3.4 Biotechnologies applied to oil and gas exploration ... · iron-reducing bacteria sulphate-reducing bacteria acetoclastic methanogens acetate acetate geogas CH 4 H 2 CO 2 CH 4

In this chapter, the term biotechnological applicationsrefers to the exploitation of biocatalyzed processes toimprove or develop new technologies for petroleumexploration or production. Petroleum is a complexmixture of hydrocarbons and represents an easysubstrate for the degrading activity of some specificmicroorganisms. In order to use the componentsneeded for cell growth, bacteria must be resistant toorganic solvents, and are characterized by theiroleophilic and solvent-resistant nature. Traditionally,important applications used in the oil industry involvethe exploitation of microbiological processes for thetreatment of contaminated land and water. Reactionsand processes linked strictly to exploration andproduction activities will also be described; theseinclude the management of microbiologicalphenomena in reservoirs at moderate temperatures.

The economic impact of H2S production bybacteria living in oil reservoirs (and the consequentsouring or acidification caused by hydrogen sulphide)makes it important for the industry to prevent thisphenomenon, which is not yet understood, much lesscontrolled. The old idea of effectively using theindigenous bacteria in reservoirs, or adding bacteriafrom outside to stimulate increased production(MEOR, Microbial Enhanced Oil Recovery) is stillcontroversial, but has never been completelyabandoned. Again in an industrial context, there isconsiderable interest in the vast reserves of methanehydrates present in abundance on the deep ocean floor,believed to be produced by methanogenic bacterialiving beneath the deposits.

The first two paragraphs provide a briefdescription of the microbial communities, active inbiotransformation of natural hydrocarbons, and adescription of the most important or recently discoveredreactions. The following section, by contrast, describessome of the applications currently in the stages of

research, development or initial field trials. In the finalchapter prospects for biological functionalization ofmethane are high-lighted.

3.4.1 Microbiology associated with hydrocarbons

Most of the regions on our planet which havereservoirs of hydrocarbons in various compositions(heavy, light, liquid or gaseous crude) are alsoinhabited by simple forms of life such as bacteria,archaeobacteria and fungi. It is estimated that most ofthe planet’s microorganisms live in the so-called deepbiosphere, where the microorganisms themselvescontribute substantially to the transformation cycles ofchemical elements such as carbon, sulphur, metals andminerals. The importance of the interactions betweenthe deep biosphere and that part of the earth’s crustwhich hosts accumulations of hydrocarbons isdemonstrated by the fact that most of the crude oildiscovered in reservoirs at a temperature below 70°Cis affected by a more or less severe history ofbiodegradation (Larter and Aplin, 2003), andbiologically induced changes of composition.

When petroleum (or some of its components)reaches the earth’s surface or the seafloor throughseepage processes or production-related activitiesunder conditions compatible with bacterial life, themain conditions for the formation of aerobiotic ormicroaerophilic ecosystems are created. These arealways based on oxidative reactions of thehydrocarbon components.

Subsurface microbiologyResearch on the biology of the subsurface is a

relatively recent and rapidly developing discipline. Interms of basic research, efforts are concentrated mainly

271VOLUME III / NEW DEVELOPMENTS: ENERGY, TRANSPORT, SUSTAINABILITY

3.4

Biotechnologies applied to oil and gas exploration,

production and conversion

on broadening our knowledge of the biodiversity andmetabolic capacities of subsurface microorganisms,and understanding the mechanisms governing theorigins of life on our planet. The favoured sites forstudies of the so-called deep biosphere are drillholes.Currently, only very few drillholes reach superdeepenvironments, and none of these were drilledexclusively for scientific purposes. The few drillingscarried out purely to explore microbial life do notexceed 1,000 m, whilst the deepest terrestrial wellsreach 12,000 m. However, distance from the earth’ssurface is not the most important limiting factor forbacterial survival; this is temperature.

Until now, the upper temperature limit measured forthe survival of a hyperthermophilic organism is 113°C,a limit which can be reached both on the ocean floorin the vicinity of hydrothermal springs, and ata depth of 10,000 m in sedimentary rock formations.Since there are extremely few wells whichreach this depth, given the high costs and technicaldifficulties involved, it is obvious that the explorationof the deep biosphere is in its infancy, and that manyaspects of microbial life in the depthsof the earth remain obscure. Specifically,one fundamental element remains to be clarifiedconcerning the metabolic state of the subsurfacemicrobial community: are intraterrestrial microbesmetabolically active at all levels in a constant way, orcan they survive in a state of quiescence for longperiods? For how long? We know that bacteria are ableto use any form of energy which is thermodynamicallyavailable in their environment. This energymay be represented by organic material deliveredfrom the earth’s surface by slow processesof water diffusion, organic carbon trapped insidesediments since their formation, the hydrocarbonspresent in the reservoirs, and flows of geogasessuch as CO2, H2 and CH4 which seep fromthe deep layers of the mantle. Potentially, these energysources represent an inexhaustible reserve for the

subsurface biomass, and may be the source ofsustenance for truly autonomous ecosystems, as shownin Fig. 1, which illustrates the carbon cycle in the deepbiosphere. This cycle does not require solar energy asan energy source. Hydrogen and CO2 from theinnermost layers of the earth’s crust are metabolized bymicroorganisms under temperature conditionscompatible with bacterial life and in the presence ofwater. Furthermore, halophilic bacteria have beenisolated in samples from wells drilled in ancient saltformations in northern Europe; they had apparentlyremained in a state of quiescence for over 250 millionyears. These observations suggest that there are somemicroorganisms in the subsurface which, under suitableconditions, can be considered immortal.

The factor currently hindering the expansion ofknowledge of the geobiosphere is the availability andquality of samples for analysis, since deep drillingprocesses are extremely expensive and technicallycomplex. The samples available often come frompetroleum wells. In the muds and formation watersfrom oil-bearing strata, it has been possible to identifyand classify different species of microbes, many ofwhich are apparently not linked to the metabolism ofhydrocarbons.

The research techniques employed are both oftraditional culture-dependent type and molecularculture-independent type. Culture-dependent methodsinvolve the isolation on a specific and selectivemedium, and subsequent classification of the bacteriastrains present in a given environment. Using cultures, itis also possible to carry out a series of experimentsaimed at the biochemical and genetic characterizationof the strains under examination. The concentration andcharacteristic set of environmental conditions(principally temperature), electron donors, salineconcentration and nutrients have considerableimportance in determining the composition of themicrobial consortium in the reservoir rock ecosystem.Under typical oxygen-free conditions, this may consist

272 ENCYCLOPAEDIA OF HYDROCARBONS

NEW UPSTREAM TECHNOLOGIES

acetogenicbacteria

iron-reducing bacteria

sulphate-reducing bacteria

acetoclasticmethanogens

acetate

acetate

geogas

CH4

H2 CO2

CH4

autotrophicmethanogens

organicpolymers

anaerobicdegradation

Fig. 1. The carbon cycle in the deep biosphere(Pedersen, 2000).

of methanogenic bacteria associated with fermentativeor acetogenic bacteria or, alternatively, consortia ofsulphate-reducing, iron-reducing and fermentativebacteria. Little is known about the sources of nitrogenand phosphorus used, although nitrogen may be fixeddirectly or used via the biodegradation of nitro-organiccomponents of crude oil. The microorganisms mostfrequently isolated in environments linked to petroleumreservoirs belong to the genera Desulphovibrio,Thermotoga (a fermentative bacterium resistant to hightemperatures and salt concentrations),Thermoanaerobacter, Geobacillus, Petrotoga,Thermosipho and Thermococcus. These are anaerobicmeso- or thermophilic microbes characterized byoptimal growth temperatures between 40 and 90°C.Some types of sulphur-reducing and fermentativeArchaea have been repeatedly isolated; these includeArchaeoglobus fulgidus, a hyperthermophilic sulphate-reducing archaeobacterium cultivated from petroleumfields where it may cause hydrogen sulphate productionphenomena at high temperatures (80-85°C) and metalliccorrosion, and which may form biofilm; andMethanococcus sp., an autotrophic anaerobicarchaeobacterium which turns CO2 and H2 into methanewhich grows optimally at 85°C, with some species ableto resist pressures of around 200 bar (Fig. 2).

However, culture-dependent methods are heavilylimited in their applications, since only a tiny fraction ofbacteria strains (0.1-1%) can be grown in the laboratory.To identify those microorganisms which cannot begrown in vitro, techniques used in molecular biology areadopted. By using DNA primers specific to given genesequences, these techniques make it possible toestablish the phylogenetic lineage of the organismspresent in the soil or water sample analysed. With thisapproach, we have been able to significantly broadenour knowledge of the microorganisms involved inreservoir microbiology, although further research isneeded to obtain a clear picture of their distribution,function and ecological interaction (Magot et al., 2000).

The approaches described above have also beenused for the metabolic and phylogeneticcharacterization of bacteria populating otherextreme environments typical of deep zones of theearth, such as mud volcanoes and the deep coldsediments of oceanic faults. Studies of the propertiesof some microorganisms capable of adapting theirmetabolism to the extremely high pressures in theseenvironments (up to 700 bar) through specificbiochemical mechanisms, which can be induced byhigh pressure conditions, have proved of particularscientific interest. The identification of species orgenes associated with the presence of hydrocarbonsin anoxic environments may be especially relevant tothe petroleum industry in the exploration sector.These can potentially be used as markers for thepresence of hydrocarbons in oxygen-poorenvironments.

The aerobic microbiology of hydrocarbonsMicroorganisms capable of using methane,

alkanes and the aromatic components of petroleumas a source of carbon in the presence of oxygen areextremely widespread in nature, both in aquatic andterrestrial environments. Specifically, molecularstudies carried out in the 1990s shed light on the‘specialized’ microbial populations which contributeto the natural response of marine and terrestrialecosystems to the accidental release of largequantities of hydrocarbons into the environment.

The use of selected consortia or strains of aerobicbacteria for the removal of hydrocarbons from theenvironment has often been called for commercially.However, the use of non-indigenous strains forenvironmental applications must be carefullyevaluated as an alternative to strategies that stimulatethe indigenous microbial flora which is generallybetter adapted to the local environment. For adescription of the types of reactions and bacteriagenera associated with the aerobic and anaerobic


BIOTECHNOLOGIES APPLIED TO OIL AND GAS EXPLORATION, PRODUCTION AND CONVERSION

A B C

Fig. 2. Microscope images of bacteria cells isolated from waters deriving from petroleum reservoirs: A, section of Thermotoga cells; B, Archaeoglobus fulgidus cells; C, Methanococcus sp.

degradation of various classes of petroleumcompounds, see below.

During the 1990s, particular interest was arousedby some specialized aerobic microorganisms capableof selectively removing organic sulphur-containingcompounds from petroleum. The bacteria most activein this type of reaction are mainly limited to groups ofmicroorganisms such as Rhodococcus, Gordonia andMycobacterium, bacteria ubiquitous on the earth’ssurface, phylogenetically closely related to oneanother, and extremely versatile from a metabolicpoint of view. For potential applications in the field ofpetroleum upgrading, see Section 3.4.3.

3.4.2 Reactions

Aerobic biotransformation of the principalcomponents of petroleum

Biodegradation of alkanesBiological systems of aerobic oxidation of alkanes

differ depending on the number of carbon atoms in thehydrocarbon chain: C1, C2-C4 (gaseous), C6-Cn

(liquid). The position of C5 (pentane) is not wellknown, since this compound is not often used inexperimental models in relevant literature.

Whereas methane is produced in abundance innature through biological pathways, no biological routefor the synthesis of ethane, propane or butane is known.For this reason, the presence of these hydrocarbons inthe environment can be associated with natural releasefrom underlying hydrocarbon deposits (microseeps) or,alternatively, contamination due to hydrocarbon use orproduction activities. The molecules most frequentlyfound in microseeps are C1-C6. Ethane is considered thebest indicator of oil, followed by propane and butane.

The microorganisms able to metabolize methaneare known as methanotrophs. The best characterizedmethanotrophs belong to two distinct groups whichdiffer in both their phylogenetic and physiologicalcharacteristics. Known species of type I belong to thegenera Methylococcus, Methylomicrobium,Methylobacter and Methylomonas; those of type II, toMethylosinus and Methylocystis. The numerous studiesundertaken have shown that methane-oxidizing bacteriaare extremely widespread in all types of environment.

