Bacterial cellulose scaffolds

Abstract

A method for the production of cellulose in a culture of one or more microorganisms in a culture medium, comprising the step of increasing / stimulating the level of biosynthesis of a macrocyclic primogenitor molecule, such as uroporphyrinogen III.

Description

[0001]F07044 / JS 1 TITLE BACTERIAL CELLULOSE SCAFFOLDS TECHNICAL FIELD The present invention relates to a method for the production of cellulose in microorganisms, such as fungi or bacteria, of high quality. PRIOR ART Cellulose is a polymer of glucose and is the most abundant biopolymer on Earth. Until recently, plants were considered the only source of cellulose available for industrial-scale applications, and the isolation of cellulose was carried out in pulp mills via mechanical and / or chemical transformation of the lignocellulosic materials that constitute most of the plant matter. These processes consume a considerable amount of energy and chemicals. However, cellulose can also be produced by microorganisms, when cultured under suitable conditions. Bacterial cellulose (BC) possesses a unique structure, as secreted nanofibers of cellulose are woven to three-dimensional reticulated network that provides it with excellent mechanical properties, high water holding capability and outstanding suspension stability. It is also characterized with high purity, high degree of crystallinity, great biocompatibility and biodegradability. Due to these advantages, BC has gained great attentions in both academic and industrial areas. BC can be synthesized by a series ofbacteria, such as bacteria from the genera Gluconacetobacter, Novacetimonas hansenii,Aerobacter, Rhizobium, Komagataeibacter rhaeticus, Sarcina, Azotobacter, Agrobacterium,Pseudomonas, and Alcaligenes. When such microorganisms are cultured statically, cellulose polymer chains secreted into the surroundings weave into a three-dimensional reticulated network of cellulose nanofibersto generate a gelatinous pellicle floating at the surface of culture media, which gelatinouspellicle can be harvested for further use. While some microorganisms that produce bacterial cellulose may be cultured in dedicated media, such as Hestrin–Schramm medium, the production of bacterial cellulose is generally F07044 / JS 2 based on agricultural and industrial waste streams, which are used as nutrient sources to reduce the cost in an industrial setting. Pellicles of bacterial cellulose have been used to produce leather-like materials that can be processed just as if they were animal-derived leather, and in essence correspond to a sheet of dewatered and dyed pellicle. It has been observed that the microorganisms that produce such cellulose pellicles sometimes suffer from slow growth and / or low cellulose yields when grown on agricultural and industrial waste. On the other hand, pellicles are not necessarily limited to being grown in planar shape, but can be produced also in more complex shapes, provided the microorganisms that produce bacterial cellulose are cultured under the right conditions and scaffolds. In order to provide bacterial cellulose that can be more efficiently produced, it is necessary to provide a solution to the above-mentioned problems by augmenting growth and / orcellulose yields, as well as the overall quality of the cellulose pellicles grown on agriculturaland industrial waste. SUMMARY OF THE INVENTION The present invention provides a method for enhancing the growth of the microorganisms that produce bacterial cellulose, as well as for enhancing the yields of bacterial cellulose.Moreover, the present invention provides a method that allows to obtain a bacterial cellulosesheet, or pellicle, that has a higher density in cellulose (w / v) and in particular, in which the cellulose has a higher degree of crystallinity. In some embodiments, the bacterial cellulose sheet is colored. Accordingly, it is an object of the present invention to provide a method for the production of cellulose in a culture of one or more microorganisms, in a culture medium, comprising the step of increasing the level of biosynthesis of a macrocyclic primogenitor molecule, in particular of uroporphyrinogen III. It is further an object of the present invention to provide a microorganism chosen from the from the genera Gluconacetobacter, Novacetimonas, Aerobacter, Rhizobium, Komagataeibacter, Sarcina, Azotobacter, Agrobacterium, Pseudomonas, or Alcaligenes,and in particular of the genus Novacetimonas or Komagataeibacter, expressing theenzymes of the C5 pathway, and preferably one or more of glutamate semialdehyde F07044 / JS 3 aminomutase (hemL) and glutamyl-tRNA reductase (hemA), or by overexpression