ABRAHAM OLAMILEKAN JAMES POST-TREATMENT OF MUNICIPAL WASTEWATER USING MICROALGAE AND ACTIVATED SLUDGE SYNERGY Bauru 2023 ABRAHAM OLAMILEKAN JAMES POST-TREATMENT OF MUNICIPAL WASTEWATER USING MICROALGAE AND ACTIVATED SLUDGE SYNERGY Dissertation presented as a requirement for obtaining the title of Master in Civil and Environmental Engineering at the São Paulo State University “Júlio de Mesquita Filho”, Area of Concentration: Sanitation. Advisor: Prof. Dr. Gustavo H. R. Silva Co-Advisor: Prof. Dr. Graziele R. L. Silva Bauru 2023 J27p James, Abraham Olamilekan Post-Treatment of Municipal Wastewater Using Microalgae and Activated Sludge Synergy / Abraham Olamilekan James. -- Bauru, 2023 124 f. : tabs., fotos Dissertação (mestrado) - Universidade Estadual Paulista (Unesp), Faculdade de Engenharia, Bauru Orientadora: Gustavo Henrique Ribeiro da Silva 1. Microalgae-Activated Sludge. 2. Nutrient Recovery. 3. Pollutant Removal. 4. Bioflocculation. 5. Hydraulic Retention Time. I. Título. Sistema de geração automática de fichas catalográficas da Unesp. Biblioteca da Faculdade de Engenharia, Bauru. Dados fornecidos pelo autor(a). Essa ficha não pode ser modificada. DEDICATION To God and my beloved mother of blessed memory, the pathfinder of this lofty height ACKNOWLEDGMENT The triumphant completion of my M. Sc. program and the successful compilation of my dissertation were achieved on the wheels of the steadfastness and supports I received from the wonderful people I sojourned with, which by extensive effect accorded a soft landing as I transitioned from environmental management to engineering; availing quality outputs and expositions without the bruising blows of trial and error. Therefore, with plesaure in my heart, I would like to express my indebtedness for the gracious supports I received from those people, I consider, as parts of God’s provisions for the success of this enviable journey. Firstly, I would like to appreciate the management of my home University (Federal University of Agriculture, Abeokuta) for considering me worthy of nomination for the Agricultural Research and Innovation Fellowship for Africa (ARIFA) scholarship, and the Tertiary Education Trust Fund (TETFund) and the Forum for Agricultural Research in Africa (FARA) for the partnership initiative. My sincere appreciation goes to my fatther (James Ologunde) brothers (Rotimi James, John James, Olasunkanmi James) and sister (Olanike Hassan) for their tireless encouragements and pragmatic display of love, albeit miles away. Moreover, my gratitude to my supervisor, Professor Gustavo Henrique Ribeiro da Silva, is immensely immeasurable. His mentoring, motivation and dedication to birthing the giant in me were as much bracing and intrumentally poising, as I have emerged a smart thinker through this process. Beyond his supervisory role, he saw to my wellbeing and sound mind, which were emotionally supportive. Equally, my elegant co-supervisor, Prof. Graziele Ruas, was superlatively complementary to my success story. Her attentiveness, openess, and brilliant injections did not just improve the quality of the work, but spurred the budding logic and science in me. The decisiveness of my supervisory team, including Prof. Maria Raya and Prof. Igor Firmino, inculcating the ideals of professional scientists as they teach and correct have contributed to my growth in no small measure. I would like to appreciate Glauco Perpetuo for his willingness to assist and provide solutions to befallen challenges all through the laboratory phase. Traversing the laboratory was made pleasurable with your support. The supports received from Thalita Santos, Gustavo Alemcar, Eduardo Miguel, Larrisa Quartaroli, Caroline Pompei, Caroline Calil during the intensive batch experiments are acknowledged and appreciated. I also acknolwedge FAPESP (2018/18367-1 and 2022/07475-3) for sponsoring the laboratory materials and equipment used for my experiment and UNESP IPMet for the gracious supply of weather data propmtly. Worthy of mention are my colleagues from Nigeria, Abayomi Bankole and Emmanuel Babajide. The brotherly love, moral supports and practically helping out through the journey emboldened my poise to pull through. Also, the feminine care received from the mother of the house, Mrs. Bankole Racheal, coupled with the unsolicited cheerful smile from baby Rejoice Camila Bankole, were tension dousing and mind attuning. And finally, I appreciate my loving and caring fiancee, Modupeola Agbeke Sarumi, for her ceaseless prayers, encouragements and moral supports throughout the jouney. i Summary O problema das águas residuárias não tratadas ou tratadas inadequadamente ainda persiste globalmente. Isso não tem apenas contribuído para o aumento das cargas de poluição ambiental, mas também falhado em aproveitar os materiais bioeconômicos presentes nas águas residuárias. Embora a ciência da tecnologia de tratamento baseada em microalgas tenha se mostrado eficiente para o tratamento e recuperação de recursos potenciais, a maioria dos estudos foi realizada sob condições controladas. Portanto, o presente estudo avaliou o desempenho do crescimento de microalgas nativas-lodo ativado (MLA) para o tratamento terciário de águas residuárias digeridas anaerobiamente em um reator anaeróbio de fluxo ascendente com manta de lodo (UASB), a partir de fotobiorreatores externos em escala laboratorial (2 L). Sete diferentes condições com proporções distintas de inóculo (Condições: 1 a 3 é MLA; 4 a 6 são controles de microalgas; 7 é sem inóculo) foram operadas em bateladas em reator em triplicata, com tempo de retenção hidráulica (TRH) de 5 dias em 11,5:12,5 Foto-horas, para identificar a melhor proporção de inóculo a ser adotada no fotobiorreator piloto do tipo flat planel. A condição 1 (0,10 g.L-1: microalga e 0,20 g.L-1: lodo ativado), com menor proporção de MLA, apresentou o melhor resultado, com produtividade de densidade celular (2,03 x 107 células.mL-1) juntamente com biomassa de 0,13 g SST.L-1.d-1, remoção de fósforo total (85,07%) e nitrogênio total (66,14%) e remoção logarítmica (Log-Re) de bactérias patogênicas (indicadores de qualidade da água), com Log- Re 3,3 para coliformes totais (137E+02 UFC.100 mL-1) e 4,7 para Escherichia coli (0,00E+00 UFC.100 mL-1). O MLA de melhor desempenho (condição 1) foi testado em um fotobiorreator piloto do tipo flat planel, ao lado de um controle inoculado com a proporção de microalgas do MLA e operado sob TDH de 5 e 3 dias. O desempenho do sistema foi determinado pela eficiência de remoção e recuperação de nutrientes, sedimentação da biomassa e remoção de Escherichia coli e coliformes totais. Os resultados mostraram que o TRH mais alto (5 dias) favoreceu o desempenho do MLA em relação ao crescimento da biomassa, com eficiência de remoção significativamente maior (p < 0,05), 88,0; 79,0; 59,5% para fósforo dissolvido total (FDT), nitrogênio amoniacal (NH+ 4-N) e demanda química de oxigênio (DQOfiltrado), respectivamente. A produtividade média de biomassa e taxa de crescimento para TRHs de 5 e 3 dias foi de 59,41±12,31 mg SST.L-1.d-1, 0,08±0,01 d-1 e ii 925,56±11,09 mg SST.L-1.d-1, 0,03±0,01 d-1, respectivamente. Os Log-Re > 4,0 UFCmL-1 e > 3,5 UFCmL-1 foram obtidos para E. coli e coliformes totais, respectivamente, em ambos os TRHs, e mostraram potencial para uso agrícola por atender ao padrão recomendado pela Organização Mundial da Saúde (OMS) de 103 MPN.100mL-1. No geral, os resultados mostraram uma viabilidade economicamente promissora do tratamento de águas residuárias à base de microalgas ao ar livre. No entanto, a eficiência de autofixação foi inversamente baixa de 33,0 e 14,0% para TRHs de 5 e 3 dias, respectivamente, e supostamente impactada pelo alto pH (> 10,0) na população bacteriana e pela dominância da não-mucilagem Cyanobium sp. (98%). iii Abstract The problem of untreated and inadequately treated wastewater still persists globally. This has not only contributed to the increasing environmental pollution loads, but has also failed to harness the bioeconomy materials present in wastewater. While the science of microalgae- based treatment technology has shown to be efficient for treatment and recovery of potential resource, the majority of the studies were conducted under controlled conditions. Therefore, this present study evaluated the performance of native microalgae-activated sludge (MAS) growth for tertiary treatment of anaerobically digested wastewater from an upflow anaerobic sludge blanket (UASB) in an outdoor lab-scale photobioreactors (2 L). Seven different conditions with distinct inoculum proportions (Conditions: 1 to 3 is MAS; 4 to 6 is microalgae controls; 7 is without inoculum) were operated in batch mode reactor in triplicate for 5-days of hydraulic retention time (HRT) at 11.5:12.5 Photo-hours, to identify the best inoculum proportion for adopted in pilot flat panel photobioreactor. Condition 1 (0.10gL-1: microalgae and 0.20gL-1: activated-sludge), with lowest MAS proportion showed the best outcome with cell density productivity (2.03 x 107 cells.mL-1) along with biomass of 0.13 g TSS.L-1.d-1 and the total phosphorus uptake (85.07%), total nitrogen uptake (66.14%), logarithmic removal (Log-Re) of bacterial pathogens (water quality indicators), which showed Log-Re (3.3 for total coliforms (137E+02 CFU.100 mL-1) and 4.7 for Escherichia coli (0.00E+00 CFU.100 mL-1)). The best performing MAS (condition 1) was tested in a pilot flat panel photobioreactor, alongside a control, inoculated with the microalgae proportion of the MAS, and operated under 5-days and 3-days HRT. The performance of the system was determined by the efficiency of nutrient removal and recovery, biomass sedimentation and removal of Escherichia coli and Total Coliforms. Results showed that high HRT favoured the performance of MAS with respect to biomass growth, with significantly higher (p < 0.05) removal efficiency, 88.0, 79.0, 59.5% for total phosphorus (TDP), ammonium nitrogen (NH+ 4-N), chemical oxygen demand (CODfiltered), respectively at 5 days HRT. The average biomass productivity and growth rate for HRTs 5 days and 3 days are 59.41±12.31 mg TSS L-1 d-1, 0.08±0.01 d-1 and 