Active Soil Nitrifying Communities Revealed by In Situ Transcriptomics and Microcosm-Based Stable-Isotope Probing.

Long-term nitrogen field fertilization often results in significant changes in nitrifying communities that catalyze a key step in the global N cycle. However, whether microcosm studies are able to inform the dynamic changes in communities of ammonia-oxidizing bacteria (AOB) and archaea (AOA) under field conditions remains poorly understood. This study aimed to evaluate the transcriptional activities of nitrifying communities under in situ conditions, and we found that they were largely similar to those of 13C-labeled nitrifying communities in the urea-amended microcosms of soils that had received different N fertilization regimens for 22 years. High-throughput sequencing of 16S rRNA genes and transcripts suggested that Nitrosospira cluster 3-like AOB and Nitrososphaera viennensis-like AOA were significantly stimulated in N-fertilized fresh soils. Real-time quantitative PCR demonstrated that the significant increase of AOA and AOB in fresh soils upon nitrogen fertilization could be preserved in the air-dried soils. DNA-based stable-isotope probing (SIP) further revealed the greatest labeling of Nitrosospira cluster 3-like AOB and Nitrosospira viennensis-like AOA, despite the strong advantage of AOB over AOA in the N-fertilized soils. Nitrobacter-like nitrite-oxidizing bacteria (NOB) played more important roles than Nitrospira-like NOB in urea-amended SIP microcosms, while the situation was the opposite under field conditions. Our results suggest that long-term fertilization selected for physiologically versatile AOB and AOA that could have been adapted to a wide range of substrate ammonium concentrations. It also provides compelling evidence that the dominant communities of transcriptionally active nitrifiers under field conditions were largely similar to those revealed in 13C-labeled microcosms.IMPORTANCE The role of manipulated microcosms in microbial ecology has been much debated, because they cannot entirely represent the in situ situation. We collected soil samples from 20 field plots, including 5 different treatments with and without nitrogen fertilizers for 22 years, in order to assess active nitrifying communities by in situ transcriptomics and microcosm-based stable-isotope probing. The results showed that chronic N enrichment led to competitive advantages of Nitrosospira cluster 3-like AOB over N. viennensis-like AOA in soils under field conditions. Microcosm labeling revealed similar results for active AOA and AOB, although an apparent discrepancy was observed for nitrite-oxidizing bacteria. This study suggests that the soil microbiome represents a relatively stable community resulting from complex evolutionary processes over a large time scale, and microcosms can serve as powerful tools to test the theory of environmental filtering on the key functional microbial guilds.

Evidence for a Growth Zone for Deep-Subsurface Microbial Clades in Near-Surface Anoxic Sediments.

Global marine sediments harbor a large and highly diverse microbial biosphere, but the mechanism by which this biosphere is established during sediment burial is largely unknown. During burial in marine sediments, concentrations of easily metabolized organic compounds and total microbial cell abundance decrease. However, it is unknown whether some microbial clades increase with depth. We show total population increases in 38 microbial families over 3 cm of sediment depth in the upper 7.5 cm of White Oak River (WOR) estuary sediments. Clades that increased with depth were more often associated with one or more of the following: anaerobes, uncultured, or common in deep marine sediments relative to those that decreased. Maximum doubling times (in situ steady-state growth rates could be faster to balance cell decay) were estimated as 2 to 25 years by combining sedimentation rate with either quantitative PCR (qPCR) or the product of the fraction read abundance of 16S rRNA genes and total cell counts (FRAxC). Doubling times were within an order of magnitude of each other in two adjacent cores, as well as in two laboratory enrichments of Cape Lookout Bight (CLB), NC, sediments (average difference of 28% ± 19%). qPCR and FRAxC in sediment cores and laboratory enrichments produced similar doubling times for key deep subsurface uncultured clades Bathyarchaeota (8.7 ± 1.9 years) and Thermoprofundales/MBG-D (4.1 ± 0.7 years). We conclude that common deep subsurface microbial clades experience a narrow zone of growth in shallow sediments, offering an opportunity for selection of long-term subsistence traits after resuspension events.IMPORTANCE Many studies show that the uncultured microbes that dominate global marine sediments do not actually increase in population size as they are buried in marine sediments; rather, they exist in a sort of prolonged torpor for thousands of years. This is because, although studies have shown biomass turnover in these clades, no evidence has ever been found that deeper sediments have larger populations for specific clades than shallower layers. We discovered that they actually do increase population sizes during burial, but only in the upper few centimeters. This suggests that marine sediments may be a vast repository of mostly nongrowing microbes with a thin and relatively rapid area of cell abundance increase in the upper 10 cm, offering a chance for subsurface organisms to undergo natural selection.

