Influence of Inorganic Nitrogen Management Regime on the Diversity of Nitrite-Oxidizing Bacteria in Agricultural Grassland Soils

To assess links between the diversity of nitrite-oxidizing bacteria (NOB) in agricultural grassland soils and inorganic N fertilizer management, NOB communities in fertilized and unfertilized soils were characterized by analysis of clone libraries and denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments. Previously uncharacterized Nitrospira-like sequences were isolated from both long-term-fertilized and unfertilized soils, but DGGE migration patterns indicated the presence of additional sequence types in the fertilized soils. Detailed phylogenetic analysis of Nitrospira-like sequences suggests the existence of one newly described evolutionary group and of subclusters within previously described sublineages, potentially representing different ecotypes; the new group may represent a lineage of noncharacterized Nitrospira species. Clone libraries of Nitrobacter-like sequences generated from soils under different long-term N management regimes were dominated by sequences with high similarity to the rhizoplane isolate Nitrobacter sp. strain PJN1. However, the diversity of Nitrobacter communities did not differ significantly between the two soil types. This is the first cultivation-independent study of nitrite-oxidizing bacteria in soil demonstrating that nitrogen management practices influence the diversity of this bacterial functional group.     

Identification and Specific Detection of a Novel Pseudomonadaceae Cluster Associated with Soils from Winter Wheat Plots of a Long-Term Agricultural Field Experiment

The genus Pseudomonas (sensu stricto) represents a group of microorganisms directly involved in functions conferring plant health. We performed a study in the DOK long-term agricultural field experiment on the basis of previously published Pseudomonas-selective PCR primers in order to investigate the community structure of the microbial groups defined by the target range of these primers. Three different agricultural management systems, i.e., conventional, biodynamic, and bio-organic, along with mineral and unfertilized controls were investigated, with each system planted with either winter wheat or a grass-clover ley. Amplified small-subunit rRNA gene fragments were analyzed using the genetic profiling techniques restriction fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE), revealing distinct differences between soils planted with winter wheat and grass clover but only minor differences between the management systems. Phylogenetic analyses of 59 clone sequences retrieved from bio-organic and unfertilized systems identified sequences related to Pseudomonas fluorescens and a novel cluster termed Cellvibrio-related Pseudomonadaceae (CRP). The CRP clones were exclusively isolated from winter wheat soil samples and were responsible for the crop-specific differences observed in RFLP and DGGE profiles. New primers were designed for the amplification of CRP targets directly from soil DNA, yielding strong signals exclusively for winter wheat soils. We concluded that crop-associated CRP exist in agricultural soils and that genetic profiling followed by specific probe design represents a valuable approach for identification as well as sensitive and rapid monitoring of novel microbial groups in the environment.     

Influence of inorganic nitrogen management regime on the diversity of nitrite-oxidizing bacteria in agricultural grassland soils

To assess links between the diversity of nitrite-oxidizing bacteria (NOB) in agricultural grassland soils and inorganic N fertilizer management, NOB communities in fertilized and unfertilized soils were characterized by analysis of clone libraries and denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments. Previously uncharacterized Nitrospira-like sequences were isolated from both long-term-fertilized and unfertilized soils, but DGGE migration patterns indicated the presence of additional sequence types in the fertilized soils. Detailed phylogenetic analysis of Nitrospira-like sequences suggests the existence of one newly described evolutionary group and of subclusters within previously described sublineages, potentially representing different ecotypes; the new group may represent a lineage of noncharacterized Nitrospira species. Clone libraries of Nitrobacter-like sequences generated from soils under different long-term N management regimes were dominated by sequences with high similarity to the rhizoplane isolate Nitrobacter sp. strain PJN1. However, the diversity of Nitrobacter communities did not differ significantly between the two soil types. This is the first cultivation-independent study of nitrite-oxidizing bacteria in soil demonstrating that nitrogen management practices influence the diversity of this bacterial functional group.     

