Bioremediation

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Environmental remediation

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The removal of contaminants and pollutants from environmental sources: soil, water and sediments, for general the protection of human health and environment

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Remediation techniques

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1. Ex-situ
2. In-situ

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Ex-situ

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1. Excavation - taking to landfill
2. Incineration - combustion of organic material
3. Pump and treat (ground water)

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In-situ

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1. Chemical oxidation by adding strong oxidants of bleach at the place of contamination
2. Introduce surfactants for aquifer (ground water) remediation

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Bioremediation

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Use of organisms (plants)/ micro-organisms (bacteria, fungus) or their derivatives to remove the contaminants from environment

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Advantages of bioremediation

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1. Permanent solution, removes all the contaminants for good, thus used in detoxification and mineralization (decompose into components that can be used by plants).
2. Eco-friendly (non-toxic by products)
3. Efficient (in-situ or ex-situ)
4. Low cost
5. Sustainable

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Disadvantages of bioremediation

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1. Hard to deal with complex contamination mixture
2. Site and Contaminant specific organisms needed
3. Slow progress
4. Long-term monitoring required

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Why use microbes for bioremediation?

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1. They have high growth rate
2. They can metabolize many organic/inorganic substances for growth
3. Highly genetically susceptible leading to more site specific
4. Inhabit various niches
5. Able to persue unusual metabolic and physiological activities

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Process of bioremediation

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Microorganism destroy contaminants while using them as their source of energy and growth.
Aerobic microbes: use oxygen as electron acceptor and contaminants as electron donor for energy.
Anaerobic microbes: same as above except use either nitrate, manganese, iron or sulphate as electron acceptor

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Bioremediation of complex molecules

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- require many organisms, each carrying out different steps in the metabolism of the molecule
- at times need to add oxygen or change temperature or add nutrients (nitrogen and phosphorus) to make the microbes work
- majority are psuedomonas

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Types of Bioremediation

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1. Intrinsic bioremediation (natural process of bioremediation)
2. Biostimulation (stimulate microbes with nutrients to allow for higher growth rates)
3. Bioaugmentation (add non-native microbes to take care of the contaminants)

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Advantages of in-situ bioremediation

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1. Less expensive
2. Decrease contaminants at the site
3. Site is not disturbed much

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Disadvantages of in-situ bioremediation

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1. Slow process
2. In compatible with certain soil types

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Advantages of ex-situ bioremediation

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1. Faster than in-situ
2. Easier to control the reaction
3. Treatment of large variety of contaminants

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Disadvantage of ex-situ bioremediation

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1. More expensive
2. Have to move the contaminated things around.. need for transportation and proper handling of the contaminants
3. Need to construct a separate place where decontamination can occur
4. Require alot of power

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Ex-situ bioremediation (slurry-phase)

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Add soil, water, oxygen, nutrients and bacterial in a bioreactor and mix them all together to treat the soil

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Ex-situ bioremediation (solid-phase)

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On large surface area soil is treated by adding moisture, heat, nutrients and oxygen supplies for bacteria. It is slower than slurry phase

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Limitation of biological treatments

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1. Complex compounds need many organism
2. If the organism is right for one molecule it will uptake the molecule and can either get through kerbs cycle or it could be misrouted.
3. the misrouted molecules can either hit a dead end with leftover metabolites or form toxic metabolites that can kill the organism or harm environment more

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Burkholderia cepacia

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1. Gram negative, soil dwelling, mostly found in plant roots.
2. It degrades toxic compounds
3. Key enzyme: Toulene ortho-monooxygenase (TOM)

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Main toxic compounds that Bc degrades

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1. 2,4,5-trichlorophenoxyacetic acid
2. Aromatic hydrocarbons (toulene, benzene and phenols)
3. Vinyl chloride (PVC pipes and bottles)
4. Naphthalene (moth balls derived from tar)
5. Trichloroethylene (TCE) - chlorinated hydrocarbons that are used as industrial solvents

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Types of protein engineering

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1. Directed evolution
2. DNA shuffling

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Directed evolution

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Process of protein engineering that harness the power of natural selection to get desired evolution of protein or RNA properties. The process includes:
1. Diversification
2. Selection
3. Amplification

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Diversification step

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Creating a library of mutants through encoding the protein of interest and mutate or recombine it at random using DNA shuffling

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DNA shuffling

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It is PCR without primers.
1. DNA fragments from various species that encode same/similar protein are mixed together.
2. Restriction digest is done
3. The fragments are then heated and cooled. Due to this the fragments will anneal to each other with complementary sequence from other specie.
4. PCR is then carried out to get full length and shuffled genes

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Selection step

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The library is tested for mutants containing the desired property through screening or selection

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Amplification

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The selected clones are replicated alot so that the DNA sequence can be understood

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Rizoremediation

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Using plants for bioremediation. The contaminant degradation occurs at the roots of the plants

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Advantages of rizoremediation

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1. Large surface area
2. Degradation occurs in roots
3. It is the natural niche and can grow wherever the plants can

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Disadvantages of rizoremediation

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1. Need adhering surfaces, so has to be a solid and static phase
2. Proliferate, conditions should be thriving for the plant
3. Need a proper functional degradative pathway

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What are organophostphates and how are they harmful?

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Hydrocarbons containing phosphates? Contain pesticide, herbicide, warfare agents. They are harmful because they cause irreversible inactivation of acetylcholine in the synapsis of neuron pathway causing fatal cholingeric reaction and neuromuscular disease

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Bioremediation of organophosphate?

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1. Flavobacterium
2. Altermonas: degrades Sarin to IMPA, which has half life of 19000 years
3. Psuedomonas testosteroni
4. Psuedomonas melophthora: Degrade IMPA to MPA and pi and MPA to pi and phosphoric acid

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Biocatalyst

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Take:
1. Organophosphate hydrolase from Flavobacterium: degrades paraxon to DEP and PNP
2. Pde from D.acidovorans and PhoA from Pseudomonas degrade DEP
3. PNP degraded by 6 enzymes in Pseudomonas

Engineer this pathway into one Pseudomona putida specie to completely degrade the contaminant paraoxon

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Technology used for using biocatalyst

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1. lypholized enzyme
2. reconstitute with water
3. spray on conminants

OR

introduce detoxifying enzyme in firefighting foam