Isolation and characterization of a chlorate-reducing bacterium Ochrobactrum anthropi XM-1

Journal of Hazardous Materials 380 (2019) 120873

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Isolation and characterization of a chlorate-reducing bacterium Ochrobactrum anthropi XM-1

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Han-Wen Chena, Meng Xua, Xi-Wen Maa, Zhong-Hua Tonga,b, , Dong-Feng Liua a b

CAS Key Laboratory of Urban Pollutant Conversion, Department of Applied Chemistry, University of Science & Technology of China, Hefei, 230026, China Anhui Province Key Laboratory of Polar Environment and Global Change, University of Science & Technology of China, Hefei, 230026, China

A R T I C LE I N FO

A B S T R A C T

Editor: E.D. Lyderatos

A Gram-negative chlorate-reducing bacterial strain XM-1 was isolated. The 16S rRNA gene sequence identified the isolate as Ochrobactrum anthropi XM-1, which was the first strain of genus Ochrobactrum reported having the ability to reduce chlorate. The optimum growth temperature and pH for strain XM-1 to reduce chlorate was found to be 30 °C and 5.0–7.5, respectively, under anaerobic condition. Strain XM-1 could tolerate high chlorate concentration (200 mM), and utilize a variety of carbohydrates (glucose, L-arabinose, D-fructose, sucrose), glycerin and sodium citrate as electron donors. In addition, oxygen and nitrate could be used as electron acceptors, but perchlorate could not be reduced. Enzyme activities related to chlorate reducing were characterized in cell extracts. Activities of chlorate reductase and chlorite dismutase could be detected in XM-1 cells grown under both aerobic and anaerobic conditions, implying the two enzymes were constitutively expressed. This work suggests a high potential of applying Ochrobactrum anthropi XM-1 for remediation of chlorate contamination.

Keywords: Chlorate-reducing bacteria Ochrobactrum anthropic XM-1 Chlorate reduction Chlorate reductase Chlorite dismutase

1. Introduction Sodium chlorate has been used for applications in bleaching industry, herbicide production, and as a chemical intermediate in the manufacture of sodium perchlorate. The global production of commercial sodium chlorate has reached 3.8 million tons in 2016 and is expected to reach 4.7 million tons by 2022 (Expert Market Research, 2019). Disinfection of drinking water with ClO2 will also produce chlorate (Rougé et al., 2018). Chlorate has shown toxic effect on plants because of its strong oxidative property and indirect toxicity due to chlorite (Borges et al., 2004). Chlorate can induce nephrotoxicity through redox imbalance, leading to DNA and membrane damage, metabolic changes and enzyme dysfunction (Ali et al., 2018). Dose dependent effects of chlorate on DNA damage and DNA-protein crosslinking have been observed in rat intestine (Ali et al., 2017). The toxicity of chlorite, a reduced product of chlorate, has also been reported inducing oxidative stress in human erythrocytes (Ali and Mahmood, 2017). Chlorate could be removed by physical (Srinivasan and Viraraghavan, 2009; Youngblut et al., 2016), chemical (Jung et al., 2017) and biological approaches (Jiang et al., 2009; Clark et al., 2016; Weelink et al., 2008). The most common method for removing chlorate

in water is biological method due to its low expense. It has been widely used to repair chlorate-contaminated water and soil taking advantages of the metabolic activity of microorganisms (Nor et al., 2011; Jiang et al., 2017). In an anaerobic environment, chlorate would be used by microorganisms as an electron acceptor and reduced to form harmless chloride (van Wijk et al., 1998). Biological method can be used independently to treat chlorate-containing wastewater, or be combined with some physical techniques to process brine water with high chlorate concentration (Carlstrom et al., 2015, 2013). In recent years, many dissimilatory perchlorate-reducing bacteria (DPRB) and dissimilatory chlorate-reducing bacteria (DCRB) have been isolated and characterized. Microorganisms with these characteristics mainly belonged to phylum Proteobacteria, including four different classes (the Alpha-, Beta-, Gamma- and Epsilonproteobacteria) (Melnyk and Coates, 2015). Among them, Pseudomonas stutzeri PDA and PDB are two chlorate respiration isolates, and they could not utilize perchlorate as an electron acceptor (Logan et al., 2001). Pseudomonas chloritidismutans AW-1T could use chlorate or oxygen as an electron acceptor but not nitrate, perchlorate or bromate (Wolterink, 2002). Recently, chlorate-reducing activity in a denitrifying haloarchaeon Haloferax mediterranei was characterized (Martínez-Espinosa et al., 2015). Degradation of benzene by Alicycliphilus denitrificans strain BC was

