The Use of a Trichoderma reesei Culture for the Hydrolysis of Wheat Straw to Obtain Bioethanol

Abstract

To reduce environmental pollution, a renewable source of energy that we may utilize is bioethanol obtained from wheat straw. Wheat straw was ground to 40–50 mm in size and heat-treated with high-pressure steam to release lignocelluloses, making them accessible to enzymes during saccharification. Through mechanical pretreatment, a substrate was obtained, which contains toxic components in concentrations that do not diminish the performance of the enzymes in the enzymatic hydrolysis phase. Through the thermal pretreatment of wheat straw, its acidity was improved, influencing the amounts of glucose, xylose, and other components emitted. Following enzymatic hydrolysis, very small concentrations of sugars were released. In order to increase the efficiency of the transformation of sugars into ethanol during the fermentation process, a strain of yeast, Trichoderma reesei multiplied in the laboratory, was added, under the conditions of temperature—28 degrees and stirring—800 rpm. Trichoderma reesei penetrated the wheat straw substrate, facilitating the subsequent hydrolysis process. The improved biodegradation of the pretreated straws was highlighted by the electron microscopy analysis.

1. Introduction

The production of energy from conventional sources, by burning fossil fuels, results in greenhouse gases that pollute the environment. One source of energy, which supplies 10–14% of the world’s energy, is biomass from wheat straw, which represents agricultural waste. To reduce environmental pollution, wheat straw is used for the production of bioethanol [1].

In the present work, wheat straw was milled and heat-treated with high-pressure steam to release the lignocelluloses, making them accessible to enzymes during saccharification. In order to increase the efficiency of the transformation of sugars into ethanol during the fermentation process, a laboratory-multiplied trichoderma strain was added.

In the biological pretreatment of wheat straw with the fungus Trichoderma reesei, the degradation of lignocellulosic biomass was improved through the secretion of hydrolyzing enzymes [2,3,4,5,6].

Renewable energy sources such as wind, solar, and hydropower, ocean and geothermal energy, biomass, and biofuels offer cleaner alternatives to fossil fuels. They reduce pollution, give us wider energy options and reducing dependence on volatile fossil fuel prices. In 2022, energy from renewable sources represented 23% of the energy consumption of the European Union [7,8].

The Renewable Energy Directive, which entered into force in November 2023, raises the European Union’s target for the share of renewable energy sources in gross energy consumption from 32% to 42.5% by 2030, and some EU countries are striving to reach a higher 45% target [7]. In the transport sector, the target is 29% for the share of energy from renewable sources by 2030, i.e., a 14.5% reduction in greenhouse gas emissions, through greater consumption of advanced biofuels and fuels from renewable sources of non-biological origins, such as hydrogen [7,8,9,10,11,12,13,14].

Decarbonization of road transport in the EU will have an increasing impact on the demand for fossil fuels and biofuels, especially after 2030 [15].

The use of biofuels would have an average contribution to reducing non-renewable energy use in 2030 of 24.5 Mtoe and in 2050 of 48.3 Mtoe, and the use of advanced biofuels would have an average contribution in 2030 of 8.7 Mtoe and in 2050 of 36.5 Mtoe, which demonstrate that the use of these biofuels will help the decarbonization of the energy sector [15].

There is an annual waste production of 3.71 × 1010 kg of nutrients for all crops combined [16]. By the year 2050, there will be an increase of over 50% in agro-waste production [17]. Burning lignocellulosic biomass produces approximately 36.73 Tera-grams of CO₂-equivalent emissions [18]. Almost 24.6 GL of bioethanol can be produced from lignocellulosic biomass per year [18].

In line with global efforts to limit global warming to 1.5 °C to 2 °C, the EU aims to become climate neutral while achieving net-zero GHG emissions by 2050. The European Commission has proposed a package plan, “Fit for 55”, to reduce gas emissions to at least 55% by 2030 compared to 1990 [19].

