61st Annual Report on Research 2016

Microbial-mediated anaerobic oxidation of methane (AOM) is an important sink for methane, a potent greenhouse gas. A group of Archaeal ANerobic MEthanotrophs (ANME) facilitate oxidation and is hypothesized to proceed via the reversal of the methanogenesis biochemical pathway (Scheller et al., 2010; Zehnder & Brock 1979). Both natural isotope abundance studies and ¹³C-labeling experiments have provided insight into the biochemistry of methane oxidation and assimilation (House et al., 2009). In addition, prior studies indicate ANME are metabolically diverse and this study confirms they possess the enzymatic machinery to assimilate carbon via more than one pathway. This plasticity would explain the ~50‰ range in Archaeal lipids from ANME dominated sediment that has been observed in the Eel River basin (House et al., 2009, Orphan et al., 2001b).

 F430 is a tetrapyrrole used in the last step of methanogenesis, and is likely to enable the first step in reverse methanogenesis. The presence and concentration of F430 in association with AOM serves as a test for the reverse methanogenesis pathway in sediment. Tetrapyrrole and archaeol lipids are formed from different biological precursors (glutamate and acetyl-CoA, respectively) and could be constructed biochemically from different carbon substrates in the sedimentary environment. In the first part of this study, we used 13C-isotopic analysis of F430, and of archaeol lipids to link biochemical compounds to the assimilation of either methane (which is naturally 13C-depleted) or dissolved inorganic carbon (which is 13C-enriched). In the second part of the study, we quantify uptake of carbon substrates (CO2 or CH4) using stable isotope label experiments.

Biomarker and Natural Abundance Isotope Data In sediment from Hydrate Ridge and the Santa Monica Basin (California, Oregon, USA) a link between F430 and AOM was established based on the observation that peak amounts of F430 were recovered where sulfide sulfate and methane concentration profiles indicate the greatest AOM activity in the sediment. These sediment horizons also contained the highest ANME-2 aggregate counts. F430 was found to be isotopically distinct from methane and archaeal lipids, but similar to DIC.

While the molecular F430 abundance data from the sediment cores strengthens the evidence for ANME oxidizing methane via the reversal of methanogenesis, the isotopic signatures reflect a more complex biochemistry. F430 in both the Hydrate Ridge and Santa Monica Basin sediment was enriched in ¹³C relative to archaeal lipids. Isotopic mass balance calculations constrain the proportion of DIC and methane assimilation needed to account for the observed isotope values of F430 and lipids. Although the ability of ANME to assimilate more than one carbon substrate has been previously demonstrated by other authors, here we linked substrates to separate biochemical pathways.

Enrichment Experiments

The linkages between substrates and biosynthetic pathways were further explored with a series of stable isotope labels (13C, 15N) applied to enrichment experiments using sediment from Hydrate Ridge and the Santa Monica Basin. Newly produced coenzyme F430 was present in experiments with methane added to the headspace, and experiments lacking methane (designed to stimulate methanogenesis) did not produce measureable quantities of F430 or methane. This further strengthens the link between F430 in the sediment and its use in AOM.

In the Hydrate Ridge experiments we observe labeled bicarbonate assimilated into archaeol, with both labeled methane and bicarbonate assimilated into F430 and lipids in the Santa Monica Basin experiments. Using 15N ammonium, we determined the percentage of new F430, which then calculated the percentage of each 13C labeled substrate (either bicarbonate or methane) converted into the produced compound associated with new biomass. Approximately 20% of F430 was built from carbon assimilated as methane, and nearly 70% was derived from carbon assimilated from a dissolved form of CO2 as inorganic carbon. Lipid carbon was derived from similar proportions of methane and inorganic carbon, although the uncertainty in this estimate is greater.

In summary, the funding for this project has allowed us to: (1) develop analytical methods to isolate F430 for high precision isotopic analyses, (2) quantify F430 in marine sediment cores associated with anaerobic oxidation of methane (AOM), and (3) test the sources of carbon assimilated into AOM biomass using isotope labels. This work uniquely demonstrated linkages between the pathway for reverse methanogenesis, and estimated approximately 1:3 of the carbon in both F430 and lipid was derived from methane versus CO2.

This funding supported the PhD thesis research of Laurence Bird, who anticipates graduating from Penn State in December, 2016, and he will submit two manuscripts based on this work for publication.

Citations

HOUSE, C. H., ORPHAN, V. J., TURK, K. A., THOMAS, B., PERNTHALER, A., VRENTAS, J. M. & JOYE, S. B. 2009. Extensive carbon isotopic heterogeneity among methane seep microbiota. Environmental Microbiology, 11, 2207-2215.

KELLERMANN, M. Y., WEGENER, G., ELVERT, M., YOSHINAGA, M. Y., LIN, Y.-S., HOLLER, T., MOLLAR, X. P., KNITTEL, K. & HINRICHS, K.-U. 2012. Autotrophy as a predominant mode of carbon fixation in anaerobic methane-oxidizing microbial communities. Proceedings of the National Academy of Sciences, 109, 19321-19326.

LLOYD, K. G., LAPHAM, L. & TESKE, A. 2006. An Anaerobic Methane-Oxidizing Community of ANME-1b Archaea in Hypersaline Gulf of Mexico Sediments. Applied and Environmental Microbiology, 72, 7218-7230.

ORPHAN, V. J., HOUSE, C., HINRICHS, K.-U., MCKEEGAN, K. & DELONG, E. 2002. Multiple Archaeal Groups Mediate Methane Oxidation in Anoxic Cold Seep Sediments. P Natl Acad Sci Usa, 99, 7663-7668.

SCHELLER, S., GOENRICH, M., BOECHER, R., THAUER, R. K. & JAUN, B. 2010. The key nickel enzyme of methanogenesis catalyses the anaerobic oxidation of methane. Nature, 465, 606-608.

ZEHNDER, A. J. & BROCK, T. D. 1979. Methane formation and methane oxidation by methanogenic bacteria. J Bacteriol, 137, 420-32.