Diversity and distribution of ammonia-oxidizing Archaea in the seasonally frozen soils in Northeastern China

The genetic diversity and distribution of ammonia-oxidizing Archaea (AOA) in nine seasonally frozen soils sampled around the city of Harbin, China, is analyzed based on archaeal amoA gene. Soil samples are divided into four groups by its properties: fertilized/unfertilized mesic (well-balanced supply of moisture) soils and fertilized/unfertilized hydric (abundant of moisture) soils. Clone libraries based on AOA amoA gene polymerase chain reaction products are constructed, and the phylogenetic analysis at 5 % cutoff level shows that AOA members mainly belong to the soil/sediment lineage which includes four clusters, and very few archaeal amoA gene sequences fall into the marine lineage. The four groups of soils have different archaeal amoA gene assemblage, and the available nitrogen and organic carbon are significantly correlated with diversity indexes. The result shows that long-term artificial amendment such as fertilization and agriculture cultivation has an important impact on AOA community shift in terrestrial environment. Moisture may drive the shape of different AOA communities by changing the aerobic environment into anaerobic. Soil composition is another noticeable factor effect AOA community, which can help the shape of a special AOA community with only two species.     

Bacterial soil community in a Brazilian sugarcane field

The assessment of bacterial communities in soil gives insight into microbial behavior under prevailing environmental conditions. In this context, we assessed the composition of soil bacterial communities in a Brazilian sugarcane experimental field. The experimental design encompassed plots containing common sugarcane (variety SP80-1842) and its transgenic form (IMI-1 — imazapyr herbicide resistant). Plants were grown in such field plots in a completely randomized design with three treatments, which addressed the factors transgene and imazapyr herbicide application. Soil samples were taken at three developmental stages during plant growth and analyzed using 16S ribosomal RNA (rRNA)-based PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and clone libraries. PCR-DGGE fingerprints obtained for the total bacterial community and specific bacterial groups — Actinobacteria, Alphaproteobacteria and Betaproteobacteria — revealed that the structure of these assemblages did not differ over time and among treatments. Nevertheless, slight differences among 16S rRNA gene clone libraries constructed from each treatment could be observed at particular cut-off levels. Altogether, the libraries encompassed a total of eleven bacterial phyla and the candidate divisions TM7 and OP10. Clone sequences affiliated with the Proteobacteria, Actinobacteria, Firmicutes and Acidobacteria were, in this order, most abundant. Accurate phylogenetic analyses were performed for the phyla Acidobacteria and Verrucomicrobia, revealing the structures of these groups, which are still poorly understood as to their importance for soil functioning and sustainability under agricultural practices.     

Effect of the Earthworms Lumbricus terrestris and Aporrectodea caliginosa on Bacterial Diversity in Soil

Earthworms ingest large amounts of soil and have the potential to radically alter the biomass, activity, and structure of the soil microbial community. In this study, the diversity of eight bacterial groups from fresh soil, gut, and casts of the earthworms Lumbricus terrestris and Aporrectodea caliginosa were studied by single-strand conformation polymorphism (SSCP) analysis using both newly designed 16S rRNA gene-specific primer sets targeting Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Bacteroidetes, Verrucomicrobia, Planctomycetes, and Firmicutes and a conventional universal primer set for SSCP, with RNA and DNA as templates. In parallel, the study of the relative abundance of these taxonomic groups in the same samples was performed using fluorescence in situ hybridization. Bacteroidetes, Alphaproteobacteria, and Betaproteobacteria were predominant in communities from the soil and worm cast samples. Representatives of classes Flavobacteria and Sphingobacteria (Bacteroidetes) and Pseudomonas spp. (low-abundant Gammaproteobacteria) were detected in soil and worm cast samples with conventional and taxon-targeting SSCP and through the sequence analysis of 16S rRNA clone libraries. Physiologically active unclassified Sphingomonadaceae (Alphaproteobacteria) and Alcaligenes spp. (Betaproteobacteria) also maintained their diversities during transit through the earthworm intestine and were found on taxon-targeting SSCP profiles from the soil and worm cast samples. In conclusion, our results suggest that some specific bacterial taxonomic groups maintain their diversity and even increase their relative numbers during transit through the gastrointestinal tract of earthworms.     

