Hiplex

Fruit by-product samples (apple, grapes, and banana) were collected in sterile containers and refrigerated at 4 °C until used to isolate ethanol-producing yeasts. Isolation was accomplished through serial dilutions up to 10⁻⁶ using the spread plate method and glucose yeast extract (GYE) agar plates for 48 h at 30 °C. The desired isolates based on their ethanol producing potential were analyzed and selected using HPLC. The colonies were introduced into a flask of 250 mL containing 50 mL GYE broth and kept at 28 °C with 150 rpm for ethanol production^{20 (link)}. The samples were collected over a 6 h period and analyzed using High Performance Liquid Chromatography (HPLC) (Agilent, HiPlex, California, USA), with a RID detector set to 55 °C and an Agilent HiPlex H column (300 mm × 7.7 mm, 8 m) set to 60 °C. The mobile phase (5 mM H₂SO₄) flow rate was 0.7 ml/min. Before HPLC, the mobile phase was degassed and filtered through a 0.22 µm nylon membrane filter (Millipore, MA). The yeast with the highest ethanol yield under mesophilic conditions was chosen. An inverted light microscope (40X) was used to examine the morphology of promising strains (Nikon Eclipse TS2, USA) and the culture was identified at a facility of Institute of Microbial Technology, Chandigarh.

For the quantitative measurement of monosaccharides and ethanol obtained during the production process, high performance liquid chromatography (HPLC) (Agilent, HiPlex, Santa Clara, California, USA) was used. For determination, a refractive index detector (RID) was used at 55 °C. The mobile phase was 5 mM H₂SO₄ at a flow rate of 0.7 ml/min in an Agilent HiPlex H analytical column (300 mm 7.7 mm, 8 m) operating at 60 °C.