GeoChip-based analysis of the functional gene diversity and metabolic potential of soil microbial communities of mangroves

Mangroves are unique and highly productive ecosystems and harbor very special microbial communities. Although the phylogenetic diversity of sediment microbial communities of mangrove habitats has been examined extensively, little is known regarding their functional gene diversity and metabolic potential. In this study, a high-throughput functional gene array (GeoChip 4.0) was used to analyze the functional diversity, composition, structure, and metabolic potential of microbial communities in mangrove habitats from mangrove national nature reserves in China. GeoChip data indicated that these microbial communities were functionally diverse as measured by the number of genes detected, unique genes, and various diversity indices. Almost all key functional gene categories targeted by GeoChip 4.0 were detected in the mangrove microbial communities, including carbon (C) fixation, C degradation, methane generation, nitrogen (N) fixation, nitrification, denitrification, ammonification, N reduction, sulfur (S) metabolism, metal resistance, antibiotic resistance, and organic contaminant degradation. Detrended correspondence analysis (DCA) of all detected genes showed that Spartina alterniflora (HH), an invasive species, did not harbor significantly different microbial communities from Aegiceras corniculatum (THY), a native species, but did differ from other species, Kenaelia candel (QQ), Aricennia marina (BGR), and mangrove-free mud flat (GT). Canonical correspondence analysis (CCA) results indicated the microbial community structure was largely shaped by surrounding environmental variables, such as total nitrogen (TN), total carbon (TC), pH, C/N ratio, and especially salinity. This study presents a comprehensive survey of functional gene diversity of soil microbial communities from different mangrove habitats/species and provides new insights into our understanding of the functional potential of microbial communities in mangrove ecosystems.     

GeoChip-based analysis of the functional gene diversity and metabolic potential of microbial communities in acid mine drainage

Acid mine drainage (AMD) is an extreme environment, usually with low pH and high concentrations of metals. Although the phylogenetic diversity of AMD microbial communities has been examined extensively, little is known about their functional gene diversity and metabolic potential. In this study, a comprehensive functional gene array (GeoChip 2.0) was used to analyze the functional diversity, composition, structure, and metabolic potential of AMD microbial communities from three copper mines in China. GeoChip data indicated that these microbial communities were functionally diverse as measured by the number of genes detected, gene overlapping, unique genes, and various diversity indices. Almost all key functional gene categories targeted by GeoChip 2.0 were detected in the AMD microbial communities, including carbon fixation, carbon degradation, methane generation, nitrogen fixation, nitrification, denitrification, ammonification, nitrogen reduction, sulfur metabolism, metal resistance, and organic contaminant degradation, which suggested that the functional gene diversity was higher than was previously thought. Mantel test results indicated that AMD microbial communities are shaped largely by surrounding environmental factors (e.g., S, Mg, and Cu). Functional genes (e.g., narG and norB) and several key functional processes (e.g., methane generation, ammonification, denitrification, sulfite reduction, and organic contaminant degradation) were significantly (P < 0.10) correlated with environmental variables. This study presents an overview of functional gene diversity and the structure of AMD microbial communities and also provides insights into our understanding of metabolic potential in AMD ecosystems.     

Dynamics of microbial community composition and function during in situ bioremediation of a uranium-contaminated aquifer

A pilot-scale system was established to examine the feasibility of in situ U(VI) immobilization at a highly contaminated aquifer (U.S. DOE Integrated Field Research Challenge site, Oak Ridge, TN). Ethanol was injected intermittently as an electron donor to stimulate microbial U(VI) reduction, and U(VI) concentrations fell to below the Environmental Protection Agency drinking water standard (0.03 mg liter(-1)). Microbial communities from three monitoring wells were examined during active U(VI) reduction and maintenance phases with GeoChip, a high-density, comprehensive functional gene array. The overall microbial community structure exhibited a considerable shift over the remediation phases examined. GeoChip-based analysis revealed that Fe(III)-reducing bacterial (FeRB), nitrate-reducing bacterial (NRB), and sulfate-reducing bacterial (SRB) functional populations reached their highest levels during the active U(VI) reduction phase (days 137 to 370), in which denitrification and Fe(III) and sulfate reduction occurred sequentially. A gradual decrease in these functional populations occurred when reduction reactions stabilized, suggesting that these functional populations could play an important role in both active U(VI) reduction and maintenance of the stability of reduced U(IV). These results suggest that addition of electron donors stimulated the microbial community to create biogeochemical conditions favorable to U(VI) reduction and prevent the reduced U(IV) from reoxidation and that functional FeRB, SRB, and NRB populations within this system played key roles in this process.     

