Screening the Pollution-Tolerant Chlorococcum sp. (Chlorophyceae) Grown in Municipal Wastewater for Simultaneous Nutrient Removal and Biodiesel Production

Abstract

Over the last few years and with increasing global climatic change, the international energy crisis and shortage of freshwater resources have raised many inquiries about global water security and energy. Therefore, finding out alternative and sustainable energy sources has become an important universal requirement. Here, we assessed the viability of exploiting municipal wastewater (WW) as a nutrient-rich growth medium for cultivating the pollution-tolerant coccoid green microalga Chlorococcum sp. (Chlorophyceae) to simultaneously remove nutrients and produce biodiesel. Chlorococcum sp. was isolated from municipal wastewater sampled from Menoufia Governorate, Egypt. Under the standard growth conditions and until reaching the late exponential growth phase, it was cultivated at different concentrations (25%, 50%, 75%, and 100%) of the secondary treated WW, and the findings were compared to the control (grown in BBM). The study results revealed that the 50% WW treatment was the most suitable approach for removing NO₃⁻, NH₄⁺, and TP with percentages of 96.9%, 98.4%, and 90.1%, respectively. Moreover, the 50% WW treatment produced the highest algal biomass (1.97 g L⁻¹) and productivity (82 mg L⁻¹ day⁻¹). In addition, it showed the highest lipid production (600 mg L⁻¹), with 25 mg L⁻¹ day⁻¹ lipid productivity and lipid yield with 30.5% of the cell dry weight (CDW). The gas chromatography–mass spectrometry (GC-MS) technique was applied to characterize fatty acid profiling, and it was found that oleic (C18:1) and palmitic (C16:0) fatty acids were present in much higher concentrations in Chlorococcum sp. cells grown in 50% WW as compared to the control, i.e., 44.43% and 27.38% vs. 36.75% and 21.36%, respectively. No big difference was present in linoleic (C18:2) fatty acid concentrations. Importantly, the biodiesel properties of our Chlorococcum sp. grown in 50% WW were consistent with the international biodiesel standards. In light of our findings, Chlorococcum sp. has a great potential for utilization as a biodiesel feedstock and for bioremediation of wastewater.

1. Introduction

During the last few decades, applications of renewable energy sources have become increasingly crucial to addressing energy demand, particularly in the developing countries. In Egypt, development of renewable energy sources is nowadays essential and is considered one of the most important priorities of the Egyptian Government because of the growing population. Egypt, therefore, has employed some ambitious strategies for establishing different renewable energy sources to integrate them with economic growth [1]. For instance, in November 2022, Egypt hosted the 27th Conference of the Parties of the United Nations Framework Convention on Climate Change (COP 27), with a view to pave the way to effectively tackle the global challenges of climate change and to increase sustainable energy resources.

Biofuel is a sustainable alternative for non-renewable fossil fuels. Biofuel production reduces CO₂ emissions that greatly contribute to global warming and climatic change. Biodiesel, biohydrogen, and bioethanol are the most popular types of biofuels made from biomass that includes oleaginous microorganisms such as algae and cyanobacteria [2]. Because of its ideal physicochemical characteristics, which are highly equivalent to those of conventional diesel, biodiesel offers, to a large extent, a possible substitute for petroleum-based fuel. Currently, researchers mainly focus on lowering the production costs of biofuels, using various green technologies and simultaneously creating more effective production methods. Microalgae can be used to produce large amounts of biofuels not only because of their high potential biomass production, but also because they can produce and store significant amounts of lipids that can be easily converted to biodiesel [3]. Importantly, microalgae and cyanobacteria can be cultivated in wastewaters rich in nutrients, such as nitrogen and phosphorus, contributing to the reduction of nutrient costs required for algal growth and bioremediation of these non-potable water sources [4]. Considering their higher biomass per unit area, i.e., approximately 7–20 times greater than corn or soybeans per land unit, microalgae can produce ~10–100 times more energy than the second-generation biodiesel crops [5,6]. Moreover, microalgae are considered an environmentally potential third-generation biodiesel feedstock [7,8]. Indeed, microalgae-derived biodiesel is less expensive when microalgae can be grown in wastewater rich in all the nutrients required for algal mass production. In addition, microalgae can be grown on lands that are unsuitable for food crops. It has been reported that microalgae can produce 5000 to 20,000 gallons of oil per acre per year [9]. Microalgae have much faster growth rates and productivity than other types of traditional agricultural crops. Microalgae need much less land area (49–132 times) than other biodiesel feedstocks of agricultural crops [10]. Moreover, CO₂ produced from industrial flue gas combustion can be employed to boost algal biomass productivity by over 30% in the field and by 100% in the laboratory setting [11]. Economically, microalgae can be grown throughout the year and their oil production was found to be much higher than any other crops [12]. In general, cultivation of microalgae in wastewater, as a supplemental source of nutrients, has dual functional advantages as they can control the environmental problems arising from nutrients discharging to freshwater bodies and they can provide good amounts of sustainable biodiesel [13,14].

