Improving the performance and yield of microalgal cultures has been identified as a prerequisite to achieving robust, sustainable microalgae-based systems. Among the strategies outlined towards achieving this, the use of co-cultures of microalgae ranks high owing to its ease of application, comparatively. However, lot of unanswered questions still surround this promising strategy. In the present study, different culture combinations of microalgae were compared with one another in terms of removal of wastewater pollutants as well as bioproducts accumulation while growing in wastewater stream. The goal was to identify the least diverse co-culture that could overcome the efficiency of the most promising monoculture.
Reduction of nutrients and organic matter from SWW
Chemical oxygen demand
All the cultures sustained the reduction of COD over the treatment period (Fig. 1A). By day 4 holding time, all the cultures yielded at least 50% COD reduction efficiency, except for the monoculture LY2 and the binary culture KY. By the 20th day, the final reduction efficiency ranged from 76.5% ± 1.441 (LY2) to 91.7% ± 0.304 (CK). This reduction efficiency of all the cultures satisfies the minimum legislative reduction recommended for urban effluents, indicating the promise of all the culture combinations in COD reductions (EU Directive 91/271/EEC 1991). This impressive performance might be related to the preference of mixotrophic microalgae for glucose, the principal organic carbon source in the synthetic wastewater (Patel et al., 2021).
Comparison among the monocultures showed that KL10 yielded the highest COD reduction efficiency of all the combinations (p < 0.05); in fact, the other two monocultures i.e., CM2 and LY2 yielded the lowest COD reduction efficiency. Similarly, among the binary combinations, CK yielded the highest (p < 0.05) reduction efficiency. Further comparison between the different culture combinations revealed that KL10 and CK achieved comparable COD reductions (p > 0.05) and that no other culture was as efficient (p < 0.05). These findings fully agree with those of Qu et al., (2019), where a pure culture of S. obliquus alongside promising binary cultures recorded higher TOC reduction efficiency than all other co-cultures. However, comparisons of grand means of the different culture combinations (i.e. means of all monocultures versus means of all binary cultures and means of the ternary culture) showed no differences among them (p > 0.05). This implies that the performance of the average cultures remained comparable and that the co-cultures did not benefit from the diverse community they harbor.
Ammonium nitrogen
Ammonium nitrogen represents the preferred form of inorganic nitrogen for microalgae (Gonçalves et al., 2017). As opposed to COD, there was an initial rise in the level of NH4-N in all the cultures, most probably due to the release of NH4-N into the cultures from the bacterial breakdown of complex nitrogenous compounds present in the growth medium (Fig. 1B). However, after the 8th day of treatment, all cultures recorded significant (p > 0.05) reduction of NH4-N (16.8% ± 3.7 to 61.5 ± 2.394) and this was sustained till the last day of treatment. At the end of the treatment, the reduction efficiency ranged from 66.9% ± 0.734 (LY2) to 99.98% (CK). When compared with the findings of Mujtaba et al., (2017), and Zheng et al., (2018), the removal of NH4-N in the cultures relatively took longer. However, in reality, the cultures removed more NH4-N than reported because of additional NH4-N input from the breakdown of organic nitrogenous compounds that constitute the synthetic wastewater. Regardless, the reduction efficiency of four cultures - CM2, KL10, CK and KY - satisfies the EU legislation (1 mg/L NH4-N) for municipal wastewater effluents. Furthermore, the final concentration of NH4-N in CY (1.04 mg/L) and CKY (1.18) cultures went very close to satisfying the legislative requirement (Council directive 96/23/EC, 2010). However, LY2 (2.72 mg/L NH4-N) was still away from that.
As observed with COD removal in the current study, the removal efficiency of NH4-N from the system also revealed the superiority and comparable performance of KL10 (99.5% ± 0.064) and CK (99.9%) amongst the monocultures and binary cultures, respectively. However, these findings were not consistent with that of Qu et al., (2019), who reported the highest NH4-N removal in a monoculture of S. obliquus compared with co-cultures. Further comparisons of the grand means of the NH4-N removal efficiencies among the culture combinations revealed the efficiency of the binary cultures over both monocultures and ternary cultures (p < 0.05), implying that on average, co-cultures with binary composition may overcome monocultures and ternary cultures in terms of NH4-N nitrogen reduction.
