Simultaneous aerobic denitrification and Cr(VI) reduction by Pseudomonas brassicacearum LZ-4 in wastewater

Bioresource Technology 221 (2016) 121–129

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Simultaneous aerobic denitrification and Cr(VI) reduction by Pseudomonas brassicacearum LZ-4 in wastewater Xuan Yu a,b,d,1, Yiming Jiang a,1, Haiying Huang a,1, Juanjuan Shi a, Kejia Wu a, Pengyun Zhang b, Jianguo Lv b, Hongli Li c, Huan He d, Pu Liu a, Xiangkai Li a,⇑ a

Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations, School of Life Science, Lanzhou University, Tianshuinanlu #222, Lanzhou, Gansu 730000, PR China Gansu Academy of Membrane Science and Technology, Duanjiatanlu #1272, Lanzhou, Gansu 730020, PR China PetroChina Lanzhou Petrochemical Company, Yumenjie #10, Lanzhou, Gansu 730060, PR China d School of Chemical Engineering and Technology, China University of Mining and Technology, Xuzhou, Jiangsu 221116, PR China b c

h i g h l i g h t s 


P. brassicacearum LZ-4 can reduce Cr(VI) and NO3

simultaneously.


Nitrate can promote Cr(VI) reduction in strain LZ-4. 1


Cr(VI) at 50 mg L

does not inhibit reduction of 100 mg L1 NO3-N. 


Strain LZ-4 efficiently removes NO3 , Cr(VI), and COD in a membrane bioreactor.

a r t i c l e

i n f o

Article history: Received 29 July 2016 Received in revised form 7 September 2016 Accepted 8 September 2016 Available online 10 September 2016 Keywords: Denitrification Cr(VI) reduction Pseudomonas Co-contaminants

a b s t r a c t Inorganic nitrogen and heavy metals pervasively co-exist in industrial and domestic wastewaters. In this work, Pseudomonas brassicacearum LZ-4 was tested for the simultaneous reduction of Cr(VI) and nitrate. Nitrate was found to be the best inorganic nitrogen source for strain LZ-4, and could promote Cr(VI) reduction. Cr(VI) had a low degree of inhibition on denitrification, and even 50 mg L1 Cr(VI) did not inhibit reduction of 100 mg L1 NO 3 -N. The capability of simultaneous reduction of Cr(VI) and nitrate was illustrated by the reductase genes contained in the LZ-4 genome. Application in a batch membrane bioreactor showed that the immobilized strain LZ-4 could remove over 95% of 500 mg L1 NO 3 -N, 80% of 10 mg L1 Cr(VI), and 96% of 5000 mg L1 COD in each batch of 46 days. In summary, the strain LZ-4 is an ideal candidate for remediation of co-contaminants. Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction Combined pollutants are the by-products of industries such as power, pesticide factory, electroplating, paints, petroleum refining, and metal smelting; they usually contain sulfur oxides, nitric oxides, organic contaminants, and heavy metals (Huang et al., 2016). The co-existence of different oxidized contaminants such as nitrate and chromate in soil or wastewater is unavoidable (Kourtev et al., 2009; Miao et al., 2015; Peng et al., 2015). Nitrate, one of the major nitrogen contaminants, can cause acute health problems, such as carcinoma, malformation, mutation, and methemoglobinemia in infants after transformation into nitrites and nitrosamines (Peng et al., 2015). Meanwhile, hexavalent chromium ⇑ Corresponding author. 1

E-mail address: [email protected] (X. Li). These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.biortech.2016.09.037 0960-8524/Ó 2016 Elsevier Ltd. All rights reserved.

(Cr(VI)) is a potent toxicant, mutagen, and carcinogen (Han et al., 2012), and is classified as the top 16th hazardous substance by the Agency for Toxic Substances and Diseases Registry (ATSDR) (Chen and Gu, 2005). Several physicochemical techniques are effective for the simultaneous removal of chromate and nitrate, including membrane filtration, reverse osmosis, electrodialysis, and ion exchange; however, the high cost of these approaches and the generated secondary wastes remain issues that need to be addressed (Li et al., 2016). Therefore, biological reduction of these oxidized contaminants to harmless or immobile forms is a cost effective and ecofriendly approach. Microbial denitrification is one of the most promising and efficient technologies for nitrate removal, but high concentrations of Cr(VI) are toxic to microorganisms and inhibit their metabolic capacity (Miao et al., 2015). Moreover, both denitrification and Cr(VI) reduction use NADH as electron donors and form a mutually competitive inhibition with each other, thus causing difficulties in the degradation of these

