IN THE NAME OF GODIN THE NAME OF GODIslamic Azad UniversityIslamic Azad University

of Falavarjanof Falavarjan

Department of MicrobiologyDepartment of Microbiology

Microbial BiotechnologyMicrobial BiotechnologyFall 2009Fall 2009

Keivan Beheshti MaalKeivan Beheshti Maal

Recent Advances in Recent Advances in Petroleum MicrobiologyPetroleum Microbiology

Jonathan D. Van Hamme, Ajay Singh and Owen P. WardJonathan D. Van Hamme, Ajay Singh and Owen P. WardUniversity of Cariboo, University of Waterloo and Petrozyme Technologies University of Cariboo, University of Waterloo and Petrozyme Technologies

Inc., CanadaInc., Canada

IntroductionIntroduction

PetroleumPetroleum Complex mixture of hydrocarbones Complex mixture of hydrocarbones

and organometal complexesand organometal complexes (Vanadium and Nickel)(Vanadium and Nickel)

Varies widely in composition and Varies widely in composition and physical propertiesphysical properties

Microbial growth substratesMicrobial growth substrates

IntroductionIntroduction Petroleum MicrobiologyPetroleum Microbiology

BiotransformationBiotransformation Use of M.O for changing cheap materials to merit productsUse of M.O for changing cheap materials to merit products

BiodegradationBiodegradation Use of M.O for degradation of petroleum and its derivativesUse of M.O for degradation of petroleum and its derivatives

BioremediationBioremediation Use of engineered petroleum degrading M.O for Use of engineered petroleum degrading M.O for

environmental clean upenvironmental clean up

BiorecoveryBiorecovery Use of M.O or their products for enhancing oil recoveryUse of M.O or their products for enhancing oil recovery

Petroleum Microbiology in a GlancePetroleum Microbiology in a Glance Study on aerobic biodegradation pathways Study on aerobic biodegradation pathways

for alkane, cycloalkane, aromatic alkane, for alkane, cycloalkane, aromatic alkane, polycyclic aromatic hydrocarbones (PAH)polycyclic aromatic hydrocarbones (PAH)

Anaerobic hydrocarbon catabolismAnaerobic hydrocarbon catabolism

Cellular and physiological adaptations to Cellular and physiological adaptations to hydrocarboneshydrocarbones

Hydrocarbone accession and uptakeHydrocarbone accession and uptake

Use of GMO for bioremediationUse of GMO for bioremediation

Petroleum Microbiology in a GlancePetroleum Microbiology in a Glance

Improvement of culture based and Improvement of culture based and culture independent methods for culture independent methods for studying hydrocarbone soils and studying hydrocarbone soils and microbial communitymicrobial community

Isolating and identifying responsible Isolating and identifying responsible bacteria, yeasts, funji and algaebacteria, yeasts, funji and algae for for hydrocarbone transformationhydrocarbone transformation

Petroleum Microbiology in a GlancePetroleum Microbiology in a Glance

Current states of oil M.Os:Current states of oil M.Os: mesophilic SRBs, thermophilic SRBsmesophilic SRBs, thermophilic SRBs methanogens methanogens mesophilic fermentative bacteriamesophilic fermentative bacteria thermophilic fermentative bacteriathermophilic fermentative bacteria

iron reducing bacteriairon reducing bacteria

Long term ecological effects of petroleum Long term ecological effects of petroleum pollution and control of deleterious microbial pollution and control of deleterious microbial activities in oil productionactivities in oil production

Current applied Researches in Petroleum Current applied Researches in Petroleum MicrobiologyMicrobiology

Oil spill remediation treatmentOil spill remediation treatment

Fermentor / wetland based hydrocarbone treatmentFermentor / wetland based hydrocarbone treatment

