Amino acid-assisted enzymatic hydrolysis of corn stover for microbial lipid production by Rhodotorula toruloides

Background

Efficient enzymatic hydrolysis of lignocellulosic biomass (LCB) is essential for maximizing the recovery of fermentable sugars for diverse biotechnological applications. However, pretreatment by-products including lignin interact with hydrolytic enzymes, blocking their access to substrates and leading to poor monomeric sugar recovery. This study evaluated the effects of all 20 exogenous amino acids (AAs) as additives to block lignin active sites and facilitate enzyme access to polysaccharide substrates for enhanced sugar recovery. The hydrolysates were subsequently tested for microbial lipid production by Rhodotorula toruloides CGMCC 2.1389 as a model application.

Results

The study found that most AAs enhanced enzymatic hydrolysis of 1% (w/v) H₂SO₄– and Na₂CO₃-pretreated corn stover (CS), with l-proline (Pro) increasing total reducing sugar (TRS) recovery by over 20%. Lipid production by R. toruloides on the hydrolysates was verified under single-stage and two-stage culture conditions. The lipid yield reached over 17 g/100 g TRS with some AAs, despite lower titers. Moreover, the dominance of C16 and C18 fatty acids in the lipids suggest no adverse effects of AAs on the yeast’s metabolism.

Conclusion

Exogenous AA addition during enzymatic hydrolysis enhanced sugar recovery; however, its impact on R. toruloides lipid production varies with culture conditions, where a two-stage process with nutrient limitation could be more favorable for high lipid production. While this strategy proved to be more effective for enhanced sugar recovery, future studies are expected to uncover the underlying mechanisms that drive this improvement.

Lignocellulosic biomass (LCB) is a renewable and low-cost source of fermentable sugars for microbial bioproduction [1, 2]. LCB is composed of 30–50% (cellulose), 15–35% (hemicellulose), and 10–30% (lignin) [3]. Cellulose, a polysaccharide composed of d-glucose units linked by β-1,4-glycosidic bonds [4], resists enzymatic hydrolysis due to its high crystallinity and extensive hydrogen bonding [5]. Hemicellulose, a branched polysaccharide of xylans, mannans, and β-(1,3 and 1,4)-glucans [6], contributes to cell wall rigidity by forming hydrogen bonds with cellulose and lignin [7]. Lignin, a phenylpropanoid polymer composed of p-coumaryl, coniferyl, and sinapyl alcohols linked via β-O-4, α-O-4, β-5, and biphenyl bonds [8], provides structural integrity to the plant cell wall. This complex polymeric matrix of LCB hinders biological conversion [9]. Celluloses and hemicelluloses can be isolated through pretreatment methods (physical and thermochemical) [10], followed by enzymatic hydrolysis into monosaccharides, primarily glucose, xylose, and arabinose [11].

Nonetheless, enzymatic hydrolysis often exhibits low efficiency due to the presence of pretreatment by-products, including lignin and pseudolignin, which limit enzyme accessibility to cellulose and hemicellulose by binding enzymes through hydrophobic, electrostatic, and hydrogen bonding interactions [12]. However, previous studies have shown that the non-productive binding of enzymes to exposed lignin can be reduced by blocking lignin’s adsorption sites with surfactants [13], and non-catalytic proteins [14, 15]. Non-ionic surfactants like Tween and polyethylene glycol (PEG) have been shown to enhance enzymatic hydrolysis of acid-pretreated LCB [16]. The mechanism behind this improvement is believed to involve the blockage of lignin adsorption sites by surfactants or non-catalytic proteins, thereby reducing the non-productive enzyme binding to surfaces of exposed lignin and enhancing enzyme availability for cellulose and hemicellulose hydrolysis [13, 17].

In addition to surfactants and proteins, exogenous AAs may prove to reduce non-productive enzyme binding by blocking adsorption sites on lignin and other pretreatment-derived by-products. AAs exhibit diverse biological and biochemical roles in both in vivo and in vitro systems. For example, Pro stabilizes proteins under stress, prevent its aggregation [18]; stabilizing it during freezing [19], elevated temperatures [20], dehydration [18], while increases its solubility [21], and scavenges reactive oxygen species (ROS) [22, 23]. Therefore, AAs can improve enzyme interactions with cellulose and hemicellulose by blocking lignin adsorption sites, which could result in enhanced sugar recovery.