The present study was performed to assess the biodiesel properties of coccoid green microalga Chlorococcum sp. (Chlorophyceae) grown in different concentrations of municipal wastewater. Another goal was to estimate its efficacy in nutrient removal from wastewaters. These promising results can be utilized as a starting point for large-scale construction of an algae-derived biodiesel production plant in Egypt.

2. Materials and Methods

2.1. Isolation and Growth Conditions of Chlorococcum sp.

Samples of municipal wastewaters that passed the secondary treatment were gathered from the treatment facilities in Egypt’s Menoufia Governorate. Sterilized plastic bottles were used to collect the water samples, which were subsequently brought to the laboratory. Large solid particles were removed using sedimentation and filtering by Whatman GF/C filter papers. To enrich the wastewater samples, 10% of culture Bold’s basal medium (BBM) and 90% of the sample were combined [15].

The batch incubation process occurred over the course of one week while being continuously illuminated by 80 µmol m⁻² s⁻¹ white light fluorescent lamps at 26 ± 2 °C and aerated by sterile filtered air. After an enrichment period, the growing microalgal species were visually examined and identified under a BEL^® photonics microscope (BEL^® Engineering, Monza, Italy). On agarized (1.5%) BBM, the growing microalgal strain Chlorococcum sp. was purified using the streaking method. The algal strain was recognized by monitoring its diagnostic morphotaxonomic features and by tracking the different stages of its life cycle [16].

2.2. Design of Nutrient Removal Experiment

The microalga Chlorococcum sp. was grown in a conical flask with 150 mL of BBM and incubated under the abovementioned growth conditions in order to be used as the inoculum for the following experiments. Four different concentrations of municipal wastewaters (25%, 50%, 75%, and 100% v/v in distilled water) were employed, with the growth medium (BBM) serving as the control. The experimental flasks containing 150 mL of each treatment were inoculated with 5 mL of microalgal growth for each treatment. Every three days, samples were evaluated, and every treatment was performed in triplicate.

2.3. Monitoring the Growth Rates of Chlorococcum sp.

This study measured the optical density at 560 nm (OD₅₆₀) over a 24-day period to evaluate the Chlorococcum sp. growth rates. Chlorophyll a was computed according to Lichtenthaler and Buschmann [17]. Following the measurement of cellular dry weight (CDW), biomass and lipid productivities were calculated [18,19]. The process adapted by Lee et al. [20] as described by Bligh and Dyer [21] was used to quantify total lipids. The Biuret assay as modified by Lowry et al. [22] was used to assess protein content. The phenol sulfuric acid method was used to measure the total amount of carbohydrates in the microalgal extract using d-glucose as a reference [23]. Every three days, samples were evaluated, and every treatment was performed in triplicate.

