Patterns of Community Change among Ammonia Oxidizers in Meadow Soils upon Long-Term Incubation at Different Temperatures

The effect of temperature on the community structure of ammonia-oxidizing bacteria was investigated in three different meadow soils. Two of the soils (OMS and GMS) were acidic (pH 5.0 to 5.8) and from sites in Germany with low annual mean temperature (about 10°C), while KMS soil was slightly alkaline (pH 7.9) and from a site in Israel with a high annual mean temperature (about 22°C). The soils were fertilized and incubated for up to 20 weeks in a moist state and as a buffered (pH 7) slurry amended with urea at different incubation temperatures (4 to 37°C). OMS soil was also incubated with less fertilizer than the other soils. The community structure of ammonia oxidizers was analyzed before and after incubation by denaturing gradient gel electrophoresis (DGGE) of the amoA gene, which codes for the α subunit of ammonia monooxygenase. All amoA gene sequences found belonged to the genus Nitrosospira. The analysis showed community change due to temperature both in moist soil and in the soil slurry. Two patterns of community change were observed. One pattern was a change between the different Nitrosospira clusters, which was observed in moist soil and slurry incubations of GMS and OMS. Nitrosospira AmoA cluster 1 was mainly detected below 30°C, while Nitrosospira cluster 4 was predominant at 25°C. Nitrosospira clusters 3a, 3b, and 9 dominated at 30°C. The second pattern, observed in KMS, showed a community shift predominantly within a single Nitrosospira cluster. The sequences of the individual DGGE bands that exhibited different trends with temperature belonged almost exclusively to Nitrosospira cluster 3a. We conclude that ammonia oxidizer populations are influenced by temperature. In addition, we confirmed previous observations that N fertilizer also influences the community structure of ammonia oxidizers. Thus, Nitrosospira cluster 1 was absent in OMS soil treated with less fertilizer, while Nitrosospira cluster 9 was only found in the sample given less fertilizer.     

Phylogenetic Diversity of Natural Populations of Ammonia Oxidizers Investigated by Specific PCR Amplification

Abstract The species composition of ammonia-oxidizing bacteria in aquatic environments was investigated using PCR primers for 16S rRNA genes to amplify specific subsets of the total ammonia-oxidizer population. The specificity of the amplification reactions was determined using total genomic DNA from known nitrifying strains and non-nitrifying strains identified as having similar rDNA sequences. Specificity of amplification was determined both for direct amplification, using the nitrifier specific primers, and with nested amplification, in which the nitrifier primers were used to reamplify a fragment obtained from direct amplification with Eubacterial universal primers. The present level of specificity allows the distinction between Nitrosomonas europaea, Nitrosomonas sp. (marine) and the other known ammonia-oxidizers in the beta subclass of the Proteobacteria. Using total DNA extracted from natural samples, we used direct amplification to determine presence/absence of different species groups. Species composition was found to differ among depths in vertical profiles of lake samples and among samples and enrichments from various other aquatic environments. Nested PCR yielded several more positive reactions, which implies that nitrifier DNA was present in most samples, but often at very low levels. Received: 25 September 1995; Revised: 15 January 1996; Accepted: 20 February 1996     

Community Analysis of Ammonia and Nitrite Oxidizers during Start-Up of Nitritation Reactors

