Biodiesel Production Processes with Yeast: A Sustainable Approach

Abstract

In recent years, renewable sources of energy have been sought due to the environmental impacts associated with fossil fuels, such as greenhouse gas emissions into the atmosphere. A promising alternative is biodiesel, particularly when obtained using yeast, as they offer certain advantages over other microorganisms due to their resilience to grow in various conditions, short reproduction times, and lower susceptibility to bacterial infections because they thrive at lower pH levels and have the ability to utilize a wide variety of substrates. Furthermore, biodiesel produced with yeast is composed of methyl ester fatty acids (FAME), providing it with good quality and performance in internal combustion engines, resulting in reduced greenhouse gas emissions compared to conventional diesel. The production of biodiesel using yeast involves six general stages, which offer various methodological alternatives with different degrees of sustainability. The objective of this review is to assess the sustainability degree of various methodologies employed in each of the stages of yeast-based biodiesel production through environmental and economic sustainability indicators.

1. Introduction

In the last century, large amounts of greenhouse gases (GHG) such as carbon monoxide (CO), carbon dioxide (CO₂), methane (CH4), nitrous oxide (N₂O), and fluorinated gases (chlorofluorocarbons, hydrofluorocarbons) have been emitted into the atmosphere. In 2021, the total emissions were 6340.2 million metric tons of carbon dioxide (CO₂) equivalents (One million metric tons is approximately equal to one trillion grams). The US inventory uses metric units for comparison and consistency with other countries. To convert gas emissions to their CO₂ equivalent, one must multiply by the Global Warming Potential (GWP) (See https://www.epa.gov/ghgemissions/overview-greenhouse-gases (accessed on 2 July 2020)), of which 79.2% corresponded to carbon dioxide, 11.5% to methane (CH4), 6.2% to (N₂O), and 3% to fluorinated gases [1]. The sectors that have contributed to these emissions are: transportation at 28%, electric power at 25%, industry at 23%, commercial and residential at 13%, and agriculture at 10% [1].

In addition to the environmental problems generated by the use of fossil fuels, their reserves are limited, and their prices are highly volatile [2]. To address this situation, the scientific community has focused on developing renewable energy sources such as biodiesel, which in this sector contributes to the reduction of greenhouse gases [3,4,5].

Biodiesel is composed of long-chain alkyl esters of fatty acids [6], which can be obtained from various raw materials such as edible vegetable oil, non-edible vegetable oil, animal fats, waste cooking oil, and microbial lipids [7]. It is used in different proportions with diesel (5–20% v/v), forming a blend that can be used as a biofuel.

The classification of biodiesel consists of four generations, which are based on the use of different types of raw materials and byproducts [8]. First-generation biodiesel is obtained from edible oils derived from various seeds such as canola or rapeseed (Brassica napus) [4], soybean (Glycine max), oil palm (Elaeis guineensis) [9], sunflower (Helianthus annus) [10], and corn (Zea mays) [8], among others. However, due to the increasing costs of edible oils and concerns about competition with the food supply chain, the development of second-generation biodiesel was promoted [11].

Second-generation biodiesel is produced from non-edible oils such as jatropha curcas oil [12], waste cooking oil [13,14], lignocellulosic residues, and industrial effluents used as substrates for the production of intracellular lipids by certain microorganisms such as fungi, bacteria, and yeast [10]. For example, among the lignocellulosic residues, we have Eucaliptus urograndis [15], wheat bran [16], rice bran [17], and corn stover [18], and among the industrial effluents, crude glycerol obtained as a byproduct of soap and biodiesel production is used [19].

The third generation is produced from lipids generated by microalgae, which are photosynthetic microorganisms that grow in ponds and use only light and carbon dioxide as their sources to grow [8].

The fourth generation is derived from genetically modified microalgae that are engineered to enhance lipid production and the biodiesel extraction process. This generation is produced through hydroprocessing technology [8].

The production of second, third, and fourth-generation biodiesel involves the use of oleaginous microorganisms that must accumulate between 20% and 70% of the lipid content in their dry weight [10,20,21]. These microorganisms include certain species of bacteria, algae, and fungi [22]. Among the fungi, there are yeasts, which are unicellular micro-organisms that reproduce asexually through budding or fission [10] and are used in the production of second-generation biodiesel.

Yeasts have advantages over the other aforementioned oleaginous microorganisms. They are more resilient to different growth conditions, have a low susceptibility to viral infections, and can control bacterial contamination due to their ability to grow at low pH levels [10]. Additionally, yeasts do not require large spaces for cultivation, have a short reproduction period [20], and are capable of metabolizing various carbon sources to produce lipids [23], such as sugars (glucose, xylose, mannose, fructose, and galactose), which can be obtained from the pretreatment of lignocellulosic residues [24,25,26,27,28,29], organic acids [30,31,32,33], or food waste rich in starch, glucose, proteins, fats, and even trace elements like potassium, calcium, and magnesium [34,35]. These food waste sources promote yeast growth at a low cost, making them an exploitable carbon source for these microorganisms.

Currently, various yeast species have been used for lipid production as a basis for biodiesel generation. These species are classified within the Phyla Ascomycota and Basi-diomycota of the Fungi Kingdom [10,36]. As shown in Figure 1 and Figure 2, the most widely used yeasts from the Ascomycota Phylum include Yarrowia lipolytica, Lipomyces starkeyi, Saccharomyces pastorianus, Candida tropicalis, and Metschnikowia pulcherrima. From the Basidiomycota Phylum, species such as Cutaneotrichosporon sp., Rhodotorula sp., Naganishia albida, Phenoliferia glacialis, and Sporobolomyces shibatanus [15,18,19,21,24,26,27,32,35,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70].

All the yeasts reported in Figure 1 and Figure 2 can produce intracellular lipids through the degradation of various substrates used as carbon sources and generate these compounds in different proportions. The lipids produced need to be extracted from the yeast biomass and then transformed into biodiesel through chemical reactions [71].

The production of biodiesel with yeasts consists of six stages, as described below: (1) Selection of the yeast strain and substrate, which in the case of the latter being lignocellulosic residue, requires pretreatment; (2) Cultivation of the selected yeast strain under optimal conditions; (3) Lipid production; (4) Extraction of lipids; (5) Transesterification reaction; and (6) Refining process of FAME to obtain biodiesel [55,72]. In each stage, there are different alternatives and approaches in the development of the methodology, depending on the yeast strains and substrates used for lipid production. This article reports on research that includes some aspects of sustainability in each of the six stages, such as energy consumption and CO₂ emissions, solvent usage, and the cost associated with biodiesel production.

The first stage of the production process involves selecting the yeast strain and substrate. The yeast strain should accumulate at least 20% of lipids in its dry biomass, and the substrate should be low-cost and readily available, such as lignocellulosic residues, industrial effluents, or food waste. All of these are carbon sources that can be utilized by yeast to produce lipids [73,74,75]. By using these substrates, a circular economy approach is promoted as waste materials are re-valorized in another process, such as lipid production with yeast.

If a lignocellulosic residue is selected, a substep is required, which is a pretreatment consisting of breaking down the outer layer of the residue’s cell wall, composed of lignin, to access the cellulose and hemicellulose. Through a hydrolysis reaction, these polysaccharides are converted into simpler sugars such as glucose and xylose, which can be utilized by the yeasts. The pretreatments can be classified as follows: (a) chemical; (b) biological; (c) oxidative; (d) green solvents; (e) mechanical; and (f) thermal. Chemical pretreatments involve the hydrolysis reaction using concentrated or diluted acids (such as sulfuric acid and nitric acid) or alkaline substances (sodium hydroxide). Concentrated acids are not commonly used due to their corrosion issues in reactors. On the other hand, diluted acids are more commonly employed because they are efficient, cost-effective, do not degrade the produced sugars, and do not cause corrosion problems in reactors. However, diluted acids can generate inhibitory compounds such as furans and phenolic compounds that inhibit microbial growth. Biological pretreatments utilize enzymes derived from white-rot fungi. They have the advantage of being environmentally friendly, efficient, and reusable. However, they are not economically viable. Oxidative pretreatments involve the use of hydrogen peroxide or peracetic acid. They have the advantage of selectively targeting the functional groups in the polymer chains but require high temperatures and produce inhibitory compounds, which makes them unsustainable. The green solvent pretreatment involves the use of mixtures composed of a hydrogen acceptor species and a hydrogen donor species. This pretreatment has the advantage of reducing the formation of inhibitory compounds, eliminating the need for high temperatures, and allowing for multiple reuses in the process. However, the cost of formulating the solvent can be high [76,77,78,79].

Mechanical pretreatments involve applying shear forces to reduce the particle size of lignocellulosic residues using grinding equipment such as blenders.

These pretreatments have the advantage of decreasing the crystallinity of lignocellulosic residues and avoiding the formation of inhibitory compounds. However, they consume a significant amount of energy and are not cost-effective. Thermal pretreatments involve subjecting the residues to temperatures higher than 150 °C, which solubilize the hemicellulose. However, thermal pretreatments are not economically viable and result in the production of inhibitory compounds [76,77,78,79].

The second stage is the cultivation of yeast under appropriate conditions that promote its growth and capacity for lipid production. The selected yeast requires a culture medium that meets its needs in terms of carbon, nitrogen, minerals, growth factors, water, and energy [80]. The composition of the culture medium is so important that if the yeast has an innate lipid-producing capability, but the composition of the medium is inadequate, it will not be able to reach its full potential [80]. During the growth phase, yeast utilizes the carbon source to meet its energy and anabolic needs, while the nitrogen source is used for protein and nucleic acid synthesis, which are essential for its cellular structure [65,81]. This stage aims to generate young and active yeast cells that will serve as inoculum for the next stage. In the work by Milessi [82], it is mentioned that substrates like rice bran function as a low-cost carbon and nitrogen source since there is no need to add additional nutrients to the culture medium. This reduces the cost of the process, contributing to its sustainability.

The third stage is the production of intracellular lipids by the yeast through two metabolic pathways: ex-novo synthesis and de-novo synthesis. The first occurs when lipid production takes place on hydrophobic substrates, and the second occurs when it takes place on hydrophilic substrates [83,84]. In the ex-novo pathway, hydrophobic substrates are degraded into free fatty acids by lipases, which are enzymes secreted extracellularly by yeast. Once they are degraded, they are incorporated into the interior of the cell. Subsequently, the modified fatty acids are converted into acetyl-CoA through β-oxidation to satisfy the energy demands for cell growth, repair, or to contribute to the formation of cellular metabolites [83]. In the de-novo pathway, lipid accumulation depends not only on the initial concentration of the carbon source but also on other components of the medium and the cultivation conditions. Nitrogen restriction is the most efficient condition in this pathway because all the available adenosine monophosphate (AMP) in the mitochondria is converted into inosine monophosphate due to an increase in the activity of the enzyme AMP deaminase [83]. Consequently, the enzyme is unavailable to convert isocitrate to alpha-ketoglutarate, resulting in the accumulation of a significant amount of isocitrate in the mitochondria. Citrate in the cytoplasm is converted to acetyl-CoA and oxaloacetate by ATP-citrate lyase (ACL). Finally, acetyl-CoA and malonyl-CoA together form the C14 to C16 long-chain fatty acids [83]. The main environmental concern at this stage is the demand for water and energy, as well as the CO₂ emissions generated during lipid production.

