Attached and unattached microbial communities in a simulated basalt aquifer under fracture- and porous-flow conditions

Bench scale column studies were used to examine the partitioning of microorganisms between groundwater and a geologic medium and to examine the effect of hydrogeology (i.e., porous- versus fracture-flow) on organism partitioning. Replicated columns were constructed with intact basalt core segments that contained natural fractures and with the same basalt crushed into particles. The columns were perfused with groundwater, and upon reaching a steady state, the columns were sacrificed and the attached and unattached communities were analyzed by multiple approaches. The analyses included the total number of cells, the phylogenetic affiliation of the cells (i.e., the alpha, beta, and gamma subclasses of the class Proteobacteria and gram positives with high G+C DNA content) by fluorescent in situ hybridization (FISH), number and taxonomic affiliation by fatty acid methyl ester profiles of culturable heterotrophs, most-probable-number estimates of methanotrophs and phenol oxidizers, and whole-community sole carbon source utilization patterns from Biolog GN microplates. In the packed columns, about 99% of the total biomass (per cubic centimeter of porous medium) was attached to the geologic medium. Lack of equitable units precluded a comparison of attached and unattached biomasses in the fractured columns where the attached biomass was expressed per unit of surface area. Compositional differences in the attached and unattached communities were evidenced by (i) the recovery of Pseudomonas stutzeri, an Enterococcus sp., and Bacillus psychrophilus from the groundwater and not from the basalt, (ii) differences between community carbon source utilization patterns, and (iii) the relative abundances of different phylogenetic groups estimated by FISH in both column types. In the packed columns, attached communities were depleted of members of the alpha- and beta-Proteobacteria subclasses in comparison to those in the corresponding groundwater. In the fractured columns, attached communities were enriched in gram-positive Bacteria and gamma-Proteobacteria and depleted of beta-Proteobacteria, in comparison to those in the corresponding groundwater. Segregation of populations and their activities, possibly modified by attachment to geologic media, may influence contaminant fate and transport in the subsurface and impact other in situ applications.     

Growth determinations for unattached bacteria in a contaminated aquifer

Growth rates of unattached bacteria in groundwater contaminated with treated sewage and collected at various distances from the source of contamination were estimated by using frequency of dividing cells and tritiated-thymidine uptake and compared with growth rates obtained with unsupplemented, closed-bottle incubations. Estimates of bacterial generation times [(In 2)/mu] along a 3-km-long transect in oxygen-depleted (0.1 to 0.7 mg of dissolved oxygen liter-1) groundwater ranged from 16 h at 0.26 km downgradient from an on-land, treated-sewage outfall to 139 h at 1.6 km and correlated with bacterial abundance (r2 = 0.88 at P less than 0.001). Partitioning of assimilated thymidine into nucleic acid generally decreased with distance from the contaminant source, and one population in heavily contaminated groundwater assimilated little thymidine during a 20-h incubation. Several assumptions commonly made when frequency of dividing cells and tritiated-thymidine uptake are used were not applicable to the groundwater samples.     

Physical, biogeochemical and isotopic processes related to heterogeneity of a shallow crystalline rock aquifer

This study deals with the chemical characterization of the biogeochemical processes occurring in a shallow aquifer in crystalline rocks. The influence of rock heterogeneity and the related physical processes on the aquifer biogeochemistry have been investigated. A hydrochemical survey (major anion and cation analysis) shows that rock heterogeneity leads to a stronger spatial than temporal variability. Some rapidly recharged and low- mineralized waters are present at the soil/rock interface. However the pumped well intersects a preferential flow path and pumps nitrate-rich water. Sulfur and oxygen isotope data from sulfates in the pumped water clearly show sulfide oxidation with only 20–30% of the oxygen atoms in sulfates formed by sulfide oxidation coming from atmospheric oxygen. This low contribution of molecular oxygen in sulfide oxidation, associated with the drastic decrease in nitrate concentration, involves a marked relationship between the nitrogen and sulfur cycles through denitrification, coupled with sulfide oxidation. Conversely, for rapidly recharged waters, the rock physical heterogeneity allows sulfide oxidation by molecular oxygen indicated by a contribution of atmospheric oxygen of nearly 70% in the newly formed sulfate. As the aquifer biogeochemistry is controlled by the physical characteristics of the rocks, pumping may overcome the natural flux pattern described previously. This anthropogenic disturbance leads to a modification of water pathways (spatial mixing or relative contribution of the fracture/matrix waters to the global fluxes) and, consequently, to a modification of the physical and biogeochemical processes occurring in the aquifer.     

Growth determinations for unattached bacteria in a contaminated aquifer.

