Bio-Augmentation of Cupriavidus sp. CY-1 into 2,4-D Contaminated Soil: Microbial Community Analysis by Culture Dependent and Independent Techniques.

In the present study, a 2,4-dichlorophenoxyacetic acid (2,4-D) degrading bacterial strain CY-1 was isolated from the forest soil. Based on physiological, biochemical and 16S rRNA gene sequence analysis it was identified as Cupriavidus sp. CY-1. Further 2,4-D degradation experiments at different concentrations (200 to 800 mg l(-1)) were carried out using CY-1. Effect of NaCl and KNO3 on 2,4-D degradation was also evaluated. Degradation of 2,4-D and the metabolites produced during degradation process were analyzed using high pressure liquid chromatography (HPLC) and GC-MS respectively. The amount of chloride ions produced during the 2,4-D degradation were analyzed by Ion chromatography (IC) and it is stoichiometric with 2,4-D dechlorination. Furthermore two different types of soils collected from two different sources were used for 2,4-D degradation studies. The isolated strain CY-1 was bio-augmented into 2,4-D contaminated soils to analyze its degradation ability. Culture independent methods like denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP), and culture dependent methods like colony forming units (CFU) and most probable number (MPN) were used to analyze the survivability of strain CY-1 in contaminated soil. Results of T-RFLP were coincident with the DGGE analysis. From the DGGE, T-RFLP, MPN and HPLC results it was concluded that strain CY-1 effectively degraded 2,4-D without disturbing the ecosystem of soil indigenous microorganisms.

Whole-Genome Sequence of Aquamicrobium sp. Strain SK-2, a Polychlorinated Biphenyl-Utilizing Bacterium Isolated from Sewage Sludge.

Here, we report the whole-genome sequence of Aquamicrobium sp. strain SK-2, a bacterium which can use 2,2',4,4',5,5'-hexachlorobiphenyl as the sole carbon source for its growth. An approximately 9.23-Mb genome sequence of SK-2 will greatly facilitate research efforts regarding the study of the polychlorinated biphenyl (PCB) degradation mechanism.

Isolation of Bacillus sp. strains capable of decomposing alkali lignin and their application in combination with lactic acid bacteria for enhancing cellulase performance.

Effective biological pretreatment method for enhancing cellulase performance was investigated. Two alkali lignin-degrading bacteria were isolated from forest soils in Japan and named CS-1 and CS-2. 16S rDNA sequence analysis indicated that CS-1 and CS-2 were Bacillus sp. Strains CS-1 and CS-2 displayed alkali lignin degradation capability. With initial concentrations of 0.05-2.0 g L(-1), at least 61% alkali lignin could be degraded within 48 h. High laccase activities were observed in crude enzyme extracts from the isolated strains. This result indicated that alkali lignin degradation was correlated with laccase activities. Judging from the net yields of sugars after enzymatic hydrolysis, the most effective pretreatment method for enhancing cellulase performance was a two-step processing procedure (pretreatment using Bacillus sp. CS-1 followed by lactic acid bacteria) at 68.6%. These results suggest that the two-step pretreatment procedure is effective at accelerating cellulase performance.

Isolation of biphenyl and polychlorinated biphenyl-degrading bacteria and their degradation pathway.

Four strains of biphenyl-degrading bacteria were isolated from a sewage and identified from the Rhodococcus genus (SK-1, SK-3, and SK-4) and Aquamicrobium genus (SK-2) by 16S rRNA sequence. Among these strains, strain SK-2 was most suitable for biphenyl degradation. When 0.65, 1.3, 2.6, or 3.9 mM of biphenyl was used, the biphenyl was completely degraded within 24 and 96 h of culture, respectively. However, in the case of 6.5 and 9.75 mM of biphenyl, the biphenyl degradation yields were about 80 % and 46.7 % after 120 h of culture, respectively. The isolated strains could degrade a broad spectrum of aromatic compounds including high-chlorinated polychlorinated biphenyl (PCB) congeners in the presence of biphenyl. In addition, strain SK-2 could utilize PCB congeners containing one to six chlorine substituents such as 2,2',4,4',5,5'-hexachlorobiphenyl. The PCB utilization rate by the strain SK-2 was increased compared to that of other PCB congener-utilizing bacteria. The four isolates metabolized 4-chlorobiphenyl to 4-chlorobenzoic acid and 2-hydroxy-6-oxo-6-(4'-chlorophenyl)-hexa-2,4-dienoic acid. These results suggest the isolated strains might be good candidates for the bioremediation of PCB-contaminated soil, especially high-saline soils.

