1. Biodegradation of MTBE and BTEX in a Full-Scale Reactor down to ppb Levels after Inoculation with a MTBE Degrading Culture Erik Arvin, DTU Environment Christopher Kevin Waul, DTU Environment Rasmus Krag, Jord·Miljoe A/S Charlotte Juhl Søegaard, Jord·Miljoe A/S Jeppe Lund Nielsen, Section of Biotechnology, AAU Natural and stimulated biological degradation – Processes and microbiology ATV Soil and Groundwater 21. April 2010

2. Content Contamination with MTBE Guidelines MTBE properties Biological degradation of MTBE Development of MTBE culture Full scale MTBE treatment plant MTBE and BTEX removal Microbiology Perspectives Conclusions

4. Oil/gasoline spill Oil, air, water Soil-zone Free oil Groundwater Dissolved hydrocarbons BTEXN

5. Aromatic hydrocarbons from gasoline, BTEXN

6. Guidelines

7. Drinking Water Requirements (DK) µg/L Benzene: 1 Alkylbenzenes: 1 Naphthalene: 2 TPH (Tot. Petroleum Hydrocarbons): 5 MTBE (Methyl tert. butyl ether): 5 Spec. phenols: 0.5

8. Groundwater & surface water requirements for MTBE (DK) Groundwater: 5 µg/L Surface water: 10 µg/L Rain water, sewers: 10 µg/L

9. MTBE is a gasoline additive! Ether compound Lead replacement Improves air quality Adds oxygen to fuel Adds octane to fuel Chemical structure of methyl tert-butyl ether Cleaner burning fuel

10. Properties of MTBE Molar weight: 88 Solubility, water: 50.000 mg/L Hc: 0.022 log Kow: ca. 1 log Koc: ca. 1 Vapour pressure: 245 mm Hg, v. 25 °C

11. Biodegradability of MTBE

12. MTBE mineralization reactions (F. Finneran and D.R. Lovley, 2003) Reactions G o (pH 7, 25C) kJ/mole MTBE Aerobic respiration C 5 H 12 O + 7.5 O 2  5 HCO 3 - + 5 H + + H 2 O -3246 Denitrification C 5 H 12 O + 6 NO 3 - + H +  5 HCO 3 - + 3 N 2 + 4 H 2 O -3055 Nitrate reduction C 5 H 12 O + 3.75 NO 3 - + 2.5 H + + 2.75 H 2 O 5 HCO 3 - + 3.75 NH 4 + -1951 Fe(III) reduction C 5 H 12 O + 30 Fe(OH) 3 + 55 H +  5 HCO 3 - + 30 Fe ++ + 76 H 2 O -347 Sulfate reduction C 5 H 12 O + 3.75 SO 4 - - + 2.5 H +  5 HCO 3 - + 3.75 H 2 S + H 2 O -275 Methanogenesis C 5 H 12 O + 2.75 H 2 O  3.75 CH 4 + 1.25 HCO 3 - + 1.25 H + -239

13. Anaerobic degradation of MTBE Probably, it does not occur in most cases. However, anaerobic abiotic hydrolysis may take place. If anaerobic biodegradation takes place, it is probably a very slow reaction.

14. Aerobic biodegradation of MTBE 1. Biodegradation of MTBE as a primary substrate 2. Biodegradation by cometabolism with lowmolecular alkanes and cycloalkanes as primary substrates

15. MTBE biomass growth rate and growth yield Doubling time: 7-20 days Growth yield: 0.1-0.2 g biomass/g MTBE Observation of no degradation may be due to the very slow biomass growth

16. Development of the MTBE degrading culture

17. Batch cultures degrading MTBE

18. Submerged biofilter for MTBE degradation Height of column 0.3 m Volume 0.46 L total 0.27 L porosity Filter material Expanded clay (Filtralite®) 1.5 – 2.5 mm

19. DTU bench scale biofilters for MTBE degradation Two columns in series Height 1 m and diameter 10 cm Volume 9.5 L each Filter material Expanded clay (Filtralite®) 2.5 – 4 mm Seeded with filter material from small column

20. Schematics of the MTBE treatment plant with biofilters BF1-BF3.

21. On-site MTBE removal in biofilter in Farum

22. Oil separator (left) and biofilters (right).

23. MTBE removal

24. BTEX removal

25. MTBE removal kinetics Data from Farum plant, Svendborg/Grubbemølle Water Works and DTU bench scale plant 1’st order removal rate constant: k 1,v = 1.7-4.4 h -1 (C_MTBE < 2000 ug/L) Variation in k 1,v probably due to differences in specific surface removal area, cultures, etc.

26. Microbiology Molecular analysis by DGGE analysis has identified a complex community structure in the biofilters. The majority were members of the phyla: Bacteroidetes, Proteobacteria, and Nitrospirae Among the genera identified were Terrimonas and Methylibium petroleiphilum (PM1) Proof of active MTBE degraders will be investigated by microautoradiography and fluorescence in situ hybridization

27. Perspectives Extremely efficient and stable MTBE and BTEX removal has been demonstrated The full scale plant removes MTBE and BTEX to below drinking water and surface water requirements Determination of the MTBE removal kinetics allows more credible plant design

28. Conclusions Successful up-scaling of MTBE removal from lab. scale to full scale biofilters MTBE removal > 99 % Effluent MTBE ~ 1 ug/L BTEX removal > 99.9 % Effluent BTEX ~< 0.01 ug/L High process stability k 1,v = 1.7-4.4 h -1 Variation in removal rate constant is probably due to differences in specific surface areas in plants Bacterial community composition is complex and under investigation

29. ACKNOWLEDGMENTS Ulrich Gosewinkel Karlson from the National Environmental Research Institute, University of Aarhus, Roskilde, Denmark, provided a mixed MTBE degrading culture for inoculation of the batch cultures that preceded the 10 L lab biofilter that was used for up-scaling. Statoil: We acknowledge very much Statoil for the opportunity to test the MTBE and BTEX removal plant in full scale.