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contaminants (He et al., 2015; Kourtev et al., 2009). Inhibition of denitrification by Cr(VI) was observed in various species, such as Shewanella oneidensis MR-1 (Viamajala et al., 2002), Geobacter metallireducens (Chovanec et al., 2012), and some soil microorganisms (Kourtev et al., 2009). To solve this problem, one study used a two-layered membrane bioreactor (MBR) for the spatial sequential reduction of chromate and nitrate (Konovalova et al., 2008). Another method for the simultaneous removal of nitrate and chromate is sulfur-based autotrophic denitrification and heterotrophic denitrification; however, this technology has the disadvantage of causing of sulfur contamination (Peng et al., 2015). Previous studies have mainly focused on simultaneous denitrification and Cr(VI) reduction by anaerobic microorganisms (Miao et al., 2015; Peng et al., 2015), whereas limited information is available regarding the involvement of aerobic bacteria. Furthermore, studies concerning the bacteria that could reduce Cr(VI) and nitrate simultaneously, will promote the application of microorganisms in multiple wastewater treatment. In this study, Pseudomonas brassicacearum LZ-4 mediated denitrification at the optimal conditions and its potential for simultaneously reducing nitrate and Cr(VI) was investigated. 2. Material and methods 2.1. Bacterial strain and medium P. brassicacearum LZ-4 was isolated from wastewater samples collected near the sewage outlet of a petrochemical company in Gansu Province, China. Denitrification medium (DM) was used for the nitrate studies and contained the following components (per liter): 3.6 g KNO3, 16.88 g sodium succinate, 50 mL trace elements solution, pH 7.5 (Ren et al., 2014). Solid media were prepared from growth media by the addition of 2% agar. Luria– Bertani (LB) medium was used for activating the strain (Duan et al., 2015). 2.2. Factors influencing nitrogen removal The nitrate reduction performance of strain LZ-4 was evaluated under different culture conditions, including different carbon sources, C/N ratios, pH, and nitrate concentrations. Different carbon sources including 10 g L1 soluble starch, sucrose, glucose, sodium acetate, sodium citrate, and sodium succinate were separately used as the sole carbon sources in the DM. To examine the effect of the C/ N ratio, the ratios were adjusted to 5, 10, 15, and 20. The initial pH was adjusted to 4, 5, 6, 7, 7.5, 8, 9, 10, and 11 by the addition of 1 mol L1 HCl or 1 mol L1 NaOH. In nitrate load experiments, the initial nitrate concentrations ranged from approximately 100– 7000 mg L1 based on the N content. To evaluate the ability of the strain LZ-4 for nitrite and ammonium removal, various concentrations of nitrite and ammonium were added to the DM, instead of using NO 3 as the sole N-source. All the experiments were conducted in 150 mL DM with an inoculation dosage of 1% (v/v). Unless the single-factor was adjusted as per the experimental design, the medium was used with a constant nitrate concentration (500 mg L1), and was incubated at 37 °C, pH 7.5, C/N 10, and 160 rpm for 48 h. Samples were collected from the flasks after 48 h to determine cell growth by measuring optical density an 600 nm (OD600). In addition, the content of nitrate nitrogen (NO 3 N), ammonium nitrogen (NH 4 -N), and nitrite nitrogen (NO2 -N) were determined. All experiments were performed in triplicate. 2.3. Simultaneous Cr(VI) reduction and denitrification by strain LZ-4 Biodegradation experiments were conducted in 250 mL Erlenmeyer flasks containing 150 mL DM, by adding different concen-

trations of nitrate as the sole nitrogen source (100 mg L1, 200 mg L1, 300 mg L1, 500 mg L1, and 700 mg L1 NO 3 -N) and different concentrations of Cr(VI) (10 mg L1, 20 mg L1, 30 mg L1, and 50 mg L1). Samples were collected from the flasks after 72 h to determine the OD600, and the NO 3 and Cr(VI) concentrations. According to the reduction efficiency of strain LZ-4, the optimal carbon source, C/N, pH, and the optimal nitrate and Cr(VI) concentrations were selected to analyze the biodegradation characteristics of the strain in DM. Samples were collected from the flasks at every 12 h to determine OD600, and the NO 3 and Cr(VI) concentrations. All experiments were performed in triplicate. 2.4. Amplification of denitrification genes 2.4.1. Genome sequencing The whole genome of strain LZ-4 was sequenced using an Illumina HiSeq 2000 sequencer. Libraries were constructed using the Nextera technology and sequenced using 2  100 nucleotide paired-end strategies. All reads were pre-processed to remove low quality of artifactual bases (Criscuolo et al., 2014). After filtering, the remaining reads were assembled using SOAPdenovo (http://soap.genomics.org.cn, version 1.05). Detailed gene sequences and functions were uploaded to the NCBI database (http://www.ncbi.nlm.nih.gov/biosample/SAMN02800876). 2.4.2. Quantitative real-time PCR (qRT-PCR) analysis of denitrifying genes The strain LZ-4 was incubated in DM with 10 mg L1 Cr(VI) and 30 mg L1 Cr(VI). When the OD600 nm reached 0.6, the cells were collected for RNA extraction according to the protocol of the Bacteria RNA Extraction Kit (OMEGA, America). Cells grown in DM without Cr(VI) were used as controls. Reverse transcription was performed with PrimeScript RT reagent kit Perfect RealTime (TAKARA, Japan). The primers for the denitrification genes and 16S rRNA were designed using DNAMAN 6.0 (Table 1). QRT-PCR analysis was performed with the SYBR Green dye method using SYBRÒ Premix Ex TaqTM GC (TAKARA, Japan) in a Real-Time PCR Detection System (Bio-Rad) with the following steps: an initial step of 30 s at 95 °C, followed by 40 cycles consisting of denaturation at 95 °C for 10 s, annealing at 60 °C for 30 s, and extension at 72 °C for 15 s. Each sample was run in triplicate, and the average values were used for quantification. The relative quantification analysis of these genes was performed using the 2DDCt method. The expression of 16S rRNA was used as the internal control. 2.5. Enzymatic assays The strain LZ-4 was cultivated in DM or LB with different concentrations of Cr(VI) and nitrate for 48 h. Cells were then collected and processed into a cell-free extract (Huang et al., 2013). Nitrate reductase activity and Cr(VI) reductase activity was determined as described previously (MacGregor et al., 1974; Park et al., 2000). Protein concentrations in the crude extract were quantified by the Bradford Reagent Kit (Sangon, Shanghai, China). The specific activity (U mg1) was defined as the amount of enzyme that catalyzed the transformation of 1 lmol of NO 3 -N or 1 nmol of Cr (VI) per minute by the amount of protein in mg. 2.6. Analytical methods Spectrophotometry was used to measure the growth of the isolated bacterial strain at a wavelength of 600 nm (OD600). Ammonium, nitrite, and nitrate concentrations were determined by Nessler’s reagent spectrophotometry, N-(1-naphthalene)diaminoethane spectrophotometry, and phenol disulfonic acid methods (APHA, 1995), respectively. Cr(VI) concentration was

