ISMOS-3 Abstract Book - Danish Technological Institute
ISMOS-3 Abstract Book - Danish Technological Institute
ISMOS-3 Abstract Book - Danish Technological Institute
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<strong>ISMOS</strong> 3<br />
International Symposium on Applied Microbiology<br />
and Molecular Biology in Oil Systems<br />
<strong>ISMOS</strong>-3 <strong>Abstract</strong> <strong>Book</strong><br />
Calgary, Canada<br />
June 13-15, 2011
3 rd International Symposium on Applied Microbiology and Molecular<br />
Biology in Oil Systems (<strong>ISMOS</strong>-3)<br />
<strong>ISMOS</strong>-3 <strong>Abstract</strong> <strong>Book</strong><br />
13-15th June 2011<br />
Symposium, Workshop & Poster Sessions<br />
www.<strong>ISMOS</strong>-3.org<br />
Editors: Sean M. Caffrey, Corinne Whitby, Torben Lund Skovhus<br />
All rights of this document belong to <strong>ISMOS</strong> TSC.<br />
The document can be cited if <strong>ISMOS</strong> is mentioned by name and webpage and only for non-profit purpose.<br />
1
TABLE OF CONTENTS<br />
Program Overview<br />
Detailed Program<br />
Welcome<br />
Contacts<br />
Workshop Agenda<br />
<strong>Abstract</strong>s<br />
3<br />
4<br />
6<br />
7<br />
8<br />
9<br />
2
Monday June 13, 2011<br />
09:00 AM Registration<br />
PROGRAM OVERVIEW<br />
09:30 AM Metagenomics Workshop (limited participation)<br />
12:30 PM Lunch<br />
01:15 PM Meeting Opening<br />
01:30 PM Opening Address<br />
02:10 PM Session 1 - Challenges in Oil Sands Development<br />
05:20 PM Poster Session 1 & Reception<br />
07:00 PM Reception Close<br />
Tuesday June 14, 2011<br />
08:30 AM Registration/Coffee/Light Breakfast<br />
09:00 AM Session 2 - MIC, Monitoring & Treatment<br />
10.50 AM Session 3 - Metagenomics & MMM<br />
12.20 PM Lunch<br />
01:20 PM Session 3 cont. - Metagenomics & MMM<br />
02:50 PM Poster Session 2<br />
04:00 PM Bus departs for conference dinner<br />
06:30 PM Conference Dinner & Awards<br />
Wednesday June 15, 2011<br />
08:30 AM Coffee/Light Breakfast<br />
09:00 AM Session 4 – Souring & Biocorrosion<br />
10:35 AM Session 5 - Biodegradation, Biofuels & Downstream Microbiology<br />
12:15 PM Lunch<br />
01:05 PM Session 5 cont. - Biodegradation, Biofuels & Downstream Microbiology<br />
02:30 PM Session 6 - Prospects for MEOR and Biodesulfurization<br />
04:30 PM Meeting Closing - Presenting <strong>ISMOS</strong>-4<br />
04:45 PM End of <strong>ISMOS</strong>-3<br />
3
Detailed Program<br />
4
Welcome to <strong>ISMOS</strong>-3 in Calgary<br />
There is an increasing need by the oil industry and downstream related industries to monitor microbial<br />
communities in 'real time' in order to address specific issues such as oil field souring, microbiologically<br />
influenced corrosion (MIC) and biofouling. Molecular Microbiological Methods (MMM) can be applied to<br />
oilfields (and other oil-related industries) to analyze and quantify the in-situ microbial communities and<br />
their activities in response to changing environmental conditions. Such information will help Operators<br />
to employ more directed and cost-effective strategies to prevent the major problems associated with<br />
deleterious microbial activities, as well as to encourage beneficial microbes.<br />
<strong>ISMOS</strong> is multidisciplinary, linking Operators, chemists, geologists, engineers and molecular<br />
microbiologists, and will include a mixture of high profile speakers from both industry and academia.<br />
The symposia will present the major industrial problems caused by microbes, (e.g. souring, MIC and<br />
biofouling) as well as beneficial activities obtained by microbes (e.g. MEOR, upgrading). The main focus<br />
of the meeting will be to explore how molecular and microbiological tools can be used to address these<br />
issues.<br />
This year <strong>ISMOS</strong> takes place in Calgary. The city is situated on the edge of the foothills, with views of the<br />
Rocky Mountains, a century ago; Calgary was a sleepy little town with a Wild West pedigree and rural<br />
roots. Today ‘Cowtown’ is one of Canada’s fastest growing cities and has been transformed into a city of<br />
over 1 million inhabitants and the center of Canada’s energy economy. Despite the economic growth<br />
the city has held on to its traditional roots as is demonstrated by its annual rodeo, the Calgary<br />
Stampede.<br />
In Calgary, <strong>ISMOS</strong>-3 takes place at the University of Calgary campus. The campus occupies a beautiful,<br />
park-like setting covering more than 200 hectares, an area larger than Calgary’s entire downtown. The<br />
main campus features over 20 academic buildings—many of which are interconnected by enclosed<br />
walkways. The MacEwan Student Centre is a hub of activity at the university, with a food court, wellness<br />
center and large concert hall. There is also a museum and art gallery, four performance theatres, two<br />
childcare centers and residences for single students and students with families.<br />
It is a great pleasure for the Technical and Scientific Committee (TSC) and the local organizers to<br />
welcome you all to <strong>ISMOS</strong>-3 in Calgary!<br />
Welcome to Calgary and <strong>ISMOS</strong>-3!<br />
Dr. Torben Lund Skovhus (DTI Oil & Gas, Denmark), Chair TSC<br />
Dr. Corinne Whitby (University of Essex, UK), Vice-Chair TSC<br />
Gijs van Rooijen & Sean M. Caffrey (Genome Alberta), Co-Organizers<br />
6
General questions regarding the technical and scientific content of <strong>ISMOS</strong><br />
please contact:<br />
<strong>ISMOS</strong> Secretariat<br />
DTI Oil & Gas<br />
Kongsvang Allé 29<br />
DK-8000 Aarhus C<br />
DENMARK<br />
Fax +45 72 20 18 70<br />
Phone: +45 72 20 18 27<br />
Email: ismos@teknologisk.dk<br />
For questions regarding registration and local issues in Calgary, please contact:<br />
Genome Alberta<br />
3553-31 Street NW<br />
Suite 115,<br />
Calgary, Alberta<br />
T2L 2K7<br />
Canada<br />
Fax: (403) 503-5225<br />
E-mail: scaffrey@genomealberta.ca<br />
7
Workshop Agenda<br />
9:30 – Biomonitoring 2.0: A high-throughput genomics approach for comprehensive<br />
biological assessment of environmental change<br />
Mehrdad Hajibabaei - Biodiversity <strong>Institute</strong> of Ontario & Integrative Biology<br />
www.ibarcode.org/hajibabaei<br />
9:45 – Introduction to pyrotags (16S rDNA community Analysis)<br />
Sean M. Caffrey – Genome Alberta<br />
www.hydrocarbonmetagenomics.com<br />
10:00 – Tools for pyrotag analysis<br />
Sean M. Caffrey – Genome Alberta<br />
Xiaoli Dong – Visual Genomics Centre<br />
10:40 – Break<br />
10:55 – Tools for pyrotag analysis continued<br />
Sean M. Caffrey – Genome Alberta<br />
Xiaoli Dong – Visual Genomics Centre<br />
11:15 – Introduction to total DNA metagenomics<br />
Sean M. Caffrey – Genome Alberta<br />
www.hydrocarbonmetagenomics.com<br />
11:30 – Computer resources for metagenomics analysis<br />
Christoph W. Sensen – Visual Genomics Centre<br />
www.visualgenomics.ca<br />
11:45 – Tools for Total DNA metagenomics<br />
Sean M. Caffrey – Genome Alberta<br />
www.hydrocarbonmetagenomics.com<br />
12:30 – Workshop end<br />
8
<strong>Abstract</strong>s<br />
Session 01- Challenges in Oil Sands Development<br />
Invited speakers:<br />
01#01<br />
Microbial Activities and Communities in Oil Sands Tailings Ponds<br />
Lisa Gieg, University of Calgary<br />
Tailings ponds are an important part of Alberta's oil sands surface mining operations as they function to<br />
store liquid and solid wastes following bitumen extraction. Such storage allows for solids to settle so that<br />
water can be recycled. Active microbial communities in oil sands tailings ponds can potentially affect gas<br />
emissions (H2S and CH4), solids densification, and the biodegradation of oil-associated compounds. To<br />
understand the roles that microbes play in the management of tailings ponds, including following pond<br />
closure, we assessed the depth-dependent microbial activities (sulfate reduction rates, SRR;<br />
methanogenesis rates, MR) and communities (via pyrosequencing) in an active (P6) and closed (P5)<br />
tailings pond treated with CaSO4 for densification. In both ponds, the highest sulfate concentrations (6-15<br />
mM) were measured at the surface, where no sulfide was detected. In P6, sulfate reducers, syntrophs<br />
and methanogens were most abundant, aligning closely with the measured SRR, MR, and levels of<br />
sulfate and sulfide as a function of depth. In P5, the SRR and MR were generally lower and confined to<br />
the upper 15 m of the pond. Methanogens were also abundant in the upper layers of P5, but the dominant<br />
bacteria included Thiobacillus, Rhodoferax, and Acidovorax spp., revealing a shift in the kinds of<br />
organisms that may proliferate following pond closure. Understanding microbial communities and<br />
activities following pond closure has important implications for reclamation efforts in the oil sands region.<br />
Ongoing work will help identify the key substrates driving the anaerobic processes in both active and<br />
closed oil sands tailings ponds.<br />
01#02<br />
Challenges in Oil Sands Tailings Pond Microbiology: False Assumptions and Flawed Logic<br />
Julia Foght, University of Alberta<br />
Beginning with the first microbial census of an oil sands tailings pond in 1983, every research advance<br />
has required reassessment and revision of assumptions about the microbiology in situ. Initially, industry<br />
discounted the potential influence of microbes on tailings, assuming that they would neither be present in<br />
appreciable numbers nor have significant activity; in fact, the tailings microbiota produce enormous<br />
volumes of methane, CO2 and H2S. Biogenic methane production in situ was predicted to disrupt settling<br />
of the solids in the ponds; instead, methanogenesis accelerates settling and recovery of pore water for reuse.<br />
To explain methane production, the default assumption was that bitumen components were the<br />
carbon source; instead, in many ponds fugitive extraction solvent components support methanogenesis.<br />
We assumed that Albian Sands' tailings pond would be analogous to Syncrude's; instead, trisodium<br />
citrate added to tailings is the primary source of methane there rather than hydrocarbons. Our initial<br />
forays into molecular biology assumed that DNA isolation would be confounded by the tailings' high clay<br />
contents and that residual bitumen would inhibit PCR amplification; eventually we developed robust<br />
methods for extracting and amplifying DNA and, through the Hydrocarbon Metagenomics Project, now<br />
use pyrosequencing to examine the prokaryotic diversity of the ponds. These recent results disprove yet<br />
another false assumption that the tailings ponds are 'extreme' environments with limited biodiversity;<br />
instead, the ponds harbour myriad species and represent a valuable resource of anaerobic metabolism<br />
genes. What other false assumptions remain to be demolished? Stay tuned.<br />
9
Offered Papers:<br />
01#03<br />
Analysis of Methanotrophic Bacteria in Surface Water of Tailing Ponds by Stable Isotope Probing<br />
and Pyrosequencing<br />
Zhiguo He, P. Dunfield, University of Calgary<br />
Processing of oilsands bitumen produces large amounts of tailings comprised of water, silt, clay, residual<br />
bitumen, and solvent used in the extraction process. Tailings are usually stored in open basins which can<br />
produce large effluxes of methane, a powerful greenhouse gas. Mildred Lake Settling Basin is the largest<br />
tailings deposit in the Syncrude oilsands operation in Northern Alberta, covering 10 square km. The basin<br />
is strongly methanogenic, with an estimated 40 million L/d of methane release to the atmosphere. We<br />
investigated the potential for methane-oxidising bacteria in surface aerobic water to attenuate methane<br />
emissions from this site. The maximum potential methane oxidation rate estimated in the laboratory was<br />
300 nmol/ml/ d. Extrapolation suggests that >50% of the methane produced in the basin could be<br />
oxidised in the aerobic layer instead of being released to the atmosphere. Microbial diversity was<br />
investigated by 454 pyrosequencing of amplified 16S rRNA genes. Methanotrophic bacteria were<br />
identified via identification of known 16S rRNA signatures, as well as by isolation in the laboratory.<br />
Predominant species included the gammaproteobacteria Methylomonas and Methylobacter as well as the<br />
alphaproteobacteria Methylosinus and Methylocystis. The most active species under different conditions<br />
were identified via stable isotope probing (SIP) of DNA, where 13 CH4 was fed to the samples and the<br />
heavy 13 C-DNA of the species utilizing the methane was then analysed via 454 pyrosequencing of 16S<br />
rRNA and methanotroph-specific pmoA genes. The results indicate that there is an active and diverse<br />
methanotrophic community in this site.<br />
01#04<br />
Oil Sand Microbial Communities Can Degrade Recalcitrant Alkyl Phenyl Alkanoic Acids<br />
Richard Johnson 1 , V. Da Fonseca 1 , J. Aubé 1 , J. Fisher 2 , C. Whitby 1 1 University of Essex; 2 DuPont<br />
Plc<br />
The vast reserves of oil sands and heavy oils have not yet been fully exploited. These resources contain<br />
complex mixtures of carboxylic acids known collectively as naphthenic acids (NAs). NAs cause major<br />
environmental and economic problems as they are recalcitrant, corrosive, toxic and need to be removed<br />
during oil processing and refining. Although aromatic compounds make up a small proportion of the NA<br />
mix, they are nonetheless highly toxic. The aims of this study were to characterise the microbial<br />
communities found in oil sands and determine the indigenous microorganisms capable of degrading the<br />
highly recalcitrant alkyl phenyl alkanoic acids; (4'-iso-butylphenyl)ethanoic acid (iso-BPEA) and (4'-tertbutylphenyl)-4-butanoic<br />
acid (tert-BPBA) as well as the more amenable (4'-n-butylphenyl)-4-butanoic acid<br />
(n-BPBA). Oil sands were enriched on the three aromatic alkanoic acids and the microbial communities<br />
analysed in relation to biodegradation rates and metabolite production by GC-MS, DGGE and sequence<br />
analysis. GC-MS analysis demonstrated that iso-BPEA was completely mineralised by day 35, whereas<br />
both n-BPBA and tert-BPBA were only partially degraded, with a concomitant production of metabolites<br />
which persisted. DGGE and 16S rRNA gene sequence analysis revealed community shifts during NA<br />
degradation, with an increase in abundance of species belonging to the beta-Proteobacteria such as<br />
Thiobacillus spp. Our findings are the first to identify microbial communities in oil sands that can<br />
completely degrade recalcitrant alkyl phenyl alkanoic acids such as iso-BPEA. These findings will allow<br />
more efficient approaches to be developed for removing NAs from oil sands wastewaters.<br />
10
01#05<br />
Microbial Communities in Oil Sands Cores Collected for Production by Steam-Assisted Gravity<br />
Drainage (SAGD) in Alberta, Canada<br />
Man-Ling Wong 1 , D. Cuthiell 2 , G. Voordouw 1 1 Petroleum Microbiology Research Group,<br />
Department of Biological Sciences, University of Calgary 2 Suncor Energy Inc.<br />
Canada's oil sands are abundant, accessible and economic. There are an estimated 1.7 trillion barrels of<br />
bitumen situated in Canada, of which only a part can be recovered with current technologies. SAGD, the<br />
predominant recovery technology for deeper bitumen deposits, is very effective but utilizes large amounts<br />
of steam which leads to significant CO2 emissions. Hence, the development of environmentally friendlier,<br />
but still economic, oil sands extraction and processing technologies is very attractive to the oil sands<br />
industry. Understanding the microbial communities and activities in Alberta's oil sands is a first step<br />
towards biotechnologies that may help in the production. Five cores collected at different locations and<br />
depths were obtained from Suncor's Firebag reservoir. Subsamples were taken every 5 cm. Microbial<br />
community composition was determined for two of these through 16S pyrosequencing surveys.<br />
Subsamples displayed diverse communities, and some of these were biased towards thermophiles. All<br />
had bacteria capable of degrading oil and methanogenic Archaea, suggesting that anaerobic degradation<br />
of bitumen with formation of methane and CO2 is still ongoing. The reasons for the observed differences<br />
in community composition on the cm scale are currently not clear. The possibility that oil composition for<br />
different sections of the cores differs needs to be investigated. The presence of thermophiles suggests<br />
that microbial activity may be stimulated by moderate heating (60-80°C) offering the opportunity to<br />
convert part of the bitumen into methane as an alternate way of producing the resource.<br />
01#06<br />
Sulfur Biogeochemistry of Syncrude's Mildred Lake Composite Tailings (CT) Deposits<br />
Lesley Warren 1 , K. Stephenson 1 , T. Penner 2 1 McMaster University 2 Syncrude<br />
Syncrude is currently building its first freshwater fen, as a large scale reclamation project for composite<br />
tailings (CT) materials at their Mildred Lake site. However, unpredicted incidents of high hydrogen sulfide<br />
gas (H2Sg) concentrations, a safety and environmental hazard, have occurred at CT dewatering wells,<br />
indicating the need to characterize the sulfur biogeochemistry in these materials and establish any<br />
impacts associated with fen development. While sulfate reducing bacteria (SRB) are a likely candidate to<br />
consider in the generation of H2S within CT, it is more than likely that a more complex series of interactive<br />
S and Fe microbial metabolisms are involved. As CT S biogeochemistry has not been evaluated to date,<br />
the objectives are to: (1) establish the occurrence of S and Fe metabolizing microbes within the CT and<br />
overlying sand cap, (2) experimentally characterize the links between CT microbial activity and H2S<br />
generation and (3) assess the S biogeochemistry of untreated CT and those underlying the fen<br />
reclamation project. Two sampling campaigns (June and Sept 2010) collected sediment cores and<br />
porewater samples for Fe/S geochemistry (solid and porewater), microbial community structure<br />
(cultivation independent molecular methods), microbial associations (variety of microscopies), and S/Fe<br />
enrichments. Sulfide was detected in all the wells, indicating widespread H2S generation within the<br />
deposit. Positive growth for S and Fe reducing and oxidizing enrichments (genetic characterization<br />
currently underway), confirm the need to consider H2S generation within a broader Fe-S linked microbial<br />
framework in order to establish the processes involved.<br />
11
01#07<br />
Ozone Treatment For Naphthenic Acid Remediation: Impacts On Indigenous Microbial<br />
Communities In Oil Sands Process-Affected Waters<br />
Lisa D. Brown 1 , L. Perez 2 , N. Wang 1 , M. Gamal El-Din 1 , J. W. Martin 2 , A. C. Ulrich 1 1 Department of<br />
Civil and Environmental Engineering, University of Alberta 2 Department of Analytical and<br />
Environmental Toxicology, University of Alberta<br />
The oil sands industry faces significant challenges in developing effective remediation technologies for<br />
process water. Naphthenic acids, a complex mixture of cycloaliphatic carboxylic acids, are of particular<br />
concern because they concentrate in tailings ponds and have been indicated as a primary source of<br />
process water toxicity. Naphthenic acids treatment via biodegradation is difficult due to their inherent<br />
recalcitrance. Ozone treatment has been demonstrated as an effective means of rapidly degrading<br />
naphthenic acids and reducing process water toxicity. In this study, two aged process waters from<br />
Syncrude experimental ponds were ozonated to reduce naphthenic acid concentration. The impact of<br />
ozone treatment on native microbial communities present in oil sands process-affected water was<br />
monitored. In addition, capacity of the rebounded microbial community or an inoculation of indigenous<br />
microbes to degrade remaining organics was assessed. Light ozone treatment reduced dissolved organic<br />
carbon (~40 mg/L) in process water by less than 10%, whereas 20% to 30% of the organics were further<br />
consumed after five weeks of aerobic incubation. Dissolved organic carbon data suggested that<br />
ozonation and subsequent biodegradation reduced naphthenic acid concentration, typically representing<br />
up to 60% of dissolved organics, by more than 30%; soon to be confirmed with UPLC-HRMS. Microbial<br />
community assessment of the experimental microcosms, using denaturing gradient gel electrophoresis<br />
and subsequent band sequencing, will identify dominant species in the degradation process. Improved<br />
understanding of the impacts of ozonating oil sands process-affected waters, and profiling indigenous<br />
microbial communities in oil sands tailings ponds will facilitate decision making regarding effective<br />
treatment processes.<br />
Posters:<br />
01#08<br />
Microsensors for Investigating Microbial Activities of Multispecies Biofilm in Biological Reactor<br />
Treating Oil Sands Tailings Water<br />
Hong Liu, K. Sharma, S. Ren, S. Tan, T. Yu University of Alberta<br />
Oil sands tailings water in Northern Alberta contains large amounts of recalcitrant and toxic organics.<br />
Although tailings water is toxic to microorganisms, researchers were able to isolate sulphate reducing<br />
bacteria (SRB), methane producing archea (methanogens) and iron reducing bacteria from matured fine<br />
tailings (MFT), where the microorganisms have ability to adapt to the toxic and harsh environments. The<br />
big challenge of treating tailings water could be overcome by exploiting microorganisms flourishing in<br />
MFT. With this objective, a bench scale biofilm reactor has been operated by feeding tailings water and<br />
MFT to grow multispecies biofilm on the media and to treat tailings water. Microsensors, as effective tools<br />
that allow measurement of chemical gradients with high spatial resolution due to their tip size from 1 to 30<br />
μm, could be used to study the microbial activities in microenvironments of the multispecies biofilm<br />
+ - - 2-<br />
treating tailings water. In our facility, we have fabricated O2, pH, NH4 , NO2 , NO3 , H2S, SO4 , ORP<br />
microsensors and used them to study structure and function of SRB, nitrifiers and denitrifiers in<br />
membrane aerated biofilm as well as in river sediments. The research on methane biosensor fabrication<br />
and application is in progress. A suite of microsensors will be used to investigate microbial activities in the<br />
multispecies biofilm treating tailings water. The microbial activities in microenvironment obtained from<br />
microsensor measurement can be used to assess reaction mechanisms and processes that further<br />
support improvements in the design and operation of reactors treating tailings water.<br />
12
01#09<br />
The Microbial Community of a Biofilm for Treatment of Oil Sands Process Water<br />
Mary Wesley, Y. Liu, M. Gamal El-Din University of Alberta<br />
Alberta Oil Sands processing is a water intensive process. Water treatment and recycle is the key to the<br />
management of water resources for oil sands development. As the water is recycled, the levels of<br />
contaminants including salts, metals, and recalcitrant organic compounds increase. This can be further<br />
exacerbated by the addition of chemicals during the oil extraction process. Demand for more efficient<br />
recycling of this water is driving the development of various wastewater treatment strategies. One of the<br />
methods being developed is the use of microbial biofilms to remove contaminants from the water. Biofilm<br />
treatment is attractive due to its ability to oxidize recalcitrant compounds and remove metals while being<br />
exposed to an extreme environment. Current studies involve the elucidation of the community members<br />
of the biofilm and their role in the oil sands process water treatment train. Molecular biology techniques,<br />
i.e. PCR-DGGE techniques, were introduced to analyze the microbial community of the oil sand process<br />
water with various treatment options. Once mechanisms of removal are determined, it will be possible to<br />
design optimal removal conditions.<br />
01#10<br />
In Situ Substrates for Methane Production and Sulphate Reduction in an Alberta Oil Sands<br />
Tailings Pond<br />
Sandra Wilson, I. Mani Bhaskar, L. Gieg University of Calgary<br />
Bitumen, naphtha, and naphthenic acids are potential microbial substrates present in Alberta's oil sands<br />
tailings ponds. Naphtha components (BTEX compounds and alkanes) have been shown to drive methane<br />
production in Syncrude tailings ponds. Considerable rates of sulfate reduction (up to 55 mmol sulfate/m 3<br />
tailings/d) and methanogenesis (up to 80 mmol CH4/m 3 tailings/d) were recently measured in Suncor<br />
tailings ponds as a function of depth, thus our goal was to determine the in situ substrates driving these<br />
activities. Three sets of incubations were established wherein tailings were amended with (1) minimal<br />
medium, (2) minimal medium containing up to 12 mM sulphate, or (3) minimal medium containing 5 mM<br />
sulphate and 5 µL naphtha. All conditions (including sterile controls) were monitored for approximately 2<br />
yr. Tailings amended with medium only demonstrated considerable methane production (up to 12<br />
µmol/mL or 12 mol/m 3 tailings), however there was no detectable volatile naphtha in the experimental or<br />
control conditions, indicating that naphtha alone is not driving methane production. The tailings amended<br />
with medium and sulphate showed lower methane amounts and only moderate loss of volatile naphtha.<br />
Incubations with medium, sulphate and naphtha showed moderate methane production (up to 1.3<br />
µmol/mL or 1.3 mol/m 3 ), sulphate reduction and partial loss of volatile naphtha. Overall, while naphtha<br />
components likely promote some of the microbial metabolic activities within the tailings ponds, other<br />
substrates such as naphthenic acids are likely also involved. Work is ongoing to identify the substrates<br />
driving the observed sulfate reduction and methanogenesis activity in the incubations.<br />
01#11<br />
An in vitro Mixed Species Biofilm Approach to Culturing Microbes Directly from an Oil Sands<br />
Tailings Pond<br />
Susanne Golby, M. Demeter, H. Ceri, T. Raymond J. University of Calgary<br />
Tailings ponds result from the extraction of bitumen from the oil sands of Northern Alberta, and currently<br />
contain millions of cubic meters of toxic waste. Tailings are a complex mixture of residual bitumen, water,<br />
sand, clay, hydrocarbons, organic acids and heavy metals. We have hypothesized that bioremediation of<br />
the tailings pond can be optimized using mixed species biofilms that are endogenous to the tailings<br />
environment. Studying natural microbial populations in vitro proves to be a challenge due to the large<br />
number of microbes that currently cannot be successfully cultured in the laboratory. Thus, using the<br />
13
Calgary Biofilm Device (CBD) as a microscale reactor, we considered if both aerobic and anaerobic<br />
mixed species biofilms could be cultivated directly from oil sands tailings pond sediments, demonstrating<br />
a cultivation technique that can be employed for subsequent bioremediation investigations in vitro. We<br />
have succeeded in culturing aerobic, microaerobic and anaerobic mixed species biofilms directly from an<br />
oil sands tailings pond under a variety of different culture conditions. Microbial diversity within biofilms<br />
was initially evaluated by denaturant gradient gel electrophoresis (DGGE) and quantified with Quantitative<br />
PCR (Q-PCR). DGGE profiles showed very diverse mixed species biofilms could be generated and each<br />
population was unique when biofilms were recovered using different incubation temperatures ranging<br />
from 4ºC to 25ºC, oxygen tensions (aerobic, microaerobic, and anaerobic) and varying growth medias<br />
and carbon sources (carbohydrates, glucose, and naphthenic acids). 454 pyrosequencing revealed that<br />
the organisms within the biofilms strongly represented the indigenous population in the tailings used as<br />
the inoculum. Furthermore, biofilms contained over 10 different genera per biofilm including organisms<br />
belonging to Pseudomonas, Thauera, Hydrogenophaga, Rhodoferax and Acidovorax. In this study, we<br />
have successfully shown that the CBD can be used to grow representative mixed species biofilms directly<br />
from the environment, and this cultivation technique recovers a more abundant and diverse population<br />
than can be seen from traditional agar plates and liquid broth cultures.<br />
01#12<br />
Linking Phylogeny to Function: Benzyl- and Alkylsuccinate Synthase Gene Homologues in<br />
Methanogenic Oil Sands Tailings Enrichment Cultures<br />
Boonfei Tan, L. Bradford, K. Bradford, S. Bradford, J. Foght University of Alberta<br />
Anaerobic biodegradation of monoaromatic hydrocarbons is initiated by benzylsuccinate synthase and its<br />
alpha-subunit, encoded by bssA. An analogous attack on n-alkanes can be accomplished by<br />
alkylsuccinate synthase and its alpha-subunit encoded by assA. Others have reported that the bssA gene<br />
is highly conserved across several species of nitrate-, sulphate-, and iron-reducers, whereas assA has<br />
been identified only in a sulphate reducing bacterial isolate. Currently, the diversity of these two genes<br />
and their functions are unknown in methanogenic cultures such as those enriched from oil sands tailing<br />
ponds exposed to naphtha, a solvent containing aromatics and short-chain alkanes. As part of the<br />
Hydrocarbon Metagenomics Project, we used degenerate PCR primers to screen for bssA and assA<br />
genes in methanogenic oil sands tailings enrichment cultures amended with short chain n-alkanes<br />
(C6,C7,C8,C10), longer chain n-alkanes (C14,C16,C18), BTEX (benzene toluene, ethylbenzene and xylene<br />
isomers), or naphtha. The resulting amplicons were cloned, sequenced and phylogenetically analyzed to<br />
