Effects of Lignocellulosic Biomass-Derived Hydrolysate Inhibitors on Cell Growth and Lipid Production During Microbial Fermentation of Oleaginous Microorganisms—A Review

Abstract

For efficient production of microbial lipids also known as single cell oil (SCO), selection of favorable growth conditions including the substrate for maximum conversion into storage lipids is imperative. Utilization of lignocellulosic biomass for microbial oil production is a promising approach as it is renewable, sustainable, and available in abundance, with a significant quantity of fermentable sugars. Because of their intricate structure and biomolecular composition, lignocellulosic substrates exhibit high recalcitrance and demand specific pretreatments to release the fermentable sugars. However, pretreating the lignocellulosic substrate not only produces assimilable sugars but also various fermentation inhibitors that can significantly impede microbial growth and/or lipogenesis. Therefore, in this review, we discuss different inhibitors present in the lignocellulosic hydrolysates, and the impact on oleaginous microbial growth and metabolic activity, particularly concerning lipid production. Furthermore, the mode of inhibition of the various inhibitors and potential strategies to detoxify these are discussed in this review.

1. Introduction

Lipids are important bulk chemicals that can act as alternative feedstocks for the oleochemical and renewable energy industries, replacing fossil fuels. However, the global lipid supply chain faces significant challenges, with a reliance on imports far exceeding that of crude oil, reaching up to 60%. Therefore, to avoid disruptions in external resource imports due to geopolitical instability, climate change, and global crises (such as pandemics and wars), there is an urgent need to develop innovate lipid production methods to ensure a stable supply of lipid resources. Utilizing lipid-producing microorganisms and leveraging inexpensive and abundant lignocellulosic biomass as raw materials for lipid biorefining is considered the most promising approach to lipid production. Microbial lipids have recently gained considerable attention as an alternative to vegetable and animal oils for producing biofuels and chemicals [1,2]. Compared to conventional lipid feedstocks, microbial lipids offer distinct advantages: they do not compete with food crops for arable land, have significantly shorter production cycles, and can be synthesized from a wide range of renewable carbon sources. Recent studies have shown that oleaginous yeasts, such as Yarrowia lipolytica, can efficiently utilize lignocellulosic biomass hydrolysates for lipid production, achieving intracellular lipid contents of up to 42% (w/w) in bioreactor cultures. This demonstrates their potential as promising candidates for sustainable microbial lipid production [3]. Moreover, Lipomyces starkeyi has shown the capability to efficiently produce lipids when cultivated on sap extracted from felled oil palm trunks, a novel and inexpensive renewable carbon source, achieving significant lipid yields under various sap medium conditions [4]. In addition to lignocellulosic materials, some organic wastes also serve as excellent raw materials, not only due to their low cost but also because of their potential as feedstock for lipid production, thereby facilitating biomass recycling [5]. For example, crude glycerol and thin stillage have been successfully used for the cultivation of Rhodotorula glutinis, resulting in a significant increase in biomass and altered lipid profiles, thus enhancing the economic competitiveness of biodiesel produced from single cell oils (SCOs) [6].

Despite these advancements, microbial lipid production still faces several key challenges, primarily arising from the presence of inhibitory compounds in lignocellulosic biomass hydrolysates. These inhibitors, generated during biomass pretreatment, negatively impact microbial growth and lipid biosynthesis, limiting overall yield and process efficiency. Microorganisms (yeasts and microalgae) have exhibited the ability to accrue lipids. Research has thoroughly validated the feasibility of producing microbial lipids from corn stover [7]. However, the transition from laboratory-scale studies to industrial-scale applications remains a significant hurdle, requiring further process optimization and economic feasibility assessments.

During the pilot phase, several critical issues were identified that require further refinement in upcoming research efforts. These findings will advance the industrialization of lipid biorefining and contribute to achieving dual carbon goals through bioenergy. Microbial oils are also known as SCOs. Due to their similar composition to that of edible plant oils, animal fats, and oils, these microorganisms can serve as effective substitutes. Moreover, oleaginous microorganisms offer an advantage because they do not compete for land resources and have much shorter growth cycles compared to traditional vegetable crops.

For the efficient production of SCO, selecting favorable growth conditions that facilitate the microorganisms’ capability to enhance/maximize the conversion of carbon substrates into storage lipids is vital. When assessing commercial applications, more emphasis should be placed on the expense of obtaining raw materials, as this typically constitutes 40–80% of overall manufacturing expenses [8]. That is why recent research has shifted towards cultivating oleaginous microorganisms using lignocellulosic substrates for microbial lipid production. Each year, around 181.5 billion tons of lignocellulosic biomass is produced across the globe [9]. Utilizing lignocellulosic biomass for producing SCO, various chemicals, fuels, food, and feed ingredients is an attractive approach as it is renewable, sustainable, and abundant, with a significant quantity of fermentable sugars [10]. However, the current research is still at the laboratory scale. To facilitate large-scale implementation, further advancements in pretreatment efficiency, fermentation strategies, and inhibitor mitigation techniques are required.

The conversion of lignocellulosic biomass into microbial lipids involves pretreatment, enzymatic hydrolysis, fermentation, and lipid extraction. During pretreatment, the breakdown of hemicellulose and lignin generates liquid-phase inhibitors such as furfural, hydroxymethylfurfural (HMF), and weak acids. While most inhibitors remain in the liquid phase, solid-phase lignin residues can also interfere with enzymatic hydrolysis, reducing sugar release. In the fermentation stage, residual inhibitors in the hydrolysate can hinder microbial growth and lipid accumulation. A clear understanding of how these inhibitors form and impact different stages is essential for optimizing lignocellulose-based lipid production.

Polysaccharides like cellulose and hemicellulose, which constitute about 60% of lignocellulosic biomass, underpin its suitability for bioprocessing. The remaining portion of the biomass primarily consists of a polyphenolic macromolecule known as lignin, which provides structural integrity to the biomass [11]. Given its intricate structure and complex biomolecular components, lignocellulosic substrates exhibit high recalcitrance.

Lignocellulosic materials must undergo a pretreatment process to break down their structure and release fermentable sugars. This can involve chemical, physical, or biological techniques [12,13,14]. Nevertheless, pretreating lignocellulosic substrate not only produces assimilable sugars but also various byproducts that can considerably impede microbial growth and/or lipogenesis [15]. These inhibitory compounds, including furfural, hydroxymethylfurfural (HMF), weak acids, and phenolics, are primarily formed in the liquid phase during biomass hydrolysis and significantly hinder microbial metabolism.