of glutamate semialdehyde aminomutase (hemL), glutamyl-tRNA reductase (hemA), and glutamyl-tRNA synthetase (gltX). Further embodiments of the invention are laid down in the dependent claims. BRIEF DESCRIPTION OF THE DRAWINGS Preferred embodiments of the invention are described in the following with reference to the drawings, which are for the purpose of illustrating the present preferred embodiments of the invention and not for the purpose of limiting the same. In the drawings,Fig. 1 shows a SEM picture of the cellulose pellicle obtained a) after and b) before themethod of the present invention. As the left image shows, cellulose fibers are more abundant and thicker after increasing the biosynthesis of the gltX, HemA, HemL, HemG and HemO, gene products in the producing strain. Accordingly,the cellulose network is more dense, which is apparent as there are less darker areas when compared to the right, where cavities are visible in the less dense cellulose network. Images were acquired on a JEOL JIB-4500 MultiBeam Scanning Electron Microscope (SEM), set to an acceleration voltage of 15.00, mag.: 5000, signal: SEI.Fig. 2 shows a map of the genetic vector (DNA plasmid) that may be used in thecontext of the present invention to increasing / stimulating the level of biosynthesis of a macrocyclic primogenitor molecule, such as uroporphyrinogen III to ultimately enhance the production of bacterial cellulose. The first gene following the ribosome binding site (RBS), is labeled PtxD, and is a gene coding for phosphite dehydrogenase. The second gene is a molecular marker (a fluorescent protein). Then, the following 5 genes (gltX, HemA, HemL, HemG and HemO) follow, which code for enzymes involved in heme synthesis via the C5 pathway. Other genes are BleoR for selection of transformants byzeocin / bleomycin screening and the Ori for plasmid replication. This can be changed or further optimized as well as the promoters used for the expression of the genes. More genes from the Heme pathway can also be included in thisor another plasmid containing the same or similar array.Fig. 3 shows plots comparing the growth measured as optic density (OD units, Y axis) F07044 / JS 4 and viability by the MTT assay (URM / OD units, Y axis) of modified strain having increased biosynthesis of the gltX, HemA, HemL, HemG and HemO, geneproducts (labeled as PT) vs the wild type (labeled as A). Time is denoted from day 1 to day 7 (D1-D7, in the X axis). DESCRIPTION OF PREFERRED EMBODIMENTS It is an object of the present invention to provide a method for the production of cellulose in a culture of one or more microorganisms, in a culture medium, comprising the step of increasing the level of biosynthesis of a macrocyclic primogenitor molecule, in particular of uroporphyrinogen III. The culture medium in which the one or more microorganisms are cultured may be chosen such as to increase the level of biosynthesis of a macrocyclic primogenitor molecule, in particular of uroporphyrinogen III. In a preferred embodiment of the method for the production of cellulose in a culture of one or more microorganisms according to a first object of the present invention, the one or more microorganisms are cultured in a culture medium that comprises, or consists of agricultural and / or industrial waste and / or post-consumer waste. From an environmental perspective, it is preferably to use such waste streams, which often are landfilled or used in the generation of bio-energy. In a preferred embodiment of the method for the production of cellulose in a culture of one or more microorganisms according to a first object of the present invention, the culturemedium is free of microorganisms other than the ones being cultured, and in particular isfree of virus particles. To that purpose, the agricultural and / or industrial waste and / or post- consumer waste is autoclaved prior to the preparation of the culture medium from it. On one embodiment, the culture medium may be obtained by comminuting the agricultural and / or industrial waste and / or post-consumer waste in the presence of water to produce an aqueous slurry comprising particles of said waste, which after autoclaving may be used as- is, or which may be filtered to provide a culture medium that is free of solid waste particles. Accordingly, the culture medium is preferably a liquid culture medium, which may or may not comprise solid particles of agricultural and / or industrial waste and / or post-consumer waste. The liquid culture medium preferably comprises a carbon source, which may be in the form of a hexose such as glucose or a tertiary alcohol such as glycol, or a mixture of F07044 / JS 5 both in an amount