TITLE

Nanocellulose

TECHNICAL FIELD

[000.1] The present invention relates to nanocellulose material, especially nanocellulose material of plant origin. The nanocellulose material is derived from plant sources and may have high hemieellulose content and/or may have a high aspect ratio. The present invention also relates to methods for producing the nanocellulose material.

BACKGROUND ART

[0002] In the last few decades, the use of natural fibres to reinforce polymer composites has been increasing because of their sustainabiiity, renewabi!ity, biodegradability, low thermal expansion, manufacturer-friendly attributes such as low density and abrasiveness, excellent mechanical properties such as very high specific stiffness and strength and consumer-friendly attributes such as lower price and higher performance. A typical natural mi ^' crofibre consists of bundles of nanofibres which in turn consist of several or more elementary (primary) nanoflbrils formed by cellulose chains (a homopolymer of glucose), concreted by/in a matrix containing lignin, hemicellulose, pectin and other components. The diameter of primary cellulose nanoflbrils is typically in the range 3-4 nm. The nanoflbrils consist of monocrystalline cellulose domains linked by amorphous domains. Amorphous regions act as structural defects and can be removed under acid hydrolysis, leaving cellulose rod-like nanocrystals, which are also called whiskers, and have a morpholog and crystallinjty similar to the original cellulose fibres. Depending on the source of cellulose, the cellulose content varies from 35 to 100%. These fibres, isolated in their primary nanofibrillar form exhibit extraordinarily higher mechanical properties (stiffness/strength) than at the microscale (as bundles of nanofibres) or in their natural state, In recent years, these nanocrystalline cellulose fibres have been explored as biologicall renewable nanomaterials that ca be applied in several engineering applications. While numerous methods have been explored for the production of microfibrillated cellulose (MFC), which by definition (Reference: Robert J. Moon, Ashlie Martini, John Nairn, John Simonsen and Jef Yoimgblood, 'Cellulose nanomaterials review: structure, properties and nanocomposites' Chem. Soc. Rev., 201 1,40, 3941-3994), consists of cellulose fibres with diameters in the range of 20-100 nm and a length in the range between 0.5 ηι and tens of microns, the production of nanofibri Hated eellulose (NFC), and eellulose nanocrystals (CNCs) is more challengin due to the requirement to separate or deco struct the eellulose fibres and/or crystals to a much greater degree. Attempts to date to produce these two types of nanocellulose (CNCs and NFCs) have focussed on the use of chemical, physical, mechanical and enzymatic steps as pre~treatments between conventional pulping processes and final mechanical defibrillation processing alone or in combinations thereof. For NFC, the prior art refers to a fibre diameter in the range of 3-20 nm and a lengt in the range between 0.5 and 2 μηι. These nanofibrils can be further made up of primary cellulose nanofibrils typically having a diameter of 3-4 mn. For example, a cellulose nanofibril wi h a diameter of 10 nm may consist of a bundle of a few primary cellulose nanofibrils with 3-4 nm diameter. For CNC, the prior art refers to fibre/crystal diameters/widths in the range of 3-20 nm and lengths of up to 500 nm. (except the special example of tunicate CNCs or t-CNCs, which have a higher aspect ratio),

[0003] A typical procedure for isolating nanocrystals of cellulose relies on acid hydrolysis using corrosive acids (like H2SO and HCl), followed by centrifugmg, dialysing, u!trasoni cation and drying (a typical flowchart showing this process is shown in Figure 1). Depending on the cellulose source and hydrqlytic conditions, cellulose nanocrystals (CNCs) with the diameter range of 3-15 nm and length in the range of 50-5QO nm are isolated. Some of these products are produced at semi-commercial scale (e.g. Ϊ tonne per day) using wood fibres as the ra material. High aspect ratio cellulose nanocrystals (with a aspect ratio of 65-100) can be obtained from rare marine animals called tunicates (urochordates), but this is not a commercially viable o sustainable route. Therefore, the sustainable production of nanocrystals that are of a higher aspect ratio, or closer to that of CNCs derived from tunicates (t-CNCs), and doing so from plant source materials, remains a challenge.

[0004] For isolatio of microfibres, which are called microfibrillated cellulose (MFC) with diameters i the range of 20-100 nm and length in the range of 0.5-10' s urn, mechanical methods such as ultrasomcation, homogenisation, milling, grinding, cryocrushing, or combinations of these are widely used to deftbrillate the macroscale bleached pulp fibres into MFC fibrils which essentially consist of bundles of nanofibrils. In order to further refine and separate the MFC into its constituent nanofibrils and to isolate these further thinner particles called nanofibri Hated cellulose (NFC) or cellulose nanofibrils (CNF), with diameters in the range of .3-20 run and lengths in the range of 500-2000 nm, a significantly larger amount of mechanical energ typically needs to be applied than that required to refine material to the microfibrillar level. In reported methods, additional chemical or enzymatic pre-treatments applied after pulping and bleaching but prior to mechanical processing are usually claimed to be beneficial for reducing both mechanical energy consumption and resultant nanoflbre diameter, as the chemical agents can aid in the removal of matrix materials such as lienin and hemi cellulose that bind the fibres together. Figures 2A and 2B compare two typical procedures used in the art for producing MFC and NFC, respectively.

[0005] Del ignifi cation and bleaching are chemical processes widely used in the paper manufacturing industry and are key steps in the pulping process.

[0006] When a large amount of mechanical energy is applied to a cellulosic feedstock or the cellulose is exposed to harsh chemical pre-treatments, the cellulose fibres can be prone t breakage, thereby reducing their length and aspect ratio. Therefore, the production of nanocellulose is typically governed by a delicate balance between the requirement to input sufficiently large amounts of energy in order to isolate the nanofibres and the propensity of this large amount of energy to break fibres, thereby reducing their length and aspect ratios. Consequently, efforts to manufacture nanocellulose at commercial scale have been hindered by the high cost introduced by these additional processing steps and the challenge of avoiding fibre breakdown during processing. In manufacturing nanocellulose, mechanical processing is typically performed by passing a cellulosic feedstock through a mechanical processing step a number of times to facilitate the gradual breakdown of the cellulose to its nanoscale fibrils. For example, cellulosic feedstock material may be passed through equipment such as a homogeniser or disc refiner several times or more before the cellulose is sufficiently separated that predominantl nanofibre are yielded. In a commercial process, this requirement to pass the material through the same step multiple times can result in high energy costs and long processing times, reducing the commercial attractiveness of the process. Some examples of typical processing conditions disclosed in the patent literature for producing cellulose nanofibrils, including the number of passes through a particular mechanical processing step are set out in Table 1 below:

Table 1 : Different mechanical methods for the production of cellulose nanofibrils

Reference Patent Methods Comments

US 4374702 Temperature assisted Obtained type: MFC (no

homogeni sation (8-20 fibre diameter is reported) passes)

US 6183596 & US 6214163 Rubbing (shear) MFC (no fibre diameter is

Supergrinding reported)

High pressure

hom ogen i sation Reference / Patent Methods Comments

US 7381294 & WO Double dis refiner MFC was produced with

2004/009902 (shearing) (up to 80 passes) diameter >0. Ι ήι after 15

passes

US 5964983 Alkaline pre-treatment and NFCs are produced , after

acid hydrolysis coupled with grinding or homogenising (8- screening or homogenisation 10 passes) after acid

hydrolysis at 60- tOO °C

WO 2007091 42 Enzymatic pre-treatment and NFCs are produced after 5

high pressure passes

homogenisation

US 2008/0057307 & US Low shear refining followed NFCs are produced after 7 7566014 by high shear refining or passes

homogenisation

WO2012/097446 & US Double disc refiner NFCs are produced after 8 201 1/0277947 passes

[0007] Some processes for the manufacture of cellulose nanofibrils use a chemical pre- treatment called TEMPO oxidation in which a cellulose pul is exposed to TEMPO [(2,2,6,6- Tetramethylpiperidin-l-oxyl (CAS No,: 2564-83-2), This pre-treatment loosens the nanofibrils, making it easier to defibrillate the fro each oilier in subsequent mechanical processing. TEMPO processing enables 3-4 nm diameter nanofibrils to be obtained, however TEMPO agents are expensive and toxic, making their use and disposal difficult In addition, the use of TEMPO agents results in conversion of the surface of the nanofibrils from one dominated by hydroxy! groups to one dominated by carboxyl groups. This can be a disadvantage when modificatio of the cellulose surface chemistry for some applications requires a hydroxy lated surface.

