bioremediation of pahs-contaminated marsh soil by white-rot fungi

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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. - PowerPoint PPT Presentation

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  • Bioremediation of PAHs-contaminated marsh soil by white-rot fungiLara Valentn Carreralaralent@usc.es

    Chemical Engineering DepartmentUniversity of Santiago de CompostelaJuly 14, 2005Chemical Engineering DepartmentVERTIMAR-2005

  • July 14, 2005Chemical Engineering DepartmentUniversidad de Santiago de CompostelaVERTIMAR-2005Bioremediation 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

  • July 14, 2005Chemical Engineering DepartmentUniversidad de Santiago de CompostelaVERTIMAR-20051.1 What is bioremediation?

  • July 14, 2005Chemical Engineering DepartmentUniversidad de Santiago de CompostelaVERTIMAR-20051.1 BioremediationTechnologies 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 ratesEx situ: 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 DepartmentUniversidad de Santiago de CompostelaVERTIMAR-2005Slurry-phase bioreactors1.1 BioremediationJuly 14, 2005

  • July 14, 2005Chemical Engineering DepartmentUniversidad de Santiago de CompostelaVERTIMAR-20051.2 Why white-rot fungi for bioremediation?

  • July 14, 20051.2 White-rot fungiLignin-degrading fungiGrowth 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.

  • July 14, 2005Chemical Engineering DepartmentUniversidad de Santiago de CompostelaVERTIMAR-20051.2 White-rot fungiExtracellular enzymes

  • July 14, 2005Chemical Engineering DepartmentUniversidad de Santiago de CompostelaVERTIMAR-20051.2 White-rot fungiRecalcitrant compounds

  • July 14, 2005Chemical Engineering DepartmentUniversidade de Santiago de CompostelaVERTIMAR-20052. Objective

  • July 14, 2005Chemical Engineering DepartmentUniversidad de Santiago de CompostelaVERTIMAR-20052. 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.

  • July 14, 2005Chemical Engineering DepartmentUniversidad de Santiago de CompostelaVERTIMAR-20053. Screening of nine strains of white-rot fungi

  • Chemical Engineering DepartmentUniversidad de Santiago de CompostelaVERTIMAR-20053. Screening of white-rot fungiMarsh soilOperational parametersMix of 4 PAHs (50 mg/kg)120 rpmFungi: 9 strains:

    Temperature: 30 C

    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 soil16 ml culture medium+4 ml blended fungusPhanerochaete chrysosporium BKM-F-1767 (ATCC 24725)Phanerochaete sordida YK-624Poliporus ciliatus ONO94-1Stereum hirsutum PW93-4Lentinus tigrinus PW94-2Bjerkandera adusta BOS55 (ATCC 90940)Irpex lacteus Fr. 238 617/93Pleurotus eryngii CBS 613.91 (ATCC 90787)Phlebia radiata WIJSTER94-6100 mL-Erlenmeyer flask

  • July 14, 2005Chemical Engineering DepartmentUniversidad de Santiago de CompostelaVERTIMAR-20053. Screening of white-rot fungiMarsh soilResults5. Lentinus tigrinus; 6. Bjerkandera adusta; 7. Irpex lacteus.

  • July 14, 2005Chemical Engineering DepartmentUniversidad de Santiago de CompostelaVERTIMAR-20054. Time course degradation of PAHs

  • July 14, 2005Chemical Engineering DepartmentUniversidad de Santiago de CompostelaVERTIMAR-20054.Time course degradation of PAHsOperational parametersMix of 4 PAHs (50 mg/kg)2 g marsh soil16 ml culture medium+4 ml blended culture120 rpmFungi:- Lentinus tigrinus PW93-4

    - Irpex lacteus Fr. 238 617/93

    - Bjerkandera adusta BOS55

    Temperature: 30 C

    Time of incubation: 60 days

    PAHs analyses: 0 (abiotic controls)

    15 days30 days

    45 days60 days

  • Results4.Time course degradation of PAHs16 21 %19 26 %26 28 %22 39 %

  • July 14, 2005Chemical Engineering DepartmentUniversidad de Santiago de CompostelaVERTIMAR-20055. Effect of salinity on the enzymes activity

