Technical Field:

Embodiments of the present invention generally relate to methods of preparing bio-based materials, and more particularly, to methods of regenerating cellulose from lignocellulosic biomass and preparing bio-based materials embedded with nanocomposites having antibacterial properties.

Background Art:

Celluloses are the most abundant renewable resource and natural biopolymer available on the earth. Moreover, celluloses are the most abundant polysaccharide material available worldwide and it is considered as an unlimited renewable organic material. Celluloses are environmental friendly and biocompatible and in cellulose, the molecular structure, P-(1→4)-D-glucan allows chain packing by strong inter and intramolecular hydrogen-bonding. As celluloses are environmentally friendly material and consequently adequate utilization of celluloses can not only save and protect the environment but also save the limited unrenewable petroleum resources available.

Currently, celluloses are far from being sufficiently utilized in chemical industry, mainly because the current processes available in the prior art for dissolving cellulose are complex in nature, and costly to manufacture. Due to stiff molecules and close chain packing via the numerous inter- and intra molecular hydrogen bonds, it is extremely hard to dissolve cellulose using water. Conventionally viscose processes have been used for producing regenerated cellulose products such as rayon, glassine paper and the like. The conventional viscose process includes reacting cellulose with CS2 (33 wt %) in the presence of strong base where the concentration of sodium hydroxide being 18 wt % to produce cellulose xanthate that is dissolved in the alkaline solution to form a viscose solution, and then spinning or casting the viscose solution of cellulose, followed by regenerating in diluent acid solution to obtain viscose fiber (rayon) or glassine paper. A great quantity of toxic gases such as CS ₂ and H ₂ S which severely pollute environment are released during the process and are harmful to human health (J. Macromol. Sci.- Rev. Macromol. Chem., 1980, C18 (1 ), 1 ).

Another prior art utilizes the cuprammonium process for producing cuprammonium rayon that suffer from the disadvantage of environmental pollution, high cost and difficulty to recover the solution. However, the prior art processes in which other organic or inorganic solvents such as dimethylsulfoxide-nitrogen oxide are utilized as disclosed in U.S. Pat. No.3236669, aqueous ZnCI ₂ solution as disclosed in U.S. Pat. No. 5,290,349, LiCI/DMAc as disclosed in U.S. Pat. No. 4,302,252 and the like are used respectively, are difficult in industrialization due to the cost involved and their complicated dissolving procedures.

Further, N-methylmorpholine oxide (NMMO) is disclosed in U.S. Pat. No.

2, 179, 181 , U.K. Patent No. GB1 144048, and U.S. Pat. No. 4,246,221 is considered as the most promising solvent for cellulose so far. However, producing it for industrial production is time-consuming due to high cost and high spinning temperature.

In recent past it has been researched that cellulose can be dissolved using specific solvents, for example in N ₂ 0 ₄ /N, N-dimethylformamide (DMF), S0 ₂ /amine, and Me ₂ SO/ paraformaldehyde (PF). Another process used to regenerate cellulose comprises reacting cellulose with urea at high temperature to obtain cellulose carbamate, and then dissolving directly in a diluent alkaline solution to obtain spinning solution (Finland Patent No. FI61003; Finland Patent No. FI62318; U.S. Patent No. US4404369). However, this process requires a great amount of urea, which subsequently leads to one or more side products, and is difficult for industrialization either.

In addition, Cellulose II can be prepared by two distinct routes: mercerization

(alkaline treatment) and regeneration (solubilization and subsequent recrystallization). Particularly, ionic liquids have been focused as solvents for cellulose processing and derivatization to produce regenerated cellulose. The most common ionic liquid is 1 - butyl-3-methylimidazolium chloride. However, recent studies have confirmed that this solvent requires a long period of time of about 10 h to completely dissolve cellulose fibres.

The raw materials available for cellulose extraction are in abundance. However, it should be noted that Malaysia is the second world largest palm oil producer after Indonesia in the year 2009 and the Oil palm fibre is rich in cellulose and lignin. Presently, the Oil Palm Empty Fruit Bunch (EFB) are waste by-products and the EFB are currently being disposed by burning due to low commercialization value. Extraction of fibre from Oil Palm Empty Fruit Bunch (EFB) can produce raw cellulose, hemicelluloses and lignin with the compositional percentage of cellulose (44.2%), hemicelluloses (33.5%), and lignin (20.4%).

Therefore, there remains a need in the art for a cost effective cellulose extraction method. Moreover, there remains a need in the art for a cost effective preparation method of preparing cellulose based materials having antibacterial properties. It is therefore an object of the present invention to provide cellulose based materials having antibacterial properties which can be obtained by a technically very simple process and do not have the abovementioned disadvantages of the prior art.

Disclosure of the Invention:

Embodiments of the present invention aim to provide a method for preparing a bio-based material which includes the steps of extracting celluloses from lignocellulosic biomass and remove lignin from the lignocellulosic biomass. After that, treating the extracted celluloses in alkali solution for a sufficient time and temperature to form a liquid solution, drying the liquid solution at a first temperature for a particular time period to form a powder material, pre-cooling one or more aqueous solvents at a first temperature and the one or more solvents are selected from a group consisting of a mixed aqueous sodium hydroxide (NaOH) solution and urea solution, a mixed aqueous lithium hydroxide solution and urea solution and any combinations thereof. Subsequently, the method includes the steps of dissolving the powder material in the pre-cooled aqueous solvent for a p re-determined time period to form a first solution. Further, the first solution is pre-cooled at a second temperature to form a solid form, and the solid form is thawed at room temperature to obtain a transparent cellulose solution. The said solution is coagulated in aqueous solution of H ₂ S0 ₄ to form a regenerated cellulose film.

In preferred embodiment of the present invention, the method further includes the steps of preparing a metal-graphene oxide nano-composite and subsequently mixing the metal-graphene oxide nano-composite into the cellulose solution to form an interaction mixture. After that, the interaction mixture is coagulated in a non-solvent coagulant, and a highly porous structure of a cellulose membrane is regenerated. Particularly, the regenerated cellulose membrane has anti-bacterial properties.

In another embodiment of the present invention, the method further includes the steps of preparing solid particulate particles via a chemical co-precipitation process wherein the chemical is selected from a group consisting of ferrous, ferric chloride and alkali hydroxide. In subsequent step, the solid particulate particles are dispersed into the cellulose solution to form a cellulose solution mixture, and the cellulose solution mixture is coagulated in aqueous solution of H ₂ S0 to form cellulose beads.

In yet another embodiment of the present invention, the method steps of extracting celluloses from the lignocellulosic biomass includes the steps of bleaching the lignocellulosic biomass to produce a dissolving lignocellulosic biomass. Particularly, the process of bleaching of lignocellulosic biomass includes the step of alkaline bleaching and chlorinating the lignocellulosic biomass with an aqueous chlorite solution, and extracting the celluloses from the lignocellulosic biomass with acetate buffer and distilled water.

According to one embodiment of the present invention, solid particulate particles are magnetite (Fe304) particles. Particularly, the cellulose beads are magnetic cellulose beads and the alkali hydroxide used in the present invention is sodium hydroxide.

In one embodiment, the bio-based materials are used as functional additives and ingredients in pharmaceutical or cosmetic preparations, food preparations, building materials, varnishes, paints, coating compounds and polymers.

While the invention is described herein by way of example using several embodiments and illustrative drawings, those skilled in the art will recognize that the invention is not limited to the embodiments of drawing or drawings described, and are not intended to represent the scale of the various components. Further, some components that may form a part of the invention may not be illustrated in certain figures, for ease of illustration, and such omissions do not limit the embodiments outlined in any way. It should be understood that the drawings and detailed description thereto are not intended to limit the invention to the particular form disclosed, but on the contrary, the invention is to cover all modification, equivalents and alternatives falling within the spirit and scope of the present invention as defined by the appended claims. The headings used herein are for organizational purposes only and are not meant to be used to limit the scope of the description or the claims. As used throughout this application, the word "may" is used in a permissive sense (i.e., meaning having the potential to), rather than the mandatory sense (i.e., meaning must). Similarly, the words "include," "including," and "includes" mean including, but not limited to. Further, the words "a" or "an" mean "at least one" and the word "plurality" means one or more, unless otherwise mentioned.

