The Influence of Technological Conditions of Co-Fermentation of Lignocellulosic and Starch Raw Materials on the Amount of Volatile By-Products Formed and the Quality of Obtained Bioethanol

Abstract

This study investigated the effect of co-fermentation of starch (1G) and lignocellulosic (2G) feedstocks on ethanol production and the profile of volatile by-products. Experiments were conducted using an integrated SHF/SSF method with separate pretreatment of each raw material. After 72 h, the ethanol concentration in the starch–lignocellulosic mash reached 49.39 g/L, which is 77% higher compared to the ethanol concentration from the lignocellulosic biomass alone (27.93 g/L). The ethanol yields were 31.32 and 17.72 L/100 kg of raw material, respectively. Co-fermentation significantly changed the profile of volatile compounds. In the starch–lignocellulosic mash, the content of aldehydes was 51.20 mg/L (43% lower vs. lignocellulose alone), higher alcohols was 2018.17 mg/L (64% lower), esters was 8.70 mg/L (73% lower), and methanol was 1.33 mg/L (98% lower). These results demonstrate that integrating 1G and 2G feedstocks reduces the formation of by-products during the fermentation process, while maintaining ethanol concentrations at an industrial profitability threshold (above 40 g/L). The findings provide important insights for optimizing integrated bioethanol production from whole corn plants, which is essential for improving the economic feasibility.

1. Introduction

Each agricultural farm generates a significant amount of post-harvest residues, the amount of which depends on the type of crops, production scale, and technologies used. Most of this biomass is still burned as a waste disposal method, whereas only a small portion is used for purposes such as mulching, animal feed, or biofuel [1,2]. This biomass has significant energy potential and should be utilized in accordance with the principles of the bioeconomy, by converting it into energy or bioproducts, depending on its physicochemical properties [3]. The high cellulose and lignin content and appropriate calorific value of this biomass allow it to be used effectively with the possibility of additional financial deductions resulting from its classification as a raw material from renewable energy sources.

Bioethanol production, currently the most commonly used biofuel, is typically carried out using sugar- and starch-rich feedstocks such as sugarcane, corn, or wheat. This is known as first-generation (1G) bioethanol production. However, increasing attention is being paid to the use of cellulosic biomass from non-edible parts of crops and dedicated energy crops, such as corn stover, rye straw, cereal husks, and switchgrass. Bioethanol production from these feedstocks is referred to as second-generation (2G) bioethanol [4,5]. Lignocellulosic feedstocks are widely available and generally cheaper than food crops, making them promising alternatives for sustainable biofuel production [6,7].

Although commercial-scale production of second-generation bioethanol has already been initiated, its development has not been fully realized. This is mainly due to several technological challenges, including high enzyme costs, the need for additional pretreatment steps, relatively low ethanol yield and productivity, and excessive water consumption [8,9]. Current research focuses on developing efficient technologies for the bioconversion of lignocellulosic biomass components into bioethanol, to achieve economic feasibility. Only a comprehensive approach to process optimization can lead to economically viable solutions. One such strategy is the integration of first- and second-generation (1G + 2G) ethanol production technologies.

Starch feedstocks (1G) such as corn grain or wheat are characterized by a high content of readily fermentable sugars, with a starch content typically exceeding 60% [10]. Lignocellulosic feedstocks (2G) generally contain lower concentrations of fermentable sugars. They also have higher levels of fermentation inhibitors, which result from the intensive thermal treatment used to degrade the lignocellulosic structure [11]. The integration of both feedstocks allows for the supplementation of their values, that is:

-

the level of fermentable carbohydrates and amino acids for yeast through using 1G raw materials,

-

the level of glucose after lignocellulose decomposition and a significant reduction in feedstock costs through using 2G raw materials.

This strategy can serve as an intermediate step toward full-scale cellulosic ethanol production, offering the potential to lower investment costs and reduce production risks compared to processes the use of only lignocellulose (2G).

The profitability of production depends not only on ethanol yield but also on its final concentration in the fermentation mash. Achievement of the highest possible ethanol concentration in the fermentation mash is a crucial requirement for the economical distillation process, which is one of the most energy-intensive steps. An ethanol concentration of 4% (w/v) is the minimal requirement for an economically feasible distillation process [9,12].

Previous studies have mainly focused on the integration of 1G and 2G ethanol production using sugarcane as a substrate [13,14,15]. The results also showed that using hydrolysates rich in glucose, such as those from wheat or corn, is advantageous because of their high nutrient content. Erdei et al. [16] conducted an experiment with the co-fermentation of a mixture of wheat straw and wheat meal, demonstrating an increase in ethanol concentration and yield as the proportion of wheat meal in the medium increased. Additionally, research has focused on process optimization, substrate feeding strategies, and the co-fermentation of glucose and xylose for ethanol production [7,16]. Tang et al. [17] showed that the addition of starch biomass can serve as an additional source of nutrients, especially nitrogen, during lignocellulosic biomass fermentation. However, increasing the utilization of 2G raw materials remains a challenge because of the complex and resistant to decomposition structure of lignocellulose [5,18].

Alcoholic fermentation is a complex metabolic process in which volatile by-products are formed alongside the main product (ethanol). Their concentration in the fermentation medium is largely determined by the process conditions and raw materials used. The main factors influencing the quantity and composition of these compounds include technological parameters such as pH, temperature, oxygen availability, and initial extract, as well as the yeast strain used for fermentation and the composition of the fermentation medium [19,20,21]. The variety of raw materials used to prepare mashes determines the availability of nutrients in the medium. This, in turn, directly affects the yeast metabolic pathways and shapes the profile and concentration of volatile by-products.

Many strategies for integrated first- and second-generation (1G + 2G) bioethanol production have been described in the literature [9,15,22,23,24]. However, there is a lack of studies on the quantitative and qualitative analysis of volatile by-products in lignocellulosic distillates and distillates from the fermentation of starch-lignocellulosic mashes. Many countries, including members of the European Union, have established strict standards for permissible contaminants in fuel bioethanol [25]. Bioethanol produced from second-generation (2G) feedstocks is classified exclusively as fuel and cannot be used for consumption. Therefore, the use of whole corn plants, that is, the lignocellulosic and starch fractions combined, will be dedicated exclusively to the production of fuel bioethanol.

