Appl Microbiol Biotechnol DOI 10.1007/s00253-014-6299-x
ENVIRONMENTAL BIOTECHNOLOGY
Abundance and diversity of polycyclic aromatic hydrocarbon degradation bacteria in urban roadside soils in Shanghai Xiaofei Li & Lijun Hou & Min Liu & Yanling Zheng & Ye Li & Xianbiao Lin
Received: 8 November 2014 / Revised: 2 December 2014 / Accepted: 4 December 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Understanding the impact of polycyclic aromatic hydrocarbons (PAHs) on soil environments is of increasingly important concern. Therefore, the microbial degradation of PAHs in soils has drawn considerable attention, but little is known about the PAH degradation genes in urban soils. In this study, we examined the diversity and abundance of the PAH degradation bacteria and evaluated whether the specific bacteria can reflect PAH contents in the soils from urban roadsides directly receiving traffic emission. The results of phylogenetic analysis indicated that low PAH degradation bacterial diversity occurred in the urban roadside soils, only including Mycobacterium sp., Terrabacter sp., and one novel cluster. The community composition diversity of PAH degradation bacteria did not show a significant difference across the sampling sites. The abundance of PAH degradation genes ranged from 5.70×106 to 6.44×107 gene copies g−1 dry soil, with an average abundance of 1.43×107 gene copies g−1 dry soil, and their spatial variations were related significantly to PAH contents in the soils. The Mycobacterium sp. was the most widely detected and estimated to occupy 65.9–100 % of the total PAH degradation bacteria at most of the soil samples, implying that
Xiaofei Li and Lijun Hou contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s00253-014-6299-x) contains supplementary material, which is available to authorized users. X. Li : M. Liu (*) : Y. Li : X. Lin College of Geographical Sciences, East China Normal University, 3663 North Zhongshan Road, Shanghai 200062, China e-mail: [email protected] L. Hou (*) : Y. Zheng State Key Laboratory of Estuarine and Coastal Research, East China Normal University, 3663 North Zhongshan Road, Shanghai 200062, China e-mail: [email protected]
the Mycobacterium sp. might play a primary role in degrading PAHs in the contaminated urban soil environments. Keywords Polycyclic aromatic hydrocarbons (PAHs) . Bacterial community . Degradation . Urban soil . Shanghai
Introduction Polycyclic aromatic hydrocarbons (PAHs), which are mainly derived from incomplete combustion of fossil fuels and biomass (Yunker et al. 1996; Liu et al. 2012), have become the focus of increased environmental and health concerns (Qin et al. 2011) because of their toxic, carcinogenic, and mutagenic characteristics (Liu et al. 2012; Heywood et al. 2006). Over the past several years, PAHs have been detected widely in urban soils due mainly to excessive traffic emission (Johnsen et al. 2005; Peng et al. 2011; Cachada et al. 2012; Meynet et al. 2012; Wang et al. 2014). As a mixture of different PAH compounds (Muangchinda et al. 2013), PAHs are favored to accumulate in various environment mediums, due to their stability which delays their degradation (Havelcová et al. 2014). Therefore, an increased soil PAH pollution would become one of the serious urban environmental problems and has potential effects on human health (Qin et al. 2011), emphasizing the need to understand PAH removal in soils. Due to the diversity and abundance of microorganism in soils, bacteria have been considered to be an important contributor to PAH degradation (Andersson et al. 2003). With the improvement of molecular techniques, polymerase chain reaction (PCR) provides an opportunity for detecting the diversity and abundance of PAH degradation genes in contaminated soil without cultivation biases (Brunk and Li AvanissAghajani 2002). Ding et al. (2010) have demonstrated that the abundance and diversity of PAH-RHDa gene were different after the same amount of phenanthrene spiking in two
Appl Microbiol Biotechnol
