Alkaline Phosphatase: An Appraisal of its Critical Role in C-Limited Deep-Sea Sediments of Central Indian Basin

Alkaline phosphatase (ALP) is a hydrolase that releases phosphate, a crucial nutrient supporting formation of many macromolecules. The oligotrophic sediments of Central Indian Basin (CIB) were examined for the extent of ALP activity in 20 cores up to 20 cm depth along with other biogeochemical parameters. Northern and southern stations distinctly clustered based on pore water phosphorus (P) concentrations. ALP appeared more active in south due to more P and organic carbon (C) limitation. Central transition profiles revealed interesting mixed diagenetic and geothermal influence. Inter- and intrazonal differences suggest that broadly established paradigms applicable to water columns may deviate in space and time in sedimentary systems. Gross differences in redox status could govern differential response in ALP activity under similar hydrostatic pressure. Hydrothermal scavenging reactions striping soluble P from pore water might be widespread in CIB albeit at extreme low intensities. ALP might redissolve the precipitated P and make it reavailable to microbes.     

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

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

Fungal Community Analysis by Large-Scale Sequencing of Environmental Samples

Fungi are an important and diverse component of soil communities, but these communities have proven difficult to study in conventional biotic surveys. We evaluated soil fungal diversity at two sites in a temperate forest using direct isolation of small-subunit and internal transcribed spacer (ITS) rRNA genes by PCR and high-throughput sequencing of cloned fragments. We identified 412 sequence types from 863 fungal ITS sequences, as well as 112 ITS sequences from other eukaryotic microorganisms. Equal proportions of Basidiomycota and Ascomycota sequences were present in both the ITS and small-subunit libraries, while members of other fungal phyla were recovered at much lower frequencies. Many sequences closely matched sequences from mycorrhizal, plant-pathogenic, and saprophytic fungi. Compositional differences were observed among samples from different soil depths, with mycorrhizal species predominating deeper in the soil profile and saprophytic species predominating in the litter layer. Richness was consistently lowest in the deepest soil horizon samples. Comparable levels of fungal richness have been observed following traditional specimen-based collecting and culturing surveys, but only after much more extensive sampling. The high rate at which new sequence types were recovered even after sampling 863 fungal ITS sequences and the dominance of fungi in our libraries relative to other eukaryotes suggest that the abundance and diversity of fungi in forest soils may be much higher than previously hypothesized.     

A Culture-Independent Approach to Unravel Uncultured Bacteria and Functional Genes in a Complex Microbial Community

Most microorganisms in nature are uncultured with unknown functionality. Sequence-based metagenomics alone answers ‘who/what are there?’ but not ‘what are they doing and who is doing it and how?’. Function-based metagenomics reveals gene function but is usually limited by the specificity and sensitivity of screening strategies, especially the identification of clones whose functional gene expression has no distinguishable activity or phenotypes. A ‘biosensor-based genetic transducer’ (BGT) technique, which employs a whole-cell biosensor to quantitatively detect expression of inserted genes encoding designated functions, is able to screen for functionality of unknown genes from uncultured microorganisms. In this study, BGT was integrated with Stable isotope probing (SIP)-enabled Metagenomics to form a culture-independent SMB toolbox. The utility of this approach was demonstrated in the discovery of a novel functional gene cluster in naphthalene contaminated groundwater. Specifically, metagenomic sequencing of the ¹³C-DNA fraction obtained by SIP indicated that an uncultured Acidovorax sp. was the dominant key naphthalene degrader in-situ, although three culturable Pseudomonas sp. degraders were also present in the same groundwater. BGT verified the functionality of a new nag2 operon which co-existed with two other nag and two nah operons for naphthalene biodegradation in the same microbial community. Pyrosequencing analysis showed that the nag2 operon was the key functional operon in naphthalene degradation in-situ, and shared homology with both nag operons in Ralstonia sp. U2 and Polaromonas naphthalenivorans CJ2. The SMB toolbox will be useful in providing deep insights into uncultured microorganisms and unravelling their ecological roles in natural environments.     