The road transport sector is a large contributor to GHG emissions, representing 22% of the total share of emissions from the entire EU industry. This explains the need for stricter policies in this sector, especially for cars, which represent 61% of road transport emissions. The proposed reduction target for the transport sector is 13%.

In the EU-28, in 2017, 27% of the total greenhouse gas (GHG) emissions from the sector resulted from international aviation above the levels of 1990 (+129%), followed by international maritime transport (+32%) and road transport (+23%). Of the total greenhouse gas emissions from road transport, 71.7% resulted as follows: from cars 43.2%, from heavy trucks and buses 18.7%, from light trucks 8.5%, and from motorcycles 0.9%. Gases also came from maritime transport 13.3%, from aviation 13.9%, from railways 0.5%, and from other means of transport 0.6% [20].

Renewable energy sources in transport help to reduce greenhouse gas emissions and dependence on fossil fuels.

The demand for gasoline and bioethanol is expected to increase and maintain this increase, as the rate of replacement of gasoline vehicles will take longer [9]. Bioethanol is the second-largest renewable energy source used in the transport sector and represents 20% of the total biofuel consumption [11].

Ethanol is obtained from lignocellulosic materials by cellulolysis (hydrolysis and fermentation). Lignocellulosic biomass is an unlimited and advantageous source of cellulose, hemicellulose, and lignin, meaning it is the raw material for the production of ecological biofuels. Lignin provides structural integrity and binds cellulose fibers. Wheat straw is composed of 15–20% lignin, polysaccharides (20–25% hemicellulose, 30–45% cellulose), phytic acid, and other organic/inorganic compounds. Figure 1 shows the inter-environment of some chemical processes: gasification and fermentation or catalytic reaction [11,12,13,14]. Wheat straw resulting from agriculture is an abundant and cheap source of lignocellulose [13].

The biological, physical, or chemical delignification of wheat straw is carried out to structurally change the lignocellulosic biomass, to make it accessible for subsequent enzymatic hydrolysis and fermentation [21,22,23,24,25,26,27]. Delignification can be performed with the help of different microorganisms, such as Pycnoporus sp., Basidiomycetous and Irpex lacteus, Aspergillus niger, and Trichoderma reesei [27]. In the present work, the delignification of wheat straw was followed by using the Trichoderma reesei fungus for enzymatic hydrolysis, due to the high yields obtained in the saccharification process.

2. Materials and Methods

In this paper, we will present the enzymatic process applied in a factory in Romania to wheat straw to obtain bioethanol with an annual theoretical production of 50,000 tons of cellulosic ethanol obtained from approximately 250,000 tons of raw material. Figure 2 shows a map made with the GIS technique, ESRI ArcMap 10.8, New York, USA. with the localities where factories supply wheat straw for ethanol products.

The wheat straw is supplied by the producers in the form of bales (L = 2400 mm, l = 1200 mm, H = 900 mm, weight 438 kg/bale) (Figure 3). Before entering the storage area, the quality of the straw bales is checked for moisture content. This parameter is important because a higher moisture content can create problems during cutting and also during thermal pretreatment.

After the quantitative and qualitative checking of the wheat straw bales, these are unloaded and stored on a platform. The quantitative check is carried out by weighing the straw bales, and the qualitative check is carried out with measurements to determine the moisture content (max 13%) and the absence of straw bodies and mold fungus. Determination of the moisture content and temperature is carried out using a temperature and moisture probe for hay and straw, Pfeuffer HFM. The next stage is the grinding. The sectioning of the straw is carried out with the help of knives at sizes smaller than 5 mm (Figure 4).

Sectioned wheat straw, once sorted and cleaned of foreign bodies and dust (Figure 5), is sent to the thermal treatment unit.

Trichoderma reesei strains were stored at −80 °C, multiplied in the laboratory, and then transferred to the plant, where their multiplication was periodically controlled until they were used. The comparative biodegradation effects of treated wheat straw on hemicellulose, lignin, and cellulose were analyzed after 35 days of fungal cultivation. Through optical microscopy analysis, the microstructural change due to biodegradation by the Trichoderma reesei fungus was highlighted.

