1. INTRODUCTION
Euglena sp. has a high potential for the utilization of biomass and primary metabolites. Microalgae have seen use as feed ingredients due to their antioxidant, neuroprotective, anti-cancer, and anti-inflammatory properties in addition to having a high nutrient content [1]. Studies have shown how Euglena affects human health. For instance, an oral dose of Euglena has been demonstrated to considerably enhance glycemic control and reduce fat buildup in the liver and abdominal regions [2]. A recent study revealed the possibility of creating unique, Euglena-based meals with anti-inflammatory and antioxidant properties rich in carotenoids, PUFAs, proteins, and other nutrients [3] and Euglena sp. is rich in active carbohydrate enzymes, complex carbohydrate synthesis, and fatty acids [4].
Food products containing Euglena extracts, such as yogurt, cookies, snacks, and beverages, are becoming increasingly common due to firms like Euglena Co. Ltd.’s successful marketing campaigns. Therefore, Euglena’s application in aquaculture is prospective because Euglena biomass in powder form improved innate immunity, disease resistance, oxidative balance, and metabolic condition in fish and bivalves [5]. While microalgae (including Euglena) are easy to cultivate, scaling that cultivation for large biorefineries is not economically feasible. Therefore, tofu whey wastewater (TWW) has the potential to replace Euglena cultivation medium and reduce production costs.
Euglena sp. cells have a high tolerance for unfavorable environments [6]. Euglena gracilis was reported to have resistance to various environmental factors such as temperatures, oxygen concentrations, salinity, and pH, against contaminant agents and tolerance to various osmolarities of medium. The Euglena cell can survive in a medium containing salinity of around 15 g/L [7]. According to Mulyadi [8], microalgae can absorb the excess minerals in TWW and utilize them as a source of nutrition for growth and development. The advantages of using TWW as a microalgae cultivation medium are its abundance, nutrient density, safety, and cost. TWW has low production costs because it can be obtained for free from tofu producers. TWW has a complete nutritional content compared to Cramer-Myers medium (CM medium), which is generally used in Euglena cultivation. TWW is enriched with the various micronutrient and macronutrient such as carbohydrates, proteins, and fats, as well as other nutrients such as P, N, Ca, K, Fe, Mg [9], ammonia (NH3), methane (CH4), oxygen (O2), hydrogen sulfide (H2S), carbon dioxide, and nitrogen (N2) [10]. However, most tofu processing industries (both small and large scale) dispose of tofu liquid waste directly into water sources, resulting in an unpleasant odor in the river and damaged river biota [11] because those high concentration of organic compound [12]. According to [13] The impact of discharge of tofu liquid waste includes such as reduced oxygen levels in water that caused rapid growth microbes, increased nutritional levels such as phosphorus and nitrogen, which can cause excessive algae growth (algal bloom) in the river. This can interfere with the balance of the ecosystem. To deal environmental pollution caused by tofu liquid waste, microalgae have a prominent role in environmental phytoremediation [14]. The development of the tofu industry in Indonesia has experienced rapid development. Tofu production grew from a small scale to a large factory scale. The process of producing tofu also produces TWW, which can harm the environment if not properly controlled [15].
[16] reported that TWW has a potential as a cultivation medium for microalgae due to its low toxicity and ability to accumulate nutrients. Chlorella pyrenoidosa cultivated in 60% TWW in autotrophic condition that obtained biomass productivity of 0.28 ± day-1 and the final biomass of C. pyrenoidosa was obtained 2.01 ± 0.12 g/L [17] reported that using 30% of tofu liquid waste concentration in the medium can produce around 9.850.000 cells/mL than the control medium (0% tofu liquid waste) around 7400,000 cells/mL in Chlorella spp. As another reported in the study of Salim [18], cultivating Scenedesmus in 20% TWW medium produced a peak cell density of 8,996.125 cells/mL. The Scenedesmus cells can perform photosynthesis faster due to the fulfillment of nutrition in the medium. C. pyrenoidosa cultivated in TWW achieved 2 into 2.3 g/L biomass and the productivity 0.64 g/L/day [19]. Syaichurrozi and Jayanudin [20] reported that the tofu liquid waste at concentrations of 0 v/v%, 2 v/v%, 4 v/v%, 6 v/v%, and 8 v/v% had a nitrogen amount of 0 mg, 3.59 mg, 7.189 mg, 10.78 mg, and 14.37 mg and in 6 v/v %, a high protein amount of 66,62% in Spirulina platensis. E. gracilis is one of various microalgae species that have highest tolerance to organic pollution which can get rid of 93% total nitrogen and 92% organic carbon [21,22]. Euglena sp. can grow in a variety of media because Euglena has capability to use up simultaneously different carbon source under distinct growth condition [23].
