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Article

Application of Proton Ionic Liquid in the Process of Obtaining Bioethanol from Hemp Stalks

by
Małgorzata Smuga-Kogut
1,*,
Bartosz Walendzik
2,
Katarzyna Lewicka-Rataj
3,
Tomasz Kogut
4,
Leszek Bychto
5,
Piotr Jachimowicz
6 and
Agnieszka Cydzik-Kwiatkowska
6,*
1
Department of Agrobiotechnology, Faculty of Mechanical Engineering, Koszalin University of Technology, Raclawicka 15-17, 75-620 Koszalin, Poland
2
Faculty of Civil Engineering, Environmental and Geodetic Sciences, Koszalin University of Technology, Sniadeckich 2, 75-453 Koszalin, Poland
3
Water Management Laboratory, Faculty of Civil Engineering, Environmental and Geodetic Studies, Koszalin University of Technology, Sniadeckich 2, 75-453 Koszalin, Poland
4
Department of Geodesy and Offshore Survey, Maritime University of Szczecin, Waly Chrobrego 1-2, 70-500 Szczecin, Poland
5
Department of Electronics, Faculty of Electronics and Computer Science, Koszalin University of Technology, Sniadeckich 2, 75-453 Koszalin, Poland
6
Department of Environmental Biotechnology, University of Warmia and Mazury in Olsztyn, Słoneczna 45G, 10-709 Olsztyn, Poland
*
Authors to whom correspondence should be addressed.
Energies 2024, 17(4), 972; https://doi.org/10.3390/en17040972
Submission received: 19 December 2023 / Revised: 15 February 2024 / Accepted: 17 February 2024 / Published: 19 February 2024
(This article belongs to the Section A4: Bio-Energy)
Browse Figures
Versions Notes

Abstract

:
In this work, hemp (Cannabis sativa L.) stalks were pretreated with pyrrolidinium acetate [Pyrr][AC] to increase the availability of cellulose for cellulolytic enzymes and thus improve the production of 2G ethanol from reducing sugars. The process was carried out under different temperature and time conditions: The control sample was raw material and deionized water, and the second sample was kept at 21 °C for 24 h. The third sample was kept at 90 °C for 30 min, and the fourth sample was kept at the same temperature for 24 h. For each pretreatment, the extraction of lignin and hemicellulose was determined, as well as the change in biomass composition before and after pretreatment. The stalks of hemp seed contained 41.54% cellulose, 18.08% lignin and 28. 87% hemicellulose. [Pyrr][AC] used to dissolve seed hemp at 90 °C for 24 h was most effective in the extraction of this biopolymer, extracting 3.1% content. After enzymatic hydrolysis, the highest content of reducing sugars was found for samples of hemp stalks that were first pretreated with proton ionic liquid (PIL) and stirred at 90 °C for 24 h. On the other hand, the highest ethanol content (5.6 g/L) after fermentation and yeast viability (56.7%) after 72 h were obtained in samples pretreated at 90 °C and for 24 h.