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.     

Metabolically active microbial communities in uranium-contaminated subsurface sediments

In order to develop effective bioremediation strategies for radionuclide contaminants, the composition and metabolic potential of microbial communities need to be better understood, especially in highly contaminated subsurface sediments for which little cultivation-independent information is available. In this study, we characterized metabolically active and total microbial communities associated with uranium-contaminated subsurface sediments along geochemical gradients. DNA and RNA were extracted and amplified from four sediment-depth intervals representing moderately acidic (pH 3.7) to near-neutral (pH 6.7) conditions. Phylotypes related to Proteobacteria (Alpha-, Beta-, Delta- and Gammaproteobacteria), Bacteroidetes, Actinobacteria, Firmicutes and Planctomycetes were detected in DNA- and RNA-derived clone libraries. Diversity and numerical dominance of phylotypes were observed to correspond to changes in sediment geochemistry and rates of microbial activity, suggesting that geochemical conditions have selected for well-adapted taxa. Sequences closely related to nitrate-reducing bacteria represented 28% and 43% of clones from the total and metabolically active fractions of the microbial community, respectively. This study provides the first detailed analysis of total and metabolically active microbial communities in radionuclide-contaminated subsurface sediments. Our microbial community analysis, in conjunction with rates of microbial activity, points to several groups of nitrate-reducers that appear to be well adapted to environmental conditions common to radionuclide-contaminated sites.     

Spatio-temporal patterns of microbial communities in a hydrologically dynamic pristine aquifer

Seasonal patterns of groundwater and sediment microbial communities were explored in a hydrologically dynamic alpine oligotrophic porous aquifer, characterized by pronounced groundwater table fluctuations. Rising of the groundwater level in consequence of snow melting water recharge was accompanied by a dramatic drop of bacterial Shannon diversity in groundwater from H' = 3.22 ± 0.28 in autumn and winter to H' = 1.31 ± 0.35 in spring and summer, evaluated based on T-RFLP community fingerprinting. Elevated numbers of bacteria in groundwater in autumn followed nutrient inputs via recharge from summer rains and correlated well with highest concentrations of assimilable organic carbon. Sterile sediments incubated to groundwater in monitoring wells were readily colonized reaching maximum cell densities within 2 months, followed by a consecutive but delayed increase and leveling-off of bacterial diversity. After 1 year of incubation, the initially sterile sediments exhibited a similar number of bacteria and Shannon diversity when compared to vital sediment from a nearby river incubated in parallel. The river bed sediment microbial communities hardly changed in composition, diversity, and cell numbers during 1 year of exposure to groundwater. Summing up, the seasonal hydrological dynamics were found to induce considerable dynamics of microbial communities suspended in groundwater, while sediment communities seem unaffected and stable in terms of biomass and diversity.     

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.     

Functional diversity changes of microbial communities along a soil aquifer for reclaimed water recharge

The physiochemical and functional diversity of soil-attached microorganisms was investigated using a stabilized laboratory-scale soil aquifer treatment (SAT) system. In this system, reclaimed water after ozonation was used as the feed water, and 60% dissolved organic carbon was removed by the unsaturated vadose layer in 0.8?days. Soil biomass (volatile solids, phospholipid extraction) and functional diversity significantly decreased from the unsaturated vadose layer to the saturated aquifer, where they maintained the same level. Using principal components analysis based on substrate utilization pattern, the vadose layer soil sample was clearly separated from the saturated layer samples. Exceptionally, the oxidation rates of esters remained stable during SAT, indicating the purification potential on certain recalcitrant organic compounds in the saturated aquifer given an adequate retention time. Correlation analysis revealed that organic carbon was the key limiting factor for microbial biomass and activity, especially for tyrosine-like aromatic proteins and soluble microbial byproduct-like materials.     

