Sustainable biodiesel production from agricultural lignocellulosic waste via oleaginous microbial processes

Abstract

Biodiesel, a renewable and eco-friendly liquid biofuel, plays a crucial role in reducing greenhouse gas emissions. Initially, biodiesel production relied on vegetable oils, non-edible oils, and waste oils. However, these sources face challenges, including high costs, labor and land requirements, and insufficient supply to meet demand, especially in the case of waste oils. Recent research highlights the potential of lignocellulosic substrates for biodiesel production via oleaginous microorganisms, which can accumulate lipids similar to those in vegetable oils under stress conditions. This review investigates various biodiesel feedstocks from microorganisms such as microalgae, fungi, yeast, and bacteria. It details the biodiesel production process from lignocellulosic substrates, biological pretreatment and bioconversion. Additionally, the review underscores the role in biofuel and biorefinery development and briefly discusses the integration of biofuels within a circular economy framework.

Introduction

Biodiesel has garnered considerable interest as a viable and renewable substitute for conventional fossil fuels. Common feedstocks employed in biodiesel production include vegetable oils from crops such as rapeseed, soybean, palm, sunflower, and other oil-producing plants, as well as animal fats [1]. The primary challenges impeding the development and widespread adoption of biodiesel are the high costs and limited availability of oil feedstocks. Furthermore, using vegetable oils as biodiesel feedstocks could lead to a scarcity of edible oils and spark debates over the competition between food and fuel [2].

Microbial oils derived from oleaginous yeasts present a promising alternative to traditional oil feedstocks for biodiesel production. These oils predominantly exist as triacylglycerols (TAGs) and, to a lesser extent, as free fatty acids (FFAs), with compositions similar to those of vegetable oils [3]. Microbial oils offer numerous advantages over vegetable oils, including shorter life cycles, reduced susceptibility to location and climate conditions, lower labor requirements, easier scalability, non-reliance on arable land, and high carbon-to-heteroatom ratios [4]. However, for these microbial oils to serve as cost-effective and sustainable feedstocks, it is crucial to identify low-cost, sustainable nutrient sources for cultivating oleaginous microorganisms.

Nevertheless, the majority of microorganisms, including oleaginous yeasts, are incapable of directly utilizing lignocellulosic biomass. Therefore, this biomass must undergo pretreatment and subsequent hydrolysis to transform its resistant polymers into monomeric carbohydrates [5]. Regarding utilizing biomass as a solid biofuel, a variety of chemical and hydrothermal pretreatment techniques have been used to reduce the amount of potassium in the material with the goal of improving its clean solid biofuel properties [6]. However, chemical pretreatment presents challenges related to handling and environmental impact, and hydrothermal pretreatment may require more energy input than the energy produced. Biological pretreatment of biomass, as opposed to conventional methods that present economic challenges, enhances the properties of the fuel and facilitates the production of value-added products before its use as a solid biofuel [7].

Numerous microorganisms, such as bacteria, yeast, and filamentous fungi, have been documented to generate hydrolytic enzymes for biological pretreatment, including xylanase, cellulase, and β-glucosidase [8]. However, fungi are the most effective producers of enzymatic cocktails due to their superior ability to produce multiple extracellular enzymes. On an industrial scale, the primary producers of efficient hydrolytic enzymes for lignocellulosic biomass are Aspergillus spp. and Trichoderma spp [9]. The incorporation of xylanases with cellulolytic enzymes is primarily regarded as a strategy for the efficient bioconversion of lignocellulosic residues, owing to their potential for synergistic interaction [10]. Through the utilization of lignocellulosic residues, enzymatic cocktails could be generated with lower costs for hydrolytic enzyme synthesis and industrial waste biovalorization, thereby lowers the cost of converting cellulose to glucose and hemicellulose to xylose. This strategy also encourages the economically viable production of bioproducts since these sugars can be used as carbon sources for cultivating bacteria [11]. The objective of this review is to examine recent studies on lignocellulosic biomass, focusing on various biological pretreatment methods involving fungi and bacteria. Additionally, the review explores the biovalorization of lignocellulosic biomass for biodiesel production using oleaginous microorganisms. Furthermore, it discusses the potential for producing biodiesel within an integrated biofuel production framework utilizing circular economy principles.

Overview of biodiesel

Currently, biodiesel is seen as a potential alternative for energy in the future and a considerable technological breakthrough to decrease the harmful environmental impacts associated with non-renewable energy sources [11]. The decreasing availability of fossil fuels and the escalating environmental damage urgently require the development of renewable and environmentally friendly fuels [12]. As a result, biodiesel is increasingly being recognized worldwide as a promising alternative for producing environmentally friendly fuel [13]. Biodiesel has been extensively adopted for use in vehicles, railways, aircraft, and generators. Additionally, it is utilized in a wide range of heavy-duty construction equipment and agricultural machinery. The attention and support for biodiesel has increased in recent years due to concerns about emerging environmental pollution and greenhouse gas (GHG) emissions, especially from vehicles.

The global energy sector is undergoing a profound transformation, driven by the collective efforts of nations and industries to advance sustainable energy systems that remain compatible with existing fossil fuel infrastructures [14]. This transition is particularly crucial given that, despite continued progress in renewable energy technologies, fossil fuels are projected to remain the dominant source of global energy demand through at least 2040 [15]. In this context, biofuels are anticipated to play an increasingly significant role. Global demand for biofuels is projected to grow by 38 billion liters between 2023 and 2028, marking a nearly 30% increase compared to the previous five-year period. By 2028, total biofuel consumption is expected to reach 200 billion liters, representing a 23% rise from current levels [16].

