Production of Biodiesel Employing Chlorella vulgaris Biomass Cultivated in Poultry Effluents

Abstract

Alternative energies have become relevant in global strategies to address climate change, and third-generation biodiesel derived from the generation of lipids from microalgae represents a viable option. This process can also be coupled with wastewater treatment to remove organic matter. To determine the effects of two catalyst levels (1 and 1.5% KOH) and two molar ratios of alcohol (methanol) with oil (1:6 and 1:9) on the conversion of lipids into FAMEs and the quality of the biodiesel produced, this work suggests a method for the ultrasonication-based extraction of lipids from C. vulgaris. It also employs an experimental 2² design and three replicates. It was found that with a molar ratio of 1:9 and a 1% catalyst, the highest yield of 98.48 ± 1.13% was achieved. The FAME profile was similar to the profiles obtained in cultures with bold basal medium or INETI. The quality of the biodiesel met ASTM standards, achieving refractive indices of 1.435–1.478. The flash point (FP) was 165 ± 18 °C, and the acid number was 0.31 ± 0.17 mg KOH/g. The viscosity ranged from 4.33 to 4.87 mm²/s. However, the rheological behavior was correlated with the Ostwald–de Waele model with pseudoplastic behavior.

1. Introduction

In recent years, green energy alternatives, such as natural gas, oil, and their derivatives, have been sought because they are cleaner and more sustainable than conventional fossil fuels concentrated only in certain regions of the world [1]. Although many countries have large oil reserves, these alternatives will not last long since most of the world’s energy needs are supplied through petrochemical sources [2]. Nowadays they are completely depleted due to high demand and the global energy transition [3].

There are different methodologies for producing liquid biofuels, classified according to the raw materials employed in their production [4]. First-generation biofuels primarily come from crops such as sugarcane and sugar beet, as well as certain types of oils like palm and sunflower [5]. On the other side, second-generation biofuels are advantageous because they generate lower greenhouse gas (GHG) emissions; they utilize residual biomass and do not require the cultivation of new raw materials [6].

Significant biological issues with third-generation biofuel production restrict them from being scaled [7]; however, they represent an avant-garde alternative for environmental care since they originate from organic matter. Additionally, they have a high degree of biodegradability [8]. Currently, microalgal biomass (MB) has been considered and investigated for its high rate of lipid production [6], which is estimated to constitute more than 70% of its total dry weight [8,9]. Therefore, people consider it a raw material to produce third-generation biofuels. Additionally, proteins, carbohydrates, and other inorganic species are present in MB [10], which means that MB is used to obtain products with high energy and economic value.

MB is an energy source that could replace fossil fuels in the long term. However, we must pursue strategies to enhance biomass production and devise biofuel conversion plans [11]. Besides that, microalgae can also produce non-combustible compounds that can be used as food supplements in the pharmaceutical industry [12], in medical fields with therapeutic and human health applications [13,14], and in various other applications [10].

Certain microalgae species, such as Chlorella vulgaris, Scenedesmus obliquus, Synechococcus sp., and Synechocystis sp., have demonstrated the ability to produce biofuels on a substantial scale. These species can produce biogas from waste [15]. Chlorella kessleri can produce biohydrogen from biomass following wastewater treatment and air purification. Additionally, researchers have employed Phaeodactylum tricornutum in the production of biodiesel from biomass [16]. Currently, researchers have identified C. vulgaris (order Chlorellales and family Chlorellaceae) as one of the most extensively researched eukaryotic species [17]. It is a microscopic spherical cell with a diameter ranging from 2.5 to 10 µm [18]. Furthermore, C. vulgaris is notable for its ability to synthesize substantial amounts of lipids in the absence of nitrogen sources [19].

