Effects of p-cresol and chlorophenols on pentachlorophenol biodegradation

The effects of three aromatic compounds, p-cresol, 2,4-dichlorophenol (DCP), and 2,4,6-trichlorophenol (TCP), on cell growth and pentachlorophenol (PCP) degradation bya Flavobacterium species were investigated. While p-cresol was not degraded by this bacterium, DCP and TCP were simultaneously degraded with PCP. Both DCP and TCP lowered cell growth and PCP degradation rate. Cell growth was modeled by cell death, because p-cresol, DCP, and TCP were toxic to the organism. A new model was used to predict cell death rate in a mixture of two toxic compounds from the cell death kinetics for each individual compound. PCP degradation rates were modeled by conventional inhibition models, but only over a small concentration range for the secondary toxic compound. However, a new empirical model described PCP degradation over a wider concentration range of the secondary toxic compound. (c) 1995 John Wiley & Sons Inc.     

Physiological properties and substrate specificity of a pentachlorophenol-degradingPseudomonas species

A bacterial strain capable of utilizing pentachlorophenol (PCP) as sole source of carbon and energy for growth was isolated from enrichment cultures containing 100 mg/l PCP in a mineral salts medium inoculated with contaminated soil from a lumber treatment waste site. The isolate, designated strain SR3, was identified as a species ofPseudomonas by virtue of its physiological and biochemical characteristics. Mineralization of PCP byPseudomonas sp. strain SR3 was demonstrated by loss of detectable PCP from growth medium, stoichiometry of chloride release (5 equivalents of chloride per mole of PCP), and formation of biomass consistent with the concentration of PCP mineralized. PCP-induced cells of strain SR3 showed elevated rates of oxygen consumption in the presence of PCP, and with different chlorinated phenols, with complete degradation of 2,3,5,6-, 2,3,6-, 2,4,6-, 2,4-, and 2,6-chloro-substituted phenols. Concentrations of PCP up to 175 mg/liter supported growth of this organism, but maximal rates of PCP removal were observed at a PCP concentration of 100 mg/liter. Based on its degradative properties,Pseudomonas sp. strain SR3 appears to have utility in bioremediation of soil and water contaminated with PCP.Abbreviations DCP dichlorophenol - TCP trichlorophenol - TeCP tetrachlorophenol Contribution No. 750 from the United States Environmental Protection Agency Environmental Research Laboratory, Gulf Breeze, FL32561, USA. A preliminary report of this work has appeared in abstract form (Resnick & Chapman 1990; Abstr. Annu Meet Amer Soc Microbiol Q-70, p. 300).     

Cytotoxic effects of environmentally relevant chlorophenols on L929 cells and their mechanisms

The chlorophenol chemicals (CPs) are a major class of widely distributed and frequently occurring persistent environmental pollutants. Pentachlorophenol (PCP) has been proposed to be procarcinogen in rodents and in possibly human beings. Human beings also easily expose to other chlorophenol chemicals, including 4-chlorophenol (CP), 2,4-dichlorophenol (DCP), 2,3,4-trichlorophenol (TCP), prompting this investigation of their comparative cytotoxic effects and cell death mechanisms, assayed in fibroblast L929 cells. The effective concentration for half-maximal response (EC50 values at 24 h for CP, DCP, TCP, and PCP are 2.18, 0.83, 0.46, and 0.11 mmol/L respectively and the EC50 values at 48 h are 1.18, 0.13, 0.08, and 0.06 mmol/L respectively by using 3-(4,5-dimethylthiazd-2-yl)-2,5-diphenyltentrazolium bromide (MTT) reduction assay. A clear structure-activity relationship was observed between toxicity of CPs and their octanol-water partition coefficients. The further studies indicate that CP, DCP, and TCP induce apoptosis in L929 cells in a concentration or time-dependent manner, but PCP mediates cell death more characteristic of necrosis than apoptosis. These results not only demonstrate that L929 cell growth inhibition bioassay may be useful to provide the comparative evaluation of toxicity of CPs in vitro, but also implicate that CP, DCP, TCP, in comparison with PCP, can induce L929 cell death by apoptosis, resulting in lower procarcinogensis, which may help to elucidate the molecular basis for the adverse health effects associated with CPs exposure.     

Effect of glutamate on the degradation of pentachlorophenol by Flavobacterium sp.