2.4. Removal of Nutrients

Through centrifugation and filtering through Whatman GF/C filter paper, the nutrient medium and microalgal biomass were separated from one another. After a 24-day growth period (the end of the late exponential growth phase), the consumption of ammonia (NH₄⁺), nitrate (NO₃⁻), total phosphate (TP), biological oxygen demand (BOD), chemical oxygen demand (COD), electrical conductivity (EC), and total dissolved solids (TDS) by the microalga Chlorococcum sp. was measured. NH₄⁺, NO₃⁻, and BOD were estimated in accordance with APHA [18], whereas TP was measured following the protocol described by Woods and Mellon [24]. COD was assessed using Mancy’s approach [25]. EC and TDS were measured using the conductivity benchtop meter. Based on the results, the removal rate (mg L⁻¹ day⁻¹) and percentage removal efficiency (%) were calculated in accordance with Eladel et al. [15].

2.5. Characterization of Fatty Acids

Before being subjected to gas chromatography analysis, the dry lipid extracts were transesterified to fatty acid methyl esters (FAMEs) [26]. In total, 0.3 mL H₂SO₄ and 1 mL methanol were added following 1 mL of the crude lipid layer collected. Additionally, 1 mL of distilled water was added, the mixture was vortexed for 3–5 min, incubated at 100 °C for 10 min, and then centrifuged at 4000 rpm for 10 min. A mass spectrometer (Thermo Scientific, Austin, TX, USA) with a TG-MS direct capillary column (30 m × 0.25 mm × 0.25 µm film thickness) was used to evaluate FAMEs utilizing gas chromatography. By matching the compounds’ mass spectra to those in the NIST/EPA/NIH mass spectral databases, the bioactive substances of Chlorococcum sp. were identified.

2.6. Characteristics of Biodiesel

According to Talebi et al. [27] and using Biodiesel Analyzer ver. 2.2 software (available at http://www.brteam.ir/biodieselanalyzer (accessed on 15 January 2023), fatty acid percentages were used to calculate the major biodiesel characteristics according to cetane number (CN), cold filter plugging point (CFPP), degree of unsaturation (DU), long-chain saturated factor (LCSF), saponification value (SV), iodine value (IV), kinematic viscosity (υ_i), density (ρ), and oxidative stability (OS).

2.7. Statistical Analysis

Each experiment was carried out in triplicate and the results are shown as means ± standard deviation (SD). Using the SPSS 16.0 software, statistical analyses were carried out. One-way analysis of variance (ANOVA) was used to compare mean values, and Duncan’s new multiple-range test was used to determine whether the results were statistically significant. At probability levels p ≤ 0.05, the differences were significant.

3. Results and Discussion

3.1. Growth Conditions

Chlorococcum sp. was grown on BBM as a control and various concentrations of secondary treated municipal wastewater (25%, 50%, 75%, and 100%). The physical and chemical properties of the secondary treated municipal wastewater are presented in

Table 1. Physical and chemical parameters of the locally produced secondary treated municipal wastewater.

Parameters	Values
pH	7.6 ± 0.07
TDS (mg L−1)	520.30 ± 47
EC (µS cm−1)	1106 ± 24.3
Temperature (°C)	26 ± 0.28
NO3− (mg L−1)	9.1 ± 1.24
NH4+ (mg L−1)	0.87 ± 0.06
TP (mg L−1)	1.39 ± 0.70
BOD (mg L−1)	12.17 ± 1.40
COD (mg L−1)	44.67 ± 3.20

Note: Data are expressed as mean ± SD (n = 3).

. Results of growth parameters (optical density, dry weight, and chlorophyll a) were obtained after 24 days of the incubation period. In comparison to the control and the other three treatments, the 50% WW treatment demonstrated the highest growth metrics as shown in Figure 1. The lowest algal growth parameters were recorded in the 25% and 75% WW treatments. Undiluted wastewater with an excessive nutrient content may be harmful to microalgae and could inhibit their growth [28]. However, excessive nutrient dilution may also prevent the formation of microalgae because of the lack of nutrients available [29]. Nutrient removal usually depends on the growth of the microalgae and the incorporation of nutrients into their biomass. In a similar study, in 40% treated wastewater, Chlorella sorokiniana biomass and total lipids were folded twice as compared to the control [30]. This finding was highly consistent with our observations, where more diluted wastewater exhibited better nutrient removal capability. In other words, 50% wastewater is the most suitable treatment for most uses, requiring a high-density microalgal culture in wastewater from municipalities. Wastewater with a high nutrient concentration can limit the growth of microalgae, whereas wastewater with a low nutritional concentration is insufficient for the algal growth. The previous work of Tan [31] also showed that C. oleofaciens can remove the nutrients TP, NO₃⁻, NH₄⁺, and COD from palm oil mill effluent.