This study presents an analysis of the impact of co-fermentation of lignocellulosic and starch raw materials on the profile of volatile by-products formed during bioethanol production and on the course of alcoholic fermentation. This study assessed the composition of distillates obtained from the fermentation of starch-lignocellulosic, starch and lignocellulosic mashes. It was hypothesized that integrating starch (1G) and lignocellulosic (2G) feedstocks in the co-fermentation process would lead to increased ethanol concentration and improved bioethanol quality through changes in the by-product profile.

2. Materials and Methods

2.1. Raw Material

Whole corn plants were used in this study, including both corn grain and lignocellulosic biomass (leaves, stalks, and cobs), which are rich in cellulose, hemicelluloses, and lignin. The biomass was harvested at full maturity from a local farm in Wielki Konopat, Poland. After harvest, the material was air-dried under shelter and stored in a dry, ventilated room at ambient temperature (15–20 °C) until used for the laboratory experiments. The lignocellulosic fraction of the biomass, expressed on a dry weight basis, consisted of approximately 75% stalks, 10% leaves, and 15% cobs.

The lignocellulosic biomass was cut into 2 cm long pieces and subsequently ground using a cutting mill (ZBPP, Bydgoszcz, Poland) to obtain particles with diameters ranging from 0.5 to 1.0 mm. After grinding, the material was dried in an oven at 50 °C for 48 h, resulting in a moisture content below 7% (w/w). The chemical composition of the lignocellulosic biomass, expressed on a dry matter basis, were determined through the analysis of fiber fractions, namely crude fiber, neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent lignin (ADL) [26]. The corn grain was milled using the same cutting mill (ZBPP, Bydgoszcz, Poland).

2.2. Pretreatment and Enzymatic Hydrolysis

2.2.1. Lignocellulosic Biomass

The lignocellulosic fraction of corn was subjected to thermohydrolysis under alkaline conditions. A calcium hydroxide solution was prepared by dissolving 0.50 g of Ca(OH)₂ in 130 mL of distilled water. Pretreatment was carried out at 150 °C and 0.4 MPa for 15 min. For each pretreatment experiment, 100 g of the lignocellulosic fraction (dry weight basis) was weighed, and 1400 mL of calcium hydroxide solution together with the biomass were introduced into a high-pressure stainless-steel reactor with a working volume of 2000 mL. The resulting solid loading was 13% (w/v).

The pretreatment was conducted at elevated pressure and high temperature, with the aim of decreasing the crystallinity and polymerization of cell wall by partially degrading their organic structure. Delignification was performed in a high-pressure stainless-steel reactor (Versoclave Type 3E, Büchiglasuster, Switzerland). The pretreatment duration was measured from the moment the reactor reached the target temperature. The target temperature in the reaction vessel was achieved using heaters integrated into the double-walled jacket of the reactor. Elevated pressure conditions were maintained using an automatic pressure control system with solenoid valves and a pressure regulator, enabling precise dosing of inert gas (N₂). The reactor was additionally equipped with a high-torque (200 N·cm) anchor-type mechanical stirrer, designed to operate under conditions up to 250 °C at a maximum pressure of 60 bar. After thermohydrolysis, the reactor content was cooled to 50 °C using a water bath. The entire slurry, without separation into liquid and solid fractions, was subsequently neutralized with 1 M H₂SO₄ to pH 5.0.

Following the pretreatment stage, the biomass was subjected to enzymatic hydrolysis to convert structural carbohydrates into fermentable sugars. The pre-saccharification process was carried out using a mixture of enzymes: Cellic^® CTec2 cellulase (15 FPU/g dry matter), Viscozyme^® L (100 FBGU/g dry matter), and Pentopan^® Mono BG (2500 U/g dry matter), all supplied by Novozymes A/S (Bagsværd, Denmark). The enzymes were applied simultaneously to ensure efficient hydrolysis of both cellulose and hemicellulose fractions. Enzymatic hydrolysis was conducted at a substrate loading of 10% (w/v) dry solids. The reactions were performed in 250 mL Erlenmeyer flasks, incubated at 50 °C and 150 rpm for 4 h on an orbital shaker (Enviro-Genie, Scientific Industries, Inc., Bohemia, NY, USA). The pH was maintained at 5.0 using 0.05 mol·L⁻¹ sodium citrate buffer.

To evaluate the effectiveness of the pretreatment process, the degree of sugar conversion and saccharification efficiency were calculated using the following formula:

$$\mathrm{Sugar} \mathrm{conversion} (\%) = ({\displaystyle \frac{{\mathrm{C}}_{\mathrm{rel}}}{{(\mathrm{C}}_{\mathrm{cellulose}}\times 1.11)+({\mathrm{C}}_{\mathrm{hemicellulose}}\times 1.14)}})\times 100$$

(1)

where C_rel—simple sugars content (g); C_cellulose—cellulose content in biomass (g/g biomass), C_{hemicellulose}—hemicellulose content in biomass (g/g biomass); 1.11 and 1.14—conversion factors for cellulose → glucose and hemicellulose (xylan) → xylose.

$$\mathrm{Saccharification} (\%)={\displaystyle \frac{\mathrm{Reducing} \mathrm{sugar} \mathrm{released} \times 0.9}{\mathrm{Carbohydrate} \mathrm{content} \mathrm{of} \mathrm{the} \mathrm{biomass}}}\times 100$$

(2)

2.2.2. Corn Grain

Starch hydrolysis of corn grain into simple sugars was performed using microbial enzyme preparations. The liquefying enzyme α-amylase (Termamyl^® SC; Novozymes A/S, Bagsværd, Denmark) and the saccharifying enzyme glucoamylase (SanSuper; Novozymes A/S, Bagsværd, Denmark) were used to hydrolyze starch into glucose monomers. Hydrolysis with α-amylase was carried out at 85 °C, whereas glucoamylase treatment was performed at 65 °C and pH 5.0. The pH of the reaction medium was adjusted using 3 M NaOH or 1 M H₂SO₄. Enzyme dosages were as follows: Termamyl^® S.C.—0.15 mL/kg of starch (assuming a product density of 1.15 g/mL), and San Super—0.6 mL/kg of starch. Enzyme dosages were applied according to the manufacturer’s recommendations per gram of starch.

