1. Biotechnological applications for environmental waste
management
What is environment?
What are remedial methods?
Natural
Why environmental problems?
Technology development?
Xenobiotic compounds
Emerging toxicants containing waste
Feb. 17, 2014

2. Major environmental issue
Impact of green house gases
Formation of dioxin-like compounds
in the environments

3. Important environmental problems
• 1. Global warming- GHG (CO2, CH4, N2O)
• 2. Energy problem- Bioethanol, biodiesel,
Hydrogen how?-a substitute• 3. Water contaminants/toxicants/eutrophication
• 4. Soil degradation/solid waste generation
• 5. Air pollutants

4. Bioremediation- A potential approach for clean
and Phytoremediation
green environment
4.1
T
Caviation methods
Ec
5. Toxicology-
Toxicogenomics
& detoxification
Reporter
Gene
nt
me
n
viro r,
En Ai
l
s oi
d
r an
te Wa
l
tura ic
Na iot
b
e no o un ds
X
p
com
try
s
mi n &
e
Chtractioysis ts
1. Ex anal utan
p
of
oll
4.2 Microbial
Bioremediation
in situ &
ex situ
Prob lar
es
bios &
ensor
2. nism
rga
roo robial
Mic Mic
gy
&
ol o
4. E
ngin
Mole eering
cu
Climate/
Climate
meteorology
5.Bioproducts
&
Bio
Chemicals biomaterials
Process
Engineering
Biotechnology
3. Molecular
BiologyCatabolic
Enzymes &
genes
Bio Environment
&
Engineering
(Environmental
Biotechnology)
6. System
6. System
approach
approach

5. Origin of Earth and Environment
The Universe created by colossal explosion that we now refer to as
the Big Bang and Planets of the solar system
The Earth
and
Environment

6. The earth
The ocean

7. Climatic changes on the Earth
Movement
of air
during
rotation
of the
Earth
and
formatio
n of
cells

8. PHYSICAL FACTORS AND BIOTIC & ABIOTIC MATERIALS
IN ENVIRONMENT

9. Origin of life on the Earth

10. Classification of living organisms
Biodiversity

11. Classification of microorganisms
Classification of plants and animals

12. Biomolecules in organisms
•
It is organic compound composed of carbon, hydrogen, oxygen, nitrogen,
sulfur, phosphorus and sometimes some other elements.
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Different types of biomolecules are:
A. Small molecules mainly include molecules like:Lipids such as phospholipids, glycolipids, sterols, and glycerolipids: Carbohydrates- provide energy and act as energy storage molecules.
Vitamins-survival and health of organisms.
Hormones, neurotransmitters and metabolites: - metabolic processes and
functions.
•
•
B. Monomers include:Amino acids: - building blocks of proteins function as genetic code and as
biomolecules, that assist in other processes such as lipid transport.
Nucleotides: - Chemical energy (ATP,GTP), assist in cellular signaling, and
enzymatic reactions (coenzyme A, flavin adenine dinucleotide, flavin
mononucleotide, nicotinamide adenine dinucleotide phosphate etc ).
Monosaccharide: - provides energy and are the building blocks of
polysaccharides.
•
•

13. Emergence of man and social environment

14. • Understanding Human Behavior and
the Social Environment
• Natural Resources:
Air, water, soil, minerals etc.
• Industrial Revolution-1760-1850 onwards

15. Environmental degradation
• The ten threats identified in 2004 by the High Level
Threat Panel of the United Nations are these:
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•
•
•
•
•
•
•
Poverty
Infectious disease
Environmental degradation
Inter-state war
Civil war
Genocide
Other Atrocities (e.g., trade in women and children for
sexual slavery, or kidnapping for body parts)
Weapons of mass destruction (nuclear proliferation,
chemical weapon proliferation,
biological weapon proliferation)
• Terrorism
• Transnational organized crime

