ENVIRONMENTAL BIOTECHNOLOGY

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ENVIRONMENTAL BIOTECHNOLOGY (BTK 4401) 4(31)
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ASSESSMENT

• TEST 1 20
• TEST 2 20
• AMALI 15
• ASSIGNMENT (SCL) 10
• FINAL 35

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INTRODUCTION ENVIRONMENTAL BIOTECHNOLOGY
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Outline of lecture

1. Environmental problems
2. Environmental pollution
3. Monitoring and measuring pollution- Bioassay
4. Cleaning up i.e. Bioremediation

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Environmental problems

• Industrial wastes
• 2. Loss of ecosystem/habitat
• 3. Overfishing-depressed fish stock
• 4. Soil erosion
• 5. Fresh water supplies
• 6. Infectious disease

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Environmental problems

• Environmental pollution Issues of most concern
• Air pollution
• Water pollution
• Toxic and heavy metal pollution
• Solid and hazardous waste

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AIR POLLUTION

• Major environmental problem in cities
• Sources-
• Vehicle emissions
• Industrial plants
• Power stations
• Oil refinery
• Domestic heating
• Cement plants

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AIR POLLUTION

• Gives rise to 3 other phenomena
• Acid rain
• Ozone depletion
• Global warming climatic change

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Ozone

• A bluish reactive gas made up of 3 oxygen atoms
• 90 found in the stratosphere a layer 10-40km
  above the earth surface
• Protects life on earth from UV light

• Problems ??
• Appearance of holes
• Holes created by gases CFC, halons, methyl
  bromide

Effects Xcess UV skin burns, skin cancer,
cataracts,
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Formation of holes through the years
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GLOBAL WARMING

• World is warming
• Cause emission of CO2 and other greenhouse gas
• Molecule per molecule methane traps gt20 times
  than CO2 more dangerous
• Consequence Temperature change 1.1oF (0.6oC)
  but the effects are quite drastic

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Global Warming Effects
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Tree-eating wood beetles are likely to benefit
from a warmer climate and reproduce in
ever-increasing numbers
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WATER POLLUTION

• Sources
• Municipal detergents/washing powder high in
  phosphates
• Industrial toxic wastes and organic substances
• Agriculture fertilizers esp. nitrates

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PESTICIDES
Problems with pesticides

• Only 0.1 reach targets the rest - 99 affects
  non-target organisms widely dispersed in the
  environment
• 10 of 80,000 pesticides used are carcinogenic
  e.g. testicular cancer
• Highly toxic to aquatic life

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PESTICDES - Effects

• Carbofuran highly toxic to ikan keli
• Affect different enzymes
• Changes in enzyme activities can be used as
  bioindicators for pesticide toxicity

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GST, GPx and EROD activities in the
hepatopancreas of Clarias gariepinus after
exposure to carbofuran at 0.8 mg/L
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AZODYES

• Synthetic colorants
• One of the oldest man-made chemicals
• Applications in textiles, food, cosmetics,
  plastics, leather, paper, color photography,
  pharmaceutical industries

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AZODYES - Problems

• Not easily degraded
• About 10 of dyestuff does not bind to fibres
  during dyeing process
• ? released into the environment accumulate in
  the biosphere
• Some are carcinogenic

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Heavy metal pollution
???????
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What are heavy metals ???

• Metallic chemicals like mercury, lead, cadmium,
  arsenic, copper and zinc that can be harmful
  pollutants when they enter soil and water
• generally toxic in low concentrations to plants
  and animals
• persist in the environment and bioaccumulate
• tend to be toxic

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Heavy metal pollution

• From extensive use in agriculture, chemical and
  industrial processes and waste disposal
• Electronic wastes TV and computer monitors
  contain between 3-5 kg of lead
• Threat to human health and limit plant
  productivity heavy metal poisoning

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Heavy metal poisoning

• Itai-itai disease due to cadmium discharged
  from mining companies
• Minamata disease mercury poisoning
• estimated that 2 million people are affected by
  eating contaminated fish
• 2,955 people died

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Effects of heavy metals - animals

• Not acting directly but disturbs other metabolism
• e.g. cadmium affects zinc and copper metabolism
• Studies with fish show
• Changes in copper containing enzymes
• Parallel changes in copper concentrations

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Effects of heavy metals - Plants

• Typified by aluminum in peat soils
• Aluminum highly toxic to plants
• Rates of DNA synthesis
• Affects photosynthesis
• Fortunately, some are tolerant to Al because they
  posses Al-tolerant functions

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Effects of Al on peanut cells grown in suspension
cultures .

• Changes in growth rate
• Morphological properties change
• Changes in protein concentration appearance of
  new proteins
• Key enzyme activities also change

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Relative growth of peanut cell suspension cultures under Aluminium stress. The toxic effects of Al on nitrate reductase activity in peanut cell suspension cultures.
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Monitoring of Toxicants in the Environment

• Difficult task
• Measure levels of toxicants
• Based on measuring chemical loads in the
  environment
• Highly technical and expensive
• Therefore, necessary to develop and test simple,
  cheap, yet accurate method
• Use biological systems

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Monitoring of Toxicants in the Environment

• Biological system bioassay
• Instrumental analysis
• classical bioassay
• modern bioassay

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1. Instrumental analysis

• Atomic Absorption Spectroscopy, Atomic flame
  spectroscopy (AFS) and atomic emission
  spectroscopy
• Accurate, specific and reproducible
• Need sophisticated instrumentation, expensive,
  require skilled technicians
• Not economical

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1. Classical Bioassay

• Various organisms used rat/mouse, Daphnia magna
  and fish (rainbow trout)
• Expensive require skilled technicians
• Not selective
• Unsuitable for large scale screening

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Daphnia magna Water fleas
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1. Modern Bioassay

