04.02.2013 Views

ISMOS-3 Abstract Book - Danish Technological Institute

ISMOS-3 Abstract Book - Danish Technological Institute

ISMOS-3 Abstract Book - Danish Technological Institute

SHOW MORE
SHOW LESS

You also want an ePaper? Increase the reach of your titles

YUMPU automatically turns print PDFs into web optimized ePapers that Google loves.

<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


Microbiology Services for the Oil and Gas Industry<br />

Organizers<br />

Sponsors<br />

Platinum<br />

Gold<br />

Silver

Hooray! Your file is uploaded and ready to be published.

Saved successfully!

Ooh no, something went wrong!