Chemical analysis and Microbial community composition of surface water samples from the Southern Lagoon of Venice

The Lagoon of Venice is a large water basin that exchanges water with the Northern Adriatic Sea through three large inlets. We examined two adjacent sites within the Southern Basin and at the Chioggia inlet in autumn 2007 and summer 2008. A pilot study in June 2007 on a surface water sample from Chioggia with a rather high salinity of 36.9 PSU had revealed a conspicuous bloom of CF319a-positive cells likely affiliated with the Cytophaga /Flavobacteria cluster of Bacteroidetes. These flavobacterial abundances were one to two orders of magnitude higher than in other marine surface waters. DAPI-stained cells were identified as bacteria with the general bacterial probe mixture EUB338 I-III. CARD-FISH counts with group-specific probes confirmed the dominance of Bacteroidetes (CF319a), Alphaproteobacteria (ALF968), and Gammaproteobacteria (GAM42a). CARD-FISH showed thatBetaproteobacteria and Planctomycetes were minor components of the bacterioplankton in the Lagoon of Venice.

Shifts in the microbial community structure in different temperatures during sample storage from IODP Hole 325-M0058A

The objective of this study was to determine shifts in the microbial community structure and potential function based on standard Integrated Ocean Drilling Program (IODP) storage procedures for sediment cores. Standard long-term storage protocols maintain sediment temperature at 4°C for mineralogy, geochemical, and/or geotechnical analysis whereas standard microbiological sampling immediately preserves sediments at -80°C. Storage at 4°C does not take into account populations may remain active over geologic time scales at temperatures similar to storage conditions. Identification of active populations within the stored core would suggest geochemical and geophysical conditions within the core change over time. To test this potential, the metabolically active fraction of the total microbial community was characterized from IODP Expedition 325 Great Barrier Reef sediment cores prior to and following a 3-month storage period. Total RNA was extracted from complementary 2, 20, and 40 m below sea floor sediment samples, reverse transcribed to complementary DNA and then sequenced using 454 FLX sequencing technology, yielding over 14,800 sequences from the six samples. Interestingly, 97.3% of the sequences detected were associated with lineages that changed in detection frequency during the storage period including key biogeochemically relevant lineages associated with nitrogen, iron, and sulfur cycling. These lineages have the potential to permanently alter the physical and chemical characteristics of the sediment promoting misleading conclusions about the in situ biogeochemical environment. In addition, the detection of new lineages after storage increases the potential for a wider range of viable lineages within the subsurface that may be underestimated during standard community characterizations.

Microbial community, biomarker concentrations and their isotopic signature in sediment of Gullfaks and Tommeliten methane seeps in the Northern North Sea

Gullfaks is one of the four major Norwegian oil and gas fields, located in the northeastern edge of the North Sea Plateau. Tommeliten lies in the greater Ekofisk area in the central North Sea. During the cruises HE 208 and AL 267 several seep locations of the North Sea were visited. At the Heincke seep at Gullfaks, sediments were sampled in May 2004 (HE 208) using a video-guided multiple corer system (MUC; Octopus, Kiel). The samples were recovered from an area densely covered with bacterial mats where gas ebullition was observed. The coarse sands limited MUC penetration depth to maximal 30 centimeters and the highly permeable sands did not allow for a high-resolution, vertical subsampling because of pore water loss. The gas flare mapping and videographic observation at Tommeliten indicated an area of gas emission with a few small patches of bacterial mats with diameters <50 cm from most of which a single stream of gas bubbles emerged. The patches were spaced apart by 10-100 m. Sampling of sediments covered by bacterial mats was only possible with 3 small push cores (3.8 cm diameter) mounted to ROV Cherokee. These cores were sampled in 3 cm intervals. Lipid biomarker extraction from 10 -17 g wet sediment was carried out as described in detail elsewhere (Elvert et al., 2003; doi:10.1080/01490450303894). Briefly, defined concentrations of cholestane, nonadecanol and nonadecanolic acid with known delta 13C-values were added to the sediments prior to extraction as internal standards for the hydrocarbon, alcohol and fatty acid fraction, respectively. Total lipid extracts were obtained from the sediment by ultrasonification with organic solvents of decreasing polarity. Esterified fatty acids (FAs) were cleaved from the glycerol head group by saponification with methanolic KOH solution. From this mixture, the neutral fraction was extracted with hexane. After subsequent acidification, FAs were extracted with hexane. For analysis, FAs were methylated using BF3 in methanol yielding fatty acid methyl esters (FAMES). The fixation for total cell counts and CARD-FISH were performed on-board directly after sampling. For both methods, sediments were fixed in formaldehyde solution. After two hours, aliquots for CARD-FISH staining were washed with 1* PBS (10mmol/l sodium phosphate solution, 130mmol/l NaCl, adjusted to a pH of 7.2) and finally stored in a 1:1 PBS:ethanol solution at -20°C until further processing. Samples for total cell counts were stored in formalin at 4°C until analysis. For sandy samples, the total cell count/CARD-FISH protocol was optimized to separate sand particles from the cells. Cells were dislodged from sediment grains and brought into solution with the supernatant by sonicating each sample onice for 2 minutes at 50W. This procedure was repeated four times and supernatants were combined. The sediment samples were brought to a final dilution of 1:2000 to 1:4000 and filtered onto 0.2µm GTTP filters (Millipore, Eschbonn, Germany).

