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2nd International Conference on Applied Microbiology

and Molecular Biology in Oil Systems

ISMOS2 Abstract Book 17-19th June 2009

Symposium, Workshops & Poster Session

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Sponsors

Display stands

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Abstract Book

ISMOS2 2009

Editors: Lars Holmkvist, Anna Krestine Nørgaard & Torben Lund Skovhus

Danish Technological Institute (DTI) − Oil & Gas Group, Teknologiparken, Kongsvang Allé 29

8000 Aarhus C, Denmark

http://www.dti.dk/ismos2

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.

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Table of Contents

Sponsors and display stands …………………………………...…………...……………..…2

Table of Contents ……………………………….………………..……………………………..4

Aims and scope……………………………………...……………………………………………6

Program overview……………….………………………………………………...……..……...7

June 17……………………………………………………………………………………....………..7

June 18……………………………………………………………………………………….…...…..8

June 19…………………………………………………………………………………………..……9

Oral presentations……………………………………………………………….…………..…10

Opening Lectures……………………………….…………………………………….………..……10

Key Notes…..…………………………..……………………………………..……….……………14

Session 1: Microbiologically Influenced Corrosion in Oil Fields…………………….………….…17

Session 2: Biofuels and Downstream Petroleum Microbiology…………………………………….20

Session 3: Status and Challenges for Microbial Enhanced Oil Recovery (MEOR)…….………..…26

Session 4: Molecular Microbiological Methods (MMM) in Oil Fields…………………………….29

Session 5: Control and Prevention of Reservoir Souring…………………………………………...34

Session 6: Practical Application of Molecular Microbiology Methods (MMM) in Oil Field

Systems………………………..…………………………………………….………..….39

Session 7: Biodegradation of Hydrocarbons in Oil Production………………………………….…43

Session 8: Microbiological Challenges in Biofuels…………………………….…………………...49

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Table of Contents

Poster presentations……….……………………..………………………….………………...53

Workshop, Lab Tour and Practical Demonstrations ……………..……………..……93

Contacts……………………………………………………………………………………….….94

Next ISMOS Conference……………………………………………………………………...94

 

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Aims and Scope

There is an increasing need by the oil industry and downstream related industries to monitor microbial communities in 'real time' in order to address specific issues such as oil field souring, microbiologically influenced corrosion (MIC) and biofouling. Molecular Microbiology Methods (MMM) can be applied to oil fields (and other oil-related industries) to analyse and quantify the in-situ microbial communities and their activities in response to changing environmental conditions. Such information will help oil companies to employ more directed, cost-effective strategies to prevent the major problems associated with deleterious microbial activities, as well as to encourage beneficial microbes.

ISMOS is multidisciplinary, linking chemists, geologists, engineers and molecular microbiologists, and will include a mixture of high profile European and overseas speakers from both industry and academia. The symposia will present the major industrial problems caused by microbes, (e.g. souring, bio corrosion) as well as beneficial activities (e.g. MEOR, upgrading). The main focus of the meeting will be to understand how molecular and microbiological tools can be used to address these issues.

The symposium will also include workshops to provide delegates with a 'hands-on' experience of specific molecular techniques and effective off-shore sampling strategies and discussions on how to implement molecular microbiology methods to monitor troublesome/beneficial microbial communities and facilitate a rapid response to troubleshoot oil field systems.

Who should attend?

This workshop is intended for professionals in the oil and gas industry and in academia who are interested in biological aspects and problems linked to exploration and production activities. The workshop is aimed at people whose principle interest falls into any of the following categories:

• Molecular Microbiology • Production Engineering • Geophysics and Geology • Biotechnology, Microbiology and Chemistry • Earth and Environmental Science Research

Welcome to Aarhus and ISMOS-2!

Dr. Torben Lund Skovhus (Danish Technological Institute, Denmark), Chair

Dr. Corinne Whitby (University of Essex, UK), Vice-Chair

Dr. Kjeld Ingvorsen (University of Aarhus, Denmark), Co-Organizer

 

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Program overview:

June 17

Welcome

Welcome and opening remark

(6:00 pm) Torben Lund Skovhus, DTI Oil & GasGroup

Opening Lectures

Chairman: Andreas Schramm

(6:05) Alexander Loy On the opportunities and challenges of diagnosing sulfate reducing microorganisms

(6:50) Light Meal

(7:30) Joseph M. Suflita The Importance of Anaerobic Hydrocarbon Biodegradation in Oil Production and Consumption Operations

(8:15) Casey Hubert Can "Misplaced Microbes" in the Ocean Serve as Vital Tracers for Oil and Gas Exploration?

(9:00 pm) Drinks Reception

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Program overview:

June 18

(8:30 am) Registration/Coffee/Tea

(8:50) Official welcome – Bo Frølund

(9:00) Key Note Bernhard Ollivier Microbiology of Oilfield Ecosystems

Session 1: Microbiologically Influenced Corrosion in Oil Fields Chairman: Marcia Lutterbach

(9:30 am) Ketil Sørensen Assessment of MIC using quantitative PCR

(9:50 am) Kathleen Duncan Biocorrosive Thermophilic Microbial Communities in Alaskan North Slope Oil Fields

(10:10 am) Coffee/Tea & Poster Session

Session 2: Biofuels and Downstream Petroleum Microbiology Chairman: Elaine McFarlane

(10:30) Elaine McFarlane Microbial Contamination of Middle Distillate Fuels: Past, Present and Future

(10:55) David Owen Biological Degradation of Biodiesel - Causes, Effects, Monitoring and Mitigation

(11:20) Margret Schmidt German Interindustry Study: Filter Blocking, Biofuels and Microbes

(11:45) Francesca De Ferra Biotechnology for Unconventional II Generation Bio-Fuel Production

(12:05) Lunch

Session 3: Status and Challenges for Microbial Enhanced Oil Recovery (MEOR) Chairman:Aleksandr Grigoryan

(1:00 pm) Janiche Beeder Aerobic Microbial Enhanced Oil Recovery - Experience from Laboratory Tests and Field Trials

(1:25) Tamara N. Nazina Microbial Ecology of the High-Temperature Petroleum Reservoir and Results of MEOR Technology Application

(1:50) Coffee/Tea & Poster Session

Session 4: Molecular Microbiological Methods (MMM) in Oil Fields Chairman: Corinne Whitby

(2:25) Catherine Joulian Bacterial Diversity of a Hypersaline Oil Reservoir Core

(2:45) Jizhong Zhou GeoChip: A High Throughput Genomics Technology for Characterizing Microbial Functional Community Structure

(3:10) Joel Kostka Quantification of Prokaryotic Gene Expression in Shallow Marine Subsurface Sediments of Aarhus Bay, Denmark

(3:30) Andrew Price Detection of SRP Activity by Quantification of mRNA for the Dissimulatory (bi) Sulphite Reductase Gene (dsrA) by Reverse Transcriptase Quantitative PCR

(3:50) Coffee/Tea & Poster Session

(4:30 - 6:00) Lab Tour and Practical Demonstrations

(8:00) Conference Dinner at Helnan Marselis Hotel

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Program overview:

June 19

(8:30 am) Coffee/Tea

(9:00) Key Note Bo Barker Jørgensen The deep sub-seafloor biosphere

Session 5: Control and Prevention of Reservoir Souring Chairman: Paul Evans

(9:30) Antje Gittel Potential of Nitrate Addition to Control the Activity of Sulfate-reducing Prokaryotes in High-temperature Oil Production Systems

(9:50) Gerrit Voordouw Use of Nitrate in Souring Control of an Oil Field with low Bottom Hole Temperature

(10:15) Aleksandr Grigoryan A Multi-disciplinary Survey to Limit Souring in Oil Reservoirs in Argentina

(10:35) Brandon Morris Isotopic Fractionation by an n-Alkane Degrading Sulfate Reducer: Implications for Monitoring Intrinsic Bioremediation and Reservoir Souring

(10:55) Coffee/Tea & Poster Session

Session 6: Practical Application of Molecular Microbiology Methods (MMM) in Oil Field Systems Chairman: Ketil Sørensen

(11:15) Geert van der Kraan Microbial Diversity of an Oil-water Processing Site and its associated Oil Field Production Water

(11:35) Pat Whalen Comparison of Biocides Using New Detection Tool

(11:55) Aleida de Vos van Steenwijk Prevention of MIC - a Case Study in Pipelines Transporting Water Condensate from Natural Gas

(12:15) Lunch

Session 7: Biodegradation of Hydrocarbons in Oil Production Chairman: Casey Hubert

(1:00 pm) Julia Foght Small Bugs in Big Ponds: Microbiology in Oil Sands Tailings

(1:25) Neil Gray Microbial Community Structure, Dynamics and Function in Methanogenic Petroleum Degrading Systems

(1:50) Dariusz Strapoc New Insight into Complexity of Subsurface Methanogenic Pathways: CO2-Reduction and Acetate-Fermentation are not alone

(2:10) Richard Johnson A Micro Solution to a Mega Problem - Can Microbes be used to clean up dirty Oil?