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Table 1 Primers used for qRT-PCR. Primers

Description of sequence (F/R)

16s nrtA narG narH narI narJ nasA napA napB nirK norB nosZ

50 -AGAGTTTGATCCTGGCTCAG-30 /50 -GGTTACCTTGTTACGACTT-30 50 -ATGAATGAAGTTCCAGCCAACCC-30 /50 -TCAGCGGCTGGCGAAGAG-30 50 -GTGAGTCATTTACTGGATCAACTG-30 /50 -TCAGTTCTCCTCGATATCGGTTG-30 50 -ATGAAAATTCGCTCACAAATCGGC-30 /50 -TCATTCCTCCCACAACTGCAC-30 50 -ATGTCTAAATGGGATCTGTTGATGTTC-30 /50 -TTAGAACTTCTGCCGCACGATTTG-30 50 -ATGAACATGCGCATTCTCAAGG-30 /50 -TTAGACATTGCCCACCTCCC-30 50 -ATGAGCCGTCAAACCACCG-30 /50 -TCACGAGACAGCGGCCAC-30 50 -ATGAGCATGACCCGCCGT-30 /50 -TCAGGCCACGCTGACCAAC-30 50 -ATGCTGCCCTTGTTTCTGCTTG-30 /50 -TCAAGGTTTCTTCGTGGCGTTG-30 50 -TTGATCGCCGTCAAGCGGGTA-30 /50 -TTGATCGCCGTCAAGCGGGTA-30 50 -ATGAGCATGGCTAATCCGCATC-30 /50 -TCAAGACGGCACCACCGC-30 50 -ATGAGCGATAAAAAAACCGAAACC-30 /50 -TCAGGCCTTTTCAACCAGCATG-30

measured by a modified 1,5-diphenyl-carbazide spectrophotometric method to which sulfamic acid was added to eliminate nitrite interference (He et al., 2015). Chemical oxygen demand (COD) was determined using the COD testing kit (Kyoritasu chemicalcheck lab, Japan). DO (dissolved oxygen), pH, and temperature were routinely measured using an oxygen electrode (DO-957, Leici, China), a pH electrode (pH400, Alalis, China), and a thermometer, respectively. 2.7. Denitrification and Cr(VI) reduction performance of the immobilized strain LZ-4 on wastewater treatment Cells of the strain LZ-4 (10 mg) were harvested and immobilized in solutions of 8% (w/v) polyvinyl alcohol (PVA) and 2% (w/ v) sodium alginate (SA) (Wu et al., 2012). The immobilized strains were added into 3 L batch member bioreactors (MBRs, 2 L of working volume) with an air supply system and a membrane module with a pore size of 0.2 lm (Fig. S1). Each batch of MBRs was operated for 48 h, and approximately 95% of the spent wastewater was replaced with fresh wastewater. The reactors were operated for 46 days at room temperature (18–23 °C) with a dissolved oxygen (DO) concentration of 4–5 mg L1. The effluent was sampled every day for contamination analysis. The synthetic medium used in the present study contained (per liter): 5000 mg glucose, 500 mg NO 3N, 10 mg Cr(VI), 300 mg MgSO4, 30 mg KH2PO4, 50 mg KHCO3, and trace element solution (Ali et al., 2015).

