Petroleum Microbiology
O P Ward and A Singh,University of Waterloo,Waterloo,ON,Canada J D Van Hamme,Thompson Rivers University,Kamloops,BC,Canada G Voordouw,University of Calgary,Calgary,AB,Canada
ª2009Elsevier Inc.All rights reserved.
Defining Statement
Introduction
Physiological Mechanisms of Accession and Efflux Metabolism
Microorganisms in Oil Recovery Oil Biorefining and Bioprocessing Biodegradation and Bioremediation Concluding Remarks
Further Reading
Glossary
一般等价物benzene,toluene,ethylbenzene,
xylenes(BTEX)Volatile monoaromatic hydrocarbons commonly found together in crude petroleum.
bioremediation Use of biological agents,such as bacteria or plants,to remove or neutralize contaminants,as in polluted soil or water.
de-emulsification Breaking of an emulsion into its separate liquid phases.
denitrogenation Removal of nitrogen atoms from nitrogen-containing petroleum hydrocarbons. desulfurization Removal of sulfur atoms from sulfur-containing petroleum hydrocarbons. dibenzothiophene(DBT)A model compound for organic sulfur in fossil fuels.
efflux Expelling from cell.
hydrocarbon accession Process whereby the cell gains access to hydrocarbon substrates.hydrodesulfurization(HDS)A catalytic chemical process widely used to remove sulfur(S)from natural gas and from refined petroleum products. methanogenic oil degradation Conversion of hydrocarbons to methane mediated by the action of syntrophs,CO2-reducing,and acetotrophic methanogens.
phytoremediation Use of plants to remove or neutralize contaminants,as in polluted soil or water. pol
ycyclic aromatic hydrocarbons(PAHs)Chemical compounds that consist of fused aromatic rings and do not contain heteroatoms or carry substituents. reservoir souring The gradual increase in H2S concentration in oil reservoirs.
surfactant A substance that reduces surface tension. taxis The responsive movement of a free-moving organism or cell toward or away from an external stimulus. volatile organic compounds(VOCs)Organic compounds that have a high vapor pressure and low water solubility.
Abbreviations
AMO anaerobic methane oxidation
BT benzothiophene
BTEX benzene,toluene,ethylbenzene,xylenes DBT dibenzothiophene
HDS hydrodesulfurization
hNRB heterotrophic nitrate-reducing bacteria MEOR microbial enhanced oil recovery NDO naphthalene dioxygenase
NR-SOB nitrate-reducing,sulfide-oxidizing
bacterium
OWC oil–water contact
PAHs polycyclic aromatic hydrocarbons
SRB sulfate-reducing bacteria
VFA volatile fatty acids
VOC volatile organic compound
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Defining Statement
Physiological aspects of petroleum microbiology include phenomena of taxis,hydrocarbon accession,transport and efflux,and pathways for aerobic and anaerobic degradation of alkane and aromatic hydrocarbons.Oil production and processing strategies include microbial enhanced oil reco
v-ery(MEOR),de-emulsification,promotion of methanogenic oil degradation and control of reservoir souring,desulfuriza-tion,denitrogenation,demetallation,biosynthesis of novel compounds,and bioremediation technology. Introduction
Petroleum comprises a very complex combination of hydro-carbons and other organic constituents including some organometallic components.Many of these compounds are metabolizable by microorganisms,and indeed some are products of microbial metabolism.Understanding the ways in which microorganisms attack,degrade,and utilize petro-leum components for growth was a strong early focus of petroleum microbiology,with a view to addressing oil spills and other environmental problems.Investigation of the kinds of microbial transformations and the associated micro-bial species,which occur in the subsurface,and indeed in the oil reservoir itself,has represented a more recent research thrust.As a result of developments in molecular techniques, we are gaining new insights into hydrocarbon catabolism and the many novel catalytic mechanisms that have been elucidated.The molecular,cellular,and physiological responses of microbes to hydrocarbon-containing environ-ments are also being intensively investigated.While applied petroleum microbiology research continues to be directed to development of improved and more reliable biodegradation and bioremediation technology,there is a growing interest in the application of microb
iology techniques to enhance oil recovery and in the onward processing and refining of petroleum products(Table1).
