Curr Microbiol (2010) 61:477–483 DOI 10.1007/s00284-010-9641-2

Identification and Analysis of Polychlorinated Biphenyls (PCBs)Biodegrading Bacterial Strains in Shanghai Jian-Jun Shuai • Yong-Sheng Tian • Quan-Hong Yao Ri-He Peng • Fei Xiong • Ai-Sheng Xiong

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Received: 19 February 2010 / Accepted: 16 March 2010 / Published online: 20 April 2010 Ó Springer Science+Business Media, LLC 2010

Abstract As one of China’s great metropolises, Shanghai is vulnerable to various forms of industrial and agricultural contamination associated with its development. Polychlorinated biphenyls (PCBs) are man-made chemicals that never existed in nature until the 1900s when they started to be released into the environment. PCBs are hazardous environmental contaminants that bind strongly to soil. In this study, four soil samples were screened for the presence of PCB-degrading bacteria. The 16 S rDNAs were amplified from those genomes and the products (*1.5 kb) were purified and sequenced for the isolation and identification of bacterial species. Four Pseudomonas strains (strain 1-212 from sample 1; strain 2-241 from sample 2; strain 3-318 from sample 3; and strain 4-150 from sample 4) were selected for analysis by HPLC. Setting the content of the biphenyl in CK as 100%, the biphenyl contents was 2.32% in 1-212, 73.11% in 2-241, 69.83% in 3-318, and 86.16% in 4-150. The results of this study suggest directions for future research, including genetic screening, cloning and restructuring, and provide guidance for the cultivation of PCBs-degrading bacteria.

Jian-Jun Shuai and Yong-Sheng Tian are contributed equally to the article. J.-J. Shuai  F. Xiong (&) College of Bioscience and Biotechnology, Yangzhou University, 88 Daxue Road, Yangzhou 225009, China e-mail: [email protected] J.-J. Shuai  Y.-S. Tian  Q.-H. Yao  R.-H. Peng  A.-S. Xiong (&) Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences, 2901 Beidi Rd, Shanghai, China e-mail: [email protected]

Introduction Polychlorinated biphenyls (PCBs), are a large group of chlorinated biphenyls with 209 possible congeners, are manufactured by the direct chlorination of biphenyl to produce complex mixtures containing up to 10 chlorines. For a long time, PCBs were used for various industrial and commercial purposes and it is estimated that more than 1.5 million tons of PCBs have been manufactured worldwide [1]. Unfortunately, due to their hydrophobic properties, PCBs are easily adsorbed by natural organic matter in soil, sludge, and aquatic sediments and they have entered the food chain. It was first recorded in the 1930s that PCBs are harmful to human health [2]. Since then, PCBs had been proved to cause cancer [3] and to have a number of serious effects on immune, reproductive, nervous, and endocrine systems [4–6]. These health concerns prioritize PCBs as major targets for environmental clean-up. Bioremediation by soil bacteria has been investigated extensively during the last few decades as an alternative, and less expensive, strategy for the destruction of PCB contaminants [7, 8]. Biodegradation of PCBs is achieved by the actions of four main types of biphenyl catabolic enzymes, including biphenyl dioxygenase (BphA), dihydrodiol dehydrogenase (BphB), 2,3-dihydroxybiphenyl dioxygenase (BphC), and hydrolase (BphD) [9–13]. The genes encoding the enzymes for PCB degradation are clustered together in a bph gene cluster [14], which can be divided into four main types on the basis of genetic organization. The first type of bph gene cluster was discovered in Burkholderia sp. LB400 [15, 16] and Pseudomonas pseudoalcaligenes KF707 [17], which contain bphR A1A2A3A4BCKHJID genes. The second type, which includes bphSEGFA1A2A3BCDA4 genes, was found in Achromobacter georgiopolitanum strain KKS102 [18, 19]

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and Alcaligenes eutrophus A5 [20–22]. The third type of cluster consists of bphA1A2A3A4CBST genes and was found in Rhodococcus sp. strain RHA1 [23]. The fourth type of bph cluster was found in Rhodococcus sp. strain R04 [24] and Rhodococcus sp. strain K37, which embraces bphBCA1A2A3A4D [25]. In this study, we investigated the distribution of PCButilizing bacteria in Shanghai from a macroscopic angle and confirmed our results by HPLC. This study will be of benefit to future research, including cloning and screening bph clusters from new species of bacteria capable of highly efficient biodegradation of PCBs.