While microbial lipid production from lignocellulosic biomass has been reviewed extensively, most existing studies focus on general lipid biosynthesis pathways or broad fermentation strategies. In contrast, this review takes a more targeted approach by examining how inhibitors generated during biomass pretreatment affect microbial metabolism and lipid accumulation. In addition, it summarizes recent advances in detoxification strategies and fermentation optimization, which are crucial for improving process efficiency. By addressing these challenges and potential solutions, this review aims to provide a clearer perspective on how lignocellulose-based microbial lipid production can be further developed for large-scale applications.

Although microbial lipid production from lignocellulosic biomass has made significant progress, several challenges still hinder its large-scale application. One major issue is the presence of inhibitors such as furfural, hydroxymethylfurfural (HMF), and weak acids, which negatively impact microbial growth and lipid synthesis. While previous studies have identified these inhibitors, the exact metabolic and genetic mechanisms by which oleaginous microorganisms tolerate and adapt to them remain insufficiently understood. Additionally, various detoxification strategies have been explored, including chemical, enzymatic, and microbial approaches, but there is still no consensus on the most efficient and cost-effective method, particularly for industrial-scale applications. Another limitation is the difficulty in fully utilizing lignocellulosic hydrolysates, as microorganisms often exhibit low conversion efficiency when metabolizing mixed sugar substrates. This leads to suboptimal lipid yields and process inefficiencies. Furthermore, most studies on microbial lipid production are conducted at the laboratory scale, often in shake flasks or small bioreactors, and the transition to industrial-scale fermentation presents challenges related to oxygen transfer, agitation, and inhibitor removal.

Therefore, this article aims to provide a thorough insight into the diverse inhibitors produced during lignocellulosic fermentation/degradation and their impact on microbial growth and metabolic activity, particularly concerning lipid production. Furthermore, it seeks to bridge the knowledge gap between biomass pretreatment, microbial inhibition, and lipid biosynthesis by discussing potential strategies to mitigate the effects of inhibitors. This will provide readers with an overview of strategies and key issues that should be considered while using lignocellulosic biomass as a feedstock for lipid production by oleaginous microorganisms.

2. Pretreatment Methods

The method of pretreatment is vital to formation of inhibitory substances. During the pretreatment process, the dissolution and degradation of hemicellulose and lignin lead to the generation of inhibitors derived from lignocellulose. The choice of pretreatment method significantly affects the type and concentration of inhibitors generated. Dilute acid hydrolysis, commonly used to break down hemicellulose and improve sugar release, often leads to the formation of furfural and hydroxymethylfurfural (HMF) due to the degradation of pentose and hexose sugars, respectively. These compounds can inhibit microbial growth by interfering with enzymatic activity and metabolic pathways. Alkaline pretreatment, which primarily targets lignin removal, results in the release of phenolic compounds. These phenolics, originating from lignin degradation, are known to disrupt microbial cell membranes and inhibit enzyme function, thereby reducing lipid production efficiency. Steam explosion and ammonia fiber expansion, while effective in disrupting biomass structure, can also generate weak acids (e.g., acetic acid), phenolics, and furan derivatives, all of which place additional stress on microbial metabolism. In contrast, ionic liquid pretreatment has gained attention as a potentially milder alternative, but residual ionic liquids may still be toxic to microorganisms, depending on their chemical composition and concentration. Understanding how different pretreatment strategies contribute to inhibitor formation is essential for optimizing lignocellulosic biomass utilization and improving microbial lipid yields.

To lessen the impact of inhibitors, two approaches involve adopting advanced hydrothermal biomass pretreatment techniques that can yield sugars suitable for fermentation while reducing material costs and minimizing toxic byproducts, and the utilization of a broader array of non-model yeasts that possess the ability to withstand elevated concentrations of inhibitors and effectively incorporate them [16,17]. Stringent pretreatment enhances biomass deconstruction but may also degrade fermentable sugars, affecting yield efficiency [18].

2.1. Alkaline/Dilute Acid Pretreatment

Alkaline pretreatment effectively extracts lignin and solubilizes a portion of hemicelluloses (usually over 40% w/w) while minimally degrading the cellulose. Alkali pretreatment uses substances like NaOH, lime, or ammonia to remove lignin and some hemicellulose. This process improves the accessibility of cellulose for enzymes, ultimately enhancing saccharification efficiency [19]. At present, several innovative alkali-based pretreatment techniques have been introduced. Alkaline pretreatment hydrolysis (such as 2% NaOH at 50 °C for 6 h) resulted in over 80% of the theoretical yield. Alkali-pretreated (ALP) corn cob was effectively converted to microbial lipids by Mycobacterium Mortierella isabellina strain DSM 1414. From the hydrolysate of this treated corn cob (LH_ALP), the maximum lipid concentration reached 11.7 g/L [20]. Results indicated that the treatment for recovering hemicellulose was at 210 °C for 2 min. The highest cellulase conversion rate reached up to 90% when a lower temperature of 200 °C was combined with a longer treatment time [21]. Compared to acid treatment, alkali treatment is most effective in breaking ester bonds between lignin, hemicellulose, and cellulose while preserving the hemicellulose polymer [22]. During this method, lignin and hemicellulose are removed, leading to improved thermodynamic stability, greater porosity, hemicellulose hydrolysis, and reduced density and crystallinity. This technique is applicable to biomass like sugarcane, bulrush residues, cotton stalks, and corn stover. Alkaline pretreatment further enhances the accessible area of cellulose and hemicellulose fibers while removing lignin. The drawbacks of this process involve salt formation, extended hydrolysis times, and elevated operational costs [23]. A recent investigation involved the application of sequential alkaline wet oxidative pretreatment (AWOP). The theoretical recovery of pentose reached 89.57%, with residual cellulose at 72.1% [24]. The yeast Rhodosporidium babjevae using an alkaline-organosolv (ethanol 60% (w/w) and NaOH 2% (w/v)) pretreatment method resulted in a lignin removal rate of 81.9%, leading to the release of 46.7 g/L of sugar through hydrolysis [25].

Acid pretreatments effectively disrupt the lignocellulosic structure by targeting the glycosidic bonds that link lignin and hemicelluloses. This process solubilizes a significant portion of hemicelluloses (typically exceeding 90% by weight), while also causing a reduction in cellulose content and partial lignin removal. Dilute acid pretreatment dissolves hemicellulose and lignin by applying temperatures between 160 and 220 °C, using 0.05% to 5% acid. The reaction time can vary, ranging from several seconds to a few minutes [21]. Regarding glucose production, it has been observed that glucose yields from corn cob under conditions of 1% H₂SO₄ at 120 °C for 20 min and 2% H₂SO₄ at 120 °C for 10 min exceeded 80% of the theoretical yield [26]. Integrating acid pretreatment with elevated temperatures or enzymes significantly contributes to achieving the desired outcomes, particularly with dilute acids. Using carbon dioxide for acid pretreatment at a temperature of 200 °C facilitates the hydrolysis of hemicellulose under acidic conditions. This process generates carbonic acid, which enhances the efficiency of enzymatic hydrolysis [27]. The acid treatment process requires numerous steps, which consequently raises both operating expenses and duration. In spite of these challenges, its high efficiency ensures its continued use in producing various biofuel products.

