Bert Engelen

Microbial Diversity in Coastal Subsurface Sediments: a Cultivation Approach Using Various Electron Acceptors and Substrate Gradients

ABSTRACT Microbial communities in coastal subsurface sediments are scarcely investigated and have escaped attention so far. But since they are likely to play an important role in biogeochemical cycles, knowledge of their composition and ecological adaptations is important. Microbial communities in tidal sediments were investigated along the geochemical gradients from the surface down to a depth of 5.5 m. Most-probable-number (MPN) series were prepared with a variety of different carbon substrates, each at a low concentration, in combination with different electron acceptors such as iron and manganese oxides. These achieved remarkably high cultivation efficiencies (up to 23% of the total cell counts) along the upper 200 cm. In the deeper sediment layers, MPN counts dropped significantly. Parallel to the liquid enrichment cultures in the MPN series, gradient cultures with embedded sediment subcores were prepared as an additional enrichment approach. In total, 112 pure cultures were isolated; they could be grouped into 53 different operational taxonomic units (OTU). The isolates belonged to the Proteobacteria, “Bacteroidetes,” “Fusobacteria,” Actinobacteria, and “Firmicutes.” Each cultivation approach yielded a specific set of isolates that in general were restricted to this single isolation procedure. Analysis of the enrichment cultures by PCR and denaturing gradient gel electrophoresis revealed an even higher diversity in the primary enrichments that was only partially reflected by the culture collection. The majority of the isolates grew well under anoxic conditions, by fermentation, or by anaerobic respiration with nitrate, sulfate, ferrihydrite, or manganese oxides as electron acceptors.

Specific Bacterial, Archaeal, and Eukaryotic Communities in Tidal-Flat Sediments along a Vertical Profile of Several Meters

ABSTRACT The subsurface of a tidal-flat sediment was analyzed down to 360 cm in depth by molecular and geochemical methods. A community structure analysis of all three domains of life was performed using domain-specific PCR followed by denaturing gradient gel electrophoresis analysis and sequencing of characteristic bands. The sediment column comprised horizons easily distinguishable by lithology that were deposited in intertidal and salt marsh environments. The pore water profile was characterized by a subsurface sulfate peak at a depth of about 250 cm. Methane and sulfate profiles were opposed, showing increased methane concentrations in the sulfate-free layers. The availability of organic carbon appeared to have the most pronounced effect on the bacterial community composition in deeper sediment layers. In general, the bacterial community was dominated by fermenters and syntrophic bacteria. The depth distribution of methanogenic archaea correlated with the sulfate profile and could be explained by electron donor competition with sulfate-reducing bacteria. Sequences affiliated with the typically hydrogenotrophic Methanomicrobiales were present in sulfate-free layers. Archaea belonging to the Methanosarcinales that utilize noncompetitive substrates were found along the entire anoxic-sediment column. Primers targeting the eukaryotic 18S rRNA gene revealed the presence of a subset of archaeal sequences in the deeper part of the sediment cores. The phylogenetic distance to other archaeal sequences indicates that these organisms represent a new phylogenetic group, proposed as “tidal-flat cluster 1.” Eukarya were still detectable at 360 cm, even though their diversity decreased with depth. Most of the eukaryotic sequences were distantly related to those of grazers and deposit feeders.

Statistical comparisons of community catabolic profiles

Comparative catabolic profiling using microtiterplates with multiple sole-carbon-sources has become a popular tool for the comparison of microbial communities with respect to their functional potential. However, the statistical treatment of such data has only been partially satisfactory so far. In this paper, a new multivariate approach developed by Lauter and coworkers [J. Lauter, Exact T and F tests for analysing studies with multiple endpoints, Biometrics 52 (1996) 964–970; J. Lauter, E. Glimm, S. Kropf, New multivariate tests for data with an inherent structure, Biometrical Journal 38 (1996) 5–23.] is applied to this problem. Using replicate observations from every community in question, this approach allows testing for significant differences between communities. It is advantageous with respect to power and maintains the α-level. Discriminating carbon-sources are found according to weighted factor loadings and univariate tests. Exemplarily, changes in microbial soil communities due to rotting of plants are analyzed. Furthermore, other statistical approaches from the literature are discussed.

