Effects of pentachlorophenol on the bacterial denitrification process

AbstractThe 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 stu...


Introduction
Pentachlorophenol (PCP), a man-made halogenated aromatic compound, has been listed as a priority pollutant by the U.S. Environmental Protection Agency for its persistence, toxicity, bioaccumulation, and potential human carcinogenicity. [1] It was used worldwide as agricultural pesticide, herbicide, wood preservative, and broadspectrum biocide, [2] as well as substance in total and bleaching effluent of pulp and paper mill industry. [3] Although the use of PCP has been banned or restricted in many countries since 1980s for its endocrine disrupting effects on the ecological environment and humans, [4] recently, it was still detected in various samples such as water, soil, sediment, aquatic organism, and human. [5][6][7] In China, the primary use of PCP and its sodium salt is to kill schistosome intermediate host snails. It was reported that the annual national output of PCP has reached approximately 3000 tons in2003, [8,9]with there-emergence of schistosomiasis in the traditional epidemic areas. [10] The increased manufacture and application of PCP likely has resulted in more environmental release. The released PCP might influence the activity of environmental microorganisms.
To our knowledge, denitrification is the respiratory reduction of N-oxides, which is a vital part of the global nitrogen cycle and the only way to transfer nitrogen from nitrate to gaseous nitrogen. [11] Denitrifying bacteria in soil are the major players in this process, any negative influence on denitrifying microorganisms will break the nitrogen balance and interfere with global climate. In the literatures, there are plenty of studies indicated that denitrification can be disturbed by several environmental pollutants, such as heavy metal and synthetic organic compounds. [12,13] For example, the Zinc oxide nanoparticles have been observed to inhibit the denitrifying reductase, which further led to more nitrate accumulation. [14] Wu et al. [15] found that the metabolic activities of enzymes involved in denitrification were influenced by ionophores. Therefore, it is necessary to investigate the influence of PCP on the metabolism and function of denitrifying bacteria.
In this paper, the effects of PCP on denitrification were investigated by using Paracoccus denitrificans (P. denitrificans) as a model denitrifying microorganism. Then, the mechanism for PCP significantly depressing denitrification were investigated from the aspects of cell membrane integrity, intracellular reactive oxygen species (ROS) production, metabolism of carbon source (namely, glycolysis), cell proliferation, generation of reducing NADH, and gene expression and activities of key enzymes involved in denitrification reactions.

Analytic methods
The N 2 O generated by P. denitrificans was determined by a gas chromatograph (gC) (Agilent 7820A, USA) with an electron capture detector. The N 2 O in gas was directly sampled and injected into the sample inlet of gC by a syringe, and the N 2 O in aqueous solution was detected after using headspace with equilibrium temperature and time of 25 °C and 3 h, respectively. [18] The detailed procedures for NAD + and NADH measurements were conducted according to the literature. [19] Duplicate samples (1 mL each) were centrifuged at 12,000 r/min for 5 min. After the supernatant was removed, 300 μL of 0.2 M HCl (for NAD + extraction) or 300 μL of 0.2 M NaOH (for NADH extraction) was added to the tube to resuspend the pellets. The extracts were bath incubated at 50 °C for 10 min, and then cooled down to 0 °C on ice. The extracts were neutralized by adding 300 μL of 0.1 M NaOH (for NAD + extraction) or 300 μL of 0.1 M HCl (for NADH extraction). The cellular debris was removed by centrifugation at 15,000 r/min for 5 min. Supernatant was transferred to a new tube for measurement immediately. The intracellular NAD + and NADH concentrations were determined by the enzymatic cycling assay. The mixture of cycling assay consisted of equal volumes of 1.0 M Bicine buffer (pH 8.0), ethanol, 40 mM EDTA (pH 8.0), 4.2 mM thiazolyl blue, and twice the volume of 16.6 mM phenazine ethosulfate. Before the experiment, the mixture was bath incubated at 30 °C for 10 min. Each reaction comprised the following: 50 μL neutralized cell extract, 300 μL ultra pure water, 600 μL the mixture, and 50 μL of alcohol dehydrogenase (500 U/mL). The reaction was employed for 10 min at 30 °C and the absorbance (570 nm) was checked at 30 s interval. The concentrations of NAD + and NADH were calibrated with 0.01-0.05 mM standard solutions of NAD + and NADH.
The crude cell extracts were prepared for the enzymes activities assays as described by the literature. [20] Briefly, at the end of the exposure experiments, cells were harvested by centrifugation at 5000 r/min for 10 min and washed twice with 0.1 M PBS (pH 7.4), and then resuspended in the same buffer. The suspension was disrupted by sonication at 20 kHz for 5 min, and then the cell debris was removed by centrifugation at 12,000 r/ min for 10 min. The above operations were carried out at 4 °C. The analyses of hexokinase (HK), 6-phosphofructose kinase (PFK), and pyruvate kinase (PK) activity were performed according to the literature. [21] The glyceraldehyde-3-phosphate dehydrogenase (gAPDH) activity was measured by the assay kit (Sciencell Research Laboratories) according to the manufacturer's instruction. The assays of nitrate reductase (NAR), nitrite reductase (NIR), nitric oxide reductase (NOR), and nitrous oxide reductase (N 2 OR) activities were carried out according to previous publication. [14] The intracellular ROS production was determined by a fluorescence assay described in the literature. [22]

