The study used an aqueous extract prepared without chemical or enzymatic pretreatment. Fresh bagasse collected from sugarcane juice vendors in Haldia, West Bengal, was cut into approximately 1-inch pieces, and 5 grams were added to 200 millilitres of distilled water. The suspension was heated at 95–100 °C for 30 minutes, cooled to room temperature and filtered through muslin cloth to remove coarse solids. Previous measurements cited by the researchers placed the extract’s reducing sugar concentration at approximately 0.764 grams per litre and its total soluble sugar concentration at about 10 grams per litre. Individual sugars such as glucose, fructose and sucrose were not separately identified in the present study, so the Lane–Eynon result was a bulk estimate of total soluble sugars rather than a detailed sugar profile. Fermentation took place at pH 4.0, 30.20 °C and 6% inoculum for 22.06 hours, using conditions established in the researchers’ earlier work.

Fourier transform infrared spectroscopy indicated that the extract contained functional groups associated with cellulose, hemicellulose and lignin. A broad band between 3200 and 3400 inverse centimetres was attributed to O–H stretching, a feature near 2920 inverse centimetres to aliphatic C–H stretching, and bands between 1000 and 1150 inverse centimetres to C–O and C–O–C vibrations associated with glycosidic linkages. These observations supported the presence of carbohydrate-rich structural material in the extract, although FTIR alone could not identify or quantify individual sugars. The extract supported some fermentation, but its limited ethanol production suggested that carbohydrate-associated components alone were insufficient to sustain high productivity under the tested conditions without additional nutrients.

Researchers screened potential supplements using a one-variable-at-a-time design while holding the fermentation conditions constant. Glucose, fructose and sucrose were each tested at 1% weight per volume as carbon sources; ammonium chloride, ammonium sulfate and urea were each tested at 1% as nitrogen sources; and magnesium sulfate, ferrous sulfate, calcium chloride, potassium chloride and sodium chloride were evaluated at 0.1 milligrams per millilitre as mineral sources. Each condition was tested in triplicate, and treatment means were compared by one-way analysis of variance followed by Fisher’s least significant difference test. An untreated, non-supplemented control was not included contemporaneously in these screening experiments, so the results established relative performance among the tested supplements rather than supplying a direct baseline for every comparison.

Sucrose performed best during the initial carbon-source screen, producing a mean ethanol concentration of 0.605 ± 0.009 grams per 100 millilitres at 1%, compared with 0.558 ± 0.01 for glucose and 0.584 ± 0.005 for fructose. When sucrose concentration was varied, ethanol increased from 0.520 ± 0.006 grams per 100 millilitres at 0.5% to 1.771 ± 0.105 at 4%. Production then declined at higher concentrations, reaching 1.633 ± 0.105 grams per 100 millilitres at 7%. The authors associated this non-linear response with possible osmotic stress or altered metabolic regulation, but they did not directly measure transporter activity, invertase function or metabolic flux, so the physiological basis for the pattern remained unconfirmed. The 4% sucrose level selected for subsequent nitrogen and mineral screening produced more ethanol than any other concentration tested in that series.

Among the nitrogen sources, ammonium sulfate gave the highest mean ethanol concentration when combined with 4% sucrose: 1.598 ± 0.189 grams per 100 millilitres, compared with 1.253 ± 0.071 for ammonium chloride and 0.425 ± 0.468 for urea. Ammonium sulfate and ammonium chloride did not differ significantly in that screening, but ammonium sulfate was carried forward because it had the higher mean. Varying its concentration produced a maximum of 1.897 ± 0.03 grams per 100 millilitres at 0.5%, while further additions reduced ethanol production. Potassium chloride similarly outperformed the other minerals under the conditions tested, yielding 1.598 ± 0.05 grams per 100 millilitres at 0.1 milligrams per millilitre, versus 1.242 ± 0.03 for magnesium sulfate, 1.138 ± 0.1 for calcium chloride, 1.092 ± 0.13 for ferrous sulfate and 0.966 ± 0.12 for sodium chloride.

The three strongest supplements—sucrose, ammonium sulfate and potassium chloride—were then optimized together through response surface methodology based on a central composite design. The researchers fitted a second-order polynomial model over 20 experimental runs and assessed it using analysis of variance, lack-of-fit testing, coefficients of determination and residual diagnostics. The model was significant, with an F value of 39.38 and P below 0.0001, and explained 97.26% of the observed variation in ethanol concentration. Its adjusted R-squared value was 0.9479, the predicted R-squared was 0.8972 and the lack-of-fit test was not significant, with F = 0.52 and P = 0.7530. The experimental coefficient of variation was 5.76%, while the adequate-precision value of 15.959 exceeded the cited adequacy threshold. Residuals were approximately normally distributed, and the Box–Cox analysis did not indicate that response transformation was needed.

