Introduction: Carbohydrates are among the most abundant and widespread components in nature, accounting for approximately 70% of the caloric intake in a typical human diet [1]. Renewable plant biomass represents the majority of organic material on Earth and serves as an almost inexhaustible source of raw materials and energy [2]. Consequently, the development of efficient strategies for its utilization is a critical priority in modern biotechnology. Currently, biomass utilization largely relies on the enzymatic degradation of polysaccharide-rich plant materials, resulting in oligosaccharides and monosaccharides that can subsequently be converted through microbial or chemical processes into a variety of valuable products, including alcohols, organic and amino acids, polymers, and feed additives [3–5].
Cellulose, the principal polymeric component of plant biomass, is the most common polysaccharide on Earth [6]. Numerous microorganisms are capable of producing enzyme systems [7] that degrade this insoluble polymer into soluble sugars, primarily cellobiose and glucose. Polysaccharides find applications across diverse fields such as medicine, the food industry, microbiology, chemistry, and agriculture.
Current trends in the selection and development of polysaccharide-producing organisms include [8]:
- Identification of strains capable of utilizing inexpensive organic substrates instead of traditional, costly sugars.
- Implementation of genetic approaches to enhance producer strains.
- Enhancement of the overall activity of known enzyme complexes, primarily through genetic engineering.
A key aspect of this research is determining the optimal qualitative and quantitative composition of enzyme preparations for efficient hydrolysis of plant polysaccharides.
Cellulose-containing raw materials (CCRM), agricultural residues, and energy crop biomass are among the most abundant renewable resources globally, with an annual production estimated at approximately 200 billion tons [9]. Non-wood plant biomass, such as agricultural straw, offers advantages due to its annual renewability and low cost. The bioconversion of renewable plant biomass into fuels, food, feed, and intermediates for chemical and microbial industries is now recognized as a pivotal area of biotechnology. The abundance and affordability of non-wood species make them attractive alternative sources for carbohydrates and bioactive compounds [10–13].
The bioconversion of lignocellulosic biomass into biofuels and industrially important products has been extensively developed worldwide. Lignocellulosic materials, including agricultural residues and wood, are inexpensive, renewable resources predominantly composed of cellulose, hemicellulose, and lignin. Effective utilization of all three components is crucial for economic viability. To maximize cellulose conversion, increasing its surface area and disrupting its microfibrillar structure is essential, making various pretreatment methods—mechanical, physical, chemical, and biological—critical to improving biomass accessibility [14]. Pretreatment of CCRM is necessary to overcome the natural recalcitrance of the polymeric matrix and determine both the economic feasibility and efficiency of enzymatic hydrolysis. Known pretreatment methods include dilute acid treatment, thermobaric processing with or without catalysts, and alkaline delignification [15,16]. Hydrothermobaric treatment (GTBT), sometimes coupled with pressure release (“explosion”), is a current focus for producing cellulose-enriched substrates [17–19].
The resistance of the lignocellulosic matrix under fermentation conditions is a major barrier to industrial-scale production of glucose and pentose hydrolysates. Therefore, investigating factors influencing the enzymatic hydrolysis of pure cellulose is crucial for optimizing hydrolysis of diverse plant materials [10–13]. While the chemical inertness of plant polysaccharides, particularly cellulose, provides structural stability and protection against premature decomposition, it complicates biotechnological processing. Developing and enhancing multi-enzyme systems capable of efficient plant cell wall degradation remains a central challenge in modern biotechnology [20].
Plant biomass is a primary resource for biotechnological applications, including the production of valuable food and feed products. When pretreated with acids, alkalis, or enzymes, plant biomass serves as an excellent substrate for microbial growth, enabling industrial-scale microbiological processes based on plant hydrolysates [21]. Native lignocellulosic materials are only partially susceptible to enzymatic hydrolysis [22], necessitating pretreatment to reduce cellulose crystallinity and increase substrate surface area. Pretreatment approaches include physical-chemical, chemical, and biological methods, with key factors influencing efficacy being biomass type, acid selection, acid concentration, reaction duration, and temperature [23]. Treatment with dilute acids enhances porosity and total surface area by partially or fully removing hemicelluloses and/or lignin [24].
