Study of the structure and characterization of membranes reinforced with CuO-NPs/Graphene Oxide using bacterial cellulose extracted from Sargassum sp. for water nanofiltration system

Bacterial cellulose membranes find extensive applications in industries involving water purification, wastewater treatment, and biomedical uses. Nevertheless, prevailing membranes suffer from drawbacks like water flow hindrances and fouling susceptibility. Hence, the pressing need for more efficient and robust BC membranes. This study aims to assess the impact of a hybrid treatment involving Copper Oxide nanoparticles (CuO-NPs) and Graphene Oxide (GO) on bacterial nanocellulose membranes. The research employed two treatments: (1) control (BNCA), and (2) Bacterial cellulose infused with 0.5 wt% CuO-NPs/GO. The nanocellulose production involved a high-pressure homogenizer, followed by acetate nanocellulose synthesis and nanocomposite membrane functionalization with CuO nanoparticles. SEM, FTIR, and XRD analyses characterized the membranes. Successfully formed seaweed-derived bacterial cellulose had a thickness of 1-3 cm. Characterization showed it belonged to Cellulose type I with a crystalline degree ranging from 82.3% to 83.1%. FTIR analysis of dry BNCA membranes indicated changes in transmittance at 1738, 1554, and 764 cm-1 due to CuO-NPs/GO addition, altering the O-H bond in bacterial cellulose. Based on the results of the above research, it is evident that the Membrane Reinforced with CuO-NPs/Graphene Oxide has been successfully developed and holds potential as a water nanofiltration system.


Introduction
Currently, it is known that aquatic ecosystems are directly or indirectly the ultimate destination for pollutants [1], and to maintain environmental quality, the management of wastewater containing heavy metals has become one of the major challenges in recent decades [2].Polluted waters pose serious problems that endanger human health and aquatic biota [3].Water quality treatment processes in fish farming installations can include physical filtration systems, chemical filtration, and biological filtration.The filters in these systems function to biologically neutralize compounds such as ammonia and other toxic substances.Filters act as devices that can trap small particles before they enter the cultivation area [4].
Water quality management for aquaculture purposes is crucial because water serves as the living medium for aquatic organisms.One method used to maintain water quality in cultivation media is through the use of filters [5].Filtration technology is also widely employed to capture and concentrate virus pathogens transmitted through water [6], from samples of drinking water, the environment, recreation, or wastewater [7,8].An environmentally friendly alternative that can be used as a filter is a material derived from bacterial nanofiber cellulose from seaweed [9,10].The application of cellulosebased materials (adsorbents, photocatalysts, and filtration membranes) for treating wastewater contaminated with harmful pollutants has been widely conducted.Generally, the affinity of native cellulose fibers for organic pollutants is 100 to 500 times lower than that of conventional nanomaterials, such as zeolite or activated carbon, due to the low number of active sites for interaction with pollutants [11].As an alternative, surface-modified nanocellulose has been tested as a support material for the adsorption of various organic pollutants [12,13].
To enhance the adsorption capacity of cellulose, it is necessary to modify the structure of bacterial cellulose by forming it into nanocomposites.Although some studies have explored cellulose-based filters, they still require improvements in performance and effectiveness.So far, the hybrid application of CuO/GO nanoparticles for filters made from acetic acid nanocellulose from sargassum has not been found.Therefore, efforts to strengthen bacterial nanocellulose for water filtration in aquaculture for water quality management by synergizing Cu and Graphene Oxide nanoparticles to form nanocomposites are needed, aiming to produce nanocomposites that can absorb microbes and water pollutants.

Materials
Sargassum, sourced from the seawaters surrounding Madura Island in East Java, Indonesia, was obtained for further processing.Acetobacter xylinum, a bacterium known for its cellulose-producing capabilities, was used in the bacterial cellulose production.Sugar (C12H22O11) and urea (CH4N2O) were utilized as fermentation reagents to support bacterial growth and cellulose synthesis.