Denitrifying bacteria
The strain of P. denitrificans used in this study was isolated from the soil, which was sampled from a 12 years bleaching effluent irrigated reed wetland in Yancheng, China. Prior to the exposure experiments, the cells grew aerobically in Difco nutrient broth at 30 °C in a shaker (150 r/min) for 24 h, then the cells were harvested by centrifuged at 5000 r/min for 10 min, and washed twice with 0.1 M phosphate buffer saline (PBS) buffer (pH 7.4), and then resuspended in the same buffer to make the cell density to 1.0 × 10 10 cell/mL.

Preparation of PCP bulk solution and mineral medium
PCP was purchased from Sinopharm Chemical Reagent Co, Ltd, China. Before the experiments the PCP stock solution (0.5 mM) were prepared by dispersing 133.17 mg of PCP to 1 L of 10% ethanol solution.
The mineral medium was prepared according to the reference with minor modification (g/L): 5 . Ammonium sulfate was added to preclude assimilatory nitrate reduction and ensure the nitrate consumption was due to the respiration. [17] To test the effects of ethanol on the denitrificans, we inoculated 1 mL of bacteria suspension into serum flask, in which containing 90 mL of mineral medium and 10 mL of ultra pure water or 10% ethanol solution. After 24 h cultivation at 30 °C, the removal efficiency of NO − 3 -N and NO − 2 -N was measured. The results showed that there was no difference on removal efficiency between water and 10% ethanol solution added (p > 0.5). Clearly, the solubilizing ethanol has no effect on the denitrificans.

Exposure experiments
In this study, P. denitrificans was exposed to 0, 10, and 50 μM of PCP. To conduct the experiments, 1 mL of bacteria suspension, 90 mL of mineral medium, and 0, 2 or 10 mL of PCP stock solution were added to serum flasks, and ultra pure water was supplemented to make the final mixture volume in each bottle to be 100 mL. The initial nitrate concentration was 250 mg NO − 3 -N/L, and the bacteria density was about 1 × 10 8 cell/mL. All bottles were bubbled with gas Argon for 10 min to ensure the anaerobic condition and covered with aluminum to avoid the possible light-induced effects. After sealing, all bottles cultured at 30 °C in a shaker (150 r/min).
To quantify of gene expression, cells were harvested by centrifugation at 5000 r/min for 10 min, and then 1 mL of RNA protect (Qiagen) was quickly added. After treatment with RNA protect, the cells were stored at −20 °C pending RNA extraction. Total RNA was extracted with RNeasy minikit (Qiagen), and the residual DNA was removed by DNase Ι treatment (Sigma). The concentration of RNA was measured by Nanodrop (Eppendorf ). The PCR primers for napA, nirS, norB, nosZ were synthesized according to the literatures. [23,24] The levels of napA, nirS, norB, and nosZ genes expressions were quantified using one-step quantitative PCR with Qiagen QuantiTect SYBR green reverse transcription-PCR kit.
The cellular growth was determined by the value of optical density OD 600 . [25,26] The glucose utilization by P. denitrificans was measured according to previous publication. [27] The lactate dehydrogenase (LDH) was measured by cytotoxicity detection kit (LDH release assay) (Roche Molecular Biochemicals) according to the manufacturer's instruction. The measurements of NO − 3 -N and NO − 2 -N were conducted by a spectrophotometer according to the Standard Methods. [28]

Statistical analysis
All tests were performed in triplicate and the results were expressed as mean ± standard deviation. An analysis of variance was used to test the significance of results and p < 0.05 was considered to be statistically significant.

PCP causes inhibition to the denitrification process
As shown in Figure 1(a), after exposure for 24 h, the concentrations of NO − 3 -N in the control, 10 and 50 μM of PCP were respectively 3.8, 32.0, and 78.2 mg/L, and the corresponding nitrate removal efficiencies were 98.5, 87.2, and 68.7%, respectively. Apparently, nitrate reduction process of P. denitrificans was significantly inhibited by PCP at the dose investigated in this study (p < 0.05). From Figure 1(a) and (b), it can be seen that the variations of NO − 2 -N and N 2 O lagged behind the tendency in the control after the additional PCP had been added, and also the highest detected N 2 O was lower than in the control (p < 0.05). However, there were no significant differences of the final nitrite and nitrous oxide in all the experiments after 24 h exposure (p > 0.05) (Figure 1(a) and (b)). To our knowledge, the slow lane for the variations of NO − 2 -N and N 2 O in the 10 and 50 μM of PCP exposure experiment was caused by the inhibited transfer of NO − 3 -N to NO − 2 -N. It was obviously that the presence of PCP did not affect the reactions involved in the reductions of NO − 2 -N and N 2 O in the process of denitrification. In the coming text the mechanisms for PCP negatively affecting nitrate reduction and marginally affecting nitrite and nitrous oxide reduction were investigated.

