Appl Microbiol Biotechnol (2015) 99:1935–1946 DOI 10.1007/s00253-014-6074-z

ENVIRONMENTAL BIOTECHNOLOGY

Abundance and diversity of soil petroleum hydrocarbon-degrading microbial communities in oil exploring areas Yuyin Yang & Jie Wang & Jingqiu Liao & Shuguang Xie & Yi Huang

Received: 8 August 2014 / Revised: 2 September 2014 / Accepted: 3 September 2014 / Published online: 20 September 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Alkanes and polycyclic aromatic hydrocarbons (PAHs) are the commonly detected petroleum hydrocarbon contaminants in soils in oil exploring areas. Hydrocarbondegrading genes are useful biomarks for estimation of the bioremediation potential of contaminated sites. However, the links between environmental factors and the distribution of alkane and PAH metabolic genes still remain largely unclear. The present study investigated the abundances and diversities of soil n-alkane and PAH-degrading bacterial communities targeting both alkB and nah genes in two oil exploring areas at different geographic regions. A large variation in the abundances and diversities of alkB and nah genes occurred in the studied soil samples. Various environmental variables regulated the spatial distribution of soil alkane and PAH metabolic genes, dependent on geographic location. The soil alkanedegrading bacterial communities in oil exploring areas mainly consisted of Pedobacter, Mycobacterium, and unknown alkBharboring microorganisms. Moreover, the novel PAHdegraders predominated in nah gene clone libraries from soils of the two oil exploring areas. This work could provide some new insights towards the distribution of hydrocarbondegrading microorganisms and their biodegradation potential in soil ecosystems.

Yuyin Yang and Jie Wang contributed equally to this study. Electronic supplementary material The online version of this article (doi:10.1007/s00253-014-6074-z) contains supplementary material, which is available to authorized users. Y. Yang : J. Wang : J. Liao : S. Xie (*) : Y. Huang (*) State Key Joint Laboratory of Environmental Simulation and Pollution Control (Peking University), College of Environmental Sciences and Engineering, Peking University, Beijing 100871, China e-mail: [email protected] e-mail: [email protected]

Keywords Alkane . Biodegradation . alkB . nah . Geographic location . Oil exploring area . Petroleum hydrocarbon . Polycyclic aromatic hydrocarbons (PAHs)

Introduction Crude oil is a complex mixture containing a variety of distinctively different chemicals, such as alkanes, aromatic hydrocarbons, and non-hydrocarbon compounds (Liang et al. 2012). Various oil industrial activities have resulted in the widespread distribution of petroleum hydrocarbons in the environment. High levels of alkanes and polycyclic aromatic hydrocarbons (PAHs) can usually be detected in contaminated soils in oil exploring areas (Liang et al. 2011, 2012; Yang et al. 2014). Petroleum hydrocarbon contamination may pose a significant threat to local ecosystem and human health (Liang et al. 2012; Zhang et al. 2012). Biodegradation using autochthonous microbiota has been accepted as a cost-effective alternative to dissipate these petroleum hydrocarbon compounds from contaminated soils (Chandra et al. 2013; Fukuhara et al. 2013; Shankar et al. 2014). Therefore, the knowledge of the hydrocarbondegradation potential in contaminated soil is crucial for the management of soils for bioremediation (Jurelevicius et al. 2012; Liang et al. 2011). In order to study the fate of petroleum hydrocarbons in soil ecosystems, numerous oil-degrading microorganisms from diverse bacterial genera have been isolated from hydrocarbon-contaminated sites, such as Pseudomonas (AlMailem et al. 2014; Pacwa-Płociniczak et al. 2014), Bacillus and Sphingobacterium (Yu et al. 2014), Streptomyces (Ferradji et al. 2014), Pestalotiopsis (Yanto and Tachibana 2013), Rhodococcus, Acinetobacter, Burkholderia, and Achromobacter (Tanase et al. 2013), and Arthrobacter and Pimelobacter (Margesin et al. 2013). However, traditional

