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Article

Investigation of the Carbon Catabolite Repression Mechanism in L-Lactic Acid Fermentation from Mixed Sugars by Bacillus coagulans DSM 2314

by
Yinan Sun
1,
Juan Wang
2,
Tong Deng
2,
Shijie Wang
3,
Jianguo Liu
1,*,
Xiaona Wang
4,* and
Qunhui Wang
4
1
Key Laboratory of Environmental Pollution Control and Remediationat Universities of Inner Mongolia Autonomous Region, College of Resources and Environmental Engineering, Inner Mongolia University of Technology, Hohhot 010051, China
2
Inner Mongolia Autonomous Region Key Laboratory of Green Construction and Intelligent Operation and Maintenance of Civil Engineering, School of Civil Engineering, Inner Mongolia University of Technology, Hohhot 010051, China
3
Laboratory of Environmental Biotechnology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China
4
School of Energy and Environmental Engineering, University of Science and Technology Beijing, Beijing 100083, China
*
Authors to whom correspondence should be addressed.
Microorganisms 2026, 14(2), 417; https://doi.org/10.3390/microorganisms14020417
Submission received: 3 January 2026 / Revised: 29 January 2026 / Accepted: 5 February 2026 / Published: 10 February 2026
(This article belongs to the Section Microbial Biotechnology)
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Abstract

Lignocellulosic hydrolysate is rich in various fermentable sugars, such as glucose, xylose, and cellobiose. Utilizing these sugars for L-lactic acid fermentation represents a promising strategy for the high-value utilization of biomass. However, when mixed sugars serve as carbon sources, microorganisms typically undergo carbon catabolite repression (CCR) at the initial fermentation stage, which significantly compromises both the yield and productivity of L-lactic acid. To clarify CCR mechanisms and explore effective mitigation strategies, Bacillus coagulans DSM 2314 was used as the fermentative strain, the effects of pH and temperature on fermentation with single and mixed carbon sources were examined, and L-lactic acid yields, productivities, and key enzymatic activities across different fermentation systems were systematically compared. The results showed that in glucose-containing mixed-sugar systems, glucose imposed strong CCR effects on both cellobiose and xylose. Under optimal conditions (initial total sugar concentration of 50 g/L, pH 7.0, and 45 °C), L-lactic acid yields increased in the following order: glucose/xylose (15.58 g/L) < glucose/cellobiose (29.65 g/L) < glucose (31.87 g/L). In contrast, in the glucose-free cellobiose/xylose system, both sugars were nearly co-consumed by B. coagulans DSM 2314, and L-lactic acid production was not significantly diminished by the mixing of carbon sources (xylose (27.45 g/L) < cellobiose/xylose (28.64 g/L) < cellobiose (29.60 g/L)). Under replicated optimal condition experiments, analyses of sugar consumption rates and enzyme activities further confirmed that the CCR between cellobiose and xylose was significantly weaker than in other mixed-sugar systems, with the L-lactic acid yield in the cellobiose/xylose system 1.61-fold higher than in the glucose/xylose system. These findings demonstrate that substituting glucose with cellobiose in mixed-sugar fermentation is an effective approach to mitigating CCR, providing a theoretical basis for efficient L-lactic acid production from lignocellulosic hydrolysates.