Summary The degradation of pentachlorophenol (PCP) by a Flavobacterium sp. was investigated by inoculating induced cells into cultures containing PCP alone or PCP and glutamate as carbon sources. Using PCP as the sole carbon source, the degradation activity increased with PCP concentration. However, a lag phase was observed and this lag was more pronounced at higher PCP concentrations. Exposure of cells to higher PCP concentrations during induction did not reduce the lag. The presence of glutamate reduced the lag in PCP degradation. Such an elimination of the lag phase appears to be due to maintaining cell viability with the presence of glutamate. Offprint requests to: W.-S. Hu     

Regiospecific dechlorination of pentachlorophenol by dichlorophenol-adapted microorganisms in freshwater, anaerobic sediment slurries.

The reductive dechlorination of pentachlorophenol (PCP) was investigated in anaerobic sediments that contained nonadapted or 2,4- or 3,4-dichlorophenol (DCP)-adapted microbial communities. Adaptation of sediment communities increased the rate of conversion of 2,4- or 3,4-DCP to monochlorophenols (CPs) and eliminated the lag phase before dechlorination was observed. Both 2,4- and 3,4-DCP-adapted sediment communities dechlorinated the six DCP isomers to CPs. The specificity of chlorine removal from the DCP isomers indicated a preference for ortho-chlorine removal by 2,4-DCP-adapted sediment communities and for para-chlorine removal by 3,4-DCP-adapted sediment communities. Sediment slurries containing nonadapted microbial communities either did not dechlorinate PCP or did so following a lag phase of at least 40 days. Sediment communities adapted to dechlorinate 2,4- or 3,4-DCP dechlorinated PCP without an initial lag phase. The 2,4-DCP-adapted communities initially removed the ortho-chlorine from PCP, whereas the 3,4-DCP-adapted communities initially removed the para-chlorine from PCP. A 1:1 mixture of the adapted sediment communities also dechlorinated PCP without a lag phase. Dechlorination by the mixture was regiospecific, following a para greater than ortho greater than meta order of chlorine removal. Intermediate products of degradation, 2,3,5,6-tetrachlorophenol, 2,3,5-trichlorophenol, 3,5-DCP, 3-CP, and phenol, were identified by a combination of cochromatography (high-pressure liquid chromatography) with standards and gas chromatography-mass spectrometry.     

Multisubstrate monod kinetic model for simultaneous degradation of chlorophenol mixtures

Chlorophenols (CPs) are persistent and highly toxic compounds rated as priority pollutants by the Environmental Protection Agency (EPA). Frequently, these compounds are present as mixtures of CPs in industrial wastewaters. Therefore the study of biodegradation on mixed pollutants is an important aspect of biodegradation and wastewater treatment. In this work, we studied the multisubstrate degradation of CPs by a mixed culture of Pseudomonas aeruginosa and a novel Acromobacter sp. capable of using pentachlorophenol (PCP), 2,4,6 trichlorophenol (2,4,6 TCP) and 2,3,5,6 tetrachlorophenol (2,3,5,6 TeCP) as the sole sources of carbon and energy. The main objective of this work was to evaluate the effect of substrate mixtures on the degradation kinetics of PCP. Batch experiments were conducted with each CP separately and in mixtures of PCP + 2,4,6 TCP, PCP + 2,3,5,6 TeCP, and PCP + 2,4,6 TCP + 2,3,5,6 TeCP. Based upon our results we have concluded that the simultaneous degradation of CPs is a key factor contributing to the improvement of PCP degradation. The kinetic parameters for PCP and 2,4,6 TCP were obtained by fitting the data to a Monod kinetics model. Using such parameters, the model was able to predict simultaneous multisubstrate degradation of PCP with others CPs.     

