Bioresource Technology 185 (2015) 346–352

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Interaction of Cr(VI) reduction and denitrification by strain Pseudomonas aeruginosa PCN-2 under aerobic conditions Da He a,b, Maosheng Zheng b, Tao Ma b, Can Li b, Jinren Ni b,⇑ a Shenzhen Key Laboratory for Heavy Metal Pollution Control and Reutilization, School of Environment and Energy, Peking University Shenzhen Graduate School, Shenzhen 518055, China b Department of Environmental Engineering, Peking University, The Key Laboratory of Water and Sediment Sciences, Ministry of Education, Beijing 100871, China

h i g h l i g h t s  Simultaneous aerobic Cr(VI) and nitrate reduction was achieved by strain PCN-2.  Coordinate presence of gene ChrR, napA, nirS, cnorB and nosZ was confirmed.  Cr(V) detection confirmed Cr(VI) reduction via initial single-electron transfer.  Cr(VI) and nitrate reduction occurred faster at higher cell amount and pH 8–9.  Increasing Cr(VI) inhibited aerobic denitrification and N2O reduction.

a r t i c l e

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Article history: Received 10 December 2014 Received in revised form 23 February 2015 Accepted 27 February 2015 Available online 6 March 2015 Keywords: Cr(VI) reduction Aerobic denitrification Pseudomonas aeruginosa Electron competition

a b s t r a c t Inhibition of efficient denitrification in presence of toxic heavy metals is one of the current problems encountered in municipal wastewater treatment plants. This paper presents how to remove hexavalent chromium (Cr(VI)) and nitrate simultaneously by the novel strain Pseudomonas aeruginosa PCN-2 under aerobic conditions. The capability of strain PCN-2 for Cr(VI) and nitrate reduction was confirmed by PCR analysis of gene ChrR, napA, nirS, cnorB, nosZ, while Cr(VI) reduction was proved via an initial singleelectron transfer through Cr(V) detection using electron paramagnetic resonance. Experimental results demonstrated that Cr(VI) and nitrate reduction by strain PCN-2 was much faster at pH 8–9 and higher initial cell concentration. However, increasing Cr(VI) concentration would inhibit aerobic denitrification process and result in an significant delay of nitrate reduction or N2O accumulation, which was attributed to competition between three electron acceptors, i.e., Cr(VI), O2 and nitrate in the electron transport chain. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Chromium exists in the environment primarily in two valence states: hexavalent chromium (Cr(VI)) and trivalent chromium (Cr(III)). Cr(VI) is intensely carcinogenic, mutagenic and teratogenic (CieslakGolonka, 1996), while Cr(III) is a crucial element having significant importance in glucose, lipid and protein metabolism (Mertz, 1993). In living organisms, Cr(VI) could cause free radicals formation and damage DNA (CieslakGolonka, 1996). Being an essential industrial material, Cr(VI) is widely employed in wood preservation, electroplating, alloy production and leather tanning, which would unavoidably enter the surrounding environment. ⇑ Corresponding author. Tel.: +86 10 62751185; fax: +86 10 62756526. E-mail address: [email protected] (J. Ni). http://dx.doi.org/10.1016/j.biortech.2015.02.109 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.

For the treatment of wastewater containing heavy metals, various conventional methods have been developed (Cheng et al., 2010; Lai et al., 2009), particularly for Cr(VI) removal processes including chemical precipitation, electrochemical treatment, reverse osmosis and ion exchange (Barrera-Diaz et al., 2012). However, disadvantages have been also reported such as high cost, energy consumption and production of chemical sludge, especially for removing low-concentrated Cr(VI) in wastewater (Ganguli and Tripathi, 2002). Due to its economical, efficient and environmental friendly advantages, biotransformation of Cr(VI) to the less toxic Cr(III) by microorganisms has been deemed as an alternative promising approach for treatment of Cr(VI) pollution (Ganguli and Tripathi, 2002). Many bacterial species able to reduce Cr(VI) to Cr(III) have been reported including Pseudomonas sp., Bacillus sp., Microbacterium sp., Shewanella oneidensis and so on (Cheung and Gu, 2007; Han et al., 2010; Salamanca et al., 2013). Some