Materials and Methods Chemicals and Media Polychlorinated biphenyl (2,20 ,3,30 -Tetrachlorobiphenyl, CAS No. 38444-93-8) (99.5% purity) and biphenyl (CAS No. 92-52-4) (99.5% purity) were purchased from the J&K Chemical Co., Ltd (Shanghai, China) and Shanghai Chemical Agent Co., Ltd (Shanghai, China). Other chemicals used in this study were of the highest purity available. Mineral salt medium (MM) contained (g): NH4NO3 1.0, KCl 0.7, KH2PO4 2.0, NaHPO4 3.0, MgSO47H2O 0.7, CaCl2 0.02, NaCl 1.0, in doubly distilled water 1 l, pH 7.0. Solid MM was MM containing agar 1.6 g/l. After autoclaving, tetrachlorobiphenyl was added as the sole carbon source. Luria–Bertani (LB) medium contained (g/l): tryptone 10.0, NaCl 10.0, and yeast extracts 5.0, pH 7.0. Isolation and Identification of PCB-Utilizing Bacterial Strains Soil samples were collected from four different sites in Shanghai, China. Sample 1 was taken from the experimental field at Shanghai Academy of Agricultural Sciences in the Minhang District. Sample 2 was taken from a riverbed near a disused chemical factory that used PCBs as raw materials in Laogang Town, Pudong New District; and sample 3 was taken from soil at the disused chemical factory. Sample 4 was taken from a riverbed at Huacao Town, Minhang District. For each sample: 10.0 g of soil was put into a sterilized mortar with 50 ml of sterile water and crushed with a sterilized pestle and then left for 5 min. A sample (1–5 ml) of the clear supernatant liquid was added to 100 ml of MM containing 0.01% tetrachlorobiphenyl as the sole carbon source, then incubated for 3 days at 28°C in the dark in an Erlenmeyer flask with shaking at 150 rpm. A 1 ml aliquot was transferred to 100 ml of fresh MM containing 0.01% tetrachlorobiphenyl and incubated under the same

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conditions. This process was repeated three times. A portion (20 ll) of the suspension was plated onto solid MM containing 0.01% tetrachlorobiphenyl as sole carbon source and incubated for 3 days at 28°C. Analysis of 16 S rDNA Sequences Genomic DNA was extracted from PCBs-utilizing bacterial strains as described [26]. Gene fragments of 16 S rDNA were amplified from genomic DNA using primers specific for Pseudomonas fluorescens. Forward: 50 -ACGGCTAC CTTGTTACGACTTC-30 ; Reverse: 50 -AGAGTTTGATCC TGGCTCAG-30 . Before PCR, the mixtures were denatured at 94°C for 10 min and the Ex-taq polymerase was added. Then PCR was done with 35 cycles of the following protocol: 94°C for 30 s, 50°C for 30 s, 72°C for 2 min, and at the end of the final cycle, 72°C for 10 min. The PCR products were ligated into the vector pMD18-T (TaKaRa, Dalian, China) and transformed into Escherichia coli strain DH5a. All the sequences were determined and aligned with BLAST on the NCBI website (http://www.ncbi.nlm.nih. gov). Phylogenetic trees were constructed with MEGA4 software (version 4.0; http://www.megasoftware.net/mega. html) [27] and the Neighbor-Joining (NJ) method with the following parameters: Poisson correction, pairwise deletion, and bootstrap (1,000 replicates). PCBs/Biphenyl Degradation Assay In order to confirm the extent of degradation, we chose four Pseudomonas sp. strains from each soil sample at random: 1-212, 2-241, 3-318, and 4-150. The four strains (100 ll) and CK (no stain) were cultured overnight at 28°C in LB medium (100 ml) in a rotary shaker at 120 rpm. A portion (1 ml) of biphenyl solution (50 mg/ml, soluble in methyl alcohol) was added into each of five Erlenmeyer flasks and the culture was continued. Four days later, 500 ll solution was placed into each of two Eppendorf tubes and then 500 ll of ether was added. The tubes were placed on an oscillator for 5–10 min, so that the biphenyl was fully extracted by the ether. The supernatants were withdrawn and mixed together in a new tube, 500 ll of methyl alcohol was added and the mixture was ready for rotary evaporation. The ether was fully evaporated and the biphenyl was kept in the methyl alcohol to prevent it adhering to the wall of the round-bottomed bottle. Five samples were membrane-filtered before HPLC analysis. A sample (10 ll) of the resulting solution was subjected to reverse-phase HPLC (1100 series, Agilent Co. USA).The separation column for the HPLC (4.6 mm 9 150 mm 9 5 lm) was filled with Kromail 100-5C18. The mobile phase consisted of acetonitrile/water (70:30, v/v) and the flow rate was 1.0 ml/min.

