Microalgae have lately attracted great interest worldwide due to their application potential in wastewater remediation, in nutraceutical, pharmaceutical, and renewable energy industries (Pahazri et al. 2016; Khan et al. 2018). Different researchers demonstrated the purification capacity of microalgae and verified that the biomass obtained had a high content of bioproducts (Tang et al. 2020).
On the other hand, the use of wastewater as a substitute for algae nutrients would significantly reduce the operational cost of the cultures. Wastewater contains phosphorus, nitrogen, carbon, and other constituents needed for microalgal growth, although some other undesirable compounds such as emerging pollutants and heavy metals can also be found (Morales-Amaral et al. 2015).
In this sense, Khan et al. (2018) described the advantages of microalgae to produce biofuels and various bioactive compounds and discussed culturing parameters. According to these authors, the most important and challenging issues are increasing microalgal growth rate and enhancing bioproduct synthesis, among others.
In the present study, we investigated the ability of C. fusca to grow in a medium composed of UW, as well as its efficiency for the removal of contaminants and its potential to produce biomass with a high lipid and protein content.
The results show that the final cell growth values in the 50% UW, 75% UW, and 100% UW treatments were higher than in the control. In cultures with 50% UW, the highest cell density and the shortest doubling time were obtained (Fig. 1). According to Katiyar et al. (2021) Chlorella minutissima and Chlorella sorokiniana showed higher growth rate, lipid content, and biomass productivity, when cultured in wastewater than in control. Similar results were reported by Singh et al. (2017), although these authors recorded a higher growth rate and biomass production, and shorter doubling time, using Parachlorella kessleri-I in 100% municipal wastewater concentration.
This greater growth in the treatments with UW is possibly because the concentration of ammonium as a nitrogen source prevents this nutrient from being limiting (Gómez Serrano 2012). Lin et al. (2007) observed that Chlorella pyrenoidosa (LK) was ammoniacal-N tolerant, with cell density increases in leachate with 405 mg l−1 of NH4+. Similar results were observed in this study, where the cell density of C. fusca increased in UW with about 620 mg l−1 of ammonium. In turn, the UWs provided a greater amount of nutrients to the treatments, including PO
3−4
, SO
2−4
, K+, Mg2+ (among others) favoring growth under these conditions. However, the nutrients are not the sole requirement for microalgal development, since temperature, light, aeration, and pH, as well as mixing, can contribute to their growth (Pahazri et al. 2016).
In algal cultures, the pH usually increases due to photosynthetic CO2 assimilation. This pH increase can be compensated by respiration (Jyoti and Awasthi 2013). According to this, in our study, pH values would be expected to increase along the experiment due to photosynthetic activity; however, the pH increased only in the control, while in cultures with UW, it went down, despite their photosynthetic activity (Fig. 2).
Nitrogen is usually present in wastewater as NH4+, contributing significantly to the changes in pH value. Assimilation of nitrate ions by algae tends to raise the pH, but if ammonia is used as nitrogen source, the pH of the medium may decrease (Kong et al. 2011; Pahazri et al. 2016). Scherholz and Curtis (2013) analyzed the influence of ammonium and pH on the growth of Chlorella vulgaris in photobioreactors and observed that cultures provided with 4.5% nitrogen from ammonium showed significant growth. At the same time, they observed a decrease in pH during the growth phase, followed by a pH rise indicating sequential ammonium and nitrate metabolism. These results are consistent with the almost exclusive consumption of ammonium instead of nitrate and corroborate the statements of previous works where it is mentioned that the use of nitrate is inhibited in the presence of ammonium (Florencio and Vega 1983).