Bacterial Diversity of Weathered Terrestrial Icelandic Volcanic Glasses

The diversity of microbial communities inhabiting two terrestrial volcanic glasses of contrasting mineralogy and age was characterised. Basaltic glass from a <0.8 Ma hyaloclastite deposit (Valafell) harboured a more diverse Bacteria community than the younger rhyolitic glass from ～150-300 AD (D?madalshraun lava flow). Actinobacteria dominated 16S rRNA gene clone libraries from both sites, however, Proteobacteria, Acidobacteria and Cyanobacteria were also numerically abundant in each. A significant proportion (15-34%) of the sequenced clones displayed <85% sequence similarities with current database sequences, thus suggesting the presence of novel microbial diversity in each volcanic glass. The majority of clone sequences shared the greatest similarity to uncultured organisms, mainly from soil environments, among these clones from Antarctic environments and Hawaiian and Andean volcanic deposits. Additionally, a large number of clones within the Cyanobacteria and Proteobacteria were more similar to sequences from other lithic environments, included among these Icelandic clones from crystalline basalt and rhyolite, however, no similarities to sequences reported from marine volcanic glasses were observed. PhyloChip analysis detected substantially greater numbers of phylotypes at both sites than the corresponding clone libraries, but nonetheless also identified the basaltic glass community as the richer, containing approximately 29% unique phylotypes compared to rhyolitic glass.     

Comparison of Soil Bacterial Communities of Pinus patula of Nilgiris, Western Ghats with Other Biogeographically Distant Pine Forest Clone Libraries

The bacterial community structure of the rhizosphere and non-rhizosphere soil of Pinus patula, found in the Nilgiris region of Western Ghats, was studied by constructing 16S rRNA gene clone libraries. In the rhizosphere and non-rhizosphere soil clone libraries constructed, 13 and 15 bacterial phyla were identified, respectively. The clone libraries showed the predominance of members of culturally underrepresented phyla like Acidobacteria and Verrucomicrobia. The Alphaproteobacteria and Acidobacteria clones were predominant in rhizosphere and non-rhizosphere soil samples, respectively. In rhizosphere, amongst Alphaproteobacteria members, Bradyrhizobium formed the significant proportion, whereas in non-rhizosphere, members of subdivision-6 of phylum Acidobacteria were abundant. The diversity analysis of P. patula soil libraries showed that the phylotypes (16S rRNA gene similarity cutoff, ≥97 %) of Acidobacteria and Bacteroidetes were relatively predominant and diverse followed by Alphaproteobacteria and Verrucomicrobia. The diversity indices estimated higher richness and abundance of bacteria in P. patula soil clone libraries than the pine forest clone libraries retrieved from previous studies. The tools like principal co-ordinate analysis and Jackknife cluster analysis, which were under UniFrac analysis indicated that variations in soil bacterial communities were attributed to their respective geographical locations due to the phylogenetic divergence amongst the clone libraries. Overall, the P. patula rhizosphere and non-rhizosphere clone libraries were found significantly unique in composition, evenly distributed and highly rich in phylotypes, amongst the biogeographically distant clone libraries. It was finally hypothesised that the phylogenetic divergence amongst the bacterial phylotypes and natural selection plays a pivotal role in the variations of bacterial communities across the geographical distance.     

Long-term effect of re-vegetation on the microbial community of a severely eroded soil in sub-tropical China

The long-term (18 years) effects of re-vegetating eroded soil on soil microbial biomass, community structure and diversity were investigated in a forest soil derived from Quaternary clay in the Red Soil Ecological Experimental Station of the Chinese Academy of Sciences. Large areas of land in this region of China have been subjected to severe soil erosion, characterised by the removal of the fertile surface soil and even the exposure of parental rock in some areas due to a combination of deforestation and heavy rainfall. The effects of planting eroded or uneroded soil with Pinus massoniana, Cinnamomum camphora or Lespedeza bicolor on the soil microbial community and chemical properties were assessed. Total soil microbial community DNA was extracted and bacterial 16 S rRNA gene fragments were amplified by PCR and analysed by terminal restriction fragment length polymorphism (T-RFLP). Microbial biomass carbon (C_mic) was measured by chloroform fumigation-extraction. Following the restoration there were significant increases in both C_mic and bacterial diversity (Shannon index), and significant changes in bacterial community structure. Erosion factors were significant only in minor dimensions suggesting that the restoration had been largely successful in terms of bacterial community structure. Compared with uneroded soil, C_mic recovered in L. bicolor and P. massoniana restored eroded plots and was significantly greater under these tree species than C. camphora, although soils in C. camphora restored plots displayed the highest bacterial diversity. The recovery of microbial biomass and diversity in the eroded plots was, to large extent, accompanied by the development of the same bacterial community structure as in the uneroded plots with erosion having relatively little effect on bacterial community structure.     

