ROLE OF MICROORGANISMS IN

THE ABATEMENT OF POLLUTANTS

by Azis Kemal Fauzie

Department of Studies in Environmental Science

University of Mysore

December 2016
OVERVIEW

• Biodegradable • Bacterial Degradation

Pollutants • Hydrocarbon-degrading bacteria
• Hydrocarbons • PAH-degrading bacteria
• PAHs • PCB-degrading bacteria
• PCBs • Pesticide-degrading bacteria
• Pesticides • Plastic-degrading bacteria
• Plastics • Dye-degrading bacteria
• Dyes • Heavy Metal-degrading bacteria
• Heavy Metals • Radionuclide-degrading bacteria
• Radionuclides
OVERVIEW... cont’d

• Other Microbial • Bioremediation &

Degradation Biodegradation
• PGPR • In-situ & Ex-situ Bioremediation
• PGPB • Role of Enzymes & Biosurfactants
• Microfungi • Aerobic & Anaerobic Degradation
• yeasts • Pathways for Degradation of
• filamentous fungi Aliphatic & Aromatic Compounds,
• Mycorrhiza PCBs
• Algae • Mechanisms in Plastic, Heavy
Metal & Radionuclide
• Protozoa
Degradation
• Genetically Engineered
• Factors affecting microbial
Microorganisms (GEMS)
degradation
 BIODEGRADABLE POLLUTANTS
Hydrocarbons, Polycyclic Aromatic Hydrocarbons (PAHs)
Polychlorinated Biphenyls (PCBs), Pesticides, Plastics, Dyes
Heavy Metals, Radionuclides

HCs, PAHs Dyes, PCBs Pesticides Plastics, Metals

SATURATES: (b) Cyclic/Naphthenes: Methylcyclohexane
(a) Aliphatic/Paraffins: CH3 CH3

Cyclopentane Methylcyclopentane
Methylcyclohexane

ASPHALTENES:

Benzaldehyde Benzoate

(Mono-) AROMATICS: RESINS:

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.
ORGANOCHLORINES: CARBAMATES:

ORGANOPHOSPHOROUS:

PYRETHRINS/PYRETHROIDS:
Fate of Pesticide in the Environment
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
 PLASTICS
Biodegradable: Non-biodegradable:
• petroleum-based • petroleum-based

Polycaprolctone (PCL) Polypropylene (PP)

Polyvinyl chloride (PVC) Polystyrene (PS)

Polyethylene
Polybutylene succinate (PBS) succinate (PES) Polyethylene terephtalate (PET)
• biomass-based • biomass-based

Polyethylene (PE)

Polyhydroxy butyrate (PHB)

Nylon 11

Acetyl cellulose (AcC)

Starch Polylactic acid (PLA)
AZO: TRIPHENYLMETHANE:
Congo Red
Methyl Orange

Bismark Brown Y

Brilliant Green Fast Green FCF

Allura Sunset
Red Yellow
Methyl Crystal
FCF
Violet Violet

Oil Red O

PHENOXAZINE: PHENOTHIAZINE: XANTHENE:

OTHER CLASSES:
Phenazine, Cyanine
Giemsa Stain Phenanthridine
Brilliant Cresyl Blue Acridine, Coumarin
Anthraquinone
Darrow Red Erythrosin Quinoline, Oxonol
Rose Bengal
Methylene Violet Tetrazolium salt
Benzofuran, Indole
Benzodiazole, Styryl
Nitro, Nitroso, Indigo
Diphenylmethane
Cresyl Violet Acetate Methylene Blue Eosin Y Rhodamine 123 Heterocycle, etc.
HEAVY METALS

RADIONUCLIDES
 BACTERIAL DEGRADATION
for Hydrocarbons, PAHs, PCBs, Pesticides, Plastics, Dyes
Heavy Metals, and Radionuclides

