Degradation Of γ-HCH Research Articles

Deletion of genomic islands in the Pseudomonas putida KT2440 genome can create an optimal chassis for synthetic biology applications

BackgroundGenome streamlining is a feasible strategy for constructing an optimum microbial chassis for synthetic biology applications. Genomic islands (GIs) are usually regarded as foreign DNA sequences, which can be obtained by horizontal gene transfer among microorganisms. A model strain Pseudomonas putida KT2440 has broad applications in biocatalysis, biotransformation and biodegradation.ResultsIn this study, the identified GIs in P. putida KT2440 accounting for 4.12% of the total genome size were deleted to generate a series of genome-reduced strains. The mutant KTU-U13 with the largest deletion was advantageous over the original strain KTU in several physiological characteristics evaluated. The mutant KTU-U13 showed high plasmid transformation efficiency and heterologous protein expression capacity compared with the original strain KTU. The metabolic phenotype analysis showed that the types of carbon sources utilized by the mutant KTU-U13 and the utilization capabilities for certain carbon sources were increased greatly. The polyhydroxyalkanoate (PHA) yield and cell dry weight of the mutant KTU-U13 were improved significantly compared with the original strain KTU. The chromosomal integration efficiencies for the γ-hexachlorocyclohexane (γ-HCH) and 1,2,3-trichloropropane (TCP) biodegradation pathways were improved greatly when using the mutant KTU-U13 as the recipient cell and enhanced degradation of γ-HCH and TCP by the mutant KTU-U13 was also observed. The mutant KTU-U13 was able to stably express a plasmid-borne zeaxanthin biosynthetic pathway, suggesting the excellent genetic stability of the mutant.ConclusionsThese desirable traits make the GIs-deleted mutant KTU-U13 an optimum chassis for synthetic biology applications. The present study suggests that the systematic deletion of GIs in bacteria may be a useful approach for generating an optimal chassis for the construction of microbial cell factories.

Interaction and coexistence characteristics of dissolved organic matter with toxic metals and pesticides in shallow groundwater

The long-term and large-scale utilization of fertilizers and pesticides in facility agriculture leads to groundwater pollution. However, the coexistence and interactions between organic fertilizers (i.e., organic matter), toxic metals, and pesticides in shallow groundwater have seldom been studied. Thus, the study sought to characterize said interactions via fluorescence, ultraviolet–visible spectroscopy (UV–Vis), and Fourier-transform infrared spectroscopy coupled with two-dimensional correlation spectroscopy and chemometric techniques. The results indicated that groundwater DOM was comprised of protein-, polysaccharide-, and lignin-like substances derived from organic fertilizers. Protein-like substances accounted for the binding of Co, Ni, and Fe, while polysaccharide- and lignin-like substances were mainly responsible for Cr and Mo complexation. Moreover, lignin- and polysaccharide-like substances played a key role in the binding of pesticides (i.e., dichlorodiphenyltrichloroethane [DDT], endosulfan, γ-hexachlorocyclohexane [γ-HCH], monocrotophos, chlorpyrifos, and chlorfenvinphos), rendering the conversion of γ-HCH to β-hexachlorocyclohexane (β-HCH) and the degradation of DDT to dichlorobenzene dichloroethylene (DDE) ineffective. However, the presence of protein-like substances in groundwater benefited the degradation and conversion of γ-HCH and α-endosulfan. Redundancy analyses showed that lignin- and polysaccharide-like matter had the most impacts on the coexistence of DOM with toxic metals and pesticides.