According to the type of feedstock used, biodiesel production is divided into many generations. Edible plants or seeds are the source of first-generation biofuels. Second-generation biofuels, on the other hand, are created from residual cooking oil, animal fats, and non-edible plants and seeds. Algae, particularly microalgae and macroalgae, are the source of third-generation biofuels. In the fourth generation, biofuels are produced by genetically modifying organisms, including cyanobacteria, yeast, fungus, and transgenic algae. Advanced biofuel production techniques use a number of modern technologies. These include of synthetic biology, genetic engineering, CRISPR-based genome editing, and computer techniques like high-throughput screening [17]. These techniques enable precise modifications at the genetic level and facilitate the development of optimized biofuel production systems.

However, the high cost of culture substrates, which accounts for 40–80% of the total biodiesel production cost, challenges the economic feasibility of microbial oils [18]. Therefore, to achieve sustainable and economically viable microbial oil production, oleaginous microorganisms should be cultivated on low-cost substrates.

Lignocellulosic biomass

Lignocellulosic biomass is predominantly obtained from agricultural, industrial, and forestry sectors. However, not all available residues are used for biodiesel production. Some are repurposed for animal feed, fertilizer production, paper manufacturing, or direct combustion as fuel. The primary obstacle in advancing biodiesel production from lignocellulosic biomass is the absence of a thorough statistical analysis of various available lignocellulosic feedstocks [19]. This lack of data impedes the scaling up of biodiesel production technologies from laboratory to pilot and industrial scales. To address this challenge, additional research is necessary to quantify and characterize the diverse lignocellulosic biomass sources suitable for biodiesel production. This would facilitate more effective utilization of these plentiful renewable resources and potentially expedite the development of large-scale biodiesel production processes using lignocellulosic feedstocks.

Lignocellulose structure and chemical composition

Cellulose, hemicellulose, and lignin contain the majority of lignocellulosic biomass, accounting for around 30–35%, 20–25%, and 15–20% of its dry weight, respectively [20]. The composition of lignocellulosic biomass can vary based on geographical location, growing conditions, and age. A comprehensive list detailing the composition of various lignocellulosic biomass types in terms of their dry weight percentages of lignin, cellulose, and hemicellulose is provided. The production of biodiesel from lignocellulosic biomass entails four essential stages: delignification, saccharification, fermentation with oleaginous microorganisms to boost lipid synthesis, and finally, transesterification for conversion [21]. Lignin is a non-carbohydrate polymer located in the primary cell wall, composed of phenyl propionic moieties. It consists of coniferyl alcohol and simply alcohol units linked by various ether bonds, providing tensile strength, microbial resistance, and impermeability. Lignin functions as an impediment to microbial and enzymatic hydrolysis due to its close association with cellulose microfibrils. The lignin content varies among different feedstocks and must be reduced through a delignification step to allow easier access to cellulose and hemicellulose polymers [22]. The principal polymer in plant cell structures, cellulose, consists mainly of D-glucose units linked by β-1,4-glycosidic linkages and has a polymerization degree of at least 10,000. Van der Waals forces and hydrogen bonds enable these cellulose polymers to bond into microfibrils, providing tensile strength, chemical stability, crystallinity, and ability to microbial degradation [23].

This inherent property of cellulose contributes to the recalcitrance of lignocellulosic biomass. In contrast to cellulose, which is a polymer composed solely of glucose units, hemicellulose is a heterogeneous polymer consisting of various monomeric units including D-xylose and L-arabinose (C5) and D-mannose, D-galactose, and D-glucose (C6) sugar [24].

Hemicellulose exhibits an amorphous structure with a polymerization degree typically below 200. As the second most prevalent polymer after cellulose, hemicellulose’s composition varies significantly across different feedstocks [25]. For instance, in softwood species, hemicellulose primarily comprises glucomannan, whereas in grasses and agricultural residues, it is predominantly composed of xylan. Hemicelluloses create an intricate network with lignin and cellulose microfibrils, which contributes to the mechanical strength of the material and affects its response to thermal and chemical treatments [24].

Potential of biological pretreatment

Due to their naturally occurring abundance, a wide variety of microorganisms are used in biological pretreatment methods. By secreting ligninolytic enzymes that break down lignin and hydrolytic enzymes, such as hydrolases, these microbes encourage the extracellular modification or decomposition of lignocellulosic material. By breaking down the structure of the cell wall, this enzymatic activity makes biopolymers easier to access to undergo further hydrolysis [26]. Cellulolytic and hemicellulolytic microbes are used in biological pretreatment to hydrolyze cellulose and hemicellulose, converting them into their corresponding monomeric sugars [27].

The simultaneous hydrolysis of lignocellulosic residual, followed by fermentation processes, leads to the production of various biofuels including ethanol, hydrogen, methane, and furfural as well as bioproducts such as various enzymes, lactate, acetate, and organic acids [28]. Several bacterial and fungal species have an impact in the hydrolysis process. The role that fungi perform in breaking down lignocellulosic residuals in their enzymes is widely recognized. These fungi are found throughout nature and are known to generate a variety of enzymes, including hemicellulolytic, ligninolytic, and cellulolytic. Lignocellulolytic fungi encompass species from several groups: ascomycetes, such as Trichoderma reesei, Pleurotus ostreatus, Tremetes versicolor, and Aspergillus sp [29, 30]., basidiomycetes, including white-rot fungi like Trametespubescens, Ganoderma adspersum, G. lipsiense, and Rigidoporus vitreus [31], and brown-rot fungi such as Serpula lacrymans [32]. Nevertheless, there has been little progress in finding fungi strains that can efficiently break down lignin while also recovering cellulose, and this problem is still not resolved for commercialized application.