1. Introduction

Hemp (Cannabis sativa L.), otherwise known as industrial hemp, is a seed-bearing plant belonging to the hemp family [1]. Cannabis sativa L. are annual plants with a vegetation period of about four months and very rapid growth. Cultivated hemp reaches a height of up to 4 m [2]. There are two forms of hemp in Poland and Europe—dioecious and monoecious. Since the 1960s, dioecious hemp has been replaced in production by monoecious varieties due to, among other reasons, their uniform maturation period that facilitates harvesting, as well as the fact that there are two separable female and male flowers on a single individual [3].
Originally, hemp was used for fiber production and medicinal purposes. Today, hemp is a raw material used in the food, textile, paper, cosmetics, construction, energy and automotive industries [3]. The main benefits of using hemp in biofuel production are on-demand storage, lower nitrogen and carbon oxides emission during the combustion process, the possibility to use reduced waste—hemp seeds are used to produce cooking oil—and a fast cultivation and harvesting process [4]. Hemp can be used multi-directionally in biofuel production, energy production by combustion and in biomass production, as well as for pressing oil from the seeds and for biodiesel production. Hemp biomass can be gasified to produce methane, or parts of the stalks can be used to produce bioethanol [2].
Second-generation bioethanol production uses lignocellulosic materials, which can be converted into ethanol by fermentation with microorganisms. Hemp stalks seem to be an ideal raw material for bioethanol production because they are easy to produce and there is the possibility of using the stalks of these plants for the production of a second-generation bioethanol. The cellulose content of the stalks of these plants, as a potential source of ethanol, can be as high as 77% [5]. To ferment cellulose into ethanol, hemp stalks must be ground to break down the raw material and separate the cellulose from the lignin and other substances that provide ballast for cellulolytic enzymes. Many further types of pretreatments for cellulosic materials can be found in the literature, such as physical (steam explosion), chemical (alkali or acid), or a combination of these. These pretreatments disrupt the compact structure of lignocellulosic biomass by removing hemicellulose/lignin and enhancing the accessible surface area of the cellulose for enzymes [6,7]. The promising pretreatments include methods using solvents that are non-toxic and recoverable after the process, making them more environmentally attractive [8,9,10,11,12]. One problem with using lignocellulosic raw materials for bioethanol production is the presence of lignin, which hinders the further conversion of complex sugars to simple sugars. The use of PILs (proton ionic liquids) which can be recovered by distillation after the process seems to be a cost-effective method for extracting lignin from biomass [13]. Studies show that dissolving biomass in PIL loosens the bonds between xylan, cellulose, and lignin, allowing its extraction with this solvent [14,15]. This process could be widely used in the production of bioethanol from lignocellulosic feedstocks and provide an alternative to more expensive imidazolium ionic liquids [16].
The applied protonic ionic liquid (PIL, Figure 1) is a salt that can be obtained through a simple and economical synthesis of a cheap acid (acetic acid) with a secondary amine (pyrrolidine). Due to the specific physical properties of this salt, it can be used at relatively low temperatures (<100 °C) as an alternative to conventional methods for lignin removal during biomass processing. Furthermore, the significant difference in volatility between the applied protonic ionic liquid and the extracted lignin allows for easy recovery of the PIL through a simple distillation, leaving the non-volatile lignin for further processing.
Bioethanol from lignocellulose is usually obtained by one of two fermentation systems: separated hydrolysis and fermentation (SHF) and simultaneous saccharification or fermentation (SSF). Separate hydrolysis and fermentation enables improved yields of fermentable sugars as it is carried out under optimal conditions for enzymes and yeast. In SSF mode, the enzymes and yeast are in the same nutrient solution, which requires the establishment of common conditions for the biocatalysts. This type of process proving is advantageous in terms of the speed of bioethanol yield and the lack of accumulation of reducing sugars, which are immediately fermented by the yeast. However, in order to test the effectiveness of pretreatment on the efficiency of the various stages of bioethanol production, the SHF process is often used [17,18].
In the present study, the authors focused on the effect of PIL on the yield of fermentable sugars, the selection of pretreatment process parameters (temperature and time), and the effect of this solvent on the physiological state of yeast in the fermentation of hydrolysates from hemp stalks.

2. Materials and Methods

The stalks of hemp (Cannabis sativa L.) used in the study came from a farm located in the Pomeranian province and were harvested in 2021. The stalks were cut and mechanically shredded. Pyrrolidinium acetate [Pyrr][AC] was used for pretreatment. Pyrrolidine, of at least 99% purity, was purchased from Sigma Aldrich/Merck (Burlington, MA, USA) and used as received. High-purity glacial acetic acid, ethanol, and sulfuric acid were used as received. Cellulase enzyme blend from Sigma Aldrich/Merck was used for enzymatic hydrolysis. The preparation contained the enzymes cellulase CelA and Cel7A. Saccharomyces cerevisiae TYPE II yeast (Sigma Aldrich/Merck) was used for alcohol fermentation. PIL [Pyrr][AC] was synthesized in a round-bottom flask according to [15]. The molar ratio of pyrrolidine and glacial acetic acid was 1:1. The flask was placed in an ice/water bath to prevent excessive heat buildup during the reaction. Stirring was continued for 24 h at room temperature to obtain the [Pyrr][AC].

