Lactic acid production from lignocellulosic biomass by a co-culture of Enterococcus casseliflavus CW3-1 and Paucilactobacillus vaccinostercus MQ2

Zayas Garriga, A., Zhang, X., Geng, A., Xie, R., and Zhu, D. (2026). "Lactic acid production from lignocellulosic biomass by a co-culture of Enterococcus casseliflavus CW3-1 and Paucilactobacillus vaccinostercus MQ2," BioResources 21(4), 10339–10352.

Abstract

Lactic acid (LA) is a growing demand platform chemical for food, pharmaceutical, and bioplastic applications. Lignocellulose offers an alternative to edible sugars as a substrate. However, its microbial conversion of mixed hexose/pentose sugars remains challenging. In this study, two new lactic acid bacteria with distinct sugar utilization profiles were isolated and evaluated for their LA production capability using a series of substrates. Enterococcus casseliflavus CW3-1 exhibited a partially homofermentative behavior and was capable of assimilating both hexose and pentose sugars. In contrast, Paucilactobacillus vaccinostercus MQ2 showed a heterofermentative manner and was a typical pentose-preferring bacterium, which did not metabolize glucose. In the simultaneous saccharification and fermentation (SSF) of alkali-pretreated corn stover and sugarcane bagasse with a commercial cellulase, co-cultivation of the two strains significantly outperformed the mono-cultures and the LA production from co-culture was slightly higher than the sum of the production from the two monocultures. Fermentation results from two types of biomasses showed that the maximum LA yield reached 0.78±0.03 g/g substrate; however, increasing the substrate concentration led to a decrease in yield. These results demonstrate the superiority of using microbial consortia for sustainable LA biorefining from lignocellulosic biomass.

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**Lactic Acid Production from Lignocellulosic Biomass by a Co-culture of Enterococcus casseliflavus CW3-1 and Paucilactobacillus vaccinostercus MQ2**

Anaiza Zayas Garriga, Xiang Zhang, Alei Geng,* Rongrong Xie, and Daochen Zhu

DOI: 10.15376/biores.21.4.10339-10352

Keywords: Lactic acid; Lignocellulosic biomass; Co-culture; Simultaneous saccharification and fermentation; Corn stover; Sugarcane bagasse

Contact information: Biofuels Institute, School of Environment and Safety Engineering, Jiangsu University, Zhenjiang, Jiangsu 212013 China; *Corresponding author: [email protected]

Graphical Abstract

INTRODUCTION

Lactic acid (LA) is a high-value organic acid that is widely used in the food, pharmaceutical, cosmetic, and polymer industries. It serves as a precursor to polylactic acid (PLA), a biodegradable bioplastic whose global demand has increased substantially due to environmental policies aimed at reducing petroleum-based plastics and promoting sustainable materials (Balasubramanian et al. 2024). As a result, LA has been established as a major platform chemical derived from biomass (Abdel-Rahman et al. 2013; Martinez et al. 2013). Currently, around 90% of LA is produced by microbial fermentation, as this route is considered more sustainable (Abdel-Rahman et al. 2013; Ojo and de Smidt 2023; Brobbey et al. 2024).

However, the reliance on edible sugars creates competition with food supplies, which has spurred research toward utilizing lignocellulosic biomass and agro-industrial wastes as feedstocks (John et al. 2007; Yankov 2022; Montipó et al. 2024). These waste-based bioprocesses are helping to advance a low-carbon bioeconomy (Blasi et al. 2023; Yue and Zhang 2023).