Synthesis of Bacterial Cellulose
The synthesis of bacterial cellulose was followed Yanuhar et al.'s study [14].To extract the juice, a blender machine was employed to crush 500 g of sargassum.Water was added to the sargassum extract to reach a total volume of 2 liters, followed by boiling it for 30 minutes. 5 grams of urea and 150 grams of sugar was dissolved in Sargassum extract and the pH was adjusted to 4.5 by adding acetic acid.After boiling, the solution was cooled to 30°C at room temperature.Subsequently, A. xylinum (20%v/v) was added into the medium, which was then fermentation was conducted for ten days.The resulting bacterial cellulose formed a clear pellicle that floated on the surface of the liquid.The pellicle was harvested and cleaned with water to eliminate any residues.It was then boiled in NaOH 1.0M at 90°C for 2 h, then it washed with distilled water until the pH reached 7.0.

Bacterial Nanocellulose Extraction
The homogenization technique was referenced from previously published articles [15].To begin, pellicles (50 g) were cleaned and then placed in a blender chamber (ICH.DS7 Fomac, China) containing 1 L water for homogenization.The pellicle was crushed for 5 min at 26,000 rpm.Subsequently, a High-Pressure Homogenization machine, specifically the AH-100D model from Berkley Scientific, was employed for the fibrillation process.This process involved 5 cycles at 150 bar.Following the fibrillation, filtration of solution was applied to obtain nanocellulose.

Nanocomposite Membrane Synthesis
A method was employed to create a BNCA-based nanocomposite.Initially, 2.5 grams of bacterial nanocellulose (BNC) and 50 milliliters of CH3COOH were combined in a glass beaker and stirred for 30 minutes at room temperature.Next, 0.32 milliliters of H2SO4 and 18 milliliters of CH3COOH were introduced to the solution and stirred for 25 minutes.The resulting solution was then filtered.Afterward, 64 milliliters of acetic anhydride were added to the filtered solution and stirred for 30 minutes.Afterwards, the solution was permitted to undergo precipitation for a duration of 14 hours.The resultant precipitate was rinsed with distilled water until achieving a pH level of 7. Subsequently, the precipitate was introduced into 200 milliliters of distilled water and stirred for 30 minutes, followed by a 30-minute session of sonication with an ultrasonic homogenizer.Elsewhere, prepare CuO-NPs/GO in various contents of 1.0%w of dry BNCA.Each CuO-NPs/GO concentration was dispersed in dH2O (80 mL) using sonication of 400 watts and 20 kHz (type UP-400S, Lawson Sci., China) for 30 min.The stirring of homogeneous suspension was conducted by 300 rpm for 2 h.Once the process was completed, cellulose acetate was mixed into the suspension and homogenized for one hour using sonication process.Subsequently, the solvent was poured into a petri dish and dried for a 24-hour at 60 C.After dry, the sample was saved in a desiccator for storage.

Observation of Morphology
Surface characteristics were assessed using Scanning Electron Microscope (SEM) analysis with an Inspect-S50 model from FEI, USA.Before the analysis, a 10 nm gold coating was applied to the BC membrane surface using an Emitech SC7-620 sputter coater.

Analysis of Structure
To examine the crystalline structure of BNC, X-ray diffraction (XRD) was employed with a PANalytical Expert-Pro instrument.The XRD analysis produced a diffractogram to determine the degree of crystallinity and crystalline index of BNC.The scanning range for XRD was set within the 2θ range of 5 degrees to 50 degrees, using CuKα (λ) radiation with a wavelength of 1.54 Å, at a current of 30 mA and a voltage of 40 kV (Suryanto et al. 2020).The Segal method was used to calculate the crystalline index (CI) and the degree of crystallinity (Cr), as illustrated in equations ( 1) and ( 2 Where I(am) is the intensity at an angle of 18 o , and I(002) is the maximum intensity at 22 o -23.

FTIR Analysis
The analysis of changes in functional groups within the BNCA membrane utilized the Fourier Transform Infra-Red spectrometer (FTIR), specifically the Shimadzu IR Prestige-21 model.In this procedure, 0.1 mg BNCA membrane samples were ground into powder and mixed with 1 mg of KBr powder.The resulting mixture was then pelletized by pressing to facilitate measurement.The FTIR analysis covered a wavenumber range of 400 to 4000 cm-1, with a rate of 4 cm-1.