2.1. Pretreatment with PIL

The hemp stalks were crushed to a fraction size of approximately 100 µm. Samples of 5 g on a dry weight basis were collected and 50 mL of [Pyrr][AC] solvent was added to each sample. The samples were then mixed and heated. Several test variants were performed: A control sample was prepared with 5 g of raw material and 100 mL of deionized water (S1). The second sample was kept at 21 °C for 24 h (S2). The third sample was kept at 90 °C for 30 min (S3), and the fourth sample was kept at the same temperature for 24 h (S4).
After heating, the samples were cooled to room temperature, and 50 mL of deionized water and 50 mL of acetone were added. The samples were then shaken at 250 rpm for 1 h and centrifuged in a laboratory centrifuge (10 min, 10,000 rpm). The supernatant was rinsed three times with water and the mixing and centrifugation step was repeated. The purified biomass was subjected to enzymatic hydrolysis.

2.2. Enzymatic Hydrolysis

Samples of hemp stalks before and after pretreatment were subjected to enzymatic hydrolysis using cellulase. First, 5% of the biomass was dissolved in 0.05 M citrate buffer (pH 4.8) with 0.2% sodium azide to prevent contamination and then cellulase was added at 28 FPU/g biomass. The prepared samples were placed in a shaking water bath at 50 °C and 200 rpm. During incubation, the samples were analyzed at specific time intervals, i.e., at 0, 12, 24, 48, and 72 h. The total content of reducing sugars was determined using the DNS method [19].

2.3. Alcohol Fermentation

Freeze-dried distiller’s yeast Saccharomyces cerevisiae type II (purchased from Sigma-Aldrich) (5%, w/v) was used to initiate ethanol fermentation after 72 h of enzymatic hydrolysis. This was afterward allowed to proceed in anaerobic conditions for four days at 35 °C. The concentration of ethanol in the samples after fermentation was determined using an enzyme assay from Megazyme that relies on UV radiation to measure ethanol concentrations in food products. Methods based on this principle have been accepted by AOAC (AOAC Method 2019.08, First Action), IFU, EBC Method 9.3.1, MEBAK, and ASBC Method Beer 4-F.

2.4. Analytical Techniques

Compositional analysis. An Ankom A200 fiber analyzer (ANKOM Technology, New York, NY, USA) was used to determine the amounts of lignin/cellulose/hemicellulose in untreated and pretreated biomass samples (with the use of filter bag encapsulation). The fiber contents were determined as follows. The neutral detergent fiber (NDF) content was determined by the Van Soest method, while the acidic detergent fiber (ADF) and acidic detergent lignin (ADL) were determined according to the standard [20]. The difference between the ADF and ADL fractional share was the cellulose content, while the difference between NDF and ADF fractional share was the hemicellulose content.
FTIR. IR spectra were recorded with a Shimadzu IRAffinity-1S spectrophotometer (Shimadzu Corp., Kyoto, Japan) equipped with an ATR attachment using the attenuated total reflection phenomenon. Individual spectra were obtained using 20 scans in the range of 4000–700 cm−1 at a resolution of 4 cm−1. Near-infrared (NIR) spectra were measured using a DLP NIRscan Nano spectrophotometer from Texas Instruments (Dallas, TX, USA), using the reflectance technique. (To obtain a uniform sample surface, the straw was homogenized and then compressed under pressure to obtain a tablet with a diameter of 10 mm and a thickness of 5 mm.) The spectra were measured in the range of 1250–1700 nm. Measurements were performed at 23 °C and a resolution of 3.5 nm. Each spectrum obtained was the average of 10 scans.
The obtained raw FTIR and NIR spectra were subjected to pre-processing. Z-score normalization was used as the main pre-processing technique. Z-score normalization refers to the process of normalizing each value in a data set so that the mean of all values is 0 and the standard deviation is 1.
To perform the normalization, the following formula was used for each value in the dataset:
NEW VALUE = (R − μ)/σ
where the variables are defined as follows:
  • R—original value
  • μ—mean of the data
  • σ—standard deviation of the data.
To distinguish hemp fiber samples pretreated with PIL, principal component analysis was performed based on the obtained FTIR and NIR spectra data.
PCA is a multivariate technique that linearly transforms the original set of variables into a much smaller set of uncorrelated variables without losing information about the data. The data for PCA are organized in a two-way matrix, in which the column vectors represent the variables and the row vectors represent the “objects” whose variables are measured [21].
Evaluation of yeast viability after fermentation. The method of staining with a 1:10,000 diluted methylene blue solution (with a concentration of 0.0001%) was used to evaluate the survival rate of yeast cells in the different variants of the conducted culture (conducted experiment). A 0.02 mL sample of the culture suspension stained with methylene blue was applied to a square of 24 × 24 mm area determined on a microscope slide. After covering with a coverslip, the slides were analyzed at 40× objective magnification using a Nikon 80i light microscope (Nikon Instruments Inc., Melville, NY, USA) and a Nikon DS.-Ri1 camera (Nikon Instruments Inc., Melville, NY, USA), which was used to archive images of 20 fields of each slide. The computer image analysis program NIS-Elements BR 3.2 was used to count the dead (stained with methylene blue) and live (colorless) yeast cells. The results were used to determine, the percentage of live yeast cells in the total number of yeast cells. The cellulose to ethanol conversion rate (%) was calculated according to the formula [22]:
Y = (C_e × V × 100)/(M × C × 1.1 × 0.51) × 100 (%)
where the variables are defined as follows:
  • Ce—ethanol concentration (g·L−1)
  • V—sample volume (L)
  • M—total amount of substrate in the sample (g s.s.)
  • C—cellulose and hemicellulose concentration in the material (%)
  • 1.1—cellulose to glucose conversion factor
  • 0.51—glucose to ethanol conversion factor

