Valérie Michotey

Determination of the bacterial processes which are sources of nitrous oxide production in marine samples.

Partial denitrification and the initial step of nitrification are the main biological processes which produce nitrous oxide. In order to determine the contribution that these processes have in nitrous oxide production, the efficiency of different inhibitors on nitrifying activity has been tested, and the effect on denitrifying activity has been investigated, using culture strains and natural marine samples. A good nitrification inhibitor should not affect denitrification. A low partial pressure of C2H2 provided the best conditions, inhibiting 75%, Nitrosococcus oceanus (culture sample) and 100% (natural sample) of the nitrifying activity and having only a small inhibitory effect (12%) on denitrifying activity. These conditions have been applied on samples from the dilution plume of the Rhĵne River, an area characterized as a source of nitrous oxide. Using these inhibitors, it has been shown that in this area, incomplete denitrification is the main process producing nitrous oxide in the surface layers at the mouth of the river and in the bottom nepheloid layer, whereas in the marine surface layer the dominant process is nitrification.

Denitrification prevails over anammox in tropical mangrove sediments (Goa, India)

Denitrification, anammox (Anx) and di-nitrogen fixation were examined in two mangrove ecosystems- the anthropogenically influenced Divar and the relatively pristine Tuvem. Stratified sampling at 2 cm increments from 0 to 10 cm depth revealed denitrification as the main process of N₂ production in mangrove sediments. At Divar, denitrification was ∼3 times higher than at Tuvem with maximum activity of 224.51 ± 6.63 nmol N₂ g⁻¹ h⁻¹ at 0-2 cm. Denitrifying genes (nosZ) numbered up to 2 × 10⁷ copies g⁻¹ sediment and belonged to uncultured microorganisms clustering within Proteobacteria. Anammox was more prominent at deeper depths (8-10 cm) mainly in Divar with highest activity of 101.15 ± 87.73 nmol N₂ g⁻¹ h⁻¹ which was 5 times higher than at Tuvem. Di-nitrogen fixation was detected only at Tuvem with a maximum of 12.47 ± 8.36 nmol N₂ g⁻¹ h⁻¹. Thus, in these estuarine habitats prone to high nutrient input, N₂-fixation is minimal and denitrification rather than Anx serves as an important mechanism for counteracting N loading.

Molecular, Biochemical, and Physiological Approaches for Understanding the Ecology of Denitrification

One of the major challenges in microbial ecology for the future is to establish links between structural and functional biodiversity. This is particularly difficult when one is interested in a phylogenetically diversified function such as denitrification. The data banks are very rich in functional gene sequences (nirS in this study), but most of them were obtained from not yet cultivated bacteria, and thus must be supplemented by sequences of organisms from the environment for which we could associate a taxonomic position and physiological characteristics. Combined analysis including molecular (16S-rRNA or nirS genes), physiological, and biochemical approaches was carried out on a bacterial set of 89 strains isolated from marine sediment. The denaturing gradient gel electrophoresis (DGGE) technique was successfully applied on unclamped polymerase chain reaction (PCR) products of nirS genes to compare the picture of the biodiversity obtained with 16S rRNA and nirS genes. The diversity of nirS genes and denitrifier characteristics were found within several of the 16S rDNA phylotypes. In contrast, the nirS phylotypes were no diverse both with respect to 16S rDNA and to physiology and biochemistry of denitrification. Sequences of the nirS PCR products were very close to marine environmental clones and were analyzed within the same phylogenetic tree.

Bacterial biogas production in coastal systems affected by freshwater inputs

Abstract The concentrations of two greenhouse gases, nitrous oxide (N 2 O) and methane (CH 4 ), and the bacterial processes involved in their production (nitrification and denitrification for N 2 O, and methanogenesis for CH 4 ), were determined in surface waters of two coastal areas under the influence of freshwater inputs, on one part in the Gulf of Lions and the Rhone River plume, in northwestern Mediterranean Sea, and on the other part in the inner Thermaikos Gulf, in Aegean Sea, eastern Mediterranean Sea. High concentrations of dissolved CH 4 and N 2 O were recorded in the surface waters of Gulf of Lions and Gulf of Thermaikos, up to 1300xa0nM for CH 4 , and 40xa0nM for N 2 O. No direct relationship could be found between the concentration and production of the biogases, as they may also be produced in deep water or bottom sediment in shallow areas, or derived from anthropogenic activity or ship contamination in polluted areas. Irrespective of the origin of CH 4 and N 2 O, the presence of extremely high concentrations of these two gases in superficial seawater implies that they can easily escape to the atmosphere; consequently, these nearshore waters enriched in greenhouse gases may play an important role in the increase in atmospheric concentration of both CH 4 and N 2 O.

Anaerobic biodegradation of squalene: Using DGGE to monitor the isolation of denitrifying Bacteria taken from enrichment cultures

Abstract The process of enrichment and subsequently isolation of squalene degrading denitrifying bacteria has been developed. The enrichment method used in this study targeted denitrifying bacteria, therefore an initial enrichment incubation using nitrate amendments under anaerobic conditions was performed before squalene amendment. Denaturant gradient gel electrophoresis (DGGE) analysis of polymerase chain reaction (PCR)-amplified DNA fragments prepared from extracted DNA was used to compare the composition of bacterial communities at various steps of enrichment cultures and the diversity of the 80 isolated strains obtained by classical culture methods. After 8 months of anaerobic incubation, the squalene biodegradation rate reached 80%. The community composition changed substantially during the incubation time. The enrichment cultures were dominated by 12 phylotypes, of which eight corresponded to cultivatable strains. Their identities were established by sequencing V3-V5 16SrRNA PCR fragments directly or after excision of DGGE bands and comparing the sequences with those available in GenBank. Most of the isolates were Proteobacteria of the gamma subgroup; among them, seven novel denitrifying bacteria which were capable of using squalene as the sole carbon source, were isolated and characterized.

