Petroleum Biotechnology - Developments and Perspectives

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Studies in Surface Science and Catalysis

Advisory Editors: B. Delmon and J.T. Yates

Series Editor: G. Centi

Vol. 151

PETROLEUM BIOTECHNOLOGY

Developments and Perspectives

Edited by

Rafael Vazquez-Duhalt

Institute of Biotechnology

National University of Mexico

Morelos Mexico

and

Rodolfo Quintero-Ramirez

Mexican Petroleum Institute

Colonia San Bartolo

Atephehuacan Mexico

ELSEVIER

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P R E F A C E

Without a doubt, historians will describe 20th and 21st centuries as the oil-based society. One

hundred years ago oil exploitation began, first as a source of energy and later to include oil as

a sourc e of raw ma terial. In addition to the 1 trillion ba rrels that have already been harve sted,

recent estimations shows that about 3 trillion barrels of oil remain to be recovered worldwide,

half from proven reserves and half from undeveloped or undiscovered sources. Oil production

is expected to peak sometime between 2010 and 2020, and then fall inexorably until the end

of this century. After the production peak, the more expensive fuel sources will come into

production. These include hard-to-extract oil deposits, tarry sands, and Synfuels from coal

that requires al ternative or complem entary to conventional oil refining techn ologies.

Our society has an inexorable challenge: to increase the production of goods and

services for people, using new process technology that should be energetically efficient and

environmental friendly. This also will be the case for the petroleum industry. Improvements

in conventional oil refining processes such as cracking, hydrogenation. isomerization,

alkylation. polymerization, and hydrodesulfurization, certainly will occur. Nevertheless, non-

conventional biotechnological processes could be implemented. In contrast to the available

processes, biological processing may offer less severe process conditions and higher

selectivity for specific reactions. Biochemical processes are expected to be low demand

energy processes and certainly environmentally compatible.

The primary target of the petroleum industry is to enhance and maintain a continuous

oil production. Preconceived ideas and misconceptions about biotechnology continue to l imit

the applications of biological processes in the chemical industry. Nevertheless, there are

biotechnological processes that have been demonstrated to be industrially successful and that

are shown to be sufficiently stable, productive and economic for commercial applications.

Even if wastewater treatment and soil bioremediation are common biotechnological

applications in the oil industry, petroleum biotechnology is still in its infancy. Doubtless,

though, biotechnology will play an increasingly important role in future industrial processes.

In this book, experts from 11 countries critically discuss the develop me nts and persp ective s of

biotechnological processes for the petroleum industry.

An integrated approach into the possibility of using petroleum biotechnology

throughout the value chain of an oil company is presented. The authors discuss the eva luation

of biotechnology as a general toolbox for solving some of the technology problems of today

and future possibilities to implement new refinery processes. Petroleum refining could be

enhanced by biochemical reactions in which the specificity exceeds by far these of chemical

reactions. The selective removal of sulfur, nitrogen, and metals from petroleum by

biochemical reactions performed by microorganisms and/or enzym es is discussed. Increasing

supply of heavy crude oils and bitumens has increased the interest in the conversion of the

high-molecular weight fractions of these materials into refined fuels and petrochemicals. This

upgrading has typically been accomplished either with high-temperature and expensive

processes thermal conversion (cracking or coking) or by catalytic hydroconversion. In

contrast to the available processes, biological processing may offer less severe process

conditions and higher selectivity to specific reactions. Enzymatic transformations of

asphaltenes in non- conventional media, and biological upgrading to improve the quality of

certain crude oils and liquid fuels could be envisaged, using biocatalysts to decrease

aromatici ty and sensit ize aromatic heterocycles to subsequent heteroatom removal.

Bioprocessing would complement conventional refining technologies and result in improved

fuel quality at lower capital and operating costs and with reduced environmental impact.

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Innovative new processes could be explored, such as methanol production from

methane. Methane monooxygenases are unique among known catalytic systems in their

ability to convert methane to methanol under ambient conditions using dioxygen as the

oxidant. The unusual reactivity and broad substrate profiles of methane monooxygenases

suggest many possible applications in the petrochemical industry. In addition, the ability of

anaerobic bacteria to convert petroleum into methane and thereby generate useful energy is a

very interesting alternative. On the other hand, biological production of hydrocarbons by

bacteria is revisited and its potential is explore d, not only as an environm entally-f riendly fuel

supply, but also as a renewable source for basic petrochem icals.

Microbial colonization of metal surfaces drastically changes the classical concept of

the electrical interface commonly used in inorganic corrosion. Corrosion is a leading cause for

pipe failure, and is a main component of the operating and maintenance costs of gas and oil

industry pipelines. The cost of corrosion to the gas and oil industries was estimated in 2001 to

be about $13.4 billion/yr and of this as much as $2 billion/yr may be due to microbially-

induced corrosion. In order to moderate the economic importance of corrosion in the oil

industry, molecular tools are used to study its microbial complexity.

The current knowledge of the indigenous deep subsurface microbial community in

petroleum reservoirs shows an enormous physiological diversi ty and consti tutes a complex

ecosystem with an active biogeochemical cycling of carbon and minerals. "Souring" of oil

reservoirs by the formation of hydrogen sulfide has been a problem since the beginning of

commercial oil production. Sulfate-reducing bacteria are the culprits that produce this noxious

gas , leading to souring. This microbial process in wastewaters and oil field waters can be

contro lled by another group of microbes, known as nitrate-reducing ba cteria. The use of

nitrate to control microbially-produced sulfide in oil fields is a proven biotechnology that is

grossly u nder-u sed by the petroleum ind ustry. Its effectiveness has been dem onstra ted in

many laboratory investigations and in some field studies. Nitrate has replaced biocides in

some of the oil fields in the North Sea, and the results have been very positive. It is now very

clear that land-based oil field operators should seriously consider using this proven

biotechnology to control, and possibly eliminate, microbially-induced souring and the

problems associated with H2S formation.

Environmentally-related biotechnological processes were pioneered in the petroleum

industry. Oil spil l bioremediation technologies epitomize modern environmental techniques,

working with natural processes to remove spilled oil from the environment while minimizing

undesirable environmental impacts. The application of biological wastewater treatment in the

frame of a process integration treatment technology will hopefully close the water cycle

allowing "zero discharge" in the petroleum industry. Nowadays, water should be considered

as one of the main raw materials of the petroleum industry and its treatment and reuse with

advanced treatment technology should be applied. On the other hand, phytoremediation is an

emerging technology that is based on sound ecological engineering principles, and that has

developed into a more acceptable technology for the remediation of soils and groundwater

polluted with residual concentrations of petroleum hydrocarbons. The advantages of using

phytoremediation include cost effectiveness, aesthetic advantages, and long-term

applicability. Finally, biological air treatment systems are among the established technologies

that can be applied to control volatile organic compounds and odor emissions, and they are

applicable for a wide range of volatile pollutants found in the petroleum industry. Biological

treatment of polluted air emissions results from the competence of active microorganisms,

including bacteria, yeast, and fungi, to transform certain organic and inorganic pollutants into

compounds with lower health and environmental impact. Their applications are growing

continually based on scientific and technological developments.

