Method for producing hydrogen gas from sweet sorghum

A method for producing hydrogen gas (H2) from sweet sorghum (Sorghum bicolor (L.) Moench) comprising steps performed in the following specific order:

• (i) preparing materials including:
• a sweet sorghum stalk juice ingredient, a sweet sorghum seed powder ingredient, an alpha-amylase enzyme ingredient, a glucoamylase enzyme ingredient, an organic acid ingredient, a dark fermentation microorganism ingredient, and a light fermentation microorganism ingredient;
• wherein the sweet sorghum stalk juice ingredient is obtained by heating the juice from sweet sorghum stalks at 60° C.-70° C. until the Brix level reaches 15-18° Bx;
• wherein the sweet sorghum seed powder ingredient is finely ground to a size of 1-2 mm and having a moisture content of less than 12%;
• wherein the organic acid ingredient is selected from the group consisting of butyric acid, acetic acid, and combinations thereof;
• wherein the dark fermentation microorganism ingredient is obtained by mixing a first increased density solution with a second increased density solution in a ratio of (1-3):(1-3);
• wherein the first increased density solution is prepared by culturing a first microorganism strain on a first growth medium comprising:
• glucose (C6H12O6) at 20 g/L, ammonium sulfate ((NH4)2SO4) at 5 g/L, potassium phosphate dibasic (K2HPO4) at 1 g/L, magnesium sulfate heptahydrate (MgSO4·7H2O) at 0.5 g/L, manganese sulfate monohydrate (MnSO4·H2O) at 0.02 g/L, cobalt chloride hexahydrate at 0.01 g/L, vitamin B12 at 0.0001 g/L, and biotin at 0.000005 g/L;
• wherein the first microorganism strain is selected from a group consisting of Clostridium acetobutylicum (ATCC 824) (with a designated identifier in the gene bank being AE001438.3), and Clostridium acetobutylicum (ATCC 4259) (with a designated identifier in the gene bank being U09978.1);
• wherein the second increased density solution is prepared by culturing a second microorganism strain on a second growth medium comprising:
• cellobiose at 5 g/L, peptone at 5 g/L, yeast extract at 5 g/L, sodium bicarbonate (NaHCO3) at 3.5 g/L, ammonium sulfate ((NH4)2SO4) at 3 g/L, resazurin at 0.001 g/L, L-cysteine hydrochloride at 0.1 g/L, vitamin B12 at 0.0001 g/L, and biotin at 0.000005 g/L;
• wherein the second microorganism strain is Caldicellulosiruptor saccharolyticus Tp8T.6331 (ATCC 43494) (with a designated identifier in the gene bank being NR_036878.1);
• wherein the dark fermentation microorganism ingredient contains the first microorganism strain with a cell density of 109 CFU/mL and the second microorganism strain with a cell density of 109 CFU/mL;
• wherein the light fermentation microorganism ingredient is obtained by culturing a third microorganism strain on a third growth medium comprising:
• glucose (C6H12O6) at 10 g/L, yeast extract at 5 g/L, peptone at 5 g/L, sodium chloride (NaCl) at 5 g/L, magnesium sulfate heptahydrate (MgSO4·7H2O) at 1 g/L, potassium dihydrogen phosphate (KH2PO4) at 1 g/L, and calcium chloride dihydrate (CaCl2·2H2O) at 0.1 g/L;
• wherein the third microorganism strain is selected from a group consisting of Rhodobacter sphaeroides KD131 (KCTC 12085) (with a designated identifier in the gene bank being CP001150.1), and Rhodobacter sphaeroides (ATCC 17029) (with a designated identifier in the gene bank being CP000579.1);
• wherein the light fermentation microorganism ingredient contains the third microorganism strain with a cell density of 109 CFU/ml;
• (ii) creating a first temporary mixture by performing steps (a) to (c), comprising:
• (a) mixing the sweet sorghum stalk juice ingredient with the sweet sorghum seed powder ingredient in a ratio of 1:(0.06-0.15) (w/w) at 70° C., then stirring well;
• (b) following the mixing and stirring step in (a), increasing the temperature to 90° C., maintaining at 90° C. for 3-5 minutes;
• and
• (c) reducing the temperature to 85° C., maintaining at 85° C. for 60 minutes;
• (iii) creating a second temporary mixture by mixing the alpha-amylase enzyme ingredient with the first temporary mixture in a ratio of (0.05-0.2):1000 (w/w), adjusting the pH to 5.5-6.5, then incubating at 90° C.-100° C. for 30-60 minutes;
• (iv) creating a third temporary mixture by performing steps (a′) to (c′), comprising:
• (a′) mixing the glucoamylase enzyme ingredient with the second temporary mixture in a ratio of (0.05-0.2):1000 (w/w), adjusting the pH to 4.0-5.0;
• (b′) incubating at 90° C.-100° C. for 30-60 minutes;
• and
• (c′) cooling to a temperature of 25° C.-30° C.;
• (v) creating a first base mixture by performing steps (a″) to (c″), comprising:
• (a″) adding the additional glucoamylase enzyme ingredient to the third temporary mixture in a ratio of (0.05-0.2):1000 (w/w) and stirring well to obtain a fourth temporary mixture;
• (b″) mixing the organic acid ingredient with the fourth temporary mixture in a ratio of (2-5):1000 (w/w) and stirring well to obtain a fifth temporary mixture;
• and
• (c″) mixing the dark fermentation microorganism ingredient with the fifth temporary mixture in a ratio of (1-2):1000 (w/w) and stirring well to obtain the first base mixture;
• (vi) fermenting the first base mixture under dark conditions at a temperature of 25° C.-30° C. for 22-26 hours to produce a first hydrogen gas and a dark fermented base mixture;
• wherein the pH is controlled at 6.0-6.5 and the dissolved oxygen concentration is controlled to be less than 1%;
• (vii) creating a second base mixture by mixing the light fermentation microorganism ingredient with the dark fermented base mixture in a ratio of (1-2):1000 (w/w) and stirring well;
• and
• (viii) fermenting the second base mixture under light conditions at a light intensity of 1500-2000 lux, a temperature of 25° C.-30° C., a stirring speed of 200-300 rpm, and for a duration of 22-26 hours to produce a second hydrogen gas;
• wherein the pH is controlled at 6.5-7.0.