Identifying the parameters of this pretreatment represents a highly promising research direction for the future. Both acid and alkaline pretreatment methods have certain limitations. To overcome these limitations, other promising methods can be integrated to improve the pretreatment efficiency, including physical techniques such as heating or applying magnetic fields.

2.2. Hydrothermal Treatment

Steam explosion is an effective pretreatment technique. It works by subjecting lignocellulosic materials to high-pressure steam and then rapidly releasing the pressure. The application of the steam alters the structure of the cell wall, breaking it down and forming a slurry [15].

Hydrothermal treatment is a method of pretreating lignocellulosic biomass using liquid- or vapor-phase water [28]. This is a comparatively gentle pretreatment technique, requiring no catalyst and causing minimal decomposition or oxidation. The process of hemicellulose dissolution is driven by hydronium ions generated through the self-ionization of water. Keeping the pH close to neutral helps reduce the inhibitors.

2.3. Ionic Liquid Pretreatment

Integrated approaches to utilizing lignocellulosic biomass have also garnered attention in recent years. A recent study pioneers a method that converts carbohydrates and lignin in corn stover into microbial lipids and advanced carbon nanofibers for energy applications using ionic liquid-based organic electrolytes [29]. The chemical sector and academic community view ionic liquids (ILs) as pivotal to the green chemistry movement. Their unique properties make them potential substitutes for toxic and environmentally harmful organic solvents [30]. A wheat straw sample (150 mg) was incubated in a mixture of 50% IL and seawater (3 g), under a nitrogen atmosphere, with continuous stirring at 150 °C and 500 rpm for 1.5 h [31]. Following pretreatment, there was a significant change in biomass composition: the polysaccharide content was boosted significantly, whereas the lignin content showed a marked reduction. Many ILs can effectively dissolve cellulose. This process has been validated through molecular dynamics studies [32], NMR [33], and simulation studies [34]. To effectively solubilize cellulose, it is essential to break the intermolecular hydrogen bonds between its chains [35]. The shape and dimensions of the anion also influence the solubilization of cellulose. Larger anions, for instance, tend to hinder solubilization in ILs due to the delocalization of charges, which impacts their performance [36]. Ionic liquids like [amim]Cl, [emim]CH₃COO, [emim]Cl, and [bmim]Cl are frequently chosen as green solvents in the pretreatment processes for dissolving lignocellulose biomass [37]. Certain ionic solvents can effectively dissolve cellulose with a yield of up to 75% within the temperature range of 50–100 °C. Additionally, increasing the temperature above 100 °C can also improve solubility for some solvents. Ionic liquids, due to their unique properties, have become a strong alternative for pretreating lignocellulosic biomass. Developing eco-friendly ionic liquids remains essential. Additionally, optimizing recovery technologies is necessary to reduce costs and minimize environmental impact. Effective adjustment of processing parameters and improving ionic liquid recovery efficiency are crucial for achieving efficient and sustainable pretreatment.

Table 1. Formation of inhibitors in hydrolysates after different pretreatments.

Lignocellulose Source	Method	5-HMF	Furfural	Levulinic Acid	Acetic Acid	Formic Acid	Phenolics	Reference
Corn stover	Acid hydrolysis	0.044 g/L	0.22 g/L	0.041 g/L	0.17 g/L	0.12 g/L	-	[38,39]
Corn stover	Acid hydolysis	15.7 mg/g biomass	7.94 mg/g biomass	3.65 mg/g biomass	34.77 mg/g biomass	3.17 mg/g biomass	-	[40]
maple wood	230 g/L with hot water at 200 °C for 20 min	4.1 g/L (5-HMF + Furfural)	4.1 g/L (5-HMF + Furfural)	-	13.1 g/L	-	-	[41]
Corn stover	Wet oxidation	0.0028 g/L	0.0065 g/L	0.0019 g/L	0.058 g/L	0.079 g/L	-	[38]
Corn stover	AFEX	0.642 mg/g biomass	0 mg/g biomass	0.024 mg/g biomass	4.61 mg/g biomass	0.91 mg/g biomass	-	[40]
Wheat straw	Steam explosion	-	0.30 ± 0.01%	-	0.72 ± 0.01%	-	1.49 ± 0.02%	[42]
Reed	Steam explosion	-	1.16 ± 0.04%	-	1.32 ± 0.00%	-	1.71 ± 0.04%	[42]
Corn stalks	Steam explosion	-	0.59 ± 0.00%	-	1.17 ± 0.01%	-	1.54 ± 0.03%	[42]
Pine	Wet oxidation	0.00064 g/L	0.0019 g/L	0.0005 g/L	0.024 g/L	0.066 g/L	-	[38]
Wheat straw	Catalyzed acid steam explosion treatment	0.9 ± 0.1 g/L (5-HMF + Furfural)	0.9 ± 0.1 g/L (5-HMF + Furfural)	-	1.9 ± 0.2 g/L	-	-	[43]

provides a summary of the formation of inhibitors from different lignocellulosic materials and under various pretreatment conditions.

3. Inhibitors and Mechanisms of Inhibition

Converting wood-based biomass into more valuable products demands pretreatment to separate the fractions of cellulose, hemicellulose, and lignin. Lignin is a complex aromatic polymer that provides rigidity and structural support to the plant cell wall. Effective pretreatment disrupts plant cell wall components, facilitating microbial conversion [44]. Specifically, the primary goal of pretreatment is to lower cellulose crystallinity, enhance the surface area for enzyme interaction with cellulose, and boost the efficiency of hydrolysis, while also lessening the influence of inhibitors [45]. As the primary constituent of lignocellulose, cellulose consists of numerous glucose molecules connected by β-1,4-glucosidic linkages. The pretreatment process facilitates the hydrolysis of cellulose polymers by breaking down the β-1,4-glucosidic bonds, which decreases crystallinity and yields glucose or oligosaccharides [1]. Additionally, depending on the pretreatment method used, the hemicellulose fraction can be recovered and utilized in various biotechnological processes, thereby enhancing the overall efficiency of lignocellulosic biomass conversion. To effectively utilize these carbon sources, it is essential to pretreat lignocellulosic biomass for biofuel-producing microorganisms [46].

