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Arsenic (As) hyperaccumulator Pteris ferns are renowned for their capacity to accumulate As and have been used to remediate As-contaminated environmental. However, there is less information on how ...

Pteris vittata is an arsenic (As) hyperaccumulator plant that accumulates a large amount of As into fronds and rhizomes (around 16,000 mg/kg in both after 16 weeks hydroponic cultivation with 30 mg/L arsenate). However, the sequence of long-distance transport of As in this hyperaccumulator plant is unclear. In this study, we used a positron-emitting tracer imaging system (PETIS) for the first time to obtain noninvasive serial images of As behavior in living plants with positron-emitting 74As-labeled tracer. We found that As kept accumulating in rhizomes as in fronds of P. vittata, whereas As was retained in roots of a non-accumulator plant Arabidopsis thaliana. Autoradiograph results of As distribution in P. vittata showed that with low As exposure, As was predominantly accumulated in young fronds and the midrib and rachis of mature fronds. Under high As exposure, As accumulation shifted from young fronds to mature fronds, especially in the margin of pinna, which resulted in necrotic symptoms, turning the marginal color to gray and then brown. Our results indicated that the function of rhizomes in P. vittata was As accumulation and the regulation of As translocation to the mature fronds to protect the young fronds under high As exposure.

Grasses are advantageous in the removal of polycyclic aromatic hydrocarbons (PAHs) in soil because of their fibrous root, high tolerance to environmental stress, and low nutritional requirements. In this study, a pot experiment was conducted to test the ability of four grasses to remove PAHs in the soil, and to investigate the corresponding bacterial community shift in the rhizosphere of each. Sudangrass achieved the maximum removal of PAHs at 98% dissipation rate after 20 days. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and next-generation sequencing revealed that sudangrass specially enriched the growth of a known PAHs degrader, Sphingomonadales, regardless of the presence or absence of PAHs in the soil. Moreover, the gene copy numbers of PAHs catabolic genes, PAH-RHDα and nidA, as measured by real time-PCR (RT-PCR) were highest in the soil planted with sudangrass. Overall, this study suggested that sudangrass further enhanced the dissipation of PAHs by enriching Sphingomonadales in its rhizosphere.

The contamination of the environment by crude oil and its by-products, mainly composed of aliphatic and aromatic hydrocarbons, is a widespread problem. Biodegradation by bacteria is one of the processes responsible for the removal of these pollutants. This study was conducted to determine the abilities of Burkholderia sp. B5, Cupriavidus sp. B1, Pseudomonas sp. T1, and another Cupriavidus sp. X5 to degrade binary mixtures of octane (representing aliphatic hydrocarbons) with benzene, toluene, ethylbenzene, or xylene (BTEX as aromatic hydrocarbons) at a final concentration of 100 ppm under aerobic conditions. These strains were isolated from an enriched bacterial consortium (Yabase or Y consortium) that prefer to degrade aromatic hydrocarbon over aliphatic hydrocarbons. We found that B5 degraded all BTEX compounds more rapidly than octane. In contrast, B1, T1 and X5 utilized more of octane over BTX compounds. B5 also preferred to use benzene over octane with varying concentrations of up to 200 mg/l. B5 possesses alkane hydroxylase (alkB) and catechol 2,3-dioxygenase (C23D) genes, which are responsible for the degradation of alkanes and aromatic hydrocarbons, respectively. This study strongly supports our notion that Burkholderia played a key role in the preferential degradation of aromatic hydrocarbons over aliphatic hydrocarbons in the previously characterized Y consortium. The preferential degradation of more toxic aromatic hydrocarbons over aliphatics is crucial in risk-based bioremediation.

Biodegradation is found to be a promising, cost-effective and eco-friendly option for the treatment of industrial wastewater contaminated by 1,4-dioxane (1,4-D), a highly stable synthetic chemical and probable human carcinogen. This study aimed to isolate, identify, and characterize metabolic 1,4-D-degrading bacteria from a stable 1,4-D-degrading microbial consortium. Three bacterial strains (designated as strains TS28, TS32, and TS43) capable of degrading 1,4-D as a sole carbon and energy source were isolated and identified as Gram-positive Pseudonocardia sp. (TS28) and Gram-negative Dokdonella sp. (TS32) and Afipia sp. (TS43). This study, for the first time, confirmed that the genus Dokdonella is involved in the biodegradation of 1,4-D. The results reveal that all of the isolated strains possess inducible 1,4-D-degrading enzymes and also confirm the presence of a gene encoding tetrahydrofuran/dioxane monooxygenase (thmA/dxmA) belonging to group 5 soluble di-iron monooxygenases (SDIMOs) in both genomic and plasmid DNA of each of the strains, which is possibly responsible for the initial oxidation of 1,4-D. Moreover, the isolated strains showed a broad substrate range and are capable of degrading 1,4-D in the presence of additional substrates, including easy-to-degrade compounds, 1,4-D biodegradation intermediates, structural analogs, and co-contaminants of 1,4-D. This indicates the potential of the isolated strains, especially strain TS32, in removing 1,4-D from contaminated industrial wastewater containing additional organic load. Additionally, the results will help to improve our understanding of how multiple 1,4-D-degraders stably co-exist and interact in the consortium, relying on a single carbon source (1,4-D) in order to develop an efficient biological 1,4-D treatment system.

Pteris vittata L., a hyperaccumulator of As, has great potential in environmental remediation. Localization analysis of As and Ca, K, S, and P in arsenic-exposed and unexposed P. vittata pinnae was performed using an electron probe micro analyzer. A comparison of section preparation methods revealed that the As in P. vittata is water-soluble and not tightly bound to the tissue. Further elemental mapping revealed that the localization of S was not consistent with that of As. Therefore, the formation of a complex of low-molecular-weight thiols and As, which is a common detoxification mechanism in plants, may be a limited in P. vittata. It was confirmed that As was localized around the cell wall when the cell was in a healthy state, and that the distribution of As expanded to the whole cell when the cell was damaged. These results suggest that P. vittata may retain As extracellularly to inhibit damage to healthy cells. However, in the periphery of pinnae, which is more susceptible to damage, As was distributed throughout the cell, eventually leading to browning.

