Isolation of a Novel Endophytic Bacillus Strain Capable of Transforming Pentachlorophenol and Structure Determination of Pentachlorophenol Phosphate Using Single-Crystal X-ray Diffraction.

A novel approach for the remediation of upland soils contaminated with pentachlorophenol (C6HCl5O; PCP) (1), a fungicide, wood perservative, and herbicide, through the exploitation of plant-endophytic bacteria may overcome the existing issues in bioaugmentaion and phytoremidiation. In this study, we isolated the endophytic Bacillus sp. strain PCP15 and determined its metabolite of PCP (1). This strain degraded 8.03 µmol L-1 PCP (1) within 24 h and generated the novel metabolite PCP phosphate (3). The PCP15 strain showed nearly complete growth inhibition of 20 µmol L-1 PCP (1). In contrast, PCP15 showed resistance to PCP phosphate (3), indicating that the phosphorylation of PCP, which has never previously been reported in organisms, contributed to the detoxification of PCP (1) in bacterial cells. Our results show the potential for practical application of this strain in hybrid remediation of PCP (1)-contaminated soils and reveal a novel PCP (1) detoxification mechanism in organisms.

Predicting rice pesticide fate and transport following foliage application by an updated PCPF-1 model.

The Pesticide Concentration in Paddy Field (PCPF-1) model has been successfully used to predict the fate and transport of granular pesticides applied to the paddy fields. However, it is not applicable for pesticides in foliar formulation while previous studies have reported that foliar application may increase the risks of rice pesticide contamination to the aquatic environment due to pesticide wash-off from rice foliage. In this study, we developed and added a foliar application module into the PCPF-1 model to improve its versatility regarding pesticide application methods. In addition, some processes of the original model such as photodegradation were simplified. The updated model was then validated with data from previous studies. Critical parameters of the model were calibrated using the Sequential Uncertainty Fitting version 2 (SUFI-2) algorithm. The calibrated model simulated pesticide dissipation trend and concentrations with moderate accuracy in the two paddy compartments including rice foliage and paddy water. The accuracy of the predicted soil concentrations could not be evaluated since no observed data were available. Although the p-factor and r-factor obtained using the SUFI2 algorithm indicated that the uncertainty encompassed in the predicted concentrations was rather high, the daily predicted pesticide concentrations in rice foliage and paddy water were satisfactory based on the NSE values (0.36-0.89). The updated PCPF-1 model is a flexible tool for the environmental risk assessment of pesticide losses and the evaluation of agricultural management practices for mitigating pesticide pollution associated with rice production.

Biochemical characterization of NADH:FMN oxidoreductase HcbA3 from Nocardioides sp. PD653 in catalyzing aerobic HCB dechlorination.

Nocardioides sp. PD653 genes hcbA1, hcbA2, and hcbA3 encode enzymes that catalyze the oxidative dehalogenation of hexachlorobenzene (HCB), which is one of the most recalcitrant persistent organic pollutants (POPs). In this study, HcbA1, HcbA2, and HcbA3 were heterologously expressed and characterized. Among the flavin species tested, HcbA3 showed the highest affinity for FMN with a K d value of 0.75±0.17 µM. Kinetic assays revealed that HcbA3 followed a ping-pong bi-bi mechanism for the reduction of flavins. The K m for NADH and FMN was 51.66±11.58 µM and 4.43±0.69 µM, respectively. For both NADH and FMN, the V max and k cat were 2.21±0.86 µM and 66.74±5.91 sec-1, respectively. We also successfully reconstituted the oxidative dehalogenase reaction in vitro, which consisted of HcbA1, HcbA3, FMN, and NADH, suggesting that HcbA3 may be the partner reductase component for HcbA1 in Nocardioides sp. PD653.

Study on the biodegradation of persistent organic pollutants (POPs).

The use of organochlorine pesticides, such as dichlorodiphenyltrichloroethane (DDT) and benzene hexachloride (BHC), have contributed substantially to the increase and stable supply of food production post-World War II. However, they have also become a major source of pollution on a global scale due to their persistence in the environment, high bioconcentration, toxicity, and their long-distance mobility. Although the use and production of these pesticides were banned over 45 years ago, they still present a risk to human health and ecosystems, and pose a threat to food safety. These pesticides were designated as persistent organic pollutants (POPs) by the Stockholm Convention in 2001, which urged the industry to reduce or eliminate them globally. The authors of this study have been involved in the research and development of bioaugmentation soil remediation technology to reduce the risk of environmental and crop contamination originating from POPs. In this paper, these studies are summarized, from basic studies (1, 2, 3) to an applied study (4), as follows: (1) use of the soil-charcoal perfusion method to explore POP-degrading bacteria, (2) bacteriological characteristics, metabolic pathways and dechlorination genes of the hexaclorobenzene (HCB)-mineralizing bacterial strain PD653, (3) characteristics and metabolic pathways of the dieldrin-degrading bacterial strain KSF27, and (4) application of these degrading bacteria for remediation of POPs-contaminated soil.

