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A novel arsenate respiring isolate that can utilize aromatic substrates

Anbo Liu , Elizabeth Garcia-Dominguez , E.D Rhine , L.Y Young
DOI: http://dx.doi.org/10.1016/j.femsec.2004.02.008 323-332 First published online: 1 June 2004

Abstract

A novel anaerobic bacterium was isolated from the sediment of Onondaga Lake (Syracuse, NY), which can use arsenate [As(V)] as a respiratory electron acceptor. The isolate, designated strain Y5 is a spore-forming, motile rod, with lateral flagella. It is Gram-negative though it phylogenetically falls within the low G + C Gram-positive organisms. In addition to the more usual electron donors such as lactate and succinate, strain Y5 also can use H2+ CO2 chemoautotrophically and metabolize aromatic compounds such as syringic acid, ferulic acid, phenol, benzoate and toluene, coupled to arsenate reduction. Aside from As(V), nitrate, sulfate, thiosulfate and Fe(III) can also serve as electron acceptors. Based on 16S rDNA phylogeny and its physiological characteristics, strain Y5 was identified as most closely related to the genus Desulfosporosinus. The ability of microorganisms to reduce arsenate for respiration appears to be widely distributed and may be relevant in the biogeochemical cycling of arsenic in environments containing mixed contaminants.

1 Introduction

Arsenic, the 20th most abundant element in the Earth's crust, is widely distributed throughout nature as a result of weathering, dissolution, fire and volcanic activity. Anthropogenic inputs include the use of arsenic in pesticides, herbicides, wood preservatives, and dye as well as arsenic-containing wastes generated during smelting and mining operations. In the environment, As(V) and As(III) comprise the bulk of the inorganic forms found in natural waters and sediments [1]. As(III) is more mobile and toxic to humans and the ecosystem, while As(V) is more absorptive to the environmental matrix. In arsenic rich environments, a major concern is the potential for mobilization and transport of this toxic element to groundwater and drinking water supplies. In natural water, reported ranges of arsenic concentrations are from 0.5 μg l−1 to greater than 5000 μg l−1[2]. Recently, the maximum contaminant level (MCL) for arsenic in the public water supply has been lowered from 50 to 10 μg/l [3].

Microorganisms are mediators in the geochemical cycling of arsenic. Bacteria and phytoplankton have been shown to be able to oxidize [4,5], methylate [6,7], and reduce arsenic [7]. Reduction of As(V) to As(III) by microorganisms can serve as a detoxification mechanism or as dissimilatory arsenate reduction (respiration) [8]. The process of arsenic detoxification has been well established in a number of organisms and is reported to be controlled by the ars genes that encode for proteins responsible for As(V) reduction followed by As(III) removal from the cell through an efflux pump [9,10].

Dissimilatory reduction, where As(V) is used as an electron acceptor for respiration and growth was described with a new isolate, Sulfurospirillum arsenophilum in 1994 [11], and since then other prokaryotes have been identified that can achieve growth via dissimilatory arsenate reduction to arsenite [1221]. Even with a limited number of species, it is clear that the phenomenon is polyphyletic. That is, these organisms are phylogenetically diverse [22]. In addition, the different strains were isolated from environments such as sediments, hot springs and mine wastes. More recently, dissimilatory arsenate reductase activity and arsenate-respiring bacteria were isolated from bovine rumen fluid, hamster feces and the termite hindgut [23].

To date the substrates on which these strains have been isolated have been simple carbon compounds such as lactate or acetate. Whether arsenate respiring microorganisms can utilize more complex substrates such as natural ligno-aromatic compounds or organics associated with hazardous wastes is addressed within this work. We report the isolation and identification of a novel anaerobic dissimilatory arsenate reducing bacterium that can use a range of aromatic compounds. This isolate appears to be physiologically and phylogenetically distinct from the other reported arsenate reducers.

