Bioremediation: how it works and Bioremediation: how it works and why to adopt thiswhy to adopt this

Microorganisms, and to a lesser extent plants, can transform and degrade many types of contaminants

These transformation and degradation processes vary, depending on the physical-chemical environment, microbial communities, and nature of the contaminant.

Bioremediation is a technology that can be used to reduce, eliminate, or contain hazardous waste

Basic concept :

Many organic contaminants such as hydrocarbon fuels can be degraded to relatively harmless products such as CO2 (the end result of the degradation process)

Microorganisms can interact with metals and convert them from one chemical form to another

Microorganisms can change the valence, or oxidation state, of some heavy metals (e.g., chromium and mercury) and radionuclides (e.g., uranium) by using them as electron acceptors.the solubility of the altered species decreases and the contaminant is immobilized in situ, i.e., precipitated into an insoluble salt in the sediment.

the solubility of the altered species increases, increasing the mobility of the contaminant and allowing it to be more easily flushed from the environment.

Both of these kinds of transformations present opportunities for bioremediation of metals and radionuclides

Pollution treatment technology that uses Pollution treatment technology that uses microbial metabolic processes to microbial metabolic processes to reduce, eliminate, contain, or transform various various contaminants present in soils, sediments, water, or air to to benign products

BioremediationBioremediation : what’s this?what’s this?

Inorganic:Inorganic: heavy metals and metalloids, heavy metals and metalloids, radionuclides, nutrients, acids and bases, radionuclides, nutrients, acids and bases,

Organic:Organic: petroleum hydrocarbons, halogenated petroleum hydrocarbons, halogenated organic compounds, pesticides, herbicides, organic compounds, pesticides, herbicides, plasticizersplasticizersXenobiotics: organic compounds, produced via chemical Xenobiotics: organic compounds, produced via chemical synthesis, which synthesis, which never before existed never before existed in natural in natural environmentsenvironments

Major Types of PollutantsMajor Types of Pollutants

ENVIRONMENTAL FATE OF RELEASED ENVIRONMENTAL FATE OF RELEASED METALS AND ORGANICSMETALS AND ORGANICS

• Abiotic FactorsAbiotic Factors• Biotic FactorsBiotic Factors

MicroorganismsMicroorganisms

•Chemical propertiesChemical properties•EnvironmentalEnvironmental mobility / persistence mobility / persistence•Toxicity Toxicity

AffectsMetals/radionuclides & organicsMetals/radionuclides & organics

• Ubiquitous • Highly adaptable and can live in most

inhospitable conditions • Vast genetic diversity • Metabolic versatility • Remarkable range of

degradative/transformative ability

Microorganisms : An Asset !Microorganisms : An Asset !

ENVIRONMENTAL FATE OF ENVIRONMENTAL FATE OF RELEASED CONTAMINANTSRELEASED CONTAMINANTS

Mobilephase

Immobilephase

Radionuclides/MetalsOrganics

Microbial activity

solubilization precipitation

Increased bioavalability and toxicity

Reduced bioavalability and toxicityMineralization

biodegradation

Why bioremediation ?Why bioremediation ?

5. Applicable for otherwise non/slowly degradable 5. Applicable for otherwise non/slowly degradable organic and inorganic contaminantsorganic and inorganic contaminants

1. Ability and versatility of microbial processes to 1. Ability and versatility of microbial processes to withstand most inhospitable conditions while tolerating withstand most inhospitable conditions while tolerating and detoxifying multiple contaminants simultaneouslyand detoxifying multiple contaminants simultaneously

2. No secondary waste generation2. No secondary waste generation

3. Useful for the high volume dilute wastes where the 3. Useful for the high volume dilute wastes where the trace contaminants ultimately limit the acceptability of trace contaminants ultimately limit the acceptability of final wastesfinal wastes

4. Economic4. Economic

Microbial diversity

Community structure Community function

Community dynamics

Biotransformation

Structural and functional diversity of Structural and functional diversity of microorganisms and bioremediationmicroorganisms and bioremediation

Bioremediation

Microbial interaction with organic Microbial interaction with organic and metallic contaminantsand metallic contaminants

Bioaccumulation / Biosorption/Bioprecipitatio

n

Metals

oxidized/reduced 

M2+

M2+M2+

M2+

M2+

Organic compounds

Biodegradation

Metals reduced/oxidized CO2, H2O, etc.

Biotransformation

Bioremediation: types and approaches

in situ and ex situ processes Intrinsic and Engineered processes

in situ and ex situ processes : Contaminants remain in place during the bioremediation or are excavated and transported to an above ground treatment system

Engineered bioremediation : employing engineering tools to greatly increase the input rates of the stimulating materials

Intrinsic bioremediation : relies on the intrinsically occurring rates of supply of substrates and nutrients as well as intrinsic population density of active microorganisms

The most important factor for any type of bioremediation is ensuring that the rate of biotransformation is first enough to meet the clean up objectives

Disadvantages : In situ processes are generally slower Mass transfer limitation and insufficient

distribution of substrates Difficult to implement in stratified soils that

hinder vertical distribution of air or other gases through contaminated zones

Inability to treat mixture of organic contaminants and metals

Bioremediation strategiesBioremediation strategies (in situ)

Intrinsic Bioremediation / Bioattenuation: Natural progress of biodegradation by indigenous microorganisms

Bioaugmentation :Introduction of microorganisms that have appropriate degradative abilities

Biostimulation : The intentional stimulation of resident bacteria to remediate the target chemicals by the addition of nutrients, water, electron donors and acceptors, etc.

Intrinsic Bioremediation

Intrinsic bioremediation occurs in situ and relies on naturally occurring biological processes carried out by indigenous microorganisms

This is a component of natural attenuation, which includes physical and chemical processes

Cleanup activities that rely on natural attenuation to reduce contaminant levels and monitoring to determine the remedial effectiveness are referred to as “monitored natural attenuation.”

To establish that intrinsic bioremediation is actually occurring at a sufficient rate in the subsurface, contaminant plume size and associated microbial activity (biodegradation and/or biotransformation) must be measured over a period of time

intrinsic bioremediation is mainly accepted for petroleum hydrocarbons and, to a limited degree, chlorinated hydrocarbons and recent time for metals and radionuclides as well

Biostimulation

For some contaminated sites, natural rates of biodegradation are inadequate

Biostimulation of indigenous microbial populations to remediate the target chemicals is employed

Natural degradative population exists within the contaminated zone but the proper environmental conditions are missing for microbial activity

Common environmental limitations

Excessively high waste concentration Lack of oxygen Unfavorable pH Lack of mineral nutrients Lack of moisture Unfavorable temperature

Variety of methods that modify environmental conditions can be employed to enhance rates of biodegradative activities by indigenous microbial polpulations

Biostimulation and Bioaugmentation

Biostimulation is the addition of nutrients (usually sources of carbon, nitrogen, and/or phosphorus), oxygen, or other electron donors or acceptors.