of 1 to 20% (w / v); a nitrogen source, which may be in the form of a ammonia salt in an amount of 0.2 to 1% (w / v); a trace metal source in the form of a salt such a magnesium salt, in an amount of 0.1 to 1% (w / v); a buffer such as acetic buffer inan amount of 1 to 3% (w / v).Agricultural waste may be plant material, or animal waste material such as dung, urine, or manure and spent bedding material, compost, whereas industrial waste may be any organic material that is a waste product of an industrial process. For example, suitable industrial waste may be a waste stream from meat processing such as offal, blood, lymph, bones and other animal tissue or from vegetable or fruit processing such as peels, seeds, juice or pulp of fruits or vegetables, or bulk spoiled or decayed fruit or vegetables. In general, thus, the culture medium may comprise any type of biomass sourced from an industrial, agricultural or post-consumer waste, and the biomass may be plant-based, animal-based, or a mixtureof both. Suitable vegetables or fruits may, as examples, be corn, squash, beans, orange,coconut, pineapple or mango. The present invention does equally include the use of algal biomass in the culture medium. For example, post-consumer waste may be green waste, such as household green waste or plant trimmings such as grass clippings, leaves and such. In a particularly preferred embodiment of the present invention, the culture medium may be based on pineapple or coconut, in the form of a pulp, juice, milk or coconut water. Furthermore, spent coffee grounds or tea leaves are suitable plant biomass that may be used in the culture medium. While from an environmental reason, the agricultural and / or industrial waste and / or post-consumer waste is preferably, it will be understood that in principle, the consumer grade products may also be used in the culture medium. In a preferred embodiment of the method for the production of cellulose in a culture of one or more microorganisms according to a first object of the present invention, the culture ofthe one or more microorganisms are aerobic or anaerobic. It is understood that culture ofthe one or more microorganisms can be adapted depending on the microorganism used inthe culture. For instance, in the case where a microorganism from the genusKomagataeibacter, such as for example Komagataeibacter xylinus, is used, the culture ofthe one or more microorganisms is aerobic.In a preferred embodiment of the present invention, the one or more microorganisms comprises bacteria from the genera Gluconacetobacter, Novacetimonas, Aerobacter,Rhizobium, Komagataeibacter, Sarcina, Azotobacter, Agrobacterium, Pseudomonas, orAlcaligenes. While the invention is not strictly limited to one of the aforementioned genera, F07044 / JS 6 these have been found to provide adequate cellulose quality. In a much preferred embodiment of the method for the production of cellulose in a culture of one or more microorganisms according to a first object of the present invention, microorganisms from the genus Komagataeibacter, such as for example Komagataeibacter xylinus, and from thegenus Novacetimonas, such as for example Novacetimonas hansenii, were found to beparticularly useful, especially when the culture medium comprises vegetable or fruit waste streams, and in particular fruit such as pineapple or coconut, for example in the form of a pulp, juice, milk, or coconut water. In a much preferred embodiment of the method for the production of cellulose in a culture of one or more microorganisms according to a first object of the present invention, the one or more microorganisms comprises fungi, such as for example basidiomycetes, preferably in the form of mycelial colonies. Alternatively, fungi from the genera Aspergillus, Trametes, Ganoderma, Schizophyllum, Phanerochaete, Lentinula, Psilocybe, Pleurotus, Ramaria,Hexagonia, Beauveria, Fomitopsis may also be used.In a preferred embodiment of the present invention, increasing the level of biosynthesis of macrocyclic primogenitor molecules, such as uroporphyrinogen, in said microorganisms is achieved by culturing the one or more microorganisms in a medium comprising a soluble metal species, where the metal is chosen from Fe, Mg, Ni, Co, Mn, Mo, Zn, or Cu. While it is understood that the medium may comprise some or even all of said metals via the use of a waste stream as discussed above, the content of said metals is generally sub-optimal for the culture of the microorganisms and may be adjusted to a suitable concentration by the addition of the relevant metals in a biologically available form. Such a form is generally a water-soluble salt or complex of said metals. It has been