3. Results and discussion 3.1. Factors affecting denitrification performance and removal performance for ammonium and nitrite by strain LZ-4 The effects of different carbon sources, C/N ratios, pH, and nitrate concentrations on nitrate removal were investigated in shaking DM cultures as shown in Fig. 1. When acetate, glucose, citrate, and succinate were used as sole carbon sources, the strain LZ-4 exhibited efficient nitrate removal ability and growth, where the removal rates of 500 mg L1 NO 3 -N were 94.8%, 85.9%, 97.9%, and 99.6%, respectively (Fig. 1a). The most optimal C/N ratio for nitrate removal was 10 (Fig. 1b). Neutral, and slightly acidic and slightly alkaline environments (pH 6.0–8.0) were suitable for denitrification and cell growth (Fig. 1c). Moreover, the strain LZ-4 could thrive in 5000 mg L1 NO 3 -N, and could remove 91.3% of 1000 mg L1 NO 3 -N (Fig. 1d). Previous studies have shown that Pseudomonas stutzeri strain T1 was able to reduce 70% of 100 mg L1 NO 3 -N (Guo et al., 2013) whereas Rhodococcus sp. CPZ24 reduced 50 mg L1 NO 3 -N with a removal ratio below 67% (Zhang et al., 2011). Thus, the strain LZ-4 was more efficient in aerobic denitrification than the other denitrifiers. The strain LZ-4 can also degrade 70–84% of 100–300 mg L1 NH+4-N, and 100% of 100–

200 mg L1 NO 2 -N in 48 h (Fig. 1e, f). These characteristics expand its scope for application in ammonium and nitrite remediation. 3.2. Growth in different concentrations of Cr(VI) and nitrate The removal ability of the strain LZ-4 with different Cr(VI) and nitrate concentrations was evaluated (Fig. 2). LZ-4 can grow in 50 mg L1 Cr(VI) and use 700 mg L1 NO 3 -N as the sole nitrogen source such that the cell concentrations in all groups were in the range OD600 of 0.8–1.1. Nitrate was found to improve Cr(VI) reduction by the strain LZ-4. Ten mg L1 Cr(VI) was completely reduced in 200 (and higher) mg L1 NO 3 -N, but only 27.9% Cr(VI) was removed in 100 mg L1 NO 3 -N (Fig. 2b). However, 50 mg/L Cr(VI) showed no inhibitory effect on reduction of 100 mg L1 NO 3 -N, but negatively affected the reduction of 200 mg L1 NO 3 -N or higher concentrations (Fig. 2c). Meanwhile, little nitrite was accumulated in 100 mg L1 NO 3 -N medium, whereas greater quantities of nitrite were detected in medium containing 200 mg L1 or more nitrate, which decreased with increase in Cr(VI). The co-removal efficiencies for nitrate and Cr(VI) were calculated by averaging the nitrate removal ratio and the Cr(VI) removal ratio (Table 2). The removal ratio for both Cr(VI) and nitrate was above 80% at 1 500 mg L1 NO Cr(VI), or 200 mg L1 NO 3 -N and 10 mg L 3 -N 1 and 20 mg L Cr(VI). This indicated that the strain LZ-4 was an efficient nitrate and Cr(VI) reducing strain, which makes it a promising candidate for treating high-strength Cr(VI) wastewater. Cr(VI) reduction by microbes is found to be co-metabolic in aerobic conditions (Thatoi et al., 2014). Sufficient nitrogen sources are necessary for cell metabolism in addition to Cr(VI) reduction. Previous study showed that, nitrate treatment increased the Cr(VI) reduction rate and cell growth comparing to the non-nitrogen group (Vatsouria et al., 2005). Nitrate elevated Cr(VI) reduction of strain LZ-4, but not cell growth. This might due to the different media was used. Previous study used non-nitrogen medium for control group that has very few nitrogen which limited cell growth (Vatsouria et al., 2005). In this study, high nitrogen medium was used as control in which nitrogen was no long limiting cell growth. Only when the nitrogen source is sufficient for metabolism, Cr(VI) can be reduced and have a negative impact on denitrification, which might result from Cr(VI) toxicity in the reduction process or its competition for electron donors with nitrate (Thatoi et al., 2014). A study reported that Cr(VI) reduction was more electroncompetitive than denitrification in which the metabolism was not affected by nitrogen (He et al., 2015). 3.3. Aerobic Cr(VI) and nitrate reduction by strain LZ-4 1 The removal characteristics of 500 mg L1 NO 3 -N and 10 mg L Cr(VI) by P. brassicacearum LZ-4 in optimal DM, with sodium succinate as carbon source, pH 7.5, C/N 10, were investigated (Fig. 3). Cell growth reached the stationary phase in 60 h as the OD600

