"BIOFUEL PRODUCTION"

FIELD OF THE INVENTION

[0001] The present invention generally relates to the production of biofuel from biomass, including plant matter, waste material, weeds and pests.

BACKGROUND OF THE INVENTION

[0002] Biofuels, such as ethanol, are fuels produced from renewable biological resources, such as commercial crops, feedstocks and agricultural waste. Production of these fuels is increasing worldwide, reducing the reliance on conventional fossil fuels.

[0003] Biofuels are often produced by me action of enzymes on a biomass. The biomass is generally pretreated before this biological action to improve the efficiency of biofuel production. Such pretreatment processes may include, for example, the addition of various chemicals (such as acids, bases and organic solvents) and heat (such as steam injection). Moreover, after the biofuel has been produced, the crude product is further refined by processes such as distillation and dehydration. As the number and complexity of processing steps increase, then it would be expected that the overall cost of biofuel production would increase as well. Relatively complex processing regimes and the relatively high cost of processing chemicals, enzyme materials and energy requirements has affected the commercial attractiveness of such biofuel production processes.

[0004] One of me most costly and important steps in producing biofuel are the pretreatment processes. Aside from the costs associated with performing the pretreatment processes, pretreatment also affects how effectively enzymes are able to produce biofuel, affecting the overall yield.

[0005] In researching biofuel production, the inventor discovered a biofuel production method which may enhance the bioavailability of the biomass for enzymatic conversion and thus reduce the time and energy required to produce the biofuel. These discoveries have been reduced to practice in a biofuel production method.

SUMMARY OF THE INVENTION

[0006] The present invention provides a method of producing biofuel, the method comprising: (a) providing a first substrate comprising biomass;

(b) reducing the particle size of the first substrate to produce a second substrate with a particle size of < 90 μπι; and

(c) contacting the second substrate with a biofuel producing enzyme; to thereby convert the second substrate into a biofuel.

[0007] In some embodiments of these methods, one or more of the following applies:

the biofuel is a bioalcohol; especially methanol, ethanol, propanol or butanol; more especially ethanol;

the first substrate is plant matter; especially commercial crops, feedstocks, wood, grasses, weeds, algae, and by-products from processes arising from processing commercial crops; more especially sugarcane bagasse, tapioca waste, artichoke thistle, water hyacinth, cumbungi, buffel grass, triticale, sweet sorghum, rice or a part thereof (e.g., rice husk), palm tree or part thereof, elephant grass or a Casuarina species; most especially water hyacinth; the particle size of the first substrate is reduced by mechanical processes, chemical processes, drying processes or combinations thereof; especially mechanical dehydration; more especially a combination of mechanical processes and oven drying;

the particle size of the first substrate is reduced by a process comprising drying the first substrate;

the particle size of the second substrate is < 85 um; especially < 80 um; especially < 75 μπι; especially < 70 um; especially < 65 um; especially < 60 μηι; especially < 55 μπι;

especially < 50 um; especially < 45 um; more especially < 40 μπι;

the particle size of the second substrate is > 0.5 μπι; especially > 1 μπν, especially > 5 μτη; especially > 10 μιη; especially > 15 μτη; especially > 20 μπι; especially > 25 um;

especially > 30 μητ, more especially > 35 μτη;

the first substrate is water hyacinth and the particle size of the second substrate is < 40 um;

the first substrate is tapioca waste and the particle size of the second substrate is < 15 μπι; the first substrate is sugarcane bagasse and the particle size of the second substrate is

< 20 μσι;

the first substrate is algae or a micro-organism and the particle size of the second substrate is < 1 μπι;

the water content (e.g. , the free or unbound water content) of the second substrate is

< 18 %, < 17 %, < 16 %, 15 %, < 14 %, < 13 %, < 12 %, < 11 %, < 10 %, especially < 8 %, more especially < 5 %, more especially < 3%, most especially < 2 %;

> 0.1 metric ton of second substrate is produced; especially > 0.25 metric ton of second substrate is produced; especially > 0.5 metric ton of second substrate is produced; more especially > 0.75 metric ton of second substrate is produced;

the producing enzyme is selected from the group consisting of endo-cellulases, exo- cellulases, thermoacidophillic cellulases and thermo-active cellulases; cellobiases;

lignocellulases; xylanases; xylosidases; chininases; amylases; glucoamylases; peroxidases; laccases; lipases; endoglucanases; pectinases; proteases; ligninases; alcohol dehydrogenases; feruloyl esterases; indole-3-acetaldehyde reductases (NADH); 3-rhethylbutanal reductases; formaldehyde dismutases; endo-xylanases; β-glucosidases; hemi-cellulases;

phosphatidylethanolamine N-methyltransferases; lignin modifying enzymes, and enzymes that act on hexoses, pentoses and disaccharides.

