Microbiology of Inorganic Arsenic: from Metabolism to Bioremediation

Journal of Bioscience and Bioengineering VOL. 118 No. 1, 1e9, 2014 www.elsevier.com/locate/jbiosc

REVIEW Microbiology of inorganic arsenic: From metabolism to bioremediation

Shigeki Yamamura1 and Seigo Amachi2,*

Center for Regional Environmental Research, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan1 and Graduate School of Horticulture, Chiba University, 648 Matsudo, Matsudo, Chiba 271-8510, Japan2

Received 29 October 2013; accepted 11 December 2013 Available online 4 February 2014 Arsenic (As) contamination of drinking water and soils poses a threat to a large number of people worldwide, especially in Southeast Asia. The predominant forms of As in soils and aquifers are inorganic arsenate [As(V)] and arsenite [As(III)], with the latter being more mobile and toxic. Thus, redox transformations of As are of great importance to predict its fate in the environment, as well as to achieve remediation of As-contaminated water and soils. Although As has been recognized as a toxic element, a wide variety of microorganisms, mainly bacteria, can use it as an electron donor for autotrophic growth or as an electron acceptor for anaerobic respiration. In addition, As detoxiﬁcation systems in which As is oxidized to the less toxic form or reduced for subsequent excretion are distributed widely in microorganisms. This review describes current development of physiology, biochemistry, and genomics of arsenic-transforming bacteria. Potential application of such bacteria to removal of As from soils and water is also highlighted. Ó 2013, The Society for Biotechnology, Japan. All rights reserved.

[Key words: Arsenate reduction; Arsenite oxidation; Arsenic contamination; Biogeochemical cycle of arsenic; Bioremediation]

Although arsenic (As) is commonly known as a toxin, it is Although As can exist in four oxidation states (V, III, 0, eIII) with ubiquitous in the environment (1); however, its abundance in the a variety of inorganic and organic forms, inorganic arsenate [As(V)] earth’s crust is low (0.0001%) and its background concentrations in and arsenite [As(III)] predominate in aquatic and soil environments soils are generally less than 15 mg kg 1 (2,3). Nevertheless, local (11e13). As(V) is present as negatively charged oxyanions ð = 2Þ concentrations can vary depending on parent materials and H2AsO4 HAsO4 at moderate pH. These oxyanions are strongly geological history of the region. For example, Himalayan-derived adsorbed to the surface of common soil minerals such as Fe and Al 0 sediment is the source of groundwater As contamination in large (hydr)oxides. As(III) primarily exists as uncharged H3AsO3 with a areas of south and southeast Asia (4). In Bangladesh and West pKa of 9.2, and is therefore less adsorptive and more mobile than Bengal, India, approximately 60e100 million people rely on As(V) in most environments (12). In aerobic environments, As(V) is drinking water that contains As in excess of the World Health Or- found to be the predominant species and immobilized in solid ganization standard (5). Direct consumption of rice irrigated with phase. In contrast, As(III) is more prevalent in anoxic environments, As-contaminated water is another signiﬁcant route of human which leads to mobilization into the aqueous phase (14). exposure (6). Accordingly, health problems associated with expo- Microorganisms can mediate redox transformations of As via sure to As are a worldwide concern. Although As has both toxic and As(V) reduction and As(III) oxidation (2). To date, a wide variety of therapeutic properties, chronic exposure to As has caused a wide As(V)-reducing and As(III)-oxidizing prokaryotes have been isolated variety of adverse health effects including dermatological condi- from various As-contaminated environments (2,15), and a recent tions and skin and internal cancers (7). In addition, recent studies study suggested that they are also distributed in the natural envi- have indicated that gestational As exposure is associated with ronment containing background levels of As (16,17). Because both increased cancer incidence in adulthood (8). As(V) reduction and As(III) oxidation directly affect the mobility and Anthropogenic discharges such as air emissions, soil amend- bioavailability of As, microbial activities play a key role in biogeo- ments, mining operations, and wood preservation have also chemical As cycling. Such microbial processes have the potential to resulted in elevated As levels (9). Indeed, As has become a prevalent promote As removal from contaminated soils/waters when used as soil contaminant throughout the world (9,10). Accumulated As in intended. This article outlines the latest physiology and phylogeny contaminated soils has the potential to leach into ground and for microbial metabolism of inorganic As and highlights their ad- surface water, and direct exposure from ingestion, inhalation, and vances as bioremediation techniques. dermal routes can impact animal and human health. In Japan, the Soil Contamination Countermeasures Law was enacted in 2003 to AS(III) OXIDATION address issues caused by harmful substances, including As.