These lipids can be valued with the production of biodiesel [20]. Biodiesel is an oxygen-rich fuel derived from various renewable sources, such as edible and non-edible oils, residual feedstocks, animal fats, and algae [21]. It has been reported that the physicochemical characteristics of biodiesel are affected by changes in the fatty acid methyl ester (FAME) profile [22], so it is essential to choose the right method for obtaining biodiesel from raw material. Between pyrolysis, transesterification, dilution, and micro-emulsification, the transesterification process stands out for achieving high-quality and high-quantity biodiesel [23].

Alkaline transesterification has demonstrated superior efficacy, even with intricate and residual lipid sources [24]. In a transesterification process, catalysts like acids or bases transform oils into alkyl esters in the presence of alcohol [23,25]. Homogeneous catalyst-assisted transesterification is the most widely employed method. Among the most often employed catalysts are acidic and alkaline types [25]. Acid catalysts have a poor reaction rate and induce technical complications in further processing, whereas alkaline catalysts are more susceptible to the concentration of free fatty acids in the oil and generate soap [26]. Alkaline-catalyzed transesterification has several advantages, including fast reaction kinetics that reduce the time required for the process and allow for simpler design [27]. Many fundamental catalysts have been used for this purpose, mainly NaOH [28], KOH [29], KBr, and CaO, which are all recognized as metallic catalysts [30,31].

The significance of catalyst concentration must be considered, as excessive catalyst in oil–methanol mixtures leads to a more concentrated solution, thereby reducing conversion efficiency. A higher catalyst leads to increased mixture viscosity, which modifies the rheological properties and may influence the final product [32,33]. Alkaline catalysts such as KOH have a multifaceted function in biodiesel generation, as demonstrated by prior research. Eze et al. [34] created a model to understand how the transesterification process works with KOH in two stages, demonstrating that other reactions, such as the saponification of triglycerides and the formation of FAME, are also involved; the balance between hydroxide and alkoxide greatly influences the neutralization of free fatty acids (FFAs). This balance affects how many active alkoxide species are available, which are important for making FAME and other reactions. While similar events have been noted in regular oil systems, there have been no additional studies on how Chlorella vulgaris behaves when grown in waste from the poultry industry. Therefore, it is crucial to analyze the influence of KOH concentration and reaction conditions on biodiesel production from microalgae [35].

This work examines a novel approach that combines wastewater treatment with fuel production through the synthesis of biodiesel by alkaline transesterification utilizing lipids obtained from C. vulgaris MB proliferating in nitrogen-dense chicken effluents rich in organic matter. This research employs a 2² factorial experimental design with three replications for each condition. The criteria examined were the concentration of the alkaline catalyst (KOH) and the molar ratio of alcohol to lipids. The generated biodiesel underwent physicochemical and rheological assessments to evaluate its qualities in accordance with regulatory norms.

2. Materials and Methods

2.1. Sample Conditioning and Biochemical Characterization of MB

This research investigated the utilization of microalgal biomass (MB) obtained by reducing chemical oxygen demand (COD) and nitrogen in chicken wastewater within 4 L Applikon photobioreactors employing C. vulgaris [19]. The microalga was produced in poultry wastewater at a concentration of 75%, equating to 1250 mg COD/L, under conditions of 9800 lux and aeration at 3.2 L/min and inoculated with C. vulgaris previously grown in bold basal medium (BBM) at a density of 3.41 × 10⁵ cells/μL in wastewater. The microalgal biomass was harvested after 16 days of growing, yielding approximately 200 g of wet biomass, and subsequently dried in a tray drier at 50 °C, followed by conditioning as detailed.

The biochemical evaluation of the MB was carried out as follows: the protein content was assessed using the Lowry method [36], the carbohydrate content was evaluated using the phenol-sulfuric method [37], and the lipid content was measured by the Folch method [38].

To facilitate cell wall disruption, pretreatment involving maceration with solvents was used. Specifically, 25 g of MB was macerated with 40 mL of 96% ethanol (Meyer^®, CAS 64-17-5) for 5 days in a 100 mL beaker (Figure 1). After maceration, the liquid was then homogenized, and the glass was sealed to prevent contamination; containers were refrigerated for future use at 4 °C ± 0.5.

