**4. Cellolignin residue of larch wood as raw material for crystalline glucose production**

The cellolignin residue is formed during the chemical processing of larch wood using the technology for obtaining dihydroquercetin and arabinogalactan [100]. CR represents larch wood chips initially extracted by ethyl acetate and hot water. The larch wood chip basically consists of cellulose, hemicellulose, and lignin. The polysaccharide content in larch wood chips is 65–75% of the mass of its absolutely dry wood (a.d.w.) [101], and the content of water-soluble substances is 10–16% (in some samples up to 30%) [102]. The content of holocellulose in CR (without water-soluble substances) is about 54% of its a.d.w. weight, whereas the content of holocellulose in the original larch wood is about 40% of its a.d.w. (also without water-soluble substances). The gain in the relative content of polysaccharides per mass of a.d.w. is about 13%, which permits one to consider the CR as a polysaccharide- "enriched" raw material from which it is possible to obtain sugar and other products by hydrolysis. Hemicellulose polysaccharides in the CR of larch wood are mainly represented by the water-soluble polysaccharide arabinogalactan and 4-O-methylglucuronoaraboxylans and galactoglucomannans [103], which are associated to differing degrees with cellulose. The yield of arabinogalactan from larch wood using dihydroquercetin production technology in which it is isolated as a by-product (without special optimization) accounts for 67% of its total content in the original raw material; therefore, the content of watersoluble substances in CR remains rather high, at 8.9%.

The data on the group composition of the components of CR were reported in [104]. The acid hydrolysis of hemicelluloses results in the formation of mono- and oligosaccharides, the presence of which substantially impairs the quality of hydrolysates and hinders the crystallization of glucose from these syrups. Therefore, it is necessary to purify CR from hemicelluloses. Hemicelluloses are commonly removed (to a particular limit) from the raw material, either by hot water extraction or by hydrolysis with diluted acid at elevated temperature.

In experiments with water hydrolysis of larch wood chips in laboratory autoclaves, the parameters of hydrolysis, i.e., temperature, hydromodulus, duration and the number of hydrolysis steps, were varied. The maximum total yield of water-soluble substances (18%) was attained based on four-step hydrolysis. However, it is economically more attractive to perform one-step hydrolysis, with the conditions specified for obtaining a maximum yield of sugars with this hydrolysis method being as follows: a gradual increase in temperature from 25 °C for 1 h and 160 °C for 1 h with a hydromodulus of 1:6. The yield of substances was 15%. As indicated by paper chromatography (PC) and thin layer chromatography (TLC), the pre-hydrolysate contained arabinose, galactose, xylose, mannose and trace amounts of glucose [105].

Mild hydrolysis of hemicelluloses of CR was performed using 1–5% sulphuric or hydrochloric acids at the boiling temperature of the solution (100–105 °C). By choosing the optimal hydrolysis conditions, a maximum yield of reducing substances (RS) in hydrolysate, 1.1%, was achieved with the use of 5% sulphuric acid, which corresponded to a 23% content of hemicelluloses or noncellulose polysaccharides in CR. The hexose content in the prehydrolysate was 33.9% of total sugars, as determined from the content of RS, and that of pentoses was 62.1%; arabinose, galactose, xylose, mannose, and glucose were identified qualitatively. Significantly less amounts of polysaccharides were hydrolyzed with the use of 2% hydrochloric acid under the same hydrolysis conditions - 9.6% .

Aqueous and acidic sugar solutions obtained during the pre-hydrolysis of CR (technological sugar solutions) contained greater amounts of fermentable sugars compared to other conifers and can be used in the production of feed for animals [106, 107] and other products.