The methane molecule is ‘activated’ and madeavailable for cellular metabolism by an oxidationreaction catalyzed by the methane-monooxygenaseenzyme. Methanotrophs of both types are able tosynthesize a methane-monooxygenase described asinsoluble (pMMO, particulateMethane-MonoOxygenase), whilst methanotrophs oftype II and Methylomonas (type I) are able tosynthesize a second soluble methane-monooxygenase(sMMO, soluble Methane-MonoOxygenase). Themethane-monooxygenases catalyze the transformationof methane into methanol; the methanol is then turnedinto formaldehyde, as shown in the diagram in Fig. 3.The formaldehyde enters the cellular metabolism cyclefollowing different routes, characteristic of thedifferent species.

Insoluble methane-monooxygenases consist of threesubunits, encoded by the genes pmoA, pmoB and pmoC.These three subunits are probably associated to form adimer (ABC)2; pmoA contains the catalytic site. Theamino acid sequence of pmoA, complete or partial, isknown for numerous species: it shares many similaritieswith the ammonium-monooxygenases which catalyzethe transformation of ammonium into hydroxylamine.

Soluble methane-monooxygenases consist of threemain components: a hydroxylase (mmoH), a reductase(mmoR) and a regulator protein, known as protein B



CH4

CytCred

CytCred XH2

CytCox

CytCox X

CH3OH

H2O

H2O

O2

O2

HCHO HCOOH CO2

sMMO

pMMO

Fig. 3. Metabolic pathway for the oxidation of methane by aerobic microorganisms. NAD�, CytCoxand X (generic compound) represent the enzymatic cofactors of the reactions in their oxidized form, shown as NADH, CytCred and XH2 in their reduced form.

(component B, mmoB). The hydroxylase, directlyresponsible for the oxidation of methane to methanol,in turn, consists of several subunits and is found in thedimeric form (abg)2. The subunit a contains thecatalytic site and is codified by a gene named mmoX:about a hundred amino acid sequences, complete orpartial, have been registered on public databases.

In some cases, the sequence of all the genes codifyingthe various subunits of methane-monooxygenase and theother ancillary proteins are known, but as far as pMMOsand sMMOs are concerned, only the sequence of thecatalytic subunit, or part of it, are known for mostspecies. Furthermore, methane-monooxygenases areextremely conserved enzymes, and the degree ofhomology among sequences from various species is high,whilst sMMO and pMMO are not homologous.

Microorganisms able to grow by using gaseousalkanes other than methane, in other words C2-C4, arepresent in the environment and are thought to beimportant indicators of the presence of petroleumreservoirs. Some of the strains able to grow onpropane and/or butane have been isolated: from thebiochemical and genetic standpoint, these are the leastwell-known alkane-oxidizers.

Alkanes can be oxidized to form either primary orsecondary alcohols, or both, depending on the type ofbacteria.

Among the systems studied, thebutane-monooxygenase of Pseudomonas (Thauera)

butanovora has been amply defined. In this bacterialspecies, the monooxygenase which catalyzes theoxidation of butane shows significant homologies tosoluble methane-monooxygenases. Recently, othersystems have been identified, which are not yetwell-characterized, in which propane and butane areused as the carbon source. A strain of Gordonia(Gordonia sp., TY-5 strain) able to use butane containsenzymes with properties similar to the family ofdiiron-monooxygenases, as in the case of thebutane-monooxygenase of P. butanovora. However, thehomologies with the butane-monooxygenase of P.butanovora are present but not high, and areconcentrated in those regions of the polypeptide chainwith specific functions, such as those able to bindFe2�/Fe3� ions, essential for their activity.Diiron-monooxygenases have been subdivided intogroups depending on both the substrates on which theyare active and the enzyme’s sequence homologies.

The microorganisms able to use non-gaseousalkanes frequently share the alk enzyme system: analkane-hydroxylase linked to the membrane, alkB,turns the alkane molecule into its primary alcohol; arubredoxin and a rubredoxin-reductase are alsoinvolved in the reaction. The system has been studiedin depth in Pseudomonas putida Gpo1; a diagram isshown in Fig. 4. Alk genes are present in numerousbacteria species and, for some, the sequence of thewhole operons which codify the proteins involved is



O

OH

OH

O

SCoA

alkJ

OalkH

alkL

alkN

alkK

alkF

alkS

sS alkSp1

alkSp2

alkBp alkBFGHJKL (alkN)

alkSTNADH

FAD

alkB

alkG

alkT

plasmaticmembrane

chemiotaxis?

cytoplasm

regulation

uptake?

b-oxidationcycle

metabolicpathway

alkanes or DCPK

alkane-hydroxylase(alkB)

rubredoxin(alkG)

rubredoxin-reductase (alkT)

Fig. 4. The alk enzyme system (van Beilen et al., 2001): metabolic pathway for the degradation of alkanes and role of alk genes. The alk operon codifies alkane-monooxygenase (alkB), two rubredoxins (alkF and alkG), an alcohol- and an aldehyde-dehydrogenase (alkJ and alkH), an alkyl-CoA-synthetase (alkK) and an external membraneprotein whose function is unknown. DCPK (DiCycloPropylKetone), a gene inducer which mimics the effects of alkanes.

known. Among these, the most widely studied is alkB.Some bacteria contain more than one copy of thecodifying gene, alkB, within their genome: up to fivein some Rhodococcus and two in Pseudomonasaeruginosa. Although alkB genes are homologous,they may differ within a single strain as much as thosebelonging to different species; it is not clear whetherthese are specific to alkanes of different length, orwhether they are activated under different conditions.However, all alkB proteins share considerablesequence homologies.

Biodegradation of aromatic compoundsUp to now, at least five different biocatalytic

systems for use with benzene have been observed inaerobic microorganisms. The introduction of one ortwo hydroxyl groups by specific oxygenases istypically the first step in the chain of reactions leadingfrom benzene, to its partial or total mineralization, towater and CO2. Similarly, the first biocatalyzed attackwhich leads to the conversion of increasingly complexpolycyclic aromatic rings is the introduction of one ortwo �OH groups, followed by the opening of thehydroxylated ring. The microorganisms in which thesereactions have been studied in greatest depth belong tothe genera Pseudomonas, Burkholderia and, in recentyears, Rhodococcus. There is considerableexperimental evidence that the genes encoding theproteins involved in the biodegradation of aromaticcompounds are present throughout the environment inbacteria which differ significantly from one another.For example, homologues of both alk genes and genesfor the degradation of aromatic compounds have beendescribed in marine microorganisms which use thesemolecules as a source of carbon in the bacteriaconsortia which evolve following the accidental releaseof petroleum into the sea (Harayama et al., 2004).

Oxidative biodesulphurizationSome bacteria strains are characterized by the

ability to use the heteroatoms present in the organic

components of petroleum to compensate for thelimited concentrations of these elements in theenvironment. Strains of Rhodococcus, Nocardia,Gordonia, Mycobacterium, Pseudomonas,Sphingomonas and others, in the absence of sulphur inthe culture medium, cxploit enzyme systemsspecialized in the oxidation of organosulphurcompounds (such as benzo- and dibenzothiophenes)and in the cleavage of the carbon-sulphur bond toconvert sulphur into sulphite or sulphate, easilyassimilated by the bacteria cell. Enzyme systems forthe utilization of the sulphur present in mercaptans arealso known. The metabolic routes for the assimilationof sulphur differ; during the 1990s, special interestwas aroused by the discovery of metabolic routeswhich preserve the carbonaceous skeleton of theorganic sulphurated compound (Fig. 5), used forbiocatalytic processes for the removal of petroleumdistillates rich in organic sulphur (Monticello, 2000).

DenitrogenationVarious microbial species are able to oxidize

organic compounds containing nitrogen, and use thenitrogenated groups for their own growth. The knownmodel compounds which can be used in this type ofreaction are pyrrole, pyrimidine, indole, quinoline andcarbazole, selectively denitrogenated by bacteriastrains belonging to the genera Pseudomonas,Comamonas, Rhodococcus and Nocardia.

Anaerobic transformation of the principalcomponents of petroleum

It is known that bacteria consortia exist which areable to oxidize methane in the absence of oxygen,although detailed information on the bacteria speciesinvolved and the possible enzyme mechanismsresponsible is not yet available. More information isavailable on alkanes with longer chains (non-gaseous)and aromatic compounds. Today, the anaerobicdegradation of hydrocarbons remains a phenomenonstudied in relation to the biodegradation of



Fig. 5. Metabolic pathwayfor the 4-S oxidation of organosulphurcompounds by the enzyme systemdszABCD. DBT,dibenzothiophene;DBTO, dibenzothiophenesulphone; DBTO2,dibenzothiophenesulphoxide; HBPS,sulphinic acid; 2-HBP, 2-hydroxybiphenyl; MO, monooxygenase.

S S

O

S

O O

S

OO�

OH

OH

DBT

DBT MOdszC

DBTO MOdszC

DBTO2 MOdszA

dszB

DBTO HBPS

2-HBP

DBTO2

hydrocarbons in reservoirs, or for applications ofenvironmental type, whilst some companies active inexploration have turned their attention to phenomenalinked to aerobic oxidation.

The scientific literature reports an increasingnumber of articles on the anaerobic oxidation ofhydrocarbons from the early 1990s onwards. Thanks tothe constant technological advances in research toolsand methods, it has been possible to isolate andcharacterize new microorganisms capable of oxidizingdifferent molecules present in petroleum, usingnitrates, sulphates, oxidized metals – such as iron(III)and manganese(IV) – and CO2 as the final electronacceptors.

Below, the principal mechanisms for the anaerobicactivation of hydrocarbons will be described, from thesimplest to the most complex compound.

Biodegradation of methaneUnderstanding the biodegradation mechanisms of

methane represents a significant challenge from thestandpoint of the subsurface ecology.

Up to now, no microorganism capable ofautonomously oxidizing methane in the absence ofmolecular oxygen as a final electron acceptor has beenisolated. However, there is strong geochemicalevidence for the potential transfer of electrons frommethane to sulphate by heterogeneous populations ofanaerobic bacteria. This evidence, supported by

experiments conducted with radioisotopes, hasrecently been confirmed using purely biologicalresearch methods. Using Fluorescent In SituHybridization (FISH) techniques, syntrophicaggregations of archaeobacteria surrounded by a layerof sulphate-reducing microorganisms have beenobserved on methane hydrates (Fig. 6). Strains ofarchaeobacteria involved in the metabolism ofmethane have been found on the surface of methanehydrates in oceanic sediments, using suitable geneticprimers.

The suggested and generally accepted reactionscheme is as follows:

CH4�SO42�� HCO3

��HS��H2O

Biodegradation of alkanesMassive and preferential degradation of alkanes in

natural biodegradation processes within oil reservoirs



5 mm

Fig. 6. Microscope image of a consortium of anaerobic methanotrophic bacteria isolated fromsediments rich in methane hydrates, stained by in situhybridization with primers specifically for archaea (red cells) and sulphate-reducers (green cells; Boetius et al., 2000).

CH3

[CO2]

CO2

H3C

A

B

C

H

H

C ( )n

H

H

C

H

H

CH3

C2

H3C C

H

H

C ( )n

C

H

C

H

OHO

H

CH3HO C

O

C ( )n

H

H

C

H

H

n-hexane (1-methylpentyl)succinate

(I)

(II)

(IV)(III) b-oxidation

catabolism

fatty acidtransformation

(e. g. chainelongation,

C10 methylation)

Fig. 7. Two alternative mechanisms for the anaerobic degradation of alkanes present in cells of strains HxN1 (A; Widdel and Rabus, 2001) and HxD3 (B; So et al., 2003).

has a significant impact on the value of the materialextracted. For years, the possibility that alkanes couldbe biologically oxidized in anoxic environments wasdebated only at a theoretical level. The experimentaldemonstration of this hypothesis was made possible bythe quantitative measurement of the consumption ofalkanes by sulphate-, nitrate- or metal-reducingbacteria.

The biochemical reactions responsible for attackson n-alkanes are still being studied. Hitherto, threemain metabolic routes have been discussed, two ofwhich are shown in Fig. 7.

The first, similar to that described below for theactivation of toluene, leads to the formation ofalkylsuccinate as a consequence of the addition of afumarate molecule to the alkane.

The reaction has been observed both insulphate-reducing strains grown on n-dodecane, and inthe nitrate-reducing strain HxN1, capable of growingon n-hexane. This reaction is complex, and it has notyet been possible to clarify all of its aspects. Analysesconducted on the fatty acids of the cells grown onalkane as the sole source of carbon showed that anorganic free radical is directly involved, and that thealkylsuccinates are present in the form of twonon-branched diastereoisomers. The biochemistrywhich follows the addition of fumarate to formalkylsuccinates is not yet understood in detail,although it is believed that this may be due to themetabolism of the fatty acids.

The second metabolic pathway for anaerobicoxidation of alkanes has been mainly developed in asulphate-reducing strain known as HxD3. Studiesconducted with radioisotopes on the fatty acids haveshown that the initial attack may involve carboxylationwith inorganic bicarbonate in the C-3 position and thesimultaneous removal of two carbon atoms from theterminal position of the alkane to form a fatty acid,with one carbon atom less than the original length.

