Regulatory effects of carbon and nitrogen nutrition on lipid accumulation by Yarrowia lipolytica cultivated with high-concentration volatile fatty acids

Abstract

Volatile fatty acids (VFAs) derived from organic waste offer promising and cost-effective carbon sources for the production of microbial lipids. This study demonstrates the significant influence of nitrogen nutrition on cell proliferation and microbial lipid synthesis in Yarrowia lipolytica during high-concentration acid cultivation. Further investigations into nitrogen sources revealed that NH₄Cl and urea are suitable options for cultivating Y. lipolytica to produce microbial lipids, resulting in lipid yields ranging from 2.00 to 2.50 g/L. Moreover, pH fluctuations were found to be influenced by both the nitrogen source and acid utilisation, with pH adaptation helping alleviate acid inhibition caused by high-concentration VFAs. Under optimised cultivation conditions, the highest yield of microbial lipids reached 4.00 g/L, accompanied by a dry cell weight of 9.91 g/L and a microbial lipid content of 40.37%, consisting predominantly of C16 ~ 18 fatty acids. These findings highlight the central role of nitrogen metabolism and pH adaptation in enhancing VFA assimilation, offering guidance for cost-effective microbial lipid production from organic waste streams.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00449-025-03263-w.

Introduction

Owing to the high cetane number, nontoxicity, low-sulfur, and nonaromatic properties, biofuels produced from microbial lipids are green alternatives to traditional fossil fuels. Volatile fatty acids (VFAs) have shown great potential for synthesising microbial lipid by oleaginous microorganisms as low-cost carbon sources [1]. Additionally, VFAs can be derived from the fermentation of organic waste, making producing microbial lipids from VFAs an environmentally friendly and effective approach for organic waste management [2].

Microbial lipid is synthesised by oleaginous microorganisms, which can accumulate more than 20% lipid of their dry cell weight, including microalgae, bacteria, yeast, and moulds [3]. As a nonconventional yeast, Yarrowia lipolytica is considered a model yeast for the production of microbial lipid and is widely studied because of its superior microbial lipid accumulation capacity, which can accumulate more than 30% of its dry cell weight, and is a well-developed genetic tool [4, 5]. Y. Lipolytica can utilise diverse carbon sources, including hydrophilic (glucose, glycerol, alcohols, etc.) and hydrophobic (fatty acids, triacylglycerols, alkanes, etc.), among which glucose is the most widely studied and utilised. However, glucose costs are approximately 60% and 80% of the total cost of production and medium, respectively [6]. The high cost of carbon sources has challenged the development and engineering application of microbial lipid production; thus, low-cost substrates need to be explored. VFAs mainly consist of short carbon chain acids (C1-C6) and can be obtained inexpensively from the fermentation of diverse organic waste, such as sludge, agro/industrial lignocellulosic waste, and food waste [7]. VFAs are promising carbon sources for oleaginous yeasts because of their shorter transformation pathway and higher theoretical microbial lipid conversion efficiency than sugar-based substrates [8]. Therefore, VFAs have been recommended as feasible low-cost carbon sources for microbial lipid production.

Several studies have shown that VFAs could be utilised as a single carbon source for Y. lipolytica cultivation. Initially, researchers commonly used VFAs at low concentrations to culture oleaginous yeasts to avoid acid inhibition. As reported in previous studies, under such conditions, the lipid yields were generally less than 1 g/L [9, 10]. Recently, increasing attention has been given to improving the lipid yield by using high-concentration VFAs [11]. However, the main problem is that high concentrations (more than 5 g/L) of VFAs inhibit the growth and lipid accumulation of oleaginous microorganisms. High concentrations of VFAs induce cytoplasmic acidification, thereby inactivating critical metabolic enzymes. Furthermore, VFAs can compromise the integrity and functionality of the cell membrane, resulting in the efflux of intracellular components and the disruption of essential ion gradients. Collectively, these effects suppress cellular growth and reduce lipid accumulation [12]. The majority of studies have primarily employed low-concentration VFAs [13] or a combination of VFAs and glucose/glycerol as carbon sources [2], but this approach necessitates an extensive duration to attain substantial enhancements in biomass and lipid concentrations, thereby rendering VFA utilisation inefficient. To address this problem, pH adaptation, inoculation enhancement, cultivation mode modification, and other cultivation conditions optimisation were attempted to avoid acid inhibition and improve the efficiency of VFA utilisation, thus increasing lipid production [14]. Gao et al. (2020) verified that alkaline conditions (pH 8) could effectively alleviate the inhibitory effect of high-concentration VFAs on Y. lipolytica and significantly improve biomass and lipid production [15].

Previous studies have investigated the effects of VFAs, pH, and nutrient conditions on Y. lipolytica growth and lipid accumulation; however, the combined regulatory influence of carbon and nitrogen sources under high-concentration VFA conditions has not been comprehensively characterised. In addition to pH, carbon sources, nitrogen sources, and their ratios play indispensable roles in the growth and lipid synthesis of Y. lipolytica. The carbon source, which determines both VFA composition and concentration, is closely associated with lipid yield and substrate inhibition effects. Nitrogen is also vital for microorganisms to synthesise cellular components (amino acids, proteins, nucleic acids, etc.) and nitrogen-containing metabolites required for proliferation [16]. Moreover, nitrogen depletion coupled with excess carbon availability stimulates de novo lipid synthesis by inhibiting isocitrate dehydrogenase activity, leading to citrate accumulation in the mitochondria and subsequent acetyl-CoA formation in the cytoplasm, the precursor of fatty acid biosynthesis [17]. Therefore, understanding the regulatory mechanism governing carbon and nitrogen metabolism is essential for improving VFA assimilation and lipid production in Y. lipolytica.

