Bioremediation:process of using biological agents to remove toxic

wastes from the environment

How do we dispose of the large quantities of wastes that are continually being produced?

How do we remove the toxic compounds that have been accumulating at dump sites, in the soil and in water systems over the last few decades?

Government’s antipollution regulations – often remain unforced

3 R’s: reduce, reuse, and recycle

Biotechnological Schemes

Problem of toxic waste disposal is enormous Most important organic pollutant in the

environment are mineral oil constituents and halogenated products of petrochemicals

Pentachlorophenol – 50,000 tons(1985, worldwide)

Incineration and chemical treatment: costly and often create new environmental problems

Soil microorganisms capable of degrading xenobiotics e.g. herbicides, pesticides, refrigerants

Members of genus Pseudomonas are the most predominant group of soil microorganisms that degrade xenobiotic compounds

Pseudomonas strains can detoxify >100 different organic compounds

The biodegradation of complex organic molecules generally requires the concerted efforts of several different enzymes

The genes that encode for the enzymes of these biodegradable pathways are sometimes located in the chromosomal DNA, although they are more often found on large plasmids (in some organisms on both chromosomal and plasmid DNA)

• Although many bacteria are able to metabolize organic pollutants, a single bacterial species does not possess the enzymatic capability to degrade all or even most of the organic compounds in a polluted soil. •Mixed microbial communities have the most powerful biodegradative potential, because the genetic information of more than one organism is necessary to degrade the complex mixtures of organic compounds present in contaminated areas.• The genetic potential and certain environmental factors such as temperature, pH, and available nitrogen and phosphorous sources seem to determine the rate and the extend of degradation.

Most rapid & complete degradation Essential characteristics of aerobic

microorganisms:a. Metabolic processes for optimizing the contact

between the microbial cells & organic pollutantsb. Initial intracellular attack on organic pollutants is

an oxidative process (by oxygenase & peroxidase)c. Peripheral degradation-TCA cycled. Biosynthesis of cell biomass from the central

precursor molecule e.g. AcetylCoA, succinate, pyruvate

• Cyclic alkanes represent minor components of mineral oil and are relatively resistant to microbial attack. •The absence of an exposed terminal methyl group complicates the primary attack. • A few species can use cyclohexane as a sole carbon source, but it is more commonly cometabolized by mixed cultures. • In general, the presence of alkyl side chains on cycloalkanes facilitates their degradation.

Degradative bacteria, in most cases, enzymatically convert xenobiotic, non-halogenated aromatic compounds to either catechol or protocatechuate.

Then, through a series of oxidative cleavage reactions, catechol and protocatechuate are processed to yield either acetyl-CoA and succinate, or pyruvate and acetaldehyde compounds that are readily metabolized by alomost all organisms

Halogenated aromatic compounds, which are the main components of most pesticides and herbicides, are converted to catechol, protocatechuate, hydroquinones, or the corresponding halogenated comounds.

Aromatic hydrocarbons, e.g., benzene, toluene, ethylbenzene and xylenes and naphthalene, belong to large-volume petrochemjcals, which are widely used as fuels and industrial solvents.

Phenols and chlorophenols are released into the environment as products and waste material from industry.

Converted enzymatically to the natural intermediates of degradation: catechol and protocatechuate.

first step(benzene) of oxidation is a hydroxylation; the product, a diol, is converted to catechol by a dehydrogenase.

The oxygenolytic cleavage of the aromatic ring occurs via ortho or meta cleavage.

1. Molecular Biotechnology, Glick & Pasternak3rd edition

2. Environmental Biotechnology, Jordening & Winter

Genetic Engineering of Biodegradative

Pathways

Despite the ability of many naturally occurring microorganisms to degrade a number of different xenobiotic chemicals, there are limitations:

i. No single microorganism can degrade all organic wastes

ii. High concentrations of some organic compounds can inhibit the activity or growth of degradative microorganisms

iii. Most contaminated sites contain mixtures of chemicals, and an organism that can degrade one or more of the components of the mixture may be inhibited by other components

iv.Many nonpolar compounds adsorbe onto particulate matter in soils or sediments and become less available to degradative microorganisms

v. Microbial biodegradation of organic compounds is often quite slow

a. Manipulation by transfer of plasmids

b. Manupulation by gene alternation

Transfer by conjugation into a recipient strain plasmid that carry genes for different degradative pathways.

If two resident plasmids contain homologous regions of DNA, recombination can occur and a single, larger ‘fusion’ plasmid with combined functions can be created

Alternatively, if two plasmids don not contain homologous regions and in addition, belong to different incompatibility groups, they can coexist within a single bacterium

Fig 13.5 (Glick, p384) Different plasmids were used to construct a bacterial strain

that degraded a number of hydrocarbon components. The strain has been called a ‘superbug’ because of its increased metabolic capabilities

The CAM plasmid was tranferred by conjugaton into a strain carrying the OCT plasmid. These 2 plasmids were incompatible and could not be maintained in the same cell as separate plasmids. However, when recombination occurred between the two plasmids, the resulting single plasmid was perpetuated and carried both CAM and OCT degradative activities

The NAH plasmid was tranferred by conjugation into a strain carrying the XYL plasmid. The NAH and XYL plasmids were compatible and could therefore coexist within the same host cell.

Finally, the CAM/OCT fusion plasmid was transferred by conjugation into the strain carrying the NAH and XYL plasmids.

Most of the degradative bacteria that have been genetically manipulated by plasmid transfer are mesophiles, organisms grow well only at temperature between 20 and 40 C.

However, rivers, lakes, and oceans that are polluted generally have temperature that range from 0 to 20 C.

To test whether bacteria with enhanced degradative abilities could be created for cold environments, a TOL plasmid from mesophilic P. putida strain was transferred by conjugation into a psychrophile (an organism with a low temperature optimum) that was able to degrade salicylate, but not toluene, and use it as a sole carbon source at temp as low as 0C.

The transformed strain carried the introduced TOL plasmid and its own SAL plasmid and was able to use either salicylate or toluene as its sole carbon source at 0C.