Pellicle comprising bacterial cellulose-bacterial spore materials

Abstract

The invention provides a pellicle (1) comprising a matrix material (50) and an activatable material (10), wherein the activatable material (10) is configured embedded in the matrix material (50), wherein the matrix material (50) comprises bacterial cellulose (55), and wherein the activatable material (10) comprises bacterial endospores (15). The pellicle (1) may especially be used in one or more of: drug delivery, biosensing, biocatalysis, skincare, and food-packaging.

Description

[0001] Pellicle comprising bacterial cellulose-bacterial spore materials

[0002] FIELD OF THE INVENTION

[0003] The invention relates to a pellicle comprising living materials and a method for the production thereof. The invention further relates to a device comprising the pellicle. The invention further relates to a kit-of-parts comprising the pellicle. The invention further relates to a method for on demand production of a biomolecule using the pellicle. The invention further relates to a method of biosensing using the pellicle.

[0004] BACKGROUND OF THE INVENTION

[0005] Engineered living materials are known in the art. For instance, EP4377468A2 describes 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.

[0006] CN118048263 A describes a bacterial cellulose live bacteria material, which is produced by constructing a co-culture system of xyloglucanacetob acter CGMCC No.15234 and spores produced by Bacillus BL-1.

[0007] WO2021 / 072399A1 describes a nanobody comprising a variable domain of an antibody, wherein the nanobody is contained within a Bacillus subtilis bacteria strain.

[0008] Yang, M. et al. (2020), “Engineering Bacillus subtilis as a Versatile and Stable Platform for Production of Nanobodies”, Appl. Environ. Microbiol. 86(8), relates to engineering the spore-forming bacterium Bacillus subtilis and converting the engineered B.subtilits to spores that are resistant to most environmental extremes.

[0009] Gilbert, C. et al. (2021), “Living materials with programmable functionalities grown from engineered microbial co-cultures”, Nature Materials 20, pp 691-700, relates to functional bacterial cellulose-based living materials using a stable co-culture of yeast and bacterial cellulose producing Komagataeibacter rhaeticus bacteria.

[0010] Florea, M. et al. (2016), “Engineering control of bacterial cellulose production using a genetic toolkit and a new cellulose-producing strain”, Microbiology 113(24), pp 3431- 3440, relates to functionalization and patterning of heterologous gene expression within the (bacterial) cellulose matrix.

[0011] Zhang, X. et al. (2020), “Applications of Bacillus subtilis Spores in Biotechnology and Advanced Materials”, Appl. Environ. Microbiol. 86(17), relates to conventional and emerging applications of B. subtilis spores, with a focus on how their unique characteristics have led to innovative applications in many areas of technology, including generation of stable and recyclable enzymes, synthetic biology, drug delivery, and material sciences.

[0012] Mohsin, M. Z. et al. (2021), “Advances in engineered Bacillus subtilis biofilms and spores, and their applications in bioremediation, biocatalysis, and biomaterials”, Synth. Syst. Biotechnol. 6(3), pp 180-191, relates to the formation of B. subtilis biofilms and spores, and summarizes the ability of B. subtilis biofilms and spores to fabricate functional living materials with self-regenerating, self-regulating and environmentally responsive characteristics.

[0013] Choi, Y.S. et al. (2002), “Development of a biological process for livestock wastewater treatment using a technique for predominant outgrowth of Bacillus species”, Water Sci. Technol. 45(12), pp 71-78, relates to simultaneous removal of organic, nitrogen and phosphorus in livestock wastewater by stimulating the growth of Bacillus species, sporeforming and aerobic or facultatively anaerobic bacteria.

[0014] Vadanan, S. V. et al. (2022), “Bacterial cellulose production, functionalization, and development of hybrid materials using synthetic biology”, Polymer Journal 54, pp 481- 492, relates to methods to improve bacterial cellulose production, functionalization, and application in synthetic biology.

[0015] Shannon, M.R. et al. (2024), “Leveraging the Power of Enzymes in Engineered Dead and Living Materials”, Advanced Functional Materials 34(44), relates to engineered dead and living materials, detailing the many methods and techniques involved in their fabrication, the key physical parameters used to control and alter enzyme function in multiple phases, and the sophisticated systems that have evolved in living systems to leverage the power of enzymes.

[0016] SUMMARY OF THE INVENTION

[0017] Living materials in nature exhibit autonomous growth and complex functions, driven from the orchestration of cells confined within or adhered to biological matrices. These properties boost the emergence of “biologically grown” engineered living materials (ELMs) built with living microbes. In such materials, living cells self-assemble building molecules to form a mechanically robust matrix, avoiding the labor for integration of living cells within manmade matrices (e.g., hydrogel). The cells confer programmable functions to the material, including sense and respond, biocatalysis, and local patterning. For example, E. coli has been engineered to secrete the amyloid protein CsgA, that self-assembles extracellularly into nanofiber networks to form biofilms. By appending functional peptide domains to CsgA, such biofilms exhibited programmable functions. However, the cells’ “livingness” within such materials poses disadvantages: the limited lifespan of cells causes faster expiration of materials unless resources are regularly provided, and their susceptibility to harsh conditions impedes practical distribution. Hence, it may be desired to provide biologically grown ELMs with dormant and resistant functionalities to meet the need of real-world applications.

[0018] Hence, it is an aspect of the invention to provide an alternative (engineered) living material, which preferably further at least partly obviates one or more of above-described drawbacks. The present invention may have as object to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

[0019] According to a first aspect, the invention provides a pellicle comprising a matrix material and an activatable material. The activatable material may especially be configured embedded in the matrix material. In embodiments, the matrix material may comprise cellulose, such as especially bacterial cellulose. Further, in embodiments, the activatable material may comprise (dormant) bacterial spores, especially bacterial endospores. Hence, in specific embodiments, the invention may provide a pellicle comprising a matrix material and an activatable material, wherein the activatable material is configured embedded in the matrix material, wherein the matrix material comprises bacterial cellulose, and wherein the activatable material comprises bacterial (endo)spores.

[0020] Such embodiments provide the advantage that the pellicle comprises a living material with programmable and dormant functionalities. Especially, the pellicle may be resistant to harsh (environmental) conditions while keeping the activatable material confined and dormant until use of their functionalities is desired. The resistance to harsh conditions (such as e.g. 70% ethanol, radiation or heating, provide the pellicle the benefit of easy (partial) decontamination, without killing the activatable material (i.e. the bacterial spores). The herein described embodiments further provide the advantage that the dormant spores maintain their functionality over time, allowing germination and activation of their functionality as desired (i.e. on demand). These materials may serve as a versatile on-demand platform for applications as biosensors, biocatalytic materials, and in situ (trans)formation of mechanically robust cellulose-based composites. The invention may thus provide a pellicle comprising bacterial cellulose-bacterial spore materials, especially bacterial cellulose-bacterial spore based materials (automatically) grown from bacterial mixtures.

[0021] The invention may thus provide a pellicle comprising a matrix material and an activatable material. The term “pellicle” may herein refer to relatively thin material product, which may typically be formed at a liquid-air interface. The “pellicle” may in embodiments be provided as “skin”, “film”, “coating”, “membrane”, “self-supporting layer”, or “sheet”. Hence, in some embodiments, the pellicle may be configured on a substrate as a skin, film or coating. In other embodiments, the pellicle may comprise a self-supporting material, such as a (dry) sheet or a (dry (or wet(ted))) membrane.

[0022] In specific embodiments, the pellicle may comprise a dry paper-like sheet. Such embodiments may be beneficial as the pellicle may be relatively easily and efficiently stored, while maintaining functionality on-demand. The pellicle may especially be dry, meaning at most 50 wt.% of the pellicle may comprise water, such as at most 40 wt.%, like at most 30 wt.%. Especially, in embodiments, at most 25 wt.%, such as at most 15 wt.%, like at most 5 wt.%, especially at most 3 wt.%, more especially at most 2 wt.% of the pellicle may comprise water. The pellicle may, in embodiments, be air-dried. In such embodiments, the pellicle may have a water content selected from the range of 0-50 wt.%, such as selected from the range of 1-40 wt.%, like from the range of 2-20 wt.%. Furthermore, in embodiments, the pellicle may be freeze-dried. In such embodiments, the pellicle may have a water content selected from the range of 0-35 wt.%, such as selected from the range of 0.1-25 wt.%, like from the range of 0.5- 10 wt.%.

[0023] Furthermore, in embodiments, the pellicle may be paper-like (or sheet-like). Especially, the pellicle may resemble the structure and / or texture of paper. Yet further, in embodiments, the pellicle may be (or resemble) a sheet. In (such) embodiments, the pellicle may be relatively thin compared to its dimensions of length and width. Especially, the pellicle may have a thickness (t), a length (L), and a width (W), wherein t<L and t<W. In embodiments, t<1.5*L, such as t<2*L, like t<5*L, especially t<10*L. Similarly, in embodiments, t<1.5*W, such as t<2*W, like t<5*W, especially t<10*W.

[0024] The pellicle may especially comprise a matrix material. The matrix material may comprise a biopolymer. In embodiments, the matrix material may be selected from the group comprising: plant cellulose, bacterial cellulose, chitin, collagen, silk fibroin, gelatin, starch, alginate, chitosan, polylactic acid, agar-agar, and polyhydroxyalkanoate. Especially, the matrix material may comprise cellulose. The term “cellulose” may herein especially refer to an organic compound with the formula (CeHio05)n, especially a polysaccharide consisting of a linear chain of several hundreds to many thousands of (1— >4) linked D-glucose units. Cellulose may be naturally produced by many plants, bacteria and algae.

[0025] The cellulose produced by, for instance, plants and bacteria may differ. In particular, bacterial cellulose, i.e. cellulose naturally produced by a bacterium, may have a relatively high purity, strength, moldability and water holding ability. Hence, in embodiments, the matrix material may comprise bacterial cellulose. In particular, bacterial cellulose (BC) may have gained interest in recent years due to its high purity (and high crystallinity), which is obtained with green processing conditions. Bacterial cellulose is an extracellular biopolymer secreted by certain microorganisms in the form of a hydrogel-like pellicle at the air-liquid interface.

[0026] In particular, bacterial cellulose may have multiple beneficial features, including its nanofibrous microstructure, light weight, low cost, biocompatibility, and biodegradability.

[0027] Hence, in specific embodiments, the matrix material may comprise bacterial cellulose (BC). Bacterial cellulose may especially refer to cellulose produced by a bacterium. In embodiments, the bacterial cellulose may be (obtainable) from a bacterium selected from a genus of the group comprising: Komagataeibacter, Acetobacter, Sarcina, Trichoderma, and Gluconacetobacter. Especially, the bacterial cellulose may be (obtainable) from a bacterium selected from the group comprising Komagataeibacter rhaeticus, Acetobacter xylinum, Sarcina ventriculi, Acetobacter hansenii, Acetobacter pasteurianus, Gluconacetobacter medellensis, Gluconacetobacter persimmonis, Gluconacetobacter sp., and Gluconacetobacter hansenii, especially from Komagataeibacter rhaeticus. Hence, in specific embodiments, the bacterial cellulose may be (obtainable) from Komagataeibacter rhaeticus. The bacterium Komagataeibacter rhaeticus may be advantageous as it is relatively easy to genetically engineer the bacterium to comprise a desired functionality.

