Hot-Water Extraction (HWE) Method as Applied to Lignocellulosic Materials from Hemp Stalk

Abstract

The article describes the process of hot water extraction treatment of a specific material—in this case, shavings of hemp shives of different thicknesses, sorted by their thickness into three different fractions of 0–4 mm, 4–8 mm, and 8–12 mm. In addition, each sample from a given fraction was separately subjected to one, two, and three extraction processes. After the material was treated with extraction, cellulose determination was performed using the Kürschner–Hoffer method in order to find out the effect that hot water extraction had on the cellulose content of the test material. This research aims to determine whether hot water extraction strongly alters the cellulose content, which may translate into a change in efficiency when producing second-generation biofuel produced from this material. The cellulose determination showed the smallest cellulose losses were in chips 4–8 mm thick, while the largest were in chips 0–4 mm thick. Each repetition resulted in a loss of cellulose, with the steepest loss occurring after the second repetition of HWE, and the smallest after the third repetition—the exception being the 4–8 fraction, in which the smallest decrease occurred after the first repetition of the HWE (Hot Water Extraction) process.

1. Introduction

The species Cannabis sativa L. (cannabis) is divided into fibrous hemp (Cannabis sativa L. var. sativa) and indica (narcotic) hemp (Cannabis sativa L. var. indica), which differ in their cannabinoid content. Hemp seeds are used in the cosmetic industry, food industry, and as an ingredient in bird food. The durability and high strength of the fibers extracted from the stalks, which are rich in cellulose, make them ideal for making rope, paper, and structural and reinforcing materials [1]. Because of the similarity between fiber hemp and cannabis, many countries have banned the cultivation of both; however, in the past 20 years, a large number of countries have re-legalized the cultivation of fiber hemp accelerating the development of research into its health properties [2,3].

The stalk of the hemp plant, known as the straw, has a core that is usually cut into pieces called shives. The mechanical processing (shelling) of hemp straw yields about 35% fiber and about 65% shive [4]. With a harvest yield of about 8 tons/ha of hemp straw, about 5–5.5 tons of wood chips can be obtained. Hemp-derived fibers belong to the group of stem-best fibers along with fibers from plants such as jute, kenaf, flax, juniper, nettle, and azole [5]. Comparing the yield of hemp, it can be seen that it is lower than that of other energy crops such as kenaf (about 24 t/ha) or miscanthus (about 30 t/ha) [6]. When hemp is grown using proper agro-technique, the crop growth is rather uniform resulting in high biomass yields of about 10–15 t/ha. Mature hemp can grow to a height of more than 2.5 m, or even up to 3.0 m [7].

In recent years, there has been increasing interest in the utilitarian, nutritional, medicinal, and construction applications of hemp Cannabis sativa L. One of the uses of hemp shive is in the construction industry, where shive is one of the components of mortar for plastering walls and pouring floors [1]. On the other hand, if treated beforehand, they have a high resistance to mold and mildew and even gain antibacterial characteristics [8]. Unlike Styrofoam, which is used all the time, this material has greater permeability, which allows air circulation and reduces the risk of fungus or moisture in buildings [9]. Another advantage is that they are also biodegradable, thus eliminating the problem of storing already used building material [10,11].

The market for certified hemp products is forecasted to grow by up to 77% by 2025, reaching a value of $166 million [12]. The biomass obtained from hemp has a high yield as an energy feedstock [13]; the growth of the plant’s stem is roughly 50 cm in one month [14]. Its most profitable part from an energy point of view is the so-called hemp shive, which is produced during the processing of hemp straw. Hemp fiber has an energy value exceeding 18 MJ/kg, unlike hemp wood which has a value of 17 MJ/kg. As for the emission values of substances during the combustion of hemp biomass, these values are about 0.8 kg CO₂/kg and CO not exceeding 0.15 kg CO/kg of raw material mass [15].

Lignocellulosic biomass has great potential for liquid biofuel production, given factors such as large-scale access, low cost, and low greenhouse gas emissions [16]. The technology uses multiple primary and secondary sources of biomass. The main primary source is the cultivation of energy crops, such as bamboo, Pennsylvania hogweed, switch millet, energy willow, and miscanthus. Residues from production processes are called secondary sources and include waste from agriculture, such as crop residues, and from forest management, such as sawdust, shavings, offcuts, bark waste, and leaves [17,18]. Additional sources may also come from the plant product processing industry (nut shells, soybeans, pomace, rice and sunflower husks, pulp, and corn cobs) [19]. A group of tertiary sources, on the other hand, will be the organic waste from municipal facilities (e.g., waste paper, dried manure) [20]. Bioethanol can be produced from lignocellulosic material with pretreatment followed by enzymatic hydrolysis and fermentation [21,22].

