Acetic acid induces strain-specific metabolic adaptation of Saccharomyces cerevisiae during pH stress

Highlights

• •Acetic acid-derived metabolic rewiring was studied depending on growth conditions.
• •Acetic acid toxicity depends on strain, pH and oxygen availability.
• •S. cerevisiae ATCC 9804 is more tolerant to acetic acid compared to ATCC 13,007.
• •Oxygen limitation amplifies growth and metabolic inhibition via acetic acid.
• •Maintaining of respirotory activity under stress stimulates higher resistance.

Abstract

This study investigates the effects of acetic acid (AA) on the central carbon metabolism of S. cerevisiae ATCC 9804 and ATCC 13,007 under aerobic or static cultivation at pH 3.0. 10 mM AA caused only minor physiological changes, whereas 20 mM AA stimulated respiratory activity, increasing oxygen consumption up to 3.0-fold. In contrast, 50 mM AA markedly impaired metabolism, reducing biomass formation ∼ 110.0-fold under static cultivation, decreasing CO₂ production ∼ 250-fold, and suppressing glucose utilization and ethanol production. ATCC 9804 maintained higher respiratory capacity, acetate utilization, biomass carbon recovery, whereas ATCC 13,007 exhibited pronounced metabolic exhaustion under static cultivation. The results demonstrate that maintenance of respiratory capacity and efficient carbon flux allocation are key determinants of AA tolerance at pH 3.0. These findings provide new insights into the metabolic mechanisms underlying weak organic acid adaptation and offer framework for engineering robust industrial yeast strains.

Graphical abstract

1. Introduction

Saccharomyces cerevisiae is one of the most prolific and industrially significant species within industrial microbiology [1]. During fermentative bioprocesses, S. cerevisiae is subjected to various environmental stressors, including high initial sugar concentrations, elevated ethanol levels in the latter stages of fermentation, oxygen limitation, acidic pH, osmotic stress, and the accumulation of inhibitory compounds, such as acetic acid (AA) [2]. AA accumulation is commonly associated with yeast-based fermentation processes and can negatively affect yeast performance in industrial applications. In alcoholic beverage production, AA is typically detected at millimolar levels, with reported concentrations ranging from 2.3 to 7.5 mM in wines and reaching up to 25 mM in sour beers, while excessive accumulation above 20 mM can negatively affect product quality. Moreover, during lignocellulosic biomass pretreatment for second-generation bioethanol production, AA concentrations can reach substantially higher levels (∼17–250 mM), being a major inhibitor of yeast fermentation [3].

The response of the yeast to AA has mainly been examined as a stress response [4], focusing on intracellular pH disruption, reactive oxygen species (ROS) production, the mechanisms of AA toxicity and adaptation pathways [5], while its metabolic regulatory mechanisms remain insufficiently explored. Our earlier study [3] showed that AA reduces cell viability, disrupts intracellular redox balance, induces oxidative stress, and impairs ADH activity and energy production, leading to reduced fermentation efficiency and ethanol yield. Moreover, the mechanism of AA toxicity was dependent on the metabolic state and strain type [3]. Consequently, the development of strategies aimed at increasing yeast tolerance to these adverse conditions is critical for improving robust fermentation and maintaining high ethanol yield [5].

Some weak organic acids (WOA), such as AA, can be used by S․ cerevisiae as alternative energy sources when fermentable sugars are limited [6]․ However, at high concentrations, WOA can function as a stressors, and its mechanism of action depends not only on extracellular pH but also on the chemical properties of the specific organic acid [7]. Previously it was shown that the inhibitory effect of WOA is stronger at low pH because the acids are mostly in an undissociated form, which more easily penetrates cells and disrupts cellular functions [6]. On the other hand, WOA, e.g. AA, lactic or sorbic acid, are key contributors of food preservation [8], thus the study of their influence depending on extracellular parameters would contribute to development of new technologies for food safety, as well as optimizing fermentation processes [8].

AA inhibits yeast growth mainly by causing acidification of the cytoplasm [9], and subsequently inactivation of phosphofructokinase and inhibition glycolysis [10]. It was shown that plasma membrane ATPase (Pma1) may consume 25–40% of cellular ATP to maintain intracellular pH homeostasis under these conditions [11]. Furthermore, it was shown that S. cerevisiae increases glycerol production and its intracellular accumulation under several stress, including osmotic and AA stress as a protective response to the impaired redox balance while the respiratory chain cannot efficiently regenerate NAD⁺ because of absence functional respiratory complex I [[12], [13], [14]]. However, despite extensive studies on AA toxicity, the combined influence of extracellular conditions and cellular metabolic state on the yeast adaptive response and fermentation performance remains insufficiently understood.

Our previous study demonstrated pronounced changes in ion fluxes and intracellular pH, revealing key biophysical adaptations to AA stress [15]․ Specifically, findings demonstrated that oxygen limitation stimulates intracellular acidification and disrupts ion flux coordination leading to non-linear Na⁺/H⁺ and K⁺/H⁺ exchange shifts, increased ion imbalance correlating with growth inhibition (unpublished data). Nevertheless, to our best knowledge, the underlying downstream proteomic analysis and metabolic regulation mechanisms remain poorly studied.

Earlier findings showed that industrial S. cerevisiae strains can differ substantially in acetate production and utilization depending on oxygen availability, suggesting strain-specific metabolic adaptation strategies [2]. Despite the limited data in the literature, it was shown that wine and beer fermentation-derived yeast strains, such as ATCC 9804 and ATCC 13,007, differ by fermentation capacity, time, temperature, as well as alcohol resistance, flavor and aroma profile, nutrient requirement and flocculation properties [2,16]. The shown differences in metabolic properties, as well as previously reported differences in AA stress resistance suggest the presence of distinct adaptation mechanisms, make them a good model for understanding of strain-dependent AA responses.

S․ cerevisiae can implement carbon source utilization and energy production by several metabolic pathways, including fermentation, respiro-fermentation or respiration [3,[17], [18], [19]], moreover, the type of metabolic pathway performed depends on environmental and nutritional conditions, such as carbon source type and concentration, nitrogen availability etc. [20]. However, the combined effects of these conditions, particularly carbon source concentration, pH, oxygen availability etc. on metabolism regulation, especially under stress conditions, have not been fully addressed yet.

Taken together, this study aimed to investigate the metabolic rewiring mechanisms of S․ cerevisiae ATCC 9804 and ATCC 13,007 strains under AA stress conditions depending on varying environmental conditions including cultivation regimes and AA concentration. The results contribute to elucidating the metabolic regulatory mechanisms and adaptive responses of S. cerevisiae under AA conditions, which can be useful for robust strains construction for industrial applications. By combining comprehensive physiological and metabolic analyses, the study provides new insights into how cultivation conditions modulate AA tolerance and central carbon metabolism in different yeast strains․

2. Materials and methods

2.1. Yeast strains and growth conditions

S. cerevisiae ATCC 9804 and ATCC 13,007 wild-type strains, with distinct AA-sensitivity to AA [3], were obtained from the Microbial Depository Centre of the Armbiotechnology Scientific and Production Centre of the National Academy of Science Republic of Armenia. S. cerevisiae ATCC 9804 strain (diploid, GenBank: Z95939.1; ENA: ERR5285363) was isolated from palm wine-fermentation environment [2], whereas S. cerevisiae ATCC 13,007 strain (prototroph, pof+, sta, diploid; ENA: ERR5358805) is derived from Irish beer-fermentation [21,22].

