INTRODUCTION

Renewable cellulosic plant biomass is a promising feedstock for the production of bioethanol and other useful products due to its low cost, abundance, and lack of food competition. The traditional technology for processing plant biomass includes grinding, pre-treatment, enzymatic hydrolysis (saccharification), fermentation, and distillation. Pretreatment is necessary to reduce the resistance of biomass to the action of hydrolytic enzymes, due to the presence of a lignin matrix and the crystalline state of cellulose, which are factors that prevent effective hydrolysis by cellulases [1]. To increase the reactivity of biomass, various methods of its pre-treatment are used, aimed at destroying the crystalline structure of cellulose, removing lignin, and increasing the surface area of cellulose available to enzymes. Despite the fact that these methods make it possible to increase the reactivity of the biomass, they are imperfect in terms of “green chemistry” and the environmental friendliness of the process [2, 3].

Pretreatment methods based on the use of deep eutectic solvents (DESs), which significantly increase the reactivity of the raw material, may be an alternative [4]. DESs have a number of advantages compared to ionic liquids (ILs) with the same characteristics [5]: simple synthesis, stability, price competitiveness (according to a rough estimate, the cost of DES synthesis is 5–10 times lower than the cost of ILs [6]), wide availability (for example, choline chloride (ChCl) is used as a feed additive for feeding farm animals and poultry), and environmental friendliness [7].

The term DES was first introduced in [8]. Depending on the type of components involved in the formation of the eutectic mixture, presently, DESs are divided into four types [9, 10]. For the purposes of biotechnology, DESs of the third type, which consist of hydrogen bond acceptors (quaternary ammonium or phosphonium salts) and hydrogen bond donors (carboxylic acids, amides, alcohols, etc.), are the most promising. Hydrogen bonds lead to charge delocalization between the donor and acceptor and the melting point of the eutectic mixture is lower compared to individual compounds. DESs that remain liquid at room temperature or moderate heating, which does not interfere with the processes of mixing and mass transfer with solid plant materials, are of greatest interest for the pretreatment of plant biomass [11].

An important characteristic of DESs is their viscosity, which can be affected by various factors, including the chemical nature of the DES components, their molar ratio, temperature, and water content. For example, the viscosity of DESs based on ChCl decreases with an increase in temperature and ChCl content within a certain range [12].

One of the requirements for application of for pretreatment of cellulose-containing biomass is its ability to dissolve lignin. Lignin limits enzymatic hydrolysis, acting as a physical barrier, preventing enzyme contact with polysaccharide substrate, and also by unproductive binding of enzymes [13, 14]. It has been shown that acid DESs (based on lactic, malic, oxalic, and other acids) are the most effective in this process and lead to the removal of more than 90% of lignin from various types of lignocellulosic biomass (corn cobs, rice straw, hardwood, and coniferous wood) [15 , 16].

Reed are cereals, thus, the content of lignin is high, from 18 to 26% in different parts of the plant. Easily hydrolysable polysaccharides (mainly xylans) account for 20–30%, while hardly hydrolyzable ones (cellulose) account for 19–37% [17]. By varying the DES composition, pretreatment temperature and time, it is possible to maximize the hydrolysis of the cane mass by reducing the lignin content in it and increasing the availability of cellulose for enzymes.

The goal of the present work was to select the conditions for pre-treatment of the southern (or common) reed, which is widespread in the lower reaches of the Volga.

MATERIALS AND METHODS

Enzyme preparations. In this work, EPs, which were lyophilized culture liquids obtained based on the strains P. verruculosum B537 (producer of cellulases) [18] and P. verruculosum F-10 (producer of β-glucosidase) [19], were used.

Reagents. Coarsely crushed common reed from the Astrakhan Oblast (Russia) was used in the work. The following substrates for determining activities was used: sodium carboxymethyl cellulose (CMC), beech xylan, p-nitrophenyl-β-glucopyranoside (pNPG) (Sigma, USA) and microcrystalline cellulose (MCC) (TU 20.16.59-001-40693384-209) (Crystacell, Russia). Buffer mixtures and saline solutions were prepared using reagents produced by Bio-Rad Laboratories (USA), Panreac (Germany), Helicon, and Reachem (Russia). To obtain DES, ChCl (Molekula, England), glycerol (Panreac Quimica, Spain), ethylene glycol (Roth, Germany), lactic acid (LA) (Acros Organics, Belgium), oxalic acid (OA) (ChimMed, Russia).