Table1Applied petroleum microbiology and biotechnology
Application Process Key microorganism/biocatalyst Role of microbes
Oil recovery Microbial-enhanced
oil recovery Acinetobacter calcoaceticus
Arthobacter paraffineus
Bacillus licheniformis
Clostridium acetobutylicum
Leuconostoc mesenteroides
Xanthomonas campestris
Zymomonas mobilis
Biosurfactants and chemicals produced by
microbes help in oil dissolution,viscosity
reduction,selective biomass plugging,
permeability increase,oil swelling,and
pressure increase
Microbial de-emulsification A.calcoaceticus
Bacillus subtilis
Corynebacterium petrophilum
Nocardia amarae
Pseudomonas aeruginosa
Rhodococcus globerulus
De-emulsification of oil emulsions,oil
地
源热泵换热
solubilization,viscosity reduction,wettingOther oil recovery applications A.calcoaceticus
RAG-1
Crude oil recovery from tank bottoms using
biosurfactants
Biorefining and bioprocessing Biodesulfurization Agrobacterium MC501
Arthrobacter sp.
Corynebacterium sp.SY1
Gordona CYKS1
Nocardia sp.
Rhodococcus erythropolis H2
Rhodococcus sp.IGTS8
Biotransformation of organic sulfur compounds,
selective removal of sulfur from crude oil or
refined petroleum products
Biodenitrogenation Comamonas acidovorans
Nocardioides sp.
P.aeruginosa
Pseudomonas ayucida
Rhodococcus sp.
韩寒Biotransformation of organic nitrogen
compounds in crude oil,nitrogen removal
Biodemetallation Bacillus megaterium
Caldariomyces fumago
Escherichia coli
Enzymatic removal of Ni and V from crude oil
using microbial enzymes chloroperoxidase,
cytochrome C reductase,and heme
oxygenase
Bioprocessing General microbial enzymes
Cytochrome p450-dependent
Monooxygenases
Dioxygenases
Lipoxygenases and peroxidases
Biotransformation of petroleum compounds to
produce fine chemicals
(Continued)
444Applied Microbiology:Industrial|Petroleum Microbiology
Physiological Mechanisms of Accession and Efflux
Hydrocarbons as Substrates
Most aerobically and anaerobically growing microorgan-isms require a reduced electron donor and a terminal electron acceptor linked by an electron transport chain in order to extract fuel from substrates for maintenance and growth(some grow fermentatively).Hydrocarbons, natural compounds found in petroleum reservoirs,as well as produced by living organisms,such as microorg
anisms, insects,and plants,are highly reduced,energy-rich molecules common in the environment–thanks to both anthropogenic and nonanthropogenic release.This structurally diverse class of molecules includes saturated and unsaturated linear,branched and cyclic alkanes, mono-and polycyclic aromatics,and nitrogen-and sulfur-containing heterocyclics that are used by eukaryo-tic and prokaryotic microorganisms for energy.In addition to energy-processing mechanisms,microorgan-isms also possess pathways to funnel catabolic products into central metabolic pathways.Given the structural similarities between some hydrocarbons and cellular macromolecules,for example,straight-chain alkanes and fatty acids,microorganisms may incorporate hydro-carbons directly into membranes and vesicles without shuttling through central pathways and typical anabolic steps.
Historical culture-based studies,and more recent molecular-based inquiries,have clearly illustrated micro-bial hydrocarbon metabolism in bacteria,cyanobacteria, yeast,fungi,and algae in a diversity of habitats.A number of obligate and near-obligate hydrocarbonoclastic micro-organisms such as Alcanivorax spp.,Cytoclasticus spp., Oleiophilus spp.,Oleispira spp.,Planomicrobium spp.,and Thalassolituus spp.have recently been isolated,and mole-cular ecology studies have found these organisms to be ubiquitous in marine environments throughout the world. As is often the case,these
organisms may be present below detectable levels prior to hydrocarbon exposure,but they achieve near dominance for periods during which pre-ferred substrates are available.