A third metabolic route, identified very recently,has been observed in the conversion of hexadecaneinto methane and CO2. These experiments have beenconducted not on samples from an oil reservoir, butfrom anoxic sediments contaminated with petroleumand lacking sulphates (less than 10 mM), nitrates (lessthan 5 mM) and with a negligible Fe(III) content. Afterrepeated transfers onto fresh medium, it was possibleto obtain a mixed culture, free of sediment and capableof converting hexadecane into methane. Theproduction of biogas is thought to be stimulated bylow concentrations of sulphate (less than 2 mM).Genetic studies on the microbial population involvedhave shown at least three groups of microorganisms:one of syntrophic acetogens which degrade thehexadecane to acetate and H2, one of archaeobacteria

which degrade the acetate to CH4 and CO2, and asecond group of archaeobacteria capable of convertingCO2 and H2 into CH4. This process has been describedas microbial alkane cracking, and is seen as thepotential source of degradation in those reservoirscharacterized by the absence of traditional electronacceptors.

Biodegradation of aromatic compoundsStudies of the anaerobic populations present

inside contaminated anoxic sediments have shownthe possibility of the degradation of benzene undersulphate- nitrate- and Fe(III)-reducing conditions.The genetic and biochemical mechanisms whichunderlie the attack and the subsequent metabolismof benzene are still unknown. However, during thedegrading phase, a transitory accumulation ofbenzoate, phenol, p-hydroxybenzoate, cyclohexane,catechol and acetate has been observed and couldcorrespond to reaction intermediates. Recently, twonitrate-reducing strains, RCB and JJ, have beenisolated, belonging to the genus Dechloromonas,capable of mineralizing benzene to CO2 in a pureculture. Dechloromonas is a type of bacteriacommonly found in anoxic aquifers.

Alongside xylenes and alkylbenzenes, toluene isthe best characterized hydrocarbon compound fromthe standpoint of the biochemistry of anaerobicbiodegradation. The identification of benzylsuccinateas the reaction intermediate in cultures of sulphate-and nitrate-reducing bacteria represented the firstimportant step towards an understanding of manymechanisms linked to the degradation of toluene andother hydrocarbons; it has been shown thatbenzylsuccinate is the first reaction intermediatebetween toluene and fumarate.

To characterize the enzymes responsible for thereaction, both genetic and biochemical approacheshave been adopted. A series of genes organized insidethe bss operon (benzylsuccinate-synthase) have beenisolated in Thauera aromatica, Geobactermetallireducens and Azoarcus sp., and characterizedfrom a structural and functional point of view. Two ofthese genes have shown a high homology with othergenes encoding known enzymes: pyruvate formate-lyase and ribonucleotide-reductase. These two proteinsare known to contain glycyl radicals inside theirpolypeptide chain.

The activity of the bss operon is shownschematically in Fig. 8. The formation ofbenzylsuccinate is followed by reactions which can beattributed to the b-oxidation of fatty acids leading tothe formation of acetyl-CoA and benzoyl-CoA. Theenzymes responsible and the genes (organized into thebbs operon) involved in their biosynthesis have been



identified. The reaction scheme from benzylsuccinateto benzoyl-CoA is shown in Fig. 9.

The subsequent degradation of the benzoyl CoAinvolves reductive dearomatization reactions, resultingin the cleavage of the aromatic ring and reactionswhich are also related to the b-oxidation of fatty acids.Two modes of anaerobic attack on ethylbenzene havebeen identified. In the first, characteristic ofnitrate-reducing bacteria, the appearance of1-phenylethanol and acetophenone as reactionintermediates is evident. The enzyme responsible forthe reaction, isolated from a strain of Azoarcus, isethylbenzene-dehydrogenase, a periplasmicmolybdenum/iron-sulphur/heme protein, whose geneshave recently been isolated.

In sulphate-reducing microorganisms, theoxidation of ethylbenzene seems to follow acompletely different route which involves theformation of (1-phenylpentyl) succinate as a specificintermediate metabolite. This reaction is similar to thatidentified for the degradation of n-alkanes andtoluene, in which a fumarate molecule is added to thehydrocarbon (see Fig. 9 again).

Despite the known resistance of PolycyclicAromatic Hydrocarbons (PAHs) to biodegradation,studies conducted on communities ofsulphate-reducing and nitrate-reducingmicroorganisms have shown the anaerobic oxidationof compounds such as naphthalene, phenanthrene,methylnaphthalene, fluorene, fluoranthene andbiphenyl to CO2. Among the reaction intermediatesidentified, 2-naphthoate and phenanthrenecarboxylicacid are the result of the incorporation of CO2 into



CH3

H2OOH

CH3

CO2

toluenexylenes

benzylsuccinate(methylbenzyl)succinates

ethylbenzene (1-phenylethyl)succinates

2-methylnaphthalene

naphthalene 2-naphthoate

ethylbenzenepropylbenzene

1-phenylethanol1-phenylpropanol

(2-naphthylmethyl)succinate

Fig. 8. Reactions of the initial oxidative attack on aromatic compounds in anaerobic microorganisms(Widdel and Rabus, 2001).

CH3

HOOC

bssABCsdh

2[H]

2[H]

bbsEF

1

COOH

COOHCOOH

2

7

bbsG

COSCoACOOH

3

2[H]

bbsCD

COSCoACOOH

5

H2O

bbsH

COSCoACOOH HO

bbsB

COSCoA

COSCoA

CoASH

COOH

6

O

4

toluene fumarate

succinate

benzylsuccinate

succinyl-CoA succinyl-CoA

benzoyl-CoA

Fig. 9. Metabolic pathway for the anaerobic transformation of toluene by bss genes (Leuthner and Heider, 2000). The numbers indicate the oxidizing passages of the overall reaction from toluene to benzoyl-CoA, catalyzed by the enzymesbssABC and bbsBCDEFGH. Sdh, succinate-dehydrogenase.

naphthalene and phenanthrene, respectively. A reactioninvolving fumarate in the initial activation process hasalso been observed in the anaerobic degradation ofPAHs: naphthyl-2-methylsuccinic acid has beenisolated as an intermediate of the degrading reactioncatalyzed by a microbial population ofsulphate-reducers growing on 2-methylnaphthalene.

Biomolecular analyses of microorganisms which usecomponents of petroleum in the environment

In environmental studies, when investigating thedistribution of species with specific properties, it ispossible to carry out research aimed at particulargenes: the DNA is extracted directly from the soilsamples and, after purification, is analysed usingspecific DNA probes.

The probes are usually designed (and synthesizedchemically) on the basis of existing knowledge ofgenes with a known function and sequence, stored inpublic databases (such as the one at the NationalCenter for Biotechnology Information or the EuropeanMolecular Biology Laboratory – EuropeanBioinformatics Institute). The amino acid (protein) andnucleotide (DNA) sequences are compared with oneanother using dedicated software; this makes itpossible to pinpoint identical, or almost identical,portions inside families of genes (or proteins). Theseportions, with identical or very similar sequences, areused for the chemical synthesis of short DNAmolecules with a specific sequence which act asprimers in the subsequent search for similar genes(homologues) in the DNA sampled. The techniqueused for this type of analysis is PCR (PolymeraseChain Reaction); the specific fragments of DNApresent in the environmental samples, even in smallquantities, are enzymatically amplified to obtain anadequate quantity for subsequent applications.

The products of PCR can be sequenced directly ifthe amplified fragments belong to a single gene, andthus present a single sequence. If the product ofamplification comes from an environmental sample,however, it generally contains fragments belonging todifferent species; in this case, the sequence is notunique and the individual fragments must be separated.Separation can be obtained by cloning on a plasmid (orother DNA vector) or with alternative techniques.DGGE (Denaturing Gradient Gel Electrophoresis)makes it possible to electrophoretically separate DNAfragments which differ even by a single nucleotide in apolyacrylamide gel; this technique is based on thepresence of denaturing agents in the gel and on hightemperature. Once separated, the fragments can becloned or sequenced directly.

Using a specific application known as quantitativeor real time PCR, the quantity of specific DNA

present in a sample can be measured indirectly. Thistechnique involves using a fluorescent molecule ableto enter the double helix as it forms during theamplification reaction; the quantity of DNA-boundmolecules is roughly proportional to the quantity ofdouble helix DNA present. Precise quantification iscarried out by constructing calibration curves withknown quantities of initial DNA.

Among the technologies which find an applicationin the field of environmental research, the use of DNAmicroarrays is extremely promising: this techniquemakes it possible to detect the presence of numerousgenes simultaneously, and to carry out asemiquantitative analysis. Although costs are currentlyhigh, this is certain to become a routine technique inthe future.

Biomolecular analysis of aerobic alkane-oxidizingbacteria

Although there are other techniques which can beused for environmental research, those deriving from theapplication of PCR currently seem to be the mosteffective for the analysis of the microbial populationsassociated with the presence of microseeps. Theavailable literature offers numerous examples of theapplication of biomolecular analysis used to characterizehydrocarbon-oxidizing microbial populations.

The most widely studied group is that ofmethanotrophic bacteria, which has been characterizedusing primers specifically for the genes of theribosomal RNA (16S rRNA) for the catalytic subunitsof methane-monooxygenases (mmoX and pmoA genes)and for methanol-dehydrogenase (mxaF). The primersmost frequently used for these studies are those for thecatalytic subunits of methane-monooxygenase,specific to this system. Primers formethanol-dehydroxygenases have a lower degree ofspecificity, since they have obvious homologies withother alcohol-dehydrogenases; in addition, they areable to recognize all methanol-oxidizing bacteria(numerous methylotrophs) which inclue a far largernumber of species than methanotrophs.Methane-oxidizing bacteria have been identified inwaters, sediments and dry soils near lakes, rivers,paddy fields, crop fields, treatment plants and oilwells at different latitudes, including the arctic regions.

Many laboratories are currently undertakingresearch on the molecular characterization of thesystems involved in exploiting gaseous alkanes. Up tonow, no environmental studies based on theapplication of biomolecular techniques have beenpublished, since primers considered sufficientlyuniversal have not been reported yet. It is possible thatpart of the species contains enzyme systemshomologous with soluble methane-monooxygenases



(family of diiron-monooxynenases). In the case of bacteria able to use alkanes with

longer chains (liquids), the specific primers for alkgenes have proved very useful since this system iswell-distributed among the species. Within the system,the optimal target gene is alkB (alkane-hydroxylase),since it is conserved at the sequence level and specificto the system. One application for primers based onalk genes has been the analysis of the bacteriapopulations which grow in areas contaminated byhydrocarbons or as a result of oil spills.

Biomolecular analysis of anaerobic microorganismsassociated with hydrocarbons

Molecular microbiology thus provides new toolsfor the identification of bacterial species which growon oil components on the oxygen-rich surface. In asimilar way, new methods are now under developmentwhich make it possible to identify species whichdegrade hydrocarbons in the absence of oxygen. A fewgenetic reference systems are known (bss genes) andcan be used as a base to identify oil-degradinganaerobic species (Nivens et al., 2004). Progress in thefields of the genomics and microbiology of thedeepest biosphere will certainly lead to significantdevelopments and applications in this sector.

3.4.3 Applications

Biotechnologies applied to exploration

BiosurveyIn recent decades, oil companies have been using

cheap techniques alongside consolidated andextremely expensive techniques in the search for bothpetroleum and gas reservoirs. Large quantities of data

have accumulated in favour of those techniquesinvolving so-called surface exploration: surfaceexploration is a more or less indirect method fordetecting the presence of underlying reservoirs, basedon the changes, known as anomalies, produced in thesurface or subsurface environment by the upwardmigration of hydrocarbons from the reservoirs. Thisrelease may occur in an overtly visible form, as inphenomena of macroseepage, or may be barelyperceptible (microseepage); in the latter case, themolecules which migrate to the surface (usuallyshort-chain alkanes in the gaseous state) can only bedetected using sensitive analytical techniques.

Numerous surface exploration techniques are inuse and can be grouped roughly into direct andindirect. Direct methods are those which make itpossible to identify and quantify the presence of targetmolecules, generally using chromatographic analyses.Indirect methods, on the other hand, aim to identifythe anomalies produced in the environment by anunusual local concentration of hydrocarbons in givenareas; these anomalies may be of geological orchemical type, or involve the presence of specificvegetation, bacteria with particular properties, etc.Anomalies are identified as localized differences inthe levels of selected parameters compared toreference baseline values obtained in adjacent areas.