[0.008] Literature published before 2011 tends to use the terms MFC and NFC interchangeably, with these terms being used for both nanofibrils and microfibrils. In this specification, we distinguish between MFCs and NFCs, using the definitions given by Moon et. al. Chem Soc, Review' 201 1, Throughout this specification, the terms "MFC" (microfibriliated cellulose) and "C'Ml ^" (cellulose microiibre) are used to describe fibrils, including bundles of nanofibrils, with a diameter above 20 nm and length in 10s of microns. The terms "NFC ^'* (nanotlbrillated cellulose) and "CNF" (cellulose nanofibre) are used to describe nanofibrils having a diameter between 3 to 20 nm. The NFCs obtained by the present invention are significantly longer than NFCs described in the prior art and may have a length above 500 nm up to 7 microns or longer. The terra "CMC" is used to describe cellulose nanocrystals, which are rod-like or whisker shaped particles that are typically produced after acid hydrolysis of bleached pulp, MFC or NFC. CNC.S with a high aspect ratio (3-5 nm diameter, 50-50.0 nm in length), are essentially 100% cellulose and are highly crystalline (54-88%). The CNCs obtained via acid hydrolysis in the present invention are longer (up to 1.5-2 microns or longer) than CNCs obtained in the prior art.

[0009] Commercial nanocellulose production largely uses wood as a source of cellulose due to wood's abundance, availability in commercial quantities and given that much of the development of nanocellulose has been supported by the forestry industry, motivated by a desire to find new applications for wood,

[0010] In one aspect, the present invention relates to producing NFC with lowest possible energy (that is generall used for MF production). As stated in paragraph [0002] of this specification, production of NFC and CMC (cellulose nanocrystals) is more difficult than production of MFC due to the requirement to separate or deconstruct the cellulose fibres to a much greater degree. This typically results in the cellulose fibres being broken, resulting in. the length of the fibres becoming significantly shorter and thus reducing the aspect ratio of the fibres.

[0011] In general, the prior art discloses that manufacturing processes requiring high energy input, disadvantaged by clogging problems during mechanical processing, complex recovery methods, harsh chemical treatments and/or high energ mechanical treatments are required to produce nanocellulose materials.

[0012] It will be clearly understood that, if a prior art publication is referred to herein, this reference does not constitute an admission that the publication forms pari of the common general knowledge in the art in Australia or in any other country.

SUMMARY OF INVENTIO

[0013] The present invention is directed to nanocellulose material of plant origin that may have a high hemi cel lulose content and/or may have a high aspect ratio.

[0014] With the foregoing in view, the present invention in one form, resides broadl in a nanocellulose material of plant origin comprising nanocellulose particles or fibres derived from a plant material having a hemi cellulose content of 30% or higher (w/w). [0015] Plant materials are composed of many different substances including waxes and resins, ash and lignocellulosic components, Lignocellulosic components are the major components of plant matter and comprise lignin. cellulose and hemicellulose. Throughout this specification, the hemicellulose content of materials is discussed in percentage terms. In all cases, the hemicellulose content is quoted as a mass percentage of the total mass of the lignocellulosic components only of the relevant material,

[0016] In a second aspect, the present invention provides a nanocellulose material of plant origin comprising nanocellulose particles or fibres having an aspect ratio of at least 250.

[0017] In another embodiment, the present invention provides a nanocellulose material of plant origin comprising nanocellulose particles or fibres derived from a plant material having a hemicellulose content of 30% or higher (w/w) and having an aspect ratio of at least 250.

[0018] The present inventors have surprisingly found that plant material derived from a plant material having a hemicellulose content of 30% or higher (w/w) could be separated into nanofibrils or nanocrystals using significantly less harsh or less energy intensive treatments than known ^' in the prior art. In one embodiment, the plant material is derived from a plant material having C4 leaf anatomy.

[0019J In another embodiment, the present invention provides a nanocellulose material derived from plants having C4 leaf anatomy comprising nanocellulose particles or fibres having an aspect ratio of at least 250. Throughout this specification,, the term "aspect ratio" is used to refer to the ratio determined from the maximum dimension of the nanocellulose particle divided by the minimum dimension of the nanocellulose particle. For nanocellulose fibres, the aspect ratio is determined b dividing the average length of the fibre by the average diameter of the fibre. The average diameter of water-washed, deligmfled, bleached, chemical and mechanical treated fibres was determined using digital image analysis (Image J), For each sample, 250 measurements of diameter were randomly selected and measured from several TEM images with the same magnification. For measuring the length of spimfex water-washed delignified, and bleached fibres, and also short cellulose nanocrystals, digital image analysis (Image J) was used. For measurement of the length of long and curly spinifex NFC two different methods were employed; (a) cryo-TEM, a 3D tomography, and (b) measurement from TEM images by

AutoCAD software. Cryo-TEM was carried out using a plunge freezing protocol. Here, 4 μΙ_ of NFC dispersion in water was transferred onto TEM holey carbon grids (C-flat and laeey carbon), in an FEI Vitrobot Mark 3 (FEI Company, Eindhoven, the Netherlands), while the chamber was set to 100 % humidity at room temperature (-22 °C). Optimal blot time was 3 - 5 seconds, and then the sample was plunged into li quid ethane. The frozen/vitrified samples were viewed on a Tecnai F30 TEM (FEI Company) operating at 300k V, and imaged at 23,000x magnification with a Direct Electron I 1 100 4k x 4k camera (Direct Electron, San Diego, United States), using low-dose mode of Serial EM image acquisition software. The reason that samples were subjected to low-dose conditions is the extreme sensiti vity of the unstained cellulose nanofibres to beam damage. This- consisted of using spot size five, making focus and exposure adjustments outside of the image capture area, creating a map of grid locations at very low magnification where area selection was based on the quality of vitreous ice rather than sample morphology, and

performing subsequent high magnification imaging via. an automated batch imaging function in SerialEM, where the total electron dose was limited to 130 electrons/A ² or less. The tilt range was +/-60 ^<> with an increment of~1.5 ^'9 to 2.5°.

[0020] For Image processing and analysis 125 2D images were captured in this instance and the raw image data was then processed using IMOD processing and modeling software. This program allows contours to be manually drawn following the non-linear path of each cellulose nanofibril. i xy space and contains tools for the subsequent calculation of contour length.

[0021] In one embodiment, the nanocellulose particles or fibres have an aspect ratio of between 250 to 10,000, or between 250 to 5000, or between 250 to 1000, or between 260 to 1000, or between 266 to 1000, or between 266 to 958.

[0022] The nanocellulose material preferably comprises cellulose nanocrystals (CNC) or nanofibril] ated cellulose (NFC).

[0023] In some embodiments, the range of the aspect ratio of the nanocellulose particles or fibres has a lower limit of 250, or 266, or 280, or 300, or 400, or 500, In some embodiments, the upper range of the aspect ratio of the nanocellulose particles or fibres is 10,000, or 5000, or 4000, or 3000, or 2000, or 1000, or 958, or 800, or 700, or 600, or 550,

[0024] The nanocellulose particles or fibres may have a diameter of up to 20nm, or up to ISnm, or up to l nm, or up to 8nm, or up to 6nm, or up to 5nm, In one embodiment, 250 individual measurements of fibre diameter were made and the following results were obtained: 1-2 nm.: 3 1 measurements, 2-3 nm: 90 measurements, 3-4 ran: 127 measurements, 4-5 nm: 1 measurements, 5-6 nm:5 measurements This is shown in Figures 6A & 9.

[0025] It will be appreciated that the fibre diameter and aspect ratio values of any given sample of nanocellulose of the present invention will be composed b a distribution of values where the value quoted approximately represents an average of values for different fibres in a sample.

