The global surge in water pollution, coupled with concerns over plastic waste and energy scarcity, necessitates the development of eco-friendly fabrication methods for renewable, bio-based materials. Biodegradable biopolymers, encompassing polyesters (like polyhydroxyalkanoates (PHA), polylactide (PLA), and polyethylene furanoate (PEF)), polysaccharides (such as cellulose, chitin, and alginate), and polyamides (including γ-poly (glutamic acid) (PGA), silk, and collagen), are attracting significant attention due to their potential in addressing these challenges. Bacterial cellulose (BC), a biopolymer produced by microbes, has emerged as a particularly promising candidate. Its biosynthesis relies on glucose, fructose, and glycerol as primary carbon sources, with the nutrient medium formulation and culture conditions (shaking or static) significantly influencing BC production, fiber morphology, and network assembly. While shaking cultures generally favor efficiency, static cultures maintain genetic stability and yield membranes with the high quality, size, and shape crucial for applications like water treatment. BC possesses unique structural and physicochemical properties, including flexibility, tensile strength (Young Modulus of 15-18 GPa), high water holding capacity (over 100 times its weight), large surface area (high aspect ratio fibers with diameters of 20-100 nm), ease of functionalization, and permeability to gases and water. These versatile properties make BC suitable for diverse applications in medicine (artificial skin, wound dressings, drug delivery systems) and various industries (food, paper, textiles, electronics). However, pure BC's use as a membrane for pressure-driven filtration is limited by lower compressive strength, lower permeance, and minimal pollutant retention. The presence of only -OH groups in its native structure, and its lack of inherent antibacterial properties, contribute to higher fouling susceptibility. Dehydration during evaporation and compressive stress leads to fiber stacking, permanently collapsing open pores, reducing flux, and increasing operational costs. Despite these limitations, BC's inherent structure—with straight-through channels formed during biosynthesis due to the natural arrangement of loose fibers—offers an ideal starting point for developing improved membranes. The abundance of hydroxyl groups facilitates hydrogen bonding, creating dense layers. However, the interlayer distance resulting from natural fiber stacking hinders the formation of straight-through passages, reducing compressive strength and fluid permeance. To address this, incorporating a reinforcing filler like graphene oxide (GO) is considered to enhance compression resistance. GO offers high surface area (2600 m²g⁻¹), favorable physicochemical and mechanical properties (Young modulus of 300 GPa), and ultrafast transport capabilities (71 LMH bar⁻¹) due to its oxygen-containing functional groups. Increasing pore numbers and reducing capillary tube length enhances permeability. This requires more pores in the X-Y direction during biosynthesis and looser fiber orientation in the Z direction. While porogens like Ca-alginate and paraffin wax enhance pore count and inhibit Z-directional stacking, they pose challenges in removal after harvesting. PEG, offering chain mobility, an excluded volume effect, high water content, and low interfacial free energy, emerges as a superior alternative porogen. Its hydrophilic nature and weakly basic ether linkages deter protein adhesion and generate nano-sized pores, improving membrane flux and fouling resistance. Many existing BC membrane development techniques utilize toxic or expensive chemicals (e.g., poly(hexamethylene guanidine hydrochloride)), complex processes (sulfonation, TEMPO-mediated oxidation, phosphorylation), or inadequate porogen removal. Many methods compromise BC's innate properties by defibrillating its native fibrillar structure. Methods to enhance BC's compressive strength, such as cross-linking with agents like epichlorohydrin and glutaraldehyde, suffer from toxicity issues. This study addresses these challenges by exploring an in-situ approach for synthesizing BC-GO composite membranes with optimized pore distribution, mechanical stability, and anti-fouling behavior for pressure-driven water filtration.