found that when the culture medium is adjusted to a concentration of cobalt inthe range of 0.0000001% - 1% by weight, and preferably in a range of 0.01% - 0.1 % byweight, the microorganisms show enhanced growth and cellulose production. In a preferred embodiment, increasing the level of biosynthesis of macrocyclic primogenitor molecules may be achieved by addition of an inorganic salt comprising cobalt (II) such as cobalt (II) chloride or by addition of a biogenic molecule such as cobalamin or a cobalamin derivative such as hydroxy-, cyano-, methyl-, or adeno-cobalamin until the above-recitedconcentration of cobalt (II) is reached. F07044 / JS 7 It has been found that when the culture medium is adjusted to a concentration ofmolybdenum in the range of 0.0000001% - 1% by weight, and preferably in a range of0.01% - 0.1 % by weight, the microorganisms show enhanced growth and celluloseproduction. In a preferred embodiment, increasing the level of biosynthesis of macrocyclic primogenitor molecules may be achieved by addition of a salt comprising molybdate suchas ammonium molybdate [(NH4)6Mo7O24 *(4H2O)] or by addition of a biogenic molecule suchas MoCo until the above-recited concentration of molybdenum (II) is reached.It has been found that when the culture medium is adjusted to a concentration of magnesiumin the range of 0.0000001% - 1% by weight, and preferably in a range of 0.01% - 0.5% byweight, the microorganisms show enhanced growth and cellulose production. In a preferred embodiment, increasing the level of biosynthesis of macrocyclic primogenitor molecules may be achieved by addition of a salt comprising magnesium such as magnesium chloride (MgCl2) or by addition of a biogenic molecule such as chlorophyll until the above-recited concentration of magnesium (II) is reached. It has been found that when the culture medium is adjusted to a concentration of copper (II)in the range of 0.0000001% - 1% by weight, and preferably in a range of 0.01% - 0.1 %by weight, the microorganisms show enhanced growth and cellulose production. In a preferred embodiment, increasing the level of biosynthesis of macrocyclic primogenitor molecules may be achieved by addition of an inorganic salt comprising copper (II), such as copper (II) sulfate or by addition of a biogenic molecule such as hemocyanin, until the above-recited concentration of copper(II) is reached. It has been found that when the culture medium is adjusted to a concentration of nickel (II)in the range of 0.0000001% - 1% by weight, and preferably in a range of 0.01% - 0.1 %by weight, the microorganisms show enhanced growth and cellulose production. In a preferred embodiment, increasing the level of biosynthesis of macrocyclic primogenitor molecules may be achieved by addition of an inorganic salt comprising nickel (II), such as nickel (II) sulfate or by addition of a biogenic molecule such as nickel-tetrapyrrole coenzymes, until the above-recited concentration of nickel(II) is reached. It has been found that when the culture medium is adjusted to a concentration of zinc (II) inthe range of 0.0000001% - 1% by weight, and preferably in a range of 0.01% - 0.1 % byweight, the microorganisms show enhanced growth and cellulose production. In a preferred F07044 / JS 8 embodiment, increasing the level of biosynthesis of macrocyclic primogenitor molecules may be achieved by addition of an inorganic salt comprising zinc (II), such as zinc (II) sulfate, until the above-recited concentration of zinc(II) is reached. It has been found that when the culture medium is adjusted to a concentration of boron inthe range of 0.0000001% - 1% by weight, and preferably in a range of 0.01% - 0.1 % byweight, the microorganisms show enhanced growth and cellulose production. In a preferred embodiment, increasing the level of biosynthesis of macrocyclic primogenitor molecules may be achieved by addition of an boric acid, until the above-recited concentration of boron is reached. It has been found that when the culture medium is adjusted to a concentration of iron (II) inthe range of 0.0000001% - 1% by weight, and preferably in a range of 0.01% - 0.5% byweight, the microorganisms show enhanced growth and cellulose production. In a preferred embodiment, increasing the level of biosynthesis of macrocyclic primogenitor molecules may be achieved by addition of an inorganic salt comprising iron (II), such as iron (II) gluconate, iron (II) chloride or by addition of a biogenic molecule hemoglobin or myoglobin, until the above-recited concentration of iron(II) is reached. In a preferred embodiment of the present invention, the method for the production of cellulose in one or more microorganisms includes