The fourth stage is the extraction of lipids, which can be achieved through chemical, mechanical, thermal, or biological processes aimed at breaking the microbial biomass cell wall to release them [10]. Chemical methods utilize sulfuric or nitric acids as agents to break the cell wall, releasing the accumulated lipid bodies in the cytoplasm. These lipids are then extracted using highly toxic solvents such as chloroform and methanol. However, less environmentally harmful solvents like n-hexane have also been used [50]. Mechanical methods involve the application of shear forces to break the cell wall [85], such as the use of an abrasive bed. Another mechanical method is ultrasound, which generates microturbulence and induces a cavitation effect to break the cell wall. These methods are fast and effective, minimizing lipid degradation, but they consume high amounts of solvents and energy, making them less sustainable [85].

Thermal methods are those that require high temperatures and pressures to break the cell wall. They have the advantage of being fast and having low solvent consumption, although they consume a lot of energy.

Biological methods act on the cell wall, composed of glucan, mannoproteins, and chitin, through the action of enzymes such as β-1,3 glucomanase, mannanase, protease, and chitinase [85]. These enzymes, through a series of consecutive reactions, solubilize the cell wall, causing its rupture. The advantages of this method are its high selectivity, eco-friendliness, and low solvent requirements. However, it is a challenging process to scale up due to its high cost and energy consumption [85].

Another method used is in situ transesterification, which is a process where cell disruption, lipid extraction, and transesterification occur in a single step. This process is fast and efficient, consuming less energy than ultrasound but more than the bead abrasion bed method. It has the disadvantage of requiring a larger number of solvents compared to biological methods; there can be impurities in the final product; and it is difficult to scale up [85]. In situ transesterification is not the most commonly used method, but it has been performed and evaluated in the studies conducted by Cheirsilp [86], Kuan [7], and Liu [87]. While the work of Khot [85] discusses the environmental advantages and disadvantages, as well as the energy requirements of each extraction technique, evaluations of costs, pollutant emissions percentage, and water consumption for each method have not been considered. Some of these aspects will be analyzed in the development of this study.

The fifth stage is the transesterification reaction, in which the extracted lipids, in the form of triglycerides, react with an excess of alcohol in the presence of an acid, base, enzymatic, or heterogeneous catalyst, resulting in the production of glycerol and fatty acid methyl esters (FAME). Once this reaction is complete, the products are separated and purified to obtain biodiesel. Transesterification is most commonly carried out using sulfuric acid [41,48,51,86,88,89], hydrochloric acid [25,61,72], sodium hydroxide, or potassium hydroxide [26,66] due to their cost-effectiveness. Enzymatic catalysts such as lipases are less frequently used [59,90] because they are more expensive compared to acid and base catalysts. However, lipases do not require high temperatures, can be reused, are efficient, and are environmentally friendly. Heterogeneous catalysts, such as hydrophobic acid and bifunctional acid superparamagnetic catalysts, may not be very economical, but they can be reused up to six times without affecting the conversion significantly, which compensates for their cost [91,92,93].

The sixth stage is the obtaining of biodiesel, which consists of separating the products of the transesterification reaction, the FAME, from the glycerol by adding organic solvents that solubilize the FAME. Once the FAME is separated from the solvent, the latter can be reused. Among the most commonly used solvents are n-hexane [26], toluene [51], or water with isooctane [25]. From an economic point of view, solvent recovery contributes to sustainability because it is reused, which allows for a reduction in the cost of the process since n-hexane and isooctane are highly flammable and highly toxic to aquatic organisms and cause severe skin and inhalation irritations in humans [94,95].

Biodiesel is composed of FAME obtained from the transesterification reaction of lipids with an alcohol (methanol or ethanol). The FAME are found in different proportions depending on the type of substrate and yeast strain used to produce lipids. They are mainly composed of a variable proportion of oleic, palmitic, and stearic acids [55,72]. This proportion influences the quality and performance of biodiesel in internal combustion engines.

The key physical properties of biodiesel are: (1) density; (2) viscosity; (3) flash point; (4) calorific value; (5) pour point; (6) oxidative stability; and (7) cetane number. (1) Density directly affects the performance of engines and is related to viscosity and the cetane number. Biodiesel with high density causes high emissions, especially particle matter (PM) and NOx emissions. (2) Viscosity. A fuel with low viscosity generates more wear and leaks in the engine, and at high viscosities, it causes incomplete combustion, generating more CO. At low temperatures, it causes engine obstruction due to an increase in viscosity. (3) The flash point is the minimum temperature at which enough vapors are released to produce an ignitable mixture. It could be influenced by the chemical composition of biodiesel, such as the total number of carbon atoms and the number of double bonds. (4) The calorific value is the amount of energy produced by combustion per kilogram of fuel. Biodiesel has a lower calorific value than diesel. Higher calorific values are required to improve the performance of fuels in engines because they increase power. (5) The pour point is the minimum temperature at which biodiesel begins to solidify. Biodiesel has a higher pour point than diesel because it has a higher number of fatty acids, which causes obstruction in engines at low temperatures. (6) Oxidative stability measures the ability of biodiesel to be stored for long periods without undergoing oxidation. (7) The cetane number determines the quality of ignition of any fuel. A high cetane number indicates a shorter ignition time, which means the engine starts quickly and runs more smoothly. In biodiesel, the cetane number increases as the amount of saturated fatty acids increases and decreases as the number of unsaturated fatty acids increases [96].

For biodiesel to function properly in internal combustion engines, it is blended with diesel in different proportions. Typically, biodiesel appears in blends ranging from 5 to 20%, while the rest is diesel [13].

The study of the performance of biodiesel blends produced with yeast and diesel in internal combustion engines and the amount of greenhouse gas emissions produced by combustion has been studied by Tamilalagan [97] and Wahlen [98]. They refer to the efficiency of biodiesel in relation to the production of greenhouse gases (GHGs) such as carbon monoxide (CO), carbon dioxide (CO₂), and nitrogen oxides (NOx) generated during the combustion of biodiesel produced by the yeasts Metschnikowia pulcherrima and Cutaneotrichosporon curvatum. They found that CO and CO₂ emissions decreased as the proportion of biodiesel in the blend increased up to 20%, but if this proportion was exceeded, NOx emissions increased. The thermal efficiency of biodiesel was maximized in a 20% blend, which was also reported with fuel consumption, which was lower up to 20% but increased beyond this value [97].

First-generation biodiesel produced with edible oils such as rapeseed [4,9,99,100,101], coconut, and soybean oil [98] reduces particle matter and smoke as the biodiesel percentage in the blend increases. However, CO₂ and NOx emissions tend to increase. Nevertheless, rapeseed oil has the least impact on CO₂ levels, with an increase of 0.418% and NO levels of 6% when a B20 blend is used [4,101].

In second generation biodiesel produced from non-edible vegetable oils [4,5,9,99,102,103,104], it has been found that emission behavior is very similar to that of first-generation and yeast derived biodiesel. The amounts of CO and hydrocarbons (HC) decrease with blends of 20% or less, while CO₂ and NOx emissions increase as this percentage goes up [5,102].

The use of biodiesel reduces greenhouse gas emissions, which is one of the goals set by the UN to counter global warming, in addition to contributing to access to affordable and reliable energy for all and the promotion of sustainable consumption and production patterns.

The objective of this review is to assess the sustainability degree of various methodologies employed in each of the stages of yeast-based biodiesel production through environmental and economic sustainable indicators. The social aspect is not included as it has not been studied in this research.

In the environmental aspect, the use of more environmentally friendly techniques and substances in the various stages of the production process has been considered, such as the pretreatment of lignocellulosic residues [28], lipid extraction [50], and transesterification [7,17,85,86,87], as well as the reuse of some chemical reagents in different stages [105].

Regarding the economic aspect, low-cost substrates and technical-economic analyses of the yeast-based biodiesel production process were considered [73,74,75]. It has been observed that some production processes have been carried out at the pilot level to evaluate the possibility of subsequently scaling them up [15,50,106,107]. This indicates that increasing the production of this biofuel could create employment opportunities.

This review contributes to a detailed description of the different stages of the biodiesel production process using various yeast strains. It establishes the sustainability level of different methods employed in each stage of the process through the evaluation of sustainable indicators. Unlike other reviews, this study achieves an ordinal classification of each method by utilizing a scale based on traffic light colors.

2. Evaluation of Sustainable Indicators in Each Stage

The evaluation of the different methods used in each stage of the biodiesel production process with yeasts is analyzed using environmental and economic sustainable indicators, both qualitative and quantitative, based on Ruiz-Mercado et al. [105].

In the stage of selection of the yeast strain and substrate, only three quantitative indicators were evaluated, such as efficiency, carbon source yield, and cost. Efficiency is assessed as the ratio between the amount of lipids produced (g lipids/L culture medium), or % ((g lipids/g dry biomass) × 100), and the fermentation time (h). A higher value for this indicator indicates a more efficient substrate. Carbon source yield is an indicator obtained as the quotient of the amount of lipids produced (g lipids/L culture medium), or % ((g lipids/g dry biomass) × 100), and the amount of carbon source used expressed in (g/L); a higher quotient indicates that the carbon source is better for lipid production. Cost is evaluated as the amount of lipids produced (g/L, or %) divided by the cost of the carbon source expressed in Mexican pesos (MXN); a higher ratio means that for every peso invested in the carbon source, more lipids are produced at a lower cost. Subsequently, the three calculated indicators were averaged, and an overall sustainable score was obtained to rank them nominally using a sustainable traffic light scale. The green color was classified as the most sustainable (above average), yellow as moderately sustainable (within the average range), and red as the least sustainable (below average).

In the substage of pretreatments for lignocellulosic residues, both qualitative and quantitative indicators were evaluated. Qualitative indicators included environmental and economic advantages and disadvantages reported in the literature. An integer value was obtained as the result of the difference between advantages and disadvantages, and these were ranked according to the sustainable traffic light system. Pretreatments with a value greater than zero were classified as green; those with values equal to zero were classified as yellow; and scores below zero were classified as red. Additionally, a quantitative indicator, efficiency, was evaluated. Efficiency was calculated as the amount of reducing sugars expressed in g reducing sugars/L of mixture, or % [(g reducing sugars/g substrate) × 100], relative to the pretreatment time expressed in hours (h). This indicator indicates that the most efficient pretreatment is the one that produces a higher amount of reduced sugar in less time. Again, they were ranked from most to least sustainable according to the traffic light scale.

In the sections on substrate selection criteria, only qualitative indicators were evaluated. This included substrate availability, accessibility, composition, and the amount of lipid produced. For yeast selection criteria, optimal growth conditions reported in the literature, such as pH, temperature, pressure, agitation speed, and oxygenation level, were considered.

In the stage of cultivation of the selected yeast strain under optimal conditions, only qualitative indicators were evaluated, such as the composition of the culture medium and optimal growth values such as pH, temperature, and agitation speed.

In the lipid production stage, qualitative indicators were evaluated, including the ratio (C/N), temperature, pH, agitation speed, inoculum size, CO₂ emissions, and wastewater discharges reported. Additionally, only the efficiency indicator was assessed, expressed as the amount of lipids (g/L or%) divided by the fermentation time (h). An overall average was obtained with this indicator, and subsequently, they were ranked according to the sustainable traffic light scale.

