5th International Symposium on Applied Microbiology and Molecular

Biology in Oil Systems (ISMOS-5)

ISMOS-5 Abstract Book

2-5th June 2015

Symposium & Poster Sessions

www.ISMOS-5.org

Editors: Catherine Boccadoro, Dominique Durand,

Corinne Whitby & Torben Lund Skovhus

All rights of this document belong to ISMOS TSC.

The document can be cited if ISMOS is mentioned by name and webpage and only for non-profit purpose.

TABLE OF CONTENTS

Program Overview .................................................................................................................................................... i

Detailed Program .................................................................................................................................................... iii

Welcome ................................................................................................................................................................ vii

Keynote .................................................................................................................................................................... 1

Abstracts .................................................................................................................................................................. 3

List of Authors........................................................................................................................................................ 61

i

Program Overview

Tue 2 June Wed 3 June Thur 4 June Fri 5 June

8am

08:30

9am Session 2 - MEOR Session 4 - Microbial biotransformations09:15

09:40

09:50

10am

10:10

10:30

11am 11:00

Session 6-Microbiology,Modelling in the O&G

11:20

11:40

12pm

12:00

1pm

13:20 Session 3 - Hydrocarbon biodegradation Session 5 - MIC, Souring of the North Sea

13:40

13:50

14:00

2pm

14:20

14:30

3pm 15:00

15:20

15:40

4pm 16:00

5pm 17:00

Session 1 - Microbiology in Arctic Oil & Gas

17:30

6pm 18:00

18:20

18:40

7pm 19:00

Coffee Break (10:30 am - 11:30 am)

Poster Session

Lunch (12:20 am - 1:20 pm)

Registration

(from 2 pm)

Welcome & Opening

Panel Debate Industry/Academia

Invited Speaker 6- IAN GATES (Univ. Calgary)

Speaker 2 - Annie An (Univ. Calgary)

Speaker 1 - Navreet Suri (Univ. Calgary) Speaker 1 - Gerrit Voordouw (Univ. Calgary)

Speaker 2 - Martin Krueger (BGS) Speaker 2 - Nicolas Tsesmetzis (Shell International

Exploration and Production)

Coffee Break (10:30 am - 11:00 am) Coffee Break (10:30 am - 11:00 am)

Speaker 1 - Jan Sunner (Univ. Oklahoma)

Speaker 3 - Vincent Bonifay (Oklahoma University)

Speaker 4 - Lei Zhu (Univ. Alberta)

Speaker 5 - Yiwei Cheng (Lawrence Berkeley

National Laboratory)

Registration (8:30am - 9:15am) Registration (8:30am - 9:15am) Registration (8:30am - 9:15am)

Invited Speaker - BRIAN CLEMENT (GLORI OIL)Invited Speaker - HANS KRISTIAN KOTLAR

(Consultant)

Speaker 2 - Anna Engelbrekston (Univ. California,

Berkeley)

Coffee Break (2:30 pm - 3 pm) Coffee Break (2:30 pm - 3 pm)

Speaker 3 - Daisuke Mayumi (AIST)

Speaker 4 - Anders Schouw (Univ. Bergen)

Speaker 5 - Ting Ma (Nankai University)

Speaker 3 - Núria Jiménez (BGS)

Speaker 4 - Daphne Jalique (Univ. Saskatchewan)

Speaker 5 - Johanna Schritter (Univ. Natural

Resources and life Sciences Vienna)

Lunch (12 am - 1:20 pm) Lunch (12 am - 1:20 pm)

Speaker 5 - Xiaoke Hu (Chinese Academy of

Sciences)

Speaker 5 - Hans Carlson (Univ. California)

Invited Speaker -AMY CALLAGHAN (University of

Oklahoma)Invited Speaker - RICHARD ECKERT (DNV GL)

Speaker 1 - Robert Duran (Pau University) Speaker 1 - Irene Roalkvam (Univ. Bergen)

Speaker 2 - Florin Musat (Helmholtz Centre for

Environmental Research)

Close of meetingSpeaker 3 - Christopher Marks (Univ. Oklahoma) Speaker 3 - Mark Conrad (Lawrence Berkeley)

ISMOS 6 announcement & venue

End of Symposium and DepartSpeaker 4 - Angela Sherry (Newcastle University) Speaker 4 - Dennis Enning (ExxonMobil)

End of Session

Conference Dinner

(Registration required) Icebreaker

(Buffet tapas)

Speaker 3- Odd Gunner Brakstad (SINTEF

Materials and Chemistry)

Poster Session (4 pm- 5 pm)

End of Session End of Session

Poster Session (4 pm- 5 pm)

Starting 5:30 pm

Social-event

(Registration required)

Visit to the Oil Museum and Dinner

Speaker 2- Kate Boccadaro (IRIS)

Speaker 1- Brett Geissler (Nalco Champion)

Invited Speaker - TIM KLETT (USGS)

PLENARY TALK

MARC STROUS

(University of Calgary)

Coffee Break (5 pm - 5:30 pm)

ii

iii

Detailed Program

Tuesday June 2

14:00 Registration 15:40 Welcome & Opening

Key Note Adddress 16:00 Marc Strous (University of Calgary) The

biogeochemical element cycles – a microbial pinball machine?

17:00 Coffee Break

S1 Microbiology in Arctic Oil & Gas Chairs: T. L. Sovhus & M. Lutterbach 17:30 Tim Klett (USGS) Assessment of Undiscovered

Oil and Gas in the Arctic 18:00 Brett Geissler (Nalco Champion) Microbial

Population in an Arctic oilfield 18:20 Kate Boccadoro (IRIS) Arctic seawater and sea-

ice microbial populations and their response to crude oil pollution

18:40 Odd Gunnar Brakstad (SINTEF Materials and Chemistry) Bacterial communities involved in oil biodegradation in cold marine environments

19:00 Icebreaker – Poster Session & Buffet tapas

iv

Wednesday June 3 08:30 Registration

S2 MEOR Chairs: R. de Paula & M. Lutterbach 09:20 Brian Clement (Glori Energy) Enhancing Oil

Recovery in Waterflooded Oil fields with a Commercial, Microbial Process

09:50 Navreet Suri (University of Calgary) Towards understanding mechanisms involved in nitrate-mediated, microbially-enhanced recovery of light oils

10:10 Martin Krueger (BGR) Characterization of indigenous oil field microorganisms for microbially enhanced oil recovery (MEOR)

10:30 Coffee Break 11:00 Daisuke Mayumi (AIST) Insight into in situ

methanogenic crude oil degradation in an oil reservoir assessed by geochemical and microbiological analyses

11:20 Anders Schouw (University of Bergen) Isolation and characterization of a novel, syntrophic alkane degrading bacterium from the Loki’s castle hydrothermal vent field

11:40 Ting Ma (Nankai University) Microbial abundance and community composition during microbial flooding in a low-temperature petroleum reservoir

12:00 Lunch

S3 Hydrocarbon Biodegradation Chairs: C. Whitby & S. Caffrey 13:20 Amy Callaghan (University of Oklahoma)

Alkylsuccinate Synthase: From Desulfatibacillum alkenivorans AK-01 to the Bottom of the Sea

13:50 Robert Duran (Pau University) Understanding microbial processes involved in hydrocarbon degradation by experimental ecology

14:10 Florin Musat (UFZ) Anaerobic degradation of gaseous alkanes by sulfate-reducing bacteria from marine gas and oil cold seeps

14:30 Coffee Break 15:00 Christopher Marks (University of Oklahoma)

Metagenome and transcript analyses of a methanogenic consortium utilizing n-octacosane

15:20 Angela Sherry (Newcastle University) Effects of micronutrients on microbial diversity and hydrocarbon biodegradation under sulphate-reducing conditions

15:40 Xiaoke Hu (Chinese Academy of Science) Bioremediation of Crude Oil by Indigenous Marine Bacteria

16:00 Poster Session 17:00 End of Session 17:30 Social event – Visit to the Oil Museum (Bus

departure from Clarion Energy at 17:00) 19:00 Dinner at Bølgen & Moi 21:30 Bus departure back to Clarion Energy

v

Thursday June 4 08:30 Registration

S4 Microbial Biotransformations Chairs: I. Head & C. Boccadoro 09:20 Hans Kristian Kotlar (Statoil) Harvesting

subsurface and oil related microbial communities for industrial use – a multidisciplinary task

09:50 Gerrit Voordouw (University of Calgary) Strategies for viscosity reduction through microbial metabolism of bitumen and heavy oil

10:10 Nicolas Tsesmetzis (Shell Int. Exploration and Production) Microbial profiling insights from an offshore oil field in South East Asia

10:30 Coffee Break 11:00 Núria Jiménez (BGR) Effects of hydraulic frac

fluids and formation waters on groundwater microbial communities

11:20 Daphne Jalique (University of Saskatchewan) Investigation of the bacterial population in a bentonite plug mimicking well cements

11:40 Joanna Schritter (Univ. of Natural Resources and Life Sciences) Geobatteries: is there a limitation to adding hydrogen to porous natural gas storages?

12:00 Lunch

S5 MIC, Souring of the North Sea and beyond

Chairs: T. Mitchell & C. Hubert 13:20 Richard Eckert (DNV GL) Advances in the

application of molecular microbiological methods in the oil and gas industry and links to microbiologically influenced corrosion

13:50 Irene Roalkvam (University of Bergen) Isolation, characterization and genome analysis of nitrate reducing Arcobacter sp. isolated from saline aquifer water

14:10 Annie An (University of Calgary) Nitrate-mediated souring control in a low temperature, saline shale oil reservoir

14:30 Coffee Break 15:00 Mark Conrad (Lawrence Berkeley National

Laboratory) Preliminary Field Results of Stable Isotope Monitoring of Reservoir Souring

15:20 Dennis Enning (Exxon Mobil) Comparing the effects of THPS and glutaraldehyde batch biocide treatment on microbial corrosion in circulating flow loops

15:40 Hans Carlson (University of California) Expanding the search for biosouring treatements: High-throughput identification of potent and specific inhibitors of sulfate-reduction

16:00 Poster Session 17:00 End of Session 19:30 Conference Dinner at Solastrand Hotel (Bus

departure from Clarion Energy at 18:45) 23:00 Bus departure back to Clarion Energy

vi

Friday June 5 08:30 Registration

09:15 Panel Debate Industry/Academia 10:30 Coffee Break & Poster Session

S6 Microbiology and Modelling in the Oil and Gas Sector

Chairs: D. Enning & T. L. Skovhus 11:30 Ian D. Gates (University of Calgary) Modelling

of Microbial Biodegradation Processes in Hetrogeneous Oil Reservoirs

12:00 Jan Sunner (University of Oklahoma) Mass Spectrometry-based Metabolomics – A Tool to Guide MIC Management in the Field

12:20 Lunch 13:20 Anna Engelbrektson (University of California)

Attenuating sulfidogenesis in a soured continuous flow column system with perchlorate treatment

13:40 Vincent Bonifay (University of Oklahoma) Metabolomic and Metagenomic Analyses of Crude Oil Production Pipelines Experiencing Differential Rates of Corrosion

14:00 Lei Zhu (University of Alberta) Microbial community analysis of a novel biological process for treating oil sands process-affected water

14:20 Ywei Cheng (Lawrence Berkeley National Lab) Next generation modeling of microbial souring – incorporating genomic information and greater complexity

Meeting Close 14:40 ISMOS-5 Summary 15:00 ISMOS-6 announcement & venue 15:30 End of Symposium

vii

Welcome to ISMOS-5, Stavanger, Norway!

The 5th International Symposium on applied Microbiology and molecular biology in Oil Systems

(ISMOS) is taking place in Stavanger, the petroleum capital of Norway, and locally organized by

The International Research Institute of Stavanger - IRIS.

The aims of ISMOS are to present the latest research on molecular biology of microbes in oil

systems in order to resolve potential challenges (e.g. biocorrosion) and encourage beneficial

activities (e.g. MEOR, hydrocarbon biodegradation for bioremediation). We are also pleased

to introduce two new sessions as part of ISMOS-5, respectively dedicated to ‘Microbiology

related to Arctic Oil and Gas’ and ‘Microbiology and Modelling in the Oil and Gas Sector’.

The symposium is multidisciplinary, linking biogeochemists, engineers, molecular biologists and

microbiologists. It will include a mixture of high profile international speakers from both

industry and academia and a panel debate, during which PhD students and early career

researchers will be encouraged to develop a dialog with a panel of representatives from industry

and academia who work in the oil and gas sector.

A number of social events is proposed to the delegates, i.e., the symposium Gala dinner at the

SolaStrand Beach Hotel, a visit to a Norwegian Petroleum Museum followed by a dinner, a cruise

on the Lysefjord and an excursion to the famous Pulpit Rock.

We are very grateful to the Technical & Scientific Committee (TSC) and the Local Organising

Committee (LOC) for their organisation and support for this conference. We also thank the

sponsors and supporters (Microbial Insights, IRIS, NECE Ltd, NCIMB, Dow Microbial Control,

Nalco Champion, Genome Alberta, The municipality of Stavanger, VIA University College, The

University of Essex and Statoil) for their support to ISMOS-5.

It is a great pleasure for the technical and Scientific Committee (TSC) and the local organizer

(IRIS) to welcome you all to ISMOS-5 in Stavanger!

Welcome to Stavanger and ISMOS-5!

Dr.Torben Lund Skovhus, VIA University College - ISMOS TSC Chair

Dr. Corinne Whitby, University of Essex - ISMOS TSC Vice Chair

Dr. Kate Boccadoro, IRIS - ISMOS-5 LOC Chair

Dr. Dominique Durand, IRIS - ISMOS-5 LOC Vice Chair

viii

General questions regarding the technical content of ISMOS

please contact

For questions regarding registration and local issues, please contact:

Henrik Dovre

Phone: +47 905 93 275

E-mail: kongress@dovre.as

Catherine Boccadoro

IRIS AS

Phone: +47 473 31 738

E-mail: cbo@iris.no

Keynote

1

Keynote The biogeochemical element cycles – a microbial pinball machine? Marc Strous University of Calgary The natural cycles of the elements have been textbook knowledge since decades. In isolation, each of these cycles tells a simple story. Together, they give rise to complex microbial communities with unpredictable outcomes. Do organizing principles exist that provide predictive understanding of, for example, well souring, nitrous oxide emissions, corrosion, etc.? In the course of the nineteenth century, a simple organizing principle emerged that is still the key to modelling natural and engineered ecosystems. This theorem, known as the microbial redox “tower”, “ladder” or “cascade”, holds that a succession (in space or time) of ecological guilds consuming electron acceptors in a thermodynamically determined order. But evidence has emerged that microbial communities do not “strictly” abide to the redox tower. In this presentation I will provide a fresh perspective on the rules of engagement for the microbial pinball machine, and on approaches to improve predictability.

Session 1 – Microbiology related to Arctic Oil and Gas

3

Abstracts

Session 1 - Microbiology related to Arctic Oil and Gas

Sessions Chairs: Torben Lund Skovhus and Marcia Lutterbach

Invited Talk:

Assessment of Undiscovered Oil and Gas in the Arctic Tim Klett U.S. Geological Survey, National and Global Assessment Project Team Among the greatest uncertainties in future energy supply and a subject of considerable environmental concern is the amount of oil and gas yet to be found in the Arctic. By using a probabilistic geology-based methodology, the United States Geological Survey has assessed the area north of the Arctic Circle and concluded that about 30% of the world’s undiscovered gas and 13% of the world’s undiscovered oil may be found there, mostly offshore under less than 500 meters of water. Undiscovered natural gas is three times more abundant than oil in the Arctic and is largely concentrated in the Russia portion above the Arctic Circle. Oil resources, although important to the interests of the respective countries, are probably not sufficient to substantially shift the current geographic pattern of world oil production. The potential for resource development is of increasing concern to the Arctic nations, petroleum companies, and to all concerned about the region’s fragile environments. These concerns have been heightened by the recent retreat of polar ice, which is changing ecosystems and improving the prospect of easier petroleum exploration and development. Limited exploration opportunities elsewhere in the world combined with technological advances make the Arctic increasingly attractive for development. To provide a perspective on the oil and gas resource potential of the region, the U.S. Geological Survey (USGS) completed a geologically based assessment of the Arctic, the Circum-Arctic Resource Appraisal (CARA), which exists entirely in the public domain. Of the 6% of Earth’s surface encompassed by the Arctic Circle, one-third is above sea level and another third is located on continental shelves beneath less than 500 m of water. The remainder consists of deep ocean basins historically covered by sea ice. Many onshore areas have already been explored; by 2007, more than 400 oil and gas fields, containing 40 billion barrels of oil (BBO), 1,136 trillion cubic feet (TCF) of natural gas, and 8 billion barrels of natural gas liquids had been developed north of the Arctic Circle, mostly in the West Siberian Basin of Russia and on the North Slope of Alaska.

Offered Talks: Microbial Population Dynamism in an Arctic oilfield Brett Geissler, Carrie Keller-Schultz, Zach Broussard, Jesus Barron-Aldana,Victor Keasler Nalco Champion, an Ecolab Company Culture-based techniques and some molecular microbiological methods of species identification have been used with some success in the oilfield. However, the decreasing technical limitations, enhanced sensitivity, and lower cost of high-volume DNA sequencing has enabled previously uncharacterized microbial populations to be studied in greater depths. Determining the specific types of bacteria and archaea present can improve the overall understanding of the microbial threats to the system and allow for tailored monitoring programs to be employed.For this study, data collected from denaturing gradient gel electrophoresis and next-generation DNA sequencing were used to survey the phylogenetic diversity within several fluid and surface sites within an oil production facility on the Alaskan North Slope in the Arctic Ocean over the course of 5 years. The results show that, while a multitude of genera encompassing several metabolic classes or bacteria and archaea were identified from these samples and that several changes to the production system occurred during the study, the overall make-up of the microbial population

Session 1 – Microbiology related to Arctic Oil and Gas

4

remained relatively unchanged over time. Because many of the microbes that persisted in these locations have also been found in neighboring assets and that these microbes have the potential to increase souring, MIC, and biofouling, continued monitoring of their incidence and abundance remains essential for the MIC management program at this facility. Arctic seawater and sea-ice microbial populations and their response to crude oil pollution Catherine Boccadoro1, Adriana Krolicka1, Mari Mæland1, Elin Austerheim1, Thierry Baussant1, Alan Le Tressoler2

1 IRIS, 2 Arctic Science Field Logistics The overall aim of this study was to investigate high Arctic seawater microbial communities and their immediate response to crude oil exposure. Pristine seawater samples were collected in Scoresbysund in Amdrup Havn, East-Greenland at different geographical locations over the iced period (April to June) and seawater crude oil exposures were set up in on-site mesocosms outdoor on the sea ice. Samples were collected at regular intervals to assess changes in bacterial abundance, composition and number of selected hydrocarbon degrading bacteria over a 3 week time period. These experiments were performed at two different conditions, during the Arctic winter at temperatures below -10°C and in early spring during the start of the sea-ice melting. An increase in the total number of organisms measured by qPCR was observed over time in both mesocosm set ups, as well as shifts in the bacterial community compositions, within days for both exposure temperatures. The ratio of Colwellia`s SSU rRNA gene copy to total number of bacterial SSU rRNA gene copy indicated an enrichment in these bacteria within 1-2 days in both experimental set-ups. Bacteria affiliated to Oleispira were also enriched following crude oil exposure in both experiments, but with a much faster response from these organisms at the higher temperatures. Interestingly, the obligate oil-degrading bacteria (OHCB) Alcanivorax and Cycloclasticus often found to be aboundant in oil polluted seawater sites were not significantly stimulated following these experimental exposures. Analysis of contribution of 16S RNA gene sequences in overall pool confirmed qPCR data indicating on rapid enrichment of Gammaproteobacteria affiliated to Oleispira, Colwellia, Glaciecola, Marinomonas and Marinobacter. Sequences assigned to Polaribacter were found to be abundant in both polluted and non-polluted samples. Bacterial communities involved in oil biodegradation in cold marine environments Odd Gunnar Brakstad1, Roman Netzer1, Synnøve Lofthus2, Anna Lewin1, Kristin Bonaunet1 1 SINTEF Materials and Chemistry, 2 Norwegian University of Science and Technology (NTNU) As the Arctic regions become of increasing interest for the oil E&P industry it is important to understand the fate processes related to oil spills in these environments. Dispersant treatment is an operational tool to treat oil spills and generates small-droplet dispersions and thereby enhances oil biodegradation in the marine water column. To study dispersed oil biodegradation and microbial communities involved in the degradation, we have developed a novel oil dispersion generator and carousel system. Studies in this system was performed with a crude oil (premixed with a dispersant) dispersed in natural seawater at 4-5°C. Comparison of GC-MS analyses and 16S rRNA gene amplicon sequencing (Illumina MiSeq) showed successions of Colwellia, Oleispira and Umboniibacter, which were associated with n-alkane degradation, followed by later abundances of Cycloclasticus and Marinospirillum during degradation of 2- to 4-ring aromatic hydrocarbon compounds. Closer examination of bacteria adhering to oil-seawater interfaces, using oil immobilized on hydrophobic adsorbents, showed that Oleispira was predominant on the oil surfaces during n-alkane biodegradation. Previous field studies of oil-contaminated marine ice (Svalbard) during a winter period (February-June) showed that Colwellia became the most abundant genus in the polluted ice. As the knowledge of cold-adapted bacterial communities and genes involved in biodegradation of specific oil compounds is increasing, microbial community and gene expression analyses will become important tools to determine the state of biodegradation and the restitution of the environment after an oil spill and during oil spill remediation actions in cold marine environments like the Arcticthe.

