Phenotypic characterization and pan-genomic analysis of a novel bioflocculant-producing Stenotrophomonas maltophilia PT-13 for wastewater treatment

Phenotypic characterization and pan-genomic analysis of a novel bioflocculant-producing Stenotrophomonas maltophilia PT-13 for wastewater treatment | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Phenotypic characterization and pan-genomic analysis of a novel bioflocculant-producing Stenotrophomonas maltophilia PT-13 for wastewater treatment Linlin Zhang, Shenghui Yang, Zhaofeng Liu, Yuqing Li, Jianjiang Lu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9636700/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Microbial bioflocculants (MBFs) are eco-friendly alternatives, yet the species-level genetic basis of heteropolysaccharide-type MBFs biosynthesis remains largely unclear. In this study, a novel high-efficiency MBF-producing bacterial strain Stenotrophomonas maltophilia PT-13 was isolated. Fermentation conditions and flocculation operational parameters were systematically optimized using single-factor experiments combined with response surface methodology (RSM). Results showed that the optimal flocculation conditions yielded a kaolin flocculation efficiency of 90.3% with RSM. Bioflocculant MBF-PT produced by S. maltophilia PT-13 was identified as an acidic heteropolysaccharide mainly composed of rhamnose, glucose and mannose, with flocculation governed by adsorption-bridging supplemented by charge neutralization. In simulated domestic wastewater, MBF-PT achieved 74.2% (chemical oxygen demand)、81.3% (turbidity) and 80.4% (suspended solids) removal efficiencies. Pan-genomic analysis revealed an open pan-genome of S. maltophilia and identified conserved core gene clusters responsible for polysaccharide synthesis, glycosyl transfer and transmembrane transport. This study provides a promising microbial resource for developing green water treatment agents and offers new insights into the species-level genetic mechanism of MBFs biosynthesis. Microbial flocculant Stenotrophomonas maltophilia Fermentation optimization Pan-genome analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Coagulation and flocculation are important technologies in water and wastewater treatment units to remove suspended particles due to its simple operation, cost-effectiveness, and wide adaptability to water quality fluctuations (Elrasoul and Yong, 2025 ; Zezulka et al., 2024 ). The traditional flocculants, including inorganic aluminum/iron salts and synthetic organic polymers, have inherent limitations despite their high efficiency (Li et al., 2025 ). Microbial flocculants (MBFs) are secondary metabolites with high flocculation activity secreted by microorganisms during growth and metabolism, mainly in the form of extracellular polymeric substances (EPS), amino acid and glycoproteins (Mnif and Ben Rebah, 2023 ; Wang et al., 2022 ). Compared with traditional chemical flocculants, MBFs exhibit advantages with excellent biodegradability, non-toxicity, no secondary pollution, and wide adaptability to complex water quality (Abu Bakar et al., 2021 ; Ben Rebah et al., 2018 ). MBFs have been extensively studied in municipal wastewater, industrial wastewater, drinking water purification, and sludge dewatering (Babiak and Krzemińska, 2021 ). Nowadays, the most reported MBF-producing strains belong to Bacillus , Pseudomonas , Klebsiella , and Serratia (Zhang et al., 2025 ; Qin et al., 2025 ; Xu et al., 2016 ). Whole-genome sequencing has been applied to explore the biosynthesis of microbial bioflocculants, enabling the identification of genes related to exopolysaccharide (EPS) synthesis, glycosyltransfer, and transmembrane transport (Fu et al., 2015 ). Genomic approaches allow precise localization of functional genetic elements, guide metabolic engineering to improve flocculant yield, and promote the mechanistic understanding of MBFs from the molecular level (Baranwal et al., 2019 ). Nevertheless, current genomic research on MBFs is overwhelmingly restricted to single-strain genomic analysis, which cannot reveal the genetic diversity, core gene conservation, and multi-gene synergy of MBFs biosynthesis at the species level. Single-genome data fail to distinguish core essential genes from accessory or strain-specific genes, leading to an incomplete picture of the complex regulatory network for heteropolysaccharide assembly and secretion (Zhong et al., 2021 ). As a powerful strategy for population-level genetic dissection, pan-genome analysis enables the identification of core, accessory, and strain-specific genes, as well as the mining of key functional gene clusters related to target phenotypes (Hu et al., 2024 ; Sutton et al., 2021 ; Srivastava et al., 2020 ). For MBF-producing bacteria, pan-genome can systematically reveal the conserved pathways for MBFs synthesis, clarify the essential genetic determinants of high-efficiency flocculant production. To date, pan-genomic analysis has rarely been employed to reveal the genetic basis of MBFs synthesis, leaving the species-level synthetic potential and functional evolutionary mechanism largely unexplored. In this study, a novel MBF-producing bacterial strain, Stenotrophomonas maltophilia PT-13, exhibiting exceptional flocculation activity was isolated and identified from algal culture broth. The fermentation conditions of strain PT-13 and the operational parameters for its flocculation application were systematically optimized using single-factor tests combined with response surface methodology (RSM). The structural properties of the purified bioflocculant MBF-PT were further characterized, and its microscale flocculation mechanism was elucidated via a suite of complementary spectroscopic and microscopic techniques. Moreover, pan-genomic analysis of representative Stenotrophomonas maltophilia genomes was conducted to uncover the genetic potential of this species for the biosynthesis of complex heteropolysaccharides at the taxonomic species level. This study provides a promising new microbial resource for the development of eco-friendly green water treatment agents. Materials and methods Isolation and screening of bioflocculant-producing bacteria The bacterial strains were isolated from the culture fluid of Phaeodactylum tricornutum which was obtained from the Institute of Oceanology, Chinese Academy of Sciences. The purified strains were inoculated into LB liquid medium and cultured at 28°C for 24 h. Then, the bacterial broth was inoculated into the fermentation medium containing C 6 H 12 O 6 ·H 2 O (10.0 g/L), CH 4 N 2 O (0.5 g/L), yeast extract (0.5 g/L), NaCl (0.1 g/L), K 2 HPO 4 (5.0 g/L), KH 2 PO 4 (2.0 g/L), MgSO 4 ·7H 2 O (0.2 g/L), pH 7.2–7.5. The flocculation activity was determined using the standard kaolin suspension method with minor modifications after culturing at 28°C and 150 rpm for 48 h. The flocculation system (100 mL) consisted of 5 mL fermentation broth, 5 mL of 1% (w/v) CaCl₂ solution, and 90 mL of 4 g/L kaolin suspension, with the pH adjusted to 7.0 ~ 8.0. T In the control group, the fermentation broth was replaced with an equal volume of sterile fermentation medium, with all other components unchanged. The mixture was stirred at 400 rpm for 5 min and allowed to stand for 5 min. The absorbance of the supernatant at 550 nm (OD₅₅₀) was measured using a UV-Vis spectrophotometer, with distilled water as the blank control. All experiments were performed in triplicate. The flocculation rate was calculated using the following formula: Flocculation activity (%) = (A − B)/A × 100 (1) where A is the OD₅₅₀ of the control group, and B is the OD₅₅₀ of the experimental group. The isolate with the highest flocculation activity was named as PT13 for subsequent experiments and stored at -80°C. Identification of the bioflocculant-producing strain The morphological characteristics of bioflocculant-producing stain PT13 was observed through colony morphology on LB agar plates and Scanning electron microscope (SEM) imaging of the bacterial cells. For SEM observation, the bacterial cells were collected by centrifugation at 10,000 rpm for 10 min, fixed with 2.5% (v/v) glutaraldehyde at 4°C for 12 h. The sample was rinsed with 0.1 M phosphate buffer saline (PBS, pH 7.0) and observed under SEM (S4800, Hitachi, Japan). The genomic DNA of the target strain was extracted using a bacterial genomic DNA extraction kit (Tiangen Beijing). 16S rRNA gene was amplified using the universal bacterial primers 27F and 1541R. The PCR products were verified by 1% agarose gel electrophoresis and sequenced bidirectionally by Tsingke Biotechnology Co., Ltd. (Qingdao, China). The obtained 16S rRNA gene sequence was aligned with the NCBI GenBank database using the BLAST tool, and a phylogenetic tree was constructed using the MEGA 6.0 software based on the maximum likelihood method. The influence of different factors on the bioflocculant production The experiments were conducted to investigate the influence of different factors on the bioflocculant production, with the OD₆₀₀ values and flocculation rate as evaluation indicators. For carbon source optimization of strain PT13, eight distinct carbon substrates (glucose, sucrose, mannose, maltose, fructose, xylose, sodium gluconate, and sodium acetate) were individually tested as the sole carbon source in the fermentation medium, with each substrate supplemented to a final concentration of 10 g/L. For nitrogen source optimization, strain PT13 was cultivated in the fermentation medium using six different nitrogen sources (yeast extract, peptone, NH₄Cl, KNO₃, (NH₄)₂SO₄, and urea), each added to a final concentration of 1.0 g/L as the sole nitrogen source. Subsequently, based on the screened optimal carbon and nitrogen sources, the carbon-to-nitrogen (C/N) ratio was further optimized by adjusting the ratio to 1, 5, 10, 15, and 20, respectively. For inoculation size optimization, the seed culture (OD₆₀₀ = 1.0) was inoculated into the fermentation medium at gradient inoculation sizes of 0.5%, 1%, 2%, 3%, 4%, and 5% (v/v), respectively. Optimization of flocculation conditions using RSM Single-factor experiments were firstly performed to screen the key variables affecting flocculation efficiency and determine the appropriate level ranges for subsequent optimization. Four variables, including rotational speed, fermentation supernatant dosage, CaCl₂ concentration, and pH, were investigated in the single-factor tests. Based on the single-factor experimental results, a three-factor, three-level Box-Behnken design (BBD) was employed to optimize the critical operational parameters of the flocculation process. The three independent variables with the most significant impact on flocculation efficiency were selected as rotational speed, fermentation supernatant dosage, and CaCl₂ concentration, while flocculation efficiency was set as the sole response value of the model. The coded values and corresponding actual levels of each independent variable are summarized in Suppl. Table S1 . A total of 17 experimental runs were designed and implemented in this study, consisting of 12 factorial points and 5 repeated center points to estimate the pure experimental error. The obtained experimental data were fitted to a second-order quadratic polynomial regression model using Design-Expert 12.0 software (Stat-Ease Inc., Minneapolis, MN, USA). Subsequently, analysis of variance (ANOVA) was conducted to assess the statistical significance of the established regression model and each individual model term. The optimal flocculation operational parameters were derived by solving the established regression model, and triplicate validation experiments were performed under the optimized conditions to confirm the reliability and predictive accuracy of the regression model. Extraction, purification and characterization of bioflocculant MBF-PT The fermentation broth of strain PT13 was centrifuged at 8,000 rpm (4°C, 10 min) to remove bacterial cells. The cell-free supernatant was mixed with 3.0-fold volumes of pre-chilled absolute ethanol, and held at 4°C for 12 h for bioflocculant precipitation. The precipitate was collected by centrifugation, washed 3 times with absolute ethanol, and lyophilized for 72 h to obtain purified bioflocculant MBF-PT. The monosaccharide composition of MBF-PT was analyzed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD, ICS-5000, Dionex, USA). Briefly, 5 mg of MBF-PT was hydrolyzed with 2 M trifluoroacetic acid (TFA) at 121°C for 2 h in a sealed tube. The hydrolysate was evaporated to dryness under nitrogen, rinsed with methanol 3 times to remove residual TFA, reconstituted in ultrapure water, and filtered