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culture-dependent methods can underestimate the diversity of hydrocarbon-degrading bacterial populations (Sun and Cupples 2012; Sun et al. 2014a; Yang et al. 2014). Moreover, microbial community plays a collaborative role in the dissipation of hydrocarbon pollutants (Fuentes et al. 2014). Molecular biology methods targeting 16S ribosomal (rDNA) genes can provide more comprehensive phylogenetic information of hydrocarbon-degrading bacterial communities in the environment (Hamamura et al. 2006; Sun et al. 2012; Ribeiro et al. 2013; Singleton et al. 2013), yet 16S methods typically cannot reveal the direct link between metabolic function and phylogenetic identity for environmental samples (Yang et al. 2014). Hydrocarbon-degrading genes can be useful biomarks for functionally characterizing bacterial community, which can present a broader estimation of the bioremediation potential of contaminated sites (Margesin et al. 2003; Liang et al. 2011; Vitte et al. 2013; Sun et al. 2014a,b; Yang et al. 2014). The alkane and PAH-metabolic genes are two classes of the commonly studied hydrocarbon-degrading genes. So far, the presence, abundance, and diversity of alkane and PAH metabolic genes have been investigated in a variety of hydrocarbon-contaminated aquatic and terrestrial ecosystems (Powell et al. 2010; Perez-deMora et al. 2011; Guibert et al. 2012; Bengtsson et al. 2013; Smith et al. 2013; Yang et al. 2014). However, the links between environmental parameters and the distribution of alkane and PAH metabolic genes still remain unclear. The coexistence of alkane and PAH metabolic genes in the environment has yielded poor attention (Margesin et al. 2003; Liang et al. 2011). In addition, although heavy crude oil contamination usually exists in soils of oil exploring area, little is known about the distribution of soil alkane and PAH metabolic genes in these areas (Liang et al. 2011; Yang et al. 2014). The alkB gene, coding for a rubredoxin-dependent alkane monooxygenase enzyme, is involved in the first step of aerobic oxidation of n-alkane compounds and can be responsible for the transformation of midchain-length n-alkanes (C5 to C16) and even longer alkanes (Perez-de-Mora et al. 2011). alkB gene is not only an ideal marker to study the potential to degrade midchain-length n-alkanes, but also the general indicator for alkane degradation in the environment (Perezde-Mora et al. 2011; Wallisch et al. 2014). Naphthalene dioxygenase gene (nah) is one of the commonly studied PAH-metabolic genes. It is known for the biodegradation of low-molecular-weight PAHs (two- to three-ring PAHs; Zhou et al. 2006). Therefore, the objective of the current study was to characterize the abundance and diversity of soil petroleum hydrocarbon-degrading bacterial community targeting both alkB and nah genes in oil exploring areas. Moreover, the factors regulating the distribution of these two hydrocarbon-degrading genes were also investigated.