Description of Drawings and Best Mode for Carrying Out the Invention:

So that the manner in which the above recited features of the present invention can be understood in detail, a more particular description of the invention, briefly summarized above, may be had by reference to embodiments, some of which are illustrated in the appended drawings. It is to be noted, however, that the appended drawings illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope, for the invention may admit to other equally effective embodiments. These and other features, benefits and advantages of the present invention will become apparent by reference to the following text figures, with like reference numbers referring to like structures across the views, wherein:

FIG. 1A illustrates a graphical representation of UV-Vis spectra of AgNP-W and AgNP-NU, in accordance with an embodiment of the present invention;

FIG. 1 B illustrates a graphical representation of UV-Vis spectra of AgGO-W and AgGO-NU, in accordance with an embodiment of the present invention;

FIG. 2A illustrates an UV-Vis spectra of AgNP-W and AgNP-NU for 1 hr and 72 hrs, in accordance with an embodiment of the present invention;

FIG. 2B illustrates an UV-Vis spectra of AgGO-W and AgGO-NU for 1 hr and 72 hrs, in accordance with an embodiment of the present invention;

FIG. 3A illustrates a TEM image of AgNP in initial state, in accordance with an embodiment of the present invention;

FIG. 3B illustrates a TEM image of AgGO in initial state, in accordance with an embodiment of the present invention;

FIG. 3C illustrates a TEM image of AgNP after being dispersed in alkaline solution, in accordance with an embodiment of the present invention;

FIG. 3D illustrates a TEM image of AgGO after being dispersed in alkaline solution, in accordance with an embodiment of the present invention;

FIG. 4 illustrates a graphical representation of XRD diffraction pattern for CM and

CM-AgGO, in accordance with an embodiment of the present invention;

FIG. 5A illustrates a FESEM image of CM at higher magnification, in accordance with an embodiment of the present invention; FIG. 5B illustrates a FESEM image of CM-AgGO at higher magnification, in accordance with an embodiment of the present invention;

FIG. 5C illustrates a FESEM image of CM-AgGO at higher magnification, in accordance with an embodiment of the present invention;

FIG. 6 illustrates a graphical representation of bacteria cell viability of gram- positive and gram-negative bacteria after treating CM and CM-AgGO with different AgGO concentrations, in accordance with an embodiment of the present invention;

FIG. 7 illustrates ATR-FTIR of films having CS/PVA ratio of (a) 100/0, (b) 99/1 , (c) 95/5, (d) 90/10 and (e) PVA, in accordance with an embodiment of the present invention;

FIG. 8 illustrates XRD patterns of films having different CS/PVA ratio, in accordance with an embodiment of the present invention;

FIG. 9 illustrates a Transmittance % of film having different CS/PVA ratio, in accordance with an embodiment of the present invention;

FIG. 10 illustrates SEM images of the top side of FD films having CS/PVA in ratio

(a) 100/0, (b) 99/1 , (c) 95/5 and (d) 90/10, in accordance with an embodiment of the present invention;

FIG. 1 illustrates SEM images of the bottom side of films having CS/PVA in ratio (a) 100/0, (b) 99/1 , (c) 95/5 and (d) 90/10, in accordance with an embodiment of the present invention;

FIG. 12 illustrates a histogram representation of tensile index of CS/PVA film with different CS/PVA ratio, in accordance with an embodiment of the present invention; FIG.13 illustrates a SEM image of untreated cotton linter, in accordance with an embodiment of the present invention;

FIG.14 illustrates a SEM image of a surface cellulose hydrogel bead, in accordance with an embodiment of the present invention;

FIG.15 illustrates a XRD pattern of cotton linter cellulose and hydrogel bead, in accordance with an embodiment of the present invention;

FIG.16 illustrates a Fourier transform infrared on Hydrogel and Cotton linter, in accordance with an embodiment of the present invention;

FIG.17 illustrates a TEM micrograph of Graphene Oxide, in accordance with an embodiment of the present invention;

FIG.18 illustrates a FESEM micrograph of GO beads, in accordance with an embodiment of the present invention;

FIG. 19 illustrates a graphical representation of TG curve of blank beads, GO beads and GO, in accordance with an embodiment of the present invention;

FIG. 20 illustrates a TG analysis of cellulose and magnetite beads, in accordance with an embodiment of the present invention;

FIG. 21 illustrates powder X-ray diffraction patterns of (a) bleached EFB, (b) bleached EFB-NaOH, (c) unbleached EFB and (d) unbleached EFB-NaOH, in accordance with an embodiment of the present invention;

FIG. 22 illustrates powder X-ray diffraction patterns of (a) bleached EFB, (b) bleached EFB-LiOH, (c) unbleached EFB and (d) unbleached EFB-LiOH, in accordance with an embodiment of the present invention; and FIG. 23 illustrates FT-IR spectra of (a) bleached EFB, (b) unbleached EFB, (c) bleached EFBNaOH, (d) unbleached EFB-NaOH, (e) bleached EFB-LiOH and (f) unbleached EFB-LiOH, in accordance with an embodiment of the present invention;

Embodiments of the present invention aim to provide a method of regenerating cellulose from lignocellulosic biomass and preparing bio-based materials embedded with nanocomposites having antibacterial properties. Particularly, the nanocomposites are metal-graphene oxide nanocomposites. The lignocellulosic biomass is selected from a group containing kenaf core powder, kenaf pulp, cotton linter and Oil Palm Empty Fruit Bunch (EFB) Pulp. In addition, the stability of Silver nanoparticles (AgNP) and silver-graphene oxide nanocomposite (AgGO) utilized in the present invention are determined by mixing it into sodium hydroxide and urea, where Silver nanoparticles (AgNP) destabilize immediately while silver-graphene oxide nanocomposite (AgGO) showed the reverse observation. Particularly, the silver-graphene oxide nanocomposite (AgGO) is used in the alkaline solution containing dissolved cellulose and regenerated in acidic coagulation bath. The antibacterial performance of the produced membrane loaded with silver-graphene oxide nanocomposite (AgGO) at different concentrations is tested against Gram-positive and Gram-negative bacteria.

In one embodiment, the lignocellulosic biomass is dissolved in an alkaline solution system containing Urea/NaOH and/or Urea/LIOH to obtain regenerated cellulose. Particularly, the present method includes the steps of dissolving kenaf core powder and kenaf pulp in Urea/NaoH and Urea/LIOH precooled system by acidic hydrolysis. In subsequent steps, the cellulose is regenerated from the kenaf cellulose solvent which is used to form beads, membrane and hydrogel. Particularly, the kenaf cellulose solvent is then added with graphene oxide or ferrites and regenerated again to form cellulose bead, hydrogel and membrane.

In accordance with an embodiment of the present invention, the present method for preparing a bio-based material which includes the steps of extracting celluloses from the lignocellulosic biomass to remove lignin from the lignocellulosic biomass and treat the extracted celluloses in alkali solution for a sufficient time and temperature to form the liquid solution. The extracted celluloses from the lignocellulosic biomass are kept in 2% sodium hydroxide for sufficient time of about 2 hrs and the temperature is raised to about 80 DEG C to form the liquid solution. Subsequently, the liquid solution is dried at a first temperature for a particular time period to form a powder material. Particularly, the liquid solution is dried at first temperature of about 105 DEG C and for a time period of about 24 h.

In one embodiment, the celluloses are extracted from the lignocellulosic biomass by bleaching the lignocellulosic biomass to produce a dissolving lignocellulosic biomass. In operation, the process of bleaching the lignocellulosic biomass includes the step of chlorinating the lignocellulosic biomass with an aqueous chlorite solution, and extracts the celluloses from the lignocellulosic biomass with acetate buffer and distilled water. The aqueous chlorite solution used in present invention is about 1.7% w/v.

In preferred embodiment, the method further includes the steps of pre-cooling one or more aqueous solvents at a first temperature and the one or more solvents are selected from a group consisting of a mixed aqueous sodium hydroxide (NaOH) solution and urea solution, a mixed aqueous lithium hydroxide solution and urea solution and any combinations thereof. Particularly, the proportion of weight ratio of the mixed aqueous lithium hydroxide solution and urea solution is about 4.6: 15 and the first temperature is about minus 13 DEG C. After that, the method includes the steps of dissolving the powder material in the pre-cooled aqueous solvent for a p re-determined time period to form a first solution and the first solution is pre-cooled at a second temperature to form a solid form. Moreover, the percentage of weight of the powder material is about 4 wt% and the powder material is stirred in the pre-cooled aqueous solvent for the pre-determined time period of about 5 min. Particularly, the second temperature is kept about minus 12 DEG C to about minus 13 DEG C to form the solid form. The solid form is thawed at room temperature to obtain a transparent cellulose solution. Further, the transparent cellulose solution is centrifuged at about 12000 rpm for about 5 minutes to remove gas bubbles and separate dissolved and undissolved cellulose from the transparent cellulose solution after the step of thawing and subsequently the transparent cellulose solution is casted on a glass plate. After that, the solution is coagulated in aqueous solution of H2SC to form a regenerated cellulose film. Subsequently, the regenerated cellulose film is washed with distilled water for removing excess chemicals and air dried on a poly(methyl methacrylate) (PMMA) sheet.

