Combinations of cellulolytic microorganisms and uses

Abstract

This disclosure relates to a combination, including a composition, of cellulolytic microorganisms, wherein each cellulolytic microorganism produces a glycosylase, said combination comprising: a first cellulolytic microorganism that produces a cellulase classifiable by EC 3.2.1.4 or EC 3.2.1.91; and a second cellulolytic microorganism that produces a cellulase classifiable by EC 3.2.1.4, EC 3.2.1.91, EC 3.2.1.21, EC 3.2.1.52, EC 3.2.1.74 or EC 3.2.1.86. Also disclosed are related methods including to produce a simple carbohydrate from cell wall cellulosic material, and a ruptured cell wall, and to retrieve intracellular materials of the ruptured cell such as lipid or protein.

Description

COMBINATIONS OF CELLULOLYTIC MICROORGANISMS AND USES TECHNICAL FIELD

[0001] This disclosure relates to a combination, including a composition, of cellulolytic microorganisms, wherein each cellulolytic microorganism produces a glycosylase, said combination comprising: a first cellulolytic microorganism that produces a cellulase classifiable by EC 3.2.1.4 or EC 3.2.1.91; and a second cellulolytic microorganism that produces a cellulase classifiable by EC 3.2.1.4, EC 3.2.1.91, EC 3.2.1.21, EC 3.2.1.52, EC 3.2.1.74 or EC 3.2.1.86. Also disclosed are related methods including to produce a simple carbohydrate from cell wall cellulosic material, and a ruptured cell wall, and to retrieve intracellular materials of the ruptured cell such as lipid or protein. BACKGROUND

[0002] Many industrial processes use simple carbohydrates, protein or lipids for the production of valuable commodities. Examples include applications in the renewable energy (e.g., biofuel), biopharmaceutical, agricultural, bioplastic manufacture, food and nutraceutical industries. For instance, in some applications, simple carbohydrates may also be used in the production of methane, hydrogen and biofertilizer using anaerobic microorganisms (fermentation), while protein may be used in feed production for livestock, and lipids may be used to produce biofuels through a transesterification process.

[0003] Traditional processes for the production of simple carbohydrates involve, for example extraction from sugarcane plants. Cultivation of sugarcane crops and extraction of simple carbohydrates is labour-intensive, demanding of natural resources (e.g., land, water) and wasteful, as are other traditional processes for the production of these materials. Traditional processes for producing lipids involve extraction primarily from oilseed plants. Cultivation of oilseed plants and lipid retrieval is similarly labour-intensive, it has high energy demand or uses harsh chemicals for mechanical or chemical cell wall disruption, and can be low-yielding. Production of protein has in the past also relied heavily on plant-based resources (e.g., legumes, cereals) and its retrieval is characterised by the same issues.

[0004] The sustainability of natural resources, environmental impact and the demands of consumers are encouraging industry to turn to ‘greener’ alternatives in the use and manufacture of commodities. One area of focus is on energy use minimisation.

[0005] Plants and algae naturally sequester carbon dioxide through the process of photosynthesis, and store carbon in their biomass. Photosynthesis allows the plant to generate energy in the form of glucose and other sugar monomers, which is stored in the plant biomass as complex carbohydrates, primarily as cell wall cellulosic material. Photosynthesis drives carbon fixation, that is, the conversion of inorganic carbon, e.g., atmospheric carbon dioxide, into organic carbon-containing compounds. This process of carbon sequestration is often referred to as ‘carbon capture’. Some plants are more efficient than others at fixingatmospheric carbon dioxide. For instance, algae tends to be more efficient than many terrestrial plants. However, while it may be possible to use plants and algae to sequester and fix atmospheric and industrial waste carbon dioxide as e.g., cell wall cellulosic material, the problem remains as to how to ‘unfix’ it in the form of simple carbohydrates for their retrieval, and for retrieval of the valuable lipid and protein commodities which the cell wall encases, in a ‘greener’ fashion.

[0006] The biomass resulting from biological carbon sequestration, largely comprising cell wall cellulosic material, and the lipid and protein the cells encased, are useful resources, more- so with improved means for producing these commodities from it, and especially in a ‘greener’ fashion without undoing so much of the environmental benefit of capturing carbon dioxide to begin with. The provision of improved means for the production of simple carbohydrates, lipid and / or protein from cell wall cellulosic materials is accordingly desirable. SUMMARY

[0007] Generally speaking, the present disclosure relates to the development of improved methods for the production of simple carbohydrates from cellulosic materials, improved methods for the retrieval of lipid and / or protein from walled cells, and materials useful in implementation of the methods. The improved methods utilise a combination of at least two cellulolytic microorganisms, where one produces a cellulase of a type that is capable of decomposing polymeric cellulosic material into a fragment, and another produces a cellulase of a type that is capable of decomposing polymeric cellulosic material or fragments thereof into simple carbohydrates. It has been found that the production of simple carbohydrates is improved, e.g., may occur at a greater rate when compared with the production capability of one cellulolytic microorganism, and that the combination may be applied to cellulosic materials of cell walls of walled cells to provide a ruptured cell wall and allow improved intracellular protein and / or lipid retrieval.

[0008] Accordingly, the present disclosure provides a combination of cellulolytic microorganisms, wherein each cellulolytic microorganism produces an extracellular glycosylase, said combination comprising: a first cellulolytic microorganism that produces a cellulase classifiable by EC 3.2.1.4 or EC 3.2.1.91; and a second cellulolytic microorganism that produces a cellulase classifiable by EC 3.2.1.4, EC 3.2.1.91, EC 3.2.1.21, EC 3.2.1.52, EC 3.2.1.74 or EC 3.2.1.86.

[0009] The present disclosure also provides a composition comprising a combination of cellulolytic microorganisms as herein described and a cellulosic material such as walled cells.

[0010] The present disclosure also provides a method of producing a simple carbohydrate by biodecomposition of a cellulosic material, comprising subjecting cellulosic material to a combination or a composition as herein described.

[0011] The present disclosure also provides a method of rupturing a cell wall of a walled cell by biodecomposition of cell wall cellulosic material, comprising: providing growth conditions to a composition comprising: a combination of cellulolytic microorganisms as herein described; and walled cells.

[0012] The present disclosure also provides a method of retrieval of intracellular lipid and / or intracellular protein from a walled cell, comprising: providing growth conditions to a composition comprising: a combination of cellulolytic microorganisms as herein described; and walled cells; to produce a simple carbohydrate and a ruptured cell wall by biodecomposition of cell wall cellulosic material; and retrieving walled cell intracellular lipid and / or walled cell intracellular protein from the composition.

[0013] The present disclosure also provides a simple carbohydrate produced by a method as herein described, a lipid retrieved by a method as herein described, and a protein retrieved by a method as herein described. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 shows a photograph as an example of the bubble column reactor set up for culturing phototrophic microorganisms.

[0015] Figure 2 A) shows a diagram of the serial dilution method and B) the plating method used to enumerate bacterial colony forming units (CFU) to determine bacterial cell concentration.

[0016] Figure 3 shows a flowchart diagram as an overview of the selection process used to identify the optimal bacterial mix for the biodecomposition of microalgae biomass.

[0017] Figure 4 shows a diagram of microorganisms distribution in soil and the distribution of characteristics associated with the microorganism that impact their agricultural significance, including: phytophathogenicity, phosphate solubilisation, nitrogen fixation, plant growthpromotion or biological control agent. The slices represent the percentages given in Figure 6; larger slice for larger percentage, smaller for smaller.

[0018] Figure 5 shows heat map diagrams of the relative abundance of certain bacterial species with either disadvantageous (5A) or advantageous (5B) characteristics.

[0019] Figure 6 shows a coded table that relates particular agriculturally significant traits with particular microorganism species.

[0020] Figure 7 shows a photograph of agar plates as an example of results generated from the cellulase activity assay.

[0021] Figure 8 shows a schematic diagram of the starch and sucrose metabolism pathway (KEGG pathway 00500) which highlights the reactions that lead to the production of D-glucose (shown in a yellow shaded box).

[0022] Figure 9 shows a graph of the total amount of algal biomass when grown in new BBM medium and singly reused BBM at the end of 7 days of culturing across four replicate experiments.

[0023] Figure 10 contains A) microscopy images and line drawings of Chlorella vulgaris cells surrounded by bacterial cells at time zero in treatments with both a 3-strain (left) and 5-strain (right) consortium, and B) biodecomposed Chlorella vulgaris cells at 24 hours in the same 5- strain treatment shown by microscopy (left) including stained with Calcofluor White (right).

[0024] Figure 11 contains photographs and line drawings of bacterial load plating-out results of the 3-strain treatment (left) and the 5-strain treatment (right). DETAILED DESCRIPTION

[0025] The following is a detailed description of the disclosure and is provided to aid those skilled in the art in practicing the present disclosure. Those of ordinary skill in the art may make modifications and variations in the embodiments described herein without departing from the spirit or scope of the present disclosure.

[0026] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the relevant art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. Definitions

[0027] As used herein, the term “and / or”, e.g., “X and / or Y” shall be understood to mean either "X and Y" or "X or Y" and shall be taken to provide explicit support for both meanings or for either meaning.

[0028] The articles “a” and “an” are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0029] The term “about” as used herein, when referring to a numerical value or range, allows for a degree of variability in the value or range, for example within 10% of a stated limit of a range.

[0030] Ranges provided herein are understood to be shorthand for all values within the range. For example, a range of 1 to 10, including a range that is specified to be “between” 1 and 10, is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Similarly, a range of 1.0 to 2.0 is understood to include any number, combination of numbers, or sub-range from the group consisting 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0.

[0031] As used herein, the phrase “at least one of”, when used with a list of items, means that different combinations of one or more of the listed items may be selected, and that only one of the items in the list may be selected. In other words, “at least one of” means any number or combination of items may be selected from the list. For example, “at least one of item A, item B, and item C” may mean item A; item A and item B; item B; item A, item B, and item C; or item B and item C.

[0032] Throughout this specification including the claims, unless the context requires otherwise the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0033] Unless otherwise indicated, terms such as “first”, “second”, “third” etc., are used herein merely as labels, and are not intended to impose ordinal, positional, or hierarchical requirements on the items to which these terms refer. Reference to a “second” etc. item does not require or preclude the existence of another item (e.g., a “first” or “third” item). That said, such terms are generally used to distinguish one item of a type from another item of that type, and the items will generally differ from one another in one or more characteristics, but without necessarily requiring that the characteristic relates to an essential feature of the present disclosure.

[0034] A “microorganism” is a noncellular or unicellular organism and is often too small to be visible to the naked human eye. Microorganisms include bacteria (including cyanobacteria), fungus, algae, spores, archaea, lichens, protozoans, mycoplasma, and parasitic organisms. One microorganism is distinguished from another at the strain level. That is, reference to a single or a first microorganism is reference to a particular strain of a species, while reference to another or a second etc. microorganism is reference to a different strain of a species. Strains are identifiable and classifiable through known taxonomic methods. A strain is defined by non- identity with others within the same species, in one or both of genotype and phenotype; i.e.,genetic makeup and observable traits (but not necessarily in respect of an essential feature of the present disclosure except as otherwise specified). When used in the singular herein, the term “microorganism” refers to a single strain.

[0035] A ”cellulolytic microorganism” is a microorganism that produces a glycosylase. Generally speaking, a cellulolytic microorganism will contain a gene or genes encoding a glycosylase and functional biological machinery necessary for its expression. Specified herein are cellulolytic microorganisms that produce an extracellular glycosylase. By producing an extracellular glycosylase is meant that the glycosylase exists extracellularly. Generally speaking, a cellulolytic microorganism that produces an extracellular glycosylase will contain functional biological machinery necessary for its secretion from the cell.

[0036] As used herein, a “simple carbohydrate” is a carbohydrate monomer (or “monomer” for short) or carbohydrate dimer (or “dimer” for short), a dimer consisting of two carbohydrate monomers linked together by an O- or S- glycosidic bond. Simple carbohydrates may also be referred to as simple sugars or simple saccharides i.e., mono- and di-saccharides. A simple carbohydrate is as distinct from an oligomer which consists of at least three carbohydrate monomers. Carbohydrate monomers may contain from four to six carbon atoms (i.e., C4, C5 or C6 sugars). Examples of carbohydrate monomers are glucose, sucrose, xylose, arabinose, rhamnose, maltose, trehalose, mannitol, sorbitol, galactose and derivative salicin. Examples of carbohydrate dimers are lactose, sucrose, maltose, cellobiose, chitobiose, trehalose and xylobiose.

[0037] As used herein, “cellulosic material” is synonymous with “polysaccharide material” and is a material comprising a backbone structure of carbohydrate monomers linked together by O- or S- 1,4-glycosidic bonds. For clarity, the term is not limited to polysaccharide materials containing necessarily containing cellulose, but is used generally to refer to polysaccharide materials. A cellulosic material being polymeric does not encompass simple carbohydrates i.e. carbohydrate monomers or dimers, but encompasses larger molecules, being oligomers of carbohydrate monomers, including trimers, tetramers etc., and encompasses complex carbohydrates which are polymers of carbohydrate monomers (i.e. “polysaccharides”), the monomers in the backbone structure being linked together by 1,4-glycosidic bonds. The backbone structure of cellulosic material may be branched. Cellulosic material may also exist as a carbohydrate component of macromolecular compounds that in addition to the carbohydrate component comprise additional structural moieties, for example proteins, nucleic acids and lipids.

[0038] Examples of cellulosic materials are hemicellulose, lignin, cellulose, glycogen, xylan and starch. These cellulosic materials are generally based on glucose monomers. For instance, starch is a complex carbohydrate consisting of amylose and amylopectin. Amylose is a straight chain polysaccharide of glucose monomers linked together by 1,4-glycosidic bonds. Amylopectin also contains a straight chain polysaccharide of glucose monomers linked together by 1,4-glycosidic bonds but is a branched polymer comprising glucose monomers(including of polymeric side chains) linked by 1,6-glycosidic bonds. Glycogen is also a branched polymer comprising a straight chain polysaccharide of glucose monomers linked together by 1,4-glycosidic bonds and branched chains of glucose monomers linked to the straight chain by a 1,6-glycosidic bond. Xylan is a branched chain polymer comprising mostly xylose monomers linked by 1,4-glycosidic bonds. Another example of a cellulosic material is chitin which contains a backbone structure of N-acetyl-glucosamines linked together by 1,4- glycosidic bonds. Examples of macromolecular compounds which are cellulosic materials include extracellular polymeric substances (EPS), glycolipids and glycoproteins. EPS are generally high molecular weight organic polymers containing a polysaccharide component which are produced by microorganisms and secreted extracellularly. In addition to the polysaccharide component, EPS may be composed of structural proteins, enzymes, nucleic acids, lipids, and other compounds such as humic acids. EPS is thought to play a role in microorganism cell adhesion, cell aggregation, nutrient entrapment, and / or protection and defence of microorganisms against environmental stressors. A wide range of microorganisms may produce EPS including many species of microalgae, bacteria, cyanobacteria, yeasts, fungi and protists.

[0039] Many to most, or all, plant, algae, oomycete and fungi cells have a cell wall. Some bacteria and archaea also have a cell wall. Cell walls are primarily composed of cellulosic materials. For instance, plant cell walls may be primarily composed of cellulose and / or lignin while fungal cell walls may be composed of chitin while algal cell walls be composed of cellulose or xylan. Cellulosic material existing in a cell wall may be referred to herein as “cell wall cellulosic material”.

[0040] A “glycosylase” is an enzyme that is capable of facilitating a chemical reaction which decomposes a cellulosic material by cleaving a 1,4-glycosidic bond, to produce two fragments from the cellulosic material, or two simple carbohydrates, or a fragment and a simple carbohydrate. As used herein, a glycosylase that is produced extracellularly is understood to be so-capable extracellularly to the cellulolytic microorganism that produces it. A “fragment” of a cellulosic material is a cellulosic material (i.e., not a simple carbohydrate) produced by a glycosylase, i.e., produced in the process of a glycosylase facilitating a chemical reaction which cleaves a 1,4-glycosidic bond.

[0041] Examples of glycosylases are cellulases, hemicellulases and amylases. A “cellulase” is capable of cleaving α- or β-1,4-glycosidic bonds between C6 sugars. A “hemicellulase” is capable of cleaving α- or β-1,4-glycosidic bonds between C5 sugars. An “amylase” is an enzyme that is capable of cleaving α- or β-1,4-glycosidic bonds (and may also be capable of cleaving α- or β-1,6-glycosidic bonds or α- or β-1,3-glycosidic bonds) between C6 sugars.

[0042] A “combination” as used herein in the context of cellulolytic microorganisms refers to the microorganisms being used simultaneously, which may be in a single composition or separate compositions or sequentially in separate compositions, such that, insofar asproduction and the biological activity of the glycosylases is concerned, use of each of the microorganisms overlaps or occurs simultaneously. Glycosylases

[0043] Glycosylases are classifiable by an Enzyme Commission (EC) number as defined by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB). An enzyme may be assigned an EC number based on the chemical reaction that it facilitates. An enzyme which has been assigned an EC number may be said to be “classified”. An enzyme which is “classifiable” by an EC number encompasses classified enzymes, and also enzymes which may not be formally classified but which facilitate the chemical reaction which the EC number defines.

[0044] In the combination of cellulolytic microorganisms described herein, the cellulolytic microorganisms suitably produce a glycosylase classifiable by an EC number prefixed by “3.2.1”. EC 3.2.1 enzymes are glycosylases capable of hydrolysing O- and S-glycosidic bonds. A cellulase, hemicellulase and amylase as a glycosylase will generally be classifiable by an EC number prefixed by 3.2.1.

[0045] EC 3.2.1.4, EC 3.2.1.91, EC 3.2.1.21, EC 3.2.1.52, EC 3.2.1.74 and EC 3.2.1.86 define cellulases according to the following reactions: ^ EC 3.2.1.4: hydrolysis of (1,4)-β-D-glycosidic linkages between C6 sugars producing dimers (e.g. cellobiose in the decomposition of cellulose) and / or trimers (e.g., cellotriose in the decomposition of cellulose). ^ EC 3.2.1.91: hydrolysis of (1,4)-β-D-glycosidic linkages between C6 sugars producing dimers (e.g., cellobiose in the decomposition of cellulose) and / or trimers. ^ EC 3.2.1.21: hydrolysis of terminal (1,4)-β-D-glycosyl linkages between C6 sugars producing monomers (e.g., glucose in the decomposition of cellulose, cellotriose or cellobiose). ^ EC 3.2.1.52: hydrolysis of terminal (1,4)-β-D-glycosyl linkages between C6 sugars of N-acetyl-β-D-hexosaminides producing N-acetyl-D-hexosamine monomers. ^ EC 3.2.1.74: hydrolysis of terminal (1,4)-β-D-glycosyl linkages between C6 sugars producing monomers (e.g., glucose in the decomposition of cellulose or cellotriose). ^ EC 3.2.1.86: hydrolysis of (1,4)-β-D-glycosyl linkages between C6 sugars of 6- phospho-oligomeric cellulosic materials producing monomers (e.g. glucose in the decomposition of 6-phospho-cellobiose.

