WO2021226094A1 - Process for conversion of cellulose recycling or waste material to ethanol, nanocellulose and biosorbent material - Google Patents

Abstract

Low dose/short duration ozone treatment of cellulosic biomass waste, such as from the paper, cardboard and cotton fabric and textile recycling and waste or from agricultural waste, is used in a process for producing ethanol, optionally including using a solid remnant byproduct of the process as a biosorbent to treat wastewater, according to the present invention or in a process for producing nanocellulose according to the present invention.

Description

PROCESS FOR CONVERSION OF CELLULOSE RECYCLING OR WASTE MATERIAL TO ETHANOL, NANOCELLULOSE AND BIOSORBENT

MATERIAL

BACKGROUND OF THE INVENTION

Field of the Invention

[0001] The present invention relates to the field of ethanol production and nanocellulose production from the cellulose-containing waste material such as paper, cardboard, cotton textile.

Description of the Related Art

[0002] Rising population growth and socioeconomic level, along with the growing popularity of e-commerce, have resulted in increased use of packaging material, mostly cardboard, and its consequent related recycling and disposal. Indeed, in the European Union alone, 87.5 million metric tons of packaging waste were produced in 2016, of which 35.4 million metric tons were paper and cardboard (41%, Eurostat webpage), with 85.6% of the paper and cardboard recycled as of March 2019. The situation in the US is similar, with ~61 million metric tons generated in 2017, of which ~40 million metric tons (66%) were recycled. Total world production of paper and cardboard is expected to reach 700-900 Mt by 2050 (Kong et ak, 2016) with an estimated 400 million tons of paper waste generated annually (Nishimura et ak, 2016).

[0003] Paper and cardboard waste are some of the most recycled materials. Paper waste is typically recycled multiple times (2.4 times in average) (Zhang 2015), but through the recycling process, the fibers are shortened by mechanical erosion, resulting in large amounts of short fibers (up to 40% of input mass) (Ochoa de Alda 2008; World Bank 2007), termed recycled paper sludge (RPS). These short fibers are rich in cellulose, but are currently disposed of in landfills (Peretz et ak, 2019), causing landfill filling, greenhouse gas emissions, and groundwater contamination (Robus et ak, 2016). Different valorization options have been suggested for RPS, among them its use as building material supplement (Frias et al., 2011 and Sutcu et al., 2009) or feedstock for nanocellulose (Peretz et al., 2019), lactic acid (Marques et al., 2008), or ethanol (Marques et al., 2008), or biogas (Xiao et al., 2019) production; nevertheless, none of these solutions was found to be viable.

[0004] Paper and cardboard are essentially made of wood feedstock, and thus are composed of 40%-80% cellulose, 5%-15% hemicellulose, and minor traces of polyphenolic lignin (Sadasivan et al., 2018). Paper waste has been used as a feedstock for applications such as energy production, nanocellulose materials for industrial applications, adhesive materials, and feedstock for microbial lipid and cellulase production (Xiao et al., 2019, Sadasivan et al., 2018; Adu et al., 2018; and Annamalai et al., 2018). Cellulose-rich waste has been demonstrated as a feedstock for ethanol production (e.g., second-generation bioethanol). However, production of ethanol from cellulose rich waste requires delignification pretreatment step for successful production of ethanol (Ximenes et al., 2011; Adani et al., 2011).

[0005] Ozonation of water- suspended biomass has been recently demonstrated as an efficient, low-energy-demanding, clean technology for biomass pretreatment prior to ethanol production (Peretz et al., 2017; Rosen et al., 2019; Sugimoto et al., 2009). Ozonation has also been successfully applied to RPS material, demonstrating lignin removal from the cellulose fibers, and thus enabling increased nanocellulose production (Peretz et al., 2019). Nevertheless, no study to date has examined the use of ozonation pretreatment of wastepaper feedstock for potential ethanol production.

[0006] To-date, -80% of the ethanol worldwide is produced by fermentation of sugars extracted from specialized crops (sugar cane in Brazil and corn in the USA) but there is much interest, both scientific and commercial in moving toward agricultural waste as feedstock, i.e., second generation ethanol. While beneficial in many ways, the production of ethanol from lignocellulosic biomass requires complicated pretreatment and thus large and expensive infrastructure, preventing decentralized, small-scale, local pretreatments and ethanol production, and resulting in shipment of large amounts of agricultural waste and hazardous materials. Thus, use of this waste as feedstock becomes less energetically, economically and environmentally feasible.

[0007] Citation of any document herein is not intended as an admission that such document is pertinent prior art, or considered material to the patentability of any claim of the present application. Any statement as to content or a date of any document is based on the information available to applicant at the time of filing and does not constitute an admission as to the correctness of such a statement.

SUMMARY OF THE INVENTION

[0008] The present invention provides a process/method for producing ethanol from cellulose recycling or waste material, such as those found in paper and cardboard waste, cotton textile waste and agricultural waste. The process/method involves treating a suspension of the cellulose material with low dose ozone followed by enzymatic hydrolysis to produce sugars that are fermented by yeast to produce ethanol. The ozonation pre-treatment of the cellulose material improved the efficiency of enzymatic hydrolysis/saccharification for the production of ethanol.

[0009] The present invention further provides a way of generating byproducts from the same cellulose material source used for producing ethanol as a resource for additional applications such as solid byproducts acting as a biosorbent. Thus, as an add-on to the process/method for producing ethanol, the solid byproduct remaining after enzymatic hydrolysis of the cellulose is applied as a biosorbent to treat wastewater.

[0010] The present invention also provides a process/method for producing nanocellulose (crystalline nanocellulose), which can serve as building blocks for various industrial applications, from cellulose recycling or waste material through treating a suspension of the cellulose material with low dose ozonation followed by acid hydrolysis with maleic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figures 1 A and IB show graphs of thermogravimetric analysis (TGA) of different paper samples before and after ozonation treatment, with thermogravimetric thermograms shown in Fig. 1 A and derivative thermogravimetric thermograms shown in Fig. IB.

[0012] Figures 2A and 2B show graphs of optimal conditions for ozone-treated recycled paper sludge (RPS) enzymatic hydrolysis. In Fig. 2A, reducing sugar content (mg/ml) is shown as a function of hydrolysis temperature (all CTec2; 10 FPU/g RPS) over time. Fig. 2B shows the effect of Tween 80 addition on enzymatic hydrolysis, where the numbers above bars denote percent of cellulose fraction hydrolyzed (all CTec2; 10 FPU/g RPS).

[0013] Figure 3 shows a graph of yeast growth in recycled paper sludge (RPS) hydrolysate enriched with yeast extract (YE) as measured by a plate reader at 600 nm. Light path length is 3 mm.

[0014] Figures 4A and 4B show UV-visible spectra of acidic dyes, Acid violet 17 (Fig. 4A) and Acid red 131 (Fig. 4B), with the continuous line representing before addition of RPS hydrolysis remnants and the dashed line representing after addition of such.

[0015] Figures 5A and 5B show graphs of streaming potential coupling coefficient (dUstr/dP) (Fig. 5A) and Fourier-transform infrared spectroscopy (FTIR) of raw recycled paper sludge (RPS; no ozone), ozone-treated RPS (15 min ozonation), and remnants of RPS hydrolysis (hydrolysis remnants).

