Amit Kumar Sharma Shraddha Sontakke Abhishek Chiyyeadu Mangesh Gaikwad Deepak Sonawane Shyamal Karanjkar

Pollutants
naturally-occurring compounds in the environment that are present in unnaturally high concentrations. Examples:
crude oil refined oil phosphates heavy metals

Xenobiotics
chemically synthesized compounds that have never occurred in nature.
Examples:
pesticides herbicides plastics

Readily degradable ____________ _

Somewhat Difficult to Generally degradable degrade recalcitrant _____________ _____________ _____________

chlorinated solvents (TCE) some pesticides and herbicides dioxins polychlorinated biphenyls (PCB)

fuel oils, gasoline creosote, coal tars ketones and alcohols monocyclic aromatics bicyclic aromatics (naphthalene) pentachlorophenol (PCP)

The use of bacteria and fungi and plants to break down or degrade toxic chemical compounds that have accumulated in the environment Requirements

Microorganism Plants - -Phytoremeditaion Fungi Mycoremediation Substrates and nutrients Electron acceptors

In situ

Ex situ

type: biosparging, bioventing, bioaugumentation, in situ biodegradation benefits: most cost efficient, noninvasive, relatively passive, natural attenuation process, treats soil and water limitations: environmental constraints, extended treatment time, monitoring difficulties factors to consider: biodegradative abilities of indigenous microorganisms, presence of metals and other inorganics, environmental parameters, biodegradability of pollutants, chemical solubility, geological factors, distribution of pollutants
efficient, low cost, can be done on site limitations: space requirements, extended treatment time, need to control abiotic loss, mass transfer

problem, bioavailability limitations type: landfarming, composting, biopiles

benefits: cost

Bioreactors:

type: slurry reactors, aqueous reactors benefits: rapid degradation kinetic, optimized environmental parameters, enhanced mass transfer, effective use of inoculants and surfactants limitations: soil requires excavation, relatively high cost capital, relatively high operating costs factors to consider: bioaugumentation, toxicity of amendaments, toxic concentration of contaminants

Cost of treating contaminated soil in place $80-$100 per ton

Cost of excavating and trucking contaminated soil off for incineration is $400 per ton.

Over 90% of the chemical substances classified as hazardous today can be biodegraded.

Aerobic
requires sufficient oxygen: Pseudomonas, Alcaligenes, Sphingomonas,Rhodococcus, Mycobacterium degrade pesticides and hydrocarbons, both alkanes and polyaromatic compounds bacteria use the contaminant as the sole source of carbon and energy no generation of methane it is a faster process

Anaerobic

Microbes substitute another chemical for O2 to degrade contaminants Nitrate, iron, sulfate, carbon dioxide, uranium, technicium, perchlorate
in the absence of oxygen, thus the energy input is slow) anaerobic bacteria are not as frequently used as aerobic bacteria anaerobic bacteria are used for bioremediation of polychlorinated biphenyls (PCBs) in river sediments, dechlorination of the solvent trichloroethylene (TCE), chloroform it may generate methane have the ability to degrade an extremely diverse range of persistent or toxic environmental pollutants (as white rot fungus Phanaerochaete chrysosporium) common substrates used include straw, saw dust, or corn cobs grow utilizing methane for carbon and energy are active against a wide range of compounds, including the chlorinated

Ligninolytic fungi

Methylotrophs

Microbes
A. eutrophus H850Lr P. putida TVA8 P. fluorescens HK44

Application
Process monitoring Process monitoring Process monitoring

Contaminants
PCB TCE, BTEX naphthalene, anthracene, phenanthrene 2,4-D BTEX Chlorobenzenes, chlorophenols, BTEX Cd, 2,4dinitrophenol,hydr oquinone

References
VAN DYKE et al., 1996 APPLEGATE et al., 1998 SAYLER et al., 1999

B. cepacia BRI6001L P. Fluorescens 10586s/pUCD607 P. Fluorescens 10586s/pUCD607

Stress monitoring Stress response Toxicity assesment

MASSON et al., 1993 SOUSA et al., 1998 BOYD et al., 1998, SINCLAIR et al., 1999; GLOVER et al., 1999 KELLY et al.,1999

Pseudomonas strain Shk1

Toxicity assesment

A. eutrophus 2050

end point analysis

nonpolar narcotics

LAYTON et al., 1999

PCB : polychlorinated biphenyls TCE : trichloroethylene BTEX : benzene, toluene, ethylbenzene, and xylene

Metabolism of a Pollutant-degrading Bacterium

Fe(III)
ACETATE *Benzoate *Toluene *Phenol *p-Cresol *Benzene *U(VI) *Co(III) *Cr(VI) *Se(VI) *Pb(II) *Tc(VII)

