FIELD OF THE INVENTION

The present disclosure relates to processes for preparing biobased bacterial cellulose materials, to biobased bacterial cellulose materials themselves, and to clothing, footwear, wearable accessories interior furnishings, automotive and aerospace fittings and upholstery, comprising the biobased bacterial cellulose materials.

BACKGROUND OF THE INVENTION

Bacterial cellulose is an organic compound with the formula (C6HioOs)n produced by certain types of bacteria. It is formed from beta-1 -4 glucan chains that are arranged into higher order structures. Individual beta-1 -4 glucan chains combine to form sub-fibrils, which crystallise into microfibrils, which in turn combine into bundles and ribbons (1 , 2). These higher order microfibrils are much narrower than plant based cellulose microfibrils and are approximately 4 nm thick x 80 nm wide (1 , 3).

Bacterial cellulose is secreted outside the cell to form an extracellular bacterial cellulose matrix (2). Bacterial cellulose demonstrates a greater degree of polymerisation and crystallisation than plant derived cellulose, leading to altered and often enhanced material properties. Bacterial cellulose films are remarkably strong, with high tensile stiffness and strength and with a high modulus of elasticity leading to desirable flexibility properties (1). Bacterial cellulose is synthesised without contaminants such as lignin or pectin as found in plants, therefore is naturally of higher purity than plant derived cellulose (3) and so does not require extensive time, energy and chemically intensive treatment to arrive at a high purity product.

There are a wide range of bacteria that produce extracellular cellulose. Specifically, prolific cellulose producing bacteria include those from the Komagataeibacter genus (strains were previously allocated as Acetobacter or Gluconacetobacter).

There are many potential applications of bacterial cellulose, including: medical applications such as temporary artificial skin due to its biocompatibility; uses in the textile industry, for example for the manufacture of paper; electronic paper displays; audio applications, for example in headphones; food applications; and in the petrochemical industry (3, 4). However, the use of bacterial cellulose in the textile / fashion industry presents some challenges, notably due to its hydrophilic properties, its natural beige colour and its tendency to be hard and brittle once dried.

The production of a dyed bacterial cellulose material is described in European patent EP3553226. The bacterial cellulose is dyed through the action of a separate microorganism. Whilst this does produce a material with a different colour compared to the original bacterial cellulose, it does not alter any existing functional properties of the original bacterial cellulose material, nor does it confer other new exotic and advantageous properties to the bacterial cellulose material. Historically, the textile industry has been heavily reliant on plant derived materials, such as cotton, hemp, flax and jute. The production of these materials is associated with a number of environmental issues including high water and energy use and the extensive use of insecticides and fertilisers during farming. Additionally, the use of arable land for the production of plants for fabrics will become an increasingly contentious issue overthe coming decades, due to the anticipated shortage of food supply due to the projected increase in the global human population.

Non-plant derived materials prominent in the textile industry include polyester, nylon and acrylic, which use raw materials derived from the petrochemical industry. The use of such materials in textiles is increasingly unpopular as consumers seek to use environmentally friendly, sustainable and biodegradable materials.

After the production of the basic textile material, there are subsequent steps often employed that affect the properties of the material including: drying the material; colouring the material; imparting water resistance to the material through processes like plasticising; improving the resilience of the material; and altering the texture of the material. These subsequent steps further require input of time and energy, and may require the addition of petrochemical derived compounds, which all further increase the environmental impact associated with the production of the material. In particular, the dying of fabrics is notoriously chemically intensive and is also associated with pollution through generating waste-water.

Therefore, it is clear that there is a need fora biobased material that overcomes the many environmental and sustainability problems associated with materials traditionally used by the textile / fashion industry. There is also a need to provide a biobased material that addresses the challenges associated with using bacterial cellulose in the textile / fashion industry, for example due to its inherent hydrophilicity, paper like texture and natural beige colour.

An aim of the present invention is to provide an environmentally friendly, sustainable, biodegradable, customisable and versatile bacterial cellulose based material, having desirable properties imparted that enables its use in the textile / fashion industry. Further objectives may include low cost and simple production of customisable bacterial cellulose based materials.

These and other uses, features and advantages of the invention should be apparent to those skilled in the art from the teachings provided herein.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a process for preparing a biobased bacterial cellulose material. The process comprising the steps of: i) providing a first microorganism, or a symbiotic culture of bacteria and yeast (SCOBY), capable of producing bacterial cellulose; ii) providing culture conditions that enable the production of bacterial cellulose by the first microorganism or by the SCOBY, so as to form a first layer comprising bacterial cellulose, wherein the first layer defines a basal layer; iii) providing a second microorganism selected from the group consisting of: one or more fungi; one or more algae; one or more bacteria; and combinations thereof; iv) providing culture conditions that enable growth of the second microorganism so as to form a second layer, wherein the second layer is grown on a surface of the first layer; to thereby form the biobased bacterial cellulose material, wherein the material has first and second major exterior surfaces formed from the first and second layers respectively, wherein the first and second surfaces exhibit different property characteristics.

In an embodiment, the properties are chosen from the group consisting of: hydrophilic; coloured; hydrophobic; water repellent; waxy surface; oily surface; hairy surface; soft surface; smooth surface; rough surface; textured surface; insulated; heat resistant; fire resistant; mould resistant; and combinations thereof.

The first microorganism may typically comprise one or more bacterial species from the genus Komagataeibacter. Suitably, the one or more bacterial species may be selected from the group consisting of: K. rhaeticus ; K. xylinus ; K. hansenir, and combinations thereof.

In embodiments, the SCOBY comprises one or more bacterial species from the genus Komagataeibacter and one or more yeast species from the genera of Saccharomyces, Saccharomycodes, Schizosaccharomyces, Zygosaccharomyces, BrettanomycestDekkera, Candida, Torulaspora, Koleckera, Pichia, Mycotorula and Mycoderma.

Suitably, the first layer comprising bacterial cellulose may be in the form of a pellicle.

In an embodiment, the first layer comprising bacterial cellulose may be grown around a patterned mesh of fibres, such that the fibres are embedded within the bacterial cellulose.

In an embodiment, after step ii) of the process and before step iii) of the process the first layer comprising bacterial cellulose may be washed and optionally pH adjusted in one or more suitable buffer solutions and/or optimal media solutions.

In an embodiment, after step ii) of the process and before step iii) of the process the first layer comprising bacterial cellulose may be pre-conditioned for growth of the second microorganism by: submerging the first layer in humectants; coating the first layer with a starch; coating the first layer with a polysaccharide; and/or coating the first layer with a protein.

Suitably, the second microorganism may comprise one or more fungi selected from the group consisting of: Ganoderma lucidem, Ganoderma tsugae, Ganoderma oregonense, Ganoderma appalantum, Fomes fomentarius, Fusarium oxysporum, Pleurotus ostreatus, Polyporus squamosus, Polyporus avleolaris, Polypores mylittae, Trametes versicolor, Piptoporous betulinus, Inonotus obliquus, Agaricus bisporus, Corprinus cinereus, Rhizopus stolonifer, Penicillium sp., Cladosporium sp., Aspergillus sp., Saccharomyces cerevisiae, Yarrowia lipolytica, Pichia pastoris, Trichoderma reesei, Neurospora crassa, Schizophyllum commune, and combinations thereof.

Suitably, the second microorganism may comprise one or more algae selected from the group consisting of: Chlamydomonas reinhardtii, Porphyridium purpureum, Himanthalia elongate, Undaria pinnatifida, Laminaria ochroleuca, Porphyra spp., Haematococcus pluvialis, Dunaliella salina, Chlorella sps., Nannochloropsis sps., Scenedesmus sps., Chlorococcum sps., Botryococcus braunii, and combinations thereof.

