Productivity and competitive dominance of freshwater filamentous macroalgal cultivars for nutrient bioremediation of primary municipal wastewater

High-rate algal ponds (HRAPs) are common outdoor pond systems utilised to improve municipal wastewater treatment (Craggs et al. 2014; Leong et al. 2021). These shallow, mixed raceway-based systems are designed to maximise algal biomass growth, microbial activity, and wastewater treatment efficiencies (Young et al. 2017; Saravanan et al. 2021; Oruganti et al. 2022). Algal–bacterial symbiosis occurs in HRAPs as algae utilise CO₂ for photosynthesis, assimilate nutrients into biomass, and release oxygen, which is utilised by bacteria for oxidising organic matter and ammonia (Oruganti et al. 2022). HRAPs were originally designed for microalgal wastewater treatment (Nurdogan & Oswald 1995). However, a major operational issue with HRAPs is the challenge and cost associated with harvesting microalgal biomass (Singh & Patidar 2018; Lane 2022). While much research has focused on ways to improve microalgal harvestability (Park et al. 2013), the cultivation of filamentous freshwater macroalgae in these systems has been proposed as an alternative solution (Liu et al. 2020).

The use of freshwater filamentous macroalgal monocultures for wastewater treatment in HRAPs is a developing area of research (Liu et al. 2020). Monocultures in HRAPs are often preferred to bicultures and polycultures as they may offer a greater ability to control and predict treatment performance through more consistent nutrient removal rates and biomass yields with more uniform biochemical composition (Liu et al. 2020; Sutherland & Ralph 2020). However, full-scale implementation of algal-based monoculture wastewater treatment systems can be challenging as large, outdoor pond systems are susceptible to contamination by wild algal strains (Newby et al. 2016). Therefore, target cultivars must achieve higher productivity compared to undesired species (Liu et al. 2020) to remain unialgal and thereby maintain consistent nutrient removal rates and prevent variation in biomass composition (Sutherland & Ralph 2020). Furthermore, municipal wastewater is highly variable, as unknown pollutants within the influent can potentially lead to cultivar die-off (Petrie et al. 2015). Therefore, target cultivars must be highly tolerant and able to withstand diurnal fluctuations in municipal wastewater composition (Gao et al. 2023). Hence, cultivar resilience is critical to the successful operation and stability of full-scale algal-based monoculture systems. Cultivar selection processes therefore must include trials in open outdoor pond mesocosms to identify cultivars that remain productive and are competitively dominant when exposed to invasion pressure and outdoor environmental conditions (Nalley et al. 2014).

To date, two studies have measured the competitive dominance of filamentous algae. Oedogonium was highly dominant compared to Cladophora and Spirogyra when grown in outdoor free-floating (tumble) bicultures and mixed polycultures (Lawton et al. 2013). Specific growth rates of Oedogonium were also higher in mixed cultures compared to monoculture, demonstrating that while fast growth rates are expected to provide a competitive advantage (Borowitzka 1992), monoculture growth rates are not an accurate measure of a target cultivar's competitive ability in the presence of other cultivars (Lawton et al. 2013). Oedogonium (tropical strain), Stigeoclonium, and Hyalotheca (both temperate strains) were also compared in competition experiments under typical seasonal conditions in the laboratory (Valero-Rodriguez et al. 2020). Growth of cultivars was highest under seasonal conditions corresponding to environments where cultivars originated, with Oedogonium dominant under warmer conditions, while Stigeoclonium was dominant under cooler conditions (Valero-Rodriguez et al. 2020). Based on these results, it was concluded that stable large open culture systems could be maintained through a seasonal rotation of monocultures, or continuous cultivation of bicultures of the most dominant cultivars (Valero-Rodriguez et al. 2020). However, establishing a dominant monoculture might be possible by selecting local cultivars that have been well-adapted to local climatic conditions (de Paula Silva et al. 2012; Bao et al. 2022). Although these studies have highlighted the importance of conducting competition experiments in the preliminary stages of cultivar selection for monoculture cultivation, they were conducted on a small scale (1 L bottles–20 L buckets) and algae were grown in dechlorinated tap water enriched with nutrient media. Therefore, cultivar growth and response to competition may vary when cultivated in municipal wastewater within a large-scale, open outdoor pond system such as a HRAP. Unlike most laboratory settings, HRAPs are constantly varying environments with limited control over environmental factors and constant exposure to potential contamination by wild algal strains, which can enter by wind or through the inflow of wastewater (Borowitzka & Moheimani 2013). Therefore, measuring target cultivar dominance and performance within an outdoor system is essential for maintaining an effective and long-lasting HRAP monoculture (Shukla et al. 2018).

