Polish Varieties of Industrial Hemp and Their Utilisation in the Efficient Production of Lignocellulosic Ethanol

2.2. Alkaline Pretreatment

The purpose of chemical pretreatment is removing lignin from materials with lignocelullose and increasing the accessibility of biomass structure. The type of a reagent used has a significant effect on the performance of the chemical pretreatment. Sodium hydroxide is one of the most popular alkaline reagents used in this process.

The optimization of the concentration of sodium hydroxide used in alkaline treatment for the four varieties of hemp biomass was carried out based on the amount of reducing sugars released after the enzymatic test. Concentrations ranging from 1.5% to 3% were tested (Table 2). It was found that, for Tygra and Białobrzeskie varieties, for 2% NaOH, the amount of released reducing sugars was about 13% higher than for 1.5%. In turn, for 3% sodium hydroxide, the content of reducing sugars was at a similar level. For Henola and Rajan varieties, a completely different correlation was observed; the lowest level of released reducing sugars was noted for the concentration of 3% NaOH, while, at the concentration of 2%, the content of reducing sugars was the highest. Moreover, it was noted that two of the varieties, i.e., Tygra and Rajan, were characterized by over 10% higher content of reducing sugars than Białobrzeskie and Henola, which proves that these are the varieties more susceptible to the alkaline pretreatment. Based on the obtained results, the concentration of sodium hydroxide at the level of 2% was selected for further research. Kumar et al. [12] conducted similar research and stated that the sodium hydroxide pretreatment of lignocellulosic biomass resulted in the highest level of delignification at 2% NaOH. In turn, Zhao et al. [10], in the research on the use of American industrial hemp for the production of bioethanol, used the alkaline treatment of 1% NaOH.

Efficient pretreatment should decrystallize cellulose, depolymerize hemicelluloses, reduce the formation of inhibitors that hinder carbohydrate hydrolysis, require low energy expenditure, and recover value-added products such as lignin.

To confirm the efficiency of the alkaline treatment, the determination of the chemical composition of hemp biomass after NaOH treatment was performed and compared to the chemical composition of the biomass before pretreatment. The results are presented in Table 3.

The analysis of the chemical composition of the hemp biomass before and after treatment showed that, in all the four varieties, the alkaline treatment resulted in a visible increase in the cellulose content (by approximately 10%) and the partial degradation of hemicelluloses (as much as 12% for the Rajan variety). The highest content of cellulose after treatment was found in the following varieties: Tygra, Białobrzeskie, and Rajan, and its level was approximately 60%. In the case of the lignin content, only for the Białobrzeskie hemp biomass, a very slight reduction was observed after the alkaline pretreatment. In the case of the remaining varieties, the tendency was contrary. Similar observations were reported by Stevulova et al. [13], who examined the chemical composition of hemp biomass before and after the pretreatment with sodium hydroxide and proved that the content of lignin after the pretreatment was 7% higher than before.

At this stage, due to better properties, availability, and higher yield, only two types of biomass of the Tygra and the Rajan varieties were selected, for which further research on bioethanol production was continued.

The effect of the alkaline treatment on Tygra and Rajan hemp biomass was also confirmed by Fourier transform infrared spectrometer (FTIR) shown in Figure 1a,b and by scanning electron microscopy (SEM) shown in Figure 2.

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Figure 2

The SEM images of hemp biomass: (a) Tygra biomass before pretreatment, (b) Tygra biomass after pretreatment, (c) Tygra biomass after enzymatic hydrolysis, (d) Tygra biomass after enzymatic hydrolysis—selected fragment in high magnification, (e) Rajan biomass before pretreatment, (f) Rajan biomass after pretreatment, (g) Rajan biomass after enzymatic hydrolysis, (h) Rajan biomass after enzymatic hydrolysis—selected fragment in high magnification.