Microbial community in the sediment of the arctic Smeerenburgfjorden

Fluorescence in situ hybridization (FISH) with 16S rRNA-targeted oligonucleotide probes were used to investigate the phylogenetic composition of a marine Arctic sediment (Svalbard). Hybridization and microscopy counts of hybridized and 4',6'-diamidino-2-phenylindole (DAPI)-stained cells were performed as described previously from Snaidr et al. (1997, http://aem.asm.org/content/63/7/2884.full.pdf). Means were calculated from 10 to 20 randomly chosen fields on each filter section, corresponding to 800 to 1,000 DAPI-stained cells. Counting results were always corrected by subtracting signals observed with the probe NON338. Formamide concentrations are given in further details. FISH resulted in the detection of a large fraction of microbes living in the top 5 cm of the sediment. Up to 65.4% ± 7.5% of total DAPI cell counts hybridized to the bacterial probe EUB338, and up to 4.9% ± 1.5% hybridized to the archaeal probe ARCH915. Besides delta-proteobacterial sulfate-reducing bacteria (up to 16% 52) members of the Cytophaga-Flavobacterium cluster were the most abundant group detected in this sediment, accounting for up to 12.8% of total DAPI cell counts. Furthermore, members of the order Planctomycetales accounted for up to 3.9% of total cell counts. In accordance with previous studies, these findings support the hypothesis that these bacterial groups are not simply settling with organic matter from the pelagic zone but are indigenous to the anoxic zones of marine sediments. Members of the gamma-proteobacteria also constituted a significant fraction in this sediment (6.1% ± 2.5% of total cell counts). A new probe (GAM660) specific for sequences affiliated with free-living or endosymbiotic sulfur-oxidizing bacteria was developed. A significant number of cells was detected by this probe (2.1% ± 0.7% of total DAPI cell counts), showing no clear zonation along the vertical profile. Gram-positive bacteria and the beta-proteobacteria were near the detection limit in all sediments.

Microbial community composition of sandy intertidal sediments of Sylt-Rømø Basin, Wadden Sea

