Biocorrosion and biofilm formation in a nutrient limited heating system subjected to alternating microaerophilic conditions.

Severe biofilm formation and biocorrosion have been observed in heating systems even when the water quality complied with existing standards. The coupling between water chemistry, biofilm formation, species composition, and biocorrosion in a heating system was investigated by adding low concentrations of nutrients and oxygen under continuous and alternating dosing regimes. Molecular analysis of 16S rRNA gene fragments demonstrated that the amendments did not cause changes in the overall bacterial community composition. The combined alternating dosing of nutrients and oxygen caused increased rates of pitting (bio-) corrosion. Detection of bacteria involved in sulfide production and oxidation by retrieval of the functional dsrAB and apsA genes revealed the presence of Gram-positive sulfate- and sulfite-reducers and an unknown sulfur-oxidizer. Therefore, to control biocorrosion, sources of oxygen and nutrients must be limited, since the effect of the alternating operational conditions apparently is more important than the presence of potentially corrosive biofilm bacteria.

Congruent phylogenies of most common small-subunit rRNA and dissimilatory sulfite reductase gene sequences retrieved from estuarine sediments.

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 produced Desulfosarcina 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.

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 x 107 to 6.7 x 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.

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).

Gracilibacillus gen. nov., with description of Gracilibacillus halotolerans gen. nov., sp. nov.; transfer of Bacillus dipsosauri to Gracilibacillus dipsosauri comb. nov., and Bacillus salexigens to the genus Salibacillus gen. nov., as Salibacillus salexigens comb. nov.

A Gram-positive, extremely halotolerant bacterium was isolated from the Great Salt Lake, Utah, USA. The strain, designated NNT (= DSM 11805T), was strictly aerobic, rod-shaped, motile by peritrichous flagella and spore-forming. Strain NNT grew at salinities of 0-20% (w/v) NaCl. A distinctive feature of strain NNT was its optimal growth in salt-free medium. The polar lipid pattern of strain NNT consisted of phosphatidyl glycerol, diphosphatidyl glycerol and two phospholipids of unknown structure. The G + C content of its DNA was 38 mol%. The morphological, physiological and, particularly, the 16S rDNA sequence data, showed that strain NNT was associated with 'Bacillus group 1'. However, the organisms showing the greatest degree of sequence similarity to strain NNT were members of the genus Halobacillus and the species Marinococcus albus, Virgibacillus pantothenticus, Bacillus salexigens and Bacillus dipsosauri. On the basis of chemotaxonomic data, strain NNT was shown to be chemically most similar to B. salexigens and B. dipsosauri, with the greatest degree of similarity being shown to the latter organism. This was consistent with the 16S rDNA sequence data. Members of the genus Halobacillus comprise a chemically distinct group and can easily be distinguished from all other organisms of 'Bacillus group 1'. On the basis of the 16S rDNA data, chemotaxonomy and the physiology of strain NNT, it is proposed that this organism is a member of a new species, within a new genus, for which the name Gracilibacillus halotolerans is proposed. It is also proposed that B. dipsosauri be transferred to this genus as Gracilibacillus dipsosauri comb. nov. and that B. salexigens be transferred to the genus Salibacillus gen. nov., as Salibacillus salexigens comb. nov. Finally, additional data is provided to support the transfer of Bacillus pantothenticus to the genus Virgibacillus, as Virgibacillus pantothenticus Heyndrickx et al. (1998).

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).

Improved Most-Probable-Number Method To Detect Sulfate-Reducing Bacteria with Natural Media and a Radiotracer

A greatly improved most-probable-number (MPN) method for selective enumeration of sulfate-reducing bacteria (SRB) is described. The method is based on the use of natural media and radiolabeled sulfate (35SO42-). The natural media used consisted of anaerobically prepared sterilized sludge or sediment slurries obtained from sampling sites. The densities of SRB in sediment samples from Kysing Fjord (Denmark) and activated sludge were determined by using a normal MPN (N-MPN) method with synthetic cultivation media and a tracer MPN (T-MPN) method with natural media. The T-MPN method with natural media always yielded significantly higher (100- to 1,000-fold-higher) MPN values than the N-MPN method with synthetic media. The recovery of SRB from environmental samples was investigated by simultaneously measuring sulfate reduction rates (by a 35S-radiotracer method) and bacterial counts by using the T-MPN and N-MPN methods, respectively. When bacterial numbers estimated by the T-MPN method with natural media were used, specific sulfate reduction rates (qSO42-) of 10(-14) to 10(-13) mol of SO42- cell-1 day-1 were calculated, which is within the range of qSO42- values previously reported for pure cultures of SRB (10(-15) to 10(-14) mol of SO42- cell-1 day-1). qSO42- values calculated from N-MPN values obtained with synthetic media were several orders of magnitude higher (2 x 10(-10) to 7 x 10(-10) mol of SO42- cell-1 day-1), showing that viable counts of SRB were seriously underestimated when standard enumeration media were used. Our results demonstrate that the use of natural media results in significant improvements in estimates of the true numbers of SRB in environmental samples.