  • July 14, 2005Chemical Engineering DepartmentUniversidad de Santiago de CompostelaVERTIMAR-20055. Effect of PAHs and salinity Operational parametersA520/A350 per day Decolorization rate520 nm350 nm

  • July 14, 2005Results5. Effect of salinity Lentinus tigrinus Irpex lacteus Bjerkandera adusta 100 % seawater50% seawater0 % seawater

  • July 14, 2005Chemical Engineering DepartmentUniversidad de Santiago de CompostelaVERTIMAR-20056. Slurry bioreactor

  • July 14, 2005Chemical Engineering DepartmentUniversidad de Santiago de CompostelaVERTIMAR-20056. Slurry bioreactorOperational parametersVolume 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 C

    Condenser water temp: 5 C

    Marsh soil: 100 g L-1

    Total Volume: 4 L

  • July 14, 2005Chemical Engineering DepartmentUniversidad de Santiago de CompostelaVERTIMAR-20056. Slurry bioreactorOperational variablesBjerkandera adusta BOS55

  • July 14, 2005Chemical Engineering DepartmentUniversidad de Santiago de CompostelaVERTIMAR-20056. Slurry bioreactorGrowth of Bjerkandera adusta BOS55Pellets 5 days (magnifying glass)Pellets 7 days (magnifying glass)Broken pellets 8 days (magnifying glass)Mycelia 9 days (microscope 40x)

  • July 14, 2005Chemical Engineering DepartmentUniversidad de Santiago de CompostelaVERTIMAR-20056. Slurry bioreactorResidual PAHs %Bjerkandera adusta BOS55

  • July 14, 2005Chemical Engineering DepartmentUniversidad de Santiago de CompostelaVERTIMAR-20056. Slurry bioreactorDecolorization of Poly-R Inoculum7 days12 days21 days26 daysBjerkandera adusta BOS55

  • July 14, 2005Chemical Engineering DepartmentUniversidad de Santiago de CompostelaVERTIMAR-20056. Slurry bioreactorGrowth of Bjerkandera adusta BOS55Pictures of Bjerkandera adusta growing on the walls of the bioreactor and on the stirrer

  • July 14, 2005Chemical Engineering DepartmentUniversidad de Santiago de CompostelaVERTIMAR-20057. Conclusions

  • July 14, 2005Chemical Engineering DepartmentUniversidad de Santiago de CompostelaVERTIMAR-20057. 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 DepartmentUniversidad de Santiago de CompostelaVERTIMAR-2005Acknowledgements to Gumersindo Feijoo, Maria Teresa Moreira, Juan Manuel Lema, Thelmo L-Chau and Alanna Malcolm.CICYT: VEM2003-20089-C02-01

    PAHs: Phenanthrene, Fluoranthene, Pyrene and and PyreneMedio kirk: 10 g/l glucose, 5 g/l peptone-ammonium tartrate, 100 ml BIII and 1 ml/L thiamine (2 mg/ml)PAHs: Dibenzothiphene, Fluoranthene, Chrysene and PyreneAll of the fungi achieved the same degradation of the 4 PAHs. Slight diferencesPAHs: Dibenzothiphene, Fluoranthene, Chrysene and PyreneKirk medium: 18 g/l de glucosa, 2g/l peptona, Mineral medium 100 ml/L, Tween y TiaminaKirkm media was addes very concentrated in 500 ml of liquid.It was used destilled water to complete the total volume.

    Red arrow indicate an addition of glucose and peptone to the reactor: 11.7, 14.6 and 17.7 daysIt was added 2 g/L of glucose and 0,4 g/l of peptone every 3 days.The samples for the microscope were agitated and diluted with water (1:4 v/v). For the magnifying glass each pellet was taken separeted. The pellets were clearly visible. The pellets started to break after 7 days.Kirk medium: 18 g/l de glucosa, 2g/l peptona, Mineral medium 100 ml/L, Tween y TiaminaKirkm media was addes very concentrated in 500 ml of liquid.It was used destilled water to complete the total volume.

    Front of the plate to show the growth of the fungi. Back of the plate to show the decolorization of the dye.