Distribution and Diversity of Nitrite-Dependent Anaerobic Methane-Oxidising Bacteria in the Sediments of the Qiantang River

Nitrite-dependent anaerobic methane oxidation (n-damo) process was reported to be mediated by “Candidatus Methylomirabilis oxyfera”, which belongs to the candidate phylum NC10. M. oxyfera-like bacteria have been detected in lake ecosystems, while their distribution, diversity and abundance in river ecosystems have not been well studied. In this study, both the 16S rRNA and the pmoA molecular biomarkers confirmed the presence of diverse NC10 phylum bacteria related to M. oxyfera in a river ecosystem—the Qiantang River, Zhejiang Province (China). Phylogenetic analysis of 16S rRNA genes demonstrated that the recovered M. oxyfera-like sequences could be grouped into several distinct clusters that exhibited 89.8 % to 98.9 % identity to the M. oxyfera 16S rRNA gene. Similarly, several different clusters of pmoA gene sequences were observed, and these clusters displayed 85.1–95.4 % sequence identity to the pmoA gene of M. oxyfera. Quantitative PCR showed that the abundance of M. oxyfera-like bacteria varied from 1.32?±?0.16?×?10⁶ to 1.03?±?0.12?×?10⁷ copies g (dry weight)^?1. Correlation analysis demonstrated that the total inorganic nitrogen content, the ammonium content and the organic content of the sediment were important factors affecting the distribution of M. oxyfera-like bacterial groups in the examined sediments. This study demonstrated the distribution of diverse M. oxyfera-like bacteria and their correlation with environmental factors in Qiantang River sediments.     

Existence of Novel Phylotypes of Nitrite-Dependent Anaerobic Methane-Oxidizing Bacteria in Surface and Subsurface Sediments of the South China Sea

Nitrite-dependent anaerobic methane oxidation (n-damo) is a recently discovered new microbial process performed by the Candidatus Methylomirabilis oxyfera with an unusual intra-aerobic pathway, but there is no report about n-damo bacteria in marine environments. M. oxyfera-like sequences were successfully retrieved for the first time from both surface and subsurface ocean sediments of the South China Sea (SCS) using both 16S rRNA and pmoA genes as biomarkers and PCR amplification in this study. The majority of M. oxyfera-like 16S rRNA gene-based PCR amplified sequences from the SCS sediments formed a new group distinctively different from those detected in freshwater habitats and the information is consistent phylogenetically with those obtained from the pmoA gene. This study showed the existence of n-damo in ocean sediments and suggests that marine sediments harbor n-damo phylotypes different from those in the freshwater. This finding here expands our understanding on the distribution of n-damo bacteria to marine ecosystem and implies their potential contribution to the marine C and N cycling.     

Molecular Existence and Diversity of Nitrite-Dependent Anaerobic Methane Oxidizing (n-Damo) Bacteria in the Lakes of Badain of the Gobi Desert

Nitrite-dependent anaerobic methane oxidation (n-damo) process, mediated by Candidatus Methylomirabilis oxyfera of the candidate phylum NC10, was discovered recently which plays an important role in coupling the global nitrogen and carbon cycles. However, the distribution and diversity of this new anaerobic methane-oxidizing microorganism have not been investigated in desert lakes yet. The present study successfully retrieved n-damo bacterial 16S rRNA and pmoA gene sequences using PCR technique from lakes in Badain Jaran Desert of China. Phylogenetic analyses showed that n-damo bacteria widely occurred in brine and freshwater lakes on the desert with high diversity, including both sediment and water samples. The results of quantitative PCR indicated that the abundance of the 16S rRNA gene in lake sediments varied from 1.12?±?0.68?×?10⁵ to 1.64?±?0.70?×?10⁵ copies g^?1 (dry weight), while that in water samples per milliliter was generally one order of magnitude lower than sediments. Correlation analyses suggested that n-damo bacterial abundance and diversity strongly depended on salinity. In lake sediments, the distribution, abundance, and diversity of n-damo bacteria were significantly associated with depth due to the concentration gradient of the NO_x^- and ammonium. This study provided new insights into both the n-damo community patterns and its interaction with ambient environmental factors in the desert lake ecosystem.     