Yeast strains were stored at −80 °C and periodically passaged on YPDA plates (yeast extract (10 g L⁻¹), peptone (20 g L⁻¹), glucose (20 g L⁻¹), agar (20 g L⁻¹) [23]. S. cerevisiae seed cultures were prepared transferring the cells in 10 mL YPD liquid medium in 50 mL Erlenmeyer flask (covered with LABOCAP screw cap (Carl Roth (Art. No. K397.1), Germany) and incubated at 30 °C with 180 rpm orbital shaking (Multitron Standard, Infors HT, Switzerland) [[23], [24], [25]]. For experiments, YPD mediums without (control) or with 10–50 mM AA were inoculated with the cells from exponential growth phase seed culture to an initial optical density (OD) of 0.2. AA was filter-sterilized (0.22 μm, VWR, USA) and added from a 10 M stock solution before the inoculation. Culture growth was monitored under aerobic cultivation (medium filled 16% of the flask volume, with rotary shaking at 180 rpm, orbital shaking diameter or throw of 25 mm) and static cultivation conditions (medium filled to 1/1.2 of flask, without shaking) at 30 °C [25]. Maier et al. investigated the maximum oxygen transfer rate in Erlenmeyer flasks ranging from 50 to 1000 mL under relative filling volumes of 4–16%, shaking diameters of 1.25–10 cm, and shaking frequencies of 50–500 rpm [26]. The applied cultivation parameters are within the range commonly used for oxygen-sufficient shake-flask cultivation [[27], [28], [29], [30]]. Medium pH was adjusted to 3.0 by 0.1 N HCl using an HI1131 pH-electrode combined to an HI3220 pH-ionometer (Hanna Instruments, USA) [2]. Under aerobic cultivation conditions biomass accumulation and growth rates were monitored using a non-invasive backscatter-based aerobic Cell Growth Quantifier (CGQ) biosensors (Scientific Bioprocessing, Germany) connected to an incubated orbital shaker (Multitron Standard, Infors, Switzerland) [3,31]. Data acquisition and growth rate analysis were performed using DOTS software (Scientific Bioprocessing, Germany). Under both aerobic and static cultivation conditions cell growth kinetics were monitored via OD₆₀₀ measurements over the growth time against sterile medium using DEN600 photometer (Biosan, Latvia) [3].

2.2. Dry biomass weight measurements

Cells were grown in 500 mL Erlenmeyer flasks under aerobic or static cultivation conditions, as described above. At 24, 48 and 72 h of growth 10 mL samples were collected from cultures grown in the presence or without 10–50 mM AA. Yeast cells were harvested at 3160xg for 10 min at 4 °C (LMC-4200R Laboratory Refrigerated Centrifuge, Biosan, Latvia) and twice washed with distilled water. Clean cells were resuspended in 5 mL of distilled water and dried at 105 °C in an oven (Wise Wen, DAIHAN, Korea) until constant weight was achieved. Dry biomass weight was expressed in g L^-1 [32].

2.3. Respirometric studies

Oxygen consumption was determined using Respirometric BOD measuring system OxiTop®-i IS 6 - WTW (Xylem Analytics, Germany) [33]. 510 mL OxiTop-i IS 6- WTW bottles containing 22.7 mL culture were incubated at 30 °C in an incubator (WiseCube, S. Korea) with a constant stirring at 200 rpm. Produced carbon dioxide was absorbed using 2 g NaOH pellets placed in a rubber sleeve inside the BOD bottles. Consumed oxygen values were automatically measured by OxiTop®-i measuring head every 24 hours based on the decrease in oxygen partial pressure in a result of its consumption by growing yeast cells. Oxygen consumption values were expressed in mg L^-1 culture and oxygen consumption rate was expressed in mg L^-1 culture h^-1 [34,35]. Oxygen consumption values normalized to cell number were expressed in mg oxygen per 10¹⁰ CFU.

2.4. Measurement of cumulative CO₂ production using gas flow monitoring

Produced carbon dioxide concentration and carbon dioxide production rate was measured in batch fermentations using a Gas Endeavour® III system (AMPTS® III, BPC Instruments, Sweden). Batch experiments were conducted under aerobic or static cultivation conditions in 1 L GL45 glass bottle reactors using YPD medium with or without 10–50 mM AA. The bottles were inoculated with seed culture to a final OD of 0.2. Following inoculation, the reactors were connected to the Gas Endeavour® system and incubated at 30 °C in a water bath with continuous mixing set to 50% motor capacity. Cumulative CO₂ production volume and rate was continuously monitored at a resolution of ≤1 mL, with data automatically recorded using bioprocess control (BPC) online monitoring Aurora™ software (BPC Instruments, Sweden). Gas volumes were measured at ambient temperature and pressure and subsequently automatically normalized to standard temperature and pressure conditions. The produced CO₂ concentration was expressed in mL L^-1 culture and carbon dioxide production rate was expressed in mL L^-1 culture h^-1 [36].

2.5. Analytical methods

2.0 mL samples were collected at 0, 3, 6, 24, 48, 72, 96, 120 h of yeast growth and stored at −20 °C before the experiments. Consequently, the samples were centrifuged at 13,500 rpm for 10 min (Biobase D2012 plus, China). After, supernatant was filtered with 0.22  μm PVDF filter and collected into the sterile Eppendorf’s tubes.

The concentrations of ethanol and AA were determined by gas chromatography (GC 7820A) combined with mass spectroscopy (MS 5977B) detector [37]. For this, samples were diluted 10-fold with methanol (99%, HPLC grade) and 1․0 µL aliquots were injected into a GC–MS system (Agilent Technologies, USA) [38]. Separation of the gases was performed using HP-5 ms capillary column (30 m × 250 µm × 0.25 µm; Agilent Technologies, USA) using helium as the carrier gas. GC conditions were as follows: injection temperature 220 °C, split mode 20:1, column flow rate 0.5 mL min⁻¹, initial oven temperature 40 °C, ramped at 5 °C min⁻¹ to 70 °C, then 10 °C min⁻¹ to 150 °C followed by 20 °C min⁻¹ to 250 °C. MS scanning was performed in the 30–150 m/z range and SIM mode was applied for ethanol (31, 45, 46 m/z) and AA (29, 43, 60 m/z). Compounds were identified by comparison with ethanol (1–100 mM) and AA (1–50 mM) standards (99.9%, HPLC grade) and NIST MS library data [39].

The concentration of glucose and organic acids (succinate, glycerol, malate, oxaloacetate) was determined by the Bio-inert high-performance liquid chromatography (Agilent 1260 Bio-inert HPLC, Agilent Technologies, Germany) with a refractive index (Agilent RID, G1362A, set on positive polarity with an optical unit temperature of 55 °C), and diode array (Agilent DAD, G7115A) detectors [40]. 10 μL 2.0-fold diluted samples were injected by Multisampler (Agilent Bio, G5668A) into MetaCarb 87H column (300 × 6.5 mm), which was kept at 60 °C using a thermostatically controlled column compartment (Agilent MCT, G7116A). The separation of organic compounds was implemented with mobile phase (5 mM sulfuric acid) in 30 min analysis time with 0.7 mL per min flow speed [40].

Chromatograms were processed, and quantitative analysis was performed using OpenLab CDS software (Agilent Technologies, Germany) using standard curves of each compound with coefficient of determination ≥0.99. Results were expressed in mmol L^-1.

2.6. Derived parameters determination

Substrate consumption and chemicals production rates were calculated as a ratio of concentration difference to the same defined period. The obtained values were expressed in mmol L^-1h^-1 [40]. Ethanol yield was defined as a ratio of produced ethanol (mmol) to the consumed glucose (mmol). Fermentation yield was determined as the ratio of the total amount of the two experimentally quantified fermentation products (ethanol and carbon dioxide mmol) to the glucose consumed (mmol), providing an estimate of the overall fermentative conversion of glucose [41]. Ethanol fermentation efficiency (η, %) was defined as the ratio between the observed and stoichiometric (Y_St = 0.511 kg ethanol and 0.489 kg carbon dioxide per kg glucose) ethanol yield coefficients and expressed in %-s [42,43]․ Biomass yield (moles of carbon, C_mole, in biomass per mole of carbon in substrate) was calculated based on dry biomass weight data and the average elemental composition of S. cerevisiae (CH_1.748N_0.148O_0.596P_0.009S_0.0019M_0.018) with molecular weight of 26.4 g mole^-1 [44,45]. Biomass carbon recovery was calculated based on biomass yields and expressed in %-s. Metabolic balance calculations were performed using carbon mass conservation during metabolism, where the combined production of chemicals was related to the amount of glucose consumed and expressed in % [40].