Suitably, the second microorganism may comprise one or more bacteria selected from the group consisting of: Bacillus subtilis, Priestia megaterium, B. atrophaeus, B. indicus, B. cibi, B. vedderi, B. jeotgali, B. okuhidensis, B. clarkii, B. pseudofirmus, B. firmus, B. marisflavi, B. altitudinis, B. safensis, Nostoc sp., Synechocystis sp. PCC6803, Escherichia coli, Hahella chejuensis, Lactobacillus acidophilus, Lactococcus lactis, Pseudoalteromonas denitrificans, Pseudomonas magnesiorubra, Pseudomonas spp., Spirulina spp., Spirulina platensis, Streptomyces coelicolor, Streptomyces coelicolor A3(2), Streptomyces spp., Streptoverticillium sp, Serratia spp., Serratia marcescens, Vibrio psychroerythrus, and combinations thereof.

Suitably, in step iv) of the process a pre-culture of the second microorganism may be inoculated onto a surface of the first layer and allowed to colonise the surface.

In an embodiment, in step iv) of the process the second layer may be grown on an upper surface of the first layer.

Suitably, step iv) of the process may further comprise growing the second microorganism into the first layer so as to integrate the first and second layers.

Suitably, the process may further comprise step v) treating the biobased bacterial cellulose material to inhibit further growth of the second microorganism and/or to bind the first and second layers together.

Suitably, the treating may comprise autoclaving the material, heat pressing the material, treating the material with alcohol, bleach (such as hypochlorite or a peroxide), or a caustic substance (such as sodium hydroxide ammonium hydroxide or potassium hydroxide), or combinations thereof.

In a second aspect, the present invention provides a biobased bacterial cellulose material prepared according to the method described above.

In a third aspect, the present invention provides a biobased composite bacterial cellulose material comprising: a) a first layer comprising bacterial cellulose; and b) a second layer comprising an organic material grown from a microorganism selected from the group consisting of: one or more fungi; one or more algae; one or more bacteria; and combinations thereof.

The bacterial cellulose may be from a bacterial species from the genus Komagataeibacter. Suitably, the bacterial species selected from the group consisting of: K. rhaeticus; K. xylinus; K. hansenir, and combinations thereof.

In an embodiment, the first layer may further comprise a patterned mesh of fibres, such that the fibres are embedded within the bacterial cellulose.

Suitably, the one or more fungi may be selected from the group consisting of: Ganoderma lucidem, Ganoderma tsugae, Ganoderma oregonense, Ganoderma appalantum, Fomes fomentarius, Fusarium oxysporum, Pleurotus ostreatus, Polyporus squamosus, Polyporus avleolaris, Polypores mylittae, Trametes versicolor, Piptoporous betulinus, Inonotus obliquus, Agaricus bisporus, Corprinus cinereus, Rhizopus stolonifer, Penicillium sp., Cladosporium sp., Aspergillus sp., Saccharomyces cerevisiae, Yarrowia lipolytica, Pichia pastoris, Trichoderma reesei, Neurospora crassa, Schizophyllum commune, and combinations thereof.

Suitably, the one or more algae may be selected from the group consisting of: Chlamydomonas reinhardtii, Porphyridium purpureum, Himanthalia elongate, Undaria pinnatifida, Laminaria ochroleuca, Porphyra spp., Haematococcus pluvialis, Dunaliella salina, Chlorella sps., Nannochloropsis sps., Scenedesmus sps., Chlorococcum sps., Botryococcus braunii, and combinations thereof.

Suitably, the one or more bacteria may be selected from the group consisting of: Bacillus subtilis, Priestia megaterium, B. atrophaeus, B. indicus, B. cibi, B. vedderi, B. jeotgali, B. okuhidensis, B. clarkii, B. pseudofirmus, B. firmus, B. marisflavi, B. altitudinis, B. safensis, Nostoc sp., Synechocystis sp. PCC6803, Escherichia coli, Hahella chejuensis, Lactobacillus acidophilus, Lactococcus lactis, Pseudoalteromonas denitrificans, Pseudomonas magnesiorubra, Pseudomonas spp., Spirulina spp., Spirulina platensis, Streptomyces coelicolor, Streptomyces coelicolor A3(2), Streptomyces spp., Streptoverticillium sp, Serratia spp., Serratia marcescens, Vibrio psychroerythrus, and combinations thereof.

Suitably, the second layer may penetrate into the first layer to integrate the layers together, thereby forming an integrated composite material.

In a fourth aspect, the present invention provides an article of clothing, an article of footwear, a wearable accessory or a fabric for home, automotive or aerospace upholstery or furnishings comprising the biobased bacterial cellulose material described above, wherein: a) the first layer forms the inner layer of the article of clothing, the article of footwear the wearable accessory or the fabric; and b) the second layer forms the outer layer of the article of clothing, the article of footwear, the wearable accessory or the fabric. In a fifth aspect, the present invention provides an article of clothing, an article of footwear, a wearable accessory or a fabric for home, automotive or aerospace upholstery or furnishings comprising the biobased bacterial cellulose material described above, wherein: a) the first layer forms the outer layer of the article of clothing, the article of footwear the wearable accessory or the fabric; and b) the second layer forms the inner layer of the article of clothing, the article of footwear the wearable accessory or the fabric.

In a sixth aspect, the present invention provides a process for manufacture of a composite non-woven textile material, wherein the composite non-woven textile material comprises at least one layer of bacterial cellulose, the process comprising the steps of: i) providing a first microbial culture capable of producing bacterial cellulose, and culturing the first microbial culture under conditions that facilitate formation of a basal layer comprised of a bacterial cellulose; and ii) providing a second microbial culture, and culturing the second microbial culture under conditions that enable growth of the second microbial culture on the basal layer; wherein the second microbial culture is comprised of a microorganism that deposits a second layer upon the basal layer, and wherein the second layer imparts a change in the physical and/or aesthetic properties of the basal layer.

In an embodiment, the first microbial culture may be comprised of a single microorganism.

In an embodiment, the first microbial culture may comprise one or more bacterial species from the genus Komagataeibacter.

Suitably, the one or more bacterial species may be selected from the group consisting of: K. rhaeticus; K. xylinus; K. hansenii ; and combinations thereof.

In an embodiment, the first microbial culture may be comprised of a symbiotic culture of bacteria and yeast (SCOBY).

Suitably, the SCOBY may comprise one or more bacterial species from the genus Komagataeibacter and one or more yeast species from the genera of Saccharomyces, Saccharomycodes, Schizosaccharomyces, Zygosaccharomyces, Brettanomyces/Dekkera, Candida, Torulaspora, Koleckera, Pichia, Mycotorula and Mycoderma.

In an embodiment, the basal layer may be in the form of a pellicle.

In an embodiment, the first microbial culture may be cultured in the presence of a mesh of fibres. Suitably, the mesh of fibres may comprise one or more fibres selected from the group consisting of: nylon; rayon; wool; silk; and cotton.

In an embodiment, the basal layer may be washed prior to step (ii) of the process.

In an embodiment, the basal layer may be pH adjusted in one or more suitable buffer solutions and/or optimal media solutions prior to step (ii) of the process.

In an embodiment, the basal layer may be pre-conditioned for growth of the second microbial culture, wherein the pre-conditioning comprises one or more of:

(a) treating the basal layer with a humectant;

(b) coating the basal layer with a starch;

(c) coating the basal layer with a polysaccharide;

(d) coating the basal layer with a protein.