2.3. Microorganisms

Fermentation experiments were conducted using the Saccharomyces cerevisiae D-2 strain, obtained from the collection of pure cultures of the Prof. Wacław Dąbrowski Institute of Agricultural and Food Biotechnology—State Research Institute. The strain is characterized by high alcohol tolerance, osmophilicity, thermophilicity, and acidophilicity. The inoculum was prepared by suspending 5 g of yeast in 30 mL of sterile 0.9% (w/v) NaCl solution, followed by rehydration for 30 min. in accordance with the manufacturer’s instructions. The resulting yeast suspension was added to the fermentation medium at 4 mL/250 mL sample or 25 mL/1500 mL sample. The D-2 strain is non-genetically modified (non-GMO) and lacks the ability to metabolize xylose.

2.4. Fermentation Process

The experiments were conducted on a laboratory scale using 0.5 L Erlenmeyer glass flasks sealed with fermentation stoppers containing glycerol. Combined SHF/SSF method was applied to increase the initial concentration of fermentable sugars. In the first stage, characteristic of SHF method, the lignocellulosic substrate was subjected to pre-saccharification at the optimal temperature for cellulolytic enzyme activity (Section 2.2.1). Subsequently, the pretreated slurry was cooled to the fermentation temperature and inoculated with Saccharomyces cerevisiae yeast. At this stage, the process became SSF, as enzymatic saccharification (due to residual cellulolytic activity) and alcoholic fermentation (initiated by yeast inoculation) occurred simultaneously under optimal conditions for fermentation. The concentration of the solid substrate was maintained at 10% (w/v). Each flask contained 250 mL of fermentation mash and was inoculated with 4 mL of yeast suspension. For ethanol quality assessment, additional larger-scale fermentations (1500 mL working volume) were performed and inoculated with 25 mL of yeast suspension. Ethanol fermentation was carried out under static conditions at 38 °C, for 72 h. The main process parameters, including hydrolysis and fermentation temperatures, were based on previous research and optimized for the specific enzymes and yeast strain employed.

2.5. Process Variants

The experiments were conducted in three process variants:

Variant 1

(1G + 2G, starch–lignocellulosic mash): starch and lignocellulosic biomasses were pretreated separately. The liquefied and saccharified corn suspension was combined with the pretreated lignocellulosic slurry, and the mixture was subjected to fermentation using the SHF/SSF method, as shown in Figure 1.

Variant 2

(1G, starch biomass): ground corn grain was liquefied with α-amylase and subsequently saccharified with glucoamylase. The resulting mash was then fermented to ethanol.

Variant 3

(2G, lignocellulosic biomass): the lignocellulosic feedstock was subjected to thermohydrolysis followed by pre-saccharification, after which the obtained slurry was fermented using the SHF/SSF method.

2.6. Sample Analysis

2.6.1. Dry Matter (%), Dry Organic Matter (% DM), pH

Dry matter and organic dry matter were determined using standard gravimetric methods. Total solids were measured by drying the samples at 105 °C to constant weight using a Radwag MAX 50 moisture analyzer (Radwag Balances and Scales, Radom, Poland). Organic dry matter was determined by incinerating of the dried samples at 550 °C for 3 h in a muffle furnace. The pH value was measured using a laboratory pH meter (Mettler Toledo, Columbus, OH, USA). The efficiency of hydrolysis was evaluated based on the concentration of released reducing sugars, which was determined using the Lane–Eynon titrimetric method.

2.6.2. Cellulose, Hemicellulose and Lignin Content

The analysis involved the extraction of NDF (neutral detergent fiber), ADF (acid detergent fiber), and ADL (acid detergent lignin), using a FOSS Fibertec^®8000 device (FOSS Analytical A/S, Hillerød, Denmark) equipped with a hot and cold extraction unit. The polysaccharides and lignin contents were determined using the detergent fiber analysis method described by Van Soest et al. [27], in accordance with ISO standards [28,29].

2.6.3. Marking of the Volatile By-Products

Following fermentation, ethanol was recovered by distillation using a glass distillation apparatus. The setup consisted of a distillation column equipped with 26 theoretical plates, a distillation flask, a dephlegmator with a water jacket and an adjustable contact thermometer for vapor temperature measurements, a Liebig condenser, and an electric heating mantle. The volatile by-products in the obtained distillates were identified using capillary gas chromatography. Analyses were performed with a gas chromatographer (HP 6890, Shimadzu, Japan) with EPC (Electronic Pneumatic Control) unit, flame ionization detector (FID), and Chrompack CP-WAX 57-CB polar capillary column (high polarity polyethylene glycol) with the dimensions of 50 m × 0.32 mm, 0.2 μm (Agilent Technologies^®, Santa Clara, CA, USA). The oven temperature was programmed to increase from 40 °C to 160 °C at a rate of 10 °C/min. The injector and detector temperatures were set to 210 °C, 250 °C, respectively. High-purity nitrogen was used as a carrier gas at a flow rate of 30.0 mL/min. Volatiles compounds, including aldehydes, higher alcohols, esters, and methanol were identified and quantified by the comparing the retention times and peak areas with those of authentic standards.

The ethanol concentration (g/L) was determined using a Carl–Zeiss refractometer and standard alcohol tables, after distillation of a 100 mL sample. All analyses were conducted in triplicate.

2.6.4. Statistical Analysis

All experiments were performed in triplicate, and the average values are presented in the studies. The experimental data were reported as mean ± standard deviation. The statistical significance of the experimental data was evaluated at 95% confidence level (p < 0.05) using Tukey’s post hoc test with Statistica 12.0 (StatSoft Inc., Tulsa, OK, USA).

3. Results

3.1. Decomposition of Lignocellulosic Biomass

The pretreatment of lignocellulosic biomass was performed in two stages. In the first stage, delignification was carried out by thermohydrolysis under alkaline conditions, leading to the initial depolymerization of polysaccharides and partial solubilization of lignin. In the second stage, enzymatic hydrolysis was applied to further decompose polysaccharides into fermentable sugars, mainly glucose and xylose.

