Torleiv Lien

Treatment of acid mine water by sulfate-reducing bacteria; results from a bench scale experiment

Overflowing water from waterfilled mines and open pits in pyrite and coal mine areas is often acid and severely polluted by metals and sulfate. The effect of stimulating bacterial sulfate reduction in such systems in order to improve water quality was examined in a bench scale experiment. Polyacrylate cylinders with a sand bed and a layer of crushed stones at the bottom were filled with acid mine water, supplied with whey to support bacterial growth and incubated for 203 d. After an initial lag phase, pH started to increase, redox potentials dropped and dissolved concentrations of copper, zinc, iron and aluminium were reduced in cylinders supplied with whey. Simultaneously, a black sludge was formed. Acetate tended to accumulate as an undesired waste product. The observations indicate that sulfate-reducing bacteria (SRB) became active in the cylinders and contributed to metal removal. Inoculation with SRB shortened the initial lag phase, but was not necessary to initiate the sulfate-reduction process. The in situ treatment concept may be useful in relatively small and well defined mine water systems, where the process can be governed properly.

Desulfotomaculum thermocisternum sp. nov., a Sulfate Reducer Isolated from a Hot North Sea Oil Reservoir

The organism described in this paper, strain ST90T (T = type strain), is a thermophilic, spore-forming, rod-shaped sulfate reducer that was isolated from North Sea oil reservoir formation water. In cultivation the following substances were used as electron donors and carbon sources: H2-CO2, lactate, pyruvate, ethanol, propanol, butanol, and C3 to C10 and C14 to C17 carboxylic acids. Sulfate was used as the electron acceptor in these reactions. Lactate was incompletely oxidized. Sulfite and thiosulfate were also used as electron acceptors. In the absence of an electron acceptor, the organism grew syntrophically on propionate together with a hydrogenothrophic methanogen. The optimum conditions for growth on lactate and sulfate were 62°C, pH 6.7, and 50 to 200 mM NaCI. The G+C content was 56 mol%, as determined by high-performance liquid chromatography and 57 mol% as determined by thermal denaturation. Spore formation was observed when the organism was grown on butyrate or propanol as a substrate and at low pH values. On the basis of differences in G+C content and phenotypic and immunological characteristics when the organism was compared with other thermophilic Desulfotomaculum species, we propose that strain ST90T is a member of a new species, Desulfotomaculum thermocisternum. D. thermocisternum can be quickly identified and distinguished from closely related Desulfotomaculum species by immunoblotting.

Thermodesulforhabdus norvegicus gen. nov., sp. nov., a novel thermophilic sulfate-reducing bacterium from oil field water

A novel gram-negative, thermophilic, acetate-oxidizing, sulfate-reducing bacterium, strain A8444, isolated from hot North Sea oil field water, is described. The rod-shaped cells averaged 1 μm in width and 2.5 μm in length. They were motile by means of a single polar flagellum. Growth was observed between 44 and 74°C, with an optimum at 60°C. Spores were not produced. Sulfate and sulfite were used as electron acceptors. Sulfur, thiosulfate, nitrate, fumarate, and pyruvate were not reduced. In the presence of sulfate, growth was observed with acetate, lactate, pyruvate, butyrate, succinate, malate, fumarate, valerate, caproate, hepatanoate, octanoate, nonadecanoate, decanoate, tridecanoate, pentadecanoate, palmitate, heptadecanoate, stearate, and ethanol. Pyruvate, lactate, and fumarate did not support fermentative growth. Cytochromes of thec-type were present. Desulfoviridin, desulforubidin, P582, and desulfofuscidin were not present. The G+C content of the DNA was 51 mol%. Sequence analysis of 16S rDNA showed that phylogenetically strain A8444 belongs to the delta subdivision of the Proteobacteria. The closest relatives areDesulfacinum infernum andSyntrophobacter wolinii. Strain A8444 is described as the type strain of the new taxonThermodesulforhabdus norvegicus gen. nov., sp. nov.