Partial nitrification of ammonium to nitrite under oxic conditions (nitritation) is a critical process for the effective use of alternative nitrogen removal technologies from wastewater. Here we investigated the conditions which promote establishment of a suitable microbial community for performing nitritation when starting from regular sewage sludge. Reactors were operated in duplicate under different conditions (pH, temperature, and dilution rate) and were fed with 50 mM ammonium either as synthetic medium or as sludge digester supernatant. In all cases, stable nitritation could be achieved within 10 to 20 days after inoculation. Quantitative in situ hybridization analysis with group-specific fluorescent rRNA-targeted oligonucleotides (FISH) in the different reactors showed that nitrite-oxidizing bacteria of the genus Nitrospira were only active directly after inoculation with sewage sludge (up to 4 days and detectable up to 10 days). As demonstrated by quantitative FISH and restriction fragment length polymorphism (RFLP) analyses of the amoA gene (encoding the active-site subunit of the ammonium monooxygenase), the community of ammonia-oxidizing bacteria changed within the first 15 to 20 days from a more diverse set of populations consisting of members of the Nitrosomonas communis and Nitrosomonas oligotropha sublineages and the Nitrosomonas europaea-Nitrosomonas eutropha subgroup in the inoculated sludge to a smaller subset in the reactors. Reactors operated at 30°C and pH 7.5 contained reproducibly homogeneous communities dominated by one amoA RFLP type from the N. europaea-N. eutropha group. Duplicate reactors at pH 7.0 developed into diverse communities and showed transient population changes even within the ammonia oxidizer community. Reactors at pH 7.5 and 25°C formed communities that were indistinguishable by the applied FISH probes but differing in amoA RFLP types. Communities in reactors fed with sludge digester supernatant exhibited a higher diversity and were constantly reinoculated with ammonium oxidizers from the supernatant. Therefore, such systems could be maintained at a higher dilution rate (0.75 day⁻¹ compared to 0.2 day⁻¹ for the synthetic wastewater reactors). Despite similar reactor performance with respect to chemical parameters, the underlying community structures were different, which may have an influence on stability during perturbations.     

基于氨单加氧酶基因的自养脱氮菌群结构分析

全程自养脱氮是一种在高氨氮低溶氧条件下完全由自养菌群作用脱除氮素的现象.以全程自养脱氮污泥为研究对象,特异性扩增氨单加氧酶活性基因amoA片段,建立克隆文库并对克隆序列进行系统发育学分析,考察全程自养脱氮系统从建立到退化过程中氨氧化菌的结构变迁.结果表明:Nitrosomonas oligotropha和Nitrosomonas europaea细菌是系统中的主要氨氧化菌,而随着系统的退化前者逐渐被后者完全取代,而氨氧化菌的种群变迁可能并不是全混流系统全程自养脱氮效率下降的原因.     

Patterns and Processes of Microbial Community Assembly

SUMMARY

Recent research has expanded our understanding of microbial community assembly. However, the field of community ecology is inaccessible to many microbial ecologists because of inconsistent and often confusing terminology as well as unnecessarily polarizing debates. Thus, we review recent literature on microbial community assembly, using the framework of Vellend (Q. Rev. Biol. 85:183–206, 2010) in an effort to synthesize and unify these contributions. We begin by discussing patterns in microbial biogeography and then describe four basic processes (diversification, dispersal, selection, and drift) that contribute to community assembly. We also discuss different combinations of these processes and where and when they may be most important for shaping microbial communities. The spatial and temporal scales of microbial community assembly are also discussed in relation to assembly processes. Throughout this review paper, we highlight differences between microbes and macroorganisms and generate hypotheses describing how these differences may be important for community assembly. We end by discussing the implications of microbial assembly processes for ecosystem function and biodiversity.     