reveal diverse sequences phylogenetically affiliated with bssA and assA genes. Biodegradation of the<br />
hydrocarbon substrates and formation of signature metabolites were analyzed using GC-MS, resulting in<br />
detection of benzylsuccinates and alkylsuccinates. These observations suggest that anaerobic<br />
biodegradation by oil sands tailings microorganisms principally involves hydrocarbon addition to fumarate.<br />
To extend our study, the 16S rRNA genes of these microbial communities were subjected to 454 FLX<br />
pyrosequencing. The community composition and the presence of bssA and assA genes are elucidating<br />
the links between phylogeny and function in these methanogenic cultures.<br />
01#13<br />
Analysis of Methanogenic Hydrocarbon-Degrading Cultures Established from Oil Sands Tailings,<br />
Using 454 FLX Pyrosequencing<br />
Kathy Semple, C. Li, J. Klassen, C. Nesbø, J. Foght University of Alberta<br />
Oil sands tailings ponds containing wastes from bitumen extraction often become methanogenic,<br />
apparently due to anaerobic biodegradation of the small proportion of hydrocarbon solvent (used during<br />
bitumen extraction) that escapes to the ponds with fine tailings. Laboratory enrichment cultures inoculated<br />
with mature fine tailings (MFT) from four tailings ponds revealed that microbes indigenous to MFT<br />
degraded a variety of hydrocarbons under methanogenic conditions. As part of the Hydrocarbon<br />
Metagenomics Project, we used 454 FLX pyrosequencing of 16S rRNA gene amplicons to characterise<br />
some of these cultures. The phylogenetic diversity of cultures incubated with authentic bitumen extraction<br />
14
solvents or with defined mixtures of hydrocarbons was analyzed using various alpha- and beta diversity<br />
statistics. In every case the Archaea were dominated by a few Euryarchaeal lineages (Methanosaeta and<br />
Methanoregula), whereas bacterial diversity was considerably higher and more variable. Cluster analysis<br />
of microbial community composition correlated best with substrate composition but also somewhat<br />
reflected MFT origin. For example, cultures enriched on long chain alkanes typically had higher<br />
proportions of Smithella whereas cultures grown with monoaromatics had higher proportions of<br />
Desulfosporosinus and/or Desulfotomaculum. In general, these taxa increased in abundance at the<br />
expense of Betaproteobacteria and other taxa. These analyses confirm that oil sands tailings ponds<br />
contain diverse microbial communities harbouring broad abilities to biodegrade hydrocarbons under<br />
methanogenic conditions. Furthermore, the ponds may represent a valuable resource of microbes<br />
capable of bioremediating diverse anaerobic hydrocarbon-impacted sites.<br />
01#14<br />
Analysis of Bacteria and Archaea in Lateral Transects of Two Tailings Ponds Using 16S rRNA<br />
Gene Pyrosequencing<br />
Carmen Li, J. Klassen, C. Nesbø, J. Foght University of Alberta<br />
Surface mining of oil sand ore produces extraction tailings: a slurry of water, sand, silt and clay particles,<br />
unrecovered bitumen and a small proportion of hydrocarbon solvent used during bitumen extraction. After<br />
deposition in tailings ponds, the tailings settle by gravity to produce anaerobic mature fine tailings (MFT)<br />
that often become methanogenic, primarily due to microbial consumption of residual solvent<br />
hydrocarbons in the MFT. As part of the Hydrocarbon Metagenomics Project, we have used 454 FLX<br />
pyrosequencing of 16S rRNA gene amplicons ('16S pyrotags') to profile the phylogenetic diversity of MFT<br />
samples collected from depth profiles along lateral transects of two tailing ponds. Confirming previous<br />
observations from clone libraries, the Archaea in all samples were dominated by a few Euryarchaeal<br />
lineages (Methanoregula, Methanolinea and Methanosaeta). In contrast, hundreds of bacterial genera<br />
were detected, with most samples dominated by Chloroflexi, Firmicutes, Deltaproteobacteria and<br />
Betaproteobacteria. We also observed varying proportions of archaeal and bacterial 16S pyrotags along<br />
the transects. Results from analysis of alpha- and beta diversity will be presented in the context of<br />
chemical metadata from the sample sites.<br />
01#15<br />
Assessing the Effect of Aerobic Microbial Community Changes in Oil Sands Outcrops on Bitumen<br />
Molecular Composition<br />
Thomas Oldenburg, M-l. Wong, G. Voordouw, S. Larter University of Calgary<br />
With the decline of conventional oil reserves in the world, oil sands bitumen is becoming more crucial for<br />
the global energy mix. Oil sands are known to have naturally in-situ biodegraded oil in them but little is<br />
known to what extent they are further degraded aerobically near the outcrop surface or whether the<br />
bitumen just reflects in-situ anaerobic processes. The objective was to determine whether aerobic<br />
hydrocarbon-degrading microorganisms from the oil sands can metabolize recalcitrant bitumen fractions,<br />
with and without the help of light. Oil sands outcrop microbial microcosms were incubated aerobically for<br />
2 years in the light and in the dark. The microbial enrichments were analyzed biologically using culturing,<br />
DGGE, and pyrosequencing as well as chemically by investigating changes in the molecular distribution<br />
of the oil sands under these conditions using an ultra-high resolution mass spectrometer (12T FT-ICR-<br />
MS). The focus of this study is on the compositional oil changes during aerobic incubation under light and<br />
in the dark. What oil sands components are utilized under these two conditions and what components are<br />
altered or newly formed, reflecting different microbial consortia being dominant and active during natural<br />
oil sands weathering. Oxygen-containing bitumen compound classes were affected by consumption of<br />
acidic compounds under light whereas in darkness, acidic components were represented by new formed<br />
hydroxy acids and dicarboxylic acids. The detailed bitumen compositional analysis will be interpreted in<br />
terms of the microbial ecology of the outcrop samples.<br />
15
01#16<br />
Effect of Calcium Ions and Biomass on Sedimentation of Oil Sands Tailings<br />
Damon Brown, E. Ramos, L. Gieg, G. Voordouw University of Calgary<br />
Oil sands tailings ponds contain large volumes (~108 m 3 ) of fine tailings, originating from bitumen<br />
production by surface mining. These sediment rapidly in dilute suspension but then form a network, which<br />
sediments much more slowly. Tailings sedimentation is monitored as the movement of a sharp boundary,<br />
separating clear supernatant fluid from the tailings network. The overall process increases solid content<br />
and is referred to as tailings densification. Addition of gypsum (CaSO4.2H2O) and formation of biomass<br />
promote densification by crosslinking small clays, increasing average size. To determine the roles of<br />
calcium ions and biomass in tailings sedimentation we placed tailings (70%) and defined medium (30%)<br />
in 24 ml anaerobic test tubes with an N2-CO2 headspace. The tubes were further amended with either 10<br />
mM CaCl2, 20 mM NaCl, 10 mM CaSO4, 10 mM Na2SO4, 10 mM Ca(NO3)2, 20 mM NaNO3 or no<br />
additions. Four replicates (tubes 1, 2, 3 and 4) were made for each condition. Following homogenization,<br />
the sedimentation in tubes 1 and 2 was monitored continuously. Tubes 3 and 4 were re-homogenized<br />
once per week after which headspace methane, and the concentrations of sulfate, sulfide, nitrate and<br />
nitrite were measured in the clear fluid. Following 2 months of monitoring, all tubes were amended with<br />
excess lactate to further boost the biomass concentration. Of all conditions tested, tubes amended with<br />
Ca(NO3)2 and lactate gave the most increased rate of tailings sedimentation. Because the biomass<br />
concentration formed in the presence of nitrate is expected to be highest, these results indicate that high<br />
biomass and calcium ions are needed for optimal tailings densification.<br />
01#17<br />
Naphthenic Acid Biodegradation by Bacteria Enriched from Oil Sands Tailings Pond Surface<br />
Water<br />
Esther Ramos-Padron, K. Nze University of Calgary<br />
Naphthenic acids (NAs) are naturally found in most crude oils. In Alberta's oil sands surface mining<br />
industry, these cyclohexane carboxylic acids (general molecular formula CnH2n+ZO2) are concentrated in<br />
tailings ponds following the caustic extraction of bitumen. Due to the zero discharge policy, the solid and<br />
liquid wastes are kept in their containment area until they can be reclaimed. However, the tailings pond<br />
water (TPW) must be treated before release to the environment as NAs have known toxicity to terrestrial<br />
and aquatic organisms. Tailings ponds harbor a wide variety of microorganisms that may serve to<br />
biodegrade NA. Here we enriched for aerobic bacterial cultures from TPW to study the microbial<br />
metabolism of 5 model monocyclic NAs ranging from cyclohexanecarboxylic acid (CHCA) to<br />
cyclohexanepentanoic acid (CHPA). The cultures grew on all of the tested NAs. In time course<br />
experiments, CHPA was metabolized via stepwise beta-oxidation to CHCA. Cyclohexene-1-carboxylic<br />
acid and cyclohexene-1, 4-dicarboxylic acid were also identified as metabolites using GC-MS. Pure<br />
cultures were obtained from the enrichments and identified by 16S rDNA gene sequencing and<br />
biochemical tests. Isolates from the genera Pseudomonas, Afipia, Xanthobacter, Acidovorax, and<br />
Sphingopyxis were capable of oxidizing the model NAs. Ongoing work with tailings enrichments<br />
incubated with complex NAs mixtures coupled with the study of model NA will help to unveil the metabolic<br />
pathways used by bacteria to biotreat NAs in TPW.<br />
01#18<br />
Identifying Key Microbes in Methanogenic Hydrocarbon-Degrading Enrichment Cultures from Oil<br />
Sands Tailings<br />
Nidal Abu Laban, K. Semple, J. Foght Department of Biological Sciences, University of Alberta<br />
Oil sands tailings settling ponds contain residual bitumen as well as solvents like naphtha. Previous<br />
studies have shown that naphtha and its constituents (particularly BTEX [benzene, toluene, ethylbenzene<br />
16
and xylenes] and C6-C10 n-alkanes) can be degraded under methanogenic conditions by microbial<br />
communities present in oil sands tailings. However, the key Bacteria initiating hydrocarbon<br />
biodegradation in tailings ponds are currently unknown. To help identify these microbes, numerous<br />
methanogenic enrichment cultures containing mature fine tailings (MFT) were incubated with naphtha,<br />
BTEX, toluene, o-xylene, C6-C10 n-alkanes, or n-octane. As part of the Hydrocarbon Metagenomics<br />
Project, 16S rRNA genes were analyzed using terminal restriction fragment length polymorphism (T-<br />
RFLP) and 454-FLX pyrosequencing. The T-RFLP profiles of naphtha- and BTEX-grown cultures were<br />
similar and were dominated by 16S rRNA gene sequences forming 157 and 159 bp T-RFs, whereas T-<br />
RFs of 121, 173, 159 and 515 bp were abundant in the o-xylene-, C6-C10 n-alkane- and n-octane-grown<br />
cultures. Pyrosequencing of enrichment cultures incubated with C6-C10 n-alkanes or BTEX showed that<br />
22% and 25% of the total sequenced operational taxonomic units (OTUs) were strongly affiliated with<br />
Syntrophobacterales and Desulfosporosinus sp., respectively. This suggests that different sulfatereducing<br />
bacteria might be important for initiating biodegradation of different classes of hydrocarbons in<br />
oil sands tailings enrichment cultures. Further investigations using stable isotope probing (SIP) and qPCR<br />
with genus- and species-specific primers are in progress to extend these observations.<br />
01#19<br />
An in vitro Mixed Species Biofilm Approach to Culturing Microbes Directly from an Oil Sands<br />
Tailings Pond<br />
Susanne Golby, M. Demeter, H. Ceri, T. Raymond J University of Calgary<br />
Tailings ponds result from the extraction of bitumen from the oil sands of Northern Alberta, and currently<br />
contain millions of cubic meters of toxic waste. Tailings are a complex mixture of residual bitumen, water,<br />
sand, clay, hydrocarbons, organic acids and heavy metals. We have hypothesized that bioremediation of<br />
the tailings pond can be optimized using mixed species biofilms that are endogenous to the tailings<br />
environment. Studying natural microbial populations in vitro proves to be a challenge due to the large<br />
number of microbes that currently cannot be successfully cultured in the laboratory. Thus, using the<br />
Calgary Biofilm Device (CBD) as a microscale reactor, we considered if both aerobic and anaerobic<br />
mixed species biofilms could be cultivated directly from oil sands tailings pond sediments, demonstrating<br />
a cultivation technique that can be employed for subsequent bioremediation investigations in vitro. We<br />
have succeeded in culturing aerobic, microaerobic and anaerobic mixed species biofilms directly from an<br />
oil sands tailings pond under a variety of different culture conditions. Microbial diversity within biofilms<br />
was initially evaluated by denaturant gradient gel electrophoresis (DGGE) and quantified with Quantitative<br />
PCR (Q-PCR). DGGE profiles showed very diverse mixed species biofilms could be generated and each<br />
population was unique when biofilms were recovered using different incubation temperatures ranging<br />
from 4°C to 25°C, oxygen tensions (aerobic, microaerobic, and anaerobic) and varying growth medias<br />
and carbon sources (carbohydrates, glucose, and naphthenic acids). 454 pyrosequencing revealed that<br />
the organisms within the biofilms represented the indigenous population in the tailings used as the<br />
inoculum. Furthermore, biofilms contained over 10 different genera per biofilm including organisms<br />
belonging to Pseudomonas, Brachymonas, Thauera, Hydrogenophaga, Rhodoferax and Acidovorax. In<br />
this study, we have successfully shown that the CBD can be used to grow diverse mixed species biofilms<br />
directly from the tailings environment that closely reflect the indigenous population.<br />
01#20<br />
Time-dependent Variation in the Anaerobic Microbial Community Structure of an Alberta Oil<br />
Sands Tailings Pond as Determined by 16S pyrosequencing<br />
Indranil Chatterjee, E. R. Padrón, L. M. Gieg, G. Voordouw Petroleum Microbiology Research<br />
Group, Department of Biological Sciences, University of Calgary<br />
Bitumen extraction from oil sands produces large volumes of tailings slurry comprising water, silt, clays,<br />
unrecovered bitumen and hydrocarbon diluent, used in the extraction process. Tailings solids settle by<br />
gravity to become denser mature fine tailings in large tailings ponds. These are highly stratified due to<br />
increasing solids content as a function of depth. The microbial community composition as a function of<br />
17
depth in an active tailings pond, receiving input of fresh tailings and treated with gypsum (CaSO4.2H2O) to<br />
accelerate densification, indicated the presence of syntrophs (Syntrophus, Smithella and<br />
Pelotomaculum), sulfate- and sulfur-reducing bacteria (Desulfocapsa and Desulfurivibrio spp.), as well as<br />
acetate- and H2-using methanogens. In the current study, we evaluated the effect of anaerobic storage<br />
(90% N2, 10% CO2) on the microbial population from this same active tailings pond. DNA was isolated<br />
from selected depths from tailings samples stored under anaerobic conditions for 1.5 years. Analysis of<br />
16S genes by pyrosequencing indicated an increase in fermentative bacteria capable of reducing nitrate<br />
(Delftia, Acidovorax, Thauera, Pseudomonas) at most of the depths along with a high abundance of<br />
methanogens (Methanosaeta, Methanobacterium, Methanolobus) at a depth of 18 m below the surface.<br />
No syntrophs or SRB were detected. UniFrac clustering analysis indicated this community profile to be<br />
similar to that of another tailings pond that no longer receives input of fresh tailings. The results indicate<br />
that tailings pond microbial communities are dynamic and change rapidly when input of fresh tailings<br />
and/or gypsum is stopped.<br />
18
Session 02 - MIC monitoring & Treatment<br />
Invited speakers:<br />
02#01<br />
An Overview of MIC in Oil Field Systems<br />
Brenda Little, J. Lee, R. Ray Naval Research Laboratory<br />
The relationship between microorganisms and corrosion in oil field systems is both predictable and<br />
complex. The numbers and types of microorganisms involved in corrosion in oil field systems, and<br />
consequently the mechanisms for microbiologically influenced corrosion (MIC), are continuously being<br />
updated. The microflora associated with oil fields is extremely diverse, including bacteria and archaea.<br />
Using molecular microbiological methods, investigators have demonstrated that the microorganisms<br />
associated with corrosion products may not be detected with culture-dependent techniques, consortia of<br />
organisms can vary with geographical location and microbial populations in produced water and scale<br />
from the same system are not necessarily similar. The ability of bacteria to produce corrosive hydrogen<br />
sulfide from the reduction of sulfate, sulfite, thiosulfate and elemental sulfur has been a chronic concern in<br />
oil field systems. These organisms are found in every anaerobic habitat examined to date and their<br />
population numbers are routinely monitored as part of integrity management efforts. They are an<br />
enormously diverse group of bacteria that can ferment both organic and inorganic compounds and use a<br />
wide variety of electron donors and acceptors. In the absence of an electron acceptor, these organisms<br />
can enter into syntrophic relationships with methanogens or other H2-consuming organisms. However,<br />
their relationship to corrosion does not depend on numbers of organisms, but on environmental<br />
conditions, including the nature of available electron acceptors and operating conditions. The<br />
circumstances that result in MIC in oil field systems will be reviewed.<br />
02#02<br />
MIC in Long Oil Pipelines: Diagnosis, Treatment and Monitoring<br />
Gary Jennerman, J. Harris, R. Webb ConocoPhillips<br />
Localized corrosion occurring in a long oil pipeline presents unique problems and challenges including the<br />
determination of its root cause through the examination of pig solids and water samples collected many<br />
kilometers from the site of corrosion. In addition, biocides needed to treat long oil pipelines must be<br />
evaluated not only on their efficacy towards control of sessile bacteria, but their compatibility with other<br />
treatment chemicals; their long term stability in produced brines; and their ability to partition favorably to<br />
the water phase, not to mention their need to satisfy local environmental and safety regulations. This<br />
paper provides laboratory and field results concerning the possible involvement of bacteria in the<br />
localized corrosion of a subsea oil pipeline containing, on average, 2% water. Both traditional and<br />
molecular microbial methods were used to help determine the cause of the corrosion and to monitor the<br />
effectiveness of chemical and physical treatments. Lab tests were conducted using planktonic and sessile<br />
field populations to select the right biocide chemistry and to ensure its chemical compatibility and efficacy<br />
under the pipeline conditions. Results indicated that biocide stability and partitioning could be an issue<br />
when treating either continuously or batchwise. Furthermore, the use of molecular methods (e.g. PLFA,<br />
qPCR, qFISH, and hydrocarbon biometabolite analyses) provided a much greater insight into the types<br />
and concentrations of bacteria present in the water and pig sludge.<br />
19
Offered Papers:<br />
02#03<br />
MIC Risk in the Halfdan Oil Export System Quantified with a DNA-based Diagnostic Tool<br />
Torben Lund Skovhus 1 , K. Rasmussen 2 , K. Andersen 2 , J. Larsen 2 1 DTI Oil & Gas 2 Mærsk Oil and<br />
Gas AS<br />
In the oil industry monitoring of Microbiologically Influenced Corrosion (MIC) has in the past been<br />
conducted mainly on sulfate-reducing Bacteria (SRB) using cultivation based techniques. However, with<br />
the introduction of novel DNA-based methods for enumeration of microbes a more accurate and fast<br />
method is now in place which is being routinely applied for better MIC control. The content of produced<br />
water in the Halfdan oil system during production is low (BS&W 1-2%) and MIC has for this reason not<br />
been considered to be a threat for the system integrity. Initial wall thickness results obtained from<br />
ultrasound scanning of the oil export spools indicated ongoing corrosion. Following replacement, cut-out<br />
sections of the spools were sampled and sent onshore for further investigation of general corrosion, MIC<br />
and scale analysis. The investigation showed that the analysed Halfdan oil export spool sections were<br />
highly corroded. This was concluded from the presence of pitting corrosion and under deposit corrosion<br />
found beneath thick layers of solid scale material. In support of the visual indications of corrosion, we<br />
demonstrated that MIC related microorganisms were present in the solids in close contact with the metal<br />
surface together with corrosion products. In particular, high numbers of hydrogen consuming<br />
methanogens (10 8 - 10 9 cells/g) were situated in the scale material directly in contact with the carbon<br />
steel pipe wall. These important conclusions could only be confirmed when results from the DNA-based<br />
enumeration methods and X-Ray analysis were combined with the visual inspections of the oil export<br />
spools. Based on the obtained results, this paper discusses how microbial numbers obtained from DNAbased<br />
enumeration methods are evaluated and interpreted in the best way with respect to general risk<br />
assessment and system integrity measures of ageing offshore assets in the North Sea region.<br />
Posters:<br />
02#04<br />
Fuel Bacteria Causes Biocorrosion on Metalic Alloys<br />
Silvia Acosta, C. Ritoles, B. Fernández, A. Cueli, F. González Cuban Petroleum Research Center<br />
The metabolic activity of microorganisms may directly or indirectly cause a deterioration of metal by<br />
corrosion processes. The aim of this study is to assess the behavior of alloy steel (20Kp), currently used<br />
for the construction of fuel storage tanks, when common spoilage bacteria like Bacillus sp., Pseudomonas<br />
aeruginosa, Bacillus sp, Sporosarcina sp., and others are isolated from these metals. Four experiments<br />
were set up in electrochemical cells for a period of 60 days. Potential points were measured and the<br />
biofilms formed on the metal surfaces were observed by optical microscopy. Bacterial growth was also<br />
monitored on nutrient media. Low corrosion rates were found ranging from 10-3 and 10-5 mm/year. The<br />
microorganisms grown on the metal showed localized corrosion and the bacterial population increased<br />
after 30 to 60 days reaching levels of up to 107 CFU/mL in study II and IV and about 108 CFU/mL in<br />
study III. The results confirmed that these bacteria were able to use the fuel as a carbon and energy<br />
source when the water was present and their growth contributed to the formation of the biofilms on the<br />
surface of metal alloys.<br />
20
02#05<br />
Investigation of Microorganisms in Water-dissolved Natural Gas Fields for Preventing the Well<br />
Clogging<br />
Ryo Wakizono 1 , Y. Sugai 1 , K. Sasaki 1 , N. Muraoka 2 , Y. Higuchi 2 1 Department of Earth Resources<br />
Engineering, Faculty of Engineering, Kyushu University 2 Godo Shigen Sangyo, Co., Ltd.<br />
The brine that is produced from water-dissolved natural gas reservoirs should be returned into reservoirs<br />
after separation of natural gas to prevent the subsidence. However, reinjection capacities of reinjection<br />
wells decline gradually and their capacities are maintained by backwashing. Biofilm-like materials can<br />
be observed in solid materials that are produced by backwashing. In this study, we investigated the<br />
relationship between microbial communities in the brine and the clogging of reinjection wells. Brine<br />
samples were extracted from several places: production wellheads, sedimentation tanks below gas<br />
separators, sump pits below the recovery plants of iodine which is also extracted from brine as a natural<br />
resource, and wellheads and bottom holes of reinjection wells. Microbial community structures in brine<br />
samples were analyzed by DGGE and nucleotide sequences of each DNA isolated by DGGE were<br />
analyzed. Microbial community structures were changed drastically after the gas separators, at where the<br />
brine began to contact air, and the iodine recovery plants, at where sulfuric acid and oxidizer were added<br />
into brine for the recovery reactions. Both sulfate-reducing bacterium Desulfovibrio sp. and sulfuroxidizing<br />
bacterium Sulfrimonas sp. were detected as dominant species from the bottom holes. Formation<br />
of microbial consortium and biofilms by those bacteria is well known in microbial corrosion. It is assumed<br />
that both oxygen and sulfate that are added through the surface process stimulate the formation of<br />
biofilms of those bacteria and it clogs the reinjection wells. Therefore, both deoxidization and<br />
desulfurization are expected to be effective for preventing the clogging the reinjection wells.<br />
02#06<br />
The Diesel and Biodiesel Fuel Biodegradation Testing and Microbial Community Fame<br />
Differentiation by IR, GC and Raman Spectrometry<br />
Vedranka Bobic, B. Spehar, L. Stajduhar INA Oil industry<br />
The microbial spoilage of petroleum products has obvious economic implications, and most research on<br />
the biodeterioration of petroleum products has been prompted by these economic considerations. The<br />
diesel/biodiesel blending increased the likelihood of microbial fuel infections and biodeterioration.<br />
Problems caused by microbial growth are: blockage of filters caused by the physical presence of<br />
microorganisms, surfactants produced by microorganisms diminishing the ability of fuel to separate from<br />
water, formation of corrosive metabolic products (organic acids and sulfide), microbial depolarization<br />
stimulating corrosion and microbial degradation of protective coatings. Today there are different<br />
approaches to microbial community assessment: cultivation -dependent and cultivation independent. To<br />
characterize microbial communities and estimate microbial biomass and community structure the profiles<br />
of fatty acids derived from phospholipids components of the microorganism's cellular membrane proved<br />
useful. The aim of this work has been to determine microbial degradation velocity of diesel and<br />
diesel/biodiesel blends and to estimate the possibilities of laboratory equipment (IR, GC, NXR FT-Raman)<br />
and analytical knowledge for a faster differentiation and identification of microbial species by profiling fatty<br />
acid methyl esters (FAME). GC analysis show different amount and different combination of isomers<br />
depending on different growth media. ATR diamond FTIR spectra of three bacterial genera show the<br />
spectral differences among these bacteria (mainly aliphatic unsaturated bonds). Comparison of bacterial<br />
spectra show differences in C - O functional groups related to type of esters and C-H bonds related to the<br />
chain structure (branching, lengths). The Raman spectra preparation technique was determined.<br />
21
02#07<br />
Laboratory Simulations on the Effect of Various Nitrate Strategies to Control Topside MIC in PWRI<br />
Systems<br />
Heike Hoffmann 1 , C. Devine 1 , G. Voordouw 2 1 Intertek Commercial Microbiology Ltd 2 University of<br />
Calgary, Petroleum Microbiology Research Group<br />
Nitrate treatment used to prevent reservoir souring can lead to an increase in the rate of both general<br />
corrosion and pitting of mild steel. In a remote offshore set-up, such as the North Sea, the supply of<br />
nitrate is not always guaranteed. This trial addresses the question - should stocks of nitrate be insufficient<br />
to meet requirements, what is the best strategy to address the problem? Options include continuing with<br />
the nitrate application at the usual concentration until stocks are depleted; reducing the dose in an effort<br />
to make the limited supply last; applying the nitrate as a batch dose on an infrequent basis or changing to<br />
a frequent batch dosing regime. The aim of this study was to determine the effect of various strategies on<br />
the injection pipework corrosion rate but also on the diversity of the sessile microbial populations. In<br />
addition to real time continuous readings of pH, redox potential and temperature; chemical,<br />
microbiological and molecular analyses on biofilms were carried out after each treatment regime.<br />
Community analysis using pyrosequencing are in progress in the moment. Data from the laboratory trial<br />
indicates that stopping nitrate application can result in a significant increase in sulphide generation. In<br />
some instances, rates of general and pitting corrosion are found to be higher than expected. So far the<br />
data indicates that the bacterial populations do not necessarily change over time with continuous<br />
treatment, which might be the reason why sulphide generation does not cease completely and corrosion<br />
is still persist.<br />
02#08<br />
Control of Souring and Microbially Influenced Corrosion in Produced Water Re-injection Systems<br />
Carsten U. Schwermer, F. K. Garshol, A. J. Dinning Aquateam Norwegian Water Technology<br />
Centre AS<br />
Biofilms and schmoo cause serious operational problems on many offshore oil field installations. They<br />
can reduce performance of produced water treatment and reservoir injectivity, and increase corrosion in<br />
topside installations often due to sulphide production by sessile sulphate reducing bacteria. Produced<br />
water re-injection (PWRI) systems are more prone to biofouling than seawater injection systems (SI) due<br />