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Fig. 1. Effects of different carbon sources (a), C/N (b), pH (c) and NO 3 -N concentrations (d) on nitrate removal and cell growth of the strain LZ-4. Utilization of ammonium (e) +  and nitrite (f) under aerobic conditions. Column, removal efficiency of NO 3 -N, NH4-N or NO2 -N; dotted line, OD600 of strain LZ-4. Values are means ± SD (error bars) of three replicates.

increased to 2.78. The strain LZ-4 could remove 80.2% nitrate within 84 h with a denitrification rate of 7.09 mg L1 h1 NO 3 -N. Cr(VI) was completely reduced within 61 h and its maximum removal rate was 0.236 mg L1 h1. Nitrite, produced from nitrate reduction, was observed throughout the culture period and was removed simultaneously. These data indicate that the strain LZ-4 is capable of simultaneous nitrate and Cr(VI) reduction. The competitive inhibition and toxicity of Cr(VI) decreased the growth rate of the bacterium and its denitrification activity as described in different reports (Kourtev et al., 2009; Vatsouria

et al., 2005). The inhibitory Cr(VI) concentration for nitrate reduction was relatively low with other denitrifiers, such as 0.5, 0.5, and 3 mg L1 in Pseudomonas aeruginosa H1, Pseudomonas stutzeri H19, and Pseudomonas fluorescens var. pseudoiodinum P-11, respectively (He et al., 2015). These bacteria started denitrification only when Cr(VI) concentration was reduced, resulting in a long delay in nitrate and Cr(VI) removal. For example, P. fluorescens var. pseudoiodinum P-11 required 12 days to reduce 10 mg L1 Cr(VI) and 200 mg L1 nitrate (Konovalova et al., 2008), and Staphylococcus epidermidis L-02 took 13 days to reduce 15.6 mg L1 Cr(VI) and

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Fig. 2. Effects of different initial nitrate concentrations (100–700 mg L1) and Cr(VI) concentrations (10–50 mg L1) on the growth profile of strain LZ-4 (a), Cr(VI) reduction (b), nitrate degradation, (c) and nitrite generation (d) in 150 ml DM. Values are means ± SD (error bars) of three replicates.

Table 2 Average co-removal rate of nitrate and Cr(VI). Cr(VI)

100 mg/L NO 3 -N

200 mg/L NO 3 -N

300 mg/L NO 3 -N

500 mg/L NO 3 -N

700 mg/L NO 3 -N

5 mg/L

83.81 (±2.90a)% 63.60 (±6.47)% 65.89 (±0.60)% 66.31 (±1.44)% 60.77 (±0.94)%

97.92 (±0.18)% 97.34 (±0.92)% 92.13 (±5.83)% 67.17 (±0.05)% 36.97 (±4.97)%

87.38 (±2.27)% 86.23 (±2.56)% 37.64 (±2.21)% 37.44 (±15.45)% 23.07 (±4.85)%

85.50 (±0.78)% 81.31 (±0.84)% 30.15 (±7.53)% 26.13 (±7.27)% 19.14 (±2.48)%

60.11 (±0.30)% 59.68 (±0.19)% 19.80 (±0.86)% 30.18 (±10.09)% 34.44 (±1.37)%

10 mg/L 20 mg/L 30 mg/L 50 mg/L a

Values are presented as means ± standard deviation of triplicates.

1

44.8 mg L nitrate in the media (Vatsouria et al., 2005). Comparatively, the efficiency of strain LZ-4 in nitrate and Cr(VI) removal was three or four times greater than that of the other strains. To date, only Pseudomonas aeruginosa PCN-2 has been reported to simultaneously reduce 5 mg L1 Cr(VI) and 200 mg L1 NO 3 -N under aerobic conditions; however, its denitrification capacity was completely inhibited by 7.5 mg L1 Cr(VI) (He et al., 2015). The results demonstrated that the strain LZ-4 could reduce nitrate and Cr(VI) simultaneously without lags, under aerobic conditions.

Fig. 3. Aerobic Cr(VI) reduction and denitrification by strain LZ-4 in 150 ml DM. Values are means ± SD (error bars) of three replicates.

3.4. Effect of Cr(VI) on expression of denitrifying genes and enzyme activity The genome of strain LZ-4 contained all the genes for denitrification, and the relative sequences were uploaded to the NCBI database (Table 3 and Text S1). Real-time quantitative PCR (qRT-PCR) was used to determine the expression of the denitrification genes under different Cr(VI) concentrations. The nitrate transporter gene

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Table 3 Putative functions of the genes involved in denitrification and Cr(VI) reduction. Locus tags

Genes

Product description

Nucleotide blast identities (%)

Length of sequence (bp)

Gene bank ID

RS08580 RS19410 RS19405 RS19395 RS19400 RS08550 RS00650 RS00645 RS01735 RS01760 RS27485 RS03595 RS05680

nrtA narG narH narI narJ nasA napA napB nirK norB nosZ nfsA nemA

Nitrate transporter Nitrate reductase alpha subunit Nitrate reductase beta subunit Nitrate reductase gamma subunit Nitrate reductase beta subunit Assimilatory nitrate reductase catalytic subunit Periplasmic nitrate reductase catalytic subunit cytochrome c-type protein NapB Nitrite reductase (NO-forming) Nitric oxide reductase subunit B Nitrous-oxide reductase Nitroreductase N-ethylmaleimide reducase

