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Article

Enhancing Docosahexaenoic Acid Production by Schizochytrium sp. via Periodic Hydrogen Peroxide and p-Aminobenzoate Control

by
Luqiang Jia
*,
Mengyao Ma
,
Xingyue Wang
,
Ruoyu Wang
and
Shuqi Xin
School of Food Science and Technology, Yangzhou University, Yangzhou 225127, China
*
Author to whom correspondence should be addressed.
Fermentation 2025, 11(10), 558; https://doi.org/10.3390/fermentation11100558
Submission received: 24 July 2025 / Revised: 11 September 2025 / Accepted: 15 September 2025 / Published: 27 September 2025
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Abstract

In producing docosahexaenoic acid (DHA) with Schizochytrium sp., the production yield of DHA can be effectively increased through using hydrogen peroxide (H2O2) and controlling its concentration at the desired level, since H2O2 is a common regulatory mediator for lipid accumulation in oleaginous microorganisms. However, when exposed to the environment of oxidative stress induced by the long-term exogenous addition of H2O2 over an extended time span, cells’ metabolic activity would be gradually decreased or even stopped, which ultimately results in a limited duration for producing DHA efficiently. In fact, the severe accumulation of ROS cannot be avoided when implementing the normal DHA fermentation batch without the use of exogenous H2O2 because of the necessity of supplying a mass of oxygen for cell respiration. Aiming to overcome these issues, a novel periodic feeding strategy for H2O2 and p-aminobenzoate was proposed, and the underlying principle of this strategy is that the substantial harm inflicted on cells due to their continuous exposure to the oxidative stress environment can be effectively alleviated through the implementation of a recovery treatment (p-aminobenzoate, reducing agent) subsequent to the environmental stimulus. When using this strategy, it was achieved that, concurrently, activities of the vital enzymes participating in lipid biosynthesis were maintained at their maximum levels and the maintenance coefficient of glucose reduced to its minimum level (0.0034 1/h vs. 0.0027 1/h) by controlling ROS concentration at lower and desired levels, and thus DHA concentration reached the maximum value of 1.49 ± 0.20 g/L, with a 49% increase compared to the control group.

Graphical Abstract

1. Introduction

As a frequently used food additive, docosahexaenoic acid (DHA), the primary supplier of omega-3 polyunsaturated fatty acids (PUFAs), has numerous positive impacts on human health, including promoting the development of infants’ brains and eyes, preventing cardiovascular diseases, having anti-inflammatory effects, and improving the cognitive functions of the elderly [1,2,3]. It was well known that DHA cannot be endogenously synthesized by humans and must be obtained by consuming foods rich in DHA [4]. Taking various factors into account, the World Health Organization (WHO) has pointed out that adults should consume an appropriate amount of at least 250 mg of DHA and EPA daily to meet the body’s demand for nutrients [5]. With the continuous improvement of living standards, the requirement for a healthy life has become higher, which has directly driven the increasing market demand for DHA. Currently, DHA is extensively utilized in functional foods and pharmaceutical formulations and increasingly incorporated into animal feed systems to enable dietary DHA enrichment. According to market analyses, the global market has increased from an estimated $2.1 billion in 2020 to a projected nearly $3.61 billion in 2028 [6]. For years, deep-sea fish were the main source of DHA, including tuna, anchovies, etc. [7,8] but this method can no longer meet people’s ever-increasing demand for DHA, and the reasons are as follows: (1) marine fish oil usually has a fishy smell, which makes it difficult for most consumers to accept; (2) the purification process of DHA is very expensive, greatly increasing the production cost [9]; (3) it leads to overfishing of deep-sea fish; (4) serious marine environmental pollution may result in contaminants such as polychlorinated biphenyls in the extracted fish oil [5]. Compared with traditional sources of DHA from deep-sea fish, Schizochytrium sp., a marine fungus, has been widely recognized as a promising and important microbial resource for producing DHA within a short duration because of the following reasons: firstly, this oleaginous microorganism is characterized by exceptional DHA biosynthetic capacity, with DHA content documented to constitute 30–40% of total fatty acids, thereby establishing it as a premium source of valuable dietary omega-3 fatty acids [10]. Secondly, superior cultivation properties are exhibited by Schizochytrium sp., including rapid growth rates and high-cell-density fermentation capability, which has been demonstrated to significantly enhance industrial production potential [11]. Compared with other microorganisms, this strain is characterized by strong fermentation adaptability and high production efficiency. Most importantly, the safety of Schizochytrium-derived DHA has been thoroughly evaluated, resulting in its designation as Generally Recognized As Safe (GRAS) status by the U.S. Food and Drug Administration (FDA) [12]. These combined advantages have positioned Schizochytrium sp. as a superior microbial platform for sustainable DHA production when compared to conventional fish oil sources.
While producing DHA from Schizochytrium sp. cells, it is commonly known that regulating the cultivation temperature to a low level of 16 °C significantly impacts the DHA production yield, increasing the DHA content from 43 to 65% of total fatty acids, compared with that obtained by controlling cultivation temperature at 28 °C [13]. Although higher DHA and biomass productivity can be achieved through low-temperature fermentation processes, during the period of large-scale industrialized fermentation production, implementing DHA fermentation with the cultivation temperature regulated at a relatively low value, for example, 16 °C, demands the utilization of a costly cooling system, rendering it economically impracticable. Besides using the lower cultivation temperature, with regard to operation cost, one available and realistic option for increasing the production yield of DHA that has not been explored is inducing the environment of oxidative stress by the use of exogenous hydrogen peroxide-H2O2 (oxidizing agent). In fact, it was widely reported that intracellular reactive oxygen species (ROS) are a common regulatory mediator for lipid accumulation in oleaginous microorganisms, and the maintenance of an appropriate level of ROS (oxidative stress conditions) is a necessary prerequisite for the effective accumulation of lipids [14,15]. For example, Yilanciooglu et al. demonstrated that exogenous application of H2O2 increased the lipid content in Dunaliella salina by up to 44% [15]. Kang et al. reported that oxidative stress triggered by TiO2 nanoparticles enhanced lipid production in Chlorella vulgaris [14]. In theory, although DHA production performance might be obviously improved by implementing the cultivation environment of oxidative stress via use of exogenous H2O2, when exposed to the environment of oxidative stress induced by the long-term exogenous addition of H2O2 over an extended time span, cells’ metabolic activity would be gradually decreased or even stopped, which ultimately results in a limited duration for producing DHA efficiently. As a result, an improved H2O2 feeding strategy with the function of eliminating the massive accumulation of ROS for further enhancing DHA production required exploration.
In theory, the heightened toxicity caused by environmental stressors can be effectively alleviated by carrying out a recovery step following exposure to environmental stimuli. In this study, aiming to further enhance the titer of DHA, a periodic feeding control strategy, namely periodic H2O2 and p-aminobenzoate control, was proposed in DHA fermentation, and the underlying principle of this strategy is that the substantial harm inflicted on cells due to their continuous exposure to the oxidative stress environment can be effectively alleviated through the implementation of a recovery treatment (p-aminobenzoate, reducing agent) subsequent to the environmental stimulus. Finally, the important fermentation parameters, such as the concentrations of DHA, ROS, and lipids, as well as the activities of key enzymes within the lipid biosynthetic route, were analyzed to verify the feasibility and validity of the proposed periodic H2O2 and p-aminobenzoate feeding strategy for increasing DHA production.