To obtain pure glucose syrups, two alternative variants of acid hydrolysis of cellulose were considered: hydrolysis of CR and of cellulose itself after delignification of CR. Using the first variant, we studied the hydrolysis of CR by diluted (1.0–5.0%) hydrochloric acid at high temperatures (160–170 °C) and by concentrated (50–85%) sulphuric acid at room temperature to compare the crystallization properties of glucose syrups obtained by the two methods. A high temperature hydrolysis of the raw material by diluted acid was carried out in laboratory autoclaves in four steps. As the acid concentration was increased from 1 to 5%, a gain in the sugar content of the hydrolysates was observed. Thus, the mass portion of RS relative to the mass of a.d.w. during hydrolysis of sawdust was 5.4% using 1% H2SO4, 7.9% with 2% H2SO4, 9.2% with 3% H2SO4, and 11.6% with 5% H2SO4. However, simultaneously to an increase in the acid concentration, the quality of the hydrolysate was markedly impaired; an intensive dark colour appeared due to the formation of sugar degradation products, and the content of colloidal impurities in syrups at later stages caused severe problems. Conversely, the low temperature hydrolysis of CR (of the same raw material) by concentrated sulphuric acid made it possible to obtain a high yield of sugar in hydrolysates with a minimum content of degradation products. The maximum yield of RS was 64% using 70% sulphuric acid at the hydromodulus (1:5) and a hydrolysis time of 5 h, and 62.6% using 80% sulphuric acid at the hydromodulus (1:5) and hydrolysis time of 2 h. Hydrolysates contained no pentoses. These characteristics meet the requirements imposed upon hydrolysates from which crystalline glucose is isolated.

178 The Complex World of Polysaccharides

amounts of glucose [105].

temperature.

The data on the group composition of the components of CR were reported in [104]. The acid hydrolysis of hemicelluloses results in the formation of mono- and oligosaccharides, the presence of which substantially impairs the quality of hydrolysates and hinders the crystallization of glucose from these syrups. Therefore, it is necessary to purify CR from hemicelluloses. Hemicelluloses are commonly removed (to a particular limit) from the raw material, either by hot water extraction or by hydrolysis with diluted acid at elevated

In experiments with water hydrolysis of larch wood chips in laboratory autoclaves, the parameters of hydrolysis, i.e., temperature, hydromodulus, duration and the number of hydrolysis steps, were varied. The maximum total yield of water-soluble substances (18%) was attained based on four-step hydrolysis. However, it is economically more attractive to perform one-step hydrolysis, with the conditions specified for obtaining a maximum yield of sugars with this hydrolysis method being as follows: a gradual increase in temperature from 25 °C for 1 h and 160 °C for 1 h with a hydromodulus of 1:6. The yield of substances was 15%. As indicated by paper chromatography (PC) and thin layer chromatography (TLC), the pre-hydrolysate contained arabinose, galactose, xylose, mannose and trace

Mild hydrolysis of hemicelluloses of CR was performed using 1–5% sulphuric or hydrochloric acids at the boiling temperature of the solution (100–105 °C). By choosing the optimal hydrolysis conditions, a maximum yield of reducing substances (RS) in hydrolysate, 1.1%, was achieved with the use of 5% sulphuric acid, which corresponded to a 23% content of hemicelluloses or noncellulose polysaccharides in CR. The hexose content in the prehydrolysate was 33.9% of total sugars, as determined from the content of RS, and that of pentoses was 62.1%; arabinose, galactose, xylose, mannose, and glucose were identified qualitatively. Significantly less amounts of polysaccharides were hydrolyzed with the use of

Aqueous and acidic sugar solutions obtained during the pre-hydrolysis of CR (technological sugar solutions) contained greater amounts of fermentable sugars compared to other conifers and can be used in the production of feed for animals [106, 107] and other products. To obtain pure glucose syrups, two alternative variants of acid hydrolysis of cellulose were considered: hydrolysis of CR and of cellulose itself after delignification of CR. Using the first variant, we studied the hydrolysis of CR by diluted (1.0–5.0%) hydrochloric acid at high temperatures (160–170 °C) and by concentrated (50–85%) sulphuric acid at room temperature to compare the crystallization properties of glucose syrups obtained by the two methods. A high temperature hydrolysis of the raw material by diluted acid was carried out in laboratory autoclaves in four steps. As the acid concentration was increased from 1 to 5%, a gain in the sugar content of the hydrolysates was observed. Thus, the mass portion of RS relative to the mass of a.d.w. during hydrolysis of sawdust was 5.4% using 1% H2SO4, 7.9% with 2% H2SO4, 9.2% with 3% H2SO4, and 11.6% with 5% H2SO4. However, simultaneously to an increase in the acid concentration, the quality of the hydrolysate was markedly impaired; an intensive dark colour appeared due to the formation of sugar degradation

