University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies The Vault: Electronic Theses and Dissertations 2013-01-03 Microbially enhanced oil recovery through stimulation of indigenous oil field microflora with nitrate or introduction of rhamnolipid producers Kryachko, Yuriy Kryachko, Y. (2013). Microbially enhanced oil recovery through stimulation of indigenous oil field microflora with nitrate or introduction of rhamnolipid producers (Unpublished doctoral thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/26902 http://hdl.handle.net/11023/380 doctoral thesis University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission. Downloaded from PRISM: https://prism.ucalgary.ca UNIVERSITY OF CALGARY Microbially enhanced oil recovery through stimulation of indigenous oil field microflora with nitrate or introduction of rhamnolipid producers by Yuriy Kryachko A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES CALGARY, ALBERTA DECEMBER, 2012 © Yuriy Kryachko 2012 Abstract Sandpack model columns filled with two kinds of heavy oil were used to study the influence of nitrate injections on oil production. Columns containing trapped oil and nitrate consistently produced more oil than columns without nitrate in anaerobic conditions under counter-diffusion-induced gas drive. The presence of nitrate likely led to an increase of biomass and amounts of produced biosurfactants, allowing a reduction of oil-water interfacial tension. Gas-driven recovery of oil rich in alkanes (EO) was enhanced to a greater extent through nitrate injection than recovery of oil rich in polyaromatic hydrocarbons (LO). A water flood following incubation of columns containing nitrate did not lead to trapped oil production indicating that microbial growth was not sufficient to plug high permeability zones. Nitrate reducers Azoarcus and Delftia, the former being from the field of origin of EO, were found to consume heptane as a sole source of carbon and energy. Bioinformatics analysis of pyrosequenced 16S rDNA of the microbial communities adhering to EO and associated with an aqueous phase of a produced water-EO mixture showed that the former community was less diverse than the latter. Many known hydrocarbon degraders, e.g. Thalassolituus, Rhodococcus and Sphingomonas, were found to adhere to EO. In contrast, all identified genera of the Deltaproteobacteria were found to reside predominantly in the aqueous phase, likely because their substrates were mostly water-soluble. Consistently with the fact that hydrogen is more soluble in oil than in water, hydrogenotrophs, e.g. Methanolobus, Methanobacterium and Acetobacterium, had higher representation in the microbial community adhering to EO than in the community associated with an aqueous phase. ii The application of a cell-containing supernatant from the culture of a native rhamnolipid producer led to a greater enhancement of water-flood-driven LO recovery than the application of chemical surfactants. The application of cell-containing supernatants from cultures of a recombinant strain carrying genes responsible for rhamnolipid production and a strain with repressed transcription of rhamnolipid production genes led to enhancement of counter- diffusion-induced-gas-driven EO recovery, suggesting that very low surfactant concentrations are required for MEOR under solution gas drive. iii Acknowledgements This work was supported by funding from Aramco Services, Baker Hughes Incorporated, British Petroleum, Intertek/CML, the Computer Modeling Group Limited, ConocoPhillips Company, YPF SA, Shell Canada Limited, Suncor Energy Developments Inc., and Yara International ASA, Alberta Innovates – Energy and Environment Solutions, Genome Canada, Genome Alberta, the Government of Alberta and Genome BC. I am thankful to my supervisor, Dr. Gerrit Voordouw for giving me an opportunity to run exciting experiments, for insightful discussions, flexibility and patience, members of my supervisory committee, Dr. Doug Storey and Dr. Lisa Gieg for numerous helpful comments and suggestions, Dr. Harvey Yarranton and Dr. Tom Jack for their recommendations regarding my research and participation in supervisory committee meetings, Dr. Christoph Sensen, Dr. Elmar Prenner, Johanna Voordouw, Patrick Lai, Safia Nathoo and Xiaoli Dong for collaboration during preparation of our articles for publication, Sheng-Hung Wang for helping analyze DNA pyrosequencing data, Dr. Akhil Agrawal, Dr. Dae-Kyun Ro, Dr. Lisa Gieg and Trinh-Don Nguyen for assistance with GCMS analysis, Shiping Lin for help with assembling sandpack columns, Dr. Brij Maini and Paul Stanislav for help with the engineering aspects of my