Spatial change of reservoir nitrite-dependent methane-oxidizing microorganisms

Nitrite-dependent anaerobic methane oxidation (n-damo), catalyzed by microorganisms affiliated with bacterial phylum NC10, can have an important contribution to the reduction of the methane emission from anoxic freshwater sediment to the atmosphere. However, information on the variation of sediment n-damo organisms in reservoirs is still lacking. The present study monitored the spatial change of sediment n-damo organisms in the oligotrophic freshwater Xinfengjiang Reservoir (South China). Sediment samples were obtained from six different sampling locations and two sediment depths (0–5 cm, 5–10 cm). Sediment n-damo bacterial abundance was found to vary with sampling location and layer depth, which was likely influenced by pH and nitrogen level. The presence of the n-damo pmoA gene was found in all these samples. A remarkable shift occurred in the diversity and composition of sediment n-damo pmoA gene sequences. A variety of distinctively different n-damo pmoA clusters existed in reservoir sediments. The pmoA sequences affiliated with Candidatus Methylomirabilis oxyfera formed the largest group, while a significant proportion of the obtained n-damo pmoA gene sequences showed no close relationship to those from any known NC10 species. In addition, the present n-damo process was found in reservoir sediment, which could be enhanced by nitrite nitrogen amendment.     

氮依赖型甲烷厌氧氧化菌的整合

氮依赖型甲烷厌氧氧化菌(nitrite-dependent anaerobic methane oxidation bacteria,n-damo细菌,属于NC10门)是最近10年来微生物生态学领域的研究热点。然而,对该类群基于现有数据的生态分布、群落结构和系统进化的整合分析还未见报道。【目的】为了更好地将近年来针对该类群的研究做一次全面梳理,本文通过整合前人已有发表数据和结合自身实验数据两方面进行。【方法】一方面,利用NCBI数据库(数据搜集到2016年11月)中所有n-damo细菌序列对其进行生物信息学分析;另一方面,对大九湖泥炭地表层泥炭利用16S rRNA二代测序技术对该类群进行检测,并同前人数据进行对比。【结果】n-damo细菌主要在沉积物、湿地和水稻土检出;基于pmo A基因的n-damo细菌的平均检出率是基于16S rRNA基因检出率的7倍,但是这两类基因分子标记物所得到的多样性指数保持相对稳定(1.4-3.4);贫氮的大九湖泥炭其NC10的丰度仅为0.067%。【结论】n-damo类群种群相对稳定,暗示其行使的生态功能相对单一;贫氮的大九湖泥炭其极低的NC10丰度暗示氮对NC10是限制因子;具有真正氮依赖型甲烷厌氧氧化细菌的Group A可能只占很少的一部分(小于20%),暗示出该类群真正的生态潜能需要进一步评估。本次整合分析为更好的理解n-damo细菌的生活环境、评估不同基因分子标记物下n-damo细菌的检出率、不同亚类群比如Group A和Group B等的丰度和真正的潜在生态功能提供参考。     

Development and Application of a Real-Time PCR Approach for Quantification of Uncultured Bacteria in the Central Baltic Sea

