Joan M. Macy

A New Chemolithoautotrophic Arsenite-Oxidizing Bacterium Isolated from a Gold Mine: Phylogenetic, Physiological, and Preliminary Biochemical Studies

ABSTRACT A previously unknown chemolithoautotrophic arsenite-oxidizing bacterium has been isolated from a gold mine in the Northern Territory of Australia. The organism, designated NT-26, was found to be a gram-negative motile rod with two subterminal flagella. In a minimal medium containing only arsenite as the electron donor (5 mM), oxygen as the electron acceptor, and carbon dioxide-bicarbonate as the carbon source, the doubling time for chemolithoautotrophic growth was 7.6 h. Arsenite oxidation was found to be catalyzed by a periplasmic arsenite oxidase (optimum pH, 5.5). Based upon 16S rDNA phylogenetic sequence analysis, NT-26 belongs to theAgrobacterium/Rhizobium branch of the α-Proteobacteria and may represent a new species. This recently discovered organism is the most rapidly growing chemolithoautotrophic arsenite oxidizer known.

Two new arsenate/sulfate-reducing bacteria: mechanisms of arsenate reduction

Abstract Two sulfate-reducing bacteria, which also reduce arsenate, were isolated; both organisms oxidized lactate incompletely to acetate. When using lactate as the electron donor, one of these organisms, Desulfomicrobium strain Ben-RB, rapidly reduced (doubling time = 8 h) 5.1 mM arsenate at the same time it reduced sulfate (9.6 mM). Sulfate reduction was not inhibited by the presence of arsenate. Arsenate could act as the terminal electron acceptor in minimal medium (doubling time = 9 h) in the absence of sulfate. Arsenate was reduced by a membrane-bound enzyme that is either a c-type cytochrome or is associated with such a cytochrome; benzyl-viologen-dependent arsenate reductase activity was greater in cells grown with arsenate/sulfate than in cells grown with sulfate only. The second organism, Desulfovibrio strain Ben-RA, also grew (doubling time = 8 h) while reducing arsenate (3.1 mM) and sulfate (8.3 mM) concomitantly. No evidence was found, however, that this organism is able to grow using arsenate as the terminal electron acceptor. Instead, it appears that arsenate reduction by the Desulfovibrio strain Ben-RA is catalyzed by an arsenate reductase that is encoded by a chromosomally-borne gene shown to be homologous to the arsC gene of the Escherichia coli plasmid, R773 ars system.

The periplasmic nitrite reductase of Thauera selenatis may catalyze the reduction of selenite to elemental selenium

Thauera selenatis grows anaerobically with selenate, nitrate or nitrite as the terminal electron acceptor; use of selenite as an electron acceptor does not support growth. When grown with selenate, the product was selenite; very little of the selenite was further reduced to elemental selenium. When grown in the presence of both selenate and nitrate both electron acceptors were reduced concomitantly; selenite formed during selenate respiration was further reduced to elemental selenium. Mutants lacking the periplasmic nitrite reductase activity were unable to reduce either nitrite or selenite. Mutants possessing higher activity of nitrite reductase than the wild-type, reduced nitrite and selenite more rapidly than the wild-type. Apparently, the nitrite reductase (or a component of the nitrite respiratory system) is involved in catalyzing the reduction of selenite to elemental selenium while also reducing nitrite. While periplasmic cytochrome C551 may be a component of the nitrite respiratory system, the level of this cytochrome was essentially the same in mutant and wild-type cells grown under two different growth conditions (i.e. with either selenate or selenate plus nitrate as the terminal electron acceptors). The ability of certain other denitrifying and nitrate respiring bacteria to reduce selenite will also be described.

Chrysiogenes arsenatis gen. nov., sp. nov., a new arsenate-respiring bacterium isolated from gold mine wastewater.