work, Ryan Ertmoed and Adriana Cavallaro for providing samples of oil and produced water, Dr. Doug Storey for providing the strains of Pseudomonas aeruginosa, Dr. Sean Caffrey for organizational efforts and recommendations on the applications of statistical methods, Dr. Hyung Soo Park, Dr. Adewale Lambo, Cameron Callbeck and Jaspreet Mand for assistance with conducting HPLC analysis, Morgan Khan for assistance with MALDI-TOF analysis, Cameron iv Callbeck and Jane Fowler for help with DGGE analysis, Dr. Rhonda Clark, Dr. Indranil Chatterjee and Dr. Dongshan An for their coordinating contributions, all sponsors of the Petroleum Microbiology Research Group, and all members of the Dr. Voordouw’s and Dr. Gieg’s laboratories at the University of Calgary with whom I worked and all of whom are my friends. Playing soccer and basketball with some of my colleagues helped me maintain myself in a good physical and mental condition during my PhD program, and I am thankful to all of them for being wonderful partners and rivals. v Table of Contents Section Page Abstract ii Acknowledgements iv Table of Contents vi List of Tables x List of Figures xi List of Abbreviations xiv Chapter 1: Overview of principles, successes and problems of MEOR- technology 1.1. Introduction 1 1.2. Alteration targets 2 1.2.1. Oil-water IFT and the role of biosurfactants in its 2 reduction 1.2.2. Wettability changes 8 1.2.3. Plugging of high permeability zones 11 1.2.4. Microbial solvent production in situ 12 1.3. Successful MEOR applications and tests 13 1.3.1. Conditions of success 13 1.3.2. Overview of some significant achievements in the history 18 of MEOR 1.4. Major problems of MEOR 20 1.5. Microbial enhancement of solution-gas-driven oil recovery 22 1.5.1. The principles of solution-gas-driven oil recovery 22 1.5.2. Enhancement of solution-gas-driven oil recovery 24 with surfactants in high and low concentrations Chapter 2: Research objectives and hypotheses 2.3. Research objectives 29 2.4. Hypotheses 29 Chapter 3: MEOR effects of nitrate injections in experiments with miniature model columns 3.1. Introduction 32 3.2. Materials and methods 34 3.2.1. Oil samples 34 3.2.2. Enrichment cultures with EO or LO as sources of carbon 34 and energy 3.2.3. Column assembly 35 3.2.4. Saturating columns with SPW 37 3.2.5. Saturating columns with oil 37 3.2.6. Eluting oil with SPW 38 3.2.7. Experiments with columns containing trapped oil 38 3.2.8. Determination of the amounts of produced oil and water 44 vi 3.2.9. Oil composition analysis 44 3.2.10. Determination of concentrations of gases produced during 46 long-time incubations 3.2.11. Enrichment cultures with heptane as a sole source of 46 carbon and energy 3.2.12. DNA pyrosequencing 47 3.2.13. Analysis of pyrosequencing data 48 3.3. Results 49 3.3.1. Oil production during long-time incubation of columns with 49 trapped EO and LO 3.3.2. Gas production during long-time incubation of columns with 50 trapped EO and LO 3.3.3. Nitrate and nitrite concentrations at the ends of long-time 55 incubation rounds 3.3.4. SPW-flood- and immiscible-gas-flood-driven oil production 55 following long-time incubation rounds 3.3.5. Compositions of EO and LO before and after long-time 56 incubation of columns with trapped oil 3.3.6. Biodegradation of heptane by NRB in enrichment cultures 64 3.4. Discussion 65 3.4.1. Mechanisms of oil production during long-time 65 incubation of columns with trapped oil 3.4.2. The mechanism of nitrate-mediated enhancement of gas- 75 driven oil recovery 3.4.3. Possible substrates for microbial growth during long-time 77 incubation of columns with trapped oil 3.4.4. NRB capable of heptane biodegradation 78 3.4.5. Long-time incubation of columns with trapped oil was not 80 accompanied by plugging of high permeability zones 3.4.6. Increased nitrate concentration did not help further enhance 81 gas-driven oil recovery 3.5. Conclusions and implications 82 Chapter 4: Microbial communities associated with oil and water in a mesothermic oil field 4.1. Introduction 84 4.2. Materials and methods 85 4.2.1. Isolation of biomass 85 4.2.2. DNA isolation 88 4.2.3. DGGE 89 4.2.4. DNA pyrosequencing 89 4.2.5. Analysis of pyrosequencing data 90 4.3. Results 91 4.3.1. DGGE community analysis 91 4.3.2. Alpha- and beta-diversity 91 4.3.3. Characterization of the OCP and the ACP 92 4.3.3.1. Phylum and class levels 92 4.3.3.2. Order and genus levels 99 4.4. Discussion 107 vii 4.4.1. Microorganisms associated with the oil phase 107 4.4.1.1. Gammaproteobacteria 107 4.4.1.2. Alphaproteobacteria 107 4.4.1.3. Betaproteobacteria 108 4.4.1.4. Actinobacteria 109 4.4.1.5. Mollicutes and Clostridia 109 4.4.2.