Biofiltration of volatile hydrocarbonesBiofiltration of volatile hydrocarbones

Microbial enhanced oil recoveryMicrobial enhanced oil recovery

Oil / fuel upgrading by desulfurizationOil / fuel upgrading by desulfurization

Oil / fuel upgrading by denitrogenationOil / fuel upgrading by denitrogenation

Coal processingCoal processing

Current applied Researches in Petroleum Current applied Researches in Petroleum MicrobiologyMicrobiology

Fine chemical productionFine chemical production

Microbial community based site assessmentsMicrobial community based site assessments

Roles and practical applications of chemical and Roles and practical applications of chemical and biological surfactantsbiological surfactants

Demetalation of distillate fractions, tar, coal Demetalation of distillate fractions, tar, coal derived liquid and synthetic fuels derived liquid and synthetic fuels

(removal of Ni and Van by Cyt-c reductase and (removal of Ni and Van by Cyt-c reductase and chloroperoxidase enzymes)chloroperoxidase enzymes)

Monitoring environmental contaminants by Monitoring environmental contaminants by biosensors biosensors

(Petroleum Metabolizing Enzymes of Petroleum (Petroleum Metabolizing Enzymes of Petroleum Degrading Bacteria ------ Electronic Systems)Degrading Bacteria ------ Electronic Systems)

MetabolismMetabolism Petroleum as a Carbon & Energy sourcePetroleum as a Carbon & Energy source

Pseudomonas putida Gpo1/pOCTPseudomonas putida Gpo1/pOCT (formerly (formerly Pseudomonas oleovoransPseudomonas oleovorans))

Plasmid OCT (Plasmid OCT (alkBFGHJKLalkBFGHJKL operon, operon, alkalk genes and genes and AlkAlk proteins proteins

Membrane responsible enzymesMembrane responsible enzymes 1. Membrane bound monooxygenase1. Membrane bound monooxygenase 2. Rubredoxin2. Rubredoxin 3. Hydroxylase3. Hydroxylase (Beta oxidation and TCA cycle )(Beta oxidation and TCA cycle )

Alkane metabolismAlkane metabolism

AlkaneAlkane alcohol alcohol alcoholalcohol aldehydealdehydealdehydealdehyde acidacidacid acid beta oxidationbeta oxidation Krebs cycleKrebs cycle

Alkane degradation in gramAlkane degradation in gramnegative bacterianegative bacteria

AlkB: alkane hydroxylaseAlkB: alkane hydroxylaseAlkF / AlkG: rubredoxinsAlkF / AlkG: rubredoxinsAlkH: aldehyde dehydrogenaseAlkH: aldehyde dehydrogenaseAlkJ: alcohol dehydrogenaseAlkJ: alcohol dehydrogenaseAlkK: acyl-coA synthetaseAlkK: acyl-coA synthetaseAlkL: outer membrane protein AlkL: outer membrane protein AlkT: rubredoxin reductaseAlkT: rubredoxin reductaseAlkN: methyl-accepting tranducer protein (chemotaxis)AlkN: methyl-accepting tranducer protein (chemotaxis)AlkS: positive regulator of alkBFGHIJKL operon and alkS/alkT genesAlkS: positive regulator of alkBFGHIJKL operon and alkS/alkT genes

Plasmid encoded hydrocarbon degradation Plasmid encoded hydrocarbon degradation gene clustersgene clusters

Chromosome encoded hydrocarbon Chromosome encoded hydrocarbon degradation gene clustersdegradation gene clusters

Petroleum hydrocarbon degrading Petroleum hydrocarbon degrading anaerobic bacteriaanaerobic bacteria

Control responses to hydrocarbonsControl responses to hydrocarbons Membrane alteration, uptake and effluxMembrane alteration, uptake and efflux 1. change in membrane architecture1. change in membrane architecture 2. change in active uptake2. change in active uptake 3. change in efflux3. change in efflux 4. change in chemotaxis4. change in chemotaxis

Hydrocarbons (lipophilic) Hydrocarbons (lipophilic) partitioning in a partitioning in a hydrophobic area in acyl chains of phospholipidhydrophobic area in acyl chains of phospholipid

