Olive-Mill Wastewater Bacterial Communities Display a Cultivar Specific Profile

Culture-dependent and -independent approaches were employed to identify the bacterial community structure from olive-mill wastewater produced from three olive-fruit varieties. The 233 bacterial isolates recovered were phylogenetically related to 38 members of Firmicutes, Actinobacteria, α-Proteobacteria, β-Proteobacteria, γ-Proteobacteria, and Bacteroidetes. Employing a novel microarray-based approach (PhyloChip) a high bacterial diversity was revealed consisting of 18 different phyla with representatives from 99 different families. The bacterial diversity in olive-mill wastewater from the three olive tree varieties was dominated by α-, β-, γ-, δ-, ε-Proteobacteria, Firmicutes, Bacteroidetes, Chloroflexi, Cyanobacteria, and Actinobacteria. This in-depth analysis of the indigenous microbiota indicated a cultivar-specific bacterial profile. Interestingly, the common bacterial taxa present in all three varieties examined were restricted indicating that the bacterial communities present in the olive-mill wastewater are greatly influenced by the olive-fruit variety.     

Cultivable Bacterial Community from South China Sea Sponge as Revealed by DGGE Fingerprinting and 16S rDNA Phylogenetic Analysis

The cultivable bacterial communities associated with four South China Sea sponges—Stelletta tenuis, Halichondria rugosa, Dysidea avara, and Craniella australiensis in mixed cultures—were investigated by microbial community DNA-based DGGE fingerprinting and 16S rDNA phylogenetic analysis. Diverse bacteria such as α-, γ-, δ-Proteobacteria, Bacteroidetes, and Firmicutes were cultured, some of which were previously uncultivable bacteria, potential novel strains with less than 95% similarity to their closest relatives and sponge symbionts growing only in the medium with the addition of sponge extract. According to 16S rDNA BLAST analysis, most of the bacteria were cultured from sponge for the first time, although similar phyla of bacteria have been previously recognized. The selective pressure of sponge extract on the cultured bacterial species was suggested, although the effect of sponge extract on bacterial community in high nutrient medium is not significant. Although α- and γ-Proteobacteria appeared to form the majority of the dominant cultivable bacterial communities of the four sponges, the composition of the cultivable bacterial community in the mixed culture was different, depending on the medium and sponge species. Greater bacterial diversity was observed in media C and CS for Stelletta tenuis, in media F and FS for Halichondria rugosa and Craniella australiensis. S. tenuis was found to have the highest cultivable bacterial diversity including α-, γ-, δ-Proteobacteria, Bacteroidetes, and Firmicutes, followed by sponge Dysidea avara without δ-Proteobacteria, sponge Halichondria rugosa with only α-, γ-Proteobacteria and Bacteroidetes, and sponge C. australiensis with only α-, γ-Proteobacteria and Firmicutes. Based on this study, by the strategy of mixed cultivation integrated with microbial community DNA-based DGGE fingerprinting and phylogenetic analysis, the cultivable bacterial community of sponge could be revealed effectively.     

Phylogenetic profiling of bacterial community from two intimately located sites in Balramgari,North-East coast of India

Microbial communities in coastal subsurface sediments play an important role in biogeochemical cycles. In this study microbial communities in tidal subsurface sediments of Balramgari in the state of Orissa, India were investigated using a culture independent approach. Two 16S rDNA cloned libraries were prepared from the closely located (100 m along the coast) subsurface sediment samples. Library I sediment samples had higher organic carbon content but lower sand percentage in comparison to Library II. A total of 310 clone sequences were used for DOTUR analysis which revealed 51 unique phylotypes or operational taxonomic units (OTUs) for both libraries. The OTUs were affiliated with 13 major lineages of domain bacteria including Proteobacteria (α, β, δ and λ), Acidobacteria, Actinobacteria, Cyanobacteria, Chloroflexi, Firmicutes, Verrucomicrobia, Bacteroidetes, Gemmatimonadetes and TM7. We encountered few pathogenic bacteria such as Aeromonas hydrophila and Ochrobactrum intermedium, in sediment from Library I. ∫-LIBSHUFF comparison depicts that the two libraries were significantly different communities. Most of the OTUs from both libraries possessed ≥85% to <97% similarity to RDP database sequences depicting the putative presence of new species, genera and phylum. This work revealed the complex and unique bacterial diversity from coastal habitat of Balramgari and shows that, in coastal habitat a variability of physical and chemical parameter has a prominent impact on the microbial community structure.     