increasing the level of biosynthesis of macrocyclic primogenitor molecules (uroporphyrinogen) in said microorganisms may be advantageously achieved by increasing the level of biosynthesis of phosphite dehydrogenase (PtxD), which is an enzyme required for regeneration of cofactors important for metabolism such as NADH and NADPH. The level of biosynthesis of phosphitedehydrogenase may be increased by overexpression of the PtxD gene by either episomal(plasmid) or integration into the genome of the microorganism via methods known to theperson of ordinary skill in the art. It is noted that the expression of phosphite dehydrogenase may be under control of a promoter, which may be constitutive or capable of induction upon exposure to a stimulus. In a preferred embodiment of the present invention, the expression product of the PtxD gene has the protein sequence according to SEQID1. In a preferred embodiment of the present invention, the method for the production of cellulose in one or more microorganisms includes increasing the level of biosynthesis of F07044 / JS 9 macrocyclic primogenitor molecules (uroporphyrinogen) in said microorganisms may be advantageously be achieved by increasing the level of biosynthesis of 5-Aminolevulinic acid (5-ALA) in said microorganisms or by increasing the expression of ALA synthase orglutamate semialdehyde aminomutase. 5-aminolevulinic acid (ALA) is a universalmetabolite in the biosynthesis of heme, and may be synthesized by one of two different routes: the C4 pathway or the C5 pathway. The C4 pathway, also called the ‘Shemin pathway’, is present in mammals, fungi and α-proteobacteria. The C5 pathway of heme biosynthesis is found in plants, most bacteria and archaea. Compared to the C4 pathway, the C5 pathway uses simple carbon sources with higher titer of heme as substrates; therefore, it is more suitable for the biosynthesis of heme in prokaryotes such as celluloseproducing species from the genus Komagataeibacter or Novacetimonas from a standpointof industrial production. However, the C5 pathway is absent in the above genera of bacteriathat produce bacterial cellulose, and the inclusion of the C5 pathway in said bacteria leadsto enhanced cellulose production. Thus, in a more preferred embodiment, the level ofbiosynthesis of 5-aminolevulinic acid (5-ALA) in said microorganisms is increased by theexpression of glutamate semialdehyde aminomutase. This may be achieved by the overexpression of the corresponding gene (hemL) in the microorganism of choice, or by overexpression of both of glutamate semialdehyde aminomutase (hemL) and glutamyl- tRNA reductase (hemA), the latter being able to generate glutamate-1-semialdehyde from glutamyl-tRNA, or by overexpression of glutamate semialdehyde aminomutase (hemL), glutamyl-tRNA reductase (hemA), and glutamyl-tRNA synthetase (gltX), the latter being able to generate glutamyl-tRNA. The overexpression of the above-mentioned enzymes allows the utilization of glutamate as a source for of heme biosynthesis in the microorganism. It is understood that the overexpression of the corresponding genes mentioned above may, and other mentioned in the present application, can be ensured, for example, by using one or more microorganisms comprising an expression vector, such as a plasmid, in which said the expression of said genes is either inducible or constitutive. The genes may be combined in one expression vector or in multiple vectors, and preferably are combined in one expression vector. In a preferred embodiment of the present invention, the one or more microorganisms thus comprise an expression vector capable of expressing aminomutase (hemL), glutamyl-tRNA reductase (hemA), and / or glutamyl-tRNA synthetase (gltX); protoporphyrinogen oxidase(HemG) and / or heme oxygenase (HemO); and / or phosphite dehydrogenase (PtxD), or anycombination of them. In a more preferred embodiment, the one or more microorganismscomprises an expression vector capable of expressing phosphite dehydrogenase (PtxD), F07044 / JS 10either alone or in addition to the above aminomutase (hemL), glutamyl-tRNA reductase(hemA), and / or glutamyl-tRNA synthetase (gltX); protoporphyrinogen oxidase (HemG) and / or heme oxygenase (HemO). In a preferred embodiment of the present invention, the expression product of the hemL gene has the protein sequence according to SEQID4. In a preferred embodiment of the present invention, the expression product of the hemA gene has the protein sequence according to SEQID3.In a preferred embodiment of the present invention, the expression product of the gltX genehas the protein sequence according to SEQID2. Furthermore, in a preferred embodiment of the present