## 3 Results and Discussion
### Tolerance of the Microalgae to Raw Whey (RW) Wastewater
Results demonstrated that _Chloerella_ and _Scenedensmus_ thrive in culture media containing moderate RW proportions (\(\leq\)RW-30%). Consequently, these RW concentrations were included in the sequential culture tests in the photobioreactor (PBR) (see Section 3.3). Results from Figure 3 seem to indicate that _Chloerella_ sp. possesses a slight competitive advantage compared to _Scenedensmus_ sp. In essence, these results underscore the effectiveness of using 10%, 20%, and 30% RW media in providing sufficient nutrients for the growth and biomass production of both microalgae compared to the control. This approach offers at least two key advantages: (i) significantly reduces the need for extensive dilution of raw RW to comply with environmental regulations, thereby minimizing the water footprint associated with microalgae-based RW treatment; (ii) eliminates the requirement for additional chemically defined nutrients, thereby reducing the environmental footprint associated with their manufacturing. Figure 3: Effect of varying proportions of raw whey (RW%) in culture medium on the growth kinetics of _Chlorella_ sp. MC18 (CH) and _Scenedensus_ sp. MJ23-R (SC) grown in flask in batch mode. **(A,B)** Natural logarithm of the normalized cell concentration (\(X/X_{0}\)) versus the culture time. **(C)** Photograph showing part of the microalgae flask cultures with RW% carried out in the cultivation system used. **(D–L)** Evaluation from a two-way ANOVA of main effects RW% and ALGA and their interaction (RW%-ALGA) on the growth kinetic parameters mean \(\mu_{max}\): **(D–F)**, \(P_{B}\): **(G–I)**, and \(Y_{B}\): **(J–L)**. Each RW% (D,G,J) and ALGA group **(E,H,K)** gathers all the results obtained for the two microalgae and for the three levels of RW% and BG11, respectively. Bars around points represent 95.0% confidence intervals. Overlapping bars indicate no significant difference. However, it is important to note that microalgae must assimilate organic material present in RW. While some microalgae species possess the metabolic machinery to utilize lactose as a carbon source, others may lack efficient mechanisms for metabolizing this sugar compound, particularly those considered obligate phototrophs relying solely on photosynthesis for energy production and growth [41; 42; 43]. For example, studies have demonstrated that species such as _Chlorella vulgaris_ and _Chlorella prototheoides_ did not show growth when cultured in lactose-supplemented medium, indicating their limited ability to metabolize lactose under mixotrophic conditions, while _Scenedesmus obliquus_ does [44]. In contrast, experiments conducted in this study with _Chlorella_ sp. MC18 and _Scenedesmus_ sp. MJ23-R strains in raw cheese whey have revealed their ability to thrive in lactose-rich environments. This suggests that these native strains possess the necessary metabolic machinery to utilize lactose as a carbon source. Furthermore, previous research has shown that certain microalgae, including _Dunaliella tertiolecta, Chlorella minutissima_, and _Nannochloropsis ocularate_[42], can metabolize lactose under mixotrophic conditions, underscoring inter- and intra-species variability in lactose utilization among microalgae. Effective lactose metabolism is associated with key traits such as the expression of \(\beta\)-galactosidase (an enzyme that hydrolyzes lactose into its constituent monosaccharides, glucose and galactose, simple sugars assimilable by microalgae), metabolic flexibility for switching between different carbon sources, and efficient sugar uptake mechanisms. Genetic and metabolic diversity within the _Chlorella_ and _Scenedesmus_ genera is likely instrumental in enabling them to metabolize lactose and other carbon sources, as evidenced by their performance in cheese-whey-based cultures. Building upon the premise established in duckweed cultures, it is plausible that the introduction of LAB into the medium used for microalgae cultures resulted in the formation of lactose-derived lactic acid.