Fine-scale vertical distribution of bacteria in the East Pacific deep-sea sediments determined via 16S rRNA gene T-RFLP and clone library analyses

Although the deep-sea sediments harbor diverse and novel bacteria with important ecological and environmental functions, a comprehensive view of their community characteristics is still lacking, considering the vast area and volume of the deep-sea sedimentary environments. Sediment bacteria vertical distribution and community structure were studied of the E272 site in the East Pacific Ocean with the molecular methods of 16S rRNA gene T-RFLP (terminal restriction fragment length polymorphism) and clone library analyses. Layered distribution of the bacterial assemblages was detected by both methods, indicating that the shallow sediments (40 cm in depth) harbored a diverse and distinct bacterial composition with fine-scale spatial heterogeneity. Substantial bacterial diversity was detected and nine major bacterial lineages were obtained, including Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Nitrospirae, Planctomycetes, Proteobacteria, and the candidate divisions OP8 and TM6. Three subdivisions of the Proteobacteria presented in our libraries, including the α-, γ- and δ-Proteobacteria. Most of our sequences have low similarity with known bacterial 16S rRNA genes, indicating that these sequences may represent as-yet-uncultivated novel bacteria. Most of our sequences were related to the GenBank nearest neighboring sequences retrieved from marine sediments, especially from deep-sea methane seep, gas hydrate or mud volcano environments. Several sequences were related to the sequences recovered from the deep-sea hydrothermal vent or basalt glasses-bearing sediments, indicating that our deep-sea sampling site might be influenced to certain degree by the nearby hydrothermal field of the East Pacific Rise at 13°N. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Microbial diversity in acid mineral bioleaching systems of dongxiang copper mine and Yinshan lead–zinc mine

To understand the composition and structure of microbial communities in acid mineral bioleaching systems, the molecular diversity of 16S rDNA genes was examined using a PCR-based cloning approach. A total of 31 Operational Taxonomic Units (OTUs) were obtained from the four samples taken from four different bioleaching sites in Yinshan lead–zinc mine and Dongxiang copper mine in Jiangxi Province, China. The percentages of overlapping OTUs between sites ranged from 22.2 to 50.0%. Phylogenetic analysis revealed that the bacteria present at the four bioleaching sites fell into six divisions, α-Proteobacteria (1.1%), β-Proteobacteria (2.3%), γ-Proteobacteria (30.8%), Firmicutes (15.4%), Actinobacteria (0.3%) and Nitrospira (50.1%). Organisms of genera Leptospirillum, Acidithiobacillus, and Sulfobacillus, which were in Nitrospira, γ-Proteobacteria, and Firmicutes divisions, respectively, were the most dominant. The results of principal component analysis based on the six phylogenetic divisions and biogeochemical data indicated that the microbial community structure of a site was directly related to the biogeochemical characteristic of that site. It follows therefore that sites with similar biogeochemical characteristics were comprised of similar microbial community structures. The results in our study also suggest that the elements copper and arsenic appear to be the key factors affecting the compositions and structures of microbial community in the four bioleaching sites. Zhiguo He, Shengmu Xiao, Xuehui Xie are equally contributed to this work.     

