Spatial analysis of archaeal community structure in grassland soil

The complex structure of soil and the heterogeneity of resources available to microorganisms have implications for sampling regimens when the structure and diversity of microbial communities are analyzed. To assess the heterogeneity in community structure, archaeal communities, which typically contain sequences belonging to the nonthermophilic Crenarchaeota, were examined at two contrasting spatial scales by using PCR-denaturing gradient gel electrophoresis (DGGE) analysis followed by unweighted pair group method with arithmetic mean analysis of 16S rRNA- and ribosomal DNA-derived profiles. A macroscale analysis was carried out with soil cores taken at 2-m intervals along triplicate 8-m transects from both managed (improved) and natural (unimproved) grassland rhizosphere soils. A microscale analysis was carried out with a single soil core by assessing the effects of both sample size (10, 1, and 0.1 g) and distance between samples. The much reduced complexity of archaeal profiles compared to the complexity typical of the bacterial community facilitated visual comparison of profiles based on band presence and revealed different levels of heterogeneity between sets of samples. At the macroscale level, heterogeneity over the transect could not be related to grassland type. Substantial heterogeneity was observed across both improved and unimproved transects, except for one improved transect that exhibited substantial homogeneity, so that profiles for a single core were largely representative of the entire transect. At the smaller scale, the heterogeneity of the archaeal community structure varied with sample size within a single 8- by 8-cm core. The archaeal DGGE profiles for replicate 10-g soil samples were similar, while those for 1-g samples and 0.1-g samples showed greater heterogeneity. In addition, there was no relationship between the archaeal profiles and the distance between 1- or 0.1-g samples, although relationships between community structure and distance of separation may occur at a smaller scale. Our findings demonstrate the care required when workers attempt to obtain a representative picture of microbial community structure in the soil environment.     

Phylogenetic analysis and characterization of lipolytic activity of halophilic archaeal isolates

Five isolates designated as B45, D83A, A206A, A85 and E49 found to possess lipolytic activities were taxonomically classified on the basis of their phylogenetic, phenotypic and chemotaxonomic characteristics. The isolates were determined to be gram-negative, catalase and oxidase positive, hydrolyzing Tween 80 and 60 but not starch, need 3.5–4 M NaCl for optimal growth and lack of anaerobic growth with arginine or DMSO. All isolates had the highest lipolytic activity at pH 8.5. Lipase and esterase activities increased with salt concentration up to 3–4.5 M NaCl, and decreased at 5 M NaCl. Esterase and lipase showed their maximal activities at 50–55°C and 60–65°C, respectively. The phylogenetic tree constructed by the neighbor-joining method indicated that the strain B45 and A85 were closely related to the members of genera Halovivax and Natrinema, respectively. The closest relative of the strain A206A and D83A were found to be Haloterrigena saccharevitans. The strain E49 displayed a more distant relationship to known strains.     

Phylogenetic analysis and characterization of lipolytic activity of halophilic archaeal isolates

Five isolates designated as B45, D83A, A206A, A85 and E49 and found to possess a activity were taxonomically classified on the basis of their phylogenetic, phenotypic and chemotaxonomic characteristics. The isolates were determined to be Gram-negative, catalase and oxidase positive, hydrolyzing Tween 80 and 60 but not starch, need 3.5-4 M NaCl for optimal growth and lack of anaerobic growth with arginine or DMSO. All isolates had the highest lipolytic activity at pH 8.5. Lipase and esterase activities increased with salt concentration up to 3-4.5 M NaCl, and decreased at 5 M NaCl. Esterase and lipase showed their maximal activities at 50-55 degrees C and 60-65 degrees C, respectively. The phylogenetic tree constructed by the neighbor-joining method indicated that the strain B45 and A85 were closely related to the members of genera Halovivax and Natrinema, respectively. The closest relative of the strain A206A and D83A were found to be Haloterrigena saccharevitans. The strain E49 displayed a more distant relationship to known strains.     

