Diversity of Sulfate-Reducing Bacteria Inhabiting the Rhizosphere of Phragmites australis in Lake Velencei (Hungary) Revealed by a Combined Cultivation-based and Molecular approach

The community structure of sulfate-reducing bacteria (SRB) associated with reed (Phragmites australis) rhizosphere in Lake Velencei (Hungary) was investigated by using cultivation-based and molecular methods. The cultivation methods were restricted to recover lactate-utilizing species with the exclusion of Desulfobacter and some Desulfobacterium species presumably not being dominant members of the examined community. The most-probable-number (MPN) estimations of lactate-utilizing SRB showed that the cell counts in reed rhizosphere were at least one order of magnitude higher than that in the bulk sediment. The number of endospores was low compared to the total SRB counts. From the highest positive dilution of MPN series, 47 strains were isolated and grouped by restriction fragment length polymorphism (RFLP) analysis of the amplified 16S ribosomal RNA (rRNA) and dsrAB (dissimilatory sulfite reductase) genes. Contrary to the physiological diversity of the isolates, the combined results of RFLP analysis revealed higher diversity at species as well as at subspecies level. Based on the partial 16S rRNA sequences, the representative strains were closely affiliated with the genera Desulfovibrio and Desulfotomaculum. The partial dsrAB sequences of the clones, recovered after isolation and PCR amplification of the community DNA, were related to hitherto uncultured species of the genera Desulfovibrio and Desulfobulbus. Nevertheless, the representative of the second largest clone group was shown to be closely affiliated with the sequenced dsrAB gene of a strain isolated from the same environment and identified as Desulfovibrio alcoholivorans. Another clone sequence was closely related to a possible novel species also isolated within the scope of this work.     

Enrichment for Estuarine Petroleum-Degrading Bacteria using Liquid and Solid Media

Bacteria in oil-contaminated and oil-free estuarine water and sediment were isolated on solid and in liquid petroleum media. A greater variety of bacteria was noted when direct plating techniques were employed, compared with liquid enrichment. In addition, strains isolated by liquid enrichment were found more frequently to be capable of utilizing model petroleum in vitro , compared with isolates obtained by direct plating.     

Ecology and taxonomy of chitinoclasticCytophaga and related chitin-degrading bacteria isolated from an estuary

A total of 103 strains of estuarine, Chitinoclastic bacteria isolated from water, and sediment samples collected from the upper Chesapeake Bay, including 17 freshwater and 11 seawater isolates, were subjected to numerical taxonomy analysis. The isolates included 44 yellow-orange pigmented strains classified asCytophaga-like bacteria (CLB) of theCytophagaceae. Salt requirement of the strains ranged from tolerance to 1% NaCl to an absolute requirement for NaCl, with 1% NaCl satisfying this requirement. The largest phenon consisted of facultatively anaerobic, oligo-nitrophilic, and flexirubin pigment-producing freshwater and estuarine isolates, and included reference strains of bothCytophaga johnsonae Stanier andCytophaga aquatilis Strohl and Tait. Other phena, containing a smaller number of strains, comprised marine and estuarine isolates which did not produce flexirubin pigments, and required organic nitrogen for growth and for production of chitinolytic enzymes. Salt-requiring, flexirubin pigment-producing, chitin-degrading strains were, on occasion, isolated from estuarine samples and represented phena found in estuaries. Most of theCytophaga isolates, as well as chitin-degrading species not of the genusCytophaga that were isolated from Chesapeake Bay, clustered in phena representing previously described species of aerobic, zymogenic, chitinoclastic bacteria. When the frequency of occurrence of features related to environmental parameters, viz., pH, salinity, temperature range of growth, and growth on media lacking organic nitrogen, was calculated, ecological groupings of strains in the 2 major phena of CLB could be distinguished among the estuarine, chitin-degrading bacteria.     