CO2
H2O
 Hydrocarbon-degrading Bacteria
Hydrocarbons Bacteria
Petroleum Bacillus, Corynebacterium, Staphylococcus, Streptococcus, Shigella,
hydrocarbons Alcaligenes, Klebsiella, Acinetobacter, Escherichia, Enterobacter, Vibrio,
Pseudomonas, Brevibacillus, Micrococcus, Nocardia, Achromobacter,
Arthrobacter, Flavobacterium, Oceanobacter kriegii
Crude oil P. aeruginosa, Bacillus subtilis, Acinetobacter lwoffi, Micrococcus roseus
Alkanes Pseudomonas, Bacillus, Micrococcus, Acinetobacter calcoaceticus, A.
venetianus, Nocardia erythroplis, Ochrobactrum, Alcaligenes sodorans,
Serratia marcescens, Arthrobacter, Rhodococcus, Marinobacter
hydrocarbanoclasticus, Alcanivorax borkumensis
Alkanes, alkenes, Methylococcus, Methylosinus, Methylocystis, Methylomonas,
cycloalkanes Methylocella, Methylobacter, Mycobacterium vaccae
Alkyl benzenes, Arthrobacter, Burkholderia, Pseudomonas, Sphingomonas,
alkanes, fatty Rhodococcus, Mycobacterium, Acinetobacter, Caulobacter
acids, cycloalkanes
Mono-aromatics Pseudomonas, Bacillus, B. stereothermophilus, Corynebacterium, Vibrio,
Brevibacillus, Achromobacter, Ochrobactrum, Thalassobacillus devorans
Benzoate Rhodopseudomonas palustris, Thauera aromatica, Azoarcus evansii
Ref: Joutey, 2013; Pandey, 2016; Kothari; Ghazali, 2014; Al-Wasify, 2014; Koch, 1993
 PAH-degrading Bacteria
PAHs Bacteria References
PAHs Rhodococcus, Corynebacterium, Achromobacter, Mrozik, 2003;
Aeromonas, Alcaligenes odorans, Sphingomonas Kelley, 1990;
paucimobilis, Bacillus, Pseudomonas, Nocardia, Bamforth &
Mycobacterium flavescens, Burkholderia cepacia, Singleton, 2005
Arthrobacter, Xanthomonas
PAHs, naphthalene, Marinobacter vinifirmus, M.alkaliphilus, Stappia Cui et al., 2008
phenanthrene, aggregate, Pseudoalteromonas ganghwensis,
pyrene Thalassospira lucentensis, Kaistia adipata
Naphthalene Pseudomonas fluorescens, Rhodococcus Bamforth, 2005
Phenanthrene Marinobacter, Pseudoalteromonas, Cycloclasticus, Melcher, 2002;
Marinomonas, Halomonas, Brevibacterium, Vibrio, Samanta, 1999;
Pseudomonas migulae, Sphingomonas yanoikuyae Haritash, 2009
Phenanthrene, Exiguobacterium, Shewanella, Methylomonas, Edlund, 2008
bromodeoxyuridine Pseudomonas, Bacteroides
PAHs, Microbacterium, Porphyrobacter, Porticoccus Gutierrez, 2012;
benzo[a]pyrene hydrocarbonoclasticus Gauthier, 2003
Anthracene Sphingomonas, Nocardia, Beijerinckia, Paracoccus, Teng, 2010
Rhodococcus
 PCB-degrading Bacteria
PCBs Bacteria References
PCBs Pseudomonas, Burkholderia, Ralstonia, Alcaligenes, Seeger, Unterman,
Achromobacter, Sphingomonas, Comamonas, Furukawa, Bedard,
Rhodococcus, Janibacter, Paenibacillus, Bacillus, Abramowicz, Petric,
Microbacterium, Acinetobacter, Corynebacterium, Abraham
Arthrobacter, Moraxella, Variovorax, Chloroflexi
Aroclor 1242 Alcaligenes odorans, A. denitrificans, Janibacter, Clark, Sierra,
Acinetobacter Brunner
Aroclor 1260 Dehalococcoides, Thermotogales, Cytophagales Tracey, Watts
Kaneclor Rhodococcus sp., Acinetobacter sp. Seeger, Furukawa
Biphenyl, Pseudomonas pseudoalcaligenes, Micrococcus, Furukawa, Ahmed,
Chlorobiphenyl Achromobacter, Acinetobacter, Arthrobacter, Benvinakatti, Focht,
Nocardia, Alcaligenes, Burkholderia xenovorans, Mondello, Baxter,
Bacillus brevis, Ralstonia eutrophus Pettigrew, Springael