Cultivable Fungal Diversity in Deep-Sea Sediment of the East Pacific Ocean

The diversity of fungi in the marine environment has been extensively studied, but their denitrification activity has rarely been reported to date. In the present study, we used six different media to isolate fungi from 10 sediment samples collected at five different locations of the East Pacific Ocean with water depths ranging from 4545 to 7068?m. Fungal identification and phylogenetic diversity analysis were conducted based on morphological characteristics and internal transcribed spacers of ribosomal DNA (ITS-rRNA) sequencing. A total of 106 fungal isolates, belonging to 12 genera, including Aspergillus (five strains), Aureobasidium (three strains), Candida (two strains), Cladosporium (56 strains), Cystobasidium (one strain), Devriesia (nine strains), Knufia (one strain), Nigrospora (three strains), Penicillium (18 strains), Rhodotorula (four strains), Sarocladium (three strains), and unclassified Xylariales (one strain) were obtained. The most dominant culturable genus was Cladosporium. One possible novel fungal strains showed less than 97% similarity to their closest matches of unclassified Xylariales in the Genbank. In addition, we used nirK gene as the molecular marker to detect denitrifying fungi among the cultivable fungal isolates. The nirK gene was detected in Aspergillus niger and Penicillium chrysogenum. Our research indicated that diverse fungi from the deep sea sediments of the East Pacific Ocean and highlighted the involvement of these fungi in denitrification process.     

Insights into Deep-Sea Sediment Fungal Communities from the East Indian Ocean Using Targeted Environmental Sequencing Combined with Traditional Cultivation

The fungal diversity in deep-sea environments has recently gained an increasing amount attention. Our knowledge and understanding of the true fungal diversity and the role it plays in deep-sea environments, however, is still limited. We investigated the fungal community structure in five sediments from a depth of ∼4000 m in the East India Ocean using a combination of targeted environmental sequencing and traditional cultivation. This approach resulted in the recovery of a total of 45 fungal operational taxonomic units (OTUs) and 20 culturable fungal phylotypes. This finding indicates that there is a great amount of fungal diversity in the deep-sea sediments collected in the East Indian Ocean. Three fungal OTUs and one culturable phylotype demonstrated high divergence (89%–97%) from the existing sequences in the GenBank. Moreover, 44.4% fungal OTUs and 30% culturable fungal phylotypes are new reports for deep-sea sediments. These results suggest that the deep-sea sediments from the East India Ocean can serve as habitats for new fungal communities compared with other deep-sea environments. In addition, different fungal community could be detected when using targeted environmental sequencing compared with traditional cultivation in this study, which suggests that a combination of targeted environmental sequencing and traditional cultivation will generate a more diverse fungal community in deep-sea environments than using either targeted environmental sequencing or traditional cultivation alone. This study is the first to report new insights into the fungal communities in deep-sea sediments from the East Indian Ocean, which increases our knowledge and understanding of the fungal diversity in deep-sea environments.     

Viral Density and Virus-to-Bacterium Ratio in Deep-Sea Sediments of the Eastern Mediterranean

Viruses are now recognized as a key component in pelagic systems, but their role in marine sediment has yet to be assessed. In this study bacterial and viral densities were determined at nine deep-sea stations selected from three main sites (i.e., the Sporades Basin, the Cretan Sea, and the Ierapetra Trench at depths of 1,232, 1,840, and 4,235 m, respectively) of the Eastern Mediterranean. The three areas were characterized by different phytopigment and biopolymeric carbon concentrations and by changes in the protein and carbohydrate pools. A gradient of increasing trophic conditions was observed from the Sporades Basin (North Aegean) to the Ierapetra Trench (South Aegean). Viral densities (ranging from 1 × 10⁹ to 2 × 10⁹ viruses ml of sediment⁻¹) were significantly correlated to bacterial densities (n = 9, r² = 0.647) and reached values up to 3 orders of magnitude higher than those generally reported for the water column. However, the virus-to-bacterium density ratio in deep-sea sediments was about 1 order of magnitude lower (range of 2 to 5, with a modal value of 2.6) than in pelagic environments. Virus density decreased vertically with depth in sediment cores at all stations and was below detection limits at the 10-cm depth of the abyssal sediments of the Ierapetra Trench. Virus density in the sediment apparently reflected a gradient of particle fluxes and trophic conditions, displaying the highest values in the Sporades Basin. The low virus-to-bacterium ratios and their inverse relationship with station depth suggest that the role played by viruses in controlling deep-sea benthic bacterial assemblages and biogeochemical cycles is less relevant than in pelagic systems.     