In yet preferred embodiment, the method further includes the steps of preparing a metal-graphene oxide nano-composite and the metal-graphene oxide nano-composite are mixed into the cellulose solution to form an interaction mixture. Particularly, proportion of weight ratio of the powder material, to sodium hydroxide solution and the urea solution is about 7:12:81 in the cellulose solution and the cellulose solution is stirred vigorously at low temperature of about minus 12 DEG C before mixing the metal- graphene oxide nano-composite. The metal-graphene oxide nano-composite formed is silver-graphene oxide nano-composite. Moreover, silver-graphene oxide nanocomposites (AgGO) have enhanced performance such as antibacterial property. The UV-Vis spectra and TEM observations results illustrate that silver-graphene oxide nanocomposite (AgGO) has relatively higher stability in alkaline solution as compared to bare Silver nanoparticles (AgNP) which is mainly due to Silver nanoparticles (AgNP) being immobilized on GO sheets. Consequently, the stabilization effect helps Silver nanoparticles (AgNP) to be incorporated into the cellulose solution. On the other hand, FESEM images reveal high porous structure of cellulose membrane containing silver- graphene oxide nanocomposite (AgGO) as compared to blank cellulose membrane.

Further, the interaction mixture is coagulated in a non-solvent coagulant, and a highly porous structure of a cellulose membrane is regenerated. The non-solvent coagulant used in the present invention is an acidic solution. Particularly, the acidic solution is acetic acid. Further, the regenerated cellulose membrane has anti-bacterial properties. Particularly, the antibacterial results proved positive inhibition of the silver- graphene oxide (AgGO) loaded membrane towards the growth of both Gram-positive and Gram-negative bacteria.

Particularly, the silver-graphene oxide (AgGO) nanocomposites are produced using microwave irradiation-assisted method, where Silver nanoparticles (AgNP) is in situ synthesized and well distributed across the GO sheets. The electrostatic interactions between oxygenated group on graphene oxide sheets (GO) and positively charged Ag ammonia complex, Ag(NH ₃ ) ₂ ⁺ , where the negatively charged site on GO serve as nucleation site for the formation Silver nanoparticles (AgNP) followed by the deposition on it. The UV-Vis spectra and TEM observations results illustrates that AgGO has relatively higher stability in alkaline solution as compared to bare Silver nanoparticles (AgNP) due to which AgNP is immobilized on GO sheets. With this stabilization effect, AgNP incorporated into cellulose solution.

On the other hand, FESEM images revealed the highly porous structure of cellulose membrane containing AgGO as compared to blank cellulose membrane.

Moreover, the high surface area to volume ratio of AgNP provides higher contact rate for the nanoparticle to interact with the bacteria. Particularly, oxidative dissolution of AgNP leads to the release of Ag ions to the environment and the induce mechanism inhibits and cause bactericide effects. The uncoated AgNP is susceptible towards destabilization and aggregation in aqueous solution affects the antibacterial performance where aggregations reduce the surface contact and render the release of Ag ions. Therefore, stabilized AgNP using surfactant or capping agent improves the antibacterial performance. Incorporation of AgNP into aqueous solutions disrupts the stability of AgNP thus leading to the aggregations due to the changes on surface chemistry.

In accordance with an embodiment of the present invention, the method further includes the steps of preparing solid particulate particles via chemical co-precipitation process. Particularly, the chemical is selected from a group consisting of ferrous, ferric chloride and alkali hydroxide. The solid particulate particles are dispersed into the cellulose solution to form a cellulose solution mixture. The solid particulate particles are magnetite (Fe ₃ 0 ₄ ) particles, and the alkali hydroxide is sodium hydroxide. Thereafter, the cellulose solution mixture is coagulated in aqueous solution of H ₂ S0 to form the cellulose beads. The concentration of the aqueous solution of H ₂ S0 is about 5%. The cellulose beads formed are magnetic cellulose beads. Particularly, the cellulose solutions functional groups are analyzed using Fourier transformed infrared spectroscopy (FTIR) and the crystallinity of cellulose is analyzed by X-ray diffraction (XRD).

The above description is not intended to limit the claimed invention in any manner, furthermore, the discussed combination of features might not be absolutely necessary for the inventive solution.

The present invention will be further illustrated in the following examples. However it is to be understood that these examples are for illustrative purposes only, and should not be used to limit the scope of the present invention in any manner.

EXAMPLES EXAMPLE 1

Materials

The chemicals used in this example are sulphuric acid (H ₂ S0 ₄ ), phosphoric acid (H ₃ P0 ₄ ), potassium permanganate (Kmn0 ₄ ), sodium hydroxide (NaOH), urea (CH ₄ N ₂ 0), graphite flakes, methylene blue (C ₁₆ H ₁₈ CIN ₃ S.3H20), cotton linter, Iron(lll) Chloride, and Iron (II) Chloride. The viscosity average molecular mass (M _n ) of cotton linter is about 1.91 10 ⁵ g/mol.

Synthesis Graphene Oxide by Hummers Method

Graphene oxide is prepared by mixing concentrated sulphuric acid (H ₂ S0 ₄ ) and phosphoric acid (H ₃ P0 ₄ ) at a ratio of about 9: 1 and stirred with the addition of 3.0 g, 1 wt equiv of graphite flakes (purity > 99.5%). After then, KMn0 ₄ , 18.0 g (6 wt equiv) is added into solution and the mixture is continued be stirred for 24 h. The solution is poured in 1 L beaker full with ice to avoid effervescence and H ₂ 0 ₂ (35%) is mixed in the beaker and the solution is continuously washed.

Washing Graphene Oxide

The washing process is divided into three steps. Firstly, the solution is centrifuged at 8000 rpm for about 15 min to form the first residue of GO. After each centrifugation, the residue adheres together and is continued with the second process. The solution of hydrochloric acid and deionized water is prepared in ratio of about 83:917 to wash the first residue GO which is centrifuged at 6000 rpm for about 15 min each time. In the last step of washing, deionized water is used for the washing process. Particularly, the washing step with deionised water is carried out for 6 times under centrifugation at about 1 1500 rpm for about 30 min in first and second washing, and 45 min for third and subsequent washings.

Preparation of Cellulose beads

For the formation of spherical beads, 0.004g of GO is added drop wise into 5 ml of cellulose solution and the mixture is stirred at 25°C for 2 min until homogenised. The process is followed by the regeneration of cellulose solvent in acid coagulant (5%

H ₂ S0 ₄ ) to produce GO encapsulate cellulose beads. The GO beads are washed several times with distilled water to remove excess acid.

Preparation of Cellulose Solution

Cotton linter is added in NaOH-Urea mixture aqueous system to produce cellulose solution. The system is precooled to -12°C in a refrigerated condition before cotton linter is added. The mixture is stir up to 1000 rpm using Teflon stirrer and centrifuged at 6000 rpm to eliminate bubbles in the viscous solution. The cellulose solution is maintained at pH 12 and stored in the refrigerator until use.

Preparation of Magnetite

Distilled water is heated up to 50°C and nitrogen gas is purged into the solution for 10 min before 100 ml of FeCI ₂ .4H ₂ 0 (0.5 mol/l) and 200 ml of FeCI ₃ (0.5 mol/l) are added. The mixture is stirred in a stirrer in 200 ml of NaOH (2.0 mol/l) at 60°C. The mixture turns to a dark colour which illustrates the formation of magnetite particles.

Preparation of Magnetic Cellulose Beads

Cellulose solution and magnetite particles are mixed in defined weight ratio as illustrated in Table 1 and are mixed with magnetic stirrer and dropped wisely in an acid coagulation solution.

Table 1 : Ratio of cellulose solution and magnetite in gram (g)

Sphere-shape beads are formed due to coagulation when the drops of cellulose solution come in contact with pH 1 sulphuric acid solution. After that, the beads are washed 3 to 5 times with distilled water to remove excess NaOH and urea. The formation of sphere- shape beads is in a ratio of 9.0:1 .0. Particularly, the magnetic embedded cellulose beads are formed when the alkaline solution is coagulated with a polar solution such as H ₂ S0 ₄ . The solvent-solvent exchange process forms the spherical porous cellulose beads. The beads possesses higher uptake due to high surface area when compared to other shapes. Moreover, the particles can be prepared successfully by the coagulating technique where the ratio of magnetite to cellulose solution is lower than 1.5%.

