Enhancing Microalgae Cultivation using Biowastes as Growth Media for High Added-Value Co-Products Generation


 This paper aims to study a new growth media using cheese whey and drainage water from agriculture for indigenous microalgae cultivation for value-added product generation. In this context, four combinations are studied beside the BG11 as reference, where BG11/Cheese whey (60/40, %v/v), drainage water 100%, drainage water/Cheese whey (60/40, % v/v), and Cheese whey 100 % have been used. Moreover, investigated parameters are biomass dry weight, pH variation, total chlorophyll and carotenoid content. Results showed that used growth media have a significant impact on microaglae culture, particularly in terms of cells growth, pigment content and pH variation. Moreover, the mixture BG11/Cheese whey (60/40, %v/v) shows the best impact for total chlorophylls and carotenoids content. Likewise, the mixture cheese whey/drainage water (60/40, %v/v) presents a positive effect on pigments content. The use of cheese whey and drainage water lead to enhance the biomass and pigment production. This study showed that using agro-industrial C-rich wastes and drainage water enhanced microalgae biomass and pigment content, thus contributing to pollution abatement. This will contribute to both reducing the cost of production and resources recycling.


Introduction
Microalgae is actually considered as a promising biocatalysts to be implemented in the increasing eld of "White Biotechnology" mainly to support the production of food, feed, ne chemicals, and diverse "green energy carriers" (Koller et al. 2014). Algae can biosynthesize an awesome variety of metabolites that could be used as biomaterials with an important potential applications in various industrial sectors (Mehta et al., 2018). According to their high market values, pigments from microalgae have received a particular interest due to its high potential for commercialization despite the need to use new systems for its extraction and puri cation to reduce its actual high production cost (Ventura et al. 2017). Three major groups of pigments are distinguished in microalgae, namely carotenoids, phycobilins, and chlorophylls; which are applied as nutrient supply, pharmaceutical purposes, as well as in cosmetic industry and food technology (Koller et al. 2014). Furthermore, chlorophyll and carotenoids from microalgae are well established, and its global market has been expanding because of its wide application in human healthcare, food industries, and cosmetics. Likewise, carotenoids are commercially available in several forms including mainly beta-carotene, lutein, lycopene, astaxanthin, zeaxanthin (Mehta et al.,2018 ;da Silva Ferreira and Sant'Anna 2017). Moreover, the addition of pure carotenoids is a common practice as sh feed for some aquaculture species since it is known to improve carcass and antioxidant capacity (Rosas et al. 2019).
Otherwise, due to its versatile composition, microalgal biomass is presented to be one of the most promising biore nery feedstock providing alternatives for different areas, such as food, feed, cosmetics and health industries, fertilizers, plastics, and biofuels. Furthermore, microalgae can also be integrated in process of wastewater treatment and CO 2 sequestration (Gorry et al. 2018). However, carbon, nutrient, and water requirements for microalgae cultivation are still a major bottlenecks for an e cient and costeffective large-scale microalgae biomass production (Kannah et al. 2018). Alternatively, agro-industrial carbon-rich wastes (Biowastes) and wastewaters as source of nutrients are representing an interesting approach to boost algae production with a particular interest for high value added co-products such as pigments, to improve the economics and the sustainability of microalgae biore nery for an e cient integration with industrial process (Salati et al. 2017); León-Vaz et al. 2019). Moreover, further optimizing culture conditions, by selecting organisms that can overcome the limitations imposed by ambient conditions and that present high pigments content could contribute to lower the cost of microalgae biomass in a biore nery frame. This paper aims to study a combination mixture BG11 growth media, cheese whey and wastewater issued from agro-industrial activities, with different proportions. These growth media have been used for indigenous microalga strain mixotrophic cultivation for high added-value co-products production especially chlorophylls and carotenoids. Therefore, cheese whey and drainage water are used as organic carbon and nutriments sources respectively for microalgae cultivation. Furthermore, this study investigates the possibility to integrate microalgae biomass production into an existing industrial sector and wastes recycling to preserve natural resources.

