1. Introduction
Surimi-based products are particularly popular among consumers due to their greater protein, lower fat, higher nutrition, and ready-to-cook or eat capability [1–2]. Surimi is pulverized and washed fish meat in the form of a stabilized myofibril protein concentrate that comes in a variety of shapes and sizes, ranging from fish balls to seafood imitations such as crab sticks [3]. In the surimi production, at least 35% of the water was used for cleaning and mincing, with the remaining 65% used for mince washing. During the mince washing process, 1 kilogram of surimi might provide 27–29 L of surimi wastewater (SWW) [4]. SWW is typically treated through aerobic or anaerobic technique. Nevertheless, according to Amenorfenyo et al. [5] and Amaro et al. [6], aerobic method has disadvantages since it not only removes organic and nutritional value but also produces more CO2 and sludge. Meanwhile, anaerobic process possesses disadvantage of not being able to remove the overall nutrients such as nitrogen and phosphorus, and requires the addition of base to adjust alkalinity [7–8]. As a result, it is a challenge to develop an appropriate technology that is both environmentally-friendly and economically effective. Improper disposal of SWW could have a harmful influence on the ecosystems since SWW had a 0.8–5.0 g/L protein, accounting for 15–30% of the original protein in fish, COD (1,000–2,000 mg/L), lipid (0.15 mg/L), vitamins, amines, colors, and enzymes depending on the fish types [9–12].
In recent years, the integration of microalgae-based wastewater treatment systems has garnered substantial recognition within the realm of sustainable environmental practices. Particularly noteworthy is the application of these systems to the management of wastewater emanating from the seafood processing industry, a sector that has long grappled with the intricacies of waste disposal. A primary advantage lies in the remarkable reduction of chemical oxygen demand (COD), a pivotal indicator of water pollution [13–14]. Additionally, this approach facilitates the recycling of essential nutrients found within wastewater, effectively converting a challenging waste stream into a valuable resource. Likewise, microalgae bioremediation offers an additional benefit to the avoidance of hazardous byproducts, which is very encouraging from an industrial perspective. To balance implementation costs and correspond to additional economic input, the biomass collected at the end of the process can be turned into products with a commercial value, such as feed, food, biofuels, or biofertilizers [15]. This duality of purpose serves as a testament to the overarching commitment to sustainability and resource optimization in contemporary environmental science [16–18].
What renders microalgae-based wastewater treatment particularly compelling is the remarkable adaptability of microalgae species. Microalgae exhibit an impressive versatility, allowing them to thrive in diverse water resources, ranging from saltwater to brackish water, and remarkably, even within the complex milieu of wastewater sources [19–21]. Currently, most research on the relationship between microalgae and wastewater treatment has concentrated on photoautotrophic cultivation [22–23]. Photoautotrophic cultivation of microalgae, on the other hand, has typically been limited by lighting and inorganic carbon sources [18–19]. Meanwhile, previous research has demonstrated that some microalgae can grow in both photoautotrophic and heterotrophic environments or mixotrophic conditions which perform well in wastewater purification [19, 20, 25]. Furthermore, some carbon dioxide produced by heterotrophic metabolism in a mixotrophic culture system can be absorbed by photoautotrophic metabolism [24]. As a result, mixotrophic production of microalgae is becoming increasingly appealing for wastewater treatment.
Organic sources have a significant impact on the growth of mixotrophic microalgae. Some common organic substances, such as glucose, acetate, and glycerol, have been shown to considerably stimulate the growth of microalgae and are thus regarded as ideal organic carbon sources for mixotrophic microalgae production. One of our experiments investigated at the effects of combining glucose and glycerol in virgin coconut oil effluent on mixotrophic culture of microalgae, and the results showed that the addition of organic carbons had a significant promotion effect on microalgal growth [19]. In this study, the effect of introducing molasses as a carbon source was proposed. Sugarcane molasses is a sugar industry waste that is high in sugars, primarily sucrose, glucose, and fructose [25]. Therefore, it is thought to be a promising feedstock for providing cheap organic carbon for microalgal development. Previous study has shown that molasses can be used efficiently for microalgal growth as a carbon source for the biomass production of Botryococcus braunii, Chlorella vulgaris, C. sorokiana, C. zofingiensis, Haemotococcus pluvialis, Scenedesmus obliquus, and Spirulina platensis [26–28]. Şimşek et al. [29] cultivated Chlorella vulgaris in the freshwater supplemented by 2 g/L molasses and resulted high cell optical density (± 0.4), protein (205.4 μg/mL) and lipid (57.15%). Scardoelli-Truzzi and Sipaúba-Tavares [30] showed that when 0.75 g/L of sugarcane molasses was added, H. pluvialis biomass increased more than when phototrophic conditions were used in terms of cell volume and dry weight. Under mixotrophic circumstances, the growth, carbohydrate, protein, and lipid of C. vulgaris after 6 days rose by approximately 94.4%, 94.37%, 98.23%, and 57.04%, respectively, corresponding to 0.45% (v/v) molasses [31].
