Effect of carbon addition and predation on acetate-assimilating bacterial cells in groundwater

Groundwater microbial community dynamics are poorly understood due to the challenges associated with accessing subsurface environments. In particular, microbial interactions and their impact on the subsurface carbon cycle remain unclear. In the present project, stable isotope probing with uniformly labeled [¹³C]-acetate was used to identify metabolically active and inactive bacterial populations based on their ability to assimilate acetate and/or its metabolites. Furthermore, we assessed whether substrate availability (bottom–up control) or grazing mortality (top–down control) played a greater role in shaping bacterial community composition by separately manipulating the organic carbon supply and the protozoan grazer population. A community fingerprinting technique, terminal restriction fragment length polymorphism, revealed that the bacterial community was not affected by changes in acetate availability but was significantly altered by the removal of protozoan grazers. In silico identification of terminal restriction fragments and 16S rRNA gene sequences from clone libraries revealed a bacterial community dominated by Proteobacteria, Firmicutes , and Bacteroidetes . Elucidation of the factors that structure the bacterial community will improve our understanding of the bacterial role in the carbon cycle of this important subterranean environment.     

Using Stable Isotope Probing to Characterize Differences Between Free-Living and Sediment-Associated Microorganisms in the Subsurface

Aquifers are subterranean reservoirs of freshwater with heterotrophic bacterial communities attached to the sediments and free-living in the groundwater. In the present study, mesocosms were used to assess factors controlling the diversity and activity of the subsurface bacterial community. The assimilation of ¹³C, derived from ¹³C-acetate, was monitored to determine whether the sediment-associated and free-living bacterial community would respond similarly to the presence of protozoan grazers. We observed a dynamic response in the sediment-associated bacterial community and none in the free-living community. The disparity in these observations highlights the importance of the sediment-associated bacterial community in the subsurface carbon cycle.     

Transport of bacteria in porous media and its enhancement by surfactants for bioaugmentation: A review

The success of bioaugmentation processes for groundwater bioremediation requires efficient transport of bacteria in the subsurface environment. In this paper, the factors that influence transport of bacterial cells in porous media are reviewed and the effects of surfactants on the transport are discussed. Movement of bacterial cells in porous media is a process driven by advection and hydrodynamic dispersion forces of fluids. Immobilization of bacterial cells takes place due to processes such as adsorption and straining. Blocking and ripening along with bacterial migration process decrease and increase the retention of cells in porous media, respectively. Physicochemical properties of the porous media, groundwater chemistry, and properties of the bacterial cells affect the transport behavior. Surfactants have the potential to modify bacterial surface properties for both bacterial cells and medium solids, and thus enhance bacterial transport.     

Coupling Between Rates of Bacterial Production and the Abundance of Metabolically Active Bacteria in Lakes, Enumerated Using CTC Reduction and Flow Cytometry

Abstract In natural bacterioplankton assemblages, only a fraction of the total cell count is active, and, therefore, rates of bacterial production should be more strongly correlated to the number of active cells than to the total number of bacteria. However, this hypothesis has seldom been tested. Herein we explore the relationship between rates of bacterial production (measured as leucine uptake) and the number of active bacteria in 14 lakes in southern Québec. Active bacteria are defined as those cells capable of reducing the tetrazolium salt CTC to its fluorescent formazan; these cells were enumerated using flow cytometry. Bacterial production varied two orders of magnitude in the lakes studied, as did the number of active bacteria, whereas the total number of bacteria varied by only sixfold. The number and proportion of active bacteria were similar among lake strata, but rates of bacterial production were highest in the epilimnion and lowest in the hypolimnion. As expected, bacterial production was better correlated to the number of active cells, and bacterial growth rates calculated for active cells ranged from 0.7 to 1.8 day⁻¹, on average threefold higher than those calculated on the basis of total bacterial abundance. Growth rates scaled to active cells were, on average, similar among lake strata and did not show any pattern along a gradient of increasing chlorophyll concentration, so there was no systematic change of bacterial growth rates with lake productivity. In contrast, growth rates scaled to the entire bacterial assemblage were positively correlated to chlorophyll, were tenfold more variable among lakes than growth rates of active cells, and showed larger differences among lake strata. Scaling bacterial production to either the total number or the number of active cells thus results in very different patterns in bacterial growth rates among aquatic systems. Received: 12 July 1996; Accepted: 24 September 1996     

Abundance and activity of Chloroflexi-type SAR202 bacterioplankton in the meso- and bathypelagic waters of the (sub)tropical Atlantic