Genome Sequence of the Marine Bacterium Marinobacter hydrocarbonoclasticus SP17, Which Forms Biofilms on Hydrophobic Organic Compounds

Marinobacter hydrocarbonoclasticus SP17 forms biofilms specifically at the interface between water and hydrophobic organic compounds (HOCs) that are used as carbon and energy sources. Biofilm formation at the HOC-water interface has been recognized as a strategy to overcome the low availability of these nearly water-insoluble substrates. Here, we present the genome sequence of SP17, which could provide further insights into the mechanisms of enhancement of HOCs assimilation through biofilm formation.

Anaerobic ammonium oxidation mediated by Mn-oxides: from sediment to strain level

Nitrite and (29)N(2) productions in slurry incubations of anaerobically sediment after (15)NO(3) or (15)NH(4) labelling in the presence of Mn-oxides suggested that anaerobic Mn-oxides mediated nitrification coupled with denitrification in muddy intertidal sediments of Arcachon Bay (SWxa0Atlantic French coast). From this sediment, bacterial strains were isolated and physiologically characterized in terms of Mn-oxides and nitrate reduction as well as potential anaerobic nitrification. One of the isolated strain, identified as Marinobacter daepoensis strain M4AY14, was a denitrifier. Nitrous oxide production by this strain was demonstrated in the absence of nitrate and with Mn-oxides and NH(4) amendment, giving indirect proof of anaerobic nitrate or nitrite production. Anaerobic Mn-oxide-mediated nitrification was confirmed by (29)N(2) production in the presence of (15)NO(3) and (14)NH(4) under denitrifying conditions. Anaerobic nitrification by M4AY14 seemed to occur only in the absence of nitrate, or at nitrate levels lower than that of Mn-oxides. Most of the other isolates were affiliated with the Shewanella genus and were able to use both nitrate and Mn-oxides as electron acceptors. When both electron acceptors were present, whatever their concentrations, nitrate and Mn-oxide reduction co-occurred. These data indicate that bacterial Mn-oxide reduction could be an important process in marine sediments with low oxygen concentrations, and demonstrate for the first time the role of bacteria in anaerobic Mn-mediated nitrification.

Nisaea denitrificans gen. nov., sp. nov. and Nisaea nitritireducens sp. nov., two novel members of the class Alphaproteobacteria from the Mediterranean Sea

Two novel Gram-negative bacteria, designated strains DR41_21(T) and DR41_18(T), were isolated from coastal, surface waters of the north-western Mediterranean Sea. The cells were motile, pleomorphic rods, 2.9 microm long and 0.9 microm wide and formed cream colonies on marine agar medium. The G+C content of the genomic DNA was 60 mol%. Phylogenetic analysis of 16S rRNA gene sequences positioned the isolates in the class Alphaproteobacteria within the family Rhodospirillaceae. The 16S rRNA gene sequence similarity of the two strains was 98.8 % but DNA-DNA hybridization indicated only 55 % relatedness. Strain DR41_21(T) was able to denitrify and possessed nirK and nosZ genes, unlike strain DR41_18(T), which possessed only nirK. These isolates represent two novel species of a new genus, Nisaea gen. nov., for which the names Nisaea denitrificans sp. nov. and Nisaea nitritireducens sp. nov. are proposed. The type strain of Nisaea denitrificans is DR41_21(T) (=DSM 18348(T)=CIP 109265(T)=OOB 129(T)) and the type strain of Nisaea nitritireducens is DR41_18(T) (=DSM 19540(T)=CIP 109601(T)=OOB 128(T)).

Thalassobaculum salexigens sp. nov., a new member of the family Rhodospirillaceae from the NW Mediterranean Sea, and emended description of the genus Thalassobaculum

A novel Gram-negative bacteria, named CZ41_10a(T), was isolated from coastal surface waters of the north-western Mediterranean Sea. Cells were motile, pleomorphic rods, 1.6 mum long and 0.7 mum wide and formed cream colonies on marine agar medium. The G+C content of the genomic DNA was 65 mol%. Phylogenetic analysis of 16S rRNA gene sequences placed the new isolate in the genus Thalassobaculum, a member of the family Rhodospirillaceae, class Alphaproteobacteria. Unlike Thalassobaculum litoreum CL-GR58(T), its closest relative, strain CZ41_10a(T) was unable to grow anaerobically and did not exhibit nitrate reductase activity. On the basis of DNA-DNA hybridization, fatty acid content and physiological and biochemical characteristics, this isolate represents a novel species for which the name Thalassobaculum salexigens sp. nov. is proposed. The type strain is CZ41_10a(T) (=DSM 19539(T)=CIP 109604(T)=MOLA [corrected] 84(T)). An emended description of the genus Thalassobaculum is also given.

The chemical-in-μwell: a high-throughput technique for identifying solutes eliciting a chemotactic response in motile bacteria.

Bacteria, and in particular marine bacteria, can be found in environments that are poor in nutrients. To survive, they are able to move toward more favorable niches by a mechanism called chemotaxis, whose first step consists in the detection of substrates by chemoreceptors. We developed a chemotactic assay enabling rapid testing of several hundred different solutes and we identified several molecules eliciting a chemotactic response from two aquatic Shewanella species. We propose that this assay be used for other bacteria to determine the repertoire of chemotactic molecules, generally not clearly elucidated.