1. Introduction
Natural polysaccharides constitute a wide class of natural biopolymers that comprise cellulose, starch, dextran, agar, xanthan, xylanes, mannanes, galactans, alginates, carrageenans, etc. Most of these polysaccharides are low crystalline or amorphous and readily soluble in water [
1]. A pronounced exception to this rule is natural cellulose, a highly crystalline and water-insoluble polysaccharide. As is known, cellulose is the most widespread natural biopolymer [
2]. Natural cellulose is a structural constituent of all land plants and many algae [
3]. In addition, cellulose occurs in shells (tunicates) of certain marine creatures [
4], and this biopolymer is also produced by some microorganisms, e.g.,
Gluconacetobacter xylinus [
5].
To isolate a pure biopolymer, the cellulose-containing raw material is treated with solutions of various chemicals, such as caustic soda, a mixture of sodium hydroxide with sodium sulfide, and sulfurous acid or its salts [
6]. Some other pulping methods are also used on a small scale. The pulping process is followed by multi-stage bleaching.
Studies have shown that macromolecules of natural cellulose may contain 2000 to 30,000 repeating anhydroglucose units (AGU), which are linked by chemical β-1,4-glycosidic bonds [
7,
8]. In nature, cellulose chains are connected in the transverse direction by hydrogen and Van der Waals bonds and form nanofibrils, which contain crystallites and non-crystalline (amorphous) domains.
Crystallites of natural celluloses may exist in two different crystal forms, triclinic CIα and monoclinic CIβ. Crystallites of algae and bacterial cellulose have CIα allomorph, while crystallites in the cellulose of land plants and tunicates have CIβ allomorph [
9,
10]. After specific physicochemical treatments of natural cellulose, other crystalline allomorphs, CII, CIII, or CIV can be obtained [
11].
Cellulose crystallites are stable constituents and therefore they are inaccessible to common organic solvents, water, and diluted water solutions of acids, alkalis, and salts [
12]. The crystallinity, i.e., mass fraction of crystallites in natural cellulose, ranges from 0.53–0.55 (cellulose of herbaceous plants) to 0.77–0.80 (tunicate cellulose) [
12,
13]. Due to increased crystallinity, natural cellulose exhibits low accessibility to reagents. In particular, it gives a small yield of glucose after acidic [
14] or enzymatic hydrolysis [
15,
16]. In addition, the high crystallinity of natural cellulose hampers dyeing, diffusion, and sorption of water vapor [
17], which negatively affects its use in textile materials.
To increase the accessibility, reactivity, and sorption ability of natural cellulose, it is necessary to destroy the crystalline structure of this biopolymer to obtain amorphized or completely amorphous cellulose.
A literature search on this problem showed that there are only research articles devoted to preparation methods of amorphized cellulose by grinding [
18] or processing with various solvents [
19,
20]. In addition, there are articles where some properties of amorphized cellulose are considered, e.g., acid hydrolyzability, thermal stability, etc. [
21]. However, no reviews were found on amorphized cellulose. Therefore, it was necessary to collect and analyze the appropriate literature, including the author’s own results, and then summarize them in a special review.
The main purpose of this review article was to discuss the main methods of cellulose amorphization, to describe methods for determining the structural characteristics of initial and amorphized cellulose, as well as to study the effect of structural changes in cellulosic materials after amorphization on various properties, including sorption, hydrolyzability, and enzymatic digestibility.
3. Methods of Cellulose Amorphization
Various physical and physicochemical methods can be used to destroy the crystalline structure of cellulose. Among physical methods, dry grinding of cellulose in ball mills is most widespread [
18,
35,
36]. For this purpose, cellulose sheets are dried, cut into small pieces, and subjected to preliminary disintegration in a knife mill. The resulting dry fibers are ground for 24 h, at least, in a ball mill using porcelain balls. The main problem of using ball milling for the amorphization of cellulose is the high cost of this process, estimated at
$10–15 per kg. In addition, the obtained amorphized cellulose has an unstable phase state and recrystallizes in a humid atmosphere or aqueous medium [
36].
