Coupling Nutrient Removal and Biodiesel Production by the Chlorophyte Asterarcys quadricellulare Grown in Municipal Wastewater

Freshwater deficiency and growing requests for renewable energy have led to increased concern about water and energy security. Therefore, the present study attempted to evaluate the feasibility of using wastewater (WW) as a culture medium for microalgal cultivation with the aim of nutrient removal and biodiesel production. The green microalga Asterarcys quadricellulare (Chlorophyta) was isolated from a wastewater treatment plant in Menoufia, Egypt, and grown for 24 days in Bold’s Basal Medium as a control and at different concentrations of secondary treated WW in distilled water (25%, 50%, 75%, and 100%). The results of 75% WW treatment recorded 96.6%, 98.4%, and 89.9% removal efficiency for nitrate, ammonia, and total phosphorus, respectively. Also, it revealed high biomass yield and biomass productivity (1.44 g L−1 and 69.0 mg L−1 day−1, respectively). In addition, it showed higher lipid production (360.6 mg L−1) and lipid productivity (17.2 mg L−1 day−1). Asterarcys quadricellulare fatty acid profile estimation revealed elevated proportions of oleic (54.92%), palmitic (20.54%), and linoleic acids (13.9%). Most of the properties of biodiesel derived from the studied microalga are consistent with those recommended by the international standards. The current study concluded that A. quadricellulare could be used for wastewater bioremediation and biomass production with high potential as biodiesel feedstock.


Introduction
In many parts of the world, the aquatic environment is at risk. Egypt suffers from freshwater stress due to lack of water supply and the increasing inhabitants, as well as increasing competition for freshwater from the Nile Basin countries (Ethiopian Renaissance Dam). Thus, the widening gap between the limited water resources and the raising ask for that water became the main challenge facing Egypt [1]. Therefore, it is necessary to seek cost-effective methods and innovative techniques for the treatment of wastewater to meet the freshwater demand for human and agriculture. In that regard, microalgae are utilized to treat wastewater due to its capacity to utilize nitrogen and phosphorus and to remove heavy metals. Moreover, it does not lead to secondary contamination from microalgae metabolites [2]. Wastewater treatment using microalgae is energy-efficient and capable of removing pollutants from wastewater compared to the traditional chemical and physical treatment methods [3]. In wastewater, excess nutrients such as nitrate and phosphate contribute to eutrophication, thus decreasing dissolved oxygen and increasing the growth of undesired vegetation in the aquatic environment. In addition, microalgae assimilate inorganic carbon from wastewater, which can lead to an increase in pH, resulting in ammonia volatilization and phosphorus precipitation [4,5]. Several earlier studies have been conducted on the nutrient removal efficiency of microalgae from multiple sorts of wastewater [6][7][8][9]. These studies concluded the feasibility of wastewater treatment using microalgae, which can decrease nutrient removal cost and water demand while improving water quality.
Due to the broad use of fossil fuels, the world faces depletion of energy sources. In this regard, biofuels are considered as a renewable alternative to fossil fuels, due to their sustainable features to overcome the global energy demand [10]. Nevertheless, due to their competition with food crops and agricultural lands, biofuel sustainability is often challenged [11]. Therefore, microalgae as a renewable source for sustainable biodiesel production are promising due to their higher oil productivity and high growth rates. They can easily be cultivated on a large scale for producing sustainable and economically feasible biofuels [12]. Moreover, they do not compete with other agricultural crops for cultivable land, because they can be grown on arid land using seawater or wastewater [13]. In contrast, while microalgae have great potential as biodiesel feedstock, there are many disputes for their large-scale application including the vast freshwater need and the costly nutrients [14]. These problems can be solved using wastewater as a growth medium for microalgae which can reduce the cost for algae cultivation to make the process of producing biofuel from algae more economic and environmentally friendly [15]. Some previous studies demonstrated that municipal wastewater is utilized to substitute synthetic growth media for algal cultivation as an alternative and inexpensive source of nutrients [16]. Microalgae were utilized for municipal wastewater treatment and simultaneous lipid accumulation with nutrient removal efficiency reaching up to 90% and lipid accumulation up to 28.5% of dry algal biomass [17]. To diminish water pollution and the reliance on fossil fuel, the present study aimed to isolate a green microalga followed by assessing its efficiency nutrient removal form wastewater. In addition, the effect of wastewater on the biomass and lipid productivity, as well as qualitative and quantitative biodiesel, was estimated.