3.2. Biomass and Biochemical Composition

The results for the Chlorococcum sp. biomass and biochemical composition are represented in

Table 2. Biomass and biochemical content of Chlorococcum sp. cultivated for 24 days in BBM as a control, along with the various concentrations of municipal wastewater of 25%, 50%, 75%, and 100%.

Parameters	Control (BBM)	25% WW	50% WW	75% WW	100% WW
biomass (g L−1)	1.84 ± 0.057 b	0.92 ± 0.041 d	1.97 ± 0.049 c	1.39 ± 0.019 a	1.46 ± 0.031 b
biomass productivity (mg L−1 day−1)	77.0± 1.99 b	38.0± 2.0 d	82.0 ± 0.987 c	58.0 ± 2.99 a	61.0 ± 2.99 b
lipid content (mg L−1)	520.6 ± 0.754 b	223.3 ± 2.88 d	600.0 ± 0.865 c	413.3 ± 0.220 a	450.7 ± 0.650 b
lipid productivity (mg L−1 day−1)	21.7 ± 0.130 a	9.3 ± 0.868 d	25.0 ± 0.125 c	17.2 ± 0.158 b	18.8 ± 0.065 ab
lipid yield (% CDW)	28.3 ± 0.760 a	24.3 ± 2.88 b	30.5 ± 0.871 b	29.7 ± 0.224 a	30.9 ± 0.650 a
protein yield (% CDW)	31.0 ± 0.112 a	31.6 ± 0.782 b	29.8 ± 0.653 b	31.1 ± 1.47 a	32.3 ± 1.29 a
carbohydrate yield (% CDW)	20.7 ± 0.568 a	26.7 ± 1.35 b	18.6 ± 0.389 b	22.5 ± 0.401 b	22.2 ± 0.376 a

Note: Each value represents the mean of three replicates ± SD. Data in the same row with the same letter reveal insignificant differences at p ≤ 0.05.

and Figure 2. The biomass productivity grown in the 50% WW treatment considerably demonstrated the maximum biomass production with a value of 82 mg L⁻¹ day⁻¹. These findings were consistent with earlier research conducted on the microalgal species Scenedesmus sp., Chlorella variabilis, and C. sorokiniana by Nagi et al. [30]. In general, the 50% WW treatment had the highest lipid content (600 mg L⁻¹), lipid productivity (25 mg L⁻¹ day⁻¹), and lipid yield (30.5% CDW), followed by the control (BBM), 100% WW, 75% WW, and 25% WW treatments. It is worth indicating that the 100% WW treatment had more or less the same lipid yield (30.9%) as the 50% WW treatment. The substantially increased biomass from this treatment at the end of the growth phase is most likely what caused the maximum lipid accumulation. It has been reported that the Chlorococcum sp. strain is a viable source for the manufacture of food, cosmetics, and biodiesel, which is in line with our findings on the Egyptian Chlorococcum sp. distinguished by its high lipid content and fatty acid composition [32]. Using FAMEs profiling, the Chlorococcum sp. strain can be exploited to produce a high-quality biodiesel [33,34]. Accordingly, our microalgal strain and other close taxa are highly recommended for biodiesel production because of their ability to grow in wastewaters and high lipid content production.

3.3. Removal of Nutrients

As shown in

Table 3. Nutrient removal rate (RR, mg L⁻¹ day⁻¹ for all the investigated parameters except EC: µS cm^−S) and removal efficiency (RE, %) of Chlorococcum sp. growing for 24 days in control (BBM) and the various municipal wastewater concentrations.