Cultivar selection is essential for optimising algal bioremediation performance in HRAP systems, making it necessary to understand how different cultivars perform within these pond systems. While existing studies suggest that monocultures can provide consistent nutrient removal (Valero-Rodriguez et al. 2020; Novak et al. 2024), there is limited research on their effectiveness in open outdoor HRAP systems, particularly concerning how target cultivars tolerate fluctuating conditions when cultivated in primary wastewater. This study aims to address these gaps by assessing the competitive dominance and resilience of various algal cultivars in outdoor high-rate filamentous algal pond (HRFAP) monocultures, with a focus on their ability to maintain effective nutrient removal under variable conditions. This study builds on recent research that developed a screening protocol to select cultivars of freshwater filamentous macroalgae for nutrient bioremediation of primary municipal wastewater (Novak et al. 2024). Based on that protocol, three freshwater filamentous cultivars – Klebsormidium flaccidum, Oedogonium calcareum, and Oedogonium sp. – were identified as potential target cultivars for primary municipal wastewater treatment in HRFAP monocultures due to their competitive dominance, high biomass productivity and bioremediation performance under local seasonal and extreme conditions (Novak et al. 2024). The objective of this study was to further quantify the growth, bioremediation performance, and competitive dominance of these target cultivars treating primary wastewater in outdoor HRFAP systems. The specific aims of this study were (i) to compare cultivar growth and nutrient removal rates when cultivated in outdoor HRFAP monocultures and (ii) to compare the dominance of cultivars when cultivated in outdoor HRFAP bicultures.

Experiments were conducted at the Te Puke municipal wastewater treatment plant (WWTP), located in the Bay of Plenty Region of New Zealand. The Te Puke municipal WWTP currently services a population of approximately 8,100 people and treats an annual average daily flow of 1,800 m³ day⁻¹. The municipal WWTP primary wastewater used throughout this experiment was taken after solids sedimentation. The average water quality variables of the primary treated wastewater were: 106 mg L⁻¹ total suspended solids (TSS), 177 mg L⁻¹ carbonaceous biological oxygen demand (cBOD₅), 43.0 mg L⁻¹ total ammoniacal-N (TAN), 1.1 mg L⁻¹ nitrate-N (⁠N), and 4.5 mg L⁻¹ dissolved reactive phosphorus (DRP). Prior to experiments, all cultivars were acclimated to primary wastewater by initially growing them at the WWTP for 4 days in primary wastewater that had been diluted 1:1 with dechlorinated tap water and then for a further 4 days in 100% primary wastewater.

Pond water temperature and light intensity were measured continuously from the bottom of three of the HRFAPs using honest observer by onset (HOBO) Pendant MX Temp/Light MX2202 water light and temperature loggers (onset). Dissolved oxygen and pH were measured every 2 days (at approximately 10 am) within each replicate at mid-HRFAP depth (12 cm) using an OxyGuard Handy Polaris 2 Dissolved Oxygen Meter and an OxyGuard Handy pH Meter. Temperature and precipitation data were obtained from the National Climate Database weather recording station located in Te Puke (−37.82455, 176.32048, data available from www.cliflo.niwa.co.nz). Experiments were conducted for a total of 24 days during spring (November) to avoid extreme seasonal effects. The maximum light intensity at the bottom of the HRFAPs ranged from 16.5 to 117.9 μmol m⁻² s⁻¹, culture water temperature ranged from 8 to 26 °C, ambient air temperature ranged from 7 to 25 °C and a total rainfall of 89.6 mm occurred across 9 days during the experiment (Supplementary material, Appendix 1, Figures A1 and A2). Three 500-mL water samples were collected from each HRFAP monoculture immediately before the final harvest during the biomass productivity and bioremediation performance experiments. These samples were used to measure concentrations of TSS, cBOD₅, chemical oxygen demand (COD), and Escherichia coli (E. coli). These analyses were conducted by Hill Laboratories in Hamilton, New Zealand, using the following methodologies: TSS APHA 2540 D (modified) 23rd ed. 2017, cBOD₅ APHA 5210 B (modified) 23rd ed. 2017, COD APHA 5220 D 23rd ed. 2017, and E. coli APHA 9222 I 23rd ed. 2017.

