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

Document Type : Research Articles

Authors

• Robabeh Golestani ¹
• Masoumeh Mehraban Sang Atash ²
• Najme Gord Noshahri ³
• Mohammad Zare Mehrjerdi ⁴
• Yaser Yousefpoor ^{5, 6}

¹ Department of Food Science and Technology, ACECR Kashmar Higher Education Institute, Kashmar, Iran

² Department of Food quality and safety, Academic Center for Education Culture, and Research (ACECR)-Khorasan Razavi Branch, Mashhad, Iran

³ Industrial Microbial Biotechnology Department, Research Institute for Industrial Biotechnology, Academic Center for Education, Culture, and Research (ACECR)-Khorasan Razavi Branch, P.O. Box 91775-1376, Mashhad, Iran

⁴ Agriculture Faculty of Shirvan, University of Bojnord, Bojnord, Iran

⁵ Department of Medical Biotechnology, School of Paramedical Science, Torbat Heydariyeh University of Medical Sciences, Torbat Heydariyeh, Iran

⁶ Research Center of Advanced Technologies in Medicine, Torbat Heydariyeh University of Medical Sciences, Torbat Heydariyeh, Iran

https://doi.org/10.22067/jcmr.2024.88136.1095

Abstract

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 10% ethanol to isolate ethanol-tolerant ones. The bioethanol-producing microorganisms were then qualitatively isolated during fermentation using high-throughput screening (HTS) based on CO₂ production and changing bromothymol blue to green color. Promising strains were selected based on the amount of ethanol production by the CO₂ flow meter. The selected strains were identified using 18S 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-25 and SCL-62 were chosen to continue based on their high ethanol production rate. The ethanol production rate for SCL-25 was 12.86%, while SCL-62 was 14.35%. After molecular characterization, it was confirmed that SCL-25 was 100% similar to Saccharomyces cerevisiae, whereas SCL-62 was 99% similar to Saccharomyces cerevisiae. The PCR products amplified by ScHO showed a 400 bp band in agarose gel electrophoresis, confirming them as S. cerevisiae. Moreover, isolate SCL-62 showed ethanol production higher than the commercial strain. The RSM optimization results showed that the growth of the SCL-62 strain increased two times at 35 °C, pH 5.0, Brix degree 20, and agitation rate 200 rpm. Therefore, the SCL-62 strain has the potential to produce efficient and cost-effective bioethanol.
 

Keywords

• Bioethanol
• high-throughput screening
• wild yeast
• CO2 production
• grape
• response surface method

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