Anaerobic Microbial Communities in Lake Pavin, a Unique Meromictic Lake in France

The Bacteria and Archaea from the meromictic Lake Pavin were analyzed in samples collected along a vertical profile in the anoxic monimolimnion and were compared to those in samples from the oxic mixolimnion. Nine targeted 16S rRNA oligonucleotide probes were used to assess the distribution of Bacteria and Archaea and to investigate the in situ occurrence of sulfate-reducing bacteria and methane-producing Archaea involved in the terminal steps of the anaerobic degradation of organic material. The diversity of the complex microbial communities was assessed from the 16S rRNA polymorphisms present in terminal restriction fragment (TRF) depth patterns. The densities of the microbial community increased in the anoxic layer, and Archaea detected with probe ARCH915 represented the largest microbial group in the water column, with a mean Archaea/Eubacteria ratio of 1.5. Terminal restriction fragment length polymorphism (TRFLP) analysis revealed an elevated archaeal and bacterial phylotype richness in anoxic bottom-water samples. The structure of the Archaea community remained rather homogeneous, while TRFLP patterns for the eubacterial community revealed a heterogeneous distribution of eubacterial TRFs.     

Microbial Community Structure and Dynamics in the Largest Natural French Lake (Lake Bourget)

We investigated the dynamics and diversity of heterotrophic bacteria, autotrophic and heterotrophic flagellates, and ciliates from March to July 2002 in the surface waters (0–50 m) of Lake Bourget. The heterotrophic bacteria consisted mainly of “small” cocci, but filaments (>2 μm), commonly considered to be grazing-resistant forms under increased nanoflagellate grazing, were also detected. These elongated cells mainly belonged to the Cytophaga-Flavobacterium (CF) cluster, and were most abundant during spring and early summer, when mixotrophic or heterotrophic flagellates were the main bacterial predators. The CF group strongly dominated fluorescent in situ hybridization–detected cells from March to June, whereas clear changes were observed in early summer when Beta-proteobacteria and Alpha-proteobacteria increased concomitantly with maximal protist grazing pressures. The analysis of protist community structure revealed that the flagellates consisted mainly of cryptomonad forms. The dynamics of Cryptomonas sp. and Dinobryon sp. suggested the potential importance of mixotrophs as consumers of bacteria. This point was verified by an experimental approach based on fluorescent microbeads to assess the potential grazing impact of all protist taxa in the epilimnion. From the results, three distinct periods in the functioning of the epilimnetic microbial loop were identified. In early spring, mixotrophic and heterotrophic flagellates constituted the main bacterivores, and were regulated by the availability of their resources mainly during April (phase 1). Once the “clear water phase” was established, the predation pressure of metazooplankton represented a strong top-down force on all microbial compartments. During this period only mixotrophic flagellates occasionally exerted a significant bacterivory pressure (phase 2). Finally, the early summer was characterized by the highest protozoan grazing impact and by a rapid shift in the carbon pathway transfer, with a fast change-over of the main predators contribution, i.e., mixotrophic, heterotrophic flagellates and ciliates in bacterial mortality. The high abundance of ciliates during this period was consistent with the high densities of resources (heterotrophic nanoflagellates, algae, bacteria) in deep layers containing the most chlorophyll. Bacteria, as ciliates, responded clearly to increasing phytoplankton abundance, and although bacterial grazing impact could vary largely, bacterial abundance seemed to be primarily bottom-up regulated (phase 3).     