Dichlorobiphenyl Pseudomonas putida, Alcaligenes eutrophus McKay, Bedard
Trichlorobiphenyl Rhodococcus globerulus, Pseudomonas sp. Furukawa, Jurcova
Tetrachloro- Comamonas testosteroni, Pseudomonas putida, Barriault, 1999;
biphenyl Corynebacterium, Alcaligenes eutrophus Unterman, 1988
 Pesticide-degrading Bacteria
Pesticides Bacteria
Glyphosate Pseudomonas, Alcaligenes, Bacillus megaterium, Rhizobium, Arthrobacter
atrocyaneus, Geobacillus caldoxylosilyticus, Agrobacterium, Flavobacterium
DDT Bacillus, Staphylococcus, Stenotrophomonas , P. fluorescens, P. aeruginosa
Endosulphan Mycobacterium, Arthrobacter
Parathion Pseudomonas sp, P. diminuta, P. stutzeri, Agrobacterium radiobacter,
Bacillus, Arthrobacter, Flavobacterium, Xanthomonas
Met. parathion Bacillus, Plesimonas, Pseudomonas sp, P. putida, Flavobacterium balustinum
Chlorpyrifos Providencia stuartii, Enterobacter, Micrococcus, Flavobacterium, P. diminuta
Coumaphos Nocardiodes simplex, Agrobacterium radiobacter, Pseudomonas monteilli,
P. diminuta, Nocardia, Flavobacterium
Monocrotophos Pseudomonas mendocina, P. aeruginosa, Bacillus megaterium, Arthrobacter
atrocyaneus, Clavibacter michiganens
Diazinon Flavobacterium, Arthrobacter, Pseudomonas
Fenitrothion Flavobacterium, Arthrobacter aurescenes, Burkholderia
Diuron, linuron Variovorax sp, Arthrobacter globiformis
Phosphotriester Pseudomonas diminuta, Agrobacterium radiobacter, Flavobacterium sp
Ref: Singh & Walker, 2006; Hernández et al., Joutey et al.
 Plastic-degrading Bacteria
Plastics Bacteria
Polyethylene (PE) Brevibacillus borstelensis, Rhodococcus rubber, Sphingomonas,
Pseudomonas chlororaphis, P. fluorescens, Comamonas acidovorans
Polylactic Acid (PLA) Amycolatopsis, Saccharrotrix, Kibdelosporangium, Streptoalloteichus
Polycaprolactone Bacillus brevis, B. pumilus, Amycolatopsis, Clostridium, Paenibacillus
Polypropiolactone Bacillus, Acidovorax, Variovorax paradoxus, S. paucimobilis
Polyhydroxybutirate Schlegelella thermodepolymerans, Pseudomonas, Ralstonia piketti,
(PHB) Acidovorax faecalis, Alcaligens faecalis, Illyobacter delafieldi,
Comamonas, Bacillus, Streptomyces, Caenibacterium thermophilum
Polycarbonates Amycolatopsis sp, Pseudomonas sp, Roseateles depolymerans,
(PHC, PBC) Chromobacterium viscosus
Polyurethane (PU) Comamonas acidovorans
PHB, PES, PCL Actinomadura, Microbispora, Streptomyces, Thermoactinomyces,
Saccharomonospora
Polyvinyl Chloride Pseudomonas putida, P. fluorescens, Ochrobactrum
PBS M. rosea, Excellospora japonica, E. viridilutea
Nylon 4, 6, 12, 66 Pseudomonas sp, Flavobacterium sp., Geobacillus thermocatenulatus
Ref: Tokiwa, 2009; Garrison, 2016; Leja & Lewandowicz, 2009
 Dye-degrading Bacteria
Bacteria Dyes
Aeromonas Crystal violet, Basic fuchsine, Brilliant green, Great red, Malachite
hydrophila green, Acid amaranth, Reactive red, Reactive brilliant blue
Citrobacter Crystal violet, Gentian violet, Methyl red, Congo red, Malachite green,
Brilliant green, Basic fuchsine
Enterobacter cloacae Reactive black, Reactive red, Acid yellow, Acid orange, Disperse yellow
Pseudomonas sp, P. Crystal violet, Reactive red, Reactive orange, Reactive blue, Direct red,
putida, P. aeruginosa, Direct orange, Acid yellow, Acid blue, Acid black, Acid orange, Blue RR,