Stable Isotope Probing Analysis of Interactions between Ammonia Oxidizers

The response of natural microbial communities to environmental change can be assessed by determining DNA- or RNA-targeted changes in relative abundance of 16S rRNA gene sequences by using fingerprinting techniques such as denaturing gradient gel electrophoresis (DNA-DGGE and RNA-DGGE, respectively) or by stable isotope probing (SIP) of 16S rRNA genes following incubation with a ¹³C-labeled substrate (DNA-SIP-DGGE). The sensitivities of these three approaches were compared during batch growth of communities containing two or three Nitrosospira pure or enriched cultures with different tolerances to a high ammonia concentration. Cultures were supplied with low, intermediate, or high initial ammonia concentrations and with ¹³C-labeled carbon dioxide. DNA-SIP-DGGE provided the most direct evidence for growth and was the most sensitive, with changes in DGGE profiles evident before changes in DNA- and RNA-DGGE profiles and before detectable increases in nitrite and nitrate production. RNA-DGGE provided intermediate sensitivity. In addition, the three molecular methods were used to follow growth of individual strains within communities. In general, changes in relative activities of individual strains within communities could be predicted from monoculture growth characteristics. Ammonia-tolerant Nitrosospira cluster 3b strains dominated mixed communities at all ammonia concentrations, and ammonia-sensitive strains were outcompeted at an intermediate ammonia concentration. However, coexistence of ammonia-tolerant and ammonia-sensitive strains occurred at the lowest ammonia concentration, and, under some conditions, strains inhibited at high ammonia in monoculture were active at high ammonia in mixed cultures, where they coexisted with ammonia-tolerant strains. The results therefore demonstrate the sensitivity of SIP for detection of activity of organisms with relatively low yield and low activity and its ability to follow changes in the structure of interacting microbial communities.Molecular characterization of natural microbial communities has demonstrated the existence of novel high-level taxonomic groups with no cultured representatives and with significant diversity within phylogenetic and functional groups already established through analysis of organisms in laboratory culture. Autotrophic ammonia-oxidizing bacteria (AOB) exemplify the latter situation. Their low growth rates and the limited number of readily measured phenotypic characteristics available for identification of these organisms necessitate the use of molecular techniques for characterization of their diversity in natural environments. Phylogenetic analysis of 16S rRNA gene sequences places the majority of cultivated autotrophic bacterial ammonia oxidizers in a monophyletic group within the Betaproteobacteria (8, 26). Amplification and phylogenetic analysis of 16S rRNA gene sequences from enrichment cultures of ammonia oxidizers and sequences of environmental clones (31) suggest the existence of novel groups with no cultivated representative and considerable diversity within those represented by pure cultures.Increased awareness of microbial diversity has raised questions regarding links between species diversity and functional diversity, functional redundancy, and the influence of environmental conditions on the activities of representatives of different phylotypes. For ammonia-oxidizing bacteria, relationships exist between broad phylogenetic groups and the environments from which laboratory isolates were obtained, which are linked, in some cases, to differences in physiological characteristics (11). There is also evidence of links between the relative abundance of different ammonia oxidizer groups and environmental conditions (1, 13, 14, 18, 21, 23, 34), suggesting selection for organisms with particular physiological characteristics. In one study (36), a combination of molecular and physiological studies has demonstrated links between species diversity, functional diversity, and soil nitrification kinetics. However, for ammonia oxidizers and other groups, there is little direct evidence about which strains within diverse communities are active under particular conditions or the extent of competition for substrates.Stable isotope probing (SIP) (24, 27) of nucleic acids provides direct evidence of which members of mixed communities are active. This involves addition of substrates labeled with a stable isotope (most commonly ¹³C), extraction of nucleic acids, separation of ¹²C- and ¹³C-labeled nucleic acids by density gradient centrifugation, and subsequent molecular analysis. Sequences amplified from ¹³C-labeled DNA or RNA are derived from organisms actively assimilating the substrate. This approach has been used to identify organisms that utilize methane or methanol (4, 19), organic compounds (15, 20), or CO₂ (6, 9) in microcosms and those that assimilate plant root exudates in the field (28). SIP therefore links phylogeny to ecosystem function and has identified established and novel groups by utilizing labeled compounds in complex soil communities. The technique also enables in situ physiological studies and investigation of interactions between organisms in mixed cultures belonging to the same functional group. For autotrophic betaproteobacterial ammonia oxidizers, amplification of 16S rRNA genes from ¹³C-labeled DNA during incubation with [¹³C]CO₂ has the potential for discriminating which strains are active under specific conditions. Assessment of the discriminatory ability of this approach in complex natural environments requires studies under controlled and well-characterized conditions. The first aim of this study was, therefore, to assess the ability of SIP to discriminate activities of different members of simple mixed communities in comparison with direct measurement of product concentration and DNA- and RNA-denaturing gradient gel electrophoresis (DGGE). The second was to determine whether the activities of members of mixed communities of ammonia-oxidizing bacteria, in particular, their ability to grow at high ammonia concentrations, could be predicted from their physiological characteristics in monoculture. Of particular interest was whether strains with low ammonia tolerance are competitive at low ammonia concentrations. Mixed cultures were assembled from pure culture representatives of Nitrosospira clusters 0, 3a, and 3b (26, 36), which are frequently found in soil environments, and from enrichment cultures containing representatives of these clusters with heterotrophic contaminants. Other criteria for choice of community members were similarities in specific growth rate and cultivation conditions to enable meaningful competition experiments.     

