Introduction

Winery wastewater (WWW) originates from various sources involved in wine production, including cleaning and pressing of grapes, water utilized for tanks, transfer lines, floor washing, and also from wine losses (Mader et al. 2022). Their chemical characteristics vary significantly according to the operating conditions applied in each winery, the working period, and the type of wine produced. The pH values range between 2.5 and 12.9, the concentrations of COD and BOD between 320 to 49,105 mg L−1 and 203 to 22,418 mg L−1, respectively, and the nitrogen concentrations between 10 and 415 mg L−1, while high ethanol, phenolic, and sugar content has also been reported (Ioannou et al. 2015). The commonly applied management practices for WWW treatment include the use of conventional physicochemical and biological processes for the removal of major pollutants and their coupling with advanced oxidation processes for the removal of color and recalcitrant compounds (Davididou and Frontistis 2021; Latessa et al. 2023). Activated sludge process that is often used for WWW treatment removes sufficiently the major contaminants, but it is characterized by high operating costs due to the large sludge production and the increased energy consumption for maintaining aerobic conditions in the bioreactors (Mamais et al. 2015). Furthermore, its by-products have no significant market value. For this reason, alternative processes are often studied in the literature for the simultaneous treatment and valorization of WWW.

Recent publications show that microalgae technology has been successfully used for the treatment of various types of agro-industrial wastewater (Asadi et al. 2019; Tsolcha et al. 2018; Zkeri et al. 2021). Concerning WWW, the co-cultivation of microalgae and cyanobacteria (Tsolcha et al. 2017; Spennati et al. 2022) and the monoculture of microalgae and cyanobacteria in winery waste (Ganeshkumar et al. 2018; Marchão et al. 2021) have been successfully tested, showing that the removal of pollutants was significant while the produced biomass could be used as a marketable product. Specifically, Makaroglou et al. (2021) reported that microalgae Stichococcous sp. could remediate WWW and produce important amounts of biomass that ranged between 25 and 32 kg d.w. per 1000 m2 of a shallow pond type reactor, after 18 days of incubation. In the case where Chlorella vulgaris and the cyanobacterium Arthrospira platensis were co-cultured, Spennati et al. (2022) reported COD removal higher than 92%. The above experiments were conducted in different batch systems (tubular photobioreactor, open pond, and column photobioreactor) of 5 and 7 L and lasted for 15 days. The initial concentration of COD was 19.7 ± 0.7 g L−1 (Spennati et al. 2022). Tsolcha et al. (2017), in experiments where the cyanobacterium Leptolyngbya sp. and the microalga Ochromonas were co-cultured, achieved almost total COD, nitrogen, and phosphate removal. These batch experiments were conducted in a 4 L photoreactor (duration: 15 days) and the initial concentrations of COD, TN, and phosphates ranged between 1732 to 2043 mg L−1, 14.9 to 21.7 mg L−1, and 2.7 to 3.6 mg L−1, respectively. When a Chlorella sp. MM3 monoculture used for winery effluents with initial TN and phosphates concentration of 11.3 mg L−1 and 3.3 mg L−1, the removal of these pollutants was equal to 98% and 36%, respectively. These batch experiments lasted for 10 days, and they were conducted in 250 mL flasks with undiluted WWW (Ganeshkumar et al. 2018). Marchão et al. (2021) used different green microalgae for WWW treatment and studied their growth under mixotrophic and heterotrophic conditions. After 8 days of incubation in batch experiments conducted in 1 L flasks with Chlorella vulgaris, the maximum removal of COD, TN, and TP was equal to 92%, 91%, and 40%, respectively. The initial concentration of COD in these experiments ranged between 595 and 2441 mg L−1 while TN between 9.8 and 11.4 mg L−1. Additionally, to the above, research results from the characterization of the produced biomass showed 22% content of lipids when Chlorella sp. MM3 was used for treatment of 100% WWW and up to 51% when a mixture of winery and piggery wastewater was used (Ganeshkumar et al. 2018). Important concentrations of carotenoids (up to 8.7 mg g−1) and chlorophyll a and b (up to 20.9 mg g−1) were also found in Chlorella vulgaris biomass (Marchão et al. 2021), while Spennati et al. (2022) reported protein and carbohydrate concentrations up to 11 g/100 g d.w. and 12 g/100 g d.w., respectively. To the best of our knowledge, there is a notable absence of data regarding the cultivation of the strain Chlorella sorokiniana in WWW as well as the impact of biomass acclimatization to different media and the addition of ammonium nitrogen on its growth and process performance. Limited information is also available in the literature on the existence of pigments and tocopherols in microalgae biomass cultivated in WWW while there is no data on its amino acid profile.

