Transcript Document

IN THE NAME OF GOD
Islamic Azad University
of Falavarjan
Department of Microbiology
Microbial Biotechnology
Fall 2009
Keivan Beheshti Maal
Recent Advances in
Petroleum Microbiology
Jonathan D. Van Hamme, Ajay Singh and
Owen P. Ward
University of Cariboo, University of Waterloo and
Petrozyme Technologies Inc., Canada
Introduction
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Petroleum
Complex mixture of hydrocarbones
and organometal complexes
(Vanadium and Nickel)
Varies widely in composition and
physical properties
Microbial growth substrates
Introduction
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Petroleum Microbiology
Biotransformation
Use of M.O for changing cheap materials to merit products
Biodegradation
Use of M.O for degradation of petroleum and its derivatives
Bioremediation
Use of engineered petroleum degrading M.O for
environmental clean up
Biorecovery
Use of M.O or their products for enhancing oil recovery
Petroleum Microbiology in a Glance
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Study on aerobic biodegradation pathways
for alkane, cycloalkane, aromatic alkane,
polycyclic aromatic hydrocarbones (PAH)
Anaerobic hydrocarbon catabolism
Cellular and physiological adaptations to
hydrocarbones
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Hydrocarbone accession and uptake
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Use of GMO for bioremediation
Petroleum Microbiology in a Glance
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Improvement of culture based and
culture independent methods for
studying hydrocarbone soils and
microbial community
Isolating and identifying responsible
bacteria, yeasts, funji and algae for
hydrocarbone transformation
Petroleum Microbiology in a Glance
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Current states of oil M.Os:
mesophilic SRBs, thermophilic SRBs
methanogens
mesophilic fermentative bacteria
thermophilic fermentative bacteria
iron reducing bacteria
Long term ecological effects of petroleum
pollution and control of deleterious microbial
activities in oil production
Current applied Researches in Petroleum
Microbiology
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Oil spill remediation treatment
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Fermentor / wetland based hydrocarbone treatment
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Biofiltration of volatile hydrocarbones
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Microbial enhanced oil recovery
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Oil / fuel upgrading by desulfurization
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Oil / fuel upgrading by denitrogenation
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Coal processing
Current applied Researches in Petroleum
Microbiology
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Fine chemical production
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Microbial community based site assessments
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Roles and practical applications of chemical and
biological surfactants
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Demetalation of distillate fractions, tar, coal
derived liquid and synthetic fuels
(removal of Ni and Van by Cyt-c reductase and
chloroperoxidase enzymes)
Monitoring environmental contaminants by
biosensors
(Petroleum Metabolizing Enzymes of Petroleum
Degrading Bacteria ------ Electronic Systems)
Metabolism
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Petroleum as a Carbon & Energy source
Pseudomonas putida Gpo1/pOCT
(formerly Pseudomonas oleovorans)
Plasmid OCT (alkBFGHJKL operon, alk
genes and Alk proteins
Membrane responsible enzymes
1. Membrane bound monooxygenase
2. Rubredoxin
3. Hydroxylase
(Beta oxidation and TCA cycle )
Alkane metabolism
Alkane
 alcohol
alcohol  aldehyde
aldehyde  acid
acid  beta oxidation
Krebs cycle
Alkane degradation in gram
negative bacteria
AlkB: alkane hydroxylase
AlkF / AlkG: rubredoxins
AlkH: aldehyde dehydrogenase
AlkJ: alcohol dehydrogenase
AlkK: acyl-coA synthetase
AlkL: outer membrane protein
AlkT: rubredoxin reductase
AlkN: methyl-accepting tranducer protein (chemotaxis)
AlkS: positive regulator of alkBFGHIJKL operon and alkS/alkT genes
Plasmid encoded hydrocarbon degradation
gene clusters
Chromosome encoded hydrocarbon
degradation gene clusters
Petroleum hydrocarbon degrading
anaerobic bacteria
Control responses to hydrocarbons
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Membrane alteration, uptake and efflux
1.
2.
3.
4.