[0026] The nanpcellulose particles or fibres may have a length that falls within the range of

[0027 j The nanocellulose material of the present invention is of plant origin and therefore is derived from plant sources. In one embodiment, the nanocellulose of the present invention is derived from plant material in which the amount of hem ί cellulose in the plant material is greater than the amount of lignin in the plant material.

[0028] In one embodiment, the plant feedstock used in the production of nanocellulose of the present invention has a hemicellulose content of at least 30 %, In some embodiments, the plant material has a hemicellulose content of from 30 to 55% w/w, or from 30 to 50% w/w, or from 36 to 48% w/w, or from 40 to 48% w/ or from 42 to 47% w/w, or any intermediate range within the ranges set out above.

[0029] In one embodiment, the plant material is derived from a grass species having C4-leaf anatomy. The present inventors believe that any plant materials from grasses having C4-leaf anatomy can be used to produce the nanocellulose material (NFC or CNC) in accordance with the present invention. Such plants can also be treated -with the low energy method or gentle chemical method described in this specification to produce the nanocellulose material.

[0030] In one embodiment, the plant material is derived from a drought-tolerant grass Species.

[0031] In one embodiment, the plant material is derived from arid grass species,

[0032] In one embodiment of the present invention, the plant material is derived from Australian native arid grass known as ''spinifex". Spinifex (also known as 'porcupine' and 'hummock' grass) is the long-established common name for three genera which include Triodi , Monodia, and Sympkctrodia (not to be confused with the grass genus Spi ifex that is restricted to coastal dune systems in Australia). Hummock grassland communities in arid Australia are dominated by spinifex species of the genus ' Ttiodkf . There are 69 described species of Triodia, which are long-lived and deep rooted allowing root growth to penetrate through tens of metres under the ground. Of the 69 species, abundant species are two soft species called T. ptmgens, T. .sMnzii and two hard species T, b s dowu. T. iongiceps. T. ptmgens has a typical composition of: cellulose (37 %), hemicellulose (36 %), lignin (25%) and ash (4 %) in the un-washe form, such that hemicellulose content makes up 37 % of the iignocellulosie content. [0033] In another aspect _s , the present invention provides a nanocellulose material produced from plant material, derived fro arid spinifex,

[0034] Without wishing to be bound by theory, the present inventors believe that, in most plant sources of nanocellulose, the cellulose molecules are biosynthesized to form long elementary fibrils. When these lon fibrils are tightly packed with either covalently or secondary bonding, the required amount of energy to separate the fibres may be higher than if the fibrils had been packed less loosely. With high energy (both mechanical and chemical) pre-treatments, the aspect ratios of these long fibrils might be decreased during the harsh processing required to isolate these tightly packed fibrils. In the case of Trkidia grass, the elementary fibrils might be loosel packed thus facilitating easier defibrillation. This can be explained with our results and prior art,

10035] Again without wishing to be bound by theory, the present inventors believe that the defibrillation of this grass is easier due to a combination of the following (1) structural morphology (loosely packed bundles of fibrils in the primary cell walls), (2) higher, hemicelluiose content (which is common in plants with C4 leaf anatomy), which ensures lower content of cementous iignin and pectin and (3) low energy pre-treatments,

[0036] In general, the cellulose fibrils that are bundled in the primary cell wall, are surrounded by hemiceUuloses and pectins. The peculiar behaviour of spinifex can be traced back to the particular morphology of spinifex which mainly consists of parenchyma tissue i the primary wall. These walls are rather fragile, as the cellulose fibrils are organized in a relatively looser network embedded in an abundant matri consisting of hemiceUuloses and pectin, while ^■ the common secondary cell walls are much stronger, due to the presence of tightly packed cellulose microfibrils with Iignin. This relatively looser arrangement and high content of hemicelluiose may be a strategy of the plant to help it retain water during periods of drought (hemicelluiose is mainly responsible for moisture absorption and retention in the plant fibre). Because of slack fibrillar interaction with the matrix in the primary cell wall, fibrils can easily be separated from one another by mechanical treatment. Cross-section SEM images of T. Ptmgens (see Figure 3) show that the bundles of cellulose microfibrils are separated by nodular structure on the surface, ensuring the slack assembly of fibres. The high hemicelluiose content may also assist in separation of fibres since hemicelluiose imparts a negative charge on the surface of the fibres, such that opposing negatively charged fibres repel each other.

[0037] Even after mild delignification, the structure of stroma lamellae is preserved and this provides a good dispersabilify of fibres in water. [0038] Similar to other grasses, spiniiex has several cell types in the leaf epidermis including epidermal cells and stomata, resin producing cells (soft species only), fibre, mesophyll, vascular tissue, multi -cellular hairs and unicellular papillae. Spi iiex grasses exhibit a 'modified C.4 leaf anatomy' which possesses two types of cells viz, outer mesophyll cells and inner spong bundle sheath ceils arranged in a circular manner like a necklace. In Triodia, the bundle of sheath cells seems to be extended beyond the vascular bundle and surrounded by mesophyll tissue (photo synthetic parenchyma cells that lie between the upper and lower epidermis layers of a leaf)- Triodia pimgens possesses a higher percentage of mesophyll tissues that ar predominantly found in the primary wall . So we believe that this structure of spinifex enables the cellulose fibrils to be "deconstructed" more easily without applying harsh treatments which cause to damage the fibrils and results in fibres with a shorter average length.

[0039] Example plants with C4 leaf anatomy that may be used in the present invention include Digitaria sanguinalis (L.) Scopoii, Pamcum coloration L, var. makarikariense Goossens, Br chiaria hriscmtha (Hochst Ex A. Rich) Stapf, D. vialascens Link, P. dichatatniflorum

Michaitx, 8, decumhem Stapf, Echinoch a crus-galli . Beam-,, P, mitiaceum L, 3. humidicola (Rendie) Schweiek , Paspakm distkhum L, B. mutica (Forsk) Stapf, Seiaria glauca (L) P. Beam', Cynodon daet lan (L.) Per soon, Panicum maximum Jaeq., S. viridis (L) P. Beam*, El usim coramna (L.) Gaerimr, UrocMoa texana (Buckley) Webster, Sorghum

sudaneme Stapf, K indica (L.) Gaerimr, Spodmpogon cotidif r (Thunb.) Haekel, Eragrostis ciikviensis(A!lioni) Vignolo-Ltitati, C Moris gay ana Ktmth, Eragrostis citrvida, Lepf loa dubia, Muhlenhergia wrightii, E. ferrnginea (Thunb.) P. Beauv., Sporobotus indicits R Br, var urpureo-mffusus (Ohwi) T. Koyama, Andropogon gerardii, Leptochioa chi nsis (L) Nees, grasses of the Miseanihus genus (elephant grass), plants of the genus Salsol including Russian Thistle, ricestraw, wheat straw, and corn stover, md Zoysia temttfolia Wittd.

[0040] Since the Triodia grasses are grown under arid conditions, the present inventors believe that other arid grasses that grow in Australia and other parts of the world may al so be used i the present invention. The most drought tolerant grass genera, in Australia, (though they need water in their first 1. or 2 years) include Anigozanthos, Austrodanthonia, Austrostipa, Baloskion pallens, Baumea juncea, Bolboschoenus, Capillipedium, Carex bichenoviana, Carec gaudiehaudiana, Carex appressa, C.tereticaulis, Caustis, Centrolepis, Chi oris truncate, Chorizandra, Conostylis, Cymbopogon, Cyperus, Desmocladus fiexuosa, Dichanthium sericeum, Dichelachne, Eragrostis, Eurychorda complanata, Evandra aristata, Firinia nodosa, Gahtiia, Gymnoschoenus sphaerocephalus, Hemarthria uncinata, Hypolaeana, Imperata cylindrical, Johnsonia, Joycea pallid, J uncus, ingia australis, Lepidosperm a, Lepironia. arti culate. Leptocarpus, Lomandra, Meeboldina, Mesoraeiaena, Neurachne alopecuroidea, Notodanthonia, Patersonia, Poa, Spini ex, Theraedo triandra, Tremulina tremula, TrigJochin. Triodia and Zanthorrhoea.