2.2. Lipid Extraction and Purification

The extraction method begins with the lysis of the MB cells, executed via ultrasonication (Model 8892, Branson, CTR Scientific, Nuevo León, Mexico) at a frequency of 400 kHz and an output power of 200 W. Then, 10 min active and 5 min inactive pulse intervals were employed. Upon completion of the sonication, the application of ultrasound induced the cellular lysis of the microalgal biomass, leading to enhanced and expedited oil extraction [39,40]. The samples were extracted from the ultrasonic bath and subjected to drying in an oven (Model 9023A, Ecoshel, Mexico City, Mexico) for 20 min at 50 °C.

The pretreatment sample was subjected to three sequential ethanol extraction processes, involving agitation and filtration, to isolate the chemicals contained in the MB. The biomass was agitated for one hour at 500 rpm at ambient temperature and thereafter filtered using a glass funnel and filter paper; the resultant alcoholic extract was gathered and preserved in a 250 mL Erlenmeyer flask. In the second step, the residual dry biomass was subjected to treatment with a hexane–isopropanol combination in a volumetric ratio of 3:2, agitated for a further 2 h under identical conditions, and then filtered; the resultant liquid fraction was combined with the same flask used in the initial step [41]. In the third phase, 30 mL of 96% ethanol was added to the resultant biomass, which was agitated for 1 h at 500 rpm and ambient temperature, followed by a final filtration. The amalgamated liquid extract was subjected to an oven at 60 °C for 24 h to facilitate solvent evaporation [42].

Purification of lipids is needed due to their residual nature; the use of adsorbent materials is among the most commonly utilized methods of lipid purification and deodorization. This was performed using 6 g of acid-treated activated carbon, which was included in the liquid samples [43].

These samples were then placed in sealed beakers to prevent solvent evaporation. The samples were allowed to macerate for 48 h in the dark at room temperature (25 ± 0.5 °C). After this period, the samples were filtered using Whatman filter paper and a separatory funnel. To facilitate the extraction of purified lipids, lipids were extracted utilizing 3 mL of chloroform as a non-polar solvent in a chloroform–methanol extraction to recover the lipids from the biomass [44] (see Figure 1).

To quantify the lipid content of the samples, the filtered sample was placed in test tubes at a consistent weight. The samples were kept for 24 h at 60 °C in a heating oven (Model 9023A, Ecoshel, Mexico City, Mexico) to remove the excess hexane; thereafter, they were weighed until a stable weight was achieved and then permitted to cool in a desiccator. Finally, the tubes were weighed to determine the proportion of pure lipids using Equation (1). The requisite production cycles were executed to generate biodiesel via alkaline transesterification in 30 g batches of lipids.

$$\% Lipids={\displaystyle \frac{Final sample weight-Initial sample weight}{Biomass weight}} \ast 100$$

(1)

Lipid Characterization

The physicochemical characterization of lipids acquired in Section 2.2 was conducted to assess their quality. To determine whether the analyzed oil qualifies as a raw material for biodiesel production, choices were made based on the prevailing Mexican regulations for fats and oils, as outlined in

Table 1. Physicochemical characterization of lipids of C. vulgaris obtained using the Soxhlet method.

Parameter	Methods
Saponification index	NMX-F-174-SCFI-2014
Acidity index	NMX-F-101-SCFI-2012
Density	NMX-F-075-SCFI-2012
Moisture and volatile material	NMX-F-211-1987
Refractive index	NMX-F-074-S-1981

[45].

