Patent Description:
Bioconversion of cellulose into glucose is an environmental approach to withdraw useful products from cotton based waste textile. Pretreatment of the feedstock is a primary and critical step to increase the cellulose accessibility to cellulase before enzymatic hydrolysis. Many pretreatment processes have been developed to reduce the recalcitrance of textile waste to enzymatic hydrolysis and furthermore decrease the consumption of cellulase. The basic approaches can be classified into four categories, i.e., chemical, biological, physical, and physico-chemical processes. Different pretreatment processes may result in different types and levels of structural modification and improve enzymatic hydrolysis after different mechanisms.

Acid pretreatment is currently the most widely studied and applied approach for the pretreatment of cellulose fibre and the other related feedstock. The key mechanisms of the acid pretreatment are the decomposition of the microstructure of cellulose fibres. The amorphous region of cellulose can be hydrolyzed while the crystalline regions with more reducing ends and non-reducing ends can be exposed to the enzymes, which facilitates the enzyme to perform degradation process. Acid pretreatment process, however, requires special reactors/container to prevent corrosion at elevated concentrations; and the acid reagents used are difficult to recycle. Both those factors can considerably increase the operation costs and environmental impacts of the overall biorefinery process. Meanwhile, high severity acid pretreatment (i.e., high acid doses, high temperature, and extended reaction time) can promote the conversion of carbohydrates to form into sugar dehydration by-products (i.e., furan-type inhibitors; <NUM>-hydroxy methyl-furfural), which are harmful to the downstream fermentation processes at high concentration.

Another pretreatment method using ionic liquid (ILs) has drawn a significant amount of attention recently. There are mainly two factors lead to the various properties of ILs: the cation structure (the symmetrical array, the influence of alkylphosphonate and hydrophobic groups) and the anion delocalization degree. Some ILs have shown outstanding characteristics for industrial application, i.e. high chemical and thermal stabilities, liquid form in wide range of temperature, low vapour pressure, and low viscosity operation which reduces the cost of mixing. ILs have been tested in pretreating certain cotton or textile wastes, although its feasibility to large scale application is still unclear and needs further investigation. Hong, Guo used a <NUM>-allyl-<NUM>-methylimidazolium chloride ([AMIM]Cl) IL to treat un-dyed <NUM>% cotton t-shorts. After <NUM> minutes of pretreatment under <NUM> a high sugar yield (<NUM>%) was achieved by using reasonable amount of cellulase. De Silva et al. used the same IL to treat a <NUM>:<NUM> blend PET/cotton yard at a longer cooking period (<NUM> hours) and higher temperature (<NUM>). The cotton was effectively dissolved and regenerated in the anti-solvent and formed into fibre films after regeneration. The PET was completely recovered after the process. The key limiting factors hindering the applicability of ILs are high production cost and environmental toxicity. The current price for ([AMIM]Cl) IL is approximately US$<NUM>-<NUM> per gram (chemical grade, Sigma-Aldrich). Furthermore, ILs have shown substantial negative influence on enzyme hydrolysis while the IL/cellulose mixture is difficult to handle in the existing reactor systems due to its high viscosity.

<NUM>-methylmorpholine <NUM>-oxide (NMMO) is another solvent that can dissolve cellulose and provide reasonable yield. In NMMO process, heating at <NUM> under low water content leads to dissolution of higher molecular weight cotton fibres. However, NMMO concentrations above <NUM> and <NUM>/L showed inhibition effects on enzymatic hydrolysis and fermentation. The treated materials must be washed before enzymatic hydrolysis and techno-economic studies showed that efficient recycling of NMMO is required in order to have an economically feasible process for pretreatment of cotton materials with NMMO. Certain side reactions present in the Lyocell process might affect the pretreatment system as well and lead to decomposition of NMMO, furthermore increasing the consumption of stabilizer. On the other hand, efficient removal of NMMO from the treated material by washing requires high amounts of water. This significant amount of water must be evaporated in an energy consuming process before reusing NMMO, which is an energy intensive process.