Bioremediation is one of the promising environment-friendly approaches to eliminate oil contamination. However, heavy oil is known to degrade slowly due to its hydrophobicity. Therefore, microorganisms capable of producing biosurfactants are gaining substantial interest because of their potential to alter hydrocarbon properties and thereby speed up the degradation process. In this study, six bacterial consortia were obtained from the oil-spilled beach areas in Miyagi, Japan, and all of which exhibited high potential in degrading heavy oil measured by gas chromatography with flame ionization detector (GC-FID). The polymerase chain reaction&mdash, denaturing gradient gel electrophoresis (PCR-DGGE) and next-generation sequencing (NGS) revealed that the diverse microbial community in each consortium changed with subculture and became stable with a few effective microorganisms after 15 generations. The total petroleum hydrocarbons (TPH) degradation ability of the consortia obtained from a former gas station (C1: 81%) and oil refinery company (C6: 79%) was higher than that of the consortia obtained from wastewater treatment plant (WWTP) (C3: 67%, and C5: 73%), indicating that bacteria present in C1 and C6 were historically exposed to petroleum hydrocarbons. Moreover, it was intriguing that the consortium C4, also obtained from WWTP, exhibited high TPH degradation ability (77%). The NGS results revealed that two bacteria, Achromobacter sp. and Ochrobactrum sp., occupied more than 99% of the consortium C4, while no Pseudomonas sp. was found in C4, though this bacterium was observed in other consortia and is also known to be a potential candidate for TPH degradation as reported by previous studies. In addition, the consortium C4 showed high biosurfactant-producing ability among the studied consortia. To date, no study has reported the TPH degradation by the combination of Achromobacter sp. and Ochrobactrum sp., therefore, the consortium C4 provided an excellent opportunity to study the interaction of and biosurfactant production by these two bacteria during TPH degradation.

Phytoremediation is a promising inexpensive method of detoxifying arsenic (As) contaminated soils using plants and associated soil microorganisms. The potential of Pteris vittata to hyperaccumulate As contamination has been investigated widely. Since As(V) is efficiently taken up by P. vittata than As(III), As speciation by associated rhizobacteria could offer enormous possibility to enhance As phytoremediation. Specifically, increased rhizobacteria mediated As(III) to As(V) conversion appeared to be a crucial step in As mobilization and translocation. In this study, Pseudomonasvancouverensis strain m318 with the potential to improve As phytoremediation was inoculated to P. vittata in a field trial for three years to evaluate its long-term efficacy and stability for enhancing As phytoextraction. The biomass, As concentration, and As accumulation of ferns showed to be increased by inoculation treatment. Although this trend occasionally declined which may be accounted to lower As concentration in soil and amount of precipitation during experiments, the potential of inoculation was observed in increased enrichment coefficients. Further, the arsenite oxidase (aioA-like) genes in the rhizosphere were detected to evaluate the influence of inoculation on As phytoremediation. The findings of this study suggested the potential application of rhizosphere regulation to improve phytoremediation technologies for As contaminated soils. However, the conditions which set the efficacy of this method could be further optimized.

The main threats to the environment from heavy metals are associated with arsenic (As), lead (Pb) and cadmium (Cd). In this study, the potential of Pteris multifida for removing As, Pb and Cd from hydroponic solution and pot soil was evaluated for the first time. Short-term (5 day) experiments were conducted to assess phytofiltration efficiency of temperate zone fern P. multifida and to compare it with mostly studied tropical zone fern P. vittata. Within 5 days, P. multifida accumulated 33% of As(III), whereas P. vittata could not accumulate that most toxic arsenic species As(III) at all. Long-term hydroponic results showed that 90% of Pb, 50% of As and 36% of Cd were removed by P. multifida. Concentration of As in the frond (22 mg/kg dw) was comparatively higher than other parts of plant and significantly higher concentration of Cd and Pb were stored in root and rhizome. Pot soil experiment of P multifida confirmed the comparative uptake and translocation of As(V), Pb and Cd from soil. Therefore, from the assessment of heavy metal accumulation capacity, translocation and healthy survival for long time, P. multifida was identified as an excellent species for the treatment of multi-metal contaminated water and soil.

A 74-year-old woman who exhibited drowsiness was referred to our hospital. Enhanced head magnetic resonance imaging (MRI) revealed multiple ring-enhancing lesions and lesions showing partial mild hemorrhaging. The patient gradually progressed to a comatose condition with notable brain deterioration of unknown cause on follow-up MRI. On day nine, the patient inexplicably died, although brain herniation was suspected. Autopsy and histopathology revealed numerous amoebic trophozoites in the perivascular spaces and within the necrotic tissue. Brain immunostaining tested positive for Balamuthia mandrillaris. Infection due to free-living amoeba is rare in Japan; however, it may increase in the near future due to unknown reasons.

The depletion of rare metals is an issue of major concern since rare metals are limited in the abundance but essential for high technology industry. However, the present rare metal recovery technical by chemical methods has high environmental impact, poor selectivity, and is too expensive to be practical. To resolve these problems, this study aimed to create a rare metal recover system using yeast, and molybdenum was selected as the first target. A molybdenum binding protein, ModE, which was derived from Escherichia coli was selected. A fusion gene was generated by linking partial modE with a secretion signal and a domain of α-agglutinin to display the ModE on the surface of yeast cells. The expression of fusion protein on the cell surface was detected by immunofluorescence labeling. As for the recovery experiment, the engineered yeast cells were incubated in 10 mM of sodium molybdate solution for 2 h, and the recovery of molybdenum ion was measured by ICP-AES. The results of fluorescence micrographs showed that the designed fusion protein was successfully expressed on yeast cell surface. According to the results of ICP-AES, the cell surface engineered yeast adsorbed molybdenum and the cells after 72~84 h incubation gave the best adsorption. Besides, the results suggested that the optimization of each functional domain in the fusion protein was important. The selectivity and the lower limit of recoverable concentration are under investigation, while this study provides a preliminary result of bio-extraction technology using cell surface engineered yeast.

A-nine month of field trail was conducted to investigate the accumulation capacity of cadmium and zinc by Arabidopsis halleri spp. germmifera. An experimental site moderately contaminated with Zn and Cd was chosen to evaluate the field traits of seed sowing and seedling transplantation. The exchangeable fraction and total Cd and Zn in the soil and the concentration in harvested plants were determined by inductively coupled plasma mass spectrometer. The shoot biomass of A. halleri ssp. germmifera increased after more than 8 months of cultivation, and it was approximately 2.13 t ha−1 in the seedling transplantation group, which was more than three times higher than in the seed sowing group. The lower ratio of Zn/Cd concentration in plants than in the soil and the higher bioaccumulation factor indicated that A. halleri ssp. germmifera has a higher uptake efficiency for Cd than for Zn. In total, A. halleri ssp. germmifera removed 18.20 kg Cd ha−1 and 27.38 kg Zn ha−1 from the soil. After nine months of growth, A. halleri ssp. germmifera extracted (22.87 ± 9.21) % of total Cd concentration and (2.99 ± 0.94) % of total Zn concentration from the soil. However, no significant decrease of exchangeable concentration was shown in soil Cd and Zn, which revealed that the uptake of Cd and Zn by A. halleri ssp. germmifera not only came from exchangeable forms but also from non-exchangeable fractions. The results of present study indicated that A. halleri ssp. germmifera can be used to efficiently remove different forms of Cd from contaminated land.