[0028] In embodiments, the pellicle may further comprise an activatable material. The activatable material may (also) comprise a bacterium. Especially, the activatable material may comprise a dormant bacterium. Hence, the activatable material may comprise a microorganism. The term “microorganism” may also refer to a plurality of microorganisms, especially a plurality of microorganisms of the same species. Moreover, in embodiments, the activatable material may comprise any type of spore-forming bacterium. Hence, in embodiments, the activatable material may comprise bacterial spores. Especially, the activatable material may comprise one or more of bacterial endospores, bacterial cysts, bacterial myxospores, and bacterial exospores.

[0029] In specific embodiments, the activatable material may comprise (dormant) bacterial endospores. Such embodiments may be beneficial as endospores may have distinct and well-characterized properties (such as e.g. the production of interesting biopolymers, see also further below). Furthermore, various methods have been developed and are well known for the characterization of the properties of such endospores. Especially good results have been achieved with bacterial endospores as such spores show high resistance to harsh conditions (such as e.g. high temperatures, drought, chemicals, and radiation), which may be advantageous for a wide variety of applications. Furthermore, the high resistance of the bacterial spores may enable long-term and thus improved shelf-life of the pellicle, while maintaining (on demand) functionality.

[0030] The activatable material may thus comprise (dormant) bacterial endospores. In embodiments, the bacterial endospores may belong to a bacterial genus selected from the group comprising: Bacillus, Sporosarcina, and Clostridium. Moreover, in embodiments, the bacterial endospores may belong to a bacterial species selected from the group comprising: Bacillus subtilis, Bacillus paralicheniformis, Sporosarcina pasleurii, Bacillus pasleurii, Bacillus megate ri m, Clostridium sporogenes, Clostridium botulinum, and Clostridium difficile. In other words, the activatable material may comprise a bacterium selected from the group comprising Bacillus subtilis, Bacillus paralicheniformis, Sporosarcina pasteurii, Bacillus pasteurii, Bacillus megaterium, Clostridium sporogenes, Clostridium botulinum, and Clostridium difficile. In specific embodiments, the bacterial endospores may belong to a bacterial species selected from Bacillus subtilis and Bacillus paralicheniformis. The bacterial species Bacillus subtilis may be advantageous as it is relatively easy to genetically engineer the bacterium to comprise a desired functionality. On the other hand the bacterial species Bacillus paralicheniformis may be advantageous as the bacterium, upon germination, may produce poly(y-glutamic acid), which may increase the toughness of the (pellicle, especially the matrix) material. Additionally, production of poly(y-glutamic acid) by Bacillus paralicheniformis may serve to improve the flexibility of the (pellicle, especially the matrix) material.

[0031] In embodiments, the activatable material may be configured embedded (or integrated) in the matrix material. The matrix material may thus, in embodiments, comprise the activatable material. Therefore, in embodiments, the bacterial (endo)spores (of the activatable material) may (be engineered to) comprise Cellulose Binding Module (CBM) functionalized bacterial (endo)spores. A Cellulose Binding Module may herein refer to a protein domain found in carbohydrate active enzymes and having an affinity towards cellulosic materials. The Cellulose Binding Module (CBM) may especially be configured on a crust (i.e. an outer shell) of the bacterial (endo)spores. In embodiments, the bacterial (endo)spores may comprise the Cellulose Binding Module obtainable through genetic engineering, especially using DNA- encoded engineering techniques (especially using DNA-encoded libraries (DELs)). Alternatively, in embodiments, such genetic engineering may comprise using high-throughput screening (HTS) technologies. Engineering of the bacterial (endo)spores to comprise a Cellulose Binding Module (CBM) may provide the benefit that the (binding) affinity of the spores towards the cellulose may be improved. As such, the bacterial (endo)spores may be more efficiently embedded into the matrix material, resulting in higher endospore concentrations (i.e. increased number of incorporated cells) in the pellicle and therewith enhanced functionality. Hence, in embodiments, the bacterial spores may comprise Cellulose Binding Module (CBM) functionalized bacterial spores (wherein the CBM may be configured on a crust of the bacterial spores).

[0032] In embodiments, the Cellulose Binding Module may comprise a Cellulose Binding Module originating (or obtainable) from a bacterium selected from the genus Clostridium. For example, in embodiments, the Cellulose Binding Module may comprise a Cellulose Binding Module originating (or obtainable) from a bacterium selected from the group comprising: Clostridium thermocellum, Clostridium josui. Clostridium cellulolyticum, and Clostridium cellulovorans. Additionally or alternatively, in embodiments, the Cellulose Binding Module may comprise a Cellulose Binding Module originating (or obtainable) from a fungus selected from the genus Trichoderma, such as e.g. a fungus from the species Trichoderma reesei. Further, in specific embodiments, when the bacterial spores comprise bacterial endospores belonging to a bacterial species selected from the group comprising: Bacillus subtilis, Bacillus paralicheniformis, Sporosarcina pasleurii, Bacillus pasleurii, and Bacillus megaterium, then the bacterial spores may optionally also comprise a bacterial species selected from the group comprising: Clostridium sporogenes, Clostridium botulinum, and Clostridium difficile. Hence, in such embodiments, the bacterial spores may additionally (to the former bacterial spores) comprise a Cellulose Binding Module producing bacterium. Furthermore, in embodiments, the pellicle may comprise a single type of bacterial (endo)spores (e.g. obtainable from a single bacterium). Alternatively, in embodiments, the pellicle may comprise multiple different types of bacterial (endo)spores (e.g. obtainable from different bacteria and / or having different functionalization (obtainable through DNA-encoded engineering)).

[0033] Yet further, in embodiments, the pellicle may comprise a bacterial (endo)spore configured to produce a biomolecule upon (especially after or in response to) germination (of the spores). Especially, in such embodiments, the biomolecule may comprise one of a drug, an enzyme, a polymer, and a chromophore (see also further below).

[0034] The pellicle may further, in embodiments, comprise one or more additives. For example, in embodiments, the pellicle may comprise one or more of a mineral and a dye (or colorant). In embodiments, the mineral may e.g. be selected from the group comprising: a carbonate (such as calcium carbonate (CaCCh) or magnesium carbonate (MgCCh)) and a chloride (such as e.g. calcium chloride (CaCh) or magnesium chloride (MgCh)). Addition of such minerals may be advantageous as the minerals may provide improved strength and fireresistance qualities to the pellicle, therewith improving its shelf-life.

[0035] The herein described embodiments of the pellicle may be obtainable via the herein described method for providing the pellicle. As a result of the method described below, in embodiments, the pellicle may further comprise (residual) density growth medium (or a density modifier). For example, the pellicle may comprise iohexol, see also further below with respect to the method of the invention. In embodiments, the pellicle may comprise a concentration (or amount) of iohexol of at least 5 ppm, such as at least 10 ppm, like at least 20 ppm, especially at least 50 ppm. Especially, in embodiments, the pellicle may comprise a concentration (or amount) of iohexol of at least 150 ppm, such as at least 200 ppm, like at least 250 ppm, especially at least 300 ppm. Moreover, in embodiments, the pellicle may comprise a concentration (or amount) of iohexol of at most 4500 ppm, such as at most 4000 ppm, like at most 3500 ppm, especially at most 3000 ppm. Hence, in embodiments, the pellicle comprises an amount of iohexol selected from the range of 5-4000 ppm, such as from the range of 10- 4000 ppm, like from the range of 200-4000 ppm. Especially, the pellicle may comprise an amount of iohexol selected from the range of 5-3500 ppm, like from the range of 250-2000 ppm.

[0036] In a further aspect, the invention may provide a device comprising the pellicle. The device may especially comprise one or more of a biosensor, a skincare product, a drug delivery device, a biocatalytic device, and a food-packaging device. In embodiments, the pellicle, upon activation (especially germination), may provide an optical change (such as a color change) dependent on its environment, therewith rendering it an efficient material for a biosensor device. For example, in embodiments, the device may be a biosensor configured for detecting contaminants in a fluid (such as a chemical sample or a body of water). In such embodiments, the device may further comprise a sensor configured to detect (and e.g. quantify) an (optical) change in the pellicle. Furthermore, in embodiments, the device may comprise one of a (color wheel) patch or cartridge configured as a reference for determining color, color intensity, or color saturation in the pellicle. Yet in another example, the device may be a biosensor configured to detecting allergens in foodstuffs or pharmaceutical products. The device may be a single-use device or may comprise a holder for hosting the pellicle and configured to allow replacement of the pellicle. In other embodiments, the pellicle as such may be the device. Some embodiments of the device are further elucidated below. Additionally or alternatively, in embodiments, upon activation (especially germination), the bacterial (endo)spores in the pellicle may provide vegetative cells configured to produce (bio)molecules, such as biomolecules interesting to skincare application or biocatalysis. The vegetative cells may also, in such embodiments, be configured to produce (bio)molecules such as drug compounds. Hence, in embodiments, the pellicle may be applied in a skincare product, a drug delivery device, and / or a biocatalytic device. It may be clear to the skilled person that the herein mentioned list of devices may not be exhaustive and further types of devices (e.g. food-packaging, contact lenses, living hydrogels, clothing items, and biocomputing devices) may herein not be excluded. Hence, in embodiments, the invention may provide a device comprising the pellicle, wherein the device may be selected from the group comprising: a biosensor, a skincare product, a drug delivery device, a biocatalytic device, and a food-packaging device.

[0037] In another aspect, the invention may thus provide a use of the pellicle as described herein in one or more of drug delivery, biosensing, biocatalysis, skincare (products), food-packaging (products). Especially, in embodiments, the pellicle may be used as a probiotic. Such embodiments may be beneficial as no germination may be required to obtain probiotic activity in the pellicle (as some bacterial endospores may provide probiotic activity). Additionally or alternatively, in embodiments, the pellicle, especially vegetative cells (upon germination) present in the pellicle, may be used in applications, such as e.g. food storage and skincare. For example, the pellicle may be used in food storage as an alternative to e.g. beeswax wraps or clingfilm. Alternatively, in embodiments, the pellicle may be embedded in a food storage container or attached to a food storage container, where it may be configured as a biosensor to indicate e.g. food freshness. Additionally or alternatively, in embodiments, the pellicle may be used in a detection device to detect the presence of allergens. In another example, the pellicle may be used in skincare e.g. as a skin (such as face) mask or as a (spot) treatment (similarly to a pimple patch). The pellicle may also be comprised by a plaster or (pressure) bandage. Yet additionally or alternatively, in embodiments, the pellicle may be used for the production of biomolecules upon germination (see also further below).