We have developed a highly sensitive approach to assess the abundance of uncultured bacteria in water samples from the central Baltic Sea by using a noncultured member of the “Epsilonproteobacteria” related to Thiomicrospira denitrificans as an example. Environmental seawater samples and samples enriched for the target taxon provided a unique opportunity to test the approach over a broad range of abundances. The approach is based on a combination of taxon- and domain-specific real-time PCR measurements determining the relative T. denitrificans-like 16S rRNA gene and 16S rRNA abundances, as well as the determination of total cell counts and environmental RNA content. It allowed quantification of T. denitrificans-like 16S rRNA molecules or 16S rRNA genes as well as calculation of the number of ribosomes per T. denitrificans-like cell. Every real-time measurement and its specific primer system were calibrated using environmental nucleic acids obtained from the original habitat for external standardization. These standards, as well as the respective samples to be measured, were prepared from the same DNA or RNA extract. Enrichment samples could be analyzed directly, whereas environmental templates had to be preamplified with general bacterial primers before quantification. Preamplification increased the sensitivity of the assay by more than 4 orders of magnitude. Quantification of enrichments with or without a preamplification step yielded comparable results. T. denitrificans-like 16S rRNA molecules ranged from 7.1 × 10³ to 4.4 × 10⁹ copies ml⁻¹ or 0.002 to 49.7% relative abundance. T. denitrificans-like 16S rRNA genes ranged from 9.0 × 10¹ to 2.2 ×10⁶ copies ml⁻¹ or 0.01 to 49.7% relative abundance. Detection limits of this real-time-PCR approach were 20 16S rRNA molecules or 0.2 16S rRNA gene ml⁻¹. The number of ribosomes per T. denitrificans-like cell was estimated to range from 20 to 200 in seawater and reached up to 2,000 in the enrichments. The results indicate that our real-time PCR approach can be used to determine cellular and relative abundances of uncultured marine bacterial taxa and to provide information about their levels of activity in their natural environment.     

Characterization of C1-Metabolizing Prokaryotic Communities in Methane Seep Habitats at the Kuroshima Knoll, Southern Ryukyu Arc, by Analyzing pmoA, mmoX, mxaF, mcrA, and 16S rRNA Genes

Samples from three submerged sites (MC, a core obtained in the methane seep area; MR, a reference core obtained at a distance from the methane seep; and HC, a gas-bubbling carbonate sample) at the Kuroshima Knoll in the southern Ryuku arc were analyzed to gain insight into the organisms present and the processes involved in this oxic-anoxic methane seep environment. 16S rRNA gene analyses by quantitative real-time PCR and clone library sequencing revealed that the MC core sediments contained abundant archaea (~34% of the total prokaryotes), including both mesophilic methanogens related to the genus Methanolobus and ANME-2 members of the Methanosarcinales, as well as members of the δ-Proteobacteria, suggesting that both anaerobic methane oxidation and methanogenesis occurred at this site. In addition, several functional genes connected with methane metabolism were analyzed by quantitative competitive-PCR, including the genes encoding particulate methane monooxygenase (pmoA), soluble methane monooxygenase (mmoX), methanol dehydrogenese (mxaF), and methyl coenzyme M reductase (mcrA). In the MC core sediments, the most abundant gene was mcrA (2.5 × 10⁶ copies/g [wet weight]), while the pmoA gene of the type I methanotrophs (5.9 × 10⁶ copies/g [wet weight]) was most abundant at the surface of the MC core. These results indicate that there is a very complex environment in which methane production, anaerobic methane oxidation, and aerobic methane oxidation all occur in close proximity. The HC carbonate site was rich in γ-Proteobacteria and had a high copy number of mxaF (7.1 × 10⁶ copies/g [wet weight]) and a much lower copy number of the pmoA gene (3.2 × 10² copies/g [wet weight]). The mmoX gene was never detected. In contrast, the reference core contained familiar sequences of marine sedimentary archaeal and bacterial groups but not groups specific to C₁ metabolism. Geochemical characterization of the amounts and isotopic composition of pore water methane and sulfate strongly supported the notion that in this zone both aerobic methane oxidation and anaerobic methane oxidation, as well as methanogenesis, occur.     

Evidence for the Cooccurrence of Nitrite-Dependent Anaerobic Ammonium and Methane Oxidation Processes in a Flooded Paddy Field