Biodegradation of 1,4-dioxane, a water contaminant of emerging concern, has drawn substantial attention over the last two decades. A number of dioxane-degraders have been identified, though many of them are unable to metabolically utilize 1,4-dioxane. Moreover, it is considered more preferable to use microbial consortia rather than the pure strains, especially in conventional bioreactors for industrial wastewater treatment. In the present study, a stable 1,4-dioxane-degrading microbial consortium was enriched, namely 112, from industrial wastewater by nitrate mineral salt medium (NMSM). The consortium 112 is capable of utilizing 1,4-dioxane as a sole carbon and energy source, and can completely degrade 1,4-dioxane up to 100 mg/l. From the consortium 112, two 1,4-dioxane-degrading bacterial strains were isolated and identified, in which the Variovorax sp. TS13 was found to be a novel 1,4-dioxane-degrader that can utilize 100 mg/l of 1,4-dioxane. The efficacy of the consortium 112 was increased significantly when we cultured the consortium with mineral salt medium (MSM). The new consortium, N112, could utilize 1,4-dioxane at a rate of 1.67 mg/l&middot, h. The results of the ribosomal RNA intergenic spacer analysis (RISA) depicted that changes in the microbial community structure of consortium 112 was the reason behind the improved degradation efficiency of consortium N112, which was exhibited as a stable and effective microbial consortium with a high potential for bioremediation of the dioxane-impacted sites and contaminated industrial wastewater.

Rhizodegradation of polycyclic aromatic hydrocarbons (PAHs) is a product of complex interactions between plant and bacteria. In this study, hydroponic culture of sudangrass was established in order to investigate the effects of the plant on PAHs degradation and vice versa through changes in rhizosphere bacterial community. Results showed a plant-induced variability in PAHs degradation dependent on a characteristic shift in bacterial community, with pH and plant age as driving factors. Moreover, bacterial communities with high diversity seemed to abate the phytotoxic effects of PAHs degradation as observed in the plant’s gross health. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and next-generation sequencing revealed that regardless of plant age and culture conditions, the increase or decrease of Sphingobium sp. could dictate the PAHs degradation potential of the bacterial consortium. Overall, this study utilized hydroponic culture of sudangrass to show that plant even of same species can suppress, support, or enhance PAHs degradation of bacteria depending on specific factors.

Pteris vittata is the most well-known arsenic (As) hyperaccumulator, while the corresponding mechanism in molecular level is still not clear. In this study, As uptake and transport in P. vittata were demonstrated by time-course analyses under As addition. Then expression of 3 genes (PvPht1;3, a phosphate (P) transporter gene; PvACR2, a AsV reductase gene; PvACR3, a AsIII transport gene) was focused on to examine their contributions on As processing in root of P. vittata. Results of As addition analyses revealed that P. vittata had high-sensitivity to even 10 ppb AsV which was quickly depleted within 6 h, while this high affinity was inhibited when coexisting with P. Analyses to As in the plant showed that in the absence of P, 99 % of the As taken up by roots was reduced to AsIII at 7 d. And 85 % of As transported to rhizomes was present as AsIII, 74 % of the As accumulated in fronds was AsIII. Results of qRT-PCR demonstrated that the transcription of PvPht1;3 was temporally induced by 100 ppb AsV without P, while 500 ppb of AsV made this induction kept through the entire period which showed 3.7-fold higher than control at 7 d. Meanwhile, PvACR2 was only induced slightly (1.15–1.45-fold) by 500 ppb AsV with a time lag, and this induction wasn’t infected by P. PvACR3 was induced by 100 ppb AsV immediately, and the strength was positively related to AsV concentration. Intriguingly, the expression of PvACR3 fitted the AsIII concentration in the root. Our results suggested a collaboration of these three genes in sensitive AsV absorption, constitutive AsV reduction and subsequent AsIII transportation which contributes to As hyperaccumulation by P. vittata.

Pteris vittata and Pteris multifida are widely studied As hyperaccumulators that absorb As mainly via roots. Hence, rhizobacteria exhibit promising potential in phytoextraction owing to their immense microbial diversity and interactions with plants. Pseudomonas vancouverensis strain m318 that contains aioA-like genes was screened from P. multifida’s rhizosphere through the high As resistance (minimum inhibitory concentrations (MICs) against As(III): 16 mM; MICs against As(V): 320 mM), rapid As oxidation (98% oxidation by bacterial cultures (OD600nm = 1) from 200 μL of 0.1 mM As(III) within 24 h), predominant secretion of IAA (12.45 mg L−1) and siderophores (siderophore unit: 88%). Strain m318 showed significant chemotactic response and high colonization efficiency to P. vittata roots, which suggested its wide host affinity. Interestingly, inoculation with strain m318 enhanced the proportion of aioA-like genes in the rhizosphere. And in field trials, inoculation with strain m318 increased As accumulation in P. vittata by 48–146% and in P. multifida by 42–233%. Post-transplantation inoculations also increased As accumulation in both ferns. The abilities of the isolated multifunctional strain m318 and the increase in the rhizosphere microbial aioA-like genes are thus speculated to be involved in As transformation in the rhizospheres and roots of P. vittata and P. multifida.

As a toxic and carcinogenic metalloid, arsenic has posed serious threat to human health. Phytoremediation has emerged as a promising approach to circumvent this problem. Arsenic uptake by Pteris vittata is largely determined by arsenic speciation and mainly occurs via roots; thus, rhizospheric microbial activities may play a key role in arsenic accumulation. The aim of this study was to investigate the potential of arsenic resistant rhizobacteria to enhance arsenic phytoextraction. A total of 49 cultivable rhizobacteria were isolated from the arsenic hyperaccumulating fern, Pteris vittata, and subjected to an initial analysis to identify potentially useful traits for arsenic phytoextraction, such as arsenic resistance and the presence of aioA(aroA)-like (arsenite oxidase-like) gene. Isolated strain r507, named as Cupriavidus basilensis strain r507, was a selected candidate for its outstanding arsenic tolerance, rapid arsenite oxidation ability, and strong colonization to P. vittata. Strain r507 was used in co-cultivation trials with P. vittata in the field for six months. Results showed that the inoculation with strain r507 potentiated As accumulation of P. vittata up to 171%. Molecular analysis confirmed that the inoculation increased the abundance of aioA-like genes in the rhizosphere, which might have facilitated arsenite oxidation and absorption. The findings of this study suggested the feasibility of co-cultivating hyperaccumulators with facilitator bacteria for practical arsenic phytoremediation.

