At the National Ethanol Conference 2022, grasses were considered affordable materials for ethanol production since they do not require subsidies, affect diversity, or contribute to deforestation [4] . Grasses have lignocellulosic biomass with a complex structure, where hemicellulose and lignin form a true physical barrier to cellulolytic enzyme penetration. Depending on the variety of grasses, they can contain from 5% to 18% lignin, from 25% to 43% hemicellulose, and from 29% to 50% cellulose [5] , so the processes for ethanol production from grass biomass must be adjusted according to the characteristics of these components [6] . Table 2 shows a list of different grasses used under various pretreatments for ethanol production.

In this regard, considering the lignocellulosic biomass of grasses as a promising raw material for ethanol production, this work presents the use of lignocellulosic biomass from Camalote grass to produce ethanol. Camalote grass (Figure 1) belonging to the Poaceae family, is a perennial, fast-growing, and invasive grass that displaces cultivated pastures, hence considered a weed. Moreover, it is not utilized as livestock feed [13] . It is abundant in the southeast of Mexico, extending to Argentina, Uruguay, and the Antilles, thus holding great potential as a raw material for ethanol production due to its high availability, easy access, and low cost.

1 m² (3.9 kg) of camalote grass (Paspalum fasciculatum Willd) was collected in the vicinity of the Universidad Popular de la Chontalpa, with coordinates 17.959 North, −93.364 West. The grass was longitudinally cut into sizes of 5 ± 1 cm to facilitate handling. Subsequently, it was air-dried for 3 days and then dried at 105˚C until reaching a constant weight.

% m o i s t u r e = ( m i − m f ) 100 (1)

where mi is the initial weight of the plant material (g) and mf is the final weight of the plant material (g).

100 g of camalote grass were placed in a 1 L beaker and 700 mL of acetone were added. It was heated to boiling for 20 minutes with continuous stirring. Subsequently, it was allowed to cool, and the resulting plant material was filtered. It was then dried at 105˚C until reaching a constant weight.

% e x t r a c t i v e s = m i − m f m i × 100 (2)

where mi is the initial weight of the plant material (g) and mf is the final weight of the plant material (g).

To 100 g of the material without extractives, 500 mL of commercial NaClO was added, and heated to boiling for 1 hour. It was then cooled and washed until neutral pH. The resulting material was air-dried for 2 days and subsequently dried at a controlled temperature of 105˚C until constant weight.

The diffraction patterns were collected using a D8 Advance Bruker with CuKα radiation (α = 1.5406 Å and energy 8.047 keV), in the range of 2θ = 7 - 60 with a 0.02 step size and an acquisition time of 5 s/step. The crystallinity percentage was calculated with Equation (2) based on the method reported by Segal (1959) [14] .

CrI ( % ) = ( 1 − I A M I 002 ) × 100 (3)

where I₀₀₂ is the maximum intensity of the crystalline peak at 22˚, and I_AM = is the minimum intensity of the crystalline peak for cellulose I.

10 g of pretreated material were placed in a 250 mL Pyrex flask and 100 mL of a 5% H₂SO₄ solution was added. This procedure was repeated varying the concentration of H₂SO₄ to 10% and 15%. Hydrolysis was carried out at 125˚C and 15 psi for 2 hours. Subsequently, the hydrolyzed juice was separated by filtration. The % Brix measurement was performed using a portable Anpro refractometer, by placing a drop on the prism surface and reading the scale.

An experimental design was conducted by varying the amount of Saccharomyces cerevisiae yeast (Fermentis®) to 0.5, 1, and 2 g 100 mL of hydrolyzed juice was placed in an Erlenmeyer flask, the pH was adjusted to 5 with NaOH 0.1 N, yeast was added, and it was allowed to ferment under anaerobic conditions for 48 hours at a temperature of 30˚C ± 1˚C.

The fermented product was placed in a simple distillation setup and heated to 78 - 80˚C for 3 hours. The % v/v of ethanol was measured using a portable refractometer for alcohol and distilled spirits with a range of 0 - 80% alcohol, by placing a drop on the prism surface and reading the scale.

It was carried out according to the Mexican Official Standard NOM-V-15-S-1980. 25 mL of bioethanol were placed in a porcelain dish, heated in a water bath to dryness, and then transferred to an oven at a temperature of 105˚C for 30 minutes. Subsequently, 50 mL of absolute alcohol was added to the residue left in the dish, and the resulting solution was poured over 250 mL of freshly boiled, cold water neutralized with NaOH 0.1 N and phenolphthalein as an indicator. The resulting solution was titrated with NaOH 0.1 N, using the indicator added to the water for neutralization. The acidity was expressed in mg of acetic acid per 100 mL referred to anhydrous alcohol through Equation (4).

FA = V × N × 60 × 100 M × 100 D . A . R (4)

The fixed acidity (FA) expressed in mg of acetic acid per 100 mL referred to anhydrous alcohol; V is the volume of the NaOH 0.1 N solution spent for the titration of the sample in mL; N is the normality of NaOH; 60 represents the meq of CH₃COOH in mg; M is the volume of the alcohol sample, in mL; and D.A.R. is the actual alcoholic degree of the sample on the Gay-Lussac scale.