Microbial Community Structure Along a Horizontal Oxygen Gradient in a Costa Rican Volcanic Influenced Acid Rock Drainage System.

We describe the geochemistry and microbial diversity of a pristine environment that resembles an acid rock drainage (ARD) but it is actually the result of hydrothermal and volcanic influences. We designate this environment, and other comparable sites, as volcanic influenced acid rock drainage (VARD) systems. The metal content and sulfuric acid in this ecosystem stem from the volcanic milieu and not from the product of pyrite oxidation. Based on the analysis of 16S rRNA gene amplicons, we report the microbial community structure in the pristine San Cayetano Costa Rican VARD environment (pH = 2.94-3.06, sulfate ~ 0.87-1.19 g L-1, iron ~ 35-61 mg L-1 (waters), and ~ 8-293 g kg-1 (sediments)). San Cayetano was found to be dominated by microorganisms involved in the geochemical cycling of iron, sulfur, and nitrogen; however, the identity and abundance of the species changed with the oxygen content (0.40-6.06 mg L-1) along the river course. The hypoxic source of San Cayetano is dominated by a putative anaerobic sulfate-reducing Deltaproteobacterium. Sulfur-oxidizing bacteria such as Acidithiobacillus or Sulfobacillus are found in smaller proportions with respect to typical ARD. In the oxic downstream, we identified aerobic iron-oxidizers (Leptospirillum, Acidithrix, Ferrovum) and heterotrophic bacteria (Burkholderiaceae bacterium, Trichococcus, Acidocella). Thermoplasmatales archaea closely related to environmental phylotypes found in other ARD niches were also observed throughout the entire ecosystem. Overall, our study shows the differences and similarities in the diversity and distribution of the microbial communities between an ARD and a VARD system at the source and along the oxygen gradient that establishes on the course of the river.

Headwater Stream Microbial Diversity and Function across Agricultural and Urban Land Use Gradients.

Anthropogenic activity impacts stream ecosystems, resulting in a loss of diversity and ecosystem function; however, little is known about the response of aquatic microbial communities to changes in land use. Here, microbial communities were characterized in 82 headwater streams across a gradient of urban and agricultural land uses using 16S rRNA gene amplicon sequencing and compared to a rich data set of physicochemical variables and traditional benthic invertebrate indicators. Microbial diversity and community structures differed among watersheds with high agricultural, urban, and forested land uses, and community structure differed in streams classified as being in good, fair, poor, and very poor condition using benthic invertebrate indicators. Microbial community similarity decayed with geodesic distance across the study region but not with environmental distance. Stream community respiration rates ranged from 21.7 to 1,570 mg O2 m-2 day-1 and 31.9 to 3,670 mg O2 m-2 day-1 for water column and sediments, respectively, and correlated with nutrients associated with anthropogenic influence and microbial community structure. Nitrous oxide (N2O) concentrations ranged from 0.22 to 4.41 µg N2O liter-1; N2O concentration was negatively correlated with forested land use and was positively correlated with dissolved inorganic nitrogen concentrations. Our findings suggest that stream microbial communities are impacted by watershed land use and can potentially be used to assess ecosystem health.IMPORTANCE Stream ecosystems are frequently impacted by changes in watershed land use, resulting in altered hydrology, increased pollutant and nutrient loads, and habitat degradation. Macroinvertebrates and fish are strongly affected by changes in stream conditions and are commonly used in biotic indices to assess ecosystem health. Similarly, microbes respond to environmental stressors, and changes in community composition alter key ecosystem processes. The response of microbes to habitat degradation and their role in global biogeochemical cycles provide an opportunity to use microbes as a monitoring tool. Here, we identify stream microbes that respond to watershed urbanization and agricultural development and demonstrate that microbial diversity and community structure can be used to assess stream conditions and ecosystem functioning.