The contribution of Chloroflexi-type SAR202 cells to total picoplankton and bacterial abundance and uptake of D- and L-aspartic acids (Asp) was determined in the different meso- and bathypelagic water masses of the (sub)tropical Atlantic (from 35 degrees N to 5 degrees S). Fluorescence in situ hybridization (FISH) revealed that the overall abundance of SAR202 was < or = 1 x 10(3) cells ml(-1) in subsurface waters (100 m layer), increasing in the mesopelagic zone to 3 x 10(3) cells ml(-1) and remaining fairly constant down to 4000 m depth. Overall, the percentage of total picoplankton identified as SAR202 increased from < 1% in subsurface waters to 10-20% in the bathypelagic waters. On average, members of the SAR202 cluster accounted for about 30% of the Bacteria in the bathypelagic waters, whereas in the mesopelagic and subsurface waters, SAR202 cells contributed < 5% to total bacterial abundance. The ratio of D-Asp : L-Asp uptake by the bulk picoplankton community increased from the subsurface layer (D-Asp : L-Asp uptake ratio approximately 0.03) to the deeper layers reaching a ratio of approximately 1 at 4000 m depth. Combining FISH with microautoradiography to determine the proportion of SAR202 cells taking up D-Asp versus L-Asp, we found that approximately 30% of the SAR202 cells were taking up L-Asp throughout the water column while D-Asp was essentially not taken up by SAR202. This D-Asp : L-Asp uptake pattern of SAR202 cells is in contrast to that of the bulk bacterial and crenarchaeal community in the bathypelagic ocean, both sustaining a higher fraction of D-Asp-positive cells than L-Asp-positive cells. Thus, although the Chloroflexi-type SAR202 constitutes a major bathypelagic bacterial cluster, it does not contribute to the large fraction of d-Asp utilizing prokaryotic community in the meso- and bathypelagic waters of the North Atlantic, but rather utilizes preferentially L-amino acids.     

Toxicity of toluene and o-xylene toAcinetobacter calcoaceticus in starvation-survival mode

Summary Toxicity of toluene and o-xylene was tested with a deep subsurface isolate during 120 day starvation period. During 120 day starvation period, toxicity appeared to increase with cell age until cells reached the starvation mode. This result may have an significant implication toin situ bioremediation applications since subsurface bacterial populations are apparently in starvation-survival mode.     

The possible origin of the first cell biosystem in the thermal subsurface environment of the earth.

Bacteria are the simplest living biosystems or organisms that exhibit all the characteristics of life. As such, they are excellent models to examine the cell as the basic unit of life and the cell theory which states that all organisms are composed of one or more similar cells. In this article I examine the hypothesis that the primordial soup so often referred to in science was possibly an oil/water interface and/or emulsion in the Earth's, warm, anaerobic subsurface. This warm subsurface location, protected from surface radiation, could have been a favourable location for the assembly of the first bacterial cells on the Earth capable of growth and controlled division or the first biosystem.     

Bacterial and Archaeal 16S rRNA Genes in Late Pleistocene to Holocene Muddy Sediments from the Kanto Plain of Japan

Microbial communities in ancient marine sediments composed of clay and silt obtained from the terrestrial subsurface were phylogenetically analyzed based on their 16S rRNA gene sequences. Chloroflexi and Miscellaneous Crenarchaeotic Group were predominant in bacterial and archaeal clone libraries, respectively. Of 44 operational taxonomic units (OTUs) that had close relatives in the database, 30 were close to sequences obtained from marine environments. Some sequences belonged to the candidate groups JS1, ANME-I, and Marine Benthic Group-C, which are typically found in marine sediments. Low chloride concentrations in the sediments suggest that these marine-affiliated sequences may not reflect currently active microbial communities. Our results indicate the existence of long-term preserved DNA or descendants of ancient oceanic microbial components in subsurface muddy sediments in a temperate region, which may reflect indigenous population of paleoenvironments.     

Denitrifying bacteria from the genus Rhodanobacter dominate bacterial communities in the highly contaminated subsurface of a nuclear legacy waste site