The medical association activity and pediatric care after the earthquake disaster in Fukushima.

On March 11, 2011, a gigantic earthquake struck eastern Japan. Utilities such as electricity, water, gas and telecommunication were interrupted. In Koriyama, the City Hall collapsed and government administration offices had to be moved to a nearby baseball stadium that had been designed to include facilities for use during a pandemic. An operations center was set up in this stadium. As members of the Koriyama Medical Association, we following the disaster protocol and set up our operations center in the Koriyama Medical Care Hospital. One large hospital with 280 inpatients and another hospital with 150 inpatients had been heavily damaged. Transfer of those patients to other hospitals without the use of telecommunications was extremely difficult. Many doctors in member hospitals and clinics went out of their way to cooperate throughout the crisis. Up to 5,000 people from the radiation evacuation zone were rushed to Koriyama. They stayed in schools and community centers, where we provided them with healthcare. Even in Koriyama, which is 60 km away from the Fukushima nuclear power plant, radiation levels were high, especially for the first few weeks. Citizens were advised to stay at home and keep their doors and windows closed. These drastic measures and frequent earthquake aftershocks were very stressful, especially for children. To help prevent children from developing posttraumatic stress disorder (PTSD), a project team composed of various groups caring for children was developed, and this team took action to protect children. Through these efforts we hoped to provide children with an appropriate environment to grow normally, even in a zone of persistent low-level radiation. We demonstrated once again that our members' long history of mutual assistance and cooperation with the administration was the main cornerstone to overcome the crisis.

Isolation and characterization of an arsenate-reducing bacterium and its application for arsenic extraction from contaminated soil.

A Gram-negative anaerobic bacterium, Citrobacter sp. NC-1, was isolated from soil contaminated with arsenic at levels as high as 5,000 mg As kg(-1). Strain NC-1 completely reduced 20 mM arsenate within 24 h and exhibited arsenate-reducing activity at concentrations as high as 60 mM. These results indicate that strain NC-1 is superior to other dissimilatory arsenate-reducing bacteria with respect to arsenate reduction, particularly at high concentrations. Strain NC-1 was also able to effectively extract arsenic from contaminated soils via the reduction of solid-phase arsenate to arsenite, which is much less adsorptive than arsenate. To characterize the reductase systems in strain NC-1, arsenate and nitrate reduction activities were investigated using washed-cell suspensions and crude cell extracts from cells grown on arsenate or nitrate. These reductase activities were induced individually by the two electron acceptors. This may be advantageous during bioremediation processes in which both contaminants are present.

Enhanced extraction of heavy metals in the two-step process with the mixed culture of Lactobacillus bulgaricus and Streptococcus thermophilus.

For biological extraction of heavy metals from chromated copper arsenate (CCA) treated wood, different bacteria were investigated. The extraction rates of heavy metals using Lactobacillusbulgaricus and Streptococcusthermophilus were highest. The chemical extraction rates were depended on the amounts of pyruvic acid and lactic acid. Especially, the extraction rates using mixed pyruvic acid and lactic acid were increased compared to those of sole one. They were also enhanced in the mixed culture of L. bulgaricus and S. thermophilus. To improve the extraction of CCA, a two-step processing procedure with the mixed culture of L. bulgaricus and S. thermophilus was conducted. A maximum of 93% of copper, 86.5% of chromium, and 97.8% of arsenic were extracted after 4 days. These results suggest that a two-step process with the mixed culture of L. bulgaricus and S. thermophilus is most effective to extract heavy metals from CCA treated wood.

Acceleration of nonylphenol and 4-tert-octylphenol degradation in sediment by Phragmites australis and associated rhizosphere bacteria.

