MBT2000 Introduction to Molecular Biotechnology

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MBT2000Introduction to Molecular Biotechnology

• Environmental Biotechnology
• Prof. K.M. Chan
• Dept. of Biochemistry and
• Environmental Science Program
• Chinese University
• Tel [phone]
• Email kingchan_at_cuhk.edu.hk

TD192.5 E58 2005 (UL Reserve 4 h) Jordening H-J
Winter J (eds.), Environmental Biotechnology
concepts and applications. Wiley-VCH, 463p.
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What was environmental biotechnology?

• Simple and traditional definition use, in a
  controlled manner, of microorganisms to degrade
  wastes
• Solving environmental problems through
  biotechnology e.g. biosensor, BioMicroElectronics
  and Nanotechnologies, Biotreatments, etc.
• International Society for Environmental
  Biotechnology, since 1992. Two streams (1)
  microbial biotechnology for environmental
  improvement (sewage treatments and
  bioremediation) and (2) chemical engineering
  related to the environment. From waste treatment
  to bioremediation.

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Recent Topics Risk Management and Biofuels

• Use of molecular techniques to protect the
  environment, including Risk assessments of GMOs
• Renewable energy and resources engineering
  plants for the production of clean energy,
  biofuel, biomass, and animals for food
  production, etc.

Environmental Biotechnology is the
multidisciplinary integration of sciences and
engineering in order to utilize the huge
biochemical potential of microorganisms, plants
and parts thereof for the restoration and
preservation of the environment and for the
sustainable use of resources.
Laboratoire de Biotechnologie Environnementale
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OUTLINE

• 1. Molecular Ecology
• 2. Bioremediation (site restoration) and
  Biotechnology for waste treatments
• 3. Biosensor (monitoring of pollution)
• 4. Environmental applications of genetically
  modified organisms and Genetic Exchange in
  Environment.
• 5. Biofuel

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1. Molecular Ecology

• Understanding nature by molecular techniques of
• DNA fingerprinting for population genetic
  studies become more important for biodiversity
  research to study kinship relationship
• Authentication inspect endangered species with
  minimal samples using non-invasive technique
• Forensic analysis, to properly identify the
  evidence for species identification

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WHAT FOR?

• Phylogenetic study e.g. horse family compare
  between species or strains.
• Population study compare within species
  collected from different locations, e,g, compare
  between Asian and African populations. Molecular
  Ecology.
• Authentication study external morphology cannot
  give positive identification of a species, e.g.
  specimen of meat samples or dried plants ground
  in powder form.

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EcoRI digestions of Tilapia genomic DNA
T W
MSL
AFD
F T
M
M (50 bp)
1
1
1
2
1
3
2
2
3
2
U
250 bp
(Chan KM, unpublished data)
galilaeus
mossam/horn
niloticus
zillii
redalli
placidus
aureus
horn
Adapted from Franck et al., 1992. Genome
35719-725.
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• RESEARCH METHODS
• Screening for micro-satellites (low Cot DNA,
  rapidly associated DNA after heat denaturation)
• Isolation of repetitive DNA (polymorphism of
  length of the repetitive DNAs)

• mitochondrial DNA (D loop or cytochrome b)
• ribosomal RNA (gaps between 16-23 s, etc)
• Highly variable gene, isoforms of HLA or MHC
  (major histocompatibility complex) loci
  polymorphism.
• RAPD, random amplification primer detection
  method

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On the use OPB-09 primer (5TGGGGGACTC) for RAPD
of different strains of O. niloticus
Adapted from Naish et al., 1995. Molecular
Ecology 4271-274.
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Different primers
AFD
MSL
Tai Wai
Fo Tan
SG
OP
SG
NS
OP
OP
NS
SG
SG
OP
NS
NS
M
M
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Bioremediation (site restoration) and
Biotechnology for Waste Treatments

• Ocean ranching for stock restoration (e.g.
  cultured salmon, grouper and abalone released to
  the sea or artificial reef).
• Recovering of damaged sites to a clean or less
  harmful site after dredging.
• Remove chemicals using biological treatments on
  site (in situ) or ex situ.
• Chemicals heavy metals, trace organics or
  mixtures.
• Bacterial or fungal degradation of chemicals
• Engineered microbes for better and more efficient
  removal of chemicals on-site

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Redox Clean-Up Reactions

• Anaerobic or aerobic metabolism involve oxidation
  and reduction reactions or Redox reactions for
  detoxification.
• Oxygen could be reduced to water and oxidize
  organic compounds. Anaerobic reaction can use
  nitrate.
• In return, biomass is gained for bacterial or
  fungal growth.
• In many cases, combined efforts are needed,
  indigenous microbes found naturally in polluted
  sites are useful.