Petrotoga mobilis sp. nov., from a North Sea oil-production well

Rod-shaped, thermophilic bacteria with a sheath-like outer structure (toga) were isolated from hot oilfield water of a North Sea oil reservoir. One of the isolates, designated SJ95(T), is an obligately anaerobic, sheathed, Gram-negative, fermentative bacterium capable of reducing elemental sulfur to hydrogen sulfide and tolerating high salt concentrations. The optimum growth conditions for this isolate are 58-60 degrees Celsius and pH 6.5-7.0 with 3-4% NaCl and 0.7% MgSO(4). 7H(2)O in the medium. Vitamins are required for growth. Growth is stimulated by yeast extract. Cells of strain SJ95(T) vary in size from 1-2 to 40-50 micron in length and are motile with a subpolar flagellation. Cels grown on xylan have xylanase activity, presumably associated with the toga, and glucose isomerase activity was detected in xylose-grown cells. The DNA G+C content is 31 and 34 mol%, determined by the thermal denaturation and HPLC methods, respectively. Phylogenetically, strain SJ95(T) is most closely related to Petrotoga miotherma with a 97.7% similarity level between their 165 rDNA sequences. The DNA-DNA reassociation value between the two DNAs was 35.6%. On the basis of differences in genotypic, phenotypic and immunological characteristics, strain SJ95t (=DSM 10674t) is proposed as the type strain of a new species, Petrotoga mobilis. It can be readily distinguished from P. miotherma by its motility.

SYNCHRONOUS GROWTH OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE): A REVIEW OF OPTIMAL CONDITIONS1

Chlamydomonas reinhardtii Dangeard was synchronized at optimal growth conditions under a 12:4 LD regime at 35 C and 20,000 lx with serial dilution to a standard starting cell density of (1.4 ± 0.2) × 106 cells/ml. Synchronous growth and division were characterized by measuring cell number, cell volume and size distribution, dry weight, protein, carbon, nitrogen, chlorophyll, carotenoids, nucleic acids, nuclear and cytoplasmic division during the vegetative life cycle. The main properties of the present system are: Exponential growth with high productivity, high degrees of synchrony and reproducibility during repeated life cycles. The degree of synchrony of this light‐dark synchronization system was evaluated and compared with those described in the literature using probit analysis of the time course of DNA synthesis, nuclear and cytoplasmic division and sporulation (increase in cell number). The results showed that the degree of synchrony is highest for cells grown under optimal conditions.

Desulfobulbus rhabdoformis sp. nov., a sulfate reducer from a water-oil separation system

A mesophilic, Gram-negative, rod-shaped, marine, propionate-oxidizing sulfate reducer (strain M16T) was isolated from a water-oil separation system on a North Sea oil platform. The optimum conditions for growth were 31 degrees C, pH 6.8-7.2 and 1.5-2.0% (w/v) NaCl and 0.1-0.3% (w/v) MgCl2.6H2O in the medium. The growth yield with sulfate was 4.6 g cell biomass (mol propionate oxidized)-1. Strain M16T is nutritionally related to members of the genus Desulfobulbus, but differs in that it has no vitamin requirement and is able to utilize fumarate and malate as carbon and energy sources. Hydrogenase activity measured as hydrogen uptake was mainly membrane-bound and varied with the growth substrate. Highest activity [28 mumol min-1 (mg protein)-1] was found in cells grown with hydrogen and lowest [50 nmol min-1 (mg protein)-1] in cells grown with propionate as electron donors for sulfate reduction. Desulforubidin, menaquinone-5(H2) and cytochromes of the c- and b-type were present. The fatty acid pattern was similar to that found for Desulfobulbus propionicus. The DNA base composition was 50.6 mol% G + C. Strain M16T is equidistantly related to D. propionicus and Desulfobulbus elongatus with 96.1% 16S rDNA similarity. On the basis of differences in genotypic, phenotypic and immunological characteristics, strain M16T (= DSM 8777T) is proposed as the type strain of a new species, Desulfobulbus rhabdoformis.

Biochemical and phylogenetic characterization of isocitrate dehydrogenase from a hyperthermophilic archaeon, Archaeoglobus fulgidus.

