As one of the major food sectors worldwide, the dairy-processing industry produces substantial amounts of waste and byproducts that can harm the environment and can be difficult to dispose of due to high nitrogen and phosphate contents (Shete and Shinkar, 2013). Therefore, the industry has been seeking green solutions for the treatment and/or utilization of its wastewater especially driven by new strict environmental regulations. Microalgae has recently gained attention as environmentally friendly wastewater bioremediation means due to their potential metabolic capabilities to treat waste streams, while at the same time produce valuable products.

Whey is known as one of the most challenging waste streams in the dairy industry. Depending on the process used to produce cheese or casein, different whey types are generated. Acid whey, as a major form of whey, is one of the by-products of acid-casein production and is a rich source of nitrogen, phosphate, and salts. It is generally further processed by filtration to obtain purified products for animal feed (Carvalho et al., 2013, Stasinakis et al., 2022). Based on the filtration process, the resulting permeate can have different compositions. The permeate derived from ultrafiltration (UF) would have its protein removed, while that derived from nanofiltration (NF) would have both protein and lactose removed. The NF-treated permeate has a relatively low biological and chemical oxygen demand as it is almost completely lactose- and protein-free, but remains rich in dissolved nitrate, phosphate, minerals, and salts (Chen et al., 2018) and still needs to be treated with conventional wastewater treatment practice such as anaerobic treatment (Demirel et al., 2005), agricultural methods (Wang and Serventi, 2019) or physico-chemical methods (Kushwaha et al., 2011). Treating this permeate, however, is difficult due to its high salinity, high nitrogen content, high phosphate content, and lack of nutrient configuration to support ideal bacterial growth in aerobic or anaerobic digestion (Prazeres et al., 2012).

One of the main issues with acid casein dairy waste is the elevated content of nitrogen, phosphate, and salt. Nitrogen and phosphate are removed by the application of biological treatments such as aerobic or anaerobic digestion. However, high nitrogen levels could significantly reduce the efficiency of nutrient removal or the capacity of the wastewater facility (How et al., 2020). Denitrification is introduced in cases that high levels of nitrogen are present in a waste stream. This process utilizes the nutrient resources in the wastewater inefficiently (Fernández-Arévalo et al., 2017), requires extra capital investment and processing costs (How et al., 2020) and generates no value-added products. In addition, high phosphate levels can lead to increased operating cost as a substantial amount of additives including ferric sulphate is needed to precipitate the phosphate out of the solution (Wang et al., 2019).

Microalgae-based methods have been introduced recently as eco-friendly, efficient, and valuable strategies for the treatment of different types of wastewater such as aquaculture waste treatment (Egloff et al., 2018, Tejido-Nuñez et al., 2019), removal of excess nitrogen sources (Qie et al., 2019), distillery wastewater treatment (Ravikumar et al., 2021), sequential bio-treatments of nutrients and metals (Rugnini et al., 2019). Microalgae are highly adaptable, having previously been shown to grow in environments with high salinity and unbalanced nutrients, such as cheese and casein powder manufacture (Qie et al., 2019, Shahid et al., 2020).

A number of studies have investigated the specific application of microalgae for the bioremediation of dairy waste. Daneshvar et al. (2018) studied freshwater (Scenedesmus quadricauda) and marine (Tetraselmis suecica) microalgae for the treatment of dairy wastewater revealing that microalgae can be utilized for effective tetracycline removal and simultaneous lipid production. Chokshi et al. (2016) employed dairy wastewater as a growth media for the cultivation of microalgae Acutodesmus dimorphus and achieved 100% ammonium reduction. Lucakova et al. (2022) cultivated Spirulina on enriched cheese whey (demineralised) and reduced the biomass production cost by 50%. de Almeida Pires et al. (2021) investigated the use of Chlorella vulgaris for the treatment of cheese-whey wastewater and demonstrated that bioremediation efficiency increased at higher inoculum ratio. Pandey et al. (2020) studied a cost-effective two-step process of coagulation followed by microalgae cultivation for simultaneous biofuel production and cheese whey wastewater treatment. Ghobrini et al. (2020) investigated the growth of Chlorella vulgaris on saline dairy wastewater and indicated that the microalgae can grow potentially on dilute and enriched waste streams. None of these studies, however, has investigated the bioremediation of wastewater derived from acid-casein production using a consortium of microalgae. Furthermore, the acid-casein wastewater presented a unique challenge due to the absence of lactose and other sugar molecules in the stream. The lack of easily accessible sugar sources, however, was compensated by the abundance of phosphate and protein in the wastewater. Microalgae cells has the ability to activate alternative hydrolytic pathways for metabolising nutrients from proteins in the wastewater in the absence of sugars (Choudhary et al., 2020). Studying the acclimation of microalgae on acid-casein wastewater can therefore reveal novel nutrient-assimilation pathways.

Microalgae single cultures may not be efficient enough to treat waste. Therefore, co-cultivation of microalgae with bacteria or with other microalgae has recently garnered attention as an innovative method for the bioremediation of different wastewater streams (Alam et al., 2022, Das et al., 2021). Co-cultures can sometimes lead to more effective nutrient utilization compared to homogenous cultures of the individual species due to beneficial interaction between species. Girard et al. (2017) showed that Scenedesmus obliquus and Chlorella protothecoides performed better in a consortium compared to the respective monocultures for the treatment of cheese by-product. Biswas et al. (2021) cultivated microalgae consortium in dairy wastewater as an eco-friendly treatment process successfully. Despite the increased bioremediation benefits conferred by co-cultivation, the nature of the synergy between species and the changes in cell population and physiology throughout the growth cycle still need further investigation (Qin et al., 2016).

Acid casein factories suffer from a large amount of nitrogen, phosphate, and salt in their wastewater with possible adverse environmental impact and inevitable limitation in the capacity of their wastewater treatment facility. There has not been a satisfactory solution reported to address this issue in the literature. Therefore, the primary aim of this study was to utilize the capabilities of different microalgae species and their consortium to assimilate excess nitrogen and phosphate simultaneously in the acid casein factory wastewater, as a green and sustainable solution. With this method, not only the environmental concerns and new strict regulations on effluent discharge can be addressed but also the improved efficiency and capacity of waste treatment facilities could be achieved by avoiding treatment bottlenecks caused by high levels of nitrogen and phosphate. For this purpose, three microalgae strains including Chlorella vulgaris, Tetradesmus obloquus, Nannochlropsis ocenica were used. This selection was made based on commercial importance of the three species and literature survey showing the complementary nature of these species in addressing the presence of phosphate, protein, and salt in the medium. Both monocultures and consortium cell cultures were studied for bioremediation of dairy waste. Flow-cytometry was employed as a tool in combination with conventional microscopic methods to track the changes in cell population and dynamics.