In an embodiment, the second microbial culture may comprise one or more fungi selected from the group consisting of: Ganoderma lucidem; Ganoderma tsugae; Ganoderma oregonense; Ganoderma appalantum; Fomes fomentarius; Fusarium oxysporum; Pleurotus ostreatus; Polyporus squamosus; Polyporus avleolaris; Polypores mylittae; Trametes versicolor; Piptoporous betulinus; Inonotus obliquus; Agaricus bisporus; Corprinus cinereus; Rhizopus tolonifera; Penicillium sp.; Cladosporium sp.; Aspergillus sp.; Saccharomyces cerevisiae; Yarrowia lipolytica; Pichia pastoris; Trichoderma reesei; Neurospora crassa; Schizophyllum commune·, and combinations thereof.

In an embodiment, the second microbial culture may comprise one or more algae selected from the group consisting of: Chlamydomonas reinhardtii; Porphyridium purpureum; Himanthalia elongate; Undaria pinnatifida; Laminaria ochroleuca; Porphyra spp.; Haematococcus pluvialis; Dunaliella salina; Chlorella sps.; Nannochloropsis sps.; Scenedesmus sps.; Chlorococcum sps.; Botryococcus braunir, and combinations thereof.

In an embodiment, the second microbial culture may comprise one or more bacteria selected from the group consisting of: Bacillus subtilis; Priestia megaterium; B. atrophaeus; B. indicus; B. cibi; B. vedderi; B.jeotgali; B. okuhidensis; B. clarkii; B. pseudofirmus; B. firmus; B. marisflavi; B. altitudinis; B. safensis; Nostoc sp.; Synechocystis sp. PCC6803; Escherichia coli; Hahella chejuensis; Lactobacillus acidophilus; Lactococcus lactis; Pseudoalteromonas denitrificans; Pseudomonas magnesiorubra; Pseudomonas spp.; Spirulina spp.; Spirulina platensis; Streptomyces coelicolor; Streptomyces coelicolor A3(2); Streptomyces spp.; Streptoverticillium sp.; Serratia spp.; Serratia marcescens; Vibrio psychroerythrus and combinations thereof.

In an embodiment, the second microbial culture penetrates into the basal layer so as to integrate the first and second layers. In an embodiment, the process may further comprise a further step of treating the composite non-woven textile material to inhibit further growth of the second microbial culture.

In an embodiment, the process may further comprise a further step of treating the composite non-woven textile material to facilitate binding of the second layer to the basal layer.

Suitably, the further step may be selected from one or more of the group consisting of: autoclaving the material; heat pressing the material; treating the material with alcohol; treating the material with bleach; treating the material with a caustic substance.

In an embodiment, the change in the physical and/or aesthetic properties of the basal layer may be selected from one or more of the group consisting of: hydrophilicity; colour; hydrophobicity; water repellence; a waxy surface; an oily surface; a hairy surface; a softer surface; a smoother surface; a rougher surface; a textured surface; thermal or electrical insulation; heat resistance; fire resistance; and mould resistance.

Suitably, the composite non-woven textile material may be more hydrophobic than the basal layer alone.

In a seventh aspect, a composite non-woven textile material obtainable via the methods described above.

BRIEF DESCRIPTION OF THE DRAWINGS

The disclosure is further illustrated by reference to the accompanying drawings in which:

Figure 1 A shows a photograph of a bacterial cellulose pellicle with a layer of Aspergillus spp., grown on its upper surface prior to treating with heat and alcohol; Figure 1B shows a photograph of the same pellicle with a layer of Aspergillus spp., grown on its upper surface after treating with heat and alcohol.

DETAILED DESCRIPTION OF THE INVENTION

All references cited herein are incorporated by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

In order to assist with the understanding of the invention several terms are defined herein.

As used herein, the term ‘comprising’ means any of the recited elements are necessarily included and other elements may optionally be included as well. ‘Consisting essentially of means any recited elements are necessarily included, elements that would materially affect the basic and novel characteristics of the listed elements are excluded, and other elements may optionally be included. ‘Consisting of means that all elements other than those listed are excluded. Embodiments defined by each of these terms are within the scope of this invention. The terms ‘bacterial cellulose’, ‘microbial cellulose’, ‘nanocellulose’, ‘bacterial nanocellulose’, ‘bacterially produced cellulose’ and ‘bacterially produced nanocellulose’ as used herein are equivalent and refer to cellulose produced by bacteria, such as species from the Komagataeibacter genus (previously known as Gluconacetobacter or Acetobacter) and others, that is characterised by high tensile strength, high tensile stiffness, high chemical purity, biocompatibility and high water-to-cellulose ratio. Suitably, such bacterial nanocellulose will be substantially pure of associated molecules typically present in plant-derived cellulose such as lignin.

The term ‘pellicle’ as used herein refers to a microbial growth produced at an air-liquid interface, typically bacterially produced nanocellulose, that has not been pulverized or otherwise chemically or physically degraded to yield reconstituted cellulose, and that thus retains its high tensile stiffness, high tensile strength, high water-to-cellulose ratio, and other properties characteristic of unmodified bacterially produced nanocellulose. A pellicle of bacterially produced nanocellulose according to the present invention provides a base layer, which can also be referred to as a basal layer, a first layer or a first substrate layer, on which a second layer of microorganisms can be grown. The pellicle may comprise a pellet, a disk or a sheet of bacterially produced nanocellulose.

The terms ‘biobased bacterial cellulose material’, ‘biobased material’, ‘biobased composite’, ‘biobased composite material’, ‘biofabricated material’, ‘biofabric’, ‘bacterial cellulose based biocomposite’, ‘biocomposite material’, ‘bacterial cellulose based biocomposite material’, ‘bacterial cellulose based material’, as used herein are equivalent and refer to biodegradable and sustainable materials, non- woven textiles, fabrics, products, articles and structures as described herein comprising bacterial cellulose, which are made from substances derived from living or once-living organisms. Suitably, the biobased bacterial cellulose materials are in the form of a layered structure, wherein one of the layers comprises bacterial cellulose. The layers have individually different physical or chemical properties that, when combined, produce a material with characteristics different from the individual components. Suitably the layer comprising bacterial cellulose is referred to as a ‘basal layer’.

The term ‘microorganism’ as used herein refers to small unicellular or multicellular living organisms that are too small to be seen with the naked eye but are visible under a microscope, and encompasses bacteria, fungi, and algae. The microorganism may be a wild-type microorganism or a genetically modified microorganism. Microorganisms are grown in microbial culture using techniques known to the skilled person.

The present invention relates to a process for preparing a biobased bacterial cellulose material.

In an embodiment of a first stage of the process, a microorganism or combination of microorganisms, also referred to herein as a first microorganism, e.g. a bacterium, is provided that is capable of producing bacterial cellulose.

Suitably, the microorganism capable of producing bacterial cellulose may be one or more bacterial species from the genus Komagataeibacter. Suitably, the bacterial species from the genus Komagataeibacter may be K. rhaeticus, K. xylinus, or K. hansenii. Single species of these bacteria can be used to produce the bacterial cellulose, e.g. pure K. rhaeticus, or combinations of these bacterial species can be used to produce the bacterial cellulose. Genetically modified versions of these bacterial species or strains may also be used. A mixture of wild-type and genetically modified bacteria may also be used.

The microorganism is then grown under culture conditions that enable the production of bacterial cellulose. Any suitable culture conditions known in the art may be used to grow the microorganism and produce the bacterial cellulose. The culturing conditions may be selected in view of the chosen microorganism in order to provide the chosen microorganism with optimal growing conditions. lOtolonifeBacterial cellulose production depends heavily on several factors such as the species or strains of bacteria used, the growth medium, pH, temperature, and oxygen. Growth media typically contain carbon, nitrogen and other macro and micro nutrients that are required for bacteria growth. Bacteria are most efficient when supplied with an abundant carbon source. Optimal temperatures for bacterial cellulose production tend to be in the range of from 28°C to 30°C, and the optimal pH for most species tends to be in the range of from 4.0 to 6.0.

