Author's personal copy

Author's personal copy

International Biodeterioration & Biodegradation 65 (2011) 1081e1086

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International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod

Nitrate-reducing community in production water of three oil reservoirs and their responses to different carbon sources revealed by nitrate-reductase encoding gene (napA) Wen-Wen Feng a, Jin-Feng Liu a, Ji-Dong Gu b, Bo-Zhong Mu a, * a

State Key Laboratory of Bioreactor Engineering and Institute of Applied Chemistry, East China University of Science and Technology, No. 130 Meilong Road, Shanghai 200237, PR China School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, PR China

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 13 March 2011 Received in revised form 15 May 2011 Accepted 15 May 2011 Available online 15 September 2011

The nitrate-reducing microbial community in oil reservoirs was examined by PCR using primers to amplify a segment of napA gene encoding for a subunit of the nitrate reductase, and the effects of different organic carbon additions on the nitrate-reducing community were also evaluated. The orders Rhodocyclales and Burkholderiales within Betaproteobacteria and Pseudomonadales within Gammaproteobacteria were recovered in production water of all three oil reservoirs. Amendment of organic acids promoted Enterobacteriales within Gammaproteobacteria and orders of Rhodocyclales, Pseudomonadales, and Burkholderiales, while alkanes favored the Rhizobiaceae family within Alphaproteobacteria and orders of Rhodocyclales and Pseudomonadales. Results indicated that the functional gene napA can be used as a valuable biomarker in analyzing the diversity of nitrate reducers in oil reservoirs. Nitrate-reducing microbial community shifts following the available carbon sources. Information about napA gene in oil reservoirs environment is scarce. This is the first study that combines molecular and culture-dependent approaches to reveal the diversity of the nitrate-reducing microbial community in production water of oil reservoirs by using the napA gene. Ó 2011 Published by Elsevier Ltd.

Keywords: Nitrate-reducing napA functional gene Oil reservoirs Community shift Organic carbon

1. Introduction Microbial denitrification is widely reported in soils (Gu et al., 2007; Wakelin et al., 2009), rumen, wastewater treatment, and activated sludge, and in wastewater (Chon et al., 2009), but hardly in production waters from oil reservoirs. Production water in the petroleum industry is wastewater produced during extraction of oil from oil reservoirs, and it contains crude oil and inorganic salts, including sulfate and chloride from the subsurface environment. Hydrogen sulfide (H2S) produced by sulfate-reducing bacteria (SRB) in oil reservoirs is a main cause of corrosion and souring. To inhibit SRBs, nitrate injection is commonly used in water-flooded oil reservoirs, and it may change the microbial community in the subsurface environment by enriching nitrate-reducing bacteria (NRB) (Hubert and Voordouw, 2007; Schwermer et al., 2010). Nitrate-reducing prokaryotes constitute a diverse group within the Alpha, Beta, and Gammaproteobacteria, gram-positive bacteria, and even Archea, sharing the common biochemical capability of obtaining energy from dissimilatory reduction of nitrate (Philippot * Corresponding author. Tel.: þ86 21 64252063; fax: þ86 21 64252485. E-mail address: [email protected] (B.-Z. Mu). 0964-8305/$ e see front matter Ó 2011 Published by Elsevier Ltd. doi:10.1016/j.ibiod.2011.05.009

et al., 2002; Baek et al., 2003; Li et al., 2011). Microbial denitrification, a respiratory process, consists of four consecutive reaction steps in which nitrate is reduced to dinitrogen (N2) gas (NO 3 / NO2 / NO / N2O / N2) (Zumft, 1997). The denitrification process involves four kinds of metalloenzymes: nitrate reductase, nitrite reductase, nitric oxide reductase, and nitrous oxide reductases (Canfield, 2010). Nitrate reductases are widespread in bacteria and three types of nitrate-reducing systems, cytoplasmic nitrate reductase, membranebound respiratory nitrate reductase, and periplasmic nitrate reductase, have been reported (Richardson et al., 2001). Nitrate reductases can reduce NO 3 to NO2 and be divided into NAR and NAP complexes. Denitrifying bacteria are reported to contain one or both of the nitrate reductases (Carter et al., 1995; Roussel et al., 2005). NAR complex is membrane-bound with the NarG catalytic protein located in the cytoplasm while NAP complex is equally membrane-bound but its NapA protein is periplasmic (Bru et al., 2007). NapA protein presents a dilemma as it can be involved in redox balancing, respiration, and NO 3 uptake (Berks et al., 1995; Carter et al., 1995; Castillo et al., 1996; Flanagan et al., 1999). Polymerase chain reaction (PCR) amplification for napA genes in nitrate-reducing bacterial communities have been reported before (Flanagan et al., 1999; Philippot et al., 2002). A gene cluster encoding components of a putative