This study aimed to systematically investigate the effects of VFA composition and concentration, nitrogen source type and level, C/N ratio, and pH on the growth and lipid synthesis of Y. lipolytica. Through mathematical modelling of biomass and lipid production, the key influencing factors were identified, and optimal cultivation conditions were determined and validated. This work contributes to a more comprehensive understanding of how coordinated carbon and nitrogen regulation enhances the utilisation of high-concentration VFAs for lipid production in Y. lipolytica.

Materials and methods

Strain, media, and test configuration

Y. lipolytica (CICC 31596) was obtained from the China Centre of Industrial Culture Collection, maintained at 4 °C on YPD agar slants (2% (w/v) agar powder), and subcultured monthly. For preculture, a loopful of yeast cells was inoculated into 100 mL of YPD medium in a 250 mL Erlenmeyer flask and incubated at 28 °C and 180 rpm for 24 h on a rotary shaker to ensure adequate aeration. The seed cells were harvested by centrifugation (3600×g, 4 °C, 5 min), washed twice with sterile saline, and resuspended in sterile distilled water to prepare the inoculum. The initial cell density of the fermentation medium was adjusted to approximately OD600 = 0.8–1.2. Subsequently, 10 mL of the inoculum was transferred into 100 mL of fermentation medium in a 250 mL Erlenmeyer flask (working volume 40%), and cultivation was performed under the same temperature and agitation conditions (28 °C, 180 rpm). The fermentation medium contained 3 g/L KH₂PO₄, 1 g/L MgSO_4·7H₂O, 15 mg/L FeCl₃·6H₂O, 7.5 mg/L ZnSO₄·7H₂O, and 0.5 mg/L CuSO₄·5H₂O, with the initial pH adjusted using 2 mol/L HCl or NaOH. All media and equipment were sterilised by autoclaving at 121 °C for 20 min before use.

To study the impacts of the composition and concentration of VFAs as carbon sources, nitrogen source types and concentrations, C/N, and pH, an orthogonal experiment including 16 tests with four factors at four levels was conducted, as shown in Table 1. After preparing the cell suspension described above, the inoculation was carried out with an inoculation ratio of 10% v/v. Specifically, 10 mL of the cell suspension was added to 100 mL of the fermentation medium in a 250 mL flask. The OD600 of the suspension after inoculation was approximately 1.0. Subsequently, the cultures were incubated as per the methods described in the previous paragraph. Samples were taken every 24 h for testing. Biomass, VFAs, nitrogen concentration, and lipid were analysed using the methods detailed in Sect. Analytical methods. Further research on specific nitrogen sources was conducted according to Table 2. Verification tests were conducted under three optimised cultivation conditions. All the tests were conducted in triplicate.

Table 1.

Test configurations of the orthogonal experiment with the factors of carbon source, nitrogen source type and concentration, C/N, and pH

No.	Initial pH	Nitrogen source	Carbon source	C/N
a1	6	NH4Cl	Acetic acid 30 g/L	5
a2	7	NH4Cl	Mixed VFAs* 30 g/L	25
a3	8	NH4Cl	Mixed VFAs 50 g/L	60
a4	9	NH4Cl	Acetic acid 50 g/L	100
b1	6	CO(NH2)2	Mixed VFAs 50 g/L	100
b2	7	CO(NH2)2	Acetic acid 50 g/L	60
b3	8	CO(NH2)2	Acetic acid 30 g/L	25
b4	9	CO(NH2)2	Mixed VFAs 30 g/L	5
c1	6	(NH4)2SO4	Acetic acid 50 g/L	25
c2	7	(NH4)2SO4	Mixed VFAs 50 g/L	5
c3	8	(NH4)2SO4	Mixed VFAs 30 g/L	100
c4	9	(NH4)2SO4	Acetic acid 30 g/L	60
d1	6	NH4NO3	Mixed VFAs 30 g/L	60
d2	7	NH4NO3	Acetic acid 30 g/L	100
d3	8	NH4NO3	Acetic acid 50 g/L	5
d4	9	NH4NO3	Mixed VFAs 50 g/L	25

Table 2.

Test configurations using different nitrogen sources to synthesise lipid from Yarrowia lipolytica under high-concentration volatile fatty acids

No.	Nitrogen source	Carbon source		C/N	pH
N-a1	NH4Cl	Acetic acid 50 g/L		5	8
N-a2	NH4Cl	Acetic acid 50 g/L		25	8
N-a3	NH4Cl	Acetic acid 50 g/L		60	8
N-a4	NH4Cl	Acetic acid 50 g/L		100	8
N-a5	NH4Cl	Acetic acid 50 g/L		150	8
N-b1	NH4NO3	Acetic acid 50 g/L		5	8
N-b2	NH4NO3	Acetic acid 50 g/L		25	8
N-b3	NH4NO3	Acetic acid 50 g/L		60	8
N-b4	NH4NO3	Acetic acid 50 g/L		100	8
N-b5	NH4NO3	Acetic acid 50 g/L		150	8
N-c1	KNO3	Acetic acid 50 g/L		5	8
N-c2	KNO3	Acetic acid 50 g/L		25	8
N-c3	KNO3	Acetic acid 50 g/L		60	8
N-c4	KNO3	Acetic acid 50 g/L		100	8
N-c5	KNO3	Acetic acid 50 g/L		150	8
N-d1	CO(NH2)2	Acetic acid 50 g/L		5	8
N-d2	CO(NH2)2	Acetic acid 50 g/L		25	8
N-d3	CO(NH2)2	Acetic acid 50 g/L		60	8
N-d4	CO(NH2)2	Acetic acid 50 g/L		100	8
N-d5	CO(NH2)2	Acetic acid 50 g/L		150	8

^* The mixed VFAs are acetic acid, propionic acid, and butyric acid mixtures with a ratio of 5:3:2.