For example, culture media suitable for growing the bacterial cellulose can comprise 5-50g/L of one or more carbon sources (e.g. stachyose, raffinose, glucose, sucrose, fructose, mannitol, galactose, maltose), 5 g/L yeast extract and 5 g/L tryptone. Alternatively, any small sugar know in the art may be used. Alternatively, a suitable exemplary culture media may be prepared using a solution of pure coconut water with 1% cider vinegar. Biowaste from a biorefinery, brewing by-products or fruit waste can be used as a carbon source.

In another embodiment of a first stage of the process, provided is bacteria capable of producing bacterial cellulose in a co-culture with yeast, i.e. a symbiotic culture of bacteria and yeast, also known as a SCOBY. Suitably, one or more bacterial species from the genus Komagataeibacter, such as K. rhaeticus, K. xylinus, or K. hansenii, can be used in the co-culture in combination with one or more yeast species from the genera of Saccharomyces, Saccharomycodes, Schizosaccharomyces, Zygosaccharomyces, Brettanomyces/Dekkera, Candida, Torulaspora, Koleckera, Pichia, Mycotorula and Mycoderma. Suitably, yeast species from the genera Saccharomyces, such as S. cerevisiae, or Zygosaccharomyces, such as Z. kombuchaensis, can be used. Genetically modified versions of the yeast species or strains may also be used. A mixture of wild-type and genetically modified bacteria and/or yeast may also be used.

Any suitable culture conditions known in the art may be used for the bacterial-yeast co-culture to produce the bacterial cellulose. The culturing conditions may be selected in view of the chosen microorganisms in order to provide the chosen microorganisms with optimal growing conditions. The culture conditions may be as described above in relation to the first microorganism.

The bacteria secrete polymerised cellulose microfibrils and these finally form a continuous highly interconnected lattice-like matrix of fibrils in the form of a dense mat. In liquid static cultures, the mat is otherwise known as a pellicle, also referred to herein as a layer or first layer of bacterial cellulose or a layer or first layer comprising bacterial cellulose. In liquid static cultures, cellulose formation occurs at the air-liquid interface and thus the pellicle forms on the upper surface of the static liquid media or broth used to culture the bacteria.

The layer of bacterial cellulose may be produced in a static culture producing a pellicle, as described above. Alternatively the bacterial cellulose producing organism may be cultured under agitated culture conditions with an air lift or stirred tank bioreactor. In an air lift reactor, air is introduced to the bottom of a column reactor to provide oxygen and mixing to the culture. In a stirred tank bioreactor air is also introduced to provide oxygen but agitation is provided by an overhead stirrer. In both of these agitated cultures, the air is bubbled through the liquid media, which increases oxygen availability and may further increase the rate of bacterial cellulose production. The bacterial cellulose in agitated cultures is produced as a viscous suspension in the liquid media, rather than as a continuous pellicle. After culturing, the bacterial cellulose product is removed and separated from spent growth media by filtration. The bacterial cellulose product may subsequently be cast into a layer of bacterial cellulose.

The microorganisms/bacteria used to produce the bacterial cellulose and the culturing conditions and/or casting process can be selected in orderto obtain the desired thickness of the bacterial cellulose matrix, layer or pellicle. Typically, the pellicle is grown or layer is cast to a thickness in the range of from 0.2 cm to 5 cm, suitably to a thickness in the range of from 1 cm to 2 cm, before being harvested.

The layer of bacterial cellulose may be in the form of a pure bacterial cellulose pellicle, as described above. Alternatively, the bacterial cellulose may be grown or cast around a patterned mesh of fibres, e.g. hydrophilic fibres such as nylon, rayon, wool, silk, or cotton, such that the fibres are embedded within the bacterial cellulose, as described in WO 2019/136036 A1 , thus forming a layer or first layer comprising bacterial cellulose and interwoven fibres. Suitably, the fibres are embedded within the bacterial cellulose layer, resulting in total encapsulation and embedment of the fibres. The patterned mesh of fibres may be woven, knitted or embroidered. The incorporation of prefabricated fibres during growth of the bacterial cellulose or during casting provides a hybrid composite material with improved tensile properties compared with pure bacterial cellulose or the fibres alone. Suitably, the fibres are hydrophilic and can include natural cellulose-based fibres (such as sisal or cotton), natural protein- based fibres (such as silk or wool) or other non-synthetic, or biobased synthetic fibres (such as rayon, viscose or lyocell), so as to provide an end product bacterial cellulose based material that is biobased, biodegradable and sustainable.

Once the layer or pellicle of bacterial cellulose has been produced, it may be removed from the liquid culture media (if present) and acclimatised or primed, e.g. washed and/or pH adjusted, for the next stage of the process. Depending on the nature of the next stage of the process, the pellicle or layer may either be washed with water, acclimatised and optionally pH adjusted in a buffer and/or optimal media solution, or it may be disinfected (suitably by alcohol, autoclaving, immersion in sodium hydroxide, bleach, methanol, isopropyl alcohol solutions, or combinations thereof) before being acclimatised and optionally pH adjusted in a buffer and/or optimal media solution. Suitably, disinfection options include treatment by autoclaving, alcohol, bleach, methanol, isopropyl alcohol solutions, or combinations thereof. Optimal media solutions will vary depending on the nature of the next stage of the process, but may include Yeast Malt Agar Medium, Saboraud’s Dextrose Agar Medium, Glucose Peptone Agar Medium, Malt Extract Agar Medium, Starch Casein Agar Medium, Potato Dextrose Agar Medium, Potato Starch Solution, unheated Starch Solution, Potato Dextrose Broth, Lysogeny Broth, yeast dextrose broth, Bristol’s Solution, Cyanophycean Agar, Erdschreiber Solution, Euglena Medium, Ochromonas Medium, Waris Solution, Bold’s Basal Medium and/or TRIS acetate phosphate. In combination with or as an alternative to the optimal media solution, the pellicle or layer may be coated with or soaked in a humectant. Humectants that may be used include sugar alcohols, such as glycerol, sorbitol, maltitol, alpha hydroxy acids or polyethylene glycols (PEG), e.g. PEG 200, PEG 400, PEG 600, PEG 1000, PEG 1500, PEG 2000, or PEG 3400. Starches, gums, polysaccharides and/or proteins may also be applied to the pellicle or layer alone or in combination with optimal media solutions and/or humectants. At least one surface of the bacterial cellulose layer may be coated with the optimal media solution, humectant, starch, gum, polysaccharide, protein, or combinations thereof. Suitably all surfaces may be coated with the optimal media solution, humectant, starch, gum, polysaccharide, protein, or combinations thereof. Suitably, the bacterial cellulose layer may be submerged in the optimal media solution, humectant, starch, gum, polysaccharide, protein, or combinations thereof. Depending on the pH preferences of the microorganisms in the next stage of the process, the bacterial cellulose layer or pellicle may be placed in buffers, e.g. PBS, at the optimal pH. The optimal pH can vary from pH 3-8, with a general preference for a pH in the range of pH 7-8. Any additional supplements that may be required to assist with the next stage of the process may be added to the optimal media solution. pH adjustment in buffers may suitably be concurrent with acclimatisation in optimal media solutions.

After the optional acclimatisation/priming step, it is possible for the layer or pellicle of bacterial cellulose to be dried before commencing the next stage of the process. However, it is preferred for the layer or pellicle of bacterial cellulose to remain in its hydrated form. The highly hydrophilic nature of the bacterial cellulose hydrogel means that it will naturally retain the liquid in which it has been grown and/or in which it has optionally been acclimatised.