A new strictly anaerobic bacterium (strain BAL-1T) has been isolated from a reed bed at Ballarat Goldfields in Australia. The organism grew by reducing arsenate [As(V)] to arsenite [As(III)], using acetate as the electron donor and carbon source; acetate alone did not support growth. When BAL-1T was grown with arsenate as the terminal electron acceptor, acetate could be replaced by pyruvate, L- and D-lactate, succinate, malate, and fumarate but not by H2, formate, citrate, glutamate, other amino acids, sugars, or benzoate. When acetate was the electron donor, arsenate could be replaced by nitrate or nitrite but not by sulfate, thiosulfate, or iron oxide. Nitrate was reduced to ammonia via nitrite. The doubling time for growth on acetate (5 mM) plus arsenate (5 mM) or nitrate (5 mM) was 4 h. The G+C content of the DNA is 49 mol%. The 16S rRNA sequence data for the organism support the hypothesis that this organism is phylogenetically unique and at present is the first representative of a new deeply branching lineage of the Bacteria. This organism is described as Chrysiogenes arsenatis gen. nov., sp. nov.

New arsenite-oxidizing bacteria isolated from Australian gold mining environments - Phylogenetic relationships

Nine novel arsenite-oxidizing bacteria have been isolated from two different gold mine environments in Australia. Four of these organisms grow chemolithoautotrophically with oxygen as the terminal electron acceptor, arsenite as the electron donor, and carbon dioxide-bicarbonate as the sole carbon source. Five heterotrophic arsenite-oxidizing bacteria were also isolated, one of which was found to be both phylogenetically and physiologically identical to the previously described heterotrophic arsenite oxidizer misidentified as Alcaligenes faecalis . The results showed that this strain belongs to the genus Achromobacter . Phylogenetically, the arsenite-oxidizing bacteria fall within two separate subdivisions of the Proteobacteria . Interestingly, the chemolithoautotrophic arsenite oxidizers belong to the f - Proteobacteria , whereas the heterotrophic arsenite oxidizers belong to the g - Proteobacteria .

Growth the Wolinella succinogenes on H2S plus fumarate and on formate plus sulfur as energy sources

Wolinella succinogenes was found to grow on H2S plus fumarate with the formation of elemental sulfur and succinate. The growth rate was 0.18 h-1 (td=3.8 h) and the growth yield was estimated to be 6.0 g per mol fumarate used. Growth also occurred on formate plus elemental sulfur; the products formed were H2S and CO2. The growth rate and estimated growth yield were 0.58 h-1 (td=1.2 h) and 3.5 g per mol formate used, respectively. These results suggest that certain chemotrophic anaerobes may be involved in both the formation and reduction of sulfur.

Bioremediation of selenium oxyanions in San Joaquin drainage water using Thauera selenatis in a biological reactor system

This report describes the use of a new selenate-respiring bacterium, Thauera selenatis, for the bioremediation of selenium (Se, as selenate) in drainage water from the Westlands Water District, San Joaquin Valley. The organism respires selenate anaerobically using acetate as the preferred electron donor. The reduction of selenate is not inhibited by nitrate; both electron acceptors are reduced concomitantly. T. selenatis was inoculated into, and was maintained in, a biological reactor system for anaerobic treatment of selenate-nitrate containing drainage water; a population of denitrifying bacteria was also present. When the pH of inflowing water was 6.9, and 2 mm acetate plus 0.56 mm ammonium chloride were fed into the reactor, selenate/selenite levels were reduced from 350–450 μg Se/l to 5.39±3.6 μg Se/l. The final product of selenate reduction was elemental Se. Analysis of reactor contents revealed that T. selenatis was the only selenate-respiring organism present in the system. Nitrate in the drainage water was also reduced in the reactor system by 98%. The lab-scale biological reactor system consisted of recycled sludge-blanket (1 l; 400 g sand) and fluidized-bed (1 l; 300 g sand) reactors. At a system flow rate of 6.5 ml/min, the retention time was 140 min.