Over the years, various methods for pretreatment have been introduced. These include chemical processes like dilute acid (DA) and alkali; physical methods such as heat, microwave, extrusion, and ultrasonic treatment; biological techniques that utilize whole cells and enzymes; and hydrothermal pretreatment methods like steam explosion, which is widely used for its efficiency in breaking down lignocellulosic structures [47]. Lignocellulosic biomass intended for liquid biofuel production is typically subjected to an acidic thermochemical pretreatment. This step improves the cellulose’s accessibility to enzymatic hydrolysis [48,49]. Nevertheless, the severity of acid pretreatment (elevated temperature or higher acid concentration) positively correlates with the formation of fermentation inhibitors. These inhibitors, including furan derivatives and phenolic compounds, significantly obstruct microbial fermentation [50]. Pretreatment of lignocellulosic biomass using chemical, physical, or biological methods significantly improves the availability of cellulose. Although acid pretreatment efficiently removes hemicellulose and lignin, and accelerates cellulose hydrolysis, it also produces fermentation inhibitors, which lower fermentation efficiency and add complexity to subsequent processing.

Pretreatment of lignocellulosic feedstock disrupts its structure and results in the production of various inhibitors [51], including furans resulting from the excessive breakdown of glucose and xylose, weak acids generated during the degradation of hemicellulose, and fatty acids that result from the transformation of furfural. Furthermore, phenolic compounds are formed through the decomposition of lignin, which includes guaiacyl and syringyl moieties found in many phenolics [52,53]. Nevertheless, certain byproducts derived from sugars, such as furfural and 5-HMF, at concentrations of 2.0 g/L and 4.0 g/L, respectively, can reduce the efficiency of subsequent enzymatic hydrolysis and hinder the growth and metabolic activities of many fermentation microorganisms [54]. To address these issues, future research needs to focus on the following directions: optimizing the conditions for acidic pretreatment to reduce inhibitor production; development of highly effective inhibitor removal technologies; and the use of advanced biotechnology, such as modifying yeast strains to improve their tolerance to inhibitors. In addition, exploring joint applications with other pretreatment methods, as well as new environmentally friendly materials and technologies, will help improve the economics and sustainability of overall biofuel production.

Inhibitory compounds derived from sugars include furfural derivatives, like furfural and HMF, which result from the dehydration processes of pentose and hexose sugars. Furfural is produced during the degradation of xylose under elevated temperatures and pressures. In contrast, HMF is generated during the breakdown of hexose. These two substances notably influence enzymatic hydrolysis, particularly when present at concentrations of >2.0 g/L for furfural and >4.0 g/L for HMF [55]. Phenolic substances are another group of inhibitors produced by the decomposition of lignin. They can appear in three distinct forms: acids, ketones, and aldehydes, including compounds such as vanillic acid, syringic acid, vanillin, and syringaldehyde. Along with other inhibitory derivatives of phenols, 4-hydroxybenzoic acid, ferulic acid, and guaiacol are prevalent products of lignocellulosic acid hydrolysis [56,57]. Figure 1 illustrates the key stages of lignocellulosic biomass (LCB) conversion into microbial lipids, highlighting the formation of inhibitors at different phases (solid and liquid) throughout the process.

4. Effects of Inhibitors on Cell Growth and Microbial Oil Production

It was found that Rhodotorula glutinis was highly tolerant to corncob hydrolysate inhibitors. In a 5 L fermenter batch culture with an optimized carbon-to-nitrogen (C/N) ratio of 75, the cells exhibited robust growth. The hydrolysate remained untreated and, as a result, the lipid titer reached 5.5 g/L, while the lipid content was measured at 36.4%. The biomass measured 70.8 g/L and the concentration of lipids was 33.5 g/L, which constituted 47.2% of the total content [58].

Oleaginous microorganisms accumulate lipids by first absorbing and utilizing sugars through glycolysis. This process is followed by lipid synthesis, which involves a series of enzymatic reactions [59]. When the solid loading rate of the pretreated biomass reached 7.5%, the lipid yield from the sugars in the biomass reached 40%. Additionally, the culture achieved a lipid concentration of 6.46 g/L [60]. For Schizochytrium sp. HX-308, cell growth and lipid accumulation remain largely unaffected when furfural concentration is below 1.2 g/L. However, as the furfural concentration increases to 1.8 g/L, the cell dry weight (CDW) and total lipids (TL) decrease by 57.7% and 58.5%, respectively. Interestingly, the addition of acetic acid at levels below 2.0 g/L led to a marked increase in both the CDW and TLs of Schizochytrium sp. HX-308 [61]. It is evident that controlling the concentration of inhibitors is essential and can even have a positive impact on microorganisms. Certain strains of oleaginous yeasts have demonstrated exceptionally high lipid titers when cultivated on lignocellulosic hydrolysates that have not undergone detoxification (39–45% of theoretical yield) [62]. Significant advancements in lipid production from cellulosic sugars were observed in three strains of Yarrowia lipolytica, including YB-392, which achieved a 64% improvement in cellulosic biomass conversion. Additionally, Candida phangngensis PT1-17 emerged as the top lipid producer, with a maximum lipid titer of 9.8 g/L [63]. Even in the presence of model-inhibiting compounds, Rhodococcus opacinus PD630 showed robust growth in R2A medium while accumulating triacylglycerol (TAG), and strain PD630 can co-metabolize furfural and 5-hydroxymethylfurfural [64]. Additionally, theoretical studies indicate that the maximum lipid yield from glucose metabolism can reach 32%, assuming all acetyl-CoA is directed toward lipid biosynthesis [65,66]. However, under practical fermentation conditions, metabolic losses, inhibitory effects, and process inefficiencies reduce actual lipid yields to approximately 22% [67]. In lignocellulose-derived hydrolysates, the presence of additional inhibitors further reduces conversion efficiency, leading to yields significantly below theoretical expectations [68].

Within oleaginous microorganisms, ATP citrate lyase (ACL) and malic enzyme (ME) are crucial for the accumulation of lipids. ACL primarily functions to produce acetyl coenzyme A, a key component necessary for lipid biosynthesis. Meanwhile, ME contributes by producing NADPH, a reducing power necessary for the biosynthesis of lipids [69]. Several byproducts from lignocellulose pretreatment inhibit the growth of oleaginous microorganisms, enzyme biocatalysts, and lipid accumulation [15,70]. Specifically, these harmful compounds hinder the growth of microorganisms involved in fermentation by disrupting their ability to absorb sugars and reducing the rates at which they produce products [71]. However, some studies have found that low concentrations of inhibitors may actually promote the growth of lipid-producing microorganisms. Controlling the concentration of inhibitors is a promising research direction for the future.