2 Materials and methods

2.1 Growth and media condition

For the initial enrichment and growth of arsenate-reducing bacteria, a mineral salts medium under anaerobic conditions was used as described by Evans et al. [24], with the exception that sodium nitrate was not included. The initial enrichment contained 10 mM As(V) as the terminal electron acceptor and 10 mM lactate as the electron donor/carbon source. The pH of the medium was adjusted to 7.2.

2.2 Source of inoculum

The inoculum for the enrichment cultures was collected from the top 20 cm of sediment of Onondaga Lake, which is a dimictic system located within metropolitan Syracuse, NY. This lake is polluted by inputs of domestic and industrial wastes, including mercury, petroleum, PCB's and other chlorinated compounds that have been discharged since the late 1800s [2527], and is designated as a Superfund site [28].

2.3 Enrichment and isolation procedures

The defined medium was prepared under strict anaerobic conditions [29]. Sediment inoculum (10% wt/vol) was added and 100 ml aliquots were anaerobically dispensed into 160 ml serum bottles with argon in the headspace. The initial lactate and As(V) concentrations were 10 and 10 mM, respectively. Based on stoichiometric calculations, the lactate was more than 5 times in excess of that needed for complete reduction of the available arsenate. The bottles were sealed with rubber stoppers and aluminum crimp seals, and incubated statically at 30 °C in the dark. To ensure anaerobic conditions, all sampling and amendments were carried out using sterile plastic syringes flushed with argon. Enrichments were established in triplicate with duplicate sterile (autoclaved three times on consecutive days) and background (no carbon source added) controls.

Reduction of 10 mM As(V) occurred in all the active bottles within two weeks of incubation. A 10% dilution of the active cultures was made into new medium and incubated as previously described. Dilution of the enrichment was carried out in this fashion 10 times over 6 months. Once the cultures were diluted to the point that there was no more sediment, an aliquot of the culture was dispensed into anaerobic shake tubes prepared with the defined medium and containing 0.8–1% (wt/vol) sterile agar (Difco, Detroit, MI). After 4 weeks of incubation at 30 °C, single colonies were picked and streaked onto new shake tubes containing 2% (wt/vol) agar twice in succession and incubated anaerobically at 30 °C to insure purity. The culture was also streaked onto the solid medium containing only lactate and no As(V) to insure that adventitious contaminants were not being carried along. The pure culture was then grown on lactate and As(V) in liquid medium anaerobically in 100 ml, then concentrated by centrifugation and preserved at −80 °C in 5 ml of 50% (vol/vol) glycerol-media solution dispensed in multiple aliquots and kept for further characterization and experiments.

2.4 Electron donors used for growth

The electron donors/carbon sources tested for their ability to support growth of strain Y5 when As(V) was present as the electron acceptor included: H2+ CO2 (10 ml at 1 atm), formate (10 mM), acetate (10 mM), H2 (10 ml at 1 atm) + acetate (10 mM), propionate (10 mM), succinate (10 mM), lactate (10 mM), glucose (10 mM), yeast extract (1 g/l), syringic acid (1 mM), ferulic acid (1 mM), phenol (1 mM), benzoate (1 mM), and toluene (0.1 mM). Balch tubes containing 15 ml liquid medium with no electron donor and 10 mM As(V), were inoculated with 1.5 ml of strain Y5 which was previously grown on 10 mM lactate and 10 mM As(V). These tubes were incubated at 30 °C for three days to allow the depletion of lactate which may have been carried over with the inoculum. After three days, the electron donors/carbon sources were added by syringe injection from sterile anaerobic stock solutions to yield the desired concentrations. Hydrogen was added by the injection of 10 ml of H2 to the headspace of culture tubes using a sterile syringe. Controls consisted of no electron donor plus As(V) and also electron donor plus no As(V). Changes in As(V) and As(III) concentration in the cultures were monitored during the incubation. Growth as turbidity was measured spectrophotometerically at 580 nm. Results were considered positive if As(V) was reduced to As(III) and growth occurred compared to sterile controls. All experiments were run in duplicate or triplicate.

