Jean-Luc Tholozan

Development and Validation of PCR Primers To Assess the Diversity of Clostridium spp. in Cheese by Temporal Temperature Gradient Gel Electrophoresis

ABSTRACT A nested-PCR temporal temperature gradient gel electrophoresis (TTGE) approach was developed for the detection of bacteria belonging to phylogenetic cluster I of the genus Clostridium (the largest clostridial group, which represents 25% of the currently cultured clostridial species) in cheese suspected of late blowing. Primers were designed based on the 16S rRNA gene sequence, and the specificity was confirmed in PCRs performed with DNAs from cluster I and non-cluster I species as the templates. TTGE profiles of the PCR products, comprising the V5-V6 region of the 16S rRNA gene, allowed us to distinguish the majority of cluster I species. PCR-TTGE was applied to analyze commercial cheeses with defects. All cheeses gave a signal after nested PCR, and on the basis of band comigration with TTGE profiles of reference strains, all the bands could be assigned to a clostridial species. The direct identification of Clostridium spp. was confirmed by sequencing of excised bands. C. tyrobutyricum and C. beijerinckii contaminated 15 and 14 of the 20 cheese samples tested, respectively, and C. butyricum and C. sporogenes were detected in one cheese sample. Most-probable-number counts and volatile fatty acid were determined for comparison purposes. Results obtained were in agreement, but only two species, C. tyrobutyricum and C. sporogenes, could be isolated by the plating method. In all cheeses with a high amount of butyric acid (>100 mg/100 g), the presence of C. tyrobutyricum DNA was confirmed by PCR-TTGE, suggesting the involvement of this species in butyric acid fermentation. These results demonstrated the efficacy of the PCR-TTGE method to identify Clostridium in cheeses. The sensitivity of the method was estimated to be 100 CFU/g.

Anaerobic Oxidation of Fatty Acids and Alkenes by the Hyperthermophilic Sulfate-Reducing Archaeon Archaeoglobus fulgidus

ABSTRACT Archaeoglobus fulgidus oxidizes fatty acids (C4 to C18) and n-alk-1-enes (C12:1 to C21:1) in the presence of thiosulfate as a terminal electron acceptor. End products of metabolism were CO2 and sulfide. Growth on perdeuterated hexadecene yielded C15- to C17-labeled fatty acids as metabolites, thus confirming the ability of A. fulgidus to oxidize alkyl chains.

Co-Culture of Microalgae, Cyanobacteria, and Macromycetes for Exopolysaccharides Production: Process Preliminary Optimization and Partial Characterization

In this study, the biomass and exopolysaccharides (EPS) production in co-cultures of microalgae/cyanobacteria and macromycetes was evaluated as a technology for producing new polysaccharides for medical and/or industrial application. Based on biomass and EPS productivity of monocultures, two algae and two fungi were selected and cultured in different co-culture arrangements. The hydrosoluble EPS fractions from mono- and co-cultures were characterized by ¹³C NMR spectroscopy and gas chromatography coupled to mass spectrometry and compared. It was found that co-cultures resulted in the production of an EPS different from those produced by monocultures, showing fungal predominance with microalgal/cyanobacterial traces. Co-cultures conditions were screened (temperature, agitation speed, fungal and microalgae inoculation rate, initial pH, illumination rate, and glucose concentration) in order to achieve maximum biomass and EPS production, resulting in an increase of 33 and 61% in exopolysaccharides and biomass productions, respectively (patent pending).

Characterization of Sporohalobacter salinus sp. nov., an anaerobic, halophilic, fermentative bacterium isolated from a hypersaline lake.

Halophilic, obligately anaerobic, Gram-stain-negative bacterial strains were isolated from a sediment sample taken from under the salt crust of El-Jerid hypersaline lake in southern Tunisia by using tryptone or glucose as the substrate. One strain, CEJFT1B(T), was characterized phenotypically and phylogenetically. Cells were non-motile, non-spore-forming, short rods. Strain CEJFT1B(T) was able to grow in the presence of 5-30 % (w/v) NaCl (optimum 20 %) and at 30-60 °C (optimum 45 °C). It grew at pH 5.5-7.8 and the optimum pH for growth was 6.8. The isolate required yeast extract for growth. Substrates utilized by strain CEJFT1B(T) as the sole carbon source included glucose, fructose, sucrose, pyruvate, Casamino acids and starch. Individual amino acids such as glutamate, lysine, methionine, serine, tyrosine, and amino acid mixtures formed by the Stickland reaction such as alanine-glycine, valine-proline, leucine-proline, isoleucine-proline were also utilized. Products of glucose fermentation were acetate (major product), butyrate, H2 and CO2. The genomic DNA G+C content of strain CEJFT1B(T) was 32.3 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain CEJFT1B(T) should be assigned to the genus Sporohalobacter. The sequence similarity between strain CEJFT1B(T) and Sporohalobacter lortetii was 98.5 %, but DNA-DNA hybridization between the two strains revealed a relatedness value of 56.4 %, indicating that they are not related at the species level. The combination of phylogenetic analysis, DNA-DNA hybridization data, and differences in substrate utilization support the view that strain CEJFT1B(T) represents a novel species of the genus Sporohalobacter, for which the name Sporohalobacter salinus sp. nov. is proposed. The type strain is CEJFT1B(T) ( = DSM 26781(T) = JCM 19279(T)).

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)).