91 92 94 88 90 90 90 84 91 92 91 90 89

1212 3774 1539 681 756 2718 2505 486 1680 1428 1938 570 1113

KU192977 KU192978 KU192979 KU192980 KU192981 KU192982 KU192983 KU192984 KU192985 KU192986 KU192988 – –

nrtA, anaerobic nitrate reducase gene narIJHG, aerobic nitrate reducase gene napAB, and assimilatory nitrate reductase gene nasA were upregulated by 3–20-fold after treatment with 10 mg L1 Cr (VI), whereas the nitrite reducase gene nirK, nitric oxide reductase gene norB, and the nitrous oxide reductase gene nosZ were upregulated by 67, 152, and 207.5-fold, respectively. They were also upregulated by 85–97-fold after treatment with 30 mg L1 Cr(VI) (Fig. 4a, b). These results indicate that Cr(VI) had a positive effect on the expression of the denitrifying genes, especially those

involved in nitrite reduction, nitric oxide reduction, and nitrous oxide reduction. Nitrate reductase and chromate reductase activities under Cr (VI)- and NO 3 -growth were also assayed. The nitrate reductase activities of the cell-free extract in medium with 0, 10 mg L1, and 30 mg L1 Cr(VI) were 0.128, 0.187, and 0.243 U mg1 protein, respectively (Fig. 4c). The Cr(VI) reductase activity was 0.730 U mg1 proteins after treatment with nitrate, whereas only 0.253 U mg1 protein was observed in the control (Fig. 4d). This

Fig. 4. Effects of Cr(VI) or nitrate on expression of denitrification genes and reductase activities. Relative fold expression of denitrification genes in strain LZ-4 under denitrifying conditions with 10 mg L1 Cr(VI) (a) and 30 mg L1 Cr(VI) (b) compared with the control. Nitrate reductase activity of cell-free extract of strain LZ-4 cultivated in different Cr(VI) concentrations (c); Cr(VI) reductase activity of cell-free extract in LB medium with 200 mg L1 NO 3 -N and in the absence of nitrate (d). Values are means ± SD (error bars) of three replicates.

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Fig. 5. Plausible mechanisms of Cr(VI) reduction and denitrification under aerobic (solid arrows) and anaerobic (dotted arrow) conditions. Aerobic Cr(VI) reductases, NfsA and NemA, require NAD(P)H as an electron donor. Anaerobic Cr(VI) reduction is associated with membrane bound reductases, such as cytochrome c (modified from Thatoi et al. (2014)). Nitrate reductases, Nar and Nap, can directly obtain electrons from the quinone pool. The cytochrome bc1 complex completes the oxidation of quinol and the transfer electrons to cytochrome c, which is an electron donor for the denitrification enzymes and cytochrome aa3 oxidase (modified from (Wehrfritz et al., 1993)). C = cytoplasm; P = periplasm.

demonstrates that Cr(VI) and nitrate mutually promoted the reductase activities. A previous study has reported strong correlations between the nitrate reduction rate and the transcript copy number of either nirS or narG, and chromate reduction was found to be synchronous with nitrate reduction (Han et al., 2010). However, direct interaction between Cr(VI) reduction and denitrifying gene expression has not been analyzed. Some reports suggested that specific denitrification genes were involved in chromate reduction (Han et al., 2010; Viamajala et al., 2002). Cr(VI) could react with various oxidoreductases in bacteria, including iron reductases, nitroreductases, glutathione reductases, lipoyl reductases, ferredoxin-NADP + reductases, and other metal reductases (Ahemad, 2014). Therefore, oxidoreductases in denitrification might be induced by the oxidation state of chromium. Chromate was also discovered to increase the nitrite reduction by cell lysates (Chovanec et al., 2012). A positive effect of Cr(VI) on gene expression and enzyme activities could promote denitrification and negate the Cr(VI)mediated inhibition. 3.5. Electron transport chain during aerobic denitrification and Cr(VI) reduction based on the genome The genome of strain LZ-4 revealed a number of oxidoreductases which were previously recognised to be associated with denitrification, Cr(VI) reduction, and aerobic respiration. The suggestive mechanism of electron translation in these three processes is shown in Fig. 5. P. brassicacearum LZ-4 possesses two types of nitrate reducases, the membrane protein Nar and the periplasmic protein Nap. Nar reduces nitrate anaerobically at the intramembrane space (Kraft et al., 2011), and Nap reduces nitrate

