Description:FIELD OF INVENTION

1. The present invention is related to a method of processing Bacterial Nano- Cellulose (BNC) substrate or cellulose sheets derived from bacteria for printing flexible electrical devices.

BACKGROUND OF THE INVENTION

2. Bacterial nano-cellulose (BNC) is a promising biocompatible substrate that is synthesised by certain bacterial strains. BNC exhibits exceptional mechanical properties, water-holding capacity and nanostructure morphology. The bio-composite and bio-polymer nature of BNC material enable it appropriate for a diverse array of uses. BNC bio-composites find application primarily in the fields of wound healing, medication delivery, and tissue regeneration. A progressive application of BNC material involves their utilisation as printable electronic substrates in the field of flexible electronics.

3. Generally, the strains of Acetobacter bacteria are employed to generate cellulose as a byproduct during their metabolic processes. These bacteria strains are capable of releasing cellulose in the form of three-dimensional web-like micro-fibrils, commonly referred to as bacterial cellulose or bacterial nano-cellulose.

4. The process of production of bacterial nano-cellulose can be easily customized to achieve the desired properties and thickness of the material, making it suitable for a wide range of applications in flexible electronics. Traditionally, synthetic and plastic substrates are used in flexible electronic devices which are derived from rapidly depleting non-renewable petroleum resources, causing serious environmental damage

5. Bacterial nano-cellulose (BNC) is a renewable and biodegradable material that can be produced using inexpensive and low-energy processes. In addition, it is non-toxic and readily recyclable or disposed of without harming the environment. It also has a low carbon impact. BNC material possess excellent durability, flexibility, mechanically strong, very transparent, low weight and have a large surface area, hence making it a suitable substrate for flexible electronic devices. Overall, BNC are sustainable and environmentally friendly materials leading to several applications in green electronics.

6. The effectiveness of BNC sheets as substrate materials for printed electronics patterns is already established in the literature. The BNC sheets demonstrated the flexibility and durability as a substrate material for the deposition of printed functional patterns using electronic inks, as well as for the subsequent thermal curing process of such patterns. Various processes, including screen printing and vacuum evaporation, were used to produce conducting patterns on BNC sheets.

7. Multiple techniques are employed for the production of BNC-based nano structures such as In-situ biological self-assembly, Membrane-liquid culture, Dynamic culture and Static culture.

8. In-situ bio-assembly, based on Acetobacter Xylinum, allows dispersion and accumulation of functional nanomaterials during the process of bacterial culture.

9. Membrane-liquid culture technique has been developed to create planar structures with precise dimension and thickness as well as uniformly distributed nanomaterials.

10. The application of shearing force in a dynamic culture environment leads to the production of cellulose, which, when combined with BNC nanofibers, results in the formation of a spheroidal shape. The proper ratio of nanomaterials is crucial for manufacturing integrated structures.

11. Static culture exhibits simple preparation, requires less labor and energy. The nano-composites that are produced demonstrate planar sheets of a precisely regulated thickness, characterised by a consistent honeycomb-like internal structure.

12. The document WO201864143 A1 discloses a method for fabricating flexible electronic devices, including a flexible substrate made of cellulose nano-fibers and a flexible device component supported by a crystalline inorganic semiconductor material. The flexible substrate is formed by a method comprising: growing a culture of a cellulose producing bacteria; harvesting the cellulose from the culture; pressing the cellulose to form a sheet of bacterial cellulose paper; and drying the sheet of bacterial cellulose paper.

13. The document US10744461B2 discloses a composition comprising cellulose and a nanomaterial graphene oxide. It involves a method of preparation of a bilayered biofilm comprising nanocellulose and a nanomaterial, the method comprising: providing a bacterial culture of Gluconacetobacter hansenii in a growth media; incubating the bacterial culture and the nanomaterial until a first biofilm layer forms.

14. However, one of the major drawbacks of these techniques is the tendency of nanomaterials with high density or poor dispersibility in the culture medium to settle or consolidate without external pressures. This leads to an uneven distribution of functional materials across the BNC network resulting in formation of wrinkles.

15. The present invention discloses a cost-effective and sustainable method for production of biocompatible BNC-based nano materials. The process eliminates the need for external pressure, ensuring the formation of wrinkle-free BNC nano materials suitable for flexible or wearable electronics.