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change
change
change
change
in
in
in
in
membrane architecture
active uptake
efflux
chemotaxis
Hydrocarbons (lipophilic)  partitioning in a
hydrophobic area in acyl chains of phospholipid
(periplasmic space in g-)
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changing
changing
changing
changing
fluidity and protein conformation
disruption of barrier
energy transduction
membrane bound enzyme activity
stress  biofilm formation
Control responses to hydrocarbons
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Partitioning lipophilic hydrocarbons in
membrane
Consequences:
 Reduction of membrane integrity
 Repair enhancing
 Phospholipid biosynthesis enhancing
 Membrane strengthening
 Intercalating inhibition
Mods of hydrocarbon uptake
1. Active uptake
 Contact with water solubilized H.C.
* Ps. aeroginosa in
Surfactant solubilized oil and in hexadecane
Limitations: - reduction of solubility
- M.W. increase
 Direct adherence to large oil droplets
* Rhodococcus in crude oil
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Encapsulating solid n-c18 and n-c-36 in
liposomes  membrane fusion  delivery to
membrane bound enzymes
2. Passive uptake
*Phenanthrene uptake by Ps. fluorescens LP6a
Microbial community analysis methods
Microbial treatment of petroleum waste
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Use of indigenous microbial population
Resistant to tidal washing
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Origin of pollution
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Crude oil recovery
Transport
Refining (production, processing, storage)
Product usage
Pollutants:
1. Lighter and toxic hydrocarbons  volatilization
into air  human and animal health threat
2. Sulfur compounds  petrochemical waste
Treatment of contaminated soils and
sludge
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Biological methods more effective
than physicochemical methods
Reasons:
1. biodegradability of major molecules
in crude oil
2. oil degrading M.O are ubiquitious
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Petroleum sludge treatment technologies
Factors affecting bioremediation of
crude oil and oily wastes
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Physical conditions and nature
Concentration
Types and amounts of various H.C.
Bioavailability of the substrate
Properties of biological system
(type, concentration/physiological conditions of M.O)
Problems: - low water solubility of majority of petroleum
hydrocarbons
- Aqueous life of microorganisms
Solution:
- use of surfactants and biosurfactants
(cell surface agents or extracellular agents)
Microorganisms major biosurfactants
Petroleum degradation processes
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Passsive bioremediation
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Landfarming of waste
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Bioreactor based process
Passive bioremediation
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Natural attenuation
The least invasive
Mediated by indigenous microbial
population
Low efficacy and so slow
Unsuitable for remediation of high
volume oily wastes
Passive bioremediation
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Hydrocarbon biodegradation by rhizospheric M.O
(phytoremediation)
Hydrocarbon uptake by plants and release to
atmosphere without transformation (phytovolatilization)
Wetland use for removal petroleum wastes
Depend on plant community, water depth and concentration of
wastes
Limitations:
1. toxicity of contaminants
2. availability of fertilizer
and oxygen
Landfarming of oily waste
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Oily sludge treatment and disposal method
in many parts of the world
(unacceptable environmentally)
Use in large refineries (200,000 -500,000
barrels/day) : 10,000 m3 sludge/year
Landfarming processes
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Contamination of large lands with oily sludges
Starting of bioremediation of less recalcitrant oil fractions
Tilling the soil to promote gas transfer
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Disadvantages:
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1.Transfer of hazardous volatile organic carbon to atmosphere
2.low rate of biodegradation
3.high rate of volatilization
4.lack of control on microbial activity
(temp.,pH, moisture, aeration, mixing and circulation)
5.effective depth: Max 10-20 cm
6.very low degradation rate: 0.5% - 1% total P.H.C / month
Landfarming examples
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Oily soil [1.3%]  treatment with nutrients,
surfactants, microbial inoculants, deep tilling
and 25 oC
Total P.H.C reduction: 90% in 34 days
Fuel oil [6%]  treatment with nutrient, M.O
moisture control and high mixing and aeration
Total P.H.C reduction: 80% - 90% in 6 months