Although biological pretreatment typically necessitates extended processing times relative to certain conventional methods, current research endeavors are directed towards optimizing microbial strains and process conditions to enhance efficiency and reduce treatment durations. This focus on innovation aims to significantly decrease treatment times, thereby improving the economic feasibility and scalability of biological pretreatment. The future outlook for this technology is promising, as ongoing advancements in microbial and enzymatic efficiency, along with the potential for co-product generation, are poised to render it an increasingly appealing and genuinely sustainable option for large-scale biofuel production. This development is expected to contribute substantially to a circular economy and diminish reliance on fossil fuels.

Oleaginous microorganisms

Oleaginous microorganisms are defined by their capacity to accumulate microbial lipids exceeding 20% of their total biomass. These microbial lipids, commonly known as single-cell oils, have attracted considerable global interest for biodiesel production. Various microorganisms, including microalgae, bacteria, fungi, and yeast, have been explored for this purpose. These microorganisms offer several advantages over traditional vegetable oils, such as requiring less space and having faster production cycles. Additionally, they are less influenced by seasonal and climatic variations. However, a significant challenge in microbial lipid production is the high cost of raw materials, which can constitute up to 70% of the total production expenses. Recent research has shifted towards utilizing various waste materials to reduce production costs. Examples of such waste materials that have been investigated for lipid production include rice straw hydrolysate, cheese whey, wheat bran, and sewage sludge.

Bacteria

Bacteria that are useful for enhancing microbial oil production typically exhibit rapid growth and are amenable to genetic modification. While bacteria are not generally known for lipid accumulation as show in Table 1, certain species like Rhodococcus opacus can store fatty acids up to 70% of their dry cell weight [33]. Furthermore, some Bacillus species, including B. cerus KM15 and B. subtilis, can accumulate over 30% lipid content [34, 35] as show in Table 1, expanding their potential in lipid production. Genetically engineered E. coli can produce fatty acids at 2.5 g/L through the deletion of the fadD gene (which encodes fatty acyl-CoA synthetase) and overexpression of acetyl-CoA carboxylase and thioesterase. This development paves the way for utilizing various metabolic tools to create efficient fatty acid-producing cells from non-oleaginous microbes. Despite their lower lipid accumulation, bacteria offer several advantages for biodiesel production: they grow rapidly (often reaching high biomass levels within 12–24 h) and are easily cultivated. Bacteria can quickly achieve high cell densities and withstand toxic or complex substances such as polycyclic aromatic hydrocarbons, phenols, and lignin compounds. These characteristics give bacteria an edge over microalgae in biodiesel production, as the latter requires larger cultivation areas and has slower growth cycles. Although bacteria generally accumulate less lipid than microalgae, this limitation can be addressed by modifying the carbon-to-nitrogen (C/N) ratio or by identifying highly oleaginous strains. Additional research is necessary to evaluate the effects of various toxic compounds found in organic wastes on the lipid yields of oleaginous bacteria [36]. Advancements in systems biology and metabolic engineering offer the potential to significantly enhance fatty acid production in common production hosts like E. coli [54].

Table 1.

A list of oleaginous microorganisms with their lipid content

	Oleaginous microorganism	Lipid content	Ref
Bacteria	Rhodococcus opacus	71%	[33]
	Bacillus cerus strain KM15	32	[34]
	Bacillus subtilis	39.8	[35]
Microalgae	Scenedesmus sp.	34.10	[38]
	Chlorella sp.	33	[39]
	Tetradesmus obliquus ACOI204/07	17.2	[40]
	Thraustochytrium sp. BM2	52	[41]
	Nannochloropsis oceanica	45	[42]
Yeast and filamentous fungi	Yarrowia lipolytica 5054	40	[44]
	Rhodotorula mucilaginosa G43	35	[45]
	Candida tropicalis X37	20	[45]
	Lipomyces starkeyi	70	[46]
	Aspergillus tubingensis TSIP9	43	[47]
	Mucor circinelloides	54.1	[48]
	Syncephalastrum racemosum	28.2	[49]
	Brevistachys sp.	21.76	[50]

Microalgae

Compared to other microbial platforms such as bacteria and yeast, microalgae are distinguished by their high photosynthetic efficiency, ease of cultivation, reduced spatial requirements, and rapid cell division, all of which contribute to increased biomass production for biodiesel generation. They can be grown using various nutritional modes, including autotrophic, heterotrophic, and photoheterotrophic mixotrophic strategies, which allow them to utilize both organic and inorganic nutrient sources [37]. Through a series of enzymatic reactions, triacylglycerols (TAGs) are synthesized in the chloroplasts and endoplasmic reticulum and are stored as glycerolipids. These TAGs accumulate in oil bodies within subcellular structures and are produced under stress conditions, serving as a neutral source of carbon and energy. The lipid content in microalgae can vary significantly, ranging from 20 to 50%. Notable microalgal genera with high lipid content include Scenedesmus sp., Chlorella sp., Tetradesmus obliquus ACOI204/07, and Thraustochytrium sp. BM2 [38–41]. Certain species of Nannochloropsis sp. are capable of storing between 40% and 80% of their dry weight as lipids within their cell [42]. Microalgae have the ability to alternate between heterotrophic and autotrophic nutritional modes and can also grow in a mixotrophic manner. During heterotrophic growth, they depend on glucose or other readily available carbon sources for their metabolism and growth. Because microalgae can be cultivated without the need for agricultural land, they do not compete with food crops, making them an advantageous feedstock for biomass production [43].