The presence of bioactive compounds such as lutein, β-carotene, chlorophyll, and tocopherols in microalgae biomass is a matter of high interest due to the heath beneficial and colorant properties of these compounds and their applications to food, pharmaceuticals, and cosmeceuticals industry (Imchen and Singh 2023). Microalgae are the fastest-growing lutein source while their cultivation has added environmental benefits over plants with higher carbon sequestration, reduced water footprint, and no pesticide use (Muhammad et al. 2024). Chlorophyll pigments are found in green algae at high concentrations, especially chlorophyll a which presents anti-inflammatory, antimutagenic, and antioxidant properties (Imchen and Singh 2023). Tocopherols have also been associated with the antioxidant activity of Chlorella sorokiniana (Napolitano et al. 2020; Matsukawa et al. 2000). Additionally to the above, essential amino acids such as leucine, valine, and threonine are also found in microalgae and are considered of high nutritional value for aquaculture (Idenyi et al. 2022). Previous studies of our research team have shown the presence of several bioactive substances and amino acids in Chlorella sorokiniana biomass cultivated in dairy wastewater (Iliopoulou et al. 2022) as well as the occurrence of δ-tocopherol, lutein, and chlorophyll in Chlorella biomass grown in solar distillates of wine lees (Mastoras et al. 2023).

Based to the above, the main objective of this study was the use of Chlorella sorokiniana for WWW treatment and valorization. Batch experiments were initially conducted to examine the effect of biomass acclimatization to a chemical medium or urban wastewater, as well as the effects of wastewater dilution and nutrient addition on the growth of microalgae and the removal of COD, NH4-N, and TP. Experiments in sequencing batch reactors (SBRs) were afterwards conducted with raw and anaerobically pretreated WWW while the performance of a two-stage and a single-stage system was compared. The produced biomass was collected and characterized for proteins, lipids, carbohydrates, amino acid profile, and the presence of bioactive pigments and tocopherols in order to evaluate the potential valorization of microalgae.

Materials and methods

Supply of winery wastewater, microalgae, and chemicals

The WWW used in the current study was collected from a winery located in Lesvos Island (Greece). They were sieved through a 1 mm mesh, centrifuged, and filtered by 1 μm pore size filter. Their characteristics are shown in Table S1. In brief, the sample was characterized by acidic pH, with concentrations of dissolved COD ranging between 4400 and 6800 mg L−1 and nondetectable NH4-N. The culture of Chlorella sorokiniana UTEX 1230 was obtained from CCAP (Oban, UK). The stock culture was grown in Bold Basal Medium with threefold Nitrogen and Vitamins (3N-BBM + V) as described by OECD (2011).

Analyses of six bioactive substances (lutein, chlorophyll a, β-carotene, α-tocopherol, γ-tocopherol, δ-tocopherol) and eighteen amino acids (Table S2) were conducted in microalgae biomass samples. Information for the supply and preparation of standards can be found in previous articles of our research team (Iliopoulou et al. 2022; Martakos et al 2019).

