Phosphate ester-linked carbonized polymer nanosheets as efficient filters for removing Vibrio parahaemolyticus from aquaculture water

Phosphate ester-linked carbonized polymer nanosheets as efficient filters for removing Vibrio parahaemolyticus from aquaculture water | 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 Article Phosphate ester-linked carbonized polymer nanosheets as efficient filters for removing Vibrio parahaemolyticus from aquaculture water Chih-Chhing Huang, Anisha Anand, Binesh Unnikrishnan, Chen-Yow Wang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4153360/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Sep, 2024 Read the published version in npj Clean Water → Version 1 posted 11 You are reading this latest preprint version Abstract In this study, we have developed a simple and low-temperature method without using toxic chemicals, to synthesize carbonized polymer nanosheets (CPNSs) that exhibit potent bacterial adsorption capabilities for their use as a filter to remove bacteria from aquaculture water. Sodium alginate (Alg), an algae-derived polysaccharide was pyrolyzed with diammonium hydrogen phosphate (DAHP) in solid state at 180 °C. Initially, Alg underwent dehydration and cross-linking via phosphate ester bonds followed by carbonization resulted in the formation of 2D structured CPNSs with distinct polymeric characteristics. The as-synthesized CPNSs demonstrate a high bacterial adsorption capability toward V. parahaemolyticus and S. aureus . Furthermore, CPNSs can be used to modify ordinary filter paper to make them effective in filtering system. Aquaculture water filtration experiments using CPNSs-modified filter paper revealed an increase in the survival rate (> 50%) of shrimp challenged with V. parahaemolyticus upon circulation through the Alg-CPNSs-modified membrane, demonstrating their potential as a viable aquaculture filter. Earth and environmental sciences/Environmental sciences/Environmental chemistry/Pollution remediation Physical sciences/Nanoscience and technology/Nanoscale materials Physical sciences/Materials science/Structural materials Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The properties and ultimate applications of carbon-based nanomaterials (CNMs) are defined by their structure and surface functionalization, hence research endeavors are directed toward fine-tuning these characteristics 1 . 2D nanosheets (NSs) are remarkable for their nanoscale thickness and high surface-to-volume ratio, attributes that render them suitable for diverse applications such as energy storage devices, optoelectronics, sensors, composite materials, water purification and filtration, and even biomedical applications 2 – 7 . Particularly, carbon nanosheets (CNSs), including graphene, graphene oxide (GO), and reduced graphene oxide (rGO), have demonstrated efficacy in areas like energy storage and batteries, fuel cells and solar cells, gas separation, and biomedical applications 4 , 8 – 11 . These benefits can be attributed to their unique honeycomb structure and the ease of surface functionalization, which enables tailoring for specific applications 1 , 4 . Besides the sheet-like structures, there has been a rise in reports on CNMs with different morphologies like carbon microtrees, flower-like carbon, carbon cones, carbon cubes, and carbon nanocontainers in the past years 12 – 16 . Despite these varied structures, 2D nanomaterials are favored for creating stable thin films and membranes due to their large surface area, customizable porosity, high chemical stability, and potential for layered arrangements 17 . CNSs have displayed potential for the adsorption of organic pollutants from water, including pesticides and pharmaceuticals 18 , 19 . Furthermore, the CNS-based nanocomposites can also function as photocatalysts, initiating the breakdown of these pollutants 20 . Additionally, CNSs have exhibited antimicrobial or bacteria adsorption properties, offering an effective method for deactivating or eliminating harmful microbes when used in water purification processes 21 – 24 . Various methods for synthesizing CNSs have been adopted, including chemical vapor deposition (CVD), mechanical/ultrasonic/chemical/electrochemical exfoliation, epitaxial growth, solvothermal synthesis, and thermal decomposition 2 , 25 – 27 . Each of these methods possesses unique requirements suited to its specific synthesis process. For instance, CVD requires hydrocarbon gases as precursors, exfoliation is most effective with raw materials featuring a multi-layered structure and weak inter-layer interactions, and solvothermal synthesis typically employs small molecules 2 . Control over the sheet size has been achieved through different parameters and approaches such as the utilization of oxidants, density gradient ultracentrifugation, pH-assisted sedimentation, and the application of sonication. However, these methods often require harsh reaction conditions, result in high costs, and lower yield 2 , 28 , 29 . Furthermore, CNSs tend to restack, compromising the unique properties of single-layered structure. The introduction of functional groups on CNS mitigates this issue 1 . Furthermore, many existing methods for synthesizing CNSs are typically constrained to small-scale laboratory processes. Scaling up these synthesis techniques while preserving the desired properties and quality of the materials remains a significant challenge. Consequently, the current research is centered on devising efficient, scalable, and economically viable methods for synthesizing stable CNSs using cost-effective precursors, such as biomass 27 , 30 , 31 . While existing methods for the synthesis of CNSs often involve high temperatures, solvents, or sophisticated instruments, our approach focuses on a simple low-temperature synthesis procedure, and the use of marine polysaccharides. In this study, we developed carbonized polymer nanosheets (CPNSs) from the polysaccharide, sodium alginate (Alg) and diammonium hydrogen phosphate (DAHP) through low-temperature synthesis in solid-state (Scheme 1 ). We proposed the formation mechanism of CPNSs could be through the cross-linking of Alg units via phosphate diester linkages. Though previous reports indicate that CNSs exhibit antibacterial activity toward both Gram-negative and Gram-positive bacterial species 32 – 35 , our CPNSs do not possess inherent antibacterial activity. Nevertheless, when adsorbed onto a filter paper support, they effectively remove bacteria via specific adsorption, and no leakage of bacteria was observed from the membrane even after 24 h. The recirculating aquaculture system (RAS) has demonstrated its eco-friendly nature, water efficiency, and exceptional productivity in farming 36 . However, if pathogenic bacteria are introduced into the RAS, they may survive and recirculate in the system which poses a potential risk to the aquatic species in the system 37 . Consequently, we employed these CPNS-modified filter papers for the successful removal of Vibrio parahaemolyticus ( V. parahaemolyticus ) from contaminated aquaculture water, which showed a marked improvement in the survival rate of the shrimp population. Results and Discussion DAHP plays a crucial role in the formation of CPNSs CPNSs obtained by heating a solid mixture of Alg and DAHP in 1:5 mass ratio at 120, 150, 180, 210, and 240°C for 3 h were denoted as CPNSs-120, CPNSs-150, CPNSs-180, CPNSs-210, and CPNSs-240, respectively. The Alg/DAHP mixture was colorless and showed a mild color change to off-white at 120°C. The mixture experienced mild dehydration at 150°C, resulting in light brown hue, and showed higher degree of carbonization at 180°C and above displaying brown or black color (Fig. 1 A). The TEM images in Fig. 1 B show that the Alg/DAHP mixture without heating had a gel-like structure, and the morphology changed to polymeric form at 120°C. From 150°C and above large sheet-like formation can be observed. At 180°C, the mixture forms 2D layered CPNSs. The resulting CPNSs-180 have sizes ranging between 200 and 500 nm, with a thickness of approximately 1.43 ± 0.25 nm and surface roughness was calculated to be 0.23 nm, as determined by atomic force microscopy (AFM) (Fig. 1 C). This thickness is considerably greater than that of single-layered graphene, which ranges from 0.4 to 1.0 nm, and GO, with a range of 0.7−1.2 nm 38 . The surface roughness (0.23 nm) is higher than that reported (0.2 nm) for single-layer free-standing chemically modified GO 39 , which suggests the presence of polymeric alginate fragments on the CPNSs. At higher carbonization temperatures of 210 and 240°C, sheet-like structures were formed; however, along with other carbonized products with different morphology and their aggregated forms were adsorbed onto the sheets (TEM images in Fig. 1 B). Alg suspended in sodium phosphate buffer (5 mM, pH 7.4) exhibits a negative charge, characterized by a zeta potential of ca. −50 mV (Supplementary Table 1 ). The zeta potential slightly changed as the synthesis temperature increased during the preparation of CPNSs to ca. −46 mV for 180°C. However, with subsequent temperature increments, the zeta potential decreased significantly to ca. −11 mV, probably due to a higher degree of carbonization resulting in elimination of carboxylate functional groups. As the synthesis temperature increased, the hydrodynamic size of the CPNSs increased significantly for 210 and 240°C. The thermal-driven dehydration and cross-linking of Alg lead to the formation of sheet-like structures. The significant carbonization at higher temperatures results in the stacking of CPNSs and other carbonized particles to form larger aggregates. The UV-vis absorption spectra of the CPNSs prepared at different temperatures are presented in Supplementary Fig. 1. CPNSs obtained at 180°C and above showed a band at ca. 285 nm and extending to the visible region of the spectra attributed to the π → π * electronic transition of the aromatic sp 2 domains of the C = C and n → π * transition of C = O and N-containing functional groups, respectively 40 . The baseline of the spectra in the entire wavelength region increased with the increase in the synthesis temperature due to the formation of larger nanosheet structures. Alg exhibited an X-ray diffraction (XRD) pattern with peaks at 2 θ of 13.7°, 21.6°, and a broad band around 39.0° corresponding to the (110) plane of polyguluronate unit (G), (200) plane of polymannuronate (M), and amorphous halo, respectively (Supplementary Fig. 2) 41 . The crystallinity of Alg is due to inter and intramolecular hydrogen bonding. When heated at 150°C and higher, the XRD peak corresponding to guluronate and mannuronate structure disappeared. Instead, a broad peak emerged, indicating the presence of carbonized nanostructures with disordered carbon phases 42 . The Raman spectra of Alg and CPNSs synthesized at different temperatures were compared with that of GO (Fig. 1 D ) . Alg did not show D and G bands, whereas, CPNSs prepared at temperatures 180°C and above showed the D band around 1350 cm – 1 and G band around 1600 cm – 1 , and their intensity and shape gradually increased and sharpened, respectively, with synthesis temperature. Nevertheless, they are still not well defined as that of GO and therefore, do not reflect structures identical to GO, due to a low degree of graphitization and ultrasmall size of the graphene-like domains 43 . Heating the precursors such as sodium alginate and sucrose at high temperatures (> 500 o C) and inert atmosphere (e.g., N 2 and Ar) produces carbon with well-defined D and G bands 44 , 45 . However, in this work, no high temperature or inert atmosphere was used. The G band is due to the formation of in-plane stretching of carbon-carbon bonds in the aromatic rings of graphene-like structures 46 , revealing that DAHP assists in the alignment of polymer chains and carbonization of Alg to form CPNSs; whereas, the D band indicates the amorphous and disordered nature of graphene-based materials 47 . The oxygen (O), nitrogen (N), and phosphorous (P) contained functional groups and dopped in the CPNSs disrupt the periodicity and long-range order of the graphene lattice, leading to a loss of crystallinity. Therefore, the CPNSs must be carbonized alginate having carbon-based structures with distinctive polymeric characteristics. To verify whether Alg could form nanosheets in the presence of other ammonium-, phosphate- or sulfate-containing compounds