(2:30) Natalya M. Shestakova Syntrophic Acetate Degradation to Methane in a High-Temperature Petroleum Reservoir

(2:50) Coffee/Tea & Poster Session

Session 8: Microbiological Challenges in Biofuels Chairman: Sune Nygaard

(3:05) Gitte Sørensen Assessing microbial spoilage of biodiesel blends under aerobic and anaerobic conditions

(3:25) Marcia Lutterbach Molecular Biology Tools in the Study of Biodiesel in Brazil

(3:45) Howard Chesneau Important Technical Considerations when Contemplating Biolumiescence as an Analytical Tool for the Determination of Microbial Contamination of Petroleum Fuels and other Matrices

(4:05) Workshop on Methods for Detection of Microbes in the Oil, Gas and Petroleum Industry Chairman: Ian Head

(4:50-5:00) Summary and Next Meeting (Torben Lund Skovhus & Corinne Whitby)

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Oral presentations:

Opening Lectures

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On the Opportunities and Challenges of Diagnosing Sulfate-Reducing Microorganisms

Alexander Loy

Department of Microbial Ecology, University of Vienna, Austria

The importance of sulfate-reducing microorganisms (SRM) for the functioning of many ecosystems, such as marine sediments, stands in contrast to the economic threat they pose to oil production. Molecular diversity surveys based on rRNA genes and dsrAB, genes that encode major subunits of the dissimilatory sulfite reductase, indicate that our picture of the natural diversity of SRM (as we know it from cultivation) is far from being complete. This enormous phylogenetic diversity complicates unbiased identification and quantification of SRM by molecular methods such as fluorescence in situ hybridization, real-time PCR or DNA microarrays. Combining these 16S rRNA and dsrAB-based molecular methods with substrate-mediated isotope labelling techniques is a potential solution for identification of yet uncultivated SRM. Once the diversity of SRM and the key players in an ecosystem of interest are determined, the existing molecular tool kit provides amble opportunities for diagnosing and monitoring of SRM in this ecosystem.

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The Importance of Anaerobic Hydrocarbon Biodegradation in Oil Production and Consumption Operations

Joseph M. Suflita

Institute for Energy & the Environment; Department of Botany and Microbiology

University of Oklahoma, Norman, OK, USA

The metabolism of hydrocarbons by anaerobic microorganisms has important ramifications for the ability of societies to meet their energy challenges. On one hand, anaerobic biodegradation is important for the intrinsic remediation of spilt fuels, for the conversion of hydrocarbons to clean burning natural gas, and for the fundamental cycling of carbon on the planet. However, the same processes can also be associated with a host of difficult problems including reservoir souring, microbial influenced corrosion, oil viscosity alteration and compromised equipment performance. Such issues may be exacerbated as newer fuels are produced and used in the existing infrastructure. Understanding the myriad of issues requires information on the fundamental mechanisms of anaerobic hydrocarbon metabolism.

Analyses by GC-MS of microbial cultures, contaminated waters, and oilfield production fluids, often with isotopically-labeled hydrocarbons, revealed a suite of signature metabolites that attest to the kinds of reactions that anaerobes are able to catalyze. Subsequent enrichment and isolation of the requisite organisms reveal that such bacteria can often participate in hydrocarbonoclastic syntrophic associations.

Recent studies demonstrate that hydrocarbons can be anaerobically metabolized by unprecedented biochemical mechanisms. In the absence of oxygen, alkylbenzenes, C1-C60 n-alkanes, cyclic alkanes and polynuclear aromatic hydrocarbons, are often activated by addition to the double bond of fumarate to form substituted succinic acid derivatives. The free radical nature of such bioconversions can be correlated with the extent of substrate transformation making reliable predictions of anaerobic fate processes possible.

Progress on the fundamental mechanisms of anaerobic hydrocarbon biodegradation has steadily emerged making generalizing principles and attendant predictions feasible. However, as central as this information is for the understanding a range of practical issues, such studies also illustrate what is yet to be discovered. By carefully following the metabolic fate of model substrates, it seems clear that multiple transformation processes have yet to be fully described.

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Can "Misplaced Microbes" in the Ocean Serve as Vital Tracers for Oil and Gas Exploration?

Casey Hubert

Max Planck Institute for Marine Microbiology, Bremen, Germany

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Key Notes

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Microbiology of Oilfield Ecosystems

Bernard Ollivier

IRD, Universités de Provence et de la Méditerranée

It is clearly established that large and diverse populations of microbes possessing a range of different metabolic activities inhabit subterranean environments, including oilfield reservoirs. Because the redox potential of the production waters is low and oxygen is generally absent, anaerobiosis is highly favored over aerobiosis in the reservoirs. In this respect, despite several aerobic microorganisms have been isolated from oilfield environments, much attention has been paid to anaerobes. This is obvious for sulfate-reducing bacteria (SRB) as they were recognised as responsible for (i) the production of H2S within reservoirs or top facilities, (ii) the reduction of oil quality, (iii) the corrosion of steel material. Bastin in 1926 first provided evidence of the existence of SRB in oil-producing wells. Besides SRB, methanoarchaea, and fermentative microorganisms have also been commonly isolated from the aqueous phase of produced fluids. All these microorganisms are believed to participate in the overall complex biogeochemistry of oil reservoirs as they possess different metabolic features ranging from autotrophy to heterotrophy. Here, we will focus on the microbiology of SRB, but also heterotrophic fermentative bacteria, some of which are able to grow by using various electron acceptors such as elemental sulfur, thiosulfate, iron and nitrate. Whether these microorganisms have an indigenous origin or have been introduced into the subsurface by drilling operations is questionable, but there is no clear answer regarding origin of microorganisms inhabiting oil reservoirs. In contrast to other better studied extreme environments, e.g., thermal springs, deep-sea hydrothermal vents, anaerobic gastro-intestinal systems, etc., our knowledge of the microbial diversity of oilfield waters is still poorly understood.

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The Deep Sub-Seafloor Biosphere

Bo Barker Jørgensen

Center for Geomicrobiology, Department of Biological Sciences, University of Aarhus, Denmark

Most bacteria and archaea on Earth live in the sub-surface world of the continents and of the seabed. Microbial cells have been found in marine deposits, more than 1.5 km deep and 110 million years old, and in the basaltic ocean crust. The energy flux available to the deep microbial communities is extremely low, yet over geological time they are responsible for extensive mineralization of buried organic matter and for important chemical changes in oil and gas reservoirs. Modeling of the turnover of electron acceptors or donors provides calculated mean rates of cellular metabolism a million-fold below those of growing cultures. This corresponds to calculated mean generation times of years to thousands of years. Such a low electron flow is difficult to reconcile with current concepts of maintenance metabolism, cell repair costs, membrane permeability, and other basic cell properties. Although buried organic matter apparently provides most of the energy for the deep biosphere, more exotic energy sources, such as hydrogen from the radiolysis of water by natural radioisotopes, make additional contributions. Most of the sub-surface bacteria and archaea are presently identified only by the genetic code of their DNA. The predominant phylogenetic lineages have no cultured or known relatives in the surface world and their functional role is therefore unknown. Studies of functional genes and their transcription in subsurface sediments, combined with other DNA/RNA based analyses, isotope probing, biomarkers, etc. are now being used to overcome this limitation.

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Session 1:

Microbiologically Influenced Corrosion in Oil Fields

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Assessment of MIC using Quantitative PCR

Ketil Sørensen1, T. Lundgaard1, U. Thomsen1, L. Holmkvist1,T.L. Skovhus1, J. Larsen2

1DTI Oil& Gas, Danish Technological Institute

2Maersk Oil

Microbiologically influenced corrosion (MIC) is widespread in oil production and distribution systems. Sulfate-reducers and methane-producers are thought to be among the main groups of troublesome microorganisms (TM) involved in MIC because both groups maintain a low hydrogen concentration at the metal surface, thus speeding up the cathode reaction. In addition sulfate-reducers produce large amounts of sulfide which is itself corrosive. Current models for MIC in oil production systems depend largely on TM data obtained from samples of produced- and injection water. Unfortunately, culturing-based enumeration techniques can rarely be applied in the surveillance of these TM in a meaningful way because the relevant organisms tend to be solidly encased in biofilms and deposits at the metal surface rather than suspended in the overlying water. Here we present a qPCR-based assay for enumeration of sulfate-reducing and methane-producing microorganisms suitable for use in water, pigging debris, as well as scale/wax samples - the latter two will far better represent the true number of TM than the water sample. Using this assay, it was demonstrated that abundant populations of TM were present at the metal surface of a steel tube exhibiting pitting corrosion. Based on the numbers of sulfate-reducers and methane-producers at the metal surface, the electron flow and thus the corrosion rates at the surface was estimated, yielding results that were consistent with the rates observed in the tube. This study demonstrates the use of qPCR for obtaining more realistic numbers of TM, resulting in improved MIC model predictions.

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Biocorrosive Thermophilic Microbial Communities in Alaskan North Slope Oil Fields

Kathleen E. Duncan1, L.M. Gieg1,3, V.A. Parisi1, R.S. Tanner1, J.M. Suflita1, S.G. Tringe2, J. Bristow2

1Dept. of Botany and Microbiology and the Institute for Energy and the Environment, University of Oklahoma

2DOE Joint Genome Institute

3Dept. Biological Sciences, University of Calgary

Corrosion of oilfield pipelines by microorganisms is a costly but poorly understood phenomenon. Through-wall breaches due to the metabolic activities of microorganisms were implicated in the 2006 Prudhoe Bay release on Alaska's North Slope (ANS) and other incidents of pipeline failure. There is no consensus on the identity of specific microorganisms responsible for corrosion or how they function to catalyze such incidents, resulting in poorly targeted efforts to monitor and combat biocorrosion.