Batch experiments

Batch experiments were initially conducted in cotton-gauze plugged 2 L flasks (final volume 1200 mL) at constant temperature (22 °C), under continuous light (24 h) and stirring to check the role of biomass acclimatization in urban wastewater and 3N-BBM + V medium as well as the role of ammonium nitrogen addition and wastewater dilution on process performance. Eight experiments were conducted in total. Their duration was 14 days, and the initial volume ratio of WWW to Chlorella sorokiniana biomass was equal to 70:30. The applied experimental conditions and the chemical characteristics at the start and at the end of each experiment are presented in Table S3 and Table S4. In experiment B1, undiluted WWW and Chlorella sorokiniana grown in Bold Basal Medium with threefold Nitrogen and Vitamins (3N-BBM + V) were used, while in experiment B2, the WWW was diluted with tap water at a ratio of 1:1. To check the role of biomass acclimatization to urban wastewater, the aforementioned conditions were also applied in experiments B3 and B4 with the difference that microalgae biomass had been previously acclimatized to urban wastewater as described by Kotoula et al. (2020). In order to examine the role of nitrogen addition, the experiments were replicated after addition of NH4-N at initial concentration of 40 mg L−1 (experiments B5 to B8, Table S4).

At the beginning of all experiments, the pH was adjusted to 7. Chlorella sorokiniana growth was monitored in a daily basis measuring optical density (OD) while wastewater samples were taken for COD, NH4-N, and TP at days 0, 3, 7, and 14. The produced biomass was collected at the end of the experiments after centrifugation; it was washed twice with deionized water, stored at − 20 °C, and freeze dried.

Sequencing batch reactor experiments

Two sets of SBR experiments were performed (Table S5). They were conducted at constant temperature (25 °C), under 16 h light/8 h dark and continuous stirring. The first experiment (experiment A) was conducted using two 2 L cotton-gauze plugged bioreactors (SBR1, SBR2) in series (Figure S1a). Diluted WWW with tap water or with medium (in ratio 1:1) was used while Chlorella sorokiniana had been acclimated to urban wastewater. In SBR1, NH4-N was added at the beginning of each cycle to achieve an initial concentration of 40 mg L−1. Eleven experimental cycles were carried out in total, and at the end of each experimental cycle, the effluent of SBR1 was transferred to SBR2 for further treatment. The final volumes of SBR1 and SBR2 were 1500 mL and 1000 mL, respectively. The hydraulic retention time (HRT) in each reactor was set to 4 days while the pH was adjusted to 7 during the experiments. In the first seven experimental cycles, the initial volume ratio of WWW to Chlorella biomass was 50:50 while diluted WWW with tap water in ratio 1:2 was used. At the last four experimental cycles, the WWW was diluted with medium in ratio 1:2.

The second experiment (experiment B) was conducted in a 2 L single-stage system (SBR3, Figure S1b) using anaerobically pretreated WWW to check the effect of wastewater pretreatment on reactor’s performance. The pretreated WWW was taken from a lab-scale anaerobic moving-bed biofilm reactor (AnMBBR) operating at ambient temperature and HRT of 36 h (Zkeri et al. 2021). The influents of the AnMBBR were prepared after WWW dilution with tap water in ratio 1:1 and pH adjustment to 7 with addition of 0.3 g L−1 Na2CO3 and NaOH solution. After anaerobic pretreatment, the average concentrations of COD in AnMBBR effluents were equal to 2269 ± 645 mg L−1. A total of eight SBR experimental cycles were carried out. The duration of each cycle was equal to 4 days while the volume ratio of anaerobically treated WWW to Chlorella biomass was equal to 50:50. The first five experimental cycles were conducted without NH4-N addition while at the beginning of the next cycles NH4-N was added to achieve an initial concentration of 27 mg L−1. The pH was adjusted to 7 during the experiments using phosphate buffer solution and NaOH solution.