Genera-Specific Immunofluorescence Labeling of Ammonia Oxidizers with Polyclonal Antibodies Recognizing Both Subunits of the Ammonia Monooxygenase

Polyclonal antibodies that recognize the two subunits AmoA and AmoB of the ammonia monooxygenase (AMO) were applied to identify ammonia-oxidizing bacteria by immunofluorescence (IF) labeling in pure, mixed, and enriched cultures. The antibodies against the AmoA were produced using a synthetic peptide of the AmoA of Nitrosomonas eutropha, whereas the antibodies against the AmoB had been developed previously is against the whole B-subunit of the AMO [Pinck et al. (2001) Appl Environ Microbiol 67:118–124]. Using IF labeling, the AmoA antibodies were specific for the detection of all species of the genus Nitrosomonas. In contrast, the antiserum against AmoB labeled all genera of ammonia oxidizers of the -subclass of Proteobacteria (Nitrosomonas, Nitrosospira, Nitrosolobus, and Nitrosovibrio). The fluorescence signals of the AmoA antibodies were spread all over the cells, whereas the signals of the AmoB antibodies were associated with the cytoplasmic membranes. The specificity of the reactions of the antisera with ammonia oxidizers were proven in pure and mixed cultures, and the characteristic IF labeling and the morphology of the cells enabled their identification at the genus level. The genus-specific IF labeling could be used to identify ammonia oxidizers enriched from various habitats. In enrichment cultures of natural sandstone, cells of the genera Nitrosomonas, Nitrosovibrio, and Nitrosospira were detected. Members of the genus Nitrosovibrio and Nitrosolobus were most prominent in enriched garden soil samples, whereas members of the genus Nitrosomonas dominated in enriched activated sludge. The antibodies caused only slight background fluorescence on sandstone and soil particles compared to oligonucleotide probes, which could not be used to detect ammonia oxidizers on these materials because of strong nonspecific fluorescence.     

Effect of Soil Ammonium Concentration on N2O Release and on the Community Structure of Ammonia Oxidizers and Denitrifiers

The effect of ammonium addition (6.5, 58, and 395 μg of NH₄⁺-N g [dry weight] of soil⁻¹) on soil microbial communities was explored. For medium and high ammonium concentrations, increased N₂O release rates and a shift toward a higher contribution of nitrification to N₂O release occurred after incubation for 5 days at 4°C. Communities of ammonia oxidizers were assayed after 4 weeks of incubation by denaturant gradient gel electrophoresis (DGGE) of the amoA gene coding for the small subunit of ammonia monooxygenase. The DGGE fingerprints were invariably the same whether the soil was untreated or incubated with low, medium, or high ammonium concentrations. Phylogenetic analysis of cloned PCR products from excised DGGE bands detected amoA sequences which probably belonged to Nitrosospira 16S rRNA clusters 3 and 4. Additional clones clustered with Nitrosospira sp. strains Ka3 and Ka4 and within an amoA cluster from unknown species. A Nitrosomonas-like amoA gene was detected in only one clone. In agreement with the amoA results, community profiles of total bacteria analyzed by terminal restriction fragment length polymorphism (T-RFLP) showed only minor differences. However, a community shift occurred for denitrifier populations based on T-RFLP analysis of nirK genes encoding copper-containing nitrite reductase with incubation at medium and high ammonia concentrations. Major terminal restriction fragments observed in environmental samples were further described by correspondence to cloned nirK genes from the same soil. Phylogenetic analysis grouped these clones into clusters of soil nirK genes. However, some clones were also closely related to genes from known denitrifiers. The shift in the denitrifier community was probably the consequence of the increased supply of oxidized nitrogen through nitrification. Nitrification activity increased upon addition of ammonium, but the community structure of ammonium oxidizers did not change.     