One of the advantages effects of using salinity stress in microalgae cultivation medium is increased lipid production, increased carbohydrate production [24-26], and capable of limiting the growth of undesirable microorganism [25]. The addition of 1.0 M NaCl concentration to the modified NORO medium at the end of the logarithmic growth phase resulted in increase in the lipid content of cells by 70.6 ± 3.9% compared to the lipid content without NaCl addition, which was 63.5 ± 1.0% [27]. Usage of high salinity has a potential to inhibit cell growth and change the morphology of the cells due to the osmotic pressure of the media and cells. Therefore, the use of salinity as a stressor must be determined specifically for the microalgae to be cultivated [25]. [28] reported that NaCl concentrations of 200 mM, 300 mM, and 400 mM could increase carbohydrate content by 20.39 ± 0.21%, 21.62 ± 0.16%, and 23.36 ± 1.10%, respectively, in S. platensis. The addition of 20% NaCl (10 mM) in Zarrouk’s medium can increase the carbohydrate content by 50% in S. platensis [29].
Based on the aforementioned background, tofu is a food that is commonly consumed in Indonesia, yet the tofu industry, both small and large scale, still generates liquid waste. This waste is often directly disposed of into rivers or other bodies of water without any treatment, resulting in an unpleasant odor and threatening the survival of aquatic biota. The liquid waste from tofu processing contains micronutrients and macronutrients such as fat, protein, and other nutrients including Mg, K, nitrogen, Fe, and others, which have the potential to serve as a medium to promote microalgae growth, specifically Euglena sp. Using tofu liquid waste as a cultivation medium, the production cost and activities in the biorefinery can be reduced. Therefore, this study aims to investigate the effect of a combination of tofu liquid waste and salinity on the growth of biomass, primary metabolites, and pigments in Euglena sp.
2. MATERIALS AND METHODS
2.1. Microalgae Strain and Inoculum
Euglena sp. was obtained from the culture collection faculty of biology, UGM. Euglena sp. was cultivated in CM medium under a 24/0-h light/dark cycle for 1 week. After that, the inoculum could be used following experiments. The CM medium composition includes the following: 1000 mg/L (NH4) 2HPO4, 1000 mg/L KH2PO4, 200 mg/L MgSO4.7H2O, 20 mg/L CaCl2.2H2O, 3 mg/L FeSO4.7H2O, 1.8 mg/L MnCl2.4H2O, 1.5 mg/L CoSO4.7H2O, 0.4 mg/L ZnSO4.7 H2O, 0.2 mg/L Na2MoO4.2 H2O, 0.02 mg/L CuSO4.5H2O, 0.0005 mg/L Vitamin B12, and 0.1 mg/L Thiamine, and 1 L of distilled water.
2.2. Preparation of CM and TWW
In this study, TWW was obtained from a local producer of tofu at the first production session on December 7, 2021, in Sleman, Yogyakarta Province of Indonesia. First, TWW was filtered using a filter paper (Whatman No.41). After that, the filtered TWW was mixed with CM medium at the percentages specified in Table 1. NaCl was added at 0, 2, and 4 g/L both to CM medium, TWW medium, or mixed medium after that, the pH was adjusted to 5.5 on all treatment and sterilization medium with an autoclave at 121°C for 20 min, and cooled medium until temperature room [30]. After that added 50 mL of Euglena sp. inoculum in 450 mL medium both to CM medium, mixed medium, or TWW medium.