As a strategy of arsenic-containing mine tailing soil, the application of arsenic hyper-accumulator plants such as Pteris vittata shows high potential. Previous studies suggest that the arsenic uptake by P. vittata is largely determined by arsenic speciation, which is strongly influenced by microbial activities. In this study, two arsenic hyper-accumulators, P. vittata that is a subtropical plant and Pteris multifida that has been found in northeastern Japan, were selected and the their rhizobacteria were investigated. Pot experiments of P. vittata and P. multifida cultivation were performed. The concentration of total arsenic in the plant samples was monitored by inductively coupled plasma - mass spectrometry. Microbes in the rhizosphere samples were investigated by 16S rDNA library analysis and arsenic resistant bacteria isolation. The results showed that both ferns accumulated As, while P. multifida was more resistant to cold weather. Two ferns presented different rhizobacterial communities and cultivatable bacteria number, but bacteria that contain As(III) oxidase gene or As(III) oxidation activity were confirmed in both rhizosphere samples. The results of present study suggested that the cultivation of ferns changed the microbial communities in soil (rhizosphere), and microbial activity in the rhizosphere played a role in As(III) oxidation. Since ferns absorbs As(III) and As(V) in different efficiency, this study provided hints when linking microbial As transformation to As uptake by ferns which is important when designing pre-treatment procedures of phytoremediation.

Stable degrading 1,2-dichlorobenzene (1,2-DCB) enrichments were generated from original contaminated soil and groundwater via enrichment procedures using a mineral salt medium containing 1,2-DCB as the sole carbon and energy source. Four transferred enrichments showed stable 1,2-DCB-degrading ability and completely degraded 1,2-DCB within 32 h. PCR-denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone library analyses indicated that two bacterial strains, belonging to Acidovorax spp. and Ralstonia spp., respectively, were the predominant organisms in each enrichment. Moreover, these strains maintained a stable coexistence in the four transferred enrichments. These two bacteria were subsequently identified as Acidovorax sp. strain sk40 and Ralstonia sp. strain sk41. Strain sk40 was more tolerant to higher concentrations of 1,2-DCB than strain sk41, while strain sk41 maintained a shorter degradation time under lower concentrations of 1,2-DCB. Notably, however, both strains exhibited similar growth rates and degradation rates in media containing 40 mg/l 1,2-DCB, as well as complete degradation of the 1,2-DCB (40 mg/l) within 32 h. It is expected that these two strains will be used in future applications of bioremediation of 1,2-DCB contamination.

On March 11, 2011, one of the negative effects of the tsunami phenomenon that devastated the Pacific coast of the Tohoku district in Japan was the deposition of a wide range of arsenic (As) contamination to the soil. To remediate such a huge area of contamination, phytoremediation by Pteris vittata, an As-hyperaccumulator, was considered. To evaluate the efficacy of applying P. vittata to the area, the salt tolerance of P. vittata and the phytoextraction of As from soil samples were investigated. For the salt tolerance test, spore germination was considerably decreased at an NaCl level of more than 100 mM. At 200 mM, the gametophytes exhibited a morphological defect. Furthermore, the growth inhibition of P. vittata was observed with a salinity that corresponded to 66.2 mS/m of electric conductivity (EC) in the soil. A laboratory phytoremediation experiment was conducted using As-contaminated soils for 166 days. P. vittata grew and accumulated As at 264 mg/kg-DW into the shoots. Consequently, the soluble As in the soil was evidently decreased. These results showed that P. vittata was applicable to the phytoremediation of As-contaminated soil with low salinity as with the contamination caused by the 2011 tsunami.

Mangroves constitute valuable coastal resources that are vulnerable to oil pollution. One of the major processes to remove oil from contaminated mangrove sediment is microbial degradation. A study on heavy oil- and hydrocarbon-degrading bacterial consortia from mangrove sediments in Okinawa, Japan was performed to evaluate their capacity to biodegrade and their microbial community composition. Surface sediment samples were obtained from mangrove sites in Okinawa (Teima, Oura, and Okukubi) and enriched with heavy oil as the sole carbon and energy source. The results revealed that all enriched microbial consortia degraded more than 20% of heavy oil in 21 days. The K1 consortium from Okukubi site showed the most extensive degradative capacity after 7 and 21 days. All consortia degraded more than 50% of hexadecane but had little ability to degrade polycyclic aromatic hydrocarbons (PAHs). The consortia were dominated by Pseudomonas or Burkholderia. When incubated in the presence of hydrocarbon compounds, the active bacterial community shifted to favor the dominance of Pseudomonas. The K1 consortium was a superior degrader, demonstrating the highest ability to degrade aliphatic and aromatic hydrocarbon compounds; it was even able to degrade heavy oil at a concentration of 15%(w/v). The dominance and turn-over of Pseudomonas and Burkholderia in the consortia suggest an important ecological role for and relationship between these two genera in the mangrove sediments of Okinawa.

In this study, the aquatic macrophyte Ceratophyllum demersum L. (coontail or hornwort) was tested for its efficiency of arsenic (As) uptake under laboratory conditions. Our results revealed that the solution pH had a significant effect on As accumulation by C. demersum (p < 0.001). The accumulation was highest at pH 5 and decreased as pH values increased. Plants that were exposed to various concentrations of arsenite (As(III)) for 24 and 48 h, exhibited tolerance and toxic responses, respectively. As accumulation by C. demersum depended on the concentrations of As(III) and the duration of exposure (p < 0.001). At 40 μM after 24 h, plants accumulated 227.5 μg As g−1 dw and showed no visible symptoms of toxicity. However, after 48 h, As level reached 302.4 μg g−1 dw and biomass production decreased significantly. Toxic effects were evident by plant necrosis and negative biomass production, leading to a decrease in the amount of accumulated As. Also, the addition of iron (Fe) into the nutrient solutions (0.18 mM) had contrasting effects on the uptake of 2 As species – the uptake of As(III) was enhanced by the presence of Fe, but the uptake of arsenate (As(V)) was considerably inhibited.

The aims of this study were to identify the bacteria responsible for the rapid degradation of aromatic hydrocarbons, and to determine the roles of bacterial species in the microbial consortium. The population changes of the four major bacterial groups, namely Burkholderia spp., Achromobacter spp./Alcaligenes spp., Cupriavidus spp., and Rhodanobacter spp. in the Y consortium that preferentially degraded petroleum oil-derived aromatic hydrocarbons were assessed using quantitative real-time polymerase chain reaction (q-PCR). P-xylene was used as a representative aromatic compound, decane as a representative aliphatic compound, p-xylene-decane as a representative hydrocarbon mixture, and kerosene as a representative petroleum-based oil. The results showed that the remarkable increase of Burkholderia spp. coincided with the rapid degradation of aromatic hydrocarbons in kerosene, and p-xylene in single and dual-substrate cultures. Cupriavidus spp. significantly increased when decane was rapidly depleted. Achromobacter spp.,/Alcaligenes spp., and Rhodanobacter spp. were initially inhibited by the presence of aromatic hydrocarbons and seemed not to be active in hydrocarbon degradation, but utilized the metabolic products. This work provided an alternative way to explain the contribution of different microbial species in the degradation of hydrocarbon fractions. The behavior of the consortium is of interest in the context of the bioremediation of the more toxic aromatic hydrocarbons in environments contaminated by petroleum products.