2% hydrochloric acid under the same hydrolysis conditions - 9.6% .

Thus, the use of concentrated sulphuric acid in the concentration range of 65–80% makes it possible to obtain hydrolysates with a maximum content of RS (up to 64%) [108]. After additional hydrolysis (the inversion stage), the hydrolysate has a pH value close to 2. On further evaporation of this hydrolysate, the acid is concentrated, which leads to further degradation of sugar; as a result, the yield of glucose decreases, and the syrup is contaminated with stained products.

Sulphuric acid was neutralized using barium acetate, sodium hydroxide and calcium hydroxide, and the contribution of each compound to formation of the mineral ash component in syrups was determined. The lowest ash content in the hydrolysate (0.2%) was achieved by applying barium acetate. However, because of the toxicity of barium and its salts, the use of barium was abandoned. Upon neutralization of sulphuric acid by calcium hydroxide the ash content in the hydrolysate was initially as high as 10%; nevertheless, preference was given to this compound alone since it is nontoxic, readily available, and convenient in operation. According to the "Glucose, crystalline hydrated" GOST 97588 Regulations [109], the ash content in the final product must not exceed 0.06–0.07%, calculated for dry substance; therefore, it was necessary to provide neutralization conditions to decrease the ash content in hydrolysates. For neutralizing sulphuric acid, pH value was brought to 4–4.5 at a temperature no higher than 80 °C. The amount of calcium hydroxide was calculated according to the sulphuric acid neutralization reaction so as to exclude the over-alkalization of the solution [110]. The resulting dihydrate gypsum crystals were filtered off. The ash content in hydrolysate decreased by up to 0.5%. In addition to glucose and mineral contaminations, the hydrolysate contained, dependent on hydrolysis conditions, the products of partial hydrolysis of the lignocarbohydrate complex of wood—mono-, di-, tri-, and oligosaccharides—as well as impurities belonging to different classes of organic compounds: acid-soluble lignin, furfurol, oxymethylfurfurol, a lignohumic complex, colloids, levulinic acid, and other organic acids [106]. At the next stage, we assessed the nature of substances by determining the colour of hydrolysates and selected how to remove them from sugar solutions [111]. It was possible to assign some impurities, that by their nature are associated with lignin, to substances based on their colouring. First of all, this was acid-soluble lignin. According to the published data, 2–3% of total lignins are dissolved in the hydrolysis of coniferous wood by a solution of 72% sulphuric acid [103].

In addition, coloured substances are formed during sugar degradation: hexosans form high molecular weight substances of brown colour, which partially precipitate from solution, and pentosans form furfurol, which imparts a yellow colour to hydrolysate. Under acidic

conditions, lignin and sugar degradation products, i.e., furfurol and oxymethylfurfurol, form condensed products in small amounts, and insoluble humic compounds; and the products of incomplete hydrolysis of polysaccharides, oligosaccharides, can be partially adsorbed by acid-insoluble lignin [106]. Hydrolysates were clarified using activated carbon BAU (Russia). The UV spectrum of a neutralisate from CR shows an absorption band at 280 nm, which disappears after treatment of the neutralisate by activated carbon. Treating the hydrolysate with dichloroethane followed by IR analysis of the concentrated extract made it possible to identify it as acid-soluble lignin. Thus, the treatment of hydrolysates with activated carbon significantly reduces the content of acid-soluble lignin in hydrolysates (from 1.4 to 0.3%).