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literatures are available about the mechanism of Cr(VI) detoxification to Cr(III) by chromate reductases from diverse bacterial species, where the best investigated chromate reductase is soluble flavoprotein ChrR from Pseudomonas putida (Cheung and Gu, 2007; Ramirez-Diaz et al., 2008). Another environmental pollutant nitrate, which is commonly used in industrial production and agricultural fertilization (Chen and Strous, 2013), would cause serious eutrophication of receiving waters (Zheng et al., 2014). Traditional process used for eradication of nitrate is biological denitrification using heterotrophic denitrifying bacteria under anoxic condition following pathway of reducing nitrate stepwise to nitrite, nitric oxide (NO), nitrous oxide (N2O) and finally nitrogen gas (N2) (Zumft, 1997). However, the denitrifying activity could be largely suppressed by the presence of oxygen due to the sensitivity of denitrifying reductase (Ferguson, 1994). In 1980s, a bacterial strain Paracoccus denitrificans (formerly Thiosphaera pantotropha) capable of respiring nitrate and oxygen simultaneously was isolated (Robertson and Kuenen, 1984). Since then, aerobic denitrification has received a lot of attentions and more novel aerobic denitrifiers with great nitrate removal performance have been isolated belonging to the genera Alcaligenes, Pseudomonas, Achromobacter, Comamonas, Bacillus and so on (Chen and Strous, 2013; Zheng et al., 2014). Recently, Cr(VI) reduction by bacteria was found to have indivisible relationships with the physiological electron acceptor (oxygen or nitrate) consumption. However, the interaction of Cr(VI) and nitrate reduction was still controversial. For example, inhibition of Cr(VI) reduction caused by nitrate or nitrite and the reverse inhibition of nitrite reduction by Cr(VI) were observed under anaerobic conditions (Viamajala et al., 2002). Others indicated that the chromate could inhibit the nitrate reduction but nitrate would not affect Cr(VI) reduction rate by Pseudomonas strains (Konovalova et al., 2008). Nevertheless, the co-metabolism of Cr(VI) and oxygen or nitrate by strain Pseudomonas stutzeri RCH2 was proposed considering the requirements of oxygen or nitrate in Cr(VI) reduction and high positive correlation of denitrification and chromate reduction rates (Han et al., 2010). Therefore, it was still not clear whether Cr(VI) and nitrate reduction were positively or negatively correlated. In addition, most of previous studies on Cr(VI) reduction were made under sole denitrifying or sole aerobic conditions, fewer were about Cr(VI) reduction under aerobic denitrifying conditions and rarely about the influence of Cr(VI) on denitrification intermediates. In this study, Cr(VI) and nitrate reduction by bacterial strain Pseudomonas aeruginosa PCN-2 under aerobic conditions were investigated with particular attention to their interaction dominated by electron transfer and competition between electron acceptors of Cr(VI), O2 and nitrate in the electron transport chain, which was of primary importance to understand Cr(VI) reduction, denitrification and N2O emission in real wastewater treatment process.

The initial concentration of ammonium nitrogen (200 mg L1) in medium was supplemented for bacterial assimilation (Zumft, 1997). All experiments were performed in duplicate. 2.2. Aerobic Cr(VI) and nitrate reduction by strain PCN-2 In order to evaluate the Cr(VI) and nitrate reduction capability under aerobic condition, strain PCN-2 was pre-cultured to late exponential growth phase and 20 mL culture was harvested, centrifuged and re-suspended in 2 mL DD H2O followed by inoculating into 100 mL BM in 250 ml shake flasks (inoculation dosage (v/v) 20%). The media were supplemented with different volumes of 1000 mg L1 Cr(VI) (K2Cr2O7) to the desired Cr(VI) concentrations. The BM containing the same concentration of Cr(VI) but without bacterial culture inoculation was set as the control. The Cr(VI) reduction performances of strain PCN-2 were also evaluated under different initial cell concentrations and various pH values. To investigate the effect of initial cell concentrations, the inoculation dosage (v/v) was adjusted to 0%, 10%, 20% and 30% at initial pH of 8. In pH experiments, the initial pH was adjusted to 5, 6, 7, 8, 9 and 10 with inoculation dosage of 20%, respectively. The initial Cr(VI) concentrations in these Cr(VI) reduction studies were set at 5 mg L1. The flasks were incubated in a rotary shaker at 150 rpm and 30 °C. 1 mL aliquots were withdrawn periodically from the shake flasks and centrifuged at 8000 rpm for 5 min at 4 °C. The supernatant was obtained for Cr(VI), total Cr and NO3-N analysis. 2.3. PCR amplification of ChrR and denitrification genes The total bacterial DNA of P. aeruginosa PCN-2 was extracted with a genomic DNA extraction kit (Beijing TianGen Biotech Co., Ltd, China). The primers for ChrR, napA, nirS, cnorB, nosZ and V3 region of 16S rRNA are shown in Table 1. Polymerase chain reaction (PCR) amplification was performed in a total volume of 25 lL containing 2 lL of DNA template, 12.5 lL Taq PCR Mastermix (Beijing TianGen Biotech Co., Ltd, China), 1 lL forward and reverse primers (10 lmol L1). PCR amplification was carried

Table 1 PCR primers used for PCR analysis of V3 region of 16 rRNA, napA, nirS, norB, nosZ and ChrR. Gene

Primer

Primer sequences (50 -30 )

References

V3 region of 16S rRNA napAa

F341 R518 napA Z3F napA Z3R nirS cd3aF nirS R3cd cnorB Z1F cnorB Z1R nosZ 1527F nosZ 1773R chrR 106F chrR 795R

