Kjeld Ingvorsen

Prokaryotic Community Structure and Sulfate Reducer Activity in Water from High-Temperature Oil Reservoirs with and without Nitrate Treatment

ABSTRACT Sulfate-reducing prokaryotes (SRP) cause severe problems like microbial corrosion and reservoir souring in seawater-injected oil production systems. One strategy to control SRP activity is the addition of nitrate to the injection water. Production waters from two adjacent, hot (80°C) oil reservoirs, one with and one without nitrate treatment, were compared for prokaryotic community structure and activity of SRP. Bacterial and archaeal 16S rRNA gene analyses revealed higher prokaryotic abundance but lower diversity for the nitrate-treated field. The 16S rRNA gene clone libraries from both fields were dominated by sequences affiliated with Firmicutes (Bacteria) and Thermococcales (Archaea). Potential heterotrophic nitrate reducers (Deferribacterales) were exclusively found at the nitrate-treated field, possibly stimulated by nitrate addition. Quantitative PCR of dsrAB genes revealed that archaeal SRP (Archaeoglobus) dominated the SRP communities, but with lower relative abundance at the nitrate-treated site. Bacterial SRP were found in only low abundance at both sites and were nearly exclusively affiliated with thermophilic genera (Desulfacinum and Desulfotomaculum). Despite the high abundance of archaeal SRP, no archaeal SRP activity was detected in [35S]sulfate incubations at 80°C. Sulfate reduction was found at 60°C in samples from the untreated field and accompanied by the growth of thermophilic bacterial SRP in batch cultures. Samples from the nitrate-treated field generally lacked SRP activity. These results indicate that (i) Archaeoglobus can be a major player in hot oil reservoirs, and (ii) nitrate may act in souring control—not only by inhibiting SRP, but also by changing the overall community structure, including the stimulation of competitive nitrate reducers.

Kinetics of sulfate uptake by freshwater and marine species ofDesulfovibrio

Apparent half-saturation constants (Km) and maximum uptake rates (Vmax) for sulfate were determined in four species ofDesulfovibrio of freshwater and marine origin using a35S-sulfate tracer technique. The lowerstKm (5 μM) was found in the freshwater speciesDesulfovibrio vulgaris (Marburg) and the highestKm (77 μM) in the marine speciesDesulfovibrio salexigens. Maximum specific rates of sulfate uptake (i.e.,Vmax) were proportional to the growth rates observed in batch cultures. The halophilicDesulfovibrio salexigens did not change itsKm andVmax between 1 and 6,000 μM SO42-, and apparently did not induce a low-affinity uptake system at high sulfate concentrations. The low half-saturation constants measured for sulfate uptake explain why high rates of bacterial sulfate reduction occur in surface sediments of freshwater lakes, and why sulfate reduction can be a quantitatively important process in anaerobic carbon mineralization in low-sulfate environments. The results shows that extremely low sulfate concentrations must occur before sulfate reduction is completely outcompeted by methanogenesis.

Halorhabdus utahensis gen. nov., sp. nov., an aerobic, extremely halophilic member of the Archaea from Great Salt Lake, Utah

Strain AX-2T (T = type strain) was isolated from sediment of Great Salt Lake, Utah, USA. Optimal salinity for growth was 27% (w/v) NaCl and only a few carbohydrates supported growth of the strain. Strain AX-2T did not grow on complex substrates such as yeast extract or peptone. 16S rRNA analysis revealed that strain AX-2T was a member of the phyletic group defined by the family Halobacteriaceae, but there was a low degree of similarity to other members of this family. The polar lipid composition comprising phosphatidyl glycerol, the methylated derivative of diphosphatidyl glycerol, triglycosyl diethers and sulfated triglycosyl diethers, but not phosphatidyl glycerosulfate, was not identical to that of any other aerobic, halophilic species. On the basis of the data presented, it is proposed that strain AX-2T should be placed in a new taxon, for which the name Halorhabdus utahensis is appropriate. The type strain is strain AX-2T (= DSM 12940T).