Anaerobic ammonium oxidation (anammox) and nitrite-dependent anaerobic methane oxidation (n-damo) are two of the most recent discoveries in the microbial nitrogen cycle. In the present study, we provide direct evidence for the cooccurrence of the anammox and n-damo processes in a flooded paddy field in southeastern China. Stable isotope experiments showed that the potential anammox rates ranged from 5.6 to 22.7 nmol N₂ g⁻¹ (dry weight) day⁻¹ and the potential n-damo rates varied from 0.2 to 2.1 nmol CO₂ g⁻¹ (dry weight) day⁻¹ in different layers of soil cores. Quantitative PCR showed that the abundance of anammox bacteria ranged from 1.0 × 10⁵ to 2.0 × 10⁶ copies g⁻¹ (dry weight) in different layers of soil cores and the abundance of n-damo bacteria varied from 3.8 × 10⁵ to 6.1 × 10⁶ copies g⁻¹ (dry weight). Phylogenetic analyses of the recovered 16S rRNA gene sequences showed that anammox bacteria affiliated with “Candidatus Brocadia” and “Candidatus Kuenenia” and n-damo bacteria related to “Candidatus Methylomirabilis oxyfera” were present in the soil cores. It is estimated that a total loss of 50.7 g N m⁻² per year could be linked to the anammox process, which is at intermediate levels for the nitrogen flux ranges of aerobic ammonium oxidation and denitrification reported in wetland soils. In addition, it is estimated that a total of 0.14 g CH₄ m⁻² per year could be oxidized via the n-damo process, while this rate is at the lower end of the aerobic methane oxidation rates reported in wetland soils.     

Occurrence and Genetic Diversity of Uncultured Legionella spp. in Drinking Water Treated at Temperatures below 15°C

Representatives of the genus Legionella were detected by use of a real-time PCR method in all water samples collected directly after treatment from 16 surface water (SW) supplies prior to postdisinfection and from 81 groundwater (GW) supplies. Legionella concentrations ranged from 1.1 × 10³ to 7.8 × 10⁵ cells liter⁻¹ and were significantly higher in SW treated with multiple barriers at 4°C than in GW treated at 9 to 12°C with aeration and filtration but without chemical disinfection. No Legionellae (<50 CFU liter⁻¹) were detected in treated water by the culture method. Legionella was also observed in untreated SW and in untreated aerobic and anaerobic GW. Filtration processes in SW and GW treatment had little effect or increased the Legionella concentration, but ozonation in SW treatment caused about 1-log-unit reduction. A phylogenetic analysis of 16S rRNA gene sequences of 202 clones, obtained from a selection of samples, showed a high similarity (>91%) with Legionella sequences in the GenBank database. A total of 40 (33%) of the 16S rRNA gene sequences obtained from treated water were identified as described Legionella species and types, including L. bozemanii, L. worsleiensis, Legionella-like amoebal pathogen types, L. quateirensis, L. waltersii, and L. pneumophila. 16S rRNA gene sequences with a similarity of below 97% from described species were positioned all over the phylogenetic tree of Legionella. Hence, a large diversity of yet-uncultured Legionellae are common members of the microbial communities in SW and GW treated at water temperatures of below 15°C.     

Linking activity,composition and seasonal dynamics of atmospheric methane oxidizers in a meadow soil

Microbial oxidation is the only biological sink for atmospheric methane. We assessed seasonal changes in atmospheric methane oxidation and the underlying methanotrophic communities in grassland near Giessen (Germany), along a soil moisture gradient. Soil samples were taken from the surface layer (0–10 cm) of three sites in August 2007, November 2007, February 2008 and May 2008. The sites showed seasonal differences in hydrological parameters. Net uptake rates varied seasonally between 0 and 70 μg CH₄ m⁻² h⁻¹. Greatest uptake rates coincided with lowest soil moisture in spring and summer. Over all sites and seasons, the methanotrophic communities were dominated by uncultivated methanotrophs. These formed a monophyletic cluster defined by the RA14, MHP and JR1 clades, referred to as upland soil cluster alphaproteobacteria (USCα)-like group. The copy numbers of pmoA genes ranged between 3.8 × 10⁵–1.9 × 10⁶ copies g⁻¹ of soil. Temperature was positively correlated with CH₄ uptake rates (P<0.001), but had no effect on methanotrophic population dynamics. The soil moisture was negatively correlated with CH₄ uptake rates (P<0.001), but showed a positive correlation with changes in USCα-like diversity (P<0.001) and pmoA gene abundance (P<0.05). These were greatest at low net CH₄ uptake rates during winter times and coincided with an overall increase in bacterial 16S rRNA gene abundances (P<0.05). Taken together, soil moisture had a significant but opposed effect on CH₄ uptake rates and methanotrophic population dynamics, the latter being increasingly stimulated by soil moisture contents >50 vol% and primarily related to members of the MHP clade.     