Yeast and fungi

Oleaginous yeasts and fungi are favored for microbial lipid production due to their high lipid content and rapid growth rates. Key oleaginous species among yeasts include those from the genera are displayed in Table 1, Yarrowia, Rhodotorula, Candida, Trichosporon, Lipomyces, and Cryptococcus [44–46]. Oleaginous filamentous fungi include Aspergillus tubingensis TSIP9, Mucor circinelloides, Syncephalastrum racemosum, and Brevistachys sp. [47–50]. Microbial lipid content in certain yeast species can reach up to 80%. These yeasts primarily use carbon sources such as glucose, sucrose, glycerol, or sugar hydrolysates [36]. On average, lipids can constitute up to 40% of their biomass, with this proportion increasing to as much as 80% under stressed conditions. Although yeast species may have similar lipid contents, their lipid profiles can vary significantly. Conversely, different lipid contents among species might result in similar lipid profiles, as observed in Rhodotorula. Yeasts are preferred over microalgae for biodiesel production due to their environmental adaptability, shorter doubling times, and ease of cultivation [51]. The production of microbial lipids using oleaginous yeasts through heterotrophic metabolism provides a stable process by reducing the effects of seasonal, climatic, and sunlight fluctuations on biomass production. Various pilot-scale systems employing oleaginous yeasts for lipid production have been evaluated, with findings supporting the potential to scale up this technology for large-scale lipid production.

Bioconversion of lignocellulosic residues to biodiesel production

The bioconversion of lignocellulose into microbial lipids entails several crucial steps: initial pretreatment of the lignocellulosic biomass, hydrolysis of structural carbohydrates into fermentable sugars, followed by microbial lipid production, and the final isolation and purification of the lipid product. Due to the absence of cellulase and hemicellulase enzymes in most oleaginous microorganisms, the structural polysaccharides within lignocellulosic biomass must first be hydrolyzed into fermentable sugars, predominantly xylose and glucose, which can then be used as a carbon source by these microorganisms. The incomplete utilization of sugars, including both hexoses and pentoses, contributes to the high costs associated with lipid production. Notably, research is progressing not only to enable microbes to ferment pentoses alongside hexoses but also to allow them to metabolize cellobiose and higher cellodextrins directly into lipid and other valuable metabolites as show in Fig. 1. Although the concept is not novel, direct fermentation of cellobiose can achieve higher conversion rates and lipid yields, holding significant potential.

Fig. 1.

Schematic diagram of lipid production from lignocellulosic biomass by oleaginous microorganisms

Fermentation

Pretreatment of lignocellulosic biomass is a crucial step in the biodiesel production process. This stage delignifies the biomass and breaks down cellulose and hemicellulose polymers, preparing them for the fermentation broth. The generated sugars are subsequently converted into biofuels and biochemicals through fermentation processes involving various microorganisms [52]. These lipids are primarily composed of fatty acids including linolenic, palmitic, and oleic acids. The conversion of these fatty acids into biodiesel is usually accomplished using acids, bases, or enzyme. The configuration of the reactor along with other factors plays a crucial role in lipid accumulation. Two main fermentation modes are operated for biodiesel production from lignocellulosic biomass: solid-state fermentation (SSF) and submerged fermentation (SmF) [53]. Each of these fermentation techniques has its own benefits and drawbacks, and the selection between them is influenced by various factors, including the type of biomass, the microorganism employed, and the desired final product. Enhancing the efficiency and scalability of biodiesel production from lignocellulosic biomass requires optimizing these fermentation processes, as well as improving pretreatment methods and reactor designs [54]. Continued research in these areas could potentially lead to more economical and sustainable biodiesel production methods, potentially increasing the feasibility of using lignocellulosic biomass as a renewable energy source.

Solid-state fermentation

SSF is a relatively straightforward and cost-effective technique that offers several advantages. It requires minimal biomass volume and has low energy requirements, making it an economical choice. One of its key benefits is the ability to utilize low-quality, natural, renewable energy crops, including by-products from food and agricultural industries [55]. However, SSF faces some challenges. A significant concern is feedback inhibition, where excess fermentable sugars can impede microbial growth and overall culture performance, ultimately affecting yield. Another drawback is the low lipid yield due to the absence of free-flowing water [55]. SSF is a widely recognized strategy for cultivating oleaginous fungi. Among these, Aspergillus sp. are well known for their ability to produce both lipids and a wide range of lignocellulolytic enzymes. These enzymes facilitate the breakdown of complex biomass into fermentable substrates by degrading lignocellulosic components. For instance, Aspergillus tubingensis TSIP9 has been reported to effectively pretreat empty fruit bunches (EFB) and simultaneously produce lipid under non-sterile SSF conditions [56]. Additionally, Aspergillus oryzae A-4 has also been shown to produce lipids using wheat straw as a substrate [57]. In addition, other fungal species such as Trichoderma sp. have also demonstrated lipid-producing capabilities [58]. Similarly, Nurika et al. [59] reported the use of Serpula lacrymans to convert oil palm empty fruit bunches (OPEFB) into lipids. To overcome the limitations of traditional SSF, researchers have proposed modifications to the process. Economou et al. [60] introduced a semi-solid-state fermentation approach by increasing the water content, which facilitated fungal growth and enhanced the production of single-cell oils [60]. Despite these improvements, this method presents new challenges during lipid extraction, as the increased water content causes the substrate to dissolve alongside the microorganisms, which may hinder the recovery of the desired product [61].