2.3. Biodiesel Production by Alkaline Transesterification

The impact of the alcohol-potassium hydroxide ratio at various concentrations on biodiesel production was assessed using the transesterification of lipids from C. vulgaris. A design experiment (2²) with three replicates was employed to facilitate the conversion of fatty acids into biodiesel. Two concentrations of catalyst (1% and 1.5% KOH) and two molar ratios of methanol to oil (1:6 and 1:9) were tested at a temperature of 60 °C. The comprehensive matrix is presented in

Table 2. Experimental matrix.

Random Sequence	Run Order	Concentrations of Catalyst (%)	Molar Ratio
7	1	1.0	1:9
5	2	1.0	1:6
11	3	1.0	1:9
10	4	1.5	1:6
1	5	1.0	1:6
3	6	1.0	1:9
2	7	1.5	1:6
12	8	1.5	1:9
8	9	1.5	1:9
4	10	1.5	1:9
6	11	1.5	1:6
9	12	1.0	1:6

. The results were analyzed with 95% confidence using the Minitab^® software, version 22.

The transesterification reaction was conducted in a 100 mL three-neck round-bottom flask fitted with a reflux condenser and thermocouple, as seen in Figure 2. Thirty grams of pre-conditioned lipids was added, along with the right amount of methanol. A catalyst was introduced and incubated at 60 °C for 1 h with continuous stirring at 600 rpm. After the reaction was completed, the mixture was transferred to a separatory funnel and allowed to settle for 12 h. The glycerin generated during the previous process was extracted, and then, 5 mL of distilled water was introduced to the biodiesel in the separating funnel. The mixture was stirred vigorously to remove any residual substances. Finally, the biodiesel was held at 120 °C in a heating oven for 1 h to eliminate any excess unreacted alcohol and residual glycerin.

Biodiesel yield was calculated by Equation (2), using data obtained through biodiesel production via alkaline transesterification [46].

$$Biodiesel Yield (\%)={\displaystyle \frac{Weight of the produced biodiesel}{Weigth of the oil}} \ast 100$$

(2)

2.4. Physicochemical and Rheological Characterization of Biodiesel

The biodiesel that was made was tested for its physical and chemical properties, as well as its flow characteristics, following the guidelines set by the American Society for Testing and Materials (ASTM) in

Table 3. ASTM standard parameters for evaluating biodiesel quality obtained via alkaline transesterification.

Parameter	Methods	References
Density	ASTM D1298	[47]
Cloud point	ASTM D2500-17	[48]
Refractive index	ASTM D1218-12	[49]
Copper foil corrosion	ASTM D130-19	[50]
Flash point	ASTM D93	[51]
Carbon residues	ASTM D4530-15	[52]
Acid number	ASTM D664 and EN 14104	[53,54]
Kinematic viscosity	ASTM D445	[55]

. This was done to confirm the quality and use of the product.

The characterization of the fatty acid methyl ester (FAME) profile was conducted using Thermo Scientific TRACE 1310 (Thermo Fisher Scientific, Waltham, MA, USA) gas chromatography equipment, integrated with a Thermo Scientific ISQ 7000 (Thermo Fisher Scientific, Waltham, MA, USA) single-quadrupole GC-MS system, employing a 30 m × 0.25 mm × 0.25 µm TG-WAXMS column. A recognized reference material, FAME C8–C22 from the Merck Supelco-CRM18920 brand, was utilized.

Rheological characterization was conducted using a viscometer Brookfield DV2T (Brookfield, WI, USA) equipped with a temperature control jacket and a “UL” needle; 15 mL was utilized for the measurement, performed at a constant temperature of 25 ± 2.0 °C, with shear rate changes ranging from 0 to 517 s⁻¹ [56]. The results were calibrated to the Herschel–Bulkley, Ostwald–de Waele, and Newton (Equations (3)–(5)) rheological models to ascertain which model most effectively elucidates the physical alterations in residues before, during, and after each stage of this process. The acquired rheological parameters included the consistency index ($k$), flow index (n), yield stress (${\tau }_o$), deformation rate ($\dot{\gamma }$), and dynamic viscosity ($\eta$). After calculating dynamic viscosity using the Newtonian model, kinematic viscosity was calculated by dividing η by ρ, where ρ represents the density of the produced biodiesel.