2. Materials and Methods

2.1. Strains

The Schizochytrium sp. S31 strain (ATCC20888) utilized for the current study was preserved at −80 °C in our laboratory.

2.2. Media

The details regarding the media employed in the current study are presented as follows. For seed cultivation, 30 g/L of glucose, 6 g/L of yeast extracts, 6 g/L of fish peptone, and 20 g/L of seawater salt, with the pH adjusted to 7.00. The medium used for batch cultivation in both fermenters and shake flasks consisted of 100 g/L of glucose, 10 g/L of yeast extract, 1 g/L of (NH4)2SO4, 12 g/L of Na2SO4, 7 g/L of K2SO4, 5 g/L of MgSO4, 2 g/L of KCl, 3 g/L of K2HPO4, and 10 mL/L of a trace mineral solution, and its pH was 7.00. The glucose-feeding medium contained 500 g/L of glucose. The medium for H2O2 feeding contained 2 mmol/L H2O2. The p-aminobenzoate feeding medium had a p-aminobenzoate concentration of 200 mg/L.

2.3. Fed-Batch Cultivation of Schizochytrium sp. for DHA Production

Fed-batch cultivation to produce DHA was carried out in a 5-L fermenting tank (BIOTECH-5BG, Baoxing Co., Shanghai, China), where the operational volume was 2.0 L. A 20% (v/v) inoculation rate was applied, and the aeration rate was fixed at 4 L/min. The cultivation temperature was maintained constantly at 28 °C, while the stirring rate remained at 500 rpm over the entire course of fermentation. The pH value was rigidly kept at 7.00 by the addition of either H3PO4 or NaOH. Upon reaching the predetermined glucose level of approximately 40 g/L, pulsed feeding of glucose medium was employed to maintain the glucose concentration in the fermentation broth at around 40 g/L until the end of fermentation, based on offline glucose concentration measurements conducted every 4 h, in order to sustain glucose concentration consistently. On the other hand, when the biomass concentration, measured as dry cell weight (DCW), attained approximately 10 g/L at nearly 20 h, the feeding of H2O2 and/or p-aminobenzoate was started, and these feeding strategies were described as follows:
Strategy A (intermittent feeding of H2O2): a certain amount of H2O2 (molar mass of H2O2/volume of fermentation broth = 2 mmol/L) was intermittently added into fermentation broth at a fixed interval of 7 h.
Strategy B (p-aminobenzoate intermittent feeding): a certain amount of p-aminobenzoate (molar mass of p-aminobenzoate/volume of fermentation broth = 200 mg/L) was intermittently added into fermentation broth at a fixed interval of 14 h.
Strategy C (periodic control): based on the above feeding strategy A and B, the fermentation process was divided into three (3) ~21 h subintervals, during which alternate intermittent feeding of H2O2 (first ~7 h) and p-aminobenzoate (later ~14 h).