• Use microorganisms, antibodies and enzymes
• Several bioassays have been developed
• Principle measure inhibition of the cells
  biochemical characteristics to quantify toxicity
• Enzymes have been used to determine heavy metals
  e.g. mercury using urease

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BIOASSAYUSING ENZYMES
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BIOASSAY KIT
NO POLLUTION
PRE-COATED ENZYME
NORMAL COLOUR FORMATION
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SUBSTRATE
SUBSTRATE
ENZYME
SUBSTRATE

SUBSTRATE
SUBSTRATE
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COLOURED PRODUCTS

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BIOASSAY KIT
POLLUTED SAMPLE
PRE-COATED ENZYME
LESS COLOUR FORMATION
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What Is Actually Happening?
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LESS COLOURED PRODUCTS
INHIBITS REACTION

ACT AS INHIBITORS
POLLUTANTS
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Bioassay using enzymes
Detection of heavy metals

• Papain and casein proteolytic assay
• Using papain IC50 for mercury, copper and
  silver is 0.20, 0.12 and 1.06mg/L sensitive
• Thus the assay system could detect levels of
  toxic heavy metals at the level of the maximum
  permissible level of toxic metals in
  Environmental quality act 1974 environmental
  quality (sewage and industrial effluents)
  regulations 1978.

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Bioassay using enzymes
Detection of pesticides - carbamate

• Current technology use acetylcholinesterse from
  Drosophila melanogaster (fruit fly)
• Expensive and foreign technology
• Present findings use acetylcholinesterase from
  Oreochromis mossambica
• Very sensitive to detect carbamates such as
  carbaryl and carbofuran (0.1mg/L 0.1 ppm)

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Bioassay using microorganisms- bacteria

• Also successfully isolated bacteria that degrade
• Petroleum products
• Acrylamide
• Detergents (SDS)

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REMEDIATION
BIO
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BIOREMEDIATION
How to remediate restore polluted environment?

• Use physical and chemical methods e.g.
• dig up contaminated soils remove it to
  landfills
• capping and containment
• use chemicals

Hence BIORMEDIATION

• Problems
• harm the habitat
• expensive
• generate other wastes

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BIOREMEDIATION

• Using living organisms to decontaminate polluted
  systems
• Living organisms

Bacteria
Fungi
Algae
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BIOREMEDIATION

• Why bacteria ?
• most common bioremediation microorganisms
• Natures recyclers e.g. Carbon, Nitrogen cycle
• Can degrade a variety of compounds as a result of
  million of years of evolution
• With genetic engineering, can be tailored to
  degrade pollutants that we want

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BIOREMEDIATION

• How do bacteria degrade pollutants ???
• produce enzymes
• break up toxic compounds to lesser or non-toxic
  compounds

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ENZYMES
POLLUTANTS

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NON-TOXIC
BIOREMEDIATION

TOXIC
NON-TOXIC
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Does Bioremediation work ??

• Exxon Valdez oil spill in Alaska in 1989
• Community of microorganisms capable of breaking
  hydrocarbons in the oil spill

? Successful example of the potential of
bioremediation
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BIOREMEDIATION Current research
HEAVY METALS
PESTICIDES
DYES
DETERGENTS
PLASTICS
OILS HYDROCARBONS
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BIOREMEDIATION

• Current findings - bacteria
• Biodegradation of pesticides carbamates
• 2 isolates of bacteria that can degrade
  carbamates have been isolated
• optimized growth conditions
• waiting to be identified

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Bacterial Growth Curve ( Isolate C1 and Isolate
C2 ) and Amount Carbofuran degraded
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BIOREMEDIATION

• Current findings - bacteria
• 2. Bioremediation of molybdenum
• A heavy metal used in pigment, electronic,
  textile and metal industries
• A bacterium was isolated that can reduce
  molybdate to molybdenum blue
• Future for bioremediation of molybdate
  contaminated aquatic bodies in Malaysia

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BIOREMEDIATION

• Current findings bacteria
• Bacteria that could degrade other xenobiotics
  were also successfully isolated
• Azodyes
• Plastics
• Petroleum products
• Detergents

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BIOREMEDIATION

• Studies also carried using fungi
• Isolated two local fungi, Isolate 5-UPM and
  Isolate 17-UPM
• Can degrade 4 different azodyes
• Work in progress using other dyes

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Clearing zones on screening medium indicating azo
dye degrading ability. (Isolate 5-UPM, 7
days old cultures)
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Azo dye degradation in static 40 ml liquid
cultures. The conical flasks contain intact (200
mg/L) azo dyes while the serum bottles degraded
azo dyes. The white mass is the mycelial mat.
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Azo dye (200 mg/L) degradation in isolate 5-UPM
static 40 ml liquid cultures. Azo dyes
concentration determined spectrophotometrically.
Ponceau 2R (P2R), Orange G (OG), Direct Blue 71
(DB71), Biebrich Scarlet (BS
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BIOREMEDIATION

• Studies carried out not confined to screening and
  isolation.
• Also include
• Optimization studies growth, effects of
  different parameters-pH, temperature, substrate,
  nutrients, etc.,etc
• Purification of the enzymes responsible
• Characterization of the enzymes

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CONCLUSION

• Bioremediation is an attractive alternative to
  traditional physico-chemical techniques for
  remediation of contaminated sites
• Cost effective
• Selectively degrade pollutants without damaging
  site indigenous flora and fauna
• Low-technology techniques
• High public acceptance

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CONCLUSION

• BUT STILL FRAUGHT WITH PROBLEMS
• Substrate and environmental variability
• Limited biodegradative potential and variability
  of naturally occurring microorganisms
• HOWEVER may be overcome by biomolecular
  engineering enhance bioremediation programs