We obtained samples from an ANS oilfield to assess the potential for biocorrosion by analysis of 16S rRNA gene sequence and dsrAB clone libraries. Targeted isolation of numerically dominant microbes was also performed. The facility has an ongoing biocorrosion control program and produces oil, gas and water from multiple hot (average temperature 68°C) reservoirs.

We found archaeal and bacterial communities comprising thermophilic and hyperthermophilic hydrogen-using methanogens, syntrophic bacteria, peptide- and amino acid-fermenting bacteria, iron reducers, sulfur/sulfate/thiosulfate-reducing bacteria and sulfate-reducing archaea. Approximately 90% of the 16S sequences originated from 10 bacterial and 3 archaeal taxa. The most abundant bacterial sequence was similar to that of Thermovirga lienii, isolated from a North Sea oil well. A thermophilic Anaerobaculum sp. was the numerically dominant heterotroph isolated from one sample. Members of the genus Anaerobaculum can reduce sulfur, thiosulfate and cysteine to H2S. dsrAB libraries were dominated by sequences similar to those of the archaeal sulfate-reducer Archaeoglobus fulgidus.

Microbes similar to those identified in the sequence libraries and by targeted isolation can stimulate metal corrosion through production of sulfides from various sulfur oxyanions, low molecular weight organic acids, CO2, hydrogen oxidation and iron reduction. Bacterial sulfate reduction may make only a minor contribution to sulfide production at this facility. The physiologies and microbial products indicate a widespread potential for biocorrosion in thermophilic oil production facilities.

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Session 2:

Biofuels and Downstream Petroleum Microbiology

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Microbial Contamination of Middle Distillate Fuels: Past, Present and Future

Elaine McFarlane

Shell Global Solutions,UK

There are three main types of spoilage and corrosive, hydrocarbon utilizing microorganisms: bacteria, yeasts and moulds (filamentous fungi). They survive and proliferate in water associated with fuel, drawing their nutrients across the fuel/water interface. This growth can lead to operational problems such as hazy fuel, filter blocking, corrosion and an unpleasant odor. Some of this has been known since 1895, but it was not until the late 1950s that microbial attack became headline news. A United States Air Force Boeing B-52 bomber was starved of fuel due to blocked filters, with disastrous results. Further problems with wing fuel tank corrosion and fuel tank gauge malfunctions were identified two years later in civil aircraft; a global survey of jet fuel supply and distribution systems was initiated to understand the root cause of the epidemic. Using traditional test methods, it was found that a mould, Cladosporium resinae (now Hormoconis resinae), was prevalent in 90% of the systems [1]. Good housekeeping (or removal of free water from storage tanks) was immediately implemented and three classes of biocides were in use by the 1970s. With jet fuel problems now relatively infrequent due to the rigorous control measures applied, the spotlight shifted to diesel fuel, particularly marine and automotive grades. In the 1980s problems with diesel vehicles were on the increase “…the contamination of middle distillate fuels by micro-organisms is an issue that has provoked considerable debate over many years” [2]. Similarly in the 1990s “The demand for reliable tests has never been more compelling in the light of increasing incidence of operational problems directly attributable to microbial contamination of fuels” [3]. Again in 2009, “…since the introduction of biodiesel blends, there is a pandemic of operational problems” [4].Fuels have undergone several compositional changes so it is unsurprising that the reasons cited for the increasing incidences have changed with each decade. Initially, reduced refinery storage tank settling times, fewer refinery staff available to control housekeeping and changes in the contaminating population of microbes from moulds to yeasts and bacteria were implicated. Then factors such as biocide resistance, increased use of additives and changes in vehicle technology were linked to problems. And now the focus is on lower sulphur fuels1 and increasing use of biocomponents. However, the microbial incidents experienced have not prompted changes in diesel fuel handling. Undoubtedly microbial growth in fuels has historically caused difficulties for fuel suppliers and looking to the future problems will continue to occur as fuel quality aligns with environmental and vehicle technology requirements. New molecular microbiological diagnostic tools will allow rapid identification of problems so unnecessary, expensive remediation can be avoided. Preventative rather than just control strategies will become of principal importance. However, the stance taken in the 1960s is still valid: housekeeping to eliminate free water and without water, no microbial growth can exist. It is effective fuel handling throughout a well designed and maintained supply and distribution that is the real key to controlling microbial growth.

                                                            1 The 2009 Euro 5 standard ensures that diesel sulphur levels are now 10ppm.

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References

1. Graef, H.W. An Analysis of Microbial Contamination in Military Aviation Fuel Systems Fuels. AFIT/GEE/ENV/03-10, Air Force Institute of Technology, (March 2003).

2. Stockdale, H. et.al., The Effects of Microbial Contamination on Middle Distillate Fuels, Petroleum Review (1994), Vol.48, No.572, 420-421.

3. Hill, G.C. & Hill, E.C., A Review of Laboratory and On-Site Tests for Micro-organisms in Fuels, 2nd International Colloquium Fuels, (1999). TAE Ostfildern, W.J. Bartz (Ed.).

4. Hill, G.C. & Hill, E.C., Green Fuels – Good for Man and the Environment – Good for Microbes, Technische Akademie Esslingen 7th International Colloquium Fuels (14th -15th January 2009).

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Biological Degradation of Biodiesel - Causes, Effects, Monitoring and Mitigation

David Owen

GE water and Process Technologies

Contamination and degradation by microorganisms is most common in middle distillate fuels derived from Petroleum or Bio origins and blends thereof. The indications of microbial growth are filter plugging, clarity and color degradation of the fuel leading to problems throughout the supply chain. The presence of free water containing fungal spores, algae and bacteria is the principal cause of such contamination. Biodiesel is on one hand more prone to biological degradation due to its relatively simple straight structure. On the other hand the solubility of water in biodiesel is very much higher than in mineral diesel and as such higher levels of water contamination are necessary to have free water. Good housekeeping and regular monitoring of any separated water for biological activity are essential tools in controlling the fuel quality. ATP-Based technology available from GE Water & Process Technologies is a rapid field-based test that can provide an indication of overall biological activity in a system within minutes. It alerts the refinery or fuel storage facility to potentially unsatisfactory levels of microbiological contamination, which may warrant further investigation. Culture-based methods are then available to determine the levels of the different organisms if necessary. It may in some cases be necessary to use broad - spectrum biocides to ensure that the fuel supply system remains free from biological infection. This paper will discuss issues around biodiesel with respect to Microbial activity and the methods to detect that activity. It will also cover options to address the bioactivity through physical and chemical means.

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German Interindustry Study: Filter Blocking, Biofuels and Microbes

Margret Schmidt

Shell Global Solutions (Deutschland)

The German interindustry DGMK study about filter blocking, biofuels and microbial contamination was initiated after occurance of scattered problems with filter clogging at retail dispensers and a few depot tanks. These problems have started after the market introduction of B5 - 5% FAME (fatty acid methyl esters) in diesel fuel. No relation of filter sticking to technical configurations of the dispenser or any regional areas was found, however, not all mineral oil retailers were likewise contributed. The impaired filters were mostly sticked with dark slime at their downstream side, all this material was heavily microbial contaminated. Only in a few cases filters were sticked with light fine deposits, investigations showed the presence of sterolglycosides. Because the main reason for filter blocking was identified as microbial contamination, the DGMK investigations focussed on: · Screening of the German fuel chains for microbial contamination and identification of microorganisms in the samples · Dedicated investigation about impact of selected fuel parameters on microbial growth The screening of market samples showed relatively low microb contamination for fuel samples out of the dispensers and in the retail tanks. However, in cases of a free water phase in fuel tanks and in all sticking material from dispenser filters high microb contamination was detected. The microb material consisted of bacteria but mainly of fungal mycelia where filamentous fungi obtained with more than 50 % of the total biomass. A lab study about the impact of selected fuel parameters on microb growth was conducted with 2 parallel inoculum streams: a mixture of 15 most typical fungi species isolated from filter slimes and another stream made from complex biomass extracted from filter slimes, both grown on Sabouraud-Agar. While B5 fuels with the 15 fungi inoculum showed clear biomass growth with increasing water content, this tendency was not seen with the complex biomass inoculum. No impact of other fuel parameters was obtained. Investigation about the impact of water configu ration (solved, microdroplets, free) in FAME containing diesel on microb growth is ongoing.

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Biotechnology for unconventional II generation bio-fuel production

Francesca de Ferra

Eni R&M Division - San Donato Milanese Research Center – Milano ITALY

New processes are needed for the large scale production of biofuels which would not impact on the food production chain. Biotechnology can be integrated with traditional approaches to develop and optimize biofuel production processes and offers new tools and ideas to tailor bio-products profile to the specifics most desirable for energy use.

A review of advancements and perspectives in this field will be discussed in the context of the evolving EU renewable energy legislative framework.