For monitoring the performance of SBRs in both experiments, measurements of pH and optical density (OD) were conducted daily, while WWW samples were taken for COD, and NH4-N at the beginning and at the end of each cycle. The produced biomass was collected at the end of the experiments, was washed twice with deionized water, stored at − 20 °C, and freeze dried.

Analytical methods

Measurements of pH, temperature (T), and electrical conductivity (EC) were performed with a Consort C932 portable electrochemical analyzer. A 6405 UV/Vis spectrophotometer (Jenway UK) was used for OD measurements at 550 nm (Gatidou et al. 2019). The closed reflux colorimetric method was used for the analysis of dissolved COD. NH4-N was analyzed with titrimetric method after distillation while the ascorbic acid method was used for TP (APHA 2005). Filtered WWW samples from the inlet and the outlet of the reactors were used for the COD, NH4-N, and TP measurements. The produced biomass was collected at the end of the experiments after centrifugation; it was washed twice with deionized water, stored at − 20 °C, and freeze dried. The freeze dried biomass samples were characterized for proteins, lipids, carbohydrates. Proteins were determined based on AOAC official Method 2001.11 (AOAC International 2002; Thiex et al. 2002) and lipids according to the gravimetric method of D’ Oca et al. (2011). Starch was measured by the anthrone method (Hansen and Møller 1975), as modified by Marshall (1986). Chlorella sorokiniana freeze-dried samples were analyzed for lutein, chlorophyll a, β-carotene α-tocopherol, γ-tocopherol, and δ-tocopherol by high performance liquid chromatography coupled to diode array detector (HPLC–DAD), after extraction with ethanol, according to relative published methods (Lourenço-Lopes et al. 2022; Martakos et al. 2019). Detailed information on the applied method is provided in Supplementary material (Sect. 3A). The amino acid profile in Chlorella sorokiniana biomass samples was determined by liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) after acidic (Papastavropoulou et al. 2022) and alkaline hydrolysis (Yust et al. 2004). Detailed information on the applied methods is provided in Supplementary material (Sect. 3B).

Results and discussion

Experiments in batch reactors

The results obtained during batch experiments are presented in Table S3. In all experiments, the average temperature was similar ranging between 21.3 ± 1.2 and 22.4 ± 0.8 °C. The average values of pH ranged between 7.5 ± 0.6 (B5) and 9.0 ± 1.6 (B8) showing an increasing trend over the course of the experiments probably due to the consumption of CO2 during microalgae photosynthesis. Αn increase in biomass was observed in all batch experiments, indicating that Chlorella sorokiniana can be cultivated in WWW. The highest growth was observed in experiment B1 when undiluted WWW were used and Chlorella sorokiniana grown in Bold Basal Medium (Figure S2, Table S3).

As for major pollutant removal, with the exception of B1 and B3 where undiluted WWW was used and the initial COD concentrations exceeded 3700 mg L−1 (Table S3), the dissolved COD was removed by more than 77% up to the end of the experiments (Fig. 1). The acclimatization of biomass to different media did not seem to affect COD removal efficiency whereas the addition of NH4-N enhanced COD removal when undiluted WWW was used. As it can be shown in Fig. 1, the removal of COD in B5 and B7 was much higher comparing to B1 and B3 reaching 80%. In all these experiments, the initial concentration of COD was in the range of 3300 to 4300 mg L−1 (Table S3).

Fig. 1
figure 1

Removal efficiency of COD, NH4-N, and TP in batch experiments. At the first four experiments, no ammonium nitrogen was found (or added) at the start of the experiments