J.-J. Shuai et al.: Analysis PCBs-Biodegradation Bacteria in Shanghai

Biphenyl was detected by monitoring absorption at a wavelength of 254 nm [14]. A solution of biphenyl (100 ll/ml) was used to determine the peak retention time.

Results Isolation of PCBs-Degrading Bacteria A total of 66 strains were isolated from four soil samples. They can be divided into 14 types on the basis of their 16 S rDNA sequences: Enterobacter sp., Pseudomonas sp., Acinetobacter sp., Sphingobacterium sp., Citrobacter sp., Rhodococcus sp., Alcaligenes sp., Aeromonas sp., Pantoea sp., Stenotrophomonas sp., Cytophaga sp., Buttiauxella sp., Xanthomonas sp., Klebsiella sp., and Ralstonia sp. The Pseudomonas spp. together accounted for 27% of the total number of bacteria. Enterobacter sp., Acinetobacter sp., Aeromonas sp., and Citrobacter sp. each accounted for a smaller proportion (about 14, 13, 13, and 10%, respectively), and the other nine strains each accounted for an even smaller proportion (*2%) (Fig. 1). Classification of PCBs-Degrading Bacteria in Four Samples Sample 1 contained nine Enterobacter sp. strains, eleven Pseudomonas sp. strains, four Acinetobacter sp. strains, and one Sphingobacterium sp. strain. Sample 2 included two Citrobacter sp. strains, five Pseudomonas sp. strains, one Rhodococcus sp. strain, one Alcaligenes sp. strain, three Aeromonas sp. strains, one Acinetobacter sp. strain, one Pantoea sp. strain, one Stenotrophomonas sp. strain, and one Cytophaga sp. strain. Sample 3 contained an Enterobacter sp. strain, two Pseudomonas sp. strains, five Aeromonas sp. strains, two Stenotrophomonas sp. strains, one Acinetobacter sp. strain, one Buttiauxella sp. strain,

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and one Xanthomonas sp. strain. Sample 4 contained five Citrobacter sp. strains, one Aeromonas sp. strain, three Acinetobacter sp. strains, one Pseudomonas sp. strain, one Klebsiella sp. strain, and one Ralstonia sp. strain. The number of strains isolated was: sample 1, 25; sample 2, 16; sample 3, 13; and sample 4, 12. There were nine types of strain isolated from sample 2, which was much more diverse than the other samples (Fig. 2). Degradation of PCBs/Biphenyl When pure biphenyl was subjected to HPLC, the highest peak appeared at a retention time of *5.8 min (Fig. 3a). The concentration of five samples was shown to be proportional to the peak area. Four Pseudomonas sp. strains were selected at random from the four soil samples: strain 1-212 from sample 1; strain 2-241 from sample 2; strain 3318 from sample 3; and strain 4-150 from sample 4. Peak height on the chromatogram decreased in the order CK [ 4-150 [ 2-241 [ 3-318 [ 1-212 (Fig. 3b, c). Setting the content of the biphenyl in CK as 100%, the biphenyl content in the strains 1–4 was 2.32% (1-212), 73.11% (2-241), 69.83% (3-318), and 86.16% (4-150), respectively. It is clear that Pseudomonas sp. strain 1-212 had degraded the biphenyl almost completely, whereas the other three strains had degraded only 15–30% of the biphenyl. This shows that all of the strains were able to degrade the biphenyl, but to very different extents. Taxonomic Identification of the Selected Strains Analysis of the 16 S rDNA gene sequence indicates that four experimental strains belong to the genus Pseudomonas. A phylogenetic tree was constructed for the taxonomic location of the four strains, which showed that the evolutionary distance among the four Pseudomonas sp. strains was very short (Fig. 4). Pseudomonas sp. strain PSD-7 was

Fig. 1 The overall situation of PCBs-degrading strains in the Shanghai area

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Fig. 2 The distribution of PCBs-degrading strains in different soil samples

the nearest to them and strains 1-212 and 2-241 are probably attributable to the same Pseudomonas sp.

