Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author. Luxury Uptake of Phosphorus by Microalgae in New Zealand Waste Stabilisation Ponds A Thesis presented in partial fulfilment of the requirements for the degree of Masters In Engineering At Massey University, Manawatu New Zealand Aidan Jeremy Crimp 2015 Thesis Abstract The discharge of phosphorus to waterways within wastewater effluent causes significant environmental damage through microalgal blooms and eutrophication. This is a particular problem for wastewater treatment plants that rely on waste stabilisation ponds (WSPs) for the bulk of their treatment. While having simple designs and low running costs, WSPs are mostly ineffective at phosphorus removal, with only 15 – 50% removal achieved on average according to some studies. The luxury uptake phenomenon within microalgae has been identified as one mechanism that could improve WSP phosphorus removal. This occurs when microalgae store phosphorus beyond what is required for their metabolism as polyphosphate, leading to phosphorus contents above the standard 1 %P/g VSS for microalgae. However, studies on this subject in full scale WSPs to date have been limited to just two different ponds. To improve knowledge on this mechanism, this study aimed to assess the impact of environmental conditions, climatic region and pond type on microalgal luxury uptake, as well as determine which specific microalgal and cyanobacterial genera were best able to perform this mechanism. To achieve these objectives, a yearlong study was conducted on 13 different WSPs from 7 sites within various climatological regions within New Zealand, as well as two pilot scale High Rate Algal Ponds (HRAPs). From this study, it was found that luxury uptake was found to occur in 56% of the WSP, with a peak phosphorus content of 3.8 %P/g VSS. Conversely, only one sample taken from the HRAPs was found to exhibit luxury uptake. Total dissolved phosphorus (TDP) concentration and rainfall were found to have a significant effect on biomass phosphorus content at a 95% confidence level, while the WSP climate was found not have an influence. There were no significant differences between the biomass phosphorus contents in primary and secondary ponds, with averages of 1.31 %P/g VSS and 1.21 %P/g VSS respectively, while HRAPs (0.71 %P/g VSS) were significantly lower due to the low TDP concentrations experienced by these ponds. 22 of the 23 identified microalgal and cyanobacterial genera were found to perform luxury uptake, at varying frequencies. The cyanobacterium Planktothrix was most effective, storing polyphosphate as granules in 84% of the samples it was identified in. Scenedesmus, Pediastrum, and Schroederia were also effective, at frequencies of 73%, 82% and 79% respectively. There was some correlation between storage of phosphorus as polyphosphate and enhanced phosphorus contents in the biomass, with nearly all samples containing no visually identifiable polyphosphate granules exhibiting phosphorus contents below 1 %P/g VSS. However, there were only limited correlations between the amount of polyphosphate and the levels of the significant variables identified previously. This research provides valuable insight into the phosphorus uptake behaviour of microalgae and cyanobacteria, and shows that there is some potential for development of a new engineered process targeting improved phosphorus removal. If the phosphorus content of 2 biomass in this new process could consistently attain a level of 3 %P/g VSS, phosphorus removal from wastewater for an average community of 500 people could be increased from 31% to 93%, thus greatly reducing the impact of wastewater discharge on the receiving environment. 3 Acknowledgements Firstly, I would like to thank my supervisors Dr. Nicola Brown and Prof. Andy Shilton for giving me this special opportunity to complete a Masters degree. Their unwavering support and faith in me during this project was greatly appreciated, and enabled me to produce the best work I possibly could. I am also grateful to the Royal Society of New Zealand for their awarding of a Marsden Research Grant to our research group, without which this research would not be possible. Thanks to Mr. Steve Nind at Manawatu District Council and Mr. Marcus Coley at Horowhenua District Council for allowing me access to their sites. Thanks to Mr. Ian Lowe, Mr. Corey Reed, Mr. Michael Lee and Mr. Daniel Wright at Gore District Council for obtaining samples at the Gore WSPs; Dr. Jason Park of NIWA for the HRAP samples; Mr. Caleb Powell of Waikato District Council for the Ngaruawahia WSP samples and Mr. Greg Timperley of Transfield Services for the Kaitaia WSP samples. Many thanks to Mrs. Ann-Marie Jackson and Mr. John Sykes at the School of Engineering and Advanced Technology (SEAT) for their assistance with laboratory work and analysis. I would also like to acknowledge all the other staff at SEAT who either directly or indirectly helped me with the completion of my thesis. Also thanks to Ms. Jordan Taylor, Ms. Niki Murray and Dr. Matthew Savoian at the Manawatu Microscopy and Imaging Centre for their frequent help with my microscopy analysis. A very special thanks must go to Mr. Ramsay Huang for his help with the chemical analysis of my samples, without which this project would have been very difficult to complete. I would like to acknowledge all my friends in the SEAT postgraduate group for all their help and assistance during the project. Lastly, thank you to my parents Jo and Paul Crimp, for supporting me since day one. 4 This thesis is dedicated to my Grandmother Helen Enid Crimp 1932 - 2015 5 Contents Thesis Abstract........................................................................................................................... 