Aerobic As(III) oxidizers The bacterial oxidation of As(III) was ﬁrst reported in 1918, although the ﬁnding went largely un- * Corresponding author. Tel./fax: þ81 47 308 8867. noticed until 1949, when Turner isolated 15 strains of heterotrophic E-mail address: [email protected] (S. Amachi). As(III)-oxidizing bacteria (18e20). Currently, physiologically

1389-1723/$ e see front matter Ó 2013, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2013.12.011 2 YAMAMURA AND AMACHI J. BIOSCI.BIOENG., diverse As(III) oxidizers are found in various groups of Bacteria and couple As(III) oxidation to nitrate reduction under anaerobic Archaea and include both heterotrophic As(III) oxidizers (HAOs) conditions. A purple sulfur bacterium, Ectothiorhodospira sp. PHS- and chemolithoautotrophic As(III) oxidizers (CAOs) (2,21,22). 1, was also isolated from red-pigmented biofilms in Mono Lake Heterotrophic As(III) oxidation is generally considered a (48). This strain can use As(III) as the electron donor for detoxification mechanism that converts As(III) into less toxic anoxygenic photosynthesis and produces As(V) anaerobically As(V), although it may be used as a supplemental energy source under light conditions. Interestingly, both of these bacteria appear (23). In contrast, CAOs use As(III) as an electron donor during to lack aioA genes and instead possess genes that are much more fixation of CO2 coupled with reduction of oxygen (24). Anaerobic closely related to arrA (49,50) (Fig. 2). This gene, designated arxA, CAOs have also recently been isolated (see below). In addition, is required for chemoautotrophic growth on As(III) and nitrate by some researchers have reported curious facultative anaerobic strain MLHE-1. In addition, arxA is strongly induced by As(III) in HAOs capable of either aerobic As(III) oxidation or anaerobic strain PHS-1. Thus, it is possible that ArxA is a novel type of As(III) As(V) reduction (25,26). In recent studies, As(III) oxidizers were oxidase that forms a distinct phylogenetic clade within the isolated from As-rich environments (27,28), as well as metal- dimethyl sulfoxide (DMSO) reductase family. arxA-like genes have contaminated soil containing low levels of As and recently been found in a nearly complete genome sequence of uncontaminated garden soil (29,30). uncultured bacterium within the candidate division OP1 (51) and Arsenite oxidase, which was first isolated in 1992 (31), has been in a reconstructed complete genome of the dominant organism identified in both CAOs and HAOs (24,32). In both cases, the (RBG-1) in deep sediment of the Colorado River, CO, USA (52). enzyme contains two subunits, a large subunit containing a As(III)-oxidizing denitrifying chemoautotrophs (strains DAO-1 and molybdopterin center and a [3Fee4S] cluster and a small subunit DAO-10 within the classes b- and a-Proteobacteria, respectively) containing a Rieske [2Fee2S] cluster (33,34). Although homologous have been isolated from As-contaminated soils, but it is still genes encoding these two subunits were formerly assigned unclear which genes are required for As(III) oxidation (53). different names (aoxB-aoxA/aroA-aroB/asoA-asoB), nomenclature for genes involved in prokaryotic aerobic As(III) oxidation was recently unified and the name aio was newly assigned; therefore, AS(V) REDUCTION the large and small subunit are denoted aioA and aioB, respectively (35). AioA is similar to the molybdenum-containing subunits in the A wide variety of bacteria known as As(V)-resistant microbes DSMO reductase family and distantly related to the catalytic sub- (ARMs) can reduce As(V) via detoxification systems (15,54). As(V) unit of respiratory As(V) reductase (ArrA) (2,21,22). usually enters bacterial cells through phosphate transporters (Pit or Homologs of genes encoding AioA are found in phylogenetically Pst). Once inside, As(V) is reduced to As(III) by a cytoplasmic As(V) diverse strains including members of a-, b-, g-Proteobacteria, Bac- reductase (ArsC) with the aid of glutathione or ferredoxin as the teroidetes, Actinobacteria, Firmicutes, Aquificae, Deinococcus-Ther- reducing power. As(III) is finally excreted out of the cells via a mus, Chlorobi, Chloroflexi, Nitrospira, and Crenarchaeota (Fig. 1). membrane efflux pump, ArsB or Acr3 (55). In