(periplasmic space in g-)(periplasmic space in g-)

changing changing fluidityfluidity and and protein conformationprotein conformation changing changing disruption of barrierdisruption of barrier changing changing energy transductionenergy transduction changing changing membrane bound enzyme activitymembrane bound enzyme activity

stressstress biofilm formationbiofilm formation

Control responses to hydrocarbonsControl responses to hydrocarbons

Partitioning lipophilic hydrocarbons in Partitioning lipophilic hydrocarbons in membranemembrane

Consequences:Consequences: Reduction of membrane integrityReduction of membrane integrity Repair enhancingRepair enhancing Phospholipid biosynthesis enhancingPhospholipid biosynthesis enhancing Membrane strengtheningMembrane strengthening Intercalating inhibitionIntercalating inhibition

Mods of hydrocarbon uptakeMods of hydrocarbon uptake1. Active uptake1. Active uptake Contact with water solubilized H.C.Contact with water solubilized H.C. * * Ps. aeroginosaPs. aeroginosa in in Surfactant solubilized oil and in hexadecaneSurfactant solubilized oil and in hexadecaneLimitations: Limitations: - reduction of solubility- reduction of solubility - M.W. increase- M.W. increase Direct adherence to large oil dropletsDirect adherence to large oil droplets * * RhodococcusRhodococcus in in crude oilcrude oil

Encapsulating solid n-c18 and n-c-36 in liposomesEncapsulating solid n-c18 and n-c-36 in liposomes membrane fusionmembrane fusion delivery to membrane delivery to membrane bound enzymesbound enzymes

2. Passive uptake2. Passive uptake *Phenanthrene uptake by *Phenanthrene uptake by Ps. fluorescens LP6aPs. fluorescens LP6a

Microbial community analysis methodsMicrobial community analysis methods

Microbial treatment of petroleum wasteMicrobial treatment of petroleum waste Use of indigenous microbial populationUse of indigenous microbial population Resistant to tidal washingResistant to tidal washing

Origin of pollutionOrigin of pollution- Crude oil recovery- Crude oil recovery- Transport - Transport - Refining (production, processing, storage)- Refining (production, processing, storage)- Product usage- Product usage

Pollutants:Pollutants:1. Lighter and toxic hydrocarbons 1. Lighter and toxic hydrocarbons volatilization volatilization into air into air human and animal health threathuman and animal health threat2. Sulfur compounds2. Sulfur compounds petrochemical wastepetrochemical waste

Treatment of contaminated soils and Treatment of contaminated soils and sludgesludge

Biological methods more effective Biological methods more effective than physicochemical methodsthan physicochemical methods

Reasons:Reasons:

1. biodegradability of major molecules 1. biodegradability of major molecules

in crude oilin crude oil

2. oil degrading M.O are ubiquitious2. oil degrading M.O are ubiquitious

Petroleum sludge treatment technologiesPetroleum sludge treatment technologies

Factors affecting bioremediation of Factors affecting bioremediation of crude oil and oily wastescrude oil and oily wastes

Physical conditions and naturePhysical conditions and nature ConcentrationConcentration Types and amounts of various H.C.Types and amounts of various H.C. Bioavailability of the substrateBioavailability of the substrate Properties of biological systemProperties of biological system (type, concentration/physiological conditions of M.O)(type, concentration/physiological conditions of M.O)

Problems:Problems: - low water solubility of majority of petroleum - low water solubility of majority of petroleum hydrocarbonshydrocarbons - Aqueous life of microorganisms- Aqueous life of microorganisms

Solution:Solution: - use of surfactants and biosurfactants- use of surfactants and biosurfactants (cell surface agents or extracellular agents)(cell surface agents or extracellular agents)