2.5 Other electron acceptors used for growth

Additional electron acceptors tested for their ability to support growth of strain Y5 included nitrate (5 mM), nitrite (5 mM), sulfate (5 mM), thiosulfate (5 mM), sulfite (5 mM), Fe(III) (5 mM) and Cr(VI) (0.15 mM). Balch tubes containing 15 ml liquid medium and 10 mM lactate were inoculated with 1.5 ml of strain Y5 that was grown on 10 mM lactate and 10 mM As(V) previously. Each electron acceptor was added from sterile anaerobic stock solution, and all cultures were incubated at 30 °C. After three weeks, cultures were sampled to detect changes in the concentration of each electron acceptor. Growth as turbidity was measured spectrophotometerically at 580 nm. Results were considered positive if the supplied acceptors were reduced and cell growth occurred. All experiments were run in triplicate.

2.6 Chemicals

All chemicals were reagent grade and were used without further purification. All solutions were prepared with sterile distilled deionized water. The As(III) stock solution was prepared from solid arsenic trioxide, As2O3 (Aldrich, A.C.S. primary standard). The As(V) stock solution was prepared from Na2HAsO4· 7H2O (Sigma) dissolved in sterile distilled deionized water.

2.7 Analyses

Liquid samples were taken periodically for analysis. Sediment slurry (from enrichment) or liquid-only (all other experiments) samples (0.5–1.0 ml) were drawn through the stoppers by using 1.0 ml sterile syringes with 16-gauge needles previously flushed with argon. The samples were transferred to spin-prep vials with 0.2 μm pore-size nylon filters (VWR Scientific, West Chester, PA) and centrifuged for 2 min in a bench top microcentrifuge to remove all suspended materials.

As(III) and As(V) concentration in the filtered samples were measured using high performance liquid chromatography (HPLC) (Shimadzu, Columbia, MD) with a Hamilton PRP-X100 anion exchange column, and detected by UV absorbance at 195 nm. The mobile phase used was 30 mM sodium phosphate buffer with pH 6.0. The flow rate was 1.0 ml/min and the injection volume was 10 μl.

Lactate, succinate and acetate concentrations were determined by HPLC (Beckman, Fullerton, CA) equipped with a 7.8 mm × 50 cm Rezex ROA organic acid column (Phenomenex, Torrance, CA) with UV detection at 210 nm. The mobile phase consisted of 0.005 N H2SO4 at a flow rate of 0.5 ml/min. The column was maintained at room temperature, and the injection volume was 20 μl.

Phenol, benzoate, syringic acid and ferulic acid concentration were measured by HPLC (Beckman, Fullerton, CA) equipped with a 4.6 mm × 25 cm Ultrasphere C18 column (Beckman) with UV detection at 280 nm. The mobile phase consisted of 60% methanol, 38% water and 2% acetic acid. The flow rate was 1 ml/min and the injection volume was 20 μl.

Sulfate, sulfite, nitrate and nitrite analyses were performed on a Dionex (Sunnyvale, CA) model 100 ion chromatograph with an IonPac AS9 column by using conductivity detection. The eluant contained 2 mM Na2CO3 and 0.75 mM NaHCO3 with a flow rate of 2.0 ml/min. The regenerant consisted of 25 mM H2SO4 under He at 8 lb/in2.

Fe(II) and Fe(III) were measured using the colorimetric technique described by Hacherl [30]. Cr(VI) reduction was determined using a standard colorimetric procedure [31].

Protein concentrations of culture samples were determined by the Bradford method [32] using a modified version of the Bio-Rad protein assay (Bio-Rad, Hercules, CA). Absorbance was read at 595 nm. A 5-ml aliquot of each sample was centrifuged at 3000 rpm for 15 min and the supernatant was discarded. The cells were washed with 5 ml phosphate buffer (50 mM, pH 7.2), centrifuged again for an additional 15 min and the supernatant was discarded. The washed cells were resuspended in 900 μl fresh phosphate buffer and 100 μl 10 N NaOH was added. The samples were boiled for 15 min and after cooling, the pH was adjusted to 7 with 5 N HCl, after which the protein concentration was determined following the Bio-Rad protocol.