[0016] Figure 6 shows a block diagram of an embodiment of the stages, elements and parameters of the present process, where each block is marked according to the legend below with blank boxes indicating no data or not applicable. The numbered diamonds represent the process number (the order of the different stages in the process).

[0017] Figure 7 is a generic schematic illustration of the present process.

[0018] Figure 8 A shows bacterial contamination on an LB plate from pieces of cotton fabric with (left side of plate) or without (right side of plate) ozone treatment and Figure 8B is a graph showing the effect of ozonation on remaining process waters by measuring total phenols released.

[0019] Figure 9 is a graph showing thermogravimetric analysis (TGA) of different cellulose-based materials (Biolose, cotton fibers with and without ozone treatment, RPS with and without ozone treatment, and mixed fibers with and without ozone treatment) for humidity release.

[0020] Figures 10A and 10B show the UV-Vis spectra (Fig. 10A) and a graph of total phenol content (Fig. 10B) of process water after ozonation of different paper models.

[0021] Figure 11 shows imagery of RPS Bacillus licheniformis contamination at different magnifications: xO, x5.8, x9, xl8 (starting at top left and going clockwise).

[0022] Figure 12 is a graph showing growth of yeasts in ammonium sulfate enriched RPS hydrolysate.

DETAILED DESCRIPTION OF THE INVENTION

[0023] Paper-recycling refuse sludge is a common waste product of cardboard and paper recycling. To date it is landfilled due to lack of feasible solution. The present invention developed from a study demonstrating that this waste can be utilized as feedstock for bioethanol production for the energy sector, as well as a low-cost bio sorbent for treatment of textile wastewater. Development of feasible integrated process may enhance utilization of industrial paper sludge and allow reduction of production. In addition, The present process/method utilizes cellulose recycling or waste material and is thus capable of reducing waste production and dumping costs such as in reducing the volume of RPS sludge, cotton textile waste, and agricultural waste. By using cellulose recycling or waste material to produce resources for other industries, the present process serves to make paper, cardboard and cotton textile production and recycling more of a circular economy.

[0024] Cardboard and paper recycling are common practices resulting in large amounts of RPS. As an example, the Hadera paper mill in Israel generates 31,000 tons of RPS per year that can be used for ethanol production, which may solve the costs for disposal of the paper waste sludge waste. However, the present process is not limited to RPS as feedstock but can be used with other cellulose-based material as feedstock, such as any type of paper, cotton textiles and fabric including cotton blends with other materials, agricultural and garden wastes such as straw, twigs, and other trimmings, and other cellulose or lignocellulosic material.

[0025] The present process/method utilizes a short ozonation pretreatment, resulting in only minor removal of lignin, and allows for efficient enzymatic conversion of the cellulosic fraction of the RPS (as one source of the cellulose recycled or waste material) to sugar and the formation of ethanol (-34% enzymatic conversion of cellulosic fraction to sugar and -15 g/L formation of ethanol or about 94 g ethanol per kg cellulose material (RPS) in one embodiment with RPS), with solid remnants further used as a bio sorbent for removal of contaminants such as dyes from a wastewater stream. The present inventors have found low dose ozonation to be an effective pre-enzymatic treatment and have demonstrated the potential contribution of RPS to the circular economy concept by reducing waste while generating a resource from that waste in the present process/method. Accordingly, ozonation of cellulosic and lignocellulosic wastes offers a simple pretreatment, which uses much less space and energy than other common pretreatment methods. Ozone can also be generated locally and on demand, thus enabling a decentralized pretreatment facility to operate near the feed source and overcome transportation costs. [0026] The process/method according to the present invention provides a novel ozone-based technology for conversion of cellulose biomass waste from various sources such as RPS, cotton textile waste, agricultural waste, etc., to ethanol and optionally but preferably to biosorbent material (i.e., for treating wastewater), using ozonation as a decentralized process, as shown schematically in Fig. 7. The present invention further provides a process for producing nanocellulose such as crystalline nanocellulose as an alternative value added product by treating the same various sources of cellulose biomass waste such as RPS, cotton textile waste, agricultural waste, etc, with ozone similar to the process for producing ethanol but without proceeding with enzymatic hydrolysis and fermentation to produce sugars and covert to ethanol and instead having different downstream processing steps.

[0027] The process/method according to the present invention includes suspending a cellulose recycling or waste material in water or a buffer solution. A buffer solution is preferred so as to provide the pH or pH range suitable for enzymatic hydrolysis of cellulose to sugars, preferably a pH in a range of 4.8 to 5.2, more preferably 4.9 to 5.1, and most preferably about 5.0. Acetate and citrate buffers are non-limiting examples of suitable buffers.

[0028] In case grinding of the cellulose recycling or waste material to small pieces is needed, this can be done prior to suspension in water or buffer solution. When grinding is necessary to reduce the size of the cellulose recycling or waste material, the grinding generates ground pieces that pass through a sieve with a screen size in a range of about 1 to 3 mm, preferably about 2 mm.

[0029] After suspending the cellulose material water or buffer solution, the suspended cellulose material is treated with ozone in a range of transferred ozone dose (TOD) of about 5 to 25 mg 0₃/g, with some preferred embodiments being in a range of, for example, about 14 to 20 mg 0₃/g or in a range of about 6 to 9 mg 0₃/g. The optimal TOD can be determined based on the type of cellulose material to be treated. For instance, for RPS materials, the TOD is preferably in a range of about 14 to 20 mg 0₃/g, most preferably about 17 mg CE/g, whereas for cotton textiles and fabrics, the TOD is preferably in a range of about 6 to 9 mg 03/g, most preferably about 7.5 mg 03/g.

[0030] Prior to enzymatic hydrolysis of the cellulose material in the process for producing ethanol according to the invention, the suspended cellulose material treated with ozone is concentrated into a slurry, such as by sedimentation (and removal of part or all the liquid supernatant layer above the sedimented solids) to preferably arrive at a suspension of cellulose material that is in a range of about 15% to 25% cellulose material in water/buffer, preferably about 20%.

[0031] The cellulose material concentrated into a slurry is enzymatically hydrolyzed with a mixture of cellulase enzymes to produce sugars in the resulting hydrolysate. The mixture of enzymes for hydrolysis of the cellulose material is a blend of several enzymes from among various cellulases, b-glucosidases, hemicellulases, exo- and endo- glucanases. Suitable commercially available enzyme blends for use in the present process include CELLIC CTec2 and CTec3 from Novozyme (sold through Sigma- Aldrich) and ACCELLERASE 1000 and 1500 from DuPont (sold through Sigma- Aldrich). The manufacturer recommended temperature for enzymatic hydrolysis of cellulose material is in a range of 50°C to 65 °C and optimal temperature for the enzymatic hydrolysis may be different depending on the source of the cellulose material. For RPS however, the temperature is preferably in a range of about 39°C to 45°C, more preferably 39°C to 42°C, most preferably 40°C to take into account that there is less of a decrease in yield of sugar over time possibly due to microbial consumption of the resulting sugar, as observed in Example 1 hereinbelow. In general though, the temperature range for enzymatic hydrolysis is in a range of 39°C to 50°C.