Finally recent activities in biofuel research in Eni will be presented.

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Session 3:

Status and Challenges for Microbial Enhanced Oil Recovery (MEOR)

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Aerobic Microbial Enhanced Oil Recovery,- experience from laboratory tests and field trials

Janiche Beeder, E. Sunde

StatoilHydro

Growth of bacteria can contribute to enhanced oil recovery either by microscopic reduction in residual oil (Sor) or enhanced sweep efficiency. Bacteria can change the interfacial properties of oil-water during growth on residual oil. Results from interfacial tension measurements (IFT) by laser-light scattering have shown reduction from 38-0.006 mN/m. Core flooding have given Sor-decline to 0.03 at the best. Based on the results from laboratory studies, AMEOR field trials have been carried out. A field in Austria showed good response and doubled the oil production in the treated well at a cost of app. 2 $/m3. The use of AMEOR has been extended in Austria and is still in use. A pilot test performed in a mature offshore field gave no response. At the Haltenbanken field Norne, the AMEOR process started full field in 2001, and the reserves has been upgraded with 900 000 m3 extra oil. AMEOR in the Norne field is still running. Other fields are currently under consideration.

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Microbial Ecology of the High-Temperature Petroleum Reservoir and Results of MEOR Technology Application

Tamara N. Nazina1, Natalya M. Shestakova1, Nadezda K. Pavlova1, Ekaterina M. Mikhailova1, Diana Sh. Sokolova1, Tamara L. Babich1, Qingxian Feng2, Fangtian Ni2, Andrei B. Poltaraus3, Sergey S. Belyaev1, Mikhail V. Ivanov1

1 Winogradsky Institute of Microbiology, Russian Academy of Sciences

2 Dagang Oilfield Company

3 Engelhardt Institute of Molecular Biology, Russian Academy of Science

Microbial communities of petroleum reservoirs have been studied by cultural, molecular-biological and biogeochemical methods. However, these approaches were often used separately. The objective of this study was to simultaneously assess the number, activity and diversity of microorganisms in the high-temperature Dagang oilfield (China), to determine the ability of microorganisms to produce oil-releasing agents from oil and to apply MEOR technology based on activation of indigenous microorganisms. Microbial diversity was characterized by analysis of 16S rRNA genes from the DNA- and RNA-based clone libraries generated from formation water. Analytical, microbiological and radioisotope methods were also applied. The Dagang oilfield was inhabited by thermophilic oil-oxidizing (Geobacillus), sulfate-reducing (Thermodesulfovibrio, Desulfotomaculum), fermentative (Thermoanaerobacter, Thermococcus, Thermovenabulum, Thermacetogenium, Thermotogales), and methanogenic microorganisms (Methanothermobacter). Bacteria of the genus Geobacillus utilized crude oil, producing biosurfactants. Fermentative and methanogenic enrichments grew in the culture liquid of the geobacilli, producing gases. Production of oil-releasing agents in the course of aerobic-anaerobic petroleum oxidation suggested the application of a MEOR technology based on the activation of formation microorganisms. The injection of oxygen (as an air-water mixture), mineral nitrogen, and phosphorous salts solution into the oilfield was accompanied by an increase of the microbial numbers, methanogenesis rate, biotransformation of oil with accumulation of bicarbonate, lower fatty acids, biosurfactants, CH4 and CO2 in formation fluids. A s a result of microbial activity, 30000 tons of additional oil was recovered at the experimental site. We experimentally proved that oilfield is an ecosystem in which a biotic community interacts with an abiotic medium. The energy fluxes are based on the biotransformation of oil in particular trophic chain and can be regulated in a goal-directed manner. The work was supported by CNPC (DFT04-122-IM-18-20RU), Russian Academy of Sciences ("Molecular and Cellular Biology"), and Ministry of Education and Science (4174.2008.4).

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Session 4:

Molecular Microbiological Methods (MMM) in Oil Fields

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Bacterial Diversity of a Hypersaline Oil Reservoir Core

Grégoire Galès, Bernard Ollivier, Didier Alazard, Bart Lomans, Dominique Morin, Jean Borgomano and Catherine Joulian

BRGM

The importance of microbiota in oil reservoirs has been recognized for over 50 years, but our knowledge of the nature, diversity of bacteria and their metabolic activities in these ecosystems are still poor. Many studies on subsurface microbial diversity have been performed on collected water from drilling tubes that can be contaminated by biofilm colonizing the tubing, thus possibly giving an erroneous insight of the subterrestrial microbial community. For this reason and with the aim to look for indigenous microorganisms, we worked on a core from a deep subsurface oil field in West Africa to avoid anthropogenic contaminations resulting from oil production. The microbial diversity was investigated by means of molecular and culture methods from a preserved core. The core was extracted from ca. 1150 m depth and was characterized by a high salt concentration close to saturation. Cultures and molecular experiments targeting various physiologic groups (sulphate-reducers, nitrate and nitrite-reducers, methanogens, fermentative and aerobic respirers), together with chemical and petrographical analyses of this core (minor and major elements) were performed. Surprisingly, halophilic aerobic taxons were predominant within the core. Neither sulfate-reducers nor methanogens were retrieved. Our results clearly indicate that bacterial activity in such environment should be moderate, because of the prominence of oxygen respirers in this typically anaerobic ecosystem. This community structure could be inherited from the time of the sediment settling.

31  

GeoChip: A High Throughput Genomics Technology for Characterizing Microbial Functional Community Structure

Jizhong Zhou

University of Oklahoma

Microarray technology provides the opportunity to identify thousands of microbial genes or populations simultaneously. A comprehensive functional gene array (GeoChip) was developed to detect and monitor microbial communities important to various biogeochemical, ecological and environmental processes. Based on the second generation of GeoChip, a new generation, GeoChip 3.0, has been developed with several new features. First, GeoChip 3.0 contains ~25,000 probes and covers ~47,000 sequences for 292 gene families. Second, the homology of automatically retrieved sequences by key words is verified by HUMMER using seed sequences so that the sequence retrieving process is automated. Third, a universal standard has been implemented so that data normalization and comparison of different microbial communities can be conducted. Fourth, a genomic standard is used to quantitatively determine absolute gene abundance. In addition, GeoChip 3.0 i ncludes phylogenic markers, such as gyrB. Finally, a software package has been developed to facilitate the management of such a complicated array, especially for data analysis and future update. GeoChips were successfully used to analyze microbial functional structure from a variety of environments such as biocorrosion, wastewater treatment, microbial fuel cells from hydrogen production, hydrothermal vents, uranium-contaminated groundwater, the distribution of microbial functional communities across different oil fields, and grassland microbial communities in response to elevated CO2 and soil warming. New insights and implications in these systems were obtained. The results also indicated GeoChip is a novel, powerful high throughput, quantitative genomics technology for characterizing microbial functional community structure from a variety of natural habitats.

32  

Quantification of Prokaryotic Gene Expression in Shallow Marine Subsurface Sediments of Aarhus Bay, Denmark

Joel Kostka1, M. Humphrys1, L. Holmkvist2, B.B. Joergensen3

1Florida State University

2Danish Technological Institute

3Max Planck Institute for Marine Microbiology

Due to limited resources for microbial growth, the shallow subsurface can serve as an ideal natural laboratory for the deep biosphere that has the advantage of being more easily accessible. Despite the global significance of the shallow subsurface, the microbiology of these environments is in its infancy. There is as yet no consensus on the predominant, "metabolically-active" microbial groups that catalyze biogeochemical cycles in situ. The objective of this study was to develop a molecular proxy for the metabolic activity of sulfate-reducing prokaryotes in the subsurface. This molecular proxy can be applied to the quantification of active sulfate-reducing prokaryotes in oil reservoirs and oil field systems.

The community abundance and diversity of mRNA transcripts directly related to sulfate reducing prokaryotes were investigated using geochemical and molecular techniques in marine subsurface sediments of Aarhus Bay, Denmark. Using geochemical techniques, determinations of sediment geochemistry, porewater chemistry, and sulfate reduction rates were performed on subsamples from sediment cores to 5 m below the sediment surface. Molecular analysis of the dissimilatory (bi) sulfite reductase (dsrAB) mRNA transcripts and 16S rRNA were performed by reverse transcription real time quantitative PCR and traditional cloning and sequencing.

The distribution of dsrAB transcripts was directly linked to both sulfate reduction rates and rRNA content. Additionally, quantitative analysis of dsrAB gene transcripts indicated the presence of active sulfate reduction at 465 cm below the sediment-water interface, where high methane concentrations persist in regions of near sulfate depletion.

These data illustrate an abundance of active bacteria in zones of high sulfate reduction and a marked decrease in zones of low sulfate reduction rates. Substantiated by geochemical and rRNA analysis, the analysis of mRNA gene transcripts serves as a versatile molecular proxy for the study of sulfate reducing communities in marine subsurface sediments.