[0038] In yet a further aspect, the invention may provide a kit-of-parts comprising the pellicle as described herein. In such embodiments, the kit-of-parts may further comprise instructions for use of the pellicle. Such instructions may, in embodiments, be included as verbal instructions, picture-based instructions, or both. The verbal and / or picture-based instructions may be included in a physical form, i.e., paper instructions. Additionally or alternatively, the verbal and / or picture-based instructions may be provided through e.g. e-mail upon purchase. Yet additionally or alternatively, in embodiments, the instructions for use may be included in the form of a reference, such as e.g. a QR code, a barcode, a geocode, a hyperlink, and a website reference. Hence, in embodiments, the invention may provide a kit-of-parts comprising a pellicle as described herein, and instructions for use and / or a reference to instructions for use.

[0039] Further, in embodiments, the kit-of-parts may comprise a holder comprising a germination fluid. The term “germination” may herein refer to the development of an (micro)organism (such as a vegetative cell) from a (seed or) spore after a period of dormancy. In embodiments, the germination fluid may thus comprise a fluid configured such that the bacterial (endo)spores may develop into vegetative cells. Especially, the germination fluid may comprise a medium suitable for germination of the bacterial (endo)spores. The medium may, in embodiments, comprise a nutrient-rich medium. Especially, in embodiments, the medium may comprise one or more nutrients selected from the group comprising: a protein (such as peptone or a growth factor), an amino acid, a vitamin, a mineral (such as a phosphate), an antibiotic (such as kanamycin), a pH agent (such as phenol red), a carbohydrate (such as glucose), and a yeast extract constituent. For instance, in embodiments, the germination fluid may comprise a carbon source for the (micro)organism. In further embodiments, the germination fluid may comprise a nitrogen source for the (micro)organism. In further embodiments, the germination fluid may comprise a phosphor source for the (micro)organism. In further embodiments, the germination fluid may comprise a sulfur source for the (micro)organism. Hence, in embodiments, the kit-of-parts may comprise a holder comprising a germination fluid, wherein the germination fluid may comprise a (nutrient-rich) medium suitable for germination of the bacterial (endo)spores. Such embodiments may be beneficial as the kit-of-parts may provide a ready-for-use combination of components. Furthermore, such a kit-of-parts may not require extensive in-depth knowledge, skills or appliances, making it highly accessible.

[0040] The invention may, in a further aspect, also provide a method for the production of a pellicle. Especially, in embodiments, the invention may provide a method for the production of a pellicle as described herein. Hence, in embodiments, the pellicle obtainable by the method may comprise bacterial cellulose and (dormant) bacterial (endo)spores, wherein the bacterial (endo)spores may be configured embedded in the bacterial cellulose. The pellicle obtainable by the method may especially be suitable for long-term storage.

[0041] In embodiments, the method may comprise a culture stage and a processing stage. The term “step”, or “stage” and similar terms used herein may refer to a (time) period (also “phase”) of a method. The different steps may (partially) overlap (in time). However, in general, steps of the invention may be executed consecutively. For instance, the culture stage may typically be completed prior to the processing stage. It will be clear to the person skilled in the art how the stages may be beneficially arranged in time.

[0042] Some living microbes can synthesize an extracellular protective matrix to improve their fitness and survival. Bacteria cellulose (BC) producing bacteria may produce highly pure BC fibers around themselves as a pellicle at air-liquid interface. The BC matrix may provide physical barrier from various stress factors. Furthermore, the bacterial cellulose matrix may have good water-holding ability, which may comfort vegetative cell-cycles of bacteria (such as germinated endospores). Furthermore, the pellicle may have good structural integrity and may form defined shapes depending on the culture conditions. The culture stage may thus comprise culturing (i.e. growing) a bacterial cellulose-producing bacterium. In embodiments, the bacterial cellulose-producing bacterium may comprise a bacterium selected from the group comprising Komagataeibacter rhaeticus, Acetobacter xylinum, Sarcina ventriculi, Acetobacter hansenii, Acetobacter pasteurianus, Gluconacetobacter medellensis, Gluconacetobacter persimmonis, Gluconacetobacter sp., and Gluconacetobacter hansenii, especially from Komagataeibacter rhaeticus. Hence, in specific embodiments, the bacterial cellulose-producing bacterium may comprise Komagataeibacter rhaeticus.

[0043] Further, in embodiments, the culture stage may comprise culturing the bacterial cellulose-producing bacterium in the presence of the (dormant, i.e., metabolically inactive) bacterial (endo)spores. In other words, in such embodiments, the bacterial cellulose-producing bacterium may be cultured in a single culture (e.g. in a petri dish) together with the bacterial (endo)spores. Especially, the bacterial cellulose-producing bacterium may grow and form a pellicle at the air-liquid interface, encapsulating spores present (i.e. floating) in the culture dish. In such embodiments, the bacterial spores may remain dormant. Therefore, in embodiments, the culture stage may comprise culturing the bacterial cellulose-producing bacterium in the presence of the bacterial (endo)spores in growth conditions suitable for (i) culturing the bacterial cellulose-producing bacterium, and (ii) maintaining (dormancy of) the bacterial (endo)spores. The phrase “growth conditions” may herein especially refer to one or more of type of medium, pH, (ambient) temperature, (ambient) oxygen level, etc. . .

[0044] Upon providing growth conditions suitable for culturing the bacterial cellulose- producing bacterium, the bacterial cellulose-producing bacteria may especially produce bacterial cellulose to provide a matrix. Such a (bacterial cellulose) matrix may especially form at an air-fluid interface, therewith providing a pellicle. In embodiments, the bacterial (endo)spores may be embedded in the produced bacterial cellulose, i.e., the bacterial endospores may be embedded in the matrix. Hence, in embodiments, the culture stage may comprise providing a pellicle comprising (dormant) bacterial (endo)spores embedded in (a) bacterial cellulose (matrix).

[0045] The method may further, in embodiments, comprise a processing stage. The processing stage may especially comprise drying the thus obtained material, therewith providing the pellicle. When drying, the weight percentage of water may be reduced. The drying may, in embodiments, comprise air-drying the material. In such embodiments, the (thus provided) pellicle may have a water content selected from the range of 0-50 wt.%, such as selected from the range of 1-40 wt.%, like from the range of 2-20 wt.%. Furthermore, in embodiments, the method may comprise freeze-drying the pellicle. In such embodiments, the (thus provided) pellicle may have a water content selected from the range of 0-35 wt.%, such as selected from the range of 0.1-25 wt.%, like from the range of 0.5-10 wt.%. Such embodiments may be beneficial as the water content in the pellicle may be further reduced, improving long-term storage stability of the pellicle. Hence, in embodiments, the invention may provide a method for the production of a pellicle comprising bacterial cellulose and bacterial (endo)spores, wherein the bacterial (endo)spores may be configured embedded in the bacterial cellulose, the method comprising: (A) a culture stage comprising culturing bacterial cellulose-producing bacterium in the presence of the bacterial (endo)spores in growth conditions suitable for (i) culturing the bacterial cellulose-producing bacterium and (ii) maintaining (dormancy of) the bacterial (endo)spores; and (B) a processing stage comprising drying at least part of the thus obtained material, therewith providing the pellicle. Such embodiments may be beneficial as the pellicle may be produced in a relatively quick singleculture using readily available materials.

[0046] In embodiments, the method may thus comprise growing bacterial cellulose- producing bacteria in a growth medium suitable for their growth. Additionally, in such embodiments, the growth medium needs to be suitable for maintaining the dormancy of the bacterial (endo)spores (i.e. the medium may allow little, especially no germination of the bacterial (endo)spores). Therefore, in embodiments, the method may further comprise a preparation step comprising preparing a growth medium suitable for culturing the bacterial cellulose-producing bacterium (during the culture stage) in the presence of the bacterial (endo)spores.

[0047] In embodiments, the growth medium may comprise a nutrient-rich medium. Especially, in embodiments, the medium may comprise one or more nutrients selected from the group comprising: a protein (such as peptone or a growth factor), an amino acid, a vitamin, a mineral (such as a phosphate), an antibiotic (such as kanamycin), a pH agent (such as phenol red), a carbohydrate (such as glucose), and a yeast extract constituent. For instance, in embodiments, the growth medium may comprise a carbon source for the (micro)organism. In further embodiments, the growth medium may comprise a nitrogen source for the (micro)organism. In further embodiments, the growth medium may comprise a phosphor source for the (micro)organism. In further embodiments, the growth medium may comprise a sulfur source for the (micro)organism.

[0048] The growth medium may, in embodiments, comprise a medium selected from the group comprising a YPD (or YEPD) medium, a Hestrin-Schramm (HS) medium, a sugarcane molasses-supplemented (SCM) medium, and a Yamanaka medium. Good results have been achieved when using a YPD (or YEPD) medium. A YPD medium may herein especially refer to a medium comprising a yeast extract, a peptone, and dextrose. In embodiments, the YPD medium may comprise 0.5-2 wt.% yeast extract relative to a total volume of the YPD medium, such as 0.5-1.5 wt.%, especially 1 wt.%. Moreover, in embodiments, the YPD medium may comprise 1-3 wt.% peptone relative to a total volume of the YPD medium, such as 1.5-2.5 wt.%, like 2 wt.%. Furthermore, in embodiments, the YPD medium may comprise 1-3 wt.% glucose relative to a total volume of the YPD medium, such as 1.5-2.5 wt.%, like 2 wt.%. In embodiments, the yeast extract may comprise essentially any type of yeast extract. In specific embodiments, the growth medium may comprise a YPD medium comprising 1 wt.% yeast extract, 2 wt.% peptone, and 2 wt.% glucose, wherein the percentages are relative to a total volume of the YPD medium.

[0049] As described above, the pellicle may form at the air-liquid interface during the culture stage. In general, the bacterial spores may have a higher density than the growth medium, causing sedimentation of the bacterial spores in a culturing mixture. Therefore, in embodiments, the growth medium may further comprise a density gradient medium. Such a density gradient medium may increase the density of the growth medium, therewith reducing (or even preventing) the sedimentation of the bacterial spores in the culturing mixture. Especially, such a density gradient medium may help increase the concentration of bacterial spores embedded into the (bacterial cellulose) matrix material. In embodiments, the density growth medium may comprise one or more of iohexol, iodixanol, silica nanoparticles, sucrose, and a sugar (such as e.g. dextran). Especially in embodiments, the growth medium may comprise iohexol in an amount selected from the range of 15-55 wt.%, such as selected from the range of 20-50 wt.%, like from the range of 25-45 wt.%, especially from the range of 30- 40 wt.% relative to the total weight of the growth medium. In specific embodiments, the growth medium may comprise 38-42 wt.% iohexol relative to the total weight of the growth medium. Hence, in embodiments, the growth medium may further comprise (a density gradient medium, wherein the density gradient medium comprises) iohexol in an amount selected from the range of 25-45 wt.% relative to the total weight of the growth medium.