upon heating at 180°C, we carbonized Alg in the presence of ammonium dihydrogen phosphate ((NH 4 )H 2 PO 4 ), phosphoric acid (H 3 PO 4 ), ammonium hydroxide (NH 4 OH), NH 4 OH/H 3 PO 4 mixture, and disodium hydrogen phosphate (Na 2 HPO 4 ) (Supplementary Fig. 3). It is noteworthy to mention that CPNSs were obtained only with (NH 4 )H 2 PO 4 . Alg did not form nanosheet structures in the presence of H 3 PO 4 , NH 4 OH, H 3 PO 4 and NH 4 OH mixture, or Na 2 HPO 4 . Also, CPNSs were not formed with Alg in the presence of other sulfates, sulfite, and ammonium-related salts such as ammonium sulfite ((NH 4 ) 2 SO 3 ), ammonium sulfate ((NH 4 ) 2 SO 4 ), ammonium chloride (NH 4 Cl), and sodium sulfite (Na 2 SO 3 ) in the same mass ratio (Supplementary Fig. 4). Thus, we conclude that solid-state heating at 180°C and DAHP have a crucial role in the formation of perfect CPNS structures. Phosphate diester linkages mediate the formation of CPNSs In order to investigate the process of CPNSs formation, we performed a time-course analysis of Alg/DAHP mixture that was heated at 180°C for a duration of 3 h (Fig. 2 ). As the heating progressed, notable changes occurred. Within 5 min, the color of the mixture shifted to a pale brown hue, followed by a transition to dark brown at 15 min (Fig. 2 A). Eventually, within 30 min, the mixture turned black due to carbonization. Concurrently, the time-course TEM analysis demonstrated the formation of supramolecular structures by Alg within the initial 5-minute heating period (Fig. 2 B). At 15 min, it tended to form thick and large sheet-like structures, which subsequently became thin at 30 min. After 3 h of heating thin-layered clean sheets of varying sizes were formed, likely due to fragmentation during the later stage of the thermal process. The time-course XRD pattern of the products and Alg are presented in Fig. 2 C. Over time, a noticeable alteration in the crystallinity of Alg becomes apparent. The crystallinity in Alg arise from the arrangement of G (2 θ = 13.7°) and M (2 θ = 21.6°) units and the amorphous halo (broad peak centered at 2 θ = 39°), which are disrupted and realigned during the heating. The amorphous halo completely disappeared after 15 min. The appearance of a broad peak centered at 2 θ of 26.2° indicates very small graphene domains in the carbonized products with highly disordered carbon 42 , 43 . This transformation, denoting carbonization, can be observed from 30 min onwards. The hydrodynamic diameter of the CPNSs by heating for different time intervals shows an increase in size to as high as ca. 2200 nm at 30 min, which decreases to ca. 440 nm after 3 h due to fragmentation of the polymer sheets during carbonization (Supplementary Table 2 ). The TEM-energy-dispersive X-ray spectroscopy (EDS) mapping of CPNSs-180 displayed in Supplementary Fig. 5A confirms the presence of nitrogen and phosphorus, and the HRTEM image and the selective area electron diffraction (SAED) pattern suggest the low crystalline nature of the CPNSs (Supplementary Fig. 5B), in agreement with the XRD pattern. If the 2D structures are not carbon nanosheets, they might instead be black phosphorous nanosheets. It is a thermodynamically stable allotrope of phosphorus with 2D structure of atomic arrangement very similar to that of graphite 48 . Black phosphorus is highly crystalline and has orthorhombic structure 49 . However, the XRD patterns in Fig. 2 C and Supplementary Fig. 2 and SAED pattern in Supplementary Fig. 5B did not show any crystalline properties corresponding to black phosphorus. Furthermore, black phosphorus is formed only at very high temperature and pressure 48 . Thus, the possibility of 2D phosphorus allotropes can be ruled out, and we believe the 2D nanostructures obtained by heating a mixture of sodium alginate and diammonium hydrogen phosphate as shown in Fig. 1 B must be CPNSs. The molecular structural changes occurring during the heating were further studied by Fourier-transform infrared spectroscopy (FTIR) (Supplementary Fig. 6 ). The FTIR spectrum of Alg exhibited specific vibrational modes such as –OH stretching at 3200−3400 cm – 1 , asymmetric stretching of –COO – at 1610 cm – 1 , the symmetric stretching of –COO – at 1412 cm – 1 , and the C–O–C (ring) vibrational modes at 1081 cm – 1 of the pyranose rings 50 . The C–O(H) symmetric vibration peak appeared at 1306 cm – 1 , and stretching vibration of the C–O–C glycosidic linkage in alginate polymer appeared at 1036 cm – 1 . FTIR spectra of Alg show significant changes in peaks at lower wavenumber region, 500–1700 cm – 1 for different durations of heating with DAHP. Notably, the C–O(H) peak at 1306 cm – 1 decreased significantly with heating time, probably due to the dehydration process. The –C–O(H) symmetric vibration peak at 1306 cm – 1 began to disappear within 5 min, meanwhile a new P = O asymmetric stretching peak emerged at 1246 cm – 1 and then started disappearing after 30 min. A peak at 1739 cm – 1 appeared after 5 min and disappeared after 30 min, indicating some new carbonyl groups of esters are formed and then degraded with time 51 . After 15 min of heating, new peaks emerged at 1054 cm – 1 , corresponding to P–O–C stretching in the phosphate ester bond. The Alg after reaction with DAHP and without heating (i.e., 0 min) showed an O–P–O bending vibration peak of the phosphate ester at 546 cm – 1 , which slightly shifted to 515 cm – 1 after 5 min onwards and decreased significantly after 1 h. A new peak emerged at 1054 cm – 1 , corresponding to P–O–C stretching in the phosphate ester bond. It can be inferred that Alg polymer chains are cross-linked via phosphate diester bonds. During the heating, (NH 4 ) 2 HPO 4 undergoes thermal decomposition to form various chemical species, such as NH 3 ( g ) , (NH 4 )H 2 PO 4 ( s ) , and H 3 PO 4 ( l ) 52 . The phosphoric acid reacts with Alg to form esters, which is in agreement with a similar work reported by Marcilla et al ., in which the various acid species react with different compounds in tobacco to form esters. Meanwhile, a portion of the NH 3 formed by the thermal degradation of (NH 4 ) 2 HPO 4 could form quaternary ammonium salts of the carboxylic acid group 52 . We conducted the 31 P nuclear magnetic resonance (NMR) spectroscopy analysis of the time course formation of CPNSs-180 (Fig. 2 D). The 31 P NMR peak of phosphate group appeared at a chemical shift of 0.87 ppm for the purified, non-heated Alg/DAHP mixture, indicating the adsorption of phosphate on the Alg. Upon heating for 1 min at 180°C, the peak shifts a little downfield (to 1.21 ppm), probably due to the formation of monoesters 53 , 54 . With a further increase in heating time to 7.5 min, peak resonances toward an up field of the central peak were observed (–5.39, − 8.50, and − 9.26 ppm), depicting the formation of phosphate diesters and pyrophosphate structures. After 15 minutes of heating, the peaks for pyrophosphate (–8.50 and − 9.26 ppm) disappeared, and with further heating the peaks 1.21 and − 5.39 ppm depicting phosphate esters, including monoesters and diesters remained. The plausible mechanism of the formation of CPNSs is illustrated in Scheme 1 . The formation of diester in solid-state will form bridges between the alginate polymer chains to form 2D polymer sheets. The previous report reveals that dry phosphorylation of starch using orthophosphate occurs through the reactive hydroxyl groups of the starch molecules to form brown-colored products at temperatures of 170°C and higher 55 . Investigation on the phosphorylation of Alg using urea/phosphate system using various NMR spectroscopy techniques revealed that the most probable site for phosphorylation is the equatorial hydroxyl group of mannuronic acid units in the polymeric chain 50 . Therefore, it is evident that heating of Alg with DAHP in a solid-state initially leads to the formation of sheet-like structures formed by the cross-linking of Alg polymer chains via phosphate diester linkages. Since this reaction system contains a mixture of monoesters, diesters, and unreacted alginate chains, the carbonized product contains CPNSs along with polymeric Alg featuring nonspecific shapes. The molecular arrangement in pure Alg is mainly due to the solid-state intra- and inter-molecular hydrogen bonding, which are disrupted upon heating above 170°C and produce carbonized products without specific shape 56 , 57 . However, the formation of cross-linking among the alginate polymer chains by phosphate ester bonds dominates the formation of a stable 2D structure, resulting in carbonized polymeric nanosheets. The elemental composition of Alg and the products obtained at various time intervals is presented in Supplementary Table S3 . The carbon content (weight percentage) of pure Alg has been found to be 29.16%, which is close to the values reported by previous studies, and some report reveals that it varies with the harvesting season of the algae 57 – 59 . The carbon and oxygen contents of Alg after reacting with DAHP (i.e., CPNSs-180 0 min) is determined to be 19.94% and 43.34%, respectively. The carbon content increased up to 38.73% and the oxygen content decreased to 33.93% after being heated at 180°C for 3 h, indicating carbonization to form slightly carbonaceous nanomaterials (i.e., CPNSs). A previous report also suggests that P/N-doped carbon dots synthesized at low temperature (90°C) possess a low degree of carbon content with a weight percentage of 8.62 60 . Carbonization of precursors in semi-closed atmosphere and temperatures as high as 900°C has been reported to yield carbonized products with higher carbon content (as high as ~ 69%) and low oxygen content (10%) 61 . The carbonized polymer products have nitrogen doping, and the final CPNSs obtained after 3 h possess 6.8% nitrogen by weight. Alginate polymer has high affinity toward phosphate ions 62 and it forms phosphate ester after reacting with the DAHP during the heating process, resulting in high phosphorous content (12.45%) in the CPNSs as determined by inductively coupled plasma optical emission spectroscopy (ICP-OES). The initial heating of Alg/DAHP mixture at 180°C leads to the crosslinking of the hydroxyl groups of the DAHP with the side chain hydroxyl group of the Alg to form phosphate ester bond 63 . With increase in the reaction time, carbonization progressed, resulting in the decrease in P content. At 3 h of heating intense phosphorylation and phosphorus doping occurred in the CPNSs due to the presence of phosphoric acid and P 2 O 5 in the system thereby increasing the P content to 12.45%. Thus, the CPNSs obtained from Alg by carbonization in the presence of DAHP are N and P co-doped. The C1s, O1s, N1s, and P2p XPS spectra of the CPNSs synthesized at 180°C for 3 h are presented in Supplementary Fig. S7 . The CPNSs have oxygen-containing functional groups and N-doping in the form of pyridinic (399.1 eV), graphitic (399.85 eV), and pyrrolic (400.66 eV) nitrogen. The deconvoluted P2p spectra of the CPNSs show peaks at 132.49, 133.01, 133.63, and 134.42 eV corresponding to the presence of P–C, P–O, P–O–C, and P = O bonding, respectively, which confirm phosphorous is incorporated in the CPNSs 64 . CPNS-modified filter paper for efficient removal of bacteria The CPNSs were tested for their antibacterial activity toward Gram-negative ( E. coli ) and Gram-positive ( S. aureus ) bacteria and toward V. parahaemolyticus , a Gram-negative bacteria that poses a significant risk in aquaculture. V. parahaemolyticus can rapidly multiply and infect cultured seafood species, which not only damages the health of these species but also increases the risk of foodborne illnesses when these products are consumed 65 . Different from the GO nanosheets which exhibit antibacterial activity through different mechanisms such as direct interaction with the bacteria through their sharp edges or by wrapping on the bacterial cell 66 , 67 , the CPNSs having 2D structure do not show antibacterial activity (Supplementary Fig. 8 ). The difference in the antibacterial behavior of the CPNSs may be ascribed to only adsorbing bacteria but could not further disrupt the bacterial membranes due to polymeric structures on the CPNSs' surfaces. It has been reported that the functional groups on GO play a crucial role in its antimicrobial activities 68 . However, these CPNSs, when modified on filter paper could effectively remove bacteria from contaminated water. Incubating the filter paper with CPNSs