some cases, an ATPase These genes are found clustered in several groups in the AioA- ArsA is bound to ArsB to facilitate As(III) efflux, conferring an based tree, with strains in the phyla including thermophiles basi- advantage to organisms exposed to high levels of As. As(III), which cally forming distinct phylogenetic branches from mesophiles. The enters bacterial cells through aquaglyceroporin, may also be major mesophile branches are divided into two groups, group I, extruded by the same system. which is mainly composed of a-Proteobacteria, and group II, which In addition to the detoxifying As(V) reduction, certain bacteria is primarily composed of b- and g-Proteobacteria. This pattern can reduce As(V) as the terminal electron acceptor for anaerobic suggests that these groups probably originated from respective respiration. Such bacteria are defined as dissimilatory As(V)- proteobacterial divisions. However, there are considerable in- reducing prokaryotes (DARPs). These bacteria are phylogenetically consistencies between the AioA-based phylogeny and 16S rRNA- diverse, including members of Firmicutes, g-, d-, and ε-Proteobac- based classification, suggesting that horizontal gene transfer plays a teria (Fig. 2) (15). The respiratory As(V) reductase (ARR) consists of role in the propagation of aio genes in prokaryotes (36). In some a larger catalytic subunit ArrA and a smaller subunit ArrB (56). cases, two identical copies of the aioA gene have been found in ArrA is a member of the DMSO reductase family containing a same strain. For example, the DGGE profile of Thiomonas arsen- molybdenum center and a [4Fee4S] cluster, while ArrB contains ivorans DSM 16361 showed two bands corresponding to two three to four [4Fee4S] clusters. ARR of Alkaliphilus oremlandii and distinct aioA-related sequences (37), whereas two copies of aioA in Shewanella sp. ANA-3, which are both As(V)-respiring bacteria, Ancylobacter sp. OL1 were clustered more closely (38). was recently found to be biochemically reversible (57), showing aioA-like genes have been amplified from a variety of As-rich both As(III) oxidation and As(V) reduction activities upon in vitro environments including mine, arsenical pesticide- or smelter- gel assay. Richey et al. (57) suggested that the physiological role of impacted sites, and geothermal sites (36,37,39e43). Additionally, ARR depends on the electron potentials of the molybdenum center Engel et al. (44) recently detected Chloroflexi and Proteobacteria- and [FeeS] clusters, additional subunits, or constitution of the related aioA sequences from the same microbial mat collected at a electron transfer chain. geyser. Taken together, these investigations suggest that the di- As sorption onto metal oxide minerals, especially on iron (hydr) versity of aioA genes in prokaryotes is wider than previously sus- oxides, is an important process controlling the dissolved concen- pected. Moreover, aioA-like genes have been obtained from soil or tration of As in various environments. As(V) is strongly associated sediments containing background levels of As (16,45), indicating with iron and aluminum (hydr)oxides, whereas As(III) is more that diverse aerobic As(III) oxidizers reside in the environment, mobile than As(V) (58). Thus, reductive dissolution of As-bearing regardless of As contamination. iron (hydr)oxides by dissimilatory iron-reducing bacteria can cause As release (59,60). In addition, direct reduction of As(V) adsorbed Anaerobic As(III) oxidizers In 2002, Oremland et al. (46) onto soil minerals by DARPs may be another important mechanism isolated an anaerobic As(III)-oxidizing bacterium, strain MLHE-1, of As mobilization (61,62). ARMs are generally not considered to be from anoxic bottom water of Mono Lake, CA, USA, which is an involved in As release because ArsC, the cytoplasmic As(V) reduc- alkaline soda lake known for its high concentration of As(V) tase, is not able to directly reduce As(V) adsorbed onto soil minerals (200 mM). Strain MLHE-1, later proposed as Alkalilimnicola ehrlichii (63). Therefore, ARR is believed to be responsible for As(V) reduc- sp. nov. (47), is a chemolithoautotrophic bacterium that can tion in solid phase, although how periplasmic ARR transfers VOL.118,2014 MICROBIOLOGY OF INORGANIC ARSENIC 3

