A Comparative Techno-Economic Analysis of Waste Cooking Oils and Chlorella Microalgae for Sustainable Biodiesel Production

Abstract

This research work presents a techno-economic assessment of biodiesel production with non-standard waste cooking oil (WCO) (brown grease of small restaurants, yellow grease of households) and semi-open Chlorella sp. microalgal cultivation, which covers the problematic areas of scale and cost-efficiency in sustainable biodiesel production. Cost-effective biodiesel feedstock research has been motivated by the urgency of finding sustainable sources of energy. With base-catalyzed transesterification optimized by ANOVA and response surface methodology (RSM), the present study recorded biodiesel yields of up to 99.08% in household WCO (at optimum conditions; 55 °C, 3.3 mg/g NaOH, ethanol) and 96.61% in restaurant WCO (at optimum conditions; 54 °C, 1.5 mg/g NaOH, methanol) compared to 28.6% in Chlorella sp. (semi-open photobioreactors). Concerning the two types of WCO feedstocks, the obtained equations are able to compute the biodiesel viscosity and yield, in good correlation with the experimental values, in relation to the temperature and ratio of catalyst to oil/alcohol solution. The assessed household WCO has better yield and quality as it contains fewer impurities, whereas the restaurant WCO needed to be further purified, driving up the prices. Although Chlorella biodiesel is carbon neutral, its production and extraction costs are higher, making it less economically feasible for biodiesel production. Economic analysis showed that the capital costs of household WCO, restaurant WCO, and Chlorella sp. are USD 190,000, USD 220,000, and USD 720,000, respectively, based on 1,000,000 L/year as biodiesel production rate. Low capital costs as well as byproduct glycerol income of the two investigated types of WCO play a role in their low payback periods (0.23–0.91 years) and high ROI (110–444.4%). The analysis highlights the economic and environmental benefits of WCO, especially household WCO, as a scalable biodiesel feedstock, which provides new insights into process optimization and sustainable biodiesel strategies. To enhance its sustainability and cost-effectiveness and contribute to the transition to renewable biofuels globally, future studies need to emphasize energy reduction in microalgae production and purification of restaurant WCO.

1. Introduction

With the rising ambitions of new energy policies in many countries, major issues have been arising, such as energy efficiency, mitigation of greenhouse gas (GHG) emissions, and improved energy independence, as well as reduced dependence on fossil fuels [1]. Bioenergy, and especially biofuels, has a potential for alleviating petroleum shortages and minimizing some of the environmental impacts, but still there are challenges to its viability of scalability, land use, and lifecycle emissions. Biofuels offer the chance to replace a sizable portion of the fossil fuels currently in use, improving supply security and reducing greenhouse gas emissions [2]. Although a variety of biofuels may be available, biodiesel and bioethanol are the main biofuels being considered globally for the transportation sector [3]. Some of the main advantages of biodiesel compared to fossil fuel sources are being renewable, lower exhaust gas emissions, being biodegradable, and having photosynthetic organic carbon. Biodiesel has replaced other biofuels, such as bioethanol, in the transportation sector because it has a higher energy density, superior lubricity, and can work with the existing diesel engines without requiring much retrofit; thus, it is especially well suited towards heavy-duty vehicles. Moreover, economic and environmental benefits are increased by the process of producing biodiesel out of waste materials, including WCO. The use of biodiesel as transportation fuel would benefit the local population and the environment by reducing carbon dioxide and sulfur levels in the atmosphere, creating jobs, and supplying rural communities with modern energy carriers. It would also prevent urban migration [4,5,6,7,8].

The base stock, geographic location, seasonal variations in crop yields, crude oil prices, and other variables all affect the price of biodiesel fuels. The cost of the biomass feedstock has a significant impact on how competitive biodiesel is. Therefore, it is critical to look for inexpensive feedstocks, like waste products, for the production of biodiesel [9,10]. Waste feedstocks such as WCO can give a more attractive, economically efficient source of feedstock to produce biodiesel, having a low cost of feedstock and fewer greenhouse gas emissions than using virgin oil. Nevertheless, biodiesel yield and applicability are decisively influenced by the quality of the waste stream. Other important constraints are inconsistent waste composition, which may involve high free fatty acid (FFA) levels, necessitating pre-treatment (e.g., esterification), which increases the production cost. Also, the scalability and reliability may be constrained by an irregular supply, possible contamination, and the logistic problems associated with surveying and shipping waste feedstocks, which are major challenges to implementing the biodiesel industry on an extensive scale. As the cost of feedstock is one of the greatest concerns in biodiesel production, waste material such as waste cooking oil will be of great benefit in boosting the economic feasibility of biodiesel production [11]. There are vast amounts of waste animal fats and cooking oils available worldwide, particularly in developed countries [12,13,14,15]. According to some reports, using inexpensive feedstocks will cut biodiesel prices by about half. Utilizing used cooking oil as a feedstock lowers the cost of producing biodiesel by roughly 60–70%. This is explained by the fact that the cost of the feedstock accounts for between 70 and 95% of the total cost of producing biodiesel [16,17,18,19,20]. Due to the above several benefits of using waste products in producing biodiesel, the objective of the present work is to provide a comprehensive comparative study for using waste cooking oils (WCOs) compared to microalgae for biodiesel production.

The most prevalent method for producing biodiesel is transesterification, which is a catalyzed chemical reaction that involves vegetable oil and an alcohol to generate fatty acid alkyl esters (biodiesel) and glycerol. The coproduct, glycerol, must be collected due to its significance as an industrial chemical [21,22,23]. Making biodiesel from waste vegetable oil (WVO) is similar to making biodiesel from straight vegetable oil, with the exception that the oil must be filtered first to remove debris, and because it has been used and most likely reheated several times, more fatty acids will be present, necessitating the addition of more NaOH (or KOH) to neutralize these acids [19]. Enzymatic catalysis, base-catalyzed transesterification, and acid-catalyzed esterification are the three fundamental processes for producing ester from oil or fat [24,25]. Because it is the most cost-effective method, the base-catalyzed transesterification technique is the most widely used method [26,27]. The transformation process, thought to be irreversible, is an equilibrium process and requires mixing of the reactants in order to convert feedstocks to biodiesel. The rate at which the transesterification reaction attains equilibrium is highly stepped up by the presence of a catalyst [28]. Waste cooking oil (WCO) is converted into methyl esters by the transesterification process, which lowers the molecular weight by about one-third, and lowers the viscosity by about one-seventh. This process also lowers the flash point, slightly raises the volatility, and significantly lowers the pour point [23]. Producing biodiesel from WCO is difficult due to the presence of undesired components such as free fatty acids (FFAs) and water [29].

Biodiesel production is more appealing and economical when it is made from low-cost feedstocks such as non-edible oils, waste cooking oil, animal fats, soap stocks, and greases. However, the current supply of waste oils and animal fats is insufficient to meet the demand for biodiesel. Thus, transitioning to second-generation biofuels, such as microalgae, is a critical strategy [30]. Microalgae are currently in the spotlight and are commonly viewed as a possible biofuel producer. They have garnered special attention in the context of biodiesel production and renewable energy generation for a decade. This is due to their capacity for rapid growth and high oil (e.g., Scenedesmus abundans and Coelastrella sp. M-60 with lipid contents up to 71% and >70% under optimized conditions, respectively). Their capacity to grow in unusual conditions, e.g., wastewater subjected to salinity stress or nutrient limitation, promotes lipid accumulation by inducing stress-induced metabolic rearrangement and lower costs of cultivation and risks of contamination. Together with their inherent carbon neutrality, these properties make microalgae an attractive feedstock source of biodiesel that can be produced in a sustainable manner [31,32,33].

Microalgae are a wide category of aquatic organisms, which do not have a complex cell structure like higher plants have. They occur in a wide range of environments, and they can survive in freshwater or in brackish waters or in salty conditions [34,35]. Autotrophic growth is currently the most popular method today of microalgae cultivation. Since microalgae are all photosynthetic, and a significant portion of microalgae are particularly effective at converting solar energy, microalgae are grown in lighted conditions either naturally or artificially. Microalgae are typically cultivated in unfenced ponds or in closed circular devices referred to as photobioreactors. The principal check on the cultivation is the introduction of adequate natural or artificial light. This enables enormous growth and high-density populations: the more light—within a certain degree, according to the species—the better the algal growth [36,37,38,39,40]. Chlorella sp. and Scenedesmus are among the most commonly used microalgae in the commercial manufacture of biodiesel. This can be ascribed to their high oil content [41]. As a result, this current research focuses also on investigating the manufacture of biodiesel from Chlorella sp. microalgae using alkali-catalyzed transesterification, as described above, for producing biodiesel from WCO. This reaction works effectively in the presence of homogeneous catalysts such as KOH or NaOH. NaOH is extensively accepted and used due to its inexpensive cost and excellent product yield [42].

The process of biodiesel production by WCO and microalgae has limitations in terms of feedstock quality, scalability, and cost-sustainability, especially when using non-standard WCO (brown grease in restaurants and yellow grease in households) and a semi-open model of microalgae growing. Such problems create logistical challenges to the feasibility of biodiesel as a renewable source of fuel. This research paper seeks to assess the techno-economic viability of biodiesel production using WCOs generated at home and in restaurants and Chlorella sp. microalgae to provide new information regarding specifications of cost-effective and scalable feedstocks to enable sustaining biofuel policies.

2. Materials and Methodology

This section addresses the materials and methodology used for producing biodiesel from two types of WCO (household and restaurant) and Chlorella sp. microalgae. The restaurant WCO was collected from local restaurants in Riyadh (Saudi Arabia), and an isolated sample of Chlorella sp. was taken from the Alex Seas Science Institute [Suez, Egypt].

2.1. Raw Materials

Six liters of waste cooking oil (WCO) were collected from a local restaurant in Riyadh (Saudi Arabia), and this oil is called restaurant oil. Also, 4 L of domicile WCO (called domestic or household oil) were collected and stored at ambient temperature for pretreatment. Methanol (CH₃OH) with a purity of 99.5% and ethanol with a purity of 99.5%, used for the esterification process, were purchased from SASOL (Johannesburg, South Africa). Potassium hydroxide (pellets, 85% extra pure) and sodium hydroxide (pellets, 98% extra pure), used as base catalysts for the esterification process, were supplied from Loba Chemie Pvt. Ltd. (Mumbai, India). Also, Ferrous sulfate heptahydrate and disodium ethylenediaminetetraacetate were purchased from Loba Chemie Pvt. Ltd. (Mumbai, India).

2.2. Production Process of Biodiesel from Microalgae

In the present work, Chlorella sp. was used as the microalgae type because it has high availability, low cost, high oil content (30–50%), and a low life cycle of 10–12 days. Its dry biomass has high economic value as it is used in a lot of fields. The Chlorella sp. production steps began by taking an isolated sample of Chlorella sp. from the Alex Seas Science Institute [Suez, Egypt]. The second step focused on the preparation of a nutrient solution required for algae growth. The nutrients required for each liter of Chlorella sp. algal culture are shown in

Table 1. Nutrients required for each liter of Chlorella sp. algal culture.

Nutrients for One Liter of Chlorella		Trace Element Solution Composition
Chemical	g L−1	Chemical	g L−1
KNO3	1.011	H3BO3	0.0618
NaH2PO4	0.0399	MnSO4·H2O	0.151
Na2HPO4	0.0709	ZnSO4·7H2O	0.2875
MgSO4·7H2O	0.0246	CuSO4·5H2O	0.0024
CaCl2·2H2O	0.0017	(NH4)6Mo7O24·4H2O	0.0135
Fe-complex	1 mL
Trace element solution	1 mL
Total volume	1000 mL
pH	6.8

. Considering the standard procedure for Chlorella culture, the culture medium used in this study is Bold’s Basal Medium (BBM), which is known as a well-studied medium for the growth of microalgae [43].

