BIOREMEDIATION. and A.K. Raymundo 3. National Institute of Molecular Biology and Biotechnology and 3 Institute of Biological Sciences

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2 BIOREMEDIATION E. J. del Rosario 1, V.P. Migo 2, M.L.F. Paje and A.K. Raymundo 3 1 Institute of Chemistry 2 National Institute of Molecular Biology and Biotechnology and 3 Institute of Biological Sciences University of the Philippines Los Banos, Laguna Philippines

3 OUTLINE OF PRESENTATION 1. Introduction 1.1. Definitions and concepts 1.2. Factors that affect bioremediation 2. Microbiological Aspects 2.1. Genetics 2.2. Physiology 2.3. Enzymology/Biochemistry

4 3. Physico-Chemical Aspects 3.1. Chemical reactions during bioremediation 3.2. Basic parameters temperature, ph, concentrations 3.3. Thermodynamic aspects 3.4. Kinetic aspects 4. Bioengineering Aspects 4.1. In situ bioremediation 4.3. Ex situ bioremediation

5 5. Policy Implications 5.1 Legislation and government policies 5.2 Research and development 5.3 Training and manpower development

6 What is Bioremediation? Bioremediation is a treatment process that uses microorganisms (bacteria, fungi or yeast) or plants to break down, or degrade,, hazardous substances into less toxic or nontoxic substances.

7 Phytoremediation Phyto means plant; remediate means to clean up The use of vegetation for in situ treatment of contaminated soils, sediments, and water through contaminant removal, degradation, or containment. It can be used to clean up metals, pesticides, solvents, explosives, crude oil, polyaromatic HC, and landfill leachates.

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10 POLLUTANT Physico-chemical Factors Temperature ph Water O 2 / Redox potential Mass transfer Solubility Microbial Parameters Genetic characteristics Physiological properties Metabolic diversity Enzymological capabilities BIOREMEDIATION PRODUCT(S) Schematic Diagram of Bioremediation Process

11 Disappearance of 2,4,5-T in soil from the Philippines (PH), Trinidad (TR), Nigeria (NI), and Puerto Rico (PR). (From Rosenberg and Alexander, Reprinted with permission from the American Chemical Society.) (Martin Alexander)

12 MICROBIOLOGICAL ASPECTS

13 Examples of Microorganisms Used for Bioremediation Caulobacter crescentus: potential for heavy-metal remediation in waste- treatment plant wastewater. Ferroplasma acidarmanus: : oxidizes iron, and transforms sulfide in metal ores to sulfuric acid. Deinococcus radiodurans: Have DNA-repa capabilities for radioactive waste cleanup.

14 Phytoextraction Phytoextraction employs hyperaccumulating plants to remove metals from the soil by absorption into the roots and shoots of the plant

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16 Genetic engineering introduction of gene(s) ) for necessary bioremediation enzyme(s) ) in genome of host microrganism.

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19 CHEMICAL ASPECTS

20 Chemical Classes and Their Susceptibility to Bioremediation CHEMICAL CLASS EXAMPLES BIODEGRADABILITY Aromatic Hydrocarbons Benzene, toluene Aerobic and anaerobic Ketones and esters Acetone, MEK Aerobic and anaerobic Petroleum hydrocarbons Fuel oil Aerobic Chlorinated solvents TCE, PCE Aerobic (methanotrophs), anaerobic (reductive dechlorination) Polyaromatic hydrocarbons Anthracene, benzo[a]pyrene, creosote Aerobic

21 Naphthalene Phenanthrene Pyrene Benzo(a)pyrene Some common polycyclic aromatic hydrocarbons (PAHs)

22 Initial Degradation of Benzene

23 OH COOH COOH CH 2 COOH OH OH OH catechol 2-hydroxymuconic semialdehyde 2-oxopent-2,4-dienoate (enol form) CH 3 COOH CH 3 COOH HO O H C O + C O 4-hydroxy-2-oxovalerate acetaldehyde CH 3 pyruvate Detoxication of Catechol: Aromatic ring cleavage