Mercerization is the treatment of cellulose with a concentrated (18–20%) solution of sodium hydroxide. This treatment is accompanied by partial decrystallization, as well as a transformation of crystalline allomorph CI into allomorph CII [
37]. Due to these structural changes, mercerization is widely used in physicochemistry and chemistry of cellulose to improve the dyeing, for determining the content of alpha-fraction, and for the preliminary activation of cellulose structure before etherification.
The treatment of cellulose with liquid ammonia, primary amines, and ethylenediamine (EDA) causes both partial decrystallization and transformation of crystalline allomorph CI into allomorph CIII [
7,
38,
39]. However, neither treatment with alkali, nor treatment with liquid ammonia, amines, or EDA leads to complete amorphization of cellulose.
It is also known that after the regeneration of cellulose from solutions, its structure is amorphized. For this purpose, you can use various cellulose solvents, such as ionic liquids (IL), NMMO, LiCl/DMAA, DMSO/PFA, DMSO/DEA/SO
2, H
3PO
4, etc. [
40,
41,
42,
43,
44,
45]. However, the application of these solvents for the amorphization of cellulose has a significant shortcoming, the quite high cost of such solvents. So, according to the Alibaba catalog, the average price of DMSO, DEA, DMAA, and H
3PO
4 is
$1.5–2 per kg, NMMO
$10–20 per kg, and IL
$100–150 per kg. In addition, the mentioned organic solvents are toxic substances, while H
3PO
4 is an inorganic acid irritating the skin and eyes. The traditional cellulose solvents, CS
2/NaOH system, and Cuproxam, used in the 20th century for the production of artificial cellulose fibers and films, are currently prohibited due to their toxicity and environmental hazard.
The cheapest cellulose solvent is probably an aqueous solution of 7% NaOH/12% Urea [
46,
47] denoted as N/U, which has a cost of
$0.05 per kg due to the low price of commercial chemicals. Urea is a harmless substance and sodium hydroxide is a non-toxic but irritating substance that nevertheless is widely used in compliance with safety rules. The problem is that only the dilute cellulose solution in this solvent can be obtained, which complicates the regeneration process and reduces the productivity of amorphized cellulose (AC). To overcome this shortcoming, the use of the cellulose swelling process in the N/U solvent instead of dissolution was proposed [
48].
The initial cellulosic pulp was placed in a glass beaker cooled with an ice/salt mixture to a temperature of −15 °C. Then, a cold N/U solvent was added at the ratio of the solvent to cellulose material (R) from 3 to 10 (v/w) while periodically stirred for 1 h, and after which left overnight in a freezer at −15 °C. The treated samples were removed from the freezer and mixed with a 10-fold volume of tap water. The swollen, gel-like samples were separated from a liquid medium on a vacuum glass filter, washed with water, neutralized with 1% HCl to pH 6–7, and then again washed with water. To study the structural characteristics and some physicochemical properties, the wet samples were rinsed with ethanol and dried at 60 °C to constant weight.
Along with the direct methods of obtaining amorphized cellulose, there are also indirect methods, e.g., by saponification of cellulose acetate in non-aqueous media [
49]. The resulting amorphous cellulose has an unstable phase state and crystallizes in a humid atmosphere or aqueous medium.
A special type of amorphous cellulose is amorphous nanocellulose (ANC) dispersed to nanosized particles. It is usually obtained through acid hydrolysis of regenerated cellulose with subsequent ultrasound or mechanical disintegration in an aqueous medium [
50]. This method comprises stages of cellulose dissolution, regeneration from solution to obtain amorphized cellulose (AC), partial acid hydrolysis of AC, washing of depolymerized AC, dilution with water, and comminuting in an aqueous media using ultrasound disintegrator, high-pressure homogenizer, microfluidizer, and some other apparatus. The main shortcomings of the common ANC production method are the loss of the solvent and part of cellulose, high consumption of chemicals and energy, as well as low productivity.
To overcome these shortcomings, a novel waste-free technology must be used [
51]. This technology includes dissolving cellulose in sulfuric acid at low temperatures, holding for partial hydrolysis and reducing the degree of polymerization, regeneration of AC by dilution with water, separation of AC from the acid solution, washing and disintegration of AC in aqueous media to obtain nanoparticles, use of dilute acid for the production of by-products, and return of used water to the production cycle.
