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).

Relative abundance of prokaryotes in sediments of the Håkon Mosby mud volcano (Z1-Z3, all habitats)

CARD-FISH was performed as previously described in Ruff et al., (2013; doi:10.1371/journal.pone.0072627) with the following modifications. 4-6 µl of 25-fold diluted sediment were used for filtration. Archaeal cell walls were permeabilized with 0.1M HCl for 2 min to detect ANME-3 cells, or Proteinase K solution (15 µg ml-1 (Merck, Darmstadt, Germany) in 0.05 M EDTA (pH 8), 0.1 M Tris-HCl (pH 8), 0.5 M NaCl) for 2-4 min at room temperature for all other archaea. Bacterial cell walls were permeabilized with lysozyme solution (1000kU/ml) for 60 min at 37°. Cells were stained with DAPI (1µg/ml), embedded in mounting medium and counted in 40-60 independent microscopic fields using an Axiophot II epifluorescence microscope (Carl Zeiss, Jena, Germany).

Use of denaturing gradient gel electrophoresis to detect Actinobacteria associated with the human faecal microbiota

With the exceptions of the bifidobacteria, propionibacteria and coriobacteria, the Actinobacteria associated with the human gastrointestinal tract have received little attention. This has been due to the seeming absence of these bacteria from most clone libraries. In addition, many of these bacteria have fastidious growth and atmospheric requirements. A recent cultivation-based study has shown that the Actinobacteria of the human gut may be more diverse than previously thought. The aim of this study was to develop a denaturing gradient gel electrophoresis (DGGE) approach for characterizing Actinobacteria present in faecal samples. Amount of DNA added to the Actinobacteria-specific PCR used to generate strong PCR products of equal intenstity from faecal samples of five infants, nine adults and eight elderly adults was anti-correlated with counts of bacteria obtained using fluorescence in situ hybridization probe HGC69A. A nested PCR using Actinobacteria-specific and universal PCR-DGGE primers was used to generate profiles for the Actinobacteria. Cloning of sequences from the DGGE bands confirmed the specificity of the Actinobacteria-specific primers. In addition to members of the genus Bifidobacterium, species belonging to the genera Propionibacterium, Microbacterium, Brevibacterium, Actinomyces and Corynebacterium were found to be part of the faecal microbiota of healthy humans.

In situ hybridization of bat chromosomes with human (TTAGGG)n probe, after previous digestion with Alu I

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

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.

NOR5/OM60 clade of the Yangtze River estuary

The phylogeny, abundance, and biogeography of the NOR5/OM60 clade was investigated. This clade includes "Congregibacter litoralis" strain KT71, the first cultured representative of marine aerobic anoxygenic phototrophic Gammaproteobacteria. Most of the NOR5/OM60 sequences were retrieved from marine coastal settings, whereas there were fewer from open-ocean surface waters, deep-sea sediment, freshwater, saline lakes and soil. The abundance of members of the NOR5/OM60 clade in various marine sites was determined by fluorescence in situ hybridization using a newly designed and optimized probe set. Relative abundances in coastal marine waters off the Yangtze estuary were up to 3% of the total 4',6-diamidino-2-phenylindole (DAPI) counts. A small cruise was undertaken from 2006-09-06 to 2006-09-08 in the Yangtze River estuary. Samples were taken from surface water, and immediately fixed with 1% paraformaldehyde (PFA) for 1 h, filtered onto polycarbonate filters (Millipore, 47 mm in diameter, 0.2 µm pore size) and stored frozen at -20 °C.

Temporal and spatial changes in milfoil distribution and biomass associated with weevils in Fish Lake, WI

During the course of an eight year monitoring effort, the Wisconsin Department of Natural Resources documented a significant decline in milfoil biomass and distribution in Fish Lake, Wisconsin. Average milfoil biomass declined by 40- 50% from 374-524 g dw m -2 during 1991-93 to 265 g dw m -2 during both 1994 and 1995. Milfoil recovered fully in 1996- 98 to 446- 564 g dw m -2 . The size of the milfoil bed, as discerned from aerial photographs, shrank from a maximum coverage of 40 ha in 1991 to less than 20 ha during 1995. During the “crash” of 1994-95, milfoil plants exhibited typical signs of weevil-induced damage, including darkened, brittle, hollowed-out growing tips, and the arching and collapse of stems associated with loss of buoyancy. Monitoring of weevils and stem damage during 1995-98 showed highest densities and heaviest damage occurred near shore and subsequently fanned out into deeper water from core infestation sites each spring. The extent of milfoil stem damage was positively correlated with weevil densities (monthly sampling). However, weevil densities and stem damage were lower during 1995 (when milfoil biomass was in decline) than during 1996-98 (when milfoil biomass was fully recovered).

Improvement in the nutritive quality of soyabean meal by co-ensiling with under-utilized fish discards

Improvement in the nutritive value of soybean meal was investigated by Co-ensiling it with underutilized trash fish discards (gizzard shad)at different proportions. The following proportions of gizzard shad to soybean meal were used; (a) 100% gizzard shad + acid combination (b)80% gizzard shad +20% soybean meal & 10% WB (c) 60% gizzard shad + 40% soybean meal & 10% WB (d) 100% gizzard shad without acid combination. Co-ensiling was achieved by adding sufficient acid to produce a paste. Products were neutralized by addition of 2% (by weight) calcium hydroxide and drying was affected by freeze-drying.The dried silage products were stored at low temperatures. Products were analysed for proximate composition and amino acid composition.The amino acid composition and ration of essential amino acid. Non essential amino acid (EAA/NEAA) was used as index of nutritive quality. Also essential amino acid profile of the co-ensiled products were compared with essential amino acid requirement of some warmwater fish species to estimate their nutritive usefulness for these species

A comparative assessment of the methods of control of water hyacinth infestation with regards to fish production

Water hyacinth (Eichhornia crassipes) has been subject of three control methods since its arrival into the Nigerian freshwater lagoon system in 1984 - mechanical, chemical and biological. An assessment of these three methods seems to suggest that mechanical and chemical control methods, both of which being costly, must be applied either solely or integrated to combat the present level of considerable infestation in Nigeria. The biological control methods are advisable for slow, sustained control and can only cope with low levels of infestation. It is thus concluded that the preliminary control method should be mechanical or chemical to effectively abate the nuisance plant, followed by biological control once infestation levels have been sufficiently reduced

A computer software package for assessing and managing risks posed by experiments with genetically modified fish and shellfish

Assessment and management of risk is needed for sustainable use of genetically modified aquatic organisms (aquatic GMOs). A computer software package for safely conducting research with genetically modified fish and shellfish is described. By answering a series of questions about the organism and the accessible aquatic ecosystem, a researcher or oversight authority can either identify specific risks or conclude that there is a specific reason for safety of the experiment. Risk assessment protocols with examples involving transgenic coho salmon, triploid grass carp and hybrid tilapia are described. In case a specific risk is identified, the user is led to consider risk management measures, involving culture methods, facilities design and operations management, to minimize the risk. Key features of the software are its user-friendly organization; easy access to explanatory text, literature citations and glossary; and automated completion of a worksheet. Documented completion of the Performance Standards can facilitate approval of a well designed experiment by oversight authorities.