With low to intermediate water solubilities,for exam-ple,from0.000282to1800mg lÀ1for tetradecane and benzene,respectively,compared to hexoses with solubi-lities of100g lÀ1or more,microorganisms have evolved mechanisms to exploit hydrocarbons as nutrients and to minimize toxic solvent effects on membrane structure and function.Mechanistically,the stages leading up to hydro-carbon catabolism may include sensing and taxis, substrate accession and uptake,and,if toxicity is an issue,efflux to maintain tolerable levels within the cell.
An excellent foundation of laboratory and field studies describing these steps is now being strengthened through genome sequencing of important petroleum-associated microorganisms(Table2)and identification of genetic elements involved in each behavior.For example,the genome of Alcanivorax borkumensis includes genes for alkane metabolism,biosurfactant production and biofilm formation,uptake of a variety of inorganic nutrients,and efflux.
Taxis
As a phenomenon,microbial taxis is movement resulting from direct or indirect response to external environmen-tal conditions.For example,chemotaxis is the response to specific chemical attractants and repellents,which relies on two-component signaling pathways consisting of a transmembrane chemoreceptor proteins,and signal kinase linked to response regulators that switch flagellar motor rotational direction.Well understood for model organisms in response to water-soluble substrates,signal cascades include a variety of other regulators that allow
Table1(Continued)
Application Process Key microorganism/biocatalyst Role of microbes
Biodegradation and bioremediation Bioremediation Acinetobacter spp.
Pseudomonas spp.
Rhodococcus spp.
Sphingomonas spp.
Some fungi
Emulsification through adherence to
hydrocarbons;dispersion;foaming agent;
detergent;soil flushing
Monitoring of
contaminated sites
Pseudomonas fluorescens HK44
Pseudomonas putida RB1401
Bacterial biosensors in monitoring of
contaminant and bioremediation progress
Biofiltration of VOC Acinetobacter spp.
Pseudomonas spp.
Rhodococcus spp.
Sphingomonas spp.
Biodegradation of volatile hydrocarbons
Biological removal of
H2S and SO x
Thioalcalobacteria spp.
Thiobacillus spp.
Thiocalovibrio spp.
Thiomicrospira spp.
Biotransformation of H2S to elemental sulfur and
sulfate
Applied Microbiology:Industrial|Petroleum Microbiology445
for a short-term memory function to ensure that micro-bial movement generally tends to more favorable locations.Other signals such as light,dark,environmental redox potential,and cellular energy levels can also direct microbial taxis.There are examples of microoganisms that exhibit chemotactic responses to a number of hydro-carbons including benzene,toluene,ethylbenzene,and naphthalene.In unmixed or poorly mixed experimental systems,hydrocarbon chemotaxis has been shown to increase substrate accession and biodegradation. Putative chemoreceptors,some linked to biodegradation genes,have been proposed for other hydrocarbon sub-strates in both aerobic and anaerobic microorganisms based on whole genome studies.For example, Pseudomonas putida strain NCIB9816-4harbors the83kb pDTG1plasmid that carries genes for naphthalene cata-bolism(nah genes)as well as the gene for NahY,which is homologous to methyl-accepting chemotaxis proteins. Mutants lacking nahY are able to metabolize naphthalene but lose chemotactic abilities.
Accession
Once a microorganism is in the vicinity of dissolved-or nonaqueous-phase hydrocarbon,the next stage prior to metabolism or efflux is accession.Distinct from uptake, that is,transport across the cell membrane,accession includes any process by which a hydrocarbonoclastic microorganism reduces mass transfer problems associated with poorly soluble substrates.To this end,microorgan-isms may produce biosurfactants to pseudosolubilize hydrocarbons in the aqueous phase,or increase cell-surface hydrophobicity in order to adhere directly to hydrocarbon droplets.