Phosphate phosphorus
The reduction of PO43−-P in all the cultures followed a similar pattern to that of NH4-N, characterized by initial spike on day 4 (Fig. 1C), i.e., negative reduction efficiency. This negative trend was sustained till day 8, except in the monoculture KL10 and binary culture CK, both of which achieved PO43—P reduction over the same time period. This implies that the removal of PO43−-P in the cultures was affected by the release of more phosphate from the breakdown of PO43−-P containing organic compounds present in the synthetic wastewater. Similar phenomenon was observed in a microalgal-bacterial system treating dairy wastewater (Tricolici et al., 2014).
At the end of the treatment (day 20), all the cultures recorded positive reduction efficiency, ranging from 43.9 ± 1.155% (CY) to 75.4% ± 4.042 (KL10). As with both COD and NH4-N, the monoculture KL10 and the binary culture CK were the best performers of their respective groups (p < 0.05), although these two could not differ performance-wise (p > 0.05). Nonetheless, the performances achieved in the current study were in most cases higher than what was reported for pure cultures grown on Bold’s basal medium (Abdelaziz et al., 2014) over a 14 day incubation period. Further comparisons between the grand mean reduction efficiencies of the culture combinations revealed reduction efficiencies in the order monoculture (p < 0.05) > binary culture (p < 0.05) > ternary culture (p < 0.05). Overall, these findings herein revealed decline in the reduction efficiencies of PO43—P with increase in species richness of the cultures. This is consistent with the findings of Qu et al., (2019), where S. obliquus axenic culture yielded higher PO43−-P reduction than all co-cultures under the same growth conditions.
Total phosphorus
All the cultures yielded impressive reduction efficiency for total phosphorus by day 4 of treatment, ranging from 52.2 ± 4.521% (CY) to 68.4% ± 0.527 (CK) (Fig. 1D). This dramatic reduction in TP supported the assertion that the rise in the level of PO43−-P in the cultures at day 4 was attributed to the release of more PO43−-P into the cultures from the breakdown of complex organic phosphorus sources in the growth medium. At day 16, up to 88.05% ± 1.319 TP reduction was recorded in the KL10 culture, which outperformed all others except CK (86.9% ± 0.603, p < 0.05). All the cultures met the minimum 80% TP reduction limit recommended by EU (Directive 1991/271/EEC, 1991; Directive 1998/15/EC, 1998) and this performance was higher than what was previously reported by similar studies (Ji et al., 2018). However, the removal of TP was not as efficient as that of both COD and NH4-N, and this would be explained by relatively lower demand for phosphorus by living cells as compared to carbon and nitrogen (Godwin et al., 2017).
It is pertinent to note that in the current study, it took shorter time (12 days) for KL10 and CK to attain this level of TP reduction, thereby separating them as the best of all. However, comparison of the grand means of the various combinations revealed no differences in their TP reduction efficiencies. These findings therefore suggest no improved performance benefits from the use of co-cultures combination in terms of TP reduction. However, this is not concordant with the finding of Gonçalves et al., (2016), who reported the superiority of binary cultures over monocultures. Their findings support the hypothetical co-operation in co-cultures to support superior performances.
Bioproducts yield of the cultures
Biomass productivity
Biomass productivity gives a reliable idea of the biomass accumulation potentials in relation to time. In the present study, the day16 incubation time was chosen because it coincided with the time when the cultures fully attained the stationery growth phase. Among all the cultures, the highest biomass productivities were yielded by CM2 (71.875 ± 2.21 mgL− 1day− 1), KL10 (75 ± 4.41 mgL− 1day− 1) and CK (78.125 ± 2.21 mgL− 1day− 1) (Fig. 2A) were comparable to one another (p > 0.05). Comparison among similar culture combinations revealed that CM2 and KL10 did better than LY2 (p > 0.05). Comparatively, while the biomass productivity of the cultures falls in the same range with what was reported in the past for monocultures and binary cultures (Gonçalves et al., 2016), it clearly surpassed what was reported by another related study (Qu et al., 2019). Similarly, the biomass productivities of CK and KY were comparable, but higher than that of CY (p < 0.05). Finally, in order to minimize perceived bias against the ternary combination, which was represented by CKY only, the grand mean biomass productivities of monocultures, and binary cultures were compared with that of CKY. The results revealed comparable biomass productivities between the monocultures and binary cultures (p > 0.05), while maintaining the superiority of the duo over the ternary culture (p > 0.05). This agrees with the findings of Qu et al., (2019), who reported superior biomass accumulation in monocultures relative to co-cultures. Still it was difficult to decide between the monocultures and the binary cultures as the best and the least performing cultures of each combination did not differ from their counterparts of the other combinations. In essence, the findings of the current study reveal no performance difference between the monocultures and the binary ones. This findings are inconsistent with those of Gonçalves et al., (2016), who reported the superior biomass productivity of binary cultures relative to monocultures.