The scheme of the acid–hydrolytic transformation of cellulose to glucose with preliminary delignification of lignocellulose raw material by industrial methods [112] is the second variant, which also makes it possible to obtain high purity glucose syrups. Its main advantage is that it enables glucose syrups to be obtained with a factor of merit of no less than 85%, which are not contaminated with colouring impurities of ligno-carbohydrate origin and ash components. It is known that, during the low temperature hydrolysis of cellulose by concentrated acids, partial destruction of cellulose with the formation of watersoluble products occurs [113]. Under optimized conditions, concentrated sulphuric acid almost completely dissolves cellulose, and the cleavage of glycoside bonds proceeds in a homogeneous medium. As a result of hydrolysis, a mixture of products differing by the polymerization degree (PD) is formed: from comparatively high molecular weight cellulose and cellodextrins (PD from 7 to 50–60) to oligosaccharides (mainly di- and trisaccharides) and glucose. The composition of the mixture and the ratio of the products in the hydrolysate depend on the hydrolysis conditions. These products also vary in water solubility. Thus, cellodextrins, oligosaccharides, cellobioses and monosaccharides are water-soluble, and part of the cellulose itself, mainly its crystalline moiety, and hydrocellulose do not dissolve in water.

Thus, the hydrolysis of cellulose enables firstly the isolatation of intermediate water-soluble hydrolysis products with simultaneous removal of sulphuric acid without its chemical neutralization, which in turns prevents the entry of mineral impurities into syrups; and, secondly, obtaining of the required monomeric sugar, i.e., glucose, in one stage, by subsequent additional hydrolysis of the intermediate product. The hydrolysis of industrial cellulose was carried out by 72% sulphuric acid at room temperature for 1 h with regular stirring of the hydrolysate mass; in this case, cellulose had completely dissolved within the first 15 min. Increasing the hydrolysis duration up to 2 and 3 h did not significantly affect the final yield of the product, which was 80–90% of the weight of absolutely dry cellulose (a.d.c.) [114].

We arbitrarily called this product the inverted polysaccharide (IPS) since this name reflects its position in the technological scheme. Dried IPS is a white or pale cream powder. The product is partially soluble in water (the insoluble fraction accounts for 43% of the weight of IPS), is soluble in aqueous alkaline solutions, and exhibits a lower PD than the starting cellulose (150; PD for starting cellulose, 573). The content of cellulose in an aqueous IPS solution was estimated, using HPLC, to be 2% of the a.d.c. A comparative analysis of IR spectra of the starting cellulose and IPS indicated that IPS is cellulose with a high degree of amorphism [94]. In particular, this is evidenced by strong changes in the IR spectrum of IPS in the region of 600–1500 cm–1, which accompanies changes in the polysaccharide hypomolecular structure, and smoothing of the intensity of so-called crystallinity bands at 1100, 1140, 1190, 1250, 1360, and 1420 cm–1 [115]. The IR spectrum of the product contained no absorption bands at 1112 and 1162 cm–1, which are typical of the spectra of a highly ordered cellulose structure. The residual sulphuric acid content in an aqueous IPS solution was 0.2%, indicating that 98% of the acid taken for hydrolysis is removed simultaneously with the isolation of IPS (without chemical neutralization).

180 The Complex World of Polysaccharides

(from 1.4 to 0.3%).