Dissipation, dehalogenation, and denitration of chloroaromatic compounds by Nocardioides sp. strain PD653: Characterization of the substrate specificity.

The substrate range of Nocardioides sp. strain PD653, capable of mineralizing hexachlorobenzene, was investigated based on the dissipation of substrates and the liberation of halogen ions. Strain PD653 dehalogenated 10 out of 18 halophenol congeners; however, it could dehalogenate only hexachlorobenzene out of seven halobenzene congeners tested. Moreover, dehalogenation activities were shown for chloronitrobenzenes, along with an increase in the number of substituted chlorine atoms except for 2,3,4,5-tetrachloro-1-nitrobenzene. These results suggested that this strain might be applicable to remediate soil contaminated with these persistent chloroaromatic compounds.

Changes in endophytic bacterial communities during different growth stages of cucumber (Cucumis sativus L.).

Endophytic bacterial diversity in plants presents the level of interaction between culturable and non-culturable endophytic bacteria, thereby providing an appropriate insight into the endophytic environment. This study was conducted to determine the trend of culturable and non-culturable endophytic bacteria at two different sites encompassing four consecutive growth stages. For culturable endophytic bacteria, isolation was carried out using the dilution plate technique, and the obtained colonies were compared using PCR-restriction fragment length polymorphism (RFLP). Different RFLP-types were identified to their nearest neighbour using 16S rRNA sequencing. The non-culturable endophytic bacterial diversity was obtained by next generation sequencing. Results suggested a similar trend among the culturable and non-culturable bacteria for observed operational taxonomic units and diversity indices. It is noticeable that the endophytic bacteria inhabiting in stage 1 disappeared, and instead, different endophytic bacteria appeared. Moreover, the temporal persistence of certain culturable and non-culturable bacteria was also observed. In conclusion, the endophytic bacterial diversity in cucumber initially increased with the plant growth and then decreased at a later stage. Furthermore, it was suggested that plants regulate the number and diversity of endophytes throughout the lifecycle of plants.

Identification of the novel hcbB operon catalyzing the dechlorination of pentachlorophenol in the Gram-positive bacterium Nocardioides sp. strain PD653.

While pcp genes are well known in Gram-negative bacteria to code for the enzymes responsible for pentachlorophenol (C6HCl5O; PCP) degradation, little is known about PCP-degrading genes in Gram-positive bacteria. Here we describe a novel gene operon possibly responsible for catalyzing the degradation of PCP in the Gram-positive bacterium Nocardioides sp. strain PD653, which is capable of mineralizing hexachlorobenzene (C6Cl6; HCB) via PCP. Transcriptome analysis based on RNA-Seq revealed overexpressed genes in strain PD653 following exposure to HCB. Based on in silico annotation, three open reading frames (ORFs) were selected as biodegrading enzyme candidates. Recombinant E. coli cells expressing candidate genes degraded approximately 9.4 µmol L-1 PCP in 2 hr. Therefore, we designated these genes as hcbB1, hcbB2, and hcbB3. Interestingly, PCP-degrading activity was recorded when hcbB3 was coexpressed with hcbB1 or hcbB2, and the function of HcbB3 was expected to be similar to chlorophenol 4-monooxygenase (TftD).

Identification of the hcb Gene Operon Involved in Catalyzing Aerobic Hexachlorobenzene Dechlorination in Nocardioides sp. Strain PD653.

Nocardioides sp. strain PD653 was the first identified aerobic bacterium capable of mineralizing hexachlorobenzene (HCB). In this study, strain PD653-B2, which was unexpectedly isolated from a subculture of strain PD653, was found to lack the ability to transform HCB or pentachloronitrobenzene into pentachlorophenol. Comparative genome analysis of the two strains revealed that genetic rearrangement had occurred in strain PD653-B2, with a genomic region present in strain PD653 being deleted. In silico analysis allowed three open reading frames within this region to be identified as candidate genes involved in HCB dechlorination. Assays using recombinant Escherichia coli cells revealed that an operon is responsible for both oxidative HCB dechlorination and pentachloronitrobenzene denitration. The metabolite pentachlorophenol was detected in the cultures produced in the E. coli assays. Significantly less HCB-degrading activity occurred in assays under oxygen-limited conditions ([O2] < 0.5 mg liter-1) than under aerobic assays, suggesting that monooxygenase is involved in the reaction. In this operon, hcbA1 was found to encode a monooxygenase involved in HCB dechlorination. This monooxygenase may form a complex with the flavin reductase encoded by hcbA3, increasing the HCB-degrading activity of PD653.IMPORTANCE The organochlorine fungicide HCB is widely distributed in the environment. Bioremediation can effectively remove HCB from contaminated sites, but HCB-degrading microorganisms have been isolated in few studies and the genes involved in HCB degradation have not been identified. In this study, possible genes involved in the initial step of the mineralization of HCB by Nocardioides sp. strain PD653 were identified. The results improve our understanding of the protein families involved in the dechlorination of HCB to give pentachlorophenol.