Isolation and Distribution of a Novel Iron-Oxidizing Crenarchaeon from Acidic Geothermal Springs in Yellowstone National Park

Novel thermophilic crenarchaea have been observed in Fe(III) oxide microbial mats of Yellowstone National Park (YNP); however, no definitive work has identified specific microorganisms responsible for the oxidation of Fe(II). The objectives of the current study were to isolate and characterize an Fe(II)-oxidizing member of the Sulfolobales observed in previous 16S rRNA gene surveys and to determine the abundance and distribution of close relatives of this organism in acidic geothermal springs containing high concentrations of dissolved Fe(II). Here we report the isolation and characterization of the novel, Fe(II)-oxidizing, thermophilic, acidophilic organism Metallosphaera sp. strain MK1 obtained from a well-characterized acid-sulfate-chloride geothermal spring in Norris Geyser Basin, YNP. Full-length 16S rRNA gene sequence analysis revealed that strain MK1 exhibits only 94.9 to 96.1% sequence similarity to other known Metallosphaera spp. and less than 89.1% similarity to known Sulfolobus spp. Strain MK1 is a facultative chemolithoautotroph with an optimum pH range of 2.0 to 3.0 and an optimum temperature range of 65 to 75°C. Strain MK1 grows optimally on pyrite or Fe(II) sorbed onto ferrihydrite, exhibiting doubling times between 10 and 11 h under aerobic conditions (65°C). The distribution and relative abundance of MK1-like 16S rRNA gene sequences in 14 acidic geothermal springs containing Fe(III) oxide microbial mats were evaluated. Highly related MK1-like 16S rRNA gene sequences (>99% sequence similarity) were consistently observed in Fe(III) oxide mats at temperatures ranging from 55 to 80°C. Quantitative PCR using Metallosphaera-specific primers confirmed that organisms highly similar to strain MK1 comprised up to 40% of the total archaeal community at selected sites. The broad distribution of highly related MK1-like 16S rRNA gene sequences in acidic Fe(III) oxide microbial mats is consistent with the observed characteristics and growth optima of Metallosphaera-like strain MK1 and emphasizes the importance of this newly described taxon in Fe(II) chemolithotrophy in acidic high-temperature environments of YNP.     

Characterization of a Highly Enriched Dehalococcoides-Containing Culture That Grows on Vinyl Chloride and Trichloroethene

A highly enriched culture that reductively dechlorinates trichloroethene (TCE), cis-1,2-dichloroethene (cDCE), and vinyl chloride (VC) to ethene without methanogenesis is described. The Dehalococcoides strain in this enrichment culture had a yield of (5.6 ± 1.4) × 10⁸ 16S rRNA gene copies/μmol of Cl⁻ when grown on VC and hydrogen. Unlike the other VC-degrading cultures described in the literature, strains VS and BAV1, this culture maintained the ability to grow on TCE with a yield of (3.6 ± 1.3) × 10⁸ 16S rRNA gene copies/μmol of Cl⁻. The yields on an electron-equivalent basis measured for the culture grown on TCE and on VC were not significantly different, indicating that both substrates supported growth equally well. PCR followed by denaturing gradient gel electrophoresis, cloning, and phylogenetic analyses revealed that this culture contained one Dehalococcoides 16S rRNA gene sequence, designated KB-1/VC, that was identical (over 1,386 bp) to the sequences of previously described organisms FL2 and CBDB1. A second Dehalococcoides sequence found in separate KB-1 enrichment cultures maintained on cDCE, TCE, and tetrachloroethene was no longer present in the VC-H₂ enrichment culture. This second Dehalococcoides sequence was identical to that of BAV1. As neither FL2 nor CBDB1 can dechlorinate VC to ethene in a growth-related fashion, it is clear that current 16S rRNA gene-based analyses do not provide sufficient information to distinguish between metabolically diverse members of the Dehalococcoides group.     