Intensive Management Affects Composition of Betaproteobacterial Ammonia Oxidizers in Turfgrass Systems

Turfgrass is a highly managed ecosystem subject to frequent fertilization, mowing, irrigation, and application of pesticides. Turf management practices may create a perturbed environment for ammonia oxidizers, a key microbial group responsible for nitrification. To elucidate the long-term effects of turf management on these bacteria, we assessed the composition of betaproteobacterial ammonia oxidizers in a chronosequence of turfgrass systems (i.e., 1, 6, 23, and 95 years old) and the adjacent native pines by using both 16S rRNA and amoA gene fragments specific to ammonia oxidizers. Based on the Shannon-Wiener diversity index of denaturing gradient gel electrophoresis patterns and the rarefaction curves of amoA clones, turf management did not change the relative diversity and richness of ammonia oxidizers in turf soils as compared to native pine soils. Ammonia oxidizers in turfgrass systems comprised a suite of phylogenetic clusters common to other terrestrial ecosystems. Nitrosospira clusters 0, 2, 3, and 4; Nitrosospira sp. Nsp65-like sequences; and Nitrosomonas clusters 6 and 7 were detected in the turfgrass chronosequence with Nitrosospira clusters 3 and 4 being dominant. However, both turf age and land change (pine to turf) effected minor changes in ammonia oxidizer composition. Nitrosospira cluster 0 was observed only in older turfgrass systems (i.e., 23 and 95 years old); fine-scale differences within Nitrosospira cluster 3 were seen between native pines and turf. Further investigations are needed to elucidate the ecological implications of the compositional differences.     

Variations of Abundance and Community Structure of Ammonia Oxidizers and Nitrification Activity in Two Paddy Soils Polluted by Heavy Metals

While microbial nitrogen transformations are sensitive indicators of trace metal toxicity in soils, studies that quantify the impacts of heavy metal pollution in polluted rice soils on microbial communities and their activities remain limited. We examined changes in the abundance, composition and activity of ammonia oxidizing communities in two paddy fields that have been polluted by metal mining and smelting activities for more than three decades. The results showed a shift in the community structure of ammonia oxidizing archaea (AOA) and, to a lesser extent, of ammonia oxidizing bacteria (AOB) under metal pollution in the soils. All the retrieved AOB sequences in this study belonged to the genus Nitrosospira. Among them, the species in Cluster 3 a.1 seemed to be more sensitive to heavy metal pollution. Both AOB abundance and nitrification activity were not affected by heavy metal pollution in the two sites; whereas, AOA abundance increased. Our results suggested an effect of metal pollutants on communities of ammonia oxidizers, the degree of which varied in accordance with the amount of metal pollution. Therefore, it is difficult to quantify the relationship between the AOB/AOA communities and nitrification activity in the polluted soil.     

Phylogenetic Characterization of a Polychlorinated-Dioxin- Dechlorinating Microbial Community by Use of Microcosm Studies

Microcosms capable of reductive dechlorination of polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDD/Fs) were constructed in glass bottles by seeding them with a polluted river sediment and incubating them anaerobically with an organic medium. All of the PCDD/F congeners detected were equally reduced without the accumulation of significant amounts of less-chlorinated congeners as the intermediate or end products. Alternatively, large amounts of catechol and salicylic acid were produced in the upper aqueous phase. Thus, the dechlorination of PCDD/Fs and the oxidative degradation of the dechlorinated products seemed to take place simultaneously in the microcosm. Denaturing gel gradient electrophoresis and clone library analyses of PCR-amplified 16S rRNA genes from the microcosm showed that members of the phyla Firmicutes, Proteobacteria, and Bacteroidetes predominated. A significant number of Chloroflexi clones were also detected. Quantitative real-time PCR with specific primer sets showed that the 16S rRNA genes of a putative dechlorinator, “Dehalococcoides,” and its relatives accounted for 0.1% of the total rRNA gene copies of the microcosm. Most of the clones thus obtained formed a cluster distinct from the typical “Dehalococcoides” group. Quinone profiling indicated that ubiquinones accounted for 18 to 25% of the total quinone content, suggesting the coexistence and activity of ubiquinone-containing aerobic bacteria. These results suggest that the apparent complete dechlorination of PCDD/Fs found in the microcosm was due to a combination of the dechlorinating activity of the “Dehalococcoides”-like organisms and the oxidative degradation of the dechlorinated products by aerobic bacteria with aromatic hydrocarbon dioxygenases.     