High Connectivity of Animal Populations in Deep-Sea Hydrothermal Vent Fields in the Central Indian Ridge Relevant to Its Geological Setting

Dispersal ability plays a key role in the maintenance of species in spatially and temporally discrete niches of deep-sea hydrothermal vent environments. On the basis of population genetic analyses in the eastern Pacific vent fields, dispersal of animals in the mid-oceanic ridge systems generally appears to be constrained by geographical barriers such as trenches, transform faults, and microplates. Four hydrothermal vent fields (the Kairei and Edmond fields near the Rodriguez Triple Junction, and the Dodo and Solitaire fields in the Central Indian Ridge) have been discovered in the mid-oceanic ridge system of the Indian Ocean. In the present study, we monitored the dispersal of four representative animals, Austinograea rodriguezensis, Rimicaris kairei, Alviniconcha and the scaly-foot gastropods, among these vent fields by using indirect methods, i.e., phylogenetic and population genetic analyses. For all four investigated species, we estimated potentially high connectivity, i.e., no genetic difference among the populations present in vent fields located several thousands of kilometers apart; however, the direction of migration appeared to differ among the species, probably because of different dispersal strategies. Comparison of the intermediate-spreading Central Indian Ridge with the fast-spreading East Pacific Rise and slow-spreading Mid-Atlantic Ridge revealed the presence of relatively high connectivity in the intermediate- and slow-spreading ridge systems. We propose that geological background, such as spreading rate which determines distance among vent fields, is related to the larval dispersal and population establishment of vent-endemic animal species, and may play an important role in controlling connectivity among populations within a biogeographical province.     

Phylogenetic Analysis of Bacterial Communities Associated with Leaves of the Seagrass Halophila stipulacea by a Culture-Independent Small-Subunit rRNA Gene Approach

Abstract The phylogenetic diversity of the bacterial community associated with leaves of the marine plant Halophila stipulacea in the northern Gulf of Elat was examined by 16S rRNA gene (rDNA) sequence analyses of a clone library. For 59 clones corresponding to 51 ARDRA (amplified rDNA restriction analysis) groups, the sequence of ∼1 kb was determined, and the fraction of the corresponding ARDRA groups of the leaf library was calculated. The class Proteobacteria was represented by 62.6% of the clone sequences. Most sequences originated from members of the γ-subclass (27.3%), affiliated with members of the genera Pseudomonas, Vibrio, Marinomonas, Oceanospirillum, and other marine groups. Affiliation to the α-subclass was determined for 24.2% of the sequences. They were related to the genera Hyphomonas, Roseobacter, Ruegeria, and Rhizobiaceae. Several α-proteobacterial sequences were distantly related to known sequences. Only 4% of the clone sequences were related to β-Proteobacteria. Additionally, 7.1% of the sequences possibly belonged to the class Proteobacteria, but branched deeply from known subclasses. Several sequences were affiliated to members of the orders Verrucomicrobiales and Planctomycetales, the Holophaga/Acidobacterium phylum, and chloroplasts of marine diatoms. Received: 20 March 1999; Accepted: 13 August 1999; Online Publication: 2 March 2000     

Reliability of CARD-FISH Procedure for Enumeration of Archaea in Deep-Sea Surficial Sediments