Supercritical fluid is a phase of reagents of which both gas and liquid phases coexist under a specific pressure and temperature. It shows liquid like density and gas like diffusing/penetrating ability to solid materials. Among all the supercritical fluids, supercritical carbon dioxide, which has a critical temperature at <NUM>, has shown to be suitable for pretreatment of cellulosic feedstock. Supercritical carbon dioxide (SC-CO<NUM>) has been widely used as an extraction solvent. In aqueous solution, CO<NUM> forms carbonic acid and can improve the hydrolysis of polymers. CO<NUM> molecule is similar in size as the molecules of water or ammonia, and therefore, they can penetrate through the same pathway to the small pores of the cellulose. CO<NUM> has even been used to modify steric structures of some cellulases to improve their stability, solvent tolerance and reactivity. Saka and Ueno investigated the direct conversion of various types of celluloses (including cotton linter) in supercritical water (<NUM>, <NUM> MPa) into glucose and found that cellulose can be hydrolyzed to a similar level as acid or enzymatic hydrolysis, without the difficulties associated with hydrolysing techniques. Muratov and Kim studied the performance of enzymatic hydrolysis of cotton fibres in supercritical CO<NUM> (<NUM> atm, <NUM>, <NUM>) and found that the productivity of the glucose increased by <NUM>% as compared to atmospheric conditions. As commercial scale of supercritical CO<NUM> treatment has been seen in textile industries (such as for dyeing), similar technology and equipment could be worthwhile to be explored further for the treatment of textile wastes in the future.

Alkaline pretreament of textile waste can also be performed. During alkaline pretreatment, the first reactions that occur are solvation and saponification, of intermolecular ester bonds cross-linking which swells the textile to expose more accessible areas for the enzymes to hydrolyse the cellulose. However, a noticeable disadvantage of alkaline pretreatment is the formation of irrecoverable salts from alkaline or the combination of salts into the biomass which hinders the enzymatic hydrolysis. And alkaline pretreatment also works as another mechanism at ambient temperature at longer pretreatment time. At high alkaline concentration dissolution step, the effect of alkaline hydrolysis surpasses the alkaline dissolution, the 'peeling' of end-groups also hydrolyse the dissolved cellulose through degradation and decomposition cause the loss of polysaccharides. This increases the chance of loss of carbon, converting to carbon dioxide.

For example <NPL>) discloses the conversion of waste cotton-polyester textile to ethanol and recovery of polyester. The cotton-polyester waste textile pretreated with different alkali solutions (NaOH, , NaOH/urea , NaOH/thiourea and NaOH/ urea/thiourea) at -<NUM>, <NUM>, <NUM>, and <NUM> for <NUM>. Subsequently, the pretreated textile samples were washed with distilled water to obtain pH <NUM> and treated with <NUM> FPU cellulase and <NUM> IU β-glucosidase per gram of cellulose.

<NPL>), discloses a method of processing cotton cellulose comprising dissolving the cotton in a NaOH/Urea solution and cooling to -<NUM> overnight. After the frozen cellulose slurry was thawed, deionized water was added to precipitate the dissolved cellulose. The mixture was filtered to collect the precipitated cellulose and washed thoroughly with deionized water. All of the washed precipitates were considered as regenerated celluloses and subjected to cellulase hydrolysis using Cellulase AP3 (Aspergillus niger strain; 10FPU/g cellulose) to produce a solution containing glucose. After <NUM>, the saccharification yield ranged from <NUM>% to <NUM>% for the regenerated celluloses while only around <NUM>% could be achieved for the untreated cellulose.

<NPL>) discloses that the cellulose obtained from citrus peel treated with alkali was insoluble in distilled H2O, acetone-H2O and in ethano|-H2O mixtures while partial solubility was observed in mild acidic H2O (pH <NUM>), acetone, ethanol and methanol-H2O mixture.

With all of the known pretreatment methods, the amount of enzyme loading required for the hydrolysis step is typically <NUM>-30FPU/g glucan. Accordingly, there remains a need for an improved textile waste treatment method that is more efficient, cheaper and which requires a lower enzyme loading.

The present disclosure provides a method of processing cotton containing textile waste comprising dissolution of textile waste in a NaOH/Urea solution at a predetermined temperature until freezing of the textile waste; adding an amount of an anti-solvent selected from boiling water or ethanol to regenerate cotton from the frozen textile waste; and hydrolysing said cotton with cellulase to produce a solution containing glucose.

The textile waste may be immersed in NaOH/Urea solution at or below <NUM>, optionally for <NUM> hours.

