Shigeki Yamamura

Microbiology of inorganic arsenic : From metabolism to bioremediation

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.

Dissimilatory arsenate reduction by a facultative anaerobe, Bacillus sp. strain SF-1

Bacillus sp. strain SF-1, isolated first as a selenate-reducing bacterium, was characterized as a novel arsenate-reducing bacterium. Strain SF-1 rapidly reduced 10 mM levels of arsenate to arsenite with concomitant cell growth and lactate oxidation under anoxic conditions, indicating that arsenate can act as the terminal electron acceptor for anaerobic respiration (dissimilatory arsenate reduction). Strain SF-1 can use various organic compounds including synthetic sewage mainly composed of peptone and meat extract as the electron donors for arsenate reduction. Although strain SF-1 can grow aerobically, which is very rare for dissimilatory arsenate-reducing bacteria, the presence of oxygen inhibited the arsenate reduction. On the other hand, the presence of nitrate or selenate, which can support the growth of strain SF-1 as electron acceptors, did not significantly inhibit the arsenate reduction. Arsenate-reducing activity, that is, arsenate reductase, was exhibited in strain SF-1 only when grown on arsenate, but the enzyme could not reduce other oxyanions including nitrate and selenate. It was presumed that arsenate reduction was carried out by an enzyme system separate from those of nitrate and selenate reduction, and the arsenate reductase was inducible and specific for arsenate. These results suggest that strain SF-1 may be utilized for extracting arsenic from contaminated soil for the purpose of bioremediation.

Pollution of montane soil with Cu, Zn, As, Sb, Pb, and nitrate in Kanto, Japan.

Soil cores and rainwater were sampled under canopies of Cryptomeria japonica in four montane areas along an atmospheric depositional gradient in Kanto, Japan. Soil cores (30cm in depth) were divided into 2-cm or 4-cm segments for analysis. Vertical distributions of elemental enrichment ratios in soils were calculated as follows: (X/Al)(i)/(X/Al)(BG) (where the numerator and denominator are concentration ratios of element-X and Al in the i- and bottom segments of soil cores, respectively). The upper 14-cm soil layer showed higher levels of Cu, Zn, As, Sb, and Pb than the lower (14-30cm) soil layer. In the four areas, the average enrichment ratios in the upper 6-cm soil layer were as follows: Pb (4.93)>or=Sb (4.06)>or=As (3.04)>Zn (1.71)>or=Cu (1.56). Exogenous elements (kg/ha) accumulated in the upper 14-cm soil layer were as follows: Zn (26.0)>Pb (12.4)>Cu (4.48)>or=As (3.43)>or=Sb (0.49). These rank orders were consistent with those of elements in anthropogenic aerosols and polluted (roadside) air, respectively, indicating that air pollutants probably caused enrichment of these elements in the soil surface layer. Approximately half of the total concentrations of As, Sb, and Pb in the upper 14-cm soil layer were derived from exogenous (anthropogenic) sources. Sb showed the highest enrichment factor in anthropogenic aerosols, and shows similar deposition behavior to NO(3)(-), which is a typical acidic air pollutant. There was a strong correlation between Sb and NO(3)(-) concentrations in rainfall (e.g., in the throughfall under C. japonica: [NO(3)(-)]=21.1 [dissolved Sb], r=0.938, p<0.0001, n=182). Using this correlation, total (cumulative) inputs of NO(3)(-) were estimated from the accumulated amounts of exogenous Sb in soils, i.e., 16.7t/ha at Mt. Kinsyo (most polluted), 8.6t/ha at Mt. Tsukuba (moderately polluted), and 5.8t/ha at the Taga mountain system (least polluted). There are no visible ecological effects of these accumulated elements in the Kanto region at present. However, the concentrations of some elements are within a harmful range, according to the Ecological Soil Screening Levels determined by the U.S. Environmental Protection Agency.