Obtaining DESs. All DESs were obtained by thermal mixing of the components with constant stirring for 6 h under the following conditions: ChCl/LA (molar ratios 1 : 2 and 1 : 5) at 40°C; ChCl/OA (molar ratios 1 : 1 and 1 : 2) at 60°C; ChCl/propionic acid (molar ratio 1 : 2) at 40°C; ChCl/glycerol (molar ratio 1 : 2) at 50°C; ChCl/ethylene glycol (molar ratio 1 : 2) at 40°C.

Reed pretreatment with DESs. To select the optimal pretreatment conditions, DESs of various compositions were used: neutral ones based on ChCl and polyhydric alcohols (glycerol and ethylene glycol) and acidic ones based on ChCl and acids (LA, OA, and propionic acid). The pretreatment time and temperature were also varied. In a typical experiment, 9.5 g of DESs (5% by weight) were added to 500 mg of reed and heated at various temperatures for 1, 2, 3, 6, 7, 9, 12, or 24 h with stirring. The solutions were cooled to room temperature and 10 mL of a 50% aqueous ethanol solution were added. The mixture was stirred and then centrifuged at 7000 g. The supernatant containing lignin and DES components was separated, and the precipitate was repeatedly washed with a water–ethanol mixture. It should be noted that the ChCl/OA DESs used in the work (molar ratio 1 : 2) solidified at room temperature, which made it difficult to separate it from the products of biomass delignification.

The yield of the substrate after pretreatment was determined as the ratio of the mass of the pretreated reed after drying to constant weight to the mass of the original reed (500 mg).

Determination of activity and concentration of enzymes. The amount of enzyme that catalyzes the formation of 1 μM of the product in 1 min was taken for 1 unit of activity.

Activity towards MCC, CMC and xylan was determined by the rate of accumulation of RS, analyzed by the Nelson-Somogyi method; activity with respect to pNPG, was determined according to the rate of accumulation of p-nitrophenol [20].

The protein content in EP was determined by the Lowry method using BSA as a standard.

Enzymatic hydrolysis of pretreated reed. Substrate hydrolysis (40 g/L by dry weight in the reaction mixture) was carried out under the action of two preparations (B537 (10 mg protein/g substrate or 0.4 mg/mL of EP reaction mixture) and β-glucosidase EP F10 (1 mg protein/g substrate or 0.04 mg/mL of the reaction mixture) in 2-mL test tubes (the volume of the reaction mixture was 1.5 mL) in a thermostatically controlled shaker. The hydrolysis process was carried out in the presence of 0.1 g/L of the antibiotic ampicillin (Belmedpreparaty, Republic of Belarus) in 0.1 M Na-acetate buffer solution pH 5.0 and 50°C.

Aliquots were taken from the reaction mixture, in which the concentration of RS was determined by the Nelson-Somogyi method and glucose content was determined using the Glucose-AGAT kit (AGAT, Russia).

The degree of enzymatic hydrolysis (DEH) was calculated as the ratio of the RS concentration after 48 h of enzymatic hydrolysis to the initial concentration of the substrate (40 g/L).

The experiment was carried out in triplicate.

RESULTS

The pretreatment of the substrate must satisfy an important condition: to effectively remove the part that is not hydrolyzable by the enzyme (lignin), while retaining cellulose and, if necessary, hemicellulose. The high values of the yield of the pretreated substrate of 90–93% when using propionic acid, glycerol, or ethylene glycol as a hydrogen bond donor in DESs (Table 1) apparently indicated that lignin was not completely removed from the reed during its pretreatment. When using LA, the substrate yield during pretreatment was 57–61%, which indicated effective delignification and, probably, partial removal of hemicellulose. The use of OA also led to the removal of lignin and part of the hemicellulose during pretreatment, and the yield of the substrate was 44–49%.