1. Introduction

Lactic acid (LA) serves as a vital intermediate in organic synthesis and is extensively applied across the pharmaceutical, food, and cosmetic industries [1]. LA has two stereoisomers, L-Lactic acid (L-LA) and D-Lactic acid (D-LA) [2]. Among them, L-LA is the metabolically acceptable isomer in humans and therefore has greater application value in the food, pharmaceutical, and polylactic acid (PLA) industries than D-LA or D-L-Lactic acid (D-L-LA) [3]. Although LA can be produced via chemical synthesis, microbial fermentation has emerged as the dominant method for industrial production due to two primary advantages: first, it allows for the selective production of optically pure L-LA; second, it offers lower energy consumption, higher cost-effectiveness, and easier access to high-purity products [4,5,6].
LA bacteria can utilize a wide range of substrates. Beyond simple sugars, they can also convert inexpensive non-food feedstocks, such as lignocellulosic waste derived from cassava, inulin, and straw. Lignocellulose is primarily composed of cellulose, hemicellulose, and lignin [7]. Cellulose is a polysaccharide consisting of numerous glucose units linked by β-1,4-glycosidic bonds [8,9]. Upon hydrolysis by cellulase, it yields glucose, which serves as a carbon source for microbial growth and fermentation [10,11]. Hemicellulose is composed of various complex heteropolymers [12,13], and its hydrolysis releases xylose, which can also be utilized as a carbon source by microorganisms [14,15]. Consequently, pretreated lignocellulose represents a low-cost raw material for microbial LA production.
However, when mixed sugars such as glucose, xylose, and cellobiose are present simultaneously, microorganisms typically prioritize the utilization of the carbon source that is most easily degradable and offers the highest metabolic efficiency for example, glucose. The metabolic products of this rapidly metabolized carbon source often inhibit or repress the expression of genes and enzymatic activities associated with the metabolism of secondary carbon sources, such as xylose and arabinose. This phenomenon, known as carbon catabolite repression (CCR), temporarily blocks the utilization of other sugars. CCR effects lead to the incomplete fermentation of non-glucose carbon sources, thereby reducing the final concentration, productivity, and yield of the product. Furthermore, the accumulation of significant amounts of residual sugars in the fermentation broth increases the downstream costs associated with LA purification [16].
To date, several researchers have investigated mixed-sugar fermentation for L-lactic acid production. Klongklaew et al. [17] screened 39 pentose-utilizing lactic bacteria from Eri silkworm, among which Enterococcus mundtii WX1 achieved glucose and xylose conversion rates of 97% and 68%, respectively. In addition, co-cultivation of E. mundtii WX1 and Lactobacillus rhamnosus SCJ9 using 30 g/L total sugars (glucose:xylose = 6:4) at 37 °C for 48 h yielded a maximum L-lactic acid concentration of 23.59 g/L and a sugar-to-lactic-acid conversion rate of 76%. Zhang et al. [18] employed poplar wood hydrolysate as a mixed-sugar substrate and co-fermented Lactobacillus brevis ATCC 367 and Lactobacillus plantarum ATCC 21028. The initial glucose and xylose concentrations were 35.4 g/L and 14.3 g/L, respectively. During early fermentation, L. plantarum first consumed glucose; after a set period, L. brevis was introduced to specifically utilize xylose. This sequential synergy improved substrate utilization and yielded 38.0 g/L L-LA and 7.2 g/L acetic acid, corresponding to a conversion rate of 80% and a productivity of 0.40 g/L/h.
Bacillus coagulans DSM 2314 is an efficient L-LA producer that forms endospores conferring strong stress resistance, including tolerance to heat, acid, and other stressors. Fermentation at elevated temperatures can significantly minimize contamination by common mesophilic contaminants, eliminating the need for strict sterile operations, thereby reducing costs and controlling risks. Ricard-Garrido et al. [19] used cow manure as a substrate for simultaneous saccharification and fermentation with B. coagulans DSM 2314, achieving a LA concentration of 13.65 g/L after 24 h with a conversion rate of 33.1%. Similarly, Pol et al. [20] reported a 74% conversion rate when converting lignocellulosic sugars to LA using this strain. Cox et al. [21] produced L-LA under non-sterile conditions using the thermophilic, homofermentative B. coagulans DSM 2314. Using pure xylose, xylose-rich bagasse, and olive pit hydrolysates as substrates in a fed-batch mode, they obtained maximum L-LA yields of 97.8 g/L, 52.4 g/L, and 61.3 g/L, respectively. Although these studies demonstrate favorable conversion performance of B. coagulans DSM 2314 using lignocellulosic hydrolysates, they did not address whether this strain also experiences carbon catabolite repression during mixed-sugar fermentation. Furthermore, whether CCR can be alleviated under suitable sugar combinations, pH, and temperature remains unclear and warrants further investigation.
In this study, B. coagulans DSM 2314 was employed as the fermentative strain. L-LA fermentation was carried out using single-sugar systems (glucose, xylose, and cellobiose) and four mixed-sugar systems (glucose/xylose, glucose/cellobiose, glucose/cellobiose/xylose, and cellobiose/xylose). L-LA yield, productivity, residual sugar concentration, enzyme activities, and other indicators were quantified. The objective was to elucidate co-fermentation behavior, characterize sugar-to-acid conversion performance in mixed substrates, and clarify both the occurrence and elimination mechanisms of the CCR effect.

2. Materials and Methods

2.1. Microorganisms and Raw Materials

Bacillus coagulans DSM 2314 was procured from the German Collection of Microorganisms and Cell Cultures. Glucose, xylose, cellobiose, sodium hydroxide (NaOH), soy peptone, agar, casein peptone, sodium chloride, and B vitamins were sourced from local (Hohhot, Inner Mongolia, China) reagent suppliers.

2.2. Seed Culture Medium and Fermentation Medium

The casein–soy peptone agar (CASO AGAR, Merck 105458) used for cultivating Bacillus coagulans consisted of casein peptone (15.0 g), soybean peptone (5.0 g), NaCl (5.0 g), and agar (15.0 g), supplemented with distilled water to a final volume of 1000 mL. The medium was sterilized at 9.8 × 104 Pa for 20 min. Cultivation was carried out in shake flasks at 55 °C and 100 rpm for 24 h to obtain the seed culture. The final inoculum was prepared following a secondary activation step of the seed culture.
For the fermentation medium, the total sugar concentration was fixed at 50 g/L for both single-sugar and mixed-sugar fermentation systems. In the mixed-sugar systems, glucose/cellobiose (G/C), glucose/xylose (G/X), and cellobiose/xylose (C/X) binary systems contained equal proportions (50% each) of the two sugars, while the glucose/cellobiose/xylose (G/C/X) ternary system contained each sugar at 33.3% of the total sugar concentration.