Submerged fermentation

Submerged fermentation is commonly employed for cultivating microalgae, whereas yeast, bacteria, and molds are typically utilized in solid-state fermentation. A key limitation of submerged fermentation is the necessity for prior extraction of fermentable sugars from biomass into a liquid medium, which is both time-intensive and costly [62]. After pretreatment, lignocellulosic biomass undergoes saccharification, facilitated by lignocellulolytic enzymes that break down lignocellulose into its monomeric components. These enzymes, predominantly found in fungi and bacteria, are categorized into two main groups: hydrolases (e.g., cellulases, hemicellulases, xylanases, proteases, and amylases) that degrade cellulose chains, and ligninases that decompose lignin structures [63].

Lignocellulolytic enzymes are advantageous due to their high specificity and ability to function under mild conditions, making them more efficient than inorganic catalysts. However, their industrial application is limited by factors such as low thermal stability, high costs associated with extraction and purification, and difficulties in recovering the enzymes from reaction mixtures [32]. Microbial fermentation-based conversion of lignocellulosic biomass has been extensively studied using various microorganisms. While S. cerevisiae is the most widely employed yeast for cellulosic residue fermentation [64], fungi from the genera Aspergillus, Mucor, Umbelopsis, Mortierella, Cunninghamella [65] as well as bacteria such as Rhodococcus, Bacillus, Streptomyce, Acinetobacter have demonstrated the capability to ferment monomeric sugars derived from lignocellulosic biomass into various valuable bioproducts [66].

In the traditional process, saccharification and fermentation are carried out in separate reactors under different conditions, a method known as separate hydrolysis and fermentation (SHF). In this approach, saccharifying enzymes such as cellulases and xylanases efficiently hydrolyze the biomass, with SHF providing the flexibility to optimize both processes under their respective ideal conditions. The primary drawback of SHF is the inhibition of cellulolytic enzyme activity caused by the accumulation of reducing sugars. Additionally, conducting the process in two separate reactors increases costs and extends processing time [67]. These limitations of SHF can be mitigated by employing a simultaneous saccharification and fermentation process, which integrates both steps into a single bioreactor. One effective method for producing lipids from lignocellulosic biomass is simultaneous saccharification and lipid production (SSLP), in which the two processes are performed in the same reactor at the same time. However, in SSLP, microbes rapidly consume the sugars that are produced during hydrolysis, reducing the inhibitory effects of glucose. Since SSLP uses a single bioreactor to complete the process, it is more economical than SHP. However, SSLP is affected by the fact that the process requires a temperature compromise because the optimal temperatures for hydrolysis and lipid production are different [68]. Intasit et al. [45] cultivated the oleaginous yeasts R. mucilaginosa G43 and Y. lipolytica 5054 using SHF with oil palm trunk hydrolysate, resulting in lipid production of 1.5 and 1.8 g/L, respectively. In contrast, SSLP improved lipid production by utilizing corn stover, where hydrolysis and lipid production occurred simultaneously. Using C. curvatus ATCC 20509 and C. tropica, lipid production increased to 8 and 15 g/L, respectively [69, 70]. Furthermore, it has been reported that fed-batch SSLP fermentation enhanced lipid production to as high as 26 g/L [71] (Table 2).

Table 2.

Microbial lipid production from lignocellulosic biomass

Feedstock	Microbial strain	Fermentation medium/mode	Lipid production (g/L)	Lipid content (%)	Ref
Oil palm trunk	R. mucilaginosa G43	SHF	1.55	30	[45]
Oil palm trunk	Y. lipolytica 5054	SHF	1.80	40	[45]
Corn stover	C. curvatus ATCC 20,509	SSLP	15.9	59.9	[69]
Corn stover	C. tropica	SSLP	8.62	-	[70]
Corn cobs	T. oleaginosus	fed-batch SSLP	26.7	50	[71]
Empty fruit bunches	Y. lipolytica Y5151	CBP	3.2	-	[47]
Empty fruit bunches	Rhizopus sp	CBP	-	51.8	[72]
cassava starch	L. starkeyi.	CBP	13.9	39	[73]
Napier grass	Cyberlindnera rhodanensis CU-CV7	CBP	1.01	26	[74]
Sugarcane Bagasse	Mortierella wolfii	CBP	1.57	41	[75]

SHF = Separate hydrolysis and fermentation

SSLP = Simultaneous saccharification and lipid production

CBP = Consolidated bioprocessing

Another approach for lipid production is consolidated bioprocess (CBP), which has become increasingly popular in the field of lignocellulosic bioethanol production. This technique simplifies the process by combining enzyme production, carbohydrate hydrolysis, and lipid production into a single step. For a CBP microorganism to be industrially viable, it must not only achieve high lipid productivity and concentration but also efficiently secrete enzyme to break down carbohydrates. Microorganisms suitable for CBP can be obtained through natural isolation or genetic engineering [27]. The development of CBP yeast strains for lignocellulosic lipid production has advanced this strategy. One approach involves conveying cellulose-degrading genes into oleaginous microorganisms through heterologous gene expression [20].

