Development of a self-flocculating <i>Zymomonas mobilis</i> biocatalyst for efficient bioethanol production from lignocellulosic biomass

Won-Gyeom Jung; Jung-In Lim; Seong Seok Choi; Hye-Sook Heo; Mi-Seon Jang; Yiu Fai Tsang; Young Jae Jeon

doi:10.4491/eer.2025.223

Environ Eng Res > Volume 31(1); 2026 > Article

Jung, Lim, Choi, Heo, Jang, Tsang, and Jeon: Development of a self-flocculating Zymomonas mobilis biocatalyst for efficient bioethanol production from lignocellulosic biomass

Research

Environmental Engineering Research 2026; 31(1): 250223.

Published online: May 9, 2025

DOI: https://doi.org/10.4491/eer.2025.223

Development of a self-flocculating Zymomonas mobilis biocatalyst for efficient bioethanol production from lignocellulosic biomass

Won-Gyeom Jung^1,^*, Jung-In Lim^1,^*, Seong Seok Choi^1,^2,^*, Hye-Sook Heo¹, Mi-Seon Jang¹, Yiu Fai Tsang³, Young Jae Jeon^1,^4,^†

¹Department of Microbiology, Pukyong National University, Busan 48513, Republic of Korea

²Industry-University Cooperation Foundation, Pukyong National University, Busan 48513, Republic of Korea

³Department of Science and Environmental Studies and State Key Laboratory in Marine Pollution, The Education University of Hong Kong, Tai po, New Territories 999077, Hong Kong Special Administrative Region

⁴School of Marine and Fisheries Life Science, Pukyong National University, Busan 48513, Republic of Korea

^†Corresponding author: E-mail: youngjaejeon@pknu.ac.kr, Tel: +82-51-629-5612 Fax: +82-51-629-5619, ORCID: 0000-0002-4867-9812

* These authors contributed equally to this work.

Received April 24, 2025 Revised May 7, 2025 Accepted May 8, 2025

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Flocculation represents a promising biological strategy to enhance biomass recovery and improve process efficiency in bioethanol production. In this study, we engineered a self-flocculating strain of Zymomonas mobilis, designated ZAM2, by inactivating the EAL domain (phosphodiesterase region) of the regulatory gene ZMO_1055. This targeted genetic modification led to enhanced cell aggregation and improved stress tolerance against common fermentation inhibitors, including acetic acid, furfural, and vanillin, which are typically generated during lignocellulosic biomass pretreatment. Compared to its parental strain ZM401, ZAM2 accumulated higher levels of intracellular c-di-GMP, exhibited reduced PDE activity, and upregulated cellulose biosynthesis genes. These changes contributed to enlarged floc size and superior fermentation performance. Notably, ZAM2 completed glucose consumption and ethanol production more rapidly than ZM401 under inhibitor-rich conditions, resulting in increased ethanol productivity. Our results demonstrate a rational genetic strategy for enhancing microbial robustness and process efficiency, offering a viable approach for sustainable, high-gravity bioethanol fermentation from lignocellulosic feedstocks.

Keywords: c-di-GMP signaling, EAL domain inactivation, Lignocellulosic ethanol fermentation, Self-flocculation, Zymomonas mobilis

Graphical Abstract

Keywords: c-di-GMP signaling, EAL domain inactivation, Lignocellulosic ethanol fermentation, Self-flocculation, Zymomonas mobilis

1. Introduction

The growing interest in sustainable and renewable energy sources has led to extensive research on the production of biofuels from various types of biomass [1–2]. Among these, lignocellulosic biomass has attracted particular attention due to its abundance, low cost, and non-competition with food resources. In addition to being renewable, lignocellulosic biomass offers notable environmental benefits by enabling the valorization of agricultural and forestry residues that are otherwise discarded, thereby reducing environmental pollution, lowering greenhouse gas emissions, and contributing to waste-to-energy strategies. Consequently, the increasing demand for sustainable and renewable energy has driven the development of microbial platforms capable of efficiently converting lignocellulosic biomass into biofuels [3–4]. Among these, Zymomonas mobilis, a Gram-negative facultative anaerobe, stands out as a promising ethanologenic bacterium due to its high ethanol yield, rapid sugar uptake, and minimal biomass formation via the Entner-Doudoroff Pathway [5–6]. Compared to conventional yeast-based fermentations, Z. mobilis offers superior theoretical conversion efficiency and reduced energy input, making it particularly suitable for second-generation bioethanol production using lignocellulosic feedstocks [7–9]. Furthermore, its nitrogen-fixing ability contributes to cost-effective fermentation by reducing the need for external nitrogen supplementation [10–11].