Preferably, the cotton may be hydrolysed using an enzyme loading of ≤ <NUM> FPU/g glucan, and preferably an enzyme loading of <NUM>-<NUM> FPU/g glucan, and more preferably an enzyme loading of <NUM>-<NUM> FPU/g glucan.

In one embodiment, an enzymatic hydrolysis yield of cotton to glucose of over <NUM>% is obtained following enzymatic hydrolysis.

Optionally, the method may further comprise the step of blending the textile waste, preferably by using a blender for <NUM>-<NUM> seconds.

In one embodiment, recovering PET fiber, and optionally recycling the recovered PET fiber may be preferred.

The foregoing and other features and advantages of the disclosure will be apparent from the following, more particular description of embodiments of the disclosure, as illustrated in the accompanying drawings.

The present disclosure provides a method of treating recycled textile waste (such as cotton/PET textile waste), which involves a modified alkaline pretreatment method. The method comprises the steps of subjecting a textile waste to alkaline pretreatment with NaOH/urea at freezing temperatures, followed by a regeneration process using boiling water and ethanol.

Advantageously, the modified method is able to enhance cellulase digestion of cellulose to glucose. With this method, substrate enzymatic digestibility (SED) is significantly increased, allowing the use of a much lower enzyme loading as compared to conventional methods. At the same time, the conversion yield of cotton to glucose could be improved to over <NUM>%, and over <NUM>% for textile waste with high cotton percentage. It is also envisaged that the residue PET fiber can be extracted for other applications.

Prior to enzymatic hydrolysis in recovery of glucose from the textile waste, the textile waste is first subjected to a pretreatment method which essentially comprises two steps as described below (and as shown in <FIG>):.

Dissolution of textile waste in NaOH/Urea solution is at freezing temperatures, for example, at or under <NUM>, and more preferably between -<NUM> to <NUM> until the textile waste is frozen. In an embodiment, the textile waste is immersed in <NUM>%NaOH/<NUM>%Urea solution at <NUM>.

This process typically takes <NUM>-<NUM> hours, and the alkaline is expected to cause a swelling effect to the cellulose, breaking down the inter and intra chains of cellulose molecules, resulting in more amorphous regions for enzymatic hydrolysis. This process reduces the DP and crystallinity index (CrI) of cellulose, increasing the cellulose accessibility to cellulase by creating more porous space for enzymatic hydrolysis.

It is envisaged that an enhancement of substrate digestibility of the waste textile is achieved through cellulose dissolution process. This process was designed based upon the cellulose dissociation theory discovered previously, as shown in <FIG>.

Regeneration process has only been used in combination with textile waste pretreatment using ionic liquid (ILs) in the past. Regeneration using adding anti-solvents such as water, ethanol, or acetone in the regeneration process is necessary when the IL pretreatment has a dissolution effect on the cellulose.

When being applied in the present method, following the above alkaline pretreatment step, thawed (previously frozen) dissolved cellulose, is regenerated using anti-solvent(s), namely boiling water or ethanol. The suitable amount of anti-solvent should be <NUM> times solvent mixture in volume.

It is expected that the regeneration of the cotton fiber is the key mechanism to increase the bioconversion efficiency of the textile waste. During the regeneration process morphological changes of cellulose can happen, with amorphous regions being increased. The dissolved cellulose can shrink to form a new allomorph structure (cellulose II). The crystallography of cellulose suggests that cellulose I comprises the parallel chains, whereas cellulose II is described as an antiparallel structure which is more enzyme accessible. The ability of NaOH to change the cellulose structure at lower temperature could be explained by a stronger binding between Na+ and OH-to water at lower temperatures, enabling the breakage of the hydrogen bonds within the cellulosic structure.

It is expected that the regenerated cellulose with a lower degree of polymerization (DP from <NUM>-<NUM>) has more β-glucosidic bonds accessible to cellulase. Regenerated cellulose is more water soluble than native cellulose prior to the regeneration. Therefore, it is expected that the regenerated cellulose can be hydrolyzed to short glucose oligomers in a shorter amount of time, contributing to an increased substrate enzyme digestibility (SED) and allowing a significantly lower enzyme loading to be used in the hydrolysis step.

Following the modified pretreatment process, the textile waste is then subjected to enzyme hydrolysis using cellulase. The hydrolysis step may be performed at any appropriate conditions, and preferable under <NUM>, at pH <NUM> for <NUM> hours.

