Alma Mater Studiorum – Università di Bologna Facoltà di Scienze Matematiche Fisiche e Naturali DOTTORATO DI RICERCA IN Scienze Ambientali: tutela e gestione delle risorse naturali Ciclo XXIV 05/A1 - Botanica BIO/01 - Botanica Generale Algal wastewater treatment and biomass producing potential: nutrient removal efficiency and cell physiological responses Presentata da: Giulia Samorì Coordinatore Dottorato Relatore Prof. Enrico Dinelli Prof.ssa Rossella Pistocchi Esame finale anno 2012 2 Table of contents 1. AIM OF THE THESIS .................................................................................................. 7 2. BACKGROUND ............................................................................................................ 8 2.1 WASTEWATER TREATMENT METHODS AND MICROALGAE APPLICATION ...................... 8 2.1.1 Conventional Biological Treatment..................................................................... 9 2.1.2 Natural biological treatment and the depuration role of microalgae ............... 12 2.2 TECHNOLOGIES FOR MICROALGAE BIOMASS PRODUCTION ......................................... 15 2.2.1 Open ponds ........................................................................................................ 16 2.2.2 Closed photobioreactors.................................................................................... 19 2.3 ALGAE BIOFUELS ....................................................................................................... 21 2.3.1 Algae biofuels: a brief history ........................................................................... 23 2.3.2 Biomass conversion ........................................................................................... 24 2.3.3 Other applications of microalgae biomass extracts .......................................... 26 2.4 BIOCHEMISTRY AND PHYSIOLOGY OF ALGAE ............................................................ 27 2.4.1 Photosynthesis in Algae ..................................................................................... 27 2.4.2 CO 2 fixation and the photosynthetic carbon reduction (Calvin) cycle .............. 28 2.4.3 Algae lipids and biosynthesis ............................................................................ 30 2.4.4 Uptake of inorganic nutrients ............................................................................ 32 3. THE CHLOROPHYCEAE GENERA SCENEDESMUS AND DESMODESMUS ............................................................................................................. 35 3.1 MICROALGAE IDENTIFICATION .................................................................................. 36 4. EXPERIMENTAL SECTION .................................................................................... 38 4.1 MATERIALS AND METHODS ....................................................................................... 38 4.1.1 Microalgae isolation and community composition ........................................... 38 4.1.2 Culture medium ................................................................................................. 40 4.1.3 Measurement of cell growth .............................................................................. 40 4.1.4 Determination of ammonia, nitrate and nitrite concentration .......................... 41 4.1.5 Determination of reactive phosphorus .............................................................. 42 4.1.6 Extraction and fractionation of total lipids ....................................................... 42 4.1.7 Determination of total fatty acids ...................................................................... 42 3 4.1.8 Determination of protein content ...................................................................... 43 4.1.9 Determination of carbohydrate content ............................................................ 43 4.1.10 Determination of chlorophyll a content ........................................................... 44 4.1.11 Biomass elemental composition....................................................................... 44 4.1.12 Induction curves .............................................................................................. 44 4.1.13 Rapid light curves and photosynthetic parameters measurements ( α, E k and rETR max ) ...................................................................................................................... 47 4.1.14 Statistical analysis ........................................................................................... 48 4.2 EFFECTS OF MUNICIPAL WASTEWATER ON CELL GROWTH AND CELL COMPOSITION OF AN AUTOCHTHONOUS STRAIN OF DESMODESMUS COMMUNIS AND OF A NATURAL MICROALGAE CONSORTIUM ............................................................................................. 49 4.2.1 Introduction ....................................................................................................... 49 4.2.2 Overview of experiments ................................................................................... 50 4.2.3 Results and Discussion ...................................................................................... 52 4.2.4 Conclusions ....................................................................................................... 74 4.3 INDUCTION OF LIPID SYNTHESIS IN DESMODESMUS COMMUNIS CULTURE GROWN IN URBAN WASTEWATERS .................................................................................................... 75 4.3.1 Introduction ....................................................................................................... 75 4.3.2 Overview of experiments ................................................................................... 76 4.3.3 Results and Discussion ...................................................................................... 76 4.3.4 Conclusions ....................................................................................................... 96 4.4 ALGAE GROWTH AND NITROGEN REMOVAL IN SMALL SCALE SEMI - CONTINUOUS DESMODESMUS COMMUNIS CULTURE IN VIEW OF URBAN WASTEWATER TREATMENT ...... 97 4.4.1 Introduction ....................................................................................................... 97 4.4.2 Overview of experiments ................................................................................... 98 4.4.3 Results and Discussion ...................................................................................... 98 4.4.4 Conclusions ..................................................................................................... 124 5. BASELINE STUDY ON THE WASTEWATER TREATMENT PERFORMANCE AND ALGAL BIOMASS PRODUCTIVITY OF AN OPEN POND PILOT PLANT .................................................................................................. 126 5.1 PILOT PLANT LAYOUT .............................................................................................. 128 5.2 ROUTING MONITORING AND MAINTENANCE ............................................................ 130 5.3 EXPERIMENTAL PROCEDURES .................................................................................. 130 4 5.4 METHODS ................................................................................................................ 132 5.4.1. Measurement of biomass production .............................................................. 132 5.4.2 Determination of ammonium ........................................................................... 132 5.4.3 Determination of nitrate and nitrite ................................................................ 132 5.4.4 Determination of reactive phosphorus ............................................................ 133 5.4.5 Biochemical oxygen demand ........................................................................... 133 5.4.6 Settling tests ..................................................................................................... 133 5.4.7 Algae observations and identifications ............................................................ 133 5.4.8 Temperature and pH ........................................................................................ 134 5.4.9 Dissolved oxygen ............................................................................................. 134 5.4.10 Weather data.................................................................................................. 134 5.4.11 Quality assurance and quality control .......................................................... 135 5.5 RESULTS AND DISCUSSION ....................................................................................... 135 5.6 CONCLUSION ........................................................................................................... 146 6. GENERAL CONCLUSIONS ................................................................................... 147 7. REFERENCES ........................................................................................................... 151 8. ACKNOWLEDGEMENTS ...................................................................................... 161 5 6 1. Aim of the thesis The concept of microalgae cultivation as an integrated system in wastewater treatment has optimized the potential of the microalgae - based biofuel production. Microalgal biomass contains lipids, polysaccharides, proteins, pigments and other cell compounds, and it can provide different kinds of biofuels such as biodiesel, biomethane and ethanol; these microorganisms can also be used to