A Pandoraea sp. strain efficiently degrades chlorobenzene via monooxygenation pathways with high potential for groundwater bioremediation

doi:10.21203/rs.3.rs-4002375/v1

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A Pandoraea sp. strain efficiently degrades chlorobenzene via monooxygenation pathways with high potential for groundwater bioremediation

https://doi.org/10.21203/rs.3.rs-4002375/v1

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Chlorobenzene (CB) is often detected in contaminated soil and groundwater at in-service petrochemical facilities. Given the high safety requirements for these petrochemical facilities, a sustainable and low-consumption microbial remediation technology is preferred. The aim of this study was to isolate an efficient chlorobenzene-degrading bacterial strain and understand its degradation mechanism to be used for in-situ bioremediation of chlorobenzene-contaminated sites in in-service petrochemical enterprises. A degrading bacterium was isolated from chlorobenzene-contaminated soil at a pesticide plant, identified as Pandoraea sp. XJJ-1 (CCTCC M 2021057). This strain completely degraded 100 mg·L^− 1 CB and showed extensive degradability across a range of pH (5.0–9.0), temperature (10°C-37°C), and CB concentrations (100–600 mg·L^− 1). Notably, the degradation efficiency was 85.2% at 15°C, and the strain could also degrade six other aromatic hydrocarbons, including benzene, toluene, ethylbenzene, and xylene (o-, m-, p-). The metabolic pathway of chlorobenzene was inferred using Ultra Performance Liquid Chromatography (UPLC), Gas chromatography-mass spectrometry (GC-MS), and genomic analysis. In strain XJJ-1, CB was metabolized to o-chlorophenol and 3-chloroxychol by chlorobenzene monooxygenase, followed by ortho-cleavage by the action of 3-chlorocatechol 1,2-dioxygenase. This is the first report of chlorobenzene monooxygenase as the rate-limiting step in Pandoraea spp.. Strain XJJ-1, which exhibits excellent degradation ability for CB at low temperatures, was isolated in this study. Moreover, the presence of the chlorobenzene monooxygenation pathway metabolism in strain XJJ-1 is reported for the first time in Pandoraea. As a biomaterial with low temperature resistance and composite pollutant degradation capacity, strain XJJ-1 has potential applications prospects in the in-situ bioremediation of chlorobenzene-contaminated sites.

Chlorobenzene

biodegradation

Pandoraea

degradation characteristics

monoo-xygenation pathway

Chlorobenzene (CB), as a significant chemical production intermediate and solvent, is widely used in the pesticide, plastic, pharmaceutical and dye industries (Sun et al. 2021). While chlorobenzene is a toxic and harmful volatile organic compound (VOC) that has been listed as a priority control pollutant by the U.S. EPA. China is the most important producer and supplier of chlorobenzenes, accounting for more than 50% of global production (Yu et al. 2018), and is therefore widespread in soil and groundwater and widely detected. A total of 13.8-1,560.0 mg·kg^− 1 chlorobenzene was detected in the soil of a solvent treating plant in Suzhou, China (Xu et al. 2019). The average concentration of chlorobenzene detected in the groundwater of a chemical plant in eastern Wuhan, China was 14.40 mg·L^− 1 (Xie et al. 2015). A total of 0.05-8,600.00 mg·kg^− 1 chlorobenzene was detected in a site of a decommissioned chlorobenzene-producing factory in Wuhan, China (Yu et al. 2018). Chlorobenzene in soil and groundwater causes serious environmental risks and human health risks through volatilization, migration and bioaccumulation (Zhang et al. 2019).

The chlorobenzene remediation technologies mainly include gas thermal remediation (Xu et al. 2019), advanced oxidation (Xie et al. 2020) and microbial degradation (Zhang et al. 2019). Thermal desorption technology is energy-intensive and costly, and the soil temperature can still be as high as 70°C after a week of restoration, which seriously damages the native environment of the soil (Xu et al. 2019). Likewise, advanced oxidation techniques require large amounts of oxidizing agents and can cause significant damage to the native environment (Xie et al. 2020). Compared to thermal desorption and advanced oxidation technologies, bioremediation technologies have the advantage of being green and low cost (Zhang et al. 2019). More importantly, bioremediation technology has the advantages of low environmental disturbance and safety, which can meet the remediation requirements of in-production enterprises (Wu et al. 2022).