[0032] For the enzymatic hydrolysis, it is preferred that a non-ionic surfactant such as polyethylene glycol sorbitan monooleate (TWEEN 80) or polyethylene glycol sorbitan monolaurate (TWEEN 20) be present to enhance enzymatic hydrolysis of cellulose. The concentration of the non-ionic surfactant, most preferably TWEEN 80, used for enhancing enzymatic hydrolysis of cellulose is in a range of about 0.03 to 0.06 g/g cellulose material. [0033] The hydrolysate that is mixed with the solid remnants of the slurry of cellulose material is filtered to separate the hydrolysate from the solid remnants in the slurry. The sugars in the filtered hydrolysate (filtrate), produced by enzymatic hydrolysis of the cellulose material, are fermented with yeast by adding growth medium components for yeast, such as for instance yeast extract, to the filtered hydrolysate and incubating at a temperature in a range of preferably 30°C to 40°C, more preferably 37°C, over a period of time in a range of 36 to 60 hours, preferably 40 to 50 hours, most preferably about 48 hours to produce ethanol by converting the sugar to ethanol. The yeast can be any yeast suitable for converting sugars to alcohol such as for example Saccharomyces cerevisiae strains and preferably a rapid fermenting, temperature tolerant strain such as the commercially available ETHANOL RED strain.

[0034] The sugars can also be fermented for the production of lactic acid (as by lactic acid bacteria) or by any other organism capable of fermentation of sugars to ethanol, butanol or any other fermentation product.

[0035] The fermentation by yeast to produce ethanol from sugars results in a mixture of ethanol and water (water/ethanol blend) which is distilled to separate ethanol from water to produce ethanol as the distillate.

[0036] While the main aspect/core of the process according to the present invention is for producing ethanol from cellulose recycling or waste material, a byproduct from a stage of this core process can also be utilized, i.e., solid remnants of enzymatic hydrolysis can be used as a biosorbent (such as to clean wastewater of contaminants, e.g., dyes from textiles, etc.), in an optional but preferable add-on stage to the core process for producing ethanol.

[0037] Accordingly, as an optional but preferable add-on stage in the process for producing ethanol, wastewater is treated with the solid remnant byproduct of the filtering step (to separate solids from the hydrolysate) acting as a biosorbent to remove contaminants such as dyes from the wastewater stream (e.g., from textile wastewater). It was discovered in the study with RPS in Example 1 that an increase in the total charge of the RPS after both ozonation pretreatment and enzymatic hydrolysis is evident by the zeta potential. The solid remnants after ethanol production exhibited high surface charge, and served as effective bio-sorbent for removal of textile dyes from wastewater. The total increase in surface charge and the changes in fiber functional groups may imply that the dye sorption is governed by ionic interaction with new binding sites on the solid remnants having been exposed by the enzymatic hydrolysis treatment.

[0038] To reduce water usage, the process water leaving a process in one or both of the steps of concentrating the suspended cellulose material and absorbing contaminants in wastewater with solids from the filtering step is recycled back for use in suspending the cellulose material in the process.

[0039] Another process in which an added value product can be prepared from the ozone treatment stage of cellulose biomass waste such as RPS, cotton textile waste, agricultural waste, etc, is for producing nanocellulose (e.g., crystalline nanocellulose), where the cellulose material, such as RPS as a preferred embodiment, after ozonation is dried, preferably oven dried at 50°C overnight. RPS as a preferred embodiment of the ozonated cellulose material is then mixed with maleic acid to form a suspension, where the suspension is incubated at about 120°C (120°C ± 3°C) for 80 to 120 minutes, preferably about 90 minutes (90 min ± 5). RPS is preferably mixed in a 10-15% suspension by weight (10-15 g solids per 100 ml) but no more than 20% solids. The acid hydrolysis reaction is terminated by diluting with added deionized water (DIW), such as with 1.5 to 2 times the volume of the suspension, and then with the solids immediately vacuum-filtered and dried, preferably oven dried at 50°C overnight. The dried acid hydrolyzed cellulose material is diluted with DIW (about 100 mL DIW per gram dried treated cellulose material), sonicated in a sonication bath (preferably for about 20 minutes), and sedimenting by centrifuged and washed, preferably several times, for example, at 12,000 rpm for approximately 12 minutes, until appearance of turbidity (indicating a turbid suspension). The turbid suspension is dialyzed against DIW for several days (such as dialysis bags with MWCO 14 kDa). The dialyzed suspension is then centrifuged at 6000 rpm for 20 minutes to remove large fibers. The nanocellulose obtained the supernatant had dimensions in a range of about 1800 nm to about 3000 nm in length (average experimentally 2431 ± 571 nm) and about 130 nm to about 200 nm in width (average experimentally 165 + 37 nm), with the average aspect ratio calculated as about 14 to 20. The supernatant fraction is sonicated again preferably for about 20 minutes to obtain a colloidal suspension of nanocellulose and produce nanocellulose. The total amount of recovered nanocellulose is determined by chemical oxygen demand (COD) readings (Wang et al., 2012), using COD tubes (Lovibond, England) containing 1,500 mg/L of the oxidation reagent potassium dichromate (K2Cr207). A 2-mL aliquot of the extracted solution is added to the tubes and heated for 2 hours in a COD reactor (Hach, DRB200, USA) at 150 °C. Then the tubes are tested in a colorimeter (Hach,

DR 1890) to determine the concentration of organic material in the sample. A calibration curve was prepared using a known Cellulose Nano Crystal Powder (Nanografi, Turkey).

[0040] The stages, elements and parameters of an embodiment of the present process for producing ethanol, including the add-on stages of utilizing the solid remnants of filtration of hydrolysate, and the present process for producing nanocellulose are shown in the block diagram of Fig. 6.

[0041] Cellulosic and lignocellulosic waste are abundant, promising and sustainable feedstock for ethanol, but require costly and polluting pretreatment, that often result in toxic byproducts. Ozonation is a nonpolluting, effective pretreatment method, but is not used commercially due to the high-energy demand for the production of the high ozone doses needed when working with gaseous phases. The present inventors have demonstrated experimentally in Example 1 that short (i.e., low dose) ozonation of water- submerged waste improved enzymatic saccharification efficiency compared to non- ozonized sample, even with minor removal of lignin, showing that there is no need for delignification (in contrast to common hypothesis) to obtain high saccharification efficiency. Ozonation was demonstrated as a superior pretreatment alternative treatment to the common treatment of acid hydrolysis, resulting in high sugar release and improved net energy balance, on Recycled Paper Sludge (RPS) waste material, a model for lignocellulosic paper waste and a preferred source of feedstock. Short ozonation treatment resulted in efficient enzymatic conversion of the cellulosic fraction of the RPS to sugar and production of ethanol. The solid remnants after ethanol production exhibited high surface charge, and served as effective bio-sorbent for removal of textile dyes from wastewater. A third application is the production of nanocellulose (NC) materials from such wastes. NC are rod-like cellulose whiskers of various dimensions (from tens of nanometers to several micrometers in length and up to 50 nm in width) and can be used as building blocks in many industrial applications. Lignin removal is a necessary step for the NC production process as lignin reduces separation efficiency of wood materials into its component. NC production from RPS material was demonstrated following short ozonation treatment.