Detoxification of hydroxylated polychlorobiphenyls by Sphingomonas sp. strain N-9 isolated from forest soil.

To examine the biodegradation of hydroxylated polychlorobiphenyls (OH-PCBs), we isolated Sphingomonas sp. strain N-9 from forest soil using mineral salt medium containing 4-hydroxy-3-chlorobiphenyl (4OH-3CB) at the concentration of 10 mg/L. Following incubation with strain N-9, the concentration of 4OH-3CB decreased in inverse proportion to strain N-9 proliferation, and it was converted to 3-chloro-4-hydroxybenzoic acid (4OH-3CBA) after 1 day. We observed that strain N-9 efficiently degraded lowly chlorinated OH-PCBs (1-4 Cl), while highly chlorinated OH-PCBs (5-6 Cl) were less efficiently transformed. Additionally, strain N-9 degraded PCBs and OH-PCBs with similar efficiencies, and the efficiency of OH-PCB degradation was dependent upon the positional relationships between OH-PCB hydroxyl groups and chlorinated rings. OH-PCB biodegradation may result in highly toxic products, therefore, we evaluated the cytotoxicity of two OH-PCBs [4OH-3CB and 4-hydroxy-3,5-dichlorobiphenyl (4OH-3,5CB)] and their metabolites [4OH-3CBA and 3,5-chloro-4-hydroxybenzoic acid (4OH-3,5CBA)] using PC12 rat pheochromocytoma cells. Our results revealed that both OH-PCBs induced cell membrane damage and caused neuron-like elongations in a dose-dependent manner, while similar results were not observed for their metabolites. These results indicated that strain N-9 can convert OH-PCBs into chloro-hydroxybenzoic acids having lower toxicity.

Isolation of endosulfan sulfate-degrading Rhodococcus koreensis strain S1-1 from endosulfan contaminated soil and identification of a novel metabolite, endosulfan diol monosulfate.

An aerobic endosulfan sulfate-degrading bacterium, Rhodococcus koreensis strain S1-1, was isolated from soil to which endosulfan had been applied annually for more than 10 years until 2008. The strain isolated in this work reduced the concentration of endosulfan sulfate (2) from 12.25 µM to 2.11 µM during 14 d at 30 °C. Using ultra performance liquid chromatography-electrospray ionization-mass spectroscopy (UPLC-ESI-MS), a new highly water-soluble metabolite possessing six chlorine atoms was found to be endosulfan diol monosulfate (6), derived from 2 by hydrolysis of the cyclic sulfate ester ring. The structure of 6 was elucidated by chemical synthesis of the candidate derivatives and by HR-MS and UPLC-MS analyses. Therefore, it was suggested that the strain S1-1 has a new metabolic pathway of 2. In addition, 6 was expected to be less toxic among the metabolites of 1 because of its higher water-solubility.

Identification and characterization of 1,4-dioxane-degrading microbe separated from surface seawater by the seawater-charcoal perfusion apparatus.

To determine the concentration of soluble 1,4-dioxane during biodegradation, a new method using of high-performance liquid chromatography equipped with a hydrophilic interaction chromatography column was developed. The developed method enabled easy and rapid determination of 1,4-dioxane, even in saline medium. Microbes capable of degrading 1,4-dioxane were selected from the seawater samples by the seawater-charcoal perfusion apparatus. Among 32 candidate 1,4-dioxane degraders,, strain RM-31 exhibited the strongest 1,4-dioxane degradation ability. 16S rDNA sequencing and the similarity analysis of strain RM-31 suggested that this organism was most closely related to Pseudonocardia carboxydivorans. This species is similar to Pseudonocardia dioxanivorans, which has previously been reported as a 1,4-dioxane degrader. Strain RM-31 could degrade 300 mg/L within 2 days. As culture incubation times increasing, the residual 1,4-dioxane concentration was decreasing and the total protein contents extracted from growth cells were increasing. The optimum initial pH of the broth medium and incubation temperature for 1,4-dioxane degradation were pH 6-8 and 25 °C. The biodegradation rate of 1,4-dioxane by strain RM-31 at 25 °C in broth medium with 3 % NaCl was almost 20 % faster than that without NaCl. It was probably a first bacteria from the seawater that can exert a strong degrading ability.