A number of chlorobenzene-degrading bacteria have been isolated from polluted environments, such as Pseudomonas sp. (Pettigrew et al. 1991), Escherichia hermanii (Kiernicka et al. 1999), Rhodococcus sp. (Rehfuss and Urban 2005), white-rot fungus (Wang et al. 2008), Pandoraea sp. (Baptista et al. 2008), Ralstonia sp. (Zhang et al. 2011), Bacillus sp. (Vyas and Murthy et al. 2015), Ochrobactrum sp. (Zhang et al. 2019). However, there are still some difficulties in the application of these microorganisms to the actual groundwater environment by bioaugmentation techniques. On the one hand, there is a difference between the culture temperature and the application temperature. The culture temperature in laboratory of the strain is 20–40°C, under which the chlorobenzene-degrading strain can degrade chlorobenzene effectively (Vyas and Murthy 2015). While the actual temperature in groundwater is usually 10–20°C, and these strains don’t adapt to the temperature. On the other hand, the types of pollutants in the actual organic contaminated site are complex and diverse (Braeckevelt et al. 2011), for example, chlorobenzene is often detected together with benzene, ethylbenzene, toluene, xylene and other pollutants (Xu et al. 2019). While most chlorobenzene-degrading strains have only been verified the characterization of chlorobenzene as single substrate. To our knowledge, there has not yet been a study of a strain can degrade six BTEX and chlorobenzene simultaneously. In general, the two main factors of low temperature and complex pollution in the groundwater environment can seriously affect the survival status and degradation performance of microorganisms. Therefore, it is very important to isolate a strain that can efficiently degrade chlorobenzene under low temperature and complex pollution conditions, which can provide microbial materials for soil and groundwater remediation of in-production enterprises.

In this study, an efficient chlorobenzene-degrading strain XJJ-1 was isolated from the chlorobenzene-contaminated soil in a pesticide plant. The work focused on the following aspects: (1) identification of the degrading strain by its bacterial morphology and 16S rRNA gene sequence analysis; (2) exploration of pH adaptation, low temperature degradation ability, high concentration chlorobenzene tolerance and BTEX complex contamination adaptation; and (3) proposing the chlorobenzene metabolic pathway by UPLC, GC-MS assay and genome analysis.

2.1 Chemicals and medium

Chlorobenzene (purity ≥ 99%) and 3-chlorocatechol (purity ≥ 95%) were acquired from Shanghai Macklin Bio-Chem Technology Co., Ltd., China. o-Chlorophenol (purity ≥ 99%) was acquired from Shanghai Aladdin Bio-Chem Technology Co., Ltd., China. All other reagents and solvents used were analytical grade and the highest purity available.

The minimal salt medium (MSM) contained (1 L): 0.023 g CaCl₂, 0.2 g MgSO₄·7H₂O, 4.5 g Na₂HPO₄·12H₂O, 2.5 g (NH₄)₂SO₄, 1.0 g KH₂PO₄, and 1 mL trace element solution. The trace element solution included (1 L): 0.15 g ZnSO₄·7H₂O, 0.26 g MnSO₄·H₂O, 0.03 g CoCl₂·6H₂O, 4.5 g FeSO₄·7H₂O, 0.02 g NiCl₂·6H₂O, 0.01 g CuCl₂, 0.1 g Na₂MoO₄·2H₂O, and 0.06 g H₃BO₃. Luria-Bertani (LB) medium contained (1 L): 10 g tryptone, 5 g yeast extract, 10 g NaCl, and 15 g agar (Chen et al. 2023).