Microbial lipids serve as a sustainable alternative to plants and fossil-based fuels, with applications in food, energy, and oleochemicals production [24, 25]. However, broader application of microbial lipids is constraint by high substrate costs and challenges in production and recovery [26]. Therefore, lignocellulosic derived sugars are considered a low-cost substrates for microbial lipids [11].

In this study, all 20 natural AAs, with a focus on Pro, were evaluated as additive to enhance enzymatic hydrolysis of corn stover (CS). The aim was to assess the influence of AAs on sugar recovery by promoting enzyme binding to cellulose and hemicellulose rather than to lignin and other pretreatment by-products. The results showed that AA addition potentially improved sugar recovery from CS pretreated with 1% (w/v) H₂SO₄ and Na₂CO₃. Lipid production from the resulting hydrolysates was confirmed using the oleaginous yeast R. toruloides.

Microorganism, media, and growth conditions

The R. toruloides CGMCC 2.1389 was acquired from China General Microbiological Culture Collection Centre. YEPD agar plates contained (20 g/L glucose, 10 g/L peptone, 10 g/L yeast extract, and 20 g/L agar) were used to maintain the strain at 4 °C, with subculturing performed twice monthly. Peptone, derived from animal tissue and containing 3% ammonium-N, 14.5% total nitrogen, and 0.14% phosphorus, and the yeast extract (3% ammonium-N, 9.0% total nitrogen, 1.3% phosphorus), were both purchased locally (Aoboxing Biotech Co. Ltd. Beijing, China). The medium for seed culturing was composed of 10 g/L yeast extract, 10 g/L peptone, and 20 g/L glucose, and had C/N and C/P molar ratios of 3.6 and 130, respectively. Sterilization (121 °C for 20 min) of all seed media was performed before use.

CS pretreatment

The CS used in this study was collected from Liaoning province, China. The CS composition was analyzed using NREL methods (NREL/TP-510-42620, and NREL/TP-510-42618) [27, 28]. The CS contained the following sugar composition: l-arabinose 4.1%, d-galactose 1.6%, d-glucose 43.0%, and d-xylose 13.2%. Elemental analysis revealed nitrogen at 0.9%, carbon at 41.5%, hydrogen at 5.9%, and sulfur at 0.03%, with C/N and C/H ratios of 43.8 and 6.9, respectively. The CS biomass with 4 mm size at 10% solids loading (100 g/L) was transferred to a 250-mL volume flask. 50 mL of 1% (w/v) Na₂CO₃– or H₂SO₄ solution was added to the flask, mixed thoroughly and steamed for 3 h at 131 °C. After steam pretreatment, the flask’s initial weight was adjusted with distilled water, and the pH was set to 4.8 using 4 M HCl. The enzymatic hydrolysis process used cellulase at a concentration of 50 μL/g CS (5.0 mg total protein per g CS) and xylanase at 2.5 mg/g CS. The cellulase, sourced from Novozymes (China) Biotechnology Co. Ltd., had an enzyme activity of 234 FPU/mL. The xylanase, with an activity of 300,000 U/g was source from Heshibi Biotechnology Co., Ltd. Moreover, AA at a concentration of 2 g/L unless specified was added to enhance enzymatic hydrolysis of CS, and ampicillin at a concentration of 0.5 mg/g CS was supplied for preventing bacterial growth during the hydrolysis process. The AAs: Asn (l-asparagine), Asp (l-aspartic acid), Val (l-valine), Ile (l-isoleucine), Arg (l-arginine), Met (l-methionine), Gln (l-glutamine), His (l-histidine), Glu (l-glutamic acid), Pro (l-proline), Ala (l-alanine), Ser (l-serine), Thr (l-threonine), Gly (l-glycine), Phe (l-phenylalanine), Cys (l-cysteine), Trp (l-tryptophan), Lys (l-lysine), Tyr (l-tyrosine), and Leu (l-leucine) were all of analytical grade with purities exceeding 98.5% and sourced from Sangon Biotech (Beijing, China). The enzymatic hydrolysis in a shaker water bath at 50 °C and 200 rpm was conducted for 48 h. Samples were withdrawn every 12 h to follow sugar evolution. Following hydrolysis, the hydrolysates were centrifuged (13,000 rpm for 20 min) for solids removal and the purified supernatants were set to pH 5.5 before sterilization (121 °C for 20 min). The sterilized hydrolysates were used for lipid production.

