Desulfovibrio marinisediminis sp. nov., a novel sulfate-reducing bacterium isolated from coastal marine sediment via enrichment with Casamino acids.

To obtain amino acid-utilizing sulfate reducers, enrichment culture was carried out with a medium containing Casamino acids and sulfate and inoculated with coastal marine sediment from the eutrophic Tokyo Bay, Japan. A sulfate reducer, designated strain C/L2(T), was isolated from the sulfide-producing enrichment culture after further enrichment with lactate and sulfate by means of the agar shake dilution method. Cells of strain C/L2(T) were vibrio-shaped, Gram-negative, motile rods (0.7-1.0 mum wide and 1.0-3.5 mum long) with single polar flagella. The optimum temperature for its growth was 37 degrees C, the optimum pH was around 7.5 and the optimum NaCl concentration was 20-25 g l(-1). Hydrogen, formate, lactate, pyruvate, fumarate, malate, succinate, ethanol, propanol, glycerol, glycine, alanine, serine, aspartate, Casamino acids, peptone and yeast extract were used as electron donors. Sulfate, sulfite and thiosulfate each served as an electron acceptor, but elemental sulfur, nitrate, fumarate, acrylate and 2,4,6-tribromophenol did not. Disproportionation of thiosulfate was not observed. Desulfoviridin, c-type cytochromes and catalase were present. The major respiratory quinone was MK-6(H(2)). The G+C content of the genomic DNA was 46.2 mol%. Comparisons based on 16S rRNA gene sequences and on dissimilatory sulfite reductase gene sequences clearly showed that strain C/L2(T) belonged to the genus Desulfovibrio: its closest relatives were the uncharacterized Desulfovibrio sp. strain TBP-1 (16S rRNA gene sequence similarity of 99.4 %) and Desulfovibrio acrylicus DSM 10141(T) (16S rRNA gene sequence similarity of 98.7 %). The level of DNA-DNA hybridization with Desulfovibrio acrylicus DSM 10141(T) was 10.3 %. On the basis of the data from this study and the physiological and phylogenetic differences that exist between the isolate and Desulfovibrio acrylicus, strain C/L2(T) represents a novel species of the genus Desulfovibrio, for which the name Desulfovibrio marinisediminis sp. nov. is proposed. The type strain is C/L2(T) (=NBRC [corrected] 101113(T)=JCM 14577(T)=DSM 17456(T)).

Dethiosulfatibacter aminovorans gen. nov., sp. nov., a novel thiosulfate-reducing bacterium isolated from coastal marine sediment via sulfate-reducing enrichment with Casamino acids.

A sulfate-reducing enrichment culture originating from coastal marine sediment of the eutrophic Tokyo Bay, Japan, was successfully established with Casamino acids as a substrate. A thiosulfate reducer, strain C/G2(T), was isolated from the enrichment culture after further enrichment with glutamate. Cells of strain C/G2(T) were non-motile rods (0.6-0.8 microm x 2.2-4.8 microm) and were found singly or in pairs and sometimes in short chains. Spores were not formed. Cells of strain C/G2(T) stained Gram-negatively, despite possessing Gram-positive cell walls. The optimum temperature for growth was 28-30 degrees C, the optimum pH was around 7.8 and the optimum salt concentration was 20-30 g l(-1). Lactate, pyruvate, serine, cysteine, threonine, glutamate, histidine, lysine, arginine, Casamino acids, peptone and yeast extract were fermented as single substrates and no sugar was used as a fermentative substrate. A Stickland reaction was observed with some pairs of amino acids. Fumarate, alanine, proline, phenylalanine, tryptophan, glutamine and aspartate were utilized only in the presence of thiosulfate. Strain C/G2(T) fermented glutamate to H2, CO2, acetate and propionate. Thiosulfate and elemental sulfur were reduced to sulfide. Sulfate, sulfite and nitrate were not utilized as electron acceptors. The growth of strain C/G2(T) on Casamino acids or glutamate was enhanced by co-culturing with Desulfovibrio sp. isolated from the original mixed culture enriched with Casamino acids. The DNA G+C content of strain C/G2(T) was 41.0 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain C/G2(T) formed a distinct cluster with species of the genus Sedimentibacter. The closest relative was Sedimentibacter hydroxybenzoicus (with a gene sequence similarity of 91 %). On the basis of its phylogenetic and phenotypic properties, strain C/G2(T) (=JCM 13356(T)=NBRC 101112(T)=DSM 17477(T)) is proposed as representing a new genus and novel species, Dethiosulfatibacter aminovorans gen. nov., sp. nov.