(a.d.c.) [114].

conditions, lignin and sugar degradation products, i.e., furfurol and oxymethylfurfurol, form condensed products in small amounts, and insoluble humic compounds; and the products of incomplete hydrolysis of polysaccharides, oligosaccharides, can be partially adsorbed by acid-insoluble lignin [106]. Hydrolysates were clarified using activated carbon BAU (Russia). The UV spectrum of a neutralisate from CR shows an absorption band at 280 nm, which disappears after treatment of the neutralisate by activated carbon. Treating the hydrolysate with dichloroethane followed by IR analysis of the concentrated extract made it possible to identify it as acid-soluble lignin. Thus, the treatment of hydrolysates with activated carbon significantly reduces the content of acid-soluble lignin in hydrolysates

The scheme of the acid–hydrolytic transformation of cellulose to glucose with preliminary delignification of lignocellulose raw material by industrial methods [112] is the second variant, which also makes it possible to obtain high purity glucose syrups. Its main advantage is that it enables glucose syrups to be obtained with a factor of merit of no less than 85%, which are not contaminated with colouring impurities of ligno-carbohydrate origin and ash components. It is known that, during the low temperature hydrolysis of cellulose by concentrated acids, partial destruction of cellulose with the formation of watersoluble products occurs [113]. Under optimized conditions, concentrated sulphuric acid almost completely dissolves cellulose, and the cleavage of glycoside bonds proceeds in a homogeneous medium. As a result of hydrolysis, a mixture of products differing by the polymerization degree (PD) is formed: from comparatively high molecular weight cellulose and cellodextrins (PD from 7 to 50–60) to oligosaccharides (mainly di- and trisaccharides) and glucose. The composition of the mixture and the ratio of the products in the hydrolysate depend on the hydrolysis conditions. These products also vary in water solubility. Thus, cellodextrins, oligosaccharides, cellobioses and monosaccharides are water-soluble, and part of the cellulose itself, mainly its crystalline moiety, and hydrocellulose do not dissolve in water. Thus, the hydrolysis of cellulose enables firstly the isolatation of intermediate water-soluble hydrolysis products with simultaneous removal of sulphuric acid without its chemical neutralization, which in turns prevents the entry of mineral impurities into syrups; and, secondly, obtaining of the required monomeric sugar, i.e., glucose, in one stage, by subsequent additional hydrolysis of the intermediate product. The hydrolysis of industrial cellulose was carried out by 72% sulphuric acid at room temperature for 1 h with regular stirring of the hydrolysate mass; in this case, cellulose had completely dissolved within the first 15 min. Increasing the hydrolysis duration up to 2 and 3 h did not significantly affect the final yield of the product, which was 80–90% of the weight of absolutely dry cellulose

We arbitrarily called this product the inverted polysaccharide (IPS) since this name reflects its position in the technological scheme. Dried IPS is a white or pale cream powder. The product is partially soluble in water (the insoluble fraction accounts for 43% of the weight of IPS), is soluble in aqueous alkaline solutions, and exhibits a lower PD than the starting cellulose (150; PD for starting cellulose, 573). The content of cellulose in an aqueous IPS solution was estimated, using HPLC, to be 2% of the a.d.c. A comparative analysis of IR In order to convert IPS to the monomeric form of sugar (glucose), an inversion was carried out at high temperature using a diluted acid. We studied the kinetics of the IPS inversion using diluted (0.075–1.5%) hydrochloric acid [116]. The choice of this acid was primarily dictated by the fact that sodium chloride formed during the neutralization of the acid by NaOH is a part of the complex composite (CC) of glucose with the formula (C6H12O6)2·NaCl·H2O, the decomposition of which results in the release of crystalline glucose. Considering that the potential yield of glucose, on inversion by 5% sulphuric acid at 100 °C for 5 h, is 1.5% in the hydrolysate, which corresponds to a glucose yield equal to 82% of the weight of a.d.c., the acid concentration of 0.125% and temperature of 170 °C represent the optimum inversion conditions under which the yield of RS in the invertion is at its maximum; the time taken for attaining the maximum yield in these conditions is minimal. Thus, during the hydrolysis of cellulose and the subsequent inversion of IPS, the main glucose content in the inverted solution is about 70% of the mass of RS; i.e., the real yield of glucose is 35–45% of a.d.c.