FTIR

FTIR result shows that both the cellulose and magnetic cellulose beads have similar spectra except weaker transmittance in the range of 700-800 cm ^"1 for magnetite beads due to physically deposition of the magnetite particles over some of the functional group.

TGA

TG analysis of cellulose and magnetite beads is conducted in the temperature range from 50-900K at a heating rate of 2K min ^"1 . Both beads show a similar weight loss profile at two different temperature ranges of about 250-450 C and about 700-800 . As illustrated in FIG.20 the first weight loss occurs from 250-400 corresponding to the decomposition of cellulose in the beads. The next significant weight loss is around 700- 800 C. The magnetic beads have greater thermal stability and higher temperature to decomposed magnetite beads when compared to cellulose beads without magnetite particles. Therefore, the magnetic cellulose beads are easily fabricated in situ by incorporating magnetite particles into microporous structure of regenerated cellulose. EXAMPLE 2

Materials

The cotton linter pulp utilized in the example is with a weight-average molecular weight ( _w ) of 9.0 x 10 ⁴ , which is determined by static laser light scattering. The analytical- grade silver nitrate (AgN0 ₃ ), sodium hydroxide (NaOH), ammonium hydroxide (NH ₄ OH, 25%) potassium permanganate (KMn0 , 99.9%), hydrogen peroxide (H ₂ 0 ₂ , 30%), sulfuric acid (H ₂ S0 ₄ , 98%) and phosphoric acid (H ₃ P0 ₄ , 85%) is used in the example. Further, glucose, urea and Graphite flakes are also utilized. All the chemical reagents used are of analytical grade and are used without further purification. GO is prepared using the simplified Hummer's method. Briefly, graphite is oxidized to graphite oxide with H ₂ S0 ₄ and KMn0 ₄ , and H ₂ 0 ₂ is added to stop the oxidation process. Graphite oxide is washed repeatedly and followed by exfoliation in an ultra-sonication bath.

Preparation of silver-graphene oxide nanocomposite (AgGO) embedded cellulose membrane and its antibacterial testing

The prepared Silver nanoparticles (AgNP) and silver-graphene oxide nanocomposite (AgGO) are centrifuged to remove excessive chemicals and concentrated for further usage. Cellulose solution (4.0 wt. %) is prepared by adding the cellulose in a pre-cooled alkaline solution containing NaOH, urea and water in a weight ratio of 7/12/81. The mixture is stirred vigorously at low temperature of about -12°C. Desired amount of the silver-graphene oxide nanocomposite (AgGO) is added into the prepared cellulose solution. The cellulose solution is prepared by casting the cellulose solutions on glass plate with a thickness of 0.5 mm and immersed into a coagulant bath containing 5 wt% of acetic acid, separately. Blank regenerated cellulose membrane (CM) without the silver-graphene oxide nanocomposite (AgGO) is also produced as control sample. The produced membranes are washed repeatedly with deionized water to remove excessive chemicals. Phenolphthalein is used to detect the excess NaOH in the filtrate.

Characterizations

The stability of AgNP and silver-graphene oxide nanocomposite (AgGO) in the alkaline NaOH-urea solution (NU) is studied using an ultraviolet-visible spectrophotometer. The AgNP and silver-graphene oxide nanocomposite (AgGO) is dispersed in water is known as AgNP-W and AgGO-W, while AgNP and AgGO that is dispersed in alkaline NU solutions is known as AgNP-NU and AgGO-NU. The UV-Vis spectrum of the AgNP and AgGO in the alkaline NaOH-urea solution is collected by obtaining the UV-Vis spectrum for 3 days. Particularly, Transmission electron microscope is used to observe the dispersion stability of AgNP and AgGO in deionized water and NU solution. CM-AgGO containing 250 pg/mL of AgGO is prepared for the following characterizations. The X- ray diffraction pattern for both CM and CM-AgGO is obtained using an X-ray diffractometer. The prepared membranes are frozen using liquid nitrogen and snapped immediately before drying in a freeze dryer. The surface morphology and structure of the freeze-dried membranes are observed using a Field emission scanning electron microscope.

Meanwhile, CM-AgGO with different concentration of 62.5 gg/ml (CM-AgGO-62.5), 125 pg/ml (CM-AgGO-125) and 250 pg/ml (CM-AgGO-250) are prepared for the antibacterial test to investigate the efficiency of the formed membrane. The CM and CM-AgGO are cut into 5 sheets (2 cm x 1 cm) and immersed into nutrient broth that is inoculated with 10 ⁵ CFU/mL of Staphylococcus aureus (S. aureus), Staphylococcus epidermidis (S. epidermidis), Escherichia coli (£. coli) and Salmonella typhi (S. typhi), respectively. A broth sample without the membrane is prepared as the control sample. All the sample are incubated in an incubate shaker at 120 rpm for 4 hrs. After 4 hrs of incubation at 37°C, the turbidity of all broth samples is obtained using spectrophotometer at the wavelength of 600 nm. Stability of AgNP and AgGO in alkaline NaOH-urea solution (NU) solution

The prepared AgNP and AgGO are stable and well dispersed in deionized water without aggregation as illustrated in FIG. 1A and FIG. 1 B of the present invention. However, AgNP turns immediately into greyish solution while AgGO remains unchanged visually after the mixing into the alkaline NU solution. The stability of AgNP and AgGO in NU solution is further examined using UV-Vis spectrophotometer and TEM. The UV-Vis spectrum of AgNP in deionized water as shown in Fig. 2A illustrates an absorption peak, A _max at 430 nm which corresponds to the localized surface plasmon resonance (LSPR) of AgNP that depends on the size and aggregation states of AgNP. However, the absorbance band decreases in intensity and broadens as the AgNP is added into the NU solution. The A _max of LSPR of AgNP in alkaline NU solution vanishes completely after 72 hrs. The TEM image of the AgNP in initial state as shown in Fig. 3A illustrates spherical AgNP are well dispersed in deionized water, whereas the AgNP in NU solution aggregates into larger particles and clusters as shown in Fig. 3C. Both observations in the UV-Vis spectra and TEM image illustrates that the destabilization of AgNP in alkaline NU solution is due to the oxidation on outer layer of particles followed with the disruption on the particles repulsion or dispersion stability of the AgNP.

On the other hand, AgGO shows greater stability in alkaline NU solution according the UV-Vis spectra as illustrated in Fig. 3B where the A _max of LSPR absorbance red-shifts from 425nm to 435nm and where the intensity is slightly decreased and broadened. Consequently, this alteration of the LSPR band of AgGO is due to the oxidation on the outer layer of AgNP. TEM images of AgGO as illustrated in Fig. 3B shows wrinkled surface of thin GO sheets which are decorated with well dispersed AgNP, while the AgGO in alkaline solution as illustrated in Fig. 3D shows slight aggregation of AgNP on the GO sheets which is in agreement with the UV-Vis spectra results. The observations on the TEM images of AgGO illustrates that AgNP are immobilized by decorated GO sheets which limits particle movement and that further prevents the oxidized AgNP from aggregating among each other as compared to bare AgNP. In addition, reversible aggregations of AgGO sample is observed after 72 hrs as the formation of brownish precipitates is observed and can be easily dispersed in alkaline solution. The UV-Vis spectrum of the AgGO after 72 hrs illustrates that LSPR band is slightly red-shifted that is due to the further oxidation of the AgNP. GO can be well dispersed in water due to the presence of rich oxygenated functional group on the basal planar. The water dispersity of the nanocomposites is unaffected. Additionally, the reduced GO has higher stability at high pH solution due to the enhanced electrostatic repulsions of the single-carbon atoms sheet. The relatively stable and reversible aggregations of GO sheets in the alkaline NU solution observed in the present invention can be served as a substrate for nanoparticle deposition and further protects it from aggregations.

Formation and characterizations of AgGO loaded regenerated cellulose membrane

The GO-stabilized AgNP is added into cellulose solution at desired amount, followed by the regeneration process in acetic acid to form the cellulose membrane. The dissolved cellulose is connected via the counter diffusion of the NaOH and urea with acetic acid. The cellulose network is able to rearrange their structure through the process. After washing, membranes are vacuum dried and subjected to characterizations such as XRD. Both diffraction pattern of CM and CM-AgGO are illustrated in Fig.4, where both samples are transformed into cellulose II with a diffraction pattern at 12°, 20° and 22° which corresponding to the (1T0), (1 10), and (200) plane. However, the absence of Ag crystal diffraction pattern for the CM-AgGO containing 250 pg/rnL of AgGO is due to the content of Ag which might be too less to be detected.