Table 1: Percentage of tofu whey wastewater with combined salinity as medium cultivated in Euglena sp.
| TWW concentration | Variant of salinity (NaCl) | ||
|---|---|---|---|
| 0 g/L | 2 g/L | 4 g/L | |
| CM (0% v/v) | CMO | CM2 | CM4 |
| MI (75% v/v) | MIO | MI2 | MI4 |
| LO (100% v/v) | LO | L2 | L4 |
TWW: Tofu whey wastewater, CM: Cramer-Myers.
2.3. Determination of Growth Euglena sp.
The determination of cell growth rate was measured by a hemocytometer (Neubauer Improved Assistant), and the monitoring of cell density was done by a microscope (Olympus CX22LED) with optilab l301. The specific growth rate was calculated using equations 1 and 2 [31].
 |
Where μ is the specific growth rate, t2 and t1 are time points 1 and 2, Nt and N0 are the density at time points 1 and 2, respectively.
2.4. Determination of Growth Kinetics Modeling
Gompertz and Logistic models were used in the growth modeling of Euglena sp. [32,33]. The Gompertz model was calculated using equations 3 and 4:
 |
Where X is cell density, Xmax is the max cell density, X0 is initial cells density, SSR is Sum Square Residual and SST is Sum Square Total, rm is maximum cell production, and tL is lag time.
The logistic model was calculated with equations 5 and 6:
 |
Where μmax is the maximum specific growth rate.
2.5. Determination of Specific Biomass Productivity
10 mL of sample was centrifuged at 3300 for 15 min. After that, the water was separated from the precipitate and dried at 30–50°C for 12 h or until the weight became constant. The biomass of Euglena sp. was calculated using equation 8.
 |
The biomass productivity was calculated using equation 8.
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When the Ax and A0 are max and initial biomass, Tx and T0 are time of max biomass and time of initial biomass, respectively.
2.6. Measurement of Lipid
Lipid was calculated as reported by the Bligh and Dyar method [34]. Cell pellet and supernatant were collected by centrifugation (4000 rpm, 10°C, 15 min). Supernatant was discharged and the pellet was re-suspended in methanol/chloroform (2:1; v/v) and homogenized with vortex. Chloroform and aquadest (1:1; v/v) were added, homogenized with vortex, and centrifuged for 15 min (4000 rpm). The bottom layer was taken by micropipette and dried for 12 h in oven. Total lipid was calculated using equation 9 [35].
 |
The lipid content (%) and lipid productivity ware calculated using equations 10 and 11 [36].
 |
When bl, Bm, and Pr are wight of lipid, weight of biomass, and biomass productivity, respectively.
2.7. Measurement of Protein
The measurement of protein is based on the Bradford method [37] with modification. The cell pellet was collected by centrifugation (3300 rpm, 10 min). The pellet was washed using distilled water and then centrifugated 3300 rpm for 10 min., after which 167 μL of phosphate buffer saline was added and centrifuged for another 15 min (10000 rpm, 4°C). The supernatant was taken, put in a microplate, and added to the Bradford solution. The red dye will turn blue when it binds to the protein. Allow color to develop for at least 10 min before reading in the absorbance at 593 nm with ELISAA READER 3300×. The standard curve in this study used BSA. The protein content (%) and protein productivity were calculated using equations 12 and 13 [36].
 |
Where bp, Bm, and Pp are weight of protein, weight of biomass, and biomass productivity, respectively.
2.8. Measurement of Carbohydrate
The carbohydrate content was quantified using phenol-sulfuric acid methods as previously described [38]. The carbohydrate content and carbohydrate productivity were calculated using equations 14 and 15 [36].
 |
Where bk, Bm, and Pk are weight of carbohydrate, weight of biomass, and biomass productivity, respectively.
2.9. Measurement of Pigment
The measurement of pigment is based on the study by method [31] where 10 mL of sample was put in 15 mL conical tube, then centrifuged (3300 rpm, 15 min). The pellet was washed with distilled water and then centrifugated (6000 rpm, 15 min). Pellets were soaked in 10 mL of methanol, wrapped in aluminum foil, and placed in a water bath (70°C, 10 min), homogenized with a vortex, and centrifuged (6000 rpm, 10 min). The supernatant was taken and transferred to a spectrophotometer cuvette and the absorbance was calculated at wavelengths of 470 λ, 646 λ, and 663 λ. The pigment of Euglena sp. was calculated using equations 16, 17, and 18.
 |
Where chl a and chl b are chlorophyll a and chlorophyll b.
