Login| Sign Up| Help| Contact|

Patent Searching and Data


Title:
BACTERIAL CELLULOSE BASED HYBRID MATERIALS
Document Type and Number:
WIPO Patent Application WO/2019/136036
Kind Code:
A1
Abstract:
Bacterial cellulose based hybrid materials are fabricated by providing a bacterial cellulose-producing bacterium; providing a suitable bacteria nutritional medium; culturing the bacterium in the nutritional medium under conditions to produce bacterial cellulose; and providing a fiber or multiple fibers in a defined pattern such that the fiber is interwoven with the bacterial cellulose.

Inventors:
KEANE, Jennifer, K. (8317 Prestwick Dr, La Jolla, CA, 92027, US)
Application Number:
US2019/012001
Publication Date:
July 11, 2019
Filing Date:
January 01, 2019
Export Citation:
Click for automatic bibliography generation   Help
Assignee:
KEANE, Jennifer, K. (8317 Prestwick Dr, La Jolla, CA, 92027, US)
International Classes:
C12P19/04; B29B15/10
Attorney, Agent or Firm:
LABATT, John, W. (Labatt, LLCPO Box 63, Valatie NY, 12184, US)
Download PDF:
Claims:
CLAIMS

What is claimed is:

1. A composition comprising:

bacterial cellulose (BC); and

a fiber fixed in a pattern wherein the fiber is interwoven with the BC

2. The composition of claim 1 wherein the pattern comprises of one or more filament fibers arranged in a defined non-random direction.

3. The composition of claim 1, wherein the pattern comprises of twisted fibers and / or yarn comprising of twisted fibers arranged in any direction.

4. The composition of claims 1-3, wherein the fiber is interwoven with the BC in situ.

5. The composition of claims 1-4, wherein the fiber pattern is arranged directly onto a frame in a single or multiple axes according to defined pattern.

6. The composition of claim 1-4, wherein the fiber pattern is a knitted, woven or nonwoven textile assembled according to any method known to the art prior to being interwoven with BC.

7. The composition of claims 1-4, wherein the fiber pattern is fabricated prior to growth phase via mechanical and/or automated fiber placement.

8. The composition of claims 1-4, wherein the fiber pattern is pre-fixed to a substrate.

9. The composition of claims 1-8, wherein the fiber pattern is fixed to a substrate by chemical adhesion.

10. The composition of claims 1-8, wherein the fiber pattern is fixed to a substrate by physical adhesion.

11. The composition of claims 1-8, wherein the fiber pattern is fabricated and/or completed during growth phase via mechanical and/or automated fiber placement.

12. The composition of claims 1-8 wherein the fiber pattern is fabricated and/or completed during growth phase via biological means such as co-culture.

13. The composition of claims 1-12, wherein the fiber comprise of a single fiber type, such as spun or filament fiber or composite fiber type.

14. The composition of claim 1-12, wherein the fibers comprise of multiple different fibers and/or yam types such as spun, filament and/or composite fiber type.

15. The composition of claims 1-14, wherein the composition further comprises a resin.

16. The composition of claim 15, wherein the resin is selected from the group consisting of biodegradable resin, water-soluble resin, natural resin, plant-based resin and non-toxic resin.

17. The composition of claim 15, wherein the resin is a petroleum-based resin.

18. The composition of claim 17, wherein the petroleum-based resin is an epoxy, vinyl, or unsaturated polyester-based resin.

19. The composition of claim 15, wherein the resin is selected from the group consisting of polyethylene oxide (PEO), polyvinyl alcohol (PVA) and polyhydroxy alkanoate (PHA).

20. A process for preparing a bacterial cellulose hybrid material, the process comprising the steps of:

(i) providing a bacterium capable of producing cellulose;

(ii) culturing the bacterium in a suitable medium in a suitable container under conditions to produce BC;

(iii) providing a fiber or combination or fibers, in a pattern

(iv) providing conditions wherein fibers are interwoven with bacterial cellulose (BC) fibers.

21. The process of claim 20, wherein the pattern comprises of one or more filament fibers arranged in a defined non-random direction.

22. The process of claim 20, wherein the pattern comprises of twisted fibers and / or yarn comprising of twisted fibers arranged in any direction.

23. The process of claims 20-22, wherein the fiber is interwoven with the BC in situ.

24. The process of claims 20-23, wherein the fiber pattern is arranged directly onto a frame in single or multiple axes according to defined pattern.

25. The process of claims 20-23, wherein the fiber pattern is a knitted, woven or nonwoven textile assembled according to any method known to the art prior to being interwoven with BC.