33  

Detection of SRP Activity by Quantification of mRNA for the Dissimulatory (bi) Sulphite Reductase Gene (dsrA) by Reverse Transcriptase Quantitative PCR

Andrew Price1, L. Alvarez2, C. Whitby2, J. Larsen3

1Oil Plus Ltd

2University of Essex

3Maersk Oil

Molecular biological methods have been used for some years to identify and quantify active microorganisms present in a commercial oil reservoir where biogenic sulphide production is routinely controlled by nitrate injection. In order to gain a more complete understanding of the effects of nitrate injection on the activity of SRP, the mRNA for dsrA present in produced water samples was quantified by RT-qPCR; mRNA for dsrA should only be produced by SRP actively reducing sulphate. The aims of this study were: to help further our understanding on the mode of action of nitrate on SRP activity e.g. competitive inhibition by NUB, nitrite toxicity, change in REDOX potential or a metabolism switch from sulphate to nitrate reduction and; to provide a rapid monitoring tool for SRP activity. Since messenger RNA is known to be unstable and is rapidly processed within cells, the first task was to design a laboratory experiment to demonstrate that mRNA for dsrA could be detected and quantified in produced water samples. Produced water samples were spiked with a SRP culture grown from the produced water sample and the mRNA for dsrA was successfully detected and quantified. For the field study, fresh produced water samples were obtained from two wells where direct seawater and nitrate breakthrough has occurred. DAPI, FISH & RT-qPCR analyses were performed directly on the water samples. This paper describes the use of RT-qPCR and detection of mRNA for dsrA as a tool for monitoring SRP activity in biogenic sulphide producing reservoirs. The technique can be used to ascertain the effects of nitrate injection on SRP populations, for instance, in the case of Desulfovibrio; do species of this bacterium preferentially reduce nitrate rather than sulphate? The technique may also be used to determine the recovery of SRP activity following nitrate or biocide dosing.

34  

Session 5:

Control and Prevention of Reservoir Souring

35  

Potential Of Nitrate Addition To Control The Activity Of Sulfate-reducing Prokaryotes In High-temperature Oil Production Systems

Antje Gittel1, K. Sørensen2, T.L. Skovhus2, K. Ingvorsen1, A. Schramm1

1Biological Sciences, Aarhus University

2 DTI Oil & Gas, Danish Technological Institute

Sulfate-reducing prokaryotes (SRP) cause severe problems like microbiologically influenced corrosion (MIC) and reservoir souring in seawater-injected oil production systems. Adding nitrate to the injection water is one strategy to control SRP activity by favoring the growth of heterotrophic, nitrate-reducing bacteria (hNRB) and nitrate-reducing, sulfide-oxidizing bacteria (NR-SOB). In this study, microbial diversity and abundance of Bacteria, Archaea and SRP as well as their potential activity were studied in production waters from the Dan and Halfdan fields. Both fields share similar physicochemical characteristics in injection water composition and reservoir conditions. However, nitrate has only been added to the injection water at Halfdan and resulted in a successful control of SRP activity. Besides 16S rRNA- and dsrAB gene-based analyses, two novel quantitative PCR assays were developed to specifically target troublesome SRP and distinguish between bacterial and archaeal dsrAB gene abundances. At both sites, Archaeoglobus-related SRP dominated the sulfate-reducing community (91-97 %). However, their relative contribution to the total prokaryotic community suggested that they were less prominent at the nitrate-treated site than at the untreated site (2.9 % and 14.1 %, respectively). Thermophilic bacterial SRP contributed for about 0.4 % to the total prokaryotic communities in both systems. The presence of active SRP (35SO42- incubations, TRI35S analysis) was demonstrated at the non-treated field only and additionally supported by growth of thermophilic bacterial SRP in enrichment cultures (60°C). No SRP activity was detected at reservoir temperature (80°C) and in samples from the nitrate-treated field. In addition, potential competitive nitrate reducers (both NR-SOB and hNRB) were more abundant at the nitrate-treated field indicating a stimulation through nitrate addition. In addition to SRP, other sulfidogenic prokaryotes (e.g. Thermococcales) were highly abundant in both production systems as well and should be considered as another clade of potentially troublesome organisms in MIC and reservoir souring.

36  

Use Of Nitrate In Souring Control Of An Oil Field With Low Bottom Hole Temperature

Gerrit Voordouw, A. Grigoryan, A. Lambo, S. Lin

University of Calgary

Production of oil by water injection often leads to souring, the production of sulfide by sulfate-reducing bacteria (SRB). These use reservoir oil organics as electron donor to reduce sulfate in the injection water to sulfide. On-land (as opposed to off-shore) reservoirs are generally subjected to produced water reinjection (PWRI) in which produced water, separated from produced oil, is mixed with make-up water to obtain injection water. We have extensively studied souring in an oil field in Alberta in which make-up water (~2 mM sulfate) is the main input of sulfate into the injection water (~0.7 mM sulfate due to mixing with sulfate-free produced water). The reservoir is at a depth of ~1000 m, has a bottom hole temperature of ~30oC and produces a heavy oil. Souring was noted several years after water injection was started. The concentrations of aqueous sulfide in produced waters vary currently from 0-0.4 mM, depending on the production well (PW). Nitrate (~2 mM) has been injected field-wide since May 2007 to bring souring under control. This stimulates heterotrophic nitrate-reducing bacteria (hNRB) and nitrate-reducing, sulfide-oxidizing bacteria (NR-SOB), which are present in all produced and injection water samples. We have monitored 4 injection wells and 16 PWs by sampling every 2 to 3 weeks. Some of the key observations made are as follows: 1. Nitrate was effectively distributed through the above ground piping system, although regular biocide treatment to prevent wall growth was essential. 2. Toluene (not VFA), present in oil at ~6 mM, appears to be the main electron donor for nitrate reduction. 3. Continued, field-wide nitrate injection lowered aqueous sulfide concentrations by ~70% of pretreatment values in most PWs within 5 weeks, followed by recovery. 4. This points to microbial stratification: SRB, growing close to the injection wellbore because of sulfate limitation, are displaced by hNRB but then grow back in regions deeper in the reservoir. 5. Nitrite, produced by hNRB and NR-SOB, helps transform iron sulfide (FeS) present downhole into greigite (Fe3S4) increasing the sulfide binding capacity of reservoir rock. 6. The microbial community present downhole, as judged by DGGE of produced waters, is diverse and changes, especially when nitrate breaks through. Nitrate injection into low temperature oil fields offers unique challenges, but is overall beneficial to production operations.

37  

A Multi-disciplinary Survey to Limit Souring in Oil Reservoirs in Argentina

Aleksandr Grigoryan, A.J. Lambo, S. Lin, T.R. Jack, A. Cavallaro, G. Voordouw

University of Calgary

Souring of oil fields due to microbial sulfide production has negative consequences for oil production. Nitrate has been used successfully for injection to contain biological sulfidogenesis in off-shore and on-shore fields. The mechanism for control of microbial souring involves in-situ stimulation of heterotrophic nitrate-reducing bacteria (hNRB), that compete with sulfate-reducing bacteria (SRB) and inhibit them metabolically. Nitrate-reducing sulfide oxidizing bacteria (NR-SOB) directly eliminate sulfide from reservoir fluids. Oil fields differ greatly in physical and geochemical parameters like temperature, porosity and permeability, salinity, as well as organic electron donor and nutrient concentrations in reservoir fluids, which influence microbial distribution and activity. Knowledge and understanding of these parameters and use of appropriate methods to determine microbial activity is required to predict success with nitr ate injection to contain reservoir souring. A combination of microbiological and geochemical surveys of seven oil fields in Argentina revealed higher microbial activities in samples with low salinity and low bottom-hole temperature. Therefore, injection waters tended to be more active than produced waters. Saline brines with a high Ca2+/Mg2+ ratio and high resident temperature depressed bacterial activity, while the synergic action of high temperature and high salinity practically eradicated microbial activity. Thermophilic microbial activity was found more readily in rich than in defined media. Use of defined media to evaluate hNRB, NR-SOB and SRB activity indicated that most produced fluids from hot, saline were sterile, supporting the conclusion that thermophilic microbial activity is absent from such fields. Activity in fields with high resident temperature is confined to the cooler injection wellbore region. Nitrate can successfully remove sulfide produced in this region. However, successful demonstration using limited injector and producer combinations can be difficult due to subsurface mixing of nitrate-treated sulfide free and sulfide-containing waters flowing in from non-treated regions of the reservoir.

38  

Isotopic Fractionation by an n-Alkane Degrading Sulfate Reducer: Implications for Monitoring Intrinsic Bioremediation and Reservoir Souring

Brandon Morris1, J. Suflita1, H-H. Richnow2

1University of Oklahoma

2Helmholtz Centre for Environmental Research - UFZ

Introduction: The complex anaerobic processes that occur in oilfield systems can exacerbate problems such as microbially-influenced corrosion, increases in oil viscosity, and reservoir souring. However, some of these same processes are also involved during in situ remediation of petroleum-contaminated environments. Part of the characterization of anaerobic metabolic processes in these systems should involve quantifying the extent of stable isotope fractionation. These data provide both biodegradation rate estimates and information about the nature of the metabolic pathway(s) involved in the oilfield system. We studied isotopic fractionation of alkanes, quantitatively the most important components of oils, with a pure culture and model substrates.

Methods: Two-dimensional isotopic analysis using GC-IRMS, which combines stable isotopic enrichment factors (ε) for carbon and hydrogen, has recently been shown to reliably predict and quantify aerobic and anaerobic biodegradation processes of aromatic hydrocarbons by both mixed consortia and pure cultures. Further application of the attendant techniques to characterize anaerobic n-alkane metabolism is reported here.