Marinobacter santoriniensis NKSG1 (ACF09051) Pseudomonas sp. 72 (ABY19330) Pseudomonas sp. 89 (ABY19328) Thiomonas arsenivorans DSM16361_band2 (ADE33057) Pseudomonas sp. 1 (ABY19326) Pseudomonas sp. 73 (ABY19327) Pseudomonas stutzeri TS44 (ACB05943) Achromobacter sp. 40AGIII (AEL22195) Achromobacter sp. NT-10 (ABD72610) Alcaligenes sp. S46 (ADF47192) Alcaligenes sp. YI13H (ABY19322) Alcaligenes sp. T12RB (ABY19321) Achromobacter sp. WA20 (ABD72615) Ralstonia sp. 22 (ABY19329) Achromobacter arsenitoxydans SY8 (ABP63660) Alcaligenes faecalis NCIB8687 (Q7SIF4) Burkholderia sp. YI019A (ABY19323) Burkholderia sp. S31R (ADF47190) β Burkholderia sp. S232 (ADF47189) -Proteobacteria Burkholderia sp. S32 (ADF47191) Burkholderia sp. S222 (ADF47188) Herminiimonas arsenicoxydans ULPAs1 (AAN05581) Thiomonas sp. WJ68 (ABY19318) Thiomonas arsenivorans DSM16361 (ABY19316) Thiomonas arsenivorans DSM16361_band1 (ADE33058) Micromonospora sp. X14 (CCD32987) Actinobacteria Thiomonas sp. NO115 (ABY19317) Acidovorax sp.75 (ABY19324) Hydrogenophaga sp NT-14 (ABD72609) Group II Hydrogenophaga sp. WA13 (ABD72613) Acinetobacter sp. WA19 (ABD72614) Limnobacter sp.83 (ABY19325) Polaromonas sp.GM1 (ABW84260) Variovorax sp. MM-1 (AFN80467) Variovorax sp. RM1 (ABD35886) Variovorax sp.4-2 (ABY19319) Leptothrix sp.S1-1 (ABY19320) Flavobacterium sp.19AAV (AEL22198) Bacteroidetes Acinetobacter sp.18AAV (AEL22197) Acinetobacter sp.16AAV (AEL22196) Ralstonia sp. R229 (CCA82914) Ralstonia solanacearum PSI07 (YP_003749888) Ralstonia syzygii R24 (CCA86643) Acinetobacter sp.33 (ABY19331) Pseudomonas sp.5AAV (AEL22194) Pseudomonas sp.D2OHCJ (ABY19332) Rhodococcus sp.46AAIII (AEL22193) Pseudomonas sp. 46 (ABY19333) Flavobacterium sp.16AGV (AEL22190) Flavobacterium sp.18AGV (AEL22191) γ Pseudomonas sp.15AGV (AEL22189) -Proteobacteria Pseudomonas sp. 20AAIII (AEL22181) Achromobacter sp. 38AGIII (AEL22188) Pseudomonas sp. 25AAIII (AEL22184) Pseudomonas sp. 25AGIII (AEL22187) Pseudomonas sp. 24AGIII (AEL22186) Bacillus sp. 21AAIII (AEL22182) Firmicutes Stenotrophomonas sp. MM-7 (AFN80468) Polymorphum gilvum (YP_004304060) Bosea sp.43AGV (AEL22179) Agromyces sp. 44AGV (AEL22180) Bosea sp. S41RM2 (ADF47199) Bosea sp. S41RM1 (ADF47198) Bosea sp.WAO (ABJ55855) Bosea sp. 7AGV (AEL22177) Bosea sp. 8AGV (AEL22178) Thiobacillus sp. S1 (ABJ55851) Hydrogenophaga sp. CL3 (ABJ55854) Ancylobacter sp. OL1_2 (ABJ55853) Ancylobacter sp. OL1_1 (ABJ55852) Ancylobacter dichloromethanicus As3-1b (CBY79896) Group I α-Proteobacteria Methylobacterium sp. S47 (ADF47200) Nitrobacter hamburgensis (YP_571843) Methylocystis sp. SC2 (CCJ06851) Aminobacter sp. 86 (ABY19334) Sinorhizobium sp. DAO10 (ABJ55850) Mesorhizobium sp. DM1 (ABD35887) Ensifer adhaerens (CBY79895) Sinorhizobium sp. IK-A2 (AGC82137) Agrobacterium sp. Ben-5 (ABD72612) Rhizobium sp. NT-26 (AAR05656) Agrobacterium tumefaciens 5A (ABB51928) Agrobacterium sp. TS45 (ACB05955) Ochrobactrum tritici (ACK38267) Sinorhizobium sp. NT-4 (ABD7261) Candidatus Nitrospira defluvii (YP_003799308) Nitrospira Chloroflexus sp. Y-400-fl (YP_002569069) Chloroflexus aurantiacus J-10-fl (YP_001634827) Chloroflexi Thermus thermophilus (BAD71923) Thermus sp. HR13 (ABB17184) Deinococcus-Thermus Thermus scotoductus SA-01 (ADW22085) Chloroflexus aggregans DSM9485 (YP_002461759) Chlorobium phaeobacteroides BS1 (YP_001960747) Chlorobium limicola DSM245 (YP_001942454) Chlorobi Sulfurihydrogenibium sp. Y04ANG1 (ACN59445) Sulfurihydrogenibium yellowstonense SS-5 (ACN59446) Aquificae Hydrogenobaculum sp. 3684 (ACJ04807) Sulfolobus tokodaii str.7 (BAB67500) Pyrobaculum oguniense TE7 (AFA38559) Aeropyrum pernix K1 (BAA81573) Crenarchaeota Aeropyrum camini SY1 (BAN91066) Roseobacter litoralis Och149 (YP_004691143) Vibrio splendidus LGP32 (YP_002395243) ArrA Chrysiogenes arsenatis (AAU11839)