⁎ Corresponding author at: CAS Key Laboratory of Urban Pollutant Conversion, Department of Applied Chemistry, University of Science & Technology of China, Hefei, 230026, China. E-mail address: [email protected] (Z.-H. Tong).

https://doi.org/10.1016/j.jhazmat.2019.120873 Received 17 February 2019; Received in revised form 4 July 2019; Accepted 5 July 2019 Available online 12 July 2019 0304-3894/ © 2019 Elsevier B.V. All rights reserved.

Journal of Hazardous Materials 380 (2019) 120873

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demonstrated under anoxic condition with chlorate as the electron acceptor (Weelink et al., 2008). The unique metabolic abilities of dissimilatory (per)chlorate-reducing bacteria, along with other aspects of metabolism, could be used for xenobiotic bioremediation (Peng et al., 2017; Wang and Coates, 2017; Bruce et al., 1999). Therefore, more chlorate-reducing bacteria are expected to be discovered for remediation of chlorate contamination. The main objective of this study was to find out chlorate-reducing bacteria with novel physiological properties. In this study, a Gram-negative, rod-shaped, facultatively anaerobic, chlorate-reducing bacterium XM-1 was identified from an anaerobic chlorate-reducing bioreactor. The optimum growth condition was determined. With 10 mM glucose as substrate, chlorate reducing activities were studied at different chlorate concentration. The activities of enzymes (chlorate reductase and chlorite dismutase) related to chlorate reducing were characterized with cell extracts grown aerobically and anaerobically. This paper increases our knowledge to the metabolic versatility of genus Ochrobactrum.

reducing and other related bacteria deposited in GenBank. The tree was built using the program MEGA6 with Kimura 2-parameter method and bootstrapping with 1000 repetitions. A sensitive recA gene-based multi-primer PCR was applied in order to complement 16S rRNA gene sequencing result. Genetically closely related Ochrobactrum anthropi, Ochrobactrum intermedium and Brucella spp. could be differentiated using the assay based on sequence variations in the recA gene (Scholz et al., 2008). Three primer pairs, O. anthropi specific Anth-f and Anth-r (544 bp amplicon), O. intermedium specific Inter-f and Inter-r (402 bp amplicon) and Brucella spp. specific Bruc-f and Bruc-r (167 bp amplicon), were used to amplify recA gene in strain XM-1 as described previously (Wozniak-Karczewska et al., 2018). PCR amplification was performed separately for each primer pair using an initial denaturation at 95 °C for 5 min, followed by 30 cycles of 94 °C for 30 s, 62 °C for 40 s, 72 °C for 60 s, and a final extension at 72 °C for 7 min. The PCR products were analyzed on a 1% agarose gel stained with ethidium bromide.

2. Materials and methods

2.3. Chlorate reducing ability

2.1. Isolation of the chlorate-reducing bacterium

Strain XM-1 was grown overnight to mid-log phase in LB broth and cells were harvested by centrifugation (6500 rpm, 5 min, 4 °C), washed with MM medium and resuspended in 10 mL MM medium. The concentrated cultures were injected into sealed serum bottles, each containing 50 mL of anaerobic MM medium with chlorate at different concentration (10 mM, 20 mM, 30 mM, 50 mM, 100 mM, 200 mM and 500 mM, respectively) and glucose (10 mM) as substrates, to a final cell density of 0.1 at OD600 and incubated with shaking at 30 °C. Samples were taken periodically to determine bacterial growth curve and concentration of chlorate. All analyses were carried out in two replicates. The following first order rate equation was used to fit chlorate reduction rate constants (kd) for different chlorate concentration (Ghosh et al., 2011):