Within the tested domain, ethanol followed curved responses to all three concentrations rather than increasing indefinitely. Sucrose performed best near 4%, ammonium sulfate near 0.5%, and potassium chloride generally performed better toward the upper part of the concentration range examined by the model. None of the pairwise interaction terms was statistically significant: sucrose by ammonium sulfate had P = 0.6630, sucrose by potassium chloride P = 0.5066, and ammonium sulfate by potassium chloride P = 0.4171. The response surface therefore attributed the main pattern to individual and quadratic effects rather than detectable interactions between nutrient pairs. The fitted model identified 4.06% sucrose, 0.5% ammonium sulfate and 0.11 milligrams per millilitre potassium chloride as the optimum, predicting 2.42 grams of ethanol per 100 millilitres with a desirability value of 0.941.

Triplicate validation at that optimum produced 2.345 grams of ethanol per 100 millilitres, equal to 96.9% of the model prediction and within its reported 95% confidence interval of 2.33–2.51 grams per 100 millilitres. Using the extract’s initial total-sugar concentration of 10 grams per litre and the added sucrose, the researchers calculated an initial supplemented sugar concentration of 5.0 grams per 100 millilitres. With a theoretical factor of 0.511 grams of ethanol per gram of sugar, that input corresponded to 2.555 grams of ethanol per 100 millilitres, making the observed value 91.8% of the theoretical estimate. On a direct mass comparison, the ethanol represented 46.9% of the initial sugar mass. Neither figure directly measured sugar consumption, however, because residual sugars, biomass, carbon dioxide and other fermentation products were not quantified after fermentation.

The results do not establish industrial competitiveness. The study’s ethanol concentration of 23.45 grams per litre was higher than the 19.9 grams per litre reported for liquid-hot-water-pretreated bagasse in the paper’s comparison, but it was below values of 44.5 and 61.3 grams per litre reported for more intensive integrated and soda-pretreatment processes. The work was conducted at laboratory scale and did not include a techno-economic assessment, downstream nutrient treatment, scale-up or a complete carbon balance. The authors also acknowledge that model validation occurred only at the predicted optimum, without multi-point testing or calculation of mean absolute error and root mean square error. The experiment therefore provides laboratory evidence that nutritional optimization can improve fermentation of a low-severity bagasse extract, rather than proof of a commercially ready waste-to-ethanol process.

Overall, the experiments show that targeted nutrient management can overcome part of the nutritional limitation of minimally processed aqueous sugarcane bagasse extract. Sucrose supplied additional fermentable carbon, ammonium sulfate supplied nitrogen, and potassium chloride supplied a mineral component, with each supplement displaying a distinct concentration-dependent response. Optimizing the three together raised the experimentally measured ethanol concentration to 2.345 grams per 100 millilitres without adding chemical or enzymatic pretreatment to the extraction step. The study did not directly track sugar consumption, carbon distribution or economic feasibility, and it did not use high-performance liquid chromatography to distinguish individual sugars. Further work involving residual-sugar measurements, individual-sugar profiling, complete carbon accounting, multi-point model validation, larger-scale fermentation and techno-economic evaluation would be required to determine whether the strategy can translate into an efficient industrial process.

Subject of Research: Nutritional optimization of Saccharomyces cerevisiae fermentation using aqueous sugarcane bagasse extract for bioethanol production

Article Title: Nutritional optimization of aqueous sugarcane bagasse extract for enhanced bioethanol production by Saccharomyces cerevisiae MTCC 180

Article References: Ray, S., Mukherjee, A., & Ghosh, U. (2026). Nutritional optimization of aqueous sugarcane bagasse extract for enhanced bioethanol production by Saccharomyces cerevisiae MTCC 180. Frontiers in Industrial Microbiology, 4, Article 1971432. https://doi.org/10.3389/finmi.2026.1971432

Image Credits: AI Generated

DOI: 10.3389/finmi.2026.1971432

Keywords: sugarcane bagasse, bioethanol, Saccharomyces cerevisiae, nutritional optimization, response surface methodology, fermentation, sucrose, ammonium sulfate, potassium chloride, waste valorization, Nutritional, optimization