16. Major environmental issues before us

17. Contd.
Pulp and paper mill effluent
Molasses from sugar cane
mill for distillation
Petroleum waste
17 million gallon oil spill under the
Greenpoint section of Brooklyn
Waste dumping
grounds in Delhi

18. Emerging industrial pollutants
Industrial sources
Pops in pulp & paper effluent
Pops in distillery effluent
• Pulp and paper
industry
lignosulphonic acid,
chlorinated
resin acid, chlorinated
phenols
dioxins, dibenzofuran,
bipheny
chlorinated hydrocarbon
Distillery industry
melanoidins
Pops in tannery effluent
Tannery industry
Chlorinated phenolics,
PCPs, chromium
Municipal
Plastic, dioxins, antibiotic
etc
Pops in municipal sludge
Transport
Metals, organics
Incineration and plastics
etc.

19. Fate of Organic Compounds in the Environment
ENVIRONMENTAL POLLUTANTS
AIR
Water
Soil

20. Major conferences and meetings
• United Nations Conference on the Human EnvironmentSweden in 1972: Declaration containing 26 principles
concerning the environment and development
6. Pollution must not exceed the environment’s capacity to clean itself
19. Environmental education is essential
20. Environmental research must be promoted, particularly in developing
countries
The United Nations Conference on Environment and development
(UNCED)- Rio Summit and- Earth Summit
United Nations Framework Convention on Climate Change-Kyoto
Protocol-reduce emissions of greenhouse gases
In Doha, Qatar, on 8 December 2012, the "Doha Amendment to the Kyoto
Protocol

22. Global warming gases in the environment

23. The potential mechanisms that regulate the
responses of GHGs (CO2, CH4 and N2O)
Production and consumption to elevated N (ANPP, aboveground net primary productivity;
BNPP, belowground net primary productivity; SOC, soil organic carbon; DOC, dissolved
23
organic carbon; DIN, dissolved inorganic nitrogen; DON, dissolved organic nitrogen).

24. Climate change and Biodiversity
Role of organisms- autotrophic & chemoautotrophic in CO2 mitigation
Carbonic anhydrase
Biosurfactants
Bioscrubbers for CO2 sequestration

25. Solid waste generation from different sources
1. Garbage- putrescible, heating value
2. Rubbish- Non putrescible, heating value
3. Pathological
4. Industrial
Municipal waste
5. Agriculture waste
6. Medical waste
7. Electronic waste
Biodegradable
Natural waste
Non biodegradable waste
Xenobiotic
Hazardous - ignitable (i.e. flammable), oxidizing, corrosivity, toxic
Radioactive, eco-toxic, explosive
•Non-hazardous waste

27. Biogas

28. Waste water treatment options
Primary
treatment
Screening
Grit removal
Equalization
Storage
Grinders
Flocculation
Sedimetation
Floatation
Coagulation
Secondary
treatment
Aerobic
Tertiary
treatment
Anaerobic
Activated
sludge process
Tricking filter
Fixed film reactor
Rotating reactor
Stabilization pond
Chemical oxidation
Filtration
Carbon adsorption
Osmosis
Electrolysis
Cavitations
Photodegradation
Upflow anaerobic sludge
blanket reactor
An. Fludized bed reactor
Anaerobic lagoons
An. Contact reactor
An baffled reactors

29. Origin of different types of chemical
compounds in the environment and their fate

30. Significance of lignocellulosics
• Total forest cover 3870 million hectares or 30% of the
earth’s land area.
• 50% 0f all biomass with an estimated annual production of
50 billion tons.
• Half of the residues remain unused while some are used
as material and energy-green manure and feed for low
producing ruminants.
• Major substrate for food, feed, energy, and other
commercial items.
• Degrading enzymes have potency for fuel, chemicals, food,
brewery and wine, animal feed, textile and laundry, pulp
and paper, agriculture and pharmaceuticals.
• Unused biomass is major source of “waste”- pose an
environmental pollution problem.