to higher electron donor availability. While nitrate is increasingly used to mitigate reservoir souring in SI<br />
systems, its addition to PWRI systems is still debated. Because nitrate provides a potent electron<br />
acceptor to nitrate utilising organisms that consequently thrive, its addition to the electron donor rich<br />
system may enhance biofouling and accelerate corrosion. In fact, accelerated corrosion during nitrate<br />
addition has previously been observed during an offshore PWRI pilot study, and has recently been<br />
confirmed in a test reactor systems in our laboratory. To limit the negative potential side effects when<br />
applying nitrate in the field, suitable procedures for biofouling and corrosion control are required. We<br />
tested the impact of a biocide (BIO) and a corrosion inhibitor (CI) on pipeline biofilms grown on mild steel<br />
coupons in anaerobic laboratory flow-cells, and pigging was simulated. Biofilms were exposed to various<br />
conditions altering between nitrate, BIO, and CI availability for 250 days. Microprofiling revealed large<br />
structural heterogeneities within small distances in all investigated biofilms. Nitrate addition enhanced<br />
biofouling that was not mitigated by biociding. In tests receiving only CI and combined CI and BIO,<br />
general corrosion was reduced while pitting corrosion might not be affected.<br />
22
02#09<br />
In Situ Description of Corrosive Biofilm with Environmental Scanning Electron Microscopy<br />
(ESEM) in Gas Pipelines<br />
Icoquih Zapata-Penasco 1 , A. Aguirre 1 , J. Chanona 2 , V. Garibay 1 , J. M. Romero 1 , J. Mendoza 2 1 IMP<br />
Mexican Petroleum <strong>Institute</strong> 2 IPN National Polytechnic <strong>Institute</strong><br />
Recently, several biofilm formation studies have been done under in vitro setting. Besides, following in<br />
situ biofilm development is a difficult achieve because of non-accessible monitor duty in real environment<br />
conditions. Since biofilm monitor is of primary importance in MIC control, testing the sessile bacteria is<br />
one of the most relevant approaches that need to be done. In this work, metal standard coupons were<br />
installed by using a bio-probe and aligned parallel to the process flow inside pipeline, in order to follow the<br />
formation of corrosive biofilm during six months of real gas pipeline operation. Coupons were observed<br />
and analyzed by using ESEM techniques; biofilm developed was described and characterized, and<br />
corrosion rates were calculated in real time operation in gas pipeline from Mexican oil exploitationproduction<br />
facilities.<br />
02#10<br />
Development of Standard Methods for the Analysis of Microbiologically Influenced Corrosion<br />
Using the Quantitative Polymerase Chain Reaction<br />
John Kilbane 1 , P. Sen 2 1 Intertek 2 Baylor University<br />
Quantitative Polymerase Chain Reaction (qPCR) has established itself as a sensitive and specific<br />
qualitative and quantitative technique that has become important to all areas of microbiology including<br />
medical microbiology, public health, food safety, cosmetics, and environmental analyses. If qPCR is to<br />
enjoy widespread use for the analysis of microbiologically influenced corrosion (MIC) in the petroleum<br />
and other industries it will be necessary to have standardized methods. Discussions are underway to<br />
revise the NACE standard TM0194 to include qPCR, however; current drafts of this document do not<br />
specify primer sequences and reaction conditions. This paper proposes a standard qPCR method for MIC<br />
analysis.<br />
02#11<br />
Microbially Influenced Corrosion (MIC) in Pipelines Transporting Brackish Water from the<br />
Subsurface to a Steam-Assisted Gravity Drainage (SAGD) Plant<br />
Hyung Soo Park, I. Chatterjee, S. Lin, S. Johnston, T. R. Jack, G. Voordouw University of Calgary,<br />
Petroleum Microbiology Research Group<br />
Production of 6000 m 3 of bitumen per day by an Alberta SAGD plant requires 10,000 m 3 of water, which is<br />
drawn from the McMurray and Grand Rapids formations. The subsurface waters are brackish with 500 -<br />
1500 mg/L of bicarbonate and 2500 to 5000 mg/L of chloride and are transported by pipelines to the<br />
SAGD plant, where they are converted into steam. Frequent corrosion problems prompted our<br />
investigation into the possibility for MIC at the request of the plant operator. Treatment to prevent<br />
corrosion includes addition of sodium bisulfite (SBS) as an oxygen scavenger. Samples of water from the<br />
Grand Rapids (E1) and the McMurray (E2) formations and combined water (E3), as well as corroded pipe<br />
sections were obtained. The latter came with pipe-associated water (PAW). Pipe-associated solids (PAS)<br />
were scrubbed from the internal pipeline surface in an anaerobic hood. Samples were analyzed for<br />
electron donors and acceptors, microbial activities, their ability to corrode iron (weight loss and linear<br />
polarization resistance) and microbial community composition by pyrosequencing of PCR-amplified 16S<br />
genes. PAS had high lactate-dependent activity of sulfate-reducing bacteria and the highest corrosion<br />
rates. Microbial community composition of pipe sections located either upstream or downstream of the<br />
SBS injection point were dominated by methanogenic Archaea or sulfite-disproportionating and sulfatereducing<br />
Deltaproteobacteria, respectively. Both of these appeared to be able to contribute to corrosion,<br />
23
i.e. iron dissolution coupled to methane production could be demonstrated. In conclusion, SBS addition<br />
changed the pipe-associated microbial community, but the main microbial groups found both contributed<br />
to corrosion.<br />
02#12<br />
dsrAB and nifD Gene Detection of Bacteria Associated with Corrosion in Mexican High Saline<br />
Water Injection Pipelines<br />
Icoquih Zapata-Penasco 1 , C. Hernandez-Rodriguez 2 , J. M. Romero 1 1 Mexican Petroleum<br />
<strong>Institute</strong> 2 National Polytechnic <strong>Institute</strong><br />
In Enhanced Oil Recovery (EOR), connate, freshwater or marine water is injected to oil reservoir through<br />
wells. Microbial corrosion control programs play a substantial role maintaining quality and standards of<br />
injected water, thus avoid great economic losses during EOR operations. Molecular tools could be used<br />
to develop specific-microbial-target strategies in biocorrosion control. Because of their polyphyletic origin<br />
of sulfate-reducing bacteria (SRB), to use functional genes is an alternative to detect them in the<br />
environment. The aim of this work was to provide molecular specific designs to detect genes of SRB and<br />
Fe (III) reducing bacteria in high saline water of injection pipelines. PCR based specific procedures were<br />
performed for dsrAB and nifD gene designs of Desulfovibrionales, Desulfobacterales and<br />
Desulfuromonadales taxonomical orders which are associated with stainless steel corrosion. DNA of<br />
reference strains and metagenomic DNA extracted from different high saline injection waters confirmed<br />
the successful pertinence of the PCR designs. These probe designs could be used to get a specific<br />
microbial diagnostic for biocide treated and non-treated injection waters in order to glance through SRB<br />
and Fe (III) reducing bacteria presence; therefore, microbial corrosion control programs could be<br />
successful maintaining quality and standards of injected water during EOR operation.<br />
02#13<br />
Determination of Successful Fracture Source Water Treatment for the Prevention of MIC and<br />
Biogenic Souring<br />
Glen Nilsen, S. Moore, R. Bosch, K. Turner Champion Technologies<br />
In recent years, hydraulic fracturing technologies in combination with advanced horizontal drilling have<br />
matured such that unlocking vast amounts of natural gas from tight shale formations is economically<br />
feasible. Termed "slickwater fracturing", the completion method is very dependent upon the availability of<br />
large volumes of fresh water, which are typically obtained from surface sources, such as ponds or rivers.<br />
These source waters generally contain large populations of microorganisms, such as sulfate-reducing<br />
prokaryotes (SRP) and acid-producing bacteria (APB). If injected into a targeted shale formation during<br />
fracturing, such microorganisms can become established and cause serious problems, including<br />
formation damage, biogenic hydrogen sulfide (H2S), microbiologically influence corrosion (MIC), and lowquality<br />
flowback water. Preventive treatment with 'on-the-fly' application of biocides to treat source water<br />
is important for controlling microorganism populations and their deleterious effects. Biocide chemistries<br />
selected for hydraulic fracturing treatments must be robust enough to eliminate the broad spectrum of<br />
bacteria which may be present in the source water, must be compatible with stimulation fluids, and must<br />
fit the economic constraints of the specific application. The current field study demonstrates a benchmark<br />
for a 'successful' biocide treatment, as determined by bacterial contamination of source and flowback<br />
waters before and after fracturing, respectively. Accuracy of the determined benchmark is supported<br />
through long-term well evaluation via industry standards for monitoring bacteria, as well as resulting<br />
cases of MIC and souring attributed to SRP. The study also discusses the rapid and costly effects of MIC<br />
and biogenic souring in unsuccessful biocide-treated or non-biocide treated wells.<br />
24
Session 03 - Metagenomics, Molecular and Microbial Methods<br />
Invited speakers:<br />
03#01<br />
Use of pyrosequencing to characterize microbial communities in hydrocarbon resource<br />
environments<br />
Gerrit Voordouw Petroleum Microbiology Research Group, Dept. of Biological Sciences,<br />
University of Calgary<br />
Novel methods to sequence DNA, like pyrosequencing, allow several hundred thousand sequences to be<br />
determined simultaneously. Because a 16S survey of a sample from an oil or gas field environment<br />
requires only about 10,000 sequences, 30-50 samples can be analyzed at once. Hence, detailed<br />
analyses of the microbial composition in samples derived from oil and gas fields can be completed rapidly<br />
and economically. Limiting factors are our ability to isolate representative DNA and possible PCR bias.<br />
Pyrosequencing of 16S amplicons has indicated that the microbial community composition of metal pipes<br />
transporting water for subsurface bitumen production is strongly influenced by the addition of sodium<br />
bisulfite (SBS) as an oxygen scavenger. SBS addition shifted the pipe-associated microbial community<br />
from methanogenic archaea to bacteria capable of disproportionating sulfite to sulfide, sulfate, sulfur and<br />
thiosulfate, as well as to sulfate-reducing bacteria. Pyrosequencing also indicated that the microbial<br />
community composition of bitumen-containing cores was surprisingly diverse as a function of distance on<br />
the cm scale, suggesting the existence of many microniches in the oil sands subsurface. Pyrosequencing<br />
of samples obtained from shale gas fields indicated dominance of microbes capable of carbohydrate<br />
fermentation at high temperatures in the subsurface and at low temperatures above ground. This is<br />
caused by extensive use of carbohydrate polymers as sand-propping agents in shale gas production.<br />
These are some examples of the range of applications for pyrosequencing surveys. The data obtained by<br />
these must of course be combined with physical, chemical and microbial physiology studies to gain more<br />
insight and understanding.<br />
03#02<br />
Experience of molecular monitoring techniques in upstream oil and gas operations<br />
Anthony Mitchell, H. Anfindsen, T. Liengen, S. Molid Statoil ASA<br />
Whilst molecular tools have been employed for a number of years within the oil industry the results have,<br />
largely, been of academic interest and of limited additional value to solving problems. More recent<br />
advances in techniques are providing scope for evaluation of potential microbially influenced problems in<br />
a variety of situations; however, there still remain challenges with interpretation of the data being<br />
generated. In many cases molecular biology enables one to confirm the presence of groups of<br />
microorganisms that may be implicated in corrosion or hydrogen sulphide generation. Nonetheless, there<br />
remains considerable work to be undertaken to define criteria for whether the corrosion events can be<br />
attributed solely to microbial activity. The paper will present case histories from water injection and oil<br />
production wells plus a multiphase flowline, review results obtained and discuss the degree to which the<br />
use of molecular monitoring has helped to more fully explain the observed events.<br />
03#03<br />
Microorganisms involved in MIC<br />
Ketil Sørensen DTI Oil & Gas<br />
Microbiologically Influenced Corrosion (MIC) is a widespread phenomenon in the oil and gas industry,<br />
where corrosive biofilms are observed throughout water injection and oil production systems. In spite of<br />
the large economical implications of the phenomenon, surprisingly little is known regarding the factors<br />
25
controlling the corrosivity of biofilms, and the link between microbial metabolism and iron dissolution from<br />
the metal surface remains vague. Microorganisms may facilitate the dissolution of Fe0 into Fe(II) or Fe(III)<br />
through e.g. hydrogen consumption, formation of pH/redox gradients, or production of corrosive<br />
metabolites. The MIC surveillance performed by the offshore oil and gas industry has historically been<br />
focused on the sulfate-reducing Bacteria (SRB) because they consume hydrogen and produce corrosive<br />
hydrogen sulfide. However, several lines of evidence indicate that other groups of microorganisms may<br />
be of equal or even larger importance to MIC in offshore production facilities in the North Sea and<br />
elsewhere; (i) Both thermophilic, sulfate-reducing Archaea (SRA) and several types of hydrogenconsuming<br />
methanogens seem to be numerically more abundant than SRB in these systems, (ii) the<br />
spatial distribution of methanogens and sulfate reducers within deposits on corroded pipings strongly<br />
indicate that mainly methanogens are found at the metal surface, and (iii) the abundances of sessile<br />
populations of methanogens are correlated with local corrosion rates. The wider implications of these<br />
findings for the MIC surveillance, risk assessment and mitigation strategies performed by the offshore<br />
industry will be discussed.<br />
Offered Papers:<br />
03#04<br />
Biogeographic patterns of soil microbial communities from different oil-contaminated fields in<br />
China<br />
Jizhong Zhou 1 , Y. Liang 1 , G. Li 2 1 University of Oklahoma 2 Tsinghua University<br />
To understand the geographic patterns and microbial functional diversity in different oil-contaminated<br />
fields, a hundred soil samples were taken from typical oil-contaminated fields located in five geographic<br />
regions of China, Daqing oilfield in northeast China, Talimu oilfield in northwest China, Shengli oilfield in<br />
North China, Jianghan oilfield in central China and Baise oilfield in South China. GeoChip 3.0, a highthroughput<br />
functional gene array, which contains 27,812 oligonucleotide (50-mer) probes, was used to<br />
study the microbial functional genes involved contaminant degradation and other major<br />
biogeochemical/metabolic processes. Our results indicated that the soil geochemical parameters were<br />
significantly different among the five fields. For example, soil nutrient level (nitrogen and phosphorous)<br />
were higher in south fields; Soil pH was around 7.5 - 8.8 in North fields and around 6 in South field; soil<br />
water content was lowest in northwest field. The overall microbial communities showed a significant<br />
geographic pattern in each oil-contaminated field that samples were clustered majorly by geographic<br />
locations. Canonical correspondence analysis (CCA) and variation partitioning analysis (VPA) indicated<br />
that the spatial pattern of soil microbial communities were highly related with field locations, oil<br />
contamination and soil geochemical variables such as nutrient content, pH. This study provided insights<br />
into the in situ microbial spatial patterns in oil-contaminated fields and discerned the linkages between<br />
microbial communities and environmental variables, which is important to the application of<br />
bioremediation in oil-contaminated sites.<br />
03#05<br />
Oil Reservoir Metagenome. New insight to the Oil Reservoir Processes<br />
Hans Kristian Kotlar 1 , A. Lewin 2 , S. Valla 2 , S. Markussen 3 1 Statoil ASA 2 NTNU, Department of<br />
Biotechnology 3 Department of Biotechnology, SINTEF Materials and Chemistry<br />
Previously, we have shown the efficiency of extremophile organisms both in bio-upgrading of oil and in<br />
heavy oil bioconversion [1]. Easily recoverable fossil fuels are declining and the petroleum industry is<br />
seeking new ways of recovering oil from reservoirs. Bioconversion of oil by extremophile microorganism<br />
communities might represent a novel method for recovery. Given the right conditions certain<br />
microorganisms can withstand and thrive in the high pressure, high temperature and high levels of<br />
potentially toxic organic as well as inorganic components found in some oil reservoirs. The oil reservoirs<br />
represent a unique habitat for microorganisms. Despite the potential environmental and economical<br />
importance of oil reservoir microbial communities, very little is still known about the ecology and in situ<br />
26
microbial processes of the oil reservoir. In order to bring insight into this particular environment, North Sea<br />
oil reservoir samples have been collected and used for direct DNA isolation and pyrosequencing using<br />
the 454 approach, aiming to characterize the microbial flora of the reservoir. The current paper will give<br />
insight into work related to oil reservoir samples, its difficulties and opportunities, as well as the results<br />
from characterizations of the different reservoir zones. Surprising variations in biodiversity are seen<br />
between wells draining from different reservoir zones. [1] Kotlar, H.K.2009. Can Bacteria Rescue the Oil<br />
Industry? The Scientist 23: 30-35.<br />
03#06<br />
Comparative community genomics of San Juan Basin coal bed methane deposits<br />
Antoine Pagé, C. Howes, Y. Song, P. Sipahimalani, E. Kuatsjah, S. Hallam University of British<br />
Columbia<br />
Long considered an unconventional form of natural gas, coal bed methane (CBM) has become an<br />
increasingly important energy resource in Canada and the United States. Although many biochemical<br />
processes and microbial players responsible for methane formation from biodegraded coal have been<br />
identified, little is known about the microbial agents or biochemical processes responsible for organic<br />
matter degradation within coal bed seams. These initial degradation processes likely impose a ratelimiting<br />
step in CBM formation and therefore limit the productivity or sustainability of proven reserves.<br />
Here, we present a comparative community genomic survey of microbial communities inhabiting coal bed<br />
deposits in the San Jaun Basin of Colorado and New Mexico to better constrain microbial assemblages<br />
involved in organic matter degradation. Microbial biomass was obtained by filtering production waters<br />
from twelve wells with differing environmental parameters. Filtered biomass was used to extract genomic<br />
DNA for use in V6 pyrotag sequencing targeting the small subunit ribosomal RNA gene. Multivariate<br />
analysis of V6 pyrotags identified four major clusters associated with differing regimes of temperature,<br />
metal or salt concentration. Representative samples from each cluster were selected for pyrosequencing,<br />
and resulting short read data sets were assembled, annotated and compared to identify common and<br />
unique metabolic subsystems. Additional data sets from related hydrocarbon environments including<br />
marine sediments and a natural asphalt lake were used to identify enzymes or subsystems diagnostic for<br />
CBM. This study opens a window into microbial life in deep subsurface environments and provides a<br />
molecular blueprint for enhanced methane recovery from coal bed seams.<br />
Posters:<br />
03#07<br />
Prospection of new genes related to denitrification process of metagenomic libraries from<br />
petroleum refinery sludge<br />
Cynthia da Silva 1 , T. Sawbridge 2 , H. Hayden 2 , P. Mele 2 , A. P. Torres 3 , V. Torres 1 1 Unicamp<br />
2 DPI/Victoria 3 Petrobras<br />
Denitrification is the dissimilatory reduction of nitrate or nitrite performed by microorganisms, with<br />
subsequent production of nitric oxide, nitrous oxide or nitrogen gas. The last step of denitrification, the<br />
conversion of N2O to N2, is catalyzed by the nitrous-oxide reductase enzyme. The N2O is a greenhouse<br />
gas which contributes to the global warming and the destruction of the ozone layer. In this sense, there is<br />
a great interest in the study of the microbial metabolism potential to improve the nitrogen removal of<br />
WWTP. The goal of this study was to evaluate the diversity of nitrous-oxide reductase genes in<br />
metagenomic libraries derived from wastewater treatment plant (WWTP) by using the pyrosequencing<br />
approach. The sludge sample was collected from a pilot membrane bioreactor (MBR) of a petroleum<br />
refinery WWTP in Brazil. Sludge DNA of high molecular weight was isolated, ligated into the fosmid vector<br />
pCC2FOS (Epicentre) and cloned into E. coli. The fosmid DNA was extracted and run on 454-FLX<br />
sequencing, yielding 322,742 reads. These data were analyzed by SEED/MG-RAST platform, which<br />
showed a broad metabolic profile, containing 428 sequences related with nitrogen metabolism. Twentyseven<br />
percent of these were affiliated with the denitrification process and 23 proteins were assigned to<br />
27
the nitrous-oxide reductase family. The phylogenetic analysis showed that nine of these proteins were<br />
grouped in a different cluster from the known sequences deposited in GenBank. The results revealed that<br />
the sludge community has a great number of potential new genes responsible for nitrous-oxide removal in<br />
petroleum refinery WWTP. Financial Support: PETROBRAS, FAPESP<br />
03#08<br />
The Analysis of Microbial Community and Organism Distribution from Polymer Flooding Daqing<br />
Oil Reservoirs<br />
Jian jun Le 1 , X. Lin 1 , J. Z. Wu 2 , Z. W. Hou 1 , J. Y. Zhang 1 , M. H. Guo 1 1 Exploration and Development<br />
Research <strong>Institute</strong> of Daqing Oil Field Company Ltd. 2 Technical Development Department of<br />
Daqing Oil Field Company Ltd.<br />
Denaturing gradient gel electrophoresis (DGGE) and conventional microbiological viable detection<br />
methods were used to analyze the indigenous microbial community from Daqing oil reservoirs after<br />
polymer flooding, the results showed that the bacterial population in produced fluid from polymer flooding<br />
reservoir were higher than that in injected water, the bacterial population in the backflashed liquid from<br />
injection well were above 2 magnitude higher than that in produced liquid,the magnitude of the bacterial<br />
population in different polymer flooding blocks varied great. The bacterial phylotypes from production well<br />
belonged to β-Proteobacteria (Azoarcus, Thauera, Petrobacter), γ-proteobacteria (Pseudomonas,<br />
Acinetobacter, Escherichia), ε-proteobacteria (Acobacter, Chloroflexi), the bacterial community varied<br />
great, dominant bacterial colonies were different. Bacterial phylotypes from injection well were<br />
represented by β-proteobacteria, γ-proteobacteria, ε-proteobacteria, Bacilli, Clostridia, and Chloroflexi,<br />
the magnitude and composition of the bacterial population varied little. Archaeal phylotypes were closely<br />
related to Methanosaeta, Methanobacterium, Methanospirillum, Methanomicrobiaceae, Crenarchaeota,<br />
and some uncultured archaea, Methanosaeta and uncultured archaea were undetected in production<br />
well; whereas Methanobacterium, Methanomicrobium, Methanospirillum and uncultured archaea were<br />
distributed both in injected well and production well, and their number in production well increased<br />
significantly. The research showed that indigenous microbial communities are inhabited in Daqing oilfield<br />
which was exploited by water flooding and polymer flooding for many years, so the reservoir was suitable<br />
to adopt the microbial stratum activation and application of MEOR technology to enhance of oil recovery.<br />
03#09<br />
Metagenomic and Proteomic Analyses to Elucidate the Mechanism of Anaerobic Benzene<br />
Degradation<br />
Nidal Abu Laban, D. Selesi, R. Meckenstock <strong>Institute</strong> of Groundwater Ecology, Helmholtz Zentrum<br />
München, German Research Center for Environmental Health, Neuherberg, Germany<br />
Anaerobic benzene degradation was studied with an iron- and a sulfate-reducing culture (BF and BPL)<br />
composed of mainly Peptococcaceae-related Gram-positive microorganisms. To elucidate the initial<br />
activation mechanism of anaerobic benzene degradation, proteomes of benzene-, phenol- and benzoategrown<br />
cells of the iron-reducing culture BF were compared by SDS-PAGE. A specific benzene-expressed<br />
protein band of 60 kDa, which could not be observed during growth on phenol or benzoate, was<br />
subjected to N-terminal sequence analysis. The first 31 amino acids revealed that the protein was<br />
encoded by ORF 138 in the shotgun sequenced genome of culture BF. ORF 138 showed 43% sequence<br />
identity to phenylphosphate carboxylase subunit PpcA of Aromatoleum aromaticum strain EbN1. A<br />
LC/ESI-MS/MS-based shotgun proteomic analysis of the 60 kDa excised band revealed other specifically<br />
benzene-expressed proteins with encoding genes located adjacent to ORF138 on the genome. The<br />
protein products of ORF137, ORF 139, and ORF 140 showed sequence identities of 37% to<br />
phenylphosphate carboxylase PpcD of A. aromaticum strain EbN1, 56% to benzoate-CoA ligase (BamY)<br />
of Geobacter metallireducens, and 67% to 3-octaprenyl-4-hydroxybenzoate carboxy-lyase (UbiD/UbiX) of<br />
A. aromaticum strain EbN1, respectively. These genes are proposed as constituents of a putative<br />
benzene degradation gene cluster (~17 Kb) composed of carboxylase-related genes. The identified gene<br />
sequences suggest that the initial activation reaction in anaerobic benzene degradation is probably a<br />
28
direct carboxylation of benzene to benzoate catalyzed by putative anaerobic benzene carboxylase (Abc).<br />
The same proteins were identified in the sulfate-reducing culture BPL suggesting an identical activation<br />
reaction.<br />
03#10<br />
A Comparison of DNA- and RNA-based Clone Libraries from Formation Water of a Hightemperature<br />
Petroleum Reservoir<br />
Natalya M. Shestakova, E. M. Mikhailova, A. B. Poltaraus, S. S. Belyaev, T. N. Nazina<br />
Microbial community of the production water and of back-flashed water from the injection well of the hightemperature<br />
Dagang oil field (P. R. China) was analyzed by DNA- and RNA-derived clone libraries. Both<br />
16S RNA genes of Bacteria, Archaea and Planctomycetes representatives and alkB (alkane<br />
monoooxygenase) genes were obtained based on DNA from injection and production wells. In RNA<br />
delivered clone libraries, only bacterial 16S rRNA phylotypes were reveled. Phylogenetic diversity in the<br />
clone libraries from injection well was higher than those from the production well. In DNA library from<br />
injection well, archaeal phylotypes similar to 16S RNA genes of Methanomethylovorans (44% of studied<br />
archaeal clones), Methanosaeta, Methanoculleus, Methanocalculus, Methanolinea, Methanococcus and<br />
Thermococcus were found. In archaeal DNA library from production water, phylotypes of H2-utilizing<br />
methanogens of the genus Methanothermobacter (50% of clones) were predominant; Methanosaeta and<br />
Halopiger phylotypes represented 26 and 24% of studied clones respectively. In the RNA-derived clone<br />
libraries, phylotypes of sulfate-reducing (Desulfomicrobium, Desulfuromonas, Desulfobulbus), Fereducing<br />
(Geobacter, Deferribacteres), denitrifying (Azoarcus, Denitromonas and Tepidiphilus) and<br />
aerobic chemolithoheterotrophic bacteria (Tepidimonas, Geobacillus, Pseudomonas) were retrieved.<br />
Phylotypes of Geobacillus constituted 64% in DNA library from backflashed water of injection well and<br />
81% in RNA library of production water. AlkB-geo1, alkB-geo4 and alkB-geo6 genes similar to those of<br />
the Geobacillus genus were determined in the DNA libraries. Comparative analysis of DNA and RNA<br />
clone libraries constructed from formation waters of the petroleum reservoir allows characterizing both<br />
genomic diversity and minor phylotypes of microbial community, which remain undetected on the DNA<br />
level.<br />
03#11<br />
Evaluation of Pure Top-down Effect (Flagellates and Viruses Predation) on Bacterial Community<br />
Structure and Hydrocarbonoclastic Activities in a Biostimulation Mesocosm Experiment<br />
Caroline Sauret 1 , D. Bottjer 2 , A. Tallarmin 3 , C. Guigue 3 , M. Goutx 3 , J-F. Ghiglione 1 1 Laboratoire<br />
d'Océanologie Biologique de Banyuls 2 University of Hawaii, Department of Oceanography 3 Centre<br />
d'Océanologie de Marseille<br />
Petroleum oil spills, such as the recent events in the Gulf of Mexico, reminds the importance of the fight<br />
against accidental and chronic pollution in marine ecosystems. Biostimulation by nutrient addition has<br />
been found to be the best remediation strategy but influence of such practice on the microbial loop has<br />
been poorly studied. Diesel was added to nutrient unlimited mesocosms containing 400L seawater and<br />
the effect of heterotrophic nanoflagellates (HNF) predation and viral lysis (top-down control) on bacterial<br />
community structure and activities was investigated. Both alkane and PAH compounds were continually<br />