Metagenomic comparison of microbial communities inhabiting confined and unconfined aquifer ecosystems

A metagenomic analysis of two aquifer systems located under a dairy farming region was performed to examine to what extent the composition and function of microbial communities varies between confined and surface-influenced unconfined groundwater ecosystems. A fundamental shift in taxa was seen with an overrepresentation of Rhodospirillales, Rhodocyclales, Chlorobia and Circovirus in the unconfined aquifer, while Deltaproteobacteria and Clostridiales were overrepresented in the confined aquifer. A relative overrepresentation of metabolic processes including antibiotic resistance (β-lactamase genes), lactose and glucose utilization and DNA replication were observed in the unconfined aquifer, while flagella production, phosphate metabolism and starch uptake pathways were all overrepresented in the confined aquifer. These differences were likely driven by differences in the nutrient status and extent of exposure to contaminants of the two groundwater systems. However, when compared with freshwater, ocean, sediment and animal gut metagenomes, the unconfined and confined aquifers were taxonomically and metabolically more similar to each other than to any other environment. This suggests that intrinsic features of groundwater ecosystems, including low oxygen levels and a lack of sunlight, have provided specific niches for evolution to create unique microbial communities. Obtaining a broader understanding of the structure and function of microbial communities inhabiting different groundwater systems is particularly important given the increased need for managing groundwater reserves of potable water.     

Adaptation to and biodegradation of xenobiotic compounds by microbial communities from a pristine aquifer

The ability of subsurface microbial communities to adapt to the biodegradation of xenobiotic compounds was examined in aquifer solids samples from a pristine aquifer. An increase in the rates of mineralization of radiolabeled substrates with exposure was used as an indication of adaptation. For some compounds, such as chlorobenzene and 1,2,4-trichlorobenzene, slight mineralization was observed but no adaptation was apparent during incubations of over 8 months. Other compounds demonstrated three patterns of response. For m-cresol, m-aminophenol, and aniline intermediate rates of biodegradation and a linear increase in the percent mineralized with time were observed. Phenol, p-chlorophenol, and ethylene dibromide were rapidly metabolized initially, with a nonlinear increase in the percent mineralized with time, indicating that the community was already adapted to the biodegradation of these compounds. Only p-nitrophenol demonstrated a typical adaptation response. In different samples of soil from the same layer in the aquifer, the adaptation period to p-nitrophenol varied from a few days to as long as 6 weeks. In most cases the concentration of xenobiotic added, over the range from a few nanograms to micrograms per gram, made no difference in the response. Most-probable-number counts demonstrated that adaptation is accompanied by an increase in specific degrader numbers. This study has shown that diverse patterns of response occur in the subsurface microbial community.     

Non-random processes determine the colonization of groundwater sediments by microbial communities in a pristine porous aquifer

Sediments accommodate the dominating share of groundwater microbiomes, however the processes that govern the assembly and succession of sediment-attached microbial communities in groundwater aquifers are not well understood. To elucidate these processes, we followed the microbial colonization of sterile sediments in in situ microcosms that were exposed to groundwater for almost 1 year at two distant but hydrologically connected sites of a pristine, shallow, porous aquifer. Our results revealed intriguing similarities between the community succession on the newly-colonized sediments and succession patterns previously observed for biofilms in other more dynamic aquatic environments, indicating that the assembly of microbial communities on surfaces may be governed by similar underlying mechanisms across a wide range of different habitats. Null model simulations on spatiotemporally resolved 16S rRNA amplicon sequencing data further indicated selection of specific OTUs rather than random colonization as the main driver of community assembly. A small fraction of persistent OTUs that had established on the sediments during the first 115 days dominated the final communities (68%–85%), suggesting a key role of these early-colonizing organisms, in particular specific genera within the Comamonadaceae and Oxalobacteraceae, for community assembly and succession during the colonization of the sediments. Overall, our study suggests that differences between planktonic and sediment-attached communities often reported for groundwater environments are not the result of purely stochastic events, but that sediment surfaces select for specific groups of microorganisms that assemble over time in a reproducible, non-random way.     