The carbohydrates in lignocellulose mainly consist of hexoses (e.g., glucose) and pentoses (e.g., xylose), both of which can serve as fermentation substrates for LA production (John et al. 2007; Agrawal and Kumar 2023). A range of microbial strains capable of utilizing lignocellulosic hydrolysates have been employed for LA fermentation, including wild lactic acid bacteria (LAB), such as Lactobacillus (Gänzle and Follador 2012) and Pediococcus spp. (Diguță et al. 2020), and metabolically engineered microorganisms (Mazzoli 2020). These strains enable the utilization of either hexoses or pentoses through diverse metabolic pathways; however, most strains cannot simultaneously utilize these two types of sugars, because of the so-called carbon catabolite repression (Gao et al. 2019; Flores et al. 2020). Strategies such as simultaneous saccharification and fermentation (SSF), staged fermentation, and microbial co-culture are employed to enhance substrate conversion rates and product yields (Sun et al. 2021; Blasi et al. 2023). Nevertheless, the process still faces key challenges, including tolerance against hydrolysis inhibitors, carbon catabolite repression, and product inhibition (Sun et al. 2021; Yankov 2022).

Microbial co-cultures represent an innovative approach to overcome carbon catabolite repression and enable simultaneous consumption of glucose and xylose from lignocellulosic biomass, a critical step toward economical biofuel and chemical production (Flores et al. 2019, 2020). This strategy employs division of labor, using engineered specialist bacterial strains that are selectively unable to metabolize either glucose or xylose (Flores et al. 2020), or employing natural co-fermenting bacteria, such as Enterococcus mundtii and Lactobacillus rhamnosus, which inherently participate in hexose and pentose utilization (Klongklaew et al. 2021, 2023). Co-culture systems have been found to increase the LA or ethanol productivity by optimizing initial inoculation ratios and medium compositions (Flores et al. 2019, 2020). Although individual performances might vary by organisms and conditions, co-culture fermentation strategies allow the simultaneous fermentation of multiple substrates (Taniguchi et al. 2005). Unlike conventional monocultures, which are often limited by substrate/product inhibition and toxic metabolite accumulation (Sun et al. 2021; Yankov 2022), synthetic microbial communities offer greater robustness and flexibility to adapt to variable substrate compositions. This positions them as promising platforms for integrated biorefinery processes (Yue and Zhang 2023). However, there is still a lack of efficient hexose- or pentose-preferring LAB strains.

In this study, two new hexose- or pentose-preferring bacteria (Enterococcus casseliflavus CW3-1 and Paucilactobacillus vaccinostercus MQ2, respectively) were isolated and their potential for LA production from lignocellulosic biomass was investigated using either a single strain or their co-cultures. Specific objectives include: (1) evaluating the effects of different carbon sources on LA yields by these strains; and (2) comparing the LA producing capability in the SSF strategy between the above-mentioned strains and their co-cultures. The findings might serve as an important reference for future co-culture research.

EXPERIMENTAL

Sample Collection and Media Preparation

A modified de Man, Rogosa and Sharpe (mMRS) medium was used for the fermentation. The composition of the medium was as follows (g/L): tryptone (1), yeast extract (2), beef extract (2), xylose (10), K₂HPO₄·3H₂O (2.9), KH₂PO₄ (1.5), NH₄Cl (0.5), MgCl₂·6H₂O (0.2), CaCl₂·2H₂O (0.075), and MnSO₄ (0.05). Where indicated, the carbon source in this medium was replaced with alternative carbohydrates. Unless otherwise stated, calcium carbonate powder was supplemented as a pH buffering agent at 10 g/L. The medium was boiled for 5 min and was then flushed with nitrogen for 5 min to remove dissolved oxygen and create anaerobic conditions. L-cysteine monohydrochloride was subsequently added as a reducing agent to a final concentration of 0.5 g/L. For the preparation of the solid medium, agar was supplemented to reach a final concentration of 15 g/L. Cow manure was collected from a local farm in Zhenjiang, China. A common local Aedes mosquito was captured using an insect net in the campus of Jiangsu University, Zhenjiang, China. Xylan was purchased from J&K Chemical, and other carbohydrates were obtained from Macklin, Shanghai, China. All the carbohydrates were of biotechnology grade with purities >99%, except for xylan (>95%).