Diversity and Activity of Bacterial Biofilm Communities Growing on Hexachlorocyclohexane

γ-Hexachlorocyclohexane (γ-HCH) persists in the environment and is recalcitrant to microbial degradation. To determine the extent of the microbial potential for the degradation of γ-HCH the diversity of bacteria from 12 soil samples collected around insecticide- and pesticide-producing factories in Egypt were assessed and compared with biofilm communities grown on γ-HCH microcrystals. From all samples, highly diverse microbes were isolated, able to grow on γ-HCH as sole source of carbon. The same soil samples were used to inoculate γ-HCH microcrystals on a substratum in microcosms to grow biofilm communities. All soil samples formed multispecies biofilms on γ-HCH. Biofilms stained with Nile Red showed distinct cell clusters of high hydrophobicity, and it is speculated that these aggregates have a substantial role in the degradation of the hydrophobic substrate. While many Bacillus species were isolated, this group was almost absent in the different biofilm communities. The finding of cells with highly hydrophobic envelopes together with the differences in species composition between isolates and interacting microbial communities points to fundamental differences in the interaction with hydrophobic substrates of single strains and microbial communities.

Biodegradation of γ-hexachlorocyclohexane by transgenic hairy root cultures of Cucurbita moschata that accumulate recombinant bacterial LinA

γ-HCH was successfully degraded using LinA-expressed transgenic hairy root cultures of Cucurbita moschata . Fusing an endoplasmic reticulum-targeting signal peptide to LinA was essential for stable accumulation in the hairy roots. The pesticide γ-hexachlorocyclohexane (γ-HCH) is a persistent organic pollutant (POP) that raises public health and environmental pollution concerns worldwide. Although several isolates of γ-HCH-degrading bacteria are available, inoculating them directly into γ-HCH-contaminated soil is ineffective because of the bacterial survival rate. Cucurbita species incorporate significant amounts of POPs from soils compared with other plant species. Here, we describe a novel bioremediation strategy that combines the bacterial degradation of γ-HCH and the efficient uptake of γ-HCH by Cucurbita species. We produced transgenic hairy root cultures of Cucurbita moschata that expressed recombinant bacterial linA, isolated from the bacterium Sphingobium japonicum UT26. The LinA protein was accumulated stably in the hairy root cultures by fusing an endoplasmic reticulum (ER)-targeting signal peptide to LinA. Then, we demonstrated that the cultures degraded more than 90% of γ-HCH (1ppm) overnight and produced the γ-HCH metabolite 1,2,4-trichlorobenzene, indicating that LinA degraded γ-HCH. These results indicate that the gene linA has high potential for phytoremediation of environmental γ-HCH.

Degradation of organochlorine pesticides (OCPs) in water by iron (Fe)-based materials

This study employees iron (Fe)-based materials which contain zero valent iron (Fe0) and magnetite (Fe3O4) for degradation of organochlorine pesticides (OCPs), such as DDT, lindane (γ-HCH) and aldrin, in water. The degradation kinetics rate, effect of iron (Fe)-based materials dosage, chloride ion (Cl−) mass balance and degradation products are investigated. The results show that iron (Fe)-based materials successfully degrade OCPs in water. After 12h reaction, the degradation of γ-HCH, p,p′-DDT and aldrin at the initial concentration of 0.001mgL−1 in water were found to be 100%, 81%, and 79%, respectively. Degradation rate order from the highest is γ-HCH, followed p,p′-DDT, and aldrin which related to their physicochemical properties. The degradation rate decreased with the increasing initial concentration of OCPs from 0.00005mgL−1 to 10mgL−1 in water. Increasing the amount of iron (Fe)-based materials clearly improves the degradation rate. Moreover, increasing free chloride ion (Cl−) for elapsed time suggests that OCPs are degraded into lower molecular compounds through dechlorination process. In addition, several degradation products of p,p′-DDT are detected, and degradation pathways are proposed, however, degradation products of γ-HCH and aldrin could not be identified in this study.