24 Microbial Degradation of HCs

25 H Cl C Cl Cl C Cl CCl 2 CCl 2 DDT Cl DDE Cl Cl Cl Cl Cl Cl Cl Cl Cl LINDANE Cl CH 3 CCOOH Cl DALAPON Cl 2,3,4,5,6-PENTACHLORO-1- CYCLOHEXENE CH 3 CCOOH O PYRUVIC ACID Detoxification of Pesticides through Dehalogenation

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29 EXXON VALDEZ (1989) Oil spill occurred in ALASKA Tanker loaded with 53 million gallons of oil Spilled about 35 million gallons Tanker was not double-hulled!!!

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31 Oil Slick from EXXON VALDEZ

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33 GUIMARAS OIL SPILL (2006) M/T Solar I tanker loaded with 2.4 million liters of BUNKER OIL Sunk on August 11, 2006 at Guimaras coast About 300,000 L oil was spilled Worst oil spill in the Philippines

34 GUIMARAS

35 GUIMARAS OIL SPILL Affected: 97% of the 220 km-long coastline 1,143 ha marine reserves 478 ha mangroves 16 m2 coral reef

36 BIOSTIMULATION Modification of the contaminated site Addition of fertilizers/nutrients Stimulates activity of indigenous HC degraders (IHDs( IHDs) Balances C:N:P ratio Not all microbes at site are IHDs!!!

37 BIOAUGMENTATION Seeding/introduction of a consortia of microorganisms to contaminated sites Microorganisms are laboratory-tested tested for hydrocarbon degradation

38 Conventional Bioremediation Enzymes released by the microbes can only attack one surface of the contaminant. This leads to slower, less effective remediation. CleanEARTH'S Enhanced Bioremediation A patented combination of surfactants and emulsifiers break the contaminant down into tiny pieces, which can then be surrounded by enzymes and quickly digested.

39 Examples of Microorganisms Used for Mine Waste Bioremediation Shewanella oneidensis MR1: : may degrade organic wastes and reduce or sequester a range of toxic metals. Thiobacillus ferrooxidans : used in mining industry to sequester iron & sulfide. Cyanobacteria: : produces cyanobacterial mats which are resistant to metals and metalloids, also remove these substances from the environment. NAST Biorem Team

40 In Situ Bioremediation of Soil In situ techniques do not require excavation of the contaminated soils so may be less expensive, create less dust, and cause less release of contaminants than ex situ techniques.

41 Ex Situ Bioremediation of Soil Ex situ techniques can be faster, easier to control, and used to treat a wider range of contaminants and soil types than in situ techniques. However, they require excavation and treatment of the contaminated soil before and, sometimes, after the actual bioremediation step. Ex situ techniques include slurry-phase bioremediation and solid- phase bioremediation.

42 POLICY IMPLICATIONS

43 PHILIPPINES: TASK FORCE ON BIOREMEDIATION (National Academy of Science and Technology) To apply scientific and technical expertise to address the problem of toxic and hazardous waste To design a research program on bioremediation To develop the capabilities of local scientists and create a pool of expertise to tackle similar problems in the future

44 R & D: Removing Heavy Metals Using Bacterial Exopolysaccharides (EPS) National Institute of Molecular Biology and Biotechnology (BIOTECH) University of the Philippines Los Baños

45 EPS is produced cheaply from indigenous bacteria (Rhizobium sp.) by fermentation in coconut wastewater medium

46 EPS removes heavy metals via adsorption or flocculation and coagulation processes. The technology is simple, fast and efficiently removes the heavy metal contaminant

47 R & D: BIOREMEDIATION OF DISTILLERY WASTES Microbial decolorization Field application as fertilizer

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49 Microbial Decolorization of Synthetic and Natural Melanoidins Synthetic Melanoidin Natural Melanoidin Absorbance (nm) Aspergillus Bacillus Aspergillus Bacillus Initial Final Decolorization %

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51 Desperate POLLUTION calls for desperate BIOREMEDIATION.

52 THANK YOU!