2.3. Fermentation Process

The fermentation media were sterilized by high-temperature and high-pressure treatment. After cooling to room temperature, the fermentation media were inoculated with an inoculum volume corresponding to 10% of the fermentation medium volume. During fermentation, the pH of the broth was intermittently adjusted every 12 h using 4 mol/L NaOH, with pH set points of 5, 6, 7, and 8. A control group without pH adjustment was included. Fermentations were conducted in a water bath shaker at 45–55 °C and 110 rpm. Samples were collected at 144 h to determine the concentrations of glucose, xylose, cellobiose, and L-LA. After identifying the optimal pH and temperature conditions, fermentation experiments for each system were repeated under the optimized conditions. Samples were collected at regular intervals (every 24 h) to measure the concentrations of glucose, xylose, cellobiose, L-LA, and the activities of enzymes.

2.4. Test Methods

The pH of the fermentation broth was measured using a Leici PHS-3C pH meter (Leici, shanghai, China). The concentrations of glucose, xylose, cellobiose, and L-LA were determined using a Shimadzu LC-20AT high-performance liquid chromatography (HPLC) system (Shimadzu, Kyoto, Japan) equipped with a Shodex Sugar SH1011 column (8.0 mm × 300 mm). The mobile phase was 0.05 mol/L H2SO4 at a flow rate of 0.6 mL/min. The column temperature was maintained at 60 °C, and the injection volume was 5 μL. Enzyme activities were quantified using ELISA kits (China) for glucose phosphate isomerase (GPI), phosphogluconolactonase (PG), xylose isomerase (XI), xylulokinase (XK), β-glucosidase (β-glu), lactate dehydrogenase (LDH), and aldehyde dehydrogenase (ALDH).

2.5. Statistical Analysis

All data were expressed as mean ± standard error (SE) of three independent fermentation batches (n = 3).
To clarify the inhibitory interactions among different substrates, the coupling interaction factor (CIF) [22], a method commonly used in fermentation studies, was employed to evaluate synergistic or inhibitory effects. Based on the mixing ratios of sugars in mixed-sugar fermentations and the L-LA yields obtained from the corresponding single-sugar fermentations, the theoretical L-LA yield for each mixed-sugar system was estimated using Equation (1). The degree of synergistic or inhibitory interaction during mixed-sugar fermentation was then quantified using Equation (2).
Mestimated = Mglucose × X1 + Mxylose × X2 + Mcellobiose × X3
where Mestimated is the estimated theoretical L-LA yield during mixed-sugar fermentation (g/L); Mglucose is the L-LA yield obtained from glucose-only fermentation (g/L); Mxylose is the L-LA yield obtained from xylose-only fermentation (g/L); Mcellobiose is the L-LA yield obtained from cellobiose-only fermentation (g/L); X1, X2 and X3 represent the proportions of glucose, xylose, and cellobiose in the mixed-sugar substrate, respectively.
The coupling interaction factor was calculated as follows:
CIF = Mactual/Mestimated × 100%
A CIF value greater than 100% indicates a synergistic effect on L-LA production, whereas a CIF value lower than 100% indicates an inhibitory effect. A CIF value equal to 100% indicates the absence of either synergistic or inhibitory interactions.

