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Oil reservoirs are specific habitats for the survival and growth of microorganisms in general. Pseudomonas stutzeri which is believed to be an exogenous organism inoculated into oil reservoirs during the process of oil production was detected frequently in samples from oil reservoirs. Very little is known, however, about the distribution and genetic structure of P. stutzeri in the special environment of oil reservoirs. In this study, we collected 59 P. stutzeri 16S rRNA gene sequences that were identified in 42 samples from 25 different oil reservoirs and we isolated 11 cultured strains from two representative oil reservoirs aiming to analyze the diversity and genomovar assignment of the species in oil reservoirs. High diversity of P. stutzeri was observed, which was exemplified in the detection of sequences assigned to four known genomovars 1, 2, 3, 20 and eight unknown genomic groups of P. stutzeri. The frequent detection and predominance of strains belonging to genomovar 1 in most of the oil reservoirs under study indicated an association of genomovars of P. stutzeri with the oil field environments.
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Pseudomonas stutzeri is a nonfluorescent widely distributed species of the genus Pseudomonas belonging to the gamma subclass of Proteobacteria (Bennasar et al. 1996; Lalucat et al. 2006). Strains of the species have been isolated from various environmental samples including marine sediments, soil contaminated with crude oil (Sikorski et al. 2002a, 2005; Mulet et al. 2011), clinical samples (Holmes 1986; Scotta et al. 2012) and bottle water (Papapetropoulou et al. 1994) among many others. The species has great physiological capacities including the ability to degrade environmental pollutants such as high-molecular-weight polyethylene glycols and other xenobiotics (Criddle et al. 1990; Chauhan et al. 1998; Coates et al. 1999) and the cleavage of C–N bonds in oil compounds (Kilbane et al. 2003). It also has been considered of relevance as a possible environmental reservoir of antibiotic resistance genes (García-Valdés et al. 2010) and in applications related to microbial enhanced oil recovery (EOR) (Keeler et al. 2013).
Diversity within the species is not limited to physiological properties but is also reflected at the genetic level. Previously, strains of P. stutzeri were classified into at least 21 genomovars (Rosselló et al. 1991; Scotta et al. 2013). The 16S rRNA gene, the internally transcribed spacer region 1 (ITS1), the genes coding for gyrase B (gyrB), and the D subunit of the sigma factor (rpoD) have been confirmed to be relevant for the phylogenetic affiliation of the species P. stutzeri (Bennasar et al. 1996; Sikorski et al. 2002b, 2005; Cladera et al. 2004). Additionally, fragments of the each locus could serve as excellent data sets to differentiate and establish the genetic diversity and population structure of the species of P. stutzeri (Yamamoto et al. 2000; Rius et al. 2001 and Cladera et al. 2004; Mulet et al. 2010, 2011).
In general, undisturbed oil reservoirs have low redox potentials and contain little oxygen and hence only strict anaerobes can be considered as truly indigenous (Magot et al. 2000). In this regard, members of the Pseudomonas sp. were not considered as indigenous to oil reservoirs (Orphan et al. 2000; Magot 2005) yet at least 10 studies of microbial community in samples from oil reservoirs reported its presence in these environments (Table S1). It is, therefore, important to note that due to drilling and oil recovery processes, producing oil reservoirs are dynamic environments that experience changing geochemical conditions such as the introduction of sulfate and oxygen ultimately resulting in changes to the indigenous microbial community structure (Youssef et al. 2009).
In such environments, local microorganisms may be continually introduced and those possessing exceptional survival abilities such as P. stutzeri can gain a lead in the formation of new ecological systems different to the original traits within surviving indigenous microbes (Youssef et al. 2009; Zhang et al. 2012). It is also well known that P. stutzeri possesses high physiological and genetic diversity which results from high rate genetic mutations, transpositions, and recombinations easily occurring in local natural environments (Ginard et al. 1997; Rius et al. 2001). Sikorski et al. (2002b) reported such complex composition, robust strain diversity, and directional selection in P. stutzeri population from marine sediment and soils while Scotta et al. (2012) demonstrated that most of the clinical strains of P. stutzeri belonged to genomovar 1. However, very little is known about the distribution and the genetic structure of P. stutzeri in the special environment of oil reservoirs.