A 0.0695 g quantity of FeSO₄·7H₂O (ferrous sulfate heptahydrate) and 0.93 g of disodium EDTA (disodium ethylenediaminetetraacetate) was dissolved in 100 mL of deionized water to make the Fe-complex. Trace element composition of a one-liter volume of nutrient medium is shown in Table 1. To make a feedstock to cultivate algae, a nutrient solution to support algal growth of 20 L was prepared by concentrating the necessary nutrients, such as the Fe-complex and trace elements. A subculture of algae was inoculated in this feedstock, and its growth was continued over a period of 7 days.

Sufficient illumination is necessary for algae growth, and an air pump was used to supply the required CO₂ during algae isolation to avoid contamination. The system was aerated at 0.5 vvm (volume of air per volume of medium per minute) through an air pump to provide good mixing and prevent sedimentation, whilst reducing the risk of contamination. CO₂ was provided to the system at a concentration of 1.5% (v/v) in the air stream to promote photosynthesis and maximize biomass growth without acidifying the medium. Cool white LED lights were also utilized to supply illumination at a light intensity of 150 µmol s⁻¹ m⁻², and it was cultivated on a light/dark cycle of 16:8 h to maximize photosynthetic capacity. The temperature was controlled at 25 ± 2 °C in order to facilitate a high metabolic rate and maximize biomass yield. After 7 days, the subculture was achieved in four glass pools, with each pool containing 125 L for a total amount of 500 L. The pools with the required nutrients were covered with plastic to prevent contamination from the outside before turning on the light, which should cover the whole area of algae. After 12 days, it was noticed that the production rate of algae increased. The second step in preparing biomass feedstock from Chlorella sp. was the algae harvesting by adding 20% w/v NaOH (20 g of NaOH per 100 mL of the total solution), which acts as a coagulant for algae settling. Additional NaOH can be added to speed up settling, but a compromise is made because of the cost factor. The precipitated algae were separated from water by siphons to get pure wet algae, which was dried by putting it on aluminum paper in a furnace maintained at 65 °C for two days. The dried algae weight was measured to calculate the amount of methanol required for the transesterification reaction.

A drying protocol of 65 °C and 48 h was selected to achieve full moisture drying of wet Chlorella sp. biomass in order to facilitate the extraction of lipid and transesterification, and to balance energy consumption costs [44]. This temperature falls into the normal range (50–70 °C) of microalgal drying, which reduces energy consumption when compared to elevated temperatures [45]. Nevertheless, extended exposure to 65 °C can result in slight oxidation of unsaturated fatty acid and can decrease the quality of lipids and account for the diminished biodiesel output. It is unlikely that saponification occurred during drying because there was no base catalyst, and water was removed to avoid this reaction. Although thermal degradation was not directly measured (e.g., radiated peroxide value), the temperature of 65 °C was selected based on literature that reports little to no lipid degradation at temperatures below 70 °C [41].

Lipid extraction was carried out through the Bligh and Dyer method that involved extraction of usable lipids in a dried Chlorella sp. biomass. In particular, 10 g of dried algae were combined with a chloroform–methanol–water mixture with proportions of 2:1:0.8, then separated into phases to bring out the chloroform layer containing lipids. Transesterification was then carried out using the extracted lipids. There were no cell disruption methods undertaken, e.g., sonication/bead milling before lipid extraction in order to make the method simpler and energy-efficient. This choice can cause a more limiting access to lipids by the strong cell walls of Chlorella sp., which can be a contributing factor to the low yields of biodiesel. The efficiency of lipid extraction was not quantified in isolation; the value reported of the biodiesel yield corresponds to the overall efficacy of the lipid extraction and transesterification.

The most common alcohols widely used in the transesterification process are methanol and ethanol. Among these two, methanol finds frequent application in commercial uses because of its low cost. The production of biodiesel from algae is similar to the production of biodiesel from WCO, as described in detail in Section 2.3.

Production of biodiesel using dry algae can be summarized into various important stages. It started with 10 g of dried algae biomass that was used as the feedstock. The algae were dehydrated prior to drying to eliminate water, which is propitious to oil extraction and reaction.

Transesterification Reaction: Per 10 g of dried algae, the alcohol constituent required in the transesterification reaction was 1 mL of methanol. NaOH (1 wt% of oil mass) was used as the catalyst to increase the rate of reaction. The dried Chlorella sp. algae contain lipids (oils) that were combined with methanol in the presence of NaOH catalyst. By this transesterification reaction, the algal oil was reacted with methanol to form fatty acid methyl esters (biodiesel) and glycerol as a byproduct.

Phase Separation: Once the transesterification reaction was completed, the reaction mixture separated into two distinct layers due to their different densities. During the process of settling, the biodiesel was separated at the bottom of the settling tank, and the glycerol and other byproducts (e.g., residual methanol, NaOH and soaps) were collected at the top. The biodiesel was then slowly decanted at the bottom of the settling tank.

Methanol Recovery: Depending on the mixing equipment, the biodiesel layer could contain residual methanol, which was removed through the washing of the biodiesel using hot water. The recovered methanol was recycled to the process for enhancing the process efficiency.

Drying: The biodiesel produced after hot water washing still had some traces of water. Thus, it was further dried to attain the required quality. This was accomplished with a vacuum dryer or by heating to remove any residual water in the achieved biodiesel. A clean biodiesel product that was prepared for use or storage was obtained by further filtering the dried biodiesel to remove any solid contaminants or particles.

2.3. Production of Biodiesel from Waste Cooking Oils

The synthesis of biodiesel production from WCO included 4 main steps: pretreatment of WCO, the transesterification process, separation of main product (biodiesel) and by-product (glycerin), and finally, separating of alcohols from biodiesel by a distillation process.

Pretreatment of WCO: The first step of pretreatment was the WCO filtration process. Filtration was used to remove food residue and other solid particle contaminants in the oil. The WCO filtration procedure depends on the oil type and its physical nature. As an example, differences in viscosity and level of impurities influenced by the frequency of use and the source of the oil make domestic WCO (commonly consisting of sunflower oil) easier to be filtered than restaurant WCO (usually palm oil). Palm oil, which is widely applied in restaurants, is highly viscous and semi-solid at room temperature, whereas sunflower oil tends to be liquid when used domestically. Palm oil has a melting point of around 35 °C, whereas the freezing point of sunflower oil is −17 °C. The elevated amount of saturated fat (about 50%) of palm oil makes palm oil very high in its melting point and viscosity as compared to sunflower oil, which only contains a low content of saturated fat (about 11%). These variations in saturated fat content affect the fluidity and filtration requirements of the oils, with palm oil having to be heated in most cases in order to undergo filtration. Household WCO needs little pretreatment, since it is less viscous and contains fewer impurities compared to restaurant WCO.

The filtration process was performed under vacuum pressure of approximately 0.3 bar through a 9 cm diameter qualitative cellulose filter paper of 5–10 µm pore size. In the case of restaurant WCO, the oil was warmed on a magnetic hot plate stirrer to near 100 °C prior to filtration, whereas the household WCO was filtered at ambient temperature. It should be noted that restaurant WCO needed preheating close to 100 °C to ensure removing the oil temperature history, which facilitates the filtration process and makes the oil handling easier without freezing. However, before filtration, some of the large food scraps such as potato pieces and some other residues were removed. After filtration, the drying process was achieved in a drying oven to eliminate any water traces from the oil. During the drying process, the oven was maintained at 110 °C. The water content of the filtered WCO was determined as received, and after the removal of water, by the Karl Fischer titration technique to ensure reduction of water content below 500 mg/kg, which is essential to avoid saponification in the transesterification process [46].

The esterification process: The important variables that affect the production of biodiesel using WCO are type of alcohol, catalyst, alcohol to oil molar ratio, and reaction temperature. These essential factors are outlined as follows:

• Type of alcohol: In the case of transesterification of domestic WCO, which commonly consists of sunflower oil, ethanol was selected over the use of methanol. This is because it is less toxic and causes less environmental and health hazards in case of spill or evaporation. But in the case of restaurant WCO, typically methanol was utilized based on its increased reactivity in transesterification and its lower cost.
• Catalyst type: The catalyst to oil molar ratio forms a very important parameter, which determines the quantity and quality of biodiesel obtained during transesterification. The transesterification reaction may be catalyzed with alkalis, acids, or enzyme catalysts. Studies have shown that the use of alkali-catalyzed reactions, usually with catalysts such as KOH and NaOH, yields a higher amount and purity of biodiesel. These reactions occur over a smaller time span as compared to acid-based or enzymatic processes [42,47]. The present study used NaOH as the base catalyst for the esterification process.
• Alcohol to oil ratio: Regarding restaurant WCO, the biodiesel yield was experimentally measured by varying the methanol/oil molar ratio using 2 wt% of NaOH catalyst at a constant temperature of 60 °C for one hour. Equation (1) represents the transesterification reaction. During this reaction, the triglyceride supplied by WCO undergoes a reaction with alcohol (methanol or ethanol) in the presence of a catalyst (NaOH) to form biodiesel (fatty acid methyl esters) and glycerol. In this reaction, the triglycerides in restaurant WCO (palm oil) and household WCO (sunflower oil) were transformed into biodiesel, where it was more efficient in the household WCO, since it is less viscous and can combine better with ethanol.

(1)

• Transesterification temperature and duration: Regarding previous research works, the esterification process using ethanol is carried out at a temperature range of 55–65 °C compared to a range of 54–60 °C in the case of using methanol [48]. Thus, for domestic WCO, transesterification reaction with ethanol (boiling point of 78.37 °C) in the presence of NaOH catalyst was performed at a temperature range of 55–65 °C. However, this reaction was carried out at a range of 54–60 °C in the case of restaurant WCO, which used methanol (boiling point of 64.7 °C) for the esterification process. The duration of the transesterification reaction for both domestic and restaurant WCO was one hour.

Synthesis and separation of biodiesel: The procedure steps for producing biodiesel are as follows:

• Considering the optimum alcohol to oil molar ratio of 6:1, 22.69 g of methanol or 32.09 g of ethanol was needed to react with 100 g of the pretreated WCO. A quantity of 100 g of the pretreated WCO was put in a 250 mL Erlenmeyer flask, while the required amount of alcohol was kept in another flask of 100 mL.
• The appropriate amount of catalyst (NaOH or KOH) was then added carefully to the alcohol (e.g., methanol or ethanol) to avoid skin contact, since alkali catalysts are potentially irritating or burn the skin. The catalyst was then added to the alcohol and thoroughly dissolved using a magnetic stirrer at a speed of around 500 rpm, and a temperature of 50 °C, to produce a homogeneous alkoxide solution. The flask holding the solution was wrapped with aluminum foil after full dissolution to avoid light and moisture exposure. In the meantime, the flask with the filtered WCO was warmed to 60 °C with mild mixing by a heated magnetic stirrer with the aim of establishing a homogenous temperature and supporting the subsequent transesterification process.
• The catalyst–alcohol solution (e.g., sodium methoxide in methanol) was slowly added to the hot WCO placed in a reaction flask by adding the prepared solution through a 90 mm funnel. Aluminum foil was then added to cover the flask and prevent exposure of the mixture to light and the moisture, after which the contents were mixed with a magnetic stirrer adjusted to around 6.5 rpm to enable mixing during the transesterification reaction.
• Once the transesterification reaction had achieved completion, the resulting reaction mixture was settled in a 250 mL separatory funnel for approximately 12 h. In this time two different layers were formed due to the lack of solubility between biodiesel and glycerol, which creates a clear separation of glycerol and biodiesel. Glycerol appeared as the lower layer due to its higher density. The lower layer was drained out of the funnel, leaving biodiesel (fatty acid methyl esters) as the upper layer in the separatory funnel to be further processed. The biodiesel was finally decanted into a round-bottom flask and heated in a heating mantle with reduced pressure. A condenser was used to recover any remaining excess methanol at about 60–70 °C, just above the methanol boiling point of 64.7 °C. This procedure extracts any remaining methanol in the biodiesel to a concentration that can be reused in the same process of biodiesel manufacturing. The obtained biodiesel was then further purified, e.g., water washed, to free it of impurities.

