1. Introduction
The changes of environmental and climate conditions as well as the economic growth and an increase in energy consumption have led to the intensification of research on the alternative fuels to replace fossil fuels [
1,
2]. One of the possibilities of improving the energy mix is the use of plant biomass for bioenergy production.
It is worth reaching for new solutions such as utilization of less popular but perspective crops, including sorghum (
Sorghum L.). Sorghum is fifth in the world in terms of volume of the production after wheat, corn, rice, and barley [
3]. Nowadays, sorghum is cultivated also in some European countries [
4,
5,
6]. The popularity of sorghum occurs due to its drought resistance [
7,
8,
9], high yield potential [
10,
11] and various applications in the economy. This plant provides a wide range of food [
12,
13,
14], industrial [
15,
16] and energy applications [
17,
18].
In comparison to corn, sorghum is able to withstand a long period without precipitation, often with almost no damage to plants. The collected data indicate that 80% of sorghum production in the world is under dryland conditions [
19]. Changing climatic conditions have been observed in European countries for several years. Drought in combination with very high temperatures are favorable conditions for sorghum development.
One of the main possibilities for the economic use of sorghum biomass is bioenergy production. Its biomass can be used for pellets and briquettes formation. These products are efficient solid biofuels because they are characterized by high calorific value [
20,
21]. In addition, stems and grain of sorghum can be effective substrates for bioethanol production [
22]. In turn, straw shives or residues from biomass processing are useful for biogas production [
23,
24,
25]. Most of the studies described in articles concern using the sorghum biomass from tropical and subtropical areas [
26,
27,
28]. For that reason, there is a lack of knowledge regarding the energy potential of sorghum straw harvested in a temperate climate.
Lignocellulosic biomass is the most widely known biomass in the world and can be found in the leaves, skins and branches of many plants. Therefore, the production of lignocellulosic bioethanol in recent years is an alternative for the still frequently used starch bioethanol. Lignocellulosic bioethanol does not compete with food production for raw materials, and moreover, it contributes to a much greater reduction of greenhouse gases emission. This type of bioethanol is particularly suitable for countries where agricultural and forestry waste is used as input materials [
29].
Lignocellulose is a complex structure found in plants and consists of cellulose, hemicellulose, lignin and other extracts and mineral traces. Lignin plays an important role in cross-linking of cellulose and hemicellulose. Due to these properties of lignin, the whole structure is more durable and resistant to external hydrolyzing agents, which in the case of bioethanol production is a significant obstacle. Hence, an important step in the production of bioethanol from plant biomass is the pretreatment to remove lignin and increase the potential for hydrolyzing agents. It is well known that the production of bioethanol from plant biomass involves deconstruction of cell walls to individual polymers and next hydrolysis of carbohydrates to monosaccharides [
30].
The sorghum biomass conversion process to bioethanol consists of three main stages. The first step is biomass preparation through the use of physicochemical pretreatment. Milling as the physical method, is aimed at to reduce the size of the substrate as well as to facilitate the access of bioactive substances to the surface, reduction of polymerization and crystallization degree of lignocellulose. In turn, chemical processes include among others acid and base treatments [
31,
32]. Depending on the method used, various changes take place within the lignocellulosic complex, e.g., alkaline and neutral pretreatment is basically a delignification process, while the acid and steam treatment dissolves most of the hemicellulose. The second step is enzymatic hydrolysis, using enzymatic preparations, which allows the release of fermentable sugars. Ethanol fermentation is the last stage in which monosaccharides are converted to ethanol by various microorganisms, including the yeast
Saccharomyces cerevisiae Meyen ex E.C. Hansen [
33].