A study investigated CBP of the yeast Y. lipolytica Y5151 using empty palm fruit bunch residues, achieving lipid production of up to 3.2 g/L [47]. Additionally, Rhizopus sp. was fermented using the CBP process, which has been reported to effectively promote lipid production, with lipid accumulation exceeding 50% [72]. Furthermore, Zhang et al. [73] demonstrated that L. starkeyi cultivated using cassava starch in a CBP system achieved a high lipid production of 13.9 g/L. In contrast, fermentation using Cyberlindnera rhodanensis CU-CV7 with Napier grass resulted in lipid production of 1 g/L [74]. For the use of sugarcane bagasse in CBP fermentation with Mortierella wolfii, lipid production of 1.57 g/L was reported, corresponding to a lipid content of 41% [75] (Table 2).

Pilot-scale production of microbial lipids

Microbial lipid production for biodiesel is still in its early stages, and several challenges must be addressed to achieve industrial-scale production. These include improving cost-effectiveness, optimizing cultivation methods, and developing new reactor technologies. While some pilot and large-scale trials have been conducted in recent years, they primarily used vegetable oils, waste fats, and animal oils instead of single-cell oils. Further research is needed to achieve market-competitive lipid production, effectively utilize base components, and select appropriate equipment and cultivation techniques [76]. Key areas for investigation include using low-cost substrates to reduce raw material expenses, selecting bacteria resistant to contamination, and improving reactor performance to minimize external interference.

Researchers are investigating alternative raw materials to lower production costs in order to overcome these issues. Using less expensive substitutes has proven to work effectively for large-scale applications, but using substrates like sugar might result in greater costs. By using pentose sugars from lignocellulosic biomass, for instance, Banerjee et al. [77] achieved their goal to successfully scale up microbial lipid production. Through fed-batch cultivation, their study was successful to achieve a lipid concentration of 1.83 g/L in a 50-L bioreactor. Huang et al. [78] successfully demonstrated pilot-scale lipid production using corn stover in a 1000-L bioreactor, with a biomass yield of 10.2 g/L and a lipid content of 76.3% under phosphorus-limited conditions. Alongside the utilization of cost-effective substrates to decrease production expenses and enhance environmental advantages, innovative technologies and manufacturing techniques are also facilitating large-scale production of microbial lipids. During operations, equipment and reactors employed in various biochemical processes are prone to external disturbances [79]. In practical industrial settings, the cultivation of oleaginous yeast for microbial lipid production occurs in open environments, where potential contamination can lead to reduced yields. Thus, minimizing the impact of external interference is crucial for increasing lipid production. A study by Abeln et al. [80] demonstrated that M. pulcherrima could be used to operate a reactor in semi-continuous culture mode for over two months, maintaining stable and uncontaminated lipid yields, with peak biomass reaching 40 g/L and lipid concentration attaining 11.6 g/L. Yeast activity has been observed to enhance microalgae biomass productivity during the lag phase. In contrast, the photosynthetic activity of microalgae increases pH and dissolved oxygen (DO) levels, producing natural bactericidal and antifungal effects. To further improve microbial lipid production, strategies such as the supplementation of micronutrients and genetic modification of oleaginous yeasts have been suggested to enhance industrial feasibility. It is also notable that most pilot-scale studies have relied on batch or fed-batch cultivation systems, while limited research has focused on continuous and semi-continuous culture approaches for scale-up processes [81]. Soccol et al. [82] efficiently produced biodiesel on a pilot scale (1000 L) using microbial lipid from R. toruloides DEBB 5533 cultivated on sugarcane juice containing 15% (w/w). The study revealed that microbial biodiesel offers a higher yield and a competitive price compared to vegetable-based biodiesel, while also reducing pollutant emissions. Similarly, Xue et al. [76] developed a cost-effective pilot-scale fermentation process (300 L) for microbial lipid production from R. glutinis by using starch wastewater as feedstock. Under non-sterile conditions and without pH control, 40 h of cultivation resulted in 40 g/L of biomass containing 35% lipids content. According to Xie et al. [83], C. vulgaris is being grown on a trial scale to cleanse actual swine effluent and reduce carbon dioxide. Wet cell disruption by enzymatic transesterification produced the maximum biodiesel conversion (93.3%).

Lipid extraction

Transesterification, lipid extraction, harvesting, and culture are the four essential upstream and downstream stages in the conversion of microbial oil into biodiesel. Nevertheless, lipid extraction from oleaginous microorganisms is part of the expensive cell disruption method, which is a major barrier in the manufacture of biodiesel on a large scale [84]. The intracellular synthesis of lipids complicates downstream processing for lipid recovery, both in laboratory and large-scale settings [85]. Lipid extraction typically follows cell disintegration through pretreatment methods, succeeded by lipid recovery using organic solvents from the lysed biomass. The energy-intensive process of cell disruption requires drying or dehydrating the biomass, which increases the ultimate cost [86]. Chloroform and methanol mixtures are used for conventional lipid extraction techniques including the Bligh & Dyer and Folch methods, which are only suitable for use in research laboratories. To increase the effectiveness of cell disruption, other issues related to these techniques, such as extraction from dry biomass and the use of harmful organic solvents, need to be resolved [87]. Currently, oleaginous microorganisms are disrupted on a laboratory scale using a variety of mechanical, chemical, and enzymatic pretreatment techniques. These techniques include bead beating, autoclaving, high-speed and high-pressure homogenization, radiation from microwaves, ultrasonication, and thermolysis. Neither of these pretreatment methods, however, work effectively for large-scale operations [88]. Lipid extraction on an industrial scale typically occurs using a solvent system, which requires the biomass to be dry; otherwise, the organic solvents’ surface charges prevent them from efficiently reaching the cells and cause them to remain in the aqueous phase [89]. Lipid extraction should preferably be conducted in wet conditions due to the costs involved in drying biomass. Alternative cell disruption methods also present limitations concerning efficiency and lipid yield. For instance, oil extraction from seeds is generally conducted using simple mechanical methods, such as oil press or expeller press, which are also applicable for extracting oils from microalgae. However, there is no documented use of this approach for lipid extraction from oleaginous bacteria and yeast [90]. Furthermore, lipid recovery remains insufficient, and the drying of biomass again results in high energy and cost demands.