It should be noted that these glucose syrups are transparent, of a light yellow colour, and are distinguished by a high factor of merit (85–90% and more) (Figure 16). In comparison, the yield of glucose from CR (the first variant of hydrolysis) is 23–25%. CG is isolated from glucose solutions either by direct or salt crystallization [117]. We studied the crystallization properties of glucose syrups obtained by hydrolysis of CR and cellulose using both the direct and salt methods. As mentioned above, the factor of merit of starting syrups must be no less than 85% for the successful crystallization of glucose using the direct method. Glucose syrups obtained by hydrolysis of cellulose completely meet this requirement. The application of activated carbon increased the quality of the syrup since direct crystallization occurred only in clarified syrups.

In order to perform direct crystallization, cellulose hydrolysis was used to obtain a hydrolysate with a RS content of 1%, pH 4.4, and a factor of merit of 94%, which was allowed to stand at room temperature for spontaneous crystallization. After two weeks, the onset of crystallization was visually observed. Crystallization by itself, without the creation of special temperature conditions, progresses slowly (taking a month and more). The method of salt crystallization of glucose has some advantages over direct crystallization. It does not require a deep purification of hydrolysates, the crystallization process is shorter and simpler (there is no need for a multiple recrystallization), and the yield of glucose increases.

**Figure 16.** Glucose syrup from larch wood CR

We studied the crystallization conditions of glucose using its CC with sodium chloride (C6H12O6)2·NaCl·H2O and examined CC crystals obtained from model mixtures of glucose, sodium chloride and water, as well as from experimental glucose syrups obtained by the hydrolysis of CR and cellulose [118].

The crystallization of CC was studied using artificial mixtures in which the NaCl content varied from 15% to a twofold excess relative to the glucose content. In all cases, a crystalline phase formed. The composition of the crystalline phase was determined by elemental analysis. We determined that the range of NaCl:glucose ratios from 0.2:1 to 0.7:1 (parts by weight) is optimal for the formation of CC. Similarly, we determined the crystallization conditions for CC in hydrolysates of CR and cellulose. CC crystals were isolated from hydrolysates; the artificial mixtures were colourless and transparent and had well-defined facets. According to the chemical analysis data, they have a composition close to being stoichiometric: C – 32.5%, H – 5.9% and Cl – 9.7%. Theoretically, CC with the general formula (C6H12O6)2·NaCl·H2O contains C – 33%, H – 6%, and O – l8.13%.

According to X-ray phase analysis, CC monocrystals synthesized from pure solutions have the unit cell parameters a = b = 16.8, c = 17.0 Å and β = 120°, and represent a hexagonal prism. Based on the symmetry of lauegrams and weissenbergograms, they belong to the diffraction class P 3 m with regular extinctions at 1 ≠ 3n. Therefore, the spatial group of CC crystals was determined as P31 12 (151) and P32 12 (153).

The set of diffraction maxima obtained by X-ray phase analysis of CC crystals and the reference indicates that, under the experimental conditions used, glucose in the presence of sodium chloride crystallizes as CC with the formula (C6H12O6)2·NaCl·H2O.

Thus, when studying the crystallization properties of glucose syrups produced by acid hydrolysis of the crystalline glucose of larch wood, we obtained CC glucose crystals with sodium chloride, upon decomposition of which D-glucose is released in crystalline form. In addition, glucose can be directly crystallized from glucose syrups produced by the hydrolysis of cellulose with a high factor of merit (more than 85%).

The ways in which the efficiency of hydrolysis of polysaccharides from wood CR can be increased are of a great interest. One method is the steam explosion hydrolysis of cellulose containing raw material, which makes possible the efficient and completely ecologically safe decomposition of lignocellulose material into its constituents: lignin, hemicellulose, and cellulose.

We studied the steam explosion hydrolysis of CR from larch wood, and showed that this method can be used for effective prehydrolysis processing of larch wood CR [119].

Hence, a laboratory scheme has been developed which will be used as the technological basis for obtaining crystalline glucose from the CR in larch wood [120].