2.2 Isolation and identification of CB degrading bacterium

The soil samples used to isolate CB-degrading bacterium were collected from chlorobenzene contaminated of a pesticide plant in Anhui Province of China. The soil (3–5 g) was inoculated in a 250 mL serum bottle with 20 mL MSM containing 100 mg·L^− 1 CB as the solo carbon source. The bacterial consortium solution was transferred every 3 days in 10% volume and incubated at 28 ^oC, 180 rpm. After 4–5 times transfers, the last solution was diluted and spread on MSM solid medium containing CB for 2–3 days at 28^oC. A single colony was selected from the MSM solid medium, which was inoculated into fresh MSM liquid medium with CB to determine the remaining CB content for testing its CB degradation ability. The bacterium was named strain XJJ-1. The 16S RNA gene was amplified (Zhang et al. 2019). The phylogenetic analysis was constructed using the neighbor-joining method of MEGA 7.0 software (Kumar et al. 2018).

2.3 Batch degradation experiments of CB by strain XJJ-1

To reveal the degradation characteristics of strain XJJ-1. The initial conditions were set to pH 7.0, temperature 28°C, initial CB concentration 100 mg·L^− 1 and initial OD_600nm 0.05. The effects of different influencing factors on strain growth and CB degradation were investigated at different pH values (5.0, 6.0, 7.0, 8.0, 9.0), temperatures (10°C, 15°C, 20°C, 28°C, 37°C) and initial CB concentrations (100 mg·L^− 1, 200 mg·L^− 1, 400 mg·L^− 1, 600 mg·L^− 1). When investigating the effect of BTEX complex pollution on strain growth and chlorobenzene degradation, the benzene, ethylbenzene, toluene and xylene concentrations were 50 mg·L^− 1 and 100 mg·L^− 1 for chlorobenzene, and total concentration was 300 mg·L^− 1. Bacterial suspensions cultured to logarithmic growth phase were added to 20 mL of sterilized MSM with CB as the sole carbon source. Three parallel and one non-biological controls (without strain XJJ-1) were set up for each group. The remaining CB was detected after incubating 11 hours at 28 ℃ and 180 rpm.

2.4 Determination degradation metabolites of CB by strain XJJ-1

The detection of chlorobenzene concentration was quantified by GC (Agilent, America). The chromatographic column was HP-5 (30 m × 0.32 mm × 0.25 µm); the column temperature was increased to 70 ℃ at the beginning, held for 1 min, with an increase rate of 10 ℃·min^− 1, and the final temperature was 110 ℃ with retention time of 1 min; the temperature of the vapor chamber was 200 ℃; the temperature of the FID detector was 250 ℃; the column flow rate was 1.5 mL·min^− 1, N₂ was the carrier gas and the sample was injected without splitting, the H₂ flow rate was 45 mL·min^− 1, the air flow rate was 450 mL·min^− 1, and the injection volume was 1 µL.

The intermediate products were detected by UPLC (Waters H-class, America) under the following chromatographic conditions: HSS T3 column (1.8 µm × 2.1 mm × 100 mm), column temperature was 35°C, detection wavelength was 210 nm, injection Volume was 10 µL, mobile phases were acetone/water (50/50), and flow rate was 0.2 mL·min^− 1 (Hu et al. 2020).

The intermediate products were identified by GC-MS (Agilent 7890A-5975C, America) under the following chromatographic conditions: HP-5MS column; carrier gas was helium, 1.5 mL·min^− 1; injector temperature was 250°C; column temperature was used with a ramp-up procedure, initial temperature 35°C, held for 10 min, 10 ℃·min^− 1 to 190℃, held for 2 min, 6 ℃·min^− 1 to 225 ℃, held 1 min; mass spectrometry using EI source, ionization energy of 70 eV, ion source temperature of 230 ℃. The intermediate product was detected and compared with the NIST standard database. Metabolic intermediates were derivatization with 450 µL N,O-bis(trimethylsilyl)-trifluoroacetamide at 60 ^oC for 1 h, then trimethylsilyl (TMS) derivatives were formed (Lee et al. 2019).

2.5 Genome sequencing and analysis

The genomic DNA of strain XJJ-1 was extracted by using Fast DNA™ Spin DNA extraction kit (MP Biomedical, USA). Illumina MiSeq and PacBio sequencing platforms were used to sequence the whole genome of strain XJJ-1 (Guangdong Magi Gene Technology Co., Ltd). Annotation was done using NR in NCBI, Swiss-Prot, KEGG and Cazy database (Choi et al. 2013).