2.7. Chemicals, data analysis, and statistics

Analytical-grade chemicals and reagents (>98%) were used for the growth, BOD and carbon dioxide production studies. HPLC-grade chemicals were used in chromatographic analyses.

Each result represents the mean value of three independent replicates, with standard deviations (SD) ≤5.0%. Data processing, analysis, and graphical representation were done using GraphPad Prism 11.0.0 program (GraphPad Software, Inc., USA). The statistical analysis was conducted using Student’s ‘t’’ and 2-way ANOVA Tukey’s tests [2]. Proposed working models and illustrations were created using BioRender.com.

3. Results

3.1. Dry biomass weight production differences of S. cerevisiae strains depending on cultivation conditions

This study investigates the rewiring of metabolism of two industrial-related strains, S. cerevisiae ATCC 9804 and ATCC 13,007 depending on environmental parameters. Our previous study showed that these strains have distinct stress tolerance and explores growth dynamics changes, specifically changes in growth phases, specific growth rate, and doubling time, antioxidant defense, and thiol groups [3]. Additionally, time-dependent biomass accumulation studies using CGQ biosensors in this study showed concentration and strain dependent changes in growth phases (Supplementary Fig. 1). To quantify the growth changes, dry biomass weight formation of S. cerevisiae was analyzed in response to varying AA (10–50 mM) concentrations under aerobic and static cultivation conditions (Fig. 1). Results indicate that maximal dry biomass weight accumulation observed within 72 h of growth of both strains. S. cerevisiae ATCC 9804 showed up to a ∼1.5-fold decrease in dry biomass weight production under 10–20 mM AA at 24 h under aerobic conditions, whereas 50 mM AA caused a ∼3.0-fold decrease in dry biomass weight production of both strains at 24 h (Fig. 1A, C).

Fig. 1.

Effect of AA on dry biomass weight of S. cerevisiae ATCC 9804 (A, B) and ATCC 13,007 (C, D). Cells were cultivated under aerobic and static cultivation conditions at pH 3.0 with 10–50 mM AA․ n = 3; ***p < 0.001, ns – not significant.

At 24 h, dry biomass weight production by S. cerevisiae ATCC 9804 decreased by ∼1.8–5.5-, and 110.0-fold compared with the control during static cultivation under 10, 20, and 50 mM AA stress, respectively (Fig. 1B). Under the same conditions, S. cerevisiae ATCC 13,007 exhibited a ∼3.8-fold reduction in dry biomass weight production at 10 mM AA relative to the control (Fig. 1D). No detectable dry biomass weight production was observed at 20–50 mM AA during 24-hour growth under same conditions, indicating a substantially lower tolerance to AA compared to ATCC 9804 under static cultivation (Fig. 1B; D).

Under aerobic conditions at 48 h, exposure to 50 mM AA resulted in similar reductions in dry biomass weight formation in both S. cerevisiae ATCC 9804 and ATCC 13,007, with a ∼1.7-fold lower dry biomass weight accumulation compared to control (Fig. 1A; C). A distinct response pattern was observed under static cultivation conditions: increasing AA concentrations to (20–50 mM), resulting in more pronounced reductions in S. cerevisiae ATCC 9804 dry biomass weight formation, reaching ∼4.6- and ∼5.8-fold lower values compared to control, respectively, (Fig. 1B). However, under control conditions during static cultivation, S. cerevisiae ATCC 13,007 strains dry biomass weight increased by only ∼17% at 48 h of growth compared to 24 h, while ATCC 9804 dry biomass weight increased by ∼92%.

At 72 h, both strains adapted to 10 mM AA independent of cultivation conditions, showing dry biomass weight yield levels like those under control conditions. Exposure to 50 mM AA, however, resulted in ∼1.9- and 2.4-fold reductions in dry biomass weight formation by S. cerevisiae ATCC 9804 and ATCC 13,007 under aerobic conditions (Fig. 1A, C), while under static cultivation conditions, dry biomass weight formation was reduced by ∼3.8-fold in S. cerevisiae ATCC 9804 and ∼1.5-fold in S. cerevisiae ATCC 13,007, respectively (Fig. 1B; D). Thus, static cultivation amplifies the inhibitory effect of high concentrations of AA on yeast dry biomass weight formation, especially in S. cerevisiae ATCC 13,007.

3.2. Oxygen consumption and carbon dioxide production studies under AA stress conditions

AA influence on the oxygen uptake kinetics by S. cerevisiae was evaluated for 120-h growth under aerobic conditions. Because of pressure increase due to rapid carbon dioxide accumulation, respirometric measurements using the OxiTop®-i IS 6 were not performed under static cultivation conditions. Results showed that only 700.0–900.0 mg L^-1 oxygen were consumed by both strains during the 24 h of growth under control conditions. Thereafter, oxygen consumption by S. cerevisiae ATCC 9804 under control conditions increased up to 96 h reaching ∼4700.0 mg L^-1, with no significant changes observed upon 10 mM AA exposure. 20 mM AA exposure leads to consistently higher oxygen consumption compared to control: during 24 h growth under 20 mM AA stress the oxygen consumption by ATCC 13,007 and ATCC 9804 strains was up to 3.0-fold higher than under control conditions. In contrast, 50 mM AA exposed cells exhibited markedly reduced oxygen uptake during the first 24 h in both strains (S. cerevisiae ATCC 9804: 110.0 ± 3.0 mg L^-1; S. cerevisiae ATCC 13,007: 59.8 ± 1.7 mg L^-1 at 24 h) correlating with reduced dry biomass weight․ Accordingly, oxygen consumption in S. cerevisiae ATCC 9804 was ∼1.9-fold higher than that observed in S. cerevisiae ATCC 13,007 under the same conditions and at the corresponding time point. However, starting from 72 h of growth sharp increase was observed in S. cerevisiae ATCC 9804 reaching to ∼3900.0 mg L^-1, suggesting a potential adaptive response to high AA stress (Fig. 2A), whereas in S. cerevisiae ATCC 13,007 oxygen consumption remained unchanged throughout the experiment. Only ∼61.0 mg L^-1 oxygen was consumed by S. cerevisiae ATCC 13,007 cells during aerobic cultivation under 50 mM AA stress conditions (Fig. 2C).

Fig. 2.

Oxygen consumption and carbon dioxide production kinetics in S. cerevisiae under AA stress. AA effects on oxygen consumption (A, C) and carbon dioxide production (B, D, E, F) kinetics were analyzed in S. cerevisiae ATCC 9804 (A, B, E) and ATCC 13,007 (C, D, F) under aerobic (A–D) and static cultivation (E–F) conditions at pH 3.0. n = 3; ***p < 0.001, ns – not significant.

The maximal oxygen consumption rate was observed at 48 h of both strains grown under control conditions (Fig. 3). Oxygen consumption rate by S. cerevisiae ATCC 9804 and ATCC 13,007 cells increased ∼3.0-fold at 48 h of growth compared to 24 h (Table 1, Fig. 3) reaching to ∼120.0 mg O₂ L^-1 culture h^-1 under the control and 10 mM AA conditions, whereas oxygen consumption rate remains constant during 24–48 h growth under 20 mM AA exposure (Fig. 3A, B, Table 1). In contrast, under 50 mM AA stress conditions, the maximal oxygen consumption rate in S. cerevisiae ATCC 9804 was observed at 72 h of growth, reaching similar the values like those observed in control, while only threshold oxygen consumption rate was observed for S. cerevisiae ATCC 13,007 cells.