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The powerful tools of molecular biochemistry can be used to improve the enzyme

stability and efficiency. These techniques may be applied to the particular needs of the

petroleum industry. In addition, the enzymes isolated from extremophilic microorganisms are

extremely thermostable and generally resistant to non-conventional conditions such as organic

solvents and extreme pH. Thus, many enzymes and enzymatic proteins are still to be

discovered.

Rafael Vazquez-Duhalt

The only way to discover the limits of the possible is to go beyond them into the impossible.

(Arthur C. Clarke).

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Table of Contents

Preface v

List of Con tributo rs xiii

Chapter 1

Use of Petroleum Biotechnology throughout the value chain of an oil company:

An integrated approach.

H.Kr. Kotlar, O.G. Brakstad, S. Markussen and A. W innberg

Statoil ASA. Trondheim, Norway

Chapter 2

Petroleum biorefining: the selective removal of sulfur, nitrogen, and metals

J.J. Kilban e II and S. Le Borgne

b

a

Gas Technology Insti tute, Il l inois U.S.A.

b

Insti tuto Mexican o del Petroleo, Mexico 29

Chapter 3

Enzymatic catalysis on petroleum products

M. Ayala and R. Vazq uez-D uhalt

b

a

lnst i tuto Mexicano del Petroleo. Mexico

b

Insti tuto de Biotecnologia, UN AM , Mexico 67

Chapter 4

Prospects for biological upgrading of heavy oils and asphaltenes

K.M. K irkwood, J .M. Foght , and M.R. Gray

Universi ty of Alberta, Canada I 13

Chapter 5

Whole-cell bio-processing of aromatic compounds in crude oil and fuels

J .M. Foght

Universi ty of Alberta, Canada 145

Chapter 6

Biocatalysis by methane monooxygenase and its implications for the petroleum

industry

T.J. Smith and H. Dalto n

3

a

Universi ty of Warwick, United Kingdom

She ffield Hal lam Universi ty, United Kingdom 177

ix

1


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Chapter 7

Biocorrosion

H.A. Videla and L.K. Herr era

h

a

Universi ty of La Plata, Argentina

Universi ty of Antioquia, Colomb ia, 193

Chapter 8

Molecular tools in microbial corrosion

X. Zhu and J.J. Kilbane II

Gas Tech nology Insti tute, Il linois U.SA. 219

Chapter 9

Potential applications of bioemulsifiers in the oil industry

H. Bach and D.L. Gutnick

1

'

b

Tel-Aviv Universi ty, Tel-Aviv, 69978, Israel

a

Ta ro Pharm aceuticals New York, U.S.A. 233

Chapter 10

Anaerobic hydrocarbon biodegradation and the prospects for microbial

enhanced energy production

J.M . Suflita , I.A. Dav ido va \ L.M. Gieg , M. Nanny and R.C. Prince

1

'

"Universi ty of Oklahoma, U.S.A.

b

Exxon Mob il Research and Engineering Co., U.S.A. 283

Chapter 11

Using nitrate to control microbially-produced hydrogen sulfide in oil field waters

R. E.

Eckford and P.M. Fedorak

Universi ty of Alberta, Edm onton, Canada 307

Chapter 12

Regulation of toluene catabolic pathways and toluene efflux pump expression

in bacteria of the genus Pseudomonas

J.L.

Ramos, E. Duque, M.T. Gallegos, A. Segura and S. Marques

Estacion Experimental del Zaidin, CSIC , Granada, Spain 341

Chapter 13

Bacterial hydrocarbon biosynthesis revisited

B. Valderrama

Instituto de Biotecnologia, UNA M. Mexico 373

Chapter 14

The microbial diversity of deep subsurface oil reservoirs

N.-K. Birkeland

Universi ty of Bergen, Norway 385

X


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Chapter 15

Biotechnological approach for development of microbial enhanced oil recovery

technique

K. Fujiwara

11

, Y. Sugai

1

', N. Yazawa

1

', K. O hn o\ C.X. Hong and H. Enomoto

1

a

Chugai Technos Co. Ltd., Japan

Akita U niversity, Japan

c

Japan National Oil Corporation, Japan

PetroChina Company Limited, China

c

Tohoku University, Japan 405

Chapter 16

Phytoremediation of hydrocarbon-contaminated soils: principles and applications

R. K amath, J. A. Ren tz, J. L. Schnoor and P. J. J. Alvarez

University of Iowa, U.S.A. 447

Chapter 1

7

Biological treatment of polluted air emissions

S. Revah* and R . Auria

a

Universidad Autonoma Metropolitana-lztapalapa, Mexico.

b

Universite de Provence, France 479

Chapter 18

Bioremediation of marine oil spills

R. C. Prince and J. R. Clark

ExxonMobil Research & Engineering Co. 495

Chapter 19

Biotreatment of water pollutants from the petroleum industry

E.

Razo-Flores, P. Olguin-Lora, S. Alcantara and M. Morales-Ibarria

Institute Mexicano del Petroleo, Mexico 513

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List of Contributors

S. Alcantara

Institute Mexicano del Petroleo

Eje Central Lazaro Ca rdenas 152. C.P. 07730, Mexico D.F.

P. J. J. A lvarez

Department of Civil and Environmental Engineering, Seamans Center

University of Iowa, Iowa City, Iowa, U.S.A. - 52242

R. Auria

Laboratoire 1RD de Microbiologie, Universi te de Provence

CESB/ESIL, Case 925, 163 Avenue de Luminy 13288, Marseil le Cedex 9 France

M. Ayala

Insti tute Mexicano del Petroleo.

Eje Central Lazaro Cardenas 152, San Bartolo Atepehuacan 07730 Mexico DF, Mexico

H. Bach

Department of Molecular Microbiology and Biotechnology, Tel-Aviv Universi ty

Tel-Aviv, 69978, Israel

N.-K. Birkeland

Department of Biology, Universi ty of Bergen, Box 7800, N-5020 Bergen, Norway

O.G. Brakstad

Sintef Materials and Chemistry, Trondheim, Norway

R. Clark

ExxonMobil Research & Engineering Co.

Annandale, NJ 08801

H. Dalton

Department of Biological Sciences, Universi ty of Warwick

Coventry CV4 7AL, United Kingdom

I.A. Davidova

Institute for Energy and the Environment and Department of Botany and Microbiology,

Universi ty of Oklahoma, Norman, OK 73019, USA.

E. Duque

Estacion Experimental del Zaidin. CS1C

C / Profesor Albareda 1, 18008 Granada, Spain


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R.E. Eckford

Department of Biological Sciences, Universi ty of Alberta

Edmonton, Alberta, Canada T6G 2E9

H. Enomoto

Department of Geoscience and Technology, Graduate School of Environmental Studies,

Tohoku Universi ty, Aramaki, Aoba-ku, Sendai 980-0845, Japan

P.M. Fedorak

Departm ent of Biological S ciences, Universi ty of Alberta

Edmonton, Alberta, Canada T6G 2E9

J. M. Foght

Department of Biological Sciences, University of Alberta

Edmonton, Alberta Canada T6G 2E9

K. Fujiwara

Chugai Technos Co. Ltd.

9-20 Yokogawa-Shinmachi Nisi-ku Hiroshima City 733-0013, Japan

M.T. Gallegos

Estacion Experimental del Zaidin, CSIC


L.M. Gieg


Universi ty of Oklahoma, Norman, OK 73019, USA.