Microalgal Isolation and Growth Conditions
Secondary treated municipal wastewater samples were collected from municipal wastewater treatment plants located in Menoufia, Egypt. Samples were collected in sterilized plastic bottles and transferred to the laboratory shortly after collection. Large solid particles in the collected wastewater were removed by sedimentation and filtration using Whatman filter paper. The collected wastewater samples were enriched with a mixture of 90% sample and 10% of cultural Bold's Basal Medium (BBM) [9]. The batch incubation process was carried out for 1 week, using continuous illumination under white light fluorescent lamps of 80 μmol m −2 s −1 at 26 ± 2 °C, and aerated using sterile filtered air. After an enrichment period, microalgal growth was visually evaluated using an optical microscope to select the pure microalgae. The fast-growing microalga was isolated and raised to pure culture through plate technique. The isolate was examined by light microscope and propagated in BBM with early growth conditions.

Experimental Design
The tested microalga was grown in a conical flask containing 150 mL of BBM and incubated under the aforementioned growth conditions to be used as the inoculum for the following study experiments. The growth medium (BBM) was used as a control, as well as four different concentrations of municipal wastewater (25%, 50%, 75%, and 100%, v/v in distilled water). For each treatment, 5 mL was used to inoculate the experimental flasks containing 150 mL/each of different treatments. All treatments were carried out in triplicates, and samples were analyzed with an interval of 3 days.

Microalgal Growth
The study was performed over a period of 24 days, in which microalgal growth was determined by measuring the optical density at 560 nm (OD 560 ). In addition, cells were counted using a hemocytometer, and chlorophyll (a) was determined according to Lichtenthaler and Buschmann [18]. In addition, cellular dry weight (CDW) was measured initially and after 24 days of incubation [19], then biomass and lipid productivities were calculated [20].

Biochemical Composition
Total lipids were determined according to the method described by Bligh and Dyer [21] with modification according to Lee et al. [22]. Protein content was determined using the Biuret reaction, adapted by Lowry et al. [23]. The total carbohydrate content in the extract was determined by the phenol sulfuric acid method using d-glucose as a standard [24].

Fatty Acid Profile
The dry lipid extracts were transesterified to fatty acid methyl esters (FAME) [25] before being analyzed by gas chromatography. One milliliter of the crude lipid layer was taken, and 1 mL methanol and 0.3 mL H 2 SO 4 were added. The mixture was vortexed for 3-5 min and incubated at 100 °C for 10 min; 1 mL distilled H 2 O was added, centrifuged for 3-5 min, and then centrifuged at 4000 rpm for 10 min. FAMEs were analyzed by gas chromatography using a Trace GC1310-ISQ mass spectrometer (Thermo Scientific, Austin, TX, USA) equipped with a TG-5MS direct capillary column (30 m × 0.25 mm × 0.25 µm film thickness).

Nutrient Removal
The nutrient medium was separated from the biomass by centrifugation after being filtered through Whatman GF/C filter paper. Consumption of ammonia, nitrite, nitrate, phosphate, biological oxygen demand (BOD 5 ), chemical oxygen demand (COD), electrical conductivity (EC), and total dissolved solids (TDS) by algae after a 24-day growth period was determined. Total phosphate (TP) was measured according to Woods and Mellon [27], while ammonia nitrogen (NH 3 -N), nitrate nitrogen (NO 3 -N), and biological oxygen demand (BOD 5 ) were determined according to APHA [19] and chemical oxygen demand (COD) was determined according to Mancy [28]. Total dissolved solids (TDS) and electrical conductivity (EC) were measured using the conductivity benchtop meter. Based on the results obtained, the percentage removal efficiency (%) and removal rate (mg L −1 day −1 ) were calculated according to Eladel et al. [9].

Statistical Analysis
All experiments were performed in triplicate, and the results are presented as means ± standard deviation (SD). Statistical analyses were performed using SPSS software 16.0. The comparisons of the mean values were conducted by oneway analysis of variance (ANOVA) followed by Duncan's new multiple-range test for statistical significance. The differences were considered significant at probability level (p) ≤ 0.05.