0.2

FIG. 1. Neighbor-joining phylogenetic tree of AioA sequences found in isolated strains. The ArrA of Chrysiogenes arsenatis was used as the outgroup. Circles and triangles at the branch nodes represent bootstrap percentages (1000 replicates): ﬁlled circles, 90e100%; open circles, 70e89%; open triangles, 50e69%. Values <50% are not shown. The scale bar represents the estimated number of substitutions per site. electrons to As(V) adsorbed onto mineral surface is still unclear. Sulfurospirillum spp. The ﬁrst DARP, strain MIT-13, was iso- Below, we describe the physiological and biochemical aspects of lated in 1994 from As contaminated sediments of the Aberjona representative DARPs, as well as the genomic organization of the Watershed, MA, USA (64). This strain, which was later proposed genes involved in respiratory As(V) reduction and As resistance. as Sulfurospirillum arsenophilum (65), is a member of the 4 YAMAMURA AND AMACHI J. BIOSCI.BIOENG.,

ArrA

ArxA

FIG. 2. Neighbor-joining phylogenetic tree of ArrA and ArxA sequences found in isolated strains. The AioA of Rhizobium sp. NT-26 was used as the outgroup. Circles and triangles at the branch nodes represent bootstrap percentages (1000 replicates): ﬁlled circles, 90e100%; open circles, 70e89%; open triangles, 50e69%. Values <50% are not shown. The scale bar represents the estimated number of substitutions per site.