In many cases, it has been noted that in the vicinityof oil or gas reservoirs, hydrocarbon degradingbacteria populations live on the surface; thesepresumably exploit the presence of the gaseous orvolatile alkanes which have migrated from theunderlying reservoirs. These observations representthe basis for microbiological prospection techniques,or biosurveys.

The detection of specific bacteria can be carriedout cheaply and extremely quickly. Currently,



1 50

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maturity

Fig. 10. Cross-section model showing oil, gas, and gas and oil reservoirs at different stages of maturity.

microbial populations are identified and characterizedfrom a qualitative and quantitative point of view,thanks to their ability to grow in cultures, usingspecific hydrocarbons as a source of carbon.

SeepageOnce produced in the source rock, oil and gas

migrate through permeable rocks in which pores andmicrofractures are interconnected, creating sufficientspace to allow for the movement of fluids. Oil and gasare less dense than the water filling the spaces in therock, and thus tend to move upwards. Duringmigration, oil and gas may become trapped if themigration is interrupted or slowed down by thepresence of impermeable rocks (Fig. 10).

In many cases, reservoirs are not perfectly sealedand the migration may continue up to the surface,depending on the characteristics of the movingmolecules and those of the upper layers. This seepagemay be visible macroscopically in areas ofaccumulation; in this case, the phenomenon isdescribed as macroseepage (macroseep). Macroseepsare generally located at the ends of faults or fractures.

In other cases, only small quantities of short-chainhydrocarbons in the gaseous state are released; thesetraces can be detected only using specific analyses. Inthis case, we speak of microseepage (microseep).

Between these two extremes, there may beintermediate phenomena, depending on the propertiesof the reservoir itself and the geological properties ofthe overlying layers. Seepages can be detected bothonshore and offshore.

Seeps are well-known phenomena in their variousforms, with varying degrees of visibility, whereas themechanisms which generate them are still widelydebated. Special attention is devoted to microseeps,since gaseous hydrocarbons move vertically above thereservoirs, allowing them to be located, with lateralmigration occurring only rarely.

Although there is as yet no model able to explainhow and at what speed hydrocarbons can reach thesurface, microseeps are usually considered potentialindicators of the presence of accumulations of oil orgas. In many cases, an anomalous presence ofhydrocarbons at the surface has been demonstrated inthe vicinity of reservoirs. In some cases, theseanomalous findings were directly above reservoirspreviously identified using seismic methodologies; inother cases, a dispersion a few hundred metres to theside of a reservoir was detected.

Surface anomaliesThe presence of seeps leads to variations of

differing types at the surface, which depend directly or



calcium and ironbicarbonates in solution

hydrocarbon trapping

(in solution) (occluded)

rainfall leached zone

active direct seepageplus hydrocarbons release

apical interstitial hydrocarbonsanomalyedge-leakage

anomaly

Delta Coccluded

hydrocarbons

Delta Coccluded

hydrocarbons

edge-leakageanomaly

old calcite plus siderite pluggedseepage paths

carbon dioxide, hydrogensulphide and water

microseeping hydrocarbonsmicroseeping hydrocarbons

new calcite plus siderite with occluded hydrocarbons

(in solution) (occluded)

hydrocarbon release

(in solution)(occluded) (rainfall)

(in solution)(occluded) (rainfall)

anaerobic:

aerobic:

solution of calcium and iron minerals

(in solution)Ca(HCO3)2

(in solution)Fe(HCO3)2

petroleumpetroleum

bacterial or chemical hydrocarbondegradation

Fig. 11. Hypothetical model of mechanisms for the formation of surface anomalies (Saunders et al., 1999). Delta C represents ferrous carbon.

indirectly on the relatively high concentrations ofhydrocarbons in the ground; these are conventionallyknown as ‘anomalies’. Some anomalies may be causedby the presence of bacteria capable of metabolizinghydrocarbons: for example, the bacterial metabolismitself may lead to variations in the environment’s redoxequilibrium and to the formation of magneticprecipitates of iron, such as magnetite (Fe3O4),maghemite (g-Fe2O3), pyrrhotite (Fe7S8) and greigite(Fe3S4). The presence of these minerals in associationwith hydrocarbon seeps has often been detected inareas above gas or petroleum accumulations. However,the presence of seeps is only one possible cause of theformation of these magnetic precipitates (Schumacher,1996). Bacteria can also play a role in the depositionof calcite (calcite cement), in the formation of pyritethrough the production of hydrogen sulphide, inlowering the level of potassium and, in some cases, inraising uranium levels (measured by gammaspectrometry). Fig. 11 summarizes some of theanomalies caused.

Petroleum exploration requires a coordinatedeffort based on the integration of geological,geophysical and geochemical knowledge. Thegeological surface survey is a useful tool forreducing exploration risk, leading to a decrease incosts and the time required. The analysis of gases inthe ground and seismic exploration techniques,above all, make it possible to: a) rapidly evaluatethe production potential of unexplored regions;b) distinguish between areas of the reservoir whichproduce oil and gas; c) integrate the geophysicaldata from earlier surveys; d ) follow thedevelopment of sites already exploited.

Direct techniques are mainly used in seeps todetect the presence of methane, ethane, propane andbutane in decreasing quantities; the latter three areconsidered the best indicators of oil. Alkanes with achain above C4 are rare, although volatiles such aspentane or hexane may be present in measurablequantities. Methane has the disadvantage of beingproduced by various microbial species, and iswidespread in the environment (biogenic origin).Anomalies in the quantity of methane may, however,be used in the search for gas reservoirs.

Until the early 1950s, the major oil companiesused gravimetric, magnetometric and seismicrefraction methods. Subsequently, following a series oftechnological innovations, the most widely usedgeophysical exploration technique has been reflectionseismics. Reflection seismics provides an image of thesubsurface which may be two-dimensional, showingdistance and depth, or three-dimensional. Seismicsurveys are considered essential for the identificationof the structures in which oil and/or gas are found.

Other indirect techniques are commonly adoptedalongside seismics, especially in the initial stages ofexploration; some of these are used depending onenvironmental conditions, whereas the realapplicability of others remains unknown.

Microbiological prospectionMicrobiological prospection applies standard

microbiological techniques to the indirectidentification of microseeps. As noted above, thepresence of bacteria leads to modifications in theenvironment, resulting in the development ofanomalies. Rather than the anomalies produced, it iseasier to identify directly the bacterial species whichproduce them, or, in an even more targeted way, thosewhich use the gases present in the seeps as a source ofcarbon for their own metabolism.

MOST (Microbial Oil Survey Technique) andMPOG (Microbial Prospection for Oil and Gas) aresimilar techniques, introduced by competingcompanies, based on the direct search for bacteria ableto use short-chain alkanes (gaseous or highly volatile).These bacteria species are present in the microseepswhere gases such as methane, ethane, propane andbutane reach the surface. The alkanes are oxidized, inthe presence of oxygen, to their respective alcohols(for example, methane to methanol). The alcoholsenter the bacterial metabolism circuit, and the cellsdraw energy and carbon for their life-cycle from them.

Alkane-oxidizing bacteria are normally present inthe environment and are not exclusively associatedwith the presence of hydrocarbons at the surface;however, it has been shown that anomalies in thepresence of hydrocarbons correspond to an anomaly inthe presence of hydrocarbon-oxidizing bacteria, tosuch an extent that it is possible to identify a positivecorrelation between the concentration of hydrocarbonsand the density of these bacteria populations.

During microbiological surveys, soil samples arecollected 20-150 cm below the surface (either onshoreor offshore). Sampling is generally carried out using agrid; the width of the mesh depends on thegeophysical and geographical characteristics of thearea, or on the aims of the sampling itself. In the caseof preventive prospections in large unknown areas, thedistance between one sample and another may be over1 km; in the case of a detailed characterization of asite, whose geophysical characteristics are alreadyknown and which is possibly already underproduction, the sampling points may be only a fewtens of metres apart. Fig. 12 shows the results providedby differently-spaced grids.

Both MOST and MPOG entail the cultivation ofthe bacteria cells present in the soil sample. Methane,propane, butane or a gas mixture are used as the sole



source of carbon; under these conditions, the speciesable to nourish themselves with these specificmolecules grow selectively.

Bacterial cells are collected by washing a standardquantity of soil with a fixed volume of an elutionliquid. The initial suspension is used to prepare aseries of dilutions. A small quantity of each dilutionmay be seeded on plates of solid culture medium,where each cell will give rise to a colony; the totalnumber of colonies will provide an indication of howmany cells are in the ground. Alternatively, thedilutions are made directly in the culture medium.Following incubation for a suitable time, all thedilutions in which at least one living cell is presentwill give rise to a visibly denser culture. The dilutionswhich do not contain any cell will show no growth. Bysuitably replicating each dilution (at least in triplicate),an estimate of the initial number of cells present in thesoil sample is obtained. The MOST technique alsoinvolves an analysis of the alkane-oxidizing activity ofthe bacteria populations: the cells collected in an areawhere surface hydrocarbons are present express theiroxidizing activity at a higher speed than those sampledin areas without significant levels of gas.

The results of the microbiological analysis aredepicted, as for geochemical data, on two-dimensionalmaps showing the area of distribution and the densityof the populations of hydrocarbon-oxidizing bacteria(sometimes separated into metabolic classes accordingto the specific molecules used as a source of carbon);these data are treated in a similar way to geochemicaland geophysical data.

The case studies reported in the literature generallyshow that microbiological techniques are extremely

effective in identifying reservoirs. For example, theresults reported from one study which involved themicrobiological survey of an unexplored area,identified as productive 13 of the 18 wells drilled – thesuccess rate in this case was 72%. The authors havestated that in the absence of MOST, the success ratewould have been 30%. A second study reported a caseof 225 wells, of which 101 were producing oil or gas,and 124 were dry wells. Anomalies were identified inthe vicinity of 83 of the producing wells, whereas 119dry wells were found to be in areas lacking anomalies.In this case, the predictive success rate based onmicrobiological methods was around 90%.

Microbiological techniques have some significantadvantages with respect to other surface surveyingtechniques, including: a) the acquisition of samplesdoes not require special tools and its environmentalimpact is practically nil; b) costs are extremely limited,for example using one of the proposed techniques, thetotal cost amounts to 100-750 dollars per linear mile;c) the absence of geological or geographical limitations;d) limited dependence on the subsurface geology andthe potential for predicting the properties of thereservoir as concerns the quality of the hydrocarbonspresent. By contrast, it is not possible to obtaininformation on the location and size of the reservoirs.

For these reasons, microbial prospection isconsidered a viable and economical alternative toother techniques in the phase preceding seismicexploration. During a recent survey in Guyana, 22microbial anomalies were located in an area of250 km2; the subsequent sorbed gas analysis showedthat these anomalies were associated with hydrocarbonmicroseepages. Unlike the microbiological study, the



sample spacing:130 . 1,600 m

sample spacing:130 . 800 m

sample spacing:130 . 130 m

Fig. 12. Diagram showingdata collected during a prospection campaign,with examples fromsamplings using grids of different sizes.

chemical analysis of the seeps had limited efficacy,due to the unusually acid nature of the soils, whichprevented extensive sampling throughout the area.

It is likely that over the coming years, biomoleculartools will be developed for onshore and offshoreexploration, due both to the speed with whichinformation is acquired and the potential for analysingthe microbial population in detail without the need forcultivation. Using suitable advanced methodologies, itis also possible to trace the presence of single genes inenvironmental samples by using specific primers. Thepotential for measuring the quantity of genesresponsible for the oxidation of alkanes present in asample with amplification techniques or, in the future,using microarrays, may render these techniques

appealing for exploration and in preliminaryprospection analysis, integrated with the indispensableseismic surveys.

Risk analysis of the biodegradation of the crudeWithin the context of petroleum exploration and

production, one of the largest risk factors is that offinding a reservoir containing oil whose quality isheavily compromised by microbial attack on its mostvaluable components, with significant impact fromboth an economic and operational point of view.

Particular attention is devoted to shallow andrelatively cold reservoirs, characterized bytemperatures no higher than 65-80oC. In theseenvironments, despite the fact that ecological



very slight

methane

ethane

propane

isobutane

n-butane

pentanes

moderate

level of biodegradation

severeheavyslight

n-alkanes

isoalkanes

isoprenoids

BTEX aromatics

alkylcyclohexanes

n-alkanes, isoalkanes

isoprenoids

phenanthrenes, DBTs

naphthalenes (C10+)

chrysenes

C1-

C5

gase

sC

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15hy

droc

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35hy

droc

arbo

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kers

regular steranes

C30-C35 hopanes

C27-C29 hopanes

triaromatic steranes

monoaromatic steranes

gammacerane

oleanane

C21-C22 steranes

tricyclic terpanes

diasteranes

diahopanes

25-nor-hopanes*

sec-hopanes*

*appearance, rather than removal of compounds (these compounds believed to be created during biodegradation)

Fig. 13. Degrees of biodegradation of crude oil based on the relative concentration of different compounds present in petroleum (Wenger et al., 2002). BTEX, benzene-toluene-ethylbenzene-xylenes; DBT, dibenzothiophene.

conditions and bacteria populations may differsignificantly from one site to another, the probabilityof finding a high biodegradation index of extractablehydrocarbons is extremely high. Typically, the rankingof degradability of the compounds present inpetroleum puts n-alkanes in the first position, followedby branched saturates, cyclic saturates, cyclic andpolycyclic aromatics, steranes, hopanes and ceranes(Fig. 13).