Session 1 – Microbiology related to Arctic Oil and Gas

5

Session 1 - Posters: P1. Analysis of bioremediation processes in oil contaminated shoreline sediments Roman Netzer, Svein Ramstad, Odd Gunnar Brakstad, Trond Størseth, Alexander Wentzel SINTEF Materials and Chemistry Accidental oil spills from petroleum production and transportation can cause damage to ocean and coastal areas. Oil spilled offshore often reaches shorelines where it can persist and have severe effects on the marine environment. Shoreline bioremediation has become a promising secondary method for the restoration of oil polluted areas. However, information about oil degradation processes during bioremediation in sediments is limited. We have used a lab-scale column system to mimic oil spill at sandy shorelines with rising and falling tide at 20°C. Biodegradation of crude oil was studied over a period of 64 days by biological and chemical methods, and the effect of biostimulation was evaluated. When additional nutrients (N and P) were added to the column system, the number of oil degrading microbes was increased by two orders of magnitude. The metabolic activity determined by O2 consumption and ATP concentrations was elevated 50- and 38-fold, respectively. Analysis of n-alkanes revealed 42% depletion without and 76% depletion with biostimulation within 64 days. 99 % of naphthalenes and 2-3 ring PAHs and 37 % of 4-6 ring PAHs were degraded under biostimulation conditions, whereas 82 % naphthalenes and 43 % 2-3 ring PAHs were biotransformed without biostimulation. No biodegradation of 4-6 ring PAHs was observed without biostimulation. Microbial community analysis by 16S rRNA amplicon sequencing revealed a high abundance of hydrocarbonoclastic bacteria over the entire experimental period in all oil contaminated samples. However, in biostimulated samples Alcanivoraceae, comprising well known n-alkane degraders, became rapidly predominant reaching a peak after 22 days. Rhodospirillaceae abundance increased significantly from day 35 while the abundance of Alcanivoraceae was declining. Rhodobacteriaceae and Piscirikettsiaceae were also among the dominating families and their abundance remained stable at levels as found in non-contaminated sediment. In non-stimulated samples Rhodobacteriaceae became predominating during the first 22 days, followed by a constant decline but still remaining in the group of dominating families. Multivariat statistical analysis compiling chemical data was used for mapping biodegradation courses under the studied conditions. Microbial community and multivariate analysis has been shown to be powerful tools for status assessment of bioremediation processes. P2. Bacterial DNA markers for crude oil pollution detection in cold seawater Adriana Krolicka, Mari Mæland Nilsen, Catherine Boccadoro, Thierry Baussant IRIS A crude oil contamination at sea causes changes in marine bacterial abundance and composition. Microorganisms that are initially undetectable in a pristine environment become prevalent following an exposure to petroleum compounds. The overarching goals of the study were 1) to examine through laboratory experiments the abundance and response time of selected bacterial DNA targets detectable in the early phases of petroleum exposure to surface and sub-surface sub-Arctic seawaters 2) to use the Environmental Sample Processor (ESP) developed by Monterey Bay Aquarium Research Institute (MBARI, CA, USA), for near-real time detection of these microbial DNA marker responses for field monitoring and surveillance of petroleum hydrocarbons. Here we present laboratory results obtained with crude oil in the range 30ppb – 2000ppb (nominal concentration) during an 11 days exposure. We also show the relationship between oil biodegradation and residual concentration and abundance of individual bacterial genus with the whole background microbial community. We selected microorganisms affiliated to obligate oil- degrading bacteria (OHCB) (Oleispira, Alcanivorax, Cycloclasticus, Marinomonas) as well as other possible targets within organisms often associated with crude oil degradation (Colwellia, Polaribacter and Glaciecola). Despite the low temperature (5°C) the ratios of C17/pristane and C18/ phytane decreased by 40 to 50 % within 4 days of incubation. After 11 days, these ratios were lower than 0.27, implying much of

Session 1 – Microbiology related to Arctic Oil and Gas

6

the compounds were being degraded. Changes in chemical composition coincided with major changes in total number of bacteria as well as in bacterial composition. The fastest response was obtained using an assay specific to Oleispira antarcitca. A significant response from these organisms was recorded within 24 hours. The abundance of microorganisms affiliated to obligate oil-degrading bacteria (OHCB) Oleispira, Alcanivorax, Marinomonas and Cycloclasticus positively correlated to the concentrations of residuals hydrocarbons. Colwellia did not show the linear dependence to oil concentration but OTU numbers affiliated to Colwellia was significantly higher in polluted seawater in all oil-polluted treatments. P3. Burrowing macrofauna stimulate PAH mineralization and bacterial community diversity in intertidal Arctic sediments Maria Granberg1, Ingela Dahllöf2

1 Norwegian Polar Institute, 2 University of Gothenburg, Sweden Bacteria mineralize polycyclic aromatic hydrocarbon (PAHs) in both sediments and soil. The efficiency is considered to depend on oxygen and nutrients availability, as well as PAH exposure history. The availability of these compounds is considered higher in surface sediments, which are thus perfunctory considered main PAH biodegradation sites. Burrowing macrofauna do however, moderate the distribution of solutes and particles through bioturbation (particle mixing and irrigation), thus affecting their availability to bacteria. Macrofauna-microbial interactions are therefore key to our understanding of, e.g. the intrinsic bioremediation capacity of natural marine sediment ecosystems. The Arctic region is one of the world’s least monitored and the functioning of marine sediments in relation to contaminants here is largely unexplored. Following climate change, this region is now facing new environmental threats associated with increasing natural resource exploitation. We investigated the PAH mineralization capacity if natural bacterial communities in intertidal Greenland sediments. The aim was to study the role of the commonly occurring burrowing polychaete Arenicola marina in regulating PAH distribution, bacterial community structure (BCS) and PAH mineralization potential (PAH-MP) in sediments. This information will help predicting environmental impact as well as unlock natural bioremediation potentials in relation to oil exposure in the Arctic. Sediments were collected at PAH contaminated and pristine sites, from surface, burrow, and bulk layers. Samples were incubated radio-respirometrically with 14C-pyrene to determine the bacterial PAH-MP. DNA based BCS was identified using PCR-DGGE on rpoB amplicons. Sediment oxygen demand, nitrification potential, nitrogen, carbon, and PAH content were determined. Surface sediment PAH concentrations were high (3,5 μg g DW-1) at the contaminated site and non-detectable at the pristine site. PAH concentrations in A. marina burrows were double compared to surface sediments, while bulk concentrations were moderate, indicating continuous bioturbation. Bacterial PAH-MP correlated with PAH exposure and were highest in surface and burrow sediments. BCS in burrows were distinct, resembling surface sediment communities more than those from the bulk. Consequently macrofaunal burrows were a functional extension of the surface at the contaminated site, with high bacterial PAH mineralization rates and higher bacterial diversity. Macrofaunal bioturbation strongly structured bacterial diversity and PAH mineralization in the sediments. P4. Microbial communities in oil and water components of reservoir fluids from two Brazilian petroleum reservoirs using metagenomics Isabel Natalia Sierra Garcia1, Ramses Capillla2, Eugenio V. Santos Neto2, Valeria Maia de Oliveira1 1University of Campinas, 2 PETROBRAS Oil reservoirs are considered poly-extreme habitats for microbial life. The interest in oil reservoir microbial communities lies in the impact of microbial activities on the oil quality, and its potential for industrial applications as Microbial Enhanced Oil Recover (MEOR) or the prospecting of thermostable biocatalysts enzymes. Several efforts using dependent or independent culture methods have been made to explore the microbial diversity inhabiting such subterranean environments. However, most of these studies have been performed using formation waters, ignoring that each component of the reservoir multiphasic fluid including crude oil, gases, and insoluble particles may act as an important habitat for the microbial growth.

Session 1 – Microbiology related to Arctic Oil and Gas

7

The present work aimed to study taxonomic and functional diversity of microorganisms from petroleum fluids in two Brazilian oil reservoirs using metagenomics. Total metagenomic DNA was extracted from both aqueous and oil phases and sequenced with paired-end 100pb libraries through Illumina HiSeq 2500 technology. A total amount of 89,442,648 reads were obtained for oil phases and 72,465,876 reads for the aqueous phase. Communities from oil and water samples were dominated by bacteria (28 phyla). Archaeal communities were composed by 5 phyla, including 9 classes in the most abundant phylum: Euryarchaeota. Functional genes involved with different stages of anaerobic degradation (peripheral and central pathways) were identified. A more detailed comparison of taxonomic and functional abundances using MG-RAST heatmaps grouped the samples differently. Taxonomy-based comparisons clustered oil samples next to each other, showing a distinct composition in the water component. Function-based comparisons showed differences according to the origin of the samples. Functional annotation have detected 4558 genes related to the KEGG category: xenobiotics biodegradation and metabolism and several enzymes related to hydrocarbon metabolism have been identified. Microbial community composition at functional and taxonomic level in each component of the reservoir fluids may reflect the relevance and potential of these communities present in the oil eservoirs.

Session 2 – MEOR

9

Session 2 - MEOR

Session chairs: Renato de Paula and Marcia Lutterbach

Invited Talk: Enhancing Oil Recovery in Waterflooded Oil Fields with a Commercial, Microbial Process Brian Clement Glori Energy, Inc. A holistic approach to enhanced oil recovery (EOR), the Activated Environment for Recovery of Oil (AERO™) process, successfully improved oil production from three different sandstone reservoirs in the USA and Canada using continuous, low-volume injection of a low-cost nutrient. Prior to deployment, nutrient formulations and application processes unique to each field were developed in a series of lab studies in order to stimulate growth of indigenous microbial populations on residual oil. When applied to the injection water in each field, the nutrient treatments improved oil production in as little as 8 weeks and increased production as much as 2.5-fold. In two of the projects, nutrient injection was continued over one year, providing sufficient data to resolve a reduced long-term decline rate. In the third project, nutrient injection was stopped after six months and the oil rate rapidly dropped back to the previous decline curve. The responses to AERO nutrient injection in these and other fields, as well as lab studies conducted on rock cores, point to a novel mechanism of oil mobilization that contrasts sharply with conventional reservoir models. We believe the primary mechanism is interfacial tension disruption due to microbial growth at the oil-water interface. While microbial growth does cause small changes in injection pressure, the low nutrient quantities utilized are only sufficient to stimulate microbial activity near the injection well-bores and the observed responses are not consistent with localized re-direction of water flow alone. Instead, the increases in production are observed in distant wells, months before the treated injection water arrives, and the response is reversed when the nutrient addition is stopped. Both features are more consistent with ongoing, dynamic alteration of oil mobility than local changes in flow direction. Overall, these observations demonstrate that stimulation of microbial processes in situ has great potential to enhance oil production and indicate that, in waterflooded formations, such microbial-driven effects are due to novel mechanism(s) we have yet to fully characterize.

Offered Talks: Towards understanding mechanisms involved in nitrate-mediated, microbially-enhanced recovery of light oils Navreet Suri, Fatma Gassara, Gerrit Voordouw University of Calgary Nitrate injection into oil reservoirs to prevent souring stimulates the growth of heterotrophic nitrate reducing bacteria (hNRB) and of sulfide-oxidizing NRB (soNRB), which couple reduction of nitrate to oxidation of oil organics and sulfide, respectively. In order to access water-insoluble oil hydrocarbons, hNRB need to establish either direct contact with the oil layer or need to produce biosurfactants to solubilize oil hydrocarbons. Cells attached to the oil layer can behave as adsorbed colloidal particles enhancing zone plugging, while biosurfactants can reduce oil-water interfacial tension (IFT). Both of these outcomes of hNRB growth, coupled with gas (N2 and CO2) production could contribute to enhanced oil recovery. The potential of these mechanisms was analyzed by using model sand-packed columns at low temperature and low pressure. Initial flooding of columns containing different light oils (viscosities ranging from 10-50 cP at 20°C) and a heavy oil (viscosity of 3400 cP at 20°C) produced 65-90% and 40-50% of oil respectively.

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Columns containing 10-35% of residual light oils and 50-60% of residual heavy oil were then further treated with an hNRB consortium and 80 mM nitrate for two weeks. No oil was produced from any of the oil columns during this incubation period, which resulted in 5-20 mM of nitrate reduction. This suggests that there was no significant effect of N2 and CO2 production by hNRB in increasing oil production. However, relative to an uninoculated control, 0-11% of additional light oils and 9-10% of additional heavy oil were recovered when flooding was resumed after the incubation period. Biomass formed within the columns may have resulted in plugging paths of least resistance in heavy oil columns diverting the flow towards oil-rich parts leading to more oil production. But this seems less likely to contribute to enhanced production of light oils, which is present as irregular patches of residual oil. Additional oil produced from residual light oil-containing columns is, therefore, likely due to attachment or biosurfactant production by hNRB and further studies are under way to define this mechanism. Characterization of indigenous oil field microorganisms for microbially enhanced oil recovery (MEOR) Martin Krueger1, Jana Sitte1, Eva Mahler2, Andrea Herold2, Nicole Klueglein2, Soujatya Mukherjee3, Hakan Alkan3 1 German Federal Institute for Geosciences and Natural Resources – BGR, 2 BASF SE, 3 Wintershall Holding GmbH Microbial activities and their resulting metabolites became a focus of attention for enhanced oil recovery (MEOR, microbial enhanced oil recovery) in the recent years. In order to develop a strategy for a MEOR application in a German oil field operated by Wintershall experiments were performed to investigate different sampling strategies and the microbial communities found in these samples. The objectives of this study were (1) to characterize the indigenous microbial communities, (2) to investigate the dependency of microbial activity/diversity on the different sampling strategies, and (3) to study the influence of the in situ pressure on bacterial growth and metabolite production. Fluids were sampled at the well head (surface) and in situ in approx. 785 m depth to collect uncontaminated production water directly from the reservoir horizon and under the in situ pressure of 31 bar (subsurface). In the lab the pressure was either released quickly or slowly to assess the sensitivity of microorganisms to rapid pressure changes. Quantitative PCR resulted in higher microbial cell numbers in the subsurface than in the surface sample. Biogenic CO2 and CH4 formation rates were determined under atmospheric and high pressure conditions in the original fluids, with highest rates found in the surface fluid. Interestingly, no methane was formed in the native fluid samples. While nitrate reduction was exclusively detected in the surface samples, sulfide formation also occurred in the subsurface fluids. Increased CO2 formation was measured after addition of a variety of substrates in the surface fluids, while only fructose and glucose showed a stimulating effect on CO2 production for the subsurface sample. Stable enrichment cultures were obtained in complex medium inoculated with the subsurface fluid, both under atmospheric and in situ pressure. Growth experiments with constant or changing pressure, and subsequent DGGE analysis of bacterial 16S rRNA genes, revealed that the pressure treatment did not affect the bacterial community composition. Our results show that bacteria in the enrichment culture can tolerate pressure changes between atmospheric and in situ reservoir pressure, which makes them promising candidates for further MEOR tests. Insight into in situ methanogenic crude oil degradation in an oil reservoir assessed by geochemical and microbiological analyses Daisuke Mayumi1, Satoshi Tamazawa1, Hideyuki Tamaki1, Haruo Maeda2, Tatsuki Wakayama2, Masayuki Ikarashi2, Koichi Nishikawa3, Hiroshi Oshibe3, Yoshikazu Shirai 3, Susumu Sakata1, Yoichi Kamagata1 1 AIST , 2 INPEX Corporation, 3 TOKYO GAS CO LTD Evidence for methanogenic crude oil degradation has been reported in several oil fields worldwide. The previous studies have shown the diverse microbial guilds involved in methanogenic crude oil degradation by enrichment culture method and molecular biological analyses. However, the evidence is still unclear for in situ methanogenic crude oil degradation in the oil reservoirs, roles of each microbe in the communities, and the metabolic pathways from hydrocarbons to methane. We investigated the signatures for in situ

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crude oil degradation in a high-temperature oil reservoir, Japan, by geochemical analyses (e.g. stable isotopic analysis of the reservoir gas sample and hydrocarbon composition analysis of crude oil). We found that carbon dioxide, propane and n-butane in the gas sample were enriched in 13C and n-alkanes were less abundant than i-alkanes in the crude oil sample, suggesting the potential for methanogenic crude oil degradation in the oil reservoir. We further conducted high-temperature and high-pressure incubation experiments mimicking the in situ oil reservoir condition (55°C, 5 MPa) in order to obtain methanogenic community responsible for crude oil degradation. During over 350 days of incubation using production water and crude oil collected from the oil field, a significant amount of methane (65 μmol/ml-crude oil) was produced. The subsequent subcultures also continued to produce methane. Stable isotope tracer experiment using [UL-13C]-toluene and change of hydrocarbon composition in crude oil during incubation indicated that the methane formed was mainly derived from alkylbenzenes such as toluene and xylenes. Molecular microbial community analysis revealed that acetoclastic methanogens dominated in archaeal community and uncultured groups including candidate class JS1 and WWE1 were predominant in bacterial community after the incubation. A functional key gene responsible for the first activation of alkylbenzenes (bssA) was also detected in this microbial community. These geochemical and microbiological analyses demonstrate that methanogenic crude oil degradation mediated by the microbial community occurs in the in situ high-temperature oil reservoir. Isolation and characterization of a novel, syntrophic alkane degrading bacterium from the Loki's castle hydrothermal vent field. Anders Schouw1, Gunhild Bødtker2, Ida Steen1, Runar Stokke1 1 University of Bergen , 2 Uni CIPR A methanogenic, oil degrading consortium was enriched from the wall of a hydrothermal chimney at the Loki's castle hydrothermal vent field on the Arctic Mid-Ocean Ridge. Molecular analysis showed that the consortium includes 6 bacterial strains and 3 strains of methanogenic archaea. Of the bacterial strains, 5 likely represent novel yet uncultivated species whereas the methanogens represent already described species. In laboratory cultures at 30°C, the consortium was shown to degrade higher hydrocarbons to methane gas. Strain L81T was isolated in pure culture from this consortium, and was shown to degrade <nC17 alkanes in syntrophy with a methanogenic archaeon under laboratory conditions. The strain was shown to produce a bio-emulsifyer, affecting the water solubility of crude oil. The emulsifier contributed to the release of substrate-bound crude oil, and the formation of a larger oil-water interface.The optimal growth was at 37°C in a marine salinity mineral medium, under strict anoxic conditions. A simple two-species syntrophic, hydrocarbon degrading consortium, like the one presented by L81T and a methanogen, represents a rare opportunity to study the syntrophic degradation of higher hydrocarbons to methane gas. The majority of hydrocarbon degrading consortia studied so far have been complex systems, where the exact role of each species is harder to determine. Growth experiments with L81T coupled with full genome sequencing, allows for a detailed study of the process in a defined system. Understanding the different processes involved in microbial hydrocarbon degradation is interesting, both from a commercial perspective, with regards to MEOR, and environmentally, with regards to the environments intrinsic capabilities to cope with hydrocarbon emissions. Cultivation based studies are hence important to improve our understanding of these processes. Microbial abundance and community composition during microbial flooding in a low-temperature petroleum reservoir Ting Ma Nankai University Indigenous microorganisms enhanced oil recovery (IMEOR) has been successfully applied in the petroleum industry, but the role of microorganisms remains poorly understood. Here, we conduct a whole IMEOR process for studying EOR mechanism in a Low-Temperature Petroleum Reservoir, China. Firstly, based on the nutrient deficiency in formation brines, improvement of gas production, stimulation of hydrocarbon-oxidizing bacteria

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(HOB), nitrate-reducing bacteria (NRB), methane-producing bacteria (MPB) and inhibition of sulfate-reducing bacteria (SRB), the injection nutrients were selected containing sources of carbon, nitrogen, phosphorus and nitrate to inhibit the growth and propagation SRB. The selected nutrients were also cost efficient, easy to store, easily accessible and without toxicity to environments. As results, oil emulsification decreased from 21-25 days to 10-15 days with the addition of molasses under aerobic conditions. According to the results of microbial stimulation and physical simulation of oil displacement in laboratory experiment, the selected nutrients contained: NaNO3, 7 g/L; Na5P3O10, 0.1 g/L, NH4Cl 3 g/L, and molasses 14g/L. Then, we investigated the relationship between microbial population dynamics and oil production performance during a water flooding process coupled with nutrient injection in the field trial. Samples were collected monthly over a two-year period. The microbial composition of samples was determined using 16S rRNA gene pyrosequencing and real-time quantitative polymerase chain reaction analyses. Our results indicated that the microbial community structure in each production well microhabitat was dramatically altered during flooding with eutrophic water. As well as an increase in the density of microorganisms, biosurfactant producers, such as Pseudomonas, Alcaligenes, Rhodococcus and Rhizobium, were detected in abundance. Furthermore, the density of these microorganisms was closely related to the incremental oil production. Oil emulsification and changes in the fluid-production profile were also observed. In addition, we found that microbial community structure was strongly correlated with environmental factors, such as water content and total nitrogen. These results suggest that injected nutrients increase the abundance of microorganisms, particularly biosurfactant producers. These bacteria and their metabolic products subsequently emulsify oil and alter fluid-production profiles to enhance oil recovery.

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Session 2 - Posters: P5. Screening of biosurfactant-producing halophilic bacteria from production water of a Brazilian oil terminal and their potential for application in MEOR Milene Gomes1, Elmer Erasmo Gonzales Limache1, Ana Paula De Melo Rodrigues1, Cynthia Canêdo Da Silva3, Maíra Paula De Sousa2, Erika Valoni2, Valeria Maia de Oliveira1 1 UNICAMP, 2 CENPES/ PETROBRAS, 3 Federal University of Viçosa (UFV) Petroleum is a major source of global energy and largely used in Brazil in diverse sectors, so in order to meet the global demand for energy, the increase in oil production is mandatory. The crude oil production from current resources enables recovery only 30% of the oil contained in the reservoir. The remaining 70% are inaccessible to recovery by conventional methods. Therefore, different techniques have been developed aiming at the recovery of residual oil from petroleum reservoirs. Among these, microbial enhanced oil recovery (MEOR) is a promising technique that employs microorganisms or their products, such as biosurfactants, to help the recovery of crude oil by. The biosurfactants are amphiphilic compounds produced on the surface or secreted from bacterial cells with the ability to reduce the surface tension (ST) and/or the interfacial tension (ITS), thus allowing the release of the oil retained in the pores of rocks by capillary forces. The aim of this work was to select and characterize biosurfactant-producing strains from a collection of halophilic bacteria isolated from production water of an oil terminal, for further evaluation in MEOR assays at lab scale. Seventy nine genetically different bacterial strains were first screened for the ability to produce biosurfactants in mineral medium with sucrose as the only carbon source. The culture supernatants were subjected to tensiometry analysis, oil spreading assay and emulsification test with four different types of oils (mineral, soybean, diesel and kerosene). Five strains showed significant ability for reduction of the surface tension (from 72 to 40 mN/m) and for emulsification (~70%). However, when these strains were grown under high concentrations of NaCl (9%), the reduction of the surface tension was around 52 mN/m. On the other hand, emulsification results were significantly higher (~76%). These results demonstrate an interesting biotechnological potential of the halophilic strains isolated from the production water. Experimental design and optimization of the growth conditions will be carried out in order to explore the full potential of such isolates in future MEOR assays. P6. Biosurfactant produced by Serratia sp for enhanced oil recovery Teresa Roldan, Patricia Olguin, Gladys Castorena Cortés, Leobardo Santiago Rosales Instituto Mexicano del Petróleo, Ingeniería de Recuperación Adicional Biosurfactants (BS) are amphiphilic molecules with surfactant properties produced by microorganisms. BS may change the surface tension (ST) and interfacial (IT) between fluids. Currently, BS have acquired great interest due to their advantages as biodegradability, low toxicity, and capability to work in drastic conditions (temperature, salinity and pH). These compounds have application in the petroleum, pharmaceutical, and food industry, and in bioremediation process. BS production is affecting by different factors as type of substrates, which can be new (not used) or wastes from industries, as waste cooking oil (WCO).The aim of this work was to evaluate BS production by IMP-X strain from soybean oil (SO, not used) and WCO, and also determinate the BS effect on crude oil recovery. IMP-X strain was isolated from a hydrocarbon-contaminated soil and identified by 16S rRNA as Serratia sp. This microorganism showed ability to produce BS using both SO or WCO as substrates with a ST of 28.3 mN/m. BS produced with these substrates was used to evaluate its effect on heavy crude oil recovery assay. The oil recovery experiment was carried out using Ottawa rock crushed and meshed to a particle size of 0.29-0.41 mm. The granular material was washed with solvent, dried under vacuum, and impregnated with heavy oil 14.7°API. In these systems were evaluated four variables: type of substrate for BS production (SO and WCO), BS concentration (900, 450, 225 mg/L), contact time (1, 12, 24 h) and incubation temperature (30, 50, 70°C), using Taguchi L9 experimental design. In each system was evaluated released oil. The assays were performed in triplicate and with its respective control. The best theoretical and experimental treatment was performed validation oil recovery. Of the four variables evaluated, the two most important for oil recovery process were contact

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time (12 h) and incubation temperature (70°C). The best BS was synthesized using WCO as substrate, with 900 mg/L and 70°C, had a greater oil recovery (95% at 9 h). This BS has an alternative potential application in the oil industry for recovery and mobilization of oil reservoirs. P7. Experimental investigation on microbial nutrients gel with core Qingxian Feng Dagang oilfield company, Petrochina Based chemical water soluble nutrients outflow and delay time short, indigenous microbes and nutrients could not reach deep of oil reservoir, microbial flooding recovery oil is not as good as polymer flooding. We create a novel microbial nutrient gel system with core. It combined chemical profile modification and microbial flooding. It promote indigenous microbe grow and metabolite highly with the gel nutrients, improve oil and water interfacial properties and enhance residual oil mobility. The system composed of nutrients, polymer, crosslinker and modificated fiber. We did experiments at 60 degrees centigrade with Dagang oilfield reservoir water (TDS is6609 mg/L) and oil (viscosity is 45 mPa.s) to evaluate the system including fiber suspension in the gel system, crosslink system optimization, nutrients and microbes influence on gel properties, compositions in gel toxicity to bacteria, gel strength and crosslink time, also dynamic gelling in the sandpacked at experimental temperature. The results show 1) optimum nutrients gel formula is 0.25% polymer, 0.03% chromium acetate, 1%modificated fiber and 0.25%complex nutrient (four kinds nutrients), 2) the system can enhanced oil recovery 16.2% higher than water flooding at same condition, 3) checked microbe number in output fluid at end of sand packed, the concentration of microbe increased 2 orders, 4) the system can gel in the sandpacked, but gel time delay 40% than static does, the gel strength is stronger than polymer gel due to contain modificated fiber which benefit to gel. It will contribute on mature oilfield and delay economic limit.

P8. Novel bioemulsifier produced by a Paenibacilus sp. strain and its applicability in microbial enhanced oil recovery Eduardo Gudiña, Lígia Rodrigues, José Teixeira University of Minho, Braga, Portugal Microbial Enhanced Oil Recovery (MEOR) is potentially useful to increment oil recovery from reservoirs beyond primary and secondary recovery operations using microorganisms and their metabolites. In situ stimulation of microorganisms that produce surface active compounds reduces the capillary forces that retain the oil inside the reservoir, thus promoting its flow and increasing oil production. Paenibacillus sp. #510, isolated from crude oil samples obtained from a Brazilian oil field, produced a bioemulsifier in a mineral medium containing sucrose as the carbon source under aerobic and anaerobic conditions, and its production was induced (up to 7.9 g/l) by the addition of paraffin or crude oil to the culture medium. It formed stable emulsions with several hydrocarbons and its emulsifying ability was not affected by exposure

to high salinities (up to 300 g/l), high temperatures (100C-121C) or a wide range of pH values (2-13). This is the first description of bioemulsifier production by a Paenibacillus strain. A preliminary chemical characterization by Fourier Transform Infrared Spectroscopy (FT-IR), proton and carbon nuclear magnetic resonance (1H NMR and 13C CP-MAS NMR) and size exclusion chromatography indicated that this new bioemulsifier is a low-molecular weight oligosaccharide-lipid complex. Mobilization of heavy crude oil by this isolate was evaluated using a core-flooding equipment working at the oil reservoir pressure (32.4 bar)

and temperature (40C). Growing in situ Paenibacillus sp. #510 for 14 days at the oil reservoir conditions using a mineral medium resulted in the mobilization of 6% of the entrapped heavy oil, confirming that this isolate can contribute to enhance oil recovery from mature reservoirs. These results will be further validated in a pilot field assay.