Strain Isolation and Identification

Samples (cow manure or the mosquito) were supplemented with 1 mL phosphate-buffered saline (PBS) and homogenized using a pestle in Eppendorf tubes. The homogenized samples were then transferred into the mMRS medium and were incubated at 37 °C under anaerobic conditions overnight. Cultures were subsequently spread onto mMRS agar plates, sealed in an AnaeroPack system (Mitsui Gas Co., Japan), and incubated at 37 °C for 24 to 48 h under anaerobic conditions. Well-isolated colonies were streaked repeatedly to ensure the purity of the isolates. Purified strains were stored at −80 °C in 20% (v/v) sterile glycerol. In addition, CW3-1 and MQ2 were deposited at Guangdong Microbial Culture Collection Center (GDMCC) under accession numbers 1.6669 and 1.6670, respectively.

To characterize the bacterial strains, morphological examination was performed using scanning electron microscopy (SEM) according to Deng et al (2021). Species identification was performed through 16S rDNA sequencing. Briefly, the 16S rDNA fragment was amplified by conventional polymerase chain reaction (PCR) employing the universal primer set Eubac27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and Eubac1492R (5′-GGTTACCTTGTTACGACTT-3′). The PCR products were sequenced by Sangon Biotech (Shanghai, China). The obtained 16S rDNA sequence was searched against the NR database of the National Center for Biotechnology Information (NCBI) for homologous sequences using the Basic Local Alignment Search Tool (BLAST).

Effect of Various Carbon Sources on the LA Production

The effects of various carbon sources (each at 25 g/L) on LA production in mMRS medium were tested, including glucose, arabinose, xylose, lactose, sucrose, starch, and xylan. Briefly, fresh one-day-old cultures were inoculated at 1% (v/v) into 15 mL of medium contained in anaerobic tubes. Fermentations were carried out at 37 °C and 180 rpm for 48 h. Finally, 1 mL of culture samples were collected and centrifuged at 10000 rpm and room temperature for 1 min. The resulting supernatant was frozen and stored for subsequent LA quantification. Meanwhile, growth behavior of the strains on either glucose or xylose was monitored over a 72 h period in the mMRS medium. Optical density was measured at 600 nm (OD₆₀₀) every 8 h.

LA Production by Mono-culture or Co-culture on Pretreated Biomass

Corn stover as well as sugarcane bagasse (< 40 mesh) was subjected to alkaline pretreatment with 7% (w/w) NaOH at 86 °C for 1 h, at a solid-to-liquid ratio of 1:7 (w/v), according to Zhang et al. (2015) . After pretreatment, the solid residue was washed with deionized water until a neutral pH was reached. The pretreated biomass was then used as carbon source to prepare the fermentation medium. LA production was carried out by simultaneous saccharification and fermentation (SSF), using individual cultures of CW3-1, MQ2, or their co-culture, in the presence of a commercial cellulase enzyme, Cellic® CTec2 (Sigma, Shanghai, China). A bacterial inoculum of 1% (v/v) and a constant enzyme loading of 20 mg of cellulase per gram of pretreated biomass were used in all experiments. For 20 and 50 g/L of pretreated biomass, the SSF were carried out within 72 h. For the higher substrate concentrations of 100 g/L, the fermentations were extended to 120 h. Calcium carbonate was added in concentrations proportional to the substrate (1 g/g substrate) for pH control during fermentation. Samples were taken at 72 and 120 h for the quantification of LA. Unless otherwise specified, all experiments were conducted in triplicate.

Chemical Analysis

The concentrations of LA were analyzed using a high‑performance liquid chromatography (HPLC) system (Shimadzu, Japan) equipped with a refractive index detector and a Bio‑Rad HPX‑87H column (Geng et al. 2019). Data are presented as mean ± standard deviation from three independent replicates. The LA contents were quantified using a standard curve constructed from lithium acetate solutions of varying concentrations. Statistical analysis was performed using Student’s t-test in Origin 2021 software (OriginLab, Northampton, MA, USA).