$$\tau =k·{\dot{\gamma }}^n$$

(3)

$$\tau ={\tau }_o+\left(k·{\dot{\gamma }}^n\right)$$

(4)

$$\tau =\eta \dot{\gamma }$$

(5)

3. Results

3.1. Lipid Characteristics

The MBs in this investigation exhibited macronutrients (proteins, carbs, and lipids) consistent with the existing literature. A protein concentration of 153.16 ± 0.30 mg/L and a carbohydrate concentration of 197.81 ± 0.48 mg/L were recorded. The characterization of the MB revealed lipid levels of 16.53 ± 0.75%, which are within the usual range exhibited by MB derived from Chlorella vulgaris, reported to be between 15% and 58% [8,9,57]. In this study, high lipid values were not observed in MB, because they are attained when their cultivation occurs under nutritional stress. This indicates that wastewater from the poultry industry sector supplies essential nutrients for crops.

3.2. Microalgal Metabolism and Biofuel Production Pathways

Lipid synthesis is influenced by the external growth circumstances experienced by microalgae, as well as by the sources of organic matter and CO₂. In comparison to conventional forests, agricultural and aquatic plants exhibit superior growth rates and carbon dioxide capture capabilities [58]. The utilization of microalgae has demonstrated efficacy and cost-effectiveness in CO₂ bio-fixation, with photosynthesis serving as the mechanism for microalgae to capture and sequester CO₂ [59]. Also, microalgae generate biomolecules, including carbohydrates, proteins, and lipids (Figure 3).

In this scenario, the Calvin cycle’s response to light generates ATP and NADPH, which are subsequently utilized to synthesize carbohydrate precursors through the assimilation of CO₂, a process enhanced by light availability. The preliminary phase in CO₂ sequestration for photosynthetic cells involves the effective transport of inorganic carbon (Ci) to the cell membrane [60]. Figure 3 illustrates the transesterification of triglycerides via alkaline transesterification using KOH and methanol, alongside the metabolism of triglycerides in C. vulgaris during the processing of chicken wastewater. The characteristics of biodiesel generated through the transesterification process are significantly influenced by the type of oil used, suggesting that the transesterification reaction occurs when fatty acid methyl esters (FAMEs) are the desired outcome of the reaction between triglycerides and methanol [61].

Figure 3. (a) Schematic representation of the triglyceride biosynthesis and accumulation pathways in Chlorella vulgaris. (b) Alkaline transesterification reaction of triglycerides with methanol (MeOH) and potassium hydroxide (KOH) to produce fatty acid methyl esters (FAMEs). Created with BioRender.com [46,62].

Figure 3. (a) Schematic representation of the triglyceride biosynthesis and accumulation pathways in Chlorella vulgaris. (b) Alkaline transesterification reaction of triglycerides with methanol (MeOH) and potassium hydroxide (KOH) to produce fatty acid methyl esters (FAMEs). Created with BioRender.com [46,62].

Using microalgae as a raw material for biodiesel production offers a significant advantage since being unicellular guarantees biomass has the same biochemical composition. It occurs with other raw materials such as plants since the compounds of these materials vary according to different parts, such as fruits, leaves, seeds, and roots [62]. The concentration of the catalyst is crucial, and a higher concentration can result in lower yields, which causes losses from unreacted alcohol, leftover catalysts, and emulsions removed during production [63]. At the transesterification stage, the methanolysis of triglycerides obtained from MB into methyl esters occurs, and a secondary reaction occurs, which consists of the saponification of glycerides (formation of soaps) and methyl esters caused by KOH [64,65]. In summary, Chlorella biomass cultivated in poultry wastewater not only demonstrates a viable macronutrient composition but also represents an exceptionally promising and sustainable option for biodiesel production through alkaline transesterification.