31. Lignocellulosic ecosystem : cellulolytic,
hemicellulolytic and liglinolytic strains

32. Structure of lignocellulose
• Cellulose : Made up of linear chains of β-1,4-linked
D-glucose residues.
• Hemicellulose : Made up of branched
heteroglycans with a backbone of β-1,4-linked Dxylopyranosyl residues with branches of α-1,3linked L-arabinofuranosyl and α-1,2-linked 4-Omethyl-glucoronic acid residues.
• Lignins is heterogeneous, three dimensional
polymer composed of oxyphenyl propanoid units
connected by c-c and c-o-c linkages. It is formed
by random coupling of coniferyl alcohol, sinapyl
alcohol and p-coumaryl alcohol.

33. Lignocellulosic components and its
importance as biomaterials
Lignocellulose
Cellulose
Pulp
Glucose
Cellulose
Hemicellulose
Furfurals
Xylose
pulp
derivatives
Fuel
Feed and
commercial
items
Single cell proteins
Xylitol
Lignin
Vanillin
Gallic acid
Phamaceuticals
Herbicides
Antifoming agents
House hold products

35. Degradation of cellulose by enzyme cellulase
Pre hydrolysis: Acid, Alkali, ammonia
Enzymes: Thermolhilic, alkalophilic, multiplicity
Products-fuel. feed, food, commercial products
Biofuels
Applications: Pulp, industries, food, feed, fuel etc.

36. Generalized mechanism of enzymatic cellulose hydrolysis
Problems:
1. End product inhibitions
Biotechnology
1. Mutants
2.Protoplast fusion
3. Genetic engineering 4. More enzyme
5. Protein engineering 6. Cellulosomemulticomponents enzyme system

37. Hemicellulose and degradation- Enzyme xylanase
•HC is homo and heteropolymer
•AnhydroB-(1,4)D-xylopyrannose, mannopyranose, glucopyranose, galactopyranose
•Monomer is D-Xylose
Applications
1. Energy
2.Food & feed industries
3. Pulp and paper- Biopulping & biobleaching
4. Waste management
5. Saccharifications of agrowaste
6. Nutritional quality 7. Enhancing texture

38. Lignin structure and degradation
Fig. 3: The
1.
2.
3.
4.
5.
6.
three common monolignols
Prior 1970- no information for degradation
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C-labelled synthetic lignin
Electron microscopy
Lignin degrading fungi-White rot, soft rot, Brown rot, other
Enzymes
Physiological parameters-oxygen, nitrogen, carbon, temp. pH, nutrients

39. Involvement of enzymes in degradation of lignin
1. Lignin peroxidase (LiP)
Extracellular, H2O2 dependent, glycosylated hemprotein,
MW 41-42 kDa,
2. Manganese peroxidase (MnP)
Extracellular, H2O2 dependent, MnII-dependent,
neutral carbohydrate, MW 41-45 kDa
3. Laccase
Extracellular, non-heme, copper containing
4. Other phenol-oxydizing enzymes
5. Glyoxal oxidase
Support oxidative turn over of LiP and MnP reduction of O2 to
H2O2 with oxidation of substrate
Applications
1. Industrial, 2.Commercial, 3. house holds, 4. waste management

40. Biodegradation and bioconversion of lignocellulosic waste
in the environment
Fermentation
Bioethanol
1.Cellulases
2.Xylanases
3.Laccase4.Lignin
peroxidase &
5.Manganese
peroxidase
Schematic diagram- ethanol production from sugarcane bagasse

44. Biotechnological innovations: biomaterials- biorefinery
• Screening for organisms with novel enzymes:
enzyme evolution-random mutagenesisrecombination-selection-screening
• Strain improvement of existing industrial organisms
and enzyme engineering
• Production and operation related factors-Process
optimization
– Substrate
– Culture conditions
– Recycling of enzymes
– Redesigning of processes
– Process optimization models and soft wares