Results: We provide the first evidence of carbon and hydrogen fractionation during anaerobic alkane degradation by a pure culture of Desulfoglaeba alkanexedens. This organism is known to initiate alkane metabolism via a fumarate-addition mechanism. Carbon and hydrogen enrichment factors for hexane were observed to be -5.52 ‰ and -43.14 ‰, respectively. Enrichment factors for octane were -5.19 ‰ for carbon and -27.77 ‰ for hydrogen. Discussion: The Rayleigh equation-based results presented here represent a new range of carbon and hydrogen values that may be expected during anaerobic alkane degradation by glycyl radical enzymes. These results serve as a basis for comparison of fractionation values observed in future field studies.

39  

Session 6:

Practical Application of Molecular Microbiology Methods (MMM) in Oil Field Systems

40  

Microbial Diversity of an Oil-Water Processing Site and its Associated Oil Field Production Water

Geert van der Kraan1, 2B. Lomans, 1M. van Loosdrecht, 1G. Muyzer

1Delft University of Technology, 2Shell Exploration & Production

Traditional community analysis of oil field production waters has limitations when determining the actual microbial composition. Therefore alternatives, like molecular methods, are required to obtain a more detailed overview of these communities. Here we present an overview of the diversity and distribution of Bacteria and Archaea in the production water from oil wells and an associated oil-water separation facility located in the western part of the Netherlands.

We used Denaturing Gradient Gel Electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments as a quick method to scan the microbial diversity in the (i) oil production wells, (ii) 2 different oil-water separators, (iii) a diluter/O2-scavenger and (iiii) water injection wells. Subsequently, we cloned and sequenced the nearly complete 16S rRNA genes to determine the diversity in more detail. Comparative sequence analysis showed significant differences in community composition between the sampled environments, (statistically validated by webLIBBSHUFF analysis).

In the production water we detected bacteria that were affiliated to those found in oil wells by other authors (e.g., Anaerobaculum thermoterrenum). In the two separators Marinobacter species were dominant, and in the desalinator the dominant bacterial species was Thermodesulfovibrio yellowstonii, which belongs to the Nitrospirales. It is known for its sulfate-reducing activity at high temperatures. The populations in the production water and in the two separators were dominated by γ-Proteobacteria. The presence of α-Proteobacteria is observed only in the production water. The overall population in the diluter was clearly sulfate reducing. Methylotrophic Archaea related to those found in high-salt environments (e.g. Methanohalophilus euhalobius) were detected in all sampled environments. Only Archaea belonging to the Methanosarcinalis and Methanomicrobiali were present in the studied environments.

The used techniques resulted in valuable information about the microbial communities. Further study into their metabolic conversions and possible impact in the oil production system is therefore advised.

41  

Comparison of Biocides Using New Detection Tool

Pat Whalen1, E. Corrin2, S. Leong3

1Luminultra Technologies

2MultiChem

3OSP Microcheck Inc

Microbial contamination in the oil and gas industry is prevalent in both downstream and upstream activities. Fouling, corrosion and all the problems associated with respiration by-products contribute to lifting and production costs. Other problems include spoilage of drilling, well stimulation and production fluids. The impact of these problems can be managed by understanding where the microbes are and when they need to be controlled. Biocide programs are one effective way to control bio-burden in produced and surface waters. There are limited methods available that will quantitatively show the direct impact of a biocide program and offer an opportunity to optimize dosage rates and application points. The most common and least expensive methods involve inoculation of a nutrient medium specific to certain types of bacteria and incubation over a period of time. Detection of viable microbes in this way results in disadvantage s, primarily the incubation time requirement which does not allow for timely adjustments to the biocide application. This paper summarizes results obtained using a novel technique for bio-burden detection via Adenosine Triphosphate (ATP) in surface and produced water from the Canadian Western Sedimentary Basin. ATP is an excellent marker for all cells and commercial detection systems have been available for several decades. The advantage of ATP detection is that results are available in minutes, rather than days or weeks. However, traditional ATP detection systems had significant drawbacks, including assay interferences and poor sampling techniques, generating results that were difficult to interpret and apply. The current protocol has been refined to eliminate these deficiencies and produce repeatable results that are comparable with industry accepted culturing methods. In addition to examining bio-burdens from representative surface and produced waters, the technique was also used to compare the efficacy of several non-oxidizing and oxidizing biocides on the microbial populations.

42  

Prevention of MIC - A Case Study in Pipelines Transporting Water Condensate from Natural Gas

Aleida de Vos van Steenwijk, J. Krooneman

Bioclear

During production of natural gas, by-products are separated and transported through water condensate pipelines to a treatment facility. The case study under investigation here was finding unexpected corrosion problems within one of these pipeline systems. The question was raised whether or not this was owing to microbiologically influenced corrosion (MIC) and if so what could be done about it. MIC occurs where micro-organisms change local conditions at or near the surface of a material and thereby induce or accelerate corrosive processes. By unraveling and understanding the underlying biological and chemical processes, MIC can be diagnosed, risks can be assessed and strategies for prevention and prognoses can be set up. The approach used in this case was to gain a detailed picture of the biological situation within the pipeline system. Molecular microbiological methods were used to detect and quantify relevant micro-organisms. The environmental conditions within the pipeline system were analyzed. Based on these analyses a risk assessment was performed. Possible strategies were investigated that could reduce risks of MIC occurring. Relevant micro-organisms were found throughout the pipeline system. Furthermore, all environmental parameters within the pipeline were favorable for growth of sulphate reducing bacteria (SRB) and thus favorable for MIC. All except for the pH, as this was lower than the optimum for SRB growth. All relevant parameters were combined in a protocol which is used to determine whether water condensate entering the pipeline system will lead to an increased risk of MIC. If risks are high, the water condensate is not allowed to enter the pipeline. The strength of this approach is that conditions within the pipeline are maintained in such a way to discourage SRB growth and as a consequence risks of MIC. Since implementation of the protocol, no internal MIC problems have been encountered in the pipeline system.

43  

Session 7:

Biodegradation of Hydrocarbons in Oil Production

44  

Small Bugs in Big Ponds: Microbiology in Oil Sands Tailings

Julia Foght1, P. Fedorak1, T. Siddique1, T. Penner2

1Univ. Alberta, Edmonton AB Canada

2Syncrude Canada Ltd.

Extraction of bitumen from surface mined oil sands ores produces enormous volumes of tailings waste comprising water, sand, silt and clay particles, unrecovered bitumen and a small proportion of hydrocarbon solvent used during extraction. The tailings are deposited into large 'ponds' where they settle slowly to generate anaerobic mature fine tailings (MFT) and an overlying layer of water. Biogenic methane bubbles observed at the surface of established and recent tailings ponds raise questions about the processes involved.

Anaerobic microcosms of ~100 mL and columns containing

45  

Microbial Community Structure, Dynamics and Function in Methanogenic Petroleum Degrading Systems

Neil Gray1, C. Aitken1, A. Sherry1, I. Head1, M. Jones1, Michael Erdmann2, J. Adams3, S. Larter3

1School of Civil Engineering and Geosciences Newcastle University

2 StatoilHydro, Research Centre Bergen

3 Geology and Geophysics University of Calgary

Geochemical evidence indicates that biological alteration of hydrocarbons occurs in anaerobic subsurface settings and has led to the World's vast but economically less valuable heavy oil deposits. However, recent data indicates these processes also played a role in the formation of associated economically valuable gas accumulations. For instance, the preferential removal of n-alkanes observed in biodegraded petroleum reservoirs is mimicked in methanogenic oil degrading laboratory microcosmsGas isotope data from biodegraded petroleum reservoirs are consistent the pathways of methanogenic oil degradation observed experimentally. Analysis of the microbial communities from methanogenic oil-degrading microcosms indicated succession in bacterial community composition with the emergence of species closely related to those identified in petroleum reservoirs, other deep subsurface environments and in other methanogenic systems exhibitin g hydrocarbon degradation. In particular Syntrophus spp. (Deltaproteobacteria) and members of the genus Anaerolineae (chloroflexi) were predominant. This congruence of species in a range of methanogenic, hydrocarbon-associated systems, suggests common mechanisms of hydrocarbon degradation exist in subsurface hydrocarbon degrading systems. Analysis of archaeal diversity in the same hydrocarbon degrading microcosms indicated selection for hydrogenotrophic methanogens suggesting that methanogenic alkane degradation was dominated by CO2 reduction linked to hydrogenotrophic methanogenesis and syntrophic acetate oxidation. Although the isotopic composition of methane and CO2 from some biodegraded petroleum reservoirs supports this notion some studies have shown dominance of acetoclastic methanogenesis with anaerobic hydrocarbon degradation. While the ecophysiology of methanogenic oil degradation is not yet fully understood it has been proposed that in situ methanogenic biodegradation of oil could be harnessed to enhance recovery of stranded energy assets from petroleum reservoirs. Methanogenic enrichments from production waters from an oil rimmed gas accumulation believed to have formed from microbial conversion of oil to methane, have established the presence of putatively indigenous fermentative bacteria and methanogens and the potential for stimulation of their activity by nutrient addition.