Fig. 3.

Maximum oxygen consumption and carbon dioxide production rates of S. cerevisiae ATCC 9804 (A, B, E) and ATCC 13,007 (C, D, F) strains during aerobic (A, B, C, D) or static cultivation (E, F) with or without AA. n = 3, ** p < 0.01; ***p < 0.001; ns- not significant.

Table 1.

Substrate consumption and metabolism products accumulation rates in S. cerevisiae cells under AA stress during 24-hour growth at pH 3.0 depending on cultivation conditions.

		Consumption rate, g L-1h-1												Production rate, g l-1h-1
		Glucose				Oxygen				AA				Ethanol					Carbon dioxide
Aerobic cultivation	AA, mM	0	10	20	50	0	10	20	50	10	20	50	0		10	20	50	0		10	20	50
	ATCC 9804	2.53b	1.46b	0.54c	0.10c	0.04c	0.04c	0.11c	0.00c	0.05c	0.13c	0.42c	0.27c		0.07c	0.02c	0.03c	0.40c		0.37c	0.28c	0.01c
	ATCC 13,007	1.81b	0.34c	0.20c	0.01c	0.03c	0.03c	0.08c	0.00c	0.03c	0.08c	0.33c	0.17c		0.03c	0.04c	0.00c	0.66c		0.59c	0.28c	0.00c

		Consumption rate, g L-1h-1												Production rate, g l-1h-1
		Glucose				Oxygen				AA				Ethanol				Carbon dioxide
	AA, mM	0	10	20	50	0	10	20	50	10	20	50	0		10	20	50	0		10	20	50
Static cultivation	ATCC 9804	0.80c	0.70c	0.25c	0.25c	n.d.				0.10c	0.16c	0.38c	0.34c		0.12c	0.01c	0.00c	0.75c		0.76c	0.60c	0.00c
	ATCC 13,007	0.90c	0.70c	0.10c	0.00c					0.03c	0.10c	0.37c	0.14c		0.01c	0.00c	0.00c	1.37b		1.05b	0.96c	0.00c

^a SD < 5 %

^bSD < 3 %

^cSD <1%, n = 3, AA- acetic acid.

To further characterize metabolic activity of strains, the carbon dioxide production kinetics were monitored under aerobic and static cultivation conditions in the presence of increasing AA concentrations (Fig. 2). Both strains exhibited high CO₂ production (∼6000 mL L^-1 culture) during 24 h aerobic growth under control conditions. Exposure to 10–20 mM AA has no significant effect on carbon dioxide production compared to the control (Fig. 2B, D). However, carbon dioxide production inhibited under 50 mM AA exposure by ∼30.0- and ∼45.0-fold in S. cerevisiae ATCC 9804 and ATCC 13,007 strains, respectively, at 24 h compared to the control. Correspondingly, these results correlate with inhibited oxygen consumption rate values under same conditions.

Under control conditions during static cultivation, CO₂ production by S. cerevisiae ATCC 9804 and ATCC 13,007 reached to ∼3000 mL L^-1 and ∼3700 mL L^-1 during 24 h of growth, respectively (Fig. 2E), remaining 1.9- and 1.6-fold lower than similar values observed during 24 h aerobic growth. 10–20 mM AA exposure under the same conditions resulted up to ∼25% increased CO₂ production relative to the control. Exposure of ATCC 9804 cells to 50 mM AA resulted in an ∼100․0-fold reduction of carbon dioxide production at 24 h. A similar trend was observed in S. cerevisiae ATCC 13,007, where CO₂ production decreased by ∼250.0-fold under 50 mM AA stress at 24 h (Fig. 2F). At 48 h, under 50 mM AA stress, ATCC 9804 was able to produce ∼1300 mL L^-1 carbon dioxide, while only ∼25 mL L^-1 carbon dioxide was produced by ATCC 13,007. At 72 h, ATCC 9804 start to extensively respire, with CO₂ production reached ∼3500 mL L^-1, while ATCC 13,007 produced only ∼150 mL L^-1 carbon dioxide.

To evaluate the physiological response of S. cerevisiae to AA stress, carbon dioxide production rate was also measured using a Gas Endeavour® III system under aerobic and static cultivation conditions (Fig. 3). In contrast to oxygen consumption rate, the maximal carbon dioxide production rate was observed within 24-hour growth in control and 10–20 mM AA conditions (Table 1). The maximum carbon dioxide production rate was 2.0-fold higher under static cultivation conditions (∼380.0 mL L^-1 culture h^-1 in ATCC 9804 and ∼700.0 mL l^-1 culture h^-1 in ATCC 13,007) compared to aerobic growth in both strains (Fig. 3A-D). Additionally, ATCC 13,007 showed up to 1.8-fold higher maximum carbon dioxide production rate compared to ATCC 9804 strain. 10–20 mM AA had no significant effect on the maximum carbon dioxide production rate of both strains under aerobic conditions, while exposure to the same concentration of AA during static cultivation resulted up to 30% inhibition of maximum carbon dioxide production rate of ATCC 13,007 strain. 50 mM AA exposure resulted in a 2.6- and 3.5-fold reduction of maximum carbon dioxide production rate in ATCC 9804 during aerobic and static cultivation, respectively, reaching the values of ∼77.0 mL L^-1 culture h^-1 under aerobic and ∼110.0 mL L^-1 culture h^-1 under static cultivation conditions (Fig. 3C). Meanwhile, the maximum carbon dioxide production rate of ATCC 13,007 strain was strongly inhibited by 99% under 50 mM AA stress conditions independent of cultivation conditions (Fig. 3D).

To further evaluate respiratory activity independently of biomass formation, the amount of oxygen consumed per colony-forming unit (mg O₂ per CFU) was calculated during aerobic cultivation (Fig. 4). Under control conditions, normalized oxygen consumption gradually increased throughout cultivation in both strains, reaching stable values of ∼26.0 mg O₂ CFU⁻¹ in S. cerevisiae ATCC 9804 and ∼24.0 mg O₂ CFU⁻¹ in S. cerevisiae ATCC 13,007 after 72 h. Exposure to 10 mM AA had no significant effect on oxygen consumption per CFU compared to the control in either strain. In contrast, 20 mM AA markedly increased oxygen consumption per CFU at 24 h, reaching ∼36.0 and ∼131.0 mg O₂ CFU⁻¹ in ATCC 9804 (Fig. 4A) and ATCC 13,007 (Fig. 4B), respectively. Thereafter, oxygen consumption per CFU in ATCC 9804 remained relatively constant (∼30.0 mg O₂ CFU⁻¹), whereas in ATCC 13,007 it gradually decreased to values comparable with the control by 72–120 h. Under 50 mM AA stress, oxygen consumption per CFU remained low in ATCC 13,007 throughout cultivation (∼9.0 mg O₂ CFU⁻¹), while ATCC 9804 exhibited a marked increase after 72 h, reaching ∼80 mg O₂ CFU⁻¹ at 120 h.

Fig. 4.

Cell population size normalized oxygen consumption by S. cerevisiae ATCC 9804 (A) and ATCC 13,007 (B) during aerobic growth under 10–50 mM AA. n = 3, ** p < 0.01; ***p < 0.001; ns - not significant.

Thus, AA showed concentration-dependent influence on oxygen consumption and carbon dioxide production kinetics in both strains. 10 mM AA showed non-significant effect on both oxygen consumption and carbon dioxide production by both strains, while 20 mM and 50 mM AA showed markedly altered metabolic activity, with 20 mM AA shifting metabolic activity toward active respiration, and 50 mM AA showed complete suppression of metabolic activity of both strains. Moreover, ATCC 13,007 showed pronounced sensitive phenotype more amplified under static cultivation.