2 Acknowledgements .................................................................................................................... 4 Table of Figures ......................................................................................................................... 9 1 Introduction ...................................................................................................................... 10 2 Literature Review............................................................................................................. 12 2.1 Waste Stabilisation Ponds ......................................................................................... 12 2.1.1 Types of Ponds ................................................................................................... 12 2.1.2 Pond Biology ..................................................................................................... 13 2.1.3 Pond Treatment Mechanisms and Performance ................................................ 16 2.1.4 Possible Upgrade Options .................................................................................. 20 2.1.5 Summary ............................................................................................................ 23 2.2 Factors Affecting Growth and Phosphorus Removal by Microalgae ....................... 23 2.2.1 Bacteria .............................................................................................................. 24 2.2.2 Light ................................................................................................................... 25 2.2.3 Temperature ....................................................................................................... 28 2.2.4 pH ....................................................................................................................... 29 2.2.5 Availability of Carbon ....................................................................................... 30 2.2.6 Dissolved Oxygen Concentration ...................................................................... 30 2.2.7 Nutrients ............................................................................................................. 30 2.2.8 Concentration of Microalgae in the Pond .......................................................... 32 2.2.9 Species of Microalgae Present ........................................................................... 32 2.2.10 Preparation of Microalgae.................................................................................. 33 2.2.11 Other Factors ...................................................................................................... 35 2.3 Phosphorus Removal by Microalgae in Natural Environments ................................ 36 2.4 Conclusions from Literature Review ........................................................................ 36 3 Methodology .................................................................................................................... 38 3.1 Site Selection ............................................................................................................. 38 3.1.1 Manawatu Sites .................................................................................................. 38 3.1.2 Nationwide sites ................................................................................................. 42 3.1.3 Different Pond Types ......................................................................................... 44 3.2 Sampling Methodology ............................................................................................. 46 3.3 Analytical Analysis ................................................................................................... 46 6 3.3.1 Volatile Suspended Solids ................................................................................. 47 3.3.2 Total Phosphorus and Total Dissolved Phosphorus ........................................... 47 3.3.3 Percentage of Phosphorus in Biomass ............................................................... 47 3.4 Environmental Variables Measured .......................................................................... 48 3.4.1 In Pond Variables ............................................................................................... 48 3.4.2 Weather Variables .............................................................................................. 48 3.5 Visual Analysis ......................................................................................................... 48 3.5.1 Sample Preparation ............................................................................................ 48 3.5.2 Staining Methodology ........................................................................................ 48 3.5.3 Visual Analysis of Samples ............................................................................... 49 3.6 Summary of Sampling Plan ....................................................................................... 50 4 Results and Discussion .................................................................................................... 52 4.1 Phosphorus Content of Biomass ............................................................................... 52 4.2 Environmental Effects on Phosphorus Content of Biomass ..................................... 53 4.2.1 TDP .................................................................................................................... 