1. ROLE OF MICROORGANISMS IN
THE ABATEMENT OF POLLUTANTS
by Azis Kemal Fauzie
Department of Studies in Environmental Science
University of Mysore
December 2016
6. Polycyclic Aromatic Hydrocarbons (PAHs)
PAHs are the class of hydrocarbons containing two or more fused
benzene rings and/or pentacyclic molecules. PAHs originate from fossil
fuels and industrial processes during coke production. PAHs are toxic
(carcinogenic, mutagenic and teratogenic) to human and animals.
9. Polychlorinated Biphenyls (PCBs)
• PCBs are organic chemicals synthesized by catalytic chlorination of biphenyls.
• First manufactured in 1929 by Monsanto. Manufacture, use, importation and
distribution of PCBs was banned in Sweden (1970), Japan (1972) and US (1976).
• Trademark: Aroclor (US), Kaneclor (Japan), Fenclor (Italy), Pyralene (France),
Clophen (Germany).
• Applications:
fluid in electrical (transformers, capacitors), heat transfer and hydraulic equipment
plasticizers in paints, plastics and rubber products
pigments, adhesives, pesticides, inks, dyes, waxes and carbonless copy paper
lubricants for turbines and pumps
• Toxicity:
Reproductive disabilities in animals and human
Nervous system and liver damage
Hepatitis, skin diseases and endocrine disrupters
Carcinogenic and allow bioaccumulation
11. AZO:
XANTHENE:
TRIPHENYLMETHANE:
Brilliant Green Fast Green FCF
Methyl Orange
Congo Red
Allura
Red
Sunset
Yellow
FCF
Bismark Brown Y
Methyl
Violet
Oil Red O
Rhodamine 123
Eosin Y
Erythrosin
Rose Bengal
Crystal
Violet
OTHER CLASSES:
Phenazine, Cyanine
Phenanthridine
Acridine, Coumarin
Anthraquinone
Quinoline, Oxonol
Tetrazolium salt
Benzofuran, Indole
Benzodiazole, Styryl
Nitro, Nitroso, Indigo
Diphenylmethane
Heterocycle, etc.
PHENOTHIAZINE:
Methylene Blue
Methylene Violet
PHENOXAZINE:
Brilliant Cresyl Blue
Darrow Red
Giemsa Stain
Cresyl Violet Acetate
25. Degradation by PGPR & PGPB
• Plant Growth Promoting Rhizobacteria (PGPR), or rhizospheric bacteria,
are naturally occurring soil bacteria that aggressively colonize plant
roots and benefit plants by providing growth promotion. The technique
to apply this in soil biodegradation is called rhizoremediation.
• Plant Growth Promoting Bacteria (PGPB), or endophytic bacteria, are
non-pathogenic bacteria that occur naturally in plants as adjuncts in
phytoremediation. They can significantly facilitate the growth of plants
in the presence of high levels of pollutants, including metals.
Pollutants Microorganisms References
Hydrocarbons Pseudomonas putida Hontzeas et al., 2004
PAHs Lysini bacillus Ma et al., 2010
PCBs Rhodococcus, Luteibacter, Williamsia Leigh et al., 2006
Malathion Azospirillum lipoferum Kanade et al., 2012
26. Degradation by Microfungi & Mycorrhiza
• Microfungi are described as a group of eukaryotic organisms that are
important part of degrading microbiota. Like prokaryotic bacteria,
they metabolize organic matter and responsible for the
decomposition of carbon in the biosphere. But fungi, unlike
bacteria, can grow in low moisture areas and in low pH solutions.
• Fungi species are ranging from unicellular yeasts to extensively
filamentous fungi or mycelial molds. Fungal metabolism can be non-
ligninolytic or ligninolytic (also known as white-rot fungi).
• Mycorrhiza is a symbiotic association between fungus and the roots
of vascular plant. It is important for mycorrhizoremediation.
• In a mycorrhizal association, the fungi colonizes the host plant's
roots, either intracellularly as in arbuscular mycorrhizal fungi (AMF),
or extracellularly as in ectomycorrhizal fungi.