In the lipid extraction stage, both qualitative and quantitative indicators were evaluated. Qualitative indicators included environmental and economic advantages and disadvantages reported in the literature. An integer value was obtained as the result of the difference between advantages and disadvantages, and these were ranked according to the sustainable traffic light system. Extraction techniques with a value greater than zero were classified as green; those with values equal to zero were classified as yellow; and scores below zero were classified as red. Quantitative indicators of efficiency were also assessed, expressed as the amount of lipids obtained (g/L or %) and the extraction time (h), and cost calculated as the amount of lipids obtained (g/L or%) divided by the cost of the extraction process in Mexican pesos (MXN). Then, the average of each indicator was calculated, and they were assigned a color based on their sustainability score. Finally, the average of the three indicators was calculated to obtain an overall sustainability score, by which they were ranked according to their level of sustainability.

In the transesterification reaction stage, qualitative and quantitative indicators were evaluated. Qualitative indicators included reaction temperature, catalyst concentration, methanol ratio, and moisture content present in the lipid. Only one quantitative indicator was assessed, which is the bioconversion of lipids to FAME (%). In the refinement process stage of FAME to obtain biodiesel, the environmental and economic impacts of purification techniques are mentioned, and emissions of CO, CO₂, HC, and NOx are also evaluated in the performance of second-generation biodiesel in engines.

In

Table 1. Evaluation of indicators in each stage.

Section or Stage	Function	Efficiency	Carbon Source Yield	Cost	Qualitative Indicators	Global Sustainable Score
Selection of the yeast strain and substrate	Provide a carbon source	$\frac{\mathrm{A}\mathrm{m}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t} \mathrm{o}\mathrm{f} \mathrm{l}\mathrm{i}\mathrm{p}\mathrm{i}\mathrm{d}\mathrm{s} \frac{\mathrm{g}}{\mathrm{L}} \mathsf{ó}\%}{\mathrm{f}\mathrm{e}\mathrm{r}\mathrm{m}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{t}\mathrm{i}\mathrm{m}\mathrm{e}\left(\mathrm{h}\right)}$	$\frac{\mathrm{A}\mathrm{m}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t} \mathrm{o}\mathrm{f} \mathrm{l}\mathrm{i}\mathrm{p}\mathrm{i}\mathrm{d}\mathrm{s} \frac{\mathrm{g}}{\mathrm{L}} \mathsf{ó}\%}{\mathrm{A}\mathrm{m}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t} \mathrm{o}\mathrm{f} \mathrm{c}\mathrm{a}\mathrm{r}\mathrm{b}\mathrm{o}\mathrm{n} \mathrm{s}\mathrm{o}\mathrm{u}\mathrm{r}\mathrm{c}\mathrm{e} (\mathrm{g}/\mathrm{L})}$	$\frac{\mathrm{A}\mathrm{m}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t} \mathrm{o}\mathrm{f} \mathrm{l}\mathrm{i}\mathrm{p}\mathrm{i}\mathrm{d}\mathrm{s} \frac{\mathrm{g}}{\mathrm{L}} \mathsf{ó}\%}{\mathrm{C}\mathrm{o}\mathrm{s}\mathrm{t} \mathrm{o}\mathrm{f} \mathrm{c}\mathrm{a}\mathrm{r}\mathrm{b}\mathrm{o}\mathrm{n} \mathrm{s}\mathrm{o}\mathrm{u}\mathrm{r}\mathrm{c}\mathrm{e} \left(\mathrm{M}\mathrm{X}\mathrm{N}\right)}$	No	Average of the indicators evaluated previously
Lignocellulosic residues pretreatments	Generate reducing sugars that serve as a carbon source	$\frac{\mathrm{A}\mathrm{m}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t} \mathrm{o}\mathrm{f} \mathrm{r}\mathrm{e}\mathrm{d}\mathrm{u}\mathrm{c}\mathrm{i}\mathrm{n}\mathrm{g} \mathrm{s}\mathrm{u}\mathrm{g}\mathrm{a}\mathrm{r}\mathrm{s} \frac{\mathrm{g}}{\mathrm{L}} \mathsf{ó}\%}{\mathrm{p}\mathrm{r}\mathrm{e}\mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{m}\mathrm{e}\mathrm{n}\mathrm{t} \mathrm{t}\mathrm{i}\mathrm{m}\mathrm{e} \left(\mathrm{h}\right)}$	No	No	-Environmental impact -Economic impact -Efficiency	Advantages - disadvantages
Selection criteria for substrate	Produce biodiesel	No	No	Qualitative (nominal)	-Availability of the substrate -Level of production of substrate -Accessibility -Composition -Production of high amount of lipids	No
Selection criteria for yeast strain	Produce biodiesel	No	No	No	-Based on the substrate -optimal growth conditions (pH, temperature, pressure, agitation speed and oxygenation level
Cultivation of the selected yeast strain under optimal conditions	Prepare the inoculum	No	No	No	-composition of the culture medium -optimal values of operating conditions (pH, temperature and stirring speed).	No
Lipid production	Obtain lipids	$\frac{\mathrm{A}\mathrm{m}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t} \mathrm{o}\mathrm{f} \mathrm{l}\mathrm{i}\mathrm{p}\mathrm{i}\mathrm{d}\mathrm{s} \frac{\mathrm{g}}{\mathrm{L}} \mathsf{ó}\%}{\mathrm{f}\mathrm{e}\mathrm{r}\mathrm{m}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{t}\mathrm{i}\mathrm{m}\mathrm{e} \left(\mathrm{h}\right)}$	No	No	-carbon/nitrogen (C/N) ratio -temperature -pH -agitation speed -inoculum size -environmental impact (CO2 emissions and wastewater produced)	No
Extraction of lipids	Breaking the cell wall of the yeast to release the intracellular lipids	$\frac{\mathrm{A}\mathrm{m}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t} \mathrm{o}\mathrm{f} \mathrm{l}\mathrm{i}\mathrm{p}\mathrm{i}\mathrm{d}\mathrm{s} \frac{\mathrm{g}}{\mathrm{L}} \mathsf{ó}\%}{\mathrm{e}\mathrm{x}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{t}\mathrm{i}\mathrm{m}\mathrm{e} \left(\mathrm{h}\right)}$	No	$\frac{\mathrm{A}\mathrm{m}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t} \mathrm{o}\mathrm{f} \mathrm{l}\mathrm{i}\mathrm{p}\mathrm{i}\mathrm{d}\mathrm{s} \frac{\mathrm{g}}{\mathrm{L}}}{\mathrm{l}\mathrm{i}\mathrm{p}\mathrm{i}\mathrm{d} \mathrm{e}\mathrm{x}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{c}\mathrm{o}\mathrm{s}\mathrm{t} \left(\mathrm{M}\mathrm{X}\mathrm{N}\right)}$	-environmental impact -economic impact -efficiency	Average of efficiency and cost indicators
Transesterification reaction	Convert lipids to biodiesel	Lipid bioconversion to FAME (%)	No	No	-temperature of reaction -catalyst concentration -methanol ratio used -moisture content present in the lipid	No

, a summary of the indicators evaluated at each stage of the process is shown.

3. Biodiesel Production Process from Yeast Lipids

The biodiesel production process using yeast consists of six stages, with a substage if a lignocellulosic residue is pretreated. This additional stage involves a pretreatment process that breaks down the lignin structure of the plant wall to access compounds such as cellulose and hemicellulose. These compounds are then depolymerized through a hydrolysis reaction to generate five and six carbon sugars, such as xylose and glucose, that can be directly used by certain yeast species.

Figure 3 outlines the six stages for producing biodiesel with yeast, which are detailed in the following sections.

3.1. Selection of the Yeast Strain and Substrate

The most commonly used substrates by oleaginous yeasts for lipid production are sugars such as glucose, fructose, cellobiose, xylose, and mannose; pure and crude glycerol, the latter coming from biodiesel production; soaps and alcoholic beverages [41,108]; industrial effluents rich in starch, glucose, proteins, and fatty acids [28], which are byproducts of the oil, fishing, confectionary, and food industries [34,35,109,110,111], and lignocellulosic residues composed of lignin, cellulose, and hemicellulose [112] from agricultural and forestry industries. It should be noted that some substrates can be used as a sole carbon source, but they can also be combined to optimize biomass and lipid production. The following sections will detail the most commonly used substrates:

3.1.1. Sugars

Sugars are divided into monosaccharides, disaccharides, trisaccharides, and polysaccharides. Sugars, mainly monosaccharides and disaccharides, can be utilized by most yeasts to produce lipids in quantities greater than 50% [25,27], but they are not economical due to being refined.

Some yeasts have a preference for certain sugars over others; for example, L. starkeyi, in the presence of cellobiose, xylose, and glucose, prefers xylose and produces 57% lipids, an amount greater than that produced when growing on glucose or cellobiose [26]. This same percentage of lipids was obtained with R. toruloides growing on glucose as a carbon source and managing a limited amount of sodium sulfate as a sulfur source to induce lipogenesis [58]. However, Amaretti [24] produced 68% of lipids using glucose, the highest amount of lipids reported using this source of carbon until now.

It is estimated that the average cost per gram of glucose is 4.11 MXN [113], xylose is USD 12.68 MXN [114], and cellobiose is 75.07 MXN [115]. Therefore, glucose is a cheaper source of sugar.

Yeasts such as R. toruloides, R. glutinis, L. starkeyi, P. glacialis, and Y. lipolytica [24,25,26,27,51] can use sugars such as glucose, xylose, cellobiose, and fructose as a carbon source for their growth; however, they cannot use some polysaccharides directly, such as cellulose and hemicellulose, because their structure is more complex and they are not fermentable sugars, like monosaccharides. For yeasts to be able to take advantage of polysaccharides, they must undergo a hydrolysis reaction to break them down into simpler sugars, which increases the cost.

Glucose is more commonly used because it can be utilized by most yeast strains, it is more cost-effective than other sugars, and it is produced in large quantities.

In the United States, it is reported that the production level of glucose syrup alone in 2019 was 5.2 million short tons [116], and because it has a majority percentage of glucose, it is widely used in industrial fermentations. It has been estimated that the production price of biodiesel would be USD 5.9/kg, considering that the cost of glucose would be $400/t [74].

3.1.2. Pure and Crude Glycerol Combined with Other Carbon Sources

In addition to sugars, other substances have also been used as pure and crude glycerol, the latter from the production of soap, stearin, alcoholic beverages, and biodiesel industries [41], which are widely used as a carbon source for the production of lipids. The yeasts Candida freyschussii [108], C. curvatum [48,50], R. toruloides [19,59], and Y. lipolytica [41,44] can be used as a single carbon source or in combination with others. Crude glycerol is generated in large quantities; it is estimated that for each ton of biodiesel produced, 100 kg of glycerol are generated [41]. Due to this, it is essential to give it a second use, obtaining a viable alternative for the production of biodiesel and also contributing to a better circular economy.

It has been observed that crude glycerol from biodiesel production yields a higher amount of lipids, exceeding 70%, compared to pure glycerol, which produces a lesser quantity [59]. The yeasts R. toruloides, Y. lipolytica, and C. curvatum are the most commonly used with both substrates, and they produce lipids exceeding 30% [44,48,50,59].

Crude glycerol is an excellent carbon source for lipid production, primarily because it is cost-effective. Unlike lignocellulosic residues, it does not require additional pretreatment or purification processes, which reduces costs. Additionally, it utilizes a byproduct that is generated in large quantities and cannot be used in the pharmaceutical and cosmetic industries due to not meeting minimum quality requirements. Economically, the purification process for crude glycerol is not feasible. Its valorization contributes to a circular economy, and there is a promising potential for scaling up production.