These amendments serve to increase the number or activity of naturally occurring microorganisms available for bioremediation.

Amendments can be added in either liquid or gaseous form, via injection.

Liquids can be injected into shallow or deep aquifers to stimulate the growth of microorganisms involved in bioremediation

Biostimulation

Oxygenation Nutrients Bioavailability

Biosparging is a type of soil venting, where air or other gases are injected below the ground into saturated sediments to minimize volatilization of contaminants, such as TCE.

Bioaugmentation

Bioaugmentation is the introduction of microorganisms that can biotransform or biodegrade a particular contaminant in a particular environment.Bioaugmentation by Dehalococcoides ethenogenes, a small obligate anaerobe that can reductively dechlorinate tetrachloroethylene to ethylene

Molecular breeding

Genetically engineered microorganisms

Adhesion deficient microorganisms

Bioaugmentation :

Molecular Breeding Molecular Breeding

Generation of spontaneous mutants with increased ability to utilize the xenobiotic compounds under a specialized enrichment procedure

Biochemical pathways are under constant evolution

Augmentation of evolution of new degradative pathways by feeding in to chemostat enrichment microorganisms hourboring the portion of desired biodegradative pathway

Exchange, recombination and amplification of genetic information under selective pressure along with spontaneous and induced mutation greatly accelerate this evolution

Genetic engineering solutions and benefitsGenetic engineering solutions and benefitsLimitation Solution BenefitIncomplete degradation

Uncoupling metabolism from degradations

Support activity with inexpensive non toxic substrates

Deregulate genetic controls Eliminate toxic –inducing substrateAchieve difficult cleanup

Low rate of degradation

Select high performance host organism

Use smaller les expensive bioreactors

Remove degradative bottlenecks Decrease fermentation/treatment costsRecalcitrant target compound

Add substitution specific functions Increase range of treatable compounds

Alter enzyme specificity Increase substrate range of a single organism

Formation of toxic intermediates

Reroute metabolites Extent treatment life

Add complementary activity /pathway

Extend range of treatable compounds

Chemical mixture Combined metabolic activitiesBroaden substrate specificity

Decrease treatment cost

Molecular engineering of microbes for improved degradative abilities

Adhesion deficient microorganismsAdhesion deficient microorganisms

Applicability of bioaugmentation is limited by the natural adhesive properties of native bacteria inhibiting their penetration through soil and rock matrices

Development and application of adhesion deficient bacteria greatly enhance their dispersion through soil matrices

Microbial interaction with Microbial interaction with organic compounds : organic compounds : Biodegradation Biodegradation

Biodegradation:Biodegradation: Biologically catalyzed reduction Biologically catalyzed reduction in complexity of organic in complexity of organic compoundscompounds

Basic facts ever-growing list of chemical contaminants released into

the environment on a large scale includes numerous aliphatic and aromatic compounds

petroleum hydrocarbons, halogenated and nitroaromatic petroleum hydrocarbons, halogenated and nitroaromatic compounds and phthalate esters are more dominantcompounds and phthalate esters are more dominant

These compounds enter the environment through many different pathsAs components of fertilizers, pesticides and herbicides some are distributed by direct applicationCombustion processes release polycyclic aromatic hydrocarbons (PAHs), dibenzo-p-dioxins and dibenzofurans.

The local concentration of a contaminant depends on

the amount present

the rate at which the compound is released

its stability in the environment under both aerobic and anaerobic conditions

the extent of its dilution in the environment

the mobility of the compound in a particular environment

its rate of biological or non-biological degradation

Two fundamental questions :Two fundamental questions :

how to dispose of the large quantities of waste how to dispose of the large quantities of waste that are continually being producedthat are continually being produced

how to remove the toxic compounds that have how to remove the toxic compounds that have been accumulating at dump sites, in the soil and been accumulating at dump sites, in the soil and in water systems?in water systems?

Practical considerations and challengesLack of stringent regulationIll defined / maintained waste dump sites

solid or liquid waste or botheither a single compound / mixture of closely related compounds / an unknown combination of unrelated substances

Technical and economic hurdles

• Ubiquitous, most abundant, highly adaptable, Ubiquitous, most abundant, highly adaptable, enormous genetic and metabolic diversityenormous genetic and metabolic diversity

• Microorganisms excel at using organic substances, Microorganisms excel at using organic substances, natural or synthetic, as sources of nutrients and energy natural or synthetic, as sources of nutrients and energy

• remarkable range of degradative abilities remarkable range of degradative abilities attributed to evolutionary coexistence of microbes with attributed to evolutionary coexistence of microbes with an immense variety of organic compounds of years with an immense variety of organic compounds of years with an immense variety of organic compoundsan immense variety of organic compounds

• The vast diversity of potential substrates for growth led The vast diversity of potential substrates for growth led to the evolution of enzymes capable of transforming to the evolution of enzymes capable of transforming many unrelated natural organic compounds by many many unrelated natural organic compounds by many different catalytic mechanisms.different catalytic mechanisms.

Microorganisms : An AssetMicroorganisms : An Asset

Depending on their behavior in the environment, organic compounds are often classified as

biodegradablepersistent recalcitrantBiodegradable : organic compound that undergoes

a biological transformationPersistent : organic compound does not undergo biodegradation in certain environmentsRecalcitrant : organic compound resists biodegradation in a wide variety of environments

• A microorganism must exist which has the A microorganism must exist which has the necessary metabolic capacity to bring about necessary metabolic capacity to bring about biodegradationbiodegradation

• Contaminant must be accessibleContaminant must be accessible– – If enzyme (s) involved in biodegradation is extracellular, If enzyme (s) involved in biodegradation is extracellular, bonds acted upon must be exposedbonds acted upon must be exposed– – If enzyme(s) involved in biodegradation is intracellular, the If enzyme(s) involved in biodegradation is intracellular, the Contaminant must be able to penetrate the cell membraneContaminant must be able to penetrate the cell membrane

• Environmental conditions must be conducive to Environmental conditions must be conducive to proliferation of biodegrading microorganismsproliferation of biodegrading microorganisms

Fundamental prerequisites for microbial Fundamental prerequisites for microbial degradationdegradation

Factors affecting biodegradation

Hydrocarbon Properties

Chemical composition Physical state Concentration Toxicity Bioavailability

Environmental Parameters

Temperature, Pressure pH, Salinity, Nutrients, Water potential Oxygen concentration Electron acceptors

Microbial Properties Specific catabolic

activity Catabolic diversity Biosurfactant production Population size

Basic Factors Regulating Rates of Organic Pollutant Biodegradation

Contaminant structure influences on bioavailability:

– Petroleum hydrocarbons: linear alkanes> branched alkanes > monoaromatics >polyaromatics

–Halogenated organics, pesticides, herbicides: presence/location of halogens, amine groups, methoxy groups, phenoxy groups affect efficiency of enzymatic attack