Growth experiments were conducted to compare biomass productivity and bioremediation performance of the three cultivars. Three replicate cultures of each cultivar were grown in HRFAPs at a stocking density of 0.105 g dry weight (DW) L⁻¹ (equivalent to 0.5–0.7 g FW L⁻¹). Biomass was harvested from each HRFAP once every 4 days for three consecutive harvest cycles (12 days total duration). Biomass was harvested by straining the entire contents of each HRFAP (culture water and algae) through a fine mesh bag. Culture water was retained in a separate container. Once excess water had drained from the bag, it was placed in a centrifugal spin dryer (Spindle NZ, SPL-265) and spun at 2,800 rpm for 4 min to remove any remaining water. The algae were then removed from the bag and weighed to determine the FW. Each HRFAP was cleaned by scrubbing the surface of the paddlewheel and the HRFAP with a brush and then each HRFAP was refilled at a 1:1 ratio of strained culture water to fresh primary wastewater from the WWTP, as described in Section 2.2. Stocking density was reset to 0.105 g DW L⁻¹ by restocking the FW equivalent of 7.35 g DW of the harvested biomass back into each replicate HRFAP. Biomass not restocked back in the HRFAP was dried in an oven at 60 °C for 48 h and reweighed to confirm the fresh weight to dry weight (FW:DW) ratio for each replicate. FW:DW ratios were used to convert the initial biomass and the harvested biomass for each replicate, which were both measured in FW, into DW. Biomass productivity (g DW m⁻² day⁻¹) was calculated for each replicate for each harvest using the equation P= (DW_f− DW_i)/A/T, where DW_f is the final algal biomass (g DW), DW_i is the initial biomass (g DW), A is the HRFAP surface area (m⁻²), and T is the number of days in culture. Growth rate (% day⁻¹) was calculated for each replicate for each harvest using the equation growth rate (GR) =((FW_f/FW_i)^1/t– 1) * 100, where FW_f is the final algal biomass (g FW), and FW_i is the initial algal biomass (g FW), and t is the number of days in culture.

A pulse amplitude-modulated (PAM) fluorometer (Junior-PAM, Heinz Walz GmbH, Effeltrich, Germany) was used to measure the effective quantum yield (Y(II)) and optimal quantum yield (F_v/F_m) of each replicate at each harvest. Y(II) was used to measure algal cell stress, by assessing changes in the chlorophyll fluorescence yield of photosystem II (PS II) (Heinz Walz GmbH 2017). F_v/F_m was measured in biomass samples that were dark-adapted for 15 min prior to analysis (Stirbet 2011). F_v/F_m was then used to estimate the maximal photochemical PSII efficiency as an indicator of photosynthetic performance (Schreiber et al. 1995; Kromkamp et al. 2008; Figueroa et al. 2013). Measurements were taken at approximately the same time of day immediately before each harvest (i.e., once every 4 days).

The bioremediation performance of cultivars was quantified by measuring concentrations of TAN, nitrate-N, and DRP in the primary wastewater and HRFAP water samples. Water samples (30 mL) of primary wastewater and water from each replicate HRFAP were taken every 2 days. Primary wastewater samples were taken in the morning at the time of peak inflow to the WWTP and HRFAP water samples were taken immediately prior to water changes. Water samples were filtered upon collection into individual sterile 50 mL clear plastic test tubes (LabServᵀᴹ) using a vacuum filtration system (Whatmanᵀᴹ GF/Cᵀᴹ, 0.22 μm) and immediately frozen. Samples were analysed within a week of collection after defrosting. Concentrations of TAN, nitrate-N, and DRP in each water sample were measured using a spectrophotometer (HACH DR 900, HACH, Loveland, CO, USA) following the United States Environmental Protection Agency (USEPA) Nessler method (HACH method 8038), the nitrate cadmium reduction method (HACH method 8039) and the ascorbic acid method (HACH method 8048), respectively. Nutrient removal rate (NR, % day⁻¹) was calculated at each water change using the equation NR = CW_f/((CW_i + PW_i)/E) *100, where CW_f and CW_i are the final and initial nutrient concentrations of the culture water, PW_i is the initial nutrient concentration of the primary wastewater and E is the proportion of water exchanged.