[0042] Process water can potentially be treated and recycled, thereby reducing water usage and contamination. In contrast to most published processes where ozone pretreatment is done on moist biomass in the gaseous phase, the present inventors utilize the high solubility of ozone in water (13 times higher in water than in air) allowing for better performance due to reduced ozone reaction time. The ozone converts to oxygen, thus making further cleaning steps unnecessary and allowing reduction of time and labor costs. Increasing ozone doses resulted in an increase of NC production.

[0043] The present invention can be used for improved conversion of cellulose material such as paper, textile and agricultural waste to ethanol as a bio-fuel or for the chemical industry and/or production of NC. The use of the remaining material (solid remnants) for either water treatment applications or NC productions, allows reducing shipping and landfilling costs. This is of interest for commercial agents for the agriculture and forestry industries, as well as for waste producers.

[0044] To date Israel has no commercial scale production of ethanol, and thus is importing -23,000 tons of ethanol for 22.5 million dollars annually (2018 data from the CBS). Using the process/method according to the present invention on cellulose- containing waste, countries can become mostly ethanol self-proficient rather than dependent on imports. Moreover, as motorized vehicles in the world will hit 1.7 million by 2035, the need for bioethanol fuel is rapidly increasing to augment the projected fuel consumption, which is expected to reach 21.9 million gallons per day by 2035. [0045] Three different products can be produced by the processes according to the present invention including the add-on for utilizing a byproduct of the process for producing ethanol:

[0046] Ethanol - as a biofuel or as medical ethanol as a disinfectant. The end user is gasoline companies or hospitals, and companies that make sanitizing products such as wet wipes, etc. Other fermentation products can also be produced (butanol, lactic acid, acetone, etc.) from sugars as well.

[0047] Low-cost bio-sorbent - water and industrial treatment for textile where the end user is water companies and filtration companies

[0048] Nanocellulose - for developing materials including thin films and nanocomposites where the end user is in biomedicines, water treatment and smart materials manufacturing.

[0049] Where the feedstock in the present process is cotton textiles, another application for the waste textile is for production of products with properties of humidity accumulation/release as part of "climate-controlling walls" - walls that can cool by releasing humidity (like out-bodies) and heat by absorbing humidity. To this end, low- cost materials that are effective in absorbing/releasing humidity are needed and hence the generation of such products from waste textile, e.g., by using the solid remnants from the present process, is of interest.

[0050] Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration and is not intended to be limiting of the present invention.

EXAMPLE 1

[0051] Four different wastepaper materials were examined — recycled paper sludge, newspaper, paper towels and printing paper — as potential feedstock for ethanol production. Recycled paper sludge was the most suitable feedstock, and ozone pretreatment and ethanol production were further examined. The goal of this study was to evaluate RPS as feedstock for ethanol production. After careful evaluation of the waste- papers’ characteristics and its reaction with ozone, the production of ethanol from ozonated RPS was demonstrated. The solid remnants were used as sorbents for textile contaminants in wastewater.

[0052] The results present below show that RPS can be used for ethanol production and wastewater treatment, promoting circular economy (CE) and sustainability principles. Short ozonation treatment resulted in enzymatic conversion of -34% of the cellulosic fraction of the recycled paper sludge to sugar and production of -15 g/L ethanol. The solid remnants, used as bio-sorbent, efficiently removed textile dyes from water.

Recycled paper sludge waste was thus a good source for both energy and water-treatment applications, increasing sustainability in the paper and cardboard recycling industry.

MATERIALS AND METHODS Paper materials

[0053] RPS was kindly provided by the Hadera Paper mill (Hadera, Israel). Paper towels (PT) were purchased from Dalas Paper Products Ltd. (Israel). Print paper (PP) was from Target Corporate (Russia) and Newspaper (NP) was a daily newspaper. All raw paper materials were dried and ground in a 250 W laboratory blade mill (MRC Ltd., Israel) and sieved through a 2-mm mesh screen (450 W, ALS Ltd., Israel).

Materials and chemicals

[0054] Sodium carbonate, sodium hydroxide, sodium azide, amylase, pullulanase, anthrone reagent, acetyl bromide, ethyl acetate, sodium chloride, hydroxylamine hydrochloride, and ammonium hydroxide were all obtained from Sigma-Aldrich (Israel). Hydrochloric acid, methanol, and acetone were from Merck (Germany). Glacial acetic acid was from Lischer Scientific (UK). Nitric and sulfuric acid were from Bio-Lab (Israel). Acidic dyes (Acid violet 17 and Acid red 131) were from Colourtex Ltd. (India). Working solutions were prepared with deionized water (DIW) (Direct-Q3 UV System, Millipore). Cellic® CTec2 was a kind gift from Novozyme.

Enzyme activity [0055] Cellulase activity was measured by the Filter Paper Assay and expressed in filter paper units (FPU), based on the protocol presented by Eveleigh et al. (2009).

Briefly, a 1 X 6 cm² strip of Whatman filter paper no. 1 was placed in a test tube containing a mixture of 0.5 mL diluted enzyme (1:100 in DDW) and 1 mL of 0.05 M sodium acetate buffer at pH 4.8. The tube was incubated at 50°C for 60 min, and the reaction terminated by adding 3 mL of Dinitro salicylic acid (DNS) reagent and boiling for 5 min at 95°C (Fernley, 1963). The samples were chilled for 5 min and 10 mL of DIW was added. Absorbance at 540 nm was read in a 96-well plate (200 pL per well) (Spark 10M, Tecan, Switzerland) and quantified using glucose standard curve treated and read the same way. One unit was defined as 2 pmol of glucose equivalents released per minute for 1 mL enzyme.

Yeasts and growth conditions

[0056] Yeasts used were Ethanol Red and were regularly grown on YPD broth (1% yeast extract, 1% peptone and 2% glucose in DIW; all w/v) or YPD agar (same with 1.5% agar added). Lor nitrogen source experiments, inoculum was grown overnight (30°C, shaking at 150 rpm, and diluted into 96- well plate wells - 10 pL inoculum, 90 pL RPS hydrolysate and 10 pL additional nitrogen source (yeast extract or ammonium sulfate). Plate was covered with a sealing breathing polyurethane membrane and placed in a plate reader. Absorbance was read at 600 nm, every 30 min for 6 h, with 10 s of shaking prior to each well reading. Atmosphere was kept at 2% oxygen-98% nitrogen by the plate reader. OD600nm at t=0 was used as blank for each well.