soils, indicating that soil types affect the PAH degradation microbial community. Numerous incubation experiments have also revealed that the microbial community structure changed after exposure to PAHs spiked in soils, and the mycorrhiza was attributed to reduction in PAHs (Joner et al. 2001; Wu et al. 2008). In addition, the content of PAHs, together with the specific environmental conditions, can influence the abundance and diversity of the ndo gene encoding naphthalene dioxygenase in mangrove sediments (Gomes et al. 2007). Therefore, the microbial degradation of PAHs, which is the best and friendly method of cleaning contaminated soils, has become a study hot spot (Lladό et al. 2009; Xu et al. 2014). So far, numerous studies of PAH degradation bacteria have focused on mangrove soils and crude oil-contaminated soils (Gomes et al. 2007; Park and Crowley 2006; Paissé et al. 2012; Meynet et al. 2012). Urban soils, as the receiver of multifarious pollutants in cities, are heavily contaminated by PAHs from vehicle emissions and atmosphere deposition, posing a potential threat to the urban environment. The soils with intensive inputs are therefore hypothesized to be PAH contamination hot spots, due to their features of long-time existence and bioaccumulation (Johnsen et al. 2005; Peng et al. 2011; Cachada et al. 2012; Meynet et al. 2012). However, few studies on the diversity and abundance of PAH degradation bacterial community have been reported in the urban soils (Johnsen et al. 2014). Furthermore, few data are available to draw conclusions on the relationships of PAH contents with associated degradation function genes in these urban environments. Shanghai is one of the most densely populated and industrialized cities in the world, and it receives substantial PAHs from increased industrial activities, vehicle emissions, and domestic emissions (Wang et al. 2014). Consequently, the PAH pollution in its environmental mediums has drawn considerable attentions. At present, many studies just focused on the spatial and temporal distributions of PAH contents (Liu et al. 2007; Yu et al. 2014; Wang et al. 2014). Thus, the microbial degradation of PAHs becomes a major concern to further understand their fate in the urban environment of Shanghai. Although the serious effects of PAHs on the urban environment have been identified, few studies have examined the distributions of PAH degradation bacteria and associated relationship with PAH contents in the PAH-enriched urban soils as yet. In this work, we investigated the distribution, biodiversity, and abundance of PAH degradation bacterial communities in the urban roadside soils of Shanghai using molecular methods. Furthermore, environmental factors were determined and compared to investigate their correlations with PAH degradation bacteria in the urban roadside soils. Another goal was to describe the inherent link between the PAH contents and the diversity and abundance of these bacteria. This work may provide a new insight into the microbial PAH
cycling in the urban soils under a heavily contaminated environment.
Materials and methods Study area and sample collection Shanghai, situated in the east of China, is one of the most densely populated cities in the world (Fig. 1). In this city, the road network is dense, with over 5000 roads and road length totaling about 17,316 km (Shanghai Statistical Bureau 2014). The total road area of the road network is 268.13 km2, equivalent to about 4.23 % of Shanghai’s total land area (6340 km2). In addition, the total number of rapidly increasing cars in Shanghai is over two million (Shanghai Statistical Bureau 2014), resulting in serious traffic congestion. The inner ring road, middle ring road, outer ring road, Yan’an highway road and north-south highway road provide together over 60 % of Shanghai’s traffic volume and constitute the backbone of the road network. The traffic characteristics, including traffic flow, vehicle type, congestion time, and frequency, of these main