The competitive dominance of the cultivars, K. flaccidum and Oedogonium sp. was assessed following completion of the biomass productivity and bioremediation performance experiment. The cultivar O. calcareum was excluded from these biculture experiments as it did not survive through to the end of the growth experiments (see Results, Section 3.1). The competitive dominance of K. flaccidum and Oedogonium sp. was measured by growing bicultures with a cultivar ratio of 1:1 at three stocking densities in HRFAPs for three consecutive harvests (12 days total). Three replicate cultures (total N = 9) were established at a stocking density of 0.25, 0.5, and 1 g FW L⁻¹ (equivalent to DW stocking densities of 0.052, 0.105, and 0.210 g DW L⁻¹) to ensure all cultures started with an equal DW biomass composition of each cultivar. Experimental protocols followed the same methodology for water changes, harvesting, biomass processing, and biomass productivity analysis, as described in Section 2.4. At each harvest, excess biomass was removed to reset stocking density, however, cultivar composition was not reset back to a 1:1 ratio to enable changes in cultivar composition, and therefore competitive dominance, to be quantified over the duration of the experiment.

Biomass samples of 0.5 g FW were collected from each replicate HRFAP on the first day of the experiment, at each harvest, and on the final day of the experiment. The cultivar composition of each biomass sample was analysed on the day of collection by photographing ten sub-samples of each biomass sample using a dissecting microscope (Olympus model CKX53) at 20× magnification. The proportional composition of each cultivar was estimated by placing a 100-point grid over each photograph and summing the number of grid points directly overlying each species. The average proportional composition across all ten photographs was then used to determine the final cultivar composition within each replicate HRFAP.

Biomass productivity, growth rate, cultivar composition, effective quantum yield (Y(II)) and optimal quantum yield (F_v/F_m) measurements were analysed using two factor repeated-measures analyses of variance (ANOVA) with cultivars and harvests as fixed factors. Nutrient concentrations were analysed using two factor repeated-measures ANOVA with cultivars and days as fixed factors. Data for each experiment were analysed separately. All analyses were conducted in SPSS Statistics (version 29). A significance level of 0.05 was used for all tests, and F-values were calculated to assess differences among groups. All data are reported as means ± S.E.

Nitrate-N concentrations in primary wastewater influent ranged between 0.95 and 1.31 mg L⁻¹. Nitrate-N concentrations in culture water varied significantly among cultivars and days (ANOVA: cultivar × day, F_10,30 = 13.74, p = <0.001; Figure 5(b)). Across all days, the highest reductions in nitrate-N on average were achieved by K. flaccidum, which reduced concentrations by 58.7% day⁻¹ (± 3.3) from 0.80 mg L⁻¹ (± 0.01) to 0.30 mg L⁻¹ (± 0.02), followed by Oedogonium sp., which reduced concentrations by 41.1% day⁻¹ (± 4.9) from 0.87 mg L⁻¹ (± 0.02) to 0.49 mg L⁻¹ (± 0.04) and O. calcareum, which reduced concentrations by 31.8% day⁻¹ (± 3.4) from 0.87 mg L⁻¹ (± 0.01) to 0.56 mg L⁻¹ (± 0.03). Nitrate-N removal rates by K. flaccidum showed an initial decline from 89.3% on day⁻¹ (± 2.2) on day 2 to a low of 10.2% day⁻¹ (± 1.9) on day 6. However, removal rates increased thereafter to 70.4% day⁻¹ (± 4.2) from 0.64 mg L⁻¹ (± 0.03) to 0.19 mg L⁻¹ (± 0.02) on day 12. Nitrate-N removal rates by Oedogonium sp. showed a decline from 87.11% on day⁻¹ (± 5.78) on day 2 to a low of 6.8% on day⁻¹ (± 3.4) on day 6. However, removal rates increased thereafter to 49.0% on day⁻¹ (± 3.1) on day 12. Nitrate-N removal rates by O. calcareum showed a sharp decline from 89.3% on day⁻¹ (± 5.8) on day 2 to a low of 3.4% day⁻¹ (± 1.7) on day 6, followed by a slight increase to 4.7% day⁻¹ (± 1.1) on day 8.