degraded during the course of our experiment (16 days), whereas two different phases occurred for the<br />
biological compartments. First, we found that diesel addition led to an important shift in both total (based<br />
on DNA) and metabolically active (based on RNA) bacterial communities resulting in an important<br />
increase in bacterial abundance after 8 days (26-fold increase. Quantitative-PCR showed that these<br />
communities presented higher abilities to degrade alkanes and polycyclic aromatic hydrocarbons (PAH).<br />
Second, peaks of HNF (657-fold increase) and viruses (40-fold increase) appeared after 12 days together<br />
with high HNF predation (dilution technique) and viral lysis (Electronic Transmission Microscopy)<br />
activities. Top-down control affected both total and active bacterial assemblages and resulted in a clear<br />
decrease of bacterial abundance. Selected bacterial populations presented high activity per cell (both<br />
bacterial production and exoenzyme activities) and conserved alkane and PAH degradation capabilities.<br />
29
Such integrative studies have important implication on understanding the impact of oil biostimulation<br />
strategies on the marine ecosystem functioning.<br />
03#12<br />
Detecting Mesophilic Thermotogales Bacteria ("Mesotogas") in Enrichment Cultures<br />
Camilla Nesbø, C. Li, S. Ebert, J. Foght University of Alberta<br />
To date, all characterized and cultivated Thermotogales bacteria have been either thermophiles or<br />
hyperthermophiles. However, previously we have shown, using metagenomic approaches, that there also<br />
are mesophilic members of this lineage inhabiting hydrocarbon-rich environments, and that these bacteria<br />
are likely to be indigenous to mesothermic oil fields. To extend these observations, we analyzed water<br />
samples collected from four North American continental oil reservoirs with in situ temperatures of 14°C -<br />
53°C, as well as sediments from two mesothermic hydrocarbon-impacted sites. Amplification using<br />
Thermotogales-specific PCR primers revealed that sequences designated 'mesotoga M1' were the<br />
dominant Thermotogales detected in samples with temperatures
03#14<br />
Expanding the Microbial Monitoring Toolkit: Evaluation of Traditional and Molecular Monitoring<br />
Methods<br />
Vic Keasler 1 , B. Bennett 1 , C. Keller 1 , P. Whalen 2 , J. Carins 2 1 Nalco 2 LuminUltra<br />
Evaluation of microbial populations in oilfield systems is critical to understand the risk of microbiologically<br />
influenced corrosion, reservoir and surface souring (hydrogen sulfide production), and biofouling.<br />
Although traditional culture based methods have dominated oilfield microbial monitoring for years, use of<br />
molecular tools is becoming more common for both field and laboratory evaluations. The implementation<br />
of these additional tools is in response to some of the disadvantages of culture-based methods such as<br />
long incubation times and underestimation of actual microbial populations. The current work provides a<br />
direct comparison of culture-based methods (serial dilution for sulfate reducing, acid producing, and<br />
general heterotrophic bacteria) with molecular methods including adenosine triphosphate (ATP) and<br />
adenosine monophosphate (AMP) quantification and quantitative polymerase chain reaction (qPCR). The<br />
results demonstrate that these technologies provide nearly identical results in untreated samples with<br />
known culturable organisms, but show some differences when used in the context of a planktonic kill<br />
study. The pros and cons of each technology will be addressed with respect to their use in field<br />
monitoring, laboratory monitoring, and microbial kill studies. The authors will convey that none of the<br />
technologies described in this work provide an all-inclusive answer, but together they provide significant<br />
insight into the microbial population in an oilfield system. In short, the authors will demonstrate that it is<br />
advantageous for oilfield stakeholders to expand their microbial monitoring toolkit with these new<br />
technologies to ensure sustainable, cost-effective operation.<br />
03#15<br />
Production Processes Affect Nitrifying Prokaryotic amoA Gene Abundance and Distribution in<br />
High-Temperature Petroleum Reservoirs of China<br />
Ji-Dong Gu 1 ,H. Li 2 , B-Z. Mu 2 1 University of Hong Kong 2 State Key Laboratory of Bioreactor<br />
Engineering and <strong>Institute</strong> of Applied Chemistry, State Environmental Protection Key Laboratory of<br />
Environmental Risk Assessment and Control on Chemical Process, East China University of<br />
Science and Technology<br />
Although the presence and activity of ammonia-oxidizing archaea (AOA) and bacteria (AOB) were<br />
observed in thermophilic habitats recently, the knowledge of them in the geothermal subterranean oil<br />
reservoirs is still not available. This study investigated the abundance and distribution of AOA and AOB in<br />
the production waters of high-temperature oil reservoirs by using real-time PCR and phylogenetic<br />
analysis based on amoA genes. The results showed the occurrence of both AOA and AOB in 9 out of a<br />
total of 17 samples. The AOA-like phylotypes were mainly clustered within two major clades of archaeal<br />
amoA sequences known from water columns, sediments and soils: clusters A and B, and few clones were<br />
related to the new genera: Candidatus 'Nitrosocaldus yellowstonii'. The AOB-like phylotypes mainly<br />
belonged to Nitrosospira and Nitrosomonas clusters, while two of them deeply branched in Nitrosospira<br />
cluster and showed no substantial alignment to the known cultured AOB, indicating the possibility of new<br />
AOB phylotypes. The abundance of AOA and AOB-like amoA gene ranged from 2.92×10 3 to 9.21×10 4<br />
and from 2.91×10 2 to 8.12×10 3 amoA gene copy numbers per ml production water with the ratios of AOA<br />
to AOB ranging from 5.10 to 95.5. Statistical analysis showed that amoA gene fell into five groups and the<br />
distribution of amoA was significantly correlated with the environmental factors, e.g., temperature and<br />
recovery process. Our study showed distribution of prokaryotic amoA gene in various oil reservoirs of<br />
China, was affected by production processes.<br />
31
03#16<br />
The Analysis of Microbial Communities and Organism Distribution of Indigenous Microbe after<br />
Polymer Flooding of Daqing Oil Reservoirs<br />
Meng Hua Guo 1 , X. L. Wu 1 , J. Z. Wu 2 , J. J. Le 1 , Z. W. Hou 1 , J. Y. Zhang 1 1 Exploration and<br />
Development Research <strong>Institute</strong> of Daqing Oil Field Company Ltd. 2 Technical Development<br />
Department of Daqing Oil Field Company Ltd.<br />
Denaturing gradient gel electrophoresis (DGGE) and conventional microbiological viable detection<br />
methods were used to analyze the growth of six types of Indigenous Microbe after polymer flooding of<br />
daqing oil reservoirs,the results showed that the bacteria population in produced fluid from polymer<br />
flooding reservoir were higher than that in injected water, the bacteria population in the backflown liquid<br />
from injection well were above 2 magnitude higher than that in produced liquid, the magnitude of the<br />
bacteria population in different polymer flooding blocks varied great. The bacteria in production well were<br />
β-proteobacteria of the Azoarcus, Thauera, Petrobacter, γ-proteobacteria of the Pseudomonas,<br />
Acinetobacter, Escherichia, ε-proteobacteria of the Acobacter and Chloroflexi, the bacterial community<br />
varied great, dominant bacterial colony were different; the bacteria in injection well were β-proteobacteria,<br />
γ-proteobacteria, ε-proteobacteria, Bacilli, Clostridia, and Chloroflexi, the species of bacteria and the<br />
magnitude of the bacteria population varied little; Archaebacteria were Methanosaeta, Methanobacterium,<br />
Methanospirillum, Methanomicrobiaceae, Crenarchaeota, and some uncultured archaebacteria,<br />
Methanosaeta and uncultured archaebacteria in the sample of production well were undetected,<br />
Methanobacterium, Methanomicrobium, Methanospirillum and uncultured archaebacteria were distributed<br />
in injected well and production well, and the number in production well increased significantly. The<br />
research showed that indigenous microbe had correlation and existing coordinated metabolism. Daqing<br />
oilfield was developed by water flooding and polymer flooding for many years, stable microbe group had<br />
formed, so the reservoirs were suitable to adopt the activating indigenous microbe recovery oil.<br />
03#17<br />
Activity of Methanosarcinales and Acetogens in Coal and Oil Reservoirs Proven by Stable Isotope<br />
Probing<br />
Martin Krüger 1 , S. Beckmann 2 , B. Engelen 2 , T. Lüders 3 , F. von Netzer 3 , H. Cypionka 2 , 1 BGR 2 ICBM<br />
3 GSF<br />
In many coal mines or oil reservoirs, methanogenic archaea are responsible for the production of<br />
substantial amounts of methane. The present study aimed to directly unravel the active methanogens<br />
being responsible for the methane release as well as the active bacteria involved in the metabolic network<br />
for hydrocarbon degradation. Therefore, the stable-isotope labeled precursors of methane, 13C-acetate<br />
and H2+ 13 CO2 as well as 13 C-hexadecane were fed to liquid cultures obtained from coal and oil reservoirs.<br />
Directed by the methane production, samples for DNA stable-isotope probing (SIP) coupled to<br />
subsequent quantitative PCR and DGGE analyses were taken over 6 months. The formation of 13 Cmethane<br />
was due to acetoclastic methanogenesis in the 13 C-acetate and H2+ 13 CO2 cultures of coal and<br />
timber. The H2+ 13 CO2 was mainly used by acetogens similar to Pelobacter acetylenicus and Clostridium<br />
species. Active Methanosarcinales, closely rel ated to Methanosarcina barkeri, rather utilize the easier<br />
accessible acetate than the more energetical efficient hydrogen. Surprisingly, we found a microbial<br />
community highly adapted to low H2 conditions in the reservoirs.<br />
32
03#18<br />
The Analysis of Microbial Communities and Organism Distribution of Indigenous Microbe in<br />
Polymer Flooding of Daqing Oil Reservoirs<br />
Xiao-lin Wu, J-Z. Wu, J-J. Le, Z. W. Hou, J-Y. Zhang, T. Ma<br />
Denaturing gradient gel electrophoresis (DGGE) and conventional microbiological viable detection<br />
methods were used to analyze the growth of sulfate-reducing bacteria, hydrocarbon degrading bacteria,<br />
saprophytic bacteria, fermentative bacteria, methanogens and denitrifying of six types of Indigenous<br />
Microbe in polymer flooding of Daqing oil reservoirs The results showed that the bacteria population in<br />
produced fluid from polymer flooding reservoir was higher than that in injected water, the bacteria<br />
population in the backflown liquid from injection well was above 2 magnitude higher than that in Produced<br />
Liquid, the magnitude of the bacteria population in different polymer flooding blocks varied greatly. The<br />
bacteria in production well were β-proteobacteria of the Azoarcus, Thauera, Petrobacter, γ-proteobacteria<br />
of the Pseudomonas, Acinetobacter, Escherichia, ε-proteobacteria of the Acobacter and Chloroflexi, the<br />
bacterial community varied greatly, dominant bacterial colonies were different; the bacteria in injection<br />
well were β-proteobacteria, γ-proteobacteria, ε-proteobacteria, Bacilli, Clostridia, and Chloroflexi, the<br />
species of bacteria and the magnitude of the bacteria population varied little; Archaebacteria were<br />
Methanosaeta, Methanobacterium, Methanospirillum, Methanomicrobiaceae, Crenarchaeota, and some<br />
uncultured archaebacteria, Methanosaeta and uncultured archaebacteria in the sample of production well<br />
were undetected, Methanobacterium, Methanomicrobium, Methanospirillum and uncultured<br />
archaebacteria were distributed in injected well and production well, and the number in production well<br />
increased significantly. The research showed that indigenous microbe had correlation and existing<br />
coordinated metabolism. As Daqing oilfield was developed by water flooding and polymer flooding for<br />
many years, stable microbe groups had formed so the reservoirs were suitable to adopt the activating<br />
indigenous microbe recovery oil.<br />
03#19<br />
Practical Molecular and Metagenomic Applications for the Identification and Quantification of<br />
Oilfield Microbes<br />
Elizabeth Summer, N. Summer, S. Campbell Ecolyse Inc<br />
Developing tools to monitor and control microbial problems in the oilfield has been hampered by a lack of<br />
knowledge about even the most common microbes in these systems. Three fundamental microbial<br />
population queries are information on the absolute abundance of microbes in the system, in terms of<br />
microbial cells per unit of sample (e.g. gram solid or ml liquid), as well as the identity and relative<br />
abundance of each component microbe. A metagenomics approach can be used to obtain robust data on<br />
the identity and relative abundance of component microbes. To be practical, the method must be suitable<br />
to analyze numerous samples from numerous locations at a reasonable cost. Recent advances in next<br />
generation, high throughput sequencing technologies has brought these methods into the realm of<br />
practicality, both in terms of ease of routine use and cost per sample analysis. Here, we describe the<br />
methodologies used to perform economical, low coverage metagenomic surveys of an oil brine and an<br />
onshore pipeline. Between 1800 and 3300 shotgun 16S rRNA pyrosequencing reads were obtained from<br />
each location. These were assigned to Genus level classification and relative abundance of all members<br />
estimated to be present at more then 1% of the starting population was analyzed. The species profiles<br />
from the onshore pipeline identified members closely related to bacteria associated with metal corrosion.<br />
The ability to accurately define the microbial consortia associated with petroleum infrastructure will enable<br />
rational choices for microbial control.<br />
33
03#20<br />
Expanded reference trees for investigating C1 transfer reactions in subsurface hydrocarbon<br />
reservoirs using MLTreeMap<br />
Young Song, K. Konwar, A. Pagé, C. Howes, S. Hallam University of British Columbia<br />
Microbial mediated anaerobic hydrocarbon degradation within subsurface reservoirs transforms light oils<br />
into more viscous and acidic forms with higher sulfur and metal content. Over geological time this<br />
conversion process leads to the formation of heavy oil or oil sands deposits, altering the composition of<br />
subsurface sediments on continental scales. Similar conversion processes have been implicated in the<br />
production of natural gas within coal bed seams and marine sediments. To better constrain and compare<br />
C1 transfer reactions mediating methane production and consumption in subsurface hydrocarbon<br />
reservoirs we developed a bioinformatics workflow integrating reference trees for<br />
tetrahydromethanopterin (H4MPT)-linked C1 transfer reactions in MLTreeMap [1]. MLTreeMap is a userextensible<br />
software framework that automates maximum likelihood analysis to recover phylogenetic or<br />
functional gene markers from environmental data sets. The resulting output files include alignments,<br />
composite trees and distribution tables mapping identified marker genes to annotated reference trees<br />
useful in assigning reads to taxonomic groups or assessing the metabolic potential of environmental<br />
samples. Application of the expanded workflow to pyrosequenced metagenomes derived from coal-bed<br />
methane deposits, marine sediments and a natural asphalt lake revealed overlapping but not identical<br />
H4MPT-linked C1 transfer profiles associated with methanogenic or reverse methanogenic phenotypes.<br />
Multivariate analysis of C1 transfer profiles identified lineage specific clusters associated with energetic<br />
substrate utilization and environmental variables including temperature, salinity and pH. This study<br />
provides a rapid and accurate method for profiling H4MPT-linked C1 transfer reactions harbored in<br />
community genomes with direct application to oil and gas exploration and monitoring efforts. Reference:<br />
1.Stark M, Berger SA, Stamatakis A and C von Mering. 2010. MLTreeMap - accurate maximum likelihood<br />
placement of environmental DNA sequences into taxonomic and functional reference phylogenies. BMC<br />
Genomics. 11:461.<br />
03#21<br />
Characterization of Subsurface Microbial Communities Within Powder River Basin Coals, United<br />
States Associated With Coal-Bed Methane<br />
Elliott Barnhart 1 , J. Wheaton 2 , A. Cunningham 1 , M. Fields 1 1 Montana State University 2 Montana<br />
Bureau of Mines and Geology<br />
A better understanding of the ecology and physiology of the methane-producing communities associated<br />
with coal-beds may promote new techniques that improve coal-bed methane (CBM) production and<br />
enhance the sustainability of the wells. We have conducted initial phylogenetic diversity studies using<br />
inoculated coal from methane producing wells in the Powder River Basin (PRB) (southeastern Montana<br />
and northeastern Wyoming). "Bug traps" loaded with coal were lowered into four wells in the PRB along a<br />
hydrogeochemical gradient and microbial communities were allowed to develop in situ for three months.<br />
Incubated material from the bug traps was used to inoculate enrichment cultures for the assessment of<br />
methane production. Nucleic acids were extracted from in situ material, well water, and enrichment<br />
cultures. Clone libraries of SSU rDNA gene sequences (both bacterial and archaeal) were used to<br />
compare community composition and structure of enrichment cultures, and 454 pyrosequencing libraries<br />
were used to characterize community composition and structure of in situ material (coal and water).<br />
Multivariate statistical methods were used to relate community composition and structure to<br />
hydrogeochemical parameters. A low sulfate well had low bacterial diversity compared to a well with<br />
intermediate sulfate levels while the archaeal diversity was higher in a low sulfate well compared to wells<br />
with higher sulfate levels. In enrichment cultures with coal, bacterial diversity was much higher than<br />
enrichments without coal. The described study aims to identify the relationships between populations of<br />
Bacteria and Archaea associated with CBM with the intent of identifying strategies for enhancement of in<br />
situ CBM production.<br />
34
03#22<br />
Molecular Characterization of Archaeal Community Structure in Water-flooding Petroleum<br />
Reservoirs with Different Temperature<br />
Li-Ying Wang 1 , S. M. Mbadinga 1 , J-D Gu 2 , B-Z. Mu 1 1 State Key Laboratory of Bioreactor<br />
Engineering and <strong>Institute</strong> of Applied Chemistry, East China University of Science and Technology<br />
2 School of Biological Sciences, The University of Hong Kong<br />
Petroleum reservoirs support the growth of diverse microbial assemblages distributed among the<br />
domains Bacteria and Archaea. Analysis of archaeal community from production water of different<br />
petroleum reservoirs at temperatures from 20.6ºC to 60ºC by 16S rRNA gene clone libraries indicates the<br />
presence of physiologically diverse and temperature-dependent archaea in these subterrestrial<br />
ecosystems. With a few exceptions (Thermocuccus, Thermoplasmatales, Halogeometricum and<br />
Thermoprotei), most of the retrieved sequences were assigned to the methanogens including<br />
methyltrophic (Methanomethylovorans), acetoclastic (Methanosaeta) and CO2-reducing methanogens<br />
(Methanothermobacter, Methanoculleus, Methanobacteria, Methanocalculus Methanocella and<br />
Methanolinea). The CO2-reducing methanogens were the most encountered in the petroleum reservoirs<br />
fluids and they may be adapted to the environmental conditions of these petroleum reservoirs and<br />
became the dominant inhabitants in subsurface environments. Our investigation is in line with others,<br />
indicating that CO2-reducing methanogens predominate in subsurface petroleum reservoirs where they<br />
may play considerable roles in anaerobic communities involved in the biotransformation of oil<br />
components to methane.<br />
35
Session 04 - Souring & Biocorrosion<br />
Invited speakers:<br />
04#01<br />
Reservoir Souring: It Is All About Risk Mitigation<br />
Cor Kuijvenhoven 1 , B. van der Linden 2 1 Shell Global Solutions (US) Inc 2 Shell International<br />
Exploration & Production<br />
There is a consensus within the oil industry that reservoir souring is invariably attributable to the activity of<br />
sulphate reducing bacteria (SRB). In anaerobic environments these bacteria metabolise small organic<br />
molecules like organic acids that can usually be found in reasonable quantities within formation waters.<br />
To support their metabolic cycle sulphate is required and, in the process, this is reduced to hydrogen<br />
sulphide. Once the risk to sour the reservoir is being identified, it is required to identify the right mitigation<br />
strategy. Historically only biocides have been applied in the field but nowadays alternatives like nitrate<br />
injection and even sulphate removal are being applied. The most suitable mitigation method is dependent<br />
on the type of field, acceptable risk and several other factors. This paper will go into more detail about the<br />
applied approach in several Shell operated fields.<br />
Offered Papers:<br />
04#02<br />
Microbial Load and Community Changes during Model Studies of Nitrate-based Produced Water<br />
Souring Mitigation<br />
Frøydis K. Garshol 1 , C. U. Schwermer 1 , M. S. Madsen 2 , E. A. Vik 1 , NK. Birkeland 2 1 Aquateam<br />
Norwegiam Water Technology Centre AS & Department of Biology, University of Bergen<br />
2 Department of Biology, University of Bergen<br />
Produced water re-injection (PWRI) is essential for future explorations of oil and gas in ecologically<br />
sensitive areas. However, PWRI is not the preferred water management solution due to several<br />
challenges, including risks of reservoir souring (RS) and microbial influenced corrosion (MIC). A key to<br />
fully understanding and successfully solving the problems of RS and MIC is through characterisation of<br />
microbial community changes when different RS mitigation methods are applied, and to determine how<br />
these changes influence operational procedures for biofouling control. Our knowledge of the mechanisms<br />
causing RS and MIC is limited. Through the characterisation of microbial communities and microbial<br />
activities it might be possible to enhance this knowledge further. A PWRI pilot study performed in the field<br />
indicates that nitrate efficiently mitigates RS, but accelerates corrosion in the top-side system. MIC was<br />
proposed to be caused by nitrate-reducing bacteria (NRB), forming partially oxidized sulphur or nitrogen<br />
compounds. RS mitigation and possible MIC was investigated in a laboratory-based anaerobic flowthrough<br />
system operating under thermophilic conditions mimicking the field conditions. In addition to<br />
monitoring corrosion and water quality, the microbe-metal interaction on the metal coupon surfaces was<br />
investigated by biofilm micro-profiling and the changes in microbial diversity were analysed by<br />
pyrosequencing of 16S rRNA gene amplicons and DGGE in biofilm samples. As observed in the field,<br />
nitrate mitigation of RS elevated corrosion. In addition to commonly found oil field organisms such as<br />
Firmicutes and Thermotogae, diversity analyses identified NRB from Deferribacteres and<br />
Epsilonproteobacteria (Sulfurospirillium). Possible MIC mechanisms will be discussed.<br />
36
04#03<br />
Influence of Extracellular Polymeric Substances on Biocorrosion<br />
Agata Wikiel, M. Vera, W. Sand University Duisburg - Essen<br />
Systems for handling stabilized oil, e.g. equipment for top side oil transport and processing plants, in<br />
Northern Europe mostly made of carbon steel, are subject to high levels of microbially influenced<br />
corrosion (MIC). This results in extensive and costly damages and additional production loss. MIC is<br />
caused by formation of biofilms - microbial communities, kept together by a matrix of extracellular<br />
polymeric substances (EPS). By immobilization, the EPS keeps cells closely together and allows genetic<br />
interactions and joint nutrient and energy source accumulation and usage. Within the "EU - Marie Curie<br />
BIOCOR Initial Training Network" performed at University Duisburg - Essen studies on the influence of<br />
EPS on biocorrosion and their role in biofilm formation in water and oil pipeline systems are done. For an<br />
improved understanding of specific problems in the offshore oil and gas industry microbial strains from<br />
seawater systems were isolated and characterized by molecular techniques such as 16s rRNA analysis<br />
(DNA isolation, PCR amplification, cloning and sequencing). Marine sulphate and nitrate reducing<br />
bacterial isolates are tested for validation of their influence on carbon steel corrosion. This includes<br />
monitoring of biofilm formation by advanced microscopic techniques (e.g. EFM, CLSM), corrosion tests<br />
and analytical assays. EPS from isolated marine bacteria are characterized by determining their<br />
constituents like sugars, proteins, DNA, uronic acids and lipids. Connecting these data with results from<br />
corrosion tests using the marine isolates will allow developing improved mitigation strategies against<br />
biocorrosion.<br />
Posters:<br />
04#04<br />
Microbial Monitoring during Hydraulic Fracturing using Molecular and Cultivation-dependent<br />
Methods<br />
Bei Yin 1 , M. Enzien 1 , D. Love 1 , M. Harless 2 , E. Corrin 2 1 Dow Chemical Company 2 Multi-Chem<br />
Production Chemicals<br />
The highly diverse metabolisms and surviving mechanisms of microorganisms allow their broad presence<br />
in a wide range of environments on earth, including oil and natural gas reservoirs. During hydraulic<br />
fracturing in unconventional natural gas operation, water from a variety of sources and fracturing related<br />
chemicals are injected into the gas reservoir which can introduce bacteria into formation, stimulate the<br />
growth of domestic microbes, and change the microbial community structure. Biocides are usually used to<br />
control microbial contamination caused by hydraulic fracturing practices, and bacteria, especially sulfate<br />
reducing bacteria and acid producing bacteria, are often monitored to evaluate the effectiveness of the<br />
biocide treatment. In this paper, we describe the microbial monitoring during a hydraulic fracturing trial<br />
which utilized a new glutaraldehyde combination product. Both cultivation-dependent and independent<br />
molecular testing methods are used and compared with the biocide effectiveness evaluation and<br />
microbial diversity exploration.<br />
04#05 #054<br />
Nitrate injection to control souring in oil bioreactors modeling a low-temperature heavy-oil<br />
reservoir in Alberta.<br />
Cameron Callbeck, Akhil Agrawal and Gerrit Voordouw. Petroleum Microbiology Research Group<br />
Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada<br />
Various nitrate injection strategies have been employed in a low-temperature field near Medicine Hat,<br />
Alberta to remediate souring, these include continuous injection of low nitrate (2 mM), pulsed injection of<br />
low nitrate (2 mM) and batch squeezes of high nitrate (>30 mM). We look to investigate the effectiveness<br />
37
of souring control from these various nitrate injection strategies in oil containing bioreactors modeling this<br />
reservoir. Five oil bioreactors have been established under the continuous injection of 2 mM sulfate, B-S-<br />
O is injected with 2 mM sulfate only (no nitrate), bioreactors B-S-LN-O and B-S-HN-O are injected with<br />
continuous low (4 mM) or high (100mM) concentrations of nitrate, respectively. While bioreactors B-S-<br />
(LN)-O and B-S-(HN)-O are pulsed with low (4 mM) or high (100 mM) nitrate, respectively.<br />
Continuous injection of low nitrate did show initial success at lowering sulfide but was followed by<br />
recovery once nitrate and later nitrite were fully reduced. Continuous or pulsed injection of high nitrate<br />
allowed nitrate and nitrite breakthrough thus achieving complete inhibition of sulfate-reduction throughout.<br />
Bioreactors B-S-O, B-S-LN-O and B-S-HN-O, were dissected into fractions along the flow-path for<br />
analysis of microbial diversity as well as for oil composition. Denaturing gradient gel electrophoresis<br />
identified species such as Desulfuromonas, Clostridum and Methanosaeta in B-S-O, B-S-LN-O contained<br />
primarily Acetobacterium sp., while B-S-HN-O contained mostly Thauera aromatica among others. Oil<br />
composition indicates that sulfate-reducing and nitrate-reducing bacteria are competing for different<br />
electron donors since bioreactor B-S-O observed degradation of n-alkanes C19-29 and O-xylene while<br />
bioreactors B-S-LN-O and B-S-HN-O used primarily N-propylbenzene, P/M-xylene, ethylbenzene and<br />
toluene.<br />
04#06<br />
Sulfate Reducing Bacteria Lower Corrosion in Sour Systems with Oxygen Ingress<br />
Shawna Johnston, H. S. Park, G. Voordouw University of Calgary<br />
Sulfate reducing bacteria (SRB), such as Desulfovibrio vulgaris strain Hildenborough (DvH) reduce sulfate<br />
to sulfide, causing reservoir souring and pipeline corrosion. SRB can be controlled with nitrate, which is<br />
reduced to nitrite by resident nitrate-reducing bacteria and is a strong inhibitor of SRB. The sulfide<br />
produced by SRB can also react chemically with nitrite or oxygen to produce precipitated sulfur (S) or<br />
dissolved polysulfide (PS). For example, S forms rapidly when sour produced waters, containing<br />
substantial sulfide concentrations, are exposed to air: 2H2S + O2 --> 2S + 2H20. S and PS (S-PS)<br />
formation is undesirable as they are strongly corrosive agents. DvH is able to prevent the formation of S-<br />