Lipid production

For a single-stage lipid production process, the flask with 45 mL sterilized hydrolysates was inoculated with 5 mL of R. toruloides seed culture (24 h old) and kept in shaker incubator (temperature 30 °C, and agitation 200 rpm). In case of two-stage lipid production, high initial cell density was employed to the CS hydrolysates. Briefly, 20 mL seed culture of R. toruloides (36 h old, cell mass 3 g/L) was extracted, followed by centrifugation (5000 rpm for 5 min) and two washes with deionized water. The pure cells were inoculated into the flask and kept in a shaker incubator (temperature 30 °C, and agitation 200 rpm) for 96 h unless specified to promote lipid production. Sugar consumption was followed every 12 h.

Analytical methods

Determination of cell mass and lipids

The determination of cell mass, lipids, lipid content, and yield followed a previous method [29]. Shortly, the cells from the culture broth were extracted through centrifugation (8000 rmp for 5 min), followed by two washes with deionized water and kept in an oven for 12 h at 105 °C to constant dry weight. The cell mass was measured gravimetrically and expressed in g/L.

Total lipid extraction involved digesting the dried cells with 4 M HCl in a shaker water bath for 1 h at 78 °C and 200 rpm followed by extraction using methanol/chloroform (1:1, v/v) [30]. After washing with 0.1% NaCl solution, the extracts were passed through an anhydrous Na₂SO₄ pad. To recover the total lipids, the solvent was evaporated under reduced pressure, and the residues were dried in an oven at 105 °C. The total lipids were measured gravimetrically and expressed in g/L, the lipid content was expressed in % and calculated as gram lipid produced per gram cell mass. Expressed in g/g TRS, the lipid yield was calculated as gram lipids produced per gram consumed total reducing sugar.

Determination of glucose and TRS

The SBA-50B glucose analyzer (Shandong Academy of Sciences, Jinan, China) was used to determine the glucose concentration. Individual sugars were analyzed through ion chromatography as described previously [31], while total reducing sugars (TRS) were quantified using a previously established 2,4-dinitrosalicylate method [32]. A Torch Combustion TOC Analyzer (Teledyne-Tekmar, OH, USA) was used to measure the total organic carbon (TOC) and total nitrogen (TN) in the enzymatic hydrolysates. Sugar yield was determined by calculating the obtained TRS per gram of CS, while sugar recovery, expressed as a percentage, was calculated as the ratio of the total recovered glucose and TRS to the total glucose and TRS content in the CS.

Fatty acid compositional profiles

The lipid samples for fatty acid profiles were determined through gas chromatography (GC). Briefly, a 70 mg lipid sample was stirred for 50 min at 65 °C with 0.5 mL of 5% KOH in methanol. Next, BF₃ diethyl etherate (0.2 mL) and methanol (0.5 mL) were added to the mixture, and refluxed for 10 min. After cooling, the mixture was diluted with deionized water and extracted with n-hexane. The organic layer underwent two washes with deionized water. Fatty acid profiling was carried with a 7890F GC system (Techcomp Scientific Instrument Co., Ltd., Shanghai, China) equipped with a (30 m × 0.32 mm × 0.4 mm) crosslinked capillary FFAP column and a flame ionization detector. Air, N₂, and H₂ were supplied at flow rates of 100 mL/min, 40 mL/min, and 30 mL/min, respectively. The injection volume was 2 μL while the temperature settings were 250 °C for the injection port, 190 °C for the oven, and 280 °C for the detector. Identification of fatty acids was based on retention times compared to standards, with quantification performed via peak area normalization.