Effect of Antibiotics on Redox Transformations of Arsenic and Diversity of Arsenite-Oxidizing Bacteria in Sediment Microbial Communities

In the present study, we investigated the effect of antibiotics on microbial arsenate (As(V)) reduction and arsenite (As(III)) oxidation in sediments collected from a small pond and eutrophic lake. The As(V)-reducing activities were less susceptible to chloramphenicol in aerobic conditions than in anaerobic conditions. Aerobic As(V) reduction proceeded in the presence of diverse types of antibiotics, suggesting that As-resistant bacteria are widely antibiotic resistant. In contrast, some antibiotics, e.g., chloramphenicol, strongly inhibited aerobic As(III) oxidation. In addition, bacterial As(III) oxidase genes were scarcely amplified and Proteobacteria -related 16S rRNA genes drastically decreased in chloramphenicol-amended cultures. Erythromycin and lincomycin, which successfully target many Gram-positive bacteria, scarcely affected As(III) oxidation, although they decreased the diversity of As(III) oxidase genes. These results indicate that the aerobic As(III) oxidizers in the sediment cultures are mainly composed of Proteobacteria and are more sensitive to certain types of antibiotics than the aerobic As(V) reducers. Our results suggest that antibiotic disturbance of environmental microbial communities may affect the biogeochemical cycle of As.

Ecological niche separation in the Polynucleobacter subclusters linked to quality of dissolved organic matter: a demonstration using a high sensitivity cultivation-based approach

The free-living, cosmopolitan, freshwater betaproteobacterial bacterioplankton genus Polynucleobacter was detected in different years in 11 lakes of varying types and a river using the size-exclusion assay method (SEAM). Of the 350 strains isolated, 228 (65.1%) were affiliated with the Polynucleobacter subclusters PnecC (30.0%) and PnecD (35.1%). Significant positive correlations between fluorescence in situ hybridization and SEAM data were observed in the relative abundance of PnecC and PnecD bacteria to Polynucleobacter communities (PnecC + PnecD). Isolates were mainly PnecC bacteria in the samples with a high specific UV absorbance at 254 nm (SUVA(254) ), and a low total hydrolysable neutral carbohydrate and amino acid (THneutralCH + THAA) content of the dissolved organic matter (DOM) fraction, which is known to be correlated with a high humic content. In contrast, the PnecD bacteria were abundant in samples with high chlorophyll a and/or THneutralCH + THAA concentrations, indicative of primary productivity. With few exceptions, differences in the relative abundance of PnecC and PnecD in each sample, determined using a high-sensitivity cultivation-based approach, were due to DOM quality. These results suggest that the major DOM component in the field, which is allochthonously or autochthonously derived, is a key factor for ecological niche separation between PnecC and PnecD subclusters.

Potential for microbially mediated redox transformations and mobilization of arsenic in uncontaminated soils.

Surface soil samples, which had no significant As contamination, were examined for As(V) reduction, As(III) oxidation and As mobilization capability. All five soil samples tested exhibited microbial As(V)-reducing activities both in aerobic and anaerobic conditions. Under aerobic conditions when As(V) reduction had almost ceased, oxidation of As(III) to As(V) occurred, whereas only As(V) reduction was observed under anaerobic conditions. In cultures incubated with As(III), As(III) was oxidized by indigenous soil microbes only under aerobic conditions. These results indicate that microbial redox transformations of As are ubiquitous in the natural environment regardless of background As levels. Mobilization through microbially mediated As(V) and Fe(III) reduction occurred both in the presence and absence of oxygen. Significant variation in dissolved As occurred depending on the Fe contents of soils, and re-immobilization of As arose in the presence of oxygen, presumably as a consequence of dissolved As(III) and Fe(II) oxidation. There was no apparent correlation between dissolved Fe(II) and As, suggesting that reductive dissolution of Fe(III) minerals does not necessarily determine the extent of As release from soils.

Microbial biomass and nitrogen transformations in surface soils strongly acidified by volcanic hydrogen sulfide deposition in Osorezan, Japan

Abstract Volcanic acidification has created unique ecosystems that have had to adapt to the acidic environments in volcanic regions. To characterize the primary microbial properties of strongly acidified soils in such environments, we investigated microbial biomass, nitrogen transformations and other relevant chemical properties in the surface soils of solfatara and forests from Osorezan, a typical volcanic region in Japan, and compared the results to common Japanese forest soils. Soil microbial biomass C (MBC) and N (MBN) were determined using the chloroform fumigation–extraction method. Potential net N mineralization and net nitrification were measured in aerobic laboratory incubations. Long-term acidification in the Osorezan soils by volcanic hydrogen sulfide deposition caused low soil pH (3.0–3.8), base cation deficiency and increased concentrations of toxic ions such as Al3+. The proportions of MBC to total carbon (MBC/TC ratio) and MBN to total nitrogen (MBN/TN ratio) were lower than those in common Japanese forest soils. The extreme acidic conditions may have inhibited microbial survival in the Osorezan acid soils. Net N mineralization occurred at rates comparable to those in common Cryptomeria japonica forest soils, probably because of the presence of acid-tolerant soil microorganisms. Net nitrification was completely inhibited and autotrophic ammonia oxidizers were not detected by the MPN method. The inhibition of nitrification prevents nitrogen leaching from the soils, thus maintaining a nitrogen cycle in the volcanic acid region in which NH+ 4 (and NH3) is recycled among microorganisms and plants.