PS through the action of nitrite reductase, as well as oxygen reductases, which reduce O2 and nitrite to<br />
H2O and ammonium, preventing their reaction with sulfide to form S-PS. DvH can also reduce S-PS to<br />
sulfide. These activities lower S-PS formation when sour waters are exposed to O2 and decrease<br />
corrosion risk. This was shown by monitoring corrosion by weight loss in experiments where 2.7 mM O2<br />
was added to DvH cultures grown to mid-log phase, where approximately 10 mM sulfide had formed. A<br />
corrosion rate of 0.064 mm/yr was observed under these conditions. However, if the culture was<br />
autoclaved prior to O2 addition, the corrosion rate was threefold higher at 0.220 mm/yr. These results<br />
indicate that while SRB known for harmful sulfide production, their oxygen reduction activity is beneficial<br />
in some situations by preventing chemical formation of corrosive sulfur products.<br />
04#07<br />
H2S Trend after Acidizing: Souring Case Study in X Field Southern Oman<br />
Ardian Nengkoda Schlumberger<br />
The ideal well stimulation activity is to increase production by improving the flow of hydrocarbons from the<br />
drainage area into the well bore. However, in one field in Southern Oman, there was a trend of increasing<br />
H2S. One common preliminary check is to review the stimulation fluid characteristics i.e. whether carrying<br />
any H2S and thus giving an impact to the wells. Some lessons we learned from the Oman study showed<br />
that the water supply sources always contained H2S. The level of H2S after acidizing showed an alarming<br />
increase over time, casting doubts on the future integrity across the processing facility. As the materials<br />
used in the X and down stream receiving facilities are not NACE 175 souring compliant and H2S release<br />
is a hazard for safety (HSE), this reservoir study initiated an integrated H2S Monitoring and implemented<br />
a Mitigation Program by improving H2S onsite measurement and method revision and established proper<br />
38
well surveillance. As a result, the cause of souring was due to SRB Bacteria and the acidizing caused by<br />
flash of Iron Sulfide as a by-product from bacterial activities. The lab screening of biocide and the<br />
application during acidizing have been conducted to see the possibility and secure acidizing job. In this<br />
kind of situation, it would be preferable to go with batch biocide injection before the acid job, then<br />
subsequent treatments to control the bacterial growth in future. H2S scavenging as part of the acidization<br />
also has been reviewed. Overall this study has improved business performance; HSE awareness and<br />
saving the plant upgrade cost.<br />
04#08<br />
New Method and Apparatus to Improve Nitrate Supply and Economics<br />
Mike Dennis 1 , D. Hitzman 2 1 LATA Group, Inc. 2 Geo-Microbial Techologies, Inc.<br />
Nitrate based treatments used in the oil and gas industry to mitigate and control biogenic H2S souring of<br />
topside and reservoir production systems have increased significantly during the last decade. These<br />
chemically "green" solutions continue to be of interest to both the scientific community and global<br />
production operators. An expanding empirical data base has demonstrated successful field operations<br />
and H2S control using nitrate injection. Also, nitrate based treatments have been employed as a nutrient<br />
source for microbial enhanced oil recovery (MEOR) in a growing number of field trials dating from the<br />
1990's. World-wide nitrate supply, storage, and economics are well established through conventional<br />
nitrate supply resources. However, many remote onshore fields and offshore operations have not been<br />
selected for nitrate treatments due to non-competitive logistical expenses. A new portable and on-site<br />
nitrate generator has been tested and demonstrated in the field. On-site production of nitrate will<br />
significantly increase logistical efficiencies and will expose remote onshore and offshore operations to<br />
enhanced H2S control (and MEOR) solutions.<br />
04#09<br />
Biosurfactants Production from Pseudomonas Aeruginosa 55F Isolated in Oil Rich Niger Delta of<br />
Nigeria<br />
Akhil Agrawal, G. Voordouw Department of Biological Sciences, University of Calgary<br />
Use of water flooding for oil recovery often leads to production of hydrogen sulfide (souring). Sulfate<br />
reducing bacteria (SRB) reduce sulfate present in formation/make-up water to sulfide, while oxidizing oil<br />
organics??? Something missing? One of the most common practices to control reservoir souring is<br />
injection of nitrate to stimulate nitrate reducing bacteria (NRB). However, this strategy is problematic in<br />
the western Canadian field due to the following reasons: (i) Low downhole temperature in these fields<br />
allowed the growth of SRB in deeper zones of the reservoir past the nitrate depletion zone. (ii) Utilization<br />
of a wide range of oil components (toluene, xylene and C6-C18 alkane components) as electron donor by<br />
SRB and of a more limited range of components (toluene and xylene) by NRB and the presence of<br />
excess oil caused both electron acceptors to become completely reduced. Hence, the sulfide<br />
concentration observed in produced water was not lowered as a result of nitrate injection under these<br />
conditions. As an alternate strategy we incubated produced water with 5 mM sulfide and 5 mM nitrate. It<br />
was observed that all the sulfide was effectively oxidized within 1 day of incubation, even when excess oil<br />
organics were present. Thus rather than treating the reservoir, nitrate injection can target souring in<br />
above ground installations<br />
39
04#10<br />
Influence of metallic iron on sulfate-reducing bacteria isolated from water-flooded oil field of<br />
Absheron Peninsula<br />
Alexander Galushko Vienna University<br />
Sulfate-reducing bacteria (SRB) have long been known as very powerful agents in corrosion of iron under<br />
anoxic conditions. Influence of corroding iron on metabolism of SRB is less investigated. Several<br />
metabolically versatile strains of sulfate-reducing bacteria were isolated from of water-flooded oil fields of<br />
Absheron Peninsula. The strains were able to grow by degradation of various organic acids. Experiments<br />
were performed to understand the influence of iron on metabolism of isolated strains. Growth and sulfide<br />
production by SRB were strongly changed in the presence of metallic iron and products of its corrosion.<br />
04#11<br />
Sulfate-reducing bacteria isolated from an oilfield in India are most closely related to strains of<br />
Desulfovibrio from geographically remote oil fields<br />
Jyoti Rao 1 , D. Ranade 2 , Y. Shouche 3 , A. Bharadwaj 4 , G. Rastogi 5 1 Modern College of Arts, Science<br />
& Commerce, India 2 Agharkar Research <strong>Institute</strong>, India 3 National Centre for Cell Science, India<br />
4 <strong>Institute</strong> of Petroleum Engineering IEOT, India 5 University of California, Davis, USA<br />
The growth of dissimilatory sulfate-reducing bacteria (SRB) in offshore oil fields results in economically<br />
and ecologically undesirable consequences. Measures to inhibit their growth in oil fields can be facilitated<br />
by the knowledge of their biochemistry and physiology. Therefore, studies on SRB present in produced<br />
water collected from an offshore oil pipeline off the west coast of India were carried out. Two strains of<br />
SRB isolated were characterized and designated as strains NUS 2 and SUS 3. These strains were<br />
halotolerant and moderately halophilic, respectively. Both were mesophilic and showed the ability to<br />
autotrophically use hydrogen as a source of energy. Neither showed the ability to metabolize alkanes<br />
from tetradecane to nonadecane. Phylogenetic analysis based on their 16S rRNA gene sequences<br />
showed that they belonged to the delta subdivision of Proteobacteria and were distantly related to each<br />
other. Based on their phenotypic characteristics and their phylogenetic affiliation, they were designated as<br />
strains of Desulfovibrio alaskensis and "Desulfovibrio capillatus", respectively. Interestingly, type strains of<br />
these species were isolated from oilfields in Alaska and Mexico, respectively. Furthermore, our strains<br />
were phylogenetically closest to isolates and clones from globally distributed, geographically remote oil<br />
fields, forming distinct clusters. This habitat-specific clustering suggests that these 16S rDNA sequences<br />
represent species whose growth may be especially supported in oil fields, irrespective of their<br />
geographical location. This stresses the need for detailed studies focused on such species, which would<br />
prove invaluable in designing efficient strategies for their detection and mitigation of oil souring and<br />
microbial corrosion.<br />
04#12<br />
Investigation of Effect of Various Nitrate Treatment Strategies to Control Souring in a Fixed Film<br />
Bioreactor System<br />
Carol Devine, H. Hoffmann Intertek Commercial Microbiology<br />
The application of nitrate treatments in seawater injection systems are frequently used to prevent and<br />
mitigate reservoir souring. However, optimising a strategy for nitrate applications for use in a PWRI<br />
system is more difficult. The relatively high concentration of water-soluble organics in produced waters<br />
can mean that, in theory, very high doses of nitrate are required to mitigate souring. Whereas previous<br />
studies have indicated that zonation occurs in low temperature areas (i.e. in the near injection well-bore<br />
area), little is published regarding the effect at higher temperatures The aim of this study was to generate<br />
data in order to determine the optimal nitrate treatment regime for this field (600C, low salinity). Analyses<br />
were also carried out to determine the effect of the various strategies on the diversity and activity of the<br />
40
microbial populations. Fixed film upflow bioreactors were used to investigate the change in the<br />
thermophilic microbial communities with various nitrate and biocide applications and the effect on the<br />
number and activity of sulphate-reducing bacteria and nitrate-reducing bacteria within those populations.<br />
The analyses carried out on the effluent from the bioreactors included bacterial enumerations (triplicate<br />
MPNs), sulphide, nitrate and nitrite. Molecular techniques (FISH and DGGE) were also utilised to give<br />
further information. The effect of continuous and batch treatments of nitrate, with and without biocide, on<br />
the generation of sulphide, will be discussed. Bioreactors receiving both continuous nitrate and batch<br />
biocide treatments were found to produce high levels of biofilm resulting in frequent blockages in the<br />
bioreactors.<br />
04#13<br />
Severe anaerobic microbial corrosion of iron under biogenic crusts<br />
Dennis Enning 1 , Hendrik Venzlaff 2 , Julia Garrelfs 1 , Achim W. Hassel 3 , Martin Stratmann 2 ,<br />
Friedrich Widdel 2 1 Max Planck <strong>Institute</strong> for Marine Microbiology 2 Max Planck <strong>Institute</strong> for Iron<br />
Research GmbH 3 Johannes Kepler University Linz<br />
Anaerobic corrosion of iron and steel causes significant damage in a number of industrial sectors (e.g. in<br />
petroleum and gas production). The process is significantly influenced by microorganisms, with sulfate<br />
reducing bacteria (SRB) being the suspected main culprits. However, the underlying electrochemical<br />
mechanisms and the relative contribution of physiologically different microorganismic groups in anaerobic<br />
biocorrosion have not been fully elucidated so far. We used the marine SRB isolates ‘Desulfopila<br />
corrodens’ strain IS4 and ‘Desulfovibrio ferrophilus’ strain IS5 as a model system in cultivation<br />
experiments to gain insights into the mode of the uptake of reducing equivalents if metallic iron (Fe 0 ) is<br />
provided as the sole source of electrons. Both strains had been originally isolated directly from corroding<br />
iron. Severe corrosion experimentally sustainable over several months at almost constant rate was<br />
observed only in the cultures of these two strains, but not in reference cultures of ‘conventional’<br />
hydrogenotrophic Desulfovibrio, Desulfobacterium and Desulfopila species. The corrosive strains<br />
deposited significant amounts of hard inorganic sulfidic crusts on the iron specimens. Scanning electron<br />
microscopy revealed a patchy distribution of the cells on the biogenic crusts that contained FeS and<br />
FeCO3 at relatively constant ratio (c. 1:3) as well as co-precipitated minerals such as CaCO3. It is likely<br />
that the surface-attached cells obtain electrons via the crust from the metal which necessarily corrodes.<br />
Biomineralization (corrosion) of iron by such and physiologically similar lithotrophic SRB may explain<br />
several cases of microbial corrosion observed in the field.<br />
04#14<br />
Anaerobic microbial corrosion of iron with limiting sulphate<br />
Julia Garrelfs 1 , Dennis Enning 1 , Hang T. Dinh 2 , Karl Mayrhofer 3 , Friedrich Widdel 1 1 Max Planck<br />
<strong>Institute</strong> for Marine Microbiology 2 University of Hanoi, Vietnam 3 Max Planck <strong>Institute</strong> for Iron<br />
Research GmbH<br />
Microbially influenced corrosion (MIC) plays a significant role in the damage of iron and steel structures<br />
exposed to seawater. MIC may even pose a threat to the environment by contributing to leaks in<br />
petroleum pipelines. In particular sulfate-reducing bacteria (SRB) are suspected to play the key role in<br />
this process. However, there is recent evidence also for an involvement of methanogenic archaea in MIC<br />
of iron. Still, their relative contribution to MIC in biofilms on iron and steel structures is currently unknown.<br />
Activity and growth of methanogens is generally expected in anoxic environments with limiting<br />
concentrations of sulfate. In 2004, Dinh et al. (Nature 427, pp 829) enriched and isolated a new<br />
physiological type of a methanogenic archaeon able to corrode iron at higher rate than could be explained<br />
by mere use of H2 as an abiotically formed intermediate (from Fe 0 and H2O). This methanogen may thus<br />
take up electrons from the iron surface in a more direct manner. Here we investigated the activity of this<br />
and newly enriched methanogens in the presence of iron-corroding SRB and different concentrations of<br />
sulfate. Fe 0 -dependent methanogenesis started only after significant decrease of the sulfate<br />
41
concentration. However, refined growth experiments revealed a more complex interaction than simply a<br />
competition governed by a threshold concentration of sulfate.<br />
04#15<br />
Detection, Identification and Quantification of Thermophilic Sulfate-Reducing Microorganisms in<br />
Cold Seawater and Marine Sediments<br />
Julia De Rezende 1 , Kasper Kjeldsen 1 , Casey Hubert 2 , Bo B. Joergensen 1 1 Center for<br />
Geomicrobiology, Aarhus University 2 Newcastle University<br />
Sulfate-reducing microorganisms (SRM) represent a concern for the offshore oil industry due to their<br />
impact on safety, oil quality, and production costs. Hydrogen sulfide production in oil fields (reservoir<br />
souring) is common following seawater injection for pressure maintenance during oil production, and is<br />
caused by introduction of high levels of sulfate and possibly also waterborne SRM. We quantified<br />
endospores of thermophilic SRM in cold sediments of Aarhus Bay using an improved most probable<br />
number (MPN) method that detected 10 0 to 10 4 endospores of thermophilic SRM per gram of sediment<br />
down to 650 cm depth. RNA- and DNA-based 16S rRNA and dsrAB gene analyses indicated >10<br />
phylotypes of Desulfotomaculum spp. were induced at 28 to 70°C, a temperature range reflecting in situ<br />
conditions in several offshore reservoirs, e.g., in the North Sea. Sediment incubation and phylogenetic<br />
analyses indicated that thermophilic Desulfotomaculum spp. likely originate from warm subsurface<br />
environments, and have been accumulating as endospores in Aarhus Bay sediments for thousands of<br />
years. A flux of SRM spores from the water column into the sediment is supported by our detection of<br />
similar SRM throughout the water column using this surveillance approach. We have identified the most<br />
abundant phylotypes in surface and deeper (650 cm) sediments that develop during incubation at 50°C,<br />
including low-abundant yet fast-growing SRM. Hence thermophilic SRM, even in low numbers in<br />
seawater, can rapidly proliferate given the right physicochemical conditions. A combination of cultivation<br />
and molecular methods can thus improve knowledge of the physiology, diversity and abundance of SRM.<br />
42
Session 05 (Biodegradation, Biofuels & Downstream Microbiology)<br />
Invited speakers:<br />
05#01<br />
Microbial ecology of methanogenic crude oil biodegradation; from microbial consortia to heavy oil<br />
Ian M. Head 1 , Carolyn M. Aitken 1 , Michael J. Maguire 1 , Angela Sherry 1 , Russell Grant 1 , Neil D.<br />
Gray 1 , Casey Hubert 1 , Cameron Callbeck, D. Martin Jones 1 , Barry Bennett 2 , Thomas B.P.<br />
Oldenburg 2 & Stephen R. Larter 2 1 School of Civil Engineering and Geosciences, Newcastle<br />
University 2 Petroleum Reservoir Group, Dept of Geology and Geophysics and Alberta Ingenuity<br />
Center for In situ Energy, University of Calgary<br />
Methanogenic crude oil biodegradation is a globally significant process and has been implicated in the<br />
development of the vast heavy oil and tar sand deposits that dominate the world’s petroleum inventory.<br />
The occurrence of gradients in the composition of oil in biodegraded petroleum reservoirs suggests that<br />
this process is driven by microbial consortia that are maximally active at the oil water transition zone<br />
(OWTZ). This model is supported by the observation that bacterial populations are elevated in the OWTZ<br />
coincident with the depletion of crude oil hydrocarbons. In many methanogenic crude oil degrading<br />
systems members of the Syntrophaceae appear to be key players in the initial anaerobic degradation of<br />
crude oil alkanes and their growth correlated with methane production from crude oil. Intriguingly, in crude<br />
oil degrading systems under sulphate-reducing conditions alkylsuccinates accumulated transiently while<br />
in methanogenic systems dominated by members of the Syntrophaceae, they did not. Does this indicate<br />
alkane activation may not proceed by fumarate addition under methanogenic conditions? This may not be<br />
the case since metagenome analysis revealed the presence of alkylsuccinate synthetase gene (assA)<br />
homologues at low frequency, an observation supported by PCR analysis using assA specific primers.<br />
Despite the importance of methanogenic crude oil biodegradation it is clear that our understanding of<br />
crude oil alkane degradation in the absence of exogenous electron acceptors is far from complete. Our<br />
current understanding of in reservoir and methanogenic crude oil biodegradation will be discussed.<br />
05#02<br />
Microbial Contamination Control in Fuels and Fuel Systems since 1970 - a Review<br />
Frederick Passman Biodeterioration Control Associates, Inc.<br />
Although the documentation of fuel biodeterioration dates back to the late 19th century, general<br />
recognition of the value of microbial contamination control evolved slowly until the 1980's. Since the early<br />
1980's a number of factors have converged to stimulate greater interest in fuel and fuel system<br />
biodeterioration. This, in turn, has stimulated applied research in the ecology of biodeteriogenic<br />
processes and biodeterioration control. This presentation reviews progress in both of these areas since<br />
1990. The aforementioned factors that have provided the impetus for improved microbial control, the<br />
evolution of our understanding of the nature of the biodeteriogenic processes will be discussed. Activities<br />
of consensus organizations to develop guidelines and practices will also be reviewed.<br />
Offered Papers:<br />
05#03<br />
Microbial Conversion of Higher Hydrocarbons to Methane in Oil and Coal Reservoirs<br />
Martin Krüger 1 , M. Siegert 1 , S. Feisthauer 2 , H-H. Richnow 2 1 BGR 2 UFZ<br />
Since almost 20 years it is known from stable isotope studies that large amounts of biogenic methane are<br />
formed in oil reservoirs. The investigation of this degradation process and of the underlying<br />
biogeochemical controls is of great economical and social importance: (1) The understanding of reservoir<br />
43
iodegradation is of great use for the exploration industry, (2) a biotechnological stimulation of the<br />
methane formation in reservoirs could provide new economical perspectives. Even under optimal<br />
conditions, today not more than 30-40 % of the total oil in a reservoir is actually recovered. The<br />
conversion of at least parts of this non-recoverable oil via an appropriate biotechnological treatment into<br />
easily recoverable methane would provide an extensive and ecologically sound energy resource.<br />
Laboratory mesocosm as well as high pressure autoclave experiments with samples from different<br />
geosystems showed high methane production rates after the addition of oils, single hydrocarbons or<br />
coals. The fingerprinting of the microbial enrichments with DGGE showed a large bacterial diversity while<br />
that of Archaea was limited to three to four dominant species. The variability of carbon and hydrogen<br />
isotopes of produced methane falls in a relative narrow range. Our stable isotope data from both, the<br />
incubations with samples from various ecosystems and from field studies, implies a common<br />
methanogenic biodegradation mechanism, resulting in consistent patterns of hydrocarbon alteration.<br />
05#04<br />
Microbial Generation of Methane in Alberta Coal Seams<br />
Karen Budwill Alberta Innovates - Technology Futures<br />
Geochemical and isotopic evidence suggest that biogenic methane production occurred and is likely still<br />
occurring, albeit at very slow rates, in many coal beds in Alberta. The microbes involved in this biogenic<br />
methane production could potentially be stimulated to increase methane yields in older or low gas content<br />
coalbed methane (CBM) wells and could be used to augment traditional CBM operations. 16S DNA<br />
pyrosequencing as well as traditional enrichment culturing of freshly-drilled coal samples have revealed<br />
that methanogenic communities can be found from different Alberta coal formations at different depths,<br />
pressures and temperatures. Biogenic methane production in the enrichment cultures could be enhanced<br />
through the addition of certain nutrients. Methane production rates at reservoir pressures and<br />
temperatures were investigated using both batch and flow-through type vessels in the laboratory. These<br />
investigations of the microbial ecology and associated methanogenesis processes in coal seams<br />
contribute to our understanding of how to increase biogenic methane production to economic levels.<br />
05#05<br />
Influence of Carbon Dioxide Storage on the Methanogenic Pathway in a High-Temperature<br />
Petroleum Reservoir<br />
Daisuke Mayumi 1 , S. Sakata 1 , H. Maeda 2 , Y. Miyagawa 2 , M. Ikarashi 2 1 National <strong>Institute</strong> of<br />
Advanced Industrial Science and Technology (AIST) 2 INPEX Corporation<br />
Deep subsurface petroleum reservoirs have been receiving attention as sites suitable for CO2 storage.<br />
Little is known, however, about the influence of CO2 storage on microbes inhabiting the reservoirs. We<br />
investigated the influence of CO2 storage on methanogenesis in a high-temperature petroleum reservoir<br />
by stable isotope (SI) tracer experiments and molecular biological analyses. The SI tracer experiments<br />
were conducted using microcosms comprised of the production water, crude oil and traces of [2- 13 C]acetate<br />
or 13 C-bicarbonate. The microcosms were pressurized with either N2 or N2+CO2 (90:10) at 5 MPa<br />
and then incubated at 55°C, simulating the conditions of in situ reservoir or CO2 storage. In all the<br />
microcosms, methane production was observed with the decrease of acetate included in the production<br />
water. Of those pressurized with N2, an immediate and distinct increase in the δ 13 C of methane was<br />
observed in the bicarbonat e-labeled microcosm, while a gradual increase in the δ 13 C of dissolved<br />
inorganic carbon was observed in the acetate-labeled microcosm, indicating that the major methanogenic<br />
pathway was acetate oxidation coupled with hydrogenotrophic methanogenesis. Of those pressurized<br />
with N2+CO2, an immediate and distinct increase in the δ 13 C of methane was observed in the acetatelabeled<br />
microcosm, indicating that the major methanogenic pathway was acetoclastic methanogenesis.<br />
Microbial community analyses showed that syntrophic acetate-oxidizing bacteria and hydrogenotrophic<br />
methanogens dominated in the microcosms pressurized with N2 while acetoclastic methanogens<br />
44
dominated in those pressurized with N2+CO2. These results clearly indicated that CO2 storage into a hightemperature<br />
petroleum reservoir would cause a drastic change in the methanogenic pathways.<br />
05#06<br />
Metagenomic and Proteomic Analyses to Elucidate the Mechanism of Anaerobic Benzene<br />
Degradation<br />
Nidal Abu Laban, D. Selesi, R. Meckenstock <strong>Institute</strong> of Groundwater Ecology, Helmholtz Zentrum<br />
München, German Research Center for Environmental Health, Neuherberg, Germany<br />
Anaerobic benzene degradation was studied with an iron- and a sulfate-reducing culture (BF and BPL)<br />
composed of mainly Peptococcaceae-related Gram-positive microorganisms. To elucidate the initial<br />
activation mechanism of anaerobic benzene degradation, proteomes of benzene-, phenol- and benzoategrown<br />
cells of the iron-reducing culture BF were compared by SDS-PAGE. A specific benzene-expressed<br />
protein band of 60 kDa, which could not be observed during growth on phenol or benzoate, was<br />
subjected to N-terminal sequence analysis. The first 31 amino acids revealed that the protein was<br />
encoded by ORF 138 in the shotgun sequenced genome of culture BF. ORF 138 showed 43% sequence<br />
identity to phenylphosphate carboxylase subunit PpcA of Aromatoleum aromaticum strain EbN1. A<br />
LC/ESI-MS/MS-based shotgun proteomic analysis of the 60 kDa excised band revealed other specifically<br />
benzene-expressed proteins with encoding genes located adjacent to ORF138 on the genome. The<br />
protein products of ORF137, ORF 139, and ORF 140 showed sequence identities of 37% to<br />
phenylphosphate carboxylase PpcD of A. aromaticum strain EbN1, 56% to benzoate-CoA ligase (BamY)<br />
of Geobacter metallireducens, and 67% to 3-octaprenyl-4-hydroxybenzoate carboxy-lyase (UbiD/UbiX) of<br />
A. aromaticum strain EbN1, respectively. These genes are proposed as constituents of a putative<br />
benzene degradation gene cluster (~17 Kb) composed of carboxylase-related genes. The identified gene<br />
sequences suggest that the initial activation reaction in anaerobic benzene degradation is probably a<br />
direct carboxylation of benzene to benzoate catalyzed by putative anaerobic benzene carboxylase (Abc).<br />
The same proteins were identified in the sulfate-reducing culture BPL suggesting an identical activation<br />
reaction.<br />
05#07<br />
Identification of Alkylsuccinate Synthase Genes and Characterization of Microbial Community<br />
Structure in Petroleum Reservoirs Fluids of China<br />
Lei Zhou 1 , C-X. Gao 1 , S. M. Mbadinga 1 ,L-Y. Wang 1 ,J-D. Gu 2 ,B-Z. Mu 11 State Key Laboratory of<br />
Bioreactor Engineering and <strong>Institute</strong> of Applied Chemistry, East China University of Science and<br />
Technology 2 School of Biological Sciences, The University of Hong Kong<br />
Anaerobic degradation of oil alkanes is now well documented. Physiological different activation strategies<br />
have been proposed, with glycyl-radical dependent fumarate addition playing a dominant role. The genes<br />
encoding the candidate alkylsuccinate synthase have been identified in most axenic cultures capable of<br />
metabolism of oil alkanes under anoxic conditions. It is now broadly accepted that the depletion of oil<br />
reserves is associated with the activities of anaerobic microorganisms thriving in these environments. In<br />
the present work, molecular phylogenetic methods were used to investigate the microbial diversity in<br />
production waters obtained from a wide range of different petroleum reservoirs in China. 16S rRNA gene<br />
libraries were generated from total community DNA, using universal archaeal or bacterial oligonucleotide<br />
primer sets. Moreover, an assay targeting a 523 bp fragment within the Ass alpha-subunit (assA) was<br />
used to detect the presence of putative alkylsuccinate synthase genes in the oil reservoirs production<br />
fluids. Our results indicate that beside the large presence of diverse physiological types of<br />
microorganisms including fermentative, nitrate-reducers, sulfidogenic, methanogens and others noncultured<br />
representatives, a high diversity of previously unidentified assA genes as well as deeply<br />
branching assA homologues were observed. We provide evidence for a previously unrecognized diversity<br />
of anaerobic oil alkane degraders using fumarate addition as the main biochemical activation strategy.<br />
These findings will be helpful in the understanding of anaerobic biodegradation processes occurring in<br />
petroleum reservoirs.<br />
45
Posters:<br />
05#08<br />
Characterization of Heavy Crude Oil Biodegradation Using GC-MS and Fourier Transform Ion<br />
Cyclotron Resonance Mass Spectrometry (FT-ICR MS) Techniques<br />
Yuehui She 1 , F. Zhang 2 , Q. Shi 3 , F. Shu 1 , Z. Wang 3 , D. Hou 4 1 Yangtze University 2 China University<br />
of Geosciences (Beijing) 3 China University of Petroleum 4 China University of Geosciences<br />
(Beijing)<br />
The mechanism of microbial enhanced oil recovery (MEOR) techniques is gaining increased attention. In<br />
this study, heavy crude oil biodegraded fractions saturates (S), aromatics (A), resins (R) and asphaltene<br />
