1. Bioremediation of a Hazardous Pollutant Kevin Drees IOFT 1101 October 15-18, 2007

2. What are hazardous pollutants? “Misplaced” chemicals that cause a harmful effect in the environment “Misplaced” chemicals that cause a harmful effect in the environment Types of harmful effects: Types of harmful effects: –Toxic (poisonous) –Carcinogenic –Mutagenic –Teratogenic (birth defects) –Flammable –Corrosive –Explosive –Endocrine Disruptive

3. Common Pollutants Petrochemicals Petrochemicals –Hydrocarbons (only carbon and hydrogen) –Mostly alkanes –Oil spills –Leaky underground storage tanks –Coal tar CH 3 – (CH 2 ) n – CH 3

4. Common Pollutants PCE (perchloroethylene), PCE (perchloroethylene), TCE (trichloroethylene) –Common drycleaning chemical Most common groundwater pollutant Most common groundwater pollutant –Used to clean circuit boards and degrease engine parts Common pollutant at electronics factories, airports, and military facilities Common pollutant at electronics factories, airports, and military facilities C = C H Cl

5. Common Pollutants Heavy metals Heavy metals –Mercury, Lead, Cadmium, Chromium, Zinc –Mine tailings –Burning coal (Mercury) –Burning leaded gasoline –Old paint (Lead)

6. Common Pollutants Polychlorinated Biphenyls (PCBs) Polychlorinated Biphenyls (PCBs) –Transformer fluid –Capacitor manufacture –Pulp bleaching –Bioaccumulates!

7. Where is the pollution? Groundwater aquifers Groundwater aquifers Soil Soil Surface waters and sediment Surface waters and sediment Air Air

8. How do we clean up pollution? Remediation Remediation Ex-situ (off-site) strategies Ex-situ (off-site) strategies –Excavation/pumping, followed by… –Landfill –Incineration –Chemical removal In-situ (on-site) strategies In-situ (on-site) strategies –Immobilization (chemicals, wells, membranes)

9. Problems with traditional remediation Expensive!!! Expensive!!! Invasive Invasive Residual contamination Residual contamination Landfilling not a permanent solution, just a delay tactic Landfilling not a permanent solution, just a delay tactic

10. Bioremediation Bacteria eat or immobilize many types of pollutants Bacteria eat or immobilize many types of pollutants Bioremediation = using bacteria to degrade or immobilize pollutants Bioremediation = using bacteria to degrade or immobilize pollutants Can be in-situ Can be in-situ –Inject nutrients organisms need to degrade pollutant into contaminated area Can be ex-situ Can be ex-situ –Excavate soil, pump water into bioreactor Can use indigenous microbes, or introduce them Can use indigenous microbes, or introduce them

11. Advantages of Bioremediation Cheap Cheap Less invasive Less invasive Long-term Long-term Can handle residual contamination Can handle residual contamination

12. How do bacteria degrade pollutants? All organisms need several things to live: All organisms need several things to live: –Chemical building blocks (containing C, N, P, O, S) –Energy to power cell biochemical reactions Breaking chemical bonds in food Breaking chemical bonds in food –Method of recycling energy-producing molecules Ex: respiration (oxygen) Ex: respiration (oxygen) –Vitamins and minerals Active sites of enzymes Active sites of enzymes Pollutants can serve as one or many of these requirements! Pollutants can serve as one or many of these requirements!

13. Ex: Petroleum Degradation Petroleum provides carbon building blocks for cells. Petroleum provides carbon building blocks for cells. In addition, cells require… In addition, cells require… –Nitrogen, phosphorous, and other nutrients –Oxygen CH 3 – (CH 2 ) n – CH 3

14. Today’s Lab Create oil-contaminated microcosms Create oil-contaminated microcosms –Sand, water, and oil in a Petri dish Introduce petroleum-degrading bacterium Introduce petroleum-degrading bacterium –Achromobacter sp. ATCC 21910 –Isolated from a crude oil-contaminated beach Introduce nutrients the bacteria need to degrade the oil Introduce nutrients the bacteria need to degrade the oil –Fertilizer (NH 4 NO 3, K 2 HPO 4 ) Carefully observe your microcosms, now and next week. Carefully observe your microcosms, now and next week. –Smell, appearance

15. Experimental Setup DishSand (30 gm) Oil (10 drops) WaterFertilizer (1 ml) Bacteria 1XX 8 ml XX 2XX 9 ml X 3XX X 4XX 10 ml

16. Procedure Label top and bottom of Petri dish with treatment number, lab group number, lab time. Label top and bottom of Petri dish with treatment number, lab group number, lab time. Weigh out 30 g sand into each dish Weigh out 30 g sand into each dish –Clean up any spilled sand! Add fertilizer, bacteria, oil at stations. Add fertilizer, bacteria, oil at stations. Add water using graduated cylinder Add water using graduated cylinder Stir sand well with glass rod. Stir sand well with glass rod. –Clean glass rod with ethanol and a tissue between dishes! Make observations Make observations Leave completed microcosms on your bench. Your TA will put them in the 30C incubator Leave completed microcosms on your bench. Your TA will put them in the 30C incubator

17. Safety Issues Oil is toxic. Avoid contact. Oil is toxic. Avoid contact. Achromobacter sp. ATCC 21910 is related to a pathogen. Achromobacter sp. ATCC 21910 is related to a pathogen. Wear labcoats, gloves, safety glasses. Wear labcoats, gloves, safety glasses. Avoid spilling/dripping bacterial culture Avoid spilling/dripping bacterial culture –Alert TA in the event of a spill, on the bench or your lab coat Wipe benches down, BEFORE AND AFTER, with disinfectant solution. Wipe benches down, BEFORE AND AFTER, with disinfectant solution. Wash your hands! Wash your hands!

18. Questions?