invention, the method for the production of cellulose in one or more microorganisms includes increasing the level of biosynthesis of protoporphyrinogen oxidase (HemG), which generates protoporphyrin IX from coproporphyrinogen III and / or of heme oxygenase (HemO), which generates biliverdin from heme. In a preferred embodiment of the present invention, the expression product of the hemG gene has the protein sequence according to SEQID5. In a preferred embodiment of the present invention, the expression product of the hemO gene has the protein sequence according to SEQID6. In a preferred embodiment of the present invention, the increasing of the level of biosynthesis of macrocyclic primogenitor molecules, such as uroporphyrinogen III, in said microorganisms, is achieved by increasing the level of biosynthesis of metal-chelatingtetrapyrroles, such as siroheme, cobalamin, coenzyme F430, chlorophyll ap, heme, heme d1or biliverdin IXα. In general, the culture of the one or more microorganisms can be carried out in suitable vessels, such as trays, which may be vertically stacked on racks to optimize footprint through a vertical production system. The culture may preferably carried out under a controlled environment, where parameters such as temperature (ºC), CO2 (ppm), air flow (cfm), F07044 / JS 11 humidity (%), light (lux), among others, are kept within optimal ranges for the microorganism being cultured. In a preferred embodiment of the present invention, the culture of one or more microorganisms in a culture medium is a static culture. In a preferred embodiment of the present invention, the culture of one or more microorganisms in a culture medium is a static culture and / or the cellulose is produced in the form of a layer and preferably of a pellicle. In a preferred embodiment of the present invention, the culture of one or more microorganisms in a culture medium is an agitated culture, such as for example shaking culture and / or agitated bioreactor culture. In a preferred embodiment of the present invention, the method for the production of cellulose in a culture comprises further the steps of : providing a floating substrate on which a layer of cellulose is produced via the one or more microorganism, preferably in the form of a pellicle, and / or harvesting the thus produced cellulose and / or optionally drying the harvested cellulose and / or densifying and tanning the harvested cellulose to provide a sheet of cellulose. EXPERIMENTS Table 1 shows the productivity (dry weight) and general mechanical properties (quality) of a 1 x 1.5m sample of dry bacterial cellulose grown using the present invention. As can be seen, the amount of cellulose produced is higher when a wildtype microorganism is genetically altered to overexpress the gene products of the following 5 genes: gltX, HemA,HemL, HemG and HemO) using a suitable plasmid, and even higher when, in addition, themedium is supplemented with trace metals. Furthermore, the mechanical properties are also enhanced, as shown in Table 1, for both breaking force and Young's Modulus, when a wildtype microorganism is genetically altered using a plasmid to overexpress the geneproducts of the following 5 genes: gltX, HemA, HemL, HemG and HemO) using a suitableplasmid, and even more so when in addition, the medium is supplemented with trace metals. The following Table 1 shows the productivity (dry weight) and general mechanical properties F07044 / JS 12 (quality) of a 1 x 1.5m sample of dry bacterial cellulose grown using the present invention. All tests were carried out following the corresponding SATRA TM29 standard test method under a universal machine, TESTEX Tensile Tester TF001. "Yong's "Breaking Dry weight Modules Thickness Strain force (kg) (MPa)" (mm) (N)" wt 0,388 155 505 0.26wt + gltX, HemA, HemL, HemG and HemO0,4328 211 1,518 0.33wt + trace metals 0,7791 172 688 0.28wt + gltX, HemA, HemL, HemG and HemO+ trace0,9824 284 2,612 0.35metals Table 1 In Table 2, the Optical Density measured using a spectrophotometer (DLAB SP-UV1100) at 600nm (OD600) of agitated cultures (250rpm) during 7 days in 50mL centrifuge tubes containing 20mL basal liquid medium is shown. As can be seen, when geneticallymodified to overexpress gltX, HemA, HemL, HemG and HemO, the microorganism is ableto multiply faster when compared with a wildtype microorganism. Culture time Strain OD600 (hours) Wildtype 0.123 24 Present Invention 0.148Wildtype 0.159 48 Present Invention 0.187Wildtype 72 0.171 F07044 / JS 13Present Invention 0.212Wildtype 0.256 96Present Invention 0.221Wildtype 0.372 120Present Invention 0.554Wildtype 0.530 144Present Invention 0.780Wildtype 0.660 172Present Invention 0.966Table 2