M.R. Gray

Department of Chemical and Materials Engineering, Universi ty of Alberta

Edmonton, Alberta, Canada T6G 2G6

D.L. Gutnick

Present address, Biotechnology Research Laboratories. Taro Pharmaceuticals U.S.A.,

3 Skyline Drive, Hawthorne, New York, 10532, U.S.A.

L.K. Herrera

b

Faculty of Engineering, Universi ty of Antioquia, Medell in, Colom bia

C.X. Hong

PetroChina Company Limited, Ji l in Oilfield Company

Jil in province, China

R. Kamath



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E. Razo-Flores,

Institute Potosino de Investigation Cienti 'fica y Tecnologica

Camino a la Presa San Jose 2055,. C.P. 78216, San Luis Potosi , SLP, Mexico.

A. Rentz


University of Iowa, Iowa City, Iowa. U.S.A. - 52242

S. Revah

Department of Process Engineering, Universidad Autonoma Metropoli tana-Iztapalapa

(UAM-I). Apdo. Postal 55-534, 09340 Mexico D.F., Mexico

J. L. Schnoor



A. Segura

Estacion Experimental del Zaidin, CSIC


T.J. Smith

Biomedical Research Centre, Sheffield Hallam University

How ard Street, Sheffield SI 1WB , United Kingdom

.I.M.

Suflita


Universi ty of Oklahoma, Norman. OK 73019, USA.

Y. Sugai

Akita Universi ty Venture Business Laboratory

1-1 Tegatagakuen-cho Akita City ,010-8502, Japan

B.

Valderrama

Departamento de Ingenieria Celular y Biocatalisis, Universidad Nacional Autonoma de

Mexico. AP 510-3. Cuernavaca, Morelos, 62250, Mexico.

R. Vazquez-Duhalt

Insti tuto de Biotecnologia, UNAM.

Apartado Postal 510-3 Cuernavaca, Morelos 62250 Mexico

H.A. Videla

Department of Chemistry. College of Pure Sciences, IN1FTA, Universi ty of

La Plata, Argentina

A. Winnberg

Department of Biotechnology, N7465 Trondheim, Norway

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N .

Yazawa

Technology Research Center, Japan National Oil Corporation

1-2-2 Hamada. Mihama-ku, Chiba 261-0025, Japan

X. Zhu

Gas Technology Institute, 1700 S. Mt. Prospect Rd., Des Plaines 1L 60018

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Studies in Surface Science and Catalysis 151

R. Vazquez-Duhalt and R. Quintero-Ramirez (Editors)

© 2004 E lsevier B.V. All rights reserved.

Chapter 1

Use of Petroleum Biotechnology throughout the value

chain of an oil company : An integrated appro ach.

H.Kr. Kotlar

a

, O.G. Brakstad , S. M arkusse n

c

and A. W innberg

c

.

a

Statoil ASA, R & D Center, Postuttak, N-7005 Trondheim, N orway

Sintef Materials and Chemistry,

b

Dept. Marine Environmental Technology,

c

Dept. Biotechnology, N7465 Trondheim, Norway

1 INTRODUCTION TO AN INTEGRATED APPROACH

The history of biotechnology goes thousands of years back in time. One of the

very first written statements of biotechnology is found in the Bible, telling that

Lot was drinking wine, made through fermentation around 2000 B.C.E. In

modern time Antoni van Leeuwenhoeck was the first to observe a micro-

organism in a primitive microscope in 1684. Louis Pasteur discovered how to

protect against diseases by vaccination, using heat-inactivated organisms,

around 1863. In 2002 the gene sequence of the human genome was completed.

Biotechnology is continuously expanding, and will play an increasingly

important role in future industrial process. Petroleum biotechnology is a very

young and exiting part of these industrial possibilities

It is well established that petroleum reservoirs contain active and diverse

populations of microorganisms. Microbial growth within oil reservoirs has

traditionally been associated with biofouling and souring. Furthermore, the

potentials for microbial improved oil recovery (MIOR) have been investigated

for many decades (see chapter 15)[1]. Recently, nitrate injection was introduced

as a method for curing reservoirs "contaminated" by sulphate-reducing

prokaryotes (see chapter 11)[2]. However, petroleum biotechnology possesses

several other opportunities besides MIOR and nitrate injection. This chapter will

focus on some of these issues.

The primary target of the petroleum industry is to enhance and maintain a

continuous oil production. In 1998/1999 Statoil initiated an R&D program

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looking into the possibility of using petroleum biotechnology as an integrated

approach throughout the value chain of the oil company.

There were three main objectives:

1: Evaluation of biotechnology as a general toolbox for solving some of the

technology problems of today.

2: Investigate future possibilities; e.g. to start refinery processes in the reservoir

using dedicated m icroorganisms.

3: To generate a resource base for new genetic information achieved from the

organisms in the reservoir.

These objectives may be achieved through focusing on biotechnology as a

new business concept of interest to the company. Coverage of all aspects of

biotechnology would be an enormous task. However, the enhanced in-house

understanding of reservoir microbiology has served as a basis for the few

selected areas described below:

. New techniques in exploration and production:

Application of molecular biology techniques as new tools for specific

identification and characterization of hydrocarbon sources during exploration

and production. Samples may come from drill cuttings from exploration

wells; produced oil and formation water; sediments from sea floor seep zones;

etc.

•

Biological well treatments (preventive medication):

Clogging of wells by scaling, hydrates, etc. may be prevented by applying

environmentally friendly biological produced chemicals. This may be

achieved by developing self-sustained, natural existing or bioengineered

microbial populations placed inside the reservoir. The target is to produce

biological substances that can replace traditional chemicals, and that this

remediation will increase treatment lifetime to ensure a continuous oil

production.

•

Bioreactors:

Low energy biological processes for up-grading of oil to improve quality and

thereby reduce penalty pricing. Various types of bioreactors and enzyme

systems can replace traditional catalysts for certain chemical reactions, waste

handling or the production of bio-energy.

•

New application of extremophiles:

New thermophilic and piezophilic enzyme system can enable new bio-

engineering processes and products for applications in the above-mentioned

areas,

or give rise to entirely new products and business opportunities.

Combined approaches of microbiology, biochemistry and DNA technology

are used to obtain microorganisms with specifically designed metabolic

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functions. Such organisms can be applied in reservoirs for the production of

various treatment products or enzymes

in situ.

Thermophilic enzymes may also

be employed to overcome possible fundamental problems related to the growth

characteristics of these microorganisms. Additionally, the "gene-pool" of the

indigenous microbial assemblages of the reservoir have direct implication to the

success of the product in the above suggested business areas.

Environmental aspects/public awareness:

Apart from providing technical

solutions, the outcome of this program will have a great impact on meeting the

environmental challenge of the future. The Norwegian authorities consider many

of the production chemicals applied in the fields today as harmful, and in the

Norwegian sector of the North Sea there is a program for phasing out such

chemicals, replacing them with more environmentally acceptable alternatives.

Biotechnology may provide us with more environmentally friendly alternatives.

Value generation:

This program will contribute to increasing and maturing

the reserve base (upstream), as well as creating business opportunities or

increasing market shares downstream . The Fig. 1 below illustrates the potential

influence of biotechnology throughout the entire value chain within an oil

company.

Fig. 1. Biotechnology throughout the value chain.