27. Degradation by Yeasts
Pollutants Yeasts References
Aliphatic/petroleum
hydrocarbons
Candida lipolytica, C. tropicalis, C. apicola,
Rhodoturula rubra, R. mucilaginosa, Geotrichum,
Aureobasidium pullulans, Trichosporon mucoides
Bartha, 1986;
Scheuer, 1998;
Was, 2001
Diesel oil Rhodotorula aurantiaca, Candida ernobii de Cássia, 2007
Alkane, fatty acids Candida maltose, C. tropicalis, Yarrowia lipolytica Iida, 2000
Phenol Trichosporon cutaneum Mörtberg, 1985
PCBs T. mucoides, Candida boidinii, C. lipolytica Sasek, Sietman
Linuron, metroburon Botrytis cinerea Bordjiba, 2001
Aniline Candida methanosorbosa Mucha, 2010
Reactive black Candida krusei, Pseudozyma rugulosa Yu & Wen, 2005
PEA, PC, PLA Candida cylindracea, Tritirachium album Tokiwa, 2009
PCB, U, Th, Co, Cr Saccharomyces cerevisiae Cabras; Brady
Chromium (VI) Pichia anomala, Cyberlindnera fabianii,
Wickerhamomyces anomalus, C. tropicalis
Bahafid, 2011,
2012
S. cerevisiae, Pichia guilliermondii, Yarrowia
lipolytica, R. pilimanae, Hansenula polymorpha
Ksheminska, 2006,
2008
Copper Schizosaccharomyces pombe Saisubhashini, ‘11
31. Degradation by Algae & Protozoa
• Reports of algae and protozoa in biodegradation are scanty.
• However, a number of cyanobacteria, green algae, brown algae,
red algae, and diatoms could oxidize naphthalene. Some studies
also reported degradation of dyes, pesticides and heavy metals.
• Protozoa are main grazer on the degrading bacteria. Protozoa help
on regulating growth of bacteria and algae populations, reducing
competition, improving turnover of nutrients, increasing space
and oxygen content, releasing excess nitrogen and special enzyme
required for biodegradation, and stimulating decomposition rates.
• For example, protozoa infusorians can accelerate biodegradation
of PAH. The degrading rate of bacteria has improved 8.5 times on
benzene and methylbenzene, and 4 times on naphthalene by the
influence of grazing bacteria of protozoa flagellate.
34. Genetically Engineered Microorganisms
(GEMs)
• Genetically Engineered Microorganisms (GEMs) or
Genetically Modified Organisms (GMOs) are microorganisms
whose genetic material have been altered using genetic
engineering techniques (known as recombinant DNA
technology) inspired by natural genetic exchange between
microorganisms and have potential capabilities of degrading
chemical contaminants useful for bioremediation.
• In 1979, Dr. Anand Mohan Chakrabarty has engineered strain
of Pseudomonas putida (called as superbug or oil eating bug)
that contains hybrid plasmids capable of degrading different
compounds i.e. CAM (camphor), OCT (octane), XYL (xylene),
and NAH (naphthalene). This superbug was used by the US
Govt. in 1990 for cleaning up oil spill in Texas.
37. Bioremediation & Biodegradation
• Biodegradation is the process by which organic substances are broken
down into smaller compounds by living organisms.
• Bioremediation is the process of utilizing microorganisms to degrade
environmental pollutants by transforming them into less toxic form.
• Methods of bioremediation strategies could be:
in-situ (at the site) or ex-situ (away from the site)
aerobic (in presence of oxygen) or anaerobic (in absence of oxygen)
enhanced by enzymes or biosurfactants
• Biodegradation can be mediated by:
Bacteria (bioremediation)
Fungi (mycoremediation)
Algae
Protozoa
Plants (phytoremediation)
38. • Natural attenuation or bioattenuation is the reduction of
contaminant concentrations in the environment through
biological processes (microbial biodegradation, plant and animal
uptake), physical phenomena (advection, dispersion, dilution,
diffusion, volatilization, sorption/desorption), and chemical
reactions (ion exchange, complexation, abiotic transformation).
• Biostimulation is the addition of soil nutrients, trace minerals,
electron acceptors, or electron donors to enhance the
biotransformation of soil contaminants by indigenous
microorganisms. It includes also bioventing and biosparging.
• Bioaugmentation is the technique for improving the capacity of a
contaminated biotope to remove pollution by the introduction of
specific competent strains of exogenous microorganisms or
genetically engineered microorganisms (GEMs).
Indigenous & exogenous
microorganisms
In-situ Bioremediation
Natural Attenuation, Biostimulation, Bioaugmentation
39. Ex-situ Bioremediation
Composting, Land farming, Biopile, Bioreactor
• Composting is a technique that involves combining contaminated
soil with non-hazardous organic amendants such as manure or
agricultural wastes.