We investigated biodegradation of technical nonylphenol (tNP) in Phragmites australis rhizosphere sediment by conducting degradation experiments using sediments spiked with tNP. Accelerated tNP removal was observed in P. australis rhizosphere sediment, whereas tNP persisted in unvegetated sediment without plants and in autoclaved sediment with sterile plants, suggesting that the accelerated tNP removal resulted largely from tNP biodegradation by rhizosphere bacteria. Three bacterial strains, Stenotrophomonas sp. strain IT-1 and Sphingobium spp. strains IT-4 and IT-5, isolated from the rhizosphere were capable of utilizing tNP and 4-tert-octylphenol as a sole carbon source via type II ipso-substitution. Oxygen from P. australis roots, by creating highly oxygenated conditions in the sediment, stimulated cell growth and the tNP-degrading activity of the three strains. Moreover, organic compounds from P. australis roots functioned as carbon and energy sources for two strains, IT-4 and IT-5, supporting cell growth and tNP-degrading activity. Thus, P. australis roots elevated the cell growth and tNP-degrading activity of the three bacterial strains, leading to accelerated tNP removal. These results demonstrate that rhizoremediation of tNP-contaminated sediments using P. australis can be an effective strategy.

Isolation and characterization of tetrachloroethylene- and cis-1,2-dichloroethylene-dechlorinating propionibacteria.

Two rapidly growing propionibacteria that could reductively dechlorinate tetrachloroethylene (PCE) and cis-1,2-dichloroethylene (cis-DCE) to ethylene were isolated from environmental sediments. Metabolic characterization and partial sequence analysis of their 16S rRNA genes showed that the new isolates, designated as strains Propionibacterium sp. HK-1 and Propionibacterium sp. HK-3, did not match any known PCE- or cis-DCE-degrading bacteria. Both strains dechlorinated relatively high concentrations of PCE (0.3 mM) and cis-DCE (0.52 mM) under anaerobic conditions without accumulating toxic intermediates during incubation. Cell-free extracts of both strains catalyzed PCE and cis-DCE dechlorination; degradation was accelerated by the addition of various electron donors. PCE dehalogenase from strain HK-1 was mediated by a corrinoid protein, since the dehalogenase was inactivated by propyl iodide only after reduction by titanium citrate. The amounts of chloride ions (0.094 and 0.103 mM) released after PCE (0.026 mM) and cis-DCE (0.05 mM) dehalogenation using the cell-free enzyme extracts of both strains, HK-1 and HK-3, were stoichiometrically similar (91 and 100%), indicating that PCE and cis-DCE were fully dechlorinated. Radiotracer studies with [1,2-¹4C] PCE and [1,2-¹4C] cis-DCE indicated that ethylene was the terminal product; partial conversion to ethylene was observed. Various chlorinated aliphatic compounds (PCE, trichloroethylene, cis-DCE, trans-1,2-dichloroethylene, 1,1-dichloroethylene, 1,1-dichloroethane, 1,2-dichloroethane, 1,2-dichloropropane, 1,1,2-trichloroethane, and vinyl chloride) were degraded by cell-free extracts of strain HK-1.

Accelerated biodegradation of pyrene and benzo[a]pyrene in the Phragmites australis rhizosphere by bacteria-root exudate interactions.

We investigated the biodegradation of pyrene and benzo[a]pyrene in Phragmites australis rhizosphere sediment. We collected P. australis plants, rhizosphere sediments, and unvegetated sediments from natural aquatic sites and conducted degradation experiments using sediments spiked with pyrene or benzo[a]pyrene. Accelerated removal of pyrene and benzo[a]pyrene was observed in P. australis rhizosphere sediments with plants, whereas both compounds persisted in unvegetated sediments without plants and in autoclaved rhizosphere sediments with sterilized plants, suggesting that the accelerated removal resulted largely from biodegradation by rhizosphere bacteria. Initial densities of pyrene-utilizing bacteria were substantially higher in the rhizosphere than in unvegetated sediments, but benzo[a]pyrene-utilizing bacteria were not detected in rhizosphere sediments. Mycobacterium gilvum strains isolated from rhizosphere sediments utilized pyrene aerobically as a sole carbon source and were able to degrade benzo[a]pyrene when induced with pyrene. Phragmites australis root exudates containing phenolic compounds supported growth as a carbon source for the one Mycobacterium strain tested, and induced benzo[a]pyrene-degrading activity of the strain. The stimulatory effect on benzo[a]pyrene biodegradation and the amounts of phenolic compounds in root exudates increased when P. australis was exposed to pyrene. Our results show that Mycobacterium-root exudate interactions can accelerate biodegradation of pyrene and benzo[a]pyrene in P. australis rhizosphere sediments.