Bacterial biodegradation of melamine-contaminated aged soil: influence of different pre-culture media or addition of activation material.

This study aimed to investigate the biodegrading potential of Arthrobacter sp. MCO, Arthrobacter sp. CSP, and Nocardioides sp. ATD6 in melamine-contaminated upland soil (melamine: approx. 10.5 mg/kg dry weight) after 30 days of incubation. The soil sample used in this study had undergone annual treatment of lime nitrogen, which included melamine; it was aged for more than 10 years in field. When R2A broth was used as the pre-culture medium, Arthrobacter sp. MCO could degrade 55 % of melamine after 30 days of incubation, but the other strains could hardly degrade melamine (approximately 25 %). The addition of trimethylglycine (betaine) in soil as an activation material enhanced the degradation rate of melamine by each strain; more than 50 % of melamine was degraded by all strains after 30 days of incubation. In particular, strain MCO could degrade 72 % of melamine. When the strains were pre-cultured in R2A broth containing melamine, the degradation rate of melamine in soil increased remarkably. The highest (72 %) melamine degradation rate was noted when strain MCO was used with betaine addition.

4,4'-DDE and Endosulfan Levels in Agricultural Soils of the Çukurova Region, Mediterranean Turkey.

Mediterranean Turkey has long been at the forefront of Turkish agriculture and the use of organochlorinated pesticides (OCPs) in this area rose considerably between the 1940s and 1980s. This study aimed to determine OCP residue levels in agricultural soils collected from the Mersin and Adana Districts, Çukurova Basin in Mediterranean Turkey. Most soil samples were contaminated with one, or both, of two OCP metabolites; 4,4'-dichlorodiphenyldichloroethylene (4,4'-DDE) and endosulfan sulfate. 4,4'-DDE occurred in 27 of the 29 samples and ranged from 6 to 1090 µg kg(-1)-dry soil (ds)(-1), while six samples contained endosulfan sulfate ranging between 82 and 1226 µg kg(-1)-ds(-1). Generally, horticultural and corn-planted soils contained only 4,4'-DDE, whereas greenhouse cultivation appeared to accumulate both residues. This study indicated that 4,4'-DDE occurred above acceptable levels of risk in agricultural soils of Mersin District and further studies on the qualitative and quantitative assessment of OCPs in other agricultural regions with intensive pesticide use are necessary to fully understand the impact of OCPs on agricultural soil in Turkey.

Biodegradation of cyromazine by melamine-degrading bacteria.

Biodegradation of cyromazine was investigated in liquid cultures using three melamine-degrading bacteria Arthrobacter sp. MCO, Arthrobacter sp. CSP and Nocardioides sp. ATD6. Experiments were performed aerobically in a mineral medium with glucose as a carbon source and cyromazine as the sole nitrogen source. All three strains of bacteria degraded cyromazine. Cyromazine at 23 mg/L completely disappeared by Arthrobacter sp. MCO within 7 days. The bacterial density of all three strains increased with degradation of the cyromazine. The cyromazine metabolite N-cyclopropylammeline was detected and identified by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). This is the first report on the use of Arthrobacter sp. and Nocardioides sp. for cyromazine degradation and the occurrence of bacterial growth with cyromazine degradation.

Mineralization of melamine and cyanuric acid as sole nitrogen source by newly isolated Arthrobacter spp. using a soil-charcoal perfusion method.

Melamine belongs to the s-triazine family, and industrially used as raw product in many ways all over the world. Melamine has been reported for human harmful effects and detected from some crops, soil and water. To remove melamine from the polluted environment, the efficient melamine-mineralizing microorganisms have been needed. We newly isolated three melamine-degrading bacteria from the same upland soil sample using soil-charcoal perfusion method. These bacteria were classified as Arthrobacter sp. MCO, Arthrobacter sp. CSP and Microbacterium sp. ZEL by 16S rRNA genes sequencing analysis. Both Arthrobacter species completely degraded melamine within 2 days, and consumed melamine as a sole nitrogen source. Both strains also grew in cyanuric acid as sole nitrogen source, and released small quantities of ammonium ions. These strains are the first identified bacteria that can mineralize both melamine and cyanuric acid as sole initial nitrogen source in Arthrobacter sp. Although ammeline and ammelide intermediates were detected, these strains possess none of the known genes encoding melamine degrading enzymes. Since the Arthrobacter strains also degraded melamine in a high pH liquid medium, they present as potential bioremediation agents in melamine-polluted environments.