26. The process of claims 20-23, wherein the fiber pattern is fabricated prior to growth phase via mechanical and/or automated fiber placement.

27. The process of claims 20-23, wherein the fiber pattern is pre-fixed to a substrate.

28. The process of claims 20-27, wherein the fiber pattern is fixed to a substrate by chemical adhesion.

29. The process of claims 20-27, wherein the fiber pattern is fixed to a substrate by physical adhesion.

30. The process of claims 20-27, wherein the fiber pattern is fabricated and/or completed during growth phase via mechanical and/or automated fiber placement.

31. The process of claims 20-27, wherein the fiber pattern is fabricated and/or completed during growth phase via biological means such as co-culture.

32. The process of claims 20-31, wherein the fiber(s) comprise of a single fiber type, such as spun or filament fiber or composite fiber type.

33. The process of claims 20-31, wherein the fibers comprise of multiple different fibers and/or yarn types such as spun, filament and/or composite fiber type.

34. The process of claims 20-33, wherein the process further comprises providing a resin.

35. The process of claim 34, wherein the resin is selected from the group consisting of biodegradable resin, water-soluble resin, natural resin, plant-based resin and non-toxic resin.

36. The process of claim 34, wherein the resin is a petroleum-based resin.

37. The process of claim 34, wherein the petroleum-based resin is an epoxy, vinyl, or unsaturated polyester-based resin.

38. The process of claim 34, wherein the resin is selected from the group consisting of polyethylene oxide (PEO), polyvinyl alcohol (PVA) and polyhydroxy alkanoate (PHA).

39. A device for preparing a bacterial cellulose (BC) hybrid material, the device comprising:

(i) a container suitable for culturing bacterium capable of producing cellulose in a suitable medium under conditions to produce BC; and

(ii) a structure for containing and/or fixing a fiber or multiple fibers in a pattern such that the fiber is interwoven with the BC.

40. The device of claim 39, wherein the pattern comprises of one or more filament fibers arranged in a defined non-random direction.

41. The device of claim 39, wherein the pattern comprises of twisted fibers and / or yarn comprising of twisted fibers arranged in any direction.

42. The device of claims 39-41, wherein the fiber is interwoven with the BC in situ.

43. The device of claims 39-42, wherein the structure comprises a frame whereupon fiber pattern is arranged directly in single or multiple axes according to defined pattern.

44. The device of claims 39-42, wherein the structure comprises a frame whereupon a fiber pattern that is a knitted, woven or nonwoven textile assembled according to any method known to the art can be fixed and interwoven with BC.

45. The device of claims 39-42 wherein the structure enables a fiber pattern that is fabricated via mechanical and/or automated fiber placement to be interwoven with BC.

46. The device of claims 39-45 wherein the structure further comprises a system for fabricating or completing the fiber pattern during growth phase.

47. The device of claims 39-46 wherein the container is further suitable for co-culturing the bacterium with other microorganisms.

48. An article of footwear comprising a Bacterial Cellulose (BC) hybrid material wherein a fiber fixed in a pattern is interwoven with the BC.

49. An article of clothing comprising a Bacterial Cellulose (BC) hybrid material wherein a fiber fixed in a pattern is interwoven with the BC.

50. A medical device comprising a Bacterial Cellulose (BC) hybrid material wherein a fiber fixed in a pattern is interwoven with the BC.

51. A wearable accessory comprising a Bacterial Cellulose (BC) hybrid material wherein a fiber fixed in a pattern is interwoven with the BC.

52. The article of any of claims 48-51, wherein the pattern comprises of one or more filament fibers arranged in a defined non-random direction.

53. The article of claim any of claims 48-51, wherein the pattern comprises of twisted fibers and / or yam comprising of twisted fibers arranged in any direction.

54. The article of claims any of claims 48-53, wherein the fiber is interwoven with the BC in situ.

55. The article of any of claims 48-54, wherein the fiber pattern is arranged directly onto a frame in a single or multiple axes according to defined pattern.

56. The article of any of claims 48-54, wherein the fiber pattern is a knitted, woven or nonwoven textile assembled according to any method known to the art prior to being interwoven with BC.

57. The article of any of claims 48-54, wherein the fiber pattern is fabricated prior to growth phase via mechanical and/or automated fiber placement.

58. The article of claims any of claims 48-54, wherein the fiber pattern is pre-fixed to a substrate.

59. The article of any of claims 48-58, wherein the fiber pattern is fixed to a substrate by chemical adhesion.

60. The article of any of claims 48-58, wherein the fiber pattern is fixed to a substrate by physical adhesion.

61. The article of any of claims 48-58, wherein the fiber pattern is fabricated and/or completed during growth phase via mechanical and/or automated fiber placement.