Densely Populated Water Droplets in Heavy-Oil Seeps.

Most of the microbial degradation in oil reservoirs is believed to take place at the oil-water transition zone (OWTZ). However, a recent study indicates that there is microbial life enclosed in microliter-sized water droplets dispersed in heavy oil of Pitch Lake in Trinidad and Tobago. This life in oil suggests that microbial degradation of oil also takes place in water pockets in the oil-bearing rock of an oil leg independent of the OWTZ. However, it is unknown whether microbial life in water droplets dispersed in oil is a generic property of oil reservoirs rather than an exotic exception. Hence, we took samples from three heavy-oil seeps, Pitch Lake (Trinidad and Tobago), the La Brea Tar Pits (California, USA), and an oil seep on the McKittrick oil field (California, USA). All three tested oil seeps contained dispersed water droplets. Larger droplets between 1 and 10 µl revealed high cell densities of up to 109 cells ml-1 Testing for ATP content and LIVE/DEAD staining showed that these populations consist of active and viable microbial cells with an average of 60% membrane-intact cells and ATP concentrations comparable to those of other subsurface ecosystems. Microbial community analyses based on 16S rRNA gene amplicon sequencing revealed the presence of known anaerobic oil-degrading microorganisms. Surprisingly, the community analyses showed similarities between all three oil seeps, revealing common OTUs, although the sampling sites were thousands of kilometers apart. Our results indicate that small water inclusions are densely populated microhabitats in heavy oil and possibly a generic trait of degraded-oil reservoirs.IMPORTANCE Our results confirmed that small water droplets in oil are densely populated microhabitats containing active microbial communities. Since these microhabitats occurred in three tested oil seeps which are located thousands of kilometers away from each other, such populated water droplets might be a generic trait of biodegraded oil reservoirs and might be involved in the overall oil degradation process. Microbial degradation might thus also take place in water pockets in the oil-bearing oil legs of the reservoir rock rather than only at the oil-water transition zone.

Comparing DNA, RNA and protein levels for measuring microbial dynamics in soil microcosms amended with nitrogen fertilizer.

To what extent multi-omic techniques could reflect in situ microbial process rates remains unclear, especially for highly diverse habitats like soils. Here, we performed microcosm incubations using sandy soil from an agricultural site in Midwest USA. Microcosms amended with isotopically labeled ammonium and urea to simulate a fertilization event showed nitrification (up to 4.1 ± 0.87 µg N-NO3- g-1 dry soil d-1) and accumulation of N2O after 192 hours of incubation. Nitrification activity (NH4+ → NH2OH → NO → NO2- → NO3-) was accompanied by a 6-fold increase in relative expression of the 16S rRNA gene (RNA/DNA) between 10 and 192 hours of incubation for ammonia-oxidizing bacteria Nitrosomonas and Nitrosospira, unlike archaea and comammox bacteria, which showed stable gene expression. A strong relationship between nitrification activity and betaproteobacterial ammonia monooxygenase and nitrite oxidoreductase transcript abundances revealed that mRNA quantitatively reflected measured activity and was generally more sensitive than DNA under these conditions. Although peptides related to housekeeping proteins from nitrite-oxidizing microorganisms were detected, their abundance was not significantly correlated with activity, revealing that meta-proteomics provided only a qualitative assessment of activity. Altogether, these findings underscore the strengths and limitations of multi-omic approaches for assessing diverse microbial communities in soils and provide new insights into nitrification.

DNA and RNA Stable Isotope Probing of Methylotrophic Methanogenic Archaea.

Methylotrophic methanogenic archaea are an integral part of the carbon cycle in various anaerobic environments. Different from methylotrophic bacteria, methylotrophic methanogens assimilate both, the methyl compound and dissolved inorganic carbon. Here, we present DNA- and RNA-stable isotope probing (SIP) methods involving an effective labeling strategy using 13C-labeled dissolved inorganic carbon (DIC) as carbon source along with methanol as dissimilatory substrate.