The general technology can be detailed by the following specific example. The initial pulp was mixed in a beaker with the predetermined amount of water. Then, 80 wt. % sulfuric acid was slowly added while cooling in an ice-water bath and stirring to obtain the required final acid concentration of 66% and acid/cellulose ratio of 10. The beaker was placed into the ice-water bath and stirred for 60 min. After acidic treatment, the contents of the beaker were poured out into a threefold volume of cold water while stirring to regenerate flocs of amorphous cellulose (AC). The flocs were separated from the dilute acid by centrifugation at 5000× g for 10–15 min; washed with distilled water to a pH of about 5, separating them from water by centrifugation. The separated flocs of AC were diluted with distilled water to a concentration of 1% and disintegrated by an ultrasound disperser “Branson S450CE” at 20 kHz for 10–15 min. The 1 wt. % water dispersion of ANC was evaporated under vacuum at 50 °C in order to obtain a hydrogel with a solids content of 10 wt. %.
The dilute acid and acidic washing water were collected together and neutralized with cheap Ca-containing compounds, e.g., calcium hydroxide, to precipitate calcium sulfate (CaSO
4) [
51]:
This by-product is widely used as a white pigment for paints, filler for polymers and paper compositions, as well as an inorganic binder for the production of putties, plasters, and drywall. Another cheap Ca-containing compound can be mineral-hydroxylapatite, Ca
5(PO
4)
3OH. As a result of treatment of the dilute acid and acidic washing water with hydroxylapatite, the sulfuric acid is almost completely utilized and turns into such valuable by-product as superphosphate fertilizer:
The wastewater was purified and returned to the technological cycle. Thus, the proposed waste-free technology ensures the production of ANC and complete utilization of solvent and reagent-sulfuric acid, by its conversion into valuable by-products, the sale of which can significantly reduce the cost of ANC. Studies have shown that ANC can be obtained with a yield of 60–65%. Particles of ANC have a round shape and average diameter of about 100 nm [
50] (
Figure 3).
The isolated ANC is characterized by a high amorphicity degree and decreased degree of polymerization, DP (
Table 2). Moreover, the ANC contains sulfonic and reducing aldehyde groups.
5. Acidic Hydrolyzability and Enzymatic Digestibility
Cellulose samples can be hydrolyzed by acids and cellulolytic enzymes. To evaluate the hydrolyzability of cellulose, the cellulose samples were treated with 2.5 M sulfuric acid at 100 °C for 4 h using acid to sample ratio of 40 [
54]. The hydrolyzate was separated from residue by centrifugation, and the acid was neutralized with calcium carbonate. The formed precipitate of calcium sulfate was separated from the aqueous phase by centrifugation, after which the liquid was analyzed to determine the concentration of glucose.
To study enzymatic digestibility, the cellulose samples were treated with a commercial cellulolytic enzyme Cellic CTec-3 (Novozymes A/S, Bagsvaerd, Denmark) [
48]. The dose of the enzyme was 30 mg per 1 g of solid sample. Hydrolysis of the samples was carried out in 50-mL polypropylene tubes. The samples containing 1 g of solid matter and 1 mL of 50 mM acetate buffer (pH = 4.8) were put into the tubes. Then, the needed amount of the enzyme was added. Further, an additional amount of the buffer was supplemented to obtain a concentration of the cellulose substrate from 50 to 150 g/L. The tubes closed with covers were placed in a shaker incubator at 50 °C and shaken for 48 h. The hydrolyzate was separated from residue by centrifugation, after which the liquid was analyzed to determine the concentration of glucose.
The concentration of glucose in hydrolyzate after acidic or enzymatic hydrolysis was determined by the HPLC-apparatus of Agilent Technologies 1200 Infinity Series. The Amines HPX-87H column was used. The main conditions of the analysis were temperature 45 °C; mobile phase 0.005 M sulfuric acid; flow rate 0.6 mL/min. The hydrolyzate was preliminarily filtered through a 0.45 μm Nylon filter. The yield of glucose (YG, %) after hydrolysis was calculated by the equation:
where C
g is the final concentration of glucose in hydrolyzate (g/L) and C
o is the initial cellulose content (g/L).