Phylogenetic analysis based evolutionary study of 16S rRNA in known Pseudomonas sp

Molecular evolution analysis of 16S rRNA sequences of native Pseudomonas strains and different fluorescent pseudomonads were conducted on the basis of Molecular Evolutionary Genetics Analysis version 5.2 (MEGA5.2). Topological evaluations show common origin for native strains with other known strains with available sequences at GenBank database. Phylogenetic affiliation of different Pseudomonas sp based on 16S rRNA gene shows that molecular divergence contributes to the genetic diversity of Pseudomonas sp. Result indicate direct dynamic interactions with the rhizospheric pathogenic microbial community. The selection pressure acting on 16S rRNA gene was related to the nucleotide diversity of Pseudomonas sp in soil rhizosphere community among different agricultural crops. Besides, nucleotide diversity among the whole population was very low and tajima test statistic value (D) was also slightly positive (Tajima׳s test statistics D value 0.351). This data indicated increasing trends of infection of soil-borne pathogens under gangetic-alluvial regions of West Bengal due to high degree of nucleotide diversity with decreased population of plant growth promoting rhizobacteria like fluorescent Pseudomonads in soil.     

Bacterial Community Diversity in the Brazilian Atlantic Forest Soils

The aim of this study was to characterize the bacterial community diversity of the Brazilian Atlantic forest soil by means of both cultivation and 16S rRNA clone libraries. A collection of 86 representative isolates, obtained from six samples of Atlantic forest soils from the National Park of Serra dos Órgãos (PARNASO), belonged to the genera Arthrobacter, Bacillus, Burkholderia, Leifsonia, Paenibacillus, Pseudomonas, Ralstonia, Serratia, and Streptomyces according to the 16S rRNA sequences. Representative isolates from the different genera degraded cellulose and lignin. The culture-independent analysis based on 894 partial 16S rRNA gene sequences revealed that the most frequently retrieved groups belonged to the phyla Acidobacteria (29–54%), Proteobacteria (16–38%), and Verrucomicrobia (0.6–14%). The majority of the sequences (82.6%) were unidentified singletons and doubletons, indicating a high diversity of rare unique sequences. Chao1 estimator disclosed a high number of phyla (41–152) and species (263–446). This is the first survey on the Atlantic Forest soils using a combination of cultivation and culture-independent approaches. We conclude that the Brazilian Atlantic Forest soil represents a vast source of novel bacteria.     

The role of Variovorax and other Comamonadaceae in sulfur transformations by microbial wheat rhizosphere communities exposed to different sulfur fertilization regimes

Sulfonates are a key component of the sulfur present in agricultural soils. Their mobilization as part of the soil sulfur cycle is mediated by rhizobacteria, and involves the oxidoreductase AsfA. In this study, the effect of fertilization regime on rhizosphere bacterial asfA distribution was examined at the Broadbalk long-term wheat experiment, Rothamsted, UK, which was established in 1843, and has included a sulfur-free treatment since 2001. Direct isolation of desulfonating rhizobacteria from the wheat rhizospheres led to the identification of several Variovorax and Polaromonas strains, all of which contained the asfA gene. Rhizosphere DNA was isolated from wheat rhizospheres in plots fertilized with inorganic fertilizer with and without sulfur, with farmyard manure or from unfertilized plots. Genetic profiling of 16S rRNA gene fragments [denaturing gradient gel electrophoresis (DGGE)] from the wheat rhizospheres revealed that the level of inorganic sulfate in the inorganic fertilizer was correlated with changes in the general bacterial community structure and the betaproteobacterial community structure in particular. Community analysis at the functional gene level (asfA) showed that 40% of clones in asfAB clone libraries were affiliated to the genus Variovorax. Analysis of asfAB-based terminal restriction fragment length polymorphism (T-RFLP) fingerprints showed considerable differences between sulfate-free treatments and those where sulfate was applied. The results suggest the occurrence of desulfonating bacterial communities that are specific to the fertilization regime chosen and that arylsulfonates play an important role in rhizobacterial sulfur nutrition.     