3.3. Carbon flux distribution in S. cerevisiae strains exposed to AA stress depending on cultivation conditions

Both strains, S. cerevisiae ATCC 9804 and ATCC 13,007, exhibited rapid glucose consumption in the control, resulting in complete glucose depletion within the first 24 h of cultivation under aerobic conditions (Fig. 5) correlated with rapid ethanol accumulation. Exposure to 10 mM AA increased has no significant effect on glucose consumption rate in ATCC 9804 cells grown for 6-hour under aerobic conditions, indicating activation of stress-tolerance mechanisms, while it decreased by 5.4-fold in ATCC 13,007 under same conditions (Fig. 5A, B, Table 1). 50 mM AA resulted inhibition of glucose consumption in both strains cultivated under aerobic conditions rigorous repression of glucose consumption, with ATCC 9804 showing 2.0-fold inhibited consumption rate during 6-hour aerobic growth and ATCC 13,007 showing no glucose consumption during 24-hour growth (Table 1).

Fig. 5.

AA effect on metabolic profiling in yeast during aerobic growth. Glucose consumption, ethanol production, and metabolic responses were analyzed in S. cerevisiae ATCC 9804 (A, C) and ATCC 13,007 (B, D) under aerobic conditions at various AA concentrations. N = 3; ***p < 0.001, ns – not significant.

These results correlated with ethanol production under aerobic conditions (Fig. 5A, B). Results indicate that both strains showed similar ethanol production tendencies with peak values (∼50.0 mM) observed within 24-h aerobic cultivation under control conditions, followed by its consumption due to glucose depletion. Similarly to glucose consumption, ethanol production yield at 24-hour was not affected by 10 mM AA under aerobic conditions by either strain (Fig. 5A, B), however the production rate decreased significantly in both strains (Table 1). 50 mM AA exposure reduced both ethanol yield by 1.7- and 4.5-fold in ATCC 9804 and ATCC 13,007, respectively, under aerobic conditions (Fig. 5A, B) and its production rate (Table 1).

To evaluate the impact of AA on central carbon metabolism, metabolite production was analyzed in S. cerevisiae strains under both aerobic conditions. Results showed that extracellular AA levels significantly altered the profile of key metabolites (Fig. 5C-D). ∼55.0% and ∼75.0% of 10 mM AA metabolized by ATCC 9804 and ATCC 13,007 cells during 6-hour aerobic growth. The increase in extracellular AA concentration to 20 mM inhibits its uptake rate by the cells with only 40.0–50.0% of AA metabolized at 6-h of both strains growth under aerobic conditions (Fig. 5C, D). At 50 mM extracellular AA concentration only ∼20.0% of AA enters the ATCC 9804 cells during 6-h growth, while in ATCC 13,007 the same value was higher (∼40.0%). Thus, more AA enters the ATCC 13,007 than ATCC 9804 cells, which correlates with reduced dry biomass weight (Fig. 1), oxygen consumption, and carbon dioxide production rates (Figs. 2, 3), inhibited glucose consumption and ethanol production (Fig. 5). Despite that, AA consumption rate was about 2.0-fold higher in ATCC 9804 compared to ATCC 13,007 (Table 1), which can be due to active metabolic activity.

Exposure to increasing extracellular AA concentrations also affects the overall metabolic activity of both strains. Thus, AA production at late growth phases under aerobic conditions decreased as the concentration of externally added AA increased. At 120 h, ATCC 9804 cells showed up to 2.7-fold decreased AA production (∼9.0 mM under stress versus ∼19.3 mM in control) under AA stress conditions compared to control conditions during aerobic growth (Fig. 5C). Meanwhile, ATCC 13,007 cells showed significantly lower AA production (∼7.0 mM) during 120-h growth under aerobic conditions (Fig. 5D).

Glycerol production depending on AA concentration displayed a clear time-dependent pattern, reaching its maximum at 24 h (10–20 mM) and 48 h (50 mM) (Fig. 5C, D). Under aerobic conditions, S. cerevisiae ATCC 9804 and ATCC 13,007 exhibited a peak in glycerol (∼114.0 mM and ∼64.0 mM, respectively) and malate (∼4.1 mM and ∼5.5 mM, respectively) production at 24 h. Glycerol accumulation in both S. cerevisiae strains exposed to 10–20 mM AA reached peak levels at 24 h, coinciding with rapid glucose consumption under aerobic conditions (Fig. 5C). However, S. cerevisiae 9804 cells exposed to 50 mM AA showed strongly reduced glycerol production, decreasing by ∼2.7-fold compared to the control at 24 h (Fig. 5A). Malate production also peaked at 24 h but showed a marked reduction under 50 mM AA. Succinate production showed a strain-dependent response to AA stress. In ATCC 9804, succinate levels were maintained under 10–20 mM AA conditions, whereas ATCC 13,007 exhibited a strong reduction in succinate production even at 10 mM AA, with ∼9.0-fold decrease observed at 24 h. At higher AA concentrations (50 mM), succinate production was strongly inhibited in both strains (Fig. 5C, D).

Differential glucose consumption and key metabolites production tendencies were observed during static cultivation (Fig. 6). Both strains utilized up to 96.0% of glucose available in the media during 24-h growth under control conditions, however, only 75% of glucose was consumed by both strains during 24-h growth under static cultivation conditions with 10–20 mM AA. For the same timepoint, 50 mM AA exposure resulted in total inhibition of glucose consumption in both strains (Fig. 6A, B). Static cultivation facilitated up to 2.0-fold lower glucose consumption rate compared to aerobic control conditions (Table 1). 10 mM AA exposure results 1.7- and 5.4- fold decrease in glucose consumption rate of ATCC 9804 and ATCC 13,007 strains, respectively, during 24-hour aerobic growth, while under static cultivation it remains unchanged (Table 1).

Fig. 6.

Metabolic profiling of S. cerevisiae under aerobic AA stress. Glucose consumption, ethanol production, and metabolic responses were analyzed in S. cerevisiae ATCC 9804 (A, C) and ATCC 13,007 (B, D) under static cultivation conditions at different AA concentrations. N = 3; ***p < 0.001, ns – not significant.

The glucose consumption kinetics were in good correlation with ethanol production results. Ethanol production reached the peak values (∼65.0 mM) at 48-hour growth in either strain under static cultivation conditions (Fig. 6A, B), which exceeds the same value observed under aerobic conditions by ∼30.0% (Fig. 5). Exposure to 50 mM AA resulted in up to 70% inhibition of ethanol production during 48-hour growth under static cultivation conditions. During static cultivation, glycerol production by S. cerevisiae ATCC 9804 at 24 h was highest in control and consistently decreased across all AA conditions. Moreover, glycerol production exhibited a concentration-dependent decline with increasing AA levels. At 24 h, glycerol levels under 10 and 20 mM AA were ∼1.2-fold higher in ATCC 9804 cells relative to control (Fig. 6C); however, 50 mM AA exhibited a delayed response, reaching maximal production (∼98.0 mM) at 96 h. From 48 h onward, citrate production by ATCC 9804 cells (∼1.6 mM) was detected only under 50 mM AA conditions. In contrast, oxaloacetate and succinate significant production were not detected across all conditions throughout the experiment (Fig. 6C). In S. cerevisiae ATCC 13,007 during static cultivation, glycerol accumulation was the highest at 24 h in the presence of 10–20 mM AA, reaching levels ∼1.3-fold higher than those observed in the control (Fig. 6D). In contrast, exposure to 50 mM AA delayed glycerol production, with maximal levels being attained only after 72 h. From 48 h onward, citrate production (∼1.6 mM) was detected only under 50 mM AA conditions as in ATCC 9804 strain (Fig. 6D).

Thus, AA exerted concentration-dependent effects on central carbon metabolism in both S. cerevisiae strains. Low AA concentrations (10–20 mM) stimulated glucose utilization in ATCC 9804 but impaired it in ATCC 13,007, whereas 50 mM AA strongly inhibited glucose consumption, ethanol production, and overall metabolic activity under both aerobic and static cultivation conditions.