SUMMARY OF THE INVENTION

16. One of the objectives of the present invention is to provide a method to prevent the formation of wrinkles during the fabrication process of BNC sheets.

17. Another objective of the present invention is to provide a means for inhibiting the rapid escape of evaporated water vapour. The present disclosure relates to a method for controlled evaporation by freeze-melt method, wherein said controlled evaporation encompasses both absorbed water and crystallised water present within the cellulose material. A method for regulating evaporation is disclosed herein, wherein the said method effectively mitigates excessive shrinkage of sheets, thereby minimising the occurrence of wrinkles along the edges. The BNC sheets obtained exhibit a uniform and homogeneous structure.

18. Yet another objective of the present invention is to provide a method that does not necessitate the application of external pressure or compression in fabrication of BNC sheet. Consequently, this method facilitates the formation of a single layer of nano-sheet exhibiting a morphology that is smooth and devoid of wrinkles.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig.1 represents the XRD of oven-dried Pristine and Processed BNC
Fig.2 represents the Scanning electron microscope image of a) pristine BNC and b) processed BNC sheet
Fig.3 represents the TGA and DTA curve of Pristine and Processed Cellulose.
Fig. 4 represents the Tensile strength of pristine and processed BNC
Fig. 5 represents Load vs. Penetration depth curves for Processed BNC and Pristine BNC film (n=5).
Fig. 6 represents the Indentation hardness and elastic modulus of processed BNC and pristine BNC film (n=5).
Fig. 7 represents the Contact angle of Pristine BNC and Processed BNC
Fig. 8 represents the Cytotoxicity studies with Alamar blue assay for 168h in processed BNC for L929 Cell Lines
Fig. 9 represents the SEM images of cell adhesion studies for 24h on processed BNC for L929 Cell Lines
Fig. 10 represents the Optical microscope image of a) pristine b) washed BNC sheets printed gridlines with Ag NP ink (1 pass)
Fig. 11 represents the Polarisation and power generated curve of BNC-based enzymatic fuel cell for non-turn over and turn over conditions (1mM -10mM).
Fig.12 represents the CV of a) Processed BNC-based EFC and b) Pristine BNC-for turnover and non-turnover (1mM-100mM) conditions.
Fig.13 represents the Michaelis-Menten fit of a) Processed BNC, b) Pristine BNC, and c) Simulation enzyme reaction kinetics

DETAIL DESCRIPTION OF THE DRAWING

19. Fig.1 gives detail about The XRD analysis of pristine BNC and processed BNC were evaluated for the crystalline structure of cellulose and to investigate the crystalline nature changes of pristine BNC after chemical treatment. The XRD patterns of oven-dried pristine and processed BNC are shown in fig.1. The pristine and processed BNC exhibits three main 2? diffraction peaks at 13.5?, 15.7?, and 21.7? which corresponds to 101 (Amorphous region), 101 ¯ (Amorphous region), and 200 (Crystalline region) crystallographic plane [1, 2]. The diffraction peaks signify cellulose 1a type having a triclinic unit cell, which is typical of bacterial cellulose.

20. The crystal structure and crystallinity specifically contribute to cellulose's mechanical and physical characteristics. Hence, the crystallinity of pristine and processed BNC was determined from the XRD patterns. The estimated degree of crystallinity was 96% for pristine BNC and 92% for processed BNC. The crystalline structure of processed BNC was not significantly changed from pristine BNC. The diffraction peaks of pristine BNC at 2? 18.5? and 24.5? could be attributed to sucrose content from the culture medium. Therefore, treatment of pristine BNC with NaOH promotes the removal of certain bacteria metabolite residues increasing viscosity, facilitating purification, and the suspension of low molecular mass cellulose which yield better characteristics of the biomaterials.

21. Fig.2 shows the SEM micrograph of the processed BNC sheet and pristine BNC sheet. The sheets show an ultrafine network of cellulose nanofibers. Pristine BNC sheets exhibit a highly porous interconnected network whereas processed BNC sheets show a denser and smoother morphology. The denser structure of processed BNC sheets could be due to the alkali treatment, which hydrolyses the cellulose fiberscross-linkers removing the bacterial residues and connecting the fibers together.