Table 1. Substrate yield after pretreatment and results of subsequent enzymatic hydrolysis of reed pretreated with DESs based on ChCl (hydrogen bond acceptor) and different hydrogen bond donors (pretreatment conditions: 7 h, 90°C)
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At the next stage, enzymatic hydrolysis of the resulting pretreated substrate was carried out. The cellulase complex of enzymes capable of deep hydrolysis of cellulose-containing raw materials to glucose should include three main groups of enzymes: cellobiohydrolases, endoglucanases, and β-glucosidases [1]. For bioconversion of pretreated reed, an EP based on the P. verruculosum B537 strain [18] containing ~60% cellobiohydrolases and ~10% endoglucanases was used. EP B537 had high specific activities with respect to MCC (860 units/g of protein) and CMC (1300 units/g of protein), due to the presence of cellobiohydrolases and endoglucanases, respectively. However, B537 EP is depleted in β-glucosidase, as evidenced by the low content of this enzyme (2%) and low activity towards the β-glucosidase-specific substrate pNPG (1800 U/g of protein). To increase β-glucosidase activity, an EP based on the P. verruculosum F10 strain [19], a β-glucosidase producer, containing about 80% of this enzyme and characterized by high activity with respect to pNPG (6110 U/g of protein) was added to the reaction mixture. Hydrolysis of cellulose-containing raw materials under the action of two EP B537 (cellobiohydrolase and endoglucanase) and EP F10 (β-glucosidase) led to high yields of glucose [21].

In addition, EP B537 contained xylanase (3%) and had a high activity towards xylan (1980 U/g of protein), which indicates the ability of this preparation to hydrolyze hemicelluloses.

It is important to note that the initial (without pretreatment) reed had a very low reactivity: after 48 h of enzymatic hydrolysis under the action of two EPs B537 and F10, the RS concentration was about 2 g/L, which corresponded to only 6% of the DEH.

When varying the DES composition, the reed was pretreated at 90°C for 7 h (Table 1). The best results were obtained for mixtures of ChCl/LA (the 1 : 5 ratio was preferable to the 1 : 2 ratio) and ChCl/OA (1 : 1 ratio). After 48 h of enzymatic hydrolysis, the RS concentration was about 18 g/L (DEH was 46%), and the glucose concentration was 16–17 g/L. When using a mixture of ChCl/OA for pretreatment with a higher proportion of OA (a molar ratio of 1 : 2), the resulting pretreated substrate had an extremely low reactivity and DEH amounted to only 9%, which may be due to excessively stringent pretreatment conditions.

Among DESs based on ChCl with organic acids, the DESs with propionic acid were the least effective: the concentrations of RS and glucose were 8.2 and 7.2 g/L, respectively, and DEH was 21%.

Pretreatment of reed with ChCl mixtures with glycerol or ethylene glycol led to an increase in the availability of the substrate for enzymes and DEH after 48 h of hydrolysis was 9 and 14%, respectively.

Thus, it is obvious that the use of organic acids as a hydrogen bond donor in DESs was more efficient than use of polyalcohols. ChCl/LA (molar ratio 1 : 5) and ChCl/OA (molar ratio 1 : 1) were optimal mixtures for pretreatment.

At the next stage, the optimal temperature and time of reed pretreatment for the selected DESs were selected. Figure 1 shows the yields of products of enzymatic hydrolysis (RS and glucose) of reed pretreated with ChCl/LA mixtures at 60, 80, and 90°C or ChCl/OA mixtures at 70, 80, and 90°C for various time intervals (1–24 h).

Fig. 1.
figure 1

The concentration of glucose (1) and RS (2) after 48 h of enzymatic hydrolysis of reed (40 g/L by dry weight) pre-treated with ChCl/LA at 60 (a), 80 (b) and 90°C (c) or ChCl/OA at 70 (d), 80 (e), and 90°C (f), after the action of EP B537 (10 mg protein/g substrate) and F10 (1 mg protein/g substrate) at 50°C, pH 5.0. The abscissa shows the pretreatment time.