Chemical hydrolysis, which converts cellulose and hemicelluloses into fermentable monosaccharides, has largely been supplanted by enzymatic hydrolysis due to various limitations. Today, enzymatic technologies are among the most effective methods for transforming diverse biological materials [25]. Enzyme-based biocatalysis expands the raw material base for the food and feed industries, increases the depth of processing, facilitates the creation of novel products, and improves digestibility and organoleptic properties [26]. Transitioning from chemical to biotechnological methods often represents the only viable approach for developing waste-free and environmentally sustainable industries [27].
Given the richness of plant biomass in polysaccharides, identifying optimal processing strategies is essential. Key criteria for waste utilization include cost, volume, availability, chemical composition, and technological characteristics. Industrial conversion of renewable plant materials into valuable products holds significant practical importance. In Kazakhstan, cellulose-containing materials are virtually unlimited, including wood, straw, cotton waste (gouza-pai), and municipal solid waste. Nevertheless, efficient conversion into biologically digestible sugars is challenging. Research continues on using microorganisms, cellulolytic enzyme complexes, and chemical hydrolyzing agents for effective transformation of non-food biomass into digestible sugars [28].
MATERIALS AND METHODS
The study utilized the following substrates: wheat straw, vegetable waste from cotton processing (guza-paya), wheat bran, and cotton cellulose (cotton wool). The bioenzymes employed were Aspergillus awamori strain F-RKM 0719, Aspergillus terreus 461, Aspergillus terreus strain 499, and Aspergillus oryzae.
Glucose concentrations, representing the cellulose hydrolysis products, were measured using several methods, including the Dubois method, GO:PO (glucose oxidase:peroxide), dinitrosalicylic acid, Bertrand method, iodine method, resorcinol method, and the Shomodi-Nelson method. Analysis of reducing sugars was conducted following the McEn-Shorel method.
Monosaccharide composition of the hydrolysates was determined by high-performance anion-exchange chromatography on a CarboPac PA-1 column (4 × 250 mm, Dionex, USA) using a pulsed amperometric detector (PAD, Dionex). Additionally, an Alliance chromatograph (Waters, USA) equipped with a refractometric detector (Waters 2414) and a Reprosil-Pur NH2 column (4 × 250 mm, Dr. Maisch GmbH, Germany) was used.
Enzymatically active cellulolytic solutions were applied for the hydrolysis of cellulose-containing substrates, such as wheat straw, wheat bran, and cotton cellulose. Prior to analysis, plant raw materials were crushed and sorted. For chemical analyses, the materials were sieved to obtain particles of 2–3 mm.
Ash content was determined by combustion of the raw material followed by calcination in a muffle furnace at 600 °C. Readily and hardly hydrolysable polysaccharides were quantified using the Kiesel and Semiganovsky method, lignin was measured by the König method in the Komarov modification using 72% sulfuric acid, and pentosans were determined based on the pentose content in hydrolysates of easily and hardly hydrolysable polysaccharides.
Acid Hydrolysis
Wheat straw underwent autohydrolysis, after which the pulp was washed to obtain a sugar solution. Up to 90% of hemicelluloses were extracted into the aqueous solution. Subsequently, lignin was removed using solvents analogous to those for native lignin, such as dioxane–water (9:1) or ethanol–water (9:1), which removed up to 90% of lignin. In some cases, a 0.4–2% NaOH solution was used. This two-step extraction yielded a cellulose-rich product, which served as the substrate for glucose production. Cellulose hydrolysis was performed at 190–250 °C with sulfuric acid concentrations of 0.6–2.5% (w/w). Optimal hydrolysis conditions were found to be 160–170 °C for 30–80 minutes. Increasing the acid concentration accelerated sugar decomposition.
Enzymatic Hydrolysis
Plant cell walls exhibit high resistance to degradation. Microorganisms that utilize cellulose have evolved complex enzymatic systems capable of hydrolyzing cellulose into glucose monomers.