62. The article of any of claims 48-58, wherein the fiber pattern is fabricated and/or completed during growth phase via biological means such as co-culture.

63. The article of any of claims 48-62, wherein the fiber comprise of a single fiber type, such as spun or filament fiber or composite fiber type.

64. The article of any of claims 48-62, wherein the fibers comprise of multiple different fibers and/or yam types such as spun, filament and/or composite fiber type.

Description:
Bacterial Cellulose based Hybrid Materials

Reference to Related Applications

[0001] The current application claims the benefit of United Kingdom Patent Application No. 1800027.3, which was filed on 2 January 2018, and which is hereby incorporated by reference.

Field of Invention

[0002] This invention relates to bio based materials and composites, in particular to bacterial cellulose (BC) based hybrid BC-textile materials, produced using an in situ self-assembly method.

Background

[0003] Bacterial cellulose (BC) fibers secreted by bacterial cellulose producing bacterium such as komagataeibacter rhaeticus , display many interesting characteristics including a high crystallinity, as well as high strength and stiffness (Young’s modulus). BC membranes produced through static fermentation have a highly porous structure, excellent biocompatibility and are completely biodegradable. Because of their unique properties, bacterial cellulose membranes are already used in numerous industrial applications such as foods, speaker diaphragms, high quality paper, textiles, artificial skin and blood vessels, as well as a binding agent for fibers and other materials.

[0004] It has also been shown that incorporation of fibers, such as sisal fibers, into the fermentation media in self-assembly approach can produce a hybrid composite material with better tensile properties compared with pure BC or sisal fibers (Qui, K.; Netravali, A. (2017). BC materials produced so far still had limited applications. Further improvement and versatility is, therefore, needed for BC materials to achieve their potential in key industries.

Summary of Invention

[0005] The invention builds and improves on this knowledge to create a new type of BC- textile hybrid material.

[0006] The invention provides a composition comprising bacterial cellulose (BC) and a fiber fixed in a pattern, wherein the fiber is interwoven with the BC.

[0007] A method for producing bacterial cellulose (BC) based hybrid BC-textile materials is also provided comprising the steps of: providing a bacterium capable of producing cellulose; culturing the bacterium in a suitable medium in a suitable container under conditions to produce BC; providing a fiber or combination or fibers in a pattern and providing conditions wherein the fiber or combination of fibers is interwoven with the bacterial cellulose (BC). [0008] The hybrid material produced retains the fiber / yarns in a predefined order and direction with bacterial cellulose acting as a binder. The hybrid BC-textile material thus offers much possibility for customization and engineering of aesthetic, mechanical and other material properties.

[0009] The invention can be employed across multiple product types in multiple industries and offers the possibility of products to be produced to shape, in or close to their final form.

[0010] The invention also provides an article of footwear, clothing, and / or wearable accessory comprising a Bacterial Cellulose (BC) hybrid material wherein a fiber fixed in a pattern is interwoven with the BC.

[0011] The natural biocompatibility properties of bacterial cellulose also allow for the invention’s potential application inside the body or for other medical purposes.

[0012] The invention also provides a medical device comprising a Bacterial Cellulose (BC) hybrid material wherein a fiber fixed in a pattern is interwoven with the BC.

[0013] In one embodiment, the pattern is comprised of one or more filament fibers arranged in a defined non-random direction.

[0014] In another embodiment, the pattern is comprised of twisted fibers and / or yarn made up of twisted fibers arranged in any direction.

[0015] In one embodiment, the fiber pattern is added to a container suitable for culturing a bacterium capable of producing BC under conditions suitable for the bacteria to produce BC. A structure such as a scaffold comprised of a yam or multiple yarns is added to the

growth/fermentation container. The fiber or yams comprised of multiple fibers are fixed above, or on the oxygenated side of, the media in a defined pattern, direction or combination of directions. The yam or yarns are thus incorporated into the BC nanofiber structure during static fermentation.

[0016] In another embodiment the fiber / yam pattern can be suspended in gel media that also provides suitable conditions for BC growth wherein the BC becomes interwoven with the fibers(s).

[0017] In one embodiment the arrangement of the yarns is prefabricated through other textile production methods such as weaving or knitting and then fixed to the‘warping frame,’ or structure that holds them in place during static growth.

[0018] In another embodiment yarns are arranged directly onto‘warping frame’ in single or multiple axes‘weave’ pattern.

[0019] The scaffold is in one embodiment comprised of a single yarn and fiber type or in another embodiment contains multiple different fibers / yarns. [0020] In another embodiment, the fiber pattern is fabricated prior to growth phase via mechanical and/or automated fiber placement.