Responses of Active Ammonia Oxidizers and Nitrification Activity in Eutrophic Lake Sediments to Nitrogen and Temperature.

Ammonium concentrations and temperature drive the activities of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB), but their effects on these microbes in eutrophic freshwater sediments are unclear. In this study, surface sediments collected from areas of Taihu Lake (China) with different degrees of eutrophication were incubated under three levels of nitrogen input and temperature, and the autotrophic growth of ammonia oxidizers was assessed using 13C-labeled DNA-based stable-isotope probing (SIP), while communities were characterized using MiSeq sequencing and phylogenetic analysis of 16S rRNA genes. Nitrification rates in sediment microcosms were positively correlated with nitrogen inputs, but there was no marked association with temperature. Incubation of SIP microcosms indicated that AOA and AOB amoA genes were labeled by 13C at 20°C and 30°C in the slightly eutrophic sediment, and AOB amoA genes were labeled to a much greater extent than AOA amoA genes in the moderately eutrophic sediment after 56 days. Phylogenetic analysis of 13C-labeled 16S rRNA genes revealed that the active AOA were mainly affiliated with the Nitrosopumilus cluster, with the Nitrososphaera cluster dominating in the slightly eutrophic sediment at 30°C with low ammonium input (1 mM). Active AOB communities were more sensitive to nitrogen input and temperature than were AOA communities, and they were exclusively dominated by the Nitrosomonas cluster, which tended to be associated with Nitrosomonadaceae-like lineages. Nitrosomonas sp. strain Is79A3 tended to dominate the moderately eutrophic sediment at 10°C with greater ammonium input (2.86 mM). The relative abundance responses of the major active communities to nitrogen input and temperature gradients varied, indicating niche differentiation and differences in the physiological metabolism of ammonia oxidizers that are yet to be described.IMPORTANCE Both archaea and bacteria contribute to ammonia oxidation, which plays a central role in the global cycling of nitrogen and is important for reducing eutrophication in freshwater environments. The abundance and activities of ammonia-oxidizing archaea and bacteria in eutrophic limnic sediments vary with different ammonium concentrations or with seasonal shifts, and how the two factors affect nitrification activity, microbial roles, and active groups in different eutrophic sediments is unclear. The significance of our research is in identifying the archaeal and bacterial responses to anthropogenic activity and climate change, which will greatly enhance our understanding of the physiological metabolic differences of ammonia oxidizers.

Comparative genomic analyses reveal trehalose synthase genes as the signature in genus Methanoculleus.

To date, the only methanoarchaea isolated directly from methane hydrate bearing sediments were Methanoculleus submarinus Nankai-1T and Methanoculleus sp. MH98A. Here, we provide the genome of Methanoculleus taiwanensis CYW4T isolated from the deep-sea subseafloor sediment at the Deformation Front offshore southwestern Taiwan, where methane hydrate deposits are likely located. Through comparative genomics analyses of nine Methanoculleus strains from various habitats, 2-3 coding genes for trehalose synthases were found in all nine Methanoculleus genomes, which were not detected in other methanogens and are therefore suggested as a signature of genus Methanoculleus among methane-producing archaea. In addition, the structural genes adjacent to trehalose synthase genes are comprised of the signaling module of Per-Arnt-Sim (PAS) domain-containing proteins, Hsp20 family proteins, arabinose efflux permeases and multiple surface proteins with fasciclin-like (FAS) repeat. This indicates that trehalose synthase gene clusters in Methanoculleus might play roles in the response to various stresses and regulate carbon storage and modification of surface proteins through accumulation of trehalose. The non-gas hydrate-associated Methanoculleus strains harbor carbon-monoxide dehydrogenase (cooS/acsA) genes, which are important for the conversion of acetate to methane at the step of CO oxidation/CO2 reduction in acetoclastic methanogens and further implies that these strains may be able to utilize CO for methanogenesis in their natural habitats. In addition, both genomes of M. bourgensis strains MS2T and MAB1 harbor highly abundant transposase genes, which may be disseminated from microbial communities in their habitats, sewage treatment plants and biogas reactors, which are breeding grounds for antibiotic resistance. Through comparative genomic analyses, we gained insight into understanding the life of strictly anaerobic methane-producing archaea in various habitats, especially in methane-based deep-sea ecosystems.