roads span a wide range depending largely on commuter time, commuting distance, and commuter crowds. Surface soil samples (5 cm deep) were collected from 12 sites from the intersections between ring roads and highway roads located in the central urban area of Shanghai (Fig. 1), including the inner ring Luban Road (NL), Yanan Road (NY), Shanghai Indoor Stadium (NS), Zhongshan Road North (NZ), middle ring Wenshui Road (ZW), Hongxu Road (ZX), Zhenbei Road (ZZ), Jinjiang Park (ZJ), outer ring Hujiang Road (WJ), Huning Road (WN), Xinzhuang Road (WX), and Huqingping Road (WQ). These selected sampling sites represent the primary intersections of backbone roads in the central urban area of Shanghai. The sampling was conducted in early spring 2014. Soil samples were collected using stainless steel cores from 8 to 12 sampling plots at each site, depending on the roadside width and intersection size. After collection, all soil samples were stored in sterile plastic bags, stored in an incubator box with ice, and transported to the laboratory within 2 h. In the laboratory, each soil sample was immediately homogenized under a nitrogen atmosphere. Subsequently, one part of the homogeneous sample was freeze-dried for measurements of PAHs and soil physiochemical characteristics, while the other portion was stored at −80 °C for the DNA extraction and molecular microbial analysis. Analysis of sediment physiochemical parameters and PAHs Soil pH was measured using a Mettler-Toledo pH meter after soil was mixed with deionized water free of CO2 at a soil to water ratio of 1:2.5 (w/v). The contents of total organic C
Appl Microbiol Biotechnol
Fig. 1 The location of Shanghai (left) and sampling sites (right) in the study area
(TOC) and total N (TN) in the soil samples were determined using a CN thermal combustion furnace analyzer (Elementar analyzer vario Max CN, Germany) after soils were leached by 1 M HCl solution. Black carbon (BC) in soil samples was determined by the K2Cr2O7 oxidation method adapted from Song et al. (2002). The PAHs in soil samples were extracted according to the US EPA method (US EPA Method 3540C 1996). In brief, 2.5 g of soil was extracted with acetone-dichloromethane (1:1, v/v) for 24 h at 65 °C. Subsequently, the extracted solutions were concentrated, exchanged to hexane, and cleaned up over amorphous sodium sulfate-alumina-silica gel columns (Wang et al. 2014). The eluates were then concentrated and solvent exchanged. Sixteen EPA priority PAHs in concentrated extracts were identified by GC-MS equipped with a DB-5 polysiloxane polymer column (30 m×250 μm×0.25 μm) (Agilent 7890A/5975C). The oven temperature was held at 55 °C for 2 min, heated to 280 °C at a rate of 20 °C min−1 and held for 4 min, and then heated to 310 °C at a rate of 10 °C min−1 and held for 5 min. The injection volume was 1 μL in a splitless mode. Five deuterated PAH mixture standard solutions were added into the each extract solution prior to the measurement of PAHs to test accuracy. DNA extraction, gene amplification, sequencing, and phylogenetic analysis The total genomic DNA of soils was extracted using Powersoil™ DNA Isolation Kits (MOBIO, USA) according to the manufacturer’s protocols. PAH degradation genes from the extracted DNA were amplified with primers Gram-
positive PAH-RHD [GP] F (5′-CGGCGCCGACAAYTTY GTNGG-3′) and PAH-RHD[GP]R (5′-GGGGAACACGGT GCCRTGDATRAA-3′), and Gram-negative PAH-RHD[GN]F (5′-GAGATGCATACCACGTKGGTTGGA-3′) and PAHRHD[GN]R (5′-AGCTGTTGTTCGGGAA GAYWGTGCMGTT-3′) (Cébron et al. 2008). The 50 μL of the PCR reaction system consisted of 5 μL of 10× PCR buffer (without MgCl2; Sangon, China), 4 μL of MgCl2 (25 mM; Sangon), 1 μL of dNTP (10 mM; Sangon), 1 μL of each primer (10 μM; Sangon), 1 μL of Taq DNA Polymerase (5 U μL−1; Sangon), and 1 μL of template DNA. Thermal cycling conditions were conducted with 5 min at 95 °C, followed by 30 cycles of 95 °C for 30 s, 54 °C for 30 s, and 72 °C for 30 s, and a final 7-min extension cycle at 72 °C. Subsequently, PCR