Abstract
In total, 435 pure bacterial strains were isolated from microtherm oil-production water from the Karamay Oilfield, Xinjiang, China, by using four media: oil-production water medium (Cai medium), oil-production water supplemented with mineral salt medium (CW medium), oil-production water supplemented with yeast extract medium (CY medium), and blood agar medium (X medium). The bacterial isolates were affiliated with 61 phylogenetic groups that belong to 32 genera in the phyla Actinobacteria, Firmicutes, and Proteobacteria. Except for the Rhizobium, Dietzia, and Pseudomonas strains that were isolated using all the four media, using different media led to the isolation of bacteria with different functions. Similarly, nonheme diiron alkane monooxygenase genes (alkB/alkM) also clustered according to the isolation medium. Among the bacterial strains, more than 24 % of the isolates could use n-hexadecane as the sole carbon source for growth. For the first time, the alkane-degrading ability and alkB/alkM were detected in Rhizobium, Rhodobacter, Trichococcus, Micrococcus, Enterococcus, and Bavariicoccus strains, and the alkM gene was detected in Firmicutes strains.
Explore related subjects
Discover the latest articles, news and stories from top researchers in related subjects.Avoid common mistakes on your manuscript.
Introduction
Along with the depletion of easily recoverable crude oil deposits, microbial enhanced oil recovery (MEOR) has been gaining increasing interest because it is environmentally friendly and cost-efficient (Lazar et al. 2007). An oil reservoir is a very special environment containing high pressure and few nutrients, which accommodates specific microorganisms. The low-temperature strata are often found in shallow oil reservoirs with depths ranging from 200 to 2,000 m. At relatively low temperatures, crude oil in the low-temperature strata is often poor in fluidity, which hampers the oil recovery. Therefore, the microbial degradation of petroleum hydrocarbons becomes more important for efficient oil recovery in microtherm oilfields.
Recently, many studies have investigated the microbial community in oil-production water from oil reservoirs (Dahle et al. 2008; Kaster et al. 2009; Li et al. 2006; Tang et al. 2012; Zhao et al. 2012). Simultaneously, many attempts have been made to isolate microbial strains from oil reservoirs as well as oil-production water (Kaster et al. 2009; Miroshnichenko et al. 2001). However, majority of the investigations and isolations were conducted on the mesotherm high-temperature oil reservoirs and oil-production water (Kaster et al. 2009; Wang et al. 2011), and only a few studies have addressed microtherm oil-production water.
Therefore, we investigated the isolation of bacterial strains from low-temperature oil-production water, which were obtained from the low-temperature oil stratum, by using four types of media at 25 °C. We also screened the strains that were able to degrade oil components and detected the phenol hydroxylase and alkane monooxygenase genes.
Materials and methods
Strain isolation and identification
The oil-production water was collected from the low-temperature oil stratum in Liuzhong Block, Karamay Oilfield, Xinjiang Uygur Autonomous Region, China, and transported to the laboratory at 4 °C. The temperature of the block strata and the production water was 20.6 °C. Other characteristics of this block have been previously described (Zhao et al. 2012).
First, cells in the production water were collected with a filter (0.22 μm pore size) under aseptic conditions. Then, the cells were resuspended with a sterile saline solution and diluted to 10−3 to make an inoculating suspension. Four types of media were used: (i) Cai medium (1,000 mL production water, 20 g agar, sterilized); (ii) CW medium (1,000 mL production water, 1.0 g NH4HO3, 1.0 g NaCl, 1.5 g K2HPO4, 0.5 g KH2PO4, 20 g agar, sterilized); (iii) CY medium (1,000 mL production water, 5.0 g yeast extract, 20 g agar, sterilized); and (iv) X medium (blood agar, Beijing Sybrisk Science & Technology Co., Ltd). The inoculating suspensions (100 μL) were plated on the four media and statically incubated at 25 °C in the dark for 9 days. All colonies that grew on the plates were picked, purified, and stored for further investigation. The isolates were named by the medium name and the colony series numbers. For example, the 1st and 10th strains isolated from Cai medium were named as Cai-1 and Cai-10, respectively.
DNA extraction, amplification, and analysis of the 16S rRNA gene from the purified isolates were conducted using previously described protocols (Wang et al. 2007). Before sequencing, the isolates were affiliated to different groups according to the colony phenotype and restriction fragment length polymorphism according to a previously described protocol (Yu et al. 2011).