through a 0.22 µm hydrophilic membrane. Separation was performed on a Dionex CarboPac PA20 column (150 mm × 3.0 mm, 10 µm) with gradient elution (0.5 mL/min, 30°C), and 15 monosaccharide reference standards were used for qualitative and quantitative analysis. Fourier transform infrared (FTIR) spectra of MBF-PT were recorded from 400 to 4000 cm⁻¹ via the KBr pellet method to identify functional groups. X-ray photoelectron spectroscopy (XPS, ESCALAB 250Xi, Thermo Fisher, USA) with monochromatic Al Kα radiation was used to analyze the surface elemental composition and chemical states of MBF-PT, raw kaolin, and kaolin flocs after flocculation. X-ray diffraction (XRD, Dmax Ultima, Rigaku, Japan) patterns were collected using Cu Kα radiation over a 2θ range of 10°–80° (scanning rate: 2°/min) to characterize the crystal structure of the samples. Scanning electron microscopy (SEM) was performed to observe the micromorphology of MBF-PT, raw kaolin, and flocculated kaolin, with samples sputter-coated with gold prior to imaging. The distribution of flocculation activity in the fermentation system was determined by measuring the flocculation rate of the original fermentation broth, cell-free supernatant, and bacterial cell resuspension, respectively. The Zeta potential of the kaolin suspension, MBF-PT solution, CaCl₂ solution, and the flocculation system at different stages was measured using a Zetasizer Nano ZS at 25°C. All measurements were performed in triplicate, and the average values were used to analyze the electrostatic interaction during the flocculation process. Treatment performance of simulated domestic wastewater The treatment performance of the bioflocculant for simulated domestic wastewater was evaluated under the optimal flocculation conditions obtained from RSM optimization. The water quality indicators, including chemical oxygen demand (COD), turbidity, and suspended solids (SS), were measured before and after treatment. The removal efficiency of each indicator was calculated. Pangenome analysis A total of 37 high-quality genome sequences of S. maltophilia (including complete genomes and high-quality scaffolds/contigs) were downloaded from the NCBI GenBank database. All genomes were re-annotated using the Prokka software for standardized open reading frame (ORF) prediction and functional annotation to eliminate systematic errors from different annotation pipelines. Pangenome analysis was executed by Integrated Prokaryotes Genome and Pangenom Analysis (IPGA) website ( https://nmdc.cn/ipga/ ) (Liu et al., 2022 ). To depict the pangenome (core and accessory genome) assessment of strains included in the comparison, a reciprocal best hit search was also performed. The accumulation curves of the pan-genome and core genome were fitted using a nonlinear regression power-law model. Statistical Analysis All data are expressed as mean ± standard error. Statistical analyses were performed using OriginPro 2021 (OriginLab, USA). Homogeneity of variances was confirmed prior to one-way ANOVA, and Duncan’s multiple range test was applied for post hoc comparisons between experimental groups. Statistical significance was set at p < 0.05, and high statistical significance was defined as p < 0.01. Results Identification and morphological characteristics of strain PT13 A total of 18 bacterial strains were isolated from the phycosphere of Phaeodactylum tricornutum , and their flocculation efficiencies were evaluated via the standard kaolin suspension assay. As shown in Fig. 1 a, the flocculation efficiencies of the isolates ranged from 18.4% to 82.2% under initial fermentation conditions. Strain PT13 exhibited the highest flocculation activity (82.2%), which was significantly higher than that of all other isolates (p < 0.05), and was thus selected as the target strain for subsequent research. In Fig. 1 (b), colonies of strain PT13 on LB agar plates were circular, pale yellow to creamy white, with smooth moist surfaces and entire margins. SEM observation confirmed that the cells were short rod-shaped, with dimensions of 0.5 µm × 2.0 µm (Fig. 1 c). Full-length 16S rRNA gene sequence alignment showed that strain PT13 shared high similarity with multiple Stenotrophomonas. maltophilia strains. Given the high conservation of the 16S rRNA gene within the Stenotrophomonas genus, the strain was conservatively designated as Stenotrophomonas. maltophilia PT13, which was further validated by phylogenetic analysis (Fig. 1 d). Optimization of fermentation conditions for flocculant production The effects of eight sole carbon sources on the growth and flocculation efficiency of strain PT13 are shown in Fig. 2 a. The high flocculation efficiencies (> 90%) were achieved with glucose, xylose, sodium gluconate, or sodium acetate as the sole carbon source. In contrast, mannose, sucrose, and maltose supported the highest biomass (OD₆₀₀ > 2.3) but significantly lower flocculation efficiencies (p < 0.05), indicating no linear positive correlation between bacterial growth and bioflocculant biosynthesis by strain PT13 under these conditions. Notably, sodium acetate, a widely used supplementary carbon source in wastewater treatment plants, also enabled 92.3% flocculation efficiency, highlighting the potential of strain PT13 for in-situ bioflocculant production and synchronous wastewater treatment. For nitrogen source optimization (Fig. 2 b), yeast extract yielded the highest flocculation efficiency and favorable bacterial growth, followed by urea, while peptone exhibited the poorest performance for both indicators. As a complex organic nitrogen source, yeast extract provides not only bioavailable nitrogen but also vitamins, amino acids, and growth factors that promote both cell growth and bioflocculant biosynthesis. As shown in Fig. 2 c, flocculation efficiency and biomass of strain PT13 increased with the C/N ratio from 1 to 15, peaked at a C/N ratio of 15, and decreased markedly at a C/N ratio of 20. At low C/N ratios, carbon was the limiting nutrient, with most carbon flux directed to cell growth rather than bioflocculant synthesis. C/N ratio of 15 was thus selected as the optimal nutritional condition. The effect of inoculum dosage on flocculation efficiency is shown in Fig. 2 d. No statistically significant difference in flocculation efficiency was observed across all tested inoculum dosage, indicating that inoculum dosage had no significant impact on the flocculation performance of strain PT13 within the tested range. Optimization of MBF-PT with RSM Single-factor pre-experiments were conducted to determine the appropriate level ranges for response surface optimization. The optimal center levels were 5 mL for fermentation supernatant dosage and 400 rpm for rotational speed. The bioflocculant maintained excellent flocculation efficiency over pH 6–10 (maximum at pH 10), thus pH was not included in subsequent optimization. A three-factor, three-level BBD was then employed to optimize the three key parameters (rotational speed, fermentation supernatant dosage, CaCl₂ dosage), with the experimental design and results summarized in Suppl. TableS2. A second-order quadratic polynomial regression model was established to correlate flocculation efficiency with the coded independent variables: Y = 88.7 + 4.01A + 1.39B + 4.71C − 0.475AB + 0.25AC − 0.45BC − 8.41A 2 −0.485B 2 −5.34C 2 where Y is the predicted flocculation efficiency (%), and A, B, C are the coded values of rotational speed (rpm), fermentation supernatant dosage (mL), and CaCl₂ dosage (mL), respectively. Analysis of variance confirmed the model was extremely statistically significant (p 0.05), indicating excellent goodness of fit and reliable predictive performance. The model had a coefficient of determination (R²) of 0.9735, with close agreement between adjusted R² (0.9435) and predicted R² (0.9347), further validating its reliability. The order of significance of the three variables on flocculation efficiency was: CaCl₂ dosage (C) > rotational speed (A) > fermentation supernatant dosage (B). Three-dimensional response surface plots and contour plots (Fig. 4 ) were used to visualize the pairwise interactive effects of rotational speed, fermentation supernatant dosage, and CaCl₂ dosage on flocculation efficiency. All response surfaces showed a convex downward-opening shape, confirming a maximum flocculation efficiency existed within the tested variable ranges. The elliptical contour plots indicated significant interactive effects between paired variables, consistent with the ANOVA results. The theoretical optimal flocculation parameters derived by solving the quadratic regression model were: rotational speed 467.8 rpm, fermentation supernatant dosage 5.3 mL, and CaCl₂ dosage 6.1 mL, with a predicted maximum flocculation efficiency of 89.6%. Triplicate independent validation experiments under these optimal conditions yielded an actual flocculation efficiency of 90.3 ± 0.98% (mean ± S.E.), which was in excellent agreement with the predicted value (p > 0.05). The adjusted R² (0.9435) and predicted R² (0.9347) showed a difference less than 0.2, indicating excellent fit and predictive performance of the model. Paired t-test showed no significant difference between the predicted (89.6%) and actual (90.3%) flocculation efficiency (t = 1.22, df = 2, p = 0.346), verifying the reliability of the model. Structural characterization of bioflocculant MBF-PT The flocculation activity distribution test showed that the cell-free supernatant had a flocculation rate of 89%, which was almost the same as that of the original fermentation broth, while the bacterial cell resuspension only had a flocculation rate of 34%. This result clearly indicated that the flocculation active substance of strain PT-13 was an extracellular secretory metabolite, rather than a cell-bound component. The monosaccharide composition of MBF-PT was shown in Suppl. TableS3. MBF-PT is a complex heteropolysaccharide composed of eight monosaccharides, with rhamnose (33.06 mol%), glucose (22.17 mol%), and mannose (18.59 mol%) as the main components, accounting for more than 70% of the total monosaccharides. In addition, it also contained glucuronic acid (7.15 mol%), ribose (6.62 mol%), galactosamine (5.48 mol%), arabinose (3.83 mol%), and glucosamine (3.09 mol%). No fucose, galactose, or xylose were detected. SEM images showed that purified MBF-PT exhibited a loose, highly porous, irregular three-dimensional network structure formed by the aggregation of fine granular and flaky subunits (Fig. 5 a, b). This unique microstructure endows MBF-PT with a large specific surface area and abundant active adsorption sites, which facilitates the capture of suspended particles and the formation of large settleable flocs via the adsorption-bridging mechanism. SEM observation of kaolin particles before and after flocculation was further performed to verify the flocculation behavior at the microscopic level. Pristine kaolin particles were discrete, irregular flakes with no obvious aggregation, whereas after flocculation treatment with MBF-PT, the particles were tightly aggregated into large, dense three-dimensional flocs (Fig. 5 c and d). This marked morphological change provides direct microscopic evidence for the excellent flocculation performance of MBF-PT. FTIR spectroscopy was performed to identify the functional groups of purified MBF-PT (Fig. 6 a). The broad intense absorption band at 3420 cm⁻¹ is attributed to O-H and N-H stretching vibrations, indicating abundant hydroxyl, amino groups and extensive hydrogen bonds in MBF-PT macromolecules. The peaks at 1645 cm⁻¹ and 1410 cm⁻¹ correspond to the asymmetric and symmetric stretching vibrations of carboxylate groups (-COO⁻), respectively, with the 1645 cm⁻¹ peak also covering the amide I band of proteins. The characteristic absorption bands at 1000–1150 cm⁻¹ are assigned to C-O-C glycosidic bond and pyranose ring C-O stretching, confirming the polysaccharide backbone of MBF-PT, consistent with monosaccharide composition analysis. These results demonstrate that MBF-PT is rich in hydroxyl, carboxyl and amino groups, which are the key active sites for bioflocculation. XRD showed that pristine kaolin exhibited well-defined characteristic crystalline diffraction peaks (Fig. 6 b). The position and profile of these peaks remained completely unchanged after flocculation with MBF-PT, with no peak shift or new crystalline phase formation observed. This result confirmed that the flocculation process mediated by MBF-PT was dominated by physical adsorption and particle aggregation, rather than chemical reactions that alter the intrinsic crystal structure of kaolin. A slight decrease in the diffraction peak