Desulfolutivibrio sulfoxidireducens gen. nov., sp. nov., isolated from a pyrite-forming enrichment culture and reclassification of Desulfovibrio sulfodismutans as Desulfolutivibrio sulfodismutans comb. nov

Two strains of sulfate-reducing bacteria (J.5.4.2-L4.2.8^T and J.3.6.1-H7) were isolated from a pyrite-forming enrichment culture and were compared phylogenetically and physiologically to the closest related type strain Desulfovibrio sulfodismutans DSM 3696^T. The isolated strains were vibrio-shaped, motile rods that stained Gram-negative. Growth occurred from 15 to 37 °C and within a pH range of 6.5–8.5. Both strains used sulfate, thiosulfate, sulfite, and dimethyl sulfoxide (DMSO) as electron acceptor when grown with lactate. Lactate was incompletely oxidized to acetate. Formate and H₂ were used as electron donor in the presence of acetate. Dismutation of thiosulfate and pyrosulfite was observed. The two new isolates differed from D. sulfodismutans by the utilization of DMSO as electron acceptor, 82% genome-wide average nucleotide identity (ANI) and 32% digital DNA-DNA hybridization (dDDH), thus representing a novel species. The type strain of the type species Desulfovibrio desulfuricans Essex6^T revealed merely 88% 16S rRNA gene identity and 49% genome-wide average amino acid identity (AAI) to the new isolates as well as to D. sulfodismutans. Furthermore, the dominance of menaquinone MK-7 over MK-6 and the dominance of ai-C_15:0 fatty acids were observed not only in the two new isolated strains but also in D. sulfodismutans. Therefore, the definition of a new genus is indicated for which the name Desulfolutivibrio is proposed. We propose for strains J.5.4.2-L4.2.8^T and J.3.6.1-H7 the name Desulfolutivibrio sulfoxidireducens gen. nov. sp. nov. with strain J.5.4.2-L4.2.8^T defined as type strain. In addition, we propose the reclassification of Desulfovibrio sulfodismutans as Desulfolutivibrio sulfodismutans comb. nov.     

Evaluation of arbitrarily primed PCR for typing of Desulfovibrio desulfuricans strains

Arbitrarily primed polymerase chain reaction (AP-PCR) method was applied to the differentiation of 15 (soil and intestinal) Desulfovibrio desulfuricans strains. The primer M 13, which is a core sequence of phage M 13, was found to be appropriate for the differentiation of isolates of this species. The analysis revealed characteristic band patterns for all of the examined strains of which two soil strains (DV-7 and DV-8) showed identical DNA fingerprints. According to Jaccard's coefficient, the soil bacterial group as well as intestinal bacterial group formed two different clusters. Furthermore, the soil strains showed greater variability than the intestinal isolates. Based on the AP-PCR fingerprints D. desulfuricans strains were differentiated depending on their origin. This study demonstrates that the typing method AP-PCR can be useful in epidemiologic investigations as a rapid and valuable tool for differentiation of the strains of D. desulfuricans species.     

Isolation and characterization of sulphate-reducing bacteria <Emphasis Type="Italic">Desulfovibrio vulgaris</Emphasis> from Vajreshwari thermal springs in Maharashtra,India

Sulphate-reducing bacteria (SRB) in the thermal springs of Vajreshwari were investigated with combined microbiological and molecular approaches. A sulphate-reducing bacteria medium containing lactate was used for enrichment and isolation, which yielded Gram negative, rod shaped, anaerobic, non-spore forming and motile bacteria capable of reducing sulphate to sulphide. These grew at temperatures ranging from 25 to 55 °C and could use pyruvate, lactate and ethanol as electron donors. Desulfoviridin was detected in all the isolates. The partial 16S rRNA and dissimilatory sulphite reductase (DSR) gene sequences of five representative isolates revealed that the strains belonged to the sulphur reducing bacterial species Desulfovibrio vulgaris.     

