Assessment of bacterial diversity in agricultural by-product compost by sequencing of cultivated isolates and amplified rDNA restriction analysis

An investigation of bacterial diversity in compost was performed using molecular chronometer in order to reveal its phylogeny. Thirty-three bacterial isolates isolated from compost were analyzed by 16S rRNA gene sequencing which revealed phylogenetic lineage of class Bacilli, γ, β-Proteobacteria, and Actinobacteria. Among these lineages, isolates belonging to class Bacilli consisted of species from genera Staphylococcus, Bacillus, Terribacillus, and Lysinibacillus. From phylum Actinobacteria: Microbacterium barkeri and Kocuria sp. were identified. Other bacterial groups had phylogenetic linkage with genera Comamonas and Acidovorax (class β-Proteobacteria); Serratia, Klebsiella, and Enterobacter (class γ-Proteobacteria). Similar isolates were analyzed through ARDRA. Amplified product of 16S rRNA gene from each isolates was subjected to cleavage by enzymes HpaII, HinfI, and MspI in separate reaction tubes. HpaII generated 2–6 bands ranging from 90–688 bp, HinfI generated 2–5 bands of 71–1,038 bp, and MspI 2–7 bands of 69–793 bp. The restriction patterns from HpaII, HinfI, and MspI were normalized separately and combined by means of pattern recognition software “Diversity Database.” HpaII had highest discrimination index (0.72) than HinfI (0.68) and MspI (0.65), and the combination of all three showed discrimination index (0.69). Numerical analysis of ARDRA patterns demonstrated sufficient phylogenetic information for characterizing bacterial diversity. Phylogenetic relationship obtained among isolates through ARDRA was compared with 16S rRNA gene sequence and ARDRA results showed sufficiently similar 16S rRNA gene sequence analysis, but not an overlapping. It has been observed that ARDRA technique facilitates the identification of bacteria in less than 36 h as compared to traditional 16S rRNA gene sequencing.     

Small core communities and high variability in bacteria associated with the introduced ascidian Styela plicata

The solitary ascidian Styela plicata is an introduced species in harbors of temperate and tropical oceans around the world. The invasive potential of this species has been studied through reproductive biology and population genetics but no study has yet examined the microbial diversity associated with this ascidian and its potential role in host ecology and invasiveness. Here, we used 16S rRNA gene tag pyrosequencing and transmission electron microscopy to characterize the abundance, diversity and host-specificity of bacteria associated with 3 Mediterranean individuals of S. plicata. Microscopy revealed low bacterial abundance in the inner tunic and their absence from gonad tissues, while pyrosequencing revealed a high diversity of S. plicata-associated bacteria (284 OTUs from 16 microbial phyla) in the inner tunic. The core symbiont community was small and consisted of 16 OTUs present in all S. plicata hosts. This core community included a recently described ascidian symbiont (Hasllibacter halocynthiae) and several known sponge and coral symbionts, including a strictly anaerobic Chloroflexi lineage. Most recovered bacterial OTUs (79.6 %) were present in single S. plicata individuals and statistical analyses of genetic diversity and community structure confirmed high variability of bacterial communities among host individuals. These results suggest that diverse and variable bacterial communities inhabit the tunic of S. plicata, including environmental and host-associated bacterial lineages that appear to be re-established each host generation. We hypothesize that bacterial communities in S. plicata are dynamic and have the potential to aid host acclimation to new habitats by establishing relationships with beneficial, locally sourced bacteria.     

A meta-analysis of the publicly available bacterial and archaeal sequence diversity in saline soils