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The main challenges are related to:

• The biological activities in a reservoir are still poorly understood. Grow th

control of reservoir microbes, and the knowledge to achieve this control, will

be crucial. In bioreactor-type processes, however, this will be possible.

. There are fundamental questions related to energy pathw ays and reaction rates

that need to be resolved. Direct use of tailor-made enzyme system might

bypass some of these obstacles.

• In bioreactors, the main challenge is to achieve sufficient reaction rates that

are required for a commercial process. This is not a challenge from the

microbiological aspect only, but also from a chemical engineering point of

view.

Acquiring new knowledge: In order to balance the beneficial and detrimental

effects of microbial growth in the reservoir, new knowledge is required. Growth

and possible excretion of products under different reservoir conditions are not

well kno wn . To date, various types of chemicals are injected into the reservoir in

order to maintain or restore oil production, e.g. to counteract or minimize the

influence of scaling, hydrate and asphaltene precipitation. Occasionally,

chemicals and antibiotics are injected to prevent microbial growth. Some of

these chemicals are known to serve as energy source for the microorganisms, i.e.

nitrogen, phosphor and carbon sources. [3-4]. Reservoir conditions vary

significantly, and thus, the microbial communities will respond differently

depending on this external influence. It is imperative to acquire in depth

understanding of the growth and production of microbial products under the

different reservoir conditions. In this respect modeling tools may be used to

simulate how the changes will influence on the indigenous microorganisms.

Joint efforts from internal experts and external collaborators are vital to the

success of this type of projects. Much knowledge on microbial technologies

already exists but the molecular biology approach represents a bold and

important step forward.

The nature of this research requires long-term commitment and support from

the R&D management. A thorough understanding and awareness of the ethical

implications is needed for all involved.

2.

MICROBIAL DNA FINGERPRINT TECHNIQUES IN EXPLORATION AND

PRODUCTION

Several studies have documented microbial communities in hot oil reservoirs

(see chapter 14)[5-9]. Indigenous microbial communities have also been

detected in core samples and water saturated regions of reservoirs [10].

Members of indigenous reservoir communities may include strictly anaerobic

sulfate-reducing prokaryotes [5, 11-12] and methanogens [13-15], as well as

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other microbes [9, 15]. Thus, one would expect to find genetic markers of

microbial activities both during exploration, drilling and production.

Statoil has filed a patent application for utilization of DNA technologies

as a tool for identification and characterization of hydrocarbon sources during

drilling or sampling from sea floor seep zones. Drill cuttings from exploration

wells,

sediments from sea floor seep zones or other specimens could be analyzed

with a selection of specific DNA probes/markers. These specific DNA probes

are taken from microbes found to be linked to different oil producing fields in

the North Sea and other sources. The energy sources for these organisms will be

constituents of the oil, gas or others, specific for the reservoir zones and

conditions of the particular field

This genetic tool may give valuable information on possible migration

routes of the hydrocarbon from the source rock. Specific recognition patterns

might also be used in monitoring different reservoir zones during production,

and further indicate the individual contribution of the particular zone to the

overall production. Possibly, sweep efficiency pattern could be calculated.

Detection of DNA from drill cuttings, sediments, or core samples during

explorative drilling may result in defined species pattern, resulting in indications

of potential hydrocarbon bearing zones (Fig. 2).

Fig. 2. System for characterization of microbes in exploration cores by culture-dependent and

-independent approaches, based on 16S rRNA gene sequencing. The sequences are used for

the generation of DNA probes to be used for screening of cores.

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2.1.

M icrobial diversity in oil reservoirs

It

is essential to establish databases of the microbial ecology in petroleum

reservoirs. Genetic tools for exploration and production can then be developed.

The knowledge of the

in situ

microbial activities should be improved through an

interdisciplinary collaboration between specialists in petroleum exploration and

production, chemists and microbiologists. Understanding the interactions

between the biosphere and the geosphere is essential.

The microbial diversity of two North Sea reservoirs (termed reservoir A and

B) has been studied in some detail [16-17]. Both a culture collection and a 16S

rDNA library have been established for these reservoirs.

2.1.1. Culture independent methods

Culture-independent methods have recently been used for the

characterization of microbial communities in some oil reservoirs [9-10]. In these

studies, DNA was extracted directly from reservoir samples (produced water,

core samples, drill cuttings etc.) This approach was used for the comparison of

microbial assemblages in some North Sea reservoirs with different reservoir

characteristics and production histories. In our studies microbial communities

differed significantly between the reservoirs (Fig. 3). Sequence studies of 16S

rDNA clones from reservoir A showed that 32 % of the clones aligned to the

sulfide reducing thermophile

Archaeoglobus fulgidus,

while bacterial clone

inserts aligned to a variety of types, including Sphingomonas, Herbaspirillum,

Nevskia, Aquabacterium, Alcanivorax, Bacillus

an d

Acetobacterium.

Clones

from reservoir B were dominated by sequences aligning to the a-proteobacteria

Erythrobacter,

the sulfide-oxidizing e-proteobacteria

Arcobacter,

the

halotolerant y-proteobacterium

Halomonas,

and the thermotogales

Geotoga.

Several of the microbial genes detected in our studies have been found in

produced fluids or enrichment cultures from oil reservoirs in the Pacific Ocean

or Canada [9, 18]. The differences in the assemblage compositions between oil

reservoirs and other subsurface structures may reflect the geochemical

influences on the community structures [19-20]. Biodegraded oils dominate the

world's petroleum inventory, and microbial activities play an essential role in

most oil reservoirs [21]. Recent studies have emphasized the impact of an active

potentially indigenous subsurface community [19].

2.1.2. Culture-based methods

Most studies of reservoir communities have been conducted by culture-

based methods [7-8, 22-23]. As a supplement to the culture-independent

characterization of the two North Sea oil reservoirs, culture-based methods were

used to study the diversity of the cultivable microbes in produced fluid from the

reservoirs. Enrichment media for fermentatives, methanogenes, sulfide-

oxidizers, sulphate-reducers and acetogenes were designed, and cultures from

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the two reservoirs showed dominance of small rods, single or in short chains,

and sheathed rods (Thermotogales like). Pure isolates were obtained from only

one of the reservoirs, reservoir A. Even though the enrichments from the other

reservoir, reservoir B, showed a variety of organisms, it was not possible to

obtain any pure isolates from these. The 16S rDNA clones from these

enrichments aligned to

Thermosipho japonicus, Bradyrhizobium

an d

Aquabacterium.

16S rDN A clones from isolates from reservoir A, showed

dominance of Archaeobglobus fulgidus, Methanococcus thermolithotrophicus,

Thermococcus sibiricus

and

Thermosipho japonicus.

Several of the sequences

abundant in the cultures were not found in the clone library from the culture-

independent approach (2.1.1). This is in accordance with other studies [9], and

suggests that several of the predominant members of the enrichment cultures

(e.g.

Thermosipho)

are not the predominant member of the reservoir

communities, but show fast-growing characteristics in several of the culture

media. Other cultures included a-, P-, s- and y-Proteobacteria

Sphingomonas,

Stenotrophomonas, Halomonas meridiana,

an d

Geospirillum,

and the Gram-

positive bacterium

Thermoanaerobacter ethanolicus.