[0046] EC 3.2.1.4 and EC 3.2.1.91 cellulases do not tend to produce carbohydrate monomers in the decomposition of cellulosic material; they tend to produce dimers or trimers. Cellulases which function in this way may be referred to herein as “Type 1” cellulases. Cellulolyticmicroorganisms which produce EC 3.2.1.4 cellulases tend to be bacterial, while cellulolytic microorganisms which produce EC 3.2.1.91 cellulases tend to be fungal.

[0047] EC 3.2.1.21, EC 3.2.1.52, EC 3.2.1.74 and EC 3.2.1.86 cellulases tend to produce carbohydrate monomers in the decomposition of cellulosic material. Cellulases which function in this way may be referred to herein as “Type 2” cellulases. Cellulolytic microorganisms which produce EC 3.2.1.21 cellulases tend to be bacterial, while cellulolytic microorganisms which produce EC 3.2.1.52 cellulases tend to be bacterial, while cellulolytic microorganisms which produce EC 3.2.1.74 cellulases may be bacterial or fungal, while cellulolytic microorganisms which produce EC 3.2.1.86 cellulases tend to be bacterial.

[0048] In preferred embodiments, when a high proportion of carbohydrate monomers is preferred, a first cellulolytic microorganism produces a cellulase classifiable by EC 3.2.1.4 or EC 3.2.1.91, and a second cellulolytic microorganism produces a cellulase classifiable by EC 3.2.1.21, EC 3.2.1.52, EC 3.2.1.74 or EC 3.2.1.86. Preferably, a first cellulolytic microorganism produces a cellulase classifiable by EC 3.2.1.4, and a second cellulolytic microorganism produces a cellulase classifiable by EC 3.2.1.21 or EC 3.2.1.74. Preferably, a first cellulolytic microorganism produces a cellulase classifiable by EC 3.2.1.4, and a second cellulolytic microorganism produces a cellulase classifiable by EC 3.2.1.21. In the decomposition of cellulosic material to carbohydrate monomers, the two types of cellulases work together: the Type 1 cellulases may decompose polymeric cellulosic materials to produce dimers or trimers, while the Type 2 cellulases may decompose polymeric cellulosic materials, dimers and trimers to produce carbohydrate monomers. It is believed that the Type 1 cellulases increase the number of cellulosic material molecules available for the Type 2 cellulases to decompose, together increasing the rate of production of carbohydrate monomers. The effect is compounded when the cellulases are produced by different cellulolytic microorganisms as explained herein below.

[0049] When a high proportion of carbohydrate dimers or trimers is preferred, used is a first cellulolytic microorganism produces a cellulase classifiable by EC 3.2.1.4 or EC 3.2.1.91, and a second cellulolytic microorganism produces a cellulase classifiable by EC 3.2.1.4 or EC 3.2.1.91 (production of Type 2 cellulases notwithstanding). A first cellulolytic microorganism may produce a cellulase classifiable by EC 3.2.1.4, and a second cellulolytic microorganism may produce a cellulase classifiable by EC 3.2.1.4. It is believed that when the cellulases are produced by different cellulolytic microorganisms the rate of production is increased as explained herein below.

[0050] Microorganisms that produce a glycosylase enzyme may also produce enzymes which phosphorylate oligomeric cellulosic materials. Representative enzymes include phosphotransferase enzymes classifiable by EC 2.7.1.205 which are generally capable of facilitating the transfer of a phosphate group from one molecule to an oligomeric cellulosic material. In the combinations described herein, EC 3.2.1.86 cellulases are particularly applicablein these embodiments for their ability to hydrolyse glycosyl linkages of phosphorylated oligomeric cellulosic materials to produce carbohydrate monomers.

[0051] Microorganisms that produce a cellulase classifiable by EC 3.2.1.4, EC 3.2.1.21, EC 3.2.1.52, EC 3.2.1.91, EC.3.2.1.74 or EC 3.2.1.86 may, and often do, produce other glycosylases including those classifiable in these same classes. For instance, a microorganism that produces a cellulase classifiable by EC 3.2.1.4 may often produce a cellulase classifiable by EC 3.2.1.21, among others. As such, single cellulolytic microorganisms may be, and often are, capable of decomposing cellulosic materials into simple carbohydrates. However, it has been found that a combination of cellulolytic microorganisms is capable of increasing the rate of production of simple carbohydrates as compared to a single microorganism (all other conditions remaining equivalent). Cellulolytic microorganisms which produce glycosylases essentially do so to produce food, i.e., cellulolytic microorganisms are capable of metabolising simple carbohydrates – it is a life-sustaining function. Without wishing to be limited by theory, it is believed that, in a combination of cellulolytic microorganisms, species to species interactions between the cellulolytic microorganisms result in the microorganisms competing with each other by changing the regulation of genes involved in the production of glycosylases and increasing the production of glycosylases, thereby increasing the rate of decomposition of cellulosic materials (and increasing the amount of ‘food’ available). It is also believed that this provides for a greater rate of decomposition of cellulosic materials than the use of enzymes alone – i.e., greater than the use of isolated glycosylase enzymes – because with enzymes alone these competitive interactions do not occur. Further, while the prevailing knowledge may generally hold that the use of microorganism combinations to effect a biological process has the drawback of reduced efficiency in terms of low specificity of biological activity (i.e., the microorganisms will undertake all life-sustaining biological functions, not just the one it is being utilised for) and consumption of nutrients (e.g., simple carbohydrates), in the present disclosure it is believed that the competitive effects result in cellulosic material biodecomposition activity that is so enhanced as to outweigh these potential drawbacks, especially with certain combinations of cellulolytic microorganisms. The greater activity or efficiency may be represented by a super-additive (i.e., synergistic) effect. A “super-additive” effect in this context means that the rate of decomposition of cellulosic material, in terms of simple carbohydrates produced over time, using a combination of cellulolytic microorganisms, at a particular total microorganism count, is greater than the added rates of decomposition achieved by the cellulolytic microorganisms singly, at the same combined total microorganism count. In other words, the combination of cellulosic microorganisms at a particular total microorganism count may produce the same amount of simple carbohydrates in a shorter time, or a greater amount in the same time, as the added amount produced by the same cellulosic microorganisms singly at the same total microorganism count.

[0052] The rate of decomposition of cellulosic material may be further increased by the inclusion of a third, fourth, fifth or subsequent cellulolytic microorganism in the combination. When included, the third, fourth or fifth cellulolytic microorganism preferably produces acellulase classifiable by EC 3.2.1.4, EC 3.2.1.91, EC 3.2.1.21, EC, 3.2.1.74, EC 3.2.1.52 or 3.2.1.86 as herein described. In these embodiments, the synergistic effect as described above may be extended to the third cellulolytic microorganism. As in, the rate of decomposition of cellulosic material, in terms of simple carbohydrates produced over time, using first, second and third cellulolytic microorganisms together, at a particular total microorganism count, is greater than the added rates of decomposition achieved by the same first, second and third cellulolytic microorganisms singly, at the same combined total microorganism count. The same may apply to any fourth or fifth cellulolytic microorganism included, that produce a cellulase classifiable by EC 3.2.1.4, EC 3.2.1.91, EC 3.2.1.21, EC 3.2.1.52, EC 3.2.1.74 or 3.2.1.86.

[0053] Accordingly, in preferred embodiments, at least a third cellulolytic microorganism which produces a cellulase classifiable by EC 3.2.1.4, EC 3.2.1.91, EC 3.2.1.21, EC 3.2.1.52, EC 3.2.1.74 or 3.2.1.86 is included in the combination.

[0054] In preferred embodiments: ^ the first cellulolytic microorganism produces a cellulase classifiable by EC 3.2.1.4; ^ the second cellulolytic microorganism produces a cellulase classifiable by EC 3.2.1.21; ^ any third cellulolytic microorganism included produces a cellulase classifiable by EC 3.2.1.4, EC 3.2.1.86, or 3.2.1.91; ^ any forth cellulolytic microorganism included produces a cellulase classifiable by EC 3.2.1.4 or EC 3.2.1.86; ^ any fifth cellulolytic microorganism included produces a cellulase classifiable by EC 3.2.1.4.

[0055] In preferred embodiments, a third, fourth and fifth cellulolytic microorganism which produce a cellulase classifiable by EC 3.2.1.4, EC 3.2.1.91, EC 3.2.1.21, EC 3.2.1.52, or 3.2.1.86 are included in the combination. Preferably, the third and fourth cellulolytic microorganisms produce cellulase classifiable by 3.2.1.4, and the fifth cellulolytic microorganism produces a cellulase classifiable by 3.2.1.86. In other words, in the preferred combinations, three cellulolytic microorganism which produce a cellulase classifiable by EC 3.2.1.4, and one each of cellulolytic microorganisms which produce a cellulase classifiable by EC 3.2.1.21, and 3.2.1.86, are present.

[0056] Similarly, microorganisms that produce cellulases classifiable by EC 3.2.1.4, EC 3.2.1.21, EC 3.2.1.52, EC 3.2.1.74 or EC 3.2.1.86, may, and often do, also produce a hemicellulase and / or an amylase.

[0057] Representative hemicellulases may be classifiable by EC 3.2.1.8 or EC 3.2.1.37 which define hemicellulases according to the following reactions:^ EC 3.2.1.8: hydrolysis of (1,4)-β-D-glycosyl linkages between C5 sugars producing fragments (e.g., fragments in the decomposition of xylan). ^ EC 3.2.1.37: hydrolysis of terminal (1,4)-β-D-glycosyl linkages between C5 sugars, producing fragments (e.g., fragments in the decomposition of xylan).

[0058] Representative amylases may be classifiable by EC 2.4.1.1, EC 3.2.1.1, EC 3.2.1.3 or EC 3.2.1.68 which define amylases according to the following reactions: ^ EC 2.4.1.1: hydrolysis of (1,4)-α-D-glycosyl linkages between C6 sugars producing phosphorylated monomers (e.g., glucose-6-phosphate in the decomposition of amylose or glycogen). ^ EC 3.2.1.1: hydrolysis of (1,4)-α-D-glycosyl linkages between C6 sugars producing dimers or fragments (e.g., maltose in the decomposition of amylose or glycogen). ^ EC 3.2.1.3: hydrolysis of terminal (1,4)-α-D-glycosyl linkages between C6 sugars producing monomers (e.g., glucose in the decomposition of amylose), hydrolysis of (1,3)- / (1,6)-α-D-glycosyl linkages producing simple carbohydrates or fragments (e.g., two fragments in the decomposition of amylopectin or glycogen). ^ EC 3.2.1.68: hydrolysis of (1,6)-α-D-glycosyl linkages between C6 sugars producing dimers or fragments (e.g., maltose in the decomposition of amylopectin).

[0059] A cellulolytic microorganism that produces a hemicellulase or an amylase may be used to advantage when the cellulosic material contains glycosidic linkages upon which these enzymes act, to further increase the production of simple carbohydrates and / or fragments upon which cellulases may act. In some embodiments, and depending on the cellulosic material to be decomposed, at least one of the cellulolytic microorganisms in the combination produces a hemicellulase classifiable by EC 3.2.1.8 or EC 3.2.1.37 and / or an amylase classifiable by EC 2.4.1.1, EC 3.2.1.1, EC 3.2.1.3 or EC 3.2.1.68.

[0060] Microorganisms that produce a glycosylase enzyme may also produce enzymes which cleave phosphate groups from oligomeric cellulosic materials or from phosphorylated simple carbohydrates. Representative enzymes include phosphatase enzymes classifiable by EC 3.1.3.58 which are generally capable of cleaving a phosphate group from a phosphorylated carbohydrates monomer. In the combinations described herein, EC 2.4.1.1 amylases are particularly applicable in these embodiments for their ability to produce phosphorylated carbohydrate monomers.

[0061] Glycosylases, being enzymes, are proteins, and may be represented by a protein sequence. Protein sequences which represent glycosylases produced by cellulolytic microorganism as described herein include:a) a cellulase classifiable by EC 3.2.1.4 is represented by SEQ ID No. 1, SEQ ID No. 2, or SEQ ID No.3; b) a cellulase classifiable by EC 3.2.1.91 is represented by SEQ ID No. 4; c) a cellulase classifiable by EC 3.2.1.21 is represented by SEQ ID No. 5; d) a cellulase classifiable by EC 3.2.1.52 is represented by SEQ ID No. 6; e) a cellulase classifiable by EC 3.2.1.86 is represented by SEQ ID No. 7; SEQ ID No. 8 or SEQ ID No.9; f) an amylase classifiable by EC 3.2.1.1 is represented by SEQ ID No.10 or SEQ ID No.11; g) an amylase classifiable by EC 2.4.1.1 is represented by SEQ ID No.12 or SEQ ID No.13; and

[0062] In preferred embodiments, a cellulase classifiable by EC 3.2.1.4, EC 3.2.1.91, EC 3.2.1.21, EC 3.2.1.52 or 3.2.1.86 is characterizable by an amino acid sequence having at least 80% sequence identity to a sequence given above for each class of cellulase. Preferably, the sequence identity is at least 85%, preferably at least 90%, preferably at least 95%, and preferably at least 98%. It has been found that enzymes representative of these sequences provide for especially efficient decomposition of cellulosic material in the combinations described herein. For a similar reason, in preferred embodiments, an amylase classifiable by EC 3.2.1.1 or EC 2.4.1.1 is characterizable by an amino acid sequence having at least 80% sequence identity to a sequence given above for each class of amylase. Preferably, the sequence identity is at least 85%, preferably at least 90%, preferably at least 95%, and preferably at least 98%.

[0063] Glycosylases often comprise a number of domains including binding domains and active sites. Active sites may be within or without binding domains. Binding domains tend to function to bind to the cellulosic material being decomposed to bring the active site into proximity with a glycosidic linkage to effect the cellulolytic reaction by the enzyme. Binding domains and active sites tend to be highly conserved, at least within a genus of microorganisms. As such, there tends to be a relationship between protein sequence encompassing active sites and binding domains, and EC classification.

[0064] Binding domains and active sites that may be, and preferably are, found in cellulase enzymes described herein and in the protein sequences provided above, include those as follows: a) carbohydrate binding type-3 domain of accession number Prosite PS51172, which may be found in cellulases classifiable by EC 3.2.1.4 and in SEQ ID Nos 1, 2 and 3; b) glycosyl hydrolase family 3 active site of accession number Prosite PS00775 which may be found in cellulases classifiable by EC 3.2.1.52 and in SEQ ID No 6;c) glycosyl hydrolase family 1 active site of accession number Prosite PS00572 which may be found in cellulases classifiable by EC 3.2.1.21 or EC 3.2.1.86 and in SEQ ID No 5 or 7; and d) carbohydrate binding type-1 domain of accession number Prosite PS51164 which may be found in cellulases classifiable by EC 3.2.1.91 and in SEQ ID No 4.

[0065] It has been found that enzymes containing these binding domains and active sites provide for especially efficient decomposition of cellulosic material in the combinations described herein.

[0066] The protein sequences provided above may be expressed as being constituted by the “conserved region” and the “non-conserved region”. The conserved region is constituted by the binding domains and active sites provided above. The non-conserved region is other than the conserved region. In which case, in preferred embodiments, a cellulase classifiable by EC 3.2.1.4, EC 3.2.1.91, EC 3.2.1.21, EC 3.2.1.52 or 3.2.1.86 is characterizable by an amino acid sequence of the non-conserved region having at least 60%, preferably at least 65%, 70%, 75%, 80% or 85%, sequence identity to the non-conserved region of a sequences given above for each class of cellulase. Similarly, in preferred embodiments, an amylase classifiable by EC 33.2.1.1 or EC 2.4.1.1 is characterizable by an amino acid sequence of the non-conserved region having at least 60%, preferably at least 65%, 70%, 75%, 80% or 85%, sequence identity to the non-conserved region of a sequences given above for each class of amylase. By the same token, a cellulase classifiable by EC 3.2.1.4, EC 3.2.1.91, EC 3.2.1.21, EC 3.2.1.52 or 3.2.1.86 or an amylase classifiable by EC 33.2.1.1 or EC 2.4.1.1 may characterizable by an amino acid sequence of the conserved region having at least 90%, preferably at least 92%, 95%, 96%, 98% or 99%, or even 100%, sequence identity to the conserved region of a sequences given above for each class of cellulase or amylase.

[0067] In preferred embodiments, including when production of a high proportion of carbohydrate monomers is preferred, each cellulolytic microorganism of the combination produces both a type 1 and a type 2 cellulase. In preferred embodiments, each cellulolytic microorganism produces a cellulase classifiable by EC 3.2.1.4 or EC 3.2.1.91, and produces a cellulase classifiable by EC 3.2.1.21, EC 3.2.1.52, EC 3.2.1.74 or EC 3.2.1.86. In preferred embodiments, each cellulolytic microorganism produces a cellulase classifiable by EC 3.2.1.4, and produces a cellulase classifiable by EC 3.2.1.21, EC 3.2.1.52 or EC 3.2.1.86. In preferred embodiments, at least one, preferably two, three or four cellulolytic microorganism of the combination produces a hemicellulase. When produced, preferably the hemicellulase is classifiable by EC 3.2.1.8. In preferred embodiments, at least one, preferably two, three, four, five or each cellulolytic microorganism of the combination produces an amylase. When produced, preferably the hemicellulase is classifiable by EC 3.2.1.1 and / or 2.4.1.1, preferably the former. In preferred embodiments, at least one, preferably two or three cellulolytic microorganisms of the combination produce an amylase classifiable by both EC 3.2.1.1 and 2.4.1.1.

[0068] Enzymes are proteins that catalyse chemical reactions. The activity of the enzyme is the degree to which it causes or accelerates a reaction. As described herein, the enzyme activity being investigated is the decomposition of cellulosic materials by glycosylase enzymes. For cellulases, the assay used to demonstrate the decomposition of cellulosic materials is preferably a carboxymethylcellulose (CMC) assay. This assay allows the visualisation of cellulose decomposition by cellulolytic microorganisms when grown on CMC agar because the agar will appear opaque when cellulose is present, but will become clear when cellulose is decomposed. As a result, a zone of clearing or transparent ‘halo’ will be visible surrounding the cellulolytic microorganism colony if the microorganism produces active cellulases. The zone of clearing radiates outwardly from the centre of the cellulolytic microorganism colony where cellulase enzymes are actively decomposing cellulose. As such, it is possible to measure the diameter of the zone of clearing and the diameter of the colony combined, and then subtract the diameter of the cellulolytic microorganisms colony to generate a ratio that expresses the index of enzyme activity (IE), as a representation of the combined activity of the functional cellulases. The larger the zone of clearing, the more cellulase activity and the higher the IE value will be. For other glycosylases a similar test is applicable using a different cellulosic material as necessary and based on the same principles.