Anaerobic Mineralization of Quaternary Carbon Atoms: Isolation of Denitrifying Bacteria on Dimethylmalonate

The microbial capacity to degrade simple organic compounds with quaternary carbon atoms was demonstrated by enrichment and isolation of five denitrifying strains on dimethylmalonate as the sole electron donor and carbon source. Quantitative growth experiments showed a complete mineralization of dimethylmalonate. According to phylogenetic analysis of the complete 16S rRNA genes, two strains isolated from activated sewage sludge were related to the genus Paracoccus within the α-Proteobacteria (98.0 and 98.2% 16S rRNA gene similarity to Paracoccus denitrificans^T), and three strains isolated from freshwater ditches were affiliated with the β-Proteobacteria (97.4 and 98.3% 16S rRNA gene similarity to Herbaspirillum seropedicae^T and Acidovorax facilis^T, respectively). Most-probable-number determinations for denitrifying populations in sewage sludge yielded 4.6 × 10⁴ dimethylmalonate-utilizing cells ml⁻¹, representing up to 0.4% of the total culturable nitrate-reducing population.     

Anaerobic oxidation of methane at different temperature regimes in Guaymas Basin hydrothermal sediments

Anaerobic oxidation of methane (AOM) was investigated in hydrothermal sediments of Guaymas Basin based on δ¹³C signatures of CH₄, dissolved inorganic carbon and porewater concentration profiles of CH₄ and sulfate. Cool, warm and hot in-situ temperature regimes (15–20 °C, 30–35 °C and 70–95 °C) were selected from hydrothermal locations in Guaymas Basin to compare AOM geochemistry and 16S ribosomal RNA (rRNA), mcrA and dsrAB genes of the microbial communities. 16S rRNA gene clone libraries from the cool and hot AOM cores yielded similar archaeal types such as Miscellaneous Crenarchaeotal Group, Thermoproteales and anaerobic methane-oxidizing archaea (ANME)-1; some of the ANME-1 archaea formed a separate 16S rRNA lineage that at present seems to be limited to Guaymas Basin. Congruent results were obtained by mcrA gene analysis. The warm AOM core, chemically distinct by lower porewater sulfide concentrations, hosted a different archaeal community dominated by the two deep subsurface archaeal lineages Marine Benthic Group D and Marine Benthic Group B, and by members of the Methanosarcinales including ANME-2 archaea. This distinct composition of the methane-cycling archaeal community in the warm AOM core was confirmed by mcrA gene analysis. Functional genes of sulfate-reducing bacteria and archaea, dsrAB, showed more overlap between all cores, regardless of the core temperature. 16S rRNA gene clone libraries with Euryarchaeota-specific primers detected members of the Archaeoglobus clade in the cool and hot cores. A V6-tag high-throughput sequencing survey generally supported the clone library results while providing high-resolution detail on archaeal and bacterial community structure. These results indicate that AOM and the responsible archaeal communities persist over a wide temperature range.     

Anaerobic Oxidation of Methane Coupled to Nitrite Reduction by Halophilic Marine NC10 Bacteria

Anaerobic oxidation of methane (AOM) coupled to nitrite reduction is a novel AOM process that is mediated by denitrifying methanotrophs. To date, enrichments of these denitrifying methanotrophs have been confined to freshwater systems; however, the recent findings of 16S rRNA and pmoA gene sequences in marine sediments suggest a possible occurrence of AOM coupled to nitrite reduction in marine systems. In this research, a marine denitrifying methanotrophic culture was obtained after 20 months of enrichment. Activity testing and quantitative PCR (qPCR) analysis were then conducted and showed that the methane oxidation activity and the number of NC10 bacteria increased correlatively during the enrichment period. 16S rRNA gene sequencing indicated that only bacteria in group A of the NC10 phylum were enriched and responsible for the resulting methane oxidation activity, although a diverse community of NC10 bacteria was harbored in the inoculum. Fluorescence in situ hybridization showed that NC10 bacteria were dominant in the enrichment culture after 20 months. The effect of salinity on the marine denitrifying methanotrophic culture was investigated, and the apparent optimal salinity was 20.5‰, which suggested that halophilic bacterial AOM coupled to nitrite reduction was obtained. Moreover, the apparent substrate affinity coefficients of the halophilic denitrifying methanotrophs were determined to be 9.8 ± 2.2 μM for methane and 8.7 ± 1.5 μM for nitrite.     