[0050] Additionally to the composition of the growth medium also other growth conditions, such as pH, (ambient) moisture content, (ambient) oxygen content, and (ambient) temperature, may be of importance. In embodiments, the growth medium may therefore be configured to have a growth medium pH suitable for (i) culturing the bacterial cellulose- producing bacterium and (ii) maintaining (dormancy of) the bacterial (endo)spores. Especially, in embodiments, the growth medium may have a growth medium pH selected from the range of 3-5, such as from the range of 3.5-4.5, like from the range of 3.7-4.3. In specific embodiments, the growth medium may have a growth medium pH of 4.0. Such a relatively low growth medium pH may be beneficial as it may provide inhibition of the germination of the bacterial (endo)spores, i.e., low growth medium pH may help maintain the dormant state of the bacterial (endo)spores. Yet such relatively low growth medium pH may still be compatible with growth and cellulose production of bacterial cellulose-producing bacteria such as ", rhaeticus.

[0051] In specific embodiments, the activatable material, especially the bacterial endospores, may comprise a bacterium selected from Bacillus subtilis and Bacillus paralicheniformis, wherein the bacterial cellulose-producing bacterium may comprise Komagataeibacter rhaeticus, wherein the growth medium may comprise a medium suitable for (i) culturing the bacterial cellulose-producing bacterium and (ii) maintaining (dormancy of) the bacterial endospores, and wherein the growth medium may have a growth medium pH selected from the range of 3.5-4.5, especially a growth medium pH of 4.0. Furthermore, in such embodiments, the growth medium may comprise 40 wt.% iohexol. More especially, in embodiments, the bacterial (endo)spores may comprise a bacterium selected from Bacillus subtilis and Bacillus paralicheniformis, wherein the bacterial cellulose-producing bacterium may comprise Komagataeibacter rhaeticus, wherein the growth medium may comprise yeast extract, peptone and dextrose (YPD / YEPD), and wherein the growth medium may have a growth medium pH selected from the range of 3.5-4.5. Such embodiments may especially be beneficial as under such growth conditions the bacterial spores may remain dormant. Furthermore, under such growth conditions the K rhaeticus may form robust BC pellicles that may be easily manipulated manually (e.g. using tweezers) and pre-treated (e.g. washing, drying). Herein, the type of activatable material, especially the type of bacterial spores) may be selected in view of desired functionalities. For example, as described above, bacterial endospores from Bacillus paralichenformis may provide the functionality of producing (in a vegetative cell-cycle) poly(y-glutamic acid) (PGA), which may improve the strength (or toughness) of the pellicle. Hence, in embodiments, the bacterial (endo)spores may have an inherent functionality. In embodiments, the bacterial (endo)spores may (also) be genetically modified, such that they may have (additional) desired functionalities. For example, in embodiments, the bacterial (endo)spores may be genetically modified to produce (in a vegetative cell-cycle) one or more biomolecules. The one or more biomolecules may, in embodiments, be selected from the group comprising: a drug, an enzyme (such as e.g. P- galactosidase), a polymer (such as e.g. y-PGA), and a chromophore (such as e.g. green fluorescent protein (GFP)). In embodiments, the one or more biomolecules may comprise an enzyme, such as P-galactosidase. Additionally or alternatively, the one or more biomolecules may comprise a chromophore, such as green fluorescent protein. However, note that, in embodiments, genetic modification may thus not always be necessary for (vegetative cells derived from) bacterial endospores to produce a biomolecule.

[0052] Further, in embodiments, the bacterial (endo)spores (of the activatable material) may (be (genetically) engineered, using DNA-encoded engineering, to) comprise Cellulose Binding Module (CBM) functionalized bacterial (endo)spores (see also further above). The Cellulose Binding Module (CBM) may especially be configured on a crust (i.e. an outer shell) of the bacterial (endo)spores. Such engineering as described here may especially be executed using DNA-encoded engineering (especially using DNA-encoded libraries (DELs)). Alternatively, in embodiments, such engineering may be executed using high-throughput screening (HTS) technologies. Engineering of the bacterial (endo)spores to comprise a Cellulose Binding Module (CBM) may provide the benefit that the (binding) affinity of the spores towards the cellulose may be improved. As such, the bacterial (endo)spores may be more efficiently embedded into the matrix material, resulting in higher spore concentrations (i.e. increased number of incorporated cells) in the pellicle and therewith enhanced functionality.

[0053] Furthermore, in embodiments, the bacterial endospores may be genetically modified, such that changes to the spore surface may be realized. In general, Bacillus spores may comprise a core and multiple coat layers, especially an inner membrane, a cortex, a coat, and a glycan layer. In embodiments, the bacterial endospores may be genetically modified, such that the glycan layer may be removed. Such embodiments may be beneficial as removal of the glycan layer may improve the efficiency of incorporating a Cellulose Binding Module on the spore coat as described above, which may result in increased numbers of spores (being) embedded in the matrix material.

[0054] Similarly to the bacterial endospores, in embodiments, the bacterial cellulose- producing bacterium may also be genetically modified. For example, in embodiments, the bacterial cellulose-producing bacterium may be genetically modified to comprise a green fluorescent protein (GFP) expressing strain. Hence, in embodiments, one or more of the following may apply: (i) the bacterial cellulose-producing bacterium may comprise genetically modified bacterial cellulose-producing bacterium, and (ii) the bacterial endospores may comprise genetically modified bacterial endospores. In other words, in embodiments, one or more of the following may apply: (i) the bacterial cellulose may be provided by a genetically modified bacterial cellulose-producing bacterium, and (i) the bacterial spores may comprise genetically modified bacterial spores.

[0055] Further, in embodiments, the method may comprise an enrichment stage. In embodiments, the enrichment stage may comprise germinating the bacterial (endo)spores to obtain vegetative bacterial cells. Germinating the bacterial (endo)spores may especially comprise providing a germination fluid (see also further above) to the pellicle. Upon germination, the bacterial (endo)spores in the pellicle may thus establish a vegetative cell-cycle.

[0056] The enrichment stage may further, in embodiments, comprise sporulating the vegetative bacterial cells to obtain an enriched pellicle. Especially, the enriched pellicle may comprise an increased viable cell count of bacterial (endo)spores (compared to a viable cell count of the pellicle prior to the enrichment stage). In embodiments, the enrichment stage may provide at least a 10-fold increase in the viable cell count of bacterial spores in the pellicle. Especially, in embodiments, the enrichment stage may provide an increase in the viable cell count of bacterial spores in the pellicle of at least 100-fold, such as selected from the range of 500-5000-fold. More especially, in embodiments, the enrichment stage may provide an increase in the viable cell count of bacterial spores in the pellicle of up to 10000-fold.

[0057] In embodiments, the pellicle (obtainable by the method for the production of a pellicle) may comprise an amount of bacterial (endo)spores selected from the range of IxlO4- IxlO7colony-forming units (or CFU). Especially, in embodiments, the pellicle (obtainable by the method for the production of a pellicle) may comprise an amount of bacterial spores selected from the range of IxlO4- 2xl06CFU, such as from the range of 8 xlO4- 8xl05CFU.

[0058] Conversely, in embodiments, the enriched pellicle (obtainable by the method for enriching a number of endospores in a (bacterial) pellicle) may comprise an amount of bacterial (endo)spores selected from the range of IxlO7- IxlO10colony-forming units (or CFU). Especially, in embodiments, the enriched pellicle (obtainable by the method for enriching a number of endospores in a (bacterial) pellicle) may comprise an amount of bacterial spores selected from the range of IxlO8- IxlO10CFU, such as from the range of 8 xlO8- 2xl09CFU. The herein indicated ranges of CFU were calculated for (enriched) pellicles having a 2.8 cm diameter, and a 1-2 mm thickness when wet (i.e. prior to drying). CFU counts were determined by plating serial dilutions of hydrolyzed pellicles.

[0059] In embodiments, sporulating the vegetative bacterial cells may comprise changing the culture conditions (e.g. medium, temperature, etc.) after germination. For example, in embodiments, a sporulation medium may be added to the pellicle after germination. Good results were obtained in embodiments where the sporulation medium comprises Schaeffer’s sporulation medium (comprising a nutrient broth, KC1, MgSO4*(7 FEO), MnC12*(4 FEO), CaCE, and FeSCU). In another example, in embodiments, a nutrient restriction may be applied to the pellicle, i.e., the pellicle may be left in the germination fluid without providing fresh medium. Additionally or alternatively, in embodiments, a nutrient shock may be applied to the bacteria. For example, particularly good results (with an increase of up to 10 times higher numbers of spores) were achieved when the pellicle was washed, for 30 min in phosphate buffered saline (PBS; 0.14 M NaCl, 2.7 mM KC1, 10 mM phosphate), between germination and sporulation.

[0060] Furthermore, in embodiments, the method may comprise washing the mixture in a wash buffer. For example, in embodiments, washing may comprise placing at least part of the mixture (comprising the pellicle) in the wash buffer for 0.5-16 hours, such as 1-12 hours, like 2-8 hours. Especially, in embodiments, washing may comprise contacting at least part of the mixture (comprising the pellicle) with the wash buffer for 8-16 hours. In embodiments, washing may be done at about room temperature (20°C). However, in specific embodiments, washing may be done at refrigerated temperature, i.e., as low as about 4-7 °C, such as especially 4 °C. Such embodiments may be beneficial as low temperatures may help retain dormancy of the bacterial spores, as germination may be difficult at such low temperatures. Furthermore, during washing the pellicle (and washing buffer) may be (gently, i.e., about 10 times per minute) shaken (e.g. using a shaker plate). Such embodiments may be beneficial as washing may remove salts, (excess) nutrients and other impurities from the pellicle, therewith reducing the risk of unwanted side-reactions during application / use of the pellicle.

[0061] In embodiments, the wash buffer may have a pH selected from the range of 3-5, such as from the range of 3.5-4.5, like from the range of 3.7-4.3. In specific embodiments, the wash buffer may have a pH of 4.0. Such a relatively low pH may be beneficial as it may provide inhibition of the germination of the bacterial (endo)spores, i.e., low pH may help maintain the dormant state of the bacterial (endo)spores. For example, in embodiments, the wash buffer may comprise a citrate (or citric acid) buffer.

[0062] Yet further, in embodiments, the method may comprise drying the washed mixture to obtain an enriched (wet) pellicle. In embodiments, the washed mixture may be dried such that a pellicle comprising a dry paper-like sheet, as described above, may be obtained.

[0063] In specific embodiments, the method may comprise an enrichment stage comprising: (i) germinating the bacterial spores (by providing a germination fluid to the pellicle) to obtain a mixture comprising the bacterial cellulose and vegetative bacterial cells; and (ii) sporulating the vegetative bacterial cells (to obtain an enriched (wet) pellicle comprising an increased viable cell count of bacterial spores); (iii) washing the mixture in a wash buffer; and (iv) drying the washed mixture to obtain an enriched pellicle. Such embodiments may be beneficial as sporulation may allow the growth of bacterial cells, resulting in an increased viable cell count embedded in the matrix material. Such an increase in viable cell count may improve the functionality (of the bacterial (endo)spores), therewith e.g. improving a biomolecule-producing efficiency (see also below) of (a device as described herein comprising) the pellicle.