Phylogenetic and Evolutionary Patterns in Microbial Carotenoid Biosynthesis Are Revealed by Comparative Genomics

Background

Carotenoids are multifunctional, taxonomically widespread and biotechnologically important pigments. Their biosynthesis serves as a model system for understanding the evolution of secondary metabolism. Microbial carotenoid diversity and evolution has hitherto been analyzed primarily from structural and biosynthetic perspectives, with the few phylogenetic analyses of microbial carotenoid biosynthetic proteins using either used limited datasets or lacking methodological rigor. Given the recent accumulation of microbial genome sequences, a reappraisal of microbial carotenoid biosynthetic diversity and evolution from the perspective of comparative genomics is warranted to validate and complement models of microbial carotenoid diversity and evolution based upon structural and biosynthetic data.

Methodology/Principal Findings

Comparative genomics were used to identify and analyze in silico microbial carotenoid biosynthetic pathways. Four major phylogenetic lineages of carotenoid biosynthesis are suggested composed of: (i) Proteobacteria; (ii) Firmicutes; (iii) Chlorobi, Cyanobacteria and photosynthetic eukaryotes; and (iv) Archaea, Bacteroidetes and two separate sub-lineages of Actinobacteria. Using this phylogenetic framework, specific evolutionary mechanisms are proposed for carotenoid desaturase CrtI-family enzymes and carotenoid cyclases. Several phylogenetic lineage-specific evolutionary mechanisms are also suggested, including: (i) horizontal gene transfer; (ii) gene acquisition followed by differential gene loss; (iii) co-evolution with other biochemical structures such as proteorhodopsins; and (iv) positive selection.

Conclusions/Significance

Comparative genomics analyses of microbial carotenoid biosynthetic proteins indicate a much greater taxonomic diversity then that identified based on structural and biosynthetic data, and divides microbial carotenoid biosynthesis into several, well-supported phylogenetic lineages not evident previously. This phylogenetic framework is applicable to understanding the evolution of specific carotenoid biosynthetic proteins or the unique characteristics of carotenoid biosynthetic evolution in a specific phylogenetic lineage. Together, these analyses suggest a “bramble” model for microbial carotenoid biosynthesis whereby later biosynthetic steps exhibit greater evolutionary plasticity and reticulation compared to those closer to the biosynthetic “root”. Structural diversification may be constrained (“trimmed”) where selection is strong, but less so where selection is weaker. These analyses also highlight likely productive avenues for future research and bioprospecting by identifying both gaps in current knowledge and taxa which may particularly facilitate carotenoid diversification.     

Culture-Independent Phylogenetic Analysis of the Microbial Community in Industrial Sugarcane Bagasse Feedstock Piles

Sugarcane bagasse is an important lignocellulosic by-product with potential for conversion to biofuels and chemicals in biorefinery. As a step towards an understanding of microbial diversity and the processes existing in bagasse collection sites, the microbial community in industrial bagasse feedstock piles was investigated. Molecular biodiversity analysis of 16S rDNA sequences revealed the presence of a complex bacterial community. A diverse group of mainly aerobic and facultative anaerobic bacteria was identified reflecting the aerobic and high temperature microenvironmental conditions under the pile surface. The major bacterial taxa present were identified as Firmicutes, Alpha- and Gammaproteobacteria, Acidobacteria, Bacteroidetes, and Actinobacteria. Analysis of the eukaryotic microbial assemblage based on an internal transcribed spacer revealed the predominance of diverse cellulolytic and hemicellulolytic ascomycota. A microbial interaction model is proposed, focusing on lignocellulose degradation and methane metabolism. The insights into the microbial community in this study provide a basis for efficient utilization of bagasse in lignocellulosic biomass-based industries.     