An integrated view of bacterial and archaeal diversity in saline soil habitats is essential for understanding the biological and ecological processes and exploiting potential of microbial resources from such environments. This study examined the collective bacterial and archaeal diversity in saline soils using a meta-analysis approach. All available 16S rDNA sequences recovered from saline soils were retrieved from publicly available databases and subjected to phylogenetic and statistical analyses. A total of 9,043 bacterial and 1,039 archaeal sequences, each longer than 250 bp, were examined. The bacterial sequences were assigned into 5,784 operational taxonomic units (OTUs, based on ≥97 % sequence identity), representing 24 known bacterial phyla, with Proteobacteria (44.9 %), Actinobacteria (12.3 %), Firmicutes (10.4 %), Acidobacteria (9.0 %), Bacteroidetes (6.8 %), and Chloroflexi (5.9 %) being predominant. Lysobacter (12.8 %) was the dominant bacterial genus in saline soils, followed by Sphingomonas (4.5 %), Halomonas (2.5 %), and Gemmatimonas (2.5 %). Archaeal sequences were assigned to 602 OTUs, primarily from the phyla Euryarchaeota (88.7 %) and Crenarchaeota (11.3 %). Halorubrum and Thermofilum were the dominant archaeal genera in saline soils. Rarefaction analysis indicated that less than 25 % of bacterial diversity, and approximately 50 % of archaeal diversity, in saline soil habitats has been sampled. This analysis of the global bacterial and archaeal diversity in saline soil habitats can guide future studies to further examine the microbial diversity of saline soils.     

Herbicides induce change in metabolic and genetic diversity of bacterial community from a cold oligotrophic lake

Pristine cold oligotrophic lakes show unique physical and chemical characteristics with permanent fluctuation in temperature and carbon source availability. Incorporation of organic toxic matters to these ecosystems could alter the bacterial community composition. Our goal was to assess the effects of simazine (Sz) and 2,4 dichlorophenoxyacetic acid (2,4-D) upon the metabolic and genetic diversity of the bacterial community in sediment samples from a pristine cold oligotrophic lake. Sediment samples were collected in winter and summer season, and microcosms were prepared using a ration 1:10 (sediments:water). The microcosms were supplemented with 0.1 mM 2,4-D or 0.5 mM Sz and incubated for 20 days at 10 °C. Metabolic diversity was evaluated by using the Biolog Ecoplate? system and genetic diversity by 16S rDNA amplification followed by denaturing gradient gel electrophoresis analysis. Total bacterial counts and live/dead ratio were determined by epifluorescence microscopy. The control microcosms showed no significant differences (P > 0.05) in both metabolic and genetic diversity between summer and winter samples. On the other hand, the addition of 2,4-D or Sz to microcosms induces statistical significant differences (P < 0.05) in metabolic and genetic diversity showing the prevalence of Actinobacteria group which are usually not detected in the sediments of these non-contaminated lacustrine systems. The obtained results suggest that contaminations of cold pristine lakes with organic toxic compounds of anthropic origin alter their homeostasis by inhibiting specific susceptible bacterial groups. The concomitant increase of usually low representative bacterial groups modifies the bacterial composition commonly found in this pristine lake.     

Bacterial communities associated with the pitcher fluids of three Nepenthes (Nepenthaceae) pitcher plant species growing in the wild

Nepenthes pitcher plants produce modified jug-shaped leaves to attract, trap and digest insect prey. We used 16S rDNA cloning and sequencing to compare bacterial communities in pitcher fluids of each of three species, namely Nepenthes ampullaria, Nepenthes gracilis and Nepenthes mirabilis, growing in the wild. In contrast to previous greenhouse-based studies, we found that both opened and unopened pitchers harbored bacterial DNA. Pitchers of N. mirabilis had higher bacterial diversity as compared to other Nepenthes species. The composition of the bacterial communities could be different between pitcher types for N. mirabilis (ANOSIM: R = 0.340, p < 0.05). Other Nepenthes species had similar bacterial composition between pitcher types. SIMPER showed that more than 50 % of the bacterial taxa identified from the open pitchers of N. mirabilis were not found in other groups. Our study suggests that bacteria in N. mirabilis are divided into native and nonnative groups.     

Bacterial and archaeal communities in the acid pit lake sediments of a chalcopyrite mine

Bacterial and archaeal community structures and diversity of three different sedimentary environments (BH1A, BH2A and BH3A) in the acid pit lake of a chalcopyrite mine at Touro (Spain) were determined by 16S rRNA gene PCR-DGGE and sequencing of clone libraries. DGGE of bacterial and archaeal amplicons showed that the sediments harbor different communities. Bacterial 16S rRNA gene sequences were assigned to Acidobacteria, Actinobacteria, Cyanobacteria, Planctomycetes, Proteobacteria, Chloroflexi and uncultured bacteria, after clustering into 42 operational taxonomic units (OTUs). OTU 2 represented approximately 37, 42 and 37 % of all sequences from sediments BH1A, BH2A and BH3A, respectively, and was phylogenetically related to uncultured Chloroflexi. Remaining OTUs were phylogenetically related to heterotrophic bacteria, including representatives of Ferrithrix and Acidobacterium genera. Archaeal 16S rRNA gene sequences were clustered into 54 OTUs. Most of the sequences from the BH1A sediment were assigned to Euryarchaeota, whereas those from BH2A sediment were assigned to Crenarchaeota. The majority of the sequences from BH3A sediment were assigned to unclassified Archaea, and showed similarities to uncultured and unclassified environmental clones. No sequences related to Acidithiobacillus and Leptospirillum, commonly associated with acid mine drainage, were detected in this study.     