25.56±11.09 mg TSS L-1 d-1, 0.03±0.01 d-1, respectively. The log-Re > 4.0 CFUmL-1 and > 3.5 CFUmL-1 were achieved for E. coli and total coliforms, respectively at both HRTs, and showed the potential for iv agricultural use for complying with the WHO recommended standard of 103 MPN.100mL- 1. Overall, results showed a promising feasibility of outdoor microalgae-based treatment of wastewater cost effectively. However, self-settling efficiency was conversely low 33.0 and 14.0% for HRTs 5 days and 3 days, respectively, and suspiciously impacted by high pH (> 10.0) on bacterial population and the dominance of non-mucilage Cyanobium sp. (98%). v List of Figures Figure 1: Agro-industrial application potential of microalgae biomass from wastewater---29 Figure 2: (a) Schematic diagram of Duran® bottle photobioreactor set-up (b) Composition of inoculum ratio for each condition evaluated---------------------------------------------------51 Figure 3: Daily average temperature and light intensity during the experiment--------------54 Figure 4: Average cell density expressed in cell mL-1 for the seven experimental conditions- ----------------------------------------------------------------------------------------------------------60 Figure 5: Log removal (Log-Re) for E. coli and total coliforms-------------------------------64 Figure 6: Framework of experimental procedure: The laboratory scale batch experiment was performed separately in previous test, explained in chapter 3----------------------------------80 Figure 7: Set-up of pilot Flat panel photobioreactors (a) Control flat panel containing microalgae (b) Experiment flat panel containing mix-culture of microalgae and activated sludge---------------------------------------------------------------------------------------------------81 Figure 8: Microalgae cell growth and environmental conditions during 5 days HRT (a & b) cell count (c) temperature (d) light intensity from solar radiation------------------------------85 Figure 9: Microalgae cell growth and environmental conditions during 3 days HRT (a) cell count (b) temperature (c) light intensity from solar radiation-----------------------------------86 Figure 10: Estimation of microalgae cell growth with total suspended solids (TSS) dry weight and optical density (OD) 680 nm (a - c) TSS, OD 680 nm and scatter plot correlation relationship between TSS and OD 680 nm, respectively for 5 days HRT---------------------87 Figure 11: Estimation of microalgae cell growth with total suspended solids (TSS) dry weight and optical density (OD) 680 nm (a - c) TSS, OD 680 nm and scatter plot correlation relationship between TSS and OD 680 nm, respectively for 3 days HRT--------------------88 Figure 12: Nitrification and assimilation of nitrogenous compounds (a) Experimental_R1 for 5 days HRT (b) Control_R2 for 5 days HRT (c) Experimental_R1 for 3 days (d) Control_R2 for 3 days HRT---------------------------------------------------------------------------------------- 922 Figure 13: (a) Log-Re for E. coli and total coliforms in 5 days HRT (b) Log-Re for E. coli and total coliforms in 3 days HRT------------------------------------------------------------------93 vi List of Tables Table 1: Mean concentration and standard deviation of substrate wastewater characteristics, being that: TN = total nitrogen; TDP = total dissolved phosphorus; DO = dissolved oxygen; TSS = total suspended solids----------------------------------------------------------------------- 50 Table 2 : Composition of operational volume per condition-------------------------------------53 Table 3: Mean and standard deviation of pH, alkalinity, biomass productivity and cell density at optical density 680nm, and nutrient removal efficiencies found in the seven conditions-57 Table 4: Correlation between TDP removal and OD680 of the seven conditions--------------62 Table 5: Geometric mean of total coliform and E. coli at D0 and D5 in the seven experimental conditions----------------------------------------------------------------------------------------------63 Table 6: P-value of mean difference for D0 and D5 of selected parameters at 95% confidence interval------------------------------------------------------------------------------------------------- 666 Table 7: Mean and Standard deviation of municipal wastewater composition---------------79 Table 8: Average dissolve oxygen concentration, pH value, removal efficiency of CODfiltered, Alkalinity, NH4 +-N , oxidation of ammonium, biomass productivity and settle ability for the 5 days and 3 days HRTs------------------------------------------------------------------------------ 900 vii List of Abbreviations CFU – Coliform Forming Unit COD - Chemical Oxygen Demand DAE - Department of Water and Sewage of Bauru DO – Dissolved Oxygen FAPESP - Fundação de Amparo à Pesquisa do Estado de São Paulo HRT – Hydraulic Retention Time IPMet - Instituto de Pesquisas Meteorológicas MAS – Microalgae and Activated Sludge n – Sample size NTK - Total Nitrogen Kjeldahl OD - Optical Density PBRs – Photobioreactors pH - Hydrogen Potential R1 – Reactor 1 R2 – Reactor 2 SDG - Sustainable Development Goals TDP - Total Dissolved Phosphorus TDN - Total Nitrogen TSS – Total Suspended Solids UASB - Upflow Anaerobic Sludge Blanket UNESP – State University “Júlio Mesquita Filho UNICEF – United Nations International Children Emergency Fund WHO – World Health Organization WW – Wastewater WWTP – Wastewater Treatment Plant viii Organization of Dissertation This thesis is organized in five chapters. Chapter 1 (Introduction) explained the background, research problem, the objectives and the hypothesis of this work. Chapter 2 (Literature review) provided the summary and highlights of relevant existing studies, and explained wastewater characteristics, importance of anaerobic sewage treatment systems, microalgae cultivations and microalgae-based system as a tertiary treatment technology, environmental factors affecting microalgae cultivation, bioeconomy, microalgae biomass recovery and industrial applications. Chapter 3 (Batch experiment study) detailed the laboratory scale experiment that evaluated the potential of microalgae-activated sludge (MAS) co-culture (seven conditions) in natural outdoor condition, and contained the introduction, methodology and results and discussion. Chapter 4 (Fed-batch experiment study) showed the performance of the best condition (identified at the laboratory scale) in pilot flat panel photobioreactor in natural outdoor condition with respect to hydraulic retention time, and contained the introduction, methodology, result and discussion and future perspective drawing from recorded findings. Chapter 5. The general conclusion of the experiments. ix Table of Contents SUMMARY ................................................................................................................................................ I ABSTRACT .............................................................................................................................................. III LIST OF FIGURES .................................................................................................................................. V LIST OF TABLES .................................................................................................................................... VI LIST OF ABBREVIATIONS ................................................................................................................. VII ORGANIZATION OF DISSERTATION ............................................................................................. VIII TABLE OF CONTENTS .......................................................................................................................... IX 1 INTRODUCTION ............................................................................................................................ 1 1.1 JUSTIFICATION OF STUDY .................................................................................................................... 6 1.2 REFERENCES ........................................................................................................................................ 8 2 OBJECTIVES ................................................................................................................................ 14 2.1 SPECIFIC OBJECTIVES ........................................................................................................................ 14 2.2 HYPOTHESES ...................................................................................................................................... 14 3 LITERATURE REVIEW .............................................................................................................. 15 3.1 INTRODUCTION .................................................................................................................................. 15 3.2 WASTEWATER CHARACTERISTICS ..................................................................................................... 15 3.3 ANAEROBIC SEWAGE TREATMENT SYSTEMS ..................................................................................... 17 3.4 POST-TREATMENT USING MICROALGAE-BASED TECHNOLOGIES ...................................................... 18 3.5 MICROALGAE CULTIVATION .............................................................................................................. 19 3.6 ENVIRONMENTAL FACTORS AFFECTING MICROALGAE CULTIVATION ............................................... 20 3.6.1 Light Intensity .......................................................................................................................... 20 3.6.2 Temperature ............................................................................................................................ 21 3.6.3 Temperature ............................................................................................................................ 21 3.6.4 Value of pH .............................................................................................................................. 