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22 Φεβ 2013 (πριν από 4 χρόνια και 8 μήνες)

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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


Petroleum


Complex mixture of hydrocarbones
and organometal complexes

(Vanadium and Nickel)



Varies widely in composition and
physical properties



Microbial growth substrates

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

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

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


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



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

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)


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 )

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


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

Control responses to hydrocarbons


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



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


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

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

Petroleum sludge treatment technologies

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)

Microorganisms major biosurfactants

Petroleum degradation processes


Passsive bioremediation



Landfarming of waste



Bioreactor based process

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

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

Landfarming of oily waste


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


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 10
-
20 cm

6.very low degradation rate: 0.5%
-

1% total P.H.C / month

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

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

Bioreactor based process examples


French Limited Crosby Tex


Indigenous M.O, mixing and aeration with pure O
2


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


Biofilters:
1. solid phase


gas phase

(gas and O
2

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
μ
g/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


S
-
oxidizing bacteria:

Thiocalovibrio


Thiocalobacteria


H2S+1/2 O2

-----


S + H2O


Efficacy:96%

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


(2000
-
2500 lb/in2) and nutrient availability

Microbial secondary recovery

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


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 :5
μ
m



Water and dirt in crude oil :


corrosion on pipeline/reactor

Microbial deemulsification

Microbial Desulfurization


[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



Rhodococous erythropolis
:dibenzothiophene(DBT)



Nocardia


Agrobacterium


Mycobacterium


Gordona


Klebsiella


Xanthomonas



Paenibacillus (Thermophil)

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)

Microbial denitrogenation


Oxygenases: Important role



N
-
utilizing Bacteria:



Burkholderia
-

Alcaligenes
-

Bacillus


Beijerinckia
-

Mycobacterium
-

Comamonas


Pseudomonas
-

Serratia
-

Xanthomonas



Biorefining microorganisms

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)

Bacterial biosensors

Microorganisms are very great

superior and powerful creatures


Thank

you