After cotton digestion, the remaining residue is mainly PET fiber, allowing simple extraction process to be performed in order to recycle the PET fiber in the textile waste.

Compared with conventional alkali pretreatment methods, the present method is able to achieve high conversion yield of cotton to glucose with a reduced enzyme loading, thereby significantly reducing overall costs. In one embodiment, an enzyme loading of as low as 5FPU/g glucan (and more preferably <NUM>- <NUM> FPU/g glucan) is sufficient to obtain a conversion yield that can normally be obtained using an enzyme loading is around <NUM>-30FPU/g glucan.

Additionally, compared with conventional methods using acid hydrolysis, NMMO and ionic liquid dissolution, the method has notable benefits as follows:.

Thse present disclosure can solve the problem of achieving high enzymatic digestibility by decreasing the crystallinity of cotton. The significant increase in the disordered or amorphous fraction enhance the enzymatic digestibility as the cellulase protein can have greater accessibility to cellulose. In particular embodiments, the enzymatic digestibility can be over <NUM>% for different cotton/PET composite fibres.

The use of NMMO in textile waste pretreatment requires a pretreatment step at an elevated temperature of <NUM> to <NUM>, as well as a step of NMMO solvent recycling, which involves high energy consumption. Both of these steps led to an increase in the total production cost.

On the other hand, the improved pretreatmemt process of the present disclosure includes a dissolution step performed at freezing temperatures (e.g. <NUM> to -<NUM>), which can be easily accomplished in a standard, non-industrial freezer or in relatively cold countries. The use of such lower temperatures not only reduces energy consumption, but also avoids side reactions that may damage the PET, allowing the recycling of PET fiber from the textile waste with a simple extraction process. Additionally, the application of boiling water or ethanol as anti-solvent can lower the distillation energy.

The new pretreatment solution is stable in room temperature and low temperature (<NUM>), therefore eliminating the need for chemical stabilizers, which are often required by other pretreatment methods. Therefore, the total cost of the chemicals required in the pretreatment process is also reduced.

The objects, advantages and features of the present disclosure will become more apparent when reference is made to the following examples taken in conjunction with the accompanying figures.

Aspects of the present disclosure demonstrated by the following examples that are demonstrative only and not intended to be limiting.

Waste textiles of five Cotton/PET ratios (i.e., <NUM>% cotton, <NUM>% cotton, <NUM>% cotton, <NUM>% cotton, and <NUM>% cotton) as list in Table <NUM> were used as starting materials. These are being referred to as Samples <NUM>-<NUM> herein. All textile scrap samples were provided by H&M Far East. The samples were cut into rectangular pieces with <NUM> in dimension before further tests or analysis. Cellic CTEC II cellulase was generously provided by Novozymes China (Beijing). The initial activities towards filter paper and cellubioase were <NUM> FPU/mL and <NUM> CBU/mL. Commercial NaOH pellet (<NUM>%) and urea pellet (<NUM>%) were purchased from Sigma without any purification before use.

<NUM> waste textile of each of the five textile waste samples is cut into smaller pieces in the size of <NUM>×<NUM> squares and immersed into <NUM> alkaline/urea solvents (NaOH/urea/water (<NUM>%/<NUM>%/<NUM>%wt), <NUM> boiling water and <NUM> <NUM>% H<NUM>SO<NUM> solution, respectively.

The samples obtained are kept under different temperatures, namely, <NUM>, <NUM> and <NUM> as shown in Table <NUM> below. Each of the five textile samples (<NUM>% cotton, <NUM>% cotton, <NUM>% cotton, <NUM>% cotton, <NUM>% cotton) are subjected to six different experiment conditions, providing a total of <NUM> test samples.

For the samples pretreated at <NUM>, the samples are placed in a freezer until it is fully frozen to dissolve the cellulose fibres (approximately <NUM> hours). As for controls, <NUM> boiling water and <NUM> <NUM>% H<NUM>SO4 solution were adopted. It is well known that dilute acid method is the most widely used method for pretreating cellulosic material (for the purpose of releasing all the glucose for measurement). This is also the standard method described in NREL measurement procedure (<NUM>% H2SO4 for <NUM> hour at <NUM>). Blending was performed under experimental conditions <NUM> and <NUM>, whereby a household blender was used to blend the substrates for <NUM>-<NUM> seconds.