An effective method for studying the structure of biomass after alkaline treatment is FT-IR [14]. Figure 1a,b show the changes in FTIR spectra after the alkali treatment of hemp biomass between 600 cm⁻¹ and 4000 cm⁻¹. On the spectra of both varieties, typical vibration bands in the cellulose molecule were observed at 3300 cm⁻¹, 2900 cm⁻¹, and 1610 cm⁻¹. The broad band in the 3600–3100 cm⁻¹ region, which was due to the OH stretching vibration, gave considerable information concerning the hydrogen bonds. The peaks characteristic of hydrogen bonds from the spectra of the Rajan variety AP became a little sharper and more intense compared to the Rajan variety BP. However, in the case of the Tygra variety, this band was less intense. The 2900 cm⁻¹ peak corresponding to the C–H stretching vibration in the case of the Tygra variety shifted to higher wavenumber values and slightly decreased in the intensity. These changes could have resulted from both the increased amount of cellulose after the treatment (2.2, Table 3) and the reduced cellulose crystallinity [15,16]. The band at 1610 cm⁻¹ from the stretching vibrations of the O–H bonds, due to the adsorbed water in the sample, decreased, especially for the Rajan variety, which could be attributed to water loss due to drying the sample [17]. The intensities of peaks at 1160−1170 cm⁻¹ (asymmetric C−O−C stretching from cellulose) and 1110−1120 cm⁻¹ (C−OH skeletal vibration in cellulose) increased after pretreatment in both types of biomass. Furthermore, the intensity at 1050−1060 cm⁻¹ (C−O−C pyranose ring skeletal vibration ascribed to cellulose) also increased slightly after alkali pretreatment. These changes were confirmed by the increase in the concentration of cellulose in the pretreated biomass in the chemical composition tests (2.2, Table 3) [18].

The vibration band visible at 1730 cm⁻¹ (C=O stretching of acetyl groups in hemicelluloses and aldehydes in lignin) [19] was reduced in both hemp varieties after alkaline treatment, while the change was much more significant for the Rajan variety where the band almost disappeared. This occurred due to the decomposition of hemicelluloses and the solubilization of lignin during alkali pretreatment. This result correlates very well with the obtained results of the chemical composition of the biomass after pretreatment. According to these studies, the content of hemicelluloses after alkaline treatment in the Rajan variety decreased by as much as 12% (2.2, Table 3).

Moreover, the obtained FTIR spectra showed that the removal of lignin in the alkaline treatment process was problematic (band at 1510 cm⁻¹). This was confirmed by the results of the chemical composition of the lignin content presented in Table 2. This problem is widely described in the literature dealing with the studies of lignocellulosic biomass by infrared spectroscopy [20,21]. The process of the degradation or fragmentation of lignin is complicated due to the presence of strong C–C bonds and other functional groups, such as aromatic groups [13].

Significant changes on the surface of the biomass were observed and presented in the SEM images taken before and after the biomass pretreatment as well as after enzymatic hydrolysis (Figure 2). In the case of both varieties of hemp biomass (Tygra and Rajan), similar changes were observed in the biomass surface, which appeared as a result of subsequent processes. However, in the case of the Rajan variety, changes were more intensive, especially after the enzymatic hydrolysis process. The untreated hemp biomass was observed to have intact, rigid, and coarse structures with smooth surface and a well-ordered fiber skeleton (Figure 2a,e). This strongly blocked access to cellulose to limit enzymatic attack [22]. As a result of pretreatment, the biomass underwent various specific structural changes. The SEM images of hemp biomass after pretreatment showed that the surface area of the biomass was partially purified (Figure 2b,f). The morphological changes that indicated damage to the structure of biomass and that increased the surface area, making it more accessible to the cellulolytic enzymes [20,23,24], were observed. The enzymatic hydrolysis of the samples subjected to the previous alkaline treatment caused further significant changes in the structure of the biomass visible in the SEM pictures (Figure 2c,d,g,h). The appearance of micropores was very characteristic here.

Undoubtedly, the opening of the hemp biomass and creating the holes all over the biomass enhanced the enzyme accessibility of the structure and facilitated biomass digestibility [25]. Moreover, it was clearly visible that enzymatic hydrolysis of the Rajan hemp biomass made the fibrous structure fragile and was more successful.