[0069] The activity of cellulases produced by cellulolytic microorganisms may achieve an IE of at least 1.05. In some embodiments, the IE is at least 1.09. In some embodiments, the IE is at least 1.15. In some embodiments the IE is at least 1.20 or 1.30. In some embodiments the IE may be at least 1.40 or at least 1.50. In some embodiments, the IE may be as high as 1.90, 1.80, 1.70, 1.60 or 1.55. Any minimum and maximum may be combined without limitation. For example, expressed as a range, the activity of cellulases produced by cellulolytic microorganisms may achieve an IE of between 1.05 and 1.90 or between 1.05 and 1.80 or between 1.05 and 1.60 or between 1.05 and 1.55 or between 1.09 and 1.90 or between 1.09 and 1.80, etc. Cellulolytic Microorganisms

[0070] The cellulolytic microorganisms may be, and preferably each is, isolated prior to forming a combination as described herein or implemented in a method as described herein. By “isolated” means that the cellulolytic microorganism is removed from its natural environment if it is naturally occurring. For example, a cellulolytic microorganism present in nature such as within soil or plant material is not isolated, but when separated from some or all of the natural material in the natural system, is isolated. In some embodiments, isolated cellulolytic microorganisms are purified to produce pure cultures such that only the desired cellulolytic microorganism is cultured (substantially or detectably). In other words, the combination of cellulolytic microorganisms may be produced from axenic cultures of individual cellulolytic microorganisms.

[0071] While applicable embodiments include the use of cellulolytic microorganisms which may produce a glycosylase intracellularly, which glycosylase may then be released, for exampleby cell lysis, it is preferred that each cellulolytic microorganism used in the combination described herein produces a glycosylase extracellularly.

[0072] Applicable embodiments include those where a microorganism has been modified to render it capable of producing a glycosylase, or to improve its glycosylase production capability. Methods for achieving this are known to those of skill in the art and generally include transfecting a microorganism with an expression cassette containing a DNA sequence encoding a glycosylase enzyme, which DNA may for example encode a protein sequence as given herein. However, in preferred embodiments, each cellulolytic microorganism is “unmodified” in terms of it being transfected with DNA, and is as naturally occurring, i.e., wild- type, though isolated.

[0073] In preferred embodiments, each cellulolytic microorganism in the combination described herein is a bacteria or a fungi. Preferably the bacteria is aerobic bacteria. Preferably the fungi is aerobic fungi. The cellulolytic microorganisms in combination may be comprised solely of bacteria, or solely of fungi, or a combination of bacteria and fungi. There are many strains of bacteria and fungi which produce glycosylases extracellularly. Most preferably, each cellulolytic microorganism in the combination described herein is an aerobic bacteria. Preferably, both the first and second cellulolytic microorganisms in the combination are of the same genus. When a third, fourth and fifth cellulolytic microorganism are used, preferably each is of the same or a related genus (i.e., of the same family). In some embodiments, each microorganism used is a different species. In some embodiments, two or more of the same species of microorganism may be used. It is believed that the competitive effects described above are particularly prevalent in embodiments using cellulolytic microorganisms of the same genus.

[0074] The cellulolytic microorganisms may be maintained in culture, for example prior to use in the methods described herein for decomposition of cellulosic material, either separately or together, usually the former. Methods of culturing cellulolytic microorganisms are generally known in the art. This may involve providing the cellulolytic microorganism with growth conditions. Growth conditions are conditions enabling the cellulolytic microorganism to survive and function as desired e.g., produce a cellulase. Growth conditions generally comprise a growth (culture) medium comprising nutrients e.g., a food source and will generally be prepared using aseptic handling techniques. Other growth conditions include appropriate light / dark, temperature, pH and atmosphere; e.g., including oxygen in the case of aerobic cellulolytic microorganisms,. Providing a cellulolytic microorganism or combination thereof to growth conditions may involve producing a growth medium containing the cellulolytic microorganism or combination thereof and subjecting the growth medium to growth conditions. Prior to use in a combination as described herein for decomposition of cellulosic materials, a cultured cellulolytic microorganism or a portion thereof, alone or together with another, may be subject to an incubation period involving culturing under growth conditions to increase the total cellulolytic microorganism count, for example to coax the cellulolyticmicroorganism growth out of a lag phase, into becoming acclimatised to growth conditions, and into an exponential growth phase where total cellulolytic microorganism count increases considerably. The preferred culturing conditions are as follows, especially in the case of the cellulolytic microorganisms each being a bacteria, and especially in the case of the preferred bacteria species and strains described below: ^ a nutrient rich aqueous medium such as peptone broth, optionally containing a source of tryptophan, for example casein digested peptone, preferably agitated; ^ the aqueous medium is maintained at a pH of between about 6.5 and about 7.5; ^ gaseous atmosphere containing oxygen at about or at least atmospheric levels, preferably provided using an aeration apparatus (which may provide agitation); and ^ a temperature maintained between about 25°C and about 38°C. Bacteria

[0075] Bacteria selected from the phyla Bacillota, Pseudomonadota and Actinomycetota are generally cellulolytic microorganisms capable of producing a cellulase classifiable by EC 3.2.1.4, EC 3.2.1.91, EC 3.2.1.21, EC 3.2.1.52 EC 3.2.1.74 or 3.2.1.86, including extracellularly. Preferably, the bacteria is selected from the genus Azospirillum, Bacillus, Bradyrhizobium, Chromobacterium, Saccharopolyspora, Bradyrhizobium and Priestia.

[0076] Preferably, the cellulolytic microorganism of the Bacillus genus is selected from the species Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumillus, Bacillus subtilis, Bacillus subtilis subsp. subtilis, Bacillus thuringiensis, Bacillus thuringiensis subsp. Kurstaki (equivalent to Bacillus thuringiensis Berliner), Bacillus toyonensis biovar Thuringiensis, and Bacillus velezensis. Preferably, the cellulolytic microorganism of the Chromobacterium genus is of the species Chromobacterium subtsugae. Preferably, the cellulolytic microorganism of the Saccharopolyspora genus is of the species Saccharopolyspora spinosa. Preferably, the cellulolytic microorganism of the Bradyrhizobium genus is selected from the species Bradyrhizobium japonicum. Preferably, the cellulolytic microorganism of the Priestia genus is selected from the species Priestia aryabhattai and Priestia megaterium.

[0077] Bacteria of these preferred species may tend to produce cellulases within about 80% sequence identify of the sequences provided herein. For instance: a) cellulolytic microorganisms of the listed Bacillus species may produce a cellulase classifiable by EC 3.2.1.4 having at least 80% sequence identify to SEQ ID No.1, 2 or 3. b) cellulolytic microorganisms of the listed Bacillus species may produce a cellulase classifiable by EC 3.2.1.21 having at least 80% sequence identify to SEQ ID No. 5. c) cellulolytic microorganisms of the listed Bacillus species may produce a cellulase classifiable by EC 3.2.1.52 having at least 80% sequence identify to SEQ ID No. 6.d) cellulolytic microorganisms of the listed Priestia species may produce a cellulase classifiable by EC 3.2.1.86 having at least 80% sequence identify to SEQ ID No. 7.

[0078] More preferably, the bacteria is selected from the genus Bacillus and Priestia including as selected from the above-preferred species. In preferred embodiments, the, two or more or each bacteria is selected from a Bacillus species which produces a cellulase classifiable by EC 3.2.1.4, preferably also 3.2.1.86, and preferably also produces an amylase classifiable by EC 3.2.1.1. In preferred embodiments, a first bacteria is selected from a Bacillus species which produces a cellulase classifiable by EC 3.2.1.4, preferably also EC 3.2.1.86, preferably also EC 3.2.1.21, and preferably also produces a hemicellulase classifiable by EC 3.2.1.8, and preferably also produces an amylase classifiable by 3.2.1.1, preferably also EC 2.4.1.1. In preferred embodiments, a second, third, fourth and / or fifth etc. bacteria is selected from a Bacillus and / or Prestia species which produces a cellulase classifiable by EC 3.2.1.4, preferably also EC 3.2.1.86, possibly also EC 3.2.1.21, and may also produces a hemicellulase classifiable by EC 3.2.1.8, and preferably also produces an amylase classifiable by 3.2.1.1.

[0079] Preferably, the bacteria is selected from the strain Bacillus amyloliquefaciens CCT 7690 (equivalent to ATCC 23845), Bacillus licheniformis CCT 2584, Bacillus pumillus CCT 2487, Bacillus subtilis ATCC 6051, Bacillus toyonensis biovar Thuringiensis CCT 7795, Bacillus velezensis CCT 7891 (equivalent to CECT 5686), Bradyrhizobium japonicum strain CCT 4065, Bacillus thuringiensis Berliner ATCC 33679 (equivalent to Bacillus thuringiensis subsp. kurstaki CCT 1306), Priestia aryabhattai strain CBMAI 1120, and Priestia megaterium strain CCT 2478 (equivalent to ATCC 14581). Each of these strains is or once was naturally-occurring. A sample of each of these strains may be found in repositories with details as follows: CCT - Tropical Culture Collection (Brazil), André Tosello Foundation, Rua Latino Coelho, 1301 Pq. Taquaral - CEP 13087-010, Campinas - SP - Brazil; CECT – Spanish Type Culture Collection, University of Valencia, Parc Científic UV Edificio 3CUE, Catedrático Agustín Escardino, 9, 46980 Paterna (Valencia) SPAIN; ATCC – American Type Culture Collection, 217 Perry Pkwy Ste 1, Gaithersburg, MD 20877, United States.

[0080] Bacteria of the listed preferred strain may tend to produce cellulases represented by the protein sequences provided herein, or within about 95% sequence identify thereto. Table 1 contains examples. Table 1. Preferred bacteria strains and cellulases produced. Strain SEQ Reference EC Accession No. Index ofB. thuringiensis 2 ATCC 33679 3.2.1.4 AGW99983 (G) 1.35

[0081] Other bacterial strains include: B. licheniformis CCT 2584 (IE of 1.12); B. pumillus CCT 2487 (IE of 1.17); B. japonicum strain CCT 4065; P. aryabhattai strain CBMAI 1120 (IE of 1.05).

[0082] Again, these cellulolytic microorganisms that produce the specified cellulases may, and often do, produce other cellulases including those classifiable by EC 3.2.1.4, EC 3.2.1.21, EC 3.2.1.52, EC 3.2.1.91, EC 3.2.1.74 or EC 3.2.1.86, but it is for the specified cellulases that these cellulolytic microorganisms may be selected. Similarly, these cellulolytic microorganisms may produce other glycosylases including a hemicellulase and / or an amylase.

[0083] The IE presented in Table 1 are measurable using the CMC assay described above. Fungi

[0084] Fungi selected from the family Cordycipitaceae, Clavicipitaceae and Hypocreaceae are generally cellulolytic microorganisms capable of producing a cellulase classifiable by EC 3.2.1.91, including extracellularly. Preferably, the fungi is selected from the genus Beauveria, Isaria, Metarhizium, Pochonia and Trichoderma.

[0085] Preferably the fungi is selected from the species Beauveria bassiana, Isaria javanica, Metarhizium anisopliae, Pochonia chlamydosporia, Trichoderma asperellum, and Trichoderma harzianum.

[0086] Fungi of these preferred species may tend to produce cellulases within about 80% sequence identify of the sequences provided herein. For instance, cellulolytic microorganisms of the species Trichoderma harzianum may produce a cellulase classifiable by EC 3.2.1.91 having at least 80% sequence identify to SEQ ID No.4.

[0087] Other Fungi species that generally produce glycosylases of interest include: Beauveria bassiana, Isaria javanica, Metarhizium anisopliae, Pochonia chlamydosporia, or Trichoderma asperellum.

[0088] Preferably, the fungi is selected from the strain Beauveria bassiana strain CCT 3161, Isaria javanica strain CCT 5823, Metarhizium anisopliae strain CCT 7442, Pochonia chlamydosporia var. chlamydosporia strain CCT 4077, Trichoderma asperellum (harzianum) strain CCT 6550, and Trichoderma harzianum strain CCT 2160 (equivalent to ATCC 48131). Each of these strains is or once was naturally-occurring. A sample of each of these strains may befound in repositories with details as follows: CCT - Tropical Culture Collection (Brazil), André Tosello Foundation, Rua Latino Coelho, 1301 Pq. Taquaral - CEP 13087-010, Campinas - SP – Brazil; ATCC - American Type Culture Collection, 217 Perry Pkwy Ste 1, Gaithersburg, MD 20877, United States.

[0089] Fungi of these preferred strains may tend to produce cellulsases represented by the protein sequences provided herein, or within about 95% sequence identify thereto. An example is shown in Table 2. Table 2. Preferred fungi strains and cellulases produced.

[0090] Again, these cellulolytic microorganisms that produce the specified cellulases may, and often do, produce other cellulases including those classifiable by EC 3.2.1.4, EC 3.2.1.21, EC 3.2.1.52, EC 3.2.1.91, EC 3.2.1.74 or EC 3.2.1.86, but it is for the specified cellulases that these cellulolytic microorganisms may be selected. Similarly, these cellulolytic microorganisms may produce other glycosylases including a hemicellulase and / or an amylase. Preferred Combinations

[0091] Preferably, at least one cellulolytic microorganism in the combination is a Bacillus species microorganism, and is preferably the cellulolytic microorganism that produces a cellulase classifiable by EC 3.2.1.4 or EC 3.2.1.91. Preferably, both the first and second cellulolytic microorganisms in the combination are a Bacillus or a Prestia species microorganism, preferably selected from the species and strains identified above. When a third, fourth and fifth cellulolytic microorganism is used, preferably each is a Bacillus or a Prestia species microorganism, preferably selected from the species and strains identified above. Preferably, every cellulolytic microorganism of the combination is a Bacillus or a Prestia species microorganism, preferably selected from the species and strains identified above. In some embodiments, each Bacillus or Prestia species microorganism used is of a different species. In some embodiments, two of the microorganism used are of the same species, being different strains. It is believed that the competitive effects described above are particularly prevalent when at least one Bacillus species / strain is used, and moreover when more than one Bacillus species / strain is used or a Bacillus species / strain is used in combination with a Prestia species / strain. Bacillus and Prestia species bacteria are found naturally in soil and water environments amongst a diverse microbiome and in resource-scarce conditions and it is believed have evolved a hyper-competitive nature. Another advantage also arises from the use of Bacillus species bacteria, including alongside Prestia species bacteria, being that the overallenzymatic activities of Bacillus species in combination are applicable to a wide array of cellulosic materials especially cell wall cellulosic materials.

[0092] In preferred embodiments, the first cellulolytic microorganism is strain Bacillus subtilis strain ATCC 6051 selected for its ability to produce a cellulase classifiable by EC 3.2.1.4, and the second cellulolytic microorganism is strain Bacillus amyloliquefaciens ATCC 23845 selected for its ability to produce a cellulase classifiable by EC 3.2.1.21. This combination is preferred for its particular capability to provide a synergistic effect in the efficiency of decomposition of cellulosic material. Preferably, a third cellulolytic microorganism is included, in which case the preferred combination comprises the first cellulolytic microorganism strain being Bacillus subtilis strain ATCC 6051 selected for its ability to produce a cellulase classifiable by EC 3.2.1.4, the second cellulolytic microorganism strain being Bacillus amyloliquefaciens ATCC 23845 selected for its ability to produce a cellulase classifiable by EC 3.2.1.21, and the third cellulolytic microorganism strain being Priestia megaterium strain ATCC 14581 selected for its ability to produce a cellulase classifiable by 3.2.1.86. This combination is preferred for its particular capability to provide a suspected synergistic effect in the efficiency of decomposition of cellulosic material. Fourth and fifth cellulolytic microorganisms may be included, in which case the fourth cellulolytic microorganism strain is Bacillus velezensis strain CECT 5686 (equivalent to CCT 7891) selected for its ability to produce a cellulase classifiable by EC 3.2.1.4, and the fifth cellulolytic microorganism strain being Bacillus thuringiensis Berliner ATCC 33679 (equivalent to Bacillus thuringiensis subsp. Kurstaki CCT 1306) selected for its ability to produce a cellulase classifiable by EC 3.2.1.4.

[0093] In preferred embodiments, the combination is free of a Klebsiella aerogenes bacteria strain. In other words, preferably the combination consists of cellulolytic microorganisms other than K. aerogenes. In other words, the combination does not include K. aerogenes. Similarly, in preferred embodiments, the combination is free of a Klebsiella genus strain. In other words, preferably the combination consists of cellulolytic microorganisms other than Klebsiella strains. In other words, the combination does not include a Klebsiella strain. Klebsiella strains are anaerobic bacteria or facultatively anaerobic bacteria. The preferred combinations of cellulolytic microorganisms herein comprise aerobic cellulolytic microorganisms only. In other words, the preferred combinations consist of aerobic cellulolytic microorganisms. Culture conditions suitable for growth of aerobic and anaerobic microorganisms are generally incompatible. This means that an aerobic microorganisms stored under anaerobic conditions, and vice versa, will not survive and / or will not perform its desired function. For instance, a Bacillus microorganism will generally not produce an extracellular cellulase under anaerobic conditions. Cellulosic Material

[0094] In preferred embodiments, the cellulosic material is of a phototrophic, heterotrophic, or mixotrophic organism. Or put generally, the cellulosic material is preferably “cellular”, meaning derived from a cell. A phototrophic organism is an organism that uses light formetabolism in the presence of water and carbon dioxide to produce cellulosic materials, by photosynthesis. Examples include species of algae, phytoplankton, bacteria and protists. A heterotrophic organism is an organism that does not use light, but uses organic compounds for metabolism to produce cellulosic materials. Examples include species of animal, bacteria, fungi and protists. A mixotrophic organism is capable of functioning both phototrophically and heterotrophically but generally only one way at a time. The way that a mixotrophic organism is functioning may be referred to as a “mode”, i.e., phototrophic mode when functioning phototrophically and heterotrophic mode when functioning heterotrophically. A mixotrophic organism when functioning in phototrophic mode may be referred to as a phototrophic organism and a mixotrophic organism when functioning in heterotrophic mode may be referred to as a heterotrophic organism. Examples of mixotrophic organisms include species of algae, terrestrial plants and protists. Preferred sources are phototrophic or mixotrophic organisms.