2.8 Transmission electron micrographs

Cells of strain Y5 were negatively strained with 2% phosphotungstic acid and were viewed and photographed with a Hitachi H-7000 transmission electron microscope.

2.9 Spore formation

To test if strain Y5 can form heat-resistant spores, triplicate cultures of Y5 were grown to stationary phase on 10 mM lactate and 10 mM As(V). The culture was incubated at 85 °C for 30 min, then transferred to fresh medium and incubated at 30 °C. The culture was sampled daily to analyze the change in the concentration of As(V), As(III) and protein.

2.1 016S rRNA gene isolation, sequencing and phylogenetic analysis

Genomic DNA was isolated from strain Y5 as previously described by Rainey et al. [33]. The 16S rRNA gene was amplified from the genomic DNA using eubacterial primers 27F and 1525R [34]. Each 50-μl PCR mixture contained the following: 5 μl of 10X PCR buffer (Invitrogen, Carlsbad, CA), 1 μl of deoxynucleoside triphosphates (10 mM of each, Invitrogen), 1.5 μl of MgCl2 (50 mM, Invitrogen), 2 μl of each primer (10 pM, Invitrogen), 0.4 μl of Taq DNA polymerase (5 U μl−1, Invitrogen), 2 μl of DNA template (50 ng), and sterile deionized water to bring to a final volume of 50 μl. The DNA was amplified in a Perkin Elmer GeneAmp PCR System 2400 with an initial denaturation step of 95 °C for 5 min followed by 30 cycles of: 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min, with a final extension for 10 min at 72 °C. Following verification of PCR product on a 1% agarose gel, the PCR product was purified using a QIAquick Spin Cleanup for PCR Purification (Qiagen, Chatsworth, CA). An ABI Prism® BigDyeTM Terminator v3.0 Ready Reaction Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) was used to directly sequence the amplicon. Each sequencing reaction contained: 4 μl of BigDyeTM Terminator v3.0 Ready Reaction Mix, 100 ng of purified PCR product, 25 ng of sequencing primer, and sterile deionized water to a final volume of 10 μl. The sequencing reactions were amplified as described by the manufacturer using eubacterial primers 27F, 530F, 926F, 519R, 1100R, and 1525R [34] to sequence both strands of DNA. Following sequencing amplification the reactions were purified, re-suspended in 15 μl of formamide, and sequenced using an ABI Prism 3100 Genetic Analyzer (Applied Biosystems).

Related 16S rRNA sequences were identified using BLAST with a 1500 base segment of the Y5 gene. Sequence alignments were done using ClustalX [36] and sequence similarity determined using PHYLIP [37]. Neighbor-joining trees and bootstrap values, expressed as percentages of 1000 replicates, were constructed using ClustalX [36]. The following sequences were obtained from GenBank and the RDP [35], GenBank accession numbers are in parentheses: Desulfomicrobium sp. ‘Bendigo B’ (AF131233), Desulfonispora thiosulfatigenes (genbank:Y18214), Dehalobacter restrictus (genbank:Y10164), Desulfitobacterium metallireducens (genbank:AF297871), Desulfitobacterium hafniense (genbank:X94975), Desulfitobacterium frappieri (genbank:U40078), Desulfosporosinus orientis (genbank:Y11571), Desulfosporosinus meridiei (genbank:AF076247), Desulfotomaculum (Desulfosporosinus) auripigmentum (genbank:U85624), Desulfotomaculum geothermicum (genbank:Y11567), Desulfotomaculum thermocistern (genbank:AF295662), Thermoterrabacterium ferrireducens (genbank:U76364), Desulfotomaculum acetoxidans (genbank:Y11566), Desulfotomaculum aeronauticum (genbank:X98407), Desulfotomaculum reducens (genbank:U95951), Succinispira mobilis (genbank:AJ006980), Anaerosinus glycerini (genbank:AJ010960), Propionispira arboris (genbank:Y18190), Centipeda periodontii (genbank:AJ010963), Anaerovibrio lipolytica (genbank:AB034191), and Anaeroarcus burkinensis (genbank:AJ010961). The 16S rRNA sequence for isolate Y5 was deposited as GenBank accession number genbank:AY233860.