P. fluorescence, P. Black B, Navy blue, Disperse yellow, Acid red, Malachite green, Fast
nitroreducens green, Orange II, Reactive black, Acid orange, Methyl red
Morganella Acid yellow, Acid orange, Reactive orange, Direct red, Acid blue
Shewanella Crystal violet, Reactive black, Direct red, Acid red, Disperse orange
Bacillus sp, B. subtilis Navy blue, Red RR, Yellow RR, Remazol black B, Acid red, Indigo blue
Clostridium Reactive red, Reactive black, Reactive yellow, Indigo blue
Actinomycetes Reactive yellow
Enterococcus Direct black
Nocardia corallina Crystal violet
Ref: Ali, 2010; Barragán et al., 2007; Shah, 2013, 2014; Bagewadi, 2011
 Metal-degrading Bacteria
Heavy metals Bacteria References
As, Cd, Cu, Co, Zn Acidithiobacillus ferrooxidans White, 1998
Cu, Zn Bacillus spp, Pseudomonas aeruginosa Philip, Gunasekaran
Cd Alcaligenes, Psedomonas, Moraxella, Bacillus cereus Springael, Gazsó
Ag, Hg P. aeruginosa, Citrobacter freundii, C. youngae, Lima e Silva, 2012
Serratia, Klebsiella pneumoniae, Enterobacter
agglomerans, Chryseobacterium
Ag P. stutzeri, Streptomyces noursei, Stenotrophomonas Lima e Silva, 2012;
maltophilia, Thiobacillus ferrooxidans, Acinetobacter Mattuschka, Gazsó
Hg(II) Alcaligenes faecalis, Bacillus pumilus, P. aeruginosa, Jaysankar, 2008;
Brevibacterium iodinum, Deinococcus radiodurans Brim, 2000
Fe, Cu, Mn, Ni Bacillus subtilis, Bacillus licheniformis Beveridge, Holan
Cr(VI), Cr(III) Enterococcus gallinarum, Achromobacter, B. subtilis, Sayel; Zhu; e Silva;
P. aeruginosa, P. putida, P. fluorescens, Burkholderia, Fernandez, 2012;
Azotobacter, Alcaligenes, Stenotrophomonas Lalithakumari, 2016
Se(IV), Ag, Hg Enterobacter cloacae Yee, 2007; e Silva
Al, Cu, Fe Microbacterium liquefaciens Kanayama, 2005
Cd, U, Pb, Co, Ni Citrobacter spp. Jain, 2014; Sar 2001
 Radionuclide-degrading Bacteria
Radionuclide Bacteria
Technetium, E. coli, Desulfovibrio desulfuricans, D. fructosovorans, D. vulgaris, Thiobacillus
99Tc(VII) ferroxidans, T. thiooxidans, Geobacter sulfurreducens, G. metallireducens
Neptunium, Shewanella putrefaciens, Citrobacter, Pseudomonas fluorescens, P. aeruginosa,
237Np(V) Micrococcus luteus, Streptomyces viridochromogenes
Plutonium, Microbacterium flavescens, Bacillus sphaericus, B. subtilis, B. circulans, B.
239Pu, 240Pu, polymyxa, E. coli, Shewanella oneidensis, S. alga, G. metallireducens,
Pu(V), Pu(VI) Clostridium, Aeromonas hydrophila
Cesium, 137Cs Rhodococcus erythropolis, E. coli, P. fluorescens, P. aeruginosa, Deinococcus
radiodurans, Arthrobacter, Nocardia
Thorium, Th P. aeruginosa, Mycobacterium smegmatis
Uranium, Arthrobacter, Acinetobacter, Anaeromyxobacter, Bacillus, Citrobacter, Geothrix,
235U(VI) Cellulomonas, Clostridium, Deinococcus, Desulfovibrio, Desulfosporosinus,
Desulfitobacterium, E. coli, Geobacter, Pseudomonas, Paenibacillus, Ralstonia,
Rahnella, Salmonella, Shewanella, Thiobacillus, Thermus, Thermoanaerobacter
Radium, Ra Desulfovibrio vulgaris, Pseudomonas aeruginosa
241Americum Serratia sp.
Iodine, 129I Desulfovibrio desulfuricans, Shewanella putrefaciens
Ref: Lloyd, 2005; Newsome, 2014; Williams, 2013; www.biorad.igib.res.in
 Streptococcus
Bacillus
Corynebacterium Staphylococcus