This method has been banned
Bioreactor based process
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Elimination of the most rate limiting and variable
factors in landfarming
Accomodation of solid contents of 5% -50% w/v
Break up solid aggregates and aqueous phase
contact increase and biodegradation enhancement
Management of volatile organic carbons (more
biodegradable, microbial growth supporter and
energizer)
Relative good duration: 1-4 months
Bioreactor based process examples
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French Limited Crosby Tex
Indigenous M.O, mixing and aeration with pure O2
300000 tons of tar like materials biodegradation in 11 months
(85% sludge destruction in 122 days)
Gulf Coast Refinery
4,000,000 liter bioreactor with float mounted aerator in 22.6 oC and total
P.H.C: [10%]
Total reduction: 50% in 90 days efficacy:90%
Petrozyme Process
8 bioreactors 1,200,000 liters
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
97% -99%
In Venezuela, U.S, Canada and Mexico
Biofiltration of volatile organic compounds
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Biofilters: 1. solid phase – gas phase (gas and O2
passes through high surface solid)
2. liquid phase – gas phase (gas and
O2 sparges through liquid)
Include N2, P,nutrients and immobilized M.O as
biofilm
Effective on benezene, tolene, ethylbenzene and
xylene as hazardous environmental pollutants
Efficacy: removal of 30 μg/h/cm2: 75%-99%
Removal of H2S
H2S /sulfide oxides :corrosive and
reservoir plugging and oil souring
 Origins:
1- petrochemical gas / liquid wastes
2- SRBs from injected of sulfate rich sea
water in secondary recovery
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S-oxidizing bacteria: Thiocalovibrio
Thiocalobacteria
H2S+1/2 O2 ----- S + H2O
Efficacy:96%
Microbial Enhanced Oil Recovery
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Injection of nutrient, indigenous or added microbes
In situ microbial growth
Generation of bioproducts
Mobilization of oil into producing well by:
1-Reservoir repressurization
2-Interfacial tension reduction
3-Oil viscosity reduction
4-Selective plugging of most permeable zones
Important physicochemical properties:
Salinity (1.3%-2.5%), temperature (70-90 oC), pH, pressure
(2000-2500 lb/in2) and nutrient availability
Microbial secondary recovery
Desirable properties of
Biopolymers
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Shear stability
High solution viscosity
Compatibility with reservoir brine
Stable viscosity over a wide range of
pH/temp/pressure
resistant to biodegradation
Microbial deemulsification
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Oilfield water in oil emulsion formed at various
stages of
Exploration
Production
Oil recovery
Emulsion :
1)Tight emulsion :100 Å
2)Loose emulsion :5μm
Water and dirt in crude oil :
corrosion on pipeline/reactor
Microbial deemulsification
Microbial Desulfurization
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[s] :0.05% - 5% in normal crude oil
: 14% in heavier oils
Most: Organically bound:
Condensed thiophens
removal by:
Expensive physicochemical methods
Aerobic Desulfurizing Organisms
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Rhodococous erythropolis
:dibenzothiophene(DBT)
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Nocardia
Agrobacterium
Mycobacterium
Gordona
Klebsiella
Xanthomonas
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Paenibacillus (Thermophil)
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Microbial denitrogenation
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[N2]: 0.5% - 2.1% in crude oil
70%-75% in form of pyrroles/ indoles/ carbazole/
pyridine / quinoline
Carbazole
 inhibitor of hydrodesufurication
 Toxic mutagenic :Air pullutant , Nitric oxide
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Removal of N-compound by expensive
physicochemical method (hydrotreatment under
high temp,pressure)
Microbial denitrogenation
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Oxygenases: Important role
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N-utilizing Bacteria:
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Burkholderia - Alcaligenes - Bacillus
Beijerinckia - Mycobacterium - Comamonas
Pseudomonas - Serratia
- Xanthomonas
Biorefining microorganisms
Bacterial biosensors
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Biosensors: Uniquely measure the interaction of spesific
compounds through highly sensitive biorecognition process
Biosensors employ:
Enzymes
Ab
Tissues
Living M.O
Properties:
1) Great sensitivity
2) Great selectivity
3) For detection/ qualification/ biodegradability determination
4) Work in mixture without pretreatment of samples providing
Fusing- a reporter gene
a promoter element (inducible by target compound)
Bacterial biosensors
Microorganisms are very great
superior and powerful creatures
Thank
you