2.4. Analysis and Statistical Experimental Design

As they minimize the number of experiments and, therefore, the time required to run these experiments, statistical procedures become increasingly common as an experimental design tool and an essential part of any laboratory study. Various factors in the reaction necessitate a large number of experiments, which is impractical due to the specific parameters that influence the reaction and its optimization [49]. Such parameters include temperature, catalyst concentration, methanol to oil ratio, reaction time, mixing intensity, and initial free fatty acid/water content. Response surface methodology (RSM) is an effective statistical tool. It has been applied in optimizing difficult processes because it reduces the number of experiments that should be conducted in order to gather enough data to achieve a statistically significant outcome [50]. RSM is a collection of statistical and mathematical tools in building experimental models. The objective is to optimize a response (an output variable), which depends on several independent variables (input variables) [51]. Biodiesel using used cooking oil has already been explored and optimized successfully using RSM.

A response surface methodology provides numerous techniques, including Box–Behnken, D-optimal methods, and central composite design. In this study, D-optimal was used, which is a direct optimization method that depends on the model to be fitted and on a chosen criterion of optimality. D-optimal design was chosen among other approaches of RSM because of its flexibility in accommodating the experimental constraints of the present study. The optimization of the process was performed with Design Expert software (version 13). Temperature and catalyst amount parameters were selected as independent variables. However, product yield, density, and viscosity were considered as dependent variables (response). The experimental design was carried out using the lower and upper extremes of the variables (catalyst mass ratio and temperature of the reaction). For the household WCO, the chosen temperature range was 55–65 °C, and the catalyst concentration was 3.3 and 6.22 mg/g solution (WCO and ethanol). The D-optimal experimental design was used to design twelve runs, as seen in

Table 2. D-optimal experimental design data for house WCO with ethanol and restaurant WCO with methanol.

Run	House WCO		Restaurant WCO
	mg Catalyst/g (WCO + Alcohol)	Temperature (°C)	mg Catalyst/g (WCO + Alcohol)	Temperature (°C)
1	3.3	60	1.5	54
2	6.22	60	2.25	57
3	6.22	55	1.88	60
4	6.22	55	2.25	60
5	6.22	65	1.5	54
6	4.76	55	2	60
7	3.3	55	1.88	57
8	4.76	65	2.25	54
9	6.22	65	1.5	60
10	4.76	60	1.88	54
11	3.3	65	2.25	54
12	3.3	55	1.5	57

. The selected temperature range of the restaurant WCO with methanol is 54–60 °C and 1.5–2.25 mg/g of catalyst concentration. The D-optimal (as illustrated in Table 2) experimental design suggested that 12 runs should be undertaken.

After the response data was entered into the experimental design structure, the next stage was response selection and analysis. The particular response to be examined was then selected. An empirical mathematical model of each response was provided and analyzed with an analysis of variance (ANOVA). The empirical mathematical model was tested with a significance level of 5% by analysis of variance (ANOVA). ANOVA compares the variation caused by random errors, which are all of the observed measurements of the generated responses, with the variation caused by the treatment (change in the combination of a variable’s levels). Considering the origins of experimental variance, such a relationship enables us to evaluate the importance of the regression applied to predict responses. The coefficient of determination is known as R². The value of R² indicates the degree to which the variation in the observed values of the response can be attributed to experimental factors and their interaction. This analysis was carried out using the “F” test and the “prob > F” value [50]. Diagnostic tools incorporated in the Design Expert software, such as residual plots and lack-of-fit tests, were used to validate the model assumptions. It was shown that residual plots indicated normality (straight-line pattern), homoscedasticity (occasional scatter), and no correlation of the errors (no trend with run order) [51].

The initial fitting of the response surface was achieved using a full quadratic model. The non-significant terms (where p > 0.05) were removed in the selective regression using Design Expert software (version 13). This provided reduced models in terms of linear and interaction terms but with parsimony and predictive accuracy confirmed using ANOVA, residual, and lack-of-fit plots.

2.5. Analytical Methods for Biodiesel Characterization

In order to verify the quality and compliance of the biodiesel made using Chlorella sp. microalgae and waste cooking oils (household and restaurant WCO), the analytical measurements were performed at the Petroleum Research Institute in Cairo, Egypt. The most important properties considered were the biodiesel yield, density, and viscosity, as shown in Table 3,

Table 4. The obtained household WCO-based biodiesel viscosity, yield, and density at different temperatures and catalyst ratios. Values are the means ± SD of triplicate measurements.

Run	Catalyst Ratio (mg/g)	Temperature (°C)	Viscosity (mPa·s)	(%) Yield	Density (g/mL)
1	3.3	60	7.06 ± 0.1	96.31 ± 0.3	0.823 ± 0.0015
2	6.22	60	6.57 ± 0.1	92.81 ± 0.3	0.849 ± 0.0015
3	6.22	55	4.82 ± 0.1	88.54 ± 0.3	0.831 ± 0.0015
4	6.22	55	4.82 ± 0.1	88.54 ± 0.3	0.831 ± 0.0015
5	6.22	65	20.75 ± 0.1	91.23 ± 0.3	0.829 ± 0.0015
6	4.76	55	5.06 ± 0.1	91.52 ± 0.3	0.814 ± 0.0015
7	3.3	55	5.48 ± 0.1	99.08 ± 0.3	0.851 ± 0.0015
8	4.76	65	7.14 ± 0.1	91.32 ± 0.3	0.836 ± 0.0015
9	6.22	65	20.75 ± 0.1	91.23 ± 0.3	0.829 ± 0.0015
10	4.76	60	7.82 ± 0.1	94.08 ± 0.3	0.843 ± 0.0015
11	3.3	65	8.05 ± 0.1	97.65 ± 0.3	0.814 ± 0.0015
12	3.3	55	5.48 ± 0.1	99.08 ± 0.3	0.851 ± 0.0015

and

Table 5. Viscosity, yield, and density for the biodiesel produced from restaurant WCO at different temperatures and catalyst ratios. Values are the means ± SD of triplicate measurements.

Run	Temperature (°C)	Catalyst Ratio (mg/g)	Viscosity (mPa·s)	Yield (%)	Density (g/mL)
1	54	1.5	14.85 ± 0.14	96.615 ± 0.5	0.949 ± 0.0021
2	57	2.25	9.0 ± 0.14	94.6 ± 0.5	0.938 ± 0.0021
3	60	1.88	8.0 ± 0.14	90.05 ± 0.5	0.8586 ± 0.0021
4	60	2.25	8.75 ± 0.14	70.82 ± 0.5	0.885 ± 0.0021
5	54	1.5	14.85 ± 0.14	96.615 ± 0.5	0.849 ± 0.0021
6	60	2.0	8.0 ± 0.14	78.94 ± 0.5	0.8455 ± 0.0021
7	57	1.88	9.8 ± 0.14	94.755 ± 0.5	0.8605 ± 0.0021
8	54	2.25	8.4 ± 0.14	88.135 ± 0.5	0.8552 ± 0.0021
9	60	1.5	8.25 ± 0.14	94.0 ± 0.5	0.82 ± 0.0021
10	54	1.88	8.7 ± 0.14	95.668 ± 0.5	0.847 ± 0.0021
11	54	2.25	8.4 ± 0.14	88.135 ± 0.5	0.8552 ± 0.0021
12	57	1.5	16.5 ± 0.14	95.515 ± 0.5	0.8877 ± 0.0021

. Biodiesel production was calculated by the yield of the purified biodiesel (fatty acid methyl esters) to the starting mass of the lipid feedstock (expressed as a percent). A digital density meter (Anton Paar DMA 4500 M) was utilized to determine density, with an accuracy of ± 0.0001 g/cm³ following the ISO 12185:2024 standard [52]. The viscosity was determined with a rotational viscometer (Brookfield DV-II+ Pro) at 40 °C with a precision of ± 1% of the full-scale range in accordance with the ISO 3104:2023 [53] standard. The measurements were performed in triplicate, and the instruments were calibrated with standard reference materials before the analysis. The repeatability and traceability of the data are provided under the industry standards based on the biodiesel characterization through these methods and device specifications.

Table 3. Characteristic of the produced biodiesel from Chlorella sp. microalgae.

Property	Unit	Measured Value	Limits		Test Method
			Min.	Max.
Minimum ester content	wt%	98.2	96.5	-	EN 14103:2020 [54]
Density at 15 °C	g/mL	0.88	0.86	0.90	ISO 12185:2024
Viscosity at 40 °C	mPa·s	3.87	2.9	6	ISO 3104:2023
Flash point	°C	152	120	-	ISO 3679:2022 [55]
Carbon residue	wt%	0.1	-	0.3	ISO 10370:2014 [56]
Water content	mg/kg	482	-	500	ISO 12937:2000 [57]
Acid value	mg KOH/g	0.42	-	0.5	EN 14104:2021 [58]
Iodine value	g I2/100 g	111.5	-	120	EN 14111:2022 [59]
Linolenic acid methyl ester	wt%	5.7	-	12	EN 14103:2020
Methanol content	wt%	0.08	-	0.2	EN 14110:2019 [60]

Table 3. Characteristic of the produced biodiesel from Chlorella sp. microalgae.

Property	Unit	Measured Value	Limits		Test Method
			Min.	Max.
Minimum ester content	wt%	98.2	96.5	-	EN 14103:2020 [54]
Density at 15 °C	g/mL	0.88	0.86	0.90	ISO 12185:2024
Viscosity at 40 °C	mPa·s	3.87	2.9	6	ISO 3104:2023
Flash point	°C	152	120	-	ISO 3679:2022 [55]
Carbon residue	wt%	0.1	-	0.3	ISO 10370:2014 [56]
Water content	mg/kg	482	-	500	ISO 12937:2000 [57]
Acid value	mg KOH/g	0.42	-	0.5	EN 14104:2021 [58]
Iodine value	g I2/100 g	111.5	-	120	EN 14111:2022 [59]
Linolenic acid methyl ester	wt%	5.7	-	12	EN 14103:2020
Methanol content	wt%	0.08	-	0.2	EN 14110:2019 [60]

3. Results and Discussion

The first part of the Results and Discussion concerns the biodiesel production from Chlorella sp. Microalgae, while the second part considers the biodiesel production from two different types of WCO. Furthermore, a comparative analysis and an economic study regarding using WCO and Chlorella sp. microalgae as feedstocks for biodiesel production will be discussed. The transesterification reaction in this study was catalyzed with alkaline catalysts (KOH and NaOH). The optimum catalyst concentration was investigated, and the results showed that for domestic WCO, the optimum NaOH concentration is varied from 3.3 to 6.22 mg/g solution, where the solution includes WCO and alcohol. On the other hand, for restaurant WCO, the optimum NaOH concentration range is 1.5–2.25 mg/g solution [42,44]. Regarding KOH catalyst, the obtained optimum concentration is 11.3 mg/g solution [42].

