Growth conditions. M. hydrocarbonoclasticus was aerobically cultivated in synthetic medium (SM) containing 1.23% (wtivol) Tris, 0.37% (wtivol) NH,Cl, 0.62% (wtkol)

Two PCR primer sets were developed for the detection and quantification of cytochrome cd 1 -denitrifying bacteria in environmental marine samples. The specificity and sensitivity of these primers were tested. Both primer sets were suitable for detection, but only one set, cd3F-cd4R, was suitable for the quantification and enumeration of the functional community using most-probable-number PCR and competitive PCR techniques. Quantification of cytochrome cd 1 denitrifiers taken from marine sediment and water samples was achieved using two different molecular techniques which target the nirS gene, and the results were compared to those obtained by using the classical cultivation method. Enumerations using both molecular techniques yielded similar results in seawater and sediment samples. However, both molecular techniques showed 1,000 or 10 times more cytochrome cd 1 denitrifiers in the sediment or water samples, respectively, than were found by use of the conventional cultivation method for counting.It is generally believed that only a small fraction of environmental bacteria are recovered by current cultivation techniques and that the quantification of microorganisms is therefore biased. The most prominent methods which have been suggested for studying this noncultivated fraction of indigenous community bacteria are based on using nucleic acids. Techniques such as most-probable-number (MPN) PCR and competitive PCR have been developed to quantify specific groups of bacteria by amplifying the 16S fragment in the ribosomal DNA (17,25,28,31) or in the functional gene (15,21,35). In using studies which target metabolic function, in some cases all the organisms of a species or genus possess the same metabolic function (nitrification or sulfate reduction, for example). Using a probe which targets a specific part of the ribosomal gene can therefore give an indication of the presence of the bacterial group which is capable of that function. On the contrary, if the function is spread among a variety of bacterial species, and only a small number of strains of each species possess that function, it is not possible to perform the classic approach of targeting the ribosomal gene. In this case the conserved region of a functional gene may serve as a suitable target. The use of a functional gene requires sufficient genetic homology of the structural genes and the availability of multiple sequences in order to reliably design primers. When the function is widely spread over the phylogenic groups, the primers used for molecular detection become more degenerated. This increases the risk of nonspecific annealing of the primer onto nontarget sequences, which in turn leads to low specificity and low sensitivity of the technique. Denitrification is a good example of a process which is performed by a great diversity of bacterial strains which come from all the major physiological groups, with the exception of Enterobacteriaceae. The nitrite reductase gene is a key enzyme for this metabolic process. Depending on the strain, denitrifying b...

The seasonal patterns of nitrification, denitrificat~on and dissimilatory ammonium production (DAP) rates were studied in the sediment of 2 stations in the Thau lagoon (south of France). The stat~on ZA was located within the shellfish farming zone and the station B was the reference site. A marked effect of shellfish farming on bacterial activities was observed. Spatial differences were associated with discrepancies in the organlc content and the reduction state of sediments, i.e. highest reductive processes (denltrificatlon and DAP) were noted In shellfish farming area, whereas the oxidative process (nitrification) was predominant outside the farming zone. At both stations, the DAP activity increased in September (autumn) concomitant with an increase of the C/N ratio in the sediment due to the sedimentation of the summer phytoplanktonic production. Nitrification and denitrification rates exhibited maxima in November (winter) corresponding to dissolved inorganic nitrogen inputs from the surrounding land. In the shellfish farming site, 98% of nitrate was reduced to NH,+ and 2 % to NzO, showing that the most of the NO3-was reduced to ammonium and remained available for the ecosystem.