[0021] In another embodiment, the fiber pattern is a knitted, woven or nonwoven textile assembled according to any method known to the art prior to being interwoven with BC.

[0022] In another embodiment, the fiber pattern is pre-fixed to a substrate.

[0023] In another embodiment, the fiber pattern is fixed to a substrate by chemical adhesion.

[0024] In another embodiment, the fiber pattern is fixed to a substrate by physical adhesion.

[0025] In another embodiment, the fiber pattern is fabricated and/or completed during growth phase via mechanical and/or automated fiber placement.

[0026] In another embodiment, the fiber pattern is fabricated and/or completed during growth phase via biological means such as co-culture.

[0027] In one embodiment, one or more of the yarns are filament yarns including but not limited to untwisted filament, twisted filament, and / or stretch filament. Any other types of filament yarns known in the art can also be used.

[0028] In one embodiment, one or more of the yarns are spun yarns including but not limited to carded spun, combed spun, ring spun, open-end spun, airjet spun, wrap spun.

[0029] Any other types of spun yarns known in the art can also be used.

[0030] In another embodiment, one or more of the yams are a woolen yarns.

[0031] In another embodiment, one or more of the yarns are worsted yarns.

[0032] In another embodiment, one or more of the yarns are braided yarns.

[0033] In another embodiment, the fibers comprising one or more of the yarns are transparent.

[0034] In another embodiment, one or more of the fibers / yarns are conductive.

[0035] In another embodiment, one or more of the fibers / yarns are biocompatible.

[0036] In another embodiment, one or more of the fibers are ingestible.

[0037] In another embodiment, one or more of the fibers are edible.

[0038] In another embodiment, the fibers in one or more of the yams comprise a natural cellulose-based or protein-based material.

[0039] In another embodiment, the natural cellulose-based material is selected from the group consisting of cotton, linen, flax, sisal, ramie, hemp, kenaf, jute, bamboo, banana, pineapple, kapok, and combinations thereof. Any other natural cellulose fibers known in the art can also be used.

[0040] In another embodiment, the natural protein-based material is selected from the group consisting of wool, silk, angora, cashmere, mohair, alpaca, milk protein, spider silk, and soy protein and combinations thereof. [0041] In another embodiment, the fibers comprise a polymeric material. In another embodiment, the polymeric material is cellulose acetate, nylon, rayon, modacrylic, olefin, acrylic, polyester, polylactic acid, polylactic-co-glycolic acid (PLGA), polyurethane, aramid (e.g. KEVLAR.RTM.), or ultrahigh molecular weight polyethylene, (e.g. SPECTRA.RTM. or DYNEEMA.RTM.). In another embodiment, the fibers comprise carbon (e.g., carbon fiber, nanotubes) or glass (e.g., fiberglass), graphite, or graphene material.

[0042] In another embodiment, the fibers comprise of or contain metal material such as aluminum, steel, silver, gold, copper.

[0043] In another embodiment, the composition further comprises a resin, which is selected from the group consisting of biodegradable resin, water-soluble resin, natural resin, plant-based resin and non-toxic resin.

[0044] The invention also provides a device for preparing a bacterial cellulose (BC) hybrid material, the device comprising of: a container suitable for culturing bacterium capable of producing cellulose in a suitable medium under conditions to produce BC; and

[0045] a structure for containing and/or fixing a fiber or multiple fibers in a pattern such that the fiber is interwoven with the BC.

[0046] In one embodiment, the structure comprises a frame whereupon fiber pattern is arranged directly in single or multiple axes according to defined pattern.

[0047] In another embodiment, the structure comprises a frame whereupon a fiber pattern that is a knitted, woven or nonwoven textile assembled according to any method known to the art can be fixed and interwoven with BC.

[0048] In another embodiment, the structure enables a fiber pattern that is fabricated via mechanical and/or automated fiber placement to be interwoven with BC.

[0049] In another embodiment, the structure further comprises a system for fabricating or completing the fiber pattern during growth phase.

[0050] In another embodiment, the container is further suitable for co-culturing the bacterium with other microorganisms.

[0051] In one embodiment, the structure comprises a base with fixed or adjustable pegs and/or raised supports. The cross section and height of the pegs/supports can be adjusted according to desired design/placement of scaffold within BC matrix.

[0052] Any bacterium known to produce BC can be used including but not limited to komagataeibacter rhaeticus, Acetobacter xylinum, or genetically modified versions of a BC- secreting genus. [0053] Any culture medium known in the art to support the selected bacteria can be used. It could be pure or have other fibers incorporated into it.