2.3. Evaluation of Enzyme Preparations

The enzymatic hydrolysis process is the second main step in the process of obtaining bioethanol from plant biomass. Enzymatic hydrolysis determines the amounts of simple sugars that are metabolized by yeast in the fermentation process. The breakdown of cellulose into simple sugars requires the synergistic action of different enzymes: cellulases, endoglucanases, cellobiohydrolases, and ß-glucosidases. First, the commercial enzyme preparations of various compositions were gained (Flashzyme Plus 200, ACx8000L, Celluclast 1.5L, Cellobiase, Xylanase), and then their cellulolytic and xylanolytic activities were determined. Taking into account evaluated activity of the tested enzymes and their commercial availability, Flashzyme Plus 200 and Celluclast 1.5L preparations were selected for further research (Table 4).

In order to select the enzyme complex for the SHF and the SSF processes, enzymatic tests were performed using selected enzymes and their supplementation with glucosidase and xylanase and were partially described in our previous studies [4]. For the SHF process in the case of the Tygra biomass, an enzyme complex was selected with the composition of Flashzyme Plus 200, glucosidase, and xylanase, while composition for the Rajan was Flashzyme Plus 200:Celluclast 1.5L hemp biomass in the proportion of 70:30. For the SSF process, the enzyme complex for the Tygra biomass was selected as Flashzyme Plus 200:Celluclast 1.5L (70%:30%) and xylanase and for the Rajan was Flashzyme Plus 200:Celluclast 1.5L hemp biomass in the 50:50 ratio.

2.4. Separate Hydrolysis and Fermentation (SHF)

To determine the optimal conditions of the enzymatic hydrolysis method as a separate process of SHF, based on the literature data and the research experience, the following parameter ranges were selected for testing with the response surface methodology (RSM): dose of the enzyme 10–30 FPU·g⁻¹ of solid, temperature 50–70 °C, pH 4.2–5.4, and time 24–72 h. The RSM method is an effective optimization tool consisting of mathematical and statistical techniques and used for the process of optimization [26,27,28].

Individual enzymatic tests of the hydrolysis process were performed for Tygra and Rajan biomasses, and the evaluation criterion was the amount of released glucose. Figure 3 presents various response surfaces and interaction effects of variables (temperature, time, enzymes’ dose, and pH) on the glucose yield. The variable that had the most significant impact on the glucose content turned out to be the temperature. The lower the temperature was, the higher the glucose content was. The pH of the solution, the process time, and the enzymes’ dose had lesser effects. However, slight differences were observed in the dependence of these variables on the glucose yield for the two biomass varieties. For the Tygra biomass, it was found that, in the SHF process, the optimal conditions for enzymatic hydrolysis were obtained for the substrate concentration of 5% using the following enzymes: Flashzyme Plus 200 30 FPU·g⁻¹ of solid, glucosidase 20 CBU·g⁻¹ of solid, and xylanase 500 XU·g⁻¹ of solid. The process parameters were: temperature 50 °C, pH 4.2, and time 48 h. These parameters provided the opportunity to obtain a maximum glucose yield, which was 36.9 ± 0.64 (g·L⁻¹). In the SHF process for the Rajan biomass, optimal enzymatic hydrolysis conditions were obtained for a substrate concentration of 5% using the Flashzyme Plus 200:Celluclast 1.5L (70:30) enzyme complex with a dose of 10 FPU·g⁻¹ of solid. The process parameters were: temperature 50 °C, pH 5.4, and time 72 h. The maximum glucose yield was 23.66 ± 0.16 (g·L⁻¹).

Similar research was conducted by Abraham [29]; during biomass hydrolysis at 50 °C and 18 FPU·g⁻¹ of solid, the highest glucose yield was obtained. Salimi and others [30] optimized the enzymatic hydrolysis of lignocellulosic biomass using the RSM method. They applied the temperature range of 45–60 °C and the pH of 4.5—6.0. They obtained the highest content of monosaccharides at 45 °C and pH 6.0. Jambo et al. [31], in turn, optimized lignocellulosic biomass using similar parameters—temperature (30–60 °C), pH (3.8–5.8), and incubation time (12–72 h)—and obtained the glucose concentration at the level 24.24 g·L⁻¹.

