Microbial Technology
Alan L. Kriz
Microorganisms have been exploited for their specific biochemical and physiological properties from the earliest times for baking, brewing, and food preservation and more recently for producing antibiotics, solvents, amino acids, feed supplements, and chemical feedstuffs. Over time, there has been continuous selection by scientists of special strains of microorganisms, based on their efficiency to perform a desired function. Progress, however, has been slow, often difficult to explain, and hard to repeat.
Recent developments in molecular biology and genetic engineering could provide novel solutions to long-standing problems. Over the past decade, scientists have developed the techniques to move a gene from one organism to another, based on discoveries of how cells store, duplicate, and transfer genetic information.
Recombinant DNA technology has vast potential benefits, and agriculture is a prime area where these modern techniques will have greatest impact. Recombinant DNA techniques may provide us with disease-resistant crops, feed crops with higher nutritional and digestive quality, improved vaccines for animal health, hormones to enhance milk and meat production, enzymes to improve digestion of feedstuffs, new ways to clean up our environment, and more efficient methods to produce chemicals.
Source: Adapted from Introduction to Plant Biotechnology, K. McPheeters, Vocational Agriculture Service (1989).
Fermentation Biotechnology
Until recently, fermentation processes depended on a few types of raw materials (substrates) and on available strains of microorganisms. But now microbes can be genetically manipulated to function more efficiently and to use a wide variety of substrates. As these microbes are re- engineered and their fermentation capabilities fully exploited, we rapidly near the day when chemicals can be produced economically and naturally.
The development of any successful industrial process ultimately depends on efficiently converting the substrate to a highly concentrated end product. Because of the potential for commercial success, research in the Department of Food Science is focused on the acetone-butanol-ethanol (ABE) fermentation. To make the ABE fermentation economically viable, several problems must be addressed.
The first problem relates to how product toxicity leads to low concentrations of butanol in the fermentation broth. The microorganism Clostridium acetobutylicum is intolerant of low concentrations of butanol; as little as 1.3 percent inhibits growth and fermentation. Increasing the butanol from 1.3 to 2 percent in the fermentation broth decreases the energy required for product recovery by 50 percent.
Another problem is the limited spectrum of substrates that can be used by the microorganism.
Work in Hans Blaschek's laboratory develops tailor-made strains of C. acetobutylicum, a microorganism that tolerates butanol and uses starch or cellulose to produce butanol. The approach involves cloning genes capable of hydrolyzing cellulose (called cellulases), and starch (amylases) into C. acetobutylicum. Although the application of genetic engineering techniques to C. acetobutylicum is still in its infancy, the potential for improving the fermentation characteristics of this microorganism is great. This technology should allow for improvements in the sequence of enzyme-catalyzed reactions, end-product tolerance, and the actual enzyme systems of this microorganism.
Potential benefits to the food industry include a practical and economical means to dispose of agricultural wastes and processing by-products. Furthermore, biomass-derived chemicals should help reduce our dependence on fossil fuels, the current source of these chemicals.
Rumen Microbiology
Ruminant animals (cattle, sheep, goats, and many other animal species) are nutritionally dependent on the activity of microorganisms present in the rumen. This dependence is based on microbial activity that degrades plant fiber, uses nonprotein nitrogen (NPN), and transforms phytotoxins or toxins produced by plants.
The approach being taken in the Department of Animal Sciences by Bryan White is to improve ruminal fiber digestion by amplifying the cellulose-hydrolyzing capabilities of predominant cellulolytic bacteria in the rumen, the Ruminococci. The complex cellulase enzyme system of Ruminococcus flavefaciens FD-1 has been studied extensively. Three synergistic cellulase components (exo- and endo-glucanases) have been purified to homogeneity, characterized biochemically, and used to elicit specific antibodies for immunological studies on enzyme location and function. These results suggest that the b-glucanases of the Ruminococci have more bond specificity than sugar specificity and probably play an important role in cell wall hydrolysis, a chemical process of decomposition.
Molecular cloning experiments of cellulases from Ruminococcus flavefaciens FD-1 have identified at least four different genes encoding for endoglucanase activity. DNA sequence analysis in progress will provide vital information on the regulatory and mechanistic elements of cellulose hydrolysis by these rumen bacteria.
Thus far, gene transfer systems have not been established in ruminal bacteria. One of the barriers is the presence of restriction-modification systems that cause extensive cutting of introduced DNA. Several restriction-modification systems have been identified in the Ruminococci and our understanding of their activity will potentially result in successful DNA transfer and allow the reintroduction of cloned (modified) genes back into the original host. Both the potential benefits and the problems involved in this research are great. Before this technology becomes a reality and genetically engineered organisms can be reintroduced into the rumen to enhance plant fiber degradation, however, a sustained and substantial commitment will be required.
Plant Pathogens
The roots and aboveground parts of a plant growing in soil are in constant contact with thousands of different microorganisms, most of which do not affect the plant in any easily observable way. Although bacteria capable of invading plant tissue and causing disease are rare, many of them are closely related to the harmless bacteria also found in soil and on plant surfaces. The plant pathogens, disease-causing agents or organisms, thus have unique properties that could be useful in devising ways to control the diseases they cause.
One species of bacteria being studied in Paul Shaw's laboratory is Pseudomonas syringae pathovar tabaci, one of about 50 different pathovars of that bacterium that cause diseases on many plants from apples to zinnias. The strain under study infects green beans and tobacco, causing a disease called "wildfire," due to the rapidity with which it can spread in a field.
A second species of bacteria is Xanthomonas campestris pathovar glycines. X. campestris also causes disease on many plants, but pathovar glycines attacks only soybeans and causes bacterial pustule, in terms of yield loss, one of the most serious bacterial diseases of soybeans in Illinois.
The initial objective of the research was to identify genes responsible for making the bacteria plant pathogens. The techniques of molecular biology are used to isolate pathogenicity genes from the bacterial chromosome. Although eight such genes have been found in P. syringae and more than 16 in X. campestris, it is not likely that they represent all the pathogenicity genes present in the bacteria. Many genes are translated into proteins or molecules responsible for carrying out most cellular functions. The next step, therefore, was to determine if the isolated genes had the potential to encode proteins. The four genes examined thus far appear to have that capability, and one likely candidate for a pathogenicity protein has been detected.
The ultimate objectives of the research are to determine the functions of the proteins and to define their roles in pathogenesis or disease origin. For example, one of the potential proteins from X. campestris is probably present in the membrane that surrounds the bacterium and might therefore be involved in transporting nutrients or other compounds into or from the bacterial cells from culture media, intercellular plant fluids, water on the plant surface, the aqueous milieu in the soil or wherever the organism is growing. Other pathogenicity proteins will be characterized so that they can become targets for disease control measures.
Hans-Peter M. Blaschek, professor of food science; Roderick I. Mackie, visiting professor of animal sciences; and Paul D. Shaw, professor of biochemistry, Department of Plant Pathology