A sludge sample taken from a wastewater treatment plant (Hefei, China) was used for the enrichment of chlorate reducing bacteria. The head space of a 100-mL serum bottle containing 50 mL mineral medium (MM medium) was purged with nitrogen gas and sealed with butyl rubber stopper. The MM medium was prepared as described in previous report (Suzuki and Giovannoni, 1996), containing (g/L): NH4Cl, 0.25; KCl, 0.1; NaH2PO4, 0.6; NaHCO3, 2.5; 10 mM chlorate and acetate was added as an electron acceptor and as an electron donor, respectively. Stock solutions of vitamins and trace metals were added at 10 mL/L respectively. The vitamin stock contained (mg/L): biotin, 2; folic acid, 2; pyridoxine HCl, 10; riboflavin, 5; thiamine, 5; nicotinic acid, 5; pantothenic acid, 5; vitamin B12, 0.1; p-aminobenzoic acid, 5; thioctic acid, 5. The trace metal stock contained (g/L): nitrilotriacetic acid, 1.5; MgSO4, 3.0; MnSO4⋅H2O, 0.5; NaCl, 1.0; FeSO4⋅7H2O, 0.1; CaCl2⋅2H2O, 0.1; CoCl2⋅6H2O, 0.1; ZnCl, 0.13; CuSO4, 0.01; AlK(SO4)2⋅12H2O, 0.01; H3BO2, 0.01; Na2MoO4, 0.025; NiCl2⋅6H2O, 0.024; Na2WO4⋅2H2O, 0.025. Approximately 5 g sludge sample was added to the bottle in an anaerobic workstation (Electrotek AW400SG, UK). The bottle was incubated at 30 °C with continuous shaking at 200 rpm for 10 days. Then, 10% (v/v) of the primary enrichment was transferred to fresh MM medium. The transfer was repeated at least 3 times until the culture in the serum bottle was turbid. Pure bacterial strains were obtained by streaking several times on LB agar plates. The chlorate-reducing abilities of the obtained bacteria were studied using chlorate (10 mM) and acetate/glucose (10 mM) as substrates. Abiotic controls (no inoculum) were prepared at the same time. A chlorate-reducing pure culture of strain XM-1 was obtained.

dC = −kd C dt

(1)

where C0 is the initial concentration of chlorate, C is the concentration at time t. The chlorate reduction rate constant kd was estimated using the linearized regression model as shown below:

ln

C = −kd t C0

(2)

The ability of strain XM-1 to reduce chlorate was also studied in the influent of a wastewater treatment plant. The influent was filtered to remove large particles. Bacterial cells of XM-1 were inoculated into sealed serum bottles containing 50 mL influent and 1.5 mM chlorate. Samples were taken at specific time to monitor chlorate reduction. 2.4. Physiological characterization of strain XM-1

2.2. Identification of strain XM-1 by 16S rRNA gene analysis and recA gene-based multi-primer PCR

Micrograph and size of XM-1 cells were characterized using scanning electron microscopy. Gram staining property was observed with a microscope (Leica ICC50 W, Japan). The strain XM-1 has been deposited at China Center for Type Culture Collection under the Budapest Treaty with accession Number CCTCC M2018048. Optimal growth pH and temperature were studied using glucose (10 mM) as the electron donor and chlorate (10 mM) as the electron acceptor. The temperatures tested were 25 °C, 30 °C, and 37 °C. To prepare culture medium at different pH, NaH2PO4 and NaHCO3 in MM medium was replaced with 100 mM phosphate buffer and the pH was adjusted to the desired pH (4.0, 4.5, 5.0, 6.0, 6.5, 7.0, 7.5, 8.0, 9.0) using HCl or NaOH. All analyses were carried out on two replicates. The ability of XM-1 to use a variety of electron acceptors and electron donors was examined. With chlorate (10 mM) as the electron acceptor, growth of the bacterium was determined using 10 mM glucose, arabinose, fructose, glycerin, sucrose, ethanol, acetate,