3.1.3. Volatile Fatty Acids (VFAs)

Short-chain fatty acids (C1–C6) are produced through anaerobic fermentation of various organic residues. These can originate from activated sludge in wastewater treatment [65], effluents from food, fruit, and vegetable waste [31,32,42,43], fermentation of brown macroalgae [33], and effluents derived from hydrogen production [30].

The most commonly used yeasts with VFAs are C. curvatum and Y. lipolytica, which produce an amount of lipids between 30 and 75% [21,30,31,33,42,49].

One disadvantage of these carbon sources is that high concentrations of VFAs (volatile fatty acids) such as acetic, propionic, and butyric acids have an inhibitory effect on microbial growth. In most yeasts, it is observed that they are more resistant to acetic acid than to propionic and butyric acids. For instance, it has been determined that C. curvatum can tolerate acetic acid concentrations of no more than 7 g/L, while Y. lipolytica can tolerate up to a concentration of 5 g/L [42,49]. However, Y. lipolytica can endure concentrations of 50 g/L of a mixture of acetic, propionic, and butyric acids in proportions of (5:2:3), respectively, resulting in a slight increase in lipid production compared to when only acetic acid is used as the sole carbon source [33].

VFAs present a promising alternative for lipid production, especially if derived from food waste, as their cost is 30 dollars per ton, which is less than 10% of the cost of a ton of glucose [31]. It is feasible to use this carbon source for yeasts if the inhibitory effect of these acids is controlled through strategies such as cultivating under alkaline conditions [31].

3.1.4. Industrial Effluents

Industrial effluents pose an environmental problem due to the emission of greenhouse gases and other substances that severely contaminate water bodies and soils. Therefore, it is important to give them a prior treatment before disposing of them or reusing them as raw materials to generate value-added products such as microbial lipids. These waste materials are an excellent source of carbon for yeast due to their high content of organic compounds.

The sugar industry generates molasses [37] and sugarcane juice [117] as by-products, which are carbon sources rich in glucose and xylose primarily. The distillery industry disposes of vinasse as effluents, which are rich in sugars, acids, and alcohols [118]. Effluents from the biodiesel industry, latex production, palm oil [86], olive oil [38], as well as the baking and confectionary industries [40,86], and wastewater from butanol fermentation are also utilized [119].

The yeasts Y. lipolytica, L. starkeyi, T. dermatis, R. glutinis, R. mucilaginosa, R. toruloides, C. tropicalis, and T. dermatis can grow and produce lipids in these effluents. C. tropicalis produced 78.7% of lipids using olive mill wastewater [38], while R. mucilaginosa, L. starkeyi, and R. toruloides produced 69.5%, 57.8%, and 50.87% of lipids while using molasses, flour-rich waste streams generated by bakery, confectionary, wheat milling, and sugar cane juice, respectively [37,40,117].

These effluents represent a viable option to be used by yeast for producing microbial lipids. One advantage they have is that they do not require pretreatment like lignocellulosic waste. They are also more cost-effective compared to using sugars such as glucose. However, it is important to consider that if these effluents are not readily available in the geographical area where lipid production is desired, the transportation cost would increase the overall process cost. Consequently, it may not be competitive with a reasonable price in the market.

3.1.5. Food Loss and Waste (FLW)

Within this category, we can find food waste that is not consumed or that is the result of the production process of food, for example, cheese whey [66,109,120], stewed rice residue [34], food waste slurry [110], and also waste cooking oil [14], which should not be used again due to the reactions of oxidation, hydrolysis, and polymerization that form toxic compounds that are very harmful to health and that are produced during the frying of oils.

These substrates can be utilized by yeast due to their rich composition of short-chain acids (acetic, propionic, and caproic), long-chain organic acids (palmitic, stearic, oleic, and linoleic), proteins, vitamins, minerals, and sugars (lactose and starch). For example, cheese whey is composed of lactose (60–80%), proteins (10–20%), and the remaining portion consists of minerals, vitamins, fats, organic acids (lactic acid), and trace elements [120]. Stewed rice residue is also rich in fats and proteins; food waste slurry (food waste) is composed of short-chain organic acids; and waste cooking oil consists of triglycerides (TAGs), which are long-chain organic acids.

The yeasts that have been used with these types of substrates include Y. lipolytica, C. curvatum, S. pararoseus, R. toruloides, and C. oleaginosum, producing lipid quantities exceeding 45%. C. oleaginosum and C. curvatum yielded 68% and 65% of lipids, respectively, when utilizing cheese whey permeate and cheese whey as substrates [66,120]. So far, these substrates have led to the highest lipid production.

Waste cooking oil has also been utilized as a substrate with Y. lipolytica, yielding 53% lipids and generating lipases [14]. These high-value enzymes can be employed in the transesterification process for biodiesel production. This approach contributes to sustainability by reducing soil and water effluent pollution using waste cooking oil. Moreover, it aligns with circular economy principles, as it adds value to waste by utilizing it to produce a high-value product like lipases.

The global annual food waste amounts to 931 million tons, with 569 million tons originating from households, 244 million tons from restaurants and food services, and 118 million tons generated in retail [121]. Disposing of this waste in landfills produces significant amounts of methane gas, which is 25 times more potent than CO₂ [122]. Given these factors, the utilization and revalorization of food waste to produce other products prove beneficial both environmentally and economically.

A comparative analysis of the utilization of sugars (glucose, cellobiose, and xylose), pure and crude glycerol, volatile fatty acids (VFAs), industrial effluents, and food loss and waste are conducted. The analysis evaluates these substrates using a selection of sustainable indicators. Efficiency is considered, which relates the amount of produced lipids and fermentation time (g lipids/L×h), as well as the amount of produced lipids and the amount of carbon source used [(g lipids/L)/carbon source (g/L)]. Additionally, the cost indicator is accounted for, which relates the amount of lipids to the cost of the carbon source used (g lipids/L×MXN); for this evaluation, it was assumed that the cost of food waste, such as waste cooking oil, cheese whey, crude glycerol, and other carbon sources from industrial effluents, is negligible. From each of these three indicators, an average score was obtained. For the efficiency indicators, the average scores were 0.17 and 0.44, and for the cost indicator, it was 0.76. Once the three aforementioned indicators were calculated, an average of these three indicators was obtained, resulting in a global sustainable score with an overall average of 0.37. Substrates scoring above the global sustainable average were classified in green (a more sustainable score), those within the average range were classified in yellow (a medium sustainable score), and those scoring below this average were classified in red (a less sustainable score).

Figure 4 displays the ordinal classification of the carbon source mentioned in this section. The numbers within each of the traffic light circles correspond to the highest global sustainable score (green), the average global sustainable score (yellow), and finally, the lowest global sustainable score (red).

The carbon sources with the highest scores are waste cooking oil [14] with 3.20, followed by glucose [24] with 1.72, and crude glycerol [108] with 1.02. Carbon sources such as food waste hydrolysate [123], molasses [37], and ricotta cheese whey [109] scored below 1, mainly because they produce a lower amount of lipids per unit of time and per amount of carbon source utilized. This impacted their global sustainable score, which was up to five times lower than that of waste cooking oil. The latter represents a very low-cost carbon source, produces a significant amount of lipids per unit of time, and is a potential alternative for use as a carbon source to produce lipids with yeast.

3.1.6. Lignocellulosic Residues

Lignocellulosic residues are any natural resource composed mainly of cellulose, hemicellulose, and lignin, and their proportion varies depending on the plant species from which they come [88]. For the production of microbial biodiesel, lignocellulosic residues or byproducts of agricultural or forestry activities are used.

Table 2. Composition of different lignocellulosic residues.

Component	Sugar Cane Bagasse	Wheat Straw	Corn Straw	Rice Straw	Barley Straw	Oat Straw	Jerusalem Artichoke	Leaves	Nutshell	Perennial Grass
Cellulose	33	38–48	36–41	33	43.3	41	20.95–25.99	15–20	25–30	31–37
hemicellulose	30	23–29	26–30	26	29.6	16	4.5–5.4	80–85	25–30	20.4–29
Lignin	29	13–19	16–21	7	7.7	11	5–5.7	0	30–40	17.6–19

Source: [105,124].

shows the composition of various residues such as sugar cane bagasse, wheat straw, corn straw, rice straw, barley straw, oat straw, Jerusalem artichoke, leaves, nutshell, and perennial grass. These residues have a cellulose content between 15–43%, hemicellulose 5–85%, and lignin 0–40% [105,124].

For yeast to use these substrates as a carbon source, it is necessary to break down the different chemical bonds that make up the plant cell wall, which is composed of lignin, cellulose, and hemicellulose. Cellulose and hemicellulose, due to their polymeric structure, must be converted into simpler sugars (glucose, xylose, mannose, galactose, and arabinose) through hydrolysis reactions so that they can be directly utilized by yeast [107].

Lignocellulosic residues are mainly composed of glucose and xylose in a 2:1 ratio [125]. Because glucose is present in greater quantities in these residues, it presents an advantage as it can be assimilated by a wide variety of yeasts. Lignocellulosic residues, such as cereal straws like wheat, corn, barley, oats, and sorghum, are considered excellent raw materials for producing biodiesel, biogas, bioethanol, and oleochemicals that have applications in the food, pharmaceutical, and biotechnology industries [126].

Most lignocellulosic residues require pretreatments that are classified as (a) mechanical, (b) thermal, (c) chemical, (d) biological, (e) oxidative, and (f) green solvents. Patel [76] analyzed the main advantages and disadvantages of these pretreatments, highlighting the environmental, economic, and energy impacts.

Table 3. Main pretreatments of lignocellulosic residues.

Pretreatments	Description	Environmental Impact	Economic Impact	Efficiency	Sustainable Score Advantages - Disadvantages	References
Chemical	Treatment with dilute or concentrated acids or alkalis at room temperature.	-No ecological at 100%	-Economical -Large-scale use	-Improve hemicellulose solubility -Salt formation, which affects the composition of hydrolysates, -Very efficient	2	[76]
Biological	Treatment with white rot fungus enzymes	-Ecological	-No economical -Reusable	-Fast and efficient	2	[77] [76]
Green solvents	Treatment with mixtures composed of a hydrogen acceptor species and a hydrogen donor species, such as choline chloride with glycerol	-Process at room temperature	-It can be reused many times in the process -The solvent formulation can be more expensive	-Reduction of inhibitors formation	2	[79]
Mechanical	Application of shear force to decrease particle size in biomass	-High energy consumption	-No economical	-Decrease crystallinity -No inhibitory compounds	0	[76]
Oxidative	Biomass treatment with oxidizing agents such as H2O2 and peracetic acid.	-Require high temperatures	-No economical	-Very selective action on polymeric chain functional groups -It can produce inhibitory compounds that affect microbial growth.	−1	[78] [76]
Thermal	Biomass heating at T > 150 °C that solubilizes hemicellulose	-Require high temperatures	-No economical	-It produces inhibitory compounds that affect other phases of the process	-2	[76]

presents a comparison of different pretreatments, evaluating the advantages and disadvantages of each one and their impact on efficiency, environmental aspects, and economics. Subsequently, a sustainable score was obtained by subtracting the number of advantages and disadvantages. Values above a score of zero were classified as green; values with a score equal to zero were classified as yellow; and scores below the average were classified as red. They were then ranked from most sustainable to least sustainable based on the sustainable traffic light colors.

Table 3 reveals that the most sustainable pretreatments, considering their environmental, economic, and efficiency impacts, are chemical pretreatments, biological pretreatments, and green solvents. The moderately sustainable options are mechanical pretreatments, while the least sustainable options are oxidative and thermal pretreatments. The latter require high temperatures, are not cost-effective, and produce inhibitory compounds for microbial growth.