Analytical methods

Biomass

To evaluate cell growth, the cell concentration was measured as the absorbance of the culture broth at 600 nm (OD600, DR-6000 spectrophotometer, HACH). Biomass was indicated by DCW (dry cell weight, g/L). The cells were harvested from 10 mL of culture medium by centrifugation at 8,000 rpm for 10 min. The cell pellet was washed twice with distilled water and dried to constant weight in an oven at 105 °C [18].

VFAs and nitrogen

After being filtered through a 0.45 μm membrane, the VFA concentrations of the culture broth were analysed via a gas chromatograph (Shimadzu, GC-2014) fitted with a capillary column (Stabilwax-DA, 30 m × 0.25 mm × 0.25 μm) and a flame ionisation detector. Reference to the temperature program is available in Gao et al. [15]. The total nitrogen and ammonium concentrations were measured according to the German standard methods for examining water, wastewater, and sludge. The free ammonia concentration was calculated according to the equations provided by Anthonisen et al. [19].

Lipid extraction and evaluation

Microbial lipid was extracted from lyophilised biomass with chloroform/methanol (2:1 v/v) in accordance with an adaptation of the method of Dyer and Bligh [18]. The extracts were centrifuged at 5000 rpm for 25 min to dissolve the lipid in the organic phase completely, and then removed and washed twice with 0.15% (w/v) NaCl solution. Microbial lipid was obtained after the purified chloroform layer was evaporated under a speed vacuum at 40 °C until a constant weight was achieved. The lipid composition was determined through gas chromatography (GC) analysis of fatty acid methyl esters (FAMEs). The methods used for FAME preparation and GC analysis were described in a previous study [20]. The lipid yield was defined as the amount of single-cell oil produced per litre of substrate (g/L), whereas the lipid content represented the proportion of lipid in Y. lipolytica cells, expressed as the percentage of lipids in the dry cell mass (% w/w).

Cell morphology

The cell morphology and intracellular lipid accumulation of Y. lipolytica were examined using Nile Red fluorescence staining combined with confocal laser microscopy. A 1 mL sample of the fermentation broth was centrifuged at 3600×g for 5 min and washed twice with phosphate-buffered saline (PBS, pH 7.2). The cells were resuspended in 1 mL of PBS, and Nile Red solution (final concentration 10 µg/mL, prepared in acetone) was added. The mixture was incubated in the dark at room temperature for 10 min to allow complete staining.

Fluorescence observation and image acquisition were performed using an LSM 980 ultra-resolution laser confocal fluorescence microscope (Carl Zeiss, Germany) equipped with an excitation wavelength of 488 nm and an emission detection range of 550–600 nm. Both differential interference contrast (DIC) and fluorescence channels were applied to visualize cell morphology and intracellular lipid droplets. Image acquisition, overlay, and fluorescence intensity analysis were conducted using ZEN Blue (Carl Zeiss) and ImageJ software.

Statistical analysis

All the statistical analyses of the experimental data were performed via Microsoft Excel 2021 and OriginPro 2024b. The collected data, which refer to VFAs, nitrogen, pH, OD600, and lipid production, represent the average of triplicate experimental measurements. For the linear models used to analyse the relationships between the influencing factors and biomass or lipid yield, a significance level of p < 0.05 was set. In addition, 95% confidence intervals were employed to evaluate the reliability of differences among various groups to determine optimal culture conditions using the SNK model.

Results and discussion

Major factor recognition

In the process of Y. lipolytica cell growth and lipid synthesis, the composition and concentration of VFAs used as carbon sources, nitrogen source type and concentration, carbon-to-nitrogen ratio, and pH are key influencing factors. Previous studies have demonstrated the significant impacts of these factors on the growth and lipid synthesis of oleaginous microorganisms [14]. For carbon sources, VFAs are chosen because they can be derived from organic waste, offering a low-cost option. Their composition and concentration affect the availability of carbon skeletons for lipid synthesis [8]. As for nitrogen sources, they are essential for the synthesis of cell substances such as amino acids, proteins, and nucleic acids. Different nitrogen sources vary in their utilisation efficiency and the resulting impact on cell growth and lipid accumulation [17]. The C/N ratio is crucial as it balances carbon and nitrogen nutrition. A proper C/N ratio is necessary for cell growth, and nitrogen limitation can promote lipid storage in cells [21]. pH influences the solubility and dissociation state of VFAs and nitrogen-containing compounds, thus affecting the uptake and metabolism of nutrients by cells [15]. As shown by the test configuration in Table 1, an orthogonal experiment with four factors and four levels was conducted to determine the significance of these factors against the background of high-concentration acids. As a carbon source, acetic acid and acid mixtures (acetic acid: propionate: butyric acid = 5:3:2) used to model the food waste hydrolysate were studied at 30 g/L and 50 g/L, respectively. NH₄Cl, NH₄NO₃, (NH₄)₂SO₄, and urea, at different concentrations, were used as nitrogen sources. C/N was set to 5, 25, 60, or 100 according to the variation in acid and nitrogen resource concentrations. In addition, the initial pH was adjusted to 6, 7, 8, or 9.