RESULTS

Isolation and Identification of Strain CW3-1 and MQ2

Strain CW3-1 and MQ2 were isolated from cow manure and a mosquito, respectively, and were identified by their morphological characteristics and 16S rDNA sequences. Based on BLAST analysis, the sequence obtained for strain CW3-1 (Genbank accession: PX899135), with a length of 1233 bp, showed 99.76% identity to three Enterococci (i.e., Enterococcus gallinarum, Enterococcus casseliflavus, and Enterococcus innesii), which share identical 16S rDNA sequences. Strain CW3-1 appeared as typical cocci, approximately 0.6 μm in diameter, and formed clusters as evaluated using SEM (Fig. 1A). CW3-1 also seemed to produce a pigment, as the culture showed a light yellow color, which matched well with the property of Enterococcus casseliflavus (Yoshino 2023). Hence, CW3-1 was assigned as E. casseliflavus CW3-1. Meanwhile, the 16S rDNA sequence of MQ2 (Genbank accession: PX899137;1428 bp) showed 99.8% identity to Paucilactobacillus vaccinostercus, enabling a trustworthy species-level assignment. MQ2 was rod-shaped and approximately 1.2 μm in length and 0.3 μm in width (Fig. 1B). In addition, MQ2 was unable to grow on glucose medium, which also matched well with the property of P. vaccinostercus (Okada et al. 1979).

Fig. 1. SEM micrographs of strains E. casseliflavus CW3-1 at 2000× magnification (A) and P. vaccinostercus MQ2 at 5000× magnification (B)

Effect of Different Carbon Sources on LA Production by CW3-1 or MQ2

Bacteria typically exhibit variable metabolic responses to different carbon sources, including hexoses, pentoses, disaccharides, and polysaccharides. The effect of these substrates on the LA production by E. casseliflavus CW3-1 and P. vaccinostercus MQ2 were evaluated. As shown in Fig. 2, LA production showed significant variation across the series of substrates at a concentration of 25 g/L.

Fig. 2. Effect of different carbon sources on the acid production by E. casseliflavus CW3-1 (A) or P. vaccinostercus MQ2 (B). Cultures were incubated at 37 °C for one day at a substrate loading of 25 g/L in a working volume of 15 mL (n = 3)

CW3-1 is a hexose-preferring LAB, as it utilized much more glucose (18.02±0.78 g/L) than xylose (5.60±0.67 g/L) as well as arabinose (10.32±0.79 g/L) within 24 h. It also produced more LA from glucose (8.28±0.50 g/L) than those from xylose as well as arabinose (5.05±0.79 and 8.18±0.45 g/L, respectively). For the disaccharide substrates, CW3-1 metabolized more sucrose than lactose, with an LA production of 6.73±0.41 g/L and 1.70±0.26 g/L, respectively. Only a low level of acetic acid was generated during the fermentation process. The final molar ratios of LA to acetic acid were 69:1 in the glucose culture and approximately 7:1 in the xylose as well as arabinose culture. Nevertheless, the LA yield from utilized glucose (0.46±0.02 g/g) was much lower than those from utilized xylose (0.90±0.03 g/g) and arabinose (0.79±0.02 g/g), probably due to the formation of intracellular polymers such as Polyhydroxyalkanoates, which has been reported in some Enterococcus (Bhuwal et al. 2013). In contrast, MQ2 is a pentose-preferring LAB, as it could not utilize glucose but was capable of nearly fully utilizing the pentose substrates within 24 h, and producing 11.89±0.33 g/L (0.47 ±0.01 g/g utilized substrate) and 10.47±0.45 g/L (0.42±0.02 g/g utilized substrate) LA from xylose and arabinose, respectively. Both strains gave LA at very low levels (< 1.7 g/L) when growing on the tested two kinds of polysaccharides (starch and xylan). Neither ethanol nor other common fermentation products were detected. In comparison, the LA production was approximately 12.1 g/L from 25 g/L of xylose by a control strain, Lactobacillus pentosus ATCC 8041, which is also a typical pentose-preferring bacterium. The LA producing capability of strain MQ2 was thus comparable to L. pentosus ATCC 8041 from the common pentose sugar, xylose.