Distribution and Diversity of Archaeal and Bacterial Ammonia Oxidizers in Salt Marsh Sediments

Diversity and abundance of ammonia-oxidizing Betaproteobacteria (β-AOB) and archaea (AOA) were investigated in a New England salt marsh at sites dominated by short or tall Spartina alterniflora (SAS and SAT sites, respectively) or Spartina patens (SP site). AOA amoA gene richness was higher than β-AOB amoA richness at SAT and SP, but AOA and β-AOB richness were similar at SAS. β-AOB amoA clone libraries were composed exclusively of Nitrosospira-like amoA genes. AOA amoA genes at SAT and SP were equally distributed between the water column/sediment and soil/sediment clades, while AOA amoA sequences at SAS were primarily affiliated with the water column/sediment clade. At all three site types, AOA were always more abundant than β-AOB based on quantitative PCR of amoA genes. At some sites, we detected 10⁹ AOA amoA gene copies g of sediment⁻¹. Ratios of AOA to β-AOB varied over 2 orders of magnitude among sites and sampling dates. Nevertheless, abundances of AOA and β-AOB amoA genes were highly correlated. Abundance of 16S rRNA genes affiliated with Nitrosopumilus maritimus, Crenarchaeota group I.1b, and pSL12 were positively correlated with AOA amoA abundance, but ratios of amoA to 16S rRNA genes varied among sites. We also observed a significant effect of pH on AOA abundance and a significant salinity effect on both AOA and β-ΑΟΒ abundance. Our results expand the distribution of AOA to salt marshes, and the high numbers of AOA at some sites suggest that salt marsh sediments serve as an important habitat for AOA.Nitrification, the sequential oxidation of ammonia to nitrite and nitrate, is a critical step in the nitrogen cycle and is mediated by a suite of phylogenetically and physiologically distinct microorganisms. The recent discovery of ammonia oxidation among Archaea (17, 38) has led to a dramatic shift in the current model of nitrification and to new questions of niche differentiation between putative ammonia-oxidizing Archaea (AOA) and the more-well-studied ammonia-oxidizing Betaproteobacteria (β-AOB). Based on surveys of 16S rRNA genes and archaeal amoA genes, it is evident that AOA occupy a wide range of niches (10), suggesting a physiologically diverse group of Archaea. Additionally, in studies where AOA and β-AOB were both targeted, AOA were typically more abundant than their bacterial counterparts (19, 21, 42). However, there are reports of β-AOB outnumbering AOA in estuarine systems (6, 33), suggesting a possible shift in competitive dominance under certain conditions.Patterns of β-AOB diversity in estuaries have been well characterized and appear to be regulated by similar mechanisms within geographically disparate systems (4, 11, 32). However, AOA distribution and their role in nitrification relative to β-AOB remain to be determined. A few studies have begun to address this question in different estuaries, but no unifying patterns or mechanisms have emerged. Although β-AOB have been well studied along estuarine salinity gradients (1, 3, 4, 7, 11, 13, 22, 33, 39) and recent studies have begun to address AOA in estuaries (1, 6, 22, 32, 33), few have investigated β-AOB in salt marshes (9), and none has included AOA.In this study, we investigated the distribution and abundance of AOA and β-AOB based on the distribution and abundance of amoA genes in salt marsh sediments dominated by different types of vegetation. Although we equate the presence of archaeal amoA genes with the genetic potential to oxidize ammonia, we acknowledge the possibility that all Archaea that have amoA genes may not all represent functional ammonia oxidizers. Vegetation patterns of New England salt marshes are strongly correlated with marsh elevation and are controlled by a combination of interspecific competition and tolerance to physico-chemical stress (28). The dominant grasses of New England salt marshes are Spartina alterniflora and Spartina patens, which typically grow as pure stands. S. alterniflora is found in two phenotypically distinct but genetically identical forms, a tall and a short growth form (34). The tall S. alterniflora grows to heights of 1 to 2 m and is typically found at the edges of the marsh and along creek banks (SAT sites), while the short-form S. alterniflora may reach heights of only 30 cm and is found in sites (SAS sites) slightly higher on the marsh where soil drainage is limited and conditions are more reduced compared to SAT sites (14). Conversely, S. patens, due to its lower tolerance of salt and more reduced conditions, is found in sites (SP sites) highest on the marsh, in areas that receive less flooding (5). Because the marsh is subjected to daily tidal fluctuations, most sites experience periods of anoxia, the degree of which depends on the marsh elevation. We hypothesized that ammonia-oxidizing communities in areas dominated by different marsh grasses would reflect the different edaphic conditions associated with each type of grass, due to differences in vertical zonation in the marsh.     