Aside from carbon source, the primary parameters influencing the growth rate and chemical compounds of microalgae are nitrogen and iron availability. Nitrogen is a macronutrient that is crucial for microalgal growth and is necessary to produce proteins, lipids, and carbohydrates [32–33]. In general, nitrogen content has a substantial impact on the high growth and biochemical compositions of microalgae such as protein and pigments [34]. Nitrogen can be assimilated by microalgae as nitrate, nitrite, urea, and ammonium. However, nitrate is more frequently employed for microalgae production than ammonium salt due to nitrate’s greater stability and lower likelihood of pH shift. Nitrate (NO3−) is usually utilized in culture conditions because ammonia (NH4+) concentrations larger than 25 M are harmful to microalgae [35]. In the meantime, microalgae have comparatively high nutritional needs. Microalgae require 56.3% of carbon, 8.6% of nitrogen, and 1.2% of phosphorus, according to Sari et al. [36]. In particular, Whitton et al. [37] found that the Redfield Ratio, or C : N : P molar ratio, for marine phytoplankton is 106 : 16 : 1. On the other hand, concentrated SWW’s N content was lower than needed (Table S1), indicating a lack of available nitrogen sources. Consequently, the medium needs to be supplemented with additional nitrate. Araujo et al. [38] reported that when sodium nitrate was raised from 25 to 75 mg/L, the biomass production of Nannochlorpsis oculata (from 0.4 to 2.2 g/L) and Chlorella vulgaris (from 1.4 to 3.4 g/L) rose significantly. On the other hand, when sodium nitrate was lowered, biomass recovery for Chlorella vulgaris and Nannochlorpsis oculata decreased by 59% and 81%, respectively. Additionally, Barman et al. [39] discovered that Tetraselmis chuii grown in a 500 mg/L sodium nitrate solution exhibited noticeably increased biomass, protein, and carbohydrate, which accounted for 0.014 g/L, 37.75%, and 4.1 mg/L, respectively. In addition, iron is another necessary metallic component that is important to numerous microalgae biological activities such as enzymatic activities, gene expression, and other metabolic processes [40]. Contrarily, excessive iron can increase the production of lipids or carbohydrates while also having an inhibiting effect on the productivity of microalgae cells [41]. According to Polat et al. [40], Auxenochlorella protothecoides showed resistance to high iron species concentrations, yielding 78.5% FAME and 1.15 g/L of biomass at 1.15 mM ferric chloride. The addition of 15 g/L ferric chloride also resulted in a considerable increase in the maximum cell density of Nannochloropsis sp., reaching 86.39×106 cell/mL compared to control [42].
Given the preceding description, microalgae have the ability to grow in SWW when molasses, sodium nitrate, and ferric chloride are also added as nutritional supplies while producing high chemical compounds such as protein, and lipid. Nevertheless, the investigation of cultivating marine microalgae in SWW has not been further upon by optimizing different quantities of molasses : ferric chloride and molasses : sodium nitrate. Therefore, this research was performed to determine the influence of molasses as source of organic carbon, nitrogen, and iron, on microalgae growth, nutrients removal and its chemical composition while cultivated in SWW mixotrophically.
2. Materials and Methods
The present study’s methodology is illustrated in Fig. S1, Fig. S2 and provided in the following description.
2.1. SWW Preparation
SWW was acquired from a surimi-producing industry with a daily waste output capacity of 275,000 – 378,000 L located in Sidoarjo East Java Indonesia. The wastewater was held at −20°C to prevent deterioration. This is a result of low temperatures inhibiting the growth of microorganisms that lead to degradation and spoiling. Nutrient degradation over time is prevented by freezing to a temperature less than −18ºC, which inactivates all bacteria, yeasts, and molds that may be present in the medium [43–44], therefore, the wastewater containers in this study were operated at −20°C. Afterwards, the particle matter in thawed SWW was filtered using GF/C glass fiber filters (Whatman, 110 mm). In order for microalgae to ingest the food sources in SWW, complex molecules including proteins, lipids, and carbohydrates in waste had to be lysed through fermentation process using effective microorganism (EM4) followed Maulana et al. method [45]. The microorganisms in EM4, Lactobacillus casei (2 × 106 cell/mL) and Saccharomyces cerevisiae (3.5 × 105 cell/mL), were previously activated by diluting it with water in a ratio of 1 : 20 (EM4 : water) for 7 d at a room temperature anaerobically. The SWW fermentation process was then carried out for 15 d at a 1 : 20 ratio (SWW : activated EM4). Afterwards, the wastewater was sterilized in an autoclave at 120°C for 30 min [17]. The SWW physicochemical characteristic is shown in Table S1.
2.2. Strain Preparation
Six strains of marine microalgae were employed in this study encompassing Spirulina sp., Chlorella vulgaris, Nannocholorpsis oculata, Chaetoceros calcitrans, Porphyridium cruentum and Tetraselmis chuii, respectively. The isolated algal strain was obtained from Institute for Mariculture Research and Development Gondol Bali Indonesia. Microalgae were grown under photoautotrophic condition in photobioreactors at 22 – 27°C and 6,000 – 8,000 lux of light with continues aeration. The medium of cultures comprised of standard Kw 21 fertilizer (consisting nitrogen (49 g/L), phosphoric acid (4 g/L), boron, manganese, iron, cobalt, zinc, EDTA, amino acid complex, vitamin complex (thiamine (vitamin B1), cyanocobalamin (vitamin B12), biotin) and 30 ppt of sea water. Likewise, 0.5 mL/L silica was added in the medium of diatom (Chaetoceros calcitrans).