2.10. Statistical Analysis
All the tests were carried out in triplicate and presented as mean ± standard error. The significance of data was analyzed by Friedman’s test and Friedman’s 2-way ANOVA (IBM® SPSS® 26.0.0.0) by ranks (P < 0.05) to determine the pairwise comparisons.
3. RESULTS
3.1. Growth and Biomass on Euglena sp.
Based on Figure 1a, the highest cell density of Euglena sp. in the CM medium (9.667 ± 0.118 cells/mL) occurred with no added NaCl (CM0 condition) then CM2 treatment (7.958 ± 0.236 cells/mL) and CM4 (7.118 ± 0.724 cells/mL). This study yielded the same results as the previously reported [24] study that Chlorella emersonii obtained the highest density in the control treatment because it was sensitive to salinity then, in MI medium [Figure 1b] was obtained in MI2 (17.625 ± 0.125), MI4 (14.188 ± 0.688), and MIO (13.938 ± 0.063), while the density on LO medium [Figure 1c] was achieved L4 (14.375 ± 0.375), L2 (13.438 ± 0.563), and LO (12.750 ± 0.750). The highest specific growth rate [Table 2] in this study was obtained on MIO treatment (0.345 ± 0.067/day), L4 treatment (0.290 ± 0.027/day), and CM2 treatment (0.285 ± 0.047/day).
 | Figure 1: The growth curve of Euglena sp. with the different percentage and salinity of tofu whey wastewater (a) 0%, (b) 75%, and (c) 100%. Each point shows the mean ± standard deviation (n = 3) (P < 0.05). [Click here to view] |
Table 2: Total content and productivity of lipid, carbohydrates, and protein with various tofu whey wastewater and salinity percentage in medium.
| Culture mode | Treatment | Specific grow rate (/day) | Content (%) | ||
|---|---|---|---|---|---|
| Lipid | Carbohydrate | Protein | |||
| Autotrophic | CMO (0 g/L) | 0.245±0.024 | 20.6±2.9 | 22.1±4.2 | 10±0.79 |
| CM2 (2 g/L) | 0.285±0.047 | 33.3±4.7 | 25.1±1.4 | 10.8±1.40 | |
| CM4 (4 g/L) | 0.241±0.043 | 34.5±2.4 | 37.3±1.6 | 12.2±1.60 | |
| MIO (0 g/L) | 0.345±0.067 | 18.3±6.46 | 36.2±2.10 | 22.3±2.13 | |
| MI2 (2 g/L) | 0.333±0.044 | 23.3±2.59 | 42.5±0.20 | 25.2±3.85 | |
| MI4 (4 g/L) | 0.273±0.052 | 28.5±4.03 | 42.8±4.30 | 23.2±3.20 | |
| L0 (0 g/L) | 0.285±0.044 | 26.8±5.35 | 32.63±3.24 | 20.88±5 | |
| L2 (2 g/L) | 0.288±0.038 | 35.7±1.23 | 45.01±2.84 | 18.13±1.95 | |
| L4 (4 g/L) | 0.290±0.027 | 44.4±0.70 | 34.43±3.61 | 22.56±2.27 | |
Each measurement shows the mean±SD (n=3) (P<0.05). SD: Standard deviation.
In Figure 2, the treatments with the highest cell density in each TWW composition group (CM, MI2, and L4) were modeled using both logistic (to determine specific growth rates (μmax)) and Gompertz (to determine cell production [rm]) models. The growth rate parameter in both the logistic and Gompertz models has the same interpretation, which is the rate of growth. However, in the logistic model, the maximum capacity parameter describes the maximum growth limit, while in the Gompertz model, the scale parameter represents the limiting factor [33]. In the TWW 0% (CM), 75% (MI2), and 100% (L4) with the logistic model, the maximum specific growth rates (μmax) acquired were 0.789/day (R2 = 0.986), 0.854/day (R2 = 0.972), and 1.663/day (R2 = 0.893), respectively. While in the Gompertz modeling, the obtained cell production (rm) of TWW 0% (CM), 75% (MI2), and 100% (L4) was 1.89 × 105 (R2 = 0.991), 4.20 × 105 (R2 = 0.967), and 3.20 × 105 (R2 = 0.986), respectively. The obtained lag time (TL) of TWW 0% (CM), 75% (MI2), and 100% (L4) Gompertz model was 1.812/day, 2.672/day, and 0.829/day, respectively. According to the Gompertz model, the concentration 100% of TWW (L4) has the optimum medium for the fastest adaptation period compared to the other treatments. Therefore, according to Figure 2, the concentration of TWW 100% (L4) medium with Gompertz model has the shortest lag phase than other media conditions and increased the adaptation of periods of Euglena sp.