The regeneration of cellulose solution in acid coagulation bath using counter diffusion acid for the removal of NaOH and urea, transforms it into cellulose II as the dissolved cellulose reconstruct cellulosic network via reconnecting inter and intra hydrogen bond. The degree of crystallinity of CM and CM-AgGO is 60.88 and 58.64. The decrease crystallinity value of CM-AgGO is due to the interruption of regeneration process for dissolved cellulose as the present GO forms intermolecular hydrogen bond with cellulose molecule and limits cellulose movement freedom during network rearrangement process.

The structure of the freeze dried CM and CM-AgGO samples are illustrated in Fig.5A-Fig.5C of the present invention. The blank CM as illustrated in Fig.5A possesses a closed structure whereas the CM-AgGO as illustrated in Fig.5B contains highly porous structure with homogenously connected network. This is attributed due to the interaction between GO sheets of the AgGO and cellulose molecular chains via intermolecular hydrogen bonding, affecting the cross linking during the coagulation process by preventing the formation of hydrogen bonds between cellulose chains. This further explains the decrease of degree of crystallinity of the membrane containing AgGO.

In addition, the FESEM image as illustrated in Fig.5C of the CM-AgGO obtained at higher magnification revealed the presence of AgNP which are attached on the cellulose network with an average particles size of 34.78 ± 3.63 nm. Therefore, GO serves as an intermediate substrate which is flexible and able to interact and attach on the cellulose network via hydrogen bond, at the same time allowing the incorporation of AgNP on the GO-cellulose structure without destabilization.

Antibacterial activity of AgGO loaded regenerated cellulose membrane

The antibacterial activity of the cellulose membrane embedded with AgGO is accessed through the turbidity of the treated broth by measuring the optical density of the broth at a wavelength 600 nm. FIG.6 illustrates a graphical representation of Bacteria cell viability of gram-positive and gram-negative bacteria after treating with CM and CM- AgGO with different AgGO concentrations, in accordance with an embodiment of the present invention. The blank CM exhibits negative antibacterial effect as the growth rate for each of the bacteria has grown over 100%. The cell viability of the bacteria is reduced significantly for the CM samples which contain AgGO. The antibacterial activity of the CM increases with increase in AgGO concentration. The viable cell of S. aureus is decreased from 56.37 %, 38.76 % and 26.40 % while S. epidermidis decrease from 65.00 %, 40.00 % and 15.00 % after treating it with CM-AgGO-62.5, CM-AgGO-125 and CM-AgGO-250. On the other hand, E. coli and S. typhi shows stronger inhibition effect as membrane containing lowest concentration of 62.5 g/mL significantly suppresses the growth of bacteria as low as 6.46% and 18.03%. As the AgGO content increases to about 250 g/ml, the cell viability for both E. coli and S. typhi is less than 1 %.

The porous structure of the membrane also allows the fluidic movement of the bacteria in the inoculated broth through the membrane where AgNP interacts directly with the bacteria. The Ag ions gradually release AgNP which undergoes oxidation dissolution upon being exposed to water or dissolved oxygen in the medium. Generally, CM-AgGO exhibits stronger antibacterial properties against gram-negative bacteria tested than that of gram positive bacteria, which is due to the thicker peptidoglycan layer on the cell membrane of gram-positive bacteria that protects the bacteria cell from the attack of Ag ⁺ ions.

Conclusion

Destabilization limits the functionality of nanoparticles in different conditions. In the present example, in situ synthesized AgGO has enhanced the AgNP dispersion stability by using GO sheets as deposition substrate for AgNP which successfully prevent the destabilization of AgNP in alkaline solution containing NaOH and urea as compared to bare AgNP. Regenerated cellulose is prepared using the same alkaline solution and embedded with AgGO exhibits positive antibacterial properties against both gram- positive and gram negative bacteria. Functionalized regenerated cellulose with AgNP is taking the advantages of utilizing reusable natural abundant resources that is potentially applicable for wound dressing materials.

EXAMPLE 3

Materials

Kenaf core (KC) powder , analytical grade lithium hydroxide monohydrate (LiOH H ₂ 0), sodium hydroxide (NaOH), urea, sulphuric acid (H ₂ S0 ₄ ), acetic acid (CH ₃ COOH) and sodium chlorite (NaCI0 ₂ ) are utilized in this example.

Cellulose Extraction

The KC powder is bleached (D) to remove the lignin by using acetate buffer, aqueous chlorite (1 .7% w/v) and distilled water. Alkali treatment (E) is conducted using 2% NaOH at 80°C for 2 h and a sequence method is performed using seven stages method (DEDEDED). Then, the sample is dried at 105 °C for 24 h. The bleached KC powder is kept in desiccator for further use. The viscosity average molecular weight (M ₀ ) in cadoxen at 25 °C is determined by Ubbelohde viscometer to be 1 .68 ^χ 0 ⁵ .

Preparation of Cellulose Films

LiOH/urea aqueous solution at a weight ratio of 4.6:15 are prepared and stored in the freezer at -13 °C. Bleached KC cellulose powder (4 wt%) is dissolved in the aqueous solution and stirred for 5 min. The solution is frozen again in the freezer at -13 °C and this process is repeated for three times. The frozen solid is thawed and stirred extensively at room temperature to obtain transparent cellulose solution. The transparent solution is centrifuged at 12000 rpm for 5 min to remove gas bubbles and separate the dissolved and undissolved cellulose. Then, the solution is cast on a glass plate and coagulated in 5% H ₂ S0 ₄ solution to form the regenerated cellulose film. The films are washed with distilled water for removing excess chemicals and air dried on a poly(methyl methacrylate) (PMMA) sheet for further characterization.

Characterizations

The surface morphology observation of the films samples are studied using scanning electron microscope (SEM). The samples are sputtered with gold, observed and photographed. The characterization of the samples are analysed using Attenuated Total Reflectance-Fourier Transform Infra Red Spectroscopy (ATR-FTIR) and X-Ray Diffraction (XRD). The transparency of films prepared from different CS/PVA ratios is measured by UV-Visible spectrophotometer at the wavelength ranging from 200 to 800 nm where the thickness of the films is 0.015 mm. The tensile strength and elongation at break of the films with different CS/PVA solution ratios in dry state are measured using a tensile machine at a speed of 10 mm min ^"1 . The samples are cut into the size of 50 mm long and 10 mm wide and three replicates of each sample are prepared.

FTIR and XRD Characterizations

FIG.7 illustrates ATR-FTIR of films having CS/PVA ratio of (a) 100/0, (b) 99/1 , (c) 95/5, (d) 90/10 and (e) PVA, in accordance with an embodiment of the present invention. Particularly, the plots of ATR-FTIR spectra of neat regenerated cellulose, cellulose/PVA films with different PVA content and neat PVA are illustrated. A broad band is observed from 3200 to 3500 cm ^-1 for cellulose, cellulose mixed PVA and neat PVA corresponding to the hydroxyls (-OH) stretching due to the strong hydrogen bond of intramolecular and intermolecular type which indicates the presence of hydroxyl groups. However, the intensity is increased as there is increase in the PVA content in the cellulose/PVA film. This may be ascribed to more and more hydrogen bonding between polymers. The intensity peak at 2929 cm ^-1 is due to the C-H stretching vibration in cellulose and hemicellulose, while the bands in the 1615 cm ^"1 region for cellulose may be attributed to C=0 stretching vibration. The bands from 1446 to 1346 cm ^-1 are associated with C-H in the plane deformation of C-H groups. The band in the region 1 160 cm ^-1 belongs to the C-0 stretching of cellulose. The peak range 894 to 902 cm ^-1 is due to β-glucosidic linkage. In this specific range, the addition of PVA in the cellulose has a slight effect on the intensity of all peaks in which the intensity peaks for all samples showed that the peak become sharper with increase in the PVA content in the CS/PVA film for spectra from (a) to (d). For neat PVA, the same intensity peaks are observed as cellulose and cellulose/PVA peaks in the range of 3200-3500 cm ^-1 but shift to the right. Peak at 1490-1340 cm ^-1 corresponds to the C-H stretching. A sharp band at 1178 cm ^-1 corresponds to an acetyl C-0 group present on the PVA backbone. The presence of these bands is attributed to the dispersion of the dissolved cellulose fiber in the mixed polymer. However, the addition of PVA in cellulose up to 10% does not showed PVA peaks in the films.