Depth-related change in archaeal community structure in a freshwater lake sediment as determined with denaturing gradient gel electrophoresis of amplified 16S rRNA genes and reversely transcribed rRNA fragments.

Vertical changes in archaeal community structure in mesophilic freshwater lake sediment were investigated using denaturing gradient gel electrophoresis of amplified 16S rRNA genes and reversely transcribed 16S rRNA fragments. Electrophoretic band pattern of archaeal community did not drastically change with depth. The archaeal 16S rRNA-based denaturing gradient gel electrophoresis band pattern was very similar to the 16S rDNA-based pattern. These results indicate that archaeal community structure does not drastically change throughout the 0-20 cm in depth, and most archaeal populations in the sediment retain extractable rRNA. Phylogenetic analysis of seven sequences retrieved from both DNA- and RNA-based denaturing gradient gel electrophoresis revealed that these sequences were divided into four major lineages; the kingdom Crenarchaeota, the order Methanomicrobiales, the family Methanosaetaceae, and an uncultured cluster within Euryarchaeota. The order Methanomicrobiales and the family Methanosaetaceae were the dominant methane-producing archaea in this profundal freshwater lake sediment. Moreover, we found Euryarchaeota that are not related to methanogens, and non-thermophilic Crenarchaeota. This suggests that archaeal populations other than methanogens in low-temperature sediment have been previously underestimated.

Amino acids as main substrates for sulfate-reducing bacteria in surface sediment of a eutrophic bay.

The inner part of Tokyo Bay, Japan, is highly eutrophicated as shown by the frequent occurrence of red tide. The bottom water is anoxic during warm seasons especially at artificially dredged sites. In the sediment slurries prepared from surface sediment samples collected from the dredged sites, substrate addition stimulated the consumption of sulfate during anaerobic incubation. Of the substrates added, the seston composed mainly of diatom stimulated consumption more than lactate and acetate. Its effect was nearly equal to that of casamino acids. Casamino acids and some amino acids also accelerated the rate of sulfate reduction measured by the tracer method in sediment samples more than lactate or acetate. Anaerobic incubation of the sediment slurry amended with casamino acids showed that the consumption of amino acids was retarded by the addition of molybdate (final concentration; 20 mM). In the slurry amended with only molybdate, glutamate was accumulated distinctively and linearly with time. Its accumulation rate in molar base was comparable to the rate of sulfate reduction. These results suggested that amino acids were the main substrates for sulfate-reducing bacteria (SRB) in the sediment. The MPN values of SRB in these sediment samples were often higher with the enumeration medium containing casamino acids instead of lactate. Furthermore, during a week incubation of sediment slurries amended with substrates, casamino acids and seston more greatly stimulated the growth of SRB enumerated by both media than lactate.

Vertical distributions of sulfate-reducing bacteria and methane-producing archaea quantified by oligonucleotide probe hybridization in the profundal sediment of a mesotrophic lake.

Abstract Vertical distributions of sulfate-reducing bacteria and methane-producing archaea were investigated in the profundal sediment of a freshwater lake using membrane-immobilized small subunit rRNA hybridization with group- and genus-specific oligonucleotide probes. The annual average of the relative abundance of small subunit rRNA hybridized with all probes for sulfate-reducing bacteria to total small subunit rRNA was 2.3% at 0-2 cm and increased with depth up to 22.9% at 8-14 cm where sulfate concentration was less than 10 nmol ml(-1) in interstitial water, suggesting that these bacteria may survive on alternative metabolisms. The signal of probe Dsv687 (the family Desulfovibrionaceae and some Geobacteraceae) was the main factor in this increase. The relative abundance of methane-producing archaea to total small subunit rRNA was highest (7.8%) at 8-14 cm, dominated by the order Methanosarcinales. The metabolic rates measured in the sediments demonstrated that the peaks of sulfate reduction and methane production were separated vertically, and were not linked to their small subunit rRNA distributions. Our data indicate that sulfate-reducing bacteria can coexist with methane-producing archaea from 0 to 20 cm in the freshwater lake sediment.