22 3.7 BIOECONOMY AND CIRCULAR ECONOMY .......................................................................................... 22 3.8 MICROALGAE BIOMASS RECOVERY ................................................................................................... 23 3.9 BIOMASS AND AGRO-INDUSTRIAL APPLICATIONS ............................................................................. 24 x 3.9.1 Biofuel Production ................................................................................................................... 24 3.9.2 Biofertilizer .............................................................................................................................. 26 3.9.3 Bioplastics ............................................................................................................................... 26 3.9.4 Cosmetics ................................................................................................................................. 27 3.9.5 Animal feeds ............................................................................................................................ 28 3.10 REFERENCES ................................................................................................................................. 30 4 LABORATORY SCALE BATCH EXPERIMENT ...................................................................... 47 4.1 INTRODUCTION .................................................................................................................................. 48 4.2 MATERIALS AND METHODS ............................................................................................................... 50 4.2.1 Anaerobically Digested Wastewater ........................................................................................ 50 4.2.2 Experimental Set-up ................................................................................................................ 51 4.2.3 Microorganisms and Culture Conditions ................................................................................ 52 4.2.4 Analytical Methods .................................................................................................................. 53 4.2.5 Statistical Analysis ................................................................................................................... 53 4.3 RESULTS AND DISCUSSION ................................................................................................................ 54 4.3.1 Influence of Temperature and Light Intensity Measurement ................................................... 54 4.3.2 Operational and Environmental Conditions ............................................................................ 56 4.3.3 Effect of Inoculum on Biomass Productivity ............................................................................ 58 4.3.4 Effect of Inoculum on Nutrients Removal ................................................................................ 60 4.3.5 Assessment of Total Coliforms and Escherichia coli Removal ................................................ 63 4.3.6 Optimum Inoculum Condition ................................................................................................. 65 4.4 CONCLUSION ..................................................................................................................................... 66 4.5 REFERENCES ...................................................................................................................................... 67 5 PILOT SCALE FED-BATCH EXPERIMENT ............................................................................. 75 5.1 INTRODUCTION .................................................................................................................................. 76 5.2 MATERIALS AND METHODS ............................................................................................................... 78 5.2.1 Anaerobically Digested Municipal Wastewater ...................................................................... 78 5.2.2 Experimental Set-up ................................................................................................................ 79 5.2.3 Microalgae and Activated sludge Inoculum ............................................................................ 81 5.2.4 Biomass Recovery .................................................................................................................... 82 5.2.5 Calculations ............................................................................................................................. 82 5.2.6 Analytical Methods .................................................................................................................. 82 5.2.7 Data Analysis ........................................................................................................................... 83 5.3 RESULTS AND DISCUSSION ................................................................................................................ 83 5.3.1 Effect of hydraulic retention time on Microalgae cell growth ................................................. 83 5.3.2 Removal efficiency of COD, Nutrients and Alkalinity ............................................................. 89 5.3.3 Escherichia coli and Total coliforms Removal Assessment ..................................................... 92 xi 5.3.4 Settleability and Biomass Recovery ......................................................................................... 94 5.3.5 Perspective for Future Study ................................................................................................... 95 5.4 CONCLUSION ..................................................................................................................................... 96 5.5 REFERENCES ...................................................................................................................................... 97 6 CONCLUSION, BENEFITS AND RECOMMENDATIONS ..................................................... 105 6.1 GENERAL CONCLUSION ................................................................................................................... 105 6.2 BENEFITS OF THE STUDY ................................................................................................................. 105 6.3 RECOMMENDATIONS ........................................................................................................................ 106 1 1 INTRODUCTION The management of wastewater is still a major challenge confronting the whole world. This could be attributed to volumetric increase of untreated wastewater from municipal, industrial, and agricultural activities, which constitutes a significant threat to the quality of water resources as well as public health (Aradhana and Kumar, 2015; Zhu et al., 2019; Pompei et al., 2022). The world population has doubled in the last 48 years, and survey has shown many of the African countries having the fastest growth rates, with the continent’s population size doubling by 2050 (Routley, 2022). This raises more concern on the capacity of these countries to treat their wastewater and invariably necessitates the need for more explorative studies to be conducted on affordable and effective alternative treatment options to bridge existential treatment gap. The growing human population has also continued to widen the supply gap of fertilizer. The survey separately conducted by the International Fertilizer Development Centre in 2010 and the United State Geological Survey in 2012 showed utilization of phosphorus would reach it peak between 2030 and 2040, with hard biting global scarcity effect and food insecurity (Desmidt et al., 2014; Neset and Cordell, 2011). Also, various authors have given different projections for outright depletion of the finite phosphate rock, ranging between 50 to 400 years (Kok et al., 2018), which is likely to have reduced due to increasing demand. Alternatively, Moges et al. (2020) showed that repurposing wastewater to produce phosphorus is capable of closing the supply loop of phosphorus, which would in turn reduce mining of phosphate rock and accompanying environmental problems. Specifically, Kok et al. (2018) revealed that phosphorus recovery can sustainably satisfy the global demand, indicating 20% potential from all urban wastewater and 90% potential from animal manure 2 for total agricultural demands, making it a good substitute for phosphorus fertilizer. For the purpose of sustainability, the decentralized wastewater treatment plants are recommended for phosphorus recovery from human waste, owing to simple treatment processes and low- cost technologies and operated in close proximity to the flow of water, energy, and organic and inorganic material (Silva et al., 2019). The war between Russia and Ukraine has further exacerbated the challenge of fertilizer scarcity and food insecurity. Recently, 20 percent hike in food prices was projected by the United Nations’ Food and Agriculture Organization hinged on dwindled availability of fertilizer and food supplies from the supplying warring nations (Lu, 2022). This further underscores the significance of recovering valuable resources from municipal wastewater, especially nitrogen and phosphorus nutrients, the main ingredient of fertilizer, while ensuring adequate treatment, before being discharged into the environment. This has the potential to reduce dependence on external supplies to meet fertilizer needs (Ashley, 2009), as prevailing social or environmental related problems may cut short supply, just as it is currently (Shaddel et al., 2019). This has the potential to drive sustainable food supply and security, and would also boost the science of circular economy (Su, 2020). Globally, approximately 80% of wastewater is released into the environment without adequate treatment (Oviedo et al., 2022). Specifically, in the Sub-Saharan and Latin America and the Caribbean between, over 50% of the wastewater generated from 2000 to 2017 was discharged into the environment without prior treatment (WHO and UNICEF, 