In Figure 5, it can be observed that these pretreatments, by acting on lignocellulosic waste and depolymerizing lignin, cellulose, and hemicellulose, generate monosaccharides as well as inhibitory compounds such as levulinic acid, formic acid, acetic acid, and phenols.

Chemical pretreatments are the most common, in which a hydrolysis reaction is carried out with concentrated or diluted acids (sulfuric acid, nitric acid) or with alkaline substances (sodium hydroxide).

Pretreatments with concentrated acids are not generally used today due to their toxicity and the fact that they generate severe corrosion problems in reactors [124]. Dilute acid pretreatment is used more frequently. It depolymerizes hemicellulose into fermentable sugars, leaving cellulignin in the substrate [82]. It has been used in food waste [127], wheat and rice bran and stubble [17,88], cob corn [112], grass, and plant residues [55,88]. This pretreatment has the advantage that it is not expensive; it is very effective because it does not degrade sugars, lignin, or hemicellulose and does not generate corrosion problems in the reactors [124]. However, it produces compounds that inhibit microbial growth, such as furan and phenolic compounds, in a range of 2–3 g/kg substrate, while organic acids are produced in a range of 2–8 g/kg substrate. However, phenolic compounds are those that are produced in smaller amounts and those that are less toxic to yeasts [105].

Sulfuric acid does not exhibit carcinogenic properties, but at concentrations greater than 15%, it is harmful to the skin, and at concentrations of 100 mg/L, it is toxic to algae and invertebrates. However, it is not bioaccumulative [128], which does not represent a threat to the food chain, nor does it harm aquatic fauna when used in concentrations ranging from 0.5 to 5% v/v.

Solarte-Toro [129] made a technical-economic evaluation of the effect of pretreatment of olive tree biomass with sulfuric acid using two scenarios, the first with a substrate load of 15% w/w with a concentration of acid at 5.9% w/w and the second scenario with a load of 35% w/w with an acid concentration of 4.9% w/w. They concluded that when the substrate load is doubled, CO₂ emissions are reduced almost threefold, and water and energy consumption are more than doubled, so if you want to reduce the environmental impact with this pretreatment, it is essential to use a load-high substrate.

It is concluded that for the other pretreatments, there is no study where CO₂ emissions, water and energy consumption, or the generation of wastewater are evaluated, so they cannot be compared from this point of view. The possibility of studying these processes from this perspective is left open to create a series of criteria that allow for establishing more sustainable operating conditions.

Alkaline pretreatment has also been studied, which is a process that breaks the lignin structure and solubilizes hemicellulose [130] using alkalis such as calcium hydroxide and sodium hydroxide. Compared to acid pretreatment, it has the advantage that it degrades fewer sugars [130], but it also forms inhibitory substances such as furans, phenolic compounds, and weak acids. These inhibitory compounds have to be removed through treatments using chemical substances such as calcium oxide [45] or through the use of activated carbon [17].

The calcium oxide treatment decreases the amount of sugars produced in the pretreatment by up to 25%, while the activated carbon treatment only decreases the total sugar content by 4.82%, so it can be established that the activated carbon is much more effective in removing inhibitory compounds derived from lignin and acetic acid [131]; it is economical and environmentally friendly compared to calcium oxide. In addition to reducing the content of sugars produced in the pretreatment, the detoxification process contributes to raising the cost of the entire pretreatment. For that reason, the effect of these inhibitory substances on the growth of yeasts and the production of lipids is to decrease process steps and reduce costs.

Ayadi [16] optimized the process of obtaining lipids by using R. mucilaginosa with wheat bran hydrolysate without carrying out the detoxification process, concluding that R. mucilaginosa has good resistance against growth-inhibiting substances, managing to degrade glucose and xylose simultaneously, a characteristic that makes it suitable for lignocellulosic substrates. This same behavior was found with C. curvatum when using wheat bran hydrolysates with and without detoxification; it was determined that neither the growth of the yeast nor the production of lipids were affected by the presence of inhibitors; even the best results were obtained in comparison with R. glutinis, L. starkeyi, Y. lipolytica, and R. toruloides [45]. However, when R. glutinis uses glucose and xylose from switchgrass (Panicum virgatum) in the presence of inhibitors such as acetic acid and furfural, it can grow using only xylose as a carbon source [29], which makes it a versatile yeast as it can take advantage of a sugar that is not as useful for other species.

Chemical pretreatments have several advantages. They are cost-effective and efficient, and their environmental impact can be reduced by using high loads of lignocellulosic residues, which decreases CO₂ emissions. Biological pretreatment involves the use of enzymes from white-rot fungi, such as cellulases obtained from the fungus Trichoderma reesei, cultivated on low-cost substrates like wheat bran and rice straw. The produced cellulases were utilized on a corn stover at a temperature of 50 °C for three days with a substrate loading of 10% (w/v), achieving a reported maximum efficiency of 60.1 g of sugars/L. The cost was reduced because there was no need to add nitrogen sources or trace elements to the fermentation medium, nor was a detoxification process required [64].

The advantage of this pretreatment is that it is environmentally friendly, it does not consume a large amount of energy, and it reduces the cost of the process by using fewer reagents and producing the enzyme directly from the fungus; however, reaction times are prolonged [124].

The pretreatment with green solvents consists of using blends composed of a hydrogen acceptor species and a hydrogen donor species. The eutectic solvent (choline chloride: glycerol) has been used with switchgrass (Panicum virgatum), obtaining an efficiency of 89% glucose with respect to the amount of lignocellulosic biomass, the highest reported in all pretreatments. Its disadvantage is that it is expensive; however, it can reduce its cost if it is reused up to four times in the process, which reduces the discharge of residual water [79]. This pretreatment must be explored in depth because it represents a sustainable alternative, yet the appropriate combination of solvents must be chosen for each type of substrate.

Mechanical pretreatment involves applying shear force to reduce the particle size of lignocellulosic residues. This pretreatment is not cost-effective due to its high energy consumption and time requirements. Nevertheless, it has been used in food waste, resulting in a 98% yield of glucose as a reducing sugar within 35 h [61].

The oxidative pretreatment consists of using oxidizing agents such as hydrogen peroxide. Annamalai [78] used this pretreatment together with enzymatic hydrolysis to treat office paper, obtaining an efficiency of 24.49 g sugars (glucose and xylose)/L. The advantage of this pretreatment is that it removes lignin without damaging the cellulose composition and can be scaled [105]. However, subsequent hydrolysis is required to improve the efficiency of the process, which increases its cost.

Thermal pretreatment involves solubilizing hemicellulose by heating the biomass at temperatures above 150 °C. This pretreatment has been conducted on wheat straw at 190 °C, resulting in a yield of 44.48 reducing sugars within 73 h [62]. Although this pretreatment is efficient, it is neither cost-effective nor environmentally friendly due to its high energy consumption and lengthy process.

One way to optimize the pretreatment of lignocellulosic substrates and fermentation is through the process of simultaneous saccharification and fermentation, which represents a great advantage by converting lignocellulosic materials to lipids in a single step. Gong [132] carried out this process with C. curvatum and corn stover, reaching a lipid production greater than 50%, even without sterilizing the culture medium, which allowed reducing the cost and time in the process. However, there are certain disadvantages that can occur during this process, such as the presence of inhibitors, a very low sugar concentration, and the ability of the yeast to use all the sugars generated during the saccharification process [133].

Lignocellulosic residues are a source of sugars from renewable resources, but they require pretreatment and a process to remove inhibitory substances through the use of alkalis or activated carbon, which ultimately increases the cost of the process [134].

Figure 6 classifies the main pretreatments used based on the assessment of the efficiency indicator, which relates the amount of reducing sugars (g/L ó %) to the duration of the pretreatment (h). The numbers within each of the traffic light circles represent the highest sustainable score (green), the average score (yellow), and the lowest score (red). Toxicity indicators were not considered because the quantity of reagents used in each of the pretreatments was not mentioned.

The most commonly used pretreatments are chemical, particularly 2% v/v sulfuric acid [17,45], 0.5% v/v nitric acid [52], and 1% v/v sodium hydroxide [63,135], followed by mechanical [61], green solvents (choline chloride and glycerol) [79], oxidative [78], and biological [62,64].

The pretreatment with sulfuric acid (2% v/v) is the most efficient, producing up to 29.2 g/L*t of reducing sugars [45], compared to mechanical, thermal, oxidative, biological, and eutectic solvent pre-treatments, which can be up to 48 times less efficient than the acid pre-treatment due to employing very long time periods. The advantage of eutectic solvents is that they are more environmentally friendly, although not economical initially. However, they can be reused up to four times in the process [79], ultimately offsetting their cost.

The search for more sustainable pretreatments from an environmental point of view is a trend in research in this field. However, it is also important to consider the environmental and economic impacts. The main challenge lies in finding a balance between these two aspects: environmental and economic. Therefore, it is essential to continue researching pretreatments that are environmentally friendly and, at the same time, economically viable.

Next, a comparison is made among different lignocellulosic residues through the evaluation of efficiency, which relates the amount of produced lipids to the time employed to produce them. Figure 7 depicts the classification of various lignocellulosic residues based on the calculation of their efficiency. The numbers within each of the traffic light circles represent the highest sustainable score (green), the average score (yellow), and the lowest score (red).

The economic indicator was not evaluated since, being lignocellulosic residues, their cost is very close to zero.

The strains most utilized with lignocellulosic residues are R. toruloides [61,62,136,137] and R. mucilaginosa [55]. Among these, R. toruloides, in combination with sugars derived from lignocellulosic biomass and hydrolysates from Jerusalem artichoke and food waste, achieved a sustainable score of up to 1.183 [61,136,137]. This underscores its exceptional capability to thrive on these substrates and produce the greatest amount of lipid in the shortest possible time. The substrates that exhibited the lowest lipid production per unit of time were the hydrolysates of corn stover, corn cob, rice bran, harmful algal, grass, and wheat straw [17,18,52,55,62,63,135].

There is a wide range of substrates, such as sugars, pure and crude glycerol, lignocellulosic residues, industrial effluents, and food waste, that yeast species such as Rhodotorula sp., C. curvatum, C. dermatis, and Y. lipolytica can utilize to produce high amounts of lipids. This versatility of yeast species allows them to adapt to various carbon sources, making them adaptable and efficient in lipid production.

Choosing substrates that require minimal pretreatment is more sustainable. Substrates like waste cooking oil, glucose, and crude glycerol from biodiesel production stand out in this regard. They yield a substantial amount of lipids at a low cost and in less time compared to other substrates. Moreover, repurposing these substrates for microbial lipid production helps reduce the environmental impact associated with their disposal.

3.2. Selection Criteria for Substrate and Yeast Strain

The criteria to select the substrates depend mainly on the cost, because this represents between 70 and 95% of the total cost of biodiesel production, which makes it a key factor in the selection of the substrate, as mentioned by Pinzi [138]. Bhuiya [139], in addition to cost, mentioned other factors to be considered: the availability of the substrate, the production in sufficient quantities, and accessibility, so its supply can be assured in a reliable, continuous way and therefore reduce transportation costs. Nevertheless, they can also be selected for their composition and production of high amounts of lipids.

Some authors choose lignocellulosic residues such as corn cob, wheat or rice bran, fruit peels (orange and banana), and starch-rich substrates like potato peels due to their low cost [16,39,60,64,112]. The same criteria have been applied when selecting industrial effluents and processed or prepared food waste [31,32,34,40,42,109].