Investigating the Diversity of Pseudomonas spp. in Soil Using Culture Dependent and Independent Techniques

Less than 1 % of bacterial populations present in environmental samples are culturable, meaning that cultivation will lead to an underestimation of total cell counts and total diversity. However, it is less clear whether this is also true for specific well-defined groups of bacteria for which selective culture media is available. In this study, we use culture dependent and independent techniques to describe whether isolation of Pseudomonas spp. on selective nutrient-poor NAA 1:100 agar-medium can reflect the full diversity, found by pyrosequencing, of the total soil Pseudomonas community in an urban waste field trial experiment. Approximately 3,600 bacterial colonies were isolated using nutrient-poor NAA 1:100 medium from soils treated with different fertilizers; (i) high N-level sewage sludge (SA), (ii) high N-level cattle manure (CMA), and (iii) unfertilized control soil (U). Based on Pseudomonas specific quantitative-PCR and Pseudomonas CFU counts, less than 4 % of Pseudomonas spp. were culturable using NAA 1:100 medium. The Pseudomonas selectivity and specificity of the culture medium were evaluated by 454 pyrosequencing of 16S rRNA gene amplicons generated using Bacteria- and Pseudomonas-specific primers. Pyrosequencing results showed that most isolates were Pseudomonas and that the culturable fraction of Pseudomonas spp. reflects most clusters of the total Pseudomonas diversity in soil. This indicates that NAA 1:100 medium is highly selective for Pseudomonas species, and reveals the ability of NAA 1:100 medium to culture mostly the dominant Pseudomonas species in soil.     

Bacterial structure and spatiotemporal distribution in a horizontal subsurface flow constructed wetland

In this study, bacterial community structure in a horizontal subsurface flow constructed wetland (HSF-CW) planted with Phragmites australis was investigated using the 16S rRNA cloning–sequencing technique. Two layer depths were considered: the rhizosphere zone (RH) and the deep-layer zone (DL) in different sampling periods. Bacteria-specific primers 008F and 1492R were used to amplify the 16S rRNA genes and construct six clone libraries. A total of 1,284 individual sequences were used to assess the HSF-CW diversity. Phylogenetic analysis of RH and DL clone libraries shows that 41.57 and 42.17 % of the 16S rRNA sequences are affiliated with the Proteobacteria in the RH and the DL, respectively. The remaining major phylogenetic groups are Bacteroidetes, Planctomycetes, and Chloroflexi with 11.78, 9.36, and 7.6 %, respectively, in the RH and 11.38, 6.48, and 7.65 % in the DL, respectively. Minor divisions such as Verrucomicrobia, TM7, Nitrospira, and Gemmatimonadetes represented <6 % of the total sequences, while 14.2 % were unidentified Bacteria. Among the Proteobacteria, the Alphaproteobacteria subclass is represented in both locations, while the Deltaproteobacteria and Gammaproteobacteria subclasses were predominant in the RH and the DL, respectively. Results suggest that Archaea and Bacteria in the HSF-CW are the essential actors in the nitrogen cycle and that the established microbial community is efficient in nitrogen removal from wastewater.     