resulted in the effective coating of the nanosheets on the fibers, as evident from the SEM images in Fig. 3 A, however, it did not form a separate layer above the filter paper. The CPNS-modified filter paper was effective in removing V. parahaemolyticus (10 5 CFU mL – 1 ) from contaminated seawater samples, with CPNSs-180 and CPNSs-210 showing superior effects (> 90%) (Fig. 3 B). Notably, neither the bacteria's morphology nor the bacterial membrane was disrupted after passing through the CPNS-modified membrane (Fig. 3 C). Adsorbing bacteria without membrane disruption is advantageous, preventing toxin release into the filtrate. For instance, disruption of V. parahaemolyticus ’ cell membrane releases toxins like PirA and PirB proteins, which induce necrosis and functional loss in the hepatopancreas of shrimp 69 . We further evaluated CPNSs-180-modified filter paper for the removal of E. coli and S. aureus . The removal efficiency for E. coli was less than 30%, and that for S. aureus was around 80% (Fig. 4 ). The difference in the bacterial removal efficiency of CPNSs-modified filter paper may be attributed to the different bacterial shapes and membrane structures 21 , 24 , 70 . The SEM image of the CPNS-modified filter paper after passing the V. parahaemolyticus bacteria solution clearly shows bacteria trapped on the membrane (Fig. 3 D). In contrast to our previous work, graphene oxide@carbon nanogels (GO@CNGs)-modified membrane reported for the removal of bacteria from contaminated water, where the efficiency of the membrane decreases with an increase in water flux 21 , the efficiency of the CPNS-modified membrane was not affected by the increase in water flux (Supplementary Fig. 9 ). Combating V. parahaemolyticus The efficiency for the removal of V. parahaemolyticus at a higher concentration (10 7 CFU mL −1 ) was further evaluated in aquarium condition (2 L water in the aquarium tank) using the CPNS-modified filter paper with a larger surface area (17.34 cm 2 ). The CPNSs-modified filter paper was effective in eliminating > 98% V. parahaemolyticus within 2 h of circulation (Fig. 5 A), and no leakage of bacteria was observed from the membrane even after 24 h. The uncoated filter paper (Ctrl) showed removal of ca. 70% within 1 h; however, it decreased with time and down to ~ 17% after 24 h, which shows that though the filter paper can adsorb bacteria, upon continuous passage of water, the bacteria are washed from the membrane back to the solution, due to the large pore size of the membrane and weak affinity toward V. parahaemolyticus . It is noteworthy that the bacterial removal efficiency for the GO-coated filter paper was ~ 74% after 4 h and remained stagnant beyond that time, probably due to the clogging of pores due to fouling of the membrane 71 ; which shows the superior efficacy of our CPNSs-modified filter paper. The decrease in water flux and removal efficiency of the membrane due to the clogging of pores of the membrane is a major drawback in membrane-based filtration systems. Therefore, we further performed the shrimp challenge experiments with the CPNSs-modified filter (Fig. 5 B). After challenging white leg shrimp ( Litopenaeus vannamei , 10 no. in 2 L sea water) with V. parahaemolyticus (10 6 CFU mL −1 ), CPNSs-180-modified filter paper was loaded onto a filter holder, and the aquarium water was circulated through it, and the results were compared with that of the control filter (filter paper without CPNSs-180 coating) and control (without any filter paper or membranes). The shrimps in the CPNSs-modified filter paper group showed 100% survival even after 48 h, while that of the other two groups decreased to 50% survival rate was observed in CPNSs-modified filter paper group. Therefore, we hope that the CPNS-modified filter may serve its use for filtering out even a very high concentration of bacteria contaminated in aquarium water. Notably, replacing the CPNSs-modified paper every 48 h after filtration could remove the bacteria completely without affecting the survival rate of the shrimp upon Vibrio infection (Supplementary Fig. 10 ). Though the control filter paper was also replaced every 48 h, only 10% survival was observed after 96 h. In summary, the methodology presented in this study, which employs low-temperature carbonization of Alg with DAHP, presents a unique approach to prepare 2D carbonized nanomaterials for filtering out pathogens from water. The advantage of this procedure is that it bypasses the need for high temperatures, sophisticated equipment, or hazardous solvents, making it more sustainable and potentially cost-effective. The decomposition of DAHP yields a phosphorylating agent, PO 4 3– , which facilitates the phosphorylation of the equatorial hydroxyl group of the mannuronic units within the alginate polymer chain. This results in the creation of phosphate diester linkages between the chains, which are more robust than the solid-state hydrogen bonds in the polysaccharides. Consequently, 2D polymer sheets are formed at the early stage of heating and are subsequently carbonized to produce CPNSs. Moreover, the study effectively demonstrates a practical application of the synthesized CPNSs, using them to enhance the survival rate of shrimp in aquaculture by removing the bacterial strain V. parahaemolyticus . These CPNSs demonstrate notable bacterial adsorption capabilities, particularly towards strains like V. parahaemolyticus . The adsorption property facilitates the development of an efficient bacterial filtration system using ordinary filter paper. Notably, our shrimp challenge experiments indicate an enhanced survival rate among shrimp exposed to V. parahaemolyticus after passing through the CPNSs-modified filter paper. This is an important contribution as it suggests the potential for Alg-CPNSs to improve aquaculture health and productivity. V. parahaemolyticus is responsible for causing acute hepatopancreatic necrosis disease (AHPND) in shrimp, a condition that leads to severe damage and dysfunction in the hepatopancreas. AHPND was accountable for a massive loss of US $ 44 billion in the global shrimp farming sector between 2010 and 2016 72 . As of the latest estimates, the annual economic losses attributed to AHPND now stand at USD 7 billion 73 . Although this study represents a promising advancement in the synthesis of CPNSs and their application in aquaculture, further exploration and validation of the findings are necessary to understand this approach's potential benefits and drawbacks fully. We have used Alg as a source of polysaccharide in this study. It would be interesting to explore whether other marine or non-marine polysaccharides can also be used as precursors in preparing CPNSs, which could potentially widen the applications and versatility of the method. On the other hand, our study shows that Alg-derived CPNSs have a high bacterial adsorption capability, but it could benefit from a more detailed examination of the mechanism behind this adsorption. Understanding this mechanism could aid in improving the efficiency and specificity of the CPNSs for bacterial adsorption. While the study focuses on the removal of specific bacterial strains, it would be valuable to examine the effectiveness of these CPNSs in adsorbing other types of contaminants in aquaculture water in the future, which could determine the broader applicability of the approach. The long-term efficacy of these Alg-CPNSs in a realistic aquaculture setting, including their durability and the need for replacement over time, is also essential in the future. While this study observes an immediate increase in the survival rate of shrimp, it might be beneficial to look at the long-term effects of using Alg-CPNSs in the future. This could include the potential impacts on the health and growth of the shrimp over time, as well as any potential environmental impacts. Finally, it would be valuable to assess the environmental impact of using Alg-CPNSs, including a lifecycle analysis, considering the source of the polysaccharides, the process of synthesizing the CPNSs, their use, and eventual disposal. Methods Synthesis of CPNSs The CPNSs were prepared by heating a solid mixture of Alg and DAHP in 1:5 mass ratio in a muffle furnace (DH 300, Dengyng, New Taipei City, Taiwan). The mixture of Alg and DAHP was blended in a coffee grinder for 5 min. 1.0 g of the mixture was then placed in 50 mL glass vials and heated in two steps: first at 60°C for 3 h followed by raising the temperature to 120, 150, 180, 210, or 240°C and heated for 3 h. The carbonized residue thus obtained was allowed to cool to room temperature, and 50 mL of deionized (DI) water was added, mixed well, and centrifuged at a relative centrifugal force (RCF) of 15,000 g for 1 h. After three centrifugation/washing cycles, the pellets were dispersed again in DI water, and the pH of the solutions were adjusted to 9 by adding NaOH solution (0.5 M), and then sonicated (100 W) for 15 min. The sonicated dispersions were centrifuged at 500 g for 15 min to remove larger carbonized particles, and the supernatants containing the CPNSs were collected and quantified by freeze-drying. The CPNSs dispersion was stored at 4°C when not in use. Preparation of CPNSs-modified Filter paper and determination of bacteria removal efficiency Qualitative filter paper (Advantec® 2, pore size 5 µm, and thickness 0.26 mm) was modified with CPNSs for the bacterial removal from water. Briefly, the filter paper (25 mm in diameter) was incubated with CPNSs solution (0.5 mg mL −1 , 5 mL) for 2 h under shaking at 150 rpm to obtain a loading of ca. 0.2 mg cm – 2 . The CPNSs-modified filter papers (area 4.91 cm 2 and active area 2.83 cm 2 ) were then placed in a syringe holder (diameter 25 mm) and washed with 50 mL of sodium phosphate buffer (5 mM, pH 7.4) to remove the unbound CPNSs (flow rate 5.8 L min – 1 m – 2 ). The CPNSs-modified filter paper was tested for the removal of Escherichia coli ( E. coli ), Staphylococcus aureus ( S. aureus ), and V. parahaemolyticus . E. coli and S. aureus were cultured overnight in Lysogeny broth (LB) medium at 37°C with shaking at 150 rpm, while V. parahaemolyticus was cultured in tryptic soy broth (TSB) containing 3% NaCl at 25°C. The bacteria cells were washed twice with sodium phosphate buffer (5 mM, pH 7.4) for E. coli and S. aureus ; sodium phosphate buffer (5 mM, pH 7.4) containing 3% NaCl for V. parahaemolyticus after removing the medium by centrifugation at 3000 g for 5 min at 25°C. The bacteria removal efficiency of the CPNSs-modified filter paper was determined using 10 5 CFU mL −1 bacteria solution by a dead-end mode filtration, using a syringe pump (KDS100, KD Scientific, Holliston, MA, USA), with a water flux of 400 mL min −1 m −2 . The filtrate was collected and diluted 100-fold, and then 100 µL of the diluted solution was spread on LB-agar plates and incubated for 12 h at 37°C (TSB agar plates were used for V. parahaemolyticus and incubated at 25°C). The liquid culture of the bacteria was also carried out by supplementing with the respective medium and incubating overnight, followed by measuring the absorbance at 600 nm (OD 600 ). Each experiment was performed in triplicate for each condition. Transmission electron microscopic (TEM; Tecnai 20 G2 S-Twin, Philips/FEI, Hillsboro, OR, USA) images were recorded to understand the morphology of the bacteria. The scanning electron microscopic (SEM; Hitachi S-4800, Hitachi High-Technologies, Tokyo, Japan) images of CPNSs-modified filter paper after passing 10 7 CFU mL −1 V. parahaemolyticus were taken to understand the bacteria removal mechanism. Declarations Competing interests The authors declare no competing interests. Author contributions J.-Y.L. and C.-C.H conceived the original idea and supervised the project from the beginning to the end, and helped in the manuscript preparation. A.A. and B.U. carried out the experiments and prepared the manuscript. C.-Y.W. and H.-J.L. helped in the manuscript preparation. A.A. and B.U. contributed equally. All authors discussed the results and contributed to the manuscript. Acknowledgements This work was supported by the National Science and Technology Council (NSTC) of Taiwan under Contract Nos. 110-2221-E-019-001, 110-2811-M-019-501, 110-2314-B-182-008-MY3, and 112-2811-B-182-022, Chang Gung Memorial Hospital, Linkou under Contract No. CMRPD2L0161, Chang Gung University under Contract No. OMRPD2N0011, and the Center of Excellence for the Oceans, National Taiwan Ocean University from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan. Data availability All data generated or analyzed during this study are included in this published article and its supplementary information files. References Speranza, G. Carbon nanomaterials: Synthesis, functionalization and sensing applications. 