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periplasmic nitrate reductase system (napAGHBFLD) has been sequenced (Simon et al., 2003; Martinez-Espinosa et al., 2006; Klotz and Stein, 2008; Bazan et al., 2008). Also, previous research indicates analysis of partial narG genes resulted in poor resolution for environmental samples, but well-defined results have been obtained from the partial napA fragments (Alcantara-Hernandez, 2009). Therefore, napA genes rather than narG genes were used to provide a sound basis for assessing the taxonomic composition of nitratereducing communities in the oilfields of this study. In this study, the objectives were to elucidate the diversity of the nitrate-reducing community in production water from three oil reservoirs using the napA functional gene as a biomarker and to evaluate the influences of organic carbon additions on the diversity of the nitrate-reducing microbial community. 2. Materials and methods 2.1. Sample collection Samples of the production water were taken from three oil reservoirs with different temperaturesdone from the Shengli oilfield, and two from the Huabei oilfield of P.R. China. The in-situ oil reservoir temperature was 60 C and total salinity was about 8425e11,196 mg l1 at the Zhan 3 unit of the Shengli oilfield, Shandong Province, P.R. China. The in-situ oil reservoir temperature and total salinity were 37 C and 1301 mg l1 at the Menggu Lin unit, and 58 C and 6199 mg l1 at the Ba 19 unit, both in the Huabei oilfield, Inner Mongolia, P.R. China. In total, 5 l of production water were taken from a central production well at each location. Half of the production waters from each site were filtered immediately after collection and the total genomic DNA was extracted from the filter and stored on dry ice in containers for transport back to the laboratory. They were stored at 20 C for analysis of the nitrate-reducing community. The other half of the collected samples were transported back to the laboratory in coolers and stored at 4 C prior to further experiments (Table 1). 2.2. Culture media and procedures The Hungate technique as described by Bryant (1972) was used in this part of the experiment. Five milliliters of the production water were inoculated into each 120-ml serum bottle containing 80 ml basal culture medium under a stream of pure N2 gas. All serum bottles were sealed with butyl rubber stoppers and aluminum seals and incubated at 37 C in the dark for 100 days to evaluate the influences of different organic carbon sources on the diversity of nitrate-reducing microorganisms. The basal culture medium contained (g l1): NaCl, 1.0; KCl, 1.3; MgCl2$6H2O, 1.0; CaCl2, 0.025; KH2PO4, 0.75; K2HPO4$3H2O, 1.16; NH4Cl, 0.5; KNO3, 1.0. Two different groups of organic carbons were used; the first contained mixed organic acids of aspartate (1.0 g l1) and citrate (8.5 g l1), while the second contained mixed alkanes (4.0 ml l1) of C15eC20 hydrocarbons.