2.4. Measurement of Concentrations of Schizochytrium sp. Cells, Lipid, DHA, and Glucose

Quantification of Schizochytrium sp. biomass, defined as dry cell weight (DCW), was performed using a gravimetric analysis, consistent with the methodology detailed in the report [16]. In brief, the fermentation broth containing Schizochytrium sp. cells was centrifuged at 10,000× g for 5 min. After removing the fermentation supernatant, the resulting precipitate was washed twice with double-distilled water and subsequently freeze-dried to a constant weight. The concentrations of lipid as well as DHA were measured using previously reported methods [17,18,19]. Briefly, the freeze-dried Schizochytrium sp. cells were subjected to hydrochloric acid hydrolysis and digestion. Subsequently, fatty acids were extracted using n-hexane, followed by methylation of the extracted fatty acids. The methylated fatty acids were then uniformly mixed with nonadecanoic acid at a concentration of 1 mg/mL. After this mixture was prepared, a 0.5 mol/L NaOH-CH3OH solution was added to initiate hydrolysis. Upon completion of the hydrolysis reaction, a 14% BF3-CH3OH solution was added for esterification. Once esterification was finished, n-hexane was added to facilitate organic phase separation, and the separated organic phase was dehydrated with anhydrous MgSO4. The concentration of DHA in the sample was determined using a Shimadzu GC-2010 (Kyoto, Japan) gas chromatograph equipped with a DB-WAX chromatographic column (30 m × 0.32 mm × 0.25 μm), with helium used as the carrier gas. DNS methodology was utilized to quantify the glucose concentration [20].

2.5. ROS and the Activities of Key Enzymes Involved in the DHA Biosynthetic Pathway Analysis

The levels of ROS were determined using the ROS ELISA kit (KT20911, Wuhan Mosak Biotechnology Co., Ltd., Wuhan, China). Its principle is as follows: purified ROS capture antibodies are coated on a microplate to form solid-phase antibodies. After sequential sample addition, binding occurs with HRP-labeled detection antibodies, resulting in the formation of an “antibody-antigen-enzyme-labeled antibody” complex. Following washing, TMB substrate is added: TMB is converted to blue via HRP catalysis and then to yellow after acid treatment. Absorbance at 450 nm is measured with a microplate reader (Infinite F50, Tecan, Männedorf, Switzerland), and ROS levels are calculated using the standard curve. The method for determining the activities of related enzymes—including malic enzyme (ME), glucose-6-phosphate dehydrogenase (G6PDH), and ATP citrate lyase (ACL)—is as follows. First, cells were resuspended in extraction buffer (100 mmol/L Tris-HCl, pH 7.4, containing 0.001 mol/L EDTA and 0.002 mol/L DTT), followed by 30-min ultrasonic disruption (455 W, 4 s on/4 s off) via a cell disrupter (JY92-IIN, Shanghai Jingqi Instruments Co., Ltd., Shanghai, China). The mixture was centrifuged at 5000× g for 15 min at 4 °C, and the supernatant was immediately collected to assay the activities of glucose-6-phosphate dehydrogenase (G6PDH), malic enzyme (ME), and ATP citrate lyase (ACL) per reported protocols [21,22]. In brief, G6PDH and ME activities were determined by monitoring the reduction of NADP+ to NADPH, quantified as the absorbance increase at 340 nm over 4 min at 25 °C. G6PDH reactions (3 mL) contained 0.1 mL Tris-HCl (1 mol/L, pH 7.4), 0.1 mL NADP+ (2 mmol/L), 0.1 mL glucose-6-phosphate (25 mmol/L), 0.1 mL MgCl2 (0.2 mol/L), 2.5 mL ddH2O, and 0.1 mL crude enzyme. ME reactions (3 mL) included 0.5 mL Tris-HCl (0.4 mol/L, pH 7.4), 0.2 mL NADP+ (3.4 mmol/L), 0.1 mL L-malate (30 mmol/L), 0.1 mL MgCl2 (0.12 mol/L), 2 mL ddH2O, and 0.1 mL crude enzyme. On the other hand, ACL activity was measured with the ATP Citrate Lyase Assay Kit (Ultraviolet Spectrophotometric, BA1920, Zhengzhou Leye-Bio Biotechnology, Zhengzhou, China). Principle: ACL cleaves citrate into acetyl-CoA, oxaloacetate, ADP, and phosphate (with ATP and coenzyme A); malate dehydrogenase then converts oxaloacetate NADH to malate NAD+, decreasing absorbance at 340 nm. ACL reactions (1 mL) comprised 0.805 mL Reagent A, 0.02 mL Reagent B, 0.1 mL Reagent C, 0.02 mL Reagent D, and 0.05 mL crude enzyme.