Microorganisms major biosurfactantsMicroorganisms major biosurfactants

Petroleum degradation processesPetroleum degradation processes

Passsive bioremediationPasssive bioremediation

Landfarming of wasteLandfarming of waste

Bioreactor based processBioreactor based process

Passive bioremediationPassive bioremediation

Natural attenuationNatural attenuation The least invasiveThe least invasive Mediated by indigenous microbialMediated by indigenous microbial

populationpopulation Low efficacy and so slowLow efficacy and so slow Unsuitable for remediation of high Unsuitable for remediation of high

volume oily wastesvolume oily wastes

Passive bioremediationPassive bioremediation

Hydrocarbon biodegradation by rhizospheric M.OHydrocarbon biodegradation by rhizospheric M.O (phytoremediation)(phytoremediation)

Hydrocarbon uptake by plants and release to Hydrocarbon uptake by plants and release to

atmosphere without transformationatmosphere without transformation (phytovolatilization)(phytovolatilization)

Wetland use for removal petroleum wastesWetland use for removal petroleum wastes Depend on plant community, water depth and concentration of Depend on plant community, water depth and concentration of wasteswastes

Limitations:Limitations:1. toxicity of contaminants1. toxicity of contaminants2. availability of fertilizer 2. availability of fertilizer and oxygenand oxygen

Landfarming of oily wasteLandfarming of oily waste Oily sludge treatment and disposal method Oily sludge treatment and disposal method

in many parts of the worldin many parts of the world

(unacceptable environmentally)(unacceptable environmentally)

Use in large refineries (200,000 -500,000 Use in large refineries (200,000 -500,000 barrels/day) : barrels/day) : 10,000 m310,000 m3 sludge/year sludge/year

Landfarming processesLandfarming processes Contamination of large lands with oily sludgesContamination of large lands with oily sludges Starting of bioremediation of less recalcitrant oil fractionsStarting of bioremediation of less recalcitrant oil fractions Tilling the soil to promote gas transferTilling the soil to promote gas transfer

Disadvantages:Disadvantages:1.Transfer of hazardous volatile organic carbon to atmosphere1.Transfer of hazardous volatile organic carbon to atmosphere2.low rate of biodegradation2.low rate of biodegradation3.high rate of volatilization3.high rate of volatilization4.lack of control on microbial activity 4.lack of control on microbial activity (temp.,pH, moisture, aeration, mixing and circulation)(temp.,pH, moisture, aeration, mixing and circulation)5.effective depth: Max 10-20 cm5.effective depth: Max 10-20 cm6.very low degradation rate: 0.5% - 1% total P.H.C / month6.very low degradation rate: 0.5% - 1% total P.H.C / month

Landfarming examplesLandfarming examples Oily soil [1.3%]Oily soil [1.3%] treatment with nutrients, treatment with nutrients,

surfactants, microbial inoculants, deep tilling surfactants, microbial inoculants, deep tilling and 25 oCand 25 oC

Total P.H.C reduction: 90% in 34 daysTotal P.H.C reduction: 90% in 34 days

Fuel oil [6%]Fuel oil [6%] treatment with nutrient, M.O treatment with nutrient, M.O moisture control and high mixing and aerationmoisture control and high mixing and aeration

Total P.H.C reduction: 80% - 90% in 6 monthsTotal P.H.C reduction: 80% - 90% in 6 months

This method has been banned This method has been banned

Bioreactor based processBioreactor based process Elimination of the most rate limiting and variable Elimination of the most rate limiting and variable

factors in landfarmingfactors in landfarming Accomodation of solid contents of 5% -50% w/vAccomodation of solid contents of 5% -50% w/v

Break up solid aggregates and aqueous phase Break up solid aggregates and aqueous phase contact increase and biodegradation enhancementcontact increase and biodegradation enhancement

Management of volatile organic carbons (more Management of volatile organic carbons (more biodegradable, microbial growth supporter and biodegradable, microbial growth supporter and energizer)energizer)