[0064] Hence, in a further aspect, the invention may also provide a method for enriching a number of (endo)spores in a (bacterial) pellicle as described herein, wherein the method comprises an enrichment stage comprising: (i) germinating the bacterial spores (by providing a germination fluid to the pellicle) to obtain a mixture comprising the bacterial cellulose and vegetative bacterial cells; and (ii) sporulating the vegetative bacterial cells (to obtain an enriched (wet) pellicle comprising an increased viable cell count of bacterial spores); (iii) washing the mixture in a wash buffer; and (iv) drying the washed mixture to obtain an enriched pellicle.

[0065] In embodiments, the invention may thus provide an enriched pellicle comprising an increased viable cell count of bacterial spores. Especially, in such embodiments, the enriched pellicle (obtainable by the method for enriching a number of endospores in a (bacterial) pellicle) may comprise an amount of bacterial spores selected from the range of IxlO8- IxlO10CFU, such as from the range of 8 xlO8- 2xl09CFU.

[0066] In a yet further aspect, the invention may provide a method for (on demand) production of a biomolecule. Especially, in embodiments, the method may comprise (on demand) production of a biomolecule using the pellicle as described above. Herein the phrase “on demand production” may refer to production of the biomolecule using the pellicle at any time that said biomolecule may be desired or needed, meaning that the pellicle may be used (i) immediately after obtaining (e.g. buying or producing) the pellicle, or (ii) after storage of the pellicle for a time period. In embodiments, the pellicle may be stored while maintaining its functionality for a time period of at least a day, such as at least a week, like at least two weeks, especially at least a month, such as at least 6 months. Moreover, in embodiments, the pellicle may be stored while maintaining its functionality for a time period of at most 100 years, for example at most 50 years, such as at most 10 years, like at most 5 years, especially at most 2 years, such as at most 12 months.

[0067] In embodiments, the method for (on demand) production of a biomolecule may comprise a preparation stage and an activation stage. The preparation stage may especially comprise providing a pellicle as described herein, such as e.g. obtainable according to the method as described further above. Especially, in embodiments, the pellicle may comprise (i) a matrix material comprising bacterial cellulose and (ii) an activatable material configured embedded in the matrix material and comprising bacterial (endo)spores. The pellicle may, in such a method, especially comprise a bacterial (endo)spore configured to produce the biomolecule upon (especially after or in response to) germination (of the spores).

[0068] Additionally, in embodiments, the preparation stage may comprise providing a germination fluid. The germination fluid may especially comprise a medium suitable for germination of the bacterial (endo)spores (in the pellicle). The medium may, in embodiments, comprise a nutrient-rich medium. Especially, in embodiments, the medium may comprise one or more nutrients selected from the group comprising: a protein (such as peptone or a growth factor), an amino acid, a vitamin, a mineral (such as a phosphate), an antibiotic (such as kanamycin), a pH agent (such as phenol red), a carbohydrate (such as glucose), and a yeast extract constituent. For instance, in embodiments, the germination fluid may comprise a carbon source for the (micro)organism. In further embodiments, the germination fluid may comprise a nitrogen source for the (micro)organism. In further embodiments, the germination fluid may comprise a phosphor source for the (micro)organism. In further embodiments, the germination fluid may comprise a sulfur source for the (micro)organism.

[0069] Further, the method may comprise the activation stage. In embodiments, the activation stage may comprise germinating the bacterial (endo)spores in the pellicle. Especially, in embodiments, the activation stage may comprise contacting (such as e.g. soaking) the pellicle with the germination fluid, such that growth-permissive conditions may be obtained and thus germination of the (endo)spores into vegetative cells may occur. Moreover, in embodiments, germination of the bacterial (endo)spores may activate production of the biomolecule, through the establishment of a metabolically active vegetative cell-cycle. Hence, in embodiments, the invention may provide a method for production of a biomolecule, wherein the method may comprise: (A) a preparation stage comprising: providing (i) a pellicle as described herein or obtainable according to the method as described herein, and (ii) a germination fluid, wherein the germination fluid may comprise a medium suitable for germination of the bacterial spores, wherein the pellicle may comprise a bacterial spore configured to produce the biomolecule after germination; and (B) an activation stage comprising germinating the bacterial spores in the pellicle by contacting the pellicle with the germination fluid to activate production of the biomolecule. Such a method may be advantageous for use in drug-delivery (e.g. in skincare) applications. Furthermore, such a method may be useful for application in biosensors. Yet further, such a method may be interesting for self-healing material applications.

[0070] In embodiments, the biomolecule especially comprises one of a drug, an enzyme, a polymer, and a chromophore. Especially, in embodiments, the biomolecule may comprise a polymer. Such embodiments may especially be beneficial as polymers may have a positive influence on mechanical properties (such as e.g. strength, elasticity, or ductility) of a material (such as a pellicle). Hence, the method may be beneficial for in situ modification of materials.

[0071] In a yet further aspect, the invention may provide a method for in situ modification of mechanical properties in a pellicle. In embodiments, the method may comprise providing a pellicle as described herein or obtainable according to the method as described herein. The pellicle may especially comprise (i) a matrix material comprising bacterial cellulose and (ii) bacterial endospores comprising Bacillus paralicheniformis. Bacterial endospores comprising Bacillus paralicheniformis may be advantageous as Bacillus paralicheniformis may be highly specialized in producing (bio)polymers, such as especially poly(y-glutamic acid) (PGA). These polymers may not only exhibit excellent mechanical properties in a dried state, but may also have multiple known roles in healthcare products, such as skincare products. Hence, the method may further, in embodiments, comprise producing a biomolecule according to the method described above. In such embodiments, the biomolecule may especially comprise a polymer configured to enhance the structural integrity of the pellicle. Especially, in embodiments, the polymer may be selected from the group comprising: poly(y-glutamic acid), poly~[3 ( 1 -6)- / V-acetylglucosamine, a biofilm surface layer protein, a silk protein, a bacterial flagella protein, elastin, keratin, melanin, hyaluronic acid, and poly-dopamine. More especially, in embodiments, the polymer may comprise poly(y-glutamic acid). Hence, in embodiments, the invention may provide a method for in situ modification of mechanical properties in a pellicle, the method comprising: (A) providing a pellicle as described herein or obtainable according to the method as described herein, wherein the pellicle may comprise a matrix material comprising bacterial cellulose and bacterial endospores comprising Bacillus paralicheniformis and (B) producing a biomolecule according to the method as described herein, wherein the biomolecule may comprise a polymer configured to enhance the structural integrity of the pellicle, wherein the polymer may be selected from the group comprising: poly(y-glutamic acid), poly-p (1-6)- A-acetylglucosa ine, a biofilm surface layer protein, a silk protein, a bacterial flagella protein, elastin, keratin, melanin, hyaluronic acid, and poly-dopamine. In embodiments, the mechanical properties may especially comprise one or more properties selected from the group comprising: elasticity, ductility, strength, flexibility, toughness etc... Such in situ modification of mechanical properties in a pellicle may especially be useful for applications relating to self- healing materials.

[0072] In a yet further aspect, the invention may provide a method of biosensing. Herein the term “biosensing” may refer to an analytical technology for detecting external stimuli (such as e.g. the presence of a molecule of interest (or target molecule) or the presence of a type of (e.g. heavy metal) ions) by applying a biosensor (e.g. a combination of a sensitive biological component (i.e., the herein described biomolecule) with a physicochemical detector) to an analyte, an aliquot, or a test sample. Hence, in embodiments, the biomolecule may be configured to induce a(n optical) change in the pellicle when exposed to another species. Especially, in embodiments, the other species may comprise one or more of a molecule of interest (or target molecule) or an ion (such as e.g. a heavy metal ion like lead). More especially, in embodiments, the other species may comprise one of a chemical species (i.e. a molecule or ion). Alternatively or additionally, in embodiments the other species may comprise a biological species (i.e. a bacterium (in embodiments especially other than the bacterial cellulose- producing bacterium and / or the bacteria comprised by the spores) or fungus).

[0073] The method may especially comprise a preparation stage and a detection stage. In embodiments, the preparation stage may comprise providing a biomolecule. Especially, the preparation stage may comprise providing a biomolecule according to the herein described method for on demand production of a biomolecule. Further, in embodiments, the detection stage may comprise detecting change, such as especially optical change, in the pellicle using the biomolecule. Additionally or alternatively, in embodiments, the detection stage may comprise detecting a change in mechanical properties of the pellicle. Therefore, in embodiments, the bacterial (endo)spore may be genetically engineered to produce a biomolecule configured to induce an optical change in the pellicle when exposed to another species. The biomolecule may especially comprise one of a drug, an enzyme, a polymer, and a chromophore. In embodiments, the one or more biomolecules may comprise an enzyme, such as P-galactosidase. Additionally or alternatively, the one or more biomolecules may comprise a chromophore, such as green fluorescent protein. Hence, in embodiments, the invention may provide a method of biosensing comprising: a preparation stage comprising providing a biomolecule using the method as described above; and a detection stage comprising detecting optical change in the pellicle using the biomolecule. The detection stage may thus comprise detecting optical change in the pellicle. For example, in embodiments, the optical change may comprise one or more of a change in color, turbidity, and dispersion.

[0074] In embodiments, the detection stage may comprise detecting optical change by eye. Additionally or alternatively, in embodiments, the detection stage may comprise detecting optical change using an optical detection method, such as optical microscopy, spectrophotometry, scanning electron microscopy (SEM), transmission electron microscopy (TEM), or (other) optical microscopic methods.

[0075] As described above, in embodiments, the biomolecule may comprise green- fluorescent protein. Such embodiments may be beneficial as it provides a real world application of the pellicle in glycerol biosensing. Glycerol is a well-known food additive (e.g., n hydration agent in sports drinks) and a by-product of fermentation (e.g., in winemaking). Both of the aforementioned applications of glycerol are highly dependent on continuous quality monitoring. The pellicle of the invention may provide a glycerol-inducible expression system. For example, as indicated below with respect to the experiments, the pellicle may provide a glycerol-inducible expression system based on the glpD promoter (named as Pgly), which is regulated by the antiterminator protein Glp. A plasmid containing this regulatory module was assembled with standardized transcriptional modules from STK as described in Koo, B.M. et al. (2017), Construction and Analysis of Two Genome-Scale Deletion Libraries for Bacillus subtilis. Cell Syst, which is hereby herein incorporated by reference, and then introduced into the B. subtilis strain. Spores from the strain were then incorporated into a bacterial cellulose matrix to produce a functional glycerol-responsive engineered living material (ELM). This ELM showed successful sensing of glycerol in drinking samples for the purpose of food quality monitoring. Hence, the invention may provide a glycerol-biosensing pellicle capable of detecting glycerol in drinking water. This system utilizes the spores of Bacillus subtilis. Once germinated, the vegetative cells may respond to the presence of glycerol by producing green fluorescent protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Embodiments of the invention will now be described, by way of example only, with reference to the accompanying schematic drawings in which corresponding reference symbols indicate corresponding parts, and in which: Figs. 1-3 schematically depict some embodiments of the system of the invention. The schematic drawings are not necessarily on scale.

[0077] DETAILED DESCRIPTION OF THE EMBODIMENTS

[0078] Fig. 1 schematically depicts an embodiment of a pellicle 1 comprising a matrix material 50 and an activatable material 10. In embodiments, the pellicle 1 may especially comprise a dry paper-like sheet.