2019). Furthermore, Brazil, being the most populous in Latin America and the Caribbean, scored fairly above half (56%) until 2017 in the treatment of domestic wastewater at secondary level, while, on the other hand, a damning 32% coverage was recorded for Nigeria for the same level of treatment of domestic wastewater. Moreover, the small percentage of treated wastewater fall short of the stipulated permissible level owing to the shortcoming of the conventional treatment technologies (Serejo et al. 2020). This has led to the discharge of wide range of pollutants (nitrate, phosphate, microplastics, metals persistent organic pollutants) and pathogenic microorganisms (bacteria, protozoa, viruses) that are usually present in wastewater in concentrations above the critical safe limits, thus increasing the pollution loads of receiving water bodies. This poses serious challenges that border on environmental pollution, ecosystem obliteration resulting from algae bloom, oxygen depletion, death of aqua species, and public health 3 epidemics from ingestion of pathogenic organisms (Akpor & Muchie, 2011; UNESCO, 2017; Ruas et al., 2017; Gonçalves et al., 2017). The disproportionate discharge of untreated wastewater into the environment may be linked to high operational cost implication of conventional treatment process, particularly for municipal wastewater. This presents a major limitation in form of high cost of energy for aeration and frequent removal of accompanying increased sludge produced during treatment (Kehrein et al., 2020; Wang et al., 2020; Molinuevo-Salces et al., 2019). Additionally, the conventional biological treatment methods fall short in the nutrients removal efficiency required, thus contributing to nutrient pollution load of receiving water bodies (Quijano et al., 2017; Wang et al., 2020). In specific terms, the technical and economic drawbacks of conventional biological treatment methods have been linked to (1) inadequate pollutant and pathogen removal (2) poor nutrient recovery (3) expensive and energy demanding, and (4) CO2 production (Molinuevo-Salces et al., 2019). The activated sludge biological treatment process is adjudged the most used of the options (Maddela et al., 2021). On the other hand, the anaerobic counterpart, which excludes the need for aeration, effectively decomposes the organic matters in wastewater, but releases effluent that is rich in nutrients (de Oliveira et al., 2021). Therefore, the highlighted limitations have made the conventional treatment methods less desirable and effective to wastewater treatment. This has the potential to undermine the realisation of the United Nations Sustainable Development Goal (SDG) 6 due to associated polluting potential and consequent reduction in water quality meant for domestic and other purposes. In response and considering that nutrients are unavoidable by-products of wastewater treatment plants and systems, biorefinery treatment technologies are being explored to ensure effective treatment. In addition to the offer of environmental protection attributable treatment efficacy, the potential resources in wastewater are concurrently recovered by biorefineries (Barros et al., 2022), thus projects an economic outlook inform of provision of materials and inputs for industrial symbiosis. The transformation of wastewater into high- value products for agriculture, aquaculture, and sundry other industrial processing inputs will incentivize maximum use of resources and ensure increased production value-chain. This will in-turn promote environmental sustainability by addressing the problems of 4 depleting natural resources and environmental pollution as well as it will improve agricultural productivity. Microalgae biorefinery has received a lot of attention in recent decades owing to the empirical evidence as an efficient treatment alternative. More importantly, high value-added products are generated through nutrient cycling and valorisation (biofertilizer, biofuel and animal feeds) while wastewater is being treated, with the removal of ammonium, nitrates and phosphorus, lipids and pigments, protein (Liu and Hong, 2021; Dourou et al., 2020; Molinuevo-Salces et al., 2019; Matamoros et al., 2016; Wang et al., 2010). These microalgae utilize the nutrients in wastewater with the aid of solar energy or artificial light source to replicate exponentially, generate high biomass growth, and concomitantly treat the fed wastewater. The nutrients are stored in their cellular composition, forming a nutrient-rich biomass that can be used as feedstock for industrial purposes (Hwang, 2016; Andrade et al., 2021). The biomass as a resource has a wide range of applicability, considering the potential of the high-value products encapsulated within the microalgae cells. The proteins, lipids, and polysaccharides contents can be used for the production of bio-based polymers; an environmentally friendly alternative packaging material due to their degradability (Sogut & Seydim, 2018). This offers the benefits of reduced environmental footprint of plastic proliferation and the corresponding municipal solid waste problem. Furthermore, it strengthens the fight against climate change, reducing the concentration of carbon emission by stalling the consumption of about 8% of the world’s oil production, meant for the manufacture of synthetic plastics (Riera & Palma, 2021). Moreover, biomass is used as feedstock for bioenergy production, in cosmetics industry, and as biofertilizers (Rumin et al. 2020). However, slow settling and consequent poor biomass harvesting of microalgae cells posits a serious challenge to biomass recovery. Microalgae are negatively charged, with small cell size (5 and 50 μm) and relatively low cell density, thus often dispersely distributed in suspension (Molinuevo-Salces et al., 2019; Quijano et al., 2017). To recover these microalgae, application of different techniques such as filtration, centrifugation, coagulation, are employed industrially (Fasaei et al., 2018), and amount to additional capital expenditure and energy consumption. Infact, the chemical harvesting method (chemical coagulants) have 5 shown the tendency of affecting the quality of harvested biomass (Quijano et al., 2017), which is liable to limit the scope of application of the biomass for industrial use. To this effect, natural coagulants and auto-flocculation have been explored to recover nutrients rich biomass. Teixeira et al. (2021) achieved better floc formation with improved biomass recovery and over 90% turbidity and optical density removal, at a much-reduced sedimentation time, with Tannin-based coagulants. Likewise, high recovery percentage of microalgae biomass anaerobically cultivated in blackwater, alongside turbidity and optical density, was reported by Silva et al. (2020). Furthermore, auto-flocculation induced by altering the pH aqueous system using NaOH achieved about 94% recovery (Ummalyma et al., 2016). Biopolymers such as chitosan, cationic starches and polyacrylamide have equally displayed a promising recovery performance of algae biomass (Matter et al., 2019), but effectiveness is pH dependent and produces good outcome at a narrow range, with low performing efficiency for some microalgae species (Matter et al., 2019). The highlighted techniques and technologies come with additional obligations in form of energy requirement, equipment and maintenance, and materials such as nature-based coagulants and NaOH in case of pH alteration, which exert cost implications. Meanwhile, the synergistic interaction of microalgae and bacteria (activated sludge) has shown to be economically viable with the aid of bio-flocculation, recovering high quality and nutirent rich biomass by sedimentation under gravity (Zhu et al., 2019). Interestingly, Quijano et al. (2017) provided a good account of how the synergy of microalgae and bacteria consortia produce stable and settleable aggregates. Microalgal-bacterial aggregates (MABAs) formed contained extracellular polymeric substances (EPS) separately produced by microalgae and bacteria, which are subsequently bonded together and mediate the formation of aggregates of efficient settling characteristics, settling out by gravity, thus yielding excellent outcome of biomass harvest. Plethora of studies have explored the pollution mitigation potential of microalgae and bacteria consortia since the realization of treatment efficiency and effectiveness. Serejo et al. (2020) reported suitable surfactant removal, and optimum removal of contaminants of emerging concern was achieved in the studies conducted by Prosenc et al. (2021) and Matamoros et al. (2016). Also, high removal efficiency of pathogenic microorganism under the influence of CO2 addition was reported by Ruas et al. (2017), the influence of temperature on treatment performance was explored by Ji et al. (2021), influence of hydraulic retention 6 time and nitrogen/carbon ration on treatment efficiency was conducted by Toledo-Cervantes et al. (2019), influence of solid retention time on the formation of microalgal-bacteria aggregate (Buitron and Coronado-Apodaca, 2022) and the defence mechanism of microalgal-bacterial granules to tetracycline (Wang et al., 2020). Notably, these studies were prominently undertaken in temperate regions and conducted largely under controlled environment in photobioreactors and High-Rate Algae Pond. Moreso, there is a dearth of information on the inoculation ratio of microalgae and activated sludge for wastewater treatment. The conducted by Su et al. (2012) in Germany with defined inoculum ratio was carried out in a controlled environment. Although, temperature, pH and nutrients richness are also essential factors for microalgae growth, but the critical role light intensity plays in photosynthetic reaction; the process that generates organic compound required for biomass growth (Prasad et al., 2021; Dolganyuk et al., 2020; Coêlho et al., 2019), justifies the predetermined amount of light energy for cultivation. Because of the externality imposed by a controlled treatment environment, it becomes imperative to explore a more energy efficient method of treatment; evaluating the performance of Microalgae Activated Sludge (MAS) under a natural outdoor condition with solar radiation as the source of light. Solar radiation as the source of light intensity for photobioreactor treatment systems will be applicable in countries with tropical climatic conditions. This study therefore assessed the performance of the synergy of microalgae and activated sludge consortia under a natural outdoor working condition and light/dark cycle in a tropical environment in a small scale, to be used later in a pilot flat panel photobioreactor, and determine the experimental operations under outdoor conditions. Assessment will be based on rate of nutrients removal and recovery, settleability, removal potential of Escherichia coli and Total Coliforms, hydraulic retention time and the best inoculation ratio. This study is expected to contribute to the application of microalgae and activated sludge consortia in tropical regions for the treatment of municipal wastewater. 