Correlation between catalysis and tertiary structure arrangement in an archaeal halophilic subtilase

Nep (Natrialba magadii extracellular protease) is a halolysin-like peptidase secreted by the haloalkaliphilic archaeon N. magadii that exhibits optimal activity and stability in salt-saturated solutions. In this work, the effect of salt on the function and structure of Nep was investigated. In absence of salt, Nep became unfolded and aggregated, leading to the loss of activity. The enzyme did not recover its structural and functional properties even after restoring the ideal conditions for catalysis. At salt concentrations higher than 1 M (NaCl), Nep behaved as monomers in solution and its enzymatic activity displayed a nonlinear concave-up dependence with salt concentration resulting in a 20-fold activation at 4 M NaCl. Although transition from a high to a low-saline environment (3–1 M NaCl) did not affect its secondary structure contents, it diminished the enzyme stability and provoked large structural rearrangements, changing from an elongated shape at 3 M NaCl to a compact conformational state at 1 M NaCl. The thermodynamic analysis of peptide hydrolysis by Nep suggests a significant enzyme reorganization depending on the environmental salinity, which supports in solution SAXS and DLS studies. Moreover, solvent kinetic isotopic effect (SKIE) data indicates the general acid-base mechanism as the rate-limiting step for Nep catalysis, like classical serine-peptidases. All these data correlate the Nep conformational states with the enzymatic behavior providing a further understanding on the stability and structural determinants for the functioning of halolysins under different salinities.     

Microbial community structure analysis of produced water from a high-temperature North Sea oil-field

Molecular and culture-based methods were used to investigate the microbial diversity in produced water obtained from the high-temperature Troll oil formation in the North Sea. 16S rRNA gene libraries were generated from total community DNA, using universal archaeal or bacterial oligonucleotide primer sets. Sequence analysis of 88 clones in the bacterial library indicated that they originated from members of Firmicutes (48 sequences), Bacteroidetes (17 sequences), δ-Proteobacteria (15 sequences), Spirochaetes (5 sequences), Thermotogales (2 sequences) and γ-Proteobacteria (1 sequence). Twenty-two sequences in the archaeal library were close relatives to members of the genera Methanococcus (18 sequences), Methanolobus (3 sequences) and Thermococcus (1 sequence). Most of the bacterial sequences shared less than 95% identity with their closest match in GenBank, indicating that the produced water harbours a unique community of novel bacterial species or genera. Members of the thermophilic genera Thermosipho, Thermotoga, Anaerophaga and Thermovirga were isolated. The Troll formations are not injected with sea water. Thus, dramatic changes of the in situ conditions have been avoided, and a common source of continuous contamination from injection water can be excluded. However, the majority of the organisms detected in the gene libraries were most closely related to cultivated organisms with optimum temperatures for growth well below the in situ reservoir temperature (70°C), indicating that produced water from the Troll platform harbours a substantial amount of non-indigenous organisms. This was confirmed by the isolation of a number of mesophilic and moderately thermophilic organisms that were unable to grow at reservoir temperature.     

Microbial diversity and community structure of a highly active anaerobic methane‐oxidizing sulfate‐reducing enrichment

Anaerobic oxidation of methane (AOM) is an important methane sink in the ocean but the microbes responsible for AOM are as yet resilient to cultivation. Here we describe the microbial analysis of an enrichment obtained in a novel submerged‐membrane bioreactor system and capable of high‐rate AOM (286 μmol g_{dry weight}^?1 day^?1) coupled to sulfate reduction. By constructing a clone library with subsequent sequencing and fluorescent in situ hybridization, we showed that the responsible methanotrophs belong to the ANME‐2a subgroup of anaerobic methanotrophic archaea, and that sulfate reduction is most likely performed by sulfate‐reducing bacteria commonly found in association with other ANME‐related archaea in marine sediments. Another relevant portion of the bacterial sequences can be clustered within the order of Flavobacteriales but their role remains to be elucidated. Fluorescent in situ hybridization analyses showed that the ANME‐2a cells occur as single cells without close contact to the bacterial syntrophic partner. Incubation with ¹³C‐labelled methane showed substantial incorporation of ¹³C label in the bacterial C₁₆ fatty acids (bacterial; 20%, 44% and 49%) and in archaeal lipids, archaeol and hydroxyl‐archaeol (21% and 20% respectively). The obtained data confirm that both archaea and bacteria are responsible for the anaerobic methane oxidation in a bioreactor enrichment inoculated with Eckernförde bay sediment.     