3. Results and Discussion

3.1. Effect of pH on Single-Sugar and Mixed-Sugar Fermentation by Bacillus coagulans DSM 2314

pH has a direct influence on microbial growth [23]. In the three single-sugar systems and four mixed-sugar systems, the fermentation broth pH was adjusted every 12 h to 5, 6, 7, or 8. After fermentation for 144 h at the same temperature, the concentrations of L-Lactic acid (L-LA) produced under different pH conditions are shown in Figure 1. When the pH was intermittently adjusted to 5, both the growth and metabolic activity of Bacillus coagulans DSM 2314, as well as L-LA production, were severely inhibited. In contrast, at pH values of 6, 7, and 8, the L-LA yields in the three single-sugar systems followed the order: glucose (G) > cellobiose (C) > xylose (X), which is shown in Figure 1A. In the three glucose-containing mixed-sugar fermentation systems, although the total sugar concentration was the same as that in the single-sugar systems (50 g/L), the L-LA yields were significantly lower than those obtained in the single-glucose system (Figure 1B). This reduction can be attributed to the lower utilization rates of cellobiose and xylose compared with glucose by B. coagulans DSM 2314. At pH 7.0, the L-LA yields ranked as follows: G/X (12.15 g/L) < G/C (20.48 g/L) < G/C/X (24.10 g/L) < glucose alone (35.37 g/L). This trend suggests that the presence of glucose adversely affected the metabolism of cellobiose and xylose, thereby producing a CCR effect.
By contrast, in the glucose-free cellobiose/xylose fermentation system, L-LA production did not differ significantly from that obtained in two single-sugar systems, following the order: xylose (23.17 g/L) < C/X (25.64 g/L) < cellobiose (29.97 g/L). It indicates that the mutual inhibitory effects between cellobiose and xylose during fermentation by B. coagulans DSM 2314 were substantially weaker than those observed in the other mixed-sugar systems examined in this study.
The cellobiose/xylose system at pH 7.0, the CIF value under pH 7 is calculated:
CIF (C/X) = 25.64/(29.97 × 50% + 23.17 × 50%) × 100% = 96.5%
By extension, the CIF values at pH 7 are calculated as follows: C/X (96.5%) > G/C/X (81.8%) > G/C (62.7%) > G/X (41.5%). This ranking indicates that when xylose or cellobiose was fermented with glucose either separately or simultaneously, the actual L-LA yields were consistently lower than the that obtained by fermenting single-sugar individually. These results further confirm that the presence of glucose inhibited the conversion of both xylose and cellobiose, consistent with CCR, and that glucose exerted a stronger inhibitory effect on xylose than on cellobiose.
Zhu et al. [24] reported that high glucose concentrations markedly upregulate the ptsH gene, which encodes the histidine-containing phosphocarrier protein in the phosphotransferase system (PTS), and the ccpA gene, which encodes the catabolite control protein A (CcpA). In this process, the histidine-containing protein (HPr) protein becomes phosphorylated and forms a complex with CcpA. The complex then binds to catabolite-responsive elements and represses the expression of xylose metabolism genes (e.g., xylA, encoding xylose isomerase, and xylB, encoding xylulose kinase). This repression results in reduced xylose conversion. Under a low-enzyme-load semi-hydrolysis strategy, the glucose concentration decreases. As a result, ptsH and ccpA expression is downregulated, the CCR effect is alleviated, and xylose conversion increases from 48.2% to 85.7%. These findings clarify the molecular mechanism of glucose-induced CCR. The changes in xylose metabolism reported in that work are consistent with the trends shown in Table 1 of our study. We will also focus on how the metabolism of cellobiose affects the regulatory network of xylose metabolism genes in future studies.
In contrast (Figure 2), in the cellobiose/xylose mixed-sugar system, the L-LA yield was comparable to those obtained from the respective single-sugar fermentations, with CIF values closest to 100%, indicating minimal inhibitory interaction. This finding suggests that cellobiose and xylose can be co-utilized nearly simultaneously during fermentation by Bacillus coagulans DSM 2314.
Microorganisms 14 00417 g002
Figure 2. Comparison of actual and estimated L-LA production in mixed-sugar fermentation at pH 7.0. Data are presented as mean ± SE (n = 3 independent fermentation batches).
Figure 2. Comparison of actual and estimated L-LA production in mixed-sugar fermentation at pH 7.0. Data are presented as mean ± SE (n = 3 independent fermentation batches).
Microorganisms 14 00417 g002

3.2. Effect of Temperature on Single-Sugar and Mixed-Sugar Fermentation by Bacillus coagulans DSM 2314

Fermentation was conducted at pH 7 over a temperature range of 40–55 °C. Within this range, variations in L-Lactic acid (L-LA) production by Bacillus coagulans DSM 2314 were less pronounced with changes in temperature than with changes in pH. Nevertheless, both single-sugar and mixed-sugar systems achieved their highest L-LA yields at 45 °C, shown in Figure 3. Consistent with previous observations, the L-LA yield in the glucose (G)-free cellobiose (C)/xylose (X) system did not differ significantly from those obtained in the corresponding single-sugar fermentations, following the order: xylose (27.45 g/L) < cellobiose/xylose 28.64 g/L (28.64 g/L) < cellobiose (29.60 g/L). Moreover, the L-LA yield in the cellobiose/xylose system was 1.84-fold higher than that in the glucose/xylose system, further demonstrating that the mutual inhibitory effects between cellobiose and xylose during fermentation by B. coagulans DSM 2314 were markedly weaker than those observed in other mixed-sugar systems examined in this study.
Similarly, calculations of synergistic and inhibitory effects for the four mixed-sugar systems indicated that at 45 °C (Figure 4), the CIF values followed the order: C/X (100.40%) > G/C (96.47%) > G/C/X (91.02%) > G/X (52.53%). These results further confirm that the presence of glucose inhibited the conversion of both xylose and cellobiose, consistent with the occurrence of CCR. In contrast, in the cellobiose/xylose fermentation system, L-LA production was comparable to that obtained from the corresponding single-sugar fermentations. Furthermore, the CIF value for the cellobiose/xylose system exceeded 100%, indicating the absence of significant inhibitory interactions under these temperature and pH conditions. This finding suggests that cellobiose and xylose can be co-utilized nearly simultaneously by Bacillus coagulans DSM 2314 during fermentation.
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Figure 4. Comparison of actual and theoretical L-LA production in mixed-sugar fermentation at 45 °C. Data are presented as mean ± SE (n = 3 independent fermentation batches).
Figure 4. Comparison of actual and theoretical L-LA production in mixed-sugar fermentation at 45 °C. Data are presented as mean ± SE (n = 3 independent fermentation batches).
Microorganisms 14 00417 g004