The following samples were used to test the acidic hydrolyzability and enzymatic digestibility:
Original chemical-grade cotton cellulose (ORC) having 98% α-fraction and DP = 2700
Mercerized cellulose (MRC)
Ammonia treated cellulose (AMC)
Ball-milled cellulose (BMC)
Cellulose materials treated with N/U-solvent at R = 5 and 10, which are completely amorphous cellulose samples (CAC)
The study of acidic hydrolyzability showed (
Figure 9) that after the amorphization of original cellulose by different methods, a significant increase in the glucose yield is observed, especially for completely amorphous cellulose samples (CAC). This phenomenon can be explained by a directly proportional relationship between the hydrolyzability and the amorphicity degree of cellulose (
Figure 10).
An exception is observed for two samples, AMC and BMC, which have an unstable phase state and partly crystallize under relatively mild reaction conditions (processing in an aqueous medium at 100 °C), and thereby their acidic hydrolyzability is reduced. In contrast to these two samples, the rest of the studied samples, including CAC and other amorphous samples obtained by regeneration from cellulose solutions, do not crystallize under the said mild reaction conditions [
49]. To crystallize CAC samples via processing in the aqueous medium, a higher temperature is required.
Since the method of cellulose swelling in N/U solvent proved to be the most effective to increase its acidic hydrolyzability, this method was also applied for pretreatment of cellulose samples intended for enzymatic hydrolysis. For such pretreatment, samples of original cotton cellulose (ORC), microcrystalline cellulose Avicel PH-101 (MCC), and mixed waste paper (MWP) were used as initial materials.
Without solvent pretreatment, the initial materials have a moderate enzymatic digestibility at C
o = 50 g/L with YG 44–48%. However, after swelling in a cold N/U-solvent at the solvent to cellulose ratio R ≥ 5, the obtained samples are hydrolyzed almost completely (
Figure 11) due to the amorphous structure of these samples (
Table 3).
Despite the high enzymatic digestibility at the content of CAC, C
o = 50 g/L, the glucose concentration in hydrolyzate does not exceed 55 g/L (5.5%), which is too low for further fermentation. To increase the concentration of glucose, the initial content of the CAC substrate should be enhanced. If C
o is increased to 150 g/L, then glucose concentration in the hydrolyzate increases to 147 g/L (
Figure 12).
6. Potential Applications of Amorphized Cellulose
Currently, amorphized cellulose (AC) is used only in laboratory research. For example, AC obtained by ball-grinding is used as an amorphous standard for structural studies of cellulose [
16]. Another example is AC regenerated from a solution in o-phosphoric acid, which is used as an amorphous standard in the enzymatic hydrolysis of cellulose and plant biomass [
45].
There are two main reasons that hinder the commercialization of amorphized cellulose. The first is the high cost of the original cellulose. So, the market price of bleached pulp is $600–800 per ton, whereas the price of cotton cellulose is even higher and reaches $1000 per ton. Therefore, it is not profitable to use them to produce cheap amorphous cellulose on a pilot or industrial scale.
The second reason is the high consumption of chemicals and energy for most of the methods of AC production. The only exception is the amortization method by swelling of the starting material in a cold N/U solvent [
48]. This process is the cheapest of all known due to the low cost of chemicals and low energy consumption.
For commercial production of amorphous cellulose, it is most advisable to use cheap raw materials such as mixed waste paper (MWP), which can be supplied for
$50 per ton. Studies have shown that if the content of MWP-based AC substrate in the reaction system reaches C
o = 150 g/L, then after 48 h of hydrolysis at the enzyme dose of 3%, a quite high glucose concentration C
g = 141 g/L can be achieved [
48]. As a result, quite cheap glucose can be released. This technical-grade product can find diverse applications in biotechnology to produce ethanol [
56]; acetic, lactic, and citric acids [
57]; proteins, yeasts, and enzymes [
58,
59,
60]; bacterial cellulose [
61], polyhydroxyalkanoates [
62], and other valuable bioproducts.
If MWP, after swelling in a cold N/U solvent, is washed with water, but not dried, then the resulting technical AC takes the form of a hydrogel. As is known, hydrogels are capable of holding large amounts of water in their three-dimensional networks [
63,
64]. The hydrogels can be used in biology, chemistry, physical chemistry, chromatography, medicine, hygiene, pharmaceutics, food processing, and other areas [
65,
66].