Genomic DNA of strain XM-1 was extracted using DNA extraction kit (Sangon Biotech, Shanghai, China) according to the manufacturer’s instructions. The 16S rRNA gene was amplified by PCR using primer set 27 F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTT GTTACGACTT-3′) as described by Suzuki and Giovannoni (1996). The PCR products were purified and ligated into the pMD19-T vector (TaKaRa, Japan) and transformed into E. coli cells (TaKaRa, Japan). Positive clone was chosen and partial 16S rRNA gene sequence was determined (Sangon Biotech, Shanghai, China). The 16S rRNA gene sequence was identified using BLAST against GenBank (Nucleotide BLAST at NCBI) and deposited in GenBank under the accession number MK284516. A neighbor-joining phylogenetic tree was constructed using the 16S rRNA gene sequence of XM-1 along with sequences of chlorate2

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enzyme activity. The activity was normalized to the total protein content and expressed as mmol O2 min−1 mg protein−1.

propionate, butyrate, lactate or citrate (supplied as sodium salts) as the electron donor. With glucose (10 mM) as the electron donor, growth of the bacterium was determined using 10 mM (supplied as sodium salts) chlorate, perchlorate, nitrate, sulfate, thiosulfate, or oxygen as the electron donor. All analyses were carried out in two replicates.

2.7. Analytical methods Chlorate concentration was measured using a Dionex ICS-1100 ion chromatograph equipped with a Duibex IonPac™ AS 20 column (4 by 250 mm) (Thermo Scientific). Gradient elution was performed at a flow rate 1.0 mL/min with 6.0 mM KOH for 0–18 min, then 40.0 mM KOH for 18–30 min, and finally 6.0 mM KOH for 30–34 min. Glucose concentration was measured using HPLC (Agilent 1260) with a differential refractive index detector and an Agilent Hi-Plex H column with a mobile phase of 5 mM H2SO4 at a flow rate of 0.95 ml/ min. Acetate concentration was determined using gas chromatography (Agilent 6890 N) as previously described (Geng et al., 2018).

2.5. Preparation of cell extracts Cell extracts of strain XM-1, grown in anaerobic MM medium with glucose as electron donor and chlorate as electron acceptor, were prepared anaerobically as previously described by Xu et al (Xu et al., 2004). Cell extracts of XM-1 grown with oxygen and/or chlorate as the electron acceptors were prepared under aerobic condition. Whole cells were harvested by centrifugation at 10,000 rpm for 10 min at 4 °C, washed and resuspended with 15 mM phosphate buffer (pH 7.2). Cells were disrupted with an ultrasonic cell disruptor (Ningbo Scientz Biotechnology Co., China) on ice for 60 cycles of 4 s pulse with 5 s intervals at a power output of 200 W. The cell homogenate was centrifuged for 10 min at 13,000 rpm at room temperature. The supernatant was recovered and stored at 4 °C before enzyme activity measurements. The protein contents of the cell free extract fraction were determined by Lowry method (Lowry et al., 1951) with bovine serum albumin as the standard.