The sheet or layer of bacterial cellulose, or pellicle, acts as a basal layer or first substrate layer on which other microorganisms can be grown. The layer of bacterial cellulose can be placed in any suitable receptacle ready for the next stage of the process.

In a next stage of the process, a microorganism or combination of microorganisms, also referred to herein as a second microorganism, is provided comprising one or more fungi, and/or one or more algae, and/or one or more bacteria.

The fungi may comprise single cell/unicellular species (e.g. yeasts) and/or multicellular fungal species (e.g. moulds). Suitably, the fungi may comprise one or more of Ganoderma lucidem, Ganoderma tsugae, Ganoderma oregonense, Ganoderma appalantum, Fomes fomentarius, Fusarium oxysporum, Pleurotus ostreatus, Polyporus squamosus, Polyporus avleolaris, Polypores mylittae, Trametes versicolor, Piptoporous betulinus, Inonotus obliquus, Agaricus bisporus, Corprinus cinereus, Rhizopus stolonifer, Penicillium sp., Cladosporium sp., Aspergillus sp., Saccharomyces cerevisiae, Yarrowia lipolytica, Pichia pastoris, Trichoderma reesei, Neurospora crassa, or Schizophyllum commune. Suitably, the fungi may comprise one or more of Ganoderma lucidem, Ganoderma tsugae, Ganoderma oregonense, Ganoderma appalantum, Pleurotus ostreatus, Agaricus bisporus, Penicillium sp., Aspergillus sp., Saccharomyces cerevisiae, Yarrowia lipolytica, Trichoderma reesei, Neurospora crassa, or Schizophyllum commune. Genetically modified versions of these species or strains may also be used. A mixture of wild-type and genetically modified fungi may also be used.

Suitably, the algae may comprise one or more of Chlamydomonas reinhardtii, Porphyridium purpureum, Himanthalia elongate, Undaria pinnatifida, Laminaria ochroleuca; Porphyra spp., Haematococcus pluvialis, Dunaliella salina, Chlorella sps., Nannochloropsis sps., Scenedesmus sps., Chlorococcum sps., or Botryococcus braunii. Suitably, the algae may comprise one or more of Porphyridium purpureum, Dunaliella salina, Chlorella sps., Nannochloropsis sps. or Botryococcus braunii. Genetically modified versions of these species or strains may also be used. A mixture of wild-type and genetically modified algae may also be used.

Suitably, the bacteria may comprise one or more of Bacillus subtilis, Priestia megaterium, B. atrophaeus, B. indicus, B. cibi, B. vedderi, B. jeotgali, B. okuhidensis, B. clarkii, B. pseudofirmus, B. firmus, B. marisflavi, , B. altitudinis, B. safensis, Nostoc sp., Synechocystis sp. PCC6803, Escherichia coli, Hahella chejuensis, Lactobacillus acidophilus, Lactococcus lactis, Pseudoalteromonas denitrificans, Pseudomonas magnesiorubra, Pseudomonas spp., Spirulina spp., Spirulina platensis, Streptomyces coelicolor, Streptomyces coelicolor A3(2), Streptomyces spp., Streptoverticillium sp, Serratia spp., Serratia marcescens, or Vibrio psychroerythrus. Suitably, the bacteria may comprise one or more of Bacillus subtilis, Priestia megaterium, Escherichia coli, Hahella chejuensis, Lactobacillus acidophilus, Lactococcus lactis, Pseudomonas magnesiorubra, Pseudomonas spp., Streptomyces coelicolor, Streptomyces coelicolor A3(2), Streptomyces spp., Streptoverticillium sp, Spirulina spp. or Spirulina platensis. Genetically modified versions of these species or strains may also be used. A mixture of wild-type and genetically modified bacteria may also be used. The bacteria used to grow the second layer is different to the bacteria used to grow the first layer comprising bacterial cellulose.

Culture conditions are then provided that enable growth of the fungi and/or algae and/or bacteria to form a layer of organic material, also referred to herein as a second layer or second substrate layer. The layer of grown organic material is grown on a surface of the basal layer of bacterial cellulose, i.e. the layer of grown organic material is grown in situ on a surface of the basal layer of bacterial cellulose.

Any suitable culture conditions known in the art may be used to grow the microorganism(s) and produce the second layer of organic material. The culturing conditions may be selected in view of the chosen microorganism or combination of microorganisms in order to provide the chosen microorganism(s) with optimal growing conditions. The optimal levels of temperature, light, humidity, oxygen, carbon dioxide and pH can be chosen accordingly depending on the microorganism(s) used.

In the case of multicellular fungi (e.g. multicellular filamentous fungi), the layer of organic material may comprise mycelium. Multicellular fungi are made up of very fine threads, hyphae, that grow and divide repeatedly and intertwine until they form a network of threads known as mycelium. In the case of unicellular fungi (e.g. yeast), some bacteria (e.g. Streptomyces) and algae cells, morphology resembles chains or pseudohyphae and these filamentous structures can grow into a network. Fungi (e.g. yeast) and most bacteria species (e.g. Bacillus) are unicellular and will form a dense lawn ora biofilm. Whether the morphology of the layer of organic material resembles unicellular lawns or more complex multicellular structures, this second layer of growth will contain cells that have unique membrane compositions (e.g. phospholipids, lipids, fatty acids, chitin, cellulose), and/or produce unique polysaccharides (agarose, alginate, cellulose, pectin, xylan, dextran, lectin), and/or produce pigments (prodigiosin, melanin, actinorhodin, carotenoids) and/or proteins (tyrosinases, hydrophobins, amyloids) that, compared to the basal layer of bacterial cellulose, create a second layer of various possible colours, textures including smooth, rough, hairy and soft surface textures, hydrophilic/hydrophobic characteristics as a result of, for example, oils and waxes, thicknesses, depending on the strain, growth factors and/or conditions used in the process, and material properties, such as14tolonifey properties, heat resistance, fire resistance and mould resistance. Suitably, the second layer of growth will have a different combination of property characteristics compared to the first layer or pellicle. The major first and second exterior surfaces of the first and second layers of the biobased bacterial cellulose material exhibit at least a different combination of property characteristics, and suitably a different combination of properties.

To enable the microorganism or combination of microorganisms to be grown on a surface of the basal layer of bacterial cellulose, a microorganism inoculation(s), i.e. a fungal and/or an algal and/or a bacterial inoculation is allowed to colonise a surface of the basal layer of bacterial cellulose and to grow so as to form a layer of organic material that covers or substantially covers the surface of the basal layer. Single strains or multiple strains of microorganisms may be grown on the basal layer or pellicle, e.g. combinations of multiple fungal, and/or algal and/or bacterial strains may be grown.

With regards to fungal (multicellular or unicellular) strains and preparations, liquid fungal pre-cultures of spores may be inoculated onto a surface of the pellicle or basal layer of bacterial cellulose. Alternatively, fungal pre-cultures may be grown to the mycelial stage and inoculated onto a surface of the pellicle or basal layer of bacterial cellulose. To assist with mycelium growth on the surface of the pellicle or basal layer of bacterial cellulose, secondary organic materials such as wood chips, organic grass fibres or coffee grounds may be scattered onto the surface of the pellicle or basal layer of bacterial cellulose. Alternatively, fungal pre-cultures may be grown to the primordial stage and inoculated onto a surface of the pellicle or basal layer of bacterial cellulose. Unicellular fungal pre-cultures may be prepared as single colonies on solid agar plates supplemented with appropriate media and then inoculated onto a surface of the pellicle or basal layer of bacterial cellulose.