Methane oxidation coupled to perchlorate reduction in a membrane biofilm batch reactor.

A specially designed CH4-based membrane biofilm batch reactor (MBBR) was applied to investigate anaerobic methane oxidation coupled to perchlorate reduction (AnMO-PR). The 0.21 mM ClO4- added in the first stage of operation was completely reduced in 28 days, 0.40 mM ClO4- was reduced within 23 days in stage 2, and 0.56 mM of ClO4- was reduced within 30 days in stage 3. Although some chlorate (ClO3-) accumulated, the recovery of Cl- was over 92%. Illumina sequencing of the 16S rRNA gene documented that the bacterial community was mainly composed by perchlorate-reducing bacteria (PRB), methanotrophic bacteria, and archaea. Real-time quantitative PCR showed the archaeal 16S rRNA and mcrA genes increased as more ClO4- was reduced, and the predominant archaea belonged to Methanosarcina mazei, which is related to ANME-3, an archaeon able to perform reverse methanogenesis. Several pieces of evidence support that ClO4- reduction by the MBBR biofilm occurred via a synergism between Methanosarcina and PRB: Methanosarcina oxidized methane through reverse methanogesis and provided electron donor for PRB to reduce ClO4-. Because methanotrophs were present, we cannot rule out that they also were involved in AnMO-PR if they received O2 generated by disproportionation of ClO2- from the PRB.

Electro-driven methanogenic microbial community diversity and variability in the electron abundant niche.

The underlying dynamics of microbial (bacteria and archaea) communities ecologically responding to an applied potential are critical to achieving the goal of enhancing bioenergy recovery but are not sufficiently understood. We built a MEC-AD mode that increased methane production rate by several times (max. 3.8 times) during the startup period compared to control AD, changed the absence or presence of external voltage to provide the pre-, dur-, and post- samples for microbial analysis. From a time and spatially dependent community analysis of electrode-respiring bacteria and methanogens, the corresponding Geobacter developed under the influence of external voltage, pairing with methanogens in the anodic and cathodic biofilm to generate methane. Additionally, at the cathode, the Alkaliphilus (basophilic bacteria) also correspondingly shifted alongside the change of external voltage. The mcrA sequencing confirmed a change in the dominant microbe from acetoclastic (mostly Methanosarcina mazei LYC) to hydrogenotrophic methanogens (mostly basophilic Methanobacterium alcaliphilum) at the cathode with 0.8 V voltage. Overall, the external voltage not only enriched the functional microbes including electrogens and methanogens but also indirectly shifted the composition of the bacterial and archaeal community via disturbing the pH condition. The predictive functional profiling indicated that the cathodic methanogenesis principally followed the metabolism pathway of the hydrogenotrophic methanogens, suggesting the F420 co-enzyme could be the key mediate for electron transfer. All data suggested that the electric stimulation would change and maintain the micro-environmental conditions to shift the bacterial/archaeal community.

Characteristics of microbial community indicate anthropogenic impact on the sediments along the Yangtze Estuary and its coastal area, China.

In the contaminated coastal sediments, variations of microbial community can reflect the impact of anthropogenic activities. The identification, evaluation and monitoring of the potential bio-indicator species and biomarker communities are vital for the ecological studies in sedimentary environments. Based on the high-throughput sequencing, the microbial communities were characterized in the sediments along the Yangtze Estuary and its coastal area. The results showed that the structure and composition of microbial communities varied greatly among different sampling sites at the phyla level, especially for Euryarchaeota. Metabolic pathway and quantitative PCR analyses suggested that the methane metabolism-related microbes were mainly included in the phylum of Euryarchaeota. Elevated abundances of methane metabolism-related microbes were found at Shidongkou (SDK) and Wusongkou (WSK), where microbes were seriously impacted by the wastewater treatment plant (WWTP) effluent and urban runoff. By comparing with the Euryarchaeota in WWTP sludge, the relatively high abundance of Euryarchaeota in sediment at SDK may be mainly related to the massive growth of indigenous species, promoted by anthropogenic nutrients. Moreover, redundancy discriminant analysis and correlation analysis revealed that methanogens and methanotrophs mainly respond to the nutrients and metals, such as total organic carbon, total phosphorus, total nitrogen, SO42-, NO2-, NH4+, Cr, and Zn, which were often related to human activities. Network analyses showed that the species related to the metabolism of methane may play a vital role in the interassociation among different microbial communities. Therefore, methanogens, methanotrophs and their community compositions could be considered as potential bio-indicator species and biomarker communities, indicating anthropogenic activities in the sediments along the Yangtze Estuary and its coastal area.

