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Composition of Bacterial Communities Isolated from Core Samples Taken from Petroleum Deposits Journal of Microbiology & Experimentation Research Article Open Access Composition of bacterial communities isolated from core samples taken from petroleum deposits Abstract Volume 1 Issue 1 - 2014 Microbial communities in the subterranean environment are unique in that they are Debarati Paul,3 Jamie Scott,1 Magan Green,2 completely isolated from biological communities that rely on photosynthesis, earth’s 2 1 atmosphere or oceans. Interestingly, bacteria found in petroleum deposits are often Lewis R Brown, Mark L Lawrence 1College of Veterinary Medicine, Mississippi State University, in a dormant form because petroleum deposits are very limited in nitrogenous and USA phosphorous-containing nutrients. These are unique populations as compared to other 2Biological Sciences, Mississippi State University, USA subsurface microbial communities and might consist of novel species, new metabolic 3Amity Institute of Biotechnology, Amity University, India capabilities, and undiscovered adaptive microbial mechanisms. The goal of the current study was to isolate and identify cultivable bacteria (mainly oligotrophs) and develop Correspondence: Mark L Lawrence, College of Veterinary a method to evaluate bacterial community structure in petroleum deposits using 16S Medicine, Mississippi State University, Mississippi State, MS- rDNA based technique. 39762, USA Email [email protected] Keywords: petroleum deposits, bacterial communities, ARDRA and sequencing Received: April 14, 2014 | Published: April 24, 2014 Introduction dwelling in any particular environment and thereby provides a global picture of the genetic structure of the bacterial community. Bacteria can exist under high pressure and temperature conditions, in low oxygen or low nutrient conditions, and sometimes are able to Identification and quantification of petroleum reservoir remain dormant for several years. Oilfields usually represent extreme microorganisms, including nitrate reducing bacteria (NRB) and environments, poor in nitrates and oxygen, but harbor a diverse sulfate reducing bacteria (SRB), has been assessed by cultivation- community of sulfate-reducing, methanogenic and fermentative dependent methods,18 and cultivation-independent methods have only bacteria, and hydrocarbon or oil-utilizing bacteria. Some of the recently been introduced into the field of reservoir microbiology.19,20 important edaphoclimatic factors that influence microbial community Considering the small number of these studies, information currently composition and diversity include pH,1 particle size,2 organic carbon,3 available on the microbial communities present in oil reservoirs is nitrogen/phosphate,4 water,5 and oxygen.6 The physiology and sparse and, most notably, the microbial diversity of drilling sample metabolic potential of microbial communities will vary greatly with cores has never been studied before, probably due to lack of location along a soil profile depending on the available water, plant accessibility and difficulties in extracting bacteria/DNA from them. derived resources (carbon, nitrogen, and other nutrients), mineralizable Most of the research so far dealt with production water from oil fields, carbon and nitrogen, and oxygen. In mines and subsurface soils, oil dumps, or from the vicinity of oil.19,21–23 these factors cause selective pressures that tend to decide the nature In the current study, we evaluated the microbial diversity directly and distribution of bacteria, especially oxygen/nutrient limitations from cores taken from an active oil well. This research is a good 7 and temperature/pressure. The most widely represented and best- example of collaborative efforts between academia and industry. As a known types are sulfate-reducing, methanogenic, and fermentative result of this study, we isolated and identified some of the hydrocarbon 8–11 bacteria. utilizing oligotrophic bacteria in petroleum reservoirs. This study Some of the bacteria in mines and petroleum deposits survive required developing methods for bacteria and DNA extraction from the extreme environment by maintaining a dormant state.8 These petroleum core samples, and to our knowledge it is the first report bacteria tend to behave like oligotrophic bacteria10 or unculturable to use culture independent study of bacterial communities from bacteria when grown in lab conditions because of long-term starvation petroleum communities using ARDRA and sequencing. We intend conditions. Oligotrophic bacteria are slow-growing and ubiquitously to compare our results with similarly deciphered communities from occur in water or soil lacking organic substances. other core samples (varying depths and regions) in the