Biosurfactant catalyzed hydrocarbon Biosurfactant catalyzed hydrocarbon uptakeuptake

Hydrocarbon

Emulsification

Micelles formation

Contact and engulfment

Bacterium

Basic Factors Regulating Rates of Organic Pollutant Biodegradation

Availability of O2:-Growth rate /yield is always maximal with O2 as the

electron acceptor-Aerobes have mono- and –dioxygenase which are

uniquely effective in oxidation of hydrocarbons (especially aromatics)

-presence of O2 can suppress degradation of halogeneated pollutants through inhibition of reductive dechlorination

Availabilityy of inorganic nutrients :-N/P availability may limit biomass production in

presence of excess C

Sorption to solid-phases decreases availabilitySorption to solid-phases decreases availability

Solubility: many organic compounds (PAHs) are Solubility: many organic compounds (PAHs) are highly insoluble and therefore difficult for highly insoluble and therefore difficult for microbes to access; Key parameter= octanol-microbes to access; Key parameter= octanol-water partition coefficient (Kwater partition coefficient (Kowow))

Non aqueous phase liquid (NAPL) associations Non aqueous phase liquid (NAPL) associations can seriously hinder solubilization / can seriously hinder solubilization / biodegradation of otherwise relatively soluble biodegradation of otherwise relatively soluble organic contaminates (e.g chlorinated solvents)organic contaminates (e.g chlorinated solvents)

Characteristics of aerobic Characteristics of aerobic microorganisms capable of degrading microorganisms capable of degrading organic pollutantsorganic pollutants

Mineral oil constituents and halogenated products of petrochemicals are most important classes of organic pollutants

Aerobic organisms are most capable for biodegradtion of such compounds

Hydrocarbon

Central metaboilc pathways

Initial attack

Hydration/reduction etc.

CO2,, H2O, CH4Biosynthesis

Oxidized electron acceptor

O2, NO3, SO4, Fe3+, Humic acid, etc.

Reduced electron acceptor

H, NO3, SO4, Fe3+, Humic acid, etc.

Oxygenation

AerobiAerobicc

AnaerobiAnaerobicc

Biodegradation : Biodegradation : Aerobic and anaerobic Aerobic and anaerobic processesprocesses

Modes of biodegradationModes of biodegradation

Growth-associated:Growth-associated: Organic pollutants are used as sole Organic pollutants are used as sole source of carbon and energy – complete mineralizationsource of carbon and energy – complete mineralization

Co-metabolism:Co-metabolism: Metabolism of organic Metabolism of organic pollutants in presence of pollutants in presence of another growth substrate another growth substrate which is used as primary which is used as primary carbon and energy sourcecarbon and energy source

Primary Growth substrateCH4

CH4

CH3OH

NADH O2

NAD, H2O

Co-substrate TCE

TCE Epoxide

Breakdown to formate, CO2,

glyoxylateGrowth

Intermediary metabolism. Reducing power generation

MethaneMonooxygenase

Key enzymatic reactions

Oxygenases : oxidoreductase that use O2 to incorporate oxygen in to the substrate Degradative bacteria need oxygen at

two metabolic sitesAt the initial attack of the substrateEnd of the respiratory chain

Peroxidases :

Degradative pathways : Degradative pathways : Aliphatic Aliphatic breakdownbreakdown

n-Alkane/alkene/alkyne

alcohol

Aldehyde

Fatty acid

Acetyl CoA

oxygenationoxygenation

Alcohol dehydrogenaseAlcohol dehydrogenase

Aldehyde dehydrogenaseAldehyde dehydrogenase

oxidation

Intermediary metabolism

carboxylation

Hydration

Anaerobic Aerobic

Aerobic degradation of an alkane yields a fatty acid (top left). The appearance of 180 in the intermediates during aerobic biodegradation confirms that the one atom of oxygen introduced into the aliphatic compound comes from molecular oxygen, Aerobic degradation of alkenes and alkynes follows the same sequence. Anaerobic degradation of alkenes also leads to fatty acids (top right). Here the oxygen introduced into the aiiphatic compound comes from H20. Anaerobic degradation of alkynes follows the same path way. Akanes appear to be anaerobically recalcitrant. The fatty acids formed by either the aerobic or anaerobic processes are further oxidized by ~-oxidation, a common pathway for both aerobic and anaerobic microorganisms. [H] indicates reducing equivalentsthat are either required or formed in each reaction

Degradation of aromatic compoundsDegradation of aromatic compounds

Aerobic biodegradation of many classes of Aerobic biodegradation of many classes of aromatic compound is common and proceeds aromatic compound is common and proceeds through the key intermediate, catecholthrough the key intermediate, catechol

First step in benzene oxidation is a hydroxylation First step in benzene oxidation is a hydroxylation catalysed by a dioxygenase forming a diolcatalysed by a dioxygenase forming a diol

The diol is then converted to catechol by a The diol is then converted to catechol by a dehydrogenasedehydrogenase

This pathway of initial hydroxylation followed by This pathway of initial hydroxylation followed by dehydrogenation is common to other aromatic dehydrogenation is common to other aromatic hydrocarbonshydrocarbons

Aromatic compounds Benzene, phenol, toluene, aniline,

phenanthrene, anthacene, naphthalene, etc.

Catechol

Cis, Cis Muconate

Muconolactone

3-Oxoadipate enol lactone

3-Oxoadipate

Succinate Acetyl-CoA

O- Cleavage

Hydroxy muconic semialdehyde

2-oxo penta 4-enoate

4-Hydroxy-2-oxo-valeriate

Acetaldehyde Pyruvate

m- Cleavage

Degradative pathways : Degradative pathways : Aromatic breakdown 1Aromatic breakdown 1

Aerobic breakdown

Reactions involved in oxidation of Reactions involved in oxidation of toluene and related arenestoluene and related arenes

Hydroxylation of methyl groups to Hydroxylation of methyl groups to corresponding alcohol corresponding alcohol

Step wise oxidation up to carboxylic acidStep wise oxidation up to carboxylic acid Final oxidation by dioxygense followed by Final oxidation by dioxygense followed by

decarboxylation to catecholdecarboxylation to catechol

Degradative pathways : Degradative pathways : Aromatic Aromatic breakdown 2breakdown 2

Anaerobic breakdown

Degradative pathways : Degradative pathways : Aromatic Aromatic breakdown 3breakdown 3

Anaerobic breakdown

Degradative pathways: Degradative pathways: Halogenated and Halogenated and nitrated organicsnitrated organics

Reductive Reductive dehalogenationdehalogenation

Hydrogenolysis Dihaloelemination Coupling Hydrolytic reduction

Nitro-eliminationNitro-elimination Oxygenation yielding nitrites reduction yielding aromatic amines Reductive elimination of nitro group yielding nitrites Partial reduction of nitro groups to hydroxyl amine

Major organic pollutants in contaminated Major organic pollutants in contaminated sitessites

Aliphatic hydrocarbons: n-alkanes, alkenes, alkynes, cycloaliphatics, ethers

Aromatic/polyaromatic: Benzene, Toluene, Ethylbenzene, Xylene (BTEX), hydrocarbons Phenol, Naphthalene, Anthracene, Phenanthrene, Fluoranthene, Pyrene, Chrysene, Benzanthracene

Halogenated aliphatics: Trichloroethylene (TCE), Tetrachloroethylene, Ethylenedibromide, etc.