Biodegradation of chlorophenols by mixed and pure cultures from a fluidized-bed reactor

An aerobic, continuous-flow fluidized-bed reactor was established with inoculum from activated sludge, and fed a mixture of 2,4,6-trichlorophenol (TCP), 2,3,4,6-tetrachlorophenol (TeCP) and pentachlorophenol (PCP) as the sole sources of carbon and energy for 2 years. Experiments with the enrichment were performed with material from the reactor. Later, degradation experiments were completed using pure cultures of bacteria that were isolated from suspended samples of the carrier biofilm. In batch-bottle bioassays, the reactor enrichment degraded PCP, TeCP and TCP both in mineral salts (MS) and tryptone-yeast extract-glucose (TGY) media. ortho-Methoxylated chlorophenols including 4,5-dichloroguaiacol (4,5-DCG), tetrachloroguaiacol (TeCG) and trichlorosyringol (TCS) resisted biodegradation by the enrichment both in MS and TGY media, whereas 5,6-dichlorovanillin (5,6-DCV) was readily transformed to an unidentified metabolite. Experiments with ¹⁴C labeled chlorophenols showed mineralization of 2,4-dichlorophenol (DCP) and 2,3,5-TCP to ¹⁴CO₂ by the enrichment. Material from the suspended biofilm after continuous chlorophenol feeding for 2 years was inoculated onto TGY-agar plates, and showed predominantly two colony, types accounting for over 99% of the total colony counts. The two colony types, were equal in abundance. Six Gram-negative, oxidase- and catalase-positive, non-fermentative small rods were isolated in TGY agar media supplemented with 10 mg/l of TeCP or PCP. All isolates formed colonies in TGY plus 150 mg/l of PCP. The isolates degraded TCP and TeCP but not PCP. In mixtures of isolated bacteria the rates of chlorophenol degradation were similar to those observed with individual isolates. Three isolates were identified as Pseudomonas saccharophila and three were an unidentified species of Pseudomonas.     

Dual substrate biodegradation of a nonionic surfactant and pentachlorophenol by Sphingomonas chlorophenolica RA2

The simultaneous biodegradation of the nonionic surfactant Tween 20 (Tw20) and pentachlorophenol (PCP) by Sphingomonas chlorophenolica sp. Strain RA2 (RA2) was measured. As a sole substrate, Tw20 biodegradation was best described by the Contois kinetic model. During concurrent biodegradation of Tw20 and PCP, the biodegradation rates of Tw20 were not significantly affected by 50 or 100 mg/L PCP, but were significantly inhibited by 500 mg/L PCP. Decreases in cell yield in the presence of PCP suggest that PCP was acting as an uncoupler. Cultures were pre-grown on PCP or Tw20 before degradation of PCP to evaluate enzyme induction effects, and long lags before PCP biodegradation after growth on Tw20 occurred. Although biokinetic models could accurately describe some of the data sets of RA2 growth and Tw20 and PCP degradation, finding a single set of kinetic parameters that predicted all dual substrate tests was not achieved. The complicating factors to modeling PCP and Tw20 interactions are described and may be more widely applicable to the biodegradation of toxic organic compounds in the presence of a biodegradable surfactant.     

Enrichment, isolation and characterization of pentachlorophenol degrading bacterium Acinetobacter sp. ISTPCP-3 from effluent discharge site

Three pentachlorophenol (PCP) degrading bacterial strains were isolated from sediment core of pulp and paper mill effluent discharge site. The strains were continuously enriched in mineral salts medium supplemented with PCP as sole source of carbon and energy. One of the acclimated strains with relatively high PCP degradation capability was selected and characterized in this study. Based on morphology, biochemical tests, 16S rDNA sequence analysis and phylogenetic characteristics, the strains showed greatest similarity with Acinetobacter spp. The strain was identified as Acinetobacter sp. ISTPCP-3. The physiological characteristics and optimum growth conditions of the bacterial strain were investigated. The results of optimum growth temperature revealed that it was a mesophile. The optimum growth temperature for the strain was 30°C. The preferential initial pH for the strain was ranging at 6.5–7.5, the optimum pH was 7. The bacterium was able to tolerate and degrade PCP up to a concentration of 200 mg/l. Increase in PCP concentration had a negative effect on biodegradation rate and PCP concentration above 250 mg/l was inhibitory to its growth. Acinetobacter sp. ISTPCP-3 was able to utilize PCP through an oxidative route with ortho ring-cleavage with the formation of 2,3,5,6-tetrachlorohydroquinone and 2-chloro-1,4-benzenediol, identified using gas chromatograph–mass spectrometric (GC–MS) analysis. The degradation pathway followed by isolated bacterium is different from previously characterized pathway.     