Claims

F07044 / JS 14 CLAIMS1. A method for the production of cellulose in a culture of one or moremicroorganisms in a culture medium, comprising the step of increasing the level of biosynthesis of a macrocyclic primogenitor molecule, such as uroporphyrinogen III.

2. A method for the production of cellulose in a culture of one or moremicroorganisms in a culture medium according to claim 1, wherein the culture medium comprises agricultural and / or industrial waste.

3. A method for the production of cellulose in a culture of one or moremicroorganisms in a culture medium according to claim 1 or 2, wherein the culture is aerobic or anaerobic, and / or the one or more microorganism is a gram-negative bacterium.

4. The method for the production of cellulose in one or more microorganisms,in a culture medium according to any one of claims 1, 2 or 3, wherein the one or more microorganisms comprises bacteria from the genera Gluconacetobacter, Novacetimonas, Aerobacter, Rhizobium, Komagataeibacter, Sarcina, Azotobacter, Agrobacterium, Pseudomonas, orAlcaligenes, and in particular wherein the one or more microorganism is Novacetimonas or Komagataeibacter.

5. The method for the production of cellulose in one or more microorganisms ina culture medium, according to any one of the preceding claims 1, 2 or 3, wherein the one or more microorganisms comprises fungi, and in particular wherein the one or more microorganism is a basidiomycete.

6. The method for the production of cellulose in one or more microorganisms ina culture medium, according to any one of the preceding claims, wherein increasing the level of biosynthesis of macrocyclic primogenitor molecules, such as uroporphyrinogen III, in said one or more microorganisms isachieved by culturing the one or more microorganisms in a mediumF07044 / JS 15 comprising one or more metals chosen from Fe, Mg, Ni, Co, Mn, Mo, Zn, or Cu.