(A) from Xinjiang (China) were analysed by GC-MS as well as electrospray ionization (ESI) coupled with<br />
high-field Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) techniques, the<br />
mechanism of transformation of heteroatoms containing nitrogen and oxygen were investigated in the<br />
range of molecule. The results showed that after biodegradation by indigenous microbial communities,<br />
the SARA in crude oil appeared changes. Relative abundances of saturated hydrocarbons and<br />
asphaltene increased, and relative abundances of aromatic hydrocarbons and resins decreased. After<br />
biodegradation by indigenous microbial communities, heteroatoms in heavy crude oil changed. Generally,<br />
relative abundances of N-species with higher molecular weight decreased, while those with lower<br />
molecular weights increased. In addition, the distribution of double-bond equivalent (DBE) and carbon<br />
number showed few changes, which indicated that side chains of N-species were metabolized<br />
preferentially, and cyclic core structure containing nitrogen were more reluctant to microbial metabolism.<br />
Molecular weight of crude oil decreased from 403 Da to 382 Da. Relative abundance of heteroatoms of<br />
O-species increased, which provide more data to support the conclusion that oxidation of hydrocarbons<br />
produced carboxylic acids. Similarity to N-species, side chains of O-species were metabolized<br />
preferentially. In addition, relative abundance of O2-species with carbon number of 16 showed an obvious<br />
increase. Key words: heavy crude oil, biodegradation, Fourier transform ion cyclotron resonance mass<br />
spectrometry (FT-ICR MS), viscosity reducing.<br />
05#09<br />
Biogenic Potential of Shale Gas and Processes that Stimulate Methanogenesis in Shales<br />
Marya Cokar, M. Kallos, I. Gates University of Calgary<br />
The Western Canadian sedimentary basin contains 550-860 Tcf of gas within shale deposits. Natural gas<br />
systems are usually stratified into three levels. The deepest level is the thermogenic kitchen where gas is<br />
produced by thermogenesis, the second level directly above this contains thermogenic gas that has been<br />
displaced, and some biogenic gas, the shallowest level (about 600 m) contains the microbe nursery, and<br />
is a favorable environment for microbes to generate methane gas. Biogenic gases originate in immature<br />
regimes from bacterially mediated anaerobic mineralization of organic matter in sediments. Biogenic gas<br />
is a product of shallow subsurface metabolism by microorganisms. The produced gas is dominantly<br />
methane, but it may contain up to 2% ethane, propane, butane and pentane. This study explores the<br />
biogenic potential of shale gas systems through a modified gas material balance theory. In addition to<br />
this, different processes that enhance methane production by microbes in-situ are also being<br />
investigated. These processes involve stimulating the methane producing bacteria in-situ by adding<br />
different nutrients to the reservoir. The analysis conducted in this research can be used to optimize<br />
productivity from these fields. This study helps reveal the key physical controls on gas production from<br />
organic shales and means to enhance it. This study is novel and significant because it further develops<br />
the underlying theory for biogenic gas transport and production from organic shales, a potentially massive<br />
gas source in Western Canada and worldwide<br />
46
05#10<br />
Metabolites and Abiotic Species in a Methanogenic Toluene Degrading Enrichment Culture<br />
S. Jane Fowler, L. Gieg University of Calgary<br />
Under methanogenic conditions, toluene degradation can occur via fumarate addition to form<br />
benzylsuccinate catalysed by benzylsuccinate synthase. Other toluene activation reactions such as ring<br />
or methyl hydroxylation by water have been proposed based on observation of cresols and benzyl alcohol<br />
in culture fluids, however the enzymes catalyzing these reactions have not been identified and the<br />
chemical feasibility of these reactions has been questioned. A methanogenic toluene degrading<br />
enrichment culture that has been maintained for many transfers was studied to investigate mechanisms<br />
of toluene activation. Metabolite analysis was performed by extraction of cultures and analysis by GC-MS.<br />
Metabolites initially detected included benzylsuccinate, o-, m-, and p-cresol, methylhydroquinone,<br />
hydroquinone, cyclohexane carboxylate and benzoic acid. The latter two products are known to be<br />
downstream metabolites of anaerobic toluene metabo lism. The presence of benzylsuccinate in the<br />
culture fluids suggested that fumarate addition was occurring. PCR amplification of benzylsuccinate<br />
synthase genes from this culture further confirmed this. To investigate the presence of alternate toluene<br />
18<br />
activation reactions such as hydroxylation, the enrichment culture was incubated with toluene and H2 O.<br />
The same hydroxylated metabolites were observed but were not 18 O-labelled. Additionally, hydroxylated<br />
metabolites were observed in abiotic controls where oxygen was added. These results suggest that<br />
hydroxylated species can be formed abiotically from toluene by exposure to trace oxygen under<br />
methanogenic conditions and are likely not direct products of toluene metabolism.<br />
05#11<br />
Investigation of the Relationship between Microbial Communities and Origin of CH4 in High CO2<br />
Content Oilfield for Post-CCS<br />
Ryo Mori 1 , Y. Sugai 1 , K. Sasaki 1 , K. Fujiwara 2 1 Department of Earth Resources Engineering, Faculty<br />
of Engineering, Kyushu University 2 Chugai Technos Corporation<br />
We are focusing on in-situ microbial conversion of CO2 that was injected into depleted oil reservoirs for<br />
CCS into CH4 by the interaction between indigenous eubacteria that produce hydrogen from residual oil<br />
and indigenous hydrogenotrophic methanogens. This process has not only the potential of reducing CO2<br />
emission but also the potential of producing CH4 in reservoirs at the same time. However, those bacteria<br />
are concerned to be affected under high CO2 condition, which causes pH reduction of the brine for<br />
example. In this study, we estimated the possibility of this process by investigating the habitations of<br />
those bacteria and origin of CH4 in a high CO2 content reservoir. Brine and gas samples were extracted<br />
from a high CO2 content reservoir in Jilin oilfield, China. Contents of CO2 and CH4 in the extracted gas<br />
were 2-60% and 20-70% respectively. Nucleotide sequences of microbial genomic DNA that were<br />
extracted from the brine were analyzed to identify the species inhabiting the reservoir. Thermotoga sp.<br />
and Thermoanaerobacter sp. that are well known as oil-degrading and hydrogen-producing bacteria were<br />
detected as a dominant in the brine. Moreover, Methanobacterium sp. and Methanothermobacter sp. that<br />
are well known as hydrogenotrophic methanogens were also detected from all the brine samples.<br />
Measurements of both 2 H/ 1 H and 13 C/ 12 C isotope ratios have revealed that a part of CH4 extracted from<br />
the reservoir originated from CO2 reduction via microbial decomposition. From these results, the<br />
possibility of microbial conversion of CO2 into CH4 under high CO2 content condition was shown.<br />
05#12<br />
Effect of Chlorination of Make-up Water on Microbial Growth in an Oil Field<br />
Yetunde Folarin, G. Voordouw Petroleum Microbiology Research Group, Department of Biological<br />
Sciences, University of Calgary, Calgary, Alberta, Canada<br />
The pressure in an oil field decreases as a result of oil production, requiring the injection of water to repressurize<br />
the reservoir. Water breakthrough causes a mixture of oil and produced water (PW) to be<br />
47
produced. In oil fields with produced water reinjection (PWRI) oil and PW are separated. The PW is mixed<br />
with make-up water (MW) in a water plant after which it is re-injected. A Southern Alberta oil field uses the<br />
effluent from a sewage treatment plant as MW. This MW is subjected to quarterly treatment with acrolein,<br />
as well as to continuous chlorination, filtration and addition of ammonium bisulfite to reduce bacterial<br />
numbers. We aimed to determine whether this treatment increases, decreases or is indifferent to bacterial<br />
counts that develop in mixtures of MW and PW further downstream. Samples of MW before and after<br />
treatment (MW-UN and MW-TR, respectively) were mixed with a PW sample under anaerobic and<br />
aerobic conditions. Nutrients (ammonium and phosphate) were added in some experiments. The<br />
microbial population was then monitored as a function of time by plating (CFU/ml) and most probable<br />
number (MPN) assays in two media for heterotrophic bacteria. Similar increases in bacterial numbers<br />
were observed when MW-UN or MW-TR were mixed with PW. Hence, treatment of MW did not influence<br />
subsequent development of bacterial numbers in mixtures of MW and PW and effects of the treatment on<br />
water quality of the injection system past the water plant are unlikely.<br />
05#13<br />
An Analysis of Anaerobic Biodegradation of Next Generation Biofuels and Corrosion<br />
Deniz Aktas 1 , J. Lee 2 , B. Little 2 , J, Suflita 1 1 University of Oklahoma 2 Naval Research Laboratory<br />
First generation biofuels are susceptible to microbial attack relative to conventional petroleum-based<br />
fuels. Next generation biofuels are often formulated with hydrogenated bio-based lipids that are<br />
chemically indistinguishable from conventional hydrocarbons. We studied the susceptibility of petrobased,<br />
first, and next generation biofuels to anaerobic biodegradation and assessed the corrosion of<br />
carbon steel. Seawater was incubated with petro- and bio-based F76 and JP5, ultralow sulfur diesel<br />
(ULSD) and soy-biodiesel under strictly anaerobic conditions and compared to less stringent anaerobic<br />
incubations. After 120d in strictly anaerobic incubations, no significant microbial metabolism of the fuels<br />
was evident except in the biodiesel incubations. However, hydrocarbon loss and biocorrosion were noted<br />
in all bio-based fuels and petro-based JP5 incubations at the end of 90d when less stringent anaerobic<br />
techniques were employed. Substituted catechols, other phenols, benzoic acid, and various alcohols and<br />
carboxylic acids were observed in non-sterile incubations. First generation biodiesels stimulated the onset<br />
of corrosion as measured by polarization resistance. These trends were not observed with next<br />
generation biofuels. In all cases, corrosion was directly related to the concentration of surface-bound<br />
sulfur (presumed sulfides). There were no significant chemical changes in petro-based F76 fuel and<br />
ULSD. We submit that the presence of oxygen stimulated the initial microbial attack of most fuel<br />
components which became amenable to anaerobic biodegradation. This ultimately exacerbated carbon<br />
steel corrosion. Since the transient exposure of fuels to oxygen is likely to be the norm in actual use<br />
scenarios, the stability of modern fuel formulations should be assessed in this context.<br />
05#14 #053<br />
Isoprenoid Biomarkers and Microbial Transformation of Deep Hydrocarbon Fluid Flows<br />
Mikhail Chudetsky Oil and Gas Research <strong>Institute</strong>, Russian Academy of Sciences<br />
A hydrocarbon-water fluid moving from higher pressure and temperature conditions to lower ones is<br />
chemically nonequilibrium with decomposing and rebuilding molecules. These features of oils cannot<br />
persist for a long time and they disappear under the effects of either abiogenic processes or<br />
microorganisms. The energy contained in such chemically nonequilibrium fluids is sufficient for anaerobic<br />
feeding of microorganisms. Isolated media having neither income nor outcome of matter are justly<br />
considered to be lifeless. The evident regularities in the chemical composition variation of oils with<br />
change of the depth have been revealed. The deepest seated oils and gas condensates contain only<br />
negligible quantities of isoprenoid biomarkers taken from the particulate organic matter. The original set of<br />
their pseudobiomarkers is similar to those discovered in the carbonaceous meteorites. The main<br />
components of this kind of oils are n-alkanes and the optical activity of such oils has not been revealed.<br />
Up-seated oils with a significant amount (up to 1% and more) of the chain isoprenoids of the phytane,<br />
pristine and their homolog types are generated during the deposit processes with the participation of<br />
48
archaea consuming geofluids as a substratum. In this case the temperature in which they exist may be as<br />
much as 100°C and even more. As a result, chain isoprenoids of the membranes of those<br />
microorganisms find themselves in the oils. It is essential that the formation of the above-mentioned<br />
biomarkers is simultaneous with the formation of oil deposits but not in their further secondary<br />
transformation.<br />
05#15<br />
Microbial Conversion of Polycyclic Aromatic Hydrocarbons to Methane<br />
Carolina Berdugo-Clavijo, L. Gieg University of Calgary<br />
Current crude oil recovery technologies can only recover up to about 50% of oil from existing deposits.<br />
Thus, new strategies are needed to extract the remaining fossil fuel resources. Recent studies have<br />
confirmed the ability of microorganisms to degrade crude oil under methanogenic conditions. Polycyclic<br />
aromatic hydrocarbons (PAH) are hazardous compounds that comprise crude oil. The study of PAH<br />
degradation in the absence of electron acceptors is important to better understand how contaminated<br />
sites may be naturally remediated and how PAH may be converted to methane. The generated methane<br />
could feasibly be recovered as an alternate energy source, or serve to help repressurize oil reservoirs for<br />
improved energy recovery. Methanogenic microbial enrichments were prepared with 2methylnaphthalene<br />
(2-MN) or 2,6-dimethylnaphthalene (di-MN) in the presence of Amberlite-XAD7, a<br />
solid adsorber resin. Microbial community members were identified by 454 pyrosequencing. In time<br />
course experiments, methane production was monitored by gas chromatography and putative metabolites<br />
were analyzed by gas chromatography-mass spectrometry. Microbial enrichments able to metabolize di-<br />
MN and 2-MN produced up to 400 μmol of methane after 15 weeks of incubation. Sequencing analysis<br />
revealed that the cultures were dominated by Clostridium, Methanosaeta and Methanoculleus species.<br />
The putative metabolites methylnaphthoic acid and 2-naphthoic acid were recovered from the di-MN and<br />
2-MN cultures, respectively. Our results show the ability of microorganisms to biodegrade PAH under<br />
methanogenic conditions. The ongoing study of these methanogenic enrichments will contribute to the<br />
elucidation of anaerobic metabolic pathways that are currently poorly understood.<br />
05#16 #070<br />
Novel Anaerobic Ammonium-oxidizing (Anammox) Bacteria Detected in High Temperature<br />
Petroleum Reservoirs of China<br />
Ji-Dong Gu 1 , H. Li 2 , S. Chen 2 , B-Z. Mu 2 1 University of Hong Kong 2 State Key Laboratory of<br />
Bioreactor Engineering and <strong>Institute</strong> of Applied Chemistry, State Environmental Protection Key<br />
Laboratory of Environmental Risk Assessment and Control on Chemical Process, East China<br />
University of Science and Technology<br />
Anaerobic ammonium-oxidizing (Anammox) process plays an important role in the nitrogen cycle of the<br />
worldwide anoxic and mesophilic habitats. Recently, the existence and activity of anammox bacteria have<br />
been detected in some thermophilic environments, but their existence in the geothermal subterranean oil<br />
reservoirs is still not reported. This study investigated the abundance, distribution and functional diversity<br />
of anammox bacteria in 9 out of 17 high temperature oil reservoirs of China by molecular ecology and<br />
functional gene analysis. High concentration (5.31-39.2 mg L-1) of ammonium was detected in the<br />
production water from these oilfields with temperature between 55 and 75°C. Both 16S rRNA and hzo<br />
molecular biomarkers indicated the occurrence of anammox bacteria in 9 out of 17 samples. Most of 16S<br />
rRNA gene phylotypes are closely related to the known anammox bacterial genera Candidatus Brocadia,<br />
Candidatus Kuenenia, Candidatus Scalindua and Candidatus Jettenia. While hzo gene phylotypes are<br />
closely related to the genera Candidatus Anammoxoglobus, Candidatus Kuenenia, Candidatus Scalindua<br />
and Candidatus Jettenia. The total bacterial and anammox bacterial densities are 6.4 ± 0.5×10 3 -2.0 ±<br />
0.18×10 6 cells ml -1 and 6.6 ± 0.51×10 2 - 4.9 ± 0.36×10 4 cell ml -1 , respectively. The cluster I of 16S rRNA<br />
gene sequences showed distant identity (
sinooilfield' has been proposed. The results extended the existence of anammox bacteria to the high<br />
temperature oil reservoirs.<br />
05#17<br />
Microbial Methane Formation from a Methanogenic, Alkane-degrading Enrichment Culture<br />
Established from Production Water of High Temperature Petroleum Reservoir<br />
Serge Maurice Mbadinga 1 , C-X. Gao 1 , L-Y. Wang 1 , L. Zhou 1 , J-D. Gu 2 ,B-Z. Mu 1 1 State Key<br />
Laboratory of Bioreactor Engineering and <strong>Institute</strong> of Applied Chemistry, East China University of<br />
Science and Technology 2 School of Biological Sciences, The University of Hong Kong<br />
Petroleum reserves, after being depleted to certain levels, are considered for anaerobic degradation to<br />
recover energy. Moreover, methane commonly associated with biodegraded oil reservoirs is believed to<br />
be a result of microbial decomposition of oil alkanes. The microbial community in a methanogenic<br />
enrichment culture derived from production water of high temperature petroleum reservoir and growing in<br />
the presence of long chain alkanes was characterized by means of 16S rRNA genes. Molecular<br />
phylogenetic analysis revealed that, after a long incubation period with concomitant production of<br />
methane, the bacterial community was represented by members of the Firmicutes, Nitrosipra,<br />
Thermotogae, Candidate division OP8 and many others non-cultured representatives. Most of these<br />
bacterial taxa are known for fermentative or syntrophic life style. The archaeal community was<br />
represented by CO2-reducing methanogens members of the genera Met hanothermobacter,<br />
Methanoculleus, Methanolinea and sulfidogenic organisms related to Archaeoglobus as well as the<br />
fermentative Thermococcus. Interestingly, we identified for the first time an organism closely related to the<br />
acetogen Moorella thermoacetica. Our results indicate that acetate methanogenesis in the oil reservoir<br />
likely proceed via syntrophic acetate oxidation linked to hydrogenotrophic methanogenesis. This extends<br />
the knowledge about the syntrophic association among microorganisms in high temperature petroleum<br />
reservoirs. Moreover, new and deeply-branching catabolic genes encoding alkylsuccinate synthase were<br />
also detected in our enrichment. The presented results shed new light on the considerable diversity of<br />
anaerobic alkane's degraders, which is of great relevance for the understanding of biodegradation<br />
processes in petroleum reservoir.<br />
05#18<br />
Microbial Community Shift Correlated With the Carbon Available Enriched from an Oily Sludge<br />
Over 1000 Days of Methanogenic Incubation<br />
L-Y. Wang 1 , C-X. Gao 1 ,S. M. Mbadinga 1 , L. Zhou 1 , J-D. Gu 2 ,B-Z. Mu 1 1 State Key Laboratory of<br />
Bioreactor Engineering and <strong>Institute</strong> of Applied Chemistry, East China University of Science and<br />
Technology 2 School of Biological Sciences, The University of Hong Kong<br />
Methanogenic microbial community responsible for oil degradation in environments is dictated by<br />
hydrocarbons available. Such information is essential for the application of methanogenic hydrocarbon<br />
degradation in remediation of crude oil contamination and also in enhancing the recovery of energy from<br />
non-producing oil reserves. In this study, detectable alkanes decay and methanogenesis in the second<br />
enrichment transfer culture with n-alkanes (C15-C20) addition suggest that an active methanogenic alkanedegrading<br />
microbial community originally from an oily sludge has been successfully established over<br />
1000 days of incubation at 37�C. Total genomic DNAs were respectively extracted from the cultures of<br />
three different stages including uncultured (original sample) oily sludge, 500 days of methanogenic<br />
incubation without any additional carbon sources and the enrichment transfer cultures amended with nalkanes<br />
and incubated for 500 days. Phylogenetic diversity of microbial community structures at different<br />
stages were assayed by means of 16S rRNA gene sequences. At the same time, catabolic genes<br />
encoding benzylsuccinate synthase (bssA) were identified only in the primary enrichment whereas genes<br />
encoding alkylsuccinate synthase (assA) could only be detected in the transfer cultures amended with nalkanes.<br />
These results provided evidence that carbon source available had a strong influence on<br />
microbial community composition at different stages; resulting in a shift from methanogenic aromatic<br />
hydrocarbon degrading consortium to a methanogenic alkane-degrading community. These findings<br />
50
indicated that methanogenic hydrocarbon degradation is a relevant process in the remediation of<br />
hydrocarbon contamination or in enhancing the recovery of energy assets.<br />
05#19<br />
Biosurfactants Production from Pseudomonas Aeruginosa 55F Isolated in Oil Rich Niger Delta of<br />
Nigeria<br />
Patience Olajide 1 , S. Ajisebutu 1 , L. B. Ogbeifun 2 1 Obafemi Awolowo University, Nigeria<br />
2 Department of Human Resources, NNPC Towers, Nigerian National Petroleum Corporation<br />
There is global concern about the liberation of hydrocarbons in the environment, both from industrial<br />
activities and from accidental spills of oil and oil-related compounds. This incited the interest to screen for<br />
bacterial strains suitable for hydrocarbon mineralization and biosurfactants production, and to study its<br />
metabolism in order to obtain these biomolecules on a commercial scale, to be safely used in pollution<br />
control. A gram-negative, rod-shaped bacterium designated 55F, identified as Pseudomonas aeruginosa<br />
was isolated in oil rich Niger Delta of Nigeria and screened for biodegradation potentials. Grown on<br />
hydrocarbon, such as diesel, petrol, kerosene, hexane, heptane and benzene, the bacterium produced<br />
surface-active compounds. The type of enzymes produced by the selected isolate was determined using<br />
standard API ZYM kit they were produced in spent media. Measurements of surface tension, critical<br />
micelle dilution and e mulsifying activity indicated that the biosurfactants is produced as a primary<br />
metabolite. The 55F culture surface-active component is mainly rhamnolipid in nature. The rhamnolipid<br />
product dropped surface tension to values below 29 mN/m and was stable during exposure to high<br />
salinity, elevated temperatures (120°C for 15 min) and a wide range of pH values. It was observed that<br />
lipase C14 was an induced enzyme on hydrocarbon as detected with the API ZYM kit. The rhamnolipid<br />
biosurfactants produced by the investigated bacterium isolate enhanced petroleum hydrocarbons<br />
degradation.<br />
05#20<br />
Microbial Contamination of Fuels: Molecular Identification of Novel Contaminants<br />
Joan Kelley, M. Ryan, A. Buddie CABI & Conidia Bioscience Ltd.<br />
Microbial contamination of fuel is well documented. Known contaminants include fungi, commonly<br />
Hormoconis resinae, yeasts including Yarrowia lipolytica or bacteria such as Pseudomonas aeruginosa.<br />
Colonisation of fuel takes place through an 'ecological succession' with a primary hydrocarbon utiliser<br />
breaking down the fuel by oxidation which yields intermediary compounds that can be utilised by other<br />
microorganisms. In this study, microbes were isolated from contaminated fuels including aviation<br />
kerosene, marine diesel and biodiesels. The microorganisms obtained were then identified by sequence<br />
analysis of parts of the ribosomal RNA gene cluster using either the internal transcribed spacer region<br />
(ITS) or partial 26/28S subunit (D1/D2) (for yeasts and filamentous fungi) or partial 16S rDNA (for<br />
bacteria). Microbes were then re-inoculated into fuel to establish whether they were primary or secondary<br />
colonisers. Secondary colonisers will not grow in pure fuel culture as they are unable to directly utilise the<br />
hydrocarbons. Organisms identified included some that had not been previously reported as<br />
contaminants from fuel. These included a novel yeast species subsequently named Candida keroseneae<br />
and the bacterium Burkholderia gladioli which were found to be primary colonisers. Other primary<br />
colonisers included the bacteria Pseudomonas aeruginosa, Methylobacter sp., Arthrobacter sp., and a<br />
particularly vigorous strain of Yarrowia lipolytica. We will discuss the significance of the changing biota of<br />
fuels plus the effects of biofuels on microbial communities and colonisation. Finally, we will consider how<br />
novel testing methods have been developed to facilitate rapid identification of the bacterial, yeast and<br />
fungal contaminants of fuel systems.<br />
51
05#21<br />
Biodegradation of Crude Oil and Core Simulation Flooding Test for MEOR in Daqing Oilfield<br />
Xiang Tingsheng 1 , C. Chunmiao 2 , S. Yuanfen 2 , L. Ruina 2 , T. Wen 1 1 Department of Geochemistry,<br />
Yangtze University, Key Laboratory of Exploration Technologies for Oil and Gas Resources of<br />
Ministry of Education of China 2 The 1st Oil Production Factory, Daqing Oilfield<br />
The high-efficient hydrocarbon degrading bacteria (strain I) has been mixed by these single strains<br />
screened from the oilfield water in Daqing and it has been used to degrade different oil<br />
samples(A(polymer flooding), B(alkali/surfactants /polymer flooding,ASP) ,C(water flooding) from different<br />
flooding test wells respectively in Daqing oilfield .Crude oils have been analyzed by GC-MS and infrared<br />
spectrum (IR) before and after degradation for studying the influence of biodegradation on the<br />
components and molecular structures of crude oil. The results showed that crude oil C was more resistant<br />
to biodegradation than A and B. The main biodegradation sequence of saturated hydrocarbons was nalkane<br />
> acyclic-isoprene > regular sterane > rearrangement sterane > hopane;pregaane ><br />
homopregnane;sesquiterpene > low molecular weight triple-terpene, tetracylic terpene > high molecular<br />
weight triple-ter- pene、tetracylic-terpene > hopane. Whether 25-norhopanoid was produced during<br />
biodegradation may influence the judgement of hopanes sequence. High molecular weight triple-terpenes<br />
and rearrangement steranes should be concerned after serious biodegradation. According to the result of<br />
IR, the molecular structure of resin and asphaltene made changes after biodegradation. During<br />
biodegradation the variation of resin was more obvious than asphaltenes, and both of them produced new<br />
substances, mainly oxygen compounds. Besides, the results of core simulation flooding test with strain I<br />
showed that MEOR increased from 12.7% to 15.9% for different permeability cores. Simulation flooding<br />
test with natural cores enhanced higher recovery than that with artificial cores.<br />
05#22<br />
Production and Partial Characterization of Biosurfactant Produced by Crude Oil Degrading<br />
Bacteria<br />
Maryam L. Ibrahim 1 , S. Umar 1 , U. J. Ijah 2 , L. S. Bilbis 1 1 Department of Microbiology, Usmanu<br />
Danfodiyo University Sokoto, Nigeria 2 Department of Biochemistry, Usmanu Danfodiyo University<br />
Sokoto, Nigeria<br />
Currently, worldwide attention has been given to enhanced oil recovery (EOR) technologies because<br />
conventional methods used in oil recovery extract only about 30-45% of oil in place. EOR can be<br />
achieved through the use of biosurfactants which are compounds capable of reducing surface and<br />
interfacial tension in hydrocarbon medium. Microbial enhanced oil recovery (MEOR) is cost effective and<br />
innovative because beneficial activities of microorganisms are being exploited using cheap substrates to<br />
overcome problems associated with oil production. Crude oil utilizing bacteria were isolated from soil by<br />
enrichment method on oil agar at 30°C for 5 days. The isolates were identified using molecular<br />
characterization of 16S rDNA gene sequence and screened for biosurfactant production using blood<br />
haemolysis, emulsification tests and Surface tension activity. IR and GC-MS analyses were carried out to<br />
detect the type of biosurfactant. The surfactant was purified and its stability at various pH, temperature<br />
and salinity were studied. The genomic DNA from the isolates was amplified using the consensus primers<br />
of the alkoxidase genes. The putative genes were gel purified using mini prep kit and cloned into TOPO-<br />
TA vector. Genomic DNA isolated revealed that the Biosurfactant producers were Achromobacter<br />
xylosoxidans subspecies Xylosoxidans, Bacillus licheniformis, Proteus vulgaris, Proteus mirabilis, Serratia<br />
marcescens, Sphingomonas paucimobilis and Micrococcus kristinae. Serratia marcescens had the<br />
highest emulsification index of 50%. GC-MS analysis indicated that the biosurfactants were lipopeptides.<br />
The amplicons were 695 bp as observed on agaose gels. The Positive white colonies subjected to miniprep<br />
revealed the 695bp amplicon. The products are being subcloned for the overexpression of the<br />
alkoxidase.<br />
52
05#23<br />
The Degradation of Propane and Butane by Sulphate Reducing Bacteria<br />
Ulrike Jaekel 1 , B. Adam 2 , N. Musat 2 , C. Vogt 3 , F. Widdel 1 , F. Musat 1 1 Dep. of Microbiology, Max<br />