The next step in the conversion of hemp biomass to bioethanol in the SHF process was ethanol fermentation. In the SHF process for the Tygra biomass, the highest concentration of ethanol was observed at 48 hours and was 10.51 g·L⁻¹. In the following hours of the process, no significant increase in ethanol concentration was observed. In turn, for the Rajan biomass, the highest ethanol concentration was noticed at 96 h, and it was only 2.76 g·L⁻¹. Such a low concentration of ethanol in this case could be attributed to various reasons, which together may have had a significant negative impact on this parameter—for example, the chemical composition, which was characterized by a higher lignin content compared to the Tygra biomass. The reason could also have been the use of a low enzyme dose (10 FPU g⁻¹ solid) in Rajan biomass as well as the low glucose concentration (23.66 g L⁻¹), which was determined immediately after the enzymatic hydrolysis step. Additionally, after optimizing this step, a pH of 5.4 was chosen based on the glucose concentration, which may have, to some extent, inhibited yeast activity in the initial phase of fermentation. Nevertheless, it was noticed that, with each passing day, the concentration of ethanol increased slightly, which ultimately indicates that the process itself was proceeding correctly. However, for Tygra biomass, no significant increase in ethanol concentration was observed with time extension of the SHF process (Figure 4). The average yield of bioethanol for the Tygra variety was equal to 253 L∙Mg⁻¹ (of hemp straw dry matter), i.e., 3.04 m³·ha⁻¹. For the Rajan variety, the average yield of bioethanol was 69 L∙Mg⁻¹ (of hemp straw dry matter), i.e., 0.80 m³·ha⁻¹. A similar study was presented by Kusmiyati et al. [32]. In their work, the conversion of the lignocelullosic biomass to bioethanol was carried out through pretreatment, saccharification, and fermentation processes. Their results showed that the SHF process gave a higher concentration of ethanol (8.11 g·L⁻¹). Fischer and others [33], in their research, dealt with lignocellulosic biomass and examined the SHF process, obtaining the ethanol concentration of 12.1 g·L⁻¹.

SHF as an alternative process in an industrial bioethanol plant manifests both potential and limitations. The main advantage of SHF is the possibility to optimize the process steps separately, especially to be able to run the enzymatic hydrolysis at an optimal temperature with respect to enzymes [34]. However, most literature reports confirm that SSF is a more promising and advantageous approach with respect to SHF because of a low production cost, less processing time, less reactor volume, higher ethanol productivity, a lower requirement of enzyme, the ability to overcome enzymatic inhibition by simultaneous end-product removal, and a lower requirement for sterile conditions, as bioethanol is produced immediately with glucose conversion [35,36].

3.2. Analytical Methods

The chemical composition of hemp biomass before and after pretreatment was determined, i.e., cellulose according to TAPPI T17 m-55 [42], hemicelluloses as the difference of holocellulose and cellulose according to TAPPI T9 m-54 [43], and lignin according to TAPPI T13 m-54 [44].

In order to provide a more complete picture of the molecular structure of hemp biomass before and after the chemical pretreatment, the analysis of FTIR spectroscopy was performed using a Fourier Transform Infrared Spectrometer ISS 66v/S (Bruker, Bremen, Germany) at infrared wavenumbers of 400–4000 cm⁻¹ [13].

The physical morphologies of hemp biomass before and after the chemical treatment and after enzymatic hydrolysis were performed using Scanning Electron Microscope S-3400N (Hitachi, Japan) in high vacuum conditions. The samples were covered with gold dust.

The contents of glucose and ethanol were determined by high performance liquid chromatography on Elite LaChrom (Hitachi, Tokio, Japan) using an RI L-2490 detector, Rezex ROA 300×7.80 mm column (Phenomenex, Torrance, CA, USA), as the mobile phase used 0.005 N H₂SO₄ at a flow rate of 0.6 mL/min at 40 °C.

3.3. Calculations

The ethanol yield from 100 g of raw material Y_s (g/100 g of raw material) was calculated according to the Equation (1) [45]:

where: Et—amount of ethanol in 1000 mL of tested sample (g); M—mass of material weighed in 1000 mL fermentation sample (g).

Then, based on the ethanol yield from 100 g of raw material, the amount of ethanol in L per ton of straw dry matter (L∙Mg⁻¹) was calculated, and on the basis of straw yield, the ethanol yield per hectare (m³∙ha⁻¹) was determined.

The study was conducted as a research project- Multiannual Program (2017–2020): Reconstruction and sustainable development of production and processing of natural fiber raw materials for the needs of agriculture and the economy was financed by the Ministry of Agriculture and Rural Development, Poland.