A Novel Community of Acidophiles in an Acid Mine Drainage Sediment

A sediment sample (pH 2.5) was collected at an acid mine drainage site in Anhui, China. The present acidophilic microbial community in the sediment was studied with a 16S rRNA gene clone library. Small-subunit rRNA genes were PCR amplified, cloned and screened by amplified rDNA restriction analysis (ARDRA). Subsequently, 10 different clones were identified and they were affiliated with Acidobacteria, β/γ-Proteobacteria, δ-Proteobacteria, Nitrospira, Candidate division TM7, and Low G + C Gram-positives. Phylogenetic analysis of 16S rRNA gene sequences revealed a diversity of acidophiles in the sediment that were mostly novel. Unexpectedly, 16S rRNA gene sequences affiliated with δ-Proteobacteria were found to constitute more than 60% of clone library. To our knowledge, this is the first occasion that bacteria of δ-Proteobacteria have been found dominant in the acidic habitat. Anaerobic sulfate- or metal reduction is the predominant physiological trait of bacteria of this subdivision. The high sulfate, ferric iron and the presence of bioavailable carbon in the anaerobic microenvironment may result in the dominance of bacteria of δ-Proteobacteria.     

Isolation of the exoelectrogenic denitrifying bacterium Comamonas denitrificans based on dilution to extinction

The anode biofilm in a microbial fuel cell (MFC) is composed of diverse populations of bacteria, many of whose capacities for electricity generation are unknown. To identify functional populations in these exoelectrogenic communities, a culture-dependent approach based on dilution to extinction was combined with culture-independent community analysis. We analyzed the diversity and dynamics of microbial communities in single-chamber air-cathode MFCs with different anode surfaces using denaturing gradient gel electrophoresis based on the 16S rRNA gene. Phylogenetic analyses showed that the bacteria enriched in all reactors belonged primarily to five phylogenetic groups: Firmicutes, Actinobacteria, α-Proteobacteria, β-Proteobacteria, and γ-Proteobacteria. Dilution-to-extinction experiments further demonstrated that Comamonas denitrificans and Clostridium aminobutyricum were dominant members of the community. A pure culture isolated from an anode biofilm after dilution to extinction was identified as C. denitrificans DX-4 based on 16S rRNA sequence and physiological and biochemical characterizations. Strain DX-4 was unable to respire using hydrous Fe(III) oxide but produced 35 mW/m² using acetate as the electron donor in an MFC. Power generation by the facultative C. denitrificans depends on oxygen and MFC configuration, suggesting that a switch of metabolic pathway occurs for extracellular electron transfer by this denitrifying bacterium.     

Changes in the bacterial populations of the highly alkaline saline soil of the former lake Texcoco (Mexico) following flooding

Flooding an extreme alkaline-saline soil decreased alkalinity and salinity, which will change the bacterial populations. Bacterial 16S rDNA libraries were generated of three soils with different electrolytic conductivity (EC), i.e. soil with EC 1.7 dS m⁻¹ and pH 7.80 (LOW soil), with EC 56 dS m⁻¹ and pH 10.11 (MEDIUM soil) and with EC 159 dS m⁻¹ and pH 10.02 (HIGH soil), using universal bacterial oligonucleotide primers, and 463 clone 16S rDNA sequences were analyzed phylogenetically. Library proportions and clone identification of the phyla Proteobacteria, Actinobacteria, Acidobacteria, Cyanobacteria, Bacteroidetes, Firmicutes and Cloroflexi showed that the bacterial communities were different. Species and genera of the Rhizobiales, Rhodobacterales and Xanthomonadales orders of the α- and γ-subdivision of Proteobacteria were found at the three sites. Species and genera of the Rhodospirillales, Sphingobacteriales, Clostridiales, Oscillatoriales and Caldilineales were found only in the HIGH soil, Sphingomonadales, Burkholderiales and Pseudomonadales in the MEDIUM soil, Myxococcales in the LOW soil, and Actinomycetales in the MEDIUM and LOW soils. It was found that the largest diversity at the order and species level was found in the MEDIUM soil as bacteria of both the HIGH and LOW soils were found in it.     