4.1. Weak Acid

Hemicellulose undergoes deacetylation, leading to the formation of acetic acid. In contrast, formic acid is generated as a secondary product during the breakdown of HMF and furfural [72,73]. The primary reason for the inhibitory impact of weak acids is the phenomenon of uncoupling. This process disrupts the normal functioning of cellular respiration, leading to decreased energy production [74]. Weak acids that remain undissociated are soluble in lipids and can readily penetrate the plasma membrane. Once they enter the cell, the higher intracellular pH causes these acids to dissociate, resulting in a reduction in cytoplasmic pH (Figure 2). This leads to the pumping out of protons, resulting in ATP depletion and anion accumulation, which negatively impact cell viability and biomass production [75]. Formic acid has a pKa of 3.75, which is lower than acetic acid’s pKa of 4.76, and that of levulinic acid, with a pKa of 4.64. At the same molar concentration, formic acid significantly affects intracellular pH, making it more toxic than acetic acid [76]. For that reason, understanding the impact of formic acid on microbial growth could inform future strategies for optimizing biomass production and improving the efficiency of oleaginous processes.

Research indicates that low levels of acetic acid and formic acid do not inhibit the growth of the oleaginous fungus Mortierella isabellina. In fact, these acids can enhance lipid production significantly. When the concentrations of acetic acid and formic acid were 4 g/L and 2 g/L, respectively, the yields reached 6.81 ± 0.07 g/L and 6.66 ± 0.33 g/L—double the amounts observed in the control group (without the addition of any weak acids) [77]. Recognized as a promising substrate or co-substrate, acetic acid also serves as a stimulant for lipid production in different types of oleaginous microorganisms, including Umbelopsis (Mortierella) isabellina, Curaneotrichosporon curvatum, Cyberlindnera (Williopsis) saturnus, Rhodotorula (Rhodosporidium) toruloides, Lipomyces lipofer, and Yarrowia lipolytica [54,72,78]. Cryptococcus curvatus can utilize 40 g/L acetic acid as the sole carbon source without significantly affecting cell growth [79]. Moreover, Cryptococcus curvatus can efficiently produce lipids from corn straw hydrolysate that contains 15.9 g/L acetate. The lipids produced from acetate exhibit a fatty acid composition that closely resembles that of vegetable oil. This similarity underscores their promising potential for biodiesel production [80]. When the concentrations of acetic acid and formic acid were set at 50.0 mM and 65.2 mM, Mortierella isabellina achieved maximum lipid concentrations of 10.13 g/L and 9.11 g/L, respectively [81]. Therefore, identifying oleaginous microorganisms with strong tolerance to lignocellulosic hydrolysis inhibitors could partially address this issue.

4.2. Furfural and 5-HMF

Dehydration of pentose and hexose sugars to furfural and HMF, respectively, is a well-documented process [54]. Saccharomyces cerevisiae is capable of metabolizing furfural in the presence of oxygen [82], and in oxygen-limited [83] and anaerobic conditions [84]. Furfural is produced from xylose-rich vegetable waste, such as corncob [85], and the hemicellulosic fraction of rice husks [86]. The pentoses produced during acid hydrolysis undergo an acid-catalyzed dehydration at elevated temperatures to generate furfural, whereas the dehydration of hexoses, primarily involving fructose and glucose, can lead to the formation of HMF, a process facilitated by acid catalysts [87]. Furfural and HMF exert detrimental effects on key enzymes critical for metabolic processes, including ethanol dehydrogenase, pyruvate dehydrogenase, and acetaldehyde dehydrogenase. They also hinder the activity of glycolytic enzymes, such as hexokinase and glyceraldehyde 3-phosphate dehydrogenase [76,79]. The primary mechanism behind the inhibitory effects of these compounds is linked to their reactive aldehyde groups, which can produce reactive oxygen species (ROS) (Figure 2). The generation of ROS contributes to a range of cellular damage, including DNA mutations, protein misfolding, and compromised cell membrane integrity [87]. The repair of these damages depletes intracellular ATP, NADH, and NADPH levels, causing growth inhibition and extended lag phases [88]. The NAD⁺/NADH ratio is altered by furfural, which subsequently inhibits cell growth and glycolytic pathways [89].

Research indicates that furfural negatively impacts various growth parameters, including the specific growth rate, ATP yield per cell mass, and overall cell volume [71,84,90]. Syringaldehyde emerged as the most harmful inhibitor among the various compounds tested, completely halting the growth of Bacillus subtilis at a concentration of 0.1 g/L. Research indicates that the combination of benzoic acid and furfural serves as the most effective inhibitor, significantly hindering the growth of Bacillus subtilis [91]. At low concentrations (<2 mM), the inhibitory effect of furans and aromatic aldehydes on fermenting Trichosporon fermentans CICC 1368 was minimal, as confirmed by sugar consumption measurements taken after 7 days of fermentation [92]. For oleaginous yeast cells of the Starkeyi species, the inhibitory effect of HMF was not significant up to a concentration of 500 mg/L. Despite the presence of inhibitors in the medium, the fatty acid profile of the lipids remained unchanged [93]. Research has shown that when the vegetative mycelium of Mortierella isabellina is used as an inoculum in place of spores, the culture exhibits enhanced resistance to furfural and HMF [20]. In the future, it would be valuable to explore whether other physical methods can detoxify these types of small molecule inhibitors. Additionally, studying the adaptability of different species of lipid-producing yeasts to these inhibitors could also be significant.

4.3. Phenols

When lignocellulosic materials are subjected to acid-catalyzed hydrolysis or pretreatment, various phenolic compounds emerge as a result of lignin breakdown. Regardless of the presence of an acid catalyst in the reaction, a range of aromatic substances is produced [52,94]. These phenols, primarily derived from hemicellulose acetyl groups, include lilac aldehyde, p-hydroxy benzaldehyde, and vanillin, among others, with vanillin being the most potent inhibitor of microbial viability [54]. Aromatic compounds, including phenols, have the potential to hinder microbial growth and reduce the overall yield of products, exhibiting widely varying effects that may be linked to specific functional groups [52,95,96]. It is important to recognize that phenolic compounds have limited water solubility, which is influenced by different functional groups. Furthermore, this solubility depends on the liquid composition and may vary between hydrolysates and defined media. Therefore, high concentrations of certain compounds may not reflect the actual levels experienced by microorganisms.