A dechlorinating consortium (designated as TES-1 culture) able to convert trichloroethene (TCE) to ethene was established from TCE-contaminated groundwater. This culture had the ability of complete dechlorination of TCE within about one month. From the clone library analysis of 16S rRNA gene, this culture was mainly composed of fermentation bacteria, such as Clostridium spp., and Desulfitobacterium spp. known as facultative dechlorinator. PCR using specific primers for Dehalococcoides spp. and the dehalogenase genes confirmed that the culture contained the Dehalococcoides spp. 16S rRNA gene and three dehalogenase genes, tceA, vcrA and bvcA. Dechlorination experiments using cis-dichloroethene (cis-DCE) at concentrations of 37-146 μM, revealed that the gene copy numbers of tceA, vcrA, and bvcA increased up to 10⁷ copy/mL, indicating that Dehalococcoides spp. containing these three dehalogenase genes were involved in cis-DCE dechlorination. However, in the culture to which 292 μM of cis-DCE was added, only the tceA gene and the Dehalococcoides spp. 16S rRNA gene increased up to 10⁷ copy/mL. The culture containing 292 μM of cis-DCE also exhibited about one tenth slower ethene production rate compared to the other cultures.

Numerous studies on the biodegradation of petroleum products using total petroleum hydrocarbons (TPH) have been carried out; however, the biodegradation of equivalent carbon number (EC) based hydrocarbon fractions in mineral oil has attracted little attention. This study investigated the ability of a microbial consortium to degrade the EC fractions in kerosene, which was used as a representative mineral oil. Based on the cloning and sequencing of the 16S rRNA gene, the microbial community was predominantly identified as Betaproteobacteria of the genera Achromobacter , Alcaligenes , and Cupriavidus . Degradation experiments in sealed 120-ml vials containing 1% (w v −1 ) kerosene revealed that aromatic fractions were degraded faster than aliphatic fractions. Aromatic fractions EC >7–8 and EC >8–10 were completely degraded after three days while aliphatic fractions EC >6–8 and EC >8–10 were only partially degraded. The aromatic EC >10–12 fraction was the third most degraded, and the aliphatic EC >10–12 and EC >12–16 fractions were the least degraded fractions. The first-order rate constants for the aromatic fractions ranged from 0.12 d −1 to 0.51 d −1 and from 0.06 d −1 to 0.32 d −1 for the aliphatic fractions. The microbial consortium preferentially utilized aromatic fractions, which are more toxic than aliphatic fractions. This finding is useful when considering risk-based bioremediation: A microbial community could potentially degrade the more toxic aromatic hydrocarbon components in petroleum-contaminated environments.

The consequence of polycyclic aromatic hydrocarbons (PAHs) in the environment is of great concern. The hydrophobic properties of PAHs significantly impact phase distribution causing limited bioavailability. Enhanced biodegradation has been extensively carried out by surfactants and the redeployment effect was recognized. However, the quantitative relationship concerning the impact of solids was rarely reported. A batch of biphasic tests were carried out by introducing Mycobacterium vanbaalenii PYR-1 and hydroxypropyl-β-cyclodextrin (HPCD) into a mixture of phenanthrene solution and various glass beads (GB37-63, GB105-125, and GB350-500). The comparative results demonstrated that HPCD had little effect on microbial growth and was not degradable by bacterium. A model was proposed to describe the biodegradation process. The regression results indicated that the partition coefficient k d (1.234, 0.726 and 0.448 L·g−1) and the degradation rate k (0 mmol·L−1: 0.055, 0.094, and 0.112; 20 mmol·L−1: 0.126, 0.141, and 0.156; 40 mmol·L−1: 0.141, 0.156 and 0.184 d−1) were positively and negatively correlated with the calculated total surface area (TSA) of solids, respectively. Degradation enhanced in the presence of HPCD, and the enhancing factor f was calculated (20 mmol·L−1: 15.16, 40.01, and 145.5; 40 mmol·L−1: 13.29, 37.97, and 138.4), indicating that the impact of solids was significant for the enhancement of biodegradation.

Pteris vittata is a fern reported to be an arsenic hyper-accumulator. To develop the practical application of the fern to a phytoremediation technique, it is necessary to explicate the effective accumulation mechanism. In this study, the arsenic distribution and the elemental correlation in the cellular level were examined in the fronds supplied with arsenate and arsenite separately via xylem vessel using an in-air micro-PIXE system at Tohoku University. The difference in transportation rate between arsenate and arsenite as well as the translocation of elements necessary for plant metabolism was revealed in different tissues of the fronds accumulating arsenic in high concentration. Hence, the in-air micro-PIXE analysis is an effective measure for undertaking phytoremediation research of hyper-accumulator plants.

This paper describes hydrogen sulfide generation, isolation and characterization of sulfate-reducing bacteria in the activated sludge obtained from wastewater treatment plant. The sludge, collected from aerobic treatment system, was used as inoculums for hydrogen sulfide generation and for isolation of sulfate-reducing bacteria. Hydrogen sulfide was generated when the sludge was inoculated into a pH controlled medium. Two strains G2 and G3 of sulfate-reducing bacteria were isolated from the sludge. The characteristics and identification of these isolates was based on culture of the experiments and molecular analysis. Reduced sulfate rates in G2 and G3 is 0.39 and 0.41 mmol/dm3 respectively. Two isolates located in the genus Desulfovibrio in the phylogenetic tree based. First one was 16S rRNA gene analysis with the highest homology, 96.6 %, as Desulfovibrio carbinoliphilus (DSM 17524T) . However, the branch of the isolates was separated from the relatives and no strain was recognized in this branch. Also the dsr gene of isolate G3 had the highest homology with Desulfovibrio fructosovorans (DSM 3604T) , 92.7 %. The isolates G2 and G3 belonged to the genus Desulfovibrio as confirmed by the results of these analyses, however, these isolates oxidized organic compounds to carbon dioxide completely although relative strain oxidized it completely. Therefore, these isolates have high possibility that they should be recognized as a new strain.