[0095] The phototrophic microorganisms may comprise lysed, ruptured or intact cells. Lysis refers to a cell membrane, whereas rupture refers to a cell wall. Both refer to a structural breakdown of the membrane or cell wall barrier such that one or more substances the passage of which across the barrier is normally controlled, is no longer fully controllable, i.e., free or less restricted passage is allowed at at least one point of the barrier. This may be by way of, for example, from creation of a pore(s) to complete or near-complete decomposition of the entire polysaccharide component of the cell wall. Intact cells containing a cell wall may be referred to as “walled cells” and are as distinct from non-walled cells. Walled cells contain cell wall cellulosic material. Generally speaking, ruptured and / or lysed cells may provide a greater amount of cellulosic materials by allowing the cellulolytic microorganisms access to intracellular cellulosic materials and access to other valuable intracellular materials including lipids and proteins – essentially every cell produces lipid and protein to some extent – which may be retrieved. However, it is traditionally difficult and costly to rupture walled cells so these embodiments are not preferred from a ‘green’ perspective. Such techniques include mechanical methods known in the art such as grinding, pressing, sonication, microwave treatment and homogenisation, and chemical methods including the use of organic solvents, surfactants, chaotropic agents, ozmolysis or acidic or alkaline reagents. That said, herein, it is preferred that the cellulosic material is cell wall cellulosic material of walled cells. This is because, in certain embodiments, the combination described herein itself provides the means for rupturing the cell wall, as described below, providing access to the valuable contents within walled cells in a milder and more energy-efficient fashion. That is, using the combination described herein for rupturing the cell wall, these traditional methods – which are energy inefficient, use harsh chemicals and many to all of which require the input of, or generate, heat to work effectively – may be avoided or used in a less intense – or ‘greener’ – fashion. For example, where traditionally homogenisation at above-physiological temperature (relative to the cells in question) may have been used to rupture walled cells, the combination described herein may achieve cell wall rupture at no higher than physiological temperature providing for a much ‘greener’ alternative, and especially if carbohydrate recycling is used. Further, wheretraditionally homogenisation at high pressure, e.g., 1070 bar for Chlorella vulgaris may have been used to rupture walled cells and lyse cell membranes to allow access to intracellular contants, the combination described herein may achieve cell wall rupture without homogentisation, i.e., at atmospheric pressure, and thereby allow access to intracellular contents using lysis methods alone. While lysis methods may employ a homogeniser, it is generally required to operate at no greater than about 100 bar, representing a >10-fold decrease and providing the companying ‘green’ benefits, and especially if carbohydrate recycling is used. Similarly, traditionally chemicals method may be performed at above- physiological temperature to rupture walled cells, and the use of traditional chemicals can require downstream purification processes at energy cost. On the contrary, the combination described herein may achieve cell wall rupture without these resource demands, and especially if carbohydrate recycling is used. In addition to the ‘green’ benefit, the reduced energy demands, use of alkaline or acidic reagents and intensive downstream purification steps may avoid degradation of lipid and / or protein molecules and allow retrieval of comparatively higher yields of structurally intact lipid and protein.

[0096] The combined cellulolytic microorganism and cellulosic material and may be said to be present together in a starting weight ratio of cellulolytic microorganism to cellulosic material, or in other words a weight ratio at time zero, i.e., t = 0, i.e., at the point in time that the cellulolytic microorganism combination and cellulosic material are brought together or provided with growth conditions such that the cellulolytic microorganisms act to biodecompose the cellulosic material. A weight ratio when referring to an organism is generally based on dry weight. A starting weight ratio is convenient to use because the ratio of cellulolytic microorganisms and cellulosic material is unlikely to be static; it will likely change over time as the cellulosic material is biodegraded. The starting (dry) weight ratio of cellulolytic microorganism to cellulosic material may be at least about 0.002 (i.e., 1 to 500), or about 0.004 or about 0.01, or at most about 0.2 or 0.1 or 0.04, or of about 0.02, or within the range defined by any of these lower and upper limits, e.g., of about 0.002 to 0.2. Preferably, the starting (dry) weight ratio of cellulolytic microorganism to cellulosic material is in the range of 0.016 to 0.03. When the cellulosic material is walled cells, the starting weight ratio may be expressed as a dry weight of cells to cells, i.e., of cellulolytic microorganism cells to cellulosic material cells and may be referred to as a starting cellular weight ratio. In these embodiments the starting (dry) weight ratio of cellulolytic microorganism to cellulosic material may be at least about 0.001 (i.e.1 to a thousand), or about 0.002 or about 0.005, or at most about 0.1 or 0.05 or 0.02, or of about 0.01, or within the range defined by any of these lower and upper limits, e.g., of about 0.001 to 0.1. Preferably, the starting cellular weight ratio of cellulolytic microorganism to cellulosic material is in the range of 0.008 to 0.015.

[0097] The preferred combinations described herein are particularly effective in the decomposition of cellulose. Accordingly, in preferred embodiments, the cellulosic material comprises cellulose, e.g., as is found in the cell wall of many walled cells.

[0098] Without being bound by theory, it is believed that the presence of EPS has the effect of upregulating the production of glycosylases by cellulolytic microorganisms and increasing the production of glycosylases, thereby increasing the rate of decomposition of cellulosic materials. Accordingly, in preferred embodiments, the cellulosic material comprises EPS. In which case, in preferred embodiments, the cellulosic material is derived from an organism which produces EPS. Examples include species of bacteria, fungi and algae. Put another way, a preferred embodiment is that the cellulosic material comprises cell wall cellulosic material of walled cells, which walled cells produce EPS.

[0099] Preferably, the organism used as the source of cellulosic material is an algae. An algae is identifiable as a photosynthetic eukaryotic non-flowering aquatic organism. Examples include red algae, green algae and cyanobacteria, which generally includes species classified as Halvaria and Hacrobia (red algae) containing the Heterokonts, Dinoflagellates, Haptophyta and Cryptomonads, as Excavata and Rhizaria (green algae) containing the Chlorarachniophytes, Euglenids and Chlorophyta, and as Primoplantae and Archaeplastida (cyanobacteria) containing the Chlorophyta and Rhodophyta. Cellulosic material, especially in the form of cellulose, tends to be a major component of the cell wall of algal cells, especially microalgae. Algal cells, especially microalgae, tend to produce EPS.

[0100] Preferably, the algae is a microalgae. A microalgae is identifiable as a unicellular algae that is often too small to be visible by the naked eye, and may otherwise be referred to as a microphyte. Microalgae may exist independently or in colonies. A microalgae is preferred as cellulosic material, especially cellulose, tends to be a major component of the cell wall of microalgal cells, and also because microalgae tend to be capable of producing EPS.

[0101] Further, microalgae tends to be capable of producing intracellular lipid and protein at a faster rate and / or in a larger proportion per cell volume or weight than many other cell types. This applies especially to mixotrophic algae, which tend to accumulate even greater lipid and protein content when grown in mixotrophic mode. In context of lipid production in the methods of the disclosure, “lipid” refers to fatty acids such as palmitic acid, oleic acid and linoleic acid, and triglycerides such as tripalmitin, triolein and trilinolein. Lipids are a valuable commodity. Microalgae also produces a significant proportion of protein which is also valuable in many industries. Accordingly, using microalgae as the source of cellulosic material provides the benefit of producing not only simple carbohydrates through the biodecomposition of cell wall cellulosic materials, but in doing so is an improved way of rupturing the cell wall allowing access to the intracellular contents, e.g., lipid, protein, cellulosic materials, and in greater quantities using microalgae in mixotrophic mode.

[0102] Preferably, the microalgae is a green algae. Preferably, the microalgae is selected from a Chlorella species, such as from the group consisting of Chlorella autotrophica, Chlorella colonials, Chlorella lewinii, Chlorella minutissima, Chlorella pituita, Chlorella pulchelloides, Chlorella pyrenoidosa, Chlorella rotunda, Chlorella singularis, Chlorella sorokiniana, Chlorella variabilis, Chlorella volutis and Chlorella vulgaris. Chlorella species are preferred for their abilityto grow rapidly and to contain significant cellulose in their cell walls and to produce significant quantities of lipid and protein. Chlorella species are also capable of producing EPS. Chlorella species may also be mixotrophic.

[0103] In preferred embodiments, the microalgae is C. vulgaris species microalgae, preferably selected from one or both of C. vulgaris species strain CS-41 or CCAP 211 / 11S, most preferably CS-41. A culture of C. vulgaris strain CS-41 is maintained at the Australian National Algae Culture Collection, CSIRO, Hobart, Tasmania, Australia, under CS number “CS-41”. A culture of C. vulgaris strain CCAP 211 / 11S is maintained at the Culture Collection of Algae and Protozoa, Oban, Scotland, UK, under accession no. MG022720, and is postulated to be the same strain as CS-41. CS-41 and CCAP 211 / 11S are mixotrophic and tend to produce EPS and significant lipid and protein.

[0104] The majority of the cellulosic material produced by C. vulgaris species microalgae (up to 55% total biomass) are polysaccharides based on glucose. These cellulosic materials tend to be sequestered in the cell wall of the microalgal cell as cellulose, hemicellulose and starch, and as exopolysaccharides which are released from the cells such as EPS. Applying the cellulolytic microorganism combinations as described herein to C. vulgaris species microalgae provides for the production of simple carbohydrates by biodecomposition of the cell wall and in doing so provides for the rupture of the cell wall for retrieval of intracellular lipid and protein. Compositions

[0105] The combination of cellulolytic microorganisms described herein may be, and preferably is, in the form of a composition. In the methods for decomposing cellulosic material described herein, generally speaking the cellulolytic microorganism are placed together with a cellulosic material. Accordingly, preferably a single composition containing each of the cellulolytic microorganisms is preferred.

[0106] The composition may be in the form of a solid or liquid culture, e.g., a growth medium. A liquid culture differs from a solid culture in that it will generally comprise a liquid medium, often aqueous. In preferred embodiments, and for applying to the methods described herein, the composition is in the form of an aqueous growth medium (e.g., growth conditions).

[0107] The composition may comprise additional components, such as nutrients, pH adjusting means (i.e., acid, alkaline species), buffer, dissolved gases, and other microorganisms including other than cellulolytic microorganisms. For instance, when the source of cellulosic material is a phototrophic organism, especially a microalgae, the other microorganisms will include the phototrophic organism or microalgae. The preferred aqueous medium is a growth medium and, especially in the case of the cellulolytic microorganisms each being a bacteria, and especially in the case of the preferred bacteria species and strains described herein, may be tryptone soy broth (TSB), brain heart infusion (BHI) broth, CMC broth and neat and diluted soy vinasse.

[0108] When placed together with a cellulosic material, the composition will also comprise a cellulosic material, and immediately upon activity by the glycosylases will also comprise simple carbohydrates, generally including carbohydrate monomer. Accordingly, in preferred embodiments, the composition comprises a first cellulolytic microorganism that produces a cellulase classifiable by EC 3.2.1.4 or EC 3.2.1.91, and a second cellulolytic microorganism that produces a cellulase classifiable by EC 3.2.1.21, EC 3.2.1.52 or 3.2.1.86. Preferably, the composition comprises a cellulosic material and a carbohydrate monomer. The composition may comprise one or more additional cellulolytic microorganisms, i.e., a third, fourth, fifth etc., as described herein.

[0109] The combination of cellulolytic microorganisms described herein may be in the form of a kit. The kit may comprise a single package or series of packages. The kit will comprise at least a first cellulolytic microorganism that produces a cellulase classifiable by EC 3.2.1.4 or EC 3.2.1.91, and a second cellulolytic microorganism that produces a cellulase classifiable by EC 3.2.1.4, EC 3.2.1.91, EC 3.2.1.21, EC 3.2.1.52, EC 3.2.1.74 or 3.2.1.86. The kit may further comprise a growth medium or ingredients for producing a growth medium. The kit may comprise instructions for using the cellulolytic microorganisms in a method for decomposing cellulosic material. Methods

[0110] In use for decomposition of cellulosic material, the combinations of cellulolytic microorganisms as described herein may need nothing further than to be placed together with a cellulosic material under growth conditions, e.g., light, temperature, pH and atmosphere; e.g., oxygen in the case of aerobic cellulolytic microorganisms. The preferred culturing conditions are as described above, especially in the case of the cellulolytic microorganisms each being a bacteria, and especially in the case of bacteria of the genus Bacillus.The glycosylases produced by the cellulolytic microorganism may then begin decomposing the cellulosic material. This is “biodecomposition” of the cellulosic material by the cellulolytic microorganisms and may be referred to as a “biodecomposition step”. Biodecomposition produces simple carbohydrates. A simple carbohydrate produced by biodecomposition may be referred to as a “biodecomposed simple carbohydrate”. Accordingly, it may be said that biodecomposition occurs in a method of producing a simple carbohydrate. Put another way, it may be said that provided herein is a method of producing a simple carbohydrate by biodecomposition using a combination of cellulolytic microorganisms as herein described. Simple carbohydrates so-produced may be, and preferably are, retrieved. In which case, it may be said that provided herein is a method of retrieval of a simple carbohydrate produced by biodecomposition of a cellulosic material using a combination of cellulolytic microorganisms as herein described.

[0111] As described above, in preferred embodiments the cellulosic material subject to the cellulase function of the cellulolytic microorganisms comprises cellulosic material of walled cells. Biodecomposition of walled cells produces a ruptured cell wall. In which case, it may besaid that biodecomposition occurs in a method of rupturing a cell wall of a walled cell. Put another way, it may be said that provided herein is a method of rupturing a cell wall of a walled cell by biodecomposition using a combination of cellulolytic microorganisms as herein described.

[0112] Rupture of a cell wall provides a means for accessing intracellular contents and especially lipid and protein. In which case, it may be said that provided herein is a method of retrieval of intracellular lipid and / or intracellular protein from a walled cell using biodecomposition of a walled cell using a combination of cellulolytic microorganisms as herein described.

[0113] In certain preferred embodiments as described above, the walled cell may be of a mixotrophic organism. Mixotrophic organisms e.g., Chlorella species are often capable of producing greater intracellular lipid and protein content when subject to heterotrophic mode. Accordingly, in such methods of retrieval, and in preferred embodiments, the walled cell is mixotrophic and subject to heterotrophic mode (or otherwise subject to a step that induces the walled cell to produce lipid and / or protein). This will generally be a step performed prior to the walled cells being subject to the combination of cellulolytic microorganism. When such a step is employed, it, or the method containing it, may be referred to as a step or method of “producing” lipid and / or protein. Put another way, it may be said that provided herein is a method of producing intracellular lipid and / or intracellular protein from a mixotrophic organism, comprising subjecting the organism to heterotrophic mode and then performing the method of retrieval as herein described.

[0114] In use for decomposition of cellulosic materials sourced from one or more phototrophic organisms, for example a microalgae such as C. vulgaris, the phototrophic organism biomass may be provided at a concentration of from about 1 g / L to about 3 g / L or about 3 g / L to about 10 g / L or at least about 15 g / L or about 30 g / L or more, in liquid culture medium.

[0115] As described above, the use of a combination of cellulolytic microorganisms provides for the production of simple carbohydrates, or biodecomposition of cellulosic material, at a greater rate when compared with the combined capability of the individual cellulolytic microorganism. Put another way, this provides for the biodecomposition of an amount of cellulosic material in a shorter time frame, or the production of an amount of simple carbohydrate in a shorter time frame. The rate may be accelerated from two cellulolytic microorganisms in the combination to three, four and five cellulolytic microorganisms in the combination..

[0116] When the cellulosic material is of walled cells, after 24 hours of biodecomposition, one or more of the following conditions may be met including when the starting cellular dry weight ratio is if from 0.008 to 0.015:a. At least 20%, preferably 30%, 40%, 50%, 55%, 60% 65% or 70%, of cell walls are ruptured, which may even be at least 75%, 80%, 85% or 90%, or may be within the range of 20% and 100%, for example between 30% and 100%, 40% and 100%, 50% and 100%, 55% and 100%, 60% and 100%, 65% and 100% or 70% and 100%; b. When the cellulosic material comprises EPS, at least 20%, preferably 30%, 40%, 50%, 60%, 70%, 80% or 90% of EPS is biodecomposed, or an amount of EPS within the range of 20% and 100%, for example between 30% and 100%, 40% and 100%, 50% and 100%, 60% and 100%, 70% and 100%, 80% and 100% or 90% and 100% is biodecomposed; c. at least 20%, preferably 30%, 40%, 50%, 55%, 60% 65% or 70%, of intracellular lipid and / or protein of the cellular cellulosic material is retrievable, which may even be at least 75%, 80%, 85% or 90%, or may be within the range of 20% and 100%, for example between 30% and 100%, 40% and 100%, 50% and 100%, 55% and 100%, 60% and 100%, 65% and 100% or 70% and 100%: i. without the application of further cell rupture techniques, or put another way in the absence of further cell rupture techniques, or put another way in a process that is free of further cell rupture techniques; ii. insofar as cell rupture and lysis techniques are concerned, by a method consisting of cell lysis or neither; iii. at no greater than physiological temperature; or iv. in the absence of highly acidic (pH < 3) or even mildly acidic (pH between 3 and 6) reagents or in the absence of highly alkaline (pH > 11) or even mildly alkaline reagents (pH between 8 and 11).

[0117] Alternatively or in addition, the amount of simple carbohydrate present after 24 hours of biodecomposition compared to the amount present just prior to the beginning of the biodecomposition step (i.e., at t = 0) may be about or at least about a two-, three-, four-, five-, six- or seven-fold increase, or up to a 12-, 11-, ten- or nine-fold increase, or between any of these lower and upper amounts without limitation, for example between a 2- and a 12-fold increase, or between a six- and a nine-fold increase, etc., including when the cellulosic material is of a cell wall, including when the starting cellular dry weight ratio is if from 0.008 to 0.015.

[0118] In preferred embodiments, simple carbohydrates are retrieved from the composition. The simple carbohydrates may be used in subsequent industrial processes including applications in the renewable energy, biopharmaceutical, agricultural, bioplastic manufacture, food and nutraceutical industries. In some applications, the simple carbohydrate may be used as a feedstock in the production of methane and / or biofertilizer in industrial processes usinganaerobic microorganisms. That said, in preferred embodiments in which the cellulosic material is of an organism, the simple carbohydrate may be used as a feedstock in the growth of that organism. For example, simple carbohydrate retrieved from the biodecomposition of one batch of cellular cellulosic material may be used as a nutrient, e.g., in a growth medium, to produce another batch of cellular cellulosic material (the same or different organism) from which simple carbohydrates may be produced by biodecomposition and retrieved, and so-on. This may be referred to as a step or method of “carbohydrate recycling”, in which simple carbohydrates produced from an organism, i.e., from cellular cellulosic material, by biodecomposition, may be fed to that organism or another organism for the production of complex carbohydrates from which simple carbohydrates can be produced by biodecomposition.

[0119] Carbohydrate recycling is particularly valuable for the commodities that may be retrieved in the process, e.g., lipid and protein, especially when the cellular cellulosic material is of a walled cell of a mixotrophic organism subject to a heterotrophic growth mode for increased production of lipid and protein. This may be referred to as a step or a method of production of lipid and / or protein by carbohydrate recycling. Put another way, simple carbohydrate produced by biodecomposition as described herein and retrieved, may be said to be used as a feedstock in the production of lipid and / or protein from walled cells. In the methods of rupturing a cell wall of a walled cell by biodecomposition and of retrieval of intracellular lipid and / or intracellular protein from a walled cell as provided herein, additional steps may be set out as follows: retrieval of biodecomposed simple carbohydrate and optionally of intracellular lipid and / or intracellular protein; and producing cells comprising a cell wall cellulosic material using a growth medium comprising the retrieved simple carbohydrate.

[0120] Put another way, in the methods of rupturing a cell wall of a walled cell by biodecomposition and of retrieval of intracellular lipid and / or intracellular protein from a walled cell as provided herein, the walled cells may be as produced using a growth medium comprising simple carbohydrate produced by a biodecomposition step.

[0121] Put another way, the following steps may occur: 1. Produce a simple carbohydrate by subjecting a cellulosic material to a combination of cellulolytic microorganisms of the present disclosure; 2. Culture a heterotrophic organism using the produced simple carbohydrate as an organic carbon source allowing the heterotrophic organism to produce cellulosic material and preferably valuable by-product (e.g., lipid, protein);3. Subject the cellulosic material (produced in step 2) to a combination of cellulolytic microorganisms of the present disclosure to produce a simple carbohydrate and preferably retrieve the valuable by-product and simple carbohydrate; and 4. Repeat steps 2 and 3 any desirable number of times.