The effect of long-term mixed-waste contamination, particularly uranium and nitrate, on the microbial community in the terrestrial subsurface was investigated at the field scale at the Oak Ridge Integrated Field Research Challenge (ORIFRC) site in Oak Ridge, TN. The abundance, community composition, and distribution of groundwater microorganisms were examined across the site during two seasonal sampling events. At representative locations, subsurface sediment was also examined from two boreholes, one sampled from the most heavily contaminated area of the site and another from an area with low contamination. A suite of DNA- and RNA-based molecular tools were employed for community characterization, including quantitative PCR of rRNA and nitrite reductase genes, community composition fingerprinting analysis, and high-throughput pyrotag sequencing of rRNA genes. The results demonstrate that pH is a major driver of the subsurface microbial community structure and that denitrifying bacteria from the genus Rhodanobacter (class Gammaproteobacteria) dominate at low pH. The relative abundance of bacteria from this genus was positively correlated with lower-pH conditions, and these bacteria were abundant and active in the most highly contaminated areas. Other factors, such as the concentration of nitrogen species, oxygen level, and sampling season, did not appear to strongly influence the distribution of Rhodanobacter bacteria. The results indicate that these organisms are acid-tolerant denitrifiers, well suited to the acidic, nitrate-rich subsurface conditions, and pH is confirmed as a dominant driver of bacterial community structure in this contaminated subsurface environment.     

Nutritional enrichment of a microbial community: The effects on activity, elemental composition, community structure and virus production

Abstract Viruses are active members of the microbial community in natural waters but little is known about the factors that regulate their activity and production. In this study we have investigated the effects of increased availability of organic nutrients and inorganic phosphate on activity, elemental composition, community structure and virus production in a natural bacterial community. The fraction of active cells in the community as estimated from microautoradiography of cells assimilating ³H-labeled thymidine ranged from 0–22%, but changes in the elemental composition of the cells indicated that more than 90% of the cells were active. The increase in carbon and energy availability stimulated virus production more than bacterial biomass production, while the increase in phosphate availability stimulated biomass production rather than virus production. A decrease in morphological diversity of the bacterial community was paralleled by a reduction in the virus-to-bacteria ratio (VBR) but the relationship between bacterial diversity and viral activity is uncertain. Our general conclusion is that nutrient availability, in addition to the bacterial activity, also affects the viral activity, and that both of these may affect the structure and diversity of the bacterial community.     

Microbial Communities of Deep Marine Subsurface Sediments: Molecular and Cultivation Surveys

Recent molecular analyses show that microbial communities of deep marine sediments harbor members of distinct, uncultured bacterial and archaeal lineages, in addition to Gram-positive bacteria and Proteobacteria that are detected by cultivation surveys. Several of these subsurface lineages show cosmopolitan occurrence patterns; they can be found in cold marine sediments and also in hydrothermal habitats, suggesting a continuous deep subsurface and hydrothermal biosphere with shared microbiota. The physiologies and activities of these uncultured subsurface lineages remain to be explored by innovative combinations of genomic and biogeochemical approaches.     

Widespread distribution and high abundance of Rhizobium radiobacter within Mediterranean subsurface sediments

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.     

Microbial diversity in alpine tundra wet meadow soil: novel Chloroflexi from a cold, water-saturated environment

Cold, water-saturated soils play important biogeochemical roles, yet almost nothing is known about the identity and habitat of microbes active under such conditions. We investigated the year-round microenvironment of an alpine tundra wet meadow soil in the Colorado Rocky Mountains, focusing on the biogeochemistry and microbial diversity of spring snowmelt--a dynamic time for alpine ecosystems. In situ measurements revealed spring and autumn periods of long-term temperature stability near 0 degrees C, and that deeper soil (30 cm) was more stable than surface soil, with more moderate summers and winters, and longer isothermal phases. The soil was saturated and water availability was limited by freezing rather than drying. Analyses of bioavailable redox species showed a shift from Mn reduction to net Fe reduction at 2-3 cm depth, elevated SO4(2-) and decreased soluble Zn at spring snowmelt. Terminal restriction fragment length polymorphism profiles detected a correlated shift in bacterial community composition at the surface to subsurface transition. Bacterial and archaeal small-subunit rRNA genes were amplified from saturated spring soil DNA pooled along a depth profile. The most remarkable feature of these subsurface-biased libraries was the high relative abundance of novel, uncultivated Chloroflexi-related sequences comprising the third largest bacterial division sampled, and representing seven new Chloroflexi subdivisions, thereby dramatically expanding the known diversity of this bacterial division. We suggest that these novel Chloroflexi are active at near -0 degrees C temperatures, under likely anoxic conditions, and utilize geochemical inputs such as sulfide from upslope weathering.     