The chemical composition of the lignocellulosic biomass, expressed on a dry matter basis, was as follows: 29.39% cellulose, 26.08% hemicellulose, 11.32% lignin and 33.21% residue (ash and extractives). The starch content of the corn grains was 60.03%.

Table 1. Characteristics of raw materials.

Lignocellulosic Biomass
Dry matter (%)	Dry organic matter (%DM)		Crude fiber (%)
90.65 ± 0.09	93.82 ± 0.11		28.43 ± 0.29
Corn grain
Dry matter (%)		Starch (%)
89.95 ± 0.06		60.03 ± 0.43

Notes: The table shows mean values and standard deviations.

presents the characteristics of the raw materials used in this study.

As a result of the hydrolysis process, simple sugars were released and subsequently utilized in ethanol fermentation. The primary monosaccharides in the integrated 1G + 2G biomass are: glucose, derived from both the starch and lignocellulosic fractions, and xylose, obtained from the hemicellulosic fraction of the biomass [30].

The study showed that delignification enhances the efficiency of enzymatic hydrolysis and increases the amount of monosaccharides. Lignin is widely recognized as a physical barrier limiting enzyme access to cellulose, and its removal from biomass significantly improves the efficiency of enzymatic saccharification [31]. The use of appropriate pretreatment methods that combine efficiency and economic viability is crucial step in the process [32]. Currently, various pretreatment strategies are used, including physical, chemical, physicochemical, and biological methods, all of which have significant impact on the overall cost of production [32,33,34,35,36].

The chemical pretreatment methods include alkaline, acid, green solvents, and organosolv methods, whereas physical pretreatment methods include microwave, thermal, and high-pressure (steam explosion) methods. The mechanical pretreatments comprise milling, extrusion and ultrasonication approaches. The biological pretreatment includes bacteria and fungi (live microorganisms), enzymes, and microorganisms (immobilized) [33,35,37,38].

Table 2. Comparison of different chemical pretreatment process [39,40,41].

Chemical Pretreatment Method	Advantages	Disadvantages	Pretreatment Effect	Reference
Acid hydrolysis	(I) Low requirements of temperatures and pressure (II) High sugar release efficiency (III) Solubilizes hemicellulose	(I) Generates toxic by-products (e.g., furfural) (II) Corrosive (III) Requires neutralization (IV) Recovery acids for further use	Hemicellulose removal: 96% Glucose yield: 94.2%	[42]
			Hemicellulose removal: 85% (leaf), 77% (stem), 75% (whole plant) Glucose yield: 89% (leaf), 43% (stem), 76% (whole plant)	[43]
			Delignification: 76% hemicellulose removal: 69% glucose yield: > 80%	[44]
			Cellulose yield: 65% Hemicellulose yield: 23% Lignin yield: 9%	[45]
			Cellulose yield: 44.43% Hemicellulose yield: 19.11%	[46]
Alkaline hydrolysis	(I) Low inhibitor formation (II) Effective lignin removal (III) Ease to recover and reuse reagents	(I) High amount of alkali solution is required (II) Requires neutralization (III) Requires long residence time	Removal of 43% lignin	[47]
			Cellulose/glucose recovery: 75.48%	[48]
			Cellulose yield: 46.8% Saccharification field: 58%	[49]
			Cellulose yield: 33.0% Hemicellulose yield: 16.5% Saccharification field: 24.7%	[50]
Organosolv pretreatment	(I) High yield of pentose sugar (II) Effective removal of lignin	(I) Requires expensive solvents	Delignification: 63% Cellulose conversion: 89.2%	[51]
			Delignification: 62% Hemicellulose removal: 75% Glucose yield: 63%	[52]
			Delignification: 75.1% Hemicellulose removal: 81.5%	[53]
Ammonia fiber explosion (AFEX)	(I) High yield of pentose sugar (II) Low formation of inhibitors	(I) Recycling of ammonia is needed (II) Less effective process with increasing lignin content (III) Highly corrosive ammonia	Delignification: 62.5% Total sugar yield: 89.4%	[54]
			Fermentable sugars conversion: 90%	[55]

shows a comparison of different pretreatment processes and their effects.

The high costs of lignocellulosic biomass pretreatment represent a significant barrier to the development of biorefinery technologies [11,56,57,58]. In this context, lime pretreatment using calcium hydroxide is increasingly being considered a favorable alternative because of its low cost, operational safety, and environmental friendliness [57,59]. Lime treatment loosens the lignocellulosic structure by breaking ester and ether bonds between lignin and carbohydrates, which significantly increases the accessibility of cellulose for enzymatic hydrolysis [56,58].

A comparison of the biomass properties before and after pretreatment revealed significant changes in the structure. During delignification, lignin is solubilized and partially transformed. Consequently, the two major polysaccharide components, hemicellulose and cellulose, were released from lignin linkages, making them more accessible for enzymatic hydrolysis. Figure 2 shows the mass balance during the pretreatment of lignocellulosic biomass.

Initial hydrolysis was carried out for 4 h under conditions optimized for the activity of the applied enzymes. Due to the presence of hemicellulose in the biomass samples, additional enzymes, namely, hemicellulase and xylanase, were employed. The broad raw material specificity of these enzymes enhances hydrolysis efficiency, resulting in a higher yield of simple sugars from decomposed polysaccharides. A high degree of sugar conversion was achieved, reaching approximately 65.78%,

Table 3. Parameters characterizing the decomposition of lignocellulosic biomass into fermentable sugars.

Component	After Thermohydrolysis	After Thermohydrolysis and Enzymatic Hydrolysis
Sugar conversion (%)	24.29 ± 0.28	65.78 ± 0.42
Total sugars (g/L)	15.13 ± 0.12	41.02 ± 0.38
Saccharification (%)	-	66.57 ± 0.54

Notes: The table shows mean values and standard deviations. Samples were analyzed in triplicate.

.