Halogenated aromatics: Polychlorinatedbiphenyls (PCB), Pentachlorophenol, Dichlorobenzene, Chlorophenoxyacetates, etc.

Nitroaromatics: Nitrophenols, Nitrobenzenes, Nitrotoluenes

Pesticides/Herbicides: Organophosphorous/organochlorine/phenolic compounds

Typical bioremediation reactions for organic Typical bioremediation reactions for organic contaminants in contaminated aquiferscontaminants in contaminated aquifers

Different zones of different degradative processes

Predominant hydrocarbon degrading Predominant hydrocarbon degrading microorganisms and enzymesmicroorganisms and enzymes

MicroorganismsPseudomonas spp.Acinetobacter spp.Alcaligenes spp.Rhodococcus spp.Sphingomonas spp.Bacillus spp.Mycobacterium spp.Nocardia spp.Arthrobacter spp.Xanthomonas spp.Comamonas spp.Burkholderia spp.Aeromonas spp.

Enzymes

DioxygenaseAlkane hydroxylasePhenol hydroxylaseSalicylate hydroxylase-Hydroxy-6-oxo-6-(20-aminophenyl)-hexadienoate hydrolase

HydroxylasesCatechol oxygenaseNapthalene dioxygenaseToluene dioxygenaseBiphenyl dioxygenase Monooxygenase

Toluene monooxygenaseXylene monooxygenase Ethene monooxygenase Fluorene monooxygenase Cyclohexanone monooxygenase

Molecular tools for in situ detection of catabolic genes

Enzyme /protein Gene Detection method

Alkane hydroxylase alk B PCR ProbesPhenol hydroxylase Dmp PCR, cPCRNaphthalene dioxygenase

pahAc, PhnAc PCR, probing

Catechol 2,3- dioxygenase

Xyl E PCR, probing

Ammonia monooxygenase

amoA PCR Cloning

Denitrification pathway Nir S and others RT PCR, CloningMethane monooxygenase

mmoX PCR Cloning

Detection by microarrays of biodegradation genes in genomes of reference microorganisms used

CometabolismTransformation of an organic compound by a microorganism which is unable to use the compound as a source of energy or as a substrate for biosynthesis

Classic example: conversion of 2,4-D to 2,4-dichlorophenol and 3,5 dichlorocatechol, discovered during search for intermediateds in 2,4-D degradation

Figure 13.1, Conversion of 2, 4-D to 2,4-dichlorophenol and 3,5-dichlorocatechol

Alexander (1999)

Cometabolism

- Trichlorethylene and - Oxygenases

tetrachloroethylene - Dehalogenases

- Chlorophenols - Hydroxylases

- Halobenzoates - Phosphatases

- Nitrobezenes - Dehydrogenases

- Various chlorinated - Deaminases

pesticides

Substrates: Enzymes:

Synergism

Interaction in which two or more microorganisms carry out a pollutant transformation that neither of which can perform alone; or in which

Biodegradation carried out by a multispecies mixture is more rapid than the sums of the rates of reactions that could be effected by the separate species

Can involve

(i) multiple organisms all gaining energy for growth from partial metabolism of compounds

(ii) combination of cometabolism and energy-generating biodegradation

Synergism Coupled to Cometabolism

Two-member associations in which the second species grows on the product of cometabolism of TCE (Uchiyama et al., 1992), DDT (Pfaender and Alexander, 1972),

Plasmid: an extrachromosomal genetic element that is not essential for growth and has no extracellular from

Plasmid-Born BiodegradationPlasmid-Born Biodegradation

Many genetic systems associated with organic pollutant biodegradation are plasmid-born

Plasmid-Born Biodegradation Plasmid-Born Biodegradation

Compounds for which plasmid-born degradative genetic/enzyme systems exist:

- Alkyl benzyl sufonatesAlkyl benzyl sufonates

-monoaronatics (e.g. Benzoate, Phenol)monoaronatics (e.g. Benzoate, Phenol)

- chlorinated monoaromatics (e.g. Chlorobenzoate, Chlorophenols)chlorinated monoaromatics (e.g. Chlorobenzoate, Chlorophenols)

- Toluene and Benzene Toluene and Benzene

- Chlorobenzenes Chlorobenzenes

- Alkanes and AlkenesAlkanes and Alkenes

- Chlorinated alkanes and alkenesChlorinated alkanes and alkenes

- PAHsPAHs

- PCBsPCBs

Reductive Dechlorination

Chlorinated monoaromatics, chloroethanes, and choroethenes can be used as electron acceptors for anaerobic microbial respiration (see Madigan et al table 17.5)- contrast with aerobic dechlorination coupled t pollutant biodegradation

Basic reaction

R-CL + 2e- + 2H+ RH + H+ + CL-

Reactions may in some cases be associated with generation of energy for growth; other not

Reductive Dechlorination

Classic example: reductive dechlorination of chloroenzoate by Desulfomonile tiedjei

C7H4O2CL- + 2e- + 2H+ C7H5O2- + H+ +CL-

2-Chlorbenzoate Benzoate

Reductive Dechlorination

More complex chlorinated compounds such as polychlorinated biphenyls (PCBs) are also subject to reductive dechlorination

Loss of CL- groups can make compound more succeptible to biodegradation

Sequential aerobic/anaerobic shifts may enhance biodegradation of highly recalcitrant polychlorinated contaminants; process may be responsible for degradation of PCBs in contaminated riverine sediments (e.g. Hudson River )

Microbial interaction with metals Microbial interaction with metals and radionuclides : and radionuclides : Bioremediation and Bioremediation and Biotechnological applicationsBiotechnological applications

Remedial goals can be achieved by:

1. the precipitation and thus immobilization of inorganic contaminants

2. the concentration and thus reduction in volume of contaminated matrices

3. the compartmentalization of metals to a part of the environment in which their harm is reduced

Bioaccumulation & Biosorption

M oxidized/reduced

 

M2+M2+

M2+M2+ M2+

M2+

Biotransformation

Extracellular complexation/precipitation

(polysaccharides, metallothionein, siderophore)

Organo-metallic compounds/metal-cyanide complex

Metal/radionuclide –Microbe Metal/radionuclide –Microbe InteractionsInteractions

BiodegradationMreduced/oxidized

CO2, H2O

Fate of accumulated metalsFate of accumulated metals

EffluxEfflux

IntracellularIntracellularCompartmentationCompartmentation/complexation/complexation