With the experimental data of % Brix and % ethanol, the average yield of each treatment was calculated using the RM ANOVA (repeated measures ANOVA) program, using biomass quantity as the fixed factor and H₂SO₄ concentration as the random factor. The size of the arithmetic mean = 3. The average values were compared by applying Analysis of Variance with a 95% Confidence Interval for the mean, and the Tukey statistical test was applied.

In the biomass’s own composition, cellulose, hemicellulose, lignin are present in larger quantities, while other organic compounds know extractives are found in low concentrations in the bark, leaves, needles, exudates, branches, flowers, fruits, and seeds, contributing to the organoleptic characteristics (flavor, odor, color) of the organic material. The amount of extractives varies according to the

species, geographical location, and time of year. Among the extractables are phenolic compounds of molecular weight that are lignin precursors, as well as aromatic aldehydes and ketones. A material balance was carried out during the biomass pretreatment, and Table 3 shows the characterization of the solid fraction of the grass. The values obtained are close to those reported for Maralfalfa grass (Pennisetum glaucum) at cutting frequencies of 90 to 180 days [15] .

During the NaClO treatment, lignin and hemicellulose were removed, as confirmed by X-ray analysis. In Figure 2(a), the diffraction patterns for natural camalote grass are shown. The intensity of the amorphous zone I_AM was located at 2θ = 10.2˚, and the intensity of the crystalline zone I₀₀₂ at 2θ = 22.6˚. In Figure 2(b), the camalote grass with NaClO is shown. The intensity of the amorphous zone I_AM was located at 2θ = 11.2˚, and the intensity of the crystalline zone I₀₀₂ at 2θ = 22.7˚, characteristic signals for cellulose I. The signal at 45.4˚ corresponds to the inorganic part of the cellulose [16] . Natural grass exhibits 42% crystallinity, and after treatment with NaClO, an increase in crystallinity to 50% is observed, attributed to the dissolution of amorphous regions (lignin and hemicellulose) in lignocellulosic biomass [17] .

The degradation of cellulose to glucose was carried out through acid hydrolysis varying the concentration of H₂SO₄. The sugar content is commonly expressed as % Brix since there is a relationship with the sugar content. Table 4 shows the results of % Brix for each of the treatments. Higher conversion of cellulose to reducing sugars was achieved with concentrations of 5% and 20%. The use of diluted acid concentrations minimizes the formation of hydroxymethylfurfural, which is a toxic compound for yeast during the fermentation process [18] .

The analysis of variance showed that there are statistical differences (p < 0.05) among the H₂SO₄ treatments (Figure 3). The treatment with a 20% acid concentration presented the highest average of 17% Brix, which was statistically superior to the treatments with 5% acid concentration of 12.3% Brix and 10% acid concentration of 10% Brix. The % Brix values obtained are within recommended ranges as a high content of soluble solids increases osmotic pressure affecting the yeast Saccharomyces cerevisiae, resulting in low ethanol production [19] .

Table 5 shows the ethanol yield obtained relative to the amount of fermentable sugars obtained in each treatment with sulfuric acid.

Figure 4 shows that all the averages are different from each other, according to the Tukey statistical test. The treatment with a 5% of H₂SO₄ concentration produced the highest average ethanol value of 10.3%, the treatment with 10% of H₂SO₄ produced 9.3% of ethanol, and the treatment with 20% of H₂SO₄ produced 6.3% of ethanol. It is established that higher acid concentrations decrease ethanol production, as it increases the formation of toxic agents for yeast [20] .

It is predicted for 2024 that ethanol consumption will be 134.5 billion liters, with Brazil being the largest producer, followed by the United States, the EU, and China [21] . Based on the results, the camalote grass produces 0.10 g ethanol/g of biomass, values comparable to the yields reported by Antonopoulou (2020) for lawn grass, which produced 0.108 g of ethanol/g of biomass; and by Yosuda (2013) for Napiergrass, which produced 0.174 g of ethanol/g of biomass. In 1 m², 3900 g of grass is obtained, therefore, 390 g ethanol/m² would be produced. So, ethanol production from camalote grass would contribute 3900000 liters of ethanol per hectare. This yield is economically viable considering that the cost of the raw material is zero.

Yieldethanol / m 2 = ( 3900gpasto 1m 2 ) ( 0 .10gethanol 1gpasto ) = 390gEthanol / m 2

Yieldethanol / 1h = ( 390gethanol 1m 2 ) ( 10000m 2 1h ) = 3900000gEthanol / h

Thereby, grasses are promising raw materials to substitute sugar cane and corn for ethanol production, as they are not affected by climatic changes. Unlike sugar cane and corn, which only thrive in tropical climates and their crops are affected in semi-arid regions, hail, and frosts.

Ethanol, to be used as fuel in gasoline, must meet certain chemical properties, including fixed acidity, which is associated with corrosiveness, taking into account the material from which the car engine is made. According to the United Nations, fuel ethanol must have a maximum acidity (acetic acid) of 30 mg/L, established by ASTM D1613 standard. The ethanol obtained from Camalote grass in the three studied H₂SO₄ treatments showed a total acidity value as acetic acid of 1.8 mg/L (Table 6), acceptable values for the specifications set by International and Brazilian Standards [22] .