As a result of raw material pretreatment and enzymatic hydrolysis, a significant reduction in the hemicellulose, cellulose, and lignin content in the lignocellulosic biomass was observed by 77%, 69%, and 54%, respectively. The decomposition of polysaccharides during enzymatic hydrolysis led to a substantial increase in the concentration of reducing sugars, which reached approximately 41.02 g/L. The saccharification efficiency was 66.57%, indicating the high effectiveness of the enzymatic system in converting complex carbohydrates into fermentable sugars. These results confirm the efficacy of combining appropriately selected pretreatment and enzymatic treatments in to prepare lignocellulosic biomass for subsequent stages of ethanol production.

3.2. The Content of Volatile Chemical Compounds in Distillates Obtained from Starch-Lignocellulosic Mashes and Mono-Feedstock

For ethanol production, the fermentation substrate was prepared by combining lignocellulosic biomass mash (stalks, leaves, and cobs) with starch mash (corn grain) at a 1:1 ratio. Due to the recalcitrant nature of second-generation feedstocks, pretreatment methods different from those used for first-generation bioethanol are required.

To enhance the effectiveness of the conventional SHF and SSF methods, a modification involving pre-saccharification was introduced, which is an initial hydrolysis at the optimal temperature for the enzymes. During this process, the temperature is initially maintained at the optimal level for enzymatic hydrolysis and then gradually lowered to a value favorable for alcoholic fermentation. The main advantage of the combined SHF/SSF method is the ability to achieve a higher ethanol yield during the early fermentation phase owing to the preliminary degradation of polysaccharides into fermentable sugars.

To evaluate the effect of feedstock composition on the course of alcoholic fermentation, experiments were conducted in three process variants: Variant 1—alcoholic fermentation of a starch–lignocellulosic mash (1:1 ratio); Variant 2—alcoholic fermentation of starch biomass; and Variant 3—alcoholic fermentation of lignocellulosic biomass. The variants were assessed in terms of ethanol concentration and yield from the feedstock,

Table 4. Ethanol concentration and yield obtained in the fermentation process under different process variants.

Variants	Ethanol Concentration (g/L)			Ethanol Yield (L/100 kg of Raw Material)
	Fermentation Time (h)			Fermentation Time (h)
	24	48	72	24	48	72
Variant 1	43.18 b ± 0.58	47.91 b ± 0.37	49.39 b ± 0.22	27.36 b ± 0.46	30.36 b ± 0.31	31.32 b ± 0.17
Variant 2	56.30 c ± 0.84	60.08 c ± 0.28	61.86 c ± 0.45	35.68 c ± 0.53	38.08 c ± 0.18	39.20 c ± 0.28
Variant 3	18.96 a ± 0.20	24.65 a ± 0.28	27.93 a ± 0.43	12.02 a ± 0.17	15.62 a ± 0.21	17.72 a ± 0.35

Notes: The table shows mean values and standard deviations. ^{a b c}: Values listed in the same column with the same letter are not statistically different at 95% confidence intervals, p < 0.05; n = 3. Variant 1: starch–lignocellulosic mash (1G + 2G), Variant 2: starch mash (1G), Variant 3: lignocellulosic mash (2G).

.

The choice of the lignocellulosic and starch biomass integration ratio was based on the need to find a balance between high process efficiency, which is typical for starch-rich raw materials, and the sustainable use of lignocellulosic biomass. Starch-based raw materials, thanks to their high content of easily fermentable sugars, are conducive to achieving high ethanol concentrations and fermentation yields. After enzymatic hydrolysis, the reducing sugar content in the starch mash was approximately 61.40 g/L, whereas in the starch–lignocellulosic mash, it was 51.21 g/L. However, the use of starch-based raw material is associated with higher costs and is primarily used by the food and animal feed sectors. In contrast, lignocellulosic biomass, although more sustainable and economically advantageous, is characterized by lower reducing sugar content (41.02 g/L) and ethanol yields.

The fermentation efficiency of lignocellulosic biomass is also influenced by the various byproducts generated during the pretreatment. These inhibitory compounds, including furfural, hydroxymethylfurfural (HMF), weak acids, and phenolics, are primarily formed during biomass hydrolysis and significantly hinder microbial metabolism and enzymatic activity [60,61]. Even at relatively low doses (usually not more than 10 g/L, inhibitors can negatively affect downstream processing or impede cell growth [61,62,63].

These compounds affect the metabolism in different ways. For example, furaldehydes, such as 5-hydroxymethylfurfural (HMF) and 2-furaldehyde (furfural), reduce the viability of S. cerevisiae, affect the fermentation rate, inhibit growth and/or prolong the lag phase, reduce intracellular NAD(P)H levels, and induce oxidative stress in yeast cells [64,65]. In addition, furfural has been shown to increase reactive oxygen species (ROS), which leads to damage to cellular structures [66]. The weak acids, primarily acetic and formic acids, reduce biomass yield [65,67]. Furthermore, the synergistic interaction of various inhibitors has been observed to amplify their toxicity compared to their individual effects, leading to more intricate reactions and more severe damage to vital cellular activities [68,69].

The analysis included both ethanol concentration (g/L) and ethanol yield per unit of raw material (L/100 kg) at three time points: 24, 48, and 72 h of fermentation. As shown in Table 4, more than 87% of the total ethanol was produced within the first 24 h of fermentation in Variants 1 and 2. The values obtained in the variant where starch-lignocellulosic mash was fermented (Variant 1) were significantly higher than in the fermentation using only lignocellulosic biomass (Variant 3). After 24 h, the ethanol concentration in Variant 1 reached 43.18 g/L, which was more than twice that of Variant 3 (18.96 g/L). This corresponded to ethanol yields of 27.36 L and 12.02 L per 100 kg of raw material, respectively, Table 4.

Significant differences between these variants persisted in the subsequent days of the process. At the end of fermentation (72 h), the ethanol concentration in the variant where starch-lignocellulosic mash was fermented was 49.39 g/L, which was 77% higher compared to Variant 3 (27.93 g/L). The corresponding ethanol yields were 31.32 and 17.72 L/100 kg of raw material, respectively.