3.1. Biodiesel Production from Chlorella sp. Microalgae

Starting from 4000 mL of Chlorella biomass, the obtained biodiesel content is 28.6% with an amount of 200.4 mL based on a biomass dry weight of 700 g. The biomass productivity was 0.15 g/L/day, which is relatively low as compared to the 0.2–0.5 g/L/day typically obtained in closed photobioreactor systems [61]. It should be mentioned that the lower growth rate and extraction coefficient obtained in this work can be attributed to the utilization of semi-open flasks to simulate an open batch photobioreactor. The quality of the obtained biodiesel was characterized using different test methods, as presented in Table 3. The specifications shown in Table 3 were measured at the petroleum research institute (Cairo, Egypt). These specifications show that all the properties of the produced biodiesel are within the standard required limits.

The triglycerides can be transformed into fatty acid methyl esters (Biodiesel) that have high ester yield content (98.2%), higher than the manageable requirements of 96.5%, as indicated in Table 3. A high amount of ester ensures good fuel performance and combustion. Such results are in line with recent studies like the work done by Milano et al. [61], whereby ester contents in excess of 96.5% are commonly found in premium microalgal biodiesel. This value means that transesterification took place efficiently, and this was likely achieved through the suitable catalyst and reaction conditions. It has a density of 0.88 g/mL, and this makes it compatible with diesel engines and at an allowable comfortable level. Fuel atomization and combustion efficiency are influenced by its density. Neag et al. [44] also stated that the densities often fall within the standard range of 0.86–0.9 g/mL (EN 14214:2012+A2:2019 [62] or ASTM D6751-23a [63]) based on fatty acid compositions of Chlorella-based biodiesel, which justifies the use of Chlorella-based biodiesel in engines, without requiring any blending with fossil diesel.

Viscosity is an important property for fuel injection and flow in diesel engines. The measured kinematic viscosity of Chlorella-based biodiesel is 3.87 mPa·s (4.4 mm²/s), which lies within the standard permitted range. This consequently proves the good performance and atomization of the considered biodiesel fuel. Low viscosity can cause inadequate lubrication, whereas high viscosity can produce ineffective combustion. According to this observation, Saber et al. [64] indicate that Chlorella biodiesel often possesses a desirable viscosity due to its balanced fatty acid composition. This is a non-flammable liquid because of its high flash point of 152 °C, superior to the minimum required, reducing the risk of ignition during transportation and storage. Owing to the low volatility, biodiesel usually has high flash points. This qualitative attribute of Chlorella biodiesel is corroborated by a study conducted by Ashour et al. [41]. This study shows that the flash points of this type of biodiesel are often higher than 150 °C. Due to a low value of carbon residue of 0.1%, the amount of carbon deposit during combustion is reduced, reducing emissions and engine wear. Sharma et al. [45] confirm this observation by noting that the carbon residue of the microalgal biodiesel is generally lower due to its cleaner combustion profile. The obtained Chlorella-based biodiesel in this study has a water content of 482 mg/kg, which satisfies the standard acceptable limits. This low water content reduces the possibility of increased microbe and corrosion development within fuel systems, thereby making it stable and compatible with diesel engines. Excessive water can affect the performance of the engines and the quality of fuel. Cazarolli et al. [46] clarify that, in order to achieve water contents lower than 500 mg/kg, the process used to produce biodiesel should be associated with efficient drying and purification. The acid value of 0.42 mg KOH/g suggests the presence of low free fatty acid content in the oil, which will reduce the chances of corrosion and damage to the engine. This value shows effective neutralization in the course of transesterification. The same is confirmed by Rasoul-Amini et al. [65] who state that Chlorella biodiesel tends to be low in acid content because of its favorable lipid profile. The extent of unsaturation that determines oxidative stability is indicated by the obtained biodiesel (from Chlorella sp.) iodine value of 111.5 g I₂/100 g. This lower value (less than 120 g I₂/100 g) is sufficient to ensure stability. Saber et al. [64] observed that the mixture of fatty acids (saturated and unsaturated) found in Chlorella biodiesel often leads to iodine values lower than 120 g I₂/100 g, meaning it is suitable to be stored.

The obtained results showed a measured value of linolenic acid methyl ester content of 5.7%, which indicates a good oxidative stability of Chlorella-based biodiesel as the value is well below the maximum limit. Too much linolenic acid can cause a shorter shelf life and promote polymerization, which can degrade fuel. According to Reference [66], a high concentration of linolenic acid methyl esters was found to adversely affect the stability of the biodiesel. This is because it is more susceptible to oxidation, whereas Chlorella as the source of biodiesel comprises a low concentration of this compound. Also, the measured methanol content of 0.08 wt% for the investigated biodiesel from Chlorella sp. is far below the stated limit of biodiesel standards (e.g., 0.2 wt% per EN 14214:2012+A2:2019 or ASTM D6751-23a). This consequently reduces the risk of corrosion due to the presence of methanol in the engine parts, which indicates safe use of biodiesel. This low methanol content indicates a good purification after the transesterification reaction. In interpreting the results, Thawornprasert et al. [67] reported the necessity of effective recovery of methanol during biodiesel production on a large scale. This will result in the desired fuel quality, confirming that the optimized production process of Chlorella-based biodiesel yields the desired fuel quality.

Analysis of the results in Table 3 showed that biodiesel produced using Chlorella sp. microalgae meets the EN 14214:2012+A2:2019 standards and therefore meets the standards required to be considered for a renewable fuel. The obtained low biodiesel yield of 28.6%, as compared to optimized systems, was due most likely to the semi-open flasks used to simulate an open batch photobioreactor. As an example, Milano et al. [61] showed that yields of more than 35% can be obtained with Chlorella under closed photobioreactor-based conditions, showing that yields could be enhanced by advanced cultivation techniques. Parameters of quality of the Chlorella-based biodiesel agree with the available literature. Similar properties were also reported by Saber et al. [64] with a density of 0.92 g/mL, a kinematic viscosity of 3.68 to 4.14 mPa·s at 40 °C, and a flash point exceeding 150 °C, as required by biodiesel standards and compatibility with diesel engines. The high quality of Chlorella-based biodiesel is further supported by the work of Sharma et al. [41] and Thawornprasert et al. [67]. Their studies focus on other quality attributes that include low carbon residue and low methanol content that lead to reducing deposits in the engine and corrosion levels, respectively.

3.2. Biodiesel from Waste Cooking Oils

This work investigates the effect of catalyst ratio and temperature on yield, density, and viscosity of biodiesel prepared from WCO. The results indicate that at methanol/oil molar ratios of 3:1, 6:1, 12:1, and 24:1, the obtained biodiesel yields are 81, 92, 85, and 80%, respectively. In order to reach the highest product yield, a ratio of 6:1 should be used. The effect of ethanol on the domestic WCO ratio was studied, and the optimum ratio was also found to be 6:1. Thus, it can be concluded that successful transesterification is achieved at an alcohol to oil ratio of 6:1 and reaction time of 60 min. This study involves the use of two catalysts (NaOH and KOH), two types of alcohol (ethanol and methanol), and two sources of WCO (households and restaurants). The investigated restaurant oil, which is often highly contaminated, is mixed with methanol and NaOH to optimize the biodiesel yield; the household oil, known to be clean and easy to handle, is treated with ethanol and NaOH [68]. Two additional tests are performed in this study involving KOH with ethanol in both household and restaurant oils to offer a comparative analysis of catalyst performance [69]. This study is contributing to the development of sustainable energy (biodiesel) sources through the possible reduction in production costs and environmental impacts by utilizing waste products and optimizing the production parameters [70].

3.2.1. Biodiesel from Household WCO and Ethanol

To investigate the production of biodiesel from household WCO, 12 experiments were performed using 100 g of waste oil and 32.09 g of ethanol to achieve the desired molar ratio (1:6). They are performed at a temperature range of 55–65 °C and catalyst ratio ranging from 3.3 to 6.22 mg/g. It should be noted that all experiments were performed in triplicate to validate the obtained results. Table 4 shows the obtained results regarding viscosity, yield, and density of the obtained biodiesel based on household WCO. It should be noted that values are the means ± SD (standard deviation) of triplicate measurements. The lower values of standard deviations denote the highest level of experimental precision. The high viscosity values of the biodiesel from household WCO can be attributed to the presence of impurities in the feedstock and challenges in the transesterification reaction.

Yield observations: Regarding Table 4, biodiesel yield is between 88.54% and 99.08%, and the highest yield of 99.08% was obtained at 55 °C and 3.3 mg/g catalyst (Run 7), the optimum condition of this setup [68]. Yield is a little lower at 65 °C (91.23%, Run 5) than at 55 °C (88.54%, Run 3), most likely because the alcohol is evaporating at the higher temperature, as was mentioned in Reference [71]. Yield is also influenced by the catalyst ratio; smaller ratios (3.3 mg/g) usually give higher yields (e.g., 99.08% in Run 7 vs. 91.23% in Run 5), indicating a high amount of catalyst might lead to reduced yield due to side reactions (e.g., saponification) [72]. These results are consistent with other previous studies, which indicate that proper catalyst levels (e.g., 0.75–5% w/w) increase yield by striking a good balance between reaction rate and byproduct generation [70,72].

As an example, for ANOVA analysis, a two-way ANOVA was used to confirm the abovementioned effects of catalyst amount and temperature on the biodiesel yield of household WCO (Table 4). This was achieved by analyzing the measured values of biodiesel yield for the selected 12 experimental runs. The analysis indicated that the main effects of catalyst amount (F(2,27) = 193.4, p < 0.001) and temperature (F(2,27) = 36.62, p < 0.001) were significant, and the interaction was significant (F(4,27) = 9.52, p = 0.0001). Post hoc Tukey HSD tests revealed that 3.3 mg/g of catalyst at 55 °C gives the highest yields (mean = 99.08, Runs 7 and 12), compared to 6.22 mg/g catalyst at 55 °C (mean = 88.537, p < 0.001), which may be attributed to less saponification at low levels of catalyst. The Shapiro–Wilk test supports the validity of these results in assuring the normality of yield data for each run (p > 0.05, range: between 0.481 and 0.801). ANOVA residuals (W = 0.968, p = 0.458) and Levene’s test indicate equality of variances (F = 1.432, p = 0.212). The achieved results confirm the validity of the robustness of the transesterification process that low doses of catalysts and moderate temperature conditions are most effective in biodiesel production, which confirms cost-effective production.

Density trends: As can be seen in Table 4, the density of the produced biodiesel (0.814–0.851 g/mL) also decreases with increasing temperature, as expected in liquids, with a decrease of approximately 0.0007 g/mL per °C [73]. As an example, density at 3.3 mg/g catalyst decreases to 0.851 g/mL at 55 °C (Run 7), to 0.823 g/mL at 60 °C (Run 1), and to 0.814 g/mL at 65 °C (Run 11), as expected based on earlier studies reporting the decreasing density of solvents upon temperature increase due to thermal expansion [69,71]. These results are a little below the typical biodiesel values (0.86–0.90 g/mL at 15 °C), presumably since the sample was measured at slightly higher temperatures (55–65 °C), which lower density [73]. The reliability of the experimental setup is supported by the constant density trend, unlike the viscosity data, which lacks consistency [74].