For example, cheese whey is selected because it is produced in large quantities in the food industry and its disposal is cost-prohibitive due to its high organic load, which can be used as a carbon source by yeasts to produce biodiesel; therefore, revaluing this waste reduces the environmental and economic impact [109].

Sugars such as glucose, xylose, and cellobiose are used because they produce an amount of lipids greater than 50% and not because of their low cost [26,27,58].

The criteria for choosing the yeast strain must be based on the substrate to be used and the optimal growth conditions for each one, such as pH, temperature, pressure, agitation speed, and oxygenation level—the latter two conditions if they are aerophilic yeasts.

Yeast strains used with lignocellulosic residues such as Rhodotorula sp. and Cutaneotrichosporon sp. have been isolated from natural environments such as soil, plant leaves (Ficus benjamina leaves), flowers (sunflower (Helianthus annus)), fruits (olive fruit), rotten fruit, and seeds (sunflower, almonds) [16,54].

Dobrowolski [41] isolated Y. lipolytica from soils contaminated with gasoline and diesel residues using crude glycerol from biodiesel production. These isolates have an environmental and economic impact by isolating yeasts that naturally grow on this type of substrate, which reduces the adaptation phase of the yeast in the culture medium and reduces the cost of lipid production.

Physicochemical parameters such as pH, temperature, pressure, and agitation speed are very specific factors according to the combination of the substrate and the yeast strain. However, it has been observed that ascomycetes such as C. tropicalis, L. starkeyi, Y. lipolytica, and basidiomycetes such as Rhodotorula sp., C. cutaneum (synonym Trichosporon cutaneum), and C. dermatis (synonym Trichosporon dermatis) are yeasts capable of growing in any substrate and whose development conditions are very similar; the optimal temperature average is 30 °C, the pH is between 5 and 6, they do not need to grow at high pressures, they have the advantage of not requiring sophisticated equipment, and for that reason, their cultivation is environmentally and economically feasible.

3.3. Cultivation of the Selected Yeast Strain under Optimal Conditions

This stage consists of providing growth to the yeast strain under optimal conditions; that is, the composition of the culture medium satisfies its needs for growth in terms of the source of carbon, water, and energy, as well as the optimal values of operating conditions such as pH, temperature, and stirring speed [80].

This stage aims to develop an inoculum, which is defined as the number of young, strong, and active cells per mL in the culture medium. For the cells to meet these characteristics, they must be in their exponential growth phase. There are no studies indicating the variation in the number of cells per mL that exists in the inoculum, but a range of $1\times {10}^7–1\times {10}^8$ cells/mL has been used [26,110,112,140]. However, this study of the optimal values of the operating conditions and the quantity of inoculum and their influence on the biodiesel production process with yeast will be explained in detail in the next stage.

The carbon source is used by the yeast for energy and anabolic processes to produce carbohydrates and lipids. The nitrogen source is used for the synthesis of proteins and nucleic acids [65,81]. Carbon sources come from refined sugars or lignocellulosic residues, industrial effluents, organic acids, and prepared food waste. Nitrogen sources come from urea, yeast extract, and peptone, which are organic nitrogen sources, while salts such as ammonium sulfate and ammonium chloride are inorganic nitrogen sources. The proportions in which these carbon and nitrogen sources are found can vary, depending on the strain of yeast and the substrate used.

Glucose is used as a basic component of the culture medium for yeast in concentrations of 10–40 g/L since it is a carbon source where the vast majority of yeasts can grow. Yeast extract and peptone are used as nitrogen sources in concentrations between 10 and 20 g/L. However, not all cases use glucose alone as the main carbon source for growth; other carbon sources can also be used in combination. For example, culture medium was supplemented with the addition of crude glycerol at a concentration of 2% without adding trace elements using Y. lipolytica [41].

However, when P. glacialis, R. mucilaginosa, C. lipolytica, C. curvatum, and C. tropicalis were used with glucose, trace elements such as potassium hydrogen phosphate and magnesium sulfate were added to the culture medium. The carbon sources used included sugars, sugarcane bagasse, and molasses, which ultimately increase the cost of the process [24,37,55,72]. The most appropriate thing is to choose the carbon source and the yeast strain, making sure that it can produce by itself all the nutrients necessary for its growth without using trace elements or supplements. Milessi [82] used rice bran, which is an economical source of carbon and nitrogen. This impacts the cost of the fermentation process by not having to add other types of carbon or nitrogen compounds.

Yeasts can grow in media whose pH ranges between 5 and 6. Nevertheless, it has been observed that R. mucilaginosa, R. glutinis, S. pastorianus, and C. cutaneum are able to grow at pH 7 in corn, rice, barley residues, wheat bran, banana peels, and starchy food residues such as potato, sweet potato, and cassava peels [39]. Moreover, R. mucilaginosa is able to grow at pH levels that are different and use lignocellulosic residues, obtaining a percentage of lipids greater than 50% [88]. R. mucilaginosa has the ability to grow in a wider pH range, and for that reason, it has great versatility to produce lipids in various conditions. Nevertheless, from an economic point of view, it is preferable to have a more acidic pH because its growth medium is less likely to be contaminated by bacteria [10], since these grow at a pH greater than 6.

Most yeasts grow at temperatures between 28 and 32 °C. However, depending on the strain and substrate, the optimum growth temperature may change. Park [49] prepared the inoculum with C. curvatum at 25 °C using a medium composed of 1% yeast extract, 2% peptone, and 2% dextrose (YPD). An advantage is observed when using this temperature because it saves energy and has environmental and economic benefits.

The agitation speed is another aspect that influences the growth and lipid production of yeast [10]. This factor has not been studied at this stage; however, speeds between 100 and 200 rpm (revolutions per minute) are used.

The optimal values of the operating conditions in the yeast culture phase are crucial as they influence the development of an inoculum that meets the desired characteristics.

The conditions for cultivating most used yeasts are within a temperature range of 28–32 °C, pH 5–6, agitation speed between 100 and 200 rpm, and an inoculum quantity ranging from 1 × 10⁷–1 × 10⁸ cells/mL.

It is observed that there are no research studies investigating the influence of operating conditions at this stage, despite the fact that the development of an inoculum with the desired characteristics relies on these factors. Therefore, an invitation can be extended to the scientific community specialized in this field to conduct research on the study of optimal operating conditions to develop a suitable inoculum for lipid production.

3.4. Lipid Production

This stage occurs through two metabolic pathways, depending on the substrate used: ex-novo and de-novo synthesis. The first occurs when lipid production takes place on hydrophobic substrates, and the second occurs when it takes place on hydrophilic substrates [83,84]. In the ex-novo pathway, hydrophobic substrates are degraded into free fatty acids by lipases, which are enzymes secreted extracellularly by yeast. Once they are degraded, they are incorporated into the interior of the cell. Subsequently, the modified fatty acids are converted into acetyl-CoA through $\mathsf{\beta }$-oxidation to satisfy the energy demands for cell growth, repair, or to contribute to the formation of cellular metabolites [83]. In the de-novo pathway, lipid accumulation depends not only on the initial concentration of the carbon source but also on other components of the medium and the cultivation conditions. Nitrogen restriction is the most efficient condition in this pathway because all the available adenosine monophosphate (AMP) in the mitochondria is converted into inosine monophosphate due to an increase in the activity of the enzyme AMP deaminase. Consequently, the enzyme is unavailable to convert isocitrate to $\mathsf{\alpha }$-ketoglutarate, resulting in the accumulation of a significant amount of isocitrate in the mitochondria. Citrate in the cytoplasm is converted to acetyl-CoA and oxaloacetate by ATP-citrate lyase (ACL). Finally, acetyl-CoA and malonyl-CoA together form the C14 to C16 long-chain fatty acids [83].

Lipid production by the de-novo pathway is affected by various factors, such as the carbon/nitrogen (C/N) ratio, which is defined as the amount of carbon source present in the fermentation medium relative to the amount of nitrogen source; temperature; pH; agitation speed; and inoculum size [134]. The carbon/nitrogen (C/N) ratio found in the culture medium to produce lipids has been analyzed in various studies and is generally highly variable depending on the yeast strain and the substrate. In the case of sugars, it has been observed that the minimum C/N ratio is from 5 to 180; in the case of glycerol and food waste, the ratio is between 30 and 100 [41,44,48,59]. In lignocellulosic residues, the range is wider; the minimum is 20 and the maximum is 300; however, in volatile fatty acids, sugarcane bagasse, and molasses, the C/N ratio has a narrower range, between 20 and 50 [30,32,49,56]. The increase in the C/N ratio has been found to enhance lipid production, but only up to a certain point. If this ratio is exceeded, it could have an inhibitory effect on yeast growth due to osmotic stress [58,108,112]. Raimondi [108] varied the C/N ratio from 8.6 to 200, finding that the optimal C/N ratio was 52 because the maximum lipid production was 33%. If this proportion was exceeded, C. freyschussii would not grow or produce lipids. This same behavior was observed when C. curvatum and R. toruloides produced higher amounts of lipids only at C/N ratios of 60 and 14.2, using corn cob hydrolysate and glucose, respectively [58,112].

The inhibitory effects of the high C/N ratio can be counteracted by using a yeast strain that can resist high concentrations of the carbon source. For example, R. glutinis is capable of growing in molasses concentrations of up to 20% and producing lipids up to 50.4% [141]. This is because the strain was isolated from soils contaminated with molasses, and the yeast developed the ability to resist high molasses concentrations. In addition, the medium was not contaminated with bacteria since molasses at concentrations greater than 8% released phenolic compounds that prevent other microorganisms, such as bacteria, from growing [141] and did not require sterilization of the medium, which benefits the economy and environment.

The temperature at which lipid production takes place in yeasts is always, on average, 30 ± 2 °C. This variation depends on the intrinsic characteristics of the yeast strain. However, there are exceptional cases, such as the psychrophilic yeast P. glacialis, which was isolated from a glacial environment at temperatures below 0 °C. Amaretti [24] observed that the yeast could grow and produce lipids in a temperature range between −3 and 20 °C, finding that the lipid production did not have a significant variation, being in a range of 4.6–5.1 g/L, which speaks of its versatility to grow and produce lipids in a wide range of temperatures; but on the other hand, it would be a disadvantage if scaling was considered, since in these processes that are exothermic, cooling water is needed to maintain the optimal temperature for the productivity of the microorganism, and this would ultimately increase its cost.

Most yeasts grow at a pH between 5 and 6. Nonetheless, for some substrates, such as volatile fatty acids (VFAs), using an alkaline pH is a good strategy to reduce their inhibitory effect on yeast growth. These conditions have also been carried out in hydrolyzed media of food waste.

It has been tested that Y. lipolytica and R. toruloides can grow and produce lipids at alkaline pH using volatile fatty acids (AGVs) and hydrolysates of food waste. Y. lipolytica produces 26.02% of lipids at a pH of 8, while R. toruloides produces 50% more lipids than at pH 4 [42,142]. This is because AGVs at alkaline pH are dissociated, whereas at low pH, yeast growth is inhibited because AGVs are not dissociated [142]. Additionally, using food waste under alkaline conditions has various advantages, such as reducing the emitted amount of methane gas compared to acidic pH [142]. It has also been estimated that for every 1000 kg of food waste, 11.10 kg of lipids can be obtained, producing 9.52 kg of biodiesel and 0.52 kg of DHA (docosahexaenoic acid), a high value nutritional product used as a dietary supplement [142].

