Optimization of Bioremediation Process

BIOREMEDIATION BIOREMEDIATION COURSECOURSE

OPTIMIZATION OF MICROBIAL OPTIMIZATION OF MICROBIAL TRANSFORMATION TRANSFORMATION

MICROORGANISMS MOSTLY MICROORGANISMS MOSTLY ACTIVE IN REMEDIATION ACTIONACTIVE IN REMEDIATION ACTION

• FUNGI• BACTERIA

– HETEROTROPH– AUTOTROPH

• Aerob: Required O2 • Anaerob: Do not need oxygen, use other

compounds as electron acceptor– Strickly anaerob: Die in the presence of O2– Facultative: Survive in the presence of O2

Microorganism in extreme Microorganism in extreme environmentenvironment

• Microorganism that live in:– High pH (Basophylic)– Low pH (Acidophylic)– High temperature (Thermophylic)– Low temperature (Cryophylic)

The normal pH: 6-8

The normal T : 25-35

Microorganism used specific Microorganism used specific substratesubstrate

• Petrophylic (used petroleum hydrocarbon for their growth)

• Sulphate Reducing Bacteria (Required sulphate in order them to grow)

• Nitrifier (bacteria responsible for nitrification)

• Denitrifier (bacteria responsible for denitrification)

How the microbe growthHow the microbe growth

• They use substrate (food)– Carbon source– Energy source

• They require nutrient– Nitrogen– Phosphor

• They required some mineral– Mono and divalent ion including some heavy

metals in a trace concentration

The growth profileThe growth profile

• Follow bell shape

Lag phase (adaptation)Exponential phaseStagnant phaseDead phase

In accordance with this growth characteristicScientist use parameters such as Ks and UmaxTo identified the ability of microbes to grow onSpecific substrate and sometimes to differentiatemicrobes

What makes microbe posses ability What makes microbe posses ability to utilize chemicals as their foodto utilize chemicals as their food

• They require pre-cursor for their cell synthesize

• Carbon, Nitrogen, and Phosphor are being main component of the cells

• They convert food to cells component with the help of biocatalyst (Enzyme)

• Microorganisms produce enzyme (extracellularly and intracellularly)

Organic PollutantOrganic Pollutant• Volatile, and Semi Volatiles Organic

Compounds– BTEX, – Halogenated organic compounds with

short C chains

• Persistent Organic Pollutants (POPs)

• Persistent Organic Pollutants (POPs) listed in The Stockholm Covention

POPs Listed in POPs Listed in The Stockholm ConventionThe Stockholm Convention

• Aldrin• Dieldrin• Endrin• Chlordane• Toxaphene• Heptachlor• Mirex• Hexa Chloro Benzene• DichloroDiphenylTrichloroethane (DDT)• PCBs• Dioksin• Furan

Persistent Organic Pollutants (POPs) Persistent Organic Pollutants (POPs) other than those in the list of other than those in the list of Stockholm ConventionStockholm Convention

• 1,2-Dichloro Ethane• 1,2-Dichloro Propilene• 1,6-DCH• 2,4-D• Epichlorohydrin• Chlorohydrin• Chloroaminobenzene• Dichlorophenol• Benzo(a)pyrene• Benzo(b)fluoranthene

Recalcitrance vs Strain DevelopmentRecalcitrance vs Strain Development

• Substances used to be classified as recalcitrance thus they were persistent:– Dalapon (2,2-DCPA, 2MCPA, MCA, 1,2-DCE,

2,4-D, Chlorophenol etc) they are now found to be amenable to biodegradation

• POPs such as DDT, PCBs, and other halogenated compounds could also be degraded by a mixed culture or consortium microorganisme resulted from careful enrichment, screening and development

Local Strains Capable of Degrading some Local Strains Capable of Degrading some relatively persistent organic compoundsrelatively persistent organic compounds

Factors that make organic pollutant Factors that make organic pollutant persistent (non-amenable to microbial persistent (non-amenable to microbial degradation)degradation)

• High molecular weight of the substances• Low solubility of the substances• Poor bioavailability• Complexity of the substances

– The 12 POPs required mixed culture and combined biochemical system to obtain transformation leading to detoxification

– Halogenated (depends on halogen type, positions, number, F>Cl>Br>I

– Petroleum Hydrocarbon: Short (Hydrolitic), Long chain (Oxygenolitic), Medium (combine), aromatic & cyclic (combined system)

• Poor environmental conditions (pH, T, Nutrients, inhibitors, oxygen)

• Lack of degradative enzyme

How can we activate the microbeHow can we activate the microbe

• Get the substrate soluble

• Bring them into the cells

• Other wise the microbe will need to excrete extracellular enzyme

• Get the enzyme active– Provide best environment conditions– Provide the right inducer– Get rid of all the inhibitors

Can microbe utilize toxic chemicals Can microbe utilize toxic chemicals

• Normally microbe will only use the food that is closest to central metabolism

• In the case of absent such food then microbe will try to use more complex substrate. At this stage specific enzyme may be required

• With careful enrichment microbe can utilize complex chemicals

Petroleum HydrocarbonPetroleum Hydrocarbon

Dissolved PHs

EmulsionMilky Emulsion

Non-milky emulsion

Film

Number of Carbon

Low (C6-C12)

Medium (C12-C25)

High (above 25)

Amenable to biological process

To be separatedby physical process

Possible biologicalprocess

Relativelybiodegradable

Lessbiodegradable

Degaradation of Some Crude Oil by Degaradation of Some Crude Oil by Microorganism (1)Microorganism (1)

EXPAN Oil

0

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2.5 5 7.5

Kadar minyak awal (% )

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Awal Akhir % Efisiensi

USED Oil

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2.5 5 7.5Kadar minyak awal (% )Ka

dar m

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)

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Degaradation of Some Crude Oil by Degaradation of Some Crude Oil by Microorganism (2)Microorganism (2)

VICO Oil

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2.5 5 7.5Kadar minyak aw al (%)

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Aw al Akhir %Efisiensi

Degaradation of Some Crude Oil Degaradation of Some Crude Oil by Bacteria and Fungiby Bacteria and Fungi

0,000,501,001,502,002,503,003,504,004,505,005,506,006,507,00

0 14 28 42 56 70 84 98 112 120

Hari ke -

TPH

(%)

1E+05

1E+06

1E+07

1E+08

1E+09

MO

TPH MO

0,000,501,001,502,002,503,003,504,004,505,005,506,006,507,00

0 14 28 42 56 70 84 98 112 120Hari ke -

TPH

(%)

1E+05

1E+06

1E+07

1E+08

1E+09

MO

TPH

MO

0,000,501,001,502,002,503,003,504,004,505,005,506,006,507,00

0 14 28 42 56 70 84 98 112 120Hari ke -

TPH

(%)

1E+05

1E+06

1E+07

1E+08

1E+09

MO

TPH

MO

pH profile during crude oil degradationpH profile during crude oil degradation

4

4.5

5

5.5

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0 20 40 60 80 100 120Hari ke-

pH

IK8IB8IBF8IF8

pH profile during crude oil degradationpH profile during crude oil degradation

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40

0 20 40 60 80 100 120Hari ke-

Tem

pera

tur

IK8IB8IBF8IF8

How do we maintain microbesHow do we maintain microbes

• In liquid medium• In solid medium• Store in the fridge• Store in the freezer (-80 centigrade)• Send to culture collection

How can we obtain microbe capable of How can we obtain microbe capable of degrading toxic chemicalsdegrading toxic chemicals

• Chose the right source for enrichment (contaminated soil)

• Design a good enrichment technique

• Use modern biotechnology to screen