Effect of rice husk gasification residue application on herbicide behavior in micro paddy lysimeter.

Effects of rice husk gasification residues (RHGR) application on the fate of herbicides, butachlor and pyrazosulfuron-ethyl, in paddy water were investigated using micro paddy lysimeters (MPLs). The dissipation of both herbicides in paddy water was faster in the RHGR treated MPL than in the control MPL. The average concentrations of butachlor and pyrazosulfuron-ethyl in paddy water in the lysimeter treated with RHGR during 21 days were significantly reduced by 51% and 48%, respectively, as compared to those in the lysimeter without RHGR application. The half-lives (DT50) of butachlor in paddy water for control and treatment were 3.1 and 2.3 days respectively, and these values of pyrazosulfuron-ethyl were 3.0 and 2.2 days, respectively. Based on this study, RHGR application in rice paddy environment is an alternative method to reduce the concentration of herbicide in paddy field water and consequently to reduce potential pollution to aquatic environment.

Arsenic biotransformation by Streptomyces†sp. isolated from rice rhizosphere.

Isolation and functional analysis of microbes mediating the methylation of arsenic (As) in paddy soils is important for understanding the origin of dimethylarsinic acid (DMA) in rice grains. Here, we isolated from the rice rhizosphere a unique bacterium responsible for As methylation. Strain GSRB54, which was isolated from the roots of rice plants grown in As-contaminated paddy soil under anaerobic conditions, was classified into the genus Streptomyces by 16S ribosomal RNA sequencing. Sequence analysis of the arsenite S-adenosylmethionine methyltransferase (arsM) gene revealed that GSRB54 arsM was phylogenetically different from known arsM genes in other bacteria. This strain produced DMA and monomethylarsonic acid when cultured in liquid medium containing arsenite [As(III)]. Heterologous expression of GSRB54 arsM in Escherichia coli promoted methylation of As(III) by converting it into DMA and trimethylarsine oxide. These results demonstrate that strain GSRB54 has a strong ability to methylate As. In addition, DMA was detected in the shoots of rice grown in liquid medium inoculated with GSRB54 and containing As(III). Since Streptomyces are generally aerobic bacteria, we speculate that strain GSRB54 inhabits the oxidative zone around roots of paddy rice and is associated with DMA accumulation in rice grains through As methylation in the rice rhizosphere.

Biodegradability and biodegradation pathways of endosulfan and endosulfan sulfate.

Endosulfan and endosulfan sulfate are persistent organic pollutants that cause serious environmental problems. Although these compounds are already prohibited in many countries, residues can be detected in soils with a history of endosulfan application. Endosulfan is transformed in the environment into endosulfan sulfate, which is a toxic and persistent metabolite. However, some microorganisms can degrade endosulfan without producing endosulfan sulfate, and some can degrade endosulfan sulfate. Therefore, biodegradation has the potential to clean up soil contaminated with endosulfan. In this review, we provide an overview of aerobic endosulfan degradation by bacteria and fungi, and a summary of recent advances and prospects in this research field.

Biodegradation of melamine and its hydroxy derivatives by a bacterial consortium containing a novel Nocardioides species.

Melamine has recently been recognized as a food contaminant with adverse human health effects. Melamine contamination in some crops arises from soil and water pollution from various causes. To remove melamine from the polluted environment, a novel bacterium, Nocardioides sp. strain ATD6, capable of degrading melamine was enriched and isolated from a paddy soil sample. The enrichment culture was performed by the soil-charcoal perfusion method in the presence of triazine-degrading bacteria previously obtained. Strain ATD6 degraded melamine and accumulated cyanuric acid and ammonium, via the intermediates ammeline and ammelide. No gene known to encode for triazine-degrading enzymes was detected in strain ATD6. A mixed culture of strain ATD6 and a simazine-degrading Methyloversatilis sp. strain CDB21 completely degraded melamine, but the degradation rate of cyanuric acid was slow. The degradation of melamine and its catabolites by the mixed culture was greatly enhanced by including Bradyrhizobium japonicum strain CSB1 in the inoculum and adding ethanol to the culture medium. The melamine-degrading consortium consisting of strains ATD6, CDB21, and CSB1 appears to be potentially safer than other known melamine-degrading bacteria for the bioremediation of farmland and other contaminated sites, as no known pathogens were included in the consortium.