Polishing of Anaerobic Secondary Effluent and Symbiotic Bioremediation of Raw Municipal Wastewater by Chlorella Vulgaris Thesis by Tuoyuan Cheng In Partial Fulfillment of the Requirements For the Degree of Master of Science King Abdullah University of Science and Technology, Thuwal Kingdom of Saudi Arabia © April 2016 Tuoyuan Cheng All rights reserved 2 EXAMINATION COMMITTEE APPROVALS FORM The thesis of Tuoyuan Cheng is approved by the examination committee. Committee Chairperson: Dr. TorOve Leiknes Committee Members: Dr. Noreddine Ghaffour, Dr. Chunhai Wei 3 ABSTRACT Polishing of Anaerobic Secondary Effluent and Symbiotic Bioremediation of Raw Municipal Wastewater by Chlorella Vulgaris Tuoyuan Cheng To assess polishing of anaerobic secondary effluent and symbiotic bioremediation of primary effluent by microalgae, bench scale bubbling column reactors were operated in batch modes to test nutrients removal capacity and associated factors. Chemical oxygen demand (COD) together with oil and grease in terms of hexane extractable material (HEM) in the reactors were measured after batch cultivation tests of Chlorella Vulgaris, indicating the releasing algal metabolites were oleaginous (dissolved HEM up to 8.470 mg/L) and might hazard effluent quality. Ultrafiltration adopted as solid-liquid separation step was studied via critical flux and liquid chromatography-organic carbon detection (LC-OCD) analysis. Although nutrients removal was dominated by algal assimilation, nitrogen removal (99.6% maximum) was affected by generation time (2.49 days minimum) instead of specific nitrogen removal rate (sN, 20.72% maximum), while phosphorus removal (49.83% maximum) was related to both generation time and specific phosphorus removal rate (sP, 1.50% maximum). COD increase was affected by cell concentration (370.90 mg/L maximum), specific COD change rate (sCOD, 0.87 maximum) and shading effect. sCOD results implied algal metabolic pathway shift under nutrients stress, generally from lipid accumulation to starch accumulation when phosphorus lower than 5 mg/L, while HEM for batches with initial nitrogen of 10 mg/L implied this threshold around 8 mg/L. HEM and COD results implied algal metabolic pathway shift under nutrients stress. Anaerobic membrane bioreactor effluent polishing showed similar results to synthetic anaerobic secondary effluent with slight inhibition while 4 symbiotic bioremediation of raw municipal wastewater with microalgae and activated sludge showed competition for ammonium together with precipitation or microalgal luxury uptake of phosphorus. Critical flux was governed by algal cell concentration for ultrafiltration membrane with pore size of 30 nm, while ultrafiltration membrane rejected most biopolymers (mainly polysaccharides). Further research would focus on balancing cell growth, specific nutrients removal, and specific COD change by utilizing rotating biological contactor. 5 ACKNOWLEDGMENTS This thesis fulfilled by Tuoyuan Cheng is kindly supervised by Prof. TorOve Leiknes, patiently supported by Dr. Chunhai, and generously allowed by the committee to revise. He is also grateful to the assistance offered by Yasmeen Najm and Ryan Lefters, as well as cooperative arrangements contributed by lab staff. 6 TABLE OF CONTENTS EXAMINATION COMMITTEE APPROVALS FORM ........................................................... 2 ABSTRACT .......................................................................................................................................... 3 ACKNOWLEDGMENTS ................................................................................................................ 5 TABLE OF CONTENTS .................................................................................................................. 6 LIST OF ABBREVIATIONS ........................................................................................................... 9 LIST OF FIGURES .......................................................................................................................... 10 LIST OF TABLES ............................................................................................................................. 12 1. Introduction ............................................................................................................................... 13 1.1 Microalgae in Wastewater Phycoremediation ................................................................ 14 1.2 Microalgae in Biofuel Production ................................................................................... 17 1.2.1 Harvesting .................................................................................................................. 20 1.2.1.1 Flocculation ....................................................................................................... 20 1.2.1.2 Flotation ............................................................................................................. 21 1.2.1.3 Gravitational and Centrifugal Sedimentation ............................................... 22 1.2.1.4 Filtration ............................................................................................................. 22 1.2.1.5 Immobilization .................................................................................................. 23 1.2.2 Drying ......................................................................................................................... 24 1.2.3 Extraction ................................................................................................................... 24 1.2.4 Downstream Processing ........................................................................................... 25 1.3 Major Factors in Algal Cultivation .................................................................................. 26 1.3.1 Microalgae Species .................................................................................................... 