MBT2000 Introduction to Molecular Biotechnology

1. MBT2000Introduction to Molecular Biotechnology Environmental Biotechnology Prof. K.M. Chan Dept. of Biochemistry and Environmental Science Program Chinese University Tel: 3163-4420 Email: kingchan@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.

2. 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.

3. 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

4. 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

5. 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

6. 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.

7. EcoRI digestions of Tilapia genomic DNA T W MSL AFD F T M U 1 2 3 1 2 1 2 3 1 2 M (50 bp) 250 bp (Chan KM, unpublished data) galilaeus mossam/horn niloticus zillii redalli placidus aureus horn Adapted from Franck et al., 1992. Genome 35:719-725.

8. 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

9. On the use OPB-09 primer (5TGGGGGACTC) for RAPD of different strains of O. niloticus Adapted from Naish et al., 1995. Molecular Ecology 4:271-274.

10. Different primers AFD MSL Tai Wai Fo Tan SG NS NS NS NS OP SG OP SG OP SG OP M M

11. 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

12. 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.

13. 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.

14. 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 < 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.

15. 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.

16. 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

17. 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.

18. 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 .

19. 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.

20. 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.

21. Dye removal and chemical removal • Azo-dye (N=N) 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.

22. 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)

23. 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.

24. 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

25. 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.

26. 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

27. Use of microarray for environmental screening and detection • NOT really quantitative, it’s 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.

28. 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.

29. Ref: Brown, K. 2001. Genetically Modified Foods: Are they safe? Scientific American 284(4):39-45.

30. Insect resistant Bt plants Herbicide-resistant Plants Adapted from Genetic Engineering News

31. BT toxins kill dipterans and unexpectedly also kill Lepidopterans. They don’t kill other insects. They are derived from Bacillus spores.

33. 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 ??

34. Bt-corn pollen • Normal corn pollen • NO pollen Five 3-day-old Monarch larvae Milkweed leave 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 399:214

35. Lower larvae survival Ate less milkweed leave • Adapted from Losey.et al.,, 1999. Nature 399: 214 Slower growth

36. However... • Laboratory test only • Duration (4 days ONLY) • don’t 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, andResearch 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) ]

37. 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.

38. 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 80’s, “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.

39. 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

40. 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.

41. 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 h–1 H2 evolution by 1 g of the microorganism at 37°C).

42. A Pathway for our Future Energy?

43. 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/biofuel_cells_contents.html

44. 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@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@hut.fi General characteristics of chemical and biological fuel cell *conversion rate 50% http://www.automation.hut.fi/research/bio/sfc00pos.htm

46. 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@hut.fi http://www.automation.hut.fi/research/bio/sfc00pos.htm

47. 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

48. 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/biofuel_cells_contents.html