intensity of kaolin in the flocs was attributed to the adsorption and surface coating of amorphous MBF-PT macromolecules, which provides direct structural evidence for the adsorption-bridging flocculation mechanism of MBF-PT. XPS was further used to characterize the elemental composition and chemical state of MBF-PT at the electronic level. The full survey spectrum (Fig. 7 ) shows that MBF-PT is mainly composed of C, O and N elements. The high-resolution C 1s spectrum was deconvoluted into three peaks at 284.8 eV (C-C/C-H), 286.3 eV (C-O/C-N) and 288.1 eV (C = O/O = C-N), corresponding to the carbon skeleton, hydroxyl/amino groups and carboxyl/amide groups, respectively. The O 1s spectrum was fitted into two peaks at 531.5 eV (C = O) and 532.8 eV (C-O), confirming carbonyl and hydroxyl groups. The N 1s spectrum was deconvoluted into two peaks at 399.8 eV (-NH₂/-CONH-) and 401.5 eV (-NH₃⁺), indicating the presence of neutral and protonated amino groups, which provides direct evidence for the charge neutralization flocculation mechanism. The XPS results are in excellent agreement with FTIR analysis, and further reveal the chemical nature of the functional groups in MBF-PT. Flocculation mechanism and sewage treatment of MBF-PT Zeta potential measurement was conducted to investigate the interfacial charge variation during flocculation and identify the dominant mechanism of MBF-PT, with results shown in Fig. 8 a. The pristine kaolin suspension had a Zeta potential of -27.0 mV, indicating a highly stable colloidal system due to strong electrostatic repulsion between negatively charged particles. The pure MBF-PT solution exhibited a Zeta potential of -12.0 mV, which is attributed to the ionization of abundant carboxyl groups in its polysaccharide backbone, which was consistent with FTIR and XPS results, rendering the flocculant macromolecules negatively charged overall. After addition of CaCl₂ alone, the Zeta potential of the kaolin suspension increased significantly from − 27.0 mV to -15.0 mV, indicating that Ca²⁺ effectively compressed the electrical double layer of kaolin particles and partially neutralized the negative surface charge, thus reducing inter-particle electrostatic repulsion. When both CaCl₂ and MBF-PT were added and floc formation was complete, the Zeta potential of the system stabilized at -10.0 mV, which did not approach the isoelectric point (IEP, 0 mV). According to classic DLVO (Derjaguin-Landau-Verwey-Overbeek) colloid stability theory, charge neutralization as the dominant mechanism would require the system Zeta potential to approach the IEP to eliminate electrostatic repulsion and achieve particle destabilization. However, efficient flocculation was achieved in this study even with the system retaining a negative surface charge, confirming that the flocculation process was dominated by the adsorption-bridging effect, with charge neutralization playing an auxiliary synergistic role. Figure 8 b shows that MBF-PT exhibits excellent purification efficiency for simulated domestic sewage. The initial water quality of the simulated domestic wastewater was as follows: COD 330 mg/L, turbidity 123 NTU, SS 92 mg/L. After treatment, the turbidity decreased to 23 NTU (removal rate 81.3%), and the SS decreased to 18 mg/L (removal rate 80.4%). This indicates that the polysaccharide network structure of MBF-PT is not severely disturbed in complex organic matrices and can still efficiently remove suspended colloids. In addition, the flocculant can effectively reduce COD, reducing it from 330 mg/L to 106 mg/L (removal rate of 74.2%). This indicates that MBF-PT can not only remove inorganic suspended solids, but also synergistically remove organic pollutants and partially dissolved COD in the aqueous phase through net capture and adsorption. In summary, MBF-PT maintains high flocculation activity and stability in the treatment of complex simulated domestic wastewater. Pan-genomic analysis of S. maltophilia To gain insights into the MBT synthesis mechanism in genus Stenotrophomonas , we analyzed the pan-genomic data of 37 different bacterial taxa. As the number of sequenced genomes increases, the pan-genome expands progressively, whereas the core genome gradually contracts (Fig. 9 a). Therefore, we speculate that the pan-genome of S. maltophilia is open. The genome size of S. maltophilia exhibits a direct positive correlation with its gene count. Specifically, S. maltophilia strain (GCF_023518235.1) possesses a maximum of 5 chromosomes, whereas S. maltophilia strain (GCA_007833655.1) harbors the largest genome. We performed pan-genome and core-genome analyses on S. maltophilia , and identified 2740 gene families that constitute the core genome (Fig. 9 b). Among these strains, S. maltophilia (GCA_900186865.1) possessed the highest number of unique genes (1677), while S. maltophilia (GCA_002025605.1) possessed the lowest number of unique genes (774). Based on topological structure and evolutionary distance, the phylogenetic tree was divided into six major taxa (Fig. 9 c). To systematically characterize the functional profiles of genes associated with bioflocculants, a comprehensive screening was conducted using pan-genome data. A total of 24 candidate genes were identified and subsequently classified into four functional categories: polysaccharide synthesis genes, glycoprotein synthesis genes, transport system genes, and other related genes (Suppl. TableS4). The identified polysaccharide synthesis genes serve as core regulators and functional enzymes involved in the biosynthesis of extracellular polysaccharides (EPS), which represent the primary component of bioflocculants. Additionally, other related genes are involved in maintaining intracellular metabolic balance and bacterial cell adhesion, thereby indirectly regulating the synthesis and function of bioflocculants. Discussion S. maltophilia PT-13 has high flocculation efficiency In this study, a novel bacterial strain S. maltophilia PT-13 with high-efficiency bioflocculant-producing capacity was isolated from the phycosphere environment. Previous studies have reported that Stenotrophomonas species exhibit strong metabolic adaptability and can secrete EPS with excellent flocculation and heavy metal adsorption properties (Chen et al., 2016 ; Qin et al., 2024 ). However, most reported bioflocculant-producing strains were isolated from activated sludge, soil, or industrial wastewater (Abu Bakar et al., 2021 ; Zhang et al., 2026 ). Single-factor optimization experiments revealed that glucose was the optimal carbon source for bioflocculant synthesis by strain PT-13, while sodium acetate, a common supplementary carbon source in wastewater treatment plants, also enabled a high flocculation efficiency of 92.3%. This result highlights the potential of strain PT-13 for in-situ bioflocculant production and synchronous wastewater treatment in practical engineering, which can reduce the production cost of MBFs by using wastewater carbon sources. Yeast extract, as a complex organic nitrogen source, achieved the highest flocculation efficiency, which is consistent with the findings in Bacillus subtilis 35A that composite organic nitrogen sources are more conducive to the synthesis of polysaccharide bioflocculants (Dai et al., 2024 ). The highly efficient floc producing bacterium Stenotrophomonas pavanii GXUN74707 isolating from municipal activated sludge showed the best fermentation and gel production efficiency when using small molecule urea as the sole nitrogen source, with a flocculation rate of up to 99.0% for kaolin suspension (Qin et al., 2024 ). The optimal C/N ratio for bioflocculant production was determined to be 15, which is different from the optimal C/N ratio of 3:1 reported in some other MBF-producing strains (Sun et al., 2025 ). The C/N ratio revealed the critical regulatory effect of nutrient balance on the secondary metabolism of bioflocculants. The inoculation dosage had no significant effect on flocculation efficiency within the tested range, which provides operational flexibility for the scale-up fermentation of strain PT-13 in industrial applications. Flocculation Optimization and Wastewater Treatment Efficiency of MBF‑PT MBF-PT maintained excellent flocculation efficiency in a wide pH range of 6 ~ 10, with the maximum flocculation activity at pH 10. This broad pH adaptability is superior to many reported MBFs, which only exhibit high activity in a narrow neutral or slightly alkaline pH range. For example, the composite flocculant MBF-06 produced by S. maltophilia ZZC-06 only reached its peak flocculation activity at pH 8 ~ 9 (Chen et al., 2016 ). MBF-PT also exhibited well pollutant removal performance in simulated domestic wastewater with complex organic components, with COD, turbidity and SS removal rates. Similarly, the microbial flocculant was employed for the treatment of tofu industrial waste water, resulting in removal rates of 75.9% for BOD and 80.2% for COD in tofu production wastewater (Ramadhani et al., 2022 ).The high turbidity and SS removal efficiencies confirmed that the polysaccharide network structure and abundant active functional groups of MBF-PT were not severely disturbed by organic components in wastewater, and could still efficiently capture and aggregate suspended colloidal particles (Li et al., 2025 ; Salehizadeh et al., 2018 ). The substantial COD reduction further indicated that MBF-PT can synergistically remove particulate organic pollutants and partial dissolved organic matter through adsorption, net capture and sweep flocculation effects. RSM was further employed to optimize the flocculation process, yielding a highly reliable regression model. The optimal operational parameters were determined as rotational speed 467 rpm, fermentation supernatant dosage 5.3 mL, and CaCl₂ dosage 6.1 mL, under which the flocculation rate of kaolin suspension reached 90.3%. The significance order of variables was CaCl₂ dosage > rotational speed > supernatant dosage. Among various metal ions, Ca²⁺ is one of the most commonly used and effective in enhancing the flocculating activity of bioflocculants, as it plays a dominant role in the bridging coordination between bioflocculants and wastewater components (Sun et al., 2025 ). For example, Ca²⁺ improved the flocculation efficiency of Bacillus megaterium BMBF to 96% (Selepe et al., 2022 ). Our results further confirmed its critical role in practical wastewater treatment applications. Structural characterization and flocculation mechanism of MBF-PT Compositional analysis and spectroscopic characterization confirmed that the bioflocculant MBF-PT is a complex acidic heteropolysaccharide mainly composed of rhamnose, glucose and mannose, with a small amount of glucuronic acid and amino sugar residues. Notably, rhamnose was the most abundant monosaccharide in MBF-PT, which is a rare feature in the EPS of Stenotrophomonas species reported in previous studies. For example, the bioflocculant produced by S. pavanii GXUN74707 was mainly composed of mannose with extremely low rhamnose content, while the high rhamnose proportion of MBF-PT is similar to that of the high-efficiency bioflocculant produced by Pseudomonas sp. HP2 (Qin et al., 2024 ; Qi et al., 2019 ). As a 6-deoxy-L-mannose, rhamnose has a deoxymethyl group that endows the polysaccharide molecule with local hydrophobic microdomains, which can reduce the surface tension of the polymer at the aqueous interface, enhance the hydrophobic interaction between the flocculant and suspended particles, and improve the adsorption capacity of the flocculant on the particle surface (Pi et al., 2020 ; Schmid et al., 2015 ). FTIR and XPS analysis confirmed that MBF-PT is rich in hydroxyl, carboxyl and amino groups, which are the key active sites for bioflocculation. The abundant hydroxyl and carboxyl groups can form hydrogen bonds with the oxygen-containing groups on the surface of kaolin particles, and also provide binding sites for divalent calcium ions. The presence of glucuronic acid and amino sugars gives MBF-PT the characteristics of an amphoteric polyelectrolyte, with both polyanionic groups and local cationic groups, which constitutes the material basis for its excellent flocculation performance (Zhang et al., 2026 ). Species-level genetic basis of bioflocculant biosynthesis in S. maltophilia Comparative pan-genomic analysis of S. maltophilia genomes was performed for the first time to reveal the genetic potential of this species for complex heteropolysaccharide bioflocculant synthesis at the population level, which fills the gap in species-level genetic dissection of MBFs biosynthesis. The pan-genome analysis revealed that S. maltophilia