Identification of Isolated Microalga
The tested microalga was isolated through streaking on agar plates and sub-culturing in liquid media. Using microscopic characterization, the isolated green microalga was morphologically identified as Asterarcys quadricellulare. It has a unicellular spherical to ovoid form, up to 10 µm in diameter, containing a single parietal lobe, sometimes lobed to fragmented chloroplast with a single prominent pyrenoid. Autosporangia containing 2-16 ovoid spores were seen, but no sexual or asexual flagellated stages were observed [29].

Growth of Asterarcys quadricellulare
A. quadricellulare was cultured on BBM as a control and different concentrations of secondary treated municipal wastewater (25,50,75, and 100%) with its physical and chemical characteristics as presented in Table 1.
The microalgal growth results presented in Fig. 1 showed the least algal growth in 25%, and 50%, WW. In agreement with the present results, 22% concentration did not increase the algal growth [30], which is explained by the relatively small increase in wastewater concentration. But using 44% WW, the cell growth was almost two times higher than 22% wastewater concentration.
Indeed, it was noticed that algae grew faster in 75% WW, where it recorded the highest growth compared to the control and the other concentrations. The use of pretreated municipal wastewater 77% concentration to grow Scenedesmus acutus in batch mode showed the highest optical density, chlorophyll (a), cell count, and dry weight of 1.23, 11.12 mg L −1 , 38.9 × 10 4 cells mL −1 , and 1.44 g L −1 , respectively [6]. Chlorophyll (a) content is an indicator of the algal growth rate, which increased with time by increasing the nutrient removal percent and algal growth biomass [31]. These results indicate that 75% WW is suitable for applications requiring high-density microalgal culture when grown in municipal wastewater. In general, wastewater with a high concentration of nutrients can inhibit the algal growth, while on the contrary wastewater with a low concentration of nutrients) is insufficient for algal growth.

Biomass and Biochemical Composition
The biomass and biochemical composition results are presented in Table 2 and Fig. 2 where the biomass productivity of A. quadricellulare recorded the highest value of 69 mg L −1 day −1 at 75% WW treatment. These results are consistent with those obtained for some other microalgae [32,33]. The recorded highest lipid content of 360.6 mg L −1 , lipid productivity of 17.2 mg L −1 day −1 , and lipid content 25.3% were recorded at 75% WW treatment. The highest lipid accumulation may be due to the highest accumulated biomass from this treatment at the end of the growth period. In line with our results, several comparable studies are presented in Table 4, where A. quadricellulare produced 0.463 g L −1 lipids, 20% DW, with lipid productivity of 19.8 mg L −1 day −1 [34]. Also, Asterarcys sp. showed the greatest biomass productivity (80 mg L −1 day −1 ) and higher lipid content (30.55%) [32]. Lipid content was 19.4% in Scenedesmus obliquus cultivated in treated urban wastewater [7]. Scenedesmus acutus showed 28.3% lipid content by cultivation using pretreated municipal wastewater as culture medium for 21 days [6].

Nutrient Removal
The removal rates and efficiency of some nutritional elements from secondary treated municipal wastewater were determined using A. quadricellulare as shown in Table 3.
The results showed that A. quadricellulare had a higher removal rate and higher efficiency for NH 3 -N and NO 3 -N, especially 75% WW. The highest NO 3 -N removal rate was 0.37 mg L −1 day −1 representing 4.6 times more than the control. It also recorded a high NH 3 -N removal rate (0.04 mg L −1 day −1 ) representing 2 times over the control. High removal efficiencies (NH 3 -N 98.41% and NO 3 -N 96.61%) were recorded at 75% WW treatment. The removal of NH3-N from the wastewater by algae can be attributed to the direct NH 3 -N and/or NH 3 stripping [5]. Ammonium is the preferred form of nitrogen for microalgal growth due  to its lower energy demand. Nutrient removal can also be increased by NH 3 drive-out or phosphorus precipitation due to the increase of pH associated with photosynthesis [4]. Regarding phosphorus uptake, the removal efficiency recorded at 75% WW was 89.9% concentration (Table 3), which is higher than the satisfactory value (80%) established by European legislation [35]. Removal percentages of this study were close to that of microalgae grown on mixed municipal and industrial wastewater [36]. The results revealed that A. quadricellulare quite has the ability to assimilate high amounts of phosphorus and nitrogen for synthesis of lipids, proteins, and carbohydrates. The specific nutrient consumption of NO 3 -N, NH 3 -N, and TP by A. quadricellulare was greater in 75% WW, while it showed the lowest specific consumption at 25% WW treatment (Fig. 3). This was attributed to the high growth of A. quadricellulare on 75% compared to other wastewater treatments and the controlled growth media.
Both COD and BOD are important in assessing water quality. As wastewater was used for the growth of Asterarcys quadricellulare at a concentration of 75% WW, COD showed high removal efficiency 84.74% and removal rate 1.23 mg L −1 day −1 and BOD with a removal efficiency of 91.52% and a removal rate of 0.38 mg L −1 day −1 ( Table 3). The higher COD and BOD values confirm the greater amount of organic matter. This result showed that Asterarcys quadricellulare resulted in increased loss in both BOD and COD values of the effluent, and this could be attributed to the increasing of algal growth rate, which implied more photosynthesis happened to produce more oxygen (Table 4). Hence, oxidation of organic matter is improved by released oxygen. Using microalgae in wastewater treatment can increase the removal efficiency of COD [37]. Biological treatment of domestic wastewater using algae indicated 68.4% BOD and 67.2% COD removal, respectively [38].