During the 16 tests, the biomass (indicated by DCW), lipid yield, and lipid content in the cells varied with pH, C/N, nitrogen type, VFA concentration, and composition, as shown in Table 3. The DCW ranging from 4.76 g/L to 20.8 g/L was significantly affected by the variation in cultivation conditions, and the lipid yield was approximately 1.68 ~ 5.08 g/L. This study employed high-concentration VFAs as the carbon source for Y. lipolytica cultivation, and lipid yields notably improved despite acid-induced growth inhibition. Moreover, acid inhibition can be alleviated by regulating pH, potentially indicating that Y. lipolytica can use high concentrations of VFAs to improve lipid production efficiency.

Table 3.

Biomass and lipid production of Yarrowia lipolytica in the orthogonal experiment

No.	DCWa g/L	Lipid Yieldb g/L	Lipid Contentc %	Y X/S g DCW/g VFAs	Y L/S g lipid/g VFAs
a1	7.87	3.55	45.13	0.26	0.12
a2	19.07	5.08	26.42	0.64	0.17
a3	20.80	4.76	22.88	0.42	0.10
a4	15.07	4.45	29.54	0.30	0.09
b1	17.73	3.91	22.04	0.36	0.08
b2	13.25	4.74	35.79	0.27	0.10
b3	7.60	4.05	53.29	0.25	0.14
b4	14.97	3.53	23.55	0.50	0.12
c1	15.73	1.79	11.37	0.32	0.04
c2	6.85	2.73	39.78	0.14	0.06
c3	12.38	2.57	20.75	0.41	0.09
c4	4.78	1.89	39.56	0.16	0.06
d1	5.71	3.07	53.81	0.19	0.10
d2	10.87	1.68	15.47	0.36	0.06
d3	11.28	2.24	19.83	0.23	0.05
d4	7.58	2.44	32.20	0.15	0.05

1. DCW refers to the dry cell weight per litre of substrate used to evaluate biomass.

2. The lipid yield refers to the production of single-cell oil per litre of substrate.

3. The lipid content refers to the proportion of lipid in Y. lipolytica cells.

In terms of lipid production, test a2 (cultivation conditions: initial pH 7, C/N 25, nitrogen source: NH₄Cl, carbon source: 30 g/L VFA with an acetic acid: propionic acid: butyric acid ratio of 5: 3: 2) performed best with a DCW of 19.07 g/L and a lipid yield of 5.08 g/L, indicating that 64% of the VFAs in the substrate were converted into Y. lipolytica cells and that 17% of the VFAs were converted to lipid. This result may be attributed to the nitrogen source and pH condition. NH₄Cl contains ammonium that can be hydrolysed into ammonia, directly available for Y. lipolytica. A neutral initial pH environment balances acid dissociation and ammonia inhibition, thereby enhancing cell metabolism and lipid synthesis.

The data analysis was further performed using linear models to comprehensively optimise cultivation conditions, with separate outputs of DCW and lipid yield. An analysis of the mathematical relationships between the four influencing factors and biomass (as shown in Table S1) revealed that the degree of influence of the factors decreased in the order of nitrogen source > VFA composition and concentration > C/N > pH, as determined by the F value. Generally, VFA composition and concentration are the most significant factors [3]; however, all the tests in this study were performed in the background of high concentrations of acids, changing within a small range. The optimal culture conditions for improving biomass based on the SNK model are NH₄Cl as the nitrogen source, 50 g/L acetic acid, pH 8, and C/N 100.

The mathematical relationships between the four influencing factors and the lipid yield (as shown in Table S2) also indicated that the nitrogen source has an important effect on the lipid yield. The optimal culture conditions for improving the lipid yield via the SNK model are NH₄Cl as the nitrogen source, 30 g/L mixed VFAs, pH 7, and C/N 60.

Based on these experiments, the SNK model with biomass output and the SNK model with lipid production output obtained three sets of cultivation conditions with different pH values, acids, and C/N ratios. Further tests were conducted under these cultivation conditions, and the results are presented in Sect. Microbial lipid production optimisation below.

Regulatory mechanism analysis

As biomass was significantly affected by the variation in cultivation conditions during the 16 tests and was directly related to lipid production, the variation in Y. lipolytica biomass during the cultivation process was tested and indicated by the OD600. Under different cultivation conditions, the lag period and logarithmic growth periods of Y. lipolytica in the tests differed; however, all the OD600 values ranged from 2.0 ± 0.4 at the end of cultivation. Y. lipolytica showed strong tolerance to high-concentration VFA or alkaline environments and carried out cell metabolism and lipid synthesis after adaptation.

pH

pH plays a crucial role in assimilating the carbon source by oleaginous microorganisms and in lipid accumulation [22, 23]. Generally, the optimal culture pH range is between 5.0 and 6.0 during lipid production from sugars by most oleaginous yeasts [24, 25]. In contrast, the optimal culture pH range is controversial when the carbon source is VFAs. Increasing the pH to higher than 7 is commonly recognised to favour acid inhibition [26]. However, as oleaginous yeasts are sensitive to alkaline environments, specific circumstances, including pH, free ammonia, nitrogen availability, acid forms and concentration, should be considered when adapting pH.

The growth curve trend revealed the important effect of pH on the cultivation of Y. lipolytica with high-concentration acids as the carbon source. As shown in Fig. 1, the growth of Y. lipolytica had a lag period of approximately 24 h in tests a1 and a4, mainly due to the high initial free ammonia concentration in these two groups. In test a1, the C/N ratio was only 5, and the nitrogen source was NH₄Cl, resulting in a high initial free ammonia concentration, whereas in test a4, the high initial free ammonia concentration was caused by the high initial pH, although the C/N ratio was only 100. After 24 h, along with the consumption of nitrogen and pH variation (pH in test a4 decreased to ± 8), ammonia inhibition occurred. After 144 h of cultivation, the growth of Y. lipolytica in tests a2, a3, and a4 reached a stable phase, whereas the multiplication rate of Y. lipolytica in test a1 increased, mainly because the C/N ratio in test a1 was the lowest and there was still sufficient nitrogen at 144 h.