Effect of l-proline on enzymatic hydrolysis

Effective LCB saccharification typically requires physical or chemical pretreatments to enhance enzymatic accessibility and facilitate the release of monomeric sugars [10]. In chemical pretreatment processes, dilute acids and alkalis are commonly employed as catalysts to solubilize the complex components of LCB [11, 33]. While each chemical pretreatment method offers distinct advantages, they also present several challenges. For instance, dilute acid pretreatment enables high recovery of hemicelluloses, improving enzymatic access to cellulose and resulting in increased net glucose yield [34, 35]. However, this method has significant drawbacks, including severe corrosion to the pretreatment equipment [34] and the production of toxic by-products in the hydrolysates, hindering enzymes activity and the following fermentation performance [35]. Similarly, alkaline pretreatment specifically targets the acetyl groups in hemicellulose and lignin–carbohydrate linkages, resulting in significant lignin removal and solubilization of the LCB matrix [36]. However, major challenges of this approach include alkali conversion into unrecoverable salts, adsorption of alkali by the biomass, and the formation of inhibitory by-products under severe pretreatment conditions [37]. Nevertheless, alkaline pretreatment, particularly with mild reagents such as sodium carbonate, provides several advantages, including increased biomass surface area, reduced crystallinity, and minimal corrosion or environmental impact [38], and supporting scalability [11].

The CS with 10% (w/v) solids loading was pretreated with either 1% (w/v) H₂SO₄ or Na₂CO₃ and subsequently hydrolyzed using cellulase and xylanase in the presence of varying concentrations of Pro. Specifically, Pro was added to the pretreated slurries at concentrations of 5.0, 10.0, 15.0, and 20.0 mg/g of CS, while the control was kept without Pro. The objective was to identify the optimal Pro concentration and assess its effect on enzymatic hydrolysis through sugar recovery. Results revealed Pro concentration-dependent enhancement in sugar release, particularly during the first 24 h, indicating improved enzyme–substrate interactions. Notably, Na₂CO₃-pretreated CS supplemented with 20 mg/g Pro yielded the highest sugar concentrations, with glucose and TRS reaching 36.6 g/L and 62.4 g/L, respectively. In contrast, the control achieved only 29.6 g/L glucose and 52.5 g/L TRS. Intermediate concentrations, such as 15 mg/g CS, also produced significant improvements, yielding 33.3 g/L glucose and 56.7 g/L TRS. These findings suggest that Pro enhances enzymatic hydrolysis efficiency by mitigating non-productive enzyme binding to lignin and other pretreatment by-products, thereby facilitating more effective interaction between enzymes and polysaccharide substrates (Fig. 1a, b).

L-Proline effects on sugar release from the acid and alkali pretreated CS hydrolysis. a Glucose and b TRS evolution from 1% Na₂CO₃ pretreated CS. c Glucose and d TRS evolution from 1% H₂SO₄ pretreated CS

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For H₂SO₄-pretreated CS, similar sugar trends were obtained. Addition of 20 mg/g CS of Pro resulted in the highest concentrations of both glucose and TRS, reaching 35.3 g/L and 61.1 g/L, respectively. This increase was considerably high than the control which was limited to glucose (27.6 g/L) and TRS (49.8 g/L). The Pro presence at 15 mg/g CS also resulted in substantial sugar release, achieving glucose (33.0 g/L) and TRS (56.2 g/L) (Fig. 1c, d). Across both applied pretreatments, the highest TRS yields were achieved at 20 mg/g CS of Pro addition: 0.624 g/g CS for Na₂CO₃-pretreated CS and 0.61 g/g CS for H₂SO₄-pretreated CS. These results indicate that Pro acts as a beneficial additive for enzymatic hydrolysis, particularly at higher concentrations, resulting in relatively enhanced glucose and TRS yields. Notably, Pro has been recognized for its protein-stabilizing properties under stressful conditions and its capacity to prevent protein aggregation [18], which may contribute to improved enzyme functionality and resilience during hydrolysis. Potentially stabilization of enzymes and reduction in non-specific binding to lignin and other inhibitory by-products, Pro facilitates a more targeted and efficient hydrolysis of cellulosic substrates, enhancing overall sugar recovery.