3. Results

3.1. Effect of PIL Pretreatment on Composition Analysis

The stalks of hemp in the present study contained 41.54% cellulose, 18.08% lignin and 22.87% hemicellulose. After pretreatment with pyrrolidinium acetate, the content of each component changed (Table 1). This depended not only on the dissolution of the stalks in [Pyrr][AC], but also on the time and temperature at which the pretreatment was carried out.
In the first variant of the study, hemp stalks were dissolved in [Pyrr][AC] and stirred at room temperature for 24 h. This reduced the lignin content by 7% compared with the untreated material. The hemicellulose content also decreased—after 24 h its content was 18%. When the hemp stalks were incubated for 30 min at 90 °C, the lignin content decreased to 8.43% and hemicellulose to 10.21% in comparison with the untreated material. However, the best results were obtained when the hemp stalks were dissolved in [Pyrr][AC] and heated at 90 °C for 24 h—extending the incubation time resulted in almost complete extraction of lignin and hemicellulose.
Ziaei-Rad et al. [16] showed that using the proton ionic liquid TEA[HSO4] to dissolve wheat straw for 3 h at 130 °C resulted in 80% delignification and 64.4% lignin removal. Chambon et al. [23] used the same ionic liquid to dissolve four types of raw material (wheat straw, sugarcane bagasse, rice hulls and rice straw) and achieved 82% lignin removal efficiency. These authors also observed improved digestibility of the pulp at further stages.
It is worth noting that lignin and hemicellulose extraction from biomass using proton ionic liquids is still being developed and researched, and that its efficiency and cost-effectiveness may depend on a number of factors, including the type of biomass, the PIL chosen, and the process conditions [15]. Therefore, it seems important to verify the content of lignin extracted with PIL, as this solvent can be recovered and reused in the biomass pretreatment process. In addition, lignin and hemicelluloses can also find use in other industries. Their presence during bioethanol production may represent an additional complication for cellulolytic enzymes when they are subjected to enzymatic hydrolysis together with cellulose. For the further stages of bioethanol production, it would be most advantageous to separate hemicelluloses and lignins from cellulose, and feed the cellulose directly to enzymatic hydrolysis and alcohol fermentation as the main substrate for bioethanol production. This simplifies and reduces the costs of further processes, i.e., enzymatic hydrolysis and alcohol fermentation.
The use of [Pyrr][AC] for the extraction of lignin and hemicellulose from hemp stalks is advantageous since the remaining biomass consists mainly of cellulose. The content of cellulose after pretreatment was 66.09% for S3 and 76.28% for S4, which is comparable to the content of this polymer in the untreated raw material. Pyrrolidinium acetate proved to be the most effective agent for lignin extraction due to its high ionicity [15]. Numerous articles can be found in the literature on pretreatment with imidazolium ionic liquids, which change the structure of cellulose from crystalline to amorphous and thus increase the concentration of reducing sugars after enzymatic hydrolysis and bioethanol after fermentation [24,25]. However, the presence of lignin and hemicellulose in the biomass intended for enzymatic hydrolysis is often the reason for low sugar conversion efficiency. The use of imidazolium ionic liquids produces chemical compounds that inhibit enzymatic hydrolysis and alcohol fermentation. Therefore, it would be better to use a solvent that extracts mainly lignin and hemicelluloses in the pretreatment stage.
The present study confirms that [Pyrr][AC] used to dissolve seed hemp at 90 °C for 24 h is most effective in the extraction of this biopolymer. To confirm the effect of proton ionic liquid in lignin extraction from hemp stalks, FTIR analysis of all samples was performed both before and after pretreatment.