 | Figure 2: The growth modeling curve of Euglena sp. with different salinity and tofu whey wastewater (TWW) composition. (a) TWW 0% with salinity 0 g/L (CM). (b) TWW 75% with salinity 2 g/L (MI4). (c) TWW 100% with salinity 4 g/L (L4). Logistic (left) and Gompertz (right). [Click here to view] |
The periodic culture biomass of Euglena sp., based on biomass curves [Figure 3a], was highest on CMO (1.55 ± 0.065 mg/mL), CM2 (1.22 ± 0.078 mg/mL), and CM4 (1.21 ± 0.082 mg/mL), on the MI treatment in [Figure 3b], the biomass obtained to MI2 (2.77 ± 0.118 mg/mL), MI4 (2.30 ± 0.123 mg/mL), and MIO (2.00 ± 0.093 mg/mL), while in the LO treatment in figure 3c was obtained L4 (2.34 ± 0.079 mg/mL), L2 (2.99 ± 0.087 mg/mL), and LO (1.35 ± 0.101 mg/mL). The total productivity of biomass can be seen in [Figure 4]. There is a difference in biomass between MI and LO medium. The right concentration was achieved when cells utilized the organic content in TWW optimally. There is a difference in biomass between MI and LO medium. The right concentration was achieved when cells utilized the organic content in TWW optimally.
 | Figure 3: The biomass curve of Euglena sp. with the different percentage and salinity of tofu whey wastewater (a) 0%, (b) 75%, and (c) 100%. Each point shows the mean ± standard deviation (n = 3) (P < 0.05). [Click here to view] |
 | Figure 4: Total productivity of biomass, lipid, carbohydrates, and protein with various tofu whey wastewater and salinity percentage in medium. Each measurement shows the mean ± standard deviation (n = 3) (P < 0.05). [Click here to view] |
3.2. Lipid, Carbohydrate, Protein, and Pigmen
For lipid production [Figure 5], the highest lipid production in each TWW group occurred in CM4 (0.35 ± 0.012), MI4 (0.46 ± 0.015), and L4 (0.81 ± 0.020). The media containing high salinity (CM4, MI4, and L4) yielded higher total lipids compared to media with lower salinities.
 | Figure 5: Total primary metabolites of Euglena sp. with mixed tofu whey wastewater and salinity medium in various percentages. Each bar shows the mean ± standard deviation (n = 3) (P < 0.05). [Click here to view] |
According to Figure 5, the total carbohydrates in the CM treatment were found in the CM4 (0.37 ± 0.037 mg/mL), CM (0.28 ± 0.017 mg/mL), and CM2 (0.23 ± 0.012 mg/mL) treatments. While in the MI and LO treatments, the total carbohydrates were achieved in MI4 (0.48 ± 0.014), MI2 (0.44 ±0.015), MI (0.42 ± 0.008) and L4 (0.39 ± 0.013), L0 (0.33 ± 0.011), and L2 (0.29 ± 0.011), respectively. In the CM treatment, the CM4 obtained the highest carbohydrate concentration due to the role of salinity. [39] reported that one of the ways to enhance carbohydrate and lipid accumulation is cultivated in stress condition. In CM4, the high NaCl concentration (4 g/L) likely caused Euglena’s high carbohydrate production.