Alcaligenes faecalis Shigella

Pseudomonas aeruginosa Escherichia coli

Acinetobacter
Klebsiella pneumoniae

Actinomycetes Enterobacter cloacae Serratia marcescens Mycobacterium

Enterococcus Aeromonas hydrophila Ralstonia Achromobacter

Shewanella

Burkholderia
Providencia Variovorax paradoxus

Streptomyces

Rhodococcus Clostridia Deinococcus radiodurans Proteus

 OTHER MICROBIAL DEGRADATION
PGPR & PGPB, Microfungi & Mycorrhiza, Algae & Protozoa,
Genetically Engineered Microorganisms (GEMS)

Bacteria Fungi Algae Protozoa

 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
 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.
 Degradation by Yeasts
Pollutants Yeasts References
Aliphatic/petroleum Candida lipolytica, C. tropicalis, C. apicola, Bartha, 1986;
hydrocarbons Rhodoturula rubra, R. mucilaginosa, Geotrichum, Scheuer, 1998;
Aureobasidium pullulans, Trichosporon mucoides 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, Bahafid, 2011,
Wickerhamomyces anomalus, C. tropicalis 2012
S. cerevisiae, Pichia guilliermondii, Yarrowia Ksheminska, 2006,
lipolytica, R. pilimanae, Hansenula polymorpha 2008
Copper Schizosaccharomyces pombe Saisubhashini, ‘11
 YEASTS

Rhodoturula Candida tropicalis Botrytis cinerea Hansenula polymorpha

Aureobasidium pullulans Saccharomyces cerevisiae Geotrichum Schizosaccharomyces pombe