Inter-field variability in the microbial communities of hydrothermal vent deposits from a back-arc basin

Diverse microbial communities thrive on and in deep-sea hydrothermal vent mineral deposits. However, our understanding of the inter-field variability in these communities is poor, as limited sampling and sequencing efforts have hampered most previous studies. To explore the inter-field variability in these communities, we used barcoded pyrosequencing of the variable region 4 (V4) of the 16S rRNA gene to characterize the archaeal and bacterial communities of over 30 hydrothermal deposit samples from six vent fields located along the Eastern Lau Spreading Center. Overall, the bacterial and archaeal communities of the Eastern Lau Spreading Center are similar to other active vent deposits, with a high diversity of Epsilonproteobacteria and thermophilic Archaea. However, the archaeal and bacterial communities from the southernmost vent field, Mariner, were significantly different from the other vent fields. At Mariner, the epsilonproteobacterial genus Nautilia and the archaeal family Thermococcaceae were prevalent in most samples, while Lebetimonas and Thermofilaceae were more abundant at the other vent fields. These differences appear to be influenced in part by the unique geochemistry of the Mariner fluids resulting from active degassing of a subsurface magma chamber. These results show that microbial communities associated with hydrothermal vent deposits in back-arc basins are taxonomically similar to those from mid-ocean ridge systems, but differences in geologic processes between vent fields in a back-arc basin can influence microbial community structure.     

Polymerase chain reaction amplification of naphthalene-catabolic and 16S rRNA gene sequences from indigenous sediment bacteria.

We report the amplification of bacterial genes from uninoculated surface and subsurface sediments by the polymerase chain reaction (PCR). PCR amplification of indigenous bacterial 16S ribosomal DNA genes was unsuccessful when subsurface sediment containing approximately 10(7) cells.g-1 was added directly to a PCR mixture. However, when 10 mg of sediment was inoculated with approximately 10(5) cells of Pseudomonas putida G7, the nahAc naphthalene dioxygenase gene characteristic of the P. putida G7 NAH7 plasmid was detected by PCR amplification. Southern blotting of the PCR amplification product improved sensitivity to 10(3) to 10(4) cells from samples inoculated with P. putida G7, but controls with no sediment added showed that the PCR was partially inhibited by the sediments. Lysozyme-sodium dodecyl sulfate-freeze-thaw DNA extraction was combined with gel electrophoretic partial purification in the presence of polyvinylpyrrolidone to render DNA from indigenous bacteria in surface or subsurface sediment samples amplifiable by PCR using eubacterial 16S ribosomal DNA primers. The nahAc gene could also be amplified from indigenous bacteria by using nahAc-specific primers when PCR conditions were modified by increasing Taq and primer concentrations. Restriction digests of the nahAc amplification products from surface and subsurface sediments revealed polymorphism relative to P. putida G7. The procedures for DNA extraction, purification, and PCR amplification described here demonstrate that the PCR is a potentially useful tool in studies of function- and taxon-specific DNA from indigenous microbial communities in sediment and groundwater environments.     

Monitoring of bacterial community in a coniferous forest soil after a wildfire

Changes in the soil bacterial community of a coniferous forest were analyzed to assess microbial responses to wildfire. Soil samples were collected from three different depths in lightly and severely burned areas, as well as a nearby unburned control area. Direct bacterial counts ranged from 3.3-22.6 x 10(8) cells/(g.soil). In surface soil, direct bacterial counts of unburned soil exhibited a great degree of fluctuation. Those in lightly burned soil changed less, but no significant variation was observed in the severely burned soil. The fluctuations of direct bacterial count were less in the middle and deep soil layers. The structure of the bacterial community was analyzed via the fluorescent in situ hybridization method. The number of bacteria detected with the eubacteria-targeted probe out of the direct bacterial count varied from 30.3 to 84.7%, and these ratios were generally higher in the burned soils than in the unburned control soils. In the surface unburned soil, the ratios of alpha-, beta- and gamma-proteobacteria, Cytophaga-Flavobacterium group, and other eubacteria groups to total eubacteria were 9.9, 10.6, 15.5, 9.0, and 55.0%, respectively, and these ratios were relatively stable. The ratios of alpha-, beta- and gamma-proteobacteria, and Cytophaga-Flavobacterium group to total eubacteria increased immediately after the wildfire, and the other eubacterial proportions decreased in the surface and middle layer soils. By way of contrast, the composition of the 5 groups of eubacteria in the subsurface soil exhibited no significant fluctuations during the entire period. The total bacterial population and bacterial community structure disturbed by wildfire soon began to recover, and original levels seemed to be restored 3 months after the wildfire.     