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P8b. The Study of Microbial Enhanced Oil Recovery in Daqing Oilfield Li Wei, Hou Zhaowei, Zhang Jiyuan, Wang Yanling, Chen Xinghong, Dou Xumou, Luo Qing, Wu XiaoLin Exploration and Development Research Institute of Daqing Oilfield Company Ltd., Daqing, 163712, China Great advancement has been achieved with polymer flooding after water flooding to enhance oil recovery in

Daqing oil field in China. By using polymer flooding, the oil recovery can be improved 12%, and the total oil

production is 10 million tons per year in Daqing oil field. However, there is still about 50% remaining oil kept in

oil-bearing formation after polymer flooding. This paper gives the method of Quarternary Oil Recovery by

using microbial enhanced oil recovery (MEOR) succeeding a polymer flooding. In this paper, several kinds of

bacteria that can grow with polymer and crude oil as the carbon source have been screened. These bacteria

are different from that of growing with carbohydrate as the only carbon source used in MEOR before.

Evaluation was made on the physiological and bio-chemical characteristics of the screened bacteria, the ability

of their degrading oil and polymer, the ability of their producing bio-surfactant, and on the core displacement

test. The results show that the light component and the rheological property of crude oil have been increased

due to the action of the selected bacteria, the paraffin content, the gel content and the freezing point of crude

oil have been decreased. Also, due to the action of the bacteria, the acid value of oil has been raised by 64.5

times and the initial boiling point has been reduced 80℃. Moreover, the Newtonian Index, Delta viscosity and

EOR Index have been increased. Core displacement shows that oil recovery can be improved 5% or 13% by

using MEOR or with the following ASP system respectively. All of the experiments can be repeated perfectly.

Session 3 – Hydrocarbon Biodegradation

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Session 3 - Hydrocarbon Biodegradation

Session Chairs: Corinne Whitby and Sean Caffrey

Invited Talk: Alkylsuccinate Synthase: From Desulfatibacillum alkenivorans AK-01 to the Bottom of the Sea Amy Callaghan University of Oklahoma Alkanes are a significant component of crude oil. Due to their chemical inertness, microbial transformation plays an important role with regard to their fate in hydrocarbon-impacted ecosystems. To date, the most well described mechanism of anaerobic alkane activation is alkane addition to fumarate (‘fumarate addition’), catalyzed by the glycyl radical enzyme, alkylsuccinate synthase (ASS) (also known as methylalkylsuccinate synthase or MAS). In conjunction with metabolite profiling and other methods, alkylsuccinate synthase has been shown to serve as a biomarker of in situ alkane degradation in groundwater, coal beds, and deep-sea sediments. Despite the ubiquitous detection of these genes in hydrocarbon-impacted environments, there is very little known about the diversity of alkylsuccinate synthases as they relate to substrate range, and there are few model organisms to interrogate. Deltaproteobacterium Desulfatibacillum alkenivorans AK-01 is a sulfate-reducer capable of utilizing non-methane alkanes (C13-C18), alkenes, fatty acids, and organic acids. Previous work demonstrated that AK-01 activates n-alkanes via ‘fumarate addition’ and that the genome has two loci encoding genes for alkylsuccinate synthases. The study herein aimed to investigate the transcriptional response of D. alkenivorans AK-01 to growth on alkanes. RT-qPCR was used to examine the role of ASS during growth on AK-01’s known alkane substrate range, and microarray analysis was used to assess whole-cell responses to growth on hexadecane vs. hexadecanoic acid. RT-qPCR experiments indicated that ass genes in cluster 1 are induced when AK-01 is grown on all tested alkanes compared to the corresponding fatty acids, whereas ass genes in cluster 2 do not appear to be induced under any of the tested conditions. Microarray analysis supported the RT-qPCR data and also indicated the upregulation of genes that may be involved in the further metabolism of methylalkylsuccinates. Ongoing work aims to further elucidate the role of ass gene cluster 2.

Offered Talks: Understanding microbial processes involved in hydrocarbon degradation by experimental ecology Robert Duran, Cristiana Cravo-Laureau Pau University To date studies of hydrocarbon biodegradation have focused mainly on simplified systems (e.g. pure strains, individual molecules), which are not representative of natural environments. As a result, little is known about whole microbial communities in their actual context, and the impact of environmental fluctuations on pollutant degradation and microbial community structure is insufficiently investigated. Notably, microbial communities in the environment are frequently exposed to varying levels of oxygen, and while the aerobic and anaerobic biodegradation of hydrocarbons is well documented, little is known as yet about degradation within the transitional zone from oxic to anoxic conditions. Oxic/anoxic oscillations are commonly found in nature as for example at water/sediment interfaces or in bioturbated sediments. This fluctuating environmental conditions influence the microbial dynamic and its role on organic compounds degradation such as hydrocarbons. It is thus important to determine the behavior of microbial communities in such oscillating conditions, especially the microbial populations involved on anaerobic metabolisms such as sulfate-reducing microorganisms. In order to decipher the mechanisms underlying microbial community

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organization after an oil spill, we develop experimental ecology approaches to manipulate the microbial communities. Maintaining sediments under conditions as close as possible to those prevailing in the environment allowed to propose a scenario describing the petroleum influence on microbial communities. After an adaptive stage, the modification of the microbial community structure occurred, concomitant with the beginning of the degradation of hydrocarbon compounds, followed by a succession of bacterial community structures along the degradation process. Submitting sediments to different oxygenation regimes in bioreactors showed the influence of the oxygenation on microbial assemblages and hydrocarbon degradation, which was favoured by oxygenation after a period of anoxia. Surprisingly, sulfate-reducing microorganisms were not affected by oscillating conditions. Applying different strategies of experimental ecology, our studies highlight the functional redundancy of microbial communities involved in hydrocarbons degradation. Better understanding of the effect of pollutants and the behaviour of microbial communities in oscillating conditions is essential to build a global view of their fate in natural environments. Anaerobic degradation of gaseous alkanes by sulfate-reducing bacteria from marine gas and oil cold seeps Florin Musat Helmholtz Centre for Environmental Research - UFZ The short-chain, gaseous alkanes ethane, propane and n-butane are the main constituents of natural gas and are also found in small quantities dissolved in crude oil. The anaerobic degradation of propane and n-butane was recently reported with a pure culture of a sulfate-reducing bacterium, strain BuS5, and with several enriched cultures. In the present study, further cultures of sulfate-reducing bacteria were enriched with propane and n-butane as substrates from marine sediments collected around gas and oil seeps in the Gulf of Mexico and at Hydrate Ridge. In temperature assays, propane- and butane-dependent sulfate-reduction peaked between 15-20°C reflecting the rather low in situ temperatures. Application of molecular biology methods showed that the enrichments were dominated (≥70% of the total cell number) by sulfate-reducing bacteria affiliated with the Desulfosarcina-Desulfococcus group of the Deltaproteobacteria. The dominant phylotype of each enrichment culture was closely related to strain BuS5, forming an apparent phylogenetic cluster of gaseous alkane degraders. Incubations with 13C-labeled propane or butane followed by hybridization with oligonucleotide probes and nanoSIMS analysis showed that the dominant microorganisms assimilated substantial amounts of 13C, while the accompanying bacteria showed little or no label incorporation. This indicated that the dominant microorganisms were indeed responsible for the short-chain alkane degradation. These or similar microorganisms may be actively involved in the in situ degradation of gaseous alkanes, offering thus an explanation for the high sulfate-reduction rates observed at some marine hydrocarbon seeps. Furthermore, using incubations of strain BuS5 with position-specific, D-labeled propane and n-butane, insights into the activation mechanism of gaseous alkanes were obtained. We were able to determine that propane activation at the primary carbon atoms, previously demonstrated by analysis of metabolites and thought to represent a side reaction, is a main route of activation along with activation at the secondary carbon atom. We estimate that about 70% of the activation events occur at the secondary carbon atom, and 30% at either of the primary carbon atoms. This finding has implications for calculation of position-specific stable isotope fractionation factors. Also, it provides support for a putative activation of ethane by addition to fumarate. Metagenome and transcript analyses of a methanogenic consortium utilizing n-octacosane Christopher Marks, Boris Wawrik, Amy Callaghan, Shane Pruit, Kathleen Duncan, Irene Davidova, Joseph Suflita University of Oklahoma An anaerobic consortium capable of mineralizing long-chain n-paraffins (C28-C50) under methanogenic conditions was enriched from San Diego Bay sediment. The phylogenetic composition and metabolic potential of the microbial community were interrogated via metagenomic sequencing, resulting in a

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~180,000 read 454 library and a ~26,000,000 paired-end read Illumina dataset. An analysis of the SSU rRNA genes recovered from metagenomic data revealed a consortium dominated by Syntrophobacterales (40%) and Methanomicrobiales (21%). Assembly of metagenomic data yielded 6 contigs containing putative genes encoding the catalytic subunit of alkylsuccinate synthase (assA), closely affiliated with requisite genes in “Smithella spp.”. A draft genome assembly of an uncultivated member within the genus Smithella was extracted from metagenome data via analysis of tetranucleotide frequencies and scaffold coverage. The “Smithella sp. SDB” draft assembly is estimated to be >93% complete and accounts for 31.2% of the reads in the consortium Illumina metagenome. A complete alkylsuccinate synthase gene cluster was identified across 4 contigs within the draft assembly. Transcription of 5 of the 6 detected assA genotypes, including the assA gene associated with the “Smithella sp. SDB” scaffold, was demonstrated through RT-PCR when the consortium was incubated with n-octacosane ± a heptamethylnonane carrier. Additionally, draft genomes for Methanoculleus and Methanosaeta, which were the dominant methanogenic lineages, were binned and represent 7.9% and 0.5% of the Illumina reads, respectively. Based on the taxa represented by the draft assemblies and the expression of assA, it is hypothesized that long-chain n-paraffins are syntrophically metabolized via ‘fumarate addition’ by "Smithella sp. SDB" coupled to the methanogenic activities of methanogens such as Methanoculleus and Methanosaeta spp.

Effect of micronutrients on microbial diversity and hydrocarbon biodegradation under sulphate-reducing conditions Angela Sherry1, Carolyn M. Aitken1, Luiza L. Andrade1, Emma J. Bowen1, Ana Suarez-Suarez1, Martin Jones1, Casey R.J. Hubert2, Neil D. Gray1, Ian M. Head1 1 Newcastle University , 2 University of Calgary Previous studies have shown that elevated concentrations of macronutrients, nitrogen (NH4

+) and phosphate (PO4

3-), on sulphate-driven oil biodegradation had a minimal effect on sulphate-reduction. Oil-amended microcosms displayed similar rates of sulphate-reduction when they were treated with nitrogen and phosphate at high (100 mM NH4

+, 10 mM PO43-), intermediate (56 mM, 5.6 mM) or low (4.7 mM, 1.5

mM) levels. However, detailed oil geochemistry performed on the microcosms indicated some effects of NH4

+ and PO43- on oil biodegradation were apparent. It has been suggested that alteration of the

concentration of micronutrients, Nickel (Ni) and Cobalt (Co), may stimulate oil biodegradation, due to the requirement of these trace metals by key enzymes involved in anaerobic hydrocarbon degradation. To determine if Ni and Co affect rates of oil biodegradation, sulphate-reducing oil-degrading microcosms, and controls, were prepared with a range of contaminated and pristine sediments (River Tyne & Amble marina, UK; Messina harbour & Gela, Sicily). Microcosms were treated with levels of Ni and Co corresponding to 0, 100, 200 and 400% of cell growth requirements (where a final concentration of 2.3 µg/l (39 nM) Ni & 1.0 µg/l (17 nM) Co corresponds to 100% cell requirement). Sulfate reduction (SR) rates were highest in River Tyne microcosms amended with oil and 100% NiCo (16.2 ±0.9 µmol SO4

2- day-1 g-1 sediment) or unamended (0% NiCo; 13.1 ±2.5 µmol SO4

2- day-1 g-1 sediment). SR rates were significantly higher than in microcosms amended with 200% NiCo (6.9 ±2.6 µmol SO4

2- day-1 g-1 sediment, p = 0.026), 400% NiCo (4.2 ±1.2 µmol SO4

2- day-1 g-1 sediment, p ≤ 0.033), and 400% NiCo killed controls (4.8 ±0.2 µmol SO42- day-1 g-1

sediment, p ≤ 0.050). By contrast, Messina and Gela sediments showed low levels of sulphate reduction (0.26 ±0.41 and 0.57 ±0.25 µmol SO4

2- day-1 g-1 sediment, respectively). Results suggest that 100% NiCo concentrations have a stimulatory effect on oil-driven sulphate reduction, whereas higher concentrations (>200% NiCo) may be inhibitory. Oil geochemistry and high-throughput DNA sequencing (16S rRNA gene) was conducted to determine the effects of NiCo on oil biodegradation and the diversity of anaerobic hydrocarbon degraders, respectively.

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Bioremediation of Crude Oil by Indigenous Marine Bacteria Xiaoke Hu, Hui Wang, Caixia Wang Yantai Institute of Costal Zone Research, Chinese Academy of Sciences Increasing exploitation, production, transportation and storage of crude oil industry have led to more accidentally spilled contaminants. Several techniques, including physical, chemical, and biological methods, are used to recover spilled oil from the environment. Bioremediation, mainly by indigenous bacteria, has been regarded as a cost-effective green alternative to clean up oil pollution after an oil spill. Moreover, the recent omics revolution has led to leaps in our understanding of microbial communities, and some of the conditions that promote predictable activity in contaminated sites and heterogeneous environments. Combinations of omics tools and new bioinformatics approaches allow us to understand integrated activity patterns between oil pollutants and microorganisms, and determine how this metaorganism can be modified to maximize growth, appropriate assembly of microbial communities, and, ultimately, bioremediation activity. In our laboratory, we have conducted omics-mediated experiments to discover the bacterial communities and the mechanisms during bioremediation. Our results revealed that (1) nutrient amendment could significantly improve the efficiency of oil-biodegradation; (2) the bacterial community structure showed dramatic changes during the bioremediation process; (3) the immobilization technique could increase the efficiency of hydrocarbon degradation; (4) the omics strategies could clarify and confirm the hydrocarbon degradation pathways. The insights and theories of our study will increase the understanding of the hydrocarbon degradation process and provide fundamental evidences for in situ bioremediation of crude oil spills.

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Session 3 - Posters: P9. Fungi in fouling, fuel degradation, and corrosion in B20 biodiesel storage systems Blake Stamps1, Oderay Andrade1, Caitlin Bojanowski2, Carrie Drake3, Pamela Lloyd3, Richard Striebich3, Oscar Ruiz3, Wendy Crookes-Goodson, Bradley Stevenson1 1 University of Oklahoma, 2 University of Dayton, 3 Air Force Research Laboratory Biodiesel, which is composed of biologically-derived fatty acid methyl esters (FAME), is more susceptible to degradation by microorganisms than fossil diesel. An investigation was conducted on B20 biodiesel (20% FAME and 80% fossil diesel) and several storage tanks in the Southwest US (SWUS) and Southeast US (SEUS) experiencing unacceptable levels of particulates in the fuel. Cultivation and molecular-based studies of the microbial assemblages were combined with characterization of fuel components, ATP measurements for microbial activity, detection of metabolites and in situ materials testing. The goal of this study was to determine if microbial fuel degradation and fouling resulted in an increased risk of corrosion. High throughput sequencing of 16S and 18S rRNA amplicon libraries indicated that the microbial assemblages were heavily dominated by fungal taxa. These were dominated entirely by Trichocomaceae at one site, while the other site was much more diversive with multiple fungal taxa from several families. 18S sequence from environmental samples and fungal isolates revealed the primary members of the microbial assemblage to be from the genera Wickerhamomyces (a synonym of Candida, in SEUS) and Byssochlamys (a member of the Trichocomaceae, in SWUS and SEUS) Representative isolates were capable of growth on B20 as the sole carbon and energy source. Metabolic activity in fuel samples, measured as ATP, correlated closely with observed microbial blooms. Analyses of fuel showed reduction in FAME concentrations over time, with an accumulation of metabolites consistent with Beta-oxidation. In situ corrosion measurements included weight loss and imaging of 1010 carbon steel coupons placed 0.5 and 30 cm off the bottom of the tank. Mass loss measurements after 3 months were negligible but surface imaging revealed many areas of pronounced localized corrosion (pitting), with both bacterial and fungal morphologies present on the steel surface. The data presented here suggests that microbial metabolism of B20 biodiesel results in microbial blooms that result in fouling of bulk fuel, probes, and dispensing hardware. Although more direct studies are needed, the biofilm associated with metal surfaces in storage tanks and the production of acidic metabolites very likely leads to a significant risk to fuel storage infrastructure. P10. New evidences on the anaerobic biodegradation pathways of hydrocarbons in subsurface oil reservoirs by detection of signature metabolites and functional genes Xin-Yu Bian1, Jing Zhou1, Serge Maurice Mbadinga1, Ji-dong Gu2, Bo-Zhong Mu1 1 East China University of Science and Technology , 2 The University of Hong Kong Anaerobic degradation of alkanes in hydrocarbon-rich environments has been documented; and different degradation strategies were proposed, of which the most commonly encountered is the addition of alkanes onto the double bond of fumarate to generate corresponding alkylsuccinates as specific chemical markers. However, little is known about the mechanisms of anaerobic degradation of alkanes in oil reservoirs, probably due to low concentrations of signature metabolites and lack of mass spectral characteristics to assist identification. This work was initiated to determine whether alkanes are biodegraded anaerobically via fumarate addition biochemical pathway in oil reservoirs. A total of twelve production fluid samples from three different oil reservoirs were collected and treated by alkali; organic acids extracted, derivatized with ethanol and determined using GC-MS. Collectively, signature metabolites of alkylsuccinates of parent compounds from C1 to C8 together with their putative downstream metabolites were detected from these samples. Additionally, metabolites indicative of the anaerobic degradation of mono- and poly-aromatic hydrocarbons (2-benzylsuccinate, naphthoate, 5, 6, 7, 8-tetrahydro-naphthoate) were also observed. The detection of alkylsuccinates and genes encoding for alkylsuccinate synthase (assA) reveals that anaerobic degradation of alkanes via fumarate addition occurs widely in oil reservoirs. This work provides strong evidences on the in situ mechanisms of anaerobic biodegradation of hydrocarbons by fumarate addition as a common biochemical reaction.

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P11. ‘Full City’ carrier oil spill near Langesund Norway: Bacterial community shifts, petroleum degrading and surfactant producing microorganisms Catherine Boccadoro, Adriana Krolicka IRIS On 31st July 2009, the “Full City” bulk carrier ran ground at Såstein, Southwest of Langesund, Norway, and an estimated 293 tons of bunker oil leaked out into the sea, causing oil slicks to be observed in the bays at Langesund, later spreading along the coast in southern Norway. The aim of this study was to evaluate both the impact of the oil on the environment and the ability of the indigenous microbial populations to degrade petroleum compounds. Seawater samples were collected at 10 different locations along the cost, at an increasing distance from the spill, including two pristine sites, 24 hrs after the initial spill. Total microbial populations were investigated by 454 pyrosequencing and key oil degrading organisms were quantified by qPCR. In addition, organisms were cultured to identify and isolate key oil degraders and evaluate their surfactant production. The composition of the bacterial communities isolated from seawater in the vicinity of the spill, as well as communities near oil drifts, were very different to those isolated from non-polluted locations, despite their geographical proximity. Bacterial communities from polluted seawater were richer in bacteria commonly associated with petroleum hydrocarbon degradation; Colwelliaceae, Oceanospiralles, Marinomonas, Cycloclasticus, Alcanivorax, Polaribacter and Glaciecola. Bacteria assigned to Marivita and Polaribacter, which are less typically characteristic of petroleum exposures in seawater, were found to contribute to 6 and 14 % respectively of the total microbial population in locations highly affected by crude oil compared to populations from pristine seawater. QPCR analysis for Alcanivorax, Cycloclasticus and uncultured Oceanospiralles confirmed pyrosequencing data, and showed high copy numbers in polluted locations while remaining undetectable in the control areas where petroleum compounds had not been reported. This result suggests significant shifts in microbial population compositions in seawater within 24 hrs of an exposure to petroleum compounds, and demonstrates the possibility of using specie identification and quantification as a basis for marine crude oil monitoring. P12. Methanogenic biodegradation of larger molecular weight hydrocarbons (3-ringed PAHs and waxy paraffins) Lisa Gieg, Courtney Toth, Lisa Oberding University of Calgary Understanding the anaerobic biodegradation of various hydrocarbons is important for applications related to oil spill clean up in anoxic aquifers and for pinpointing the microbial processes occuring in crude oil reservoirs that lead to heavy oil formation or enhanced energy production. The methanogenic biodegradation of alkanes, monoaromatic hydrocarbons such as toluene, and more recently, 2-ringed polycyclic aromatic hydrocarbons (PAH), have been reported in several studies. In order to determine whether larger molecular weight hydrocarbons may also be susceptible to methanogenic decay, we are examining the biodegradation of a model 3-ringed PAH (phenanthrene) and a model paraffin (octacosane) by methanogenic consortia. In the phenanthrene-amended enrichment, enhanced levels of methane and hydrocarbon depletion were observed relative to controls, providing evidence for phenanthrene degradation coupled with methane production. Microbial community analysis using 16S rRNA gene pyrosequencing revealed Rikenellaceae, Clostridium, Spirochaeta, and Proteiniphilim as the dominant bacterial genera along with mainly hydrogenotrophic methanogens in the phenanthrene-utilizing culture. GC-MS analysis of culture supernatants revealed several putative metabolites including decahydro-2-naphthoic acid and a variety of branched fatty acids that were not detected in control incubations providing clues to how phenanthrene may be metabolized methanogenically. In separate, newer enrichments amended with the paraffin octacosane (supplied in an inert hydrocarbon layer), signficantly enhanced levels of methane were detected compared to substrate-free controls suggesting that this paraffin was bioconverted to methane. Interestingly, when sulfate was also added as an electron acceptor (in separate incubations), the rate and extent of methane production increased and occurred at the same time as

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sulfate consumption. Ongoing studies to determine the predominant taxa and putative biodegradation genes operating during methanogenic paraffin metabolism (e.g., assA gene for alkylsuccinate synthase) will help more clearly define how this methanogenic consortium consumes paraffins. Overall, our studies have revealed that larger molecular weight hydrocarbons such as 3-ringed PAH and paraffin waxes are susceptible to methanogenic biodegradation, findings that have important implications for bioremediating spilled fuels and managing oilfield operations. P13. A mechanistic approach to microbial degradation of crude oil: The importance of interfacial processes at the microscale Gabriel Juarez, Roman Stocker MIT The degradation of oil in the ocean is a process of immense societal and ecological importance, whose efficiency ultimately hinges on fundamental problems in colloidal and interface physics, because degradation occurs when individual bacteria attach to individual oil droplets. Here, we present experimental and theoretical results on the effect of oil drop diameter on the attachment of marine bacteria to the oil-water interface. Using time-lapse phase-contrast and fluorescence microscopy, we directly observed the dynamics of marine bacteria in the presence of oil droplets of varying diameter within microfluidic devices. We find that microbial growth and degradation become strongly limited for drops smaller than a critical diameter. A theoretical model of attachment supports this finding, suggesting that rendering oil droplets too small by the excessive addition of dispersants, as often done in oil spills, might be a counterproductive strategy, and that deeper understanding of the fundamental oil-microbe interactions should instead be pursued and guide oil spill management. These observations provide a new framework for understanding how the interplay between physical, chemical, and biological processes at interfaces shape the dynamics of microbial biodegradation of crude oil in the ocean. P14. The effects of Corexit 9500 on oil biodegradation in cold temperatures Krista Kaster, Andrea Bagi , Mona Ulas, Roald Kommedal University of Stavanger Constituents from crude oil are major sources of marine pollution and despite there being a natural presence of crude oil hydrocarbons in the marine environment, anthropogenic activity causes major contributions to the total release of hydrocarbons into the marine environment. Efforts in removal of hydrocarbon pollution are based on the natural weathering mechanisms, specifically biodegradation, and the chemical enhancement of this process. Chemical dispersants have been developed that serve to disperse spilled oil rapidly and extensively into the water column, making it more accessible for biodegrading microorganisms. Here the effect of the chemical dispersant Corexit 9500 was studied on the degradation of crude oil from the Ekofisk oil field at 3 different temperatures. The biodegradation of the dispersant alone was also investigated. Both chemical and microbiological methods were used to analyse the effects at 3, 8 and 15°C. Experiments were carried out using seawater samples in 2013 and again in 2014. Biological oxygen consumption analysis showed increased biodegradation rates with increasing temperature. The addition of Corexit insignificantly increased biodegradation rates of crude oil during both trials. Total hydrocarbon analysis via GC-FID of the samples form 2013 revealed that between 82 and 95% of hydrocarbons in the C10-C40 n-Alkane were degraded over a period of 46 days. The addition of Corexit showed an increase in hydrocarbon removal of 2% at 8 and 15°C, and 10% at 3°C. Microbial community analysis using DGGE revealed a change between the control samples to the microbial communities seen in the oil exposed communities. Different microbial communities were seen in the samples where either only oil or only Corexit was added. The microbial community containing both Corexit and oil contained both Corexit and Oil degrading microbial communities.