2.3. Microalgae Screening
Each microalga in exponential phase was introduced to the 1 L flask in triplicate at a concentration of 20% (v/v), to which SWW and sterile salt water at five different quantities (v/v) were added (0% (0 : 0.8 L); 5% (0.05 : 0.75 L); 10% (0.1 : 0.7 L); 15% (0.15 : 0.65 L); 20% (0.2 : 0.6 L)). To calculate the growth rate and biomass, the optical density of the cells was measured at 750 nm every day with a Shimadzu UV-1201 spectrophotometer for 10 d (see analysis section below). The COD, nitrate, and phosphorus concentrations were also determined (see the analysis section below). At the end of the 10-d incubation, the highest final biomass in the culture was analyzed by centrifugation and spectrophotometric detection; in contrast, protein, and lipid contents in the pellet was determined (see analysis section below).
2.4. Mixotrophic Cultivation
Microalgae with the highest biomass and growth rate at certain SWW concentration were re-cultured under the same conditions as in Section 2.2. The microalgae were cultivated in a mixotrophic condition by applying molasses : sodium nitrate and molasses : ferric chloride. Sugarcane molasses had a pH of 5.2 and contained water, fructose (24.18%), glucose (24.18%), sucrose (29.64%) and metal ions such calcium, potassium, sodium, iron, magnesium, copper, and others [31]. Crude molasses was diluted in distilled water and autoclaved for 30 min at 1 atm. The fluid was then utilized in mixotrophic culture experiments. The goal was to find the optimum concentration at which microalgae grew successfully with high biomass production, nutrient reduction, and chemical compounds concentrations namely lipid, and protein (see analysis section below). As a result, with 13 total experimental runs, the optimum growth condition, significance, and interaction effects of molasses : sodium nitrate and molasses : ferric chloride on biomass production and nutrient removal productivity were investigated using the central composite design (CCD) response surface methodology (RSM). The ranges employed in these studies were 1, 2, and 3 g/L for molasses (X1), 50 and 100 mg/L for sodium nitrate (X2), and 50 and 100 mg/L for ferric chloride addition (X2). The empirical form of the second order polynomial model (Eq. 1) can be expressed in Eq. (1).
where y is the predicted value; β0, βi, βii, and βij are a constant, linear, quadratic, and the interaction coefficient, respectively; and xi and xj are independent variables of the model.
2.5. Analytical Method
2.5.1. Measurement of specific growth rate
Growth rate and biomass measurements were employed for 10 d to evaluate the effect of SWW on molasses, sodium nitrate and ferric chloride on microalgae growth. The method was in accordance with previous research [46] where as much as 3 mL of sample was taken and filtered with GF/C glass fiber filters (Whatman, 110 mm) every day. Afterwards, the supernatant was resuspended in 3 mL of ultrapure water (4 ppt salinity) to prevent color interferences between SWW, molasses, and microalgae absorption. The filtrate was then placed in the cuvette and analyzed at 750 nm using Shimadzu UV-1201 spectrophotometer. This procedure was performed twice. The specific growth rate (μ) was calculated in conformity within Eq. (2).
where OD1 and OD0 are the optical density on day 1 (t1) and day 0 (t0) accordingly.
2.5.2. Measurement of biomass
Biomass was determined by measuring the optical density of microalgae every day. As per Dewi et al.’ method [19], the microalgal biomass content was determined by utilizing the optical density of the culture. Five Erlenmeyer flasks with five distinct strain concentrations (10 – 50% v/v) were prepared to achieve this connection. A spectrophotometer was used to measure each flask’s optical density. To obtain the biomass, the microalgae were then harvested, dried, and weighed. For the other flasks, the identical process was followed. There is a direct relationship between optical density and weight of microalgae biomass through a standard curve which is connected by a linear equation as shown in Eq. (3) – Eq. (8).
where x denotes biomass (g/L) and OD750 denotes the optical density at 750 nm for 10 d.
2.5.3. Biomass harvesting
During the auto flocculation, NaOH solution was utilized as a coagulant followed Dewi et al.’ method [19]. When the pH approached 11, the microalgae succumbed automatically, causing them to clump together. The biomass in the flask settled the next day and was separated using centrifugation and filtration with Whatman GF/CTM (25 mm) paper. To determine the ultimate weight, the biomass was dried at 100°C until it reached a consistent weight.
2.5.4. Nutrient removal analysis
Sample was collected daily from each flask for nutrient removal analyses. The Salifert Profi Test was used to properly assess the sample for nitrate (NO3−) and phosphorus (PO43−) followed Falconer’s method [47] in duplicate. A total of 1 mL of sample was placed in a flask, along with 4 drops of NO3− solution and 0.1 g of NO3− solid powder. The liquid was then thoroughly agitated for 30 s, and the color change was watched for 3 min. The color of the sample was then calibrated on a standard paper which has a NO3− concentration range of 0, 2, 5, 10, 25, 50 and 100 mg/L. Meanwhile, phosphorus analysis was carried out by collecting 10 mL of sample and mixing it with 4 drops of PO43− solution. After stirring for 10 s, 0.1 g of PO43− powder was added. The color change after 3 min was then calibrated using standard paper to estimate the sample’s phosphate concentration ranging from 0, 0.03, 0.1, 0.25, 0.5, 1 to 3 mg/L. Other than that, COD analysis was performed according to Dewi et al. method [19]. The analysis was conducted by taking 10 mL of sample in an Erlenmeyer using 0.01 N potassium-permanganate, 4 N sulfuric acid and 0.01 N oxalic acid reagents. The titration was undergone at 70–80°C. Eventually, nutrient removal efficiency (%) was calculated according to [17] equation as follows in Eq. (9).
where Ri denotes the removal efficiency (%) of substrate, i (NO3−, PO43−, or COD), Si0 and Sit denote the beginning and final concentrations of i during the culture phase, respectively.