Ion chromatography analysis of the hydrolysates, as shown in Fig. 2, confirms an increase in individual sugar concentrations, particularly glucose and xylose, with increasing Pro concentrations. The data indicate that high Pro concentrations, especially 10 mg/g, 15 mg/g, and 20 mg/g CS, led to relatively high sugar release compared to the control. The concentrations of glucose and xylose show a consistent upward trend with increased Pro, suggesting enhanced enzymatic activity and access to the target material. Glucose and xylose recovery rates reached the maximum values at Pro concentration of 20 mg/g CS. While arabinose levels also rose with Pro addition, there was a slight decrease in arabinose levels at Pro concentration of 20 mg/g CS compared to 15 mg/g CS using 1% (w/v) H₂SO₄ pretreated CS. Nonetheless, arabinose levels were still notably higher than in the control. These results align with the observed trend in the total sugar yield, supporting the positive role of Pro in improving enzymatic hydrolysis of acid and alkali pretreated CS. Compared with 42% total sugar recovery on control, 29% increase was achieved in total sugar recovery in the presence 20 mg/g CS of Pro using 1% Na₂CO₃ pretreated CS. Since the TRS recoveries were 46.2%, 52.9%, 59.8% with the highest 71.3% in the presence of 5.0 mg/g, 10.0 mg/g, 15.0 mg/g and 20.0 mg/g CS of Pro, respectively.

Effects of Pro on sugar release from CS hydrolysis. a Concentration and b recovery of glucose and xylose on 1% Na₂CO₃ pretreated CS. c Concentration and d recovery of glucose and xylose on 1% H₂SO₄ pretreated CS

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Similar phenomenon was observed in the case of 1% H₂SO₄ pretreated CS, where Pro unlocked high glucose and xylose release (Fig. 2c), resulting in their increased recoveries (Fig. 2d). These outcomes reveal that the effects of Pro are a common occurrence, not limited to the type of pretreatment used (acidic or alkaline). As previously discussed, Pro known for its protein-stabilizing and aggregation-preventing properties under stressful conditions [18], likely contributed to enhanced enzyme functionality and structural resilience during hydrolysis. These observations are consistent with previous studies that demonstrated similar effects using non-catalytic proteins such as bovine serum albumin (BSA), which reduced non-productive enzyme binding by preferentially adsorbing to lignin surfaces, thereby improving sugar yields [39]. Similarly, Tween 80 and PEG have been shown to enhance enzymatic saccharification by disrupting lignin–enzyme interactions, and stabilizing enzyme conformation [13]. The observed effects of Pro may be attributed to similar mechanisms namely, its interaction with lignin or other inhibitory by-products, reduction of non-productive adsorption of cellulolytic enzymes, and maintaining enzyme accessibility to polysaccharides substrates. Further analysis of the hydrolysates for TOC and TN confirmed the beneficial effects of Pro on the enzymatic hydrolysis. The narrow increase in TOC contents confirms high carbon contents of the hydrolysates with increase in the concentration of Pro (Table 1), while the increase in TN could be attributed to efficient hydrolysis of the CS and the addition of exogenous Pro.

Effects of enzymes loading on CS hydrolysis in the presence of l-proline

The 1% H₂SO₄ pretreated-CS biomass slurry was hydrolyzed with various concentration of enzymes (Table S1). Cellulase at a concentration of 1.25, 2.25, 3.75, and 5.0 mg/g, respectively, was used while the xylanase concentration was kept at 0.62, 1.25, 1.87 and 2.50 mg/g C, respectively. Pro (20 mg/g CS) was added along with each concentration of the enzymes while the control groups with the same enzymes concentrations were kept without Pro addition. The experiment aimed to optimize the enzymes concentration and evaluate Pro effects at low enzymes loading.