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P15. Targeting bacterial adaptive traits for mitigation of fuel biodeterioration Oscar Ruiz1, Thusitha Gunasekera2, Richard Striebich2 1 U.S. Air Force Research Laboratory, 2 University of Dayton Research Institute Microbial colonization of hydrocarbon fuels can affect fuel systems, vehicle performance and fuel quality. To develop effective mitigation approaches to treat and prevent fuel biofouling, better understanding of microbial adaptive strategies and their effects in fuel systems is required. We have combined fuel degradation assays with genomics, transcriptomics and detailed chemical analysis to characterize the genetic and metabolic response of bacteria and their unique hydrocarbon degradation profiles. Our results showed that upon exposure to fuel, bacteria activate a number of genes involved in hydrocarbon degradation, exopolysaccharide and biofilm formation, iron and phosphorus acquisition, and toxic compound resistance (efflux pumps and porins). It was observed that n-alkanes are the preferred food source for many common fuel degraders but that aromatics and branched alkanes may be effectively degraded by specialized bacteria. During bioreactor tests, bacteria mix cultures were able to completely degrade many hydrocarbon components in fuel. Currently, we are using the acquired knowledge to develop detection and treatment methods to mitigate fuel biodeterioration. P16. Removal of TPH from soil by Pseudomonas aeruginosa TGC01 stimulated by Gossypium sp. (malvaceae) cake Diogo dias1, Thiago Cavalcant2, Ulrich Vasconcelos2 1 Universidade Federal do Rio de Janeiro, 2 Universidade Federal da Paraíba Soil is a complex matrix highly prone to contamination by hydrocarbons. This sort of contamination is largely due to anthropogenic activities, which requires specific intervention and remediation of the contaminated matrix. Bioremediation is one of the most accepted techniques for the removal of toxic contaminants employing microorganisms. However, the recalcitrant nature of some hydrocarbons represents a serious difficulty in devising a bioprocess for the recovery of matrices such as soil. In this context, agroindustrial co-substrates and co-products may be used as adjuvants in the bioremediation process. The aim of this work was to investigate the best strategy to efficiently remediate sand soil contaminated with approximately 16,000mg of Total Petrol Hydrocarbons (TPH) per Kg of soil supplemented with 250mg/Kg of cotton (Gossypium sp.). To this end the following conditions were evaluated: biostimulation with the cotton cake, bioaugmentation using Pseudomonas aeruginoaTG01 isolated from petrol station soil, and biostimulation associated with bioaugmentation. A bioreactor containing the bacterial suspension was used in order to observe the role of the microorganism in the remediation process. All conditions were assayed in duplicates. Abiotic losses were estimated using sterilized soil in the bioreactor. In a 30-day period, the highest TPH removal (34.5±1.3%) was achieved with the biostimulation associated with the bioaugmentation, revealing a daily degradation rate of 201 mg/Kg/d. Biostimulation was capable of removing 31.1±1.1% do TPH, with a daily degradation rate of 182 mg/Kg/d. Bioaugmentation was the least effective strategy with a removal of 22.1±1.4% of the initial TPH contamination. The cottoncakeprovided substrates for the biomass production and energy acquisition and improved bacterial growth, which was observed by an increase of two orders of magnitude in heterotrophic bacterial count compared to the assays in the absence of the cake. Higher bacterial growth in the processes employing bioaugmentation and biostimulation associated with bioaugmentation was also observed by a higher CO2 emission (8% and 23%, respectively). Ecotoxicity assays using previously validated plant seeds indicated that the soil amended with the cotton cake significantly reduced the phytotoxicity of the contaminants to Artemisia dracunculus and Brassica nigra, but not to Cucumis anguria, considered the best bioindicator of this study.

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P17. Potential of bacteriophage as an inhibitor of microbial contamination of fuels Caitlin Bojanowski1, Wendy Crookes-Goodson2, Jayne Robinson1 1 University Of Dayton , 2 Air Force Research Laboratory Microbial contamination and biofilm formation are especially troubling in fuel tanks where they can lead to degradation of tank coatings, corrosion of metallic tank components, degradation of fuel quality, and fouling of fuel system components. To evaluate the potential use of bacteriophage in the mitigation of bacterial contamination of fuel systems, we examined the stability of bacteriophages in fuel and determined their impact on planktonic and biofilm-associated populations grown in fuel tank microcosms containing Jet A aviation fuel. Two phages were chosen for this study: UT1, a pseudolysogenic phage isolated from fresh water, and phage PEV2, an obligately lytic phage isolated from sewage. The stability of these phages was assessed over time in the presence of aviation fuel in sterile microcosms containing a layer of Jet A aviation fuel over Bushnell-Haas (BH) media to model fuel tank bottoms. Identical microcosms inoculated with Pseudomonas aeruginosa, an organism readily isolated from soils and previously identified in fuels, were challenged with these phages to assess the impact of phage on biofilm formation. Bacteriophages UT1 and PEV2 were found to retain their infectivity in the presence of aviation fuel. In inoculated microcosms, the presence of bacteriophage UT1 resulted in 1.7 log reduction in the number of planktonic cells and a 3.3 log reduction in the number of biofilm associated cells compared to unexposed controls. The presence of bacteriophage PEV2 did not result in a significant reduction in the number of planktoninc cells, however, there was a significant 2.1 log reduction in the number of biofilm associated cells. These findings demonstrate that there is a potential to use bacteripohage as a treatment for microbial contamination in fuel storage systems. P18. Bioremediation of waste oil by mix bacterial isolates in anaerobic condition Ahmed Alshehhi Masdar Institute Petroleum hydrocarbons such as waste oil have been known to be a serious environmental problem. Concerns have been raised by government and environmental authorities in different countries about these problems. Different methods have been used to treat contaminated sites such as incineration, pyrolysis and landfilling. These methods are costly and they alter the general ecosystem in soils and water. Biodegradation, a cost effective method which involves the use of microorganisms to degrade these pollutants have been looked into in this study. Biodegradation of waste oil in liquid media was carried out in the laboratory using mix bacterial isolates from waste oil contaminated soil and marine environment. To improve bioremediation of waste oil nutrient is adding in the reactor. Using TPH as measurement of degrading of hydrocarbon before and after the reactor.

P19. Anaerobic oxidation of short-chain hydrocarbons in marine sediments – microbial targets for oil & gas exploration Antje Gittel, Johanna Donhauser, Kasper Kjeldsen, Hans Røy, Bo B. Jørgensen Aarhus University The understanding that hydrocarbons migrate from marine subsurface petroleum reservoirs to the sediment surface has recently resulted in the use of gas seep detection as a prospecting tool in the oil and gas industry. However, the turnover of gaseous C2-C4 short-chain alkanes (SCA) in cold surficial sediments and the capacity of the indigenous anaerobic microbial community to degrade these compounds have received little attention so far. Local elevated abundances of anaerobic SCA degraders in marine sediments may be diagnostic for seepage from underlying petroleum reservoirs, rendering SCA degraders promising targets in microbial prospecting for oil and gas. We investigated the diversity and abundance of anaerobic SCA degraders and determined in situ concentrations of SCA (ethane, propane and butane) in surficial sediments with and without gas seepage. SCA degraders were assessed at the functional gene level, targeting the masD gene (encoding the large subunit of the 1-methylalkyl succinate synthase) which

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represents a marker gene for the anaerobic degradation of aliphatic hydrocarbons. Because existing masD gene primer pairs do not comprehensively target the known environmental masD gene diversity, we developed an improved detection assay and applied it to seepage and non-seepage sediment samples. First results showed that seepage sites were clearly distinguishable from non-seepage sites as shown by higher SCA concentrations. Anaerobic SCA degraders were found in all samples, including reference sites that are currently not impacted by hydrocarbon seepage. This finding indicates that anaerobic SCA degraders are ubiquitous members of marine sediment microbial communities, and in the event of seepage, might grow to represent a larger fraction of the community. Further data analyses are currently performed to assess differences in seep-specific masD diversity and to link increased SCA concentrations to elevated abundances of the SCA-degrading microorganisms. Eventually, our study will provide a detailed quantitative and qualitative picture of the SCA-degrading community in anoxic marine sediments, direct future research on the turnover of SCA in this environment and facilitate the application of microbial prospecting tools in marine oil and gas exploration. P20. Biodegradation approach for soil petroleum spill using bacterial consortia Servio Cassini, Regina Keller Universidade Federal ES (UFES) Petroleum hydrocarbons contain a complex mixture of compounds and have been a major concern for environmental pollution. Characterization of spilled oil is very important in order to predict the behaviour and fate in the environment trying to select the proper cleaning methods. Generally, the complex mixture of hydrocarbons are removed with different rate, which depends of several factors linked to chemical structure, concentration and environmental conditions. In order to verify the biodegradation rate of each petroleum fraction of a complex hydrocarbon mixture, a set of experiments were conducted with several autochtonous bacterial isolates and components of petroleum hydrocarbon mixture, using the soil analogue conditions as main substrate. The experiment were conducted in two phases a) isolation and bacterial isolates screening for efficiency and b) testing the best isolates over 4 petroleum fractions looking for strain specificity for petroleum fractions biodegradation. The petroleum fractions were correspondents to saturated, aromatics, resin and asphaltene fractions. Biodegradation analysis was performed through respirometry by the release of carbon dioxide generated by the bacteria in aerobic system. Data were analysed and adjusted for the calculation of the biodegradation rate according the best fit to kinetic model. The results shown that of the various petroleum fractions, n-alkanes of intermediate length (C10-C25) are the preferred for microorganisms and tend to be the most readily degradable, whereas shorter chain compounds are rather more toxic. Longer chain alkanes (C25-C40) are hydrophobic solids and consequently are difficult to degrade due to their low bioavailability, and branched chain alkanes and cycloalkanes are also degraded more slowly than the corresponding normal alkanes. Highly condensed aromatic and cycloparaffinic structures, tars, bitumen and asphaltic materials, have the highest boiling points and exhibit the greatest resistance to biodegradation. It has been suggested that the residual material from oil degradation is analogous to, and can even be regarded as, humic material. Also, the best strain regarded as Pseudomonas sp. was effective to remove almost 72% of original saturated fraction over a 15 day period. In conclusion, we can infer that bacterial consortia isolated from contaminated soil can be very effective in petroleum fraction removal from impacted environment. P21. Biodegradation of crude oil by the bacteria MCC0016 and MCC0034 isolated from a coal mine: Impact of oil concentration, nutrient addition and bioaugmentation Oghenekume Edeki Rhodes University Petroleum hydrocarbons are major pollutants of both soil and marine environments. A promising approach used for the treatment of these contaminated environments has been highlighted by researchers. Bioremediation; a treatment that uses naturally occurring organisms to break down hazardous substances into less toxic or non-toxic substances has been accepted globally due to the fact that the ecosystem is not

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altered negatively during this process. In the present study, naturally occurring bacteria were isolated from coal slurry at a coal mine in the Witbank region of the Mpumalanga Province in South Africa. A total of 52 bacterial isolates were obtained and these were subjected to screening for their hydrocarbon degradation competence and characterized. Amongst the 52 isolates, 2 strains (MCC0016 and MCC0034) were able to effectively proliferate in hydrocarbon media and this was confirmed by plate counts and biomass accumulation in the media. Soil microcosm studies were set up, and monitoring and estimation of crude oil degradation rates was carried out for a 45 d period in a polycarbonate-covered tunnel in order to investigate an effort to increase the suite of microorganisms available for use in rehabilitation practices. The impact of oil concentrations and nutrient addition to the medium was also evaluated. GC-MS analysis was used to monitor changes that took place in the soil environment during the degradation process. Studies described in the present work showed the effective degradation of diesel by the two bacterial strains with strain MCC0016 (with nutrient addition) degrading n-alkanes faster and at a higher rate compared to polyaromatic hydrocarbons. However, strain MCC0034 was able to effectively degrade both n-alkanes and polyaromatic hydrocarbons (with nutrient addition). In the case where nutrients were not added to the media, a slow degradation process was observed. At the end of the study, it was concluded that nutrient amendment enhance the biodegradation rates of petroleum hydrocarbon. P22. Biodiversity in the Dagang oil field: Degradation of hydrocarbons under methanogenic conditions Nontje Straaten1, Núria Jiménez1, Hans H. Richnow2, Jun Yao3, Martin Krueger1 1 German Federal Institute for Geosciences and Natural Resources - BGR, 2 Helmholtz Centre for Environmental Research – UFZ, 3 University of Science and Technology Beijing In a world with increasing demands for fossil fuels and decreasing resources it is important to understand the processes involved in oil degradation. Biodegraded oil is found in many reservoirs worldwide. Consequently, it is very important to get insight in the microbial communities and metabolic processes involved in hydrocarbon degradation. Due to the lack of alternative electron acceptors in hydrocarbon-rich geosystems, degradation often takes place under methanogenic conditions. The studied samples originate in the Gangxi zone in the Dagang oil field. The Dagang oil field is one of the largest in the world and is in operation since several decades. The moderate temperatures (30° to 70°C) and the ongoing secondary recovery, like water flooding, are an ideal environment for microbial growth and biodegradation. Therefore the aim of the present study is to identify the autochthonous microorganisms of the oilfield and mechanisms involved in the degradation of complex hydrocarbons. For this purpose the original samples were analysed using molecular biological techniques, like next generation sequencing and quantitative PCR, to identify and quantify the microbiota in the reservoir geosystem. Moreover, injection and production water as well as oil and gas samples of Dagang were analysed for evidence for the in situ occurrence of methanogenic oil degradation. The gas data provided in several cases hints for a recent biological origin of the methane present. First results of the microbial community analysis showed in the environmental samples high numbers of Bacteria and Archaea, especially a high proportion of Methanogens (105 – 106 gene copy numbers per ml). Results of clone libraries and pyrosequencing confirmed a diverse community of methanogenic Archaea. The analyses of the bacterial sequences revealed a large quantity belong to the families Pseudomonadaceae and Campylobacteraceae.. In summary, the Dagang reservoir system provides a suitable habitat for diverse prokaryotic communities and thus a promising environment to test MEOR approaches, like an in situ stimulation of methanogenic Archaea. P23. Harnessing environmental microbial communities as biofilms for polycyclic aromatic hydrocarbon bioremediation Marc Demeter1, Joe Lemire1, Sean Mercer2, Raymond Turner 1 1 University of Calgary, 2 Imperial Defined as aromatic hydrocarbons with two or more fused benzene rings, and formed from fossil-fuel combustion and petroleum refining, polycyclic aromatic hydrocarbons (PAHs) are ubiquitously found in all environmental spheres. Due to their toxic, mutagenic and carcinogenic nature, both the US EPA and the

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European Union have identified PAHs as priority pollutants. PAHs accumulate in the environment due to their low water solubility, and complex chemical structure. One method to mitigate PAH pollution is biodegradation. Biodegradation of low-molecular weight PAHs has been extensively studied. However, the difficulties associated with degradation of persistent, high-molecular weight PAHs, along with the variable extent and rate of microbial PAH degradation, has thus far prevented bacterial-mediated PAH bioremediation from developing into a mature biotechnology. Factors that affect the rate and extent of PAH bioremediation include the nutrient accessibility, PAH bioavailability, and microbial composition. Having previously demonstrated that we can grow upwards of 70% of an environmental microbial community as a multispecies biofilm, our working hypothesis is that we can use our novel methods to culture environmental biofilms from site-samples contaminated with PAHs, and subject them to various nutrients, and PAH-stress conditions aimed at producing a microbial biofilm with enhanced PAH degradation capacity. Known for their pollutant tolerance, biofilms of a multispecies consortium may also possibly stimulate coordinate metabolic activities resulting in enhanced PAH degradation not observed in pure cultures. Here, we explore how using a modified MBEC device and scaled flask-reactors, we cultured multispecies biofilms from a PAH-contaminated fluid industrial tailings waste, and three different PAH-contaminated soil samples in a minimal medium spiked with 16 EPA identified PAHs at concentrations relative to those found in gasoline. The effect of nutrient accessibility was examined by supplementing the media with either yeast extract or glucose. Biofilm growth was observed using confocal microscopy, and quantified via qPCR. GC-FID was used to examine PAH degradation. Multispecies environmental biofilms were compared to biofilms of Rhodococcus opacus R7 (a known naphthalene degrader), the results of which demonstrate as a whole that tailored biofilm communities can better degrade PAHs. Comparisons between multispecies inoculum sources reveals more refined differences in PAH degradation abilities. P24. Microbial Contamination Thriving in Alternative Jet Fuels compared to the Jet A-1 Mohammed Yahyaoui, Thierry Lemettais, Isabelle Lombaert-Valot Aibrus Group Innovations The aim of the present work is to assess the behaviour of the new alternative fuels with biological contaminants.The aviation fuel systems can be an ideal environment for micro-organism growth, due to the presence in some areas of good conditions for thriving: water, carbon source, and with an ideal ground temperature in some regions of the world. To anticipate the use of alternative fuels in the aeronautic transport, knowledge have to be acquired concerning micro-contaminant proliferation in these fuels and the suitability of current procedures (tests checking periodicity, biocidal treatment and dose, etc.). Several alternative aviation fuels with different compositions have been tested in comparison with the fossil jet fuel. The effect of hydrocarbon families contained in the aviation fuels is discussed. The paraffin families seem to favour the micro-organisms growth in the jet fuel. Since alternative fuels are mainly composed by paraffin hydrocarbons, more frequent and regular check of the microbial contamination should be done when alternative fuels are used in order to avoid undesirable effects of corrosion or biofilm formation. The second part of this work was focused on the use of some biocides against microbial contamination in altrantive fuels compared to the Jet A-1. One more time, an easier and stronger microbial thriving has been observed within alternative jet fuels compared to the Jet A-1. The fuels were contaminated by 6 micro-organisms (5 fungus and 1 bacteria) usually identified in the jet fuel. The characterisation of the microbial contamination has been done by ATMmetry (bioluminescence detection). The kinetic of microbial contamination evolution has been done by measuring the thriving within time (T0, T0+t1, etc.).

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P25. Microbial diversity in methanogenic hydrocarbon-degrading enrichment cultures isolated from a water-flooded oil reservoir (Dagang oil field, China) Núria Jiménez1, Minmin Cai3, Nontje Straaten1, Jun Yao2, Hans H. Richnow3, Martin Krueger 1 1 German Federal Institute for Geosciences and Natural Resources-BDR, 2 University of Science and Technology Beijing, 3 Helmholtz Centre for Environmental Research – UFZ Microbial transformation of oil to methane is one of the main degradation processes taking place in oil reservoirs, and it has important consequences as it negatively affects the quality and economic value of the oil. Nevertheless, methane could constitute a recovery method of carbon from exhausted reservoirs. Previous studies combining geochemical and isotopic analysis with molecular methods showed evidence for in situ methanogenic oil degradation in the Dagang oil field, China. However, the main key microbial players and the underlying mechanisms are still relatively unknown. In order to better characterize these processes and identify the main microorganisms involved, laboratory biodegradation experiments under methanogenic conditions were performed. Microcosms were inoculated with production and injection waters from the reservoir, and oil or 13C-labelled single hydrocarbons (e.g. n-hexadecane or 2-methylnaphthalene) were added as sole substrates. Indigenous microbiota were able to extensively degrade oil within months, depleting most of the n-alkanes in 200 days, and producing methane at a rate of 76 ± 6 µmol day-1 g-1 oil added. They could also produce heavy methane from 13C-labeled 2-methylnaphthalene, suggesting that further methanogenesis may occur from the aromatic and polyaromatic fractions of Dagang reservoir fluids. Microbial communities from oil and 2-methyl-naphthalene enrichment cultures were slightly different. Although, in both cases Deltaproteobacteria, mainly belonging to Syntrophobacterales (e.g. Syntrophobacter, Smithella or Syntrophus) and Clostridia, mostly Clostridiales, were among the most represented taxa, Gammaproteobacteria could be only identified in oil-degrading cultures. The proportion of Chloroflexi, exclusively belonging to Anaerolineales (e.g. Leptolinea, Bellilinea) was considerably higher in 2-methyl-naphthalene degrading cultures. Archaeal communities consisted almost exclusively of representatives of Methanomicrobia (mainly belonging to genera Methanosaeta and Methanoculleus). As both syntrophic Bacteria and methanogenic Archaea are abundant in Dagang, the studied areas of this oil field may have a significant potential to test the in situ conversion of oil into methane as a possible way to increase total hydrocarbon recovery. P26. Microbial Community Structure Succession in the Methanogenic Metabolism Process from Petroleum Hydrocarbon Degradation

Jin Rui， Hou Zhaowei， Liu Yang，Chen Xinghong，Guomenghua Exploration and Development Research Institute of Daqing Oilfield Company Ltd., Daqing 163712

At present the prominent issue faced by most continental oilfield in China is that there hasn’t an effect method found to deal with the quite amount of residual oil remained in the reservoirs. Therefore, if the crude oil in the depleted reservoir, or in the high water-cut reservoir or in the low permeability reservoir can be converted into gas such as methane easier to develop by microbial degradation, the life of oilfield would be further prolonged. In this study, a complex microbial system that can use crude oil as the sole carbon source was separated from the sample water coming from producing wells. The complex microbial system can degrade the crude oil and produce gas such as the methane under 45℃ while having been cultured for 2 months. The bacteria can invert one gram of crude oil into 200ml methane approximately in a year. After microbial treatment, the phenomenon of emulsified oil was very obvious and the viscosity of crude oil decreased from 50mPa·s to 30mPa·s, the interfacial tension between oil and water decreased from 63mN/m to 41mN/m. At the same time, the crude oil was degraded into fatty acid mainly including acetic acid, propionic acid, and other short chain fatty acid (C4-C9). Using the technology of constructing microbial 16S rRNA gene clone library, the composition and dynamic change of microbial community in the process of culture were also analyzed. At the beginning of culture, the dominant bacteria was capable of degrading crude oil and produced a lot of polar oxygenated chemicals which provided the substrate for final methanogenesis. Then the microbial community

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composition was predominantly represented by hydrogenotrophic methanogens and aceticlastic methanogens reducing CO2, formic acid and acetic acid. According to the metabolic function, the whole course of methane formation was a result of the cooperation between fermentative bacteria, syntrophic bacteria and aceticlastic methanogens. The conversion of residual oil to natural gas by anaerobic microorganisms could substantially improve the exploitation and utilization of oil resources. The recovery of methane gas as an alternate form of energy from unrecoverable crude oil may offer a route to economic production of energy from petroleum reservoirs. This paper gives the change of microbial community during the conversion of petroleum hydrocarbon to methane gas and discusses the feasibility and potential for energy recovery via methanogenesis of residual oil.

Session 4 – Microbial Biotransformations

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Session 4 - Microbial Biotransformations Session Chairs: Ian Head and Catherine Boccadoro

Invited Talk: Harvesting subsurface and oil related microbial communities for industrial use - a multidisciplinary task. Hans Kristian Kotlar Private consultant, ex. senior specialist Statoil A new “gold rush” is on – not for the yellow metal, but for new genes coding for substances giving new potentials in industrial processes. Our search has been in a variety of harsh environments, but with a special emphasize on hyperthermophiles of oil reservoirs, 1 – 3 km subsurface. In the deep biosphere, sedimented or buried 200 – 500 million years ago, extraordinary “new” types of organisms can be found. These organisms will be polyextremophiles to the 4th power, e.g. highly thermotolerant, halophilic, piezophilic and solventophilic, i.e. they tolerate a high load of organic solvents. With the completely new and so far unevaluated candidates discovered from these extreme subsurface habitats, bioprocesses can be performed at temperatures and pressure conditions never before considered possible. These organisms are found both in the bacterial and archaeal kingdom. By DNA sequencing of all DNA present in the oil reservoir habitat, one can from the sequencing data, construct a metagenome DNA library of that particular habitat. Another way of making a library is to take this isolated DNA, from the subsurface community and break it down to smaller pieces. These smaller pieces of DNA can be cloned into a host organism and make a functional- or a fosmid library. Making both DNA libraries and functional libraries, we can do bioprospecting and bio-mining for genes with desired properties. Tremendous possibilities – spin offs for technical solutions to many manmade problems could be sought. This approach has proved successful in search for efficient bio-based EOR, finding microbial communities with high kinetics for biotransformation of oil, boosting the recovery factor significantly. Furthermore, highly termotolerant enzymes for the CO2 capture and storage, CCS, process have been produced. However, in order to be successful in using these “new” technologies one have to think outside the box and bring in a multidisciplinary approach. You must have it all!