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Concerning the removal of NH4-N, important differences were observed between experiments B5 and B8 where ammonium nitrogen was added at the start of the experiments (Fig. 1). It should be noted that the removal of ammonium nitrogen is not presented for B1 to B4 experiments as no NH4-N was added in these cases. The highest removals were achieved when microalgal biomass had been previously acclimatized to urban wastewater, reaching 81% in B8 (initial NH4-N: 35.9 mg L−1) and 100% in B7 (initial NH4-N: 45.9 mg L−1). On the other hand, much lower removal efficiencies (< 66%) were observed in B5 (initial NH4-N 33.5 mg L−1) and B6 (initial NH4-N: 42.9 mg L−1) where Chlorella sorokiniana was acclimatized in medium (Table S4). In these cases, ammonium nitrogen and nitrate nitrogen, which was originally contained in 3N-BBM + V medium, co-existed in the experimental flasks. As a result, the available amount of total nitrogen at the start of these experiments could be in excess with concentrations higher than 70 mg L−1 (36.9 mg L−1 as NO3-N contained in medium + 33.5 mg L−1 as NH4-N for B7 and 42.9 mg L−1 for B8). This could potentially result to a lower removal efficiency of NH4-N by microalgae. In the literature, it is generally reported that microalgae prefer ammonium as a nitrogen source as the metabolic cost for its conversion to organic matter is lower comparing to that from the conversion of other forms of nitrogen (Salbitani and Carfagna 2021; Ortíz-Sánchez et al. 2023). Other alternative rationales for the reduced nitrogen removal observed in these two experiments could be the lower concentration of microalgae biomass in these flasks (Table S3) as well as the co-existence of microalgae with bacteria, which consume NH4-N via nitrification, in experiments B7 and B8 where Chlorella had been acclimatized to urban wastewater (Table S4).

As regards the removal of TP, it was ranged between 29% (experiment B5, initial TP 15.5 mg L−1) and 90% (experiment B3, initial TP 10.6 mg L−1). The acclimatization of microalgae to urban wastewater seems to enhance their ability to remove P. According to Fig. 1, higher removals (> 67%) were observed in all flasks where microalgae had been previously acclimatized to urban wastewater (B3, B4, B7, and B8). The ability of Chlorella sorokiniana to grow in different types of wastewater and remove efficiently phosphorus has also been shown in previous studies (Lee et al. 2022). The main mechanisms which are responsible for the removal of TP in these experiments are the assimilation of orthophosphates by microalgal components and the formation of polyphosphate granules into microalgal cells. In experiments B4 and B8 where average pH values higher than 8.5 were noticed, TP removal via precipitation is also possible (Kotoula et al. 2020).

Experiments in sequencing batch reactors

Taking into account the results of the batch experiments, acclimatized biomass to urban wastewater was used for the operation of SBRs. The environmental conditions applied during the experiments and the characteristics of raw and treated WWW are shown in Table 1. As reported in the “Sequencing batch reactor experiments” section, in experiment A, SBR1 and SBR2 were operated in series to examine the performance of a two-stage system with microalgae. The total HRT applied in the system was 8 days while the removal of COD and NH4-N was equal to 85 ± 9% (initial COD concentration 2503 ± 1215 mg L−1) and 91 ± 20% (initial NH4-N concentration 41.8 ± 21.2 mg L−1), respectively. As shown in Figure S3a, both reactors contributed to COD and NH4-N removal. Similar COD (78 ± 9%; initial COD concentration 996 ± 256 mg L−1) and NH4-N removal (95 ± 9%; initial NH4-N concentration 25.3 ± 6.8 mg L−1) was also observed when a single-stage SBR system with a HRT of 4 days and feeding on anaerobically pretreated WWW was used (experiment B, Figure S3b).

Table 1 Operational conditions and characteristics of winery wastewater (WWW) and Chlorella sorokiniana biomass in sequencing batch reactor experiments. SBR1 and SBR2 were connected in series; the hydraulic retention time in each of them was 4 days. SBR3 was fed on anaerobically pretreated WWW; the hydraulic retention time was set to 4 days. Eleven experimental cycles were run for SBR1 and SBR2, whereas eight were run for SBR3. Samples were analyzed in each experimental cycle, and mean ± SD values were calculated (n = 11 for SBR1 and SBR2, n = 8 for SBR3)
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Concerning the removal of major pollutants during the different experimental cycles of experiment A, it was observed that the average COD and NH4-N removal was 68 ± 9% and 88 ± 9%, respectively, in the first seven experimental cycles were WWW diluted with tap water was used (Fig. 2a). During the last four experimental cycles, WWW was diluted with medium 3NBBM + V, the average COD removal was increased to 79 ± 1% while the average ammonium nitrogen removal was reduced to 46 ± 3%. A similar reduction of ammonium nitrogen removal was also observed in the batch experiments contained 3NBBM + V medium and it has been discussed in the “Experiments in batch reactors” section.