Although sugar release increased with higher enzyme concentrations across all groups, the Pro-added groups consistently yielded higher TRS than their corresponding controls (Fig. 3a–h). The effect of Pro was especially noticeable at lower enzyme loadings, suggesting that Pro may enhance the catalytic efficiency or stability of enzymes under suboptimal conditions. At 24 h, the differences in sugar release were already significant, indicating that Pro likely facilitated more rapid enzyme–substrate interactions. The beneficial effects can be attributed to Pro’s previously discussed known effects which helped maintain non-productive adsorption of enzymes onto lignin and other inhibitory surfaces, thereby increasing the concentration of free enzymes available for polysaccharides hydrolysis. Although sugar release was gradually increased with the increase in enzymes concentration, but the Pro added groups were with higher sugars release compared with the control (Fig. 3a–h). At 24 h of hydrolysis, the difference in sugar release was more significant at all enzymes level in the presence of Pro compared with control which suggest Pro encouraged enzymes access to cellulosic and hemicellulosic substrates. A slow sugar release was observed on control which suggest that less hydrolysis time is required with Pro addition. The final TRS was 53.2 g/L on control while it reached 63 g/L with 20 mg/g CS Pro addition (Fig. 3h). These findings suggest that Pro enables similar or greater hydrolysis efficiency at lower enzyme dosages, a phenomenon comparable to previous reports where non-catalytic proteins enhanced saccharification efficiency under reduced enzyme loadings [17].

Effects of enzymes concentration on CS hydrolysis. a, c, e, g Glucose and b, d, f, h TRS evolution at enzyme concentrations (cellulase:xylanase) of 1.25:0.62, 2.25:1.25, 3.75:1.87, and 5.0:2.5 mg/g CS, respectively

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Two-stage lipid production on CS hydrolysates containing l-proline

The hydrolysates obtained from both strategies of Pro supplementation, applied either before or after steam pretreatment, were evaluated for two-stage lipid production (Fig. 4). The hydrolysis results are shown in Table S2 and Fig. S1. Pro addition before pretreatment led to increased cell mass across all Pro concentrations, with a maximum of 15.7 g/L at 20 mg/g CS, compared to 14.4 g/L in the control. However, lipid production decreased at higher Pro levels, i.e., only 5.5 g/L lipid titer (35.5% lipid content) were obtained at 20 mg/g CS, while 5 mg/g CS Pro resulted in highest lipids of 9.0 g/L, lipid content of 60% and lipid yield of 0.165 g/g TRS.

Lipid production on enzymatic hydrolysates with Pro added either before or after steam pretreatment. a, c, e Show results for Pro supplementation before steam pretreatment, and b, d, f for Pro supplementation after steam pretreatment. a, b Consumption of glucose. c, d Consumption of TRS, and e, f cell mass, lipid yield, and lipid content. Culture conditions: 30 °C, 200 rpm, 72 h

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Similar patterns were observed when Pro was added post-pretreatment. Cell mass reached 19.3 g/L at 20 mg/g CS, but lipid accumulation was again lowest (5.2 g/L, 27.3%). In contrast, 5 mg/g CS resulted in the highest lipid of 9.2 g/L, lipid content of 56.8%, and yield of 0.16 g/g TRS. The reduced lipid production at higher Pro concentrations likely results from increased nitrogen input, which lowers the C/N ratio and shifts metabolism toward cell mass rather than lipid production. At lower concentrations, Pro may act as an osmoprotectant and antioxidant, supporting red yeast lipid accumulation under stress without disrupting nutrient balance [40].

