Evaluation of the Use of Microalgae as a Novel Process for the Valorisation of Nutrient Rich Digestate in a Context of Circular Economy

Nutrient rich digestate resulting from the anaerobic digestion of waste from different feedstocks (kitchen waste, manure, agricultural waste, etc.) is mainly used in Northwest Europe as a fertiliser, but its heavy usage has led to soil eutrophication and its spreading onto arable land is now restricted under the Nutrient Vulnerable Zones policy. Consequently, digestate has become an underused resource and novel technologies to remediate this waste are now needed. In this thesis, microalgae were evaluated for their potential to bioremediate digestate, within the context of a circular economy. Chapter 1 presents a summary of digestate characteristics and its current use and discusses the circular economy concept focusing on microalgae as a bioremediation tool. Digestate is characterised by its dark colour and high dry matter content, which can be bottlenecks to light penetration and consequently microalgal growth, hence treatment is essential to allow for microalgal growth on this waste. Chapter 2 investigated mechanical and biological processing of a digestate from kitchen waste and aimed to assess its potential to support microalgal growth. Digestate was shown to be a suitable nutrient source for Chlorella vulgaris, following membrane filtration, which was highly efficient at separating liquid and solid fractions of digestate. 2.5% of digestate was the optimal concentration used in this work, as higher levels could lead to ammonium toxicity, therefore, this chapter also highlighted the bottlenecks of digestate utilisation for microalgal cultivation. To improve digestate uptake by microalgae, chapter 3 tailored different digestate sources to Chlorella vulgaris and Scenedesmus obliquus while maintaining a low pH to increase ammonium availability by reducing evaporation. Digestate from pig manure provided the best growth results in both strains, and while ammonium availability was increased, acclimation of strains to high ammonium levels was still necessary, limiting the use of high concentrations of digestate in cultures. Composition analysis of the microalgal biomass showed that nitrogen starvation, caused by higher pHs and reduced ammonium lead to lipid increase. This chapter demonstrated the importance of digestate and strain pairing to improve remediation and enhance scalability of valorising digestate within a circular economy. Growth of microalgae on digestate was demonstrated in chapters 2 and 3, to pursue the circular economy approach and use the produced biomass, chapter 4 subsequently aimed to assess the feasibility of using a microalgal hydrolysate derived from digestate as a feed ingredient in the feed of Nile tilapia. Enzymatic hydrolysis was used to alleviate palatability issues encountered when whole cells are incorporated into feed. An incorporation of 10% of the waste-derived hydrolysate did not convey advantages in terms of the fish growth but increased fatty acid content demonstrating a commercial advantage of the ingredient in terms of flesh quality. Increasing digestate uptake by microalgae is essential to increase viability of the circular economy concept presented in chapter 2, 3 & 4. Consequently, chapter 5 investigated the potential of microalgae-bacteria consortia for this purpose, by studying the bacterial community associated to Chlorella vulgaris and the influence of nutrient availability on this dynamic association. Sequencing analysis revealed that there was a likelihood of growth-promoting and other advantages conveyed by bacteria growing concomitantly with microalgae and the use of bacteria to increase digestate remediation by microalgae was discussed. The different lines of research investigated in this thesis demonstrated the feasibility of valorising nutrient rich digestate using microalgae by developing a circular economy approach. My thesis has also highlighted some of the bottlenecks to the establishment of a sustainable and commercially viable microalgal industry in the UK and globally, and additional research contributing to bridging this gap were discussed in Chapter 6, giving an outlook on future research prospects in the field of microalgae biorefineries

Valorising nutrient-rich digestate: Dilution, settlement and membrane filtration processing for optimisation as a waste-based media for microalgal cultivation

