Environ. Sci. Technol. 1982, 16, 300-301
Microorganisms Responsible for the Oxidation of Carbon Monoxide in Soil Gene W. Bartholomew and Martln Alexander* Laboratory of Soil Microbiology, Department of Agronomy, Cornell University, Ithaca, New York 14853
The oxidation of CO by Williamson silt loam does not result in increases in numbers of CO oxidizers, enhancement of C02fixation, microbial assimilation of carbon from CO, or enhancement of the oxidation of subsequent increments of CO. The likeliest explanation for these findings is that CO oxidation in this soil is a cometabolic process and not one effected by autotrophs or heterotrophs using CO as a carbon source. W
Introduction Soils have the capacity to destroy CO, and because this activity is abolished if samples of soil are sterilized (1,2), the process is apparently microbial. Several microorganisms in axenic culture are able to bring about CO oxidation (3,4),but the identities of the microorganisms responsible for the process in nature are unknown. The responsible populations may be chemoautotrophic because the conversion of CO to COBis an exergonic reaction, and autotrophic bacteria using this oxidation to provide energy for growth have been isolated (5,6). However, the transformation could also be brought about by heterotrophs using CO as a carbon source or acting on it cometabolically. This study was designed to determine which types of microorganisms are responsible for CO oxidation in soil. Methods Labeled CO was prepared by dehydrating H14COONa (New England Nuclear Corp., Boston MA, specific activity 58 mCi mmol-l) with concentrated H2S04( 4 ) . Unlabeled CO was prepared similarly using H12COONa. Labeled C02 was prepared by oxidizing [U-14C]glucose (California Bionuclear, Sun Valley, CA., specific activity 240 mCi mmol-') with a concentrated HZSO4/85% H3P04 (6:4) solution saturated with K2Cr20,. The microbial oxidation of 14C0was measured by trapping the 14C02formed in the reaction and subsequently measuring its radioactivity. Fixation of l4COZby soil suspensions was measured by the addition of 2 N H2S04to the soil suspension after the test period and subsequent direct counting of radioactivity in the suspension. Details of these methods have been previously published (1). For the determination of numbers of CO oxidizers in Williamson silt loam (pH 5.8, 2.6% organic matter, and moisture content of 20%), the five-tube most-probablenumber (MPN) technique (7) was used. Serial dilutions were made of soil samples, and 25-mL Erlenmeyer flasks (in place of the tubes usually used in MPN counts) containing 4.0 g of soil sterilized by autoclaving were inoculated with these dilutions. After 1 week of incubation, during which time the flasks were amended twice with 0.5 pL of CO, each of the samples was assayed for the ability to oxidize CO as previously mentioned. The incubation period allowed for growth of CO oxidizers, thus permitting easy assessment of which flasks contained such organisms. Results and Discussion It is experimentally possible to distinguish the three categories of organisms that may bring about CO oxidation in soil: (i) A heterotrophic population using CO as a C source will incorporate some of the carbon into cellular material, its size will increase as CO is being metabolized, 300
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and the rate of CO oxidation in a soil containing these as the dominant CO-oxidizingorganisms will be greater if the soil was previously exposed to the gas than if not so exposed. (ii) If chemoautotrophs were responsible, CO oxidation would be paralleled by a rise in numbers of CO oxidizers, and second increments would be metabolized more readily than the first. In this instance, however, carbon from CO would not be incorporated into cells, but concomitant with CO oxidation would be the fixation of COP,which is the carbon source for the autotrophs. (iii) If the active organisms were oxidizing CO by cometabolism, carbon from CO should not appear in the cells, CO oxidation would not promote COz fixation or population increases, and prior exposure of a soil containing such organisms should not lead to an enhanced rate of transformation of subsequent increments of CO. These several characteristics were examined experimentally to determine the type of reaction occurring in Williamson silt loam. In our previous investigation of CO transformation in soil, CO was found to be rapidly oxidized to COz aerobically or anaerobically, but the amount of carbon from CO that was incorporated into soil organic matter, which includes the microbial cell carbon, was several orders of magnitude less than that converted to C02 ( I ) . Thus, heterotrophs using the gas as a carbon source for growth are not important in the soil tested. To determine if incubation of soil with CO increased its population of CO oxidizers, 1.0-mL portions of a 10% suspension of Williamson silt loam in 122-mL milk dilution bottles were amended with 2.44 pL of l2C0 L-l on 8 separate days. Identical suspensions received no CO. During this incubation period, which was sufficient for all the added CO to be oxidized, the amended suspensions were exposed to sufficient CO to result in the fixation of 350 ng of CO, carbon, assuming that the ratio of oxidized CO to fixed C02 carbon was 30:l (6) and that the oxidation is growth linked. This amount of fixed carbon is sufficient to increase the population of CO oxidizers IO-fold. The numbers of CO oxidizers were the same (4.1 X lo58-l of soil) in the amended and unamended soils after incubation, indicating that no growth of CO oxidizers had occurred despite the presence of the substrate. One might argue that the 8-day incubation period was too short for proliferation of slow-growingautotrophs, yet it seems likely that slow-growing organisms would not participate in the destruction of a substrate that is metabolized as rapidly as has been shown for CO (1)if the slow-growing bacteria used that substrate as a source of energy. Such autotrophs may indeed have been present in the test soil and they are known to be widespread ( 6 ) , but ubiquity does not denote abundance or even activity in a particular ecosystem. The incorporation of 14C02into organic matter was not enhanced when the soil was metabolizing CO as compared to soil not exposed to CO (1). On the assumption that autotrophs might require high CO levels for replication, 5.0 mL of a soil suspension (10% soil in water) was exposed to a much wider range of CO levels than previously used; 14C02was also present. Immediately before addition of l2C0 and 14C02,the suspension was bubbled for 4 h with C02-free air to reduce the amount of 12C02in the flasks. The samples were then incubated for 24 h in the presence
0013-936X/82/0916-0300$01.25/0
0 1982 American Chemical Society
Table I. Incorporation of I4CO, into Organic Matter by Soil Suspensions Exposed to Various Concentrations of W O radioactivity in organic matter, cpm CO level, pL L-I 0 841 (190)“ 100 2800 (2000) 500 1000 (180) 1000 1000 (90) 10000 920 (150) a Numbers reported are means of at least three replicates. Values in parentheses represent one standard deviation.