CCTACGGGAGGCAGCAG ATTACCGCGGCTGCTGG CGCGAACAAGCTGATGAAGG

Muyzer et al. (1993)

nirS

2. Methods cnorBa

2.1. Bacterial strain and medium The bacterial strain P. aeruginosa PCN-2 used in this study was isolated from a landfill leachate treating reactor. The composition of basal medium (BM) used for bacteria cultivation and performance evaluation was as follows (per liter): 6.0 g C6H12O6, 1.2 g NaNO3, 0.76 g NH4Cl, 1.0 g K2HPO43H2O, 0.20 g MgSO47H2O, 0.02 g CaCl2, 0.005 g FeSO47H2O and 1.0 mL trace elements solution (Yao et al., 2013). All media were adjusted to pH 8 and autoclaved at 105 °C for 20 min where the relative low sterilizing temperature was adopted to avoid the carbonization of glucose.

nosZ

ChrR

AAGATCATCGGGATGTCGGC GTSAACGTSAAGGARACSGG

Throback et al. (2004)

GASTTCGGRTGSGTCTTGA CGTCGGTCAGATCCTCTTCG GCGATGATCACGTAGAGCCA CGCTGTTCHTCGACAGYCA

Throback et al. (2004)

ATRTCGATCARCTGBTCGTT GACTGGCAYCTSGTGCACCT

Han et al. (2010)

GGADGASACGTCGA TCAGGT

a Designed by primer designing tool in NCBI according to napA and cnorB sequence of P. stutzeri A1501.

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out in a thermal cycler (Bio-Rad, USA) with the following settings: initial denaturation at 94 °C for 5 min; 30 cycles of denaturation at 94 °C for 30 s, annealing at 54 °C (nosZ and nirS) or 60 °C (napA, cnorB, V3 and ChrR) for 15 s and extension at 72 °C for 1 min; followed by a final extension step of 72 °C for 10 min.

O2, N2O were measured and calculated as described in the literature (Zheng et al., 2014). 3. Results and discussion 3.1. Aerobic reduction of Cr(VI) and nitrate by P. aeruginosa PCN-2

2.4. Cr(V) detection by electron paramagnetic resonance (EPR) To detect the intermediate of Cr(V) formed during Cr(VI) reduction by P. aeruginosa PCN-2, the electron paramagnetic resonance (EPR) spectrum was recorded by an Electron Spin Resonance spectrometer (JES-FA200, Japanese electronics co., LTD). The measurement conditions were 100-kHz field modulation, 1.0-G modulation amplitude, 9.05-gHz microwave frequency and 4.0-mW power at room temperature. 2.5. Interaction between Cr(VI) and nitrate reduction studies To investigate the effect of initial nitrate concentrations on Cr(VI) reduction performances, the different initial nitrate concentrations from 0, 100, 200 and 400 mg L1 were selected. The inoculation dosage (v/v) was increased to 50% to reduce the influence of bacterial growth under different nitrate concentrations. Other operational parameters were same as that in the aerobic Cr(VI) and nitrate reduction studies. To evaluate the effect of initial Cr(VI) concentrations on aerobic denitrification process, 50 mL BM in 300 mL glass serum bottles with gas-impermeable rubber stoppers were inoculated at a dosage (v/v) of 20%. The initial Cr(VI) concentrations were adjusted to 0, 2.5, 5.0, 7.5 and 10.0 mg L1, respectively. Atmospheric air (approximately 21% O2) in the headspace was not replaced to ensure aerobic condition. The bottles were incubated in a rotary shaker at 150 rpm and 30 °C. At regular time 100 lL gas samples were collected using a gas-tight syringe for detection of N2O and O2. Simultaneously, 1 mL aliquots were withdrawn from the bottles by syringe for the measurement of cell optical density (OD600) followed by centrifuged at 8000 rpm for 5 min at 4 °C. The supernatant was obtained for Cr(VI), NO2-N, and NO3-N analysis.

Fig. 1 demonstrated that Cr(VI) and nitrate reduction commenced after the bacterial culture of strain PCN-2 was inoculated and almost depleted at 13.5 and 9.5 h, respectively, while the concentrations of Cr(VI) and nitrate in the controls were kept stable. This indicated the removal was ascribed to the bacterial degradation. Only a slight decrease of total Cr was observed in the solutions even as Cr(VI) was almost completely reduced, indicating a large proportion (>68%) of Cr(VI) was transformed into Cr(III). Conventionally, Cr(III) would precipitate as Cr(OH)3 at neutral pH (Barrera-Diaz et al., 2012), however, there was no visible Cr(III) precipitate in the solutions. It was found the end-product of microbial Cr(VI) reduction was existed as soluble organoCr(III) complexes which would not precipitate (Puzon et al., 2005). The fate of the rest reduced Cr(VI) may be transformed to insoluble organo-Cr(III) complexes (Puzon et al., 2005) or adsorbed on the cells (Kang et al., 2007). It was notable that the whole process of Cr(VI) and nitrate reduction was under aerobic condition, which indicated P. aeruginosa PCN-2 performed simultaneous aerobic denitrification and Cr(VI) reduction, i.e., O2, nitrate and Cr(VI) severed as electron acceptors for bacterial metabolism at the same time. 3.2. PCR amplification of ChrR, denitrification genes and Cr(V) detection As shown in Fig. 2(a), PCR amplification resulted in the bands of 303, 425, 637, 246 and 732 bp products for napA, nirS, cnorB, nosZ and ChrR genes, respectively. ChrR encoded by gene ChrR is a soluble, flavin mononucleotide-binding protein (Eswaramoorthy et al., 2012), also named as NAD(P)H-dependent FMN reductase family which functions as a 50-kDa dimer (Ackerley et al., 2004)