3.2. Fourier Transform Analysis of Infrared Spectra (FTIR)

Figure 2 shows the FTIR spectra of the analyzed samples. All hemp fibers treated with [Pyrr][Ac] showed high similarity in the presence of individual functional groups. A broad adsorption band in the range of 3648–3000 cm−1 was recorded, which is attributed to the stretching vibrations of the O-H bonds in the polysaccharides [26]. A broad peak at 3336 cm−1 is also attributed to inter- and intramolecular vibrations of hydrogen bonds in cellulose [27,28]. The intensity of this peak, as well as the peak at 1157.8 cm−1, was highest for sample S4, indicating a higher exposure of the cellulose after applying a higher pretreatment temperature [29].
Two small absorption bands present in the range 2960–2800 cm−1 correspond to the C-H bond stretching vibrations characteristic to all hydrocarbon substituents in polysaccharides [26,30]. Typical bands assigned to cellulose were observed in the region 1650–875 cm−1. The peak observed at 1650 cm−1 can be attributed to water–cellulose interactions [26,30,31]. The absorption bands at 1421 cm−1 and 1032 cm−1, on the other hand, belong to the stretching and bending vibrations of the -CH2, C-H and -OH bonds also present in cellulose [32,33]. These bonds allow for the ordering of the molecule’s structures and preserve its thermal stability [27]. The peaks at 1160 cm−1 (asymmetric vibrations of C-O-C bonds) and 897 cm−1 (characteristic vibrations of β-1,4-glycosidic bonds) also demonstrate the presence of cellulose molecules in all the samples analyzed [34,35]. Peaks indicating the presence of hemicellulose and lignin compounds were observed for each sample at 1745 cm−1 (stretching vibrations of the C=O bonds of hemicellulose) and 1558 cm−1 (stretching vibrations of the C=C bonds of lignin). Their intensity was lower in sample S4, which could also indicate a lower content of both compounds in this sample [36].

3.3. Analysis of NIR Spectra

As the results show, the NIR spectra in the ranges of 1250–1700 nm, although monotonic, can also provide information about changes in the structure of the hemp fibers.
The most important absorption peaks of the NIR range for PIL-treated hemp fibers are shown in Figure 3. The characteristic wavelengths at 1380 nm, which are common to hemicellulose, cellulose and lignin, correspond to both the stretching and deformation of the C-H groups in -CH3 (the first overtone band) [37]. The broad wavelength range between 1400–1550 nm is associated with the presence of a band characteristic of stretching vibrations of the O-H groups of hemicellulose and cellulose (first overtone band). The signals in the range of 1640–1700 nm are attributed to the vibrations of C-H groups (first overtone band) [38,39].
In our study, we applied PCA analysis to the FTIR spectra of hemp samples differing in the pretreatment process. The purpose of this method was to try to classify the analyzed samples in terms of their possible similarities or differences. The analysis was performed on the basis of a correlation matrix. The number of principal components characterizing the data set used in the analysis was determined using the Keiser criterion, according to which only factors with eigenvalues greater than 1 should be retained.
For the entire spectrum (4000–700 cm−1), the first and second principal components (PC1 and PC2) described 95.8% of the total variation (92% and 3.8%, respectively), as shown in Figure 4.
According to this criterion, the first two factors (PC1 and PC2) were selected and they “explained” more than 95% of the total variability. (The PC1 and PC2 factors account for 92 and 3.8% of the variance, respectively.) The analysis revealed that all variables convey important information and can significantly contribute to the further classification of sites.
To determine any similarities between the samples, an observation graph (Figure 3) was drawn up, showing the position of the grouping variables in a new coordinate system, defined by the PC1 and PC2 components determined during the analysis. The measure of similarity during the graphical interpretation of the results was the Euclidean distance. Analysis of the graph indicates the existence of two fully separated sets. The results of the analysis suggest that the hemp samples treated with PIL at 90 °C and for 24 h were significantly different from the other samples. This difference was mainly due to the chemical composition (different contents of cellulose, hemicellulose, and lignin).