extraction. Microbial biomass of 20 ml amended culture from incubation was collected by centrifugation at 13,000 x g for 1 min. Total genomic DNA of both sets of samples was extracted with the AxyPrep Bacterial Genomic DNA Miniprep kit (Axygen Biosciences, California, U.S.) following the manufacturer’s instructions. 2.4. PCR amplification of napA genes Primers for the detection of napA genes were based on those previously described (Alcantara-Hernandez, 2009) and synthesized (GenScript, New Jersey). However, the PCR mixture, and the protocol of PCR amplification of napA genes were modified according to the production water samples from oil reservoirs. Primer sequences used to amplify napA genes were: napAf1: 50 -C TGG ACI ATG GGY TTI AAC CA-30 ; napAr1: 50 eCC TTC YTT YTC IAC CCA CAT-30 . The PCR fragments’ expected size was 492 bp. Each 25-ml PCR mixture for amplification of total genomic DNA obtained from both production water and amended cultures contained 12.5 mM of each primer, 50 ng DNA, 10 ml 2x PCR master mix (Lifefeng Biotechnology, Shanghai, China), which was pre-mixed with Mg2þ, deoxynucleotide triphosphate, Taq polymerase, and PCR buffer. The PCR amplification protocol for the napA gene consisted of an initial denaturation at 95 C for 4 min, followed by 35 cycles at 94 C for 1 min, at 52 C for 1 min, and at 72 C for 1 min, with a final extension step at 72 C for 10 min. Thermal cycling was carried out with a thermal cycler (Bio-Rad Laboratories, California). PCR products were detected by agarose (1.0%) gel electrophoresis and UV transillumination after ethidium bromide staining. 2.5. Cloning and sequencing Total genomic DNA of samples was used as template in the PCR assay. One hundred nanograms of each napA gene fragment was inserted into 1 ml pMD 19-T Simple Vector (TaKaRa Biotechnology, Dalian, China), and transferred into 100 ml competent cell DH5a (Tiangen Biotech, Beijing, China) to establish a napA gene library. The universal primers M13 for PCR amplification (GenScript, New Jersey) were: M13f: 50 -TGT AAA ACG ACG GCC AGT-30 ; M13r: 50 -CGC CAG GGT TTT CCC AGT CAC GAC-30 . Positive clones of napA library were sequenced (GenScript, New Jersey). 2.6. Phylogenetic analysis VecScreen software was used to remove the sequence segments of vector origin. The OTUs of the sequences were calculated by FastGroupII; the cutoff similarity value used here was 97%. Types of sequences were detected by ORF finder to find all open reading frames. The deduced protein sequences were compared to those available at NCBI Protein Blast. 2.7. Nucleotide sequence accession numbers

2.3. DNA extraction Production water (2.5 l) from these oil reservoirs was filtered on site and microbial biomass was collected for total genomic DNA

The partial sequences (w500 bp) obtained in this study were deposited in the GenBank database under the Accession Numbers HQ727696eHQ727722.

Table 1 Clones obtained and sequenced from different sources. Production waters Oil reservoirs Clones Total amount

Menggu lin 81 258

Organic acids enrichments Zhan 3 87

Ba 19 90

Menggu lin 136 362

Zhan 3 91

Alkane enrichments Ba 19 135

Menggu lin 94 274

Zhan 3 88

Ba 19 92



3. Results 3.1. Nitrate-reducing community of production waters The deduced NapA protein fragment sequences of the total 258 sequenced clones were used to construct a consensus phylogenetic tree (Figs. 1 and 4). The 258 fragments fell into six OTUs, with three predominant OTUs accounting for 63.2% of the clone library. The Betaproteobacteria group is represented by four types with 209 clones accounting for 82.2% of the clone library. Three types (M-ENV-1, Z-ENV-1, B-ENV-2) with 163 fragments accounting for 63.2% of the clone library and relatedness of 100% identity to NapA fragment of Rhodomonadales order and 1 type (M-ENV-2) of 49 fragments, accounting for 19.0% of the library identical to NapA fragment from Burkholderiales order. Two types (Z-ENV-2 and B-ENV-1) with 46 fragments, accounting for 17.8% of the library, were related to the Gammaproteobacteria group (96% identity) to NapA fragments from Pseudomonadales order. The most abundant type in the library, B-ENV-2 with 74 fragments (28.7% of the library), is identical to the periplasmic nitrate reductase large subunit from Thauera sp. MZ1T (YP 002890969). 3.2. Effect of organic acids on the nitrate-reducing community A total of 362 clones were sequenced and their deduced protein fragments were used to construct another phylogenetic tree (Figs. 2 and 4). The 362 fragments fell into eight OTUs, with three predominant OTUs accounting for 58.8% of the clone library. The Gammaproteobacteria group, with 209 fragments represented by nine types, accounts for 57.7% of the clone library. Seven types (MNO3-1, M-NO3-2, M-NO3-3, M-NO3-4, M-NO3-8, M-NO3-9, B-NO33) with 147 fragments, accounting for 40.6% of the clone library, exhibited >86% identity to NapA fragment of Enterobacteriales order and another two types (B-NO3-1, B-NO3-2) with 62 fragments, accounting for 17.1% of the library, exhibited 98% identity to NapA fragment of Pseudomonadales order. Five types with 153 clones, accounting for 42.3% of the library, fell into the Betaproteobacteria group. Four types (M-NO3-5, M-NO3-6, M-NO3-7, Z-NO3-1) with 140 fragments, accounting for 38.7% of the library, are identical to NapA fragment of Rhodomonadales order and one type (Z-NO3-2) with 13 fragments, accounting for 3.6% of the library, is 100%