Appl Microbiol Biotechnol (2015) 99:1935–1946

Materials and methods Study sites and sampling Soil samples (0–5 cm) in triplicate were collected from two large oil exploring areas in different geographic regions in China, namely Karamay Oil Field (KOF) and Daqing Oil Field (DOF; Fig. 1). KOF (84°42′N, 45°36′E), located in the Xinjiang Uygur Autonomous Region (northwest China), has a temperate continental arid climate, with a mean annual rainfall of 108.9 mm and a mean annual temperature of 8.6 °C. DOF (46°35′N, 125°18′E), located in the Daqing City (northeast China), has a temperate continental monsoon climate, with a mean annual rainfall of 427.5 mm and a mean annual temperature of 4.2 °C. Soil samples were collected adjacent to crude oil pumping wells. The five sampling sites in the same oil exploring area were distributed in an area of 8– 10 km2. Soil samples were placed in sterile plastic bags, sealed and transported to laboratory on ice in nearly 24 h after collection. The levels of hydrocarbons and other physicochemical parameters of these soil samples are shown in Table 1. Molecular analyses Soil DNA was extracted using the PowerSoil DNA extraction kit (Mo Bio Laboratories, USA) following the manufacturer’s instructions. Each replicate soil DNA sample was individually subjected to quantitative PCR assays. The specific primers for quantification of alkB and nah genes were selected according to the literatures (Baldwin et al. 2003; Perez-de-Mora et al. 2011), alkB-F (5′-AAYACIGCICAYGARCTIGGICAYAA3′), alkB-R (5′-GCRTGRTGRTCIGARTGICGYTG-3′), NAH-F (5′-CAAAA(A/G)CACCTGATT(C/T)ATGG-3′), and NAH-R (5′-A(C/T)(A/G)CG(A/G)G(C/G)GACTTCTT TCAA-3′). The PCR conditions were as previously described (Baldwin et al. 2003; Perez-de-Mora et al. 2011). Standard curve was obtained using serial dilutions of linearized plasmids (pGEM-T, Promega) containing cloned alkB and nah genes amplified from soil. The amplification efficiency and coefficient (r2) for alkB and nah genes were 96 % and 0.997, and 97 % and 0.999, respectively. For the determination of the diversities of alkB and nah genes, the primer pairs alkB-F/alkB-R and NAH-F/NAH-R were also used for further clone library analysis according to the literatures (Perez-de-Mora et al. 2011; Yang et al. 2014). Chimera-free sequences were grouped into the operational taxonomic units (OTUs) using a 97 % similarity cutoff. OTU-based community Shannon index and rarefaction curve of each soil sample were generated using the MOTHUR program (Schloss et al. 2009). Phylogenetic analysis of the compositions of petroleum hydrocarbon-degrading bacterial communities was performed using MEGA software version

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Fig. 1 Schematic representation of the sampling sites in two oil exploring fields. Sampling sites DQ1–DQ5 were located in Daqing Oil Field (in Daqing City), while sampling sites XJ1–XJ5 in Karamay Oil Field (in Xinjiang Uygur Autonomous Region)

6.0 (Tamura et al. 2013). Jackknife Environment Clusters analysis was performed with the online UniFrac program for comparison of alkB and nah gene assemblages (Lozupone et al. 2006). Moreover, Pearson’s correlation analysis of the abundances and diversities of alkB and nah genes with the soil physicochemical parameters was further conducted using SPSS 20.0 software. The sequences obtained in this study were submitted to GenBank under accession numbers KJ684789–KJ684949, and KJ653680–KJ653808 for alkB gene, and KJ371605–KJ371691, KJ371762– KJ371799, KJ371804–KJ371851, and KM222729– KM222790 for nah gene.

Results Abundances of alkB and nah genes In this study, the abundances of petroleum hydrocarbondegrading bacterial communities targeting alkB and nah genes in KOF and DOF soils were estimated using quantitative PCR assays. A large variation in the densities of both alkB and nah genes occurred in the studied soil samples from either of the two oil exploring areas (Fig. 2). The alkB gene copy number varied from 2.56×106 to 9.37×107 copies per gram dry soil in DOF soils, while 7.48×105 to 6.63×107 copies per gram dry

Table 1 Physicochemical features of soil samples Sample

WC (%)

pH

Salinity (g kg−1)

TN (g kg−1)

TP (g kg−1)

TK (g kg−1)

TOC (g kg−1)

C/N

OC (g kg−1)

ALH (g kg−1)

ARH (g kg−1)

PA (g kg−1)

AS (g kg−1)