3.1 Isolation and identification of strain XJJ-1

Through multiple transfers, a single colony could grow with chlorobenzene as the solo carbon source and showed obvious degradation ability by determining the remaining chlorobenzene content. The strain XJJ-1 on LB solid medium showed regular colony morphology, yellow, raised, opaque and smooth, and were rod-shaped by scanning electron microscopy (Figure S1). The 16S rRNA gene of strain XJJ-1 has 99% similarity sequence identity with Pandoraea pnomenusa I3-10 (GenBank accession number: KU252675), Pandoraea fibrosis AO4A4 (GenBank accession number OP018832) and Pandoraea fibrosis 6399 (GenBank accession number KX712083), the phylogenetic tree reveals that the strain is also closed to Pandoraea sp..(Fig. 1)and named as Pandoraea sp. XJJ-1 ( GenBank accession number: MW453046 ). As a result, it was initially assigned as Pandoraea sp. and has been deposited in the China Center for Type Culture Collection with accession number CCTCC M 2021057.

3.2 Growth conditions and CB degradation abilities by strain XJJ-1

To confirm the growth conditions and the degradation abilities of environmental variables (pH, temperature, CB concentration and BTEX complex pollution) of CB by strain XJJ-1 was investigated and summarized in Fig. 2. Strain XJJ-1 had a more than 80% degradation efficiency in the pH range of 5.0–9.0, with a final OD_600nm of more than 0.15 (Fig. 2a). The optimum pH was 7.0, and the degradation efficiency reached 100% with a final OD_600nm of 0.182. The degradation efficiency was more than 90% in the range of 20–37°C, and the most suitable temperature was 28°C, with a 100% degradation efficiency (Fig. 2b). The degradation efficiency was approximately 60% and 85% at low temperatures of 10 and 15°C, respectively, and there was a 27% inhibition of the final OD_600nm of the strain at 10°C compared to 28°C. Although the growth and degradation of microorganisms were greatly inhibited by high concentrations of chlorobenzene, strain XJJ-1 still had some degradation and growth ability in solutions up to 600 mg·L^− 1 chlorobenzene, and the degradation efficiency and growth increase of the strain in 11 h were 10.2% and 56%, respectively (Fig. 2c). When the initial concentrations of benzene, ethylbenzene, toluene and xylene were 50 mg·L^− 1 and the concentration of chlorobenzene was 100 mg·L^− 1, strain XJJ-1 still had good degradation ability in the complex pollution system of BTEX and chlorobenzene, with removal efficiencies of 45.3%, 36.9%, 13.9%, 10.2% and 52.3%, respectively, and the final OD_600nm was 0.123 (Fig. 2d). The optimal pH and temperature of growth and degradation were 7.0 and 28^oC, respectively. The optimal concentration of CB was 100 mg·L^− 1. At the optimal conditions the degradation ability of strain XJJ-1 was tested. Stain XJJ-1 completely degrade 100 mg·L^− 1 chlorobenzene within 11 h (Fig. 3a).

3.3 Metabolic intermediates of CB by strain XJJ-1

By UPLC detection, the retention times of the authentic standards 3-chlorocatechol, o-chlorophenol and chlorobenzene were 1.95 min, 2.70 min and 6.78 min, respectively (Figure S2). By comparing the retention times of the authentic standards, chromatograms of the changes in 3-chlorocatechol, o-chlorophenol and chlorobenzene were obtained (Fig. 3b). With the decrease in chlorobenzene concentration, the concentrations of o-chlorophenol and 3-chlorocatechol showed a trend of increasing and then decreasing, reaching maximum concentrations of 4.82 mg·L^− 1 and 9.78 mg·L^− 1 at 7 h and 11 h, respectively, and o-chlorophenol and 3-chlorocatechol were completely degraded within 20 h.