Offered Talks: Strategies for viscosity reduction through microbial metabolism of bitumen and heavy oil. Gerrit Voordouw University of Calgary Anaerobic microbial activity in oil reservoirs catalyzes the water-mediated, slow conversion of light oil to heavy oil to bitumen over geological time. The reasons for this seemingly inevitable sequence are well known. Microbes metabolize the low molecular weight (alkanes, aromatics), but not the high molecular weight components of oil, which accumulate as a result. These accumulating fractions (e.g. naphthenic acids, asphaltenes, resins) are recalcitrant because of their structural diversity. If a fraction consists of thousands of components, then the concentration of each individual one is small. Synthesizing an enzyme, catalyzing the initial metabolic attack of one of these, requires an investment of 4n ATP molecules, where n is the number of amino acids in the enzyme catalyst. This investment must be recouped by metabolizing sufficient substrate molecules, but this is a tough task when their concentration is low and when there are many similar molecules, which could act as competitive inhibitors. A solution to this problem is to use another strategy, as used by white-rot fungi for the degradation of lignin. In this strategy oxygen is activated, often extracellularly, to reactive oxygen species (ROS), which then randomly attack all organic carbon. The probability of sustaining a hit increases with molecular weight and so this strategy would, in

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principle, remove high molecular weight components preferentially. We have found that aerobic degradation of bitumen in oil sands outcrops near Fort McMurray, Alberta is catalyzed by a community of fungi and hydrocarbon-degrading bacteria. Metagenomic analyses indicate that the former catalyze the random, ROS-mediated degradation of hydrocarbon with the degradation products being further degraded and mineralized in a way that benefits the entire consortium. Higher temperatures (up to 60°C) favour aerobic bitumen degradation, presumably because bitumen is less viscous (10-4 centipoise) at 60°C than at 20°C (10-6 centipoise). The extent to which limited oxygenation promotes random attack at the expense of subsequent mineralization of degradation products to CO2 is not known and understanding this balance is likely at the heart of defining strategies for achieving a successful microbially-mediated decrease of the viscosity of bitumen or heavy oil. Microbial profiling insights from an offshore oil field in South East Asia Nicolas Tsesmetzis1, Eric Alsop2, Peter Marks1, Michael Maguire3, Ian M. Head3, Fons Marcelis1, Cor Kuijvenhoven1, Joseph Westrich1, Bart Lomans1 1 Shell International Exploration and Production , 2 Joint Genome Institute, 3 Newcastle University Three hydrocarbon reservoir cores were obtained from a non-flooded field in South East Asia. All three cores were taken from the same well and the top and bottom cores were spaced 23 and 33 meters respectively (TVD) from the middle one. Based on their CT scans, the top and bottom cores were very homogeneous whereas the middle one showed some signs of bioturbation. In order to understand their in-situ microbial communities, DNA isolation was attempted from the inner section of each core under sterile conditions. Despite the low biomass and the high concentration of hydrocarbons present sufficient DNA was recovered for all three cores which was suitable for downstream processing. Amplicons from the V4-V5 variable regions of 16S rRNA marker gene were produced from each reservoir core and sequenced using the Illumina sequencing platform (2x300bp). The microbial profiles deduced from these amplicons identified a high proportion of members from the Oxalobacteraceae family (31-50 %) followed by members from the Comamonadaceae family (7-24%). Members from both of these families are known to be able to reduce nitrate (and/or chlorate) under anaerobic conditions. Such finding might suggest a faster response of any in situ NRBs to any potential nitrate injections. On the contrary, very few delta-Proteobacteria (and SRBs) were detected in these cores (<0.01%) suggesting that the current reservoir conditions might be unfavorable to SRB growth. However, if any seawater injection takes place in the future, the microbial abundance and proportion of these SRBs may well change if no nitrate is also injected. The findings from this study were also used for defining a baseline for modeling and prediction of souring of this particular field using the SourSim toolkit. More specifically; based on these results souring simulation runs were conducted assuming higher temperature thresholds than the default (70°C). The reservoir temperature of this field is 105°C. Effects of hydraulic frac fluids and formation waters on groundwater microbial communities Núria Jiménez, Martin Krueger German Federal Institute for Geosciences and Natural Resources-BGR Shale gas is being considered as a complementary energy resource to other fossil fuels. Its exploitation requires using advanced drilling techniques and hydraulic stimulation (fracking). During fracking operations, large amounts of fluids (fresh water, proppants and chemicals) are injected at high pressures into the formations, to create fractures and fissures, and thus to release gas from the source rock into the wellbore. The injected fluid partly remains in the formation, while up to 40% flows back to the surface, together with reservoir waters, sometimes containing dissolved hydrocarbons, high salt concentrations, etc. The aim of our study was to investigate the potential impacts of frac or geogenic chemicals, frac fluid, formation water or flowback on groudnwater microbial communities. Laboratory experiments under in situ conditions (i.e. at in situ temperatures, with high pressure, etc.) were conducted using groundwater samples from three different locations. Series of microcosms (3 of each kind) containing R2 broth medium or groundwater spiked with either single frac chemicals (including biocides), frac fluids, artificial reservoir water, NaCl, or

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different mixtures of reservoir water and frac fluid (to simulate flowback) were incubated in the dark. Controls included non-amended and non-inoculated microcosms. Classical microbiological methods and molecular analyses were used to assess changes in the microbial abundance, community structure and function in response to the different treatments. Microbial communities were quite halotolerant and their growth benefited from low concentrations of reservoir waters or salt, but they were negatively affected by higher concentrations of formation waters, salt, biocides or frac fluids. Changes on the microbial community structure could be detected by T-RFLP. Single frac components like guar gum or choline chloride were used as substrates, while others like triethanolamine or light oil distillate hydrogenated prevented microbial growth in groundwaters. Ongoing work will provide information on potential transformations of frac or geogenic chemicals by groundwater microbiota and their lifetime. Investigation of the bacterial population in a bentonite plug mimicking well cements Daphne Jalique, Alexander Grigoryan, Simcha Stroes-Gascoyne, Darren Korber University of Saskatchewan Cementing is currently one of the most conventional completion methods for vertical and directional wells. Used for a number of different reasons, cementing protects and seals the wellbore. The poor quality of cements reduces their performance and therefore poses an engineering problem. Microbial contamination is one such quality hazard. Bacteria produce a number of deleterious by-products (i.e. acids, gases, etc.), which can compromise well-seal integrity. However, there is a lack of information on microbial distribution in sealing blends and environmental factors controlling viability and propagation of microorganisms in cemented systems over long time periods comparable to an oil-well lifecycle. Here, we report on the culturability and phylogenetic diversity of aerobic organotrophic bacteria in a compacted bentonite (85% smectite) plug, which had been continuously infused with high-pressure distilled water for a period of ~8 years, mimicking some oil-well compacted cement and clay sealants. Background levels of bacterial counts in the commercial bentonite used in this study were ~102 CFU/g. Throughout the long-term experiment, the bentonite dry density and swelling pressure attained 1.8 g/cm3 and 23 MPa, respectively, and water activity was <0.96. The number of culturable aerobes from the interfacial and internal regions of the plug was 104 CFU/g and 10^2 CFU/g, respectively. 16S rDNA analysis determined that microorganisms isolated from the plug were affiliated with endospore-forming bacteria of the families Bacillaceae and Paenibacillaceae: i.e. Bacillus niacini, B. soli, B. drentensis, B. firmus, B. licheniformis, Paenibacillus naphthalenovorans, Pb. borealis, Pb. wynnii and Brevibacillus limnophilus. We hypothesize that during long-term experimentation, bacteria indigenous to the commercial bentonite proliferated on the interfaces of highly-compacted material; however, bacterial activity inside the plug was negligible due to adverse environmental factors (i.e. low water activity, high swelling pressure, etc.). Microorganisms with dormant forms, such as the identified spore-forming bacilli, were found to exclusively survive these stressful conditions. It is proposed that the observations from the bentonite plug model can be extended to microbial processes in other artificial (i.e. cement) or natural (i.e., geological) highly-compacted porous sealing media, where low water activity and high hydrostatic pressure, along with other environmental stresses (i.e. high pH), control microbial activity. Geobatteries: is there a limitation to adding hydrogen to porous natural gas storages? Johanna Schritter1, Kerstin Scherr1, Robert Komm1, Diana Backes1, Markus Pichler2, Stephan Bauer 2, Andreas Loibner1 1 BOKU University of Natural Resources and Life Sciences, 2 RAG Electricity production form renewable sources are of increased importance for mitigating effects of fossil energy production on climate change. However, peak production is just partly in line with peak consumption. While conversion of renewable electricity to hydrogen (Power-to-Gas) is broadly discussed throughout Europe, efficient storage possibilities for hydrogen are still a matter of investigation. In the Underground-Sunstorage research project, possibilities to store hydrogen blended with (renewable) methane, as a chemically conserved renewable energy in depleted porous underground gas reservoirs are

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explored. However, it is expected that biogeochemical transformation processes in reservoirs will interfere with hydrogen in terms of well clogging, acidification, pressure loss, and MIC. Thus, the objective of this study is to characterize microbial and geochemical processes associated to the introduction of hydrogen into natural gas reservoirs, e.g. hydrogenotrophic and acetoclastic methanogenesis, homoacetogenesis, sulphate- and iron-reduction, on a qualitative and quantitative level. To simulate storage of hydrogen, cores (diameter ~10 cm, length ~20 cm) from an underground porous gas storage were placed in corrosion resistant bioreactors and operated at reservoir conditions (46 bar, 45°C). Gas reservoir formation water was used for inoculation. The reactors were initially operated under a methane atmosphere to simulate reservoir conditions for two months. Subsequently, the 10 bioreactors (including 2 abiotic controls) were loaded with several gas mixtures containing different concentrations of hydrogen (4-10%), methane and carbon dioxide. Prior to and after 6 months of exposure, cores and reactor fluids were analysed with respect to hydrochemical and microbiological characteristics: pH, conductivity, microbial electron acceptors, nutrients, and a comprehensive list of elements. Eubacterial and archaeal communities were profiled. During gas exposure, temperature, partial pressure of methane, hydrogen, carbon di- and monoxide, hydrogen sulphide and acetic acid were monitored. During incubation, distinct changes in the gas composition were observed. Microbial processes were found to be the driving force, with background geochemical processes. Quality and quantity of transformation processes were found to be highly dependent on microbial and petrographical reservoir properties and are thus highly reservoir-specific. With respect to these boundaries, the storage of hydrogen in porous gas storages is a promising approach to solving imminent energy issues.

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Session 4 - Posters: P27. Anaerobic microbial communities and their potential for bioenergy production in heavily biodegraded crude oil reservoirs Julia R. de Rezende1, Angela Sherry1, Tetyana Korin1, Ian M. Head1, Alexander Grigoryan2, Gerrit Voordouw3, Casey Hubert3 1 Newcastle University, 2 University of Saskatchewan, 3 University of Calgary Most of the oil in low temperature, non-uplifted reservoirs is biodegraded due to millions of years of microbial activity. To assess the potential of heavy oil for bioenergy production through methanogenesis, we combined basal water and surface-mined bitumen from an Athabasca oil sands reservoir (Alberta, Canada) in microcosms that were incubated for 3000 days under different redox conditions, with bitumen as the only organic substrate. Maximal rates of methanogenesis were below 15 nmol/day/g oil sands. This is 10 to 1000x lower than other published reports of methanogenesis from lighter crude oils. Methanogenesis was eventually observed in microcosms originally set up under sulfate- or oxygen-reducing conditions suggesting that these electron acceptors were consumed first. Interestingly, in sulfate-reducing microcosms methanogenesis took place in the presence of >20 mM sulfate that had not been removed. No methanogenesis was observed in nitrate-reducing microcosms. Ion torrent sequencing of 16S rRNA genes in the 3000-day microcosms revealed significant differences in community composition between methanogenic and non-methanogenic microcosms. The presence and high relative abundance of Methanosarcinaceae, Syntrophaceae, Desulfobulbaceae, Desulfovibrionaceae, Desulfomicrobiaceae and Geobacteraceae in libraries from methanogenic microcosms point to possible syntrophic partnerships involved in methane generation from bitumen. These results demonstrate that microbial communities in Athabasca oil sands are capable of accessing a limited pool of organic carbon present in severely biodegraded heavy oil as substrates for further biodegradation resulting in methanogenesis, but that rates are relatively low.

P28. Microbial communities responsible for CO2 fixation in petroleum reservoirs with different temperatures revealed by using mcrA, cbbM, cbbL, fthfs, fefe-hydrogenase genes as molecular biomarkers Jin-Feng Liu1, Guang-Chao Yang1, Serge Maurice Mbadinga1, Ji-dong Gu2, Bo-Zhong Mu1 1 East China University of Science and Technology, 2 The University of Hong Kong Sequestration of CO2 in oil reservoirs is one of the feasible options for mitigating atmospheric CO2 building up. The in situ bioconversion of sequestrated CO2 to methane by microorganisms inhabiting the oil reservoirs is believed to be active. To evaluate the potential of in situ microbial fixation and conversion of CO2 into CH4 in oil reservoir system, a comprehensive molecular survey was performed on microbial

communities inhabiting four oil reservoirs with different temperatures from 21C to 90C by analysis of functional genes involved in the biochemical pathways of CO2 fixation and bioconversion, namely cbbM, cbbL, fthfs, [FeFe]-hydrogenase encoding gene, and mcrA. A rich diversity of these functional genes was found in all the samples with both high and low temperatures, which were affiliated to members of the Proteobacteria (cbbL and cbbM, fthfs), Firmicutes and Actinobacteria (fthfs), uncultured bacteria ([FeFe]-hydrogenase), and Methanomirobiales, Methanobacteriales and Methanosarcinales (mcrA). The predominant methanogens were all identified to be hydrogenotrophic CO2 -reducing ones. These results showed that the occurrence of functional microbial communities universally in oil reservoirs, suggesting the potential capability for microbial fixation and bioconversion of CO2 into methane, which is important to not only microbial recycling of sequestrated CO2 but also production of additional methane in oil reservoirs.

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P29. Sophorolipid production using cotton seed oil and its activity against KB cancer cell line Farazul Haque Institute of microbial technology,Chandigarh Now a day’s sophorolipid (SL) production is the most thriving field and tempted many researchers towards it. Due to biomedical application potentials and its superior surface active properties, SL is considered to be one of the most promising biosurfactant with much industrial importance. SL is produced by yeast via fermentation of sugars with suitable hydrocarbons or oils as lipid precursors but requires lots of efforts, money and time. In the present study, we approached cost effective production of SL using cheapest available vegetable oils in a lesser period of time. Among all other oils used Cotton seed oil gives immensely high amount of SL in shake flask as well as in fermentor i.e 88.5 g/l and 213 g/l respectively . Interrelation between production and other growth parameters were also keenly analysed in the presence of each oils used. Quality of produced SL was checked by performing bioactivity assay on KB cancer cell line. This is the first report using cotton seed oil as a cheapest substrate and its activity on KB cell line.

P30. The characterization of a novel bioemulsifier-producing bacterium, Geobacillus stearothermophilus DM-2 isolated from the oil reservoir Ting Ma, Jiefang Zhou, Guoqiang Li Nankai University This article evaluated the potential of biosurfactant production when the Geobacillus stearothermophilus DM-2 growing at different carbon sources, especially in case of cheap carbon sources. The biosurfactant produced by strain DM-2 were partially purified and the structural were fully characterized. Meanwhile, the application of the biosurfactant on enhanced oil recovery was investigated. Geobacillus stearothermophilus DM-2 was isolated from produced water of Dagang Oilfield, China. It was a thermophilic bacterium, and could produce surface active agent when growing at 40-70 °C. The maximum yield of biosurfactant could be achieved with the cheap carbon source-sodium acetate added. Composition analysis of the biosurfactant indicated that the biosurfactant was a novel glycoprotein emulsifier, and the linkage of sugar and polypeptide was N-connection. The emulsifier composed by 72% sugar content, among them, that monosaccharide elements were glucose (31.9%), mannose (37.8%), galactose (28.0%) and glucuronic acid (2.3%), and the protein content was 27%, from 17 kinds of polar and non-polar amino acids. The emulsifier could emulsify various hydrocarbons. Emulsion stability test showed that the bioemulsifier is still effective when the salt concentration reached 25%, the temperature reached 70°C. Particularly noteworthy, at the range of pH1-14, its activity could maintain 100% without loss. The crude oil recovery studies using the bioemulsifier produced by Geobacillus stearothermophilus DM-2 suggested its potential applications in the field of oil exploration, especially the high temperature reservoir exploration.

P31. Bio-Electrochemical conversion of carbon dioxide to methane by using indigenous microbes in subsurface reservoir Haruo Maeda1, Masayuki Ikarashi1, Naoya Fukushima2, Hajime Kobayashi 2, Kozo Sato2 1 INPEX Corporation, 2The University of Tokyo Recently, the use of depleted oil-gas reservoirs and aquifer has been proposed, as potential CO2 geological storage site in CCS, The long-term aim of this research is to establish a biotechnological system to microbiologically convert geologically stored CO2 into methane, as energy resource. To develop a means for the conversion, we focus on technological application of bio-electrochemical reaction using microbially catalyzed electrode. On the electrode surface, methanogenic microorganisms (as biocatalysts) utilize electrical current (electrons) to reduce CO2 (CO2+ 8H+ + 8e- → CH4 + 2H2O). For such system, recruitment of microorganisms indigenous to the reservoir as the biocatalysts will be a better choice, as they are more likely to maintain the activity within the reservoir. Toward technological application of the bio-electrochemical system in CO2 geological reservoir, we examined bio-electromethanogenic activity of subsurface microbial consortium for the first time. Indigenous microorganisms originated from a formation

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water of a domestic depleted oil reservoir were inoculated into electrochemical-cultivation reactors. Upon application of constant voltage of -0.80 V, the reactor inoculated with reservoir-indigenous microorganisms produced methane at maximum rate of 1103 mmol day-1m-2 (cathode surface area), which was the highest electromethanogenic production rate so far documented. Moreover, current-to-methane conversion efficiency of the reaction was almost 100% with all applied voltages tested. Thus, it was suggested that subsurface microbes have high ability to catalyze electromethanogenesis. P32. Freezing bacterial communities affecting oil and gas industry Iris Porat, William Morris Kemira Kemira’s microbial biocide studies depend on the supply of healthy and consistent communities of bacteria affecting the oil and gas industry. Usually, a customer sample is obtained and the microbial community including sulfate-reducing bacteria (SRB) and acid producing bacteria (APB)/general heterotrophic bacteria (GHB) is grown followed by biocide efficacy testing. Different samples contain different mixture of bacteria. In order to compare different biocide treatments, it is important to have the same bacterial mixture as biocide treatment target. Bacteria mixture could be stored at 4oC for a few days. However for long time

storage, the microbial community would need to be frozen and stored at -80C. This study tested the possibility of freezing microbial communities and compares the bacterial composition of the mixture before and after the freezing treatment. Bacteria communities from before and after freezing treatment were identified by pyrosequencing; the DNA from the bacteria mixture were isolated and pyrosequencing, targeting the gene 16S rRNA, was used. SRB and APB/GHB were grown in Modified Posgate B media (MPB)

and Phenol Red Dextrose media (PRD), respectively, at 3% salinity at 32C. The dominants strains detected from PRD growths where Citrobacter amalonaticus, Rhodobacter capsulatus, Vibrio furnissii, Enterococcus sp. and Clostridium boliviensis. The species R. capsulatus, C. amalonaticus and Enterococcus sp. were found in abundance before and after freezing treatment. The dominants strains detected from MPB growth were Alkaliphilus sp, Citrobacter amalonaticus, two species of Clostridium, three species of Desulfovibrio, Dethiosulfovibrio peptidovorans, Fusibacter paucivorans, Rhodobacter capsulatus, Sedimentibacter sp. and Vibrio vulnificus. Seven of the species grown on MPB media, were found in abundance before and after freezing treatment. In spite the differences found in the microbial communities when comparing the samples before and after freezing treatment, the method could be used to supply a consistent microbial community for biocide experiments. P33. Microbial community composition of two natural asphalt seeps from the Kurdistan region of Iraq Adris Shlimon1, Kasper Kjeldsen2, Kai Finster2 1 Soran University, 2 Aarhus University The aim of this study was to identify microorganisms populating natural asphalt seeps with a potential role in heavy oil formation. The microbial community composition of two geographically separate natural asphalt seeps, from the Kurdistan region of Iraq, were compared to the community composition in the soil underlying and flanking one of the seeps. For community profiling an approximately 280 bp long 16S rRNA gene fragment was PCR amplified from DNA directly extracted from the samples using an universal prokaryotic primer pair and sequenced on an IonTorrent sequencing platform. The microbial community composition of the two asphalt samples was more similar to each other than to any of the soil samples, both with respect to OTU composition and their overall taxonomic composition. The two asphalt samples were dominated by sequences affiliated the bacterial genus Deferribacter constituting 30% and 69% of the total number of reads in these libraries. Another group of bacteria, which was highly enriched in the asphalt samples, was the family Thermaceae (Phylum Deinococcus- Thermus), which accounted for 8-9% of the reads in both libraries. Furthermore, Proteobacteria were abundant in both asphalt samples with putative sulfate-reducing Deltaproteobacteria accounting for 12% the library reads. In agreement 0.6-2.0 mM sulfate was present in the water phase of the asphalt from the two seeps and. Archaea including methanogens were rare (<1%) in all samples analyzed. In conclusion Asphalt seeps harbor a unique

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microbial community dominated by Deferribacter, Delta- and Gamma Proteobacteria and members of Thermaceae family. Anaerobic mineralization of asphalt components may proceed via sulfate reduction and reduction of unknown electron acceptors used by Deferribacter, as members of this group are currently not known to reduce sulfate. In the next step the functional role of the dominant microbial community members will be addressed.

P34. Microbiological quantification and evaluation Of biocorrosion In biofilm samples from the walls of Jardim Da Princesa, Rio De Janeiro Diogo Dias1, Ulrich Vasconcelos2, Eliana Sérvulo1 , Márcia Lutterbach3 1 Universidade Federal do Rio de Janeiro, 2 Universidade Federal da Paraíba, 3 Instituto Nacional de Tecnologia The three main factors which may influence the deterioration of cultural heritage are weathering, the natural or anthropogenic pollution and by biodeterioration processes. The third one is related to biofilm formation which implies efforts in order to protect buildings, bridges as well as monuments. Cultural heritage comprises a people or country’s identity composed by largely archaeological and historical monument and statues. Its material may deteriorate and despite aesthetical matters, the historical value of such monuments encourages several studies particularly in developed countries. Only in few years researches on such issue have been achieved in Latin countries, such as Brazil. In the context of Brazilian heritage preservation, the goal of this study was to investigate biofilm composition and its implication on the deterioration of concrete. The study was set in the Jardim da Princesa at the National Museum in Rio de Janeiro. A detailed scanning was performed in order to know the state of conservation as well as to characterize weathering role on the deterioration. The buildings were also photographed. Samples from adhered slime on walls were gently collected for the microbiological analysis involving eight groups: total aerobes heterothrophic, precipitating-iron bacteria, acid-producing bacteria, total anaerobes, sulphate reducing bacteria, actinobacteria, algae and total fungi. The biofilm formation was assessed using coupons composed by a mixture of sand, clay and cement. SEM analysis verified integrity and characterized the microbial diversity which varied according sample sites as well as the humidity, despite the coating material on walls and weather conditions were the same.

Session 5 – MIC, Souring of the North Sea and beyond

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Session 5 - MIC, Souring of the North Sea and beyond

Session Chairs: Tony Mitchell and Casey Hubert

Invited Talk:

Advances in the application of molecular microbiological methods in the oil and gas industry and links to

microbiologically influenced corrosion

Richard Eckert1, Torben Lund Skovhus2 1 DNV GL, 2 VIA University College, Research Group for Energy and Environment While the oil and gas industry has witnessed increased applications of molecular microbiological methods (MMM) for diagnosing and managing microbiologically influenced corrosion (MIC) in the past decade, the process for establishing clear links between microbiological conditions and corrosion mechanisms is still emerging. Different MMMs provide various types of information about microbial diversity, abundance, activity and function, all of which are quite different from the culture-based results that are familiar to many industry corrosion professionals. In addition, a multidisciplinary process for establishing the significance of molecular microbiological data in regard to corrosion threat identification, mitigation and monitoring has yet to be clearly established. As a result, the benefits of employing MMMs for MIC management are not yet being fully realized or appreciated. Regardless of advances in technology, the microbiological insights being afforded by MMMs will not be embraced by many operators until their significance relative to corrosion management and asset integrity are made more transparent. Achieving these goals will require continuing close collaboration between the disciplines of microbiology, corrosion and materials, chemical mitigation, and asset operations. Further, some type of initiative is needed to bring these different corrosion and microbiological monitoring technologies and disciplinary experts together to reach a common understanding and to provide a clear path for broader industry engagement.