For the bioethanol production process, the high content of lignin in the lignocellulosic biomass used is a factor that hinders the process of producing the said bioethanol [23,24,25]. The compound has considerable resistance to chemical and biological degradation, blocking hydrolytic enzymes from accessing polysaccharides [26]. Hot water extraction is classified as a hydrothermal process and is readily used in the cellulosic industry [27]. During the HWE (Hot Water Extraction) process, lignocellulosic biomass is immersed in hot water while using high pressure. This results in a high efficiency of pentose recovery after enzymatic hydrolysis and a dissolution rate of 4–22% for cellulose and 35–60% for lignin. In addition, a low number of inhibitors is generated [28].

The HWE method was developed by SUNY EFS in the United States and is based on the separation of hemicellulose and other undesirable compounds from biomass. A key finding of this research is that extracted wood chips may contain a large amount of readily available cellulose. The remaining polysaccharides present in wood chips can be hydrolyzed to convert them into simpler sugars, which in turn can be fermented to produce ethyl alcohol (bioethanol). Due to current climate policy, the introduction and diffusion of the HWE method in the production of bioethanol using new raw materials such as hemp, can potentially increase access to high-quality alternative fuel.

These studies aim to determine whether hot water extraction strongly alters the cellulose content of the material treated with the HWE process, which may translate into a change in efficiency during the production of second-generation biofuel produced from this material. For the study, chips sorted into three fractions due to their thickness were used in order to compare where the greatest changes in the content of the target occur. In addition, the amount of substances leached from the chips from the distilled water used during the HWE process was also examined.

2. Materials and Methods

2.1. Material

The tested raw material was supplied by Cannabotanique. Hemp obtained for research was grown in Poland, more precisely in the Mazowieckie Voivodeship on a plantation located in its central part. According to the regulations in force in Poland, hemp (Cannabis sativa L. var. sativa) was grown on a plantation, in which the THC content cannot exceed 0.2%. Cultivation took place in conditions of a temperate climate; average daytime temperatures during cultivation were in the range of 15–24 °C. After the crops matured, the plants were harvested using specialized agricultural machinery.

2.2. Physical Properties of the Raw Material

2.2.1. Division of Chips into Fractions by Their Thickness

Material was crushed and then sorted into fractions using a sorting machine A C.B.K.O Hydrolab orbital shaker was used for this purpose. The chips used in the subsequent testing processes were divided according to the diagonal mesh of the sieve, resulting in 3 fractions:

• Finest (p < 0 ÷ 4) mm.
• Middle (p < 4 ÷ 8) mm.
• Coarsest (p < 8 ÷ 16) mm.

2.2.2. Material Moisture Measurement

The samples were dried in a laboratory chamber at 105 °C, until absolutely dry. After drying, each sample was weighed (m_s). After weighing, the samples were placed in a laboratory cuvette to standardize the moisture content for the samples taken out of the dryer and were measured. After two weeks, a moisture Material Moisture Measurement content of 12% was recorded [29,30,31]. After this time, their mass (m_w) was measured to check the moisture content, using the formula for absolute moisture content. The moisture content (M) of the chips was tested for the original distribution of the material. The value was determined according to Equation (1):

$$M=\frac{m_w-m_s}{m_s}\cdot 100\%$$

(1)

where:

m_w—the mass of wet chips.

m_s—the mass of absolutely dry chips (dried to 0% moisture content).

2.3. HWE—Hot Water Extraction

As shown in Figure 1, before the HWE process was treated, it was divided into fractions; in addition, after the HWE process, cellulose determination was carried out.The 10 g of material was put into the chip container each time, while 400 g of distilled water was used for extraction. The extraction process was carried out at a temperature of 120 °C and a pressure of 2 MPa; its time was 30 min each time. To carry out the HWE process, a device with a cylindrical shape, shown in Figure 2, was used. Looking from the bottom of the figure, 3 main parts can be seen on it: At the very bottom you can see the reactor into which distilled water was poured each time. Then at the top of the reactor, you can see the container for the raw material undergoing the HWE process. In the center of the cross-section of the container there is a tube extending towards the bottom. At the very top, there is a lid with a hole used for pressure drainage.