 Degradation by Filamentous Fungi
Pollutants Fungi References
Aliphatic & Amorphoteca, Neosartorya, Talaromyces, Aspergillus, Chaillan,
aromatic HCs Cunninghamella, Penicillium, Fusarium, Cephalosporium Singh, Steliga
Toluene Cladophialophora, Exophiala, Leptodontium, Francesc,
Pseudeurotium zonatum 2001
PAHs Pleurotus ostreatus, Cunninghamella elegans, Okparanma,
Chrysosporium pannorum, Aspergillus niger, 2011;
Phanerochaete chrysosporium, Antrodia vaillantii Maigari, 2015
Hydrocarbons, Cladosporium cladosporioides Steliga, Chen,
pesticide, gelatin Abruscia
Chlorpyrifos P. chrysosporium, Hypholama, Coriolus, Trichoderma Singh &
Glyphosate Penicillium, Trichoderma, A. niger, Alternaria alternata Walker, 2006
Malachite green Acremonium kiliense, Aspergillus flavus, A. solani Youssef, Ali
PEA, CPAE, PBC Penicillium, Rhizopus delemar, R. arrhizus Tokiwa, 2009
Polycaprolactone Penicillium funiculosum, A. flavus, R. arrhizus, R. delemar
Polyurethane Rhizopus delemar, Curvularia senegalensis
Uranium, Thorium Rhiloprzs juvanicus Sears
PCBs, Cd, Pb Aspergillus niger, Rhizopus arrhizus Pal, 2010
 Alternaria alternata Fusarium

Penicillium chrysogenum
Trichoderma

Pleurotus ostreatus Exophiala Cladosporium cladosporioides

Rhizopus

Aspergillus niger Aspergillus ustus Aspergillus nidulans Aspergillus versicolor

 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.
 Degradation by Algae & Protozoa
Pollutants Microorganisms References
Hydrocarbons Prototheca zopfii Walker, 1975
PAHs, pyrene, Chlorella vulgaris, Scenedesmus platydiscus, S. Wang, 2007;
fluoranthene quadricauda, Selenastrum capricornutum Ueno, 2008
Monocrotophos Aulosira fertilissima, Nostoc muscorum Megharaj, 1987
Azo dyes C. vulgaris, C. pyrenoidosa Jinqi, 1992
Methyl red, orange C. vulgaris, Lyngbyala gerlerimi, Nostoc lincki, El-Sheekh,
II, G-Red, basic Oscillatoria rubescens, Elkatothrix viridis, Volvox 2009
cationic, b. fuchsin aureus
Amido Black Chroococcus minutus Parikh, 2005
Sky Blue, Acid Red Gloeocapsa pleurocapsoides, Phormidium ceylanicum
Heavy metals Chlorella, Anabaena inacqualis, Westiellopsis Dwivedi, 2012
prolifica, Stigeoclonium lenue, Synechococcus sp.
Cr(VI), Cd(II), Cu(II) Scenedesmus incrassatulus Castro, 2004
Cr(III) Chlorella sorokiniana Akhtar, 2008
241 Am, 237Pu(III-VI) Thalassiosira pseudonana Fisher,1983
Benzene, Heteromita globosa (protozoa/grazing bacteria) Mattison, 2005
methylbenzene
 ALGAE

Chlorella Nostoc Synechococcus PROTOZOA

Scenedesmus quadricauda Stigeoclonium Oscillatoria Ciliate protozoa

surrounding bacteria

Volvox aureus Prototheca Anabaena

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.
 Degradation by GEMs
Pollutants GEMs References
Aliphatic, aromatic, Pseudomonas Markandey, 2004;
terpenic, PAHs, PCBs Erickson, 1993
TCE, BTEX, salicylate, Pseudomonas putida TOL, RA500, pAC25, Sayler, ‘00; Panke,
naphthalene, benzoate pKF439, KT2442, TVA8 ‘98; Applegate, ‘98
BTEX, naphthalene, Pseudomonas fluorescens HK44, Sayler, 2000;
anthracene 10586s/pUCD607 Sousa, 1997
TCE, toluene, benzene P. pseudoalcaligenes KF707-D2 Suyama, 1996
Chlorobiphenyls Comamonas testosteroni VP44 Hrywna, 1999
Chromium, PCBs, Alcaligenes eutrophus AE104/pEBZ141, Srivastava, Dyke,
narcotics H850Lr, 2050 Layton,
2,4-D Bacillus cepacia BRI6001L, A. paradoxus Masson, 2002
Toluene, mercury Deinococcus radiodurans Brim, 2000
Hg2+ Rhodopseudomonas palustris Xu & Pei, 2011
PCBs, heavy metal R. eutropha A5, Achromobacter sp. LBS1C1, Menn, 2008
A. denitrificans JB1, R. eutropha CH34
Cd2+ Mesorhizobium huakuii subsp. rengei strain Sussman, 1988
B3, Astragalus sinicus, Arabidopsis thaliana
BIOREMEDIATION & BIODEGRADATION
The principles, methods, strategies, mechanisms,
and limiting factors
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)
 In-situ Bioremediation
Natural Attenuation, Biostimulation, Bioaugmentation