This paper described that relationship between Acidithiobacillus feroooxidans T23-3 and fungus TEF1-1 in an enrichment culture. In order to evaluate the relationship, some culture experiments using cell-free extracts from T23-3 and TEF1-1 were carried out. Chemolitotrophic iron-oxidizing bacterium, A. ferrooxidans T23-3, was able to grow using inorganic compounds in cell-free extract of fungus TEF1-1 or A. ferrooxidans T23-3. Since the fungus TEF1-1 was able to grow using cell-free extract of the both microorganisms at pH 1.5, the fungus used some organic compounds in the cell-free extract as energy and carbon sources. When the cell-free extract of TEF1-1 used for the cultivation of A. ferrooxidans T23-3, lag-phase was observed because of lack of magnesium ion. Minimum concentration of magnesium ion for complete growth of A. ferrooxidans T23-3 was found to be 2.0 x 10-5 mol/dm3 from the experiments using synthetic medium. Also the cell-free extract contained enough nutritious components except magnesium ion for the growth of A. ferrooxidans T23-3. It was suggested that fungi grew using organics and inorganic ions released from the cell of chemolithotrophic iron-oxidizing bacteria by bacteriolysis and/or metabolisum and that the bacteria grew using inorganic ions from the lysis of fungi cells in the enrichment culture of iron-oxidizing bacteria. In addition, chemolithotrophic iron-oxidizing bacterium fixes carbon dioxide to convert some organic compounds in the culture. Therefore, it was evaluated that the relationship between chemolithotrophic iron-oxidizing bacteria and fungi was mutualistic symbiosis in the enrichment culture.

By means of functional screening using the cadmium (Cd)-sensitive ycf1 yeast mutant, we have isolated a novel cDNA clone, DcCDT1, from Digitaria ciliaris growing in a former mining area in northern Japan, and have shown that it confers Cd tolerance to the yeast cells, which accumulated almost 2-fold lower Cd levels than control cells. The 521 bp DcCDT1 cDNA contains an open reading frame of 168 bp and encodes a deduced peptide, DcCDT1, that is 55 amino acid residues in length, of which 15 (27.3%) are cysteine residues. Five DcCDT1 homologs (here termed OsCDT1-OsCDT5) have been identified in rice, and all of them were up-regulated to varying degrees in the above-ground tissues by CdCl(2) treatment. Localization of green fluorescent protein fusions suggests that DcCDT1 and OsCDT1 are targeted to both cytoplasmic membranes and cell walls of plant cells. Transgenic Arabidopsis thaliana plants overexpressing DcCDT1 or OsCDT1 displayed a Cd-tolerant phenotype and, consistent with our yeast data, accumulated lower amounts of Cd when grown on CdCl(2). Collectively, our data suggest that DcCDT1 and OsCDT1 function to prevent entry of Cd into yeast and plant cells and thereby enhance their Cd tolerance.

The copper extraction yield from thermophilic bioleaching of chalcopyrite depends on temperature, pH, and the oxidation-reduction potential (ORP), as well as on the activity of the thermophile used. The copper extraction yields obtained with three thermophiles under various pH and temperature conditions and with different initial amounts of Fe 3+ were studied. The results indicated that because of the low ability of Acidianus brierleyi to leach iron as Fe 3+ , high biomass concentrations were reflected by ORP close to a critical value (450 mV, Ag 0 /AgCl reference), at which copper extractions were highest. By contrast, because of the higher ability of Sulfolobus metallicus and Metallosphaera sedula to leach iron as Fe 3+ , high biomass concentrations were reflected by high ORP which in combination with the precipitation of Fe 3+ as jarosite attenuated the leaching rates. Therefore, optimum temperatures for the growth of thermophiles did not always mean high copper extraction yields. Generally, highest copper extractions were obtained at initial pH 1.5. However, higher copper extractions were observed at initial pH 2.5 than at pH 2.0, suggesting that at high pH the bioleaching of chalcopyrite is controlled by the ORP rather than by the pH or temperature. The bioleaching capacity of A. brierleyi was reduced or suppressed when insufficient initial Fe 3+ was provided to trigger the leaching reaction, whereas S. metallicus and M. sedula were less sensitive to the initial availability of Fe 3+ . This result confirmed that a direct enzymatic attack on the mineral surface could initiate the leaching reaction, but later ORP governed the leaching rate of chalcopyrite.

We have developed an in-air particle-induced x-ray emission(PIXE) analysis system that provides elemental distribution images in a region of 3 X 3 cm 2 with a spatial resolution of ∼0.5 mm. We call this system an in-air submilli-PIXE camera. This system consists of a submilli-beam line, beam scanners and a data acquisition system for elemental mapping. We applied the in-air submilli-PIXE camera to phytoremediation research. Phytoremediation is a technology for cleaning metal-contaminated soils using plant physiology. Pteris vittata, which is known as a hyperaccumulator of arsenic, was analyzed by the PIXE camera. Elemental images of laminas were obtained in vivo without sample preparation. The elemental map of the laminas in several centimeters region showed that arsenic was accumulated in the edges of P. vittata fronds and redistributed in the laminas at different growth stages. The submilli-PIXE camera is an effective tool for undertaking phytoremediation research.

This paper describes about microbial diversity in an iron oxidation tank of an AMD treatment plant established at an abandoned sulphur mine in Japan. Since the mining operation was stopped, this mine has produced strong acidic mine drainage, pH 1.8, including a high concentration of ferrous iron, 301mg/L, and the flow rate was about 4m3/min. In 2006, a pilot scale microbial iron oxidizing system was installed to remove total iron more easily from the AMD by oxidizing ferrous iron to ferric iron. From the start of this pilot operation, microbial diversity in the iron oxidation tank was investigated using a PCR-DGGE method for about two months. In the PCR, V3 region of 16S rRNA gene for Bacteria was amplified. The profiles of DGGE showed that there were three dominant species in the iron oxidation tank through the experimental period. Number of bands on DGGE profiles decreased with dates of sampling so that the microbial population became less diverse because of the iron oxidizing operation. There was a wide variety of bacterial species of even though conditions were strongly acidic.

In this study, we found that high-performance hydroponics of arsenic hyperaccumulator fern Pteris vittata is possible without any mechanical aeration system, if rhizomes of the ferns are kept over the water surface level. It was also found that very low-nutrition condition is better for root elongation of P. vittata that is an important factor of the arsenic removal from contaminated water. By the non-aeration and low-nutrition hydroponics for four months, roots of P. vittata were elongated more than 500 mm. The results of arsenate phytofiltration experiments showed that arsenic concentrations in water declined from the initial concentrations (50 μg/L, 500 μg/L, and 1000 μg/L) to lower than the detection limit (0.1 μg/L) and about 80% of arsenic removed was accumulated in the fern fronds. The improved hydroponics method for P. vittata developed in this study enables low-cost phytoremediation of arsenic-contaminated water and high-affinity removal of arsenic from water. The water level condition affects the plant growing for arsenic hyperaccumulator P. vittata in non-aerated and low-nutrition hydroponic system.