After being placed in the chip container, the material was subjected to a single HWE process; then after replacing the material and distilled water, there were two repetitions of the HWE process in a row on the new material. After the second repetition of the HWE process on the material, there was another replacement of the material and distilled water, followed by 3 repetitions of the HWE process in a row. The 3 different fractions of chips were used for extraction—0–4, 4–8, and 8–16. After the extraction process with distilled water, the chips were transferred to a labeled container and put into a dryer to dry the chips. On the other hand, the distilled water used after extraction was poured into a marked and weighed flask. The flask with the overflowed distilled water was then subjected to an evaporation process to obtain the mass of the precipitate. The precipitate obtained after the evaporation process was the substances washed out of the chips by the distilled water used during the HWE process.

2.4. Extraction and Determination of Cellulose (Native and HWE)—Post-Extraction Chips

For extraction, a mixture of chloroform–ethanol in a mass ratio of 93:7 was prepared. Each sample was further divided into 3 series of approximately 2.7 g, additionally labeled with the letters A, B, C. For example, the sample obtained after one repetition of the hot water extraction process was then divided into 3 separate samples to obtain 3 test series for the material treated with one repetition of the hot water extraction process. The chips were then poured into the prepared extraction thimbles; then the thimbles already filled with chips were placed in the soufflés. The next step was pouring the extraction thimbles with the already prepared chloroform–ethanol mixture. Then the heating bowls heating the round-bottomed flasks were connected to electricity, and their power was adjusted so that one overflow of the mixture in the soxhlet extractor took about 7 min. The entire extraction process took a total of 10 h for a single thimble.

After extraction, the soxhlet extractor was disconnected from the power supply, and we then waited about 20–30 min for the soxhlet extractor to cool down. After the soxhlet extractor cooled, the flasks were unplugged and subjected to an evaporation process. The evaporation process had two purposes: to regenerate the chloroform–ethanol mixture and to obtain the mass of the precipitate in the flasks. After the evaporation process, the mass of the flasks with the precipitate remaining in them was weighed, and the mass of the empty flask before the extraction process was subtracted from it; this was to obtain the mass of substances washed out of the chips during extraction with the chloroform –ethanol mixture (the mass of the precipitate). The process of cellulose determination was carried out according to a similar procedure presented in a book by Krutul [32].

After the chip extraction process in a chloroform-ethanol mixture, the Kürschner–Hoffer method was used to determine cellulose content. The extracted samples were dried in a vacuum dryer (temperature 60 °C, pressure 0.4 kPa) to a constant weight. The samples thus prepared were destined for the determination of cellulose content. First, about 0.7 g of the test material of each was weighed, which was transferred to a 300 cm³ conical flask. To the flask, 20 cm³ of ethanol (ether, Chempur company, Piekary Śląskie, Poland) was first added, and then 5 cm³ of 65% nitric acid (pure, Chempur company) was added slowly, in portions and while stirring. The flask with the contents was heated in a water bath at about 85 °C. The heating time was calculated from the moment of boiling and was 60 min.

After this time, the liquid contents of the flask were drained on a previously dried and weighed Schott G3 filter, and the residue in the flask was poured with another portion of ethanol and nitric acid. In this way, 3 one-hour boiling cycles were performed in the Kürschner–Hoffer mixture. Then the sample in a conical flask was poured with distilled water (about 25 cm³) and boiled for a period of 30 min. After this time, the liquid contents of the flask were drained on a Schott G3 filter, while the residue in the conical flask was again flooded with distilled water (about 25 cm³) and boiled for a period of 30 min. After undergoing two 30-min boiling cycles in distilled water, the contents of the flask were transferred to a Schott filter and the precipitate was washed with hot distilled water until it reached a neutral pH. The cellulose precipitate thus obtained was subjected to drying at 103 ± 2 °C; then the dried precipitate was weighed to obtain m_c:

$$C=\frac{m_c}{m_0}\ast 100\%$$

(2)

where:

C—Percentage of cellulose [%].

m_c—the amount of obtained dried cellulose [g].

m₀—the mass of absolutely dry extracted sawdust [g].

3. Results

3.1. Extraction and Determination of Cellulose (Native and HWE)—Post-Extraction Chips

Chip moisture content remains around 12% for all samples. Moistures for individual samples are shown in

Table 1. Material moisture.