3.3. Variation of Carbon Catabolite Repression During Mixed-Sugar Fermentation

To further investigate the differences in metabolic rates of glucose (G), cellobiose (C), and xylose (X) across different systems, fermentation experiments for all systems were repeated at pH 7.0 and 45 °C. The consumption profiles of single sugars and mixed sugars during 144 h of fermentation were compared (Figure 5). In the early stages of fermentation in the G/X, G/C, and G/C/X mixed-sugar systems, the presence of glucose exerted a negative effect on the consumption rates of both xylose and cellobiose (Figure 5A–C). In contrast, in the C/X mixed-sugar fermentation system, the metabolic rates of xylose and cellobiose exhibited minimal deviation from those observed in their respective single-sugar fermentations (Figure 5D). In this system, the small amount of glucose present in the C/X system serves as an intermediate product of cellobiose hydrolysis. Due to its low concentration (<2.5 g/L), it did not inhibit xylose metabolism nor significantly suppress L-Lactic acid (L-LA) production (Figure 6C). Based on these findings, in the practical bioconversion of lignocellulosic residues, the cellulose fraction can first be converted into the intermediate product cellobiose, which can then be co-fermented with xylose. Acting as a slow-release carbon source of glucose, cellobiose not only enhances fermentation efficiency but also effectively alleviates CCR caused by the presence of high glucose concentrations during mixed-sugar fermentation.
Figure 5D further shows that in the C/X mixed-sugar system, the glucose produced from cellobiose hydrolysis was consistently maintained at a low concentration throughout fermentation. This low glucose level had no significant effect on fermentation efficiency or L-LA production, indicating that CCR does not occur under low-glucose conditions.
Figure 6 compares the variation in L-LA concentration over fermentation time for three single-sugar systems and their corresponding mixed-sugar systems. The results further demonstrate that the C/X system exhibited the smallest difference in L-LA yield relative to its corresponding single-sugar systems (Figure 6C), indicating that cellobiose does not significantly inhibit the metabolic pathway of xylose conversion to L-LA by Bacillus coagulans. Conversely, the G/X system showed the largest deviation from its corresponding single-sugar fermentations (Figure 6A). Therefore, during the hydrolysis of lignocellulosic residues, cellulase alone may be applied while reducing or even avoiding the addition of β-glucosidase, allowing cellulose to be partially hydrolyzed into a mixed-sugar solution dominated by cellobiose and oligosaccharides, supplemented by glucose, thereby mitigating the CCR effect. This result is consistent with the findings reported by Zhu et al. [24,25], who observed that the CCR effect in the cellobiose/xylose system was markedly alleviated during the semi-hydrolysis process of food waste and garden waste.