Unique Microbial Signatures of the Alien Hawaiian Marine Sponge Suberites zeteki

Invasive species poses a threat to the world’s oceans. Alien sponges account for the majority of introduced marine species in the isolated Hawaiian reef ecosystems. In this study, cultivation-dependent and cultivation-independent techniques were applied to investigate microbial consortia associated with the alien Hawaiian marine sponge Suberites zeteki. Its microbial communities were diverse with representatives of Actinobacteria, Firmicutes, α- and γ-Proteobacteria, Bacteroidetes, Chlamydiae, Planctomycetes, and Cyanobacteria. Specifically, the genus Chlamydia was identified for the first time from marine sponges, and two genera (Streptomyces and Rhodococcus) were added to the short list of culturable actinobacteria from sponges. Culturable microbial communities were dominated by Bacillus species (63%) and contained actinobacterial species closely affiliated with those from habitats other than marine sponges. Cyanobacterial clones were clustered with free-living cyanobacteria from water column and other environmental samples; they show no affiliation with other sponge-derived cyanobacteria. The low sequence similarity of Planctomycetes, Chlamydiae, and α-Proteobacteria clones to other previously described sequences suggested that S. zeteki may contain new lineages of these bacterial groups. The microbial diversity of S. zeteki was different from that of other studied marine sponges. This is the first report on microbial communities of alien marine invertebrate species. For the first time, it provides an insight into microbial structure within alien marine sponges in the Hawaiian marine ecosystems.     

Incorporation of 3H-Thymidine by Different Prokaryotic Groups in Relation to Temperature and Nutrients in a Lacustrine Ecosystem

The incorporation of [³H-methyl] thymidine (³H-TdR) by Eubacteria, bacterial groups (α- and β-Proteobacteria, Cytophaga–Flavobacter), and Archaea was measured according to temperature (7 and 17°C) and nutrient levels (nitrogen, phosphorus, and carbon) in a lacustrine system (Sep, France). Short-term incubation was performed using a combination of microautoradiography and fluorescent in situ hybridization. Irrespective of the temperatures and nutrients studied, all the major phylogenetic bacterial groups assimilated ³H-TdR, and in most of the treatments studied, the proportion of β-Proteobacteria taking up ³H-TdR was higher than those in the other bacterial groups. The proportion of Bacteria and different bacterial groups studied incorporating ³H-TdR were significantly increased, approximately 1.5-fold, by temperature except for α-Proteobacteria (7.6-fold). The nutrient effect was not the same for the different bacterial groups according to the temperatures studied. The proportions of α-Proteobacteria (at both temperatures) and Cytophaga–Flavobacter (at 7°C) taking up ³H-TdR were significantly decreased and increased by adding N and P, respectively. Also, adding N, P, and C increased and decreased the percentage of β-Proteobacteria incorporating ³H-TdR at 7 and 17°C, respectively. The archaeal community showed a similar proportion of active cells (i.e., ³H-TdR) to the bacterial community, and uptake of ³H-TdR by Archaea was significantly increased (P < 0.05) by both temperature and nutrients. Thus, the assimilation of ³H-TdR by bacterial groups and Archaea in lacustrine system is significantly controlled by both temperature and nutrients.     

Archaeal and bacterial communities of heavy metal contaminated acidic waters from zinc mine residues in Sepetiba Bay

Mining of metallic sulfide ore produces acidic water with high metal concentrations that have harmful consequences for aquatic life. To understand the composition and structure of microbial communities in acid mine drainage (AMD) waters associated with Zn mine tailings, molecular diversity of 16S genes was examined using a PCR, cloning, and sequencing approach. A total of 78 operational taxonomic units (OTUs) were obtained from samples collected at five different sites in and around mining residues in Sepetiba Bay, Brazil. We analyzed metal concentration, physical, chemical, and microbiological parameters related to prokaryotic diversity in low metal impacted compared to highly polluted environments with Zn at level of gram per liter and Cd–Pb at level of microgram per liter. Application of molecular methods for community structure analyses showed that Archaea and Bacteria groups present a phylogenetic relationship with uncultured environmental organisms. Phylogenetic analysis revealed that bacteria present at the five sites fell into seven known divisions, α-Proteobacteria (13.4%), β-Proteobacteria (16.3%), γ-Proteobacteria (4.3%), Sphingobacteriales (4.3%), Actinobacteria (3.2%) Acidobacteria (2.1%), Cyanobacteria (11.9%), and unclassified bacteria (44.5%). Almost all archaeal clones were related to uncultivated Crenarchaeota species, which were shared between high impacted and low impacted waters. Rarefaction curves showed that bacterial groups are more diverse than archaeal groups while the overall prokaryotic biodiversity is lower in high metal impacted environments than in less polluted habitats. Knowledge of this microbial community structure will help in understanding prokaryotic diversity, biogeography, and the role of microorganisms in zinc smelting AMD generation and perhaps it may be exploited for environmental remediation procedures in this area.     