Degradation of petroleum components by bacterial strains
After cells of different strains were grown in liquid LB medium (5.0 g L−1 yeast extract, 10.0 g L−1 tryptone, 10 g L−1 NaCl; pH 7.0) at 25 °C for 2 days, they were harvested as pellets after centrifugation (2,000×g at 4 °C for 10 min). Then, the pellets were washed twice and resuspended in an aseptic saline solution to prepare the inoculating suspension for further physiological experiments.
To investigate the petroleum degrading ability of different strains, the inoculum suspensions were added to 30 mL of mineral salt medium (MSM: 1.0 g L−1 NH4NO3, 1.0 g L−1 NaCl, 1.5 g L−1 K2HPO4, 0.5 g L−1 KH2PO4, 0.2 g L−1 MgSO4·7H2O) supplemented with phenol (100 mg L−1, final concentration), n-hexadecane (100 mg L−1, final concentration), or phenanthrene (100 mg L−1, final concentration) as the sole carbon and energy source. Then, the cultures were incubated at 25 °C and shaken at 150 rpm in the dark. On the 5th day, the cultures were sampled for the detection of residual organic compounds as well as cell growth.
The concentrations of phenol and n-hexadecane were detected by using the HPLC–UV and GC-FID with the previously described methods and protocols (Sun et al. 2011; 2012). The residual phenanthrene in the culture was extracted with dichloromethane. Briefly, the samples were thoroughly extracted with an equal volume of dichloromethane. From the dichloromethane layer, 2 mL of the mixture was collected and dried by anhydrous Na2SO4. Then, 1 mL of the dried mixture was transferred into a new tube for volatilization. The dichloromethane residue was dissolved in 0.5 mL of methanol. Then, methanol was used to determine the concentration of phenanthrene following the same protocol as that used for determining the phenol concentration except that the results were recorded at a wavelength of 250 nm.
Analysis of phenol hydroxylase and alkane monooxygenase genes
The previous described primers used to amplify phenol hydroxylase genes (pheN) as well as alkane monooxygenase genes, including nonheme diiron alkane monooxygenase gene (alkM/alkB), Cytochrome P450 enzymes gene (CYP153A), and Flavin-binding monooxygenase gene (almA) (Kloos et al. 2006; Wang et al. 2010; Wang and Shao 2011; Xu et al. 2001) and are listed in Table 1. DNA fragments corresponding with the correct target size for each gene were cloned into the pMD19-T vector (TaKaRa Biotechnology (Dalian) Co. Ltd., Dalian, China) and sequenced.
The obtained DNA sequences were aligned in GenBank by using the BLAST tools (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The amino acid sequences of alkane monooxygenase and phenol hydroxylase were translated using the MEGA software package version 5.0 according to the universal codon (Tamura et al. 2011). The reference sequences were retrieved from GenBank. After multiple sequence alignment of the sequences by CLUSTAL X and manually correction, the phylogenetic tree based on the phenol hydroxylase or alkane monooxygenase gene sequences were constructed using neighbour-joining method (Saitou and Nei 1987) in the MEGA software package version 5.0 (Tamura et al. 2011). The stability of tree topology was evaluated with maximum-likelihood and maximum parsimony algorithms.
Results
Phylogenetic distribution of the isolates from different media
After cultivation on four different kinds of media at 25 °C in the dark, X and Cai media led to the fastest and slowest growth, respectively, whereas CY and CW media showed the highest number of colonies (Fig. S1). At the end of the cultivation period (9 days), 435 colonies were isolated and purified from CY (134 isolates), CW (140 isolates), X (119 isolates), and Cai (42 isolates) media. Screened according to colony topologies, these 435 isolates were classified into 246 representative strains. Then, 246 representative strains were selected for further analyses including 16S rRNA gene sequencing. These 246 strains were classified into 61 groups according to 16S rRNA gene sequences and 16S rRNA gene fragment restriction digestion patterns, which could be further assigned to 32 genera belonging to the Actinobacteria, Firmicutes, Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria (Fig. 1).