in the periplasm irrespective of oxygen (Cartron et al., 2002). Both Nar and Nap accept electrons from quinone and reduce nitrate to nitrite. NirK, a homotrimeric copper-containing enzyme, catalyzes the reduction of nitrite to nitric oxide. Cytochrome C550 (used name C551) and pseudoazurin can provide electrons to NirK and other denitrification enzymes, so that nitrite can be sequentially reduced to NO, N2O, and N2. Meanwhile, electron transfer from NADH to O2 passes via the cytochrome bc1 complex, cytochrome C, and cytochrome aa3 oxidase in the respiratory chain (Wehrfritz et al., 1993). Microbial Cr(VI) reduction occurs in both aerobic and anaerobic reduction systems. Anaerobic Cr(VI) reduction by bacteria occurs in the periplasmic space by membrane bound reductases. Studies have indicated that cytochrome C is involved in the electron transfer to chromate under anaerobic conditions (Wang et al., 1991). The competitive inhibition between denitrification and Cr(VI) reduction has been pervasively observed in anaerobic bacteria (Kourtev et al., 2009). They might compete for the electron from cytochrome C in periplasmic space. Aerobic Cr(VI) reduction is generally associated with soluble proteins present in the cytoplasm. NAD(P)H serves as the electron donor for Cr(VI) reduction by these soluble enzymes. Different chromate reductases such as ChrR, YieF, NemA, NfsA, and LpDH, have been identified from bacterial sources (Thatoi et al., 2014). The genome of strain LZ-4 revealed the chromate reductase genes nemA and nfsA with identification rates of 89% and 90% (Table 3). NfsA, a major oxygen-insensitive nitroreductase, is a flavoprotein that can reduce Cr(VI) to Cr(III). Cr(VI) reduction by NsfA involves single-electron transfer, giving rise to reactive oxygen species (ROS) and Cr(V) as intermediates (Ackerley et al., 2004). Nethylmaleimide reductase (NemA) is a member of the ‘‘old yellow

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enzyme” family of flavoproteins. It can catalyze chromate reduction through the addition of one or two electrons from the cofactors, NADH or NADPH (Robins et al., 2013). Aerobic denitrification and Cr(VI) reduction use quinone/cytochrome c and NAD(P)H as electron donors, respectively, which might decrease the competition for electrons. Moreover, these two processes occur in separate spaces in the bacteria. Theoretically, simultaneous Cr(VI) reduction and denitrification might be easier when conducted by bacteria aerobically rather than anaerobically. A soil experiment discovered that the inhibitory effect of Cr(VI) on denitrification is more serious under anaerobic conditions than under aerobic conditions (Kourtev et al., 2009). (Chovanec et al., 2012) found that the growth of a Nar-employing bacterium, Geobacter metallireducens on nitrate was completely inhibited by Cr(VI), whereas the growth of a Nap-employing bacterium, Sulfurospirillum barnesii on nitrate was not affected by Cr(VI), and Cr (VI) was reduced to Cr(III) in this case. Some reports have also recorded that aerobic Cr(VI) reduction was not inhibited by NO 3 (Ishibashi et al., 1990). Under aerobic conditions, reduction of 100 mg L1 nitrate by the strain LZ-4 was not inhibited even at 50 mg L1 Cr(VI) (Fig. 2), indicating that the electron competition between the denitrification and Cr(VI) reduction processes was indirect. In addition, the electron competition depended on nitrogen concentrations. Denitrification of 100 mg/L NO 3 -N was preferentially conducted to Cr(VI) reduction, and Cr(VI) reduction was only performed when the nitrogen concentration was above 200 mg/L (Fig. 2). Thus, there must be a special mechanism to regulate the electron transfer between these two processes in order to achieve simultaneous Cr(VI) and nitrate reduction under aerobic conditions.

3.6. Immobilized strain LZ-4 continuously and simultaneously degrades nitrate and chromate in MBR

Fig. 6. Cr(VI), nitrate, and COD removal from wastewater by the immobilized strain LZ-4 in a membrane bioreactor. (a) Degradation characteristics of Cr(VI), NO 3 -N, and COD in the first batch reaction, (b) pH, DO, and temperature were monitored throughout the process. The ability of removing COD (c), nitrate, (d) and Cr(VI) (e) for strain LZ-4 in MBR.

Previous results have shown that the strain LZ-4 can reduce high concentrations of nitrate and Cr(VI). A laboratory scale batch-MBR was set up to analyze its implementation potential in artificial industrial wastewater, including 500 mg L1 NO 3 -N, 10 mg L1 Cr(VI), and 5000 mg L1 COD. The operational conditions in this process were monitored as shown in Fig. 6b. Taking the first batch as an example, the degradation characteristics of COD, nitrate and hexavalent chromium in the bioreactor were analyzed by function fitting (Fig. 6a). COD was decreased rapidly from 5000 mg L1 to 25 mg L1 in 48 h with a fitting curve of y = 4629.94ex/2.94 + 369.76 (R2 = 0.9974). COD removal was mainly focused in the 12 h at the beginning with the maximum COD removal ratio of 1574 mg L1 h1. Meanwhile, the nitrate degradation curve was fitted as a linear function y = 9.7706x + 501.47 (R2 = 0.993), and the maximum removal ratio was 9.77 mg L1 h1 NO 3 -N. Cr(VI), initially at 10 mg/L, declined to 2 mg/L after 48 h of operation, and its degrading curve was fitted as y = 10.153  0.019x  0.003x2 (R2 = 0.9994), resulting in a maximum Cr(VI) removal ratio of 0.277 mg L1 h1 at the end of the fermentation period. PVA (polyvinyl alcohol) is a preferable immobilization filler because its porous structure allows the substrate and oxygen to diffuse into the internal pores and facilitates biodegradation (Cheng et al., 2012). Upon immobilization, the strain LZ-4 was able to degrade over 95% nitrate, 80% Cr(VI), and 96% COD continuously, without any decrease in its properties over 46 days (Fig. 6c–e). These results indicated that the immobilized strain LZ-4 could bring about high efficiency reduction of Cr(VI) and nitrate in an MBR bioreactor over a long time.