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When reed was pretreated with a mixture of ChCl/LA, its hydrolyzability increased with an increase in the pretreatment time (up to 24 h). The highest yield of enzymatic hydrolysis products was obtained for the substrate pretreated at 80°C for 24 h: the concentrations of RS and glucose were 32 and 27 g/L, respectively, DEH was 80% (Fig. 1b). After 24 h of reed pretreatment at 90°C, subsequent enzymatic hydrolysis was less efficient, DEH was 60% (Fig. 1c). However, a short-time (3–6 h) pretreatment at a temperature of 90°C was more efficient than that at 80°C. The pretreatment of cane with the ChCl/LA mixture at 60°C was ineffective: the DEH of the substrate pretreated for 24 h was only 21% (Fig. 1a).

The dependence of the degree of hydrolysis of reed polysaccharides on the time of its pretreatment with the ChCl/OA mixture at 80 and 90°C was bell-shaped with maxima at 6 h at 80°C and 2–3 h at 90°C (Figs. 1e and 1f). At the same time, the highest yield of enzymatic hydrolysis products was observed for the substrate pretreated for 6 h and 80°C: the concentration of RS and glucose was 34 and 33 g/L, respectively; DEH was 86% (Fig. 1e). At a higher pretreatment temperature (90°C) for 2–3 h, 25 g/L of RS and 24 g/L of glucose were formed from the resulting substrate; DEH was 63% (Fig. 1f). With a decrease in the pretreatment temperature to 70°C, in order to obtain a greater yield of sugars, it was necessary to increase the processing time to 24 hours; as a result, the concentrations of RS and glucose after the enzymatic hydrolysis of the substrate were 29 and 28 g/L, respectively; DEH was 73%.

DISCUSSION

Currently, research in the field of synthesis and properties of DES, as well as the search for opportunities for their industrial application are at the initial stage. The attractive side of DESs is the fact that they belong to “green” solvents, which have such advantages as simple synthesis, low cost (relative to ionic liquids), biodegradability, and nontoxicity [4, 5, 7]. DESs can play a crucial role in the selective solubilization and removal of lignin from plant biomass, while keeping cellulose and hemicellulose intact and suitable for their further processing [22].

It is believed that the increase in the efficiency of enzymatic hydrolysis of biomass after its pretreatment with DESs is primarily due to delignification and destruction of the crystalline structure of cellulose [23]. In [24], sugar yields were compared after enzymatic hydrolysis of several types of plant raw materials before and after pretreatment with three mixtures of ChCl/boric acid, ChCl/glycerol, and betaine/glycerol. Without pretreatment, the hydrolysis efficiency decreased in the following order: eucalyptus cellulose (pulp) (DEH was 62%)—MCC (DEH was 49%)—wheat straw (DEH was 18%)—spruce sawdust (DEH was 8%). Pretreatment with DESs led to an improvement in the yields of enzymatic hydrolysis for all substrates, while the trend remained the same: the maximum yields of hydrolysis products of pretreated raw materials were 100% for eucalyptus cellulose (pulp), 65% for MCC, 33% for wheat straw, and 20% for spruce sawdust. Thus, mild DES pretreatment was effective for cellulosic substrates (eucalyptus cellulose, MCC), but was relatively ineffective for wheat straw and spruce sawdust.

In the present work, the conditions for the pretreatment of cane with DESs were selected, in which DESs in terms of the yield of RS increased from 6% for the original reed without pretreatment to 80–86% for that pretreated under optimal conditions. At the same time, glucose was the main product of hydrolysis: it accounted for 84 and 97% of all RS for the ChCl/LA and ChCl/OA mixtures, respectively, which were used as DES.