Bacterial Diversity and Bioprospecting for Cold-Active Lipases,Amylases and Proteases,from Culturable Bacteria of Kongsfjorden and Ny-Ålesund,Svalbard, Arctic

Culturable bacterial diversity of seven marine sediment samples of Kongsfjorden and a sediment and a soil sample from Ny-Ålesund, Svalbard, Arctic was studied. The bacterial abundance in the marine sediments of Kongsfjorden varied marginally (0.5 × 10³–1.3 × 10⁴ cfu/g sediment) and the bacterial number in the two samples collected from the shore of Ny-Ålesund also was very similar (0.6 × 10⁴ and 3.4 × 10⁴, respectively). From the nine samples a total of 103 bacterial isolates were obtained and these isolates could be grouped in to 47 phylotypes based on the 16S rRNA gene sequence belonging to 4 phyla namely Actinobacteria, Bacilli, Bacteroidetes and Proteobacteria. Representatives of the 47 phylotypes varied in their growth temperature range (4–37°C), in their tolerance to NaCl (0.3–2 M NaCl) and growth pH range (2–11). Representatives of 26 phylotypes exhibited amylase and lipase activity either at 5 or 20°C or at both the temperatures. A few of the representatives exhibited amylase and/or lipase activity only at 5°C. None of the phylotypes exhibited protease activity. Most of the phylotypes (38) were pigmented. Fatty acid profile studies indicated that short chain fatty acids, unsaturated fatty acids, branched fatty acids, the cyclic and the cis fatty acids are predominant in the psychrophilic bacteria.     

Profiling Bacterial Diversity and Taxonomic Composition on Speleothem Surfaces in Kartchner Caverns, AZ

Caves are relatively accessible subterranean habitats ideal for the study of subsurface microbial dynamics and metabolisms under oligotrophic, non-photosynthetic conditions. A 454-pyrotag analysis of the V6 region of the 16S rRNA gene was used to systematically evaluate the bacterial diversity of ten cave surfaces within Kartchner Caverns, a limestone cave. Results showed an average of 1,994 operational taxonomic units (97 % cutoff) per speleothem and a broad taxonomic diversity that included 21 phyla and 12 candidate phyla. Comparative analysis of speleothems within a single room of the cave revealed three distinct bacterial taxonomic profiles dominated by either Actinobacteria, Proteobacteria, or Acidobacteria. A gradient in observed species richness along the sampling transect revealed that the communities with lower diversity corresponded to those dominated by Actinobacteria while the more diverse communities were those dominated by Proteobacteria. A 16S rRNA gene clone library from one of the Actinobacteria-dominated speleothems identified clones with 99 % identity to chemoautotrophs and previously characterized oligotrophs, providing insights into potential energy dynamics supporting these communities. The robust analysis conducted for this study demonstrated a rich bacterial diversity on speleothem surfaces. Further, it was shown that seemingly comparable speleothems supported divergent phylogenetic profiles suggesting that these communities are very sensitive to subtle variations in nutritional inputs and environmental factors typifying speleothem surfaces in Kartchner Caverns.     

