--> Realistic Rates of Biological Methane Production in Hydrate Bearing Sediments, by F.S. Colwell, S. Boyd, M.E. Delwiche, and D.R. Reed; #90035 (2004)

Datapages, Inc.Print this page

REALISTIC RATES OF BIOLOGICAL METHANE PRODUCTION IN HYDRATE BEARING SEDIMENTS

F. S. Colwell, S. Boyd, M. E. Delwiche, and D. R. Reed
Idaho National Engineering and Environmental Laboratory (INEEL), Idaho Falls, Idaho

Stable isotope data suggests that the methane in natural gas hydrates in marine sediments is largely biogenic, a product of the activity of methane-producing microorganisms called methanogens. Current models used to predict hydrate distribution and concentration in marine sediments lack accurate estimates of microbial methane production rates. This is because the activities of methanogens in subsurface environments are notoriously low and difficult to estimate. We are determining the methanogen biomass in sediments from Hydrate Ridge and the minimal rates of activity for a methanogen in laboratory experiments to better estimate the bulk methane production rates in hydrate-bearing sediments. A recently identified species of methanogen from deep marine sediments is being incubated in a bioreactor that simulates some of the in situ conditions of the hydrated sediments. We anticipate that bulk methane production rates derived from these experiments will more accurately represent in situ activities, be lower than previous estimates, and provide more accurate data for use in models describing the origin of the methane in natural hydrates.

We have developed a quantitative polymerase chain reaction (q-PCR) method in order to enumerate the biomass of methanogens in marine sediments. Degenerate primers were designed to target the methyl Co-M reductase gene (mc), a gene that is largely confined to methanogens. Twenty-six MCR subunit genes representing unique methanogen species were aligned using Clustal W to identify conserved nucleotide regions. These conserved regions were used to design primers targeting a 280 base pair amplicon of mc. The primers successfully amplified DNA from all five of the known methanogenic taxonomic orders but did not amplify DNA from closely related archaeal cells. Using known quantities of DNA from Methanocaldococcus jannaschii (a model methanogen) experiments suggested that mc-specific DNA can be detected and quantified at concentrations as low as approximately 100 methanogen cells.

To test the method with field samples, sediments collected from 1 to 277 meters below seafloor (mbsf) were obtained from six methane hydrate coring sites on Hydrate Ridge of the Cascadia Margin (Ocean Drilling Program Leg 204). After core recovery, samples were rapidly frozen and stored at -80°C prior to DNA extraction. Of the 48 Hydrate Ridge samples that have been evaluated approximately 25% showed detectable quantities of mc DNA using q-PCR, each sample yielding a value of approximately 100 to 1000 methanogens per g of sediment. Samples testing positive for methanogen presence came from within and outside the hydrate stability zone.

To determine the minimal rates of methanogenic activity for methanogens, we are using a biomass recycle reactor (4) operated in fed-batch mode and maintained within an anaerobic glove bag. Methanoculleus submarinus, a methanogen cultured from sediments collected 247 mbsf in the Nankai Trough, is being sustained in the reactor using carbon dioxide and hydrogen as the carbon and energy sources, respectively. After approximately 20 weeks M. submarinus biomass within the reactor became constant suggesting that the cells were no longer dividing. This condition, where microbial cells are using energy only to maintain their biomass without cell growth, may simulate microbial survival under subsurface conditions. Cells in this starved state were then used for short-term experiments to determine the rates at which they made methane. Preliminary studies indicted that these cells produce methane at a rate of approximately 0.06 fmol methane per cell per day. This is considerably lower than rates reported for methanogens in lake sediments (31.5 fmol methane per cell per day) (5) and anaerobic reactors (108.8-135.0 fmol methane per cell per day) (6) when a biomass recycle reactor was not used to starve the cells. This new value for specific (i.e., per cell) methane production rates when methanogens are starved is useful for calculating in situ rates of methanogenesis.