55 4.2.2 Rainfall ............................................................................................................... 56 4.3 Climatic Effects on Biomass Phosphorus Content .................................................... 57 4.4 Effect of Pond Type on Phosphorus Content of Biomass ......................................... 59 4.4.1 Primary vs. Secondary Ponds ............................................................................ 59 4.4.2 High Rate Algal Ponds....................................................................................... 60 4.5 Luxury Uptake of Different Microalgal and Cyanobacterial Genera ....................... 61 4.5.1 Commonality of Microalgal and Cyanobacterial Genera and Frequency of Luxury Uptake ................................................................................................................. 61 4.5.2 Granule Scores for each Microalgal and Cyanobacterial Genus ....................... 63 4.5.3 Granule Score and Biomass Phosphorus Content .............................................. 65 4.5.4 Influence of Environmental Variables on Granule Storage by Scenedesmus and Chlamydomonas/Cryptomonas ........................................................................................ 66 5 Conclusions ...................................................................................................................... 70 5.1 Phosphorus Content of WSP Biomass. ..................................................................... 70 5.2 Environmental Effects on Phosphorus Uptake .......................................................... 70 5.3 Climatic Effects on Phosphorus Uptake .................................................................... 70 5.4 Effect of Different Pond Types ................................................................................. 70 5.5 Luxury Uptake by Different Microalgal and Cyanobacterial Genera ....................... 71 5.6 Potential for Future Application of Findings ............................................................ 71 7 6 Bibliography .................................................................................................................... 72 Appendix 1 – Error Bounds in Total Phosphorus Analysis ..................................................... 79 Appendix 2 – Effect of Lugol’s Iodine on Total Phosphorus Analysis ................................... 79 Appendix 3 – Summary of Variables Measured in Study ....................................................... 80 Appendix 4 – Full Stepwise Regression Output ...................................................................... 80 8 Table of Figures Figure 1: Outline of the different zones found within facultative ponds ................................. 12 Figure 2: Examples of microalgae, taken from Landcare Research.. ...................................... 14 Figure 3: Diagram of the symbiotic relationship between microalgae and aerobic bacteria ... 15 Figure 4: Chemical reaction pathway for alum in wastewater, from Edzwald and Kaminski (2008). ..................................................................................................................................... 21 Figure 5: Outline of the EBPR process .................................................................................... 22 Figure 6: Photosynthesis vs. irradiance response curve for microalgae. ................................. 26 Figure 7: Aerial photo of Halcombe WSPs.. ........................................................................... 39 Figure 8: Aerial photo of Sanson WSPs. ................................................................................. 40 Figure 9: Aerial photo of Foxton Beach WSPs.. ..................................................................... 41 Figure 10: Aerial photo of Rongotea WSPs.. .......................................................................... 42 Figure 11: Aerial view of Gore WSPs.. ................................................................................... 43 Figure 12: Aerial view of Kaitaia WSPs.. ................................................................................ 44 Figure 13: Twin HRAPs located at NIWA.. ............................................................................ 45 Figure 14: WSP at Ngaruawahia.............................................................................................. 46 Figure 15: Residual plots for stepwise regression model ........................................................ 54 Figure 16: Plot of TDP against biomass phosphorus content .................................................. 55 Figure 17: Monthly average rainfall versus phosphorus content of WSP biomass ................. 56 Figure 18: Average phosphorus content of WSP biomass with confidence interval at each geographical location in the study. .......................................................................................... 58 Figure 19: Plot of average polyphosphate granule scores for each genus of microalgae found in the study.. ............................................................................................................................. 64 Figure 20: Phosphorus content of WSP biomass versus the average sample granule score .... 66 Figure 21: Scenedesmus (top graph) and Chlamydomonas/Cryptomonas (bottom graph) granule score against TDP ....................................................................................................... 67 Figure 22: Scenedesmus (top) and Chlamydomonas/Cryptomonas (bottom) granule scores against rainfall levels ............................................................................................................... 69 9