Compositional and carbon-to-nitrogen ratio analyses

[0057] Cellulose and lignin content were determined in triplicates by the protocol of Poster et al. (2010a, 2010b). Two mg dry material was weighed into 2-mL screw caps tube, 1 mL acetic acid-water-nitric acid mixture (8:2:1, v/v) added, and the suspension heated in a boiling water bath for 30 min. After cooling, the tubes were centrifuged (10,000 rpm, 15 min) and supernatant discarded. Pellet was thoroughly washed once by resuspended and centrifugation with 1.5 mL DIW, and three more times with 1.5 mL acetone. The pellets were dried overnight at 50°C and dry pellet was mixed with 175 pL of 72% (v/v) sulfuric acid, and the suspension incubated for 45 min. Volume of 825 pL DIW was added, and cellulose content assayed using the colorimetric anthrone assay. A 100-pL aliquot was transferred to each well of flat-bottom 96-well plate (Costar, USA), 200 pL anthrone reagent added, the plate mixed and heated to 80°C for 30 min. Absorbance at 625 nm was measured using microplate reader. A calibration curve was prepared using glucose.

[0058] Soluble lignin content was determined in triplicates by the acetyl bromide method, which has proven to be better, simpler and faster than other lignin-recovery methods, and to exhibit higher recovery yields (Moreira-Vilar et al., 2014). Two mg material was weighed into screw cap tube and 100-pL volume of freshly made acetyl bromide solution (25% v/v acetyl bromide in glacial acetic acid) was added. The samples were heated at 50°C for 3 h, and after cooling 400 pL of 2 M sodium hydroxide and 70 pL of freshly prepared 0.5 M hydroxylamine hydrochloride were added, and the tubes filled with glacial acetic acid to final volume of 2 mL. A 200-pL aliquot of the resulting solution of acetyl bromide-soluble lignin (ABSL) transferred into UV-transparent flat- bottom 96- well plate and absorbance at 280 nm was read using microplate reader. Percent ABSL was determined by:

where ‘abs’ is the absorbance at 280 nm, Coeff. = 18.21, and the number 0.526 the path length (which depends on plate type and fill volume, here 96-well and 200 pL).

[0059] Acid-insoluble lignin was determined (in triplicates) according to Petti et al. (2013). Briefly, 300 mg of dried material was placed in a 50-mL test tube and hydrolyzed with 3 mL of 72% sulfuric acid for 2 h, in a 30°C water bath. Tubes were stirred every 15 min with a glass rod. After hydrolysis, the mixtures were transferred to 250-mL Erlenmeyer flasks and autoclaved at 121°C for 1 h. DIW (84 mL) was added and the diluted hydrolyzed sample vacuum filtered through pre-weighed Whatman GF/A filter. The filter was dried overnight at 105°C, weighted again, and then placed in a furnace at 575°C for 4 h and cooled in a desiccator for an additional 1 h. Final weight was recorded for the determination of total acid-insoluble lignin (i.e., weight lost in the furnace).

[0060] Ash content was analyzed according to TAPPI (2002). Briefly, the samples were weighed and heated in a furnace at 525 ± 25°C for 6 h, and then cooled in a desiccator. Once cooled to room temperature, the samples were reweighed. The ash content was calculated as:

where A and B are the sample weights (g) before and after the procedure, respectively.

[0061] Carbon- to-nitrogen ratio was determined in triplicate using a Thermo Flash

EA 112 elemental analyzer equipped with CHNS and O reactors (Thermo Fischer Scientific, USA), after drying and grinding to a particle size of 0.2-0.5 mm diameter using liquid nitrogen and pastel and mortar. Sample of 2-3 mg was wrapped in tinfoil capsule and purged with helium, which served as the carrier gas. Samples were dropped into a vertical quartz tube maintained at 1 ,020°C and packed with two separate layers of catalyst, serving as combustion reactor for the oxidation and reduction stages. A mixture of combustion gases was formed, pushed through a layer of closely packed pure-copper wire to remove excess oxygen and reduces nitrogen oxide, and carried to a chromatographic column where nitrogen, carbon dioxide, water and sulfur dioxide were separate. Components were detected by a thermal conductivity detector connected to the gas pathway after the column.

Thermogravimetric Analysis

[0062] Thermogravimetric analysis (TGA) was conducted on the RPS as is or after 15 minutes ozonation. For post-ozonation, the slurry was allowed to settle, the liquid phase discarded, and the wet solids dehydrated at 105°C overnight and milled with coffee grinder. Analyses were performed with a high sensitivity thermogravimetric analyzer (Q5000 TGA-IR, TA Instruments) operating from ambient temperature to 800 °C at a heating rate of 10 °C min-1, with nitrogen purging. Initial sample weight was 8-13 mg (all according to Rosen et al., 2019). The output was plotted as derivative weight loss vs temperature (differential thermogravimetric kinetic curve, hereafter, differential curve).

To determine specific components, the results were compared to those previously reported in Rosen et al., 2019.

Ozonation treatment

[0063] Ozone treatments were performed in a semi-continuous batch reactor with ozone gas generated by oxygen-fed ozone generator (up to 4 g/h; BMT 802N, Germany). Twenty grams of paper material were suspended in 500 mL of 0.1 M acetate buffer pH 5 (made in tap water) and the oxygen-ozone mixture bubbled directly into the glass reactor using 7.95 cm³ diffuser with nominal pore size of 25 pm with gas flow of 0.35 L/min. Reactor dimensions were 15 cm in height and 9 cm in diameter as demonstrated by Peretz et al. (2017). pH levels were adjusted as needed with 1 M HC1. Accumulated ozone reacting with the material was determined as transferred ozone dose (TOD; equation 3 below) by continuous measurements of ozone concentrations in the in-gas and off-gas (Peretz et al., 2019):

where Co3_, in is ozone concentration in the in-gas, Co3_, out is ozone concentration in out-gas (i.e., unreacted ozone), and dt is the time interval between measurements (here, 1 min).

Total phenol content

[0064] Total phenol content was determined using the Folin-Ciocalteu (F-C) method (Carnegie Institute of Science, 2011). Briefly, a 100-pL sample of ozonation process water was placed in a 2-mL tube and mixed with 200 pL of 10% (v/ v) F-C reagent in DIW. Samples were covered and incubated for 30 min at room temperature, mixed with 800 pL aqueous 700 mM sodium carbonate, incubated at room temperature for another 2 h, and 200-pL aliquot transferred to a 96-well plate. Absorbance was determined at 735 nm. A calibration curve was prepared using gallic acid and phenol concentrations were reported as gallic acid equivalents.

Isolation and identification of microbial contaminants in recycled paper sludge

[0065] Raw and ozone treated RPS were stirred in sterile ultra-pure water for 10 min, followed by water sampled with sterile microbial loop and streaked on lysogenic broth (LB) agar plates. The plates were incubated for 3 days at 50°C, the temperature at which the drop in reducing sugars was observed. Colonies were isolated five times on LB agar, 16S and rpoB genes were amplified from a signal colony, and the PCR products were sequenced (Ben-Gad et al., 2017) and compared to the genebank using BLAST.

Enzymatic hydrolysis

[0066] After ozonation pretreatment, the solid concentration was increased to 20% w/v by allowing solids to settle and decanting buffer at a final volume of 100 mL. Cellic CTec2 enzyme and Tween 80 were added to the slurry. Hydrolysis was performed at 40°C temperature, with frequent adjustment of the pH to 5 using 1 M HC1. After enzymatic hydrolysis, solid remnants were collected by vacuum filtering through Whatman GF/A filter. Alternatively, for smaller volumes, hydrolysate samples were centrifuged (10,000 rpm,10 min).