3.4. PCA Analysis of NIR Spectra

Near-infrared spectra were also used to discriminate between hemp samples. For the entire spectrum (1250–1700 nm), the first and second principal components (PC1 and PC2) described 98% of the total variation (80% and 18%, respectively), as shown in Figure 5.
Based on the NIR data, we distinguished two groups. The first group had higher PC2 values (S1, S2 and S3). The second group, which consisted of samples of hemp fibers treated with PIL for 24 h and at 90 °C (S4), had lower PC2 values. However, concerning the PC1 component, the S4 sample was most consistent with the S1 sample (raw hemp stalks). The results of the above analysis also confirm the differences in the chemical composition of PIL-treated hemp fibers.

3.5. Effect of Using PIL for Pretreatment of Hemp Stalks on the Enzymatic Hydrolysis and Alcohol Fermentation Process

Enzymatic hydrolysis of hemp stalks both before and after pretreatment lasted 72 h at 50 °C. During the process, the concentration of total reducing sugars was measured to test the effectiveness of the process. Figure 6 shows graphs of reducing sugars in hemp samples tested at the beginning of the process, after 12, 24, 48 and 72 h.
The best results were achieved in samples that were incubated at 90 °C. The incubation time had an impact on the yield of reducing sugars in the samples, however, considering the cost of running the process. Incubation for 30 min at 90 °C resulted in an increase in reducing sugars after enzymatic hydrolysis of 7.5 g/L. In the best variant, 8.6 g/L of reducing sugars were obtained in samples incubated for 24 h. With possible scaling up of the process, shortening the incubation time of biomass in PIL to achieve better results for enzymatic hydrolysis of complex sugars could be considered.
It is known that the native complex structure of biomass, as well as the presence of lignin, hemicellulose and other compounds, hinders cellulase access to the cellulose substrate, leading to poor hydrolysis efficiency [40,41,42]. In their study, Reis et al. [43] used cashew apple baggase, which was purified with PIL and subjected to enzymatic hydrolysis. They obtained the highest glucose concentration after hydrolysis in biomass samples that had been pretreated with PIL and incubated for 24 h at 130 °C. Their study confirms that the removal of lignin is a key factor in increasing the efficiency of enzymatic hydrolysis by promoting access of the cellulase enzyme to cellulose [44,45].
The results of the conversion of biomass to ethanol are presented in Figure 7.
The post-fermentation ethanol content was highest in samples pretreated for 24 h at 90 °C. An average of 5.6 g/L of ethanol was obtained—about four times more than in the variant I of the study, in which the biomass was not pretreated with PIL (Figure 8). Yeast viability tested after 72 h of fermentation was also highest in these samples, averaging 57%. Das et al. [46] fermented a mixture of woody biomass (almond/walnut/pine) that had been pretreated with [Ch][Lys]. Fermentation of this hydrolysate using a Saccharomyces cerevisiae strain utilizing C5 sugars yielded a maximum ethanol concentration of 17.9 g/L for the biomass hydrolysate mixture, corresponding to a fermentation efficiency of 60.8%.
It is important to investigate the effectiveness of the pretreatment and enzymatic hydrolysis in terms of the alcohol fermentation process. Previously produced chemical compounds such as furfural, and polyphenols, for example, or insufficient breakdown of cellulose into fermentable sugars can harm yeast metabolism [20,47]. There are methods to remove fermentation inhibitors and better adapt the yeast to the hydrolysate environment, but this requires the introduction of an additional 2G ethanol production step, thus increasing production costs [48].
Biomass treatment with [Pyrr][Ac] contributed to better enzymatic hydrolysis and increased yeast survival during fermentation, which is important. The lowest yeast viability was in the untreated sample, averaging 31.7%. On the other hand, it was similar in the [Pyrr][AC] pretreated samples (58.1% for samples incubated at 21 °C for 24 h, 54.6% for samples incubated at 90 °C for 30 min, and 56.7% for samples incubated for 24 h at 90 °C; Figure 9). The presence of lignin in the samples reduced enzyme activity, which is associated with a lack of reducing sugars for the yeast, causing it to enter a starvation state, resulting in inefficient bioethanol production. The sugar conversion efficiencies and final ethanol concentrations are summarized in Table 2.
Geshwend et al. [54], Malaret et al. [55] and Nakasu et al. [56] showed that pretreatment with PIL does not negatively affect enzymatic hydrolysis and alcohol fermentation. When PIL is used to dissolve biomass, virtually no alcohol fermentation inhibitors are produced, which could reduce yeast viability. On the other hand, insufficient leaching of PIL from the pulp can result in reduced enzymatic activity and yeast performance. PIL residues such as acetate can cause inhibition of yeast growth.