The high efficiency of acid DESs for pretreatment of plant biomass was noted in many works [16, 24]. The present work is no exception: DESs with glycerol or ethylene glycol were less effective than DESs with organic acids. It is believed that the stronger the acid is, the greater the effectiveness of a DES is. Thus, during the pretreatment of pine wood, the highest extraction of lignin and the yield of products of enzymatic hydrolysis were observed when using a mixture of ChCl with formic acid (pKa 3.75) than with LA (pKa 3.86) or acetic acid (pKa 4.75), all other conditions being equal [25]. In the pretreatment of wheat straw, the mixture of ChCl with OA (pKa 1.2) was preferable to LA [23]. The reed pretreatment carried out in this work also confirmed this regularity.

The molar ratio of hydrogen bond donors to acceptors also plays an important role in the efficiency of biomass pretreatment. For example, when a mixture of cellulose and lignin was used as a substrate, the solubility of the lignin was improved with an increase in the acid content in the ChCl/LA mixture from 1 : 1 to 1 : 9 [26]. Similarly, a higher solubilization of lignin from rice straw (from 51 to 60%) was observed with an increase in the proportion of acid in the ratio from 1 : 2 to 1 : 5 in ChCl/LA mixtures [22]. In another study, an increase in the ChCl/LA molar ratio from 1 : 2 to 1 : 15 led to an improvement in the extraction of lignin from corn cobs from 65 to 93%, but there was no significant increase in the yield of sugars during enzymatic hydrolysis (from 79 to 84%), from which it was concluded that the removal of 70% of lignin was sufficient to achieve the optimal yield of hydrolysis products [16]. In the present work, a slight increase in reed conversion with an increase in the proportion of acid in the ChCl/LA mixture (from 1 : 2 to 1 : 5) was shown, while at the same time, an increase in the proportion of acid in the ChCl/AO mixture (from 1 : 1 to 1 : 2 ) led to a very significant decrease in the reactivity of the reed.

One of the main advantages of using DESs for pretreatment of plant biomass is the moderate heating of the reaction mixture (up to 80–150°C), which minimized the formation of toxic substances (furfural derivatives, hydroxy acids, and aliphatic carboxylic acids) from lignin [27]. Delignification processes and their effect on the yield of enzymatic hydrolysis of corn cobs pretreated in the presence of DESs were studied in this temperature range [28]. When using a mixture of ChCl/glycerol with an increase in the pretreatment temperature from 80 to 150°C, an increase in the DEH of the substrate from 40 to 92% was observed. When a ChCl/urea mixture was used, the yield of hydrolysis products also increased, but to a significantly lesser extent: from 51 to 59% with an increase in the pretreatment temperature from 80 to 115°C. However, in the pretreatment temperature range of 80–150°C, the yield of products of enzymatic hydrolysis of corn cobs treated in a ChCl/imidazole mixture remained almost unchanged and was equally high, 92–95%. In a similar study [16], it was reported that an increase in delignification from 18 to 96% was observed with an increase in the temperature of pre-treatment of corn cobs with a mixture of ChCl/LA from 70 to 110°C. However, an increase in the yield of hydrolysis products (from 45 to 79%) was observed only in the range from 70 to 90°C, and then remained at the level of 78–80%. In the present work, the change in the temperature and time of pretreatment showed that the optimal temperature for treating cane with a mixture of ChCl/LA was 80°C, and the pretreatment time should be at least 24 h, while the DEH of the resulting substrate into soluble sugars was 80%. When using a mixture of ChCl with OA (a stronger acid than LA), in order to obtain the highest yield of enzymatic hydrolysis products, it was necessary either to limit the pretreatment time at 80°C to 6 h, or to reduce the temperature to 70°C, while maintaining the pretreatment time of at least 24 h. In the first case, the DEH of the obtained substrate was 86%, in the second, it reached 73%.

Thus, the possibility of efficient pretreatment of the common reed with DESs based on ChCl as a hydrogen bond acceptor and LA and OA as hydrogen bond donors has been shown. Reed pretreatment conditions were optimized, that is, the ratio of DES components, temperature and process time. This made it possible to achieve high DEH of pretreated reed with EP B537 (cellobiohydrolase and endoglucanase) and EP F10 (β-glucosidase), which were 80 and 86%, after pretreatment with DESs based on ChCl/LA and ChCl/OA mixtures, respectively.