Isolation and characterization of a Gram-positive polyphosphate-accumulating bacterium.

A Gram-positive polyphosphate-accumulating bacterium was isolated from phosphate-removal activated sludge using pyruvate-supplemented agar plates. The isolate was oval or coccobacilli (0.4-0.7 x 0.5-1.0 mm) that occurred singly, in pairs or irregular clumps. Polyphosphate granules in the cells were observed by toluidine blue staining. The pure culture of the isolate rapidly took up phosphate (9.2 mg-P/g-dry weight) in the 3-h aerobic incubation without organic substrates, after anaerobic incubation with organic substrates containing casamino acids. When acetate was the sole carbon source in the anaerobic incubation, the isolate did not remove phosphate. These physiological features of the isolate were similar to those of Microlunatus phosphovorus. However, unlike M. phosphovorus the P-removal ability of the isolate was relatively low and was not accelerated by repeating the anaerobic/aerobic incubation cycles. Phylogenetic analysis and comparison of several characteristics showed that the isolate was identified as Tetrasphaera elongata which was recently proposed as a new polyphosphate-accumulating species isolated from activated sludge. As the isolate contained menaquinone (MK)-8(H(4)) as the predominant isoprenoid ubiquinone, it may be significantly responsible for phosphate removal, because MK-8(H(4)) has reportedly been found in fairly high proportions in many phosphate-removing activated sludges.

Polyphasic approaches to the identification of predominant polyphosphate-accumulating organisms in a laboratory-scale anaerobic/aerobic activated sludge system.

By combination of denaturing gradient gel electrophoresis of PCR-amplified 16S rDNA (PCR-DGGE), quinone profiling, and 16S rRNA-targeted fluorescence in situ hybridization (FISH), a polyphosphate-accumulating organism (PAO) responsible for phosphate (P)-removal was identified in activated sludge with high P-removal ability from a laboratory-scale anaerobic/aerobic continuous flow reactor. The DNA fragment from the most dense band on the DGGE gel was closely related to that of 'Candidatus Accumulibacter phosphatis' (beta-Proteobacteria). Quinone profiling also suggested the predominance of beta-Proteobacteria. FISH with a specific oligonucleotide probe designed for the sequence showed that the targeted bacterium was dominant in the activated sludge, and the accumulation and consumption of polyphosphate were observed by dual staining with 4',6-diamidino-2-phenylindole. The bacterium was concluded to be the responsible PAO in the reactor. However, when the P-removal ability per cell slightly decreased, the dominance of the PAO greatly diminished in the activated sludge. Such sludge might be dominated by other types of PAOs.

Characterization by denaturing gradient gel electrophoresis of bacterial communities in deep groundwater at the Kamaishi Mine, Japan.

Bacterial communities in groundwater collected from five different sites at the Kamaishi Mine were investigated by using denaturing gradient gel electrophoresis (DGGE). The bacterial cells in groundwater were collected on Millipore filters, and their nucleic acid was extracted by freeze-thaw cycles. A partial 16S rRNA gene was amplified by using a universal primer set by PCR. The PCR products were analyzed by DGGE. The band pattern of DGGE was essentially identical between two samples obtained from different depths in the same borehole (KH-1). Samples from the other sites differed from one another. The partial sequences of 16S rRNA genes (about 350 base pairs) isolated from bands were determined and analyzed for phylogenetic position. Almost half the sequences from two samples of the KH-1 belonged to the cluster of spore-forming, gram-positive sulfate reducer, Desulfotomaculum. The other bands also were related to those of obligate anaerobes. This suggests that the environment in both sites of KH-1 was highly anaerobic. Although only a few sequences were retrieved from the other sites, they were phylogenetically distanced from known isolates.