Fraction	Number of Repetitions of the HWE Process	Series	Absolute Dry Chip Weight [g]	Wet Chip Weight [g]	Moisture Content of Chips before HWE [%]
0–4	0	A	13.7	15.4	12.4
		B
		C
	1	A	13.9	15.5	11.7
		B
		C
	2	A	14.2	16.0	12.7
		B
		C
	3	A	13.5	15.2	12.3
		B
		C
4–8	0	A	13.1	14.7	12.2
		B
		C
	1	A	12.1	13.6	12.5
		B
		C
	2	A	14.7	16.5	12.0
		B
		C
	3	A	13.8	15.5	12.6
		B
		C
8–16	0	A	13.7	15.4	12.4
		B
		C
	1	A	14.1	15.9	12.6
		B
		C
	2	A	13	14.5	11.5
		B
		C
	3	A	12.7	14.3	12.3
		B
		C

. In two cases, chip moisture content was just below 12%, in one case for fractions 0–4 and in another case for fractions 8–16. The rest of the results were in the 12–13% range. The moisture content for the chips in each fraction ranged from 11.5 to 12.7%. The moistures for individual samples are shown in Table 1.

3.2. HWE—Hot Water Extraction

During the research, the amount of sediment remaining after the evaporation of the distilled water used for the HWE process was analyzed. It was noticed that the amount of sludge is the smallest in chips of fractions 0–4, then increases in fractions 4–8, and is the largest in fractions 8–16. The mass of sludge after the evaporation of the water used for HWE is presented in

Table 2. Mass of sludge after evaporation of water used for HWE.

Fraction	Number of Repetitions of the HWE Process on the Material	Mass of Chips Treated with the HWE Process [g]	Mass of Sludge Remaining after Evaporation of Distilled Water Used for the HWE Process [g]	Percentage of Residual Sludge after Evaporation of Distilled Water Used in the HWE Process [%]
0–4	1	10	0.143	1.43
	2		0.216	2.16
	3		0.262	2.62
4–8	1		0.154	1.54
	2		0.217	2.17
	3		0.052	0.52
8–16	1		0.163	1.63
	2		0.235	2.35
	3		0.375	3.75

.

A relationship can be seen here, according to which, with each repetition of the HWE process the amount of sludge increases; the only exception here is in fractions 4–8 after three repetitions of the HWE process. The increasing values of the leached substances during the HWE process as a result of the increasing number of repetitions of the HWE process on the material are shown in Figure 3.

3.3. Extraction and Determination of Cellulose (Native and HWE)—Post-Extraction Chips

According to the results shown in

Table 3. Mass of sediment after evaporation of the chloroform–ethanol mixture.

Fraction	Number of Repetitions of the HWE Process on the Material	Series	Weight of Shavings Used for Extraction [g]	Mass of Precipitate after Evaporation of the Chloroform Mixture-Ethanol from the Flask [g]	Percentage of Sediment after Evaporation of Chloroform Mixture-Ethanol from Flask [%]	Sum of Percentage Sediment Content after Evaporation of Chloroform-Ethanol Flask Mixture [%]	Sum of Percentage Sediment Content after Evaporation of the Chloroform-Ethanol Mixture for the Entire Chip Fraction [%]
0–4	0	A	2.688	0.026	0.967	3.738	22.682
		B	2.715	0.013	0.479
		C	2.793	0.064	2.291
	1	A	2.713	0.052	1.917	5.221
		B	2.692	0.062	2.303
		C	2.697	0.027	1.001
	2	A	2.871	0.066	2.299	6.389
		B	2.789	0.054	1.936
		C	2.785	0.06	2.154
	3	A	2.74	0.036	1.314	7.334
		B	2.635	0.122	4.630
		C	2.805	0.039	1.390
4–8	0	A	2.721	0.028	1.029	3.340	20.381
		B	2.715	0.031	1.142
		C	2.737	0.032	1.169
	1	A	2.727	0.033	1.210	4.258
		B	2.785	0.026	0.934
		C	2.79	0.059	2.115
	2	A	2.714	0.032	1.179	5.556
		B	2.752	0.063	2.289
		C	2.73	0.057	2.088
	3	A	2.712	0.099	3.650	7.226
		B	2.726	0.048	1.761
		C	2.7	0.049	1.815
8–16	0	A	2.714	0.023	0.847	2.938	17.947
		B	2.743	0.028	1.021
		C	2.711	0.029	1.070
	1	A	2.781	0.033	1.187	4.438
		B	2.784	0.033	1.185
		C	2.71	0.056	2.066
	2	A	2.716	0.039	1.436	4.627
		B	2.72	0.047	1.728
		C	2.733	0.04	1.464
	3	A	2.717	0.062	2.282	5.943
		B	2.72	0.055	2.022
		C	2.745	0.045	1.639