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Impacts of acute hepatopancreatic necrosis disease on commercial shrimp aquaculture. Rev. Sci. Tech. 38, 477–490 (2019). Tang, K.F.J. et al. Shrimp acute hepatopancreatic necrosis disease strategy manual. FAO Fisheries and Aquaculture Circular No. 1190, Rome, FAO (2020). Scheme 1 Scheme 1 is available in the Supplementary Files section. Additional Declarations (Not answered) Supplementary Files CPNSsSI202403222.docx floatimage1.png Scheme 1 | Heating of Alg/DAHP mixture in solid-state at 180 °C leads to phosphorylation at the equatorial hydroxyl groups of the mannuronic units in the Alg chain resulting in cross-linking and 2D polymer sheet formation via phosphate ester linkage, followed by carbonization to form CPNSs. Cite Share Download PDF Status: Published Journal Publication published 05 Sep, 2024 Read the published version in npj Clean Water → Version 1 posted Editorial decision: revise 13 May, 2024 Review # 3 received at journal 12 May, 2024 Review # 1 received at journal 01 May, 2024 Reviewer # 3 agreed at journal 30 Apr, 2024 Review # 2 received at journal 29 Apr, 2024 Reviewer # 2 agreed at journal 24 Apr, 2024 Reviewer # 1 agreed at journal 24 Apr, 2024 Reviewers invited by journal 24 Apr, 2024 Submission checks completed at journal 25 Mar, 2024 Editor assigned by journal 23 Mar, 2024 First submitted to journal 23 Mar, 2024 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. 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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-4153360","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":294841955,"identity":"cfc5679f-d0b7-4d59-9649-9e3f7bf7dbcc","order_by":0,"name":"Chih-Chhing Huang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIiWNgGAWjYBACAxjJDxVgbCBai2QDM0wLMzFaQIwDxGoxl0h+9vBLwR27zbf7Dz7mYbCR3XCA/5gEPi2WM9LMjWUMniVvu3OY2ZiHIc14wwFmNrxaDG4kmElLGBxONruRzCbNw3A4EaTlBn4t6d/AWoxngLX8J0ZLjpnkB4PDdgYSYC0HiNBy5k2ZNIPB4QSJG8nGhnMMko1nHmY2/4FXy/H0bZI//hy255+R+PDBmwo72b7jjY8N8GkBAWYeBobEBogJIC4h9UDACHSHPRHqRsEoGAWjYKQCAL8FSU2L0au5AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-0363-1129","institution":"National Taiwan Ocean University","correspondingAuthor":true,"prefix":"","firstName":"Chih-Chhing","middleName":"","lastName":"Huang","suffix":""},{"id":294841956,"identity":"d91d9169-c505-4297-bec1-3b57c0415246","order_by":1,"name":"Anisha Anand","email":"","orcid":"","institution":"National Taiwan Ocean University","correspondingAuthor":false,"prefix":"","firstName":"Anisha","middleName":"","lastName":"Anand","suffix":""},{"id":294841957,"identity":"0cfc1c13-1192-45a5-9e94-0dbde5081223","order_by":2,"name":"Binesh Unnikrishnan","email":"","orcid":"","institution":"National Taiwan Ocean University","correspondingAuthor":false,"prefix":"","firstName":"Binesh","middleName":"","lastName":"Unnikrishnan","suffix":""},{"id":294841958,"identity":"1f65567f-54d6-40ea-9902-99a07ef51db1","order_by":3,"name":"Chen-Yow Wang","email":"","orcid":"","institution":"National Taiwan Ocean University","correspondingAuthor":false,"prefix":"","firstName":"Chen-Yow","middleName":"","lastName":"Wang","suffix":""},{"id":294841959,"identity":"2d4d892b-6a0b-4d67-a700-94ba948d0622","order_by":4,"name":"Jui-Yang Lai","email":"","orcid":"","institution":"Chang Gung University","correspondingAuthor":false,"prefix":"","firstName":"Jui-Yang","middleName":"","lastName":"Lai","suffix":""},{"id":294841960,"identity":"94122fa1-83d7-4269-a36c-e36bd05a626e","order_by":5,"name":"Han-Jia Lin","email":"","orcid":"","institution":"National Taiwan Ocean University","correspondingAuthor":false,"prefix":"","firstName":"Han-Jia","middleName":"","lastName":"Lin","suffix":""}],"badges":[],"createdAt":"2024-03-23 08:00:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4153360/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4153360/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41545-024-00378-7","type":"published","date":"2024-09-05T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":55501115,"identity":"3c46ebbb-7ac0-49ed-b7cc-0c7f3f505e93","added_by":"auto","created_at":"2024-04-29 10:01:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":967302,"visible":true,"origin":"","legend":"\u003cp\u003eDry heating of a mixture of Alg and DAHP in 1:5 ratio (A) photographs and (B) TEM images of products obtained by heating at different temperatures. (C) AFM image of CPNSs-180. The horizontal line and the rectangle box indicate the thickness mapping and roughness, respectively, calculated using NanoScope Analysis 2.0 software. (D) Raman spectra of GO, Alg, and CPNSs prepared at different temperatures.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4153360/v1/1b8cb8014a257c35d9617631.png"},{"id":55501117,"identity":"926d6207-da8a-414a-9d4a-9434c1d5cdc0","added_by":"auto","created_at":"2024-04-29 10:01:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":644240,"visible":true,"origin":"","legend":"\u003cp\u003eTime course analysis of CPNSs formation from a mixture of Alg and DAHP in 1:5 mass ratio at 180 °C up to 3 h. (A) photographs, (B) TEM images, (C) XRD patterns, and (D) \u003csup\u003e31\u003c/sup\u003eP NMR spectra.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4153360/v1/55ecf5ef73ed5648aa06661f.png"},{"id":55501120,"identity":"99764462-45ab-46b1-9dbd-deac48ab0dc4","added_by":"auto","created_at":"2024-04-29 10:01:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1092293,"visible":true,"origin":"","legend":"\u003cp\u003eCPNS-modified filter paper for the removal of \u003cem\u003eV. parahaemolyticus\u003c/em\u003e. (A) Photographs, SEM images, and agar plates showing bacterial removal efficiency of CPNS-modified filter paper (0.2 mg cm\u003csup\u003e–2\u003c/sup\u003e) as compared with that of control. (B) Bacterial removal efficiency of \u003cem\u003eV. parahaemolyticus\u003c/em\u003e after passing through paper membrane coated with CPNSs (0.2 mg cm\u003csup\u003e–2\u003c/sup\u003e) at a water flux of 400 mL min\u003csup\u003e-1\u003c/sup\u003e m\u003csup\u003e-2\u003c/sup\u003e. Error bars in (B) represent the standard deviations from triplicate experiments. (C) \u003cem\u003eV. parahaemolyticus \u003c/em\u003e(a)\u003cem\u003e \u003c/em\u003ebefore and (b) after passing through CPNSs-180 modified filter paper. (D) SEM images of CPNSs-modified filter paper after passing 10 mL 10\u003csup\u003e7\u003c/sup\u003e CFU mL\u003csup\u003e–1\u003c/sup\u003e solution of \u003cem\u003eV. parahaemolyticus.\u003c/em\u003e Magnification: (a) × 1.0 K and (b) × 5.0 K. The red circles indicate the bacteria adsorbed on the CPNSs-coated filter.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4153360/v1/b56a947a5bfb889e3cdd2eae.png"},{"id":55501323,"identity":"6b43c973-442c-4c37-a0d1-9da353b8f472","added_by":"auto","created_at":"2024-04-29 10:09:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":858560,"visible":true,"origin":"","legend":"\u003cp\u003eCPNS-modified filter paper for the removal of \u003cem\u003eE. coli \u003c/em\u003eand\u003cem\u003e S. aureus\u003c/em\u003e. (A, B) The bacterial removal efficiency of the CPNS-modified filter papers (0.2 mg cm\u003csup\u003e-2\u003c/sup\u003e) toward 10\u003csup\u003e5\u003c/sup\u003e CFU mL\u003csup\u003e–1\u003c/sup\u003e \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e at a water flux of 400 mL min\u003csup\u003e-1\u003c/sup\u003e m\u003csup\u003e-2\u003c/sup\u003e.\u003cem\u003e \u003c/em\u003e(C) TEM images \u003cem\u003eof E. coli \u003c/em\u003eand \u003cem\u003eS. aureus \u003c/em\u003ebefore and after passing through CPNS-180-modified filter paper. Error bars in (B) represent the standard deviations from triplicate experiments.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4153360/v1/c7c9c88f2f04e696e3d03902.png"},{"id":55501322,"identity":"5fb02bc7-4213-46ee-b055-e7c996b64677","added_by":"auto","created_at":"2024-04-29 10:09:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":85426,"visible":true,"origin":"","legend":"\u003cp\u003eRemoving of \u003cem\u003eV. parahaemolyticus\u003c/em\u003e in shrimp cultured aquarium water by CPNSs-180-coated filter paper. (A) Relative removal \u003cem\u003eof V. parahaemolyticus \u003c/em\u003e(10\u003csup\u003e7\u003c/sup\u003e CFU mL\u003csup\u003e–1\u003c/sup\u003e) from an aquarium tank at different time periods upon passing through control membrane (uncoated filter paper), CPNSs-180- and GO-modified filter papers, at a flow rate of 1150 L min\u003csup\u003e–1\u003c/sup\u003em\u003csup\u003e–2\u003c/sup\u003e.\u003cstrong\u003e \u003c/strong\u003e(B) The survival of shrimp infected with \u003cem\u003eV. parahaemolyticus\u003c/em\u003e (10\u003csup\u003e6\u003c/sup\u003e CFU mL\u003csup\u003e-1\u003c/sup\u003e)\u003cem\u003e \u003c/em\u003eand circulating water through control membrane (uncoated filter paper) and CPNSs-180-coated filter paper. Error bars represent the standard deviation of triplicate experiments.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4153360/v1/b769f1cc121384b540590e68.png"},{"id":64071048,"identity":"cc9ed79d-1b38-4ce4-b89f-267611e2c582","added_by":"auto","created_at":"2024-09-06 07:09:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5254587,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4153360/v1/7e7afe2e-fd28-437a-9dcb-00097b7bf2a3.pdf"},{"id":55501122,"identity":"f8f6d6f2-fd29-409d-a372-a46280d4fd87","added_by":"auto","created_at":"2024-04-29 10:01:09","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2869627,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"CPNSsSI202403222.docx","url":"https://assets-eu.researchsquare.com/files/rs-4153360/v1/d41ce6690abd12c0c8d5d5cf.docx"},{"id":55501118,"identity":"8b279eb7-4fce-40da-b593-39ab2abbe7fa","added_by":"auto","created_at":"2024-04-29 10:01:09","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":334601,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1 |\u003c/strong\u003e Heating of Alg/DAHP mixture in solid-state at 180 °C leads to phosphorylation at the equatorial hydroxyl groups of the mannuronic units in the Alg chain resulting in cross-linking and 2D polymer sheet formation \u003cem\u003evia\u003c/em\u003e phosphate ester linkage, followed by carbonization to form CPNSs.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4153360/v1/33d0af80e966a88bb64de0e9.png"}],"financialInterests":"(Not answered)","formattedTitle":"Phosphate ester-linked carbonized polymer nanosheets as efficient filters for removing \u003ci\u003eVibrio parahaemolyticus\u003c/i\u003e from aquaculture water","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe properties and ultimate applications of carbon-based nanomaterials (CNMs) are defined by their structure and surface functionalization, hence research endeavors are directed toward fine-tuning these characteristics\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. 