2.11 RuBisCO gene analysis

To further evaluate the ability of strain Y5 to grow autotrophically, both Type I (cbbL) and Type II (cbbM) genes were looked for by PCR amplification of the ribulose-1,5′-biphsophate carboxylase/oxygenase (RuBisCO) as described by Elsaied and Naganuma [38]. An 800-base pair segment of the cbbL gene was amplified using the forward (5′-GACTTCACCAAAGACGACGA-3′) and reverse primers (5′-TCGAACTTGATTTCTTTCCA-3′). For the Type I cbbL gene, the initial denaturing step was at 94 °C for 2 min, followed by denaturation at 94 °C for 1 min, annealing at 62 °C for 1 min, and extension at 72 °C for 3 min for 30 cycles, with a final extension for 15 min at 72 °C. Results were negative for the cbbL gene.

The primer set used to amplify the Type II gene included forward primer (5′-ATCATCAARCCSAARCTSGGCCTGCGTCCC-3′) and the reverse primer (5′-MGAGGTGACSGCRCCGTGRCCRGCMCGRTG-3′), resulting in a 400-base pair fragment from the cbbM gene [38]. The cbbM gene was amplified by initial denaturation at 94 °C for 2 min, followed by 30 cycles of denaturation at 94 °C for 1 min, annealing at 62 °C for 1 min, and extension at 72 °C for 3 min, with a final extension for 15 min at 72 °C. The amplified cbbM gene was then sequenced and the sequence confirmed with known RuBisCO Type II cbbM gene sequences found in GenBank.

3 Results

3.1 Isolation and morphological characteristics of the isolate

The isolate, designated strain Y5, is a motile, rod-shaped bacterium possessing one to two laterally attached flagella and produce endospores (Fig. 1(a)). Cells of strain Y5 were Gram-negative with some variability. Electron micrographs of negative stained cells also showed invaginated cell walls typical of Gram-negative organisms. The average cell size of strain Y5 was about 2 μm in length and 0.5 μm in width. On solid media, strain Y5 colonies appeared initially white with a yellow pigment produced after about three weeks of incubation. The colonies are approximate 0.5–1.5 mm in diameter and have rounded edges (Fig. 1(b)).

1

Colonies and electron micrograph of strain Y5. (a) Electron micrograph of strain Y5. (b) Colonies of strain Y5 grown anaerobically on solid medium containing 10 mM lactate and 10 mM arsenate.

Cultures that were heat treated to kill viable cells were incubated in media with 10 mM lactate and 10 mM As(V). After 7 days incubation at 30 °C, complete reduction of As(V) to As(III) was achieved. Spores were also observed when cultures were exposed to oxygen.

3.2 Biological reduction of As(V) to As(III)

Fig. 2 shows the cell protein concentration of strain Y5 after incubation for 14 days in the defined medium with approximately 0.5 mM of syringic acid in the absence and the presence of 6 mM As(V). Following the 14 day incubation period, the protein concentration for strain Y5, when 6 mM As(V) was added to the media, was 290.00 μg/ml. The final protein concentration when no As(V) was added was 27.70 μg/ml, and 24.59 μg/ml protein was calculated for the autoclaved sterile controls which received 6 mM As(V) prior to incubation. Hence, growth of strain Y5 was arsenic dependent.

