Characterization of the depth-related changes in the microbial communities in Lake Hovsgol sediment by 16S rRNA gene-based approaches

The undisturbed sediment of Lake Hovsgol (Mongolia) is scientifically important because it represents a record of the environmental changes that took place between the Holocene (the present age) and Pleistocene (the last ice age; 12,000 14C years before present day). Here, we investigated how the current microbial communities change as the depth increases by PCR-denaturing gradient gel electrophoresis (DGGE) analysis of the 16S rRNA genes of the microbial communities. The microbial diversity, as estimated by the Shannon index, decreased as the depth increased. In particular, significant changes in archaeal diversity were observed in the middle depth (at 39-42 cm depth of total 60 cm depth) that marks the border between the Holocene and Pleistocene. Phylotype belonging to Beta-and Gamma-Proteobacteria were the predominant bacteria and most of these persisted throughout the depth examined. However, as the depth increased, some bacteria (some genera belonging to Beta-Proteobacteria, Nitrospira, and OP8-9) were not detectable while others (some genera belonging to Alpha-, Beta-, Gamma-Proteobacteria) newly detected by DGGE. Crenarchaea were the predominant archaea and only one phylotype belonging to Euryarchaea was found. Both the archaeal and bacterial profiles revealed by the DGGE band patterns could be grouped into four and three subsets, respectively, subsets that were largely divided by the border between the Holocene and Pleistocene. Thus, the diversity of the current microbial communities in Lake Hovsgol sediments decreases with increasing depth. These changes probably relate to the environmental conditions in the sediments, which were shaped by the paleoclimatic events taking place between the Holocene and Pleistocene.     

16S rDNA-based characterization of BTX-catabolizing microbial associations isolated from a South African sandy soil

In the presence of different selection pressures, particularly pHand electron donor concentration, indigenous microbial associations which catabolize selected petroleum hydrocarbon components (benzene, toluene and o-, m- and p-xylene (BTX)) were enriched and isolated from a petroleum hydrocarbon-contaminated KwaZulu-Natal sandy soil. Electron microscopy revealed that, numerically, rods constituted the majority of the populations responsible for BTX catabolism. Molecular techniques (polymerase chain reaction (PCR) and 16S rDNA fingerprinting by denaturing-gradient gel electrophoresis (DGGE)) were employed to explore the diversities and analyze the structures of the isolated microbial associations. Pearson product-moment correlation indicated that the different, but chemically similar, petroleum hydrocarbon molecules, effectedthe isolation of different associations. However, some similar numerically-dominant bands characterized the associations. A 30% similarity was evident between the m- and o-xylene-catabolizing associations regardless of the molecule concentration and the enrichment pH. PCR-DGGE was also used to complement conventional culture-based microbiological procedures for environmental parameter optimization. Band pattern differencesindicated profile variations of the isolated associations which possibly accounted for the growth rate changes recorded in response to pH and temperature perturbations.     

Study of nitrogen fixation in microbial communities of oil-contaminated marine sediment microcosms

Aerobic microbial degradation of pollutant oil (petroleum) in aquatic environments is often severely limited by the availability of combined nitrogen. We therefore studied whether the microbial community enriched in marine sediment microcosms with an added oil layer and exposure to light harboured nitrogenase activity. The acetylene reduction (AR) assay indeed indicated active nitrogenase; however, similar activity was observed in oil-free control microcosms. In both microcosms, the AR rate was significantly reduced upon a dark shift, indicating that enriched cyanobacteria were the dominant diazotrophs. Analysis of structural dinitrogenase reductase genes (nifH) amplified from both microcosms indeed revealed NifH sequences related mostly to those of heterocystous cyanobacteria. NifH sequences typically affiliating with those of heterotrophic bacteria were more frequently retrieved from the oil-containing sediment. Expression analyses showed that mainly nifH genes similar to those of heterocystous cyanobacteria were expressed in the light. Upon a dark shift, nifH genes related to those of non-heterocystous cyanobacteria were expressed. Expression of nifH assignable to heterotrophs was apparently not significant. It is concluded that cyanobacteria are the main contributors of fixed nitrogen to oil-contaminated and pristine sediments if nitrogen is a limiting factor and if light is available. Hence, also the oil-degrading heterotrophic community may thus receive a significant part of combined nitrogen from cyanobacteria, even though oil vice versa apparently does not stimulate an additional nitrogen fixation in the enriched community.     