2D nanosheets (NSs) are remarkable for their nanoscale thickness and high surface-to-volume ratio, attributes that render them suitable for diverse applications such as energy storage devices, optoelectronics, sensors, composite materials, water purification and filtration, and even biomedical applications\u003csup\u003e\u003cspan additionalcitationids=\"CR3 CR4 CR5 CR6\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Particularly, carbon nanosheets (CNSs), including graphene, graphene oxide (GO), and reduced graphene oxide (rGO), have demonstrated efficacy in areas like energy storage and batteries, fuel cells and solar cells, gas separation, and biomedical applications\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. These benefits can be attributed to their unique honeycomb structure and the ease of surface functionalization, which enables tailoring for specific applications\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Besides the sheet-like structures, there has been a rise in reports on CNMs with different morphologies like carbon microtrees, flower-like carbon, carbon cones, carbon cubes, and carbon nanocontainers in the past years\u003csup\u003e\u003cspan additionalcitationids=\"CR13 CR14 CR15\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Despite these varied structures, 2D nanomaterials are favored for creating stable thin films and membranes due to their large surface area, customizable porosity, high chemical stability, and potential for layered arrangements\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. CNSs have displayed potential for the adsorption of organic pollutants from water, including pesticides and pharmaceuticals\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Furthermore, the CNS-based nanocomposites can also function as photocatalysts, initiating the breakdown of these pollutants\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Additionally, CNSs have exhibited antimicrobial or bacteria adsorption properties, offering an effective method for deactivating or eliminating harmful microbes when used in water purification processes\u003csup\u003e\u003cspan additionalcitationids=\"CR22 CR23\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eVarious methods for synthesizing CNSs have been adopted, including chemical vapor deposition (CVD), mechanical/ultrasonic/chemical/electrochemical exfoliation, epitaxial growth, solvothermal synthesis, and thermal decomposition\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Each of these methods possesses unique requirements suited to its specific synthesis process. For instance, CVD requires hydrocarbon gases as precursors, exfoliation is most effective with raw materials featuring a multi-layered structure and weak inter-layer interactions, and solvothermal synthesis typically employs small molecules\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Control over the sheet size has been achieved through different parameters and approaches such as the utilization of oxidants, density gradient ultracentrifugation, pH-assisted sedimentation, and the application of sonication. However, these methods often require harsh reaction conditions, result in high costs, and lower yield\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Furthermore, CNSs tend to restack, compromising the unique properties of single-layered structure. The introduction of functional groups on CNS mitigates this issue\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Furthermore, many existing methods for synthesizing CNSs are typically constrained to small-scale laboratory processes. Scaling up these synthesis techniques while preserving the desired properties and quality of the materials remains a significant challenge. Consequently, the current research is centered on devising efficient, scalable, and economically viable methods for synthesizing stable CNSs using cost-effective precursors, such as biomass\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWhile existing methods for the synthesis of CNSs often involve high temperatures, solvents, or sophisticated instruments, our approach focuses on a simple low-temperature synthesis procedure, and the use of marine polysaccharides. In this study, we developed carbonized polymer nanosheets (CPNSs) from the polysaccharide, sodium alginate (Alg) and diammonium hydrogen phosphate (DAHP) through low-temperature synthesis in solid-state (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We proposed the formation mechanism of CPNSs could be through the cross-linking of Alg units \u003cem\u003evia\u003c/em\u003e phosphate diester linkages. Though previous reports indicate that CNSs exhibit antibacterial activity toward both Gram-negative and Gram-positive bacterial species\u003csup\u003e\u003cspan additionalcitationids=\"CR33 CR34\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, our CPNSs do not possess inherent antibacterial activity. Nevertheless, when adsorbed onto a filter paper support, they effectively remove bacteria \u003cem\u003evia\u003c/em\u003e specific adsorption, and no leakage of bacteria was observed from the membrane even after 24 h. The recirculating aquaculture system (RAS) has demonstrated its eco-friendly nature, water efficiency, and exceptional productivity in farming\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. However, if pathogenic bacteria are introduced into the RAS, they may survive and recirculate in the system which poses a potential risk to the aquatic species in the system\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Consequently, we employed these CPNS-modified filter papers for the successful removal of \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e (\u003cem\u003eV. parahaemolyticus\u003c/em\u003e) from contaminated aquaculture water, which showed a marked improvement in the survival rate of the shrimp population.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDAHP plays a crucial role in the formation of CPNSs\u003c/h2\u003e \u003cp\u003eCPNSs obtained by heating a solid mixture of Alg and DAHP in 1:5 mass ratio at 120, 150, 180, 210, and 240\u0026deg;C for 3 h were denoted as CPNSs-120, CPNSs-150, CPNSs-180, CPNSs-210, and CPNSs-240, respectively. The Alg/DAHP mixture was colorless and showed a mild color change to off-white at 120\u0026deg;C. The mixture experienced mild dehydration at 150\u0026deg;C, resulting in light brown hue, and showed higher degree of carbonization at 180\u0026deg;C and above displaying brown or black color (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The TEM images in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB show that the Alg/DAHP mixture without heating had a gel-like structure, and the morphology changed to polymeric form at 120\u0026deg;C. From 150\u0026deg;C and above large sheet-like formation can be observed. At 180\u0026deg;C, the mixture forms 2D layered CPNSs. The resulting CPNSs-180 have sizes ranging between 200 and 500 nm, with a thickness of approximately 1.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25 nm and surface roughness was calculated to be 0.23 nm, as determined by atomic force microscopy (AFM) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). This thickness is considerably greater than that of single-layered graphene, which ranges from 0.4 to 1.0 nm, and GO, with a range of 0.7\u0026minus;1.2 nm\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. The surface roughness (0.23 nm) is higher than that reported (0.2 nm) for single-layer free-standing chemically modified GO\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, which suggests the presence of polymeric alginate fragments on the CPNSs. At higher carbonization temperatures of 210 and 240\u0026deg;C, sheet-like structures were formed; however, along with other carbonized products with different morphology and their aggregated forms were adsorbed onto the sheets (TEM images in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAlg suspended in sodium phosphate buffer (5 mM, pH 7.4) exhibits a negative charge, characterized by a zeta potential of ca. \u0026minus;50 mV (Supplementary \u003cb\u003eTable\u0026nbsp;1\u003c/b\u003e). The zeta potential slightly changed as the synthesis temperature increased during the preparation of CPNSs to ca. \u0026minus;46 mV for 180\u0026deg;C. However, with subsequent temperature increments, the zeta potential decreased significantly to ca. \u0026minus;11 mV, probably due to a higher degree of carbonization resulting in elimination of carboxylate functional groups. As the synthesis temperature increased, the hydrodynamic size of the CPNSs increased significantly for 210 and 240\u0026deg;C. The thermal-driven dehydration and cross-linking of Alg lead to the formation of sheet-like structures. The significant carbonization at higher temperatures results in the stacking of CPNSs and other carbonized particles to form larger aggregates.\u003c/p\u003e \u003cp\u003eThe UV-vis absorption spectra of the CPNSs prepared at different temperatures are presented in Supplementary Fig.\u0026nbsp;1. CPNSs obtained at 180\u0026deg;C and above showed a band at ca. 285 nm and extending to the visible region of the spectra attributed to the \u003cem\u003eπ\u003c/em\u003e\u0026rarr;\u003cem\u003eπ\u003c/em\u003e* electronic transition of the aromatic sp\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e domains of the C\u0026thinsp;=\u0026thinsp;C and \u003cem\u003en\u003c/em\u003e\u0026rarr;\u003cem\u003eπ\u003c/em\u003e* transition of C\u0026thinsp;=\u0026thinsp;O and N-containing functional groups, respectively\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. The baseline of the spectra in the entire wavelength region increased with the increase in the synthesis temperature due to the formation of larger nanosheet structures. Alg exhibited an X-ray diffraction (XRD) pattern with peaks at 2\u003cem\u003eθ\u003c/em\u003e of 13.7\u0026deg;, 21.6\u0026deg;, and a broad band around 39.0\u0026deg; corresponding to the (110) plane of polyguluronate unit (G), (200) plane of polymannuronate (M), and amorphous halo, respectively (Supplementary Fig.\u0026nbsp;2)\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. The crystallinity of Alg is due to inter and intramolecular hydrogen bonding. When heated at 150\u0026deg;C and higher, the XRD peak corresponding to guluronate and mannuronate structure disappeared. Instead, a broad peak emerged, indicating the presence of carbonized nanostructures with disordered carbon phases\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. The Raman spectra of Alg and CPNSs synthesized at different temperatures were compared with that of GO (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. Alg did not show D and G bands, whereas, CPNSs prepared at temperatures 180\u0026deg;C and above showed the D band around 1350 cm\u003csup\u003e\u0026ndash;\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e and G band around 1600 cm\u003csup\u003e\u0026ndash;\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, and their intensity and shape gradually increased and sharpened, respectively, with synthesis temperature. Nevertheless, they are still not well defined as that of GO and therefore, do not reflect structures identical to GO, due to a low degree of graphitization and ultrasmall size of the graphene-like domains\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Heating the precursors such as sodium alginate and sucrose at high temperatures (\u0026gt;\u0026thinsp;500 \u003csup\u003eo\u003c/sup\u003eC) and inert atmosphere (e.g., N\u003csub\u003e2\u003c/sub\u003e and Ar) produces carbon with well-defined D and G bands\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. However, in this work, no high temperature or inert atmosphere was used. The G band is due to the formation of in-plane stretching of carbon-carbon bonds in the aromatic rings of graphene-like structures\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e, revealing that DAHP assists in the alignment of polymer chains and carbonization of Alg to form CPNSs; whereas, the D band indicates the amorphous and disordered nature of graphene-based materials\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. The oxygen (O), nitrogen (N), and phosphorous (P) contained functional groups and dopped in the CPNSs disrupt the periodicity and long-range order of the graphene lattice, leading to a loss of crystallinity. Therefore, the CPNSs must be carbonized alginate having carbon-based structures with distinctive polymeric characteristics.\u003c/p\u003e \u003cp\u003eTo verify whether Alg could form nanosheets in the presence of other ammonium-, phosphate- or sulfate-containing compounds upon heating at 180\u0026deg;C, we carbonized Alg in the presence of ammonium dihydrogen phosphate ((NH\u003csub\u003e4\u003c/sub\u003e)H\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e), phosphoric acid (H\u003csub\u003e3\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e), ammonium hydroxide (NH\u003csub\u003e4\u003c/sub\u003eOH), NH\u003csub\u003e4\u003c/sub\u003eOH/H\u003csub\u003e3\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e mixture, and disodium hydrogen phosphate (Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e) (Supplementary Fig.