[0041 ) Arid grasses that grow in other parts of the world that may also be used in the present invention include Aristida pollens (Wire grass), Andropogon gerardii (Big bluestem), Bouieloua eriapoda (Black grama), Ch!oris mxburgkkma (Horsetail grass), Themeda triandra (Red grass), Panicum virgatum (Switch grass), Petmis tum ciliaris (Buffel grass), Schimchyrhtm scoparium (Little bluestem), Sorghatrum nutans (Indian grass), AmmophUa arenaria (European beach grass) and Stipa t iacissima (Needle grass),

[0042] The present inventors have also discovered that nanocellulose particles or fibres can be produced from species of spinifex using methods that general iy involve less harsh chemical treatments and/or less energy intensive mechanical treatments than have been used in the processes disclosed in the prior art. This has apparent beneficial implications in respect of the consumption of chemicals and energy consumption in the manufacture of the nanocellulose particles or fibres.

[0043] In another aspect, the present invention provides a method of producing nanocellulose particles or fibres from plant material derived from plant having a hemicellulose content of 30 % (w/w) or higher comprising the ste s of deli giiifi cation and optionally bleaching the plant material, followed by separating the plant material into nanofibrils or natiocrystals, wherein the step of separating the plant material into nanofibrils or nanocrysials is selected from: a) a low energy mechanical separation; and/or b) a mild chemical treatment step.

[0044] Importantly _. , the method of the present invention does not require the use of a pre- treatment step after the pulping steps of del igmfi cation and optionally bleaching and before the step(s) carried out to separate the pulp into constituent nanofibrils,

[0045] The pulping steps of delignification and bleaching are well known to those skilled in the art of paper manufacture,

[0046] With regard to plant material derived from arid spinifex, it seems that the hemicellulose (especially xylars) remained associated with the microfibrils after delignification chemical treatments (based on N MR results), and this is likely to be responsible for the easy cell wall disruption during a subsequent mechanical treatment, and also for the specific properties of the spinifex cellulose microfibrils when they are homogenized or milled and suspended in water.

[0047] In one embodiment, the plant material having a lesser amount of lignin than beraicellulose comprises a plant material derived from arid grasses. In another embodiment, the plan material having a lesser amount of lignin than hemicellulose is derived, from Australian arid grass spinifex of the genus Triodia. In a further embodiment, the plant material comprised plant material from Triodia pi ge .

[0G48J In one embodiment, the plant material having a hemicellulose content of 30 % (w/w) or greater comprises a plant material derived from arid grasses. In another embodiment, the plant material having a hemicellulose content of 30 % (w/w) or greater is derived from Australian arid grass spi nifex of the genus Triodia, In a further embodiment, the plant material comprised plant material from Triodia pungem.

[0049] In one embodiment, the low energy mechanical separation comprises homogemsation of the plant material by subjecting the plant material to 5 passes or less, or 3 passes or less, preferably 2 passes or less, through a homogenisation step. The hom ogenisati on step may comprise passing the material through a high pressure homogenizer.

[0050} Treating fibres with a homogeniser typically comprises submitting a dilute fibre suspension in water to a homogeniser in order to del animate fibres. Applying a large pressure drop under high shear forces and impact forces,, for example, against a valve and a ring leads to splitting or fibrillating the fibres into nanofibrils. In order to increase the degree of fibrillation, prior art processes cycled the fibres through a high pressure homogenize using approximately 10-30 passes. It is obvious that with increasing the number of passes, the required energy for fibrillation is markedly increased. The other disadvantage of using a homogenizer to cause fibrillation is that "system clogging" by the long fibres is a common problem which requires disassembly of equipment and operator time to unciog. In contrast, in the present invention, passing the fibres through the homogeniser 5 times or fewer has been found to be sufficient to produce nanocelluiose particles or fibres such that the occurrence of clogging is lessened. Others have reported as few as 5 passes. The present inventors have successfully produced NFC of the present invention using as few as 1 single pass through a homogeniser. The present inventors have used a pressure as low as 150 bar, where others have generally required 500 to 2500 bar. Surprisingly, the present inventors were able to produce nanofibrils of the present invention from spinifex using both the lowest pressure and fewest number of passes in combination, when compared to prior art processes. [0051 j For experiments conducted by the present inventors, the range of applied pressure of homogenizer is 150-1500 bar and the number of passes is in the range of 1- 15,

[0052] The minimum applied pressure in our work was 15.0 bar. In some experiments conducted by the present inventors, using a pressure of 150 bar and I -3 passes through the homogenizer resulted in production of NFC with 3-5nm average fibre diameters. In. a .further embodiment, the range of applied pressure is 200-700 bar or preferabl 250-650 or preferably 300-600 bar or more preferably 350-550 bar.

[0053] h another embodiment, the low energy mechanical separation step comprises bead milling, ball milling, disk-rotator or stator refining, cr o-crushing, steam explosion, grinding, refining, high intensity ultrasonic, microf!uidisation, high shear processing such as that performed with Silverson type mixers, processing with other rotor-stators or micronizmg or a combination of these. Other separation methods known to those skilled in the art may also be used.

[0054j Any combination of mechanical processing treatments described above may he used to process the eellulosic pul to nanocellulose. For example, if high pressure homogenisation is used to process the cellulose to the final nanofibril product, the pulp material may first be passed through a Silverson type mixer to partially fibril] ate the cellulose bundles, allowing even lower energy processing in a high pressure homogeniser had the Silverson processing not been performed. In doing so, the cellulose bundles are partially fibrillated i the first processing step and then fibrillation is complete in the second processing step. In cases where there is a cost difference between two mechanical processing methods that can be used i fibrillation, the use of a low cost processing step (such as Silverson or rotor-stator fibrillation) can reduce the amount of energy or processing time required in a subsequent and more expensive step such as high pressure homogenisation, lowering the processing cost overall. The first mechanical step can also act to pre-homogenise the pulp before processing at the second mechanical step, reducing the chances of cloggin problems during second stage processing which lead to equipment downtime.

[0055] hi a further embodiment, the mild chemical treatment comprises an acid hydrolysis step conducted using an acid solution having an acid concentration below 45%, the acid hydrolysis step being conducted at a temperature below 50°C. In one embodiment, the acid hydrolysis step may be conducted at an acid concentration of about 35% to 40% and a temperature of about 45°C. The acid may comprise sulphuric acid, hydrochloric acid or any other suitable acid. Typically, with other nanocellulose production methods, harsh chemical treatment conditions are required in order to sufficiently separate the cellulose fibres into fibres with nanometre scale diameters. ^' However, such harsh conditions also tend to cause breakage of the fibres such that there lengths are reduced. In doing so, the production of high aspect ratio cellulose nanocrystals and/or nanofibrilJated cellulose has been challenging. The abov combination of especially mild acid concentration and treatment temperatures has not previously been reported as being suitable for obtaining nanofibres or nanocrystals of cellulose with the aspect ratios of the present invention,

[00561 The present inventors also added a harsh ultrasoni cation treatment after acid hydrolysing (70% amplitude for 20 minutes), and yet the spinifex nanofibres did not break to shorter fibres and still retained a high aspect ratio. The average diameter of acid treated fibres was 4±1.4 nni

|005?j In other embodiments, the plant material may be separated into nanofibrils or nanoparticles using carboxymethylation, or 2,2,6,6-tetram.ethylpiperidinyl-t-oxyl (TEMPQ)- mediated oxidation or enzyme-treatment. lit another embodiment, a steam explosion process may be used for converting lignocellulosic biomass with the final aim of separating nanofibrils

[0058] It is preferred that the plant material is subject to pulping in which the plant material is pulped by deiignifi cation and optionally bleaching prior to separating the plant material into nanofibrils or nanocrystals. Pulping steps of deiignifi cation and bleaching are commonly used and a skilled perso would readily understand that there are a number of deiignifi cation steps and bleaching methods that may be used in the present invention.

[0059] Delignification ma be achieved by contacting the plant material with alkaline reagents such as sodium hydroxide or potassium hydroxide, or by contacting the plant material with organic solvents such as ethanol, acetone, toluene and/or methanol, typically at elevated temperatures and possibly elevated pressures. Bleaching will typically involve contacting the plant material with an oxidising agent such as peroxide, sodium chlorite or sodium hypochlorite, often i n the presence of other chemical s and at el evated temperatures.