Molecular biological methods were used to investigate the microbial diversity and community structure in intertidal sandy sediments near the island of Sylt (Wadden Sea) at a site which was characterized for transport and mineralization rates in de Beer et al., (2005, hdl:10013/epic.21375). The sampling was performed during low tide in the middle of the flat, approximately 40 m in the offshore direction from the high water line on October 6, 1999, March 7, 2000, and July 5, 2000. Two parallel cores were collected from each season for molecular analyses. Within 2 h after sampling the sediment cores were sub-sampled and fixed in formaldehyde for FISH analysis. The cells were hybridized, stained with 4',6'-diamidino-2-phenylindole (DAPI) and microscopically counted as described previously [55]. Details of probes and formamide concentrations which were used are shown in further details. Counts are reported as means calculated from 10-15 randomly chosen microscopic fields corresponding to 700-1000 DAPI-stained cells. Values were corrected for the signals counted with the probe NON338. Fluorescence in situ hybridization (FISH)with group-specific rRNA-targeted oligonucleotide probes were used to characterize the microbial community structure over depth (0-12 cm) and seasons (March, July, October). We found high abundances of bacteria with total cell numbers up to 3×109 cells ml-1 and a clear seasonal variation, with higher values in July and October versus March. The microbial community was dominated by members of the Planctomycetes, the Cytophaga/Flavobacterium group, Gammaproteobacteria, and bacteria of the Desulfosarcina/Desulfococcus group. The high abundance (1.5×10**7 - 1.8×10**8 cells/ml accounting for 3-19% of all cells) of presumably aerobic heterotrophic polymer-degrading planctomycetes is in line with the high permeability, deep oxygen penetration, and the high rates of aerobic mineralization of algal biomass measured in the sandy sediments by de Beer et al., (2005, hdl:10013/epic.21375). The high and stable abundance of members of the Desulfosarcina/Desulfococcus group, both over depth and season, suggests that these bacteria may play a more important role than previously assumed based on low sulfate reduction rates in parallel cores de Beer et al., (2005).

Microbial community composition in fresh water at different stations

Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes was used to investigate the phylogenetic composition of bacterioplankton communities in several freshwater and marine samples. An average of about 50% of the cells were detected by probes for the domains Bacteria and Archaea. Cells were concentrated from water samples (1 to 100 ml) on white polycarbonate filters (diameter, 47 mm; pore size, 0.2 mm; type GTTP 4700 [Millipore, Eschborn, Germany]) by applying a vacuum of <25 kPa. They were subsequently fixed by covering the filter with 3 ml of a freshly prepared, phosphate-buffered saline (pH 7.2)-4% paraformaldehyde (Sigma, Deisenhofen, Germany) solution for 30 min at room temperature. Airdried filters are ready for hybridization and can be stored at 220°C or room temperature for several months without showing apparent changes. Probes BET42a, GAM42a, and PLA886 were used with competitor oligonucleotides as described previously amongst others in Manz et al., (1992; doi:10.1016/S0723-2020(11)80121-9). The filters were transferred to a vial containing 50 ml of prewarmed (48°C) washing solution (70 mM NaCl, 20 mM Tris-HCl [pH 7.4], 5 mM EDTA, 0.01% sodium dodecyl sulfate) and incubated freely floating without shaking at 48°C for 15 min. The filter sections were dried on Whatman 3M paper (Whatman Ltd., Maidstone, United Kingdom) and covered with 50 ml of DAPI solution (1 mg/ml in distilled water filtered through at 0.2-mm filter) for 5 min at room temperature in the dark. For each sample and probe, more than 500 cells were enumerated; for the DAPI examination, more than 1,500 cells were counted per sample. All probe-specific cell counts are presented as the percentage of cells visualized by DAPI. The mean abundances and standard deviations were calculated from the counts of 10 to 20 randomly chosen fields on each filter section. All counts were corrected by subtracting the counts obtained with the negative control NON338. Mean and standard deviation were calculated from the counts of 10 to 20 randomly chosen fields on each filter section.