High abundance of heterotrophic prokaryotes in hydrothermal springs of the Azores as revealed by a network of 16S rRNA gene-based methods

Two hydrothermal springs (AI: 51 °C, pH 3; AIV: 92 °C, pH 8) were analysed to determine prokaryotic community composition. Using pyrosequencing, 93,576 partial 16S rRNA gene sequences amplified with V2/V3-specific primers for Bacteria and Archaea were investigated and compared to 16S rRNA gene sequences from direct metagenome sequencing without prior amplification. The results were evaluated by fluorescence in situ hybridization (FISH). While in site AIV Bacteria and Archaea were detected in similar relative abundances (Bacteria 40 %, Archaea 35 %), the acidic spring AI was dominated by Bacteria (68 %). In spring AIV the combination of 16S rRNA gene sequence analysis and FISH revealed high abundance (>50 %) of heterotrophic bacterial genera like Caldicellulosiruptor, Dictyoglomus, and Fervidobacterium. In addition, chemolithoautotrophic Aquificales were detected in the bacterial community with Sulfurihydrogenibium being the dominant genus. Regarding Archaea, only Crenarchaeota, were detected, dominated by the family Desulfurococcaceae (>50 %). In addition, Thermoproteaceae made up almost 25 %. In the acidic spring (AI) prokaryotic diversity was lower than in the hot, slightly alkaline spring AIV. The bacterial community of site AI was dominated by organisms related to the chemolithoautotrophic genus Acidithiobacillus (43 %), to the heterotrophic Acidicaldus (38 %) and to Anoxybacillus (7.8 %). This study reveals differences in the relative abundance of heterotrophic versus autotrophic microorganisms as compared to other hydrothermal habitats. Furthermore, it shows how different methods to analyse prokaryotic communities in complex ecosystems can complement each other to obtain an in-depth picture of the taxonomic composition and diversity within these hydrothermal springs.     

Diversity,cold active enzymes and adaptation strategies of bacteria inhabiting glacier cryoconite holes of High Arctic

Cryoconite holes have biogeochemical, ecological and biotechnological importance. This communication presents results on culturable psychrophilic bacterial diversity from cryoconite holes at Midre Lovénbreen (ML), Austre Brøggerbreen (AB), and Vestre Brøggerbreen (VB) glaciers. The culturable bacterial count ranged from 2.7 × 10³ to 8.8 × 10⁴ CFUs/g while the total bacterial numbers ranged from 5.07 × 10⁵ to 1.50 × 10⁶ cells at the three glaciers. A total of 35 morphologically distinct bacterial isolates were isolated. Based on 16S rRNA gene sequence data, the identified species belonged to eight genera namely Pseudomonas, Polaromonas, Micrococcus, Subtercola, Agreia, Leifsonia, Cryobacterium and Flavobacterium. The isolates varied in their growth temperature, NaCl tolerance, growth pH, enzyme activities, carbon utilization and antibiotic sensitivity tests. Fatty acid profiles indicate the predominance of branched fatty acids in the isolates. To the best of our knowledge, this is the first record of culturable bacterial communities and their characterization from glacier cryoconites from High Arctic. High amylase and protease activities expressed by Micrococcus sp. MLB-41 and amylase, protease and lipase activities expressed by Cryobacterium sp. MLB-32 provide a clue to the potential applications of these organisms. These cold-adapted enzymes may provide an opportunity for the prospect of biotechnology in Arctic.     

Diverse cellulolytic bacteria isolated from the high humus, alkaline-saline chinampa soils

Aiming at learning the functional bacterial community in the high humus content, saline-alkaline soils of chinampas, the cellulolytic bacteria were quantified and 100 bacterial isolates were isolated and characterized in the present study. Analysis of 16S-23S IGS (intergenic spacer) RFLP (restriction fragment length polymorphism) grouped the isolates into 48 IGS types and phylogenetic analysis of 16S rRNA genes identified them into 42 phylospecies within 29 genera and higher taxa belonging to the phyla Actinobacteria, Firmicutes and Proteobacteria, dominated by the genera Arthrobacter, Streptomyces, Bacillus, Pseudomonas, Pseudoxanthomonas and Stenotrophomonas. Among these bacteria, 63 isolates represent 26 novel putative species or higher taxa, while 37 were members of 17 defined species according to the phylogenetic relationships of 16S rRNA gene. Except for the novel species, the cellulolytic activity was not reported previously in 9 of the 17 species. They degraded cellulose in medium at pH?4.5–10.0 or supplied with NaCl up to 9 %. In addition, 84.8 and 71.7 % of them degraded xylan and Avicel, respectively. These results greatly improved the knowledge about the diversity of cellulolytic bacteria and demonstrated that the chinampa soils contain diverse and novel cellulolytic bacteria functioning at a wide range of pH and salinity levels, which might be a valuable biotechnological resource for biotransformation of cellulose.     