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identical to the NapA fragment of the Burkholderiales order. The most abundant in the clone library, Z-NO3-1, has 78 fragments accounting for 21.5% of the whole library, and is identical to Azoarcus sp. BH72 (YP935444), a mutualistic, N2-fixing grass endophyte (Krause et al., 2006). The second most abundant, B-NO3-3 with 73 clones, accounting for 20.2% of the library, is closely related to nitrate reductase catalytic subunit from Escherichia coli O127 (YP002329855) with 87% similarity, an enteropathogen (Iguchi et al., 2009). 3.3. Effect of alkanes on the nitrate-reducing community A total 274 clones were sequenced, and their translated protein fragments were used to construct a phylogenetic tree (Figs. 3 and 4), and categorized on the basis of their similarity at the 97% level. All of the 274 fragments fell into six OTUs, with two predominant OTUs accounting for 62.0% of the library. Five types (Z-ALK-3, B-ALK-1, M-ALK-1) with 184 fragments, accounting for 67.2% of the library, fell into the Gammaproteobacteria group with >98% identity to NapA fragment of Pseudomonadales order. Two types (Z-ALK-1 and M-ALK2) with 60 fragments, accounting for 21.9% of the library, belong to the Betaproteobacteria group with identical NapA fragment of Rhodomonadales. Two types (Z-ALK-2, Z-ALK-4) with 30 fragments, accounting for 10.9% of the library, fell into the Alphaproteobacteria group with 88% identity to NapA fragment of Rhizobiaceae family. The most abundant in the library, B-ALK-1, with 92 fragments and accounting for 33.6% of the library, is closely related (98% identity) to Pseudomonas stutzeri A1501(YP001171804), a nitrate reductase catalytic subunit from a root-associated bacterium (Yan et al., 2008). Representing 30 fragments, the types Z-ALK-2 and Z-ALK-4 are related (88% identity) to the uncultured bacterium (ACB06438), an uncultured bacterium from saline alkaline soil of Mexico City. 4. Discussion 4.1. Diversity of nitrate-reducing bacteria in production water from oil reservoirs Most of the research attention on nitrate-reducing communities with the napA gene has been on soil, plants (Gu et al., 2007; Wakelin et al., 2009), lakes, and wastewater (Chon et al., 2009); there is no

Fig. 1. The diversity of nitrate-reducing bacteria in production water from three oil reservoirs. The phylogram for the napA gene is based on deduced partial protein fragments. The neighbor-joining likelihood phylogenetic tree was constructed from deduced protein fragments; fragments from three libraries were analyzed: Menggu Lin of Huabei oilfield (M), Zhan 3 of Shengli oilfield (Z), and Ba 19 of Huabei oilfield (B). Reported fragments from amendment and incubation under three conditions are included; the percentage support values were above 50% and all clusters are indicated.