The monosaccharides were analyzed with the help of HPLC—high-performance liquid chromatography—which includes a degasser, a pump, an autosampler, a column, an oven, and an RI detector. The separation column is a lead column (Aminex HPX-87 P, Bio-Rad, Hercules, CA, USA). The eluent is water. The calculation is performed by the Chromeleon program according to the internally determined calibration factor. The results are expressed in mg/mL

In thermal pretreatment, wheat straw is physically pretreated at certain temperature values between 50 and 240 °C and at certain pressure values for a certain period of time, in order to positively change the lignocellulosic composition in ways that lead to an increase in bioethanol yield [21,22,23,24].

In this work, by treating the straw with high-pressure steam, of 13–15 bar, at a temperature of 201 °C, the straw was softened and the straw fibers were decomposed, creating a co-rectified substrate with 17.3% dry matter (DM), for analysis in the laboratory (Figure 6). The image presents a substrate sample obtained from straw cut to the size considered optimal for an 80% saccharification yield. The substrate represents the by-product obtained after the thermal treatment of cut straw. This substrate treatment with Trichoderma reesei for 72 h allowed us to obtain the hydrolysate, i.e., sugar solution. The yield in saccharification, also called enzymatic hydrolysis, is calculated based on the concentrations of glucose and xylose obtained.

To demonstrate the importance of heat pretreatment in the enzymatic process, we studied three batches of straw harvested and stored on the factory platform. The straws were subjected to mechanical treatment, grinding, and heat treatment with high-pressure steam (

Table 1. The conditions in which the straw samples are subjected to thermal treatment.

Lot of Straw	Raw Material	Steam Pressure [bar]	Thermal Treatment Time [min]	Water Added [lt H2O]	Process Time [s]	pH Before Thermal Treatment	pH After Thermal Treatment
1	Wheat straw/1 t	11.5	10	175	72	4.19	4.91
2	Wheat straw/1 t	11.5	10	175	72	4.19	4.88
3	Wheat straw/1 t	11.5	10	175	72	4.19	4.85

), to make the lignocelluloses more accessible to enzymes during saccharification, thus increasing the contact surface of enzymes compared to raw, untreated straw.

The evidence of microstructural changes as a result of the fungal biodegradation of untreated and biotreated wheat straw was obtained through scanning electron microscopy with a magnification of ×1.00 kx.

3. Results

3.1. Thermal Pretreatment of Wheat Straw

The experiments were performed in triplicate according to the data below (

Table 2. Experiments performed on three lots of substrate straw.

Batch Straw Substrate	Glucose [mg/g]	Xylose [mg/g]	Sodium Lactate (NaDL) [mg/g]	Acetic Acid [mg/g]
1	55.96	27.02	0.75	5.50
	56.04	26.52	0.59	5.34
	59.67	29.07	0.72	5.65
Average value [mg/g]	57.22	27.54	0.69	5.49
Standard deviation (SD)	2.12	1.35	0.85	0.15
2	54.73	26.76	0.26	5.04
	54.61	26.57	0.25	5.03
	54.47	26.71	0.26	5.04
Average value [mg/g]	54.60	26.68	0.26	5.04
Standard deviation (SD)	0.13	0.098	0	0
3	55.54	26.17	0.19	5.31
	56.78	27.33	0.21	5.35
	55.43	26.22	0.30	6.28
Average value [mg/g]	55.92	26.57	0.23	5.65
Standard deviation (SD)	0.75	0.65	0.06	0.55

).

During the preparation of the sample, there is the probability that the hydrolysis will continue with the formation of glucose and xylose, so that there is a greater deviation between the results than in the case of the sodium lactate and acetic acid compounds.

The results obtained after the thermal pretreatment for the three batches of straw are presented in

Table 3. The conditions in which the straw samples are subjected to heat treatment.