Vertical and temporal shifts in microbial communities in the water column and sediment of saline meromictic Lake Kaiike (Japan), as determined by a 16S rDNA-based analysis, and related to physicochemical gradients

The vertical and temporal changes in microbial communities were investigated throughout the water column and sediment of the saline meromictic Lake Kaiike by PCR-denaturing gradient gel electrophoresis (DGGE) of 16S rDNA. Marked depth-related changes in microbial communities were observed at the chemocline and the sediment-water interface. However, no major temporal changes in the microbial community below the chemocline were observed during the sampling period, suggesting that the ecosystem in the anoxic zone of Lake Kaiike was nearly stable. Although the sequence of the most conspicuous DGGE band throughout the anoxic water and in the top of the microbial mat was most similar to that of an anoxic, photosynthetic, green sulphur bacterium, Pelodyction luteolum DSM273 (97% similarity), it represented a new phylotype. A comparison of DGGE banding patterns of the water column and sediment samples demonstrated that specific bacteria accumulated on the bottom from the anoxic water layers, and that indigenous microbial populations were present in the sediment. The measurements of bicarbonate assimilation rates showed significant phototrophic assimilation in the chemocline and lithoautotrophic assimilation throughout the anoxic water, but were not clearly linked with net sulphide turnover rates, indicating that sulphur and carbon metabolisms were not directly correlated.     

Metabolically active microbial communities in marine sediment under high-CO2 and low-pH extremes

Sediment-hosting hydrothermal systems in the Okinawa Trough maintain a large amount of liquid, supercritical and hydrate phases of CO₂ in the seabed. The emission of CO₂ may critically impact the geochemical, geophysical and ecological characteristics of the deep-sea sedimentary environment. So far it remains unclear whether microbial communities that have been detected in such high-CO₂ and low-pH habitats are metabolically active, and if so, what the biogeochemical and ecological consequences for the environment are. In this study, RNA-based molecular approaches and radioactive tracer-based respiration rate assays were combined to study the density, diversity and metabolic activity of microbial communities in CO₂-seep sediment at the Yonaguni Knoll IV hydrothermal field of the southern Okinawa Trough. In general, the number of microbes decreased sharply with increasing sediment depth and CO₂ concentration. Phylogenetic analyses of community structure using reverse-transcribed 16S ribosomal RNA showed that the active microbial community became less diverse with increasing sediment depth and CO₂ concentration, indicating that microbial activity and community structure are sensitive to CO₂ venting. Analyses of RNA-based pyrosequences and catalyzed reporter deposition-fluorescence in situ hybridization data revealed that members of the SEEP-SRB2 group within the Deltaproteobacteria and anaerobic methanotrophic archaea (ANME-2a and -2c) were confined to the top seafloor, and active archaea were not detected in deeper sediments (13–30 cm in depth) characterized by high CO₂. Measurement of the potential sulfate reduction rate at pH conditions of 3–9 with and without methane in the headspace indicated that acidophilic sulfate reduction possibly occurs in the presence of methane, even at very low pH of 3. These results suggest that some members of the anaerobic methanotrophs and sulfate reducers can adapt to the CO₂-seep sedimentary environment; however, CO₂ and pH in the deep-sea sediment were found to severely impact the activity and structure of the microbial community.     