Novel cyanide-hydrolyzing enzyme from Alcaligenes xylosoxidans subsp. denitrificans.

A cyanide-metabolizing bacterium, strain DF3, isolated from soil was identified as Alcaligenes xylosoxidans subsp. denitrificans. Whole cells and cell extracts of strain DF3 catalyzed hydrolysis of cyanide to formate and ammonia (HCN + 2H2O----HCOOH + NH3) without forming formamide as a free intermediate. The cyanide-hydrolyzing activity was inducibly produced in cells during growth in cyanide-containing media. Cyanate (OCN-) and a wide range of aliphatic and aromatic nitriles were not hydrolyzed by intact cells of A. xylosoxidans subsp. denitrificans DF3. Strain DF3 hydrolyzed cyanide with great efficacy. Thus, by using resting induced cells at a concentration of 11.3 mg (dry weight) per ml, the cyanide concentration could be reduced from 0.97 M (approximately 25,220 ppm) to less than 77 nM (approximately 0.002 ppm) in 55 h. Enzyme purification established that cyanide hydrolysis by A. xylosoxidans subsp. denitrificans DF3 was due to a single intracellular enzyme. The soluble enzyme was purified approximately 160-fold, and the first 25 NH2-terminal amino acids were determined by automated Edman degradation. The molecular mass of the active enzyme (purity, greater than 97% as determined by amino acid sequencing) was estimated to be greater than 300,000 Da. The cyanide-hydrolyzing enzyme of A. xylosoxidans subsp. denitrificans DF3 was tentatively named cyanidase to distinguish it from known nitrilases (EC 3.5.5.1) which act on organic nitriles.

Microbial hydrolysis of organic nitriles and amides.

Nitrile-hydrating enzymes produced by bacteria and fungi catalyse the conversion of a large number of chemically diverse nitriles, including many economically important compounds used industrially for chemical synthesis of amides and acids. This paper presents data on two new, highly different nitrile-hydrolysing enzymes which were isolated in connection with our studies on enzymic nitrile transformations. Particular attention was paid to the enzymes' substrate specificities and sensitivity to substrate/product inhibition. One of our microbial isolates was a Rhodococcus sp. (strain CH5). This strain produces a constitutive hydratase that has a broad substrate spectrum, including aliphatic and aromatic nitriles, mononitriles and dinitriles, hydroxynitriles and amino-nitriles. It also produces a constitutive amidase of equally low substrate specificity. The hydratase/amidase system catalysed the hydrolysis of D,L-aminonitriles into racemic mixtures of amino acids. Strain CH5 is able to produce high concentrations of malonic acid monoamide from malononitrile and malonamide. The other isolate, Alcaligenes sp. (strain I4), can convert high concentrations of cyanoacetate into malonic acid, presumably by means of an aliphatic nitrilase that is specific for cyanoacetate. Enzyme kinetic experiments have shown that this enzyme is very resistant to both substrate and product inhibition.

Kinetics of Sulfate and Acetate Uptake by Desulfobacter postgatei.

The kinetics of sulfate and acetate uptake was studied in the sulfate-reducing bacterium Desulfobacter postgatei (DSM 2034). Kinetic parameters (K(m) and V(max)) were estimated from substrate consumption curves by resting cell suspensions with [S]sulfate and [C]acetate. Both sulfate and acetate consumption followed Michaelis-Menten saturation kinetics. The half-saturation constant (K(m)) for acetate uptake was 70 muM with cells from either long-term sulfate- or long-term acetate-limited chemostat cultures. The average K(m) value for sulfate uptake by D. postgatei was about 200 muM. K(m) values for sulfate uptake did not differ significantly when determined with cells derived either from batch cultures or sulfate- or acetate-limited chemostat cultures. Acetate consumption was observed at acetate concentrations of

Dynamics of bacterial sulfate reduction in a eutrophic lake.

Bacterial sulfate reduction in the surface sediment and the water column of Lake Mendota, Madison, Wis., was studied by using radioactive sulfate (SO(4)). High rates of sulfate reduction were observed at the sediment surface, where the sulfate pool (0.2 mM SO(4)) had a turnover time of 10 to 24 h. Daily sulfate reduction rates in Lake Mendota sediment varied from 50 to 600 nmol of SO(4) cm, depending on temperature and sampling date. Rates of sulfate reduction in the water column were 10 times lower than that for the surface sediment and, on an areal basis, accounted for less than 18% of the total sulfate reduction in the hypolimnion during summer stratification. Rates of bacterial sulfate reduction in the sediment were not sulfate limited at sulfate concentrations greater than 0.1 mM in short-term experiments. Although sulfate reduction seemed to be sulfate limited below 0.1 mM, Michaelis-Menten kinetics were not observed. The optimum temperature (36 to 37 degrees C) for sulfate reduction in the sediment was considerably higher than in situ temperatures (1 to 13 degrees C). The response of sulfate reduction to the addition of various electron donors metabolized by sulfate-reducing bacteria in pure culture was investigated. The degree of stimulation was in this order: H(2) > n-butanol > n-propanol > ethanol > glucose. Acetate and lactate caused no stimulation.