With regards to bacterial strains and preparations, bacterial pre-cultures may be prepared as single colonies on solid agar plates supplemented with appropriate media and then inoculated onto a surface of the pellicle or basal layer of bacterial cellulose. Alternatively, bacterial pre-cultures may be prepared as liquid cultures grown to a dense OD600 (approximately OD600=1) and then inoculated onto a surface of the pellicle or basal layer of bacterial cellulose. Alternatively, bacterial pre-cultures may be prepared as spores which are then inoculated onto a surface of the pellicle or basal layer of bacterial cellulose.

With regards to algal strains and preparations, algal pre-cultures may be prepared by any suitable method known in the art and then inoculated onto a surface of the pellicle or basal layer of bacterial cellulose.

The microorganisms that grow to form the layer of organic material may be grown on an upper surface of the pellicle or basal layer of bacterial cellulose, namely the surface of the pellicle nearest or at the air interface, or a lower surface of the pellicle or basal layer of bacterial cellulose, namely the surface of the pellicle furthest from the air interface (e.g. the surface of the pellicle adjacent to the bottom of the receptable in which the pellicle is contained). For example, anaerobic organisms may suitably be grown on a lower surface of the pellicle or basal layer of bacterial cellulose. However, most bacteria and fungi, due to their oxygen requirements, would benefit from being grown on an upper surface of the pellicle or basal layer of bacterial cellulose. The upper surface of the pellicle or basal layer of bacterial cellulose may be otherwise be referred to as the top of the pellicle/layer of bacterial cellulose or the top surface of the pellicle/layer of bacterial cellulose, and the lower surface of the pellicle or basal layer of bacterial cellulose may otherwise be referred to as the bottom of the pellicle/layer of bacterial cellulose or the bottom surface of the pellicle/layer of bacterial cellulose. Suitably, the microorganisms that form the layer of organic material are grown on an upper surface of the pellicle or basal layer of bacterial cellulose, namely the surface of the pellicle nearest or at the air interface.

The inoculations of fungi and/or algae and/or bacteria may also be allowed to grow into the pellicle or basal layer of bacterial cellulose, to thereby integrate the layer of organic material produced by the microorganisms with the layer of bacterial cellulose. For example, mycelium may be allowed to grow into the basal layer of bacterial cellulose to integrate the layers together. However, growth of the mycelium into the basal layer will suitably be of a limited extent such that the final material will still be predominantly cellulose based. In this embodiment, the major first and second exterior surfaces of the first and second layers of the biobased bacterial cellulose material exhibit at least a different combination of property characteristics, and suitably a different combination of properties.

In an embodiment, the pellicle or basal layer of bacterial cellulose is placed in a liquid pre-culture containing fungal and/or algal and/or bacterial cultures and these cultures are allowed to colonise the pellicle/layer of bacterial cellulose. Shaking cultures may be used to allow for oxygenation when colonising with aerobic organisms. The fungi and/or algae and/or bacteria can then be allowed to additionally grow into the pellicle or layer of bacterial cellulose, as described above. In this embodiment, growth of the second microorganism may occur on all surfaces of the basal layer of bacterial cellulose or pellicle, although the resultant product will still be in the form of a sheet of material.

In an embodiment, instead of the second layer of organic material being grown on a surface of the basal layer of bacterial cellulose, i.e. grown in situ, the second layer of organic material can be grown in isolation from / separately from the basal layer of bacterial cellulose, and the second layer, once grown, may be applied to or placed on a surface of the basal layer of bacterial cellulose without allowing for any additional growth of the second microorganism into the pellicle or basal layer of bacterial cellulose. Alternatively, once the second layer of organic material has been grown and applied to a surface of the basal layer of bacterial cellulose, the second microorganism may be allowed to additionally grow into the pellicle or basal layer of bacterial cellulose, to thereby integrate the layer of organic material produced by the microorganisms with the layer of bacterial cellulose.

The layer of organic material grown from / originating from the microorganism(s) may be applied to or placed on an upper surface of the pellicle or basal layer of bacterial cellulose, namely the surface of the pellicle nearest or at the air interface, or a lower surface of the pellicle or basal layer of bacterial cellulose, namely the surface of the pellicle furthest from the air interface (e.g. the surface of the pellicle adjacent to the bottom of the receptable in which the pellicle is contained). Suitably, the layer of organic material grown from the microorgan ism(s) is applied to or placed on an upper surface of the pellicle or basal layer of bacterial cellulose, namely the surface of the pellicle nearest or at the air interface.

Where one or more fungi are used to grow the second layer, and to assist with mycelium growth, the second microorganism layer grown in isolation from the layer of bacterial cellulose may be grown on one or more secondary organic materials, such as wood chips, organic grass fibres or coffee grounds, to assist with growth ofthe microorganism, priorto applying the layerto a surface of the layer of bacterial cellulose. Once grown, and as described above, this microorganism layer may then be applied to or placed on a surface of the basal layer of bacterial cellulose without allowing for any additional growth of the second microorganism into the pellicle or basal layer of bacterial cellulose, or it may be applied to a surface of the basal layer of bacterial cellulose and the second microorganism may be allowed to additionally grow into the pellicle or basal layer of bacterial cellulose to thereby integrate the layer of organic material produced by the microorganisms with the layer of bacterial cellulose.

Once the second microorganism, and thereby the second layer, has reached an optimal or suitable growth end point (e.g. the second layer has reached an optimal colour, an optimal density, or optimal coverage of the bacterial cellulose base layer), growth of the second microorganism can be halted/terminated. In the scenario where the second layer is grown in isolation from the bacterial cellulose layer, growth ofthe second layer is suitably terminated afterthe second layer has been applied to a surface of the bacterial cellulose layer, and suitably after optional growth of the second microorganism into the bacterial cellulose layer has occurred. Termination of growth can be achieved by various methods including, treating the layered bacterial cellulose based material (i.e. the layered biobased bacterial cellulose material) with alcohol or bleach or NaOH, autoclaving the layered bacterial cellulose based material, heat pressing the layered bacterial cellulose based material, or combinations thereof. Suitably, the termination of growth is achieved by treating the layered bacterial cellulose based material with alcohol, bleach, autoclaving the bacterial cellulose based material, heat pressing the bacterial cellulose based material, or combinations thereof. The skilled person in the art will be familiar with suitable autoclaving and heat pressing techniques for this application. These treatments act to terminate biological activity of the layered bacterial cellulose based material and help to bind or bond the two layers, i.e. the bacterial cellulose base layer and the second layer of organic material, together permanently. Heat pressing is especially preferable in embodiments where the second layer comprises mycelium. The layered bacterial cellulose based material may be soaked in humectant prior to or after the treatment with an alcohol, a bleach, a caustic substance such as NaOH, autoclaving or heat pressing. Humectants that may be used include sugar alcohols, such as glycerol, sorbitol, maltitol, alpha hydroxy acids or polyethylene glycols (PEG), e.g. PEG 200, PEG 400, PEG 600, PEG 1000, PEG 1500, PEG 2000, or PEG 3400.

If required, the layered bacterial cellulose based material can be further dried. Drying may include pressing the layered material at a pressure of from 10 psi to 2000 psi, suitably 350 psi to 500 psi, and at a temperature of from 20°C to 550°C until dry. Alternatively, drying may include leaving the layered material to dry out at room temperature. Alternatively, drying may include using a dehumidifier at a temperature in the range of from 20°C to 80 °C.

The steps of the process outlined above suitably occur sequentially, such that the basal layer of bacterial cellulose is formed / produced prior to the second layer being grown on the basal layer or the second layer being applied to the basal layer. However, in the instance where the second layer is grown in isolation from the first layer, it is possible for the second layer to be grown concurrently with yet separately from the first layer, so long as the first layer is formed by the time the second layer is applied to it.