Microbial biomass and community structure of a phase-separated methanogenic reactor determined by lipid analysis

Summary An anaerobic phase-separation biomass reactor was established on cellulose with the hydrolysis and fermentation steps occurring in the first stage, and acetogenesis and methanogenesis in the second stage. Based upon lipid biomarker analysis, eubacterial and eukaryotic cells accounted for approximately 6% of the volatile solids of the first stage and 17% of the second, while methanogens were approximately 1% of the volatile solids in the first stage and 9% of the second. Clustering the polar lipid fatty acids into groups based upon their distributions between the two stages of the reactor clarified the differences in community structure caused by phase-separated operation. Although inoculated from the same source, the two stages maintained very different microbial communities. Signature fatty acids known as indicators of unbalanced growth in eubacteria were significantly higher in the first stage of the reactor.     

Microbial community composition in sediments resists perturbation by nutrient enrichment

Functional redundancy in bacterial communities is expected to allow microbial assemblages to survive perturbation by allowing continuity in function despite compositional changes in communities. Recent evidence suggests, however, that microbial communities change both composition and function as a result of disturbance. We present evidence for a third response: resistance. We examined microbial community response to perturbation caused by nutrient enrichment in salt marsh sediments using deep pyrosequencing of 16S rRNA and functional gene microarrays targeting the nirS gene. Composition of the microbial community, as demonstrated by both genes, was unaffected by significant variations in external nutrient supply in our sampling locations, despite demonstrable and diverse nutrient-induced changes in many aspects of marsh ecology. The lack of response to external forcing demonstrates a remarkable uncoupling between microbial composition and ecosystem-level biogeochemical processes and suggests that sediment microbial communities are able to resist some forms of perturbation.     

Salt tolerance of archaeal extremely halophilic lipid membranes

The membranes of extremely halophilic Archaea are characterized by the abundance of a diacidic phospholipid, archaetidylglycerol methylphosphate (PGP-Me), which accounts for 50-80 mol% of the polar lipids, and by the absence of phospholipids with choline, ethanolamine, inositol, and serine head groups. These membranes are stable in concentrated 3-5 m NaCl solutions, whereas membranes of non-halophilic Archaea, which do not contain PGP-Me, are unstable and leaky under such conditions. By x-ray diffraction and vesicle permeability measurements, we demonstrate that PGP-Me contributes in an essential way to membrane stability in hypersaline environments. Large unilamellar vesicles (LUV) prepared from the polar lipids of extreme halophiles, Halobacterium halobium and Halobacterium salinarum, retain entrapped carboxyfluorescein and resist aggregation in the whole range 0-4 m NaCl, similarly to LUV prepared from purified PGP-Me. By contrast, LUV made of polar lipid extracts from moderately halophilic and non-halophilic Archaea (Methanococcus jannaschii, Methanosarcina mazei, Methanobrevibacter smithii) are leaky and aggregate at high salt concentrations. However, adding PGP-Me to M. mazei lipids results in gradual enhancement of LUV stability, correlating with the PGP-Me content. The LUV data are substantiated by the x-ray results, which show that H. halobium and M. mazei lipids have dissimilar phase behavior and form different structures at high NaCl concentrations. H. halobium lipids maintain an expanded lamellar structure with spacing of 8.5-9 nm, which is stable up to at least 100 degrees C in 2 m NaCl and up to approximately 60 degrees C in 4 m NaCl. However, M. mazei lipids form non-lamellar structures, represented by the Pn3m cubic phase and the inverted hexagonal H(II) phase. From these data, the forces preventing membrane aggregation in halophilic Archaea appear to be steric repulsion, because of the large head group of PGP-Me, or possibly out-of-plane bilayer undulations, rather than electrostatic repulsion attributed to the doubly charged PGP-Me head group.     

