Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Biogeosciences Discuss., 9, 5177–5203, 2012 www.biogeosciences-discuss.net/9/5177/2012/ Biogeosciences doi:10.5194/bgd-9-5177-2012 Discussions BGD © Author(s) 2012. CC Attribution 3.0 License. 9, 5177–5203, 2012 This discussion paper is/has been under review for the journal Biogeosciences (BG). The microbial Please refer to the corresponding final paper in BG if available. communities of petroleum reservoirs fluids Molecular analysis of the microbial L.-Y. Wang et al. community structures in water-flooding petroleum reservoirs with different Title Page temperatures Abstract Introduction Conclusions References L.-Y. Wang1, R.-Y. Duan1, J.-F. Liu1, S.-Z. Yang1, J.-D. Gu2, and B.-Z. Mu1 Tables Figures 1State Key Laboratory of Bioreactor Engineering and Institute of Applied Chemistry, East China University of Science and Technology, Shanghai 200237, China J I 2School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China J I Received: 6 April 2012 – Accepted: 16 April 2012 – Published: 27 April 2012 Back Close Correspondence to: B.-Z. Mu ([email protected]) Full Screen / Esc Published by Copernicus Publications on behalf of the European Geosciences Union. Printer-friendly Version Interactive Discussion 5177 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Abstract BGD Temperature is one of the most important environmental factors regulating the activ- ity and determining the composition of the microbial community. Analysis of microbial 9, 5177–5203, 2012 communities from six water-flooding petroleum reservoirs at temperatures from 20 to ◦ 5 63 C by 16S rRNA gene clone libraries indicates the presence of physiologically di- The microbial verse and temperature-dependent microorganisms in these subterrestrial ecosystems. communities of In high-temperature petroleum reservoirs, most of the archaeal sequences belong to petroleum reservoirs the thermophilic archaea including the genera Thermococcus, Methanothermobacter fluids and Thermoplasmatales, most of the bacterial sequences belong to the phyla Firmi- 10 cutes, Thermotogae and Thermodesulfobacteria; in low-temperature petroleum reser- L.-Y. Wang et al. voirs, most of the archaeal sequences are affiliated with the genera Methanobacterium, Methanoculleus and Methanocalculus, most of the bacterial sequences to the phyla Proteobacteria, Bacteroidetes and Actinobacteria. Canonical correspondence analy- Title Page sis (CCA) revealed that temperature, mineralization, ionic type as well as volatile fatty Abstract Introduction 15 acids showed correlation with the microbial community structures. These organisms may be adapted to the environmental conditions of these petroleum reservoirs over Conclusions References geologic time by metabolizing buried organic matter from the original deep subsurface Tables Figures environment and became the common inhabitants in subsurface environments. J I 1 Introduction J I 20 Petroleum reservoirs represent extreme anaerobic environments because of the tem- Back Close perature, pressure and salinity with multiphase fluids of oil, gas and water. Microorgan- isms in such subterranean play an important role in energy flow and nutrients cycling. Full Screen / Esc The microbial activity, in particular sulfate-reducing bacteria (SRBs) not only causes is- sues like reservoir souring and corrosion of drilling equipment with H2S, but potentially Printer-friendly Version 25 can also be used to our advantage, e.g., microbial-enhanced oil recovery (MEOR) ap- plications. Extraction of currently usable energy from marginal petroleum reservoirs by Interactive Discussion 5178 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | microbial conversion of residual oil to methane (natural gas) has received renewed attention in the past decade (Parkes, 1999; Suflita et al., 2004; Gieg et al., 2008; BGD Jones et al., 2008; Wang et al., 2010, 2011; Mbadinga et al., 2011, 2012). Microor- 9, 5177–5203, 2012 ganisms with diverse physiological and metabolic abilities and phylogenetic affiliations 5 have been recovered from oil reservoirs by culture-dependent and culture-independent approaches since the first sulfate-reducing bacteria (SRB) was isolated from production The microbial water (Bastin et al., 1926). Though isolation efforts have identified numerous bacterial communities of and archaeal species that are capable of mediating various metabolic processes oc- petroleum reservoirs curring in oil fields, culture-independent 16S rRNA gene- and functional genes-based fluids 10 investigations have provided new information on the microbial community composition in such deep-subsurface ecosystem (Li et al., 2010, 2011). L.