, the most precipitate remained after the evaporation of the mixture with chips on which the HWE process was carried out three times, while the least was with native chips. The same relationship can be seen here for each fraction; with each successive repetition of the HWE process, the amount of sludge remaining after the extraction process of the chloroform–ethanol mixture also increases. The largest amount of sludge remained after the extraction of the chips with fractions 0–4, while the smallest was for fractions 8–16 and fractions 4–8 are in the middle.

Looking at the graph presented in Figure 4, we can observe the relationship, according to which, with the increasing number of repetitions of the HWE process on the material, the amount of substances washed out by the chloroform–ethanol mixture increases. It is also easy to see that most substances were leached from fraction 0–4, but the least were from fraction 8–16.

Analyzing the percentage of cellulose content shown in

Table 4. Percentage cellulose content.

Fraction	Number of Repetitions of the HWE Process on the Material	Series	Mass of Dry Extracted Sawdust [g]—mo	The Amount of Obtained Dried Cellulose [g]—mc	Percentage of Cellulose Content [%]—C	Average Percentage of Cellulose Content [%]—C [%]	Loss of Cellulose Compared to Previous Extraction [%]—Cn–Cn-1	Loss of Cellulose Compared to Cellulose Content in Native Material [%]
0–4	0	A	0.723	0.377	52.144	60.071
		B	0.731	0.49	67.031
		C	0.77	0.47	61.039
	1	A	0.714	0.413	57.843	56.145	−3.926	−3.926
		B	0.719	0.397	55.216
		C	0.744	0.412	55.376
	2	A	0.714	0.342	47.899	51.241	−4.904	−8.830
		B	0.73	0.407	55.753
		C	0.703	0.352	50.071
	3	A	0.735	0.351	47.755	48.359	−2.882	11.712
		B	0.747	0.353	47.256
		C	0.757	0.379	50.066
4–8	0	A	0.728	0.348	47.802	48.925
		B	0.726	0.358	49.311
		C	0.737	0.366	49.661
	1	A	0.727	0.355	48.831	47.778	−1.147	−1.147
		B	0.713	0.337	47.265
		C	0.76	0.359	47.237
	2	A	0.766	0.306	39.948	43.186	−4.591	−5.739
		B	0.742	0.326	43.935
		C	0.74	0.338	45.676
	3	A	0.774	0.301	38.889	40.713	−2.473	−8.211
		B	0.726	0.31	42.700
		C	0.762	0.309	40.551
8–16	0	A	0.755	0.445	58.940	53.456
		B	0.79	0.452	57.215
		C	0.717	0.317	44.212
	1	A	0.72	0.418	58.056	50.021	−3.435	−3.435
		B	0.747	0.35	46.854
		C	0.784	0.354	45.153
	2	A	0.736	0.336	45.652	45.741	−4.280	−7.715
		B	0.725	0.341	47.034
		C	0.723	0.322	44.537
	3	A	0.735	0.339	46.122	43.381	−2.360	10.075
		B	0.72	0.334	46.389
		C	0.76	0.286	37.632

, we can see that the HWE process resulted in a decrease in cellulose content with each repetition of the HWE process. The total range within which there was a decrease in cellulose content after each repetition of the HWE process is from about 2 to 5%, with the range varying slightly in each fraction. The largest decrease in cellulose occurred in chips of fractions 0–4, while the smallest was in fractions 4–8. In fractions 8–16, there was a decrease that was smaller than in fractions 0–4 but greater than in fractions 4–8. The largest decrease in cellulose content occurred under the second repetition, while the smallest was after the third repetition; this relationship is the same for chip fractions 8–16 and 0–4.

Analyzing the percentage of cellulose content shown in Figure 5, we can see that the largest total decrease in cellulose content occurred in chips of fraction 0–4 and that it is after three repetitions, by as much as 11.7%. In contrast, the smallest occurred in fractions 4–8, by 8.21%. With the increasing number of repetitions of the HWE process on the material, an increasing depletion in cellulose content can be observed compared to the native material.