future. To explore bacterial diversity in various environments/niches, Materials and methods 16S rRNA amplification and sequencing methods are commonly and efficiently used.12–15 Amplified ribosomal DNA restriction analysis Sample collection 12 (ARDRA) is one of the simplest and most cost-effective techniques Cores were obtained from approximately 700m below surface in 16 for culture-independent description of bacterial communities. For an oil-bearing formation in Brookhaven, Alabama. Each core was several environmental samples such as soil or seawater, the proportion stored in a BBL Gaspak container to keep it in anaerobic conditions of cells that can be cultured is estimated to be 0.1% or at most 10% at low temperatures (0-4°C). Each core was cut into 4 inch sections 17 of the total population, and few data are available concerning how using sterile steel under an atmosphere of nitrogen. The center of the closely they reflect the actual composition of these communities. core was placed in a sterile stainless steel crusher and then sieved. ARDRA allows ‘genetic fingerprinting’ of the bacterial community Submit Manuscript | http://medcraveonline.com J Microbiol Exp. 2014;1(1):1‒7. 1 © 2014 Paul et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and build upon your work non-commercially. Copyright: Composition of bacterial communities isolated from core samples taken from petroleum deposits ©2014 Paul et al. 2 DNA extraction from the core samples was calculated as the total number of distinct RFLP patterns in a core. The Shannon–Weiner diversity index25 was calculated as follows: DNA was isolated directly from crushed core for metagenomic H=S(ri)(log2r_i), where r represents the proportion of a distinct analysis. The PowerMax Soil DNA Isolation Kit (Mo Bio RFLP pattern relative to the sum of all distinct patterns. Evenness was Laboratories, Carlsbad, CA) was adapted for use with a Barocycler calculated from the Shannon–Weiner diversity index: E =H/Hmax (Pressure Biosciences, South Easton, MA). One gram of the crushed where Hmax= log2(S). Partial 16S rRNA gene sequences (at least 400 core material was vortexed with Power Bead buffer then mixed with bp) of one representative member of each phylotype were obtained lysis buffer and vortexed. The PCT (Pressure Cycling Technology) using M13F primer with the ABI PRISM Big Dye Terminator Cycle tube was loaded into the PCT machine and run for 80 cycles (35kpsi Sequencing kit and an ABI 310 automated DNA sequencer (Applied for 20sec, atmospheric pressure 5sec). This helped in lysis of the Biosystems, Foster City, CA). These sequences were used to construct bacteria using high pressure. DNeasy spin column (QIAGEN) was phylogenetic trees to understand phylogenetic relations between the used to purify the DNA from cell debris. The DNA was quantified and phylotypes. diluted with water for PCR. Data analysis PCR and cloning Partial 16S rRNA gene sequences were initially analyzed using Universal primers 27F (E. coli numbering 8~27; BLAST search (www.ncbi.nlm.nih.gov/blast/blast.cgi) and RDPII 5’-AGAGTTTGATCMTGGCTCAG-3’) and 1492R (E. coli analysis software (www.ce.msu.edu/RDP/html/analyses.html). numbering 1492~1510; 5’-GGTTACCTTGTTACGACTT-3’) The sequences were submitted to the NCBI (National Centre for (Cheneby et al., 2000) were used for amplification of bacterial 16S Biotechnology and Biotechnology Information), and GenBank rRNA genes. PCR amplification of nearly ~500bp of 16S rRNA gene accession numbers were obtained for all of them. The closest match was performed in 25μl reaction mixtures. Reaction tubes contained 25 and GenBank accession numbers of each sequence are listed in Table ng DNA, 1UTaq DNA Polymerase (New England Biolabs, Beverly, 1 & 2. Clustal X26 was used to align these sequences. Sequence MA), 1Xbuffer (10mM Tris-HCl [pH 9.0], 1.5mM MgCl , 500mM 2 dissimilarities were converted to evolutionary distances according KCl, 10mM deoxynucleoside triphosphate, and 20pmol of each -1 to the method of Jukes and Cantor (1969). Dendrograms were primer ml . Initial DNA denaturation and enzyme activation steps constructed with a neighbor-joining algorithm using NJ PLOT.27 were performed at 95°C for 30s, annealing at 50°C for 1min, and extension at 72°C for 2min, with a final extension for 10min at 72°C. Culturing some of the aerobic microorganisms The presence and yield of PCR product was monitored by 1% agarose (oligotrophs) for identification gel electrophoresis at 200V for 1h in 1X Tris-acetate-EDTA buffer and stained with GELSTAR. Fifty grams of crushed core material was suspended in simulated production water,28 thoroughly mixed, and streaked on either Bacto- 16S rRNA gene clone libraries were constructed for elucidating Tryptic Soy Agar (TSA) or Bacto-Plate
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