ATP

CO2

Fe(II)
*CCl4 *Cl-ethenes *Cl-aromatics *Nitro-aromatics

Use of Plants to remove or degrade contamination from soils and surface waters Its cheap, sustainable, effective, and environmentally friendly alternative to conventional remediation technologies Use of Solar energy to extract chemicals from the soil and to deposit them in the above-ground part of their bodies, or to convert them to a less toxic form Harvesting and removal of pollutants

Phytoextraction or phytoaccumulation

Phytotransformation or phytodegradation Phytostabilization

the plants accumulate contaminants into the roots and aboveground shoots or leaves saves tremendous remediation cost by accumulating low levels of contaminants from a widesp read area produces a mass of plants and contaminants (usually metals) that can be transported for disp osal or recycling uptake of organic contaminants from soil, sediments, or water and, subsequently, their transfo rmation to more stable, less toxic, or less mobile form plants reduce the mobility and migration of contaminated soil leachable constituents are adsorbed and bound into the plant structure so that they form a st able mass of plant from which the contaminants will not re-enter the environment breakdown of contaminants through the activity existing in the rhizosphere, due to the presen ce of proteins and enzymes produced by the plants or by soil organisms such as bacteria, yeast, and fungi is a symbiotic relationship that has evolved between plants and microbes: plants provide nutrients necessary for the microbes to thrive, while microbes provide a healthier soil environment is a water remediation technique that involves the uptake of contaminants by plant roots is used to reduce contamination in natural wetlands and estuary area plants evaportranspirate selenium, mercury, and volatile hydrocarbons from soils and ground water rainwater from soil is evaportranspirated by plants to prevent leaching contaminants from disposal sites

Phytodegradation or rhizodegradation

Rhizofiltration

Phytovolatilization
Vegetative cap

high tolerance to the pollutant ability to either degrade or concentrate the contaminant at high levels in the biomass extensive root systems the capacity to absorb large amounts of water from the soil fast growth rates and high levels of biomass.

Three classes of proteins :

Metallothioneins Phytochelatins Glutathione

Eg .

shrub tobacco (Nicotiana glauca) Arabidopsis Indian mustard and tobacco

Transgenic Arabidopsis plants expressing a selenocysteine methyltransferase (SMTA) gene from the selenium hyper accumulator Landmines - Arabidopsis whose roots change color when they come into contact with degradation products of landmines have been developed.

Herbicides - Mammalian P450 cytochrome genes have been used to confer herbicide resistance to crop plants, which can be used in herbicide rotation systems designed to delay the evolution of herbicide resistance in weeds, and to reduce the environmental load of agricultural chemicals Herbicide resistance is also provided by the overexpression of the maize glutathione S-transferase I (GSTI) gene Explosive - Tobacco plants engineered with the bacterial gene for a NADPH-dependent nitroreductase tolerate and degrade high levels of TNT Arabidopsis plants carrying the xplA gene from Rhodococcus bacteria are highly resistant to of RDX

Mercury - Detoxification of organomercurials has been achieved in transgenic plants by transforming Arabidopsis, tobacco, poplar trees, Indian mustard, and eastern cotton wood with two bacterial genes, merA and merB The combined actions of merA and merB transform methylmercury to the volatile elemental form, which is 100 times less toxic, and is released by the plant to the atmosphere at non-toxic concentrations through transpiration Arsenic - Scientists have engineered Arabidopsis plants with arsenic tolerance by introducing two bacterial genes: arsC and y-ECS. arsC converts arsenate, the arsenic form absorbed by plants, to arsenite. Double transgenics are not only highly tolerant of arsenic, they also have improved cadmium tolerance, and a six-fold increase in the level of biomass compared to wild-type controls

Pollutants discharged from the industries Textile industries - enzymes, acids, alkali, alcohols, phenols, dyes, heavy metals, radionucliods, etc Traces of zinc, cadmium, mercury, copper, chromium, lead are found in dyes Actinomycetes show a higher capacity to bind metal ions as compared to fungi and bacteria

Dead biomass immobilized on polymeric membrane absorbs uranium Immobilized Aspergillus oryzae cells on reticulated foam particles have been used for Cd removal Aspergillus niger biomass contains up to 30 per cent of chitin and glucan Chitin phosphate and chitosan phosphate of fungi absorb greater amount of U than Cu, Cd, Mn, Co, Mg and Ca

Degradation through reduction azo-reductase of Pseudomonas strains in the chemostat culture This enzyme catalyses azo-linkage of the dye. During degradation process of azo, NAD(P) acts as electron donor degradation of black liquor pulp mill effluents by the strains of Pseudomonas putida Some anaerobic bacteria,Streptomyces and fungi (e.g. Phaenerochaete chrysosporium) have been characterized for decoloration of chromogenic dyes. The enzymes involved in dye degradation are lignases (lignin peroxidase), Mn (II) dependent peroxidase and glyoxal oxidase. These enzymes are well associated with lignin degrading system