2.5.5. Chemical composition analysis
The highest biomass production obtained at certain concentrations from Section 2.3 and Section 2.4 was then further analyzed for chemical compound content including protein and lipid. Protein content was determined by multiplying the nitrogen content by 6.25 based on the Indonesian National Standard SNI-01.2354.4-2006 [48]. Nitrogen compounds were extracted from 0.3 g microalgae cells by subjecting them to 410ºC for ± 2 hours of heat-induced destruction with the addition of 15 mL concentrated sulfuric acid (97%) and 3 mL hydrogen peroxide (30%) to form ammonium sulfate. Ammonium hydroxide was then produced by adding sodium hydroxide to the ammonium sulfate. Distillation was used to separate the ammonium hydroxide in order to extract the ammonia component. Ammonia was eventually bound by boric acid to produce ammonium borate, which was then titrated with hydrochloric acid. Crude protein content was calculated stoichiometrically as follows in Eq. (10).
where A is the volume (mL) of the sample titration. B is the volume (mL) of the blank titration. N is the normality of the HCl standard. W is the weight of the sample (g). 14.007 and 6.25 are nitrogen atomic weight and protein conversion factor, respectively.
Lipid content was extracted by mixing 2 g of sample with chloroform and methanol (1:2 v/v) solvent for 10 mins at 5000 rpm, followed by adding more chloroform (10 mL) and re-homogenized for 30 s at 5000 rpm in accordance with the Indonesian National Standard SNI-01-2354.3-2006 [49]. Homogenates were filtered and furtherly evaporated at 50–70ºC for 1–3 hrs until a consistent lipid weight was achieved. The percentage of fat content was measured as follows in Eq. (11).
where A is the weight of the empty dish, B is the sample weight, and C is the final weight of the dish and extracted sample.
2.6. Data Analysis
Experimental design and statistical analyses were carried out using IBM SPSS Statistic Version 26, IBM Inc., Chicago, and Response Surface Methodology utilizing Minitab 20. For attribution of causes of variance, a One-Way ANOVA was performed with α = 0.05 followed by post hoc test (Tukey). The central composite design (CCD) model was used to generate optimization trials. To understand the most influential factors and the connections between parameters, Pareto charts and 3D plots were used.
3. Result and Discussion
3.1. Effect of SWW Fraction
Fig. 1 depicts the concentrations of microalgal biomass in various diluted SWW. Six strains of marine microalgae were cultivated for 10 d in a batch reactor resulting significant biomass concentrations (P < 0.05). The results also demonstrate that marine microalgae could adapt effectively in SWW which is in accordance with prior research that microalgae grew well in the fishery wastewater [20, 50–51]. In addition, some microalgae such as Chlorella vulgaris, Porphyridium cruentum and Chaetoceros calcitrans undergo lag phase faster than other strains which fell on day 1 – 2. The experiment’s feedstock of marine microalgae was cultured when it reached the exponential phase, which may be responsible for faster adaptability. Following that, microalgae growth accelerated and attained maximum biomass production during the exponential phase which occurred on different days (between 5 – 7 d) depending on the microalgae species and SWW concentrations. The highest biomass production at the exponential phase was achieved by Nannochloropsis oculata (5.25 ± 0.77 g/L) on day 6 at 20% SWW and Spirulina sp. (5.16 ± 0.20 g/L) on day 3 at 20% SWW in respective. The subsequent days showed a drop in the biomass output of microalgae, indicating that the stationary and decline phases happened one after the other.
In terms of SWW fraction effect, two microalgae yielded the highest biomass on the final day at a concentration of 10% wastewater accounting for 4.77 ± 0.53 g/L (Porphyridium cruentum) and 5.15 ± 1.27 g/L (Nannochloropsis oculata) (P < 0.05) exhibiting that both strains in 10% fraction were able to use the waste to its fullest potential. The same findings were also obtained in the previous research that microalgae could utilize the nutrient in the wastewater such as swine effluent diluted in fishery wastewater (1.95 g/L using Tribonema sp. and Chlorella zofingiensis) [17], municipal wastewater (1.5 g/L using Chlorella pyrenoidosa) [52], and fishponds wastewater (1.85 g/L using Chlorella sorokiniana CMBB276) [51]. Porphyridum cruentum has been shown to thrive in a variety of wastewater, including swine wastewater, and it generates more biomass (5.1 ± 2.3 mg/L/day) than controls (3.3 ± 1.93 mg/L/day) [53]. This research also supports previous finding by Araujo [54] that Nannocholorpsis oculata resulted the highest growth rate compared to Chlorella vulgaris, Tetraselmis chuii and Tetraselmis tetrathele while cultivated in the artificial wastewater.