Enzyme kinetics experiments were conducted using wheat straw. The wheat straw was ground, sieved, and dried at 120 °C for 2 h to constant weight. Prior to enzymatic hydrolysis, the material was autoclaved at an overpressure of 0.05–0.1 MPa for 0.5–1 h. Fermentolysis was performed according to the specific characteristics of the enzyme preparations, maintaining pH 4.9–5.0 and a temperature of 49 °C. The duration of enzymatic hydrolysis ranged from 7 to 10 h.
Results and Discussion
Based on published data regarding chemical composition (Table 1) and the annual volume of generated waste, wheat straw was identified as a suitable feedstock for polysaccharide depolymerization.
Table 1. The chemical composition of wheat straw, in wt.
| Name of components | Content, % |
| Ash materials | 2,3 |
| Easily hydrolysable polysaccharides | 25,7 |
| Hardly hydrolysable polysaccharides | 41,3 |
| Gecosans | 35,3 |
| Pentozans (without uronic acids) | 24,5 |
Among the methods used for hydrolysis, the primary approaches are acid hydrolysis and enzymatic hydrolysis. Although sulfuric, hydrochloric, and phosphoric acids exhibit relatively high catalytic activity, their use in lignocellulose hydrolysis remains economically challenging due to their strong corrosive nature, high cost, and the expenses and environmental impact associated with neutralizing excess acid in hydrolysates. Sulfuric acid, however, presents a promising option, as its recovery can reduce the overall consumption of the hydrolyzing agent.
Consequently, identifying optimal pretreatment conditions using sulfuric acid, as well as studying the effect of these conditions on the efficiency of enzymatic hydrolysis of wheat straw, is of considerable importance. Developing integrated processing methods for wheat straw can enhance environmental sustainability while simultaneously generating raw materials and value-added products for industry.
In the study, straw was processed at temperatures ranging from 190°C to 250°C with sulfuric acid concentrations varying from 0.6% to 2.5% by weight. Increasing the temperature reduced the processing time required to achieve the maximum yield of reducing substances (RS) more significantly than the polysaccharide decomposition reactions. Consequently, the yield of polysaccharides increased with higher reaction temperatures. At temperatures below 150°C, the effect of sulfuric acid concentration was more pronounced; however, this influence diminished as the temperature reached 160°C.
The optimal temperature and duration for hydrolysis of straw with sulfuric acid were found to be 160–170°C and 30–80 minutes, respectively. Higher concentrations of sulfuric acid accelerated sugar decomposition, with the optimum concentration determined to be 1.77% by weight. Pretreatment of straw under varying hydromodules (1:3 to 1:5) was conducted at 1.6% sulfuric acid and 150°C. The highest yields of reducing substances were achieved with hydromodules of 1:3.5, 1:5, and 1:5.8, corresponding to 26.8%, 27.0%, and 29.2%, respectively. The hydrolysates’ polysaccharide composition was predominantly glucose, reaching concentrations of up to 25 g/L.
Thus, treating straw with 1.35% sulfuric acid at 150°C for 60 minutes and a hydromodule of 1:3 produced hydrolysates containing up to 7.6% reducing substances, suitable for further use in microbiological applications. Using a hydromodule of 1:4.5, the maximum reducing substances concentration was achieved at 160°C with 1.6% sulfuric acid, yielding 25.57% of the absolutely dry substance. Across all hydrolysis experiments, the most favorable results were consistently obtained with 1.6% sulfuric acid at 150–160°C. Experimental results are summarized in Table 2.
Table 2. Results of fermentolysis of wheat straw in a fermenter
| Process number | Number of enzymes, units of activity | The maximum concentration of RS,% | Yield of RS,% |
| 1 | 62-75,8 | 0,43 | 4,3 |
| 2 | 62-75,8 | 0,46 | 4,6 |
| 3 | 6,2-17,6 | 0,46 | 4,6 |
| 4 | 31-37,9 | 0,48 | 4,8 |
| 5 | 62-75,8 | 0,32 | 3,2 |
| 6 | 1,6-1,9 | 0,26 | 2,6 |
| 7 | 3,0-3,8 | 0,26 | 2,6 |
| 8 | 4,6-5,7 | 0,22 | 2,2 |
Model experiments for the study of enzyme kinetics were carried out using paper and cotton wool as the source of cellulose. In the experimental processes of fermentolysis, the wheat straw was preliminarily ground, sifted and dried to a constant value in a drying oven at a temperature of 120 °C for 2 h, which was previously welded in an autoclave at an excess pressure of 0.05-0.1 MPa for 0.5 – 1 h.