Material And Methods
The microalgae used in these study was an indigenous strain of Chlorella sp., previously isolated from a local environment and maintained in Erlenmeyer asks with BG11 medium. Erlenmeyer asks of 500 mL were incubated on an orbital shaker under 150 rpm continuous agitation (DaihanScient c and SHR Digital Shaker, Korea), light intensity of 6000 Lux measured on the external surface of the asks using a photo ux meter (Model Testo 545 GmbH and CO, Germany). The initial cellular concentration was set approximately to 2 x 10 6 cells/mL. The pH of the culture broth was measured using a digital pH meter (Starter3100, OHAUS corporation, USA).
Four combinations of growth media are studied beside the BG11 growth media as a reference. aiming the production of high bene t co-products. In order to prevent bacteria contamination, the DW was ltered with 0.45 µm vacuum lter holder (Sartorius stedium biotech GMBH-D-37070, Goettingen) and the CW was autoclaved at 120°C for 20 minutes.
The mixotrophic and autotrophic cultures were kept under continuous illumination. The cultures were sampled every two days to monitor growth. Biomass concentration was estimated by measuring the optical density at 680 nm with UV/Visible spectrophotometer (Lambda 25 UV-visible spectrophotometer, PerkinElmer life and Analytical, UK).Cells were washed 3 times with an equivalent volume of distilled water to avoid excess of sugars and salts (centrifuged for 5 min at 4000 rpm between each washing). Culture samples were then dried for 24 h at 70°C. A good linear regression t was obtained between the dry weight and OD measurements at 680 nm (R 2 value of 0.86). The nal biomass yield was calculated subtracting the initial biomass used as inoculum and the nal biomass at the end of the culture.
The Pigment content determination was carried out according to (Pruvost et  under mixotrophic conditions while carbon availability (BOD 5 /COD) did not cause any differences in algae kinetic growth parameters and nal 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 3 rd 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 e ciency (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 4 th 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 de ciency, whether organic or not. In this context. García-Cubero et al. 2018 have found that the highest biomass productivity and CO 2 xation 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

Final Biomass production
Obtained results for biomass production expressed in terms of dry weight (g/L) showed a good growth with a high nal biomass, which could reach ve times more the biomass yield of BG11 growth media. According to gure 3, CW100 culture media showed the best nal 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 ( gure 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 e ciency (Nagarajan et al. 2019).

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 gure 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 (Sa 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 8 th 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 de ciency in these two elements can induce respectively 64% and 67% reduction in chlorophyll content (da Silva Ferreira and Sant'Anna 2017).

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 Microalgae are considered as excellent candidates for bioactive compounds, yet microalgal residues remaining after the extraction of one or two compounds are usually discarded, which is not economical. The aim of this work was to evaluate new growth media combination issued from waste sources, used for microalgae cultivation for biomass and pigments production the objective to decrease the cost of production and wastes recycling, thus preserving natural resources. Results showed that new growth media used for microalgae cultivation have a signi cant impact on microaglae culture, particularly in terms of growth, pH evolution, chlorophylls and carotenoids concentration. Moreover, the mixture of BG11/Cheese whey (60/40% vv) shows the best performance for both total chlorophylls and carotenoid content. Likewise, pH evolution presents a good stability, which is an important factor for microalgae cultivation. In addition, using the Cheese whey could be a good growth media to support microalgae cultivation for biomass production. The mixture cheese whey/drainage water (60/40 % vv) present a signi cant impact on the pigment content of microalgae biomass.
Finally, the use of cheese whey and drainage water lead to enhance the biomass and pigment production.
Using agro-industrial carbon-rich biowastes and wastewater as growth media for chlorophyll and carotenoid production under alkaline conditions represents a promising approach to boost microalgae integration in existing industries in a more sustainable way. Moreover, the use of high-performance strains with a high added-value products potential alongside the use of waste carbon and nutriment rich combination for mixotrophy microalgae cultivation would offer the most economic, environmentalfriendly and sustainable algae-re nery. Nevertheless, further optimizing culture conditions by selecting strains is still needed to overcome the limitations imposed by ambient conditions.

Declarations
Funding: This study was funded by Direction Générale de la Recherche Scienti que et Développement Technologique of Algeria.  Carotenoids content under mixotrophic culture using different growth media