On a whole,biosurfactants have a range of physiological functions including roles in cell signaling,biofilm formation, cellular differentiation,motility,and,of particular interest here,increasing substrate bioavailability.Analogous to their chemical counterparts,biosurfactants may increase the apparent solubility of hydrocarbons by decreasing interfacial tensions between oil–water phases and via micellization. These effects are achieved due to the amphipathic nature of these cell-associated or extracellular compounds,which may be composed of a combination of fatty acids,peptides, and carbohydrates.A dissolved hydrocarbon,with or with-out low concentrations of biosurfactant present,may be directly accessible to a microorganism in the aqueous phase and,once in close contact,the hydrocarbon may partition into the interior of the cell membrane via passive uptake.In some cases,initial oxidation steps are carried out by membrane-associated enzymes whereby the su
bstrate is oxygenated and passed into the cytoplasm for further meta-bolism,the AlkB monooxygenase being a well-characterized example as described below.As the biosurfactant concentra-tion increases above the critical micellization concentration, a microorganism with a hydrophilic cell surface may interact
Table2Complete genomes available for important petroleum-related microorganisms
Organism Properties GenBank no.
Alcanivorax borkumensis SK2Aerobic saturated hydrocarbon metabolism;marine
environments;biosurfactant and biofilm formation
AM286690
Arthrobacter sp.F824Xylene metabolism CP000454
Azoarcus sp.EbN1Aromatic hydrocarbon metabolism;denitrifying NC006513;NC006823–NC006824 Dechloromonas aromatica Anaerobic benzene metabolism CP000089
Desulfovibrio vulgaris Hildenborough Pipeline corrosion;sulfate-reducing bacterium AE017285.1–AE017286.1 Geobacter metallireducens GS15Participate in anaerobic aromatic hydrocarbon
metabolism
CP000148to CP000149
Geobacter sulfurreducens Participate in anaerobic aromatic hydrocarbon
metabolism
AE017180.1
Marinobacter aquaeoli Aerobic hexadecane,pristane,crude oil metabolism;
moderate halophile
CP000514–CP000516 Methylobium petrophilum MTBE metabolism to CO2;benzene,toluene,
ethylbenzene metabolism
CP000555–CP000556
Mycobacterium sp.KMS PAH metabolism CP000518–CP000520 Mycobacterium sp.MCS PAH metabolism CP000384–CP000385 Mycobacterium vanbaalenii strain全后汉文
PYR-1
High molecular weight PAH metabolism(pyrene)CP000511
Polaromonas naphthalenivorans CJ2Aerobic naphthalene metabolism;<20 C CP000529–CP000537 Pseudomonas putida F1Benzene,toluene,ethylbenzene metabolism CP000712
P.putida Naphthalene degradation plasmid AF491307
NCIB9816-4
Rhodococcus sp.RHA1Aerobic alkyl benzene metabolism(best known for
PCB degradation)
CP000431.1–CP000434.1
Rhodopseudomonas palustris Anaerobic aromatic hydrocarbon metabolism;
facultative phototroph CP000250
446Applied Microbiology:Industrial|Petroleum Microbiology
上海
电视大学浦东分校with hydrophilic micellar head groups and access nonpolar hydrocarbons partitioned in the micellar core.It is possible for the biosurfactant micelle to recombine with the cell membrane at this point and deliver hydrocarbon into the cell.If the micelle concentration becomes too high,then mass transfer may be limited by substrate dilution in the micellar phase.
If a microorganism has expressed a hydrophobic cell surface in order to adhere directly to the oil–water inter-face,then a biosurfactant may disrupt cell–hydrocarbon contact,especially in mixed communities.There are many examples of microorganisms that become highly hydrophobic when growing on hydrocarbons.For exam-ple,some Rhodococci adhere so tightly to oil–water interfaces that centrifugation will not remove them. Rhodococci are well known to produce complex cell walls that include nonpolar materials such as mycolic acids and have been shown to directly incorporate unal-tered and partially oxidized alkanes into the cell wall. Finally,a microorganism with a hydrophobic cell surface may shed outer biosurfactant-type cell wall material in order to make the cell surface more hydrophilic and detach from a used oil droplet.