Despite these advantages, industrial application of Z. mobilis is often hindered by the presence of fermentation inhibitors such as acetic acid, furfural, and vanillin, which are generated during lignocellulosic biomass pretreatment and can negatively impact microbial growth and ethanol productivity [12–15]. One promising approach to address these challenges is microbial flocculation, the self-aggregation of cells into settleable floc [16]. Flocculating strains are known to exhibit enhanced stress tolerance [17–18], enable simplified downstream processing, and facilitate semi-continuous or cell-recycling fermentation systems [12, 19]. The flocculating strain Z. mobilis ZM401 has been shown to produce extracellular cellulose that promotes aggregation, a process regulated by intracellular levels of cyclic-di-GMP (c-di-GMP), a ubiquitous bacterial second messenger [20–23].

Intracellular c-di-GMP levels are governed by the activity of diguanylate cyclases (DGCs) and phosphodiesterases (PDEs), which contain GGDEF and EAL domains respectively [24–27]. The gene ZMO_1055, encoding a bifunctional GGDEF-EAL protein in Z. mobilis, has been implicated in the modulation of c-di-GMP turnover and flocculation behavior [20, 21, 28–30].

In this study, we genetically inactivated the EAL domain of ZMO_1055 to disrupt its PDE activity and promote the accumulation of intracellular c-di-GMP. The resulting mutant strain, ZAM2, demonstrated enhanced self-flocculation, improved tolerance to fermentation inhibitors, and increased ethanol productivity under high-glucose and stress-inducing fermentation conditions. These findings suggest that rational modulation of intracellular signaling pathways can serve as an effective strategy for improving microbial robustness and bioethanol production.

Our approach employs a targeted genetic strategy to inactivate the EAL domain of a specific regulatory gene, enabling precise control of c-di-GMP levels and more predictable phenotypic outcomes. This strategy not only provides mechanistic insights into flocculation regulation but also offers a more rational and reproducible framework for strain engineering. By advancing the understanding of flocculation regulation in Z. mobilis, this work contributes to the development of robust microbial platforms for sustainable bioenergy production from lignocellulosic waste, in line with the goals of environmental sustainability and the circular bioeconomy.

2. Materials and Methods

2.1. Strains and Media

The Z. mobilis strains used in this study—wild-type ZM4 (ATCC 31821), its flocculating mutant ZM401 (ATCC 31822), and the engineered knockout strain ZAM2—were cultivated at 30°C in rich medium (RM) [31] for flocculation and ethanol fermentation experiments. RM contained glucose (20 g/L), yeast extract (10 g/L), KH₂PO₄ (2 g/L), (NH₄)₂SO₄ (1 g/L), and MgSO₄ (1 g/L). Recombinant strains were selected using 100 μg/mL chloramphenicol (Sigma-Aldrich, USA).

Escherichia coli strains used for plasmid construction were grown in Luria-Bertani (LB) medium [32], consisting of tryptone (10 g/L), yeast extract (5 g/L), and NaCl (10 g/L), and supplemented with 50 μg/mL ampicillin (Sigma-Aldrich, USA) when necessary to maintain plasmid selection. All strains were stored at −70°C in 80% (v/v) glycerol for long-term preservation. Strain and plasmid details are provided in supplementary materials (Table S1).

2.2. Genetic Engineering for Enhanced Flocculation in Z. mobilis

2.2.1. Cloning of the EAL domain region for targeted gene disruption

To construct a knockout vector targeting the phosphodiesterase-encoding EAL domain of ZMO_1055, genomic DNA and plasmid DNA were extracted and manipulated using standard molecular biology protocols [33–34]. PCR products were purified from agarose gels, followed by restriction digestion and ligation to assemble the pRBC::pde::cat plasmid. The ligation product was subsequently introduced into E. coli for propagation and sequence confirmation using the Sanger method. Detailed primer sequences, PCR conditions, and reagent compositions are provided in supplementary materials (Table S2, S3).

