Microorganisms from Anaerobic, Gas Condensate-Contaminated Sediments that Degrade Acetate, Butyrate, and Propionate Under Methanogenic and Sulfate-reducing Conditions

Abstract

Acetate, butyrate, and propionate, are important intermediates that are produced as a result of the anaerobic degradation of organic matter in methanogenic and sulfate-reducing ecosystems. Despite the importance of these compounds in methanogenic and sulfate-reducing ecosystems, it is often unclear what populations of microorganisms are involved in the degradation of acetate, butyrate, and propionate. In order to understand the microbial populations involved, the anaerobic metabolism of these fatty acids was studied using sediments and groundwater from a gas condensate-contaminated aquifer near Denver, Colorado. This particular site was chosen for this study because previous work showed that methanogenesis and sulfate reduction were important terminal-electron accepting processes at this site.

Most probable number (MPN) dilutions with acetate indicated that there was no significant difference in the number of acetate degraders under methanogenic and sulfate-reducing conditions at this site. Acetate loss was coupled to methane production in all MPN dilution tubes regardless of whether sulfate was present or not. Higher quantities of 14CH4 than 14CO2 were observed in microcosms that contained either 14CH3COOH or 14CH314COOH in the presence or absence of sulfate. This 14CH4 accounted for 70-100% of the total labeled gas in these [14C] acetate microcosms regardless of whether sulfate was present or not. Denaturing gradient gel electrophoresis (DGGE) of the acetate microcosms both with and without sulfate using Archaea-specific primers showed that identical predominant bands, which had 99% sequence similarity to acetate-degrading methanogens from the family Methanosaetaceae, were present in all of these microcosms. Analysis of clone libraries of archaeal 16S rRNA gene sequences amplified from sediments collected in the contaminated portion of the aquifer showed that 180 of the 190 sequenced clones were similar to acetate-using methanogens from the family Methanosaetaceae.

The most probable number of syntrophic butyrate-degraders (MPNs that were amended with Methanospirillum hungatei or Desulfovibrio vulgaris strain G11) was similar to the number of sulfate-reducing, butyrate-degraders (MPNs with sulfate but without a hydrogen-user). Butyrate loss was coupled to methane production in butyrate-amended microcosms without sulfate, and to sulfate reduction in microcosms amended with butyrate and sulfate. The addition of 2-bromoethanesulfonic acid (BESA) inhibited butyrate degradation in methanogenic microcosms, which was restored upon the addition of a hydrogen-using sulfate reducer and 5 mM sulfate, but not when only 5 mM sulfate was added. The addition of carbon monoxide, which inhibits hydrogenases, to the headspace of sulfate-reducing microcosms inhibited butyrate metabolism and caused the hydrogen partial pressure to increase to levels that would make syntrophic butyrate degradation thermodynamically unfavorable (-5 to +3 kJ mol-1 of butyrate). Inhibition of butyrate metabolism was not observed in control microcosms with butyrate and sulfate that were amended with nitrogen gas. Approximately thirty percent of the 16S rRNA gene sequences in clone libraries from the MPN cultures grouped with members of the Syntrophomonadaceae. DGGE analysis of butyrate enrichments with sulfate detected an identical predominant band whose sequence was closely related to butyrate-degraders from the family Syntrophaceae. 16S rRNA sequences related to the Syntrophaceae were also present in clone libraries prepared from the contaminated sediment. 16S rRNA sequences related to Desulfovibrio accounted for 75% of the total number of sequences affiliated with sulfate reducers in clone libraries from MPN cultures.

Propionate was indirectly degraded to acetate and carbon dioxide in anoxic sediments and groundwater from a hydrocarbon-contaminated aquifer where geochemical evidence implicated sulfate reduction and methanogenesis as the predominant terminal electron-accepting processes. The most probable number of propionate-degraders from hydrocarbon-contaminated sediments was significantly higher (p >0.05) in cultures with propionate and sulfate that contained hydrogen-using microorganisms compared to cultures with propionate and sulfate but without the hydrogen-user added, suggesting that syntrophic propionate degraders were more numerous than sulfate-reducing propionate degraders. However, propionate degraders were not detected in MPNs that contained propionate and a hydrogen-using methanogen, but were not amended with sulfate. A new propionate-degrading, sulfate-reducing bacterium, with less than 96% sequence similarity to all described Desulfobulbus spp., was isolated from MPN enrichments that contained propionate and sulfate. Propionate loss by the pure culture and in microcosms with propionate and sulfate was coupled to sulfate loss and acetate accumulation. Acetate was converted to methane by aceticlastic methanogens in microcosms with propionate and sulfate. 16S rRNA gene sequences related to propionate-degrading, sulfate reducers from the genus Desulfobulbus were detected in all MPNs with sulfate, all propionate-degrading microcosms except those with molybdate added, and the contaminated sediments by using group specific PCR primers. Desulfobulbus sequences accounted for approximately four percent of the total 16S rRNA genes sequences in clone libraries prepared with DNA from the contaminated sediment. Sequences related to microorganisms capable of syntrophic propionate degradation were not detected in sediment clone libraries. This work shows that sulfate reduction was the dominant fate of propionate at this site and suggests that a new species of Desulfobulbus was involved in propionate degradation at this site.

The results of the work presented in this dissertation showed that aceticlastic methanogenesis and syntrophic metabolism can occur in sulfate-reducing ecosystems. These results are surprising since kinetic and thermodynamic comparisons of isolated species of aceticlastic methanogens, syntrophic microorganisms, and sulfate-reducing bacteria suggest that sulfate reducers should dominate in sulfate-reducing ecosystems. However, this kinetic and thermodynamic information is based on a relatively small number of isolates. The results of this study suggest that this kinetic and thermodynamic information cannot always be used to predict what microorganisms are involved in the degradation of acetate, butyrate, and propionate in contaminated aquifers.