\u0026nbsp;3). It is noteworthy to mention that CPNSs were obtained only with (NH\u003csub\u003e4\u003c/sub\u003e)H\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e. Alg did not form nanosheet structures in the presence of H\u003csub\u003e3\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, NH\u003csub\u003e4\u003c/sub\u003eOH, H\u003csub\u003e3\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e and NH\u003csub\u003e4\u003c/sub\u003eOH mixture, or Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e. Also, CPNSs were not formed with Alg in the presence of other sulfates, sulfite, and ammonium-related salts such as ammonium sulfite ((NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e3\u003c/sub\u003e), ammonium sulfate ((NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e), ammonium chloride (NH\u003csub\u003e4\u003c/sub\u003eCl), and sodium sulfite (Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e3\u003c/sub\u003e) in the same mass ratio (Supplementary Fig.\u0026nbsp;4). Thus, we conclude that solid-state heating at 180\u0026deg;C and DAHP have a crucial role in the formation of perfect CPNS structures.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePhosphate diester linkages mediate the formation of CPNSs\u003c/h2\u003e \u003cp\u003eIn order to investigate the process of CPNSs formation, we performed a time-course analysis of Alg/DAHP mixture that was heated at 180\u0026deg;C for a duration of 3 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). As the heating progressed, notable changes occurred. Within 5 min, the color of the mixture shifted to a pale brown hue, followed by a transition to dark brown at 15 min (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Eventually, within 30 min, the mixture turned black due to carbonization. Concurrently, the time-course TEM analysis demonstrated the formation of supramolecular structures by Alg within the initial 5-minute heating period (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). At 15 min, it tended to form thick and large sheet-like structures, which subsequently became thin at 30 min. After 3 h of heating thin-layered clean sheets of varying sizes were formed, likely due to fragmentation during the later stage of the thermal process. The time-course XRD pattern of the products and Alg are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC. Over time, a noticeable alteration in the crystallinity of Alg becomes apparent. The crystallinity in Alg arise from the arrangement of G (2\u003cem\u003eθ\u0026thinsp;=\u003c/em\u003e\u0026thinsp;13.7\u0026deg;) and M (2\u003cem\u003eθ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;21.6\u0026deg;) units and the amorphous halo (broad peak centered at 2\u003cem\u003eθ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;39\u0026deg;), which are disrupted and realigned during the heating. The amorphous halo completely disappeared after 15 min. The appearance of a broad peak centered at 2\u003cem\u003eθ\u003c/em\u003e of 26.2\u0026deg; indicates very small graphene domains in the carbonized products with highly disordered carbon\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. This transformation, denoting carbonization, can be observed from 30 min onwards. The hydrodynamic diameter of the CPNSs by heating for different time intervals shows an increase in size to as high as ca. 2200 nm at 30 min, which decreases to ca. 440 nm after 3 h due to fragmentation of the polymer sheets during carbonization (Supplementary \u003cb\u003eTable\u0026nbsp;2\u003c/b\u003e). The TEM-energy-dispersive X-ray spectroscopy (EDS) mapping of CPNSs-180 displayed in Supplementary Fig.\u0026nbsp;5A confirms the presence of nitrogen and phosphorus, and the HRTEM image and the selective area electron diffraction (SAED) pattern suggest the low crystalline nature of the CPNSs (Supplementary Fig.\u0026nbsp;5B), in agreement with the XRD pattern. If the 2D structures are not carbon nanosheets, they might instead be black phosphorous nanosheets. It is a thermodynamically stable allotrope of phosphorus with 2D structure of atomic arrangement very similar to that of graphite\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Black phosphorus is highly crystalline and has orthorhombic structure\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. However, the XRD patterns in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and Supplementary Fig.\u0026nbsp;2 and SAED pattern in Supplementary Fig.\u0026nbsp;5B did not show any crystalline properties corresponding to black phosphorus. Furthermore, black phosphorus is formed only at very high temperature and pressure\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Thus, the possibility of 2D phosphorus allotropes can be ruled out, and we believe the 2D nanostructures obtained by heating a mixture of sodium alginate and diammonium hydrogen phosphate as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB must be CPNSs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe molecular structural changes occurring during the heating were further studied by Fourier-transform infrared spectroscopy (FTIR) (Supplementary \u003cb\u003eFig.\u0026nbsp;6\u003c/b\u003e). The FTIR spectrum of Alg exhibited specific vibrational modes such as \u0026ndash;OH stretching at 3200\u0026minus;3400 cm\u003csup\u003e\u0026ndash;\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, asymmetric stretching of \u0026ndash;COO\u003csup\u003e\u0026ndash;\u003c/sup\u003e at 1610 cm\u003csup\u003e\u0026ndash;\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, the symmetric stretching of \u0026ndash;COO\u003csup\u003e\u0026ndash;\u003c/sup\u003e at 1412 cm\u003csup\u003e\u0026ndash;\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, and the C\u0026ndash;O\u0026ndash;C (ring) vibrational modes at 1081 cm\u003csup\u003e\u0026ndash;\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e of the pyranose rings\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. The C\u0026ndash;O(H) symmetric vibration peak appeared at 1306 cm\u003csup\u003e\u0026ndash;\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, and stretching vibration of the C\u0026ndash;O\u0026ndash;C glycosidic linkage in alginate polymer appeared at 1036 cm\u003csup\u003e\u0026ndash;\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. FTIR spectra of Alg show significant changes in peaks at lower wavenumber region, 500\u0026ndash;1700 cm\u003csup\u003e\u0026ndash;\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e for different durations of heating with DAHP. Notably, the C\u0026ndash;O(H) peak at 1306 cm\u003csup\u003e\u0026ndash; \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e decreased significantly with heating time, probably due to the dehydration process. The \u0026ndash;C\u0026ndash;O(H) symmetric vibration peak at 1306 cm\u003csup\u003e\u0026ndash;\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e began to disappear within 5 min, meanwhile a new P\u0026thinsp;=\u0026thinsp;O asymmetric stretching peak emerged at 1246 cm\u003csup\u003e\u0026ndash;\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e and then started disappearing after 30 min. A peak at 1739 cm\u003csup\u003e\u0026ndash;\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e appeared after 5 min and disappeared after 30 min, indicating some new carbonyl groups of esters are formed and then degraded with time\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. After 15 min of heating, new peaks emerged at 1054 cm\u003csup\u003e\u0026ndash;\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, corresponding to P\u0026ndash;O\u0026ndash;C stretching in the phosphate ester bond. The Alg after reaction with DAHP and without heating (i.e., 0 min) showed an O\u0026ndash;P\u0026ndash;O bending vibration peak of the phosphate ester at 546 cm\u003csup\u003e\u0026ndash;\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, which slightly shifted to 515 cm\u003csup\u003e\u0026ndash;\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e after 5 min onwards and decreased significantly after 1 h. A new peak emerged at 1054 cm\u003csup\u003e\u0026ndash;\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, corresponding to P\u0026ndash;O\u0026ndash;C stretching in the phosphate ester bond. It can be inferred that Alg polymer chains are cross-linked \u003cem\u003evia\u003c/em\u003e phosphate diester bonds. During the heating, (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e undergoes thermal decomposition to form various chemical species, such as NH\u003csub\u003e3 (\u003cem\u003eg\u003c/em\u003e)\u003c/sub\u003e, (NH\u003csub\u003e4\u003c/sub\u003e)H\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4 (\u003cem\u003es\u003c/em\u003e)\u003c/sub\u003e, and H\u003csub\u003e3\u003c/sub\u003ePO\u003csub\u003e4 (\u003cem\u003el\u003c/em\u003e)\u003c/sub\u003e\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. The phosphoric acid reacts with Alg to form esters, which is in agreement with a similar work reported by Marcilla \u003cem\u003eet al\u003c/em\u003e., in which the various acid species react with different compounds in tobacco to form esters. Meanwhile, a portion of the NH\u003csub\u003e3\u003c/sub\u003e formed by the thermal degradation of (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e could form quaternary ammonium salts of the carboxylic acid group\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe conducted the \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003eP nuclear magnetic resonance (NMR) spectroscopy analysis of the time course formation of CPNSs-180 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). The \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003eP NMR peak of phosphate group appeared at a chemical shift of 0.87 ppm for the purified, non-heated Alg/DAHP mixture, indicating the adsorption of phosphate on the Alg. Upon heating for 1 min at 180\u0026deg;C, the peak shifts a little downfield (to 1.21 ppm), probably due to the formation of monoesters\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. With a further increase in heating time to 7.5 min, peak resonances toward an up field of the central peak were observed (\u0026ndash;5.39, \u0026minus;\u0026thinsp;8.50, and \u0026minus;\u0026thinsp;9.26 ppm), depicting the formation of phosphate diesters and pyrophosphate structures. After 15 minutes of heating, the peaks for pyrophosphate (\u0026ndash;8.50 and \u0026minus;\u0026thinsp;9.26 ppm) disappeared, and with further heating the peaks 1.21 and \u0026minus;\u0026thinsp;5.39 ppm depicting phosphate esters, including monoesters and diesters remained.\u003c/p\u003e \u003cp\u003eThe plausible mechanism of the formation of CPNSs is illustrated in Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The formation of diester in solid-state will form bridges between the alginate polymer chains to form 2D polymer sheets. The previous report reveals that dry phosphorylation of starch using orthophosphate occurs through the reactive hydroxyl groups of the starch molecules to form brown-colored products at temperatures of 170\u0026deg;C and higher\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Investigation on the phosphorylation of Alg using urea/phosphate system using various NMR spectroscopy techniques revealed that the most probable site for phosphorylation is the equatorial hydroxyl group of mannuronic acid units in the polymeric chain\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Therefore, it is evident that heating of Alg with DAHP in a solid-state initially leads to the formation of sheet-like structures formed by the cross-linking of Alg polymer chains \u003cem\u003evia\u003c/em\u003e phosphate diester linkages. Since this reaction system contains a mixture of monoesters, diesters, and unreacted alginate chains, the carbonized product contains CPNSs along with polymeric Alg featuring nonspecific shapes. The molecular arrangement in pure Alg is mainly due to the solid-state intra- and inter-molecular hydrogen bonding, which are disrupted upon heating above 170\u0026deg;C and produce carbonized products without specific shape\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. However, the formation of cross-linking among the alginate polymer chains by phosphate ester bonds dominates the formation of a stable 2D structure, resulting in carbonized polymeric nanosheets.