AU2010100669A4

AU2010100669A4 - Biofuel Production

Info

Publication number: AU2010100669A4
Application number: AU2010100669A
Authority: AU
Inventors: Dudi Djuhdia Sastraatmadja
Original assignee: Pt Endugo Enzimes Int
Current assignee: PT ENDUGO ENZIMES INTERNATIONAL
Priority date: 2010-06-25
Filing date: 2010-06-25
Publication date: 2010-07-22
Anticipated expiration: 2018-06-25
Also published as: WO2011160192A1

Description

Regulation 3:2 AUSTRALIA Patents Act 1990 ORIGINAL COMPLETE SPECIFICATION INNOVATION PATENT Applicant: PT. ENDUGO ENZIMES INTERNATIONAL Actual Inventor: Dudi Djuhdia Sastraatmadja Address for Service: DAVIES COLLISON CAVE Patent & Trade Mark Attorneys Level 3, 303 Coronation Drive Milton QLD 4064 Invention Title: "Biofuel production" The following statement is a full description of this invention, including the best method of performing it known to us: C:\NRPonbl\DCC\JXT\3030057_1 DOC - 25/6/10 TITLE OF THE INVENTION "BIOFUEL PRODUCTION" FIELD OF THE INVENTION [00011 The present invention generally relates to the production of biofuel from 5 biomass, including plant matter, waste material, weeds and pests. BACKGROUND OF THE INVENTION [00021 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. 10 [00031 Biofuels are often produced by the 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 15 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. 20 [00041 One of the 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. [00051 In researching biofuel production, the inventor discovered a biofuel 25 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 [00061 The present invention provides a method of producing biofuel, the method 30 comprising: -1- (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 jim; and (c) contacting the second substrate with a biofuel producing enzyme; 5 to thereby convert the second substrate into a biofuel. [00071 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; 10 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, elephant grass or a Casuarina species; most especially water hyacinth; 15 the particle size of the first substrate is reduced by mechanical dehydration; 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 20 substrate; the particle size of the second substrate is < 85 jim; especially S 80 4m; especially 5 75 im; especially : 70 ptm; especially 65 ptm; especially 60 ptm; especially 5 55 pm; especially < 50 ptm; especially < 45 ptm; more especially < 40 pim; the particle size of the second substrate is > 0.5 ptm; especially > 1 jim; especially > 5 25 pim; especially > 10 jim; especially 15 jim; especially > 20 jim; especially > 25 jim; especially 30 jim; more especially > 35 jim; the first substrate is water hyacinth and the particle size of the second substrate is < 40 pim; the first substrate is tapioca waste and the particle size of the second substrate is 30 <l 5 pm; -2the first substrate is sugarcane bagasse and the particle size of the second substrate is S20 ptm; the first substrate is algae or a micro-organism and the particle size of the second substrate is S I tm; 5 the water content of the second substrate is < 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; 10 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-methylbutanal reductases; 15 formaldehyde dismutases; endo-xylanases; p-glucosidases; hemi-cellulases; phosphatidylethanolamine N-methyltransferases; lignin modifying enzymes, and enzymes that act on hexoses, pentoses and disaccharides. 100081 In some embodiments, the method further comprises refining the biofuel; especially by distillation; more especially by distillation and dehydration. 20 DETAILED DESCRIPTION OF THE INVENTION 1. Definitions [00091 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 25 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. 100101 The articles "a" and "an" are used herein to refer to one or to more than one 30 (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. -3 - 100111 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. 5 [00121 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 10 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 15 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 20 [00131 The term "biofuel" 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. [00141 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%, 25 40%, 35%, 30%, 25%, 20%, 15%, 10%, 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 100151 The first substrate comprises biomass. The term "biomass" relates to any 30 material derived from one or more living organisms. This includes any plant matter, such as commercial crops and feedstocks, wood and wood chips, grasses, forest residue (such as dead trees, tree stumps and branches), weeds, fungi, algae, food processing waste stream material, -4including 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, miscanthus, sugarbeet, sorghum, grain sorghum, switchgrass, barley, hemp, kenaf, potatoes, sweet potatoes, cassava, sunflower, 5 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, 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 10 sewage. [00161 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 (a weed). Advantageously, when such weeds are used to produce biofuel, land that would be used to produce other crops may be unaffected. Examples of such weeds include aquatic and semi 15 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 20 an aquatic weed, especially water hyacinth. [00171 In some embodiments, the first substrate comprises biomass that comprises at least 95%, 90%, 85%, 80%, 75% or 70% intact plant cells. [00181 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. 25 [00191 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 [00201 Reducing the particle size of the first substrate to < 90 ptm produces a 30 second substrate. In some embodiments, the particle size is reduced to < 85 jim, < 80 pim, 5 75pm,570pm,S65pm,560 m,555pm,<50 m,<45pmor<40pm. Insome -5embodiments, the particle size of the second substrate is > 0.5 pm, > I pm, > 5 pm, > 10 pm, 15 pm, > 20 pm, > 25 pm, 30 pm, or > 35 pm. 10021] In some embodiments, the particle size of the second substrate is between I pm and 90 pm, between 5 pm and 85 pm, between 10 pm and 80 pm, between 15 ptrm and 5 75 pm, between 20 pm and 70 pm, between 25 pm and 65 pm, between 30 pm and 60 pm, between 35 pm and 55 pm. 100221 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. 10 [00231 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 15 embodiments, the bioavailable surface area to the enzymes is increased a factor of at least 3, leading to a corresponding increase in yield. [0024] 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 20 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. [00251 For example, for water hyacinth (Eichhornia crassipes) the particle size is especially < 40 pm, for tapioca waste (Manihot esculenta) the particle size is especially < 15 25 pm, and for sugarcane bagasse the particle size is especially 5 20 pm. For algae and other micro-organisms, the particle size may be < 1 pm. [00261 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 intra 30 cellular materials more bio-accessible for enzymatic processing and may result in more rapid processing and higher yields. -6- [0027] 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 and the 5 particle size of the biomass would be lower than if sugarcane itself were used. [00281 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. 100291 The particle size of the first substrate may be reduced by various methods or 10 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. 15 100301 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 20 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. 100311 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 25 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. [00321 In some other embodiments, a hammer mill may be used to reduce the 30 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 - 7sufficiently 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. 100331 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 5 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 10 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. [0034] 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 15 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. 