26 1.3.2 Carbon Source and Energy Source ......................................................................... 27 1.3.3 Nutrient Source ......................................................................................................... 28 1.3.4 Light Intensity ............................................................................................................ 29 1.3.5 Cell Density ................................................................................................................ 30 1.3.6 pH ................................................................................................................................ 30 1.3.7 Temperature ............................................................................................................... 31 1.3.8 Dissolved Oxygen (DO) .......................................................................................... 31 1.3.9 Biotic Factors ............................................................................................................. 32 1.3.10 Reactor Configuration .............................................................................................. 32 1.3.11 Mixing ......................................................................................................................... 33 1.3.12 Hydraulic Retention Time (HRT) ........................................................................... 34 1.3.13 Operation Mode ........................................................................................................ 35 7 1.4 Scope of this Research ...................................................................................................... 35 2. Materials and Methods ............................................................................................................. 37 2.1 Inoculum Preparation ....................................................................................................... 37 2.2 Reactor Design and HRT ................................................................................................. 37 2.3 Carbon Source and pH ..................................................................................................... 38 2.4 Energy Source and Light Intensity .................................................................................. 39 2.5 Feed Water Composition and Seeding ........................................................................... 40 2.6 Water Quality Characterization ....................................................................................... 41 2.7 Filtration Process and Critical Flux ................................................................................. 44 2.8 Data Processing ................................................................................................................. 45 3. Results and Discussion ............................................................................................................. 46 3.1 Algal Growth and Nutrients Removal............................................................................ 46 3.1.1 Algal Growth and Nutrients Removal in Synthetic Anaerobic Secondary Effluent Polishing ..................................................................................................................... 46 3.1.2 Algal Growth and Nutrients Removal in AnMBR Effluent Polishing ............. 56 3.1.3 Algal Growth and Nutrients Removal in Symbiotic Bioremediation ................ 57 3.2 COD, HEM and LC-OCD Analysis .............................................................................. 58 3.2.1 COD, HEM and LC-OCD Analysis in Synthetic Anaerobic Secondary Effluent Polishing ..................................................................................................................... 58 3.2.2 COD, HEM and LC-OCD Analysis in AnMBR Effluent Polishing ................ 63 3.2.3 COD, HEM and LC-OCD Analysis in Symbiotic Bioremediation ................... 65 3.3 Particle Size Distribution and Critical Flux ................................................................... 67 3.3.1 Critical Flux in Synthetic Effluent Polishing ......................................................... 68 3.3.2 Critical Flux in AnMBR Effluent Polishing .......................................................... 70 3.3.3 Critical Flux in Symbiotic Bioremediation ............................................................. 70 4. Conclusion ................................................................................................................................. 71 5. Appendices ................................................................................................................................. 74 5.1 MATLAB Codes for Data and Plotting ......................................................................... 74 5.2 Summary of Synthetic Anaerobic Secondary Effluent Polishing ............................... 78 5.3 EPA Method 1664A–Extraction of Oil and Grease from Water Samples Using Solid-Phase Extraction (SPE) Cartridge Configuration, Application Note 817 (Eric Francis 2012) ...................................................................................................................................... 80 5.3.1 Introduction ............................................................................................................... 80 5.3.2 Equipment .................................................................................................................. 80 5.3.3 Solvents ....................................................................................................................... 80 8 5.3.4 SPE Material .............................................................................................................. 80 5.3.5 Oil and Grease Cartridge Configuration Method ................................................. 