Fig. 2
figure 2

Removal of COD and NH4-N in the different experimental cycles of experiment A where SBR1and SBR2 operated in series (a) and in experiment B where SBR3 operated with anaerobically pretreated WWW (b)

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Study on the removal of major pollutants in the different experimental cycles of experiment B showed that during the first five experimental cycles, the COD removal gradually increased from 65 to 90% (mean value 80 ± 11%) while the addition of ammonium nitrogen in the last three experimental cycles resulted to a slight decrease of COD removal between 71 and 76% (mean value 74 ± 3%) (Fig. 2b). Concerning the ammonium nitrogen, high removal rates, which ranged between 85 and 100%, were observed in the three experimental cycles when NH4-N was added (mean value 95 ± 8%).

The removal efficiencies achieved in the SBR experiments of the current study are similar or even higher than those observed in previous articles with WWW and microalgae. Specifically, in the study of Marchão et al. (2021) where Chlorella vulgaris were used for WWW treatment under mixotrophic and heterotrophic conditions, it was reported that the removal of major pollutants after 8 days of incubation in batch experiments conducted in 1 L flasks was 92% for COD (initial COD concentration 595 to 2441 mg L−1) and 40% for TN (initial TN concentration 9.8 to 11.4 mg L−1). Ganeshkumar et al. (2018) showed that Chlorella sp. MM3 removed 98% of TN from undiluted WWW with initial concentration of 11.3 mg L−1 after 10 days of incubation in batch experiments conducted in 250-mL flasks. Finally, Tsolcha et al. (2017) observed COD and TN removal up to 86% and 84%, respectively, when cyanobacterium Leptolyngbya sp. and microalgae Ochromonas were used for WWW treatment in batch experiments conducted for 15 days in a 4 L photoreactor. The initial concentrations of COD and TN in these experiments were ranged between 1732 and 2043 mg L−1 and 21.7 and 14.9 mg L−1, respectively.

Proteins, lipids, and carbohydrates in the produced biomass

Βiomass samples were collected at the end of batch and SBR experiments and analyzed for proteins, starch, and lipid content. For SBR experiments, Chlorella biomass used as inoculum was also collected at the start of experiment A and characterized for the aforementioned parameters.

According to the results, important differences were observed on the protein content of collected biomass in different batch experiments, ranging between 13.5% (B4) and 58.8% (B5) (Fig. 3). In general, the addition of ammonium nitrogen and the growth of biomass to 3NBBM + V medium enhanced the protein content found in microalgal biomass (B5, B6). Concerning the SBR experiments, almost 13% increase of total proteins was observed comparing the protein content of initial biomass (33.8%) and that analyzed at the end of experiment A in SBR1 (47.6%) and SBR2 (46.7%). A slight increase was also observed when anaerobically pretreated WWW was used in SBR3 (Fig. 3). It should be noted that ammonium nitrogen was added in all experimental cycles of experiment A, while in experiment B, ammonium nitrogen was added only at the last part of the experiments. So far, there is only one study where the total proteins of microalgal biomass were analyzed in experiments with WWW. Specifically, Spennati et al. (2022) reported concentrations up to 11 g/100 g d.w. in a co-culture of Chlorella vulgaris and Arthrospira platensis. These concentrations were achieved in a column photobioreactor treating diluted WWW (20% w w−1). In a previous study of our research team, Iliopoulou et al. (2022) reported that the protein content of Chlorella sorokiniana ranged between 28.6 and 52.8% when grown in dairy wastewater. In a recent study where the diluted solar distillate of wine lees was used as substrate for Chlorella sorokiniana growth, its protein content ranged between 25.9 and 42.8% depending on the dilution rate with 3 N-BBN + V medium (Mastoras et al. 2023).