It will be appreciated that the order of growth of the first layer followed by growth of the17tolonnd layer is of importance to the method of the invention. The Komagataeibacter species ideally used to produce the first layer of bacterial cellulose are highly aerobic and grow best at an air interface - they would not grow so well downwardly into a layer of already grown second microorganism where the oxygen content would be lower. It is also beneficial to ensure the bacterial cellulose base layer is solid and of good quality before growing a second microorganism on it.

The resultant product is a bacterial cellulose based biocomposite material / biobased bacterial cellulose material, in the form of a layered structure, e.g. a sheet-like structure, comprising a layer or base layer of bacterial cellulose and a layer or upper layer of grown microorganism, e.g. organic material produced by or grown from one or more fungi, and/or one or more algae, and/or one or more bacteria.

The present invention also relates to the biobased bacterial cellulose materials themselves.

The biobased bacterial cellulose material as described herein advantageously has properties that differ from that of its individual components. Bacterial cellulose is strong, biodegradable and can be produced by bacteria that are capable of growing on food waste, however it is also innately light or beige in colour, hydrophilic, and when dried can be hard and brittle (if it remains untreated), which can limit its use, particularly in the fashion industry. For example, bacterial cellulose requires additional chemical treatments to make it water resistant. By combining bacterial cellulose with one or more fungi, and/or one or more algae, and/or one or more bacteria, grown on or placed on the basal layer of bacterial cellulose, it is possible to add advantageous properties of these materials to that of the basal layer of bacterial cellulose. The properties are chosen from the group consisting of: hydrophilic; coloured; hydrophobic; water repellent; waxy surface; oily surface; hairy surface; soft surface; smooth surface; rough surface; textured surface; insulated; heat resistant; fire resistant; mould resistant; and combinations thereof. Suitably, the properties are chosen from the group consisting of: hydrophilic; hydrophobic; water repellent; waxy surface; oily surface; hairy surface; soft surface; smooth surface; rough surface; textured surface; insulated; heat resistant; fire resistant; mould resistant; and combinations thereof.

For example, grown fungi (including yeast and mycelium), algae and bacteria, may innately have water repellent properties (i.e. they are hydrophobic). So, by combining a layer of these grown microorganisms with a layer of bacterial cellulose, it is possible to create a biocomposite material that contains beneficial properties from both, e.g. with a strong bacterial cellulose layer on one side of the material and a water repellent grown fungal, and/or algal, and or bacterial layer on the other side.

In addition, bacterial cellulose has a flat, paper-like texture when dry. This is unsuitable for applications where the handfeel of the material is a feature. Other grown organic materials may have a different surface texture, providing a smooth handfeel, or 3 dimensional structures imparting hairiness, softness, roughness or alternative textures. Texture layers of grown organic material typically do not have significant tensile strength. By creating a biocomposite material, the strength and structure of the bacterial cellulose layer is complemented by the texture of the second grown organic material layer.

In the embodiment where the second microorganism is allowed to colonise all surfaces of the layer of bacterial cellulose, a sheet of material can be produced that is, for example, coloured, textured, hydrophobic and/or water-repellent on all sides, with the benefit of having a strong bacterial cellulose core.

The particular microorganism, or combination of microorganisms, used to grow / form the second layer can be chosen based on its properties. For example, since bacterial cellulose innately has a beige / light colour, a pigment producing microorganism can be chosen to form the second layer so as to create a biocomposite material with a strong bacterial cellulose layer on one side of the material and a desirably coloured layer on the other side. Any suitable pigment producing microorganism or combination of microorganisms can be chosen. Bacterial cellulose also has a specific texture, so a microorganism with a different desired texture can be chosen to form the second layer so as to create a biocomposite material with a strong bacterial cellulose layer one on side and a desirably textured layer on the other side. Any suitably textured microorganism or combination of microorganisms can be chosen.

For example, the basal layer of bacterial cellulose can be inoculated on its upper surface or lower surface with a liquid yeast culture of S. cerevisiae. The yeast can be genetically modified to produce a pigment so as to create a coloured layer of growth. For example, the basal layer of bacterial cellulose can be inoculated on its upper surface with a liquid yeast culture of Yarrowia lipolytica to develop a lipid-dense layer of grown yeast material. The yeast can be genetically modified to further enhance the properties of the bacterial cellulose basal layer.

For example, the basal layer of bacterial cellulose can be inoculated on its upper surface with Ganoderma lucidem spores or liquid culture to develop a layer of grown mycelium material that is hydrophobic, coloured and texturally different to the bacterial cellulose basal layer.

For example, the basal layer of bacterial cellulose can be inoculated on its upper surface with liquid bacterial cultures of native or genetically modified B. subtilis, E. coli or S. coelicolor strains and the bacteria allowed to grow and produce coloured pigments or molecules. These pigments/molecules may include carotenoids, melanins, actinorhodin, coelimycin, or chromophores, for example. Growth of the bacterial layer can be halted once colouration has reached an optimal level.

For example, the basal layer of bacterial cellulose can be inoculated on its upper surface with a liquid culture or colonies of Chlamydomonas reinhardtii to develop a layer of grown algal material that is hydrophobic, coloured and/or texturally different to the bacterial cellulose basal layer.

By choosing which particular microorganism or combinations of microorganisms form the second layer, the material of the invention can be tailored or customised in any manner desired, e.g. in terms of aesthetic, physical or other material properties. The material of the invention can differ from the basal bacterial cellulose material in its insulation potential, heat resistance, fire resistance, mould resistance, texture, colour and hydrophobicity, for example. The material of the invention is also advantageously biodegradable, sustainable, durable, strong and flexible.

The material can be grown to any size that suits its ultimate intended use. For example, the material can be grown to a size typical of material sizes suitable for use in the fashion industry.

Many different types of dyes can be produced by the microorganism(s) used to form the second layer. For example, the dyes produced may include violacein, carotenoids (e.g. lycopene, carotene), melanins, actinorhodin, coelimycin, chromophores, undecylprodigeosin, actinorhodin, coelimycin, indigo and tripyrrole.

The biobased bacterial cellulose material of the invention can be utilised for many applications in multiple types of industries. These applications can be of any kind where biodegradable, sustainable, and customisable materials are needed. Suitable applications include use of the material in the fashion /textile industry, in medical applications, in packaging applications, in the automobile industry, in high- performance materials, and in furniture covering applications, for example. The ability to tailor / customise the properties of the second layer of the material of the invention, makes it a particularly suitable material for use in the fashion industry for various items of apparel and clothing, as well as footwear and wearable accessories. The present invention thus further relates to articles of clothing, articles of20tolonifar, wearable accessories and fabrics for home, automotive and aerospace upholstery and furnishings comprising the biobased bacterial cellulose material described herein.

In an embodiment, the second layer of the biobased bacterial cellulose material forms the outer or outermost layer of the article of clothing or apparel, article of footwear or wearable accessory, i.e. it forms the external visible side of the material when an article of clothing, article of footwear or wearable accessory is worn. Thus, the visible side of the material is the customisable side of the material. The first layer of the biobased bacterial cellulose material forms the inner or innermost layer of the article of clothing, article of footwear or wearable accessory, i.e. it forms the internal, not typically visible side of the material when an article of clothing, article of footwear or wearable accessory is worn. Such a configuration may be advantageous when an article of clothing, article of footwear or wearable accessory with a water repellent outer surface is desired, for example.