has a highly open pan-genome structure and has strong genomic plasticity and environmental adaptability, which is consistent with its wide distribution in various ecological niches and strong metabolic versatility (Brooke, 2021 ; Sutton et al., 2021 ). The targeted genomic mining anchored the core gene clusters governing the synthesis of rhamnose and amino sugars, as well as highly amplified glycosyltransferases and the Wzx/Wzy-dependent EPS transmembrane transport assembly system in the core genome (Fu et al., 2020 ; Guo et al., 2022 ). Specifically, the abundant glycosyltransferases are responsible for the sequential assembly of different monosaccharide units into the polysaccharide main chain, which determines the complex monosaccharide composition of MBF-PT (Takeo et al., 2018 ). The Wzx/Wzy-dependent transport system is responsible for the transmembrane transport and extracellular assembly of polysaccharide subunits, which is the key genetic basis for the secretion of extracellular bioflocculants by strain PT-13. These candidate genes for bioflocculant synthesis provide key targets for subsequent metabolic engineering modification to improve the yield of MBF-PT and reduce its production cost. Conclusion In this study, S. maltophilia PT-13 with exceptional bioflocculant-producing capacity was isolated. The optimal fermentation conditions for bioflocculant production were determined as glucose as the carbon source, yeast extract as the nitrogen source, and C/N ratio of 15, under which the fermentation broth exhibited the highest flocculation activity. The optimal flocculation application parameters optimized by RSM were initial pH 6 ~ 10, fermentation supernatant dosage 5.3 mL, CaCl₂ dosage 6.1 mL, and rotational speed 467 rpm. Under these conditions, the flocculation rate of kaolin suspension stably reached 90.3%. MBF-PT also exhibited excellent pollutant removal performance in simulated domestic wastewater, with COD, turbidity and SS removal rates. MBF-PT is a complex acidic heteropolysaccharide mainly composed of rhamnose, glucose and mannose, with abundant hydroxyl, carboxyl and amino active groups. Comparative pan-genomic analysis revealed that S. maltophilia has a highly open pan-genome structure. The core gene clusters governing rhamnose synthesis, glycosyl transfer and Wzx/Wzy-dependent EPS transmembrane transport was widely conserved in the species. Future research will focus on the metabolic engineering modification of strain PT-13 to improve the yield of MBF-PT, and carry out pilot-scale test verification in actual wastewater treatment projects. Declarations Author contributions Linlin Zhang: Writing–original draft. Shenghui Yang: Methodology. Zhaofeng Liu: Conceptualization. Yuqing Li: Supervision. Jianjiang Lu: Supervision, Writing–review & editing. Funding This work was supported by Project supported by the Open Fund of the State Key Laboratory of Water Resource Protection and Utilization in Coal Mining (Grant No. NICE_RD_2024_297). National Natural Science Foundation of China (Grant No. 52400048), the China Postdoctoral Science Foundation (Grant No. 2024M761847) and the Natural Science Foundation of Shandong Province (Grant No. ZR2024QE482), Linyi City Key Research and Development Plan (Special Project for Industry-Academia-Research Collaboration, Grant No. 2025003). Data availability Data summarized in tables are drawn from the relevant references and can be accessed there. Supplementary Information The online version contains supplementary material available at website. Ethical approval Not applicable. Competing interests The authors declare no competing interests. References Abu Bakar S, Abu Hasan H, Abdullah S, Kasan N, Muhamad M, Kurniawan B (2021) A review of the production process of bacteria-based polymeric flocculants. 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Comput Struct Biotec 19:1458–1466. https://doi.org/10.1016/j.csbj.2021.02.021 Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterials.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 14 May, 2026 Reviewers agreed at journal 10 May, 2026 Reviewers invited by journal 08 May, 2026 Editor assigned by journal 08 May, 2026 Submission checks completed at journal 08 May, 2026 First submitted to journal 06 May, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9636700","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":640086145,"identity":"9563e6c9-fea1-4683-898f-2c29d31b3333","order_by":0,"name":"Linlin Zhang","email":"","orcid":"","institution":"Shandong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Linlin","middleName":"","lastName":"Zhang","suffix":""},{"id":640086146,"identity":"4bdaa943-1408-40a4-83bc-ed8e5feba449","order_by":1,"name":"Shenghui Yang","email":"","orcid":"","institution":"Shandong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Shenghui","middleName":"","lastName":"Yang","suffix":""},{"id":640086147,"identity":"bf107a9a-2630-4450-8c8e-e1c348a96ed4","order_by":2,"name":"Zhaofeng Liu","email":"","orcid":"","institution":"National Institute of Low Carbon and Clean Energy","correspondingAuthor":false,"prefix":"","firstName":"Zhaofeng","middleName":"","lastName":"Liu","suffix":""},{"id":640086148,"identity":"d27adcfa-cacc-4ba8-93c4-091d8318e78a","order_by":3,"name":"Yuqing Li","email":"","orcid":"","institution":"Shandong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Yuqing","middleName":"","lastName":"Li","suffix":""},{"id":640086150,"identity":"647e3eaf-a2a4-4f51-8f73-c58a103e530e","order_by":4,"name":"Jianjiang Lu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABA0lEQVRIie3RMUvEMBTA8VcK6fKut3bQqx+hh+uBX+WJcF0iOEkHNyEup7Nwx/ktxPGFQKcqjgUdKrcqnNs5CCbL4XLpKpj/UNImP0IagFDoD5YCEAMIdC+8rrKRSC7ZS8QvEunbZnKYYk19ZFtsBmp6vMzkgZ9k9MZfD+necH71bIkpFUiATXXvI6SvG4HZa3OmF0/mVMEjR7PmxUt4oOxZWkn8fm5JdENxpPxEf1uSO4LClCLGopcYt0vRlqxRTUmIPoIdmX1Lxq0E95PHCgVp31mGiTz5/FD10agtV2t7lXl+t9LdptpNAJDso3aDYvuNPettiZu/cIPOvzAUCoX+bT8qFl595pn6RAAAAABJRU5ErkJggg==","orcid":"","institution":"Shandong University of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Jianjiang","middleName":"","lastName":"Lu","suffix":""}],"badges":[],"createdAt":"2026-05-07 03:39:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9636700/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9636700/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109489944,"identity":"0c885f90-de92-4557-b413-23d955350898","added_by":"auto","created_at":"2026-05-18 17:25:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":723116,"visible":true,"origin":"","legend":"\u003cp\u003eScreening and identification of the bioflocculant-producing strain PT-13. (a) Flocculation efficiency of 18 isolated bacterial strains towards kaolin suspension, data are presented as mean ± standard error (n=3). (b) Colony morphology of strain PT-13 on LB agar plate. (c) SEM image of strain PT-13. (d) Phylogenetic tree based on 16S rRNA gene.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-9636700/v1/9e072cd1ebddb8e61c2ccc43.png"},{"id":109489937,"identity":"bda5753e-e865-40f9-96da-9c487cf446e5","added_by":"auto","created_at":"2026-05-18 17:25:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":503486,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of flocculation efficiency and bacterial growth of strain PT13 in different (a) carbon sources, (b) nitrogen sources, (c) C/N ratio and (d) dosage.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-9636700/v1/205e5ffefa6e65505e3349cd.png"},{"id":109759618,"identity":"b533bf1e-4c8f-4e44-85af-da4c63f8447d","added_by":"auto","created_at":"2026-05-22 07:27:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":74743,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different factors on the flocculation efficiency. (a) CaCl₂ concentration, (b) inoculum size, (c) rotational speed and (d) pH.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-9636700/v1/ec976f1a59b63f4ee0d247bd.png"},{"id":109489939,"identity":"cd8fa56e-bebc-4b72-b09b-c46d7db01bc0","added_by":"auto","created_at":"2026-05-18 17:25:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":629951,"visible":true,"origin":"","legend":"\u003cp\u003eThe response surface plot and contour plot of different factors\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-9636700/v1/af25360d0be72d4fd31838df.png"},{"id":109489945,"identity":"f002e2f3-a299-40c8-9187-ba036297bb58","added_by":"auto","created_at":"2026-05-18 17:25:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2673888,"visible":true,"origin":"","legend":"\u003cp\u003eSEM image of microbial flocculant at different magnifications: (a) 1000× and (b) 2000×. SEM images of kaolin particles before (c) and after (d) flocculation with MBF-PT.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-9636700/v1/4cb4ed34d46e4f8fc20ef6c7.png"},{"id":109489940,"identity":"ed687198-a72b-4ff3-8763-e7edf42386e8","added_by":"auto","created_at":"2026-05-18 17:25:56","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":139711,"visible":true,"origin":"","legend":"\u003cp\u003e(a) FTIR spectra and of MBF-PT, kaolin suspension, and kaolin flocs. (b) X-ray XRD patterns of MBF-PT, kaolin suspension, and kaolin flocs.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-9636700/v1/a9526f75cc85b223e15b3672.png"},{"id":109489941,"identity":"f7ee3b0f-91c6-4371-a5c9-cc2ff5e8eedc","added_by":"auto","created_at":"2026-05-18 17:25:56","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":264685,"visible":true,"origin":"","legend":"\u003cp\u003eHigh-resolution XPS spectra of the surface of kaolin particles before and after flocculation. (a) C 1s, (b) N 1s, and (c) O 1s spectra of raw kaolin; (d) C 1s, (e)N 1s, and (f) O 1s spectra of kaolin flocs.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-9636700/v1/0d0f289a662872e0a923bf62.png"},{"id":109760633,"identity":"18e1c673-d1f8-445d-a1d3-3a5949abe5f8","added_by":"auto","created_at":"2026-05-22 07:28:56","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":91045,"visible":true,"origin":"","legend":"\u003cp\u003eZeta potentials of different flocculation systems (a). Treatment effect of MBF-PT on simulated domestic wastewater (b).\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-9636700/v1/8ff1f6f8fa28420b62d2d6ac.png"},{"id":109489943,"identity":"f33bb0be-880d-4382-9395-7eb39bdc65f0","added_by":"auto","created_at":"2026-05-18 17:25:56","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":430486,"visible":true,"origin":"","legend":"\u003cp\u003ePan-genome accumulation curve and core genome decay curve (a). Pan-genome flower plot (b). Genome phylogenetic analysis of\u003cem\u003e S. maltophilia\u003c/em\u003e (c)\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-9636700/v1/f49be9983d1f62d70ab15780.png"},{"id":109764121,"identity":"815ff0ce-97d4-4d81-8bb1-811b45eb3da1","added_by":"auto","created_at":"2026-05-22 07:36:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5445035,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9636700/v1/60fbf67d-fd73-4e3e-8680-3312bedb3587.pdf"},{"id":109489936,"identity":"84230ae5-7481-457b-9453-fa621b484de7","added_by":"auto","created_at":"2026-05-18 17:25:56","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":21684,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-9636700/v1/0761931d737d74088b07cad5.