ε-Proteobacteria that can use As(V) as the electron acceptor when Mn(IV), fumarate, elemental sulfur, sulfite, and thiosulfate (75).In lactate is supplied as the electron donor and carbon source. a genome of D. hafniense DCB-2, arsenic-metabolizing genes are Several other members of Sulfurospirillum spp., including encoded on an arsenic island (Fig. 3). In addition to arrA and arrB Sulfurospirillum barnesii, S. multivorans, and S. halorespirans,are genes, genes encoding a NrfD-like membrane protein (ArrC), a also capable of dissimilatory As(V) reduction (66). Many strains of TorD-like chaperone protein (ArrD), and putative regulatory Sulfurospirillum spp. can use nitrate, nitrite, Fe(III), elemental proteins involved in the two-component signal transduction sulfur, thiosulfate, and oxygen (microaerobic) as the electron system (ArrR, ArrS, and ArrT) are present. Furthermore, As acceptors in addition to As(V). S. barnesii has been found to detoxification genes encoding a putative transcriptional repressor reduce As(V) adsorbed on ferrihydrite as well as on aluminum (ArsR), an As(III) chaperone protein (ArsD), and an ATPase (ArsA) hydroxide (62). S. arsenophilum also released As from sterilized are present, but are encoded on the opposite strand. Aberjona sediments (61). Chrysiogenes arsenatis C. arsenatis, which is a phylogeneti- Shewanella sp. ANA-3 Shewanella sp. ANA-3 was isolated cally unique member of the phylum Chrysiogenetes, was isolated from an As-treated wooden pier located in a brackish estuary (76). from Australian gold mine wastewater as an acetate-using This organism can also use nitrate, Fe(III), Mn(IV), thiosulfate, dissimilatory As(V)-reducer that also uses nitrate and nitrite as fumarate, and oxygen as electron acceptors. Because Shewanella electron acceptors in the presence of acetate (67). ARR of spp. are genetically tractable, comprehensive molecular C. arsenatis has been purified and characterized (68). Specifically, characterization of dissimilatory As(V) reduction has been performed using this strain. ARR of Shewanella sp. ANA-3 is a it is a heterodimer (a1b1) consisting of two subunits (ArrA and ArrB) that is located in the periplasmic space. The expression of periplasmic enzyme that is expressed during the exponential and fi ARR is induced in the presence of As(V). stationary phases (77). Interestingly, ARR is nally released from the cells into the surrounding environment (77). The expression fi Bacillus spp. To date, at least ve Bacillaceae, Bacillus arsen- dynamics of arrA in strain ANA-3 were determined and compared icoselenatis (69), B. selenitireducens (69), B. macyae (70), by Saltikov et al. (78), and the results revealed that it is only B. selenatarsenatis (71), and B. beveridgei (72), have been isolated expressed anaerobically, while it is repressed by other electron as dissimilatory As(V)-reducing bacteria. Among these, acceptors such as oxygen, nitrate, and fumarate. Conversely, arsC B. selenatarsenatis strain SF-1 is able to release As from iron and is expressed under both aerobic and anaerobic conditions. arrA is aluminum (hydr)oxides coprecipitated with As(V), as well as from also induced by 100 nM As(III), while 1000 times more As(III) is As-contaminated soils (73). The membrane-bound ARR of required for the induction of arsC. A gene encoding a membrane- fi B. selenitireducens has been puri ed and characterized (74). associated tetraheme c-type cytochrome, cymA, is required for Desulfitobacterium spp. Niggemyer et al. (75) isolated a ANA-3 to grow on As(V) (79). This cytochrome is also dissimilatory As(V)-reducing bacterium, Desulfitobacterium sp. indispensable for growth on Fe(III), Mn(IV), and fumarate. When strain GBFH, from As-contaminated sediments. They also incubated with As(V)-adsorbed ferrihydrite, a Fe(III) reduction demonstrated that Desulfitobacterium hafniense DCB-2 and deficient mutant of strain ANA-3 effectively reduced solid-phase D. frappieri PCP-1, which are both known to be reductively As(V), while an As(V) reduction deficient mutant did not (80). dechlorinating bacteria, can also respire As(V). These These findings strongly suggest that the Fe(III) reduction pathway Desulfitobacterium strains are metabolically versatile and can use is not required for ferrihydrite-adsorbed As(V) reduction. The a wide variety of electron acceptors including Fe(III), Se(VI), organization of arrA and arrB genes in the genome of strain ANA- VOL.118,2014 MICROBIOLOGY OF INORGANIC ARSENIC 5

arsA arsD arrTarrS arrR arrC arrAarrB arrD arsR

arsC arsB arsA arsD arrA arrB

acr3 arsR arrT arrS arrR arrF arrA arrB arrD arrE

acr3 arsR arrAarrB arrD arrE arrF

FIG. 3. Arsenic islands found in the genomes of Desulﬁtobacterium hafniense DCB-2 (110), Shewanella sp. ANA-3, Geobacter lovleyi SZ (111), and Geobacter uraniireducens Rf4. Gene organizations surrounding respiratory As(V) reductase genes (arr) are shown. Note that D. hafniense DCB-2 and Shewanella sp. ANA-3 are dissimilatory As(V)-reducing bacteria (75,76), while the other two Geobacter strains are not able to grow using As(V) as the electron acceptor (86). The arr genes and putative functions of their products are described according to Reyes et al. (112). arrA, respiratory As(V) reductase catalytic subunit; arrB, respiratory As(V) reductase small subunit; arrC, a NrfD-like membrane protein that may function as an anchor for ArrAB; arrD, a TorD-like chaperone that may be involved in cofactor insertion into ArrA; arrE, a putative FeeS protein; arrF, a putative multiheme c-type cytochrome; arrR, arrS, and arrT, putative regulatory proteins involved in two-component signal transduction systems; arsA, an ATPase bound to ArsB; arsB and acr3, membrane- bound As(III) efﬂux proteins; arsC, a cytoplasmic As(V) reductase; arsD, a chaperone protein that transfers As(III) to the ArsAB pump; arsR, a putative transcriptional repressor. An asterisk indicates that arrB of G. uraniireducens Rf4 contains a stop codon (86).