Following the alkaline/urea pretreatment process, the dissolved cellulose of all samples (with the exception of the controls) are regenerated by using boiling water as anti-solvent (amount of boiling water being <NUM> folds of solvent mixture in volume). The regenerated solids are then stirred using a <NUM>-L lab-scale blender. Finally, the substrate is washed with deionised water until the sample pH is neutral, whereby the mixture is then filtrated to collect the solid, which is stored at <NUM> for further use. The overall pretreatment and regeneration process is shown in <FIG>.

The pretreated substrate is freeze-dried and stored in a desiccator for following steps. The experiment design is shown in Table <NUM>.

Compositional analysis for all five samples is carried out by using the NREL procedure following by a HPAEC-PAD analysis. The untreated textile samples and pretreated substrates were ground into powder (by passing through a <NUM>-mesh screen). In summary, acid insoluble content was calculated gravimetrically after digestion, and the concentrations of monosaccharides was determined by using a high-performance liquid chromatography (HPLC, Shimadzu) equipped with refractive index detector (RID). The monosaccharides (i.e., glucose),) were analyzed using an Aminex HPX-87P column (Bio-Rad) at <NUM> with <NUM>/min eluent of deionized water. The glucose concentration of the five samples following dissolution under different parameters are measured through HPLC and the results are shown in Table <NUM>. The conversion of glucose can be calculated by using the equation <NUM> below.

<MAT> Where G is glucose (mg/ml) concentration from HPLC analysis, V is the initial volume of biomass slurry (mL), W is the initial dry biomass weight. <NUM> is the conversion factor of glucose to equivalent glucan.

<FIG> showed the cellulose content in different types of textile waste by using NREL standard procedure.

Enzymatic hydrolysis of lignocellulosic substrates was conducted at <NUM>% (w/v) in <NUM> total volume which contains <NUM> <NUM> acetate buffer (pH <NUM>), <NUM> Tetracycline in a shaking incubator at <NUM> and <NUM> rpm. The CTecII loading was <NUM> FPU/g glucan. Aliquots of <NUM> Samples were taken were taken periodically (<NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>) for glucose analysis. Glucose concentration in hydrolysate was then determined by the glucose oxidase-peroxidase system using a commercial enzymatic assay kit.

<NUM>% cotton textile waste was pretreated as mentioned above using six different dissolution parameters and regenerated with boiling water as anti-solvent. The solid was further hydrolysed as mentioned in section <NUM>. The glucose obtained during enzymatic hydrolysis was measured by a commercial enzymatic assay kit. The substrate digestability at different time points (<NUM>, <NUM>, <NUM> and <NUM> hours) were recorded. The substrate enzymatic digestibility means the percentage of glucan enzymatically hydrolyzed to glucose in <NUM> at a <NUM>% w/w substrate solid loading. The results are shown in Table <NUM>.

Textile waste of <NUM>%/<NUM>% cotton/elastane was pretreated as mentioned above using six different dissolution parameters and regenerated with boiling water as anti-solvent. The solid was further hydrolysed as mentioned above. The glucose obtained during enzymatic hydrolysis was measured by a commercial enzymatic assay kit. The substrate digestability at different time points (<NUM>, <NUM>, <NUM> and <NUM> hours) were recorded. The results are shown in Table <NUM>.

Cotton/PET <NUM>/<NUM> textile waste was pretreated as mentioned above using six different dissolution parameters and regenerated with boiling water as anti-solvent. The solid was further hydrolysed as mentioned in Section <NUM>. The glucose obtained during enzymatic hydrolysis was measured by a commercial enzymatic assay kit. The substrate digestibilities at different time points (<NUM>, <NUM>, <NUM> and <NUM> hours) were recorded. The results are shown in Table <NUM>.

Cotton/PET <NUM>/<NUM> textile waste was pretreated as mentioned above using six different dissolution parameters and regenerated with boiling water as anti-solvent. The solid was further hydrolysed as mentioned in Section <NUM>. The glucose obtained during enzymatic hydrolysis was measured by a commercial enzymatic assay kit. The substrate digestibility at different time points (<NUM>, <NUM>, <NUM> and <NUM> hours) were recorded. The results are shown in Table <NUM>.