[0060] Following harvesting, the plant material ma be subject to a size reduction step, such as by chopping or grinding the plant material to obtain particles of plant material having a longest dimension of less than 10 mm, prior to pulping. This step makes the long grass easier to process by enhancing flowability but is not considered essential to the production of nanocel!ulose of the present invention. [0061] The plant feedstocks used to produce nanocellulose according to the present invention may have a high hemicellulose content, of 30 % or greater. In such cases, this high hemicellulose content may be also observed in the resulting nanocellulose materials. Accordingly, one embodiment of the inventio consists in nanocellulose materials which have a hemicellulose content of 30 % (w/w) or greater. Whereas cellulose is a strong and crystalline material, hemicellulose has an amorphous structure with little strength. Within plants, hemicellulose exists between adjacent cellulose fibrils and providing some binding capacity, acting as a glue. Without being limited by theory, the inventors believe that the high hemicellulose content in the nanocellulose of the present invention may contribute to the observed flexibility of the cellulose nanofihrils and nanocrystals and the high toughness of the nanocellulose and interlocking networks formed from the nanocellulose such as those found in paper sheets made from the nanocellulose of the present invention . Here, hemicellulose may be acting as a glue or cross-linker between cellulose fibres or crystal s, increasing the toughness and energy required to bring about mechanical failure of the material.

[0062] The different processing steps involved in the manufacture of the nanocellulose of the present invention may cause a change in the hemicellulose content of the material as it moves through chemical and mechanical processing steps. In general, hemicellulose content may be altered during delignification and bleaching steps however mechanical processing is less likely to cause a change in hemicellulose content. As a consequence, the hemicellulose content of the nanocellulose final product can be the same as the processed pulp prior to mechanical processing.

[0063] Accordingly, another embodiment of the present inventio consists in nanocellulose with a hemicellulose content of 30% (w/w) or greater, hi some embodiments, the nanocellulose has a hemicellulose content of from 30 to 55% w/w, or from 35 to 50% w/w, or from 37 to 48% w/w, or from 38 to 46% w/w or from 40 to 44% w/w, or any intermediate range within the ranges set out above.

[0064] In another embodiment, the present invention consists in nanocellulose with- a hemicellulose content of 30 % (w/w) or greater and an average fibre or particle diameter of 10 nm or lower. The average diameter of the nanocellulose fibres or particles may be within the ranges given in paragraph [0023] above. The aspect ratio may be greater than 250 and within the ranges given in paragraph [0020] or [0022] above. [0065] Table 2 below compares the chemical composition of NFC materials obtained from a. number of different plant feedstocks and using different processing routes as reported in the scientific literature.

Table 2, Comparison chemical composition of NFC obtained from different sources and treatments of cellulose.

Source of Mechanical NFC FC Cellulose Heraicellulose Ligiiin Ref cellulose treatment

Diameter (%) (%) (%)

(tint)

Bamboo fibre Ultrasonication 30 - 80 93.3 6.7 0.1 1

Sugar-beet Ultrasoni catio 2 -15 80.33 6.9 7 pulp and

homogenization

Sugar beet Horn ogeni zati on 20 - 70 82.83 7,01 0 3 pulp

Abaca Homogenization 20 86,84 13.5 - 4

Hemp Homogenization 30 - 50 91.50 8.51 - 4

Bleached Ultrasonication 1000 - 80.35 13.52 0.20 5 eucalyptus and

5000

fibre homogenization

Softwood TEMPO- 3-4 90 10 6-8 bleached kraft oxidation and

pulp ultrasonication

homogenization

Bleached Disc refiner , 20-50 85.2 13.9 0.1 9 eucalyptus knife mil] and

pulp soni cation

Empty Palm PFI mill and 75,8 6.2 9 10 Fruit Bunch microflouidizati

Fibers on

Wheat straw Cryoerushing 10-80 84.6 6 9.4

and

homogenization

Wood pulp Masuko grinder 4-5 41.8 26.9 30.6 11

Holocellulose Masuko grinder 10-20 77 21.4 0.1 n pulp

Alkali treated Masuko gri der 10-20 82.5 17.4 0.1 1 1 pulp Source of Mechanical NFC/MFC Cellulose Hemicellulose Lignin Ref cellulose treatment

Diamete (%) (%) (%)

(am)

70% TEMPO- 78, 1 22 12

Eucalyptus oxidation and

nitens and homogenization

30%

Eucalyptus

globulus

Pi us Radiata TEMPO- 87.8 12.2 12 fibres oxidation and

hornogenization

References of Table 2.

1. Chen, W.; Yu, H. ; Liu, Y Carbohydrate Polymers 2011, 86, (2), 453-461.

2. Agoda-Tandja a, G.; Durand, S.; Berot, S.; Blassel, C; Gaillard, C; Gamier, C;

Doublier, J. L Carbohydrat Polymers 2010, 80, (3), 677-686.

3. Li, M,; Wang, L.-j .; Li, D ; Cheng, Y.-L,; Adhikari, B. Carbohydrate Polymers 2014, 102, (0), 136-143.

4. Alila, S,; Besbes, I,; Vilar, M. R.; Murje, P.; Boufi, S. Indmtiial Craps and Products 2013, 41, (0), 250-259.

5. Urnxzola, I.; Serrano, L,; Llano-Ponte, R.; Angeles de Andres, M,; Labidi, J. Chemical Engineeririg Journal 2013, 229, (0), 42-49,

6. Qua, E. H.; Hornsby, P. R ; Sharma, H. S. S.; Lyons, G. Journal of Materials Science 2011, 46, (18), 6029-6045. ^'

7. isogai, T.; Saito, T.; Isogai, A. Cellulose 201 1 , 18, (2), 421 -431.

8. Fukuzumi, H ; Saito, T.; Iwata, T.; uraamoto, Y.; Isogai, A. Biomacromolecitks 2008, 10, (1), .162-165.

9. Toftoii, G. H. D.; Teixeira, E, M,; Correa, A. C; Marconcmi, 3, M.; Caixeta, L. A,;

Pereira-da-Silva, M. A.; Mattoso, L, H. C. Carbohydrate Polymers 2012, 89, (1), 80-88.

10. Ferrer, A.; Filpponen, I,; Rodriguez, A.; Laine, J.; Rojas, O. J . Bioresource Technology 2012, 125, 249-255.

11. I amoto, S.; Abe, K.; Yano, H. Biomacrotnokcules 2008, 9, (3), 1022-1026.

12. Spence, K, L; Venditti, R. A.; Rojas, O. J.; Habibi, Y.; Pawlak, J, J. Cellulose 2011, 18, (4), 1097-1 1 1. [0066] Hero i cellulose content and the content of other lignoceUulosic components was measured b chemical analysis. Specifically, TAPPI standard methods were used to characterize the components present in spinifex grass samples before and after pulping. Initially, spinifex water-washed grass and fibre samples were ground to 60 mesh fibre size using a small Wiley mill. Then the ground fibre samples were extracted with ethanol in a Soxhlet apparatus (Tecator Soxtec System Mode! HT 1043, from Foss,- Denmark) for one hour followed, by rinsing with water for another hour. The total lignin content was determined using the standard methods (TAPPI, Acid-insoluble lignin in wood and pulp, modified method based on Test Method T-222 orn-88, 1988; TAPPI, Acid-soluble ligni in wood and pulp, Useful Method UM-250, 1991). Monomenc sugars also were determined by ion chromatography according to the reference Pettersen, R. C ; Schwandt, V. H . Journal ofwoodehemistry and technology 1991, 1 1 , (4), 495- 501.

[0067J In their natural state, cellulosie materials including nanocellulose have a surface chemistry dominated by hydroxy! (OH) groups, as these are natural constituents of the polysaccharides that make up cellulose. Some chemical treatments used by other groups to facilitate the production of cellulose nanofibrils from plant feedstocks result in modification of the surface functionality of the cellulose. For example, the TEMPO oxidation process results in a highly carboxylated cellulose nanofibrils surface. Since the process of the present invention ma be performed without the use of harsh chemical agents, the hydroxy! ated surface of natural cellulose may be retained in the product cellulose nanofibrils,

[0068] Accordingly, one embodiment of the invention consists in nanocellulose with high aspect ratio, a mean fibril diameter of 10 nra or lower and a surface dominated by hydroxy 1 groups.