Abstract A thermostable homodimeric isocitrate dehydrogenase from the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus was purified and characterized. The mol. mass of the isocitrate dehydrogenase subunit was 42 kDa as determined by SDS-PAGE. Following separation by SDS-PAGE, A. fulgidus isocitrate dehydrogenase could be renatured and detected in situ by activity staining. The enzyme showed dual coenzyme specificity with a high preference for NADP+. Optimal temperature for activity was 90° C or above, and a half-life of 22 min was found for the enzyme when incubated at 90° C in a 50 mM Tricine-KOH buffer (pH 8.0). Based on the N-terminal amino acid sequence, the gene encoding the isocitrate dehydrogenase was cloned. DNA sequencing identified the icd gene as an open reading frame encoding a protein of 412 amino acids with a molecular mass corresponding to that determined for the purified enzyme. The deduced amino acid sequence closely resembled that of the isocitrate dehydrogenase from the archaeon Caldococcus noboribetus (59% identity) and bacterial isocitrate dehydrogenases, with 57% identity with isocitrate dehydrogenase from Escherichia coli. All the amino acid residues directly contacting substrate and coenzyme (except Ile-320) in E. coli isocitrate dehydrogenase are conserved in the enzyme from A. fulgidus. The primary structure of A. fulgidus isocitrate dehydrogenase confirmes the presence of Bacteria-type isocitrate dehydrogenases among Archaea. Multiple alignment of all the available amino acid sequences of di- and multimeric isocitrate dehydrogenases from the three domains of life shows that they can be divided into three distinct phylogenetic groups.

Desulfobacter vibrioformis sp. nov., a sulfate reducer from a water-oil separation system

A mesophilic, gram-negative, vibrio-shaped, marine, acetate-oxidizing sulfate reducer (strain B54) was isolated from a water-oil separation system on a North Sea oil platform. The optimum conditions for growth were 33 degrees C, pH 6.8 to 7.0, and concentrations of NaCl and MgCl2.6H2O of at least 1 and 0.3%, respectively. Of various organic acids tested, only acetate was used as an electron and carbon source. The presence of 2-oxoglutarate:dye oxidoreductase suggests acetate oxidation via an operative citric acid cycle. Even though growth of most Desulfobacter strains (including strain B54) did not occur on hydrogen, hydrogenase was detected at low activity. The growth yields were 4.6, 13.1, and 9.6 g of (dry weight) cells per mol of acetate oxidized with sulfate, sulfite, and thiosulfate, respectively, as electron acceptors. Strain B54 was able to fix dinitrogen. Desulforubidin and cytochromes of the c and b types were present. The G+C content of the DNA was 47 mol%. Strain B54 is most closely related to Desulfobacter latus, with a 16S rDNA sequence similarity of 98.1%. The DNA-DNA relatedness between them was 40.5%. On the basis of differences in genotypic, phenotypic, and immunological characteristics, we propose that strain B54 is a member of a new species, D. vibrioformis. It can be easily identified and distinguished from other Desulfobacter species by its large, vibrioshaped cells.

Properties and primary structure of a thermostable L-malate dehydrogenase from Archaeoglobus fulgidus.

Abstract A thermostable l-malate dehydrogenase from the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus was isolated and characterized, and its gene was cloned and sequenced. The enzyme is a homodimer with a molecular mass of 70 kDa and catalyzes preferentially the reduction of oxaloacetic acid with NADH. A. fulgidusl-malate dehydrogenase was stable for 5 h at 90° C, and the half-life at 101° C was 80 min. Thus, A. fulgidusl-malate dehydrogenase is the most thermostable l-malate dehydrogenase characterized to date. Addition of K2HPO4 (1 M) increased the thermal stability by 40%. The primary structure shows a high similarity to l-lactate dehydrogenase from Thermotoga maritima and gram-positive bacteria, and to l-malate dehydrogenase from the archaeon Haloarcula marismortui and other l-lactate-dehydrogenase-like l-malate dehydrogenases.

Purification of a derepressible arylsulfatase from Chlamydomonas reinhardti: Properties of the enzyme in intact cells and in purified state

Arylsulfatase (aryl-sulfate sulfohdydrolase, EC 3.1.6.1) has been purified from SO4-2-minus-starved cells of Chlamydomonas reinhardti. The enzyme was isolated from acetone-powder extract by (NH4)2SO4 precipitation, Sephadex G-200 filtration and ion-exchange chromatography. Only one fraction of aryl-sulfatase was found. The final preparation was homogenous by the criteria of sedimentation, diffusion and polyacrylamide gel electrophoresis. The purified enzyme had a molecular weight of about 150 000, estimated by ultracentrifugation and gel filtration, and an isoelectric point of 9.0. The properties of the enzyme as investigated in intact cells and in the purified state were found to be very similar except for the temperature optimum. Imidazole strongly increased the enzyme by increasing the V, but reduced the affinity for the substrate. The enzyme activity was competitively inhibited by borate with a greater affinity for borate than for the substrate. The Chlamydomonas enzyme is a Type I arylsulfatase since it was inhibited by CN-minus, but not SO4-2-minus and phosphate.