Study of bacterial communities in Antarctic coastal waters by a combination of 16S rRNA and 16S rDNA sequencing

An ecological study on distribution of Antarctic bacterial communities was determined by 16S-based phylogenetic analyses of clone libraries derived from RNA and DNA extracted from two different marine areas and compared between each other. Superficial seawater samples were collected from four stations in Ross Sea, three of them located in Rod Bay and one in Evans Cove; for each station two clone libraries (16S rDNA and 16S rRNA) were prepared and evident divergences between DNA and RNA libraries of each site were obtained. Of all phylotypes 93.6% were found in RNA libraries; in contrast, only 31 phylotypes (70.5%) were retrieved from total microbial community (DNA libraries). DNA and RNA sequences related to gamma-Proteobacteria and Bacteroidetes groups, typical for Antarctic sea-ice bacterial communities, were detected in analysed sites. 16S rDNA and rRNA libraries derived from the two different areas were enriched by picophytoplanktonic 16S sequences of plastid and mitochondrion origins, reflecting that the algal blooms occurred during sampling (Antarctic summer 2003). The finding in Rod Bay libraries of high percentage of DNA clones apparently affiliated with beta-Proteobacteria typical for activated sludges and well water could be explained by the presence of a sewage depuration system at this site. Obtained results clearly demonstrate that combination of 16S rDNA and 16S rRNA gene sequencing is preferred approach to have a more reliable vision on the composition of microbial communities.     

Multiple detection of food-borne pathogenic bacteria using a novel 16S rDNA-based oligonucleotide signature chip

There have been many attempts to develop sensitive and accurate techniques for the detection and diagnosis of pathogenic bacteria using nucleic acid-based technology. To achieve efficient multiple detection of seven selected food-borne pathogens, we assessed the respective 16S rDNA pathogen specific sequences using an oligonucleotide-based signature array. Strategic optimal design of specific capture probes was achieved by using the characteristic first variable region. To assess the specificity of this pathogen detection system, we employed a two-step experimental strategy. Under conditions established through experiments with chemically synthesized model targets comprising both conserved and variable regions of 16S rDNA, we confirmed the validity of this system using real 16S rDNA targets. Detection with real targets was successfully performed using our system, and better specificity was obtained compared to experiments with model targets. Moreover, the subtypes of Vibrio pathogens were successfully classified. We developed a two-dimensional visualization plot tool for positive control and specific spots, which allowed facile and minute differentiation between spot intensities. Repeated array formats were employed to ensure experimental uniformity, and included the statistical p-value criterion for pathogen discrimination. The present results thus indicate that our novel oligonucleotide-based signature chip detection system can be employed for the effective detection of multiple pathogens.     

Shifts in coastal Antarctic marine microbial communities during and after melt water-related surface stratification

Antarctic coastal waters undergo major physical alterations during summer. Increased temperatures induce sea-ice melting and glacial melt water input, leading to strong stratification of the upper water column. We investigated the composition of micro-eukaryotic and bacterial communities in Ryder Bay, Antarctic Peninsula, during and after summertime melt water stratification, applying community fingerprinting (denaturing gradient gel electrophoresis) and sequencing analysis of partial 18S and 16S rRNA genes. Community fingerprinting of the eukaryotic community revealed two major patterns, coinciding with a period of melt water stratification, followed by a period characterized by regular wind-induced breakdown of surface stratification. During the first stratified period, we observed depth-related differences in eukaryotic fingerprints while differences in bacterial fingerprints were weak. Wind-induced breakdown of the melt water layer caused a shift in the eukaryotic community from an Actinocyclus sp.- to a Thalassiosira sp.-dominated community. In addition, a distinct transition in the bacterial community was found, but with a few days' delay, suggesting a response to the changes in the eukaryotic community rather than to the mixing event itself. Sequence analysis revealed a shift from an Alpha- and Gammaproteobacteria to a Cytophaga-Flavobacterium-Bacteroides-dominated community under mixed conditions. Our results show that melt water stratification and the transition to nonstabilized Antarctic surface waters may have an impact not only on micro-eukaryotic but also bacterial community composition.     