Fig. 1.

Yarrowia lipolytica growth curve according to the OD600 in the orthogonal experiment a test a1-4 using NH₄Cl as the nitrogen source, b test b1-4 using urea as the nitrogen source, c test c1-4 using (NH₄)₂SO₄ as the nitrogen source, d test d1-4 using NH₄NO₃ as the nitrogen source

When Y. lipolytica is cultured under high-concentration acids, the uncoupling induces cytoplasmic acidification and inhibits the metabolism of cells [15]. Therefore, high concentrations of acids inhibit the growth and lipid synthesis of Y. lipolytica, as proven by tests b1, c1, and d1, which clearly inhibited the growth of Y. lipolytica at a low initial pH of 6. In the tests of this orthogonal experiment, the pH of the substrate was initially adjusted to 6, 7, 8, or 9, and no further intervention was made during cultivation. The final pH in all tests was approximately 9 ± 0.5 (as depicted in Figure S1), which is consistent with the previous publications [27, 28], which reported that the final pH was approximately 9.0 ~ 9.5 when Cryptococcus curvatus was cultivated with 20 ~ 30 g/L acetic acid as the carbon source. In addition, in the four tests with an initial pH of 8, the pH fluctuation was low; when the initial pH was 9, the pH decreased rapidly within 24 h and eventually approached ± 9. Under the conditions of this study, a pH of 8 was more suitable than a pH of 9. In terms of the relationship between pH variation and acid consumption, when the initial pH was acidic or neutral, the general trend of pH change was to increase first and then stabilize, and the inflection point was consistent with that of acetic acid consumption; that is, the inflection point of pH appeared when acetic acid was exhausted, and the consumption of acetic acid had a greater impact on the pH of the medium than did the consumption of propionic acid or butyric acid.

pH variation reflects the metabolism of microorganisms and indicates that Y. lipolytica can survive in a mildly alkaline environment. Therefore, pH regulation can alleviate the persistent effect of acid on Y. lipolytica growth, as shown by test a3 in this study, in which Y. lipolytica did not have a lag phase when the initial pH was adjusted to 8, in accordance with Kuttiraja et al.’s conclusions [29]. A previous study revealed that acetic acid in its dissociated form is much less toxic than its undissociated form [30]. When the culture pH was 5.5, approximately 15% acetic acid was in the undissociated form, while 99% acetic acid was dissociated into acetate anions [27]. High-concentration VFAs cause cytoplasmic acidification in Y. lipolytica. This is mainly because VFAs can freely diffuse across the cell membrane in their undissociated form. Once inside the cell, they dissociate, releasing protons and thereby decreasing the intracellular pH. Disturbance of intracellular pH triggers cellular acid–base homeostasis responses. To restore cytoplasmic pH, Y. lipolytica activates ATP-dependent proton pumps (H⁺-ATPases) and ion exchange systems on the plasma and vacuolar membranes. This process consumes additional ATP, altering the intracellular ATP/AMP ratio and affecting the redox balance (NADH/NAD⁺). The resulting changes in energy metabolism and cofactor availability can further influence acetyl-CoA flux and lipid biosynthetic activity, thereby linking extracellular pH regulation with intracellular acid–base and energy homeostasis [31, 32]. Thus, pH regulation works partly by converting acetic acid to its dissociated form. This study further suggested that pH adaptation would cause ammonia inhibition, as the free ammonia concentration would increase with increasing pH; for example, in test d4, with an initial pH of 9 and a C/N of 25, free ammonia clearly inhibited the growth of Y. lipolytica, leading to a lag phase of 144 h.

Acid utilisation

In terms of the carbon source used, the acid preference of Y. lipolytica was not affected by differences in cultivation conditions. The acid consumption was analysed when (NH₄)₂SO₄ or NH₄NO₃ was used as the nitrogen source. As illustrated in Fig. 2, tests c1, c4, d2, and d3, utilising acetic acid as the sole carbon source, exhibited similar acid consumption profiles despite variations in other cultivation conditions. This suggests that acetic acid consumption was rapid during the first 24 h and subsequently decelerated, leading to complete exhaustion at 120 h.

Fig. 2.

Acid utilisation during the cultivation process of Yarrowia lipolytica in the orthogonal experiment arbon sources: c1 50 g/L acetic acid, c2 50 g/L acid mixture, c3 30 g/L acid mixture, c4 30 g/L acetic acid, d1 30 g/L acid mixture, d2 30 g/L acetic acid, d3 50 g/L acetic acid, d4 50 g/L. The ratio of the acid mixture was acetic acid: propionic acid: butyric acid = 5:3:2. The nitrogen sources of c1-4 and d1-4 are (NH₄)₂SO₄ and NH₄NO₃, respectively

In other tests in which mixed acids were used as the carbon source, the utilisation of acetic acid was similar to that in the first 24 h, as the initial acetic acid concentration was rather low. Acetic acid is initially consumed at a high rate, and the uptake rate by Y. lipolytica subsequently decreases due to the reduction in the driving force for acetic acid transport into the cell, which is determined by the concentration gradient. Additionally, after the initial rapid consumption of acetic acid, Y. lipolytica may undergo metabolic adjustments similar to diauxic growth. Initially, the cell prefers to utilise acetic acid as an energy and carbon source [33]. As acetic acid becomes scarce, the cell shifts its metabolic pathways to adapt to the remaining propionic acid and butyric acid in the medium [8]. This metabolic shift requires time for the cell to adjust enzyme production and activity, leading to a slower consumption rate of acetic acid.

