3.1 Chlorella sp. growth under different growth media
The cultivation was conducted in the objective to investigate the impact of real growth media on the cell growth and adaptation capacity relatively to conventional BG11 growth media. Chlorella microlagae culture under different growth media shows that chlorella sp. is well adapted to the CW100, CW/DW growth media, where the maximum growth is achieved after the 4th day of culture. On the other hand, all growth media showed a positive effect on the growth of chlorella sp. comparatively to reference BG11 media (Figure1). Moreover, the good growth of chlorella sp. on the CW100 growth media is explained by its high concentration of phosphorus and ammonium (Data are not shown). In this respect, Salati et al., 2017 found that total phosphorus content in the medium was the driving factor affecting algae growth under mixotrophic conditions while carbon availability (BOD5/COD) did not cause any differences in algae kinetic growth parameters and final biomass concentration. However, (Girard et al. 2014) found that the microalgae growth is also affected by the lactose hydrolysis which makes glucose available as carbon source in the growth media since the 3rd day of culture thus increasing biomass production.
It should be noticed that culture growth was undertaken under ambient temperature where its range was from 10 °C to 18 °C (winter saison). In this regard, temperature is an important factor affecting microalgal growth and most microalgae prefer an ambient temperature of about 25 °C to 30 °C for maximal biomass production without affecting the photosynthetic efficiency (Nagarajan et al. 2019). Likewise, temperature was found to have an impact on the biomass production and microalgae growth. This factor could explain the falling of growth since the 4th day due to temperature decreasing where microalgae cultivation is undertaken at the ambient temperature, where it could be seen the variation of biomass production which is decreased for CW100 media when the temperature is about 14 °C. In addition, others growth media show a less microalgae growth due to carbon deficiency, whether organic or not. In this context. García-Cubero et al. 2018 have found that the highest biomass productivity and CO2 fixation rates were noticed in the range of 15–25 °C.
3.2 Effect of pH growth media on microalgae growth
The pH evolution during culture shows a good stability for all growth media. Figure 3 shows a rapid increase in pH to stabilize near to 11 for both BG11 and BG11/CW growth media, while for others pH is stabilized between 9 and 10. Moreover, pH culture evolution indicates the capacity of this local microalgae to support alkaline conditions. In this regard, Vadlamani et al. 2017 have reported that alkaline conditions permit to improve the CO2 dissolution and to keep an acceptable contamination level avoiding culture failure at large scale.
Chlorella sp. has showed a good adaptation with high value of pH where it went-up rapidly as showed in figures 1 and 2. In comparable conditions of high pH culture, Vadlamani et al. 2017 have found an equivalent biomass productivity in the range of 1.5 to 2 g/L despite low temperature during the culture in the CW100 medium. Likewise, (Qu and Miao 2021) have found that microalgae cultivation under alkaline pH of 10 conducts to increase biomass yield of 14.1 % comparatively to pH of 7.5.
3.3 Final Biomass production
Obtained results for biomass production expressed in terms of dry weight (g/L) showed a good growth with a high final biomass, which could reach five times more the biomass yield of BG11 growth media. According to figure 3, CW100 culture media showed the best final biomass concentration followed by BG11/CW. This result indicates that the CW100 growth media could be used as an alternative for microalgae cultivation and the Chlorella sp. algae is showing a good use of organic carbon which presents a good ability for mixotrophic culture.
On the other hand, BG11/CW growth media showed a biomass concentration better than that obtained using DW100 and CW/DW growth media (figure 3). All the combinations growth media used in this study are presenting the highest biomass production comparatively to BG11 growth media. Final biomass yield in mixotrophic cultivation condition using cheese whey as carbon source was obtained by (Girard et al. 2014) to be 3.6 g/L after 13 days of culture. In this study, the obtained biomass yield in CW100 growth media is 1.2 g/L. This value may be due to low temperature during the cultivation period, knowing that temperature is a crucial factor for microalgae growth. In fact, a maximum biomass production is obtained for a temperature between 25 °C and 30 °C without affecting the photosynthetic efficiency (Nagarajan et al. 2019).
3.4 Pigments content determination
3.4.1 Total Chlorophylls
Chlorophyll is one of the valuable bioactive compounds that can be extracted from microalgal biomass. It is used as a natural food coloring agent and has antioxidant as well as antimutagenic properties (Hosikian et al. 2010). As showed in figure 4, chlorophylls content under mixtrophic culture is more important where it could reach 50 mg/g dry weight for both CW/DW and BG11/CW. Furthermore, DW100, CW100, and BG11 growth media showed also a good total chlorophylls content, with values in the range of 10 mg/g to 30 mg/g dry weight. Based on these results, using carbon rich biowastes combined to a source of mineral nutrients would improve chlorophylls content.
As reported in literature, total chlorophylls content for Chlorella sp. is found in the range of 22.6 to 32.4 mg/g dry weight (Figueroa-Torres, Gonzalo M. et al. 2020). Moreover, the most abundant pigment in Chlorella vulgaris is chlorophyll, which can reach 1-2% dry weight, also the content of total chlorophylls varies from 0.3-15.4 mg/g dry weight (Safi et al. 2014).
The high content in total chlorophylls is due to the supplementation of glucose derived from cheese whey degradation as a carbon source at low concentration (4 g/L) which promotes the biosynthesis of chlorophylls. In addition, it has been found that all glucose could be exhausted within 7 days, which explains the maximum content observed during the 8th day of culture and the decreasing of the chlorophylls after this period maintaining its content almost the same with others growth media (Chai et al. 2018).
Many Strategies have been reported in order to promote the chlorophyll content in microalgae including variation in light intensity, culture agitation, changes in temperature and nutrient availability. Nitrogen and phosphorus are essential nutrients for microalgae culture and then critical for the synthesis of chlorophyll molecules. A deficiency in these two elements can induce respectively 64% and 67% reduction in chlorophyll content (da Silva Ferreira and Sant’Anna 2017).
3.4.2 Total Carotenoids
Fig.5 shows the evolution of total carotenoids content under different growth media. Carotenoids content evolution showed practically a good tendency for all growth media until the 10th day, which could reach more than 1% of dry weight even if temperature is very low. However, it was found that growth media composed of BG11/CW presents the best content after the 10th day which are in the same range of those reported in literature where quantity of carotenoids (lutein) extracted from Chlorella sp. in mixtrophic cultivation was found to be about 6.83 mg/g dry weight (Gong and Huang 2020). Furthermore, total carotenoids of 2.7-8.3 mg/g dry weight for chlorella genus were reported in literature (Figueroa-Torres, Gonzalo M. et al. 2020).
The high content of carotenoids in BG11/CW and BG11 growth media is probably due to their nitrogen availability, where nitrogen is a major essential element for both cell growth and lutein accumulation. This latter is a primary carotenoid under no stress conditions, where a content of 8.39 mg/g of lutein is reported for Chlorella sorokenia (Xie et al. 2019). Moreover, it has been reported that increasing in glucose concentration increase lutein content (Minhas et al. 2016). Likewise, glucose derived from cheese whey degradation stimulates the microalgae to produce carotenoids (Chen et al. 2018; Gong and Huang 2020).