By combining our estimates of specific methanogenic rates (ca. 0.06 fmol methane per cell per day) and methanogenic numbers (<100-1000 methanogens per g sediment for most samples) we have derived a lower end estimate of approximately 6 x 10-6 nmol methane produced per g sediment per day for four of the Hydrate Ridge sediment samples. Previous determinations of the rate of methanogenesis in samples from hydrate-bearing sediments have yielded higher values using traditional microbiological methods of rate determination. For example, deep sediments within or near hydrates have yielded methane production rates of approximately100 to 103 (Blake Ridge; (10)), 10-3 to 10-2 (Cascadia Margin; (2)), 10-1 (Japan Sea; (1)), and 10-1 (Nankai Trough; (9)) nmol methane produced per g sediment per day. That this new approach has yielded methanogenic rates that are lower than previously measured is expected based on a general understanding that microbial activities in subsurface materials low (3, 7, 8).

We expect that additional experiments with cells starved in biomass recycle reactors can determine even lower rates of methanogenesis for use in models. The combined factors of in situ temperature, pressure, and substrate/product concentrations when controlled in the reactor may cause our estimates of maintenance level methane production rates to decrease further. Also, more accurate determinations of the numbers of methanogens and the locations of those cells in marine sediments will supplement existing data on the supply of biogenic methane along continental margins. These data will help to improve models intended to predict the location and distribution of hydrates in these systems.

References

1. Cragg, B. A., S. M. Harvey, J. C. Fry, R. A. Herbert, and R. J. Parkes, 1992. Bacterial biomass and activity in the deep sediment layers of the Japan Sea, Hole 798B-ODP Leg 128. Proc. ODP Sci. Results 127/128:761-775.

2. Cragg, B. A., R. J. Parkes, J. C. Fry, A. J. Weightman, P. A. Rochelle, and J. R. Maxwell, 1996. Bacterial populations and processes in sediments containing gas hydrates (ODP Leg 146: Cascadia Margin). Earth Planet. Sci. Lett. 139:497-507.

3. D'Hondt, S., S. Rutherford, and A. J. Spivak, 2002. Metabolic activity of subsurface life in deep-sea sediments. Science 295:2067-2070.

4. Konopka, A., 2000. Microbial physiological state at low growth rate in natural and engineered ecosystems. Curr. Opin. Microbiol. 3:244-247.

5. Lay, J.-J., T. Miyahara, and T. Noike, 1996. Methane release rate and methanogenic bacterial populations in lake sediments. Water Research 30:901-908.

6. Li, Y. Y., and T. Noike, 1992. Upgrading of anaerobic digestion of waste activated sludge by thermal pretreatment. Wat. Sci.Technol. 26:857-866.

7. Onstott, T. C., T. J. Phelps, T. Kieft, F. S. Colwell, D. L. Balkwill, J. K. Fredrickson, and F. Brockman, 1999. A global perspective on the microbial abundance and activity in the deep subsurface, p. 487-500. In J. Seckbach (ed.), Enigmatic microorganisms and life in extreme environments. Kluwer Academic Publishers, Netherlands.

8. Phelps, T. J., E. M. Murphy, S. M. Pfiffner, and D. C. White, 1994. Comparison between geochemical and biological estimates of subsurface microbial activities. Microb. Ecol. 28:335-349.

9. Reed, D. W., Y. Fujita, M. Delwiche, D. B. Blackwelder, P. P. Sheridan, T. Uchida, and F. Colwell, 2002. Microbial communities from methane hydrate-bearing deep marine sediments in a forearc basin. Appl. Environ. Microbiol. 68:3759-3770.

10. Wellsbury, P., K. Goodman, T. Barth, B. A. Cragg, S. P. Barnes, and R. J. Parkes, 1997. Deep marine biosphere fueled by increasing organic matter availability during burial and heating. Nature 388: 573-576.