Research suggests that phenolic compounds can be more toxic than other strong inhibitory substances (such as furfural, weak acids, and various degradation byproducts). Their small molecular size enables them to easily pass through cell membranes, disrupt internal structures, and co-precipitate with cellulase, resulting in alterations to cell morphology [15,71,97,98,99]. Specifically, phenols contain hydroxyl, carboxyl, and formyl groups that can compromise cell membranes, consequently hindering cell growth and glycation [100]. Research has also explored how phenolic compounds inhibit cellulase activity during hydrolysis [101]. Certain experiments with phenols suggest that they induce protein precipitation as one of the mechanisms affecting proteins [41]. Phenolic compounds, including vanillin, can lead to the breakdown of biofilms, impacting their function as barriers and substrates for enzymes [76]. Interestingly, phenolic compounds with higher molecular weights tend to be less harmful to microorganisms compared to those with lower molecular weights [57].

Among various inhibitors, phenolic compounds stand out as the most effective in hindering the growth and lipid accumulation of Mortierella isabelline. In phenolic compounds, ferulic acid exhibited the least inhibition on lipid synthesis, followed by 4-hydroxybenzaldehyd [20].

4.4. Effect of Combinations of Aldehydes on Cell Growth and Lipid Accumulation

Lignocellulosic hydrolysates typically contain multiple inhibitors, and the interactions between these different inhibitors can be complex [102,103]. The inhibitors alone may not have a strong effect on the fermentation microbe, but their combination can greatly hinder the fermentation reaction. Oleaginous yeast Rhodosporidium toruloides Y4 is a potential oleaginous microorganism, able to accumulate more than 70% lipid with a titer of more than 100 g/L [104].

For the oleaginous yeast Rhodosporidium toruloides Y4, the following combinations: 8 mM HMF and 9 mM vanillin or 9 mM vanillin exhibit a relative growth ratio close to zero compared to the additive effect of individual inhibitors, indicating strong synergistic interactions of these combinations [105]. These findings highlight the importance of developing strategies to enhance yeast tolerance against such inhibitory compounds. Future research could focus on screening for novel yeast strains with high lipid production efficiency or employing random mutagenesis techniques combined with rapid screening methods to identify robust, high-performing microbial strains.

On the other hand, the combination of furfural and 5-HMF showed less inhibition of the oleaginous yeast Trichosporon fermentans CICC 1368 compared to each inhibitor used alone [92], which is contrary to the findings for ethanologenic Escherichia coli [106]. If we can combine high resistance to inhibitors with high oil production efficiency, it will significantly enhance the industrial potential of SCO.

Table 2. Effects of different inhibitors on the growth and lipid production of oleaginous microorganisms.

Fermentation Process	Microbial	Inhibitor	Inhibitor Concentration	Biomass Decline (%)	Lipid Concentration Decreased (%)	References
Microbial growth	Yarrowia lipolytica	acetic acid	75 mM	100	-	[107]
	Yarrowia lipolytica	formic acid	37.5 mM	100	-	[107]
	Rhodosporidium fluviale DMKU-SP314	formic acid	0.5 g/L	100	-	[72]
	Rhodosporidium fluviale DMKU-SP314 Rhodosporidium toruloides Rhodosporidium toruloides Rhodosporidium toruloides	acetic acid formic acid acetic acid furfural	1.0 g/L 2, 4 g/L 5, 10, 20 g/L 1.0 g/L	72 25, 40 15.6, 50, 100 60	97 - -	[53,72]
	Rhodosporidium toruloides Y4 Mortierella isabelline DSM 1414 Mortierella isabelline NRRL 1757 Mortierella isabelline NRRL 1757	furfural furfural furfural 5-HMF	1 mM 21.8 mM 2.0 g/L 2.0 g/L	45.5 77 11 25	26.5 84 3 23	[20,105,108]
Lipid accumulation	Trichosporon fermentans CICC 1368	furfural	2.1, 4.7 mM		25, 50	[92]
	Trichosporon fermentans CICC 1368	HMF	15.1, 37.7 mM		25, 50	[92]

shows a summary of various studies that examine how different inhibitors affect the growth of oleaginous microorganisms.

5. Strategies for Detoxification of Lignocellulosic Biomass-Derived Inhibitors

5.1. Enzymatic Detoxification

Utilizing enzymes for detoxification offers a valuable biotechnological approach to treat lignocellulosic hydrolysates. Enzymatic biodetoxification methods are receiving attention as a more environmentally friendly strategy that can be substrate-specific and offer the possibility of increasing the rate of glycosylation and fermentation, thereby reducing processing time without consuming carbohydrates and under mild reaction conditions [109]. Laccase can oxidize various phenolic and non-phenolic compounds. Additionally, it helps to reduce lignin content before pretreatment methods, enhancing lignocellulose hydrolysis and the release of fermentable sugars [110]. To detoxify willow hemicellulose hydrolysate, enzymes such as laccase and peroxidase were extracted from Trametes versicolor [15]. Both the enzymes could effectively remove phenols and other acidic compounds [52]. Reports indicate that laccases from Gluconacetobacter xylinus are capable of detoxifying furan and phenolic derivatives; additionally, when redox mediators are present, these enzymes can also interact with phenolic ketone derivatives [111].

Treatment with laccase and pre-fermentation with Trichoderma reesei, a filamentous fungus, can reduce the negative effects of inhibition in fermentation to varying degrees. The process of detoxifying hemicellulose hydrolysates employs enzymes such as peroxidase (derived from Armoracia rusticana) and superoxide dismutase (sourced from Coptotermes).

5.2. Combination/Integration of Different Strategies/Measures

Various alternative strategies can be implemented to mitigate the challenges posed by inhibitors. The concentration of these inhibitors, as well as the sugars present in the hydrolysate, is affected by the type of raw material and the conditions applied during both pretreatment and hydrolysis. Therefore, it may be possible to choose less recalcitrant raw materials and use milder pretreatment conditions.

The fermentation process can be designed to address inhibition challenges. Implementing simultaneous saccharification and fermentation (SSF) is a practical strategy that effectively prevents sugar-induced inhibition of cellulolytic enzymes. Alternatively, employing batch feeding or continuous culture methods can be more beneficial than using conventional batch processing [112]. The fermentation process of Lipomyces starkeyi may be conducted by involving a method of recovering spent cell masses, mineral nutrients, and water. It was demonstrated that hydrolysates derived from spent cell masses provide an ideal medium for the growth of various oleaginous yeast cells [113]. Research has shown that using oleaginous yeasts Lipomyces starkeyi and Rhodotorula toruloides in two-stage culture mode is a novel and efficient way to produce microbial lipids in D-xylose solution [114]. When choosing fermentation processes, key factors include achieving high yields and productivity, ensuring a high product titer, and allowing for the potential recycling of water.