Activity of sulfate-reducing bacteria in human periodontal pocket

Samples of subgingival dental tissues were examined for the presence of sulfate-reducing bacteria (SRB). Using enrichment cultures, SRBs were detected in 9 of 17 individuals. A pure culture of SRB was obtained from one sample collected from a patient with type IV periodontal disease. The characterization of this isolate showed that it belongs to the genus Desulfovibrio. The isolate used pyruvate, lactate, glucose, fructose, and ethanol as the sole source of carbon. However, the isolate was unable to use acetate and methanol as a carbon source, indicating it as an incomplete oxidizer unable to carry out the terminal oxidation of substrates. Apart from using sulfate as electron acceptor, the isolate also used thiosulfate and nitrate as an electron acceptor. It has the ability to use a variety of nitrogen sources, including ammonium chloride, nitrate, and glutamate. The optimum growth temperature of the isolate was 37 degrees C and the optimum pH for growth was 6.8. The SRB isolate contained the electron carrier desulfoviridin. The numbers of SRB in the mouth are assumed to be limited by sulfate. Potential sources of sulfate in the subgingival area include free sulfate in pocket fluid and glycosaminoglycans and sulfur-containing amino acids from periodontal tissues.     

Diversity of bacteria and Archaea in sulphate-reducing enrichment cultures inoculated from serial dilution of Zostera noltii rhizosphere samples

We have analysed the diversity of culturable sulphate-reducing bacteria (SRB) in Zostera noltii colonized sediments from Bassin d'Arcachon (France). Four organic substrates have been tested as well as the combination of H2 and CO2 to select for lithotrophic SRB. All energy sources were supplied in parallel cultures that were amended with yeast extract plus NH4+ and prepared without a source of combined nitrogen, the latter to select for diazotrophic SRB. The 10 different enrichment media were inoculated from serial dilution of rhizosphere samples. The highest dilution cultures yielding positive growth (i.e. 10-7) were studied by molecular techniques (16S rDNA clone libraries, RISA and ARDRA). Lactate as a single organic substrate in combination with a source of combined nitrogen resulted in selection of members of the Desulfovibrionaceae. Surprisingly, when lactate was added without a source of combined nitrogen, Desulfobacteriaceae were selected. A strong influence of the presence or absence of combined nitrogen was also observed for the substrates sucrose and fructose. Whereas the liquid culture growing on sucrose and NH4+ systematically yielded 16S rDNA clones related to an environmental unidentified green sulphur bacterium (OPS185), on plates we were able to isolate a SRB related to Desulfovibrio dechloracetivorans, which likely represents a non-described species. Under diazotrophic conditions, sucrose selected for SRB clones related to the cluster formed by Desulfovibrio zosterae, Desulfovibrio salexigens and Desulfovibrio bastinii. The corresponding isolate obtained on plates showed only low sequence similarity with this closest neighbour (93.8%), and we suggest that it also represents a non-described species. Surprisingly, a 16S rDNA sequence corresponding to an archaeon, i.e. a non-extremophile Crenoarchaeota, was retrieved from several of the SRB enrichment cultures even after subsequent transfers.     

Isolation of sulfate-reducing bacteria from the terrestrial deep subsurface and description of Desulfovibrio cavernae sp. nov

Deep subsurface sandstones in the area of Berlin (Germany) located 600 to 1060 m below the surface were examined for the presence of viable microorganisms. The in situ temperatures at the sampling sites ranged from 37 to 45 degrees C. Investigations focussed on sulfate-reducing bacteria able to grow on methanol and triethylene glycol, which are added as chemicals to facilitate the long-term underground storage of natural gas. Seven strains were isolated from porewater brines in the porous sandstone. Three of them were obtained with methanol (strains H1M, H3M, and B1M), three strains with triethylene glycol (strains H1T, B1T, and B2T) and one strain with a mixture of lactate, acetate and butyrate (strain H1-13). Due to phenotypic properties six isolates could be identified as members of the genus Desulfovibrio, and strain B2T as a Desulfotomaculum. The salt tolerance and temperature range for growth indicated that the isolates originated from the indigenous deep subsurface sandstones. They grew in mineral media reflecting the in situ ionic composition of the different brines, which contained 1.5 to 190 g NaCl x l(-1) and high calcium and magnesium concentrations. The Desulfovibrio strains grew at temperatures between 20 and 50 degrees C, while the Desulfotomaculum strain was thermophilic and grew between 30 and 65 degrees C. The strains utilized a broad spectrum of electron donors and acceptors. They grew with carbon compounds like lactate, pyruvate, formate, n-alcohols (C1-C5), glycerol, ethylene glycol, malate, succinate, and fumarate. Some strains even utilized glucose as electron donor and carbon source. All strains were able to use sulfate, sulfite and nitrate as electron acceptors. Additionally, three Desulfovibrio strains reduced manganese oxide, the Desulfotomaculum strain reduced manganese oxide, iron oxide, and elemental sulfur. The 16S rRNA analysis revealed that the isolates belong to three different species. The strains H1T, H3M and B1M could be identified as Desulfovibrio indonesiensis, and strain B2T as Desulfotomaculum geothermicum. The other Desulfovibrio strains (H1M, H1-13, and B1T) showed identical 16S rDNA sequences and similarities as low as 93% to their closest relative, Desulfovibrio aminophilusT. Therefore, these isolates were assigned to a new species, Desulfovibrio cavernae sp. nov., with strain H1M as the type strain.     