Microbial activity may thus have a significantimpact on the essential parameters of the quality ofproducible oil, including: lowering of API (AmericanPetroleum Institute) gravity, increase in viscosity andthe concentration of undesired elements such as heavymetals (especially nickel, vanadium and iron),asphaltenes, waxes and sulphur. An oil with theseproperties has a low commercial value due to lowdistillation yields and the consequent increase ofresidues, the significant presence of naphthenic acids(a cause of corrosion and emulsions) and numerousrefining problems resulting from the presence of heavymetals and sulphur. In degraded oils, sulphur is presentboth in organic compounds (due to their resistance tomicrobial attack) and in the inorganic form as H2S,which represents the final product of microbialsulphate-reduction. Hydrogen sulphide is toxic, causessignificant corrosion problems, and forms ironsulphide precipitates which make the separation ofoil/water emulsions difficult, and reduce thepermeability of the reservoir rocks if water is injectedduring the extraction phase. Furthermore, highviscosity has a detrimental impact on the productivityof wells and on the reservoir’s recovery factor, makingit less profitable than an accumulation of light oil.

Some research and development activitiessponsored by the petroleum industry concentrate onthe possibility of understanding the mechanisms, thebiological players involved and the environmentalconditions necessary for the activation of anaerobichydrocarbonoclastic microorganisms. The objective ofthis research effort is the development of mathematicalmodels able to predict, at the simple survey level, theoil’s degree of degradation as accurately as possible.The development of this type of tool may make itpossible to evaluate new exploration areas better,resulting in a significant reduction in heavy drillingcosts.

This goal is certainly ambitious, since theecological niche occupied by bacteria capable ofexploiting hydrocarbons as a source of carbon andenergy for their own growth is strictly correlated withan environment whose exploration presents numerousproblems from a technical standpoint (such as theavailability of samples and their handling), and is stilllargely unknown.

Until the end of the 1980s, the scientificcommunity agreed that the oxidation of hydrocarbonscould occur only under aerobic conditions, through theconstant supply of molecular oxygen transported bymeteoric waters. This principle was later refuted, fromboth a geochemical and biological point of view, bythe discovery of deep reservoirs (thus not exposed tofresh oxygenated waters) with a high degree ofbiodegradation. Since the early 1990s, microorganismsable to oxidize hydrocarbons in absolute anaerobiosishave been isolated. The latter discovery had asignificant impact on the criteria applied bygeochemists, and paved the way for new studies by thenumerous researchers and technicians working on themicrobiology of the subsurface and the still largelyunknown reservoir ecosystem.

In the ExxonMobil model (Wenger et al., 2002),the hypothesis of the anaerobic degradation ofpetroleum is accepted because it is consistent withsituations encountered during offshore exploration.The recognized limit factors for biodegradationinclude not only temperature (agreed at a limit ofabout 80oC) and the availability of oxidants – forexample O2, Fe(III), SO4

2�, HCO3� – and nutrients

(N, P, K), but also salinity (with a possible limitof 150 g/l, Total Dissolved Solids), acidity, porosity(surface area) and the permeability of the rocks.Specifically, the possibility that biodegradation maydevelop in reservoirs beyond the zone of contact withthe water table (thanks to the water which is present inthe pores) has been taken into serious consideration.Wenger et al., have developed their own scale ofincreasing biodegradation for oils. This scale is basedon the presence of reference compounds, detectable aspeaks in analyses chosen according to the nature ofthese compounds: GC (Gas Chromatography)for hydrocarbons, GC/MS, Mass Spectrometry(gas chromatography combined with massspectrometry), and GC/MS/MS for biomarkers.

A comparison with the Peters and Moldowan scale(1993) is of special interest; compared to this, that ofWenger et al. differs for the simplification of the levelsof degradation (five rather than ten) and, at the sametime, for the larger number of hydrocarbon species andbiomarkers considered, and the greater detail in thedefinition of the hydrocarbon components. Thesequence of increasing resistance to biodegradation ofthe chemical species is, in general terms, identical tothat of Peters and Moldowan(n-paraffins�isoprenoids�hopanes anddiasteranes�aromatic steroids), but attention has nowshifted towards the initial phases of biodegradation.Unlike the Peters and Moldowan scale, to define theboundary of the first stage of biodegradation, the useof light C6-C15 isoalkanes is suggested (in addition to



the lighter isoprenoids), whilst the specific attack onsome cyclic and aromatic hydrocarbons (alongsideheavier isoprenoids) denotes and distinguishesintermediate levels of biodegradation (for example:BTEX�alkylcyclohexanes;naphthalenes�phenanthrenes anddibenzothiophenes�chrysenes). The attack onbiomarkers defines the most severe stage ofbiodegradation, with possible distinctions between thedifferent species, though these are believed to be lesssignificant for applications in production (see below).The appearance of the series of 25-nor-dimethylhopanesis considered a product of biodegradation at its mostadvanced stage, and is thus an important marker forrecognizing, for example, cases of refreshing (renewalof the liquid phase with new fluids) of reservoirsbiodegraded in preceding eras.

As always, the sequence described is not to beinterpreted rigidly, since a certain degree of specificityis recognized in the biodegradation mechanisms ofdifferent types of oils. Using biodegradation indicesremains critical, however, in relation to the possiblesequence over time – on a geological scale – ofprocesses such as the recharging (recharging of theliquid phase with new fluids) of reservoirs, waterwashing, phase separation, gravitational segregation,the precipitation of asphaltenes or thermal cracking.Future developments will involve the analysis oforganic acids, especially naphthenic acids (well-knownand feared products of biodegradation), to define newindicators of ongoing bioalteration in reservoirs.

There is an obvious effort to improve theapplicability of biodegradation scales to theproduction sector. Indeed, the gradual disappearanceof whole hydrocarbon classes (such as n-alkanes orisoprenoids, not to mention biomarkers) is generallydetected by gas chromatography when biodegradationhas already long affected the quality of the oil – inother words, when the properties that are important forproduction have already been at least partiallycompromised. However, the production context is alsoimportant: in the case of deep waters, the geothermalgradients are small, and differences of a few APIgrades, caused by biodegradation still in its initialstage, often lead to the profitability threshold for theexploitation of a reservoir being exceeded. On theother hand, moderate biodegradation may significantlyimprove some properties of oils with a high paraffincontent, such as the pour point and the tendency toform waxes (a common situation in the oil fields ofSouth-East Asia).

As far as the biodegradation of natural gas isconcerned, the model suggests that during the initialphase, the bacteria principally attack propane, which isdegraded in preference to n-butane, leading to

measurable effects as concerns different parameters:decrease of the GOR (Gas/Oil Ratio) for the associatedoil; increase in the relative concentration of methaneand, frequently, of CO2 (a by-product ofbiodegradation); isotopic fractionation (the gas not yetbiodegraded – typically propane – graduallyaccumulates in the fraction richest in 13C, whilst the by-products – typically CO2 – show an enrichment in 12C).

More recently, for offshore and deep waterenvironments, important elements have emerged for thedevelopment of reference systems on which to baserisk analyses, based on the biodegradation of thehydrocarbons present in seeps at the sea floor level. Inthis environment, biodegradation has differentproperties as compared to those of onshore seeps andthe underlying reservoirs (for example, the series of25-nor-hopanes has never been detected, even in casesof extremely severe biodegradation). In the case ofsubsea seeps, it is always necessary to distinguish thecontribution of ‘recent’ and indigenous organicmaterial to the sediments; however, the identification ofbiodegradation series exclusive to the sea floor hasmade it possible to calibrate molecular parameters(biomarkers) which can be used well in advance asreliable indicators of the origin, maturity and propertiesof the oil present in the underlying reservoirs. Thecalibration is of a local nature because there is evidenceof activity by different bacteria strains, with differentbiodegradation routes for different families of oils.

It has also been observed that the level ofbiodegradation on sea floors affected by seepagesincreases with the total amount of hydrocarbons in thesediments and, perhaps, with the size of the underlyingreserves; the existence of a threshold for the flow ofhydrocarbons below which biodegradation processesbecome unsustainable has also been suggested.Bacterial activity is also clearly influenced by thetypology of the sediments (porosity and permeability).

The most significant factors for the developmentof new biodegradation models, defined during recentstudies and industrial projects, can be summarized asfollows:• Biodegradation of the hydrocarbons in the

reservoir is mainly an anaerobic process, andexposure to oxygenated waters is thereforeunnecessary.

• The estimated flows of degradation are in the orderof 10-4 kg/m2/y and vary depending on thetemperature of the reservoir, with values close tozero at around 80°C.

• Over 50% of the oil fraction above 12 carbonatoms can be used at different levels ofbiodegradation.

• Biodegradation of oil in relatively cool reservoirsor in seeps from deep layers is a process which can



be prevented by the preceding ‘pasteurization’ ofthe petroleum which occurred when the oiloccupied deeper layers where the temperaturesexceeded 85°C.

• The oil/water contact seems to be the most probablelocalization for degradation activity; the electrondonors are provided by the oil and the finalacceptors, alongside the nutrients, by the water.Recent developments in this field, which mainly

concern the discovery of new anaerobicmicroorganisms active in the subsurface in theoxidation of hydrocarbons, the relevant metabolicdegradation mechanisms and the interaction betweengeochemistry and the microbial physiology of thesubsurface, will have a significant impact on theformulation of new predictive models. The recentdiscovery of reaction intermediates in the hydrocarbonoxidization processes in anaerobiotic environments(Aitken et al., 2004) is an example of the developmentof new indicators useful in the evaluation of areasidentified for petroleum exploration.

Biotechnologies applied to production

Prevention of the production and accumulationof hydrogen sulphide (souring) in reservoirs

The presence of hydrogen sulphide in oil wells hasbeen detected since 1920, but only in 1926 was itunderstood that at temperatures below 100°C this

could be attributed to the activity of sulphate-reducingbacteria present in the wells themselves. There is alsoa ‘thermogenic’ H2S production mechanism in thedeepest layers of the earth’s crust, but usually in colderreservoirs, the origin of this gas is mainly biological.

The significant damage affecting reservoirs andproduction plants contaminated by populations ofsulphate-reducing bacteria active in the production ofH2S must be kept under control, both with expensivemaintenance operations and high cost treatmentsrequiring the use of biocides and anti-corrosionagents. Hydrogen sulphide is a compound which ishazardous to human health, induces metal corrosion,and in association with crude oil during the productionphase, significantly lowers its quality and requiresspecialized removal technologies. This compound isproduced by a large variety of sulphate-reducingmicroorganisms (SRB, Sulphate-Reducing Bacteria) inthe absence of oxygen, following the enzymatictransformation of sulphates, sulphites andthiosulphates. These microorganisms may be native tothe reservoir, or come from the water injected duringthe secondary extraction phase, which may containboth the bacteria and the quantities of sulphate neededto sustain their activity. The mechanisms governingsouring phenomena and their propagation through thereservoir have not yet been clarified, and there arecontrasting models for the prediction of the kinetics ofH2S development during the productive life of



A B

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SRB activity off SRB activity on simulator

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formation water

convection, diffusion, adsorption

conc

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Fig. 14. Comparison between two alternative models of reservoir souring: A, the mixing zone model (Lingthelm et al., 1991); B, the biofilm model (Sunde et al., 1993). The yellow coloured zone indicates the area of activity of sulphate-reducing bacteria and synthesis of H2S.

reservoirs affected by the phenomenon. Diagrams oftwo alternative models are shown in Fig. 14.

Traditional technologies for controlling souringphenomena involve the use of broad-spectrum biocidesor inhibitors of the sulphate-reduction by the SRB.Treatment with chemical agents such as HCl, aldehydesand amines is expensive, often ineffective, especially inthe long term, and not ecocompatible. Additionally, theproducts of the degradation of the biocides themselvesmay supply additional substrates for the SRBs.Alternative technologies involve the removal bynanofiltration of the sulphate present in the water usedin the water flooding process. Due to its high costs, thismethod is applied only rarely, mainly when filtration isnecessary to prevent scaling phenomena.