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Phenotypic characterization and pan-genomic analysis of a novel bioflocculant-producing Stenotrophomonas maltophilia PT-13 for wastewater treatment","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCoagulation and flocculation are important technologies in water and wastewater treatment units to remove suspended particles due to its simple operation, cost-effectiveness, and wide adaptability to water quality fluctuations (Elrasoul and Yong, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Zezulka et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The traditional flocculants, including inorganic aluminum/iron salts and synthetic organic polymers, have inherent limitations despite their high efficiency (Li et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Microbial flocculants (MBFs) are secondary metabolites with high flocculation activity secreted by microorganisms during growth and metabolism, mainly in the form of extracellular polymeric substances (EPS), amino acid and glycoproteins (Mnif and Ben Rebah, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Compared with traditional chemical flocculants, MBFs exhibit advantages with excellent biodegradability, non-toxicity, no secondary pollution, and wide adaptability to complex water quality (Abu Bakar et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Ben Rebah et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). MBFs have been extensively studied in municipal wastewater, industrial wastewater, drinking water purification, and sludge dewatering (Babiak and Krzemińska, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNowadays, the most reported MBF-producing strains belong to \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003ePseudomonas\u003c/em\u003e, \u003cem\u003eKlebsiella\u003c/em\u003e, and \u003cem\u003eSerratia\u003c/em\u003e (Zhang et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Qin et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Xu et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Whole-genome sequencing has been applied to explore the biosynthesis of microbial bioflocculants, enabling the identification of genes related to exopolysaccharide (EPS) synthesis, glycosyltransfer, and transmembrane transport (Fu et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Genomic approaches allow precise localization of functional genetic elements, guide metabolic engineering to improve flocculant yield, and promote the mechanistic understanding of MBFs from the molecular level (Baranwal et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Nevertheless, current genomic research on MBFs is overwhelmingly restricted to single-strain genomic analysis, which cannot reveal the genetic diversity, core gene conservation, and multi-gene synergy of MBFs biosynthesis at the species level. Single-genome data fail to distinguish core essential genes from accessory or strain-specific genes, leading to an incomplete picture of the complex regulatory network for heteropolysaccharide assembly and secretion (Zhong et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). As a powerful strategy for population-level genetic dissection, pan-genome analysis enables the identification of core, accessory, and strain-specific genes, as well as the mining of key functional gene clusters related to target phenotypes (Hu et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Sutton et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Srivastava et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). For MBF-producing bacteria, pan-genome can systematically reveal the conserved pathways for MBFs synthesis, clarify the essential genetic determinants of high-efficiency flocculant production. To date, pan-genomic analysis has rarely been employed to reveal the genetic basis of MBFs synthesis, leaving the species-level synthetic potential and functional evolutionary mechanism largely unexplored.\u003c/p\u003e \u003cp\u003eIn this study, a novel MBF-producing bacterial strain, \u003cem\u003eStenotrophomonas maltophilia\u003c/em\u003e PT-13, exhibiting exceptional flocculation activity was isolated and identified from algal culture broth. The fermentation conditions of strain PT-13 and the operational parameters for its flocculation application were systematically optimized using single-factor tests combined with response surface methodology (RSM). The structural properties of the purified bioflocculant MBF-PT were further characterized, and its microscale flocculation mechanism was elucidated via a suite of complementary spectroscopic and microscopic techniques. Moreover, pan-genomic analysis of representative \u003cem\u003eStenotrophomonas maltophilia\u003c/em\u003e genomes was conducted to uncover the genetic potential of this species for the biosynthesis of complex heteropolysaccharides at the taxonomic species level. This study provides a promising new microbial resource for the development of eco-friendly green water treatment agents.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIsolation and screening of bioflocculant-producing bacteria\u003c/h2\u003e \u003cp\u003eThe bacterial strains were isolated from the culture fluid of \u003cem\u003ePhaeodactylum tricornutum\u003c/em\u003e which was obtained from the Institute of Oceanology, Chinese Academy of Sciences. The purified strains were inoculated into LB liquid medium and cultured at 28\u0026deg;C for 24 h. Then, the bacterial broth was inoculated into the fermentation medium containing C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u0026middot;H\u003csub\u003e2\u003c/sub\u003eO (10.0 g/L), CH\u003csub\u003e4\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO (0.5 g/L), yeast extract (0.5 g/L), NaCl (0.1 g/L), K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e (5.0 g/L), KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e (2.0 g/L), MgSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO (0.2 g/L), pH 7.2\u0026ndash;7.5. The flocculation activity was determined using the standard kaolin suspension method with minor modifications after culturing at 28\u0026deg;C and 150 rpm for 48 h. The flocculation system (100 mL) consisted of 5 mL fermentation broth, 5 mL of 1% (w/v) CaCl₂ solution, and 90 mL of 4 g/L kaolin suspension, with the pH adjusted to 7.0\u0026thinsp;~\u0026thinsp;8.0. T In the control group, the fermentation broth was replaced with an equal volume of sterile fermentation medium, with all other components unchanged. The mixture was stirred at 400 rpm for 5 min and allowed to stand for 5 min. The absorbance of the supernatant at 550 nm (OD₅₅₀) was measured using a UV-Vis spectrophotometer, with distilled water as the blank control. All experiments were performed in triplicate. The flocculation rate was calculated using the following formula:\u003c/p\u003e \u003cp\u003eFlocculation activity (%) = (A\u0026thinsp;\u0026minus;\u0026thinsp;B)/A \u0026times; 100 (1)\u003c/p\u003e \u003cp\u003ewhere A is the OD₅₅₀ of the control group, and B is the OD₅₅₀ of the experimental group. The isolate with the highest flocculation activity was named as PT13 for subsequent experiments and stored at -80\u0026deg;C.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIdentification of the bioflocculant-producing strain\u003c/h3\u003e\n\u003cp\u003eThe morphological characteristics of bioflocculant-producing stain PT13 was observed through colony morphology on LB agar plates and Scanning electron microscope (SEM) imaging of the bacterial cells. For SEM observation, the bacterial cells were collected by centrifugation at 10,000 rpm for 10 min, fixed with 2.5% (v/v) glutaraldehyde at 4\u0026deg;C for 12 h. The sample was rinsed with 0.1 M phosphate buffer saline (PBS, pH 7.0) and observed under SEM (S4800, Hitachi, Japan). The genomic DNA of the target strain was extracted using a bacterial genomic DNA extraction kit (Tiangen Beijing). 16S rRNA gene was amplified using the universal bacterial primers 27F and 1541R. The PCR products were verified by 1% agarose gel electrophoresis and sequenced bidirectionally by Tsingke Biotechnology Co., Ltd. (Qingdao, China). The obtained 16S rRNA gene sequence was aligned with the NCBI GenBank database using the BLAST tool, and a phylogenetic tree was constructed using the MEGA 6.0 software based on the maximum likelihood method.\u003c/p\u003e\n\u003ch3\u003eThe influence of different factors on the bioflocculant production\u003c/h3\u003e\n\u003cp\u003eThe experiments were conducted to investigate the influence of different factors on the bioflocculant production, with the OD₆₀₀ values and flocculation rate as evaluation indicators. For carbon source optimization of strain PT13, eight distinct carbon substrates (glucose, sucrose, mannose, maltose, fructose, xylose, sodium gluconate, and sodium acetate) were individually tested as the sole carbon source in the fermentation medium, with each substrate supplemented to a final concentration of 10 g/L. For nitrogen source optimization, strain PT13 was cultivated in the fermentation medium using six different nitrogen sources (yeast extract, peptone, NH₄Cl, KNO₃, (NH₄)₂SO₄, and urea), each added to a final concentration of 1.0 g/L as the sole nitrogen source. Subsequently, based on the screened optimal carbon and nitrogen sources, the carbon-to-nitrogen (C/N) ratio was further optimized by adjusting the ratio to 1, 5, 10, 15, and 20, respectively. For inoculation size optimization, the seed culture (OD₆₀₀ = 1.0) was inoculated into the fermentation medium at gradient inoculation sizes of 0.5%, 1%, 2%, 3%, 4%, and 5% (v/v), respectively.\u003c/p\u003e\n\u003ch3\u003eOptimization of flocculation conditions using RSM\u003c/h3\u003e\n\u003cp\u003eSingle-factor experiments were firstly performed to screen the key variables affecting flocculation efficiency and determine the appropriate level ranges for subsequent optimization. Four variables, including rotational speed, fermentation supernatant dosage, CaCl₂ concentration, and pH, were investigated in the single-factor tests. Based on the single-factor experimental results, a three-factor, three-level Box-Behnken design (BBD) was employed to optimize the critical operational parameters of the flocculation process. The three independent variables with the most significant impact on flocculation efficiency were selected as rotational speed, fermentation supernatant dosage, and CaCl₂ concentration, while flocculation efficiency was set as the sole response value of the model. The coded values and corresponding actual levels of each independent variable are summarized in Suppl. Table\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eA total of 17 experimental runs were designed and implemented in this study, consisting of 12 factorial points and 5 repeated center points to estimate the pure experimental error. The obtained experimental data were fitted to a second-order quadratic polynomial regression model using Design-Expert 12.0 software (Stat-Ease Inc., Minneapolis, MN, USA). Subsequently, analysis of variance (ANOVA) was conducted to assess the statistical significance of the established regression model and each individual model term. The optimal flocculation operational parameters were derived by solving the established regression model, and triplicate validation experiments were performed under the optimized conditions to confirm the reliability and predictive accuracy of the regression model.\u003c/p\u003e\n\u003ch3\u003eExtraction, purification and characterization of bioflocculant MBF-PT\u003c/h3\u003e\n\u003cp\u003eThe fermentation broth of strain PT13 was centrifuged at 8,000 rpm (4\u0026deg;C, 10 min) to remove bacterial cells. The cell-free supernatant was mixed with 3.0-fold volumes of pre-chilled absolute ethanol, and held at 4\u0026deg;C for 12 h for bioflocculant precipitation. The precipitate was collected by centrifugation, washed 3 times with absolute ethanol, and lyophilized for 72 h to obtain purified bioflocculant MBF-PT. The monosaccharide composition of MBF-PT was analyzed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD, ICS-5000, Dionex, USA). Briefly, 5 mg of MBF-PT was hydrolyzed with 2 M trifluoroacetic acid (TFA) at 121\u0026deg;C for 2 h in a sealed tube. The hydrolysate was evaporated to dryness under nitrogen, rinsed with methanol 3 times to remove residual TFA, reconstituted in ultrapure water, and filtered through a 0.22 \u0026micro;m hydrophilic membrane. Separation was performed on a Dionex CarboPac PA20 column (150 mm \u0026times; 3.0 mm, 10 \u0026micro;m) with gradient elution (0.5 mL/min, 30\u0026deg;C), and 15 monosaccharide reference standards were used for qualitative and quantitative analysis.\u003c/p\u003e \u003cp\u003eFourier transform infrared (FTIR) spectra of MBF-PT were recorded from 400 to 4000 cm⁻\u0026sup1; via the KBr pellet method to identify functional groups. X-ray photoelectron spectroscopy (XPS, ESCALAB 250Xi, Thermo Fisher, USA) with monochromatic Al Kα radiation was used to analyze the surface elemental composition and chemical states of MBF-PT, raw kaolin, and kaolin flocs after flocculation. X-ray diffraction (XRD, Dmax Ultima, Rigaku, Japan) patterns were collected using Cu Kα radiation over a 2θ range of 10\u0026deg;\u0026ndash;80\u0026deg; (scanning rate: 2\u0026deg;/min) to characterize the crystal structure of the samples. Scanning electron microscopy (SEM) was performed to observe the micromorphology of MBF-PT, raw kaolin, and flocculated kaolin, with samples sputter-coated with gold prior to imaging.\u003c/p\u003e \u003cp\u003eThe distribution of flocculation activity in the fermentation system was determined by measuring the flocculation rate of the original fermentation broth, cell-free supernatant, and bacterial cell resuspension, respectively. The Zeta potential of the kaolin suspension, MBF-PT solution, CaCl₂ solution, and the flocculation system at different stages was measured using a Zetasizer Nano ZS at 25\u0026deg;C. All measurements were performed in triplicate, and the average values were used to analyze the electrostatic interaction during the flocculation process.