Surface Morphology of Membrane
The addition of CuO-NPs/GO to BNC results in changes to the surface morphology of the membrane, as depicted in Figure 1.This morphology reveals a smooth surface with small pores (Figure 1A).The addition of 1.0%wt CuO-NPs/GO produces fibrous cellulose on the surface of the BNC membrane (Figure 1B).In addition to CuO-NPs/GO clustering on the surface, they are mixed with BNC within the membrane, and a greater accumulation of CuO-NPs/GO is evident.This morphology presents a smooth surface with small pores.The addition with 1.0%wt CuO-NPs/GO results in fibrillated cellulose fibers on the surface of the BNC membrane (Figure 2).In addition to the CuO-NPs/GO clusters on the surface, CuO-NPs/GO are also blended with BNC inside the membrane, leading to increased CuO-NPs/GO agglomeration.

The Results of the XRD Analysis
Based on the diffraction analysis, there are several main peaks that can be observed at 2θ angles.These peaks are located at angles of 14.31°, 16.61°, 22.63°, 35.41°, and 38.69°.A study carried out by Suryanto et al. (2018) [16] identified the peak structure of bacterial cellulose within the diffraction angle range of 22° to 23°, indicating the presence of type I cellulose (natural/native cellulose).While the peaks of Graphene's diffraction pattern at 2θ are 13.7° and 26.5°.This peak of Graphene is covered by a crystalline plane of cellulose (1"1" 0) at a large and intense 14.7° angle [17].The reduction in crystallinity can be attributed to the interaction between CuO-NPs/GO and the cellulose matrix, potentially affecting hydrogen bonding and intermolecular interactions [18].Figure 2 depicts the configuration of both the BNCA membrane and the BNCA when combined with CuO-NPs/GO.FTIR tests were performed on different BNCA membranes to examine the variations in functional groups pre and post reinforcement.The range spanning from 4000 to 500 cm−1, is typically linked to the functional group region.Within this range, different chemical bonds and functional groups display characteristic absorption bands, enabling researchers to identify specific chemical groups in the sample.The FTIR spectrum analysis of BNCA reveals peaks at 1738, 1554, and 764 cm−1, indicating CuO-NPs/GO interaction.The addition of Graphene to the nanocomposite membrane causes a reduction in the intensity of the O-H group.Moreover, no additional peaks were observed in the spectrum of BC Graphene, which is an indication of the absence of chemical interactions between BC and Graphene.It can be concluded that the interaction between BC and Graphene is only on the mechanical properties, which is the bundling of Graphene nanosheets by BC nanofibers in BC-Graphene [19].
Based on the results of the above research, it is evident that the Membrane Reinforced with CuO-NPs/Graphene Oxide has been successfully developed and holds potential as a water nanofiltration system.An alternative environmentally friendly material that can be utilized as a filter is material derived from bacterial cellulose nanofibers from seaweed.The advantage of cellulose nanofibers lies in their very high surface area-to-volume ratio, where one of the key triggering factors to enhance the interaction between adsorbents and metal ions is the surface area.Therefore, efforts to strengthen bacterial nanocellulose as a water filtration medium in aquaculture for water quality management, by synergizing copper nanoparticles and graphene oxide nanoparticles to form a nanocomposite, aim to produce a nanocomposite capable of absorbing microbes and water pollutants.

Conclusion
The results of this study have successfully synthesized a CuO-NPs/GO-reinforced BNCA nanocomposite membrane.The addition of stabilizing agents influences their morphology.XRD analysis revealed distinct peaks at 2θ angles of 14.31, 16.61, 22.63, 35.41, and 38.69.FTIR analysis of the BNCA membrane revealed peaks at 1738, 1554, and 764 cm−1, signifying the CuO-NPs/GO interaction.

Figure 2 .
Figure 2. Diffraction pattern of the BNCA membrane and the BNCA nanocomposite membrane strengthened with CuO-NPs/GO through acetate treatment