20 100351 To obtain a desired particle size for the second substrate a mesh 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 5 90 pm, 5 85 pm, 5 80 ptm, 5 75 pim, 5 70 pm, 5 65 gm, 5 60 pm, 5 55 pm, < 50 pm, 5 45 pm or < 40 pm to pass through the mesh or mesh sieve. 25 [00361 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. 30 [00371 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 - 8under 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. 100381 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 5 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. 100391 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. 10 100401 The second substrate will generally have a lower water content then the first substrate. Lowering the 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 15 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. 100411 In some embodiments, the water content of the second substrate is S 10 %, 20 especially 5 8 %, more especially < 5 %, more especially < 3%, most especially 5 2 %. [00421 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. 25 4.1 Mechanical dehydration 100431 In one embodiment, the particle size of the first substrate is reduced by mechanical dehydration. This means that the 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 ptm. 30 100441 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 -9they 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 5 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. 100451 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 10 substrate has been previously processed, such as for sugarcane bagasse, then the particle size may not need to be reduced prior to drying. [00461 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 15 crusher to further reduce the particle size. [00471 A further example is squeezing the fluids out of the first substrate, reducing the 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. [00481 In these examples, the time required for each step could be determined by a 20 person skilled in the art, and depends upon the biomass being treated and the equipment used. 5. Contacting the substrate with a biofuel producing enzyme [00491 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 25 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. 100501 In some embodiments, the biofuel producing enzyme may include one or 30 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; - 10proteases; ligninases; alcohol dehydrogenases; feruloyl esterases; indole-3-acetaldehyde reductases (NADH); 3-methylbutanal reductases; formaldehyde dismutases; endo-xylanases; p-glucosidases; hemi-cellulases; phosphatidylethanolamine N-methyltransferases; lignin modifying enzymes, and fermentation enzymes to convert hexoses, pentoses and 5 disaccharides to biofuels such as ethanol. 100511 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, 10 Aspergillus awamori, Aspergillus brevipes, Aspergillus candidus, Aspergillus carbneus, Aspergillusflavus, Aspergillusfumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Aspergillus parasiticus, Aspergillus phoenicis, Aspergillus terreus; Bacillus sp., especially Bacillus agaradhaerens, Bacillus amyloliquefaciens, Bacillus cellulyticus K-12, Bacillus circulans, Bacillus lichenformis, Bacillus pumilus, Bacillus sphaericus, Bacillus 15 subtillis; Butyrivibibrofibrisolvens; Candida rugosa; Candida shehatae; Cellulomonas sp.; Chaetomium thermophile; Clostridium jungdahlii; Clostridium papyrosolvens; Clostridium thermocellum; Cellvibriojaponicus; Escherichia coli; Fusarium oxysporum; Geobacillus sterothermophilus; Humicola grisea; Klebsiella oxytoca; Neospora crassa; Pachysolen tannophilus; Paenibacillus macerans; Pichia stipitis; Piromyces equi; Phanerochaete 20 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 thermophilus; Streptomyces sp., especially 25 Streptomyces albaduncus, Streptomyces lividans, Streptomyces olivochromogenes, Streptomyces reticule, Streptomyces rochei; Talaromyces stipitatus; Trichoderma sp., especially Trichoderma citrinoviride, Trichodermafasciculatum, Trichoderma harzianum, Trichoderma koningii, Trichoderma lignorum, Trichoderma longibrachiatum, Trichoderma mobilis, Trichoderma reesei, Trichoderma virens, Trichoderma viride; Torula thermophila 30 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, 403-465. - 11 - 100521 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, 5 generally the pH of the solution in which the treatment occurs may affect the effectiveness of the enzyme. [00531 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 10 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). 15 [00541 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 20 temperature may be between about 40 and 90 'C. 100551 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 25 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. [00561 Advantageously, reducing the particle size of the first substrate, as discussed 30 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. - 12- 6. Refining the biofuel [0057] 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. 100581 For example, an initial refining step may comprise filtering the crude 5 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. [00591 In order that the invention may be readily understood and put into practical 10 effect, particular preferred embodiments will now be described by way of the following non limiting examples. EXAMPLES EXAMPLE 1 - PRETREATMENT [0060] Water hyacinth (Eichhornia crassipes) was collected from infested 15 environmental sources. It was washed to remove adhering soil and debris before draining to remove excess water. [0061] The plant material was then chopped into smaller pieces (-1 0cm) 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%, 20 as determined by Karl Fischer titration over a number of successive batches. [0062] 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 I 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 25 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 5 40 gm, which was determined microscopically. EXAMPLE 2 - ENZYME PREPARATION [0063] Micro-organisms were selected for their ability to secrete extracellular 30 enzymes useful for demethoxylation, decarboxylation, hydroxylation and aromatic ring opening. This included: ligninolytic enzymes, dioxygenases, amylases, lactases, ligninolytic - 13peroxidases, manganese peroxidases, lignin peroxidases, pectinases, cellulases, endoglucanases, glucoamylases, xylanases, pectinases, chitinases, other lignin-degrading enzymes and cellulase-free xylanases. [00641 Separate cultures were growth on Potato Dextrose Slant Agar for 7 days. 5 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 10 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. [00651 The liquid fraction containing the enzymes was strained and the liquid 15 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. [00661 The activity of the enzymatic extract was verified using a refractometer to 20 estimate the concentration of glucose and ethanol via an in vitro assay. EXAMPLE 3 - YEAST INOCULUM [00671 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) 25 slants and stored at 4C. [00681 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 30 100691 Ethanol was produced from 132 kg dried, milled 40 pm Eichhornia crassipes powder in a single 1000 L bio-reactor. The powder was incubated in acetate buffer - 14 - (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. 5 100701 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. [00711 After 48 hours analysis of the reaction fluid broth showed that up to 25.5% 10 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 :S 40 pLm. [0072] 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. 15 [00731 The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety. [0074] 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. 100751 The reference in this specification to any prior publication (or information 20 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. [00761 Throughout the specification the aim has been to describe the preferred 25 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 30 claims. - 15-