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eTreatment performance of simulated domestic wastewater\u003c/h2\u003e \u003cp\u003eThe treatment performance of the bioflocculant for simulated domestic wastewater was evaluated under the optimal flocculation conditions obtained from RSM optimization. The water quality indicators, including chemical oxygen demand (COD), turbidity, and suspended solids (SS), were measured before and after treatment. The removal efficiency of each indicator was calculated.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePangenome analysis\u003c/h3\u003e\n\u003cp\u003eA total of 37 high-quality genome sequences of \u003cem\u003eS. maltophilia\u003c/em\u003e (including complete genomes and high-quality scaffolds/contigs) were downloaded from the NCBI GenBank database. All genomes were re-annotated using the Prokka software for standardized open reading frame (ORF) prediction and functional annotation to eliminate systematic errors from different annotation pipelines. Pangenome analysis was executed by Integrated Prokaryotes Genome and Pangenom Analysis (IPGA) website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://nmdc.cn/ipga/\u003c/span\u003e\u003cspan address=\"https://nmdc.cn/ipga/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Liu et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). To depict the pangenome (core and accessory genome) assessment of strains included in the comparison, a reciprocal best hit search was also performed. The accumulation curves of the pan-genome and core genome were fitted using a nonlinear regression power-law model.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error. Statistical analyses were performed using OriginPro 2021 (OriginLab, USA). Homogeneity of variances was confirmed prior to one-way ANOVA, and Duncan\u0026rsquo;s multiple range test was applied for post hoc comparisons between experimental groups. Statistical significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, and high statistical significance was defined as p\u0026thinsp;\u0026lt;\u0026thinsp;0.01.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eIdentification and morphological characteristics of strain PT13\u003c/h2\u003e \u003cp\u003eA total of 18 bacterial strains were isolated from the phycosphere of \u003cem\u003ePhaeodactylum tricornutum\u003c/em\u003e, and their flocculation efficiencies were evaluated via the standard kaolin suspension assay. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, the flocculation efficiencies of the isolates ranged from 18.4% to 82.2% under initial fermentation conditions. Strain PT13 exhibited the highest flocculation activity (82.2%), which was significantly higher than that of all other isolates (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and was thus selected as the target strain for subsequent research. In Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(b), colonies of strain PT13 on LB agar plates were circular, pale yellow to creamy white, with smooth moist surfaces and entire margins. SEM observation confirmed that the cells were short rod-shaped, with dimensions of 0.5 \u0026micro;m \u0026times; 2.0 \u0026micro;m (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Full-length 16S rRNA gene sequence alignment showed that strain PT13 shared high similarity with multiple \u003cem\u003eStenotrophomonas. maltophilia\u003c/em\u003e strains. Given the high conservation of the 16S rRNA gene within the \u003cem\u003eStenotrophomonas\u003c/em\u003e genus, the strain was conservatively designated as \u003cem\u003eStenotrophomonas. maltophilia\u003c/em\u003e PT13, which was further validated by phylogenetic analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eOptimization of fermentation conditions for flocculant production\u003c/h2\u003e \u003cp\u003eThe effects of eight sole carbon sources on the growth and flocculation efficiency of strain PT13 are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea. The high flocculation efficiencies (\u0026gt;\u0026thinsp;90%) were achieved with glucose, xylose, sodium gluconate, or sodium acetate as the sole carbon source. In contrast, mannose, sucrose, and maltose supported the highest biomass (OD₆₀₀ \u0026gt; 2.3) but significantly lower flocculation efficiencies (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating no linear positive correlation between bacterial growth and bioflocculant biosynthesis by strain PT13 under these conditions. Notably, sodium acetate, a widely used supplementary carbon source in wastewater treatment plants, also enabled 92.3% flocculation efficiency, highlighting the potential of strain PT13 for in-situ bioflocculant production and synchronous wastewater treatment. For nitrogen source optimization (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), yeast extract yielded the highest flocculation efficiency and favorable bacterial growth, followed by urea, while peptone exhibited the poorest performance for both indicators. As a complex organic nitrogen source, yeast extract provides not only bioavailable nitrogen but also vitamins, amino acids, and growth factors that promote both cell growth and bioflocculant biosynthesis. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, flocculation efficiency and biomass of strain PT13 increased with the C/N ratio from 1 to 15, peaked at a C/N ratio of 15, and decreased markedly at a C/N ratio of 20. At low C/N ratios, carbon was the limiting nutrient, with most carbon flux directed to cell growth rather than bioflocculant synthesis. C/N ratio of 15 was thus selected as the optimal nutritional condition. The effect of inoculum dosage on flocculation efficiency is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed. No statistically significant difference in flocculation efficiency was observed across all tested inoculum dosage, indicating that inoculum dosage had no significant impact on the flocculation performance of strain PT13 within the tested range.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eOptimization of MBF-PT with RSM\u003c/h2\u003e \u003cp\u003eSingle-factor pre-experiments were conducted to determine the appropriate level ranges for response surface optimization. The optimal center levels were 5 mL for fermentation supernatant dosage and 400 rpm for rotational speed. The bioflocculant maintained excellent flocculation efficiency over pH 6\u0026ndash;10 (maximum at pH 10), thus pH was not included in subsequent optimization. A three-factor, three-level BBD was then employed to optimize the three key parameters (rotational speed, fermentation supernatant dosage, CaCl₂ dosage), with the experimental design and results summarized in Suppl. TableS2.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA second-order quadratic polynomial regression model was established to correlate flocculation efficiency with the coded independent variables:\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eY\u0026thinsp;=\u0026thinsp;88.7\u0026thinsp;+\u0026thinsp;4.01A\u0026thinsp;+\u0026thinsp;1.39B\u0026thinsp;+\u0026thinsp;4.71C\u0026thinsp;\u0026minus;\u0026thinsp;0.475AB\u0026thinsp;+\u0026thinsp;0.25AC\u0026thinsp;\u0026minus;\u0026thinsp;0.45BC\u0026thinsp;\u0026minus;\u0026thinsp;8.41A\u003csup\u003e2\u003c/sup\u003e\u0026minus;0.485B\u003csup\u003e2\u003c/sup\u003e\u0026minus;5.34C\u003csup\u003e2\u003c/sup\u003e\u003c/h2\u003e \u003cp\u003ewhere Y is the predicted flocculation efficiency (%), and A, B, C are the coded values of rotational speed (rpm), fermentation supernatant dosage (mL), and CaCl₂ dosage (mL), respectively.\u003c/p\u003e \u003cp\u003eAnalysis of variance confirmed the model was extremely statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), with a non-significant lack of fit (p\u0026thinsp;=\u0026thinsp;0.9507, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), indicating excellent goodness of fit and reliable predictive performance. The model had a coefficient of determination (R\u0026sup2;) of 0.9735, with close agreement between adjusted R\u0026sup2; (0.9435) and predicted R\u0026sup2; (0.9347), further validating its reliability. The order of significance of the three variables on flocculation efficiency was: CaCl₂ dosage (C) \u0026gt; rotational speed (A) \u0026gt; fermentation supernatant dosage (B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThree-dimensional response surface plots and contour plots (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) were used to visualize the pairwise interactive effects of rotational speed, fermentation supernatant dosage, and CaCl₂ dosage on flocculation efficiency. All response surfaces showed a convex downward-opening shape, confirming a maximum flocculation efficiency existed within the tested variable ranges. The elliptical contour plots indicated significant interactive effects between paired variables, consistent with the ANOVA results. The theoretical optimal flocculation parameters derived by solving the quadratic regression model were: rotational speed 467.8 rpm, fermentation supernatant dosage 5.3 mL, and CaCl₂ dosage 6.1 mL, with a predicted maximum flocculation efficiency of 89.6%. Triplicate independent validation experiments under these optimal conditions yielded an actual flocculation efficiency of 90.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98% (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.E.), which was in excellent agreement with the predicted value (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The adjusted R\u0026sup2; (0.9435) and predicted R\u0026sup2; (0.9347) showed a difference less than 0.2, indicating excellent fit and predictive performance of the model. Paired t-test showed no significant difference between the predicted (89.6%) and actual (90.3%) flocculation efficiency (t\u0026thinsp;=\u0026thinsp;1.22, df\u0026thinsp;=\u0026thinsp;2, p\u0026thinsp;=\u0026thinsp;0.346), verifying the reliability of the model.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStructural characterization of bioflocculant MBF-PT\u003c/h2\u003e \u003cp\u003eThe flocculation activity distribution test showed that the cell-free supernatant had a flocculation rate of 89%, which was almost the same as that of the original fermentation broth, while the bacterial cell resuspension only had a flocculation rate of 34%. This result clearly indicated that the flocculation active substance of strain PT-13 was an extracellular secretory metabolite, rather than a cell-bound component. The monosaccharide composition of MBF-PT was shown in Suppl. TableS3. MBF-PT is a complex heteropolysaccharide composed of eight monosaccharides, with rhamnose (33.06 mol%), glucose (22.17 mol%), and mannose (18.59 mol%) as the main components, accounting for more than 70% of the total monosaccharides. In addition, it also contained glucuronic acid (7.15 mol%), ribose (6.62 mol%), galactosamine (5.48 mol%), arabinose (3.83 mol%), and glucosamine (3.09 mol%). No fucose, galactose, or xylose were detected.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSEM images showed that purified MBF-PT exhibited a loose, highly porous, irregular three-dimensional network structure formed by the aggregation of fine granular and flaky subunits (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b). This unique microstructure endows MBF-PT with a large specific surface area and abundant active adsorption sites, which facilitates the capture of suspended particles and the formation of large settleable flocs via the adsorption-bridging mechanism. SEM observation of kaolin particles before and after flocculation was further performed to verify the flocculation behavior at the microscopic level. Pristine kaolin particles were discrete, irregular flakes with no obvious aggregation, whereas after flocculation treatment with MBF-PT, the particles were tightly aggregated into large, dense three-dimensional flocs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec and d). This marked morphological change provides direct microscopic evidence for the excellent flocculation performance of MBF-PT.