Digestate produced from the anaerobic digestion of food and farm waste is primarily returned to land as a biofertiliser for crops, with its potential to generate value through alternative processing methods at present under explored. In this work, valorisation of a digestate resulting from the treatment of kitchen and food waste was investigated, using dilution, settlement and membrane processing technology. Processed digestate was subsequently tested as a nutrient source for the cultivation of Chlorella vulgaris, up to pilot-scale (800L). Dilution of digestate down to 2.5% increased settlement rate and induced release of valuable compounds for fertiliser usage such as nitrogen and phosphorus. Settlement, as a partial processing of digestate offered a physical separation of liquid and solid fractions at a low cost. Membrane filtration demonstrated efficient segregation of nutrients, with micro-filtration recovering 92.38% of phosphorus and the combination of micro-filtration, ultra-filtration, and nano-filtration recovering a total of 94.35% of nitrogen from digestate. Nano-filtered and micro-filtered digestates at low concentrations were suitable substrates to support growth of Chlorella vulgaris. At pilot-scale, the microalgae grew successfully for 28 days with a maximum growth rate of 0.62 day−1 and dry weight of 0.86 g⋅L−1. Decline in culture growth beyond 28 days was presumably linked to ammonium and heavy metal accumulation in the cultivation medium. Processed digestate provided a suitable nutrient source for successful microalgal cultivation at pilot-scale, evidencing potential to convert excess nutrients into biomass, generating value from excess digestate and providing additional markets to the anaerobic digestion sector

Microalgae Cultivation on Nutrient Rich Digestate: The Importance of Strain and Digestate Tailoring under PH Control

The bioremediation of digestate using microalgae presents a solution to the current eutrophication issue in Northwest Europe, where the use of digestate as soil fertiliser is limited, thus resulting in an excess of digestate. Ammonium is the main nutrient of interest in digestate for microalgal cultivation, and improving its availability and consequent uptake is crucial for optimal bioremediation. This work aimed to determine the influence of pH on ammonium availability in cultures of two green microalgae, additionally screened for their growth performances on three digestates produced from different feedstocks, demonstrating the importance of tailoring a microalgal strain and digestate for bioremediation purposes. Results showed that an acidic pH of 6–6.5 resulted in a better ammonium availability in the digestate media, translated into better growth yields for both S. obliquus (GR: 0.099 ± 0.001 day−1; DW: 0.23 ± 0.02 g L−1) and C. vulgaris (GR: 0.09 ± 0.001 day−1; DW: 0.49 ± 0.012 g L−1). This result was especially true when considering larger-scale applications where ammonium loss via evaporation should be avoided. The results also demonstrated that digestates from different feedstocks resulted in different growth yields and biomass composition, especially fatty acids, for which, a digestate produced from pig manure resulted in acid contents of 6.94 ± 0.033% DW and 4.91 ± 0.3% DW in S. obliquus and C. vulgaris, respectively. Finally, this work demonstrated that the acclimation of microalgae to novel nutrient sources should be carefully considered, as it could convey significant advantages in terms of biomass composition, especially fatty acids and carbohydrate, for which, this study also demonstrated the importance of harvesting time

Microalgae Cultivation on Nutrient Rich Digestate: The Importance of Strain and Digestate Tailoring under PH Control

The bioremediation of digestate using microalgae presents a solution to the current eutrophication issue in Northwest Europe, where the use of digestate as soil fertiliser is limited, thus resulting in an excess of digestate. Ammonium is the main nutrient of interest in digestate for microalgal cultivation, and improving its availability and consequent uptake is crucial for optimal bioremediation. This work aimed to determine the influence of pH on ammonium availability in cultures of two green microalgae, additionally screened for their growth performances on three digestates produced from different feedstocks, demonstrating the importance of tailoring a microalgal strain and digestate for bioremediation purposes. Results showed that an acidic pH of 6-6.5 resulted in a better ammonium availability in the digestate media, translated into better growth yields for both S. obliquus (GR: 0.099 +/- 0.001 day(-1); DW: 0.23 +/- 0.02 g L-1) and C. vulgaris (GR: 0.09 +/- 0.001 day(-1); DW: 0.49 +/- 0.012 g L-1). This result was especially true when considering larger-scale applications where ammonium loss via evaporation should be avoided. The results also demonstrated that digestates from different feedstocks resulted in different growth yields and biomass composition, especially fatty acids, for which, a digestate produced from pig manure resulted in acid contents of 6.94 +/- 0.033% DW and 4.91 +/- 0.3% DW in S. obliquus and C. vulgaris, respectively. Finally, this work demonstrated that the acclimation of microalgae to novel nutrient sources should be carefully considered, as it could convey significant advantages in terms of biomass composition, especially fatty acids and carbohydrate, for which, this study also demonstrated the importance of harvesting time