This study demonstrates that utilizing microalgae as a feedstock for biodiesel synthesis is beneficial, as its unicellular structure guarantees a uniform biochemical composition throughout the biomass. This differs from traditional plant-based raw materials, where composition varies between tissues, including fruits, leaves, seeds, and roots, resulting in unpredictable yields and processing difficulties [62]. The experimental findings demonstrated that catalyst concentration significantly influences biodiesel yield; elevated concentrations correlated with diminished yields, presumably due to enhanced losses of unreacted alcohol, residual catalyst, and the development of emulsions that are eliminated during processing [63]. In the transesterification phase, the methanolysis of triglycerides derived from Chlorella biomass resulted in the production of methyl esters, alongside a secondary saponification process that generated soaps from both glycerides and methyl esters in the presence of KOH [64,65]. These findings corroborate prior observations and underscore the necessity of meticulously adjusting catalyst concentration to prevent side reactions and material losses. The cultivated Chlorella biomass exhibited a satisfactory macronutrient composition and significant potential as a sustainable feedstock for biodiesel production through alkaline transesterification, especially when cultivated in poultry wastewater, thereby aiding in renewable energy generation and wastewater bioremediation.

3.3. Biodiesel Yields and Characterization

Yields obtained from the production of biodiesel from lipids extracted from the MB of C. vulgaris from a culture in wastewater from the poultry industry were higher than 94% (Figure 4) when different molar ratios were applied through alkaline transesterification. The highest yields were obtained with 1% KOH at molar ratios of 1:6 and 1:9. The results align with those reported by López, Bocanegra, and Malarón-Romero [66], indicating that a 1% catalyst yields the highest outputs. This is due to elevated KOH concentrations, which enhance the saponification reaction, thereby hindering the transesterification process and reducing biodiesel conversion efficiency [67]. In this work, the highest yield achieved was 98.48 ± 1.13%, which is higher than obtained from cooking oil [68].

A catalyst’s ideal concentration is determined by the lipid composition of the substrate. Ashouri [69] demonstrated that a maximum biodiesel conversion yield of 94% was achieved with an ideal catalyst weight percentage of 0.01%. Conversely, Ulukardesler [70] attained a peak conversion rate (91%) with a catalyst concentration of 2%. This signifies that a characterization must be performed before the transesterification procedure.

3.4. FAME Characterization of Biodiesel

The FAME profile revealed the presence of myristic acid (14:0), palmitic acid (16:0), palmitoleic acid (16:1), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2), and linolenic acid (18:3). The profiles obtained through the characterization of biodiesel agrees with the literature, and they are between 80% and 85% of the DMARDs generated from lipids obtained from C. vulgaris. The ratio of unsaturated acids–saturated acids was 1.9142, which is close to those generated in a culture with bold basal medium (1.7986) [71] and INETI 58 medium (1.3580) [72], This indicates that the culture derived from poultry wastewater serves as a suitable medium for producing lipids using the microalga C. vulgaris.

Table 4. Comparison of FAME profiles obtained from lipids of C. vulgaris in different culture media.

FAME	Culture Medium
	FAME Obtained (%)	Bold Basal (%) [71]	INETI 58 (%) [72]
14:0	1.74	2.5	3.07
16:0	25.08	25.20	25.07
16:1	1.63	1.80	5.25
16:3	0.31	0	1.27
16:4	1.02	0	4.06
18:0	1.75	8.75	0.63
18:1	27.75	22.5	12.64
18:2	11.98	17.25	7.19
18:3	20.08	18.5	19.05
20:0	0	0	0.09
20:1	0.23	0	0.93
20:3	0.21	0	0.83
20:4	0	0	0.23
20:5	0.12	0	0.46
Saturated	28.57	36.45	28.86
Unsaturated	63.33	60.05	51.91

shows the FAME profile of biomass cultured in poultry wastewater and two similar profiles reported in the literature for synthetic culture media.