The utilisation of acetic acid, propionic acid, and butyric acid by Y. lipolytica occurred simultaneously; however, their consumption rates differed, with acetic acid, propionic acid, and butyric acid. This finding further supports directed acid production from organic wastes as the upstream unit of the microbial oil production process.

Nitrogen type and C/N

Nitrogen is an essential nutrient for the growth and proliferation of Y. lipolytica. Furthermore, on the basis of the factor analysis of the orthogonal experiment, nitrogen source type significantly impacts biomass and lipid production in the context of high concentrations of acid. Nitrogen in the substrate exists in the forms of ammonia, ammonium, nitrate, nitrite, and organic nitrogen. Ammonia is one of the most efficient forms for taking in nitrogen by microorganisms (Fig. 3). Nitrate is reduced to nitrite by the reductase system in microorganisms and further converted into ammonia, which is ultimately used to synthesise cell components. Urea can be hydrolysed into two ammonia molecules to supply nitrogen to microorganisms.

Fig. 3.

Schematic illustration of nitrogen assimilation and lipid biosynthesis from volatile fatty acids (VFAs) in Yarrowia lipolytica

Under high-concentration VFA conditions, the nitrogen metabolism of Y. lipolytica is intertwined with its overall metabolic state. Nitrogen is not only crucial for cell growth but also has a significant impact on lipid synthesis. When Y. lipolytica is cultured with high-concentration VFAs, the cell needs to balance the utilisation of carbon from VFAs and nitrogen for various metabolic processes. At the same time, nitrogen also participates in regulating lipid-synthesis-related genes. Previous studies have shown that nitrogen availability can affect the expression of genes encoding key enzymes in lipid synthesis pathways, like acetyl-CoA carboxylase [21, 34]. Under high-concentration VFA, the cell might adjust the expression of these genes based on the nitrogen supply, thereby influencing lipid synthesis.

According to Table 3, regarding biomass accumulation and lipid production, NH₄Cl demonstrated the most favourable performance, while (NH₄)₂SO₄ exhibited the least satisfactory results among the 16 tests. As there are many variables in the orthogonal test, it is difficult to analyse the influence of different nitrogen sources fully; thus, 20 tests were conducted with varying sources of nitrogen and C/N ratios. Considering the various types of nitrogen sources and the unsatisfactory performance of (NH₄)₂SO₄ in the orthogonal experiments, NH₄Cl, NH₄NO₃, KNO₃, and urea were utilised as nitrogen sources at C/N ratios of 5, 25, 60, 100, and 150, as shown in Table 2.

When NH₄Cl is used as the nitrogen source, nitrogen exists in the forms of ammonium and ammonia, as ammonium can be hydrolysed into ammonia. Y. lipolytica can directly utilise ammonia; however, a high free ammonia concentration inhibits cell activity, disrupting the cytoplasm’s acid‒base balance when it enters cells. As shown in Fig. 4, the initial free ammonia concentration reached 1 g/L (calculated via the Anthonisen equation) when the C/N ratio was 5, which inhibited the growth of Y. lipolytica and led to low biomass with a DCW of 4.40 g/L. In addition, when the C/N ratio was greater than 100, cell growth and multiplication were restricted by an insufficient nitrogen source, indicating that the OD600 decreased after 168 h of cultivation. When N-a1 was tested, along with the consumption of nitrogen and the adaptability to ammonia of Y. lipolytica, the OD600 increased rapidly in the later stage of culture and even continued to rise after 216 h. In test N-a5, with the highest C/N of 150, nitrogen limitation promoted the ability of Y. lipolytica to store lipids in the cell, resulting in a high lipid content of 42%; thus, test N-a5 presented a high lipid yield of 2.20 g/L, as shown in Fig. 5, although nitrogen limitation also decreased the biomass.

Fig. 4.

Yarrowia lipolytica growth curve in tests for investigating the impacts of different nitrogen sources a N-a1 to N-a5 were tested with NH₄Cl as the nitrogen source, b N-b1 to N-b5 were tested with NH₄NO₃ as the nitrogen source, c N-c1 to N-c5 were tested with KNO₃ as the nitrogen source, and d N-d1 to N-d5 were tested with urea as the nitrogen source. The C/N ratio was changed from 5 to 150 in all the groups

Fig. 5.

Biomass and lipid production of Yarrowia lipolytica in tests for investigating the impacts of different nitrogen sources a N-a1 to N-a5 were tested with NH₄Cl as the nitrogen source, b N-b1 to N-b5 were tested with NH₄NO₃ as the nitrogen source, c N-c1 to N-c5 were tested with KNO₃ as the nitrogen source, and d N-d1 to N-d5 were tested with urea as the nitrogen source. The C/N ratios were 5, 25, 60, 100, and 150 for all the groups

.

When the nitrogen came from NH₄NO₃, the nitrogen existed in the forms of ammonium, nitrate, and ammonia; the ammonia inhibition could be ignored, as half of the nitrogen existed in the form of nitrate, and the initial ammonia concentration was less than 0.05 g/L. With sufficient nitrogen in the test with a C/N of 5, Y. lipolytica grew and proliferated well, obtaining an OD600 of 10.18 (highest during the 20 tests); however, Y. lipolytica did not tend to accumulate lipids under these conditions, leading to a very low lipid contest of 8.74 g/L; therefore, the lipid yield was only 0.89 g/L, similar to that in test N-a1. In addition, the presence of nitrate also affected the pH variation, as shown in Figure S2. When all the nitrogen sources were ammonium, the pH first increased rapidly to the peak value (9.5 ~ 10.0) and then decreased to ± 9.0; however, when parts of the nitrogen source were nitrate, the pH continued to increase and reached a maximum value of 8.2 ~ 9.0. In the test in which NH₄NO₃ was used as the nitrogen source, the lipid yield and lipid content of Y. lipolytica were lower than those when NH₄Cl was used as the nitrogen source, probably because nitrate cannot be directly metabolised by Y. lipolytica. N-c1 to N-c5 tests using KNO₃ further revealed that nitrate is less suitable than ammonium for cultivating Y. lipolytica for lipid production.