By GC‒MS detection, TMS derivatives of o-chlorophenol and 3-chlorocatechol were detected (Figure S3), and the mass spectra were confirmed by matching against the NIST library. The TMS derivative of o-chlorophenol gave a molecular ion at m/z 200, and major fragments at m/z 93, m/z 149 and m/z 185. The TMS derivative of 3-chlorocatechol gave a molecular ion at m/z 288.1 and a major fragment at m/z 73.1. Based on the findings of Zhang (2011), the isolation of o-chlorophenol and 3-chlorocatechol provided clear evidence that o-chlorophenol and 3-chlorocatechol were produced from chlorobenzene.

3.4 Genomic analysis of strain XJJ-1

The whole genome of Pandoraea sp. XJJ-1 contains a 5378876 bp chromosome (GenBank accession number CP113827) and a 51847 bp plasmid (GenBank accession number CP113828) with an average GC content of 64.00%. The coding sequence coverage of the whole genome was 87.1%, with a total of 4937 predicted protein coding sequences (CDS). In addition, the whole genome contained 12 rRNAs, 66 tRNAs and 38 ncRNAs, accounting for 0.50% of the whole genome. The whole genome sequence map of Pandoraea sp. XJJ-1 is shown in Figure S4.

The amino acid sequence comparison results of the related enzymes are shown in Table 1. The gene XJJ_1002003 has 57% identity with the toluene 2-monooxygenase gene of Burkholderia vietnamiensis G4 (Nelson 1987), which was initially inferred to encodes the chlorobenzene monooxygenase. The gene XJJ_1004912 has more than 65% identity with the 3-chlorocatechol 1,2-dioxygenase gene of Ralstonia sp. JS705, Pandoraea pnomenusa MCB032, Pseudomonas chlororaphis RW71, and Pseudomonas sp. P51 (Van der Meer 1998; Chao 2019; Potrawfke 1998; Werlen 1996), which was initially inferred to encode 3-chlorocatechol 1,2-dioxygenase. The gene XJJ_1004911 has more than 70% identity with the chloromuconate cycloisomerase gene of Pandoraea pnomenusa MCB032, Pseudomonas chlororaphis RW71, and Pseudomonas sp. P51, which was initially inferred to encode the chloromuconate cycloisomerase. The gene XJJ_1004909 has more than 50% identity with the dienelactone hydrolase gene of Pandoraea pnomenusa MCB032 and Pseudomonas chlororaphis RW71, which was initially inferred to encode the dienelactone hydrolase. The gene XJJ_1004908 has 55% identity with the maleylacetate reductase gene of Pseudomonas sp. P51 and Pseudomonas chlororaphis RW71, which was initially identified as encoding the maleylacetate reductase.

Table 1

BLASTP search results for deduced amino sequence
Gene location	Proposed function	Organism	Accession number	Amino acid identity (%)
XJJ_1002003	Chlorobenzene monooxygenase	Burkholderia vietnamiensis G4	ABO60467.1	57
XJJ_1004912	Chlorocatechol 1,2-dioxygenase	Ralstonia sp. JS705	CAA06968.1	97
XJJ_1004912	Chlorocatechol 1,2-dioxygenase	Pandoraea pnomenusa MCB032	WP_063599759.1	99
XJJ_1004912	Chlorocatechol 1,2-dioxygenase	Pseudomonas chlororaphis RW71	CAB52140.1	67
XJJ_1004912	Chlorocatechol 1,2-dioxygenase	Pseudomonas sp. P51	P27098.1	67
XJJ_1004911	Chloromuconate cycloisomerase	Pandoraea pnomenusa MCB032	ABS81347.1	100
XJJ_1004911	Chloromuconate cycloisomerase	Pseudomonas chlororaphis RW71	CAB89822.1	70
XJJ_1004911	Chloromuconate cycloisomerase	Pseudomonas sp. P51	P27103.1	76
XJJ_1004909	Dienelactone hydrolase	Pandoraea pnomenusa MCB032	ABS81349.1	100
XJJ_1004909	Dienelactone hydrolase	Pseudomonas chlororaphis RW71	CAB89824.1	52
XJJ_1004908	Maleylacetate reductase	Pseudomonas sp. P51	P27101.1	60
XJJ_1004908	Maleylacetate reductase	Pseudomonas chlororaphis RW71	CAB89825.1	59

