UNIVERSITY OF PANNONIA GEORGIKON FACULTY FESTETICS DOCTORAL SCHOOL School leader: Dr. habil. Angéla Anda, DSc THE BASICS OF RESEARCH IN MICROBIOLOGICAL SOIL REMEDIATION PRACTICES DOCTORAL (PHD) THESIS Written by: Nikoletta Horváth Thesis supervisors: Dr. Borbála Biró, DSc Dr. Péter Budai, PhD 2017 KESZTHELY HUNGARY CONTENTS 1. ABSTRACTS............................................................................................................................................... 1 1.1. Abstract (English) ................................................................................................................................. 1 1.2. Kivonat (Hungarian) ............................................................................................................................. 2 1.3. Abstract (German) ................................................................................................................................ 3 2. INTRODUCTION AND IMPORTANCE OF THE TOPIC ................................................................... 4 2.1. Research background ............................................................................................................................ 4 2.2. Basis of the research ............................................................................................................................. 4 3. OBJECTIVES ............................................................................................................................................. 6 4. LITERATURE REVIEW........................................................................................................................... 7 4.1. Soil characteristics and soil pollution ................................................................................................... 7 4.2. Biodegradation of soil pollutants by microorganisms .......................................................................... 8 4.2.1. Bacteria in bioremediation ............................................................................................................ 9 4.2.2. Fungal mycoremediation applications ........................................................................................ 12 4.2.3. The aspects of choosing microorganisms for bioremediation ..................................................... 12 4.3. Bioremediation practices .................................................................................................................... 12 4.3.1. Environmental Monitoring .......................................................................................................... 13 4.3.2. Bioremediation methods .............................................................................................................. 14 4.3.3. Advantages and disadvantages of bioremediation ...................................................................... 20 4.3.4. Phytoremediation ........................................................................................................................ 21 4.3.5. Advantages and disadvantages of phytoremediation................................................................... 23 5. METHODS AND MATERIALS ............................................................................................................. 24 5.1. Research sites ...................................................................................................................................... 24 5.1.1. Gardermoen, Norway (de-icing chemical contamination) .......................................................... 24 5.1.2. Trecate, Italy (crude-oil contamination) ..................................................................................... 28 5.1.3. Őrbottyán, Hungary (Manganese treatment) .............................................................................. 31 5.2. List of the performed analytical tests .................................................................................................. 35 5.3. Microbial counts using traditional Plate Count method ...................................................................... 37 5.4. The MPN method ................................................................................................................................ 38 5.4.1. Monitoring of the rate of degraders in Gardermoen ................................................................... 39 5.4.2. Manganese tolerance tests in the soil of Őrbottyán .................................................................... 39 5.5. Total Organic Carbon measurements .................................................................................................. 39 5.6. Enzymatic activity .............................................................................................................................. 39 5.7. Column experiments ........................................................................................................................... 41 5.8. Molecular methods.............................................................................................................................. 43 5.8.1. Population analysis in propylene-glycol contaminated soil ........................................................ 44 5.8.2. Isolation and identification of abundant microorganisms from crude-oil contaminated soil ..... 45 5.8.3. Isolation and identification of Manganese tolerant strains from Őrbottyán ............................... 47 5.9. Antibiotic susceptibility testing .......................................................................................................... 52 6. RESULTS AND DISCUSSION ............................................................................................................... 54 6.1. Results of the propylen-glycol contaminated soils of Gardermoen .................................................... 54 6.1.1. Vertical and horizontal microbial abundance in PG contaminated soils .................................... 54 6.1.2. Microbial enzymatic activity in propylene-glycol contaminated soils ........................................ 58 6.1.3. Organic carbon content in propylene-glycol contaminated soil ................................................. 60 6.1.4. Simulation of de-icing chemical contamination and potential microbial adaptation ................. 60 6.1.5. Summary of the research in the deicing-fluid contaminated Gardermoen site ........................... 66 6.2. Results of the crude-oil contaminated Trecate site ............................................................................. 67 6.2.1. Microbial abundance in crude-oil contaminated soils ................................................................ 67 6.2.2. Microbial enzymatic activity in crude-oil contaminated soils ..................................................... 67 6.2.3. Identification of isolates from the crude-oil contaminated soil ................................................... 71 6.2.4. Summary of the research in the crude-oil contaminated Trecate site ......................................... 75 6.3. Results of the Manganese treated soil of Őrbottyán ........................................................................... 76 6.3.1. Biomass of the Energy grass on Manganese treated soil ............................................................ 76 6.3.2. The effect of Manganese on microbial abundance ...................................................................... 77 6.3.3. Microbial enzymatic activity after Manganese treatment ........................................................... 78 6.3.4. The appearance of Manganese tolerance .................................................................................... 79 6.3.5. Identification of Manganese tolerant strains ............................................................................... 80 6.3.6. Summary of the research in the Manganese treated soil of Őrbottyán ....................................... 84 7. CONCLUSIONS AND FUTURE ASPECTS ......................................................................................... 85 8. NEW SCIENTIFIC RESULTS ................................................................................................................ 89 9. ACKNOWLEDGEMENTS...................................................................................................................... 90 10. PUBLICATIONS ...................................................................................................................................... 91 10.1. Scientific articles (English) ................................................................................................................. 91 10.2. Presentations published in full length (English) ................................................................................. 91 10.3. Presentations published as abstracts (English) .................................................................................... 91 10.4. Contributions to other publications not connected to the topic (Hungarian) ...................................... 92 11. REFERENCES .......................................................................................................................................... 93 LIST OF FIGURES 1. Figure: The connection of organisms, soils and contaminants, and their effect on each other .................................. 9 2. Figure: Design of a biopile: the contaminated soil mixed with amendments is palced on a treatment area, where oxygen is supplied with pipes