Planck <strong>Institute</strong> for Marine Microbiology 2 Dep. of Biogeochemistry, Max Planck <strong>Institute</strong> for Marine<br />
Microbiology 3 Dep. of Isotope Biogeochemistry Helmholtz Centre for Environmental Research -<br />
UFZ<br />
Short-chain alkanes (C2-C5) are major constituents of natural wet gas reservoirs and also present in crude<br />
oil reservoirs. In places where tectonic plate movement causes the formation of fractures in deep rocks,<br />
these alkanes seep upwards as dissolved components of geothermal fluids through the sediment and<br />
come into contact with the biosphere. The anaerobic degradation of propane and butane was recently<br />
reported with a pure culture (Strain BuS5) and several enrichment cultures of sulfate-reducing bacteria<br />
from marine deep sea hydrocarbon seeps. However, the diversity of anaerobic propane and butane<br />
degrading bacteria is largely unknown. In this study, enriched cultures of sulfate-reducing bacteria which<br />
can degrade propane and butane were obtained from marine sediments collected around natural gas and<br />
oil seeps in the Gulf of Mexico, and at Hydrate Ridge. 16S rRNA gene libraries revealed that most<br />
phylotypes affiliated with the Desulfosarcina/Desulfococcus cluster of the Deltaproteobacteria. Whole-cell<br />
hybridization with sequence-specific probes showed that each enrichment culture was dominated by a<br />
distinct phylotype, related to Strain BuS5. Incubations of the enriched cultures with 13 C-labeled propane<br />
or butane followed by Halogen In Situ Hybridization-Secondary Ion Mass Spectroscopy analysis showed<br />
substantial 13C-assimilation by the dominant phylotype in each enrichment culture. Two-dimensional<br />
compound specific stable isotope fractionation analysis was performed with several cultures and resulted<br />
in C and H fractionation values which are specific for the degradation of propane and butane via fumarate<br />
addition. These could, in principle, serve as tools to assess the anaerobic transformation processes for<br />
gaseous alkanes in situ.<br />
05#24<br />
Microbiological and Geochemical Characterization of Microorganisms Occuring in Oil Field<br />
Waters (SE Poland)<br />
Ludwina Jarzynowska 1 , D. Wolicka 1 , A. Borkowski 2 , A. Gójska 1 1 <strong>Institute</strong> of Geochemistry,<br />
Mineralogy and Petrology, Faculty of Geology, University of Warsaw 2 Department of<br />
Environmental Protection and Natural Resources, Faculty of Geology, University of Warsaw<br />
Oil field waters constitute an environment with extreme properties. Very high pressures and temperatures<br />
and a high salinity, reaching well over 10%, pH in situ (3-8) are parameters which influence bacterial<br />
activity. In spite of this, it has been possible to isolate and identify very many different groups of bacteria<br />
from this environment. Tested oil field waters are presented by sodium carbonates. Microorganisms were<br />
isolated from oil field waters from 12 mines: oil, oil-gas and gas-mines. The high count microorganisms<br />
were isolated over pH ranges from 6.6 to 8.3 and salinity from 1 (oil mines) - 7% (gas mines). The waters<br />
were contaminated by petroleum products especially benzene, toluene, ethylobenzene and toluene<br />
(BTEX). The aim of this study was the isolation and characterisation of microorganisms occurring in oil<br />
field water and the show its role in biodegradation BTEX and biotransformation of chlorides. Aerobic<br />
bacterial communities were isolated with M9 medium and incubated for 2 weeks at 18oC. The anaerobic<br />
bacterial communities were isolated using the microcosms method into glass bottles with a barren<br />
modified Postgate C medium (without lactate, citric acid and yeast extract). The cultures were incubated<br />
for 2 weeks at ca. 18°C. The results of a gene analysis indicate the presence of aerobic bacterial<br />
communities mainly belonging to Pseudomonas sp., Ralstonia sp., Geobacillus sp., Bacillus sp.,<br />
Stenotrophomonas sp. Xanthomonas sp. The anaerobic bacteria were represented by sulphate reducing<br />
bacteria belonging to Desulfovibrio, Desulfohalobium, Desulfuromonas, Desulfonatronospira, and<br />
Desulfomicrobium and methanogenic archaea Metyhanosarcina, Methanococcoides and iron (III)<br />
reducing bacteria like Shewanella sp.<br />
53
05#25<br />
Microbial characterization of anaerobic consortia's isolated from crude oil (SE Poland)<br />
Agnieszka Gójska 1 , D. Wolicka 1 , L. Jarzynowska 1 , A. Borkowski 2 , 1 <strong>Institute</strong> of Geochemistry,<br />
Mineralogy and Petrology, Faculty of Geology, University of Warsaw 2 Department of<br />
Environmental Protection and Natural Resources, Faculty of Geology, University of Warsaw<br />
Crude oil is an extreme environment for the occurrence of microorganisms but it is possible to isolate and<br />
identify many different groups of bacterial communities from it. The most frequent groups of bacteria<br />
isolated from crude oil are anaerobic communities containing sulphate-reducing bacteria (SRB),<br />
fermenting bacteria and methanogenic archaea. The paper presents the results of a gene analysis (16S<br />
rDNA gene) carried out in cultures incubated in conditions favouring the development of sulphidogenic<br />
bacterial communities. The microorganisms were isolated from paraffin 10 crude oil from south-eastern<br />
Poland. Anaerobic bacteria communities were isolated using the microcosms method under anaerobic<br />
conditions using 15 ml of crude oil introduced into glass bottles with a barren modified Postgate C<br />
medium (without lactate, citric acid and yeast extract). The cultures were incubated for 6 weeks at ca.<br />
18°C. The genetic analysis of the microbial communities isolated from crude oil is very interesting. For<br />
many reasons, crude oil is not a good environment to isolate genetic material in and may cause many<br />
procedural problems. Results of gene analysis indicate the presence of SRB, mainly belonging to<br />
Desulfovibrio, Desulfohalobium, Desulfuromonas, Desulfonatronospira, and Desulfomicrobium,<br />
methanogenic archaea Metallospharea, Methanococcus as well as accompanying microflora, whose<br />
genes show similarities to Geobacter sp., Bacillus sp., Pseudomonas sp., Thioalkalivibrio sp., Ralstonia<br />
sp., Shewanella sp. The reports on the presence of various microorganism groups in crude oil and<br />
deposit fuels data on the isolation of microorganisms from this environment from reservoirs in Poland are<br />
scarce. With the increasing application of microorganisms in bioremediation, studies of various crude oil<br />
reservoirs with regard to their microbiology seem justified.<br />
05#26<br />
Application of aerobic bacteria in bioremediation of environments contaminated by BTEX<br />
Dorota Wolicka 1 , L. Jarzynowska 1 , A. Gójska 1 , A. Borkowski 2 , 1 <strong>Institute</strong> of Geochemistry,<br />
Mineralogy and Petrology, Faculty of Geology, University of Warsaw 2 Department of<br />
Environmental Protection and Natural Resources, Faculty of Geology, University of Warsaw<br />
Pollution of the environment by oil-derived products still remains an unsolved issue, most probably linked<br />
to its multi-aspect nature. This results e.g. from the common application of oil-derived products, their<br />
hazardous influence on living organisms and specific abilities linked with their distribution and long-term<br />
durability in the environment. This in turn points to a multidisciplinary approach to the issue and the need<br />
to involve not only scientists working on bioremediation such as e.g. microbiologists, but also specialists<br />
in geology, soil science and hydrogeology. This paper is focused on the isolation of active autochtonous<br />
aerobic microorganisms from soils contaminated by BTEX, able to biodegrade benzene, toluene,<br />
ethylobenzene and xylene as the sole carbon sources. Samples of soil were inserted in 300 ml flasks and<br />
covered with medium M9 containing benzene (0.5g/L), toluene (0.5g/L), ethylobenzene (0.5g/L) and<br />
xylene (0.5g/L) as a soul source of carbon. The cultures were vortexed and the resulting supernatant<br />
became the inoculum for stationary cultures and field experiments. The cultures were incubated under<br />
aerobic conditions at ca. 18°C. BTEX reduction was observed in all cultures: benzene 88%, toluene 86%,<br />
ethylbenzene 90%, m-p- xylene 91% and o-xylene 90%, compared to an abiotic control batch. Bacterial<br />
strains were assigned to particular species by analysis of their 16S rDNA gene sequence. Results of<br />
gene analysis indicate the presence of microorganisms, mainly belonging to Pseudomonas, Bacillus,<br />
Flavobacterium, Brevundimonas and Ralstonia The isolated microorganism communities were then used<br />
for in situ bioremediation of an area polluted by BTEX. Under field conditions, the total reduction of BTEX<br />
was ca. 98%.<br />
54
05#27<br />
Study and Isolation of Aerobic Hydrocarbon-Degrading Bacteria from a Shoreline in Cuba<br />
Yaima Barrios-San Martín, S. Acosta-Díaz, F. González-Hernández Cuban Petroleum Research<br />
Center (CEINPET)<br />
In Cuba, one of the technologies used for the oil decontamination of coasts and seas, is the use of<br />
microorganisms. It is a cost-effective technique that offers big expectations at large scales throughout the<br />
country. In the present work, water and sediment samples were collected from the coast of Felton in the<br />
province of Holguin, Cuba. A first screening using a Bushnell-Haas medium and light crude oil as a<br />
carbon source yielded 27 bacteria isolates. These were subsequently subjected to the same medium but<br />
supplemented with yeast extract and/or heavy crude oil resulting in a total of 3 strains as the best<br />
degraders in 45 days. Pure culture of these strains: F9S, F1FLC, and F10S1, were further used in crude<br />
oil biodegradability assays. The total petroleum hydrocarbons (TPH) degradation was evaluated using<br />
S.A.R.A. analysis, and the aliphatic and aromatic hydrocarbon studies were carried out using gas<br />
chromatography coupled with F ID detector (GC-FID) and FT-IR respectively. All three strains removed<br />
more than 60 % of the TPH but F10S1 showed the best degradation percentages with numbers above<br />
65% for the entire hydrocarbon fraction, except resins. They were also able to decrease C17:Pr and<br />
C18:Ph ratios to less than 50 % in comparison to the abiotic control. The strains were phenotypically<br />
identified as Bacillus sp. for F1FLC and F9S and Alcaligenes sp. for F10S1. New studies have already<br />
started with these strains, taking advantage of their degradability potentials, as efficient biological<br />
products to clean contaminated sea coasts in a near future.<br />
05#28<br />
Microbes or mass transport - what are the real barriers to production scale microbial gasification<br />
of oil or coal to produce methane or hydrogen in subsurface reservoirs?<br />
Steve Larter 1 , I. Head 2 , M.M. Hamedi 2 , I. Gates 1 1 PRG U of Calgary 2 CEGS U of Newcastle<br />
Low carbon emission energy sources are needed for sustainable development but the timescales of<br />
technology replacement mean fossil fuels will be burnt for decades. A successful sociotechnical finesse,<br />
switching from direct use of high carbon coal or oil, to in situ biologically or thermally generated natural<br />
gas for home, industrial and transportation would permit rapid reduction in emissions while maintaining<br />
infrastructure, thus facilitating real change. Advances in understanding subsurface microbial processes,<br />
large scale reactive reservoir simulation with optimization of complex chemical and biological processes,<br />
active sensing of recovery process operations, and ability to simulate complex heterogeneous reservoirs<br />
permits many new potential approaches to insitu gasification. Petroleum hydrocarbon biodegradation in<br />
subsurface reservoirs is now well understood with Methanogenic Alkane Degradation by Carbon dioxide<br />
Reduction (MA DCOR), producing both hydrogen and methane in abundance in natural settings. Some of<br />
the principle organisms are identified and in heavy oil reservoirs, nutrient supply from water legs appears<br />
to be rate limiting on geological timescales. Laboratory data indicates field scale engineering of nutrient<br />
delivery via water injection can accelerate gas production to commercially interesting levels, however<br />
mass transport, both diffusive-dispersive and free phase flow, of water and gas are likely to limit<br />
production rates in real reservoirs. In contrast, the mechanisms of coal biodegradation are poorly<br />
understood and it is less clear what the rate limiting steps are although mass transport, of microbial<br />
growth substrates from nanoscale to macroscale pores, is also likely to limit gas production. We review<br />
progress in engineering accelerated microbial gasification of coal and heavy oil to rates sufficient to<br />
significantly replace conventional oil or coal production with microbial gas production.<br />
55
Session 06 - Prospects for MEOR and Biodesulfurization<br />
Invited speakers:<br />
06#01<br />
Biosurfactants and their Role in Oil Recovery<br />
Michael McInerney University of Oklahoma<br />
Biosurfactant-mediated oil recovery may be an economic approach to recover the large amounts of oil<br />
that remain entrapped in oil reservoirs. The lipopeptide biosurfactants made by members of the<br />
Bacillus subtilis/licheniformis group lower the interfacial tension between oil and water sufficiently<br />
(
Offered Papers:<br />
06#03<br />
Problems and Opportunities of Microbial Methane Production in Oil Reservoirs<br />
Alexander Grigoryan, A. Sen, C. Ezeuko, I. Gates Department of Chemical and Petroleum<br />
Engineering, Schulich School of Engineering, University of Calgary<br />
Transformation of hydrocarbons in petroleum reservoirs by endogenous prokaryotes has two<br />
contradictory effects. First, on the negative side, it yields large concentrations of heavy fractions in crude<br />
oil due to biodegradation of light oils which increase recovery and refining costs and associated<br />
environmental impact particularly with current recovery and upgrading technologies. Second, on the<br />
positive side, the net result of the biological conversion of subsurface organic matter is methane gas<br />
realized with relatively high energy efficiency and low emissions when compared to equivalent energy<br />
recovered in the form of oil by steam-based recovery and conventional upgrading methods. Thus,<br />
understanding the process of oil bioconversion to methane offers a potential path to feasible production of<br />
green energy from oilfields. The complex functionality of oilfield methane-producing microbial consortia,<br />
uncertainty of transformation routes and slug reaction kinetics limits biotechnological process design,<br />
robustness, and management. Here we provide a critical review on recent knowledge gained from<br />
laboratory tests, computer models, field pilots and economic data to identify the most sustainable<br />
application of microbial activity for enhancement of methane recovery from oil-bearing horizons. Primary<br />
reservoir, geochemical, microbiological and modeling requirements for successful treatment design of<br />
biologically advanced methane production in-situ are outlined. Biogenic methane production in a<br />
waterflood oilfield, treated as a multistage solid-liquid process that consists of an open hydrocarbon<br />
hydrolyzing methane producing reactor is evaluated. Future perspectives on biotechnology development<br />
and modification for accelerated gaseous hydrocarbon production are briefly proposed.<br />
06#04<br />
Molecular and Risks-Based Approach to Nutrient Development for a Proposed Sub-Surface Coal-<br />
Bed Methane Biogasification Field Trial<br />
Adewale Lambo 1 , D. Strapoc 1 , M. Pittenger 1 , L. Wood 2 , M. Ashby 2 , B. Huizinga 1 1 ConocoPhillips<br />
Company 2 Taxon Biosciences<br />
Several studies of various coal-derived gas fields and coal-bed methane (CBM) reservoirs have shown<br />
that significant proportions of their gas reserves have a biogenic origin, where the biomethane is formed<br />
at slow rates over a geological time-frame. In contrast, recent laboratory studies using microbial consortia<br />
from a coal-derived gas reservoir show that this process can occur at significantly faster rates when<br />
optimized conditions (e.g. nutrients, water chemistry, etc) are provided and maintained. Therefore a<br />
dominant control of in-situ biomethane production is the supply of nutrients and the maintenance of<br />
appropriate water-chemistry conditions for the targeted biomethane-forming consortia. For this reason,<br />
nutrient amendment is being explored for commercial-scale enhanced biomethane formation in coaly<br />
reservoirs. In our work, specific biogenic gas fields have been studied in order to obtain detailed<br />
information on their microbial community composition, the geochemical conditions, and uniqueness of the<br />
identified microbial associations, which would allow development and injection of appropriate nutrients<br />
during a proposed field trial. In addition to this, evaluation of the likely risks, due to nutrient injection, was<br />
carried out in order to help mitigate and prevent such risks during a proposed field trial. The initial<br />
approach was to identify the indigenous methanogenic pathway showing the most favorable biomethaneproducing<br />
rate, and which is to be optimized for enhanced biomethane production while avoiding<br />
deleterious effects. To accomplish the goals, microbial genomic DNA was extracted from water samples<br />
obtained from the fields and microbial DNA sequences were determined using PCR-amplified 16s RNA<br />
following established protocols. This microbial sequence information, with the results of bench-scale<br />
studies, was subsequently used to identify the targeted methanogenic pathway for enhanced in-situ<br />
biomethane production. These correlations identify Archaeal-bacterial candidates associated with the<br />
methanogenic pathway, the effective members of which are confirmed by additional bench-testing and<br />
57
the use of FISH technique. Optimized nutrient and water-chemistry conditions were developed for the<br />
targeted biomethane-forming consortia using a combination of field geochemical data and tests,<br />
laboratory culturing, and literature review. The preliminary nutrient formulation was further modified based<br />
on the potential risks associated with injecting nutrients into the gas reservoir. The potential risks that<br />
were considered included several biological factors (e.g. biofilm plugging, microbial sulfide production,<br />
etc) and physicochemical factors (e.g. scaling, produced-gas specifications, etc).<br />
06#05<br />
Experimental Investigation of Microbial Enhanced Oil Recovery Effectiveness in Omani Oil Field<br />
S. Al-Bahry, Y. Al-Wahaibi, A. Elshafie, A. Al-Bemani, H. Al-Sulaimani, S. Joshi Sultan Qaboos<br />
University<br />
An extensive research has been carried out in the last four years at Sultan Qaboos University (SQU) to<br />
experimentally investigate the potential of Microbial Enhanced Oil Recovery (MEOR) in some of the<br />
Omani oil fields. The goal of this project was to test different applications and mechanisms of MEOR.<br />
Both in situ and ex situ processes were investigated using indigenous microbes isolated from one of the<br />
Omani oil fields and exogenous microbes isolated from oil contaminated soil samples from different parts<br />
of the Sultanate. This paper describes the key laboratory tests used to evaluate these mechanisms and<br />
the lessons learned from this lab experience. Sampling techniques were standardized for anaerobic<br />
sampling from the field. Using Denaturation Gradient Gel electrophoresis (DGGE), PCR and DNA<br />
sequencing, the microbial community/consortia of one of the oilfields were identified. Some novel<br />
microorganisms were found that had great potential with useful bio-products for MEOR such as biogases<br />
and biomass. For the ex-situ process, Bacillus strains isolated from oil contaminated soil samples were<br />
found to produce excessive biosurfactants that could reduce the interfacial tension against n-heptane<br />
from 46.6 mN/m to 3.28 mN/m in less than 24 hours. The yield of the biosurfactant product was 2.5 g/l<br />
and it showed high stability under wide range of temperature, pH and salinities. The interaction of the<br />
biosurfactant product with porous media was tested. This was done by series of core flooding<br />
experiments where the biosurfactant was tested as a tertiary recovery stage. The results showed high<br />
potential of using this bioproduct during ex-situ process where a total of 23% of residual oil was<br />
recovered. Interesting results were found when testing the potential of mixing the biosurfactant with<br />
commercial chemical surfactants at different concentrations where residual oil recovery increased up to<br />
50% when applying a mixture of (25:75) and (50:50) of the (chemical surfactants: biosurfactants)<br />
respectively. Overall, the ultimate objective was to evaluate the performance of these various MEOR<br />
techniques in the lab-scale to better understand the mechanisms and their potential in enhancing oil<br />
recovery prior to field implementation.<br />
Posters:<br />
06#06<br />
The Impact of Biofilm Growth on the Flow Properties within a Porous Medium<br />
Ali Bozorg, A. Sen, A. Grigoryan, I. Gates University of Calgary<br />
Biopolymeric substances secreted by microorganisms adhered to a solid surface can accumulate and<br />
form a three-dimensional extracellular matrix within which the microbes can replicate. These complex<br />
dynamic systems, which are referred to as biofilm, are ubiquitous in nature and are of interest because<br />
they can negatively impact human health and decrease the efficiency of industrial processes such as<br />
water treatment, oil recovery, and food processing. Their presence in porous media is of special concern<br />
because their growth can lead to a progressive decrease in permeability of flow channels (i.e. reduction in<br />
hydraulic conductivity), eventually resulting in complete clogging. To better understand the impact of<br />
biofilms in porous media, we have developed a novel macroscopic computer model that treats biofilm as<br />
a viscous fluid. To experimentally validate our model, we report here on a minimally invasive method that<br />
uses a packed column to investigate the spatiotemporal development of the biofilm phase in a saturated<br />
porous medium. Using the bacterium Pseudomonas fluorescence HK44 as a model organism, we have<br />
58
discovered that pressure changes are correlated to biofilm saturation as a function of distance from the<br />
column inlet. Tracer experiments have allowed visualization of the biofilm and indirect measurement of<br />
hydraulic properties. We were able to determine key parameters such as effective permeability and<br />
mobility under different operating conditions. Our experimental results have allowed us to better<br />
understand biofilm behaviour in porous media, and to further validate our new computer model to<br />
simulate microbial processes in the oil reservoirs.<br />
06#07<br />
A New Modelling Approach to Simulate Biofilm Phase Growth and Evolution in Porous Media<br />
Ali Bozorg, A. Sen, I. Gates University of Calgary<br />
Biofilms are communities of microorganisms embedded in a self-generated extracellular polymeric matrix<br />
(EPM) which adhere to external surfaces. They are present in aqueous environments, and as such, can<br />
impact important industrial processes including water distribution systems, sewage treatment facilities, oil<br />
recovery systems, and food processing facilities. In processes containing porous media, biofilm growth<br />
can lead to complex biophysical phenomena such as bioclogging (local hydraulic conductivity reduction).<br />
Since this can significantly affect flow characteristics, and thus system efficiencies, it is important to study<br />
the impact of biofilms in porous media. Current models of biofilm development in porous media treat<br />
biofilm as a solid phase. Growth of biofilm, therefore, translates to changes in porosity. We report here a<br />
novel alternative approach by considering biofilm as an incompressible and highly viscous fluid. Using a<br />
commercially available simulator (CMG-2009) we developed an innovative multi-phase, multi-component<br />
flow model with biological reactions to simulate biofilm behavior and related phenomena in porous media.<br />
The model took into account biofilm growth, decay, attachment, detachment, and nutrient consumption.<br />
Bioclogging was simulated by describing the relative permeability as a function of biofilm saturation. The<br />
model was validated using published experimental data. Comparison of simulation and experimental<br />
results revealed that the model could accurately describe observed biofilm dispersion and the impact of<br />
clogging, such as changes in two-dimensional nutrient transport. This study provides a basis for future<br />
studies aimed at further understanding the impact of biofilm formation in porous media. An experimental<br />
program has been initiated to further validate this approach.<br />
06#08<br />
Production of Biosurfactants by Crude Oil Degrading Bacteria and Their Possible Use in Recovery<br />
of Residual Oil<br />
Lami Ibrahim 1 , S. Umar 1 , U. Ijah 2 1 Department of Microbiology, Usmanu Danfodiyo University<br />
Sokoto, Nigeria 2 Department of Microbiology, Federal University of Technology, Minna, Nigeria<br />
Currently, worldwide attention has been given to enhanced oil recovery (EOR) technologies because<br />
conventional methods used in oil recovery extract only about 30-45% of oil in place. EOR can be<br />
achieved through the use of biosurfactants which are compounds capable of reducing surface and<br />
interfacial tension in hydrocarbon medium. Microbial enhanced oil recovery (MEOR) is cost effective and<br />
innovative because beneficial activities of microorganisms are being exploited using cheap substrates to<br />
overcome problems associated with oil production. Crude oil utilizing bacteria were isolated from soil by<br />
enrichment method on oil agar at 30ºC for 5 days. The isolates were identified using molecular<br />
characterization of 16S rRNA gene sequence and screened for biosurfactant production using blood<br />
haemolysis, emulsification tests and surface tension activity. GC-MS analyses were carried out to detect<br />
the type of biosurfactant. The surfactant was purified and its stability at various pH, temperature and<br />
salinity were studied. In addition, potential application of the biosurfactants in oil recovery was<br />
demonstrated using sand packed column. Six isolates were identified as biosurfactant producers and they<br />
were Achromobacter xylosoxidans subspecies Xylosoxidans, Bacillus licheniformis, Proteus vulgaris,<br />
Proteus mirabilis, Serratia marcescens, Sphingomonas paucimobilis and Micrococcus kristinae.<br />
Emulsification test (E24) revealed that Serratia marcescens had the highest emulsification index of 50%.<br />
GC-MS analysis indicated that the biosurfactants were glycolipid. Stability test and PCR amplification and<br />
59
sequencing of the genes encoding for biosurfactant production in the organism as well as the oil recovery<br />
potential of the biosurfactants are being studied.<br />
06#09<br />
The Prospect of a Novel Oil-Viscosity-Reducing Bacteria for MEOR<br />
Yuichi Sugai, I. Purwasena, K. Sasaki Department of Earth Resources Engineering, Faculty of<br />
Engineering, Kyushu University<br />
A novel thermophile having a sheath-like structure called "toga" was isolated from Yabase oilfield in<br />
Japan. According to its 16S rDNA sequence, this isolate is related to the members of the genus<br />
Petrotoga. The potential of this isolate as a candidate for MEOR was investigated by cultivation<br />
experiments in this study. This isolate can grow and reduce oil viscosity in brine medium supplemented<br />
with crude oil. Therefore, the enhancement of oil recovery can be expected by this isolate. Influences of<br />
cultivation conditions such as nitrogen source and reservoir conditions such as temperature, salinity and<br />
pressure on the isolate were investigated in this study. A small amount of yeast extract was effective for<br />
stimulating the growth of the isolate as a nitrogen source. The viscosity of crude oil incubated with the<br />
isolate was 46.3%, 51.6% and 65.9% lower than that of control after 3 weeks incubation at 50°C, 60°C<br />
and 70°C respectively. The isolate reduced the oil viscosity effectively under the salinity up to 3% while it<br />
can grow under the salinity up to 9%. Moreover, the isolate reduced oil viscosity to 35.1% of its original<br />
viscosity under high pressure condition, 700 psi. The results of GC analyses revealed that the isolate<br />
converted heavier components of crude oil into lighter components. These results indicate that the isolate<br />
achieves economically feasible MEOR because it doesn't need costly nutrients such as molasses but<br />
some nitrogen sources. In addition, the isolate can be applied to a wide range of reservoirs for MEOR.<br />
06#10<br />
Estimation of the Possibility of MEOR Using a Strain Belonging to the Genus Petrotoga by<br />
Investigating Its Habitations in Several Oilfields<br />
Isty Purwasena, Y. Sugai, K. SasakiDepartment of Earth Resources Engineering, Faculty of<br />
Engineering, Kyushu University<br />
A talented microorganism belonging to the genus Petrotoga was isolated from Yabase oilfield in Japan.<br />
The isolate can grow in the brine supplemented with crude oil and reduce oil viscosity to 40-50% of its<br />
original viscosity at a temperature between 50 and 80°C, a salinity up to 9%, and a pressure up to 700<br />
psi, therefore, it is expected to be applied to MEOR in many oilfields. Possibility of field application of this<br />
isolate was considered by investigating the habitation of the genus Petrotoga in several oilfields where<br />
