Jeremy Ayre

Algae-based technologies offer various solutions to mitigate the environmental impacts of mining during operations and after closure. Algae sequester CO2 into biomass, allowing possible offsetting of carbon emissions. Algae can also facilitate the removal of various contaminants from mine water, dust suppression, stabilisation of mine waste, and mine-site rehabilitation. The cultivation of algae at mine sites may also provide opportunities for the manufacture of valuable products such as pigments, bioplastics, biofuels, and animal feed. This presents the potential to establish a bio-based economy, creating job opportunities for local communities. The feasibility of cultivating and utilising algae requires a case-by-case evaluation of technical performance, local conditions, regulatory constraints, economic feasibility, and environmental impacts. This study aims to explore the potential of various algae-based technologies designed for cultivating, harvesting, and utilising algae at mine sites for beneficial purposes.

Anaerobic digestate animal effluent (ADAE) contains high N and P nutrients which need to be treated. In this study, an integrated process was proposed using a microalgae consortium of Chlorella and Scenedesmus. The system was designed for 71 m3/d (medium-sized) and 355 m3/d (large-sized) animals of ADAE. Process simulation estimated to produce 83–417 kg d-1 of microalgae biomass which can be used as further products. As much as 2 kg of animal feed and 36–180 L/d of bio-oil can be produced during the treatment of 1 m3/d of ADAE. The produced biogas can generate 247–1,217 MWh y-1 of electricity. Likewise, the process can reduce greenhouse gas emissions by 2 kg-CO2eq kg−1 of hot standard carcass weight (HSCW). This integrated system offers merits in treating ADAE as well as producing chemicals and energy with low environmental burdens.

Algal cultivation for treating agricultural waste offers distinct advantages over conventional treatment options. These include the capacity for carbon dioxide capture and the potential reuse of nutrients through the production of valuable algal biomass. Piggery wastewater (WW) is of particular interest due to its characteristically extreme pollutant potential, typically exceeding concentration levels of other agricultural effluents, and a rich history of experimental work going back to the 1970s. The associated challenges and potential for success in this field also have important ramifications for the processing of many other agricultural, industrial, and commercial WWs. This review takes into account research involving raw effluent, the development of anaerobic digestate (AD) as the preferred first‐stage treatment approach, current challenges such as water conservation via minimizing dilution while coping with excessively high ammonia concentrations, and the potential for nutrient limitations such as phosphorous. Other aspects of algal cultivation such as the choice of photobioreactor and some of the relevant economic factors are also discussed.

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.

Within the biorefinery concept, microalgal cultivation has potential as one component of the wastewater treatment toolkit for anaerobic digestates. Recovering nutrients from digestate such as anaerobic digestate of piggery effluent, has been well demonstrated with Scenedesmus sp. and Chlorella sp. in mixed cultures. Less understood during microalgae cultivation, is the participation of bacterial communities as they play a fundamental role in biological nutrient cycling processes with potential to optimise algal productivity and nutrient recovery. To this end, we batch cultivated microalgae on increasing concentrations of digestate (250, 500 and 890 mg N NH4+ L−1), took samples under time series and quantified culture conditions including water chemistry properties with a focus on nitrogen values during treatment. Chlorophyll and dry weight were measured to provide reasonable estimates of the health of the microalgal culture. We additionally characterised the bacterial community using next generation 16S rRNA sequencing on the ION torrent, followed by an in-silico analysis of functional nitrogen and carbon cycling genes using PICRUSt. Our data suggest the microalgae form symbiotic relationships with a number of bacterial groups including Bacteriodetes, Cyanobacteria, nitrifying and N-fixing bacteria. These microalgae-microbial consortia favour NH4+ and NO2− removal possibly via nitrification and nitrifier denitrification pathways while accumulating NO3− in the inoculated diluted digestate treatment systems. In the absence of inoculation and at high ammonium concentrations in the digestate, almost all NH4+, NO2− and NO3− are driven from the system, largely due to stripping and are unable to be captured for any further use. Thus, a microalgae-microbial consortia-driven digestate treatment system offers the potential to recapture and recover N, enabling production of N fertiliser. These data demonstrate the integral role of syntrophic relationships for microalgae and bacteria in third generation biorefinery concepts.