Functional redundancy of seasonal vitamin B12 biosynthesis pathways in coastal marine microbial communities

Vitamin B₁₂ (cobalamin) is a major cofactor required by most marine microbes, but only produced by a few prokaryotes in the ocean, which is globally B₁₂-depleted. Despite the ecological importance of B₁₂, the seasonality of B₁₂ metabolisms and the organisms involved in its synthesis in the ocean remain poorly known. Here we use metagenomics to assess the monthly dynamics of B₁₂-related pathways and the functional diversity of associated microbial communities in the coastal NW Mediterranean Sea over 7 years. We show that genes related to potential B₁₂ metabolisms were characterized by an annual succession of different organisms carrying distinct production pathways. During the most productive winter months, archaea (Nitrosopumilus and Nitrosopelagicus) were the main contributors to B₁₂ synthesis potential through the anaerobic pathway (cbi genes). In turn, Alphaproteobacteria (HIMB11, UBA8309, Puniceispirillum) contributed to B₁₂ synthesis potential in spring and summer through the aerobic pathway (cob genes). Cyanobacteria could produce pseudo-cobalamin from spring to autumn. Finally, we show that during years with environmental perturbations, the organisms usually carrying B₁₂ synthesis genes were replaced by others having the same gene, thus maintaining the potential for B₁₂ production. Such ecological insurance could contribute to the long-term functional resilience of marine microbial communities exposed to contrasting inter-annual environmental conditions.     

Characterization of microbial communities in anaerobic bioreactors using molecular probes

The microbial community structure of twenty-one single-phase and one two-phase full-scale anaerobic sewage sludge digesters was evaluated using oligonucleotide probes complementary to conserved tracts of the 16S rRNAs of phylogenetically defined groups of methanogens and sulfate-reducing bacteria. These probe results were interpreted in combination with results from traditional chemical analyses and metabolic activity assays. It was determined that methanogens in healthy mesophilic, single-phase sewage sludge digesters accounted for approximately 8–12% of the total community and thatMethanosarcinales andMethanomicrobiales constituted the majority of the total methanogen population.Methanobacteriales andMethanococcales played a relatively minor role in the digesters. Phylogenetic groups of mesophilic, Gram-negative sulfate-reducing bacteria were consistently present at significant levels:Desulfovibrio andDesulfobulbus spp. were the dominant sulfate-reducing populations,Desulfobacter andDesulfobacterium spp. were present at lower levels, andDesulfosarcina, Desulfococcus, andDesulfobotulus spp. were absent. Sulfate reduction by one or more of these populations played a significant role in all digesters evaluated in this study. In addition, sulfate-reducing bacteria played a role in favoring methanogenesis by providing their substrates. The analysis of the two-phase digester indicated that true phase separation was not accomplished: significant levels of active methanogens were present in the first phase. It was determined that the dominant populations in the second phase were different from those in the single-phase digesters.     

Occurrence of nitric and nitrous oxides in a coastal marine sediment.

The distribution of denitrification activity in a coastal marine sediment was determined by the acetylene inhibition technique and compared to concentration profiles of NO3-, NO2-, NO, and N2O. The bulk of the denitrification activity was associated with the accumulation of NO3- in the oxidized surface zone of the sediment, but a secondary denitrification zone was occasionally found in the deeper layers where oxidized patches had been introduced by the burrowing activity of the macrofauna. Maxima of NO and N2O were not associated with the peak activity of denitrification in the surface zone but were located at the lower edge of the activity profile. Significant accumulation of NO was found at the redox transition zone towards the deeper, sulfide-rich layers.     

Analysis of bacterial and archaeal diversity in coastal microbial mats using massive parallel 16S rRNA gene tag sequencing

Coastal microbial mats are small-scale and largely closed ecosystems in which a plethora of different functional groups of microorganisms are responsible for the biogeochemical cycling of the elements. Coastal microbial mats play an important role in coastal protection and morphodynamics through stabilization of the sediments and by initiating the development of salt-marshes. Little is known about the bacterial and especially archaeal diversity and how it contributes to the ecological functioning of coastal microbial mats. Here, we analyzed three different types of coastal microbial mats that are located along a tidal gradient and can be characterized as marine (ST2), brackish (ST3) and freshwater (ST3) systems. The mats were sampled during three different seasons and subjected to massive parallel tag sequencing of the V6 region of the 16S rRNA genes of Bacteria and Archaea. Sequence analysis revealed that the mats are among the most diverse marine ecosystems studied so far and consist of several novel taxonomic levels ranging from classes to species. The diversity between the different mat types was far more pronounced than the changes between the different seasons at one location. The archaeal community for these mats have not been studied before and revealed a strong reaction on a short period of draught during summer resulting in a massive increase in halobacterial sequences, whereas the bacterial community was barely affected. We concluded that the community composition and the microbial diversity were intrinsic of the mat type and depend on the location along the tidal gradient indicating a relation with salinity.     