Microbial Ecology of an Extreme Acidic Environment, the Tinto River

The Tinto River (Huelva, southwestern Spain) is an extreme environment with a rather constant acidic pH along the entire river and a high concentration of heavy metals. The extreme conditions of the Tinto ecosystem are generated by the metabolic activity of chemolithotrophic microorganisms thriving in the rich complex sulfides of the Iberian Pyrite Belt. Molecular ecology techniques were used to analyze the diversity of this microbial community. The community's composition was studied by denaturing gradient gel electrophoresis (DGGE) using 16S rRNA and by 16S rRNA gene amplification. A good correlation between the two approaches was found. Comparative sequence analysis of DGGE bands showed the presence of organisms related to Leptospirillum spp., Acidithiobacillus ferrooxidans, Acidiphilium spp., “Ferrimicrobium acidiphilum,” Ferroplasma acidiphilum, and Thermoplasma acidophilum. The different phylogenetic groups were quantified by fluorescent in situ hybridization with a set of rRNA-targeted oligonucleotide probes. More than 80% of the cells were affiliated with the domain Bacteria, with only a minor fraction corresponding to Archaea. Members of Leptospirillum ferrooxidans, Acidithiobacillus ferrooxidans, and Acidiphilium spp., all related to the iron cycle, accounted for most of the prokaryotic microorganisms detected. Different isolates of these microorganisms were obtained from the Tinto ecosystem, and their physiological properties were determined. Given the physicochemical characteristics of the habitat and the physiological properties and relative concentrations of the different prokaryotes found in the river, a model for the Tinto ecosystem based on the iron cycle is suggested.     

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 β-Proteobacteria. Similarly, the hematite-associated community formed in the contaminated area was populated in large part by β-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.     

Functional Groups and Activities of Bacteria in a Highly Acidic Volcanic Mountain Stream and Lake in Patagonia,Argentina

Acidic volcanic waters are naturally occurring extreme habitats that are subject of worldwide geochemical research but have been little investigated with respect to their biology. To fill this gap, the microbial ecology of a volcanic acidic river (pH approximately equal to 0-1.6), Rio Agrio, and the recipient lake Caviahue in Patagonia, Argentina, was studied. Water and sediment samples were investigated for Fe(II), Fe(III), methane, bacterial abundances, biomass, and activities (oxygen consumption, iron oxidation and reduction). The extremely acidic river showed a strong gradient of microbial life with increasing values downstream and few signs of life near the source. Only sulfide-oxidizing and fermentative bacteria could be cultured from the upper part of Rio Agrio. However, in the lower part of the system, microbial biomass and oxygen penetration and consumption in the sediment were comparable to non-extreme aquatic habitats. To characterize similarities and differences of chemically similar natural and man-made acidic waters, our findings were compared to those from acidic mining lakes in Germany. In the lower part of the river and the lake, numbers of iron and sulfur bacteria and total biomass in sediments were comparable to those known from acidic mining lakes. Bacterial abundance in water samples was also very similar for both types of acidic water (around 10(5) mL(-1)). In contrast, Fe(II) oxidation and Fe(III) reduction potentials appeared to be lower despite higher biogenic oxygen consumption and higher photosynthetic activity at the sediment-water interface. Surprisingly, methanogenesis was detected in the presence of high sulfate concentrations in the profundal sediment of Lake Caviahue. In addition to supplementing microbiological knowledge on acidic volcanic waters, our study provides a new view of these extreme sites in the general context of aquatic habitats.     

Cyanophage Diversity, Inferred from g20 Gene Analyses, in the Largest Natural Lake in France, Lake Bourget

The genetic diversity of the natural freshwater community of cyanophages and its variations over time have been investigated for the first time in the surface waters of the largest natural lake in France. This was done by random screening of clone libraries for the g20 gene and by denaturing gradient gel electrophoresis (DGGE). Nucleotide sequence analysis revealed 35 distinct cyanomyovirus g20 genotypes among the 47 sequences analyzed. Phylogenetic analyses showed that these sequences fell into seven genetically distinct operational taxonomic units (OTUs). The distances between these OTUs were comparable to those reported between marine clusters. Moreover, some of these freshwater cyanophage sequences were genetically more closely related to marine cyanophage sequences than to other freshwater sequences. Both approaches for the g20 gene (sequencing and DGGE analysis) showed that there was a clear seasonal pattern of variation in the composition of the cyanophage community that could reflect changes in its biological, chemical, and/or physical environment.     