Enzyme genes encoding chlorobenzene ortho-modified degradation pathways are distributed in two gene clusters (Fig. 4a), one is the chlorobenzene monooxygenase gene cluster and the other is the 3-chlorocatechol dioxygenase gene cluster. The chlorobenzene monooxygenase gene cluster contains the gene XJJ_1002003 encoding chlorobenzene monooxygenase. The 3-chlorocatechol dioxygenase gene cluster contains the genes XJJ_1004912, XJJ_1004911, XJJ_1004909 and XJJ_1004908, which were initially inferred to encode the enzymes 3-chlorocatechol 1,2-dioxygenase, chloromylate cyclase, lactone hydrolase and maleacetoacetate reductase, respectively.

Based on the 3-chlorocatechol 1,2-dioxygenase gene sequence alignment of strain XJJ-1, the phylogenetic tree is shown in Fig. 4b. The 3-chlorocatechol 1,2-dioxygenase gene of strain XJJ-1 has more than 95% identity with Ralstonia sp. JS705 (GenBank accession number CAA06968) and Pandoraea pnomenusa MCB032 (GenBank accession number WP_06399759), and has more than 60% identity with Pseudomonas sp. P51 (GenBank accession number P27098), Pseudomonas sp. RW71 (GenBank accession number CAB52140) and Alcanivorax sp. HA03 (GenBank accession number AFK13024). As a result, the gene XJJ_1004912 was deduced to encode 3-chlorocatechol 1,2-dioxygenase.

3.5 The CB degradation pathway of strain XJJ-1

By UPLC assay, GC‒MS assay and genome analysis, o-chlorophenol and 3-chlorocatechol were detected and the chlorobenzene monooxygenase gene cluster and the 3-chlorocatechol 1,2-dioxygenase gene cluster were identified. The possible pathway for the degradation of chlorobenzene by strain XJJ-1 was speculated (Fig. 5). Chlorobenzene first forms o-chlorophenol and 3-chlorocatechol under the action of chlorobenzene monooxygenase, and then ortho-cleavage under the action of 3-chlorocatechol 1,2-dioxygenase to form 2-chloromuconic acid, which is then dechlorinated by lactonization under the action of the chloromyucucoate cyclase to form 4-carboxymethyl-2-butene-4-internal lipid. Then, under the action of the lactone hydrolase and the maletoacetate reductase, it is gradually reduced to the maleoyl acetate and β-ketoadipic acid, and finally goes into the TCA cycle.

Chlorobenzene is common pollutants of soils and groundwater due to be widely used as intermediate and solvent in the petrochemical industry and other industries. In addition, Chlorobenzene and BTEX are commonly found in the groundwater organic-contaminated petrochemical at the same time. The occurrence of compound pollution mainly chlorobenzene has been of great concern because they had both inhibitory and anesthetic effects on the central nervous system in humans.

In this study, we isolated a Pandoraea strain able to degrade chlorobenzene and BTEX, even has degraded the chlorobenzene in 10℃ and pH 5.0–9.0. In actual groundwater, temperature and pH is vital factor affecting microbial growth and degradation performance because the tow could affect enzyme activity. Under the conditions of pH 5.0 and pH 9.0, the removal efficiency of chlorobenzene of Ralstonia pickettii L2 is lower than 20% (Zhang et al. 2011), and under the condition of 15℃, the removal efficiency of chlorobenzene is lower than 15%, which is far lower than the removal efficiency of strain XJJ-1.

Microorganisms with higher chlorobenzene tolerance would presumably are more effective in real contaminated sites. The chlorobenzene tolerance concentration of Ralstonia pickettii L2 is only 250 mg·L^− 1 (Zhang et al. 2011). Compared to strain L2, strain XJJ-1 had better chlorobenzene tolerance performance, which may be due to the type and activity of the relevant metabolic enzymes of the microorganism. There was a 3 h lag period in the degradation of chlorobenzenes by strain XJJ-1 because chlorobenzene-related enzymes are inducible enzymes. Similarly, Enterobacter sp. CB-2 was able to use 100 mg·L^− 1 chlorobenzene as the sole carbon source at pH 7.0 and 30°C.

