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Bioremediation of PAHs-contaminated marsh soil by white-rot fungi

Bioremediation of PAHs-contaminated marsh soil by white-rot fungi . Lara Valentín Carrera laralent@usc.es Chemical Engineering Department University of Santiago de Compostela. Chemical Engineering Department VERTIMAR-2005. July 14, 2005. Bioremediation of PAHs by white rot fungi.

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Bioremediation of PAHs-contaminated marsh soil by white-rot fungi

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  1. Bioremediation of PAHs-contaminated marsh soil by white-rot fungi • Lara Valentín Carrera • laralent@usc.es • Chemical Engineering Department • University of Santiago de Compostela Chemical Engineering Department VERTIMAR-2005 July 14, 2005

  2. Bioremediation of PAHs by white rot fungi Introduction Objective Screening of nine strains of white-rot fungi (WRF) Time course degradation of PAHs Effect of salinity on the enzymes activity Slurry bioreactor Conclusions Chemical Engineering Department Universidad de Santiago de Compostela VERTIMAR-2005 July 14, 2005

  3. 1.1 What is bioremediation? Chemical Engineering Department Universidad de Santiago de Compostela VERTIMAR-2005 July 14, 2005

  4. 1.1 Bioremediation Technologies • Bioremediation attempts to use plants and microbes (bacteria, fungi and algae) to enhance the natural processes for removing or decomposing the unwanted substances (Cheng and Mulla, 1999). • Classification of bioremediation technologies (Bonten, 2001) On site: Biopiles and landfarming In situ: Natural attenuation and Bioaugmentation Ex situ: Slurry-phase bioreactors Degradation rates Chemical Engineering Department Universidad de Santiago de Compostela VERTIMAR-2005 July 14, 2005

  5. 1.1 Bioremediation Slurry-phase bioreactors • Certain amount of water is added to the contaminated soil. The soil-water mixture is mixed and aerated. • Solid content of 10 to 20 weight percentage. • Operated continuously or semi-continuously. • Aerobic conditions (frequently) or anaerobic. • High contact microorganisms – contaminant. • High mass transfer rates. • High degradation rates. • Constant control of the degradation process. Chemical Engineering Department Universidad de Santiago de Compostela VERTIMAR-2005 July 14, 2005

  6. 1.2 Why white-rot fungi for bioremediation? Chemical Engineering Department Universidad de Santiago de Compostela VERTIMAR-2005 July 14, 2005

  7. 1.2 White-rot fungi Lignin-degrading fungi • Group of basidiomycetes which produce a group of extracellular enzymes involve in the degradation of the most recalcitrant layer of the plant cell wall (lignin). • WRF colonize dead or dying tree trunks and stumps causing white rot via the utilization of hemicellulose and cellulose during the degradation of lignin. Growth of Bjerkandera adusta on a trunk Molecular structure of lignin July 14, 2005

  8. 1.2 White-rot fungi Extracellular enzymes Chemical Engineering Department Universidad de Santiago de Compostela VERTIMAR-2005 July 14, 2005

  9. 1.2 White-rot fungi Recalcitrant compounds Chemical Engineering Department Universidad de Santiago de Compostela VERTIMAR-2005 July 14, 2005

  10. 2. Objective Chemical Engineering Department Universidade de Santiago de Compostela VERTIMAR-2005 July 14, 2005

  11. 2. Objective • To develop a slurry-phase bioreactor technology operated with white-rot fungi for the treatment of marine sites contaminated with fuel oil derivatives. • The study was focused on the aromatic fraction of the fuel, especially on the PAHs since they have a recalcitrant and toxic nature. Chemical Engineering Department Universidad de Santiago de Compostela VERTIMAR-2005 July 14, 2005

  12. 3. Screening of nine strains of white-rot fungi Chemical Engineering Department Universidad de Santiago de Compostela VERTIMAR-2005 July 14, 2005

  13. 3. Screening of white-rot fungiMarsh soil Operational parameters Fungi: 9 strains: Temperature:30 ºC Time of incubation:30 days PAHs analyses:0 (abiotic controls) 30 days PAHs extraction: - 40 ml Hexane : Acetone (1:1) - Shaking at 300 rpm for 2 h - HPLC • Phanerochaete chrysosporium BKM-F-1767 (ATCC 24725) • Phanerochaete sordida YK-624 • Poliporus ciliatus ONO94-1 • Stereum hirsutum PW93-4 • Lentinus tigrinus PW94-2 • Bjerkandera adusta BOS55 (ATCC 90940) • Irpex lacteus Fr. 238 617/93 • Pleurotus eryngii CBS 613.91 (ATCC 90787) • Phlebia radiata WIJSTER94-6 Mix of 4 PAHs (50 mg/kg) 100 mL-Erlenmeyer flask 16 ml culture medium + 4 ml blended fungus 2 g marsh soil 120 rpm Chemical Engineering Department Universidad de Santiago de Compostela VERTIMAR-2005

  14. Ct=0 Ct=30 1 2 3 4 5 6 7 8 9 Ct=0 Ct=30 1 2 3 4 5 6 7 8 9 3. Screening of white-rot fungiMarsh soil Results 5. Lentinus tigrinus; 6. Bjerkandera adusta; 7. Irpex lacteus. Chemical Engineering Department Universidad de Santiago de Compostela VERTIMAR-2005 July 14, 2005