FIG. 8 illustrates XRD patterns of films having different CS/PVA ratio of (a) 100/0, (b) 99/1 , (c) 95/5 and (d) 90/10, in accordance with an embodiment of the present invention. All the cellulose/PVA film samples exhibits a crystalline structure with a peak angle around 2Θ = 12.4°, 20.3° and 22.2° which can be assigned to cellulose II crystal planes of (1 T 0), (1 1 0) and (2 0 0) respectively. In contrast, these films are different with native cellulose in which the crystal plane of (1 T 0), (1 1 0), (2 0 0) and (0 0 4) exhibited diffraction peaks 2Θ = 14.9°, 16.3°, 22.6° and 34.5° which are assigned cellulose I plane. This indicates that the original cellulose has been transformed into cellulose II crystal state. The crystallinity index (Crl) of cellulose decreases from 66.8% (PVA0) to 53.2% (PVA10). These results indicate that the addition of PVA into cellulose solution destroyed the ordered packing of mixed polymer. In addition, the overlapping of cellulose and PVA peaks occurred which leads to decrease in the intensity of the sample.

Transparency

FIG. 9 illustrates a Transmittance % of film having different CS/PVA ratio of 100/0, 99/1 , 95/5 and 90/10, in accordance with an embodiment of the present invention. The transmittance of the samples with different CS/PVA ratio is analysed using UV-Vis spectrophotometer. The highest transmittance shown by the film with 10% PVA exhibits up to 45% and subsequently decreases as the content of the PVA in the samples is decreased. The miscibility of the polymer can be explained from the transparency of the film produced. The miscibility of the polymers may be due to the formation of hydrogen bonding between the hydroxyl group of cellulose and hydroxyl group of PVA. Therefore, as the PVA content is increased in the mix polymer, the formation of hydrogen bond further increases. From the details of the transmittance percentage, the higher the transparency miscibility of the polymer is better with higher PVA content due to more polymer interaction. The crystalline region in the membrane resulted in the loss of light transparency. It can be proved by XRD spectra in which the crystallinity index decreases by increasing PVA, and therefore increases the transparency of the films.

Particularly, the SEM images illustrated in FIG.10 and FIG.1 1 illustrates the morphology of top side of FD films having CS/PVA in ratio (a) 100/0, (b) 99/1 , (c) 95/5 and (d) 90/10 are contacted with the coagulant and bottom side of films having different CS/PVA in ratio (a) 100/0, (b) 99/1 , (c) 95/5 and (d) 90/10 are contacted with glass plate of the films. As illustrated in FIG.10 and FIG.1 1 , the films illustrate porous structure which is due to the freeze drying process. As PVA content is increased, it is observed that the average pore size of the films surface is also increased. This is due to more polymer interaction between the PVA and the cellulose. The interaction is mainly from the hydrogen bonding which is formed between the glucose ring ether oxygen and hydroxyl group (OH) in PVA while the other bond is formed between secondary OH of C2 or C3 with OH in PVA. Moreover, the interaction leads to expansion of the pore size of the films. However, smooth surface is observed in different SEM images of FIG.1 1 where the diffusion of acid during the coagulation process is slower at the surface which is contacted to the plate as compared to (a) and (b) SEM images of Fig.10. During the coagulation process, the phase separation starts from the surface contacted with acid coagulant and slowly penetrates into the cross section and the bottom side of the film. The difference in the diffusion between solvent in the cellulose solution into coagulation bath brings about a significant variation of the pore size between the surface and the bottom side of the films. The surface is directly contacted to the coagulant and it subsequently results in bigger pore size and coarse surface.

Tensile Strength

FIG.12 illustrates a histogram representation of tensile index of CS/PVA film with different CS/PVA ratio, in accordance with an embodiment of the present invention. The strength of conditioned neat cellulose and CS/PVA is evaluated and tensile index results are illustrated in FIG.12. The histogram illustrates the tensile index for different PVA content in the polymer solution. The tensile index is reduced by addition of PVA and further the strength is reduced due to increase in the hydroxyl group between cellulose and PVA polymer. By increasing the PVA solution in the mixed polymer, the hydrogen bonding is increased further. Particularly, it creates more water hydrate leading to decrease in the total concentration of the mixed polymer solution. Consequently, it will lead to more water absorption and moisture content in the films. Increase in the amorphous region increase the degree of disorder in the polymer and cause the band tail to increase which leads to decrease in the strength at break. Therefore, from the XRD results the crystallinity of the films decreases as the PVA content in the film is increased.

Conclusion

Cellulose/PVA film having different CS/PVA ratio are prepared using pre-cooled and casting method. It is observed that as the PVA content increases in the mix polymer, the crystallinity decrease and from the UV-Vis transmittance data it illustrates that transparency increases. From the SEM images, as observed from the top side of the film which is contacted to the coagulant, the pore size of the film increases with increase in the PVA content. However, as observed from the bottom side which is contacted to the glass plate, the surface is smooth and the pore size is very small. Further, the tensile strength properties of the film illustrate opposite trend which is decreased as the PVA is increased.

EXAMPLE 4

SCANNING ELECTRO MICROSCOPY (SEM)

FIG.13 illustrates a SEM image of untreated cotton linter, in accordance with an embodiment of the present invention. The SEM image of untreated cotton linter clearly illustrates that the surface of untreated cotton linters is smooth, compact and almost free of trenches. In contrast, modified cellulose in FIG.14 illustrates the SEM image of the surface cellulose hydrogel beads prepared by freezing methods. The fiber like structure is due to a slow and strong self-association of the cellulose chains at low temperature. The result also shows a homogeneous structure in the surface suggesting a certain level of miscibility between cellulose and the system NaOH/Urea.

X-Ray Diffraction (XRD) Cellulose is recognized by forming extended crystalline regions. The crystalline state has a lower energy than the amorphous one. Therefore, the crystalline state of cellulose should be more difficult to dissolve than the amorphous one. Modification done on cellulose tends to make it more soluble in water. FIG.15 illustrates the diffraction peaks at 2Θ = 14.8°, 16.5°, 22.8°, 34.5° for (1 0 0), (1 1 0), (2 0 0), and (0 0 4) planes which are characteristic of cellulose I crystal (cotton linter).

Fourier transform infrared (FTIR)

The Fourier transform infrared (FT-IR) spectrum of the cotton linter and hydrogel bead are illustrated in FIG.16, in accordance with an embodiment of the present invention. The band at 1 1 14 cm ^"1 is stronger in the spectrum of cotton linter, and it appears as a shoulder in band near 1067 cm ^"1 . The major peak for pure cellulose centered at 3436cm ^"1 corresponds to the stretching vibration of OH. The band at 894 cm ^"1 belong to β-anomers or β-linked glucose polymers, and the absorption peak in hydrogel bead is more intense and sharper than the corresponding one in cotton linter. Particularly, the peak at 1482 cm ^"1 is attributed to the methyl groups of ammonium, and indicates the presence of urea in hydrogel beads networks. The peaks at 3300-3450 cm ^"1 corresponds to stretching vibration of hydroxyl groups of cellulose moves to higher wavenumber and becomes broader, and indicates a strong interaction between the groups of cellulose for the formation of hydrogel beads.

Transmission electron microscopy (TEM)

FIG.17 illustrates a TEM micrograph of a Graphene Oxide, in accordance with an embodiment of the present invention. The morphology and structure of the aqueous dispersion of GO illustrates large flakes of GO that are not crumpled, and no multiplicity of oxygen functionalities are observed. However, in TEM image the presence of topological features along with overlapping area of GO reveals that they are highly dispersed in water.

Field Emission Scanning Electron Microscopy (FESEM)

FIG.18 illustrates a FESEM micrograph of GO beads illustrating the presence of GO in the cellulose beads which results in some hollow space and subsequently produce GO beads with great porous structure on the surface.

Thermogravimetric Analysis (TGA)

FIG. 19 illustrates the TGA of the samples GO, cellulose blank and cellulose GO is heated from room temperature to 800°C. At first gradient loss mass 19.71 % at approx 100 °C is observed in the GO sample due to water solvent molecules absorbed into the reduced GO bulk material. The major mass reduction at ~ 200 °C is caused by pyrolysis of the oxygen-containing functional groups, and generates CO, CO ² and stream. Further decomposition take place upto 800°C. Cellulose beads without GO (blank sample) illustrate a very fast thermal degradation. The amount of water absorbed in the sample is higher compared to samples encapsulated with GO. The thermal degradation temperature up to 100°C (for both samples) shows the removal of water. Particularly, sample with GO possesses higher thermal stability, where it requires higher temperature to degrade as compared to the blank sample. The residual content for GO cellulose beads is higher which show some of the residual (GO) remained after the heating process is completed. Further weight loss of 50.0 % and 60.0 % is observed for cellulose beads without GO and cellulose beads with GO, when heated from 251 to 580 °C. The different in profile appears to indicate that the cellulose without GO is less thermally stable than cellulose with GO with mass loss occurring at slightly lower temperature. This is due to the enhanced thermal conductivity of GO aiding bond cleavage. Moreover, cellulose beads without GO (blank sample) showed a very fast thermal degradation. The amount of moisture in the sample is higher compared to samples encapsulated with GO. The thermal degradation is at around 100°C (for both samples) shows the removal of moisture. The cellulose beads with GO showed higher thermal stability, where it requires higher temperature to degrade as compared to the blank beads. The residual content for GO cellulose beads is higher which illustrates some of the residual (GO) remained after the heating process is completed.