Parameters	Control (BBM)		25% WW		50% WW		75% WW		100% WW
	RR	RE%	RR	RE%	RR	RE%	RR	RE%	RR	RE%
NH4+	0.02 ± 0.0	90.9 ± 1.38	0.01 ± 0.0	72.88 ± 5.42	0.02 ± 0.0	98.38 ± 1.93	0.03 ± 0.01	90.20 ± 1.0	0.04 ± 0.0	93.33 ± 2.78
NO3−	0.08 ± 0.01	91.44 ± 1.11	0.10 ± 0.01	80.42 ± 1.09	0.21 ± 0.01	96.90 ± 1.72	0.21 ± 0.01	90.74 ± 1.2	0.36 ± 0.01	91.42 ± 1.19
TP	0.05 ± 0.0	79.82 ± 1.51	0.01 ± 0.0	63.07 ± 1.48	0.03 ± 0.0	90.11 ± 1.27	0.04 ± 0.01	82.23 ± 1.95	0.05 ± 0.0	83.70 ± 0.85
COD	1.45 ± 0.12	89.94 ± 5.31	0.35 ± 0.06	57.82 ± 7.57	0.98 ± 0.02	91.73 ± 2.93	1.17 ± 0.07	80.19 ± 1.12	1.73 ± 0.04	88.90 ± 1.56
BOD	0.29 ± 0.02	86.56 ± 1.38	0.08 ± 0.02	52.41 ± 5.36	0.26 ± 0.03	96.67 ± 3.09	0.34 ± 0.01	81.55 ± 1.77	0.46 ± 0.05	87.04 ± 6.17
TDS	4.12 ± 0.19	53.66 ± 3.12	2.17 ± 0.19	34.20 ± 3.05	8.55 ± 0.21	76.16 ± 2.94	8.65 ± 0.25	51.52 ± 1.3	13.33 ± 0.05	58.93 ± 0.59
EC	7.13 ± 0.35	58.52 ± 2.84	3.94 ± 0.40	28.96 ± 3.95	14.75 ± 0.52	56.80 ± 1.62	14.08 ± 0.42	35.31 ± 0.91	19.63 ± 0.64	37.64 ± 1.05

Note: Each value represents the mean of three replicates ± SD.

and Figure 3, nutrient removal rates and efficiency for the secondary treated municipal wastewater by Chlorococcum sp. were discussed. The highest NH₄⁺ removal rates were found in treatments using 100% WW and 75% WW, with values of 0.04 mg and 0.03 mg L⁻¹ day⁻¹, respectively. These values represent 1.75- and 1.25-times higher removal rates than the control (BBM), respectively. Likewise, the rates of NO₃⁻ elimination followed the similar pattern, measuring 0.36, 0.21, and 0.21 mg L⁻¹ day⁻¹ for 100% WW, 75% WW, and 50% WW, respectively. In general, and based on our findings, Chlorococcum sp. grown in 50% WW concentration showed the highest removal efficiency for NH₄⁺ and NO₃⁻ with values of 98.4% and 96.9%, respectively, followed by 100% WW (93.3% and 91.4% for NH₄⁺ and NO₃⁻, respectively). The Chlorococcum sp. investigated in this work had the highest removal efficiency for TP with values of 90.1% at concentration 50% WW with a final value of 0.19 mg L⁻¹, followed by 83.7% (0.23 mg L⁻¹) at concentration 100% WW and 82.2% (0.20 mg L⁻¹) at concentration 75% WW. According to our findings, at some wastewater concentrations, NH₄⁺ removal was slightly higher than NO₃⁻ removal. This might be explained by direct NH₄⁺ utilization by Chlorococcum sp. or by simply stripping ammonia gas [35]. In line with our observations, Tan et al. [31] reported that C. oleofaciens could eliminate more than 98% of TN and NH₄⁺ from the lab-scale palm oil mill effluent. As regards TP removal efficiency, it ranged from 63% to 90%. The findings of this study were consistent with those of other earlier studies where it was postulated that Chlorella vulgaris exhibited a good TP removal from undiluted cattle farm wastewater by 85.29% [36]. In addition, 97.7% phosphorous in domestic wastewater was eliminated by microalgal treatment [37].