Microbial community structure and biogeochemistry of Miocene subsurface sediments: implications for long-term microbial survival

Thirty closely spaced cores were obtained from Miocene-aged fluvial, lacustrine and palaeosol subsurface sediments ranging in depth from 173 to 197 m at a site in south-central Washington to investigate the size and composition of the microbial community in relation to sediment geochemical and geophysical properties. Total phospholipid fatty acid (PLFA) analysis indicated that the greatest concentrations of microbial bio-mass were in low-permeability lacustrine sediments that also contained high concentrations of organic carbon. Community structure, based on lipid analyses and on in situ hybridization of bacterial cells with 16S RNA-directed DNA probes, also revealed the presence of metabolically active bacteria that respire sulphate and/or Fe(III) in the lacustrine sediments. Concentrations of pore water sulphate were low (4–8 mg/L) and HCI-extractable Fe was predominantly Fe(II) in the same samples where total biomass and organic carbon were highest. The low hydraulic conductivity (10^-6 to < 10^-9 cm/s) of these sediments has likely contributed to the long term maintenance of both bacteria and organic carbon by limiting the supply of soluble electron acceptors for microbial respiration. These results suggest that the current subsurface microbial population was derived from organisms that were present during lake sedimentation = 6–8 million years ago.     

On Comparison Between Fungal and Bacterial Pretreatment of Coal for Enhanced Biogenic Methane Generation

Owing to better understanding of subsurface geochemical carbon recycling and real-time active methanogenesis in major coal basins around the globe, substantial share of subsurface methane generation is attributed to biogenic origin. Since coal, being complex geopolymer, does not appear to be a favorable microbial substrate, enhancement in biogenic methane yield depends on its degradation into simpler organic substrates. This review puts forward a comparative analysis of fungal and bacterial pretreatment for determining the extent of facilitation in initial degradation of coal, which is still rate limiting step in overall conversion of coal into methane. Primarily, the initial fungal degradation of coal differs from bacterial pretreatment of coal in terms of the nature of released organics. On the basis of previous reports, fungal pretreatment of coal yields, majorly, polyaromatic hydrocarbons, however, bacterial pretreatment results in the generation of mixed organics pool of aromatics and aliphatics. The presence of aliphatics may be prospected for achieving greater conversion rates of coal conversion into methane. Considering the criticality of preliminary degradation of coal and associated issues, the fate of commercial biogenic methane generation would be dictated by the factors pertaining to geological considerations and reservoir geology, chemistry of coal and associated water tables, geomicrobial considerations and economic viability.     

Phylogenetic and Functional Diversity of Microbial Communities Associated with Subsurface Sediments of the Sonora Margin,Guaymas Basin

Subsurface sediments of the Sonora Margin (Guaymas Basin), located in proximity of active cold seep sites were explored. The taxonomic and functional diversity of bacterial and archaeal communities were investigated from 1 to 10 meters below the seafloor. Microbial community structure and abundance and distribution of dominant populations were assessed using complementary molecular approaches (Ribosomal Intergenic Spacer Analysis, 16S rRNA libraries and quantitative PCR with an extensive primers set) and correlated to comprehensive geochemical data. Moreover the metabolic potentials and functional traits of the microbial community were also identified using the GeoChip functional gene microarray and metabolic rates. The active microbial community structure in the Sonora Margin sediments was related to deep subsurface ecosystems (Marine Benthic Groups B and D, Miscellaneous Crenarchaeotal Group, Chloroflexi and Candidate divisions) and remained relatively similar throughout the sediment section, despite defined biogeochemical gradients. However, relative abundances of bacterial and archaeal dominant lineages were significantly correlated with organic carbon quantity and origin. Consistently, metabolic pathways for the degradation and assimilation of this organic carbon as well as genetic potentials for the transformation of detrital organic matters, hydrocarbons and recalcitrant substrates were detected, suggesting that chemoorganotrophic microorganisms may dominate the microbial community of the Sonora Margin subsurface sediments.     

Deep Sequencing of Subseafloor Eukaryotic rRNA Reveals Active Fungi across Marine Subsurface Provinces

The deep marine subsurface is a vast habitat for microbial life where cells may live on geologic timescales. Because DNA in sediments may be preserved on long timescales, ribosomal RNA (rRNA) is suggested to be a proxy for the active fraction of a microbial community in the subsurface. During an investigation of eukaryotic 18S rRNA by amplicon pyrosequencing, unique profiles of Fungi were found across a range of marine subsurface provinces including ridge flanks, continental margins, and abyssal plains. Subseafloor fungal populations exhibit statistically significant correlations with total organic carbon (TOC), nitrate, sulfide, and dissolved inorganic carbon (DIC). These correlations are supported by terminal restriction length polymorphism (TRFLP) analyses of fungal rRNA. Geochemical correlations with fungal pyrosequencing and TRFLP data from this geographically broad sample set suggests environmental selection of active Fungi in the marine subsurface. Within the same dataset, ancient rRNA signatures were recovered from plants and diatoms in marine sediments ranging from 0.03 to 2.7 million years old, suggesting that rRNA from some eukaryotic taxa may be much more stable than previously considered in the marine subsurface.