3.4. Relationship Between Enzyme Activities and L-LA Production in Mixed-Sugar Fermentation Systems

As shown in Figure 5, in the three types of mixed-sugar fermentation systems, the sequence of carbon-source utilization was characterized by the rapid preferential consumption of glucose (G), whereas the consumption of xylose (X) and cellobiose (C) was markedly delayed. Only after glucose was depleted at approximately 72 h did the consumption rates of xylose and cellobiose increased substantially. By fitting the sugar consumption curves in Figure 5 together with the L-Lactic acid (L-LA) production curves in Figure 6 in two distinct phases, the average consumption rates of each sugar during the early fermentation stage (0–72 h) and late fermentation stage (72–144 h) were obtained for both single-sugar and mixed-sugar systems (denoted as V72 h, E and V72 h, L, respectively), the results are summarized in Table 1.
Table 1. Sugar Consumption Rates and L-LA Production Rates before and after 72 h of Fermentation in Single-Sugar and Mixed-Sugar Systems (g/L/h).
Table 1. Sugar Consumption Rates and L-LA Production Rates before and after 72 h of Fermentation in Single-Sugar and Mixed-Sugar Systems (g/L/h).
Fermentation SystemsInitial Sugar Concentration (g/L)Average Sugar Consumption Rate (g/L/h)Average L-LA Production Rate (g/L/h)
V72 h, E *V72 h, L *V72h, E *V72h, L *
Glucose/XyloseGlucose-Based Mixed-Sugar Systems25.000.2920.0300.1020.121
Xylose-Based Mixed-Sugar Systems25.000.0530.242
Glucose/CellobioseGlucose-Based Mixed-Sugar Systems25.000.2120.0970.1960.213
Cellobiose-Based Mixed-Sugar Systems25.000.0390.158
Glucose/Cellobiose/XyloseGlucose-Based Mixed-Sugar Systems16.670.2040.0160.2070.149
Xylose-Based Mixed-Sugar Systems16.670.0430.133
Cellobiose-Based Mixed-Sugar Systems16.670.0320.101
Cellobiose/XyloseXylose-Based Mixed-Sugar Systems25.000.0850.1940.1560.202
Cellobiose-Based Mixed-Sugar Systems25.000.1210.145
Single-Sugar FermentationGlucose-Based Single-Sugar Systems50.000.5860.0540.4360.057
Xylose-Based Single-Sugar Systems50.000.3620.2720.1740.137
Cellobiose-Based Single-Sugar Systems50.000.3600.2600.2150.187
* V72 h, E: The average consumption rates of each sugar and average L-LA production rate during the early fermentation stage (0–72 h). V72 h, L: The average consumption rates of each sugar and average L-LA production rate during the late fermentation stage (72–144 h).
Regardless of whether glucose was fermented as a single carbon source or within mixed-sugar systems, its consumption rate during the first 72 h of fermentation (V72 h, E) was markedly higher than that during the subsequent 72 h (V72 h, L). For xylose and cellobiose in single-sugar fermentations, the average consumption rate during the early phase (V72 h, E) was slightly higher than that during the late phase (V72 h, L). In contrast, when co-fermented with glucose, V72 h, E for both xylose and cellobiose were significantly lower than their corresponding V72 h, L. Specifically, in the G/X system, the V72 h, E of xylose was 78.1% lower than its V72 h, L, while in the G/C system, the V72 h, E of cellobiose was 75.3% lower than its V72 h, L. These results further demonstrate that in glucose-containing mixed-sugar fermentation systems, glucose was preferentially utilized, thereby suppressing the early-stage consumption of xylose and cellobiose. Consequently, the metabolism of xylose and cellobiose exhibited a strong and typical CCR effect, which in turn significantly reduced the L-LA yield and productivity in the G/X and G/C systems (Figure 6A,B).
Notably, in the C/X mixed-sugar system, no significant difference was observed between V72 h, E and V72 h, L. To further examine whether CCR was absent in this system, Table 2 compares the activities of key enzymes, including xylulokinase (XK), β-glucosidase, and lactate dehydrogenase (LDH), across the mixed-sugar fermentation systems. XK represents one of the final and key rate-limiting steps by which xylose enters central carbon metabolism and therefore directly determines the flux and efficiency of xylose degradation [26]. As shown in Table 2, the XK activity in the C/X system was the highest, reaching 678.4 U/L, which was 30.4% and 18.0% higher than those observed in the G/X and G/C/X systems, respectively. This increase indicates an enhanced flux and efficiency of xylose metabolism.
The primary function of β-glucosidase is to catalyze the hydrolysis of cellobiose into two molecules of glucose [27]. In the C/X system, β-glucosidase activity reached 117.8 U/L, representing the highest level among the mixed-sugar systems. However, this activity was comparable to that of the two glucose-containing systems (G/C and G/C/X), showing only a slight increase of 4–7%. This result suggests that the rate of cellobiose consumption is less significantly affected by glucose than by xylose. When considered together with the nearly simultaneous consumption of both sugars in the C/X system (Figure 5D) and the absence of significant differences in L-LA yield compared with the corresponding single-sugar fermentations (Figure 1 and Figure 6C), multiple lines of evidence confirm that CCR between xylose and cellobiose was substantially weaker than that observed in the other mixed-sugar systems investigated in this study.
In addition, lactate dehydrogenase (LDH), the key enzyme catalyzing the conversion of pyruvate to LA [28], also exhibited its highest activity in the C/X system (108.5 U/L), which was significantly greater than those measured in the other three glucose-containing mixed-sugar systems. This further supports the feasibility of constructing mixed-sugar fermentation systems by substituting glucose with cellobiose to alleviate carbon catabolite repression, in practical bioconversion of lignocellulosic residues, incomplete hydrolysis can be achieved through strategies such as mild pretreatment and the use of purified exo-cellulases lacking β-glucosidase activity. This approach results in cellulose hydrolysates dominated by cellobiose rather than glucose, enabling cellobiose to substitute for glucose in mixed-sugar fermentation. Such a strategy not only improves fermentation efficiency but also effectively alleviates CCR commonly induced by high glucose concentrations during mixed-sugar fermentation. The experimental results in Figure 6 further confirm that the C/X system achieved a 1.61-fold increase in L-LA yield compared to the G/X system.