Lot of Straw	Raw Material	Dry Component [%]	Glucose [g/L]	Xylose [g/L]	Furfural [g/L]
1	Average wheat straw/1 t	48.74	62.77	28.94	0.23
2	Average wheat straw/1 t	48.74	64.52	29.25	0.22
3	Average wheat straw/1 t	48.74	67.38	30.48	0.22

, where it can be observed that the acidity of wheat straw after pretreatment influences the amounts of glucose, xylose, and other components emitted.

The same straws, cut to the dimensions previously described, released very low concentrations of sugars following standard enzymatic hydrolysis (Table 3). This aspect leads to the conclusion that the lignocellulosic fiber must be vulnerable both mechanically and thermally for the process of obtaining bioethanol from the lignocellulosic mass to be considered useful.

The composition of the hydrolysate obtained from straw cut to sizes smaller than 50 mm (20–30 mm) and straw cut to sizes larger than 50 mm (70 mm–90 mm) and subjected to heat treatment was analyzed, and the results are presented in

Table 4. The composition of the hydrolysate obtained from straw cut to different sizes.

Wheat Straw Size [mm]	Glucose [g/L]	Xylose [g/L]	Lactate (NaDL) [g/L]	Acetic Acid [g/L]	Hydroxymethyl Furfural (HMF) [g/L]	Furfural [g/L]
20–30	55.67	18.85	1.25	8.75	1.20	1.15
	54.76	18.76	1.23	8.67	1.3	1.1
	55.87	18.67	1.22	8.6	1.2	1.3
Average [g/L]	55.43	18.76	1.23	8.67	1.23	1.18
Standard deviation	0.59	0.09	0.015	0.075	0.06	0.10
70–90	54.52	19.25	0.65	6.44	0.19	0.84
	54.23	19.56	0.45	6.34	0.18	0.77
	53.87	18.78	0.55	6.21	0.17	0.85
Average [g/L]	54.21	19.19	0.55	6.33	0.18	0.82
Standard deviation	0.32	0.39	0.1	0.11	0.01	0.04
50	64.89	29.56	0.67	4.58	0.17	0.22
	63.78	28.987	0.56	4.87	0.15	0.18
	64.78	27.98	0.62	4.54	0.16	0.23
Average [g/L]	64.48	28.84	0.62	4.66	0.16	0.21
Standard deviation	0.61	0.80	0.05	0.18	0.01	0.02

.

Straws cut shorter than the length considered optimum (50 mm) produced an agglomerated substrate, with higher concentrations of the toxic components acetic acid, hydroxymethyl furfural, and furfural. Their presence in higher concentrations had a negative impact on the enzymes in the enzymatic hydrolysis process, leading to low concentrations of glucose and xylose (Table 3).

Straws cut to sizes longer than the optimal 50 mm resulted in a substrate containing non-disintegrated lignocellulosic material, which resulted in low yields of fermentable sugars after enzymatic hydrolysis.

In conclusion, for the material used in this analysis, the optimal straw size after cutting, before heat treatment, was 40–50 mm. This ensured we obtained a substrate with toxic components in concentrations that did not diminish the performance of the enzymes in the enzymatic hydrolysis phase.

3.2. Enzymatic Hydrolysis

Lignin can be eliminated through the enzymatic saccharification of wheat straw, which supports the inhibition of polysaccharides, the accessibility of enzymes, and a high yield of biofuels [25,26,27,28,29,30,31,32,33,34]. Thus, following enzymatic pretreatment of lignocellulosic biomass for 90 min, an 83% cellulose content was obtained, 81% of lignin was decomposed, and there was a 10.5% hemicellulose content [26].

In the enzymatic hydrolysis step, the substrate obtained after heat pretreatment is converted into fermentable sugars of types C6 (glucose) and C5 (xylose, arabinose) with the help of enzymes produced by a mold fungus, which ensures the production of glycosidases and glucanases necessary to convert lignocellulosic material from the substrate into fermentable sugars (glucose, xylose, and arabinose).