2.6. Analytical methods and calculations Cr(VI) concentration was measured by a modified 1,5-diphenylcarbazide spectrophotometric method. It was observed that 5 mM nitrite would cause the diphenylcarbazide unresponsive to Cr(VI) and make the DPCI spectrophotometric method completely invalid (Han et al., 2010). In this study, sulphamic acid was adopted as a masking agent, which was proved to be an efficient way to eliminate the interference of nitrite (Table 2). Total chromium was measured using an inductively coupled plasma-atomic emission spectroscopy (ICP-AES, Prodigy, Leeman, USA) after filtration with 0.22 lm hydrophilic filter membrane. The OD600, NO2-N, NO3-N, Table 2 DPCI method testing the response to Cr(VI) in the presence of nitrite and with the masking agent of sulphamic acid. Cr(VI) concentration

Measured Cr(VI) without nitrite

Measured Cr(VI) + 60 mg L1 nitrite

Measured Cr(VI) + 60 mg L1 nitrite + 800 mg L1 sulphamic acid

0.2 0.4 0.6 0.8 1.0

0.206 0.398 0.606 0.790 1.003

0.001 0.003 0.014 0.003 0.001

0.202 0.402 0.607 0.802 0.993

Fig. 1. Cr(VI) and nitrate reduction by strain PCN-2 under aerobic condition. Symbols: h, Cr(VI) or nitrate in control group; d, Cr(VI) or nitrate; N, Total Cr.

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Fig. 2. (a) PCR amplification of napA, nirS, cnorB, nosZ, V3 and ChrR genes in P. aeruginosa PCN-2 (DNA Marker DL2000); (b) EPR spectrum of Cr(V) generated by the bioreduction of Cr(VI) by P. aeruginosa PCN-2.

and shows a quinone reductase activity (Eswaramoorthy et al., 2012). ChrR could transfer one electron to Cr(VI) and generate the intermediate Cr(V), which is subsequently reduced by some biomolecules to the stable Cr(III) (Cheung and Gu, 2007). Periplasmic nitrate reductase encoded by gene napA is insensitive to O2 which could be expressed under aerobic condition and catalyze nitrate reduction (Reyes et al., 1996). Therefore, the presence of ChrR and denitrification genes ensured the capability of aerobic Cr(VI) and nitrate reduction simultaneously by P. aeruginosa PCN-2. The process of Cr(VI) reduction was investigated with EPR test and a signal of g value 1.975 was detected as shown in Fig. 2(b) which confirmed that Cr(V) was produced during Cr(VI) reduction (Myers et al., 2001; Wani et al., 2007) and suggested that the reduction by strain PCN-2 was via an initial single-electron transfer under the catalysis of ChrR. Cr(VI) received one electron and generated the intermediate Cr(V) which was subsequently reduced to the final stable product Cr(III). This pathway was consistent with that demonstrated in several other Cr(VI) degrading bacterial stains, like Shewanella putrefaciens MR-1 (Myers et al., 2001) and Burkholderia cepacia MCMB-821 (Wani et al., 2007).

Cr(VI) and nitrate reduction were not observed when the medium was not inoculated. When the inoculation dosage was 10%, Cr(VI) concentration decreased from 4.8 to 0.88 mg L1 after 15 h and nitrate decreased from 200 mg L1 to about 5.83 mg L1 after 11.5 h. As the inoculation amount increased from 10% to 30%, Cr(VI) and nitrate were reduced more rapidly, indicating that the Cr(VI) and nitrate reduction activities were positively correlated with bacterial amount. The pH of medium also played an important role in Cr(VI) and nitrate reduction. As shown in Fig. 3c and d, strain PCN-2 could effectively remove both Cr(VI) and nitrate at pH from 8 to 9, in which more than 80% of Cr(VI) removal was achieved within 15 h and nitrate was depleted in 9 h. The precipitation of produced Cr(III) was not observed even at pH 9, which further reinforced the fact that Cr(VI) was reduced to organo-Cr(III) complexes (Puzon et al., 2005). In addition, the alkaline condition could help to neutralize H+ produced during glucose consumption (Zheng et al., 2014), which contributed to the growth of strain PCN-2. However, strong alkaline (pH > 10) or acidic (pH < 6) distinctly defeated the Cr(VI) and nitrate reduction ability since the growth and activities of strain PCN-2 were significantly suppressed.