Diversity of alkane hydroxylase genes on the rhizoplane of grasses planted in petroleum-contaminated soils

The study investigated the diversity and genotypic features of alkane hydroxylase genes on rhizoplanes of grasses planted in artificial petroleum-contaminated soils to acquire new insights into the bacterial communities responsible for petroleum degradation in phytoremediation. Four types of grass (Cynodon dactylon, two phenotypes of Zoysia japonica, and Z. matrella) were used. The concentrations of total petroleum hydrocarbon effectively decreased in the grass-planted systems compared with the unplanted system. Among the representative alkane hydroxylase genes alkB, CYP153, almA and ladA, the first two were detected in this study, and the genotypes of both genes were apparently different among the systems studied. Their diversity was also higher on the rhizoplanes of the grasses than in unplanted oil-contaminated soils. Actinobacteria-related genes in particular were among the most diverse alkane hydroxylase genes on the rhizoplane in this study, indicating that they are one of the main contributors to degrading alkanes in oil-contaminated soils during phytoremediation. Actinobacteria-related alkB genes and CYP153 genes close to the genera Parvibaculum and Aeromicrobium were found in significant numbers on the rhizoplanes of grasses. These results suggest that the increase in diversity and genotype differences of the alkB and CYP153 genes are important factors affecting petroleum hydrocarbon-degrading ability during phytoremediation.

Effect of extracellular electron shuttles on arsenic-mobilizing activities in soil microbial communities

Microbially mediated arsenate (As(V)) and Fe(III) reduction play important roles in arsenic (As) cycling in nature. Extracellular electron shuttles can impact microbial Fe(III) reduction, yet little is known about their effects on microbial As mobilization in soils. In this study, microcosm experiments consisting of an As-contaminated soil and microbial communities obtained from several pristine soils were conducted, and the effects of electron shuttles on As mobilization were determined. Anthraquinone-2,6-disulfonate (AQDS) and riboflavin (RF) were chosen as common exogenous and biogenic electron shuttles, respectively, and both compounds significantly enhanced reductive dissolution of As and Fe. Accumulation of Fe(II)-bearing minerals was also observed, which may lead to re-immobilization of As after prolonged incubation. Interestingly, Firmicutes-related bacteria became predominant in all microcosms, but their compositions at the lower taxonomic level were different in each microcosm. Putative respiratory As(V) reductase gene (arrA) analysis revealed that bacteria closely related to a Clostridia group, especially those including the genera Desulfitobacterium and Desulfosporosinus, might play significant roles in As mobilization. These results indicate that the natural soil microbial community can use electron shuttles for enhanced mobilization of As; the use of this type of system is potentially advantageous for bioremediation of As-contaminated soils.

Unexpected Diversity of pepA Genes Encoding Leucine Aminopeptidases in Sediments from a Freshwater Lake

We herein designed novel PCR primers for universal detection of the pepA gene, which encodes the representative leucine aminopeptidase gene, and investigated the genetic characteristics and diversity of pepA genes in sediments of hypereutrophic Lake Kasumigaura, Japan. Most of the amino acid sequences deduced from the obtained clones (369 out of 370) were related to PepA-like protein sequences in the M17 family of proteins. The developed primers broadly detected pepA-like clones associated with diverse bacterial phyla—Alpha-, Beta-, Gamma-, and Deltaproteobacteria, Acidobacteria, Actinobacteria, Aquificae, Chlamydiae, Chloroflexi, Cyanobacteria, Firmicutes, Nitrospirae, Planctomycetes, and Spirochetes as well as the archaeal phylum Thaumarchaeota, indicating that prokaryotes in aquatic environments possessing leucine aminopeptidase are more diverse than previously reported. Moreover, prokaryotes related to the obtained pepA-like clones appeared to be r- and K-strategists, which was in contrast to our previous findings showing that the neutral metalloprotease gene clones obtained were related to the r-strategist genus Bacillus. Our results suggest that an unprecedented diversity of prokaryotes with a combination of different proteases participate in sedimentary proteolysis.