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Problems with bioremediation

• Work in vitro, may not work in large scale. Work
  well in the laboratory with simulation, may not
  work in the field. Engineering approach is
  needed.
• Alternatively, select adapted species on site
  (indigenous species) to remediate similar damage.
• Most sites are historically contaminated, as a
  results of the production, transport, storage or
  dumping of waste. They have different
  characteristics and requirements.
• Those chemicals are persistent or recalcitrant to
  microbial breakdown.

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Use of bacteria in bioremediation

• Greatly affected by unstable climatic and
  environmental factors from moisture to
  temperature.
• For examples, pH in soil is slightly acidic
  petroleum hydrocarbon degrading bacteria do not
  work well lt 10 C.
• These microbes are usually thermophilic
  anaerobic.
• Fertilizers are needed. Seeding or
  bioaugmentation could be useful too.
• They contain monooxygenases and dehydrogenases to
  break down organic matters including most toxic
  substances.

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Pseudomonas

• Genetically engineered bacteria (Pseudomonas)
  with plasmid producing enzymes to degrade octane
  and many different organic compounds from crude
  oil.
• However, crude oil contains thousands of
  chemicals which could not have one microbe to
  degrade them all.
• Controversial as GE materials involved.

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Use of fungi in bioremediation

• Lipomyces can degrade paraquat (a herbicide).
• Rhodotorula can convert benzaldehyde to benzyl
  alcohol.
• Candida can degrade formaldehyde.
• Gibeberella can degrade cyanide.
• Slurry-phase bioremediation is useful too but
  only for small amounts of contaminated soil.
• Composting can be used to degrade household wastes

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White rot fungi

• White rot fungi can degrade organic pollutants in
  soil and effluent and decolorize kraft black
  liquor, e.g. Phanerochaete chrysosporium can
  produce aromatic mixtures with its lignolytic
  system.
• Pentachlorophenol, dichlorodiphenyltrichloroethane
  (e.g. DDT), even TNT (trinitrotoluene) can be
  degraded by white rot fungi.

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Phyto-remediation

• Effective and low cost
• Soil clean up of heavy metals and organic
  compounds.
• Pollutants are absorbed in roots, thus plants
  removed could be disposed or burned.
• Sunflower plants were used to remove cesium and
  strontium from ponds at the Chernobyl nuclear
  power plant.
• Transgenic plants with exogenous metallothionein
  (a metal binding protein) used to remove metals .

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Waste water treatments

• Bioremediation of water or groundwater or
  materials recovered from polluted sites.
• Ex situ As many bacteria work better in
  controlled conditions, e.g. anaerobic, higher
  temperature, effluent (sewage treatment) or solid
  materials (composting) can be treated with
  bacteria to decompose organic matters.
• Primary treatment screening and emulsification.
• Secondary treatments Nutrient removal and
  chemical removal.

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Nutrient removal

• Phosphate removal by polyphosphate accumulating
  organisms and glycogen accumulating organisms.
• Nitrogen removal by Nitrosomonas which denitrify
  nitrite to nitrogen gas. Anaerobic ammonium
  oxidation is also important.
• Algae could absorb many nutrients and pollutants.
  Dunaliella. Chlorella and Spirulina are valuable
  species.

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Dye removal and chemical removal

• Azo-dye (NN) removal
• Sensitive to redox and anaerobic treatments can
  decolorize azo dyes
• Specific reductase enzymes are needed to detoxify
  the dye after discoloration
• Chemical treatment or biological treatment, e.g.
  Candidatus Brocadia Anammoxidans for ammonia
  removal.

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3. Biosensor(monitor pollution)

• Measurement of mutagenic activity (microtox and
  mutatox tests with lux gene from Vibrio)
• Biomarkers of exposures to pollutants (stress
  proteins)
• Detection of pathogens by multiplex-PCR
• Detection of toxins (Ciguatoxin)

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Ames Tests
Ames 1973 developed a rapid screening method
based on mutation of Salmonella typhimurium. The
mutant strains used in the Ames Tests are
histidine defective (unable to synthesize
histidine). Back mutation make them able to
survive on plates without histidine.
Adapted from Lowy, D.R. 1996 The Causes of
Cancer. In American Scientific Molecular
Oncology. Sci. Amer., Inc., New York, pp41-59.
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BioDetection Systems

• CALUXR Bioassay
• A sensitive bioassay for exposure to dioxins and
  related compounds
• Synthetic gene promoter was created and linked to
  a reporter gene which gives colour when the gene
  promoter is turned on
• The synthetic gene promoter contains multiple
  cis-acting elements responsible for dioxin (DRE)
  and dioxin receptor (Ah receptor) binding.
• The reporter gene is tranfected into a cell-line
  for the bioassay.

http//www.biodetectionsystems.com/caluxd_bc.html
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Stress Proteins

• Metallothionein for exposure to heavy metals
• Cytochrome P450 (CYP) IA1 for exposures to trace
  organics
• Vitellogenin (an egg yolk protein) for exposure
  to environmental estrogens
• Heat shock protein for general stress conditions
• Q These biomarkers are NOT biomarkers of toxic
  effects. They are biomarkers of exposures.
  Still controversial
• Biomarkers have biological relevance and usually
  less expensive than chemical analyses. Data could
  be diagnostic and indicative.