80 5.3.6 Conclusion .................................................................................................................. 82 5.3.7 References .................................................................................................................. 82 5.3.8 List of Manufacturers ............................................................................................... 82 REFERENCES .................................................................................................................................. 84 9 LIST OF ABBREVIATIONS AnMBR Anaerobic membrane bio-reactor BBM Bold’s basal medium CF Critical flux COD Chemical oxygen demand dCOD The change of COD DO Dissolved oxygen GHGs Greenhouse gases HEM n-Hexane extractable material HRAP High rate algal ponds HRT Hydraulic retention time LCA Life cycle analysis LC-OCD liquid chromatography-organic carbon detection MF Microfiltration MLVSS Mixed liquor volatile suspended solids NH -N Ammonium nitrogen 4 NO -N Nitrite nitrogen 2 NO -N Nitrate nitrogen 3 OD Optical density OND Organic nitrogen detector PBR Photo bio-reactor PO -P Phosphate-phosphorus 4 PSD Particle size distribution PVDF Polyvinylidene fluoride sCOD Specific COD change rate sN Specific nitrogen removal rate sP Specific phosphorus removal rate SPE Solid phase extraction TAG Triacylglycerol THF Tetrahydrofuran TMP Transmembrane pressure TN Total nitrogen TSS Total suspended solids UF Ultrafiltration UVD UV254 detector 10 LIST OF FIGURES Fig.1 Schematic presentation of an overall microalgae production, harvesting and recovery process ................................................................................................................................................. 18 Fig. 2 TAG and transesterification .................................................................................................. 19 Fig. 3 Potential algal biomass conversion processes ..................................................................... 26 Fig. 4 MLVSS-OD691.5nm relationship ........................................................................................ 42 Fig. 5 Illustration of improved flux step method .......................................................................... 45 Fig. 6 Final VSS against initial N and initial P for synthetic anaerobic secondary effluent polishing............................................................................................................................................... 47 Fig. 7 Generation time against initial N and initial P for synthetic secondary effluent polishing............................................................................................................................................... 47 Fig. 8 Final N against initial N and initial P for synthetic secondary effluent polishing ......... 49 Fig. 9 Final P against initial N and initial P for synthetic secondary effluent polishing .......... 49 Fig. 10 Specific nitrogen removal rate against initial N and initial P for synthetic secondary effluent polishing ................................................................................................................................ 50 Fig. 11 Specific phosphorus removal rate against initial N and initial P for synthetic secondary effluent polishing ............................................................................................................. 50 Fig. 12 Nutrients removal ratio against initial N and initial P for synthetic secondary effluent polishing............................................................................................................................................... 51 Fig. 13 Relationship between specific nutrients removal rate for synthetic secondary effluent polishing............................................................................................................................................... 51 Fig. 14 Schematic of lipid accumulation triggered by nitrogen deficiency................................. 52 Fig. 15 Nitrogen removal rate against initial N and initial P for synthetic secondary effluent polishing............................................................................................................................................... 54 Fig. 16 Phosphorus removal rate against initial N and initial P for synthetic secondary effluent polishing ................................................................................................................................ 55 Fig. 17 Nutrients removal rate against initial nutrients ratio for synthetic secondary effluent polishing............................................................................................................................................... 56 Fig. 19 Specific COD change against initial N and initial P for synthetic secondary effluent polishing............................................................................................................................................... 60 Fig. 20 HEM for synthetic secondary effluent polishing (measured for batches with initial N=10 mg/L) ....................................................................................................................................... 61 Fig. 21 LC-OCD results for treated synthetic secondary effluent (with initial N=10 mg/L, P=8 mg/L) .......................................................................................................................................... 64 Fig. 22 LC-OCD results for treated AnMBR effluent .................................................................. 66 Fig. 23 LC-OCD results for treated synthetic raw municipal wastewater ................................. 67 Fig. 24 Particle size distribution ....................................................................................................... 68 Fig. 25 Critical flux for synthetic secondary effluent polishing ................................................... 69