Growth rates of unattached bacteria in groundwater contaminated with treated sewage and collected at various distances from the source of contamination were estimated by using frequency of dividing cells and tritiated-thymidine uptake and compared with growth rates obtained with unsupplemented, closed-bottle incubations. Estimates of bacterial generation times [(In 2)/mu] along a 3-km-long transect in oxygen-depleted (0.1 to 0.7 mg of dissolved oxygen liter-1) groundwater ranged from 16 h at 0.26 km downgradient from an on-land, treated-sewage outfall to 139 h at 1.6 km and correlated with bacterial abundance (r2 = 0.88 at P less than 0.001). Partitioning of assimilated thymidine into nucleic acid generally decreased with distance from the contaminant source, and one population in heavily contaminated groundwater assimilated little thymidine during a 20-h incubation. Several assumptions commonly made when frequency of dividing cells and tritiated-thymidine uptake are used were not applicable to the groundwater samples.     

Composition and diversity of microbial communities recovered from surrogate minerals incubated in an acidic uranium-contaminated aquifer

Our understanding of subsurface microbiology is hindered by the inaccessibility of this environment, particularly when the hydrogeologic medium is contaminated with toxic substances. In this study, surrogate geological media contained in a porous receptacle were incubated in a well within the saturated zone of a pristine region of an aquifer to capture populations from the extant communities. After an 8-week incubation, the media were recovered, and the microbial community that developed on each medium was compared to the community recovered from groundwater and native sediments from the same region of the aquifer, using 16S DNA coding for rRNA (rDNA)-based terminal restriction fragment length polymorphism (T-RFLP). The groundwater and sediment communities were highly distinct from one another, and the communities that developed on the various media were more similar to groundwater communities than to sediment communities. 16S rDNA clone libraries of communities that developed on particles of a specular hematite medium incubated in the same well as the media used for T-RFLP analysis were compared with those obtained from an acidic, uranium-contaminated region of the same aquifer. The hematite-associated community formed in the pristine area was highly diverse at the species level, with 25 distinct phylotypes identified, the majority of which (73%) were affiliated with the beta-Proteobacteria. Similarly, the hematite-associated community formed in the contaminated area was populated in large part by beta-Proteobacteria (62%); however, only 13 distinct phylotypes were apparent. The three numerically dominant clones from the hematite-associated community from the contaminated site were affiliated with metal- and radionuclide-tolerant or acidophilic taxa, consistent with the environmental conditions. Only two populations were common to both sites.     

Spatial heterogeneity in sediment-associated bacterial and eukaryotic communities in a landfill leachate-contaminated aquifer

Heterogeneity in eukaryotic and bacteria community structure in surface and subsurface sediment samples downgradient of the Banisveld landfill (The Netherlands) was studied using a culturing-independent molecular approach. Along a transect covering the part of the aquifer most polluted by landfill leachate, sediment was sampled at 1-m depth intervals, until a depth of 5.5 m, at four distances from the landfill. Two drillings were placed in a nearby clean area as a reference. Denaturing gradient gel electrophoresis banding patterns revealed high bacterial and eukaryotic diversity and complex community structures. Bacteria and eukaryotic community profiles in polluted samples grouped different from those in clean samples. Bacteria community profiles in surface samples clustered together and separately from subsurface community profiles. Subsurface bacteria profiles clustered in a location-specific manner. Eukaryotic community structure did not significantly relate to distance from the landfill or depth. No significant spatial autocorrelation of bacteria or eukaryotic communities was observed over 1-m depth intervals per sampling location. Spatial heterogeneity in sediment-associated bacterial communities appears to be much larger than in groundwater. We discuss how on the one hand, spatial heterogeneity may complicate the assessment of microbial community structure and functioning, while on the other it may provide better opportunities for natural attenuation.     

Attached and Unattached Bacterial Communities in a 120-Meter Corehole in an Acidic, Crystalline Rock Aquifer