Degradation kinetics of pentachlorophenol by Phanerochaete chrysosporium

The extracellular enzymes and cell mass from the pregrown Phanerochaete chrysosporium cultures were used for the degradation of PCP. The use of both extracellular enzymes and cell mass resulted in extensive mineralization of PCP, while the action of only the crude extracellular enzymes led to the formation of a degradation intermediate (TCHD). A kinetic model, which describes the relationship among PCP degradation, initial PCP concentration, dosage of extracellular enzymes, and cell mass concentration, was developed. Based on this model, various effects of initial PCP concentration, dosage of extracellular enzymes, and cell mass concentration were evaluated experimentally. It was found that when initial PCP concentration is lower than 12 mumol/L, the model of a parallel-series first-order reaction is sufficient to describe the degradation process. PCP disappearance and mineralization were enhanced by increasing either the extracellular enzyme concentration or the cell mass concentration. As high as 70% of PCP mineralization could be obtained by using a higher dosage of extracellular enzymes and cell mass. Various parameters of the kinetic model were determined and the model was verified experimentally. Simulation using this model provided the criteria needed to choose rational dosages of extracellular enzymes and cell mass for the degradation of PCP. Data reported allow some insight into the function of the extracellular enzymes and cell mass of P. chrysosporium in degradation processes of toxic pollutants and assist in the design and evaluation of practical bioremediation methods.     

Investigation of the biodegradation of pentachlorophenol by the predominant bacterial strains in a mixed culture

A pentachlorophenol (PCP) degrading mixed culture contained three predominant strains identified as Flavobacterium gleum, Agrobacterium radiobacter and Pseudomonas sp. The relative abilities of the three strains to degrade PCP were tested individually and in combination. Rates of PCP degradation by individual isolates were lower than that observed for the three isolates combined. Of the individual strains, Flavobacterium gleum manifested highest PCP degradation ability. A biodegradation medium inoculated with a combination of the three isolates exhibited PCP degradation patterns similar to the original mixed culture. Varying low amounts of tetrachlorophenol were found in degradation medium inoculated with individual isolates, but this intermediate was absent from media inoculated with the mixed culture.     

Molecular analysis of pentachlorophenol degradation

A limited number of microorganisms have been described for their ability to partially degrade pentachlorophenol (PCP), or to completely mineralize it. Several years ago we chose one of these microorganisms,Flavobacterium sp. strain ATCC 39723, for use in a detailed molecular analysis of the catabolism of PCP. This strain was chosen because it had previously been studied in great detail for its growth characteristics in relation to degradation of PCP. In this paper we provide an overview of the degradation pathway of PCP to 2,6-dichloro-p-hydroquinone byFlavobacterium. The specific biochemical reactions and the genes encoding the enzymes are reviewed. The successful transformation and site specific mutagenesis ofFlavobacterium, as well as the discovery of two newpcp alleles is also presented.     

Degradation of pentachlorophenol by <Emphasis Type="Italic">Kocuria</Emphasis> sp. CL2 isolated from secondary sludge of pulp and paper mill

A pentachlorophenol (PCP) degrading bacterium was isolated and characterized from sludge of pulp and paper mill. This isolate used PCP as its sole source of carbon and energy and was capable of degrading this compound, as indicated by stoichiometric release of chloride and biomass formation. Based on morphology, biochemical tests, and 16S rRNA gene sequence analysis this strain was identified as Kocuria sp. CL2. High Performance Liquid Chromatography (HPLC) analysis revealed that this strain was able to degrade PCP up to a concentration of 600 mg/l. This is first time we are reporting the degradation of PCP by the Kocuria species. This isolate was also able to remove 58.64% of PCP from the sludge within two weeks. This study showed that the removal efficiency of PCP by CL2 was found to be very effective and can be used in degradation of PCP containing pulp paper mill waste in the environment.     

Microbial degradation of pentachlorophenol

Pentachlorophenol (PCP) was the most prevalent wood preservative for many years worldwide. Its widespread use had led to contamination of various environments. Traditional methods of PCP clean-up include storage in land-fill sites, incineration and abiotic degradation processes such as photodecomposition. Some aerobic and anaerobic microorganisms can degrade PCP under a variety of conditions. Axenic bacterial cultures, Flavobacterium sp., Rhodococcus sp., Arthrobacter sp., Pseudomonas sp., Sphingomonas sp., and Mycobacterium sp., and fungal cultures, Phanerochaete sp. and Trametes sp. exhibit varying rates and extent of PCP degradation. This paper provides some general information on properties of PCP and reviews the influence of nutrient amendment, temperature and pH on PCP degradation by various aerobic and anaerobic microorganisms. Where information is available, proposed degradation pathways, intermediates and enzymes are reviewed.     