As represented on Figure 5 that the CMO (0.13 ± 0.013 mg/mL) treatment exhibits higher total protein than CM4 (0.12 ± 0.016 mg/mL) and CM2 (0.10 ± 0.012 mg/mL) in CM medium. While in the TWW medium, MI4 (0.14 ± 0.05 mg/mL), MI2 (0.13 ± 0.009 mg/mL), MI (0.12 ± 0.009 mg/mL), L4 (0.09 ± 0.006 mg/mL), L2 (0.05 ± 0.007 mg/mL), and LO (0.04 ± 0.004 mg/mL).
According to Figure 6, in the CM medium, CMO produced the highest total chlorophyll A (2.52 ± 0.146) and B (0.34 ± 0.018) concentrations than CM2 chlorophyll A (2.16 ± 0.129) and chlorophyll B (0.25 ± 0.021), and CM4 chlorophyll A (2.121 ± 0.223) and chlorophyll B (0.18 ± 0.018). The high chlorophyll A and B occurred in the death phase when microalgae were at maximum growth. The duration of cultivation affects the amount of chlorophyll A and B levels in Euglena sp. because chlorophyll will increase in line with the increasing cell growth. This is same opinion as Komarawidjaja [40] that the high cell growth will lead to high chlorophyll content in microalgae. In CM medium, the CMO treatment produced high chlorophyll A and chlorophyll B than the CM2 and CM4 treatment, because CM treatment did not get salinity stress so that maximum growth is obtained compared to the CM2 and CM4 treatments. According to the CM2 treatment, the total chlorophyll was obtained at a higher than CM4 due to the CM4 contained higher salinity stress then CM2 medium.
 | Figure 6: Total chlorophyll A, chlorophyll B, and carotenoids with various tofu whey wastewater and salinity percentage in medium. Each bar shows the mean ± standard deviation (n = 3) (P < 0.05). [Click here to view] |
As shown in Figure 6, CM4 achieved carotenoid concentrations higher than CM2 and CM media (0.56 ± 0.042, 0.37 ± 0.007, and 0.15 ± 0.040 mg/L, respectively), likely due to salinity stress caused by the increased NaCl concentration.
In the MI medium, the highest chlorophyll A and B was obtained in MI2, MI4, and MI medium. The chlorophyll A in 6.32 ± 0.32, 4.94 ± 0.18, and 2.88 ± 0.16 and in chlorophyll B was obtained 1.33 ± 0.01, 1.09 ± 0.13, and 0.93 ± 0.06, respectively. The carotenoid MI4 was obtained higher than MI2 and MIO, which are 0.96 ± 0.04, 0.90 ± 0.02, and 0.67 ± 0.08, respectively, while in LO medium, L4 medium performed the highest chlorophyll A and B than the others treatment. The value of chlorophyll A and B is 10.70 ± 0.14, 8.40 ± 0.52, 5.89 ± 0.32, 2.53 ± 0.07, 2.05 ± 0.14, and 1.91 ± 0.04, respectivelyin L4 in figure 5 was obtained highest carotenoids compared to MI2 and MIO with a value of 2.57 ± 0.002, 2.17 ± 0.002, and 1.74 ± 0.05, respectively. Apriati [41] reports that the addition of 25% of tofu liquid waste yielded a high chlorophyll A (3.53 mg/L) and B (3.72 mg/L) compared to controls (chlorophyll A (0.671 mg/L) and B (0.726 mg/L).
4. DISCUSSION
In the TWW, there are contained organic matters as phosphate and ammonium ions than can use absorbed cells in microalgae. The phosphate was the essential matters that have a function for replication and metabolism cells, while the ammonium has a function for photosynthesis cells [42]. In CM medium, the CMO treatment exhibited the highest growth rate compared to the others treatment (Cm2 and CM4) which can be attributed to the role of salinity. The salinity has the potential to significantly inhibit the growth of microalgae cells, as it affects both morphology and cell growth due to osmotic pressure between the media and cells. Microalgae cells exposed to salinity can undergo several adaptive mechanism, including ions regulation at the cell membrane, the accumulating protective fluid to counteract osmotic pressure, the accumulating of stress proteins, and enhancement of turgor pressure [25].