Ethanol determination

[0067] Ethanol concentration was determined by GC-FID according to Gerchman et al. (2012). Briefly, 1 mL of fermentation was centrifuged, and 0.495 mL supernatant was moved to a new Eppendorf tube; five pL n-Butanol added (as internal standard), and the mixture extracted by adding 0.5 mL ethyl acetate, vortexing 5 min, and waiting for phase separation. One pL of the upper (organic) phase was injected into a GC-FID (SRI- GC 8610) equipped with on-column injector and a 60 m capillary MTX-1 column (Restek, USA), 0.53 mm ID and 5 pm coating. Temperature program started at 70°C, raised 10°C/min to 120°C and held for 1 min. Nitrogen (99.999%) was used as the carrier gas. Ethanol, n-butanol and ethyl acetate retention times were determined by injecting standards, and calibration curve by extracting and injecting known solutions of ethanol in water (with n-butanol internal standard). Ethanol peak to n-butanol peak ratio was used all over.

Dye removal from water with recycled paper sludge or its remnants

[0068] Different RPSs were tested for dye adsorption in duplicates: raw RPS, RPS after ozonation, and RPS solid remnants after enzymatic hydrolysis. All RPSs were dried overnight and ground as above. For each tested adsorbent 0.1 g were mixed with 10 mL of 100 ppm dye solution in 15-mL centrifuge tubes. Tubes were mixed for 30 min and then centrifuged (15 min, 6,000 rpm) for solids removal. Supernatant absorbance was analyzed for each dye using the characteristic absorbance peak wavelengths (550 nm for Acid red 131 and 540 nm for Acid violet 17). Calibration curves were plotted for each dye.

Determination of zeta potential

[0069] Zeta potentials were determined from streaming potential measurements according to Peretz et al. (2019). Analyzer was SurPASSTM 3 electrokinetic (Anton Paar GmbH, Austria) equipped with a conductivity probe and a pH electrode. Samples were dried overnight in 50°C oven, placed in cylindrical measuring cell and compressed to create a sample plug with controlled permeability to water flow. Aqueous 0.01 M NaCl was passed through the sample plug, generating a streaming potential. Pressure difference between the two ends of the sample started at 600 mbar and lowered continuously to 200 mbar, with measurement starting at high pressure. Streaming potential measurements at various pHs (in triplicates) was done by automated titration with 0.05 M HC1 from high to low pH to avoid effects of acid or changing ionic strength. Zeta potential was calculated from streaming potentials using the Helmholtz-Smoluchowski equation (Luxbacher et al, 2016):

where g is the zeta potential (mV), dU_str (mV) is the streaming potential, dP (mbar) is the pressure gradient, h and £ are the viscosity (mPa s) and dielectric coefficient of water, respectively, £o is the vacuum permittivity (8.854 x 10 ¹² As/Vm), and I _B is the electrical conductivity (mS/m).

Fourier- transform infrared spectroscopy

[0070] Fourier-transform infrared (FTIR) analysis was conducted using attenuated total reflection (ATR)-FTIR spectroscopy (Tensor 27-IR, Bruker, USA).

RESULTS AND DISCUSSION Compositional analysis

[0071] The properties of the RPS materials were found to be as follows (all % of dry weight): 40.74 ± 0.24% carbon and <0.1% nitrogen; 75.32 ± 20.21% cellulose; 18.04 ± 2.22% total lignin, out of it 10.71 ± 2.21% was acetyl bromide-soluble lignin (ABSL) and 7.34 ± 0.22% non- ABSL soluble; 15.45 ± 0.44% ash. The isoelectric point of both RPS and NP (i.e., pH~2) were in the range of those reported for pure cellulose (Ingle sby et al, 2005; Pothan et al, 2002) and higher for PP (~3), probably due to the presence of additives (Table 1).

Table 1

Properties of paper towels (PT), printing paper (PP), and newspaper (NP).

data; TOD, transferred ozone dose.

[0072] Those results were confirmed by thermogravimetric analysis, that demonstrated a strong peak at 366°C (Fig. 1), correlating to cellulose (Rosen et al, 2019). Given that RPS material exhibited the highest cellulose and lowest total lignin content (Table 1), and given that RPS is the end-product of the paper-recycling process, and landfills are the only option currently practiced for this waste., RPS was chosen as the feedstock to explore for ethanol-production testing, with the properties of RPS shown below in Table 2.

Table 2

Properties of recycled paper sludge (RPS)

ABSL, acetyl bromide-soluble lignin; IEP, isoelectric point; NA, no data; TOD, transferred ozone dose.

Transferred ozone dose and effect of ozonation treatment on paper models [0073] The Transferred Ozone Dose (TOD) — the accumulated amount of ozone transferred to liquid phase in the ozonation process, is the ozone available for reaction. RPS and PT accumulated the lowest TOD (Table 1), while NP and PP exhibited high TOD probably due to the reaction of ozone with the ink (in NP)(von Sonntag et al., 2012) and with optical whitener (PP) (Shadkami et al., 2011). When the process waters were analyzed at the end of the ozonation process, the UV-visible spectra showed notable increase in absorbance, with peaks at 206 and 280 nm (Fig. 10A), suggesting release of organic and aromatic compounds. Indeed, when total phenolic concentration was determined by the F-C method, an increase was found in all experiments (Fig. 10B), supporting degradation of lignin and release of small phenolic substances from the paper wastes during ozonation (Rosen et al., 2019; Peretz et al., 2017). It is noteworthy that an especially high increase in phenol content was observed with NP (Figs. 10A and 10B), in good agreement with its high lignin content and the presence of ink.

Determination of optimal conditions for enzymatic hydrolysis

[0074] Hydrolysis conditions were optimized for RPS as the most suitable waste. Recommended temperatures for Cellic CTec2 (59.08 ± 2.53 FPU/mL) are 50-65°C although in some cases lower temperatures were found to produce higher sugar yields (Li et al., 2012). To determine the optimal temperature, 1 g of ozone treated RPS was suspended in 50 mL of 0.1 M acetate buffer and mixed with 0.04 mL (2.5 FPU/g RPS) of CTec2, and further shaken at 40°C, 50°C or 60°C (each in triplicate). Reducing sugar content was evaluated by the DNS method (Fig. 2A). Hydrolysis at 40°C and 50°C showed similar yield after the 4-h mark, which was higher than at 60°C. However, at both 50°C and 60°C, but not at 40°C, reducing sugar concentration decreased between 4 and 20 h, suggesting sugar consumption. Interestingly, RPS steam-sterilized prior to hydrolysis showed similar hydrolysis but no decrease in sugar concentration at 50°C hydrolysis, suggesting a microbial consumption of the resulting sugar. Indeed, when raw RPS sample was suspended in sterile water, and water samples plated on LB agar, bacterial growth of single morphotype was observed (Fig. 11). The bacteria were identified by 16S and rpoB amplification and sequencing as Bacillus licheniformis, a common environmental spore -producing bacterium (RPS is stored in open piles) with optimal growth temperature of 50°C. As sterilization of large amounts of RPS sample is both time- and energy- consuming, all further hydrolysis experiments were carried out at 40°C.