Invasion of Spartina alterniflora on Zostera japonica enhances the abundances of bacteria by absolute quantification sequencing analysis

Plant invasion can alter soil organic matter composition and indirectly impact estuary ecology; therefore, it is paramount to understand how plant invasion influences the bacterial community. Here, we present an absolute quantification 16S rRNA gene sequencing to investigate the bacterial communities that were collected from Zostera japonica and Spartina alterniflora covered areas and Z. japonica degradation areas in the Yellow River Estuary. Our data revealed that the absolute quantity of bacteria in the surface layer was significantly (p < .05) higher than that in the bottom and degradation areas. Following the invasion of S. alterniflora, the abundances of Bacteroidia, Acidimicrobiaceae, and Dehalococcoidaceaewere enriched in the S. alterniflora sediment. In addition, variations in the composition of sediment bacterial communities at the phylum level were the most intimately related to total organic carbon (TOC), and the content of heavy metals could reduce the abundance of bacteria. This study provided some information to understand the effects of S. alterniflora invasion on Z. japonica from the perspective of microbiome level.     

Stratification of Archaeal communities in shallow sediments of the Pearl River Estuary,Southern China

Microorganisms are known to play fundamental roles in the biogeochemical cycling of carbon in the coastal environments. To get to know the composition and ecological roles of the archaeal communities within the sediments of the Pearl River Estuary, Southern China, the diversity and vertical distribution of archaea in a sediment core was reported based on the 16S rRNA and mcrA genes for the first time. Quantitative PCR analysis revealed that archaea were present at 10⁶–10⁷ 16S rRNA gene copies/g (wet weight) in the sediment core, and the proportion of mcrA versus 16S rRNA gene copies varied from 11 to 45%. 16S rRNA gene libraries were constructed and analyzed for the top layer (0–6 cm), middle layer (18–24 cm), sulfate-methane transition zone (SMTZ, 32–42 cm), and bottom layer (44–50 cm) sediments. The results indicated that Miscellaneous Crenarchaeotal Group (MCG) was the main component in the sediments. The MCG archaea could be further divided into six subgroups: MCG-A, B, C, D, E, and F. On the other hand, mcrA sequences from methanogens related to the order Methanomicrobiales and ANME-2 methanotrophs were detected in all sediment layers. Taken together, our data revealed a largely unknown archaeal community in which MCG dominated within the Pearl River estuarine sediments, while methanogens and methane-oxidizing archaea putatively involving in methane metabolism, were also found in the community. This is the first important step towards elucidating the biogeochemical roles of these archaea in the Pearl River Estuary.     

Phylogenetic Study of Methanol Oxidizers from Chilika-Lake Sediments Using Genomic and Metagenomic Approaches

Group-wise diversity of sediment methylotrophs of Chilika lake (Lat. 19°28′–19°54′N; Long. 85°06′–85°35′E) Odisha, India at various identified sites was studied. Both the culturable and unculturable (metagenome) methylotrophs were investigated in the lake sediments employing both mxaF and 16S rRNA genes as markers. ARDRA profiling, 16S rRNA gene sequencing, PAGE profiling of HaeIII, EcoRI restricted mxaF gene and the mxaF gene sequences using culture-dependent approach revealed the relatedness of α-proteobacteria and Methylobacterium, Hyphomicrobium and Ancyclobacter sp. The total viable counts of the culturable aerobic methylotrophs were relatively higher in sediments near the sea mouth (S3; Panaspada), also demonstrated relatively high salinity (0.1 M NaCl) tolerance. Metagenomic DNA from the sediments, amplified using GC clamp mxaF primers and resolved through DGGE, revealed the diversity within the unculturable methylotrophic bacterium Methylobacterium organophilum, Ancyclobacter aquaticus, Burkholderiales and Hyphomicrobium sp. Culture-independent analyses revealed that up to 90 % of the methylotrophs were unculturable. The study enhances the general understandings of the metagenomic methylotrophs from such a special ecological niche.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-015-0510-3) contains supplementary material, which is available to authorized users.     