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFTIR spectroscopy was performed to identify the functional groups of purified MBF-PT (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). The broad intense absorption band at 3420 cm⁻\u0026sup1; is attributed to O-H and N-H stretching vibrations, indicating abundant hydroxyl, amino groups and extensive hydrogen bonds in MBF-PT macromolecules. The peaks at 1645 cm⁻\u0026sup1; and 1410 cm⁻\u0026sup1; correspond to the asymmetric and symmetric stretching vibrations of carboxylate groups (-COO⁻), respectively, with the 1645 cm⁻\u0026sup1; peak also covering the amide I band of proteins. The characteristic absorption bands at 1000\u0026ndash;1150 cm⁻\u0026sup1; are assigned to C-O-C glycosidic bond and pyranose ring C-O stretching, confirming the polysaccharide backbone of MBF-PT, consistent with monosaccharide composition analysis. These results demonstrate that MBF-PT is rich in hydroxyl, carboxyl and amino groups, which are the key active sites for bioflocculation.\u003c/p\u003e \u003cp\u003eXRD showed that pristine kaolin exhibited well-defined characteristic crystalline diffraction peaks (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). The position and profile of these peaks remained completely unchanged after flocculation with MBF-PT, with no peak shift or new crystalline phase formation observed. This result confirmed that the flocculation process mediated by MBF-PT was dominated by physical adsorption and particle aggregation, rather than chemical reactions that alter the intrinsic crystal structure of kaolin. A slight decrease in the diffraction peak intensity of kaolin in the flocs was attributed to the adsorption and surface coating of amorphous MBF-PT macromolecules, which provides direct structural evidence for the adsorption-bridging flocculation mechanism of MBF-PT.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eXPS was further used to characterize the elemental composition and chemical state of MBF-PT at the electronic level. The full survey spectrum (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e) shows that MBF-PT is mainly composed of C, O and N elements. The high-resolution C 1s spectrum was deconvoluted into three peaks at 284.8 eV (C-C/C-H), 286.3 eV (C-O/C-N) and 288.1 eV (C\u0026thinsp;=\u0026thinsp;O/O\u0026thinsp;=\u0026thinsp;C-N), corresponding to the carbon skeleton, hydroxyl/amino groups and carboxyl/amide groups, respectively. The O 1s spectrum was fitted into two peaks at 531.5 eV (C\u0026thinsp;=\u0026thinsp;O) and 532.8 eV (C-O), confirming carbonyl and hydroxyl groups. The N 1s spectrum was deconvoluted into two peaks at 399.8 eV (-NH₂/-CONH-) and 401.5 eV (-NH₃⁺), indicating the presence of neutral and protonated amino groups, which provides direct evidence for the charge neutralization flocculation mechanism. The XPS results are in excellent agreement with FTIR analysis, and further reveal the chemical nature of the functional groups in MBF-PT.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eFlocculation mechanism and sewage treatment of MBF-PT\u003c/h2\u003e \u003cp\u003eZeta potential measurement was conducted to investigate the interfacial charge variation during flocculation and identify the dominant mechanism of MBF-PT, with results shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea. The pristine kaolin suspension had a Zeta potential of -27.0 mV, indicating a highly stable colloidal system due to strong electrostatic repulsion between negatively charged particles. The pure MBF-PT solution exhibited a Zeta potential of -12.0 mV, which is attributed to the ionization of abundant carboxyl groups in its polysaccharide backbone, which was consistent with FTIR and XPS results, rendering the flocculant macromolecules negatively charged overall.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter addition of CaCl₂ alone, the Zeta potential of the kaolin suspension increased significantly from \u0026minus;\u0026thinsp;27.0 mV to -15.0 mV, indicating that Ca\u0026sup2;⁺ effectively compressed the electrical double layer of kaolin particles and partially neutralized the negative surface charge, thus reducing inter-particle electrostatic repulsion. When both CaCl₂ and MBF-PT were added and floc formation was complete, the Zeta potential of the system stabilized at -10.0 mV, which did not approach the isoelectric point (IEP, 0 mV).\u003c/p\u003e \u003cp\u003eAccording to classic DLVO (Derjaguin-Landau-Verwey-Overbeek) colloid stability theory, charge neutralization as the dominant mechanism would require the system Zeta potential to approach the IEP to eliminate electrostatic repulsion and achieve particle destabilization. However, efficient flocculation was achieved in this study even with the system retaining a negative surface charge, confirming that the flocculation process was dominated by the adsorption-bridging effect, with charge neutralization playing an auxiliary synergistic role.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb shows that MBF-PT exhibits excellent purification efficiency for simulated domestic sewage. The initial water quality of the simulated domestic wastewater was as follows: COD 330 mg/L, turbidity 123 NTU, SS 92 mg/L. After treatment, the turbidity decreased to 23 NTU (removal rate 81.3%), and the SS decreased to 18 mg/L (removal rate 80.4%). This indicates that the polysaccharide network structure of MBF-PT is not severely disturbed in complex organic matrices and can still efficiently remove suspended colloids. In addition, the flocculant can effectively reduce COD, reducing it from 330 mg/L to 106 mg/L (removal rate of 74.2%). This indicates that MBF-PT can not only remove inorganic suspended solids, but also synergistically remove organic pollutants and partially dissolved COD in the aqueous phase through net capture and adsorption. In summary, MBF-PT maintains high flocculation activity and stability in the treatment of complex simulated domestic wastewater.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePan-genomic analysis of\u003c/b\u003e \u003cb\u003eS. maltophilia\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo gain insights into the MBT synthesis mechanism in genus \u003cem\u003eStenotrophomonas\u003c/em\u003e, we analyzed the pan-genomic data of 37 different bacterial taxa. As the number of sequenced genomes increases, the pan-genome expands progressively, whereas the core genome gradually contracts (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea). Therefore, we speculate that the pan-genome of \u003cem\u003eS. maltophilia\u003c/em\u003e is open. The genome size of \u003cem\u003eS. maltophilia\u003c/em\u003e exhibits a direct positive correlation with its gene count. Specifically, \u003cem\u003eS. maltophilia\u003c/em\u003e strain (GCF_023518235.1) possesses a maximum of 5 chromosomes, whereas \u003cem\u003eS. maltophilia\u003c/em\u003e strain (GCA_007833655.1) harbors the largest genome. We performed pan-genome and core-genome analyses on \u003cem\u003eS. maltophilia\u003c/em\u003e, and identified 2740 gene families that constitute the core genome (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eb). Among these strains, \u003cem\u003eS. maltophilia\u003c/em\u003e (GCA_900186865.1) possessed the highest number of unique genes (1677), while \u003cem\u003eS. maltophilia\u003c/em\u003e (GCA_002025605.1) possessed the lowest number of unique genes (774). Based on topological structure and evolutionary distance, the phylogenetic tree was divided into six major taxa (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003eTo systematically characterize the functional profiles of genes associated with bioflocculants, a comprehensive screening was conducted using pan-genome data. A total of 24 candidate genes were identified and subsequently classified into four functional categories: polysaccharide synthesis genes, glycoprotein synthesis genes, transport system genes, and other related genes (Suppl. TableS4). The identified polysaccharide synthesis genes serve as core regulators and functional enzymes involved in the biosynthesis of extracellular polysaccharides (EPS), which represent the primary component of bioflocculants. Additionally, other related genes are involved in maintaining intracellular metabolic balance and bacterial cell adhesion, thereby indirectly regulating the synthesis and function of bioflocculants.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cb\u003eS. maltophilia\u003c/b\u003e \u003cb\u003ePT-13 has high flocculation efficiency\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn this study, a novel bacterial strain \u003cem\u003eS. maltophilia\u003c/em\u003e PT-13 with high-efficiency bioflocculant-producing capacity was isolated from the phycosphere environment. Previous studies have reported that \u003cem\u003eStenotrophomonas\u003c/em\u003e species exhibit strong metabolic adaptability and can secrete EPS with excellent flocculation and heavy metal adsorption properties (Chen et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Qin et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). However, most reported bioflocculant-producing strains were isolated from activated sludge, soil, or industrial wastewater (Abu Bakar et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2026\u003c/span\u003e). Single-factor optimization experiments revealed that glucose was the optimal carbon source for bioflocculant synthesis by strain PT-13, while sodium acetate, a common supplementary carbon source in wastewater treatment plants, also enabled a high flocculation efficiency of 92.3%. This result highlights the potential of strain PT-13 for in-situ bioflocculant production and synchronous wastewater treatment in practical engineering, which can reduce the production cost of MBFs by using wastewater carbon sources. Yeast extract, as a complex organic nitrogen source, achieved the highest flocculation efficiency, which is consistent with the findings in \u003cem\u003eBacillus subtilis\u003c/em\u003e 35A that composite organic nitrogen sources are more conducive to the synthesis of polysaccharide bioflocculants (Dai et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The highly efficient floc producing bacterium \u003cem\u003eStenotrophomonas pavanii\u003c/em\u003e GXUN74707 isolating from municipal activated sludge showed the best fermentation and gel production efficiency when using small molecule urea as the sole nitrogen source, with a flocculation rate of up to 99.0% for kaolin suspension (Qin et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The optimal C/N ratio for bioflocculant production was determined to be 15, which is different from the optimal C/N ratio of 3:1 reported in some other MBF-producing strains (Sun et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The C/N ratio revealed the critical regulatory effect of nutrient balance on the secondary metabolism of bioflocculants. The inoculation dosage had no significant effect on flocculation efficiency within the tested range, which provides operational flexibility for the scale-up fermentation of strain PT-13 in industrial applications.\u003c/p\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eFlocculation Optimization and Wastewater Treatment Efficiency of MBF‑PT\u003c/h2\u003e \u003cp\u003eMBF-PT maintained excellent flocculation efficiency in a wide pH range of 6\u0026thinsp;~\u0026thinsp;10, with the maximum flocculation activity at pH 10. This broad pH adaptability is superior to many reported MBFs, which only exhibit high activity in a narrow neutral or slightly alkaline pH range. For example, the composite flocculant MBF-06 produced by S. maltophilia ZZC-06 only reached its peak flocculation activity at pH 8\u0026thinsp;~\u0026thinsp;9 (Chen et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). MBF-PT also exhibited well pollutant removal performance in simulated domestic wastewater with complex organic components, with COD, turbidity and SS removal rates. Similarly, the microbial flocculant was employed for the treatment of tofu industrial waste water, resulting in removal rates of 75.9% for BOD and 80.2% for COD in tofu production wastewater (Ramadhani et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).The high turbidity and SS removal efficiencies confirmed that the polysaccharide network structure and abundant active functional groups of MBF-PT were not severely disturbed by organic components in wastewater, and could still efficiently capture and aggregate suspended colloidal particles (Li et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Salehizadeh et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The substantial COD reduction further indicated that MBF-PT can synergistically remove particulate organic pollutants and partial dissolved organic matter through adsorption, net capture and sweep flocculation effects.