To make it possible to extract fermentable sugars from cellulose during the hydrolysis process, the factory has developed through genetic engineering a strain of mold fungus capable of breaking the cellulose macromolecule into simple sugars (hexoses and pentoses) that can be metabolized later in the fermentation process.

This strain of mold fungus will be used in the production process under closed and isolated conditions. This genetically modified microorganism belongs to risk group 1, genetically modified microorganisms that pose no or negligible risk to human health and the environment.

At the same time, the lignin is separated from the cellulose fibers. Enzymatic hydrolysis of the substrate, using the enzymes produced by the mold fungus, takes place in six large vessels located on the plant platform, each with a capacity of 3584 m³.

Each enzymatic hydrolysis vessel is provided with agitators, recirculation pumps, and automation elements to maintain the optimal temperature and pH for the enzymatic activity. In each hydrolysis vessel, the following steps are carried out: mixing (substrate, process water, enzymes), enzymatic hydrolysis, thermal inactivation of enzymes after the maximum yield of sugars has been obtained, emptying, and washing. In the mixing and hydrolysis step, the liquid suspension (pretreated substrate plus water, enzymes) is continuously mixed to ensure the homogeneity of enzyme activity conditions. At the end of the enzymatic hydrolysis step, a solution rich in fermentable sugars of the C6 and C5 types (glucose, xylose, and arabinose) and lignin is obtained. After hydrolysis, under the influence of temperature (50 °C) and a long retention/contact period (3 days), all enzyme-producing microorganisms are inactivated. The suspension obtained in this stage is later pumped to the filtration line.

Following enzymatic hydrolysis, a suspension containing an aqueous solution rich in sugars is obtained (Figure 7) and a solid part, insoluble in water, called lignin. The hydrolysis of the substrate is reflected not only in the formation of sugars but also in the transformation of the material from a solid, the substrate, into a liquid containing a suspension, i.e., lignin.

The purpose of the filtration stage is to separate the insoluble components (lignin) from the aqueous solution containing the sugars needed for the fermentation process. Following the filtration process, the insoluble solid component—lignin (with 60% dry matter)—and the filtrate, called the hydrolysate, are finally obtained. The hydrolysate is sent either directly to the unit for ethanol fermentation, to the yeast production unit, or to the hydrolysate concentration unit, to be concentrated and then used as a nutrient for enzyme production.

The lignin obtained is a biomass with a high content of dry matter (over 60%), and due to its high calorific value, it will be used as a fuel to obtain the energy needed for the production process. The lignin is transported using a system of conveyor belts to the thermal plant serving the factory, where it is burned. The energy obtained is used in the production of the thermal agent (steam) and electricity needed for the factory.

In order to increase the efficiency of the transformation of sugars into ethanol during the fermentation process, a yeast strain, Trichoderma reesei multiplicated in the laboratory, is added (Figure 8, under conditions of temperature—28 degrees and stirring—800 rpm. The image of Trichoderma reesei was obtained with a Zeiss optical microscope.

During this stage, the hydrolysate is mixed with top fermentation yeast capable of metabolizing both glucose and the pentoses xylose and arabinose present in the hydrolysate. Following the metabolic activity of the yeast, the fermentable sugars in the hydrolysate such as glucose, xylose, and arabinose are transformed into ethanol, higher alcohols, secondary reaction products, and carbon dioxide. Heat is also released during the fermentation process. The fermentation process takes place in batches, with each batch taking place in a fermentation vessel.