From the results of Table <NUM>-<NUM>, pretreatment method using freezing soda/urea in combination with a regeneration step, followed by blending showed the highest substrate digestibility. Over <NUM>% of cotton cellulose has been digested within <NUM>-<NUM> hours for all <NUM> different Cotton/PET samples. Noticeably, in regards to the <NUM>%/<NUM>% cotton/PET waste textiles (sample <NUM>) and the <NUM>%/<NUM>% cotton/PET textile waste (sample <NUM>), it was found that <NUM>% and <NUM>% of cotton cellulose were digested within <NUM> hours, respectively.

As shown in <FIG>, even without the blending step, substrate digestibilities were found to be consistently higher when the pretreatment method combining a dissolution step using freezing soda/urea and a regeneration step was adopted (Samples <NUM>-<NUM>).

Other pretreatment methods using water with high temperature or dilute acid showed low degree of enzymatic digestion.

Therefore, the textile wastes treated with this novel pretreatment process demonstrate better effect on increasing the cotton digestibility and further allow the PET residue to be more easily collected after the digestion of cotton. As noted in Table <NUM>, as compared to NaOH/urea pretreatment at <NUM>, the new pretreatment process can be performed at a more efficient manner, since the textile wastes are only immersed in NaOH/urea for <NUM> hours.

<FIG> shows comparison on substrate enzymatic hydrolysis of different textile with different pretreatments within <NUM> hours.

The following experiment was conducted to demonstrate the effect of the regeneration process on the efficiency of cellulose recovery. After dissolving the textile waste in NaOH/urea as described in Example <NUM> above using experiment condition <NUM> (<NUM>%NaOH/<NUM>%Urea/Blend), frozen cellulose/ dissolved textile solvent was first thawed, subsequently, boiling water, room temperature (RT) water or ethanol was slowly poured into the thawed solvent respectively and stirred for <NUM> minutes. The samples were then washed to pH=<NUM> by using deionized water. The control experiment of regeneration were conducted by directly adjusting the thawed solvent to neutral by <NUM> HCl solution for further use.

Table <NUM> below shows the glucan content of the samples after different solvent methods. It was observed that the HCL neutralization method resulted in the highest loss of glucan content (around <NUM>%) due to the missing step of regeneration. By contrast, the boiling water regeneration process recovered the highest content of the glucan.

To demonstrate the significance of the regeneration process, enzymatic hydrolysis analysis of the <NUM>/<NUM> textile waste sample pretreated under experimental condition <NUM> (<NUM>%NaOH/<NUM>%Urea/Blend) was conducted to compare the substrate enzymatic digestibility (hydrolysis yield) of different samples when being regenerated using different anti-solvents. The results are shown in <FIG>.

It can be observed that freezing soda process largely increased the substrate enzymatic digestibility regardless of the anti-solvent selected for regeneration. However, regeneration using boiling water and ethanol are found to be the most effect way to further increase the enzymatic hydrolysis yield.

Although HCl neutralization resulted in high enzymatic hydrolysis yield, it should be noted that the glucan content that can be recovered using this method is much lower (see Table <NUM>).

As further illustrated in <FIG>, when pretreatment is carried out using NaOH/urea at freezing temperature and when boiling water is selected as the anti-solvent for regeneration, an enzyme loading at <NUM> FPU/g glucan is sufficient to achieve over <NUM>% substrate enzyme digestibility. This further highlights the unexpected superior SED of textile waste achieved by using the pretreatment process as disclosed herein.

<FIG> is a set of photos recording the textile appearance throughout the pretreatment process.

A <NUM>% cotton/<NUM>%PET sample has been observed under microscope to investigate the difference in appearance before and after NaOH/urea pretreatment (<NUM>% NaOH/<NUM>%Urea, under <NUM> with blending operation). As demonstrated in <FIG>, the photos showed that after enzymatic digestion, the residue PET fiber was still preserved, indicating the potential of such being recycled for other applications.

Claim 1:
A method of processing cotton containing textile waste comprising:
dissolution of textile waste in a NaOH/Urea solution at a predetermined temperature until freezing of the textile waste;
adding an amount of an anti-solvent selected from boiling water and ethanol to regenerate cotton from the frozen textile waste; and
hydrolysing said cotton with cellulase to produce a solution containing glucose.