Diverse microbial communities and numerous energy-yielding activities occur in deeply buried sediments of the eastern Pacific Ocean. Distributions of metabolic activities often deviate from the standard model. Rates of activities, cell concentrations, and populations of cultured bacteria vary consistently from one subseafloor environment to another. Net rates of major activities principally rely on electron acceptors and electron donors from the photosynthetic surface world. At open-ocean sites, nitrate and oxygen are supplied to the deepest sedimentary communities through the underlying basaltic aquifer. In turn, these sedimentary communities may supply dissolved electron donors and nutrients to the underlying crustal biosphere.
Summary Eastern Mediterranean sediments are characterized by the occurrence of distinct, organic‐rich layers, called sapropels. These harbour elevated microbial numbers in comparison with adjacent carbon‐lean intermediate layers. A recently obtained culture collection from these sediments was composed of 20% of strains closely related to Rhizobium radiobacter , formerly classified as Agrobacterium tumefaciens . To prove and quantify the in situ abundance of R. radiobacter , a highly specific quantitative polymerase chain reaction (PCR) protocol was developed. To convert quantification results into cell numbers, the copy number of rrn operons per genome was determined. Southern hybridization showed that our isolates contained four operons. Finally, quantitative PCR was applied to 45 sediment samples obtained across the eastern Mediterranean. Rhizobium radiobacter was present in 38 of 45 samples indicating an almost ubiquitous distribution. In total, 25–40 000 cells per gram of sediment were detected, corresponding to 0.001–5.1% of the bacterial cells. In general, the relative and absolute abundance of R. radiobacter increased with depth and was higher in sapropels than in intermediate layers. This indicates that R. radiobacter forms an active population in up to 200 000 years old sapropels. The present study shows for the first time that a cultivated subsurface bacterium is highly abundant in this environment.
Vertical distribution of sulfate-reducing bacteria (SRB) and sulfate reduction rates were investigated in littoral and profundal sediments of the oligotrophic Lake Stechlin. SRB counted by the most probable number (MPN) technique showed two peaks, one at the oxic-anoxic interface, and the second deeper in the reduced sediment layer. Sulfate reduction rates determined as potential rates in anoxic sediment slurries showed a similar vertical distribution with the highest values in the anoxic zones. The highest cell numbers observed were 1.3×105 cells cm−3 with lactate as substrate. SRB were also found in oxic sediment layers in numbers similar to those detected in anoxic zones. From the highest positive MPN tubes 34 pure cultures were isolated. Physiological characterization including nutritional behaviour, tests for desulfoviridin and spore formation turned out that nearly all isolates belonged to the genera Desulfovibrio, Desulfomicrobium, and Desulfotomaculum. SRB from the oxic sediment layers revealed a higher oxygen tolerance and capacity of oxygen respiration than isolates from the anoxic sediment layers. However, no sulfate reduction was observed in the presence of oxygen, since oxygen was preferentially reduced.
Bacterial spores are widespread in marine sediments, including those of thermophilic, sulphate-reducing bacteria, which have a high minimum growth temperature making it unlikely that they grow in situ. These Desulfotomaculum spp. are thought to be from hot environments and are distributed by ocean currents. Their cells and spores upper temperature limit for survival is unknown, as is whether they can survive repeated high-temperature exposure that might occur in hydrothermal systems. This was investigated by incubating estuarine sediments significantly above (40-80 °C) maximum in situ temperatures (∼ 23 °C), and with and without prior triple autoclaving. Sulphate reduction occurred at 40-60 °C and at 60 °C was unaffected by autoclaving. Desulfotomaculum sp. C1A60 was isolated and was most closely related to the thermophilic D. kuznetsovii(T) (∼ 96% 16S rRNA gene sequence identity). Cultures of Desulfotomaculum sp. C1A60, D. kuznetsovii(T)and D. geothermicum B2T survived triple autoclaving while other related Desulfotomaculum spp. did not, although they did survive pasteurisation. Desulfotomaculum sp. C1A60 and D. kuznetsovii cultures also survived more extreme autoclaving (C1A60, 130 °C for 15 min; D. kuznetsovii, 135 °C for 15 min, maximum of 154 °C reached) and high-temperature conditions in an oil bath (C1A60, 130° for 30 min, D. kuznetsovii 140 °C for 15 min). Desulfotomaculum sp. C1A60 with either spores or predominantly vegetative cells demonstrated that surviving triple autoclaving was due to spores. Spores also had very high culturability compared with vegetative cells (∼ 30 × higher). Combined extreme temperature survival and high culturability of some thermophilic Desulfotomaculum spp. make them very effective colonisers of hot environments, which is consistent with their presence in subsurface geothermal waters and petroleum reservoirs.