Seasonal variations in bacterioplankton community structures in two small rivers in the Himi region of central Japan and their relationships with environmental factors

The aim of this study was to improve our understanding of seasonal variations and the effects of physicochemical conditions on the bacterioplankton communities in two small rivers, the Moo and Nakayachi Rivers in the Himi region of central Japan. These rivers are inhabited by unionid freshwater mussels, which are used for oviposition by the endangered Itasenpara bitterling (Acheilognathus longipinnis). Water samples were collected every month between March 2011 and February 2012. Changes in bacterioplankton community structures were analysed using an approach that did not require cultivating the bacteria and involved PCR and denaturing gradient gel electrophoresis. The bacterioplankton community structures in the two rivers were similar in all seasons except winter. The bacterial sequences identified were dominated by typical freshwater Actinobacteria, Bacteroidetes, Cyanobacteria, α-Proteobacteria, and β-Proteobacteria bacterioplankton. Many β-Proteobacteria species were detected in all seasons, but Bacteroidetes species were dominant in the winter. The bacterioplankton community structures were affected by biochemical oxygen demand, chemical oxygen demand, chlorophyll-a concentration, water depth, and water temperature. These results provide a foundation for a more detailed understanding of the conditions that provide a suitable unionid habitat.     

Microbial diversity in Tunisian geothermal springs as detected by molecular and culture-based approaches

Prokaryotic diversities of 12 geothermal hot springs located in Northern, Central and Southern Tunisia were investigated by culture-based and molecular approaches. Enrichment cultures for both aerobic and anaerobic microorganisms were successfully obtained at temperatures ranging from 50 to 75°C. Fourteen strains including four novel species were cultivated and assigned to the phyla Firmicutes (9), Thermotogae (2), Betaproteobacteria (1), Synergistetes (1) and Bacteroidetes (1). Archaeal or universal oligonucleotide primer sets were used to generate 16S rRNA gene libraries. Representative groups included Proteobacteria, Firmicutes, Deinococcus-Thermus, Thermotogae, Synergistetes, Bacteroidetes, Aquificae, Chloroflexi, candidate division OP9 in addition to other yet unclassified strains. The archaeal library showed a low diversity of clone sequences belonging to the phyla Euryarchaeota and Crenarchaeota. Furthermore, we confirmed the occurrence of sulfate reducers and methanogens by amplification and sequencing of dissimilatory sulfite reductase (dsrAB) and methyl coenzyme M reductase α-subunit (mcrA) genes. Altogether, we discuss the diverse prokaryotic communities arising from the 12 geothermal hot springs studied and relate these findings to the physico-chemical features of the hot springs.     