Fig. 3
figure 3

Total proteins, carbohydrates, and lipids in Chlorella sorokiniana biomass collected from batch and SBR experiments. Due to the limited amount of collected biomass, lipids were not analyzed in biomass samples from B5, B6, and SBR1 Final. Similarly, for carbohydrates and biomass from experiment B6

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In all batch experiments, high biomass lipid content was observed ranging between 35.8 (B8) and 48.2% (B3) (Fig. 3). This high lipid content is probably related with Chlorella sorokiniana starvation during batch experiments. In the SBR experiments, the lipid content of the biomass at the start of the experiment was low (23.8%) and remained almost stable in SBR2. On the other hand, a significant decrease was observed in SBR3, reaching 3.9% at the end of the experiments (Fig. 3). In a previous study where Chlorella sp. MM3 was used, Ganeshkumar et al. (2018) reported that the biomass contained 22% lipids when 100% WWW was used. Tsolcha et al. (2017) reported 13% lipid content in cyanobacterium Leptolyngbya sp. biomass used for combined winery and raisin wastewater treatment. Finally, Spennati et al. (2021) measured lipid content higher than 20 g/100 g d.w. in biomass originated from co-culture of Arthrospira platensis and Chlorella vulgaris in WWW.

As regards the concentrations of carbohydrates in Chlorella sorokiniana biomass, with the exception of B1, carbohydrate content higher than 30% was observed in all other batch experiments (Fig. 3). In SBR experiments, no significant change in the percentage of carbohydrates was observed between the initial and final biomass in SBR1 and in SBR2. On the other hand, the amount of carbohydrates at the end of the experiments conducted in SBR3 was reduced to 16.2% (Fig. 3). To the best of our knowledge, there is only one study where carbohydrates have been analyzed in microalgal biomass cultivated in WWW. Specifically, Spennati et al. (2022) observed carbohydrates concentrations up to 12 g/100 g d.w. in co-culture of Chlorella vulgaris and Arthrospira platensis. When Chlorella sorokiniana was cultivated in diluted solar distillate of wine lees, its starch content ranged between 21.2 and 56.9% (Mastoras et al. 2023).

Pigments, tocopherols, and amino acids in the produced biomass

The concentrations of lutein, β-carotene, chlorophyll, and of α-tocopherol, γ-tocopherol, δ-tocopherol and the sum of tocopherols in Chlorella sorokiniana biomass samples are summarized in Table S6.

Figure 4 illustrates the concentrations of lutein in Chlorella sorokiniana biomass obtained after the different batch and SBR experiments. It is observed that the concentrations of lutein in the batch experiments (B1–B7) ranged between 3.7 and 29.3 mg kg−1, whereas in the batch experiment B8, a significantly increased concentration of 338 mg kg−1 was determined, indicating that the simultaneous addition of NH4-N and dilution of the WWW favor the biosynthesis of lutein by Chlorella sorokiniana. Lutein concentrations of the same order to B8 were obtained for SBR1 and SBR2 experiments while the conditions applied to SBR3 experiment appear to enhance even more the production of lutein reaching a concentration of 1075 mg kg−1 at the end of the experiment. Specifically, in our recent study where Chlorella sorokiniana was cultivated in dairy wastewater, the concentrations of lutein in the microalgal biomass ranged between 442 and 508 mg kg−1 (Iliopoulou et al. 2022). Additionally, Mastoras et al. (2023) reported lutein concentrations up to 342 mg kg−1 in Chlorella sorokiniana biomass grown in wine lees solar distillates. According to a recent review paper on the production of lutein from microalgae, the most critical parameters that enhance the lutein content in microalgal biomass are the temperature, the applied light wavelength, and the harvesting of biomass (Muhammad et al. 2024). Further research is required to find the optimal conditions for the production of microalgae biomass with high lutein content that has been cultivated in WWW.