\u003c/p\u003e \u003cp\u003eThe elemental composition of Alg and the products obtained at various time intervals is presented in Supplementary \u003cb\u003eTable S3\u003c/b\u003e. The carbon content (weight percentage) of pure Alg has been found to be 29.16%, which is close to the values reported by previous studies, and some report reveals that it varies with the harvesting season of the algae\u003csup\u003e\u003cspan additionalcitationids=\"CR58\" citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. The carbon and oxygen contents of Alg after reacting with DAHP (i.e., CPNSs-180 0 min) is determined to be 19.94% and 43.34%, respectively. The carbon content increased up to 38.73% and the oxygen content decreased to 33.93% after being heated at 180\u0026deg;C for 3 h, indicating carbonization to form slightly carbonaceous nanomaterials (i.e., CPNSs). A previous report also suggests that P/N-doped carbon dots synthesized at low temperature (90\u0026deg;C) possess a low degree of carbon content with a weight percentage of 8.62\u003csup\u003e60\u003c/sup\u003e. Carbonization of precursors in semi-closed atmosphere and temperatures as high as 900\u0026deg;C has been reported to yield carbonized products with higher carbon content (as high as ~\u0026thinsp;69%) and low oxygen content (10%)\u003csup\u003e61\u003c/sup\u003e. The carbonized polymer products have nitrogen doping, and the final CPNSs obtained after 3 h possess 6.8% nitrogen by weight. Alginate polymer has high affinity toward phosphate ions\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e and it forms phosphate ester after reacting with the DAHP during the heating process, resulting in high phosphorous content (12.45%) in the CPNSs as determined by inductively coupled plasma optical emission spectroscopy (ICP-OES). The initial heating of Alg/DAHP mixture at 180\u0026deg;C leads to the crosslinking of the hydroxyl groups of the DAHP with the side chain hydroxyl group of the Alg to form phosphate ester bond\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. With increase in the reaction time, carbonization progressed, resulting in the decrease in P content. At 3 h of heating intense phosphorylation and phosphorus doping occurred in the CPNSs due to the presence of phosphoric acid and P\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e in the system thereby increasing the P content to 12.45%. Thus, the CPNSs obtained from Alg by carbonization in the presence of DAHP are N and P co-doped. The C1s, O1s, N1s, and P2p XPS spectra of the CPNSs synthesized at 180\u0026deg;C for 3 h are presented in Supplementary \u003cb\u003eFig. S7\u003c/b\u003e. The CPNSs have oxygen-containing functional groups and N-doping in the form of pyridinic (399.1 eV), graphitic (399.85 eV), and pyrrolic (400.66 eV) nitrogen. The deconvoluted P2p spectra of the CPNSs show peaks at 132.49, 133.01, 133.63, and 134.42 eV corresponding to the presence of P\u0026ndash;C, P\u0026ndash;O, P\u0026ndash;O\u0026ndash;C, and P\u0026thinsp;=\u0026thinsp;O bonding, respectively, which confirm phosphorous is incorporated in the CPNSs\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCPNS-modified filter paper for efficient removal of bacteria\u003c/h2\u003e \u003cp\u003eThe CPNSs were tested for their antibacterial activity toward Gram-negative (\u003cem\u003eE. coli\u003c/em\u003e) and Gram-positive (\u003cem\u003eS. aureus\u003c/em\u003e) bacteria and toward \u003cem\u003eV. parahaemolyticus\u003c/em\u003e, a Gram-negative bacteria that poses a significant risk in aquaculture. \u003cem\u003eV. parahaemolyticus\u003c/em\u003e can rapidly multiply and infect cultured seafood species, which not only damages the health of these species but also increases the risk of foodborne illnesses when these products are consumed\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. Different from the GO nanosheets which exhibit antibacterial activity through different mechanisms such as direct interaction with the bacteria through their sharp edges or by wrapping on the bacterial cell\u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e,\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e, the CPNSs having 2D structure do not show antibacterial activity (Supplementary \u003cb\u003eFig.\u0026nbsp;8\u003c/b\u003e). The difference in the antibacterial behavior of the CPNSs may be ascribed to only adsorbing bacteria but could not further disrupt the bacterial membranes due to polymeric structures on the CPNSs' surfaces. It has been reported that the functional groups on GO play a crucial role in its antimicrobial activities\u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. However, these CPNSs, when modified on filter paper could effectively remove bacteria from contaminated water. Incubating the filter paper with CPNSs resulted in the effective coating of the nanosheets on the fibers, as evident from the SEM images in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, however, it did not form a separate layer above the filter paper. The CPNS-modified filter paper was effective in removing \u003cem\u003eV. parahaemolyticus\u003c/em\u003e (10\u003csup\u003e5\u003c/sup\u003e CFU mL\u003csup\u003e\u0026ndash;\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e) from contaminated seawater samples, with CPNSs-180 and CPNSs-210 showing superior effects (\u0026gt;\u0026thinsp;90%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Notably, neither the bacteria's morphology nor the bacterial membrane was disrupted after passing through the CPNS-modified membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Adsorbing bacteria without membrane disruption is advantageous, preventing toxin release into the filtrate. For instance, disruption of \u003cem\u003eV. parahaemolyticus\u003c/em\u003e\u0026rsquo; cell membrane releases toxins like PirA and PirB proteins, which induce necrosis and functional loss in the hepatopancreas of shrimp\u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe further evaluated CPNSs-180-modified filter paper for the removal of \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e. The removal efficiency for \u003cem\u003eE. coli\u003c/em\u003e was less than 30%, and that for \u003cem\u003eS. aureus\u003c/em\u003e was around 80% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The difference in the bacterial removal efficiency of CPNSs-modified filter paper may be attributed to the different bacterial shapes and membrane structures\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. The SEM image of the CPNS-modified filter paper after passing the \u003cem\u003eV. parahaemolyticus\u003c/em\u003e bacteria solution clearly shows bacteria trapped on the membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). In contrast to our previous work, graphene oxide@carbon nanogels (GO@CNGs)-modified membrane reported for the removal of bacteria from contaminated water, where the efficiency of the membrane decreases with an increase in water flux\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, the efficiency of the CPNS-modified membrane was not affected by the increase in water flux (Supplementary \u003cb\u003eFig.\u0026nbsp;9\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCombating\u003c/b\u003e \u003cb\u003eV. parahaemolyticus\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe efficiency for the removal of \u003cem\u003eV. parahaemolyticus\u003c/em\u003e at a higher concentration (10\u003csup\u003e7\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;1\u003c/sup\u003e) was further evaluated in aquarium condition (2 L water in the aquarium tank) using the CPNS-modified filter paper with a larger surface area (17.34 cm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e). The CPNSs-modified filter paper was effective in eliminating\u0026thinsp;\u0026gt;\u0026thinsp;98% \u003cem\u003eV. parahaemolyticus\u003c/em\u003e within 2 h of circulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), and no leakage of bacteria was observed from the membrane even after 24 h. The uncoated filter paper (Ctrl) showed removal of ca. 70% within 1 h; however, it decreased with time and down to ~\u0026thinsp;17% after 24 h, which shows that though the filter paper can adsorb bacteria, upon continuous passage of water, the bacteria are washed from the membrane back to the solution, due to the large pore size of the membrane and weak affinity toward \u003cem\u003eV. parahaemolyticus\u003c/em\u003e. It is noteworthy that the bacterial removal efficiency for the GO-coated filter paper was ~\u0026thinsp;74% after 4 h and remained stagnant beyond that time, probably due to the clogging of pores due to fouling of the membrane\u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e; which shows the superior efficacy of our CPNSs-modified filter paper. The decrease in water flux and removal efficiency of the membrane due to the clogging of pores of the membrane is a major drawback in membrane-based filtration systems. Therefore, we further performed the shrimp challenge experiments with the CPNSs-modified filter (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). After challenging white leg shrimp (\u003cem\u003eLitopenaeus vannamei\u003c/em\u003e, 10 no. in 2 L sea water) with \u003cem\u003eV. parahaemolyticus\u003c/em\u003e (10\u003csup\u003e6\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;1\u003c/sup\u003e), CPNSs-180-modified filter paper was loaded onto a filter holder, and the aquarium water was circulated through it, and the results were compared with that of the control filter (filter paper without CPNSs-180 coating) and control (without any filter paper or membranes). The shrimps in the CPNSs-modified filter paper group showed 100% survival even after 48 h, while that of the other two groups decreased to \u0026lt;\u0026thinsp;50%. After 72 h, the survival rate of the shrimp decreased to 10% and 20% for the control and the control filter paper group, respectively; \u0026gt;50% survival rate was observed in CPNSs-modified filter paper group. Therefore, we hope that the CPNS-modified filter may serve its use for filtering out even a very high concentration of bacteria contaminated in aquarium water. Notably, replacing the CPNSs-modified paper every 48 h after filtration could remove the bacteria completely without affecting the survival rate of the shrimp upon \u003cem\u003eVibrio\u003c/em\u003e infection (Supplementary \u003cb\u003eFig.\u0026nbsp;10\u003c/b\u003e). Though the control filter paper was also replaced every 48 h, only 10% survival was observed after 96 h.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn summary, the methodology presented in this study, which employs low-temperature carbonization of Alg with DAHP, presents a unique approach to prepare 2D carbonized nanomaterials for filtering out pathogens from water. The advantage of this procedure is that it bypasses the need for high temperatures, sophisticated equipment, or hazardous solvents, making it more sustainable and potentially cost-effective. The decomposition of DAHP yields a phosphorylating agent, PO\u003csub\u003e4\u003c/sub\u003e \u003csup\u003e3\u0026ndash;\u003c/sup\u003e, which facilitates the phosphorylation of the equatorial hydroxyl group of the mannuronic units within the alginate polymer chain. This results in the creation of phosphate diester linkages between the chains, which are more robust than the solid-state hydrogen bonds in the polysaccharides. Consequently, 2D polymer sheets are formed at the early stage of heating and are subsequently carbonized to produce CPNSs. Moreover, the study effectively demonstrates a practical application of the synthesized CPNSs, using them to enhance the survival rate of shrimp in aquaculture by removing the bacterial strain \u003cem\u003eV. parahaemolyticus\u003c/em\u003e. These CPNSs demonstrate notable bacterial adsorption capabilities, particularly towards strains like \u003cem\u003eV. parahaemolyticus\u003c/em\u003e. The adsorption property facilitates the development of an efficient bacterial filtration system using ordinary filter paper. Notably, our shrimp challenge experiments indicate an enhanced survival rate among shrimp exposed to \u003cem\u003eV. parahaemolyticus\u003c/em\u003e after passing through the CPNSs-modified filter paper. This is an important contribution as it suggests the potential for Alg-CPNSs to improve aquaculture health and productivity. \u003cem\u003eV. parahaemolyticus\u003c/em\u003e is responsible for causing acute hepatopancreatic necrosis disease (AHPND) in shrimp, a condition that leads to severe damage and dysfunction in the hepatopancreas. AHPND was accountable for a massive loss of US\u003cspan\u003e$\u003c/span\u003e44\u0026nbsp;billion in the global shrimp farming sector between 2010 and 2016\u003csup\u003e72\u003c/sup\u003e. As of the latest estimates, the annual economic losses attributed to AHPND now stand at USD 7 billion\u003csup\u003e \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e \u003c/sup\u003e. Although this study represents a promising advancement in the synthesis of CPNSs and their application in aquaculture, further exploration and validation of the findings are necessary to understand this approach's potential benefits and drawbacks fully.