Anaerobic oxidation of o-xylene, m-xylene, and homologous alkylbenzenes by new types of sulfate-reducing bacteria

Various alkylbenzenes were depleted during growth of an anaerobic, sulfate-reducing enrichment culture with crude oil as the only source of organic substrates. From this culture, two new types of mesophilic, rod-shaped sulfate-reducing bacteria, strains oXyS1 and mXyS1, were isolated with o-xylene and m-xylene, respectively, as organic substrates. Sequence analyses of 16S rRNA genes revealed that the isolates affiliated with known completely oxidizing sulfate-reducing bacteria of the delta subclass of the class Proteobacteria. Strain oXyS1 showed the highest similarities to Desulfobacterium cetonicum and Desulfosarcina variabilis (similarity values, 98.4 and 98.7%, respectively). Strain mXyS1 was less closely related to known species, the closest relative being Desulfococcus multivorans (similarity value, 86.9%). Complete mineralization of o-xylene and m-xylene was demonstrated in quantitative growth experiments. Strain oXyS1 was able to utilize toluene, o-ethyltoluene, benzoate, and o-methylbenzoate in addition to o-xylene. Strain mXyS1 oxidized toluene, m-ethyltoluene, m-isoproyltoluene, benzoate, and m-methylbenzoate in addition to m-xylene. Strain oXyS1 did not utilize m-alkyltoluenes, whereas strain mXyS1 did not utilize o-alkyltoluenes. Like the enrichment culture, both isolates grew anaerobically on crude oil with concomitant reduction of sulfate to sulfide.     

Microbial communities in the saturated groundwater environment II: Diversity of bacterial communities in a Pleistocene sand aquifer and their in vitro activities

Bacterial cell numbers obtained from 103 water and sediment samples from a Pleistocene sandy aquifer in the Lower Rhine region (Bocholt, FRG) were determinated on P-agar and by direct count. Below 5 m under the surface, colony-forming unit (cfu) numbers in water samples were less than 100/ml, and in many cases less than 50/ml. In sediment samples, they were 10- to 100-fold higher (10²–10⁴ cfu/g dry wt), but changing markedly between different depths. Direct cell counts yielded numbers two to three orders of magnitude higher.About 2,700 strains of bacteria from 60 samples were isolated randomly and characterized by morphological and physiological properties. Of all the isolates, 71.6% were gram-negative, and 52.2% were gram-negative straight rods. Water communities, with one exception, had low proportions of gram-positive bacteria (<11%), whereas in all but one of the sediment communities percentages of gram-positive isolates were three- to sevenfold higher (35–43%). Water and sediment communities, as well as communities from different sampling sites and communities from different depths of the same sampling site, differed in their qualitative and quantitative morphotype composition and physiological capabilities.The in vitro activities of strains within a single community were quite different, indicating that each community is composed of many diverse bacteria, several having extremely different capabilities. Thus, each community has its own specific activity pattern. Gram-positive bacteria showed on an average lower total activities than did gram-negative bacteria. Grampositive bacteria as well as gram-negative bacteria from sediment had higher values of in vitro activities than the corresponding groups isolated from water. Many water and sediment bacteria preferred the same substrates which were utilized at high rates. However, there were differences in the degradation of the various other substrates present, and each community showed preferences for particular substrates, which they degraded best.The results of cell morphology and physiology studies indicated that all eight characterized communities were very different from one another and very diversely structured.     