The growth behavior of strains MQ2 and CW3-1 was monitored for 72 h in mMRS medium supplemented with specific sugars at 25 g/L. Both strains grew rapidly within the first 16 h, after which their growth slowed down and gradually reached a plateau (Fig. 3). MQ2 seemed to grow much faster than CW3-1 on xylose substrate, as it had a higher slope in the growth curves during the first 16 h and it also gave a significantly higher OD value than that of CW3-1at 16 h in both glucose and xylose medium, reaching a maximum of 6.5±0.4 (p < 0.01, n=3). Meanwhile, the growth rates of CW3-1 on glucose and xylose substrates were quite similar, with the maximum OD₆₀₀ of about 4.0±0.7. The growth curve of MQ2 on glucose was omitted because it does not metabolize glucose. These results indicated that MQ2 is a pentose-preferring fermenter.

Fig. 3. Growth curve of E. casseliflavus CW3-1 and P. vaccinostercus MQ2 in 25 g/L of xylose- or glucose-mMRS media at 37 °C in a working volume of 15 mL (n = 3)

LA Production by Mono-culture or Co-Culture of CW3-1 and MQ2 on Pretreated Corn Stover in the Presence of CTec2

The LA production from alkali-pretreated corn stover was assessed via SSF using individual cultures of CW3-1 and MQ2, as well as their co-culture, in the presence of a commercial cellulase, CTec2. At a concentration of 20 g/L of substrate, the co-culture produced 15.53±0.61 g/L of LA after 72 h, significantly exceeding the values obtained by the CW3-1 (9.63±0.19 g/L) and MQ2 (5.50±0.73 g/L) mono-cultures (p < 0.01, n=3; Fig. 4A). Neither acetic acid nor ethanol was detected in the CW3-1 mono-culture, while some acetic acid was accumulated in the MQ2 mono-culture as well as the co-culture at a concentration of 1.52±0.05 and 1.20±0.11 g/L, respectively. In addition, both the residual glucose (0.47±0.05 g/L) and xylose (0.10±0.01 g/L) concentration in the co-culture were significantly (p < 0.01, n=3) lower than those of the mono-cultures (3.23±0.32 and 1.19±0.15 g/L, respectively, for CW3-1; and 7.15±0.61 and 0.65±0.04 g/L, respectively, for MQ2; Fig. 4B), suggesting the superiority of the co-culture in substrate assimilation.

Fig. 4. LA production (A) and the residual free sugars (B) in the SSF of pretreated corn stover by E. casseliflavus CW3-1, P. vaccinostercus MQ2, and their co-culture. Fermentation was carried out at 37 °C for 72 h in the presence of the CTec2 cellulase at 20 mg/g substrates and in a working volume of 15 mL (n = 3). The initial substrate loading was 20 g/L, and calcium carbonate was supplemented at 1 g/g of substrate for pH neutralization.

Fig. 5. SSF of pretreated corn stover for LA production by the co-culture of E. casseliflavus CW3-1 and P. vaccinostercus MQ2. Fermentation was carried out at 37 °C in the presence of the CTec2 cellulase at 20 mg/g substrates and in a working volume of 15 mL (n = 3). The calcium carbonate was supplemented at 1 g/g of substrate for pH neutralization. Fermentation was carried out for 3 days for the substrate loadings of 20 and 50 g/L, and 5 days for the 100 g/L of substrates.