Effects of AAs on CS hydrolysis

Proteins are soluble in the native folded state in aqueous solution. Proteins exposed to destabilizing conditions can negatively impact their function [41]. Such abnormal conditions include physical degradation, e.g., surface adsorption [42]. Unfortunately, during the course of biomass pretreatment, soluble inhibitors are generated which hamper enzymatic hydrolysis [43]. These inhibitors include cellulose derived sugars [44, 45], furan derivatives (such as hydroxymethylfurfural and furfural), and organic acids (including acetic acid, formic acid, and levulinic acid) [43], lignin-derived phenolics [46], and reactive oxygen species, etc. These all can bind or oxidize proteins on sensitive sites, which lower enzymatic functions, or limit enzymes binding to the target substrate causing poor hydrolysis of the biomass. To prevent enzyme binding, AAs supplementation during enzymatic hydrolysis, can play multiple chemical and physical roles to mitigate these inhibitory effects. These include; preferential adsorption to lignin, thereby reducing non-productive enzyme–lignin binding similar to BSA [39]. Acting as osmolytes or stabilizers, protecting enzyme structure and function under stress (e.g., Pro) [18]. Forming hydrogen bonds or ionic interactions with reactive lignin phenolics or organic acids, neutralizing their inhibitory action. Our results demonstrated that several AAs notably improved total reducing sugars (TRS) release, indicating their positive effect on enzymatic saccharification (Fig. 5). Except for Asn, Arg, and Ile, all tested AAs increased TRS relative to the control. In particular; Leu, Cys, Tyr, Ser, Thr, Asp, Met, and Ala enhanced TRS by ~ 5–10%. Glu, His, and Trp resulted in increases of 11%, 13%, and 15%, respectively. Lys, Gly, and Phe showed even higher improvements (16–18%). The most pronounced increases were observed with Gln (24%) and Pro (28%), as detailed in Table S3.

AAs effect on the CS enzymatic hydrolysis. a Concentration of glucose. b Concentration of TRS. c TRS yield

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The superior performance of Pro and Gln may stem from their known functions as protein stabilizers and osmoprotectants. Pro, in particular, has a cyclic structure that restricts conformational flexibility, making it especially effective at preventing protein unfolding under stress [18, 47]. It also possesses a zwitterionic nature, allowing it to interact with both hydrophilic and hydrophobic molecules, potentially binding or neutralizing inhibitors like phenolics or furans. Interestingly, most sugar increases were seen in non-glucose reducing sugars, suggesting that AAs might enhance xylanase or accessory enzyme activities, or protect these enzymes from inhibitors. Chemically, Lys and Arg, due to their basic side chains, might act by neutralizing acidic inhibitors (e.g., acetic acid), while Cys may form thiol-based interactions with lignin phenolics. Aromatic AAs like Phe and Trp could interact via π–π stacking with lignin aromatic structures, limiting lignin–enzyme interactions [48]. These results parallel findings from studies using non-catalytic proteins (e.g., BSA) [39], and surfactants (e.g., Tween 80) [13], which also mitigate non-productive enzyme adsorption and improve saccharification efficiency by stabilizing enzyme structures or modifying lignin surface properties.

Although this study did not quantify changes in LCB pretreatment by-products, e.g., furfural, HMF, or other soluble inhibitors after AAs supplementation, it is plausible that AAs may mitigate inhibitor toxicity through adsorption, binding, or redox-related interactions. This potential needs further exploration in future studies to clarify the mechanisms through which AAs improve LCB hydrolysis. Similarly, most AAs proved beneficial for enzymatic hydrolysis, applying 20 mg/g CS may be economically impractical at scale compared to other similar enzymes promoting cheap agents (Table S4). To enhance feasibility, AAs could be sourced from low-cost, protein-rich industrial by-products such as agro-industrial residues (e.g., corn steep liquor, soybean meal hydrolysates), food processing and meat wastes, wastewater-grown protein-rich microalgae biomass, and fermentation by-products. These alternative sources offer a cost-effective option compared to using pure commercial-grade AAs.

Lipid production on hydrolysates containing AAs

The hydrolysates obtained were used for lipid production. Unfortunately, the presence of AAs in the hydrolysates reduced lipid accumulation by R. toruloides compared to the control (Fig. 6). The low lipid production on hydrolysates with AAs was likely caused by the high nitrogen content, which altered the C/N ratio favorable for lipid accumulation, as indicated by the high cell mass production. The yeast showed higher growth on hydrolysates with AAs; however, in the case of cysteine, cell mass production was lower than in the control. This reduction may be due to cysteine’s known detrimental effects on yeast cell growth when present in excess [49, 50]. Similarly, no increase in cell mass was observed with Met, Ile, Phe and Glu, suggesting that the concentrations of these AAs had little or no effect on cell mass production, although all AAs resulted in reduced lipid production in R. toruloides. Except for His, Trp and Tyr, all other AAs led to comparatively lower lipid levels in R. toruloides. Hydrolysates containing Arg supported cell mass, yet the lipid as well as lipid content was the lowest (Table 2). In fact, the production of microbial lipids generally requires growth conditions that are “imbalanced”, with an abundance of extracellular organic carbon and restricted nitrogen availability [51]. The presence of AAs decreased C/N of the hydrolysates, which likely hindered the accumulation of yeast lipids. High nitrogen availability promotes cell growth over lipid accumulation by upregulating protein and nucleotide synthesis pathways, diverting metabolic resources toward cell mass production rather than lipids.