The literature extensively covers the use of bacterial cellulose (BC) and graphene oxide (GO) in various applications, but their combination for water filtration membranes requires a more focused review. Studies on BC highlight its biocompatibility, high water absorption capacity, and tunable mechanical properties, making it attractive for biomedical applications and tissue engineering. However, its inherent weaknesses, such as low mechanical strength and susceptibility to fouling, limit its use in water filtration. Modifications to overcome these limitations include chemical functionalization, the incorporation of other polymers or nanomaterials, and the use of different biosynthesis techniques. Graphene oxide (GO), on the other hand, is well-established for its superior mechanical properties, high surface area, and tunable surface chemistry. Its use in membrane technology has focused on enhancing the performance of existing polymeric membranes, improving their water permeability, and reducing fouling. However, the direct incorporation of GO into BC for membrane fabrication is relatively unexplored, with limited studies focusing on the optimization of GO loading and the impact on membrane properties. This gap in research highlights the novelty and importance of this study, which aims to combine the unique properties of BC and GO to create a high-performance, sustainable water filtration membrane.

This study employed an in-situ synthesis method to fabricate BC-GO composite membranes. The process began with the revival of *Komagataeibacter hansenii* (ATCC 53582), a cellulose-producing bacterium, from a frozen vial using Hestrin-Schramm (HS) medium. *E. coli* DH5a was also used for bacterial retention tests. Graphene oxide (GO) nanosheets were synthesized from graphite powder using a modified Hummer's method, followed by preparation of GO dispersions in deionized (DI) water and poly(ethylene glycol) (PEG-400) at various concentrations. The in-situ synthesis involved adding these GO dispersions to the inoculated HS medium in sterile Petri dishes, followed by static incubation at 26 °C. The BC-GO composite membranes were harvested at different time intervals, purified by rinsing with DI water and 0.1 M NaOH treatment, and then air-dried. BC yield was determined gravimetrically, measuring the dry weight of BC per volume of medium. Membrane morphology was analyzed using Scanning Electron Microscopy (SEM), while pore size distribution was determined via the molecular weight cut-off (MWCO) method using PEG and polyethylene oxide (PEO) solutes. Physicochemical characterizations included Fourier Transform Infrared (FTIR) spectroscopy, Raman spectroscopy, Thermogravimetric analysis (TGA), streaming potential measurements, contact angle measurements, and compression measurements. Membrane filtration performance was assessed using a cross-flow filtration setup, evaluating pure water flux (PWF), fouling behavior using a synthetic natural organic matter (NOM) solution (BSA, SA, and HA), and bacterial retention using *E. coli* solutions. Flux recovery ratio (FRR) was calculated after cleaning with DI water. Bacterial concentrations were determined using plate counting and flow cytometry (FCM) methods, analyzing intact and membrane-compromised cell counts. Statistical analysis involved one-way and two-way ANOVA tests. Specific details of the characterization techniques include: * **SEM:** FEI Nova NanoSEM 650 Scanning Electron Microscope (5 kV electron beam energy, 100 µA emission current, <10 mPa chamber vacuum). Gold coating (7 nm) was applied for improved conductivity. * **MWCO:** Cross-flow filtration setup (2 bar pressure, 25 °C) using PEG and PEO solutes (10,000–300,000 g/mol), analyzed with a TOC analyzer. * **FTIR:** PerkinElmer Frontier spectrometer (4000–500 cm⁻¹ wavenumber range, 4 cm⁻¹ resolution, 32 scans). * **Raman:** WITec Alpha 300 R confocal micro-Raman spectrometer (532 nm laser excitation). * **TGA:** NETZSCH High Temperature TGA (20–800 °C at 10 °C/min, nitrogen gas flow). * **Streaming Potential:** Anton Paar SurPASS3 Electrokinetic Analyzer (10 mM KCl background solution). * **Contact Angle:** Krüss GmbH DSA25 drop shape analyzer (5 µL DI water droplets). * **Compression:** HAAKE Mars III Rheometer (25 °C, 100 µm/s strain rate). * **Flow Cytometry:** Accuri C6+ flow cytometer (propidium iodide and SYBR Green 1 staining).