The merC gene from Acidithiobacillus ferrooxidans functions as a mercury uptake pump. MerC protein localizes in the cytoplasmic membrane of plant cells. When Arabidopsis thaliana and tobacco plants were transformed with the merC gene under the control of the Cauliflower mosaic virus 35S promoter, the resulting overexpression of merC rendered the host plants hypersensitive to Hg2+ and they accumulated approximately twice as much Hg2+ ion as the wild type plants. Thus, bacterial mercuric ion transporters such as MerC may be useful molecular tools for producing transgenic plants that hyperaccumulate Hg2+ ion.

This paper describes that evaluation of soil remediation contaminated with oil on quicklime process and the effect of the treatment for oil degradable bacteria in the soil. In this paper, two different contaminated sites were used. When quicklime method was applied for a contaminated site, the temperature of soil increased over 60 °C by reaction heat and the pH value also rose to about 12. Although the density of bacteria in the contaminated soil decreased from 40 to 5 - 10 x 1010 cells/g-dry soil, they could survive against the increasing of heat and alkalinity caused by the treatment. Some oil degradable bacteria were isolated from original contaminated and treated soil and the abilities of oil degradation were evaluated. These bacteria might contribute the degradation of remained oil in the treated soil. On another site, the oil concentration in soil decreased from 3,555 to 1,430 mg-oil/kg-soil by the treatment. In this case, some light parts of oil, C6 to C10 and C10 to C28, were almost volatized to below 5 and 35% respectively, but the heavy part, C28 to C44 was still remained. However the remained oil decreased gradually after the treatment to 250 mg-oil/kg-soil within 6 month. This shows the high possibility of oil remediation by oil degradable bacteria which survive in contaminated soil against the treatment.

Bactrerium inhabiting in Amarume crude oil of Yamagata prefecture was identified as Pseudomonas mendocina. In the first step, biodegradation experiments were conducted by using the bacteria of 0.5106 cells / ml and 300 ppm of chlorobenzene in aqueous solution under aeration and irradiation. After 6 hours, chlorobenzene was perfectly decomposed. Next, the mixtures of synthesized polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) were degraded with the bacteria of 0.5108 cells / ml, where the initial total concentrations of PCDDs and PCDFs were 1,400 ppm and 180 μg-TEQ / ml. After 24 hours, about 70 % of PCDDs and 60 % of PCDFs were biodegraded. The biodegradation rates of PCDD / Fs were much faster than those reported in literatures with other bacteria.

The purA gene of Thiobacillus ferrooxidans encoding adenylosuccinate synthetase [EC 6.3.4.4] was identified in the upstream region of the iro gene encoding Fe(II)-oxidase (J. Biol. Chem 267:11242-11247, 1992). The purA gene consisted of 1290 base-pairs, which translated into a 29-amino-acid protein. The gene is functionally active, because it is able to complement an Escherichia coli purA-deficient strain. The deduced gene product has a high degree (60.9%) of sequence identity with that (432 aa) of E. coli purA gene, and both the products share GDEGKGK-DETG-TKLD sequences which are supposed to be GTP-binding domain. The downstream region of the iro gene contained another open-reading frame (ORF) of 1218 bp, and this showed high homology (56.6% over 249 bp) with E. coli ORF-II, which is found as a second ORF and truncated form in the downstream region of the purA gene. Comparison of the gene organization in the flanking region of purA gene between T. ferrooxidans and E. coli is also described.

The physiological responses of the arsenic-hyperaccumulator, Pteris vittata, such as arsenic uptake and chemical transformation in the fern, have been investigated. However, a few questions remain regarding arsenic treatment in hydroponics. Incubation conditions such as aeration, arsenic concentration, and incubation period might affect those responses of P. vittata in hydroponics. Arsenite uptake was low under anaerobic conditions, as previously reported. However, in an arsenite uptake experiment, phosphorous (P) starvation-dependent uptake of arsenate was observed under aerobic conditions. Time course-dependent analysis of arsenite oxidation showed that arsenite was gradually oxidized to arsenate during incubation. Arsenite oxidation was not observed in any of the control conditions, such as exposure to a nutrient solution or to culture medium only, or with the use of dried root; arsenite oxidation was only observed when live root was used. This result suggests that sufficient aeration allows the rhizosphere system to oxidize arsenite and enables the fern to efficiently take up arsenite as arsenate. X-ray absorption near edge structure (XANES) analyses showed that long-duration exposure to arsenic using a hydroponic system led to the accumulation of arsenate as the dominant species in the root tips, but not in the whole roots, partly because up-regulation of arsenate uptake by P starvation of the fern was caused and retained by long-time incubation. Analysis of concentration-dependent arsenate uptake by P. vittata showed that the uptake switched from a high-affinity transport system to a low-affinity system at high arsenate concentrations, which partially explains the increased arsenate abundance in the whole root.

Arsenic accumulation by hyperaccumulator, Pteris vittata, when exposed to arsenite in hydroponics is comparable to that when exposed to arsenate while arsenite uptake is considerably slower than arsenate uptake. Arsenite species around root and that in the incubating solution were analyzed to investigate arsenite oxidation in hydroponics. XANES analysis of the fern roots showed that arsenate predominated in all parts tested, root tip, middle, and root top, even an hour after the addition of arsenite, suggesting arsenite oxidation in whole rhizosphere. Although arsenite in the rhizosphere is rapidly oxidized to arsenate within an hour, arsenate was not observed in the bulk solution and was gradually increased during 18 days-incubation, supporting the involvement of rhizosphere in arsenite oxidation. Arsenite speciation of medium during arsenite uptake experiment showed that in 7 days, the most arsenite was oxidized and remained as arsenate with a fern conditioned with 1mM phosphate (Pi), whereas the most arsenite was decreased with a fern conditioned with 10 M Pi, suggesting arsenite oxidation and subsequent phosphorus-starvation-dependent uptake of the arsenate.

An arsenic-hyperaccumulater, Pteris vittata, has been considered as an environmental friendly-method to clean up arsenic-contaminated water. Although P. vittata accumulates high amount of arsenic up to approximately 2% of its dry-weight, the influence of senescence and As accumulation on each other, especially the extent the fern accumulates arsenic without cell death is not clearly understood. XSAM analysis showed that As accumulation in a mature leaflet was not higher than that in a young leaflet. Chlorophyll content and Ca level of the frond (leaf) was measured as indicators of (metal) metalloid stress. In the results, there was correlation of Ca level and As accumulation while there was no correlation between chlorophyll content and As accumulation. XANES analysis showed that at root top, arsenate was dominant species among ferns incubated for various periods of time. On the other hand, at root tips, arsenate abundance was gradually increased as incubation period was increased.