Lipid production on hydrolysates containing AA by R. toruloides. a Cell mass concentration. b Lipid concentration. c Lipid content

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Conversely, lipid yields on some AAs-containing hydrolysates were higher than the control, although most AAs-containing hydrolysates prevented high lipid yields. The lowest lipid yield of 8 g/100 g TRS was observed on Arg-containing hydrolysates followed by 8.3, 9.4 and 9.5 g/100 g TRS on Gln, Cys and Met containing hydrolysates, respectively. In contrast, the highest lipid yields of 17.3, 17.3 and 17.9 g/100 g TRS were achieved on Phe-, Tyr- and His-containing hydrolysates, respectively. These high lipid yields indicate the potential for lipid production on certain AAs-containing hydrolysates, which could be further optimized with effective culture strategies such as two-stage culture [52] or phosphate limitation [11, 53]. Additionally, removing residual proteins through precipitation from the enzymatic hydrolysates could help in achieving a favorable C/N ratio, potentially enhancing the lipid titer [2]. Moreover, precise molecular studies involving transcriptomics or metabolomics analyses are needed to understand each AAs effects on the lipid production.

Compositional profiles of fatty acid

The GC-FID analysis of the transesterified lipid samples showed that fatty acids with 16 and 18 carbon atoms were the predominant fractions in all lipid samples (Fig. 7). Oleic acid (C18:1) was the main fatty acid, with palmitic acid (C16:0) and stearic acid (C18:0) being the next most abundant. It is noteworthy that oleaginous yeasts store neutral lipids in the form of triacylglycerols in their lipid bodies. Myristic acid (14:0), palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), and linoleic acid (C18:2) can all be synthesized by the red yeast R. toruloides [54]. While the fatty acid profiles might be influenced by the carbon substrate and culture conditions, the general composition tends to remain same. These neutral lipids, with 16 and 18 carbon-containing fatty acids as the major components, are highly similar to vegetable oils and are viewed as a potential feedstock for the production of biodiesel [54, 55]. These results indicate that AAs did not influence compositional profile of the fatty acid produced by R. toruloides while the lipid produced on the CS hydrolysates can serve as a valuable feedstock for biodiesel and oleochemicals production.

Fatty acids produced by R. toruloides on AAs-containing CS hydrolysates

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Authors and Affiliations

1. Guangdong Technology Research Center for Marine Algal Bioengineering, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen, 518055, China

   Rasool Kamal, Jiaxi Luo & Chaogang Wang

2. Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Optoelectronic Engineering, Shenzhen University, Shenzhen, 518060, China

   Rasool Kamal & Chaogang Wang

3. Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai, 264003, China

   Qitian Huang

4. Laboratory of Biotechnology, Dalian Institute of Chemical Physics, CAS, 457 Zhongshan Road, Dalian, 116023, People’s Republic of China

   Qitian Huang & Aabid Manzoor Shah

5. Laboratory of Fisheries and Aquaculture, Department of Zoology, Kohat University of Science and Technology, Kohat, 26000, Khyber Pakhtunkhwa, Pakistan

   Farman Ullah Dawar

Contributions

Rasool Kamal designed and performed this research; Luo Jiaxi, Aabid Manzoor Shah and Farman Ullah Dawar helped in data analysis and manuscript revision; Qitian Huang and Wang Chaogang gave funding support and advice.

Corresponding author

Correspondence to Chaogang Wang.

Ethics approval and consent to participate

No animals or human subjects were used in the above research.

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Our manuscript does not contain any individual data in any form.

Competing interests

The authors declare no competing interests.

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