The authors, based on previous study on the process of obtaining bioethanol from sorghum biomass, general concluded that sorghum biomass may be a valuable raw material for obtaining lignocellulosic bioethanol [
34]. Moreover, no literature has been published before in relation to possibility of effective cultivation of sorghum as a second crop and utilization its biomass for bioethanol production above 50 degrees north latitude. Therefore, the aim of this presented research is made to determine the yield of bioethanol produced from three varieties of sorghum in the main and secondary crops, which can be grow in temperate climate typical of Central and Eastern Europe.
2. Materials and Methods
2.1. Sorghum Biomass
The raw materials used in the study were three varieties of sorghum biomass: Sucrosorgo 506, Santos and Rona 1 (AgriSem GmbH, Einbeck, Germany). The field research was conducted in a three-year period in Stary Sielec in Poland (51°39′ N, 17°10′ E) in the Experimental Farm of the Institute of Natural Fibers and Medicinal Plants.
The experiments were carried out on a soil characterized by abundance of nutrients and medium agricultural usefulness. The plots were located next to the last position annually, but not in the same place to avoid the monoculture. The forecrop was rye, which was harvested in the milk-wax phase of seed maturity for animal feed or harvested for grain, depending on the cultivation variant provided for sorghum. There were two terms of sorghum sowing accordingly: in mid-May (as a main crop) and in the end of June (as a second crop). Sorghum seeds were sown at the following level of fertilization (in kg∙ha−1): 120 N, 60 P2O5, 100 K2O and 30 MgO in the amount of 10 kg∙ha−1. The surface of the experimental plot was 50 m2 each and for every variety it was repeated four times a year. The cultivation was carried out in accordance with the principles of good agricultural practice. The row spacing was 40 cm. After sowing, the herbicide Afalon was applied in the amount of 1 kg∙ha−1. Since plants have shown a few green sprouts, no chemical plant protection products were used. The weeds were removed mechanically at the initial stage of growth.
During the growing season, the dates of sowing, germination, appearance of stems, panicles, flowering were noted. Under the climatic conditions of Greater Poland, the tested varieties do not produce fully mature seeds. The number of plants for 1 m2 in each plot was measured after sowing and immediately before harvesting. Also, the height of randomly selected 10 plants in each plot was checked before harvesting. Sorghum was harvested in the last days of September, in the early stages of seed maturity, onset of the first autumn frost. During the biomass harvest, sorghum straw samples were collected from each plot. Then, a reference amount of raw material was taken from the mixed samples of each variety to obtain and determine bioethanol.
2.2. Bioethanol Production Process
2.2.1. Pretreatment
The chemical pretreatment of the sorghum biomass was performed by using 1.5% sodium hydroxide after 5 h treatment at 90 °C. After a 5-h incubation, the process was stopped by filtration of the biomass suspension under reduced pressure. The filtered biomass was rinsed with portions of distilled water until a neutral pH was reached. The filtrate prepared in this way was used as a substrate in enzymatic hydrolysis and ethanol fermentation.
2.2.2. Enzymatic Hydrolysis
In a subsequent step enzymatic hydrolysis of the sorghum biomass was carried out. Flashzyme Plus 200 (AB Enzymes, Darmstadt, Germany) in an amount of 30 FPU g−1 was added to the 10% hydrolysate. The process was carried out at 50 °C, for 72 h, at a pH value of 4.2.
2.2.3. Ethanol Fermentation
The sorghum biomass after enzymatic hydrolysis was sterilized by autoclaving in 121 °C for 15 min and used as a fermentation medium. The medium was inoculated with Saccharomyces cerevisiae cells from the inoculum culture (1 × 107 cfu/mL).
S. cerevisiae Meyen ex E.C. Hansen yeast (commercial strain Ethanol Red) was obtained from Lessafre Fermentis (Lille, France). The microorganisms were stored on yeast extract peptone dextrose (YPD) medium with the addition of 1% yeast extract (w/v), 2% peptone (w/v), 2% glucose (w/v) and 2% (w/v) agar-agar kept at the temperature of 4–8 °C.