Based on [24], salinity affects enhanced lipid production by cells that are cultivated under salinity stress. It will affect recovering turgor pressure so that the accumulation will be restrained and impact the release and absorption of ions in the cell membrane. As a result, it will trigger osmosis in the cell and an increase in lipid content. Under stress conditions, cultivated microalgae accumulate lipids as an alternative nutrition to survive. In this study, cultivation was carried out by exposure light (24 h). This has the potential to increase lipid productivity levels in each treatment due to the role of the acetyl CoA carboxylase enzyme, which is both influenced by light and a precursor for lipid formation [43]. According to Takagi and Yosida [27], adding NaCl concentration 0.1 M can produce higher lipid content in cells cultivated amount 67% than with NaCl 0.5 M amount 60% in Dunaliella tertiolecta ATCC 30929.
The combination of salinity and nutrients in the media can alter the metabolism of microalgae by shifting the protein content to energy storage compounds such as carbohydrates [44]. In this study, MI and LO medium were used to evaluate carbohydrate production, with the MI medium showing greater yields than the LO medium. These results suggest that the MI medium achieved the optimal concentration of total wastewater (TWW) compared to the LO medium. This finding is consistent with the study by Syaichurrozi and Jayanudin [20], who reported that the addition of TWW at a concentration of 2 v/v% produced a higher amount of carbohydrates (61.23%) than a concentration of 8 v/v% (56.22%) in S. platensis.
In the protein, the CM medium was carried out like the study of [28] that the protein content in the salinity stress treatment of 200 mM, 300 mM, and 400 mM NaCl was obtained yield under control media in S. platensis. The MI (75% of TWW) medium was carried out like [16] that the addition of 60% TWW concentration can increase protein productivity by 97.2 ± 6.2 mg/L/h in C. pyrenoidosa. In the MI medium, higher protein was obtained than in LO medium. Because TWW medium, especially in LO, contained higher nitrogen than MI medium, while nitrogen was the most important source for increasing protein [45], high nitrogen caused beneficial effects.
In the CM medium, the CM4 treatment showed lower levels of chlorophyll A and B compared to the other treatments (CMO and CM2) due to the effect of salinity. Be and He [25] reported that high salinity can significantly inhibit cell growth. However, in terms of carotenoid content, the CM4 treatment exhibited the highest level compared to the CMO and CM2 treatments. Carotenoids play a protective role by guarding cells against oxidative damage by reactive oxygen species, which can cause enzymatic inactivation and cell death [46]. [47] suggested that carotenoids accumulate in organisms under stress, resulting in the synthesis of carotenoids in the chloroplast. Elloumi et al. [48] reported that under stress conditions (10 g/L NaCl), the accumulation of carotenoids increased relative to the control in Scenedesmus (3295.84 ± 5.750 and 2997.45 ± 2.01 μg/mL, respectively).
5. CONCLUSION
Based on this study, TWW could be used as an alternative medium for the growth of Euglena sp. Since TWW contains some essential nutrients such as N, C, P, Ca, K, and Fe, using it to enrich the growth media could enhance 0the metabolism of microalgae. A TWW: CM medium ratio of 3:1 (v/v) or TWW alone, combined with a specific salinity level (2 g/L and 4 g/L), could produce a higher growth rate, biomass, total lipid, total carbohydrate, total protein, and pigment then the sole CM medium. The higher grow rate obtained in MI2 treatment, biomass in MI2 treatment, total lipid in L4 treatment, total carbohydrate in MI4 treatment, and total protein in MI4 treatment. Our study resulted that TWW can be used to economically replace synthetic commercial media in large-scale cultivation to reduce the production cost in producing biorefinery activities.
6. ACKNOWLEDGEMENT
The manuscript was a part of first author thesis. Authors thank to Universitas Gadjah Mada of Indonesia for facilitating this research.
7. AUTHORS’ CONTRIBUTIONS
All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agreed to be accountable for all aspects of the work. All the authors are eligible to be an author as per the International Committee of Medical Journal Editors (ICMJE) requirements/guidelines.
8. FUNDING
There is no funding to report.
9. CONFLICTS OF INTEREST
The authors report no financial or any other conflicts of interest in this work.
10. ETHICAL APPROVALS
This study does not involve experiments on animals or human subjects.
11. DATA AVAILABILITY
All data generated and analyzed are included within this research article.
12. PUBLISHER’S NOTE
This journal remains neutral with regard to jurisdictional claims in published institutional affiliation.
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