Meanwhile, Chlorella vulgaris and Nannochloropsis oculata were also found to survive in a higher SWW concentration (20%) resulting 4.28 ± 0.54 g/L and 4.10 ± 0.43 g/L of biomass. Chlorella is phytoplankton that can grow in extreme conditions and tolerate external impacts for extended periods of time [55]. Under comparison to other strains, some research found that Chlorella vulgaris can live under all conditions, including untreated wastewater [56–57]. Whilst Nannochloropsis oculata biomass output was lower than Chlorella vulgaris because this strain was discovered to only adapt when the nutrients in the medium were sufficient by nourishing more food in the wastewater [58]. Nevertheless, the addition of 20% SWW rendered some microalgae to respond after day 4 – 6, severely limiting or creating decreased biomass which was equal to 3.24 ± 0.01 (Spirulina sp.); 3.86 ± 0.07 (Tetraselmis chuii); 2.85 ± 0.17 g/L (Chaetoceros calcitrans) (P < 0.05). This finding was consistent with previous research that some marine algae could endure a wide variety of salinities but could not survive prolonged exposure to low salinity or freshwater [59–60]. As indicated in Table S1, the salinity of SWW is only 4.5 ppt, despite the fact that marine microalgae grow best with a salinity of 30 ppt. As a result, higher SWW fraction prevents some microalgae from growing properly with the final salinity in the medium was only 25–29 ppt (Table S2). In addition, the light intensity was limited at >10% SWW entering the medium since the turbidity caused by wastewater also increased [61]. Wang et al. [62] also reported that excess wastewater proved difficult to digest since the hazardous metabolites in the medium of microalgae could serve as inhibitors.
Eventually, the fermented SWW could be utilized for several marine microalgae growth. at a larger scale, the fermentation process for treating SWW does not face significant challenges because many surimi companies have already installed aerobic or anaerobic facilities that can function as fermenters. Future wastewater treatment facilities can be integrated with microalgae bioremediation to address present SWW treatment and produce value-added compounds from microalgae biomass.
Through experiments in SWW, it was demonstrated that microalgae could efficiently treat the wastewater by clearly lowering the levels of typical SWW pollutants such NO3− (P > 0.05), PO43− (P > 0.05), and COD (P < 0.05) as shown in Fig. 2 – Fig. 4. However, owing to the testing instrument’s constrained concentration range, the nitrate and phosphorus content analysis findings displayed a stepwise pattern. A range of nutrient concentrations in the sample is thus represented by the values on the graph, which is described in Section 2.5.4. According to this study, the removal effectiveness of NO3− content in SWW was inversely proportional to SWW concentration, meaning that a higher fraction of SWW would result in a poorer efficiency of nutrient removal for most strains (Table 1). The majority of nitrogen in SWW was in the form of NO3− (Table S1). Nevertheless, Cheng [17] mentioned that a high concentration of NO3− is harmful to the development of microalgae. Therefore, NO3− removal effectiveness would vary depending on NO3− concentrations at various levels of wastewater fractions. In addition, it takes more energy for nitrate reduction by the microalgae absorption process than other types of N preventing the microalgae from entirely removing these contents over a 10-day timeframe [63]. Under different wastewater fractions as shown in Table 1, some microalgae were still able to remove 100% of nutrients at 0 – 5% SWW, however at 10 – 20% SWW, that ability substantially dropped to 50 – 80% leaving 5 – 10 mg/L of NO3− in the medium (P > 0.05).
Similar observation was also reported by Enwere et al. [64] that Tetradesmus obliquus, Heterochlorella luteoviridis and Chlamydomonas reinhardtii succeeded removing up to 99% of NO3− at modified fish farm wastewater (dilution with distilled water) compared to unmodified ones (without dilution). In this study, the most effective at consuming NO3− were Nannochloropsis oculata (80 – 100%) followed by Porphyridium cruentum (50–100%) and Chlorella vulgaris (50 – 100%) (P > 0.05) (Table 1). In comparable, Gao et al. [20] noted that Scenedesmus obliquus and Chlorella vulgaris grown in aquaculture effluent possessed nutrient reduction ability accounting for 86.1%, in contrast, Nasir et al. [65] resulted in 87 – 95% removal efficiency by Chlorella sp.
In this study, the highest effectiveness of PO43− decrease was attributed to Nannochloropsis oculata, Porphyridium cruentum, and Chlorella vulgaris, respectively, accounting for 88 – 100%, 75 – 100%, and 75 – 100% (Table 1). Cheng [17] supported this finding that Tribonema sp. and Chlorella zofingiensis in swine wastewater had higher phosphorus removal efficiencies at 5 times dilution, reaching 80.5%, 69.5%, 84.7%, 73.1%, and 75.9%, respectively. The efficiency of phosphorus removal also improved once more; at a 10-times dilution, phosphorus removal peaked at 97.0%. In the meantime, Bhuyar [66] grew Chlorella vulgaris in tilapia fish wastewater for 12 days, and the outcome was 0.22 mg/L of PO43−, which is comparable to our discovery that PO43− still remained in the medium at a concentration of 15 – 20% SWW on day 10. The starting concentration of nutrients in the wastewater and the microalgal strains used affect nutrient removal efficiency (Fig. 3) [67]. PO43− reduction shared the same pattern as NO3− removal in which at higher SWW fractions, the removal ability tended to decrease (P > 0.05). While nitrogen is crucial for the synthesis of proteins and genetic material, phosphorous is a major component of adenosine triphosphate (ATP), which is necessary for the storage and transfer of short-term energy [68–69].