Biodiversity of diazotrophic bacteria within the soil, root and stem of field-grown maize

Recent studies suggest a high diversity of diazotrophic bacteria in maize. However, none of these works have been based on a sufficient number of samples to provide reasonable quantitative estimates of diazotrophic bacterial diversity. Here we present the use of molecular tools and statistical inference to assess diazotrophic bacterial diversity within rhizosphere soils, roots and stems of field grown maize. DNA was isolated from the latter collected from six maize growing regions within the southern most state in Brazil, Rio Grande do Sul. Using conserved primers, nifH Cluster I gene fragments were amplified from each of the three zones, and the products cloned and sequenced. The majority of the sequences were classified within the Proteobacteria with the α-proteobacteria and β-proteobacteria being the most abundant in the rhizosphere soil and stem samples. The γ-proteobacteria were most abundant in rhizosphere soils, less so in roots, and least in the stem samples. According to three different diversity measures, the rhizosphere soil samples possessed greater diazotrophic bacterial diversity than the roots and stems of the maize plants. Only two genera, Azospirillum and Azotobacter, were found in virtually all samples at an abundance of over 1% of the total nifH sequences obtained. Other genera were largely restricted to soil (Methylocystis, Beijerinckia, Geobacter, Rhodovulum, Methylobacterium, Gluconacetobacter, Methylocella, and Delftia), roots (Dechloromonas), or stems (Methylosinus, Raoultella, and Rhizobium). Three genera, Herbaspirillum, Ideonella, and Klebsiella, appeared to dominate in the interior of the plant but were much rarer in soil.     

Culture-independent and -dependent methods to investigate the diversity of planktonic bacteria in the northern Bering Sea

Planktonic bacteria are abundant in the Bering Sea. However, very little is known about their diversity and the roles of various bacteria in the ocean. Bacterioplankton diversity in the northern Bering Sea was investigated using a combination of molecular and cultivation-based methods. Community fingerprint analysis using polymerase chain reaction-denaturing gradient gel electrophoresis revealed an apparent difference in the bacterioplankton community composition between sampling locations in the area. The bacterial communities were characterized by two 16S rRNA gene clone libraries for surface and bottom water at shallow station NEC5 (<60 m in depth) on the continental shelf. Sequences fell into 21 major lineages of the domain Bacteria, including Proteobacteria (Alpha, Beta, Gamma, and Delta), Bacteroidetes, Actinobacteria, Firmicutes, Acidobacteria, Planctomycetes, Verrucomicrobia, Fusobacteria, Chlamydiae, Chloroflexi, Chlorobi, Spirochaetes, Cyanobacteria (or algal chloroplasts), and candidate divisions OP8, OP11, TM6, TM7, and WS3. Significant differences were found between the two clone libraries. Actinobacteria formed the dominant bacterial lineage in both surface and bottom water, and the Alphaproteobacteria was another dominant fraction in surface water. A total of 232 heterotrophic bacterial strains were isolated and 81% showed extracellular proteolytic activity. Phylogenetic analysis revealed that the isolates fell into three bacterial groups, including the Gammaproteobacteria, Actinobacteria, and Firmicutes. The most common genus in both the bacterial isolates and protease-producing bacteria was Pseudoalteromonas. Divergence of bacterial community composition in the northern Bering Sea was mainly characterized by the dominance of Actinobacteria and reflected a bacterial community different from that currently known for marine bacterioplankton communities in other polar regions.     

