o Data matching to outdoor high rate algal ponds and valorisation of the biomass has yet to be investigated, before the stage is set for the industrial implementation of MaB-floc ponds. o Further research is needed to clarify whether flue gas sparging inhibits bacterial ammonium oxidation, enhances microalgal nitrate uptake and/or decreases the ammonium inhibition of nitrate uptake in light conditions. o Bioflocculation can lower the biomass harvesting costs by providing a nearly cost-free first harvesting step, as only (a short) time is the prerequisite. The need and choice of a second harvesting step, such as centrifugation, will depend on the application and composition of the biomass. o More accurate methods for the determination of microalgae and bacteria are needed, especially in mixed and flocculating biomass samples. - Van Den Hende et al. 2014: industrial w.w. in sequencing batch reactors. o Phosphorous was limiting the nitrogen removal in manure treatment wastewater. The addition of dark-coloured CAS influent to light-coloured CAS effluent of the manure treatment could enhance the TN:TP ratio, but will also lead to a darker wastewater colour, thereby affecting autotrophic microalgae growth. A balanced mixture for manure treatment wastewaters needs to be determined. o Further research is needed to confirm whether this was because UASB sludge particles were removed during settling in the SBR but not oxidised during BOD5 determination, and/or photosynthesis-induced metabolism occurred which resulted in some surplus TCOD that was vulnerable to biologic degradation. o On average more TN was removed compared to TC, as can be seen from the low TC removal rate: TN removal rate ratio. Further research is needed to confirm whether this is due to
ammonia volatilisation at a pH level of above 9 or
denitrification during anoxic/anaerobic dark periods. o It should be verified if discharge norms can be met for the nutrient-rich wastewater of the food-processing industry by increasing the HRT. - Van Den Hende et al. 2014: up-scaling o This means that for each mol TN removed, 1.52 1.43 mol TC and 2.38 1.33 mol TOC was removed. The latter values are low compared to the TC:TN ratio of micro-organisms of approximately 6 (Geider and La Roche, 2002). This suggests that the N removal could not be due to biomass growth only. More research is needed to confirm whether denitrification in anoxic microniches of the flocs, as in activated sludge was an additional mechanism for N removal. o This means that not all C and N removal could have been due to biomass growth. More research is needed to confirm which of the following removal mechanisms play a role in this: TN removal via denitrification, VSS removal by predators, TC removal via CO2 emission to air and CaCO3 precipitation. o The nitrate concentration of the effluent (55.0 8.60 mg N NO3 L1) was largely above the current discharge norm of 33.9 mg NNO3 L1. The question remains whether this was due to nitrification, excretions by MaB-floc predators and/or lysis of organic compounds catalyzed by enzymes excreted by cyanobacteria, such as protease and urease. o The supersaturated DO values up to 24 mg L1 (Fig. 5) demonstrate photosynthetic aeration by MaBflocs and BOD5 underloading of 12M. These supersatured DO values can be toxic for microalgae (Richmond, 2004). Therefore, during further optimisation research should investigate the potential of increased BOD5 loadings on the reactor performance, for example by altering the influent pre-treatment step. o Negative NO2 REs were obtained, despite the aerobic conditions needed for nitrification. Further optimization to lower the NO2 concentrations is crucial. Since the NO2
concentration in the indoor fish tanks was lower than 1 mg L1,
the NO2 must have increased during storage in the influent buffer tanks. Therefore a possible strategy to enhance the NO2 of the effluent would be to decrease the HRT of the influent buffer tanks by discontinuous feeding of the MaB-floc SBR during the day. o Conclusion: Firstly, up-scaling to outdoor conditions strongly increased the effluent pH. Outdoors, flue gas sparging was needed to obtain an effluent pH below the discharge norm. Despite being advantageous in terms of greenhouse gas mitigation, flue gas sparging in wastewater treatment raceways represents an extra cost (up to 20% of the capital expenditures; Zamalloa et al., 2011 ). In certain countries, such as Belgium, this cost cannot be compensated by revenues for CO2 credits because no such credits can be obtained for flue gas scrubbing in open ponds. Third, up-scaling increased the HRT to 4 days. This means that per m3 indoor fish tank, a microalgae raceway pond area of 1 m2 would be needed (daily discharge of 10% of the fish tank water). To overcome potential problems of regarding shortage of availability of land, raceway ponds on the roof of the indoor aquaculture facility could be a worthwhile option to investigate. If year round wastewater treatment is targeted, pond heating with waste heat will be needed. o Microscopic observations showed that a large amount of crystals were present in the MaB-flocs (Fig. A1). Further research is needed to confirm the nature of these crystals. o Future research should focus on nitrogen removal and biomass valorisation. - Sutherland et al. 2015: enhancing microalgae o Organic nutrient loading rate, hydraulic retention time, pond depth, CO2 supply, as well as vertical and horizontal mixing