Furthermore, the co-culture still maintained effective fermentative capacity at high substrate loadings. Overall, the acid production increased as the substrate loadings were raised from 20 to 100 g/L. The LA production increased from 15.53±0.61 to 26.48±0.18 and 43.32±0.83 g/L as the substrate loadings of corn stover were increased from 20 to 50 and 100 g/L, respectively (Fig. 5A). Meanwhile, the acetic acid production also increased from 1.20±0.11 to 3.33±0.35 and 5.96±0.68 g/L, respectively. Similarly, the acid production from pretreated sugarcane bagasse by the co-culture of CW3-1 and MQ2 were very close to those of corn stover, with a maximum production of LA at 42.09±0.41 g/L and acetic acid at 8.80±0.63 g/L under the same condition (Fig. 5B). However, the LA yield was quite high at the low substrate loading (20 g/L) but gradually deceased as the loadings were increased. The LA yield reached 0.78±0.03 and 0.62±0.04 g/g biomass for 20 g/L of corn stover and sugarcane bagasse, respectively. The value decreased to 0.53±0.01 and 0.43±0.01 g/g biomass for 50 g/L of corn stover and sugarcane bagasse, respectively, and maintained at about 0.43±0.01 g/g biomass for 100 g/L of substrates.

DISCUSSION

The newly isolated strains E. casseliflavus CW3-1 and P. vaccinostercus MQ2 exhibited a broad substrate spectrum, capable of metabolizing not only several common monosaccharides and disaccharides but also some polysaccharides such as starch and xylan. This metabolic versatility distinguishes them from many reported LAB with more limited substrate ranges, particularly regarding the direct utilization of complex polysaccharides (Gänzle and Follador 2012). This characteristic significantly expands their potential application scenarios for LA production, especially in processes utilizing unrefined or non-food biomass feedstocks containing starchy or hemicellulosic components, where pretreatment or enzymatic hydrolysis steps could potentially be simplified.

The establishment of a stable co-culture or artificial microbial consortium often relies on niche differentiation, with carbon source specialization being a key factor. However, due to the lack of efficient and substrate-specific microbes, most consortia for LA production were not composed of strictly hexose- and pentose-specific bacteria (Flores et al. 2020; Klongklaew et al. 2021, 2023). In this study, MQ2 demonstrated rapid growth on xylose but negligible growth on glucose, while CW3-1 efficiently consumed glucose. This complementary carbon utilization profile formed a stable ecological basis for co-cultivation (Geng et al. 2024). Consequently, this co-culture is particularly suited for the simultaneous fermentation of hexoses and pentoses present in lignocellulosic hydrolysates. By avoiding direct competition for the same primary carbon sources, the two strains could coexist stably, enabling simultaneous conversion of hexoses and pentoses.

Division of labor within microbial consortia is a recognized strategy to enhance metabolic efficiency and product yields. The current study’s SSF experiments clearly demonstrated that the co-culture of CW3-1 and MQ2 achieved a significantly higher LA production compared to either mono-culture (Fig. 4A). This synergistic effect can be partially attributed to their complementary roles: the simultaneous and more thorough consumption of glucose (from cellulose hydrolysis) and xylose (from hemicellulose hydrolysis), as significantly lower residual free sugars were detected in the residual broth (Fig. 4B). The similar results have also been observed in other reports. For instance, Klongklaew et al. (2021, 2023) obtained 59 g/L of LA from mixed sugars by a co-culture, compared to 15.51 g/L by a monoculture. This concurrent sugar uptake likely alleviates product inhibition on hydrolytic enzymes (e.g., cellulases and xylanases) by preventing the accumulation of free sugars, which are known inhibitors of these enzymes (Flores et al. 2019). The reduced feedback inhibition on hydrolysis, coupled with efficient parallel fermentation pathways, ultimately led to the observed increase in overall LA yield.