[0122] In preferred embodiments, retrieval of lipid and / or protein is performed at no higher than physiological temperature. Depending on the cells in question, this may be a temperature of no higher than 70oC, or no higher than 65, 60, 55, 50, 45, or no higher than 40 or 35oC for the preferred walled cell cellulosic materials, and may be performed at a temperature of no lower than 0oC, or no lower than 5, 10 or 15oC, or no lower than 20oC for the preferred walled cell cellulosic materials, and may be performed at a temperature within any range compiled thereby. Using the preferred walled cell cellulosic materials, retrieval of lipid and / or protein is performed at about room temperature, or standard laboratory conditions, or within the range of about 15oC to 40, 35 or 30oC.

[0123] The biodecomposition of an amount step may be performed for a time period sufficient to decompose all or a desired quantity of the cellulosic material. This may be a time period of from about 2 hours to about 72 hours or of about 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66 or 70 hours or within any range compiled thereby. That said, given that the biological function of the cellulolytic microorganisms in the production of cellulases is to produce simple carbohydrates essentially for food, the cellulolytic microorganisms are generally capable of consuming the simple carbohydrates. As in preferred embodiments retrieval of simple carbohydrates is desired, it is preferred that the biodecomposition step be performed for a time period that reduces or minimises consumption of simple carbohydrates. In some embodiments, this may be a time period of from about 2 hours to about 36 hours or of about 4, 6, 8, 10, 12, 14 ,16, 18, 20 or 22 hours to about 34, 32, 30, 28 or 26 hours, or within any range compiled thereby, or of a time period of about 24 hours, say of about 23 to about 25 hours. When the cellulosic material is of walled cells, it has surprisingly been found that it is possible to disrupt cell walls such that one or more of the conditions described above are met, including with a when the starting cellular dry weight ratio is if from 0.008 to 0.015, within 24 hours.

[0124] Methods for producing cellular cellulosic materials, e.g., walled cells, including of mixotrophic organisms, including microalgae are otherwise generally known in the art.

[0125] In the case of mixotrophic organisms and microalgae in particular, the preferred phototrophic mode culturing methods, including for C. vulgaris, may be as described in the specification published under number WO WO / 2024 / 168400 which is herein incorporated by reference in its entirety. In brief, this culturing method involves growing the organism phototrophically in an aqueous medium under growth conditions with nutrients, specified photoperiod, light intensity, temperature, pH and gaseous atmosphere, including an above- atmospheric concentration of carbon dioxide for carbon dioxide sequestration, and the use ofan organism adapted to above-atmospheric concentrations of carbon dioxide. When the organism culture reaches a desired biomass concentration, the organism or a portion of the culture may be removed and used a source of cellulosic material as described herein. The preferred growth conditions include: ^ the aqueous medium is selected from one or both of Bold Basal media (BBM) or Blue- Green 11 media (BG-11), preferably BG-11; ^ using a bioreactor, the volume of microalgae and aqueous medium is between about 50% and 70% of bioreactor capacity; ^ the photoperiod is between about 15 h and about 17 h, preferably of about 16 h; ^ the light is of an intensity of between about 4500 lux and about 5500 lux, or preferably about 5000 lux; ^ the temperature is maintained between about 27°C and about 29°C, and more preferably at about 28°C ^ the aqueous medium is maintained at a pH of between about 6.5 and about 7.5, preferably at a pH of about 7; ^ the gaseous atmosphere has a concentration of carbon dioxide of between about 5% and 25%. ^ the gaseous atmosphere is provided at a flow rate of between about 0.9 VVM and 1.0 VVM, preferably of about 1 VVM. ^ The phototrophic organism is cultured until a biomass concentration of between 5.0 g / L and 5.3 g / L is achieved and a portion of biomass is removed to facilitate continuous culturing.

[0126] These conditions may be used to provide for a continuous feedstock source of phototrophic organisms for use as the source of cellulosic material for the purposes of the present disclosure.

[0127] When the phototrophic organism is mixotrophic, and especially when production of lipid and / or protein is desired, this culturing method may comprise a heterotrophic growth mode by simply turning out the lights and providing the organism with a source of organic carbon, e.g., simple carbohydrates. The preferred heterotrophic mode growth conditions, especially for microalgae and especially C. vulgaris, include the (practicably) absence of light, an aqueous growth medium containing an organic carbon source, and suitable temperature, pH and gaseous atmosphere, usually comprising oxygen. Carbohydrate recycling is applicable, i.e., simple carbohydrates produced by biodecomposition as described herein may be used in the growth medium for the phototrophic growth of the organism. When the organism culturereaches a desired biomass concentration, the organism or a portion of the culture may, optionally, be removed, and used as a source of cellulosic material as described herein.

[0128] It is optional for organism to be removed from culture as the source of cellulosic material because it is applicable for the cellulolytic microorganism combination to be added to the same culture e.g., along with simple carbohydrates and while switching off the lights. Put another way, growing an organism as the source of cellulosic material and the biodecomposition step may be a one-pot or two-pot process (two-pot meaning “two or more pots”). In a two-pot process, a step of collecting heterotrophic organism and separately subjecting it to biodecomposition, e.g., by transfer to a fresh growth media containing cellulolytic microorganisms. A one-pot process does not prevent lipid and protein production. This is because the organic carbon in the medium acts as an inhibitor to the production of cellulases by the cellulolytic microorganisms, e.g., food is plentiful such that it is not necessary for the cellulolytic microorganisms to activate their machinery, i.e., produce cellulases, to produce it from the heterotrophic organism and hereby decreasing its viability and lipid / protein production potential. The heterotrophic organism and cellulolytic microorganisms will consume the simple carbohydrates and increase biomass and produce lipid and protein. When the growth conditions become depleted of simple carbohydrate, the cellulolytic microorganisms will then begin producing cellulases to produce simple carbohydrates. It is believed, however, and without wishing to be limited by theory, that when the organism is a walled cell which produces extracellular cellulosic materials such as EPS, biodecomposition of the cell wall by cellulases is not immediate or at least is not initially swift, but rather that the extracellular cellulosic materials are swiftly biodecomposed which provides additional simple carbohydrates for the organism to metabolise and gain biomass before its cell wall has been disrupted. Further, it is believed that some cellulolytic microorganisms are capable of producing an organic carbon source in situ from an inorganic carbon source, e.g., carbon dioxide, which may also contribute to the simple carbohydrate available for metabolism. This would be to benefit e.g. of lipid and protein production, and it is thought outweigh the drawback of loss of simple carbohydrates consumed by the cellulolytic microorganisms even when retrieval of simple carbohydrates is desired, e.g., for carbohydrate recycling. That said, when lipid and / or protein production is desirable, it is preferred that the heterotrophic organism is given time to culture with simple carbohydrate before being subjected to a combination of cellulolytic microorganisms of the present disclosure, as it is believed that it leads to increased production of lipid and protein, especially in the case of microalgae and especially C. vulgaris. Put another way, preferably a two-pot process is used.

[0129] When heterotrophic mode is available, it is preferred to use a combination of phototrophic mode and heterotrophic mode culturing. Phototrophic mode, especially with microalgae, results in increased algal biomass through increased cell numbers, i.e., higher cell count. Heterotrophic mode, on the other hand, results in increased algal biomass through increased cell size and increased content of valuable by-product. This has been found to maximise the production efficiency of valuable by-product, e.g., lipid and / or protein.

[0130] Put another way, the following steps may occur: 1. Produce cellular cellulosic material by culturing a mixotrophic organism in phototrophic mode; 2. Produce cellular cellulosic material by culturing the mixotrophic organism in heterotrophic mode using produced simple carbohydrate as an organic carbon source; 3. Subject the mixotrophic organism to the combination of cellulolytic microorganisms to produce simple carbohydrate and ruptured cell walls by biodecomposition of the cellulosic material; 4. Retrieve lipid, protein and / or simple carbohydrate and repeat steps 1 to 4 any desirable number of times using retrieved simple carbohydrate as the organic carbon source in step 2.

[0131] When the cellulosic material is in the cell wall of a welled cell, a ruptured cell wall results by biodecomposition of the cell wall cellulosic materials by the cellulolytic microorganisms. This leaves the intracellular contents of ruptured cells generally encased by only a cell membrane which may be lysed to gain access to the intracellular contents, e.g., for retrieval of lipid and protein. Cell membrane lysis may be achieved using a mechanical and / or chemical lysis method. Mechanical methods are known in the art and include grinding, pressing, sonication and homogenisation. Chemical methods are also known in the art and include the use of organic solvents, surfactants, chaotropic agents and ozmolysis. When a walled cell is used as the source of cellulosic material, especially microalgae e.g., C. vulgaris, preferably a combination of mechanical and chemical lysis methods are used, and in particular sonication or homogenisation coupled with organic solvent with or without a surfactant. The organic solvent may be isopropanol, ethyl acetate, acetone, chloroform, toluene, dichloromethane, an alkane, a dialkyl ether, acetonitrile, cyclohexane and the like. Preferably the organic solvent is of comparatively low toxicity. The surfactant may be non-ionic, anionic, cationic or zwitterionic. Examples of cationic surfactants are quaternary ammonium salts such as cetyltrimethylammonium bromide (CTAB), dodecyltrimethylammonium chloride (DTAC), benzalkonium chloride (BAC), cetrimonium bromide (CTAB), tetramethylammonium hydroxide, behentrimonium chloride and stearyldimethylbenzylammonium chloride. Examples of anionic surfactants are: sulfates such as sodium dodecyl sulfate (SDS), ammonium lauryl sulfate (ALS) and sodium laureth sulfate (SLES); sulfonates such as dodecylbenzenesulfonate (DBS), linear alkylbenzene sulfonate (LAS) and toluene sulfonate; carboxylates such as sodium stearate and sodium oleate; and phosphates such as alkyl phosphates. Examples of nonionic surfactants are: polyoxyethylene ethers (ethoxylates) such as triton x-100 (polyethylene glycol octylphenyl ether), tween 20 (polysorbate 20), tween 80 (polysorbate 80) and brij series (e.g., brij-35); glycosides such as octyl glucoside and decyl glucoside; and alkanolamides such as cocamide MEA and cocamide DEA. Examples of zwitterionic surfactants are: betaines such as cocamidopropyl betaine and lauryl betaine; sulfobetaines (sultaines) sich as chaps, chapso andlauryl hydroxysultaine; amino acid derivatives such as sarcosinates (e.g., sodium lauroyl sarcosinate) and taurates; and phospholipids such as lecithin and phosphatidylcholine.

[0132] It has been found that the method herein applied to walled cells provides for the efficient rupture of cell walls compared to energy-intensive traditional methods, and for the efficient retrieval of intracellular commodities including lipid and / or protein, especially when utilising carbohydrate recycling which further reduces the energy input required to run the system.

[0133] Methods for identifying cellulolytic microorganisms for use in the combinations describe herein may involve one or more of the steps of genetic analysis, metabolic analysis and enzyme activity analysis.

[0134] Genetic analysis may involve identifying a cellulolytic microorganism based on it possessing a gene encoding for a glycosylase. This may be performed using bioinformatics techniques, or physically using, for example a hybridisation probe for binding to and detecting the presence of a gene in a microorganism. Bioinformatics and physical techniques for detecting genes are generally known in the art. Both techniques are principally similar, in that they utilise a reference DNA sequence or probe to detect the same or a complementary gene or portion thereof. The reference gene sequence will generally be derived from a known sequence from a glycosylase gene. The reference sequence may be derived from one or more of the protein sequences as described herein (i.e. by reverse translation). In preferred embodiments, the reference gene sequence is (Genbank accession NC_000964), which encodes for SEQ ID NO. 1. There is an immense body of electronically stored and accessible microorganism DNA sequence data and as such bioinformatics genetic analysis is preferred.

[0135] Metabolic analysis may involve identifying a cellulolytic microorganism based on its ability to produce a celullase. This may be performed using bioinformatics techniques, or physically using, for example an affinity probe for binding to and detecting the presence of a glycosylase in a microorganism. Bioinformatics and physical techniques for detecting glycosylases are generally known in the art. Bioinformatics techniques are principally similar to those for genetic analysis, in that they utilise a reference protein sequence to detect the same or a complementary protein sequence or portion thereof. The reference protein sequence may be one or more of the protein sequences as described herein or a portion thereof. In preferred embodiments, the reference protein sequence is UniProt accession number P10475 and Brenda enzyme database accession GUN2_BACSU). Physical techniques may utilise, for example, affinity probes which are complementary to a binding domain of a glycosylase. There is an immense body of electronically stored and accessible microorganism protein sequence data and as such bioinformatics metabolic analysis is preferred.

[0136] Enzyme activity analysis may subject a microorganism to an assay to test for the presence of a glycosylase and its functional efficiency. This is preferably performed physically and generally involves subjecting a cellulosic material to a microorganism or a glycosylasederived there from. Decomposition of the cellulosic material may be measurable as an indication of the glycosylase activity. The preferred enzyme activity analysis technique is a carboxymethylcellulose hydrolysis assay as described herein.

[0137] All three analyses may be performed: genetic, metabolic and enzyme activity analysis. In preferred embodiments, the steps of genetic or metabolic analysis are performed, along with a step of enzyme activity analysis.

[0138] Once a cellulolytic microorganism has been identified, it may be selected for use in the combinations described herein. Carbon dioxide sequestration

[0139] One or more of the cellulolytic microorganisms included in the combination may be capable of fixing carbon dioxide, meaning converting carbon dioxide into another molecule. At least the preferred species and strains of cellulolytic microorganisms described herein are so-capable. In which case, the method may also be referred to as a method of sequestering carbon dioxide from a source of carbon dioxide. EXAMPLES

[0140] Reference will now be made in detail to exemplary embodiments of the disclosure. While the disclosure will be described in conjunction with the exemplary embodiments, it will be understood that it is not intended to limit the disclosure to those embodiments. To the contrary, it is intended to cover alternatives, modifications, and equivalents as may be included within the spirit and scope of the disclosure as defined by the appended claims. Organisms

[0141] The organisms used in the following examples are listed in Table 3 (photosynthesis step) and Table 4 (biodecomposition step). Table 3. Photosynthesis step – phototrophic organism used as the source of cellulosic material. Species Strain Source Chlorella vulgaris CS-41 Australian National Algae Culture Collection, CSIRO, Hobart, Tasmania, Australia, under CS number “CS-41”. A culture of C. vulgaris strain CCAP 211 / 11S is maintained at the Culture Collection of Algae and Protozoa, Oban, Scotland, UK, under accession no. MG022720, and is postulated to be the same strain as CS-41.Table 4. Biodecomposition step – aerobic cellulolytic microorganisms used to decompose cellulosic material. Species Strain Source / availability Bacillus subtilis ATCC 6051 American Type Culture Collection Priestia megaterium ATCC 14581 American Type Culture Collection Bacillus thuringiensis subsp. kurstaki CCT 1306 American Type Culture Collection (equivalent to Bacillus thuringiensis Berliner ATCC 33679) Bacillus velezensis CCT 7891 Spanish Type Culture Collection - CECT (equivalent to CECT 5686) Bacillus pumillus CCT 2487 Tropical Culture Collection (Brazil) Priestia aryabhattai CBMAI 1120 Brazilian Collection of Environmental and Industrial Microorganisms (CBMAI) Bacillus amyloliquefaciens ATCC 23845 American Type Culture Collection Bacillus licheniformis CCT 2584 Tropical Culture Collection (Brazil)

[0142] The experiments were performed using the microalgae Chlorella vulgaris CS-41 (Table 3), which was cultured phototrophically in a photosynthesis step, and then in some instances heterotrophically, to produce an organic feedstock being a source of cellulosic material, which was then subject to a biodecomposition step using cellulolytic microorganisms. Said microorganisms used were aerobic microorganisms capable of decomposing cellulosic materials (e.g., cellulose and hemicellulose) found in the cell walls of the microalgae, to simple carbohydrates (e.g., glucose). The aerobic biodecomposition generated a feedstock for downstream applications, comprising one or more of simple carbohydrates, lipid and / or protein. Photosynthesis step Initial Culture Handling

[0143] Initially, C. vulgaris CS-41 stocks were resuscitated from -80°C and re-cultured in 1^L conical flask with a working volume of 400 mL in Bold’s Basal Medium (BBM) media at 28^°C. The culture was exposed to fluorescent lamps to provide the irradiance of approximately 4000 LUX until the sufficient cell density (~5-7 g / L) was attained using a purge gas of 10% (v / v) carbon dioxide and 90% air. This initial culture was used as an inoculum for subsequent studies. General Culturing Method

[0144] C. vulgaris was cultured in an aqueous culture medium in a bioreactor in the presence of light and a carbon dioxide source. After investigating the following parameters: culture medium (Bold’s Basal Media, BBM; soybean vinasse), reactor type (airlift bioreactor; bubble column reactor), centrifugation conditions to harvest algae and potential reuse of BBM media, the preferred culturing method was developed as described below.