4. Conclusions

(1) Under fermentation conditions with an initial total sugar concentration of 50 g/L, pH 7.0, and a temperature of 45 °C, the fermentation of Bacillus coagulans DSM 2314 was utilized for L-LA production. In all glucose-containing mixed-sugar systems, the L-LA yields were lower than obtained in the single-glucose system, following the order G/X (15.58 g/L) < G/C/X (26.95 g/L) < G/C (29.65 g/L) < glucose (31.87 g/L). These results indicate that glucose exerted a pronounced CCR effect on both cellobiose and xylose. In contrast, in the C/X mixed-sugar system, both sugars were utilized nearly simultaneously, and L-LA production was not significantly reduced by carbon-source mixing (xylose (27.45 g/L) < C/X (28.64 g/L) < cellobiose (29.6 g/L)).
(2) In replicate experiments conducted under the optimal fermentation conditions, analyses of sugar consumption rates revealed that in glucose-containing mixed-sugar systems, glucose metabolism was characterized by a rapid initial consumption followed by a slower phase, with glucose being almost completely depleted after 72 h. In contrast, the consumption of xylose or cellobiose generally exhibited a slow initial phase followed by an accelerated consumption rate at later stages. Enzyme activity analyses further demonstrated that in the glucose-free C/X system, the activities of XK and β-glucosidase were the highest, reaching 678.4 U/L and 117.8 U/L, respectively. In this system, the two sugars were co-utilized nearly, and the L-LA yield did not differ significantly from those obtained with the corresponding single-sugar systems. From a metabolic perspective, these findings confirm that CCR between cellobiose and xylose was markedly weaker than in the other mixed-sugar systems investigated.
(3) Under identical fermentation conditions, the L-LA yield in the C/X system was 1.61-fold higher than that in the glucose/xylose system. These results demonstrate that substituting glucose with cellobiose in mixed-sugar fermentation constitutes an effective strategy for alleviating CCR; moreover, the strategy of incomplete cellulose hydrolysis provides a theoretical basis for the efficient fermentation of lignocellulosic hydrolysates toward L-LA production; in future work, L-lactic acid production can be further improved through fed-batch fermentation with semi-hydrolysate supplementation, combined with high-sugar-loading and cell-recycling strategies.

Author Contributions

Conceptualization, Y.S. and Q.W.; methodology, J.L.; software, T.D.; validation, Y.S.; formal analysis, Q.W.; investigation, S.W.; resources, J.W.; data curation, X.W.; writing—original draft preparation, Y.S.; writing—review and editing, Q.W.; visualization, Y.S.; supervision, J.L.; project administration, J.L.; funding acquisition, J.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Major Demonstration Special Project for Scientific and Technological Innovation in Inner Mongolia Autonomous Region (Grant No. 2025ZDSF0027); National Natural Science Foundation of China (Grant NO. 52470137 and 52170121) and Program for Young Talents of Science and Technology in Universities of Inner Mongolia Autonomous Region (Grant No. NJYT 22083).

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 authors.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
CCRCarbon catabolite repression
LALactic acid
L-LAL-Lactic acid
D-LAD-Lactic acid
D-L-LAD-L-Lactic acid
NaOHSodium hydroxide
G/CMixed-sugar systems, glucose/cellobiose
G/XMixed-sugar systems, glucose/xylose
C/XMixed-sugar systems, cellobiose/xylose
G/C/XMixed-sugar systems, glucose/cellobiose/xylose
GPIGlucose phosphate isomerase
PGPhosphogluconolactonase
XIXylose isomerase
XKXylulokinase
β-gluβ-glucosidase
LDHLactate dehydrogenase
ALDHAldehyde dehydrogenase
CIFCoupling interaction factor
V72h, EThe average consumption rates of each sugar during the early fermentation stage (0–72 h)
V72h, LThe average consumption rates of each sugar during the late fermentation stage (72–144 h)
PTSPhosphotransferase system
CcpACatabolite control protein A
HPrHistidine-containing protein