3.3. Effect of inoculation dosage, pH on Cr(VI) and nitrate reduction

3.4. Effect of nitrate concentration on aerobic Cr(VI) reduction

The cell concentration was an important factor to influence the rates of Cr(VI) and nitrate reduction by strain PCN-2 (Fig. 3a and b).

To reveal the relationship between Cr(VI) reduction and denitrification under aerobic condition, the nitrate effect was

Fig. 3. Cr(VI) and nitrate removal performances of strain PCN-2 under different inoculation dosages (ab) and pH (cd). Symbols: (ab): h, Cr(VI) or nitrate, 0%; d, Cr(VI) or nitrate, 10%; N, Cr(VI) or nitrate, 20%; ., Cr(VI) or nitrate, 30%; J, Total Cr, 10%; I, Total Cr, 20%; , Total Cr, 30%; (cd): j, pH = 5; d, pH = 6; N, pH = 7; ., pH = 8; J, pH = 9; I, pH = 10.

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experimentally investigated at varying the initial concentrations of 0, 100, 200 and 400 mg L1. As results, the reduction of nitrate and Cr(VI) commenced immediately after inoculation at all nitrate concentrations (Fig. 4). Although the nitrate reduction rate increased significantly with increasing nitrate concentration within 9 h (Fig. 4a), whereas no change in reduction rate of Cr(VI) was observed (Fig. 4b), indicating the nitrate reduction rate had no influence on Cr(VI) reduction. This phenomenon was consistence with a previous research (Wang and Xiao, 1995) but conflicted with another which reported the high correlation between chromate reduction and denitrification rates (Han et al., 2010). As both nitrate and Cr(VI) reduction need NADH as electron donor (Chen and Strous, 2013; Ramirez-Diaz et al., 2008), they may compete for electrons and have some impact on each other. The possible reason for this result is that Cr(VI) reductase is more competitive for NADH which caused the aerobic Cr(VI) reduction cannot be affected by the high nitrate reduction rate.

3.5. Effect of Cr(VI) concentration on aerobic denitrification The effect of Cr(VI) concentration on nitrate reduction and intermediates accumulation was also investigated to further explore the relationship between Cr(VI) reduction and aerobic denitrification. The initial Cr(VI) concentrations were set at 0, 2.5, 5.0, 7.5 and 10 mg L1. With the increasing initial Cr(VI) concentration, the residue Cr(VI) concentrations were 0, 0.01, 0.59 and 2.64 mg L1 after 33 h incubation, respectively (Fig. 5a). At the same time, the growth of strain PCN-2 was slightly depressed due to the toxicity of Cr(VI) (Fig. 5e). When Cr(VI) was absent in the solution, the nitrate reduction commenced immediately after inoculation and depleted completely at 6 h (Fig. 5b). The high nitrate reduction rate resulted in 3.93 mg L1 of nitrite accumulation at 3 h, which subsequently decreased to near zero at 6 h (Fig. 5f). When nitrate and nitrite were exhausted, the gaseous denitrification intermediate N2O accumulated to maximal 65.06 mg L1 at 6 h, accounting for 31.70% of denitrified nitrate, and then completely reduced to N2 at 12 h (Fig. 5d). At the initial