2

Protein concentration of strain Y5 after growth for 14 days in the absence and the presence of As(V). The initial concentration of As(V) and syringic acid was 6 and 0.5 mM, respectively. As(V) was completely reduced to As(III) in 14 days. Data reported are averages of triplicates, and error bars are representative of standard deviations. Sterile controls were inoculated and autoclaved.

3.3 As(V) reduction coupled to the degradation of aromatic compounds

Strain Y5 was found to be able to degrade a series of aromatic compounds coupled to As(V) reduction. The compounds tested included syringic and ferulic acids, phenol, benzoate and toluene. Fig. 3(a) illustrates that after a lag, complete reduction of 6 mM As(V) to As(III) takes place, while 0.5 mM syringic acid is consumed as an electron donor/carbon source, over a period of 14 days. No As(V) reduction was detected in the sterile controls, in which the inoculated bacteria had been autoclaved. The reduction of As(V) generated stoichiometric amounts of As(III) and no elemental As or AsH3 was detected, hence As(V) was completely reduced to As(III) by isolate Y5. When 1.5 mM of ferulic acid was supplied as the sole electron donor/carbon source, 7.5 mM As(V) was stoichiometrically reduced to As(III) within 21 days (Fig. 3(b)), and reduction of As(V) was concurrent with loss of ferulic acid. The relative rates of arsenate reduction for the different aromatic compounds tested are in the order of syringic acid > ferulic acid > phenol/benzoate (data not shown).

3

Arsenate reduction coupled to the degradation of ligno-aromatic compounds. (a) syringic acid, (b) ferulic acid. Data reported are averages of triplicates, and error bars are representative of standard deviations.

Toluene was also tested as a possible C-source for growth by isolate Y5 coupled to respiratory As(V) reduction. Fig. 4 illustrate As(V) reduction and the degradation of toluene by strain Y5 over a 17 day incubation period, where 2.68 mM of As(V) was completely reduced to As(III) and 0.237 mM toluene was utilized as the sole C-source. Other arsenic species were not detected. When toluene was present but no As(V) was added, there was no apparent loss of toluene, indicating that toluene metabolism was dependent on As(V) reduction. Further support for the loss of toluene coupled to As(V) reduction was inferred from the stoichiometry of the reaction using the following equation: Embedded Image 1 If it assumed that the toluene lost (0.237 mM) was completely metabolized to CO2, then the predicted amount of As(III) formed should be 3.32 mM. This is close to the actual concentration measured (2.68 mM).

4

Arsenate reduction coupled to the degradation of toluene. Data are averages of triplicates, and error bars are representative of standard deviations.

3.4 Growth with other electron donors and electron acceptors

Carbon source and electron donor utilization profiles of Y5 are summarized in Table 1. The following electron donors and carbon sources supported growth: lactate, H2, H2+ acetate, succinate, yeast extract, syringic acid, ferulic acid, phenol, benzoate, and toluene. Formate, acetate, propionate and glucose did not support growth. When inorganic-CO2 was supplied as the sole C-source, strain Y5 was able to grow autotrophically and use H2 to reduce As(V). Positive PCR amplification showed that strain Y5 contained the Type II RuBisCO gene, cbbM but not the Type I cbbL gene (data not shown). The sequence of cbbM was 91% (618/674 bases matched) similar to Thiobacillus denitrificans (accession number genbank:L37437) (data not shown).

View this table:
1

Compounds tested as electron donors for strain Y5, as compared to Desulfosporosinus auripigmentum[18] and Desulfosporosinus meridiei[41]

Electron donors (mM)Y5D. auripigmentumD. meridiei
Syringic acid (1 mM)+ND+
Ferulic acid (1 mM)+NDND
Phenol (1 mM)+ND
Benzoate (1 mM)+
Toluene (0.1 mM)+ND
Formate (10 mM)NDND
Acetate (10 mM)
Propionate (10 mM)NDND
Succinate (10 mM)+ND
H2 (1 atm)+Acetate (10 mM)+++
Glucose (10 mM)ND
Yeast extract (1 g/l)+ND
Lactate (10 mM)+++
H2 (1 atm) + CO2++ND
  • Symbols (+) means growth and/or loss of electron donor, (−) means no growth or loss of electron donor.