Overall, the achieved results indicate that low catalyst amount (3.3 mg/g) and moderate temperatures (55 °C) have the highest biodiesel yield with the lowest density, as well as an acceptable viscosity [68]. These obtained results are consistent with efforts being made to convert WCO to sustainable fuels, which lead to less environmental issues and less cost of production [70]. Temperature and catalyst ratio optimization are an essential consideration in industrial scale applications because viscosity and density impact fuel injection and diesel engine performance [69].

3.2.2. Biodiesel from Restaurant WCO and Methanol

This section considers biodiesel production using WCO of local restaurants through reaction with methanol in the presence of NaOH as a catalyst. Similarly, as was done for household WCO, the selected optimum molar ratio of WCO to methanol is fixed at 1:6, and the reaction time is one hour. Table 5 includes 12 experimental runs showing the effect of temperature and catalyst amount on the achieved biodiesel viscosity, yield, and density at temperatures ranging from 54 to 60 °C and catalyst ratios varying from 1.5 to 2.25 mg/g of solution (WCO and alcohol). It is worth noting that the presented values in Table 5 are the means ± SD (standard deviation) of triplicate determinations. The lower values of standard deviations indicate the good accuracy of the experiments.

Viscosity observations: Table 5 shows that the viscosity ranges from 8.0 mPa·s (Runs 3 and 6) to 16.5 mPa·s (Run 12). Raising the temperature from 54 °C (Runs 1, 5: 14.85 mPa·s) to 60 °C (Run 9: 8.25 mPa·s) significantly reduces viscosity at a constant catalyst ratio of 1.5. This suggests that by reducing the quantity of unreacted triglycerides, which increase viscosity, higher temperatures enhance reaction kinetics [75]. Raising the catalyst ratio from 1.5 (Runs 1, 5: 14.85 mPa·s) to 2.25 (Runs 8, 11: 8.4 mPa·s) reduces viscosity at 54 °C. Higher catalyst ratios likely promote full transesterification by lowering the viscosity of the final biodiesel [76]. With catalyst ratios of 1.88 (Run 3) and 2.0 (Run 6), the lowest viscosity (8.0 mPa·s) is recorded at 60 °C, suggesting that moderate catalyst ratios and higher temperatures are ideal for reducing viscosity.

The literature data agree with the inverse viscosity–temperature and viscosity–catalyst ratio relationships. Increasing temperatures enhance the reaction between high-viscosity triglycerides and lower-viscosity fatty acid methyl esters (FAMEs), which in turn reduce the biodiesel viscosity [77]. Similarly, increasing the catalyst concentration enhances the completion of the reaction by reducing any escape of high-viscosity components [78]. The restaurant WCO likely required further purification or improved conditions to achieve the required biodiesel viscosity ranges (1.9–6.0 mPa·s according to EN 14214:2012+A2:2019), since it contained more impurities (viscosity readings are more than 16.0 mPa·s in Run 12) compared to household WCO [79].

Yield Trends: Table 5 shows the trends of biodiesel (from restaurant WCO) yield as the value ranges between 70.82% (Run 4) and 96.615% (Runs 1, 5). At a catalyst ratio of 1.5 mg/g, increasing the temperature from 54 °C (Runs 1, 5: 96.615%) to 60 °C (Run 9: 94%) also slightly decreases yield. As the temperature increases, the yield drops sharply, 88.135% at 54 °C (Runs 8, 11) versus 70.82% at 60 °C (Run 4) at the catalyst ratio of 2.25 mg/g, which suggests that temperatures above 60 °C may increase side reactions, e.g., saponification [49]. The yield is decreased at 54 °C when the catalyst ratio is increased from 1.5 (Runs 1, 5: 96.615%) to 2.25 mg/g (Runs 8, 11: 88.135%). These results indicate that an excess of catalyst may lead to the formation of soap, which will reduce the effective yield [80]. It is proposed that lower temperatures and lower catalyst ratios are ideal conditions for restaurant WCO, since the highest biodiesel yield (96.615%) is achieved at 54 °C and a 1.5 mg/g catalyst ratio (Runs 1, 5).

It was found in the earlier research that excessive heat or catalyst may yield side reactions, like saponification, which consume reactants and reduce yield. This is evidenced by the negative relation between yield and temperature as well as catalyst ratio [81]. In comparison, at 54 °C and a catalyst ratio of 1.5 mg/g, optimum yield is comparable with literature values (e.g., 95.99% for optimized biodiesel from WCO) [42]. The higher level of impurity in restaurant WCO could be the possible reason of the decreased yields at higher temperatures and catalyst ratios, since this will promote side reactions [82].

Density patterns: The obtained density ranges between 0.82 g/mL (Run 9) and 0.949 g/mL (Run 1). Density is reduced at a catalyst ratio of 1.5 mg/g when the temperature is raised from 54 °C (Run 1: 0.949 g/mL) to 60 °C (Run 9: 0.82 g/mL). At a catalyst ratio of 2.25 mg/g, density slightly decreases by 0.03 g/mL by increasing temperature from 54 °C (Runs 8, 11) to 60 °C (Run 4). At a temperature of 54 °C, density decreases as the amount of catalyst increases, i.e., at 1.5 mg/g catalyst ratio, the density is 0.949 g/mL (Run 1), and a 2.25 mg/g catalyst ratio gives a density of 0.8552 g/mL (Run 8). It is noticed that with increasing temperature this trend decreases. The achieved lowest density (0.82 g/mL) is very close to the lower limit of the EN 14214:2012+A2:2019 standard range (0.86–0.90 g/mL) at 60 °C and a catalyst ratio of 1.5 mg/g (Run 9) [79].

Unlike viscosity and yield, density is less sensitive to the temperature and catalyst ratio. This agrees with previous studies, indicating that the major factor used in predicting the density of biodiesel is the fatty acid profile of the feedstock rather than the reaction conditions [83]. The obtained density range (0.82–0.949 g/mL) is slightly higher than the typical biodiesel standards [84], perhaps due to the effect of the contaminants of the restaurant WCO on the molecular composition of the biodiesel. The lower density at increased temperatures and catalyst ratios indicates better conversion to FAME, which has a lower density than unreacted oils [85].

The above results indicate that temperature and catalyst ratio influence the quality of biodiesel generated by restaurant WCO. Density is less sensitive, achieving an optimal density of 0.82 g/mL at 60 °C and catalyst ratio of 1.5 mg/g. However, low viscosity (8.0 mPa s) and high yield (96.615%) were obtained at 54 °C and a catalyst ratio of 1.5 mg/g. These findings emphasize the challenges of using restaurant WCO, which possesses more impurities than domestic WCO, thus resulting in a slightly lower yield and showing increased viscosity.

Free fatty acid (FFA) and water are major issues in biodiesel production from WCO, especially by using a base catalyst in transesterification. Restaurant WCO, where FFA levels may be high because of repeated heating and oxidation, can react with the alkaline catalyst (NaOH) to form soaps. This, in turn, leads to lower yields of biodiesel and complicates phase separation. In this current study, restaurant WCO had a greater amount of FFA compared to household WCO, which required more pretreatment. Another key parameter is water content; this is determined through Karl Fischer titration, which is reduced to <500 mg/kg after drying. This facilitates hydrolysis of triglycerides, which raise FFA levels, leading to saponification. To help overcome such problems, the research procedure involved 0.3 bar vacuum filtration with the use of 5–10 micrometer cellulose filter paper. The product containing water and solid contaminants was dried at 110 °C. In the case of restaurant WCO, it was necessary to preheat to 100 °C in order to lower the viscosity and facilitate filtering. However, in case of WCO containing more than 2–3% FFA, another acid-involving esterification may be used before transesterification. Sulfuric acid (H₂SO₄) and methanol are used to react and convert FFAs into methyl esters to reduce soap production and yield. More FFAs can be converted by applying an acid where the end product is the desired methyl ester and not the soap byproduct. Although it requires an extra twostep process and increases the operational procedure cost, this procedure becomes more realistic in processing highly degraded WCO. Such strategies guarantee compliance with the biodiesel quality standards (EN 14214:2012+A2:2019) and improve the process efficiency.

3.3. RSM and ANOVA Analysis

Incorporating response surface methodology (RSM), as used in prior studies, could optimize the biodiesel production parameters more effectively [71]. Thus, the following subsections will focus on using RSM and ANOVA analysis to investigate the combined effects of temperature and catalyst ratio on biodiesel viscosity, yield, and density. The RSM and ANOVA analyses were conducted using Design Expert software. The version employed in this study is the recent version of Design Expert (version 13), which allows complex experimental designs and statistics, and thus this version can be used to optimize biodiesel production parameters.

3.3.1. Combined Effect of Temperature and Catalyst Ratio on Biodiesel Viscosity

The interaction of level of temperature, catalyst ratio, and viscosity of biodiesel prepared using the considered two types of WCO is illustrated in Figure 1. Regarding Figure 1a, the results for household WCO indicate that viscosity and temperature are directly proportional, where higher temperatures result in an increased viscosity; e.g., at 60 °C, the viscosity of 6.57 mPa·s at 6.22 mg/g catalyst ratio is increased to 7.06 mPa·s at 3.3 mg/g catalyst ratio. This seems unreasonable, since in a usual scenario, viscosity reduces with increasing temperature due to the decrease of the number of molecular interactions [86]. Such deviation may originate in reaction-specific factors that alter the composition of biodiesel, such as incomplete transesterification or the production of byproducts at elevated temperatures. Alternatively, viscosity and catalyst ratio have an inverse relationship. The optimum viscosity of 4.82 mPa·s is obtained at 55 °C and 6.22 mg/g catalyst ratio, which falls into the required standard range (2.9–6 mPa·s) of EN 14214:2012+A2:2019 [87].

The two-factor interaction model of the ANOVA is an indication that high data variability is being registered, since it is associated with a standard deviation of 2.79. The literature viscosity value of 4.92 mPa·s obtained at 40 °C is so low compared to the present study average viscosity of 8.65 mPa·s at 25 °C, which means that the investigated WCO could have impurities or that the reaction was not complete [88]. The obtained determination coefficient (R² = 0.8473) indicates a good fit with a reasonable agreement between the adjusted R² (0.7818) and the predicted R² (0.5721). The “prob > F” value of 0.003 (<0.05) proves the importance of the model. The generated Equation (2) can be utilized to calculate the viscosity of the obtained biodiesel based on household WCO as a feedstock.

Viscosity = 102.80863 − 1.71266*T − 28.8225*C_R + 0.510908*T*C_R

(2)

where T is the temperature in °C, and C_R is the catalyst ratio in mg/g. The interaction term (TC_R) implies the idea of combined effect, whereas the negative coefficient against C_R indicates the reverse relationship with the catalyst ratio. Restaurant WCO shows a lower standard deviation of 1.41 compared to 2.79 for the household WCO. Similarly, it shows a quadratic effect compared to a linear effect in household WCO. The viscosity at 25 °C is 10.23 mPa·s, which is greater than the value recorded in the literature at 40 °C (5.16 mPa·s), signifying poor optimization [89]. Although the predicted R² (0.1692) indicates a potential issue with the model or data, the achieved adjusted R² (0.8154) and the “prob > F” (0.0127) values prove model significance. The derived Equation (3), which is depicted in ANOVA by a quadratic equation type, can be employed in order to determine the viscosity of biodiesel produced using WCO in a relation with temperature (T) and catalyst ratio (C_R).

Viscosity = −386.45616 + 19.32579*T − 149.01087*C_R + 1.39751*T*C_R
−0.195477*T² + 17.01833*C_R²

(3)

At elevated temperatures (114.81 °C), more recent works, such as that by Ahmad et al. [86], show viscosities of 4.34 mm²/s (approximately 3.7 mPa·s) of biodiesel based on WCO by applying RSM. This implies that the investigated temperature range (55–60 °C) might affect the viscosity optimization in this study. Likewise, the WCO composition can influence the biodiesel viscosity because, according to Nabizadeh et al. [87], the obtained result of 6.9 mm²/s (approximately 6.1 mPa·s) slightly exceeds the standard limits.