3.5. Statistical Analysis of Quality Biodiesel Production

Table 5. Physical and chemical properties of biodiesel produced.

Test (% KOH, Molar Ratio Methanol to Oil)	Density (g/mL)	Refractive Index (-)	FP (°C)	Carbon Residue (-)	Acid Number (mg KOH/g)
1.0%, 1:6	0.863 ± 0.005 a	1.476 ± 0.003 a	161.333 ± 5.033 ab	0.035 ± 0.005 b	0.263 ± 0.041 b
1.0%, 1:9	0.829 ± 0.016 b	1.447 ± 0.011 b	152.666 ± 3.214 b	0.024 ± 0.004 a	0.393 ± 0.031 a
1.5%, 1:6	0.879 ± 0.010 a	1.435 ± 0.002 b	179.666 ± 5.033 a	0.046 ± 0.006 b	0.216 ± 0.058 b
1.5%, 1:9	0.879 ± 0.011 a	1.445 ± 0.009 b	160.666 ± 12.503 ab	0.043 ± 0.002 a	0.473 ± 0.011 a

The same lowercase letters by row represent non-significant differences (Tukey, p < 0.05).

presents the physical and chemical properties of biodiesel produced under varying conditions. Specifically, it shows how different percentages of KOH catalyst and molar ratios of methanol to oil affect the properties of the resulting biodiesel. The data includes mean values and standard deviations for each test condition. Biodiesel samples produced with varying amounts of KOH and molar ratios had an average density of 0.8629 ± 0.05 g/mL. The results adhere to the specifications of ASTM D1298, which sets the limits at 0.86 to 0.9 g/mL. Analysis of variance (ANOVA) with a 95% confidence interval indicates that catalyst concentration significantly influenced biodiesel density (p = 0.001), molar ratio (p = 0.034), and their interaction (p = 0.037). The cloud point ranged from −3 to −8 °C, aligning with the standards set out in ASTM D2500-17, which specifies a range of −3 to −12 °C. This parameter was strongly influenced by molar ratio (p = 0.001) and by the interaction between molar ratio and catalyst concentration (p = 0.027).

The refractive index measurements in this study varied from 1.435 to 1.478, consistent with ASTM D1218-12, which specifies a maximum value of 1.479. Concentration (p = 0.001) and the interaction between concentration and molar ratio (p = 0.002) significantly influence the refractive index. The absence of corrosion on copper in the biodiesel produced in this study indicates a lack of contaminants. This lack of corrosion can be attributed to factors such as glycerol, alcohol, free acids, water, or catalysts [73].

A flash point (FP) value of 165 ± 18 °C was recorded, adhering to ASTM D4530-15, which stipulates a minimum threshold of 130 °C. Catalyst concentration (p = 0.015) and molar ratio (p = 0.012) significantly influence the flash point (FP). The flash point of biodiesel derived from C. vulgaris lipids is above that which was reported in prior studies, measuring 143 °C [74,75]. The carbon residue value was 0.035 ± 0.014. The catalyst concentration significantly influenced carbon residue (p = 0.001), whereas the molar ratio also had a notable effect (p = 0.044). The molar ratio strongly influenced the acid number, which ranged from 0.31 to 0.17 mg KOH/g (p = 0.001). The molar ratio of lipids to alcohol strongly influences the qualitative parameters of biodiesel.

These findings underscore the increasing apprehension over fossil fuel burning and its environmental repercussions, emphasizing that microalgae possess a substantial concentration of oils and starches suitable for the development of high-quality biofuels [76]. Biodiesel in this instance conformed to both American and European standards, while concurrently providing advantages in wastewater treatment and carbon dioxide sequestration akin to those realized with other microalgal species such as Tribonema minus [77].