EXAMPLE 5

Bleached and unbleached EFB pulp is dissolved to form cellulose solution under an alkaline system including NaOH and LiOH mixed in urea. The cellulose solution formed is then treated with cross linking agent to form a hydrogel film. The reaction observed is based on different solubility and viscosity between the bleached and unbleached EFB fibre dissolved in different alkaline systems. The water retention of hydrogel film formed is measured.

Materials

EFB fibres used are obtained from soda pulping process from pulp mill. The pulp fibres are dried at 105°C for 12 h and stored in a desiccator. The chemical used for solvent such as, LiOH (reagent grade, >98%), NaOH, urea and epichlorohydrin (analytical grade). Bleaching Process

There are four stages of bleaching process. EFB pulp fibres are bleached with CI0 ₂ (D stage), where the chlorine dioxide is obtained from the reaction of 1 M sodium chlorite with water. The oven dried pulp used for bleaching is 10 %. The temperature of the D stage is at 60 °C for 2 h which is treated in water bath shaker. The pre-treated pulps followed with treatment of 4% wt NaOH (E stage) solution at 60 °C for 30 min. The samples are subsequently washed with excess distilled water in order to remove the impurities and neutralize the pulps. The second E stage is carried out with treatment of 6 % wt NaOH solution at 70°C for 30 min. Then, further treatment is carried out by bleaching with second D stage at 70°C for 1.5 hours duration. The extracted cellulose is then washed with excess distilled water and dried at 105°C for 24 h. The effectiveness of chlorine dioxide used is about 58.65 g/l.

Preparation of Cellulose Solution

Two types of aqueous solution are prepared with different weight ratio of urea, NaOH and LiOH. Two homogeneous solutions are obtained by mixing 7 wt % NaOH/ 12 wt% urea/ 81 wt % distilled water which are measured by weight. The prepared solution is stirred and stored in a refrigerator until pre-cooled to a low temperature of about -12 to - 10 °C for about 4 h because the ability of the solvent to dissolve can be improved at low temperature. This is due to active NaOH which helps the cellulose to swell by forming a new hydrogen bonded structure in the solution at decreased temperature. 4% of cellulose prepared from bleached EFB and unbleached EFB cellulose fibre (cellulose I) are dissolved into each solution under vigorous stirring for 10 min at maintained temperature to obtain transparent cellulose solution (cellulose II). The temperature maintained at about -4 to -2°C by using salt ice bath. The same procedure is applied to 4.6 wt % LiOH/ 15 wt % urea/ 80.4 wt% distilled water. The resultant solution is subjected to centrifugation at 10000 rpm for 10 min to degasify and exclude the slightly remaining undissolved part. The undissolved part is washed to extract the NaOH and then filtered to get the pure undissolved cellulose fibre. Subsequently, the extracted cellulose is dried in an oven at 105 °C for 24 h. The solubility (Sa) of the EFB-bleached and EFB-unbleached cellulose fibre in both solvent system are measured by:

S = ^w - a JJ (1 )

where W1 is weight of dissolved cellulose and W2 is weight of original cellulose.

Hydrogel Film Formation

The cellulose solution formed is then added with a cross linker. In the present example epichlorohydin is used as the cross linking agent. The cellulose solution formed is added with 9 wt% epichlorohydrin that is stirred for 1 h to form a gel like structure and is dried in an oven at 50 °C for 12 h to form the dried hydrogel film.

Characterization

The bleached and unbleached EFB fibres cellulose fibre and transparent cellulose solution are dried in an oven before characterization. The crystallinity of the samples is analyzed using X-ray diffraction (XRD) measurement. The radiation used in the XRD pattern is Cu Ka (1 .5406 x 10-10 m) at 40 kV and 30 mA is recorded in the range of 9- 77° at a scanning speed of 27 min. Comparison of Solubility bleached EFB and unbleached EFB

The solubility of bleached EFB and unbleached EFB in NaOH and LiOH systems are illustrated in Table 2 and Table 3.

Table 2: The solubility of EFB-bleached and EFB-unbleached in NaOH/urea/H20 bleached EFB unbleached EFB Volume of Solution (ml) 50 50

Weieht ofFibre (a) 2.0029 2.0037

of EFB used 4.09 4.01

Solubility (° o) 402 34.2

Table 3: The solubility of EFB-bleached and EFB-unbleached in LiOH/urea/H20 bleached EFB unbleached EFB

Volume of Solution (ml) 50 50

Weight of Fibre (g) 2.0028 2 2.0082

° o of EFB used 4.00 4.00

Solubility (° o) 25.3 33.4

The results show that solubility of NaOH/urea/H20 system for bleached EFB is higher than that of the unbleached EFB, while the solubility in LiOH/urea/H20 for unbleached EFB is higher than the bleached. However, the solubility between both solutions is not different much due to the lignin content in the bleached EFB and unbleached EFB is only 0.3% and 6.1 %. These results illustrated above show that the lignin does not affect much on the dissolution of the cellulose in the system. Crystallinity of Cellulose Fibre and Regenerated Cellulose

There are various ordered crystalline arrangements of cellulose due to the hydroxyl groups present in cellulose macromolecules which are involved in a number of intra and intermolecular hydrogen bonds. XRD is used to evaluate the Crystallinity Index (CI) of cellulose. FIG. 21 illustrates powder X-ray diffraction patterns of (a) bleached EFB, (b) bleached EFB-NaOH, (c) unbleached EFB and (d) unbleached EFB-NaOH. The diffraction peaks observed at 2Θ= 14.8°, 16.3°, and 22.6° for (1 T 0), (1 1 0) and (0 0 2) planes are characteristic for cellulose I crystal, and those at 2Θ= 12. , 19.8 ^° , and 22.6 ^° for (1 T 0), (1 1 0) and (0 0 2) planes are characteristic for cellulose II crystal. The cellulose raw material as illustrated in Fig.21 (a) and Fig. 21 (c) have typical crystalline peaks of cellulose I, whereas the regenerated cellulose Fig.21 (b) and Fig. 21 (d) samples exhibits peaks of the cellulose II. The crystallinity index (CI) of EFB cellulose is calculated by using XRD peak height method developed by Segal and coworkers (1962), and expressed by following equation where CI expresses the apparent crystallinity (%) defined by Segal and coworkers, l ₀ 02 gives the maximum intensity of the peak corresponding to the plane in the sample with the Miller indices (0 0 2) at a 2Θ of between 22-24° and l _am represents the intensity of diffraction of the amorphous materials, which is taken at an angle of about 18° 2Θ in the valley between the peaks. Interestingly, the crystallinity index of the regenerated cellulose samples (b) and (d) are higher than both cellulose samples, bleached EFB and unbleached EFB.

As shown in Table 4 below, the crystallinity index of EFB cellulose (bleached EFB and unbleached EFB) has different crystallinity index value. The crystallinity value of bleached EFB (58.4%) is higher than unbleached EFB (52.0%). However, regenerated cellulose shows that the crystallinity index has increased for bleached EFB and unbleached EFB cellulose is treated with NaOH and LiOH.

Table 4 Crystallinity Index of EFB Pulp Cellulose

Samples Crystallinity Index (° o) bleached EFB 5 S .4

unbleached EFB 52.0

bleached NaOH 79.2

unbleached aOH 87.0

bleached LiOH 64. 1

unbleached LiOH 7S .6

■ ^■ —

FTIR Spectra Analysis

From the FTIR spectra for bleached EFB and unbleached EFB which are cellulose I, there are 3 common peaks observed, i.e., 3300-3400 cm ^"1 , 2800-2900 cm ^"1 regions and 1630- 1640 cm ^"1 peak. The major peaks for cellulose I is located at 3329 cm ^"1 and is attributed to O-H stretching vibration. The region 2800-2900 cm ^"1 refers to the stretching vibration of C-H molecules in the fibres and the peak region 1630- 640 cm ^"1 belongs to C=0 stretching of acetyl groups of hemicellulose. The difference between bleached EFB and unbleached EFB is at a peak of about 280 cm ^"1 which appears for bleached EFB. This peak indicates the presence of ether linkage of carboxylic group of hemicelluloses. For cellulose II, there are sharp peaks which are observed at 3199 cm ^"1 and 3428 cm ^"1 . Both peaks indicate the existence of N-H stretch.