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Fig. 2. The diversity of nitrate-reducing bacteria after 100 days of incubation with nitrate as nitrogen source and citrate and aspartate as the major carbon sources. (Additional information is identical as that in Fig. 1.)

information available on oilfields (Li et al., 2010). Previously, Rhodocyclales and Pseudomonadales orders were detected in anaerobic environments with molecular approaches (Lorenzo et al., 2006). Burkholderiales were found in complex petroleum hydrocarbon mixtures, such as natural asphalts (van Beilen and Funhoff, 2007), and forest surface soil (King, 2006). When analyzing nitrate reducers based on napA genes, the Rhodobacterales order and Rhizobiaceae family of Alphaproteobacteria have been found in addition to those in the production water samples (Philippot, 2005). In this study, Burkholderiales, Rhodomonadales, and Pseudomonadales were detected in production water from all three oil reservoirs with different temperatures based on napA genes. Two dominant clusters, Beta and Gammaproteobacteria, possessing nitrate reductase or subunit of nitrate reductase, were recovered with napA gene as a biomarker,

while nitrate reductase negative bacteria were not detected, showing that functional gene napA-based methods can be used in analyzing the diversity of nitrate reducers from oil reservoirs. Results of napA fragments analysis showed that the diversity of nitratereducing communities in production water from different oil reservoirs is high. However, all the napA fragments analyzed related to mesophilic bacteria; none of them, even those from the hightemperature oil reservoirs, were thermophilic bacteria. This may be due to the contamination when injecting water into oil reservoirs, or more probably it may be related to the gradient temperature of oil reservoirs, as these mesophilic bacteria could exist in water close to the surface of the oil reservoirs, where a temperature lower than 45 C is generally expected. According to the results, 37 C was chosen to enrich nitrate reducers in the laboratory.

Fig. 3. The diversity of nitrate-reducing bacteria after 100 days of incubation with nitrate as nitrogen source and a mixture of alkanes as the carbon source. The NarG fragment from Enterobacteriales is used as outgroup. Clone designations M, Z and B are for the three oil reservoirs investigated as described above and also in the Materials and methods section. (Additional information is identical to that in Fig. 1.)



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Fig. 4. The community of nitrate-reducing bacteria from oil reservoirs. Each piece in the pie graph was categorized on the basis of its percentage of identical fragments. Fragments from three different sources were analyzed: production waters (inner), incubation with nitrate as nitrogen source and citrate and aspartate as the major carbon sources (middle), and incubation with nitrate as nitrogen source and a mixture of alkanes as the carbon source (outer).

4.2. Organic carbons affect diversity of nitrate-reducing bacteria

Acknowledgments

Different organic carbons may influence the diversity of nitratereducing communities; amendment and incubation were carried out under laboratory conditions to assess the microbial response to intervention. Organic acids, such as aspartate and citrate, were generally used as carbon and energy sources in enriching nitratereducing bacteria, and alkanes were commonly found in production water from oil reservoirs as intermediate metabolites of biodegradation. Therefore, these organic carbons were used to affect the nitrate-reducing microbial community separately. Organic carbons favored the order Enterobacteriales in Gammaproteobacteria in addition to the Rhodocyclales, Burkholderiales, and Pseudomonadales orders. Enterobacteriales could be previously found in oilfield (Wunch, 2010) and acidic conditions (pH 4.5) (Collavino et al., 2010), but their role in hydrocarbon degradation has not been substantiated (Rauch et al., 2006). The orders Rhodocyclales and Pseudomonadales remained in the incubation culture when alkanes were supplied as the sole carbon source, while Burkholderiales and Enterobacteriales were not detectable. The Rhodocyclales order includes the genera Azoarcus and Thauera, which are known to be metabolically very versatile with respect to substrate utilization and capable of using aromatic hydrocarbons as their sole sources of carbon and energy (Widdel and Rabus, 2001; Callaghan et al., 2010). The order Pseudomonadales has been detected in oilfields and confirmed to be related to degradation of C5eC16 alkanes, fatty acids, alkylbenzenes, and cycloalkanes (van Beilen and Funhoff, 2007). A new group of bacteria, the Rhizobiaceae family within Alphaproteobacteria, appeared when hydrocarbons were degraded. The Rhizobiaceae family of bacteria has been shown to be related with nutrient cycling for dinitrogenfixation and alkane degradation (Kvennefors et al., 2010). PCR primer based on napA gene can be used to investigate the microbial community in oil reservoirs and different organic carbons affect the diversity of nitrate reducers and the community structure by enriching those utilizing organic acids or alkanes amended. However, the biochemical mechanism of alkane degradation by these bacteria is still not well known and should be further studied.

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