Degradation of pentachlorophenol by the white rot fungus Phanerochaete chrysosporium grown in ammonium lignosulphonate media

Removal and degradation of pentachlorophenol (PCP) by Phanerochaete chrysosporium in static flask cultures was studied using ammonium lignosulphonates (LS), a waste product of the papermill industry, as a carbon and nitrogen source. After 3 days, cultures of P. chrysosporium grown in either a 2% LS (nitrogen-sufficient) medium or a 0.23% LS and 2% glucose (nitrogen-deficient) medium removed 72 to 75% of PCP, slightly less than the 95% removal seen using nitrogen-deficient glucose and ammonia medium. PCP dehalogenation occurred despite the fact that extracellular enzyme (LiP) activity, measured by a veratryl alcohol oxidation assay and by PCP disappearance in cell-free extracts, was inhibited by LS. This inactivation of LiP likely contributed to the lower percent of PCP dehalogenation observed using the LS media. In order to better understand the relationship between PCP disappearance and dehalogenation, we measured the fate of the chlorine in PCP. After 13 days, only 1.8% of the initial PCP added was recoverable as PCP. The remainder of the PCP was either mineralized or transformed to breakdown intermediates collectively identified as organic halides. The largest fraction of the original chlorine (58%) was recovered as organic (non-PCP) halide, most of which (73%) was associated with the cell mass. Of the remaining chlorine, 40% was released as chloride ion, indicating a level of dehalogenation in agreement with previously reported values.     

Effect of chlorophenols on the membrane lipids of bacterial cells

Chlorophenols, widespread soil and water contaminants and often degradation products of some pesticides, are a potential stress factor for survival of environmental bacteria. The effect of pentachlorophenol (PCP) and 2,4-chlorophenol (2,4-CP) on the growth, amount of lipid, and fatty acid composition in the membrane lipids was examined in a strain of the bacterium Kocuria varians, able to degrade chlorophenols. The index of fatty acid unsaturation in two main membrane lipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) decreased in the presence of chlorophenols. Transformation of stearic acid into oleic acid was significantly increased by PCP addition only in PE, but conversion of oleic acid into linoleic acid was blocked by PCP and 2,4-CP in both PC and PE. This observation may indicate that while Δ⁹ desaturase was sensitive mainly to 2,4-CP, activity of Δ¹² desaturase was inhibited by both PCP and 2,4-CP.     

Adsorption capacity as a key parameter for enzyme induction and pentachlorophenol degradation in Mycobacterium chlorophenolicum PCP-1.

Adsorption of pentachlorophenol (PCP) on induced cells of Mycobacterium chlorophenolicum PCP-1 and its influence on enzyme induction and PCP degradation of this strain were studied. Compared to non-induced cells, induced degrading cells had a lower adsorption capacity (q(ads)), particularly at prolonged induction and low PCP concentration. Unlike the effects of pH and biomass concentration previously reported for non-induced cells, the variation of q(ads) of induced cells was associated with changes of both the capacity and intensity constants of the Freundlich equation which was used to describe PCP adsorption on M. chlorophenolicum PCP-1. This indicated changes of cell surface properties during enzyme induction and PCP degradation. The latter was shown in turn to be affected by several parameters such as PCP concentration, pH value and induction time. Interestingly, irrespective of the pH and PCP concentration, the specific PCP degradation rate (q(t)(PCP)) at a given induction time was found to be solely a function of q(ads), revealing that adsorption capacity is an inherent key parameter for enzyme induction and PCP degradation. Based on this knowledge, a kinetic model was developed for q(t)(PCP) which used only q(ads) and induction time as variables. The model considered inhibition of PCP on both enzyme induction and enzyme activity and described the experimental data at different PCP concentrations and pH values well. q(ads) also turned out to be a useful criterion for choosing optimum induction concentration of PCP. Irrespective of pH and biomass concentration, an initial adsorption capacity of 2-3 micromol PCP/g cells was found to be optimum for enzyme induction in M. chlorophenolicum PCP-1.     