2.2.2. Assembly of EAL knockout vector

To enhance the self-flocculating phenotype in Z. mobilis, a gene knockout strategy targeting the EAL domain of ZMO_1055 was employed. Gene disruption was performed via homologous recombination using the suicide vector pRBC::pde::cat constructed in E. coli DH5α. The ZMO_1055 region, including flanking sequences, was PCR-amplified from ZM401 genomic DNA using primers ZMO1055F2 and ZMO1055DW, generating a 2.9 kb fragment. This fragment was cloned into the TA cloning vector pRBC (RBC, Taiwan), yielding the construct pRBC::pde, verified by blue-white screening and PCR with M13 primers.

The chloramphenicol acetyltransferase (cat) gene was amplified from pLysS using primers CatFNsiI and CatRNsiI, generating a 1 kb product. The cat cassette was inserted into pRBC and verified via PCR and antibiotic resistance. Both pRBC::pde and pRBC::cat were digested with NsiI (NEB, UK), gel-purified, and ligated to generate the final construct pRBC::pde::cat. The assembled plasmid was transformed into E. coli DH5α and verified by colony PCR and chloramphenicol resistance (34 μg/mL). This plasmid was later introduced into Z. mobilis ZM401 for chromosomal integration to construct the engineered strain ZAM2, following previously described methods [35–36]. Primer sequences and cloning details are provided in supplementary materials (Table S2).

2.2.3. Transformation of ZM401 and mutant verification

To improve flocculation and inhibitor resistance in Z. mobilis, targeted inactivation of the phosphodiesterase (PDE)-encoding EAL domain in ZMO_1055 was performed. The suicide vector (pRBC:: pde::cat) was introduced into electrocompetent Z. mobilis ZM401 cells prepared as previously described [35–36]. Electroporation was performed using a Gene Pulser® (1.6 kV/cm, 25 μF, 200 Ω), and transformants were selected on chloramphenicol-supplemented RM agar. Putative knockout mutants (designated ZAM2) were confirmed by PCR using primer sets flanking the EAL region (ZMO1055F2, ZMO1055DW, ZMO1054F, ZMO1056R). Primer sequences and electroporation conditions are listed in supplementary materials (Table S2, S4).

2.3. Characterization of Engineered Strains

2.3.1. Quantification of intracellular c-di-GMP

To elucidate the molecular mechanism underlying enhanced flocculation in engineered Z. mobilis strains, intracellular concentrations of cyclic-di-GMP (c-di-GMP), a key bacterial second messenger regulating cellulose synthesis and cell aggregation, were quantified using a previously reported LC–MS/MS method [37]. Cells were harvested during the exponential growth phase and lysed via bead homogenization. After protein removal and ethanol precipitation, the soluble fractions were filtered and analyzed using a UPLC–Xevo TQ-S micro system (Waters, USA) operating in positive ionization mode. Chromatographic separation was performed on an ACQUITY UPLC BEH C₁₈ column. Quantification was based on an external calibration curve prepared with analytical-grade c-di-GMP (Sigma-Aldrich).

Measured c-di-GMP levels were normalized to total protein concentration and used to compare intracellular signaling activity among the engineered strain (ZAM2), the spontaneous mutant (ZM401), and the wild-type strain (ZM4). Key analytical parameters and instrument settings are detailed in supplementary materials (Table S5).

2.3.2. Measurement of intracellular PDE activity

To validate the hypothesis that disruption of phosphodiesterase (PDE) activity leads to intracellular accumulation of c-di-GMP and promotes enhanced flocculation, total PDE activities were measured in crude protein extracts from Z. mobilis strains ZM4, ZM401, and ZAM2. All strains were cultivated to mid-exponential growth phase, lysed via bead homogenization, and the soluble protein fractions were collected for enzymatic analysis. PDE activity was quantified using a colorimetric assay with bis(p-nitrophenyl) phosphate as a substrate, and the release of p-nitrophenol was monitored at 410 nm [38]. Bis(p-nitrophenyl) phosphate was used as a chromogenic substrate to enable reliable colorimetric quantification of total PDE activity, as it facilitates comparative analysis of enzymatic activity across different strains [39]. Enzyme activity was normalized to total protein concentration. Key assay buffers, substrate concentrations, and spectrophotometric parameters are provided in supplementary materials (Table S6). All measurements were performed in triplicate, and differences in enzymatic activity were evaluated using one-way ANOVA with a significance threshold of p < 0.05.