1.1 Justification of Study The Sub-Saharan Africa and Latin America and the Caribbean still grapple with the menace of municipal wastewater treatment. According to the joint monitoring program report of the World Health Organization and UNICEF, 2019 over 50% of wastewater are 7 discharged into the environment without any form of treatment in many parts of the regions. This poses a potential risk of increased public health burden that may arise from the consumption of water contaminated with pathogens and environmental pollution. Moreover, the widely practised conventional treatment technology lacks the capacity to stem the tide due to apparent disadvantages of high operation cost resulting from high energy consumption, resultant sludge production, inadequate removal and loss of nutrient. Although, the microalgal-based treatment technologies have been explored and proven to be an efficient alternative owing to comparatively low cost of operation, excellent nutrients removal and cycling, which conform with the principle of circular economy (Teixeira et al., 2021), but there exists limited information on the performance of photobioreactors under a natural tropical condition. Many of the microalgae-based studies so far conducted were carried out in the temperate region and under a controlled environment. Also, these studies have been widely demostrated at the laboratory scale (Min et al., 2022), which presents a question around the feasibility in real-life operationality. Based on the established robust potential benefits of microalgae-based treatment options, it is essential to optimise the treatment process and advance the recovery of accompanying high-value products, which are proven means of providing economically viable resources in an environmentally friendly manner, that supports the resilience of overburdening natural systems (World Bank, 2020; Chrispim et al., 2020). Therefore, it becomes imperative to evaluate the performance of microalgae- activated sludge synergy for the treatment of municipal wastewater in pilot flat panel photobioreactors, under a natural tropical outdoor environment for treatment and recovery nutrient rich biomass which could be used as inputs for industrial processes. This would offer the benefits of providing information on the experimental design for adoption in tropical regions and as well bridge the wide treatment gap, especially in the Sub-Saharan Africa, which has posed serious environmental and public health threats. This also translates into recovery of bioeconomy potential products such as nitrogen and phosphorus nutrients, which can alleviate the burden of food scarcity and inflation linked to dwindled fertilizer provision from supplying countries due to prevailing social crisis. 8 1.2 References Akpor, O. B. & Muchie, M., 2011. Environmental and public health implications of wastewater quality. African Journal of Biotechnology, 10 (13), pp. 2379-2387. https://doi.org/10.5897/AJB10.1797. Andrade, D. S., Amaral, H. F., Gavilanes, F. Z., Morioka, L. R. I., Nassar, J. M., de Melo, J. M., Silva, H. R. & Telles, T. S. (2021). Microalgae: Cultivation, Biotechnological, Environmental, and Agricultural Applications. In Maddela, N. 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Singerpore: Spinger https://doi.org/10.1007/978-981-13-2264-8. https://doi.org/10.1016/j.algal.2019.101672 https://doi.org/10.1016/j.biortech.2016.03.114 https://unesdoc.unesco.org/ark:/48223/pf0000247153?1=null&queryId=af4727d7-c9aa-4f55-8d94-94bee4dad76d https://unesdoc.unesco.org/ark:/48223/pf0000247153?1=null&queryId=af4727d7-c9aa-4f55-8d94-94bee4dad76d https://doi.org/10.1016/j.biortech.2020.123605 https://www.worldbank.org/en/topic/water/publication/wastewater-initiative https://apps.who.int/iris/handle/10665/329370 https://doi.org/10.1007/978-981-13-2264-8 14 2 OBJECTIVES The general of objective of this study was to investigate the best inoculum proportion of microalgal-activated sludge consortia in a flat panel photobioreactor for the treatment of anaerobic municipal wastewater, in terms of nutrients removal, pathogens removal, biomass productivity and excellent settling characteristics. 2.1 Specific Objectives The specific objectives are outlined as follows: • To determine the best inoculum proportion for microalgae and activated sludge consortia in a lab-scale under outdoor condition; • To investigate settleability of biofloc (microalgal-bacteria aggregate); • To investigate the Escherichia coli and Total coliforms removal potential of microalgae-activated sludge consortia. • To determine the best hydraulic retention time for removal and recovery of nutrients under outdoor condition. 2.2 Hypotheses • Increased biomass settleability will be achieved in photobioreactor with Microalgae- bacteria consortia. • Increased nutrients recovery rate will be in photobioreactor with Microalgae-bacteria consortia. • Photobioreactors with Microalgae-bacteria consortia will produce excellent sedimentation. 15 3 LITERATURE REVIEW 3.1 Introduction The increasing rise of human population and the accompanying behavioural change of civilization are translating to increase in wastewater generation, owing to increased anthropogenic activities bordering on agricultural practices, industrialization and urbanization, in pursuit of meeting insatiable needs (Gonçalves, et al., 2017). The resultant wastewater has been shown to contain high levels of personal care products, pesticides, industrial chemicals (Rizzo et al., 2018) and organic loads and nutrients, particularly ammonia, which are known deleterious potential when discharged into the environment without treatment (EPA, 2007 ; Rizzo et al., 2015). According to Gonçalves et al. (2017), discharge of untreated wastewater into the environment will lead to the enrichment of nitrogen and phosphorus of the receiving water bodies, and cause a condition that is known as eutrophication. This manifests varying consequences ranging from depletion of dissolved oxygen, death of aqua lives to degradation of the affected water body, and by extension poses threat to public health. Although, microalgae consortium and microalgal and activated sludge consortia have been adjudged efficient alternative to the conventional treatment methods, resulting from their metabolic flexibility and ensuing excellent treatment outcomes of wastewater from different sources (Wollmann et al., 2019), it is essential to examine the various secondary treatment technologies and varying microalgal treatment operating conditions, in relation to different environmental conditions. 3.2 Wastewater Characteristics According to Sperling (2007), the characteristic nature of wastewater is defined by the presence of some pollutants (suspended solids, biodegradable organic matter, nitrogen 16 and phosphorus nutrients, pathogenic organisms, pesticides, detergents and metals), which varies across different locations, based on the degrees of activities of the contributing settlements. In addition to the aforementioned, pollutants of emerging concerns pharmaceutical and personal care products are also commonly found in wastewater (Chen et al., 2022; Shi et al., 2021). Primarily, nutrients are unavoidable constituents of wastewater and are present in form of nitrogen and phosphorus. Nitrogen occurs in organic or inorganic forms, ammonium (NH4 +-N), nitrate (NO3 --N), and nitrite (NO2 --N), and the ammonium and nitrate being the most assimilable forms for microorganisms, while phosphorus is present in inorganic form, orthophosphate (PO4 3--P) (Li et al., 2022; Wang et al., 2021; Chen et al., 2022). The advent of circular economy initiative and the implementation in wastewater treatment process, using microalgae biomass recovery for example, has heralded quite a number of demonstrations to obtain the bioeconomic compounds present in wastewater such as nutrients, carbohydrate, protein etc., which also presents the prospect of environmental sustainability (Gao et al., 2021; Vargas-Estrada et al., 2021). Based on this, the looming exhaustion of phosphate rock, due to the rising demand for phosphorus, being the consequent effect of growing human population may be averted, exploring this environmentally friendly alternative of phosphorus recovery (Liu et al., 2021). Moreso, the dissolved organic matter in wastewater also serves as a source of energy for microalgae (Lyu et al., 2021). Biochemical oxygen demand, chemical oxygen demand and total organic carbon make up the organic matter component of wastewater, which is a prominent part of wastewater properties (Kim et al., 2022; Gao et al., 2020). Also, the recovery of micronutrient metals such as Copper (Cu), Cadmium (Cd), Manganese (Mn), Chromium (Cr), Iron (Fe), Nickel (Ni) and Zinc (Zn) in wastewater by microalgae is essential as they are easily replenished into the soil by using microalgae biomass as biofertilizer (Huang et al., 2922; Liu et al., 2021). In particular, the significant role of Zn in agriculture has been established (Muhammad et al., 2022), however, the bioremediating and nutrients recovery potential of microalgae can be affected by the coexistence of high Zn concentration and nutrients in culture broth (Oliveira et al., 2023). Therefore, it is essential to study the compromising influence of Zn on microalgae growth and the overall treatment performance of wastewater. 