tropicalis, or Torula cremoris) can convert the sugar to various products without altering the other nutrients in whey; this has become a commercial process for producing lactose-free whey and ethanol (80 to 90 per cent conversion of the lactose).
Several hundred-thousand tons of yeast for baking, feed, and food supplements have been manufactured for many years, utilizing low-grade sugars as a substrate; the demand for such protein is increasing. Recently a new, continuous-flow, closed-system plant has been put into operation to produce the lactose-fermenting yeast Candida utilis from whey. The plant is capable of manufacturing 7,500 tons of yeast annually.
Juices from various fruits, leaves, and stalks of plants contain sugars that can be grouped in categories I and II (Table 1). Many of the materials are abundant and cheap, and could be readily converted by microbial processes to useful substances. One example may be mentioned.
Agave juice from plants growing on arid lands has been used experimentally as a substrate for SCP production (8). Both pure cultures of yeast (Saccharomyces carbajali, Candida utilis, etc.), and mixed cultures of yeast, fungi (Ustilago maydis), and bacteria (Corynebacterium glutamicum, Brevibacterium flavum) were used to produce the SCP biomass. The yields of high-quality microbial protein obtained were good (20 g/l) from a 24hour semi-continuous operation. Indications are that a plant would have considerable socioeconomic impact on production in Mexico, where protein feed and food are badly needed.
There is extensive literature on the utilization of starch-containing materials by microorganisms. Although not al) microbes are capable of producing enzymes (amylases) that attack starch, amylases have been found in many species of bacteria, streptomyces, yeasts, and moulds. The following species appear to be the most active (9). Bacteria:
a-amylase: Bacillus subtilis, B. macerans, B. amyloliquefaciens, B. stearothermophilus, Clostridium acetobutylicum
b-amylase: Bacillus cereus, B. megaterium, B. polymyxa
Moulds: Aspergillus oryzee, A. niger, A. fumigatus
Two examples may be mentioned briefly where substances rich in starch are converted by the organisms mentioned above to useful products.
Aspergillus fumigatus, a thermophilic mould, has been used to make single-cell protein from cassava (10). Because this process is not complex and produces good yields of protein, it could be adapted to rural areas. Cellulolytic fungi (Trichoderma viride, basidiomycetes) have been employed with commercial amylases to enhance the saccharification of cassava starch; the hydrolysate served as a better substrate for the alcoholic fermentation by yeast (1 1).
The second example is currently a successful commercial process, but because of its nature it could possibly be adapted to the production of sugar "sweetener" in rural regions. In the United States, in 1977, over 2 million tons of fructose-sweetener corn syrup were manufactured from corn starch, using over 1,000 tons of microbial amylases and glucose isomerases.
The manufacture of high fructose corn syrup is now a continuous process, employing immobilized enzymes. The saccharification of the starch is accomplished by the combined action of acid and microbial amylases from bacilli, and the resulting maltose-glucose solution is then subjected to isomerization to yield fructose (42 per cent), glucose (50 per cent), and some higher saccharides (Figure 2). Several commercial processes employ preparations of isomerase from Streptomyces sp. (S. albus, S. olivaceus, S. wedmorensis, and mutants of several kinds), but several bacterial species (Bacillus coagulans, Pseudomonas hydrophilia, Escherichia freundii, Nocardia asteroides, etc.) and aquatic actinomycetes (Actinoplanes missouriensis) yield considerable amounts of glucose isomerase (12, 13).
Figure. 2. Flow Chart for the Production of High-Fructose Corn Syrup from Cornstarch (From Mermelstein )
Many other agricultural residues and agro-industrial wastes belong to this group of substances, which are rich in starch, pectin, sugars, organic acids, and even some nitrogenous compounds. They include cull and wash materials from fruit, vegetables, meat, and other foods being processed. In the United States, Pimentel and associates (3) estimate these materials to be several million tons annually (Table 3). For example, Aspergillus niger will convert 97 per cent of the sugars from brewery-spent grain liquor to fungal mass suitable for feeding purposes (14). Similarly large quantities of wastes occur from washing and pulp waters from coffee processing in Central and South American, Asian, and African nations (1,15-17) These substances offer considerable challenge and promise for future developments, and micro-organisms play a part in their utilization. According to a review by Han and Smith, they can best be utilized for what they call one of the five F's: fuel, fibre, fertilizer, feed, and food (18).
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