[0054] The hybrid material could be bleached or dyed, dried pure, plasticized using any commercially available plasticizers such as glycerol or impregnated with resin to form a composite.

Figures

[0055] The invention is described herein with reference to the accompanying drawings. It is to be understood that in some instances, various aspects of the invention may be shown exaggerated or enlarged to facilitate an understanding of the invention.

[0056] Figure 1 depicts two examples of warping frame support pegs.

[0057] Figure 2 depicts an example of spun yarns held in place on a warping frame support peg at a notch.

[0058] Figure 3 depicts an angled view of one embodiment of an assembled warping frame with multiple support pegs and an example of applied single yarn scaffold.

[0059] Figure 4 depicts an example of a warping frame with an irregular shape and rectangular pegs.

[0060] Figure 5 depicts an example of a warping frame with a square shape and rectangular pegs.

[0061] Figure 6 depicts an example of a warping frame with a circle shape, and round pegs.

[0062] Figure 7 depicts an example of an embodiment where a single yarn is arranged directly on the warping frame.

[0063] Figure 8 depicts an example of an embodiment where a multiple different yarns are arranged directly on the warping frame.

[0064] Figure 9 is a perspective view of one embodiment of a fermentation container.

[0065] Figure 10 is the top view of one embodiment of a fermentation container.

[0066] Figure 11 is the side view of one embodiment of a fermentation container.

[0067] Figure 12 shows perspective view of positioning of warping frame placement in fermentation container.

[0068] Figure 13 shows top view of positioning of warping frame placement in fermentation container.

[0069] Figure 14 is a side view of the of fermentation container with inoculated media and warping frame in starting setup.

[0070] Figure 15 is a side view of the of fermentation container and pellicle growth after 2-4 days. [0071] Figure 16 is a side view of the of fermentation container and pellicle growth after 4-8 days.

[0072] Figure 17 is a side view of the fermentation container and pellicle growth after 7-15 days, or when material is fully-grown.

[0073] Figure 18 shows the top view of one embodiment of a scaffold fixed to a warping frame before being added to growth/fermentation container for BC production.

[0074] Figure 19 shows the top view of fully grown BC- textile hybrid pellicle removed from growth/fermentation container.

[0075] Figure 20 shows a cross section of one embodiment of the BC - textile hybrid material (wet) where yarns are concentrated at the midpoint of the pellicle.

[0076] Figure 21 shows a cross section of one embodiment of the BC - textile hybrid material (wet) where yarns are concentrated at bottom of the pellicle.

[0077] Figure 22 shows a cross section of one embodiment of the BC - textile hybrid material (wet) where yarns are spread throughout thickness of the pellicle.

[0078] Figure 23 shows a cross section of one embodiment of the BC - textile hybrid material (wet) where yarns are spread throughout thickness of the pellicle, and parts of the yarn extend outside of the pellicle.

Detailed Description

[0079] For clarity of disclosure, and not by way of limitation, the detailed description of the invention and process for producing it are divided into the subsections set forth below.

[0080] “Pattern”, in reference to the pattern of fibers is a non-random and intentional arrangement of fibers in one, multiple or a combination of directions.

1. Warping Frame and Scaffold Preparation

[0081] The purpose of the warping frame is to provide support to the scaffold or matrix of yarns during scaffold assembly as well as during fermentation stage and hybrid material production. The warping frame is composed of a base with fixed or adjustable pegs and/or raised supports.

[0082] Figure 1 shows examples of two embodiments of pegs. Number 1 depicts a round peg that is fixed to the base with nut and bolt. Number 2 depicts a laser cut peg with a rectangular cross section, which is fixed with glue to the base. The cross section and height of the pegs/supports can be adjusted according to desired design/placement of scaffold within BC matrix. What is important is that a yam or multiple yarns can be easily fixed in one or more places 3 on the support. Figure 2 depicts one embodiment of a spun yarn 1, or yarn composed of twisted fibers, fixed to a preferred peg embodiment 2 where a notch 3 holds the yarn in place.

[0083] Figure 3 shows one embodiment of an assembled warping frame with multiple support pegs 2 with yarn 1 fixed at the notch 3 in each peg.

[0084] Figure 4 depicts an example of a warping frame 1 with an irregular shape and rectangular pegs 2. Figure 5 depicts an example of a warping frame with a square shape 1 and rectangular pegs 2. Figure 6 depicts an example of a warping frame with a circle shape 1, and round pegs 2. There is no limit in shape, size or positioning of base plate and pegs for warping frame embodiments.