In an alternative embodiment, the second layer of the biobased bacterial cellulose material forms the inner or innermost layer of the article of clothing, article of footwear or wearable accessory, i.e. it forms the internal (typically non-visible) side of the material when an article of clothing, article of footwear or wearable accessory is worn. Thus, the internal side of the material is the customisable side of the material. The first layer of the biobased bacterial cellulose material forms the outer or outermost layer of the article of clothing, article of footwear or wearable accessory, i.e. it forms the external, visible side of the material when an article of clothing, article of footwear or wearable accessory is worn. Such a configuration may be advantageous when an article of clothing, article of footwear or wearable accessory with a soft suede-like inner lining (mycelium can feel like suede) is desired, for example.

The articles of clothing may include any type of garment, such as trousers, coats, t-shirts, and sportswear. Likewise, the articles of footwear may include any type of footwear, such as trainers, boots, and shoes. Likewise, the wearable accessories may include any type of wearable accessory, such as bags, purses, wallets, hats, and mobile phone covers.

The biobased bacterial cellulose material of the invention is manufactured in an environmentally friendly way, and avoids the use of harsh chemicals used in known manufacturing and dying processes typically used in the fashion / textile industry.

EXAMPLES

The invention will now be further illustrated by way of the following non-limiting examples.

Example 1— K. rhaeticus alone (control)

A K. rhaeticus pre-culture was inoculated into fresh coconut water media. Static growth at 30°C and 40- 70% humidity for 3-7 days followed, enabling a thick bacterial cellulose pellicle to be grown (1-2 cm thickness). The pellicle was treated in 1 % bleach solution for 24 hours to terminate growth. The pellicle was washed with distilled water to remove any remaining traces of the bleach solution. Alternative methods include but are not limited to autoclaving and immersion in sodium hydroxide, bleach, methanol, or isopropyl alcohol solutions. The material was then oven dried at 30°C for 24 hours to reduce the water content of the pellicle.

The material was assessed to determine its hydrophilic/hydrophobic properties. A 10pl_ liquid droplet of distilled water was dispensed onto the surface of the material and photographed after 1-10s. A photograph was taken and subsequently processed using ImageJ to determine the contact angle. Increased contact angles indicate the surface exhibits greater hydrophobicity. The contact angle was 72.7° indicating that the surface displayed hydrophilic properties.

The texture of the material was determined haptically and was found to exhibit a dry, papery, brittle property.

The colouration of the material was determined manually and was found to be a beige.

Example 2- K. rhaeticus and Aspergillus sp.

A K. rhaeticus pre-culture was inoculated into fresh coconut water media. Static growth at 30°C and 40- 70% humidity for 3-7 days followed, enabling a thick bacterial cellulose pellicle to be grown (1-2 cm thickness). The pellicle was treated in 1 % bleach solution for 24 hours to terminate growth. The pellicle was washed with distilled waterto remove any remaining traces of the bleach solution. The upper layer of the pellicle was then inoculated with Aspergillus sp. spores and was incubated at 30°C for 7 days, allowing growth across the surface of the pellicle. A small area of the Aspergillus layer was scrapped off to show the bacterial cellulose layer below (as shown in Figure 1A). Once the Aspergillus had covered the upper surface of the pellicle, growth of the Aspergillus was halted by pressing the material at 100 - 200°C and treating with 70% isopropanol. Alternative methods include but are not limited to autoclaving and immersion in sodium hydroxide, bleach, methanol, or isopropyl alcohol solutions. The material was then air dried.

The material was assessed to determine its hydrophilic/hydrophobic properties. A 10pL liquid droplet of distilled water was dispensed onto the surface of the material and photographed after 1-10s. A photograph was taken and subsequently processed using ImageJ to determine the contact angle. Increased contact angles indicate the surface exhibits greater hydrophobicity. The contact angle was 84.2° indicating that the surface was more water repellent than the material formed from K. rhaeticus alone (Example 1); hence, the surface displayed hydrophobic properties.

The layers of the resultant biocomposite material were inseparable. The second layer was assessed haptically and was found to exhibit a soft texture. The colouration of the material was determined manually and had green-brown colouration (as shown in Figure 1B).

Example 3- K. rhaeticus and A. oryzae

A K. rhaeticus pre-culture was inoculated into fresh coconut water media. Static growth at 30°C and 40- 70% humidity for 3-7 days followed, enabling a thick bacterial cellulose pellicle to be grown (1-2 cm thickness). The pellicle was treated in 1% bleach solution for 24 hours to terminate growth. The pellicle was washed with distilled waterto remove any remaining traces of the bleach solution. The upper layer of the pellicle was then inoculated with Aspergillus oryzae spores which were allowed to colonise the surface of the pellicle at 30°C and 65% humidity for 4 days. Growth of the Aspergillus was halted by treating the sample with 70% ethanol treatment. Alternative methods include but are not limited to autoclaving and immersion in sodium hydroxide, bleach, methanol, or isopropyl alcohol solutions. The material was then air dried.

The material was assessed to determine its hydrophilic/hydrophobic properties. A 10pL liquid droplet of distilled water was dispensed onto the surface of the material and photographed after 1-10s. A photograph was taken and subsequently processed using ImageJ to determine the contact angle. Increased contact angles indicate the surface exhibits greater hydrophobicity. The contact angle was 89.1° indicating that the surface was more water repellent than the materials formed from K. rhaeticus alone (Example 1) and K. rhaeticus and Aspergillus sp. (Example 2); hence, the surface displayed enhanced hydrophobic properties.

The layers of the resultant biocomposite material were inseparable. The second layer was assessed haptically and was found to exhibit a gritty texture.

The colouration of the exterior surface of the second layer was determined manually and had green- brown colouration.

Example 4— K. rhaeticus and A. oryzae with starch pre-treatment

A K. rhaeticus pre-culture was inoculated into fresh coconut water media. Static growth at 30°C and 40- 70% humidity for 3-7 days followed, enabling a thick bacterial cellulose pellicle to be grown (1-2 cm thickness). The pellicle was treated in 1% bleach solution for 24 hours to terminate growth. The pellicle was washed with distilled waterto remove any remaining traces of the bleach solution.

The pellicle was partially oven-dried for 30 min at 30°C to partially reduce the water content. A 15% (w/v) potato starch solution was prepared by heating the solution to 60-80°C. A layer less than 1 mm of the potato starch solution was applied to the surface of the pellicle and cooled to below 40°C. Alternative methods include but are not limited to using an unheated starch solution, potato dextrose broth, starch casein agar media, malt extract agar, and other standard nutritional media. In addition, the modification of pH was possible using buffers to reach pH < 7.

The upper layer of the pellicle was then inoculated with Aspergillus oryzae spores which were allowed to colonise the surface of the pellicle at 30°C and 65% humidity. Once the Aspergillus had covered the upper surface of the pellicle, growth of the Aspergillus was halted by treating the sample with 70% ethanol treatment. Alternative methods include but are not limited to autoclaving and immersion in sodium hydroxide, bleach, methanol, or isopropyl alcohol solutions. The material was then air dried.

The material was assessed to determine its hydrophilic/hydrophobic properties. A 10pL liquid droplet of distilled water was dispensed onto the surface of the material and photographed after 1-10s. A photograph was taken and subsequently processed using ImageJ to determine the contact angle. Increased contact angles indicate the surface exhibits greater hydrophobicity. The contact angle was unable to be measured due to the distilled water being absorbed, indicating the surface was more hydrophilic than the materials formed from K. rhaeticus alone (Example 1), K. rhaeticus and Aspergillus sp. (Example 2) and K. rhaeticus and A. oryzae (Example 3); hence, the surface displayed enhanced hydrophilic properties.

The layers of the resultant biocomposite material were inseparable. The second layer was assessed haptically and was found to exhibit a soft texture.

The colouration of the exterior surface of the second layer was determined manually and had predominantly white colouration, with white filaments and dark green spores.

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