2.6. Measurements of Specific Rates of Cell Growth and Glucose Consumption

The method for estimating the specific glucose consumption rate is as follows: an electronic balance (CN-LQC3002, Kunshan Ante Metrology Equipment Co., Ltd., Kunshan, China) was used to measure the glucose feeding amount (g/L) every 4 h by monitoring the weight loss of the glucose feeding reservoir. Subsequently, glucose consumption was quantified by combining this data with changes in glucose concentration in the fermentation broth. Taking fermentation time (t) as the independent variable, the glucose consumption amount was smoothed using the corresponding quadratic polynomials; the glucose consumption rate at specific time points was then calculated by differentiating the consumption data with respect to t. Finally, the specific glucose consumption rate at each time point was derived by incorporating the cell concentration at the corresponding time point. On the other hand, the estimation method for the specific cell growth rate is as follows: with fermentation time (t) as the independent variable, cell concentration was fitted using a quadratic polynomial, and the cell growth rate at each time point was obtained by differentiating the fitted curve. The specific cell growth rate at each time point was then determined by combining the cell concentration at the corresponding time point.

2.7. Statistical Analysis

The experimental results are presented as the mean value ± standard deviation (SD), as the data were obtained from three (3) independent replicates. SPSS 20.0 software (IBM, Chicago, IL, USA) was used for statistical analysis based on one-way analysis of variance (ANOVA), with significant differences defined as p < 0.05.

3. Results and Discussion

3.1. Effects of Exogenous H2O2 on DHA Fermentation Performance

It is widely reported that intracellular reactive oxygen species (ROS) are a common regulatory mediator for lipid accumulation in oleaginous microorganisms, and maintaining an appropriate ROS level (oxidative stress) is a necessary prerequisite for effective lipid accumulation [14,15]. Therefore, aiming to enhance the production yield of DHA, the comparisons of different H2O2 concentrations (a typical oxidizing agent) on DHA fermentation performance in shake cultures were explored in this study. As shown in Table 1, the glucose consumption rate could be obviously enhanced with the increase in H2O2 concentration and reached its peak value of 1.18 ± 0.10 g/L/h when controlling H2O2 concentration at the optimal level of 2 mmol/L. Correspondingly, the lipid yields also reached their highest value of 1.16 ± 0.10 g/L, representing a 45.0% increase compared to the control group. However, once the H2O2 concentration surpassed 2 mmol/L, both the glucose consumption rate and lipid yield began to decline with further increases in H2O2 levels. Among the four fermentation batches, the lowest glucose consumption rate (0.74 ± 0.06 g/L/h) and lipid yield (0.82 ± 0.07 g/L) were observed when the H2O2 concentration was set at 4 mmol/L. In addition, the cultivation environment of oxidative stress induced by exogenous addition of H2O2 at the optimal levels of 2 mmol/L, namely strategy A, was also carried out in a 5-L fermenter. As shown in Figure 1, with the strategy A, DHA concentration rapidly rose to 0.84 ± 0.13 g/L in the first 50 h, which was a 42% increase over that obtained in the control group at the same fermentation instant (Figure 1F and Figure 2A). However, after that, the continuous biosynthesis of DHA was obviously destroyed and even stopped along with the fermentation time (Figure 1F), which might be due to the following reason: oxidative stress induced by intermittent feeding of H2O2 (Strategy A) was gradually strengthened with prolonged fermentation as evidenced by the rising levels of reactive oxygen species (ROS) (Figure 1E), and this increase in ROS would result in a decline in cellular metabolic activity, ultimately restricting the period for efficient DHA production (Figure 1C,F).

3.2. Effects of Exogenous p-Aminobenzoate on DHA Fermentation Performance

Although the production performance of DHA could be obviously improved in the first 50 h when cells underwent exposure to a proper and sufficient oxidative stress environment with strategy A, the entire DHA biosynthesis after 50 h was destroyed by the severe accumulation of ROS when implementing strategy A (Figure 1). In fact, it has been reported that severe accumulation of ROS is unavoidable during normal DHA fermentation batches without exogenous H2O2, as large amounts of oxygen must be supplied to meet the demands of cellular respiration [23,24,25]. To solve these questions, a two-point addition strategy for ascorbic acid, a typical reducing agent, was carried out in the previous report, and the results suggested a 44% enhancement in DHA production yield compared to the control group, achieved by mitigating serious ROS accumulation through a two-point addition strategy for ascorbic acid [26]. As a result, the effects of different concentrations of p-aminobenzoate (a reducing agent) on DHA fermentation performance in shake cultures were also explored. As shown in Table 1, both the glucose consumption rate and dry cell weight (DCW) increased significantly with rising concentrations of p-aminobenzoate, reaching peak values of 1.25 ± 0.10 g/L/h and 6.80 ± 0.21 g/L, respectively, when the p-aminobenzoate concentration was controlled at the optimal level of 200 mg/L. Correspondingly, the lipid yield also reached its highest value of 1.18 ± 0.10 g/L, representing a 22.9% increase compared to the control group, and the reason why the exogenous addition of 200 mg/L p-aminobenzoate enhanced lipid production was that p-aminobenzoate promoted glucose catabolism in glycolysis while increasing the mevalonate pathway and weakening the tricarboxylic acid (TCA) cycle; moreover, p-aminobenzoate enhances NADPH generation by activating the pentose phosphate pathway (PPP), ultimately redirecting metabolic flux toward lipid synthesis [27]. After that, the yield of lipid tended to decline with further increasing the concentration of p-aminobenzoate. Furthermore, the exogenous addition of the optimal level of 200 mg/L p-aminobenzoate, namely strategy B, was also implemented in a 5-L fermenter. As shown in Figure 1, the serious accumulation of ROS (strategy B, 320.07 ± 54.49 pg/mL vs. control group 582.72 ± 54.58 pg/mL) was efficiently repressed with strategy B, resulting in a higher final DHA concentration of 1.19 ± 0.15 g/L, representing a 19% increase over the control group.