The metabolic pathway is a key factor affecting the yield of LA from the fermentation of hexoses and pentoses. The LA yield of the homofermentative pathway is generally higher than that of the heterofermentative pathway, because the latter is accompanied by the generation of by-products such as acetate, ethanol, and CO₂, which reduces the carbon conversion efficiency. Notably, some homofermentative microorganisms can convert pentoses into hexose metabolic intermediates, such as fructose-6-phosphate, via the pentose phosphate pathway, thereby directing pentose metabolism into the efficient homolactic fermentation route (Cubas-Cano et al. 2018). This conversion mechanism not only expands the substrate utilization range but also avoids the undesirable side reactions of heterofermentation, making it a decisive factor in improving the lactic acid yield from pentose fermentation. In this work, CW3-1 showed high ratios of LA to acetic acid (69:1) in the fermentation of glucose, suggesting that it could metabolize glucose nearly in a homofermentative manner, and it was probably capable of partially directing pentose into the pentose phosphate pathway, thus resulting in a relative high ratio of LA to acetic acid at approximately 7:1. In comparison, MQ2 exhibited a typical heterofermentative pattern, as the molar ratio of LA to acetic acid from pentose metabolism was close to 1:1. Nevertheless, the heterofermentative fermentation generates more ATP than the homofermentative pathway (Cubas-Cano et al. 2018), which might explain the robust growth of MQ2 compared to CW3-1 (Fig. 3).

High-solid fermentation remains challenging due to a series of factors. It was reported that the inhibiting chemicals (from biomass pretreatment), end-product inhibition, as well as restricts enzyme adsorption are primary obstacles for lignocellulosic LA production (Cao et al. 2026). In this study, the LA yield was quite high under conditions with low biomass loadings (20 g/L biomass; 0.62±0.04 to 0.78±0.03 g/g biomass). However, a further increase to 50 and 100 g/L of biomass led to a notable drop in yield to 0.42±0.01 to 0.53±0.01 g/g biomass, suggesting that inhibition mechanisms became significant at this concentration. The yields obtained in this work (0.42 to 0.78 g/g) are comparable to those reported in the literature for LA production from various types of biomass (as summarized in Table 1), confirming the competitive potential of the CW3-1/MQ2 consortium. Future work could focus on strain mutation to further improve productivity at high substrate loadings.

Table 1. Literature Reported for LA Production by Individual Strains or Co-cultures

CONCLUSIONS

Enterococcus casseliflavus CW3-1 and Enterococcus casseliflavus CW3-1 are hexose- and pentose-preferring LAB, respectively. E. casseliflavus CW3-1 exhibited a partially homofermentative behavior, and the products showed a high LA to ac etic acid ratio even in the fermentation of pentoses. Meanwhile, P. vaccinostercus MQ2 exclusively utilized pentoses and is a typical heterofermentative bacterium.
The co-culture of CW3-1 and MQ2 confirmed that employing bacteria with distinct substrate preferences is an effective strategy for constructing artificial microbial consortia. Co-culturing of CW3-1 and MQ2 resulted in LA production slightly higher than the sum of the individual monoculture production. The co-culture also demonstrated superior substrate assimilation compared to the mono-cultures. The SSF of pretreated corn stover for LA production by the co-culture of CW3-1 and MQ2 reached up to 0.78 g /g biomass.
Artificial microbial consortia with complementary hexose- and pentose-utilizing strains shed light on industrial lignocellulosic biorefineries. Future scale-up efforts should focus on maintaining the optimal spatial and temporal distribution of these two populations to ensure sustained cooperative performance in large-volume fermenters.

ACKNOWLEDGMENTS

This work was financially supported by the Zhenjiang key research and development project (CN2023001), and the Drug Efficacy and Health Risk Assessment Key Laboratory of ZhenJiang (SS2024006).

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Article submitted: June 27, 2026; Peer review completed: August 1, 2026; Revised version received: August 12, 2026; Accepted: August 14, 2026; Published: September 4, 2026.

DOI: 10.15376/biores.21.4.10339-10352