4. Conclusions

The large proportion of cellulose in hemp stalks makes this waste attractive for use in the production of bioethanol from lignocellulosic raw material. The use of [Pyrr][AC] for the pretreatment of hemp is relevant to the process of bioethanol production. PIL allows the extraction of lignin and part of the hemicellulose from hemp stalks so that the resulting supernatant is mainly cellulosic fibers, which in a further stage of enzymatic hydrolysis using commercial enzyme preparations, i.e., cellulase, can be broken down to reducing sugars. Such hydrolysates are a good medium for the yeast Saccharomyces cerevisiae, which will convert them into ethanol during the fermentation process. PIL pretreatment is ineffective at dissolving biomass at room temperature even for 24 h. Only heating at 90 °C for 30 min causes lignin to be extracted, which increases the cost of the process. The highest content of reducing sugars (8.5 g/L on average) was obtained in samples of hemp stalks previously purified with pyrrolidinium acetate and incubated at 90 °C for 24 h, and in these samples, the ethanol content after fermentation was also the highest, averaging 5.7%. The efficiency of the present process depends on the pretreatment used, including the time and temperature at which the biomass is incubated in the solvent, with longer pretreatments at higher temperatures allowing more efficient separation of cellulose from the other polymers. This also has the effect of increasing the viability of yeast in alcohol fermentation. In addition, pyrrolidinium acetate can be recycled after the process by distillation, allowing it to be reused.

Author Contributions

Conceptualization, methodology, results and formal analysis, and writing—original draft preparation: M.S.-K., B.W., K.L.-R., A.C.-K. and P.J.; results analysis, and plotting figures of the obtained analytical and numerical data: T.K., B.W., L.B. and M.S.-K.; writing—review and editing: A.C.-K., T.K., L.B. and P.J. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