Cr(VI) concentration of 2.5 mg L1, Cr(VI) reduction was fast and totally depleted at 9 h (Fig. 5a) but nitrate reduction commenced after a 3-h time lag (Fig. 5b). Although a certain degree of inhibition of nitrate reduction by Cr(VI) was observed, nitrate was completely reduced within 12 h (Fig. 5b). Moreover, owing to the relatively lower nitrate reduction rate, there was no nitrite accumulation (Fig. 5f). However, the N2O accumulated to maximal 131.86 mg L1 at 21 h, then reduced to 114.74 mg L1 (Fig. 5d), accounting for 56.96% of denitrified nitrate, indicating that only 43.04% of nitrate was finally reduced to N2. At the initial Cr(VI) concentration of 5 mg L1, Cr(VI) was totally depleted in 21 h (Fig. 5a) and nitrate reduction commenced after a 6-h time lag when the Cr(VI) concentration was relatively low (2.57 mg L1, Fig. 5b). The denitrification process was obviously inhibited and nitrate was completely exhausted until 33 h (Fig. 5b) without nitrite accumulation (Fig. 5f). Consistent with nitrate reduction, the N2O accumulated to the maximal 103.94 mg L1 at 33 h (Fig. 5d), accounting for 53.22% of denitrified nitrate, indicating that the process of N2O reduced to N2 was largely suppressed by Cr(VI). At the initial Cr(VI) concentration of 7.5 mg L1, only 8.99% nitrate was denitrified (Fig. 5b) and the N2O was accumulated to 15.48 mg L1 at 33 h (Fig. 5d). Nitrate reduction was completely inhibited by Cr(VI) at initial concentration of 10 mg L1 while Cr(VI) was still reduced. Therefore, with the increasing Cr(VI) concentration, aerobic denitrification of strain PCN-2 was inhibited more and more seriously. Moreover, it seems that the process of N2O reduction was more sensitive to Cr(VI) than nitrate reduction and nitrite reduction was not affected by Cr(VI) since no nitrite was accumulated when Cr(VI) existed (Fig. 5f). At the same time, the O2 decreasing profile had slight differences (Fig. 5c) under different initial Cr(VI) concentration conditions, which was probably due to the slight depression of bacterial growth (Fig. 5e), indicating electron accepted by O2 was not influenced much by Cr(VI). So it can be inferred that when Cr(VI), O2 and nitrate served as electron acceptors simultaneously, Cr(VI) and O2 tended to be used in preference while nitrate only be used when the Cr(VI) was decreased to a low concentration. The significant lag and inhibition of nitrate reduction herein was ascribed to the electron competition between Cr(VI) and nitrate. Consistent with the former experiment, the nitrate inhibition indicated that the Cr(VI) reductase, ChrR has more competition capability for electrons than nitrate reductase. It was found that the nitrate reduction would be inhibited by a chromate concentration above 500 mg L1 (Salamanca et al., 2013). In soil microcosms nitrate reduction was dependent on the reduction of added Cr(VI), which may serve as a competing electron acceptor and a toxic metal inhibiting the nitrate reduction (Kourtev et al., 2009). However, few researches focused on N2O reduction by denitrifying bacteria in the presence of Cr(VI). This study demonstrated that the concentration of Cr(VI) which would cause inhibition of N2O reduction was much lower than that of nitrate reduction. Considering the important role of N2O in ozone depletion and greenhouse effect, the effect of Cr(VI) on inhibition of N2O reduction is of great significance. One possibility responsible for this considerable N2O accumulation was ascribed to the severe suppression of the activity of N2O reductase by Cr(VI). Another was that the Cr(VI) reduction process gave rise to reactive free radicals, such as Cr(V) and O2, which further caused oxidative damage on DNA, RNA and proteins of the bacterial cells (Valko et al., 2005) and severely influenced the enzyme synthesis and bacterial metabolism. 3.6. Electron transport chain during aerobic Cr(VI) reduction and denitrification

Fig. 4. Nitrate and Cr(VI) removal performances of strain PCN-2 under different nitrate concentrations. Symbols: j, 0 mg L1; d, 100 mg L1; N, 200 mg L1; ., 400 mg L1.

An electron transport chain was proposed when Cr(VI), O2 and nitrate served as electron acceptors simultaneously (Fig. 6).

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Fig. 5. Nitrate and Cr(VI) removal performances of strain PCN-2 under different Cr(VI) concentration. Symbols: j, 0 mg L1; d, 2.5 mg L1; 10 mg L1.

NADH was originated from the organic substrates and functioned as a quinone oxidoreductase involved in bacterial electron transport (Brandt, 2006). The produced NADH was assumed to be digested by three electron transfer pathways: aerobic respiration; denitrification respiration and chromate reduction. In aerobic respiration pathway, electrons are transferred from NADH to complex IV (cytochrome a and cytochrome a3) via ubiquinone, cytochrome b and cytochrome c, then the complex IV transduces the electrons from cytochrome c to oxygen (Chen and Strous, 2013; Wilson and Bouwer, 1997). In denitrification respiration, NOx reductases (nitrate, nitrite, nitric oxide and nitrous oxide reductases) accept electrons transferred from NADH via ubiquinone, complex b and cytochrome c, then transduce to nitrate, nitrite, nitric oxide and nitrous oxide (Chen and Strous, 2013). In addition, Cr(VI) also serves as the terminal electron acceptor with chromate reductase with NADH acting as electron donors (Park et al., 2000). ChrR

351

N, 5 mg L1; ., 7.5 mg L1; J,

would receive electrons from NADH and transfer one electron to Cr(VI) and generate the intermediate Cr(V), which subsequently reduced to the Cr(III) (Cheung and Gu, 2007). In previous aerobic denitrification researches, it has been suggested that electron acceptors are usually utilized in a specific order or hierarchy and O2 is the preferred acceptor than nitrate under aerobic condition (Unden and Bongaerts, 1997). Generally, the denitrifying activity of most denitrifying bacteria would be suppressed by oxygen (Ferguson, 1994), but aerobic denitrifiers belonging to several bacterial species could respire O2 and nitrate simultaneously (Chen and Strous, 2013). Strain PCN-2 also possesses the capability respiring both oxygen and nitrate, but the denitrifying activity was suppressed at the presence of Cr(VI) (Fig. 5), indicating that Cr(VI) accepted electrons in priority than nitrate. With the increasing Cr(VI) concentration, more electrons were drawn into the chromate reduction and the inhibition of aerobic denitrification became more serious step by step (Fig. 5). Moreover, the electron competition and cytotoxicity of Cr(VI) would result in a production of large amount of N2O, which should be paid more attention due to the high ozone depleting and global warming potential of N2O. Therefore, considering the potential N2O emission caused by Cr(VI), this simultaneous reduction of Cr(VI) and nitrate needs more investigation when applied in practical wastewater treatment.

