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452 BIOCHEMICAL SOCIETY TRANSACTIONS MICROBIAL CATABOLISM: ITS ROLE IN THE CARBON CYCLE AND ENVIRONMENTAL SIGNIFICANCE : a Colloquium organized by P. A. Williams (Bangor) Introduction: Anaerobic and Aerobic Environments W. CHARLES EVANS Department of Biochemistry and Soil Science, University ColIege of North Wales, Bangor, Gwynedd LL57 2UW, Wales, U.K. Life on Earth depends on a dynamic balance between living organisms possessing a diversity of biochemical competence, and the environment. This ‘throughput’ of chemicals from inanimate materials (rocks, soils, natural waters and air, with the aid of solar radiation) to their participation in biochemical processes and their subsequent mineralization again, has evolved over a geological time-scale and under a variety of physical conditions. The constituents ofliving organisms must be susceptible to degradation or they would eventually accumulate on the surface of the earth and thus lock the chemicals in an unavailable form. Micro-organisms are the scavengers par excellence in maintaining the carbon cycle, producing eventually COz and methane from all naturally occurring organic compounds. Man is introducing increasingly large amounts of synthetic organic chemicals into the environment, particularly as herbicides, pesticides, detergents and the unwanted byproducts of industry. Some of these molecules are recalcitrant to microbial attack and therefore accumulate in the biosphere; their effects (apart from any direct toxicity to man) on normal food-chains are not easy to predict, but have been disastrous in some instances. Biodegradability is clearly obligatory for soluble, potentially toxic, organic chemicals if they are to be used by man for whatever purpose, and are likely to be disseminated in the environment. Microbial catabolism is a wide topic; it seems appropriate to focus attention on some of the fundamental biochemical aspects of the central ‘carbon’ cycle. Guiding principles may emerge that have a bearing on biodegradability in Nature. Carbon cycIe Scheme 1 illustrates the biological carbon cycle. The principal geochemical reservoirs of carbon have been quoted as follows (Doetsch & Cook, 1973): Atmosphere = 2.3 x 10i8g(as COz) Biosphere = 2.6 x lO”g (inorganic and organic C) Lithosphere = 1.2 x 1Oz2g(sedimentary rocks and carbonates) = 3.6 x loz1g (fossil fuels: coal, petroleum and kerogen) = 1.3 x 1Oz2g(igneous and metamorphic rocks) The smallest segment with respect to the amount of this element is the biosphere. Carbon enters the biosphere primarily by photosynthesis (terrestrial plants, aquatic algae and especially the unicellular marine diatoms and dinoflagellates), i.e. by the reduction of COz; the contribution of photosynthetic and chemolithotrophic bacteria is thought to be relatively small in comparison. Estimates of COz fixation vary widely; possibly 5 x 1016-15x 10l6gof carbon enters the biosphere annually by photosynthesis 1976 453 562nd MEETING, BANGOR Carbonates Volcanoes, magma, Igneous rock deposits Preclplratlon Scheme 1. Carbon cycle e) (i.e. 8-23 %ofthe total carbon in the atmosphere). O2is also released from water in this process, and it is apparent that cycles of C and Ot must be inter-related. Transformations of carbon in the biosphere may be represented as follows : Heterotrophs Animals Micro-organisms Auto trophs Micro-organisms Plants Organic compounds Photosynthesis H20 O2 Mineralization Vol. 4 454 BIOCHEMICAL SOCIETY TRANSACTIONS As is well known, three general methods exist by which organic compounds are catabol ized . (a) Aerobic respiration. Here, the oxidation stages occur at the expense of O2 as the terminal electron acceptor; O2is also a molecular reactant of microbial oxygenase action. The energy produced comes mainly from oxidative phosphorylation, although the complexity of the electron-transport system varies among different micro-organisms. The availability of O2is often a limiting factor in mineralization. (b) Anaerobic respiration. Some prokaryotes are able to utilize certain inorganic electron acceptors, by a process confusingly called anaerobic respiration. Thus NO3- is reduced to NH3, N20 or N2, SO4’- is reduced to S2- and COz to methane. Since these inorganic electron acceptors have higher redox potentials than oxygen, less ATP will usually be produced compared with aerobic respiration. Although pathways of substrate degradation are usually identical in aerobic and anaerobic respiration, this is not the case for aromatic compounds. (c)Fermentation. Here, no external electron acceptor is required. Instead, the C source is metabolized by a series of reactions that release energy by substrate-level phosphorylation. There is