Phylogenetic and Physiological Diversity of Cultured Deep-Biosphere Bacteria from Equatorial Pacific Ocean and Peru Margin Sediments

During ODP Leg 201 microbial communities in Eastern Equatorial Pacific Ocean and Peru Margin sediments were investigated. The sediment layers sampled extended down to 420 m below the sea floor, with estimated ages of up to 40 million years. Contamination-free anoxic slurries were inoculated into media containing different substrate combinations, all at micromolar concentration. These culture media were designed for a broad spectrum of physiological groups. A total of 162 pure cultures were isolated that could be grouped into 19 different phylotypes based on 16S rRNA gene analysis. The isolates belonged to the Alpha-, Gamma- and Deltaproteobacteria, the Firmicutes, Actinobacteria, and Bacteroidetes. The genera most frequently isolated were Bacillus (68 isolates) and Rhizobium (40 isolates). Comparison of strains with the same phylotypes by enterobacterial repetitive intergenic consensus (ERIC-PCR) analysis revealed the presence of several subgroups that did not correlate with medium, sediment depth or sampling site. The majority of the isolates, although obtained from anoxic environments and isolated under strictly anoxic conditions, turned out to be facultativly aerobic. Physiologically, the isolates were characterized as generalists, able to utilize a broad variety of electron donors with either oxygen, nitrate and in some cases manganese oxides as electron acceptors. The diversity inferred from physiological tests was even higher than that on the phylogenetic or genomic level. The outcome of the contamination tests, the isolation of close relatives of already known subsurface bacteria, the repeated finding of the same phylotype from different sites and the level of diversity present in the culture collection strongly suggest that indigenous deep-biosphere bacteria had been isolated.

Fluids from the Oceanic Crust Support Microbial Activities within the Deep Biosphere

The importance of crustal fluid chemical composition in driving the marine deep subseafloor biosphere was examined in northeast Pacific ridge-flank sediments. At IODP Site U1301, sulfate from crustal fluids diffuses into overlying sediments, forming a transition zone where sulfate meets in situ-produced methane. Enhanced cell counts and metabolic activity suggest that sulfate stimulates microbial respiration, specifically anaerobic methane oxidation coupled to sulfate reduction. Cell counts and activity are also elevated in basement-near layers. Owing to the worldwide expansion of the crustal aquifer, we postulate that crustal fluids may fuel the marine deep subseafloor biosphere on a global scale.

Trends in Basalt and Sediment Core Contamination During IODP Expedition 301

Perfluorocarbon tracers (PFTs) are used during cruises of the Ocean Drilling Program (ODP) and Integrated Ocean Program (IODP) to measure sample contamination with drilling fluid. Drilling fluid is supplied with a constant PFT concentration that can then be detected and quantified in sediment and basalt core samples. During IODP Expedition 301, we used washing (2×) and flaming to effectively remove PFT from the exterior of basalt rocks. Near-complete removal from the exterior allowed us to demonstrate that the interior of basalts was only minutely, if at all, contaminated with drilling fluid. We examined horizontal and vertical trends in sediment core contamination. Contamination decreased greatly between the core exterior to halfway along the core radius, and slightly from halfway to the center of cores, and was generally very low in halfway and center portions. Clay cores were, on average, more contaminated than cores with fine sand. Contamination was typically highest in the two uppermost sections (sections 1 and 2) and lower below (sections 3–5). There was no relationship between depth of core origin and contamination. To determine mechanisms of contamination in halfway and interior parts of cores, we estimated the diffusive flux of PFT from the core liner towards the core center. Based on conservative estimates, we concluded that diffusion did not account for any of the PFT measured in halfway and interior parts of cores in this study. Any measurable PFT concentrations in halfway and center parts of cores were caused by advection.

Acetogens and Acetoclastic Methanosarcinales Govern Methane Formation in Abandoned Coal Mines

ABSTRACT In abandoned coal mines, methanogenic archaea are responsible for the production of substantial amounts of methane. The present study aimed to directly unravel the active methanogens mediating methane release as well as active bacteria potentially involved in the trophic network. Therefore, the stable-isotope-labeled precursors of methane, [13C]acetate and H2-13CO2, were fed to liquid cultures from hard coal and mine timber from a coal mine in Germany. Guided by methane production rates, samples for DNA stable-isotope probing (SIP) with subsequent quantitative PCR and denaturing gradient gel electrophoretic (DGGE) analyses were taken over 6 months. Surprisingly, the formation of [13C]methane was linked to acetoclastic methanogenesis in both the [13C]acetate- and the H2-13CO2-amended cultures of coal and timber. H2-13CO2 was used mainly by acetogens related to Pelobacter acetylenicus and Clostridium species. Active methanogens, closely affiliated with Methanosarcina barkeri, utilized the readily available acetate rather than the thermodynamically more favorable hydrogen. Thus, the methanogenic microbial community appears to be highly adapted to the low-H2 conditions found in coal mines.