The results from the screening of terminal electron acceptors are summarized in Table 2. When strain Y5 was grown in minimal medium with lactate as the electron donor and carbon source, nitrate, sulfate, thiosulfate and Fe(III) could be used as electron acceptors. Nitrite, sulfite and Cr(VI) did not support growth.

View this table:
2

Compounds tested as electron acceptors for strain Y5, as compared to Desulfosporosinus auripigmentum[18] and Desulfosporosinus meridiei[41]

Electron acceptorsY5D. auripigmentumD. meridiei
As(V) (10 mM)++
Nitrate (5 mM)+
Nitrite (5 mM)NDND
Sulfate (5 mM)+++
Sulfite (5 mM)++
Thiosulfate (5 mM)+++
Fe (III) (5 mM)++
Cr(VI) (0.15 mM)NDND
  • Symbols (+) means growth and/or loss of acceptor, (−) means no growth or loss of acceptor.

Tables 1 and 2 also list the electron donor profile and the electron acceptor profile of bacterial strains to which strain Y5 is most closely related, and this will be discussed further.

3.5 Phylogenetic characterization

The BlastN search result of the 16S rRNA gene for isolate Y5, based on a 1500 nucleotide segment, showed a close relationship to Desulfosporosinus meridiei (97% sequence similarity, 1407/1447 bases matched) and Desulfotomaculum auripigmentum (96% sequence similarity, 1223/1269 bases matched), a known arsenate-respiring bacterium [18], which has recently been reclassified as Desulfosporosinus auripigmentum[39]. A phylogenetic dendrogram based on a 1300 nucleotide segment of the 16S rRNA gene was constructed to compare the sequence of isolate Y5 to other members of the δ-Proteobacteria class (Fig. 5). Based on the phylogenetic dendrogram, isolate Y5 belonged to the Peptococcaceae group and branched between the species Desulfosporosinus meridiei and Desulfosporosinus auripigmentum (68% and 70% bootstrap values, respectively).

5

Phylogenetic dendrogram based on 16S rDNA sequence comparison of 1300 bases. The neighbor-joining tree was derived using ClustalX, bootstrap values (expressed as percentages of 1000 replicates) are shown at the branch points, and the bar equals 2% difference. Based on 16S rDNA sequence comparison, isolate Y5 belongs in the Peptococcaceae group, and is closely related to Desulfosporosinus meridiei and Desulfosporosinus auripigmentum.

4 Discussion

Isolated from Onondaga Lake, a designated Superfund site, strain Y5, a strictly anaerobic respiratory As(V) reducing bacterium, is motile with 1 to several peritrichous lateral flagella and produces spores. Interestingly, it stains Gram-negative and appears to have the invaginated outer cell wall typical of Gram-negative organisms, though it phylogenetically places in the spore forming Clostridium/Bacillus subphylum of the Gram-positive bacteria. Recently, Desulfotomaculum auripigmentum, another known arsenate-respiring isolate that stains Gram-negative but phylogenetically clusters within the low G + C Gram-positive Peptococcaceae group, has been reclassified as Desulfosporosinus auripigmentum[39]. Although this may appear paradoxical, other examples of anaerobic microorganisms that are Gram-negative spore-formers can be found in the literature and include Pelospora glutarica[40], Cetobacterium ceti[41], and Propionispora vibroides[42]. Based on phylogenetic and physiological characterization, strain Y5 is most closely related to the genus Desulfosporosinus[43]. This is consistent with its ability to reduce sulfate and thiosulfate to sulfide, and the observation that it readily produces spores when exposed to oxygen.