Strains of P. stutzeri isolated through culture-dependent methods may provide information on the morphological, physiological, and chemical characteristics of the species in addition to the diversity of the species in the local populations. However, such information solely obtained from cultivable P. stutzeri strains does not provide a complete picture of the species compositional distribution in a given environment. The use of a culture-independent molecular method of construction of a clone library based on 16S rRNA gene, a widely accepted tool for molecular identification in bacterial community, therefore, became an informing supplement. 16S rRNA gene sequences of P. stutzeri consequently have been frequently detected in oil reservoirs without the need for culture isolation (Table 1).
Table 1. Chemical and physical characteristics of the oil reservoirs surveyed for the presence of Pseudomonas stutzeri in literatures and in this study.
|Location||Oil field||Oil reservoir||EOR practiceda||Reservoir characteristics||Samples||Relative abundanceb (%)||Reference|
| || || ||(Year)||Water-cut||Temp (°C)||Depth (m)||pH||Cl− (mg/L)|| || || |
|Malaysia||Bokor||104SL||Gas lift with CO2 and CH4 (NA)||70–80%||50||733–751||7.5||13500|| || ||Li et al. (2012)|
| ||104SS||Gas lift with CO2 and CH4 (NA)||70–80%||47||619–706||7.5||13200|| || ||Li et al. (2012)|
|Alaska||Schrader bluff||GMR75||Water-flooded||NA||27||1000||7.4–7.7||6141||PW(1)||NA||Pham et al. (2009)|
|Brazil||Potiguar||NA||NA||80–90%||42.2||535.5–540.5||NA||30000||PW (1)||0.76||Silva et al. (2013)|
|China||Shengli||Gudao||Water-flooded (1974)||95%||69||1173–1230||7.2–7.5||3138||PW (1)||66.7||Ren et al. (2011)|
|China||Shengli||Menggulin||Water-flooded (1989)||95%||37||806||8.2–8.6||NA||PW (1)||6.7||Tang et al. (2012)|
|China||Shengli||Baologe||Water-flooded (2001)||78%||58.4||1380||8.4–9.2||NA||PW (2)||55.5, 82.6||Tang et al. (2012)|
|China||Huabei||Block M||Water-flooded (NA)||NA||37||NA||6.7||502.5||PW (1)||8.8||Zhao et al. (2012)|
|China||Huabei||Block B||Water-flooded (NA)||NA||58.4||NA||7.2||507.8||PW (1)||8.1||Zhao et al. (2012)|
|China||Kalamay||Block Q||Water-flooded (1974)||NA||32||NA||7.6||4550||PW (1)||3.7||Zhao et al. (2012)|
|China||NA||Qinghuang||Water-flooded (2003)||NA||65||1100–1300||7.1–7.6||NA||PW (1)||NA (<1)||Li et al. (2007)|
|China ||Xinjiang||No. 6||Water-flooded (1973)||69.5%||25||800||7.5||3012||IW (1)||3||Zhang et al. (2012)|
|China||Dagang||Kong 2||Water-flooded (1975)||94.9%||55||1400||7.2||4617|| || ||Zhang et al. (2012)|
|China||Henan||V4||Water-flooded (1977)||95%||70||1355||7.3||7995|| || ||Zhang et al. (2012)|
|Middle east||Arabian||NA||NA||NA||NA||NA||NA||NA||Oil (1)||5.3||Yamane et al. (2008)|
|Japan||Minami-Aga ||NA||NA||NA||NA||NA||NA||NA||Oil (1)||5.2||Yamane et al. (2008)|
|Japan||Sagara||NA||NA||NA||NA||>600||NA||NA||Core (7)||100, 22.2, 100, 4.3, 6.3, 7.7, 26.3||Nunoura et al. (2006)|
|China||Qinghai||Gasi||Water-flooded (NA)||70%||55||2200||7.6||91425||PW (1)||3.7||This study|
|China||Jianghan||Wangxie||Water-flooded (1973)||86%||80||1500−1800||7.9||150513||PW (1)||3.1||This study|
|China||Tuha||Qiuling||Water-flooded (1995)||87%||35||2600–2800||7.5||5346||IW (2)||62.8, 34.4||This study|
|China||Dagang||Yangerzhuang||Water-flooded (1975)||95%||55||1800||7.2||4761||PW (2)||31, 12.2||This study|
|China|| ||Yangcong||Water-flooded (1975)||94.1%||50||2000||7.3||5024||PW(2)||60.5, 61.2||This study|
|China|| ||Kongdian||Water-flooded||93%||50–60||1300–1400||7.2||5362||PW (2)||48.2, 85.6||This study|
|China||Shengli||Gudao||Water-flooded (1983) Polymer-flooded (1993)||96%||70||1100–1240||7.6||3875||PW (1)||71.1||This study|
|China||Daqing||N2||Water-flooded (1978) Polymer-flooded (1995–2003)||97%||40–45||1300||7.9||525||PW (3)||64.4, 50, 39.3||This study|
The main aim of this study was to determine the distribution and the genomovar assignment for P. stutzeri in the oil reservoirs environment by grouping 16S rRNA gene sequences of P. stutzeri, obtained using both culture-dependent and culture-independent methods.