The degradation rate of microorganisms is an important factor limiting their scale-up applications. Strain XJJ-1 had a maximum average degradation rate of 9.09 mg·L^− 1·h^− 1 under optimal conditions, second only to Ochrobactrum sp. ZJUTCB-1 (Zhang et al. 2019). Performance of CB degradation by various microorganisms are shown in Table 2. Planococcus sp. ZD22 removed 67.3% of chlorobenzene in 120 h with an average degradation rate of 0.63 mg·L^− 1·h^− 1 (Li et al. 2006). Acidovorax avenae completely removed chlorobenzene in 24 h with an average degradation rate of 0.318 mg·L^− 1·h^− 1 (Monferran et al. 2005). A mixed fungal-bacterial consortium removed 93% of chlorobenzene in 21.6 h with an average degradation rate of 2.155 mg·L^− 1·h^− 1 (Cheng et al. 2017). Ralstonia sp. DSM 8910 was able to completely remove chlorobenzenes in 60 h with an average degradation rate of 3.666 mg·L^− 1·h^− 1 (Kaschl et al. 2005). The average degradation rate of strain XJJ-1 was at least twice as high as that of the above microorganisms. This shows that strain XJJ-1 has high efficiency in the degradation of chlorobenzene under optimal conditions. Interestingly, strain XJJ-1 removed 85.2% of chlorobenzene in 11 h with an average degradation rate of 7.745 mg·L^− 1·h^− 1 at 15 ℃, which has a great advantage in adapting to the actual groundwater environment.

Table 2

Performance of CB degradation by various microorganisms
Microorganisms	Concentration (mg·L^− 1)	Time (h)	Temperature (℃)	Removal efficiency (%)	Average degradation rate (mg·L^− 1_·h^− 1)	Reference
Planococcus sp. ZD22	112.5	120	20	67.3	0.63	(Li et al. 2006)
Acidovorax avenae	7.65	24	25	100	0.318	(Monferran et al. 2005)
Fungal-bacterial consortium	50	21.6	20	93	2.155	(Cheng et al. 2017)
Ralstonia sp. DSM 8910	220	60	30	100	3.666	(Kaschl et al. 2005)
Pandoraea sp. XJJ-1	100	11	28	100	9.09	This work

The metabolic pathway of chlorobenzene has been widely studied and is mainly determined by chlorobenzene oxygenase and 3-chlorocatechol dioxygenase, which can be roughly divided into four pathways. In the first and main metabolic pathway, chlorobenzene is first formed into dihydrodiols by the action of chlorobenzene dioxygenase, followed by the formation of the key intermediate 3-chlorocatechol in the presence of dehydrogenase, ortho-cleavage in the presence of 3-chlorocatechol 1,2-dioxygenase and subsequent gradual reductive dechlorination. The degradation of chlorobenzene by Pseudomonas putida, Pandoraea sp. MCB032 and strain WR1306 follow this metabolic pathway (Nishino et al. 1992; Jiang et al. 2009; Reineke and Knackmuss 1984). In the second metabolic pathway, chlorobenzene first forms 3-chlorocatechol under the action of chlorobenzene monooxygenase and then undergoes ortho-cleavage under the action of catechol 1,2-dioxygenase. The degradation of chlorobenzene by Bacillus subtilis strain DKT, Delftia tsuruhatensis LW26 and Ralstonia pickettii L2 follows this metabolic pathway (Nguyen and Ha 2019; Ye et al. 2017; Zhang et al. 2011). The third metabolic pathway was found in Pseudomonas putida GJ31, degrading chlorobenzene via chlorobenzene dioxygenase and catechol 2,3-dioxygenase (Mars et al. 1997). The fourth metabolic pathway was found in Ochrobactrum sp. ZJUTCB-1, degrading chlorobenzene via chlorobenzene monooxygenase and catechol 2,3-dioxygenase (Zhang et al. 2019).