Bead beating, a mechanical method, obviates the need for a drying step, thereby reducing the overall cost of extraction. In this technique, a wet biomass slurry is agitated in a high-speed rotator containing fine beads. However, bead beating is limited to small sample sizes, rendering its application on a larger scale challenging [84]. The limitations associated with conventional methods can be addressed through alternative physical methods, such as microwave irradiation and ultrasonication. Ultrasonication is a widely employed pretreatment method for disrupting the cellular integrity of oleaginous microorganisms. This technique utilizes mild pressures and temperatures, making it straightforward, environmentally friendly, and time-efficient [91]. Additionally, it operates without the need for beads or chemicals. Nonetheless, a significant drawback of this technique is the generation of free radicals after extended treatment, which may adversely affect the quality of the extracted lipids. Cell disruption occurs due to the pressure exerted on the cell wall by vapors generated from the water within the cell. As a result, microwave radiation makes membranes permeable and is essential to the extraction of lipids. However, this approach is costly for commercial-scale applications due to its high-power costs [84]. Thus, scientists have looked at biological approaches as a viable and affordable substitute for mechanical techniques.

Fatty acid profile of oleaginous microorganisms cultivated on various lignocellulosic biomass

The fatty acid composition of oleaginous microorganisms cultivated on lignocellulosic biomass plays a crucial role in determining the efficacy of biofuel production. These lipid-accumulating microorganisms are employed in the conversion of plant-derived materials into biodiesel and other biofuels. The resultant fatty acid profiles exhibit variability based on several factors, including the specific lignocellulosic substrate, fermentation parameters, and yeast strain employed. Lignocellulosic biomass, encompassing agricultural byproducts, forestry residues, and other plant-based materials, represents a sustainable and abundant resource for microbial cultivation. Through various pretreatment methodologies, this biomass is transformed into fermentable sugars, which are then utilized by oleaginous yeasts for lipid accumulation [92]. These accumulated lipids, predominantly fatty acids, serve as the primary component in biodiesel production. The analysis of fatty acid compositions in yeasts grown on diverse lignocellulosic substrates offers valuable insights for optimizing lipid yields and enhancing biodiesel quality. The source of biomass can significantly influence the types of fatty acids produced, which in turn affects the performance characteristics of the resulting biodiesel. These characteristics include fuel stability, combustion efficiency, and environmental impact. Consequently, research focusing on the fatty acid profiles of oleaginous yeasts cultivated on lignocellulosic biomass is of paramount importance for advancing sustainable biofuel production technologies [93]. This investigation can potentially lead to the development of more efficient and environmentally friendly biofuel production processes, contributing to the global effort towards sustainable energy solutions.

Lipid-accumulating yeasts predominantly store fats in the form of diacylglycerols (DAG) and triacylglycerols (TAG). The typically produced fatty acids include myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), and linoleic acid (C18:2). The growth medium and cultivation conditions have an impact on the particular fatty acid content of these bacteria. For example, R. toruloides Y4 produced different amounts of C14:0, C16:0, C18:0, C18:1, C18:2, and C18:3 when cultivated in glucose synthetic medium including inhibitors [94]. Conversely, research has shown that R.kratochvilovae fermented mostly C14:0, C16:0, C18:0, C18:1, C18:2, and trace levels of C18:3 on a variety of substrates [95].

Several significant physicochemical properties were observed in the single-cell oil produced from fungi Mortierella sp. C16:0 (19.6%), C16:1 (0.3%), C18:0 (11.5%), C18:1 (38.2%), C18:2 (4.8%), C20:0 (3.3%), γ-linolenic acid (GLA) (8.1%) were among the lipid composition [96]. In addition, the FAME profile of fungal oils from A. tubingensis TSIP9 was mainly long chain fatty acids with C16-C18. These include 60.96% C16:0, 19.69% C18:1, 8.28% C18:0, and 4.55% C18:2 [56]. C. pyrenoidosa resulted in lipid compositions including C16:0, C16:1, C18:0, C18:1, C18:2, and C18:3, which accounted for 96.28–98.61% of the total fatty acids [97]. Additionally, Kumsiri et al. [98] reported that the fatty acids of microbial lipids from the co-cultivation of Piscicocus intestinalis WA3 with microalga Tetradesmus obliquus AARL G022 are C16-C18 and include γ-linolenic acid (C18:3n6, 1.96%), oleic acid (18:1n9, 23.02%), linoleic acid (C18:2n6c, 23.43%), linolenic acid (C18:3n3, 2.22%), and palmitic acid (C16:0, 29.61%). Patel et al. [99] found that Auxenochlorella protothecoides, when grown on hydrolysates from Norway spruce and silver birch, produced lipids at a concentration of 5.6 g/L. The lipid composition was mainly of C16:0 (9.3%), C18:0 (2.85%), C18:1 (70%), C18:2 (13.6%), and C18:3 (1.74%). Additionally, Chlorella vulgaris and Scenedesmus obliquus are acknowledged for their proficiency in lipid production. The study by Sibi et al. [100] involved fermenting these microalgae with hydrolysates from sweet sorghum and rice straw. The qualitative analysis of fatty acids revealed particularly elevated levels of stearic acid and palmitic acid, as shown in Table 3.