Patterns in the Composition of Microbial Communities from a Subtropical River: Effects of Environmental,Spatial and Temporal Factors

Microbes are key components of aquatic ecosystems and play crucial roles in global biogeochemical cycles. However, the spatiotemporal dynamics of planktonic microbial community composition in riverine ecosystems are still poorly understood. In this study, we used denaturing gradient gel electrophoresis of PCR-amplified 16S and 18S rRNA gene fragments and multivariate statistical methods to explore the spatiotemporal patterns and driving factors of planktonic bacterial and microbial eukaryotic communities in the subtropical Jiulong River, southeast China. Both bacterial and microbial eukaryotic communities varied significantly in time and were spatially structured according to upper stream, middle-lower stream and estuary. Among all the environmental factors measured, water temperature, conductivity, PO₄-P and TN/TP were best related to the spatiotemporal distribution of bacterial community, while water temperature, conductivity, NO_x-N and transparency were closest related to the variation of eukaryotic community. Variation partitioning, based on partial RDA, revealed that environmental factors played the most important roles in structuring the microbial assemblages by explaining 11.3% of bacterial variation and 17.5% of eukaryotic variation. However, pure spatial factors (6.5% for bacteria and 9.6% for eukaryotes) and temporal factors (3.3% for bacteria and 5.5% for eukaryotes) also explained some variation in microbial distribution, thus inherent spatial and temporal variation of microbial assemblages should be considered when assessing the impact of environmental factors on microbial communities.     

Microscale Analyses of the Formation and Nature of Microbial Biofilm Communities in River Systems

The review covers aspects of biofilm cultivation, laser scanning microscopy, molecular probes and digital image analyses. This is accomplished through an overview of selected studies which illustrate the application of the microscale approach and laser microscopy techniques to the study of river biofilms and the results obtained.     

Microbial Ecology and Association of Bacillus thuringiensis in Chicken Feces Originating from Feed

To explain the association of Bacillus thuringiensis (Bt) with animal feces, an ecological analysis in chickens was conducted by introducing a cry ^? strain marked by production of green fluorescent protein (GFP). After feeding with the tagged Bt strains, the feces of the tested chickens were collected at different times, isolated, and the morphology of Bt was observed. It was shown that Bt strain HD-73GFP in spore form could be isolated from feces of chickens for a period of 13 d, and then it disappeared thereafter. Bt could be detected only up to day 4 (but not thereafter), when chickens were fed with vegetative cells of HD-73GFP. To confirm the source of newly isolated strains, the gfp gene was examined by polymerase chain reaction (PCR), which showed that all the isolated strains harbored the marker gene. Recent data from isolation and PCR had suggested that fecal Bt strains had originated from food. Chicken tissues were thus dissected to isolate Bt strains and to investigate whether Bt could be located in vivo. Bt was located within the duodenum in spore form. Compared to the morphology of the isolated strains at different growth times, the growth rates of all the tested Bt had little changes when passing through the digestive system to the feces. Dissection of the chickens confirmed that Bt was safe for the tested animal.     

Resource-based adaptive divergence in the freshwater fish Telmatherina from Lake Matano, Indonesia

Adaptive radiations are an important source of biodiversity, but resolving which ecological pressures seed these processes in natural systems remains difficult. Here the adaptive radiation among Telmatherina, a genus of freshwater fish endemic to an ancient lake in central Sulawesi, Indonesia, was examined to determine its causal root. We demonstrate that all Telmatherina in this lake can be categorized into three lineages each possessing specialized skull shapes and pharyngeal jaw bones allowing them to exploit different resources. These data demonstrate a natural example of how resource partitioning has likely initiated adaptive radiation in a resource limited environment.     