3.3. Increasing DHA Production with the Periodic Hydrogen Peroxide and p-Aminobenzoate Feeding Strategy

To harmonize the beneficial effects of hydrogen peroxide (H2O2, an oxidizing agent) and p-aminobenzoic acid (a reducing agent) in enhancing DHA production by using Schizochytrium sp. S31, a novel periodic H2O2 and p-aminobenzoate feeding strategy, designated as Strategy C, was proposed and carried out in this study. As shown in Figure 3, this periodic feeding strategy partitioned the fermentation process into three (3) ~21-h stages, featuring cyclic feeding of H2O2 for the first ~7 h and p-aminobenzoate for the following ~14 h within each interval, and the principle of this strategy is that the substantial harm inflicted on Schizochytrium sp. S31 cells, due to their continuous exposure to the oxidative stress environment induced by the use of exogenous addition of H2O2, can be effectively alleviated through the implementation of a recovery treatment (p-aminobenzoate, reducing agent) subsequent to the environmental stimulus. Figure 3 depicts DHA fermentation performance under strategy C, indicating that the accumulation of ROS induced by exogenous H2O2 was effectively alleviated (772.73 ± 56.35 pg/mL vs. 547.03 ± 49.21 pg/mL) compared with strategy A (intermittent feeding of H2O2). As a result, the concentrations of lipid and DHA consistently rose throughout the fermentation period, achieving the highest concentrations of 2.18 ± 0.20 g/L and 1.49 ± 0.20 g/L, exceeding the control group by 29% and 49%, respectively (Figure 2 and Figure 3).

3.4. Enhancement of Catalytic Performance of Key Enzyme in DHA Biosynthesis with the Periodic Hydrogen Peroxide and p-Aminobenzoic Acid Feeding Strategy

It has been reported that when oxidative stress exceeds the buffering capacity of the cell’s antioxidant system, it induces damage to intracellular lipids (e.g., peroxidation of unsaturated fatty acids in cell membranes), proteins (e.g., conformational denaturation), carbohydrates, nucleic acids, and other organelles, ultimately leading to an obvious decrease in cell viability [26,28,29]. To further verify the mechanism by which oxidative damage is alleviated via the periodic control strategy C, thereby enhancing the activity of key enzymes (essentially proteins) in the DHA biosynthesis pathway, as well as ultimately increasing DHA yield, a comparative analysis of the activity changes of key enzymes in the DHA biosynthesis pathway under different control strategies was carried out. The main metabolic pathways involved in DHA production by Schizochytrium sp., as well as the associated key enzymes of these pathways, were summarized in Figure 4. Specifically, acetyl-CoA and NADPH are key precursors for lipid biosynthesis (including DHA) [30,31]. The functions of the key enzymes involved in the biosynthesis of these two precursors are as follows: glucose-6-phosphate dehydrogenase (G6PDH) and malic enzyme (ME) are two key enzymes in the NADPH regeneration process, and both play a pivotal role collectively; the synthesis of acetyl-CoA is catalyzed by ATP citrate lyase (ACL). Figure 5 demonstrates that the periodic control strategy C sustained the peak activities of G6PDH, ME, and ACL. This result suggests that strategy C ensured sufficient availability of acetyl-CoA and NADPH, essential precursors for lipid biosynthesis, leading to a maximum lipid yield of 2.18 ± 0.20 g/L—a 29% improvement compared to the control (Figure 1, Figure 2 and Figure 3). It should be noted that NADPH is not only utilized as a precursor for lipid synthesis (including DHA) but is also involved in the cellular antioxidative system to eliminate the impact of accumulated ROS on normal cellular metabolism (Figure 4). Compared with the control strategy A, ROS accumulation can be effectively alleviated through using the periodic control strategy C, which results in a certain reduction in the amount of NADPH directed to the oxidative defense system. This, in turn, leads to a further increase in the proportion of NADPH flowing into the lipid synthesis pathway, with the ultimate effect of further enhancing DHA production yield under the periodic control strategy.
There was some doubt about why the different treatments produced similar trends of the enzyme activities; the reasons can be attributed as follows: under the traditional batch fermentation mode, cell growth could be basically divided into four stages: lag phase, logarithmic growth phase, stationary phase, and death phase. Correspondingly, a changing trend will also be exhibited by cellular activity (including enzyme activity): starting from low levels, progressing to rapid growth, then tending to stabilize, and finally declining gradually. Therefore, a certain similarity was shown by the changing trend of enzyme activity under different control strategies in this study.