3. Results and discussion 3.1. Enrichment and Identification of chlorate-reducing bacteria After 10 days incubation, 10% of the primary enrichemnt was transferred into fresh MM medium. The transfer was repeated until good growth was observed. Six bacterial strains were obtained by streaking several times on LB agar plates. Sequence analysis of their 16S rRNA genes using NCBI BLAST algorithm revealed high identity to the gene sequence of Pseudomonas stutzeri DSM 4166 (99.8%), Morganella morganii strain W1 (99.8%), Citrobacter freundii strain UMH19 (99.9%), Pseudochrobactrum saccharolyticum (99.9%), Klebsiella michiganensis strain AR375 (100%) and Ochrobactrum sp. LX3 (100%), respectively. However, only two of the strains demonstrated the ability to reduce chlorate using acetate/glucose as electron donor. One isolate, designated as strain XM-1, was identified as belonging to the genus Ochrobactrum, whose ability to reduce chlorate has never been reported. Another isolate, designated as XM-2, is closely related to species Pseudomonas stutzeri, whose chlorate-reducing ability has been reported previously (Clark et al., 2016; Logan et al., 2001). Strain XM-1 is a Gram-negative and rod-shaped bacterium with a dimention of ˜0.30.5 × 1–2 μm (Fig. 1a). The strain forms circular, non-pigmented and mucoidy colonies on LB agar plates (Fig. 1b). Previous studis have shown that Ochrobactrum and Brucella are closely related genera of the family Brucellaceae (Velasco et al., 1998), and they are difficult to be differentiated by 16S rRNA gene analysis and biochemical assay systems (Vila et al., 2016). To solve this problem, a recA gene-based multi-primer PCR was developed by Scholz et al. (2008) using three primer pairs specific for O. anthropi, O. intermedium and Brucella spp. respectively, which allowed the identification of isolated strains at species level. Therefore, stain XM-1 was further identified by PCR amplification with each primer pair separately. The PCR products were observed only in the reaction with O. anthropispecific primer set (Supplementary information, Fig. S2), and matched the size of amplicons described by Scholz et al. (2008). No PCR

2.6. Enzyme activity measurements Chlorate reductase (Clr) activity was determined spectrophotometrically as described by Lindqvist et al (Lindqvist et al., 2012). The reaction mixture (3 mL final volume) contained 15 mM phosphate buffer solution, 5 mM methyl viologen (MV) and 5 μL appropriate amount of cell extract. Freshly prepared 0.2 M dithionite solution (2 μL) was added into the reaction mixture to reduce MV to reach an absorbance of 1.5, and then 5 μLof 1 M sodium chlorate was added to start the reaction. The oxidation of reduced MV by chlorate was monitored by recording the optical density of the reaction mixture at 578 nm and 30 °C. Controls without electron acceptor or without cell extract were included. The enzyme activity was calculated from the linear decrease in absorbance at 578 nm using an extinction coefficient of 1.3 × 104 M−1 cm−1 (Thorneley, 1974), normalized to the total protein content and expressed as μmol oxidized MV min−1 mg protein−1. Chlorite dismutase (Cld) activity was determined by monitoring oxygen evolution using a portable DO/pH meter (HQ 40 d, Hach Co., USA) in a 55-mL glass reactor (Supplementary information, Fig. S1). The glass reactor was filled with about 54 mL reaction solution containing 0.25 mM sodium chlorite and 15 mM phosphate buffer (pH 7.2), and deoxygenated until dissolved oxygen (DO) reading approached zero. The glass reactor was then sealed with a rubber stopper and aluminum cap, and placed in a 30 °C water bath. After temperature stabilization, the reaction was initiated by adding 1 mL of 3000 mg L−1 cell extract (prepare fresh) into the reaction mixture using a syringe. DO measured and the initial linear part was used to calculate the rate of

Fig. 1. SEM image of strain XM-1 cells (a) and colony morphology of strain XM-1 on LB plates (b). 3

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Fig. 2. Phylogenetic tree of partical 16S rRNA gene sequences, showing the relationships between strain XM-1 and closely related species. Bootstrap confidence levels (replicate 1000 times) greater than 50% are indicated at the nodes. GeneBank accession numbers are indicated. The bar indicates 5% divergence.

and the remaining 42.5% might convert to some intermediate metabolites.