Abyssal community analysis from replicate box cores in the central North Pacific

A 0.25 m**2 United States Naval Electronics Laboratory box corer was used to take replicate samples from an oligotrophic bottom under the North Pacific Central Water Mass (~28°N, 155°W). The bottom is a red clay with manganese nodules at a depth of 5500-5800 m. Macrofaunal density ranges from 84 to 160 individuals per m**2 and is therefore much the same as in Northwest Atlantic Gyre waters. Of the macrofaunal taxa, polychaetes dominate (55 %), followed by tanaids (18 %), bivalves (7 %), and isopods (6 %). Meiofaunal taxa were only partially retained by the 297 µm screen used in washing. Even then, they are 1.5-3.9 times as abundant as the macrofaunal taxa, with nematodes being numerically dominant by far. Foraminifera seem to comprise an important portion of the community, but could not be assessed accurately because of the inability to discriminate living and dead tests. Remains of what are probably xenophyophoridans are also very important, but offer the same problem. Faunal diversity is extremely high, with deposit feeders comprising the overwhelming majority. Most species are rare, being encountered only once. The distributions of only three species show any significant deviation from randomness. The polychaete fauna from box cores collected from 90 miles to the north was not significantly different from that of the principal study locality. Concordance appeared at several taxonomic levels, from species through macrofaunal/meiofaunal relationships. As a result, the variation in total animal abundance shows aggregation among cores. We discuss Sokolova's concept of a deep-sea oligotrophic zone dominated by suspension feeders, and reconcile it with our present findings. The high diversity of the fauna combined with the low food level contradict theories that relate diversity directly with productivity.

Microbial community composition and bacterioplankton at time series station Helgoland Roads, North Sea

A process of global importance in carbon cycling is the remineralization of algae biomass by heterotrophic bacteria, most notably during massive marine algae blooms. Such blooms can trigger secondary blooms of planktonic bacteria that consist of swift successions of distinct bacterial clades, most prominently members of the Flavobacteriia, Gammaproteobacteria and the alphaproteobacterial Roseobacter clade. This study explores such successions during spring phytoplankton blooms in the southern North Sea (German Bight) for four consecutive years. The surface water samples were taken at Helgoland Island about 40 km offshore in the southeastern North Sea in the German Bight at the station 'Kabeltonne' (54° 11.3' N, 7° 54.0' E) between the main island and the minor island, Düne (German for 'dune') using small research vessels (http://www.awi.de/en/expedition/ships/more-ships.html). Water depths at this site fluctuate from 6 to 10 m over the tidal cycle. Samples were processed as described previously (Teeling et al., 2012; doi:10.7554/eLife.11888.001) in the laboratory of the Biological Station Helgoland within less than two hours after sampling. Assessment of absolute cell numbers and bacterioplankton community composition was carried out as described previously (Thiele et al., 2011; doi:10.1016/B978-0-444-53199-5.00056-7). To obtain total cell numbers, DNA of formaldehyde fixed cells filtered on 0.2 mm pore sized filters was stained with 4',6-diamidino-2-phenylindole (DAPI). Fluorescently labeled cells were subsequently counted on filter sections using an epifluores-cence microscope. Likewise, bacterioplankton community composition was assessed by catalyzedreporter deposition fluorescence in situ hybridization (CARD-FISH) of formaldehyde fixed cells on 0.2 mm pore sized filters.

Microbial community composition in sediments of the Hydrate Ridge (Cascadian Margin) collected during RV SONNE cruises SO143 (1999) and SO148-1 (2000)

Cold seep environments such as sediments above outcropping hydrate at Hydrate Ridge (Cascadia margin off Oregon) are characterized by methane venting, high sulfide fluxes caused by the anaerobic oxidation of methane, and the presence of chemosynthetic communities. This investigation deals with the diversity and distribution of sulfate-reducing bacteria, some of which are directly involved in the anaerobic oxidation of methane as syntrophic partners of the methanotrophic archaea. The composition and activity of the microbial communities at methane vented and nonvented sediments are compared by quantitative methods including total cell counts, fluorescence in situ hybridization (FISH). Bacteria involved in the degradation of particulate organic carbon (POC) are as active and diverse as at other productive margin sites of similar water depths. The availability of methane supports a two orders of magnitude higher microbial biomass (up to 9.6×10**10cells/cm**3). Sediment samples were obtained during RV SONNE cruises SO143-2 and SO148-1 at the crest of southern Hydrate Ridge at the Cascadia convergent margin off the coast of Oregon. Sediment cores of 20 - 40 cm length were obtained using a video-guided multiple corer from gas hydrate bearing sediments and from reference sites not enriched in methane in the surface sediments. Samples for total cell counts were obtained from 1 cm core slices, fixed with 2% formaldehyde and stored cold (4°C) and the quantification of aggregates was done via epifluorescence microscopy after staining the sediments with Acridine Orange Direct Counts (AODC) according to the method of Meyer- Reil (1983, doi:10.1007/BF00395813). Total cell counts were defined as the sum of single cells plus the aggregated cells in the syntrophic consortia. DAPI staining was used to measure ANME2/DSS aggregate sizes via epifluorescence microscopy of FISH-treated samples. For FISH, subsamples of sediment cores were sliced into 1 cm intervals and fixed for 2-3 h with 3% formaldehyde (final concentration), washed twice with 1×PBS (10 mM sodium phosphate; 130 mM NaCl), and finally stored in 1×PBS/EtOH (1:1) at -20°C.