Claims

1. A method of producing a biofuel, the method comprising (a) providing a first substrate comprising biomass; (b) reducing the particle size of the first substrate to produce a second 5 substrate with a particle size of 5 90 pm; and (c) contacting the second substrate with a biofuel producing enzyme; to thereby convert the second substrate into a biofuel.

2. The method according to claim 1, wherein the biofuel comprises ethanol.

3. The method according to claim I or claim 2, wherein the first substrate 10 comprises biomass selected from commercial crops, feedstocks, sewage, wood, grasses, weeds, algae and by-products from processes arising from processing commercial crops.

4. The method according to any one of claims I to 3, wherein the first substrate comprises biomass selected from water hyacinth, cumbungi, artichoke thistle, 15 tapioca waste, buffet grass, triticale, sweet sorghum, elephant grass and a Casuarina species.

5. The method according to any one of claims 1 to 4, wherein the particle size of the second substrate is < 80 pm.

6. The method according to any one of claims I to 5, wherein the particle size of 20 the second substrate is < 70 pm.

7. The method according to any one of claims 1 to 6, wherein the particle size of the second substrate is < 60 pm.

8. The method according to any one of claims 1 to 7, wherein the particle size of the second substrate is S 50 pm. 25

9. The method according to any one of claims I to 8, wherein the particle size of the second substrate is < 40 pm.

10. The method according to any one of claims 1 to 9, wherein step (b) comprises drying.

11. The method according to any one of claims I to 9, wherein step (b) comprises 30 mechanical dehydration.

12. The method according to any one of claims 1 to 9, wherein step (b) comprises mechanical processing. - 16-

13. The method according to any one of claims I to 9, wherein step (b) consists essentially of mechanical processing.

14. The method according to any one of claims I to 9, wherein step (b) consists of mechanical processing. 5

15. The method according to any one of claims I to 11, wherein step (b) comprises a combination of mechanical processing and drying.

16. The method according to any one of claims 1 to 11, wherein step (b) consists essentially of mechanical processing and drying.

17. The method according to any one of claims 1 to 11, wherein step (b) consists of 10 mechanical processing and drying.

18. The method according to any one of claims I to 17, wherein the first substrate is water hyacinth and the particle size of the second substrate is S 40 pm.

19. Biofuel when produced by the process of any one of claims I to 18. - 17-