Bacteria, algae, fungi, actinomycetes, higher plants Algae

The species of Chlorella, Anabaena inaequalis, Westiellopsis prolifica, Stigeoclonium tenue, Synechococcus sp. tolerate heavy metals However, several species of Chlorella, Anabaena, marine algae have been used for the removal of heavy metals

Fungi also are capable of accumulating heavy metals in their cells

Metabolism-independent accumulation - The positively charged ions in the solution are attracted to negatively charged ligands in cell materials. Bio sorption of metal ion occurs on microbial cell surface but composition of biomass and other factors affect bio sorption

 Metabolism-dependent accumulation - In fungi and yeast, heavy metal ions are transported into the cells through cell membrane. However, as a result of metabolic processes ions are precipitated around the cells, and synthesized intracellularly as metal-binding proteins. Moreover, intracellular uptake is influenced by certain external factors such as pH, anions, cations and organic materials, growth phase, etc Metal uptake by growing batch culture was found maximum during lag phase and early log phase in Aspergillus niger, Penicillium spinulosum andTrichoderma viride

 Extracellular precipitation and complexation - extracellular products which can complex or precipitate heavy metals Siderophores In Saccharomyces cerevisiae removal of metals is done by their precipitation as sulphides e.g.Cu2+ is precipitated as CuS

Bioremediation of Coal Wastes through VAM Fungi

Measuring Redox in soil and groundwater, together with pH, temperature, oxygen content, electron acceptor/donor concentrations, and concentration of breakdown products (e.g. carbon dioxide)

Process
Aerobic Denitrification Manganese reduction Iron reduction Sulphate reduction Fermentation

Reaction
O2 + 4e + 4H+ 2H2O 2NO3 + 10e + 12H+ N2+ 6H2O
MnO2 + 2e + 4H+ Mn2+ + 2H2O Fe(OH)3 + e + 3H+ Fe2+ + 3H2O SO42 + 8e +10 H+ H2S + 4H2O 2CH2O CO2 + CH4

Redox Potential (E in mV)

600 ~ 400 500 ~ 200 400 ~ 200 300 ~ 100 0 ~ -150 150 ~ -220

Microbial Growth until critical biomass is reached Mutation and horizontal gene transfer Enzyme induction Enrichment of the capable microbial populations Production of toxic metabolites Environmental Depletion of preferential substrates Lack of nutrients Inhibitory environmental conditions Substrate Too low concentration of contaminants Chemical structure of contaminants Toxicity of contaminants Solubility of contaminants

Biological aerobic vs anaerobic process Oxidation/reduction potential Availability of electron acceptors Microbial population present in the site Growth substrate vs co-metabolism Type of contaminants Concentration Alternate carbon source present Microbial interaction (competition, succession, and predation) Physico-chemical bioavailability of pollutants Equilibrium sorption Irreversible sorption Incorporation into humic matters Mass transfer limitations Oxygen diffusion and solubility Diffusion of nutrients Solubility/miscibility in/with water

most hydrocarbons and organic compounds will be mineralized intrinsic microbes (those already found in the soil) will mostly be able to acclimatize to the contaminants instead of transferring contaminants from one environmental medium to another, the complete destruction of target pollutants is possible it usually does not produce toxic by-products is usually less expensive than other technologies it can be used where the problem is located, often without causing a major disruption of normal activities

is limited to those compounds that are biodegradable short supply of substrate, electron acceptors, or nutrients will hinder bioactivity high levels of organic contaminants may be toxic to the microbes heavy metals may inhibit the microbial activity the contaminant must be provided in an aqueous environment the lower the temperature, the slower the degradation the process must be carefully monitored to ensure the effectiveness it is difficult to extrapolate from bench and pilotscale studies to full-scale field operations often takes longer than other actions

permanence
contaminant is degraded

potentially low cost

60-90% less than other technologies

Many factors control biodegradability of a contaminant in the environment Before attempting to employ bioremediation technology, one needs to conduct a thorough characterization of the environment where the contaminant exists, including the microbiology, geochemistry, mineralogy, geophysics, and hydrology of the system

 Bioremediation , Montana University

Wikipedia www. eplantscience.com Review paper, Factors limiting bioremediation technologies, R. Boopathy, Department of Biological Sciences, Nicholls State University, Thibodaux, LA 70310, USA Maria Gavrilescu, Environmental Biotechnology : Achivesments, Oppurtunities and Challenges