Offered Talks: Isolation, characterization and genome analysis of a nitrate reducing Arcobacter sp. isolated from saline aquifer water Irene Roalkvam, Karine Drønen, Runar Stokke, Ida Steen University of Bergen Corrosion of mild steel is a pervasive problem in the oil industry, and the replacement of pipes and installations is costly. While sulfate reducing bacteria are well studied, less is known about other co-occurring microbial taxa involved in microbially induced corrosion (MIC). To expand our knowledge on this, a nitrate reducing Arcobacter strain was isolated from the Oilfield A in the North Sea, where saline aquifer water is added to the injection water. Recent studies showed that the aquifer water has a great corrosion potential, and the corrosion rate increased considerable in nitrate-amended experiments (Drønen et al., 2014). Physiological and metabolic characterization of the Arcobacter isolate revealed a mesophilic microorganism capable of respiration with nitrate, sulfur, ferric iron and oxygen (3-10% O2). Organic substrates such as acetate, lactate, peptone, pyruvate, tryptone, xylan and yeast extract are utilized, as well as H2, H2S and thiosulfate. In order to identify the genes encoding these processes, the genome of the isolate was sequenced using the SMRT technology from Pacific Biosciences. Preliminary genome analyses include genes for flagellar motility, chemotaxis and biofilm formation, in addition to genes encoding metabolic pathways corresponding to the substrates mentioned above. Overall, the Arcobacter strain has a

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great MIC potential as a result of its capacity for biofilm formation and wide metabolic range. This includes production of corrosive agents like nitrite and H2S, or maintaining favorable growth conditions for SRB by O2 removal, detoxification of H2S and H2 scavenging from their fermentation. The Arcobacter strain might also play different roles at different MIC situations, depending on environmental conditions and co-occurring taxa in the biofilm. Nitrate-mediated souring control in a low temperature, saline shale oil reservoir Annie (Biwen) An, Priyesh Menon, Gerrit Voordouw University of Calgary Souring in oil reservoirs is caused by sulfate reducing bacteria (SRB) through their reduction of sulfate to hydrogen sulfide. The formation of sulfide will not only decrease the value of the produced oil, but will also damage the oil reservoir facilities through microbially influenced corrosion (MIC). SRB in oil reservoirs are either indigenous or introduced during water flooding for secondary oil recovery. One way to inhibit sulfide production is through the injection of nitrate, which stimulates the growth of nitrate reducing bacteria (NRB). NRB can reduce nitrate to nitrite and then to N2 or ammonium. Nitrite competitively inhibits dissimilatory sulfite reductase (Dsr), the key enzyme catalyzing the reduction of sulfite to sulfide as the last step of the sulfate reduction pathway. However, the reduction of nitrate to nitrite is only a partial reaction. The end products (N2 or ammonium) of nitrate reduction do not inhibit sulfide production. Samples obtained from a saline (~2.3 M NaCl), low temperature, shale oil reservoir in Saskatchewan, Canada showed halophilic SRB and NRB activities. The low salt (~0.7 M NaCl) injection water used for secondary oil recovery from the shale oil reservoir contained high levels of sulfate (~28 mM), which stimulated the growth of halophilic SRB causing souring in this field. Microbial community analysis showed the presence of halophilic SRB such as Desulfohalobium, as well as other anaerobic halophiles (Methanohalophilus, Halanaerobium). In high salt enrichments, halophilic NRB activity was also observed. Interestingly, nitrate reduction terminated at nitrite under high salt conditions allowing effective inhibition of sulfate production by low concentrations of added nitrate. We are currently working on determining the species of halophilic NRB involved and their potential for souring control under various saline conditions using microcosms and model bioreactor studies. Preliminary Field Results of Stable Isotope Monitoring of Reservoir Souring Mark Conrad1, Christopher Hubbard1, Markus Bill1, Ken Wunch2, Martin Carrera2 1 Lawrence Berkeley National Laboratory, 2 BP The stable isotopic composition of sulfate and sulfide in produced water present a promising tool for monitoring microbial sulfate reduction in oil reservoirs. Many oil reservoir formation waters have low concentrations of sulfate, particularly when compared with seawater that is often used for water injection to support production, especially in off shore fields. Following seawater breakthrough at a production well, the isotopic composition of the sulfate will therefore tend towards a seawater-dominated signature (δ34S ~21‰, δ18O ~9‰). Microbial sulfate reduction will result in progressive increases in the isotopic composition of the produced water sulfate, potentially allowing early detection of souring before sulfide breakthrough. The isotopic composition of the sulfate is relatively insensitive to factors such as precipitation of minerals (e.g., iron sulfides, barite) that can affect the concentrations of sulfate in the produced water. However, this simple model may be complicated if, (i) the reservoir formation water has high sulfate, (ii) different water sources are used for water injection e.g. produced water reinjection, (iii) chemical treatments shift the injection sulfate isotopic composition, and (iv) the reservoir is naturally sour. We will present preliminary results from isotopic analyses of waters from reservoirs at different stages of souring and under different injection regimes. This dataset will be used to test the robustness of stable isotope monitoring for tracking reservoir-souring processes. Produced water samples from sour reservoirs have sulfate δ34S values as high as 40‰ confirming the expected trend. In another case, the δ34S of sulfate from a reservoir not considered sour has been shifted to a higher value, suggesting that microbial sulfate reduction is occurring in the reservoir. Additional data from other reservoirs will be presented and

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discussed. We will also show how this data can be combined with water isotopes and chemistry to constrain the extent of souring. Complementary to our field campaign, we are conducting experiments to constrain the magnitude of isotope fractionation for reservoir-relevant processes, and integrating isotopes into reservoir models for a powerful predictive tool and hypothesis-testing framework. Comparing the effects of THPS and glutaraldehyde batch biocide treatment on microbial corrosion in circulating flow loops Dennis Enning, Ramsey Smith, Jennifer Hornemann, Sanket Desai ExxonMobil Upstream Research Company Microbiologically Influenced Corrosion (MIC) is one of the main types of corrosion with a profound impact on materials integrity in the oil and gas industry. Of particular interest in this context is the internal corrosion of large carbon steel pipelines, the replacement and repair of which can be very costly as well as disruptive to production. Pipelines for which a susceptibility to MIC has been identified are usually treated with biocides which are injected periodically (e.g. weekly) into the lines for a designated period of time (usually a few hours). These treatments are intended to kill or inhibit steel-attached corrosive microorganisms with the aim of slowing down overall pipe wall loss and preventing localized ‘pitting’. The comparative effectiveness of different biocides is usually determined in the laboratory. Most commonly this involves microbial ‘kill tests’ that evaluate the degree to which microbes in a biofilm can be killed or inactivated. However, the distinction between live and dead microorganisms in a complex biofilm is not always straightforward and results of such ‘kill tests’ do not necessarily translate directly into effective MIC control. We recently developed a methodology to test biocides with respect to their capability to reduce microbial corrosion rates (rather than kill efficiency) as a function of biocide type, treatment duration and treatment frequency. This presentation elaborates on the novel methodology and compares the effects of glutaraldehyde and THPS batch treatments on microbial corrosion rates, corrosion morphology and microbial community composition. It was found that under the tested conditions, highly corrosive (up to 80 mpy) SRB-dominated biofilms were controlled more effectively and more reliably with weekly treatments of THPS. Expanding the search for biosouring treatments: High-throughput identification of potent and specific inhibitors of sulfate-reduction Hans Carlson, John Coates University of California, Berkeley Despite the global environmental and economic cost of microbial sulfidogenesis in oil recovery operations, very few compounds have been identified as specific inhibitors of respiratory sulfate reducing microorganisms (SRM), and fewer studies have systematically and quantitatively evaluated the selectivity and potency of SRM inhibitors. We have developed a robust and general high-throughput screening strategy to quantitatively evaluate inhibitor potency and selectivity in a model sulfidogenic microbial ecosystem and inhibitor specificity for the sulfate reduction pathway in a model SRM. We have screened panels of inorganic oxyanions, biocides and small molecule libraries and have identified many novel and/or poorly characterized specific inhibitors of sulfidogenesis. None of the commonly utilized oil industry biocides (THPS, formaldehye, bronopol, benzalkonium chloride) displayed selectivity for SRM. In contrast, monofluorophosphate (FPO3

2-), an often overlooked sulfate analog, is a potent and selective inhibitor of SRM with a unique mechanism of action and other favorable chemical characteristics. In the >30,000 small molecules we have screened, several are potent and selective SRM inhibitors. We have also evaluated inhibitor synergism for a number of the screen hits. Using tagged-transposon pools of a model SRM, Desulfovibrio alaskensis G20, chemogenomic profiling of screen hits has provided insights into the mechanism of action of inhibitors, and 16S amplicon sequencing has provided insights into microbial community shifts upon inhibitor amendment. Our high-throughput workflow is general and our results show that it can be used successfully to identify new candidates for the treatment of biosouring in oil reservoirs and other industrial ecosystems.

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Session 5 - Posters: P35. Understanding microbiologically-influenced corrosion of metallic materials in marine environments: are –omics the answer? Iwona B Beech1, Zakari Makama1, Recep Avci2, Jan Sunner1 1 University of Oklahoma, 2 Montana State University Mechanisms by which bacterial biofilms thriving on metallic surfaces influence near-surface electrochemical processes leading to corrosion of these materials are subject of extensive studies. Deterioration of a metal due to microbial presence and/or activity is referred to as biocorrosion or microbiologically-influenced corrosion (MIC). The necessity of cells being present on the surface has been demonstrated as a prerequisite for MIC, and sampling of biofilm populations is advocated in order to diagnose biocorrosion. There is, however, a tendency to implicate MIC as the cause of corrosion failures, based solely on sampling of bulk fluids, i.e. by examining planktonic bacterial populations, and mitigation strategies are often designed based on planktonic data. This approach can, perhaps, be partially justified when applied to systems where a fluid, e.g. seawater, is re-circulating over an extended time period (years). Although planktonic and biofilm populations in such systems are, most likely, not too dissimilar, surface-associated population may still have markedly different phylogenetic and/or metabolic profile than the community present in the bulk phase. In open environments, structures of biofilm and planktonic populations vary considerably and laboratory testing of MIC using planktonic organisms can be misleading. A combination of electrochemical measurements, genomics, metabolomics, imaging and mineralogical analyses have been employed to study MIC of carbon steel and 70/30 CuNi alloys in marine environments. These laboratory investigations revealed that differences in (i) community structure between biofilm and planktonic bacterial populations; (ii) metabolic activities of these communities and (iii) chemical composition of corrosion products accumulating on biofilmed versus non-colonized surfaces, had a pronounced effect on corrosion. Indisputably, –omics have offered a powerful tooll allowing to gain unprecedented insight into mechanisms of biofilm-driven corrosion processes. However, an integrated approach, i.e. combining –omics with other techniques, in particular materials and surface science, is strongly advocated when diagnosing and mitigating MIC.

P36. Influence of microorganisms on corrosion of welded area Vitor Liduino1, Márcia Lutterbach2, André Ferreira3, Jorge Ribeiro3, Eliana Sérvulo1 1 Federal University of Rio de Janeiro, 2 Instituto Nacional de Tecnologia, 3 National Industrial Training Service The welding sector is in evidence due the construction of new plants of mining and energy, as well as uncountable developments of naval, oil, gas and petrochemical industries. Nevertheless, welded regions deserve special attention as deterioration may occur by various environmental factors, the major one corresponding to the high temperature cycles used in welding processes that results in the modification of the microstructure steel. Moreover, the ubiquity of microorganisms in natural and industrial environments makes it possible the biofilm formation on around (Heat Affected Zone) and over weld bead. In the biofilm may co-exist several microbial species, whose activity can result in the production of enzymes, EPS, organic and inorganic acids, which in turn may affect the cathodic and/or anodic reactions on metals, increasing the speed of electrochemical processes in biofilm/metal interface. But, researches in this subject are still scarce, so the importance of the study. The aim of this work was to investigate the corrosion by microbial activity on welded carbon steel coupons by using the coated electrode technique. For soldering, electrode 7018 (2.5 mm) and API 5L X65 steel coupons (6 cm2 area) were used. The tests were performed for 7 days in static reactor of 1000 mL capacity filled with 800 mL of polluted seawater, from Guanabara Bay (Rio de Janeiro, Brazil). For comparison, simultaneous tests with sterile seawater were performed under same conditions. The coupons with biofilms showed a visibly greater degradation than those in sterile condition. Quantitative microbiological analysis of the biofilm indicated iron-oxidizing bacteria with 1010 order of magnitude, 2 orders smaller than total heterotrophic aerobic bacteria. For coupons exposed to sterile seawater, the mean weight loss of circa 0.1016 mm/year is representative of moderate corrosion, while high corrosion rate (0.1462 mm/year) was evidenced for fresh seawater assays.

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P37. Enzyme-based hydrogen sulfide mitigation for oil wells and water treatment Prasad Dhulipala, Grahame Taylor, Nadjmeh Doostdar, Charles Armstrong Baker Hughes Acidithobacillus ferrooxidans (A. ferrooxidans) is a gram-negative, acidophilic and chemolithotropic bacterium that utilizes oxidation of ferrous ions, hydrogen and reduced inorganic sulfur compounds such as H2S as sources of energy. Sulfur oxidation in A. ferroxidans is catalyzed by the sulfide-quinone reductase (SQR) enzyme system. The initial step of the SQR reaction is the oxidation of sulfide to elemental sulfur or to the less toxic polysulfide. H2S may be further oxidized to intermediate sulfite and then to its final oxidation state sulphate by other enzymes in the sulfur metabolic pathway. SQR could prevent the formation of H2S in waters and reservoirs contaminated with sulfur reducing bacteria (SRB). H2S causes reservoir souring and corrosion and present danger to oilfield personnel due to its inherent toxicity. SQR would not destroy the SRBs already present in the system, but catalytically attacks the H2S and H2S precursors that are produced by SRBs. As an enzyme, SQR it is a greener, sustainable, and catalytic solution to a industry growing problem with regards to H2S mitigation. The SQR gene was cloned from A. ferrooxidans and the protein was expressed in E.Coli. Functional studies conducted to show that the enzyme is stable in brine solutions containing up to a 2% sea salt or KCl and 200°F. However, the enzyme showed narrow pH sensitivity of pH 6 to 7. Treatment of soured well water with SQR revealed a 25-fold reduction of H2S by SQR. Further testing of this enzyme to reduce sulfide in the standard sodium sulfide solution confirmed that SQR does indeed remove H2S in liquids. Evaluation of the SQR enzyme confirmed that the enzyme has the potential of acting as a barrier to prevent H2S from spreading in the well. Similarly, further testing of SQR to investigate its ability to reduce H2S in the head space of equilibrated aqueous and gaseous sulfide environments showed that the enzyme reduced H2S in both phases, as confirmed by gas chromatography and colorimetric analysis. In summary, the results suggest that SQR may be used as a novel and more environmentally compliant H2S scavenger.

P38. Heavy nitrate corrosion in anarobe aquifer injection water biofilm: Process modeling Karine Drønen1, Irene Roalkvam2, Janiche Beeder3, Terje Torsvik1, Ida Steen2, Arne Skauge1, Turid Liengen3 1 UniResearch CIPR, 2 University of Bergen, 3Statoil Nitrate treatment is well documented as a mitigation method for sea water based injection water. However, heavy carbon steel corrosion developed during nitrate mitigation of a flow rig connected to a water injection pipeline flowing anaerobe saline aquifer water. ER and LPR probes demonstrated a 50-days initial phase with relative stable corrosion rate <0.5 mm/year. This was followed by an extremely corrosive phase lasting 15 days before a terminal phase with slowly chasing corrosion. In the phase with heaviest corrosion, the corrosion rate showed erratic behaviour, with peaks of several hundred mm/year and a mean of 3.7 mm/year. Furthermore, a 1-2 mm thick deposit layer pile don the coupons having a black inner core with a greyish layer on top. X-ray diffraction analysis of the film demonstrated the presence of greigite (Fe3S4) and siderite (FeCO3). In the nonamended parallel rig, there was a long-lasting stable corrosion situation, with low to moderate (<0.1 mm/year) corrosion rates. No corrosion products were observed on the coupons. Genera- specific. QPCR primers quantified 74% of the microbial biofilm community in the nitrate-amended rig, and further 87% of the community of the nonamended parallel rig. The nonamended biofilm hosted 6.3 × 106 SRB cells/cm2 and the S35- sulfate-reduction rate was 1.1 μmol SO4

2-/cm2/day, being congruent with the estimated SRB biomass formation and the sulfate areal flux. Nitrate amendment caused an 18-fold smaller SRB population, but up to 44 times higher sulfate reduction rates. This H2S formation was insufficient to form the observed Fe3S4 layer. Additional H2S was provided by microbial disproportionation of sulfur, also explaining the increased accessibility of sulfate. The reduced nitrate specie nitrite inhibited the dominating H2-scavenging Desulfovibrio population, and sustained the formation of polysulfide and Fe3S4, herby also dissolved sulfur. This terminated the availability of acetate in the inner biofilm and caused cell starvation that initiated growth upon metallic electrons, probably by the sulfur- reducing Desulfuromonas population. On the basis of these observations we propose a model of heavy nitrate corrosion where three microbiological processes of nitrate reduction, disproportionation of sulfur, and metallic electron growth are nicely woven into each other.

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P40. Development of an activity based qPCR assay for the detection of viable corrosion causing microoganisms Sarah Eisenlord, Al Darzins, Ernest Lever, Amanda Harmon Gas Technology Institute Quantitative polymerase chain reaction (qPCR) is a valued, genetic-based technique used in the detection and quantification of a diverse set of microorganisms known to cause microbially-influenced corrosion (MIC) on the surface of steel pipelines used in the natural gas, oil, and water industries. However, the current MIC qPCR assay cannot differentiate between live and dead cells. Propidium monoazide (PMA), a DNA interchelating dye, when used in conjunction with real time qPCR has proven useful for differentiating live from dead cells in environmental, food and clinical samples but has not been evaluated for MIC microorganisms. PMA selectively enters membrane compromised dead cells where it binds to DNA with high affinity. With light activation the azidide group creates covalent cross-linkages within DNA to inhibit amplification, leaving only the live cell DNA to be targeted for qPCR. We describe the development of an activity based qPCR assay which can distinguish live from dead acid producing bacteria (APB), sulfate reducing bacteria (SRB), iron oxidizing bacteria (IOB), denitrifying bacteria (DNB), and archaea (AR): all organisms known to contribute to corrosive biofilms. We optimized the use of PMA on (1) pure cultures of Clostridium acetobutilicum (APB), Desulfovibrio vulgaris (SRB), Sphaerotilus natans (IOB), Pseudomonas aeruginosa (DNB), Pseudomonas putida (DNB), Shaewanella putrificans (BA) and Haloferax volcanii (AR) and (2) industry supplied water condensate samples from natural gas pipelines containing a mixed community of microorganisms. Standardization of the methods were performed on pure, heat killed, isopropanol killed, and biocide killed (XC-370) cultures and verified with viable plate cell counts. We detected up to a 6 log decrease in viable bacterial cells, and a 7.8 log decrease of Archaeal cells using our optimized PMA-qPCR methods with pure culture samples. Additional experiments using industry generated samples prove our methods are capable of distinguishing the active microbial population from the total organisms present in five major groups of MIC Bacteria and Archaea. We will discuss the limitations of the developed PMA-qPCR assay as well as the development of a preliminary corrosion Risk Assessment tool generated from a Bayesian network using corrosive failure analysis and qPCR distribution data collected over a four year period. P41. The corrosion of carbon steel in suboxic/sulfidogenic fuel/seawater environments: the role of metallurgy Recep Avci1, Joshua Martin1, Mark Wolfenden1, Bret Davis1 , Kilean Lucas1, Iwona Beech2 1 Montana State University, 2University of Oklahoma This presentation focuses on the role that the metallurgical preparation of carbon steel plays in its corrosion. When a micro- or nano-size phase (such as pearlite or MnS) is introduced into a pure Fe matrix it is expected that the immediate surroundings of these islands will distort the pure Fe lattice, giving rise to localized dislocations, which in turn cause localized plastic strain. We hypothesize that this strain increases carbon steel's propensity toward corrosion. This hypothesis was verified experimentally using carbon steel (1018) coupons cut and polished parallel or perpendicular to their rolling direction. The coupons were exposed to sulfidogenic environments under anaerobic or suboxic conditions. The cultures of interest were D. alkanenexedens, a hydrocarbon-degrading organism, in anaerobic artificial seawater and mixtures of Marinobacter and D. indonensiensis in a suboxic fuel/seawater environment. Predetermined areas were mapped using backscattered electron diffraction before corrosion. After corrosion the same areas were analyzed with and without the corrosion products using electron microscopy and atomic force microscopy. Corrosion was monitored in situ with electrochemical measurements. Matlab codes were written to compare corrosion rates of strained areas and unstrained areas. Predicted carbon steel corrosion rates were compared to actual corrosion rates determined from AFM depth measurements. A close correlation was observed between corrosion rate and plastic strain left from metallurgical processes. Areas containing a carbide phase (pearlite) within the iron lattice appear to be the most strained. Quantitative agreement was found between predicted and observed corrosion rates of strained areas on carbon steel. A galvanic potential difference of

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about 10-20 mV was predicted by the model calculations. This is in agreement with the experiments carried out with 1018 carbon steel and pure Fe subjected to the same MIC environment. Carbide and MnS phases in carbon steel introduce local strains within the carbon steel matrix, creating a 3-D network of galvanic cells between the strained and unstrained areas of the Fe lattice, which increases the metal’s propensity toward localized corrosion. Dissolution of MnS at low pH conditions gives rise to localized high concentrations of H2S, HS- and S, which fuels the localized corrosion. P42. Targeted Microbial Control for the Reservoir: Novel Thermophilic Testing Capabilities Identify New Biocide Actives for Souring Control Bei Yin, Terry Williams, Thomas Koehler, Brandon Morris, Kathleen Manna The Dow Chemical Company

Subsurface microbiology studies have demonstrated that thermophilic microorganisms, including bacteria and archaea, thrive underground in extreme environments, where hydrocarbon reservoirs exist. These thermophiles, particularly sulfide producers, can cause reservoir souring during hydrocarbon recovery operations such as waterflooding and hydraulic fracturing. To control microbial contamination and souring, biocides may be used in these challenging environments but have not been extensively studied under these conditions. Conventional oilfield biocide efficacy evaluations assess performance against mesophilic bacteria at temperatures much lower than actual reservoir temperatures. However, due to their variations in thermal stability and modes of actions, biocides may behave differently at elevated temperature. In addition, mesophilic bacteria are not representative of thermophilic prokaryotes, which are often the dominant microorganisms in deep hydrocarbon reservoirs. In this paper, we describe a multiple-challenge biocide

efficacy study against a thermophilic sulfide-producing bacterium Thermotoga petrophila at 75C for 16 days.

A mesophilic sulfide-producing bacterium Desulfovibrio longus was tested with the same treatments at 35C for comparison. It was found that several commonly used biocides such as glutaraldehyde, tetrakis(hydroxymethyl)phosphonium sulfate (THPS), and a glutaraldehyde-quaternary ammonium compound blend (Glut-Quat) were very effective against both mesophilic and thermophilic bacteria. However, their

efficacies persisted for shorter periods at 75C as compared to 35C. Non-traditional oilfield biocides such as cis-1-(3-chloroallyl)-3,5,7-triaza-1-azoniaadamantane chloride (CTAC), 4,4-dimethyloxazolidine (DMO), and tris

(hydroxymethyl) nitromethane (THNM) showed enhanced performance at 75C versus 35C and their efficacy persisted longer than the traditional oilfield biocides at elevated temperature. Further evaluations are being conducted in different matrices which mimic different field environments as well as against additional thermophiles including sulfide producing archaea. This study represents a significant advancement in expanding the understanding and validating the efficacy of alternative biocides in subsurface environments with challenging temperature characteristics.

P43. Using sand packed bioreactors to demonstrate the impact of downhole pressure on the sulphate-reducing microbial community Matt Streets, Bob Eden Rawwater Engineering Company Ltd The impact of downhole pressure on the microbial consortium and its souring capability was determined using sand packed, oil saturated, pressurized bioreactors to simulate injection into the oil leg of a reservoir at pressures ranging from 0psig to 3000psig. This paper will demonstrate how using sulphide concentration analysis in conjunction with Molecular Microbiological Methods (MMM) it is possible to determine the effects of downhole pressure on biological sulphide production and its impact on the diversity of reservoir microbial communities.