[0085] The material composition of one warping frame embodiment is acrylic.

[0086] In another embodiment, stainless steel is used.

[0087] The only limiting factor of the material composition of the warping frame is it should be as inert as possible as to not corrode, rust or react with the liquid chemical composition of the media or material processing solutions such as but not limited to NaOH solution, bleach, or solvents. The material should also not be harmful to living organisms such as bacteria or fungi. Also for many sterilization and finishing treatments it may need to withstand high heats.

[0088] Yarn is applied to the warping frame by hand in one embodiment but in other embodiments by machine, robotic arm or automated thread placement mechanism.

[0089] In another embodiment, the fibers comprising the yarn are transparent.

[0090] In another embodiment, the yarn is conductive.

[0091] In another embodiment, the fibers comprise a natural cellulose-based or protein-based material.

[0092] In another embodiment, the natural cellulose-based material is selected from the group consisting of cotton, linen, flax, sisal, ramie, hemp, kenaf, jute, bamboo, banana, pineapple, kapok, and combinations thereof. Any other natural cellulose fibers known in the art can also be used.

[0093] In another embodiment, the natural protein-based material is selected from the group consisting of wool, silk, angora, cashmere, mohair, alpaca, milk protein, spider silk, and soy protein and combinations thereof.

[0094] In another embodiment, the fibers comprise a polymeric material. In another embodiment, the polymeric material is cellulose acetate, nylon, rayon, modacrylic, olefin, acrylic, polyester, polylactic acid, polylactic-co-glycolic acid (PLGA), polyurethane, aramid (e.g. KEVLAR.RTM.), or ultrahigh molecular weight polyethylene, (e.g. SPECTRA.RTM. or DYNEEMA.RTM.). In another embodiment, the fibers comprise carbon (e.g., carbon fiber) or glass (e.g., fiberglass).

[0095] The scaffold could be comprised of a single yarn and fiber type or could contain multiple different yarns in a single hybrid material. The arrangement of the yams could be prefabricated through other textile production methods such as weaving or knitting and fixed to ‘warping frame’ or can be arranged directly onto‘warping frame’ in single or multiple axes weave pattern. Figure 7 depicts an example of an embodiment where a single yarn 2 is arranged directly on the warping frame 1. Figure 8 depicts an example of an embodiment where a multiple different yarns 2,3,4 are arranged directly on the warping frame 1.

[0096] As yarns often contain contaminants and/or other microbes or can easily become contaminated during handling. For this reason, it is important to sterilize the frame and yam after yarn is applied to the frame, and/or prevent pre-sterilized elements from being contaminated during assembly. Sterilization method depends on the composition of the warping frame as well as the yarn used. Sterilization methods include but are not limited to soaking in ethanol or bleach solution, dry heat, autoclave, exposure to UV light, or other chemical sterilization methods.

2. Bacteria and Culture Media for BC Production

[0097] Methods are provided herein for producing bacterial cellulose (BC). In one

embodiment, the method comprises providing a bacterium wherein the bacterium is a bacterial cellulose-producing bacterium; providing a bacteria nutritional medium; culturing the bacterium in the bacteria nutritional medium under conditions to produce BC.

[0098] Many strains of bacteria that synthesize cellulose can be used to synthesize BC including but not limited to komagataeibacter rhaeticus, Acetobacter xylinum, or genetically modified versions of BC secreting genus. In a preferred embodiment, komagataeibacter rhaeticus is used.

[0099] In one embodiment, Acetobacter xylinum, ATCC 23769, can be used (American Type Culture Collection (ATCC), Manassas, Va.).

[0100] In other embodiments, the following can be used.

[0101] Gluconacetobacter hansenii ATCC 53582

[0102] Gluconacetobacter xylinus ATCC 23767

[0103] Acetobacter pasteurianus ATCC 10245

[0104] Acetobacter liquefaciens ATCC 14835

[0105] Any culture medium known in the art to support the selected bacteria can be used. For example, culture media that can be used can consist of 5-50 g/l, of one or more carbon sources (e.g., stachyose, raffmose, glucose, sucrose, fructose, mannitol, galactose, maltose), 5 g/L yeast extract and 5 g/L tryptone. In one embodiment, 25 g/L is used. Any small sugar known in the art can also be used. In a preferred embodiment, media is prepared using a solution of pure coconut water with 1% AspalTs organic Cyder Vinegar. The coconut water media is sterilized in microwave as in cannot be autoclaved.

[0106] In one embodiment a liquid medium is used, in another embodiment the growth medium may be a gel.