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Pathogen detection

• Bacteria coli form bacteria, salmonella,
  Legionella, Vibrio, etc.
• Virus Influenza, SARS, hepatitus, polio, etc.
• Algae dinoflagellates, diatoms, toxic algae,
  ciguatoxin, etc.
• Multiplex technology is being developed one run
  for many pathogens.
• Collection with minimal amount of samples water,
  soil, or air.
• Use PCR or real-time PCR techniques

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Use of microarray for environmental screening and
detection

• NOT really quantitative, its qualitative.
• A rapid screening procedure for pathogens or
  multiple biomarkers to monitor or identify the
  problem. Require later verification and real-time
  PCR detection with antibody confirmations.
• Array of probes (biomarkers or pathogens) placed
  on a piece of glass or other solid surface. DNA
  or RNA from a test environmental sample, is then
  applied to the solid surface and wherever there
  is a match with a probe sequence, specific and
  sensitive hybridization occurs, resulting in the
  generation of a signal.
• Methods are still under development.

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4. Environmental applications of genetically
modified organisms

• Insect Bt resistance, producing a bacterial toxin
  called bacillus toxin (Bt) so that insects
  (dipterans) die when eating the plants
• Extensively used in the past 20 years
• Green groups complained that this is gene
  pollution

• New Traits
• 74 Herbicide resistant
• 19 Insect resistant
• 7 Both
• Major GM crops
• 58 Soybean
• 23 corn
• 12 cotton
• 6 Canola

Ref Brown, K. 2001. Genetically Modified Foods
Are they safe? Scientific American 284(4)39-45.
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Ref Brown, K. 2001. Genetically Modified Foods
Are they safe? Scientific American 284(4)39-45.
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Insect resistant Bt plants
Herbicide-resistant Plants
Adapted from Genetic Engineering News
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BT toxins kill dipterans and unexpectedly also
kill Lepidopterans. They dont kill other
insects. They are derived from Bacillus spores.
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(No Transcript)
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GM plants with Bt toxins
Ref Brown, K. 2001. Genetically Modified Foods
Are they safe? Scientific American 284(4)39-45.
Bt-pollens kill Monarch Larvae ??
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• Milkweed leave

• Bt-corn pollen
• Normal corn pollen
• NO pollen

Five 3-day-old Monarch larvae
Dusted with the same densities visually
Fed for 4 days

• Milkweed leave consumption
• Larval survival
• Final larval weight

Adapted from Losey et al., 1999, Nature 399214
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Lower larvae survival
Ate less milkweed leave
Slower growth

• Adapted from Losey.et al.,, 1999. Nature 399 214

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However...

• Laboratory test only
• Duration (4 days ONLY)
• dont know the amount of pollen added
• Sample size (5 larvae in each group)
• CANNOT simulate natural Environment
• No choice of diet in lab test
• UV, humidity, Wind, Monarch behaviour
• Unknown Bt-pollen concentration
• majority of Bt pollen (90) falls within 5
  meters, not 10 m as they claimed (Field trial is
  underway to prove BT plants are save)
• Last yr, Monarch butterflies ?30, Bt-corn ?40
• Data from Monarch Watch, and Research findings
  presented at the Monarch Butterfly Research
  Symposium, Chicago, 1999 by Dr. Richard
  Helmich, USDA, lowa State University Dr. Galen
  Dively, University of Maryland, And Dr. John
  Pleasants, lowa States University)

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Genetic Exchange in the Environment

• Risk Assessments and Biotechnology Regulations
  (e.g. environmental use permits).
• To detect the 35s CaMV (Cauliflower mosaic virus)
  promoter sequence or NOS (nopaline synthase gene
  terminator) DNA sequence by Quantitative PCR for
  GMO detection.
• GMOs Bacteria is associated with disease and
  hence is always held up by fears. E.g. antibiotic
  resistance.
• GEM The concern is antibiotic resistant plasmid
  horizontally transferred to other microorganisms.

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GEMS in the environment

• Genetically Engineered Microorganisms (GEMs)
• Many pollutant degradation genes or resistance
  genes are in plasmids inside bacteria
• By cloning, we can insert genes into plasmid for
  gene transfer to different bacteria
• In 80s, ice minus was release of Pseudomonas
  syringae and P. fluorescens, had lead to concerns
  over release of GEMS.
• Another GEM in the environment is the
  combinations of different BT genes released in
  the field.
• Has to be reviewed case by case and become very
  unpopular, worse than GMOs, thus inhibiting
  further field trials of GEMs.