Among the 61 groups of bacterial strains, 13, 15, 27, and 25 groups were isolated from Cai, CW, CY, and X media, respectively (Table 2; Figs. S2–S6). Furthermore, 1 (3 isolates), 4 (6 isolates), 3 (6 isolates), and 2 (2 isolates) groups isolated from Cai, CW, CY, and X media, respectively, which accounts for 7.7, 26.7, 11.1, and 7.7 % of the total groups in the corresponding media, potentially represented novel bacterial taxonomical groups because they shared <98 % 16S rRNA gene sequence identity with validly published bacterial species (Supplementary Table S2). Considerably more bacteria belonging to Proteobacteria were isolated from the high-nutrient (CY and X) media than from the relatively low-nutrient (Cai and CW) media. Acinetobacter strains were isolated only from Cai medium. Sanguibacter, Exiguobacterium, and Sphingobium strains were isolated only from CW agar. Microbacterium strains were only isolated from Cai and CW media, whereas no Betaproteobacteria strains were obtained from these 2 media. Halolactibacillus, Enterococcus, Planomicrobium, Paracoccus, and one Actinomycetaceae strains (with the 16S rRNA gene sequence identities <94 % with all of the known species) were unique species isolated from CY agar. Cellulosimicrobium, Micrococcus, Paenibacillus, Sphingobium, Delftia strains, in addition to 2 strains belonging to the genera Erysipelotrichaceae and Alcaligenaceae (with identical 16S rRNA gene sequence identities with 2 known genera of Alcaligenaceae: Kerstersia and Bordetella) were unique species isolated from X agar.
Three genera, Dietzia (18 isolates), Rhizobium (30 isolates), and Pseudomonas (66 isolates), could be isolated from all four media. In all, 4, 6, 5, and 3 Dietzia strains; 16, 7, 6, and 1 Rhizobium strains; and 2, 45, 12, and 7 Pseudomonas strains isolated from Cai, CW, CY, and X agars, respectively (Table S2).
Distributions of isolates with different hydrocarbon-degrading abilities as well as alkane monooxygenase and phenol hydroxylase genes
Among the 61 representative strains, four strains from CY and X media belonging to Alcaligenes, Comamonas, and Enterococcus genera could use phenol as the sole carbon source for growth (Table S1). In contrast, no strains from Cai and CW agars showed phenol-degrading ability. Five strains belonging to Halolactibacillus, Exiguobacterium, Paenibacillus, Pseudomonas, and Alcaligenaceae (a potential novel genus) and isolated from the four media could use phenanthrene as the sole carbon and energy source for growth (Tables 2, S1). It is notable that no study has described Enterococcus spp. using phenol and Exiguobacterium and Halolactibacillus spp. using phenanthrene as the sole carbon and energy sources for growth. Four phenol hydroxylase genes (pheN) responsible for the hydroxylation of phenolic compounds were obtained from four strains isolated from CY and X media that belonged to Alcaligenes, Sphingobium, and Comamonas. The four pheN were grouped into different clusters in the phylogenetic tree (Fig. S7). Although the pheN gene was PCR amplified from strain Sphingobium sp. X-b4, no phenol-degrading ability was detected. In contrast, no phe gene was detected in the phenol-degrading Enterococcus sp. CY-29 (Table S1).
Compared with the small amount of strains able to degrade phenol and phenanthrene, 46.2, 40.0, 33.3, and 24.0 % of the total strains from Cai, CW, CY, and X media were able to degrade n-hexadecane, respectively (Table S2). These included Planococcus, Rhizobium, Acinetobacter, Pseudomonas, Rhodobacter, and Dietzia strains from Cai medium; Pseudomonas, Rhodobacter, Planococcus, and Dietzia strains from CW medium; Pseudomonas, Paracoccus, Planococcus, Trichococcus, Enterococcus, Bavariicoccus, and Dietzia strains from CY medium; and Pseudomonas, Acidovorax, Micrococcus, Dietzia strains from X medium. The genes encoding alkane monooxygenase, which is responsible for alkane hydroxylation, were also detected using PCR amplification from the isolates. The alkB/alkM could be clustered into three groups (Fig. 2). Cluster I represents the alkB genes from all of the Dietzia strains. Cluster II represents the alkM genes from Trichococcus, Micrococcus, Enterococcus, Paracoccus, Pseudomonas, and Bavariicoccus, which is also clustered with alkM from Acinetobacter strains (Ratajczak et al. 1998; Sun et al. 2012). Cluster III represents the alkB genes from Rhizobium, Rhodobacter, and Acinetobacter, which is also clustered with the alkB gene from Pseudomonas putida Gpo1 (van Beilen et al. 2001). CYP153A, another medium-chain alkane hydroxylation gene (Nie et al. 2013b), and almA, a long-chain alkane hydroxylation genes, were detected only in Dietzia strains (data not shown).