Relative good duration: 1-4 monthsRelative good duration: 1-4 months

Bioreactor based process examplesBioreactor based process examples French Limited Crosby TexFrench Limited Crosby Tex Indigenous M.O, mixing and aeration with pure OIndigenous M.O, mixing and aeration with pure O22

300000 tons of tar like materials biodegradation in 11 months300000 tons of tar like materials biodegradation in 11 months (85% sludge destruction in 122 days)(85% sludge destruction in 122 days)

Gulf Coast RefineryGulf Coast Refinery 4,000,000 liter bioreactor with float mounted aerator in 22.6 oC and total 4,000,000 liter bioreactor with float mounted aerator in 22.6 oC and total P.H.C: [10%]P.H.C: [10%] Total reduction: 50% in 90 days efficacy:90%Total reduction: 50% in 90 days efficacy:90% Petrozyme ProcessPetrozyme Process 8 bioreactors 1,200,000 liters8 bioreactors 1,200,000 liters temp: 28-32 / pH:6.4-7.6 / sparged air lift aeration incubation:10-12 temp: 28-32 / pH:6.4-7.6 / sparged air lift aeration incubation:10-12

days / total P.H.C:[10%] / degradation rate : 1%/day degradation rate days / total P.H.C:[10%] / degradation rate : 1%/day degradation rate 97% -99%97% -99%

In Venezuela, U.S, Canada and MexicoIn Venezuela, U.S, Canada and Mexico

Biofiltration of volatile organic compoundsBiofiltration of volatile organic compounds

Biofilters: Biofilters: 1. solid phase – gas phase1. solid phase – gas phase (gas and O (gas and O22 passes through high surface solid)passes through high surface solid)

2. liquid phase – gas phase2. liquid phase – gas phase (gas and O2 (gas and O2 sparges through liquid)sparges through liquid)

Include N2, P,nutrients and immobilized M.O as Include N2, P,nutrients and immobilized M.O as biofilmbiofilm

Effective on Effective on benezene, tolene, ethylbenzene benezene, tolene, ethylbenzene andand xylenexylene as as hazardous environmental pollutantshazardous environmental pollutants

Efficacy: removal of 30 Efficacy: removal of 30 μμg/h/cm2: 75%-99%g/h/cm2: 75%-99%

Removal of H2S Removal of H2S

H2S /sulfide oxides :H2S /sulfide oxides :corrosivecorrosive and and reservoir pluggingreservoir plugging and and oil souringoil souring

Origins:Origins: 1- petrochemical gas / liquid wastes1- petrochemical gas / liquid wastes 2- SRBs from injected of sulfate rich sea 2- SRBs from injected of sulfate rich sea

water in secondary recoverywater in secondary recovery

S-oxidizing bacteria:S-oxidizing bacteria: Thiocalovibrio Thiocalovibrio ThiocalobacteriaThiocalobacteria H2S+1/2 O2H2S+1/2 O2 ----- ----- S + H2OS + H2O

Efficacy:96%Efficacy:96%

Microbial Enhanced Oil RecoveryMicrobial Enhanced Oil Recovery

Injection of nutrient, indigenous or added microbesInjection of nutrient, indigenous or added microbes In situ microbial growthIn situ microbial growth Generation of bioproductsGeneration of bioproducts Mobilization of oil into producing well by:Mobilization of oil into producing well by: 1-Reservoir repressurization1-Reservoir repressurization 2-Interfacial tension reduction2-Interfacial tension reduction 3-Oil viscosity reduction3-Oil viscosity reduction 4-Selective plugging of most permeable zones4-Selective plugging of most permeable zones

Important physicochemical properties:Important physicochemical properties: Salinity (1.3%-2.5%), temperature (70-90 oC), pH, pressure Salinity (1.3%-2.5%), temperature (70-90 oC), pH, pressure (2000-2500 lb/in2) and nutrient availability (2000-2500 lb/in2) and nutrient availability