  15. 4. Time course degradation of PAHs Chemical Engineering Department Universidad de Santiago de Compostela VERTIMAR-2005 July 14, 2005

  16. 4.Time course degradation of PAHs Operational parameters Fungi: - Lentinus tigrinus PW93-4 - Irpex lacteus Fr. 238 617/93 - Bjerkandera adusta BOS55 Temperature:30 ºC Time of incubation:60 days PAHs analyses:0 (abiotic controls) 15 days 30 days 45 days 60 days Mix of 4 PAHs (50 mg/kg) 16 ml culture medium + 4 ml blended culture 2 g marsh soil 120 rpm Chemical Engineering Department Universidad de Santiago de Compostela VERTIMAR-2005 July 14, 2005

  17. 4.Time course degradation of PAHs Results 19 – 26 % 16 – 21 % 26 – 28 % 22 – 39 %

  18. 5. Effect of salinity on the enzymes activity Chemical Engineering Department Universidad de Santiago de Compostela VERTIMAR-2005 July 14, 2005

  19. 5. Effect of PAHs and salinity Spectrum of Poly-R before degradation 0,35 0,3 0,25 5 solutions of seawater 0,2 Absorbance 100 % seawater 0 % seawater 0,15 0,1 0,05 0 250 350 450 550 650 Wavelength (nm) Spectrum of Poly-R after degradation 2 agar-plugs of fungus 15 ml culture medium 0.02 % Poly-R Operational parameters 350 nm 520 nm Decolorization rate A520/A350 per day Chemical Engineering Department Universidad de Santiago de Compostela VERTIMAR-2005 July 14, 2005

  20. 5. Effect of salinity 1 day 10 days 22 days Results 100 % seawater 50% seawater 0 % seawater Lentinus tigrinus■ Irpex lacteus ♦ Bjerkandera adusta ▲ July 14, 2005

  21. 6. Slurry bioreactor Chemical Engineering Department Universidad de Santiago de Compostela VERTIMAR-2005 July 14, 2005

  22. 6. Slurry bioreactor Operational parameters Volume of the reactor: 5 L Fungus: Bjerkandera adusta BOS55 Initial biomass concent:0.69 g L-1 Initial concentration of PAHs: 50 mg kg-1 Initial glucose concent: 18 g L-1 Air flow: 4 L min-1 Stirring: 250 rpm Temp:30 ºC Condenser water temp: 5 ºC Marsh soil: 100 g L-1 Total Volume: 4 L Chemical Engineering Department Universidad de Santiago de Compostela VERTIMAR-2005 July 14, 2005

  23. 6. Slurry bioreactor Operational variables Bjerkandera adusta BOS55 Chemical Engineering Department Universidad de Santiago de Compostela VERTIMAR-2005 July 14, 2005

  24. 6. Slurry bioreactor Growth of Bjerkandera adusta BOS55 Pellets – 5 days (magnifying glass) Pellets – 7 days (magnifying glass) Broken pellets – 8 days (magnifying glass) Mycelia – 9 days (microscope 40x) Chemical Engineering Department Universidad de Santiago de Compostela VERTIMAR-2005 July 14, 2005

  25. 6. Slurry bioreactor Residual PAHs % Bjerkandera adusta BOS55 Chemical Engineering Department Universidad de Santiago de Compostela VERTIMAR-2005 July 14, 2005

  26. 6. Slurry bioreactor Decolorization of Poly-R Bjerkandera adusta BOS55 Inoculum 7 days 12 days 21 days 26 days Chemical Engineering Department Universidad de Santiago de Compostela VERTIMAR-2005 July 14, 2005

  27. 6. Slurry bioreactor Growth of Bjerkandera adusta BOS55 Pictures of Bjerkandera adusta growing on the walls of the bioreactor and on the stirrer Chemical Engineering Department Universidad de Santiago de Compostela VERTIMAR-2005 July 14, 2005

  28. 7. Conclusions Chemical Engineering Department Universidad de Santiago de Compostela VERTIMAR-2005 July 14, 2005

  29. 7. Conclusions • All of the WRF degraded PAHs in small scale slurry-phase bioreactors. • Lentinus tigrinus, Bjerkandera adusta and Irpex lacteus were selected for further experiments. • No effect of salt conditions on the enzyme activity of WRF. • Scale-up of the bioreactor did not affect the growth of Bjerkandera adusta. • Bjerkandera adusta produced pellets in the beginning of process. After 8 days the pellets broke, however the degradation continued. • The activity of the fungus was probed by the decolorization of Poly-R plates. • A basidiomycetes fungus is able to resist the slurry-phase conditions (stirring, aireation, water and solid content) and to degrade PAHs after 26 days. Chemical Engineering Department Universidad de Santiago de Compostela VERTIMAR-2005 July 14, 2005

  30. Acknowledgementsto Gumersindo Feijoo, Maria Teresa Moreira, Juan Manuel Lema, Thelmo Lú-Chau and Alanna Malcolm.CICYT: VEM2003-20089-C02-01 Chemical Engineering Department Universidad de Santiago de Compostela VERTIMAR-2005

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