[0008] In some embodiments, the method further comprises refining the biofuel; especially by distillation; more especially by distillation and dehydration.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 shows a design plan for a plant that can be used to reduce the particle size of a first substrate in order to produce or obtain a second substrate with a particle size of < 90 um for use in a particularly preferred embodiment of the invention. DETAILED DESCRIPTION OF THE INVENTION

1. Definitions

[0010] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. 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. For the purposes of the present invention, the following terms are defined below.

[0011] 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.

[0012] The term "about" is used herein to refer to conditions (e.g., amounts, concentrations, time, etc.) that vary by as much as 30%, preferably by as much as 20%, and more preferably by as much as 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% to a specified condition.

[0013] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. Thus, use of the term "comprising" and the like indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By "consisting of is meant including, and limited to, whatever follows the phrase "consisting of. Thus, the phrase "consisting of indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

2. Biofuel

[0014] The term "biofueF refers to a fuel produced from a biomass. Biofuels may include solid, liquid or gaseous fuels. Examples of biofuels include biodiesel and bioalcohols, such as methanol, ethanol, propanol and butanol, especially ethanol.

[0015] In some embodiments, the biofuel produced by the method of the present invention may comprise at least 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, or 5% fuel. In some embodiments, the biofuel produced by the method of the present invention may comprise 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% of fluids other than fuel, especially water.

3. First Substrate

[0016] The first substrate comprises biomass. The term "biomass" relates to any material derived from one or more living organisms. This includes any plant matter, such as commercial crops and feedstocks, wood and wood chips, grasses, palm trees and parts thereof (such as palm trunks or palm fronds), forest residue (such as dead trees, tree stumps and branches), weeds, fungi, algae, food processing waste stream material, including spoiled foods and peelings derived from food manufacturing processes (for example, banana skins, nut shells, tea leaves and pomace from vineyards). Commercial crops and feedstocks includes, for example, sugarcane, rice, miscanthus, sugarbeet, sorghum, grain sorghum, switchgrass, barley, hemp, kenaf, potatoes, sweet potatoes, cassava, sunflower, fruit, molasses, corn, stover, grain, wheat, straw, soybean, cotton, triticale and sweet sorghum. Biomass also includes by-products arising from processing such commercial crops, such as sugarcane bagasse, rice husk, tapioca waste, corn stalks, wheat straw and rice straw, as well as crops grown specifically for the purpose of field reconditioning and or bio-processing feedstock materials. The biomass may also include animal matter, such as animal excreta, especially sewage.

[0017J In some embodiments, the first substrate is a pest plant species or is derived from a pest plant species, including a fast-growing pest plant species (e.g. , a weed).

Advantageously, when such weeds are used to produce biofuel, land that would be used to produce other crops may be unaffected, or may be improved for example by removal of the weeds. Examples of such weeds include aquatic and semi-aquatic weeds such as water hyacinth {Eichhornia crassipes) and cumbungi (Typha domingensis), and terrestrial weeds such as artichoke thistle (Cynara cardunculus), buffel grass (Cenchrus ciliaris), elephant grass (Pennisetum purpureum, Saccharum ravennae or Miscanthus sinensis) and Casuarina species, especially Casuarina glauca, Casuarina cunninghamiana and Casuarina

equisetifolia. In specific embodiments, the first substrate is an aquatic weed, especially water hyacinth.

[0018] In some embodiments, the first substrate comprises biomass that comprises at least 95%, 90%, 85%, 80%, 75% or 70% intact plant cells. [0019] In some embodiments, the biomass may be harvested plant material that has not been subjected to any chemical treatment steps or processes such as crushing or grinding.

[0020] In some embodiments, the first substrate comprises biomass comprising plant material but not comprising by-products from processes arising from processing commercial crops; including but not limited to sugarcane bagasse and tapioca waste.

4. Reducing the particle size of the first substrate

[0021] Reducing the particle size of the first substrate to < 90 um produces a second substrate. In some embodiments, the particle size is reduced to < 85 um, < 80 um, < 75 um, < 70 μηι, < 65 um, < 60 um, < 55 um, < 50 μτη, < 45 μπ. or < 40 u . In some embodiments, the particle size of the second substrate is > 0.5 um, > 1 um, > 5 um, > 10 um, > 15 μιη, > 20 μπι, > 25 um, > 30 um, or > 35 um.

[0022] In some embodiments, the particle size of the second substrate is between 1 μτη and 90 μπι, between 5 μτη and 85 μτη, between 10 μηι and 80 m, between 15 μτη and 75 μτη, between 20 μπι and 70 um, between 25 μm and 65 um, between 30 um and 60 um, between 35 um and 55 μm.

[0023] The reduced particle size makes the second substrate easier to handle, by improving blending, mixing, aerating and distribution properties. In some embodiments, the second substrate may behave as a pseudo-liquid.

