2.4. Characterization of Stalk Residues of Chiquere and Gebabe Varieties
This protocol is extracted from research article:
Bioethanol Production from Stalk Residues of Chiquere and Gebabe Varieties of Sweet Sorghum
Int J Microbiol, Feb 18, 2021; DOI: 10.1155/2021/6696254

The empty evaporated dishes were weighed on an electronic balance before adding the sample. Then, 3 g of the sample powder was placed in an empty evaporated dish. The weighed samples were placed in an oven at 105°C for 3 hours to vaporize the water. Then, the evaporated dish was transferred into a desiccator and allowed to cool before determining its weight. The dried samples were weighted three times, and the mean of weighted sample was determined [23]. Moisture content was calculated using the following formula:

where W1 = weight of the evaporated dish with sample before drying (3 g); W2 = weight of the evaporated dish and sample after drying.

The empty crucibles were weighed on an electronic balance before adding the sample. Then, 3 g of the sample was added in it. The sample and the crucible were placed in a muffle furnace for 30 min at 600°C. The crucible was removed from the furnace and placed in a desiccator to cool, and then the weight was reweighed until constant weight was obtained [23]. The volatile content was determined as follows:

where W1 = original weight of the sample (3 g); W2 = weight of the sample after cooling.

6 g of the oven dried sample was weighed and placed into Trimble, which is plugged with filter paper and placed in a Soxhlet extraction tube. An exhaustive ethanol extraction was completed in 6 hours using the Soxhlet method. The residue was then dried in the oven at 105°C for 4 hours at a constant weight and cooled at room temperature in a desiccator and reweighed. The weighed difference between the varieties of sweet sorghum sample before and after leaching was the amount of the extractives [23]. The extractive contents in the raw sweet sorghum variety sample were calculated as follows:

where W1 = weight of the origin sample (5 g); W2 = weight after drying.

1g of extractive-free sample was placed in the conical flask and 12 ml of 72% sulfuric acid was added. 100 mL of distilled water was added to the mixture. After distilled water addition, the mixture was boiled for 2 hours and cooled at room temperature. The insoluble material (lignin) was filtered by using Whatman paper (no. 42). The lignin was washed with distilled water until neutralized with H2SO4 and then oven dried at 105°C for 10 hours at a constant weight and cooled down in a desiccator and weighed [7, 24]. Lignin content was calculated as follows:

where W1 = oven dried sample (1 g); W2 = extracted residues.

2 g of the extractive-free dried sample was placed in a conical flask and 150 mL of NaOH solution (20 g/L) was added to the sample. The mixture was boiled for 3 hours, filtered, and washed with distilled water to remove the NaOH solution. The residue was then dried in an oven for 5 hours at 105°C at a constant weight, cooled at room temperature in a desiccator, and reweighed (W2). The difference between the sample weight before and after treatment was the hemicellulose content [23]. The hemicellulose content sweet sorghum variety was calculated as follows:

where W1 = weight of the sample before drying (2 g); W2 = weight of the sample after drying.

The cellulose content was calculated in different ways, assuming that extractive hemicellulose and lignin are the only components of the entire sweet sorghum variety stalk residues [23] as follows:

where WC, WH, WL, and WE are cellulose, hemicellulose, lignin, and extractive content, respectively.

The total reducing sugar contents of Chiquere and Gebabe varieties were determined through phenol sulfuric acid method by taking anhydrous glucose as the standard. To prepare stock solution, 0.5 g of glucose was dissolved into 100 ml of distilled water in a volumetric flask. Six test tubes were prepared for standard solution; one test tube for blank and the five test tubes were for glucose standard of 20 mg/ml, 40 mg/ml, 60 mg/ml, 80 mg/ml, and 100 mg/ml. 1 ml of phenol solution (5 g/100 mL) and 1 ml of the sample were added into the test tubes. Then, 5 ml of 96% concentrated sulfuric acid was added rapidly to produce a good mixture. The test tubes were allowed to stand for 10 min to develop [13]. The absorbance of the sample was measured at 490 nm by using a UV-visible spectrophotometer using glucose as the standard [25]. A graph was plotted against the standard to get the total sugar.

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