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Appl Microbiol Biotechnol (2014) 98:875–884 DOI 10.1007/s00253-013-4854-5 ENVIRONMENTAL BIOTECHNOLOGY Bacterial polycyclic aromatic hydrocarbon ring-hydroxylating dioxygenases in the sediments from the Pearl River estuary, China Peng Wu & You-Shao Wang & Fu-Lin Sun & Mei-Lin Wu & Ya-lan Peng Received: 17 December 2012 /Revised: 10 March 2013 /Accepted: 11 March 2013 /Published online: 5 April 2013 # Springer-Verlag Berlin Heidelberg 2013 Abstract Bacterial community compositions were charac- Introduction terized using denaturing gradient gel electrophoresis anal- ysis of bacterial 16S rRNA gene in the sediments of the Polycyclic aromatic hydrocarbons (PAHs) are a class of Pearl River estuary. Sequencing analyses of the excised ubiquitous hydrophobic organic pollutants with known or bands indicated that Gram-negative bacteria, especially potential toxic, mutagenic, or carcinogenic properties Gammaproteobacteria, were dominant in the Pearl River (DeBruyn et al. 2007; Zhou et al. 2006). Due to the low estuary. The diversity of polycyclic aromatic hydrocarbon solubility of PAHs in water, the sediment represents a major ring-hydroxylating dioxygenase (PAH-RHD) gene in this sink for PAHs released to the environment (Pohlman et al. estuary was then assessed by clone library analysis. The 2002). The Pearl River Delta (PRD) region, one of the phylogenetic analyses showed that all PAH-RHD gene economically fastest growing regions in China, has under- sequences of Gram-negative bacteria (PAH-RHD[GN])were gone rapid urbanization and industrialization (Guan et al. closely related to the nagAc gene described for Ralstonia sp. 2009). Eventually, the contamination of PAHs has been U2 or nahAc gene for Pseudomonas sp. 9816–4, while the reported in various environmental media in the Pearl River PAH-RHD gene sequences of Gram-positive bacteria (Liu et al. 2011; Luo et al. 2008; Mai et al. 2001). Based on (PAH-RHD[GP])atsamplingsiteA1showedhighsequence the ability of particular bacteria to degrade PAHs, bioreme- similarity to the nidA gene from Mycobacterium species. diation is proposed as a convenient tool for the cleanup of Meanwhile, molecular diversity of the two functional genes contaminated sites (Doyle et al. 2008). The diversity infor- was higher at the upstream of this region, while lower at the mation of bacteria involved in PAH decontamination can downstream. Redundancy analysis indicated that environmen- provide the basic knowledge for PAH bioremediation tech- tal factors, such as NH4-N, ∑PAHs, pH, SiO3-Si, and water nology in a natural environment. depth, affected the distribution of the PAH-RHD[GN] gene in The primary step of PAH metabolism commonly occurs via the Pearl River estuary. the incorporation of molecular oxygen into the aromatic nu- cleus by a multicomponent PAH ring-hydroxylating Keywords Polycyclic aromatic hydrocarbons . dioxygenase (PAH-RHD) enzyme (Ding et al. 2010;Penget Ring-hydroxylating dioxygenase . The Pearl River estuary . al. 2010). Iwai et al. (2011) have analyzed all the existing Bacterial community primer systems targeting the PAH-RHD enzymes and found that those enzymes can be divided into two subclades: PAH- : : : : RHD enzyme from Gram-negative bacteria (PAH-RHD[GN]) P. Wu Y.-S. Wang F.-L. Sun M.-L. Wu Y.-l. Peng and PAH-RHD enzyme from Gram-positive bacteria (PAH- State Key Laboratory of Tropical Oceanography, South China Sea Institute of Oceanology, Chinese Academy of Sciences, RHD[GP]). Many previous studies demonstrated that positive Guangzhou 510301, China amplicons of the PAH-RHD gene can be obtained only after additionally amending PAHs in sediments or soils (Bordenave : * : : : P. Wu Y.-S. Wang ( ) F.-L. Sun M.-L. Wu Y.-l. Peng et al. 2008; DeBruyn et al. 2007;Dingetal.2010;Meynetet Daya Bay Marine Biology Research Station, Chinese Academy of Sciences, Shenzhen 518121, China al. 2012;NiChadhainetal.2006;Pengetal.2010;Zhouetal. e-mail: [email protected] 2006). However, only several studies focused on the diversity 876 Appl Microbiol Biotechnol (2014) 98:875–884 of the PAH-RHD gene in the natural environment (Flocco et al. 2009; Gomes et al. 2007; Jurelevicius et al. 2012). Because the catabolic potential of the PRD region is essentially un- known, culture-independent methods targeting the PAH-RHD gene were employed to provide for a broader estimation of the biodegradation potential in this region. Bacterial community structure can be significantly influenced by environmental variables, and environmental heterogeneity can drive a taxa–area relationship of bacteria (Cao et al. 2012; Hong et al. 2011; Horner-Devine et al. 2004; Meynet et al. 2012; Zhang et al. 2009). For example, Cao et al. (2012) reported that water depth and temperature were major factors shaping the community structure of ammonia-oxidizing β-Proteobacteria from the Pearl River Delta to the South China Sea. Our previous study has also shown that NH4-N and SiO3-Si can obviously influence the bacterial community composition in the Pearl River estuary (Sun et al. 2011). Moreover, available