DRP concentrations in primary wastewater influent ranged between 3.53 and 5.45 mg L⁻¹. DRP concentrations in culture water varied significantly among cultivars and days (ANOVA: cultivar × day, F_10,30 = 2.87, p = 0.012; Figure 5(c)). Across all days, the highest reductions in DRP on average were achieved by K. flaccidum, which reduced concentrations by 15.2% day⁻¹ (± 2.1) from 4.20 mg L⁻¹ (± 0.03) to 3.52 mg L⁻¹ (± 0.07), followed by Oedogonium sp., which reduced concentrations by 8.5% day⁻¹ (± 1.7) from 4.44 mg L⁻¹ (± 0.04) to 4.04 mg L⁻¹ (± 0.09) and O. calcareum, which reduced concentrations by 7.7% day⁻¹ (± 1.1) from 4.75 mg L⁻¹ (± 0.02) to 4.35 mg L⁻¹ (± 0.05). DRP removal rates by K. flaccidum showed an initial decline from 32.0% day⁻¹ (± 2.9) on day 2 to a low of 5.3% day⁻¹ (± 2.5) on day 4. However, removal rates increased thereafter to 17.4% day⁻¹ (± 1.8) on day 6, followed by 12.2% day⁻¹ (± 1.1) on day 12. DRP removal rates by Oedogonium sp. declined from 20.8% day⁻¹ (± 3.4) on day 2 to a low of 2.0% day⁻¹ (± 1.0) on day 4. Removal rates increased thereafter to 10.0% day⁻¹ (± 1.3) on day 12. DRP removal rates by O. calcareum showed a sharp decline from 23.8% day⁻¹ (± 1.9) on day 2 to 2.5% day⁻¹ (± 1.0) on day 4. Removal rates continued to decline to a low of 1.3% day⁻¹ (± 0.6) on day 8.

Water quality variables of the HRFAP culture water varied between cultivars (Table 1). The concentration of TSS in K. flaccidum culture water was 53.8% lower than in the primary wastewater, whereas in Oedogonium sp. culture water, it was 9.4% higher. Carbonaceous biochemical oxygen demand (cBOD₅) of the K. flaccidum and Oedogonium sp. cultures were 92.7 and 74.6% lower, respectively, compared to the primary wastewater. COD of the K. flaccidum and Oedogonium sp. cultures were 73.9 and 43.3% lower, respectively, compared to the primary wastewater. The concentration of E. coli in the K. flaccidum and Oedogonium sp. culture water were both 1 log lower than that of the primary wastewater. Water quality variables were not measured for O. calcareum as complete die-off had occurred during experiments.

We assessed the productivity and bioremediation performance of three freshwater filamentous macroalgal cultivars (K. flaccidum, O. calcareum, and Oedogonium sp.) grown in outdoor HRFAP mesocosms treating primary municipal wastewater. K. flaccidum was identified as the most suitable cultivar for bioremediation of primary municipal wastewater due to its superior biomass productivity and nutrient removal rates, and competitive dominance at low stocking densities. Previous research has compared biomass productivity and bioremediation performance of Klebsormidium sp. and Oedogonium sp. within tertiary-treated municipal effluent (Lawton et al. 2021). Oedogonium sp. demonstrated greater biomass productivity and bioremediation performance compared to Klebsormidium sp., thereby identifying Oedogonium sp. as a target cultivar for bioremediation of tertiary-treated WWTP effluent (Lawton et al. 2021). However, compared to primary municipal wastewater, tertiary-treated WWTP effluent has low turbidity, permitting high penetration of light, which may have contributed to the superior performance of Oedogonium sp. Conversely, our study measured cultivar productivity in highly turbid (TSS 106 g m⁻³) primary municipal wastewater. This high turbidity reduced light availability within the HRFAPs (maximum light intensity 16.5–117.8 μmol m⁻² s⁻¹) and may have led to the lower biomass productivity of Oedogonium sp. as this species exhibits optimal growth under moderate to high light conditions (Cole et al. 2018). In contrast, Klebsormidium sp. demonstrated high productivity in primary municipal wastewater despite reduced light availability, further demonstrating that this genus has low light requirements for growth and photosynthesis (Karsten & Rindi 2010), making it a suitable candidate for cultivation in wastewaters with high turbidity.