EnzymaticEnzymaticconversionconversion

Biosorption:Biosorption: Metabolism independent Metabolism independent physicochemical uptake of metal ionphysicochemical uptake of metal ion

M+M+

M+M+

M+

M+M

+

M+ M+

M+

M+

M+

M+

M+ M+

M+

M+

GroupGroup LocationLocationCarboxylCarboxyl Uronic acidUronic acidSulphonateSulphonate Cysteic acidCysteic acidPhosphatePhosphate PolysaccharidesPolysaccharidesHydroxylHydroxyl Tyrosine-phenolicTyrosine-phenolicAminoAmino CytidineCytidineIminoImino PeptidePeptideImidazoleImidazole HistidineHistidine

Ion ExchangeIon Exchange : Binding of metal : Binding of metal ions with stoichiometric release of ions with stoichiometric release of a previously bound ion – over all a previously bound ion – over all neutrality maintainedneutrality maintained

Adsorption & microprecipitationAdsorption & microprecipitation : : Binding of electrically neutral Binding of electrically neutral material without involving release material without involving release of any stoichiometric amount of of any stoichiometric amount of previously bound ionpreviously bound ion

Microbially catalyzed redox transformationsMicrobially catalyzed redox transformations

Oxidized Oxidized metals metals /radionuclides and sulfate /radionuclides and sulfate U(VI), Tc(VII), Cr(VI)U(VI), Tc(VII), Cr(VI)

Reduced Reduced metals/radionuclidesmetals/radionuclidesU(IV), Cr(III), Tc(0), HU(IV), Cr(III), Tc(0), H22SS

Reduced Reduced metals/radionuclides, metal metals/radionuclides, metal sulfides U(IV), CdS, ZnSsulfides U(IV), CdS, ZnS

Oxidized Oxidized metals metals /radionuclides and sulfate /radionuclides and sulfate U(VI), CdSOU(VI), CdSO44, CuSO, CuSO44

ReductionReduction OxidationOxidation

M + H2S = MS

Microbially catalyzed redox reaction Microbially catalyzed redox reaction that lead to metal mobilization : that lead to metal mobilization : BioleachingBioleaching

Metal oxidizing bacterium

Reduced metals, metal sulfides

Oxidized metals, metal sulfate, H2SO4

e-

O2, CO2H2OOrganic matter

Direct oxidationDirect oxidationBacterial cells are in physical contact with mineral Bacterial cells are in physical contact with mineral surface surface FeS2 + O2+ H2O Fe2(SO4)3 + H2SO4

BacteriaMeS + O2 MeSO4 + O2 Covelite CuS, Chalcocites Cu2S, Sphalerite ZnS, Galena PbS, Cobaltite CoS, Millerite NiS, etc. can be used by the metal oxidizing strains

Indirect oxidationIndirect oxidationBacterial cells generate a lixiviant that chemically Bacterial cells generate a lixiviant that chemically oxidizes the sulfide mineraloxidizes the sulfide mineral MeS + Fe2(SO4)3 MeSO4 + FeSO4 + S

Metal/radionuclide Metal/radionuclide complexation/precipitation by extracellular complexation/precipitation by extracellular microbial productsmicrobial products

Polysaccharides :

Polysaccharides : Extremely hydrated polymeric anionic carbohydrate substances Alginic acids, Chitin/Chitosan ; Pseudomonas spp., Algae, Fungi Siderophores :Low molecular weight iron binding molecules ; Enterobactin, Pyochelin, Pseudobactin etc. ; E.coli, Pseudomonas Proteins/peptides :Copper/ cadmium binding proteins ; Vibrio alginolyticus ;Pseudomonas putida. Organic acids : Gluconic acid, protocatechuic acid, oxalic acid;Aspergillus niger, Penicillium sp.

Inorganic ligands : Produced via enzymatic breakdown of organic phosphates;Citrobacter sp. Pigments :Melanin, Tanin, Flavin; Aureobasidium sp. Saccharomyces sp. Candida sp.

Metal/radionuclide complexation/precipitation by Metal/radionuclide complexation/precipitation by extracellular microbial productsextracellular microbial products

Inorganic ligandInorganic ligandMetal Phosphate precipitationMetal Phosphate precipitation

Organic phosphate (GP, TBP, etc.)

HPO42- + Metal

Metal phosphate

Phosphatase*

Microbially Enhanced Microbially Enhanced Chemisorption of MetalsChemisorption of Metals Flowthrough Bioreactor system Flowthrough Bioreactor system developed to remove and recover developed to remove and recover U, Th, Pu, Am, Np, Cd, Cu, Ni U, Th, Pu, Am, Np, Cd, Cu, Ni ( ~900g g( ~900g g-1-1 metal loading). metal loading).

P-limiting condition

Pseudomonas aeruginosa

MetalPolyphosphate kinase

Citrobacter sp.

Polyphosphate bodies

Precipitation- immobilization of contaminantsPrecipitation- immobilization of contaminants

Concentration-contaminated waste volume reductionConcentration-contaminated waste volume reduction

Compartmentalization of metals to a part of Compartmentalization of metals to a part of environment in which their harm is reduced environment in which their harm is reduced

Biodegradation - mineralization of organic Biodegradation - mineralization of organic contaminants and reduction of their mobility/toxicitycontaminants and reduction of their mobility/toxicity

How to achieve remedial goal ?How to achieve remedial goal ?

Self replenishingContinuous metabolic uptake of metals is

possibleSimultaneous removal of co contaminants

possiblePotential for optimization through

development of resistant species

Bioremediation with Living cells :Bioremediation with Living cells :

Mender System - Algae & Bacteria - Lead Mine EffluentAlgal Pond System- Cyanobacteria & Bacteria - Uranium Mine Effluent-Use of metal resistant bacteria : Use of metal resistant bacteria : Operates at high metal concentrationCSTR system : Bacterial consortia - Ag, Cu & Cd removalContinuous Flow system : Pseudomonas strains- Hg- removal

Natural isolates :Natural isolates : Operates at subtoxic metal concentration

Genetically engineered strains for Genetically engineered strains for improved metal sequestrationimproved metal sequestration

Use of metallothioneinUse of metallothionein

metallothionein

Nix A Ni2+Ni2+Cd2+

Cd2+

Cd2+

Cd2+

In situIn situ approach for metal immobilization approach for metal immobilization in soil using designed bacteriain soil using designed bacteria

3. same soil inoculated with engineered R. metalliduransdisplaying MT on cell surface

1. Cd2+ -polluted peat soil2. same soil inoculated with wild R. metallidurans

Use of novel metal binding ligands : Use of novel metal binding ligands :

Modified metallothioneinsModified metallothioneinsTandem repeats of Tandem repeats of N. crassaN. crassa MT expressed in MT expressed in E. coli E. coli showed ~7 fold increase in Cdshowed ~7 fold increase in Cd2+ 2+ removalremoval.