2.3.3. Gene expression analysis of flocculation-related pathways

To investigate the molecular basis underlying enhanced flocculation and inhibitor tolerance in engineered Z. mobilis strains, the expression levels of key regulatory genes involved in cellulose biosynthesis (bcsB, ZMO_1084) and flagellar assembly (flgB, ZMO_ 0614) were quantified via real-time quantitative PCR (qPCR). Total RNA was extracted using the HiYield™ Total RNA Mini Kit (Real Biotech Corporation, Taiwan) following the manufacturer’s protocol. RNA concentration and purity were assessed by spectrophotometry at 260/280 nm (MECASYS, Korea). Genomic DNA was removed using RQ1 RNase-Free DNase (Promega, USA), and cDNA was synthesized from 1 μg of total RNA using the PrimeScript™ RT Master Mix (TaKaRa, Japan). Synthesized cDNA was used as a template for downstream qPCR analysis.

qPCR reactions were carried out using the QuantStudio 6 Flex Real-Time PCR system (Applied Biosystems, USA) with AccuPower® 2X GreenStar qPCR Master Mix (Bioneer, Korea). Each 20 μL reaction contained 50 ng of cDNA, 10 μL of master mix, 1 μL each of forward and reverse primers (10 pmol), and nuclease-free water. The amplification protocol consisted of 40 cycles at 95°C for 15 s and 60°C for 30 s.

All reactions were performed in triplicate. Relative expression levels were calculated using the 2^−ΔΔCt [40], with 16S rRNA as the internal reference gene and wild-type ZM4 as the calibrator. Primer sequences are listed in supplementary materials (Table S2). Statistical comparisons were performed using Student’s t-test, with p < 0.05 considered statistically significant.

2.3.4. Floc size distribution analysis

To evaluate the self-flocculating behavior and settling potential of Z. mobilis strains under conditions relevant to industrial bioethanol production, floc size distribution was quantitatively analyzed using a laser diffraction particle size analyzer (LS 13320, Beckman Coulter, USA). This measurement provides insight into the uniformity and average diameter of cell aggregates, which are key parameters influencing biomass recovery and cell recycling efficiency in high-cell-density fermentation systems.

All strains were cultured overnight at 30°C in 20 mL of rich medium (RM) in 50 mL conical tubes. Cell suspensions were then analyzed using the liquid module of the particle size analyzer under default settings. The frequency distribution of floc diameters was recorded for each strain.

2.4. Comparative Ethanol Fermentation Under Lignocellulose-derived Inhibitors

To evaluate high-gravity fermentation performance and inhibitor tolerance, batch fermentations were conducted using the engineered strain ZAM2, along with wild-type ZM4 and flocculating mutant ZM401, in the presence of key toxic compounds commonly found in lignocellulosic biomass hydrolysates. Fermentations were carried out in 250 mL Erlenmeyer flasks with a 100 mL working volume containing 100 g/L glucose, 10 g/L yeast extract, 2 g/L KH₂PO₄, 2 g/L (NH₄)₂SO₄, and 1 g/L MgSO₄. Furfural (1.7 g/L), acetic acid (12 g/L), and vanillin (0.5 g/L) (all from Sigma-Aldrich, USA) were added to simulate typical inhibitor concentrations derived from lignocellulosic biomass pretreatment [12, 41]. The initial pH was adjusted to 6.0 using 2 N KOH.

Seed cultures were prepared by inoculating a single colony into 20 mL of RM broth and incubating overnight at 30°C to reach mid-exponential phase (OD₆₆₀ ≈ 1.0). For fermentation, 10 mL of seed culture was transferred to 90 mL of fresh fermentation medium containing the inhibitors. Batch fermentations were performed at 30°C with shaking at 100 rpm.

Glucose consumption and ethanol production were quantified using HPLC (Dionex™ UltiMate™ 3000, Thermo Fisher Scientific, USA), while biomass concentrations were determined gravimetrically. All HPLC conditions were as described previously [36]. Kinetic parameters such as maximum specific growth rate, ethanol yields and dry cell weight were calculated following standard protocols [20, 36].