products were separated by electrophoresis in 1 % agarose gels and purified using Gel Advance-Gel Extraction System (Viogene, China). Eventually, a positive amplification gene was obtained from all samples, whereas that of the negative amplification gene was not found at all samples. The purified fragments were cloned using the TOPO-TA cloning kit (Invitrogen, USA) in accordance with the manufacturer’s instructions. The ABI Prism genetic analyzer was run to sequence screened clones (Applied Biosystems, Canada). The PAH degradation gene sequences in this study have been deposited in GenBank under accession numbers KP050795 to KP051278. All the sequences and their relatives obtained from the NCBI were aligned by using the ClustalX2 program (version 2.1) (Thompson et al. 1997). The sequences with more than 97 % identity were grouped into one operational taxonomic unit (OTU) using the Mothur program (version
Appl Microbiol Biotechnol
1.31.2; USA) by the furthest neighbor approach. Phylogenetic trees were created using the MEGA software (version 5.1) by neighbor-joining (NJ) method (Kumar et al. 2004). The relative confidence of the tree topologies was evaluated by performing 1000 bootstrap replicates (Tamura et al. 2007). Real-time quantitative PCR Plasmids of the PAH degradation gene fragment were extracted from Escherichia coli hosts using a Plasmid Mini Preparation Kit (Tiangen, China) to construct standard curves. Concentrations of plasmid DNA were measured using a SMA4000 Spectrophotometer (Merinton, China). The standard curves spanned tenfold concentration gradients ranging from 4.55×102 to 4.55×109 copies per μL for the PAH degradation genes. The gene copy numbers of each sample in triplicate were identified by an ABI 7500 Sequence Detection System (Applied Biosystems, Canada) using the SYBR Green quantitative PCR (qPCR) method. The 25-μL qPCR mixture contained 12.5 μL of Maxima SYBR Green/Rox qPCR Master Mix (Fermentas, Lithuania), 1 μL of each primer (10 μM), 1 μL template DNA, and 9.5 μL of sterile water without DNA. All reactions were performed in eightstrip thin-walled PCR tubes with ultraclean cap strips (ABgene, UK). The qPCR cycling conditions were as follows: 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles of 30 s at 95 °C, 30 s at 54 °C, and 30 s at 72 °C. Three negative controls of sterile water without DNA were performed to detect and exclude any possible pollutant. A strong linear relationship between the threshold cycle (CT) and the natural log10-transformed value of gene copy number (r2 =0.994) was obtained in the standard curve assay. The amplification efficiencies were 98.4 % for PAH degradation genes. In addition, only one observable peak at a melting temperature (Tm =87.55) and no detectable peaks associated with primer-dimer artifacts or other nonspecific PCR amplification products were observed through melting curve analyses. The gene abundances of each DNA template were calculated based on the constructed standard curve and then converted to copies per gram of dry soil, assuming 100 % DNA extraction efficiency (Zheng et al. 2014). Statistical analysis The Mothur program (version 1.31.2; USA) was performed to generate rarefaction curves and to determine biodiversity indicators (Shannon and Chao 1) of PAH degradation genes. The coverage of each clone library was estimated by the percentage of the OTUs divided by Chao 1. The correlations between bacterial communities with environmental factors were determined by redundancy analysis (RDA) using the Software for Canonical Community Ordination (ter Braak and Šmilauer 2002). Pearson correlation analyses were
performed by SPSS program (version 19.0) to examine correlations between abundance and diversity of degradation gene and PAH contents. In addition, one-way analysis of variance (ANOVA), followed by Tukey’s honestly significant difference (HSD) test, was conducted to analyze the significant differences of abundance and diversity of PAH degradation genes among the sites.