17 3.3 Anaerobic Sewage Treatment Systems According to de Oliveira et al. (2021), anaerobic treatment technology is one of the most common treatment process of wastewater. Particularly, this treatment method is used in tropical regions due to the favourable temperature. It is not suitable for the temperate regions owing to poor performance of anaerobic microorganisms at less than 20oC and the lack of feasibility to be heated (Chernicharo, 2007). Up-flow Anaerobic Sludge Blanket (UASB) is popular among the anaerobic treatment metheds (Chernicharo, 2007) and this may be attributable to the conservation potential of the nutrients by converting to stable form that is recoverable through post-treatment technology. Up-flow Anaerobic Sludge Blanket (UASB) reactor has a favourable temperature condition that makes it suitable for use in warm-climate condition (tropics), especially for the treatment of high organic loads wastewater from slaughter house, agricultural industry, food and beverage industry, urban settlements (Chernicharo, 2007). Hellal et al., (2021) reiterated that the operating temperature and condition of UASB makes it readily adoptable to the treatment of wastewater by tropical and sub-tropical countries and coupled with its cost effectiveness in terms of low operating cost and energy consumption, and biogas production which could serve as fuel. Furthermore, while it was shown that UASB can effectively treat biodegrade organic and ensure significant removal of BOD and COD, the need for a post-treatment with an additional treatment technology that is capable of removing nutrients (Nitrogen and Phosphorus), heavy metals, microorganisms and emerging contaminants, in compliance with stipulated standard was established (Hellal et al., 2021). In a bid to upscale the operation of UASB, de Oliveira et al. (2021) integrated micro aeration into the operation mechanism of UASB and increased organic matter removal and methane production was recorded due to enhanced hydrolysis of the organic that was attributed to the micro-aeration. However, this did not overrule the need for post-treatment owing to the presence of high concentration of nutrients in the effluent, which can serve as alternative source of fertilizer when recovered. On the other hand, aside the failings of aerated treatment technology highlighted above and the possibility of ecosystem chemical imbalance from inorganic chemical use, nutrients (phosphorus and nitrogen) are consumed during the treatment process, particularly phosphorus (Amenorfenyo et al., 2019). This presents a negative implication by reducing 18 the recovery rate of nutrients, loss of high-valued products and unsuitable for the circular economy model that prioritizes recovery of high-valued products and environmental sustainability. Removal of high amount of organic matter from wastewater by UASB than other anaerobic treatment technology is ascribed to high concentration of microorganisms, but it is restrained by the accumulation of Volatile Fatty Acids (VFA) (Hu et al., 2019). 3.4 Post-Treatment using Microalgae-Based Technologies Up-flow Anaerobic Sludge Blanket (UASB) reactor has demonstrated capacity as an excellent alternative to conventional Septic tank wastewater treatment due to significant removal of organic matter and suspended solids (Vassalle et al., 2020). It was further shown that effluent from UASB reactor is not suitable for direct discharge into the environment due to high concentration of nutrients (nitrogen and phosphorus), microorganisms and emerging pollutants, which makes post-treatment compelling, aimed at improving the quality of effluent. The poor removal of nutrients from anaerobic treatment (UASB) was linked to hydrolysis of organic nitrogen and phosphorus to ammonia and phosphate respectively, which are not removable from the system and as such become more concentrated (Khan et al., 2011). In this light, microalgae-based treatment technologies (open or closed system) were incorporated for the treatment of secondary effluent as they have shown to adequately remove the remaining contaminants. While this generates effluent that is fit for disposal into the environment without a damaging potential, it has paved room for circular economy through recovery of nutrients, carbohydrate, protein, lipids, cellulose carotenoids and reusability of generated water (Liu and Hong, 2021 ; Nagarajan et al., 2020; Speranza et al., 2022). Microalgae based technologies operate as a complete system, breaking down organic matter and consuming nutrients by photodegradation, bioadsorption and biodegradation, with self-replenishing gaseous exchange between photosynthetic microalgae and heterotrophic bacteria, generating oxygen and carbon dioxide respectively (Vassalle et al., 2020; Wieczorek et al., 2015). 19 3.5 Microalgae Cultivation A wide variety of culture methods is used to produce microalgae. Microalgae can be produced indoor where illumination, temperature, nutrient level, contamination are subject to control or under less predictable outdoor condition, and open culture systems like the High-Rate Algae Pond (HRAP) or tanks or closed culture systems like tubes, flasks, flat panels, or carboys (FAO, 1996). Juxtaposition of open and close photobioreactors showed that the former is prone to conditions that may undermine treatment efficiency when deployed under open roof and, which are (1) contamination with undesired microorganisms (2) dilution during heavy down pour (3) heat loss and evaporation and subsequent concentration of the effluent (Sepúlveda-Muñoz et al., 2020). Therefore, closed photobioreactor, by virtue of the design, enjoys a relatively high temperature condition that influence biomass growth and removal of organic matter and nutrients in return (Sirohi et al. 2022). However, temperature for optimal growth and performance in photobioreactors is specie specific, but it is worth mentioning that the microalgae have the capability to adapt after a lag phase, which also varies, before commencing treatment operation (Sirohi et al., 2022; Sepúlveda-Muñoz et al., 2020). The pH condition of both open and closed photobioreactors tends towards alkaline due to the disintegration of volatile fatty acid and consumption of carbon dioxide, and it is relatively higher in the open system resulting from readily diffusion of carbon dioxide into the atmosphere (Sepúlveda-Muñoz et al., 2020). Hence, the limitation of carbon source and pH variability can interfer with photosynthesis and subequently limit microalgae cell growth in both photobioreactors and with CO2 supplementation (Fernández et al., 2012). However, a closed photobioreactor of microalgae culture could accumulated dissolved oxygen when may inhibit microalgae growth at > 250% air saturation (Solimeno et al., 2017). Notably, penetration of light either from solar or artificial source, usually fluorescent lamps, may be minimized by microalgae fouling in closed photobioreactors by sticking to light receiving surfaces (Sirohi et al., 2022). For efficiency in terms of biomass production, gas exchange and energy transfer, wastewater treatment and nutrients removal, high light receiving surface to volume ratio is essential (Lam et al., 2019). The cubical shape and depth of flat-panel reduces the extent of light penetration, covering a short distance, but the translucent nature on both sides of the flat-panel compensate for it (Sirobi et al., 2022). 20 Considering the inhibitory impact of shadow by reducing the intensity of light, spacing range of 0.2 m to 0.4 m between flat-panels was recommended (Huang et al., 2019). Importantly, culturing microalgae in high-rate wastewater as substrate will lead to high yield of biomass and nutrients recovery, which will produce high value-added end product, usable for soil conditioning (Silver et al., 2019). 3.6 Environmental Factors Affecting Microalgae Cultivation The environmental factors are critical to microalgae growth process and biomass productivity, hence the need for a review of the factors in order to identify means to optimise the influence of the factors on microalgae growth. 3.6.1 Light Intensity Light, as the main source of energy input, plays a crucial role in microalgae cultivation, and has been shown to have a proportional impact on microalgae growth rate (Chowdury et al., 2020). In other words, increasing light intensity influences photosynthetic activities of microalgae which invariably increases microalgae productivity (Richmond, 2004; Chisti, 2007; Rai & Gupta, 2017). However, there are certain levels of intensity, although different across specie line, microalgae growth may be inhibited as due to exceedance of the maximum saturation level (Vejrazka, 2011; Vejrazka, 2012; Acien Fernandez et al., 2013). Also, light saturation requirement of a particular microalgae strain is location specific, considering the influence of environmental ambience on irradiance level, and as such location specific evaluation should be carried out for actual level of light requirement (Chowdury et al., 2020). Besides the influencing impact of microalgae culturing ambience, exposure quality in terms of wavelength and source of light and quantity in terms of light intensity and duration of photoperiod are contributory to influencing microalgae growth rate (Schulze et al., 2014). The light source can either be natural or artificial and the photosynthetically active radiation usable for microalgae growth ranged from 400 to 700 nm (Scott et al., 2010). The artificial light from light emitting diode (LED) and fluorescent lamp sources have been shown to enhance microalgae biomass production (Chowdury et al., 2020). From previous explorative studies of light intensity’ influence of microalgae growth, the optimum intensity that fostered microalgae growth ranged from 26 to 400 µmol photon m2 s-1, with only a few species such as Dunaliella salina, displaying the trait of 21 photoprotection mechanism for surviving under light intensity of 1000 µmol photon m2 s-1 (Maltsev et al., 2021). Additionally, lipid synthesis could be induced by varying the intensity of light in relation to the light requirement of the microalgae specie in use, with exposure to a range of 60 to 700 µmol photon m2 s-1 (Maltsev et al., 2021). It is essential to determine the optimum working light intensity for the microalgae specie to be used for treatment purpose, especially when dealing with a mix community of species, to achieve a desirable outcome. 