All experiments were designed to simulate industrial stress conditions relevant to lignocellulosic fermentation and to evaluate the robustness of the engineered strain. Statistical significance was evaluated using a paired two-tailed t-test (GraphPad Prism 8.0), with p < 0.05 considered significant.

3. Results and Discussion

3.1. Genetic Confirmation and Construction of EAL Knockout Strain ZAM2

The EAL domain of the regulatory gene ZMO_1055 was successfully inactivated in Z. mobilis ZM401 via homologous recombination, generating the engineered strain ZAM2. PCR analysis using multiple primer sets confirmed the insertion of the cat cassette within the EAL domain region, as presented in the supplementary materials (Fig. S1(a)). Agarose gel electrophoresis confirmed the expected amplicon sizes corresponding to successful recombination events in ZAM2 but not in the parental strain, which is shown in the supplementary materials (Fig. S1(b)). These results demonstrate the precise genetic disruption of the EAL domain, supporting the downstream investigation of its functional consequences on flocculation and fermentation [21].

3.2. Enhanced Flocculation Phenotype of ZAM2

The engineered strain ZAM2 exhibited significantly enhanced self-flocculation in rich medium (RM), forming large, dense aggregates that were visually more prominent than those observed in ZM401, whereas ZM4 remained fully planktonic, as shown in Fig. 1(a–c). Microscopic examination further revealed tightly clustered cells in ZAM2 cultures, in contrast to the dispersed morphology observed in ZM4, as shown in Fig. 1(d–f).

These phenotypic differences were quantitatively supported by laser diffraction particle size analysis, as illustrated in Fig. 2. Over 86% of ZAM2 cells fell into floc size groups D and E (>10 μm), with an average floc diameter of 30 μm, larger than ZM401 (17 μm) and significantly greater than the non-flocculating ZM4 (<1 μm) (Table 1).

These results suggest that targeted disruption of the EAL domain in ZMO_1055 enhances cell aggregation by elevating intracellular c-di-GMP levels. This phenotype aligns with established models in which high c-di-GMP promotes cellulose biosynthesis and represses motility, thereby reinforcing stable floc formation.

3.3. Fermentation Performance under Lignocellulose-derived Inhibitor Stress

Fermentation experiments conducted in glucose-rich media containing acetic acid, furfural, and vanillin revealed that ZAM2 outperformed both ZM401 and ZM4 under inhibitory conditions. Based on one-way ANOVA analysis, ZAM2 exhibited significantly faster glucose consumption (p < 0.05), completing the process within 18 hours compared to 21 hours for ZM401 and even longer for ZM4, as shown in Fig. 3(a). Correspondingly, ZAM2 achieved an ethanol productivity of approximately 2.33 g/L/h, surpassing that of ZM401 (1.90 g/L/h). Ethanol production was also significantly more rapid in ZAM2 (p < 0.05), reaching 42 g/L within 18 hours, as shown in Fig. 3(b). Biomass accumulation was significantly higher in ZAM2 than ZM401 (p < 0.05), indicating robust growth despite the presence of fermentation inhibitors. Notably, the ethanol yield of ZAM2 (0.38 g/g) was marginally lower than in ZM401 (0.41 g/g), potentially reflecting increased carbon flux toward cellulose biosynthesis. This tradeoff suggests that while enhanced flocculation in ZAM2 confers improved resistance to inhibitors—resulting in faster sugar consumption, higher ethanol productivity, and increased biomass formation—it may also divert carbon flux into cellulose biosynthesis or other stress-response pathways, thereby slightly lowering ethanol yield [21]. These findings, illustrated in Fig. 3(c), support the hypothesis that enhanced flocculation improves inhibitor tolerance.

Most importantly, the significantly elevated ethanol productivity of ZAM2, achieved through accelerated glucose conversion, demonstrates the practical value of engineered flocculation in bioethanol fermentation. This performance advantage underscores the dual role of flocculation in stress shielding and metabolic resilience, reinforcing its utility in industrial-scale lignocellulosic bioprocesses [12, 26, 42].