46  

New Insight into Complexity of Subsurface Methanogenic Pathways: CO2-Reduction and Acetate-Fermentation are not alone

Dariusz Strapoc1, M. Ashby2, R. Levinson1, B. Huizinga1

1ConocoPhillips

2Taxon Biosciences

Methanogenesis is considered the main terminal process of subsurface anaerobic organic matter degradation. Previous studies reported CO2-reducing and acetoclastic methanogenesis as dominant subsurface methanogenic pathways for primary and secondary biogenic gas generation, (i.e. in oil biodegradation or coalbed methane settings). In lab-scale experiments, methanogens were shown to be able to utilize a wider variety of substrates, typically containing methyl groups, i.e. dimethyl sulfide (DMS), methyl amines, formate, and methanol. Our recent observations from a single biogenic gas field in Alaska suggested a significant contribution of another methanogenic pathway in the subsurface environment. Low maturity organic debris in fluvial sediments is being degraded to methane via three different methanogenic pathways: CO2, acetate, and methanol utilizing. The addition of a third subsurface-operative methanogenic pathway (methanol utilizing) provides new insight into stable isotopic classification of biogenic gases. Clone libraries suggest equal importance of each of the three methanogenic pathways (CO2, acetate, and methanol utilizing) as a source of methane for this gas field. The distribution of microbes involved in the dominant methanogenic pathway varies from well to well and appears to be controlled by local geochemical conditions within the field. The main environmental controls are reservoir temperature and water chemistry (i.e. pH and salinity). Statistical analyses of integrated microbial and geochemical data suggest variation of methanogenic communities with environmental reservoir conditions.

47  

A Micro Solution to a Mega Problem - Can Microbes Be Used to Clean up Dirty Oil?

Richard Johnson1, B. Smith2, K. Robinson2, S. Rowland3, C. Whitby1

1Department of Biological Sciences, University of Essex

2Oil Plus Ltd.

3University of Plymouth

There are vast reserves of tar sands and heavy oils that have not yet been fully exploited. These tar sands contain complex mixtures of predominantly cycloaliphatic or alicyclic carboxylic acids known as naphthenic acids (NAs). These NAs cause major environmental and economic problems as they are recalcitrant, corrosive, and toxic and need to be removed during refining. Aromatic compounds make up a small, but so far unexplored fraction of this mix. This project aims to characterize aromatic NA-degrading microorganisms in relation to NA biodegradation rates, metabolite production and NA structure. Enrichment cultures were set up on four aromatic NAs that differed in the branching structure of the alkyl side chain. Environmental samples from a contaminated coal tar site were inoculated into minimal medium containing individual NAs as the sole carbon source. GC-MS analysis of the enrichment cultures demonstrated complete metabolism of the least branched compound (4-(4'-n-butylphenyl)butanoic acid) after 14 days and consequent mineralization within 49 days. The other NAs tested were recalcitrant and resulted in persistent metabolites, which remained after 125 days incubation. PCR and 16S rRNA gene clone libraries demonstrated a shift in community composition during NA degradation. In addition, an environmental isolate (with 99% 16S rRNA gene sequence identity to Mycobacterium fluoranthenivorans) also demonstrated the ability to metabolize aromatic NAs within 7 days. Our findings suggest that the presence of specific microorganisms as well as NA structure dictates the degree to which NAs are biodegradable. This novel research will enable further mechanistic studies to be performed to identify the NA biodegradation pathways and the genes involved. This will allow the development of more efficient approaches to removing NAs from heavy oils.

48  

Syntrophic Acetate Degradation to Methane in a High-Temperature Petroleum Reservoir

Natalya M. Shestakova1, T.N. Nazina1, Q. Feng2, F. Ni2, T.P. Tourova1, A.B. Poltaraus3, S.S. Belyaev1, M.V. Ivanov1

1Winogradsky Institute of Microbiology, Russian Academy of Sciences

2Dagang Oilfield Company, Tianjin, China 3Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Two mechanisms of methane formation from acetate have been described. The first one is aceticlastic, which is carried out by representatives of Methanosarcinaceae or Methanosaetaceae. The second mechanism is syntrophic, which is performed by an association of acetate-oxidizing bacteria (strain AOR or Thermacetogenium phaeum) and hydrogenotrophic methanogens. The aim of the present work was to study the microbial community from the high-temperature Dagang oilfield (China) and the processes of acetate degradation to methane. The diversity and activity of microorganisms of the high-temperature Dagang oilfield were studied by radioisotope, culture-based, and 16S rRNA gene approaches. The processes of methanogenesis from NaH14CO3 and 14CH3-COONa were registered in formation waters by radioisotope methods. Microorganisms producing methane both from Н2+CO2 and acetate were also detected in the microbial community by cultural methods. However, pure cultures of thermophilic aceticlastic methanogens were not obtained. 16S rDNA analysis of the formation water and methanogenic enrichments (more than 800 archaeal clones) revealed that representatives of the order Methanobacteriales (Methanothermobacter thermautotrophicus) were predominant in the microbial community. 16S rRNA genes of acetate-utilizing methanogens (5 phylotypes) were detected only after the increase of acetate concentration in formation water. Phylotypes of fermentative bacteria and archaea, sulfate-reducing and syntrophic (Thermacetogenium) bacteria were revealed. Pure cultures of M. thermautotrophicus and Thermoanaerobacter ethanolicus were isolated from the methanogenic enrichments grown o n acetate. It was shown that a syntrophic association of T. ethanolicus and the H2-utilizing methanogen M. thermautotrophicus carried out the reaction of acetate degradation to methane. Thus, the terminal stages of oil biodegradation and the ecological function of a widespread Thermoanaerobacter-Caldanaerobacter group were defined in the high-temperature petroleum reservoir. The work was supported by the Dagang Oil field Company (DFT04-122-IM-18-20RU), Russian Ministry of Education and Science (4174.2008.4), and the Russian Academy of Sciences (Programs No 14 and "Molecular and Cellular Biology").

49  

Session 8: Microbiological Challenges in Biofuels

50  

Assessing Microbial Spoilage of Biodiesel Blends under Aerobic and Anaerobic Conditions

Gitte Sørensen, K. Sørensen, U.S. Thomsen, T.L. Skovhus, H.O. Hansen, S.D. Nygaard

DTI Oil & Gas, Danish Technological Institute

Micro organisms are known to grow in fuel tanks where they may affect both fuel quality and the integrity of storage facilities. Mixing with sustainable, biomass-derived fuel (biodiesel) is suspected to increase these problems, but the underlying, microbiological mechanisms remain poorly understood. In this study, five different mixtures (0, 2, 5, 10, and 20 % biodiesel mixed with petrochemical diesel) were incubated with microbially contaminated water under anaerobic and aerobic conditions for 51 and 29 days, respectively, and changes in chemical properties of the fuel as well as in the microbial communities were monitored. The results indicate that rapid microbial growth occurred in all mixtures. Anaerobic mixtures contained mainly fermentative bacteria, and methane-producers were less abundant (< 1% of the population). This is significant since methanogens are thought to increase rates of corrosion in oil installations by maintaining a low hydrogen concentration. Aerobic mixtures contained large amounts of fungi, and both growth and diversity of bacteria was limited, possibly as a consequence of bacteriocides excreted by the fungi or competition for organic substrates. Several conclusions may be drawn from this study: (i) in spite of significant microbial growth, the chemical properties of the fuel was not significantly altered, (ii) the groups of bacteria and fungi identified in this study should be employed in future evaluation of biocidal fuel additives rather than the standard laboratory strains, and (iii) a better understanding of the implication of fungi and fermentative bacteria in biofilm formation and MIC is urgently needed.

51  

Molecular Biology Tools in the Study of Biodiesel in Brazil

Marcia Lutterbach, L. Contador, M. Galvão, V. Oliveira

National Inst. of Tech.

Biodiesel is a renewable and biodegradable fuel composed of alkyl esters derived from vegetable oils or animal fats. The blend of biodiesel and diesel is mandatory in several countries, as USA and EU. In Brazil, the diesel fuel must have 2% of biodiesel since 2008. The Brazilian program for development of biofuels encourages the diversification of raw materials used in the production of biodiesel. Due to its biodegradability, biodiesel is probably more vulnerable than traditional diesel to microbial deterioration, but there are still few studies on this subject. The Laboratory of Biocorrosion and Biodegradation (LABIO) works with microbial contamination of biodiesel since 2004, through traditional culture methods and molecular biology analysis. According to our results, the anaerobic bacteria are predominant in biodiesel samples compared to aerobic bacteria. Bacterial strains isolated from biodiesel are different from the bacteria retrieved from diesel samples. The strain Cupriavidus pauculus has been detected in different samples of biodiesel from different raw materials. Molecular biology tools are being employed in the microbial identification and in the analysis of microbial community diversity for a better understanding of the biodeterioration of biodiesel, diesel and mixtures.

52  

Important Technical Considerations when Contemplating Bioluminescence as an Analytical Tool for the Determination of Microbial Contamination of Petroleum Fuels and Other Matrices

Howard Chesneau

Fuel Quality Services, Inc.