Fig. 3. DGGE analysis of PCR-amplified 16S rDNA sequences from two North Sea oil

reservoirs, reservoir A (1, 2) and reservoir B (3, 4, 5). Only sample 2 contained fluids with

seawater penetration.

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Thermophilic species of

Thermotogales, Archaeoglobus, Thermoanaero-

bacter, Methanococcus

and

Thermococcus

have been reported from high-

temperature oil reservoirs [6-9, 14]. Several of these microbes are typical sulfur-

utilizers, being active in desulphurization of crude oil. These microbes may be

the predominant sources for H

2

S generation rather than typical sulphate-

reducing bacteria, and interestingly several of them were enriched in culture

media designed for SRB.

2.1.3. Detection o f specific micro bes

Monitoring of microbes in the oil reservoir has traditionally been

accomplished by culture methods, e.g. MPN methods for quantification of

viable sulphate-reducing bacteria (SRB), as recommended by the American

Petroleum Institute [24]. Some commercial techniques have also been

introduced, for instance a commercialized immunoassay for semi-quantification

of the SRB-specific enzyme APS reductase [25]. Monitoring may also include

molecular biology methods. Currently, two RNA-based methods are

investigated, fluorescence in-situ hybridization (FISH) and nucleic acid

sequence-based amplification (NASBA). By using RNA detection mainly the

metabolic active cells are assessed. The FISH methods include fluorescence-

labeled DNA probes for the targeting of specific microbes. An example is given

in Fig. 4 where bacteria, archaea, Archaeoglobus, Arcobacter and Erythobacter

are enumerated in production fluids from two reservoirs. These methods may be

further refined for offshore analysis by using field equipment, e.g. the Microcyte

fluorescence cell counter. NASBA is an isothermic alternative to PCR [26].

Real-time miniaturized lab-on-a-chips systems are currently under development

with the NASB A technology as basis [27].

2.1.4. Characterization ofmicrobial dynamics by microarrays

Nucleic acid microarrays have recently been introduced for phylogenetic

identification in microbial ecology. Basically, microarrays consist of series of

specific DNA probes (grabber probes) that are printed on glass slides. Sample

nucleic acids are extracted and labeled (e.g. by fluorescence) and incubated on

the slides, followed by recording. Labeled detector probes may be used for

detection as alternatives or supplements to labeled target DNA [28]. The

microarrays are made quantitative by employing reference DNA to normalize

variations in spot size and hybridization (29). The methods provide a powerful

tool for parallel detection of 16S rRNA genes [30-31] and may be particularly

useful for environmental studies of phylogenetically diverse groups. Although

most arrays are based on the PCR amplification of target genes prior to array

hybridization, systems have also been described where direct profiling of

extracted rRNA from en vironmental sam ples have been used [32 ]. Printed slides

may be broug ht offshore and target genes quantified directly on the platforms by

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portable devices. Arrays have also been established for the assessment of

functional gene diversities and distribution, for instance with genes from the

nitrogen cycling [33-34]. For offshore conditions the sulphur and nitrogen

cycles may be ad dressed during curing of biological sou ring by nitrate injection.

3.

BIOREACTOR: POTENTIAL USE OF BIOCATALYSTS IN CRUDE

OIL UP-GRADING AND REFINING

Until recently, research within oil biotechnology mainly focused on bio-

degradation and bioremediation in connection with clean up after oil spills, and

less on the application of microbial systems in industrial processes. However,

the interest in the latter has been growing the last years, addressing problems

like asphaltenes, high sulfur content, the poor transportability of heavy crudes

due to high viscosity, the presence of heavy metals and polyaromatic/

heterocyclic compounds (see chapters 2, 3, 4 and 5).

The aim of our activity is to use biotechnological processes in up-grading of

"problem" oils/heavy oil and refinery fractions. The overall scope is to define

microb ial/biotechnological technologies along the crude oil value chain that will

give the potential highest cost-benefits, competing with or being superior to

existing methods, or even better, provide solutions where no acceptable methods

exist. In the current program there has been focused on:

Fig. 4. FISH enumeration of the total concentrations of cells (DAPI), bacteria (EUB338),

archaea (ARCH915), Arcoglobus (ARGLO605) and thermotogales (THERSI672) in produced

fluids from tw o North Sea reservoirs, Reservoir A and Resevoir B wl and w2.

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Reduction of the viscosity of heavy crudes through partial degradation of

waxes and/or asphaltenes, thereby increasing the transportability.

Microbial or enzymatic ring opening of polyaromatic hydrocarbons in

refinery distillates in order to increase the fraction of aliphatic com ponents.

Removal of heavy metals such as nickel and vanadium from crude oils

through microbial sequestering, thereby simplifying the subsequent refining

of the crude.

Although chemical means to tackle the above problems exist, they are often

relatively expensive and may lead to pollution of the environment.

Biotechnological processes may represent new and more environmentally

friendly alternatives for value enhancement of heavy oils and partially distilled

petroleum products.

3.1. Pre-refining

Up-grading of crude oils by biocatalytic processes may take place anywhere

from dow n-hole to the refinery; in the reservoir, at the wellhead, d uring tanking,

transport and storage. The pre-refining opportunity is to utilize the time slot

from the start of drainage in the reservoir to the crude reaches the refinery stage.

At any of these stages, a specially designed biocatalyst could be introduced (see

Fig. 1). Although there will be considerable differences between traditional

crude oils and the heavy crudes in physical handling as well as refinery

processes, the chemistry of the compounds that need to be bio-converted could

be close relatives within the same classes.

3.1.1.

Increased transportability by biocatalytic cleavage of heavy com pounds

Extraction, transportation and handling of heavy oils often represent a

problem due to high viscosity. Several classes of molecules are important in

building viscosity. These are asphaltenes, waxes and the more heavy fractions of

polyaromatics. Controlled biodegradation of asphaltenes and waxes in heavy

crudes are highly desirable, as these processes could lead to a substantial

economical gain (see chapter 4).

Wax is degraded by several bacterial species that use the degradation

produ cts for their metabo lic pathways [35-36]. Efficient m ethods for isolation of

wax-utilizing microorganisms with the help of selective media, bacteriophages,

and paraffin wax baiting system have been developed [37-38]. Although the

enzymology of the wax degradation is not understood, some clues have been

obtained through studies of wax biosynthesis by certain bacteria, such as

Acinetobacter spp. [39-40].


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Biodegradation of asphaltenes seems to represent a more challenging

problem - very few publications is found on this subject. However, several

studies have shown that biodegradation of asphaltenes occurs in nature [41], and

that certain bacteria, such as Acinetobacter an d Providencia, proliferate in

environments containing high amounts of asphaltenes [42]. Fungi capable of

"erosion" of hard coal due to the cleavage of asphaltenes have also been

reported [43], as well as combined steam/bacteria treatment of asphaltene

depositions [44]. In addition, biodegradation of bitumen has been observed [45],

and bacteria like

Pseudomona s, Flavobacterium, Acinetobacter,

and

Caulobacter growing on bitumen-contaminated surfaces have been described

[46].

Potential processes are not limited to the natural occurring microorganisms

and their native enzymes. By gene technology it is possible to improve key

enzymes by rational engineering and by use of "gene shuffling" techniques.