In the production stage, the content of a multiplication batch obtained in the laboratory in a hermetic bottle is inoculated under sterile conditions in the first yeast multiplication vessel (Figure 9), in which the culture medium (hydrolysate and nutrients) was previously introduced. Yeast multiplication takes place in a cascade, with the contents of the first vessel being transferred to the next one, of a larger volume. The transfer is conducted through a closed system of pipes using centrifugal pumps. The culture medium is dosed in stages or continuously, through an automatically controlled flow, in order to ensure the necessary nutrients for the multiplication of microorganisms. To maintain the optimal temperature in the yeast production vessels, they are equipped with special double cooling walls. In each vessel, there is a system of continuous, controlled aeration, in which a flow of sterile air is introduced, thus ensuring the oxygen necessary for the multiplication of yeasts. The optimum pH value for yeast development is maintained by a controlled dosage of acidic or basic medium.

Transfer to the hydrolysis vessel, in order to mix with the substrate, to obtain the hydrolysate takes place in the last fermentation reactor (Figure 10). The medium used for preparation contains a source of sugar and a source of salts and minerals so that the final pH does not exceed the value of 5.

The wheat straw substrate was physico-chemically pretreated [35,36] to facilitate microbial biodegradation. We followed the digestion of untreated wheat straw by co-cultivating the fungus Trichoderma reesei, on 5 g of straw substrate, at 30 °C temperature, with pH 4.5. Enzyme activity was measured at 72, 90, 120, 140 and 160 h of cultivation. Most fungus cultures were placed at 94 h after cultivation on a bed of wheat straw (200 U/mL), with a decrease over time, up to 160 h, reaching 160 U/mL. Alternatively, they increased, which was due to the conditions of pH 4.5 or the carbon source [37,38,39]. Higher values of cellulase and xylanase were obtained at 94 h, decreasing at 160 h.

An impact of the co-cultivation of the fungal strain on the amount of total proteins and total reducing sugars released during hydrolysis was also observed. During the 150 days of mushroom cultivation, an increase in sugars (from 200 to 410 mg/g) and total protein concentration (from 100 to 151 μg/mL) was observed, until day 24, followed by a slow decrease on the other days. The effectiveness of the fungus in decomposing the wheat straw substrate was observed. The composition of biodegraded wheat straw after 30 days of Trichoderma reesei cultivation and without biodegradation is shown in

Table 5. Composition analysis of untreated/biotreated WS samples.

Treatment	Moisture [%]	Lignin [%]	Hemicellulose [%]	Cellulose [%]
Untreated WS	9.7	17.8	24.6	39.3
Trichoderma reesei-treated WS	9.9	11.06	20.9	33.6

. It can be seen that in the case of biotreated straw, the contents of lignin, hemicellulose, and cellulose decreased.

Scanning electron microscope (SEM) images (magnification 1.00 kx) of untreated and biotreated wheat straw at 30 days of cultivation are shown in Figure 11. The biodegraded structure can be observed after fungal pretreatment. We used microscope JSM-6360LVPRIME, JEOL^® [2].

The biodegradability of wheat straw was morphologically analyzed by SEM electron microscopy, before and (30 days) after the cultivation of the Trichoderma reesei fungus. We observed, in the case of untreated wheat straw, a compact, rough, and continuous surface, which was inaccessible for enzymatic saccharification and required treatment. And with the biotreated straws, the surface showed irregularities. The fungal strain penetrated the wheat straw substrate, facilitating the subsequent hydrolytic process.

4. Conclusions

Based on the experimental results presented in this paper, we found the following:

The efficiency of enzymatic hydrolysis is influenced by the quality of the substrate and enzymes.

We considered a qualitatively variable substrate (three lots from three geographical areas in Dolj county: Băilești, Radovan, and Calafat) and a single enzyme, Trichoderma reesei.

We varied the size of the straws and determined, based on the contents of cellulose and hemicellulose, the concentration of sugars.

In reality, the values obtained were lower than those in the laboratory, which leads to the conclusion that the biotechnological process is influenced by several factors differing from the laboratory to production.

This study confirms that the yield when transforming vegetable materials, wheat straw in this case, into bioethanol can be positively influenced by the ability of enzymes to trigger enzymatic hydrolysis. Utilizing their ability should be the goal of biotechnological research developing microorganisms adaptable to plant raw materials.