Results Soil physicochemical variables Soil pH varied from 7.16 to 7.78 at the study area, with the highest pH value at site ZH and the lowest pH value at site NJ (Table 1). Higher concentrations of organic carbon were detected at the outer ring roadside sites (ranging from 30.63 to 36.66 mg g−1), while relatively lower concentrations at the middle ring roadside sites (ranging from 24.01 to 26.26 mg g−1) (dry weight, similarly hereinafter). The concentrations of total nitrogen in soils ranged from 1.37 to 2.85 mg g−1 with high spatial heterogeneity. The molar ratios of C to N (C:N) in soils ranged from 12.12 to 22.10 with lower ratios at the high-nitrogen sampling sites. Concentrations of black carbon in soils were in the range of 11.50– 25.71 mg g−1, which exhibited a positive correlation with organic carbon concentrations (r=0.934, P0.63, P
ENVIRONMENTAL BIOTECHNOLOGY
Abundance and diversity of polycyclic aromatic hydrocarbon degradation bacteria in urban roadside soils in Shanghai Xiaofei Li & Lijun Hou & Min Liu & Yanling Zheng & Ye Li & Xianbiao Lin
Received: 8 November 2014 / Revised: 2 December 2014 / Accepted: 4 December 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Understanding the impact of polycyclic aromatic hydrocarbons (PAHs) on soil environments is of increasingly important concern. Therefore, the microbial degradation of PAHs in soils has drawn considerable attention, but little is known about the PAH degradation genes in urban soils. In this study, we examined the diversity and abundance of the PAH degradation bacteria and evaluated whether the specific bacteria can reflect PAH contents in the soils from urban roadsides directly receiving traffic emission. The results of phylogenetic analysis indicated that low PAH degradation bacterial diversity occurred in the urban roadside soils, only including Mycobacterium sp., Terrabacter sp., and one novel cluster. The community composition diversity of PAH degradation bacteria did not show a significant difference across the sampling sites. The abundance of PAH degradation genes ranged from 5.70×106 to 6.44×107 gene copies g−1 dry soil, with an average abundance of 1.43×107 gene copies g−1 dry soil, and their spatial variations were related significantly to PAH contents in the soils. The Mycobacterium sp. was the most widely detected and estimated to occupy 65.9–100 % of the total PAH degradation bacteria at most of the soil samples, implying that
Xiaofei Li and Lijun Hou contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s00253-014-6299-x) contains supplementary material, which is available to authorized users. X. Li : M. Liu (*) : Y. Li : X. Lin College of Geographical Sciences, East China Normal University, 3663 North Zhongshan Road, Shanghai 200062, China e-mail: [email protected] L. Hou (*) : Y. Zheng State Key Laboratory of Estuarine and Coastal Research, East China Normal University, 3663 North Zhongshan Road, Shanghai 200062, China e-mail: [email protected]
the Mycobacterium sp. might play a primary role in degrading PAHs in the contaminated urban soil environments. Keywords Polycyclic aromatic hydrocarbons (PAHs) . Bacterial community . Degradation . Urban soil . Shanghai
Introduction Polycyclic aromatic hydrocarbons (PAHs), which are mainly derived from incomplete combustion of fossil fuels and biomass (Yunker et al. 1996; Liu et al. 2012), have become the focus of increased environmental and health concerns (Qin et al. 2011) because of their toxic, carcinogenic, and mutagenic characteristics (Liu et al. 2012; Heywood et al. 2006). Over the past several years, PAHs have been detected widely in urban soils due mainly to excessive traffic emission (Johnsen et al. 2005; Peng et al. 2011; Cachada et al. 2012; Meynet et al. 2012; Wang et al. 2014). As a mixture of different PAH compounds (Muangchinda et al. 2013), PAHs are favored to accumulate in various environment mediums, due to their stability which delays their degradation (Havelcová et al. 2014). Therefore, an increased soil PAH pollution would become one of the serious urban environmental problems and has potential effects on human health (Qin et al. 2011), emphasizing the need to understand PAH removal in soils. Due to the diversity and abundance of microorganism in soils, bacteria have been considered to be an important contributor to PAH degradation (Andersson et al. 2003). With the improvement of molecular techniques, polymerase chain reaction (PCR) provides an opportunity for detecting the diversity and abundance of PAH degradation genes in contaminated soil without cultivation biases (Brunk and Li AvanissAghajani 2002). Ding et al. (2010) have demonstrated that the abundance and diversity of PAH-RHDa gene were different after the same amount of phenanthrene spiking in two