velocities are the main operational parameters that can be
modified in a HRAP. In particular, given that reported photosynthetic efficiency is, at best, only 1/5 of the theoretical maximum, further research is necessary to understand how the operational parameters impact on the physiology, photosynthetic capability and productivity of HRAP microalgae. Mixing frequency, as well as the mixing velocity, is likely to play a critical role for maintaining desirable large colonies in HRAPs. Understanding how the frequency of mixing events affects the performance of microalgae, including photosynthesis, productivity, nutrient removal efficiency, as well as physiological and morphological adaptations, is important for enhancing wastewater treatment and biomass yields. Under medium frequency light fluctuations (ranging from seconds to minutes), such as those expected in fullscale wastewater HRAPs, the effects of enhanced vertical mixing on photosynthesis and productivity have been less conclusive. While the effects of HRT and nutrient loads have been investigated with respect to nutrient removal and biomass production, their effects on microalgal physiology have not been investigated, to date. Increased HRT would lead to increased biomass concentration and therefore reduced pond light climate. This may negatively impact on light absorption, photosynthesis and ultimately biomass production as microalgae become low light adapted. o Available land is often limited in most countries and operating sufficient numbers of HRAPs at the shallowest pond depth may not be economically, or socially, viable. In order to reduce their footprint, wastewater HRAP systems have been designed to retrofit into existing facultative pond systems. There is a need to understand the performance of HRAPs with respect to optimizing microalgal photosynthesis, nutrient removal
efficiency and areal productivity and the trade-offs with
increasing pond depth. o While there have been some mesocosm and pilot-scale studies evaluating ways to increase both light and carbon in wastewater HRAPs (as discussed above), there has been a lack of research in this regard at full-scale, to date. Research at full-scale on both improved light and carbon availability should be the main focus of future studies. Full-scale research would help to answer the question of whether implementing operational modifications can be done cost-effectively. CO2 addition and enhanced mixing, while identified as beneficial to microalgal productivity at pilot-scale, could substantially increase capital and operational costs and the benefits, in terms of biomass yield, may not out way the costs unless improvements in these technologies are made. - Su et al. 2012: algae/bacteria ratios. o Some algae-based bio-systems were used to treat highly concentrated municipal wastewater and the corresponding COD was removed from initial 2500 mg/l to around 250 mg/l (Zhou et al., 2011; Li et al., 2011). The performance of the algae sludge system proposed in this study for high concentration wastewater treatment could be our further research goal. - Park et al. 2011: wastewater treatment HRAPs for biofuel o Since wastewater treatment HRAP systems are already an economically viable technology for efficient tertiary-level wastewater treatment, they could potentially be the testing ground to develope large-scale algal production, harvest and biofuel conversion technologies that will be latter used when higher fossil fuel costs make purpose built algal biofuel production systems economical. Therefore, both fundamental and field-scale research is urgently needed to optimise algal production and harvest from wastewater treatment HRAPs, however for these systems this must be achieved while maintaining high effluent water quality.
o Further research is required in large-scale wastewater treatment
HRAPs using cheap/free CO2 sources (flue gas or biogas) to minimize operational costs. o The influence of zooplankton grazing and parasitism (fungal and viral) on HRAP wastewater treatment and algal production requires further study. A greater understanding of how these organisms interact with HRAP algae may lead to the development of effective control methods. o The physiological properties of colonial algae and exact mechanisms of the aggregation have yet to be determined. - Garcia et al. 2000: HRAP for nitrogen removal. o From an engineering point of view, the optimal size of a HRAP for nitrogen removal requires a mathematical modeling that correlates effluent TN with influent TN, through environmental conditions and HRT. The design models normally available are based on BOD5 loading; a further refinement should be made to include nitrogen removal processes, as indicated in this study.