3.5. Reducing Glucose Distribution for Cell Maintenance, the Periodic Hydrogen Peroxide and p-Aminobenzoic Acid Feeding Strategy

It is well known that oxidative stress induces a series of issues, such as damage to intracellular proteins (e.g., conformational denaturation), carbohydrates, nucleic acids, and other organelles [26,28,29]. Under this condition, to maintain normal cell’s metabolism, the carbon source (glucose) flux directed toward the maintenance metabolic pathway (i.e., sustaining the cell’s basic metabolic activities) increases significantly. It should be noted that maintenance energy is not a constant value; it fluctuates dynamically with environmental conditions. Specifically, when Schizochytrium sp. is exposed to stressful environments (e.g., abnormal temperatures, pH fluctuations, carbon/nitrogen source deficiency, oxidative stress), the cell’s basic metabolic consumption increases accordingly to maintain physiological homeostasis (e.g., repairing damaged organelles, regulating intracellular osmotic balance), which in turn leads to an elevation in the maintenance coefficient. Conversely, in a suitable fermentation environment, the maintenance coefficient remains at a stable “minimum threshold” level. In order to investigate the effects of oxidative stress on Schizochytrium sp. cells under different control strategies, the values of maintenance energy (m), which represents the amount of substrate consumed per unit biomass per unit time for maintenance, were calculated under different feeding strategies using a widely recognized equation by plotting the specific growth rate (1/h) on the abscissa and the specific glucose consumption rate (1/h) on the ordinate.
q glu = 1 Y X / S μ + m
Here, qglu, μ, and YX/S represented the specific rate of glucose consumption (1/h), the specific rate of Schizochytrium sp. cells growth (1/h), and the yield of biomass on glucose (-), respectively. The variations of glucose metabolism patterns under different control strategies were presented in Figure 6. The results indicate that, under the periodic feeding strategy C, the metabolic flux of glucose dedicated to cellular maintenance remained at a minimal value of 0.0027 1/h, indicating that the impact of oxidative stress on Schizochytrium sp. cells is significantly alleviated. As a result, the metabolic flux of glucose toward the pathway of cell assimilation, including the pathway of DHA biosynthesis, would be obviously enhanced with strategy C and thus a maximum DHA concentration of 1.49 ± 0.20 g/L was achieved (Figure 2 and Figure 3).

3.6. The Advantages of the Proposed Periodic Control

From the perspective of fermentation optimization, maintaining specific state and operating variables such as concentrations of dissolved oxygen (DO) and nitrogen source at target constant levels is a commonly used approach, and this method of regulating fermentation processes by stabilizing key parameters provides a relatively stable cultivation environment for microbial growth and product synthesis, thereby reducing the interference of fluctuations on fermentation efficiency [32]. Nevertheless, the constant control strategy cannot be realized in large-scale microbial bioprocesses, and it is well-documented that concentration gradients in the liquid phase of large-scale microbial bioprocesses exert a significant influence on cellular physiology [33,34,35,36]. To explore the operational dynamics of large-scale microbial bioprocesses and examine how these dynamics affect microbial physiology and process efficiency, laboratory studies have extensively utilized scale-down techniques to investigate the impacts of fluctuating cultivation conditions—such as substrate concentrations (e.g., glucose) and dissolved oxygen (DO)—on cell physiology and the yields of target products [34,36]. Previous research has indicated that during fed-batch cultivation of recombinant E. coli, cells exposed to oscillating environments (characterized by varying DO and glucose levels) maintained higher cell viability compared to those in laboratory-scale cultures [34,36]. In the present study, the proposed periodic feeding strategy for H2O2 and p-aminobenzoate presented certain similarities to the previously reported scale-down approaches, and thus, efficient production of DHA by Schizochytrium sp. via periodic hydrogen peroxide and p-aminobenzoate control was expected.
In fact, the traditional oscillatory or pulsed strategies are typically implemented through the feeding of a single substance (e.g., carbon and nitrogen sources) and fail to be adjusted adaptively based on the dynamic changes in the metabolic environment during the microbial fermentation process [26,37,38]. Compared with the traditional oscillatory or pulsed strategies, the periodic control is typically defined as a process where the control variable(s) undergo alternating changes within predefined time intervals, thereby inducing transitions in the physiological state of the fermentation system between distinct steady states [39]. In this study, aiming to further enhance the production yield of DHA, based on strategies A and B, a comparable periodic feeding strategy for H2O2 and p-aminobenzoate was proposed and carried out based on the dynamic changes in the metabolic environment during the process of DHA production. Specifically, the periodic control strategy was employed in this study, where the entire fermentation process was divided into three sub-phases (each lasting approximately 21 h), and periodic regulation was achieved through the alternating feeding of hydrogen peroxide (H2O2) and p-aminobenzoate. Based on the variation characteristics of the metabolic environment during DHA fermentation and the properties of different feeding substances, the sub-phases were divided to enable the application of oxidative stress (via H2O2 addition) and oxidative stress recovery treatment (via p-aminobenzoate addition) to Schizochytrium sp. cells in an alternating manner; thus, more precise regulation of the fermentation process was realized. Furthermore, reducing fermentation costs for DHA remains a key goal. Russo et al. [40] demonstrated that using food waste in Aurantiochytrium sp. cultivation reduces operating costs by 35%, raises ROI by up to 8%, and can lower unit production costs by up to 38%. Beyond low-cost feedstocks, the periodic control strategy in this study—pulsed feeding of H2O2 and p-aminobenzoate—also enhances DHA yield and offers a viable cost-reduction approach, guiding future research and industrial applications.