[0024] Reducing the particle size also increases the surface area of the second substrate, and thus may enhance the bioavailability of the second substrate during enzymatic digestion, and reduce biofuel production time (for example through fermentation). The increased surface area of the second substrate may also permit a greater amount of biomass to be treated in a given reaction volume than if the particle size was not reduced. In some embodiments, the bioavailable surface area to the enzymes is increased a factor of at least 3, leading to a corresponding increase in yield.

[0025] In some embodiments the optimum particle size of the second substrate is where at least 30%, 40%, 50 %, 60%, 70%, 80%, 90%, or 95% of the cell walls of the biomass are physically broken. In one embodiment, substantially all the cell walls of the biomass are physically broken. The optimum particle size differs between types of biomass, but in one embodiment the particle size is selected by choosing a size that is equal to or marginally less than the average dry cellular size of the biomass. [0026] For example, for water hyacinth Eichhornia crassipes) the particle size is especially < 40 um, for tapioca waste (Manihot esculenta) the particle size is especially < 15 μι ^η , and for sugarcane bagasse the particle size is especially < 20 um. For algae and other micro-organisms, the particle size may be < 1 um.

[0027] In some embodiments, where the biomass comprises plant matter, the reduction in particle size significantly disrupts the lignin barrier surrounding the cells, and the interstitial spaces (which also contain lignin) are opened. This makes the inter- and intracellular materials more bio-accessible for enzymatic processing and may result in more rapid processing and higher yields.

[0028] The first substrate may have previously been subjected to processing steps.

Consequently, and especially if the particle size of the biomass in the first substrate has already been reduced, fewer processing steps may be required to provide the second substrate. For example, if sugarcane bagasse is the biomass, then the both the water content (e.g. , the free or unbound water content) and the particle size of the biomass would be lower than if sugarcane itself were used.

[0029] If an unprocessed first substrate is used, it may be necessary to wash the biomass to remove any residual detritus such as soil prior to reducing the particle size of the biomass.

[0030] The particle size of the first substrate may be reduced by various methods or combinations of methods known to a person skilled in the art. For example, mechanical processes may be used, including abrasion, centrifugation, chipping, chopping, crushing, cutting, extruding, grinding, macerating, milling, screening, shearing, shredding and/or sieving in sequential continuous flow or batched operations. Ultrasonic and other energised physical means may also be used.

[0031] The force applied in such mechanical processes may be compression, impact, or shear, and both the magnitude of the force and the time of application affect the resultant particle size. For efficient particle size reduction, the energy applied to the first substrate may exceed, by only a small margin, the minimum energy needed to rupture the cell walls as excess energy is lost as heat. The energy required to rupture the cell walls depends upon the hardness of the material and also its friability. The required energy will therefore vary from batch to batch, especially for substrates in various stages of growth or where multiple types of biomass are used. [0032] In some embodiments, a ball mill may be used to reduce the particle size of the first substrate. In a ball mill, the first substrate is enclosed in a horizontal cylinder or a cone and tumbled with a large number of steel balls, natural pebbles or artificial stones. In some other embodiments, an edge runner mill may be used, especially in an initial grinding process. The edge runner mill has a heavy, broad wheel running round a circular trough to grind the substrate.

[00331 hi some other embodiments, a hammer mill may be used to reduce the particle size of the first substrate. In a hammer mill material is crushed and pulverized between the hammers and the casing. This material is retained in the mill until it is sufficiently fine to pass through a size exclusion screen or mesh at the bottom of the mill housing. In further embodiments, a fixed head or plate mill may be employed.

[0034] Fixed head mills typically utilise a shearing action between a fixed casing and a rotating head, with only fine clearances between the faces. In plate mills the substrate is fed in through two circular plates, one of which is fixed and the other rotating to achieve the necessary shear force. The substrate enters close to the central axis of rotation and is sheared and crushed as it transitions to the exit at the edge of the plates. The plates can be mounted horizontally (as in the traditional Buhr stone that is used for grinding corn) or mounted vertically. A colloidal mill may also be used, in which very fine clearances and very high speeds are used to produce particles of colloidal dimensions. In some embodiments, a near colloidal mill may be used to reduce the particle size of the first substrate.

[0035] When reducing the particle size of the first substrate by mechanical processes, the relative proportion of a certain particle size may increase in the mixture and become the predominant size fraction. For example, after initial crushing a wide range of particle sizes may be present, but after further grinding the predominant fraction may pass through a 250 mm sieve, while being retained on a 125 mm sieve. This predominant fraction may build up however long the grinding continues, so a secondary in-line milling operation may be employed. In one embodiment, a combination of mechanical processes may be used to reduce the particle size of the first substrate.

[0036] To obtain a desired particle size for the second substrate a mesh or mesh sieve of a particular size may be used. In some embodiments, the particle size of the second substrate is such that >95%, >90%, >85%, >80%, >75%, or >70% of the particles pass through a mesh or mesh sieve that permits particles < 90 μπι, < 85 um, < 80 μιη, < 75 um, < 70 urn, < 65 μι ^η , < 60 μτη, < 55 um, < 50 urn, < 45 um or < 40 um to pass through the mesh or mesh sieve.