Remediation HCHs POPs-contaminated soil by activated persulfate technologies: Feasibility, impact of activation methods and mechanistic implications

Hexachlorocyclohexanes (HCHs) waste stockpiles are the single largest source of POPs. Sites contaminated with high levels of HCHs pose serious threats to public health and environmental safety, but there remains a lack of proper environmentally sound solution. To provide guidance on this issue, the remediation of contaminated soil with high levels of γ-HCH (∼800mg/kg) by persulfate with three commonly applied field activation methods, namely heat activation at 40°C, high pH and Fe(III)-EDTA activation, was investigated. The effects of activation method and persulfate dosage on the degradation and dechlorination of γ-HCH were studied, and degradation pathways were proposed. The results show that heat-activated persulfate is highly effective for the remediation of γ-HCH, achieving complete degradation and dechlorination of γ-HCH in 14days. Under high pH conditions, γ-HCH rapidly dehydrochlorizes into trichlorobenzenes, which are further degraded by persulfate. On the other hand, persulfate alone at 20°C and Fe(III)-EDTA activated persulfate are not effective for the degradation of γ-HCH, due to limited generation of reactive radical species resulting from the inefficient activation of persulfate, and the competition of soil organic materials and Fe(III)-EDTA with γ-HCH for reactive radicals.

Biodegradation of Hexachlorocyclohexane (HCH) Isomers by White Rot Fungus, Pleurotus florida

Hexachlorocyclohexane (HCH) isomers are reported to persist in the environment long after their usage is discontinued. Pleurotus florida, a white rot fungus, was found to degrade not only γ-HCH but also its more persistent isomers individually as well as a mixture in a Raper’s complete medium. Within 30 days γ-HCH degraded completely, whereas other 3 isomers degraded 92-99%. The degradation rate was γ-HCH>β-HCH>α- and δ-HCH. The mycelium biomass was free from γ-HCH residues but accumulated about 3% residues of other 3 isomers. Presence of intermediate metabolites was not detected indicating complete mineralization of HCH isomers. Ability of P. florida to degrade HCH isomers was further studied in soil by amendment with spent mushroom substrate (SMS). SMS addition could marginally increase degradation of α-, β- and δ-HCH, but significantly increased degradation of γ-HCH. When the study was repeated similar trend was observed. The half-life of degradation of γ-HCH was 439-570 days in un-amended soil while 37-42 days in SMS amended soil. For other 3 stable isomers the half-life was reduced from 686-828 to 88-125 days by SMS amendments. These results indicate that SMS from P. florida cultivation can be utilized for bioremediation of HCH contaminated site.

An integrated (nano-bio) technique for degradation of γ-HCH contaminated soil

We have evaluated the effect of an integrated (nano-bio) technique involving the use of stabilized Pd/Fe0 bimetallic nanoparticles (CMC-Pd/nFe0) and a Sphingomonas sp. strain NM05, on the degradation of γ-HCH in soil. Factors affecting degradation such as pH, incubation temperature and γ-HCH initial concentration were also studied. The results revealed that γ-HCH degradation efficiency is ~ 1.7–2.1 times greater in integrated system as compared to system containing either NM05 or CMC-Pd/nFe0 alone. The integration showed synergistic effect on γ-HCH degradation. Further, cell growth studies indicated that NM05 gets well acclimatized to nanoparticles, showing potential growth in the presence of CMC-Pd/nFe0 with respect to control system. This study signifies the potential efficacy of integrated technique to become an effective alternative remedial tool for γ-HCH contaminated soil. Further research in this direction could lead to the development of effective remediation strategies for other isomers of HCH and other chlorinated pesticides as well.

Mechanochemical degradation of γ-hexachlorocyclohexane by a planetary ball mill in the presence of CaO

Although the use of the insecticide γ-hexachlorocyclohexane (HCH) is now prohibited in many countries because of its hazardousness, stockpiles of γ-HCH still exist. In this study, we subjected γ-HCH to mechanochemical (MC) treatment with a planetary ball mill in the presence of CaO to investigate the feasibility of using this method for the treatment of γ-HCH stockpiles. We confirmed the degradation of γ-HCH and investigated the degradation mechanism. The major intermediates were identified to be 1,3,4,5,6-pentachlorocyclohexene (γ-PCCH) and chlorobenzenes (CBzs). Analysis of the steric structure of γ-HCH and identification of the degradation intermediates suggested that successive dehydrochlorination led to the formation of trichlorobenzenes. Products of further degradation (dichlorobenzenes, monochlorobenzene, and benzene) were also detected. Surprisingly, methane and ethane were also detected, which suggests cleavage of the C–C bonds of the cyclohexane ring and hydrogenation. All of the chlorine atoms in the γ-HCH could be transformed into inorganic chloride compounds by the MC treatment with CaO. Our results indicate that γ-HCH can be completely dechlorinated by MC treatment.