amplification occurred with primer sets targeting O. intermedium and Brucella spp. Based on these results, strain XM-1 could be attributed to O. anthropic. A rooted dendrogram was constructed to show the phylogenetic position of strain XM-1 among closely related species based on the partial 16S rRNA gene sequences (Fig. 2). The ability of XM-1 to reduce chlorate was confirmed by growing the bacteria in MM medium with glucose as the sole electron donor and chlorate as the electron acceptor. Substrate utilization and chlorate reduction over time were observed with increases in chloride concentration and bacterial biomass (Fig. 3), indicating that the XM-1 could utilize glucose as electron donor during growth and chlorate was reduced to chloride. As shown in Fig. 3, glucose concentration decreased 5.17 mM within 240 h, resulted in the reduction of 9.50 mM chlorate and the formation of 9.45 mM chloride. No reduction of chlorate occurred in the abiotic control. The amount in chlorate reduction is highly consistent with the amount of chloride formation, suggesting the reduced chlorate was eventually converted to chloride. Theoretically, complete conversion of 1 mol of glucose could reduce 4 mol of chlorate: 4 ClO3− + C6H12O6 → 4 Cl− + 6CO2 + 6H2O. Combining with the concentration changes of chlorate and glucose, we would therefore postulate that about 45.9% of the metabolized glucose was used to reduce chlorate. Meanwhile, results show that the concentration of ammonium nitrogen (NH4+-N) was reduced by 0.721 mM (Supplementary information, Table S1). If all NH4+-N can be transformed to protein (C5H7NO2 as formula) (Zhang et al., 2009) and finally to biomass, 11.6% of the glucose would be used for biomass increase

3.2. Physiological characterization of strain XM-1 The effects of temperature, pH, electron donors and electron acceptors on bacterial growth were investigated in50 ml MM medium. Temperature affects the activity of enzymes in microorganisms, which in turn affects the electron transport process in chlorate-reducing bacteria (Zhu et al., 2016). Chlorate-reducing bacteria have different optimum growth temperatures and most of them have been reported to be around 30 °C (Weelink et al., 2008; Wolterink, 2002; Clark et al., 2014). A few chlorate-reducing bacteria have higher optimum temperature for growth, such as Sedimenticola NSS, which has an optimum temperature of 37–42 °C (Carlstrom et al., 2013). The strain XM-1 was cultured at three temperature. Results show that bacteria grew well at 25 °C and 30 °C, while growth at 37 °C was significantly inhibited (Fig. 4). Moreover the growth at 30 °C was better than at 25 °C, so the optimum growth temperature for XM-1 was 30 °C which is same as that for most chlorate reducing bacteria. Generally, pH is an important factor affecting bacterial growth and physiological functions. Degradation abilities of Ochrobactrum sp. have been demonstrated at a neutral pH in several studies (Talwar et al., 2014; Liu et al., 2017). Different optimum pH range has also been reported for the genus Ochrobactrum. Bacterium Ochrobactrum sp. QZ2 was able to reduce nitrite anaerobically using sulfide with an optimum

Fig. 3. Growth curve and chlorate reduction by strain XM-1 on 10 mM glucose with 10 mM chlorate as the electron acceptor. Concentration changes of chlorate and glucose in abiotic controls were also monitored.

Fig. 4. Effects of temperature on the growth of strain XM-1. 4

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Fig. 5. Effect of pH on the growth of strain XM-1 (a) and chlorate reduction (b). Fig. 6. Growth of strain XM-1 with different electron donors (a) and electron acceptors (b).