3.6. Rheological Characterization of Biodiesel

Figure 5 shows graphs of biodiesel made through alkaline transesterification using different amounts of KOH compared to oil and methanol. These illustrate the characteristics of shear stress and dynamic viscosity. The dynamic viscosity profiles of biodiesel decreased as the shear rate increased. This behavior is typical of biofuel and has been previously documented [78]. Newtonian behavior has been established in biodiesel samples [79], serving as a critical determinant for identifying its characteristics.

The biodiesel made showed non-Newtonian behavior, as shown by the flow properties in

Table 6. Rheological parameters of biodiesel produced obtained using Herschel–Bulkley, Ostwald–de Waele, and Newton models.

Test (% KOH, Oil–Methanol Ratio)	Herschel–Bulkley				Ostwald–de Waele			Newton
	${\mathit{\tau }}_0$	$\mathit{k}$	$\mathit{n}$	${\mathit{R}}^2$	$\mathit{k}$	$\mathit{n}$	${\mathit{R}}^2$	$\mathit{\eta }$	${\mathit{R}}^2$
1.0, 1:6	0.0042 a	0.0081 a	0.8554 a	0.9991	0.0077 a	0.8657 a	0.9991	3.99 × 10−3 a	0.9900
1.5, 1:6	0.0043 a	0.0085 a	0.8541 a	0.9995	0.0080 a	0.8644 a	0.9995	4.15 × 10−3 a	0.9902
1.0, 1:9	0.0020 b	0.0080 a	0.8450 a	0.9992	0.0082 a	0.8400 a	0.9992	3.74 × 10−3 a	0.9857
1.5, 1:9	0.0001 c	0.0080 a	0.8660 a	0.9995	0.0080 a	0.8657 a	0.9995	4.21 × 10−3 a	0.9905

${\tau }_0 \left(\mathrm{P}\mathrm{a}\right)$ is the yield stress, $k (\mathrm{P}\mathrm{a}·{\mathrm{s}}^{\mathrm{n}})$ is the consistency index, $n$ is the flow index, and $\eta \left(\mathrm{P}\mathrm{a}·\mathrm{s}\right)$ is the viscosity. The same lowercase letters by row represent non-significant differences (Tukey, p < 0.05).

. Given that the flow index value in this case remained below 1, we can determine that the fluid is pseudoplastic (n > 1). Then, three different models were used to study the data, and the Herschel–Bulkley model showed slightly better correlation coefficients (R²) than the Ostwald–de Waele model.

The pattern was similar in all biodiesel samples, indicating no significant differences in rheological characteristics (p > 0.05). The dynamic viscosity of the fluid was ascertained using Newton’s model. The dynamic viscosity values ranged from 3.74 × 10⁻³ to 4.21 × 10⁻³ Pa∙s, while the kinematic viscosity ranged from 4.33 to 4.87 mm²/s. The numbers match those found in previous studies, like biodiesel made from leftover sunflower oil, which had a kinematic viscosity of 4.71 mm²/s [80], and rapeseed oil, which started at 4.3 mm²/s [81].

The cultivation of microalgae from residual substrates alters their lipid profile, which, according to Zakaira [82], correlates with the characteristics of the methyl esters produced via transesterification, exhibiting non-Newtonian behavior, in contrast to the Newtonian behavior observed in conventional biodiesel production methods.

4. Conclusions

This study demonstrates that lipids extracted from the microalga C. vulgaris, which is cultivated in chicken industry effluent, are suitable for biodiesel production. The optimal yields were achieved using 1% KOH as a catalyst with molar ratios of 1:6 and 1:9, resulting in 98% yields in both cases. The molar ratio significantly affected different properties, including density, cloud point, refractive index, flash point, carbon residue, and acid number. The biodiesel produced meets the quality standards set by the ASTM and exhibits non-Newtonian pseudoplastic behavior, characterized by a decrease in viscosity when force is applied. The fatty acid methyl ester (FAME) analysis showed that methyl esters are present, providing more proof that C. vulgaris is effective for making biodiesel.