Bacterial communities in sediments of the shallow Lake Dongping in China

Aims: The purpose of this study was to discuss how the environmental inputs and anthropogenic activities impact bacterial communities in the sediments of a shallow, eutrophic and temperate freshwater lake. Methods and Results: Sediment cores were collected from Lake Dongping, located in Taian, Shandong, China. All samples were processed within 4 h of collection. Total nitrogen, total phosphorus (TP), total organic carbon, ammonium nitrogen and nitrate nitrogen content of samples were measured by Kjeldahl determination, sulphuric acid–perchloric acid digestion and molybdenum blue colorimetry, potassium dichromate titration, Nessler’s reagent colorimetric and the phenol disulphonic acid colorimetric method, respectively. Seasonal and temporal diversity of sediment bacterial communities at six stations in Lake Dongping were investigated using molecular approaches (terminal restriction fragment length polymorphism and 16S rDNA clone libraries). Noticeable seasonal and temporal variations were observed in bacterial diversity and composition at all six stations. Sediment bacterial communities in Lake Dongping belonged to 16 phyla: Proteobacteria (including α‐Proteobacteria, β‐Proteobacteria, δ‐Proteobacteria, ε‐Proteobacteria, γ‐Proteobacteria), Acidobacteria, Planctomycetes, Bacteroidetes, Firmicutes, Verrucomicrobia, Nitrospira, Chloroflexi, Gemmatimonadetes, Chlorobi, Cyanobacteria, Deferribacteres, Actinobacteria, OP8, Spirochaetes and OP11. Members of β‐, δ‐ and γ‐Proteobacterial sequences were predominant in 11 of 12 clone libraries derived from sediment samples. Sediment samples collected at stations 1 and 4 in July had the greatest bacterial diversity while those collected at station 2 in October had the least diversity. TP concentration was significantly correlated with the distribution of bacterial communities. Conclusions: Our results suggested that different environmental nutrient inputs contribute to seasonal and temporal variations of chemical features and bacterial communities in sediments of Lake Dongping. TP concentration was significantly correlated with the distribution of bacterial communities. Significance and Impact of the Study: This study has an important implication for the optimization of integrated ecosystem assessment of shallow temperate freshwater lake and provides interesting information for the subsequent of the ecosystem.     

Community Composition of Marine Bacterioplankton Determined by 16S rRNA Gene Clone Libraries and Fluorescence In Situ Hybridization

We determined the compositions of bacterioplankton communities in surface waters of coastal California using clone libraries of 16S rRNA genes and fluorescence in situ hybridization (FISH) in order to compare the community structures inferred from these two culture-independent approaches. The compositions of two clone libraries were quite similar to those of clone libraries of marine bacterioplankton examined by previous studies. Clones from γ-proteobacteria comprised ca. 28% of the libraries, while approximately 55% of the clones came from α-proteobacteria, which dominated the clone libraries. The Cytophaga-Flavobacter group and three others each comprised 10% or fewer of the clone libraries. The community composition determined by FISH differed substantially from the composition implied by the clone libraries. The Cytophaga-Flavobacter group dominated 8 of the 11 communities assayed by FISH, including the two communities assayed using clone libraries. On average only 10% of DAPI (4′,6′-diamidino-2-phenylindole)-stained bacteria were detected by FISH with a probe for α-proteobacteria, but 30% of DAPI-stained bacteria appeared to be in the Cytophaga-Flavobacter group as determined by FISH. α-Proteobacteria were greatly overrepresented in clone libraries compared to their relative abundance determined by FISH, while the Cytophaga-Flavobacter group was underrepresented in clone libraries. Our data show that the Cytophaga-Flavobacter group can be a numerically dominant component of coastal marine bacterioplankton communities.     

Responses of Salt Marsh Plant Rhizosphere Diazotroph Assemblages to Changes in Marsh Elevation,Edaphic Conditions and Plant Host Species

An important source of new nitrogen in salt marsh ecosystems is microbial diazotrophy (nitrogen fixation). The diazotroph assemblages associated with the rhizospheres (sediment directly affected by the roots) of salt marsh plants are highly diverse, somewhat stable, and consist mainly of novel organisms. In Crab Haul Creek Basin, North Inlet, SC, the distribution of plant types into discrete zones is dictated by relatively minor differences in marsh elevation and it was hypothesized that the biotic and abiotic properties of the plant zones would also dictate the composition of the rhizosphere diazotroph assemblages. Over a period of 1 year, rhizosphere sediments were collected from monotypic stands of the black needlerush, Juncus roemerianus, the common pickleweed, Salicornia virginica, the short and tall growth forms of the smooth cordgrass Spartina alterniflora, and a mixed zone of co-occurring S. virginica and short form, S. alterniflora. DNA was extracted, purified and nifH sequences PCR amplified for denaturing gradient gel electrophoresis (DGGE) analysis to determine the composition of the diazotroph assemblages. The diazotroph assemblages were strongly influenced by season, abiotic environmental parameters and plant host. Sediment chemistry and nitrogen fixation activity were also significantly influenced by seasonal changes. DGGE bands that significantly affected seasonal and zone specific clustering were identified and most of these sequences were from novel diazotrophs, unaffiliated with any previously described organisms. At least one third of the recovered nifH sequences were from a diverse assemblage of Chlorobia, and γ-, α-, β- and δ-Proteobacteria. Diazotrophs that occurred throughout the growing season and among all zones (frequently detected) were also mostly novel. These significant sequences indicated that diazotrophs driving the structure of the assemblages were diverse, versatile, and some were ubiquitous while others were seasonally responsive. Several ubiquitous sequences were closely related to sequences of actively N₂ fixing diazotrophs previously recovered from this system. These sequences from ubiquitous and versatile organisms likely indicate the diazotrophs in these rhizosphere assemblages that significantly contribute to ecosystem function.     