growth pH at 6.5–7.0 (Mahmood et al., 2009). Ochrobactrum sp. strain CSCr-3 could reduce Cr(VI) efficiently from pH 7 to 11 with an optimum at pH 10 (He et al., 2009). These results indicate that genus Ochrobactrum have a wide range of optimum pH. By measuring bacterial growth in the studied pH range, it was found that growth of strain XM-1 occurred at a pH range from 4.5 to 8.0 with a better growth at pH 5.0 to 7.5 (Fig. 5a), indicating it is more resistant to acidic environments compared with previously isolated chlorate-reducing bacteria which grew optimally in the vicinity of neutral pH (Weelink et al., 2008; Carlstrom et al., 2015, 2013). Meanwhile, XM-1 completely reduced chlorate at pH 5.0 to 8.0, but only reduced 74% of chlorate at pH 4.5 (Fig. 5b). These results show that XM-1 can tolerate a wide range of pH, and its ability to withstand acidic conditions is significantly stronger than previously isolated chlorate-reducing bacteria. Most chlorate-reducing bacteria can utilize a wide variety of organic and inorganic electron donors (Zhu et al., 2016; Xu et al., 2015). Acetate were usually utilized as a single organic electron donor. To investigate the range of energy source for XM-1, bacteria cells were clutivated in MM medium containing 10 mM of electron donor for bacterial reproduction. As shown in Fig. 6a, glucose was an excellent electron donor for chlorate reduction. XM-1 could also use many different electron donors, including arabinose, fructose, sucrose and glycerol. However, it could not utilize acetate, propionate butyrate and lactate for chlorate reduction. Fig. 6b shows strain XM-1 could grow with oxygen, chlorate and nitrate as electron acceptors, but not perchlorate, sulfate or thiosulfate. These results shared some similarity in metabolic characters of previously isolated species of genus Ochrobactrum (Zuo et al., 2008; Cheng et al., 2009, 2010), such as chromium (VI) reduction and exoelectrogenic ability. When strain XM-1 was grown in MM medium in the presence of both oxygen and chlorate, only oxygen was used as the electron acceptor, which is consistent with the charateristic of chlorate-respiring bacterium Pseudomonas sp. PDA (Xu et al., 2004). The ability of strain XM-1 to reduce different concentration of chlorate was studied. XM-1 can grow with 10 to 200 mM chlorate as

Fig. 7. Growth (a) and reduction of chlorate (b) at different chlorate concentrations.

5

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(Bardiya and Bae, 2011). However, an exception to this rule was found in 2008 for strains of the genus Arthrobacter, which was able to reduce perchlorate grown under aerobic condition (Shete et al., 2008). The Cld activities of Ochrobactrum anthropic XM-1 were examined for the cells grown both aerobically and anaerobically. The enzyme activity in cells grown anaerobically was measured to be 0.47 μmol O2 min−1 mg−1 (Table 1), which is much lower than that in previous studies (Lindqvist et al., 2012; Chaudhuri et al., 2002). The low enzyme activity could be a result of low enzyme concentration. Mehboob et al (Mehboob et al., 2009) have reported that Cld could be inactivated by chlorite when the enzyme concentration was insufficient in the assay mixture. Table 1 also shows that Cld activity in anaerobically grown cells was two times lower than that in cells grown aerobically. Previous study showed that Clr and Cld activities were higher in cells grown anaerobically and the presence of chlorate had no inducing effect on either of the two enzymes under aerobic conditions (Lindqvist et al., 2012). The Clr activity in strain XM-1 showed similar results. However, difference was found for Cld activity in XM-1 which could be ascribed to the extremely low Cld activity grown anaerobically. The highest kd for chlorate reduction in Fig. 3 was calculated to be 0.029 h−1, while the highest kd for chloride formation was 0.0094 h−1. These results are highly consistent with the enzyme activities.

Table 1 Enzyme activities in the cell extacts of XM-1 cultures (average of duplicate). Activities

ClO3− grown

O2 grown

O2+ ClO3− grown

Clr (μmol MV min−1 mg−1) Cld (μmol O2 min−1 mg−1)