The enumeration of Archaea in deep-sea sediment samples is still limited, although different methodological procedures have been applied. Among these, catalysed reporter deposition-fluorescence in situ hybridisation (CARD-FISH) technique is a promising tool for estimation of archaeal abundance in deep-sea sediment samples. Comparing different permeabilisation treatments, the best results obtained both on archaeal pure cultures and on natural assemblages were with hydrochloric acid (0.1 M) and proteinase K (0.004 U/ml) treatments. The application of CARD-FISH on deep-sea sediments revealed that Archaea reach up to 41% of total prokaryotic cells. Specific probes for planktonic Archaea showed that marine Crenarchaea dominated archaeal seafloor communities. No clear bathymetric trends were observed for archaeal abundances and the morphology of continental margin (slope vs. canyon) seems not to have a direct influence on archaeal relative abundances. The site-specific sediment habitat—both abiotic environmental setting and sedimentary organic matter quality—explain up to 65% of variance of archaeal, crenarchaeal and euryarchaeal relative abundance, suggesting a wide ecophysiological adaptation to deep-sea benthic ecosystems. The findings demonstrate that Archaea are an important component of benthic microbial assemblages so far neglected, and hence they lay the groundwork for more focused research on their ecological importance in the functioning of deep-sea benthic ecosystems.     

Population Structure and Phylogenetic Characterization of Marine Benthic Archaea in Deep-Sea Sediments

During the past few years Archaea have been recognized as a widespread and significant component of marine picoplankton assemblages and, more recently, the presence of novel archaeal phylogenetic lineages has been reported in coastal marine benthic environments. We investigated the relative abundance, vertical distribution, phylogenetic composition, and spatial variability of Archaea in deep-sea sediments collected from several stations in the Atlantic Ocean. Quantitative oligonucleotide hybridization experiments indicated that the relative abundance of archaeal 16S rRNA in deep-sea sediments (1500 m deep) ranged from about 2.5 to 8% of the total prokaryotic rRNA. Clone libraries of PCR-amplified archaeal rRNA genes (rDNA) were constructed from 10 depth intervals obtained from sediment cores collected at depths of 1,500, 2,600, and 4,500 m. Phylogenetic analysis of rDNA sequences revealed the presence of a complex archaeal population structure, whose members could be grouped into discrete phylogenetic lineages within the two kingdoms, Crenarchaeota and Euryarchaeota. Comparative denaturing gradient gel electrophoresis profile analysis of archaeal 16S rDNA V3 fragments revealed a significant depth-related variability in the composition of the archaeal population.     

Culture-Independent Microbial Community Analysis Reveals that Inulin in the Diet Primarily Affects Previously Unknown Bacteria in the Mouse Cecum

Inulin is a well-known fructose-based prebiotic which has been shown to stimulate the growth of bifidobacteria, a bacterial group generally considered beneficial for intestinal health. In the present study, we analyzed inulin-associated shifts in the total bacterial community of wild-type mice and mice carrying a genetically inactivated adenomatous polyposis coli tumor suppressor gene by using DNA-based approaches independent of bacterial culturability. Mice were fed a high-fat, nonfiber diet with or without inulin inclusion at a 10% (wt/wt) concentration. Cecal contents were analyzed after 0, 3, and 9 weeks on the experimental diets. Inulin inclusion significantly affected the total bacterial community structure of the cecum as determined by both a nonselective percent-guanine-plus-cytosine-based profiling analysis and a more specific 16S ribosomal DNA sequence analysis. The shifts included stimulation of bifidobacteria and suppression of clostridia, but sequence comparison revealed that the major shifts were within previously unknown bacterial taxa. Concomitantly, significantly higher bacterial densities, determined by flow cytometry, were observed with the inulin-amended diet, and the metabolism of the cecal bacterial community was altered, as indicated by higher levels of residual short-chain fatty acids, particularly lactic acid. With regard to all of the microbiological parameters measured, the wild-type mice and mice carrying a genetically inactivated adenomatous polyposis coli tumor suppressor gene were essentially identical. Studies of the implications of pre- and probiotics may need to be expanded to include careful analysis of their effects on the entire microbial community, rather than just a few well-known species. Further studies are needed to increase our understanding of the possible roles of currently unknown gastrointestinal bacteria in health and disease.     