Sulfate Reduction Dynamics and Enumeration of Sulfate-Reducing Bacteria in Hypersaline Sediments of the Great Salt Lake (Utah, USA)

Bacterial sulfate reduction activity (SRA) was measured in surface sediments and slurries from three sites in the Great Salt Lake (Utah, USA) using radiolabeled 35S-sulfate. High rates of sulfate reduction (363 ± 103 and 6,131 ± 835 nmol cm-3 d-1) were measured at two sites in the moderately hypersaline southern arm of the lake, whereas significantly lower rates (32 ± 9 nmol cm-3 d-1) were measured in the extremely hypersaline northern arm. Bacterial sulfate reduction was strongly affected by salinity and showed an optimum around 5-6% NaCl in the southern arm and an optimum of around 12% NaCl in the more hypersaline northern arm of the lake. High densities of sulfate-reducing bacteria (SRB) ranging from 2.2 × 107 to 6.7 × 108 cells cm-3 were determined by a newly developed tracer MPN-technique (T-MPN) employing sediment media and 35S-sulfate. Calculation of specific sulfate reduction rates yielded values comparable to those obtained in pure cultures of SRB. However, when using a conventional MPN technique with synthetic media containing high amounts of Fe(II), the numbers of SRB were underestimated by 1-4 orders of magnitude as compared to the T-MPN method. Our results suggest that high densities of slightly to moderately halophilic and extremely halotolerant SRB are responsible for the high rates of sulfate reduction measured in Great Salt Lake sediments.

Evaluation of Three Automated Genome Annotations for Halorhabdus utahensis

Genome annotations are accumulating rapidly and depend heavily on automated annotation systems. Many genome centers offer annotation systems but no one has compared their output in a systematic way to determine accuracy and inherent errors. Errors in the annotations are routinely deposited in databases such as NCBI and used to validate subsequent annotation errors. We submitted the genome sequence of halophilic archaeon Halorhabdus utahensis to be analyzed by three genome annotation services. We have examined the output from each service in a variety of ways in order to compare the methodology and effectiveness of the annotations, as well as to explore the genes, pathways, and physiology of the previously unannotated genome. The annotation services differ considerably in gene calls, features, and ease of use. We had to manually identify the origin of replication and the species-specific consensus ribosome-binding site. Additionally, we conducted laboratory experiments to test H. utahensis growth and enzyme activity. Current annotation practices need to improve in order to more accurately reflect a genomes biological potential. We make specific recommendations that could improve the quality of microbial annotation projects.

Congruent Phylogenies of Most Common Small-Subunit rRNA and Dissimilatory Sulfite Reductase Gene Sequences Retrieved from Estuarine Sediments

ABSTRACT The diversity of sulfate-reducing bacteria (SRB) in brackish sediment was investigated using small-subunit rRNA and dissimilatory sulfite reductase (DSR) gene clone libraries and cultivation. The phylogenetic affiliation of the most commonly retrieved clones for both genes was strikingly similar and producedDesulfosarcina variabilis-like sequences from the inoculum but Desulfomicrobium baculatum-like sequences from a high dilution in natural media. Related organisms were subsequently cultivated from the site. PCR bias appear to be limited (or very similar) for the two primersets and target genes. However, the DSR primers showed a much higher phylogenetic specificity. DSR gene analysis is thus a promising and specific approach for investigating SRB diversity in complex habitats.

Desulfobacter halotolerans sp. nov., a halotolerant acetate-oxidizing sulfate-reducing bacterium isolated from sediments of Great Salt Lake, Utah

Summary Dissimilatory acetate-oxidizing sulfate-reducing bacteria were enriched from hypersaline sediments of the Great Salt Lake (Utah, USA) in a synthetic medium. Three mesophilic rod-shaped strains were isolated from deep-agar dilution series, and one strain (GSL-Ac1) was chosen for further studies. Strain GSL-Ac1 showed a high degree of salt tolerance (up to 13% NaCl and 4.5% MgCl 2 · 6H 2 O), but displayed optimum growth with only 1–2% NaCl in the medium. 16S rDNA sequencing placed GSL-Acl in the genus Desulfobacter . On the basis of DNA-DNA hybridization studies and physiological differences, we describe strain GSL-Acl as a new species, Desulfobacter halotolerans sp. nov. Strain GSL-Acl showed the highest NaCl-tolerance reported for any member of the genus Desulfobacter .