of 14C02and varying amounts of l2C0. The incorporation of 14C02into microbial cells was determined by measuring the radioactivity in the suspension after acidification. The data show that C02 fixation into microbial cells (which would be in the soil organic fraction) was not significantly different in Williamson silt loam whether CO was present or not (Table I). These results confirm that CO oxidation in soil is apparently not an autotrophic process. It is possible in the previous experiment that 12C02was formed from the added l2C0 so that a CO-dependent COz fixation could not be demonstrated by the methods used. However, before the level of 12C02generated from the added l2C0became high enough to dilute the radioactivity of the added 14C02,sufficient 14C02should have been fixed were autotrophs involved in the oxidation. Thus, in effect, the 12C02from l2C0 oxidation appears too late to dilute the 14C02to a significant extent (the 14C02level was about 2 ppm or lo6 dpm). One might also argue that the local concentration of CO in soil is much higher than in the atmosphere, the putative autotrophs growing by using the higher levels of CO in situ than were used in this study. However, Conrad and Seiler (8) showed that CO consumption exceeds CO production in soil, so that the existence of locally high CO concentrations seems likely. Their data also suggest that both eucaryotes and procaryotes are involved in CO oxidation. Williamson silt loam was preincubated with various levels of CO to determine if prior exposure of soil to the gas would increase the subsequent rate of CO oxidation. Portions (5.0g) of soil incubated in 25-mL flasks at 22 OC received various amounts of l2C0on 8 separate days. After a total incubation period of 14 days, all the samples were flushed with air and received 14C0to a final concentration of 20 pL L-l, and the soils were incubated for 1 h, after which the amount of 14C02formed from 14C0was determined. The results in Table I1 show that the quantities of CO oxidized were not influenced by preincubation with any of these CO concentrations. Thus, a population does not appear to be responding, presumably by growth, to prior exposures to CO. A possibility exists that the responsible organisms are mixotrophs that use organic matter of the soil as a source of carbon and derive energy by oxidizing CO. However, inasmuch as soil organic matter was present, one might expect that such organisms would have replicated when their energy source was provided and that their activity would have increased by prior exposure of the soil sample to CO, but an increase in number was not found nor was the rate of CO oxidation enhanced by prior exposure to the gas.
Table 11. Effect of Preincubation with ‘,CO on Rate of I4CO Oxidation CO added, pL “TO, formed, L-I cpm x IO3
0 93 (6)“ 20 97 (5) 40 96 (6) 60 99 (7) 100 97 (4) a Numbers reported are means of at least three replicates. Values in parentheses represent one standard deviation.
The lack of increase in abundance of CO oxidizers and in rates of metabolism of second increments of CO could result from an attack on the responsible species by protozoa or by lytic microorganisms. However, the steadystate populations of prey of some, although not all, species coexisting with protozoa are much higher than those observed here for CO oxidizers (9),and it would be a remarkable coincidence for lysis and protozoan predation to reduce the population precisely back to the level in the soil not supplemented with CO. The likeliest explanation for these findings is that CO oxidation in Williamson silt loam is cometabolic. In contrast, Spratt and Hubbard (IO)concluded that autotrophic microorganisms were responsible for CO oxidation in a few soils near a road. The contrasting results may indicate that the types of microorganisms oxidizing CO vary with the soil type and environmental conditions. However, the unnaturally high levels of CO (200 pL L-l) used by Spratt and Hubbard (IO)in the enrichments prior to their determination of the type of oxidation may have stimulated autotrophic organisms or artificially selected for them. Neverthelkss, the organisms involved in the reaction and the contribution they make to the global CO cycle are unknown.
Literature Cited Bartholomew, G. W.; Alexander, M. Appl. Environ. Microbiol. 1979,37,932-937. Inman, R. E.; Ingersoll, R. B.; Levy, E. A. Science (Washington, D.C.) 1971,172,1229-1231. Ferenci, T.;Stram, T.;Quayle, J. R. J . Gen. Microbiol. 1975, 91,79-91. Fuchs, G.; Schnitker, U.; Thauer, R. K. Eur. J . Biochem. 1974,49,111-115. Meyer, 0.; Schlegel, H. G. J . Bucteriol. 1979,137,811-817. Zavarzin, G. A.; Nozhevnikova, A. N. Microb. Ecol. 1977, 3,305-326. “Standard methods for the examination of water and wastewater”;American Public Health Association: Washington, D. C., 1975. Conrad, R.; Seiler, W. Appl. Environ. Microbiol. 1980,40, 437-445. Habte, M.; Alexander, M. Arch. Microbiol. 1977, 113, 181-183. Spratt, H. G.;Hubbard, J. S. Appl. Environ. Microbiol. 1981,41,1192-1201. Received for review March 6,1981.Revised manuscript received December 21,1981. Accepted January 29,1981. This study was supported by Public Health Service training Grant E807052from the Division of Environmental Health Sciences.
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