3.4. Metabolic performance of S. cerevisiae ATCC 9804 and ATCC 13,007 cells under AA stress depending on cultivation conditions

AA stress affected metabolic performance in both S. cerevisiae strains depending on oxygen availability, strain, and AA concentration (Table 2, column 1). Under aerobic conditions, fermentation yield remained stable at 10–20 mM AA but decreased by up to 7.0-fold at 50 mM AA. Under static cultivation conditions, fermentation yields were 1.6- and 1.4-fold lower in ATCC 9804 and ATCC 13,007 strains, respectively, compared to aerobic cultivation. 50 mM AA severely impaired at, reaching ∼0.3 and ∼0.001 in ATCC 9804 and ATCC 13,007, respectively.

Table 2.

Metabolic performance indicators during AA stress in S. cerevisiae cells grown at pH 3.0 for 24 h under aerobic and static cultivation.

		Fermentation yield				Ethanol yield				Fermentation efficiency, %				Biomass carbon recovery, %				Carbon metabolic balance, %
Aerobic cultivation	AA, mM	0	10	20	50	0	10	20	50	0	10	20	50	0	10	20	50	0	10	20	50
	ATCC 9804	2.8b	2.8b	2.6b	0.4c	0.4c	0.3c	0.3c	0.3c	70.3a	69.4a	65.2a	8.8 a	17.2a	14.4a	11.5a	6.2a	98a	71a	58a	22a
	ATCC 13,007	3.0a	2.9b	2.9b	0.7c	0.5 c	0.5c	0.2c	0.2c	75.4a	71.4a	71.4a	16.3a	14.6a	11.6a	5.8a	n.d.	67a	67a	60a	12a

		Fermentation yield				Ethanol yield				Fermentation efficiency, %				Biomass carbon recovery, %				Carbon metabolic balance, %
Static cultivation	AA, mM	0	10	20	50	0	10	20	50	0	10	20	50	0	10	20	50	0	10	20	50
	ATCC 9804	1.8b	2.0b	1.8b	0.3c	0.3c	0.5c	0.3c	0.0c	44.2b	50.7b	45.6c	6.3a	6.6b	4.9a	1.6b	0.8 b	80a	60a	51a	25a
	ATCC 13,007	2.1b	2.1b	2.1b	0.0c	0.5c	0.4c	0.4c	0.0c	53.0b	52.1c	52.0b	0.0	9.4b	2.6b	0.01c	n.d.	71a	62a	55a	10a

n.d.- not detectable.

^aSD < 5 %

^bSD < 3 %

^cSD <1%.

Ethanol yield showed strain- and condition-dependent responses to AA (Table 2, column 2). Under aerobic conditions, ethanol yield remained constant in ATCC 9804 (∼0.3–0.4) across the tested AA concentrations, whereas ATCC 13,007 exhibited a 2.5-fold reduction at 20–50 mM AA. Under static cultivation conditions, ethanol yield increased by ∼40.0% in ATCC 9804 at 10 mM AA, however higher AA concentrations affect the ethanol yield. In both strains, ethanol production was completely abolished at 50 mM AA under static cultivation conditions.

Changes in ethanol production were reflected in fermentation efficiency (Table 2, column 3). Under aerobic cultivation, fermentation efficiency remained high (∼75.0%) at 10–20 mM AA. However, exposure to 50 mM AA caused a pronounced decline to ∼8.8% and ∼16.3% in ATCC 9804 and ATCC 13,007 strains, respectively. Static cultivation resulted in lower fermentation efficiencies overall (∼55%). with no significant changes under 10–20 mM AA and decreasing to ∼6.3% in ATCC 9804 and fully abolished in ATCC 13,007, respectively, at 50 mM AA.

Biomass carbon recovery decreased progressively with increasing AA concentration in both strains (Table 2, column 4). Under aerobic conditions, biomass carbon recovery gradually declined from 17.2% to 6.2% in ATCC 9804 and from 14.6% to nearly 0% in ATCC 13,007 as AA concentration increased from 0 to 50 mM under aerobic conditions. The effect was more pronounced under static cultivation conditions, where biomass carbon recovery decreased from 6.6% to 0.8% in ATCC 9804 and from 9.4% to approximately 0.01% in ATCC 13,007.

Carbon metabolic balance was also negatively affected by AA stress (Table 2, column 5). During 24-hour growth metabolic balance in ATCC 9804 cells decreased from ∼98.0% in the absence of AA to ∼2% at 50 mM AA. Similarly, ATCC 13,007 exhibited a reduction from ∼70% to 12%. Comparable trends were observed under static cultivation conditions. Overall, increasing AA concentrations reduced substrate conversion efficiency, biomass-associated carbon recovery, and carbon balance, with the strongest effects observed at 50 mM AA regardless of cultivation conditions.

4. Discussion

This study investigates the impact of AA on the central metabolic response of two wine and beer industrial fermentation-derived, S․ cerevisiae strains, with distinct AA sensitivity [3] depending on cultivation conditions. Most of the yeast-based industries currently perform in semi-aerobic conditions to enhance yeast biomass production [46]. Therefore, it is important to study the impact of static cultivation with reduced oxygen transfer and mixing on yeast metabolism and adaptation pathways to AA, especially under low pH.

AA toxicity was shown to be strongly influenced by the strain background, AA concentration, and cultivation conditions. The observed reduction in dry biomass weight formation indicates that AA imposes a strong inhibitory effect on S. cerevisiae growth, with S. cerevisiae ATCC 9804 showing higher biomass yield and tolerance than S. cerevisiae ATCC 13,007, particularly under aerobic condition, while static cultivation significantly enhanced toxicity in both strains, especially at higher AA levels (Fig. 1) [3]. The strain- and concentration-dependent differences in dry biomass weight formation under AA stress may also be associated with alterations in central carbon metabolism and energy generation efficiency, as AA has been shown to disrupt intracellular homeostasis from growth-associated respiration toward stress-adaptive responses [47]. This metabolic shift may come at the expense of biomass yield, as efficient growth requires channeling carbon flux through oxidative pathways rather than toward fermentative by-products such as ethanol and acetaldehyde [48]. Although H⁺-ATPase activity was not directly determined in the present study, the delayed increase in biomass observed at 50 mM AA may indicate gradual physiological adaptation to acid stress. This adaptation may involve activation of cellular pH homeostasis mechanisms, including plasma membrane H⁺-ATPase (Pma1), together with recovery of cellular metabolism following the initial inhibitory phase [9]. In addition, differences in the timing of the diauxic shift among the experimental conditions may also contribute to the observed growth patterns [49]. In addition, it was shown that the expression of key monocarboxylate transporters, Jen1 and Ady2 can be also increased during prolonged growth, which enables to utilize acetate as a carbon source by respiration. The regulation of these transport systems depends on the carbon source conditions and nutrient availability, which may influence acetate utilization. Therefore, modulation of acetate transport and metabolism could contribute to improved adaptation to AA stress [50]. These changes in transporters can contribute to an increase in extracellular pH [30], which can stimulate the increased growth after diauxic shift. However, at higher AA concentrations, these mechanisms may still be activated but become insufficient to fully counteract the stronger acid stress, resulting in delayed growth recovery and lower biomass production because of membrane damage caused by high AA exposure and consequent ROS generation [51].

