We devised a microbial culture apparatus capable of maintaining sub-nanomolar H2 concentrations. This apparatus provides a method for study of interspecies hydrogen transfer by externally fulfilling the thermodynamic requirement for low H2 concentrations, thereby obviating the need for use of cocultures to study some forms of metabolism. The culture vessel is constructed of glass and operates by sparging a liquid culture with purified gases, thereby removing H2 as it is produced. We used the culture apparatus to decouple a syntrophic association in an ethanol-consuming, methanogenic enrichment culture, allowing ethanol oxidation to dominate methane production. We also used the culture apparatus to grow pure cultures of the ethanol-oxidizing, proton-reducing Pelobacter acetylenicus (WoAcy 1), and to study the bioenergetics of growth.

Hydrogen production was studied in four species of methanogens (Methanothermobacter marburgensis, Methanosaeta thermophila, Methanosarcina barkeri, and Methanosaeta concilii) under conditions of low (sub-nanomolar) ambient hydrogen concentration using a specially designed culture apparatus. Transient hydrogen production was observed and quantified for each species studied. Methane was excluded as the electron source, as was all organic material added during growth of the cultures (acetate, yeast extract, peptone). Hydrogen production showed a strong temperature dependence, and production ceased at temperatures below the growth range of the organisms. Addition of polysulfides to the cultures greatly decreased hydrogen production. The addition of bromoethanesulfonic acid had little influence on hydrogen production. These experiments demonstrate that some methanogens produce excess reducing equivalents during growth and convert them to hydrogen when the ambient hydrogen concentration becomes low. The lack of sustained hydrogen production by the cultures in the presence of methane provides evidence against "reverse methanogenesis" as the mechanism for anaerobic methane oxidation.

The role of methane clathrate hydrates in the global methane budget is poorly understood because little is known about how much methane from decomposing hydrates actually reaches the atmosphere. In an attempt to quantify the role of water column methanotrophy (microbial methane oxidation) as a control on methane release, we measured water column methane profiles (concentration and δ13C) and oxidation rates at eight stations in an area of active methane venting in the Eel River Basin, off the coast of northern California. The oxidation rate measurements were made with tracer additions of 3H-CH4. Small numbers of instantaneous rate measurements are difficult to interpret in a dynamic, advecting coastal environment, but combined with the concentration and stable isotope measurements, they do offer insights into the importance of methanotrophy as a control on methane release. Fractional oxidation rates ranged from 0.2 to 0.4% of ambient methane per day in the deep water (depths >370 m), where methane concentration was high (20-300 nM), to near-undetectable rates in the upper portion of the water column (depths <370 m), where methane concentration was low (3-10 nM). Methane turnover time averaged 1.5 yr in the deep water but was on the order of decades in the upper portion of the water column. The depth-integrated water column methane oxidation rates for the deep water averaged 5.2 mmol CH4 m-2 yr-1, whereas the upper portion of the water column averaged only 0.14 mmol CH4 m-2 yr-1; the depth-integrated oxidation rate for deep water in the 25-km2 area encompassing the venting field averaged 2 x 106 g CH4 yr-1. Stable isotope values (δ13C-CH4) for individual samples ranged from -34 to -52‰ (vs. PDB, Peedee belemnite standard) in the region. These values are isotopically enriched relative to hydrates in the region (δ13C-CH4 about -57 to -69‰), further supporting our observations of extensive methane oxidation in this environment. Copyright © 2001 Elsevier Science Ltd.