High-density PhyloChip profiling of stimulated aquifer microbial communities reveals a complex response to acetate amendment

There is increasing interest in harnessing the functional capacities of indigenous microbial communities to transform and remediate a wide range of environmental contaminants. Information about which community members respond to stimulation can guide the interpretation and development of remediation approaches. To comprehensively determine community membership and abundance patterns among a suite of samples associated with uranium bioremediation experiments, we employed a high-density microarray (PhyloChip). Samples were unstimulated, naturally reducing, or collected during Fe(III) (early) and sulfate reduction (late biostimulation) from an acetate re-amended/amended aquifer in Rifle, Colorado, and from laboratory experiments using field-collected materials. Deep community sampling with PhyloChip identified hundreds-to-thousands of operational taxonomic units (OTUs) present during amendment, and revealed close similarity among highly enriched taxa from drill core and groundwater well-deployed column sediment. Overall, phylogenetic data suggested that stimulated community membership was most affected by a carryover effect between annual stimulation events. Nevertheless, OTUs within the Fe(III)- and sulfate-reducing lineages, Desulfuromonadales and Desulfobacterales, were repeatedly stimulated. Less consistent, co-enriched taxa represented additional lineages associated with Fe(III) and sulfate reduction (e.g. Desulfovibrionales; Syntrophobacterales; Peptococcaceae) and autotrophic sulfur oxidation (Sulfurovum; Campylobacterales). Data implies complex membership among highly stimulated taxa and, by inference, biogeochemical responses to acetate, a nonfermentable substrate.     

Adaptation to and biodegradation of xenobiotic compounds by microbial communities from a pristine aquifer.

The ability of subsurface microbial communities to adapt to the biodegradation of xenobiotic compounds was examined in aquifer solids samples from a pristine aquifer. An increase in the rates of mineralization of radiolabeled substrates with exposure was used as an indication of adaptation. For some compounds, such as chlorobenzene and 1,2,4-trichlorobenzene, slight mineralization was observed but no adaptation was apparent during incubations of over 8 months. Other compounds demonstrated three patterns of response. For m-cresol, m-aminophenol, and aniline intermediate rates of biodegradation and a linear increase in the percent mineralized with time were observed. Phenol, p-chlorophenol, and ethylene dibromide were rapidly metabolized initially, with a nonlinear increase in the percent mineralized with time, indicating that the community was already adapted to the biodegradation of these compounds. Only p-nitrophenol demonstrated a typical adaptation response. In different samples of soil from the same layer in the aquifer, the adaptation period to p-nitrophenol varied from a few days to as long as 6 weeks. In most cases the concentration of xenobiotic added, over the range from a few nanograms to micrograms per gram, made no difference in the response. Most-probable-number counts demonstrated that adaptation is accompanied by an increase in specific degrader numbers. This study has shown that diverse patterns of response occur in the subsurface microbial community.     

Testing the functional significance of microbial composition in natural communities

Ecologists have long studied the relationship between biotic composition and ecosystem functioning in larger organisms; however, only recently has this relationship been investigated widely in microorganisms. Recent studies are reviewed within a framework of three experimental approaches that are often used to study larger organisms: environmental treatment, common garden, and reciprocal transplant experiments. Although the composition of microorganisms cannot be easily manipulated in the field, applying these approaches to intact microbial communities can begin to tease apart the effects of microbial composition from environmental parameters on ecosystem functioning. The challenges in applying these approaches to microorganisms are highlighted and it is discussed how the experimental approach and duration affects a study's interpretation. In general, long-term environmental treatment experiments identify correlative relationships between microbial composition and ecosystem functioning, whereas short-term common garden experiments demonstrate that microbial composition influences ecosystem functioning. Finally, reciprocal transplants simultaneously test for interactive effects of the environment and composition on functioning. The studies reviewed provide evidence that, at least in some cases, microbial composition influences ecosystem functioning. It is concluded that whole-community experiments offer a way to test whether information about microbial composition will help predict ecosystem responses to global change.     