Pandoraea sp. XJJ-1 formed 3-chlorocatechol in the presence of a chlorobenzene monooxygenase gene cluster, different from the chlorobenzene dioxygenase gene cluster reported in Pandoraea sp. MCB032 (Jiang et al. 2009), which is the first report of chlorobenzene monooxygenase in Pandoraea spp. Chlorobenzene monooxygenase exists in Bacillus subtilis DKT, Delftia tsuruhatensis LW26, Ralstonia pickettii L2 and Ochrobactrum sp. ZJUTCB-1 (Nguyen and Ha 2019; Ye et al. 2017; Zhang et al. 2011; Zhang et al. 2019). However, research on the coding gene of chlorobenzene monooxygenase is still unclear. To our knowledge, chlorobenzene-degrading genes probably originate from toluene-degrading genes (van der Meer 1997; van der Meer et al. 1998). As an example, the chlorobenzene dioxygenase gene cluster of strain JS705 has approximately 90% identity with the toluene dioxygenase gene cluster of strain F1 (van der Meer et al. 1998; Wackett and Gibson 1988). The gene XJJ_1002003 has 57% identity with toluene 2-monooxygenase of Burkholderia vietnamiensis G4 (Nelson et al. 1987). Therefore, the gene XJJ_1002003 was initially inferred to be a possible gene encoding chlorobenzene monooxygenase.

A new CB-degrading strain Pandoraea sp.XJJ-1 was isolated and identified from the chlorobenzene-contaminated soil. The strain was capable of complete degradation of 100 mg·L-¹ CB within 11 h, and the degradation rate of chlorobenzene at 10℃ and 15℃ can reach 60.2% and 85.2%, respectively. It could also simultaneously degrade BTEX with a degradation rate of 10.2%-52.3% within 11 h, which also indicates that the strain has a prominent broad-spectrum of degradation substrate types compared to other CB-degrading strains. Moreover, during the metabolism of chlorobenzene by strain XJJ-1, o-chlorophenol and 3-chlorocatechol were determined as major intermediates. Combined with genome analysis, the 3-chlorocatechol dioxygenase gene cluster of strain XJJ-1 has more than 95% identity with Pandoraea sp. MCB032. Therefore, it was inferred that the pathway of strain XJJ-1 metabolizing chlorobenzene was via o-chlorophenol into 3-chlorocatechol, followed by the ortho-cleavage pathway for further metabolism. The adaptability characteristics of strain XJJ-1 in low temperature and composite pollutants were conducive to its application in in-situ groundwater remediation of in-production enterprises.

Ethics approval and consent to participate

This article does not contain any studies with human or animal subjects performed by any of the authors.

Consent for publication

The author agrees to publish.

Competing interests

The authors declare no competing interests.

Funding

This work was supported by the Science and Technology Commission of Shanghai Municipality (No.23HC1400300).

Author’s contributions

Changzheng Cui and Jie Yang conceived the idea and designed the experiments. Lixu Pan and Bo Yuan conducted the experiments, analyzed the data, and prepared first draft of the manuscript. Qingqing Li, Ji Ouyang, Yan Zhou and Changzheng Cui reviewed and revised the manuscript. All authors have reviewed and approved the final manuscript.

Acknowledgements

No applicable.

Data Availability

Data available on request from the authors.

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A Pandoraea sp. strain efficiently degrades chlorobenzene via monooxygenation pathways with high potential for groundwater bioremediation

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Chemicals and medium

2.2 Isolation and identification of CB degrading bacterium

2.3 Batch degradation experiments of CB by strain XJJ-1

2.4 Determination degradation metabolites of CB by strain XJJ-1

2.5 Genome sequencing and analysis

3. Results

3.1 Isolation and identification of strain XJJ-1

3.2 Growth conditions and CB degradation abilities by strain XJJ-1

3.3 Metabolic intermediates of CB by strain XJJ-1

3.4 Genomic analysis of strain XJJ-1

3.5 The CB degradation pathway of strain XJJ-1

4. Discussion

5. Conclusions

Declarations

References

Supplementary Figure

Additional Declarations

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