• Land farming is a simple technique in which contaminated soil is
excavated and spread over a prepared bed and periodically tilled
until pollutants are degraded.
• Biopile is a hybrid of land farming and composting constructed as
aerated composted piles to control physical losses of the
contaminants by leaching and volatilization.
• Bioreactor or slurry reactor is a containment vessel used to
create a three-phase (solid, liquid, gas) mixing condition to
increase the bioremediation rate of soil-bound and water-soluble
pollutants as a water slurry of the contaminated soil and biomass
(microorganisms) capable of degrading target contaminants.
40. Role of Enzymes in Biodegradation
The degradation of pollutants can be mediated or catalyzed by specific
enzymes secreted by the microorganisms like (mono- or di-) oxygenases,
peroxidases, oxidoreductases, hydrolases, hydroxylases, dehalogenase,
dehydrogenases, esterases, phosphotriesterases, etc.
41. Role of Biosurfactants in Biodegradation
Biosurfactants or bioemulsifiers are biological surface-active agents
that have both hydrophilic and hydrophobic moieties.
Biosurfactants are produced by either degrading or non-degrading
microorganisms to help on metabolizing carbon and energy source.
Biosurfactants can act by:
- forming micelles or microdroplets of pollutants
- reducing surface tension in chemical compounds
- increasing surface area of hydrophobic substrates
- increasing mixing of aqueous and non-aqueous fluid phases
- increasing rate of transfer into or through aqueous media
- increasing bioavailability of the compounds.
Low molecular weight biosurfactants include:
glycolipids (rhamnolipid, trehalose lipids, and sophorolipids)
or lipopeptides (surfactin, gramicidin S, and polymyxin).
High molecular weight biosurfactants include:
polysaccharides, proteins, lipopolysaccharides, lipoproteins
or complex mixtures of these biopolymers.
42. R
C
H
C
H
R
OH
OH
R
OH
OH
R
OH
COOH
O
O
COOH
RCOOH
NAD+ NADH
NADH NAD+
O2 O2
+
Alkylbenzene Dihydrodiol Ring fission
product 2-Oxopenta-
4-enoate
2,3-Dihydroxy-
alkylbenzene
Smith & Ratledge 1989
E2 E3 E4
E1
E1 = Alkylbenzene dioxygenase
E2 = cis-alkylbenzene glycol dehydrogenase
E3 = 2,3-dihydroxyalkylbenzene 1,2-dioxygenase
E4 = ring fission product-hydrolysing enzyme
Aerobic Degradation:
C
H2
CH3
Ethylbenzene
C
H
CH3
O
H
1-Phenylethanol
CH3
O
Acetophenone
O O
CH2
O
Benzoylacetate
O O
CH2
O
S
CoA
Benzoylacetate-CoA
O S
CoA
Benzoyl-CoA
O
H2 CO2 CoASH CoASH Acetyl-CoA
Ethylbenzene
Dehydrogenase
1-Phenylethanol
Dehydrogenase
Acetophenone
Carboxylase
Benzoylacetyl-CoA
forming enzyme
Benzoylacetyl-CoA
CoA thiolase
2[H]
2[H]
Anaerobic Degradation:
Heider et al. 1999
43. The degradation of a straight chain hydrocarbon:
Pathway for Degradation of Aliphatic Compounds
Bacteria involved:
Pseudomonas putida
Fungi (yeast) involved:
Candida maltosa, Candida tropicalis, Candida apicola
The degradation of a cyclic hydrocarbon:
Enzymes involved:
E1 = alkane monooxygenase
E2 = fatty alcohol dehydrogenase
E3 = fatty aldehyde dehydrogenase,
(Harayama et al. 1999)
E1 E2
E3
44. Pathway for Degradation of Aromatic Compounds
The microbial degradation of catechol
Benzene Arene oxide
cis/trans-dihydrodiol Cathecol
Naphthalene cis-1,2-naphthalene 1,2-dihydroxynaphthalene Salicylic acid
dihydrodiol
Bacteria involved:
Pseudomonas, Rhodococcus, Mycobacterium
Fungi (yeast) involved:
Pleurotus ostreatus
45. Pathway for Degradation of PCBs
Organisms involved:
Achromobacter,
Beijerinckia,
Pseudomonas
putida
Pathway for anaerobic dechlorination of a highly chlorinated congener (Fish & Principe, 1994).