[0145] Once the resuscitated stock culture was acclimatised to culture conditions, the inoculum for the experiments described in the present set of examples was prepared in pre- established volumes, with known concentration under aseptic conditions. The initial culture conditions used in the experiments of the example (unless stated otherwise) are given in Table 5 and the components of the media are provided in Table 6. Table 5. C. vulgaris culture conditions Inoculum volume 1% v / v of working volume (i.e., the volume of aqueous media) Culture medium BBM (available from e.g., Austratec, product ID B1675) Reactor type Multiphase reactor, e.g. bubble column (Figure 1) Reactor volume 2000 mL Reactor working volume 1800 mL Aeration Yes Flow rate 1VVMaFlow composition 10% CO2+ 90% air Air source Instrumental grade CO2source Industrial grade pH 7 Light type LED Photoperiod 12 h Light intensity 4,000 lux Temperature 28°C Runtime 7 daysaVVM, gas volumetric flow rate per unit volume of culture medium. Table 6. Composition of Bold’s Basal Media (BBM) culture media Chemicals BBM (g / L) Potassium Phosphate, Dibasic 0.075 Potassium Phosphate, Monobasic 0.014 Magnesium Sulfate•7H2O 0.075 Sodium nitrate 0.09 Dihydrated calcium chloride 0.025 Sodium chloride 0.025 EDTA-Na40.05 Ferrous Sulfate•7H2O 0.00498 Boric acid 0.01142 Manganese Chloride•4H2O 0.00023 Zinc Sulfate•7H2O 0.00141 Cupric Sulfate•5H2O 0.00025 Sodium Molibdate•2H2O 0.00019 Cobalt chloride•6H2O 0.00001 Yeast extract 0.3Preparation of concentrated organic feedstock

[0146] Once the desired algal biomass level was achieved, the C. vulgaris cells were harvested by centrifugation at 5^C for 10 minutes at 6000 rpm in 50 mL aliquots. The supernatant was removed and stored for potential reuse. The pelleted biomass was used, or stored frozen until needed. Unfrozen or defrosted biomass was used in the aerobic biodecomposition step. General Analytical Methods

[0147] The read-out parameters used to monitor and measure phototrophic culture performance included: pH, biomass concentration, and CO2absorption. The methodologies used for evaluating each parameter is summarised below. As used for these experiments, biomass refers to the C. vulgaris yield. Accordingly, biomass in these examples illustrates the generation of an organic feedstock. Determination of algal biomass by dry weight

[0148] The amount of algal biomass present in the aqueous media in the reactor was measured by dry weight, according to known methodology. Briefly, an aliquot of the aqueous medium was collected from each reactor in a collection tube and the cells were harvested by centrifugation at 6000 rpm / 10 min. The supernatant was discarded, and the pellet was washed twice with distilled water. Finally, the washed pellet was transferred to a petri dish (previously dried in an oven at 105°C / 15 hours and weighed) and taken to dry in an oven at 105°C for 15 hours, until a constant weight was achieved. To determine the amount of biomass (g / L) the following formula (Equation 1) was used: Biomass ൌ ^Pa-Pp^ x Va (Equation 1) where: Pa is the weight of the sample already dried; Pp is the weight of dry petri dish; Va is the sample volume (L) used. Determination of algal cell concentration

[0149] The number of algal cells in a known volume was determined by counting individual cells using a neubauer counting chamber (hemocytometer cell counting chamber), according to known methods. Briefly, microalgae cultures were enumerated at the end of the 7-day cultivation time. A standard curve of known cell densities as determined by spectrophotometry (wavelength of 600 nm) was also used to determine algal cell concentration (g / L). The microalgae cultures were transferred into 3 × 50 mL centrifuge tubes and centrifuged at 4700 rpm for 10 minutes to separate the supernatant and pellet. Serial dilution was performed on the pellets resuspended in a known volume of deionized distilled water. The cells present in an appropriate dilution were counted using a Neubauer chamber aid in optical microscope with 400x increase magnification.Aerobic Biodecomposition Step General culturing method

[0150] Pure cultures of bacteria were prepared by being resuscitated from a pure stock and incubated for 18 hours at 35°C in TSB broth (see Table 7). Cell densities were measured spectrophotometrically at 580 nm, using known methods. The bacterial cell densities were standardised to achieve a bacterial concentration of approximately at least 1x106CFU / mL. Culturing conditions for mixed cultures

[0151] The standardised pure cultures were mixed as equal parts and grown in tryptic soy broth (TSB) available from, e.g, Becton Dickinson (catalogue number 257107). The composition of TSB media is listed in Table 7. The media was pH adjusted to between a range of from pH 6.5 to pH 7.5 and was sterilised by autoclaving prior to inoculation. The bacterial suspensions were cultured in 500 mL schott bottles or in bench bioreactors. Table 7. Composition of TSB media Chemicals g / L Tryptone (pancreatic digest of casein) 17 Peptone (Peptic digest of soybean meal) 3.0 Glucose (dextrose) 2.5 Sodium Chloride 5.0 Dipotassium phosphate, dibasic 2.5

[0152] General culturing conditions used to grow aerobic bacterial cultures in a bottle reactor are listed in Table 8. Table 8. General aerobic bacterial culturing conditions using a bottle reactor Parameters Types and Values Bioreactor type Schott bottle Bioreactor volume 500 mL Bioreactor working volume 100 mL Light type NA* pH 7,0 Temperature 35 °C Culture media TSB Inoculum size 10% Cultivation time per batch 24 hours Agitation 100 RPM in shaker table Determination of CO2absorption

[0153] An elemental analyser was used to analyse the content of carbon, hydrogen, and nitrogen in the biomass. A known volume of culture was withdrawn at the beginning and theend of the cultivation cycle, the biomass was harvested by centrifugation and analysed for carbon, hydrogen, and nitrogen content. Inductively coupled plasma-optical emission spectroscopy (ICP-OES) was used to analyse the element present in liquid media, separated after centrifugation.

[0154] The pH of the cultures was analysed every day using a pH meter according to known methods. The pH of the cultures was also maintained within the specified range (7.0 ± 0.5) using 0.1 M NaOH. Two CO2sensors (model no. GMP251) using Vaisala Insight PC Software were installed into the inlet and outlet gas lines of the reactors as shown in Figure 1 to record CO2concentration every 5 seconds for the entire cultivation cycle. The change in the CO2concentration recorded by these sensors was also used to establish mass balance and calculate the bio-fixation of CO2. Determination of bacterial cell concentration

[0155] Cell concentration was determined by serial dilution in 0.85% saline, and enumeration of colony forming units (CFU) by culturing a known volume of serially diluted bacterial suspensions on agar plates using known methods, see Figures 2A and 2B. Briefly, 100 µL of a serially diluted bacterial suspension (e.g., 10 fold dilutions from 10-1to 10-4) were transferred to an agar plate. The plate was tilted to allow the bacterial suspension to run down the surface of the agar allowing the suspension to separate into individual colonies to facilitate colony counting. The inoculated plates were incubated at 30-35^C for 24 h. The bacterial colony forming units (CFU) were enumerated to determine the total bacterial concentration using the following formula (Equation 2). ^^^^ ∗ ^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^(Equation 2)where: MC is the average count ID the inverse of the dilution selected and; VG drop volume, in mL, used to inoculate the agar plate for bacterial CFU enumeration. Cellulase activity assay

[0156] Cellulase activity of aerobic bacteria was evaluated on carboxymethylcellulose (CMC) agar in the presence of Gram’s iodine. Briefly, a volume of 5 µL of a suspension of each bacterial strain was inoculated onto the centre of a separate CMC agar plate. The composition of CMC agar is provided in Table 9. CMC agar was prepared by adding 15 g / L of bacteriological agar. The plates were incubated at 28^C for 48 h. Then, the plates were flooded with Gram’s iodine (2.0 g of potassium iodine and 1.0 g of iodine in 300 ml of distilled water) for 3 to 5 minutes to allow the zone of clearing to develop. The zones of clearing were measured, and the cellulolytic potential of each species was calculated according to Equation 3: ^^^^ ൌ ^^^^ / ^^^^ (Equation 3)where: IE is the index of enzymatic activity; DT the total diameter (mm) of the colony plus the inhibition zone and; DC the diameter (mm) of the colony. The diameter of the hydrolysis zone is indicative of the level of cellulase activity, i.e., the higher the IE, the greater the cellulolytic activity of the bacterium.

[0157] A zone of clearing in the agar surrounding the point of inoculum is indicative of cellulase activity because the clearing is caused by the hydrolysis of cellulose in the agar due to the cellulolytic enzymes produced by the bacteria. That is, CMC agar combined with Gram’s iodine form a blue-black colour in the presence of cellulose, but become clear when cellulose is hydrolysed. The diameter of the zone of clearing is indicative of the level of enzyme activity. Table 9. Composition of CMC agar for cellulase activity assays Chemicals g / L Yeast extract 6 Glucose 1 Carboxymethylcellulose 0,5 Ammonium sulfate (NH4)2SO4) 0,1 Sodium Chloride 0,3 Magnesium Sulfate 0,1 Calcium Carbonate 0,02 Iron Sulfate 0,001 Zinc Sulfate 0,0009 Manganese Sulfate 0,0002 Example 1. Identification of cellulolytic microorganisms

[0158] The approach taken to identify cellulolytic microorganisms for use in aerobic biodecomposition combined phenotypic and bioinformatic analysis. An overview of this approach is shown in Figure 3. Known microorganisms with publicly available genome sequence data were investigated to identify microorganism species that encode cellulases. The focus was on agriculturally important microorganisms. A diagram depicting the distribution (relative abundance as determined by 16S fragment sequencing) of microorganisms of agricultural importance is shown in Figure 4. The slices represent the percentages given in Figure 6.

[0159] Another consideration in the selection of cellulolytic microorganisms was that any potentially phytopathogenic cellulolytic microorganisms were not considered for further investigation. Phytopathogenicity status was assessed based on publicly available characterised genetic data using known methods. The abundance of potentially phytopathogenic microorganisms of agricultural significance is shown as a heat map in Figure 5A, wherein the microorganisms are listed by genus. The abundance of microorganisms of agricultural significance with potentially crop enhancing properties are shown as a heat map in Figure 5B, wherein the microorganisms are listed by genus. Attributes of individualmicroorganisms of agricultural significance are listed by genus and their abundance in soil is represented as %DNA / g soil as shown in Figure 6.

[0160] A list of potentially suitable aerobic biodecomposition microorganisms was generated, and publicly available strains representing microorganisms species of interest were identified, see Table 10. The secondary consideration of potential to enhance crop performance also contributed to microorganism selection. Where possible, each microorganism of interest was located in a publicly accessible culture collection. A selection of strains that are suitable for culturing in a laboratory environment were sourced for phenotypic characterisation to confirm cellulolytic activity using the cellulase activity assay described above. Table 10. Putative cellulolytic microorganismsharmful microorganisms. Additionally, theDSMExample 2. Investigation of cellulase enzymes present in microorganisms

[0161] A selection of microorganisms were obtained from culture collections (Table 4) and phenotypic analysis was performed to determine their cellulolytic potential using the cellulase activity assay described above. Bacillus spp. were selected for further investigation because they were thought to possess superior cellulase activity compared to other microorganisms, are readily culturable in a laboratory environment. Phenotypic analysis

[0162] The cellulase activity of a range of Bacillus spp. was measured, the IE calculated, and the results are shown in Table 11. An example of cellulase activity results with a selection of Bacillus spp. plated on CMC agar with Gram’s iodine with zones of clearing indicative of cellulase activity are shown in Figure 7. Cellulase activity was observed in all strains tested. A range of cellulase activity levels were observed across a range of Bacillus spp., ranging from IE 1.05 to IE 1.52.Table 11. Cellulase activity results for select Bacillus spp. represented as an index of enzyme activity (IE) Bacillus spp. IE Bacillus subtilis 1.15 Priestia megaterium 1.09 Bacillus thuringiensis Berliner1.35Bacillus velezensis 1.44 Bacillus pumillus 1.17 Priestia aryabhattai 1.05 Bacillus amyloliquefaciens 1.52 Bacillus lichiniformis 1.12 Bioinformatics analysis

[0163] To expand the search for potential cellulolytic microorganisms, genome sequence data from Bacillus spp. identified in the phenotyping studies described above were investigated using a biomining process to identify cellulase encoding genes in other potentially cellulolytic microorganisms. Genome database biomining

[0164] The publicly available genome sequences of Bacillus spp. were searched for genes encoding cellulases capable of catalysing reactions that decompose cellulosic material (e.g., cellulase and hemicellulose) to carbohydrate monomers, including reactions that result in trimers, dimers and monomers of C4, C5 and / or C6 sugar residues. Starting with the extensively characterised reference genome of Bacillus subtilis strain 168 (available at Bacillus subtilis subsp. subtilis str. 168 genome assembly ASM904v1 – NCBI – NLM (nih.gov), accessed on 25the enzyme database BRENDA for B. subtilis strain 168 (available at https: / / www.brenda-

[0165] Enzymes are classifiable by an Enzyme Commission (EC) number as defined by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB). Using the BRENDA database, the EC 3.2.1.4 class of enzymes produced by B. subtilis 168 and capable of cellulosic material decomposition was identified (UniProt accession P10475). Specifically, this glycosylase (Type 1 cellulase) catalyses, for example the decomposition of cellulosic material including: cellohexaose to cellotriose (trimeric saccharide), or cellodextrin to cellobiose (dimeric saccharide). The identification of this Type 1 cellulase in a cellulolytic microorganisms allowed further searching to identify similar enzymes in other potential cellulolytic microorganisms, as shown in Figure 3 (primary strains – grey box).These strains included, for example: B. subtilis ATCC 6051, Priestia megaterium ATCC 14581 (formerly classified as Bacillus megaterium) and Bacillus thunrigiensis ATCC 33679.

[0166] Further bioinformatic analysis was performed to identify amino acid sequences that are closely related to EC 3.2.1.4 amino acid sequences. This was achieved by using the identified EC 3.2.1.4 reference sequences (Table 12) to search the BLASTp protein sequence database (available at https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE=Proteins) to identify proteins with an aminoat least 75% coverage of the full protein sequence. This process identified relevant glycosylases of different EC classes, for example EC 3.2.1.91, EC 3.2.1.21, EC 3.2.1.52, EC 3.2.1.74 and EC 3.2.1.86. The search resulted in 346 hits that were then assessed for agricultural significance and limited to Bacillus spp, example search results are shown in Table 12. Table 12. Putative cellulase biomining hit results using reference sequences Reference sequenceaEC 3.2.1.4 EC 3.2.1.21 (GUN2_BACSU) (J9ZZW8_BACAM) Strains Query %identity Query %identity coverage coverage B. subtilis ATCC 6051 100% 100% 28% 80.15% P. megaterium ATCC 14581 0% 0% 100% 56.34% B. thuringiensis ATCC 33679 93% 92.69% 99% 67.80% B. amyloliquefaciens ATCC 23845 0% 0% 100% 96.99% B. velezensis CCT 7891 100% 93.39% 99% 54.70%aReference sequences: Brenda accession GUN2_BACSU, Bacillus subtilis strain 168; UniProtKB accession J9ZZW8_BACAM, Bacillus amyloliquefaciens ATCC 23844.

[0167] In addition, the pathways related to carbohydrate metabolism were investigated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) enzyme pathway database (Starch and Sucrose Metabolism pathway rn00500, available at https: / / www.kegg.jp / pathway / rn00500+R11307). The function of selected EC classes identified as a result of biomining the BLASTp database (Table 13) and as depicted in KEGG pathway rn00500 confirmed their cellulolytic potential, see Figure 8. Reactions of interest yield D- glucose monomers (Figure 8 – shaded yellow box) and are catalysed by cellulases produced by cellulolytic microorganisms. Secondary strains with cellulolytic potential identified by the biomining process (Figure 3, white box) included, Bacillus amyloliquefaciens ATCC23845 and Bacillus velezensis CCT 7891. Table 13. Identification of EC classes within Bacillus spp. biomining search results Strain EC class Glycosylase accession number Bacillus subtilis ATCC 6051 EC 3.2.1.4 WP_003231540 Priestia (Bacillus) megaterium ATCC 14581 EC 2.7.1.205 BG04_3062 EC 3.2.1.86 BG04_270Bacillus thuringiensis subsp. Berliner ATCC EC 3.2.1.4 AGW99983 33679 Bacillus amyloliquefaciens ATCC 23845 EC 3.2.1.21 WP_013354123.1 Bacillus velezensis (CCT 7891) EC 3.2.1.4 WP_012117671.1 Bacillus subtilis strain 168 EC 3.2.1.8 P18429|XYNA_BACSU EC 3.2.1.86 P42403|BGLC_BACSU EC 3.2.1.1 P00691|AMY_BACSU EC 2.4.1.1 A0A6M4JMW5 Bacillus thuringiensis ATCC 10792 EC 3.2.1.1 A0A0B5NG27 Bacillus thuringiensis subsp. Berliner ATCC EC 2.4.1.1 Q29SF6_BACTK 33679 Sequence similarity analysis

[0168] Amino acid sequences that are representative of Bacillus spp. glycosylases of a range of EC classes were analysed to evaluate the degree of sequence similarity of a reference sequence compared to amino acid sequences of other Bacillus spp. The analysis was performed using NCBI BLASTp with the standard non-redundant protein sequences (nr) database selected. The results shown in Table 14 are the percentage of sequence identity based on the Blastp alignments of a reference amino acid sequence to the amino acid sequence of similar proteins present in other Bacillus spp.,%s s,%:si.%,%o yr..hai,%sseh,:%si .%yr .%e :s.%aB9 nE=NGATKT3 rA1WLGRD Yn2i4t8 US G AS S 08 SWG A 4n2 USiKaF F PWKVn1eP CA0LG KQ3s(B4 G NV Y RM4P C rt HTWHAs( S Y Re86 _2V VGA T V V(Q8 B_siK KlPG VL Drp1 A2LA GS6Cit FssN= F N T Y 1L b S PIS TGF L Fn iiliYXX N|ON VY PWAsiliGB|usEKR VRett ebu 92 )8KIN I RYQ Wtbu 30sulP TF Y PEKorcpnss48 61KfeFGT YT T Nss42lic HV AYK I Lorull1P|ni KGGRGF SGull4PaQB DLH TIyetfic paite srtF FT i |D P WT L c p =HSIWG HS CnRaB >s(MT S S R NaBs>OMVML Yed i8.–C9e1. Ec28 On. Ne -3uaqe etO ) - D Ie CN esβS s.a4lb1y-E al-o es4s QE,1eDIullhp ae-sae d Sscs is68aleolc o nQ i ohoc.1 ullbyld alEm aePul.2ecTGnEyx S ( h-6 g.3 -GT W 1A = A HQAT FTI SA G GI ANQDFY96UEP F LN AN RN H YG AS VA MSEV S F 00 S E L I G CG KP TEMVSDITWKWS P CAy PL T E H LYGYDP VS R AYHDSK ,8 BmaLG KMT SW HR N SF C K6 A RTN1_Y=S SVEWFQNSS S H S LKPE Y K sGGL ilMN TKiAI Rtb |1G8KIQ KS NF Q EFFG NI TIENV S AVFGNLR AT T F0F P IPI QGTWP N GL P QLG LKu9 P QTY V NYD Lss6034R A S E S T TA T QNE V DulG Sl0icP|22KA Tp = FLT TK NSDEQ V QGM VLSVAL LDVKD GG ITIGE T D Y KG TV GN KTIT a s X LS IF GKD S B > OMN A Y N N D N D R A G D S I ACE01OeNsa DlIymQaE-Sesah1. alyp 1l.A2.m 3a-1 G Y DA R V0J4 1R AS GCN =FRC5 LW N H AFG G AN EBNGLNGYNIN D DYRND D8Mn6 iK Aa LT V L NTA08 V Ds WYV E LPNTiAKRN L KS K 61ArtFEV S KLs 0 24 Vn A|1 SHADY R HLVQ IR IL G I D G D Y KMF AL NIni0As( L I DDTT ar |5 sili DWRL Wei72 =XNG GY N G E F EAVT A TAtVsWtb AD V FQg niGruN O Vh5si D GQ SD DLHL YLI L T silMJusF A G C EPs IS VKVDDI YG Ai LPGN t 4 s R SIL ALt B n TI PEW s0DH IulAeiAK A L N DH N NLPDS TbuM ull EYNA ET Y KNI K INsQs 6Aic KSEML Vi0 gN AL FK ul 0 aRLRl ncA|i K Y Rarru FQ GVNAN l BSGQBt>htW T EI NMTA NDFLA TR D i AYD IW KHGc|r = FVN L HMEQMP L V TaBt>SO M MQ G GCE11 na C 2Oc E 1ulOeNgNsea DlI sya DImQlaE ey-S s4, rQoE ea1al1- hS sp 1alh .p1 y al.mh s .o1 y.m A2.3ap-lAhp4.2a-E RET Q LKS IDL S.p KusT DQ I E SAY VFIKVI KA NHVF sbTSCsi PRH N GI Q D TEK I KN IPAQF YuL AKLKFGY YG sAsnT Y LW I V GV FIRMsB_ei EYLD AE INL S P YISS L K I ED Y Sis 6FgF GE EIA TQNYFQD LKQAR DH EH ne Snir G E Y E I L KLP T SS GYNR Y ETRNA ig92uV Sh RM HNEDF VHPNK YAGS H InirQ| tVPVEWY ANQ I S YI KNA QYSMI R PN u6 su AAAP STYL VI ht FSlliV E RL IMTH FLTPKR KKKI I ADAVKFL QHSVLGL S s9 caEI E EPLG L EA RAGSuHllLVDP GDENi2QB A R ME L L P EM K NNIR K Y GN D Ec|r =SLK RD PDYK T LVWF K A N N EFI SaBt>O G YVI L K H F K N R Kna C 3cE 1ulOgNesa DlI4,yrQ1-oE eahS shpalps1.1 ylo .Ahp4.m 2a-BesA L G LMQHD TRQ L IBAadT N NL PWE I D YP A YHs insAPeA F MY H GioccG DIE KANN LL D GYA P EDTRRauflgG DRT YVL LH K Q TKEP NL Y KSeu-aWLT D Y FKEiWSM A T Nq tEl eol bFH A IK P F I I GT YMYR TM RR Ey1. G4FIDPSVRTNT KS L Y Na1T AATmT V TMGD T As7PSF R P F Q T Y TKDS Iul46 RMFEIVKAGMPQAVlicSF IKATN ASI PD ISDaG Q R EBA>K Y MI TV KMD F H A GMS Y Q G L YCEesadisoculg1-2a.t1e.B2.3]siliG RE LNKREK L KR AK HITIAY Gt F FAbFuFR IMTAQ YL KSI E TI K GPCD T GP LTVDL Y G T T TKAQYM K KssuSIlMI IAL H DV VLS LEDVF TKI A Vi L G SGS I SI AGlcFL G L D I K S ERY Sa LMG GE LVER F V AG PREA K P V PBF SL AEATVFAI[VQ Q QK PPNMV IDDAGGTL F LKQQVb YD IL F S L EKT LVbSLYLK KISI EGYQG AINHEEA A P1I.L E K L SRH VM ED NI Q YP HWK LA T99 P D VL MGTN4S S H K EAQ VQ SA TK EG9F LV S S LPQ SDTR GQG VI KSSEFK1AI YF VLAEVF PAPAM BRSML KLRVEK A D VD N K>PR TV GVPIIKIRQSIA AMN D SNI V DMT A D K S S T A Qesadinimasox 2e5.hl 1y.t2.e3caCETTC S A NT L RWITG F A A QG QA Y A T SK S G P D Q E S YQWVF C C GY T CY DD V S FPWT QPL QMS E R VV D WQPP N]T NL G LT QD Smu P D L S C C D T T L A A LC N LKNIS PV R VTnP Y A V Y S SY K T M G T Qa CiH C D D GzrT N G V T G T T N V S S TD G H G T G LS V S T YahE TT ST F Y G G DL Y TS G GaAQS T S K G C A S M PG C P Smre QK V FE A V SdD F G G S N ACo T T G N G GA T G S V QG S RhC DH Y ST G A N S G QF R G TcirV T G S N A C P A A KA T PT[Q LeQWS T G N M G NP K G N GTsa R Y F P N T YNGlARF QI P GWN E YK SS QS QQT T Word AN A QS P G T F A SA S GTyh AA G YT QE VT N K L TGIGoiAD DL S G WE G V GbLFL C D G G C L G G A P GR N K NGColl AVV C N D E P YQD TFQec SSNA A VH PPVT PYRIG1I .V GKAM D NTNYF TNY1LMAYS SSH93A QCYLW T T6 L3QTLSVQADRSG DYI Q FK PARFGNESCI GL VTARA Y T>CDSY II GSFWL KAM GDGLP V A FKSNDG PD ATSM NTP LCEesalordyhoib1o9l.l1e.c2.3