In Variant 2, where only starch biomass was subjected to fermentation, the ethanol concentration (61.86 g/L) was approximately 25% higher than that in the starch–lignocellulosic mash and more than two times higher than that in the lignocellulosic biomass (72 h).

Variant 2, which involved fermentation of starch biomass alone, was characterized by the highest ethanol content and yield after 72 h of fermentation: 61.86 g/L and 39.20 L/100 kg, respectively. However, the high cost of the 1G feedstock makes the integration of starch and lignocellulosic feedstocks an equally cost-effective solution. Compared to the fermentation of lignocellulosic biomass alone, the use of a starch-lignocellulosic mash allows for a higher ethanol concentration and improved process efficiency.

The differences in results between variants using starch-lignocellulosic mash and lignocellulosic mash were statistically significant (p < 0.05). This indicates that the combination of raw materials provides a compromise between high yield and the utilization of difficult to decompose lignocellulosic raw materials. This is important from a technological and economic perspectives, as it allows for satisfactory yields at relatively low raw material cost.

The experiments analyzed the effect of the type of mash used: starch–lignocellulosic, starch, and lignocellulosic on the profile of volatile by-products formed during the ethanol fermentation process. This analysis allows for an understanding of metabolic pathways and their modifications in response to different feedstocks, which is essential for optimizing biotechnological processes. During the distillation process, volatile compounds such as aldehydes, higher alcohols, and esters can co-distill with ethanol. This occurs partly because these compounds can form azeotropes or exhibit similar volatility characteristics in hydro-alcoholic solutions [70]. This phenomenon causes compounds that would not normally distill under simple conditions to be transferred into the distillate along with ethanol. Their quantity and composition depend on the type of raw material, yeast strain, and technological conditions, including pretreatment, fermentation, and distillation. Lignocellulosic raw materials are characterized by the presence of phenolic compounds, furfural, and organic acids, which can undergo further transformations into volatile secondary metabolites. In contrast, during the fermentation of starchy raw materials, higher alcohols predominate, formed through amino acid catabolism via the Ehrlich pathway, along with aldehydes and esters produced as by-products of yeast metabolic activity [71,72]. A high content of volatile compounds is an indicator of the deterioration of the quality of the produced bioethanol.

The distillation process was carried out using a distillation set consisting of distillation column, distillation flask, and a dephlegmator equipped with an adjustable electric contact thermometer. The volatile compounds in the distillates (aldehydes, higher alcohols, esters, and methanol) were identified and quantified using gas chromatography. Nine volatile substances were identified and classified into three product groups based on their chemical classes.

Analysis of the composition of distillates obtained from fermentation showed that the proportion of starch biomass in the starch–lignocellulosic mixture gave the effect of obtaining lower amounts of acetaldehyde, propionaldehyde, and ethyl acetate. Figure 3 presents a comparison of the concentrations of these volatile compounds across the tested variants, indicating a relationship between the proportion of raw materials and the by-product profile in the obtained distillates.

Acetaldehyde is the most important carbonyl by-product formed during alcoholic fermentation by yeast metabolism of sugars via the action of pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH). Acetaldehyde functions as a terminal electron acceptor, playing a crucial role in ethanol production yields, product stabilization, and toxicology. In the presence of alcohols, acetaldehyde can react with the amino groups of nucleosides to produce acetal mixtures [72,73,74].

In distillates from the starch-lignocellulosic mash (Variant 1), the acetaldehyde concentration was 85.13 mg/L, which was 48% lower compared to distillates from the lignocellulosic mash (Variant 3—162.67 mg/L). The lower level of acetaldehyde in variants containing starch indicates higher activity of alcohol dehydrogenase (ADH), the enzyme responsible for reducing aldehyde to ethanol [75,76]. Factors that potentially affect ADH activity include the composition of the raw material, fermentation conditions (pH, temperature), and the presence of inhibitory compounds formed during pretreatment of lignocellulosic biomass [77].

The highest concentration of this compound was recorded in the early stages of fermentation, after which some acetaldehyde was converted to ethanol. Compared to distillates from starch feedstock (Variant 2—ca. 51 mg/L), the acetaldehyde concentration in starch-lignocellulosic mash (Variant 1) was ca. 66% higher, Figure 3. Higher acetaldehyde concentrations in Variants 1 and 3 may be related to the presence of inhibitors, such as furan aldehydes, which inhibit ADH activity, affecting both the volatile by-product profile and ethanol production yield [77,78]. Elevated acetaldehyde levels may also result from lower zinc ion concentrations in the fermentation media. Zinc ions are an essential metallic cofactor for alcohol dehydrogenase (ADH), an enzyme that catalyzes the reduction of acetaldehyde to ethanol [79]. Zinc functions as an electron attractor, which gives rise to an increased electrophilic character of the aldehyde, consequently facilitating the transfer of a hydride ion to the aldehyde [80,81]. Lower acetaldehyde concentrations were observed in raw materials richer in zinc, such as corn grain, indicating a more effective conversion of this compound to ethanol. These results highlight the significant role of trace elements in yeast enzymatic activity and in shaping the profile of volatile by-products during alcoholic fermentation.

In Variant 3, where only lignocellulosic biomass from whole corn plants was subjected to fermentation, the furfural concentration in the distillate was 72.40 mg/L. In contrast, this compound was not detected in Variants 1 and 2.

Analysis of the distillates showed that the concentrations of propionaldehyde and ethyl acetate in the distillate from the starch-lignocellulosic mash were 5.13 mg/L and 32.37 mg/L, respectively, Figure 3. These values were approximately 55% and 35% lower compared to Variant 3, in which only lignocellulosic biomass was fermented. In Variant 2, where only starch feedstock was used for fermentation, the ethyl acetate concentration was even lower, at ca. 8.70 mg/L. However, the presence of propionaldehyde was not detected in this variant.

Ethyl acetate is an ester formed from the esterification of acetic acid and ethanol, and it is a common product of fermentation [82]. Further analysis indicated that the high concentration of ethyl acetate in distillates obtained from lignocellulosic biomass fermentation may result from the use of highly active cellulases during enzymatic hydrolysis. This increased cellulase activity can enhance the permeability of the yeast cell wall, introducing metabolic stress. This, in turn, can affect the activity of alcohol acetyltransferases (AAT), the enzymes responsible for ethyl acetate synthesis [83]. Consequently, this leads to an increased concentration of this compound during alcoholic fermentation.