According to Fig. 4, the COD concentration had the highest clearance rate of 69% (424 mg/L) in Nannocholorpsis oculata inoculum at 15% SWW, which was marginally similar to the values of Porphyridium cruentum at 10% that took into account of 66% (244 mg/L) (P < 0.05). COD removal efficiency differed with microalgae stains, accounting for 64% in 10% SWW (782 mg/L) for Chlorella vulgaris, 64% in 5% SWW (670 mg/L) for Spirulina sp., 58% in 20% SWW (796 mg/L) for Teraselmis chuii, and 58% in 10% SWW (554 mg/L) for Chaetoceros calcitrans (Table 1). The removal effectiveness of COD was lower for the majority of microalgae under conditions with greater SWW fractions (Fig. 4). Microalgae used COD from SWW, which also contained a significant amount of organic pollutants, to create biomass [70]. While decreasing the need for additional nutrients for microalgae to create biomass, organic compounds in wastewater can also be used as a source of energy [19]. According to research by Mennaa et al. [71] and Molinuevo-Salces et al. [70], different microalgae strains require varied rates of nutrient absorption. Some strains may result in low nutrient removal, while others may provide superior nutrient elimination. Because some resistant organic chemicals are typically hardly degradable by microalgal cultivation, total COD elimination is unlikely to be achieved [72]. The COD removal efficiency in this study was far less than previous research by Cheng [17] which found Tribonema sp. and Chlorella zofingiensis could remove 95.9% organic content since they use co-culture cultivation. Whereas Calderini et al. [16] cultivated Chlorella vulgaris in standing water obtained > 90% COD removal due to the organic content in the medium was way lower (85.6 mg/L) compared to this study (6,084.5 mg/L).
3.2. Effect of Molasses, Sodium Nitrate, and Ferric Chloride
The present study shows that Nannochlorpsis oculata could optimally grow in 10% of SWW supplemented with molasses : sodium nitrate and molasses : ferric chloride for 10 d. Nannochloropsis oculata grew differently when grown in a mixotrophic environment with sugarcane molasses as a carbon source and sodium nitrate as a nitrogen source. Fig. 5 reveals that the highest biomass was obtained when the concentration of molasses remained low and sodium nitrate was high, accounting for 5.79 ± 0.24 g/L (at 1 g/L of molasses and 50 mg/L of sodium nitrate), which was higher than previous cultivation (see Section 3.1). At greater molasses addition, Nannochloropsis oculata resulted lower biomass since they lacked a particular sucrose transporter or were unable to extracellularly hydrolyze sucrose into monosaccharide, the algal species had trouble using sucrose [62]. The biomass reduced by 1-1.5-fold which was solely 2.6 ± 0.21 – 3.83 ± 0.16 g/L when the molasses ranged between 3 – 3.4 g/L. Yan [73] also added that disaccharides like sucrose and lactose are rarely absorbed by green microalgae. The turbidity caused by molasses could also be responsible for the fall of [25]. However, at the right amount of molasses, they play the same impact as other carbon sources in the growth and cellular composition of microalgae in mixotrophic cultivation [28]. Glucose and fructose in sugarcane molasses have attested that mixotrophic cultivation in a variety of microalgal strains, including Scenedesmus sp. [74], Chlorella sp. [75], and Monoraphidium sp. [76] could perform well at certain concentrations.
On the contrary, nitrate seemed to boost up the growth of microalgae. Numerous studies have connected the decline in protein and enzyme content in nitrogen-limited cultures to the reduction in biomass productivity. The production of microalgae is dependent on the availability of nitrogen since it is a necessary component of the structures of proteins, amino acids, and enzymes [77–78]. In this study, at low concentration of sodium nitrate (1.72 – 10 mg/L) the biomass production of Nannochloropsis oculata was remarkably low which was 2.88 ± 0.11 – 3.42 ± 0.08 g/L. According to Xi et al. [79], lowering the nitrogen concentration below 40 mg/L is a growth limiting factor for Chlorella vulgaris. Likewise, Zarrinmehr [33] discovered that when Isochrysis galbana was grown in low nitrogen concentrations (36 mg/L), it produced less biomass. The abovementioned research suggested that the growth of microalgae can be boosted up by adding 72 – 144 mg/L of sodium nitrate at least. Nevertheless, this study found out that the highest of biomass could be achieved at lower nitrogen concentration (30 – 50 mg/L) since the cultivation was undergone simultaneously within molasses and sodium nitrate, which could be linked to this phenomenon. Sari [80] mentioned that the nutritional needs of microalgae are relatively high which are 56.3% of carbon, 8.6% of nitrogen and 1.2% of phosphorus on a weight basis. Meanwhile, Whitton et al. [37] reported that the Redfield Ratio, or 106 : 16 : 1, is the molar ratio for C : N : P that has been determined to be the internal composition of marine phytoplankton. In contrast, the C : N : P ratio of concentrated SWW, as shown in Table S1 was only 918 : 5 : 1, indicating a paucity of nitrogen source.
The C : N : P ratio switched to 210 : 37.5 : 1 (weight basis) at 10% SWW with the addition of molasses and sodium nitrate, providing adequate nutrients for microalgae growth. However, if the supply of nutrients is excessive, it will precipitate into poison since it will not be absorbed properly, resulting in a loss in growth [33, 80]. The addition of molasses and sodium nitrate was observed to be significantly affecting the biomass fraction as shown in the Pareto chart (Fig. 5). The statistical analysis confirmed that the model in this approach was very well fit to the second-order polynomial formula as defined in Eq. (12).