Lipid productivity
Lipid productivity gives assessment of the dry weight lipid content in relation to time. The range of lipid content of the cultures 20.01 ± 1.03 (CY) to 38.01 ± 0.84 mgL− 1day− 1 (CK) compared favorably with past studies (Gonçalves et al., 2016; Qu et al., 2019). The lipid productivity of all the three monocultures remained comparable (p > 0.05) to one another, while for binary cultures, CK and KY edged out CY (p < 0.05) (Fig. 2B). Interestingly, the lipid productivity of the CK binary culture overcame what has been reported for Chlorella pyrenoidosa (37.01 mgL− 1d− 1) cultivated in an acetate plus iron-enriched growth under mixotrophic conditions. Further comparisons of the grand means of the monocultures and binary cultures with the ternary culture revealed the superiority (p < 0.05) of the binary combinations over the ternary combinations, whereas the monocultures were comparable to the ternary culture (p > 0.05). Comparison of the individual cultures revealed, the binary culture CK and the monoculture KL10 were outstanding. This was however concordant with the findings of Gonçalves et al., (2016), who observed the improved lipid productivity of the best binary cultures over their monocultures counterparts. The findings herein therefore further point towards the superior lipid productivity of binary cultures, although this could still be challenged by the best monocultures. In a nutshell, increased species richness to binary level might improve lipid productivity; however, further increase to ternary level might be counterproductive.
Protein content
There was not much variation (p > 0.05) in the protein content of the strains regardless of the culture combination (Fig. 2C). However, KL10 and CK were the cultures whose protein contents were comparatively higher (p < 0.05) than the lowest of all (CM2). Comparison of the average protein content of the three culture combinations. Compared with previous studies, the range of protein content of the cultures ranked one of the best and also showed minimal variation from one culture to another (Duong et al., 2015; Schenk, 2016). This coupled with the minimum variation in the protein content of the cultures indicated possible inherent protein-rich attribute of the individual strains. Species of Scenedesmus, such as Scenedesmus sp. NT1d, S. dimorphus NT8c, have been infamous for their high protein content (Duong et al., 2015; Schenk, 2016). Although this appears true with the Scenedesmus sp. (Scenedesmus spp. LY2), in the present study, the results further reveal that allied strains and their co-culture combinations might also guarantee similar performance.
Pigments content
At individual culture level, KL10 stood out in terms of chlorophyll a content; although this was still comparable to CK and KY (p > 0.05). However, it was probable that the high chlorophyll a content of the two binary cultures was due the presence of KL10 as one of species in the community of two binary cultures, whereas the means of the three culture combinations did not differ significantly (p < 0.05) (Fig. 5). Further comparison of the grand means of the various combinations revealed no differences between them (p > 0.05). These observations were more or less the same with carotenoid content of the cultures, suggesting possible association between chlorophyll a and carotenoids. On the contrary, the ternary culture CKY overcame all in terms of chlorophyll b content; other cultures appeared comparable to one another (p > 0.05). The high chlorophyll b content of the CKY culture would certainly be attributed to the ternary combination, as the monocultures of the individual strains of the ternary cultures yielded low chlorophyll b content.