22. Fig.3 illustrates the thermal properties analysis which is a vigorous approach to studying the material's response to a temperature change. Thermogravimetry (TG) and differential thermal analysis (DTA) were commonly used to determine polymers' thermal stability and pyrolysis behaviour under different thermal conditions. TG curves show the change in the weight loss percentage of the materials during the heating process. The DTA curves show the difference in temperature as an exothermic or endothermic reaction in a sample.

23. Fig.3 illustrates the initial mass losses start at ?34?C for both processed and pristine BNC showing evaporation of moistures that are loosely bound on the BNC surface. The intermolecularly hydrogen-bonded water responsible for crystallisation is evaporated at around 150?C. The degradation of Pristine BNC starts at 175?C whereas that of Processed BNC at 230?C. This is because of the controlled evaporation mitigated by the freeze-melt method in processed BNC, by which the water of crystallisation escaped along with absorbed water which is loosely bound to the surface. Processed BNC showed two-stage degradation whereas pristine BNC showed three-stage degradation at 252?C. This is due to the supplementary residue from the culture medium and the water of crystallisation, which altered the BNC nanofibers' molecular weight, crystallinity, and orientation.

24. The first stage of degradation from 34?C - 230?C was attributed to the weight loss of water molecules – both absorbed and the crystalline, in the BNC. The second degradation stage appears in the temperature range 230?C - 354?C, showing a significant increase in weight loss assigned to the degradation of the structural compositions of BNC. The gradual decrease in mass at this stage may further involve dehydration, depolymerization, decomposition of glycosyl units, and the formation of a charred residue. The char formation of pristine BNC (10%) is higher than processed BNC (7%) may be due to its higher crystalline nature which increases the proportion of carbon; therefore, the formation of char residue increases as carbon content increases.

25. DTA traces show an endothermic peak at 350?C of the processed BNC indicating the fusion or melting of crystallites and the nature of decomposition [5]. Since maximum decomposition temperature is a criterion of thermal stability [7] processed BNC exhibit better stability than pristine BNC.

26. Fig.4 As shown in fig.4 the mechanical properties of pristine BNC and processed BNC were evaluated in terms of tensile stress and tensile strain. In this study, the pristine BNC film showed a tensile stress of 58.55MPa and a tensile strain of 6.5%, the corresponding value for the processed BNC was 41.66MPa and 3.8% respectively. The tensile stress strain in pristine BNC is higher as compared to the processed BNC. This decreases in tensile properties of processed BNC could be attributed to the chemical treatment in purifying the cellulose sheets which denature the cross linking network of cellulose nanofibers, hence poor network formation of BNC sheets. The poor network formation of processed BNC results in reducing the stress transfer between the BNC fibers.

27. Processed BNC sheets owing to the poorer BNC network formation having lower grammage and less degree of hornification, could likely have further deformation and assimilate more energy upon tensile loading, whereas pristine BNC with higher grammage imparts structural reinforcement to the BNC sheet. Pristine BNC showing lower ductility could be delineated by its more uniform network formation, which is presumed to acquire additional physical cross linking between the nanofibers [8]. Therefore, the nanofibers in the pristine BNC sheets when subjected to a tensile loading do not reform easily, leading to exhibiting a higher degree of elongation at break.

28. Fig.5 shows the indentation tests have been performed with the Ultra Nanoindentation tester. The load vs. penetration depth curves shown in fig.5 obtained on pristine BNC film are more superimposed making the homogeneity of the surface compared to processed BNC film

29. Fig.6 illustrates the results showing the highest hardness and modulus of elastic values for processed BNC film compared to pristine BNC film. Processed BNC shows the mean hardness and elastic modulus values of 69.505 MPa and 832.835 MPa respectively with the penetration depth at a maximum load of ~3.5µm. As the penetration depth of the pristine BNC increases to ~4.4µm, the obtained mean hardness and elastic modulus values are 49.612 MPa and 520.671 respectively. The obtained hardness and elastic modulus values are comparable to the bacterial cellulose films reported by Muling Zeng et. al. The better mechanical property of the processed BNC could be attributed to its higher purity on alkali treatment. Therefore, it exhibits better resistance to plastic deformation at a given load.

30. The contact angle is an important factor to characterize the printability of substrates. From the contact angle, we can understand the wettability and spreading properties of the substrate which contribute to printing quality. The contact angle of <90% affirms the wettability in contact with the substrate surface.