环己酮降解细菌的筛选和系统发育分析

通过以环己酮为唯一碳源的选择培养基富集培养和细菌环己酮降解能力的测定,从巴陵石化公司环己酮生产车间排水口的污泥样品中分离到12株降解环己酮性能强的细菌菌株。根据形态观察、部分生理生化试验和16SrRNA基因序列的比对分析,初步确定这些菌株代表8个物种,属于3个大的系统发育类群/门(Actinobacteria,Proteobacteria,Bacteroidetes)的5个科、7个属;大多数菌株与其系统发育关系最密切的典型菌株之间存在一定的遗传差异。结果表明降解环己酮性能强的细菌具有较丰富的系统发育多样性。     

Genetic Diversity among Thermophilic Bacteria Isolated from Geothermal Sites by Using Two PCR Typing Methods

Microbial communities thriving at two hot springs, Hammam Pharaon (Pharaoh's Bath) and Oyoun Mossa (Moses springs), in Egypt was studied by cultural and molecular methods. Thirteen morphologically distinct strains of facultative anaerobic thermophilic bacterial isolates have been characterized and identified using phenotypic and genotypic characters including RAPD-PCR, ERIC-PCR typing, plasmid analysis and 16S rRNA sequencing. All isolates produced plasmid DNA with various sizes ranging from 0.7 kb to a larger plasmid 7.2 kb. The bacterial strains could tolerate a temperature range between 45 to 85°C and a pH between 4–11. Also, sulphate-reducing bacteria (SRB) in the thermal springs were investigated with combined biochemical and molecular approaches. A sulphate-reducing bacteria medium containing lactate was used for enrichment and isolation, which yielded Gram negative, rod shaped, anaerobic, non-spore-forming and motile bacteria capable of reducing sulphate to sulphide. These grew at temperatures ranging from 30 to 50°C and could use pyruvate, lactate and ethanol as electron donors. The dissimilatory sulphite reductase (DSR) gene sequences of eleven representative isolates revealed that the strains belonged to the sulphur reducing bacterial species Desulfovibrio vulgaris. 16S rRNA gene partial sequence results indicated the presence of novel or existing species of Bacillus (one species), Anoxybacillus (four species) and Geobacillus (eight species). In this study phenotypic and genotypic diversity were applied for the first time to differentiate thermophilic bacteria of such geothermal sites in Sinai, Egypt.     

Growth of sulfate-reducing bacteria with sulfur as electron acceptor

In addition to three new isolates, six strains of representative species of sulfate-reducing bacteria were tested for their capacity to use elemental sulfur as an electron acceptor for growth. There was good growth and sulfide production by strain Norway 4 and the three isolates, two of which had been enriched with sulfur flower and one isolated from a culture with green sulfur bacteria. Slow but definite growth was observed with Desuflovibrio gigas. The type strains of Desulfovibrio desulfuricans, D. vulgaris, and Desulfotomaculum nigrificans as well as Desulfomonas pigra did not grow with sulfur. The four strains that grew well with sulfur flower were straight, nonsporulating rods and did not contain desulfoviridin.     