During the 1990s, new technologies based ontreating the water with nitrates were usedexperimentally to control and prevent souring.Adequate concentrations of nitrate lead to alterationsin the composition of the microbial populations in thereservoir environment, and the creation of so-calledbiocompetitive exclusion, in other words, competitionbetween SRB and NRB (Nitrate-Reducing Bacteria).

Different competing mechanisms have beensuggested which make biocompetition possible:• The activity of the SRB is inhibited directly by the

production of toxic intermediates that derive fromthe reduction of nitrate (for example NO2, N2O).

• The nitrate-reducing bacteria compete efficientlywith the sulphate-reducers for commonelectron-donors and for potential sources of C.

• The presence of sulphide-oxidizing NRB(NR-SOB, Nitrate-Reducing Sulphide OxidizingBacteria) inhibits the growth of the SRB and thusthe production of H2S, probably due to the increaseof the environmental redox potential (the SRBneed a very negative redox potential for optimalgrowth).

• In the presence of sufficient nitrate, the H2S alreadypresent is reoxidized via the chemical route, partlyto elemental sulphur and partly to sulphate.The presence of a nitrite-reductase activity may

represent a factor of resistance in the bacteriapopulations treated, eliminating one of the possibleintermediates which inhibit the activity and growth ofthe SRB population.

The mechanisms of the phenomenon are thuswell-studied in model systems under controlledconditions, but the results of actual treatments maydepend significantly on the different compositions ofthe consortia present in the reservoir and on conditionsin the surrounding environment (concentration ofsulphate and volatile fatty acids). For these reasons,applications of this technology in the field are oftenlinked to contingencies in the site.

After the first experimental evidence, numerousexperimental treatments with nitrate have been carriedout or are ongoing in the field and in model systems,some examples of which are discussed below(Hitzman and Dennis, 2004).

Statoil-Norsk Hydro. Laboratory experiments on asmall-scale model had shown the possible inhibition ofH2S formation after the addition of nitrate. Thisexperiment was then repeated in the field, at Veslefrikkin the North Sea, after water flooding, replacingglutaraldehyde biocide treatments with nitrate. A fewmonths after injection, a 20,000-fold reduction wasobserved in the activity of the SRB (measured as theproduction of H2S) and, simultaneously, an increase inNRB. The corrosion of metal samples decreased from0.7 to 0.2 mm/y.

Treatment at Skjold. This gas field in Denmark hasan associated production of H2S which, over the years,has risen from 100 to 700 kg/d. To keep thisphenomenon under control, THPS –Tetrakis(Hydroxymethyl)Phosphonium Sulphate �was used, a bactericide which can reduce the amountof H2S produced by up to 40%, and reduce thephenomena of contamination and damage caused bythe growth of microorganisms (biofouling) in theinjection system. The bactericide was replaced with250-150 mg/l of nitrate for one or two months in twodifferent injection wells in the same field. Thistreatment led to a reduction of about 10 kg/d in theH2S produced. After the treatment ended, the activityof the SRB resumed. More prolonged treatments mayhave a more marked effect on the whole field.

Phillips Petroleum Treatment. The Coleville fieldin Canada was subjected to continuous treatment for50 days with 500 ppm of nitrate, resulting in totalcontrol over the production of H2S in one well and areduction of 50-60% in the two adjacent wells.

It is generally thought that the most favourableconditions for treatments may be linked to the‘prevention’ of the microbiological development ofhydrogen sulphide; the existence of predictive modelsfor the souring phenomenon in reservoirs is thereforeimportant.

Processes for removing H2S from gas mixturesBiological processes for the removal of H2S from

gas mixtures during production are based on the abilityof some aerobic strains to oxidize sulphur according tothe reaction H2S�1/2O2�

��S�H2O. The hydrogen

sulphide, dissolved in water in the form of HS�, isoxidized by microorganisms to elemental sulphur and,as such, accumulates outside the cells and is separatedphysically from the aqueous medium. Variants of thisprocess differ depending on the metabolic properties ofthe strains used as biocatalysts (Thiobacillus,



Thioalkalivibrio and Thioalkalimicrobium). For adetailed discussion of the applicability of these recentlydeveloped processes, see chapter 3.3.

Microbial enhanced oil recoveryMEOR (Microbial Enhanced Oil Recovery) makes

it possible to exploit microbial activity to facilitate therecovery of the petroleum fraction not produced byreservoirs which have reached the end of the primaryand secondary production phases. These techniques,first proposed several decades ago, are rarely appliedon a large scale, due to the high risk of estimatedfailure resulting from the poor understanding of theunderlying mechanisms. Despite this, MEOR has, inrecent years, met with renewed interest from operators,especially in China, South America and the northernoceans, probably due in part to knowledge acquired inthe field of the microbiology of extreme environmentssuch as petroleum reservoirs. MEOR modifies themicrobial populations present in the reservoir bystimulation with nutrients or the addition of bacteriacultures of suitable type, is potentially inexpensivecompared to other types of tertiary treatment, and hasno environmental impact (Brown et al., 2002).

The hypothetical functioning of MEOR isgenerally based on complex and diverse mechanisms:according to one of the most reliable mechanisms (atthe current state of knowledge), the growth of biomassin the most permeable layers of the reservoir, probablyin association with the production of cellularexopolymers (alginates, polysaccharides, pullulanes),may facilitate the selective plugging (or obstruction)of permeable strata. Controlled in vitro systems basedon the activity of selected strains such as Leuconostochave shown that these phenomena are possible. On theother hand, the cellular exopolymers such as xanthangum produced by cells cultivated ‘at the surface’ havetraditionally been used with varying degrees ofsuccess in injection treatments using water-basedsolutions of the polymer (polymer flooding) to inducemodifications in the reservoir’s permeability profiles.

An alternative and potentially successful mechanismis based on the production of biosurfactants by residentmicroorganisms, able to induce an increase inproduction based on the displacement of residual oilfrom the pores by lowering the surface tension of theoil/water contact. This is an interesting phenomenon intheory, but the actual potential (both in size and activity)of the bacteria populations resident in situ to synthesizesufficient quantities of surfactants has been questioned(Bryant and Lockhart, 2000). However, in some reports,the chemico-physical properties of the oil blendsproduced after MEOR stimulation appear to showmodifications which support this hypothesis. Otherproducts of the bacterial cellular metabolism which may

theoretically influence the mobilization of residual crudeoil are gases (CO2, methane) and acids which, underfavourable conditions, may induce the solubilization ofmatrices containing carbonates, and lead tomodifications in the water/oil mobilization profiles.

As for treatments to remove contaminants fromsubsurface aquifers by stimulating the native microbialpopulations, it can be assumed that treatments must beadapted to the properties of the site (or reservoir)under examination. This imposes significantlimitations on the applicability of the technology,which requires significant characterization andpre-feasibility studies.

In the attempt to improve the prospects for MEORtreatments, current research activities, both in thelaboratory and in the field, are based on definingschemes to inject limiting nutrients, generally scarcein the subsurface, such as nitrogen and phosphorus, tostimulate microbial growth and activity in ageneralized way. The research activities are also basedon the cultivation and characterization of strainsselected in situ for their properties, favouring desirableactivities such as: a) decreasing the viscosity of theoil; b) production of biosurfactants; c) production ofexopolymers; d ) production of organic acids from oil.A proposal recently presented by Statoil is based onthe stimulation of native or resident microbial activityfollowing the injection of seawater by oxygenating thewater. A different approach involves engineeringsurfactant-producing bacteria to adapt their geneticexpression to the recovery phase following injectioninto the reservoir.

Any treatment method based on the addition of non-native populations must take into account the additionalproblems linked to the survival of the bacteria strainsinjected in the environmental conditions of the reservoirand competition with resident populations, as well asthe limitations due to poor bacteria flow in porousmedia. After several decades of relatively empirical andpoorly controlled MEOR interventions, it is possiblethat the development of methods for characterizingmicrobial populations in the environment may lead to anew phase in the knowledge of the phenomena involvedin the mechanisms of interaction between water/oilsystems and the microbial populations of thesubsurface. However, the inherent difficulties ofmanaging complex biomasses, physiologically poorlycharacterized and probably diversified even within thesame site, remain considerable.

Upgrading

In situ methanizationThe difficulty of recovering most of the crude oil in

reservoirs has encouraged proposals and projects for the



development of new technologies in the secondary andtertiary exploitation of reservoirs. Among these, a selectgroup of technologies, presented in the form of articlesand patents, are based on the exploitation ofmicroorganisms able to transform the hydrocarboncomponents of petroleum (and bitumen sands, shalerocks or coal) into methane. Anderson and Lovley(2000) have suggested the existence in the deepbiosphere, and especially in reservoirs, of mechanismsfor the transformation of alkanes and aromatichydrocarbons into methane by bacterial communitieswhich include methane-producing (methanogenic)bacteria. In general terms, the coexistence of fermentingbacteria which can turn hydrocarbons into smallermolecules such as alcohols, acids, hydrogen and CO2,with methanogenic bacteria, leads to the transformationof some of the crude oil into methane. The conversionvelocities are still a subject of debate (Anderson andLovley, 2000), but are estimated to range fromextremely slow to manageable on the exploitation scale.

As for other biotic processes, microbial conversionsare often limited by low concentrations of nutrients orby dynamics of competition with other trophic groups.Thus, the theoretical possibility exists of manipulatingthe ‘reservoir’ system, and managing it on a rationalbasis to favour the accelerated production of methanefrom alkanes (biocracking). These processes, whichcurrently exist only at the level of proposals in thetechnical literature and patents, require a completecharacterization of the microbial communities present,and an understanding of the complex interactionsbetween them and environmental conditions(temperature, water, nutrients, salts, gas, etc), difficultto obtain with currently available technologies.

In bioremediation processes, rational interventionmay modify biotransformation of contaminants by theindigenous microorganisms of the subsurface. In thesame way, it may be possible to manipulate the

environmental conditions of the reservoir andencourage processes aimed at recovering the fractionof crude which remains trapped in the reservoir byconverting liquid hydrocarbons into more easilyrecoverable products (methane, hydrogen). Thesetechniques might be developed for the reservoirs withthe most favourable properties in terms of temperatureand salinity. Fig. 15 shows a diagram of a possible insitu transformation process which can be exploited forsecondary recovery.

Fluidization and dewaxingThe technical literature of this sector often describes

applications based on the modification of the chemico-physical parameters of the crude oil by microbiologicaltreatments. In particular, localized treatments at theindividual well scale may lead to the selective removal ofprecipitates of long-chain paraffins. Also reported arecases in which the presumed accumulation of theproducts of cellular metabolism – such as proteins orbiosurfactants – may lead to a local lowering of thesurface tension of the oil-water contact. While these maybe real phenomena, since they reflect a spectrum ofreactions already observed in controlled systems, or aretheoretically possible, this field of application, which hassignificant impacts on the problem of transporting thecrude oil, still remains unsupported by evidence, and isoften linked to the local properties of the system treated.

Desulphurization, denitrogenationand demetallization

In the recent past, the tightening of the limitationswhich regulate fuel emissions has led to the design anddevelopment, up to the pilot scale, of sophisticatedbiocatalyzed processes to remove organiosulphurcompounds from petroleum products. Despiteadvances in this sector, mainly due to the developmentof new and efficient engineered strains, the cost limit



hexadecane(C16H34)

acetate(CH3COOH)

CH4

H2S

acetogens(3 types)

methanogen(1 type)

methanogens(2 types)

sulphate-reducingbacteria (1 type)

Fig. 15. Biocracking:diagram of the hypotheticalinteractions between theseven types of bacteriapresent in a consortiumactive in the conversion of hexadecane to methane.The bacteria types are inblue, whereas the substratesand products are in green;red shows the predominantmetabolic pathways(Parkes, 1999).

imposed on upgrading processes for petroleumproducts (a few tenths of a dollar cent per litre ofdiesel treated) has heavily limited their applicability.Similar considerations affect potential processes fordenitrogenation and the removal of metals andbenzene, which remain theoretically interesting only inthe absence of alternative chemical processes, andwhich have as their characteristic advantage theirability to exploit the extreme specificity ofbiocatalyzed reactions. However, it is possible that thedevelopment of anaerobic processes catalyzed byconsortia of highly oleophilic species, active directlyon crude oil, will provide a new approach to theproblem of treating the ‘difficult’ crudes which areincreasingly produced in large amounts.

3.4.4 Biological activation of methane

Methanol is a highly interesting synthetic energyvector, as it can be produced from synthesis gasobtained from various fossil sources (natural gas,refinery residues, coal). It is liquid at ambienttemperatures, has a favourable balance between carbonand hydrogen atoms and good calorific value. Inprinciple, the prospect of producing methanol by directoxidation of methane appears very promising, but ashas been seen, no catalysts exist able to bring about thisreaction efficiently on an industrial scale. Therefore,selective functionalization of methane remains one ofthe great challenges of contemporary chemistry.