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P44. Use of Iron Beads to Determine MIC Under Flow Conditions Tijan Pinnock, Gerrit Voordouw University of Calgary

Carbon-steel corrosion costs the oil and gas industry billions of dollars each year. Microbially influenced corrosion (MIC) in oil and gas facilities is due in part to the activities of sulfate reducing bacteria (SRB). Some of these can remove electrons from iron directly to reduce sulfate to sulfide, accelerating corrosion rates. Although iron coupons are traditionally used to study MIC, we have explored the use of iron beads (55.0 ± 0.3

mg, ∅ = 0.238 cm). In SRB batch cultures, significant corrosion rates of up to 0.1 mm/yr have been observed during one month incubations. In order to study and understand SRB-mediated MIC in flowing systems, like those observed in oil and gas facilities and pipelines, an SRB consortium from an oilfield in southern Alberta was pumped into an up-flow column containing 35 iron and 35 similarly-sized glass beads. The SRB consortium was grown in a chemostat system in order to maintain constant conditions and flow of bacteria. SRB activity was monitored by determining sulfide, sulfate, and organic electron donor concentrations at different points along the flow path. Little ferrous iron was found in the column effluent, indicating that most was retained in the column as FeS and FeCO3. After 256 days of continuous flow, the carbon-steel beads were removed from the column and treated with strong acid followed by base (NACE RP0775-2005) to remove corrosion products from the steel-surface. Weight loss was used to determine the corrosion rate over the 8 month experiment. An overall industrially significant corrosion rate of 0.11 mm/yr was obtained. The residual weight of the beads ranged from 36.5 to 51.4 mg, with an average of 44.0 + 3.3 mg. The most heavily corroded beads showed significant pitting. The significant 11-fold increase in standard deviation from 0.3 to 3.3 mg is likely a good proxy for the unevenness of corrosion and, therefore, for pitting corrosion. As a result an effective system for studying SRB mediated MIC under flow conditions has been designed and this is now being used to determine the efficacy of various biocides and metabolic inhibitors in preventing general and pitting corrosion.

P45. Using Aramid Polymer Film Impressions to Determine Spatial Organization of Corrosion Coupon Biofilms Kerry Sublette1, Dora Ogles2, Richard Eckert3 1 University of Tulsa, 2 Microbial Insights, Inc., 3 DNV GL Advances in molecular biological tools have allowed investigators to remove the culture bias in microbial sampling of corrosive environments. DNA can be readily extracted from field samples to allow both qualitative [denaturing gradient gel electrophoresis (DGGE)] and quantitative analysis [quantitative polymerase chain reaction (qPCR)] of taxonomic and functional genes indicative of MIC-related organisms in biofilms associated with surface corrosion. However, a sampling bias remains in the analysis of biofilms. MIC biofilms are microbiologically diverse and structurally complex. Standard sampling of a biofilm on a corrosion coupon typically consists of DNA extraction of a coupon swab or whole coupon extraction. A sampling method that accounts for the spatial heterogeneity of biofilms has the potential to significantly improve MIC diagnosis. We have developed a method to address this spatial bias in the sampling of corrosion coupon biofilms which allows the biofilms to be sampled in layers parallel to the metal surface. A solution of an aramid polymer in dimethylacetamide is applied to the surface of the corrosion coupon in a thin layer. The coupon is then immersed in sterile distilled water causing the polymer to precipitate. The precipitating polymer entraps a layer of the biofilm matrix. The aramid polymer layer is removed then the process is repeated until all the biofilm has been recovered. Each of these layers can be extracted for DNA and analyzed using qPCR and/or DGGE. In this way the biofilm is fractionated in layers from outside to inside.This method has been tested on corrosion coupons from two field sites, a natural gas well drip line and an offshore seawater injection system. qPCR and DGGE analysis of bioflm layers from these samples have shown that 1) DNA can be readily extracted from the Nomex films; 2) whereas swabs of biofilms produced smeared DGGE banding patterns of little use in qualitative analysis, banding patterns of the Nomex layers were sharp and distinct; 3) for a given coupon the microbial community structure collected by the Nomex peels varied with depth in the biofilm, and 4) known MIC-associated organisms were found deep in the biofilms near the coupon surface.

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P46. Assessing sulfate-reducing bacteria in clay additives for oil and gas industry Alexander Grigoryan, Daphne Jalique, Kara Friesen, Prabhakara Medihala, Darren Korber University of Saskatchewan

The control of microbial contamination in water additives used at different stages of a well lifecycle is a significant part of the sustainable performance of oil and gas production systems. Detrimental microorganisms, like sulfate-reducing bacteria (SRB), introduced into a reservoir with water and additives can facilitate biogenic sulfide production, resulting in souring of the production fluids and gas, iron sulfide formation, and SRB-associated microbiologically influenced corrosion. Clay, i.e. bentonite, is one such component that is required for drilling and cementing operations. Thus, there is a strong rationale to characterize the distribution of SRB and other bacteria in clays that are used for hydrocarbons production. Here we report on the abundance and activity of SRB in several commercial bentonites frequently used in water-based mud or cementing systems.Preliminary results from microcosm experimentation indicate that our commercially available clays a colonised by metabolically-versatile bacteria. The number of mesophilic aerobic organotrophs, anaerobes with fermentative metabolism, and sulfate-reducers in the tested clays ranged from 10^2-10^3 CFU/g, ≥10^4 cell/g 102-103 CFU/g and ≥104 cells/g, respectively. In microcosms containing 10% clay slurry, the rates of sulfide production and the quantity of sulfide produced in different bentonites was shown to be dependent on temperature. Lactate was the preferential substrate for microbial sulfidogenesis in comparison with acetate. Data revealed that both complete lactate- and acetate-oxidizers proliferated at 15°C, though only incomplete oxidizers of these electron donors dominated at 37°C. A 16S rDNA PCR-DGGE-based community analysis of the bentonite microcosms revealed that bacterial genera included the Desulfobulbus, Desulfuromonas, Pelobacter and Pseudomonas. Some unidentified physico-chemical properties of the different clays, which resulted in different initial bacterial numbers, were presumably the cause for the variance in bacterial activity (i.e. sulfidogenesis) in the different microcosms. Overall, commercial bentonites are, not surprisingly, a source of diverse microorganisms. Therefore, an antibacterial treatment may be warranted in order to mitigate unwanted bacterial activity in different water- and bentonite-based blends that are intended for use in well drilling, completion, production or abandonment.

P47. Biofilm initiation and cell density is independent of planktonic cell density Damon Brown1, Shawna Johnston1, Raymond Turner2, Pat Teevens1 1 Broadsword Corrosion Engineering , 2 University of Calgary Quantifying biofilm-associated microbes is of great importance in both medical and industrial fields, where biofilms can have effects ranging from chronic infections to metal corrosion. Bacteria have been associated with corrosion, known as microbially-influenced corrosion (MIC). Bacteria are able to cause corrosion indirectly through the production of corrosive agents such as hydrogen sulfide, through attack and removal of protective coatings or directly through consumption of electrons from iron. The production of biofilms can also directly contribute to corrosion by creating differential concentration cells on the surface of metals. Biofilms have been reported as either a causative agent of corrosion or a protective agent under different environmental conditions. However, the unpredictability of biofilm growth makes predicting the effects of specific environments very difficult. Many approaches are used to quantify biofilm initiation (cell attachment), cell growth from microcolony formation to fully mature structured biofilms. It is of great importance to interest groups to be able to accurately quantify biofilm-associated cells and understand how the difference in planktonic cell populations can affect the initiation of biofilms and their proliferation. Here, we study the initiation of biofilm growth in terms of biofilm-associated cells of single-species biofilms with respect to planktonic inoculation levels. An aerobic soil species (Pseudomonas fluorescens) and an anaerobic species (Geoalkalibacter subterraneus) known to be in association with oil pipeline corrosion were studied using the Calgary Biofilm Device™ with sonication disruption and CFU plate counting along with confocal imaging. Results indicate that for P. fluorescens and G. subterraneus, a minimum concentration of planktonic cells are required to initiate biofilm formation, then biofilms reach a maximum CFU count. Following biofilm formation and maturation, the concentration of biofilm-associated cells remains constant regardless of the planktonic

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cell concentration. These results show that an established biofilm will reach a steady-state cell concentration where biofilm-associated cells remain constant regardless of planktonic cell densities. This finding has great significance for the oil and gas industry; as it suggests that traditional planktonic cell counts are an inaccurate way of estimating microbial numbers. This may lead to inaccurate risk assessments of microbially influenced corrosion. P48. Composition and corrosivity of extracellular polymeric substance from the hydrocarbon-degrading sulfate-reducing bacterium Desulfoglaeba alkanexedens Tiffany Lenhart, Irene Davidova, Ravindranath Garimella, Sylvie Le Borgne, Mark Nanny, Joseph Suflita University of Oklahoma Planktonic and biofilm-associated anaerobes, most notably the sulfate reducing bacteria, have long been implicated in metal biocorrosion processes. Some anaerobes produce extracellular polymeric substances (EPS) and thereby provide a matrix for cell aggregation and biofilm formation. Further, bacterial EPS was directly implicated in the corrosion of mild steel. To our knowledge, the composition and corrosivity of EPS from anaerobic microbes growing on hydrophobic substrates has not been well studied. Yet, all known anaerobic hydrocarbon-degrading bacteria have a tendency to aggregate and to produce EPS. We isolated EPS from the hydrocarbon-degrading and corrosive sulfate reducing bacterium, Desulfoglaeba alkanexedens, strain ALDC. When cultivated on either decane or butyrate, the resulting EPS was largely composed of a polysaccharide with oxygenated alkyl functional groups bonded to the saccharide ring via carbon-carbon bonding. Negligible amounts of proteins and lipids were detected. The largely saccharide nature of EPS was confirmed by liquid 1H nuclear magnetic resonance spectroscopy experiments (1-D, COSY, DOSY). The corrosivity of the D. alkanexedens EPS to carbon steel coupons was also assessed as evidenced by weight loss determinations, total iron measurements, and scanning electron microscopy. Our results suggested that, relative to filter-sterilized culture fluids or live cultures, the EPS of this organism is not particularly corrosive and may actually protect the metal. This study is an initial characterization of EPS from an anaerobic hydrocarbon-degrading bacterium and suggests that the biofilm matrix produced by this organism is less important for carbon steel corrosion than the metabolic products of hydrocarbon metabolism. P50. Experimental Constraints on Isotope Fractionation During Microbial Souring Christopher Hubbard1, Jil Geller1, Markus Bill1, Lauren Tom1, Yvette Piceno1 , Anna Engelbrektson2, Pravin Shrestha2, John Coates2, Gary Andersen1 , Mark Conrad1 1 Lawrence Berkeley National Laboratory, 2 University of California, Berkeley Microbial sulfate reduction is well known to impart large, characteristic shifts in the sulfur and oxygen isotope compositions of the residual sulfate and produced sulfide, and may therefore be a useful tool for monitoring oil reservoir souring. The magnitude of these shifts is controlled by isotope fractionation factors which, for δ34S, have been experimentally shown to vary between -4‰ and +66‰ based on factors such as sulfate concentration, type and availability of electron donors, and composition of the microbial community. It is clearly essential to better constrain the magnitude of fractionation factors for oil reservoirs if we are to use isotope monitoring as a fully quantitative and predictive technique. Parallel to our field sampling efforts, we are conducting a range of batch and continuous flow experiments under reservoir-relevant conditions to constrain fractionation factors. Initial batch experiments carried out with a saline microbial community and crude oil as the electron donor exhibited classic Rayleigh fractionation with a 30‰ fractionation. Sulfate oxygen isotope values also increased by 10‰, suggesting partial re-equilibration with water. Ongoing batch experiments are also being conducted using enrichment cultures from oil reservoirs for comparison with these initial results. Increasing in complexity, continuous flow experiments can be used to explore how perturbations in conditions may change isotope fractionation. A good example for oil reservoirs is sulfate reduction linked to volatile fatty acid (VFA) oxidation, where the production of VFAs for sulfate reduction may be limited by the rates of degradation of more complex hydrocarbon components. This scenario would effectively create electron donor limitation when the rate of VFA utilization exceeds VFA formation. For a microbial community using acetate as an electron donor, we observed an increase in fractionation factor from ~28‰ to ~40‰ as

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sulfate reduction rates decreased by an order of magnitude following increased electron donor limitation. Work is ongoing to expand this approach to other electron donors (e.g. propionate) and extend the range of sulfate reduction rates examined. Finally, we plan to study how the addition of non-lethal concentrations of inhibitors (e.g. in the diffusive front of a treatment) further change sulfate reduction fractionation factors.

P51. How does the microbial community change in a soured well after a prolonged period of shut-in? Heike Hoffmann Intertek Reservoir Souring is the unplanned production of increased concentrations of hydrogen sulphide (H2S) in well-stream fluids from production wells that are subjected to water-injection. This is typically associated with the activity of a specialised group, the sulphate-reducing Bacteria (SRB). However, in recent years, various other micro-organisms are believed to be involved in souring, e.g. sulphate reducing Archaea (SRA). Various reasons can lead to well shutdown, e.g. maintenance, scale removal or when hydrogen sulphide gas is produced and escapes into the environment (e.g. offshore installation). When soured production wells, which have been shut-in for a period of time, are reopened do they show the same rate of sulphide production, lower or higher sulphide production compared to the rates detected before the shutdown? How does the shut in period affect the microbial community? In this study fixed film up flow bioreactors (FFBUR) were utilized to assess the potential for H2S production or changes in such H2S production, when seawater is injected into a North Sea oil reservoir. Analysis of microbial community composition by pyrosequencing indicated that although overall numbers of microbes present did not change significantly, the microbial community showed a distinct change towards a community primarily consisting of Desulfobacteracea, a group of complete-oxidizing SRB (belonging to the δ –proteobacteria); Desulfovibrionaceae, and Thermotogales. P52. Impact of light oil toxicity on souring by acetate-utilizing SRB Priyesh Menon, Gerrit Voordouw University of Calgary Souring the reduction of sulfate to sulfide by sulfate reducing bacteria (SRB) is a major concern in the oil and gas industry. SRB are a diverse group of anaerobes that couple the reduction of sulfate to the oxidation of organic-carbon including hydrocarbons. Souring has severe impacts on both onshore and offshore oil production operations. Interestingly, although acetate can be used as an electron donor for sulfate reduction by Desulfobacter and other completely-oxidizing SRB, it often accumulates in produced waters of water flooded oil fields, including seawater-injected oil fields, which have significant sulfate concentrations throughout. We therefore hypothesized that light oil components could be toxic to Desulfobacter species. Despite the fact that microorganisms can easily utilize low molecular weight hydrocarbon at low concentrations, the high concentration of these compounds in light oil can make them toxic preventing degradation due to their accumulation in the lipid bilayer of their cytoplasmic membranes. Experiments were done by inoculating an oil field sample into medium with acetate and sulfate in the presence or absence of light oil. It was observed that SRB oxidized acetate and reduced sulfate to sulfide in the absence, but not in the presence of light oil, suggesting that Desulfobacter species were inhibited by light oil toxicity. This observation was confirmed by repeating the same experiment with an inoculum of Desulfobacter postgatei (DSM 2034 strain 2ac9) with either light oils (30-40 ̊API), with heavy oil (16 ̊API), or with no oil. Sulfide production was observed only in the absence of oil and in the presence of heavy oil, but not in the presence of light oils, suggesting that light oils are toxic to Desulfobacter postgatei. We are currently conducting further experiments with different concentrations of light oil in an inert carrier phase to observe which concentrations and which components of light oil are toxic. Overall this work indicated toxicity of light oil to Desulfobacter species. Thus, in oilfield operations with high acetate and sulfate concentrations in produced waters, souring by Desulfobacter species may not start until the oil has been removed.

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P53. Multifaceted Approach to Biocide Selection Reveals Resistance Mechanisms and Survival Strategies Found in Oilfield Microbial Populations Timothy Tidwell, Zach Broussard, Victor Keasler, Renato De Paula, Brett Geissler NALCO Champion, an Ecolab Co.

Microorganisms present multiple challenges in oilfield systems such as microbiologically influenced corrosion (MIC), reservoir souring, and biofouling. The selection of chemistries to minimize microbial risk has traditionally relied on culture-based methods such as serial dilution to determine biocide efficacy. However, the diversity of microbes and the unique conditions found in each oil and gas asset make it very challenging to obtain reliable results through growth assays in the culture media commonly used. Our research has shown that a single method for performance evaluation does not always provide the definitive information required to select a biocide for field usage. In this study, we used a multitude of culture-independent methods, including flow cytometry, ATP/ADP/AMP quantification (adenylate energy charge), qPCR, and next-generation DNA sequencing coupled with cell sorting to correctly identify the highest risk organisms present in samples from a production system in the Eagle Ford Shale. Together, these evaluation methods identified a combination of chemistries to reduce plugging from suspended solids and mitigate the risk of corrosion failures at this sampling location. This multifaceted approach has revealed multiple resistance mechanisms and survival strategies found in microbial populations that have allowed us to design custom biocide treatment strategies to mitigate microbial risks in oilfield production systems.

P54. A Novel, Rapid Method of Total Bacteria Detection James R. Fajt M-I SWACO, A Schlumberger Company Bacteria that generate hydrogen sulfide or slime can cause corrosion or operational safety risks, resulting in significant negative economic impact on oil and gas production. We address the three most commonly used methods of detecting bacteria type and population—serial serum dilution, adenosine-triphosphate (ATP) testing, and plate-count determination—and detail each method’s theory of detection, limitations, and merits. Then, we compare these three methods with a novel, rapid-detection methodology. The three conventional methods discussed in the paper suffer long lead times, rendering them ill-suited as process-control aids. The new rapid bacteria test put forth in this paper overcomes this constraint and can produce reliable results within minutes compared with days when using traditional methods. The presented method provides a measure of total bacteria without differentiating bacteria type and has proven to reliably and quickly deliver bacterial status information, to help the user select appropriate disinfection strategies. We present supporting data on the use of this compact, user-friendly, accurate, and cost-efficient method of bacterial analysis in the field or the laboratory.

P55. Assessing the role of sulfide-oxidizing nitrate-reducing Epsilonproteobacteria in oil field corrosion

Sven Lahme.1, Eland L.E.2, Wipat A. 2, Head I.M.1, Curtis T.P.1 and Hubert C.R.J.1,3 1 School of Civil Engineering and Geosciences, Newcastle University, Newcastle upon Tyne NE1 7RU, UK, 2 School of Computing Science, Newcastle University, Newcastle upon Tyne NE1 7RU, UK 3 Department of Biological Sciences, University of Calgary, Calgary T2N 1N4, Canada Microbiologically-influenced corrosion (MIC) of metal infrastructure is a multibillion £ problem. In the oil industry MIC is often linked to the production of hydrogen sulfide (H2S) by sulfate(SO4

2−)-reducing microorganisms (SRM), e.g. due to injection of sulfate-rich seawater into oil reservoirs to maintain in-situ pressure. Nitrate (NO3

−) injection, as a ‘green’ bioengineering strategy, is often used to counteract souring by promoting: i) organotrophic nitrate-reducing microorganism (oNRM), competing with SRM for oil organic carbon sources, ii) sulfide-oxidizing NRM (soNRM), consuming the souring agent H2S and iii) NRM-mediated production of nitrite (NO2

−), as a potent SRM inhibitor. However, soNRM can produce corrosive

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sulfur intermediates such as elemental sulfur (S0), thiosulfate (S2O32−) or polysulfides (Sn-S2−).

Epsilonproteobacterial soNRM are frequently detected in oil reservoirs and have been linked to MIC during souring control by nitrate injection. The oil-field soNRM Sulfurimonas sp. strain CVO initially converts sulfide to elemental sulfur prior to further oxidation to SO4

2–. NO3–-dosing regimes at sour oil fields could

therefore affect soNRM metabolism and influence corrosion. Strain CVO was incubated with iron coupons at different initial N/S ratios ranging from 6.0–0.9. High corrosion rates of 0.23–0.27 mm/y were observed when nitrate was high relative to sulfide (ratios 6.0–1.5) whereas at a lower relative nitrate dose (ratio 0.9) corrosion was only 0.14 mm/y. Time-dependent examination of corrosion coupons revealed initially corrosion at a high rate (0.37-0.48 mm/y), which decreased around 80% after 50 h (0.08-0.09 mm/y). The high initial rate coincided with maximal abundance of S0 and therefore it may play a key role in the observed high corrosion of strain CVO. Sulfurimonas spp. contain sox genes for oxidation of reduced sulfur compound, and their expression may govern the differential accumulation of S intermediates in response to varying nitrate dose regimes.

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Session 6 - Microbiology and Modelling in the Oil and Gas Sector Session Chairs: Dennis Enning and Torben Lund Skovhus

Invited Talk:

Modelling of Microbial Biodegradation Processes in Heterogeneous Oil Reservoirs Ian D. Gates University of Calgary Subsurface microbial communities in petroleum systems operate as large-scale reactive processes where microbial activities compete with transport limitations. Over geological time scales, these processes can consume oil components and water to yield hydrogen, carbon dioxide, and methane resulting in heavy oil or extra heavy oil (bitumen). The complexity of these reactive-transport processes arises due to their multiscale nature. Microbes exist and cause reactions at pore scale whereas mass transport occurs at similar scales (diffusion) to much larger length scales associated with dispersion, oil migration, and water and gas transport. Geological heterogeneity has length scales ranging from pore scale to kilometer scales. In this talk, approaches to model multiscale microbial phenomena within oil reservoirs are described that integrate geological heterogeneity of the systems.

Offered Talks: Mass Spectrometry-based Metabolomics - A Tool to Guide MIC Management in the Field Jan Sunner, Iwona, B Beech University of Oklahoma Metabolic profiling of solid and liquid specimens from 4 different oilfields, located in the USA and oversees, where microbially influenced corrosion (MIC) was suspected was performed using HPLC coupled to an ultra-high resolution mass spectrometer (MS). A larger number (15-22) of produced water samples were collected from two of the investigated fields. In addition, abiotic and aerobic laboratory experiments were carried out to determine corrosiveness of 0.2 μm filter-sterilized produced water (SPW) using 1018 carbon steel coupons

submerged in 15 ml of SPW for 30 days at 21C. It was found that not only the overall corrosion damage, but also the severity of pitting, varied considerably between different samples obtained from the same field Principal component analyses of the respective metabolomes demonstrated that “low corrosion” and “high corrosion” samples were separated by principal components 1 and 2 (PC1 and PC2). Thus, the combination of molecular features that determines the severity of MIC in the field is a dominating aspect of the overall variability of the field metabolome. The laboratory corrosion rates highly statistically correlated with available field corrosion data. SPW from well-heads that experienced severe MIC caused more rapid corrosion of 1018 carbon steel in the laboratory experiments than SPW recovered from well heads at which corrosion was “low” or “as expected”, This observation demonstrates that, not only is the metabolome representative of the microbial activities associated with varying degrees of corrosion, but that the produced metabolomes are contributing to, if not causing, such variations. Results of metabolomic investigations support “chemical model of MIC” according to which changes to the chemistry in the near-surface environment, which, are largely caused by microbial metabolic activity, are of key importance to corrosion. The study revealed that metabolomic monitoring using HPLC/MS methods have considerable potential to guide MIC management in oilfield installations.

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Attenuating sulfidogenesis in a soured continuous flow column system with perchlorate treatment Anna Engelbrektson1, Christopher Hubbard2, Yvette Piceno2, Yong Tae Jin1, Lauren Tom2, Mark Conrad2, Gary Andersen2, John Coates1 1 University of California, 2 Lawrence Berkeley National Laboratory Hydrogen sulfide biogenesis in oil reservoirs is a primary cause of souring and of associated costs in reservoir and pipeline maintenance. Amending these environments with perchlorate resolves these problems. Flow through columns packed with bay sediment were flushed with bay water ([SO4=] = 25-30 mM) containing 50 mM inhibitor concentrations (perchlorate or nitrate) decreasing to 25 mM and finally 12.5 mM. Geochemistry was monitored and DNA was prepared from the sediment bed for microbial community analysis. Souring (H2S production) was reversed by all treatments (at 50 mM) compared to the control columns. Nitrate treated columns began to re-sour when treatment concentration was decreased to 25 mM but treatment had to be decreased to 12.5 mM before the perchlorate treated columns began to re-sour. All treated columns re-soured to a lesser extent than the control columns. Microbial community analyses indicate community shifts and clustering by treatment. High concentrations of perchlorate most enriched for Proteobacteria (Gamma, Alpha, and Delta), and Fusobacteria. The decrease in treatment further enriched for Gammaproteobacteria and Alphaproteobacteria and the final decrease further enriched for Firmicutes, Actinobacteria, and Proteobacteria (Gamma, Delta, and Alpha). Deltaproteobacteria seem to be particularly involved in sulfur cycling. Within this class, perchlorate addition stimulated sulfide oxidizing Desulfobulbaceae (50 mM and 12.5 mM) and elemental sulfur reducing Desulfomonadaceae (25 & 12). Sulfate reducing families Desulfovibrionaceae (enriched in 25 mM perchlorate), Nitrospiraceae (enriched in 50 mM perchlorate), Syntrophaceae (enriched in 25 mM perchlorate), and Desulfobacteraceae (enriched in 50 mM and 25 mM perchlorate) contain known sulfate reducing organisms suggesting that effluent sulfide concentrations may be controlled through sulfur redox cycling in addition to toxicity and biocompetitive exclusion. This study indicates that perchlorate shows great promise as an inhibitor of sulfidogenesis in communities and provides insight into which organisms are involved in this process. Metabolomic and Metagenomic Analyses of Crude Oil Production Pipelines Experiencing Differential Rates of Corrosion Vincent Bonifay, Boris Wawrik, Egemen Aydin, Emily, C West, Kathleen Duncan, Athenia, L Oldham, Amy Callaghan, Iwona, B Beech, Jan Sunner University of Oklahoma

Microbially influenced corrosion (MIC) is a process in which metal corrosion is initiated or enhanced by microbial activity. The processes specific to MIC are not well understood, and management of MIC represents a serious challenge. Efforts to utilize molecular techniques for MIC prediction have, to date, met with mixed success. This is likely because the organic chemical environment at the metal surface plays a critical role in regulating the trajectory, extent, and nature of corrosion. Organic matter composition in metal biofilms is, however, not independent of microbial activity, and both molecular and metabolomic data are therefore useful when studying MIC in situ. Pig envelope material was sampled from two pipelines, each carrying crude oil from one of two adjacent North Sea oilfields but experiencing different rates of corrosion. Both have been treated with nitrate, the low corrosion (LC) pipeline continuously from early operation and the high corrosion (HC) only in recent years. 16S rRNA gene and metagenomic data indicated that the HC pigging material was dominated by Thermotogae, Deltaproteobacteria, Firmicutes and Methanococci, whereas Gammaproteobacteria, closely related to marine Pseudomonas, were dominant in the samples from low corrosion (LC) pipelines. Metagenomic data further indicated that the Pseudomonas in LC samples contained the genetic potential for denitrification as well as aerobic hydrocarbon degradation. Anaerobic hydrocarbon degradation genes were not detected in LC or HC samples, though HC samples contained large proportions of bacteria with sulfidogenic potential. Approximately one thousand metabolites were detected in each sample via liquid chromatography/mass spectrometry (LC-MS) for metabolic profiling. Significant differences were observed between the metabolomes of the HC and the LC pipeline samples. The two pipeline systems therefore differ in the presence of strictly anaerobic (HC) vs. facultative denitrifying (LC) microbes as well as in

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aerobic (LC) and anaerobic (HC) hydrocarbon degradation metabolic intermediates. It is hypothesized that the LC pipeline but not the HC pipeline, experiences incursions of oxygen and that the differential amounts of oxygen govern community ecology in these pipelines Metagenomic and metabolomic data were mutually supportive, thus offering a promise of developing integrated –omic approach to MIC mitigation.