Table 5. Concentration of higher alcohols in the obtained distillates.

Variants	1-Propanol, (mg/L)	Isobutanol, (mg/L)	n-Butanol, (mg/L)	2-Methyl-1-Butanol, (mg/L)
1	3365.03 c ± 29.86	1690.27 b ± 41.75	233.43 b ± 12.20	336.57 a ± 8.16
2	365.10 a ± 7.10	1100.50 a ± 11.10	5.90 a ± 0,30	546.67 b ± 5.70
3	2945.50 b ± 50.24	1978.13 c ± 32.84	296.10 c ± 17.10	622.23 c ± 11.86

Notes: The table shows mean values and standard deviations. ^{a b c}: Values listed in the same column with the same letter are not statistically different at 95% confidence intervals, p < 0.05; n = 3. Variant 1: starch–lignocellulosic mash (1G + 2G), Variant 2: starch mash (1G), Variant 3: lignocellulosic mash (2G).

shows the concentrations of 1-propanol, isobutanol, n-butanol, and 2-methyl-1-butanol, which belong to the group of higher alcohols, also known as fusel alcohols. Yeasts produce higher alcohols during fermentation, either directly from sugars or indirectly from amino acids via the Ehrlich reaction. After the initial transamination of the amino acids, the resulting α-keto acids are subsequently decarboxylated by the yeast cells and reduced to higher alcohols or acids. Their concentrations in sugarcane distillates are influenced by factors such as the yeast strain, and fermentation conditions such as temperature and aeration [72,82].

Lower concentrations of higher alcohols, such as isobutanol and 2-methyl-1-butanol, in distillates from the starch-lignocellulosic mash (Variant 1) compared to those from the lignocellulosic mash (Variant 3) more favorable fermentation conditions. Isobutanol and 2-methyl-1-butanol are formed in the Ehrlich pathway from valine and isoleucine, respectively, through transamination, decarboxylation, and reduction [71]. Excessive production of these metabolites typically results from a deficiency in available mineral nitrogen or other nitrogen sources in the medium. Under these conditions, yeast are forced to catabolize amino acids to acquire nitrogen. In contrast, starch-based raw materials provide not only carbohydrates but also proteins and peptides, which are hydrolyzed during mashing and fermentation to readily assimilable nitrogen compounds (amino acids and free ammonium nitrogen). The availability of these nitrogen sources reduces the need for yeast to degrade branched-chain amino acids (valine, isoleucine) through the Ehrlich pathway, thereby lowering the formation of higher alcohols. Yeast metabolism focuses on ethanol production rather than amino acid degradation [84,85].

In distillates from the starch-lignocellulosic mash, the concentrations of isobutanol (1690.27 mg/L) and 2-methyl-1-butanol (336.57 mg/L) were lower than in distillates from the lignocellulosic mash (1978.13 mg/L and 622.23 mg/L, respectively), corresponding to reductions of approximately 15% and 46%, respectively. This favorable change in the impurity profile resulted from the fact that the starch-lignocellulosic mash (1G + 2G) was characterized by a better nutrient balance. The availability of simple sugars from the degraded starch fraction directed yeast metabolism primarily toward ethanol production, thereby limiting the amino acids catabolism in the Ehrlich pathway. In the starch-based mash, despite the higher sugar concentration, a higher level of 2-methyl-1-butanol (546.67 mg/L) was recorded compared to the starch–lignocellulosic mash. This can be attributed to the greater content of isoleucine from grain proteins and potential deficiency of mineral nitrogen. The addition of the lignocellulosic fraction in Variant 1 improved the overall balance of nitrogen sources, resulting in lower production of 2-methyl-1-butanol and a more favorable effect on the quality of the distillate.

In the distillates, n-butanol concentrations were also measured which varied significantly depending on the type of mash. In the starch-lignocellulosic mash, the concentration was 233.43 mg/L (Variant 1), in the starch mash 5.90 mg/L (Variant 2), and in the lignocellulosic mash 296.10 mg/L (Variant 3). The compound n-butanol is formed mainly as a result of sugar fermentation and partially through the Ehrlich pathway. However its biosynthesis is more complex and depends on fermentation conditions and the presence of inhibitors [86].

The biosynthesis of 1-propanol follows a different mechanism than that of branched-chain alcohols, and is dependent mainly on carbohydrate metabolites rather than amino acids. The concentrations of this alcohol in the distillates were as follows: starch-lignocellulosic mash—3365.03 mg/L, starch mash—365.10 mg/L, and lignocellulosic mash—2945.50 mg/L, Table 5. The highest concentration of 1-propanol in the starch–lignocellulosic mash (Variant 1) resulted from the synergistic effect of both fractions, which provided more fermentable sugars, thereby stimulating the metabolic pathways leading to 1-propanol production more effectively than in the lignocellulosic mash (Variant 3). In the lignocellulosic mash, the presence of inhibitors formed during pretreatment could have partially limited the biosynthesis of this alcohol. In contrast, in the starch mash, although the sugar content was the highest, the concentration of 1-propanol was the lowest. This is likely due to the metabolic preference of the yeast—in glucose-rich conditions, metabolism is primarily directed towards ethanol production, thereby limiting 1-propanol production. These results indicate that the biosynthesis of higher alcohols depends selectively on the availability of sugars and amino acids, as well as on fermentation conditions.

As shown in

Table 6. Concentrations of volatile by-products in the different test variants.

Variants	Aldehydes (mg/L)	Higher Alcohols (mg/L)	Esters (mg/L)	Methanol (mg/L)
1	90.27 b ± 0.95	5625.30 b ± 91.55	32.37 b ± 0.45	54.11 b ± 0.44
2	51.20 a ± 2.30	2018.17 a ± 12.20	8.70 a ± 0.20	1.33 a ± 0.03
3	246.40 c ± 3.20	5840.41 b ± 46.10	50.27 c ± 0.70	61.91 c ± 1.55

Notes: The table shows mean values and standard deviations. ^{a b c}: Values listed in the same column with the same letter are not statistically different at 95% confidence intervals, p < 0.05; n = 3. Variant 1: starch–lignocellulosic mash (1G + 2G), Variant 2: starch mash (1G), Variant 3: lignocellulosic mash (2G).