[0037] In some embodiments, the present invention employs the use of more than one mechanical process and more than one mesh or mesh sieve, wherein the first substrate is passed along a passage (e.g. , a conduit, cylinder, tunnel) where the first substrate encounters a first means for mechanically reducing the particle size and subsequently a first mesh or a first mesh sieve that permits a specific particle size to pass therethrough (e.g., < 200 μπι, < 150 M , < 25 um, < 100 um, < 95 um, < 90 um, < 85 um). This first mesh or first mesh sieve prevents particles of a designated size from passing further along the passage. The substrate that passes through the first mesh or first mesh sieve then encounters a second means for mechanically reducing the particle size and subsequently a second mesh or mesh sieve that permits a specific particle size to pass therethrough, wherein the size of the particles permitted through the second mesh or second mesh sieve is smaller than the first mesh or first mesh sieve (e.g., < 125 um, < 100 um, < 95 um, < 90 um, < 85 um, < 75 um, < 70 um,). The passage may comprise further means for mechanically reducing the particle size and further meshes or mesh sieves such where the size of the particles permitted through the further mesh(es) or mesh sieve(s) is smaller for each progressive sieve. Suitably, the first substrate is passed along the passage through use of force (e.g. , gravity, air blower, vacuum) so that substrate reduced to a particle size that passes through a mesh or mesh sieve is passes through the passage to the next mechanical processing means (or the end of the passage). Suitably, substrate not reduced to a particle size that can pass through a mesh or mesh sieve is further processed by the mechanical processing means preceding the mesh or mesh sieve and processed to a particle size that passes through a mesh or mesh sieve.

[0038] Chemical processes may also be employed to facilitate the reduction in particle size. This may include acid (such as acetic, formic, nitric, sulfuric or perchloric acid) or basic (such as sodium hydroxide) treatment, which may be at elevated temperatures. Such chemical processes may promote hydrolytic reactions to cleave internal bonds in lignin and to cleave glycosidic linkages in both hemicellulose and cellulose.

[0039] Drying processes may also be used to facilitate the reduction in particle size. This may include sun drying, air drying, oven drying, evaporative drying and freeze drying under reduced atmospheric pressure (such as between 0 and -20 °C). In one embodiment, when the biomass is plant matter it is heated to no greater than 80 °C. Suitably, the drying process reduces the water content (e.g. , the free or unbound water content) of the first substrate to less than 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, or 9%.

[0040] The first substrate may also be placed in a liquid medium (such as in water or in an organic solvent), and/or heated to facilitate the particle size reduction. Water has been reported to lower the softening point of lignin, which allows easier separation of plant fibers. Processes such as steam injection and autoclaving may also be employed.

[0041] Combinations of processes may also be used. For example, in sugarcane processing the sugarcane is crushed, shredded and mixed with water. Other suitable processes are outlined, for example, in Lynd, Annu. Rev. Energy. Environ. 1996, 21, 403-465. The present invention specifically contemplates combinations of processes including both at least one drying process and at least one mechanical process wherein the at least two processes are performed simultaneously or sequentially. In exemplary embodiments, the first substrate is subjected to a drying process followed by a mechanical process. In other exemplary embodiment, the first substrate is subjected to a mechanical process followed by a drying process. The drying process when used in combination with the mechanical process may assist in reducing the energy required to reduce the particle size by way of the mechanical process.

[0042] The second substrate will generally have a lower water content (e.g. , the free or unbound water content) then the first substrate. Lowering the water content (e.g. , the free or unbound water content) is advantageous as the presence of water may enable some undesirable biofuel production processes to commence prematurely. Water may also allow suboptimal biofuel production processes by biofuel producing enzymes which may then generate a wider range of undesired cellular and bacterial materials. The presence of water may also may increase the mechanical strength required to physically produce the second substrate at the desired particle size. Drying may also facilitate storage and handling of the second substrate, as wet mass tends to clump and aggregate whereas dry mass flows more readily.

[0043] In some embodiments, the water content (e.g. , the free or unbound water content) of the second substrate is < 18 %, < 17 %, < 16 %, 15 %, < 14 %, < 13 %, < 12 %, < 11 %, < 10 %, especially < 8 %, more especially < 5 %, more especially < 3%, most especially < 2 %. [0044] These processes allow biofuel to be produced on an industrial scale, for example producing > 0.1 metric ton of second substrate; especially > 0.25 metric ton of second substrate; especially > 0.5 metric ton of second substrate; more especially > 0.75 metric ton of substrate. 4.1 Mechanical dehydration

[0045] In one embodiment, the particle size of the first substrate is reduced by mechanical dehydration. This means that the water content (e g., the free or unbound water content) of the biomass is reduced, and a mechanical process (for example, as outlined above) is used to reduce the particle size of the first substrate to < 90 um.