The processes of fermentolysis were carried out while maintaining active acidity in the pH of 4.9-5 units of and temperature of 49 °C. The duration of the fermentolysis processes was 7-10 h.
Studies of the kinetics and stoichiometry of the reactions of enzymatic hydrolysis of disperse solid-phase vegetable substrates were carried out by a vibatory fermentolizer with automated pH adjustment and thermostating36. The results of the experiment are given in Table 3.
Table 3. Results of fermentolysis of wheat straw in a fermenter
| Process number | Number of enzymes, units of activity | The maximum concentration of RS,% | Yield of RS,% |
| 1 | 62-75,8 | 0,43 | 4,3 |
| 2 | 62-75,8 | 0,46 | 4,6 |
| 3 | 6,2-17,6 | 0,46 | 4,6 |
| 4 | 31-37,9 | 0,48 | 4,8 |
| 5 | 62-75,8 | 0,32 | 3,2 |
| 6 | 1,6-1,9 | 0,26 | 2,6 |
| 7 | 3,0-3,8 | 0,26 | 2,6 |
| 8 | 4,6-5,7 | 0,22 | 2,2 |
In order to reduce the error and verify the action of the enzyme on single-component substrates, a series of experiments was carried out on fermentolysis of crushed, buffered paper in fermentolysis with a volume of 6 liters. The results of comparative processes of fermentolysis of straw and paper are shown in Fig. 1.
Fig. 1. Variation in the concentration of RS in the processes of fermentolysis of paper and wheat straw
Since the paper is nearly pure cellulose, using the same kinetic parameters for fermentolysis, the amount of sugars produced from straw would theoretically be 1.49 times lower than from paper. However, in practice, straw exhibits a more compact fiber structure and contains other interfering components, which slows down the fermentolysis process by a factor of 2.5.³⁷
CONCLUSION
Analysis of the experimental results indicates that enzymatic hydrolysis is the most effective method for producing glucose from plant waste. This approach is promising for the development of low-waste technologies, provides high yields of the target product (up to 90%), and operates under mild conditions, such as low temperature and atmospheric pressure.
Following the cultivation of microorganisms, experiments were conducted to compare the efficiency of acid and enzymatic hydrolysis in producing hexoses (glucose) from wheat straw. The degree of conversion of plant biomass to monosaccharides was measured at various time intervals after the start of hydrolysis: 3, 24, 48, and 72 hours.
The optimal conditions for acid hydrolysis of wheat straw were found to be a temperature of 160–170 °C and a duration of 30–80 minutes. Increasing the concentration of sulfuric acid accelerated sugar decomposition, with the optimum concentration determined to be 1.6% by weight. Pretreatment of wheat straw with different hydromodules (1:3 to 1:5) was carried out under these conditions at 150 °C. The maximum yield of reducing sugars (PB) was achieved with hydromodules of 1:3.5, 1:5, and 1:5.8, resulting in yields of 26.8%, 27.0%, and 29.2%, respectively.
A comparison of the efficiency of acidic and enzymatic hydrolysis revealed that the Aspergillus awamori strain F-RKM 0719 was the most effective for wheat straw. Even so, enzymatic hydrolysis consistently outperformed acid hydrolysis in cellulose conversion. Enzymatic hydrolysis of wheat biomass provided the highest conversion rates of polysaccharides to monosaccharides, demonstrating its potential as a method for obtaining glucose and sorbitol. Under optimal conditions, enzymatic hydrolysis achieved a conversion degree of 90%.
Wheat straw is therefore a suitable raw material for enzymatic depolymerization of polysaccharides. The optimal enzymatic hydrolysis parameters were determined to be: enzyme dosage of 0.05 g/g substrate, temperature of 50 °C, pH 5.0, and hydromodule of 1:30. Under these conditions, a glucose yield of 63.97% of the raw material content was obtained.
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