Microbial community in black rust exposed to hot ridge flank crustal fluids.

ABSTRACT During Integrated Ocean Drilling Program Expedition 301, we obtained a sample of black rust from a circulation obviation retrofit kit (CORK) observatory at a borehole on the eastern flank of Juan de Fuca Ridge. Due to overpressure, the CORK had failed to seal the borehole. Hot fluids from oceanic crust had discharged to the overlying bottom seawater and resulted in the formation of black rust analogous to a hydrothermal chimney deposit. Both culture-dependent and culture-independent analyses indicated that the black-rust-associated community differed from communities reported from other microbial habitats, including hydrothermal vents at seafloor spreading centers, while it shared phylotypes with communities previously detected in crustal fluids from the same borehole. The most frequently retrieved sequences of bacterial and archaeal 16S rRNA genes were related to the genera Ammonifex and Methanothermococcus, respectively. Most phylotypes, including phylotypes previously detected in crustal fluids, were isolated in pure culture, and their metabolic traits were determined. Quantification of the dissimilatory sulfite reductase (dsrAB) genes, together with stable sulfur isotopic and electron microscopic analyses, strongly suggested the prevalence of sulfate reduction, potentially by the Ammonifex group of bacteria. Stable carbon isotopic analyses suggested that the bulk of the microbial community was trophically reliant upon photosynthesis-derived organic matter. This report provides important insights into the phylogenetic, physiological, and trophic characteristics of subseafloor microbial ecosystems in warm ridge flank crusts.

Microbial Methane Formation from Hard Coal and Timber in an Abandoned Coal Mine

About 7% of the global annual methane emissions originate from coal mining. Also, mine gas has come into focus of the power industry and is being used increasingly for heat and power production. In many coal deposits worldwide, stable carbon and hydrogen isotopic signatures of methane indicate a mixed thermogenic and biogenic origin. In this study, we have measured in an abandoned coal mine methane fluxes and isotopic signatures of methane and carbon dioxide, and collected samples for microbiological and phylogenetic investigations. Mine timber and hard coal showed an in-situ production of methane with isotopic signatures similar to those of the methane in the mine atmosphere. Enrichment cultures amended with mine timber or hard coal as sole carbon sources formed methane over a period of nine months. Predominantly, acetoclastic methanogenesis was stimulated in enrichments containing acetate or hydrogen/carbon dioxide. Molecular techniques revealed that the archaeal community in enrichment cultures and unamended samples was dominated by members of the Methanosarcinales. The combined geochemical and microbiological investigations identify microbial methanogenesis as a recent source of methane in abandoned coal mines.

High virus-to-cell ratios indicate ongoing production of viruses in deep subsurface sediments

Marine sediments cover two-thirds of our planet and harbor huge numbers of living prokaryotes. Long-term survival of indigenous microorganisms within the deep subsurface is still enigmatic, as sources of organic carbon are vanishingly small. To better understand controlling factors of microbial life, we have analyzed viral abundance within a comprehensive set of globally distributed subsurface sediments. Phages were detected by electron microscopy in deep (320 m below seafloor), ancient (∼14 Ma old) and the most oligotrophic subsurface sediments of the world’s oceans (South Pacific Gyre (SPG)). The numbers of viruses (104–109 cm−3, counted by epifluorescence microscopy) generally decreased with sediment depth, but always exceeded the total cell counts. The enormous numbers of viruses indicate their impact as a controlling factor for prokaryotic mortality in the marine deep biosphere. The virus-to-cell ratios increased in deeper and more oligotrophic layers, exhibiting values of up to 225 in the deep subsurface of the SPG. High numbers of phages might be due to absorption onto the sediment matrix and a diminished degradation by exoenzymes. However, even in the oldest sediments, microbial communities are capable of maintaining viral populations, indicating an ongoing viral production and thus, viruses provide an independent indicator for microbial life in the marine deep biosphere.