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Collection and Microbiological Analysis of Gas Hydrate Cores
Project Number
FWP-4340-60 AND FWP-42C1-01

Determine the presence and activity of methanogens in methane hydrate-bearing sediments.


Idaho National Engineering and Environmental Laboratory (Ineel) – Sample Collection and Analysis


Idaho Falls, ID 83415

Demonstration projects are located in Japan, offshore Oregon, Mackenzie Delta, NWT, Canada, and the Gulf of Mexico. The Mallik Project is on Richards Island in the Beaufort Sea offshore Canada.


The project was set up to determine a fundamental modeling parameter — the amount of methane generated in deep sediments by methanogenic microorganisms. This would allow methane distribution models of gas hydrate reservoirs to accurately reflect an unknown volume and the distribution of biogenic methane within in a reservoir. The personnel at INEL have experience in similar biologic research and are considered to be experts by their global peers.


The testing for biological activity in the sediments associated with gas hydrate is supported by the international methane research community. This project will provide better understanding of the source rocks, migration and entrapment of gas in the hydrate reservoirs, and aid in future E & P activities by industry. Results to date indicate that most of the gas trapped in the permafrost hydrate bearing sediments is thermogenic and has migrated up-structure through faults into a hydrate stability zone. It is expected that similar geological settings would be the targets for industry in the future.


  • Devised a sampling procedure for Mallik 5L-38 core sediments that would enable microbiological properties to be evaluated.
  • Collected microbiology core samples from the Mallik 5L-38 (23 cores; February, 2002; for five research teams) and Ocean Drilling Program Leg 204 (63 cores; July-September, 2002 at Hydrate Ridge) boreholes,
  • Developed primers and probes to allow detection and quantification of methanogens using real time polymerase chain reaction (PCR),
  • Identified lack of evidence of methanogens in Mallik 5L-38 samples, and
  • Identified molecular evidence of methanogens in Leg 204 corehole.

A sampling procedure was devised for the Mallik 5L-38 research well core sediments that would enable microbiological properties to be evaluated. This procedure was developed under this project and then iterated with microbiologists, molecular biologists, and organic chemists from India, Germany, and Japan who would receive the samples.

Methanogen biomass recycle  reactor (BRR) Determines  energy requirements for  methanogens Courtesy INEEL
Methanogen biomass recycle 
reactor (BRR) Determines 
energy requirements for 
Courtesy INEEL

Primers and probes were developed to allow detection and quantification of methanogens using real time polymerase chain reaction (PCR). These molecular primers and probes are specific to the methanogenic methyl CoM reductase (MCR) gene that is integral to methanogenic metabolism of C-1 compounds. The MCR gene is broadly believed to be present in all methanogens and its sequence is highly conserved.

Both direct polymerase chain reaction (PCR) amplification and quantitative, real time PCR of DNA extracted from the Mallik well sediments failed to show evidence of methanogenic DNA. These results are consistent with an absence of biogenic methane production in enrichments (data from T. Phelps, ORNL) and an absence of known methanogen 16S rDNA sequences when DNA is extracted from the sediments, amplified, cloned, and sequenced (data from T. Nunoura, JAMSTEC). These data contrast with methanogenic coenzyme M (CoM) measurements on Mallik 5L-38 samples that suggest as many as 105 methanogens per g of sediment in some samples. Taken together the evaluations suggest that Mallik samples contain exceedingly low levels of methanogens.

In contrast to Mallik sediments, some of the samples from Leg 204 show molecular evidence of methanogenic DNA. Samples from Leg 204 corehole 1245 at 16 m below the seafloor (8 m below the sulfate-methane interface) contain methanogenic 16S rDNA when nested PCR is used to detect these cells.

Researchers completed the methanogen biomass estimates for samples collected from Mallik 5L-38 and from Hydrate Ridge using real time quantitative PCR. Catabolic rate experiments were completed using a biomass recycle reactor to grow Methanoculleus submarinus under simulated in situ conditions. This new species of methanogen is the first to be isolated from sediments containing hydrates. Subsequently, the combined sediment biomass and catabolic rate data were used to generate numerical rate expressions for biogenic methane production in hydrate sediments. These rate expressions can be used to model methane hydrate distribution and production.

Current Status

Work on this project has been completed.

Project Start
Project End
DOE Contribution

$30,000 (FWP-4340-60)   $300,000 (FWP-42C1-01)

Performer Contribution


Contact Information

NETL – Thomas Mroz ( or 304-285-4071)
INEEL – Rick Colwell ( or 208-526-0097)

Pertinent Publications
Colwell, F., M.E. Delwiche, D. Blackwelder, M. Wilson, R.M. Lehman, and T. Uchida. 1999. Microbial communities from core intervals, JAPEX/JNOC/GSC Mallik 2L-38 gas hydrate research well, pp. 189-195. In (S.R. Dallimore, T. Uchida, and T.S. Collett, eds.), Scientific Results from JAPEX/JNOC/GSC Mallik 2L-38 Gas Hydrate Research Well, Mackenzie Delta, Northwest Territories, Canada, Geological Survey of Canada, Bulletin 544.

Colwell, F., M. Delwiche, D. Blackwelder, R. Cherry, J. Mikucki, Y. Liu, D.R. Boone, and T. Uchida. 2002. Evidence of broad thermal tolerance of methanogens in sediments containing gas hydrates, pp 19-24. Proceedings of the 4th International Conference on Gas Hydrates. Yokohama, Japan.

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.

Mikucki, J.A., Y. Liu, M. Delwiche, F.S. Colwell, and D.R. Boone. 2003. Isolation of a methanogen from deep marine sediments that contain methane hydrates, and description of Methanoculleus submarinus sp. nov. Appl. Environ. Microbiol. 69: 3311-3316.

Colwell, F.S. and R. P. Smith. Unifying principles of the deep terrestrial and deep marine biospheres. Amer. Geophys. Union Monograph - Subseafloor Biosphere at Mid-Ocean Ridges. In press.

Colwell, F., R. Matsumoto, and D.W. Reed. Gas hydrates and the geology and biology of the Nankai Trough. Chem. Geol. In revision.

Nunoura, T., F.S. Colwell, S. Boyd, M.E. Delwiche, R.M. Lehman, K. Takai, K. Horikoshi, T. J. Phelps. Molecular biology and microbiology of the core samples from the Mallik 5L-38 gas hydrate research well. In preparation.