These methods make it possible to rapidly "adapt" a given enzyme to new

substrates, or dramatically change the enzyme's properties such as

K

m

,

pH and

tempe rature optimum [47 ]. The modified enzyme(s) may then be introduced

into the appropriate microorganism(s) and its over-production, may greatly

enhance the ability of this microbe(s) to reduce the viscosity of heavy oils.

3.1.2. Demineralization - Biosorption of heavy metals

Demineralization of heavy oils that contain considerable amounts of Ni and

V is an important issue for oil industry due to refinery stage catalyst poisoning.

Several reports describing the use of microorganisms for bioremediation of

environments polluted with heavy metals, suggest that the use of microbes for

demineralization of heavy oils is possible [48-49].

Six mechanisms for microbial resistance to heavy metals have been

described: exclusion by a permeability barrier, intra- and extra-cellular

sequestration, active transport by efflux pumps, enzymatic detoxification, and

reduction of sensitivity of cellular targets to metal ions [50]. For

demineralization of heavy oils, sequestration and enzymatic detoxification seem

to be the most relevant mechanisms to study. In our current work we have just

entered this particular field of research.

3.2. Biocatalytic refining, distillate quality improvements

Perio-refining or post-refining technologies might also be of interest.

Although some of these areas have been addressed elsewhere in this book, we

would like to convey some of our own work (see chapters 2, 3, 4 and 5).


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3.2.1.

Selective ring opening

The mechanisms, the biochemical pathways, and the genetics of degradation

and bioconversion of hydrocarbons in general, and polycyclic aromatic

hydrocarbons in particular have been extensively studied [51-53]. The research

has mainly concentrated on biodegradation and bioremediation in connection

with cleanup after oil spills etc., and less on the application of these systems in

proce sses. How ever, the interest in the latter has been grow ing the last years. In

the petroleum industry there is a desire for products with a larger fraction of

aliphatic components, and thus a higher H/C-ratio, and microbial/enzymatic ring

opening of aromatics may be used to achieve this (see chapter 5).

Development of biocatalysts for aromatic- and heterocyclic ring opening,

including nitrogen compounds such as the polycyclic compound carbazole is of

particular interest. Middle distillate fractions from thermochemical conversion

of heavy oils contain di- and tricyclic aromatics with low fuel value. These are

currently upgraded by expensive high pressure-high temperature chemical

hydrogenation. A Canadian research group [54-55] has suggested an alternative

to thermochemical cracking: "microbial cracking" - a two-step process where

the aromatic rings first are cleaved enzymatically by a blocked mutant under

"near ambient conditions", followed by hydrogenation of the oxygenated

product under mild chemical conditions. Our group is currently engaged in a

project, "Upgrading of crude oils and refined products" involving selective ring

opening of aromatic distillates. In this work, a blocked mutant of

Sphingomonas

is used for studies of bioconversion of aromatic distillates in a bioreactor [56].

Bioconversion of aromatic compoun ds in a real feedstock from crude oil in a

bioreactor system.

The content of polyaromatic hydrocarbons (PAH's) in the

diesel fuels contribute to low cetane numbers and particle emissions from

combustion. The present study focuses on the use of a continuous bioreactor

system for up-grading of light gas oil (LGO) feed stock from the refinery as a

potential industrial process. This is done by biocatalytic ring opening of the

PAH's to generate a more paraffmic diesel fuel.

Two different bacterial strains, Sphingomonas yanoikuyae N2 and

Pseudomonas fluorescence

LP6a 21-41 (donated by Dr. Julia Foght, University

of Edmo nton Cana da), and a mixed blend of six different strains were com pared

for biocatalysis of the PAH's in the LGO feed stock using a fed batch

reactor/semi-continuous reactor.


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Fig. 6. Bioconversion of light gas oil by the specially designed Sphingo monas spp. N2.

In order to apply the concept to a real industrial process, higher degrees of

conversion of the more substituted aromatic compounds are necessary. The

enzyme systems in the PAH degrading pathway of N2 were found to be too

specific. Using the mixed biocatalytic blend a broader range of substrate

conversion was observed. More than 30 % of both the di- and the tri aromatic

compounds were removed from the LGO feedstock; in addition, approximately

30 % of the sulfur containing substrates was removed (Fig. 7). As already

mentioned, the mixed blend had not been genetically modified to terminate the

degradation of PAH's after the ring-opening step. The further uses of this mixed

biocatalytic blend with respect to developing an industrial process; will demand

genetic modification of the strains

The results achieved in the fed batch reactor are now being verified in a

continuous bioreactor to mimic a potential industrial process. Figure 10 shows

the schematic outline of the continuous bioreactor.

In conclusion, microorganisms with biocatalytic pathways that will

selectively convert aromatic compounds in a crude hydrocarbon mixture without

degrading aliphatic compounds exist. Such strains have been used as model

systems for studies of bioconversion of aromatic distillates (LGO) from the

refinery in a bioreactor system.


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Fig. 7. Efficient bioconversion by a m ixed biocatalyst.

The PAH degradation pathway of

Sphingomonas yanoikuyae

DSM 6900

have been genetically modified in order to obtain a recombinant strain that

terminates the PAH degradation after the ring-opening.

The LGO feedstock from the refinery has been shown to have no toxic

effects on the tested organisms,

S . yanoikuyae

mutant N2 and

Pseudomonas

fluorescence

LP6a mutant

21-41,

in concentrations up to 50 vol%. This is of

vital significance, because in an overall technological process it will be of

importance to keep the water volumes as low as possible.

The uptake mechanism and also the substrate specificity differ between the

two strains. The substrate specificity seems to be rather narrow for each

(both) of the strains, non- or mono substituted PAH's were the preferred

substrates.

Importantly, no C is lost by breaking the C - C chains in the blocked

mutants. The organisms are not gaining energy by the reaction. It is of value

that neither the fuel properties nor the cetan number are lost.

A broader range of substrate specificity was observed with a mixed

biocatalytic blend. More than 30% of both the di- and tri aromatic


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compounds and approximately 30 % of the sulfur containing substrates were

removed from the LGO feedstock in a continuous bioreactor system.

In future refinery processes this might replace the energy-expensive

distillation processes. These results suggest that bioreactor systems have the

potential for up-grading of hydrocarbon refinery fractions, heavier distillates and

possibly crude oils. In the years to come governmental regulations will be very

strict on both PAH and sulfur content in the diesel fuel. These preliminary

studies are thought as initial steps in a process of making a more environmental

acceptable diesel fuel with dramatic reduction in both PAH's and sulfur content,

while still maintaining adequate fuel combustion values (Fig. 8). This will be a

bio-upgraded environmental friendly diesel.

Bioconversion for more

environmental friendly diesel fuel

Fig. 8. Bio-reactor for conversion of PAH 's in a real feedstock from crude oil


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Study of pure enzyme vs. whole cell based biocatalysts.

In future investigations

this will include "the aromatic ring opening dioxygenase system". The

Sphingomonas yanoikuyae N2 will be used as a mod el system for comp aring

enzyme and whole cell biocatalysts. In many instances it is an advantage to use

pure enzyme systems instead of whole cells as biocatalysts (see chapter 3).