As regards COD, Chlorococcum sp. reduced COD concentrations in the 100% WW treatment to 4.97 mg L⁻¹ (88.9% removal efficiency and 1.73 mg L⁻¹ day⁻¹ removal rate). Similarly, BOD concentrations were also reduced to 1.57 mg L⁻¹ with removal efficiency of 87.0% and a removal rate of 0.46 mg L⁻¹ day⁻¹. Furthermore, the 50% WW treatment caused the maximum reduction of BOD and COD with 96.7% and 91.7% removal rates, respectively. The increase in algal growth, which resulted in increased photosynthesis to produce more oxygen, could explain this action [38]. Therefore, the released oxygen promoted the oxidation of organic materials. This indicated that, when compared to COD, microalgae are more effective at removing BOD. The elevated wastewater COD values, which are unaffected by microflora, are most likely the main reason because of the high concentration of organic molecules [39]. Our Chlorococcum sp. revealed the highest TDS and EC removal efficiency (76.2% and 56.8%, respectively) with removal rates 8.55 and 14.75 mg L⁻¹ day⁻¹, respectively, in the 50% WW treatment (Table 3). In general, the incorporation of nutrients into algal biomass and the subsequent microalgal growth are what determine the process of nutrient removal. The study found that the 50% WW treatment had the highest ability to remove nutrients. Based on this observation, in the second phase of this study, Chlorococcum sp. was applied in the 50% WW treatment to conduct additional research in order to be used as a feedstock for the biodiesel production.

3.4. Fatty Acid Composition

As mentioned in the previous section, Chlorococcum sp. demonstrated high lipid productivity in the 50% WW treatment. Gas chromatography−mass spectrometry (GC−MS) was used to analyze the fatty acid profile and compare it to the control. As shown in

Table 4. Fatty acid profile (as % percentage composition) of Chlorococcum sp. cultured for 24 days in control (BBM) and 50% WW treatment.

Fatty Acids	Control (BBM)	50% WW Treatment
Saturated fatty acids (SFAs)
Pentadecenoic acid (C15:0)	0.42 ± 0.02	−
Palmitic acid (C16:0)	21.36 ± 1.8	27.38 ± 1.4
Stearic acid (C18:0)	0.93 ± 0.02	0.56 ± 0.04
Behenic acid (C22:0)	0.38 ± 0.01	−
Octacosanoic acid (C28:0)	0.41 ± 0.01	−
Monounsaturated fatty acids (MUFAs)
Palmitoleic acid (C16:1)	−	1.54 ± 0.1
Oleic acid (C18:1)	36.75 ± 3.0	44.43 ± 3.7
Polyunsaturated fatty acids (PUFAs)
Hexadecadienoic acid (C16:2)	3.49 ± 0.7	2.92 ± 0.5
Linoleic acid (C18:2)	23.45 ± 2.9	21.02 ± 2.4
Hexadecatrienoic acid (C16:3)	4.93 ± 0.6	−
α-Linolenic acid (C18:3)	0.98 ± 0.08	−
Hexadecatetraenoic acid (C16:4)	4.85 ± 0.95	1.50 ± 0.08
Stearidonic acid (C18:4)	2.06 ± 0.4	0.66 ± 0.06
SFAs	23.50	27.94
MUFAs	36.75	45.97
PUFAs	39.75	26.09
Total	100.00	100.00

Note: Each value represents the mean of three replicates ± SD.

and Figure 4, the 50% WW treatment had higher values of monounsaturated and saturated fatty acids as compared to the control, i.e., 45.97% and 27.94% vs. 36.75% and 23.50%, respectively. It is worth noting that a much lower value of polyunsaturated fatty acids was recorded in the 50% WW treatment (26.09%) than in the control (39.75%). The fatty acid profile of Chlorococcum sp. in general had carbon chains ranging from C15 to C18. The monounsaturated oleic acid (C18:1) was the predominant fatty acid species in both the control and the 50% WW treatment. However, its relative content in the 50% WW algal treatment (44.43%) was higher than the control (36.75%). It is worth indicating that Chlorococcum sp. revealed a higher content of fatty acids in the wastewater treatment than in the control, except for linoleic acid (C18:2). Our findings indicated that the fatty acids profile of the tested microalgal strain made it a good choice for a feedstock in the production of biodiesel. Microalgae growing in wastewater have an advantage because of the rise in fatty acids (C16−C18) in 50% WW. Because of the predominance of saturated fatty acids, the fuel shows oxidative stability [40].