Microalgal biomass grown in wastewater can be a sustainable source of animal feedstock. We have previously shown the feasibility of mass algal cultivation on undiluted anaerobic digested piggery effluent (ADPE). In this study, we evaluated the nutritional value, pathogen load, in vitro digestibility and potential physiological energy (PPE) of ADPE-grown microalgae as a potential feedstock for pigs. Pathogen load of ADPE-grown microalgae was within regulatory limits. Crude protein of ADPE-grown microalgae was higher than full fat soybeans but was much lower than conventional soybean meals (SBM) currently employed as a source of protein in pig feeds. The essential amino acid content of the microalgae was also lower than SBM. Fatty acid composition of the microalgae was favourable with an omega-3:omega 6 ratio of ~1.9, which may offer potential for value-adding use in some diets. In vitro digestibilities were higher in faeces than at the ileum and were lower for the defatted microalgal biomass. The (theoretical) net energy values of ground and bead-milled algae samples were found to be comparable to that of deshelled sunflower meal used as a feeding ingredient for pigs, but were lower than SBM.

Anaerobic digestate of piggery effluent (ADPE) is extremely high in ammonia toxic to many microorganisms. Bioprospecting and nutrient enrichment of several freshwater and wastewater samples combined and further acclimation resulted in a mixed culture containing at least three microalgae species capable of growing on undiluted ADPE. Outdoor growth of the mixed culture using raceway ponds showed potential for up to 63.7 ± 12.1 mg N-NH4 + L −1 d −1 ammonium removal from the ADPE. The microalgal consortium was dominated by Chlorella sp. and was stable at between 800 and 1600 mg N-NH4 + L −1. Regulation of CO2 addition to the ponds to maintain a pH of 8 increased chlorophyll content of the microalgal consortium. Average microalgal biomass productivity of 800 mg N-NH4 + L −1 culture conditions during five weeks semicontinuous growth was 18.5 mg ash-free dry weight L −1 d −1. Doubling the ammonium concentration from 800 to 1600 mg N-NH4 + L −1 resulted in a 21% reduction of productivity, however the culture grown at 1600 mg N-NH4 + L −1 with the addition of CO2 by keeping pH at pH = 8 led to a 17% increase in biomass productivity.

The overwhelming interest in the use of microalgae to handle associated nutrient surge from anaerobic digestion technologies for the treatment of wastewater, is driven by the need for efficient nutrient recovery, greenhouse gas mitigation, wastewater treatment and biomass reuse. Here, the feasibility of growth and ammonium nitrogen removal rate of semi-continuous mixed microalgae culture in paddle wheel-driven raceway pond and helical tubular closed photobioreactor (Biocoil) for treating sand-filtered, undiluted anaerobic digestion piggery effluent (ADPE) was compared under outdoor climatic conditions between June and September 2015 austral winter season. Two Biocoils, (airlift and submersible centrifugal pump driven) were tested. Despite several attempts in using airlift-driven Biocoil (e.g. modification of the sparger design), no net microalgae growth was observed due to intense foaming and loss of culture. Initial ammonium nitrogen concentration in the Biocoil and pond was 893.03 ± 17.0 mg NH4 +-N L-1. Overall, similar average ammonium nitrogen removal rate in Biocoil (24.6 ± 7.18 mg NH4 +-N L-1 day-1) and raceway pond (25.9 ± 8.6 mg NH4 +-N L-1 day-1) was achieved. The average volumetric biomass productivity of microalgae grown in the Biocoil (25.03 ± 0.24 mg AFDW L-1 day-1) was 2.1 times higher than in raceway pond. While no significant differences were detected between the cultivation systems, the overall carbohydrate, lipid and protein contents of the consortium averaged 29.17 ± 3.22, 32.79 ± 3.26 and 23.29 ± 2.15% AFDW respectively, revealing its suitability as animal feed or potential biofuel feedstock. The consortium could be maintained in semi-continuous culture for more than three months without changes in the algal composition. Results indicated that microalgae consortium is suitable for simultaneous nutrient removal and biomass production from piggery effluent.