Microbial Community Composition and Ecology of an Acidic Aquatic Environment: The Tinto River, Spain

We studied the correlation between physicochemical and biological characteristics of an acidic river, the Tinto River, in Southwestern Spain. The Tinto River is an extreme environment characterized by its low pH (mean of 2.2) and high concentrations of heavy metals (Fe 2.3 g/L, Zn 0.22 g/L, Cu 0.11 g/L). These extreme conditions are the product of the metabolic activity of chemolithotrophic microorganisms, including iron- and sulfur-oxidizing bacteria, that can be found in high concentrations in its waters. The food chain in the river is very constrained and exclusively microbial. Primary productivity in the Tinto River is the sum of photosynthetic and chemolithotrophic activity. Heterotrophic bacteria and fungi are the major decomposers and protists are the major predators. A correlation analysis including the physicochemical and biological variables suggested a close relationship between the acidic pH values and abundance of both chemolithotrophic bacteria and filamentous fungi. Chemolithotrophic bacteria correlated with the heavy metals found in the river. A principal component analysis of the biotic and abiotic variables suggested that the Tinto River ecosystem can be described as a function of three main groups of variables: pH values, metal concentrations, and biological productivity.     

Nitrogen Fixation in Microbial Mat and Stromatolite Communities from Cuatro Cienegas,Mexico

Nitrogen fixation (nitrogenase activity, NA) of a microbial mat and a living stromatolite from Cuatro Cienegas, Mexico, was examined over spring, summer, and winter of 2004. The goal of the study was to characterize the diazotrophic community through molecular analysis of the nifH gene and using inhibitors of sulfate reduction and oxygenic and anoxygenic photosynthesis. We also evaluated the role of ultraviolet radiation on the diazotrophic activity of the microbial communities. Both microbial communities showed patterns of NA with maximum rates during the day that decreased significantly with 3-3,4-dichlorophenyl-1′,1′-dimethylurea, suggesting the potential importance of heterocystous cyanobacteria. There is also evidence of NA by sulfur-reducing bacteria in both microbial communities suggested by the negative effect exerted by the addition of sodium molybdate. Elimination of infrared and ultraviolet radiation had no effect on NA. Both microbial communities had nifH sequences that related to group I, including cyanobacteria and purple sulfur and nonsulfur bacteria, as well as group II nitrogenases, including sulfur reducing and green sulfur bacteria.     

A Microbial Arsenic Cycle in Sediments of an Acidic Mine Impoundment: Herman Pit,Clear Lake,California

The involvement of prokaryotes in the redox reactions of arsenic occurring between its +5 [arsenate; As(V)] and +3 [arsenite; As(III)] oxidation states has been well established. Most research to date has focused upon circum-neutral pH environments (e.g., freshwater or estuarine sediments) or arsenic-rich “extreme” environments like hot springs and soda lakes. In contrast, relatively little work has been conducted in acidic environments. With this in mind we conducted experiments with sediments taken from the Herman Pit, an acid mine drainage impoundment of a former mercury (cinnabar) mine. Due to the large adsorptive capacity of the abundant Fe(III)-rich minerals, we were unable to initially detect in solution either As(V) or As(III) added to the aqueous phase of live sediment slurries or autoclaved controls, although the former consumed added electron donors (i.e., lactate, acetate, hydrogen), while the latter did not. This prompted us to conduct further experiments with diluted slurries using the live materials from the first incubation as inoculum. In these experiments we observed reduction of As(V) to As(III) under anoxic conditions and reduction rates were enhanced by addition of electron donors. We also observed oxidation of As(III) to As(V) in oxic slurries as well as in anoxic slurries amended with nitrate. We noted an acid-tolerant trend for sediment slurries in the cases of As(III) oxidation (aerobic and anaerobic) as well as for anaerobic As(V) reduction. These observations indicate the presence of a viable microbial arsenic redox cycle in the sediments of this extreme environment, a result reinforced by the successful amplification of arsenic functional genes (aioA, and arrA) from these materials.     