31. Fig.7 shows the water’s contact angle with respect to time, showing the stability of the wetting on processed BNC and pristine BNC as of the depicted time. Processed BNC shows a contact angle of ~64.72?, which has good surface wettability and high surface energy. Pristine BNC showing contact angle ~97.93?>90? is a poor surface wettability and has low surface energy. If the surface contact angle is too low leading to very high surface energy and high absorption of liquid on the substrate, there will be too much spreading of ink resulting inadequate printing consistency and resolution. The results suggest that the as-prepared processed BNC is a suitable substrate for printing.

32. Fig.8 As processed BNC sheet aims to be used as a wearable substrate it is necessary to investigate biocompatible properties. The biocompatibility studies for the processed BNC are assessed using alamar blue assay through cell viability of L929 fibroblast cell lines. The cell adhesion studies are also done with alamar blue assay.

33. Fig.9 As shown in fig.9 the cell viability % against time shows that the cell viability of processed BNC sheet is >80% for a time interval of 24h 10 168h. Processed BNC sheet also promotes cell adhesion and cell growth as shown in fig.12. The grown cells orient to form a cellular network on the processed BNC surface. Hence, processed BNC is biocompatible to be used as a substrate.

34. Fig.10 Commercial AgNP ink was printed over the washed and pristine BNC sheets using a Tvasta Origin extrusion printer with a needle of gauge 0.2 mm. The extrusion amount was kept at 0.5 nl/mm with a bed temperature of 40oC. The printed patterns were UV cured for 1h and the sheet resistance was measured to be 2.0±0.4?/sq. and 7.0±0.9?/sq. for Ag ink printed on processed and printed BNC sheets, respectively. From fig.10 a uniform printed layer of Ag ink has been observe on processed BNC sheets with Ag layer thickness of 0.3mm and substrate thickness of 1mm. The porosity and hydrophilicity of the processed nano-cellulose make BNC nanosheets a good flexible substrate for printed wearables.

35. To evaluate the property as a printable, flexible, and biocompatible substrate for electrochemical sensing applications, an enzymatic fuel cell electrode was designed and printed on the processed BNC and pristine BNC substrate.

36. Fig.11 shows the polarisation curve and power-generated curve of BNC-based enzymatic fuel cells for non-turnover conditions and varying glucose concentration (1mM-10mM) for turnover conditions. Compared to the non-turnover condition the power output and current output generated for the turnover condition is higher, it increases with the increase in the concentration of the glucose loading. The power output is highest at the externally applied resistance of 100kO. A linear increase of power from 1mM to 9mM decreases at 10mM.The maximum power generated is ~70nW which is comparatively higher than the paper-based EFC reported by Christopher F et.al.

37. Fig.12 The sensitivity of the BNC-based enzymatic fuel cell towards glucose is further evaluated by Cyclic voltammetry in PBS 0.01M. The enzyme catalytic activity shows no significant peak current from glucose concentration of 1mM-8mM. As shown in Fig. 12, the oxidation peak current appears in the presence of 9mM glucose and increases with the glucose concentration. This correlates with maximum power output at 9mM.

38. Fig.13 To understand the enzymatic reaction occurring at the anode, the affinity of GOxon glucose is determined by fitting the Michaelis-Menten equation as shown in Fig. 13(a,b).The Kmis the Michaelis constant. It is the substrate concentration that gives rise to a reaction velocity that is 0.5Vmax. The Km and Vmax value obtained from the fitted Michaelis-Menten equation is used for enzyme kinetic simulation. The simulated graph shown in Fig. 13cdemonstrates the saturation of the enzyme and hyperbolic relationship. The obtained Km of pristine BNC and processed BNC are 59.36 mM and 35.38 mM respectively. Thus, processed BNC-based EFC shows better sensitivity and a higher reaction rate. The result shows that GOx on reaching its steady-state level cannot catalyze all the glucose present in the PBS electrolyte for both pristine BNC and processed BNC-based EFC. The reaction rate no longer increases with the increase in the substrate concentration. Hence, the BNC-based EFC glucose sensing is in accordance with Michaelis-Menten kinetics.

DETAILED DESCRIPTION OF THE INVENTION

39. The present invention discloses a cost-effective and sustainable method for production of biocompatible BNC-based nano-cellulose matrix. The method eliminates the need for external pressure, ensuring the formation of wrinkle-free BNC nano-cellulose substrate suitable for flexible or wearable electronics.