Enzymes involved in the biodegradation of hexachlorocyclohexane: A mini review

The scope of this paper encompasses the following subjects: (i) aerobic and anaerobic degradation pathways of γ-hexachlorocyclohexane (HCH); (ii) important genes and enzymes involved in the metabolic pathways of γ-HCH degradation; (iii) the instrumental methods for identifying and quantifying intermediate metabolites, such as gas chromatography coupled to mass spectrometry (GC-MS) and other techniques. It can be concluded that typical anaerobic and aerobic pathways of γ-HCH are well known for a few selected microbial strains, although less is known for anaerobic consortia where the possibility of synergism, antagonism, and mutualism can lead to more particular routes and more effective degradation of γ-HCH. Conversion and removals in the range 39%-100% and 47%-100% have been reported for aerobic and anaerobic cultures, respectively. Most common metabolites reported for aerobic degradation of lindane are γ-pentachlorocyclohexene (γ-PCCH), 2,5-dichlorobenzoquinone (DCBQ), Chlorohydroquinone (CHQ), chlorophenol, and phenol, whereas PCCH, isomers of trichlorobenzene (TCB), chlorobenzene, and benzene are the most typical metabolites found in anaerobic pathways. Enzyme and genetic characterization of the involved molecular mechanisms are in their early infancy; more work is needed to elucidate them in the future. Advances have been made on identification of enzymes of Sphingomonas paucimobilis where the gene LinB codifies for the enzyme haloalkane dehalogenase that acts on 1,3,4,6-tetrachloro 1,4-cyclohexadiene, thus debottlenecking the pathway. Other more common enzymes such as phenol hydroxylase, catechol 1,2-dioxygenase, catechol 2,3-dioxygenase are also involved since they attack intermediate metabolites of lindane such as catechol and less substituted chlorophenols. Chromatography coupled to mass spectrometric detector, especially GC-MS, is the most used technique for resolving for γ-HCH metabolites, although there is an increased participation of HPLC-MS methods. Scintillation methods are very useful to assess final degradation of γ-HCH.

Simultaneous adsorption and degradation of γ-HCH by nZVI/Cu bimetallic nanoparticles with activated carbon support

Cu amended zero valent iron bimetallic nanoparticles were synthesized by doping Cu on the surface of iron. They were incorporated with granular activated carbon (AC) to prepare supported particles (AC–Fe0–Cu), which were used to remove γ-HCH. Cu on the surface of iron enhanced the dechlorination activity of Fe0. The dechlorination rate constant (kobs) increased with the Cu loading on the surface of iron and the maximum was achieved with 6.073% Cu. AC as a support was effective for increasing the dispersion of the nanoparticles and avoiding the agglomeration of the metallic nanoparticles. The simultaneous adsorption of γ-HCH on AC accelerated the degradation rate of γ-HCH by the bimetals. After reaction for 165 min, around 99% of γ-HCH was removed by the solids of AC–Fe0–Cu. In addition, AC could adsorb the degradation products. The degradation of γ-HCH was mainly through dehydrochlorination and dichloroelmination based on the intermediate products detected by GC/MS.

Application of zerovalent iron (Fe 0) to enhance degradation of HCHs and DDX in soil from a former organochlorine pesticides manufacturing plant