In the 1970s, the worldwide energy crisis encouraged the use of microalgae as renewable and sustainable sources to produce biofuels. Depending on the type of wastewater used in the cultivation of microalgae, the lipid percentage obtained will be different. In agricultural wastewaters Chlorella sp. showed 9% DW (Jacobson and Alexander 1981) and 13.6% DW (Wang et al. 2010a) of lipid content. In industrial effluents, Chlorella saccharophila was obtained with 18.10% DW of lipid content (Chinnasamy et al. 2010). In this study, the lipid percentage was 14.7% DW (25% UW), 15.5 DW (50% UW), 16% DW (75% UW), and 16.7% DW (100% UW). These values are comparable to those obtained by Jebali et al. (2015) and Hernández et al. (2016). Hempel et al. (2012) showed that the strains with the highest lipid content were Chlorella sp. 589 (30.2% DW), C. saccharophila 477 (27.6% DW), and Chlorella sp. 800 (24.4% DW).
It is known that the production of lipids increases up to 65% by the nutritional deprivation of microalgae (Markou and Nerantzis 2013). In our case, we observed a smaller increase. This is because, despite being the original purpose, the high concentration of ammonium in the UW and the ability of C. fusca to metabolize it showed it was not in a status of nutritional nitrogen deprivation. The increase in lipid production is possibly related to the stress conditions induced by physicochemical conditions of the UW. Probably the solution may be to perform assays in two phases: the first to decrease the concentration of nutrients, essentially nitrogen, and a second crop in which, with nitrogen deficiency, lipid production increases. This solution was also given by Cai et al. (2013).
Moreover, it has been recorded that the maximum accumulation of lipids in Chlorella sp. is related to the pH of the medium, the optimum being pH values between 7.0 and 8.5 (Wang et al. 2010b; Sakarika and Kornaros 2016). In our study, the pH range in the treatments with UW was 6.4 – 8.1, therefore this parameter should be considered in later studies, in order to increase the productivity of lipids in C. fusca.
The cultivation of algae has expanded to new fields, such as feed and food, cosmetics, and biopharmaceutical products (Khan et al. 2018). In this regard, different authors investigated biomass productivity and the synthesis of amino acids on microalgae strains. Hempel et al. (2012) identified strains with an amino acid content of more than 40% DW: Spirulina platensis reached a protein content of 46.8% DW, Chlorella sp. 589 of 44.3% DW, and C. saccharophila 477 of 42.4% DW. In our study, a higher concentration of UW in the cultures leads to an increase in the production of proteins, implying a greater assimilation of nitrogen, which in the UW is mainly ammonium (Peralta López 2013). Given the high proportion of protein accumulated in 75% UW treatment (51%), this system can be considered as a form of protein production for commercial use.
In addition to demonstrating that the biomass of microalgae obtained in cultures with wastewater has a high content of bioproducts, these authors verified their capacity and efficiency for bioremediation. Although C. fusca is not a species commonly used in the remediation of UW (Pahazri et al. 2016), we demonstrate that it has the capacity to grow mixotrophically and accumulate nutrients from it. In this study, the maximum efficiency of DOC removal was obtained on the tenth day of exposure, with removal efficiency ranges that vary between 23.35% and 45.48% (Fig. 4). Katiyar et al. (2021) reported a higher total organic Carbon removal efficiency by C. minutissima and C. sorokiniana (95% and 98%, respectively) in wastewater collected from India (500 ml, for 12 days), compared to that recorded in this study.
This trend is justified by different authors: Eny (1951) observed that the metabolic route of Chlorella sp. could be altered with the supply of organic substrates such as glucose or organic acids, which means that they can perform not only autotrophic but also heterotrophic growth. The organic compounds can be used as an essential nutrient (Sachdev and Clesceri 1978) or as an accessory growth factor (Saunders 1957). The heterotrophic growth of microalgae (including Chlorella sp.) can be rapid, from the incorporation of organic substrates in the oxidative assimilation process for storage material production (Burrell et al. 1984).
