Characterization of Microbial Community Structure in Gulf of Mexico Gas Hydrates: Comparative Analysis of DNA- and RNA-Derived Clone Libraries

Abstract

The characterization of microbial assemblages within solid gas hydrate, especially those that may be physiologically active under in situ hydrate conditions, is essential to gain a better understanding of the effects and contributions of microbial activities in Gulf of Mexico (GoM) hydrate ecosystems. In this study, the composition of the Bacteria and Archaea communities was determined by 16S rRNA phylogenetic analyses of clone libraries derived from RNA and DNA extracted from sediment-entrained hydrate (SEH) and interior hydrate (IH). The hydrate was recovered from an exposed mound located in the northern GoM continental slope with a hydrate chipper designed for use on the manned-submersible Johnson Sea Link (water depth, 550 m). Previous geochemical analyses indicated that there was increased metabolic activity in the SEH compared to the IH layer (B. N. Orcutt, A. Boetius, S. K. Lugo, I. R. Macdonald, V. A. Samarkin, and S. Joye, Chem. Geol. 205:239-251). Phylogenetic analysis of RNA-and DNA-derived clones indicated that there was greater diversity in the SEH libraries than in the IH libraries. A majority of the clones obtained from the metabolically active fraction of the microbial community were most closely related to putative sulfate-reducing bacteria and anaerobic methane-oxidizing archaea. Several novel bacterial and archaeal phylotypes for which there were no previously identified closely related cultured isolates were detected in the RNA-and DNA-derived clone libraries. This study was the first phylogenetic analysis of the metabolically active fraction of the microbial community extant in the distinct SEH and IH layers of GoM gas hydrate.Marine gas hydrates, which are ice-like crystalline solids, are composed of rigid water molecules with trapped gas molecules, primarily methane and other hydrocarbons. Gas hydrate reservoirs, which are distributed in the sediments of active and passive continental slope margins, as well as in terrestrial (i.e., permafrost) regions (38), are a proposed fossil fuel energy source (10). Additionally, the estimated global volume of submarine methane hydrates exceeds 10 16 m 3 (7, 10), highlighting the impact of hydrates on global carbon cycling, climate conditions, and seafloor stability (16,18,28,31,35). The formation of gas hydrates is dependent upon suitable gas, temperature, and pressure conditions (reviewed in reference 38). Geological and chemical conditions in the northern continental slope of the Gulf of Mexico (GoM) promote the formation of gas hydrates where seepage of hydrocarbon gases forms extensive surface-breaching mounds on the seafloor, as well as vast veinfilling hydrates in hemipelagic sediments (27).Geochemical characteristics, including gas composition and isotopic ratios of surface breaching hydrate, in the GoM have been well documented (19,33,34,38). Growth and dissolution of GoM hydrate mounds have also been observed, with changes in mound size and shape evident over a period of months (19). Such hydrate growth patterns increase fluid and solid (i.e....

Tracing carbon flow from microphytobenthos to major bacterial groups in an intertidal marine sediment by using an in situ 13C pulse‐chase method

“…The total RNA extracted from the sediment with the chloroform-phenol method (0 h sample) and captured 16S rRNA were reverse-transcribed with reverse primer DXR518 (Nogales et al 1999). The reverse transcripts were amplified by polymerase chain reaction (PCR) using primers 27F-DXR518 (Mills et al 2005;Martinez et al 2006). The PCR products were ligated into pGEM T-easy vector (Promega) and transformed into Escherichia coli JM109 competent cells following the manufacturer's instructions.…”

Evaluating rRNA as an indicator of microbial activity in environmental communities: limitations and uses

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Activity profiles for marine sponge-associated bacteria obtained by 16S rRNA vs 16S rRNA gene comparisons

“…Activity profiles for marine sponge-associated bacteria J Kamke et al Bacterial 16S rRNA gene vs 16S rRNA comparisons in sponges Earlier studies have successfully used the 16S rRNA gene vs rRNA approach to investigate the activity of, for example, marine plankton, sediment and gas hydrate communities (Moeseneder et al, , 2005Mills et al, 2005;Gentile et al, 2006;RodriguezBlanco et al, 2009 Agelas dilatata clone AD004, EF076125 Tethya aurantium clone TAA-10-101, AM259897…”

“…The analysis of 16S rRNA genes through clone libraries and fingerprinting approaches such as denaturing gradient gel electrophoresis (DGGE) has greatly extended our knowledge about the phylogenetic richness of sponge-associated bacteria and archaea (Webster et al, 2001(Webster et al, , 2004Hentschel et al, 2002;Taylor et al, 2004Taylor et al, , 2007bHolmes and Blanch, 2006;Longford et al, 2007;Schmitt et al, 2007Schmitt et al, , 2008Thiel et al, 2007;Mohamed et al, 2008b;Zhu et al, 2008). Researchers in other systems have taken this approach one step further, yielding insights into both richness and activity by comparing 16S rRNA gene-and 16S rRNA-derived sequences, respectively (Moeseneder et al, , 2005Winter et al, 2001;Troussellier et al, 2002;Mills et al, 2005;Gentile et al, 2006;Martinez et al, 2006;Brinkmann et al, 2008;McIlroy et al, 2008;West et al, 2008;Rodriguez-Blanco et al, 2009). In general, cellular concentrations of rRNA are correlated with growth rate and activity (DeLong et al, 1989;Poulsen et al, 1993), hence-with acknowledgement of certain caveats (e.g., for ammoniaoxidizing bacteria; Morgenroth et al, 2000)-the rRNA itself can yield useful information about which community members are active.…”