Isolation and characterization of 4-tert-butylphenol-utilizing Sphingobium fuliginis strains from Phragmites australis rhizosphere sediment.

We isolated three Sphingobium fuliginis strains from Phragmites australis rhizosphere sediment that were capable of utilizing 4-tert-butylphenol as a sole carbon and energy source. These strains are the first 4-tert-butylphenol-utilizing bacteria. The strain designated TIK-1 completely degraded 1.0 mM 4-tert-butylphenol in basal salts medium within 12 h, with concomitant cell growth. We identified 4-tert-butylcatechol and 3,3-dimethyl-2-butanone as internal metabolites by gas chromatography-mass spectrometry. When 3-fluorocatechol was used as an inactivator of meta-cleavage enzymes, strain TIK-1 could not degrade 4-tert-butylcatechol and 3,3-dimethyl-2-butanone was not detected. We concluded that metabolism of 4-tert-butylphenol by strain TIK-1 is initiated by hydroxylation to 4-tert-butylcatechol, followed by a meta-cleavage pathway. Growth experiments with 20 other alkylphenols showed that 4-isopropylphenol, 4-sec-butylphenol, and 4-tert-pentylphenol, which have alkyl side chains of three to five carbon atoms with α-quaternary or α-tertiary carbons, supported cell growth but that 4-n-alkylphenols, 4-tert-octylphenol, technical nonylphenol, 2-alkylphenols, and 3-alkylphenols did not. The rate of growth on 4-tert-butylphenol was much higher than that of growth on the other alkylphenols. Degradation experiments with various alkylphenols showed that strain TIK-1 cells grown on 4-tert-butylphenol could degrade 4-alkylphenols with variously sized and branched side chains (ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, tert-pentyl, n-hexyl, n-heptyl, n-octyl, tert-octyl, n-nonyl, and branched nonyl) via a meta-cleavage pathway but not 2- or 3-alkylphenols. Along with the degradation of these alkylphenols, we detected methyl alkyl ketones that retained the structure of the original alkyl side chains. Strain TIK-1 may be useful in the bioremediation of environments polluted by 4-tert-butylphenol and various other 4-alkylphenols.

Isolation and characterization of a novel 2-sec-butylphenol-degrading bacterium Pseudomonas sp. strain MS-1.

A novel bacterium capable of utilizing 2-sec-butylphenol as the sole carbon and energy source, Pseudomonas sp. strain MS-1, was isolated from freshwater sediment. Within 30 h, strain MS-1 completely degraded 1.5 mM 2-sec-butylphenol in basal salt medium, with concomitant cell growth. A pathway for the metabolism of 2-sec-butylphenol by strain MS-1 was proposed on the basis of the identification of 3 internal metabolites-3-sec-butylcatechol, 2-hydroxy-6-oxo-7-methylnona-2,4-dienoic acid, and 2-methylbutyric acid-by gas chromatography-mass spectrometry analysis. Strain MS-1 degraded 2-sec-butylphenol through 3-sec-butylcatechol along a meta-cleavage pathway. Degradation experiments with various alkylphenols showed that the degradability of alkylphenols by strain MS-1 depended strongly on the position (ortho >> meta = para) of the alkyl substitute, and that strain MS-1 could degrade 2-alkylphenols with various sized and branched alkyl chain (o-cresol, 2-ethylphenol, 2-n-propylphenol, 2-isopropylphenol, 2-sec-butylphenol, and 2-tert-butylphenol), as well as a dialkylphenol (namely, 6-tert-butyl-m-cresol).

Biodegradation of bisphenol A and bisphenol F in the rhizosphere sediment of Phragmites australis.

The accelerated removal of bisphenols A and F (BPA, BPF) was observed in the rhizosphere sediment of Phragmites australis, while they persisted in the absence of P. australis. A BPA-degrading bacterium, Novosphingobium sp. strain TYA-1, and a BPF-degrading bacterium, Sphingobium yanoikuyae strain TYF-1, were isolated from the rhizosphere of P. australis. The results suggested that interactions between P. australis and these bacteria can accelerate the removal of bisphenols from sediment.

Enrichment of bacteria possessing catechol dioxygenase genes in the rhizosphere of Spirodela polyrrhiza: a mechanism of accelerated biodegradation of phenol.

