Halanaerobacter jeridensis sp. nov., isolated from a hypersaline lake.

An obligatory anaerobic, moderately halophilic bacterium, designated strain CEJFG43(T), was isolated from a sample of sediment collected below the salt crust on the hypersaline El Jerid lake, in southern Tunisia. The cells of this novel strain were Gram-staining-negative, non-sporulating, motile, short rods. They grew in media with 6-30% (w/v) NaCl (optimum 15%), at 20-60 °C (optimum 45 °C) and at pH 5.5-9.5 (optimum pH 8.3). The micro-organism fermented glucose, fructose, ribose, raffinose, galactose, mannose, sucrose, maltose, xylose, mannitol, pyruvate and glycerol. The products of glucose fermentation were lactate, ethanol, acetate, H(2) and CO(2). The genomic G+C DNA content of strain CEJFG43(T) was 33.3 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain CEJFG43(T) belonged in the genus Halanaerobacter and was most closely related to Halanaerobacter lacunarum DSM 6640(T) (95.3% gene sequence similarity) and Halanaerobacter chitinivorans DSM 9569(T) (95.3%). The predominant cellular fatty acids were non-branched (C(16:0) and C(16:1)). Based on the phylogenetic and phenotypic evidence, strain CEJFG43(T) represents a novel species in the genus Halanaerobacter for which the name Halanaerobacter jeridensis sp. nov. is proposed. The type strain is CEJFG43(T) ( = DSM 23230(T) = JCM 16696(T)).

Characterization of Halanaerocella petrolearia gen. nov., sp. nov., a new anaerobic moderately halophilic fermentative bacterium isolated from a deep subsurface hypersaline oil reservoir : New taxa: Firmicutes (Class Clostridia, Order Halanaerobiales, Halobacteroidaceae, Halobacteroides).

An anaerobic, halophilic, and fermentative bacterium, strain S200(T), was isolated from a core sample of a deep hypersaline oil reservoir. Cells were rod-shaped, non-motile, and stained Gram-positive. It grew at NaCl concentrations ranging from 6 to 26% (w/v), with optimal growth at 15% (w/v) NaCl, and at temperatures between 25 and 47°C with an optimum at 40-45°C. The optimum pH was 7.3 (range 6.2-8.8; no growth at pH 5.8 and pH 9). The doubling time in optimized growth conditions was 3.5 h. Strain S200(T) used exclusively carbohydrates as carbon and energy sources. The end products of glucose degradation were lactate, formate, ethanol, acetate, H(2), and CO(2). The predominant cellular fatty acids were non-branched fatty acids C(16:1), C(16:0), and C(14:0). The G + C mole% of the DNA was 32.7%. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain S200(T) formed a distinct lineage within the family Halobacteroidaceae, order Halanaerobiales, and was most closely related to Halanaerobaculum tunisiense DSM 19997(T) and Halobacteroides halobius DSM 5150(T), with sequence similarity of 92.3 and 91.9%, respectively. On the basis of its physiological and genotypic properties, strain S200(T) is proposed to be assigned to a novel species of a novel genus, for which the name Halanaerocella petrolearia is proposed. The type strain of Halanaerocella petrolearia is strain S200(T) (=DSM 22693(T) = JCM 16358(T)).

Desulfovibrio marinus sp. nov., a moderately halophilic sulfate-reducing bacterium isolated from marine sediments in Tunisia.

Two novel sulfate-reducing bacterial strains, designated E-2(T) and IMP-2, were isolated from geographically distinct locations. Strain E-2(T) was recovered from marine sediments near Sfax (Tunisia), whereas strain IMP-2 originated from oilfield production fluids in the Gulf of Mexico. Cells were Gram-negative, non-sporulated, motile, vibrio-shaped or sigmoid. They were strictly anaerobic, mesophilic and moderately halophilic. Sulfate, sulfite, thiosulfate and elemental sulfur served as electron acceptors, but not nitrate or nitrite. H(2) (with acetate as carbon source), formate, fumarate, lactate, malate, pyruvate, succinate and fructose were used as electron donors in the presence of sulfate as terminal electron acceptor. Lactate was oxidized incompletely to acetate. Fumarate and pyruvate were fermented. Desulfoviridin and c-type cytochromes were present. 16S rRNA gene sequence analysis of the two strains showed that they were phylogenetically similar (99.0 % similarity) and belonged to the genus Desulfovibrio, with Desulfovibrio indonesiensis and Desulfovibrio gabonensis as their closest phylogenetic relatives. The G+C content of the DNA was respectively 60.4 and 62.7 mol% for strains E-2(T) and IMP-2. DNA-DNA hybridization experiments revealed that the novel strains had a high genomic relatedness, suggesting that they belong to the same species. We therefore propose that the two isolates be affiliated to a novel species of the genus Desulfovibrio, Desulfovibrio marinus sp. nov. The type strain is strain E-2(T) (=DSM 18311(T) =JCM 14040(T)).