[0079] The activatable material 10 may especially be configured embedded in the matrix material 50. Furthermore, the matrix material 50 may comprise bacterial cellulose 55. Especially, in embodiments, the bacterial cellulose 55 may be (obtainable) from one or more of Komagataeibacter rhaelicus. Acetobacter xyHnum. Sarcina ventriculi, Acetobacter hansenii, Acetobacter pasleiirianus, Gluconacetobacter medellensis, Gluconacetobacter persimmonis, Gluconacetobacter sp., and Gluconacetobacter hansenii, such as especially Komagataeibacter rhaeticus. Conversely, the activatable material 10 may comprise (dormant) bacterial (endo)spores 15. Especially, in embodiments, the bacterial endospores 15 belong to a bacterial species selected from the group comprising Bacillus subtilis, Bacillus paralicheniformis, Sporosarcina pasteurii, Bacillus pasteurii, Bacillus megaterium, Helicobacter pylori, Pseudomonas aeruginosa, Clostridium sporogenes, Clostridium botulinum, and Clostridium difficile, such as especially a bacterial species selected from Bacillus subtilis and Bacillus paralicheniformis.

[0080] Furthermore, the invention may provide a method for the production of a pellicle 1 (suitable for long-term storage) comprising bacterial cellulose 55 and (dormant) bacterial (endo)spores 15. The method is schematically depicted in Fig. 1. As depicted in Fig. 1, the method may comprise a culture stage comprising culturing a bacterial cellulose- producing bacterium 51 (during the culture stage) in the presence of the bacterial spores 15. Such culturing may especially be executed in growth conditions (such as a growth medium 3 and a growth medium pH) suitable for (i) culturing the bacterial cellulose-producing bacterium and (ii) maintaining (dormancy of) the bacterial spores 15. Subfigure I schematically depicts a holder 6, such as e.g. a test tube, comprising a mixture of a growth medium 3, the matrix material 50 comprising the bacterial cellulose 55, and the activatable material 10, comprising the bacterial spores 15. In specific embodiments, (i) the bacterial (endo)spores 15 may comprise a bacterium selected from Bacillus subtilis and Bacillus paralicheniformis, (ii) the bacterial cellulose-producing bacterium 51 may comprise Komagataeibacter rhaelicus. (iii) the growth medium 3 may comprise yeast extract, peptone and dextrose, and (iv) the growth medium 3 may have a growth medium pH selected from the range of 3.5-4.5, such as especially a growth medium pH of 4.0.

[0081] The bacterial spores 15 may be embedded in the bacterial cellulose 55. However, due to a difference in density between the bacterial spores 15 and the growth medium 3, the bacterial spores 15 may tend to sediment. To prevent sedimentation, i.e. to keep the bacterial spores 15 afloat, the growth medium 3 may comprise (a density gradient medium comprising) iohexol in an amount selected from the range of 25-45 wt.%, such as especially 40 wt.%.

[0082] Subfigures II and III schematically depict the holder 6 comprising the mixture over time. As can be derived from the figure, the bacterial cellulose-producing bacterium 51 may form the matrix material 50, especially the bacterial cellulose 55, at an interface between the growth medium 3 and the (ambient) air 4. Especially, the bacterial cellulose 55 may be formed at the air-liquid interface.

[0083] After pellicle 1 formation in the culture stage the method may comprise a processing stage. In embodiments, the processing stage may comprise drying at least part of the thus obtained material, thereby providing the pellicle 1. In embodiments, the method may comprise one of air drying or freeze drying, such as especially freeze drying. In embodiments, the pellicle 1 may comprise an amount of bacterial spores 15 selected from the range of IxlO4- 2xl06CFU.

[0084] Fig. 2 schematically depicts a method for on demand production of a biomolecule 20. As depicted in subfigure I, the method may comprise a preparation stage comprising providing the pellicle 1. In embodiments, the pellicle 1 may comprise a bacterial (endo)spore 15 configured to produce the biomolecule 20 upon (especially after or in response to) germination. As depicted in subfigure II, the preparation stage may further comprise providing a germination fluid 2. In embodiments, the germination fluid 2 may comprise a (nutrient-rich) medium suitable for germination of the bacterial spores 15. Especially, as depicted in subfigure II, the method may further comprise an activation stage comprising germinating the bacterial spores 15 in the pellicle 1. Germinating the bacterial spores 15 may, in embodiments, be achieved by contacting (such as e.g. soaking) the pellicle 1 with the germination fluid 2 to activate production of the biomolecule 20 (in the pellicle 1). In embodiments, the biomolecule may comprise one of a drug, an enzyme, a polymer, and a chromophore.

[0085] In specific embodiments, the invention further provides a method of biosensing. The method may comprise a preparation stage comprising providing a biomolecule 20 as described above. Furthermore, as depicted in subfigure III, the method of biosensing may comprise a detection stage comprising detecting optical change in the pellicle 1 using the biomolecule 20. Therefore, in embodiments, the bacterial spore 15 may be genetically engineered to produce a biomolecule 20 configured to induce an optical change in the pellicle

[0086] 1 when exposed to another species 7 (such as e.g. the presence of a molecule of interest or a (heavy metal) ion). In embodiments, the biomolecule 20 may be produced and kept inside the bacterial spore 15. Additionally or alternatively, in embodiments, the biomolecule may be secreted outside the bacterial spore 15, i.e., such as depicted with the triangles depicted in Fig.

[0087] 2 subfigure III. Figure 2 may thus schematically illustrate how the incorporated Bacillus endospores 15 may confer a sense-and-respond function to the pellicle 1. The bacterial endospores 15 may undergo germination under growth-permissive conditions, and the resulting vegetative cells may produce the biomolecule 20 (such as e.g. a chromophore like green fluorescent protein) after specific stimulation (with a molecule of interest 7, such as e.g. a stimulus).

[0088] Further, the invention may provide a method for in situ modification of mechanical properties in a pellicle 1. The method may, in embodiments, especially comprise providing a pellicle 1 as described above. Especially, in embodiments, the pellicle 1 may comprise a matrix material 50 comprising bacterial cellulose 55 and bacterial endospores 15 comprising Bacillus paralicheniformis. The method may further, in embodiments, comprise producing a biomolecule 20, wherein the biomolecule 20 may comprise a polymer configured to enhance the structural integrity of the pellicle 1. Especially, in embodiments, the polymer (depicted as the triangles in Fig. 2 subfigure III) may be selected from the group comprising: poly(y-glutamic acid), poly-p (l-6)-A-acetylglucosamine, a biofilm surface layer protein, a silk protein, a bacterial flagella protein, elastin, keratin, melanin, hyaluronic acid, and polydopamine.

[0089] The invention provides a use of the pellicle 1 as described herein in one or more of: drug delivery, biosensing, biocatalysis, skincare (products), food-packaging (products). Fig. 3 A therefore schematically depicts examples of a device 100 comprising the pellicle 1. Especially, the device 100 may be selected from the group comprising a biosensor, a skincare product, a drug delivery device, a biocatalytic device, and a foodpackaging device.

[0090] Fig. 3B further schematically depicts, a kit-of-parts 105 comprising (the device 100 comprising) the pellicle 1 and (verbal and / or picture-based) instructions for use and / or a reference (such as a QR code or hyperlink) to instructions for use. Moreover, in embodiments, the kit-of-parts may comprise a holder 6 comprising a germination fluid 2. The germination fluid 2 may, in embodiments, comprise a (nutrient-rich) medium suitable for germination of the bacterial (endo)spores 15. As depicted here, in embodiments, the kit-of- parts may further comprise a container 9 configured to host one or more of the pellicle 1, the holder 6, and the (reference to) instructions for use.

[0091] Fig. 4 schematically depicts (genetic) modification of the bacterial (endo)spores 15. In embodiments, the bacterial cellulose-producing bacterium 51 may comprise genetically modified bacterial cellulose-producing bacterium. Additionally or alternatively, in embodiments, the bacterial (endo)spores 15 may comprise genetically modified bacterial (endo)spores.

[0092] For example, in embodiments, the bacterial endospores 15 may be genetically modified, such that changes to the spore surface may be realized. Especially, in embodiments, the bacterial endospores 15 may (be engineered, using DNA-encoded engineering, to) comprise Cellulose Binding Module 40 functionalized bacterial endospores.

[0093] As schematically depicted in step I of Fig. 4, bacterial endospores 15 (especially Bacillus spores) may generally comprise a core 151 and multiple coat layers, especially an inner membrane 152, a cortex 153, a coat 154, and a glycan layer 155. In embodiments, the bacterial endospores 15 may be genetically modified, such that the glycan layer 155 may be removed to improve the efficiency of incorporating the Cellulose Binding Module 40 on the spore coat 154. Hence, in step II, Cellulose Binding Module 40 expression may be induced, such that the bacterial endospore 15 depicted in step III comprising the Cellulose Binding Module 40 configured on the spore coat 154 may be obtained. Further, for step IV, a technique such as e.g. gene deletion may be applied to obtain the bacterial endospore 15 depicted in step V where the glycan layer 155 is removed.

[0094] Figs. 5 schematically depict a method for enriching a number of bacterial (endo)spores 15 in a (bacterial) pellicle 1. Fig. 5 A subfigure I especially schematically depicts the pellicle 1, obtainable by the above described method, comprising the bacterial cellulose 55 and an initial amount of bacterial (endo)spores 15. Conversely, Fig. 5 A subfigure II schematically depicts an enriched pellicle 11, obtainable by the method for enriching a number of bacterial (endo)spores 15, comprising the bacterial cellulose 55 and an enriched (i.e. increased) amount of bacterial (endo)spores 15.

[0095] The method may especially comprise an enrichment stage comprising germinating the bacterial spores 15 (in the pellicle 1 further comprising bacterial-cellulose 55) to obtain a mixture comprising the bacterial cellulose 55 and vegetative bacterial cells 25. As depicted in Fig. 5B, germination may especially comprise providing the germination fluid 2 to the pellicle 1. Subsequently, as depicted in the next step in the flow diagram of Fig. 5B, the enrichment stage may comprise sporulating the vegetative bacterial cells 25 (to obtain an enriched (wet) pellicle 11 comprising an increased viable cell count of bacterial spores 15). For example, in embodiments, a sporulation medium 8 may be added to the mixture (comprising the pellicle 1) after germination. For example, in embodiments, the sporulation medium 8 may comprise Schaeffer’s sporulation medium (8 g / L Difco nutrient broth, 1 g / L KC1, 1 g / L MgSO4*(7 H2O), 0.002 g / L MnCl2*(4 H2O), 0.5 mM CaCl2, 1 pM FeSO4). After sporulation, in embodiments, the method may comprise washing the mixture in a wash buffer 5. Following the washing, in embodiments, the method may comprise drying the washed mixture to obtain an enriched pellicle 11. In embodiments, the pellicle 1 may thus be replaced with the enriched pellicle 11. Alternatively, in embodiments, the pellicle 1 may comprise the enriched pellicle 11. In embodiments, the enriched pellicle 11 may comprise an amount of bacterial spores (15) selected from the range of IxlO7- IxlO10CFU.