Microbial assessments of soil with a 40-year history of reclaimed wastewater irrigation.

The long-term effects on soil microorganisms from 40 years of irrigating soil with reclaimed wastewater was investigated by determining the quantity, composition, and inter-species connection of microorganisms. No significant difference in microbial quantity and composition were identified in the reclaimed wastewater- and groundwater-irrigated soils. The dominant bacterial phylum in both irrigation water sources and soils was Proteobacteria, which commonly exists in soil. From the analysis of four (4) alpha diversity metrics, including the observed number of operational taxonomic units (OTUs), Chao1, and the Shannon and Simpson diversity, there was no significant difference between the reclaimed wastewater- and groundwater-irrigated soils. Three zones (shallow, medium and deep) were identified in the reclaimed wastewater- and groundwater-irrigated soils based on the taxonomic networks and clusters generated by graphical lasso and random walk algorithm. The cluster profiles (shallow, medium and deep zones) appear to be different in the reclaimed wastewater- and groundwater-irrigated soils. Soil irrigated with reclaimed wastewater showed less depth of clustered profile in medium zone than that in soil irrigated with groundwater (20-60 cm of reclaimed wastewater-irrigated soil compared to 20-100 cm of groundwater-irrigated soil), although the significance of such a variance (the depth of medium zone of reclaimed wastewater-irrigated soil decreased 40 cm than that of groundwater-irrigated soil) is not clear at this time. Positive influence has been identified in the growth and yield of eggplant, tomato and cucumber between the reclaimed wastewater- and groundwater-irrigated soils, suggesting that reclaimed wastewater irrigation can potentially substitute groundwater irrigation, despite the variance in inter-species clustering profiles in soil microbes in certain soil zones. Nevertheless, the possible negative influence of pathogens, organic compounds and pharmaceuticals should be seriously considered during the reclaimed wastewater irrigation.

Diet Influences Early Microbiota Development in Dairy Calves without Long-Term Impacts on Milk Production.

Gastrointestinal tract (GIT) microorganisms play important roles in the health of ruminant livestock and affect the production of agriculturally relevant products, including milk and meat. Despite this link, interventions to alter the adult microbiota to improve production have proven ineffective, as established microbial communities are resilient to change. In contrast, developing communities in young animals may be more easily altered but are less well studied. Here, we measured the GIT-associated microbiota of 45 Holstein dairy cows from 2 weeks to the first lactation cycle, using Illumina amplicon sequencing of bacterial (16S rRNA V4), archaeal (16S rRNA V6 to V8), and fungal (internal transcribed region 1 [ITS1]) communities. Fecal and ruminal microbiota of cows raised on calf starter grains and/or corn silage were correlated to lifetime growth as well as milk production during the first lactation cycle, in order to determine whether early-life diets have long-term impacts. Significant diet-associated differences in total microbial communities and specific taxa were observed by weaning (8 weeks), but all animals reached an adult-like composition between weaning and 1 year. While some calf-diet-driven differences were apparent in the microbiota of adult cows, these dissimilarities did not correlate with animal growth or milk production. This finding suggests that initial microbial community establishment is affected by early-life diet but postweaning factors have a greater influence on adult communities and production outcomes.IMPORTANCE The gut microbiota is essential for the survival of many organisms, including ruminants that rely on microorganisms for nutrient acquisition from dietary inputs for the production of products such as milk and meat. While alteration of the adult ruminant microbiota to improve production is possible, changes are often unstable and fail to persist. In contrast, the early-life microbiota may be more amenable to sustained modification. However, few studies have determined the impact of early-life interventions on downstream production. Here, we investigated the impact of agriculturally relevant calf diets, including calf starter and corn silage, on gut microbial communities, growth, and production through the first lactation cycle. Thus, this work serves to further our understanding of early-life microbiota acquisition, as well as informing future practices in livestock management.