3.3.2. The Combined Effect of Catalyst Ratio and Temperature on Biodiesel Yield

Figure 2a shows that the yield of biodiesel obtained using household WCO is inversely proportional to the catalyst ratio. At 60 °C and catalyst ratios of 3.3 mg/g and 6.22 mg/g, the achieved yields are 96.31% and 92.815%, respectively. At higher concentrations of catalyst during transesterification for biodiesel production, there would also be a corresponding increase in the glycerol byproduct formation. Nevertheless, increasing concentrations of catalyst may lead to side reactions (i.e., saponification), which in turn may complicate separation processes and reduce the measured yield [69]. The optimal yield (99.08%) achieved at 55 °C and a 3.3 mg/g catalyst ratio is slightly higher than the yield range (95–99%) reported in the literature. As the temperature change is rather low, the effect of temperature on yield seems to be much less important than the effect of the catalyst concentration [90].

The ANOVA indicates the presence of a linear model and a low standard deviation (1.98), which means that the data are concentrated around the mean yield of 93.45%. The “prob > F” lower value (0.0017) validates significance, and R² (0.7959), adjusted R² (0.7448), and predicted R² (0.5589) all indicate good fit. Equation (4) represents the derived yield equation that follows the linear form and adequately agrees with the experimental biodiesel yields obtained from household WCO.

Yield = 103.35803 + 0.044645*T − 2.557*C_R

(4)

It should be noted that the negative coefficient for C_R implies the catalyst inverse effect.

The maximum biodiesel production (97.5%) of restaurant WCO was obtained at 54 °C and a 1.5 mg/g catalyst ratio. The lower temperature and lower catalyst ratio can lead to higher biodiesel production, as presented in Figure 2b. The experimental results of the study revealed that higher yields of biodiesel were obtained at the optimal temperatures (55 °C for household WCO and 54 °C restaurant WCO) and catalyst ratios (3.3 mg/g for household WCO and 1.5 mg/g restaurant WCO). Regarding biodiesel from restaurant WCO, there is increased variability in the levels of the standard deviation (5.51) and the mean yield (90.32%). Even though the significance is registered by the lower “prob > F” value (0.019), the lower value of predicted R² (0.0769) denotes that this model is inadequate, and also the R² (0.6289) and the adjusted R² (0.5361) values are found to be low. The derived equation given in Equation (5) can be utilized to determine the yield of biodiesel generated from restaurant WCO.

Yield = 196.75306 − 1.40933*T − 14.38448*C_R

(5)

Like the results obtained in the current study, Danane et al. [69] found that up to 99.38% of biodiesel can be produced out of waste cooking oil using RSM, but the concentration of catalyst was determined to be the main factor affecting the transesterification process. Biodiesel output rises with rising concentration of catalyst to optimum level, after which saponification and other side processes diminish the effectiveness of transesterification, as reflected during experimental optimization of household and restaurant WCO transesterification in the present study [91].

3.3.3. The Combined Effect of Catalyst Ratio and Temperature on Biodiesel Density

Concerning the biodiesel produced through household WCO, the temperature and catalyst ratio did not make much influence on density (Figure 3a). At 25 °C, the average density is 0.83 g/mL, close to the literature’s value of 0.878 g/mL at 15 °C [92]. The density of the measured biodiesel is slightly lower than it should be, which is in line with the overall trend of density with temperature (generally drops by about 0.0007 g/mL per °C), possibly affected by the fatty acid content of the feedstock as well as the circumstances of measurement. For example, the elevated temperature of measurement (25 °C rather than 15 °C) makes direct comparison with standard biodiesel specifications difficult and necessitates extra cooling or adjustment procedures during the process, both of which consume more energy. These augmentations may affect the economic feasibility of WCO for biodiesel production, as it increases the cost of operation relative to processes that have been optimized under normal conditions. These limitations could therefore be mitigated by optimization of reaction and measurement conditions, including alignment with standard temperatures. For biodiesel from household WCO, the mean model of ANOVA shows that the data tend to cluster closely around the mean density (0.8334 g/mL) with a very low standard deviation (0.014). Since the effect of the density is so small, no particular equation is provided.

Regarding Figure 3b, biodiesel based on restaurant WCO shows density variation with a mean density of 0.8709 gm/mL and a lower standard deviation of 0.0347. The lower significance of the model could be attributed to the higher “prob > F” value (0.3002) with a negative value of predicted R² (−1.6351). This implies that the overall mean may be a better predictor of the experimental results than the current model. Also, the values of R² (0.6212) and adjusted R² (0.2424) are low. The achieved quadratic Equation (6) can be used to obtain the density of biodiesel produced from restaurant WCO.

Density = −12.29225 + 0.52544*T − 1.82845*CR + 0.025898*T*CR
−0.005065*T² + 0.1016*CR²

(6)

The obtained density at 15 °C is in good agreement with the biodiesel standard specifications (0.86–0.90 g/mL) [87]. These results agree with those of recent research works that indicate a density of 0.86 g/mL for WCO biodiesel, as observed by Nabizadeh et al. [87]. The consistency in density across conditions as measured by Ahmad et al. [86] using RSM to attain similar densities is a very positive aspect relating to practical applications.

3.4. Comprehensive Analysis of WCOs and Chlorella sp. Microalgae as Feedstocks for Biodiesel Production

This paper provides a comparison of the nature, processing methodologies, biodiesel production, quality, economical, and environmental aspects of the three investigated feed stocks (household WCO, restaurant WCO, and Chlorella sp. microalgae) used to produce biodiesel.

Properties of feedstock: WCO generated by households is cleaner and contains fewer impurities such as residual food, water, and degradation products compared with commercial sources [93]. Regarding household WCO, the cleaning process is less efficacious, since it is less utilized in home cooking. Its lower quantity of impurities facilitates its handling and processing; it requires simple filtration to remove the solid particles prior to transesterification. This implies that household WCO is suitable in producing biodiesel at a small scale. Household WCO would be available in residential locations more readily than restaurant WCO, so it can be processed on a local scale [94,95]. On the other side, restaurant WCO, or brown grease, is obtained from small local restaurants of Riyadh (Saudi Arabia). It is referred to as heavy polluted oil due to its increased impurity and products of degradation as a result of repeated usage during commercial cooking purposes. It constitutes a big percentage of the waste oil produced in the region. Because of high contamination, restaurant WCO necessitates additional processes for decontamination to prevent issues such as saponification during transesterification, which can reduce yield and quality [93,94]. Restaurant WCO is more widespread than domestic WCO, and hence may become a good source of large-scale biodiesel production, provided the issues with processing are resolved [95].

Due to their high oil content (30–50 wt%), as indicated in

Table 6. Comparison summary between the three considered feedstocks for producing biodiesel.

Aspect	Household WCO	Restaurant WCO	Chlorella sp. Microalgae
Characteristics	Cleaner, less contaminated	More polluted, higher impurities	High oil content (30–50%), cultivated
Alcohol Used	Ethanol	Methanol	Methanol
Temperature Range	55–65 °C	54–60 °C	~60 °C
Catalyst Concentration	3.3–6.22 mg/g (NaOH)	1.5–2.25 mg/g (NaOH)	NaOH (unspecified)
Biodiesel Yield	Up to 99.08%	Up to 96.61%	28.6% (semi-open)
Biodiesel Viscosity	4.82–5.49 mPa·s	8.25 mPa·s	Within EN 14214
Biodiesel Density	0.851 g/ml	0.82–0.949 g/ml	Within EN 14214
Processing Challenges	Slight pretreatment	Extensive purification needed	Complex cultivation and extraction
Economic Viability	High (low preprocessing cost)	Moderate (higher processing cost)	Low (high cultivation cost)
Environmental Impact	Reduces waste, low emissions	Reduces waste, low emissions	Carbon neutral, high energy use

, and ease of adaptation to various conditions, such as freshwater and salty environments, Chlorella microalgae are cultivated in semi-open systems that resemble open batch photobioreactors [96]. They are promising for the environment, with high rates of growth due to their carbon neutrality [97]. The regulated conditions, including the presence of sufficient light and nutrients, increase the complexity of its cultivation and add more cost. It was noticed that oil extraction and transesterification in the case of Chlorella microalgae demands a greater amount of energy (1.81 MJ/MJ of biodiesel as the lowest energy demand for Chlorella biodiesel production based on the nitrogen starvation scenario) compared to WCO processing (0.46 MJ/MJ of biodiesel) [98]. In contrast to WCO, microalgae cannot be readily grown in nature and would require infrastructure to grow, thus constraining immediate supply, but would be highly scalable when it comes to investment [99].

Processing Techniques: The cleaner feedstock is compatible with ethanol, which leads to high biodiesel yields and quality and a limited number of adverse reactions. Therefore, the recommended alcohol to use in the transesterification of the household WCO is ethanol [91,100]. Owing to the higher boiling point of ethanol (78 °C) as compared to that of methanol (64.7 °C), chances of unsafe reactions are reduced. The primary catalyst used in this study is sodium hydroxide (NaOH), which is used most efficiently at concentrations between 3.3 and 6.22 mg/g solution (alcohol and WCO). Potassium hydroxide (KOH) was less efficient, with a yield of 85.4% at a catalyst concentration of 11.38 mg/g.

Restaurant WCO uses methanol, possibly due to its lower cost or its greater compatibility with higher levels of impurities in brown grease [95]. Whereas the low boiling point of methanol increases the rate of reactions, it can also increase safety concerns. Nonetheless, methanol is very toxic to human beings, where blindness or even death can occur in case of ingestion, inhalation, or even when absorbed through the skin. In relation to these risks, a high level of safety precautions should be put in place, which involves the use of fume hoods, personal protection (protective clothing, gloves, glasses, etc.), and good ventilation when handling, and effective recovery of the unused methanol to reduce the amount of residual methanol in the end biodiesel product. The obtained results of the current study show that NaOH is suggested with an optimal concentration range of 1.5–2.25 mg/g for restaurant WCO. This confirms the superiority of NaOH, as KOH at 11.38 mg/g yields a lower biodiesel yield of 86.09%. Similar to the household WCO, the molecular ratio of methanol to oil is 6:1, and the temperature at which the reaction occurs is 54–60 °C. In spite of the process being similar to that of household WCO, additional purification might be required because of the presence of impurities. The impurities in restaurant WCO are higher in content as compared to those of household WCO and lower the effectiveness of the transesterification process, leading to a slightly reduced yield and quality of biodiesel [94].

Methanol is generally used in the transesterification of Chlorella microalgae due to its low costs and compatibility with algal oil, similar to restaurant WCO. Nonetheless, at a lower popularity level, ethanol could be explored regarding quality. Although the conditions are very close to those of WCO transesterification (e.g., 6:1 methanol to oil ratio, 60 °C), NaOH is the catalyst of choice due to its low cost and the fact that it produces a high yield of biodiesel product [94]. The extraction of oil and transesterification follow the cultivation of Chlorella sp. in semi-open photobioreactors with the controlled rates of the input light and nutrients [99]. It is also more complex and requires harvesting and drying the biomass prior to oil extraction. The achieved biodiesel meets the quality requirements, but the complex process of cultivation and extraction results in low yields of 28.6% compared to WCO [94].