[0169] Different EC classes of glycosylases are found across a range of Bacillus spp. with a range of sequence similarities. This information supports the general observation that a combination of, for example, different Bacillus spp. that are grown as a mixed culture can provide a comprehensive array of glycosylases that complement each other for the purpose of biodecomposing an algal biomass. Example 3. Production of an organic feedstock using C. vulgaris

[0170] The phototrophic microalgae C. vulgaris was cultured in a bubble column reactor and biomass generation and CO2absorption was measured as described in the general analytical methods above to evaluate reproducibility. To reduce cost and water usage in the photosynthesis step, reuse of the BBM media was also investigated. Used BBM media was generated during the algal biomass harvesting step, where the algal cells of a previous C. vulgaris culture step were harvested by centrifugation and the supernatant was retained using aseptic techniques and reused (once only) in a subsequent C. vulgaris culture step.

[0171] The cultures were prepared according to the culture conditions listed in Table 5. The algal cell concentration was determined as described above at the beginning of the experiment (T0– concentration of algae in the BBM media just after the inoculum was added) and again after 7 days of growth (T1). These results are shown in Table 15 for 5 replicate experiments (TH005A, TH006A, TH007A, TH008A and TH009A). Table 15. Cell concentration of C. vulgaris cultures when grown in BBM media either used fresh or reused once. Cell concentration (Log cells / mL) CO2 fixed daily (g / L) Experiment BBM (fresh) BBM (reused) BBM BBM reference(fresh) (reused) TH005A 4.78 5.02NT NT0.551NTTH006A 6.60 7.33 6.53 7.26 0.396 0.400 TH007A 6.20 7.95 5.36 5.67 0.470 0.459 TH008A 4.67 6.11 5.77 5.21 0.419 0.395 TH009A 5.60 5.17 5.45 5.36 0.498 0.506 NT = not tested.

[0172] The photosynthesis step culture was run for 7 days, at which point the total amount of algal biomass was measured according to the method described above. The algal biomass was measured in four replicate experiments (TH006A, TH007A, TH008A and TH009A), see Figure 9. Figure 9 shows that the levels of biomass production across each of the four replicate experiments were not significantly different irrespective of whether fresh or reused BBM media was used. The levels of biomass generated were comparable across each of the four experimental replicates, achieving between approximately 210 g / L and 280 g / L biomass after 7 days of culturing.Example 4. Aerobic biodecomposition of a microalgae organic feedstock Initial culturing of bacteria for use in aerobic biodecomposition

[0173] To establish a benchmark, Bacillus subtilis strain 6051 was grown in TSB broth without the algal organic feedstock added. The resulting cell density measured by serial dilution at 12 and 24 hours post inoculation is shown in Table 16. The pH was measured at the time of inoculation and at 24 h post inoculation, also shown in Table 16. Table 16. Cell growth data for B. Subtilis strain 6051 in TSB Duration of growth (h) Cell Concentration (Log10CFU / mL) pH 0 5.98 7.1 12 7.67 24 7.88 7.7 Optimisation of bacterial culture conditions – media type

[0174] Two different Bacillus spp were cultured in a range of culture media types, including TSB (composition is provided in Table 7) and soybean vinasse (prepared at a range of dilutions in water: including 20% v / v, 40% v / v and undiluted). The composition of soybean vinasse is provided in Table 17. Soybean vinasse, a viscous, nutritious, dark liquid was investigated because it can be used as a liquid fertiliser to enhance crop performance and was therefore of interest as a potentially advantageous component of a biofertiliser product generated downstream of the aerobic biodecomposition process. Table 17. Composition of soybean vinasse culture media Chemicals Soybean vinasse (g / L) Calcium 5.67 Phosphorus 8.9 Magnesium 4.48 Potassium 69.52 Sulphur 5.13 Nitrogen 32.76 Iron 0.299 Manganese 0.01 Copper 0.065 Zinc 0.203 Boron 0.041 Total sugars 0.387 Organic carbon 3.977 Organic matter 6.84

[0175] The results listed in Table 18 are bacterial cell concentrations and pH values of the bacterial culture media collected immediately after inoculation (initial) and at the end of the incubation period (final, 24 h). The bacteria were cultured under the conditions described inTable 8 above. Across the two Bacillus spp. tested in this experiment, TSB achieved an optimal cell yield and maintained an acceptable pH buffering capacity when compared to soy vinasse, especially at the higher concentration (40% v / v and undiluted). TSB also has the advantage of being a commercially available and consistent quality tested product. Table 18. Comparison of different media types for culturing Bacillus spp. B. megaterium ATCC 14581 B. thuringensis CCT 1306Microalgae decomposition

[0176] Two aerobic biodecomposition experiments were performed to evaluate the microalgae aerobic biodecomposition process and select the optimal culture conditions and bacterial mix. The conditions used in each trial are provided in Table 19. Table 19. Microalgae decomposition assay conditions Trial Microalgae biomass Bacterial culture conditions reference Media Concentration TH004B BBM Not Triple mix: The decomposition stage of the concentrated microalgae was performed in culture bottles with a 20% working volume, incubated for 24 hours, no photoperiod, aeration of 1VVM, at a temperature of 35°C. The bacterial mix (B. subtilis, P. aryabhattai and B. pumulis) was co-cultured in CMC medium. Bacterial cell densities were measured spectrophotometrically at 24 h post inoculation. The experiment was performed in triplicate (technically and biologically). TH005D BBM 6 g / L Triple mix: The decomposition stage of the microalgae was performed in culture bottles with a 20% working volume, incubated for 24 hours, no photoperiod, aeration of 1VVM, at a temperature of 35°C. The bacterial mix (B. subtilis, P. aryabhattai and B. pumulis) was co-cultured in CMC medium. Total bacterial cell counts were determined on samples collected at 24 h post inoculation. TH009C BBM 2.5 g / L or Quintuple mix: The decomposition stage of the BBM 10 g / Lmicroalgae was performed in culture bottles with a20% working volume, incubated for 24 hours with shaking at 100 rpm, no photoperiod, aeration of 1VVM, at a temperature of 35°C. The bacterial mix (B. subtilis, P. megaterium, B. thuringiensis Berliner, B. amyloliquefaciens and B. velezensis) was co-cultured in CMC medium. Total bacterial cell counts were determined on samples collected at 24 h post inoculation.

[0177] The cell concentrations of each inoculum used in the microalgae aerobic biodecomposition study TH009C are provided in Table 20. An inoculum of 10% v / v of approximately 1 x 108cfu / mL was used for each trial. Total bacterial cell concentrations were determined for the microalgae decomposition assay bacterial mix at initial (immediately post inoculation) and final (24 h post inoculation) time points, and the results are provided in Table 21. Table 20. Bacterial inoculum grown in CMC medium and used in trial TH009C Species Inoculum (Log CFU / mL) B. subtilis 8,88 P. megaterium 8,57 B. thuringiensis Berliner 8,29 B. amyloliquefaciens 8,74 B. velezensis 8,17 Table 21. Total bacterial cell concentrations of mixed cultures at the beginning and end of the microalgae biodecomposition assay Trial reference (study descriptor) Total bacterial cell concentration (Log CFU / mL) Initial Final TH004B (microalgae biomass not concentrated) 6.31 6.70 TH005D (microalgae biomass 6 g / L) 6.55 6.98 TH009C (microalgae biomass 2.5 g / L) 7.95 9.05 TH009C (microalgae biomass 10 g / L) 7.03 8.93 Confirmation of cellulase activity

[0178] The cellulase activity of each bacterial strain used in TH009C was determined using the cellulase activity assay as described herein, with results shown Table 22. Table 22. Cellulase activity of each bacteria included in TH009C Bacillus spp. IE Bacillus subtilis 1.15 Priestia megaterium 1.09Bacillus thuringiensis Berliner 1.35 Bacillus velezensis 1.44 Bacillus amyloliquefaciens 1.52 Optimisation for improved runtime and biomass biodecomposition

[0179] The biodecomposition of microalgae studies were repeated in a different laboratory under further optimised conditions, including using TSB culture media and a higher starting inoculum. The data shown in Table 23 shows that the aerobic biodecomposition process can be completed in a runtime of 24 h instead of 3 days when the optimal 5 bacterial strains are used together in a co-culture, and can process larger amounts of microalgae biomass, up to 30 g / L. Superior cellulase activity demonstrated by the optimal 5 bacterial strains (Table 22) aligns with the improved microalgae biodecomposition results shown in Table 23. Table 23. Repeat of microalgae biodecomposition assay Species B. subtilis, B. megatherium, and B. subtilis, B. megatherium, B. B. thunrigiensis thunrigiensis, Bacillus amyloliquefaciens, and Bacillus Conditions velezensis Culture medium TSB TSB Working volume 80% 80% Inoculum volume 15% working volume (5:5:5) 28% Inoculum conc. 109CFU / mL (1:1:1) 109CFU / mL Aeration Yes Yes Flow rate 0.5 VVM 0.15-0.5 VVM Flow composition Air Air pH 7 7 Temperature 38oC 38oC Runtime 3 d 24 h Stage 1 feedstock 85% working volume 33% Total biomass 7 g / L 30 g / L

[0180] At 24 hours, it is believed that about 70% of cells were ruptured using the quintuple bacteria mix as compared to about 40% being ruptured using the triple mix. Based on the bacterial cell concentrations and the cellulase activity assay results obtained with TH009C and the repeated assays (Table 23), the bacterial mix of B. subtilis, P. megaterium, B. thuringiensis, B. amyloliquefaciens and B. velezensis were selected because of the surprisingly advantageous efficiency for microalgae biodecomposition and apparent synergistic affect. Carbon analysis

[0181] A preliminary CO2absorption and retention test was performed, (TH005F). In this assay the following biomass formulation was used: algal biomass in new and concentrated BBM medium at 6 g / L combined with 1% v / v B. subtilis, 1% v / v B. megaterium, 1% v / v B.thuringiensis, 1% v / v B. velezensis and 1% v / v B. amyloliquefaciens, grown in TSB. The study was performed according to the following methodology.

[0182] Civil construction sand with granulometry from 1.2 to 0.42 mm was used to simulate a biocrust. This sand was sifted to remove the larger dirt and debris, such as branches and grass, etc. After cleaning, the material was arranged in plastic trays with dimensions 0.6 x 0.4 x 0.1 m (LxWxD), so that each tray had 16 kilos of sand, being measured with a capacity scale of 30 kilos.

[0183] After weighing the sand, it was transferred to autoclave bags and sterilized for 20 minutes at 121ºC and 1 BAR, to avoid introducing contamination microorganisms from the sand into the experiment. The sterilised sand was transferred to trays and the biomass formulation or water (negative control) were added to sterile trays, the treatment groups were performed in triplicate. For each of the treatment trays, a volume of 12 mL of biomass mixed with 88 mL of water was sprinkled over the surface of the sand. For the water only control, a volume of 100 mL of was similarly sprinkled over the sand.

[0184] Samples were collected soon after the biomass formulation or water were added to the sand to establish a baseline. Approximately 500 grams of sand were collected from each tray, collecting material from the surface and bottom levels in the tray to perform the determination of CO2initially fixed.

[0185] The biomass formulation was used weekly, always in the same way described above. At the end of the fourth application (1 month of experiment) samples of the sand were collected again for microbiological analysis and tested for CO2absorption. Further samples were collected and analyses, using the same method, at the end of 2 months of the experiment (treatment and control samples), another sand sample was collected to verify the retention of CO2. To determine the concentration of sequestered organic C, the following equation was used (Equation 4 below). SC= (SOCE- SOCI) ×SBDx D (Equation 4) where SC is the amount of C sequestered during the experimental time period (g m−2); SOCEand SOCIare the amount of soil organic carbon (g C kg−1 soil ) at the end and initial time of the experiment period. Respectively; SBDis the bulk density of the soil; and D is the depth of the sampled soil.

[0186] To estimate the potential CO2removed from the atmosphere, the following equation was used (Equation 5 below). RCO2= SC × I3.67(Equation 5) where RCO2is the potential amount of CO2removed from the atmosphere and soils during the experimental period (g m−2), SC is the amount of C sequestered, and theconstant number I3.67is based on the co-mass molar2(one carbon atom and two oxygen atoms = [1 × 12.0107 g mol−1] + [2×15.9994gmol−1] = 44.0095 g mol−1).

[0187] Thus, 1 g of C in CO2in the soil is ~3.67 g of CO2removal from the atmosphere (=44 / 12). Carbon analysis of the phototrophic culture step and the microalgae biodecomposition by aerobic bacteria step confirmed that there is an exchange of carbon from solid to liquid phase, approximately an increase of 1.36 (Table 24). This shift is concurrent with the change of %C in the solid phase, implying the decomposition caused by the mix of cellulolytic microorganisms in the microalgae biodecomposition step has successfully released the stored cellulosic materials in the microalgae biomass (Table 24), as demonstrated by the % of C in total mass decreasing in the biomass / solid material, while the % of C in total mass increasing in the culture liquid from the phototrophic step to the end of the aerobic biodecomposition step. Table 25 shows the physio-chemical analysis, performed according to standard known methods, of the biomass composition for the phototrophic and aerobic biodecomposition steps. Table 24. Carbon content analysis of solid-liquid phases of the phototrophic microalgae biomass production step and before and after the microalgae biodecomposition step. % of C inTable 25. Physio-chemical analysis of phototrophic step and aerobic biodecomposition step. Origin of biomass Carbohydrates% Proteins% Lipids% Stage 1 (rep 1) 0.045 0.052 0.070 Stage 1 (rep 2) 0.042 0.050 0.059 Stage 1 (rep 3) 0.048 0.059 0.075 Stage 1 (rep 4) 0.053 0.070 0.067Stage 2 (rep 2) 0.376 1.993 0.019Stage 2 (rep 3) 0.348 2.097 0.014 Stage 2 (rep 4) 0.357 2.092 0.021

[0188] Interestingly, when the mass of carbon in solution is calculated from the total biomass (Table 26), the amount of carbon increases (with and without the carbon derived from media added to process). Furthermore, the rate of change for the biomass is negative, indicating less and less biomass is added as the process proceeds from Stage 1 to stage 3, in contrast the rate of change of C% is positive, indicating more and more carbon is added into the system; this increase in C is unaccounted for even when the IN flows for stage 2 and stage 3 are considered. As such, the second derivative of BiomassTotal ≠ second derivative of CarbonTotal and is therefore considered not to be directly proportional. Evidence suggests that the aerobic biodecomposition step is capable of fixing CO2as a result of the cellulolytic microorganisms. Agricultural field data has shown this capacity to be not only present but also functional. Farmland emissions dropped from 18.2 kg / ha / d to 0.5 kg / ha / d. As such, the data suggests that biomass is able to improve the soil carbon storage efficiency and can be used to enhance the carbon capture capacity of agriculture, mine sites and processing plant tailings (Industry targets). Table 26. Elemental analysis and respective mass from each reactor step Stage 1 Stage 2 Stage 3

[0189] The process is responsible for decomposing the starch, hemicellulose and cellulose components of the microalgae and thereby releasing the carbohydrate monomers (C4, C5 and C6) sugars into a free-form useful in several industries such as nutraceutical, fermentation, and bioplastic. Example 5: CO2absorption and retention - Organic material vs Total Organic Carbon.

[0190] Experiments were conducted to evaluate if the biomass generated in the aerobic biodecompostion step could sequester carbon dioxide. For the carbon dioxide absorption and retention experiment, civil construction sand was used with granulometry from 1.2 mm to 0.42 mm. This sand was sifted to remove the larger dirt. such as branches, pieces of grass, and other debris. All sand used in this experiment was sterilised by autoclave for 20 minutes at 121ºC.After sifting, the material was arranged in trays with dimensions 0.6m x 0.4 m x 0.1 m (LxWxH) so that each tray contained 16 kg of sand.