The stirring speed in the medium is a very important factor at this stage and has a very wide range. The minimum reported is 100, and the maximum is 1000 rpm [44,143]. These factors depend on the type of yeast strain, the substrate, the volume of medium, and the equipment where the process is carried out. When lipid production is carried out in flasks, the minimum reported speed is 100 rpm [86], and the maximum is 240 rpm [41]. However, when bioreactors have capacities of 2 L or greater, the maximum speed reported is 1000 rpm. It has been found that with different speeds, lipid percentages greater than 50% can be achieved. The inoculum size is generally between 1–20% v/v [48,50,108]. Rakicka [44] studied the effect of the inoculum size, managing two levels: low and high density (the amount was not specified). They found that at a high density, the growth of the yeast (Y. lipolytica) was fast; however, there was not a good production of lipids. But when a low inoculum density was used, the lipids increased from 11 to 15.5 g/L, which had a very positive impact on production. This can be an advantage because larger amounts of cells are not required to achieve a high percentage of lipids; therefore, it also represents an economic and environmental advantage.

Depending on the feeding strategy of the carbon source that is carried out in the production process, it can be carried out in three modalities: (a) batch, (b) fed batch, and (c) continuous [144].

(a)

Batch: in this type of production, the carbon source is fed at the beginning of the process, and the system is kept closed until the reaction is complete [21,38,62,63,135].

(b)

Fed batch: in this modality, the carbon source is administered throughout the process, and when it reaches a minimum concentration, it is fed back into the system, repeating this process each time the concentration level decreases to such a level that the growth of the yeast is inhibited by the scarcity of the carbon source. This modality allows mitigating the inhibitory effect of a high initial concentration of the carbon source, from 35% to 80%, which makes the process more efficient [25,33,108,117,137].

(c)

Continuous: in this mode, the carbon source is fed continuously at a specific dilution rate, which is generally equivalent to one third of the growth rate of the microorganism [140].

Though the most commonly used modalities in lipid production are batch and fed-batch, which have been optimized by conducting the fermentation process in two stages, the first stage involves generating the maximum amount of cellular biomass possible using a carbon source different from the one used for lipid production. For example, in the first stage, cellular biomass can grow using a carbon source such as sugars (glucose and xylose), and then in the second stage, it can grow using a different carbon source such as cheese whey, VFAs, and other sugars derived from lignocellulosic residues [43,66,136]. With this strategy, it has been observed that the fermentation time is reduced to 48 h, achieving up to 61.3% of lipids using R. toruloides and sugars derived from lignocellulosic residues as a carbon source [136].

Figure 8 classifies the main fermentation modes using an efficiency indicator that relates the amount of lipids produced (g/L ó %) and the fermentation time (h). The numbers within each of the traffic light circles represent the highest sustainable score (green), the average score (yellow), and the lowest score (red).

The most sustainable fermentation processes are batch and fed-batch processes using stewed rice residue [34], molasses [37], xylose [26], lignocellulosic residues such as Jerusalem artichoke [137], and flour-based industrial waste streams [40] because they achieved an efficiency score of up to 0.589 g/L*t. The least sustainable processes were batch and fed-batch in two stages using crude glycerol [48], cheese whey [109], food waste hydrolysate [123], VFAs from food residues [21,43], and rice straw hydrolysate [49], whose lowest score was 0.014 g/L*t.

This indicates the need to increase lipid yields and reduce fermentation times, which impact process efficiency and cost.

Another strategy that has been used is the reuse of lysed cells (cells with broken cell walls) obtained from lipid extraction as a nitrogen source for yeast growth in a new batch process [50]. This strategy reduces the process cost and contributes to a circular economy by valorizing these biological waste materials.

The repeated batch fermentation medium has also been used, which consists of using the fermentation medium from a previous batch as an inoculum for the next. This strategy has the advantages of reducing costs and even doubling the amount of lipids compared to batch processing [33,46,50].

Microbial consortia have also been used, made up of the species of the microalgae Chlorella sp. and of the yeast Saccharomyces cerevisiae, R. glutinis, which can increase the production of lipids from 40 to 50%. This is due to the fact that microalga generates the oxygen that the yeast uses for its growth and mitigates the inhibitory effect that the organic acids in the medium generate on the yeast. Simultaneously, the yeast provides CO₂ that the microalga uses as the only source of carbon to grow since it is a photoautotrophic microorganism [145,146]. The advantages of using microbial consortia are that the depletion of the nitrogen source occurs earlier than in pure cultures, thus increasing the efficiency of lipid production [145].

On the other hand, advances in metabolic engineering have promoted genetic manipulation in yeasts, and strains have been obtained that exceed the lipid production capacity of those that are natural producers of these compounds [147]. For example, to improve lipid synthesis, optimization focuses on increasing lipid precursors, such as ATP citrate lyase (ACL) and malic enzymes, and inhibiting the degradation of the lipids produced [148]. The malic enzyme plays an important role in the regulation of fatty acid biosynthesis, and therefore research related to this enzyme represents a promising path for the improvement of the production of lipids in oleaginous yeasts [149].

It is estimated that the fermentation process accounts for 33.4% of the total electrical energy in the process, indicating that it is the second stage that requires the highest amount of energy after the thermal pretreatment [73]. Some authors mention the type of reactor used for the fermentation process. With this information, the amount of electrical energy consumed in the fermenters was determined, finding that the minimum consumption was 57.6 kWh and the maximum was 375 kWh [27,44], which corresponded to a cost of $110.99 MXN and $722,625 MXN, respectively. Reducing the operating time of the fermenters is a determining factor in reducing the cost of biodiesel production.

Another aspect of sustainability that must be considered are the CO₂ emissions and the wastewater produced at this stage. Through stoichiometric calculations based on the generalized fermentation reaction model, it was calculated that for each mole of sucrose, 288.68 g of CO₂ and 124.47 g of waste water are produced [75]. This indicates that the amount of CO₂ that is produced is more than twice the amount of water that is discarded. Thus, from a sustainable point of view, this stage is one of the ones that contributes the most to the emission of greenhouse gases, so it is essential to reduce the operating times of the fermenters to reduce the consumption of electrical energy and the cost of the process.

The most commonly used modes of lipid production are batch and fed-batch, and this stage has been optimized by using a two-stage fermentation system, handling non-aseptic conditions, and reusing lysed yeast from a previous batch for another batch, which translates to energy and cost savings. However, this stage produces twice the amount of CO₂ emissions compared to the wastewater produced by the reaction itself. In addition, to make scaling up potential profitable, it is necessary to simultaneously produce other high-value nutritional and pharmaceutical products, such as docosahexaenoic acid (DHA).

3.5. Extraction of Lipids

The fourth stage consists of breaking the cell wall of the yeasts to release the intracellular lipids produced during fermentation and subsequently continuing with the transesterification reaction, which leads to the production of biodiesel.

There are several cell disruption techniques that can be grouped into the following categories: (a) mechanical, (b) thermal, (c) chemical, (d) biological, (e) in situ transesterification, and (f) the combination of two or more of these [150].

In

Table 4. Cell disruption techniques.

Cell Disruption Techniques	Description	Environmental Impact	Economic Impact	Efficiency	Sustainable Score Advantages - Disadvantages	References
Thermal	High temperatures and pressure to break down the cell wall.	-High energy consumption	-Low solvent consumption -Scale up potential	-Less time required	2	[85]
Mechanical	Shear force applications such as bead-milling, abrasive beads, high-pressure homogenization, and ultrasound	-High energy consumption	-High solvent consumption -Scale up potential	-Fast -Effective -Minimize lipid degradation	1	[85]
Chemical	hydrochloric and sulfuric acid applications, which react with the cell wall to release glucose, mannose, and glucosamine monomers.	-Corrosion problems in reactors for long time -No eco-friendly	-No sophisticated equipment is required -Economical	No reported	0	[85]
Biological	They use enzymes such as glucomanases and proteases, which act on the $\beta$-glucan, mannan, and protein layers, solubilizing the cell wall.	-Eco-friendly	-Low solvent consumption -High cost -Hard to scale up	-High selectivity -High time consumption	0	[85]
Transesterification in situ	Breaking cells, lipid extraction, and transesterification happen in one single step.	No reported	-High solvents comsumption -Hard to scale up	-Fast -Impurities can be extracted during the process	−2	[85]

, a comparison is made between different lipid extraction techniques, evaluating their advantages and disadvantages as well as their impact on efficiency, environmental aspects, and economics. A sustainable score is then calculated by subtracting the number of advantages and disadvantages. Values above a score of zero are classified as green, values with a score equal to zero as yellow, and scores below the average as red, following a sustainable traffic light system. Finally, the techniques are ranked from most sustainable to least sustainable based on the assigned colors.

The most widely used treatments are chemical; acids such as hydrochloric or sulfuric are generally used to break the cell wall [16,17,18,54,55], but the chloroform-methanol mixture is also used both to break the cell wall [112] and to extract the lipids released from the biomass in two proportions, 2:1 (v/v) and 1:1 (v/v). Mechanical treatments with chemicals are also used, for example, ultrasound with a chloroform-methanol mixture (2:1) [39] and in situ transesterification, where lipid extraction and transesterification occur in just one step.

Figure 9 classifies the different extraction techniques, evaluating efficiency and cost indicators. Efficiency was assessed by considering the amount of extracted lipids (g/L) and the time required for each pretreatment. The economic indicator was calculated by relating the amount of extracted lipids (g/L) to the cost of the reagents used (MXN). Subsequently, an average of the two aforementioned indicators was obtained, resulting in a global sustainable score that ordinarily classified the extraction techniques from most to least sustainable. In Figure 9, the numbers in the traffic light circles represent the highest score (green), the average score (yellow), and the lowest score (red).

Lipid extraction techniques such as ultrasound [39] and chemical extraction [112] using a chloroform-methanol mixture have the highest scores (8.966), compared to techniques using chemical methods with hydrochloric acid (4M) [16,55,88] and in situ transesterification [7]. The difference lies in the fact that techniques with higher scores use a larger quantity of reagents, resulting in higher costs but significantly reduced processing time. On the other hand, acid-based techniques are less expensive, but the processing time can be up to 20 times longer compared to ultrasound and solvent mixture methods.

In situ transesterification, due to its prolonged duration, significantly increases the operational costs of equipment, reaching into the thousands [74]. However, this is compensated by the additional time that the other techniques need to achieve the conversion of lipids to FAME, which takes an average of 20 h to achieve complete transesterification. The advantage of this technique is that FAME yields of up to 111% have been obtained [7], and solvents as toxic as chloroform are not used. Nonetheless, its scaling is difficult since the initial investment to install a biorefinery with a capacity of 10,000 tons of microbial lipid utilizing glucose as a substrate and using in situ transesterification is M USD 33.1, while the investment using indirect transesterification is M USD 2.15. This is explained because in the first case, the cost of installation and operation of the fermenters is high due to the consumption of steam to recover the solvents and the cost of electricity in the fermenters, which is 12.5 kWh/kg biodiesel, which is 416 times higher than using indirect transesterification [74].

On the other hand, the techniques that require more energy consumption are ultrasound with 183.6 MJ/kg lipid extracted, followed by in situ transesterification with 147.96 MJ/kg lipid, and finally chemical treatment with a consumption of 4.4–8.8 MJ/kg biomass [85].