Selecting suitable strains is also an effective strategy. Lipomyces starkeyi is able to produce microbial lipids effectively by using an amalgamation of cellobiose and xylose as carbon sources. This method of co-fermentation not only mitigates glucose-induced inhibition but also aims to enhance the economic viability of lipid extraction from lignocellulosic biomass [115]. Rhodococcus opacus PD630 can use vanillic acid and trans-p-coumaric acid (TPCA) to promote cell growth and accumulate triacylglycerol (TAG). These results indicate that this strain can effectively utilize hydrolyzed materials derived from grass, corn straw, and sorghum for TAG production [64].

The effluent from palm oil mills (POME) contains high levels of chemical oxygen demand (COD) and is highly effective in promoting microbial oil production. This not only facilitates lipid generation but also aids in waste management. Justine et al. found optimal conditions for COD reduction and lipid production at a POME concentration of 59.88%, with a yeast-to-microalgae ratio of 9:25 over 18 days, achieving a 72.27% reduction in COD and a lipid productivity of 0.012 mg/L/d [116]. This research offers an effective approach for managing waste sustainably within the palm oil sector. promoting the eco-friendly production of integrated biofuels.

5.3. Microbial Genetic Engineering

Through genetic and metabolic engineering, promising oleaginous microorganisms have been created. Identifying the genes involved in lipid production within a system and enhancing their expression through recombinant DNA technology is an effective strategy to boost lipid production in microbial cells [117]. For instance, mutants E15-11, E15-15, and E15-25 of the oleaginous yeast Lipomyces starkeyi, which were isolated from UV-exposed cells using Percoll density gradient centrifugation, demonstrated approximately 4.5 times higher lipid productivity than the wild type [118]. Additionally, the deletion of the LsSNF1 gene in Lipomyces starkeyi has been shown to enhance lipid accumulation, highlighting the gene’s role in lipid production and carbon-source utilization [119]. In other studies, integrating and expressing the genes for expressing manganese peroxidase (MNP) and versatile peroxidase (VP) into the genome of Rhodosaccharomycete NP11 demonstrated enhanced growth in the presence of vanillin and 5-HMF. In particular, strain VP18 showed a 30% and 25% increase in cell mass and lipid content, respectively [120]. Also, Kang et al. (2017) improved lipid production in Nannochloropsis salina by incorporating the AtWRI1 transcription factor sourced from Arabidopsis thaliana [121]. This transcription factor regulates Wrinkled 1 (WRI1), a key player in lipid accumulation within Arabidopsis seeds. WRI1, in turn, controls genes involved in fatty acid biosynthesis.

Additionally, the lipids from the oleaginous yeast Trichosporon dermatis were generated through sequential plasma and chemical mutagenesis, using enzymolysis products extracted from pretreated bagasse under various scales and cultivation methods. In shaker flasks, the DE-mutated strain L7 produced 26.7 g/L of biomass and 14.0 g/L of lipids, while achieving 34.9 g/L of biomass in batch culture using a hydrolysate containing 80 g/L of glucose and xylose in a 2:1 mass ratio. During fed-batch cultivation, L-cells grown in a medium comprising 120 g/L of glucose and xylose (in the same ratio) resulted in a lipid yield of 20.2 g/L [122]. A strain known as Kurthia huakuii was identified by Wang et al. (2024) for its ability to break down common phenolic and furfural inhibitors present in lignocellulosic process water (LPW), which also exhibited antagonistic effects in combination with these inhibitors [123].

Moreover, adaptive laboratory evolution (ALE) is also an effective strategy for cultivating yeast in the presence of inhibitors. During ALE experiments, yeast cells undergo random genetic mutations, with the more adaptive strains proliferating more extensively and eventually dominating the population [124]. By combining whole-genome sequencing with reverse engineering, ALE provides a robust framework for identifying and validating adaptive alleles, enabling the exploration of the genetic basis underlying yeast responses to lignocellulosic hydrolysates [125]. ALE experiments targeting oleaginous microorganisms represent a highly promising area for future research.

5.4. Improvement of Fermentation Environment

Cell growth in lignocellulosic hydrolysates is significantly influenced by pH levels [126]. Therefore, it is possible to feed in batches or monitor pH in real time to keep cell growth in good condition. If medium conditions are balanced, oleaginous microorganisms do not accumulate large amounts of lipids. However, when the nitrogen substrate is scarce, microorganisms will utilize the carbon source to produce lipids as a protective mechanism [127].

To achieve significant lipid accumulation within cells, it is essential to limit nitrogen availability. Consequently, media with elevated carbon-to-nitrogen (C/N) ratios are typically utilized to enhance the lipid content within the cells [128]. The maximum lipid yield of the microbe Mortierella isabellina at a low C/N ratio is 0.19 g of lipid per gram of consumed substrate (Y_P/S), representing 61% of the theoretical lipid yield from glucose (Y_P/S = 0.31 mol/mol) [20]. The balance between carbon and nitrogen content in the growth medium significantly affects both cell biomass and lipid production levels. When the C/N ratio is lower, ranging from 5 to 25 mol/mol, it encourages cell growth. In contrast, a higher C/N ratio, exceeding 50 mol/mol, supports lipid accumulation but adversely affects fungal growth [20]. Recent studies have demonstrated that the gene responsible for lipid transport, known as MMF1 and belonging to the major facilitator superfamily, was successfully expressed in the model green microalga Chlamydomonas reinhardtii using genetic engineering techniques [129]. Notably, nitrogen starvation significantly increased the total lipid content in MMF1 mutant cells, with levels nearly doubling in cultures expressing MaMMF1 compared to the control strain after two days of nitrogen deprivation.

The yeast Cryptococcus curvatus can withstand degradation inhibitors like furfural and HMF within specific concentration ranges [130]. Among various oleaginous yeasts, including Yarrowia lipolytica, Rhodotorula glutinis, Rhodotorula toruloides, and Lipomyces starkeyi, Rhodotorula glutinis has shown significant potential in biodiesel conversion from microbial oils, particularly under varying dissolved oxygen (DO) levels, which influence lipid accumulation. Studies indicate that low DO levels retard cell growth but enhance lipid accumulation in R. glutinis, making it a promising candidate for lipid production under controlled DO conditions [131]. The production of lipids was performed with Lipomyces starkeyi AS 2.1560 in a glucose-based medium that lacked micronutrients, utilizing a two-step fermentation process. This method achieved a lipid productivity rate of 2.0 g/L·h during the first 16 h, with an overall yield of 1.6 g/L·h for the entire fermentation duration. Importantly, the processes for cell growth and lipid accumulation can be independently optimized, allowing for further improvements in both aspects [132].