Coding sequence for Mtt1 monomer

Metallothionein oligomer assembly, n = 0-12

Protein designing and selection of novel metal-binding Protein designing and selection of novel metal-binding peptides for increased ligand stability and peptides for increased ligand stability and affinity/selectivity towards heavy metalsaffinity/selectivity towards heavy metals

Synthetic peptides :Synthetic peptides :

Tandem repeat of peptide Tandem repeat of peptide Cys-Gly-Cys-Cys-Gly Cys-Gly-Cys-Cys-Gly exhibited high exhibited high HgHg2+2+ and Cd and Cd2+ 2+ binding in binding in E. coliE. coli

Poly histidine residues Poly histidine residues Gly-His-His-Pro-His-Gly Gly-His-His-Pro-His-Gly expressed in expressed in E. E. colicoli showed a 12-15 fold high metal binding showed a 12-15 fold high metal binding

Synthetic phytochelatins (Synthetic phytochelatins (Glu-CysGlu-Cys))22 Gly Gly (n = 2-11) displayed (n = 2-11) displayed

over bacterial surface showed a 12-20 fold high Hgover bacterial surface showed a 12-20 fold high Hg2+2+ and Cd and Cd2+ 2+

binding binding

Expression of a Cd-binding peptide on the surface of E. coli

USE OF MERCURY REDUCTASE SYSTEMUSE OF MERCURY REDUCTASE SYSTEM

Mer P

Mer T

Hg2+

Hg2+Mer A reductase

Organo- Hg Lyase

Hg0

Organic HgVolatile Hg

merR OP merT merP merA merB

Cytoplasm

Cell envelop

Engineered radioresistant Deinococcus radiodurans (in-situ bioremediation)Engineered E. coli (ex-situ bioremediation)Engineered higher plants (phytoremediation)

1 neutralization tank; 2 bioreactor; 3 activated carbon filter, 4 bioreactor inflow valve; 5 control of bioreactor inflow valve; 6 bypass; 7 sodium hydroxide tank; 8 medium tank; Hg automated continuous mercury measurement; O2 oxygen probe; c conductivity probe; Cl2 chlorine probe; pH pH-probe; r redox potential probe; T temperature measurement. Source : Dobler et al, 2000. Env. Sci. Technol 34. 4628

Scheme of pilot plant for microbial Scheme of pilot plant for microbial mercury remediationmercury remediation

Application of biosorptionApplication of biosorption

Biosorption can concentrate metals/ radionuclides several Biosorption can concentrate metals/ radionuclides several thousand fold : recovery of precious metals from sea water and from thousand fold : recovery of precious metals from sea water and from process solutionprocess solutionBiosorption is particularly suited as a polishing step : Wastewater Biosorption is particularly suited as a polishing step : Wastewater with low-medium metal concentration (< 100 ppm) is purified to with low-medium metal concentration (< 100 ppm) is purified to drinking water qualitydrinking water quality

High effluent qualityHigh effluent quality

High efficiency of metal / radionuclide removalHigh efficiency of metal / radionuclide removal

No toxic secondary waste generationNo toxic secondary waste generation

Broad operating conditions (pH, Temp. metal conc. other ions. etc.Broad operating conditions (pH, Temp. metal conc. other ions. etc.

Cost effectiveCost effective

Application of metal transformationApplication of metal transformation

•BioremediationBioremediation•Mineral processingMineral processing

Bioremediation :Metal precipitation by DMRB and SRBMetal precipitation by DMRB and SRB

Biotechnological potential of SRBBiotechnological potential of SRB Sulfides of many environmentally important metals are insoluble. Sulfides of many environmentally important metals are insoluble.

Sulfide precipitation is effective over broad pH rangeSulfide precipitation is effective over broad pH range

They can utilize a wide range of organics as carbon source. They can utilize a wide range of organics as carbon source. Simultaneous degradation of organics and removal of toxic metals Simultaneous degradation of organics and removal of toxic metals is possible is possible (Benzoate, phenol, catechol, (Benzoate, phenol, catechol, EDTA, NTA, TBP , etc. EDTA, NTA, TBP , etc. degradation and radionuclide /metal precipitation)degradation and radionuclide /metal precipitation)

Removal of sulfate reduces acidity and salinityRemoval of sulfate reduces acidity and salinity

Microbes in metal environment

Application in mineral processing :Application in mineral processing : Recovery of metals from oresRecovery of metals from ores

Treatment of metal & radionuclide containing wastes Treatment of metal & radionuclide containing wastes

Applicable to low grade ores (0.03%-0.3%)Applicable to low grade ores (0.03%-0.3%) In situIn situ bioleaching saves the cost of bringing vast amount of bioleaching saves the cost of bringing vast amount of

ores outores out Possible to recover metals/radionuclides from recalcitrant oresPossible to recover metals/radionuclides from recalcitrant ores Controlled bioleaching and recovery of metals/radionuclides Controlled bioleaching and recovery of metals/radionuclides

results in minimum environmental pollutionresults in minimum environmental pollution

Advantages of biomining/bioleachingAdvantages of biomining/bioleaching

Metal/radionuclide bioleached using Thiobacillus ferrooxidans

Amount recovered (Tons)

   

Uranium 300 (annually, only in US)

Copper 1000000 (annually, world wide)

Gold 115-150 (per day, South Africa & Australia)

Combination of biooxidation and bio Combination of biooxidation and bio precipitation for metallic waste treatmentprecipitation for metallic waste treatment

Bioleaching Stage :Sulfur oxidizing bacteriaM-sulfide (insoluble) ------M-sulfate( Soluble)

Contaminated Soil

Bioprecipitation Stage :Sulfur Reducing BacteriaM-sulfate( Soluble)------M-sulfide (precipitate)

Soil leachate

Solid metal sulfides Metal free effluent

Biotechnological applicationBiotechnological applicationDevelopment of biopolymer based metal sorbents Designer polymer synthesis through polymer engineering for specific / high recovery of valuable and strategic metals Source of polymer Metal binding capacity Optimum conditions

 

Zoogloea ramigera (zooglan)

880 mg U/g dry weight323 mg Cu / g223 mg Cd / g

Flocculative processBatch process effective

Acenetobacter(engineered polymer)

>800 mg U / g Efficient sorption-desorption process

Rhizopus arrhizus (chitin, chitosan)

180 mg U / g 8-10 sorption-desorption cycle

Siderophore based metal/radionuclide removalSiderophore based metal/radionuclide removal

Plutonium removal by siderophore (Desferrioxamine) Plutonium removal by siderophore (Desferrioxamine) of of Microbacterium flavescensMicrobacterium flavescens

Immobilization of biomassImmobilization of biomass

Advantages: Advantages: • Enhanced microbial cell stabilityEnhanced microbial cell stability• Easy separation and reuse of biosorbentEasy separation and reuse of biosorbent• Continuous process operationContinuous process operation

Criteria of good immobilization• High sorption capacity (minimum matrix) • Fast kinetics (hydrophilic, high porosity) • Smooth flow dynamics (minimum pressure drop)• Recovery, regeneration and reuse• Cost effective

Pseudomonas cells

Biobeads : Immobilized cells

Free and Immobilized Pseudomonas biomass

Cross sectional view

Surface view

Scanning Electron Micrographs of Biobead

Metal resistance genes in biosensor developmentMetal resistance genes in biosensor developmentMetal resistance genes are tightly regulated and Metal resistance genes are tightly regulated and induced specifically in presence of particular metal induced specifically in presence of particular metal

Lights off : non specific Lights on : specific

The promoter and regulator genes of metal resistance operon fused with suitable reporter gene enable to produce signal specifically against the target metal

Highly specific biosensors are developed for Hg, Cr, Al, Ni, Cd, As, etc.