3.4. Intracellular c-di-GMP Concentration and Phosphodiesterase Activities of Z. mobilis Strains

Quantification of intracellular c-di-GMP levels revealed a clear distinction among the strains. ZAM2 exhibited the highest concentration (111.6 pmol/μg), followed by ZM401 (99.9 pmol/μg) and ZM4 (43.7 pmol/μg), as shown in Fig. 4(a). This trend indicates that deletion of the EAL domain in ZAM2 effectively inhibited PDE activity, allowing c-di-GMP to accumulate. Importantly, ZM4, as the wild-type strain, exhibited the lowest c-di-GMP level, highlighting its native PDE activity that maintains intracellular c-di-GMP at a baseline level. The approximately 2.3-fold increase in ZM401 and 2.6-fold increase in ZAM2 compared to ZM4 demonstrate the progressive accumulation of c-di-GMP as a result of EAL domain disruption. Elevated c-di-GMP levels are known to promote cellulose synthesis and floc formation in various bacterial species, which is consistent with the observed phenotype in ZAM2 [20–21]. These findings suggest that the EAL domain of ZMO_1055 plays a functional role in modulating intracellular c-di-GMP turnover, directly influencing cell aggregation behavior.

Enzymatic assays measuring total PDE activity showed significantly reduced activity in both ZM401 and ZAM2 compared to ZM4 (p < 0.005), as shown in Fig. 4(b). This finding confirms that the EAL domain in ZMO_1055 encodes active PDE functionality, and its disruption in ZAM2 reduces c-di-GMP degradation, contributing to the higher intracellular concentrations of this signaling molecule. Consistent with the c-di-GMP data, ZM4 exhibited the highest PDE activity among the three strains, underscoring its role as a physiological baseline. The significantly lower PDE activity in ZM401 and ZAM2 reinforces the conclusion that reduced degradation capacity leads to intracellular c-di-GMP accumulation. Interestingly, the presence of residual PDE activity in both ZM401 and ZAM2, despite EAL disruption, suggests functional redundancy within the Z. mobilis genome, which contains other c-di-GMP turnover proteins. Identified GGDEF and/or EAL domain-containing proteins in the Z. mobilis genome, in addition to ZMO_1055, include ZMO_0401, ZMO_0919, ZMO_1365, and ZMO_1487. These findings align with observations in other bacteria where multiple GGDEF and EAL domain proteins contribute to complex regulation of intracellular signaling [14, 21, 43].

3.5. Gene Expression and Domain Context of Cellulose Signaling

qRT-PCR analysis revealed significant upregulation of bcsB (ZMO_1084), involved in cellulose biosynthesis, in ZAM2 and ZM401—3.2-fold and 3.8-fold increases, respectively, compared to ZM4. In contrast, flgB (ZMO_0614), a key flagellar assembly gene, was downregulated by approximately 50% in both mutant strains, as shown in Fig. 5. These expression patterns are consistent with the regulatory effects of high c-di-GMP levels, which promote biofilm formation and flocculation while repressing motility-related genes. Similar patterns have been observed in other biofilm-forming bacteria, further validating the regulatory role of c-di-GMP in floc formation [12, 20].

Additionally, the Z. mobilis genome encodes several other GGDEF and/or EAL domain-containing proteins, such as ZMO_ 0401, ZMO_0919, ZMO_1365, and ZMO_1487, which may also contribute to intracellular c-di-GMP regulation in parallel with ZMO_1055 [21, 44–46]. Their potential roles in modulating stress responses and flocculation merit future investigation, especially in the context of optimizing ethanol production traits.

4. Conclusion

In this study, we developed and characterized a genetically engineered strain of Z. mobilis (ZAM2) with enhanced self-flocculation and stress tolerance by disrupting the EAL domain of the regulatory gene ZMO_1055. The resulting strain exhibited significantly elevated intracellular c-di-GMP levels, suppressed phosphodiesterase activity, increased expression of cellulose biosynthesis genes, and downregulated expression of motility-associated genes. These molecular changes collectively contributed to enhanced floc formation, improved tolerance to lignocellulose-derived fermentation inhibitors, and accelerated ethanol production.

Our findings underscore the value of engineering intracellular signaling pathways to enhance microbial robustness and process efficiency. This strategy provides a rational framework for the development of next-generation microbial biocatalysts tailored for high-performance bioethanol production from renewable lignocellulosic feedstocks.

