Biotechnological applications for environmental waste management

Natural Xenobiotic compounds

Emerging toxicants containing waste

Feb. 17, 2014

What is environment? Why environmental problems?

What are remedial methods? Technology development?

Major environmental issueImpact of green house gases

Formation of dioxin-like compounds in the environments

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

3. Molecular Biology-Catabolic

Enzymes & genes

1. Chemistry-

Extraction &

analysis

of pollutants

2.

Microorganism

& Microbial

Ecology

5. T

oxic

olog

y-To

xico

geno

mic

s&

det

oxifi

catio

nR

epor

ter

Gen

e

4. E

ngin

eerin

gM

olec

ular

Pr

obes

& b

iose

-ns

or

Environment

Air,

Water and soil

-Natural

Xenobiotic

compounds

T

BioChemicalsProcess

Engineering

Biotechnology

Bio Environment&

Engineering(Environmental

Biotechnology)

4.2 Microbial Bioremediation

in situ & ex situ

4.1 PhytoremediationCaviation methods

Bioremediation- A potential approach for clean and green environment

5.Bioproducts &

biomaterials

6. System approach

Climate Climate/meteorology

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

The earth

The ocean

Climatic changes on the Earth

Movement of air during rotation of the Earth and formation of cells

PHYSICAL FACTORS AND BIOTIC & ABIOTIC MATERIALS IN ENVIRONMENT

Origin of life on the Earth

Classification of living organisms

Biodiversity

Classification of microorganisms

Classification of plants and animals

Biomolecules in organisms• It is organic compound composed of carbon, hydrogen, oxygen, nitrogen,

sulfur, phosphorus and sometimes some other elements.

• 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.  

Emergence of man and social environment

• Understanding Human Behavior and the Social Environment

• Natural Resources:Air, water, soil, minerals etc.

• Industrial Revolution-1760-1850 onwards

Environmental degradation • The ten threats identified in 2004 by the High Level

Threat Panel of the United Nations are these:• 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

Major environmental issues before us

Pulp and paper mill effluent Molasses from sugar cane mill for distillation

17 million gallon oil spill under the Greenpoint section of Brooklyn

Waste dumping grounds in Delhi

Petroleum waste

Contd.

Emerging industrial pollutantsIndustrial sources• Pulp and paper

industry lignosulphonic acid,

chlorinated resin acid, chlorinated

phenolsdioxins, dibenzofuran,

biphenychlorinated hydrocarbonDistillery industry melanoidinsTannery industryChlorinated phenolics,PCPs, chromiumMunicipalPlastic, dioxins, antibiotic

etcTransportMetals, organics

Incineration and plastics etc.

Pops in distillery effluent Pops in pulp & paper effluent

Pops in tannery effluent

Pops in municipal sludge

Fate of Organic Compounds in the Environment

AIR

Water Soil

ENVIRONMENTAL POLLUTANTS

Major conferences and meetings• United Nations Conference on the Human Environment-

Sweden in 1972: Declaration containing 26 principles concerning the environment and development 6. Pollution must not exceed the environment’s capacity to clean itself19. Environmental education is essential20. 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

Global warming gases in the environment

The potential mechanisms that regulate the responses of GHGs (CO2, CH4 and N2O)

23

Production and consumption to elevated N (ANPP, aboveground net primary productivity; BNPP, belowground net primary productivity; SOC, soil organic carbon; DOC, dissolved organic carbon; DIN, dissolved inorganic nitrogen; DON, dissolved organic nitrogen).

Climate change and BiodiversityRole of organisms- autotrophic & chemoautotrophic in CO2 mitigation

Carbonic anhydrase Biosurfactants Bioscrubbers for CO2 sequestration

Solid waste generation from different sources

1. Garbage- putrescible, heating value 2. Rubbish- Non putrescible, heating value

3. Pathological4. Industrial Municipal waste5. Agriculture waste6. Medical waste7. Electronic waste

Biodegradable Non biodegradable waste

Natural waste Xenobiotic Hazardous- ignitable (i.e. flammable), oxidizing, corrosivity, toxic Radioactive, eco-toxic, explosive

Non-hazardous waste

Biogas

Waste water treatment options

Primary treatment

ScreeningGrit removalEqualizationStorageGrindersFlocculationSedimetationFloatationCoagulation

Secondary treatment

Aerobic Anaerobic

Tertiarytreatment

Chemical oxidationFiltrationCarbon adsorptionOsmosisElectrolysisCavitationsPhotodegradation

Activated sludge processTricking filterFixed film reactorRotating reactorStabilization pond

Upflow anaerobic sludge blanket reactorAn. Fludized bed reactorAnaerobic lagoonsAn. Contact reactorAn baffled reactors

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

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.

Lignocellulosic ecosystem : cellulolytic, hemicellulolytic and liglinolytic strains

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 D-xylopyranosyl residues with branches of α-1,3-linked L-arabinofuranosyl and α-1,2-linked 4-O-methyl-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.