Properties of Desulfovibrio carbinolicus sp. nov. and Other Sulfate-Reducing Bacteria Isolated from an Anaerobic-Purification Plant

Several sulfate-reducing microorganisms were isolated from an anaerobic-purification plant. Four strains were classified as Desulfovibrio desulfuricans, Desulfovibrio sapovorans, Desulfobulbus propionicus, and Desulfovibrio sp. The D. sapovorans strain contained poly-beta-hydroxybutyrate granules and seemed to form extracellular vesicles. A fifth isolate, Desulfovibrio sp. strain EDK82, was a gram-negative, non-spore-forming, nonmotile, curved organism. It was able to oxidize several substrates, including methanol. Sulfate, sulfite, thiosulfate, and sulfur were utilized as electron acceptors. Pyruvate, fumarate, malate, and glycerol could be fermented. Because strain EDK82 could not be ascribed to any of the existing species, a new species, Desulfovibrio carbinolicus, is proposed. The doubling times of the isolates were determined on several substrates. Molecular hydrogen, lactate, propionate, and ethanol yielded the shortest doubling times (3.0 to 6.3 h). Due to the presence of support material in an anaerobic filter system, these species were able to convert sulfate to sulfide very effectively at a hydraulic retention time as short as 0.5 h.     

Characterization of sulfate-reducing bacteria isolated from Senegal ricefields

Abstract Sulfate-reducing bacteria were enumerated in soils and water samples from Senegal ricefields using lactate and sulfate as substrates. When rice plants were severely injured by sulfides, maximum densities ranged from 10⁷ to 10⁹ bacteria g⁻¹ of dry spermosphere or rhizosphere soil. Seven non-sporulating, mesophilic strains were isolated. The strains had motile curved cells and stained Gram-negative. Lactate, pyruvate, H₂+ CO₂, malate, fumarate, or ethanol could serve as electron donors. Organic acids were incompletely oxidized to acetate. Alcohols were degraded to the corresponding fatty acids. Sulfate, sulfite, or thiosulfate could serve as electron acceptors and were reduced to sulfide. Vitamins, yeast extract, Biotrypcase, or additional NaCl were not required for growth. On the basis of morphological and physiological properties, and the G + C mol % of the DNA, six isolates were identified as Desulfovibrio vulgaris and one as Desulfovibrio desulfuricans . The comparison of their main physiological properties with the physico-chemical properties of sampling sites indicated that they were better adapted to conditions prevailing in the rice rhizosphere than to those prevailing in the bulk of soil.     

Gas vacuolate bacteria obtained from marine waters of Antarctica

Several strains of heterotrophic, gas vacuolate bacteria were isolated from marine waters of Antarctica. To our knowledge these are the first marine forms of gas vacuolate bacteria to be reported. Current isolates are all Gram-negative rods. All isolates are psychrophiles that grow at temperatures between –1.5°C and 7°C, with none growing at temperatures higher than 14°C. All can grow at salt concentrations of sea water, although some can grow at 1/16th that concentration. Two of the strains produce orange colored pigments, whereas all heterotrophic gas vacuolate bacteria known to date are nonpigmented. The two pigmented filamentous strains grow well at 5.0% NaCl and have a specific sodium ion requirement. Isolates differ in the substrates they use for growth. Organic acids, amino acids, and sugars are used depending upon the strain. All isolates appear to be microaerophilic and oxidase and catalase positive. All grow well at pH 7.0. The mol% G+C of the pigmented strains is 31 and that of the non-pigmented strains, 52–57.     

The reductive dechlorination of 2,3,4,5-tetrachlorobiphenyl in three different sediment cultures: evidence for the involvement of phylogenetically similar Dehalococcoides-like bacterial populations

Anaerobic cultures capable of reductively dechlorinating 2,3,4,5-tetrachlorobiphenyl (CB) were enriched from three different sediments, one estuarine, one marine and one riverine. Two different electron donors were used in enrichments with the estuarine sediment (elemental iron or a mixture of fatty acids). The removal of doubly flanked meta and para chlorines to form 2,3,5-CB and 2,4,5-CB was observed in all cultures. Bacterial community analysis of PCR-amplified 16S rRNA gene fragments revealed different communities in these cultures, with the exception of one common population that showed a high phylogentic relatedness to Dehalococcoides species. No Dehalococcoides-like populations were ever detected in control cultures to which no PCBs were added. In addition, the dynamics of this Dehalococcoides-like population were strongly correlated with dechlorination. Subcultures of the estuarine sediment culture demonstrated that the Dehalococcoides-like population disappeared when dechlorination was inhibited with 2-bromoethanesulfonate or when 2,3,4,5-CB had been consumed. These results provide evidence that Dehalococcoides-like populations were involved in the removal of doubly flanked chlorines from 2,3,4,5-CB. Furthermore, the successful enrichment of these populations from geographically distant and geochemically distinct environments indicates the widespread presence of these PCB-dechlorinating, Dehalococcoides-like organisms.     

