You can use HPLC to analyze the ethanol production in your fermentation. For example: Article Use of post-harvest sugarcane residue for ethanol production

Hi,

You can use either GC FID or HPLC.

GC FID: alcohols present in the spent media were estimated by Gas chromatograph (GC, Agilent Technology, 7890A, U.S.A) equipped with a flame ionization detector (FID) and capillary column (Nitroterephthalic-acid-modified polyethylene glycol (PEG) column). The FID operational temperatures of the injection port, detector, and programmed column were 220 °C, 240 °C and 130–175 °C, respectively. A mixture of hydrogen and air (air - 40 mL min−1 and H2 - 400 mL min−1) at a flow rate of 30 mL min−1 was used for flame generation Article Biological hydrogen production via dark fermentation: A holi...

HPLC: A Waters high-performance liquid chromatography (HPLC) system (Waters e2695 Separation Module, Waters Corporation, Milford, MA, USA) with a 2414 refractive index detector and Aminex HPX-87H column (300 × 7.8 mm, 9 µm particle size; Bio-Rad Laboratories, Hercules, CA, USA) was used for the quantification of glucose, xylose, arabinose, acetic acid, furfural, 5-HMF, levulinic acid, formic acid and ethanol. Samples were eluted with 5 mM H2SO4 at a flow rate of 0.6 mL/min at 65℃. An Aminex HPX-87P (300 × 7.8 mm, 9 µm particle size; Bio-Rad Laboratories, Hercules, CA, USA) column was used for the quantification of sucrose, glucose, and fructose, with samples eluted with Milli-Q water (18.2 MΩ) at 85 °C and 0.6 mL/min. The RI detector was maintained at 30℃ (https://doi.org/10.1016/j.fuel.2024.134254).

THANKS

If you are using water-lock (or glycerol lock) flasks you can estimate the ethanol based on weight loss as the CO2 escapes and then calculating back. Best to confirm with actual measurements at the end though. There are HPLC methods, but also enzymatic assays if you don't have HPLC or GC or other equipment.

Depending on the volume of your biomass, a cheap method would be to use a distillation process by heating the mixture and cooling it down with a condenser to isolate and collect the clear liquid, which is 96% ethanol.