Bacterial diversity of extremely alkaline bauxite residue site of alumina industrial plant using culturable bacteria and residue 16S rRNA gene clones

Bauxite residue (red mud), generated during the extraction of alumina from bauxite ore is characterized by high pH, high concentrations of soluble ions with low or virtually no organic matter. These extreme conditions along with numerous nutrient deficiencies, limit the microbial growth and vegetation establishment. In the present study, diversity of both cultivable and non-cultivable bacteria present in the red mud was investigated by 16S rDNA sequence analyses. The cultivable bacteria were identified as Agromyces indicus, Bacillus litoralis, B. anthracis, Chungangia koreensis, Kokuria flava, K. polaris, Microbacterium hominis, Planococcus plakortidis, Pseudomonas alcaliphila and Salinococcus roseus based on their 16S rDNA sequence analysis. These isolates were alkali tolerant, positive for one or more of the enzyme activities tested, able to produce organic acids and oxidize wide range of carbon substrates. For non-cultivable diversity of bacteria, DNA was extracted from the bauxite residue samples and 16S rDNA clone library was constructed. The 16S rDNA clones of this study showed affiliation to three major phyla predominant being betaproteobacteria (41.1 %) followed by gammaproteobacteria (37.5 %) and bacteroidetes (21.4 %). We are reporting for the first time about the bacterial diversity of this unique and extreme environment.     

Bacterial Diversity Analysis of Zhenjiang Yao Meat During Refrigerated and Vacuum-Packed Storage by 454 Pyrosequencing

Zhenjiang Yao meat is a traditional variety of cooked marinated and jellied pork food in China. It is usually stored refrigerated to prevent gelatine liquefaction. Our understanding of the bacterial populations found in Zhenjiang Yao meat is limited. This study was designed to explore both the bacterial diversity and the main bacterial flora of Zhenjiang Yao meat using pyrosequencing of tagged amplicons from the V3 and V9 regions of the 16S rRNA gene. A total of 53,363 bacterial sequences from five samples and an average 10,672 reads of each sample were acquired and used in the analysis of microbial diversity. The bacterial diversity was observed to increase weekly, and the main bacterial flora changed significantly under refrigerated vacuum-packaged storage. The predominant bacteria were Vibrio during the first 7 days of storage, whereas Shewanella, Lactobacillus, Lactococcus, Yersinia, and other Enterobacteriaceae members were the main groups after 15 days in refrigerated storage. The bacterial population changed significantly after 15 days, and the bacterial communities in the 30 days sample were significantly different from all other samples. In comparison with culture-dependant method, pyrosequencing provides a more comprehensive estimate of bacterial diversity and more close to the real bacterial composition in Zhenjiang Yao meat.     

Modulation of microbial consortia enriched from different polluted environments during petroleum biodegradation

Environmental microbial communities are key players in the bioremediation of hydrocarbon pollutants. Here we assessed changes in bacterial abundance and diversity during the degradation of Tunisian Zarzatine oil by four indigenous bacterial consortia enriched from a petroleum station soil, a refinery reservoir soil, a harbor sediment and seawater. The four consortia were found to efficiently degrade up to 92.0% of total petroleum hydrocarbons after 2 months of incubation. Illumina 16S rRNA gene sequencing revealed that the consortia enriched from soil and sediments were dominated by species belonging to Pseudomonas and Acinetobacter genera, while in the seawater-derived consortia Dietzia, Fusobacterium and Mycoplana emerged as dominant genera. We identified a number of species whose relative abundances bloomed from small to high percentages: Dietzia daqingensis in the seawater microcosms, and three OTUs classified as Acinetobacter venetianus in all two soils and sediment derived microcosms. Functional analyses on degrading genes were conducted by comparing PCR results of the degrading genes alkB, ndoB, cat23, xylA and nidA1 with inferences obtained by PICRUSt analysis of 16S amplicon data: the two data sets were partly in agreement and suggest a relationship between the catabolic genes detected and the rate of biodegradation obtained. The work provides detailed insights about the modulation of bacterial communities involved in petroleum biodegradation and can provide useful information for in situ bioremediation of oil-related pollution.     