temperature and salinity were different in this study. Brine samples were extracted from high temperature<br />
(80°C) and high salinity (7%) reservoirs in Oman, high temperature (60-80°C) and low salinity (0.2-1.0%)<br />
reservoirs in Japan, China and Indonesia, and low temperature (45°C) and low salinity (0.2%) reservoirs<br />
in China. Nucleotide sequences of each DNA extracted from brine samples and isolated by DGGE were<br />
analyzed to identify the species. Petrotoga sp. was detected in the brine of Japanese oilfield and<br />
Indonesian oilfield as a dominant species. Thermotoga sp. which is biosystematically in the same family<br />
with Petrotoga sp. and both of them can grow under the similar environment was detected in Chinese<br />
high temperature and low salinity oilfield. On the other hand, Petrotoga sp. and/or its closely related<br />
species were not detected in the brine of other oilfields. These results indicate that the isolate can be<br />
applied more easily to reservoirs which were characterized as high temperature and low salinity reservoir<br />
for MEOR.<br />
60
06#11<br />
Investigation of Microbial Communities in Typical Oil Reservoirs with Different Temperatures and<br />
Geological Conditions<br />
Yuehui She, F. Shu, Z. Wang, S. Kong Yangtze University<br />
Microbial enhanced oil recovery (MEOR) techniques are gaining increased attention due to their<br />
economic, environmentally-friendly and simpler application advantages. In this study, microbial<br />
communities in typical oil reservoirs with different temperatures and geological conditions in Xinjiang<br />
(China) were analyzed. The results of microbial communities of hyperthermal condensate gas reservoir<br />
(110℃) showed that microbial populations in water samples were dominant by Pseudomonas sp.<br />
(AY486375) of 41% Shewanella sp. (FM887036) of 22% and Enterobacter sp. (GU086162) of 14%. The<br />
microbial populations in two condensate oil samples were dominant by Pseudomonas aeruginosa<br />
(HM582426) of 67.82% and Vibrio sp. (FJ457366) of 56.32%. Detected sequences had high similarity<br />
(more than 99%) with cultured Acinetobacter, Pseudomonas and Sphingobacterium which are related to<br />
production of biosurfactant. Results also showed that dominant bacteria in the production well in a heavy<br />
oil reservoir with low temperature (20°C) were uncultured Desulfobacterales bacterium (GQ354918)<br />
covering 66.4% of the total microbial population while the dominant bacteria in the water injection well<br />
were uncultured bacterium clone (AY327241) covering 47% of the total microbial population. Overlapping<br />
microbial communities were Uncultured Desulfuromusa sp. (EU283459), Denitrovibrio acetiphilus<br />
(NR027535), Syntrophus sp. (AJ133795), Uncultured Syntrophus sp. (GU112190), and Uncultured<br />
Desulfobacterales bacterium (GQ354918). Results of microbial communities showed that bacteria having<br />
potential application on MEOR inhabited all oil reservoirs with different temperatures.<br />
06#12<br />
The Impact of Biofilm on Porous Media Permeability and Implications for MEOR<br />
Cosmas Ezeuko, I. Gates, A. Sen, A. Grigoryan University of Calgary<br />
Microbial methods have enormous potential for sustained economic production of oil fields via favourable<br />
modification of petrophysical properties of the porous medium, reduction of interfacial tension via<br />
biosurfactants, conversion of oil components to gas thus enlivening the oil phase yielding both oil phase<br />
viscosity reduction and solution-gas drive, and bioclogging shut-off technologies to improve sweep<br />
efficiency. In this study, we present a 3D pore network simulator to predict biofilm evolution in porous<br />
media under flow conditions. The model consists of water and permeable biofilm phases enclosed within<br />
pores of a solid phase. Nutrient transport in the aqueous phase and across the water/biofilm interface<br />
occurs by advection and diffusion with reactions to account for nutrient consumption. A dual-diffusion<br />
coefficient, mass transfer model is introduced to handle simultaneous mass transfer in both liquid and<br />
biofilm. Inclusion of growth, decay, and shear stress dependent detachment permits examination of<br />
interaction between biofilm evolution and changes in hydraulic properties of the porous media. The<br />
results are supported by experimental observations and demonstrate the impact of biofilm growth on<br />
permeability of the porous media. In addition to inlet concentration, the results reveal that biofilm growth<br />
depends on the limiting shear stress which depends in turn on the physiology of the biofilm. Inoculation of<br />
microbes at the inlet yields larger reduction of network permeability compared to inoculation away from<br />
the inlet, for the same biomass volume. This study reveals that systematic management of biofilm growth<br />
can be achieved to yield desired outcomes in a porous medium.<br />
61
06#13<br />
Production of Rhamnolipids in Recombinant E. coli as a Possible Approach for Improvement of<br />
MEOR and Bioremediation Technologies<br />
Yuriy Kryachko, S. Nathoo, P. Lai, G. Voordouw, J. Voordouw, E. Prenner University of Calgary,<br />
Department of Biological Sciences<br />
Rhamnolipids have been known as potent biosurfactants that could be used for enhanced oil recovery<br />
and bioremediation. They are less toxic and easier degradable than chemically synthesized surfactants.<br />
Using engineered microorganisms can be the way to making oil recovery and bioremediation<br />
technologies more environmentally friendly and, possibly, more efficient. To develop a recombinant E. coli<br />
strainable to produce rhamnolipids, Pseudomonas aeruginosa PA14 rhlAB gene cluster responsible for<br />
production of mono-rhamnolipids was PCR amplified with two sets of primers inserted into pNOT19 vector<br />
to produce two recombinant plasmids, pF1bR4 and pF4R4 that were used for transformation of E. coli<br />
TG2 to get two recombinant strains, E. coli TG2-0 and E. coli TG2-F4R4. The recombinant strains were<br />
grown at 37°C and centrifuged at 3000 rpm for 10 minutes. Cell free supernatants from both recombinant<br />
strains emulsified hexadecane, unlike the supernatant obtained from the control strain. However,<br />
measurements performed with a Langmuir trough showed that supernatants from the recombinant strains<br />
had surface tension that was lower than surface tension of the control, but higher than surface tension of<br />
the supernatant from Pseudomonas aeruginosa PA14. These differences could be possibly due to high<br />
solubility of mono-rhamnolipids or their micelles in water or low biosurfactant yield. Nevertheless, the<br />
transformation of E. coli as a model microorganism, as well as microorganisms naturally inhabiting oil<br />
reservoirs and oil contaminated sites with the plasmids containing not only rhlAB, but also rhlC genes<br />
may lead to production of di-rhamnolipids that could have greater potential for lowering surface tension.<br />
06#14<br />
Biophysical Characterization and Screening of Biosurfactant Activity in Experiments on vitro<br />
Tailing Pond Sedimentation and Enhanced Oil Release from Drilling Cores<br />
Patrick Lai, S. Nathoo, Y. Kryachko, T. Jack, G- Voordouw, E. Prennner University of Calgary<br />
Synthetic or biological surfactants can be powerful tools to enhance oil recovery and decrease the<br />
environmental footprint of the oil sands. Biosurfactants are economic, biodegradable and low toxic agents<br />
that work by altering the surface tension between contact surfaces. This work examined the possibility of<br />
using biosurfactants to improve the sedimentation of tailing pond samples by facilitating the release of<br />
bound water. Moreover, the potential use of biosurfactants to enhance the release of hydrocarbon from<br />
drilling core samples was studied. Biosurfactants isolated from cultures of Pseudomonas aeruginosa<br />
PA14 and Pseudomonas aeruginosa PA01 were compared to synthetic surfactants. In addition, two E.<br />
coli recombinant strains, TG2-0 and TG2-F4R4 containing the rhlAB gene cluster responsible for<br />
rhamnolipid production were tested. The presence of surface active compounds in organic extracts of<br />
these supernatants was assessed by their emulsifications properties. Hydrocarbon release was<br />
monitored over time by photographic imaging and spectroscopic measurements. Sedimentation of tailing<br />
pond samples was also determined by comparing the height of the compact pellet to the overall water<br />
level in the sample container. Surface activity tests for analyzing large numbers of samples in a microtiter<br />
plate format were established within comprehensive screening efforts of the Hydrocarbon Metagenomics<br />
project supported by Genome Canada. The ultimate goal is developing a novel fully automated highthroughput<br />
screening approach to identify biosurfactants in cultures originating from environmental<br />
samples.<br />
62
06#15<br />
Enrichment and Characterization of Thermophilic Microbes Growing on Heavy Oils<br />
Odd Gunnar Brakstad 1 , S. Markussen 1 , M. Frenzel 1 , K. Bonaunet 1 , S-H. Vang 1 , H. K. Kotlar 2<br />
1 SINTEF Materials and Chemistry, Dept. Marine Environmental Technology 2 Statoil Research<br />
Centre<br />
Biotechnological processes may be used as one of several tools for increased oil recovery and upgrading<br />
of heavy oils. In order to generate enrichment cultures for potential heavy oil bioconversion a horizontal<br />
flow-through column system was established for enrichment of microbes attaching to heavy oils. The<br />
system included a heavy oil stationary phase, with salt medium mobile phase which was continuously<br />
circulated across the heavy oil surface. Bacterial inocula originating from natural hydrocarbon seeps were<br />
introduced to the system and incubated at 55-60°C for 2-4 weeks. Enrichment cultures growing in liquid<br />
face and on the oil surface were isolated and propagated separately for storage. PCR-DGGE analyses of<br />
16S rRNA gene sequences revealed different bacterial and archaeal banding profiles for enrichments<br />
from the oil surfaces and the liquid phase of the flow-through column system. Sequence analyses of<br />
bacterial and archaeal 16S rRNA genes showed closest matches of the cultures to microbial groups<br />
typically originated from oil reservoirs and hydrocarbon seeps. Bacterial enrichment cultures from oil<br />
surfaces included a number of proteobacteria and bacilli, of which members of the genus Geobacillus<br />
were abundant. Enrichment cultures were able to grow at temperatures up to 80°C, and the enrichment<br />
cultures attached well to heavy oil surfaces. These cultures have now been used for studies of<br />
bioconversion of heavy oils and bitumen.<br />
06#16<br />
Isolation of Biosurfactant-Producing Microorganisms from Oil Samples for Use in Microbial<br />
Enhanced Oil Recovery<br />
Eduardo J. Gudiña 1 , L. R. Rodrigues 1 , J. F. B. Pereira 2 , J. A. P. Coutinho 2 , J. A. Teixeira 1 1 IBB -<br />
<strong>Institute</strong> for Biotechnology and Bioengineering, Centre of Biological Engineering, University of<br />
Minho 2 CICECO - Chemistry Department, University of Aveiro<br />
Microbial Enhanced Oil Recovery (MEOR) is an important tertiary oil recovery process where<br />
microorganisms and their metabolites are used to retrieve unrecoverable oil from a reservoir after<br />
application of primary and secondary recovery techniques. Stimulation of bacterial growth and<br />
biosurfactant production by indigenous microorganisms can reduce the capillary forces that retain the oil<br />
into the reservoir. MEOR offers major advantages over conventional EOR, namely low amounts of energy<br />
consumption and independence of the price of crude oil. In this work we have been addressing the<br />
isolation and identification of microorganisms capable of producing biosurfactants under conditions<br />
existent in oil reservoirs. Biosurfactant production by microorganisms isolated from crude oil samples was<br />
evaluated by measuring surface tension and emulsification activity. Among the isolated microorganisms,<br />
seven Bacillus strains were able to grow and produce extracellular biosurfactants at 40ºC under<br />
anaerobic conditions in medium supplemented with hydrocarbons. Three isolates were selected as the<br />
higher biosurfactant producers; biosurfactants produced by those isolates reduce the surface tension of<br />
water from 72 to 30 mN/m, exhibit emulsifying activity and are not affected by exposure to high<br />
temperatures (121ºC), which make them good candidates for use at the extreme conditions usually<br />
existent in oil reservoirs. The results obtained show that those isolates exhibit potential for the<br />
development of enhanced oil recovery processes.<br />
63
06#17<br />
Bioconversion of Heavy Oil - Characterization and Screening Microbes Growing on Heavy Oil<br />
Sidsel Markussen 1 , A. Winnberg 1 , O. G. Brakstad 1 , A. Brunsvik 1 , T. Ellingsen 1 , H. K. Kotlar 2<br />
1 SINTEF Materials and Chemistry, Dept. Biotechnology 2 Statoil Research Center<br />
Heavy oil contains high concentrations of resins, asphaltenes and oil fractions with polar and highmolecular<br />
weight compounds. The high viscosity of heavy oil makes the oil difficult to produce, transport<br />
and refine by conventional methods. The use of biotechnological processes, alone or in combination with<br />
other conventional oil recovery processes represents interesting possibilities for development of novel<br />
methods for e.g. increased oil recovery and upgrading of oil. Statoil's in-house strain collection comprises<br />
extremohilic microorganisms isolated from a wide variety of sources. The isolates are able to survive and<br />
grow under selected heavy oil reservoir conditions (e.g. temperature, salinity and presence of oil). The<br />
present work concerns the use of biocatalytical processes in order to increase recovery and upgrading of<br />
heavy oil. It focuses on screening isolates for possible applications related to increased oil recovery and<br />
upgrading. Preliminary biocatalysis experiments have shown a distinct difference between inoculated and<br />
un-inoculated controls, fingerprint analysis using LC-MS/Q-TOF spectra compiled samples into distinct<br />
groups distinguishing controls from bioconverted oil samples. Screening and characterization of single<br />
isolates and microbial assemblages for bioconversion of heavy oil and bitumen using a novel screening<br />
system together with the use of fingerprint analysis will be presented.<br />
06#18<br />
Evaluation of the Potential of Microorganisms Isolated from Brazilian Oils for Microbial<br />
Degradation of Heavy Oil<br />
Jorge F. B. Pereira 1 , J. A. P. Coutinho 1 , E. J. Gudiña 2 , L. R. Rodrigues 2 , J. A. Teixeira 2 1 CICECO -<br />
Chemistry Department, University of Aveiro 2 IBB - <strong>Institute</strong> for Biotechnology and Bioengineering,<br />
Centre of Biological Engineering, University of Minho<br />
Microbial Enhanced Oil Recovery (MEOR) is potentially useful to recover incremental oil from a reservoir<br />
beyond primary and secondary recovery operations. In this work we address the isolation and<br />
identification of microorganisms from Brazilian heavy oil samples capable of promoting the degradation of<br />
heavy oil fractions, in particular long-chain hydrocarbons. Crude oil samples obtained from four oil<br />
reservoirs were used for the isolation of microorganisms. Most of the isolated microorganisms were<br />
Pseudomonas and Bacillus strains. The growth of different microbial isolates was studied under both<br />
aerobic and anaerobic conditions at 40ºC, and various parameters were evaluated, such as nutritional<br />
composition of the medium, incubation time and paraffinic composition of the mixture aiming at the<br />
optimization of the parameters to allow a stimulation of the degradation of the heavy oil fractions. Results<br />
show that some strains display a capacity to degrade, aerobically and anaerobically, the large alkyl<br />
chains (C18<br />
+ ) and reduce the viscosity of hydrocarbon mixtures. In the anaerobic growth, the addition of<br />
nitrates allows the enhancement of the degradation of the heavy hydrocarbon fraction. The results<br />
gathered in this work suggest that consortia of the isolated microorganisms have interesting<br />
characteristics to be applied for MEOR.<br />
06#19<br />
Biocatalytic Conversion of Heavy Oil - Effects on Reservoir Recovery?<br />
Hans Kristian Kotlar 1 , S. Markussen 2 , A. Winnberg 2 1 Statoil ASA 2 Department of Biotechnology,<br />
SINTEF Materials and Chemistry<br />
2/3 of the world's extractable fossil fuels lay within the category of heavy to extra heavy oil. The world's<br />
average recovery rate from this type of oil reservoirs are at about 7%. Technologies that could boost<br />
these recoveries would have a tremendous economical interest. Today, different process technologies<br />
are developed to extract these oils, like steam assisted gravity drainage (SAGD), vapour extraction<br />
64
(VAPEX) and cold heavy oil extraction with sand (CHOPS). These are all high cost, high energy and high<br />
emission technologies and are also associated with other environmental concerns. Combinations of<br />
conventional methods with biotechnological processes represent a completely novel approach to<br />
overcome many of these obstacles. The present paper focuses on the use of extremophile<br />
microorganisms as in situ biocatalysts for conversion of heavy oils. Experimental set ups designed to<br />
mimic reservoir conditions, with special emphasis on the biocatalytic processes involved in reducing the<br />
viscosity of the heavy oil components, are given. From a production technological aspect, however, a<br />
major challenge is the control and the regulation of these in situ bio-processes in the oil reservoir. By use<br />
of the Statoil internal strain collection (the BTC collection) and also high throughput screening<br />
approaches, enrichment cultures have been assayed for catalytic activities to the heavy constituents of<br />
various heavy oil types. The BCT collection includes both Achaea and bacteria associated with oil<br />
reservoirs. Moreover, different other terrestrial samples associated with effects on hydrocarbons are part<br />
of the collection. Several different types of heavy oil have been tested. Depending on the type of oil, the<br />
viscosities have been reduced 2 to 3 times. At reservoir-like conditions this has given between 2 to 3 fold<br />
increases in recovery. Various types of extremophile microorganisms have been found to be involved in<br />
the bioconversion and the reactions are tested at 55-60°C. Careful characterization of the influence of the<br />
bioconversion on the heavy oil constituents have been investigated by MS-QTOF in a petroleomics<br />
approach.<br />
06#20<br />
Bacillus Cereus, a Choice for Upgrading Vacuum Distillation Residue<br />
Mitra Sadat Tabatabaee 1 , M. Mazaheri Assadi 2 1 Islamic Azad University/ Central Tehran<br />
branch/Faculty of Science 2 Iranian Research Organization for Science and<br />
Technology/biotechnology section<br />
Vacuum distillation residue, the heaviest structure of crude oil is a net sample of problematic components<br />
of crudes in refinery. Biological processing of it may propose an alternative or complementary process to<br />
upgrade heavy fractions of crude oil or biorefine it. A sample of VR-contaminated soil, from Tehran<br />
refinery distillation unit, was enriched for a month with VR in a minimal salt medium to screen the bacteria<br />
able to use VR as the sole source of carbon. The selected organism was studied for its ability to utilize<br />
paraffin and antheracene; and also VR as both sources of carbon and sulfur. It’s capability in producing<br />
biosurfactant and emulsification was studied. Finally its ability to use VR as lonely source of nutrition in<br />
saline was investigated. The VR structure after bacterial treatment was measured with SARA test. The<br />
physicochemical endurance of the bacterium has also been investigated. The selected bacterium was<br />
identified molecularly. The isolate was a biosurfactant producing bacillus cereus closely related to the<br />
strain 03BB102 which was able to grow in a wide range of pH from 5 to 11, salinity up to 3.5% and<br />
temperature from 20°C to 45°C. It used Paraffin as long chain alkenes and anthracene as a sample of<br />
polycyclic aromatic hydrocarbon. It particularly utilized VR as its sole source of nutrition and consequently<br />
degraded it. It could decrease the asphaltene content of the VR up to 53.4%. Remarkable abilities of this<br />
microorganism, proposed the possibility of its application to upgrade Heavy distillates.<br />
06#21<br />
Study of Complex Microbial Nutrient and Its Effects on EOR<br />
Qing-xian Feng 1 , R-w. Zhou 1 , T. Ma 2 , Z. Zhang 3 . H-i. Cheng 1 1 Dagang Oilfield, Petrochina 2 Nankai<br />
University,China 3 China University of Petroleum,Beijing<br />
In our previous filed, we found that nutrient solution lost due to presence of thief zone, which led to a<br />
incompletely utilized by indigenous microbe????. As a result, the bacteria active was weaker and oil<br />
recovery was less. In this study, novel multifunctions microbial nutrients were developed to provide both<br />
profile control and recovery agent. The nutrients consist of three kinds of cellulose components which are<br />
by-product from agriculture. Experiment results under reservoir temperature (58℃) with produced water<br />
containing indigenous bacteria and crude oil from target oilfield showed the nutrient (1) delays release of<br />
component from 3 to 5 months; stimulates bacteria growth quickly and the peak bacteria concentration<br />
65
(1000 million cells/ml) shortened 2 days; (2) produces biogas around 232ml per gram nutrient;<br />
rhamnoilpid is main biosurfactant with concentration 1650ppm, (3) reaches 12.9% recovery enhanced, at<br />
water cut 90% by sand pack flooding test with its complex effects from microbes and fluid diversion to low<br />
permeation area. The dilatability, suspensibility and injectivity of the nutrients were increased. The pilot<br />
flooding test was carried out in 1 block with 4 injectors and 7 producers (reservoir temperature are 52°C<br />
and 60°C individually) from March 2008 to now. The nutrient solution, 175 tons, was injected first,<br />
followed by air monthly with 82000 cubic meters. The monitor result shows bio-parameters and reservoir<br />
environment was modified, water injection pressure was increased 2.2MPa, and 15075 bbl of oil was<br />
increased up to now.<br />
06#22<br />
Structure of Subsurface Microbial Communities and Activities Related to Carbon Capture and<br />
Storage<br />
Sylvain Bordenave, Y. Folarin, G. Voordouw University of Calgary<br />
Carbon capture and storage (CCS) in the subsurface is a novel and promising technology. The role of<br />
microorganisms in CO2 storage is still unknown. If H2 were to diffuse into geological layers where CO2 is<br />
stored, microorganisms can act by converting CO2 to acetate and water or to methane and water. In order<br />
to predict which of these processes may occur it is important to understand the types of microorganisms<br />
present at CCS sites and their metabolic capacities. Samples have been obtained from different potential<br />
or actual subsurface CO2 injection sites and analyzed for their microbial CO2 fixation or transformation<br />
activities with a focus on homoacetogenic bacteria and methanogens. Physiological analyses have been<br />
conducted with laboratory enrichment cultures. High acetogenic activity and low hydrogenotrophic and<br />
acetotrophic methanogenic activities were observed in waters from an oil field subjected to water<br />
injection. Waters from an oil field subjected to both water and CO2 injection for oil production gave<br />
comparable acetogenic activity, but no hydrogenotrophic and acetotrophic methanogenic activities.<br />
Community analyses to try to understand these differences are in progress. Overall these data will<br />
uncover the roles of these microbial activities in CCS and will fill a gap in knowledge on the influence of<br />
microorganisms on CO2 injected into the subsurface.<br />
06#23<br />
Oil Recovery in Granular Systems by a Mixed Culture Isolated from Mexican Reservoir<br />
Gladys Castorena-Cortés, I. Zapata-Peñasco, T. Roldán-Carrillo, J. Reyes-Avila, M. Mayol-Castillo,<br />
P. Olguín-Lora Instituto Mexicano del Petróleo<br />
Mexico is one of the most important oil producers and their main oil reserves are in carbonate reservoirs,<br />
which turn into a complex process for the remaining oil extraction. Enhanced recovery process such as<br />
microbial enhanced oil recovery (MEOR), could be useful for oil extraction of this type of reservoir. The<br />
application feasibility of MEOR depends on adequate understanding of the system components: oil,<br />
microorganism, and porous media. Oil samples were collected from a carbonate oil reservoir in Cordoba<br />
Platform, Veracruz, Mexico. Geochemical analysis of the oil samples presented: API gravity 14.5-10.7°,<br />
sulfur content 3-5 %, aromatic hydrocarbons 36.58-54.06%, polar products (resins plus asphaltenes)<br />
>43%, and the δ 13 C isotopic values in total oil were -23.98 to -24.24 (0/00). Anaerobe, thermophile,<br />
halotolerant and fermentative enrichment cultures were obtained from the oil samples. Metabolites as<br />
CO2, CH4, ethanol, aceto ne, acetate and biosurfactants were detected. The selected culture grew<br />
between 50-80°C, 5-35 g L -1 NaCl and 0.8-145 kg cm -2 pressure. Analysis of V3 region 16S rRNA gene of<br />
the culture showed that the predominant taxon was Thermoanaerobacter. Phylogenetic approximations<br />
demonstrated that similitude percentage of 99.9% corresponded to T. ethanolicus, 99.6% to T.<br />
pseudethanolicus and 98.9% to T. brockii and T. finii. The feasibility of mixed culture for MEOR<br />
application was evaluated in oil-impregnated granular porous media. Culture enhanced recovery up to<br />
12% of a heavy oil (11.16 oAPI) in oil impregnated carbonate granular porous media; production of<br />
metabolites was detected in the inoculated system, while in the control only 0.4% recovery was obtained.<br />
66
06#24<br />
Activation of Stratal Microflora and Physical Simulation Experiments of Microbial Displacement<br />
Lin-xin Huang 1 , L. Yu 1 , C-g. Zheng 2 , J. Wang 2 1 Research <strong>Institute</strong> of Petroleum exploitation and<br />
development (Lang fang), China National Petroleum Corporation 2 Tianjin <strong>Institute</strong> of Industrial<br />
Biotechnology, PR China<br />
Activation of stratal microflora recovery (ASMR) is a novel technology which directly employs the existing<br />
indigenous microorganisms in oil reservoirs for the purpose of enhancing oil recovery and the technology<br />
exhibits a series of advantages, including perfect reservoir adaptability, environmental friendship and et<br />
al. Many pilot field tests have been carried out in oil reservoirs in China and Russia in recent years and<br />
gained significant increase in oil recovery. In order to investigate the activation of indigenous microbial<br />
characteristics and oil displacement mechanisms, physical simulation experiments were employed under<br />
aerobic and anaerobic conditions in our laboratory study. The results showed that metabolic rates of<br />
microorganisms in porous media were much lower than those cultivated in the shake flasks. Oxygen<br />
introduction can further improve the microbial activities and in turn enhance the total oil displacement<br />
efficiency. An enhanced oil recovery (EOR) of 8.65% was achieved without oxygen introduction while the<br />
recovery was 14.53% with air supplement after the water-flooding. Therefore, it can be concluded that air<br />
supplement was essential for in-situ ASMR applications.<br />
06#25<br />
A potent Biosurfactant-Producing Bacterium, Pseudomonas Aeruginosa MA01 Isolated from<br />
Spoiled Apples: Preliminary Rheological Characteristics of Isolated Biosurfactant<br />
Kambiz Akbari Noghabi 1 , H. Abbasi 2 , M.M. Hamedi 2 , H. Shahbani Zahiri 1 , H. Sharafi 1 , N.<br />
Masoudzadeh 1 1 National <strong>Institute</strong> of Genetic Engineering and Biotechnology (NIGEB) 2 Department<br />
of Food Science and Engineering, College of Agriculture and Natural Resources, University of<br />
Tehran<br />
An extensive investigation was conducted to isolate indigenous bacterial strains with outstanding<br />
performance for biosurfactant production from different types of spoiled fruits, food-related products and<br />
food processing industries. Three isolates were selected from 800 by the highest biosurfactant yield in<br />
soybean oil medium and one of them was identified by 16S rRNA and the two most relevant<br />
hypervariable regions of this gene; V3 and V6 as Pseudomonas aeruginosa MA01. The isolate was able<br />
to produce 12 g/l of a glycolipid-type biosurfactant and generally less efficient to emulsify vegetable oils<br />
compared to hydrocarbons and could emulsify corn and coconut oils more than 50%. However,<br />
emulsification index (E24) of different hydrocarbons including hexan, toluene, xylene, brake oil, kerosene<br />
and hexadecane was between 55.8 - 100%. The surface tension of pure water decreased gradually with<br />
increasing biosurfactant concentration to 32.5 mN m -1 with CMC value of 34 mg/l. Among all carbon<br />
substrates examined, vegetable oils were the most effective on biosurfactant production. The strain<br />
sweep experiment for measuring the linear viscoelastic of biosurfactant showed that typical behavior<br />
characteristics of a weak viscoelastic gel, with G' (storage modulus) greater than G'' (loss modulus) at all<br />
frequencies examined, both showing some frequency dependence. Results also showed that the MA01<br />
biosurfactant has shear thinning performance. A decrease in viscosity with respect to the time at constant<br />
shear rate demonstrating the thixotropic behavior of biosurfactant. MA01 biosurfactant can be desirable<br />
candidate for potential applications in food and cosmetic industries.<br />
67
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