Entrapment of anaerobic thermophilic and hyperthermophilic marine micro-organisms in a gellan/xanthan matrix.

AIMS: The aims of this study were (i) to develop a protocol for the entrapment of anaerobic (hyper)thermophilic marine micro-organisms; (ii) to test the use of the chosen polymers in a range of physical and chemical conditions and (iii) to validate the method with batch cultures. METHODS AND RESULTS: The best conditions for immobilization were obtained at 80°C with gellan and xanthan gums. After 5-week incubation, beads showed a good resistance to all tested conditions except those simultaneously including high temperature (100°C), low NaCl (<0∙5 mol l(-1) ) and extreme pH (4/8). To confirm the method efficiency, batch cultures with immobilized Thermosipho sp. strain AT1272 and Thermococcus kodakarensis strain KOD1 showed an absence of detrimental effect on cell viability and a good growth within and outside the beads. CONCLUSION: This suggests that entrapment in a gellan-xanthan matrix could be employed for the culture of anaerobic (hyper)thermophilic marine micro-organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: (Hyper)thermophilic marine micro-organisms possess a high biotechnological potential. Generally microbial cells are grown as free-cell cultures. The use of immobilized cells may offer several advantages such as protection against phage attack, high cell biomass and better production rate of desired metabolites.

Cultivation of an immobilized (hyper)thermophilic marine microbial community in a bioreactor.

Cultivation in a bioreactor of immobilized deep-sea hydrothermal microbial community was tested in order to assess the stability and reactivity of this new system. A community composed of eight hydrothermal strains was entrapped in a polymer matrix that was used to inoculate a continuous culture in a gas-lift bioreactor. The continuous culture was performed for 41 days at successively 60°C, 55°C, 60°C, 85°C and 60°C, at pH 6.5, in anaerobic condition and constant dilution rate. Oxic stress and pH variations were tested at the beginning of the incubation. Despite these detrimental conditions, three strains including two strict anaerobes were maintained in the bioreactor. High cell concentrations (3 × 10(8) cells mL(-1)) and high ATP contents were measured in both liquid fractions and beads. Cloning-sequencing and qPCR revealed that Bacillus sp. dominated at the early stage, and was later replaced by Thermotoga maritima and Thermococcus sp. Acetate, formate and propionate concentrations varied simultaneously in the liquid fractions. These results demonstrate that these immobilized cells were reactive to culture conditions. They were protected inside the beads during the stress period and released in the liquid fraction when conditions were more favorable. This confirms the advantage of immobilization that highlights the resilience capacity of certain hydrothermal microorganisms after a stress period.

Cloning of DNA fragments carrying hydrogenase genes of Rhodopseudomonas capsulata.

A cosmid library of Rhodopseudomonas capsulata DNA was constructed in Escherichia coli HB101 using the broad-host-range cosmid vector pLAFR1. More than ninety per cent of the clones in the bank contained cosmids with DNA inserts averaging 20 kilobase pairs in length. Mutants deficient in uptake hydrogenase (Hup-) were obtained from R. capsulata strain B10 by ethylmethylsulfonate (EMS) mutagenesis. The content of hydrogenase protein in Hup- mutant cells was tested by rocket immunoelectrophoresis. Hup- mutants (Rifr) were complemented with the clone bank by conjugation and, from the transconjugants selected by rifampicin and tetracycline resistance, Hup+ transconjugants were screened for the ability to grow photoautotrophically and to reduce methylene blue in a colony assay. The recombinant plasmid pAC57 restored hydrogenase activity in the Hup- mutants RCC8, RCC10, RCC12 and ST410 whereas pAG202 restored that of IR4. The cloned R. capsulata DNA insert of pAC57 gave 5 restriction fragments by cleavage with EcoRI endonuclease. Fragment 1 (7 kb) restored hydrogenase activity in Hup- mutant strains RCC12 and ST410 and fragment 5 (1.3 kb) in strains RCC8 and RCC10. Since the 2 cosmids pAC57 and pAG202 are different cosmids, as indicated by restriction analyses and absence of cross hybridization, it is concluded that at least two hup genes are required for the expression of hydrogenase activity in R. capsulata.

Physiology and continuous culture of the hyperthermophilic deep-sea vent archaeon Pyrococcus abyssi ST549.

The deep-sea vent archaeon Pyrococcus abyssi strain ST549 was grown in batch cultures in closed bottles and by continuous culture in a gas-lift bioreactor, both in the presence and in the absence of elemental sulfur. Growth on carbohydrates, proteinaceous substrates and amino acids was investigated. The disaccharides maltose and cellobiose were shown not to be able to enhance growth suggesting that P. abyssi ST549 is unable to use them as carbon sources. By contrast, proteinaceous substrates such as peptone and brain heart infusion were shown to be very good substrates for the growth of P. abyssi ST549 and allowed growth at high steady-state cell densities in continuous culture. Growth on brain heart infusion was shown to require additional nutrients when sulfur was not present in the culture medium. Growth on amino acids only took place in the presence of sulfur. These results indicate that sulfur plays an important role in the metabolism and energetics of P. abyssi ST549.