When urea was used as the nitrogen source, Y. lipolytica utilised ammonia from urea hydrolysis for metabolism, and the initial ammonia concentration was approximately 0.045 g/L under a background C/N of 5, which wouldn’t cause ammonia inhibition. Due to urea hydrolysis characteristics, little variation in the ammonia concentration occurred with nitrogen consumption. When the C/N ratio was 5, the C/N_ammonia ratio was approximately 150, promoting lipid storage in Y. lipolytica cells via ammonia nitrogen limitation and increasing the lipid content to 52.82%. An increase in the C/N ratio further promoted cell proliferation but did not promote lipid conversion. The tests using urea as the nitrogen source obtained lipid yields ranging from 2.43 ~ 2.64 g/L.

Overall, NH₄Cl and urea are suitable nitrogen sources for Y. lipolytica cultivation for lipid production, resulting in lipid yields ranging from 2.00 ~ 2.50 g/L. NH₄Cl can be utilised more directly and efficiently by Y. lipolytica than urea and nitrate. It was reported that nitrogen availability is crucial for gene expression (especially that of hexokinase), which has a higher impact than carbon source variation [34]. Additionally, ammonium assimilation releases protons, causing extracellular acidification that activates ATP-consuming proton pumps (V-ATPase) to maintain pH. This increases energy demand, lowering the ATP/AMP ratio and triggering AMPK activation and TOR inhibition. Consequently, carbon flux shifts from growth to triacylglycerol storage, linking nitrogen source, pH homeostasis, and energy metabolism as a key regulatory axis in lipid remodelling of oleaginous yeasts [35]. However, NH₄Cl is more likely to cause ammonia inhibition than urea. Urea can be hydrolysed into ammonia, a nitrogen source for Y. lipolytica. When urea is used as the nitrogen source, the initial ammonia concentration is relatively low, which won’t cause ammonia inhibition. As the culture progresses, the slow release of ammonia from urea hydrolysis maintains a relatively stable nitrogen supply. It creates a nitrogen-limited environment, further promoting the accumulation of lipids and resulting in a high lipid content. This study suggested that NH₄Cl and urea were advantageous nitrogen sources for lipid production.

Nitrogen availability regulates the transcription of key genes involved in fatty acid biosynthesis and triacylglycerol (TAG) assembly, such as ACC1 (encoding acetyl-CoA carboxylase) and DGA1 (encoding diacylglycerol acyltransferase), which are upregulated under nitrogen-limited conditions, thereby promoting lipid accumulation. Conversely, when nitrogen is abundant, lipid synthesis is repressed due to reduced expression of these genes and enhanced activity of enzymes in amino acid biosynthesis [5, 36]. Therefore, in terms of nitrogen utilisation, both the nitrogen source type and the C/N ratio need to be carefully considered. C/N affects Y. lipolytica in three main aspects: (1) balanced carbon and nitrogen nutrients are necessary for cell growth and proliferation, (2) nitrogen limitation promotes the ability of Y. lipolytica to store lipids in cells, and (3) free ammonia might inhibit the activity of Y. lipolytica. Nitrogen is an important element for cell growth and proliferation. It is generally believed that the suitable C/N for microbe growth and proliferation is 20 ~ 30 [37], and increasing the C/N is conducive to synthesising oil and storing it in the cell [38, 39]. Under nitrogen-deficient conditions, the activity of isocitrate dehydrogenase is inhibited, leading to the accumulation of citrate in the mitochondria, which is then translocated to the cytoplasm, where it is released by adenosine triphosphate citrate lyase and the formation of acetyl-CoA, the precursor of fatty acids [40]. However, providing adequate nitrogen nutrition for the growth and proliferation of Y. lipolytica is still important, as the biomass would be reduced if lacking nitrogen nutrition; e.g., nitrogen deficiency has limited the growth and proliferation of Y. lipolytica when the C/N ratio was greater than 100 in this study.

Microbial lipid production optimisation

Three sets of cultivation conditions with different pH values, acids, and C/N ratios were obtained according to Sect. Major factor recognition, and tests were further conducted under these conditions, as shown in Table 4. The lipid yields ranged from 3.34 ~ 4.00 g/L in the tests. Consistent with the findings presented in Sect. Major factor recognition, the cultivation conditions employed in the FT1 test promoted the growth and proliferation of Y. lipolytica, resulting in the highest biomass. Conversely, these conditions were not conducive to lipid accumulation, leading to the lowest lipid content. FT2 test obtained the highest lipid yield, 4.00 g/L.

Table 4.