The bacterial community structure in bulk water and in rhizosphere fractions of giant duckweed, Spirodela polyrrhiza, was quantitatively and qualitatively investigated by PCR-based methods using 6 environmental water samples to elucidate the mechanisms underlying selective accumulation of aromatic compound-degrading bacteria in the rhizosphere of S. polyrrhiza. S. polyrrhiza selectively accumulated a diverse range of aromatic compound-degrading bacteria in its rhizosphere, regardless of the origin of water samples, despite no exposure to phenol. The relative abundances of the catechol 1,2-dioxygenase (C12O) gene (C12O DNA) and catechol 2,3-dioxygenase (C23O) gene (C23O DNA) were calculated as the ratios of the copy numbers of these genes to the copy number of 16S rDNA and are referred to as the rhizosphere effect (RE) value. The RE values for C12O DNA and C23O DNA were 1.0 x 10(1)-9.3 x 10(3) and 1.7 x 10(2)-1.5 x 10(4) times as high, respectively, in rhizosphere fractions as in bulk water fractions, and these higher values were associated with a notably higher sequence diversity of C12O DNA and C23O DNA. The RE values during phenol degradation were 3.6 x 10(0)-4.3 x 10(2) and 2.2 x 10(0)-1.7 x 10(2), respectively, indicating the ability of S. polyrrhiza to selectively accumulate aromatic compound-degrading bacteria in its rhizosphere during phenol degradation. The bacterial communities in the rhizosphere fractions differed from those in the bulk water fractions, and those in the bulk water fractions were notably affected by the rhizosphere bacterial communities. S. polyrrhiza released more than 100 types of phenolic compound into its rhizosphere as root exudates at the considerably high specific release rate of 1520mg TOC and 214mg phenolic compounds/d/g root (wet weight). This ability of S. polyrrhiza might result in the selective recruitment and accumulation of a diverse range of bacteria harboring genes encoding C12O and C23O, and the subsequent accelerated degradation of phenol in the rhizosphere.

Potential of a new biotreatment: Sphingomonas cloacae S-3T degrades nonylphenol in industrial wastewater.

Sphingomonas cloacae S-3(T), a nonylphenol (NP)-degrading bacterium, was evaluated for its utility in the remediation of NP-contaminated wastewater. In flask-scale experiments, S-3(T) cells immobilized on porous polypropylene carriers (beads) efficiently degraded NP to concentrations routinely measured in aquatic environments [a few parts per billion (ppb), or micrograms per liter). Therefore, we constructed and evaluated a laboratory-scale wastewater treatment system with a 3-l carrier-filled column. The system worked properly and consistently removed several hundred ppb of NP to ecologically safe concentrations of less than 10 ppb in industrial wastewater without the addition of nutrients. The effect of wastewater pH on the system performance was also evaluated; and wastewater samples with pH values of 6 or 8 were treated efficiently without pH adjustment. These results suggest that a biotreatment system using NP-degrading bacteria can efficiently remediate industrial wastewater and contribute to the preservation of aquatic environments.

Novosphingobium tardaugens sp. nov., an oestradiol-degrading bacterium isolated from activated sludge of a sewage treatment plant in Tokyo.

An oestradiol-degrading bacterium isolated at a sewage treatment plant in Tokyo was studied phenotypically, genotypically and phylogenetically. Analysis of its 16S rDNA sequence, DNA base composition, whole-cell fatty acid profile and isoprenoid quinone composition, as well as the presence of sphingoglycolipid, revealed that the isolate is a member of the genus Novosphingobium. However, the sequence similarity of its 16S rDNA to those of known Novosphingobium species was no higher than 97%, implying that the isolate is distinctive. The results of DNA-DNA hybridization experiments and physiological characterization also indicated that the isolate represents a novel Novosphingobium species, for which the name Novosphingobium tardaugens sp. nov. is proposed; strain ARI-1T (=JCM 11434T =ATCC BAA-531T =IFO 16725T) is the type strain.

Degradation of 17beta-estradiol by a gram-negative bacterium isolated from activated sludge in a sewage treatment plant in Tokyo, Japan.

A 17beta-estradiol (E2)-degrading bacterium was isolated from activated sludge in a sewage treatment plant in Tokyo, Japan. The isolate was suggested to be a new Novosphingobium species. Gas chromatography-mass spectrometry and (1)H nuclear magnetic resonance analyses of the metabolites of E2 degradation suggested that no toxic products accumulated in the culture medium.