Appl Microbiol Biotechnol
soils, indicating that soil types affect the PAH degradation microbial community. Numerous incubation experiments have also revealed that the microbial community structure changed after exposure to PAHs spiked in soils, and the mycorrhiza was attributed to reduction in PAHs (Joner et al. 2001; Wu et al. 2008). In addition, the content of PAHs, together with the specific environmental conditions, can influence the abundance and diversity of the ndo gene encoding naphthalene dioxygenase in mangrove sediments (Gomes et al. 2007). Therefore, the microbial degradation of PAHs, which is the best and friendly method of cleaning contaminated soils, has become a study hot spot (Lladό et al. 2009; Xu et al. 2014). So far, numerous studies of PAH degradation bacteria have focused on mangrove soils and crude oil-contaminated soils (Gomes et al. 2007; Park and Crowley 2006; Paissé et al. 2012; Meynet et al. 2012). Urban soils, as the receiver of multifarious pollutants in cities, are heavily contaminated by PAHs from vehicle emissions and atmosphere deposition, posing a potential threat to the urban environment. The soils with intensive inputs are therefore hypothesized to be PAH contamination hot spots, due to their features of long-time existence and bioaccumulation (Johnsen et al. 2005; Peng et al. 2011; Cachada et al. 2012; Meynet et al. 2012). However, few studies on the diversity and abundance of PAH degradation bacterial community have been reported in the urban soils (Johnsen et al. 2014). Furthermore, few data are available to draw conclusions on the relationships of PAH contents with associated degradation function genes in these urban environments. Shanghai is one of the most densely populated and industrialized cities in the world, and it receives substantial PAHs from increased industrial activities, vehicle emissions, and domestic emissions (Wang et al. 2014). Consequently, the PAH pollution in its environmental mediums has drawn considerable attentions. At present, many studies just focused on the spatial and temporal distributions of PAH contents (Liu et al. 2007; Yu et al. 2014; Wang et al. 2014). Thus, the microbial degradation of PAHs becomes a major concern to further understand their fate in the urban environment of Shanghai. Although the serious effects of PAHs on the urban environment have been identified, few studies have examined the distributions of PAH degradation bacteria and associated relationship with PAH contents in the PAH-enriched urban soils as yet. In this work, we investigated the distribution, biodiversity, and abundance of PAH degradation bacterial communities in the urban roadside soils of Shanghai using molecular methods. Furthermore, environmental factors were determined and compared to investigate their correlations with PAH degradation bacteria in the urban roadside soils. Another goal was to describe the inherent link between the PAH contents and the diversity and abundance of these bacteria. This work may provide a new insight into the microbial PAH
cycling in the urban soils under a heavily contaminated environment.
Materials and methods Study area and sample collection Shanghai, situated in the east of China, is one of the most densely populated cities in the world (Fig. 1). In this city, the road network is dense, with over 5000 roads and road length totaling about 17,316 km (Shanghai Statistical Bureau 2014). The total road area of the road network is 268.13 km2, equivalent to about 4.23 % of Shanghai’s total land area (6340 km2). In addition, the total number of rapidly increasing cars in Shanghai is over two million (Shanghai Statistical Bureau 2014), resulting in serious traffic congestion. The inner ring road, middle ring road, outer ring road, Yan’an highway road and north-south highway road provide together over 60 % of Shanghai’s traffic volume and constitute the backbone of the road network. The traffic characteristics, including traffic flow, vehicle type, congestion time, and frequency, of these main roads span a wide range