(0069) Nanocellulose i accordance with the present invention may be used in many applications, including in materials, composites, packaging materials, and coatings. Numerous other applications may afso be available.

[0070] Nanocellulose material in accordance with the present invention may be particularly useful for manufacturing composite materials (including composites made by mixing the nanocellulose material with polymeric material to form a polymeric composite material) and paper made from the nanocellulose material.

[0071] Any of the features described herein .can be combined in any combination with any one or more of the other features described herein within the scope of the inventi n. [007.2] The reference to any prior art in this specification is not, and should not be taken as an acknowledgement or any form of suggestion that the prior art forms part of the common general knowledge.

BRIEF DESCRIPTION OF DRAWINGS

[0073] Figure 1 is a flowchart showing the most commonly used general protocol for producing CNC in the prior art;

[0074] Figures 2A and 2B show a comparison between common established procedures for producing MFC (Fig 2 A) and common established procedures for producing NFC (Fig 2B);

[0075] Figure 3 shows SEM micrographs showing cross -sections of a native Triodi a fibre; a) low magnification overview, b) parenchyma cells showing a high surface area, "flaky" morphology, and c) nodular structures;

[0076] Figure 4 shows an example process for plant feedstock preparation and pulping in the ease that both del ignifi cation and bleaching are carried out, using plant material from Triodia Pungens. ;

[0077] Figures 5 A and SB show TEM images of cellulose nanocrystals/fibrils obtained from bleached spinitex pulp via acid hydrolysis (40 % sulphuric acid, at 45° C for 3h) (scale bar: 200nm), The average diameter of acid treated fibres in Figure 5 A is 4±L4 nm. The nanocellulose of Figure SB had an. average diamete of 3.4 ± 0.78 nm.

[0078] Figure 6A shows a TEM image of the nanofibers obtained from spimfex after pass through a HPH with the pressure of 1500 bar and Figure 6B shows a TEM image of the nanofibers obtained from spinite after 5 passes througli HPH with the pressure of 1500 bar (scale bar is 200nm in both eases);

[0079] Figure 7 shows a TE image of the suspension obtained after milling a spimfex bleached pulp at 1500 rpm for 30 min i circulating setup in a ball mill, The nanofibers have a diameter of 42 ± 24 nm and a length of a few microns (scale bar: 2μηι);

[0080] Figure 8 shows a TEM image of the suspension obtained after milling a spimfe bleached pulp at 3000 rpm for 20 min in batch setup showing nanofibers with a diameter of 8 ± 2 nm and length of 341 ± 100 nm (scale bar: 200 nm);

[0081] Figure 9 is a graph showing dispersity of dimensions (diameter) of spinifex derived nanofibrils obtained with only a single pass through high pressure homogenizer, based on a total of 250 measurements;

[0082] Figures IQA and lOB are process flowcharts showing a comparison of a commonly established procedure for producing NFC (Figure I QA) and a process for producing NFC from spinifex grass using a method in accordance with an embodiment of the present invention (Figure 10B);

[0083] Figure 11 shows a graph of dispersity of dimensions (diameter) of fibrils obtained from a spinifex bleached pulp via high energy milling (see Example 5);

[0084] Figures 12 shows an SEM image of spinifex grass deligmfied via alkali treatment at 80 °C;

[0085] Figure 13 shows SEM images of the deligmfied spinifex grass obtained via orgartosolv treatment at 185 °C;

[0086] Figure 14 shows a tensile curve of spinifex NFC paper produced by homogenization and dried with a hot-press at 103 C for 2h with no significant force applied during drying;

[0087] Figure 15 shows ATR FTIR spectra of T. pungens fibres in their virgin state (water washed), after alkaline delignificati on and after bleaching;

[0088] Figure 16 shows cellulose nanofibrils produced by processing bleached pulp of Triodia Pungens in a Silverson rotor-stator unit. The scale bar is 200 nm.

[0089] Figure 17 shows cellulose nanofibrils produced by processing bleached pulp of Triodia Pungens in a Silverson rotor-stator unit followed by further mechanical processing in a high pressure homogeniser. The scale bar is 1000 nm.

[0090] Figure 18 shows cellulose nanofibrils from Triodia Pungens grass processed by alkaline deligniflcation, followed by high pressure homogenisation at 500 bar pressure (no bleaching). The scale bar is 500 nm.

[009Ϊ ] Figure 19 shows cellulose nanofibrils from Triodia Pungens grass processed by alkaline delignjfication, followed by high pressure homogenisation at 700 bar pressure (no bleaching). The scale bar is 500 nm. EXAMPLES

Pulping

[0092 j The following examples used plant material derived from Australian arid spinifex (Triodia pungens). For pulping the plant material, the material was chopped to a particle size of less than 10 mm to enable easier processing and the natural fibre or cellulose fibre part was separated by delignification. In some eases del .igni.fi cation was followed by a. bleaching treatment. The general process for pulping the plant material is shown in Figure 5. For delignification, the- fibres were first treated with either alkaline solution of low concentration or organosolv at higher temperature.

Delignificatio of spinifex grass

[0093] Delignification was done using two different methods;

[0094 j Organosols: In this treatment a 40 w/v% ethanol solution is combined with the grass in a 2.5: 1 solvent:grass (v/w) ratio at 185 C was applied for 2 hours under the pressure in an autoclave then washing was performed using a I M NaOH solution and finally water. This procedure was repeated once more in order to pull out the residual lignin from between the fibrils.

[0095] Alkaline treatments: In alkali treatment spinifex grass was subjected to alkaline solution of 2 wt % NaOH with the solvent to grass ratio of 10: 1 at 80 °C for 2 hours, then filtered a d washed with water.

[0096} Bleaching spinifex grass

[0097] For bleaching delignified fibres, a 1 wt% aqueous solution of sodium chlorite at 70 ^Q C and pH=4 (pH adjusted using glacial acetic acid) was used for an hour with 30: 1 solvent to grass mass ratio under stirring until white point which the coloured substances were removed from the grass.

[0098] Table 3 below shows the composition of lignocellulosic components in Triodia pungens grass following washing in water and different stages in the pulping process. It was observed that prior to chemical processing (pulping) the Spinifex grass had a hemiceliulose content of 44 % (w/w) and this reduced to 43 % and 42 % following delignificaiion and bleaching steps respectively. In all cases, these percentage amounts are percentages of the total lignocellulosic mass of the material . Interestingly, the hemiceliulose content does not decrease significantly on deiignification or bleaching of the water-washed grass, allowing the high hemicellulose content of the grass to be carried through to the final nanocellulose product. This may be a result of the very mild deiignification and bleaching conditions used in the method of the present invention.

: Composition of lignocellulosic components in water-washed and pulp of Γ. pimge

The ATR FTIR spectr for water-washed, delignified and bleached T. pungens fibres shown in Figure 15 feature a main broad peak within the wave number range of 3000 - 3650 cm-1 , which confirms stretching vibrations of hydroxyl (OH) groups as the principal functional group in these 1 i gnocell ul osi c material s .

EXAMPLE 1 -Higher aspect ratio nanofibers from spinifex via acid hydrolysis.

[0099] Sulphuric acid hydrolysis is a suitable chemical method for isolating cellulose nanocrystals, due to high yield and the surface charges (sulphate) created after the hydrolysis, which can facilitate the dispersion i water and other polar solvents. In a typical prior art procedure, the acid concentration varies from 35 to 65% and the temperature varies from 40 to 100 C, depending on the source. In general, if a low range of acid concentration is used, a higher temperature is used, and if a low temperature is used, a higher acid concentration is used. With spinifex grass, use of an acid concentration above 45 % and a temperature above 50 °C resulted in detrimental effect on the hydrolysis, either charring or complete hydrolysis into lo molecular sugars.

