Identification of isolated microalga
The tested microalga was isolated through streaking on agar plates and sub-culturing in liquid media. Using microscopic characterization, a green microalga (chlorophyta) was morphologically identified as Asterarcys quadricellulare (Fig. 1). It has a unicellular spherical to ovoid form, up to 10 µm in diameter, containing a single parietal, 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 .
Growth of Asterarcys quadricellulare in municipal wastewater
A. quadricellulare was cultured on BBM as control and different concentrations of secondary treated municipal wastewater (25, 50, 75 and 100%WW) with its physical and chemical characteristics are presented in Table 1.
Growth was assessed using optical density (OD), chlorophyll (a) content and cells count. The results of microalga growth presented in fig. 2 showed that the least algal growth in 25%, 50% and 100%WW. Agreement with the study results, 22% concentration did not increase algal growth, that might be due to the relatively small increase in wastewater concentration. But when used 44% concentration, cell growth was almost two times higher than 22% wastewater concentration .
Indeed, it was noticed that algae grew faster in the 75%WW, where it recorded the highest growth as compared with control and the other concentrations. Using pre-treated municipal wastewater 77% concentration to grow Scenedesmus acutus in batch mode . At 75%WW optical density, chlorophyll (a), cells count and dry weight recorded the highest values was 1.23, 11.12 mg L-1, 38.9x104 cells mL-1 and 1.44 gL-1, respectively. Chlorophyll (a) content is sign of algal growth rate, which increased with time by increasing nutrients removal percent and algal growth biomass . This indicating that 75%WW is suitable for applications requiring high-density microalga culture when grown in municipal wastewater. Generally, wastewater with high nutrient concentrations could inhibit algal growth, while on the other hand wastewater with low nutrient concentrations could insufficient for algal growth.
Biomass and biochemical composition
Results for biomass and biochemical composition presented in Table 2 and Fig. 3 showed that biomass productivity of A. quadricellulare recorded highest value 69 mgL-1 day-1 at 75%WW treatment. These results are consistent with those obtained for some other microalgae [32, 33]. Lipid accumulation by Asterarcys quadricellulare cultivated on different concentrations of municipal wastewater and BBM (control) are presented in Table 2 and Fig. 3. The results recorded high lipid content with value 360.6 mgL-1, lipid productivity 17.2 mgL-1day-1 and lipid yield was 25.3% for 75%WW treatment. The highest lipid accumulation may be due to the highest accumulated biomass from this treatment at the end of growth period. In line with our results several studies were presented in Table 4, Asterarcys quadricellulare microalga produce 0.463 g/L lipid, 20% DW, with lipid productivity of 19.8 mg/L-1day-1 . Also, Asterarcys sp. showed the greatest biomass productivity (80 mgL-1d-1) and higher lipid content (30.55%) . lipid content was 19.4% after Scenedesmus obliquus cultivation using treated urban wastewater . Scenedesmus acutus had a 28.3% lipid content on cultivation using pretreated municipal wastewater as the culture medium for 21 days .
The removal rates and efficiencies of some nutritional elements from secondary treated municipal wastewater were determined using Asterarcys quadricellulare as presented in Table 3. The result showed that Asterarcys quadricellulare had higher removal rate and efficiency to NH3-N and NO3-N, especially at concentration 75%WW. The highest NO3-N removal rate in 75%WW was 0.37 mg L-1 day-1 with 4.6 times over control. It also recorded high NH3-N removal rate as 0.04 mgL-1 day-1 indicating 2 times over control. High removal efficiencies (NH3-N 98.41%, NO3-N 96.61%) were recorded at 75%WW treatment. Removal of NH3-N from the wastewater by algae can be due to direct use as NH3-N and/or NH3 stripping . Ammonium is the preferred form of nitrogen for microalgae growth due to its lower energy demand. Nutrient removal can also be increased by NH3 drive out or phosphorus precipitation due to the increasing of the pH associated with photosynthesis .
Regarding phosphorus uptake, the recorded removal efficiency at concentration 75% WW with a value 89.9% (Table 3). was higher than satisfactory value (80%) established by European legislation . Removal percentages of this study were closed to that of microalgae grown on mixed municipal and industrial wastewater . The results revealed the ability of Asterarcys quadricellulare to assimilate high amounts of phosphorus and nitrogen for the synthesis of lipid, protein, and carbohydrates of microalga dry weight. The specific nutrient consumption values of NO3-N, NH3-N and TP by microalga were greater recorded in 75%, while 25%WW showed the lowest specific consumption (Fig. 4). This is attributed to the higher growth of Asterarcys quadricellulare on 75% in comparison to other wastewater concentrations and the control growth media.
Both COD and BOD are important in assessing water quality. As wastewater used for the growth of Asterarcys quadricellulare at concentration 75%WW, COD showed high removal efficiency 84.74% and removal rate 1.23 mgL-1 day-1 and BOD with removal efficiency 91.52% and removal rate 0.38 mgL-1 day-1 (Table 3). The higher COD and BOD values confirm the greater amount of organic matter. This result showed that Asterarcys quadricellulare prompted increasing the loosing 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 producing more oxygen. Hence, oxidation of organic matter is improved by released oxygen. Using microalgae in wastewater treatment can increase the removal efficiency of COD . Biological treatment of domestic wastewater using algae indicated 68.4% BOD and 67.2% COD removal, respectively .
Fatty acids profile
Lipid assessment results of Asterarcys quadricellulare microalga recorded its high lipid productivity at 75%WW concentration. Thus, its Fatty acids profile were analyzed using GC and compared to the control (BBM). It was revealed that A. quadricellulare fatty acids profile mainly consisting of monounsaturated fatty acid (MUFA), followed by polyunsaturated fatty acid (PUFA) and saturated fatty acid (SAF) shown in Fig. 5 and Table 5. The total MUFA content showed a significant increase in 75%WW treatment with 23.2% higher than 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, a high proportion of Palmitic acid, Oleic acid and Linoleic acid were found in 75%WW and control treatments (Table 5). Microalgal lipids which have high proportion of C16:0 and C18:0 fatty acids are proper feedstock for biodiesel production [45, 46]. The dominant fatty acids in the microalgae were Palmitic, Stearic and Oleic acids . Thus, this microalga strain is a promising candidate as feedstock for biodiesel production. The increase in the fatty acids (C16-C18) in 75%WW may refer to the composition balance of this medium.
Biodiesel properties of Asterarcys quadricellulare grown in BBM and 75%WW treatments were determined as shown in Table 6. Iodine value is an indicating characteristic of the degree of unsaturation of fatty acid which influences the viscosity and cold filter plugging point. Iodine value of control treatment is 84.09, and of 75%WW has 77.08. Because 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 a biodiesel. Biodiesel with high amounts of saturated fatty acids will have a higher cetane number, while biodiesel with high amounts of unsaturated fatty acids will have a lower CN. Cetane number was 58.66 for control and 58.70 for 75%WW for Asterarcys quadricellulare biodiesel, it was found to be higher than both the standards ensuring good ignition quality low nitrous oxide emissions, less occurrence of knocking and easier engine start-up . Our results are agreeing with the literature, which report that most properties of biodiesel derived from the studied microalga species already meet with the limit values established by the ASTM D6751 and EN 14214 biodiesel standards [47, 49]. Also, most of the biodiesel properties represented in Table 6 for Asterarcys quadricellulare in both studied treatments complied with those of another study .