In this study, TN decreased at the end of the incubation period (14th day) in the 50% UW and 100% UW treatments, obtaining a removal efficiency between 55% and 24.6%. Similar percentages were reported by Katiyar et al. (2021), with TN removal rates (12 days) for C. minutissima and C. sorokiniana as 28.46% and 40% respectively. An opposite tendency was observed by Lin et al. (2007), who reported that the relative NH4+ removal rate in lower leachate concentrations (10% and 30%) was higher than that in higher concentrations (50%, 80%. and 100%). In this sense, C. fusca is considered suitable microalgae for the degradation and elimination of nitrogenous waste present in UW.
The fact that microalgal cultures subjected to a high concentration of UW maintain their capacity for carbon removal is crucial for their possible use as a bioremediation system. The UW contains a microorganism cocktail (bacteria, protozoa, rotifers, among others) that feed on organic matter giving rise to a very high BOD5 (1000 mg l−1) (Madoni 2011). During the experiment, in the treatment with 100% UW, BOD5 decreased by 75% (from 1000 mg l−1 to 250 mg l−1). The dissolved organic matter in the UW is probably assimilated by microalgae and is thus eliminated. He et al. (2013) reported that the microalgae–bacteria consortium present in wastewater is more efficient in removing BOD (97%).
The dynamics of photosynthetic pigment content, changes in their ratio, as well as rETR and Fv/Fm associated with PSII, indicate C. fusca adaptation in response to the impact of UW addition to the culture medium. Different authors reported that the accumulation of Chl a is related to nitrogen metabolism (Rüdiger and López-Figueroa 1992). Chu et al. (2015) found that Chl a content in C. vulgaris cultures increased on the first days of incubation, when there was enough N in the medium, to decrease later when N was running low. In our case, the trend was similar, decreasing from day 10 until the end of the experiment. At the same time, in 50% UW and 75% UW concentrations, the carotenoid concentration increased at the end of the incubation period; similar results were reported by Kiran et al. (2014).
In this work, the increase in UW concentration was accompanied by a reduction of the Chl a/Chl b ratio and an increase in the pigment index (carotenoids/Chl a) in C. fusca. The Chl a/Chl b ratio can characterize the photochemical potential and biosynthetic activity of algae, controlling the absorbed light intensity (Tanaka and Melis 1997). Thus, under stress action, a decrease in Chl a content takes place, and accordingly the ratio between these two types of pigment decreases. When this happens, the pigment index increases due to the formation of carotenoids that perform a supporting and protective role in the photosynthesis. Chl a is the part of reaction centers and peripheral complexes of photosystems I and II, and Chl b is the component of the light-collecting complex of photosystem II. Therefore a change in the Chl a/Chl b ratio may indicate a shift of stoichiometric balance between the reaction center complexes of both photosystems and the light-collecting complex of photosystem II (Bodnar et al. 2016).
Also, the quality of the light inside each reactor was affected by the presence of UW, which possibly has a negative impact on cultures (Peralta et al. 2019). In this sense, the characteristics of the medium with different UW concentrations conditioned the biomass optical properties and consequently, the ETRmax and Fv/Fm. The UW treatments showed initial Fv/Fm values lower than the control, demonstrating the stress induced by the culture medium with UW at the beginning of the experiment, improving their yield at the end when manage to acclimate to the conditions of the culture medium. Similar results were obtained by Peralta et al. (2019), who reported that Fv/Fm was correlated with nitrogen content and maximal rETR with photosynthetic performance and nitrogen metabolism. The UW treatments reached the lowest rETR values at the end of the experiment in relation to the control. This indicates that microalgae could not achieve optimal photosynthetic activity, possibly due to the toxicity and turbidity of UW.
The cultivation of microalgae in UW is a promising alternative for its treatment, at the same time as it provides a culture medium with nutritional properties. Even though UW affected the photosynthetic activity of microalgae, they were able to grow and synthesize lipids and proteins. The TN, DOC, and BOD5 reduction efficiency increased with the exposure time, showing the potential of C. fusca to remove these compounds from effluents on a laboratory scale. Therefore, this is recommended as an eco-friendly method that should be tested for wastewater treatment on a larger scale, improving the factors limiting the performance of these microalgae-based wastewater treatment systems (Acién et al. 2016).