The choice of not separately quantifying the oil extracted from Chlorella sp. upstream of transesterification was chosen to simplify the process and avoid energy-intensive stages, which fits within the proposed study aiming to produce biodiesel at a low cost. Direct extraction of the lipids using the Bligh and Dyer technique gave 28.6% as a combined extraction and conversion efficiency for Chlorella sp. biodiesel production. The extracted oil may be separated to improve knowledge of lipid yield, but this will lead to additional costs in energy and operations, and this would result in a higher net production cost. The residual algal biomass left by the lipid extraction would still be viable as animal feed, biofertilizers, or biogas production, leading to byproduct revenue of USD 0.10–0.20/L (see Table 7). This valorization increases the sustainability of Chlorella biodiesel, even though it has lower yields than those of WCOs, given the circular economy approach.

In this work, feedstock specific yields and feasible processing were optimized through the manipulation of transesterification conditions. Standardization to methanol (1 wt% NaOH, 60 °C) could marginally decrease production of household WCO biodiesel. It does not have an indicative impact on biodiesel production based on restaurant WCO. It does not affect positively the Chlorella sp. biodiesel production, which is constrained by the upstream processes of cultivation and extraction.

Biodiesel Yields: The obtained results showed that at optimal conditions (55 °C, 3.3 mg/g NaOH, ethanol), the household WCO can achieve a maximum biodiesel yield of 99.08%. The low content of impurities in the feedstock reduces saponification reactions. The high yield is attributable to the effectiveness of ethanol during the transesterification process. The applicability of these conditions was justified by the optimization achieved in other studies by applying RSM [94,101]. At the obtained optimal conditions (54 °C, 1.5 mg/g NaOH, methanol), it is possible to attain a biodiesel yield of 96.61% based on the restaurant WCO. The somewhat lower yield is likely due to a high content of impurities, which may result in reactions such as soap formation and decreasing the efficiency of the transesterification process. The previous studies have shown that these issues can be more pronounced at higher temperatures and higher catalyst ratios [93,94]. Under semi-open systems, Chlorella sp. microalgae can achieve a biodiesel yield of 28.6%, significantly lower than WCO. Optimized closed photobioreactors are able to achieve up to a 35% biodiesel yield. The reduced production is due to problems in oil extraction effectiveness and the microalgae production system (e.g., light and nutrient constraints). The high content of oil (30–50%) in Chlorella sp. microalgae is offset by energy-intensive processing [99].

The biodiesel generated by the domestic WCO has viscosity ranges from 4.82 to 5.49 mPa·s, which falls within the ISO 3104:2023 standard of 1.9 to 6.0 mPa s [94]. Its lower viscosity allows it to be suitable to be directly used in diesel engines, enhancing the comparative flow as well as performance of diesel engines [102]. Restaurant WCO generates biodiesel with a viscosity of 8.25 mPa·s, exceeding the standard range, and which may require additional purification to be suitable to use in diesel engines [93]. The possible causes of the raised viscosity are incomplete transesterification and residual contaminants. Chlorella microalgae produce lower yields, but the resulting biodiesel has a viscosity of 3.87 mPa·s, which is within the specifications of ISO 3104:2023 standards; this means that it has good flow properties [94].

The household WCO exhibits consistent biodiesel quality with its constant density of 0.851 g/mL, representing a value close to the ISO 12185:2024 standard of 0.86 to 0.90 g/mL at 15 °C [102]. The biodiesel from restaurant WCO has a variable range of density (0.82–0.949 g/mL), with the best density of 0.82 g/mL. This variance indicates the challenge of keeping a consistent degree of biodiesel quality as a result of the composition of the feedstock [93]. The density of biodiesel created by the Chlorella microalgae is 0.88 g/mL, and it is within the ISO 12185:2024 standard range, which implies reliable quality in terms of engine usage [94].

The additional microalgae quality parameters are as follows: The flash point of 152 °C implies exceptionally safe handling and storage. The carbon residue is 0.1%, indicating minimum engine deposits. The water content is 482 mg/kg, with a framework of stability. The acid value of 0.42 mg KOH/g is an indication of the low corrosivity of the obtained biodiesel [94].

Environmental concerns: In comparison with fossil fuels, the considered household and restaurant WCOs can minimize carbon and sulfur gas emissions and waste disposal issues [103]. Household WCO is more ecofriendly as it has less byproducts since it is clean. Nevertheless, in spite of the advantages, restaurant WCO can be more environmentally costly, as it needs more energy to be purified [95]. The intrinsic carbon neutrality of Chlorella sp. microalgae is achieved by the possibilities of carbon sequestration through its cultivation. Nevertheless, the energy costs of planting and extracting (which are intensive) can counter these environmental gains [97,98].

In this study, RSM and ANOVA were employed to optimize the conditions of biodiesel production. The results demonstrated that household WCO had higher performance compared to restaurant WCO and Chlorella sp. microalgae in a number of significant parameters [94,101]. The main differences between the three feedstocks considered to produce biodiesel are summarized in Table 6.

Strategies to Improve Biodiesel Quality from WCO: Contamination of WCO by food wastes, water, and free fatty acids (FFAs) as well as the byproducts of oxidation, peroxides, and aldehydes developed through repeated cooking could have negative impacts on biodiesel quality, increasing viscosity and acid values and decreasing oxidative stability. In the current research, household WCO contained lower concentrations of impurities, so that the respective biodiesel had a viscosity of 4.82–5.49 mPa and density of 0.851 g/mL, which match well with EN 14214:2012+A2:2019 standards. Conversely, higher levels of impurities in restaurant WCO generated a biodiesel whose viscosity was out of the standard range (1.9–6.0 mPa s), which is indicative of either incomplete transesterification or contaminants. In order to enhance the quality of the biodiesel, extra purification procedures were tested. Post transesterification phase of water washing successfully produced residual catalysts, soaps, and glycerol-free biodiesel with an acid value of 0.42 mg KOH/g in the case of household WCO biodiesel. In the case of restaurant WCO, it was suggested that dry washing using adsorbent magnesium silicate (0.5–1 wt%) could be used as a means of eliminating polar impurities while not creating any wastewater and improving the viscosity to be within 4.5–6.0 mPa s [93]. Subsequent vacuum distillation at 60–70 °C further depleted methanol to <0.2 wt%, increasing fuel safety as well as EN 14110:2019 compliance. In an effort to overcome oxidative stability, especially of restaurant WCO, which is subjected to higher peroxide values due to thermal degradation, antioxidants such as butylated hydroxytoluene (BHT) at 200–500 ppm were examined to lower the iodine value to 105–110 g I₂/100 g and increase shelf life [94]. Although these purification and stabilization methods cause a cost escalation of operations, they ensure high quality biodiesel can be used as diesel engine fuel and aid the process of scalability due to the ability to minimize engine wear and emissions.

3.5. Economic Comparison of Biodiesel Production from WCO and Microalgae

Based on the results of the current research work, Table 7 provides the economic assessment of the three different feedstocks (Chlorella microalgae, restaurant WCO, and household WCO biodiesel) used for producing biodiesel with a commercial production scale of 1,000,000 L/year. Household WCO is less costly in processing because of its lower impurities, but because of the high cost of collection at the household level, there are logistical constraints. Restaurant WCO can be collected in large quantities, which is plentiful but rich in impurities and requires long purification, raising the cost of processing. Chlorella sp. microalgae are technically carbon-neutral but have high extraction and cultivation costs compared to WCO [98]. The costs, revenues, ROI, and payback periods were estimated based on a biodiesel selling price ranging from USD 0.7/L to USD 1/L, energy cost of USD 0.1/kWh, and 330 days of operation per year [104,105,106]. The costs and prices are in US dollars. It should be noted that the obtained results were achieved by integrating the experimental results with the literature data [107,108,109,110,111,112].

Table 7. Economic analysis of biodiesel production from WCOs and microalgae.

Parameter	Household WCO	Restaurant WCO	Microalgae (Chlorella)
Capital Costs
Filtration Systems	USD 60,000 [107]	USD 80,000 [107]	-
Heating Units	USD 30,000 [107]	USD 40,000 [107]	-
Transesterification Reactors	USD 100,000 [107]	USD 100,000 [107]	USD 100,000 [108]
Photobioreactors/Glass Pools	-	-	USD 480,000 [108]
Harvesting Equipment	-	-	USD 140,000 [108]
Total Capital Costs	USD 190,000	USD 220,000	USD 720,000
Operational Costs (per L)
Feedstock	USD 0.05 [109]	USD 0.07 [109]	USD 0.10–0.20 [108]
Pretreatment	USD 0.05–0.10 [109]	USD 0.10–0.15 [109]	-
Transesterification	USD 0.10–0.20 [109]	USD 0.10–0.20 [109]	USD 0.10–0.20 [109]
Cultivation (Nutrients, CO2, Light)	-	-	USD 0.70–0.90 [108]
Harvesting and Drying	-	-	USD 0.30–0.50 [110]
Energy (Pretreatment and Processing)	USD 0.05–0.10 [106]	USD 0.05–0.10 [106]	USD 0.20–0.30 [106]
Total Operational Costs (per L)	USD 0.25–0.45	USD 0.32–0.52	USD 1.40–2.10
Byproduct Revenue (per L)
Glycerol	USD 0.05–0.07 [111]	USD 0.05–0.07 [111]	USD 0.05–0.07 [111]
Algal Biomass	-	-	USD 0.10–0.20 [112]
Total Byproduct Revenue (per L)	USD 0.05–0.07 [111]	USD 0.05–0.07 [111]	USD 0.15–0.27 [111,112]
Net Production Costs (per L)	USD 0.20–0.38 [109,111]	USD 0.27–0.45 [109,111]	USD 1.25–1.83 [108,110,112]
Biodiesel Revenue (per L)	USD 0.70–1.00 [105]	USD 0.70–1.00 [105]	USD 0.70–1.00 [105]
Annual Cash Flow	USD 320,000–800,000	USD 250,000–730,000	(−USD 1,130,000)–(−USD 250,000)
ROI (%)	168.42–421.05%	113.64–331.82%	(−156.94)–(−34.72)
Payback Period (Years)	0.24–0.59	0.3–0.88	Negative cash flow

Table 7. Economic analysis of biodiesel production from WCOs and microalgae.