Sulfur is almost insoluble in water at ambient temperatures, and therefore polysulfide (S(n)(2-)) has been considered as a possible intermediate that is used directly by bacteria in sulfur respiration. Sulfur-reducing reductases have been purified and characterized from a few sulfur reducers. However, polysulfide reduction has only been confirmed in Wolinella succinogenes. In our previous study, the direct production of hydrogen sulfide from polysulfide was confirmed by an enrichment culture obtained from natural samples under sulfate-reducing conditions. The present study attempted to isolate and identify polysulfide-reducing bacteria from the enrichment cultures. Almost all the isolated strains were classified into the genus Clostridium, based on 16S rRNA gene sequence analysis. The isolates, and some closely related strains, were able to reduce polysulfide to hydrogen sulfide. During production of 1 mol of hydrogen sulfide, approximately 2 mol of lactate was converted to acetate. Thus, dissimilatory polysulfide reduction occurred using lactate as an electron donor. The ability to reduce elemental sulfur was also examined with the isolates and the related strains. Although elemental sulfur reducing strains can reduce polysulfides, not all polysulfide-reducing strains can reduce elemental sulfur. These results demonstrate that the conversion of elemental sulfur to polysulfide seems to be important in the reduction process of sulfur.

More than 100 independent strains ofThiobacillus ferrooxidans were isolated from six different domestic mining sites. Although there was some variation according to sampling site, about 73% of all strains carried more than one plasmid ranging in size from about 2.0 to 30 kilobase-pairs(kb). Among these, four plasmids of 2.4, 4.7, 5.1, and 8.9 kb, designated pTNA33, pTSY91, pTSB121, and pTSB122, respectively, were cloned intoEscherichia coli plasmids. pTSB121 and pTSB122, originated from the sameT. ferrooxidans strain, showed weak homology by Southern blotting, whereas pTSB121 showed high homology with pTSY91 from a different strain. It seems that the occurrence of the plasmid homologous to pTSB121 or pTSB122 is more ubiquitous inThiobacillus. On the other hand, pTNA33 is a unique plasmid because it showed no significant homology with other plasmids.

Mercuric reductase activity determined by the Thiobacillus ferrooxidans merA gene (cloned and expressed constitutively in Escherichia coli) was measured by volatilization of 203Hg2+. (The absence of a merR regulatory gene in the cloned Thiobacillus mer determinant provides a basis for the constitutive synthesis of this system.) In the absence of the Thiobacillus merC transport gene, the mercury volatilization activity was cryptic and was not seen with whole cells but only with sonication-disrupted cells. The Thiobacillus merC transport function was compared with transport via the merT-merP system of plasmid pDU1358. Both systems, cloned and expressed in E. coli, governed enhanced uptake of 203Hg2+ in a temperature- and concentration-dependent fashion. Uptake via MerT-MerP was greater and conferred greater hypersensitivity to Hg2+ than did uptake with MerC. Mercury uptake was inhibited by N-ethylmaleimide but not by EDTA. Ag+ salts inhibited mercury uptake by the MerT-MerP system but did not inhibit uptake via MerC. Radioactive mercury accumulated by the MerT-MerP and by the MerC systems was exchangeable with nonradioactive Hg2+.

An anaerobic mixed microbial culture was enriched from soil and groundwater taken from a site contaminated with trichloroethene (TCE). This enrichment culture could dechlorinate TCE sequentially to cis‐dichloroethene (cis‐DCE), vinyl chloride (VC), and ethene rapidly within 2 weeks. This enrichment culture could utilize various organic compounds, such as methanol, ethanol, and sodium acetate and so on, as electron donor. This culture had maintained high ability of TCE dechlorination for about 3 years since the start of enrichment cultivation. The optimum pH value for the dechlorination activity of this culture, which reacts from TCE to ethene, was 6.7. However above the pH value 7.1, it lost the dechlorination ability of cis‐DCE and VC. So cis‐DCE was remained at that pH conditions. From the DNA sequencing analysis of 16SrRNA gene, this enrichment culture includes Dehalococcoides sp. which has the ability to dechlorinate TCE to VC completely with hydrogen. It suggested that this Dehalococcoides sp. takes part in the dechlorination of chloroethenes.

To find an efficient bacterium, which has the strong capacity to produce hydrogen sulfide from polysulfide as the waste of process generating hydrogen from hydrogen sulfide by photocatalytic reaction using sun light, is very important for constructing hydrogen producing system. 10 strains of sulfate‐reducing bacteria (SRB), which can reduce polysulfide directly, have been isolated from various natural samples such as TCE contaminated soil, soil and sludge around hot spring environment, and the cooling tower of a geothermal plant. This study describes physiological and phylogenetic characterization of SRB which can reduce polysulfide. All of isolates had the ability to reduce polusulfide but these reduction rates were difference depend on isolates. Phylogetetically, all of isolates located difference position for general SRB including Desulfovibrio desulfuricans, which is used standard strain in this study, so they do not belong to Proteobacteria. These have close relation to the genus Desulfotomaculum which can reduce elemental sulfur. It suggests that the ability of reducing elemental sulfur is important for reducing polysulfide to hydrogen sulfide.

A heterotrophic bacterium, designated as HIB4, having an ability to oxidize ferrous iron was isolated from the sample of an enrichment culture with 9K medium, by using the modified WAYE (washed agarose/yeast extract) medium with ferrous sulphate. This isolate was identified as Alicyclobacillus disulfidooxidans from 16S rDNA sequence analysis. The isolate grew and oxidized ferrous iron in an inorganic medium containing 0.02 % (w/v) of yeast extract and Ferrous iron oxidation occurred at the almost end of its logarithmic phase. Yeast extract was an essential substrate for the isolate because the isolate could not grow or oxidize ferrous iron without yeast extract. However, higher concentration of yeast extract inhibited the growth of the isolate. On the other hand, it was confirmed that the isolate was able to grow without ferrous ion so that it did not get any energy by ferrous ion oxidation. Its optimum concentration of yeast extract was 0.02% (w/v) at the concentration of ferrous ion 0.08mol/liter. Its o...

The merC gene of Thiobacillus ferrooxidans was overexpressed in Escherichia coli under the control of the tac promoter. MerC protein synthesized in E. coli has a N-terminal amino acid sequence of S-A-I-X-R-I-I-D-K-I-G-I-V-G-, which agrees with the amino acid sequence deduced from its nucleotide sequence except that an initiating methionine residue was removed. The MerC protein was localized in the particulate (membrane) cell fraction, and not in the soluble cytoplasmic fraction. E. coli cells carrying a plasmid containing the tac promoter-directed merC showed 203Hg2+ uptake in an isopropyl-1-thio-beta-D-galactopyranoside (IPTG)-dependent manner.