Moreover, optimizing bioreactor design plays a critical role in improving microbial lipid production from lignocellulosic hydrolysates. Various reactor configurations, including fed-batch systems and continuous stirred-tank reactors (CSTRs), have been explored to enhance process efficiency. Fed-batch bioreactors enable controlled substrate feeding, reducing inhibitor toxicity and improving lipid yields [133]. Furthermore, membrane bioreactors incorporating simultaneous saccharification, filtration, and fermentation (SSFF) have demonstrated potential in maintaining high cell density while mitigating inhibitory effects, making them promising candidates for large-scale applications [133].

Recent research has shown that directly adding H₂O₂ can enhance algal lipid production, although it may also lead to decreased biomass levels. The effects of H₂O₂ and variations in nutrient levels on total lipid accumulation in Scenedesmus sp. were found [134]. Future research may explore the potential of these findings by testing them in optimized conditions aimed at large-scale biodiesel production. This has the potential to be a key factor in alleviating the energy crisis.

6. Challenges and Future Perspective

Recent large-scale research initiatives have focused on overcoming economic and environmental challenges in microbial lipid production. The DOE Center for Advanced Bioenergy and Bioproducts Innovation (CABBI) has been actively developing engineered yeast strains to enhance lipid accumulation and process efficiency in bio-based refineries. Additionally, studies on economic feasibility suggest that scaling up production to 48,000 tons per year could lower microbial lipid costs to USD 1.20/kg, making it competitive with conventional oils [135]. Furthermore, research efforts emphasize the utilization of agricultural residues such as sugarcane bagasse and sweet sorghum hydrolysates to enhance lipid yields. Life Cycle Assessment (LCA) studies indicate that microbial oils offer a reduced carbon footprint compared to plant oils, though further advancements in cell disruption and lipid extraction technologies are necessary to improve sustainability.

Inhibitors remain a major challenge for microbial lipid production, requiring further research to enhance microbial tolerance and process optimization. However, there are more research inputs needed to identify the potential inhibitors and understand their effects on oleaginous microbe growth to devise a strategy to overcome this problem. There is a strong need to look for strategies to develop oleaginous strains with the ability to grow in hydrolysates derived from different substrates containing different inhibitory compounds. Most of the studies performed focused on using single strains; however, more studies should be carried out with microbial consortia. The synergistic activity of combinations of several microorganisms should be studied in different lignocellulosic hydrolysates. Also, different approaches to enhance their activity should be applied to these consortia to make them more resilient and efficient in producing microbial oils. Furthermore, the focus should be placed on reducing the cost of inputs, such as the pretreatment chemicals used for lignocellulosic biomass conditioning, detoxification agents, and nutrient supplementation required for microbial growth, while simultaneously increasing productivity. Although many of the discussed detoxification methods are available, research is needed to find a robust method of conditioning the lignocellulosic biomass, which could generate fewer inhibitors and favor the growth of microbes and lipogenesis. More studies on optimization of pretreatment approaches are required to decrease byproducts and enhance sugar concentration. Similarly, emerging research has identified new combinations of raw materials and lipid-producing microorganisms. For example, Russo et al. (2024) conducted research on growing Porphyridium cruentum with alternative substrates sourced from agricultural waste [136]. They specifically focused on using beet molasses and corn steep liquor (CSL). The findings indicated that the best growth conditions were reached with about 1.78 g/L of molasses and 1.89 g/L of CSL. This combination resulted in a cell concentration of 12.1 × 10⁶ cells/mL and a lipid content of 24.48%. This represents a highly promising new research direction. In addition to strain engineering and inhibitor mitigation strategies, optimizing bioreactor design is another critical aspect of scaling up microbial lipid production. Reactor configurations, including batch, fed-batch, and continuous systems, influence key factors such as oxygen transfer, agitation, and inhibitor degradation, all of which play a fundamental role in microbial growth and lipid accumulation. Understanding the reaction kinetics in microbial lipid production using lignocellulosic hydrolysates is crucial for optimizing process efficiency. The presence of inhibitors in hydrolysates can significantly affect microbial metabolism, leading to reduced lipid yields. Studies have shown that the kinetics of microbial growth and lipid accumulation are influenced by factors such as substrate concentration, inhibitor presence, and oxygen availability. Developing kinetic models that accurately describe these interactions can aid in predicting process performance and designing effective bioreactor systems. For instance, incorporating inhibition kinetics into models can help in understanding how different pretreatment methods affect microbial activity and lipid production. Future research should focus on refining these models to account for the complex nature of lignocellulosic substrates and the dynamic conditions within bioreactors. Additionally, integrating real-time monitoring data with kinetic models could enable adaptive control strategies, enhancing overall process robustness and efficiency.

All these research activities will help in devising an economically feasible and sustainable approach for the production of microbial oils by oleaginous microbes utilizing lignocellulosic biomass substrate. Combined with new technological developments, this should lead to the sustainable conversion of lignocellulosic biomass to SCO, which can cater to the needs (food, chemicals, biofuels, etc.) of the rising population.

7. Conclusions

The renewable and readily accessible nature of lignocellulosic materials has positioned them as a plentiful and cost-effective resource for biofuel production. To make the complex structure of lignocellulose more manageable, various pretreatment methods are essential. These processes break down the lignocellulosic matrix, resulting in a sugar solution that is suitable for fermentation by oleaginous microorganisms. However, pretreatment not only produces monosaccharides but also produces some inhibitors that are harmful to the growth of oleaginous microorganisms and the process of lipid production, including furfural, 5-HMF, formic acid, acetic acid, and levulinic acid. To improve conversion efficiency, future research should focus on optimizing bioreactor conditions, including controlled oxygen transfer, nutrient supplementation, and real-time process monitoring, to dynamically adjust fermentation parameters. Additionally, detoxification strategies must be further refined to selectively remove inhibitors while preserving sugar integrity. Enhancing microbial strain resilience through metabolic engineering and adaptive evolution can further mitigate the inhibitory effects, allowing more efficient lipid accumulation. Finally, integrating pretreatment and fermentation strategies, such as sequential hydrolysis-fermentation approaches, can help balance sugar recovery and lipid productivity, ultimately making microbial lipid production more economically viable. This review attempted to discuss all these aspects, but still there is a need to work on different aspects to make the process of producing microbial oil using lignocellulosic biomass as a substrate both sustainable and economically feasible.