Brown algae belong to a phylogenetic lineage distantly related to green plants and animals, and are found predominantly in the intertidal zone, a harsh and frequently changing environment. Because of their unique evolutionary history and of their habitat, brown algae feature several peculiarities in their metabolism. One of these is the mannitol cycle, which plays a central role in their physiology, as mannitol acts as carbon storage, osmoprotectant, and antioxidant. This polyol is derived directly from the photoassimilate fructose-6-phosphate via the action of a mannitol-1-phosphate dehydrogenase and a mannitol-1-phosphatase (M1Pase). Genome analysis of the brown algal model Ectocarpus siliculosus allowed identification of genes potentially involved in the mannitol cycle. Among these, two genes coding for haloacid dehalogenase (HAD)-like enzymes were suggested to correspond to M1Pase activity, and thus were named EsM1Pase1 and EsM1Pase2, respectively. To test this hypothesis, both genes were expressed in Escherichia coli. Recombinant EsM1Pase2 was shown to hydrolyse the phosphate group from mannitol-1-phosphate to produce mannitol but was not active on the hexose monophosphates tested. Gene expression analysis showed that transcription of both E. siliculosus genes was under the influence of the diurnal cycle. Sequence analysis and three-dimensional homology modelling indicated that EsM1Pases, and their orthologues in Prasinophytes, should be seen as founding members of a new family of phosphatase with original substrate specificity within the HAD superfamily of proteins. This is the first report describing the characterization of a gene encoding M1Pase activity in photosynthetic organisms.

The pelagic realm of the dark ocean is characterized by high hydrostatic pressure, low temperature, high-inorganic nutrients, and low organic carbon concentrations. Measurements of metabolic activities of bathypelagic bacteria are often underestimated due to the technological limitations in recovering samples and maintaining them under in situ environmental conditions. Moreover, most of the pressure-retaining samplers, developed by a number of different labs, able to maintain seawater samples at in situ pressure during recovery have remained at the prototype stage, and therefore not available to the scientific community. In this paper, we will describe a ready-to-use pressure-retaining sampler, which can be adapted to use on a CTD-carousel sampler. As well as being able to recover samples under in situ high pressure (up to 60 MPa) we propose a sample processing in equi-pressure mode. Using a piloted pressure generator, we present how to perform sub-sampling and transfer of samples in equi-pressure mode to obtain replicates and perform hyperbaric experiments safely and efficiently (with <2% pressure variability). As proof of concept, we describe a field application (prokaryotic activity measurements and incubation experiment) with samples collected at 3,000m-depth in the Mediterranean Sea. Sampling, sub-sampling, transfer, and incubations were performed under in situ high pressure conditions and compared to those performed following decompression and incubation at atmospheric pressure. Three successive incubations were made for each condition using direct dissolved-oxygen concentration measurements to determine the incubation times. Subsamples were collected at the end of each incubation to monitor the prokaryotic diversity, using 16S-rDNA/rRNA high-throughput sequencing. Our results demonstrated that oxygen consumption by prokaryotes is always higher under in situ conditions than after decompression and incubation at atmospheric pressure. In addition, over time, the variations in the prokaryotic community composition and structure are seen to be driven by the different experimental conditions. Finally, within samples maintained under in situ high pressure conditions, the active (16S rRNA) prokaryotic community was dominated by sequences affiliated with rare families containing piezophilic isolates, such as Oceanospirillaceae or Colwelliaceae. These results demonstrate the biological importance of maintaining in situ conditions during and after sampling in deep-sea environments.

Earlier observations in mangrove sediments of Goa, India have shown denitrification to be a major pathway for N loss1. However, percentage of total nitrate transformed through complete denitrification accounted for <0–72% of the pore water nitrate reduced. Here, we show that up to 99% of nitrate removal in mangrove sediments is routed through dissimilatory nitrate reduction to ammonium (DNRA). The DNRA process was 2x higher at the relatively pristine site Tuvem compared to the anthropogenically-influenced Divar mangrove ecosystem. In systems receiving low extraneous nutrient inputs, this mechanism effectively conserves and re-circulates N minimizing nutrient loss that would otherwise occur through denitrification. In a global context, the occurrence of DNRA in mangroves has important implications for maintaining N levels and sustaining ecosystem productivity. For the first time, this study also highlights the significance of DNRA in buffering the climate by modulating the production of the greenhouse gas nitrous oxide.

Monitoring photooxidative and salinity-induced bacterial stress in the Canadian Arctic using specific lipid tracers,

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