depending largely on commuter time, commuting distance, and commuter crowds. Surface soil samples (5 cm deep) were collected from 12 sites from the intersections between ring roads and highway roads located in the central urban area of Shanghai (Fig. 1), including the inner ring Luban Road (NL), Yanan Road (NY), Shanghai Indoor Stadium (NS), Zhongshan Road North (NZ), middle ring Wenshui Road (ZW), Hongxu Road (ZX), Zhenbei Road (ZZ), Jinjiang Park (ZJ), outer ring Hujiang Road (WJ), Huning Road (WN), Xinzhuang Road (WX), and Huqingping Road (WQ). These selected sampling sites represent the primary intersections of backbone roads in the central urban area of Shanghai. The sampling was conducted in early spring 2014. Soil samples were collected using stainless steel cores from 8 to 12 sampling plots at each site, depending on the roadside width and intersection size. After collection, all soil samples were stored in sterile plastic bags, stored in an incubator box with ice, and transported to the laboratory within 2 h. In the laboratory, each soil sample was immediately homogenized under a nitrogen atmosphere. Subsequently, one part of the homogeneous sample was freeze-dried for measurements of PAHs and soil physiochemical characteristics, while the other portion was stored at −80 °C for the DNA extraction and molecular microbial analysis. Analysis of sediment physiochemical parameters and PAHs Soil pH was measured using a Mettler-Toledo pH meter after soil was mixed with deionized water free of CO2 at a soil to water ratio of 1:2.5 (w/v). The contents of total organic C
Appl Microbiol Biotechnol
Fig. 1 The location of Shanghai (left) and sampling sites (right) in the study area
(TOC) and total N (TN) in the soil samples were determined using a CN thermal combustion furnace analyzer (Elementar analyzer vario Max CN, Germany) after soils were leached by 1 M HCl solution. Black carbon (BC) in soil samples was determined by the K2Cr2O7 oxidation method adapted from Song et al. (2002). The PAHs in soil samples were extracted according to the US EPA method (US EPA Method 3540C 1996). In brief, 2.5 g of soil was extracted with acetone-dichloromethane (1:1, v/v) for 24 h at 65 °C. Subsequently, the extracted solutions were concentrated, exchanged to hexane, and cleaned up over amorphous sodium sulfate-alumina-silica gel columns (Wang et al. 2014). The eluates were then concentrated and solvent exchanged. Sixteen EPA priority PAHs in concentrated extracts were identified by GC-MS equipped with a DB-5 polysiloxane polymer column (30 m×250 μm×0.25 μm) (Agilent 7890A/5975C). The oven temperature was held at 55 °C for 2 min, heated to 280 °C at a rate of 20 °C min−1 and held for 4 min, and then heated to 310 °C at a rate of 10 °C min−1 and held for 5 min. The injection volume was 1 μL in a splitless mode. Five deuterated PAH mixture standard solutions were added into the each extract solution prior to the measurement of PAHs to test accuracy. DNA extraction, gene amplification, sequencing, and phylogenetic analysis The total genomic DNA of soils was extracted using Powersoil™ DNA Isolation Kits (MOBIO, USA) according to the manufacturer’s protocols. PAH degradation genes from the extracted DNA were amplified with primers Gram-
positive PAH-RHD [GP] F (5′-CGGCGCCGACAAYTTY GTNGG-3′) and PAH-RHD[GP]R (5′-GGGGAACACGGT GCCRTGDATRAA-3′), and Gram-negative PAH-RHD[GN]F (5′-GAGATGCATACCACGTKGGTTGGA-3′) and PAHRHD[GN]R (5′-AGCTGTTGTTCGGGAA GAYWGTGCMGTT-3′) (Cébron et al. 2008). The 50 μL of the PCR reaction system consisted of 5 μL of 10× PCR buffer (without MgCl2; Sangon, China), 4 μL of MgCl2 (25 mM; Sangon), 1 μL of dNTP (10 mM; Sangon), 1 μL of each primer (10 μM; Sangon), 1 μL of Taq DNA Polymerase (5 U μL−1; Sangon), and 1 μL of template DNA. Thermal cycling conditions were conducted with 5 min at 95 °C, followed by 30 cycles of 95 °C for 30 s, 54 °C for 30 s, and 72 °C for 30 s, and a final 7-min extension cycle at 72 °C. Subsequently, PCR products were separated by electrophoresis in 1 % agarose gels and purified using Gel Advance-Gel Extraction System (Viogene, China). Eventually, a positive amplification gene was obtained from all samples, whereas that of the negative amplification gene was not found at all samples. The purified fragments were cloned using the TOPO-TA cloning kit (Invitrogen, USA) in accordance with the manufacturer’s instructions. The ABI Prism genetic analyzer was run to sequence screened clones (Applied Biosystems, Canada). The PAH degradation gene sequences in this study have been deposited in GenBank under accession numbers KP050795 to KP051278. All the sequences and their relatives obtained from the NCBI were aligned by using the ClustalX2 program (version 2.1) (Thompson et al. 1997). The sequences with more than 97 % identity were grouped into one operational taxonomic unit (OTU) using the Mothur program (version