Pentachlorophenol (PCP) dechlorination in horizontal-flow anaerobic immobilized biomass (HAIB) reactors

This study verifies the potential applicability of horizontal-flow anaerobic immobilized biomass (HAIB) reactors to pentachlorophenol (PCP) dechlorination. Two bench-scale HAIB reactors (R1 and R2) were filled with cubic polyurethane foam matrices containing immobilized anaerobic sludge. The reactors were then continuously fed with synthetic wastewater consisting of PCP, glucose, acetic acid, and formic acid as co-substrates for PCP anaerobic degradation. Before being immobilized in polyurethane foam matrices, the biomass was exposed to wastewater containing PCP in reactors fed at a semi-continuous rate of 2.0 μg PCP g⁻¹ VS. The applied PCP loading rate was increased from 0.05 to 2.59 mg PCP l⁻¹ day⁻¹ for R1, and from 0.06 to 4.15 mg PCP l⁻¹ day⁻¹ for R2. The organic loading rates (OLR) were 1.1 and 1.7 kg COD m⁻³ day⁻¹ at hydraulic retention times (HRT) of 24 h for R1 and 18 h for R2. Under such conditions, chemical oxygen demand (COD) removal efficiencies of up to 98% were achieved in the HAIB reactors. Both reactors exhibited the ability to remove 97% of the loaded PCP. Dichlorophenol (DCP) was the primary chlorophenol detected in the effluent. The adsorption of PCP and metabolites formed during PCP degradation in the packed bed was negligible for PCP removal efficiency.     

Degradation of p-nitrophenol and pentachlorophenol mixtures by Sphingomonas sp. UG30 in soil perfusion bioreactors

The degradation of mixtures of pentachlorophenol (PCP) and p-nitrophenol (PNP) were evaluated in pure cultures of Sphingomonas sp. UG30, statically incubated soils (60% water-holding capacity) and soil perfusion bioreactors where encapsulated cells of UG30 were used as a soil inoculant. In pure-culture studies, conditions were optimized for mineralization of PCP and PNP mixtures at concentrations of 30 mg l⁻¹ each. Optimum in vitro mineralization of PCP and PNP mixtures by UG30 was facilitated using ammonium phosphate as a nitrogen source, while inhibition was observed with ammonium nitrate. The bioreactor system used columns containing soil treated with mixtures of 100, 225 or 500 mg kg⁻¹ of PCP and PNP. Rapid dissipation of both substrates was observed at the 100 mg kg⁻¹ level. Inoculation with UG30 enhanced PCP degradation at the 100 mg kg⁻¹ level in bioreactors but not in static soil microcosms. At higher PCP and PNP concentrations (225 mg kg⁻¹), occasional complete degradation of PNP was observed, and PCP degradation was about 80% compared to about 25% in statically incubated soils after 20 days at 22°C. There was no additional degradation of the PCP and PNP mixtures attributable to inoculation with encapsulated cells of UG30 in either soil system at concentrations of 225 or 500 mg kg⁻¹. Journal of Industrial Microbiology & Biotechnology (2000) 25, 93–99. Received 25 February 2000/ Accepted in revised form 07 June 2000     

Effects of pentachlorophenol on the bacterial denitrification process

The use of pentachlorophenol (PCP) was banned or restricted in many countries worldwide because of its adverse influences on the ecological environment and humans. However, the potential disrupting effects of PCP on denitrifying microorganisms have warranted more analysis. In this study, the impacts of PCP on denitrification were investigated by using Paracoccus denitrificans as a model denitrifying bacterium. Compared with the control, the presences of 10 and 50 μM of PCP were found to significantly decrease the denitrification efficiencies from 98.5 to 87.2% and 68.7%, respectively. The mechanism studies showed that PCP induced the generation of reactive oxygen species, which decreased the vital enzymes activities related to glycolysis process, causing the disturbance of the metabolism of P. denitrificans utilizing carbon source (glucose) and the growth of the cell, and subsequently the generation of electron donor (NADH) for denitrification via NAD⁺ reduction was severely depressed. Further studies indicated that PCP also decreased the genes expression of several key enzymes responsible for denitrification, such as napA of nitrate reductase (NAR), nirS of nitrite reductase, norB of nitric oxide reductase, and nosZ of nitrous oxide reductase; however, there was only the enzyme activity of NAR was remarkably inhibited.