\u003c/p\u003e \u003cp\u003eWe have used Alg as a source of polysaccharide in this study. It would be interesting to explore whether other marine or non-marine polysaccharides can also be used as precursors in preparing CPNSs, which could potentially widen the applications and versatility of the method. On the other hand, our study shows that Alg-derived CPNSs have a high bacterial adsorption capability, but it could benefit from a more detailed examination of the mechanism behind this adsorption. Understanding this mechanism could aid in improving the efficiency and specificity of the CPNSs for bacterial adsorption. While the study focuses on the removal of specific bacterial strains, it would be valuable to examine the effectiveness of these CPNSs in adsorbing other types of contaminants in aquaculture water in the future, which could determine the broader applicability of the approach. The long-term efficacy of these Alg-CPNSs in a realistic aquaculture setting, including their durability and the need for replacement over time, is also essential in the future. While this study observes an immediate increase in the survival rate of shrimp, it might be beneficial to look at the long-term effects of using Alg-CPNSs in the future. This could include the potential impacts on the health and growth of the shrimp over time, as well as any potential environmental impacts. Finally, it would be valuable to assess the environmental impact of using Alg-CPNSs, including a lifecycle analysis, considering the source of the polysaccharides, the process of synthesizing the CPNSs, their use, and eventual disposal.\u003c/p\u003e \u003c/div\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eSynthesis of CPNSs\u003c/h2\u003e \u003cp\u003eThe CPNSs were prepared by heating a solid mixture of Alg and DAHP in 1:5 mass ratio in a muffle furnace (DH 300, Dengyng, New Taipei City, Taiwan). The mixture of Alg and DAHP was blended in a coffee grinder for 5 min. 1.0 g of the mixture was then placed in 50 mL glass vials and heated in two steps: first at 60\u0026deg;C for 3 h followed by raising the temperature to 120, 150, 180, 210, or 240\u0026deg;C and heated for 3 h. The carbonized residue thus obtained was allowed to cool to room temperature, and 50 mL of deionized (DI) water was added, mixed well, and centrifuged at a relative centrifugal force (RCF) of 15,000 \u003cem\u003eg\u003c/em\u003e for 1 h. After three centrifugation/washing cycles, the pellets were dispersed again in DI water, and the pH of the solutions were adjusted to 9 by adding NaOH solution (0.5 M), and then sonicated (100 W) for 15 min. The sonicated dispersions were centrifuged at 500 \u003cem\u003eg\u003c/em\u003e for 15 min to remove larger carbonized particles, and the supernatants containing the CPNSs were collected and quantified by freeze-drying. The CPNSs dispersion was stored at 4\u0026deg;C when not in use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of CPNSs-modified Filter paper and determination of bacteria removal efficiency\u003c/h2\u003e \u003cp\u003eQualitative filter paper (Advantec\u0026reg; 2, pore size 5 \u0026micro;m, and thickness 0.26 mm) was modified with CPNSs for the bacterial removal from water. Briefly, the filter paper (25 mm in diameter) was incubated with CPNSs solution (0.5 mg mL\u003csup\u003e\u0026minus;1\u003c/sup\u003e, 5 mL) for 2 h under shaking at 150 rpm to obtain a loading of ca. 0.2 mg cm\u003csup\u003e\u0026ndash;\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The CPNSs-modified filter papers (area 4.91 cm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e and active area 2.83 cm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e) were then placed in a syringe holder (diameter 25 mm) and washed with 50 mL of sodium phosphate buffer (5 mM, pH 7.4) to remove the unbound CPNSs (flow rate 5.8 L min\u003csup\u003e\u0026ndash;\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e m\u003csup\u003e\u0026ndash;\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e). The CPNSs-modified filter paper was tested for the removal of \u003cem\u003eEscherichia coli\u003c/em\u003e (\u003cem\u003eE. coli\u003c/em\u003e), \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (\u003cem\u003eS. aureus\u003c/em\u003e), and \u003cem\u003eV. parahaemolyticus\u003c/em\u003e. \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e were cultured overnight in Lysogeny broth (LB) medium at 37\u0026deg;C with shaking at 150 rpm, while \u003cem\u003eV. parahaemolyticus\u003c/em\u003e was cultured in tryptic soy broth (TSB) containing 3% NaCl at 25\u0026deg;C. The bacteria cells were washed twice with sodium phosphate buffer (5 mM, pH 7.4) for \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e; sodium phosphate buffer (5 mM, pH 7.4) containing 3% NaCl for \u003cem\u003eV. parahaemolyticus\u003c/em\u003e after removing the medium by centrifugation at 3000 \u003cem\u003eg\u003c/em\u003e for 5 min at 25\u0026deg;C. The bacteria removal efficiency of the CPNSs-modified filter paper was determined using 10\u003csup\u003e5\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;1\u003c/sup\u003e bacteria solution by a dead-end mode filtration, using a syringe pump (KDS100, KD Scientific, Holliston, MA, USA), with a water flux of 400 mL min\u003csup\u003e\u0026minus;1\u003c/sup\u003e m\u003csup\u003e\u0026minus;2\u003c/sup\u003e. The filtrate was collected and diluted 100-fold, and then 100 \u0026micro;L of the diluted solution was spread on LB-agar plates and incubated for 12 h at 37\u0026deg;C (TSB agar plates were used for \u003cem\u003eV. parahaemolyticus\u003c/em\u003e and incubated at 25\u0026deg;C). The liquid culture of the bacteria was also carried out by supplementing with the respective medium and incubating overnight, followed by measuring the absorbance at 600 nm (OD\u003csub\u003e600\u003c/sub\u003e). Each experiment was performed in triplicate for each condition. Transmission electron microscopic (TEM; Tecnai 20 G2 S-Twin, Philips/FEI, Hillsboro, OR, USA) images were recorded to understand the morphology of the bacteria. The scanning electron microscopic (SEM; Hitachi S-4800, Hitachi High-Technologies, Tokyo, Japan) images of CPNSs-modified filter paper after passing 10\u003csup\u003e7\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;1\u003c/sup\u003e \u003cem\u003eV. parahaemolyticus\u003c/em\u003e were taken to understand the bacteria removal mechanism.\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eJ.-Y.L. and C.-C.H conceived the original idea and supervised the project from the beginning to the end, and helped in the manuscript preparation. A.A. and B.U. carried out the experiments and prepared the manuscript. C.-Y.W. and H.-J.L. helped in the manuscript preparation. A.A. and B.U. contributed equally. All authors discussed the results and contributed to the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis work was supported by the National Science and Technology Council (NSTC) of Taiwan under Contract Nos. 110-2221-E-019-001, 110-2811-M-019-501, 110-2314-B-182-008-MY3, and 112-2811-B-182-022, Chang Gung Memorial Hospital, Linkou under Contract No. CMRPD2L0161, Chang Gung University under Contract No. OMRPD2N0011, and the Center of Excellence for the Oceans, National Taiwan Ocean University from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan.\u003c/p\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eAll data generated or analyzed during this study are included in this published article and its supplementary information files.\u003c/p\u003e \u003c/div\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSperanza, G. 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FAO Fisheries and Aquaculture Circular No. 1190, Rome, FAO (2020).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-clean-water","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"npjcleanwater","sideBox":"Learn more about [npj Clean Water](http://www.nature.com/npjcleanwater/)","snPcode":"41545","submissionUrl":"https://mts-onc.nature.com/cgi-bin/main.plex","title":"npj Clean Water","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4153360/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4153360/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn this study, we have developed a simple and low-temperature method without using toxic chemicals, to synthesize carbonized polymer nanosheets (CPNSs) that exhibit potent bacterial adsorption capabilities for their use as a filter to remove bacteria from aquaculture water. Sodium alginate (Alg), an algae-derived polysaccharide was pyrolyzed with diammonium hydrogen phosphate (DAHP) in solid state at 180 \u0026deg;C. Initially, Alg underwent dehydration and cross-linking \u003cem\u003evia\u003c/em\u003e phosphate ester bonds followed by carbonization resulted in the formation of 2D structured CPNSs with distinct polymeric characteristics. The as-synthesized CPNSs demonstrate a high bacterial adsorption capability toward \u003cem\u003eV. parahaemolyticus\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e. Furthermore, CPNSs can be used to modify ordinary filter paper to make them effective in filtering system. Aquaculture water filtration experiments using CPNSs-modified filter paper revealed an increase in the survival rate (\u0026gt;\u0026thinsp;50%) of shrimp challenged with \u003cem\u003eV. parahaemolyticus\u003c/em\u003e upon circulation through the Alg-CPNSs-modified membrane, demonstrating their potential as a viable aquaculture filter.\u003c/p\u003e","manuscriptTitle":"Phosphate ester-linked carbonized polymer nanosheets as efficient filters for removing Vibrio parahaemolyticus from aquaculture water","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-29 10:01:04","doi":"10.21203/rs.3.rs-4153360/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2024-05-13T13:06:33+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-05-12T17:11:23+00:00","index":3,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-05-01T11:13:08+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-04-30T22:41:16+00:00","index":3,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-04-29T06:24:40+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-04-24T07:57:18+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-04-24T06:37:02+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2024-04-24T06:33:11+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-03-25T11:37:16+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-23T07:57:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Clean Water","date":"2024-03-23T07:56:59+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-clean-water","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"npjcleanwater","sideBox":"Learn more about [npj Clean Water](http://www.nature.com/npjcleanwater/)","snPcode":"41545","submissionUrl":"https://mts-onc.nature.com/cgi-bin/main.plex","title":"npj Clean Water","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b785591a-c473-42fe-b71d-437a3569bdf9","owner":[],"postedDate":"April 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":31083509,"name":"Earth and environmental sciences/Environmental sciences/Environmental chemistry/Pollution remediation"},{"id":31083510,"name":"Physical sciences/Nanoscience and technology/Nanoscale materials"},{"id":31083511,"name":"Physical sciences/Materials science/Structural materials"}],"tags":[],"updatedAt":"2024-09-06T07:09:25+00:00","versionOfRecord":{"articleIdentity":"rs-4153360","link":"https://doi.org/10.1038/s41545-024-00378-7","journal":{"identity":"npj-clean-water","isVorOnly":false,"title":"npj Clean Water"},"publishedOn":"2024-09-05 04:00:00","publishedOnDateReadable":"September 5th, 2024"},"versionCreatedAt":"2024-04-29 10:01:04","video":"","vorDoi":"10.1038/s41545-024-00378-7","vorDoiUrl":"https://doi.org/10.1038/s41545-024-00378-7","workflowStages":[]},"version":"v1","identity":"rs-4153360","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4153360","identity":"rs-4153360","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}