Pathway for aerobic degradation of PCBs into chlorobenzoates (Sylvestre & Sandossi, 1994).
Organisms involved:
Dehalococcoides,
Thermotogales,
Chloroflexi
47. Mechanism in Heavy Metal Degradation
Mechanisms of heavy metal bioremediation by
microorganisms include bioleaching, biomineralization,
biosorption, bioaccumulation, and biotransformation.
- Bioleaching: heavy metal mobilization through methylation reactions or
excretion of organic acids.
e.g. Acidithiobacillus ferrooxidans, Leptospirillum ferriphilum
- Biomineralization: heavy metal immobilization through formation of
insoluble sulfides, phosphates, carbonates, hydroxides or polymeric
complexes in response to localised alkaline conditions at cell surface.
e.g. Serratia, Citrobacter
- Biosorption: passive uptake of metals to the surface of living or dead
microbial cells by physico-chemical mechanisms including absorption,
adsorption, ion exchange, surface complexation and precipitation.
e.g. Bacillus subtilis, Rhizopus arrhizus
- Bioaccumulation: active uptake of essential elements (particularly heavy
metals) within the cell of microorganisms.
e.g. Pseudomonas, Arthrobacter
- Biotransformation: metabolic activity of microorganisms on metal ions
through enzyme-catalyzed redox reactions.
e.g. Geobacter, Thermoterrabacterium ferrireducens
48. Mechanism in Radionuclide Degradation
Mechanisms of radionuclide bioremediation by micro-
organisms include bioreduction, biomineralisation,
bioaccumulation, and biosorption.
49. Factors affecting microbial degradation
• Biological factors
— competition between organisms for limited carbon sources
— antagonistic interactions between microorganisms
— predation of microbes by protozoa and bacteriophage
• Physical factors
— temperature
— pH
— moisture
• Environmental factors
— soil type and porosity
— soil organic matter
— soil oxidation-reduction potential
51. References
• Joutey et al., Biodegradation: Involved Microorganisms & GEMs, 2013.
• Das & Chandran, Microbial Degradation of Petroleum Hydrocarbon Contaminants: An
Overview, 2011.
• Maigari & Maigari, Microbial Metabolism of PAHs: A Review, 2015.
• Hernández et al., Pesticide Biodegradation: Mechanisms, Genetics & Strategies to
Enhance the Process, 2013.
• Borja et al., Polychlorinated Biphenyls & Their Biodegradation, 2005.
• Furukawa & Fujihara, Microbial Degradation of PCBs: Biochemical & Molecular Features,
2008.
• Tokiwa et al., Biodegradability of Plastics, 2009.
• Leja & Lewandowicz, Polymer Biodegradation & Biodegradable Polymers – A Review, 2010.
• Ali, Biodegradation of Synthetic Dyes - A Review, 2010.
• Meenambigai et al., Biodegradation of Heavy Metals – A Review, 2016.
• Girma, Microbial Bioremediation of Some Heavy Metals in Soils: An Updated Review, 2015.
• Newsome et al., The Biogeochemistry & Bioremediation of Uranium & Other Priority
Radionuclides, 2014.
52. References
• Vidali, Bioremediation: An Overview, 2001.
• Kothari et al., Microbial Degradation of Hydrocarbons.
• Pandey et al., Microbial Ecology of Hydrocarbon Degradation in the Soil: A Review, 2016.
• Harayama et al., Petroleum Biodegradation in Marine Environments, 1999.
• Zacharia & Tano, Identity, Physical & Chemical Properties of Pesticides.
• Singh & Walker, Microbial Degradation of Organophosphorus Compounds, 2006.
• Abraham et al., PCB-degrading Microbial Communities in Soils & Sediments, 2002.
• Garrison et al., Bio-Based Polymers with Potential for Biodegradability, 2016.
• Dussud & Ghiglione, Bacterial Degradation of Synthetic Plastics, 2014.
• Barrágan, Biodegradation of Azo Dyes by Bacteria Inoculated on Solid Media, 2007.
• Jain et al., Review on Bioremediation of Heavy Metals with Microbial Isolates &
Amendments on Soil Residue, 2014.
• Gazsó, The Key Microbial Processes in the Removal of Toxic Metals & Radionuclides from
the Environment, 2001.
• Lloyd & Renshaw, Bioremediation of Radioactive Waste: Radionuclide–Microbe Interactions
in Laboratory & Field-Scale Studies, 2005.