Relationships between the microalgae cultures, pollutants removal efficiency and bioproducts yield
In order to discern the potential linkage between the microalgal cultures, their efficiency in nutrients and organic matter removal vis-a-viz bioproducts yield, principal component analysis was carried out. The PCA percent variance explained by the biplot (Fig. 4) were 65.89 (PC1) and 12.86 (PC2). All the vectors of nutrients and organic matter removal efficiency projected towards the same direction and were linked by obtuse angles, implying positive correlation between them. However, the most significant of these correlations were between biomass productivity, lipid productivity, chlorophyll a and carotenoids (r = 0.683 to 0.922, p ≤ 0.05). Furthermore, these vectors projected farthest away from the monoculture KL10 and the binary culture CK, implying that the removal efficiency of the nutrients and organic matter was highest in the two cultures. Conversely, except for chlorophyll b, the vectors of the bioproducts accumulation in the system pointed in the same direction and opposite to that of nutrients and organic matter removal efficiency. These were also positively correlated with one another (r = 0.624 to 0.883, p ≤ 0.05). The projection of the vectors in space was closest to the monoculture KL10 and the binary cultures CK and KY, indicating the superiority of the three cultures in accumulating bioproducts. All in all, the PCA biplot reaffirms the superiority of the KL10 and CK culture in yielding the highest nutrients and organic matter removal efficiency while simultaneously accumulating bioproducts in the system.
Gonçalves et al., (2016) reported that in co-cultures comprising of two species of microalgae, competition for nutrients (nitrogen and phosphorus) between the individual species will lead to faster removal of nutrients. Since these nutrients are necessary for the synthesis of microalgal macromolecules, increased uptake of nutrients would result in increased microalgal biomass and/or macromolecular composition. Although the promising performance of the CK binary and, to a lesser extent, KY binary culture have supported this assertion, the sterling performance of the monoculture KL10 proves otherwise. Apparently when paired with any of the two other monocultures (CM2 or LY2), the performance of the resulting binary cultures (CK and KY) improved compared with the individual cultures. It is also obvious that both CM2 and LY2 performed well when paired with KL10, but failed to do the same in those co-cultures that combined them together. This therefore highlights the importance of prior consideration of species compatibility while constructing microalgal co-cultures. Overall, these findings of the current study suggest that compatible binary cultures may at least challenge the best monocultures in terms of yield and performance. Further understanding of the ecological interactions of microalgal communities would be key to ecological engineering of microalgal communities for better productivity.
Fatty acids composition of the cultures
Fatty acids profiling is paramount in deciding the suitability of microalgal biomass for a particular application. In the current study, the fatty acids composition of the cultures is represented as the percentage of the total fatty acids (FAs) composition (Fig. 4). The cultures did not differ from one another in terms of the types of fatty acids detected. Similarly, for all the culture combinations, the chain length of the FAs ranges from C14 to C20. Notably, palmitic acid (C16:0), oleic (C18:1) and linoleic acid (C18:2) accounted for the major proportion (77.81 to 80.11%) of the total FAs. Furthermore, there was no significant difference (p > 0.05) between the culture combinations in terms of average total FAs, palmitic, oleic and linoleic acid content. Among the individual FAs, the proportion of oleic acid alone was in excess of 40% of the total FAs in some of the cultures. High content of oleic acid in microalgal FAs is considered a desirable trait as for high-quality biodiesel production as it enhances fuel qualities, such as ignition quality, oxidative stability and viscosity (Hwang et al., 2016). Similarly, for all the combinations, the degree of saturation of the FAs varies considerably from C:0 to C:4, following the order saturated > monounsaturated > polyunsaturated. In all the cultures, the unsaturated FAs (monounsaturated and polyunsaturated FAs combined) had higher proportion (≥ 65%) than the saturated FAs, revealing similarities in the degree of saturation irrespective of the species diversity of the culture. The high unsaturated FA content might be attributed to high load of organics in the cultivation media as observed by Dang & Lee, (2018) and Chinnasamy et al., (2010), who both reported unsaturated FAs in excess of 65% from microalgal biomass. In particular, glucose supplementation has been reported to increase the degree of unsaturation of microalgal FAs (Pang et al., 2019). Putting these together, the FAs composition of the various culture combinations revealed considerable similarities and presents promising profile for biodiesel production.