Enzyme reactions are specific and easy to control, they can be carried out in

non-aquatic environments, and enzymes, as other chemical catalysts, will not

consume carbon i.e. the carbon content in the fuel will be preserved. The

opening of the aromatic ring (e.g. naphthalene, Fig. 9) is a four step enzymatic

process starting with a dioxygenase reaction, then a dehydrogenation followed

by a second dioxygenase reaction and finally an isomerization. The first

oxygenation requires NADH, but the formed NAD

+

is recycled to NADH in the

dehydrogenation reaction. The challenge is to develop a system where this

mu ltistep enzym e reaction could proceed efficiently in a cell free system.

Fig. 9: Metabolic pathway of naphthalene showing the enzymes involved See reference [57],


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3.2.2. Bioreactors

Bioconversion of refinery fractions may take place using growing or resting

cells, "dead" cells, or immobilized cells or enzymes as biocatalysts. Aromatic

ring-opening involves a multistep metabolic pathway. Multistep enzymatic

reactions often require co-factors and/or reducing power (NAD (P) H) that has

to be regenerated or supplied for the enzymatic reaction to take place. Thus,

whole cells, rather than pure enzymes, are often required. The biocatalysts are

usually contained in the aqueous phase and the reaction take place either in this

phase or at the interface between the aqueous and the organic/oil phase. The

components in the refinery fraction that are being up-graded usually show low

water solubility, while the converted products usually are more soluble in the

aqueous phase than in the organic/oil phase. Mass transfer of substrates and

products between the water and oil phase is a major challenge. To achieve

adequate mass transfer, reactors capable of generating a large interface between

oil and water should be chosen. Various types of bioreactors have been

employed by others [58], including stirred tank reactors, airlift reactors,

emulsion phase contactors reactor and fluidized bed reactors. The current

investigation has used stirred tank reactors run in batch, fed-batch and

continuous mode with free growing or resting cells. However, immobilized cells

and enzymes are included in the next phase of studies.

Fig. 10. Schematic of a bioreactor for continuous feed of LGO.


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Continuous processes are well suited for multiphase processes. In the

continuous bioreactor based on a stirred tank reactor in fig. 10, a continuous

stream of substrate (oil phase) is run through the reactor while the biocatalyst (in

the water phase) is recycled. Recycling of the biocatalysts reduces the amount of

water needed in the process. The overall economy of the process is also

dependent upon the lifespan of the biocatalyst and their stability in water/oil

media. In a continuous reactor it is possible to regenerate or boost the

biocatalyst. In the current studies, problems have been encountered connected to

formation of stable emulsions. The emulsion increases mass transfer, but the

stable emulsions made phase separation problematic. Currently, different

approaches are explored to solve this problem.

Enzymes or cells may be immobilized by binding or adsorption to

mem brane surfaces or beads , or by entrapment in a matrix. In a continuous

reactor with an immobilized biocatalyst, it is possible to have a higher

biocatalyst concentration, little or no water in the reactor, and the product

separation is easy. Reactions with purified enzymes might be easier to control

compared to whole cell biocatalysts (see chapter 3). Whole cells may contain

different metabolic pathways and could lead to production of several unwanted

by-products. By co-immobilization of series of enzymes in the water phase of

the reactor, it might be possible to run multi step enzymatic reactions.

Realistic cost of developing new technology.

New technologies are often

met with obstructive arguments. Sentences like "it cannot be done" and "it is

impossible" are customary. Such arguments are "progress killers", and within

the oil industry, new techniques will have to compete with traditional

technolo gy that has been o ptimized for the last 50 years. A lesson can be learned

from the Canadians. None of their syncrude technologies for mining bitumen

would have been available today if they had listened to the "wise guys" 14 years

ago. At that time the operational cost of the technology was more than 30

US$/bbl, today the operational cost is down to around 10 US$/bbl.

The OPEX (operational expenditure) profile (Fig. 11) illustrates the cost

developments in developing new technology for mining bitumen. This curve

profile is believed to be quite universal for most new technology

implementations.

4.

WEL L TREATME NTS TO SECURE CONTINUOUS PRODUC TION

BY PREVENTIVE MEDICATION.

MICROBE S AS SELF-GENERATING SYSTEMS

Preventive medication could be defined as intelligent treatment concepts

performed in advance during the complementation phase, before the impairment


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in productivity occur in the well. The preventive actions are to avoid the onset of

these predicted situations.

With the advance in drilling and completion, increasing number of complex

and expensive wells are being installed, e.g. multilateral, multi-zones, sidetrack

and horizontal. The infrastructures that are in place, such as flow lines and

platforms, also enable the targeting and drainage o f the additional reserves found

near the exiting fields. Very often these additional oil and/or gas are produced

via tieback and satellite facilities. Successful treatments of stimulation, scale

squeeze and tubing deposit removal in these wells can no longer rely on the

traditional method of bullheading. Special tools such as coil tubing and

inflatable plug will be needed to place the chemicals accurately down-hole.

Intervention in these wells will be prohibitory expensive due to tools hire,

personnel and extended period of deferred oil production (tools run). It is

important to realize that for certain type of completion, well re-entry is almost

impossible despite accepting the financial penalty. There is clearly a need to

develop an intervention free system for these wells that allow the flow of oil

unhindered and preferably with the chemicals pre-delivered down-hole.

Syncrude Canada OPEX

Fig. 11. OPEX profile in developments of new technology for mining bitumen. The curve

shows the measured cost until 1998, then the further projection. T he bars in 99 , 00 and 01 are

the actual cost. (M aurice B. Dusseault, personal communication).


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4.1. Preventive treatment: Increased productivity by self-generating- or

more en vironmental friendly treatment system/processes (scale, hydrate,

asphaltenes, w a x , etc.)

The generation of effective production chemicals could be achieved using a

self-sustained, natural existing or bio-engineered, microbial population. This

will protect and free the well from most other intervention treatment and could

be of great economical interest to an oil company, enhancing both well recovery

and well productivity.

This will imply the search for microbes that have the genetic machinery to

produce certain treatment chemicals (i.e. organic acids, enzymes, surfactants,

antifreeze-proteins etc). Alternatively, genetic engineering could be used to

introduce this capability to the organisms. Such organisms could be introduced

to the near well bore area by various means (i.e. squeezed with/without solid

support, immobilized, combined with nutrients, etc), to produce the treatment

chemicals.

If the organism is not fit for life under the reservoir conditions, the bacteria

can be used in bioreactors to produce the desired product.

Bypassing the problems of placements:

Correct placement of the treatment

fluids is of crucial importance to the overall treatment success. Numerous

treatments have failed due to poor placement. Nonetheless, in many wells,

especially in gravel packed wells, uniform placement is difficult to achieve.

With this new technology placement should no longer be the problem.

The strategies of this new technolog y are illustrated in fig. 12 and include:

Placement of the treatment during the completion stage. This can be done

either by bullheading the specially designed organism together with nutrients

into the formation, or by coiled tubing (CT) deployment.

Use of porous particles soaked with the product placed inside the gravel

packs at the completion face.

Use of micro encapsulation, with the desired microorganism together with

nutrition inside the capsules. Inject far beyon d the critical matrix in the well.

If successful, this concept constitutes the only possible self sustained and

lasting method by which production chemicals can be produced in situ and to

allow wells to operate free of most interventions.


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In situ production of treatment chemicals

Fig. 12. Schematic view of in situ production of treatment che micals.