A comparison of culture-dependent and culture-independent techniques used to characterize bacterial communities on healthy and white plague-diseased corals of the Montastraea annularis species complex

Diseases of hermatypic corals pose a global threat to coral reefs, and investigations of bacterial communities associated with healthy corals and those exhibiting signs of disease are necessary for proper diagnosis. One disease, commonly called white plague (WP), is characterized by acute tissue loss. This investigation compared the bacterial communities associated with healthy coral tissue (N = 15), apparently healthy tissue on WP-diseased colonies (N = 15), and WP-diseased tissues (N = 15) from Montastraea annularis (species complex) colonies inhabiting a Bahamian reef. Aliquots of sediment (N = 15) and water (N = 15) were also obtained from the proximity of each coral colony sampled. Samples for culture-dependent analyses were inoculated onto one-half strength Marine Agar (½ MA) and Thiosulfate Citrate Bile Salts Sucrose Agar to quantify the culturable communities. Length heterogeneity PCR (LH-PCR) of the 16S rRNA gene characterized the bacterial operational taxonomic units (OTU) associated with lesions on corals exhibiting signs of a white plague-like disease as well as apparently healthy tissue from diseased and non-diseased conspecifics. Analysis of Similarity was conducted on the LH-PCR fingerprints, which indicated no significant difference in the composition of bacterial communities associated with apparently healthy and diseased corals. Comparisons of the 16S rRNA gene amplicons from cultured bacterial colonies (½ MA; N = 21) with all amplicons obtained from the whole coral-associated bacterial community indicated ≥39 % of coral-associated bacterial taxa could be cultured. Amplicons from these bacterial cultures matched amplicons from the whole coral-associated bacterial community that, when combined, accounted for >70 % total bacterial abundance. An OTU with the same amplicon length as Aurantimonas coralicida (313.1 bp), the reported etiological agent of WPII, was detected in relatively low abundance (<0.1 %) on all tissue types. These findings suggest a coral disease resembling WP may result from multiple etiologies.     

Culture-Dependent and Independent Studies of Microbial Diversity in Highly Copper-Contaminated Chilean Marine Sediments

Cultivation and molecular-based approaches were used to study microbial diversity in two Chilean marine sediments contaminated with high (835 ppm) and very high concentrations of copper (1,533 ppm). The diversity of cultivable bacteria resistant to copper was studied at oxic and anoxic conditions, focusing on sulfate-, thiosulfate-, and iron-reducing bacteria. For both sediments, the cultivable bacteria isolated at oxic conditions were mostly affiliated to the genus Bacillus, while at anoxic conditions the majority of the cultivable bacteria found were closely related to members of the genera Desulfovibrio, Sphingomonas, and Virgibacillus. Copper resistance was between 100 and 400 ppm, with the exception of a strain affiliated to members of the genus Desulfuromonas, which was resistant up to 1,000 ppm of copper. In parallel, cloning and sequencing of 16S rRNA was performed to study the total bacterial diversity in the sediments. A weak correlation was observed between the isolated strains and the 16S rRNA operational taxonomic units detected. The presence of copper resistance genes (copA, cusA, and pcoA) was tested for all the strains isolated; only copA was detected in a few isolates, suggesting that other copper resistance mechanisms could be used by the bacteria in those highly copper-contaminated sediments.     

Microbial diversity in the coralline sponge Vaceletia crypta

Coralline sponges of the genus Vaceletia are regarded as ‘living fossils’, the only recent members of the so-called ‘sphinctozoan-type’ sponges that contributed to reef-building during the Palaeozoic and Mesozoic eras. Vaceletia species were thought to be extinct until the discovery of Vaceletia crypta in the 1970s. Here, we used molecular methods to provide first insights into the microbial diversity of these coralline sponges. Both denaturing gradient gel electrophoresis (DGGE) analyses of 19 Vaceletia specimens and the analysis of 427 clones from a bacterial 16S rRNA gene clone library of a specimen of V. crypta from the Great Barrier Reef (Australia) revealed high diversity and a complex composition with a relatively uniform phylogenetic distribution. Only a single archaeal 16S rRNA phylotype was recovered. The most abundant bacteria were the Chloroflexi (35 %). Of the microbial community, 58 % consisted of the Gammaproteobacteria, Gemmatimonadetes, Actinobacteria, Nitrospira, Deltaproteobacteria, Deferribacteres and Acidobacteria, with nearly equal representation. Less abundant members of the microbial community belonged to the Alphaproteobacteria (3 %), as well as to the Poribacteria, Betaproteobacteria, Cyanobacteria, Spirochaetes, Bacteroidetes, Deinococcus-Thermus and Archaea (all together 4 %). Of the established 96 OTUs, 88 % were closely related to other sponge-derived sequences and thereof 71 OTUs fell into sponge- or sponge-coral specific clusters, which underscores that the “living fossil” coralline sponge Vaceletia shares features of its microbial community with other sponges. The DGGE cluster analysis indicated distinct microbial communities in the different growth forms (solitary and colonial) of Vaceletia species.     