Lignocellulosic components and its importance as biomaterials

Lignocellulose

Cellulose Hemicellulose

LigninVanillin

Gallic acidPhamaceuticals

HerbicidesAntifoming agents

House hold productsPulp

GlucoseCellulosederivatives

Fuel

Feed and commercial

items

FurfuralsXylosepulp

Single cell proteinsXylitol

Degradation of cellulose by enzyme cellulase

Pre hydrolysis: Acid, Alkali, ammoniaEnzymes: Thermolhilic, alkalophilic, multiplicityProducts-fuel. feed, food, commercial productsBiofuels

Applications: Pulp, industries, food, feed, fuel etc.

Generalized mechanism of enzymatic cellulose hydrolysis

Problems:1. End product inhibitions

Biotechnology1. Mutants 2.Protoplast fusion3. Genetic engineering 4. More enzyme5. Protein engineering 6. Cellulosome-

multicomponents enzyme system

Hemicellulose and degradation- Enzyme xylanase

•HC is homo and heteropolymer•AnhydroB-(1,4)D-xylopyrannose, mannopyranose, glucopyranose, galactopyranose•Monomer is D-Xylose

Applications1. Energy 2.Food & feed industries3. Pulp and paper- Biopulping & biobleaching4. Waste management5. Saccharifications of agrowaste6. Nutritional quality 7. Enhancing texture

Lignin structure and degradation

1. Prior 1970- no information for degradation2. 14C-labelled synthetic lignin3. Electron microscopy4. Lignin degrading fungi-White rot, soft rot, Brown rot, other5. Enzymes6. Physiological parameters-oxygen, nitrogen, carbon, temp. pH, nutrients

Fig. 3: The three common monolignols

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. LaccaseExtracellular, non-heme, copper containing

4. Other phenol-oxydizing enzymes

Applications1. Industrial, 2.Commercial, 3. house holds, 4. waste management

5. Glyoxal oxidaseSupport oxidative turn over of LiP and MnP reduction of O2 to H2O2 with oxidation of substrate

Bioethanol

1.Cellulases2.Xylanases3.Laccase-4.Lignin peroxidase &5.Manganese peroxidase

Biodegradation and bioconversion of lignocellulosic waste in the environment

Fermentation

Schematic diagram- ethanol production from sugarcane bagasse

Biotechnological innovations: biomaterials- biorefinery

• Screening for organisms with novel enzymes: enzyme evolution-random mutagenesis-recombination-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

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

Process optimization Bioreactors

Liquid stateFermentation

-Homogeneous -Heterogeneous

Stirred tank reactorAir-lift or bubble-column

reactor

Solid state Fermentation

FlaskTray

Packed bedTunnelPaddle

Rotating drumTower

Batch

Continuous

Fed-batch

laboratory scalePilot scale

Industrial scale

Biofuel Production and integrated pollution control using microalgae

• Microalgal Farming and CO2Mitigation• 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

Anthropogenic chemical compounds in environemnt

Persistent organic pollutants in environment• Wide distribution- POPs detected from soil, water, food

items, commercial products

• Sources- Mostly chlorinated organic compounds formed unintentionally- industries, commercial, agriculture, military, other human activities, and natural sources

• Insufficient data- No reliable data for their persistence in Indian environment- No management practices

• Problems in detection methods- Methods for detection and degradation not up to the mark.

• Highly toxic and recalcitrant- ultimate formation of- tetrachlorodibenzo-p-dioxin and furan-like compounds-complete physiological impairment.

• Tremendous scope for medical diagnostics and therapy and products.

• Therefore, methods & technology for detection, bioremediation and detoxification is required.

Degradation of aromatic compounds

Key component: POPs

Emerging environmental contaminant in present scenario

Classification of POPs•  Dirty Dozen - UNEP Stockholm Convention on Persistent Organic Pollutants - 2001

aldrin dieldrin toxaphenechlordane endrin mirexpolychlorinated biphenyls heptachlor DDTpolychlorinated 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

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.

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.

• No reliable data from developing countries including India.

• Detection methods.

• Instrument development.

• Thermokinetic modelling, equilibrium modelling, statistical determinations and others.

• Field validation.

• Laboratory

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

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

mineralization

Catabolic gene in degradation of alkanesDegradation of methane

Remediation of POPs in waste sites

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.

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.

BIOAUGUMENTATIONBIOAUGUMENTATION

BIOSTIMULATIONBIOSTIMULATION

Enzymatic methodsEnzymatic methods

Ex situ BioremediationEx situ Bioremediation

In situ Bioremediation In situ Bioremediation

Bioremediation

Importance of soil–plant–microbial interactions in bioremediation

Soil–plant–microbial interactions in remediation of pollutants in environment

Biocolloid formation in metal bioremediationColloidal 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

Technologies in BioremediationEx situ bioremediation• 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• Bioventing• Bioslurping• Biopiling

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

Biosafety assessment of leachate after biological treatments

EstrogenicityGenotoxicityCytotoxicity

MTT Assay Comet Assay E-Screen Assay

(Nwagbara et al. 2007)

(Singh et al. 1988)

(Vanparys et al. 2006)

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.

*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.

Molecular Probes for tracking

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.

System biology approaches

The four-step paradigm for metabolic systems biology

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.

• Thanks