Microbial community analysis of a novel biological process for treating oil sands process-affected water Lei Zhu, Yan Zhang, Tong Yu University of Alberta With rapidly development of Canadian oil sands industry, the third-largest proven crude oil reserve in the world, concerns about adverse impacts of oil sands process-affected water (OSPW) on aquatic resources are magnified. Environmental management of OSPW becomes a pressing challenge for regulators, engineers and researchers. We engineered a novel biological process that is composed of a moving bed biofilm reactor (MBBR), an ozonation unit, and a membrane aerated biofilm reactor (MABR). This process has demonstrated effective removal of organic chemicals, alkalinity and toxicity in laboratory scale operation over 2 year. In this study, Illumina high-throughput sequencing was adopted to explore the bacterial communities in suspended biomass and biofilms of the bioreactors as well as in raw OSPW. The diversity in biofilms is more complex than that in suspended biomass and in raw OSPW based on analysis of the 3,608,551 16S rRNA gene sequences obtained. MABR biofilm was revealed possessing the most complex microbial community. Proteobacteria was the majority class and existed in all six tested samples; the percentages of α, β, γ-proteobacteria of raw OSPW were higher than that of all reactor samples. Flavobacteria was reported to show great resistance under toxic and alkaline environment, which existed in MBBR suspended biomass. Ignavibacteria found in MABR biofilm belongs to Chlorobi phylum, which relates to sulfur metabolism. Nitrospira that were related to nitrification process existed only in biofilm. Nine common genus, 3_genus_incertae_sedis, Caldilinea, Falvobacterium, Gp4, Hydrogenophaga, Opitutus, Prosthecobacter, Pseudomonas, Rhodobacter, were founded in six samples. The number of common OTUs between raw OSPW and sample from MBBR decreased during operation, which may correspond to the changing of operational conditions. Due to the nutrients conditions, toxicity level and operational conditions, the microbial community in raw OSPW was different from that inside the bioreactors. However, bioreactors could select and enrich specific microorganisms belonging to classes such as Pseudomonas, Falvobacterium and Rhodobacter, which showed great resistance to harsh environments and the capability of degrading carboxylic group of the main carbon chain. Bioaugmentation happening inside MBBR and MABR made the biodegradation of recalcitrant organic chemicals in OSPW faster and the acceleration of end-pit lake reclamation promising. Next generation modeling of microbial souring – incorporating genomic information and greater complexity Ywei Cheng1, Nicholas Bouskill1, Liange Zheng1, Christopher Hubbard1, Li Li2, Anna Engelbrektson3, John Coates3, Jonathan Ajo-franklin1 1 Lawrence Berkeley National Lab, 2 Pennsylvania State University, 3 University of California, Berkeley Microbially mediated sulfate reduction is the major metabolic process that leads to the production of hydrogen sulfide (H2S) in oil reservoirs. Biogenesis of H2S (souring) has detrimental impacts on oil production operations and can cause significant environmental and health problems. Understanding the processes that control the rates and patterns of sulfate reduction is a crucial step in developing a predictive understanding of reservoir souring and associated mitigation processes. In this study, we describe the development of a microbial trait-based model coupled to a multi-phase reactive transport model. The model represents several anaerobic microbial functional guilds with different resource (e.g., electron donor, sulfate) acquisition traits. These trait values are derived from information encoded in genomic data. The integrated model was used to simulate the temporal and spatial evolution of the primary chemical species (e.g. sulfate, sulfide, nitrate, chlorate and perchlorate) and the microbial community dynamics involved in the souring and desouring processes as revealed in a recent laboratory column experiment comparing the effectiveness of nitrate,

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chlorate and perchlorate treatments as souring control strategies. Simulation of the laboratory results shows that the model captured the spatiotemporal trend of the chemical species and microbial guilds during both souring and desouring. Subsequent field-scale model simulations across a set of reservoir relevant environmental conditions are also conducted. This integrated model demonstrates that intra-guild interactions between individual SRBs, and inter-guild interactions between the SRBs and other heterotrophs can impact the occurrence and extent of H2S production.

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Session 6 - Posters: P56. Bioremediation of O&G produced water: Cupriavidus metallidurans and its potential for heavy metal removal at industrial scale Milica Markovic1,Catherine Boccadoro2, Arild Johannessen3, Andreas Rag2, Juliana Americo1, Henrique Pinheiro1, Mauro Rebelo1 1 Federal University of Rio de Janeiro (UFRJ), 2 IRIS, 3 Biosentrum AS Wastewater produced from O&G installations and petrochemical plants is a key focus area due to strict regulation. Effluents needs treatment before being discharged, and while main strategies to date have focused on the pre-treatment, degradation and/or removal of PAHs, a more persistent problem is caused by heavy metals. The aim of this project is to develop a new bioremediation technology for wastewater treatment using Cupriavidus metallidurans to recover heavy metal ions by expressing a protein with high affinity to heavy metals. These characteristics make it applicable to for even wider usage, depending on its successful growth optimization at an industrial scale. The main goal of this work was to investigate the growth of a strain of Cupriavidus metallidurans LMG1195 in a fermentor, and up-scale the fermentation process from 8L to 100L. The results showed better growth in the fermentor of working volume of 8 L, compared to growth in an Erlenmeyer flask. 12 hours fermentation in the 8 L fermentor was enough to obtain the necessary inoculum for a 100 L fermentor (OD 1.54; cell number of 10*6 CFU/mL; dry weight 42.27 g/L). Fermentation in a 100 L fermentor was feasible, and after 31 hours of process, the bacteria grew up to OD 0.83, with the cell number of 10*8 CFU/mL, and dry weight of 32.42 g/L. The results suggest that C. metallidurans LMG1195 can be grown at a pilot scale and be used for industrial applications.

P57. Contribution of qPCR-based methods to the studies of microbial contamination in fuel systems Pedro Maria Martin-Sanchez1, Jörg Toepel1, Hans-Jörg Kunte1, Anna A. Gorbushina1,2 1 BAM Federal Institute for Materials Research and Testing, 2 Freie Universität Berlin Microbial contamination of fuels is a phenomenon widely reported in the literature. A variety of microorganisms, fungi and bacteria, can contaminate fuels during distribution and then develop abundant biomass in the storage tanks. Contaminant populations cause fuel deterioration and dramatic infrastructure problems such as blockage pipelines and filters and microbiologically influenced corrosion (MIC). These damages produce important economic losses to the oil industries.The overarching goal of this study was to develop robust and reliable biological molecular methods to detect and quantify the microbial contamination. The methods should improve control procedures. In this regard, a variety of novel real-time quantitative PCR (qPCR) protocols were established to analyze total bacteria, total fungi and specifically the species Hormoconis resinae. This filamentous fungus is traditionally considered one of the main microbial pollutants in fuels, responsible for degradation of kerosene and diesel. The qPCR results for a selection of diesel samples confirmed the validity of these protocols. Analyses of total bacteria and fungi were useful tools to quantify contamination levels. Their implementation in analytic procedures in the distribution chain will allow the early detection of microbial outbreaks in fuel systems. The H. resinae qPCR showed a remarkably specificity and sensitivity, detecting this fungus in samples without visual evidence of contamination. Additional studies should be conducted in order to determine the current incidence of this fungus in the cases of biological fuel contamination. P58. Feasibility of a bioremediation process coupled to CO2 fixation using green microalgae for hydrocarbon contaminated soil Abdeldjalil Abid1,2, Yasmine Souissi 3, Moktar Hamdi 1,2

1 National Institute of Applied Science and Technology (INSAT), 2 University of Carthage, 3 Free University of Tunis Petroleum industries’ activities, such as oil exploration, production transportation and refining raise serious threats on human health and environment. The hydrocarbon spreads on the ground water surface and can

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also be conducted to ground water as well as on soil particles surfaces. Bioremediation consists in a process that uses microorganisms in order to mineralize hydrocarbon contaminant. In the other hand this mineralization it is important raison for greenhouse effect through the CO2 that result. In the current study, biodegradation activity of hydrocabonoclastic microflora and biostimulation with inorganic nutrient (NPK) were investigated through the determination of optimal conditions which can improve bioremediation process. It has been noticed that after 56 days, about 98% of the total petroleum hydrocarbon’s (TPH) removal rate in the soil treated by microcosm’s technique was observed relative to approximately 2.22×107CFU/g soil of bacterial number. This performance was achieved with microbial metabolism is concomitant with hydrocarbons biodegradation. Moreover, hydrocarbon fractions including alkanes and aromatic compounds were degraded faster than the other complex fractions according to GC-MS and FT-IR analysis. In order to fix the CO2 resulting from mineralization and to reduce the greenhouse effect, a new strategy based on microalgae growth in the microflora involved in the contaminated hydrocarbon was established. The result showed a good growth of microalgae associated with a high removal rate of hydrocarbon and minerals amount in treated soil. P59. Microbial communities in and around hydrocarbon-contaminated sediments, and possible implications for microbial prospecting for oil and gas Morten Poulsen1, Steffen Sanvig Bach2, Dorthe Skou Pedersen1 1 Danish Technological Institute , 2 Maersk Oil Research and Technology Centre Sediments associated with oil- or gas seeps may contain unique microorganisms that are able to utilize and break down hydrocarbons. Information on such indicator microorganisms would be valuable in oil exploration and bioremediation. Here we present an initial study applying 454-pyrosequencing of 16S rRNA genes to identify indicator microorganisms of hydrocarbon-contamination. Eight sediment cores were obtained from areas around oil-producing platforms in the Danish Sector of the North Sea. Four of the cores were from areas with higher concentration of total hydrocarbon content (THC) as a result of oil and gas drilling activities and four of the cores were from reference sites with no direct contamination. Measured total hydrocarbon content (THC) varied from 4.8 to 90 ppm at contaminated sites, and from <1 to 5.9 ppm at reference sites. Bacterial and archaeal 16S rRNA genes from 0-1 cm and 3-6 cm depth in each core were amplified and sequenced. Multivariate analysis did not reveal significant differences in total communities of bacteria or archaea in sediment samples with high THC compared to reference samples. However, in 3D PCoA plots, bacterial communities of hydrocarbon-contaminated samples from the area around the Gorm field grouped together separately from Gorm reference samples. A detailed analysis of the communities of these samples revealed that a family of order Xanthomonadales comprising up to 5.9% of the total bacterial community was significantly overrepresented in contaminated Gorm samples compared to the Gorm reference samples. Interestingly, Xanthomonadales organisms have previously been isolated from oil-contaminated soils. Furthermore, an uncultivated group belonging to proposed order NB1-j within class Deltaproteobacteria was significantly overrepresented in all reference sediments, and hence useful as indicator organisms of uncontaminated sediments. This study demonstrates the potential of using new sequencing technologies for identifying microorganisms, which may act as suitable indicators of presence of hydrocarbons. P60. Can we maximize the data from coupons to include bacterial indicators, like ATP and DNA? Kim Dockens

OSP Microcheck

Ever-increasing demands are being placed on oil and gas operators to become more efficient, cost-effective and environmentally compliant. As the media has brought greater scrutiny to the activities within the industry and every failure is being examined under the microscope, it is apparent that monitoring and controlling microbes is a priority. Traditionally culture media used to measure planktonic bacteria was the most common method available and there wasn’t much confidence that this data could predict, with any certainty, what was taking place on the surface of the equipment. Coupon data and pitting rates can give some indication of the

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effects of sessile biofilms on the rates of corrosion, but there is still some doubt about whether it is true microbial influence corrosion, (MIC). Bacterial cultures can be obtained from a technique of swabbing coupons, but correlations between the pitting rates and bacterial population isn’t clear. Swabbing of the coupon may disrupt bacterial activity and enumeration through serial dilution isn’t reliable, though it does identify some types of living bacteria that may be present. This indicates a need for another method to verify the presence of biofilms on the coupons. Adenosine Triphosphate (ATP) measurement has been utilized in the oil and gas industry for approximately 6-7 years with increasing confidence in the results. The second generation ATP assay provides a preparation for enumeration of total living cells. It hasn’t though, been utilized to its fullest potential which is to measure sessile populations. Also it doesn’t identify which bacteria are present. Metagenomics, a new trend in bacteria identification, may be beneficial in tandem use with the ATP assay for more complete analysis. This paper will evaluate several case studies for the potential of expanding coupon analysis to include ATP and DNA information and where possible to look for correlation with pitting rates.

P61. Diagnosing the oilfield microbial profile: An optimized ion torrent 16S rRNA gene-based analysis protocol using 400 bp chemistry Indranil Chatterjee, Varsha Shukla, Victor Keasler

Nalco Champion, an Ecolab Company Microbial communities present in oil and gas reserves are frequently contained within production fluids. Culture-dependent methods employed to detect and enumerate these microorganisms are not always successful. 16S rRNA sequencing is a fast, sensitive, inexpensive profiling technique based on variation in the microbial 16S ribosomal RNA (rRNA) gene. Recently, it has been used as a comprehensive tool to determine the composition of microbial communities in the complex oilfield resource environments. Our understanding of metagenomic diversity is based predominantly on studies of community DNA. ‘Universal’ and domain-specific rDNA gene PCR primers have historically been used for the estimation of microbial diversity without adequate regard to the degree of specificity of primer pairs to different prokaryotic and archaeal groups. In a reassessment of the published primers of 200 bp commonly used for ‘universal’ 16S rDNA sequence amplification, we found that substantial variations in specificity exist. In the present study, primer pairs of ~400 bp specifically designed for ‘universal’ (archaeal and bacterial) 16S rDNA sequence amplification, with homology to major microbial taxa, have been designed. Here, we present an overview for primers upon algorithm development for bioinformatics of metagenome, on estimation of microbial genome plasticity and diversity, work on a bioinformatics pipeline for the analysis of microbial metagenome and also work on phylogenetic clustering of metagenomic fragments using Ion TorrentTM platform (Personal Genome Machine - PGM) 400 bp chemistry. The key output of this work implicated that an increment of the in-depth coverage of the 16S rDNA have increased our overall specificity for detection of the oilfield microbial population, hence keep us better prepared to intervene with microbial corrosive and undesirable loss to asset integrity. Our results also showed that microbial community analyses using the PGM platform with Ion Torrent’s new 400-base chemistry improved the species-level discriminatory power of 16S rRNA profiling and provided a low cost, scalable and high throughput solution for oilfield’s related metagenomic analyses. Altogether, this research work will enhance our current DNA sequencing based detection and monitoring capabilities, thus contributing to more specific and improved ways to control microbially-related problems in the oil and gas industry.

P62. Analysis of cpn60 sequences reveals incompleteness of oil field microbial community compositions determined through 16S rDNA sequence analysis Yuriy Kryachko, Diana Semler, John Vogrinetz, Randy Irvine, John Davidson, Matthew Links, Luke McCarthy, Brenda Haug, Sean Hemmingsen National Research Council Canada

While various 16S rDNA primer sets have been known to be biased for or against certain taxonomic groups, in most studies a single primer set is used. Using multiple 16S rDNA primer sets may address the issue. However, targeting an additional gene is a more objective and reliable approach. The cpn60 gene is the

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only target other than the 16S rRNA gene that can be accessed with “universal” PCR primers. In addition, a curated sequence database (cpnDB, http://www.cpndb.ca) is available. In this study, the microbial communities from five wells of a Canadian oil field were characterized using sequencing of 16S rRNA and cpn60 gene fragments. DNA was isolated from biomass from the samples of oil-containing produced water, and universal bacterial and archaeal 16S rDNA and cpn60 primers were used to amplify the target fragments. MiSeq (Illumina, Inc.) was employed for DNA sequencing. Analysis of 16S rDNA fragments showed that Ralstonia, Arcobacter, Halanaerobium, Bacillus and Sulfurospirillum spp. were abundant in most of the samples. Methanohalophilus, Methanocalculus and Methanolobus spp. were found to be the most abundant among methanogens. Analysis of cpn60 DNA fragments confirmed that Bacillus, Methanolobus and Sulfurospirillum spp. were well represented in the samples, and showed that Bacillus mojavesis, known to be a biosurfactant producer, represented the genus Bacillus. In addition, as a result of cpn60 sequencing, it was found that the Deltaproteobacteria, particularly Pelobacter carbinolicus, were abundant in the samples. Analysis of 16S rDNA fragments amplified with primers preferentially targeting the Deltaproteobacteria confirmed the abundance of the representatives of this class. Hence, likely due to the bias of the used 16S rDNA universal primer set, Pelobacter sp. were missed and some other Deltaproteobacteria were either missed or underrepresented when the analysis relied on sequencing of 16S rDNA fragments. On the other hand, analysis of cpn60 sequences failed to identify Ralstonia sp., other representatives of the Betaproteobacteria and some Epsilonproteobacteria, such as Arcobacter. This might occur, likewise, because of the cpn60 primer set bias and/or due to the cpnDB being still not as well developed as databases carrying information on the 16S rRNA genes. Thus, our findings suggest that complementing 16S rDNA sequencing analysis with the information based on cpn60 sequencing is valuable for the analysis of oil field microbial community compositions.

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List of Authors Annie (Biwen) An Abdeldjalil Abid Carolyn Aitken Jonathan Ajo-franklin Hakan Alkan Ahmed Alshehhi Eric Alsop Juliana Americo Gary Andersen Luiza Andrade Oderay Andrade Charles Armstrong Elin Austerheim Recep Avci Egemen Aydin Diana Backes Andrea Bagi Jesus Barron-Aldana Stephan Bauer Thierry Baussant Iwona Beech Janiche Beeder Xin-Yu Bian Markus Bill Catherine Boccadoro Caitlin Bojanowski Kristin Bonaunet Vincent Bonifay Nicholas Bouskill Emma Bowen Odd Gunnar Brakstad Zach Broussard Damon Brown Gunhild Bødtker Minmin Cai Amy Callaghan Cynthia Canêdo Da Silva Ramses Capillla Hans Carlson Martin Carrera Servio Cassini Gladys Castorena Cortés Thiago Cavalcant Indranil Chatterjee Ywei Cheng Brian Clement John Coates Mark Conrad Cristiana Cravo-Laureau

Wendy Crookes-Goodson T.P. Curtis Ingela Dahllöf Al Darzins Irene Davidova John Davidson Bret Davis Ana Paula De Melo Rodrigues Renato De Paula Maíra Paula De Sousa Marc Demeter Sanket Desai Prasad Dhulipala Diogo Dias Kim Dockens Johanna Donhauser Nadjmeh Doostdar Carrie Drake Karine Drønen Kathleen Duncan Robert Duran Richard Eckert Oghenekume Edeki Bob Eden Sarah Eisenlord L.E. Eland Anna Engelbrektson Dennis Enning James Fajt Qingxian Feng André Ferreira Kai Finster Kara Friesen Naoya Fukushima Ravindranath Garimella Fatma Gassara Ian Gates Brett Geissler Jil Geller Lisa Gieg Antje Gittel Milene Gomes Elmer E. Gonzales Limache Anna Gorbushina Maria Granberg Neil Gray Alexander Grigoryan Ji-dong Gu Eduardo Gudiña

Thusitha Gunasekera Guomenghua Moktar Hamdi Farazul Haque Amanda Harmon Brenda Haug Ian Head Sean Hemmingsen Andrea Herold Heike Hoffmann Jennifer Hornemann Xiaoke Hu Christopher Hubbard Casey Hubert Masayuki Ikarashi Randy Irvine Daphne Jalique Núria Jiménez Yong Tae Jin Zhang Jiyuan Arild Johannessen Shawna Johnston Martin Jones Gabriel Juarez Bo Jørgensen Yoichi Kamagata Krista Kaster Victor Keasler Regina Keller Carrie Keller-Schultz Kasper Kjeldsen Tim Klett Nicole Klueglein Hajime Kobayashi Thomas Koehler Robert Komm Roald Kommedal Darren Korber Tetyana Korin Hans Kotlar Adriana Krolicka Martin Krueger Yuriy Kryachko Cor Kuijvenhoven Hans-Jörg Kunte Sven Lahme Sylvie Le Borgne Alan Le Tressoler Thierry Lemettais

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Joe Lemire Tiffany Lenhart Ernest Lever Anna Lewin Guoqiang Li Li Li Vitor Liduino Turid Liengen Matthew Links Jin-Feng Liu Pamela Lloyd Synnøve Lofthus Andreas Loibner Bart Lomans Isabelle Lombaert-Valot Kilean Lucas Márcia Lutterbach Ting Ma Haruo Maeda Michael Maguire Eva Mahler Zakari Makama Kathleen Manna Fons Marcelis Milica Markovic Christopher Marks Peter Marks Joshua Martin Pedro Maria Martin-Sanchez Daisuke Mayumi Serge Maurice Mbadinga Luke McCarthy Prabhakara Medihala Priyesh Menon Sean Mercer Brandon Morris William Morris Bo-Zhong Mu Soujatya Mukherjee Florin Musat Mari Mæland Nilsen Mark Nanny Roman Netzer Koichi Nishikawa Lisa Oberding Dora Ogles Athenia Oldham Patricia Olguin Valeria Maia de Oliveira Hiroshi Oshibe Yvette Piceno Markus Pichler

Henrique Pinheiro Tijan Pinnock Iris Porat Morten Poulsen Shane Pruit Luo Qing Andreas Rag Svein Ramstad Mauro Rebelo Julia de Rezende Jorge Ribeiro Hans Richnow Irene Roalkvam Jayne Robinson Lígia Rodrigues Teresa Roldan Jin Rui Oscar Ruiz Hans Røy Susumu Sakata Leobardo Santiago Rosales Eugenio Santos Neto Steffen Sanvig Bach Kozo Sato Kerstin Scherr Anders Schouw Johanna Schritter Diana Semler Eliana Sérvulo Angela Sherry Yoshikazu Shirai Adris Shlimon Pravin Shrestha Varsha Shukla Isabel Natalia Sierra Garcia Jana Sitte Arne Skauge Dorthe Skou Pedersen Torben Lund Skovhus Ramsey Smith Yasmine Souissi Blake Stamps Ida Steen Bradley Stevenson Roman Stocker Runar Stokke Nontje Straaten Matt Streets Richard Striebich Simcha Stroes-Gascoyne Marc Strous Trond Størseth

Ana Suarez-Suarez Kerry Sublette Joseph Suflita Jan Sunner Navreet Suri Hideyuki Tamaki Satoshi Tamazawa Grahame Taylor Pat Teevens José Teixeira Timothy Tidwell Jörg Toepel Lauren Tom Terje Torsvik Courtney Toth Nicolas Tsesmetzis Raymond Turner Mona Ulas Erika Valoni Ulrich Vasconcelos John Vogrinetz Gerrit Voordouw Tatsuki Wakayama Caixia Wang Hui Wang Boris Wawrik Li Wei Alexander Wentzel Emily West Joseph Westrich Terry Williams A. Wipat Mark Wolfenden Ken Wunch Wu XiaoLin Chen Xinghong Dou Xumou Mohammed Yahyaoui Guang-Chao Yang Liu Yang Wang Yanling Jun Yao Bei Yin Tong Yu Yan Zhang Hou Zhaowei Liange Zheng Jiefang Zhou Jing Zhou Lei Zhu

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