40. The present invention is advantageous as Bacterial nanocellulose (BNC) substrate is a biodegradable, sustainable and environmentally friendly materials leading to several applications in green electronics.

41. Culture Preparation: As outlined above, a process for producing bacterial nano-cellulose (BNC) is herein presented, the BNC sheets are prepared from the symbiotic culture of bacteria and yeast (SCOBY) cultivated from the kombucha culture. The culture medium is prepared with 1 L of deionized water, 50g of sucrose, and 5g of tea, wherein 87g of SCOBY and 50 ml of mother broth are added.

42. Collection and Purification: The culture is maintained at room temperature, and after 7 days the cellulose pellicles formed were collected with a thickness of 3-5mm. For pristine BNC sheets, the collected pellicles were washed several times with DI water removing the bacteria residue to neutral pH. The pellicles were treated with NaOH (2%) to processed BNC sheets.

43. Freezing and Melting: Both pristine and processed pellicles were frozen for 16 hours at 4°C and kept for melting at room temperature until all the absorbed water melts along with the water of crystallisation bound intermolecularly with the nanofibers. The controlled evaporation in processed bacterial nanocellulose (BNC) is effectively regulated through the freeze-melt technique. This technique allows the escape of both crystallisation water and the absorbed water, which is loosely bound to the surface of the BNC material.

44. Drying: The thin sheet floating on the water is collected and dried in a vacuum oven at 80°C for 2hr. The obtained freeze-melt processed BNC and the pristine BNC were used to study their physicochemical properties and their compatibility as a flexible substrate.

45. XRD (X-ray Diffraction): XRD was used to analyze the crystallinity of the BNC sheets by measuring the diffraction patterns generated using a specific X-ray source and calculating crystallinity percentage using the formula CrI(%).

46. SEM (Scanning Electron Microscopy): FE-SEM Quanta 3D FEG was employed to study the surface morphology of freeze-dried BNC sheets.

47. TGA (Thermo Gravimetric Analysis): A Thermo gravimetric analyzer was used to assess the thermal stability of BNC samples by heating them from 50°C to 650°C at a specific heating rate.

48. Tensile Strength Test: The mechanical properties of BNC sheets were determined through tensile stress-strain tests conducted in accordance with ASTM D88 standards, using a Universal Tensile Tester.

49. Hardness Test: Indentation tests were used to measure the elastic modulus, hardness, and penetration depth of BNC films using an Ultra Nanoindentation tester.

50. Absorption Studies: The swelling property of cellulose sheets was investigated by measuring the weight difference before and after immersing them in different solutions (phosphate buffer saline, DI water, and NaCl) at specific conditions.

51. Contact Angle Measurement: Contact angles were measured to assess the wetting behavior of the BNC sheets using a tangent fitting method.

52. Biodegradability Studies: Biodegradability was evaluated by incubating BNC sheets in a lysozyme solution and observing the weight changes over time.

53. Biocompatibility Studies: Cell adhesion and cytotoxicity studies were conducted by seeding L929 cells on BNC sheets and measuring cell viability using the Alamar blue assay, as well as taking SEM images to assess cell adhesion.
, Claims:CLAIMS
We claim,

1. A method for the production of bacteria-derived nano-cellulose (BNC) sheets suitable for flexible electronics devices, the method comprising the steps of:
a) Preparing a kombucha culture,
b) Collecting bacteria-derived nano-cellulose (BNC),
c) Purifying the collected BNC, and
d) drying the purified BNC.

2. The method as claimed in claim 1, additionally comprising the step of freezing the purified BNC overnight, followed by melting the frozen BNC at ambient room temperature for a duration of 2 hours.
3. The method as claimed in claim 1, wherein excess water, including crystallized water and absorbed water, is removed from the BNC sheets by controlled evaporation after melting, and the BNC sheets are subsequently dried in an oven at 80°C.
4. The method as claimed in claim 1, wherein the bacterial nano-cellulose BNC is obtained through the cultivation of a symbiotic culture comprising bacteria and yeast.
5. The method as claimed in claim 1, wherein the BNC sheets exhibit a wrinkle-free characteristic.
6. A method as claimed in claim 1, wherein a single-layer nanosheet substrate produced demonstrates exceptional printability, rendering it suitable for the fabrication of flexible electronics devices.