Data is contained within the article.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. PIL: pyrrolidine [Pyrr]+ and acetate [Ac]−.
Figure 1. PIL: pyrrolidine [Pyrr]+ and acetate [Ac]−.
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Figure 2. FTIR spectra of hemp fiber samples (in the 4000–700 cm−1 wavenumber ranges) after z-score normalization, measured by the reflection technique.
Figure 2. FTIR spectra of hemp fiber samples (in the 4000–700 cm−1 wavenumber ranges) after z-score normalization, measured by the reflection technique.
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Figure 3. Absorption spectra of hemp fiber samples in the NIR range (1250–1700 nm) after z-score normalization, measured using the reflection technique.
Figure 3. Absorption spectra of hemp fiber samples in the NIR range (1250–1700 nm) after z-score normalization, measured using the reflection technique.
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Figure 4. Observation graph. Projection of FTIR spectra of hemp fibers onto the space defined by the principal components PC1 and PC2.
Figure 4. Observation graph. Projection of FTIR spectra of hemp fibers onto the space defined by the principal components PC1 and PC2.
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Figure 5. Observation graph. Projection of NIR spectra of hemp fibers onto the space defined by principal components PC1 and PC2.
Figure 5. Observation graph. Projection of NIR spectra of hemp fibers onto the space defined by principal components PC1 and PC2.
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Figure 6. Content of reducing sugars tested in samples of hemp stalks before and after pretreatment with [Pyrr][AC].
Figure 6. Content of reducing sugars tested in samples of hemp stalks before and after pretreatment with [Pyrr][AC].
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Figure 7. Ethanol fermentation efficiency.
Figure 7. Ethanol fermentation efficiency.
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Figure 8. Concentration of bioethanol obtained from hemp stalks.
Figure 8. Concentration of bioethanol obtained from hemp stalks.
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Figure 9. Yeast viability results.
Figure 9. Yeast viability results.
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Table 1. Chemical composition of hemp stalks before and after pretreatment with PIL (Sample: S1—untreated materials; S2—hemp stalks after PIL pretreatment at 21 °C for 24 h; S3—hemp stalks after PIL pretreatment at 90 °C for 30 min.; S4—hemp stalks after PIL pretreatment at 90 °C for 24 h).
Table 1. Chemical composition of hemp stalks before and after pretreatment with PIL (Sample: S1—untreated materials; S2—hemp stalks after PIL pretreatment at 21 °C for 24 h; S3—hemp stalks after PIL pretreatment at 90 °C for 30 min.; S4—hemp stalks after PIL pretreatment at 90 °C for 24 h).
SampleCelulose [%]Lignin [%]Hemicelulose [%]
S141.5418.0822.87
S254.4111.0818.00
S366.098.4310.21
S476.283.104.32
Table 2. Enzymatic saccharification and fermentation of lignocellulosic biomass after pretreatment.
Table 2. Enzymatic saccharification and fermentation of lignocellulosic biomass after pretreatment.
FeedstockPretreatedFermentable Sugar Content (g/L)Ethanol Concentration (g/L)References
Sweet sorghum juiceSqueezing in a juice extractor20037.47–72.69 at 37 °C
16.52–53.89 at 40 °C		[18]
Spent coffee groundsAcid hydrolysis50 11.7[49]
Sorghum stoverAlkaline treatment20056–68[50]
Coconut fiber matureHydrothermal pretreatment
catalyzed with sodium hydroxide (HPCSH)		5010.81–11.65[51]
Wheat strawLow cost ionic liquid/ultrasound irradiation
IL/US		10038.9 after 15 min treatment
42.0 after 30 min treatment		[16]
Corn stoverExtremely low-liquid ammonia (ELLA)27.914.5[52]
Industrial hemp stemStem with SO212.7Separated fiber 21.3
Whole slurry 18.4		[53]
Industrial hemp silageStem with SO210.8Separated fiber 20.3
Whole slurry 15.4		[53]
Industrial hempLiquid hot water (LHW)21.5910.9[1]
Industrial hempH2SO4 treatment28.0413.8[1]
Industrial hempNaOH treatment40.0820.3[1]
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Smuga-Kogut, M.; Walendzik, B.; Lewicka-Rataj, K.; Kogut, T.; Bychto, L.; Jachimowicz, P.; Cydzik-Kwiatkowska, A. Application of Proton Ionic Liquid in the Process of Obtaining Bioethanol from Hemp Stalks. Energies 2024, 17, 972. https://doi.org/10.3390/en17040972

AMA Style

Smuga-Kogut M, Walendzik B, Lewicka-Rataj K, Kogut T, Bychto L, Jachimowicz P, Cydzik-Kwiatkowska A. Application of Proton Ionic Liquid in the Process of Obtaining Bioethanol from Hemp Stalks. Energies. 2024; 17(4):972. https://doi.org/10.3390/en17040972

Chicago/Turabian Style

Smuga-Kogut, Małgorzata, Bartosz Walendzik, Katarzyna Lewicka-Rataj, Tomasz Kogut, Leszek Bychto, Piotr Jachimowicz, and Agnieszka Cydzik-Kwiatkowska. 2024. "Application of Proton Ionic Liquid in the Process of Obtaining Bioethanol from Hemp Stalks" Energies 17, no. 4: 972. https://doi.org/10.3390/en17040972

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