Discussions
It is believed that environmental conditions drive microbial community to evolve relevant functions or to select special microorganisms to adapt the environments. In a microtherm oil reservoir, such as the Liuzhong Block of the Karamay Oilfield, microorganisms should be able to use the crude oil components for growth and adapt to temperatures as low as 22 °C. Therefore, it is reasonable that more than 24 % of the bacteria isolated from this oil-rich environment have the ability to degrade petroleum hydrocarbons, including n-hexadecane and phenanthrene. Among them, strains belonging to Rhizobium, Rhodobacter, Trichococcus, Micrococcus, Enterococcus, and Bavariicoccus genera were detected, for the first time, with the ability to degrade n-hexadecane, as well as contain the alkB/alkM genes. Phylogenetically distant but related bacteria, including Micrococcus, Trichococcus and Enterococcus, Paracoccus and Bavariicoccus, and Pseudomonas, had closely related alkB/alkM genes (Fig. 2). In addition, alkM genes, usually detected in Acinetobacter (Ratajczak et al. 1998; Sun et al. 2012), Actinobacteria (Alonso-Gutiérrez et al. 2011; Shen et al. 2010), and Proteobacteria (Tesar et al. 2002; Wang et al. 2010), were first detected in Firmicutes strains. It is notable that although Planococcus spp. CW-123 and CY-b41 as well as Pseudomonas spp. CW-122 and X-b2 could use n-hexadecane as the sole carbon and energy source for vigorous growth, none of the alkB/alkM, CYP153A and almA genes could be detected by using PCR method.
It is interesting that so many Rhizobium strains were isolated from the microtherm oil-rich environment, which is in consistent with the results analyzed by clone library analysis of the 16S rRNA genes (Zhao et al. 2012). Rhizobium strains are usually isolated from soil or aquatic environments, especially in the plant rhizosphere (Yoon et al. 2010), although a few Rhizobium strains were recently obtained from a bioreactor (Hunter et al. 2007; Quan et al. 2005; Wen et al. 2011). Recently, some Rhizobium strains were reported to be capable of utilizing aromatic compounds such as phenanthrene (Wen et al. 2011; Zhang et al. 2012). In the present study, most of the Rhizobium strains isolated from Cai medium could efficiently degrade n-hexadecane and harbored a special alkane monooxygenase gene.
These phenomena may be ascribed to the horizontal transfer of the alkane monooxygenase genes between different bacteria, as argued by Nie et al. (2013a). However, further investigation is needed to explain the phenomena as well as to understand the behaviors of bacterial strains under mesophilic conditions.
As artificial environmental selecting pressure, each medium led to the isolation of different and unique bacterial strains. For example, seven groups of bacteria were isolated only from X medium, 6 groups from CY medium, three groups from CW medium, and one group from Cai medium (Table 2). In addition, crude oil constituents from oil production could enhance the ability of Cai, CW, and CY media to grow more bacterial strains that are able to degrade petroleum hydrocarbons such as n-hexadecane and phenanthrene. In contrast, the yeast extract in CY and X media resulted in the isolation of phenol-degrading strains. A similar pattern was also found in the distribution of the alkB/alkM genes. Except for those from the Dietzia and Rhizobium strains (isolated from four media simultaneously), alkB genes clustered with the media: Cai-strains harbored Cluster III (alkB) nonheme diiron alkane monooxygenase and CY-strains harbored Cluster II (alkM) nonheme diiron alkane monooxygenase, regardless of the phylogenetic differences among the host bacterial strains. Further investigation is needed to address whether the medium selection of the isolates with specific functions is a universal phenomenon.