Table 3.

Fatty acid composition of microalgae from waste fermentation

Organism	C14:0	C16:0	C16:1	C18:0	C18:1	C18:2	C18:3	C20:0	C22:6	C24:1	Ref.
R. toruloides Y4	3	34.3	1.9	4.8	46.2	7.8	-	-	-	-	[94]
R. kratochvilovae	-	18.6	0.5	5.7	3.8	13.3	5.6	-	-	-	[95]
Mortierella sp.	-	19.6	0.3	11.5	38.2	4.8	-	3.3	-	-	[96]
A. tubingensis TSIP9	-	60.9		8.3	19.7	4.6	-	-	-	-	[56]
C. pyrenoidosa	1.3	3.1	6.0	-	3.7	2.5	-		35.5	37.	[97]
Piscicoccus intestinalis +  T. obliquus	-	29.6	-	-	23.0	23.4	-	-	-	-	[98]
A. protothecoides	0.7	9.3	0.26	2.8	70	13.6	1.7	-	-	-	[99]
C. vulgaris	11.4	23.5	0.4	28.4	16.5	0.2	-	8.0	-	6.3	[100]
S. obliquus	9.7	26.2	2.0	31.0	19.1	0.1	-	10.1	-	5.9	[100]

Microbe-derived oils are particularly high in saturated fatty acids. Cetane number (CN), iodine value (IV), cold filter plugging point (CFP), oxidative stability (OS), and other metrics are used to evaluate the quality of biodiesel. These measurements are frequently used to assess whether microbial oils are suitable as feedstocks for biodiesel. The IV indicates the possible production of partly oxidized chemicals that might result in deposits in engine injection systems by measuring the amount of unsaturated fatty acids in biodiesel samples. Biodiesel-derived oils usually satisfy IV international requirements. The CN value evaluates biodiesel’s combustion characteristics in relation to its fatty acid makeup. The length of the fatty acid chain and the degree of unsaturation have a significant impact. The degree of saturated fatty acids in biodiesel determines its filterability at low temperatures, as shown by the CFP value. The OS rating indicates how resistant the biodiesel is to deterioration over time. An IV of 19.83 (g I₂/100 g) was obtained from studies employing T. fermentans in glycerol minimum media with sweet potato vine hydrolysate [101]. The saponification value (SV) of bacterial lipids derived from lignin compounds was higher, ranging between 231 and 238 mg KOH/g of oil. SVs of 160.054 and 157.757 mg KOH were observed in biodiesel produced from Y. lipolytica cultivated in detoxified and non-detoxified wheat straw hydrolysate, respectively. The maximum SV of 203.958 mg KOH was shown by L. starkeyi ATCC 56304 grown in a biphasic system [102]. Microbial lipids from residues biomass had comparable higher heating values (HHVs), which ranged from 38.4 to 40.4 MJ kg^− 1. Oil having a density of 0.877 g cm⁻³ was generated by C. curvatus ATCC 20509 utilizing volatile fatty acids as a substrate [103]. Based on the composition of fatty acids, which are suitable for aviation fuel and found in microorganisms capable of producing lipid, numerous studies have further developed these lipids towards producing aviation fuel using microorganisms [104]. This is achieved through process innovation involving fermentation and catalytic upgrading. Typically, a two-step process is employed: (1) microbial fermentation to produce fatty acids or alcohols and (2) hydro-processing to convert these intermediates into drop-in jet fuels [105].

Conclusions and future prospects

The principal challenge in large-scale biofuel production is the substantial cost. Current feedstocks, including non-edible plant oils and lipids derived from microalgae, macroalgae, and yeast, encounter several limitations, such as low yields, slow growth rates, climate dependency, and processing difficulties. Consequently, these feedstocks are not yet economically competitive with fossil fuels. As a result, considerable research efforts are directed towards addressing these challenges. Future objectives aim to achieve cost-effective and environmentally sustainable biofuel production, with promising advancements being made towards these goals. Oleaginous microorganisms are being considered as promising non-plant feedstock candidates due to their high lipid bioaccumulation capabilities. The primary expense in yeast cultivation necessitates the development of cost-effective strategies, such as open pond cultivation, to reduce production costs. Utilizing low-cost substrates, often derived from waste streams, could significantly lower fermentation costs. Furthermore, engineering yeast strains to efficiently utilize xylose will further enhance this approach. Post-fermentation, the spent medium, which still contains nutrients, minerals, and residual sugars, must be appropriately treated before disposal to prevent water body eutrophication. Streamlining downstream processing steps, coupled with the valorization of waste in line with the “zero waste” concept, can significantly reduce overall production costs. Additionally, innovative biorefinery models have the potential to generate new business opportunities, create job openings, and enhance the economic status of the country while simultaneously supporting global environmental goals. Subsequently, it is essential to convert diverse waste streams into high-value products in order to balance the costs associated with low-value, high-volume products. This step should be succeeded by a life cycle assessment (LCA) to identify key unit operations and processes that significantly impact the overall environmental footprint, thus providing guidance for future enhancements. Advances in process conditions, energy inputs, and solvent utilization can enhance the ecological performance of the process.

Author contributions

R.I.: Conceptualization, Investigation, Data curation, Writing – original draft, B.S.K.: Conceptualization, Funding acquisition, Writing – review & editing.

Funding

This research was supported by the National Research Foundation of Korea (NRF-2019R1I1A3A02058523).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Competing interests

The authors declare no competing interests.