7. The method for the production of cellulose in one or more microorganisms,in a culture medium, according to any one of the preceding claims, wherein the increasing the level of biosynthesis of macrocyclic primogenitor molecules, such as uroporphyrinogen III, in said microorganisms is achieved by overexpression of uroporphyrinogen III synthase (hemD) or bilirubinogen deaminase (hemC) or phorphobilinogen synthase (hemB).

8. The method for the production of cellulose in one or more microorganisms,in a culture medium, according to any one of the preceding claims, wherein the increasing the level of biosynthesis of macrocyclic primogenitor molecules, such as uroporphyrinogen III , in said microorganisms is achieved by increasing the level of biosynthesis of 5-aminolevulinic acid (5-ALA), in particular by overexpression of ALA synthase or of glutamate semialdehyde aminomutase (hemL) and / or increasing the level of biosynthesis ofmacrocyclic primogenitor molecules by overexpression of and glutamyl-tRNA synthetase (gltX), glutamyl-tRNA reductase (hemA), glutamate semialdehyde aminomutase (hemL), protoporphyrinogen oxidase (HemG),and heme oxygenase (HemO).

9. The method for the production of cellulose in one or more microorganismsaccording to claim 8, wherein the increasing the level of biosynthesis of macrocyclic primogenitor molecules, such as uroporphyrinogen III , in said microorganisms, is achieved by increasing the level of biosynthesis of 5- aminolevulinic acid (5-ALA) by overexpression of glutamate semialdehyde aminomutase (hemL).

10. The method for the production of cellulose in one or more microorganismsaccording to claim 8, wherein the increasing the level of biosynthesis of macrocyclic primogenitor molecules, such as uroporphyrinogen III , in said microorganisms, is achieved by increasing the level of biosynthesis of 5- aminolevulinic acid (5-ALA) by overexpression of glutamate semialdehyde aminomutase (hemL) and glutamyl-tRNA reductase (hemA), or by overexpression of glutamate semialdehyde aminomutase (hemL), glutamyl-F07044 / JS 16 tRNA reductase (hemA), and glutamyl-tRNA synthetase (gltX).

11. The method for the production of cellulose in one or more microorganisms,in a culture medium, according to any one of the preceding claims, wherein the increasing the level of biosynthesis of macrocyclic primogenitor molecules, such as uroporphyrinogen III, in said microorganisms, is achieved by increasing the level of biosynthesis of metal-chelating tetrapyrroles, such as siroheme, cobalamin, coenzyme F430, chlorphyll ap, heme, heme d1or biliverdin IXα.

12. The method for the production of cellulose in one or more microorganisms,in a culture medium, according to any one of the preceding claims, wherein the culture of one or more microorganisms in a culture medium is a static culture or agitated culture.

13. The method for the production of cellulose in one or more microorganisms,in a culture medium, according to any one of the preceding claims, wherein the one or more microorganisms comprises an expression vector capable of expressing aminomutase (hemL), glutamyl-tRNA reductase (hemA), and / or glutamyl-tRNA synthetase (gltX).

14. The method for the production of cellulose in one or more microorganisms,in a culture medium, according to any one of the preceding claims, wherein the method for the production of cellulose in a culture comprises further the steps of : -providing a substrate on which a layer of cellulose is produced via theone or more microorganism, preferably in the form of a pellicle, and / or -harvesting the thus produced layer of cellulose, and / or- optionally drying the harvested cellulose, and / or- densifying and optionally tanning the harvested cellulose.

15. A microorganism chosen from the from the genera Gluconacetobacter,Novacetimonas, Aerobacter, Rhizobium, Komagataeibacter, Sarcina, Azotobacter, Agrobacterium, Pseudomonas, or Alcaligenes, and in particularof the genus Novacetimonas or Komagataeibacter, expressing one or moreof glutamate semialdehyde aminomutase (hemL) and glutamyl-tRNAF07044 / JS 17reductase (hemA), or expressing glutamate semialdehyde aminomutase(hemL), glutamyl-tRNA reductase (hemA), and glutamyl-tRNA synthetase

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