Seasonal and regional diversity of maple sap microbiota revealed using community PCR fingerprinting and 16S rRNA gene clone libraries

An arbitrary primed community PCR fingerprinting technique based on capillary electrophoresis was developed to study maple sap microbial community characteristics among 19 production sites in Québec over the tapping season. Presumptive fragment identification was made with corresponding fingerprint profiles of bacterial isolate cultures. Maple sap microbial communities were subsequently compared using a representative subset of 13 16S rRNA gene clone libraries followed by gene sequence analysis. Results from both methods indicated that all maple sap production sites and flow periods shared common microbiota members, but distinctive features also existed. Changes over the season in relative abundance of predominant populations showed evidence of a common pattern. Pseudomonas (64%) and Rahnella (8%) were the most abundantly and frequently represented genera of the 2239 sequences analyzed. Janthinobacterium, Leuconostoc, Lactococcus, Weissella, Epilithonimonas and Sphingomonas were revealed as occasional contaminants in maple sap. Maple sap microbiota showed a low level of deep diversity along with a high variation of similar 16S rRNA gene sequences within the Pseudomonas genus. Predominance of Pseudomonas is suggested as a typical feature of maple sap microbiota across geographical regions, production sites, and sap flow periods.     

Comparative Diversity of Ammonia Oxidizer 16S rRNA Gene Sequences in Native, Tilled, and Successional Soils

Autotrophic ammonia oxidizer (AAO) populations in soils from native, tilled, and successional treatments at the Kellogg Biological Station Long-Term Ecological Research site in southwestern Michigan were compared to assess effects of disturbance on these bacteria. N fertilization effects on AAO populations were also evaluated with soils from fertilized microplots within the successional treatments. Population structures were characterized by PCR amplification of microbial community DNA with group-specific 16S rRNA gene (rDNA) primers, cloning of PCR products and clone hybridizations with group-specific probes, phylogenetic analysis of partial 16S rDNA sequences, and denaturing gradient gel electrophoresis (DGGE) analysis. Population sizes were estimated by using most-probable-number (MPN) media containing varied concentrations of ammonium sulfate. Tilled soils contained higher numbers than did native soils of culturable AAOs that were less sensitive to different ammonium concentrations in MPN media. Compared to sequences from native soils, partial 16S rDNA sequences from tilled soils were less diverse and grouped exclusively within Nitrosospira cluster 3. Native soils yielded sequences representing three different AAO clusters. Probes for Nitrosospira cluster 3 hybridized with DGGE blots from tilled and fertilized successional soils but not with blots from native or unfertilized successional soils. Hybridization results thus suggested a positive association between the Nitrosospira cluster 3 subgroup and soils amended with inorganic N. DGGE patterns for soils sampled from replicated plots of each treatment were nearly identical for tilled and native soils in both sampling years, indicating spatial and temporal reproducibility based on treatment.     

Identification and isolation of a Castellaniella species important during biostimulation of an acidic nitrate- and uranium-contaminated aquifer

Immobilization of uranium in groundwater can be achieved through microbial reduction of U(VI) to U(IV) upon electron donor addition. Microbial community structure was analyzed in ethanol-biostimulated and control sediments from a high-nitrate (>130 mM), low-pH, uranium-contaminated site in Oak Ridge, TN. Analysis of small subunit (SSU) rRNA gene clone libraries and polar lipid fatty acids from sediments revealed that biostimulation resulted in a general decrease in bacterial diversity. Specifically, biostimulation resulted in an increase in the proportion of Betaproteobacteria (10% of total clones in the control sediment versus 50 and 79% in biostimulated sediments) and a decrease in the proportion of Gammaproteobacteria and Acidobacteria. Clone libraries derived from dissimilatory nitrite reductase genes (nirK and nirS) were also dominated by clones related to Betaproteobacteria (98% and 85% of total nirK and nirS clones, respectively). Within the nirK libraries, one clone sequence made up 59 and 76% of sequences from biostimulated sediments but only made up 10% of the control nirK library. Phylogenetic analysis of SSU rRNA and nirK gene sequences from denitrifying pure cultures isolated from the site indicate that all belong to a Castellaniella species; nearly identical sequences also constituted the majority of biostimulated SSU rRNA and nirK clone libraries. Thus, by combining culture-independent with culture-dependent techniques, we were able to link SSU rRNA clone library information with nirK sequence data and conclude that a potentially novel Castellaniella species is important for in situ nitrate removal at this site.     