3.6.2 Temperature Alongside light intensity, temperature is considered an environmental factor critical to microalgae growth. Usually, light irradiation confers a level of temperature increment within a photobioreactor, which jointly influence biomass productivity (Deb et al., 2017). However, this can as well cause overheating of in the reactor which may pose a negative implication to microalgae cells, although more preponderant in large scale reactors, as the lab-scale is usually compensated by convection with ambient air (Chowdury et al., 2020). As a form of solution, dark-coloured netted shading, water sprinkling, partial water immersion of reactors and heat exchange have been recommended in places with low air humidity to whittle the possibility of reactor overheating (Becker, 1994; Torzillo, 1997; Watananbe et al., 2011). However, the tendency of net shading to over protect and reduce light irradiance was noted, which was implicated for reducing biomass productivity. On the other hand, water sprinkling guarantees good biomass productivity but imposes cost implications on microalgae cultivation (Chowdury et al., 2020). Notably, microalgae grow significantly at the optimum temperature which ranged between 20 to 35°C, while the mesophilic species can thrive at 40°C (Chowdury et al., 2020). Therefore, this implies that microalgae culture may underperform below the optimum temperature range while the microalgae cells may be lysed at high temperature, leading to cell death (Sanchez et al., 2008; Bernard & Fernandez, 2012). 3.6.3 Nutrients Nutrients are vital to microalgae growth and must be available is sufficient amount in culture medium for biomass productivity. Nutrients are presented largely in form of carbon, nitrogen and phosphorus, and metals such as iron, zinc etc., in wastewater culture medium with the proportion of carbon being the highest, as the microalgae biomass has 22 shown to contain about 50% of carbon (Richmond, 2004; Acien Fernandez et al., 2013). Culture medium can be enriched with inorganic compounds such as CO2 and bicarbonates (HCO3 −) or organic compounds such as sugars, acids and alcohols as sources of carbon (Acien Fernandez et al., 2013) while nitrogen is presented in form of proteins, nucleic acids, vitamins as assimilable as NH4 +-N , NO3 −-N, NO2 −-N (Perez-Garcia et al., 2011), and phosphorus is presented in polyphosphate, pyrophosphate, orthophosphate, and metaphosphate (Markou et al., 2014) for microalgae uptake and growth. The proportionate ration of these nutrients, particularly nitrogen and phosphorus for optimum microalgae growth differs for different microalgae species (Silva et al., 2015), and must be evaluated to achieve the desire productivity outcome. 3.6.4 Value of pH The influence of pH on microalgae culture is manifest in microalgae growth, dissolution of minerals and carbon dioxide (CO2) (Qui et al., 2017), and ranged from 6.0 to 8.0 on the average for microalgae (Rai & Gupta, 2017; Zhu, 2015), although some species have been noted to thrive at high alkaline pH of 10.0 and some at acidic pH level of 3.0 (Lu et al., 2014). pH tolerance level differs for different microalgae species and respective pH level in cultures is determined by buffering capacity, CO2 dissolution, temperature and metabolic activities (Singh & Dhar, 2011). Also, the pH of microalgae culture increases during the fixation of CO2 by photosynthesis due to the release of hydroxide ion (OH-) and subsequent accumulation (Richmond, 2004). 3.7 Bioeconomy and Circular Economy This is an emerging concept that is engineered towards the recovery of materials of economic value from products intended for disposal, and as such advancing the re-use and recycle prevention protocols, and by extension, reducing over reliance and exploitation on finite natural capital, which in turns promotes environmental sustainability. Puyol et al. (2017) provided a detailed account of the valued end products, among of which are, nutrients (phosphorus and nitrogen), biofuels (methane, diesel) and trace metals, that are derivable from waste products, with corresponding treatment technologies, and microalgae-based treatment technology was ranked among the most prominent and growing options. The nitrogen and phosphorus content of municipal wastewater, excluding waste from animal production, can potentially cater for 20% of global nitrogen and phosphorus needs, 23 which really represents a sustainable alternative source of supplies. This was sustained in Kok et al. (2018) indicating the potential of 20% global agricultural needs from the nutrients load of urban wastewater and a humongous proportion, 90%, derivable from animal manure. Harnessing this resource from wastewater would prevent over stretching the finite natural phosphate rock which is projected to go out of supply in 50 to 400 years, and readily provide alternative means of fertilizer, particularly for the Sub-Saharan Africa, and other regions with known deficiency soil fertility (Kok et al., 2018). A possibility of 100% recovery of nutrients (phosphorus and nitrogen) and significant proportions of other high-valued products (carbon, potassium, copper, cobalt, zinc, iron, polysaturated acids, pigments, carotenoids etc.) from wastewater was shown through microalgae assimilation (Fernandes et al., 2022). This further gives credence to microalgae-based treatment technology. Also in the same vein, Silva et al (2019) corroborated the high-rate removal potential of micronutrients and macronutrients by microalgae, making it suitable for agricultural soil conditioning and improved soil productivity, and by extension this will reduce the environmental impacts that accompany extraction of phosphate rock. 3.8 Microalgae Biomass Recovery Microalgae treatment efficiency is detailed in extant literatures (Molinuevo-Salces et al., 2019; Quijano et al., 2017). The drawback of microalgae axenically for wastewater treatment is linked to poor biomass settling which is due to negatively charged of the micro cells and preventing coalescing into sizes large enough to settle under gravity (Yan et al., 2022; Molinuevo-Salces et al., 2019). To overcome this challenge, filtration, centrifugation and chemical coagulation are deployed industrially (Fasaei et al., 2018), while nature-based coagulants are being explored to reduce cytotoxicity (Teixeira et al., 2022; Silva et al., 2020), but these methods come with the implications of high energy consumption and high cost of implementation (equipment, maintenance, operation, personnel). However, the symbiosis of microalgae and activated sludge bacteria presents energy efficient biomass recovery option, which is also accompanied with improved purification and biomass production (Yan et al., 2022). Chemical exchanges were shown to be parts of the symbiotic interaction, which have been very beneficial to both microalgae and bacteria, and ultimately producing good quality outcome (Yan et al., 2022). Indole-3-acetic acid 24 (IAA) and N-acyl-homoserine lactones (AHLs) chemicals produced by bacteria spike microalgae growth and gaseous exchange alternatively improve the activities of microalgae and bacteria, with increased biomass production (Chan et al., 2022; Barreiro-Vescovo et al., 2021). Aside the increased biomass production induced by the symbiotic interaction of microalgae and bacteria, which is put at 10 to 70%, substantial amount of extracellular polymeric substances (EPS) is secreted by bacteria and cause the formation of aggregates (bioflocs) that are large enough to settle out of gravity, with efficient settling rate (95–99%) and high biomass yield (Zhang et al., 2021), providing and the economic value of the high- valued products (biodiesel, feed, fertilizer, and cosmetics). The EPS contains proteins and polysaccharides and are produced under reflex conditions of environmental stress and also without a precursor, which are produced in significant concentration to initiate the agglomeration of cells and subsequent settling (Wang et al., 2022). 3.9 Biomass and Agro-Industrial Applications In general microalgae biomass has a number of potential uses in various industries such as biofuel production, bioplastics, cosmetics, animal feeds, etc. Wastewaters of various origins often contain abundant energy and nutrient sources that can be recovered and utilized within a circular bioeconomy standpoint, although, not usable in cosmetics and animal feeds due to probable presence of toxic substances. Microalgae cultivation can aid the recovery and reuse of recyclable nutrients from secondary sources, contributing significantly to global sustainable demands (Nagarajan et al., 2020). Microalgae serve as a rich source of lipids, proteins, carbohydrates, pigments, nutrients, cellulose, carotenoids which are synthetically processed and converted into the various agro-industrial uses (Fig. 1). These resources embedded in the cells of the microalgae biomass, and as a result, they are dewatered and dried after sedimentation, to obtain the bioproducts for their respective uses (Speranza et al., 2022). 