Studies of the microbial diversity on analyzed samples from from Peru Margin, Equatorial Pacific, Hydrate Ridge, and Juan de Fuca Ridge

Marine sediments harbor an enormous quantity of microorganisms, including a multitude of novel species. The habitable zone of the marine sediment column begins at the sediment-water interface and probably extends to depths of several thousands of meters. Studies of the microbial diversity in this ecosystem have mostly relied on molecular biological techniques. We used a complementary method - analysis of intact polar membrane lipids - to characterize the in-situ microbial community in sediments covering a wide range of environmental conditions from Peru Margin, Equatorial Pacific, Hydrate Ridge, and Juan de Fuca Ridge. Bacterial and eukaryotic phospholipids were only detected in surface sediments from the Peru Margin. In contrast, deeply buried sediments, independent of their geographic location, were dominated by archaeal diether and tetraether lipids with various polar head groups and core lipids. We compared ring distributions of archaeal tetraether lipids derived from polar glycosidic precursors with those that are present as core lipids. The distributions of these related compound pools were distinct, suggestive of different archaeal sources, i.e., the polar compounds derive from sedimentary communities and the core lipids are fossil remnants from planktonic communities with possible admixtures of decayed sedimentary archaea. This in-situ production of distinct archaeal lipid populations potentially affects applications of the TEX86 paleotemperature proxy as demonstrated by offsets in reconstructed temperatures between both pools. We evaluated how varying cell and lipid stabilities will influence the sedimentary pool by using a box-model. The results are consistent with (i) a requirement of continuous inputs of freshly synthesized lipids in subsurface sediments for explaining the observed distribution of intact polar lipids, and (ii) decreasing lipid inputs with increasing burial depth.

Table 1 Microorganisms, identified with DOGE analysis. 16S rDNA sequences, obtained from sequencing of DOGE bands, were assigned to the corresponding microorganisms

Microorganisms play an important role in the transformation of material within the earth's crust. The storage of CO2 could affect the composition of inorganic and organic components in the reservoir, consequently influencing microbial activities. To study the microbial induced processes together with geochemical, petrophysical and mineralogical changes, occurring during CO2 storage, long-term laboratory experiments under simulated reservoir P-T conditions were carried out. Clean inner core sections, obtained from the reservoir region at the CO2 storage site in Ketzin (Germany) from a depth of about 650 m, were incubated in high pressure vessels together with sterile synthetic formation brine under in situ P-T conditions of 5.5 MPa and 40°C. A 16S rDNA based fingerprinting method was used to identify the dominant species in DNA extracts of pristine sandstone samples. Members of the alpha- and beta-subdivisions of Proteobacteria and the Actinobacteria were identified. So far sequences belonging to facultative anaerobic, chemoheterotrophic bacteria (Burkholderia fungorum, Agrobacterium tumefaciens) gaining their energy from the oxidation of organic molecules and a genus also capable of chemolithoautotrophic growth (Hydrogenophaga) was identified. During CO2 incubation minor changes in the microbial community composition were observed. The majority of microbes were able to adapt to the changed conditions. During CO2 exposure increased concentrations of Ca**2+, K**+, Mg**2+ and SO4**2- were observed. Partially, concentration rises are (i) due to equilibration between rock pore water and synthetic brine, and (ii) between rock and brine, and are thus independent on CO2 exposure. However, observed concentrations of Ca**2+, K**+, Mg**2+ are even higher than in the original reservoir fluid and therefore indicate mineral dissolution due to CO2 exposure.