Bioaugmentation strategies for the treatment of pesticide waste streams Eng. Pieter Verhagen Promotors: Prof. dr. ir. Nico Boon Department of Biochemical and Microbial Technology Laboratory of Microbial Ecology and Technology Ghent University Prof. dr. ir. Leen De Gelder Department of Applied Biosciences Laboratory for Environmental and Biomass Technology Ghent University Dean: Prof. dr. ir. Marc Van Meirvenne Rector: Prof. dr. Anne De Paepe Bioaugmentation strategies for the treatment of pesticide waste streams Eng. Pieter Verhagen Thesis submitted in fulfillment of the requirements for the degree of Doctor (PhD) in Applied Biological Sciences Titel van het doctoraat in het Nederlands: Bioaugmentatie strategieën voor de behandeling van pesticide afvalstromen To refer tot his work: Verhagen, P (2015) Bioaugmentation strategies for the treatment of pesticide waste streams, Ph.D. thesis, Ghent University, Belgium ISBN: 978-90-5989-839-4 This work was funded by Ghent University (Assistant 2008-2014). The author and the promotors give the authorization to consult and to copy parts of this work for personal use only. Every other use is subject to copyright laws. Permission to reproduce any material contained in this work should be obtained from the author Notation index 2,4-D dichlorophenoxy acetic acid 3-CA 3-chloroanline AMO ammonium monooxigenase AMPA aminomethylphosphonic acid AOB ammonium oxidizing bacteria AOP advanced oxidation processes BPS biopurification systems CIPC chloropropham DGGE denaturing gradient gel electrophoresis EPS extracellular polymeric substance DNA deoxyribonucleic acid HPLC high performance liquid chromatography LB medium Luria Bertani medium MOB methane oxidizing bacteria MM minimal incubation medium MMO methane monooxygenase MPN most probable number PCR polymerase chain reaction pMMO particulate methane monooxygenase sMMO soluble methane monooxigenase VSS volatile suspended solids Table of contents Chapter 1: Introduction ....................................................................................................... 1 1 Pesticides: use, risks, regulations ............................................................................ 1 2 Routes of pesticide emission into the environment ................................................. 4 3 Treatment of pesticide point source contaminations ............................................... 7 3.1 Physico-chemical treatment methods ................................................................... 7 3.2 Biological treatment methods .............................................................................. 8 4 Bioaugmentation strategies to improve pesticide degradation inside a bps .......... 14 4.1 Strategy 1: metabolic approach: bioaugmentation strategies to enhance pesticide removal inside a bps ................................................................................... 15 4.2 Strategy 2: cometabolic degradation of pollutants using nitrifying and methane oxidizing bacteria ...................................................................................................... 19 4.3 Conclusion ......................................................................................................... 22 5 Objectives and outline ........................................................................................... 23 Chapter 2: Planktonic versus biofilm catabolic communities: importance of the biofilm architecture for species selection and pesticide degradation ............................................ 25 Abstract ......................................................................................................................... 25 1 Introduction ........................................................................................................... 26 2 Material and methods ............................................................................................ 27 3 Results ................................................................................................................... 33 4 Discussion .............................................................................................................. 43 Chapter 3: Inoculation with a mixed degrading culture improves the pesticide removal of an on-farm biopurification system .................................................................................... 47 Abstract ......................................................................................................................... 47 1 Introduction ........................................................................................................... 47 2 Material and methods ............................................................................................ 49 3 Results ................................................................................................................... 53 4 Discussion .............................................................................................................. 59 Chapter 4: spatial heterogeneity in degradation characteristics and microbial community composition of pesticide biopurification systems ............................................................ 61 Abstract ......................................................................................................................... 61 1 Introduction ........................................................................................................... 62 2 Material and methods ............................................................................................ 64 3 Results ................................................................................................................... 68 4 Discussion .............................................................................................................. 75 Chapter 5: Pesticide degradation using methanotrophs: influence of pesticide concentration and the microbial community composition ............................................... 79 Abstract ......................................................................................................................... 79 1 Introduction ........................................................................................................... 80 2 Materials and methods ........................................................................................... 81 3 Results ................................................................................................................... 84 4 Discussion .............................................................................................................. 90 Chapter 6: Discussion, conclusions and perspectives ...................................................... 94 1 Positioning of this work ......................................................................................... 94 2 Degradation by biofilm cultures results in less formation of metabolites ............. 96 3 Other carrier materials are possible ....................................................................... 97 4 Co-metabolism is a promising strategy to enhance biodegradation inside a BPS …………………………………………………………………………………..98 5 Guidelines to secure maximal pesticide degradation efficiency in biopurification systems ........................................................................................................................ 100 6 Suggestions for further Research ......................................................................... 101 Appendices ..................................................................................................................... 104 1 Abstract ................................................................................................................ 104 2 Samenvatting ....................................................................................................... 107 3 Bibliography ........................................................................................................ 110 4 Curiculum vitae ................................................................................................... 129 OUTLINE Recently, on-farm biopurification systems were developed to prevent pesticide point source contamination of ground- and surface water. However, some pesticides and their degradation products are highly recalcitrant and are not degraded inside a BPS. Two bioaugmentation strategies to enhance the biodegradation potential of biopurification systems were proposed in this work. (i) The usage of the metabolic capabilities of heterotrophic bacteria to improve pesticide degradation. (ii) Employing the pollutant degradation capabilities of the monooxygenase enzyme of methane oxidizing bacteria to co-metabolically degrade pesticides. In Chapter 1 we introduce the topic and provide enough information to enable the reader to comprehend the significance of the research questions. At the end of this chapter, the objectives of this thesis are given. In Chapter 2, we try to obtain pesticide degrading biofilm cultures and compare their degradation characteristics and microbial community structure with their planktonic counterparts. We also investigate the influence of the biofilm architecture on biodegradation characteristics. In Chapter 3, pilot scale BPS systems were inoculated using different types of inocula and substratum materials. We investigate the effect of the different inocula and substratum materials on the removal efficiency of the BPS. In Chapter 4, we investigate the