3. Growth/Fermentation Container Design and Preparation

[0107] Details are provided here for building a device for preparing a bacterial cellulose (BC) hybrid material, the device comprising of: a container suitable for culturing bacterium capable of producing cellulose in a suitable medium under conditions to produce BC;

[0108] and a structure for containing and/or fixing a fiber or multiple fibers in a pattern such that the fiber is interwoven with the BC.

[0109] The fermentation container should be made out of an inert and easily sterilized material. In a preferred embodiment, the container is clear glass to allow for better monitoring of fermentation/growth but in other embodiments is opaque. Figures 9-11 show top (Figure 10), side (Figure 11) and perspective (Figure 9) views of one embodiment of a fermentation container. Import features include two separate sections, a material production compartment 1 and a media addition compartment 2. These are separated by a divider 3 that prevents liquid from flowing easily between the areas except for through a narrow opening 4 at the bottom of the divider. The shape of the container and subsequent compartments can be changed to suit desired size and shape of hybrid pellicle. The pellicle will take on the shape of the material production compartment 1. Container has a lid 5 to protect from contamination by other organisms during fermentation but that allows or promotes oxygen flow to the culture.

[0110] In one embodiment, a paper towel is used as a lid.

[0111] In another embodiment, a sterile cloth is used as a lid.

[0112] In another embodiment, lid is sealed onto container with tube inserted to control gas flow in and out of the container.

[0113] In another embodiment, container is to be sterilized prior to growth. Sterilization methods include but are not limited to soaking in ethanol or bleach solution, dry heat, autoclave, exposure to UV light, or other chemical sterilization methods. Most effective sterilization method should be determined based on material composition of the container. 4. Hybrid textile-BC pellicle production

[0114] Figures 12 and 13 show top (Figure 13) and perspective (Figure 12) views of how the assembled scaffold/warping frame is placed within fermentation container. Figure 14 shows the side view of set up fermentation container where number 1 indicates the yarn scaffold and number 2 indicates the media. Once warping frame is in place, prepared media and starter bacteria culture are added slowly into media addition compartment. The media and bacteria travel 3 through the gap at the bottom of the barrier until liquid media equalizes and reaches the predetermined fill line 4 in all compartments. It is important that the container remains level throughout full fermentation stage.

[0115] Figure 15 shows BC pellicle growth after 2-4 days, number 5 indicates visible layer of BC. Number 3 indicates how media in all compartments remains at equilibrium during fermentation.

[0116] Figure 16 shows BC pellicle after 4-8 days when surface of the pellicle reaches the scaffold and starts to incorporate the yams into its structure.

[0117] Media is continually added in small amounts to media addition compartment throughout fermentation so that media remains at fill line in equilibrium throughout entire fermentation. This provides conditions for the pellicle in the material production area to grow evenly with minimal imperfections.

[0118] A pellicle also forms in the media addition compartment 6 but usually will grow unevenly or in layers as the addition of media disturbs its growth.

[0119] In another embodiment, media is applied in the form of a fine mist to the top (or oxygen exposed side) of the growing pellicle to help stimulate growth.

[0120] Figure 17 shows final BC-textile hybrid pellicle after yarns are fully incorporated, usually within 7 to 15 days depending on growth conditions. Hybrid pellicle is still fixed in place on warping frame at the end of this phase. In one embodiment the hybrid pellicle is removed gently from the warping frame before post processing. In another embodiment it remains on the frame for post processing.

[0121] When removed from the frame, both BC fibers and fibers characterizing the yams might shift slightly but yams should still remain completely or partly fixed within the BC matrix. Figure 18 shows the top view of one embodiment of a scaffold comprised of a yarn 1 fixed to a warping frame 2 before being added to growth/fermentation container for BC production. Figure 19 shows the top view of the fully grown BC- textile hybrid pellicle removed from

growth/fermentation container and warping frame, number 1 indicates area where yarn fibers are concentrated and 2 denotes BC growth between the yams and fibers of the yams and where growth was not inhibited by shape of the container other means.

[0122] Figure 20 shows a cross section of one embodiment of the BC - textile hybrid material (wet) where yarns 1 are concentrated at the midpoint of the BC pellicle 2.

[0123] Figure 21 shows a cross section of one embodiment of the BC - textile hybrid material (wet) where yarns 1 are concentrated at bottom of the pellicle 2.

[0124] Figure 22 shows a cross section of one embodiment of the BC - textile hybrid material (wet) where yarns 1 are spread throughout thickness of the pellicle 2.

[0125] Figure 23 shows a cross section of one embodiment of the BC - textile hybrid material (wet) where yarns 1 are spread throughout thickness of the pellicle, and parts of the yarn extend outside of the pellicle 2.