Bioluminescence has become a very versatile analytical tool used for the assay of microbial ATP. With the availability of high purity and commercially pure prepared reagents, highly sensitive photomultiplier tubes (PMT), software, miniaturization of portable power supply and equipment, this analytical technique has moved from the research laboratories and universities and into the field where technicians and laypersons with a minimum amount of training can quantify the microbial load of fuel samples from pipelines, storage tanks, vehicles and aircraft on a nearly real time basis. As such this technique has earned its rightful place as a valid analytical tool used to quantify the microbial load present in petroleum fuels, some biofuels and their respective fuel associated bottom water. Since 1947 when William D. McElroy isolated and purified the heat-stable luciferin and labile enzyme luciferase from the firefly, many analytical challenges have been identified and resolved in order that bioluminescence could become a reputable and reliable analytical tool. For example, in order to ensure optimal light emissions proportional to the ATP concentration the chemicals and solutions used to duplicate reactions found in nature has to be commercially available; in order to ensure the maximum optical output of photons from the cuvette to the detector, the cuvette material has to be of a certain minimum optically quality; temperature compensation and computer algorithms has to be available to ensure results are corrected to the optimal temperature; and the detector has to be of a specific design capable of sensing low light/photon output in the spectrum of 460nm to 580nm from the bioluminescence reaction over an interval of several seconds as well as managing background noise levels. This presentation will address the important considerations when contemplating bioluminescence as a valid analytical test method for the determination of microbial contamination of petroleum fuels and other matrices as well as appropriate biocide remediation strategies in dealing with contamination.

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Posters presentations:

(Note that the posters below are listed alphabetically after the name of first author)

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An Example Of The Use Of Molecular Microbiological Methods For Detection, Monitoring And Control Of Biological Processes

Aleida de Vos van Steenwijk, B. Geurkink, I. Dinkla, J. Krooneman

Bioclear

Application of molecular microbial methods provides vital information needed for monitoring and control of biological processes. Sometimes to encourage these processes such as biological remediation of oil spills, sometimes to discourage them, such as microbiologically induced corrosion (MIC). In this presentation an approach is described that is used in practice for monitoring and control of biological processes. Generally speaking a combination of two techniques is used to gain insight in a particular biological process. DGGE (Denaturing Gradient Gel Electrophoresis) is used to visualize populations and population shifts. This technique also provides information on which micro-organisms are relevant in a process. Quantitative Polymerase Chain Reaction (Q-PCR) is used to accurately detect and quantify these specific (groups of) micro-organisms or to detect and quantify critical functions within a process. This technique relies on the detection and quantification of genetic material (DNA and/or RNA). The combination of Q-PCR and DGGE is particularly valuable as it allows quantitative detection/monitoring tools to be set up for micro-organisms that have been identified as being relevant in a process. Using this approach molecular detection tools have been set up and applied in practice for a variety of biological processes. Examples include monitoring of biological remediation, detection of plant pathogens and diagnosing of problems in waste water treatment plants. One specific example is given in which a broad set of Q-PCR analyses was developed for the detection and quantification of micro-organisms relevant in MIC. These include analyses for specific groups of micro-organisms (sulphate reducing bacteria, sulphur oxidizing bacteria, iron reducing bacteria, iron oxidizing bacteria, methanogens and total bacteria/archaea) as well as specific functions (sulphate reduction, sulphur oxidation, methanogenesis). Combined with chemical data from a particular location, these analyses provide a strong tool for the detection, monitoring and control of MIC.

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Identification of a New Freshwater Sulfate-Reducing Bacterium Degrading 2-Methylnaphthalene in the Enrichment Culture by Combined Cultivation and Molecular Approaches

Alexander Galushko1, J. Loncar2, U. Kiesele-Lang3, B. Schink2

1MPI Marine Microbiology

2Konstanz University

3Grundstücksgesellschaft Metallhüttengelände mbH

2-methylnaphthalene (2MN) is a bicyclic aromatic hydrocarbon that occurs in crude oil. In last decade degradation of 2MN by sulfate-reducing bacteria (SRB) was demonstrated in anoxic marine and freshwater environments. Several pure cultures of marine 2MN-degrading SRB were isolated and phylogenetically characterized. They form a distinct cluster of closely related bacteria within the delta group of Proteobacteria. Several cultures of freshwater SRB degrading 2MN were recently enriched but phylogeny of the bacteria was still unknown. We obtained a highly enriched culture of freshwater SRB (named as RS2MN) growing on 2MN. Numerous attempts to isolate a pure culture of bacterium degrading 2MN from the enrichment culture RS2MN using several different organic substrates were unsuccessful. Therefore, we applied a combination of microbiological and molecular methods to investigate bacteria in the enrichment culture. We identified and characterized 16S rRNA gene and dsrAB genes of a sulfate-reducing bacterium which utilizes 2MN in the enrichment culture. It is a bacterium which oxidizes organic substrates completely and the closest cultivated relatives are not marine 2MN-degrading SRB but the Desulfosarcina species. One additional sulfate-reducing bacterium closely related to the species of the Desulfomicrobium genus was present in the enrichment culture. It was isolated in pure culture (named as strain RSPyr) and turned out not to be a hydrocarbon-degrading microorganism. Physiological role of this bacterium in the enrichment culture remained unclear. The enrichment culture did not contain any non-SRB. A comparison of physiological reactions of strain RSPyr and the enrichment culture upon addition of various electron donors and abiotic and biotic electron acceptors allowed us to conclude that the enrichment culture degrades 2MN not via syntrophic cooperation of bacteria but by the activity of a single su lfate-reducing bacterium of the family Desulfobacteraceae.

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The microbiota associated to tropical Loricariidae catfishes as sources of new and promising cellulases to the biofuels industry

Alexandre Rosado, A. Castro,R. Peixoto, E. Bon

UFRJ

Catfishes Loricariidae are endemic of tropics and developing a role in nature by reducing the turnover of carbon to the atmosphere by recycling refractory material. These fish species have a peculiar microbial community associated involved in the processing of detritus. Such bacterial groups, in a similar way, could be used in the industry of biofuels in the degradation of plant biomass. The goal of this work was to determine the main plant energy sources of the tropical detritivorous fishes Parotocinclus maculicauda and Hypostomus punctatus and make an overview on the microbial structure and its potential use in the biotechnology industry. The isotopic analysis were performed using 13C as a tracer to determine the main sources of plant to the species. Were also performed the detection of endoglucanase activity of microorganisms isolated from intestinal samples and the molecular characterization of the structure of microbial comm unities through to PCR/DGGE. The obtained results showed that the sources of energy were diverse. The isolated strains analyzed showed high endoglucanase activity, with four new cellulolytic bacteria species. The DGGE assay reveals different structure of bacterial communities from the two fish species indicating a host selection. Altogether, these informations were important to define the energy plant sources for both species, besides the functional role of microorganisms in recycling of refractory material. Moreover, it is emphasized the importance of the bacterial endosymbionts of this fish group as a source of new and efficient enzymes that could be used in the conversion of plant biomass into biofuels, generating alternative energy sources.

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Molecular Detection Of Genes Involved In Anaerobic Alkane Degradation In Hydrocarbon-Impacted Environments And Enrichment Cultures

Amy Callaghan, L.M. Gieg, R.S. Norman, V. Parisi, I. Davidova, J.M. Suflita, J.J. Kukor, and B.Wawrik

University of Oklahoma

Anaerobic microbial processes have important implications for the oil and gas industry. Dissimilatory sulfate reduction, coupled to hydrocarbon degradation, produces sulfide, which can lead to oil and gas reservoir souring as well as corrosion within oil pipelines. Metabolites of anaerobic biotransformation may also serve as substrates for microorganisms catalyzing metal reduction/oxidation reactions within pipelines. Given the economic incentive to optimize production processes and reduce pipeline corrosion, a better understanding of the biochemistry of sulfate-reducing and anaerobic alkane-degrading microorganisms is desirable. Desulfatibacillum alkenivorans AK-01 is a model organism for studying sulfate reduction and anaerobic alkane metabolism. Activation of alkanes by AK-01 proceeds via sub-terminal addition of alkanes to fumarate. This reaction is catalyzed by the glycyl radical enzyme, alkylsuccinate synthase (As s). The assA gene was deemed a suitable functional biomarker for anaerobic alkane activation because it codes for the catalytic subunit of Ass and is well conserved. Degenerate assA-targeted primer sets were tested in order to survey the diversity of alkane-degrading organisms in hydrocarbon-impacted environments and enrichment cultures. DNA was extracted from contaminated river and aquifer sediments, paraffin-degrading enrichment cultures, and oil field samples. assA genes were amplified, cloned, and sequenced. Sequence analysis revealed novel clades of assA genotypes. Several OTUs, originating from oil-contaminated sediments were most similar to assA genes found in strains ALDC and HxN1, which are known to degrade C6 to C12 and C6 to C8 alkanes, respectively. One genotype, originating from a methanogenic, paraffin-degrading culture, was most similar to the assA genes found in sediment from a gas-condensate contaminated aquifer. The involvement of these newly-discovered assA genotypes in hydrocarbon degradation remains to be demonstrated. However, the detection of these genes, in combination with metabolite profiling and future attempts to assay mRNA levels, may facilitate in situ monitoring of alkane degradation and corrosion in production facilities.