In particular, the self-flocculating phenotype of ZAM2 offers advantages for industrial-scale applications by facilitating protection against fermentation inhibitors through cell aggregation, which supports faster sugar consumption and increased ethanol production rates. Such traits could be leveraged in continuous fermentation, integrated cell recycling, or downstream separation processes to improve operational stability and reduce production costs.

Supplementary Information

eer-2025-223-supplementary.pdf

Notes

Acknowledgement

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (2015R1D1A1A01056794 and 2018R1D1A1A09084264) and the Pukyong National University Industry-university Cooperation Foundation’s 2024 Post-Doc. support project.

Conflict of Interest

The authors declare no competing financial or commercial interests related to this work.

Author Contributions

W.G.J. (Master’s student) constructed mutant strains, conducted fermentation experiments, analyzed data, and contributed to manuscript writing. J.I.L. (Master’s student) constructed mutant strains, conducted fermentation experiments, analyzed data, and contributed to manuscript writing. S.S.C. (Ph.D.) performed qRT-PCR analysis, analyzed data, and co-wrote the manuscript. H.S.H. (Master’s student) carried out c-di-GMP quantification and enzyme activity assays. M.S.J. (Master’s student) performed qRT-PCR and particle size analysis. Y.F.T. (Professor) supervised c-di-GMP quantification and enzyme activity assays. Y.J.J. (Professor) conceived and supervised the project, analyzed data, and co-wrote the manuscript.

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Fig. 1

Comparison of macroscopic and microscopic flocculation phenotypes in Z. mobilis strains. Macroscopic images of Z. mobilis strains grown on 90 mm Petri dishes: (a) wild-type ZM4 (negative control), (b) parental strain ZM401, and (c) engineered strain ZAM2. Microscopic images of the corresponding strains captured at 1000× magnification (Scale bars represent 20 μm.): (d) wild-type ZM4 (negative control), (e) parental strain ZM401, and (f) engineered strain ZAM2.

Fig. 2

Particle size distribution of Z. mobilis strains ZM4, ZM401, and ZAM2. ZM4 (purple line) exhibited a narrow distribution with particles predominantly below 1 μm. In contrast, ZM401 (blue line) and ZAM2 (green line) showed broader distributions with major populations in the 10–100 μm range. Notably, ZAM2 exhibited a higher degree of cell aggregation than ZM401.

Fig. 3

Fermentation profiles of ZM4 (●), ZM401 (■), and ZAM2 (◆) under lignocellulosic inhibitor stress. Batch fermentations were performed in 100 g/L glucose medium containing 12 g/L acetic acid, 1.7 g/L furfural, and 0.5 g/L vanillin at 30°C and 100 rpm. Time-course data include: (a) residual glucose concentration, (b) ethanol titer, (c) cell biomass (OD₆₀₀). Statistical significance among strains was assessed using one-way ANOVA (p < 0.05).

Fig. 4

Intracellular c-di-GMP levels and phosphodiesterase activities in Z. mobilis strains. (a) Total intracellular c-di-GMP concentrations in Z. mobilis strains ZM4, ZM401, and ZAM2. (b) Total phosphodiesterase activities measured in crude protein extracts of the same strains. Phosphodiesterase activity is expressed as the amount of p-nitrophenol released. Bars represent mean ± SD from three independent biological replicates.

Fig. 5

Relative expression of flagellar gene flgB and cellulose synthesis gene bcsB in ZM4, ZM401, and ZAM2 strains. Expression levels normalized to 16S rRNA and represented as fold change relative to ZM4. The error bars indicate mean ± SD from three independent replicates.

Table 1

Particle size distribution of cell floc in strains of Z. mobilis ZM4, ZM401, and ZAM2. Cells were grouped into five particle size populations based on laser diffraction analysis: Population A (<1 μm), B (1–2.5 μm), C (2.5–10 μm), D (10–100 μm), and E (>100 μm). Particle sizes greater than 10 μm (Populations D and E) are typically associated with effective floc formation, whereas smaller particles (<2.5 μm) are indicative of planktonic, single-cell populations.

Group (%)	Particle size range (μm)	The strains of Z. mobilis

		ZM4	ZM401	ZAM2
Population A	1>	100	0	1.6
Population B,	1 – 2.5	0	0	2.5
Population C,	2.5–10	0	20.9	9.6
Population D,	10–100	0	73.2	86.1
Population E,	100<	0	5.9	0.3

Total (%)		100	100	100