[0075] Tween 80 surfactant has been shown to enhance the enzymatic breakdown of cellulose (Kurakake et al., 1994; Kleingesinds et al., 2018). Hence the effect of different doses of Tween 80 on the enzymatic hydrolysis of ozone treated RPS was tested, with the finding that 0.03 g/g increased total reducing sugar content by 50% (Fig. 2B).

Enzymatic hydrolysis and the effect of nitrogen addition

[0076] As all paper wastes were found to be very low in nitrogen (<0.1% DW; Table 1), the effect of nitrogen addition was tested. To this end, ozone-treated RPS was hydrolyzed under optimal conditions (40°C; 0.03 g/g Tween 80; solid concentration 20% w/v; 7.5 FPU/g CTec2; 48 h mixing at 300 rpm), resulting in 51.43 ± 5.03 g/L of reducing sugars (by DNS), indicating conversion of 34.3 ± 3.35% of the cellulose fraction to sugars. After separation by filtration, the hydrolysate was used to test the effect of addition of nitrogen (yeast extract or ammonium sulfate) on growth of the yeast at micro- aerobic conditions, as an indicator of fermentation capacity. The results presented in Fig.

3 show that addition of yeast extract had a positive dose-dependent effect on growth, while ammonium sulfate had no effect (Fig. 12).

Fermentation experiments

[0077] Fermentation experiments were performed on RPS hydrolysate prepared as before and enriched with 2% w/v yeast extract. To this end 40 mL of hydrolysate were placed in 50 mL glass bottle and 5 mL of each 20% yeast extract and 5 mL yeast culture grown over-night in YPD were added. The bottle was incubated for 48 h at 37°C with mixing at 150 rpm, resulting in 14.90 ± 0.02 g/L ethanol (94.42 ± 0.13 g/kg RPS), better than previously reported for RPS after steam sterilization/pretreatment (Schroeder et al., 2017), although direct comparison is difficult due to differences in RPS origin and composition.

Removal of textile dye from water with hydrolyzed recycled paper sludge remnants

[0078] In many countries textile industry use and release of large amount of dyes in its wastewater (Gupta 2009). Different dyes are used, but water-soluble reactive dyes and acidic dyes were found to be persistent in conventional wastewater-treatment systems (Colak et al., 2009). Accordingly, different techniques are sought for the removal of these dyes, among them sorption (Gupta 2009; Fernandez et al., 2010), sometimes to lignocellulosic material (Demir et al., 2008; Mahmoodi et al., 2011; Namasivayam et al., 1996). In this context the use of industrial byproducts/wastes as adsorbents is desirable, as these are low cost and within the "green industry" concept of reduced waste generation (Gupta 2009; Hao et al., 2017; Carpenter et al., 2015). The use of raw, ozone treated, and RPS hydrolysis remnants were tested as sorbent for two industrial textile acidic dyes:

Acid violet 17 and Acid red 131. Dye removal and uptake by RPS, presented in Table 3 below and in Figures 4 A (Acid violet 17) and 4B (Acid red 131), where the continuous line representing before addition of RPS hydrolysis remnants and the dashed line representing after addition of such, demonstrate that ozonation of RPS alone had no effect on dye removal (compared to raw RPS), while the RPS hydrolysis solid remnants showed much better dye uptake for both dyes, with little preferences for Acid red 131.

Table 3

Dye removal and uptake by recycled paper sludge (RPS)

Values with different superscript letters are significantly different by one-way ANOVA with post-hoc (p < 0.05); calculated separately for % removal and % uptake. [0079] To further investigate the differences in sorption ability, zeta potentials were determined for the three RPS stages (Fig. 5A). The streaming potential coupling coefficient dUstr/dP was observed as a function of pH at different stages of the RPS treatment (Peretz et al., 2019; Jacobasch et al., 1985). All RPSs showed negative streaming potential coupling coefficients, and negative zeta potential, with the RPS solid remnants having the highest negative charge. A plateau was observed at pH > 4, and an increase in dUstr/dP with decreasing pH, correlating well with previous work on RPS (Peretz et al., 2019). The evolution of the streaming potential coupling coefficient with pH for raw RPS was similar to that seen with cotton, regenerated cellulose, and lignocellulosic fibers (Pusic et al., 1999; Bismarck et al., 2001 and 2002). The IEP for all RPS samples remained almost unaffected at pH 2, while dUstr/dP and the apparent zeta potential showed more negative values with every additional treatment. The shift in the apparent zeta potential is attributed mainly to changes in the surface charge of the fibers due to ozonation and hydrolysis, since fiber structure is less likely to be affected (Peretz et ak, 2019).

[0080] To better understand the ozonation and hydrolysis effects, FTIR analysis was applied to the different RPSs. Ozone treatment removed the absorbance peak at 3222 cm-1 and somewhat dampened the peak at 3470 cm-1, characteristic of alcoholic and phenolic OH groups, whereas the enzymatic hydrolysis remnants showed decreased absorbance at 1000 to 1200 cm-1, characteristic of cellulose (Cintron et al., 2015). Interestingly, the enzymatic treatment also resulted in changes in 2841 cm-1 and 2400 to 1500 cm-1 (aromatic rings), and at 1270 cm-1 (guaiacyl rings), characteristic of lignin (Bykov 2008). Since changes in fiber morphology and electrokinetic characteristics of the fiber are directly correlated (Stana-Kleinschek et al., 1999), ultimately affecting and increasing sorption ability. These results fit well with the increase in the total charge of the RPS after both ozonation pretreatment and enzymatic hydrolysis evident by the zeta potential. The total increase in surface charge and the changes in fiber functional groups may imply that the dye sorption is governed by ionic interaction with new binding sites exposed by the enzymatic treatment.

[0081] Cardboard and paper recycling are common practices resulting in large amount of RPS. Here the present inventors showed that a short ozonation pretreatment, with only minor removal of lignin, allows efficient enzymatic conversion of -34% of the cellulosic fraction of RPS to sugar, and the formation of -15 g/L ethanol; where the solid remnants are further used as a bio-sorbent for dye removal from water. Thus, ozonation was found to be an effective pre-enzymatic treatment and demonstrates the contribution that RPS can make to the circular economy concept by reducing waste while generating a resource from that waste. The treated water can be recycled, thereby reducing water usage and contamination.

EXAMPLE 2

The impact of ozone on Cotton

[0082] Ozonation was performed on 100% cotton in semi-batch ozonation as described above in Example 1. Ozonation was also shown as a good disinfectant on cotton. Pieces of cotton before and after ozonation were placed carefully on LB plates (on right and left sides of the plate, respectively). Then heated DI water was dripped on the fabrics, and the fabrics were removed. The LB plates were incubated for three days at 50°C. As shown in Lig. 8A, the right side of the plates (fabric with no ozone treatment) showed higher contamination and growth compared to the left side (fabric after ozonation). The effect of the ozonation on the remaining process waters was also investigated. UV-vis scan (200-800 nm) was applied and also the L-C test for total phenols. In general, cotton appears to be affected by ozonation, releasing higher amounts of phenols into the water (Lig. 8B).