[0126] Wash BC crude pellicle successively with water and 1% (w/v), aqueous NaOH, and then remove all microbial product contaminants by deionized water (BC wet pellicle).

[0127] The hybrid material could be bleached or dyed, dried pure, plasticized using any commercially available plasticizers such as glycerol or impregnated with resin to form a composite.

[0128] Remove water from the BC either by pressing, air dry, oven dry or freeze dry (for BC film), or combination there of.

[0129] In one embodiment the material is impregnated with resin.

[0130] In another embodiment, the resin is transparent.

[0131] In another embodiment, the resin is selected from the group consisting of natural resin, plant-based resin and non-toxic resin.

[0132] In another embodiment, the resin is biodegradable.

[0133] In another embodiment, the resin is water-soluble.

[0134] In another embodiment, the natural or plant-based resin is a soy -based resin.

[0135] In another embodiment, the resin is a petroleum-based resin.

[0136] In another embodiment, the petroleum-based resin is an epoxy, vinyl, or unsaturated polyester-based resin.

[0137] In another embodiment, the resin is polyethylene oxide (PEO).

[0138] In another embodiment, the resin is polyvinyl alcohol (PVA).

[0139] In another embodiment, the resin is polyhydroxy alkanoate (PHA).

EXAMPLE PROTOCOL

[0140] Listed below is an example protocol for the production of one embodiment of the invention. [0141] Media solution is prepared using coconut water (100%) with Aspall’s organic Cyder Vinegar. Media is sterilized by microwaving in a covered glass container (but not sealed). For 500ml of solution it is heated in the microwave for 6 minutes then allowed to cool (still covered) naturally to room temperature (22-25 degrees celcius).

[0142] Bacteria used is komagataeibacter rhaeticus.

[0143] Started culture is prepared in small sterilized glass jar (5cm diameter) by adding bacteria to about 3cm depth of sterilized media solution. It is covered with a paper towel and culture is left static at 22 degrees C for about 7 days or at least until a pellicle of about 6mm is formed at the surface.

[0144] Construct warping frame using laser cut acrylic base and support pegs. See Figure 2 for shape of example peg. In this example, 3mm thick sheet of clear acrylic is used for the pegs and 5mm clear acrylic for the base. Each peg is identical with a height of 40mm and notch at height of 25mm. Base is l50mm x l50mm square with 40, 3mm by 4mm, rectangular holes, 10 on each side of the square at a distance of 9mm apart. 40 pegs are secured to the based by insertion into the holes and fixed perpendicular to the base using dichloromethane. See Figure 18 for reference of peg spacing.

[0145] Apply 16/2 organic cotton yarn directly to the warping frame by fixing yarn to each peg at its notch. The thread is tied off at first and last peg to avoid unraveling. See Figure 18 for reference of yam arrangement in this example.

[0146] See Figures 9-11 as reference for growth/fermentation container design. Whereas the outside of the container is made of clear vacuum formed plastic and the divider laser cut acrylic.

[0147] The assembled frame and container are both sterilized by short soak in ethanol and air dried (covered) until ethanol fully evaporates.

[0148] See Figures 12-13 as reference for how the frame is placed into the growth container.

[0149] The entire contents of starter culture jar are carefully (to avoid contamination) added to the media addition compartment of the container and sterilized media is added to the same side slowly until equilibrium is met at fill line is met (see Figure 14). In this example the fill line is set at a height of 23mm or 2mm below yam scaffold.

[0150] Container is covered with a paper towel and left static on a level surface. Incubate at 22-25 degrees Celsius. Throughout fermentation process the media level will drop and it is imperative it is monitored and media be added (to media addition compartment only) throughout growth so media level stays at equilibrium at fill line (number 4 in Figures 14-17).

[0151] Allow fermentation to occur for 10-15 days or until yarn is fully incorporated into BC and hybrid pellicle is about 6mm thick. [0152] Remove frame from container but leave pellicle on warping frame.

[0153] Wash BC crude pellicle successively with water and 1% (w/v), aqueous NaOH at room temperature C. for 15 min, and then remove all microbial product contaminants by deionized water (BC wet pellicle).

[0154] Then soak pellicle in water and 5% (w/v), aqueous glycerol solution for 8 hours.

[0155] Remove pellicle carefully from frame and press out excess water by placing it between

2 sheets of fine metal mesh and wooden boards to which even pressure is applied. Let further air dry until about 2mm thick. Place between two sheets of wax paper, surround with paper towel and place between 20mm plywood boards. Apply even pressure using nuts and bolts to clamp boards together and leave until hybrid material is fully dried.