Microbial community and function of enrichment cultures with methane and toluene

The interaction effect of co-existence of toluene and CH₄ on community and activity of methanotrophs and toluene-degrading bacteria was characterized in three consortia enriched with CH₄ and toluene (MT), toluene (T), and CH₄ (M), respectively, in this study. The CH₄ oxidation activity in the enrichment culture of MT was significantly lower than that of M at the end of the experiment (P?=?0.001). The toluene degradation rate could be enhanced by continuous addition of CH₄ and toluene in the initial days, but it was inhibited in the later days. Phylogenetic analysis of 16S rRNA genes showed that Proteobacteria and Bacteroidetes were dominant in the three enriched consortia, but the community of methanotrophs and toluene-degrading bacteria was significantly affected by the co-existence of CH₄ and toluene. Both Methylosinus (91.8 %) and Methylocystis (8.2 %) were detected in the enrichment culture of MT, while only Methylocystis species were detected in M. The toluene-degrading bacteria including Burkholderia, Flavobacteria, Microbacterium, and Azoarcus were all detected in the enrichment culture of T. However, only Azoarcus was found in the enrichment culture of MT. Significantly higher contents of extracellular polymeric substances polysaccharose and protein in the enrichment culture of MT than that of T and M suggested that a higher environmental stress occurred in the enrichment culture of MT.     

Production of halocin is a practically universal feature of archaeal halophilic rods

Antagonisms among members of nine phenons of halobacteria were detected by combining two methods based on the double layer technique. Inhibitory activities were not due to phages. The protein nature of the inhibitors indicated that they were halocins. With only one exception, all strains tested exhibited inhibitory activity against other halobacteria due to the production of halocins. A very wide range of activity spectra was detected and the numerical comparative analysis showed little grouping, due to the scarce similarities between them. This indicates that many different halocins are produced by this heterogeneous group of micro-organisms. Our results show that halocin production should be considered as a practically general feature of halobacteria.     

Microbial community structure and global trace gases

Global change can affect soil processes by either altering the functioning of existing organisms or by restructuring the community, modifying the fundamental physiologies that drive biogeochemical processes. Thus, not only might process rates change, but the controls over them might also change. Moreover, previously insignificant processes could become important. These possibilities raise the question ‘Will changes in climate and land use restructure microbial communities in a way that will alter trace gas fluxes from an ecosystem?’ Process studies indicate that microbial community structure can influence trace gas dynamics at a large scale. For example, soil respiration and CH₄ production both show ranges of temperature response among ecosystems, indicating differences in the microbial communities responsible. There are three patterns of NH₄⁺ inhibition of CH₄ oxidation at the ecosystem scale: no inhibition, immediate inhibition, and delayed inhibition; these are associated with different CH₄ oxidizer communities. Thus, it is possible that changes in climate, land-use, and disturbance regimes could alter microbial communities in ways that would substantially alter trace gas fluxes; we discuss the data supporting this conclusion. We also discuss approaches to developing research linking microbial community structure and activity to the structure and functioning of the whole ecosystem. Modern techniques allow us to identify active organisms even if they have not been cultivated; in combination with traditional experimental approaches we should be able to identify the linkages between these active populations and the processes they carry out at the ecosystem level. Finally, we describe scenarios of how global change could alter trace gas fluxes by altering microbial communities and how understanding the microbial community dynamics could improve our ability to predict future trace gas fluxes.     

Microbial community analysis by FISH for mathematical modelling of selective enrichment of gel-entrapped nitrifiers obtained from domestic wastewater