Hydrocarbons in Deep-Sea Sediments following the 2010 Deepwater Horizon Blowout in the Northeast Gulf of Mexico

The Deepwater Horizon (DWH) spill released 4.9 million barrels of oil into the Gulf of Mexico (GoM) over 87 days. Sediment and water sampling efforts were concentrated SW of the DWH and in coastal areas. Here we present geochemistry data from sediment cores collected in the aftermath of the DWH event from 1000 – 1500 m water depth in the DeSoto Canyon, NE of the DWH wellhead. Cores were analyzed at high-resolution (at 2 mm and 5 mm intervals) in order to evaluate the concentration, composition and input of hydrocarbons to the seafloor. Specifically, we analyzed total organic carbon (TOC), aliphatic, polycyclic aromatic hydrocarbon (PAHs), and biomarker (hopanes, steranes, diasteranes) compounds to elucidate possible sources and transport pathways for deposition of hydrocarbons. Results showed higher hydrocarbon concentrations during 2010-2011 compared to years prior to 2010. Hydrocarbon inputs in 2010-2011 were composed of a mixture of sources including terrestrial, planktonic, and weathered oil. Our results suggest that after the DWH event, both soluble and highly insoluble hydrocarbons were deposited at enhanced rates in the deep-sea. We proposed two distinct transport pathways of hydrocarbon deposition: 1) sinking of oil-particle aggregates (hydrocarbon-contaminated marine snow and/or suspended particulate material), and 2) advective transport and direct contact of the deep plume with the continental slope surface sediments between 1000-1200 m. Our findings underline the complexity of the depositional event observed in the aftermath of the DWH event in terms of multiple sources, variable concentrations, and spatial (depth-related) variability in the DeSoto Canyon, NE of the DWH wellhead.     

The Bacterial Community of the Lithistid Sponge Discodermia spp. as Determined by Cultivation and Culture-Independent Methods

The marine lithistid sponge Discodermia spp. (Family Theonellidae) contains many types of associated bacteria visible in the mesohyl while biofilms cover the pinacoderm. This study determined the identity of bacteria associated with members of the genus Discodermia using microbial culture, 16S rRNA gene clone libraries and fluorescence in situ hybridization. Four samples of Discodermia spp. were collected at depths between 24–161?m near Grand Bahama Island and Cay Sal Bank, Bahamas. A total of 80 unique isolates and 94 different clone sequences from at least eight bacterial classes were obtained. It appeared that Discodermia spp. may have a core community of bacteria that is common to all sponges of this genus. Species of at least six different classes of bacteria were regularly found in most of the sponge specimens collected, irrespective of collection depth or location. This indicates that a diverse spectrum of bacteria is associated with lithistid sponges irrespective of the transient seawater community that enters the sponge.     

Culture-Independent Analysis of Probiotic Products by Denaturing Gradient Gel Electrophoresis

In order to obtain functional and safe probiotic products for human consumption, fast and reliable quality control of these products is crucial. Currently, analysis of most probiotics is still based on culture-dependent methods involving the use of specific isolation media and identification of a limited number of isolates, which makes this approach relatively insensitive, laborious, and time-consuming. In this study, a collection of 10 probiotic products, including four dairy products, one fruit drink, and five freeze-dried products, were subjected to microbial analysis by using a culture-independent approach, and the results were compared with the results of a conventional culture-dependent analysis. The culture-independent approach involved extraction of total bacterial DNA directly from the product, PCR amplification of the V3 region of the 16S ribosomal DNA, and separation of the amplicons on a denaturing gradient gel. Digital capturing and processing of denaturing gradient gel electrophoresis (DGGE) band patterns allowed direct identification of the amplicons at the species level. This whole culture-independent approach can be performed in less than 30 h. Compared with culture-dependent analysis, the DGGE approach was found to have a much higher sensitivity for detection of microbial strains in probiotic products in a fast, reliable, and reproducible manner. Unfortunately, as reported in previous studies in which the culture-dependent approach was used, a rather high percentage of probiotic products suffered from incorrect labeling and yielded low bacterial counts, which may decrease their probiotic potential.     