On the other hand, the biocatalysts that carry out theoxidation of alkanes (including methane) enable a greatvariety of microorganisms to use these hydrocarbons inorder to grow. In fact, the natural activity of thesemicroorganisms influences the carbon cycle on ourplanet in a significant way and indirectly controls theclimate, insofar as this is determined by theconcentrations of certain greenhouse gases. It isestimated that the capture and turnover of methane by thebacteria that consume methane (methanotrophs)contribute towards the consumption of a large part of themethane emitted or produced on the Earth’s surface,thereby participating in a decisive manner in determiningthe level at equilibrium of this gas in the atmosphere.

The great variety of enzymatic systemscharacterized by an oxidative capacity on alkanes hasaroused curiosity and inspired attempts to mimic themexperimentally because of their catalyzing capacityunder extremely easy conditions of pressure andtemperature. On the other hand, their low turnover rate,the low stability of the enzymes and the complexity ofsystems with multicomponents have limited theirindustrial application to just a few examples of

synthesis high value-added compounds. In the lastdecade, the application of innovative techniques ofenzymatic evolution in vitro has permitted the synthesisof more efficient variants of the natural enzymes. Thestudy of the obtained in vitro variants of the biocatalystof the methane oxidation reaction promises, at least, tobridge a gap in knowledge between the mechanisms ofthe C�H bond selective activation and thedevelopment of convenient applicative processes. Thissection briefly reports on the biological process ofoxidative conversion of methane, on biocatalysts, onpast attempts at mutagenesis and experimental mimics,and on alternative biological processes for theproduction of methanol.

Methane-monoxygenaseThere are two types of biocatalysts dedicated to the

functionalization of methane: particulateMethane-MonOxygenase (pMMO) and solubleMethane-MonOxygenase (sMMO). Both enzymesform part of the group of monoxygenases with twoiron atoms. No other oxygenases capable of acceptingmethane as substrate are known, while MMOs canaccept and functionalize different substrates, such asalkanes of various length and chlorinatedhydrocarbons (this makes the use of methanotrophicbacteria interesting for practical applications to restorecontaminated sites). Both enzymes have beencrystallized: sMMO in various conformations in thecourse of the last decade (Rosenzweig et al., 1993),while pMMO only recently (Lieberman and



Fe

MMOB

MMOH

e�

MMOR

CH4�O2

CH3OH�H2O

CH4�O2�2H��2e� CH3OH�H2O

NADH

NAD�

O

O

O

Fe

Fe Fe

H

regulation

Fig. 16. Structure of the enzymatic complex sMMO and reaction scheme (Merkx et al., 2001).

Rosenzweig, 2005). The bacteria that use theseenzymes (aerobic methanotrophs) live in environmentson the border between aerobic and anoxic zones,where the necessary levels of methane and molecularoxygen coexist (stagnant groundwaters or surfacewaters, shallow sediments). sMMO include threedifferent protein components (Fig. 16), each of whichis essential to guarantee an efficient level of catalysis.

pMMO, in its turn, is a trimer, in which eachcomponent is formed by three different sub-units, withthe catalysing sub-unit containing two metallic centreswith one and two copper atoms, respectively.

The catalytic activity carried out by all of theMMOs consists of the selective oxidation of methaneto methanol according to the reaction:

CH4�NADH�H��O2�� CH3OH��NAD��H2O

where NADH and NAD� represent the enzymaticco-factors respectively reduced and oxidized (Fig. 3).The natural enzymatic system is complex, formed byenzymes consisting of a number of sub-units anddependent on co-factors, oxidized and reduced in theirturn according to complex metabolic cellular routes. It

is precisely this complexity which limits themanipulation of the natural catalytic systems aimed atincreasing efficiency and the possibility of industrialapplication. The ‘historic’ leaders of research in thisfield (Merkx et al., 2001; Astier et al., 2003; Urlacheret al., 2004) report that, rather than directly applyingenzymes on an industrial scale, understanding theenzymatic mechanism of reaction may lead towardsthe development of more efficient synthetic catalyststhan those now in existence.

Action mechanism and active site of sMMOThe catalytic mechanism of sMMO has been

studied in depth in the last decade and a good level ofdetailed knowledge has been gained on the sequenceof reactive states of the catalytic centre, formed by twoiron atoms in the hydrolytic sub-unit. The essentialrole of the two auxiliary components is the transfer ofelectrons, via NADH, by reductase; the third sub-unitplays a regulatory and stabilizing role, according to amechanism that is still obscure. The catalytic site withtwo iron atoms complexed by carboxylic groups is alsopresent in other enzymes, which catalyze a greatvariety of different reactions (reversible link with O2,specific desaturation of a double bond of a fatty acid,oxidation and capture of iron, oxygen detection). Thecycles of oxygen bonding and activation take placethrough a sequence of transient intermediate stages,some of which have been portrayed by ElectronParamagnetic Resonance (EPR), Mossbauer andExtended X-ray Absorption Fine Structure (EXAFS)spectroscopic surveys, in combination with otherspectroscopic techniques. Much still remains to beunderstood about the mechanisms of interaction withmethane of the reactive intermediate state of thecatalytic centre (MMOHQ), the structure of which isshown in Fig. 17, and about the mechanism of theC�H bond scission reaction.

MutagenesisMMOs deservedly have the reputation of being

‘difficult’ enzymes. So far, attempts at (direct) genemutagenesis have been hindered by the lack of suitablegenetic instruments for the selection of large numbersof mutants; moreover the great majority of themutations described have not resulted in any significantincreases in enzymatic activity or in the acquisition ofcharacteristics favourable for industrial uses such as, forexample, mutations that make the enzymes independentof external co-factors. Therefore, the mutagenesis ofMMO has hitherto clarified certain aspects of themechanism of action of these enzymes, but has not yetled to the synthesis of variants of industrial interest. Theliterature on the subject does not indicate that it has yetbeen possible to apply the systems of directed evolution



substrate

FeFe

Fig. 17. Biferric catalytic centre in the MMOHQstate in relation to a molecule of ethane as substrate (Guallar et al., 2002).

in vitro to MMOs because of the absence of efficientsystems of expression and screening of the genesencoding these enzymes.

Mutagenesis of P450 oxygenasesIn the group of enzymes able to oxidize

hydrocarbon compounds (but not methane), the P450class of oxygenases is distinguished by a number ofcharacteristics that make it an advantageous prototypefor future industrial applications.

In the first place, the activity of some P450s (P450BM-3) is independent of the presence of co-factors,the enzyme does not consist of different sub-unitswhich have to interact with large losses in efficiencyand the oxidative activity on natural substrates of theP450s is hundreds of times greater than that of otheralkano-monoxygenases. The catalytic site of the P450oxygenases contains a porphyrinic ring and thesubstrate oxidation mechanism differs from that of theMMOs (Fig. 18). In Fig. 19 there is a schematiccomparison of a number of essential passages of theoxidative reaction catalysed by the two enzymes.

In the second place, as opposed to the MMOs, inthe P450s there are adequate gene expression systemsto make their engineering and their evolution in vitropossible. On the basis of these premises, new P450variants have been generated by means of evolution invitro which are capable of oxidizing C3-C8, a substratenot recognized by the natural enzyme, with greateractivity than the known alkano-monoxygenases(Glieder et al., 2002). The engineering of thisbiocatalyst has thus made it possible to direct theadvantageous characteristics of the oxidativechemistry of the P450s towards the selective use of

short alkanes. The next attempt will be the creation ofvariants able to catalyze the methane-methanol andethane-ethanol oxidative reactions; indeed, theisolation of the first active P450 mutants on ethane hasrecently been reported (Meinhold et al., 2005).

Biological production of methanolBiological production processes of alcohols are

often conditioned by conversion yields, by the toxicnature of the product for the biocatalyst and by thehigh costs of fermentation. Methanol is the product ofthe first reaction in the methane utilization chain bymethanotrophs and can be found in the aqueousculture medium of the bacteria which expressinsufficient levels of the second enzyme in themetabolic chain or in which this activity is partlyinactivated. The typical conversion levels described forthe MMOs are quite low (turnover rate of around 200molecules of oxidized substrate per minute) and theaccumulation of reaction intermediates is limited inthe first place by the inefficiency of this first passageof the metabolic chain and secondly by the need forfurther energy to recycle the oxidation/reduction of theco-factors. Within these limits, a process has beenproposed in which the methanol-dehydrogenaseactivity of the cells is inhibited by high concentrationsof CO2 (the end product of the reaction). In theseconditions, stable production of methanol from cells ofMethylosinus trichosporium of 0.13 mmol/h (aboutone-quarter of the methane-oxidative activity of thecells used; Xin et al., 2004) has been reported. Themethane-oxidation activity of the strain used is,however, less than one-hundredth part of that observedin other strains of the same species, e.g. in



O

SCys

H H

H2O

H2O

HOOH

H2O

O2

O

SCys

O

SCys

Fe(III)

SCys

Fe(II)

O

SCys

O HO

SCys

a b c

f e d

Fe(V)

Fe(III)

Fe(II)Fe(III)

Fig. 18. Mechanism of hydroxylation of the substratecatalyzed by P450oxygenase: the squarerepresents the hemegroup of the catalyticcentre (Urlacher et al.,2004).

Methylosinus trichosporium 11131, of which theproduction of 100 mmol/h of methanol fromimmobilized cells has, in fact, been reported (Methaet al., 1991). Osaka Gas has patented a process basedon the use of methanotrophs in which the furtheroxidation of methanol to formaldehyde is partlyinhibited by the fermentative conditions of the process(Tsubota et al., 2002). In a similar manner thecontinuous production from cultures of Methylosinustrichosporium of about 8 mM of methanol in 36 hourshas been obtained (using formate in the culturemedium and 1:4 methane/air) by inhibiting methanolconsumption with high salt concentrations(Lee et al., 2004).

Clark and Roberto (2003) report a fermentativemethod for the production of hydroxylated alkaneswhich uses a fraction of apolar solvent as a carrier forthe hydrogen necessary to regenerate the reducedco-factors which assist enzymatic activity.

The processes that use variants (developed in vitro)of P450 type oxygenase by the mechanisms described

by the Caltech group (Glieder et al., 2002; Meinholdet al., 2005) and mentioned in the preceding sections,fall into a first group of fairly general patents whichcontain methods usable in the process of identifyingmutants more active in alkane oxidation reactions.

The development of processes based on mutants invitro for synthesizing oxygenated compounds fromalkanes and other derivatives of fossil origin is also oneof the declared objectives of long-term links between thebiotech industry and some petrochemical companies.

Biological production of methanol and of other oxygenated compounds from agricultural waste products

The need to make use of the waste products ofhuman activities as a potential source of energy is atbasis of the reassessment of initiatives developed inpast years, which were then to be abandoned for theexclusive exploitation of oil derivatives as an energysource. It could be recalled here that the production ofmethanol by thermocombustion of wood was a process



Fe(II)

Fe(II) Fe(II)

Fe(II) Fe(II)

Fe(II)

Fe(I)

Fe(I)Fe(I)

Fe(I)

Fe(I)

Fe(I)

C

C

C

C

C CC C

CC

C

O

O

O OO

O

O

O

O

O

O O

O

O

O

O

O

OO

O

O

O O

O

O

O

O

O

H H

H

HH

H

HH

H H

H

HH

H

H

H

C

Hred Hperoxo

R3COH

R3CH

Fe(III)

SR

O O

OH H

Fe(II)

SR

R3CH

O

Fe(IV)

SR

R3COH

Fe(III)

SR

Hox Q

�1

oxygenated substrate

Fig. 19. Comparison of the catalytic mechanism of substrate oxidation in MMO and P450 oxygenase (Guallar et al., 2002).

used in the Twentieth century, then set aside in favourof chemical production from methane. More recently,production processes have been developed based on thefermentation of methanol or ethanol from sugar-richagricultural products, e.g. agricultural wastes having ahigh cellulose content deriving from the processing ofmaize and sugar cane. Of the two alcohols, theproduction of ethanol from wheat is favouredcompared to that of methanol from the same source.

The production of methanol from sugar-beet waste(a non-food use) is being intensively researched byAtlantic Biomass Conversions in collaboration withvarious universities and with the United StatesDepartment of Agriculture (USDA) in a projectcentred on the farming industry in North Dakota(USA). The process, for which the installation ofprototypes in beet sugar refineries is foreseen by theend of 2007, is based on the use of bacterial strainsdeveloped in vitro for the efficient use of waste rawmaterials. A quantity of methanol for energy use,equal to 350 million litres per year, could be producedfrom the wet waste fraction in United States refineries,according to Clark and Roberto (2003), which iscomparable to what could be obtained in Europeanrefineries. In Europe, the process is under study bybiotechnological companies such as Biopract.