3 is shown in Fig. 3. Genes encoding ArsD, ArsA, ArsB (a membrane flooded soils and anoxic sediments (87). Preliminary draft genome efflux pump), and ArsC are also present, which may confer strain analysis of strain OR-1 identified two arsenic islands containing ANA-3 As resistance (76). multiple arsA and arsD genes (Ehara and Amachi, unpublished data). Geobacter spp. Geobacter spp., which are the most common and ubiquitous iron-reducing bacteria, play a pivotal role in Anaeromyxobacter sp. PSR-1 Anaeromyxobacter dehalo- dissimilatory iron reduction in terrestrial and freshwater environ- genans was isolated by Sanford et al. (88) as a reductively ments (81). Recent studies have demonstrated that Geobacter spp. dechlorinating d-Proteobacterium that grows using 2- also function as dissimilatory As(V)-reducing bacteria, and may chlorophenol as an electron acceptor. This organism has a gliding play a role in As reduction and mobilization. Islam et al. (60) motility, forms a spore-like structure, and can also use nitrate, found that As release from West Bengal sediments occurred Fe(III), U(VI), Se(IV), fumarate, and oxygen (microaerobic) as e when they were incubated anaerobically with acetate as the electron acceptors (89 91). Kudo et al. (92) recently isolated a electron donor, and subsequent 16S rRNA gene clone library dissimilatory As(V)-reducing bacterium, strain PSR-1, from As- analysis indicated that 70% of clones in the acetate-amended contaminated soil and found that it had 99.7% 16S rRNA gene sediments were affiliated with the family Geobacteraceae. Similar similarity with A. dehalogenans. As release was observed when results were observed upon microbial community analysis of strain PSR-1 was incubated with As(V)-adsorbed ferrihydrite or Cambodian sediments (82). In addition, dissimilatory As(V) sterile As-contaminated soil. Multiple attempts to amplify the reductase genes (arrA) closely related to Geobacter spp. have putative arrA gene from genomic DNA of strain PSR-1 were frequently been detected in As-contaminated sediments (83e86). unsuccessful, indicating that strain PSR-1 may harbor an atypical fi Until recently, only two Geobacter species, Geobacter arrA gene that cannot be ampli ed using previously designed uraniireducens Rf4 and G. lovleyi SZ, had been known to possess degenerate primers (92). Although complete genome sequences putative arrA and arrB genes in their genomes. However, neither of several strains of Anaeromyxobacter spp. have been released, of these strains are able to grow using As(V) as the electron none possess putative genes for arrA and arrB. acceptor, even though resting cells of G. lovleyi SZ reduce As(V) in the presence of acetate (86). Various arr genes with an operon- APPLICATION TO BIOREMEDIATION like structure are present in the G. lovleyi SZ genome, but it appears to lack the arsA and arsD genes (Fig. 3). An arsenic island Removal from water As described above, As(III) is less found in the genome of G. uraniireducens Rf4 also lacks the arsA adsorptive than As(V); therefore, As(III) oxidation is an important and arsD genes. Considering that both of these As detoxifying pretreatment process for adsorption and coprecipitation using Al/ genes are frequently found in other dissimilatory As(V)-reducing Fe(III) minerals. Because chemical oxidation of As(III) via oxygen is bacterial genomes, their incapacity to grow on As(V) might be in very slow (93), application of aerobic As(III) oxidizers can be an part due to the absence of arsA and arsD genes. effective remediation strategy for removal of As from In 2013, Ohtsuka et al. (87) isolated a new dissimilatory As(V)- contaminated water. For example, Ike et al. (94) successfully reducing bacterium, Geobacter sp. OR-1, from As-contaminated obtained an enrichment culture with a soil free from As Japanese paddy soil. Strain OR-1 also used nitrate, Fe(III), and contamination that showed high As(III)-oxidizing activity and fumarate as electron acceptors and catalyzed the release of As from greatly enhanced As adsorption by activated alumina. Three As(V)-adsorbed ferrihydrite or sterilized paddy soil. Furthermore, aerobic HAOs, presumably classified as Haemophilus spp., As K-edge X-ray absorption near-edge structure (XANES) analysis Micrococcus spp., and Bacillus spp., were isolated from their suggested that strain OR-1 reduced As(V) directly in the soil solid enrichment culture. Andrianisa