Thermococcus fumicolans sp. nov., a new hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent in the north Fiji Basin.

An extremely thermophilic archaeon, strain ST557T (T = type strain), was isolated from a deep-sea hydrothermal vent in the North Fiji Basin. This strain is a strictly anaerbic coccus whose cells are about 0.8 to 2 microns in diameter. The optimum temperature, pH and sea salt concentration for growth are 85 degrees C, 8.5, and 20 to 40 g/liter, respectively. Strain ST557T grows preferentially in the presence of elemental sulfur on proteinaceous substrates and on a mixture of 20 amino acids. It grows slowly on pyruvate and maltose. Growth is inhibited by rifampin. The DNA G + C content is 54 to 55 mol%. Sequencing of the 16S rRNA gene revealed that strain ST557T belongs to the genus Thermococcus. We propose that this organism should be placed in a new species, Thermococcus fumicolans.

Thermococcus hydrothermalis sp. nov., a new hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent.

An extremely thermophilic archaeon, strain AL662T, was isolated from a deep-sea hydrothermal vent located on the East Pacific Rise at a latitude of 21 degrees N. This strain is a strictly anaerobic coccus, and its cells range from 0.8 to 2 microns in diameter. The optimum temperature, pH, and Sea Salt concentration for growth are 85 degrees C, 6, and 20 to 40 g/liter, respectively. Strain AL662T grows preferentially on proteolysis products, on a mixture of 20 amino acids, and on maltose in the presence of elemental sulfur. The membrane lipids consist of di- and tetraether glycerol lipids. The DNA G+C content is 58 mol%. Sequencing of the 16S rRNA gene showed that strain AL662T belongs to the genus Thermococcus. On the basis of hybridization results, we propose that this strain should be placed in a new species, Thermococcus hydrothermalis.

Marinitoga camini gen. nov., sp. nov., a rod-shaped bacterium belonging to the order Thermotogales, isolated from a deep-sea hydrothermal vent.

A thermophilic, anaerobic, chemo-organotrophic sulfur-reducing bacterium, designated MV1075T, was isolated from a deep-sea hydrothermal chimney sample collected on the Mid-Atlantic Ridge. Cells were rod-shaped with a sheath-like outer structure, motile with polar flagella and stained Gram-negative. They appeared singly, in pairs or in short chains. The temperature range for growth was 25-65 degrees C, with an optimum at 55 degrees C. Growth was observed from pH 5 to pH 9, and the optimum pH was around 7. The salinity range for growth was 15-70 g sea salt l(-1) (corresponding to 10-45 g NaCl l(-1)), with an optimum at 30 g l(-1) (20 g NaCl l(-1)). The isolate was able to grow on a broad spectrum of carbohydrates or complex proteinaceous substrates. Sulfur was not necessary for growth. Growth was inhibited by H2, but, in presence of sulfur, this inhibition was removed and H2S was produced. The G+C content of the genomic DNA was 29 mol %. Phylogenetic analyses of the 16S rRNA gene located the strain within the order Thermotogales, in the domain Bacteria. On the basis of 16S rDNA sequence comparisons, in combination with morphological and physiological characteristics, it is proposed that the isolate should be described as a novel species of a new genus, Marinitoga gen. nov., of which Marinitoga camini sp. nov. is the type species. The type strain is MV1075T (= CNCM 1-2413T = DSM 13578T).

Pyrococcus glycovorans sp. nov., a hyperthermophilic archaeon isolated from the East Pacific Rise.

A hyperthermophilic archaeon, strain AL585T, was isolated from a deep-sea hydrothermal vent located on the East Pacific Rise at latitude 13 degrees N and a depth of 2650 m. The isolate was a strictly anaerobic coccus with a mean cell diameter of 1 micron. The optimum temperature, pH and concentration of sea salt for growth were 95 degrees C, 7.5 and 30 g l-1. Under these conditions, the doubling time and cell yield were 0.5 h and 5 x 10(8) cells ml-1. Strain AL585T grew preferentially in media containing complex proteinaceous carbon sources, glucose and elemental sulfur. The G + C content of the DNA was 47 mol%. Sequencing of the 16S rDNA gene showed that strain AL585T belonged to the genus Pyrococcus and was probably a new species. This was confirmed by total DNA hybridization. Consequently, this strain is described as a new species, Pyrococcus glycovorans sp. nov.