45. Strain improvement of existing industrial
organisms and enzyme engineering
• Hyper producer organisms
• Robust organisms
– Culture conditions: isolation of 1% strains-Great
culture plate enigma
– Biomining through:
• Genomics-complete blue print of the organism
• Metagenomics-genomics with functional aspects at
community level
– Necessity of discovering unique gene, cloning,
quantitative analysis, and expression

46. Process optimization
Bioreactors
laboratory scale
Pilot scale
Industrial scale
Liquid state
Fermentation
-Homogeneous
-Heterogeneous
Stirred tank reactor
Air-lift or bubble-column
reactor
Batch
Continuous
Fed-batch
Solid state
Fermentation
Flask
Tray
Packed bed
Tunnel
Paddle
Rotating drum
Tower

47. •
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Biofuel Production and integrated pollution
control using microalgae
Microalgal Farming and CO Mitigation
2
Microalgal Farming using Wastewater
Microalgal Farming using Marine Microalgae
Possible routes to
energy products
Basic overview of the pathway of
carbon capture and lipid biosynthesis

48. Anthropogenic chemical compounds in environemnt

49. Persistent organic pollutants in environment
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Wide distribution- POPs detected from soil, water,
food items, commercial products
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Sources- Mostly chlorinated organic compounds
formed unintentionally- industries, commercial,
agriculture, military, other human activities, and
natural sources
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Insufficient data- No reliable data for their persistence
in Indian environment- No management practices
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Problems in detection methods- Methods for detection
and degradation not up to the mark.
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Highly toxic and recalcitrant- ultimate formation oftetrachlorodibenzo-p-dioxin and furan-like
compounds-complete physiological impairment.
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Tremendous scope for medical diagnostics and
therapy and products.
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Therefore, methods & technology for detection,

50. Degradation of aromatic compounds

51. Key component: POPs
Emerging environmental contaminant in present scenario

52. Classification of POPs
• Dirty Dozen - UNEP Stockholm Convention on Persistent Organic Pollutants - 2001
aldrin
dieldrin
toxaphene
chlordane
endrin
mirex
polychlorinated biphenyls
heptachlor
DDT
polychlorinated
dibenzo-p-dioxins
polychlorinated
dibenzofurans
hexachlorobenzene
• UNEP has added nine new chemicals (all are poly haloginated compounds) to the
"dirty dozen" list of restricted or banned toxic chemicals in 2009.
• Some other organic pollutants that may be persistent or lead to formation of dioxin
like compounds in the environment include:
Poly Aromatic
Hydrocarbons
Aromatic amines
Pyrethroids
Volatile Organic
Compounds
Metabolites of VOCs
Phthalates

53. Biomagnification
>Biomagnification, also known as bioamplification, or biological
magnification is the increase in concentration of a substance, such as
the pesticide DDT, that occurs in a food chain as a consequence of:
Food chain energetics
>Low (or nonexistent) rate of excretion/degradation of
the substance.

54. Persistence and detection of dioxin-like POPs
•
Dioxin detected from food items, human exposure, milk and its
products, environmental sources from US, Japan and EU countries.
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No reliable data from developing countries including India.
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Detection methods.
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Instrument development.
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Thermokinetic modelling,
equilibrium modelling,
statistical determinations
and others.
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Field validation.
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Laboratory

55. Biodegradation strategies for removal of
organic compounds in environment
• Possible use of biodegradation processes
-Indigenous microorganisms
-Genetically modified microorganism
-Continuous enrichment of microorganism
#Culture dependent and culture independent
microorganisms-Metagenomic approach

56. Fate of organic compounds in the uptake into
the cells and degradation, assimilation and
mineralization

57. Degradation of methane
Catabolic gene in degradation of alkanes

58. Remediation of POPs in waste sites

59. What is Bioremediation?
• Bio
= living
• Remediate = to bring the sites and affairs
•
into the original states
• Bioremediation can be defined as any process
that uses microorganisms, green plants or their
enzymes to return the environment altered by
contaminants to its original condition.
• Bioremediation technology using
microorganisms was reportedly invented by
George M. Robinson.
• Use of biological sciences and technology for
metals and organic compounds remediation.