Degradative capacities and 16S rRNA-targeted whole-cell hybridization of sulfate-reducing bacteria in an anaerobic enrichment culture utilizing alkylbenzenes from crude oil.

A mesophilic sulfate-reducing enrichment culture growing anaerobically on crude oil was used as a model system to study which nutritional types of sulfate-reducing bacteria may develop on original petroleum constituents in oil wells, tanks, and pipelines. Chemical analysis of oil hydrocarbons during growth revealed depletion of toluene and o-xylene within 1 month and of m-xylene, o-ethyltoluene, m-ethyltoluene, m-propyltoluene, and m-isopropyltoluene within approximately 2 months. In anaerobic counting series, the highest numbers of CFU (6 x 10(6) to 8 x 10(6) CFU ml-1) were obtained with toluene and benzoate. Almost the same numbers were obtained with lactate, a substrate often used for detection of the vibrio-shaped, incompletely oxidizing Desulfovibrio sp. In the present study, however, lactate yielded mostly colonies of oval to rod-shaped, completely oxidizing, sulfate-reducing bacteria which were able to grow slowly on toluene or crude oil. Desulfovibrio species were detected only at low numbers (3 x 10(5) CFU ml-1). In agreement with this finding, a fluorescently labeled, 16S rRNA-targeted oligonucleotide probe described in the literature as specific for members of the Desulfovibrionaceae (suggested family) hybridized only with a small portion (< 5%) of the cells in the enrichment culture. These results are consistent with the observation that known Desulfovibrio species do not utilize aromatic hydrocarbons, the predominant substrates in the enrichment culture. All known sulfate-reducing bacteria which utilize aromatic compounds belong to a separate branch, the Desulfobacteriaceae (suggested family). Most members of this family are complete oxidizers. For specific hybridization with members of this branch, the probe had to be modified by a nucleotide exchange. Indeed, this modified probe hybridized with more than 95% of the cells in the enrichment culture. The results show that completely oxidizing, alkylbenzene-utilizing sulfate-reducing bacteria rather than Desulfovibrio species have to be considered in attempts to understand the microbiology of sulfide production in oil wells, tanks, and pipelines when no electron donors other than the indigenous oil constituents are available.     

Identification and characterization of humic substances-degrading bacterial isolates from an estuarine environment

Bacterial isolates were obtained from enrichment cultures containing humic substances extracted from estuarine water using an XAD-8 resin. Eighteen isolates were chosen for phylogenetic and physiological characterization based on numerical importance in serial dilutions of the enrichment culture and unique colony morphology. Partial sequences of the 16S rRNA genes indicated that six of the isolates were associated with the alpha subclass of Proteobacteria, three with the gamma-Proteobacteria, and nine with the Gram-positive bacteria. Ten isolates degraded at least one (and up to six) selected aromatic single-ring compounds. Six isolates showed ability to degrade [(14)C]humic substances derived from the dominant salt marsh grass in the estuary from which they were isolated (Spartina alterniflora), mineralizing 0.4-1.1% of the humic substances over 4 weeks. A mixture of all 18 isolates did not degrade humic substances significantly faster than any of the individual strains, however, and no isolate degraded humic substances to the same extent as the natural marine bacterial community (3.0%). Similar studies with a radiolabeled synthetic lignin ([beta-(14)C]dehydropolymerisate) showed measurable levels of degradation by all 18 bacteria (3.0-8.8% in 4 weeks), but mineralization levels were again lower than that observed for the natural marine bacterial community (28.2%). Metabolic capabilities of the 18 isolates were highly variable and generally did not map to phylogenetic affiliation.