AA exposure caused a concentration-dependent reduction in oxygen uptake (Fig. 2). At 20 mM AA, oxygen consumption was higher than in control and 10 mM AA conditions in both strains, whereas at 50 mM AA, oxygen uptake was markedly suppressed. This indicates that although inhibiting biomass yield, 10 mM AA had only minor effects on metabolic performance, suggesting that both strains were able to effectively compensate for the imposed stress. Increased oxygen uptake at 24 hours under 20 mM AA indicates shift of the metabolic flux from fermentation toward respiration. Previous studies also indicate that WOS, e.g. AA, represents a major class of microbial fermentation inhibitors that are widely encountered in food preservation, fermentation, and biotechnological processes [10]. The increased oxygen consumption reflects an early shown adaptive metabolic response in which functional mitochondria enhance respiratory activity to meet the elevated ATP demand required for proton extrusion, intracellular pH homeostasis, ion transport, and other energy-dependent stress adaptation processes [5]. This may also explain the poor cellular performance observed at 50 mM AA, which, as shown, results in complete inhibition of not only fermentative but also respiratory activity of the yeast (Figs. 2, 3, Tables 1, 2).

Normalization of oxygen consumption to viable cell number further demonstrated that AA induced marked changes in cellular respiratory activity (Fig. 4). While 10 mM AA had no significant effect on oxygen consumption per CFU, exposure to 20 mM AA substantially increased the amount of oxygen consumed by individual cells despite the reduced biomass yield. This observation further supports the conclusion that moderate AA stress redirects cellular metabolism toward enhanced respiratory activity, allowing cells to meet the increased ATP demand associated with maintenance of intracellular pH homeostasis and activation of energy-dependent stress response mechanisms. Under 50 mM AA stress, the surviving S. cerevisiae ATCC 9804 population exhibited a pronounced increase in oxygen consumption per CFU after 72 h, whereas ATCC 13,007 maintained only basal respiratory activity throughout cultivation. The elevated oxygen consumption per viable cell in ATCC 9804 likely reflects the selection and adaptation of a small subpopulation of metabolically active cells capable of maintaining mitochondrial function under severe AA stress. In contrast, the inability of ATCC 13,007 to increase respiration on a per-cell basis is consistent with its higher sensitivity to AA and the absence of an effective adaptive response, supporting the physiological differences observed between the two strains. These results are in good conformity with previous study showing that exposure to higher AA concentrations eventually lead to mitochondrial dysfunction, loss of respiratory control, and programmed cell death [52].

The metabolic performance analysis further supports this interpretation. Under aerobic conditions, fermentation yields and fermentation efficiencies remained constant at 10–20 mM AA in both strains (Table 2), despite reduced growth and altered respiratory activity. This indicates that low-to-moderate AA concentrations do not completely disrupt carbon conversion into ethanol but increase the energetic cost of maintaining cellular homeostasis. In contrast, exposure to 50 mM AA resulted in a dramatic decline in fermentation yield and efficiency in both strains, demonstrating that carbon utilization becomes increasingly diverted from dry biomass weight formation and ethanol production toward maintenance-associated processes. The effect was even more pronounced under static cultivation conditions, where fermentation efficiencies were inherently lower and were completely abolished at 50 mM AA. These findings suggest that oxygen availability is a critical factor enabling metabolic adaptation to AA stress.

Higher ATP demand under AA stress can also be due to intracellular pH regulation, especially under low extracellular pH, for which plasma and vacuolar membrane H⁺-ATPases (Pma1 and Vma1) expend most of intracellular ATP levels [53]. The increased oxygen consumption reflects a metabolic shift toward oxidative phosphorylation to meet the higher ATP demand for pH homeostasis, which in turn is consistent with enhanced respiratory activity during AA-induced stress; however, the observed increase in carbon dioxide production may originate not only from the TCA cycle but also from pyruvate decarboxylation during fermentative metabolism, indicating a combined contribution of respiratory and fermentative pathways in the overall stress response. The inability of S. cerevisiae to consume oxygen at 50 mM AA, combined with the severe reduction in carbon dioxide production, indicates a comprehensive failure of both respiratory and fermentative pathways. This metabolic shutdown suggests that at 50 mM the toxic concentration of AA to compromises mitochondrial function and glycolytic flux, preventing the strain from maintaining the energy production required for cellular survival [54]. Overall, increasing AA concentration led to a dose-dependent decrease in carbon dioxide production in both strains. Notably, strain S. cerevisiae ATCC 9804 exhibited higher tolerance at 20 mM AA stress, while both strains were strongly inhibited at 50 mM AA. The high sensitivity of ATCC 13,007 strain can also be due to combined effects of pH, reduced oxygen-transfer, nutrient distribution, mixing associated with static cultivation and AA stress. The reduced biomass formation observed under high AA concentrations may be associated not only with growth inhibition but also with decreased cell viability. Previous studies have demonstrated that severe AA stress can induce programmed cell death and loss of viability in S. cerevisiae [55,56]. Therefore, the lower biomass observed at elevated AA concentrations in our study may reflect a combination of impaired growth and potential viability loss.

The glucose consumption and ethanol production profiles indicate substantial reorganization of carbon flux under AA stress. While S. cerevisiae ATCC 9804 maintained efficient glucose utilization at 10–20 mM AA, the reduced ethanol production rates and increased oxygen consumption suggest a redistribution of carbon and energy toward maintenance metabolism rather than biomass formation or fermentative product synthesis. This interpretation is supported by the progressive decline in biomass carbon recovery (Table 2), which indicates that a smaller fraction of consumed substrate was converted into new cellular material as AA concentration increased. Such carbon reallocation is consistent with the elevated ATP requirements associated with intracellular pH regulation, proton extrusion, membrane repair, and activation of stress-response pathways. While 10 - 20 mM concentrations had minimal impact on early metabolic activity, 50 mM AA impaired both substrate consumption and product formation under aerobic conditions (Fig. 5). The 1.7-fold reduction in ethanol for the S. cerevisiae ATCC 9804 strain suggests a controlled redirection of carbon flux toward stress-defense mechanisms, whereas the 4.5-fold reduction and abrupt inhibition of glucose uptake in S. cerevisiae ATCC 13,007 point to a systemic failure of glycolytic and respiratory pathways once the AA load exceeds the strain homeostatic capacity.

Supplementary Table 1 showed biomass yield changes of S. cerevisiae ATCC 9804 and ATCC 13,007 under 10–50 mM AA during aerobic or static cultivation. Under control conditions, depending on oxygen availability, both strains showed 0.15–0.17 biomass yield (Cmol of biomass/C mol of glucose consumed), while under static cultivation the values dropped to 0.07 and 0.09 biomass yield in ATCC 9804 and ATCC 13,007 strains, relatively. Both observed values under aerobic and static cultivation conditions consistent with glucose fermentative conditions due to Crabtree effect. Previous studies also showed that yeast biomass yield can significantly vary depending on growth conditions, carbon source type and concentration, pH and oxygen availability [44,57]. The biomass carbon recovery data derived from Supplementary Table 1 showed that the values dropped from ∼17.2% to ∼6.2% aerobically and reached zero under static cultivation (Table 2). The pronounced decline in biomass carbon recovery, particularly under static cultivation conditions in ATCC 13,007, indicates that carbon assimilation into new biomass was far more sensitive to AA stress than ethanol production, highlighting growth as one of the most energetically demanding processes during adaptation. Furthermore, the observed amplification of stress-sensitivity under static cultivation can also be the effect of reduced oxygen transfer and absence of agitation, which limits nutrient availability.

S. cerevisiae ATCC 9804 exhibits a resilient metabolic profile, although glucose consumption is initially delayed, the strain successfully achieves substrate consumption, indicating the effective activation of adaptive stress-response mechanisms [54]. Conversely S. cerevisiae ATCC 13,007 exhibits metabolic exhaustion, evidenced by its failure to initiate significant ethanol production for 48 hours and its inability to recover ethanol yields even after extended cultivation (120 h).