Shewanella litorisediminis sp. nov., a gammaproteobacterium isolated from a tidal flat sediment

A Gram-negative, facultatively anaerobic, non-motile and rod-shaped bacterial strain, designated SMK1-12^T, was isolated from a tidal flat sediment on the western coast of Korea. Phylogenetic analyses based on 16S rRNA and gyrB gene sequences showed that strain SMK1-12^T belonged to the genus Shewanella, clustering with the type strain of Shewanella amazonensis. Strain SMK1-12^T exhibited the highest 16S rRNA gene sequence similarity value (97.0 %) and the highest gyrB sequence similarity value (87.8 %) to S. amazonensis SB2B^T, respectively. Strain SMK1-12^T contained simultaneously both menaquinones and ubiquinones; the predominant menaquinone was MK-7 and the predominant ubiquinones were Q-7 and Q-8. The major fatty acids (>10 % of the total fatty acids) detected in strain SMK1-12^T were the MIDI system summed feature 3 (iso-C_15:0 2-OH and/or C_16:1 ω7c), iso-C_15:0, C_17:1 ω8c and C_16:0. The DNA G+C content of strain SMK1-12^T was 58.0 mol% and its mean DNA–DNA relatedness value with S. amazonensis ATCC 700329^T was 15 ± 4.6 %. Differential phenotypic properties, together with the phylogenetic and genetic distinctiveness, revealed that strain SMK1-12^T is distinguishable from recognized Shewanella species. On the basis of the data presented, strain SMK1-12^T is considered to represent a novel Shewanella species, for which the name Shewanella litorisediminis sp. nov. is proposed. The type strain is SMK1-12^T (=KCTC 23961^T = CCUG 62411^T).     

Thalassomonas fusca sp. nov., a novel gammaproteobacterium isolated from tidal flat sediment

A Gram-negative, aerobic, non-motile, dark brown-coloured and rod-shaped bacterial strain, designated G-MB1^T, was isolated from a tidal flat sediment of the South Sea, South Korea. Strain G-MB1^T was found to grow optimally at 25 °C, at pH 7.0–8.0 and in the presence of 2.0 % (w/v) NaCl. A neighbour-joining phylogenetic tree based on 16S rRNA gene sequences revealed that strain G-MB1^T fell within the clade comprising Thalassomonas species, clustering with the type strains of Thalassomonas agarivorans, Thalassomonas loyana, Thalassomonas ganghwensis and Thalassomonas agariperforans, with which it exhibited 16S rRNA gene sequence similarity values of 96.0–96.9 %. The 16S rRNA gene sequence similarity values between strain G-MB1^T and the type strains of the other Thalassomonas species were 94.6–95.1 %. Strain G-MB1^T was found to contain Q-8 as the predominant ubiquinone and C_16:0, C_17:1 ω8c, C_16:1 ω9c, C_12:0 3-OH and summed feature 3 (C_16:1 ω7c and/or C_16:1 ω6c) as the major fatty acids. The major polar lipids of strain G-MB1^T were phosphatidylglycerol, phosphatidylethanolamine and one unidentified aminolipid. The DNA G+C content of strain G-MB1^T was determined to be 42.4 mol%. Differential phenotypic properties, together with the phylogenetic distinctiveness, revealed that strain G-MB1^T is separated from other Thalassomonas species. On the basis of the data presented, strain G-MB1^T is considered to represent a novel species of the genus Thalassomonas, for which the name Thalassomonas fusca sp. nov. is proposed. The type strain is G-MB1^T (=KCTC 32499^T = NBRC 109830^T).