Shift in microbial population in response to crystalline cellulose degradation during enrichment with a semi-desert soil

Addition of crystalline cellulose to semi-desert soil shifts the microbial population; this was assessed by following the 16S rRNA gene, glycosyl hydrolase, and measuring its functional diversity in the bacterial population. Quantification of the glycosyl hydrolase gene showed an increase from 1 × 10⁴ g⁻¹ of unamended soil to 3 × 10⁴ g⁻¹ of crystalline-cellulose-amended soil by the 15th day of crystalline cellulose utilization. The indigenous glycosyl hydrolase community in unamended soil was dominated by the clone families that were closely related to the glycosyl hydrolases from Betaproteobacteria and Firmicutes. The addition of crystalline cellulose induced a shift in the glycosyl hydrolase population toward an increase in the relative abundance of the glycosyl hydrolase that was consistent with those of Bacteroidetes and Flavobacteria. The population shift of glycosyl hydrolase was also supported by the comparison of the 16S rRNA gene families in unamended and crystalline-cellulose-amended soil libraries. The most abundant 16S rRNA gene sequences retrieved in the unamended soil were identical to Pseudomonas, Massilia, Paenibacillus, and Bacillus spp., while Cytophaga and Flavobacterium spp. dominated in crystalline-cellulose-amended soil.     

Identification and Isolation of a Castellaniella Species Important during Biostimulation of an Acidic Nitrate- and Uranium-Contaminated Aquifer

Immobilization of uranium in groundwater can be achieved through microbial reduction of U(VI) to U(IV) upon electron donor addition. Microbial community structure was analyzed in ethanol-biostimulated and control sediments from a high-nitrate (>130 mM), low-pH, uranium-contaminated site in Oak Ridge, TN. Analysis of small subunit (SSU) rRNA gene clone libraries and polar lipid fatty acids from sediments revealed that biostimulation resulted in a general decrease in bacterial diversity. Specifically, biostimulation resulted in an increase in the proportion of Betaproteobacteria (10% of total clones in the control sediment versus 50 and 79% in biostimulated sediments) and a decrease in the proportion of Gammaproteobacteria and Acidobacteria. Clone libraries derived from dissimilatory nitrite reductase genes (nirK and nirS) were also dominated by clones related to Betaproteobacteria (98% and 85% of total nirK and nirS clones, respectively). Within the nirK libraries, one clone sequence made up 59 and 76% of sequences from biostimulated sediments but only made up 10% of the control nirK library. Phylogenetic analysis of SSU rRNA and nirK gene sequences from denitrifying pure cultures isolated from the site indicate that all belong to a Castellaniella species; nearly identical sequences also constituted the majority of biostimulated SSU rRNA and nirK clone libraries. Thus, by combining culture-independent with culture-dependent techniques, we were able to link SSU rRNA clone library information with nirK sequence data and conclude that a potentially novel Castellaniella species is important for in situ nitrate removal at this site.     

Diazotrophic diversity, nifH gene expression and nitrogenase activity in a rice paddy field in Fujian, China

The diazotrophic communities in a rice paddy field were characterized by a molecular polyphasic approach including DNA/RNA-DGGE fingerprinting, real time RT-PCR analysis of nifH gene and the measurement of nitrogen fixation activities. The investigation was performed on a diurnal cycle and comparisons were made between bulk and rhizosphere / root soil as well as between fertilized / unfertilized soils. Real time RT-PCR showed no significant difference in the total quantity of nifH expression under the conditions investigated. The functional diversity and dynamics of the nifH gene expressing diazotroph community investigated using RT-PCR-DGGE revealed high diurnal variations, as well as variation between different soil types. Most of the sequence types recovered from the DGGE gels and clone libraries clustered within nifH Cluster I and III (65 different nifH sequences in total). Sequence types most similar to Azoarcus spp., Metylococcus spp., Rhizobium spp., Methylocystis spp., Desulfovibrio spp., Geobacter spp., Chlorobium spp., were abundant and indicate that these species may be responsible for the observed diurnal variation in the diazotrophic community structure in these rice field samples. Previously described diazotrophic cyanobacterial genera in rice fields, such as Nostoc and Cyanothece, were present in the samples but not detectable in RT-PCR assays.