Appl Microbiol Biotechnol
1.31.2; USA) by the furthest neighbor approach. Phylogenetic trees were created using the MEGA software (version 5.1) by neighbor-joining (NJ) method (Kumar et al. 2004). The relative confidence of the tree topologies was evaluated by performing 1000 bootstrap replicates (Tamura et al. 2007). Real-time quantitative PCR Plasmids of the PAH degradation gene fragment were extracted from Escherichia coli hosts using a Plasmid Mini Preparation Kit (Tiangen, China) to construct standard curves. Concentrations of plasmid DNA were measured using a SMA4000 Spectrophotometer (Merinton, China). The standard curves spanned tenfold concentration gradients ranging from 4.55×102 to 4.55×109 copies per μL for the PAH degradation genes. The gene copy numbers of each sample in triplicate were identified by an ABI 7500 Sequence Detection System (Applied Biosystems, Canada) using the SYBR Green quantitative PCR (qPCR) method. The 25-μL qPCR mixture contained 12.5 μL of Maxima SYBR Green/Rox qPCR Master Mix (Fermentas, Lithuania), 1 μL of each primer (10 μM), 1 μL template DNA, and 9.5 μL of sterile water without DNA. All reactions were performed in eightstrip thin-walled PCR tubes with ultraclean cap strips (ABgene, UK). The qPCR cycling conditions were as follows: 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles of 30 s at 95 °C, 30 s at 54 °C, and 30 s at 72 °C. Three negative controls of sterile water without DNA were performed to detect and exclude any possible pollutant. A strong linear relationship between the threshold cycle (CT) and the natural log10-transformed value of gene copy number (r2 =0.994) was obtained in the standard curve assay. The amplification efficiencies were 98.4 % for PAH degradation genes. In addition, only one observable peak at a melting temperature (Tm =87.55) and no detectable peaks associated with primer-dimer artifacts or other nonspecific PCR amplification products were observed through melting curve analyses. The gene abundances of each DNA template were calculated based on the constructed standard curve and then converted to copies per gram of dry soil, assuming 100 % DNA extraction efficiency (Zheng et al. 2014). Statistical analysis The Mothur program (version 1.31.2; USA) was performed to generate rarefaction curves and to determine biodiversity indicators (Shannon and Chao 1) of PAH degradation genes. The coverage of each clone library was estimated by the percentage of the OTUs divided by Chao 1. The correlations between bacterial communities with environmental factors were determined by redundancy analysis (RDA) using the Software for Canonical Community Ordination (ter Braak and Šmilauer 2002). Pearson correlation analyses were
performed by SPSS program (version 19.0) to examine correlations between abundance and diversity of degradation gene and PAH contents. In addition, one-way analysis of variance (ANOVA), followed by Tukey’s honestly significant difference (HSD) test, was conducted to analyze the significant differences of abundance and diversity of PAH degradation genes among the sites.
Results Soil physicochemical variables Soil pH varied from 7.16 to 7.78 at the study area, with the highest pH value at site ZH and the lowest pH value at site NJ (Table 1). Higher concentrations of organic carbon were detected at the outer ring roadside sites (ranging from 30.63 to 36.66 mg g−1), while relatively lower concentrations at the middle ring roadside sites (ranging from 24.01 to 26.26 mg g−1) (dry weight, similarly hereinafter). The concentrations of total nitrogen in soils ranged from 1.37 to 2.85 mg g−1 with high spatial heterogeneity. The molar ratios of C to N (C:N) in soils ranged from 12.12 to 22.10 with lower ratios at the high-nitrogen sampling sites. Concentrations of black carbon in soils were in the range of 11.50– 25.71 mg g−1, which exhibited a positive correlation with organic carbon concentrations (r=0.934, P0.63, P
