Jeremy Ayre

Microalgae cultivation for treating anaerobic digestates provides advantages over many other treatment options, however the limitations of this emerging technology currently prevent implementation in many cases where it is needed most. Agricultural systems present some of the greatest need. One of the most prominent is in the context of pig farming, which has already seen decades worth of investigation worldwide in this area. Much of this research is yet to realise it’s full potential but seems tantalising close to fruition. The high concentration of nutrients, such as nitrogen - mostly in the form of ammonium which is very volatile and toxic - is both problematic and a rich fertiliser which under the right conditions can enable growth and cultivation of microalgae. The conceptual framework for a system which incorporates uptake of wastewater nutrients and production of harvestable and useful microalgal biomass is sometimes referred to as the third generation biorefinery. Anaerobic digestate of piggery effluent (ADPE) is a very appealing target for such a biorefinery system. The introduction to this dissertation – Chapter 1 looks at a wide range of literature that covers the topic of wastewater treatment using microalgae, with a particular focus toward ADPE specific treatments and concerns. Amongst the microalgae with greatest promise in this context include Scenedesmus sp. and Chlorella sp. varieties. As detailed in Chapter 2, it was found through bioprospecting and outdoor growth investigations that microbial consortia containing these species could grow on undiluted piggery effluent with very high ammonium concentrations up to 1600 mg N NH4 L−1. These experiments demonstrated five weeks of semicontinuous growth using sand-filtered, undiluted ADPE as growth media and found growth rates of around 18.5 mg ash-free dry weight L−1 d−1 and ammonium removal rates up to 63.7 ± 12.1 mg N NH4 L−1. Carbon dioxide addition as a pH control measure was also tested and shown to enhance growth performance by around 17% under these outdoor growth conditions. Further experiments using a closed to the atmosphere laboratory environment demonstrated clean and simple methods to retain ammonium during ADPE microalgae cultivation and prevent ammonia vapour escaping and threatening harm to human and wildlife health. Findings from this research are detailed in Chapter 3. The closed system tested the use of deionised water and recirculated airflow in order to successfully retain virtually all of the ammonia gas which would otherwise be lost. Lower starting pH conditions provided the benefit of keeping more ammonia in the form of less toxic ammonium dissolved in ADPE. Surprisingly, this system was also found to have sufficient carbon reserves within the ADPE growth media, negating the need to add an extrinsic carbon source. Additional indoor growth experiments investigated relationships between bacteria and microalgae during this microalgae cultivation wastewater treatment process. These findings – documented in Chapter 4 revealed dynamic changes across many bacterial phyla. Assessments of functional genes predicted during cultivation were also performed using the in-silico toolkit - Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt), which was able to inform of the role bacteria play in cycling of nitrogen and carbon compounds during the wastewater treatment process. These data offer insights into the microbial population dynamics which include the revelation of dominant bacterial phyla Bacteroidetes and Proteobacteria, decreases in bacterial richness and diversity as wastewater treatment proceeded and indicators of symbiotic relationships forming with a number of bacterial phyla including Bacteriodetes and Cyanobacteria. Additionally the key pathways favoured by the microalgae-microbial consortia are NH4 and NO2− removal possibly via nitrification and nitrifier denitrification pathways while accumulating NO3− in inoculated diluted digestate treatment systems. In the absence of inoculation and at high ammonium concentrations in the digestate, NH4 , NO2− and NO3− are almost completely eliminated from the system via a combination of microbial N assimilation and denitrification. Finally, in Chapter 5 investigations into the nutritional profile and potential use of the harvested and dried microalgae biomass for application as a feed for livestock, aquaculture or other uses are outlined. Pathogenic test results were favourable, crude protein of ADPE-grown microalgae was higher than full fat soybeans but was much lower than conventional soybean meals, net energy values of ground and bead-milled algae samples were found to be comparable to that of deshelled sunflower meal commonly used in pig feed, and favourable omega-3:omega-6 ratio of ∼1.9 was found, indicating suitability for inclusion into pig or other animal diets. General conclusions which are informed by these experiments and summarised in Chapter 6 find that not only are Scenedesmus sp. and Chlorella sp. currently among the optimal candidates for treatment of minimally or undiluted piggery digestate, but that cultivation systems can be customised toward specific needs such as closed growth conditions which allow for very high proportions of ammonium capture, or larger scale outdoor growth systems where ammonia losses might be less of a concern for some cultivation environments. Under long term outdoor conditions the benefits of incorporating carbon capture and pH adjustments using carbon dioxide have been demonstrated and provide a good foundation for further research using these methods. These studies have also shown that beneficial relationships can form between microalgae and bacterial populations, and these interactions may be a fruitful target for research which aims toward optimisation of health and stability of microalgae based wastewater treatment cultivation conditions. In addition, the microalgae of harvested and dried algal biomass shows indications of being an adequate supplement for inclusion into animal feed. Overall, this PhD dissertation addresses many critical points of concern regarding the treatment of high strength anaerobic digestates such as ADPE using microalgae cultivation.