Growth and chemotactic behavior in oxic-anoxic gradients were studied with two freshwater and four marine strains of sulfate-reducing bacteria related to the genera Desulfovibrio, Desulfomicrobium or Desulfobulbus. Cells were grown in oxygen-sulfide counter-gradients within tubes filled with agar-solidified medium. The immobilized cells grew mainly in the anoxic zone, revealing a peak below the oxic-anoxic interface. All tested strains survived exposure to air for 8 h and all were capable of oxygen reduction with lactate. Most strains also oxidized sulfide with oxygen. Desulfovibrio desulfuricans responded chemotactically to lactate, nitrate, sulfate and thiosulfate, and even sulfide functioned as an attractant. In oxic-anoxic gradients the bacteria moved away from high oxygen concentrations and formed bands at the outer edge of the oxic zone at low oxygen concentration (<5% O2 saturation). They were able to actively change the extension and slope of the gradients by oxygen reduction with lactate or even sulfide as electron donor. Generally, the chemotactic behavior was in agreement with a defense strategy that re-establishes anoxic conditions, thus promoting anaerobic growth and, in a natural community, fermentative production of the preferred electron donors of the sulfate-reducing bacteria.
Deep subseafloor sediments may contain depressurization-sensitive, anaerobic, piezophilic prokaryotes. To test this we developed the DeepIsoBUG system, which when coupled with the HYACINTH pressure-retaining drilling and core storage system and the PRESS core cutting and processing system, enables deep sediments to be handled without depressurization (up to 25 MPa) and anaerobic prokaryotic enrichments and isolation to be conducted up to 100 MPa. Here, we describe the system and its first use with subsurface gas hydrate sediments from the Indian Continental Shelf, Cascadia Margin and Gulf of Mexico. Generally, highest cell concentrations in enrichments occurred close to in situ pressures (14 MPa) in a variety of media, although growth continued up to at least 80 MPa. Predominant sequences in enrichments were Carnobacterium, Clostridium, Marinilactibacillus and Pseudomonas, plus Acetobacterium and Bacteroidetes in Indian samples, largely independent of media and pressures. Related 16S rRNA gene sequences for all of these Bacteria have been detected in deep, subsurface environments, although isolated strains were piezotolerant, being able to grow at atmospheric pressure. Only the Clostridium and Acetobacterium were obligate anaerobes. No Archaea were enriched. It may be that these sediment samples were not deep enough (total depth 1126-1527 m) to obtain obligate piezophiles.
Summary Microbial populations exist to great depths on Earth, but with apparently insufficient energy supply. Earthquake rock fracturing produces H 2 from mechanochemical water splitting, however, microbial utilization of this widespread potential energy source has not been directly demonstrated. Here, we show experimentally that mechanochemically generated H 2 from granite can be directly, long‐term, utilized by a CH 4 producing microbial community. This is consistent with CH 4 formation in subsurface rock fracturing in the environment. Our results not only support water splitting H 2 generation as a potential deep biosphere energy source, but as an oxidant must also be produced, they suggest that there is also a respiratory oxidant supply in the subsurface which is independent of photosynthesis. This may explain the widespread distribution of facultative aerobes in subsurface environments. A range of common rocks were shown to produce mechanochemical H 2 , and hence, this process should be widespread in the subsurface, with the potential for considerable mineral fuelled CH 4 production.
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