Xylanase production by a novel halophilic bacterium increased 20-fold by response surface methodology

Abstract Medium optimisation for a novel halophilic eubacterium, strain SX15, resulted in a 20-fold increase of extracellular xylanase activity. This facilitates the purification of xylanase produced by this strain. Prior experiments revealed that xylan concentration, source and concentration of nitrogen and salinity were important variables for xylanase production. Based on this, we adopted a fractional factorial design to determine the best combinations and approximate levels of these values. Subsequently, response surface (RS) methodology was applied to locate the optimal levels of xylan, NH 4 Cl and salinity.

Nitrile hydrolysis by Rhodococcus erythropolis BL1, an acetonitrile-tolerant strain isolated from a marine sediment

A number of physiologically different nitrile-hydrolysing bacteria were isolated from coastal marine sediments in Denmark by enrichment culture. One strain, BL1, identified as Rhodococcus erythropolis, grew on acetonitrile as sole carbon and nitrogen source in a defined medium. Growth occurred between 0 and 8% NaCl with an optimum around 2%, thus reflecting the marine origin of the isolate. Intact cells of R. erythropolis BL1 could hydrolyse a large variety of saturated and unsaturated aliphatic nitriles to their corresponding acids. Benzonitrile and benzylcyanide were not hydrolysed, whereas some aromatic compounds containing a -CN group attached to a C3 or C4 aliphatic side chain were accepted as substrates. The substrate spectrum of R. erythropolis BL1 was thus markedly different from those of other Grampositive nitrile-hydrolysing bacteria isolated from non-marine environments. Nitrile hydrolysis during growth and in resting cell suspensions usually occurred without intermediate accumulation of amide outside the cells. Detailed studies, however, showed that nitrile hydrolysis by strain BL1 was due to a nitrile hydratase/amidase enzyme system. Nitrile hydratase activity was found to be inducible whereas amidase activity was constitutive. The amidase activity of cells could, however, be enhanced manyfold by growth in media containing acetamide or acetonitrile. In most cases amides were hydrolysed at a much higher rate than the corresponding nitriles, which explained why amides were rarely detected in the surrounding medium during nitrile hydrolysis. R. erythropolis BL1 exhibited the highest tolerance towards acetonitrile ever reported for a nitrile-hydrolysing bacterium, as demonstrated by its ability to grow exponentially in the presence of 900 mM acetonitrile.

Desulfocella halophila gen. nov., sp. nov., a halophilic, fatty-acid-oxidizing, sulfate- reducing bacterium isolated from sediments of the Great Salt Lake

A new halophilic sulfate-reducing bacterium, strain GSL-But2T, was isolated from surface sediment of the Southern arm of the Great Salt Lake, UT, USA. The organism grew with a number of straight-chain fatty acids (C4-C16), 2-methylbutyrate, L-alanine and pyruvate as electron donors. Butyrate was oxidized incompletely to acetate. Sulfate, but not sulfite or thiosulfate, served as an electron acceptor. Growth was observed between 2 and 19% (w/v) NaCl with an optimum at 4-5% (w/v) NaCl. The optimal temperature and pH for growth were around 34 degrees C and pH 6.5-7.3, respectively. The generation time under optimal conditions in defined medium was around 28 h, compared to 20 h in complex medium containing yeast extract. The G+C content was 35.0 mol%. 16S rRNA gene sequence analysis revealed that strain GSL-But2T belongs to the family Desulfobacteriaceae within the delta-subclass of the Proteobacteria and suggested that strain GSL-But2T represents a member of a new genus. The name Desulfocella halophila gen. nov., sp. nov. is proposed for this organism. The type strain of D. halophila is strain GSL-But2T (= DSM 11763T = ATCC 700426T).