3.5. Biodiesel Properties

Chlorococcum sp. grown in BBM and the 50% WW treatment were assessed for their biodiesel characteristics as indicated in

Table 5. Biodiesel characteristics based on the fatty acid profile of Chlorococcum sp. grown for 24 days in the control (BBM) and 50% WW treatment, compared to international standards.

Predicted Biodiesel Characteristics	Chlorococcum sp.		International Standards of Biodiesel
	Control	50% WW	ASTM D6751-08 [45]	EN 14214:2012+A2:2019 [46]
Degree of unsaturation (DU)	89.73	89.33	–	–
Saponification value (mg g−1) (SV)	175.90	195.68	–	–
Iodine value (gI 100 g−1) (IV)	85.78	81.99	–	less 120
Cetane number (CN)	58.03	55.74	above 47	minimum 51
Long-chain saturated factor (LCSF)	3.17	3.02	–	–
Cold filter plugging point (°C) (CFPP)	−6.52	−7.0	−5 to −13	5 to −20
Cloud point (°C) (CP)	6.24	9.41	−3 to 12	4
Pour point (°C) (PP)	−0.04	3.39	−15 to 10	–
Allylic position equivalent (APE)	89.73	87.79	–	–
Bis-allylic position equivalent (BAPE)	25.41	21.02	–	–
Oxidation stability (h) (OS)	7.42	8.20	3	8
Higher heating value (HHV)	34.04	37.69	–	–
Kinematic viscosity (mm2 s−1) (υi)	3.13	3.59	1.9–6	3.5–5
Density (g cm−3) (ρ)	0.76	0.84	0.88	0.86–0.90

. When assessing the stability of biodiesel, iodine value (IV) is a key factor [41]. The higher the cetane number (CN), which indicates better fuel and vice versa, the lower the iodine value. The unsaturation and sensitivity to oxidation and rancidification increase with increasing iodine value. The expected iodine value of biodiesel produced from the control treatment was 85.78 gI 100 g⁻¹, while the 50% WW treatment had 81.99 gI 100 g⁻¹. Both values were lower than the European and American standards. Another conducted study reported the significance of the high value of CN for the synthesis of high-quality biodiesel, whereas a low CN of the fuel is characterized by the high ignition delay period [42]. In this study, Chlorococcum sp. had a cetane number value of 55.74 in the 50% WW treatment. Therefore, cetane number values best met the standard ASTM D6751; this is greater than 47 and seems to have high fuel ignition quality. Our microglial strain met the requisite CN value because of their high contents of MUFAs and SFAs. Accordingly, its biodiesel properties generated from growing in the wastewater in this study can be applied to modern engines. Generally, the current findings shown in Table 5 were supported by the literature, which claimed that the majority of biodiesel properties derived from the investigated microalga species complied with international standards and met the limit values set by ASTM D6751 and EN 14214:2012+A2:2019 biodiesel standards [42,43,44].

4. Conclusions

In this study, cultivation of the coccoid green microalga Chlorococcum sp., using the 50% WW treatment, is the suggested best approach for the production of high algal biomass, lipid content, productivity, elimination of nutrients, and biodiesel generation. In addition, it was revealed that the biodiesel properties of Chlorococcum sp. grown in 50% WW conditions are highly consistent with the international biodiesel standards, making it a good candidate for the large-scale production of biodiesel. Lastly, the cultivation of microalgae in municipal wastewater is an alternative and sustainable green technology with a dual function.