Effects of PCP on LDH release and ROS production
It was reported that the biological function of cells could be affected after cell membrane integrity was damaged. [29] In this study the cell membrane integrity was measured by LDH release. As seen in Figure 2 the presence of PCP induced the similar LDH release to the control, suggesting that PCP did not cause the damage of P. denitrificans cell membrane integrity at these concentrations. However, with the additional 10 and 50 μM PCP had been added, the ROS production were respectively increased to 113.5 and 136.4% of the control (Figure 2). In the literature it was reported that the increase of ROS production could inhibit the energy production process (such as glycolysis). [30] It seems that the presence of PCP were investigated. Kinase, a type of important enzyme transferring phosphate groups from high-energy donor molecules to specific substrates, is in charge of energy transform and generation during cell growth and metabolism. From the data in Figure 3(b) it can be seen that the presence of 10 or 50 μM of PCP caused significant loss of the activities of HK, PFK, and PK. For example, these enzymes were respectively decreased to 76.5, 74.2, and 81.5% of the control in the test of 50 μM of PCP. The inhibition towards these key enzymes suppressed glycolysis process, and the utilization of glucose was therefore hindered by the presence of PCP.
Since the presence of PCP did significant adverse effects on glucose metabolism of P. denitrificans, the cellular growth was also possibly influenced. As shown in Figure 3(c), the bacteria exposed to 10 or 50 μM of PCP inhibited the growth of the bacterial. After attention was first called to the toxicity of PCP by Bechold and Ehrlich in 1906, [32] many investigations of its toxic properties and its metabolic fate have been reported. It was found that the bacterial PCP resistance and sensitivity were related to many factors such as bacterial caused a more oxidative environment than the control, and finally led to the lower denitrification efficiency.

Effects of PCP on P. denitrificans glucose metabolism and cell growth
It is well known that the Embden-Meyerhof pathway (namely glycolysis) undertakes important role in denitrification bacteria during anoxic spells, such as the generating of NADH and ATP. [20,31] The utilized glucose by P. denitrificans was calculated and shown in Figure  3(a). After 24 h cultivation, less glucose was utilized by P. denitrificans in the presence of 10 μM of PCP, and the inhibitory effect was further enhanced when the dose of PCP was 50 μM (Figure 3(a)), which confirmed that the adverse effect of PCP on P. denitrificans glucose metabolism was significant at the dose investigated in this study (p < 0.05).
To illuminate the details for PCP inhibiting glycolytic activity, three vital kinases (i.e. HK, PFK, and PK), catalyzing three irreversible reactions in glycolysis process,   Figure 1). However, the result of genes expression showed that there were inhibitions of the expression of napA, nirS, norB and nosZ ( Figure 5(b)). Thus, the decreased biological nitrogen removal at these does of PCP was attributed to the inhibition of the enzyme activity of NAR.

Conclusions
In conclusion, the exposure of PCP to P. denitrificans induced the generation of ROS, which decreased the key enzymes activities related to glycolysis process, caused the disturbance of the metabolism of glucose utilization and the cell growth, and subsequently disturbed the generation of electron donor (NADH) for denitrification via NAD + reduction. Also, it was found that PCP would severely depress the genes expression of key enzymes responsible for denitrification. However, further investigation revealed that there was only the enzyme activity of NAR has been remarkably inhibited at this does of PCP. Denitrification process was significantly inhibited by the PCP at higher concentration of PCP, which would further disturb the nitrogen cycle in soil.

Disclosure statement
No potential conflict of interest was reported by the authors.  [33] In this study, the final OD 600 value was 1.475 in the control test, which was respectively decreased to 1.320 and 1.097 in the presence of 10 and 50 mg/L of PCP. The introduction of PCP significantly disturbed the growth of P. denitrificans.

Effects of PCP on the transfer of NADH/NAD +
It was well known that the respiratory reduction of substrates depends on the proper electron transport systems, and NADH is the most important electron donor in the reducing process of nitrogen oxides. [34] In the glycolysis process, the only step generating NADH was catalyzed by gAPDH, in which accompanied with the converting of glyceraldehyde 3-phosphate (g-3-P) to 1,3-biphosphoglycerate (1,3-BPg), oxidized NAD + was transfer to reduced NADH. It is critically important for the mutual transformation between NADH and NAD + to maintain the redox balance, and the NADH/ NAD + ratio is a vital parameter to measure the redox statusin bacteria. [35] As seen in Figure 4, the treatment of 10 and 50 μM of PCP caused the inactivation of gAPDH, which was decreased to 72.0 and 43.8% of the control, respectively. And the corresponding intracellular NADH/NAD + ratios were also significantly decreased (p < 0.05), which indicated that PCP induced a more oxidative environment, and finally led to the lower denitrification efficiency.

Effects of PCP on activities and genes expression of denitrification enzymes
Biological denitrification is mainly catalyzed by NAR, NIR, NOR, and N 2 OR. [11,36] As shown in Figure 5 Figure 5. effects of pCp on the activities of key enzymes involved in denitrification (a), and the genes expression of denitrification enzymes (b). asterisks indicate statistical differences (p < 0.05) from the control. error bars represent standard deviations of triplicate tests.