-Y. Wang et al. Culture-independent surveys of high-temperature oil reservoirs have been con- ducted in oil fields in continental and offshore California (Orphan et al., 2000, 2003), Title Page an offshore oil field in Qinghuang, China (Li et al., 2007b), Huabei oil field in conti- 15 nental China (Li et al., 2006, 2007a), Dagang oil field in China (Nazina et al., 2006), Abstract Introduction and in Troll oil formation in the North Sea (Dahle et al., 2008). Bacterial sequences affiliated with Firmiucutes are the most frequency detected in these high-temperature Conclusions References oil reservoirs. In addition, the discovery of moderately thermophilic members of the Tables Figures Bacteroidetes (genus Anaerophaga) in Troll oil formation in the North Sea and sul- 20 fate reducing Nitrospira (genus Thermodesulfovibrio) in the Huabei and Qinghuang J I oil fields in China have not been previously isolated from oil reservoirs. Most of Ar- chaeal belong to methanogens including methyltrophic, acetoclastic as well as CO2- J I reducing methanogens and CO2-reducing methanogens are the most common mem- Back Close bers in high-temperature oil reservoirs. Moreover, methanogenesis from acetate driven 25 by syntrophic acetate oxidation has been documented in high-temperature reservoir Full Screen / Esc (Nazina et al., 2006). Compared with high-temperature oil reservoirs, few 16S-based analysis of the mi- Printer-friendly Version crobial community in low-temperature oil reservoirs have been reported. The bacte- rial diversity in a low-temperature, low-salinity, non-water flooded oil reservoir (Pelikan Interactive Discussion 5179 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | lake oil field) in Western Canada was extremely low with only one phylotype related to the genus Arcobacter (ε-Proteobacteria) (Grabowski et al., 2005). Several poten- BGD tially metabolic active fermentative and/or acetogenic microorganisms, sulfide-oxidizers 9, 5177–5203, 2012 and sulfate-reducers were identified from a low-temperature oil reservoir in Western 5 Canada by 16S rRNA gene clones library analysis (Voordouw et al., 1996). The ecological processes of these microorganisms play considerable important roles The microbial in energy flow and nutrients cycling in subsurface ecosystem. The distribution of differ- communities of ent microbial community structures in petroleum reservoirs depends entirely on their petroleum reservoirs adaption to the in situ physical and chemical variables, including temperature, pH, and fluids 10 salinity. Despite of the available studies on microbial population both in high- or low- petroleum reservoirs, microbial populations in reservoirs from low to high temperatures L.-Y. Wang et al. has not been studied in a single investigation. In this report, the distribution of microbial community in petroleum reservoirs from production water of several petroleum reser- Title Page voirs at temperature of 20, 32, 37, 45, 58 and 63 ◦C was investigated by 16S rRNA 15 gene library analysis. Microbial community data were also correlated with environmen- Abstract Introduction tal factors using canonical correspondence analysis (CCA). Conclusions References 2 Materials and methods Tables Figures 2.1 Collection of sample and nucleic acid extraction J I The production water samples of six production oil wells (S1–S6) from six Block affil- J I 20 iated with 3 oil fields of China: Well Z3-26 belongs to Zhan 3 of Shengli oil field; Well Back Close B18-43 to Ba 19, Well B51-45 to Ba 51 and Well M17-10 to Menggulin of Huabei oil field; Well 7222 to No. 7, Well 6190 to No. 6 of Kelamayi oil field. The temperature Full Screen / Esc and the mineralization of these six petroleum reservoirs sampled range from 20 to ◦ −1 63 C and 1301 to 11196 mgl , respectively and the pH of these production waters Printer-friendly Version 25 were neutral or slightly alkaline. The characterization of the petroleum reservoirs water sampled are listed in Table 1. In order to characterize the microbial community from Interactive Discussion 5180 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | the different temperature petroleum reservoir, these samples were grouped into two classes: high-temperature (45 ∼ 63 ◦C) and low-temperature (20 ∼ 37 ◦C). BGD Ten liters of production water at wellhead were taken from each of the six production 9, 5177–5203, 2012 oil wells from six petroleum reservoirs. Samples were collected into sterile bottles to 5 full capacity after discarding the initial oil/water mixture. The bottles were tightly sealed to avoid oxygen intrusion and immediately transported to the laboratory and filtered The microbial directly to minimize the chance of community changes. During filtration procedures, communities of the residual