3.9.1 Biofuel Production Microalgae is a third-generation source of biofuels (Noraini et al., 2014) and it guarantees clean energy production and promotes environmental conservation, with significant reduction in greenhouse gas emission and reduced air pollution problems (Gavilanes et al., 2017; Peng et al., 2020). The first-generation (edible materials like corn, 25 palm, soybeans, beets, sugarcane, etc.) and second-generation (lignocellulosic material like jatropha and grass) sources were remarkedly disadvantaged for low lipid yields, low growth rate, low CO2 mitigation potential, low tolerance to wastewater, large expanse of land (Noraini et al., 2014; Bennion et al., 2015; Bharadwaj et al., 2020; Goh et al. 2019; Yin et al. 2020). Based on the potential of microalgae biomass, they are used as feedstock for the production of various biofuels such as biodiesel, bioethanol, biohydrogen and biogas. Although, the best production route for optimum yield is yet to be defined, anaerobic digestion has shown to be the least complex, but microalgae strain is an essential factor that determines productivity (González-Fernández et al. 2012; Ayala-Parra et al. 2017). Moreover, the pH and temperature of substrate in anaerobic digestion process influence the production of biogas (methane) (Chu and Phang 2019). The alkaline range produces highly pure biogas due to the solubility of CO2 from biogas as dissolved carbonate. High microalgae biomass with high content of lipid within the cells increases the oil production strength of microalgae (Chisti, 2007). By dry weight, the lipid contents of microalgae biomass ranged between 2% and 41% (Gouveia et al. 2008), and they can be transformed into fatty acids methyl esters which serves as a feedstock for the production of biodiesel (Soares et al. 2019). However, if the proportion of free fatty acids > 5%, although often constitutes about 85% of lipid content depending on microalgae strain and condition of cultivation (Chen et al. 2012; Krohn et al. 2011), triggers saponification and renders it unsuitable for biodiesel (Huang et al. 2010). As a solution, two steps treatment processes are advised, first is to convert of free fatty acids to fatty acids methyl esters with methanol and followed with transesterification (Dong et al., 2013). Another biofuel derivative is bioethanol. Bioethanol production from microalgae biomass is specie specific, and the species that produce high level of carbohydrates as reserve polymers instead of lipids are considered the ideal candidates (Andrade et al., 2020), as they can be extracted to produce fermentable sugars, to obtain bioethanol (Mussatto et al. 2010). Some of the highlighted species that stand out for this purpose are Sargassum fulvellum, Hizikia fusiformis, Chlorella vulgaris, Chlorella sorokiniana, etc. (Andrade et al., 2020). 26 3.9.2 Biofertilizer Microalgae biomass as a source of fertilizer serves an environmentally friendly alternative for producing healthy foods in a sustainable manner by the release of nutrients for plant uptake from biomass, and potentially reducing the implication of sole reliance on chemical fertilizer (Singh t al., 2011). Additionally, Rachidi et al. (2020) showed that the metabolites such as phytohormones, polysaccharides, amino acids from green microalgae and cyanobacteria provides the benefits of high agricultural yields with nutritional values. Also, micronutrients such as zinc, copper manganese, iron from green microalgae and cyanobacteria enrich food crops, and are crucial for the nourishment of humans and animals (Renuka et al., 2018). The potential of microalgae-based biofertilizer for high agricultural yields have been demonstrated for different food crops. High maize yields with high nutritional content were reported by Dineshkumar et al. (2019), celery plant (Rashad et al., 2019) and rice cultivation (Chittapun et al., 2018). Specifically, biomass Acutodesmus dimorphus microalgae specie have the potential of increased productivity in Roma tomato plant (Garcia-Gonzalez & Sommerfeld, 2016). In a comparative study, utilizing microalgae-based biofertilizer in crop production has yielded results that are similar to those of commercial treatment (Coppens et al., 2016). According to Renuka et al. (2016), reliance on agrochemicals can be reduced by 75% with the use of microalgae-based biofertilizer. If the production of microalgae biomass is fully optimised for increased productivity, for application as biofertilizer, it will reduce excessive use of agrochemical, promotes sustainable crop production and minimise associated environmental degradation (Abideen et al., 2022). 3.9.3 Bioplastics The popularity of bioplastics as an alternative to the synthetic plastic from fossil fuel is due to environmental pollution, high carbon footprint, non-degradability, toxicity ascribed to the later (Cinar et al., 2020). As at 2019, production of bioplastics stood at 1% of 2.11 MMt global production (European Bioplastics 2019; Payne et al. 2019). This is still at the infancy stage with a huge gap, and more work is needed to be done to match production demand from the fast-rising human population. It is important to decrease the production of 27 fossil fuel-based plastics as this will contribute towards ensuring environmental sustainability. Among the bioplastic sources, microalgae appear to be the most preferred because of the advantages of less space for cultivation, which does not compete for land with food crop, growing on wastewater, high lipid yields (Rahman & Miller, 2017; Hempel et al., 2011). Moreso, microalgae-based bioplastics showed higher mechanical strength (stress and elongation) when compared with the conventional synthetic plastics (Abdo & Ali, 2019). However, the performance of different strains of microalgae for use as feed for bioplastics may differ in terms of biomass yields and accumulation of required high-valued products such as lipids, carbohydrate and polyhydroxybutrate (Abdo & Ali, 2019). The feasibility of producing bioplastic from microalgae biomass, using the high- valued products derivatives such as lipids, cellulose, carbohydrate, protein polyhydroxybutrate was established in (Khoo et al., 2019). However, most of the studies so far conducted were at the lab scale, and therefore, the challenge of the economic viability bordering on large scale cultivation, harvesting, extraction and pre-treatment needs to be explored for the establishment of a promising pathway. In the light of this, microalgae biofilm technology, which is still at the infancy stage, is proposed owing to the advantages of low cost of production, low consumption of energy for harvesting and increased productivity over cultivation ponds (Chong et al., 2022). 3.9.4 Cosmetics There are several bioactive compounds from microalgae and cyanobacteria biomass such as Polysaccharides, Methanolic extracts of exopolysaccharides, Chrysolaminarin, Sulfated polysaccharides, ß-1,3-Glucan, ß-carotenes, Asthaxanthin, Phycocyanobilin phycoerythrobilin, β-Cryptoxanthin, Chlorophyll, etc., that have potential uses for cosmetic products (Mourelle et al., 2017). This is due to their pharmacological activities capable of protecting and improving the appearance of human skin and hair (Mourelle et al., 2015; De Luca et al., 2021). In more specific terms, these bioactive compounds have both protective and healing potentials, correcting dermatitis (dandruff), preventing inflammation, external aging, and are moisturizing (Kim et al., 2008), but wastewater cultivated microalgae may not be applicable for this purpose. 28 Additionally, polysaccharides obtained from microalgae and cyanobacteria have potential for free-radical collection and immunostimulaton, which make them a good fit for use in skincare cosmetics (Koller et al., 2014), and offers the benefits to stand in gap, protecting the skin against oxidative stress and damaging free-radicals due to compromising susceptibility of the skin’s natural antioxidant system to external factors like UV from sunlight (Mourelle et al., 2017). Also, carotenoids, one of the bioactive substances from microalgae biomass have shown numerous skincare potentials such as photoprotection, antiallergen, antioxidant, etc. (Heydarizadeh et al., 2013; Rumin et al., 2020). The interlinking beneficial effects of skin care products and formulations containing these bioactive compounds, which cut across pharmaceuticals and cosmetics, with medical and drug-like properties has led to the creation of cosmeceuticals in skincare parlance (De Luca et al., 2021). This reflects the appreciation of the benefits in the advancement of the cosmetics industry and as shown by Sathasivam et al. (2018), the naturally formulated cosmetic products enjoy more preference by consumers. This portends a fast-rising global demand and more research on how to optimize production and match demand should be conducted. 3.9.5 Animal feeds Microalgae biomass have demonstrated the potential to sustainably provide livestock, poultry and aquaculture animals (Viegas et al., 2021). Microalgae grown in municipal wastewater can as well be used for this purpose considering the richness in protein, carbohydrates, lipids, carotenoids and microelements (Saadaoui et al., 2021). However, the biomass to be used for this purpose must be sure to be free from toxins and pathogens (Ahmad et al., 2022). Moreso, biomass harvested with the aid of chemical coagulants may not be suitable for this purpose, as the presence of chemical residue reduces the quality of biomass, rendering it toxic (Silver et al., 2020). The nutritional benefits of microalgae biomass are evident in the sound physiological functioning, optimum growth and good health, and the overall product quality of animals fed with a blend of small portion of commercial feed with Chlorella, Scenedesmus and Arthrospira microalgae (Kotrbáček et al., 2015). This provides a basis for the increasing interest in microalgae feed-supplements for animals in countries like United State of America, India, Japan, China, Korea, Thailand, United Kingdom, Philippines, with the Asian countries taking the lead (Saadaoui et al., 2021). As a matter of fact, 30% of microalgae 29 biomass production is currently being used as animal feed-supplement globally (Becker, 2007; Dineshbabu et al., 2019). Notably, accruable nutritional benefits from microalgae biomass differ across specie line, due to differences in growth conditions such as illumination, temperature, and location, etc. (Saadaoui et al., 2021). The understanding of the cultivation condition will aid the optimization growth process, to achieve a sustainable animal feed alternative that is economically viable. Figure 1: Agro-industrial application potential of microalgae biomass from wastewater 30