4. Conclusions

Fig. 6. Electron transport chain of aerobic denitrification and chromate reduction (adapted from (Chen and Strous, 2013; Ramirez-Diaz et al., 2008; Wilson and Bouwer, 1997)), Nar: Nitrate reductase; Nir: Nitrite reductase; Nor: Nitric oxide reductase; Nos: Nitrous oxide reductase; Cyt, cytochrome.

In this study, efficient simultaneous reduction of Cr(VI) and nitrate with help of P. aeruginosa PCN-2 under aerobic condition was achieved at alkaline condition (pH = 8–9) and higher initial cell concentration. Cr(V) was generated as an intermediate during Cr(VI) reduction. The presence of denitrifying and chromate reductase genes interpreted the capability of the strain to utilize O2, nitrate and Cr(VI) as electron acceptors at the same time. However, Cr(VI) would inhibit aerobic denitrification process and

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result in N2O accumulation due to the competitions between the three electron acceptors in the electron transfer chain.

Acknowledgements Financial support from National Natural Science Foundation of China (Grant No. 21261140336/B070302) and Collaborative Innovation Center for Regional Environmental Quality is very much appreciated. Support from National Research Foundation and the Economic Development Board (SPORE, COY-15-EWI-RCFSA/N1971) is also acknowledged.

References Ackerley, D.F., Gonzalez, C.F., Park, C.H., Blake 2nd, R., Keyhan, M., Matin, A., 2004. Chromate-reducing properties of soluble flavoproteins from Pseudomonas putida and Escherichia coli. Appl. Environ. Microbiol. 70, 873–882. Barrera-Diaz, C.E., Lugo-Lugo, V., Bilyeu, B., 2012. A review of chemical, electrochemical and biological methods for aqueous Cr(VI) reduction. J. Hazard. Mater. 223–224, 1–12. Brandt, U., 2006. Energy converting NADH: quinone oxidoreductase (Complex I). Annu. Rev. Biochem. 75, 69–92. Chen, J., Strous, M., 2013. Denitrification and aerobic respiration, hybrid electron transport chains and co-evolution. Biochim. Biophys. Acta 1827, 136–144. Cheng, J., Zhu, X.P., Ni, J.R., Borthwick, A., 2010. Palm oil mill effluent treatment using a two-stage microbial fuel cells system integrated with immobilized biological aerated filters. Bioresour. Technol. 101, 2729–2734. Cheung, K.H., Gu, J.D., 2007. Mechanism of hexavalent chromium detoxification by microorganisms and bioremediation application potential: a review. Int. Biodeterior. Biodegrad. 59, 8–15. CieslakGolonka, M., 1996. Toxic and mutagenic effects of chromium(VI). A review. Polyhedron 15, 3667–3689. Eswaramoorthy, S., Poulain, S., Hienerwadel, R., Bremond, N., Sylvester, M.D., Zhang, Y.B., Berthomieu, C., Van Der Lelie, D., Matin, A., 2012. Crystal structure of ChrRa quinone reductase with the capacity to reduce chromate. PLoS One 7, e36017. Ferguson, S.J., 1994. Denitrification and its control. Anton. Leeuw. Int. J. G. 66, 89– 110. Ganguli, A., Tripathi, A.K., 2002. Bioremediation of toxic chromium from electroplating effluent by chromate-reducing Pseudomonas aeruginosa A2Chr in two bioreactors. Appl. Microbiol. Biotechnol. 58, 416–420. Han, R., Geller, J.T., Yang, L., Brodie, E.L., Chakraborty, R., Larsen, J.T., Beller, H.R., 2010. Physiological and transcriptional studies of Cr(VI) reduction under aerobic and denitrifying conditions by an aquifer-derived pseudomonad. Environ. Sci. Technol. 44, 7491–7497. Kang, S.Y., Lee, J.U., Kim, K.W., 2007. Biosorption of Cr(III) and Cr(VI) onto the cell surface of Pseudomonas aeruginosa. Biochem. Eng. J. 36, 54–58. Konovalova, V., Nigmatullin, R., Dmytrenko, G., Pobigay, G., 2008. Spatial sequencing of microbial reduction of chromate and nitrate in membrane bioreactor. Bioprocess Biosyst. Eng. 31, 647–653. Kourtev, P.S., Nakatsu, C.H., Konopka, A., 2009. Inhibition of nitrate reduction by chromium(VI) in anaerobic soil microcosms. Appl. Environ. Microbiol. 75, 6249–6257.