Transport in the Cell
With respect to uptake,that is,passage of substrate across the cell membrane,it was historically believed that microorgan-isms rely solely on passive hydrocarbon partitioning into the cell rather than by facilitated diffusion or active uptake. However,bacteria and yeast have long been observed to store unaltered hydrocarbons in membrane-bound vesicles, which may indicate a more active process,especially in Gram-negative bacteria where the lipopolysaccharide layer creates a barrier to the passage of hydrophobic molecules.In addition,some metabolic flux rates have been calculated to be greater than passive partitioning rates,and selective uptake and storage of specific alkanes from alkane mixtures has been observed.Experiments employing electron trans-port chain inhibitors,uncouplers,and ATP synthase inhibitors indicate good evidence for active uptake of naphthalene and phenanthrene.
Genome studies have revealed putative hydrocarbon transporters,mostly related to the FadL long-chain fatty acid transporter in Escherichia coli,in a number of micro-organisms.Evidence for facilitated diffusion of toluene by TbuX and TodX,m-xylene by XylN,and for n-hexadecane,phenanthrene,and dibenzothiophene (DBT)is available,although no transport proteins have been isolated to date.The FadL transporter has been crystallized and structural studies indicate
that this ß-barrel protein contains a hatch that opens sponta-neously due to conformational changes upon substrate binding.StyE is a facilitated uptake membrane protein for styrene related to FadL,and other putative permeases have been proposed for related compounds such as benzoate,phthalate,and -hexachlorocyclohexane.For the latter,the putative transporter coded by linKLMN appears to consist of a permease,ATPase,periplasmic protein,and lipoprotein.
It has been proposed that AlkL is involved in alkane uptake in Pseudomonas and that pbhD in Sphingomonas paucimobilis var.EPA505may code for a fluoranthene metabolite transporter.Of course,the transport of other limiting nutrients such as nitrogen and phosphorus is also important in oil-impacted environments.The genome of the obligate hydrocarbonoclast A.borkumensis carries approximately50permeases,many of which are pre-sumed to be associated with hydrocarbon uptake.
In contrast to uptake,a number of efflux pumps have been described that are able to expel hydrocarbons from the cell cytoplasm or perhaps directly from the cell mem-brane.Related to antibiotic efflux pumps,these pumps can often efflux both hydrocarbons and antibiotics;hydro-carbon
efflux pumps are constructed of an outer membrane protein linked to a pump protein via a peri-plasmic channel protein.Pumps for ,srpABC, ttgABC,and ttgGHI),some of which also pump styrene, m-xylene,ethylbenzene,and propylbenzene,have been characterized.Phenanthrene,anthracene,and fluor-anthene(emhB)pumping has also been observed in aerobes.Antibiotic efflux pumps have been identified in medically important anaerobes,and putative efflux genes in anaerobic hydrocarbon degrading microorganisms have been discovered through genome sequencing.
For a nonhydrocarbon-metabolizing microorganism, being able to efflux membrane-disrupting hydrocarbons is essential.For hydrocarbonoclasts,efflux pumps are important for maintaining homeostatic levels of metabo-lizable substrate,and may also serve to excrete nonmetabolizable hydrocarbons from mixtures. Metabolism
Aerobic Alkane Metabolism
As a catabolic group,microorganisms possess metabolic tools to exploit a diverse range of hydrocarbons as sources of energy,carbon,nitrogen,and sulfur.Microorganisms have been isolated that are able to metabolize C1to C40 alkanes,cycloalkanes,benzene,toluene,ethylbenzene, xylenes(
BTEX)and related compounds,two-to six-ring polyaromatic hydrocarbons,and N-and S-containing heterocyclics such as carbazole and DBT.Generally, hydrocarbon catabolism begins with mono-and dioxy-genase reactions involving molecular oxygen under aerobic conditions,and via addition of other groups such
Applied Microbiology:Industrial|Petroleum Microbiology447
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