Metabolic profiling indicates that AA stress triggers a profound suppression of the TCA cycle under aerobic conditions (Fig. 5). The near-complete depletion of citrate and oxaloacetate, alongside the marked reduction in succinate, provides compelling evidence of a systemic suppression of central carbon metabolism. These findings highlight that under high AA stress, cell primary respiratory and energetic pathways are essentially halted, reflecting a metabolic failure that S. cerevisiae ATCC 13,007 is unable to overcome. The reduction in succinate production together with the absence of detectable citrate and oxaloacetate suggests diminished flux through central respiratory metabolism under severe AA stress. These observations are consistent with the marked reduction in oxygen consumption and biomass formation, indicating substantial impairment of energy-generating pathways. Previous studies have suggested that AA stress may impair succinate dehydrogenase activity, limiting the conversion of succinate to fumarate and consequently reducing electron transport chain activity and oxidative ATP generation, which reduces FADH₂ formation, disrupts the electron transport chain, and ultimately limits oxidative ATP production [54]. The suppression of succinate dehydrogenase can result in a failure of electron transport chain facilitated by the absence of functional proton pumping respiratory complex I [58].

The near-total depletion of these early-stage intermediates confirms an abrupt arrest of the TCA cycle, cohering with glucose repression and Crabtree effect stimulation [59]. Increasing the capacity of cells to assimilate acetate through overexpression of acetyl-CoA synthetase enhances acetate uptake and improves cellular tolerance, thereby promoting its incorporation into central carbon metabolism in S. cerevisiae [60]. Our results also support that more tolerant strain, ATCC 9804, has higher AA consumption rate (Table 1) compared to ATCC 13,007.

Carbon metabolic balance analysis revealed an additional consequence of AA stress (Table 2). Under control conditions, most consumed carbon could be accounted for through biomass and measured metabolites in ATCC 9804, while in ATCC 13,007 it accounted for only ∼70%, indicating strain-specific differences in carbon partitioning even in the absence of AA stress. However, increasing AA concentrations progressively reduced carbon recovery in both strains. The marked decline in carbon balance at 50 mM AA suggests the formation of unquantified metabolites, increased maintenance-associated carbon expenditure, or extensive metabolic rerouting during stress adaptation. Together with the reduction in biomass carbon recovery and fermentation efficiency, these results demonstrate that severe AA stress compromises not only growth and product formation but also the overall efficiency of carbon utilization. Notably, ATCC 9804 consistently exhibited higher biomass carbon recovery, fermentation performance, oxygen consumption under AA stress than ATCC 13,007 (Tables 1, 2). These observations indicate that tolerance to AA is associated not simply with the maintenance of growth inhibition but with the capacity to maintain metabolic activity and efficient carbon flux distribution under stress. Static cultivation amplified the negative effects of AA on all measured parameters, highlighting the importance of respiratory metabolism for sustaining ATP generation and supporting adaptation mechanisms under acidic conditions. Together, these findings indicate that maintenance of respiratory capacity is a major determinant of AA tolerance, particularly under low-pH conditions. Our previous data also supports that yeast tolerance to AA at low pH is stimulated under respiratory conditions compared to respirofermentative and fermentative metabolism [3].

The observed higher sensitivity of the strains to AA under static cultivation may be conditioned also due to the absence of agitation. Lack of stirring can result in reduced mass transfer, leading to gradients in nutrient and metabolite concentrations, as well as localized accumulation of inhibitory compounds such as ethanol. Moreover, although oxygen limitation typically favors fermentative metabolism in S. cerevisiae, complete absence of agitation can also restrict efficient heat and substrate distribution, potentially lowering overall fermentation performance [42,61]. Furthermore, it was shown that only half of the nitrogen available is consumed in static fermentation, while this nutrient is fully depleted in stirred fermentation [61] .

The stronger dependence of AA tolerance on cultivation conditions observed in this study may also be linked to limitations in anabolic metabolism. The high respiratory activity can be crucial for AA adaptation not only because of energetic demands, but also due to anabolic processes. Under low pH conditions, mitochondrial activity may be reduced thereby limiting anabolic metabolism [30]. Krebs cycle intermediates serve as important precursors for amino acid biosynthesis [62], and impaired respiratory activity may therefore restrict cellular growth not only through ATP limitation but also through reduced precursor availability. Besides, previously was shown that due to glucose repression under fermentative conditions, pentose phosphate pathway (PPP) can also be disrupted [63], and, as a result, due to insufficient NADPH and riboso-5-phosphate generation, the efficient cell duplication and antioxidant defense can be abolished.

The present study was conducted exclusively in shake-flask cultures, which are widely used for preliminary screening and optimization of microbial growth and metabolite production because they provide a simple and reproducible cultivation platform. Shake flasks differ substantially from stirred-tank bioreactors in terms of oxygen transfer, mixing efficiency, pH control, dissolved oxygen regulation, and shear environment. Consequently, the fermentation performance observed in shake flasks should be considered indicative of the strain's physiological potential rather than directly predictive of industrial-scale productivity. Translation of the optimized conditions identified here to a bioreactor would require further investigation, including optimization of engineering parameters such as agitation, aeration, and oxygen transfer coefficient, which strongly influence microbial metabolism during scale-up [64,65]. Previous studies using controlled bioreactor cultivations have shown that S. cerevisiae produces the same major fermentation-related metabolites, including ethanol, glycerol, and acetate, but their relative amounts depend on cultivation conditions, such as oxygen availability, mixing, process control and metabolic state [66]. Nevertheless, shake-flask optimization represents the first stage of bioprocess development and provides the basis for subsequent validation and scale-up in controlled bioreactor systems [67] . Furthermore, this study investigates cultivation condition-, AA concentration- and strain-dependent changes in process performance under AA stress, which, indeed, will be valuable for scale-up strategies development.

To characterize the effects of studied strain, cultivation condition, and AA concentration, as well as their combined effects on ATCC 9804 and ATCC 13,007, the observed metabolic differences and interactions among these factors are summarized in Fig. 7. Overall, the results demonstrate that the physiological response of S. cerevisiae to AA stress is determined by the interplay between strain background, cultivation conditions, and AA concentration rather than by any single factor alone. While each factor independently influenced specific physiological traits, their combined interactions ultimately governed respiratory activity, carbon flux distribution, biomass formation, and the overall adaptive capacity of the two strains.

Fig. 7.

General and specific stress responses of S. cerevisiae under AA exposure depending on cultivation conditions, strain, and AA concentration. The graphical illustration was prepared using BioRender.com.

Summarizing, AA induced concentration-dependent alterations in carbon metabolism, respiration, and biomass formation in both S. cerevisiae strains. The more tolerant strain, ATCC 9804, maintained higher respiratory activity, acetate utilization, carbon recovery, and biomass production, whereas ATCC 13,007 exhibited a progressive loss of metabolic performance, particularly under static cultivation conditions. These findings demonstrate that maintenance of respiratory capacity and efficient carbon flux distribution are key determinants of AA tolerance under low-pH conditions (Fig. 7). Thus, this study demonstrates that AA has a pronounced concentration- and strain-dependent effect on S. cerevisiae physiology and central metabolism (Fig. 8). These findings demonstrate that maintenance of respiratory capacity and efficient carbon flux distribution are key determinants of AA tolerance under low-pH conditions. The results contribute to elucidating the metabolic regulatory mechanisms and adaptive responses of S. cerevisiae under AA conditions, which can be useful for robust strains construction for industrial applications.

Fig. 8.

Proposed model describing the metabolic responses of S. cerevisiae under combined stress conditions. Abbreviations: TCA- tricarboxylic acid cycle; ↓↓ inhibition; × suppression․ The graphical illustration was prepared using BioRender.com.

CRediT authorship contribution statement

Gohar Azbekyan: Writing – original draft, Investigation, Conceptualization. Anahit Shirvanyan: Writing – original draft, Methodology, Investigation, Data curation, Conceptualization. Nicoletta Guaragnella: Writing – review & editing, Visualization, Validation, Supervision, Methodology, Conceptualization. Karen Trchounian: Writing – review & editing, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.