[00100] Different methods have been applied in the prior art to prepare cellulose nanocrystals. Each of these lead to different types of nanomateriai (e.g., shape, length, and diameter), depending on the source of the cellulose and the degradatio process (e.g., controlled time, temperature and acid concentration), and also the applied pre-treatmen The main process in the preparation of cellulose nanocrysial (CNCs) is based on strong acid hydrolysis under strictly controlled conditions of temperature, agitation, and time to remove amorphous, disordered or para-crystalline regions and isolate crystalline domains with higher resistance to acid attack. Removing the amorphous region has shown improvement in the crystallinity and thermal stability of extracted rod-like nanocrystals.

[00101] Different concentrations of sulphuric acid solution at different temperatures were used for different times to characterize the effect of hydrolysis parameters on cellulose fibres properties,

[00102] The experimental results showed that we could successfully produce cellulose nanocrystals from spinifex grass using the minimum acid concentration and lowest temperature together (we used 35% sulphuric acid at 45°C - the difference with our work is that we used the minimum for both conditions). Applying harsh treatment, such as more than 40% sulphuric acid (mostly 64% is using to hydrolysis different source of cellulose for producing cellulose nanocrystals) and higher temperature (above 50 °C), to spinifex-derived plant material damages the fibres and hydrolyses the cellulose into low molecular sugars glucose. It is worth noting that the nanocrystals obtained from spinifex grass have a ver long length while the nanocrystals from the other sources of cellulose are short and straight. The highest known aspect ratio cellulose nanocrystals are obtained from marine animals called tunicates.. Because of their rarity, the production of high aspect ratio CNCs is limited at a commercial scale Production of high aspect ratio CNCs derived from plant sources was unknown prior to the present invention,

[00103] Figures 5A and 5B show TEM image of cellulose nanocrystals/fibrils obtained from bleached spinifex pulp via acid hydrolysis (40 % sulphuric acid, at 45° C for 3h) (scale bar: 2 μηι) as used i Example 1. The average diameter of acid treated fibres in Figure 5A is 4±1.4 nm, A measurement of shorter fibres of an acid treated fibres which we could find the start and end point in Figure 5B shows the average diameter of 3.4±0.?8 nm.

EXAMPLE 2: Small diameter nanofibers from spinifex via homogenisation

[00104] To obtain nanofibrillated cellulose (NFC), aqueous suspensions of delignified (alkaline route) and bleached spinifex pulps (42 % hemicel!ulose content) were homogenized using a high pressure homogeniser (EmulsiFl x~C5 homogenizer) at different solids loadings (QJ, 0,3 and 0.7% w/v) and at different pressures (1500, 1000,350 bar). _: Figure 6 shows the TEM iniages of the obtained nanofiber-s with the average width of about 3,5 am after 1 pass (Figur 6A) and 5 passes (Figure 6B), through the homogeniser. Hemieellulose content was 42 ¾,

[00105] Obtaining a homogeneous suspension of nanotibers within a few number of passes with 100% yield has been beneficial. Unlike fibres from other source, there was no clogging issue encountered even after increasing the number of passes up to 15, suggesting the nanofibres could be obtained with lower energy consumption, Efficiency of this process may be further increased by increasing the solid content in suspension.

EXAMPLE 3 - Agglomerated nanofibres from spinifex via high-energy ball milling.

[00106] As a scalable method to produce cellulose nanofibres (fibrils/crystals), vve investigated high-energy ball milling (Netsch-Labstar 10, diameter of the milling chamber: 97 mm, volume of balls: 400 ml (including the interstitial space between the balls), media for grinding: water, loading of suspension; 400 ml, volume of grinding chamber: 620ml). There have been few reports made using lab-scaled to 5 g scale) ball milling, Our method/set-up relies on large-scale processing, Figure 7 shows the nanofibres (width 42 ± 24 nm) obtained from milling a delignified (alkaline) and bleached pulp (42 % hemieellulose content) at 1500 rpm (lower energy) after 30 min of circulation.

[00107] In another example, a pulped suspensio was milled at 3000 rpm for 20 min of batch setup (high energy). Figure 8 TEM of the resulting suspension showing the nanofibres (width was 8 ± 2 nm, length was .341 ± 100 nm) (scale bar: 20 nm). This also suggests that by applying higher energy we could further break down the nanofibres into shorter nanocrystals.

EXAMPLE- 4 - NFC preparation using high-pressure homogenizer.

[00108] A slurry of Spinifex pulp (alkaline delignified and bleached fibres) (42 % hemieellulose) was passed through a high-pressure homogenizer (EmulsiFlex-C5. Homogenizer). This homogeniser rapidly reduces particles size from micron to nanometer scale based on the principle of dynamic high-pressure homogenisation. During the preparation of NFCs, it was found that high-pressure homogenization. has a noticeable effect on the diameter of fibres. The NFCs showed a complex, web like structure. Different shape of twisted/untwisted, and curled/straight naiiofibrils have the diameter less than 7 nm and several microns length even after only 1 pass through homogenizer.

[00109] Applying different pressure, different slurry concentration and also different numbers of passes exhibited almost the same results on fibrils diameter and length (Table 4), All nanocel!ulose products had 42 % hemicellulose content. In case of higher pressure, more fibrillation was observed. The most important issue when thinking about an up-sealing of the nanofibrillated cellulose production in industry is the energy consumption. So recently, several researchers have fooussed on the development of less energy consuming disintegration methods using enzymatic, chemical or mechanical pre-treatment In our process with spinifex grass, homogenisation of bleached pulp produces NFC even at first pass through the homogenizer without an clogging issues. Since the fibres were already well -fibril lated into nanoscale material at first pass, further homogenisation did not show any clogging and it only heiped to fibriliate into a few nanometers, In other words, it was easier to fibnllate into nanoscale fibres at first pass (Figure 9) whereas in the reported papers, a minimum of 6 passes or treating with acid/alkaline/poiyeleetrolyie was usually performed to reduce the higher number of passes which is crucial in terms of energy consumption of the process 2-4,

Table 4. Average diameter of nanofibres obtained by homogenization

[001 10} Process flowcharts in Figures 1Q and 10B showing a comparison of the common established procedures for producing NFC and a process for producing NFC from spinifex grass using a method in accordance with an embodiment of the present inventio As can be seen, in the process of the present invention (Figure 10B), the steps of chemical pre-treatment, mechanical pre-treatment and/or enzymatic pre-treatment can be omitted.

[00111] With bleached ethanol and alkali treated spinifex feedstock, the present inventors were able to produce NFCs using considerably fewer (1 ) passes compared with -20 passes for cotton -derived feedstock prepared using much harsher multistep pretreatment steps, i.e. an order of magnitude larger energ and time required to obtain a much lower aspect ratio product. Furthermore, with spinifex the EPE could practically be run at higher suspension concentration without clogging, meaning much higher potential yields,

EXAMPLE 5 - MFC preparation using high-energy milling, and resultant dimensions

[00112] The milling of spinifex grass was performed in which a slurry of bleached spinifex pulp in only water was subjected to the high-energy milling (Netzsch Laboratory agi tator based mill LABSTAR). Figure 11 shows a graph of dispersity of dimensions (diameter) of fibrils obtained via high-energy milling.

[00113} This example demonstrates extraction of cellulose from spinifex grass using a high- energy milling while the crystallinity of obtained nanofibrils and structure of cellulose didn't change. The nanofibrils have the diameter in the range of below 40 nm and several microns length (Figure 11).

[00114] Without wishing to be bound by theory, the present inventors believe that the milder conditions or lower energy (for chemical or mechanical methods) required for defibrillating/microflising the spinifex grass fibers into nanofibers/nanocrystals is. likely to be attributable to the structural morphology of the fibers. Figure 12 and 13 show SEM images of spinifex fiber after delignifi cation via alkali and organosolv treatment respectively. The morphology of the fibers suggests the elementary fibrils are intertwined and stacked to form microfibres which are connected together with hollow tube-like channel.

[00115] It is assumed that fibres with this morphology may have evolved to adapt to the harsh drought conditions and to reduce the water evaporation.