Remediation of pesticide-polluted soil is particularly challenging when pesticides in soil are aged and a mixture of pesticides is present. Application of zerovalent iron (Fe 0) was investigated to accelerate the degradation of HCHs (α-, β-, γ- and δ-hexachlorocyclohexane) and DDX (DDT, DDE and DDD) in the soil from a former organochlorine pesticide manufacturing plant. Ultrasonic extraction was used extract the organochlorine pesticides from soil. The identification and quantification of organochlorine pesticides in the extracts were accomplished by gas chromatograph. The addition of Fe 0 facilitated the degradation of the β-HCH isomer, but had little effect on the degradation of α-HCH, γ-HCH and δ-HCH. Zerovalent iron significantly increased the degradation of p,p′-DDT and o,p′-DDT in soil, and the percentage degradation of p,p′-DDT and o,p′-DDT increased with increased Fe 0 concentration during the first period of incubation. However, the amount of p,p′-DDD, the main dechlorinated product of p,p′-DDT, basically kept increased except in the unamended soil. The addition of Fe 0 therefore did not increased the percent degradation of ∑DDT (p,p′-DDT + p,p′-DDD + p,p′-DDE) markedly.

Photodegradation of γ-HCH by α-Fe 2O 3 and the influence of fulvic acid

The purpose of this study was to examine photodegradation of γ-hexachlorocyclohexane (γ-HCH) by α-Fe 2O 3 and the influence of fulvic acid (FA) in this photochemical process. A 300 W high-pressure mercury lamp was employed as the light source. The result showed that the UV light alone could play an important role in the degradation of γ-HCH. The presence of α-Fe 2O 3 was found to promote the photodegradation of γ-HCH. The photodegradation of γ-HCH by α-Fe 2O 3 follows pseudo-first-order reaction kinetics. The effect of the FA on the photodegradation of γ-HCH was also performed. The result revealed that the FA slowed the photodegradation rate of γ-HCH. Oxidation products were also identified by gas chromatography with a mass spectroscopy detector. This process is of interest in both a remedial process induced by iron oxides in natural water surfaces and α-Fe 2O 3-mediated photodegradation method for purifying natural water containing γ-HCH and humic substances.

Degradation of soil-applied isomers of hexachlorocyclohexane by a Pseudomonas SP

A Pseudomonas sp., that readily degraded α-,β- and γ-isomers of hexachlorocyclohexane (HCH) in a mineral salts medium under aerobic conditions, was tested for its ability to degrade these isomers after their application to two soils under flooded or nonflooded conditions. Interestingly, the concentration of soil-applied α- and γ-HCH decreased from the original concentration of 5 μg g −1 soil to undetectable amounts within 10–20 days in soils inoculated with Pseudomonas sp., and the loss of both isomers was more pronounced under nonflooded conditions than under flooded conditions. During the corresponding period, there was no appreciable loss of either isomer from uninoculated soils. The degradation of β-HCH was not accelerated by bacterial inoculation of the soils under either water regime despite the ability of the bacterium to degrade this isomer when added to the mineral salts medium. The rapid degradation of α- and γ-HCH, and not β-HCH, in inoculated soils is attributed to the ability of the bacterium to utilize α- and γ-HCH, and not β-HCH, for growth. Addition of sodium acetate to the mineral salts medium enhanced the bacterial degradation of β-HCH implicating cometabolism and retarded the degradation of γ-HCH.

Accelerated aerobic degradation of ?-hexachlorocyclohexane in suspensions of flooded and non-flooded soils pretreated with hexachlorocyclohexane

A commercial wettable-powder formulation (50% a. i.) of hexachlorocyclohexane (HCH) was applied to unplanted and planted (to rice) soils under flooded and non-flooded conditions at 1 kg a. i. ha−1 at 15-day intervals. A mineral salts medium supplemented with γ-HCH as a sole source of C was inoculated with suspensions from HCH-treated and untreated soils (unplanted or planted to rice) and incubated under aerobic conditions. γ-HCH disappeared completely within 10 days from the aerobically incubated medium inoculated with the suspension from the treated soil compared to less than 30% loss from the uninoculated medium or from the medium inoculated with the suspension from the untreated soil, during the corresponding period. Soil samples from HCH-treated flooded pots lost their capacity for accelerated degradation of γ-HCH after autoclaving. The addition of HCH clearly stimulated aerobic degradation of γ-HCH, even in predominantly anaerobic flooded soil. The factor responsible for the accelerated degradation of γ-HCH in pretreated soil was not detected in the deeper layers (>10 cm) of flooded soil and in fields that had dried following the rice harvest.