60. Bioremediation
Potential alternative for conservation and management of
environment
Bio
= living
Remediate = to bring the sites and affairs into the original states
Bioremediation can be defined as any process that uses
microorganisms, fungi, green plants or their enzymes to return
the environment altered by contaminants to its original
condition.
BIOAUGUMENTATION
BIOSTIMULATION
Enzymatic methods
Ex situ Bioremediation
In situ Bioremediation

61. Soil–plant–microbial interactions in remediation of
pollutants in environment
Importance of soil–plant–microbial interactions in bioremediation

62. Biocolloid formation in metal bioremediation
Colloidal aggregation–flocculation or attachment to inorganic and
organic particles in water can lead to settling and removal of
metals from the water column to the bottom sediment

63. Technologies in Bioremediation
Ex situ bioremediation
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Electro kinetically enhanced remediation
Soil Washing
Soil mound Bioxidation ProcessDispersing by Chemical Reaction
Biocolloid formation
Bioreactors
Land Treatment
Composting
Lagoons (aerobic/ anaerobic)
Partial peroxidation
In situ bioremediation
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Bioventing
Bioslurping
Biopiling

64. Limitation of in-situ removed by Enhancement
of Bioremediation
Use of microorganisms to degrade contaminants in saturated soils
and groundwater obtaining harmless chemicals as end products

65. Biosafety assessment of leachate after biological
treatments
Cytotoxicity
Genotoxicity
Estrogenicity
MTT Assay
Comet Assay
E-Screen
Assay
(Nwagbara et al.
2007)
(Singh et al.
1988)
(Vanparys et al.
2006)
*Huh 7 cell line is used for evaluating cytotoxicity and genotoxicity as hepatocytes express many
nuclear receptor proteins that regulate the expression of xenobiotic metabolizing enzymes like CYP
1A1.
*An estrogen receptive cell line MCF 7 is used for E-Screen assay.
References:1) Nwagbara O, Darling-Reed SF, Tucker A, Harris C, Abazinge M, Thomas RD and Gragg RD. 2007. Induction of cell death, DNA strand breaks, and cell cycle
arrest in DU145 human prostate carcinoma cell line by benzo[a]pyrene and benzo[a]pyrene-7,8-diol-9,10-epoxide. International Journal of Environmental Research and Public
Health.4: 10–14.
2) Singh NP, McCoy MT, Tice RR and Schneider EL. 1988.A simple technique for quantitation of low levels of DNA damage in individual cells.Experimental Cell
Research.175: 184-191.
3) Vanparys C, Maras M, Lenjou M, Robbens J,Van Bockstaele D and Blust R. 2006.Flow cytometric cell cycle analysis allows for rapid screening of estrogenicity in MCF-7
breast cancer cells. Toxicology in Vitro.20:1238–1248.

66. Molecular Probes for tracking

67. Miniaturized ecogenomic sensors to measure
microbial activity-carbon sequestration
•
The sensors could be installed
into advanced ocean
observatories to monitor DNA
and RNA from diverse microbial
communities.
•
Subsystems for monitoring, data
management and communication,
and data modelling would be
incorporated for data
contextualization.
•
The sensors would report to a
worldwide network of
laboratories in real time by
satellite telemetry.
•
Culturable and nonculturable
(metagenomics) bacteria for
degradation of organic
compounds & carbon
concentrating mechanisms and
value added products.

68. System biology approaches
The four-step paradigm for metabolic systems biology

69. Conclusion
• POP/ DF and its congeners are difficult to
detect in the environment.
• Degradation of POP/DF in several steps by
formation of intermediary metabolites.
• Degrading genes are present in various
locations.
• Bioremediation difficult.
• Bioassay methods are useful which may be
optimized and developed.
• System approach is recent days methods.

70. • Thanks