[0046] In some embodiments, a chemical process is not used. Not using chemical processes is advantageous as the use of chemicals may be environmentally undesirable and they may alter the native structure of the plant material which makes it less susceptible to microbiological and enzymatic degradation. Chemicals may also add to the cost of production due to the cost of the chemicals and the time required to perform the chemical steps, Furthermore, if chemical processes are used the complexity of the pretreatment process may be increased. For example, if an acid treatment step is employed, then the acid may need to be neutralised before further processes are performed.

[0047] For example, the particle size of the first substrate may be reduced to a size where it can be effectively dried, after which a drying step may be performed, and then the particle size may be further reduced, to thereby produce the second substrate. If the first substrate has been previously processed, such as for sugarcane bagasse, then the particle size may not need to be reduced prior to drying.

[0048] Examples of mechanically dehydrating the first substrate include repeatedly crushing and straining the first substrate until the desired particle size is obtained and also chopping the biomass into small pieces, drying these pieces in an oven, and then using a crusher to further reduce the particle size.

[0049] A further example is squeezing the fluids out of the first substrate, reducing the water content (e.g., the free or unbound water content). Following this the squeezed material is dried in an oven, and then a mill is used to further reduce the particle size to the desired size.

[0050] In these examples, the time required for each ste could be determined by a person skilled in the art, and depends upon the biomass being treated and the equipment used. 5. Contacting the substrate with a bio fuel producing enzyme

[0051] The term "biofuel producing enzyme" refers to an enzyme that can assist in converting the second substrate into biofuel. The biofuel producing enzyme may comprise an isolated enzyme or an organism that contains an enzyme, such as a microorganism. It may be necessary to employ a variety of biofuel producing enzymes or microorganisms to convert components of the second substrate such as cellulose and lignin to biofuel such as ethanol. Accordingly, the second substrate may be contacted with one or more isolated enzymes, and/or one or more organisms containing an enzyme.

[0052] In some embodiments, the biofuel producing enzyme may include one or more of the following: endo-cellulases, exo-cellulases, thermoacidophillic cellulases and thermo-active cellulases; cellobiases; lignocellulases; xylanases; xylosidases; chininases; amylases; glucoamylases; peroxidases; laccases; lipases; endoglucanases; pectinases;

proteases; ligninases; alcohol dehydrogenases; feruloyl esterases; indole-3-acetaldehyde reductases (NADH); 3-methylbutanal reductases; formaldehyde dismutases; endo-xylanases; β-glucosidases; hemi-cellulases; phosphatidylethanolamine N-methyltransferases; lignin modifying enzymes, and fermentation enzymes to convert hexoses, pentoses and

disaccharides to biofuels such as ethanol.

[0053] Microorganisms that may be used to break down such complex compounds may include naturally occurring or modified microorganisms, such as one or more of the following: Acetivibrio celluloyticus; Acetobacter xylinum; Acidothermus cellulolyticus;

Alcaligenes sp.; Arthrographis sp.; Aspergillus sp., especially Aspergillus aculeatus,

Aspergillus awamori, Aspergillus brevipes, Aspergillus candidus, Aspergillus carbneus, Aspergillus flaws, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Aspergillus parasiticus, Aspergillus phoenicis, Aspergillus terreus; Bacillus sp., especially Bacillus agaradhaer ns, Bacillus amyloliquefaciens, Bacillus cellulyticus K-12, Bacillus circulans, Bacillus lichenformis, Bacillus pumilus, Bacillus sphaericus, Bacillus subtillis; Butyrivibibro flbrisolvens; Candida rugosa; Candida shehatae; Cellulomonas sp.; Chaetomium thermophile; Clostridium ljungdahlii; Clostridium papyrosolvens; Clostridium thermocellum; Cellvibrio japonicus; Escherichia coli; Fusarium oxysporum; Geobacillus sterothermophilus; Humicola grisea; Klebsiella oxytoca;, Neospora crassa; Pachysolen tannophilus; Paenibacillus macerans; Pichia stipitis; Piromyces equi; Phanerochaete chrysosporium; Penicillium sp., especially Penicillium brasilianum, Penicillium decumbens, Penicillium funiclosum, Penicillium Notatum; Pseudomonas sp., especially Pseudomonas Fluroscenes, Pseudomonas Putida; Pseudoalterromonas haloplanktis; Rhizopus oryzae; Saccharomyces sp., especially Saccharomyces cerevisiae; Sporotrichum pruinosum;

Sporotrichum thermophile; Streptococcus thermophilics; Streptomyces sp., especially

Streptomyces albaduncus, Streptomyces lividans, Streptomyces olivochromogenes,

Streptomyces reticule, Streptomyces rochei; Talaromyces stipitatus; Trichoderma sp., especially Trichoderma citrinoviride, Trichoderma fasciculatum, Trichoderma harzianum, Trichoderma koningii, Trichoderma lignorum, Trichoderma longibrachiatum, Trichoderma mobilis, Trichoderma reesei, Trichoderma virens, Trichoderma viride; Torula thermophila and Zymomonas mobilis. Other suitable microorganisms are known to a person skilled in the art. Various microorganisms and ethanol production processes are outlined, for example, in Lynd, Annu. Rev. Energy. Environ. 1996, 21, 03-465.