Metal accumulating microorganism in Metal accumulating microorganism in NanotechnologyNanotechnologyMetal accumulating microorganism can be used as a tool Metal accumulating microorganism can be used as a tool to produce nanoparticles and their assembly for the to produce nanoparticles and their assembly for the construction of new advanced materialsconstruction of new advanced materials

TEM of Pseudomonas stutzeri AG259 cell grown on a 50 mM Ag+ containing agar substrate. Silver-based single crystals with different size and morphology are associated with the cell.

A variety of silver (Ag) crystal morphologies. The particles are a selection of different crystal morphologies that are found in whole cell preparations and thin sections.

SEM of a cross section of a thinfilm on a glass substrate. The film is prepared from the biomass of theAg-accumulating bacterial strain Pseudomonas stutzeriAG259following heat treatment for 1h at 400 ｰ C. Small granular silver particles are embedded in the carbonaceous host matrix.

Source : Klaus-Joerger et al, 2001, TIBTECH, 19, 15

Liquid outlet

Soil to drying

Temperature control

AgitatorVapor out

Air inlet

Nutrient

Contaminated soil

Contaminated liquid

Bioremediation strategyBioremediation strategy (ex situ) Bioreactor operation

Microorganism

Bioremediation strategiesBioremediation strategies (ex situ)

Biopiling

Land farming

Composting

The need for designed organismsThe need for designed organisms

6. Improving the process relevant properties of 6. Improving the process relevant properties of microorganismsmicroorganisms

Bacteria designed for bioremediationBacteria designed for bioremediation

1. Creating new metabolic routes1. Creating new metabolic routes

2. Expanding the substrate range of existing pathways2. Expanding the substrate range of existing pathways

3. Avoids substrate misroute in to unproductive routes or to 3. Avoids substrate misroute in to unproductive routes or to toxic intermediatestoxic intermediates

4. Increased genetic stability of catabolic activities4. Increased genetic stability of catabolic activities

5. Increasing the bioavailability of hydrophobic pollutants5. Increasing the bioavailability of hydrophobic pollutants

Bacteria designed for bioremediation Bacteria designed for bioremediation cont.cont.

Strategies for designing new/improved Strategies for designing new/improved organism /systemorganism /system

1. Making of a consortium through the addition of 1. Making of a consortium through the addition of ‘specialist’ organisms‘specialist’ organisms2. Molecular breeding2. Molecular breeding3. Development of combined aerobic-anaerobic processes ( 3. Development of combined aerobic-anaerobic processes ( for PCB degradationfor PCB degradation))4. Genetic engineering4. Genetic engineering

Bioremediation process developmentBioremediation process development

Site characterization

Treatment evaluation

Process scale up

• Pollutant • Environmental • Microbiological

• Biotreatability studies • Process evaluation

Process efficacy evaluationProcess monitoring

Investigation and optimization of microbial Investigation and optimization of microbial remediationremediation

Samples

Determination of biodegradation potential

Structural and functional analysis of microbial communitiesIdentification and characterization of functionally important microbial population

Identification and characterization of catalytically important enzymes

Optimization of biodegradation processes

in situ process developmentex situ process development

Full scale bioremediation

Experimental approachExperimental approachEnvironmental sample

Culture independent methods

Culture dependent methods

Pure culture

Extraction of community DNA

PCR

Cloning

Amplification of the cloned rRNA gene

Sequence analysis

Phylogenetic analysis

Purification

Purification

Confirmation of insert size

Selection of clones

ARDRASelection of dominant OTUs

Sequencing

PCR

Cloning

Microbial diversity

Physiochemical analysis

Molecular analysis of Molecular analysis of microbial diversitymicrobial diversity

The great plate count anomaly

0.01 – 1% of total bacteria are cultivable

~50% of total bacterial phyla do not have any cultivable representative

Our understanding of microbial diversity is not represented by the cultured fraction of the diversity

The cultured microorganisms represent only a small fraction of natural microbial communities and hence the microbial diversity in terms of species richness and species abundance is grossly underestimated

Isolation of community DNAIsolation of community DNA

A 13 121110 9 8 6 5 4 3 2 B

10 kb 4 kb 2 kb

1kb

500 bp

1.5kb

850bp400bp200bp 50 bp

Sample 1

Community DNA isolated from different Community DNA isolated from different samplessamples

PCR amplification of 16S PCR amplification of 16S rRNA generRNA gene

PCR protocolPCR protocol

Temperature program: 30 cycle: 940 C for 5 min, 940 C for 30 sec, 580 C for 30 sec, 720 C for 30 sec, 720 C for 7 min

Performed in 50µl reaction volume

M 2 5 6 7 8 9 10 11 13 15 16 17 18 19 21 22

PCR Sample No .2

1.5 kb

CloningCloning

The vector used for cloningThe vector used for cloning

pGEM®-T Easy Vector pGEM®-T Easy Vector circle mapscircle maps

The promoter and multiple cloning sequence of the pGEM®-T Easy Vector. The top strand of the sequence shown corresponds to the RNA synthesized by T7 RNA Polymerase. The bottom strand corresponds to the RNA synthesized by SP6 RNA Polymerase.

Cloning of 16s rDNACloning of 16s rDNA

Amplified ribosomal DNA from three samples [Bagjata Amplified ribosomal DNA from three samples [Bagjata (2-(2-10), 10), Turamdihi Turamdihi (1-12)(1-12) and Jadugoda and Jadugoda (1-5(1-5 ))] were selected for ] were selected for cloningcloning

All three samples were cloned using pGEM-T Easy vector All three samples were cloned using pGEM-T Easy vector system using system using E. coliE. coli- JM109 and positive clones were - JM109 and positive clones were selectedselected

Insert size within these clones were checked (initially with Insert size within these clones were checked (initially with plasmid isolation and restriction digestion) by colony PCR plasmid isolation and restriction digestion) by colony PCR using vector specific SP-6 and T-7 primers using vector specific SP-6 and T-7 primers

Results of the colony PCR of selected clonesResults of the colony PCR of selected clones

1.5 kb 850bp 400bp

200bp

50bp

M 1 2 3 4 5 6 7 8 9 101% agarose Gel:Run time: 40 min,Volt: 80V1-49 Different colony, M= DNA Rular

1.5kb

850bp400bp200bp50bp

11 12 15 20 21 24 25 26 29 28 30 44 47 49 M

1 2 3 4 5 6 7 8 9 10 M 11 12 13 14 15 161718 19 20 21 22 232425 26272829 30 31 32 33 34 35 M

1-35 different colony from sample 5

1% agarose gel. 40mins. Volt: 80V.