\u003c/p\u003e \u003cp\u003eRSM was further employed to optimize the flocculation process, yielding a highly reliable regression model. The optimal operational parameters were determined as rotational speed 467 rpm, fermentation supernatant dosage 5.3 mL, and CaCl₂ dosage 6.1 mL, under which the flocculation rate of kaolin suspension reached 90.3%. The significance order of variables was CaCl₂ dosage\u0026thinsp;\u0026gt;\u0026thinsp;rotational speed\u0026thinsp;\u0026gt;\u0026thinsp;supernatant dosage. Among various metal ions, Ca\u0026sup2;⁺ is one of the most commonly used and effective in enhancing the flocculating activity of bioflocculants, as it plays a dominant role in the bridging coordination between bioflocculants and wastewater components (Sun et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). For example, Ca\u0026sup2;⁺ improved the flocculation efficiency of \u003cem\u003eBacillus megaterium\u003c/em\u003e BMBF to 96% (Selepe et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Our results further confirmed its critical role in practical wastewater treatment applications.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eStructural characterization and flocculation mechanism of MBF-PT\u003c/h2\u003e \u003cp\u003eCompositional analysis and spectroscopic characterization confirmed that the bioflocculant MBF-PT is a complex acidic heteropolysaccharide mainly composed of rhamnose, glucose and mannose, with a small amount of glucuronic acid and amino sugar residues. Notably, rhamnose was the most abundant monosaccharide in MBF-PT, which is a rare feature in the EPS of \u003cem\u003eStenotrophomonas\u003c/em\u003e species reported in previous studies. For example, the bioflocculant produced by \u003cem\u003eS. pavanii\u003c/em\u003e GXUN74707 was mainly composed of mannose with extremely low rhamnose content, while the high rhamnose proportion of MBF-PT is similar to that of the high-efficiency bioflocculant produced by \u003cem\u003ePseudomonas\u003c/em\u003e sp. HP2 (Qin et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Qi et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). As a 6-deoxy-L-mannose, rhamnose has a deoxymethyl group that endows the polysaccharide molecule with local hydrophobic microdomains, which can reduce the surface tension of the polymer at the aqueous interface, enhance the hydrophobic interaction between the flocculant and suspended particles, and improve the adsorption capacity of the flocculant on the particle surface (Pi et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Schmid et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). FTIR and XPS analysis confirmed that MBF-PT is rich in hydroxyl, carboxyl and amino groups, which are the key active sites for bioflocculation. The abundant hydroxyl and carboxyl groups can form hydrogen bonds with the oxygen-containing groups on the surface of kaolin particles, and also provide binding sites for divalent calcium ions. The presence of glucuronic acid and amino sugars gives MBF-PT the characteristics of an amphoteric polyelectrolyte, with both polyanionic groups and local cationic groups, which constitutes the material basis for its excellent flocculation performance (Zhang et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2026\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eSpecies-level genetic basis of bioflocculant biosynthesis in\u003c/b\u003e \u003cb\u003eS. maltophilia\u003c/b\u003e\u003c/p\u003e \u003cp\u003eComparative pan-genomic analysis of \u003cem\u003eS. maltophilia\u003c/em\u003e genomes was performed for the first time to reveal the genetic potential of this species for complex heteropolysaccharide bioflocculant synthesis at the population level, which fills the gap in species-level genetic dissection of MBFs biosynthesis. The pan-genome analysis revealed that \u003cem\u003eS. maltophilia\u003c/em\u003e has a highly open pan-genome structure and has strong genomic plasticity and environmental adaptability, which is consistent with its wide distribution in various ecological niches and strong metabolic versatility (Brooke, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Sutton et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The targeted genomic mining anchored the core gene clusters governing the synthesis of rhamnose and amino sugars, as well as highly amplified glycosyltransferases and the Wzx/Wzy-dependent EPS transmembrane transport assembly system in the core genome (Fu et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Guo et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Specifically, the abundant glycosyltransferases are responsible for the sequential assembly of different monosaccharide units into the polysaccharide main chain, which determines the complex monosaccharide composition of MBF-PT (Takeo et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The Wzx/Wzy-dependent transport system is responsible for the transmembrane transport and extracellular assembly of polysaccharide subunits, which is the key genetic basis for the secretion of extracellular bioflocculants by strain PT-13. These candidate genes for bioflocculant synthesis provide key targets for subsequent metabolic engineering modification to improve the yield of MBF-PT and reduce its production cost.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, \u003cem\u003eS. maltophilia\u003c/em\u003e PT-13 with exceptional bioflocculant-producing capacity was isolated. The optimal fermentation conditions for bioflocculant production were determined as glucose as the carbon source, yeast extract as the nitrogen source, and C/N ratio of 15, under which the fermentation broth exhibited the highest flocculation activity. The optimal flocculation application parameters optimized by RSM were initial pH 6\u0026thinsp;~\u0026thinsp;10, fermentation supernatant dosage 5.3 mL, CaCl₂ dosage 6.1 mL, and rotational speed 467 rpm. Under these conditions, the flocculation rate of kaolin suspension stably reached 90.3%. MBF-PT also exhibited excellent pollutant removal performance in simulated domestic wastewater, with COD, turbidity and SS removal rates. MBF-PT is a complex acidic heteropolysaccharide mainly composed of rhamnose, glucose and mannose, with abundant hydroxyl, carboxyl and amino active groups. Comparative pan-genomic analysis revealed that \u003cem\u003eS. maltophilia\u003c/em\u003e has a highly open pan-genome structure. The core gene clusters governing rhamnose synthesis, glycosyl transfer and Wzx/Wzy-dependent EPS transmembrane transport was widely conserved in the species. Future research will focus on the metabolic engineering modification of strain PT-13 to improve the yield of MBF-PT, and carry out pilot-scale test verification in actual wastewater treatment projects.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e Linlin Zhang: Writing\u0026ndash;original draft. Shenghui Yang: Methodology. Zhaofeng Liu: Conceptualization. Yuqing Li: Supervision. Jianjiang Lu: Supervision, Writing\u0026ndash;review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e This work was supported by Project supported by the Open Fund of the State Key Laboratory of Water Resource Protection and Utilization in Coal Mining (Grant No. NICE_RD_2024_297). National Natural Science Foundation of China (Grant No. 52400048), the China Postdoctoral Science Foundation (Grant No. 2024M761847) and the Natural Science Foundation of Shandong Province (Grant No. ZR2024QE482), Linyi City Key Research and Development Plan (Special Project for Industry-Academia-Research Collaboration, Grant No. 2025003).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e Data summarized in tables are drawn from the relevant references and can be accessed there.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Information\u0026nbsp;\u003c/strong\u003eThe online version contains supplementary material available at website.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e The authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbu Bakar S, Abu Hasan H, Abdullah S, Kasan N, Muhamad M, Kurniawan B (2021) A review of the production process of bacteria-based polymeric flocculants. 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Comput Struct Biotec 19:1458\u0026ndash;1466. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.csbj.2021.02.021\u003c/span\u003e\u003cspan address=\"10.1016/j.csbj.2021.02.021\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"world-journal-of-microbiology-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wibi","sideBox":"Learn more about [World Journal of Microbiology and Biotechnology](https://www.springer.com/journal/11274)","snPcode":"11274","submissionUrl":"https://submission.nature.com/new-submission/11274/3","title":"World Journal of Microbiology and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Microbial flocculant, Stenotrophomonas maltophilia, Fermentation optimization, Pan-genome analysis","lastPublishedDoi":"10.21203/rs.3.rs-9636700/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9636700/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMicrobial bioflocculants (MBFs) are eco-friendly alternatives, yet the species-level genetic basis of heteropolysaccharide-type MBFs biosynthesis remains largely unclear. In this study, a novel high-efficiency MBF-producing bacterial strain \u003cem\u003eStenotrophomonas maltophilia\u003c/em\u003e PT-13 was isolated. Fermentation conditions and flocculation operational parameters were systematically optimized using single-factor experiments combined with response surface methodology (RSM). Results showed that the optimal flocculation conditions yielded a kaolin flocculation efficiency of 90.3% with RSM. Bioflocculant MBF-PT produced by \u003cem\u003eS. maltophilia\u003c/em\u003e PT-13 was identified as an acidic heteropolysaccharide mainly composed of rhamnose, glucose and mannose, with flocculation governed by adsorption-bridging supplemented by charge neutralization. In simulated domestic wastewater, MBF-PT achieved 74.2% (chemical oxygen demand)、81.3% (turbidity) and 80.4% (suspended solids) removal efficiencies. Pan-genomic analysis revealed an open pan-genome of \u003cem\u003eS. maltophilia\u003c/em\u003e and identified conserved core gene clusters responsible for polysaccharide synthesis, glycosyl transfer and transmembrane transport. This study provides a promising microbial resource for developing green water treatment agents and offers new insights into the species-level genetic mechanism of MBFs biosynthesis.\u003c/p\u003e","manuscriptTitle":"Phenotypic characterization and pan-genomic analysis of a novel bioflocculant-producing Stenotrophomonas maltophilia PT-13 for wastewater treatment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-18 17:25:51","doi":"10.21203/rs.3.rs-9636700/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"147083034685113972850224923323171476978","date":"2026-05-14T05:53:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"138243417761941742556662256289669709354","date":"2026-05-11T00:07:04+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-05-08T07:42:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-05-08T07:24:36+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-05-08T06:33:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"World Journal of Microbiology and Biotechnology","date":"2026-05-07T03:31:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"world-journal-of-microbiology-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wibi","sideBox":"Learn more about [World Journal of Microbiology and Biotechnology](https://www.springer.com/journal/11274)","snPcode":"11274","submissionUrl":"https://submission.nature.com/new-submission/11274/3","title":"World Journal of Microbiology and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"dc254cd9-42da-498d-a3ed-068d5b346704","owner":[],"postedDate":"May 18th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"147083034685113972850224923323171476978","date":"2026-05-14T05:53:04+00:00","index":15,"fulltext":""},{"type":"reviewerAgreed","content":"138243417761941742556662256289669709354","date":"2026-05-11T00:07:04+00:00","index":13,"fulltext":""},{"type":"reviewersInvited","content":"7","date":"2026-05-08T07:42:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-05-08T07:24:36+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-05-08T06:33:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"World Journal of Microbiology and Biotechnology","date":"2026-05-07T03:31:32+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-18T17:25:52+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-18 17:25:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9636700","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9636700","identity":"rs-9636700","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}