Nitrifying activated sludge from natural domestic sewage was entrapped in hydrogel beads, which were subsequently enriched for nitrifiers in a continuous stirred tank reactor (CSTR). Fluorescently labelled, 16S rRNA-targeted oligonucleotide probes specific for ammonia and nitrite oxidisers were used in combination with DAPI staining to monitor the selectivity of the enrichment process. The growth of both nitrifying and heterotrophic bacteria was more pronounced in the periphery of the beads, leading to a biofilm-like stratification of the biomass during the enrichment. Quantitatively, the relative number of nitrifiers increased from 20% immediately after immobilisation up to 64% after 30 days, but decreased again due to extensive heterotrophic growth. These changes were accompanied by an increase in nitrifying activity for about 30 days, whereupon it reached a stable level. This selective enrichment was mathematically modelled by applying finite difference techniques to the diffusion-reaction mass balances of all soluble substrates relevant in the nitrification process. To model biomass growth and spreading, balanced by both decay and detachment at the surface of the beads, the differential methods were combined with a descrete cellular automaton approach. The spatially two-dimensional model was used to calculate radial concentration profiles within a gel bead, as well as to estimate the corresponding total activity of the reactor. Qualitatively, this model could simulate all essential aspects observed experimentally. However, more and better population data as well as independent estimates of decay and hydrolysis rates are needed to refine and verify the quantitative model. In conclusion, even in the absence of an external carbon source and with excess ammonium, it was only possible to obtain a moderate enrichment of nitrifying cells compared to heterotrophs. Under long-term cultivation, the biofilm-like structure developed in the outer gel layers led to a vigorous competition between auto- and heterotrophs for space, and thereby, access to oxygen. FISH analysis in combination with mathematical modelling constitute a suitable toolbox for analysing the population dynamics and biocatalytic performance of such an ecosystem based on lithoautotrophic primary production.     

PCR biases distort bacterial and archaeal community structure in pyrosequencing datasets

As 16S rRNA gene targeted massively parallel sequencing has become a common tool for microbial diversity investigations, numerous advances have been made to minimize the influence of sequencing and chimeric PCR artifacts through rigorous quality control measures. However, there has been little effort towards understanding the effect of multi-template PCR biases on microbial community structure. In this study, we used three bacterial and three archaeal mock communities consisting of, respectively, 33 bacterial and 24 archaeal 16S rRNA gene sequences combined in different proportions to compare the influences of (1) sequencing depth, (2) sequencing artifacts (sequencing errors and chimeric PCR artifacts), and (3) biases in multi-template PCR, towards the interpretation of community structure in pyrosequencing datasets. We also assessed the influence of each of these three variables on α- and β-diversity metrics that rely on the number of OTUs alone (richness) and those that include both membership and the relative abundance of detected OTUs (diversity). As part of this study, we redesigned bacterial and archaeal primer sets that target the V3-V5 region of the 16S rRNA gene, along with multiplexing barcodes, to permit simultaneous sequencing of PCR products from the two domains. We conclude that the benefits of deeper sequencing efforts extend beyond greater OTU detection and result in higher precision in β-diversity analyses by reducing the variability between replicate libraries, despite the presence of more sequencing artifacts. Additionally, spurious OTUs resulting from sequencing errors have a significant impact on richness or shared-richness based α- and β-diversity metrics, whereas metrics that utilize community structure (including both richness and relative abundance of OTUs) are minimally affected by spurious OTUs. However, the greatest obstacle towards accurately evaluating community structure are the errors in estimated mean relative abundance of each detected OTU due to biases associated with multi-template PCR reactions.     

Microbial diversity and community structure in Fynbos soil

The Fynbos biome in South Africa is renowned for its high plant diversity and the conservation of this area is particularly important for the region. This is especially true in the case of endangered vegetation types on the lowlands such as Sand Fynbos, of which only small fragments remain. The question is thus whether the diversity of the above‐ground flora is mirrored in the below‐ground microbial communities. In order to determine the relationship of the above‐ and below‐ground communities, the soil community composition of both fungal and bacterial groups in Sand Fynbos was characterized over space and time. A molecular approach was used based on the isolation of total soil genomic DNA and automated ribosomal intergenic spacer analysis of bacterial and fungal communities. Soil from four different sites was compared to resolve the microbial diversity of eubacterial and fungal groups on a local (alpha diversity) scale as well as a landscape scale (beta diversity). The community structures from different sites were compared and found to exhibit strong spatial patterns which remained stable over time. The plant community data were compared with the fungal and the bacterial communities. We concluded that the microbial communities in the Sand Fynbos are highly diverse and closely linked to the above‐ground floral communities.     