4. Conclusions

DHA production yield can be effectively increased by using H2O2 as a common regulatory mediator for lipid accumulation in oleaginous microorganisms. But continuous long-term exposure of cells to oxidative stress leads to a limited duration of efficient DHA production. Aiming to further enhance the production yield of DHA, a novel periodic feeding strategy for H2O2 and p-aminobenzoate was proposed, and this strategy divided the entire DHA fermentation process into three ~21 h subintervals, during which alternate intermittent feeding of H2O2 (first ~7 h) and p-aminobenzoate (later ~14 h). This novel periodic feeding strategy simultaneously achieved peak activities of key enzymes in the DHA biosynthesis and minimized the distribution of glucose metabolic flux dedicated to cellular maintenance (0.0027 1/h) and thus the lipid and DHA consistently rose throughout the fermentation period, achieving the highest levels of 2.18 ± 0.20 g/L, and 1.49 ± 0.20 g/L, exceeding the control group by 29%, and 49%, respectively.

Author Contributions

L.J.: Conceptualization, Validation, Data curation, Supervision, Funding acquisition, Writing—original draft, Writing—review and editing; M.M.: Investigation, Methodology, Validation, Data curation, Writing—original draft, Writing—review and editing; X.W., R.W. and S.X.: Investigation. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (32302024), China Postdoctoral Science Foundation (2023M732991), and Jiangsu Provincial Natural Science Foundation of Universities (22KJB180008).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare that there are no conflicts of interest.