[0096] Materials and Methods

[0097] Unless specified otherwise, the experiments described herein were performed using the following materials and methods.

[0098] Engineering culture conditions to grow BC-spore living materials - A bacteria mixture of K. rhaeticus wildtype (WT) cells and Bacillus spores was cultured in YPD medium acidified to pH 4.0 and supplemented with 40 wt. % iohexol. Bacillus spores from two species (B. subtilis NS1, B. paralicheniformis WT) stayed dormant at this pH. The B. subtilis strain NS1 was obtained from Piersma, S. et al. (2013), TLM-Quant: An Open-Source Pipeline for Visualization and Quantification of Gene Expression Heterogeneity in Growing Microbial Cells. PLoS One, which is hereby herein incorporated by reference. B. subtilis containing a genomically inducible green fluorescent protein (GFP) was used, because wild-type B. subtilis did show some evidence of germination at pH 4.0. The iohexol supplementation increased the number of spores incorporated by 4.5-fold for B. subtilis NS1 and 49-fold for B. paralicheniformis WT, by delaying the sedimentation of spores. The bacteria mixture formed a BC pellicle which could be readily worked by hand.

[0099] The cell number and their spatial distribution within BC-spore living materials were characterized, using a GFP expressing strain of K. rhaeticus (K. rhaeticus GFP) and fluorescently dyed spores. K. rhaeticus GFP cells were dominant in total number (108) compared to B. subtilis NS1 (8* 105) and B. paralicheniformis WT (6x 105) spores.

[0100] Once growth-permissive conditions are available, the encapsulated spores should germinate and reproduce. As proof-of-concept BC-spore living materials were incubated in lysogeny broth (LB) medium, and viable Bacillus cells per pellicle were counted by plating serial dilutions after hydrolyzing the pellicles. After 24 hours of incubation, viable cell counts revealed an increase of colony -forming units (CFU) per pellicle for both B. subtilis NS1 (4.1 >< 105to 6.3>< 106CFU) and / f paralicheniformis WT (5.4x l05to 1.7x l06CFU). These results indicate that Bacillus spores within materials may switch to a metabolically active state and initiate a vegetative cell-cycle.

[0101] Experiments

[0102] Programmable spore functionality - To investigate whether Bacillus cells, after germination, can endow programmable functionalities to the pellicles, a function of sense and respond was conferred to the pellicles by integrating spores with inducible GFP expression in pellicles grown by K. rhaeticus WT. To this end, two Bacillus strains, B. subtilis NS1 and B. paralicheniformis GFPind were used. The former strain contained a GFPmut2 sequence under control of a IPTG-inducible promoter, which was inserted into the amyE site in the genome. The latter strain was prepared by transforming wild-type B. paralicheniformis with plasmid, pAD-sfGFP-Pman, harboring a sfGFP sequence under the control of a mannose-inducible promoter. The GFP expression conditions of both strains were optimized for the concentration of the inducers. The plasmid pAD-sfGFP-Pmanhas a sequence according to SEQ ID No. 2. Moreover, the plasmid pAD-sfGFP-Pmanis described in Yi, Y., Frenzel, E., et. al. (2018), “Optimized fluorescent proteins for the rhizosphere-associated bacterium Bacillus mycoides with endophytic and biocontrol agent potential”, Environmental microbiology reports, IO( \ ), 57-74, which is hereby herein incorporated by reference.

[0103] The spores were germinated within pellicles, an inducer was added, and fluorescence of whole pellicles was recorded. The results revealed a clear signal from the pellicles for both strains compared to controls (no induction). Consistent with those results, confocal imaging exhibited that individually distributed Bacillus cells clearly expressed GFP within the pellicles. These results confirmed that programmable functions may be conferred to the pellicle by integrating the spores from engineered Bacillus cells.

[0104] Resistant spore functionality - To investigate if the Bacillus spores’ resistant and dormant properties are maintained in the BC-spore material, pellicles were treated with GFP inducible spores under harsh conditions. The fold-change was evaluated using green fluorescence intensity. The materials’ functionality was mostly maintained after 1 h treatment with 70% ethanol, and 30 min of illumination with UV-A (wavelength distributed around 365 nm and intensity of 21.8 mW / cm2), but not after 1 h of illumination with UV-A. It should be noted that all pellicles were heated to 70 °C prior to germination to eliminate ", rhaeticus cells. In addition, pellicles were air- or freeze-dried, transforming them to thin paper-like materials. After germination freeze-dried pellicles exhibited fold-changes comparable to untreated ones, whereas the fold-change was reduced for air-dried pellicles. The air-drying method caused less porosity and swelling of the cellulose fiber networks compared to freeze-drying. To test dormant functionalities of the materials, air- or freeze-dried pellicles with 7>. paralicheniformis GFPind or B. subtilis NS1 spores were stored for up to 6 months at room temperature. After 6 months, the pellicles still displayed spore germination and a GFP signal was successfully detected. These results confirm that the integrated spores can survive under the tested conditions and keep their dormancy, enabling various treatments and on-demand use of the BC-spore material.

[0105] Utilization of BC-spore enzyme-functionalized pellicle - Living materials’ functionality mainly relies on the living components’ specialties. B. subtilis is well- characterized as microbial specialist for high-level protein secretion. By leveraging the protein secretion system of B. subtilis, the pellicles were functionalized as catalytic materials by enzyme secretion. As a proof of concept, B. subtilis NS1 was engineered to secrete P- galactosidase fused to the PhoD secretion peptide under a PIP promoter, from a newly designed plasmid pFJOSJL -LacZ (B. subtilis U \ P-gal). The plasmid pFJOSJL -LacZ has a sequence according to SEQ ID No. 1. The enzyme activity within cellulose was confirmed using chromogenic substrate X-P-gal, that was degraded into a dark blue compound.

[0106] Pellicles were prepared using a mixture of K. rhaeticus WT and the spores from B. subtilis NS1 P-gal. Following the germination of spores and the growth of vegetative cells within the pellicle, the enzyme activity from pellicles was confirmed, as indicated by the color change. These results indicated that the BC can be functionalized by enzymes secreted by B. subtilis NS1 P-gal. Further, it was observed that increased incubation times of pellicles in medium, increases the catalytic activity. Engineering spores ’binding affinity to the matrix material - It was assumed that pellicles with a higher number of incorporated spores might display a decreased functionalization time, by decreasing the growth time to achieve sufficient biomass within the pellicle for production of reporter molecules or enzymes. As proof-of-concept the spores’ binding affinity to BC was modified by both modifying the spore coat (i.e. a crust layer of coat protein) and removing the outermost glycan layer. B. subtilis spores have hierarchical proteinaceous layers (core, inner membrane, cortex, and outer coat or crust), in which CgeA plays as a linker between the crust and the encapsulating glycan (or polysaccharide) layer. A cellulose binding module (CBM) from Clostridium thermocellum was displayed on the crust proteinaceous layer by fusing the domain with CotY, which is the one of main crust components, and mScarletl, a red fluorescent protein for confirmation of expression. The synthetic gene circuit was integrated in the ThrC ectopic site, yielding strain B. subtilis NS2. The cgeA deletion in B. subtilis NS2 was conducted with the insertion of kanamycin resistance cassette into the cge gene clusters, creating strain B. subtilis NS3. It was confirmed that the engineered strain B. subtilis NS3 expressed CBM and that no halo like-structure of polysaccharide was visible on the surface.

[0107] Interestingly, there was a striking improvement in BC-integration efficiency when the polysaccharide layer was removed in B. subtilis strain NS3 (CBM expression, with cgeA deletion), reaching an incorporation efficiency of 10%. This engineered strain displayed similar spore germination properties and spore density, and the vegetative cells showed no difference in growth kinetics compared to its parental strain NS1. These results indicated that removal of the polysaccharide layer can improve the affinity between an incorporation matrix and spores.

[0108] In situ modification of BC-spore composites- Among genus Bacillus, B. paralicheniformis are highly specialized in producing gamma-polyglutamic acid (y-PGA). These polymers not only exhibit excellent mechanical properties as dried state, but also have multiple roles in healthcare products. BC-PGA composites were made by leveraging the PGA production capability of B. paralicheniformis WT within pellicles.

[0109] First, the spore germination and growth conditions of B. paralicheniformis WT in medium E, known as PGA production media, were characterized. B. paralicheniformis WT spores successfully germinated in medium E, but exhibited barely any growth compared to normal medium. Interestingly, the germinated cells could produce PGA, as indicated by glutamate conversion in medium E. Then, PGA production within BC containing B. paralicheniformis WT spores was characterized. The pellicles were incubated within medium E for spore germination, taken out, and incubated within petri-dishes. The residual glutamate within the pellicles was monitored. The residual glutamate concentration decreased daily, and was mostly depleted after 3 days. The stiffness of BC pellicles also decreased due to slight cellulase activity from the vegetative cells, but it doesn’t affect the further manipulation of pellicles.

[0110] Having established PGA production conditions, BC-PGA film was prepared and its mechanical properties were characterized. In comparison, the mechanical properties of the materials were highly improved compared to normal dried BC films. BC film exhibited less than 1% elongation at break, however, the BC-PGA film showed more than 50-fold increase of elongation.

[0111] Glycerol detection - To demonstrate a real -world application of BC-spore materials as a biosensor, a k subtilis strain was engineered to detect the presence of glycerol.

[0112] For these experiments, cgeA was deleted in B. subtilis NS1, yielding strain B. subtilis NS4. Using Golden Gate assembly, a plasmid (pKM_bsO2) containing a glycerolinducible promoter (Pg / y) driving the expression of GFP (GFPmut3), was constructed. This plasmid was transformed into the B. subtilis strain NS4 to enable GFP expression in response to glycerol.

[0113] To construct the inducible reporter expressing plasmids, 10 pl Golden Gate reaction containing 50 fmol of the expression vector (STK201) and 50 fmol of the STK basic parts were mixed with 0.5 pl (10 units) of Bsal (a Type Ils restriction enzyme) and 0.5 pl (200 units) of T4 ligase in 1* T4 ligase buffer. The reaction mixture was cycled 30 times at 37 °C for 2 min and 16 °C for 5 min. The reaction mixture was then incubated at 65 °C for 5 min to denature the enzymes and was directly used to transform E. coli and plated on LB agar plates with 100 pg / ml ampicillin. Successful transformants were indicated by non-fluorescent colonies. Glycerol-inducible promoter (pGly) was used as a promoter, and GFPmut3b was used as a fluorescence reporter. As a ribosomal binding site (RBS), optimal RBS + 5 A was used. A strong synthetic terminator (BBa_B0015) was used for transcriptional termination. Final constructs were verified via full plasmid sequencing. The resulting plasmid pKM_bsO2 has a sequence according to SEQ ID No. 3.