4.2 Green treatment products

In order to prove the basic concept of the above-mentioned technology of

preventive medication for secured production, the following approaches have

been made:

A synthetic gene, coding for polyaspartate (polyAsp), has been cloned in

E.coli. In a construct with 75 basepairs, coding for 25 am ino acids, with a fusion

protein included, the polyAsp polypeptide was expressed in the host cell.

Most service companies in the oil industry are supplying polyAsp as a

combined scale - and corrosion inhibitor. Recently, polyAsp has also proved to

be an efficient bridging agent, boosting the squeeze lifetime of traditional scale

inhibitor jobs. PolyAsp is classified as a green treatment product, being more

than 60 % biodegradable and non-toxic. From 2005 the Norwegian government,

through chart 12 and the Norwegian Pollution Authorities, SFT, will implement

a "zero harmful discharge" policy for the Norwegian sector of the North Sea.

This will focus the search for more environmental friendly treatment products.

On shore bioreactor: E. coli

will not survive during reservoir conditions.

However, the bacteria can be used in bioreactors to produce the desired product.

Bioreactor production of PolyAsp might prove to be economically feasible.


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Down hole:

Work is in progress, introducing the corresponding synthetic

gene construct into a vector, compatible with extremop hiles. This is a first step

towards down hole application

5. NEW APPLICATION O F EXTREMO PHILES IN OIL RELATED

INDUSTRY

5.1. Bioprospecting of the gene pools

Oil quality may be linked to microbial growth in oil reservoirs. This has

been substantiated in fields with biodegraded heavy oils. Although biogenic

reservoir processes seem to be slow [21] oil is utilized as carbon source and

water as a source of inorganic nutrition. The reservoir microbes, acting at high

temperature and pressure, have preferences or tolerance for these extreme

conditions. Enzymes from extremophilic microbes may be tailor-made for

industrial systems run at high temperature and pressures, i.e. systems in which

enzymes from mesophilic microbes will not function. Such enzyme systems

may be utilized inside the reservoir, in bioreactors, in waste handling or in

energy processes. DNA technology may be used to link appropriate enzyme

systems to microbes growing at relevant temperature and/or pressure conditions.

An immediate prerequisite for the utilization of microbes and enzymes from

the hot oil reservoirs will be to perform surveys of the genetic pools within the

reservoirs. The knowledge about microbial species in these environments is

constantly increasing, but the understanding of the interactions between the

microbes and their environments is still limited. It will be essential to

characterize active enzyme systems in the reservoirs. Complete genomes have

been sequenced for several microbes detected in oil reservoirs, including

Archaeoglobus fulgidus an d Methanococcus jannaschi [59-60 ]. Recent progress

in molecular microbial ecology has revealed that traditional culturing methods

fail to represent microbial diversity in nature, since only a small proportion of

viable microorganisms in most environmental samples are recovered by

culturing techniques. Methods to investigate the full extent of microbial

genomes in nature include the use of BAC (bacterial artificial chromosome)

vectors or random shotgun sequencing techniques [61-62]. These approaches

also have potentials for characterization of the complete genomic structures in

oil reservoirs. Besides explaining microbial structure-function relationships in

the reservoirs, the genomic libraries may be excellent tools for prospecting of

novel biocatalysts [63].

5.2.

Thermophilic/extremophilic enzymes

New application of extremophilic/thermophilic enzyme systems:

The concept

is to investigate the commercial utilization of thermophiles. These organisms

have enzyme systems working at high temperature, and often at high pressure.


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Such enzymes are tailor-made as catalysts in industrial processes performed at

extreme conditions. Enzymes from most mesophilic microbes will not function

as the high temperature will denaturate their proteins (e.g. the enzymes). Such

enzyme systems will work placed either inside the reservoir, in bioreactors, in

waste handling or in energy processes.

5.3. Future prospective

The petroleum biotechnology is still in its infancy and will play an

increasingly important role in the future industrial processes. Within the oil

company it will have a substantial economical impact throughout the value

chain. This will influence on the development of:

New techniques in exploration and production

Biological well treatments (Preventive medication)

Biocatalytic up-grading of oil

New application of extremophiles

Acknowledgement

The authors would like to thank Statoil for the permission to publish this

book chapter and for their support in the "Applied Biotechnology" program.

Many thanks to our special adviser, Hakon Rueslatten, for valuable help and

discussions.

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Studies in Surface Science and Catalysis 151

R. Vazquez-Duhalt and R. Quintero-Ramirez (Editors)

©2 004 Published by ElsevierB.V. 29

Chapter 2

Petrole um biorefining: th e selective remo val of sulfur,

nitrogen, and m etals

J.J. Kilbane II

a

and S. Le Borgne

a

Gas Technology Institute, 1700 S. Mt. Prospect Rd., Des Plaines, Illinois 60018

b

Instituto Mexicano del Petroleo, Eje Central Lazaro Cardenas 152, Col. San

Bartolo Atepehuacan, 07730 Mexico D.F., Mexico

1. INTRODUCTION

The quality of petroleum is progressively deteriorating as the highest quality

petroleum deposits are preferentially produced. Consequently the concern about

the concentrations of compounds/contaminants such as sulfur, nitrogen, and

metals in petroleum will intensify. These contaminants not only contribute to

environmental pollution resulting from the combustion of petroleum, but also

interfere with the processing of petroleum by poisoning catalysts and

contributing to corrosion. The selective removal of contaminants from

petroleum while retaining the fuel energetic value is a difficult technical

challenge. New processes are needed and bioprocesses are an option. Existing

thermo-chemical processes, such as hydrodesulfurization, can efficiently remove

much of the sulfur from petroleum but the selective removal of sulfur from

compounds such as dibenzothiophene, the removal of organically bound

nitrogen, and the removal of metals cannot be efficiently accomplished using

currently available technologies. The specificity of biochemical reactions far

exceeds that of chemical reactions. The selective removal of sulfur, nitrogen,

and metals from petroleum by biochemical reactions performed by

microorganisms and/or enzymes has been demonstrated. However, further

research is needed before biorefining technology can be commercialized. This

chapter reviews the status of biorefining and discusses topics requiring further

research.


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30

The geochemical conversion of organic matter into petroleum is a slow

and inefficient proce ss. It is estimated that 23.5 tonnes of plant m aterial/biomass

are required to form a single liter of petroleum during geological periods of time

[1]. Moreover, the current rate of energy consumption is 400 times greater than

the capacity of the planet to produce biom ass. It beho oves us to utilize our fossil

fuel legacy as efficiently as possible while avoiding environmental damage.

Environm ental regu lations limit the amount of sulfur oxides em itted to the

atmosphere by the combustion of fossil fuels by regulating the concentration of

sulfur in these fuels. In particular, transportation fuels are severely regulated.

For example, the permissible concentration of sulfur in diesel has been

progressively decreased over the past decade from 500 ppm to 10 to 15 ppm [2].

Environmental regulations do not specify concentration limits for nitrogen and

metals in transportation fuels, but such regulations may be forthcoming as these

compounds inhibit the catalytic converters used to cleanse exhaust gases from

vehicles. The future use of petroleum products to power fuel cells may provide a

further impetus to decrease the sulfur, nitrogen, and metal content of petroleum

derived fuels as reforming catalysts and fuel cell electrodes are sensitive to

Petroleum Biotechnology - Developments and Perspectives

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