A High-Throughput Screening Procedure (Py-Fe3+) for Enhancing Ethanol Production by Saccharomyces cerevisiae Using ARTP Random Mutagenesis

Abstract

Saccharomyces cerevisiae is an important microbial organization involved in ethanol synthesis. Mutant strains that can withstand multiple pressures during this process are critical to the industrial development of biofuels. In this study, a dual high-throughput screening method of Triphenyl-2H-tetrazoliumchloride (TTC)-based macroscopic observation and the reaction of ferric nitrate with pyruvate (or pyruvate radical ion) in fermentation broth was used. Using this, an S. cerevisiae mutant library that could tolerate 381 g/L sucrose was established by ARTP random mutation and adaptive evolution to select the best strain; its ethanol yield was increased by an additional 20.48%; and the sucrose utilization rate was 81.64%. This method is specific to the selection of strains with increased ethanol production.

Key role of K+ and Ca2+ in high-yield ethanol production by S. Cerevisiae from concentrated sugarcane molasses

“…Thus, S. cerevisiae GJ08 was subjected to ARTP mutagenesis and ALE under varying circumstances. Previously described [ 2 ] high-throughput approaches of triphenyl‑2 H‑tetrazoliumchloride (TTC)‑based macroscopic observation and the reaction of ferric nitrate with pyruvate (or pyruvate radical ion) in fermentation broth (Py-Fe 3+ ) were used to screen the target strains (Table 1 ).…”

“…According to our previous research results, the yeast with active cell growth has a stronger ethanol metabolism capacity, and its ethanol production is inversely proportional to the content of the precursor pyruvate. Therefore, TTC (triphenyl-2 H-tetrazoliumchloride) and Py-Fe3 + -based screening was carried as described in previous work [ 2 ]. So, after 24 h of cultivation, 20 mL of TTC solution was introduced to react for 5 min, and the yeast strains with the earliest red and darkest color were selected.…”

“…Recently, bioethanol has attracted much attention because it can achieve green biosynthesis. Apart from being a fuel with high octane number that can be mixed with existing energy substances, in this era of constant threats to human health, ethanol is also used widely as a medicine and food additive [ 1 , 2 ]. Bioethanol is synthesized primarily via fermentation by Saccharomyces cerevisiae using various substrates such as glucose, sucrose, and starch.…”

“…Mutagenesis is an important means of tolerance engineering and is a highly promising technique for improving ethanol production of S. cerevisiae , especially under high-gravity fermentation conditions [ 21 , 22 ]. In the study reported herein, we applied ARTP (atmospheric and room-temperature plasma) mutagenesis, ALE (adaptive laboratory evolution), and high-throughput screening (Py-Fe 3+ ) [ 2 ] to obtain strains with robust performance against high sugar concentration (400 g/L total fermentable sugars (TFS)), high temperature (37℃), high alcohol level (10%(v/v)), and of 16 g/L K + , 8 g/L Ca 2+ , 16 g/L&8 g/L K + &Ca 2+ , and high sugarcane molasses (300 g/L TFS), and then ethanol production was performed in molasses broth containing 250 g/L TFS, and omics were studied. Herein, to the best of our knowledge and based on experimental verification, the major restricting factors of S. cerevisiae for high-concentration ethanol fermentation from sugarcane molasses are reported for the first time.…”

Ethanol Synthesis by <em>Saccharomyces cerevisiae</em>: Summary, Discovery, and Prospects

“…With the deepening of people's understanding of microorganisms, ethanol, as the product of microbial fermentation, has become one of the oldest forms reflecting the application of microbial biotechnology [1]. Today, the use of ethanol is no longer limited to the beverage; the high octane number of ethanol means that can be mixed with existing fuel sources and is an important part of the solution to the energy crisis and environmental problems [2].…”