2.31 ± 0.01 0.47 ± 0.05

1.08 ± 0.13 1.13 ± 0.05

0.83 ± 0.15 1.00 ± 0.09

electron acceptor, and the maximum growth occurred with 50 mM and 100 mM chlorate (Fig. 7a). When the concentration exceeded 200 mM, no growth was observed. Consistent with the growth of bacteria cells, strain XM-1 can reduce chlorate at concentrations up to 200 mM (Fig. 7b). As shown in table S2 (Supplementary information), the highest reduction rate constant (kd) was 0.039 h−1 for initial chlorate concentration at 20 mM, which is about 1.7 time of that with 10 and 30 mM chlorate. The Kd decreased from 0.0061 to 0.002 h−1 with the increase in chlorate concentration from 50 to 200 mM. Extremely low Kd was obtained with 500 mM chlorate. These results indicate that high concentration of chlorate is toxic to bacteria (van Wijk et al., 1998; Feretti et al., 2008), and the growth of XM-1 cell was almost completely inhibited by 500 mM of chlorate. Previous studies have shown that most dissimilatory (per)chlorate-reducing bacteria can not grow in salinities greater than 2% (Coates and Achenbach, 2004). The high concentration of chlorate may also contributes to the medium salinity that strain XM-1 could not tolerate. However, the tolerance of XM-1 for chlorate was higher than other chlorate-reducing bacteria isolated from freshwater environment (Ghosh et al., 2011). We also studied the ability of XM-1 for chlorate reduction in the influent of a wastewater treatment plant. Chlorate is one of the substances found in the effluents from pulp and paper industries with a concentration from 100 mg/L to nearly 350 mg/L (Cabrera et al., 2019). Therefore, 1.5 mM chlorate was amended to the influent in our assay. Results show that chlorate concentration decreased 0.92 mM within 12 h and achieved a 100% chlorate removal within 72 h (Supplementary information, Fig. S3). While in the influent without XM-1 inoculation, obvious chlorate removal was observed after 36 h and only 36% chlorate was removed at 72 h. These results suggest the potential for strain XM-1 used in chlorate remediation.

4. Conclusion In this study, a Gram-negative, rod-shaped, facultative anaerobe having chlorate reducing ability was isolated and identified as Ochrobactrum anthropi XM-1 based on 16S rRNA gene sequence and recA gene-based multi-primer PCR. Compared with other chlorate-reducing bacteria, XM-1 has a high tolerance for acidic conditions and it was able to grow well at pH 5–8. Under anaerobic condition, strain XM1 can utilize chlorate up to 200 mM as electron acceptor, which is promising for high-strength chlorate reduction. Oxygen and nitrate could also be used as electron acceptors. Activities of chlorate reductase and chlorite dismutase could be detected in XM-1 cells grown under both aerobic and anaerobic conditions. Acknowledgment

3.3. Characterization of enzyme activities

The authors acknowledge support from the National Natural Science Foundation of China (NSFC) (21477121, 51538012).

The pathway for (per)chlorate reduction has been proposed to proceed according to ClO4− → ClO3− → ClO2− → Cl2 + O2 (Rikken et al., 1996). During chlorate reduction, chlorate is reduced by chlorate reductase to harmful chlorite which finally disproportionates by chlorite dismutase into chloride and oxygen. Therefore, enzyme activity assays were performed on XM-1 cells grown aerobically with and without chlorate, and on cells grown anaerobically with chlorate. The cultured cells were harvested in mid-log phase, and the enzymes activities were measured in cell extracts. The chlorate reductase (Clr) activity of strain XM-1 with chlorate as the sole electron acceptor was 2.31 μmol min−1 mg−1 (Table 1), Similarly, the Clr activity in the cell extracts from chlorate-grown Pseudomonas sp. PDA was 5.85 μmol min−1 mg−1 (Xu et al., 2004), and 2.4 μmol min−1 mg−1 for the cell extracts from Ideonella dechloratans (Lindqvist et al., 2012). Studies have reported that Clr was constitutively expressed in most chlorate-reducing bacteria (Xu et al., 2004; Lindqvist et al., 2012; Mehboob et al., 2016). Our results show that aerobically grown XM-1 cells synthesized Clr no matter the presence of chlorate (Table 1), suggesting strain XM-1 could constitutively express Clr. The Clr activity in cells grown aerobically was less than half of that derived from anaerobically grown cells. The presence of oxygen might affect the expression of Clr at transcriptional level (Xu et al., 2004) or directly inhibit the enzyme activity. As oxygen has a higher electron affinity than chlorate, XM-1 will not reduce chlorate in the presence of oxygen, and similar results were found for most (per)chlorate respiring bacteria

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