et al. (95) demonstrated that an phase. Because strain OR-1 is the first Geobacter species with the activated sludge collected from a treatment plant receiving no As- capacity to grow using As(V) as the sole electron acceptor, it may be contaminated wastewater rapidly oxidized As(III). Furthermore, useful as a model microorganism influencing mobilization of As in they isolated an aerobic CAO through subculture of the sludge 6 YAMAMURA AND AMACHI J. BIOSCI.BIOENG., without an organic carbon source. They also found that biological As-contaminated soil and developed a mathematical model that As(III) oxidation can occur in a full-scale plant based on field provides a framework for understanding and predicting the investigations of an oxidation ditch activated sludge process dissolution of As from soil. Lee et al. (109) applied a combination of receiving As-contaminated wastewater. In their investigation, microbial As mobilization and electrokinetic remediation to As- oxidized As in the effluent was shown to be effectively removed by contaminated mine tailing soils. In both batch- and column-type coprecipitation with ferric hydroxide. In a recent study by the bioleaching reactors, stimulation of indigenous bacteria by the in- same group, a continuous-flow bioreactor with immobilized jection of organic carbon sources led to the reductive dissolution of aerobic As(III)-oxidizing bacteria was performed as a pretreatment As. Subsequent electrokinetic treatment enhanced As removal ef- step for As removal from groundwater (96). ficiency, and the combined process achieved about 67% removal of Anaerobic CAOs can be also used as an alternative process for As from soil containing 4023 mg/kg of As. Although similar removal pre-oxidation of As(III). Sun et al. (97) constructed two different efficiency was only obtained during electrokinetic treatment, the continuous bioreactors with a denitrifying granular sludge and a preliminary microbial As mobilization reduced the duration of methanogenic granular sludge. The anoxic oxidation of As(III) electrokinetics, with the combined process resulting in a 26.4% cost linked to chemolithotrophic denitrification was continuously reduction. Because the treatment methods available for As- observed in both bioreactors. They inoculated the As(III)-oxidizing contaminated soils are mainly soil replacement, containment, and denitrifying granular biofilms into continuous flow columns packed solidification/stabilization, these studies indicate a great potential with activated alumina and found that nitrate-dependent oxidation for application of DARPs as a novel bioremediation strategy for As of As(III) enhanced adsorption and immobilization of As onto removal from soils. activated alumina (98). In another study, simultaneous oxidation of In conclusion, the history of the interaction between As and As(III) and Fe(II) linked to denitrification was performed in prokaryotes considerably exceeds that of its interaction with hu- continuous flow sand columns inoculated with As(III)-oxidizing man beings. Although the microbiological study of As is a century denitrifying sludge (99). The oxidation of soluble Fe(II) led to for- old, we still have only a limited understanding of the ancient pro- mation of a mixture of Fe(III) oxides dominated by hematite, cesses by which prokaryotes utilize or tolerate As. The more we resulting in enhanced immobilization of As in the column. These learn about them, the more complex they appear to become. investigations imply that anaerobic treatment has the potential for However, we believe that the development of research in this field both nitrate and As removal from water in a single system. can provide the key to handling this toxic and ubiquitous metalloid. Under greater reducing conditions, the opposite approach, namely As(V) reduction, can be used to remove As from the aqueous phase because As(III) forms precipitates with sulfide (100). ACKNOWLEDGMENTS Formation of As sulfides or decreases in aqueous As resulting from microbial As(V) and sulfate reduction have been observed in several This work was partly supported by JSPS KAKENHI Grant bioreactors (101e103). Additionally, Upadhyaya et al. (102) Numbers 23681005 for S. Y. and 23580103 for S. A. In addition, S. A. demonstrated simultaneous removal of nitrate and As from a is grateful to A. Ehara and M. 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