Fig. 5 summarizes the change in biomass production. The combination of molasses:sodium nitrate and molasses : ferric chloride had a strikingly similar trend. In this situation, Nannochloropsis oculata produced 5.29 ± 0.07 g/L of biomass (at 1 g/L of molasses and 50 mg/L of ferric chloride), which was scantly lower than the additional nitrate. This figure was just slightly higher than the photoautotrophic condition, which was 5.15 ± 1.27 g/L (see Section 3.1). Dou et al. [81] discovered that iron ranging from 0 to 117 mM increased the dry cell weight of Nannochloropsis oculata by about 1.1-fold. The fact that iron is required for cytochrome, ferredoxin, and several enzymes, including nitrate and nitrite reductase, explains the rise in dry cell weight of Nannochloropsis oculata [81]. Furthermore, under iron-depleted conditions, biomass reduced 2.3-fold in our study. Botebol et al. [82] discovered that the microalgal cells died after two days in an iron-depleted state in the investigation with Ostreococcus tauri. Polat [40] also added that Auxenochlorella protothecoides grew optimally at 0.58 mM (equal to 65 mg/L) resulting 1.15 g/L without the addition of wastewater and molasses. Nevertheless, at more than 30 – 40 mg/L of ferric chloride, the biomass tended to decrease since it prevented the growth of algae and produced hazardous byproducts [65, 83]. In this current study, molasses took a role in providing organic carbon source while assimilating with ferric chloride to achieve higher biomass. The statistical analysis also showed that all the factors significantly affected the biomass (Fig. 5) with the second-order polynomial equation shown in Eq. (13).
In terms of nutrient removal, the addition of molasses : sodium nitrate and molasses : ferric chloride on NO3−, PO43− and COD varied differently (Fig. 6). As previously mentioned, the nitrate and phosphorus concentrations do not precisely match the sample concentration due to the analytical device’s concentration range, which is described in Section 2.5.4. Additionally, it is evident that the removal of NO3− tended to decrease at maximal molasses supplementation, suggesting that complex molecules and turbidity from molasses were taken into consideration at this stage and impeded the proliferation of microalgae. Under comparison to other treatments, less nitrate was consumed under dark or turbid circumstances. This suggests that the dark medium may operate as a stressor and slow down the rate of cell growth [25]. On the contrary, the NO3− removal successfully decreased, leaving 0 mg/L fraction, when the medium was supplied with 1 g/L of molasses with increased sodium nitrate (more than 10 mg/L). Jacobus [84] conducted a similar study in which he used sodium acetate and sodium nitrate to cultivate Nannochloropsis oculata in a mixotrophic environment. The results showed increased biomass and nutrient removal productivity at a lower sodium acetate and a higher sodium nitrate percentage, which accounted for about 30 × 106 cells/mL. Uniformly, ferric chloride addition achieved 100% NO3− elimination when molasses concentration range between 1 and 2 g/L. Conversely, NO3− reduction was reduced when the medium was supplied with a greater molasses proportion mixed with more than 30 mg/L ferric chloride. As previously indicated, the dark medium caused by molasses may be responsible for this phenomenon. In addition, Polat [40] reported that microalgae can sustain 0.07 – 21.6 mM of ferric chloride, whereas, Botebol et al. [82] added that most microalgae species respond favorably to iron concentrations up to 2 mg/L, but that adverse effects are typically seen when iron concentrations surpass 30 – 40 mg/L. In this research, the elimination of NO3− peaked at extensive ferric chloride concentration (1.72 – 68.8 mg/L) as long as the medium was coalesced with low molasses concentration (1 – 2 g/L).
To embark on PO43− removal, the result experienced a scantly different trend to NO3− that the addition of higher molasses (3 g/L) deterred the growth indicating by a lower phosphorus omission rate. At low molasses fraction (1 – 2 g/L), higher sodium nitrate addition boosted up the PO43− removal leaving 0 mg/L in the medium. Huang [85] similarly reported that as the concentration of sodium nitrate grew, so did the rate of phosphorus removal by Chlorella vulgaris cultured in microbial fuel cell wastewater due to higher growth rate that supported higher nutrient adsorption. Thereunto, increased ferric chloride had the same effect as nitrate removal since it also led to a drop in phosphorus elimination, which peaked when the molasses content increased. When molasses was supplied in small amounts, adding ferric chloride at different concentrations did not create a barrier to decreasing PO43−.
The reduction in COD content rose dramatically when sodium nitrate was added in greater amounts and molasses was added at a very low concentration. Vice versa, because nitrogen is a necessary component for cell division, nitrogen limitation generally prevented microalgal cells from dividing and growing leading to lower nutrient adsorption [86]. The greatest reduction in COD was observed at a concentration of 1 g/L for molasses and 30–50 mg/L for sodium nitrate; the end level was 510 – 512 g/L, or 80.38%. According to Molinuevo-Salces et al. [70], while the efficiency of nutrient absorption in waste also rose, the COD content obtained decreased with increasing numbers of microalgae cells. Similarly, the addition of 0 – 30 mg/L ferric chloride only generated a drastic decrease of COD content when the molasse was at a low state accounting for 1 mg/L. Prior research investigated that high ferric chloride concentrations hindered the growth of Auxenochlorella protothecoides and resulted in a 5.5-fold reduction in biomass [40]. This restriction can be linked to the iron uptake processes of Auxenochlorella protothecoides and can be explained by the harmful action of ferric chloride whilst reducing nutrient adsorption. In conclusion, the hazardous nature of iron sources can be influenced by iron concentration, ion dissolution, medium chemical composition, iron chemistry, and iron speciation [82]. Therefore, the lowest COD concentration was achieved by 740 mg/L at 0.76 g/L molasses and 30 mg/L of ferric chloride. According to earlier research by Cheng et al. [17], the use of Tribonema sp. and Chlorella zofingiensis could minimize COD in swine mixed fishing effluent by as much as 91.5%. Molinuevo-Salces et al. [70] highlighted that microalga required COD from wastewater, which also contained a high concentration of organic pollutants, in order to create biomass. Since some resistant organic chemicals are typically not easily broken down by microalgal culture, it is unlikely that total COD removal will be accomplished as a whole [72].