Methane emission from feather moss stands.

Data from remote sensing and Eddy towers indicate that forests are not always net sinks for atmospheric CH4 . However, studies describing specific sources within forests and functional analysis of microorganisms on sites with CH4 turnover are scarce. Feather moss stands were considered to be net sinks for carbon dioxide, but received little attention to their role in CH4 cycling. Therefore, we investigated methanogenic rates and pathways together with the methanogenic microbial community composition in feather moss stands from temperate and boreal forests. Potential rates of CH4 emission from intact moss stands (n = 60) under aerobic conditions ranged between 19 and 133 pmol CH4 h-1 gdw-1 . Temperature and water content positively influenced CH4 emission. Methanogenic potentials determined under N2 atmosphere in darkness ranged between 22 and 157 pmol CH4 h-1 gdw-1 . Methane production was strongly inhibited by bromoethane sulfonate or chloroform, showing that CH4 was of microbial origin. The moss samples tested contained fluorescent microbial cells and between 104 and 105 copies per gram dry weight moss of the mcrA gene coding for a subunit of the methyl CoM reductase. Archaeal 16S rRNA and mcrA gene sequences in the moss stands were characteristic for the archaeal families Methanobacteriaceae and Methanosarcinaceae. The potential methanogenic rates were similar in incubations with and without methyl fluoride, indicating that the CH4 was produced by the hydrogenotrophic rather than aceticlastic pathway. Consistently, the CH4 produced was depleted in 13 C in comparison with the moss biomass carbon and acetate accumulated to rather high concentrations (3-62 mM). The δ13 C of acetate was similar to that of the moss biomass, indicating acetate production by fermentation. Our study showed that the feather moss stands contained active methanogenic microbial communities producing CH4 by hydrogenotrophic methanogenesis and causing net emission of CH4 under ambient conditions, albeit at low rates.

Microbial diversity in a permanently cold and alkaline environment in Greenland.

The submarine ikaite columns located in the Ikka Fjord in Southern Greenland represent a unique, permanently cold (less than 6°C) and alkaline (above pH 10) environment and are home to a microbial community adapted to these extreme conditions. The bacterial and archaeal community inhabiting the ikaite columns and surrounding fjord was characterised by high-throughput pyrosequencing of 16S rRNA genes. Analysis of the ikaite community structure revealed the presence of a diverse bacterial community, both in the column interior and at the surface, and very few archaea. A clear difference in overall taxonomic composition was observed between column interior and surface. Whereas the surface, and in particular newly formed ikaite material, was primarily dominated by Cyanobacteria and phototrophic Proteobacteria, the column interior was dominated by Proteobacteria and putative anaerobic representatives of the Firmicutes and Bacteroidetes. The results suggest a stratification of the ikaite columns similar to that of classical soda lakes, with a light-exposed surface inhabited by primary producers and an anoxic subsurface. This was further supported by identification of major taxonomic groups with close relatives in soda lake environments, including members of the genera Rhodobaca, Dethiobacter, Thioalkalivibrio and Tindallia, as well as very abundant groups related to uncharacterised environmental sequences originally isolated from Mono Lake in California.

Microbial community and performance of slaughterhouse wastewater treatment filters.