RNA microarray for estimating relative abundance of 16S rRNA in microbial communities

We developed an RNA microarray protocol in which total RNA from a microbial community was attached to a slide glass, and rRNA was detected by fluorescently labeled oligonucleotide probes. The RNA microarray requires only 4 h for hybridization and enables double staining and estimating relative abundance of rRNA.     

Characterization of microbial community structure and population dynamics of tetrachloroethene-dechlorinating tidal mudflat communities

Tetrachloroethene (PCE) and trichloroethene (TCE) are common groundwater contaminants that also impact tidal flats, especially near urban and industrial areas. However, very little is known about dechlorinating microbial communities in tidal flats. Titanium pyrosequencing, 16S rRNA gene clone libraries, and dechlorinator-targeted quantitative real-time PCR (qPCR) characterized reductive dechlorinating activities and populations in tidal flat sediments collected from South Korea’s central west coast near Kangwha. In microcosms established with surface sediments, PCE dechlorination to TCE began within 10 days and 100% of the initial amount of PCE was converted to TCE after 37 days. cis-1,2-Dichloroethene (cis-DCE) was observed as dechlorination end product in microcosms containing sediments collected from deeper zones (i.e., 35–40 cm below ground surface). Pyrosequencing of bacterial 16S rRNA genes and 16S rRNA gene-targeted qPCR results revealed Desulfuromonas michiganensis-like populations predominanted in both TCE and cis-DCE producing microcosms. Other abundant groups included Desulfuromonas thiophila and Pelobacter acidigallici-like populations in the surface sediment microcosms, and Desulfovibrio dechloracetivorans and Fusibacter paucivorans-like populations in the deeper sediment microcosms. Dehalococcoides spp. populations were not detected in these sediments before and after incubation with PCE. The results suggest that tidal flats harbor novel, salt-tolerant dechlorinating populations and that titanium pyrosequencing provides more detailed insight into community structure dynamics of the dechlorinating microcosms than conventional 16S rRNA gene sequencing or fingerprinting methods.     

Characterization of depth-related bacterial communities and their relationships with the environmental factors in the river sediments

To better understand the bacterial processes in river sediments, it is necessary to investigate the depth-related bacterial communities in the whole sediment profile. Sediment samples were collected to a depth of 25 cm from the Pearl River. Bacterial abundance, activity, cell-specific respiration rate, and diversity were measured, respectively, by 4′, 6-diamidino-2-phenylindole direct count, 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) staining, electron transport system by CTC reduction, and denaturing gradient gel electrophoresis analysis of 16S rRNA amplification fragments. Results showed that the bacterial metabolism activities decreased with the sediment depth. The total bacterial abundance was highest in the surface sediment with 65.1 × 10⁷ cells g⁻¹, and decreased to 11.1 × 10⁷ cells g⁻¹ below 20 cm in the sample location that suffered from heavy sewage inputs. The active bacteria accounted for 7.50–46.7% of the total bacterial number and decreased with the sediment depth. Electron transport system by the CTC reduction showed that bacterial respiration rate declined from 1.093 μmol CTC-formazan h⁻¹ g⁻¹ in the surface sediment to a half in the bottom sediment, while the cell-specific respiration increased significantly with the depth from 3.56 to 93.75 fmol CTC-formazan cell⁻¹. The bacterial diversity also changed with the depth. Beta-Proteobacteria were the dominant species in the surface sediment, whereas Delta-Proteobacteria were the main species below 10 cm. Results of canonical correspondence analysis (CCA) indicated that the distribution of bacteria was affected by the combined effect of various dissolved inorganic matter, while the respiration rate was independent of the nutrient conditions. The specific bacterial distribution contributed to not only the nutrient cycle but also enhanced pollutant decomposition in sediment of the Pearl River. The results showed that some specific bacterial species had a strong activity in the deeper layers. Therefore, the metabolic functions of the deeper bacterial species should not be neglected.