Although four kinds of media were used, they were obviously not enough to isolate all the bacteria in the production water. As revealed by the clone library analyses, some bacterial lineages could not be isolated by the four media, including bacteria belonging to Deltaproteobacteria and Spirochaetes (Zhao et al. 2012). In contrast, Betaproteobacteria and Actinobacteria which were isolated by cultivation were not detected by the clone library analyses. In addition, the Clostridia relatives were only detected by the culture-independent method, while Bacilli and Erysipelotrichi strains were isolated (Fig. 1). The common predominant bacteria detected with both methods were Pseudomonas.
In summary, 435 pure bacterial strains were isolated from microtherm oil-production water by using four different media, which were affiliated with 61 phylogenetic groups belonging to phyla of Actinobacteria, Firmicutes, and Proteobacteria. Only Rhizobium, Dietzia, and Pseudomonas strains were commonly isolated from the 4 media. Different medium selected bacterial strains with different n-hexadecane degradation abilities with alkB/alkM genes being clustered according to the media. In addition, the alkane-degrading abilities and alkB/alkM genes were detected in Rhizobium, Rhodobacter, Trichococcus, Micrococcus, Enterococcus, and Bavariicoccus strains.
References
Alonso-Gutiérrez J, Teramoto M, Yamazoe A, Harayama S, Figueras A, Novoa B (2011) Alkane-degrading properties of Dietzia sp. H0B, a key player in the Prestige oil spill biodegradation (NW Spain). J Appl Microbiol 111:800–810
Dahle H, Garshol F, Madsen M, Birkeland N-K (2008) Microbial community structure analysis of produced water from a high-temperature North Sea oil-field. Antonie Van Leeuwenhoek 93:37–49
Hunter W, Kuykendall L, Manter D (2007) Rhizobium selenireducens sp. nov.: a selenite-reducing α-Proteobacteria isolated from a bioreactor. Curr Microbiol 55:455–460
Kaster K, Bonaunet K, Berland H, Kjeilen-Eilertsen G, Brakstad O (2009) Characterisation of culture-independent and -dependent microbial communities in a high-temperature offshore chalk petroleum reservoir. Antonie Van Leeuwenhoek 96:423–439
Kloos K, Munch JC, Schloter M (2006) A new method for the detection of alkane-monooxygenase homologous genes (alkB) in soils based on PCR-hybridization. J Microbiol Meth 66:486–496
Lazar I, Petrisor IG, Yen TF (2007) Microbial enhanced oil recovery (MEOR). Petrol Sci Technol 25:1353–1366
Li H, Yang S-Z, Mu B-Z, Rong Z-F, Zhang J (2006) Molecular analysis of the bacterial community in a continental high-temperature and water-flooded petroleum reservoir. FEMS Microbiol Lett 257:92–98
Miroshnichenko M, Hippe H, Stackebrandt E, Kostrikina N, Chernyh N, Jeanthon C, Nazina T, Belyaev S, Bonch-Osmolovskaya E (2001) Isolation and characterization of Thermococcus sibiricus sp. nov. from a Western Siberia high-temperature oil reservoir. Extremophiles 5:85–91
Nie Y, Fang H, Li Y, Chi C-Q, Tang YQ, Wu XL (2013a) The Genome of the moderate halophile Amycolicicoccus subflavus DQS3-9A1T reveals four alkane hydroxylation systems and provides some clues on the genetic basis for its adaptation to a petroleum environment. PLoS One 8(8):e70986
Nie Y, Liang J-L, Fang H, Tang Y-Q, Wu X-L (2013b) Characterization of a CYP153 alkane hydroxylase gene in a Gram-positive Dietzia sp. DQ12-45-1b and its “team role” with alkW1 in alkane degradation. Appl Microbiol Biotechnol. doi:10.1007/s00253-013-4821-1
Quan Z-X, Bae H-S, Baek J-H, Chen W-F, Im W-T, Lee S-T (2005) Rhizobium daejeonense sp. nov. isolated from a cyanide treatment bioreactor. Int J Syst Evol Microbiol 55:2543–2549
Ratajczak A, Geißdörfer W, Hillen W (1998) Alkane hydroxylase from Acinetobacter sp. strain ADP1 is encoded by alkM and belongs to a new family of bacterial integral-membrane hydrocarbon hydroxylases. Appl Environ Microbiol 64:1175–1179
Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425
Shen F-T, Young L-S, Hsieh M-F, Lin S-Y, Young C–C (2010) Molecular detection and phylogenetic analysis of the alkane 1-monooxygenase gene from Gordonia spp. Syst Appl Microbiol 33:53–59
Sun J-Q, Xu L, Tang Y-Q, Chen F-M, Liu W-Q, Wu X-L (2011) Degradation of pyridine by one Rhodococcus strain in the presence of chromium (VI) or phenol. J Hazard Mater 191:62–68
Sun J-Q, Xu L, Tang Y-Q, Chen F-M, Wu X-L (2012) Simultaneous degradation of phenol and n-hexadecane by Acinetobacter strains. Bioresource Technol 123:664–668
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739
Tang Y-Q, Li Y, Zhao J-Y, Chi C-Q, Huang L-X, Dong H-P, Wu X-L (2012) Microbial communities in long-term, water-flooded petroleum reservoirs with different in situ temperatures in the Huabei Oilfield, China. PLoS One 7:e33535
Tesar M, Reichenauer TG, Sessitsch A (2002) Bacterial rhizosphere populations of black poplar and herbal plants to be used for phytoremediation of diesel fuel. Soil Biol Biochem 34:1883–1892
van Beilen JB, Panke S, Lucchini S, Franchini AG, Röthlisberger M, Witholt B (2001) Analysis of Pseudomonas putida alkane-degradation gene clusters and flanking insertion sequences: evolution and regulation of the alk genes. Microbiology 147:1621–1630
Wang W, Shao Z (2011) Diversity of flavin-binding monooxygenase genes (almA) in marine bacteria capable of degradation long-chain alkanes. FEMS Microbiol Ecol 80:523–533
Wang YN, Cai H, Yu SL, Wang ZY, Liu J, Wu XL (2007) Halomonas gudaonensis sp. nov., isolated from a saline soil contaminated by crude oil. Int J Syst Evol Microbiol 57:911–915
Wang L, Wang W, Lai Q, Shao Z (2010) Gene diversity of CYP153A and AlkB alkane hydroxylases in oil-degrading bacteria isolated from the Atlantic Ocean. Environ Microbiol 12:1230–1242
Wang X-B, Chi C-Q, Nie Y, Tang Y-Q, Tan Y, Wu G, Wu X-L (2011) Degradation of petroleum hydrocarbons (C6–C40) and crude oil by a novel Dietzia strain. Bioresource Tech 102:7755–7761
Wen Y, Zhang J, Yan Q, Li S, Hong Q (2011) Rhizobium phenanthrenilyticum sp. nov., a novel phenanthrene-degrading bacterium isolated from a petroleum residue treatment system. J Gen Appl Microbiol 57:319–329
Xu Y, Fang X, Chen M, Zhang W, Li J, Lin M (2001) The detection of phenol degrading strain in environment with specific primer of phenol hydroxylase gene. Acta Microbiologica Sinica 41:298–303 (in Chinese)
Yoon J-H, Kang S-J, Yi H-S, Oh T-K, Ryu C-M (2010) Rhizobium soli sp. nov., isolated from soil. Int J Syst Evol Microbiol 60:1387–1393
Yu S, Li S, Tang Y, Wu X (2011) Succession of bacterial community along with the removal of heavy crude oil pollutants by multiple biostimulation treatments in the Yellow River Delta, China. J Environ Sci 23:1533–1543
Zhang X, Li B, Wang H, Sui X, Ma X, Hong Q, Jiang R (2012) Rhizobium petrolearium sp. nov., isolated from oil-contaminated soil. Int J Syst Evol Microbiol 62:1871–1876
Zhao L, Ma T, Gao M, Gao P, Cao M, Zhu X, Li G (2012) Characterization of microbial diversity and community in water flooding oil reservoirs in China. World J Microb Biot 28:3039–3052
Acknowledgments
This research was supported by the National Natural Science Foundation of China (31200100, 31070107, and 31225001) and National High Technology Research and Development Program of China (2009AA063501 and 2013AA064401).
Author information
Authors and Affiliations
Corresponding author
Additional information
Ji-Quan Sun and Lian Xu have contributed equally to this work.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Sun, JQ., Xu, L., Zhang, Z. et al. Diverse bacteria isolated from microtherm oil-production water. Antonie van Leeuwenhoek 105, 401–411 (2014). https://doi.org/10.1007/s10482-013-0088-x
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10482-013-0088-x