References

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Fermentation 11 00558 g001
Figure 1. Production performance profiles in DHA fermentation under different feeding strategies. (A–C): Control group; (D–F): Strategy A (H2O2 intermittent feeding); (G–I): Strategy B (p-aminobenzoate intermittent feeding). (A,D,G): ♦: cell concentration; Fermentation 11 00558 i001: glucose concentration;Fermentation 11 00558 i002: glucose addition; Fermentation 11 00558 i003: p-aminobenzoate addition; Fermentation 11 00558 i004: p-aminobenzoate addition; (B,E,H): ■: ROS concentration; (C,F,I): ❚: DHA concentration; ❚: lipid concentration.
Figure 1. Production performance profiles in DHA fermentation under different feeding strategies. (A–C): Control group; (D–F): Strategy A (H2O2 intermittent feeding); (G–I): Strategy B (p-aminobenzoate intermittent feeding). (A,D,G): ♦: cell concentration; Fermentation 11 00558 i001: glucose concentration;Fermentation 11 00558 i002: glucose addition; Fermentation 11 00558 i003: p-aminobenzoate addition; Fermentation 11 00558 i004: p-aminobenzoate addition; (B,E,H): ■: ROS concentration; (C,F,I): ❚: DHA concentration; ❚: lipid concentration.
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Fermentation 11 00558 g002
Figure 2. Comparisons of key fermentation data across different control strategies. Error bars represent SDs of 3 replicates. Bars with different letters in the same color are significantly different at p < 0.05. (A): Comparisons of key fermentation data between the H2O2 intermittent feeding control strategy and the control group at 50 h of the fermentation process. (B): Comparisons of key fermentation data at the end of fermentation under the control strategy, p-aminobenzoate intermittent feeding strategy, and periodic control strategy. A: H2O2 intermittent feeding. B: p-aminobenzoate intermittent feeding. C: periodic control. (A,B): ❚: cell concentration; ❚: lipid concentration; ❚: DHA concentration.
Figure 2. Comparisons of key fermentation data across different control strategies. Error bars represent SDs of 3 replicates. Bars with different letters in the same color are significantly different at p < 0.05. (A): Comparisons of key fermentation data between the H2O2 intermittent feeding control strategy and the control group at 50 h of the fermentation process. (B): Comparisons of key fermentation data at the end of fermentation under the control strategy, p-aminobenzoate intermittent feeding strategy, and periodic control strategy. A: H2O2 intermittent feeding. B: p-aminobenzoate intermittent feeding. C: periodic control. (A,B): ❚: cell concentration; ❚: lipid concentration; ❚: DHA concentration.
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Fermentation 11 00558 g003
Figure 3. Production performance profiles in DHA fermentation under the periodic feeding strategy for hydrogen peroxide and p-aminobenzoate. (A): ♦: cell concentration; Fermentation 11 00558 i001: glucose concentration; Fermentation 11 00558 i003: p-aminobenzoate addition; Fermentation 11 00558 i004: H2O2 addition; Fermentation 11 00558 i002: glucose addition; (B): ■: ROS concentration; (C): ❚: DHA concentration; ❚: lipid concentration.
Figure 3. Production performance profiles in DHA fermentation under the periodic feeding strategy for hydrogen peroxide and p-aminobenzoate. (A): ♦: cell concentration; Fermentation 11 00558 i001: glucose concentration; Fermentation 11 00558 i003: p-aminobenzoate addition; Fermentation 11 00558 i004: H2O2 addition; Fermentation 11 00558 i002: glucose addition; (B): ■: ROS concentration; (C): ❚: DHA concentration; ❚: lipid concentration.
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Figure 4. Basic diagram of glucose metabolic pathways during the production of DHA with Schizochytrium sp.
Figure 4. Basic diagram of glucose metabolic pathways during the production of DHA with Schizochytrium sp.
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Figure 5. Changes in enzymatic activities associated with DHA production under the four different feeding strategies. (A–C): ♦: Control group; ■: Strategy A-“H2O2 intermittent feeding”; ▲: Strategy B-“p-aminobenzoate intermittent feeding”; ●: Strategy C-“periodic control”.
Figure 5. Changes in enzymatic activities associated with DHA production under the four different feeding strategies. (A–C): ♦: Control group; ■: Strategy A-“H2O2 intermittent feeding”; ▲: Strategy B-“p-aminobenzoate intermittent feeding”; ●: Strategy C-“periodic control”.
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Figure 6. Glucose metabolism patterns under the four different feeding strategies. (A–C): ♦: Control group; ●: Strategy C—“periodic control”; (D): ❚: Control group; ❚: Strategy A—“H2O2 intermittent feeding”; ❚: Strategy B—“p-aminobenzoate intermittent feeding”; ❚: Strategy C—“periodic control”.
Figure 6. Glucose metabolism patterns under the four different feeding strategies. (A–C): ♦: Control group; ●: Strategy C—“periodic control”; (D): ❚: Control group; ❚: Strategy A—“H2O2 intermittent feeding”; ❚: Strategy B—“p-aminobenzoate intermittent feeding”; ❚: Strategy C—“periodic control”.
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Table 1. The effects of exogenous addition of H2O2 and p-aminobenzoate on DHA fermentation.
Table 1. The effects of exogenous addition of H2O2 and p-aminobenzoate on DHA fermentation.
RunH2O2 Concentration (mmol/L)p-Aminobenzoate Concentration (mg/L)Glucose Consumption Rate
(g/L/h)		Dry Cell Weight
(g/L)		Lipid Concentration
(g/L)
1000.87 ± 0.06 bc5.34 ± 0.29 c0.80 ± 0.05 e
2100.91 ± 0.07 b5.21 ± 0.27 c0.92 ± 0.07 cde
3201.18 ± 0.10 a5.12 ± 0.29 c1.16 ± 0.10 ab
4300.88 ± 0.06 bc4.96 ± 0.16 c0.82 ± 0.07 de
5400.74 ± 0.06 d4.22 ± 0.15 d0.58 ± 0.05 f
601000.94 ± 0.08 b6.18 ± 0.20 b0.96 ± 0.08 cd
702001.25 ± 0.10 a6.80 ± 0.21 a1.18 ± 0.10 a
803001.11 ± 0.09 a6.30 ± 0.20 b1.02 ± 0.09 bc
Values corresponding to distinct letters within the same column exhibit statistically significant differences at p < 0.05.
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MDPI and ACS Style

Jia, L.; Ma, M.; Wang, X.; Wang, R.; Xin, S. Enhancing Docosahexaenoic Acid Production by Schizochytrium sp. via Periodic Hydrogen Peroxide and p-Aminobenzoate Control. Fermentation 2025, 11, 558. https://doi.org/10.3390/fermentation11100558

AMA Style

Jia L, Ma M, Wang X, Wang R, Xin S. Enhancing Docosahexaenoic Acid Production by Schizochytrium sp. via Periodic Hydrogen Peroxide and p-Aminobenzoate Control. Fermentation. 2025; 11(10):558. https://doi.org/10.3390/fermentation11100558

Chicago/Turabian Style

Jia, Luqiang, Mengyao Ma, Xingyue Wang, Ruoyu Wang, and Shuqi Xin. 2025. "Enhancing Docosahexaenoic Acid Production by Schizochytrium sp. via Periodic Hydrogen Peroxide and p-Aminobenzoate Control" Fermentation 11, no. 10: 558. https://doi.org/10.3390/fermentation11100558

APA Style

Jia, L., Ma, M., Wang, X., Wang, R., & Xin, S. (2025). Enhancing Docosahexaenoic Acid Production by Schizochytrium sp. via Periodic Hydrogen Peroxide and p-Aminobenzoate Control. Fermentation, 11(10), 558. https://doi.org/10.3390/fermentation11100558

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