Cloning and Sequencing of the Genes Encoding the Periplasmic-Cytochrome B-Containing Selenate Reductase of Thauera selenatis

The periplasmic selenate reductase (Ser) of Thauera selenatis is a component of the electron transport chain catalyzing selenate reduction with acetate as the electron donor (i.e., selenate respiration). The purified enzyme consists of three subunits (SerA, SerB and SerC). Using transposon (i.e., Tn5) mutagenesis selenate reductase mutants were isolated. Junction fragments of DNA adjacent to the integrated Tn5 were used, together with oligonucleotides derived from the N-termini of SerA and SerB, to clone from a gene bank a DNA fragment that contained the corresponding genes. After sequencing, ser A, serB and serC were identified by sequence comparison with the N-termini of the three subunits. The genes are arranged in the order serA, serB, serC; a fourth open reading frame (serD) in between, but overlapping serB and serC, is also present. The serA gene product contains an apparent leader peptide with a twin-arginine motif. The remainder of the translated amino acid sequence is similar to that of a number of prokaryotic molybdenum-containing enzymes (e.g., nitrate reductases and formate dehydrogenases of Escherichia coli). The serB gene product contains four cysteine clusters and is similar to various iron-sulfur protein subunits. The serC gene product contains a putative Sec-dependent leader peptide, but there are no similarities between the remainder of the translated protein and other protein subunits. The SerC contains two histidine and four methionine residues, and these may noncovalently bind heme b—which is a component of the active selenate reductase. The serD gene product encodes a putative protein that shows no significant sequence similarities to other proteins. However, the location of the serD within the other ser genes is similar to that of narJ within the E. coli narGHJI operon (nitrate reductase A); thus suggesting that the role of SerD may be similar to that of NarJ, which is a system-specific chaperone protein.

Bioremediation of selenite in oil refinery wastewater

Selenium-oxyanion-containing wastewater, with levels of selenite as high as 3690 μg Se/l and very low levels of selenate, was treated in a laboratory-scale biological reactor system inoculated with the selenate-respiring bacterium Thauera selenatis. The wastewater contained selenite that had been removed from refinery effluent wastewater using iron-coprecipitation followed by selenite release to yield a more concentrated selenium-containing wastewater. The reactor system consisted of recycling sludge-blanket (500 ml; 200 g sand) and fluidized-bed reactors (500 ml; 150 g sand). The flow rate through the reactor system was 3.5 ml/min. The carbon source fed into the reactor was acetate (3 mM); nitrate was also present (3 mM). The selenium oxyanion levels in the wastewater were reduced by 95%. T. selenatis was the only selenate-reducing bacterium detected in the reactor system and it presumably reduced a portion of the selenate present in the water to selenite. The selenite present in the water, and that formed by selenate reduction, was reduced both by the Thauera and by a population of denitrifying bacteria also present in high numbers in the reactor system.

Isolation of a New Arsenate-Respiring Bacterium--Physiological and Phylogenetic Studies

A new strictly anaerobic arsenate-respiring bacterium has been isolated from arseniccontaminated mud obtained from a gold mine in Bendigo, Australia. This organism, designated JMM-4, was found to be a Gram-positive, spore-forming rod, 0.6 2 2.5-3 w m, motile by means of flagella that are subpolar or along one side of the cell. JMM-4 grows using arsenate as the terminal electron acceptor and lactate as the electron donor. Arsenate is reduced to arsenite and the lactate is oxidized to CO2 via the intermediate, acetate. The doubling time for exponential growth with arsenate (5 mM) and lactate (5 mM) was 4.3 - 0.2 h. Alternative electron donors used by JMM-4 when grown with arsenate as the terminal electron acceptor are acetate, pyruvate, succinate, malate, glutamate, and hydrogen (with acetate as carbon source). Apart from arsenate, nitrate can serve as an alternative electron acceptor.Optimal growth occurs at pH 7.8 with a sodium chloride concentration of 1.2 g · l -1 . Based upon 16S rRNA gene sequence analysis, JMM-4 falls within the low G + C, Gram-positive, aerobic, spore-forming bacilli cluster and is most closely related to the previously described haloalkalophilic arsenate/selenate respiringbacterium Bacillus arsenicoselenatis . The physiological differences between JMM-4 and B. arsenicoselenatis however suggest that JMM-4 is a new species of Bacillus .