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IN THE NAME OF GOD Islamic Azad University of Falavarjan

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Presentation on theme: "IN THE NAME OF GOD Islamic Azad University of Falavarjan"— Presentation transcript:

1 IN THE NAME OF GOD Islamic Azad University of Falavarjan
Department of Microbiology Microbial Biotechnology Fall 2009 Keivan Beheshti Maal

2 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

3 Introduction Petroleum
Complex mixture of hydrocarbones and organometal complexes (Vanadium and Nickel) Varies widely in composition and physical properties Microbial growth substrates

4 Introduction 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

5 Petroleum Microbiology in a Glance
Study on aerobic biodegradation pathways for alkane, cycloalkane, aromatic alkane, polycyclic aromatic hydrocarbones (PAH) Anaerobic hydrocarbon catabolism Cellular and physiological adaptations to hydrocarbones Hydrocarbone accession and uptake Use of GMO for bioremediation

6 Petroleum Microbiology in a Glance
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

7 Petroleum Microbiology in a Glance
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

8 Current applied Researches in Petroleum Microbiology
Oil spill remediation treatment Fermentor / wetland based hydrocarbone treatment Biofiltration of volatile hydrocarbones Microbial enhanced oil recovery Oil / fuel upgrading by desulfurization Oil / fuel upgrading by denitrogenation Coal processing

9 Current applied Researches in Petroleum Microbiology
Fine chemical production Microbial community based site assessments Roles and practical applications of chemical and biological surfactants 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)

10 Metabolism 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 )

11 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

12 Plasmid encoded hydrocarbon degradation gene clusters

13 Chromosome encoded hydrocarbon degradation gene clusters

14 Petroleum hydrocarbon degrading anaerobic bacteria

15 Control responses to hydrocarbons
Membrane alteration, uptake and efflux 1. change in membrane architecture 2. change in active uptake 3. change in efflux 4. change in chemotaxis Hydrocarbons (lipophilic)  partitioning in a hydrophobic area in acyl chains of phospholipid (periplasmic space in g-)  changing fluidity and protein conformation  changing disruption of barrier  changing energy transduction  changing membrane bound enzyme activity stress  biofilm formation

16 Control responses to hydrocarbons
Partitioning lipophilic hydrocarbons in membrane Consequences: Reduction of membrane integrity Repair enhancing Phospholipid biosynthesis enhancing Membrane strengthening Intercalating inhibition

17 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 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

18 Microbial community analysis methods

19 Microbial treatment of petroleum waste
Use of indigenous microbial population Resistant to tidal washing Origin of pollution - 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

20 Treatment of contaminated soils and sludge
Biological methods more effective than physicochemical methods Reasons: 1. biodegradability of major molecules in crude oil 2. oil degrading M.O are ubiquitious

21 Petroleum sludge treatment technologies

22 Factors affecting bioremediation of crude oil and oily wastes
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)

23 Microorganisms major biosurfactants

24 Petroleum degradation processes
Passsive bioremediation Landfarming of waste Bioreactor based process

25 Passive bioremediation
Natural attenuation The least invasive Mediated by indigenous microbial population Low efficacy and so slow Unsuitable for remediation of high volume oily wastes

26 Passive bioremediation
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

27 Landfarming of oily waste
Oily sludge treatment and disposal method in many parts of the world (unacceptable environmentally) Use in large refineries (200, ,000 barrels/day) : 10,000 m3 sludge/year

28 Landfarming processes
Contamination of large lands with oily sludges Starting of bioremediation of less recalcitrant oil fractions Tilling the soil to promote gas transfer Disadvantages: 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 cm 6.very low degradation rate: 0.5% - 1% total P.H.C / month

29 Landfarming examples 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

30 Bioreactor based process
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

31 Bioreactor based process examples
French Limited Crosby Tex Indigenous M.O, mixing and aeration with pure O2 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: / pH: / 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

32 Biofiltration of volatile organic compounds
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%

33 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 S-oxidizing bacteria: Thiocalovibrio Thiocalobacteria H2S+1/2 O  S + H2O Efficacy:96%

34 Microbial Enhanced Oil Recovery
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 ( lb/in2) and nutrient availability

35 Microbial secondary recovery

36 Desirable properties of Biopolymers
Shear stability High solution viscosity Compatibility with reservoir brine Stable viscosity over a wide range of pH/temp/pressure resistant to biodegradation

37 Microbial deemulsification
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

38 Microbial deemulsification

39 Microbial Desulfurization
[s] :0.05% - 5% in normal crude oil : 14% in heavier oils Most: Organically bound: Condensed thiophens removal by: Expensive physicochemical methods

40 Aerobic Desulfurizing Organisms
Rhodococous erythropolis :dibenzothiophene(DBT) Nocardia Agrobacterium Mycobacterium Gordona Klebsiella Xanthomonas Paenibacillus (Thermophil)

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

42 Microbial denitrogenation
Oxygenases: Important role N-utilizing Bacteria: Burkholderia - Alcaligenes - Bacillus Beijerinckia - Mycobacterium - Comamonas Pseudomonas - Serratia Xanthomonas

43 Biorefining microorganisms

44 Bacterial biosensors 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)

45 Bacterial biosensors

46 Microorganisms are very great superior and powerful creatures
Thank you


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