As(V) is used as a respiratory electron acceptor for growth by strain Y5. Growth is dependent on As(V) reduction and no growth occurred when As(V) was not provided (Fig. 2). Furthermore, stoichiometric conversion of As(V) to As(III) takes place (Fig. 3).

Compared to the previously described dissimilatory As(V) reducers which were all screened on lactate or acetate, it is especially interesting to note that strain Y5 has a broad substrate range which includes the ligno-aromatic compounds syringic and ferulic acids, and aromatic compounds such as phenol, benzoate and the gasoline component toluene (Table 1). To our knowledge, use of aromatic carbon sources coupled to As(V) reduction has not been previously reported. Also, strain Y5 is capable of autotrophic growth on H2/CO2 and has the Type II RuBisCO gene, cbbM. It has been reported that anaerobic CO2 fixing microorganisms commonly have the Type II form of RuBisCO but not the Type I [38].

In addition, strain Y5 is further distinguished from other dissimilatory As(V) reducers in the range of other electron acceptors it can use. Arsenate reducers described in the literature appear to also reduce either nitrate [10,13,14,41] or sulfate [15,17], while strain Y5 can reduce both, in addition to a series of other electron acceptors (Table 2). Furthermore, since both As(V) and Fe(III) can be reduced by strain Y5, this can affect the distribution and transport of arsenic species in anoxic environments. Sorption of As(V) to hydrous manganese, aluminum and Fe(III) is known to occur in sediments [44,45]. An organism such as strain Y5, by reducing Fe(III) to Fe(II), potentially could release the Fe(III) bound As(V), making it available for further chemical or biological attack. In fact, mobilization of As(V) in sediment by Fe(III)-reducing bacteria has been reported [46] and proposed as the source of arsenic in the wells of Bangladesh [47]. Strain Y5 could affect arsenic transport first by releasing bound As(V) from the Fe(III) oxyhydroxides through Fe(III) reduction and second by mobilizing arsenic by reducing As(V) to the more soluble and mobile As(III).

Phylogenetically, strain Y5 is most closely related to Desulfosporosinus meridiei and the recently reclassified Desulfosporosinus auripigmentum (formerly Desulfotomaculum auripigmentum) [39]. Strain Y5 shares some common physiological characteristics with each, however, differences are also notable. First, D. meridiei cannot reduce As(V), while strain Y5 and D. auripigmentum can. Strain Y5 is able to use nitrate and Fe(III) as electron acceptors while D. auripigmentum cannot. All 3 strains can utilize sulfate and thiosulfate. On the other hand, strain Y5 cannot use sulfite while D. auripigmentum and D. meridiei both can. With respect to the organic substrates, phenol, benzoate and toluene can be used as electron donors and growth substrates by strain Y5 and cannot be used by D. auripigmentum. Unlike D. auripigmentum, strain Y5 is similar to D. meridiei in its ability to produce spores.

The ability of strain Y5 to couple As(V) reduction to the degradation of aromatic compounds is especially interesting in light of the fact that many Superfund sites (based on a query of the EPA Superfund database, http://www.epa.gov/superfund) contain both arsenic and aromatic organic contaminants. Biodegradation of the latter may be a benefit; on the other hand, reduction of As(V) to the more mobile and toxic As(III) would be undesirable. Microorganisms able to reduce arsenate appear to be widely distributed, and there are likely to be even more strains of greater metabolic and physiological diversity which have yet to be described. As a consequence, the role of anaerobes in the fate, transport and geochemical cycling of arsenic in the environment has yet to be fully appreciated.

Acknowledgements

This work was supported in part by the NIEHS-Superfund Basic Research Program (P42ES10344) and in part by NSF EMSI (CHE9810248). We thank Valentin Starovoytov for transmission electron microscopy assistance and Maria Rivera, Dr. Craig Phelps, Kateri Finger, José Peréz-Jimenéz, Nicholas de Vito and Fang Liu for technical support and advice.

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View Abstract