3.3. Chemical Composition of Microalgae
The chemical makeup of different microalgae strains grown on 10% SWW concentration (P < 0.05) is displayed in Fig. 7a. According to the bar chart, the maximum protein was obtained by Spirulina sp. (28.5 ± 0.16%), Chlorella vulgaris (26.66 ± 1.14%), and Chaetoceros calcitrants (32.84 ± 0.51%). Conversely, Porphyridium cruentum (27.93 ± 0.22%), Nannochloropsis oculata (25.42 ± 0.78%), and Chaetoceros calcitrants (21.55 ± 0.61%) produced larger amounts of lipid. The lipid and protein contents in the microalgae was way lower than when it is cultivated without wastewater. The microalgae’s chemical composition is impacted by the fact that the CNP requirements of the algae cannot be satisfied based on the Redfield Ratio, as indicated by Table 1 and Table S2. Sisman-Aydin and Sismek [87] also mentioned that the protein content in synthetic medium were higher than in the wastewater cultivation. This was also evident in the protein and lipid content; the protein and lipid contents of C. sorokiniana cultivated in aquaculture effluent were 24.57% and 30.15%, respectively. These values were lower than those of the biomass produced in synthetic medium, which had 28.64% protein and 35.75% lipid [88]. In addition, turbidity in microalgae reduces light penetration, which in turn lowers the productivity of the algae (photosynthesis)—including its lipid and protein content [89–90].
When 1 g/L and 50 mg/L sodium nitrate was added, Nannochloropsis oculata’s protein content increased significantly by 32.41 ± 2.21%, but the algae’s lipid content decreased by 20.05 ± 1.36% (Fig. 7b). Araujo et al. [54] mentioned that nitrogen limitation proved to be highly effective in causing lipid buildup. Microalgae have a tendency to store energy in the form of chemicals during periods of hunger, which is why the cells’ lipid content increases [78]. Due to their density, hydrophobicity, and capacity to aid in cell survival, lipids serve as the main components for storage under stressful circumstances [91]. Meanwhile, sodium nitrate is reported to be able to enhance protein content as previously conducted by Barman et al. [39] that the protein content of Tetraselmis chuii cultivated under the 500 mg/L of sodium nitrate concentration was substantially higher compared to control. The results aligned with the findings of Kim et al. [92] who also documented a similar pattern for the marine chlorophyte Tetraselmis sp. During nitrogen-deficient conditions, protein concentrations gradually declined while carbohydrate production rose to a maximum of 55%. However, the addition of 1 g/L of molasses and 50 mg/L ferric chloride markedly enhanced the amount of protein (27.15 ± 0.88%) and fat (27.38 ± 1.17%) in Nannochloropsis oculata specimens. In addition to being a crucial electron acceptor for photosynthetic processes, iron (Fe) can enhance algal nitrogen fixation and reduction capabilities. Microalgae growth and proliferation were aided by an appropriate concentration of iron ions while producing high biomass and its chemical compounds [93]. There was shown to be a significant positive correlation between ferric chloride and the protein/lipid ratio [40]. Similar to this study, Auxenochlorella protothecoides had the maximum lipid content of 34.3% upon the addition of 6.47 mM ferric chloride [40].
4. Conclusions
The study involved the cultivation of marine microalgae in varying ratios of sodium nitrate, ferric chloride, and molasses by using SWW as the medium. The results of microalgae growth on SWW were 4.35 ± 0.18 g/L at 15% (Spirulina sp.), 4.28 ± 0.54 g/L at 20% (Chlorella vulgaris), 4.77 ± 0.53 g/L at 10% (Porphyridium cruentum), 3.86 ± 0.07 g/L at 20% (Tetraselmis chuii), 5.15 ± 1.27 g/L at 10% (Nannochloropsis oculata) and 3.98 ± 0.06 g/L at 10% (Chaetoceros calcitrants). Other than that, microalgae have been shown to be capable of removing nutrients in SWW. Nannochlorpsis oculata was able to remove more NO3− (80–100%) and PO43− (100%) from the 10% SWW. In the meantime, Chlorella vulgaris eliminated COD at a rate of 49 – 64%. When 1 g/L molasses : 50 mg/L sodium nitrate and 1 g/L molasses : 50 mg/L ferric chloride were added to Nannochloropsis oculata medium during mixotrophic conditions, the biomass generated was significantly larger than it was during the previous stage, accounting for 5.79 ± 0.24 g/L and 5.15 ± 1.27 g/L, respectively. The result also showed that the addition of sodium nitrate and ferric chloride significantly increased the protein content resulting 32.41 ± 2.21% and 27.15 ± 0.88%, accordingly. While lipid content only increased by the addition of ferric chloride accounting for 27.38 ± 1.17%. In the future, it is recommended to investigate the effect of untreated SWW, hydrolyzed molasses at various nitrates, and iron sources on microalgae growth and nutrient removal as well as chemical compounds.
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