Anaerobic treatment of cassava starch extraction wastewater using a horizontal flow filter with bamboo as support.

Small-scale sour starch agroindustry in Colombia suffer from absence of water treatment. Although starch processing plants produce diluted wastewater, it is a source of pollution and cause environmental problems to the nearby rural population. A laboratory scale anaerobic horizontal flow filter packed with bamboo pieces was evaluated for the treatment of cassava starch extraction wastewater. The wastewater used in the experimentation was the draining water of the starch sedimentation basin. The reactor was operated for 6 months. It was inoculated with a semi-granular sludge from an anaerobic UASB reactor of a slaughterhouse. Maximum organic loading rate (OLR) applied was 11.8g COD/L d without dilution of the wastewater. At steady state and maximum OLR applied, 87% of the COD was removed and a gas productivity of 3.7L/L d was achieved. The average biogas yield was 0.36L/g COD removed. Methane content in the biogas was in the range of 69-81%. The total suspended solids (TSS) removed were 67%. The relative high lactic acid content did not negatively influence the performance of the reactor. No perturbation due to cyanide (3-5mg/L) was observed during the reactor operation. The results obtained indicated that the anaerobic horizontal flow filter could be used efficiently for the treatment of wastewater from Colombian starch processing small-scale agroindustry.

Desulfovibrio hydrothermalis sp. nov., a novel sulfate-reducing bacterium isolated from hydrothermal vents.

Mesophilic, hydrogenotrophic, sulfate-reducing bacteria were isolated from a deep-sea hydrothermal chimney sample collected at 13 degrees N on the East-Pacific Rise at a depth of 2,600 m. Two strains (BL5 and H9) were found to be phylogenetically similar to Desulfovibrio profundus (similarity >99%), whereas two other strains (H1 and AM13T) were found to be phylogenetically distinct (similarity 96.4%) from Desulfovibrio zosterae, their closest relative. Strain AM13T was characterized further. It was a barophilic, Gram-negative, non-sporulating, motile, vibrio-shaped or sigmoid bacterium possessing desulfoviridin. It grew at temperatures ranging from 20 to 40 degrees C, with an optimum at 35 degrees C in the presence of 2.5% NaCl. The pH range for growth was 6.7-8.2 with an optimum around 7.8. Strain AM13T utilized H2/CO2, lactate, formate, ethanol, choline and glycerol as electron donors. Electron acceptors were sulfate, sulfite and thiosulfate, but not elemental sulfur or nitrate. The G + C content of DNA was 47 mol%. Strain AM13T (= DSM 14728T = CIP107303T) differed from D. zosterae not only phylogenetically, but also genomically (DNA-DNA reassociation value between the two bacteria was 23.8%) and phenotypically. This isolate is therefore proposed as the type strain of a novel species of the genus Desulfovibrio, Desulfovibrio hydrothermalis sp. nov.

Identification of A2-restricted hepatitis C virus-specific cytotoxic T lymphocyte epitopes from conserved regions of the viral genome.

We have focused on conserved regions of the hepatitis C Virus (HCV) genome to identify viral peptides that contain HLA class I binding motifs and bind with high affinity to the corresponding purified HLA molecules. Accordingly, we have identified 31 candidate epitopes in the HCV that have the potential to be recognized by either HLA-A1, A2.1-, A3, A11- or A24-restricted cytotoxic T lymphocytes (CTL). Twelve conserved peptides that bind HLA-A2.1 with high or intermediate affinity were tested for immunogenicity in vitro in human primary CTL cultures and in vivo by direct immunization of HLA-A2.1/Kb transgenic mice. Six HLA-A2.1-restricted CTL epitopes were immunogenic in both systems. At least three of these peptide epitopes were endogenously processed and presented for CTL recognition. Overall, these data illustrate the value of this approach for the development of virus-specific, peptide-based vaccines.