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Microorganisms 14 00417 g001
Figure 1. L-LA production in single-sugar (A) and mixed-sugar (B) fermentation systems at different pH levels. Data are presented as mean ± SE (n = 3 independent fermentation batches).
Figure 1. L-LA production in single-sugar (A) and mixed-sugar (B) fermentation systems at different pH levels. Data are presented as mean ± SE (n = 3 independent fermentation batches).
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Figure 3. L-LA production in single-sugar (A) and mixed-sugar (B) fermentation systems at different temperature levels. Data are presented as mean ± SE (n = 3 independent fermentation batches).
Figure 3. L-LA production in single-sugar (A) and mixed-sugar (B) fermentation systems at different temperature levels. Data are presented as mean ± SE (n = 3 independent fermentation batches).
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Microorganisms 14 00417 g005
Figure 5. Changes in Sugar Concentrations during Fermentation with Single Sugars and Corresponding Mixed-Sugar Systems: (A) Glucose- or xylose-based single-sugar and the G/X mixed-sugar system; (B) Glucose- or cellobiose-based single-sugar and the G/C mixed-sugar system; (C) Glucose-, cellobiose-, or xylose-based single-sugar and the G/C/X mixed-sugar system; (D) Cellobiose- or xylose-based single-sugar and the C/X mixed-sugar system. Data are presented as mean ± SE (n = 3 independent fermentation batches).
Figure 5. Changes in Sugar Concentrations during Fermentation with Single Sugars and Corresponding Mixed-Sugar Systems: (A) Glucose- or xylose-based single-sugar and the G/X mixed-sugar system; (B) Glucose- or cellobiose-based single-sugar and the G/C mixed-sugar system; (C) Glucose-, cellobiose-, or xylose-based single-sugar and the G/C/X mixed-sugar system; (D) Cellobiose- or xylose-based single-sugar and the C/X mixed-sugar system. Data are presented as mean ± SE (n = 3 independent fermentation batches).
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Microorganisms 14 00417 g006
Figure 6. Changes in L-LA Concentration during Fermentation with Single Sugars and Corresponding Mixed-Sugar Systems: (A) Glucose- or xylose-based single-sugar and the G/X mixed-sugar system; (B) Glucose- or cellobiose-based single-sugar and the G/C mixed-sugar system; (C) Cellobiose- or xylose-based single-sugar and the C/X mixed-sugar system. Data are presented as mean ± SE (n = 3 independent fermentation batches).
Figure 6. Changes in L-LA Concentration during Fermentation with Single Sugars and Corresponding Mixed-Sugar Systems: (A) Glucose- or xylose-based single-sugar and the G/X mixed-sugar system; (B) Glucose- or cellobiose-based single-sugar and the G/C mixed-sugar system; (C) Cellobiose- or xylose-based single-sugar and the C/X mixed-sugar system. Data are presented as mean ± SE (n = 3 independent fermentation batches).
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Table 2. Enzyme activity of β-glucosidase, xylokinase, and lactate dehydrogenase at 72 h in a mixed-sugar fermentation system. Data are presented as mean ± SE (n = 3 independent fermentation batches).
Table 2. Enzyme activity of β-glucosidase, xylokinase, and lactate dehydrogenase at 72 h in a mixed-sugar fermentation system. Data are presented as mean ± SE (n = 3 independent fermentation batches).
Mixed-Sugar Systemsβ–glucosidase (U/L)Xylulokinase (U/L)Lactate Dehydrogenase (U/L)
Types of
Enzymes
C/X117.8 ± 0.57678.4 ± 2.49108.5 ± 1.30
G/C112.7 ± 0.76ND72.4 ± 1.26
G/C/X110.0 ± 0.92574.9 ± 2.9792.1 ± 1.27
G/XND520.1 ± 2.5879.1 ± 1.02
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MDPI and ACS Style

Sun, Y.; Wang, J.; Deng, T.; Wang, S.; Liu, J.; Wang, X.; Wang, Q. Investigation of the Carbon Catabolite Repression Mechanism in L-Lactic Acid Fermentation from Mixed Sugars by Bacillus coagulans DSM 2314. Microorganisms 2026, 14, 417. https://doi.org/10.3390/microorganisms14020417

AMA Style

Sun Y, Wang J, Deng T, Wang S, Liu J, Wang X, Wang Q. Investigation of the Carbon Catabolite Repression Mechanism in L-Lactic Acid Fermentation from Mixed Sugars by Bacillus coagulans DSM 2314. Microorganisms. 2026; 14(2):417. https://doi.org/10.3390/microorganisms14020417

Chicago/Turabian Style

Sun, Yinan, Juan Wang, Tong Deng, Shijie Wang, Jianguo Liu, Xiaona Wang, and Qunhui Wang. 2026. "Investigation of the Carbon Catabolite Repression Mechanism in L-Lactic Acid Fermentation from Mixed Sugars by Bacillus coagulans DSM 2314" Microorganisms 14, no. 2: 417. https://doi.org/10.3390/microorganisms14020417

APA Style

Sun, Y., Wang, J., Deng, T., Wang, S., Liu, J., Wang, X., & Wang, Q. (2026). Investigation of the Carbon Catabolite Repression Mechanism in L-Lactic Acid Fermentation from Mixed Sugars by Bacillus coagulans DSM 2314. Microorganisms, 14(2), 417. https://doi.org/10.3390/microorganisms14020417

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