a wide range of fermentation end-products; anaerobic growth is much less efficient energetically than aerobic respiration. In practice all naturally occurring carbon compounds are susceptible to attack in the biosphere by one or other of the above methods; that great reservoir, soil humus, has a definite, if slow, turn-over rate (Jenkinson, 1968). Elemental C, whether amorphous (carbon black) or crystalline (graphite, diamonds), however, is apparently not metabolized by living organisms. Anaerobic and aerobic environments exist in the biosphere, and biochemical degradative reactions occur in both; their relative magnitude is a matter for conjecture (Zobell, 1964). Although aerobic processes are of major importance in microbial catabolism, O2is also necessary to complete that part of the carbon cycle initiated under anaerobic conditions, e.g. CH4 + Organic c Methane-utilizing bacteria These aerobic pathways will receive prime consideration at this Symposium. Perhaps therefore it is opportune to draw attention to the anaerobic metabolism of aromatic substrates, even if only as a counterpoise. Certain of the photosynthetic bacteria Athiorhodaceae (e.g. Rhodopseudomonas palustris) photometabolize many aromatic compounds under anaerobic conditions by the reductive pathway shown in Scheme 2 for benzoate (Dutton & Evans, 1969; Guyer & Hegeman, 1969; P. J. Whittle & W. C. Evans, unpublished work). Thioesterification apparently allows the aromatic ring to be reduced to cyclohexanoyl-CoA; this intermediate then undergoes a B-oxidation sequence culminating in ring cleavage with the production of pimelate-diCoA ester, suitable for energy-yielding reactions by its subsequent metabolism. Pseudomonas sp. (PNl) and a Moraxe//a sp. isolated from soil by Taylor et a/. (1970) and Williams & Evans (1975) respectively, metabolize benzoate anaerobically by nitrate respiration. The latter organism employs a reductive pathway similar to that shown in Scheme 2 except that decarboxylation occurs and adipate is the ring-fission product. A ‘consortium’ of bacteria occur in anaerobic environments, e.g. rumen and intestinal contents, sewage sludge from anaerobic digesters etc., that, in the absence of 02,NO3‘ or produce methane and C 0 2 from benzoate according to the equation: 4CsHSC02H+ 18H20 -+ 15CH4+ 13C02 (Clark & Fina, 1952; Fina & Fiskin, 1960; Roberts, 1962; Nottingham & Hungate, 1969; Ferry, 1974). M. Balba & W. C. Evans (unpublished work) have recently detected 1976 562nd MEETING, BANGOR COA-SH “60 OC-S-COA HzO L -7-’ 455 @ OC-S-COA @ OC-S-COA 7-+ NAD+ 2WHz) FAD J OC--S--CoA I c OC-S-COA I OC-S-COA Metabolic pool Scheme 2. Anaerobic photometabolicpathway of benzoate b y Rhoabpseudomonaspalustris cyclohex-l-enecarboxylate,cyclohexanecarboxylate and adipate in the fermentation liquor metabolizing benzoate. Propionate and acetate were also identified, as reported previously by Roberts (1962) and Ferry (1 974) respectively. o-Chlorobenzoate inhibits benzoate utilization without affecting methane production from acetate (Ferry, 1974). Therefore it seems likely that the methanogenic fermentation of benzoate occurs by the co-operation of bacteria in the consortium that accomplish the reductive pathway of metabolism, affording aliphatic acids and eventually acetate for the methane bacteria to convert this to methane and COs. Clark, F. M.& Fina, L.R. (1952) Arch. Biochem. Biophys. 36, 26-32 Doetsch, R.N. & Cook, T. M.(1973) in Bacteria and their Ecobiology, pp. 294-295, University Park Press, Baltimore Dutton, P. L. & Evans, W. C. (1969) Biochem. J. 113, 525-536 Ferry, J. G. (1974) Ph.D. Thesis, University of Illinois Fina, L. R. & Fiskin, A. M. (1960) Arch. Biochem. Biophys. 91, 163-165 Guyer, M. & Hegeman, G. (1969) J. Eacleriol. 99, 906-907 Jenkinson, D. S. (1968) Biochem. J. 109, 2~ Nottingham, P. M.& Hungate, R. E. (1969) J. Bacteriol. 98, 1170-1172 Roberts, F. F. (1962) M.Sc. Thesis, Kansas State University Taylor, B. F., Campbell, W.L. & Chinoy, I. (1970) J. Bacferiof.102, 430-436 Williams, R.J. & Evans, W. C. (1975) Biochem. J. 148, 1-10 Zobell, C. (1964) in Proc. Ruablfs’ Research ConJ, Rutgers, Tie State Univ., New Brunswick, NJ, U.S.A.:PrinciplesandApplicationsin Aquatic Microbiology (Heukelekian, H. & Dondero, N. C., eds.), pp. 337-339, John Wiley and Sons,New York, London and Sydney Microbial Catabolism and the Carbon Cycle STANLEY DAGLEY Department of Biochemistry, University of Minnesota, St. Paul, MN 55108, U.S.A. It was possible for living forms to evolve in all their diversity when they became capable of harnessing energy released by the aerobic oxidation of biochemicals. These compounds were replenished by the reactions of photosynthesis that were initiated on this planet by Vol. 4