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 July 14, 2005 Chemical Engineering Department VERTIMAR-2005

2. July 14, 2005 Chemical Engineering Department Universidad de Santiago de Compostela VERTIMAR-2005 Bioremediation of PAHs by white rot fungi 1. Introduction 2. Objective 3. Screening of nine strains of white-rot fungi (WRF) 4. Time course degradation of PAHs 5. Effect of salinity on the enzymes activity 6. Slurry bioreactor 7. Conclusions

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

4. July 14, 2005 Chemical Engineering Department Universidad de Santiago de Compostela VERTIMAR-2005 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 Degradation rates Ex situ: Slurry-phase bioreactors

5.  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 Slurry-phase bioreactors 1.1 Bioremediation July 14, 2005

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

7. July 14, 2005 1.2 White-rot fungi Lignin-degrading fungi Growth of Bjerkandera adusta on a trunkMolecular structure of lignin  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.

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

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

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

11. July 14, 2005 Chemical Engineering Department Universidad de Santiago de Compostela VERTIMAR-2005 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.

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

13. Chemical Engineering Department Universidad de Santiago de Compostela VERTIMAR-2005 3. Screening of white-rot fungi Marsh soil Operational parameters Mix of 4 PAHs (50 mg/kg) 120 rpm 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 2 g marsh soil 16 ml culture medium + 4 ml blended fungus 1.Phanerochaete chrysosporium BKM-F-1767 (ATCC 24725) 2.Phanerochaete sordida YK-624 3.Poliporus ciliatus ONO94-1 4.Stereum hirsutum PW93-4 5.Lentinus tigrinus PW94-2 6.Bjerkandera adusta BOS55 (ATCC 90940) 7.Irpex lacteus Fr. 238 617/93 8.Pleurotus eryngii CBS 613.91 (ATCC 90787) 9.Phlebia radiata WIJSTER94-6 100 mL-Erlenmeyer flask

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

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

16. July 14, 2005 Chemical Engineering Department Universidad de Santiago de Compostela VERTIMAR-2005 4.Time course degradation of PAHs Operational parameters Mix of 4 PAHs (50 mg/kg) 2 g marsh soil 16 ml culture medium + 4 ml blended culture 120 rpm 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

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

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

19. July 14, 2005 Chemical Engineering Department Universidad de Santiago de Compostela VERTIMAR-2005 5. Effect of PAHs and salinity Operational parameters 5 solutions of seawater 0 % seawater 100 % seawater 2 agar-plugs of fungus 15 ml culture medium 0.02 % Poly-R A 520 /A 350 per day Decolorization rate Spectrum of Poly-R after degradation 0 0,05 0,1 0,15 0,2 0,25 0,3 0,35 250350450550650 Wavelength (nm) Absorbance Spectrum of Poly-R before degradation 520 nm 350 nm

20. July 14, 2005 Results 5. Effect of salinity Lentinus tigrinus ■ Irpex lacteus ♦ Bjerkandera adusta ▲ 100 % seawater50% seawater0 % seawater 1 day10 days22 days

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

22. July 14, 2005 Chemical Engineering Department Universidad de Santiago de Compostela VERTIMAR-2005 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

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

24. July 14, 2005 Chemical Engineering Department Universidad de Santiago de Compostela VERTIMAR-2005 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)

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

26. July 14, 2005 Chemical Engineering Department Universidad de Santiago de Compostela VERTIMAR-2005 6. Slurry bioreactor Decolorization of Poly-R Inoculum 7 days12 days21 days26 days Bjerkandera adusta BOS55

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

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

29. July 14, 2005 Chemical Engineering Department Universidad de Santiago de Compostela VERTIMAR-2005 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.

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