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5. Bio-fuels

• Plant-derived fuels plant species for
  hydrocarbon (oil) production, e.g. rape-seed,
  sunflower, olive, peanut oils. Or ethanol
  production of sugars (or cellulose) derived from
  plants.
• Conversion of used cooking oil to bio-fuel
  (called bio-diesel)
• Biogas gases from composts or landfill, but
  methane is a green house gas

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Bioethanol and biofuel cell

• Sugar cane, sugar beet wastes, high starch
  material (cassava, potatoes, millet) to be
  hydrolyzed by starch hydrolyzing enzyme to
  convert sucrose or glucose to ethanol. Mainly
  used in Brazil.
• Corn ethanol 22 less carbon emission, used in
  the US.
• Bio-diesel 68 less carbon emission oils from
  soybean (US) or canola oil (Germany)
• Cellulosic ethanol 91 less carbon emission, but
  difficult to change cellulose to ethanol
• Hydrogen energy however is the trend of future
  renewable energy without carbon emission a
  journey to forever.
• Problem is how to generate the hydrogen too
  costly with conventional chemical methods or
  reverse osmosis.

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A Journey forever?

• Various bacteria and algae, for example
  Escherichia coli, Enterobacter aerogenes,
  Clostridium butyricum, Clostridium
  acetobutylicum, and Clostridium perfringens have
  been found to be active in hydrogen production
  under anaerobic conditions.
• The most effective H2 production is observed upon
  fermentation of glucose in the presence of
  Clostridium butyricum (strain IFO 3847, 35 mmol
  h1 H2 evolution by 1 g of the microorganism at
  37C).

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A Pathway for our Future Energy?
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A microbial biofuel cell
(A) With a microbial bioreactor providing fuel
separated from the anodic compartment of the
electrochemical cell.
(B) With a microbial bioreactor providing fuel
directly in the anodic compartment of the
electrochemical cell. 
http//chem.ch.huji.ac.il/eugeniik/biofuel/biofue
l_cells_contents.html
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Adapted from STUDY OF BIOLOGICAL FUEL CELLS
Aarne Halme, Xia-Chang Zhang and Anja Ranta
Automation Technology Laboratory, Helsinki
University of Technology, P.O. Box 5400,
FIN-02015 HUT, ESPOO, FINLAND email
anja.ranta_at_hut.fi
The Working principle Of An Enzyme Fuel Cell
                                                  
                                                  
                                               
                                                  
                                                  
                                                  
                                        
The enzyme and mediator are immobilized on the
anode.                                       
                        Rough layout of the
anode structure
http//www.automation.hut.fi/research/bio/sfc00pos
.htm
45
Adapted from STUDY OF BIOLOGICAL FUEL CELLS by
Aarne Halme, Xia-Chang Zhang and Anja Ranta
Automation Technology Laboratory, Helsinki
University of Technology, P.O. Box 5400,
FIN-02015 HUT, ESPOO, FINLAND email
anja.ranta_at_hut.fi
General characteristics of chemical and
biological fuel cell  
conversion rate 50
http//www.automation.hut.fi/research/bio/sfc00pos
.htm
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Theoretical energy content of methanol, ethanol,
and glucose. The calculation is based on the
assumption of complete conversion the likely
conversion rate in practice is around 50 .  
Adapted from STUDY OF BIOLOGICAL FUEL CELLS by
Aarne Halme, Xia-Chang Zhang and Anja Ranta
Automation Technology Laboratory, Helsinki
University of Technology, P.O. Box 5400,
FIN-02015 HUT, ESPOO, FINLAND email
anja.ranta_at_hut.fi
http//www.automation.hut.fi/research/bio/sfc00pos
.htm
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CONCLUSIONS

• Different aspects of environmental biotechnology
  were elaborated from species identification
  (molecular ecology) to bioremediation
  development of bio-fuel and hydrogen energy
• For molecular biotechnology development Cloned
  enzymes could be modified, immobilized and become
  more useful
• Combination of biotechnology and engineering or
  nano technology is essential

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References

• Thieman, W.J., and Palladino, M.A. 2004.
  Introduction to Biotechnology. Pearson Ed., Inc.
  Benjamin Cummings. 304p. (Chapter 9
  Bioremediation, pp 185-204).
• Wainwright, M. 1999. An Introduction to
  Environmental Biotechnology. Kluwer Academic
  Publishers, Boston/Dordrecht/ London,171p.
• http//chem.ch.huji.ac.il/eugeniik/biofuel/biofue
  l_cells_contents.html