Structural Stability,Microbial Biomass and Community Composition of Sediments Affected by the Hydric Dynamics of an Urban Stormwater Infiltration Basin

The sedimentary layer deposited at the surface of stormwater infiltration basins is highly organic and multicontaminated. It undergoes considerable moisture content fluctuations due to the drying and inundation cycles (called hydric dynamics) of these basins. Little is known about the microflora of the sediments and its dynamics; hence, the purpose of this study is to describe the physicochemical and biological characteristics of the sediments at different hydric statuses of the infiltration basin. Sediments were sampled at five time points following rain events and dry periods. They were characterized by physical (aggregation), chemical (nutrients and heavy metals), and biological (total, bacterial and fungal biomasses, and genotypic fingerprints of total bacterial and fungal communities) parameters. Data were processed using statistical analyses which indicated that heavy metal (1,841 μg/g dry weight (DW)) and organic matter (11%) remained stable through time. By contrast, aggregation, nutrient content (NH₄⁺, 53–717 μg/g DW), pH (6.9–7.4), and biological parameters were shown to vary with sediment water content and sediment biomass, and were higher consecutive to stormwater flows into the basin (up to 7 mg C/g DW) than during dry periods (0.6 mg C/g DW). Coinertia analysis revealed that the structure of the bacterial communities is driven by the hydric dynamics of the infiltration basin, although no such trend was found for fungal communities. Hydric dynamics more than rain events appear to be more relevant for explaining variations of aggregation, microbial biomass, and shift in the microbial community composition. We concluded that the hydric dynamics of stormwater infiltration basins greatly affects the structural stability of the sedimentary layer, the biomass of the microbial community living in it and its dynamics. The decrease in aggregation consecutive to rewetting probably enhances access to organic matter (OM), explaining the consecutive release of NH₄⁺, the bloom of the microbial biomass, and the change in structure of the bacterial community. These results open new perspectives for basin management since the risk of OM and pollutant transfer to the aquifer is greatly affected by alternating dry and flood periods.     

Microbial Communities in the Chemocline of a Hypersaline Deep-Sea Basin (Urania Basin, Mediterranean Sea)

The Urania basin is a hypersaline sulfidic brine lake at the bottom of the eastern Mediterranean Sea. Since this basin is located at a depth of ~3,500 m below the sea surface, it receives only a small amount of phytoplankton organic carbon. In the present study, the bacterial assemblages at the interface between the hypersaline brine and the overlaying seawater were investigated. The sulfide concentration increased from 0 to 10 mM within a vertical interval of 5 m across the interface. Within this chemocline, the total bacterial cell counts and the exoenzyme activities were elevated. Employing 11 cultivation methods, we isolated a total of 70 bacterial strains. The 16S ribosomal DNA sequences of 32 of the strains were identical to environmental sequences detected in the chemocline by culture-independent molecular methods. These strains were identified as flavobacteria, Alteromonas macleodii, and Halomonas aquamarina. All 70 strains could grow chemoorganoheterotrophically under oxic conditions. Sixty-six strains grew on peptone, casein hydrolysate, and yeast extract, whereas only 15 strains did not utilize polymeric carbohydrates. Twenty-one of the isolates could grow both chemoorganotrophically and chemolithotrophically. While the most probable numbers in most cases ranged between 0.006 and 4.3% of the total cell counts, an unsually high value of 54% was determined above the chemocline with media containing amino acids as the carbon and energy source. Our results indicate that culturable bacteria thriving at the oxic-anoxic interface of the Urania basin differ considerably from the chemolithoautotrophic bacteria typical of other chemocline habitats.