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Pseudomonas stutzeri habited a wide range of oil reservoirs with different characteristics. Oil recovery processes seemed to be the main factor for its appearance in oil reservoirs. Two studies of phylogenetic diversity of microbial community in the non-flooded oil reservoirs in Niibori oil field in Japan (Kobayashi et al. 2012) and the Troll C platform (Dahle et al. 2008) showed no single clone of P. stutzeri detected. It is important to note that oil recovery processes could not be considered as an absolute indicator for the existence of P. stutzeri in oil reservoirs. Other complex, correlative, and invisible characteristics of the producing oil reservoirs need to be taken into consideration, which may account for the many perplexing appearances of phylogenetic diversity of microbial communities. For example, P. stutzeri had high abundances in some samples but was not detected in others which were collected from oil wells in the same oil reservoir after undergoing water-flooding (Ren et al. 2011; Tang et al. 2012).
This is the first study on the compositional distribution and the genomovar assignment of P. stutzeri in the environments of oil reservoirs. On the basis of 16S rRNA gene sequence analysis of numerous P. stutzeri detected in the oil reservoirs under this study, P. stutzeri inhabited in these oil reservoirs included sequences affiliated with four known genomovar 1, 2, 3, and 20 and eight genomic groups that may represent new genomovars which require further taxonomic studies involving DNA–DNA hybridization, sequencing of internally transcribed 16S–23S rRNA gene spacer region (ITS1), and basic physiological properties (Sikorski et al. 2005). There were complex diversity associated with P. stutzeri detected in oil reservoirs and those belonging to genomovar 1 were detected in almost all of the oil reservoirs. The results obtained show an association of genomovars of P. stutzeri with the particular geographical environment of oil reservoirs as the genomovar three members preferentially exist in marine and the genomovar seven members mainly in soil habitats contaminated by petrochemicals or other pollutions (Sikorski et al. 2002a).
While interpreting the existence of complex diversity of P. stutzeri in the environment of oil reservoirs, we should take into account two main factors. The first one is the origination of P. stutzeri strains in oil reservoirs. Notably, it was suggested that they are exogenous organisms which were introduced to oil reservoirs during drilling and oil recovery procedures and survived gradually (Magot et al. 2000; Orphan et al. 2000; Youssef et al. 2009). Therefore, their origin may be derived from inoculation of oil reservoirs with surface P. stutzeri within the injecting water and recycled water after being exposed to surface conditions. Cells of some genomovars (genomovar 1 in this study) might be physiologically flexible which allows them to successfully occupy the habitat of oil reservoirs and survive (Sikorski et al. 2002a). Alternately the second factor may be based on the special stresses in oil reservoirs including abiotic factors or community-related conditions which increase mutation frequencies (Finkel and Kolter 1999; Radman 1999) and transposition speed (Oliver et al. 2000) of P. stutzeri, which may enhance the selection chance of mutant strains (Papadopoulos et al. 1999). These kinds of gene variation mechanisms may frequently occur in P. stutzeri which is a species with high rearranged chromosomes and no long-range conservation of genetic map (Ginard et al. 1997). Moreover, P. stutzeri is capable of natural transformation resulting in genomic diversification by recombination (Carlson et al. 1983; Sikorski et al. 2002a,b).