FIG. 23 illustrates the FTIR spectra of (a) bleached EFB, (b) unbleached EFB, (c) bleached EFBNaOH, (d) unbleached EFB-NaOH, (e) bleached EFB-LiOH and (f) unbleached EFB-LiOH, in accordance with an embodiment of the present invention. A broad absorption band is observed around 3100-3500 cm ^"1 and the formation of two peaks is around 3199 and 3428 cm ^"1 for the derivatives are ascribable to the stretching frequency of the -OH and -NH ₂ groups, which becomes stronger than the band of the stretching vibration of -OH of the native cellulose in (c), (d), (e) and (f). The peak at wavenumber 1717 cm ^"1 for both bleached EFB and unbleached EFB represents amide formed after cellulose I is treated with NaOH and urea. The band at 1427 (bleached EFB-NaOH) and 1428 cm ^"1 (unbleached EFB-NaOH) is involved in the change of the conformation of CH ₂ OH at the C6 position in cellulose. The peak at 2165 cm- ¹ is attributed to the formation of C Ξ H after being treated with LiOH and urea as compared to cellulose I where there is no significant peak observed at these peak ranges. The sharp peak at 1653 cm ^"1 in treated cellulose indicates the formation of NH ₂ for both bleached EFB-LiOH and unbleached EFB-LiOH.

In the above example, cellulose is extracted from palm oil EFB fibres and processed using soda pulping process from pulp mill to form EFB pulp where the EFB pulp is bleached to obtain the delignified fibres. The improvements of cellulose are attributed by the removal of lignin from the sample which is confirmed by the FTIR analysis. The crystallinity index of EFB fibre cellulose is increased in crystallinity from cellulose I (bleached EFB and unbleached EFB) to cellulose II from treated EFB pulp in NaOH and LiOH and urea system. From the results obtained, there is no significant difference in terms of degree of dissolution either the EFB fibre bleached or unbleached.

EXAMPLE 6

Materials

EFB pulp is obtained from a pulping mill after the soda pulping. The pure cellulose fibres are obtained from quantitative filter papers ash less grades (Whattman). Cellulose is extracted from EFB pulp, dried at 105 °C for 24 h and stored in a desiccator before used. NaOH, thiourea, and urea (99> purity were analytical grade) are used in this example.

Preparation of EFB fibre

EFB pulp is immersed in excessive water and disintegrates before producing EFB fibre. For bleaching process, there are four stages to bleach the EFB fibre. The EFB pulp is bleached with chlorine dioxide (D stage), and chlorine dioxide is produced through reaction between sodium chloride and water where the sodium chloride molarity is 1 M at about 60 °C for 2 h using water bath shaker. Then, the process is followed by E stage with 4% NaOH solution at 60°C for 30 min. EFB pulp is washed with excess distilled water to remove all the impurities and unreacted NaOH/thiourea/urea. Pre treatment is continued with second stage with 6% NaOH (E stage) at 70 °C for 30 min. Last stage for bleaching treatment is using chlorine dioxide at 70 °C for 1.5 h. The removed lignin is washed with excess distilled water and dried at 105 °C for 24 h.

Preparation of cellulose solution

A 100 ml solution of NaOH (8 w†%), urea (8 wt%) and thiourea (6.5 wt%) is prepared in a beaker. The solvent prepared are pre-cooled to -10°C for 24 h. About 1 g of cellulose sample is added into the solvent and stirred to obtain transparent cellulose solution. The temperature of the solvent is maintained using a salt ice bath. After vigorous stirring the temperature of solution is increased to approximately 0°C. The cellulose solution is then centnfuged at 10000 rpm for 10 min to separate the undissolved fraction. The undissolved fraction is washed with excess distilled water to remove excess NaOH/urea/thiourea to determine the percentage of dissolution. The dissolved fraction of cellulose solution is then dried in an oven at 105°C and kept for further characterizations.

Characterization

The cellulose powder is analyzed using an X-ray diffractometer in the chamber at a temperature at which it can perform X-ray diffraction techniques on sample at high temperature up to 2300°C and low until 263°C. The radiation used in the XRD pattern is Cu Ka (1.5406 x 10-10 m) at 40 kV and 30 mA is recorded in the range of 9-77° at a scanning speed of 2 min. Fourier transform infra red-attenuated total reflectance (ATR) model Perkin Elmer Spectrum 400 GX is used to characterize the functional groups in the determined sample. The sample is taken at random from cellulose solution in deionized water. The residual water on the surface of samples is removed using filter paper (FP). In direct contact with Ge crystal, data of the sample are collected over 32 scans at 4 cm ^"1 resolution using a variable-angle ATR at a nominal incident angle of 45°.

Comparison of Solubility EFB and Filter Paper (FP)

The solubility of EFB and FP in NaOH/urea/thiourea systems is shown in Table 5 below. Table 5: The Solubility of EFB and FP

The results show that solubility of NaOH/urea/thiourea system for FP is higher than the EFB, this is because FP is pure cellulose meanwhile EFB might still consists of small amount of lignin of about 0.3% after the bleaching process. For each determination, the lignin content is calculated in the test specimen as follows:

( I )

100

Lignin. ⁰ o = A

W where A is the weight of lignin, g and W is the weight of the specimen after oven dry, g. Meanwhile, the solubility is calculated from the following formula:

S _fl = i- x l00° o (2) where Fi denotes the weight (g) of the insoluble cellulose fibre.

In the above example, NaOH/urea/thiourea is dissolved in EFB pulp and FP to compare the solubility and regenerated cellulose at low temperature. The low temperature condition for dissolution of cellulose avoids the evaporation of the chemical agents and qualifies the aqueous NaOH/urea/thiourea system as a green solvent. From the dissolution, the results illustrate that EFB fibers can get up to 40% solubility compared to FP which is used as pure cellulose. The crystallinity index of EFB pulp cellulose is increased in crystallinity from cellulose I to cellulose II after being treated in NaOH/urea/thiourea system. Moreover, FTIR results showed there is some lignin attached on EFB pulp that decreases the solubility.

Therefore, the present invention provides a rapid dissolution method for cellulose at low temperature using a mixture of urea and alkaline medium such as sodium hydroxide or lithium hydroxide. In addition, graphene and ferrites are added to the system. Particularly, the regenerated cellulose is dissolved in alkaline solution containing sodium hydroxide (NaOH) and urea at low temperature. Silver-graphene oxide (AgGO) nanocomposite is prepared and mixed into the cellulose solution followed with the regeneration of the mixture into cellulose membrane.

Particularly, the transparent dissolved cellulose solution prepared in the present invention is transformed into various form of regenerated cellulose such as hydrogel, wet-spun fibers and membrane. The regenerated cellulose membrane is prepared via coagulation process in non-solvent coagulant such as acidic solution. Moreover, the method of hydrolysis is used to utilize kenaf powder and kenaf pulp to reduce the molecular weight and generate regenerated cellulose. By utilizing the dissolution and regeneration process of natural abundant cellulose and further combining it with functionalized nanomaterials such as silver nanoparticle (AgNP) is used to produce value-added bio-based materials in the present invention. Moreover, the kenaf core powder, kenaf pulp and Oil Palm Empty Fruit Bunch (EFB) Pulp which are waste materials are utilised to produce useful product material having anti-bacterial properties. Particularly, the utilisation of the kenaf core will increase value to the present invention. Furthermore, the bio-based material having anti-bacterial properties can be used for many applications such as in cosmetic, medical, agriculture, absorption, and magnetic technology industry fields. In addition, the bio-based materials are used as functional additives and ingredients in pharmaceutical or cosmetic preparations, food preparations, building materials, varnishes, paints, coating compounds and polymers. In addition, unbleached EFB fibres are directly used in the dissolution process using alkali and urea. Consequently, the cost of the whole method is reduced and the present method can be used in industrial production of cellulose based materials.

It should be understood that all value ranges in the description and claims are intended to include their end values and all sub ranges within these ranges.

Although the present invention is illustrated and described with reference to the illustrative examples, those skilled in the art would understand that the present invention could be varied in manners and details without departing from the spirit and scope of the present invention. While an illustrative embodiment of the present has been shown in the drawings and described in considerable detail, it should be understood that there is no intention to limit the invention to the specific form disclosed.

On the contrary the intention is to cover all modifications, alternative constructions, equivalents and uses falling within the spirit and scope of the invention as expressed in the appended claims. The protection scope of the present invention is defined as claimed in the appended claims.