[0054] The time required for the enzyme treatment, and the conditions under which the treatment occurs may be determined by a person skilled in the art. When determining the ideal conditions, the temperature and pH should be considered. Generally, at a lower temperature the stability of the enzyme is higher, but the enzyme is less effective. Similarly, generally the pH of the solution in which the treatment occurs may affect the effectiveness of the enzyme.

[0055] In some embodiments, different biofuel producing enzymes may be contacted with the second substrate at the same or different times. Separate use of different biofuel producing enzymes may be advantageous, as this allows different processes to be conducted at, for example, different temperatures or at a different pH. For example, it may be advantageous to perform an initial treatment with a first biofuel producing enzyme at a higher temperature (for example when hydrolysing cellulose), and performing a subsequent treatment at a lower temperature with a second biofuel producing enzyme (for example during fermentation).

[0056] The second substrate may be contacted with the biofuel producing enzyme at a temperature of between about 20 °C and 60 °C. The temperature at which the . second substrate is contacted with a biofuel producing enzyme may depend upon, for example, the organism that contains the enzyme. For example, if a mesophilic organism is used, then the temperature may be between about 15 and 40 °C. If a thermophilic organism is used, then the temperature may be between about 40 and 90 °C.

[0057] The biofuel producing enzyme may also be contacted with the second substrate in a buffered solution. If different biofuel producing enzymes are used at different times, the pH of the solution at these times may be the same. In one embodiment, the buffered solution is mildly acidic, for example at about pH 5. In this embodiment, an acetate buffer may be used. In another embodiment, the buffered solution has a pH of between about 6.0 and 8.0. In this embodiment, a phosphate buffer may be used. In some embodiments, an enzymatic co-factor may be used, such as a divalent cation. In these embodiments, a buffer with a low metal binding characteristics may be used, such as PIPES, HEPES and HPPS.

[0058] Advantageously, reducing the particle size of the first substrate, as discussed herein, may allow a reduction in the time required for enzymatic processing, for example, from 24 to 72 hours, to around 8 to 30 hours. Furthermore, by reducing the particle size of the first substrate production of the biofuel may occur in a more efficient manner, including reduced time and/or energy expenditure.

6. Refining the biofuel

[0059] The biofuel produced by the method of the present invention may be refined. Such refining processes are known to a person skilled in the art.

[0060] For example, an initial refining step may comprise filtering the crude product and then distilling the filtrate. Various distillation processes may be used, such as fractional distillation, and azeotropes may also be used to assist in this process. Dehydration processes may also be employed. Molecular sieves may also be used to remove impurities, such as water.

[0061] In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non- limiting examples.

EXAMPLES

EXAMPLE 1 - PRETREATMENT

[0062] Water hyacinth (Eichhornia crassipes) was collected from infested environmental sources. It was washed to remove adhering soil and debris before draining to remove excess water.

[0063] The plant material was then chopped into smaller pieces (~10cm) by hand. The chopped material was then dried using a commercial oven with a maximum drying temperature of 80 °C. The water content of the oven dried substrate was in the range of 5-8%, as determined by Karl Fischer titration over a number of successive batches. [0064] The cut and dried water hyacinth was fed into a hopper using a screw conveyer powered by a 3 kW motor. The material was passed through a mechanical 1 kW cutter, with the resultant fragments collected in a hopper. To produce a powder, the contents of the hopper were then conveyed into two, sequential ball mills which were powered by a 3 kW motor. The powder was collected via suction into a receptacle container. The powder was then passed through the ball mills twice more to provide a particle size of < 40 um, which was determined microscopically.

EXAMPLE 2 - ENZYME PREPARATION

[0065] Micro-organisms were selected for their ability to secrete extracellular enzymes useful for demethoxylation, decarboxylation, hydroxylation and aromatic ring opening. This included: ligninolytic enzymes, dioxygenases, amylases, lactases, ligninolytic peroxidases, manganese peroxidases, lignin peroxidases, pectinases, cellulases,

endoglucanases, glucoamylases, xylanases, pectinases, chitinases, other lignin-degrading enzymes and cellulase-free xylanases.

[0066] Separate cultures were growth on Potato Dextrose Slant Agar for 7 days.