Dynamics of a benthic microbial community in a riverine environment subject to hydrological fluctuations (Mulargia River,Italy)

Temporary rivers are characterized by recurrent dry phases, and global warming will stress their hydrology by amplifying extreme events. Microbial degradation and transformation of organic matter (OM) in riverbed sediment are key processes with regard to carbon and nutrient fluxes. In this study, we describe structural and functional changes of benthic microbial communities in a riverine environment subject to hydrological fluctuation. Sampling was carried out in the outlet section of the Mulargia River (Sardinia, Italy) under various water regimes, including one flood event. Overall, sediments were characterized by low bacterial cell abundance (range 0.6–1.8 × 10⁹ cell g⁻¹) as a consequence of their low nutrient and OM concentrations. No major differences were found in the community composition. Alpha-Proteobacteria dominated during the whole year (range 21–30%) followed by Beta-Proteobacteria, Gamma-Proteobacteria, and Cytophaga-Flavobacteria which always contributed <18%. Planctomycetes and Firmicutes were found in smaller amounts (<7%). In spring, when the highest total organic carbon content was also detected (0.42% w/w), both bacterial abundance and C production (BCP, 170 nmol C h⁻¹ g⁻¹) reached relatively high values. During the flood event, an increase in BCP and the highest values of community respiration (CR, 74 nmol C h⁻¹ g⁻¹) were observed. Moreover, most of the extracellular enzyme activities (EEA) changed significantly during the flood. The variation of the water flow itself can explain part of these changes and other factors also come into play. The presence of different patterns of functional parameters could suggest that the quality of the OM could be the major driving force in nutrient flux.     

Characterization of Ni-resistant bacteria in the rhizosphere of the hyperaccumulator <Emphasis Type="Italic">Alyssum murale</Emphasis> by 16S rRNA gene sequence analysis

The diversity of 184 isolates from rhizosphere and bulk soil samples taken from the Ni hyperaccumulator Alyssum murale, grown in a Ni-rich serpentine soil, was determined by 16S rRNA gene analysis. Restriction digestion of the 16S rRNA gene was used to identify 44 groups. Representatives of each of these groups were placed within the phyla Proteobacteria, Firmicutes and Actinobacteria by 16S rRNA gene sequence analysis. By combining the 16S rRNA gene restriction data with the gene sequence analysis it was concluded that 44.6% (82/184) of the isolates were placed within the phylum Proteobacteria, among these 35.9% (66/184) were placed within the class α-Proteobacteria, and 20.7% (38/184) had 16S rRNA gene sequences indicative of bacteria within genera that form symbioses with legumes (rhizobia). Of the remaining isolates, 44.6% (82/184) and 5.4% (10/184) were placed within the phyla Actinobacteria and Firmicutes, respectively. No placement was obtained for a small number (10/184) of the isolates. Bacteria of the phyla Proteobacteria and Actinobacteria were the most numerous within the rhizosphere of A. murale and represented 32.1% (59/184) and 42.9% (79/184) of all isolates, respectively. The approach of using 16S rRNA gene sequence analysis in this study has enabled a comprehensive characterization of bacteria that predominate in the rhizosphere of A. murale growing in Ni-contaminated soil.