Parameter	Household WCO	Restaurant WCO	Microalgae (Chlorella)
Capital Costs
Filtration Systems	USD 60,000 [107]	USD 80,000 [107]	-
Heating Units	USD 30,000 [107]	USD 40,000 [107]	-
Transesterification Reactors	USD 100,000 [107]	USD 100,000 [107]	USD 100,000 [108]
Photobioreactors/Glass Pools	-	-	USD 480,000 [108]
Harvesting Equipment	-	-	USD 140,000 [108]
Total Capital Costs	USD 190,000	USD 220,000	USD 720,000
Operational Costs (per L)
Feedstock	USD 0.05 [109]	USD 0.07 [109]	USD 0.10–0.20 [108]
Pretreatment	USD 0.05–0.10 [109]	USD 0.10–0.15 [109]	-
Transesterification	USD 0.10–0.20 [109]	USD 0.10–0.20 [109]	USD 0.10–0.20 [109]
Cultivation (Nutrients, CO2, Light)	-	-	USD 0.70–0.90 [108]
Harvesting and Drying	-	-	USD 0.30–0.50 [110]
Energy (Pretreatment and Processing)	USD 0.05–0.10 [106]	USD 0.05–0.10 [106]	USD 0.20–0.30 [106]
Total Operational Costs (per L)	USD 0.25–0.45	USD 0.32–0.52	USD 1.40–2.10
Byproduct Revenue (per L)
Glycerol	USD 0.05–0.07 [111]	USD 0.05–0.07 [111]	USD 0.05–0.07 [111]
Algal Biomass	-	-	USD 0.10–0.20 [112]
Total Byproduct Revenue (per L)	USD 0.05–0.07 [111]	USD 0.05–0.07 [111]	USD 0.15–0.27 [111,112]
Net Production Costs (per L)	USD 0.20–0.38 [109,111]	USD 0.27–0.45 [109,111]	USD 1.25–1.83 [108,110,112]
Biodiesel Revenue (per L)	USD 0.70–1.00 [105]	USD 0.70–1.00 [105]	USD 0.70–1.00 [105]
Annual Cash Flow	USD 320,000–800,000	USD 250,000–730,000	(−USD 1,130,000)–(−USD 250,000)
ROI (%)	168.42–421.05%	113.64–331.82%	(−156.94)–(−34.72)
Payback Period (Years)	0.24–0.59	0.3–0.88	Negative cash flow

The economic analysis results listed in Table 7 show that the production of biodiesel using household WCO, restaurant WCO, and microalgae varies widely. The USD 60,000 and USD 80,000 are filtered costs for household and restaurant WCO, respectively, which are derived by Haas et al. [107] and adjusted to 100,000 L/year for a biodiesel plant. The price of the restaurant WCO is higher because of the necessity of stronger filtration in order to process higher impurities (e.g., food debris, FFAs). The heating unit costs of household WCO (USD 30,000) and restaurant WCO (USD 40,000) have been deduced based on Haas et al.’s [107] research work. The increased cost for restaurant WCO compared to household WCO is based on a greater thermal load and extra heating needs, resulting in higher impurity rates. The volume of a photobioreactor was estimated at 889 m³ to produce 100,000 L/year of biodiesel with Chlorella as the production organism based on a productivity of 25 g/m²/day, 30% lipid content, and 300 days operation per year, and requiring a surface area of 44,440 m². The cost, when estimated at USD 120/m², was USD 480 000, converted to 2023 USD with an inflation factor of 1.4. The harvesting equipment (using centrifugation) cost of 140,000 was estimated based on 333,333 kg/year of biomass, scaled to the 100,000 L/year scale, and adjusted to inflationary conditions [108]. It should be noted that all costs are adjusted to 2023 USD (inflation factor 1.4).

The equations used to calculate return on investment (ROI), payback period, annual cash flow, and net production costs are presented in Equations (7)–(10). It should be noted that the annual cash flow and net production costs (USD/L) will be multiplied by the biodiesel production rate of 1,000,000 L/year.

$$\mathbf{R}\mathbf{O}\mathbf{I} (\%)=(\mathbf{A}\mathbf{n}\mathbf{n}\mathbf{u}\mathbf{a}\mathbf{l} \mathbf{C}\mathbf{a}\mathbf{s}\mathbf{h} \mathbf{F}\mathbf{l}\mathbf{o}\mathbf{w}/\mathbf{T}\mathbf{o}\mathbf{t}\mathbf{a}\mathbf{l} \mathbf{C}\mathbf{a}\mathbf{p}\mathbf{i}\mathbf{t}\mathbf{a}\mathbf{l} \mathbf{C}\mathbf{o}\mathbf{s}\mathbf{t}\mathbf{s})\times 100$$

(7)

$$\mathbf{P}\mathbf{a}\mathbf{y}\mathbf{b}\mathbf{a}\mathbf{c}\mathbf{k} \mathbf{P}\mathbf{e}\mathbf{r}\mathbf{i}\mathbf{o}\mathbf{d} \left(\mathbf{Y}\mathbf{e}\mathbf{a}\mathbf{r}\mathbf{s}\right)=\mathbf{T}\mathbf{o}\mathbf{t}\mathbf{a}\mathbf{l} \mathbf{C}\mathbf{a}\mathbf{p}\mathbf{i}\mathbf{t}\mathbf{a}\mathbf{l} \mathbf{C}\mathbf{o}\mathbf{s}\mathbf{t}\mathbf{s}/\mathbf{A}\mathbf{n}\mathbf{n}\mathbf{u}\mathbf{a}\mathbf{l} \mathbf{C}\mathbf{a}\mathbf{s}\mathbf{h} \mathbf{F}\mathbf{l}\mathbf{o}\mathbf{w}$$

(8)

$$\mathbf{A}\mathbf{n}\mathbf{n}\mathbf{u}\mathbf{a}\mathbf{l} \mathbf{C}\mathbf{a}\mathbf{s}\mathbf{h} \mathbf{F}\mathbf{l}\mathbf{o}\mathbf{w}=\mathbf{B}\mathbf{i}\mathbf{o}\mathbf{d}\mathbf{i}\mathbf{e}\mathbf{s}\mathbf{e}\mathbf{l} \mathbf{R}\mathbf{e}\mathbf{v}\mathbf{e}\mathbf{n}\mathbf{u}\mathbf{e}-\mathbf{N}\mathbf{e}\mathbf{t} \mathbf{P}\mathbf{r}\mathbf{o}\mathbf{d}\mathbf{u}\mathbf{c}\mathbf{t}\mathbf{i}\mathbf{o}\mathbf{n} \mathbf{C}\mathbf{o}\mathbf{s}\mathbf{t}\mathbf{s}$$

(9)

$$\mathbf{N}\mathbf{e}\mathbf{t} \mathbf{P}\mathbf{r}\mathbf{o}\mathbf{d}\mathbf{u}\mathbf{c}\mathbf{t}\mathbf{i}\mathbf{o}\mathbf{n} \mathbf{C}\mathbf{o}\mathbf{s}\mathbf{t}\mathbf{s}=\mathbf{T}\mathbf{o}\mathbf{t}\mathbf{a}\mathbf{l} \mathbf{O}\mathbf{p}\mathbf{e}\mathbf{r}\mathbf{a}\mathbf{t}\mathbf{i}\mathbf{o}\mathbf{n}\mathbf{a}\mathbf{l} \mathbf{C}\mathbf{o}\mathbf{s}\mathbf{t}\mathbf{s}-\mathbf{T}\mathbf{o}\mathbf{t}\mathbf{a}\mathbf{l} \mathbf{B}\mathbf{y}\mathbf{p}\mathbf{r}\mathbf{o}\mathbf{d}\mathbf{u}\mathbf{c}\mathbf{t} \mathbf{R}\mathbf{e}\mathbf{v}\mathbf{e}\mathbf{n}\mathbf{u}\mathbf{e}$$

(10)

Household WCO has the lowest net production cost (USD 0.20–0.38/L) compared to restaurant WCO. Household WCO is the most cost-effective feedstock due to the higher yield (99.08%) and reduced energy needs to run the processing [109]. Restaurant WCO has a more expensive pretreatment cost due to the high free fatty acid (FFA) content, requiring more catalyst inputs, although it is also competitive (USD 0.27–0.45/L) [113]. Both types of WCO have low capital costs (USD 190,000–220,000) and glycerol byproduct revenues rate (USD 0.05–0.07/L), which leads to a short payback duration (0.24–0.88 years) and high ROI (113.64–421.05%) [107,111].

Nevertheless, the major reasons behind the high net production costs of microalgae biodiesel (USD 1.25–1.83 to produce 1 L of biodiesel) are the cost of cultivation (50 to 60% of the total costs, or USD (0.7–0.9)/L) and the harvesting and drying costs (USD 0.30 to USD 0.50 or more per liter) [108,110]. It is also limited in economic viability by the large amount of capital investment (USD 720,000) needed to build glass pools or photobioreactors [108]. At the studied conditions, in the case of microalgae biodiesel production, no payback period can be feasible, and their production will produce negative cash flows and ROI, even taking into consideration the extra revenue provided by algal biomass (USD 0.1–0.20/L) [107]. Despite the lower cost of the semi-open flask cultivation method used in this study in comparison to the closed photobioreactors, the achieved biodiesel yield is only 28.6%. The recent research works show that the current prices of microalgae could be reduced to USD 1–1.5/L through the use of renewable energy and optimization of harvesting techniques (like bioflocculation), which would make it more competitive [110,114]. Kumar and Singh in their research work gave an example by noting that the economic feasibility of microalgae may be enhanced by an open-pond system and coproduct valorization [114].

The economic advantage of using WCO, which also advocates the concepts of a circular economy, is due to its more straightforward processing and utilizing waste streams in its routines [111]. However, it is not scalable because it requires regional organizations to supply and collect [115]. Even though microalgae are carbon-neutral and scalable, they require significant cost improvement necessities [114,116]. This research is more relevant to the creation of sustainable biofuels due to its focus on using non-standard WCOs and semi-open Chlorella growth, which provides new opportunities for local small-scale production.

4. Conclusions

The aim of the current study is to evaluate the production of biodiesel using non-standard sources of WCO (local restaurants and homes) and semi-open production of Chlorella sp. microalgae, providing novel visions on the cost-effective and scalable production of biodiesel to meet the needs of a small-scale or commercial production. The introduced techno-economic analysis strongly supports the viability of these feedstocks, with important consequences in the realm of energy sustainability. Household WCO has the optimal biodiesel yield at 99.08% at 55 °C using 3.3 mg/g NaOH as a catalyst ratio and ethanol due to its fewer impurities and low preprocessing requirements. The combination of this high yield, a viscosity of 5.45 mPa·s, and a density of 0.88 g/mL meets the standards (EN 14214:2012+A2:2019) requirements of biodiesel and a high-quality fuel with the least side reactions, such as saponification. Restaurant WCO, having a higher level of impurities, has an optimum biodiesel yield of 96.61% at 54 °C with a 1.5 mg/g NaOH catalyst ratio and methanol. Yet, extra purification is needed, which adds to the cost, because it is thick and contains a lot of contaminants. Despite the high energy input required to grow and extract the algae, microalgae Chlorella grown in semi-open photobioreactors only produced 28.6%, with an acid value of 0.42 mg KOH/g and an iodine value of 111.5 g I₂/100 g, indicating good quality.

The obtained results were validated by ANOVA and RSM. The household WCO showed a significant model fit (prob > F = 0.003) of viscosity and a strong determination coefficient (R² = 0.8473). The average viscosity for restaurant WCO at 40 °C is 10.3 mPa·s, which needed to be optimized to attain the standard range (1.9–6.0 mPa·s). The economic feasibility of generating biodiesel from the three investigated feedstocks is scaled up to a 1,000,000 L/year as a commercial production capacity. The economic advantage of the household WCO compared to restaurant WCO is its cheaper preprocessing cost (USD 190,000 overall capital cost) and better yield, which makes it more economical. The purification expenses offset the abundance of restaurant WCO and lead to an increase of the total capital cost (USD 220,000). Even though carbon neutrality has many benefits, the economically viable use of microalgae Chlorella may be limited by unreasonably high cultivation costs, which contributes to the higher capital costs of USD 720,000. Although energy requirements partially offset Chlorella carbon sequestration, both forms of WCO reduce carbon and sulfur emissions, and household WCO produces fewer byproducts. The obtained results of ROI for biodiesel production from household WCO, restaurant WCO, and Chlorella sp. microalgae are (168.42–21.05%), (113.643–31.82%), and (−156.94–(−34.72)), respectively. The payback periods are (0.24–0.59) and (0.3–0.88) for biodiesel production from household WCO and restaurant WCO, respectively. These results suggest that household WCO should be prioritized as a high-yield, cost-effective feedstock for the production of biodiesel. Additionally, increasing microalgae growth in a way that benefits and supports the scalability of biofuel production and sustainable biofuel development in accordance with a circular economy should be a top priority.