(sample-1- 12 and 1-5)(sample-1- 12 and 1-5)

Amplified Ribosomal DNA Amplified Ribosomal DNA Restriction Analysis Restriction Analysis (ARDRA)(ARDRA)

RsaI digestionThe lane number corresponds to the colony no. M-low range marker

M 2 3 4 5 6 7 8 10 1213 1415 16 17 18 19 20 21 22 23 24 2526 27 28 29 30 31 32

1.5kb

850bp

400bp

200bp

50bp

ARDRA pattern of 1-5ARDRA pattern of 1-5

1.5kb

850bp

400bp

200bp

50bp

M 2 3 4 5 6 7 8 10 1213 1415 16 17 18 19 20 21 22 23 24 2526 27 28 29 30 31 32

ARDRA pattern of 1-5ARDRA pattern of 1-5

SummarySummary of 1-5 resultsof 1-5 results Total no of clones selected :Total no of clones selected : 73 73

Number of OTUs :Number of OTUs : 4747

Frequency plots of different operation taxonomic units Frequency plots of different operation taxonomic units (OTU’s)(OTU’s)

Sequencing 16S rRNA genes Sequencing 16S rRNA genes clonesclones

Serial no

Original clone no

Clone ID

1 14 A22 5 A33 8 A44 29 A55 10 A66 2 A77 60 A88 36 A99 44 A1010 3 A1111 20 A1212 25 A1413 31 A1514 40 A16

Serial no

Original clone no

Clone ID

15 56 A1716 57 A1817 77 A1918 78 A2019 8 B120 25 B221 4 B322 1 B423 2 B524 5 B625 63 C226 65 C327 70 C428 72 C5

Clones selected for sequencingClones selected for sequencing

Sequence alignment Sequence alignment

Matching the databasesMatching the databases

Ribosomal Database Project IIRibosomal Database Project II

Phylogram of B1 clonePhylogram of B1 clone

Similarity search in NCBISimilarity search in NCBI

NCBI

Clone ID

Phylum Class Order Family GenusA2 Gemmatimo

nadetesGemmatimonadetes

Gemmatimonadales

Gemmatimonadaceae

Gemmatimonas

A3 Proteobacteria

Eammaproteobacteria

Ehromatiales Ectothiorhodospiraceae

Alkalispirillum

A4 Proteobacteria

Betaproteobacteria

Burkholderiales Incertae sedis Leptothrix

A5 Bacteroidetes

Sphingobacteria Sphingobacteriales

Flexibacteraceae Arcicella

A6 Gemmatimonadetes

Gemmatimonadetes

Gemmatimonadeles

Gemmatimonadaceae

Gemmatimonas

A7 Proteobacteria

Alphaproteobacteria

Rhodospirillales Acetobacteriaceae

Acidisphaera

A8 Proteobacteria

Gammaproteobacteria

Aeromonadales Succinivibrionaceae

Succinivibrio

A9 Proteobacteria

Alphaproteobacteria

Sphingomonadales

Sphingomonadaceae

Erythromicrobium

A10 Proteobacteria

Betaproteobacteria

Burkholderiales Incertae sedis Leptothrix

Summary of the phylogenetic analysesSummary of the phylogenetic analyses

Clone ID

Phylum Class Order Family GenusA12 Proteobacte

riaDeltaproteobacteria Bdellovibrional

esBdellovibrionaceae

Bdellovibrio

A14 Proteobacteria

Deltaproteobacteria Myxococcales Cystobactericeae

Anaeromyxobacter

A15 Proteobacteria

Gammaproteobacteria

Alteromonadales

Incertae sedis Alishewanella

A16 Bacteroidetes

Flavobacteria Flavobacteriales

Cryomorphaceae

Cryomorpha

A17 Bacteroidetes

Gammaproteobacteria

Oceanospirillales

Saccharospirillaceae

Saccharospirillum

A18 Bacteroidetes

Sphingobacteria Sphingobacteriales

Flexibacteriaceae

Sporocytophaga

A19 Acidobacteria

Acidobacteria Acidobacteriales

Acidobacteriaceae

Acidobacterium

A20 Proteobacteria

Betaproteobacteria Burkholderiales Incertae sedis Schlegelella

Summary of the phylogenetic analyses Summary of the phylogenetic analyses cont.cont.

Phylogenetic relationship among the dominant clones from 1-5Phylogenetic relationship among the dominant clones from 1-5

Lovely (2003) Nature reviews Microbiology

Microbial functions at contaminated Microbial functions at contaminated environment : Genome enabled modelenvironment : Genome enabled model

Molecular approaches for detection and identification of xenobiotic-degrading bacteria and their catabolic genes from environmental samples

The diversity of microbial biocatalytic processes can be exploited for : Bioremediation of toxic organics, metals and radionuclides Bioprospecting for novel bioprocesses Nanobiotechnolgy Astrobiology Bioleaching/biomining of metals and radionuclids

Conclusion :Conclusion :

Screening and molecular characterization of microorganisms from contaminated sites

Analyses of catalytic genes involved in contaminants biotransformation

Metagenomics and in silico biology Application of genetic and polymer engineering Collaboration between microbiologist, environmental/chemical

engineers and geochmists for effective bioremediation

The way ahead :

Screening and molecular characterization of microorganisms from Screening and molecular characterization of microorganisms from contaminated sitescontaminated sites

Analyses of catalytic genes involved in contaminant Analyses of catalytic genes involved in contaminant biotransformationbiotransformation

Metagenomics and Metagenomics and in silicoin silico biology biology

Application of genetic and polymer engineeringApplication of genetic and polymer engineering

Collaboration between microbiologist, environmental/chemical Collaboration between microbiologist, environmental/chemical engineers and geochmists for effective bioremediation engineers and geochmists for effective bioremediation

The way ahead :The way ahead :

A few examples :A few examples :

Groundwater remediation at NetherlandsGroundwater remediation at NetherlandsZinc contaminated groundwater was treated with SRB and methonogens in a 1800 m3 concrete reactor

Pilot scale selenium bioremediation by Pilot scale selenium bioremediation by Thauera selenatis Thauera selenatis at California at California Using packed bed reactors selenium contaminated drainage water was treated to ppb level

Cleanup program for US DOE sites Cleanup program for US DOE sites contaminated with a huge number of metals contaminated with a huge number of metals and radionuclides (U, Tc, Pu, etc, ) is currently and radionuclides (U, Tc, Pu, etc, ) is currently carried outcarried out