4. Discussion

Analyzing the mass of sludge remaining after the evaporation of the distilled water used for the HWE process, one can observe its increasing value with each successive repetition of HWE; the only exception here is the mass of sludge after the third repetition of HWE in fraction 4–8. In addition, its highest values are found in the thickest fraction of chips 8–16; in fractions 4–8 they are smaller, while in the thinnest fraction, they are the lowest. The occurrence of this relationship may be due to the fact that thicker chips release more substances than thinner ones [33].

After analyzing the mass of sludge remaining after the evaporation of the chloroform –ethanol mixture, the same relationship can be observed as in the case of the mass of sludge remaining after the evaporation of the distilled water consumed for the HWE process, namely, with each successive repetition of the HWE process, the mass of sludge also increases. However, here there is no longer any deviation as before in fractions 4–8 after the third repetition of HWE. In addition, its highest values occur in the thinnest fraction of chips 0–4; in fractions 4–8 they are smaller, while in the thickest fraction 8–16, they are smallest [34]. Here, the reason may be that finer chips have a larger contact area with the solvent, which may contribute to more efficient extraction of the substance.

After seeing the percentage content, one can see the decreasing relationship of the cellulose za-value after the HWE process. The decrease in cellulose content as a result of HWE may be related to its decomposition at high temperatures. The HWE process requires an increased temperature and pressure, which may affect the degradation of cellulose. In addition, water is used during HWE extraction, which can also affect cellulose degradation [35]. A decrease in cellulose content can also result from the selective extraction of other wood components that are near or on the cellulose surface. In the case of HWE extraction, water heated to high temperatures can affect the leaching of substances such as lignin and hemicelluloses, which are present in the wood alongside the cellulose.

In addition, after the second repetition of the HWE process, an even greater loss of cellulose can be observed relative to the loss after the one-time HWE process. This may be due to the fact that there is a greater loss of cellulose after the second HWE process compared to the first process due to changes in the chip structure caused by the previous extraction. As a result of the extraction, not only the extracted compounds may be dissolved but also other components such as lignin, hemicelluloses, or pectin, which may affect the structure of the chips [36]. The change in chip structure can lead to a greater susceptibility to dissolution by subsequent HWE extractions and a greater loss of cellulose [37]. An interesting relationship can be observed after the third repetition of HWE, namely, the loss here is smaller relative to the previous extraction; the reason for this may be a reduction in the amount of easily extractable compounds during the first two HWE processes.

5. Conclusions

The results of the percentage of cellulose content removed by the HWE process that is carried out on the shavings of hemp shives show a rather high loss of cellulose. According to the results, the thickness of the shavings is important when carrying out the HWE process; in this case, the 4–8 fraction shows the least loss of cellulose compared to others. It should be remembered that cellulose is one of the main components of lignocellulose, which is a potential feedstock for biofuels such as bioethanol or biobutanol [38]. If more cellulose is lost, the amount of remaining biomass that can be processed into biofuels will be less.

The high loss of cellulose during the HWE process can also affect the quality of the final product obtained. Cellulose is one of the basic components of lignocellulose which is the raw material for hydrolyzing enzymes, and these enzymes are crucial for bioethanol production. When the cellulose content of biomass is low, the hydrolysis process can be significantly impeded, which can lead to lower biofuel production yields [39]. Therefore, minimizing the loss of cellulose during the HWE process is important from the point of view of biofuel production, as it can affect the quantity and quality of the final products obtained.

It should also be noted that the coarsest fraction of chips had the largest mass of precipitate found in distilled water. In the case of the HWE-processed material, which are shavings made from hemp shive, the possible substances that are useful for biofuel production and can dissolve in the water used for the process are primarily cellulose, hemicelluloses, and lignin. It is important that the HWE process be effective in extracting these substances, as they are a potential source of substrate for biofuel production [40]. The loss of these substances during the HWE process can lead to a reduction in the efficiency of biofuel production from plant material and thus an increase in production costs.

In order to determine what substances have been washed away by the distilled water in the HWE process, it would be necessary to conduct additional studies examining the composition of these substances. In addition, by analyzing the loss of cellulose content, one could consider changing the parameters of the HWE process to be less aggressive so that its loss could decrease. However, it is worth remembering that reducing the aggressiveness of the process may result in a decrease in efficiency in the extraction of other substances, such as hemicelluloses and lignin, which in turn may affect the efficiency of biofuel production. Therefore, changing HWE process parameters should be carefully considered and based on experimental studies.