Enzyme production was then stimulated for each of the selected micro-organisms. This was achieved by collecting liquid aliquots of each of the actively growing cultures and transferring them to organism specific enzyme promoting growth media, in which starch and similar simple carbohydrates were the predominant nutrient source. This growth media was used as a promoter-substrate for enzyme production over 2-4 days. During this phase the culture organisms preferentially produce proteolytic enzymes at lower temperatures (about 25 to 30 °C), and preferentially produce cellulose and amylase enzymes at slightly elevated temperatures (about 30 to 35 °C). Over the 2-4 day period the temperature was maintained at >30 °C, after which the enzymes were harvested.

[0067] The liquid fraction containing the enzymes was strained and the liquid fraction collected. The retained viable culture material was added to fresh enzyme growth promoting media and returned to temperature controlled incubation conditions. This process of straining and returning the culture material to fresh enzyme growth promoting media was repeated for a total of three successive harvests.

[0068] The activity of the enzymatic extract was verified using a refractometer to estimate the concentration of glucose and ethanol via an in vitro assay. EXAMPLE 3 - YEAST INOCULUM

[0069] A glucose fermenting yeast, Saccharomyces cerevisiae, was used. The stock cultures were maintained on Malt extract- Yeast extract-Glucose-Peptone (MYGP) agar (3 g/L malt extract, 3 g L yeast extract, 15 g/L glucose, 10 g/L peptone, and 20 g/L agar, pH 6.0) slants and stored at 4°C.

[0070] Yeasts from these agar slants were suspended aseptically in 100 mL liquid MYGP medium (pH 5.0) and incubated at 30 °C for 24 h, with agitation at 150 rpm. These suspension cultures of yeast were then used as inocula for fermentation.

EXAMPLE 4 - ETHANOL PRODUCTION

[0071 J Ethanol was produced from 132 kg dried, milled 40 μπι Eichhornia crassipes powder in a single 1000 L bio-reactor. The powder was incubated in acetate buffer (pH 5) and the mixture heated to 60 °C. Once this temperature had been achieved, the enzymatic extract from a facultative thermopile was added, and aerobic and thermophilic conditions (60 °C) were maintained for 6 hours. After this, the temperature was reduced to 32 °C.

[0072] After cooling to 32 °C, Simultaneous Saccharification and Fermentation (SSF) methodology was employed. Extracts from the cultures of mesophilic organisms were added to the bio-reactor along with viable Saccharomyces cerevisiae. Anaerobic conditions were then maintained for between 24 and 48 hours.

[0073] After 48 hours analysis of the reaction fluid broth showed that up to 25.5% of the available carbohydrates were converted to ethanol. This compares to only 15.0% being converted in the absence of drying and particle size reduction to < 40 um.

[0074] Ethanol yields of up to 45.5 g/L have been obtained via this method.

Processing using essentially identical enzymic extracts and fermentative processes provided a maximum ethanol yield of 25.5 g/L.

EXAMPLE 5 - EXAMPLE OF PARTICLE SIZE REDUCTION

[0075] Figure 1 shows a design plan for a plant that can be used to reduce the particle size of a first substrate in order to produce or obtain ^' a second substrate with a particle size of < 90 μπι in accordance with the present invention.

[0076] As can be seen in the Figure, the first substrate enters this plant on the left hand side of the drawing, and moves to the right through the processors or processing steps indicated in the drawing. At the right hand side of the drawing (i.e., the end of the processing), the second substrate has been produced.

[0077] In the first step, the first substrate (e.g., sugarcane bagasse) enters a sawdust crusher mainframe (A) which crushes the first substrate into smaller size pieces of substrate. These smaller size pieces are then transferred into a collecting chamber (C) by way of a crusher blower (B).

[0078] In a second step, the smaller size pieces of substrate are transferred by way of a first bucket elevator (D) into a first storage bin (E) and then discharged along a first screw conveyer (F) which transfers the material from the first storage bin (E) and into a drying machine (G).

[0079] The drying machine (G) applies heat to the smaller size pieces of substrate so as to remove moisture from therein. Suitably, the drying machine reduces the water content (e.g. , the free or unbound water content) of the smaller size pieces by 1 %, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, or 85%.

[0080] The smaller size pieces of substrate leave the drying machine (G) by way of a second screw conveyer (H) which transports the material to the second bucket elevator (I) while at the same time cooling the material before it is further processed.

[0081] The second bucket elevator (I) transports the substrate into a second storage bin (J) which is used to hold the substrate until the next processing step occurs. A

discharging screw conveyer (K) is then used to transport the substrate from the second storage bin (J) to a processors) (L) that reduces the particle size of the substrate. The processors) (L) may be any suitable processor, including a hammer mill, grinder, or powder machine. The processors) (L) may be more than one processor that reduces the particle size of the substrate, for example, two processors acting in concert to reduce the particle size of the substrate. The grinding processors) (L) reduces the particle size of the substrate to a particle size of < 90 um, . e. , the second substrate.

[0082] The second substrate is then transported by way of a third bucket elevator (M) to a third storage bin (N).