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.     

Using Phylogenetic,Functional and Trait Diversity to Understand Patterns of Plant Community Productivity

Background

Two decades of research showing that increasing plant diversity results in greater community productivity has been predicated on greater functional diversity allowing access to more of the total available resources. Thus, understanding phenotypic attributes that allow species to partition resources is fundamentally important to explaining diversity-productivity relationships.

Methodology/Principal Findings

Here we use data from a long-term experiment (Cedar Creek, MN) and compare the extent to which productivity is explained by seven types of community metrics of functional variation: 1) species richness, 2) variation in 10 individual traits, 3) functional group richness, 4) a distance-based measure of functional diversity, 5) a hierarchical multivariate clustering method, 6) a nonmetric multidimensional scaling approach, and 7) a phylogenetic diversity measure, summing phylogenetic branch lengths connecting community members together and may be a surrogate for ecological differences. Although most of these diversity measures provided significant explanations of variation in productivity, the presence of a nitrogen fixer and phylogenetic diversity were the two best explanatory variables. Further, a statistical model that included the presence of a nitrogen fixer, seed weight and phylogenetic diversity was a better explanation of community productivity than other models.

Conclusions

Evolutionary relationships among species appear to explain patterns of grassland productivity. Further, these results reveal that functional differences among species involve a complex suite of traits and that perhaps phylogenetic relationships provide a better measure of the diversity among species that contributes to productivity than individual or small groups of traits.     

Responses of Active Ammonia Oxidizers to Eutrophication and Oxygen Statuses in Taihu Freshwater Sediments

Abstract

Here, we employed DNA-based stable isotope probing (SIP) and molecular biology methods to investigate active ammonia oxidizer communities in suboxic sediments (0 to –2?cm) at the micromolar oxygen level and layers (–2 to –5?cm) at nanomolar oxygen concentrations from meso-eutrophic and light-eutrophic locations in Taihu Lake. The results revealed that ammonia-oxidizing archaea (AOA) were less active in the anoxic layer of meso-eutrophic sites, while ammonia-oxidizing bacteria (AOB) were less active in suboxic sediments of light-eutrophic sites after 8?weeks of incubation. The active AOA in the meso- and light-eutrophic sediments belonged to the Nitrosopumilus, Nitrosotalea, and Nitrososphaera clusters and the Nitrosopumilus and Nitrososphaera clusters, respectively, with Nitrosopumilus cluster as the predominant AOA, which took up a higher ratio in the light-eutrophic and suboxic layers than their counterparts. The advantageous active AOB were numerically predominated by the Nitrosomonas cluster in the suboxic layers, and the Nitrosospira cluster in the anoxic layers, respectively, both of which were distributed in diverse frequencies in different eutrophication statuses. The role and community composition diversities of active ammonia oxidizers in freshwater sediments were attributed to the different eutrophication (including nitrogen and organic carbon content) and oxygen statuses.     

Microbial Community Composition of the Danshui River Estuary of Northern Taiwan and the Practicality of the Phylogenetic Method in Microbial Barcoding

In this study, the microbial community in a mangrove ecosystem was surveyed and used to test the eligibility of 16S rDNA library and neighbor-joining method for the purpose of estimating microbial composition. Genetic diversity (π) and four other diversity indices (Simpson’s unbiased, Shannon-Wiener, Evenness, and Chao1 indices) were applied to estimate the adaptive lineages of microorganisms in the mangrove ecosystem. The results indicated that γ-Proteobacteria is the most diverse taxon, while the most abundant family is Rhodobacteraceae (α-Proteobacteria), followed by Comamonadaceae (β-Proteobacteria). This result may imply the existence of a graded distribution of microbial diversity across a spectrum of different salinities in the waterbody of this estuary ecosystem. Furthermore, at least 500–1,000 bps of the posterior portion of 16S rDNA is required as a marker to profile the microbial diversity in a microcosm of interest using phylogenetic methods, according to the results of our sliding window analyses for the measurements of π, consistency index, and retention index.