Enumeration and Characterization of Iron(III)-Reducing Microbial Communities from Acidic Subsurface Sediments Contaminated with Uranium(VI)

Iron(III)-reducing bacteria have been demonstrated to rapidly catalyze the reduction and immobilization of uranium(VI) from contaminated subsurface sediments. Thus, these organisms may aid in the development of bioremediation strategies for uranium contamination, which is prevalent in acidic subsurface sediments at U.S. government facilities. Iron(III)-reducing enrichment cultures were initiated from pristine and contaminated (high in uranium, nitrate; low pH) subsurface sediments at pH 7 and pH 4 to 5. Enumeration of Fe(III)-reducing bacteria yielded cell counts of up to 240 cells ml⁻¹ for the contaminated and background sediments at both pHs with a range of different carbon sources (glycerol, acetate, lactate, and glucose). In enrichments where nitrate contamination was removed from the sediment by washing, MPN counts of Fe(III)-reducing bacteria increased substantially. Sediments of lower pH typically yielded lower counts of Fe(III)-reducing bacteria in lactate- and acetate-amended enrichments, but higher counts were observed when glucose was used as an electron donor in acidic enrichments. Phylogenetic analysis of 16S rRNA gene sequences extracted from the highest positive MPN dilutions revealed that the predominant members of Fe(III)-reducing consortia from background sediments were closely related to members of the Geobacteraceae family, whereas a recently characterized Fe(III) reducer (Anaeromyxobacter sp.) and organisms not previously shown to reduce Fe(III) (Paenibacillus and Brevibacillus spp.) predominated in the Fe(III)-reducing consortia of contaminated sediments. Analysis of enrichment cultures by terminal restriction fragment length polymorphism (T-RFLP) strongly supported the cloning and sequencing results. Dominant members of the Fe(III)-reducing consortia were observed to be stable over several enrichment culture transfers by T-RFLP in conjunction with measurements of Fe(III) reduction activity and carbon substrate utilization. Enrichment cultures from contaminated sites were also shown to rapidly reduce millimolar amounts of U(VI) in comparison to killed controls. With DNA extracted directly from subsurface sediments, quantitative analysis of 16S rRNA gene sequences with MPN-PCR indicated that Geobacteraceae sequences were more abundant in pristine compared to contaminated environments,whereas Anaeromyxobacter sequences were more abundant in contaminated sediments. Thus, results from a combination of cultivation-based and cultivation-independent approaches indicate that the abundance/community composition of Fe(III)-reducing consortia in subsurface sediments is dependent upon geochemical parameters (pH, nitrate concentration) and that microorganisms capable of producing spores (gram positive) or spore-like bodies (Anaeromyxobacter) were representative of acidic subsurface environments.     

Functional Gene Differences in Soil Microbial Communities from Conventional,Low-Input,and Organic Farmlands

Various agriculture management practices may have distinct influences on soil microbial communities and their ecological functions. In this study, we utilized GeoChip, a high-throughput microarray-based technique containing approximately 28,000 probes for genes involved in nitrogen (N)/carbon (C)/sulfur (S)/phosphorus (P) cycles and other processes, to evaluate the potential functions of soil microbial communities under conventional (CT), low-input (LI), and organic (ORG) management systems at an agricultural research site in Michigan. Compared to CT, a high diversity of functional genes was observed in LI. The functional gene diversity in ORG did not differ significantly from that of either CT or LI. Abundances of genes encoding enzymes involved in C/N/P/S cycles were generally lower in CT than in LI or ORG, with the exceptions of genes in pathways for lignin degradation, methane generation/oxidation, and assimilatory N reduction, which all remained unchanged. Canonical correlation analysis showed that selected soil (bulk density, pH, cation exchange capacity, total C, C/N ratio, NO₃⁻, NH₄⁺, available phosphorus content, and available potassium content) and crop (seed and whole biomass) variables could explain 69.5% of the variation of soil microbial community composition. Also, significant correlations were observed between NO₃⁻ concentration and denitrification genes, NH₄⁺ concentration and ammonification genes, and N₂O flux and denitrification genes, indicating a close linkage between soil N availability or process and associated functional genes.     