Fig. 4
figure 4

Lutein and chlorophyll concentration in Chlorella sorokiniana samples collected from batch and SBR experiments

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As regards the concentrations of chlorophyll, in batch experiments, concentrations up to 490 mg kg−1 were found (B8) (Fig. 4). In a recent study, Marchão et al. (2021) reported concentrations of chlorophyll α and β up to 20.9 mg g−1 in Chlorella vulgaris biomass used for WWW treatment. The operation of SBR systems resulted again to the increase of chlorophyll concentrations with the highest concentrations noticed in SBR2 (313 mg kg−1). A similar trend was also observed for α-tocopherol, γ-tocopherol, δ-tocopherol, and β-carotene where the cultivation of microalgae under sequencing batch conditions enhanced the concentration levels of these compounds. It worth to be mentioned that at the end of the experiment with SBR3, the sum of tocopherols at the microalgal biomass was equal to 131.2 mg kg−1 (Table S6). Tocopherols are lipid-soluble antioxidants that are synthesized only by photosynthetic organisms and have vitamin E activity. Previous articles have shown that the tocopherols content of microalgae depends on the concentration and the form of available nitrogen as well as on microorganisms’ growth phase (Durmaz 2007).

The total amino acids concentrations in batch experiments ranged between 9.4 g/100 g and 19 g/100 g (Fig. 5). Higher amounts were measured in B1 and B2 biomass samples where Chlorella grown in medium 3N-BBM + V was used than in B3 and B4 where Chlorella had been previously acclimatized in urban wastewater (Fig. 5, Table S7). Comparison of amino acid concentration levels in biomass collected from B7 and B8 with those collected from B3 and B4 showed that the ammonium nitrogen addition enhanced total amino acids’ production. Concerning the operation of the SBRs, a slight increase of amino acids was observed in the two-stage system, reaching 26.3 g/100 g in SBR2 whereas no important differences were noticed in SBR3 (Fig. 5). Among different amino acids, the highest concentrations were measured for glutamic acid (3.21 g/100 g in SBR2; 2.82 g/100 g in SBR3) and aspartic acid (2.99 g/100 g in SBR2; 2.61 g/100 g in SBR3) (Table S7). It is the first time that the concentrations of amino acids are measured in microalgal biomass cultivated in WWW. In a previous study of our research team, the concentrations of amino acids in Chlorella sorokiniana biomass cultivated in dairy wastewater ranged between 18.86 and 40.22 g/100 g dried biomass (Iliopoulou et al. 2022).

Fig. 5
figure 5

Total amino acids concentrations in Chlorella sorokiniana samples collected from batch and SBR experiments

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Conclusion

The results of this study showed that the microalgae Chlorella sorokiniana can be cultivated in WWW. The acclimatization of biomass to urban wastewater enhanced the removal of NH4-N and TP whereas it did not affect the removal of COD. The SBR experiments showed that the use of the two-stage system resulted to efficient removal of COD and NH4-N while the produced Chlorella biomass had high protein and chlorophyll content. The single-stage system removed also efficiently the conventional pollutants at a lower HRT while the produced biomass was characterized by higher concentration of lutein and tocopherols. The results of the present study indicate that the best method for the treatment and valorization of WWW is the operation of a single-stage SBR at HRT of 4 days using anaerobically pretreated wastewater as a feed as it removes efficiently the major pollutants while the produced biomass contains high concentrations of bioactive substances. Further research could lead to a larger-scale production of these high value ingredients by Chlorella sorokiniana cultivation in WWW contributing to the sustainable management of winery waste and circular economy.