The bacteria colonizing geologic core sections (attached) were contrasted with those found suspended in the groundwater (unattached) by examining the microbiology of 16 depth-paired core and groundwater samples using a suite of culture-independent and culture-dependent analyses. One hundred twenty-two meters was continuously cored from a buried chalcopyrite ore hosted in a biotite-quartz-monzonite porphyry at the Mineral Park Mine near Kingman, Ariz. Every fourth 1.5-m core was acquired using microbiologically defensible methods, and these core sections were aseptically processed for characterization of the attached bacteria. Groundwater samples containing unattached bacteria were collected from the uncased corehole at depth intervals corresponding to the individual cores using an inflatable straddle packer sampler. The groundwater was acidic (pH 2.8 to 5.0), with low levels of dissolved oxygen and high concentrations of sulfate and metals, including ferrous iron. Total numbers of attached cells were less than 10⁵ cells g of core material⁻¹ while unattached cells numbered about 10⁵ cells ml of groundwater⁻¹. Attached and unattached acidophilic heterotrophs were observed throughout the depth profile. In contrast, acidophilic chemolithotrophs were not found attached to the rock but were commonly observed in the groundwater. Attached communities were composed of low numbers (<40 CFU g⁻¹) of neutrophilic heterotrophs that exhibited a high degree of morphologic diversity, while unattached communities contained higher numbers (ca. 10³ CFU ml⁻¹) of neutrophilic heterotrophs of limited diversity. Sulfate-reducing bacteria were restricted to the deepest samples of both core and groundwater. 16S ribosomal DNA sequence analysis of attached, acidophilic isolates indicated that organisms closely related to heterotrophic, acidophilic mesophiles such as Acidiphilium organovorum and, surprisingly, to the moderately thermophilic Alicyclobacillus acidocaldarius were present. The results indicate that viable (but possibly inactive) microorganisms were present in the buried ore and that there was substantial distinction in biomass and physiological capabilities between attached and unattached populations.     

Plankton communities of an acidic,polymictic, brown-water lake

The plankton of an acidic, polymictic, brown-water lake was investigated over a one-year period. The phytoplankton community was dominated by Chlamydomonas sp., Melosira varians and Peridinium pusillum during different times of the year. Densities were abnormally low, with a maximum of only 13,781 individuals per liter during the March bloom. The water column was nearly void of phytoplankton during the fall. These low densities were attributed to several of the physical and chemical characteristics of the water, including low pH, low levels of nutrients and light limitation. The latter factor was important because of low light transmission into the highly colored water, shading from macrophytes and the nearly continuous transport of the phytoplankton into the aphotic zone due to mixing of the water column by sea breezes.The zooplankton was dominated by Diaptomus floridanus, Polyarthra vulgaris, Keratella cochlearis and Daphnia ambigua. The observed densities were typical of southeastern oligotrophic lakes. The zooplankton community did not exhibit a decrease in density that corresponded with the observed low phytoplankton densities, suggesting the possibility that they relied heavily on bacterial and detrital food sources.     

Microbial communities in the saturated groundwater environment II: Diversity of bacterial communities in a Pleistocene sand aquifer and their in vitro activities

Bacterial cell numbers obtained from 103 water and sediment samples from a Pleistocene sandy aquifer in the Lower Rhine region (Bocholt, FRG) were determinated on P-agar and by direct count. Below 5 m under the surface, colony-forming unit (cfu) numbers in water samples were less than 100/ml, and in many cases less than 50/ml. In sediment samples, they were 10- to 100-fold higher (10²–10⁴ cfu/g dry wt), but changing markedly between different depths. Direct cell counts yielded numbers two to three orders of magnitude higher.About 2,700 strains of bacteria from 60 samples were isolated randomly and characterized by morphological and physiological properties. Of all the isolates, 71.6% were gram-negative, and 52.2% were gram-negative straight rods. Water communities, with one exception, had low proportions of gram-positive bacteria (<11%), whereas in all but one of the sediment communities percentages of gram-positive isolates were three- to sevenfold higher (35–43%). Water and sediment communities, as well as communities from different sampling sites and communities from different depths of the same sampling site, differed in their qualitative and quantitative morphotype composition and physiological capabilities.The in vitro activities of strains within a single community were quite different, indicating that each community is composed of many diverse bacteria, several having extremely different capabilities. Thus, each community has its own specific activity pattern. Gram-positive bacteria showed on an average lower total activities than did gram-negative bacteria. Grampositive bacteria as well as gram-negative bacteria from sediment had higher values of in vitro activities than the corresponding groups isolated from water. Many water and sediment bacteria preferred the same substrates which were utilized at high rates. However, there were differences in the degradation of the various other substrates present, and each community showed preferences for particular substrates, which they degraded best.The results of cell morphology and physiology studies indicated that all eight characterized communities were very different from one another and very diversely structured.     