[00116] For a spinifex NFC sample prepared via 1 pass through the high-pressure homogenizer at 1500 bar, the following dimensions were measured. The average aspect ratio of nanofibrils with an average width/diameter of 3,2±0.7 nm and an average length of 1686±591 nm is 527± 18.5 (with lengths ranging between 266 and 958, as measured from TEM images taken at a higher magnification— noting some higher aspect ratio nanofibrils could not be measured due to the limited field of view). The average aspect ratio of larger diameter nanofibrils (or rather, larger bundles comprising several nanofibrils) with an average width/diameter of 1G,69±3.9 nm and an average length of 577Q.-i-.17D0 nm is 540±166 ( ranging from 305 to 727, as measured from TEM images taken at. low magnification to cover the whole length - ^■ also noting that at low magnification, the measured average diameter ma be overestimated, due t limited resolution, but still the NFC bundles which were visible showed an average 10.7 nm. width). EXAMPLES - Preparation of cellulose nanopaper

100117] Spinife cellulose nanopaper was produced from an aqueous NFC suspension after vacuum filtration on a Buehner funnel fitted with a cellulose acetate membrane filter (pore size: 0.45μπι, diameter: 47mm). The filtration was continued until the wet sheet of NFC was formed. The wet sheet was then dried using hot press trying at a temperature of 103. °C for 2 hours.

[00118] Mechanical testing of the spinife nanopaper was performed at room temperature using an Instron model 5543 universal testing machine fitted with a 500 N load cell. A total of five replicates of each sample with dimensions of 25 mm in length and 6 mm in width were tested at hum / mi ^' n strain rate with a 10 mm gauge length. The Young ^' s modulus was determined from the slope of the initial linear region of the stress-strain curves. Maximum tensile strength is the largest stress that a film is able to sustain against applied tensile stress before the film tears. Elongation at break is the maximum percentage change in the original film length before breaking, and work to fracture is measured as the area under the stress-strain curve.

[00119J The density of nanopaper was calculated by measuring dried paper's weight and dividing it by its volume calculated from the thickness by digital micrometer and its area. The corresponding porosity was estimated as the following Eq. (1); pNFCpaper

[00120] Porosity = 1

pcellulose ( I)

[00121] Here pmc paper 8n pceiiutose represent density of the obtained NFC films and neat cellulose (1460 kg/m3), respectively.

[00122] Mechanical properties of the nanopaper made from spinifex nanofibrils (42 % hemi cellul se content) are set out in the Table 5:

Table 5: Mechanical properties of spinifex nanopaper produced by homogenization and dried with a hot-press at 103 C for 2h with no significant force

[00123] Figure 14 shows tensile curves of spinifex NFC paper produced by vacuum filtration of homogenized nanofibrils and dried with a hot-press at 103 C for 2h. For a given nanopaper density, the present inventors believe that the overall toughness of this spinifex derived material (i.e, area under the tensile curve) is very impressive due to the entanglements of the long fibrils enabling quite a high plastic deformation before breakage.

EXAMPLE 7- Silverson processi ng of bleached pulp

[00124] An aqueous suspension of deiignified (alkaline) and bleached spinifex pulp (42 % hemicelluiose) was subjected to processing through a Silverson rotor stator homogenising unit for 5 minutes at room temperature. As shown in Figure 16, long cellulose nanofibrils with a diameter of 5,5 ±7.3 nm and 42 % hemicelluiose content were produced,

EXAMPLE 8- HPH processing of Silverson processed pulp

[00125] An aqueous suspension of deiignified (alkaline) and bleached spinifex pulp (42 % hemicellui se) was subjected to processing through a Silverson rotor stator homogenising unit for 5 minutes at room temperature followed by a single pass through a high pressure homogeniser at 500 bar pressure. As shown in Figure 17, long cellulose nanofibrils with a diameter of S.7±3 nm and 42 % hemicelluiose content were produced. Scale bar is 1000 nm.

EXAMPLE 9- Cellulose nanofibrils produced without bleaching

[00126] A sample of Triodia pungens grass was subjected to alkaline delignifi cation. The deiignified pulp (43 % hemicelluiose) was then passed through a high-pressure homogeniser at 500 bar pressure for a single pass only, The pulp was not bleached. As shown in Figure 18, long cellulose nanofibrils with diameter of 6.8.±0.23 nm and 43 % hemicelluiose content were obtained,

EXAMPLE 1.0- Cellulose nanofibrils produced without bleaching

[00127] A sample of Triodi pungens grass was subjected to alkaline delignifi cation. The deiignified pulp (43 % hemicelluiose) w¾s then passed through a high-pressure homogeniser at 700 bar pressure for a single pass only. The pulp was not bleached As show in Figure 19, long cellulose nanofibrils with diameter of 3.9 ±1.3 u and 43 % hemicelluiose content were obtained.

ASPECT RATIO METHOD OF MEASUREMENT

[00128] In the examples given in this specification, the following method was used to measure or determine aspect ratio: [00129] Samples of spinifex. cellulose nanofibrils in water were sonicated and Ι μΐ was spotted onto formvar coated Cu/Pd 200 mesh grids and allowed to dry, Samples were then stained wit 2 % uranyl acetate (aq) for 10 minutes in the absence of light then excess UA was removed and grids were allowed to dry. Grids were then examined on a JEOL 1011 TEM operating at 100 KV and captured on a SIS Morada 4 CCD camera system,

[00130| For each sample, 250 measurements of diameter were randomly selected and measured from several TEM images using digital image analysis (Image J).

[00131} For measuring the length of fibres, each ^" TEM image was processed using AutoCAD software. This program allows contours to be manually drawn following the non-linear path of each cellulose nanofibre in xy space and contains tools for the subsequent calculation of contour length,

[00132] Throughout this specification, the following terms have the following meanings:

[00133] Microfibrillated cellulose (MFC): MF is produced via mechanical refining of highly purified WF and PF pulps, have a high aspect ratio (20-100 nm wide, 0.5-10's μηι in length), are 100% cellulose, and contain both amorphous and crystalline regions.

[00134] Wood fibre (WF),

(a) (Bot.) Fibrovascular tissue.

(b) Wood comminuted, and reduced to a powdery or dusty mass.

[00135] Plant fibre (PF) - 1 : fibre derived from plants [syn: plant fibre, plant fibre]

[00136] Nanofibrillated cellulose (NFC): NFC particles are finer cellulose fibrils produced when specific techniques to facilitate fibrillation are incorporated in the mechanical refining of WF and PF have a high aspect ratio (3-20 nra wide, 500-2000 am in length), are 100% cellulose and contain both amorphous and crystalline regions,

[00137] Cellulose nanocrystals (CMC): CNCs are rod-like or whisker shaped particles remaining after acid hydrolysis of WF, PF, MCC, MFC, or NFC, These particles have also been named nanocrystalline cellulose, cellulose whiskers, cellulose nanowhi skers and cellulose niierocrystals (in the early literature). CNCs have a high aspect ratio (3-5 nm wide, 50-500 nm in length), are 1.00% cellulose, are highly crystalline (54-88%) [00138] Tunicate cellulose iianocrystals (t-CNC): Particles produced from the acid hydrolysis of tunicates are called t-CNCs. The ribbon-like shaped t-CNC s have a height of B8 ran, a width of B20 nm, a length of 100-4000 nm (typical aspect ratios 70-100), :are 100% cellulose, are highly crystalline (85-100%).

[00139] Micfocrystalline cellulose (MCC): Cellulose micropartiel.es produced commercially via regular pre-treatments (delignification, bleaching, grinding and/or acid hydrolysis and back- neutralization with alkali). Their width 10-50 μηΐ and length 10- 500 um.. They are the current commercial source for producing MFC, NFC and CN ^' Cs,

[00140] In the present specification and claims (if any), the word 'comprising' and its deri vati ves including 'comprises' and 'comprise' include each of the stated integers but does not exclude the inclusion of one or more further integers.

[0 141] Reference throughout this specification to One embodiment' or 'an embodiment' means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearance of the phrases 'in one embodiment'' or 'in a embodiment' in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more combinations.

[00142] In compliance with the statute _* the invention has been described in language more or less specific to structural or methodical features. It is to be understood that the invention is not limited to specific features shown or described since the means herein described comprises preferred forms of putting the invention into effect. The invention is, therefore, claimed in any of its forms or modifications within the proper scope of the appended claims (if any) appropriately interpreted by those skilled in the art.