Lai, P., Zhao, H.Z., Zeng, M., Ni, J.R., 2009. Study on treatment of coking wastewater by biofilm reactors combined with zero-valent iron process. J. Hazard. Mater. 162, 1423–1429. Mertz, W., 1993. Chromium in human nutrition: a review. J. Nutr. 123, 626–633. Muyzer, G., Dewaal, E.C., Uitterlinden, A.G., 1993. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl. Environ. Microbiol. 59, 695–700. Myers, C.R., Carstens, B.P., Antholine, W.E., Myers, J.M., 2001. Chromium(VI) reductase activity is associated with the cytoplasmic membrane of anaerobically grown Shewanella putrefaciens MR-1. J. Appl. Microbiol. 88, 98– 106. Park, C.H., Keyhan, M., Wielinga, B., Fendorf, S., Matin, A., 2000. Purification to homogeneity and characterization of a novel Pseudomonas putida chromate reductase. Appl. Environ. Microbiol. 66, 1788–1795. Puzon, G.J., Roberts, A.G., Kramer, D.M., Xun, L., 2005. Formation of soluble organochromium(III) complexes after chromate reduction in the presence of cellular organics. Environ. Sci. Technol. 39, 2811–2817. Ramirez-Diaz, M.I., Diaz-Perez, C., Vargas, E., Riveros-Rosas, H., Campos-Garcia, J., Cervantes, C., 2008. Mechanisms of bacterial resistance to chromium compounds. Biometals 21, 321–332. Reyes, F., Roldan, M.D., Klipp, W., Castillo, F., MorenoVivian, C., 1996. Isolation of periplasmic nitrate reductase genes from Rhodobacter sphaeroides DSM 158: structural and functional differences among prokaryotic nitrate reductases. Mol. Microbiol. 19, 1307–1318. Robertson, L.A., Kuenen, J.G., 1984. Aerobic denitrification: a controversy revived. Arch. Microbiol. 139, 351–354. Salamanca, D., Strunk, N., Engesser, K.-H., 2013. Chromate reduction in anaerobic systems by bacterial strain Pseudomonas aeruginosa CRM100. Chem. Ing. Tech. 85, 1575–1580. Throback, I.N., Enwall, K., Jarvis, A., Hallin, S., 2004. Reassessing PCR primers targeting nirS, nirK and nosZ genes for community surveys of denitrifying bacteria with DGGE. FEMS Microbiol. Ecol. 49, 401–417. Unden, G., Bongaerts, J., 1997. Alternative respiratory pathways of Escherichia coli: energetics and transcriptional regulation in response to electron acceptors. Biochim. Biophys. Acta 1320, 217–234. Valko, M., Morris, H., Cronin, M.T., 2005. Metals, toxicity and oxidative stress. Curr. Med. Chem. 12, 1161–1208. Viamajala, S., Peyton, B.M., Apel, W.A., Petersen, J.N., 2002. Chromate/nitrite interactions in Shewanella oneidensis MR-1: evidence for multiple hexavalent chromium [Cr(VI)] reduction mechanisms dependent on physiological growth conditions. Biotechnol. Bioeng. 78, 770–778. Wang, Y.T., Xiao, C.S., 1995. Factors affecting hexavalent chromium reduction in pure cultures of bacteria. Water Res. 29, 2467–2474. Wani, R., Kodam, K.M., Gawai, K.R., Dhakephalkar, P.K., 2007. Chromate reduction by Burkholderia cepacia MCMB-821, isolated from the pristine habitat of alkaline Crater Lake. Appl. Microbiol. Biotechnol. 75, 627–632. Wilson, L.P., Bouwer, E.J., 1997. Biodegradation of aromatic compounds under mixed oxygen/denitrifying conditions: a review. J. Ind. Microbiol. Biotechnol. 18, 116–130. Yao, S., Ni, J., Ma, T., Li, C., 2013. Heterotrophic nitrification and aerobic denitrification at low temperature by a newly isolated bacterium, Acinetobacter sp. HA2. Bioresour. Technol. 139, 80–86. Zheng, M., He, D., Ma, T., Chen, Q., Liu, S., Ahmad, M., Gui, M., Ni, J., 2014. Reducing NO and N2O emission during aerobic denitrification by newly isolated Pseudomonas stutzeri PCN-1. Bioresour. Technol. 162, 80–88. Zumft, W.G., 1997. Cell biology and molecular basis of denitrification. Microbiol. Mol. Biol. Rev. 61, 533–616.

Interaction of Cr(VI) reduction and denitrification by strain Pseudomonas aeruginosa PCN-2 under aerobic conditions.

Inhibition of efficient denitrification in presence of toxic heavy metals is one of the current problems encountered in municipal wastewater treatment...
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