Iranian Grape Waste: A High Potential Source for Screening of Bioethanol-Producing Microorganisms

The demand for bioethanol as a renewable energy source is rising. This study screened high-ethanol-producing microorganisms found in grapes to reduce production costs and compete with other fuels. The grapes and their waste samples were collected from Iranian vineyards. Microorganisms were initially screened by growing them in a glucose-enriched culture medium containing ۱۰% ethanol to isolate ethanol-tolerant ones. The bioethanol-producing microorganisms were then qualitatively isolated during fermentation using high-throughput screening (HTS) based on CO۲ production and changing bromothymol blue to green color. Promising strains were selected based on the amount of ethanol production by the CO۲ flow meter. The selected strains were identified using ۱۸S rRNA sequencing and PCR by the S. cerevisiae species-specific primers (ScHO). Finally, the growth of the most promising strain was optimized using the response surface methodology (RSM) in a shaking incubator. A total of one hundred isolates were tested using HTS devices. Out of these, nine strains were quantitatively screened, and SCL-۲۵ and SCL-۶۲ were chosen to continue based on their high ethanol production rate. The ethanol production rate for SCL-۲۵ was ۱۲.۸۶%, while SCL-۶۲ was ۱۴.۳۵%. After molecular characterization, it was confirmed that SCL-۲۵ was ۱۰۰% similar to Saccharomyces cerevisiae, whereas SCL-۶۲ was ۹۹% similar to Saccharomyces cerevisiae. The PCR products amplified by ScHO showed a ۴۰۰ bp band in agarose gel electrophoresis, confirming them as S. cerevisiae. Moreover, isolate SCL-۶۲ showed ethanol production higher than the commercial strain. The RSM optimization results showed that the growth of the SCL-۶۲ strain increased two times at ۳۵ °C, pH ۵.۰, Brix degree ۲۰, and agitation rate ۲۰۰ rpm. Therefore, the SCL-۶۲ strain has the potential to produce efficient and cost-effective bioethanol.

کلیدواژه ها:

Bioethanol ، high-throughput screening ، wild yeast ، CO۲ production ، Grape ، Response surface method