• Natural attenuation or bioattenuation is the reduction of

contaminant concentrations in the environment through
biological
Indigenous processes (microbial biodegradation, plant and animal
& exogenous
uptake), physical phenomena (advection, dispersion, dilution,
microorganisms
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).
 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.
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.
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.
 Aerobic Degradation:
R R R R RCOOH
NADH NAD+ H OH
C NAD+ NADH OH O +
COOH COOH
E1 C
H OH E2 E3 E4
O2
OH O2 OH O
Alkylbenzene Dihydrodiol 2,3-Dihydroxy- Ring fission
alkylbenzene product 2-Oxopenta-
4-enoate

E1 = Alkylbenzene dioxygenase
E2 = cis-alkylbenzene glycol dehydrogenase
E3 = 2,3-dihydroxyalkylbenzene 1,2-dioxygenase
E4 = ring fission product-hydrolysing enzyme Smith & Ratledge 1989

Anaerobic Degradation:
CoA
S

Heider et al. 1999 O O O O

CoA

CH3 HO CH3 O CH3 O CH2 O CH2 O S

H
H2C C

Ethylbenzene 1-Phenylethanol Acetophenone Benzoylacetyl-CoA

Dehydrogenase Dehydrogenase Benzoylacetyl-CoA
Carboxylase forming enzyme CoA thiolase

Ethylbenzene Acetophenone Benzoylacetate Benzoylacetate-CoA Benzoyl-CoA

1-Phenylethanol

H2O 2[H] CO2

2[H] CoASH CoASH Acetyl-CoA
 Pathway for Degradation of Aliphatic Compounds

The degradation of a straight chain hydrocarbon:

E1 E2
E3

Bacteria involved: Enzymes involved:

Pseudomonas putida E1 = alkane monooxygenase
Fungi (yeast) involved: E2 = fatty alcohol dehydrogenase
Candida maltosa, Candida tropicalis, Candida apicola E3 = fatty aldehyde dehydrogenase,

The degradation of a cyclic hydrocarbon:

(Harayama et al. 1999)

 Pathway for Degradation of Aromatic Compounds
Bacteria involved:
Pseudomonas, Rhodococcus, Mycobacterium
Benzene Arene oxide
Fungi (yeast) involved:
Pleurotus ostreatus

cis/trans-dihydrodiol Cathecol

Naphthalene cis-1,2-naphthalene 1,2-dihydroxynaphthalene Salicylic acid

dihydrodiol

The microbial degradation of catechol

 Pathway for Degradation of PCBs

Organisms involved:
Dehalococcoides,
Thermotogales,
Chloroflexi

Pathway for anaerobic dechlorination of a highly chlorinated congener (Fish & Principe, 1994).

Organisms involved:
Achromobacter,
Beijerinckia,
Pseudomonas
putida

Pathway for aerobic degradation of PCBs into chlorobenzoates (Sylvestre & Sandossi, 1994).
Mechanism in Plastic Degradation
 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
Mechanism in Radionuclide Degradation
Mechanisms of radionuclide bioremediation by micro-
organisms include bioreduction, biomineralisation,
bioaccumulation, and biosorption.
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
 Temp.

(Bodishbaugh, 2006)

pH
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.
 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.