In vitro induction of primary, antigen-specific CTL from human peripheral blood mononuclear cells stimulated with synthetic peptides.

A protocol for in vitro induction of primary, antigen-specific CTL from human peripheral blood mononuclear cells (PBMCs) was developed. Antigen presenting cells (APCs) consisted of Staphylococcus aureus Cowan-I (SAC-I) activated PBMCs treated with a citrate-phosphate buffer at pH 3 to release endogenous peptides bound to surface MHC. This treatment resulted in transient expression of empty class I molecules which could be subsequently stabilized with peptide and beta 2-microglobulin (beta 2m). SAC-I activated PBMCs from HLA-A2.1 normal donors loaded with HBV core 18-27 peptide following acid treatment were used to stimulate PBMCs depleted of CD4+ T cells, in the presence of recombinant interleukin-7 (rIL-7). After 12 days, cells were restimulated with autologous, peptide-pulsed, adherent cells and tested for CTL activity 7 days later. In 23 independent experiments from 13 different HLA-A2.1 donors, this protocol resulted in induction of primary CTL more than 90% of the time. As indicated by both the frequency and magnitude of the response against peptide-sensitized target cells, SAC-I activated PBMCs treated with acid were the most efficient stimulator APC. Thirteen per cent of the cultures generated were capable of lysing target cells transfected with the HBV core antigen and, in general, these CTL cultures exhibited high avidity for the HBV core peptide. This protocol is generally applicable to different antigens and class I alleles, and thus, may be utilized to screen large numbers of peptides to identify human CTL epitopes.

Involvement of a single periplasmic hydrogenase for both hydrogen uptake and production in some Desulfovibrio species.

Various sulphate-reducing bacteria differing in the number of genes encoding hydrogenase were shown to ferment lactate in coculture with Methanospirillum hungatei, in the absence of sulphate. The efficiency of interspecies H2 transfer carried out by these species of sulphate-reducing bacteria does not appear to correlate with the distribution of genes coding for hydrogenase. Desulfovibrio vulgaris Groningen, which possesses only the gene for [NiFe] hydrogenase, oxidizes hydrogen in the presence of sulphate and produces some hydrogen during fermentation of pyruvate without electron acceptor. The hydrogenase of D. vulgaris was purified and characterized. It exhibits a molecular mass of 87 kDa and is composed of two different subunits (60 and 28 kDa). D. vulgaris hydrogenase contains 10.6 iron atoms, 0.9 nickel atom and 12 acid-labile sulphur atoms/molecule, and the absorption spectrum of the enzyme is characteristic of an iron-sulphur protein. Maximal H2 uptake and H2 evolution activities were 332 and 230 units/mg protein, respectively. D. vulgaris cells contain exclusively the [NiFe] hydrogenase, whatever the growth conditions, as shown by biochemical and immunological studies. Immunocytolocalization in ultrathin frozen sections of cells grown on lactate and sulphate, on H2 and sulphate and on pyruvate showed that the [NiFe] hydrogenase was located in the periplasmic space. Labelling was enhanced in cells grown on H2 and sulphate and on pyruvate. The results enable us to conclude that D. vulgaris Groningen contains a single hydrogenase of the [NiFe] type, located in the periplasmic space like that described for D. gigas. This enzyme appears to be involved in both H2 uptake and H2 production, depending on the growth conditions.

Stem and Root Nodulation in Aeschynomene spp.

Nodulation ability of 15 Rhizobium strains isolated from root and stem nodules of tropical Aeschynomene species was studied on 20 different Aeschynomene species and four other legumes-Arachis hypogaea, Stylosanthes guianensis, Macroptilium atropurpureum, and Sesbania rostrata. The results of this investigation showed that Aeschynomene species could be divided into three groups according to the cross-inoculation group concept.