Across all three nutrients analysed (TAN, nitrate-N, and DRP), K. flaccidum consistently exhibited higher bioremediation performance compared to O. calcareum and Oedogonium sp. Nutrient removal rates by K. flaccidum compared with Oedogonium sp. and O. calcareum were 32 and 94% greater, respectively, for TAN, were 43 and 85% greater, respectively, for nitrate-N, and 78 and 98% greater, respectively, for DRP. These findings align with previous laboratory-scale research identifying Klebsormidium sp. as a key target cultivar for bioremediation of primary municipal wastewater (Novak et al. 2024). Klebsormidium sp. has previously demonstrated reductions in nutrient concentrations, when cultivated in primary municipal wastewater under summer conditions, with TAN reduced by 94% from 10.7 to 0.6 mg L⁻¹ (± 0.07), nitrate-N by 64% from 1.1 to 0.4 mg L⁻¹ (± 0.10) and phosphate by 91% from 1.3 to 0.1 mg L⁻¹ (± 0.00) (Novak et al. 2024). Similarly, Klebsormidium sp. cultivated in urban wastewaters with varying nutrient concentrations (total nitrogen (TN): 10–50.7 mg L⁻¹, TP: 3.2–10.7 mg L⁻¹), achieved nutrient removal rates of 63–96% for TN and 69–74% for TP (La Bella et al. 2023). K. flaccidum has also demonstrated high nutrient removal rates of TAN (28 mg L⁻¹) and phosphate (15 mg L⁻¹) from synthetic municipal wastewater (Umetani et al. 2023). However, previous studies have not extensively reported improvements in water quality variables of wastewater resulting from filamentous algae cultivation. In this current study, monocultures of K. flaccidum and Oedogonium sp. were found to have large effects on water quality variables, resulting in improved water quality. However, removal rates varied between cultivars, with cultivation of K. flaccidum resulting in greater reductions in TSS, cBOD₅, and COD, while both K. flaccidum and Oedogonium sp. produced comparable reductions in E. coli. The effectiveness of different species of filamentous algae in improving water quality variables in wastewater is yet to be thoroughly compared, and therefore, cultivar-specific mechanisms and physico-chemical bioremediation performance are not well understood. Regardless, these results further support the selection of K. flaccidum as a target cultivar for bioremediation of primary municipal wastewater.

In addition to demonstrating superior biomass productivity and nutrient removal performance, K. flaccidum was also competitively dominant, however this dominance was not consistent across all three stocking densities. K. flaccidum was most dominant under lower stocking densities of 0.25 and 0.5 g FW L⁻¹, however, no cultivar was dominant after 12 days at the stocking density of 1 g FW L⁻¹. Contrary to previous studies, monoculture biomass productivity was a reliable predictor of biculture performance. For example, monocultures of Oedogonium sp. at a stocking density of 0.5 g FW L⁻¹ and bicultures at a stocking density of 0.5 g FW L⁻¹ had similar biomass productivities of 1.07 DW m⁻² day⁻¹ and 1.09 g DW m⁻² day⁻¹, respectively, on day 12. Stocking density also had a significant effect on productivity, as biculture biomass productivities of Oedogonium sp. were 34.4 and 62.7% higher on day 12 at a stocking density of 0.25 and 1 g FW L⁻¹, respectively, compared to the monocultures at a stocking density of 0.5 g FW L⁻¹. However, the proportional composition at various stocking densities may not have reached equilibrium. Therefore, future assessments of competitive dominance should be undertaken for longer durations to increase the reliability of findings. Experiments should also be conducted across multiple seasons to provide insight into cultivar dominance under varying light and temperature conditions, as the biomass productivities of individual cultivars may vary from season to season (Ranjan et al. 2019; Novak et al. 2024).

Primary municipal wastewaters often contain a wide variety of micropollutants from domestic and industrial inputs, including pesticides, personal care products, pharmaceuticals, polycyclic hydrocarbons, plasticisers, and surfactants (Rout et al. 2021), which may be toxic to certain algal species (Rydh Stenström et al. 2021; Othman et al. 2023). It is likely that a micropollutant was present in the wastewater during the initial experiment investigating biomass productivity and bioremediation performance of monocultures as frothy water occurred in the HRFAPs on day 8, indicating the likely presence of a surfactant within the culture water. At this time, all replicate cultures of O. calcareum had completely died off, highlighting the potential toxic effect of primary wastewater on cultivar growth and cell health. In contrast, while biomass productivity of K. flaccidum had declined by day 8 relative to day 4, maximal quantum yields were maintained throughout the experiment. Biomass productivity of Oedogonium sp. remained consistent throughout the experiment, but maximal and optimal quantum yields declined at day 8 relative to day 4 measurements, before returning to optimal levels at day 12. These results demonstrate the robustness of K. flaccidum and Oedogonium sp. and their capacity for growth under highly unpredictable and potentially toxic conditions. Prior research has often measured the nutrient bioremediation performance of filamentous algae when cultivated in nutrient-rich synthetic wastewater under small-scale laboratory conditions (Liu & Vyverman 2015; Umetani et al. 2023). However, the current results show that previous assessments of cultivar performance within synthetic wastewater and treated wastewater effluents are not applicable when selecting target cultivars for primary wastewater treatment. Instead, the nutrient bioremediation performance of cultivars should be assessed using the actual wastewater in outdoor HRFAPs, with long-term exposure to detect tolerance to stochastically occurring micropollutants (Sabatte et al. 2024).