The performance of anaerobic filter bioreactors (AFs) is influenced by the composition of the substrate, support medium, and the microbial species present in the sludge. In this study, the efficiency of a slaughterhouse effluent treatment using three AFs containing different support media was tested, and the microbial diversity was investigated by amplified ribosomal DNA restriction analysis and 16S rRNA gene sequencing. The physicochemical analysis of the AF systems tested suggested their feasibility, with rates of chemical oxygen demand removal of 72±8% in hydraulic retention times of 1 day. Analysis of pH, alkalinity, volatile acidity, total solids, total volatile solids, total Kjeldahl nitrogen, and the microbial community structures indicated high similarity among the three AFs. The composition of prokaryotic communities showed a prevalence of Proteobacteria (27.3%) and Bacteroidetes (18.4%) of the Bacteria domain and Methanomicrobiales (36.4%) and Methanosarcinales (35.3%) of the Archaea domain. Despite the high similarity of the microbial communities among the AFs, the reactor containing pieces of clay brick as a support medium presented the highest richness and diversity of bacterial and archaeal operational taxonomic units.

RNA processing in the minimal organism Nanoarchaeum equitans.

BACKGROUND: The minimal genome of the tiny, hyperthermophilic archaeon Nanoarchaeum equitans contains several fragmented genes and revealed unusual RNA processing pathways. These include the maturation of tRNA molecules via the trans-splicing of tRNA halves and genomic rearrangements to compensate for the absence of RNase P. RESULTS: Here, the RNA processing events in the N. equitans cell are analyzed using RNA-Seq deep sequencing methodology. All tRNA half precursor and tRNA termini were determined and support the tRNA trans-splicing model. The processing of CRISPR RNAs from two CRISPR clusters was verified. Twenty-seven C/D box small RNAs (sRNAs) and a H/ACA box sRNA were identified. The C/D box sRNAs were found to flank split genes, to form dicistronic tRNA-sRNA precursors and to be encoded within the tRNAMet intron. CONCLUSIONS: The presented data provide an overview of the production and usage of small RNAs in a cell that has to survive with a highly reduced genome. N. equitans lost many essential metabolic pathways but maintains highly active CRISPR/Cas and rRNA modification systems that appear to play an important role in genome fragmentation.

Molecular analysis of 16S rRNA genes identifies potentially periodontal pathogenic bacteria and archaea in the plaque of partially erupted third molars.

PURPOSE: Small subunit rRNA sequencing and phylogenetic analysis were used to identify cultivable and uncultivable microorganisms present in the dental plaque of symptomatic and asymptomatic partially erupted third molars to determine the prevalence of putative periodontal pathogens in pericoronal sites. MATERIALS AND METHODS: Template DNA prepared from subgingival plaque collected from partially erupted symptomatic and asymptomatic mandibular third molars and healthy incisors was used in polymerase chain reaction with broad-range oligonucleotide primers to amplify 16S rRNA bacterial and archaeal genes. Amplicons were cloned, sequenced, and compared with known nucleotide sequences in online databases to identify the microorganisms present. RESULTS: Two thousand three hundred two clones from the plaque of 12 patients carried bacterial sequences from 63 genera belonging to 11 phyla, including members of the uncultivable TM7, SR1, and Chloroflexi, and difficult-to-cultivate Synergistetes and Spirochaetes. Dialister invisus, Filifactor alocis, Fusobacterium nucleatum, Porphyromonas endodontalis, Prevotella denticola, Tannerella forsythia, and Treponema denticola, which have been associated with periodontal disease, were found in significantly greater abundance in pericoronal compared with incisor sites. Dialister invisus and F nucleatum were found in greater abundance in sites exhibiting clinical symptoms. The archaeal species, Methanobrevibacter oralis, which has been associated with severe periodontitis, was found in 3 symptomatic patients. CONCLUSIONS: These findings have provided new insights into the complex microbiota of pericoronitis. Several bacterial and archaeal species implicated in periodontal disease were recovered in greater incidence and abundance from the plaque of partially erupted third molars compared with incisors, supporting the hypothesis that the pericoronal region may provide a favored niche for periodontal pathogens in otherwise healthy mouths.

Dynamics and persistence of Dead Sea microbial populations as shown by high-throughput sequencing of rRNA.

16S rRNA amplicon libraries from a haloarchaeal bloom in the hypersaline Dead Sea in 1992 were analyzed together with the 2007 residual population and simulated blooms in experimental mesocosms. Significant population shifts were observed during the bloom, and surprisingly a signature from the bloom was retained 15 years later.