[0191] A biomass feedstock (4.30E+06 CFU / mL total bacteria, containing 1.50E+06 CFU / mL Bacilli mix) was inoculated into trays containing sand as prepared above, at a rate of 100 mL / m². Over a period of 30 days, 3 inoculations were applied to the trays. A sample of sand was collected at 9 weeks post initial inoculation and divided into 10 subsamples (10cm profile). The samples were analysed to determine CO2content using the equations described in Example 4 above with the results given in Table 27.Table 27. Results of carbon dioxide absorption and retention tests – Biocrust simulation15 / 06 / 2022 18 / 07 / 2022 T1-1apl Control T2 - 3 apl Control Nitrogen N g / Kg 0.93 0.84 0.83 0.56 Total Phosphorus P g / Kg 1.05 0.98 1.21 1.22 Potassium K+ g / Kg 2.00 2.03 1.58 1.76 Calcium Ca2+ g / Kg 1.54 1.46 1.73 1.58Zinc Zn mg / Kg 16.51 8.22 11.59 6.11 Boro B mg / Kg 7.55 7.92 8.39 8.20 Organic carbon COT % 0.11 0.16 1.29 0.16 Organic matter To % 0.18 0.28 1.54 0.28 Moisture N.A % 0.53 0.79 0.15 0.09 pH CaCl2 N.A N.A 7.87 7.79 7.74 7.92 C / N ratio N.A N.A 1 / 1 2 / 1 9 / 1 3 / 1 Granulometry N.A mm 0.42-1.2 0.42-1.5 0.42-1.2 0.42-1.9 Example 6. Aerobic biodecomposition of a microalgae organic feedstock Heterotrophic growth mode

[0192] A media containing biodecomposed microalgae and cellulolytic microorganisms was prepared as per Example 4. The working volume was centrifuged at 6000 rpm for 3 minutes to separate the biomass and collect the supernatant. The supernatant was sterilized using autoclave. The glucose content was tested and adjusted, if necessary, to 1 g / L by adding glucose. To this supernatant was added to a microalgae organic feedstock produced phototrophically as per Example 3, in a 5 L bottle to make up 4 L. The bottle was incubated overnight at 28°C in the absence of light with continuous mixing. Samples taken every 2 hours were analysed for glucose content using using a Megazyme D-Glucose Assay kit measuring asper the manufacturer instructions. The glucose concentration decreased over time and was substantially depleted after 8 hours accompanied by a 1.5 time increase in microlagal biomass weight indicating microalgal growth by consumption of glucose. Microalgae decomposition

[0193] Two distinct bacterial mixtures were prepared: a 3-strain consortium comprising Bacillus subtilis, Bacillus megaterium, and Bacillus thuringiensis, and a 5-strain consortium including in addition Bacillus licheniformis and Bacillus amyloliquefaciens. A total of 800 mL of Chlorella vulgaris culture prepared as above using heterotrophic growth mode, with an initial dry weight of 11 g / L, was treated separately with each bacterial consortium (i.e., the media was inoculated with bacteria mixes) and incubated under standard laboratory conditions. The detailed optical density (OD), estimated colony-forming units (CFU), and volume used for each bacterial strain are summarized in Table 28. Table 28. OD, CFU and volume of bacterial strains used. OD CFU VolumeB. subtilis 9.26 >1099 ml

[0194] Microscopy and cell wall analysis were conducted at 18- and 24-hours post- inoculation. Samples were stained with Calcofluor White, a fluorescent dye that binds to cell wall components, and examined using fluorescence microscopy to assess the integrity of Chlorella vulgaris cell walls. After 24 hours of incubation, samples were plated on PCA (Plate Count Agar) to assess bacterial load. The plates were incubated at 37^°C for 18 hours.

[0195] After introducing the bacterial consortia into the algal cultures, microscopy images were captured. Microscopy images taken at time zero showed intact Chlorella vulgaris cells surrounded by bacterial cells in both the 3-strain and 5-strain treatments (Figure 10 A)). Decomposed or algal cell walls appear under microscopy as dispersed, hazy structures lacking distinct circular morphology of healthy cells. At 24 hours, both treatments showed cell wall biodecomposition observed under fluorescence microscopy (Figure 10B)).

[0196] Plating results after 24 hours showed a significantly higher number of bacterial colonies in the 5-strain group compared to the 3-strain group, despite equal inoculum volumes (Figure 11). This indicated differences in bacterial growth rate and survivability between the consortia.A reducing sugar assay was performed using the 5-strain bacterial consortium and showed an increase in sugar concentration from 1.302 g / L at the start to 1.629 g / L after 24 hours. This study demonstrated that the 5-strain Bacillus consortium was more effective in degrading the cell wall of Chlorella vulgaris than the 3-strain consortium. Example 7: Lipid and Protein Retrieval

[0197] A microalgae organic feedstock, especially when coupled with a heterotrophic growth mode, may contain about 36-40% w / w of protein and also 40% w / w lipids being comprised of fatty acids and triglycerides. Both are valuable commodities, especially lipids for producing biofuels. However, extraction has traditionally been difficult as disruption of the cell wall is required. This is improved herein by biodecomposition of microalgae as per Examples 4 and 6 which disrupts the cell wall allowing for milder and more energy efficient lipid extraction methods, and in the absence of conditions or chemicals which may lead to lipid degradation or require involved downstream purification processes. Described herein are different lipid extraction methods performed on biodecomposed microalgae produced as per Example 6, including mechanical methods (ultrasonic) and chemical methods (surfactant, and / or solvent extraction). Materials and Methods

[0198] The algal biomass had been subject to biodecomposition of its cell wall cellulosic material as per Example 6. Dried algal biomass was grinded down with a mortar and pestle.

[0199] In each extraction method, the lipid content was estimated with the assumption that the dry weight of the solvent layer consists of mostly lipids and that is taken as 100% lipid content. The lipid content of the solvent layer was estimated with the following equation:

[0200] The protein content of the water or sediment layer was estimated with the following equation:

[0201] For Soxhlet extraction cell disruption (conventional solvent extraction), 1g of biomass was kept in a Whatman 603 thimble (33 mm x 100 mm).200 mL of chloroform:methanol (2:1) solution was used as the solvent. The solvent was heated up to 110 °C in the Soxhlet extractor equipment and let to run for a minimum of 6 hours. The solvent with the extracted biomass content in the round bottom flask was then removed. MilliQ water was then added at a ratio of 1:5 of MilliQ to methanol to wash the solvent and remove water-soluble content, such as protein and carbohydrate content. The solvent was then transferred to an Eppendorf centrifuge tube and centrifuged at 2000g for 5 minutes. This resulted in two separate layers,where the bottom layer contained lipids solubilised in chloroform and the top layer contained proteins solubilised in a mixture of methanol and water. The separated layers were then transferred to a rotary evaporator to remove the solvent and quantify the lipid and protein content. The interface between the two different liquid systems was discarded to ensure the purity of the lipid and protein extracted.

[0202] For probe sonication cell disruption, 1g of dried algal biomass was weighed in a glass vial and 10 mL of MilliQ water was added to the glass vial. The content in the glass vial was shaken for a minute. The probe (VCX 130, Sonics & Materials, Inc., Connecticut, USA) was placed in the biomass solution and the solution was let to sonicate for 30 minutes. The glass vial was placed within an ice bath to prevent the solution from overheating and the probe sonicator settings were as follows: 40% amplitude, 5 seconds on, and 5 seconds off.

[0203] Two different conditions which were completed for the use of solvent and prolonged sonication time: a. Mixture of ethyl acetate and water: 30 mL of ethyl acetate and 10 mL of MilliQ were added to 1 g of dried algal biomass. The biomass solution was then vortexed for 1 minute before sonicated with the probe sonicator at 40% amplitude (5 seconds on and 5 seconds off cycle) in an ice bath. The resulting biomass was centrifuged at 10,000 g for 30 minutes. The top layer (lipids in solvent) was removed for solvent removal with rotary evaporator and the bottom layer (proteins in water) was removed for freeze drying and protein content analysis. The interface between the two different liquid systems was discarded to ensure the purity of the lipid and protein extracted. b. The addition of ethyl acetate after probe sonication: 20 mL of MilliQ was added to 1 g of dried algal biomass. The biomass solution was then vortexed for 1 minute before sonicated with the probe sonicator at 40% amplitude (5 seconds on and 5 seconds off cycle) in an ice bath.60 mL of ethyl acetate was added to the resulting biomass and vortexed for a minute. The resulting biomass was centrifuged at 10,000 g for 30 minutes. The top layer (lipids in solvent) was removed for solvent removal with rotary evaporator and the bottom layer (proteins in water) was removed for freeze drying and protein content analysis. The interface between the two different liquid systems was discarded to ensure the purity of the lipid and protein extracted.

[0204] For surfactant cell disruption, 1g of dried algal biomass was weighed in a 50 mL centrifuge tube. The biomass was solubilised in 20 mL of 10mM CTAB solution and mixed for a minute. The tube was then placed on a tube shaker and allowed to shake at 99 rpm for 5 hours. The solution was then mixed with 60 mL of ethyl acetate and vortexed for a minute. The resulting solution was centrifuged at 10,000 g for 30 minutes. This resulted in 3 separate layers, the top layer consisted of lipids solubilised in ethyl acetate, the bottom layer consisted of waterand water-soluble biomass content, and sediment (solids). The solvent layer was used in a rotary evaporator to remove the ethyl acetate. The water layer and the sediment layer were placed in a -80 °C freezer and then freeze dried. The interface between the two different liquid systems was discarded to ensure the purity of the lipid and protein extracted.

[0205] Microscopy images of the liquid biomass and liquid supernatant were taken with the Olympus BX51. For CHNS analysis, the total protein content of the dried algal biomass was determined with the Thermo Scientific FlashSmart CHNS analyser (Waltham, United States). Approximately 4 mg of biomass sample was used for each measurement and all measurements were completed in triplicate. The standard used for determination of protein content was BBOT and the general nitrogen conversion factor of 6.25 was used to estimate the protein content of algal biomass. Results

[0206] Protein solubility of algal biomass: An experiment to induce isoelectric precipitation of the protein content showed stable protein content in solution suggesting that the algal membranes in the biomass are not disrupted and still intact.

[0207] Conventional solvent extraction: The overall solvent soluble content of the dried biomass was found to be approximately 53% w / w in methanol / chloroform. The lipid content of the algal biomass was estimated to be approximately 39% w / w of the dried algal biomass weight through Soxhlet extraction with chloroform and methanol, whereas the polar biomass content (such as proteins and carbohydrates) was approximately 14% w / w. This suggests that most of the protein content that was present within the algal biomass was insoluble and was not efficiently extracted through solvent extraction. Major drawbacks of using organic solvents such as chloroform and methanol are such as the toxicity and the flammability of these solvents and the energy intensive process of solvent recovery.

[0208] Sonication: Microscopy and fluorescence images of algal structures showed release of lipid content suggesting that at least partial disruption of the cell membranes had occurred. The dried supernatant layer consisted of both proteins and lipids. This is due to the water- soluble properties of algal proteins. In one way to separate the lipid and protein content, ethyl acetate was added to the algal biomass solution before sonication. The biomass solution appeared homogenous after 30 minutes of probe sonication and was centrifuged to assist in the separation of the lipid (ethyl acetate fraction) and protein content (water fraction). The protein and lipid content estimated in the separated biomass content, as w / w of starting biomass, were: 13.3% lipid in the solvent layer, 15.7% protein in the water layer, and 8.2% protein in the sediment layer. In another way to separate the lipid and protein content, ethyl acetate was added to the algal biomass solution after sonication. The two liquids did not form a homogeneous solution and separated easily. The protein and lipid content estimated in the separated biomass content, as w / w of starting biomass, were: 12.2% lipid in the solvent layer, 25.2% protein in the water layer, and 13.5% protein in the sediment layer.

[0209] Surfactant: The effect of CTAB and ethyl acetate in combination was investigated and microscopy confirmed the release of lipid content from the algal membranes using CTAB and extraction from the biomass solution using ethyl acetate, with higher retrieval. The protein and lipid content estimated in the separated biomass content, as w / w of starting biomass, were: 23.2% lipid in the solvent layer, 13.7% protein in the water layer, and 12.9% protein in the sediment layer. Conclusion

[0210] Different methods of cell disruption were investigated for the extraction of lipid and protein from biodecomposed algal biomass. The use of a mild solvent (ethyl acetate) at room temperature was found to be applicable for lipid extraction in place of toxic chemicals such as chloroform and methanol at high temperatures. While the yield was not as high, the process was significantly faster. Yield was improved using a cationic surfactant (CTAB) with ethyl acetate, also at room temperature and much faster.

[0211] It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art, in Australia or any other country.

Claims

1. THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A combination of cellulolytic microorganisms, wherein each cellulolytic microorganism produces an extracellular glycosylase, said combination comprising: a first cellulolytic microorganism that produces a cellulase classifiable by EC 3.2.1.4 or EC 3.2.1.91; and a second cellulolytic microorganism that produces a cellulase classifiable by EC 3.2.1.4, EC 3.2.1.91, EC 3.2.1.21, EC 3.2.1.52, EC 3.2.1.74 or EC 3.2.1.

86.

2. A combination according to claim 1, further comprising a third cellulolytic microorganism that produces a cellulase classifiable by EC 3.2.1.4, EC 3.2.1.91, EC 3.2.1.21, EC 3.2.1.52, EC 3.2.1.74 or 3.2.1.

86.

3. A combination according to claim 2, further comprising a fourth cellulolytic microorganism that produces a cellulase classifiable by EC 3.2.1.4, EC 3.2.1.91, EC 3.2.1.21, EC 3.2.1.52, EC 3.2.1.74 or 3.2.1.

86.

4. A combination according to claim 3, further comprising a fifth cellulolytic microorganism that produces a cellulase classifiable by EC 3.2.1.4, EC 3.2.1.91, EC 3.2.1.21, EC 3.2.1.52, EC 3.2.1.74 or 3.2.1.

86.

5. A combination according to any one of claims 1 to 4, wherein at least one cellulolytic microorganism is a bacteria selected from the species Bacillus velezensis and Bacillus thuringiensis.

6. A combination according to claim 5, wherein each cellulolytic microorganism is a bacteria selected from the species: Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumillus, Bacillus subtilis, Bacillus subtilis subsp. subtilis, Bacillus thuringiensis, Bacillus thuringiensis Berliner, Bacillus thuringiensis subsp. Israelensis, Bacillus toyonensis and Bacillus velezensis; Bradyrhizobium japonicum; Chromobacterium subtsugae; Saccharopolyspora spinosa; and Priestia aryabhattai and Priestia megaterium.

7. A combination according to any one of claims 1 to 6, wherein at least one cellulolytic microorganism is a bacteria selected from the strain: Bacillus amyloliquefaciens CCT 7690, Bacillus licheniformis CCT 2584, Bacillus pumillus CCT 2487, Bacillus thuringiensis subsp. Israelensis CCT 7795, Bacillus thuringiensis subsp. Berliner ATCC 33679, and Bacillus velezensis CCT 7891; Bradyrhizobium japonicum strain CCT 4065; and Priestia aryabhattai strain CBMAI 1120.

8. A combination according to any one of claims 1 to 7, wherein: a) the first cellulolytic microorganism is selected from Bacillus velezensis and Bacillus thuringiensis, preferably Bacillus velezensis CCT 7891, Bacillus thuringiensis subsp. Israelensis CCT 7795, and Bacillus thuringiensis subsp. Berliner ATCC 33679;b) the second cellulolytic microorganism is selected from Bacillus subtilis, preferably strain Bacillus subtilis ATCC 6051; and c) the combination comprises a third cellulolytic microorganism selected from Bacillus amyloliquefaciens, preferably Bacillus amyloliquefaciens ATCC 23845, wherein the combination preferably further comprises a fourth cellulolytic microorganism selected from Priestia megaterium, preferably Priestia megaterium strain ATCC 14581, and wherein the combination preferably further comprises a fifth cellulolytic microorganism selected from Bacillus subtilis, preferably B. subtilis 168 ATCC 23857.

9. A composition comprising: a combination of cellulolytic microorganisms, wherein each cellulolytic microorganism produces an extracellular glycosylase, said combination comprising: a first cellulolytic microorganism that produces a cellulase classifiable by EC 3.2.1.4 or EC 3.2.1.91; and a second cellulolytic microorganism that produces a cellulase classifiable by EC 3.2.1.4, EC 3.2.1.91, EC 3.2.1.21, EC 3.2.1.52, EC 3.2.1.74 or EC 3.2.1.86; and walled cells.

10. A composition according to claim 9, wherein the walled cells are microalgae.

11. A composition according to claim 10, wherein the algae is a Chlorella spp., preferably Chlorella vulgaris.

12. A composition according to any one of claims 9 to 11, in the form of a growth medium.

13. A composition according to any one of claims 9 to 12, further comprising simple carbohydrate and wherein the walled cells are ruptured walled cells.

14. A method of rupturing a cell wall of a walled cell by biodecomposition of cell wall cellulosic material, said method comprising: providing growth conditions to a composition comprising: a combination of cellulolytic microorganisms, wherein each cellulolytic microorganism produces an extracellular glycosylase, said combination comprising: a first cellulolytic microorganism that produces a cellulase classifiable by EC 3.2.1.4 or EC 3.2.1.91; anda second cellulolytic microorganism that produces a cellulase classifiable by EC 3.2.1.4, EC 3.2.1.91, EC 3.2.1.21, EC 3.2.1.52, EC 3.2.1.74 or EC 3.2.1.86; and walled cells.

15. A method according to claim 14, further comprising retrieval from the composition of one or more of: walled cell intracellular lipid, walled cell intracellular protein, and biodecomposed simple carbohydrate.

16. A method according to claim 14 or 15, further comprising producing walled cells using a growth medium comprising simple carbohydrate produced by the biodecomposition.

17. A method according to any one of claims 14 to 16, wherein the walled cells are produced using a growth medium comprising simple carbohydrate produced by biodecomposition of cellulosic material.

18. A method according to claim 15, wherein retrieval of walled cell intracellular lipid is performed by extraction using an organic solvent and / or a surfactant.

19. A method of retrieval of intracellular lipid and / or intracellular protein from a walled cell, said method comprising: providing growth conditions to a composition comprising: a combination of cellulolytic microorganisms, wherein each cellulolytic microorganism produces an extracellular glycosylase, said combination comprising: a first cellulolytic microorganism that produces a cellulase classifiable by EC 3.2.1.4 or EC 3.2.1.91; and a second cellulolytic microorganism that produces a cellulase classifiable by EC 3.2.1.4, EC 3.2.1.91, EC 3.2.1.21, EC 3.2.1.52, EC 3.2.1.74 or EC 3.2.1.86; and walled cells; to produce a simple carbohydrate and a ruptured cell wall by biodecomposition of cell wall cellulosic material; and retrieving walled cell intracellular lipid and / or walled cell intracellular protein from the composition.

20. A lipid and / or protein retrieved by the method according to claim 19.

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