[0114] Constructed plasmids were transformed into Bacillus subtilis strain NS4 using a modified MC medium -based transformation protocol taken from Koo, B.M. et al. (2017), Construction and Analysis of Two Genome-Scale Deletion Libraries for Bacillus subtilis. Cell Syst, which is hereby herein incorporated by reference. Transformants were selected on LB agar containing 5 pg / mL erythromycin. The glycerol-responsiveness of pellicles was tested in the following samples: (1) LB with and without glycerol, (2) drinking water with and without 0.5% glycerol, and (3) drinking water with 5% ethanol, with and without 0.5% glycerol. Pellicles containing B. subtilis NS4 pKM_bsO2 were placed in LB medium (7 mL in a 6-well plate, 37°C, 80 rpm) for 8 hours to induce germination of the spores and growth of vegetative cells. For condition (1), glycerol or MilliQ water (negative control) was added directly to the LB medium. For conditions (2) and (3), half of the LB medium was replaced with the respective drinking water samples containing glycerol or drinking water alone as a negative control. Following an additional 8- hour incubation, GFP expression in the full pellicles was measured using the Amersham Typhoon Scanner (Cytiva, Marlborough, USA) with 488 nm excitation and 525 / 20 nm emission.

[0115] Vegetative cells incubated with and without glycerol showed a significant difference in GFP expression, through a fluorescence intensity difference between the two samples of at least 3000 a.u.. Furthermore, the experiments showed that B. subtilis spores embedded in pellicles could germinate, respond to glycerol induction, and produce fluorescence. A clear GFP signal was observed in induced samples compared to non-induced controls. To simulate the detection of 0.5% glycerol in hydration drinks and wine, as model environments drinking water and drinking water with 5% ethanol were used. Under these conditions, BC-spore materials still produced detectable GFP signals, suggesting their potential applicability in food and beverage quality monitoring.

[0116] The term “plurality” refers to two or more. Furthermore, the terms “a plurality of’ and “a number of’ may be used interchangeably. The terms “substantially” or “essentially” herein, and similar terms, will be understood by the person skilled in the art. The terms “substantially” or “essentially” may also include embodiments with “entirely”, “completely”, “all”, etc. Hence, in embodiments the adjective substantially or essentially may also be removed. Where applicable, the term “substantially” or the term “essentially” may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%. Moreover, the terms ’’about” and “approximately” may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%. For numerical values it is to be understood that the terms “substantially”, “essentially”, “about”, and “approximately” may also relate to the range of 90% - 110%, such as 95%-105%, especially 99%-l 01 % of the values(s) it refers to. The term “comprise” also includes embodiments wherein the term “comprises” means “consists of’. The term “and / or” especially relates to one or more of the items mentioned before and after “and / or”. For instance, a phrase “item 1 and / or item 2” and similar phrases may relate to one or more of item 1 and item 2. The term "comprising" may in an embodiment refer to "consisting of but may in another embodiment also refer to "containing at least the defined species and optionally one or more other species". Use of the verb "to comprise" and its conjugations does not exclude the presence of elements or steps other than those stated in a claim. Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, “include”, “including”, “contain”, “containing” and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”. The article "a" or "an" preceding an element does not exclude the presence of a plurality of such elements.

[0117] Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

[0118] The devices, apparatus, or systems may herein amongst others be described during operation. As will be clear to the person skilled in the art, the invention is not limited to methods of operation, or devices, apparatus, or systems in operation.

[0119] The term “further embodiment” and similar terms may refer to an embodiment comprising the features of the previously discussed embodiment, but may also refer to an alternative embodiment.

[0120] It should be noted that the above-mentioned embodiments illustrate rather than limit the invention, and that those skilled in the art will be able to design many alternative embodiments without departing from the scope of the appended claims. In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim.

[0121] The invention may be implemented by means of hardware comprising several distinct elements, and by means of a suitably programmed computer. In a device claim, or an apparatus claim, or a system claim, enumerating several means, several of these means may be embodied by one and the same item of hardware. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.

[0122] The invention also provides a control system that may control the device, apparatus, or system, or that may execute the herein described method or process. Yet further, the invention also provides a computer program product, when running on a computer which is functionally coupled to or comprised by the device, apparatus, or system, controls one or more controllable elements of such device, apparatus, or system.

[0123] The invention further applies to a device, apparatus, or system comprising one or more of the characterizing features described in the description and / or shown in the attached drawings. The invention further pertains to a method or process comprising one or more of the characterizing features described in the description and / or shown in the attached drawings. Moreover, if a method or an embodiment of the method is described being executed in a device, apparatus, or system, it will be understood that the device, apparatus, or system is suitable for or configured for (executing) the method or the embodiment of the method, respectively. The various aspects discussed in this patent can be combined in order to provide additional advantages. Further, the person skilled in the art will understand that embodiments can be combined, and that also more than two embodiments can be combined. Furthermore, some of the features can form the basis for one or more divisional applications.

[0124] The project leading to this application has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No 101071159.

Claims

CLAIMS:

1. A pellicle (1) comprising a matrix material (50) and an activatable material (10), wherein the activatable material (10) is configured embedded in the matrix material (50), wherein the matrix material (50) comprises bacterial cellulose (55), wherein the bacterial cellulose (55) is from Komagataeibacter rhaelicus. and wherein the activatable material (10) comprises bacterial spores (15), wherein the bacterial spores (15) belong to a bacterial species selected from Bacillus paralicheniformis.

2. The pellicle (1) according to claim 1, wherein the bacterial spores (15) comprise Cellulose Binding Module (40) functionalized bacterial spores.

3. The pellicle (1) according to any one of the preceding claims, wherein the pellicle (1) is a paper-like sheet.

4. The pellicle (1) according to any one of the preceding claims, wherein the pellicle (1) comprises a bacterial spore (15) configured to produce a biomolecule (20) upon germination, wherein the biomolecule (20) comprises one of a drug, an enzyme, a polymer, and a chromophore.

5. The pellicle (1) according to claim 4, wherein the biomolecule comprises poly- y-glutamic acid.

6. The pellicle (1) according to any one of the preceding claims, wherein the pellicle (1) comprises a concentration of iohexol of >5 ppm.7 The pellicle (1) according to any one of the preceding claims, wherein the pellicle (1) comprises an amount of bacterial spores (15) selected from the range of IxlO4- 2xl06CFU.

8. The pellicle (1) according to any one of the preceding claims, wherein one or more of the following applies: (i) the bacterial cellulose (55) comprises bacterial cellulose froma genetically modified bacterial cellulose-producing bacterium, and (i) the bacterial spores (15) comprise genetically modified bacterial spores.

9. A device (100) comprising the pellicle (1) according to any one of the preceding claims, wherein the device (100) is selected from the group comprising a biosensor, a skincare product, a drug delivery device, a biocatalytic device, and a food-packaging device.

10. A kit-of-parts (105) comprising: a pellicle (1) according to any one of the preceding claims 1-8; and instructions for use and / or a reference to instructions for use.

11. The kit-of-parts (105) according to claim 10, further comprising a holder (6) comprising a germination fluid (2), wherein the germination fluid (2) comprises a medium suitable for germination of the bacterial spores (15).

12. A method for the production of a pellicle (1) comprising bacterial cellulose (55) and bacterial spores (15), wherein the bacterial spores (15) are configured embedded in the bacterial cellulose (55), the method comprising: a preparation step comprising preparing a growth medium (3) suitable for culturing the bacterial cellulose-producing bacterium (51) in the presence of the bacterial spores (15), wherein the growth medium (3) further comprises 25-45 wt.% iohexol relative to the total weight of the growth medium (3); a culture stage comprising culturing bacterial cellulose-producing bacterium (51) in the presence of the bacterial spores (15) in growth conditions suitable for (i) culturing the bacterial cellulose-producing bacterium (51) and (ii) maintaining the bacterial spores (15); and a processing stage comprising drying at least part of the thus obtained material, thereby providing the pellicle (1).

13. The method according to claim 12, wherein the bacterial spores (15) comprise a bacterium selected from Bacillus subtilis and Bacillus paralicheniformis, wherein the bacterial cellulose-producing bacterium (51) comprises Komagataeibacter rhaeticus.

14. The method according to any one of the preceding claims 12-13, wherein one or more of the following applies: (i) the bacterial cellulose-producing bacterium (51) comprises genetically modified bacterial cellulose-producing bacterium, and (i) the bacterial spores (15) comprise genetically modified bacterial spores.

15. The method according to any one of the preceding claims 12-14, wherein the bacterial spores (15) comprise Cellulose Binding Module (40) functionalized bacterial spores; and wherein the growth medium (3) comprises yeast extract, peptone and dextrose, and wherein the growth medium (3) has a growth medium pH selected from the range of 3.5 -4.5.

16. The method according to any one of the preceding claims 12-15, wherein the bacterial spores (15) are configured to produce a biomolecule (20) upon germination, wherein the biomolecule (20) comprises one of a drug, an enzyme, a polymer, and a chromophore.

17. The pellicle (1) according to claim 16, wherein the biomolecule comprises poly- y-glutamic acid.

18. The method according to any one of the preceding claims 12-17, wherein the method comprises freeze drying the pellicle (1).

19. The method according to any one of the preceding claims 12-18, further comprising an enrichment stage, wherein the enrichment stage comprises: germinating the bacterial spores (15), by providing a germination fluid (2) to the pellicle (1), to obtain a mixture comprising the bacterial cellulose (55) and vegetative bacterial cells (25); sporulating the vegetative bacterial cells (25); washing the mixture in a wash buffer; and drying the washed mixture to obtain an enriched pellicle (11).

20. The method according to claim 19, wherein the enriched pellicle (11) comprises an amount of bacterial spores (15) selected from the range of IxlO7- IxlO10CFU.

21. A method for on demand production of a biomolecule (20), wherein the method comprises:a preparation stage comprising: providing (i) a pellicle (1) according to any one of the preceding claims 1-8 or obtainable by the method according to any one of the preceding claims 12-20, and (ii) a germination fluid (2), wherein the germination fluid (2) comprises a medium suitable for germination of the bacterial spores (15); wherein the pellicle (1) comprises a bacterial spore (15) configured to produce the biomolecule (20) upon germination; an activation stage comprising germinating the bacterial spores (15) in the pellicle (1) by contacting the pellicle (1) with the germination fluid (2) to activate production of the biomolecule (20).

22. The method according to claim 21, wherein the biomolecule comprises poly-y- glutamic acid.

23. The method according to claim 22, wherein the biomolecule comprises one of a drug, an enzyme, a polymer, and a chromophore.

24. A method of biosensing, wherein the method comprises: a preparation stage comprising providing a biomolecule (20) using the method according to any one of the preceding claims 21-23; and a detection stage comprising detecting optical change in the pellicle (1) using the biomolecule (20).

25. The method according to claim 24, wherein the bacterial spore (15) is genetically engineered to produce a biomolecule (20) configured to induce an optical change in the pellicle (1) when exposed to another species.

26. The method according to claim 25, wherein the biomolecule comprises poly-y- glutamic acid.

27. Use of the pellicle according to any one of the preceding claims 1-7 in one or more of: drug delivery, biosensing, biocatalysis, skincare, and food-packaging.

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