Impact of ozonation on humidity release for climate control

[0083] In addition to the above previous results demonstrating RPS remnants for water treatment application, a test for humidity release was performed. This is important for climate control applications in buildings. Different cellulose-based materials were absorbed with maximal humidity. The materials were then tested via TGA analysis for weight reduction (release of water vapors). Cellulose and cotton-based materials after ozone showed increased humidity release (Fig. 9).

[0084] Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation.

[0085] While this invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications.

This application is intended to cover any variations, uses, or adaptations of the inventions following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth as follows in the scope of the appended claims.

[0086] All references cited herein, including journal articles or abstracts, published or corresponding U.S. or foreign patent applications, issued U.S. or foreign patents, or any other references, are entirely incorporated by reference herein, including ah data, tables, figures, and text presented in the cited references. Additionally, the entire contents of the references cited within the references cited herein are also entirely incorporated by references.

[0087] Reference to known method steps, conventional methods steps, known methods or conventional methods is not in any way an admission that any aspect, description or embodiment of the present invention is disclosed, taught or suggested in the relevant art.

[0088] The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art (including the contents of the references cited herein), readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art.

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Claims

WHAT IS CLAIMED IS:

1. A process for producing ethanol from cellulose recycling or waste material, comprising: suspending a cellulose recycling or waste material in water or a buffer solution; treating the suspended cellulose material with a low dose of ozone; concentrating the suspended cellulose material into a slurry; hydrolyzing the cellulose material in the slurry enzymatically with a mixture of cellulase enzymes to produce sugars in a resulting hydrolysate; filtering to separate the hydrolysate from solids in the slurry; fermenting the filtrate, which is filtered hydrolysate, with yeast to convert the sugars to ethanol; and distilling the filtered hydrolysate to separate ethanol as the distillate from water to produce ethanol.

2. The process of claim 1, further comprising grinding the cellulose recycling or waste material before suspending in water or a buffer solution.

3. The process of claim 1 or 2, further comprising treating wastewater with solids separated from the hydrolysate in the filtering step as bio- sorbents to absorb contaminants from the wastewater.

4. The process of any one of claims 1-3, wherein the water or aqueous solution resulting from one or more of the steps of concentrating, distilling and absorbing contaminants in wastewater with the solids from the filtering step is recycled back for use in the suspending step.

5. The process any one of claims 1-4, wherein the pH hydrolyzing the cellulose material enzymatically is in a range of 4.8 to 5.2, preferably 4.9 to 5.1, most preferably 5.0.

6. The process any one of claims 1-5, wherein the pH is maintained with an acetate or citrate buffer.

7. The process of any one of claims 1-6, wherein the temperature for hydrolyzing the cellulose material is in a range of 39°C to 50°C, preferably 39°C to 45°C, more preferably 39°C to 42°C, most preferably 40°C.

8. The process of any one of claims 1-7, wherein, in the step of hydrolyzing the cellulose material enzymatically, a non-ionic surfactant is added to the slurry.

9. The process of claim 8, wherein the non-ionic surfactant is polyethylene glycol sorbitan monooleate (TWEEN 80; CAS Number 9005-65-6) or polyethylene glycol sorbitan monolaurate (TWEEN 20; CAS Number 9005-65-5).

10. The process of any one of claims 1-9, wherein the low dose of ozone in the treatment of the suspended cellulose material is in a range of transferred ozone dose (TOD) of about 5 to 25 mg 0₃/g.

11. The process of any one of claims 1-10, wherein the step of fermenting the filtrate is carried out at a temperature preferably in a range of 30°C to 40°C, more preferably 37°C, over a period of time preferably in a range of 36 to 60 hours, more preferably 40 to 50 hours, most preferably about 48 hours, to produce ethanol by converting the sugar to ethanol.

12. The process of any one of claims 1-11, wherein the yeast is a rapid fermenting, temperature tolerant yeast strain such as ETHANOL RED.

13. The process of any one of claims 1-12, wherein the cellulose recycling or waste material is Recycled Paper Sludge (RPS).

14. The process of claim 13, wherein the temperature for hydrolyzing RPS is preferably in a range of 39°C to 45°C, more preferably 39°C to 42°C, most preferably 40°C.

15. The process of claim 13 or 14, wherein the low dose of ozone in the treatment of the suspended cellulose material is preferably in a range of transferred ozone dose (TOD) of about 14 to 20 mg 0₃/g, most preferably about 17 mg 0₃/g.

16. The process of any one of claims 13-15, wherein about 34% of the cellulosic fraction of RPS is enzymatically converted to sugar.

17. The process of any one of claims 13-16, wherein the process produces about 94 g ethanol per kg RPS.

18. The process of any one of claims 1-12, wherein the cellulose recycling or waste material is cotton fabrics and textiles.

19. The process of claim 18, wherein the low dose of ozone in the treatment of the suspended cellulose material is preferably in a range of transferred ozone dose (TOD) of about 6 to 9 mg 0₃/g, most preferably about 7.5 mg 0₃/g.

20. The process of any one of claims 1-12, wherein the cellulose recycling or waste material is agricultural waste such as straw, twigs and plant trimmings.

21. A process for producing crystalline nanocellulose from cellulose recycling or waste material, comprising: suspending a cellulose recycling or waste material in water or a buffer solution; treating the suspended cellulose material with a low dose of ozone; drying the suspended cellulose material treated ozone; suspending the dried treated cellulose material in a solution of maleic acid and acid hydrolyzing the suspension at a temperature in the range of 100°C ± 3°C; terminating the acid hydrolyzing step by diluting with water to stop acid hydrolysis; filtering to obtain solids and drying into a dried acid hydrolyzed cellulose material; adding water and sonicating the dried acid hydrolyzed cellulose material; sedimenting treated cellulose by centrifugation; dialyzing the sedimented treated cellulose in a suspension against water; sedimenting the dialyzed treated cellulose by centrifugation to remove large fibers above 3000 nm in length and obtain a supernatant fraction; sonicating the supernatant fraction to obtain a colloidal suspension of nanocellulose and produce crystalline nanocellulose.

22. The process of claim 21, further comprising grinding the cellulose recycling or waste material before suspending in water or a buffer solution.

23. The process of claim 21 or 22, wherein the low dose of ozone in the treatment of the suspended cellulose material is in a range of transferred ozone dose (TOD) of about 5 to 25 mg 03/g.

24. The process of any one of claims 21-23, wherein the cellulose recycling or waste material is Recycled Paper Sludge (RPS).

25. The process of claim 24, wherein the low dose of ozone in the treatment of the suspended RPS is preferably in a range of transferred ozone dose (TOD) of about 14 to 20 mg 03/g, most preferably about 17 mg 03/g.

26. The process of any one of claims 21-23, wherein the cellulose recycling or waste material is cotton fabrics and textiles.

27. The process of claim 26, wherein the low dose of ozone in the treatment of the suspended cellulose material is preferably in a range of transferred ozone dose (TOD) of about 6 to 9 mg 03/g, most preferably about 7.5 mg 03/g.

28. The process of any one of claims 21-23, wherein the cellulose recycling or waste material is agricultural waste such as straw, twigs and plant trimmings.