To date, few studies have demonstrated the potential of filamentous algae for primary municipal wastewater treatment (Neveux et al. 2016; Ge et al. 2018; Kube et al. 2022). Among these studies, two were conducted under controlled laboratory conditions utilising photobioreactors (Kube et al. 2022) and flat-plate aquariums (Ge et al. 2018), while one study utilised 20 L outdoor cylindrical tanks and pilot-scale aerated open pond systems (with a total volume of 10 m³) (Neveux et al. 2016). While these studies successfully demonstrated the potential of integrating monocultures of freshwater filamentous algae into wastewater treatment operations, it is important to note that the choice of cultivation system significantly influences biomass yields and bioremediation performance (Ge et al. 2018; Sabatte et al. 2024). Factors such as depth (Sutherland et al. 2014), surface area (Sutherland et al. 2020), mixing/turbulence (Grobbelaar 2010), and grazer pressure (Smith & Mcbride 2015) all play pivotal roles in determining algal production rates. Furthermore, outdoor climatic conditions are critical for algal cultivation, as water bodies are highly responsive to environmental change (Meerhoff et al. 2012). Shallow ponds (with operating depths of 15–30 cm) commonly used in algal cultivation are particularly sensitive to changes in light and temperature (Smith & Mcbride 2015). Further research is required before filamentous HRAP systems can be widely implemented into mainstream municipal wastewater treatment operations (Liu et al. 2020; Sabatte et al. 2024). Hence, future studies should prioritise evaluating cultivar performance within outdoor HRAPs at large-scale to confirm their feasibility as a treatment system.

This study confirms K. flaccidum as a target cultivar for nutrient bioremediation of primary municipal wastewater based on superior biomass productivity, high nutrient bioremediation performance, improvements in selected water quality variables, and competitive dominance. K. flaccidum outperformed the other cultivars by significantly reducing TAN, nitrate-N, and DRP concentrations as well as achieving notable reductions in TSS, cBOD_5, and COD, while also reducing E. coli concentrations by 98%. Additionally, K. flaccidum demonstrated increased competitive dominance over time in bicultures, establishing itself as a key target cultivar for primary municipal wastewater bioremediation. The results of this study support the use of our previously developed screening protocol, which identified K. flaccidum as a key target cultivar, therefore, this current study has confirmed the screening protocol as an accurate tool for selecting suitable target cultivars for the bioremediation of primary municipal wastewater. Our experimental design effectively assessed cultivar productivity and nutrient bioremediation in primary wastewater over a short period. Future research now needs to focus on assessing competitive dominance and seasonal variability in cultivar performance over annual time scales. Future research should also identify optimal operational parameters, which are necessary for improving biomass productivity and bioremediation efficiency in larger systems.

The authors would like to thank Logan Forsythe and Ari Brandenburg for their assistance with experiments and Western Bay of Plenty District Council for allowing the experiments to be undertaken at the Te Puke wastewater treatment plant. Graphical abstract illustrations were created with Biorender.com under license number: SW278IT4WH.

This research was funded by the New Zealand National Institute of Water and Atmospheric Research through a Ministry of Business, Innovation, and Employment endeavour research programme contract number C01X1912.

I.N.N. developed methodology, rendered support in investigation, data curation, formal analysis, and wrote the original draft of the article. R.J.L. conceptualised and supervised the work, wrote the review and edited the article, and rendered support in funding acquisition. M.M. conceptualised and supervised the work, wrote the review and edited the article, and rendered support in funding acquisition. R.J.C. conceptualised and supervised the work, wrote the review and edited the article, and rendered support in funding acquisition.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.