1. Remediation of organic pollutants by solid phase capture Coleen Murty Supervisor: Vernon Phoenix

2. Contents Background o Organic pollutants o Existing remediation strategies o Solid phase capture overview Project overview and hypothesis Research strategy and analytical techniques

3. Background Organic pollutants Toxic chemical compounds Bioaccumulators Hydrophobic and fat-soluble Persistent (stable) Volatile Range of anthropogenic sources pesticides e.g. DDT and chlordane industrial chemicals e.g. Hexachlorobenzene and polychlorinated biphenols Gas and petrol station sources e.g. Hydrocarbons Petrol canister

4. landfill leakage Sources of groundwater contamination pesticide run-off Industrial storage leaks

5. Some of the current remediation strategies A few existing techniques used to clean-up organic waste include: Chemical oxidation processes Pump and treat technologies Newly emerged technologies include bioremediation methods: Phytoremediation - use of plants to degrade pollutants Bioventing - in situ simulation of microorganisms in order to biodegrade organic contaminants

6. Solid phase capture Calcite precipitation is a new strategy being developed for remediation of contaminated groundwater Mechanism involves capture of pollutant into calcite crystal as it precipitates Already been used to successfully immobilize inorganic pollutants e.g. metals Immobilization of organic pollutants by this method is yet to be explored

8. + + + + Different immobilization mechanisms Adsorption Co-precipitation by isomorphic replacement Lattice incorporation (defect vacancies) calcite crystal interstitial positions replaces Ca 2 ⁺

9. Project overview This project will assess calcites ability to capture proteins within its molecular structure as it precipitates Proteins used as model organic pollutants The formation of calcite will be induced by both chemical and microbial methods Hypothesis being tested: Organic pollutants can be immobilized from groundwater into a calcite crystal as it precipitates

10. Chemically induced calcite precipitation Addition of calcium hydroxide to solution then stirred with carbon dioxide: Ca(OH) 2 + CO 2  CaCO 3 + H 2 O calcium hydroxide calcium carbonate increase pH increase in calcite saturation Atmospheric

11. Calcite precipitation system Cloudy solution = CaCO 3

12. Microbially-induced calcite precipitation Ureolytic bacteria will be used to facilitate calcite precipitation (bacteria containing urease enzyme) This reaction is called ureolysis and involves the microbial degradation of urea Results in an overall increase in pH and higher calcite saturation levels S. Pasteurii Urease positive

13. Ureolysis During this reaction urea will be hydrolysed to form ammonia and carbonic acid: CO(NH 2 ) 2 + 2H 2 O  2NH 3 + H 2 CO 3 urea ammonia carbonic acid These then equilibrate in water to become: H 2 CO 3  HCO 3 ⁻ + H ⁺  CO 3 2 ⁻ carbonic acid bicarbonate carbonate Urease enzyme

14. Ureolysis cont.. Ammonia also equilibrates with water to ammonium and hydroxide ions: 2NH 3 + H 2 O  NH 4 ⁻ + OH ⁻ ammonia ammonium hydroxide Bicarbonate from reaction 2 interacts with calcium input and increases calcite saturation Ca 2 ⁺ + HCO 3 ⁻  CaCO 3 + H ⁺ calcium bicarbonate calcium carbonate

15. SEM images of forming calcite precipitates around bacteria, A (left image) shows initial stages of precipitation and B (right) shows bacteria buried within growing calcite precipitate (Mitchell and Ferris, 2005)

16. Research strategy Calcite precipitation experiments will take place in glass beakers containing proteins Concentration of proteins free in solution will be monitored pH and amounts of Ca 2 ⁺ and NH 4 ⁻ ions in solution will also be measured

17. Protein assay The protein assay used will be used to measure the amount of protein free in solution Involves the addition of a series of chemicals Added chemicals form complexes with the peptide bonds present within protein molecule Peptide bonds in protein react with Cu 2 ⁺ from protein assay

18. Methods UV/VIS spectrophotometer used in conjunction with protein assay Determines proteins free in solution Detects presence of peptide bonds Colour change dependant on number of peptide bonds Intense colour change = more protein molecules

19. Thermodynamic modelling Results derived from experiments will be run through a thermodynamic software called Satistica Various reaction rates will be determined by calculations: Rates of ureolysis Rates of calcite precipitation Rate of protein uptake Gives more insight into reaction kinetics

20. Conclusions Solid phase capture is shown to be a promising strategy for the sequestration of inorganic pollutants The investigation of calcites ability to capture proteins will open gateways to further research prospects Thank you

21. References Carlini, C.R. 2014. Bacillus pasteurii. [image online] [Accessed 20th November 2014]http://www.ufrgs.br/laprotox/en/research-lines/ureases-soil/bacillus-pasteurii-and- bpu Huling, S.G. 2014. ISCO. [image online] Available at: http://www.epa.gov/ada/gw/isco.html [Accessed 20th November 2014]http://www.epa.gov/ada/gw/isco.html Lauchnor, E.G., Schultz, L.N., Bugni, S., Mitchell, A.N., Cunningham, A.B., and Gerlack, R. 2013. Bacterially Induced Calcium Carbonate Precipitation and Strontium Coprecipitation in a Porous Media Flow System. Environmental Science and Technology. 47: 1557-1564 Mitchell, A.C., and Ferris, F.G. 2005. The coprecipitation of Sr into calcite precipitates induced by bacterial ureolysis in artificial groundwater: Temperature and kinetic dependence. Geochimica et Cosmochimica Acta. 69: 4199-4210 Tobler, T.J., Cuthbert, M.O., Greswell, R.B., Riley, M.S., Renshaw, J.C., Sidhu, S.H., and Phoenix, V.R. 2011. Comparison of rates of ureolysis between Sporosarcina pasteurii and an indigenous groundwater community under conditions required to precipitate large volumes of calcite. Geochimica et Cosmochimica Acta. 75: 3290-3301 Wang, H., Liu, S., and Du, S. 2013. Groundwater Pollution. [image online] Available at: [Accessed 19th November 2014]http://www.intechopen.com/books/organic- pollutants-monitoring-risk-and-treatment/the-investigation-and-assessment-on-groundwater-organic-pollution Warren, L.A., Maurice, P.A., Parma, N., and Ferris, F.G. 2001. Microbially Mediated Calcium Carbonate Precipitation: Implications for Interpreting Calcite Precipitation and for Solid-Phase Capture of Inorganic Contaminants. Geomicrobiology Journal. 18: 93-115