Recently, much attention has been paid to the use of hydrogels in agriculture and soil technology [
67,
68,
69,
70,
71,
72]. However, agricultural applications are limited by the high cost of synthetic polymeric hydrogels, their low production volume, and biostability.
To solve these problems, it is necessary to use cheap, available, and biodegradable natural material, such as MWP, for the production of hydrogels. Such hydrogels can be produced by the swelling of MWP in a cold N/U-solvent at R ≥ 5 followed by washing [
73]. Another technology of hydrogel aimed especially for growing plants was the following: after MWP swelling in the cold N/U solvent, the swollen sample was treated with phosphoric acid to obtain a hydrogel containing phosphate salt and urea serving as PN fertilizer. This hydrogel enriched with PN fertilizer is highly effective in seed germination, and after usage, it can be completely decomposed under the action of microorganisms present in the soil [
73].
Undried amorphous nanocellulose (ANC) can also form a hydrogel. It was found that the hydrogel of ANC can be used as an effective thickener. Therefore, a small addition of this hydrogel significantly increases the phase stability of aqueous dispersions of some drugs, e.g., “Maalox” (
Figure 13).
Particles of ANC can also find application in medicine and cosmetics as a carrier of therapeutically active substances (TAS). Due to the increased content of acidic sulfonic and reducing functional groups (
Table 2), ANC can serve as a nano-carrier of various TAS. For example, sulfonic groups of ANC can attach some bactericides, e.g., Ag-cations and ZnO [
74]:
In addition, acidic functional groups of ANC contribute to the ion binding of amino acids containing basic amino groups, e.g., arginine, lysine, or histidine, by the following scheme:
In particular, the obtained ANC-Arginine conjugate can be used for the effective treatment and regeneration of damaged skin tissues, cure of herpes simplex virus, and in other arginine-therapy areas [
75,
76].
The presence in ANC reducing groups provides additional opportunities for attaching TAS. For example, the joining of a proteolytic enzyme like trypsin to nanoparticles of ANC can be expressed by the scheme:
Along with trypsin, some others enzymes, such as chymotrypsin, papain, collagenases, lysoamidases, lysozymes, etc., can be attached to ANC–nanocarrier and used for the treatment of wounds and burns [
77].
7. Conclusions
Cellulose is the most abundant and widely used crystalline biopolymer. However, due to increased crystallinity, cellulose exhibits low accessibility to reagents. In particular, it gives a small yield of glucose after acidic or enzymatic hydrolysis. To increase the accessibility and reactivity of natural cellulose, it is necessary to destroy the crystalline structure of this biopolymer. Various physical and physicochemical methods can be used for the amorphization of cellulose structure, such as dry grinding, mercerization, treatment with liquid ammonia, primary amines, EDA, and diverse solvents, etc. The problem is that most of these methods are quite expensive and cannot be used for cellulose decrystallization on a large scale.
The exception is a quite cheap solvent that is an aqueous system of 7% NaOH/12% Urea (N/U). It has been found that after cellulose treatment with cold N/U-solvent at the solvent to cellulose ratio R ≥ 5, a completely amorphous sample (CAC) can be obtained. Structural studies showed that amorphous cellulose contains mesomorphous clusters with an average size of 1.85 nm and specific gravity of 1.49 g/cm3. Furthermore, each cluster consists of about five glucopyranose layers with an average interlayer spacing of 0.45 nm.
Amorphous cellulose is characterized by increased hydrophilicity, accessibility, reactivity, and enzymatic digestibility. Due to its amorphous structure, the CAC is converted to glucose almost completely in a short time under the action of a relatively small dose of cellulolytic enzymes. Such a CAC sample can be used as an amorphous standard, and as a substrate for the commercial production of glucose, which can find application in biotechnology as a promising nutrient for various microorganisms. In addition, the hydrogel of amorphous cellulose can be applied in agriculture.
The amorphous nanocellulose (ANC) having round particles with an average diameter of about 100 nm can be prepared using waste-free technology. The hydrogel of ANC can be used as a thickener. Moreover, due to the presence of acidic sulfonic and reducing functional groups, ANC can serve as a nano-carrier of various therapeutically active substances.