The fermentation process was performed in a 250 mL Erlenmeyer flask and incubated in a rotary shaker at 140 rpm for 120 h at 37 °C, and pH value 4.8, with fermentation tubes. The prepared hydrolysate was adjusted to the desired pH with 10% sulfuric acid and 10% sodium hydroxide.
2.3. Analytical Methods
The chemical components of sorghum biomass were evaluated i.e., cellulose acc. to TAPPI T17 m-55, hemicellulose as the difference holocellulose acc. to TAPPI T9 m-54 and cellulose, as well as lignin acc. to TAPPI T13 m-54 [
35,
36,
37], moisture by the dried (gravimetric) method, and ash according to Tappi DIN 51,731 [
38].
The content of ethanol concentration was determined by high performance liquid chromatography (HPLC, Agilent Technologies 1200, Santa Clara (California), USA). For this purpose, a Rezex ROA column (Phenomenex, Aschaffenburg, Germany) was used, at 40 °C, in 0.005 N H2SO4, as an eluent at the flow rate of 0.6 mL∙min−1.
2.4. Calculations
Ethanol yield from 100 g of raw material Y
s (g/100 g of raw material) was calculated according to the Equation (1) [
39]:
where: Et—amount of ethanol in 1000 mL of tested sample (g), M—mass of material weighed in 1000 mL fermentation sample (g).
Then, based on the ethanol yield from 100 g of raw material, the amount of ethanol in L per ton of straw dry matter (L∙Mg−1) was calculated, and on the basis of straw yield, the ethanol yield per hectare (m3∙ha−1) was determined.
2.5. Statistical Analysis
All experiments, both for main and second crop were carried out in a completely randomized design with four independent repetitions. The Shapiro-Wilk W-test was used to test for normality. If the hypothesis of normality was not rejected, an analysis of variance (ANOVA) was performed and Tukey’s post hoc test was used. Otherwise, the nonparametric Kruskal-Wallis (K-W) rank sum test and multiple comparisons of mean ranks were calculated. p-values indicating whether the general hypothesis tests are statistically significant were marked with a standard significance code (*** for a significance level of 0.001; ** for a significance level of 0.01; * for a significance level of 0.05).
General sample testing using the principal component analysis (PCA procedure) was done by a singular value decomposition of the scaled data matrix.
In order to measure the relationships between the observed variables, Pearson’s correlation coefficients were determined for the linear association and Spearman’s rank correlations were determined for the monotonic association. The strength of the correlation was described using a scale suggested by Evans [
40]. Statistical analysis was performed using R software (R version 3.5.2, Microsoft Corporation, Washington (District of Columbia) USA). It should be added that PCA analysis used yield and bioethanol data for fresh and dry straw mass.
4. Conclusions
Based on the obtained results it can be concluded that Rona 1 and Sucrosorgo 506 straw had higher ethanol values harvested as a main and also second crops compared to Santos variety, while for all of the three tested cultivars higher amounts of ethanol per ton from the main crop than from the second crop were observed. Experiments and statistical analysis of the results showed that Santos variety is different from the other analyzed sorghum varieties.
Moreover, on the basis of the study concluded that Sucrosorgo 506 is the most effective cultivar for the production of lignocellulosic ethanol in both periods of cultivation. Furthermore, Rona 1 was characterized by the highest resistance to drought as a main crop, both in terms of yield and the amount of obtained bioethanol.
The production of bioethanol from sorghum biomass is possible in temperate climate and it does not compete with the production of food due to the possibility of growing sorghum after rye.
The results of study indicate the possibility of effective sorghum cultivation as a second crop and utilization of its biomass for the bioethanol production. However, techno-economic analysis should be performed to popularize the sorghum cultivation in Central and Eastern Europe.
Due to breeding progress, new sorghum varieties, which give fully matured grains in a temperate climate, have already appeared. Therefore, it is planned to determine in the future the effectiveness of obtaining bioethanol from waste biomass of this sorghum cultivars after deseeding.