Influences of Infaunal Burrows on the Community Structure and Activity of Ammonia-Oxidizing Bacteria in Intertidal Sediments

Influences of infaunal burrows constructed by the polychaete (Tylorrhynchus heterochaetus) on O₂ concentrations and community structures and abundances of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) in intertidal sediments were analyzed by the combined use of a 16S rRNA gene-based molecular approach and microelectrodes. The microelectrode measurements performed in an experimental system developed in an aquarium showed direct evidence of O₂ transport down to a depth of 350 mm of the sediment through a burrow. The 16S rRNA gene-cloning analysis revealed that the betaproteobacterial AOB communities in the sediment surface and the burrow walls were dominated by Nitrosomonas sp. strain Nm143-like sequences, and most of the clones in Nitrospira-like NOB clone libraries of the sediment surface and the burrow walls were related to the Nitrospira marina lineage. Furthermore, we investigated vertical distributions of AOB and NOB in the infaunal burrow walls and the bulk sediments by real-time quantitative PCR (Q-PCR) assay. The AOB and Nitrospira-like NOB-specific 16S rRNA gene copy numbers in the burrow walls were comparable with those in the sediment surfaces. These numbers in the burrow wall at a depth of 50 to 55 mm from the surface were, however, higher than those in the bulk sediment at the same depth. The microelectrode measurements showed higher NH₄⁺ consumption activity at the burrow wall than those at the surrounding sediment. This result was consistent with the results of microcosm experiments showing that the consumption rates of NH₄⁺ and total inorganic nitrogen increased with increasing infaunal density in the sediment. These results clearly demonstrated that the infaunal burrows stimulated O₂ transport into the sediment in which otherwise reducing conditions prevailed, resulting in development of high NH₄⁺ consumption capacity. Consequently, the infaunal burrow became an important site for NH₄⁺ consumption in the intertidal sediment.     

Detection and Quantification of Geobacter lovleyi Strain SZ: Implications for Bioremediation at Tetrachloroethene- and Uranium-Impacted Sites

Geobacter lovleyi strain SZ reduces hexavalent uranium, U(VI), to U(IV) and is the first member of the metal-reducing Geobacter group capable of using tetrachloroethene (PCE) as a growth-supporting electron acceptor. Direct and nested PCR with specific 16S rRNA gene-targeted primer pairs distinguished strain SZ from other known chlorinated ethene-dechlorinating bacteria and closely related Geobacter isolates, including its closest cultured relative, G. thiogenes. Detection limits for direct and nested PCR were approximately 1 × 10⁶ and 1 × 10⁴ 16S rRNA gene copies per μl of template DNA, respectively. A quantitative real-time PCR (qPCR) approach increased the sensitivity to as few as 30 16S rRNA gene copies per μl of template DNA but was less specific. Melting curve analysis and comparison of the shapes of amplification plots identified false-positive signals and distinguished strain SZ from G. thiogenes when analyzed separately. These indicators were less reliable when target (strain SZ) DNA and nontarget (G. thiogenes) DNA with high sequence similarity were mixed, indicating that the development of qPCR protocols should not only evaluate specificity but also explore the effects of nontarget DNA on the accuracy of quantification. Application of specific tools detected strain SZ-like amplicons in PCE-dechlorinating consortia, including the bioaugmentation consortium KB-1, and two chlorinated ethene-impacted groundwater samples. Strain SZ-like amplicons were also detected in 13 of 22 groundwater samples following biostimulation at the uranium- and chlorinated solvent-contaminated Integrated Field-Scale Subsurface Research Challenge (IFC) site in Oak Ridge, TN. The numbers of strain SZ-like cells increased from below detection to 2.3 × 10⁷ ± 0.1 × 10⁷ per liter groundwater, suggesting that strain SZ-like organisms contribute to contaminant transformation. The G. lovleyi strain SZ-specific tools will be useful for monitoring bioremediation efforts at uranium- and/or chlorinated solvent-impacted sites such as the Oak Ridge IFC site.