Biofuels (H2, CH4, Ethanol) Waste Biomass Energy and Waste based Environmental Treatment Biorefnery Sustainability Biochemicals (organic acids) Anish Ghimire Joint PhD degree in Environmental Technology Docteur de l’Université Paris-Est Spécialité : Science et Technique de l’Environnement Dottore di Ricerca in Tecnologie Ambientali Degree of Doctor in Environmental Technology Thèse – Tesi di Dottorato – PhD thesis Anish Ghimire Dark fermentative biohydrogen production from organic waste and application of by-products in a biorefinery concept Defended on December 17th, 2015 In front of the PhD committee Prof. Dr. Ing. Massimiliano Fabbricino Reviewer Prof. Dr. Ing. Raffaella Pomi Reviewer Dr. Hab. E.D. van Hullebusch Examiner Prof. Dr. Ing. Giovanni Esposito Promotor Prof. Dr. Ir. Piet N.L. Lens Co-promotor Prof. Dr. Ing. Francesco Pirozzi Co-promotor Prof. Michel Madon Co-promotor Erasmus Joint doctorate programme in Environmental Technology for Contaminated Solids, Soils and Sediments (ETeCoS3) i Thesis committee Thesis Promotor Prof. Dr. Ing. G. Esposito, Associate Professor of Sanitary and Environmental Engineering University of Cassino and Southern Lazio, Cassino, Italy Thesis co-promotors and supervisors Prof. Dr. Ir. P.N.L. Lens Professor of Biotechnology UNESCO-IHE, Delft, The Netherlands Prof. Dr. Ing. Francesco Pirozzi Professor of Sanitary and Environmental Engineering University of Naples Federico II, Naples, Italy Dr. Ing. L. Frunzo Assistant Professor in Applied Mathematics University of Naples “Federico II”, Naples, Italy Dr. R. Escudié Research Scientist Laboratory of Environmental Biotechnology (LBE), INRA, Narbonne, France Dr. E. Trably Research Scientist Laboratory of Environmental Biotechnology (LBE), INRA, Narbonne, France Prof. Michel Madon Professor University of Paris-Est, France Other members Prof. Dr. Ing. Massimiliano Fabbricino Associate Professor of Sanitary and Environmental Engineering University of Naples Federico II, Naples, Italy Dr. Ing. Raffaella Pomi, Assistant Professor of Sanitary and Environmental Engineering Sapienza University of Rome, Rome, Italy Dr. Hab. E.D. van Hullebusch Associate Professor of Biogeochemistry University of Paris-Est, France This research was conducted under the auspices of the Erasmus Mundus Joint Doctorate Environmental Technologies for Contaminated Solids, Soils, and Sediments (ETeCoS3). ii iii TABLE OF CONTENTS TABLE OF CONTENTS ......................................................................................... iii LIST OF FIGURES ................................................................................................. vi LIST OF TABLES ................................................................................................... ix ABSTRACT ............................................................................................................ xi SINTESI ................................................................................................................. xii RÉSUMÉ ............................................................................................................... xiv SAMENVATTING ................................................................................................. xv ACKNOWLEDGEMENT ...................................................................................... xvi CHAPTER 1 ................................................................................................................. 1 Introduction .................................................................................................................. 1 1.1 Background and problem statement ............................................................... 2 1.2 Scope of the PhD thesis ................................................................................. 5 REFERENCES ......................................................................................................... 7 CHAPTER 2 ................................................................................................................. 9 State of the art in dark fermentation of complex waste biomass by mixed culture and utilization of dark fermentation effluents in photo fermentation .................................... 9 2.1 Dark fermentative biohydrogen production from organic biomass ............... 10 2.1.1 Introduction ............................................................................................. 10 2.1.2 Microbiology and biochemical pathways of DF ....................................... 14 2.1.3 Potential sources of organic biomass for fermentative biohydrogen production ........................................................................................................... 18 2.1.4 Factors affecting DF pathways and H2 yield ............................................. 25 2.1.5 Inoculum and enrichment methods........................................................... 26 2.1.6 Design and operation of bioreactors ......................................................... 32 2.1.7 Substrate pre-treatment for enhanced H2 yield ......................................... 41 2.1.8 Use of by-products ................................................................................... 45 2.1.9 Pilot scale applications ............................................................................. 57 2.1.10 Challenges and future prospects ............................................................... 57 2.1.11 Conclusions ............................................................................................. 61 2.2 Valorization of dark fermentation effluents via photo fermentative production of biohydrogen and biopolymers ............................................................................. 62 2.2.1 Introduction ............................................................................................. 62 2.2.2 Microbiology and phototrophic metabolism of PNSB .............................. 65 2.2.3 Operating conditions of PF ...................................................................... 71 iii 2.2.4 PBR systems ............................................................................................ 85 2.2.5 Design considerations for PBRs ............................................................... 90 2.2.6 Mathematical modeling of growth and product kinetics of PNSB............. 91 2.2.7 Future perspectives .................................................................................. 94 REFERENCES ....................................................................................................... 97 CHAPTER 3 ............................................................................................................. 124 Operational Strategies to improve dark fermentative H2 production using complex waste biomass ..................................................................................................................... 124 3.1 Evaluation of methods for obtaining H2 producing seed inoculum for dark fermentation .......................................................................................................... 125 3.1.2 Introduction ........................................................................................... 125 3.1.3 Materials and methods ........................................................................... 126 3.1.4 Results and discussions .......................................................................... 128 3.1.5 Conclusions ........................................................................................... 132 3.2 Effects of operational parameters on dark fermentative H2 production ....... 133 3.2.1 Introduction ........................................................................................... 133 3.2.2 Materials and methods ........................................................................... 136 3.2.3 Results ................................................................................................... 141 3.2.4 Discussion ............................................................................................. 150 3.2.5 Conclusion............................................................................................. 155 REFERENCES ..................................................................................................... 157 CHAPTER 4 ............................................................................................................. 163 Long-term operation of hydrogen-producing continuous reactors .............................. 163 4.1 Continuous H2 production from food waste at low organic loading rates .... 164 4.1.1 Introduction ........................................................................................... 164 4.1.2 Materials and methods ........................................................................... 167 4.1.3 Results and discussion ........................................................................... 170 4.1.4 Conclusion............................................................................................. 176 4.2 Co-fermentation of cheese whey and buffalo manure for pH control .......... 177 4.2.1 Introduction ........................................................................................... 177 4.2.2 Materials and methods ........................................................................... 179 4.2.3 Results and discussion ........................................................................... 181 4.2.4 Conclusion............................................................................................