Rapid degradation of γ-HCH in upland soil after multiple applications

Since 1973, the authors have carried out a long-term upland field experiment by applying γ-HCH (γ-BHC, lindane) every year in the same field in order to examine how the fate of γ-HCH was afrected in the long run. The disappearance-rate of γ-HCH increased with the increasing period of application. After the third and fourth applications of γ-HCH, more than 80% of the applied γ-HCH was lost within 1 month. The rapid loss of γ-HCH in the field was considered to be principally due to the degradation by microorganisms accumulated in the field for the long-term experiment. This asssumption was supported by several laboratory experiments: which indicated that the γ-HCH-decomposing microorganisms could survive and decompose γ-HCH in other soils to which γ-HCH had never been applied and that the active γ-HCH-decomposing microorganisms were aerobic or facultative anaerobic bacteria.

Influence of processing on the degradation of pesticides in meat products

The influence of processing on the degradation of C-14 labelled DDT, γ-hexachlorocyclohexane (γ-HCH) and monolinuron (MLN) was investigated. In culture media micrococci and yeasts metabolised DDT, mainly to DDD, within two weeks, whereas γ-HCH and MLN were negligibly degraded. Only small amounts of the pesticides were metabolised by lactobacilli. Meat of rabbits fed with C-14 labelled pesticides was used to prepare various meat products. In dry sausages with nitrite-curing salt (NCS) up to 60% residual DDT and 70% γ-HCH were recovered. In the presence of a starter culture and nitrate the rate of degradation was lower, 76% DDT (after 38 days) and 77% of γ-HCH (after 30 days) of the initial concentrations were found in the final products. In M. Long. dorsi 61% DDT and 80% γ-HCH were still present after curing. In cured, cooked meats 87% DDT but only 35% γ-HCH of the initial amounts were left. In the case of MLN about 50% of the radioactivity was removed by curing, hot-smoking or cooking; only 2% of the radioactive material was extracteble. The enzymic degradation of DDT, γ-HCH and MLN, respectively, by muscle mitochondria (released after freezing) amounted to about 10% on average. In liver sausage mixtures with sodium chloride, DDT and γ-HCH were metabolised to a greater extent than in mixtures containing NCS or NCS and sodium ascorbate (NaASC). A modified ascorbic acid oxidation system did not enhance the degradation of the pesticides investigated to a significant extent in a cervelat type sausage.

Comparative stimulation of γHCH metabolism by pretreatment of rats with γHCH, DDT, and DDT + γHCH

Pretreatment of rats with γHCH, DDT, and DDT + γHCH accelerates the metabolism of γHCH. A single oral dose of 14C-γHCH was administered to all animals after 2 wk of pretreatment. The treated rats excreted significantly more radioactivity and stored significantly less 14C-γHCH than the control animals. In addition to significantly larger livers, the treated rats exhibited higher cytochrome P-450 content and greater in vitro enzyme activity than the control animals. The self-induction of γHCH metabolism, which has not previously been reported, could be of major importance in establishing a steady-state relationship between exposure and storage of such pesticides by mammals. Both quantitative and qualitative differences in γHCH metabolism are observed between the treated groups of rats. DDT pretreatment results in a 35% higher excretion of radioactivity than γHCH pretreatment. From the distribution of radioactivity it is concluded that DDT pretreatment produces proportionately more neutral and weakly polar but less free acid-type γHCH metabolites than γHCH pretreatment. Microsomal mixed function oxidases appear to be primarily responsible for the 3- to 4-fold increase in γHCH metabolism by the DDT-pretreated rats. However, the γHCH-pretreated rats exhibit significantly higher glucuronyl transferase activity, an enhanced GSH-dependent degradation of γHCH, and a lower level of the mixed function oxidases than DDT-pretreated rats. Results of this study suggest that the mechanism by which γHCH induces its own degradation differs from the mechanism through which DDT stimulates γHCH metabolism.