Biomass and lipid production of Yarrowia lipolytica cultivated under optimal conditions

No.	pH	Nitrogen source	Carbon source	C/N	DCWa g/L	Lipid Yieldb g/L	Lipid contentc %
FT1	7	NH4Cl	VFAsd 30 g/L	25	14.09	3.34	23.68
FT2	7	NH4Cl	VFAsd 30 g/L	60	9.91	4.00	40.37
FT3	8	NH4Cl	acetic acid 50 g/L	100	8.17	3.50	42.87

1. DCW refers to the dry cell weight per litre of substrate used to evaluate biomass.

2. The lipid yield refers to the production of single-cell oil per litre of substrate.

3. The lipid content refers to the proportion of lipid in Y. lipolytica cells.

4. VFAs contain mixed acids with a ratio of acetic acid: propionate: butyric acid = 5:3:2.

In addition to the above quantitative methods for evaluating biomass and lipid production, the cell morphology of Y. lipolytica on days 3 and 6 was photographed via an LSM 980 ultra-resolution laser confocal fluorescence microscope. The intracellular lipid was stained with Nile red, and the bright green area in the cell was the lipid, as shown in Fig. 6. A comparison of the images taken on the third and sixth days revealed that the cell morphology changed to a fuller shape during the cultivation process, which was especially noticeable in the FT3 test, in which the cells on the third day were slender and columnar and became more rounded and rod-shaped on the sixth day. In addition, the images revealed that lipid accumulation in Y. lipolytica occurred later than cell growth and proliferation did, as indicated by the increased bright green area in the sixth-day images across all tests. Based on the evaluation of DCW, the biomass of Y. lipolytica was the highest in the FT1 test because of the fastest growth, as shown by the images showing that the cells in Fig. 6-a were fuller in shape than those in Fig. 6 -b and -c. In terms of the images on the sixth day, the bright green spots in Fig. 6 -e and -f were stronger, indicating higher lipid contents in the FT2 and FT3 cells, which was consistent with the results of the lipid content calculated on the basis of the DCW and lipid yield measurements.

Fig. 6.

Images of Yarrowia lipolytica cultivated under optimal conditions by LSM 980 ultra-resolution laser confocal fluorescence microscopy. The bright green area in the cell is single-cell oil. a Day 3, test FT1; b day 3, test FT2; c day 3, test FT3; d day 6, test FT1; e day 6, test FT2; f day 6, test FT3

As shown in Fig. 7, the compositions of lipid obtained under the three different groups of cultivation conditions were similar, as the lipids were composed of C14 ~ C20 fatty acids, and the main fatty acids were C16 and C18, which were the same as the fatty acids of ordinary vegetable oil and soybean oil. The highest content was C18:1 (oleic acid)-based unsaturated fatty acids, which are conducive to improving the dew point of refined oil to increase its fluidity and obtain higher-quality refined oil. However, the difference in carbon source caused variation in the contents of odd-carbon fatty acids. The carbon sources of FT1 and FT2 were acetic acid, propionic acid and butyric acid, whereas the carbon sources of FT1 were all acetic acid, lacking odd-carbon acids, resulting in the contents of C15 and C17 fatty acids in FT1 and FT2 being greater than those in FT3. These odd-carbon fatty acids are important in cosmetics, pesticides, health care products, and other fields. In addition, linoleic acid (C18:2) content in FT3 was relatively high, which plays an important role in preventing cardiovascular diseases.

Fig. 7.

Composition of lipid obtained from Yarrowia lipolytica cultivated under optimal conditions

To further interpret these results, comparisons with glucose-based lipid synthesis in Y. lipolytica were made based on previous studies. Carsanba et al. (2020) investigated several Y. lipolytica strains under glucose-based cultivation and found that their lipid profiles were consistently dominated by C16:0, C18:0, C18:1, and C18:2 fatty acids. This result indicates that glucose supports a typical de novo lipid synthesis pathway in Y. lipolytica, leading mainly to even-numbered long-chain fatty acids [41]. Furthermore, Gao et al. (2021) reviewed the metabolic mechanisms underlying glucose-based de novo lipid synthesis in Y. lipolytica and confirmed that acetyl-CoA is the main precursor, inherently limiting the biosynthesis to C16–C18 fatty acid chains [42]. In contrast, the lipid composition observed in this study under VFA-based cultivation (acetic, propionic, and butyric acids) was also dominated by C16 and C18 fatty acids, similar to those obtained under glucose-based conditions. However, it exhibited a broader fatty acid spectrum, including a considerable proportion of odd-chain (C15:0, C17:0, C17:1) and medium-chain fatty acids. This compositional shift suggests that VFAs, particularly propionic and butyric acids, can be directly incorporated into the fatty acid elongation pathway, thereby bypassing the chain-length restriction of conventional de novo synthesis. These findings highlight the enhanced metabolic flexibility of Y. lipolytica when utilising VFAs as carbon sources, enabling the production of structurally diverse lipids with potential industrial applications.

Conclusions

High-concentration VFAs (30 g/L) enhanced the lipid yield of Y. lipolytica, with optimised conditions alleviating acid inhibition. Acetic, propionic, and butyric acids were co-utilised, though degraded at different rates (acetic > propionic > butyric). Nitrogen sources significantly influenced growth and lipid synthesis; NH₄Cl was more direct and efficient than urea but more prone to ammonia inhibition. Alkaline pH adaptation mitigated acid effects. Using high VFAs, lipid yield reached 4.00 g/L with 42.87% content, mainly C14–C20 fatty acids. However, achieving simultaneous biomass and lipid improvement remains challenging and warrants further study.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

X.W. and M.L. conceived the study and designed the methodology. X.W. performed the investigation, project administration, software analysis, and wrote the original draft. S.S. contributed to methodology. G. Y., S.C. and L.C.C. revised the manuscript, with L.C.C. also providing supervision. Z.L. acquired funding and supervised the project. All authors reviewed and approved the final manuscript.

Funding

This study was supported by the National Key Research and Development Program of China (2024YFD1600201) and the International Exchange and Growth Program for Young Teachers Project (QNXM20250004).

Data availability

The authors declare that the data supporting the findings of this study are available within the paper and its Supplementary material file. Should any raw data files be needed in another format, they are available from the corresponding author upon reasonable request.

Declarations

Conflict of interest

The authors declare no competing interests.