Seasonal changes in nitrogen-cycle gene abundances and in bacterial communities in acidic forest soils

The abundance of genes related to the nitrogen biogeochemical cycle and the microbial community in forest soils (bacteria, archaea, fungi) were quantitatively analyzed via real-time PCR using 11 sets of specific primers amplifying nifH, bacterial amoA, archaeal amoA, narG, nirS, nirK, norB, nosZ, bacterial 16S rRNA gene, archaeal 16S rRNA gene, and the ITS sequence of fungi. Soils were sampled from Bukhan Mountain from September of 2010 to July of 2011 (7 times). Bacteria were the predominant microbial community in all samples. However, the abundance of archaeal amoA was greater than bacterial amoA throughout the year. The abundances of nifH, nirS, nirK, and norB genes changed in a similar pattern, while narG and nosZ appeared in sensitive to the environmental changes. Clone libraries of bacterial 16S rRNA genes were constructed from summer and winter soil samples and these revealed that Acidobacteria was the most predominant phylum in acidic forest soil environments in both samples. Although a specific correlation of environmental factor and gene abundance was not verified by principle component analysis, our data suggested that the combination of biological, physical, and chemical characteristics of forest soils created distinct conditions favoring the nitrogen biogeochemical cycle and that bacterial communities in undisturbed acidic forest soils were quite stable during seasonal change.     

Stability of interstitial crustacean communities in an isolated alluvial aquifer

The temporal stability of interstitial crustacean communities was investigated in relation to environmental variability in a perched aquifer, characterized by limited connection with surface waters. The author hypothesized that strong physico-chemical stability would enhance persistence and affect dominance patterns for groundwater fauna. Fifteen wells were sampled four times over a two-year period in order to assess the global stability of ground waters and to evaluate temporal changes in species assemblages. For each station, environmental patterns displayed only minor changes, while between-site differences were much larger. At the scale of the aquifer, strong temporal stability was found for groundwater physical and chemical variables. Interstitial crustaceans displayed diverse assemblages dominated by cyclopoid taxa. High persistence characterized community composition and structure (species relative abundance patterns, species richness, Shannon's diversity index) over the study period. Most well faunas were dominated numerically by a single species, whether micro- or macrocrustacean assemblages were considered. Faunal persistence observed in the aquifer was greater than that reported in many surface waters and was probably related to the strong environmental stability.     

Insect-associated bacterial communities in an alpine stream

Hydrobiologia - The roles of macroinvertebrate and microbial communities in stream ecosystems are recognized to be important to energy flow and nutrient cycling. While the linkages of these major...     

Molecular diversity of bacterial communities from subseafloor rock samples in a deep-water production basin in Brazil

The deep subseafloor rock in oil reservoirs represents a unique environment in which a high oilcontamination and very low biomass can be observed. Sampling this environment has been a challenge owing to the techniques used for drilling and coring. In this study, the facilities developed by the Brazilian oil company PETROBRAS for accessing deep subsurface oil reservoirs were used to obtain rock samples at 2,822-2,828 m below the ocean floor surface from a virgin field located in the Atlantic Ocean, Rio de Janeiro. To address the bacterial diversity of these rock samples, PCR amplicons were obtained using the DNA from four core sections and universal primers for 16S rRNA and for APS reductase (aps) genes. Clone libraries were generated from these PCR fragments and 87 clones were sequenced. The phylogenetic analyses of the 16S rDNA clone libraries showed a wide distribution of types in the domain bacteria in the four core samples, and the majority of the clones were identified as belonging to Betaproteobacteria. The sulfate-reducing bacteria community could only be amplified by PCR in one sample, and all clones were identified as belonging to Gammaproteobacteria. For the first time, the bacterial community was assessed in such deep subsurface environment.     

Identification and isolation of a Castellaniella species important during biostimulation of an acidic nitrate- and uranium-contaminated aquifer

Immobilization of uranium in groundwater can be achieved through microbial reduction of U(VI) to U(IV) upon electron donor addition. Microbial community structure was analyzed in ethanol-biostimulated and control sediments from a high-nitrate (>130 mM), low-pH, uranium-contaminated site in Oak Ridge, TN. Analysis of small subunit (SSU) rRNA gene clone libraries and polar lipid fatty acids from sediments revealed that biostimulation resulted in a general decrease in bacterial diversity. Specifically, biostimulation resulted in an increase in the proportion of Betaproteobacteria (10% of total clones in the control sediment versus 50 and 79% in biostimulated sediments) and a decrease in the proportion of Gammaproteobacteria and Acidobacteria. Clone libraries derived from dissimilatory nitrite reductase genes (nirK and nirS) were also dominated by clones related to Betaproteobacteria (98% and 85% of total nirK and nirS clones, respectively). Within the nirK libraries, one clone sequence made up 59 and 76% of sequences from biostimulated sediments but only made up 10% of the control nirK library. Phylogenetic analysis of SSU rRNA and nirK gene sequences from denitrifying pure cultures isolated from the site indicate that all belong to a Castellaniella species; nearly identical sequences also constituted the majority of biostimulated SSU rRNA and nirK clone libraries. Thus, by combining culture-independent with culture-dependent techniques, we were able to link SSU rRNA clone library information with nirK sequence data and conclude that a potentially novel Castellaniella species is important for in situ nitrate removal at this site.     