Our results indicate that P. stutzeri belonging to genomovar 1 predominates appearing most frequently in oil reservoirs with different geographical locations (Table 1). Although strains of genomovar 1 were isolated from soil as well as aquatic habits, their frequent appearance in oil reservoirs attracted our attention to the common geological characteristics of oil reservoirs. When strains of P. stutzri are introduced into oil reservoirs with injected fluids, the physico-chemical characteristics of oil reservoirs, such as temperature, pH, and electron donors and acceptors would surely have an effect on their survival, abundance, and diversity. In addition to the likelihood of originating from injected liquids, common geological characteristics of oil reservoirs must be taken into account to interpret the frequent detection of P. stutzeri strains belonging to genomovar 1.
Oil reservoirs are mainly subsurface environments with low redox potentials due to isolation from surface water (Magot et al. 2000). This means the available electron donors can be limited to hydrogen, volatile fatty acids (Fisher 1987), petroleum hydrocarbons, and inorganic electron donors (e.g., sulfide) while electron acceptors minimally include sulfate, carbonate, and iron (III), moreover, nitrate and oxygen are limiting in most oil reservoirs unless added with injected fluids (Youssef et al. 2009). It appears members belonging to P. stutzeri genomovar 1 may possess a greater ability to acclimatize to the special niches of oil reservoirs or have higher advantages to be able to achieve such capacity than other genomovars of P. stutzeri.
Members of different genomovars of P. stutzeri from the similar geographical environment belong to different ecological subpopulations inhabiting own ecological niches and constitute different evolving units (Palys et al. 1997). Our results showed sequences of P. stutzeri only in respective oil reservoirs which could be considered to be evolving units in own ecological niches. Cluster H (Fig. 1) included two sequences from Bokor oil field which is an oil reservoir not being subjected to water-flooding but gas lift with CO2 and CH4. Cluster G comprising sequences only from Dagang oil field. Notably, cluster C and D mainly consisted of sequences from oil reservoirs subjected to polymer flooding with polyacrylamide. Cluster B, E, and F, each, only contained one sequence. All these representative sequences seemed to associate with some unusual characteristics in these producing oil fields. These unusual characteristics, therefore, may act as the special stress factors for the evolution of P. stutzeri in the natural habitats, resulting in the genomic diversity of the species in the environment of oil reservoirs.
In this study, strains of P. stutzeri isolated in our laboratory were grouped into genomovar 1, cluster D and G, which seemed roughly to be consistent with the distribution of P. stutzeri in the oil fields of Dagang and Daqing. Notably, not a single strain of P. stutzeri was isolated from the sample of D2 collected from Daqing oil field where P. stutzeri of genomovar 1 was noticed to be dominant in the clone library constructed from the sample of D2. This demonstrated the belief that compositional distribution of microorganisms in natural habits could not be solely established based on culture-dependent methods as it does not take into account the discrepancies in growth environments between nature and laboratories (Ward et al. 1990; Singleton et al. 2001) leading to detectable microorganisms not yet cultured. Although culture-dependent methods are not sufficient to obtain a full picture of microbial ecology, further investigation of microbial metabolism activity and determination of the genomovars of the unassigned groups must depend on these isolates.
Our results were based on the analysis of 16S rRNA gene sequences of P. stutzeri in 16S rRNA gene clone libraries. Although 16S rRNA gene sequences of P. stutzeri do not provide high resolution of all genomovars as would using special genes of P. stutzeri such as the rpoD (Cladera et al. 2004; Mulet et al. 2011; Scotta et al. 2012, 2013), our results show that using of 16S rRNA gene sequences seemed to be applicable for the purpose due to the following reasons: (1) with the studies in microbial ecology of oil reservoirs, more 16S rRNA gene data of microbial community detected in oil reservoirs were reported, which provided a good resource for us to collected information worldwide. (2) 16S rRNA gene clone library, showing a clear picture of a microbial community of bacteria (Palys et al. 1997), can provide information concerning the relative abundance of different bacterial groups. In this study, P. stutzeri was dominant in some samples and were slight in others, which easily demonstrated relative abundance perception of P. stutzeri in oil reservoirs. (3) 16S rRNA gene sequences used in the study were obtained by culture-independent methods, which reflect the microbial community more realistically than would culture-dependent methods due to uncultivable characteristic of microbes in laboratories.