The energy yields of biofuels (including additivesand biodiesel) from agricultural crops, hotly debatedbetween supporters and detractors, are always linkedto local circumstances on the whole, and in varioussituations they seem to indicate the possiblecompetitiveness of biofuels with other fossil energysources. Initiatives with adequate quantities ofbiomass locally available must, however, be taken intoconsideration to be competitive.

Bioinorganic mimetic systemsTo obviate some of the natural limits of MMO

systems, hybrid experimental systems (Biomethanol[...], 2004) or completely inorganic ones (Astier et al.,2003) have been designed, aimed at the selectiveoxidation reaction of methane under mild conditions.One of these systems involves the direct transfer ofelectrons from an electrode to the catalytic sub-unit ofthe purified sMMO. Under controlled conditions(presence of an enzyme that regulates the hydrogenperoxide level), the reaction can take place at a ratecomparable with that of the native enzyme. While thisis an advantage, reducing dependence of the productivesystem on the co-factor NADH, the synthesis methodremains based on purified enzymes, hence not verystable, liable to oxidation and short lived.

New hybrid systems based on the use of electronsshuttle such as sepulchrated cobalt trichloride (II) orrodium compounds have been experimented on, with

good results, in combination with otherNADH-dependent oxygenases, such as a number ofP450s (Schwaneberg et al., 2000) and could also beused with methane oxygenases.

Bioconversion of methane in the absence of oxygenThe natural oxidation of methane in anoxic

environments is mediated by microorganisms andfunctions as an important control reaction of the flowof methane from deep marine sediments to theatmosphere. It is estimated that this process,performed by consortia of microbes that use thesulphate as an oxidant, consumes 5-20% of the globalflow of methane into the atmosphere (20-100�106 t/y).In addition to the impact of this reaction on a globalscale on the methane cycle in our age, the sameprocess is also assumed to have played a fundamentalrole during the planet’s geochemical evolution, whenhigh atmospheric concentrations of greenhouse gasescounterbalanced scanty irradiation from the Sun. Thehypothesis is, in fact, that in that period theatmospheric gas mainly responsible for the greenhouseeffect was methane (while geological finds show lowconcentrations of CO2) and therefore the methaneanaerobic oxidation process was the main modulatorof the Earth’s climate.

In spite of extensive studies, the bases of the naturalprocess remain elusive, the reaction mechanism is notyet clear and the main hypotheses are still open(Valentine and Reeburgh, 2000). In recent years,important progress has, however, been made; studies onmarine sediments show evidence of the participation ofnew classes of microorganisms, belonging to theArchaea group and close philogenetically to theMethanosarcinales, as primary catalysts in thefunctionalization of methane. This reaction appears tobe closely interdependent on the presence and on theactivity of consortia of archaebacteria and sulphate-reducing bacteria; the chemical species that foster theinterdependence of these components of the microbicconsortium are not yet known, but they probablyinvolve hydrogen and perhaps acetate or acetic acid.The reactions at the basis of sulphate-dependentmethanation, assuming the interspecies transfer ofhydrogen in acetic acid, are:

2CH4�2H2O�� CH3COOH�4H2

4H2�SO42��H�� HS��4H2O

CH3COOH�SO42�� 2HCO3

��HS��H�

2CH4�2SO42�� 2HS��2HCO3

��2H2O

The thermodynamic yield of the complex ofreactions as a whole is rather low, but would be helpedby the high methane concentrations characteristic ofthe gaseous methane sources on the ocean bed.



In conclusion, the biological oxidation ofnatural gas in anoxic sediments is a fundamentalprocess on a global level, attributed toarchaebacteria that live in syntrophic consortiawith sulphate-reducing bacteria and probablylinked to energy-conserving reactions andbacterial-growth. Until now, however, it has not yetbeen possible to study the reactions identified inthe laboratory in isolated species and the mostimportant observations are, for the moment, at thelevel of field measurements. Therefore, theexploitation of the (bio)catalysts involved in theanaerobic methane functionalization processdepends closely on the results of further studiesnecessary to understand this process.

ProspectsThe use of whole microorganisms to biocatalyze

oxidative reactions suffers from certain limitations ofthe natural enzymatic systems: the dependence of thereaction on reduced co-factors and the complexity ofmulti-component enzymatic systems. Thebiotechnological response to these limitations istwofold and concerns: the development of engineeredhost systems to optimize the cellular environment andfoster the oxidative reaction, for example by optimizingthe levels of expression of co-factors and reductases;the creation of more active ‘evolved’ enzymaticvariants, or enzymes more active on substrates ofinterest not recognized by the natural enzyme.

In the case of methane-monoxygenases, bothpoints need to be developed, with the possibleexception of the recent synthesis of interesting variantsof the P450 B-3 oxygenase, active over short alkanesand which can be subjected to mutagenesis torecognize methane. It is also possible to envisagepossible developments in the system of expression ofthe enzymes themselves, a necessary step for MMOengineering exploiting, for example, systems ofexpression of similar oxygenases(propane-monoxygenase) in species that can easily beengineered.

Lastly, the oxidative reactions of methane in theabsence of oxygen are potentially open to exploitationfor the synthesis of derivatives, in which the carbon isincluded in intermediates, the nature of which has notyet been precisely established.

References

Aitken C.M. et al. (2004) Anaerobic hydrocarbon biodegradationin deep subsurface oil reservoirs, «Nature», 431, 291-295.

Anderson R.T., Lovley D.R. (2000) Biogeochemistry.Hexadecane decay by methanogenesis, «Nature», 404, 722-723.

Astier Y. et al. (2003) Cofactor independent oxygenationreactions catalyzed by soluble methane monooxigenase atthe surface of a modified gold electrode, «European Journalof Biochemistry», 270, 539-544.

Beilen J.B. van et al. (2001) Analysis of Pseudomonas putidaalkane-degradation gene clusters and flanking insertionsequences: evolution and regulation of the alk genes,«Microbiology», 147, 1621-1630.

Biomethanol from sugar beet pulp (2004), «Energy andSustainable Development Magazine», May/June, 3, 15.

Boetius A. et al. (2000) A marine microbial consortiumapparently mediating anaerobic oxidation of methane,«Nature», 407, 623-627.

Brown L.A. et al. (2002) Slowing production decline andextending the economic life of an oil field: new MEORtechnology, «SPE Reservoir Evaluation and Engineering»,5, 33-41, SPE 75355.

Bryant S.L., Lockhart T.P. (2000) Reservoir engineeringanalysis of MEOR, in: Proceedings of the Society ofPetroleum Engineers annual technical conference andexibition, Dallas (TX), 1-4 October, SPE 63229.

Clark T.R., Roberto F.F. (2003) US Patent 2003203456.Glieder A. et al. (2002) Laboratory evolution of a soluble,

self-sufficient, highly active alkane hydroxylase, «NatureBiotechnology», 20, 1135-1139.

Guallar V. et al. (2002) Quantum chemical studies of methanemonooxigenase: comparison with P450, «Current Opinionin Chemical Biology», 6, 236-242.

Harayama S. et al. (2004) Microbial communities in oil-contaminated seawater, «Current Opinion in Biotechnology»,15, 205-214.

Hitzman D.O., Dennis M. (2004) New nitrate-based treatmentscontrol hydrogen sulfide in reservoirs, «World Oil», 225,51-55.

Larter S., Aplin A. (2003) Mechanism of petroleumbiodegradation and of caprock failure: new insights,applications of reservoir geochemistry, in: Cubbitt J. et al.(edited by) Conference abstracts. Geochemistry of reservoirsII. Linking reservoir engineering and geochemical models,Geological Society of London, London, 3-4 February.

Lee S.G. et al. (2004) Optimization of methanol biosynthesisfrom methane using M. trichosporium OB3b, «BiotechnologyLetters», 26, 947-950.

Leuthner B., Heider J. (2000) Anaerobic toluene catabolismof Thauera Aromatica: the bbs Operon Codes for enzymesof beta oxidation of the intermediate benzylsuccinate,«Journal of Bacteriology», 182, 272-277.

Lieberman R.L., Rosenzweig A.C. (2005) Crystal structureof a membrane bound metallo-enzyme that catalyzes thebiological oxidation of methane, «Nature», 434, 177-182.

Lingthelm D.J. et al. (1991) Reservoir souring: an analyticalmodel for H2S generation and transportation in an oilreservoir owing to bacterial activity, in: Proceedings of theOffshore European conference, Aberdeen (UK), 369-378,SPE 23141.

Magot M. et al. (2000) Microbiology of petroleum reservoirs,«Antonie van Leeuwenhoek», 77, 103-116.

Meinhold P. et al. (2005) Direct conversion of ethane toethanol by engineered cytochrome P450 BM3,«ChemBioChem», 6, 1765-1768.

Merkx M. et al. (2001) Dioxygen activation and methanehydroxylation by sMMO: a tale of two irons and three



proteins, «Angewandte Chemie. International Edition inEnglish», 40, 2782-2807.

Metha P.H. et al. (1991) Methanol biosynthesis by covalentlyimmobilized cells of Methylosimus trichosporium: butchand continuous studies, «Biotechnology and Bioengineering»,37, 551-556.

Monticello D.J. (2000) Biodesulfurization and the upgradingof petroleum distillates, «Current Opinion in Biotechnology»,11, 540-546.

Nivens D.E. et al. (2004) Bioluminescent bioreporter integratedcircuits: potentially small, rugged and inexpensive whole-cell biosensors for remote environmental monitoring,«Journal of Applied Microbiology», 96, 33-46.

Parkes J. (1999) Cracking anaerobic bacteria, «Nature», 401,217-218.

Pedersen K. (2000) Exploration of deep intraterrestrial life:current perspectives, «Microbiology Letters», 185, 9-16.

Peters K.E., Moldowan J.M. (1993) The biomarker guide,Englewood Cliffs (NJ), Prentice Hall.

Rosenzweig A.C. et al. (1993) Crystal structure of a bacterialnon-haem iron hydroxy lase that catalyses the biologicaloxidation of methane, «Nature», 366, 537-543.

Saunders D.F. et al. (1999) Model for hydrocarbon microseepageand related near-surface alterations, «American Associationof Petroleum Geologists Bulletin», 83, 170-185.

Schumacher D. (1996) Hydrocarbon induced alteration of soilsand sediments, in: Schumacher D., Abrams M.A. (edited by)Hydrocarbon migration and its near surface expression.Outgrowth of the American Association of PetroleumGeologists research conference, Vancouver (Canada), 24-28 April 1994, Tulsa (OK), AAPG Memoir 66, 71-89.

Schwaneberg U. et al. (2000) P450 in biotechnology: zincdriven omega-hydroxylation of p-nitrophenoxydodecanoic

acid using P450BM-3 F8-7A as a catalyst,«Journal ofBiotechnology», 84, 249-257.

So C.M. et al. (2003) Anaerobic transformation of alkanes tofatty acids by a sulfate-reducing bacterium, Strain Hxd3,«Applied and Environmental Microbiology», 69, 3892-3900.

Sunde E. et al. (1993) Field-related mathematical model topredict and reduce reservoir souring, in: Proceedings ofthe Society of Petroleum Engineers international symposiumon oilfield chemicals, New Orleans (LA), 1-5 March, 449-456, SPE 25197.

Tsubota J. et al. (2002) Japanese Patent JP 2003235546 toOsaka Gas co.

Urlacher V.B. et al. (2004) Microbial P450 enzymes inbiotechnology, «Applied Microbiology and Biotechnology»,64, 317-325.

Valentine D.L., Reeburgh W.S. (2000) New perspectiveson anaerobic methane oxydation, «EnvironmentalMicrobiology», 2, 477-484.

Wenger L.M. et al. (2002) Multiple controls on petroleumbiodegradation and impact on oil quality, «SPE ReservoirEvaluation & Engineering», October, SPE 80168.

Widdel F., Rabus R. (2001) Anaerobic biodegradation ofsaturated and aromatic hydrocarbons, «Current Opinionin Biotechnologies», 12, 259-276.

Xin J. et al. (2004) Production of methanol from methaneby methanotrophic bacteria, «Biocatalysis andBiotransformation», 22, 225-229.

Francesca de FerraEniTecnologie

San Donato Milanese, Milano, Italy



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3.4 Biotechnologies applied to oil and gas exploration ... · iron-reducing bacteria sulphate-reducing bacteria acetoclastic methanogens acetate acetate geogas CH 4 H 2 CO 2 CH 4