[0083] The second substrate may be unloaded from the third storage bin ( ) by way of the storage bin unloader (O) and then contacted with a biofuel producing enzyme at any suitable time to thereby convert the second substrate to a biofuel. EXAMPLE 6 - ETHANOL PRODUCTION FROM RICE HUSKS

[0084] A pilot scale byconversion of rice husk substrate into weak ethanol was undertaken.

(0085] A first substrate comprising rice husk was obtained. The first substrate was waste material derived from rice manufacture. The rice hull or husk is a hard, indigestible protective outer layer removed when the grain is milled for preparation of food stuffs, and often utilised for construction and industrial purposes. In this case, the first substrate was air dried and then the particle size of the first substrate was reduced via colloidal milling to produce a second substrate with an particle size capable of passing through a mesh sieve that permits passage of particles < 46 um. This resulted in a uniform power with an appearance of coarse flour.

[0086] Bioconversion of the second substrate was achieved at 30 °C to 55 °C through the qualitative combination of 2 parts warm water and 1 part second substrate into a. vessel. To this suspension was added a combination of enzymes, containing cellulase, xylanase and Iigninase activities. The reaction within the vessel was mixed, intermittently over 24 hours.

[0087] After 24 hours, gas was vented from the vessel and the ethanol content measured as being 10.5 % using an Atago Pocket refractometer (PAL-34S).

EXAMPLE 7 - ETHANOL PRODUCTION FROM SUGARCANE BAGASSE

[0088] Bioconversion of sugarcane bagasse into ethanol was undertaken.

[0089] A first substrate comprising sugarcane bagasse was obtained. Sugarcane bagasse is a highly fibrous waste material, that remains following juicing of cane in order to produce cane sugar. In this case, the sugarcane bagasse was air dried and then shredded to approximately 1cm long fibres before employing a hammer mill to obtain a second substrate with an approximately uniform particle size, wherein all of the second substrate passed through a mesh sieve that permits particles with < 37 um to pass through, and 90% of the second substrate passed through a mesh sieve that permits particles with < 46 um to pass through mesh.

[0090] Bioconversion of the second substrate into weak ethanol was achieved in 24 hours, at 30 °C to 55 °C through the qualitative combination of 2 parts warm water and 1 part milled bagasse. To this suspension was added a combination of enzymes, containing cellulase, xylanase and ligninase activities. The reaction was mixed, intermittently. After 14 hours, gas was vented from vessel and the ethanol content measured as being 5 % using an Atago Pocket refractometer (PAL-34S). After 24 hours, the ethanol content measured as being 10.8 % using an Atago Pocket refractometer (PAL-34S). EXAMPLE 8 - ETHANOL PRODUCTION FROM PALM TREE TRUNK

[0091 J Palm tree trunk was supplied in sections of approximately 4 x 40cm lengths each, comprising both the hard outer surface and the inner fibrous material. The palm trunk sections had been oven dried to a constant mass. The sections were then reduced in size using crushing and milling, to very small fibres resembling fine saw dust, 100% of fibres passing through a mesh sieve that permits particles of < 105 m to pass through and >99% of the fibres passing through a mesh sieve that permits particles of < 88 μπι to pass through.

[0092] A bioconversion reactor was set up using processes described above for Example 7, and similar results were obtained following 14 and 24 hours. In this case the fermentation was allowed to continue with warming and mixing for a further 24 hours, with ethanol content measured as being 24% after a total of 48 hours fermentation.

EXAMPLE 9 - ETHANOL PRODUCTION FROM WHEATEN FLOUR

[0093] Commercially available coarse wheaten flour is required by Codex to have a maximum particle size of 212 um, whereas fine wheaten flour has a typical particle size of 30 to 70 um, and ultra fine wheaten flour has a particle size of typically less than 15 um.

[0094] One part commercially available when fine wheaten flour (particle size of

30 to 70 μιη) was combined with two parts warm water, and a suitable quantity of mixed enzymes, and then allowed to ferment, with mixing at 30-55 °C for 24 hours. Following 24 hours, the ethanol content of the mixture was measured as being 24 %, and the sugar reading 6 % using an Atago Pocket refractometer (PAL-34S). EXAMPLE 10 - ETHANOL PRODUCTION FROM RED SORGHUM

[009S] Conversion of cellulose material incorporated in red sorghum to simple sugar and alcohol was also demonstrated.

[0096] Commercially prepared red sorghum flour, prepared using sequential drying, crushing and colloidal milling in 1 part was mixed with 4 parts warm water, and a suitable volume of mixed action enzymes were added. The particle size of the flour was such that 100% of fibres passed through a mesh sieve that permits particles of < 90 um to pass through.

[0097] Fermentation was allowed to proceed at 32 °C. After 8 hours, 9.5% ethanol was measured, and after 48 hours 17 % ethanol was measured.

[0098] The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.

[0099] The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application.

[0100] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

[0101] Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Those of skill in the art will therefore appreciate that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention. All such modifications and changes are intended to be included within the scope of the appended claims.