The in-situ synthesis method yielded BC-GO composite membranes with significantly improved properties compared to pristine BC membranes. The optimal GO loading was determined to be 2 mg/mL, resulting in a nearly twofold increase in water flux (380 L m⁻² h⁻¹) and a sixfold increase in wet compression strength. The composite membranes exhibited excellent flux recovery (over 95%) when challenged with synthetic organic foulants and bacterial solutions. Rejection rates for synthetic NOM and bacterial solutions were also above 95%, indicating enhanced fouling resistance and selectivity. Morphological analysis revealed that at lower GO concentrations (0.5–2 mg/mL), GO nanosheets were uniformly dispersed within the BC matrix, creating a percolated network structure. At higher concentrations, GO nanosheets aggregated, negatively impacting membrane properties. The MWCO method showed a reduction in average pore size from 64 nm in pristine BC to 42 nm in BC-GO (2 mg/mL) composites, yet with a broader pore size distribution, ranging from 20 to 90 nm. Porosity increased by 32–35% for the optimal GO loading. FTIR and Raman spectroscopy confirmed the interaction between GO and BC, with changes in peak intensity and the appearance of new bands indicating hydrogen bonding. TGA showed enhanced thermal stability of the BC-GO composites. Streaming potential measurements revealed a significantly more negative surface charge for BC-GO composites, contributing to their anti-fouling properties. Water contact angle measurements demonstrated superhydrophilicity for BC-GO composites, further enhancing anti-fouling performance. Compression modulus of BC-GO (2 mg/ml) was fourfold higher than Pristine-BC. Filtration performance testing showed a superior PWF and stable flux for the BC-GO composite membranes compared to pristine BC. The BC-GO composites showed a higher flux recovery ratio (FRR) during both NOM and bacteria filtration experiments, indicating superior antifouling properties. Flow cytometry analysis demonstrated that BC-GO membranes achieved a log reduction of five for *E. coli*, corresponding to a 99.99% rejection rate, significantly higher than pristine BC. Post-filtration SEM images revealed fewer adherent bacteria on the BC-GO membranes, highlighting their self-cleaning ability.

The results demonstrate that the in-situ incorporation of GO into the BC matrix, using PEG-400 as a porogen, is an effective strategy for creating high-performance water filtration membranes. The enhanced flux and fouling resistance observed in the BC-GO composite membranes can be attributed to a combination of factors. The improved mechanical strength, due to GO reinforcement, prevents pore collapse under pressure, maintaining high permeance. The increased hydrophilicity, owing to the incorporation of both GO and PEG, creates a hydration layer that inhibits foulant adsorption. Furthermore, the negative surface charge of the BC-GO membranes enhances electrostatic repulsion, further reducing fouling. The enhanced negative charge density increases the repulsion against the negative charged foulants, thereby mitigating their deposition onto the membrane surface. The superior bacterial rejection observed suggests that GO also contributes to antimicrobial activity, likely through its interaction with bacterial cell membranes. The broader pore size distribution observed in the BC-GO membranes might be beneficial for reducing both pore blocking and cake layer formation, contributing to the superior antifouling performance. The combination of enhanced mechanical strength, hydrophilicity, and electrostatic repulsion offers a synergistic effect, leading to the observed improvements in flux and fouling resistance. These findings are significant because they demonstrate the potential of this novel membrane material to provide a sustainable and effective solution for various water purification applications, particularly those requiring high flux and high fouling resistance.

This study successfully demonstrated the fabrication of high-performance BC-GO composite membranes using a novel in-situ synthesis method. The resulting membranes exhibited significantly enhanced water flux, mechanical strength, and antifouling properties compared to pristine BC membranes. The optimal GO loading of 2 mg/mL resulted in superior performance in terms of flux, flux recovery, and bacterial rejection. The findings highlight the potential of these membranes for various water treatment applications. Future research could focus on optimizing the biosynthesis process, exploring different GO and PEG concentrations, and investigating the long-term stability and performance of these membranes in real-world wastewater treatment scenarios. Further investigation into the antimicrobial mechanism of the GO-BC composite is also warranted.

This study primarily focused on laboratory-scale experiments using synthetic NOM and *E. coli* solutions. The performance of these membranes in real-world wastewater with complex mixtures of foulants needs further investigation. The long-term stability and durability of the membranes under continuous operation also need to be assessed. While the study demonstrated effective cleaning with DI water, the effectiveness of other cleaning methods and the potential for membrane degradation during cleaning should be explored. Finally, a more comprehensive life cycle assessment considering the energy consumption and environmental impact of membrane production is needed to fully evaluate its sustainability.