Microbial community structure in polluted Baltic Sea sediments

Nearly half the seabed of the Baltic Proper is incapable of supporting life of higher organisms as a consequence of oxygen depletion resulting from eutrophication. However, these areas are actually teeming with microbial life. Here we used terminal-restriction fragment length polymorphism (T-RFLP) to investigate the dominant archaeal and bacterial groups, with respect to community structure, in surface layers of bottom sediments of the Baltic Sea along a coastal pollution gradient. Both archaeal and bacterial communities formed distinct clusters along the pollution gradient and the community compositions were different at the polluted sites compared with the relatively clean reference sites. The structures of the bacterial communities were most strongly correlated to water depth, followed by organic carbon, oxygen, salinity and silicate levels. In contrast, the structures of the archaeal communities were most strongly correlated to oxygen, salinity, organic carbon, silicate and nitrate levels. Some members of the microbial communities were identified using a combination of traditional and molecular approaches. Isolates obtained on different culture media were identified by partial sequencing of their 16S rRNA genes and some novel species were found. In addition, we developed a computer program, aplaus, to elucidate the putative identities of the most dominant community members by T-RFLP.     

Microbial community structure and trichloroethylene degradation in groundwater

Trichloroethylene (TCE) is a prevalent contaminant of groundwater that can be cometabolically degraded by indigenous microbes. Groundwater contaminated with TCE from a US Department of Energy site in Ohio was used to characterize the site-specific impact of phenol on the indigenous bacterial community for use as a possible remedial strategy. Incubations of 14C-TCE-spiked groundwater amended with phenol showed increased TCE mineralization compared with unamended groundwater. Community structure was determined using DNA directly extracted from groundwater samples. This DNA was then analyzed by amplified ribosomal DNA restriction analysis. Unique restriction fragment length polymorphisms defined operational taxonomic units that were sequenced to determine phylogeny. DNA sequence data indicated that known TCE-degrading bacteria including relatives of Variovorax and Burkholderia were present in site water. Diversity of the amplified microbial rDNA clone library was lower in phenol-amended communities than in unamended groundwater (i.e., having Shannon-Weaver diversity indices of 2.0 and 2.2, respectively). Microbial activity was higher in phenol-amended ground water as determined by measuring the reduction of 2-(p-iodophenyl)-3(p-nitrophenyl)-5-phenyl tetrazolium chloride. Thus phenol amendments to groundwater correlated with increased TCE mineralization, a decrease in diversity of the amplified microbial rDNA clone library, and increased microbial activity.     

Microbial community structure of a pilot-scale thermophilic anaerobic digester treating poultry litter

The microbial community structure of a stable pilot-scale thermophilic continuous stirred tank reactor digester stabilized on poultry litter was investigated. This 40-m³ digester produced biogas with 57 % methane, and chemical oxygen demand removal of 54 %. Bacterial and archaeal diversity were examined using both cloning and pyrosequencing that targeted 16S rRNA genes. The bacterial community was dominated by phylum Firmicutes, constituting 93 % of the clones and 76 % of the pyrotags. Of the Firmicutes, class Clostridia (52 % pyrotags) was most abundant followed by class Bacilli (13 % pyrotags). The bacterial libraries identified 94 operational taxonomic units (OTUs) and pyrosequencing identified 577 OTUs at the 97 % minimum similarity level. Fifteen OTUs were dominant (≥2 % abundance), and nine of these were novel unclassified Firmicutes. Several of the dominant OTUs could not be classified more specifically than Clostridiales, but were most similar to plant biomass degraders, including Clostridium thermocellum. Of the rare pyrotag OTUs (<0.5 % abundance), 75 % were Firmicutes. The dominant methanogen was Methanothermobacter which has hydrogenotrophic metabolism, and accounted for >99 % of the archaeal clones. Based on the primary methanogen, as well as digester chemistry (high VA and ammonia levels), we propose that bacterial acetate oxidation is the primary pathway in this digester for the control of acetate levels.