nutrients (N/P), pH, and salinity were considered as the important factors in in Fig. 1 The sampling sites in the Pearl River estuary situ bioremediation of PAHs (Lu et al. 2011). Therefore, further studies should be conducted to determine the rela- tionships between environmental factors and the diversity of Guangzhou Channel, the Shiziyang Channel, the Lingding the PAH-RHD gene in the Pearl River estuary. Bay, and the northern South China Sea, respectively. In Surface sediments across a five-site transect along the order to investigate the relationship between PAHs and the upstream to the downstream of the Pearl River estuary were PAH-RHD gene, a field background value of PAHs was examined. The bacterial composition was firstly detected conducted by gas chromatography/mass spectrometry based on denaturing gradient gel electrophoresis (DGGE) (GC/MS) (N6890/5975B, Agilent, USA) following the analysis of bacterial 16S rRNA gene (16S-DGGE). methods EPA 3550C and EPA 8270D. The total concentra- Secondly, the PAH-RHD gene was amplified to determine tions of 16 priority PAHs (∑PAHs) at sites A1, A8, A10, C1, the diversity and distribution of PAH-degrading bacteria. and C5 were 2.09, 1.11, 0.48, 0.32, and 0.17 mg/kg, respec- Finally, statistical analysis was employed to investigate the tively. The results further showed that naphthalene, phenan- main potential physicochemical contributors to the distribu- threne, pyrene, and benzo[a]pyrene were the dominant tion of the PAH-RHD[GN] gene. components. Previous studies also demonstrated that PAHs were higher at upstream, but lower at the downstream of the Pearl River estuary (Luo et al. 2008; Mai et al. 2001). Material and methods Sample collection Characteristics of the Pearl River estuary Surface sediment samples (0–10 cm) were collected by a static The Pearl River estuary, a subtropical estuary, is the second gravity corer in August, 2010. When the sediments were re- largest in China in terms of discharge volume. This estuary trieved, air-exposed outer surface of the sediments was can be roughly divided into four parts—Guangzhou Channel, discarded and the central cores were taken with a sterile stainless Shiziyang Channel, Lingding Bay, and the northern South steel knife. Then, the sediment samples were stored at −20 °C in China Sea. The Guangzhou Channel drains over the city of sterile collection bags. Meanwhile, the overlying water from the Guangzhou, the most urbanized and heavily populated district same sampling site was collected, filtered through 0.45 μm in the PRD region. The Shiziyang Channel receives inflows membranes, and preserved at −20 °C for biogeochemical from the Guangzhou Channel and the East river. The Lingding analysis. Physicochemical parameters of the overlying water Bay, a funnel-shaped estuary, receives about 1.7×1014 L/a were determined according to Wang et al. (2008). fresh water runoff and nearly 3×108 tons/a of suspended particles from four outlets, i.e., Humen, Jiaomen, DNA extraction Hongqilimen, and Hengmen (Mai et al. 2001). In the present study, five sampling sites are selected (Fig. 1). Site A1, site Total community DNA (TC-DNA) of each sampling site A8, sites C1 and A10, and site C5 are located on the was extracted directly from 0.5 g sediment in triplicate using Appl Microbiol Biotechnol (2014) 98:875–884 877 TM the E.A.N.A. Soil DNA Kit (OMEGA, USA) following al. 2008). However, for the PAH-RHD[GP] gene, the first the manufacturer’s instructions. TC-DNA was visualized round of PCR was with primers Nid-for and Nid-rev1, while with 1 % agarose gel electrophoresis to assess its integrity. primers Nid-for and Nid-rev2 were then selected and used in All DNA preparations were stored at −20 °C prior to PCR the second round (Zhou et al. 2006). A high fidelity Ex Taq amplification. (Takara, Japan) was employed for amplification of the PAH- RHD gene. Positive PCR products were confirmed with elec- 16S-DGGE analyses of sediment bacterial community trophoresis on a 1.0 % agarose. Eventually, positive amplifi- cation of the PAH-RHD[GN] gene was obtained from all the A nested PCR was used for amplification of bacterial 16S five samples, whereas that of the PAH-RHD[GP] gene was rRNA gene. Briefly, the first round of PCR was with primers only found at site A1. 27 F and 1492R (Suzuki and Giovannoni 1996), and the second round with primers 341 F (with a GC clamp attached Clone library analyses of the PAH-RHD gene to the 5′ end) and 907R (Muyzer and Ramsing 1995). At this stage, the PCR products obtained from the different triplicates The PCR products obtained from each sampling site in of each sampling site were individually pooled together before triplicate were pooled together and purified. Purified PCR loading on DGGE gel. products of the PAH-RHD gene were individually DGGE was performed using Dcode universal mutation inserted into the pMD18-T vector (Takara, Japan) for detection system as described in the manufacturer’s manual constructing clone libraries, and then positive clones (Bio-Rad, Hercules, CA, USA).

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