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Simultaneous reduction of Cr(VI) and nitrate generally incorporates sulfur oxidizing denitrifiers, heterotrophic denitrifiers, and Cr (VI) reducing bacteria (Peng et al., 2015). Denitrifiers have a lower tolerance to Cr(VI), and so Cr(VI) reducing bacteria can defeat other species and have negative effect on microbial abundance (Li et al., 2016; Peng et al., 2015), resulting in poor removal efficiencies for nitrate and Cr(VI) in these systems. The strain LZ-4 could survive under high concentrations of Cr(VI), and could reduce nitrate and Cr(VI) simultaneously. It could improve the degradation of cocontaminants and resolve the efficiency loss problems resulting from interspecific competition in wastewater treatment. An excellent strain needs to remove contamination chronically under industrial applications (Yang et al., 2011). This study suggests that the strain LZ-4 is applicable and promising for high-strength wastewater treatment. 4. Conclusions Pseudomonas brassicacearum LZ-4 can simultaneously reduce nitrate and Cr(VI) at a rate of 7.09 mg L1 h1 and 0.236 mg L1 h1, respectively, at the optimal concentrations 500 mg L1 N and 10 mg L1 Cr(VI). Nitrate positively affects Cr(VI) reduction, and Cr(VI) positively affects the expression of denitrifying genes and nitrate reductase activity. The genome of strain LZ-4 contains denitrifying and chromate reductase genes, implying its capability to reduce Cr(VI) and nitrate simultaneously under aerobic conditions. The strain LZ-4 was capable of persistent and stable aerobic denitrification and Cr(VI) reduction as demonstrated in the membrane bioreactors. Acknowledgements The present study was supported by the National Natural Science Foundation grants 31470224 and 31400430, MOST international cooperation grant 2014DFA91340, and Gansu Provincial International Cooperation grant 1504WKCA089-2. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2016.09. 037. References Ackerley, D., Gonzalez, C., Keyhan, M., Blake, R., Matin, A., 2004. Mechanism of chromate reduction by the Escherichia coli protein, NfsA, and the role of different chromate reductases in minimizing oxidative stress during chromate reduction. Environ. Microbiol. 6 (8), 851–860. Ahemad, M., 2014. Bacterial mechanisms for Cr(VI) resistance and reduction: an overview and recent advances. Folia Microbiol. (Praha) 59 (4), 321–332. Ali, M., Oshiki, M., Rathnayake, L., Ishii, S., Satoh, H., Okabe, S., 2015. Rapid and successful start-up of anammox process by immobilizing the minimal quantity of biomass in PVA-SA gel beads. Water Res. 79, 147–157. APHA, 1995. Standard Methods for the Examination of Water and Wastewater, nineteenth ed. American Public Health Association, Washington, DC. Cartron, M.L., Roldán, M.D., Ferguson, S.J., Berks, B.C., Richardson, D.J., 2002. Identification of two domains and distal histidine ligands to the four haems in the bacterial c-type cytochrome NapC; the prototype connector between quinol/quinone and periplasmic oxido-reductases. Biochem. J. 368 (2), 425– 432. Chen, Y., Gu, G., 2005. Preliminary studies on continuous chromium(VI) biological removal from wastewater by anaerobic-aerobic activated sludge process. Bioresour. Technol. 96 (15), 1713–1721. Cheng, Y., Lin, H., Chen, Z., Megharaj, M., Naidu, R., 2012. Biodegradation of crystal violet using Burkholderia vietnamiensis C09V immobilized on PVA-sodium alginate-kaolin gel beads. Ecotoxicol. Environ. Saf. 83, 108–114. Chovanec, P., Sparacino-Watkins, C., Zhang, N., Basu, P., Stolz, J.F., 2012. Microbial reduction of chromate in the presence of nitrate by three nitrate respiring organisms. Front Microbiol. 3, 416. Criscuolo, A., de la Blanchardière, A., Coeuret, S., Passet, V., Saguet-Rysanek, V., Vergnaud, M., Verdon, R., Leclercq, A., Lecuit, M., Brisse, S., 2014. Draft genome

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