Discussion Polychlorinated biphenyls are environmental pollutants, which are distributed widely in the world. These compounds have been shown to undergo biodegradation under a variety of conditions in the laboratory and in the environment. The two distinct biological systems (aerobic oxidative processes and anaerobic reductive processes) have been identified to be capable of biodegrading PCBs [28–30]. Numerous reports of PCBs-degrading bacterial strains have been documented on isolation and biodegradation PCBs, many of which are common soil organisms. Shanghai has developed rapidly in recent decades and along with growth of the economy, environmental pollution is a subject worthy of concern. The city suffers from the impact of manufacture and using of chemical industrial products, including PCBs [31, 32]. PCBs are industrial pollutants that can impact on organisms through the food chain, and are recognized carcinogenic agents. PCB pollution of the environment has attracted much attention. The current approaches to the degradation of PCBs can be divided into heat, chemical, and microbiological. Of these, biodegradation, including microbial degradation and plant degradation, is the most promising strategy because of the relatively low cost and the absence of secondary pollution [30, 33]. In this study, PCB-degrading bacteria were isolated and characterized in soil samples taken from four sites in Shanghai. Both the number of all bacteria and the number of bacteria types were high, which might be associated with industrial development. The number and the types of

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PCB-degrading bacteria varied among the four soil samples. The number of strains isolated from sample 1 was greater and the types of PCBs-degrading bacteria were fewer than those in the other samples. The strains of bacteria were more varied in soil samples 2 and 4, which were collected from a riverbed, where the environment can change rapidly. In recent years, site 3, a disused chemical factory that used PCBs in the past, underwent enormous changes with rapid growth in PuDong New District. The PCBs-degrading ability of the bacteria in sample 3 was not strong, probably due to the change of the land. Overall, the types of PCBs-degrading bacteria in Shanghai were relatively abundant. Given the distribution of the bacteria, the fact that only Pseudomonas sp. strains were found in all four samples might because they can grow in simple nutritional conditions. The PCBs-degrading ability of four Pseudomonas sp. strains was analyzed. HPLC showed that strain 1-212 degraded *97% of biphenyl with respect to CK, which was set at 100%, whereas strain 3-318 degraded 21% of biphenyl and strains 2-241 and 4-150 degraded about 15% of biphenyl. Those four PCBs-degrading bacteria belong to the same type of Pseudomonas. To compare the sequences of the four Pseudomonas sp. strains isolated in this study with known Pseudomonas sp. strains, we constructed phylogenetic tree and found that they have a very close genetic relationship. Furthermore, the phylogenetic tree analysis indicated that they belong to the same branch. The strain 1-212, which isolated from experimental field, degraded significantly more biphenyl than the other three strains. The degradation efficiency of the other three stains, Fig. 3 The results of HPLC chromatogram. a The HPLC chromato- c gram of pure biphenyl (100 lg/ml); b The HPLC chromatogram of each sample. From top to bottom: CK, 1-212, 2-241, 3-318, 4-150. c Comparison of the four experimental samples and CK

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Fig. 4 The 16 S rDNA sequence homology analysis of four Pseudomonas sp. strains. The phylogenetic tree was constructed with MEGA4 software

which from riverbed and chemical industrial zone, is similar. Those results confirmed that the screened bacteria really were capable of degradation of PCBs; in addition, external conditions influenced the PCBs-degrading ability. Here, the abundance, identities, and degradation abilities of indigenous PCB-degrading bacteria from four potential PCBs contaminated sites in Shanghai were investigated. Our future work will include screening bacterial strains for efficient PCBs degradation, cloning the gene cluster from those new strains, restructuring more efficient bioengineered strains, and applying them to the biodegradation of environmental PCB contamination. Acknowledgments The research was supported by the Hi-tech Research and Development 863 Program of China (2008AA10Z401); Shanghai Rising-Star Program and Natural Science Foundation (08QH14021).

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