Chemical extraction is cost-efficient and suitable for large-scale production, while techniques such as ultrasound and in situ transesterification are more suitable for laboratory and pilot-scale production processes since they have a high cost due to the operation of the equipment and the amount of solvents used, but they have a high lipid yield and are a little more friendly to the environment.

3.6. Transesterification Reaction

The transesterification reaction is the way in which biodiesel is synthesized, in which a triacylglyceride reacts with an alcohol (methanol or ethanol) in the presence of a catalyst that can be acid [25,41,43,48,51,63,86,87,88], alkaline [26,37,66,110], or enzymatic (lipases) [59,90], or using heterogeneous catalysts (hydrophobic acid catalyst and bifunctional acid superparamagnetic catalyst), such as (FDCA/SA-Hf) composed of 2.5-furandicarboxilic acid, stearic acid, and hafnium; FS-B-L-PILS, which is a combination of acid poly ionic liquids (PILS) and Fe₃O₄-SiO₂; and 30%Sn-MMT-SO₃H, composed of 30% tin, montmorillonite (MMT), and the SO₃H group [91,92,93].

The transesterification reaction is affected by temperature, catalyst concentration, the proportion of methanol used, the moisture content present in the lipid, and reaction time. Acidic or alkaline catalysts (sulfuric acid and sodium hydroxide) are commonly used for commercial biodiesel production due to their high efficiency and low cost [151], typically in a temperature range of 50 to 90 °C [63,86]. However, alkaline catalysis with potassium hydroxide has been carried out at room temperature [66], which has positive impacts on the economic and environmental aspects by saving energy. However, these catalysts have the disadvantage of not being easily reusable [152].

On the contrary, heterogeneous catalysts can be reused up to six times in the transesterification process. Generally, they are not economical, but supported catalysts on biomass-derived matrices, such as FDCA/SA-Hf, have been designed, which may be less expensive. Heterogeneous catalysts are used in a temperature range of 49–150 °C, making them versatile for different reaction conditions.

Alkaline and acid catalysts are typically used in concentrations between 2–10% w/v and 1–9 wt% in heterogeneous catalysts [91]. It has been found that higher catalyst concentrations increase the conversion of lipids to FAME, but only up to a certain point. For example, in the case of sulfuric acid, a FAME yield of 103% was achieved at a concentration of 0.6 M. Yet, if this concentration were increased to 0.8 M, the FAME yield percentage would decrease due to polymerization reactions that occur at this concentration [7].

In the case of heterogeneous catalysts, it has been observed by the majority that conversions exceeding 90% are achieved. Even when utilizing 4.1 wt% of hydrophobic acidic catalysts (FDCA/SA-Hf), conversions of up to 98.6% can be attained [92]. This represents a very high conversion, especially considering that it is a reusable and environmentally friendly catalyst, as it is manufactured using biomass-derived components.

The same behavior has been observed with the methanol-to-oil ratio, where increasing the ratio also increases the FAME yield, but only up to a ratio of 1:20. If the ratio is exceeded, the excess methanol dilutes the catalyst concentration and the lipids, resulting in a decrease in FAME production [7]. In heterogeneous catalysts, the same behavior has been observed in the methanol-to-oil ratio. If the ratio exceeds between 15:1 and 20:1, the biodiesel conversion is reduced to as low as 10% [93].

The water content is a factor that must be avoided since it favors the generation of free fatty acids. These must be in concentrations less than 0.5% in the lipid [6], otherwise the reaction of saponification (soap formation) is favored and decreases the production of FAME. Kuan [7] determined that for every 10% increase in humidity, FAME decreased by more than 40%. The advantage of enzymatic catalysis in this regard is that the moisture content does not affect their action in transesterification and gives conversions close to 90% [90].

The average reaction time in transesterification using acid and alkaline catalysts ranges from 6 min to 24 h [66], heterogeneous catalysts range from 6 to 10 h, and enzymatic catalysts require a reaction time of 48 h. The FAME yield increases as the reaction time increases. For example, when using a basic catalyst like sodium hydroxide, the reaction time can be half compared to sulfuric acid, but the latter can achieve a FAME yield of 111% with a reaction time of 20 h [7]. However, alkaline catalysts have the disadvantage that if the moisture content is equal to or greater than 0.1%, the conversion of lipids to FAME is reduced due to decreased catalyst effectiveness and the formation of saponification products (soap) [153].

From a sustainability standpoint, it can be preliminarily concluded that sulfuric acid is a catalyst that, at a concentration of 0.6 M, produces yields of FAME greater than 100%, although its reaction time is longer compared to alkaline catalysts. On the other hand, alkaline catalysts can be used at room temperature with a reaction time as short as 6 min. Heterogeneous catalysts, while not as economical, have the advantage of being easily recoverable in the process, achieving conversions of up to 90% after being reused six times. This ability to be easily recovered from the reaction system can offset their cost. Enzymatic catalysts are more environmentally friendly, but their reaction times can be longer and they are more costly.

3.7. Refining Process of Fatty Acid Methyl Ester (FAME) to Obtain Biodiesel

The biodiesel refining process consists of removing the impurities generated in the transesterification reaction, such as glycerol, traces of soap, alcohol excess, and catalyst residues [153], in order to meet quality standards for use in internal combustion engines.

There are several purification techniques, such as the use of (a) liquid-liquid extraction, in which deionized water and organic solvents such as n-hexane, toluene, and isooctane are used [25,26,51,89] to remove residues of soap, catalyst, alcohol, and other contaminants present in biodiesel [154]; and (b) absorbent materials such as silicate of magnesium (Magnesol) [117] are also used, which separates polar substances such as glycerol and methanol through absorption on the surface of the material [154].

Purification techniques with deionized water and organic solvents are very efficient, but when 3–10 L of water are used for each liter of biodiesel, this has an impact on cost, and, furthermore, this equivalent volume of water is what must be treated later [154], so this option is not economically and environmentally viable.

The use of absorbent materials (magnesium silicates) has the advantage of requiring less time than liquid-liquid extraction methods; there is no risk of traces of moisture in the biodiesel, but they have certain disadvantages because impurities such as glycerides and free fatty acids can sometimes not be absorbed by the material, and their commercial cost is also high [155]. However, it does not pose a threat to health or the environment.

4. Evaluation of Greenhouse Gas Emissions and Efficiency in the Use of Second-Generation Biodiesel in Engines

Biodiesel is blended with diesel in various proportions to be used in internal combustion engines. Typically, it is used in blends ranging from 5% (B5) to 20% (B20), with the remainder of the volume being diesel fuel. One way to evaluate the performance of these blends in internal combustion engines is by measuring the emissions of greenhouse gases (GHGs) produced during combustion.

The assessment of GHGs has been studied by various authors for second-generation biodiesel. In the case of oleaginous yeasts, it has been observed that CO and CO₂ emissions decrease as the proportion of biodiesel in the blend increases to 20%. However, if this proportion is exceeded, NOx emissions increase using biodiesel produced by yeasts such as M. pulcherrima and C. curvatum [97,98]. CO₂ emissions increase up to a B20 blend and then decrease in B30. CO emissions decreased by 2.6% and 21.4% with B10 and B20 blends, respectively, compared to conventional diesel. Unburned hydrocarbons (HC) decrease as the biodiesel proportion in the blend increases, with reductions of 15.97%, 37.2%, and 18.34% for B10, B20, and B30, respectively. NOx emissions increase as the biodiesel proportion in the blend increases, with increases of 15.5% for B10, 31.47% for B20, and 22.4% for B30 [97].

The thermal efficiency of biodiesel is also maximized in the B20 blend. Similarly, fuel consumption was reported to be lower, up to 20%, but if this proportion is exceeded, fuel consumption increases [78]. Therefore, using blends higher than B20 is not economical.

The GHG of biodiesel produced from edible and non-edible oils has also been evaluated. Edible oils such as canola oil [4] and non-edible oils such as karanja, palm, jatropha, and polanga oil [13,156] reduce the amount of CO, HC, particulate matter, and smog but increase the levels of NOx and CO₂. The increase in CO₂ is due to the oxygen content of biodiesel, which promotes complete combustion [9]. However, using karanja oil in a B100 blend can also increase CO₂ emissions because it reduces the thermal efficiency of biodiesel. On the other hand, the emissions of methane (CH₄) and nitrous oxide (N₂O) decrease significantly [102].

When using a 10% biodiesel blend (B10) with waste cooking oil, the amount of CO is reduced to the maximum, while the greatest reduction in NOx is achieved with the B20 blend [13]. However, with canola oil, all blends from B5 to B100 increase NOx emissions [4].

Biodiesel produced with yeast contributes to a lesser extent to global warming by reducing CO₂, CO, HC, and NOx emissions, making it an excellent choice as a renewable fuel. The most suitable blend is B20.

There are no studies that assess the performance of yeast-based biodiesel in internal combustion engines, leaving room for further research and promoting the use of this biofuel.

5. Conclusions

This research, which aimed to assess the sustainability level of various methodologies used in each stage of the biodiesel production process with yeasts, leads to the following conclusions:

• In the first stage, which is the selection of yeast strain and substrate, yeasts such as Y. lipolytica, R. toruloides, R. glutinis, R. mucilaginosa, L. starkeyi, and C. curvatum have proven to be excellent lipid producers. This is attributed to their capability to grow on a wide variety of low-cost substrates. The most sustainable include used cooking oil, glucose, and sugars derived from lignocellulosic residues.
• In the substage of lignocellulosic residue pretreatments, the most sustainable pretreatment in terms of efficiency involves sulfuric acid (2% v/v). However, this pretreatment emits a significant amount of CO₂, which can be reduced if the solid load is doubled.
• In the stage of cultivating the selected yeast strain, it has been observed that the growth pH range of most yeast strains is between 5 and 6, the temperature is between 25 and 32 °C, the stirring speed is between 100 and 200 rpm, and the inoculum size is between $1\times {10}^7–1\times {10}^8$ cells/mL. Yeast growth at a pH below 6 inhibits bacterial contamination, and growth at room temperature benefits economic and environmental aspects as there is energy savings.
• In the lipid production stage, the most efficient fermentation modes are batch and fed-batch. This stage has been optimized using a two-stage fermentation system, managing non-aseptic conditions, and reusing lysed yeasts from one batch to another, resulting in energy and cost savings. However, this stage produces twice the amount of CO₂ emissions compared to the wastewater produced by the reaction itself. Additionally, for scaling up to be potential and profitable, it is necessary to simultaneously produce other high-value nutritional and pharmaceutical products, such as docosahexaenoic acid (DHA).
• In the lipid extraction stage, the most sustainable technique in terms of cost and efficiency is ultrasound because it is fast and does not require a large amount of solvent. This technique can be twice as efficient as solvent extraction but is currently only used on a laboratory scale.
• In the transesterification reaction stage, both acid and alkali catalysts are more economical and efficient due to their shorter times, and the conversion of FAME exceeds 100%. Yet, heterogeneous catalysts proved to be a promising alternative because they can be reused many times in the process without significantly affecting their high conversions. This results in a reduction in the overall processing cost.
• In the FAME refining stage to obtain biodiesel, magnesium silicates are the most sustainable, as they have the advantage of being more efficient regardless of the moisture content in the biodiesel.
• Yeast-derived biodiesel is a good alternative for use in engines, mixed in proportions with diesel (10 and 20%), because its composition is very similar to vegetable oils, it reduces the emission of greenhouse gases, and it has good performance in engines. However, the technology is not yet fully developed so that it can fully replace fossil fuels and meet the required demand.