, for Variant 2, that is fermentation of starch biomass, the methanol concentration in the obtained distillate was 1.33 mg/L, whereas in the remaining variants the methanol content ranged from 54.11 to 61.91 mg/L. Higher methanol levels were recorded in Variants 1 and 3, in which the proportion of lignocellulosic biomass ranged from 50% to 100%. The by-products present in the mashes also include acetic acid and inorganic acids used to regulate the pH of the environment. The acidity of all the analyzed distillates was approximately 30–59 mg/L.

4. Discussion

The results of the analysis confirmed that the raw material composition of the mashes significantly affected the profile of volatile by-products in distillates. The co-fermentation of lignocellulosic and starch mashes selectively influenced the concentrations of acetaldehyde, propionaldehyde, ethyl acetate, and higher alcohols. These mechanisms were linked to both the availability of fermentable sugars and the amino acids and microelement content. Our observations indicate that obtaining bioethanol from first- and second-generation feedstocks (1G + 2G) in a 1:1 ratio leads to a lower concentration of undesirable volatile by-products than ethanol production from a lignocellulosic monosubstrate.

The integration of first-generation (1G) and second-generation (2G) ethanol pro-duction processes can be implemented at several stages in the process, from directly after pretreatment, to the downstream processes, for example, in the distillation or evaporation steps. Thus, several different process configurations are possible, which should be flexibly selected, depending on market factors such as the price of raw materials and bioethanol, as well as the presence and value of by-products [4].

The economics of fuel ethanol production are significantly influenced by operating costs, including the price of feedstock, enzymes, and the wholesale price of electricity. Among these, the cost of enzymes has been found to have the greatest effect on the cost of 2G bioethanol production, followed by the electricity selling price and, to a lesser extent, the cost of transportation [87]. Therefore, profitability analysis requires consideration of both technological aspects and economic forecasts. The economic analysis indicates that the integration of 1G and 2G feedstocks for ethanol production showed significant economic benefits (up to 50% improvement), compared to 2G-only ethanol production, thereby lowering the cost of lignocellulose conversion [23]. Enzymatic hydrolysis of 2G biomass requires the application of high enzyme doses to effectively release fermentable sugars, which significantly increases process costs. In contrast, in co-fermentation, part of the sugars is derived from the starch contained in cereal grains, which requires simpler processing and is more easily assimilated by yeast. This makes it possible to partially reduce the overall enzymatic hydrolysis costs per unit of ethanol [23,88]. Moreover, co-fermentation of starch and lignocellulosic biomass improves fermentation efficiency through improved biomass utilization, dilution of pretreatment inhibitors, and lower enzyme and energy requirements [88].

Enhanced ethanol concentration achieved during co-fermentation (1G + 2G) could lower downstream distillation costs. In addition, co-fermentation of cellulosic biomass with cereal grains such as corn or sorghum can be easily adopted by the existing ethanol industry based on starch feedstock [74,89]. Integration can be achieved either by designing a completely new combined plant, or by installing a 2G unit at an existing 1G plant [4].

Ayodele et al. [90] reviewed integration opportunities for sustainable bioethanol production from first- and second-generation feedstocks. They reported that 1G + 2G integration can not only improve economic performance but also reduce environmental burden compared to 2G bioethanol production facilities. Joelsson et al. [4] presented a feasibility assessment of ethanol production in an integrated 1G + 2G plant using cereals and wheat straw as feedstocks. Macrelli et al. [87] developed fifteen scenarios for integrated ethanol production from sugarcane, indicating that proper optimization can significantly reduce costs and increase energy efficiency of the process. Similar conclusions were presented by Furlan et al. [91] who demonstrated a 1G + electric energy and 1G + 2G ethanol biorefineries. They found that the 1G + 2G ethanol biorefinery is closer to feasibility than the conventional 1G + electric energy industrial plant.

Despite these potential benefits, current integration processes face challenges related to high costs, feedstock recalcitrance, and scalability issues [92]. The critical barriers primarily concern difficulties in maintaining optimal hydrodynamic and mass transfer conditions. Although these parameters are easily controlled under laboratory conditions, they tend to cause heterogeneity in the fermentation environment on an industrial scale. Additional challenges include the high cost and limited recovery of enzymes and/or solvents, as well as the increased risk of inhibitor formation during large-scale operations [93].

5. Conclusions

Bioethanol production based on co-fermentation of 1G and 2G feedstocks in a 1:1 ratio contributed to a reduction in undesirable volatile by-products compared to fermentation of a lignocellulosic mono-feedstock.

The analyses revealed that in distillates from the co-fermentation of lignocellulosic and starch biomass (Variant 1), the concentration of aldehydes was 63% lower compared to that in distillates obtained from 100% lignocellulosic biomass (Variant 3). Similarly, for other groups of volatile by-products lower levels were observed in distillates from the starch–lignocellulosic mash (Variant 1): esters by 36%, and higher alcohols by 4%, compared to lignocellulosic mono-feedstock (Variant 3). These differences can be attributed to the higher availability of fermentable sugars and the varying amino acid and microelement content in the respective fermentation variants.

The use of starch–lignocellulosic feedstocks allow for maintaining a high ethanol yield.

The integration of lignocellulosic and starch raw materials is a promising strategy for increasing ethanol concentration and yield, reducing energy consumption during distillation, and improving the overall profitability of bioethanol production compared with lignocellulosic mono-feedstock. Utilizing surplus lignocellulosic biomass in combination with starch feedstock provides an attractive method for reducing investment costs.

The technology described in this study may serve as a transitional step toward full-scale production of cellulosic ethanol.

The results provide new data on the amounts of volatile by-products formed when using multi-component mash. This complements the limited knowledge on how these feedstocks influence the quality of the resulting bioethanol and indicates the potential for using starch–lignocellulosic mashes in bioethanol production.