Biocatalysis and Biodegration: Microbial Transformation of Organic Compounds

This chapter presents a historical view of microbial biodegradation and biocatalysis. One indicator of the importance of oxygenases in microbial catabolism, and in the reactions principally studied for biodegradation and biocatalysis, is their high level of occurrence in the Biocatalysis/Biodegradation Database. As the 20th century proceeded, biochemical investigations of microbes revealed two important and seemingly contradictory views of the biochemistry of life. First, the idea of the unity of biochemistry was established. At the same time, the diversity of microbial metabolism was continuously recorded as new microbes were identified and studied biochemically. The role of oxygen in microbially mediated transformation of chemicals was perhaps first suggested by the work of Jean Jacques Schloesing and A. Muntz. A classic treatment of the thermodynamics of anaerobic metabolism was published by Rolf Thauer and colleagues in 1977. This review also elegantly described the thermodynamic logic behind interspecies hydrogen transfer. While anaerobic metabolism was important in commercial fermentation processes, the details of anaerobic reactions were not well known because of difficulties in culturing anaerobic bacteria in pure culture. This derived partly from the predilection of microbiologists for agar plate culturing and the difficulty of maintaining plates under sufficiently anoxic conditions. Experiments examining the expression of metabolic activities in microbial cells led to the idea of catabolite control, or induction, of enzyme activity.

This chapter focuses on microbial diversity and the catabolism of nonintermediary chemical compounds by disparate genera of prokaryotes. It describes the role that prokaryotes and fungi play collectively in biodegradation and how their extensive biocatalytic potential derives from a long evolutionary history. But microbial diversity also implies that individual bacteria and fungi are metabolically unique. In this context, it is useful to think of microbes as metabolic machines, dependent on gathering chemicals from their environment to obtain carbon, other elements, and energy to compete favorably against other microbes. Fungi are prominent in many environmental biodegradation processes and also in industrial biocatalysis. Four of the five major phyla of fungi are commonly used in industry. Of the ascomycota, Saccharomyces and Schizosaccharomyces are the major fungal genera used in alcoholic-beverage fermentations. Many of the best-studied prokaryotes are aerobic proteobacteria and the high-G + C gram-positive bacteria. These, along with the fungus Cunninghamella elegans, are the examples discussed in the chapter. It is important to point out that some of the extensive catabolic activities of this class are based on the broad substrate specificities of a few oxygenases and related enzymes which handle the oxygenated intermediates.

This chapter focuses on single-enzyme-catalyzed biodegradation reactions and other physiological processes that microbes use to compete successfully for scarce nutritional resources in soil and water. It is likely that a lot of reactions that fall under the general heading of biodegradation are fortuitous. There are numerous examples of this with insects or fungi that biosynthesize broad-specificity enzymes, such as cytochrome P450 monooxygenases, for detoxifying biological toxins. For example, plant-pathogenic fungi are sometimes warded off with toxic chemicals manufactured in the leaves of the plant being attacked. Fungal cytochrome P450 monooxygenases oxidize an enormous array of compounds, some of which are unlikely to prove toxic, and thus these reactions may well fall into the fortuitous category. Catabolic enzymes are so useful in large part because many have been found. In fact, catabolic enzymes may be the major group of enzymes catalyzing unique reactions found on the Earth. Additionally, there are the known catabolic enzymes which catabolize industrial chemicals. The role of biosurfactants in microbial metabolism has been investigated primarily with petroleum or with purified alkanes. Surfactants are compounds which are amphipathic; that is, they contain hydrophilic and hydrophobic chemical groups linked together in the same molecule. People use surfactants as soaps and detergents and as emulsifying agents in food. The sensing of chemical compounds starts with binding at the cell membrane to a methyl-accepting chemotaxis protein (MCP). The extracellular sensing is transmitted through the MCP, which spans the membrane, to its cytoplasmic domain.

This chapter makes extensive use of visual images of chemical compounds, in a familiar format, to highlight the underlying patterns of catabolism. Intermediary metabolism is defined as enzyme-catalyzed reactions common to most living things: bacteria, eukaryotic single-celled life, plants, and animals. More specifically, intermediary metabolism deals with (i) common energy metabolism—for example, the catabolism of glucose by the glycolytic, or Embden-Meyerhof, pathway—and (ii) common biosynthetic pathways, such as those that generate necessary amino acids, lipids, and carbohydrates. This is illustrated, in the form of an electronic circuit diagram of the intermediary metabolism of Escherichia coli. Catabolic pathways funnel into a limited set of key intermediates, getting the most metabolic bang from the limited genetic buck. Thus, a new pathway is not required for each compound the organism might metabolize if there is redundancy by generating a common intermediate. This is well illustrated in aromatic-ring metabolism, which often proceeds through catecholic intermediates aerobically and benzoylcoenzyme A (CoA) anaerobically. These points are illustrated in the chapter by using a series of metamaps. The metamaps discussed include C1 metamap, C2 metamap, cycloalkane metamap, BTEX metamap, PAH metamap, heterocyclic-ring metamap, triazine-ring metamap, organohalogen metamap, and organometallic metamap.

Certain large-scale fermentation processes persisted for some time, but after World War II, there was a tremendous rise in the size and influence of the commodity chemical industry. The industry has increasingly looked into alternatives, and slowly but surely, biotechnology is making inroads into commodity chemical production. For specialty chemical production, many different microorganisms are used, for example, Streptomyces sp. for antibiotics and Corynebateria sp. for amino acids. For many years the herbicide was marketed as the racemate, (R,S)- dichloroprop. The (S)-isomer is inactive as a herbicide. As a result, application of the racemate imposed an additional and unnecessary burden on the environment for the biodegradation of the herbicide. In the South Dakota field test, it was decided to implement several treatments. The statistical software used to deal with the large variations was MacAnova, a package developed at the University of Minnesota. The goal of this statistical treatment was to attain a final atrazine level in the treated soils of 2,000 ppm or lower. This study demonstrated that a combination of laboratory pretesting, innovative technology, and regulatory agency-industry cooperation can result in the management of an accidental spill. In the past, applications of biodegradation and biocatalysis for chemical manufacture, even while using similar enzymes, have been carried out by different practitioners. This is changing as industry seeks cleaner practices and strives for greater competitiveness.

It is often speculated that every non-polymeric compound conceived of by humans and occurring in nature will be metabolized by some microorganism somewhere in the soil or water of the Earth. Is this true? Can it be studied systematically? This chapter provides answers to these questions. The beginnings of an answer are first addressed with existing knowledge. Then, ideas are presented as to how the far reaches of microbial metabolism can be further identified. This effort is also linked to the widespread enterprise of microbial functional genomics. The total extent of genomic diversity is unknown, but comparative genomic analysis to date suggests that genetic diversity in prokaryotes is greater than previously anticipated. This, along with other evidence presented in the chapter, suggests that there is much catalytic diversity yet to be discovered. In the wealth of catalytic diversity possessed by microbial enzymes, two issues are relevant. First, it is unlikely that we have uncovered all of the catalytic potential of these known cofactors. Moreover, it is unclear how many new cofactors, metals, and modified amino acids remain to be discovered in microbial systems. To determine the potential for microbes to transform the exotic compounds for which metabolism is unknown, several compounds containing such functional groups were put into standard enrichment cultures in which the compounds were used as sole carbon or nitrogen sources to support growth. Most of these yielded actively growing cultures of microorganisms, and the target compound was transformed in the cultures, as demonstrated by high-pressure liquid chromatography.