DQ1 DQ2 DQ3 DQ4 DQ5 XJ1

8.59 12.53 4.82 2.88 6.15 2.11

9.02 8.96 8.74 8.71 8.32 7.53

0.14 0.22 0.1 0.15 0.04 0.44

1.41 0.66 0.56 1.55 0.51 0.84

0.17 0.25 0.34 0.29 0.18 0.8

13.1 12.1 2.8 7.9 11.6 6.3

77 32.5 95.4 146 9.8 30

54.6 49.2 170.4 94.2 19.2 35.7

9.62 5.7 52.584 112.36 0 4.39

4.0 3.7 18.1 54.5 0 0.6

2.2 4.8 13.1 27.8 0 1.3

7.2 3.2 12.4 31.6 0 2.1

3.2 1.2 8.7 21.2 0 0.7

XJ2 XJ3 XJ4 XJ5

2.35 2.58 2.35 2.26

7.77 7.64 8.5 8.01

0.57 0.57 0.19 2.33

0.42 0.42 1.13 0.54

0.14 0.11 0.67 0.6

6.8 5.6 12.7 13.6

17.9 51.4 50.3 88.6

42.6 122.4 44.5 164.1

1.8 10.024 7.21 5.47

2.2 8.2 4.0 35.1

1.3 2.9 2.9 9.4

0.7 6.5 5.1 5.4

1.1 2.5 1.1 4.7

WC water content, TN total nitrogen, TN total phosphorus, TK total potassium, TOC total organic carbon, C/N ratio of TOC to TN, OC oil content, ALH aliphatic hydrocarbons, ARH aromatic hydrocarbons, PA polar aromatics, AS asphaltenes

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soil in KOF soils. For DOF soil samples, the highest abundance of alkB gene was detected in sample DQ4 (Table 1). A low density of alkB gene occurred in sample DQ3. In contrast, a high density of alkB gene was detected in sample DQ5. Moreover, for the KOF soil samples, sample XJ5 was collected from a contaminated site with the highest level of aliphatic hydrocarbons, and it had a much higher abundance of alkB gene than the other four soils (samples XJ1–XJ4). The nah gene copy number varied from 2.76×106 to 2.29× 7 10 copies per gram dry soil in DOF soil samples, while 5.65×105 to 1.93×107 copies per gram dry soil in KOF soil samples. For DOF soils, sample DQ4 had the largest number of nah gene copies. Sample DQ3 had a much higher level of aromatic hydrocarbons but a lower abundance of nah gene, compared with sample DQ1. Moreover, sample XJ5 had a relatively higher density of nah gene compared with the other four KOF soil samples (samples XJ1–XJ4). In addition, a large shift in the ratio of alkB gene to nah gene was also found in both KOF and DOF soils (Figure S1). In this study, Pearson’s correlation analysis was conducted to illustrate the relationships between the abundances of alkB and nah genes and the determined soil physicochemical parameters. The nah gene abundance of DOF soil was positively correlated with the level of total nitrogen (p0.05; Table 2). Moreover, the abundances of both alkB gene and nah gene of KOF soils showed significant positive correlations with salinity and the levels of aliphatic hydrocarbons, aromatic hydrocarbons and asphaltenes (p < 0.05; Table 3). Diversities of alkB and nah genes In this study, a total of 290 alkB gene sequences and 235 nah gene sequences were retrieved from the KOF and DOF soils. These alkB and nah gene clone libraries were composed of 5– 24 and 2–11 OTUs at 97 % similarity level, respectively (Table 4). The rarefaction curves for most of the alkB and nah gene clone libraries nearly leveled off (Figure S2), suggesting that these hydrocarbon-degrading bacterial communities were well sampled. A marked variation in the alkB gene community diversity was observed in the studied soils from the two oil exploring areas, with the values of Shannon index=1.37–3.16 and 1.12–2.48 for DOF soils and KOF soils, respectively. A large difference in the nah community diversity was also found in different soil samples from either of the two oil exploring areas. Sample DQ1 had a much lower nah diversity (Shannon index=0.32) than the other four DOF soil samples (Shannon index=0.86–1.23). Sample XJ1 had a much higher nah diversity (Shannon index=2.03) than the other four KOF soil samples (Shannon index=0.17–0.98).

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Moreover, except sample XJ1, the studies soils had a higher alkB Shannon diversity than nah diversity. Pearson’s correlation analysis indicated that the DOF soil alkB Shannon diversity was positively correlated with the level of total potassium (p