Evaluation of Aurantiochytrium mangrovei biomass grown on digestate as a sustainable feed ingredient of sea bass, Dicentrarchus labrax, juveniles and larvae

The use of microalgae as a sustainable source of n-3 long-chain polyunsaturated fatty acids (LC-PUFA) as an alternative to fish oils from small pelagic fish (e.g., anchovy, sardine) has received growing interest in the past few years. The present study aimed to: (i) produce Aurantiochytrium mangrovei biomass by heterotrophic fermentation using a medium containing anaerobic digestion liquid effluent, and (ii) evaluate a biomass rich in n-3 LC-PUFA and good quality proteins as a feed ingredient for sea bass juveniles and larvae. Two 800 L bioreactors were used to produce Aurantiochytrium biomass in non-axenic conditions. Biomass was then filtered through a crossflow filtration system (300 Kda ceramic membrane) and freeze-dried. Sea bass juveniles (32.7 +/- 4.2 g) were fed both a control diet and a diet containing 15% of freeze-dried A. mangrovei biomass for 38 days. Juvenile survival percentage was 90% on average in both dietary conditions. Similar growth was observed between fish fed with both diets, demonstrating the feasibility to replace 15% of a standard fish feed by Aurantiochytrium biomass. The liver of sea bass juveniles fed with the A. mangrovei diet contained significantly higher proportions of 22:6n-3, 22:5n-6, and 20:4n-6 than those fed with the control diet, while the proportions of 16:0, 16:1n-7, and 18:1n-9 were significantly lower. The secondary oxidation, as measured by malonylaldehyde (MDA) content, in the liver and muscle of juveniles fed with the microalgae diet tended to be higher than in fish fed the control diet, but the differences were not statistically significant. Although the larvae survival percentage was low for all the tanks after 41 days of rearing, the inclusion of 15% of hydrolyzed A. mangrovei biomass in the larvae micro-diet did not impair the development of sea bass larvae and only marginally affected their lipid composition. In the future, we have to further optimize a sustainable workflow between Aurantiochytrium cultivation and fish feed production and confirm the zootechnical and biochemical results

Evaluation of Aurantiochytrium mangrovei Biomass Grown on Digestate as a Sustainable Feed Ingredient of Sea Bass, Dicentrarchus labrax, Juveniles and Larvae

The use of microalgae as a sustainable source of n-3 long-chain polyunsaturated fatty acids (LC-PUFA) as an alternative to fish oils from small pelagic fish (e.g., anchovy, sardine) has received growing interest in the past few years. The present study aimed to: (i) produce Aurantiochytrium mangrovei biomass by heterotrophic fermentation using a medium containing anaerobic digestion liquid effluent, and (ii) evaluate a biomass rich in n-3 LC-PUFA and good quality proteins as a feed ingredient for sea bass juveniles and larvae. Two 800 L bioreactors were used to produce Aurantiochytrium biomass in non-axenic conditions. Biomass was then filtered through a crossflow filtration system (300 Kda ceramic membrane) and freeze-dried. Sea bass juveniles (32.7 ± 4.2 g) were fed both a control diet and a diet containing 15% of freeze-dried A. mangrovei biomass for 38 days. Juvenile survival percentage was 90% on average in both dietary conditions. Similar growth was observed between fish fed with both diets, demonstrating the feasibility to replace 15% of a standard fish feed by Aurantiochytrium biomass. The liver of sea bass juveniles fed with the A. mangrovei diet contained significantly higher proportions of 22:6n-3, 22:5n-6, and 20:4n-6 than those fed with the control diet, while the proportions of 16:0, 16:1n-7, and 18:1n-9 were significantly lower. The secondary oxidation, as measured by malonylaldehyde (MDA) content, in the liver and muscle of juveniles fed with the microalgae diet tended to be higher than in fish fed the control diet, but the differences were not statistically significant. Although the larvae survival percentage was low for all the tanks after 41 days of rearing, the inclusion of 15% of hydrolyzed A. mangrovei biomass in the larvae micro-diet did not impair the development of sea bass larvae and only marginally affected their lipid composition. In the future, we have to further optimize a sustainable workflow between Aurantiochytrium cultivation and fish feed production and confirm the zootechnical and biochemical results