Influence of Land Use,Nutrients, and Geography on Microbial Communities and Fecal Indicator Abundance at Lake Michigan Beaches

Microbial communities within beach sand play a key role in nutrient cycling and are important to the nearshore ecosystem function. Escherichia coli and enterococci, two common indicators of fecal pollution, have been shown to persist in the beach sand, but little is known about how microbial community assemblages are related to these fecal indicator bacteria (FIB) reservoirs. We examined eight beaches across a geographic gradient and range of land use types and characterized the indigenous community structure in the water and the backshore, berm, and submerged sands. FIB were found at similar levels in sand at beaches adjacent to urban, forested, and agricultural land and in both the berm and backshore. However, there were striking differences in the berm and backshore microbial communities, even within the same beach, reflecting the very different environmental conditions in these beach zones in which FIB can survive. In contrast, the microbial communities in a particular beach zone were similar among beaches, including at beaches on opposite shores of Lake Michigan. The differences in the microbial communities that did exist within a beach zone correlated to nutrient levels, which varied among geographic locations. Total organic carbon and total phosphorus were higher in Wisconsin beach sand than in beach sand from Michigan. Within predominate genera, fine-scale sequence differences could be found that distinguished the populations from the two states, suggesting a biogeographic effect. This work demonstrates that microbial communities are reflective of environmental conditions at freshwater beaches and are able to provide useful information regarding long-term anthropogenic stress.     

Ecological Assessment and Geological Significance of Microbial Communities from Cesspool Cave,Virginia

Microbial mats from hydrogen sulfide-rich waters and cave-wall biofilms were investigated from Cesspool Cave, Virginia, to determine community composition and potential geomicrobiological functioning of acid-producing bacteria. Rates of microbial mat chemoautotrophic productivity were estimated using [ 14 C]-bicarbonate incorporations and microbial heterotrophy was determined using [ 14 C]-leucine incubations. Chemoautotrophic fixation was measured at 30.4 - 12.0 ng C mg dry wt -1 h -1 , whereas heterotrophic productivity was significantly less at 0.17 - 0.02 ng C mg dry wt -1 h -1 . The carbon to nitrogen ratios of the microbial mats averaged 13.5, indicating that the mats are not a high quality food source for higher trophic levels. Ribosomal RNA-based methods were used to examine bacterial diversity in the microbial mats, revealing the presence of at least five strains of bacteria. The identity of some of the strains could be resolved to the genus Thiothrix and the Flexibacter - Cytophaga - Bacteriodes phylum, and the identity of the remaining strains was to either the Helicobacter or Thiovulum group. Two of 10 sulfur-oxidizing, chemoautotrophic pure cultures of Thiobacillus spp. (syn. Thiomonas gen. nov.) demonstrated the ability to corrode calcium carbonate, suggesting that the colonization and metabolic activity of these bacteria may be enhancing cave enlargement.     

Ecology of the Major Periphytic Diatom Communities from the Mesta River,Bulgaria

Epilithon, epiphyton, epipelon, epipsammon and plocon diatom samples and water chemistry samples were collected bimonthly from 11 stations along the Mesta River, Bulgaria between December 1989 and April 1991. Principal component analysis (PCA), correlation, and dominance analysis were employed for describing the seasonal dynamics of diatom assemblages and estimating the correlation between diatom distribution and the physico-chemical parameters. All periphytic communities were compared by PCA, MANOVA and Fisher's LSD multiple comparison test. Ionic strength and eutrophication were the major abiotic factors affecting diatom distribution in the epilithon, epiphyton, epipelon and epipsammon. Current velocity had an important influence on the diatom assemblages from the epilithon and epiphyton. Epilithon showed the lowest correlation with most of the environmental factors and had significantly lower species diversity than epiphyton, epipelon and epipsammon. Multivariate analysis of the diatom data suggested that in all benthic habitats of the Mesta River seasonal replacement of diatom assemblages was controlled by the water chemistry rather than substratum, current velocity or discharge. The oligotrophic, mesotrophic and eutrophic diatom communities in the Mesta River comprised early-, mid- and late-successional colonizers respectively. Multiple comparisons of all periphytic communities showed that habitat specificity was positively correlated with current velocity.