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.     

Applications of functional gene microarrays for profiling microbial communities

Functional gene arrays (FGAs) have been considered as a specific, sensitive, quantitative, and high throughput metagenomic tool to detect, monitor and characterize microbial communities. Especially GeoChips, the most comprehensive FGAs have been applied to analyze the functional diversity, composition, structure, and metabolic potential or activity of a variety of microbial communities from different habitats, such as aquatic ecosystems, soils, contaminated sites, extreme environments, and bioreactors. FGAs are able to address fundamental questions related to global change, bioremediation, land use, human health, and ecological theories, and link the microbial community structure to environmental properties and ecosystem functioning. This review focuses on applications of FGA technology for profiling microbial communities, including target preparation, hybridization and data processing, and data analysis. We also discuss challenges and future directions of FGA applications.     

Stimulating the in situ activity of Geobacter species to remove uranium from the groundwater of a uranium-contaminated aquifer

The potential for removing uranium from contaminated groundwater by stimulating the in situ activity of dissimilatory metal-reducing microorganisms was evaluated in a uranium-contaminated aquifer located in Rifle, Colo. Acetate (1 to 3 mM) was injected into the subsurface over a 3-month period via an injection gallery composed of 20 injection wells, which was installed upgradient from a series of 15 monitoring wells. U(VI) concentrations decreased in as little as 9 days after acetate injection was initiated, and within 50 days uranium had declined below the prescribed treatment level of 0.18 micro M in some of the monitoring wells. Analysis of 16S ribosomal DNA (rDNA) sequences and phospholipid fatty acid profiles demonstrated that the initial loss of uranium from the groundwater was associated with an enrichment of Geobacter species in the treatment zone. Fe(II) in the groundwater also increased during this period, suggesting that U(VI) reduction was coincident with Fe(III) reduction. As the acetate injection continued over 50 days there was a loss of sulfate from the groundwater and an accumulation of sulfide and the composition of the microbial community changed. Organisms with 16S rDNA sequences most closely related to those of sulfate reducers became predominant, and Geobacter species became a minor component of the community. This apparent switch from Fe(III) reduction to sulfate reduction as the terminal electron accepting process for the oxidation of the injected acetate was associated with an increase in uranium concentration in the groundwater. These results demonstrate that in situ bioremediation of uranium-contaminated groundwater is feasible but suggest that the strategy should be optimized to better maintain long-term activity of Geobacter species.     

恩诺沙星对土壤微生物群落功能多样性的影响

为了评价恩诺沙星(enrofloxacin)对土壤微生物群落的影响,借助BIOLOG检测法比较了不同浓度恩诺沙星影响下的土壤微生物的群落特征。结果表明,各添加药物组与空白对照组的土壤微生物群落代谢多样性差异显著(P<0.05),空白对照组土壤微生物在BIOLOG微平板上的平均每孔颜色变化率(AWCD)和微生物代谢多样性指数(丰富度和多样性)显著高于添加药物组;药物显著影响微生物群落对BIOLOG微平板中碳源的利用能力,较低浓度的恩诺沙星(0.01μg/g)就显著影响了土壤微生物群落对α-Ketobutyric Acid、L-Phenylalanine、1-Erythritol、D-Xylose等碳源的利用代谢能力(P<0.05),且土壤微生物群落利用各类碳源的能力随药物浓度加大而降低。由此可见,恩诺沙星在土壤中残留时对土壤微生物的影响不容忽视。