Macroscopic streamer growths in acidic, metal-rich mine waters in north wales consist of novel and remarkably simple bacterial communities

The microbial composition of acid streamers (macroscopic biofilms) in acidic, metal-rich waters in two locations (an abandoned copper mine and a chalybeate spa) in north Wales was studied using cultivation-based and biomolecular techniques. Known chemolithotrophic and heterotrophic acidophiles were readily isolated from disrupted streamers, but they accounted for only <1 to 7% of the total microorganisms present. Fluorescent in situ hybridization (FISH) revealed that 80 to 90% of the microbes in both types of streamers were beta-Proteobacteria. Terminal restriction fragment length polymorphism analysis of the streamers suggested that a single bacterial species was dominant in the copper mine streamers, while two distinct bacteria (one of which was identical to the bacterium found in the copper mine streamers) accounted for about 90% of the streamers in the spa water. 16S rRNA gene clone libraries showed that the beta-proteobacterium found in both locations was closely related to a clone detected previously in acid mine drainage in California and that its closest characterized relatives were neutrophilic ammonium oxidizers. Using a modified isolation technique, this bacterium was isolated from the copper mine streamers and shown to be a novel acidophilic autotrophic iron oxidizer. The beta-proteobacterium found only in the spa streamers was closely related to the neutrophilic iron oxidizer Gallionella ferruginea. FISH analysis using oligonucleotide probes that targeted the two beta-proteobacteria confirmed that the biodiversity of the streamers in both locations was very limited. The microbial compositions of the acid streamers found at the two north Wales sites are very different from the microbial compositions of the previously described acid streamers found at Iron Mountain, California, and the Rio Tinto, Spain.     

Planktonic and sediment bacterial communities in an integrated mariculture system

An integrated multi-trophic aquaculture (IMTA) system, with one fish cage model surrounded by an island and shellfish rafts, was used in the current study. Planktonic and sediment bacterial communities in the IMTA system were monitored over four seasons in 2019. In both plankton and sediment samples, the most dominant phyla were Proteobacteria and Bacteroidota. Sediment bacterial samples were more similar and had higher levels of biodiversity than planktonic bacterial samples. Obvious seasonal variations were found in plankton samples, but not in sediment samples. No obvious inter-site variations in planktonic and sediment bacteria (fish cages, shellfish rafts and control sites) were found and the results suggested that no obvious impact of feeding operations in fish culture cage model on bacterial communities in the IMTA system was observed in this study. Based on the sequence data, some faecal indicator bacteria and potentially pathogenic bacterial species were detected. According to the results, the bacterial water quality in the IMTA system was acceptable. PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) analysis revealed that the primary difference in potential functional roles of planktonic and sediment bacteria was amino acid transport and metabolism, which was active in different seasons.     

Effects of abiotic factors on the phylogenetic diversity of bacterial communities in acidic thermal springs

Acidic thermal springs offer ideal environments for studying processes underlying extremophile microbial diversity. We used a carefully designed comparative analysis of acidic thermal springs in Yellowstone National Park to determine how abiotic factors (chemistry and temperature) shape acidophile microbial communities. Small-subunit rRNA gene sequences were PCR amplified, cloned, and sequenced, by using evolutionarily conserved bacterium-specific primers, directly from environmental DNA extracted from Amphitheater Springs and Roaring Mountain sediment samples. Energy-dispersive X-ray spectroscopy, X-ray diffraction, and colorimetric assays were used to analyze sediment chemistry, while an optical emission spectrometer was used to evaluate water chemistry and electronic probes were used to measure the pH, temperature, and E(h) of the spring waters. Phylogenetic-statistical analyses found exceptionally strong correlations between bacterial community composition and sediment mineral chemistry, followed by weaker but significant correlations with temperature gradients. For example, sulfur-rich sediment samples contained a high diversity of uncultured organisms related to Hydrogenobaculum spp., while iron-rich sediments were dominated by uncultured organisms related to a diverse array of gram-positive iron oxidizers. A detailed analysis of redox chemistry indicated that the available energy sources and electron acceptors were sufficient to support the metabolic potential of Hydrogenobaculum spp. and iron oxidizers, respectively. Principal-component analysis found that two factors explained 95% of the genetic diversity, with most of the variance attributable to mineral chemistry and a smaller fraction attributable to temperature.