Microbial secondary recoveryMicrobial secondary recovery

Desirable properties of BiopolymersDesirable properties of Biopolymers

Shear stabilityShear stability High solution viscosityHigh solution viscosity Compatibility with reservoir brineCompatibility with reservoir brine Stable viscosity over a wide range of Stable viscosity over a wide range of

pH/temp/pressurepH/temp/pressure resistant to biodegradationresistant to biodegradation

Microbial deemulsificationMicrobial deemulsification Oilfield water in oil emulsion formed at various Oilfield water in oil emulsion formed at various

stages of stages of ExplorationExploration Production Production Oil recoveryOil recovery

Emulsion :Emulsion : 1)Tight emulsion :100 Å1)Tight emulsion :100 Å 2)Loose emulsion :52)Loose emulsion :5μμmm

Water and dirt in crude oil :Water and dirt in crude oil :

corrosion on pipeline/reactor corrosion on pipeline/reactor

Microbial deemulsificationMicrobial deemulsification

Microbial DesulfurizationMicrobial Desulfurization

[s] :0.05% - 5% in normal crude oil [s] :0.05% - 5% in normal crude oil : 14% in heavier oils: 14% in heavier oils

Most: Organically bound:Most: Organically bound: Condensed thiophensCondensed thiophens

removal by: removal by: Expensive physicochemical methodsExpensive physicochemical methods

Aerobic Desulfurizing OrganismsAerobic Desulfurizing Organisms

Rhodococous Rhodococous erythropolis :dibenzothiophene(DBT)erythropolis :dibenzothiophene(DBT)

NocardiaNocardia AgrobacteriumAgrobacterium MycobacteriumMycobacterium GordonaGordona KlebsiellaKlebsiella XanthomonasXanthomonas

Paenibacillus (Thermophil)Paenibacillus (Thermophil)

Microbial denitrogenationMicrobial denitrogenation

[N2]: 0.5% - 2.1% in crude oil[N2]: 0.5% - 2.1% in crude oil

70%-75% in form of pyrroles/ indoles/ carbazole/ 70%-75% in form of pyrroles/ indoles/ carbazole/ pyridine / quinoline pyridine / quinoline

CarbazoleCarbazole inhibitor of hydrodesufurication inhibitor of hydrodesufurication Toxic mutagenic :Air pullutant , Nitric oxideToxic mutagenic :Air pullutant , Nitric oxide

Removal of N-compound by expensive Removal of N-compound by expensive physicochemical method (hydrotreatment under physicochemical method (hydrotreatment under high temp,pressure)high temp,pressure)

Microbial denitrogenationMicrobial denitrogenation

Oxygenases: Important roleOxygenases: Important role

N-utilizing Bacteria:N-utilizing Bacteria:

Burkholderia - Alcaligenes - BacillusBurkholderia - Alcaligenes - Bacillus Beijerinckia - Mycobacterium - ComamonasBeijerinckia - Mycobacterium - Comamonas Pseudomonas - Serratia - XanthomonasPseudomonas - Serratia - Xanthomonas

Biorefining microorganismsBiorefining microorganisms

Bacterial biosensorsBacterial biosensors Biosensors: Uniquely measure the interaction of spesific Biosensors: Uniquely measure the interaction of spesific

compounds through highly sensitive biorecognition processcompounds through highly sensitive biorecognition process

Biosensors employ: Biosensors employ: EnzymesEnzymes AbAb TissuesTissues Living M.OLiving M.O

Properties:Properties: 1) Great sensitivity1) Great sensitivity 2) Great selectivity2) Great selectivity 3) For detection/ qualification/ biodegradability determination3) For detection/ qualification/ biodegradability determination 4) Work in mixture without pretreatment of samples providing4) Work in mixture without pretreatment of samples providing

Fusing- a reporter geneFusing- a reporter gene a promoter element (inducible by target compound) a promoter element (inducible by target compound)

Bacterial biosensorsBacterial biosensors

Microorganisms are very great superior and powerful creatures

ThankThank

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