Fatty Acid Profile
A. quadricellulare recorded its high lipid productivity at 75% WW as discussed in the previous section. Therefore, the fatty acid profile was analyzed using GC compared with the control (BBM). It was revealed that the A. quadricellulare fatty acid profile mainly consisted of monounsaturated fatty acids (MUFA), followed by polyunsaturated fatty acids (PUFA) and saturated fatty acids (SAF) as shown in Fig. 4 and Table 5. The total MUFA content showed a significant increase using 75% WW, 23.2% higher than the control and a significant reduction in total PUFA content by 34.37% below control. The main fatty acids in A. quadricellulare microalga were 16-carbon and 18-carbon, and high proportions of palmitic acid (20.54%), oleic acid (54.92%), and linoleic acid (13.9%) were found in 75% WW (Table 5). Microalgal lipids which have a high proportion of C16:0 and C18:0 fatty acids are proper feedstock for biodiesel production [43,44]. Previous studies confirmed that the dominant fatty acids in the microalgae were palmitic, stearic, and oleic acids [45]. Thus, this microalgal strain is a promising candidate as a feedstock for biodiesel production. The increase in the fatty acids (C16-C18) in 75% WW may indicate a balanced composition of this medium.

Biodiesel Properties
Biodiesel properties of Asterarcys quadricellulare grown in BBM and 75% WW were determined as shown in Table 6. The iodine value is a property indicating the degree of unsaturation of fatty acid which influences the viscosity and cold filter plugging point. The iodine value of the control and that grown in 75% WW were 84.09 gI/100 g, and that grown in 75% WW has 77.08 gI/100 g. Since the melting point and oxidative stability are related to the degree of unsaturation, the greater the iodine value, the more unsaturation and the  higher the susceptibility to oxidation. The lower the iodine value, the better the fuel will be as biodiesel. Biodiesel with large amounts of SFA have a higher cetane number, while biodiesel with high amounts of unsaturated fatty acids have a lower CN. The cetane number was 58.66 for the control and 58.70 for 75% WW, and it was found to be higher than both standards ensuring good ignition quality low nitrous oxide emissions, lower knocking occurrence, and easier engine start-up [46]. The present results are consistent with the literature, which reports that most properties of biodiesel derived from the studied microalgae species already meet the limit values established by the ASTM D6751 and EN 14,214 biodiesel standards [45,47]. Also, most of the biodiesel properties represented in Table 6 for Asterarcys quadricellulare in both studied treatments agree with those of other studies [25,41].

Conclusion
Due to the existing mineral nutrients, secondary treated municipal wastewater could be used by microalgae as a lowcost growth medium to produce biomass-based biofuels. In conclusion, nitrogen and phosphorus were efficiently eliminated during the study experiment, which may increase the economical production of the lipid-rich Asterarcys quadricellulare biomass for biodiesel production by saving water and nutrients. Moreover, the current study recorded the highest percentage of C16-C18 fatty acids with a concentration containing a higher proportion of secondary treated wastewater. Our results offer a primary stage for isolation of microalgal species convenient for local conditions, and further studies are needed to enhance the growth and lipid productivity of microalgae grown on wastewater as abundant cheap cultivation medium in either lab-scale or large-scale cultivation conditions.