Study On Ultrasound-assisted Ethanol Fermentation Of Saccharomyces Cerevisiae And Its Mechanism

Ethanol is an organic compound that has been widely used in food industry,agriculture,chemical industry,medical and health care.The producing methods of ethanol mainly include synthetic and fermentation technologies.Due to its low costs and easy process,the fermentation method is widely used.Currently,Saccharomyces cerevisiae is mostly used in ethanol fermentation because of its strong ferment ability,short growth cycle,and the easiness for large-scale cultivating.With the worldwidely growing demand for ethanol,promotion technologies development of ethanol generation by Saccharomyces cerevisiae fermentation is of great research significance.It was found that low-intensity ultrasound could significantly promote the ethanol fermentation of Saccharomyces cerevisiae,promote the proliferation of Saccharomyces cerevisiae and increase the ethanol production.However,the current research on ultrasonic assisted ethanol fermentation is limited to shake flask culture,which can not meet the needs of industrial application and development.Therefore,it is necessary to explore the ultrasonic assisted ethanol fermentation technology and its mechanism at the fermentation tank level.In this study,the components of the fermentation medium were optimized,and the ultrasonic treatment conditions were screened.A 7.5 L automatic ultrasonic-assisted fermentation tank was used to perform external-circulation ultrasonic treatment on the fermentation broth of Saccharomyces cerevisiae,and the changes of fermentation indexes before and after the ultrasonic treatment were measured.Through scanning electron microscopy,intracellular Ca²⁺concentration and extracellular nucleic acid protein content changes,the effect of ultrasound on cell membrane permeability were explored.By employing of transcriptomics and metabolomics analysis the molecular level mechanism of ultrasound's promotion effect on ethanol production,and the effects of ultrasound on the activities of key enzymes in glycolysis,the central metabolic pathway of glucose decomposition,were explored to reveal the influence mechanism of ultrasound on ethanol fermentation.The research content and results of this article are as follows:(1)Study on the ultrasonic fermentation process of Saccharomyces cerevisiae ethanol fermentation.A single factor experiment was used to optimize the composition of the fermentation medium to obtain the optimal medium composition.The optimal concentrations of medium ingredients for the maximal fermentation process were:255.07 g/L of glucose,25.67 g/L of yeast powder,20.31 g/L of tryptone,2 g/L of ammonium sulfate,10 g/L of potassium dihydrogen phosphate,0.5 g/L of Mg SO₄,and0.15 g/L of calcium chloride.The ultrasonic treatment conditions were screened,and the optimal ultrasonic treatment parameters were obtained with ethanol output as an indicator:ultrasonic frequency was 28 k Hz,power density was 180 W/L,and treatment time was 24 h.The optimized fermentation medium and ultrasonic treatment conditions were used to process the ethanol fermentation process of Saccharomyces cerevisiae.Compared with the non-sonicated group,the final ethanol production was 10.48%(v/v),which was 30.79%higher than that of the non-sonicated group.After treatment,the dry weight of the bacteria reached 10.24 g/L,which was 24.10%higher than that of the non-sonicated group.The residual sugar consumption rate of the ultrasonic-treated group was 22.89%higher than that of the non-sonicated group.In terms of cell membrane permeability,the increased rate of extracellular nucleic acid and protein of Saccharomyces cerevisiae in the ultrasonic treatment group increased by 8%and 12%,respectively.Scanning electron microscope observation revealed that the surface of Saccharomyces cerevisiae after ultrasonic treatment appeared wrinkles.Using an inverted fluorescence microscope,it was found that the fluorescence intensity of intracellular Ca²⁺was enhanced after ultrasonic treatment,indicating that ultrasonic waves increased the permeability of the cell membrane of Saccharomyces cerevisiae.(2)The effect of ultrasonic treatment on the gene expression of Saccharomyces cerevisiae by using transcriptomics analysis.61 differentially expressed genes(47 up-regulated and 14 down-regulated genes)were identified.There are a total of 44differential genes annotated in GO.A total of 44 differential genes were annotated into146 GO entries.GO functional enrichment cluster analysis and KEGG metabolic pathway analysis show that there are significant differences in the three categories of biological processes,cell functions,and molecular functions before and after ultrasonic treatment.In the process of serine catabolism,gluconeogenesis,ascus wall assembly,protein folding,ATP binding,metal ion binding,helicase activity,pyridine phosphate binding,cell composition,cell metabolism,genetic information processing,environmental information processing and cellular processes,etc.All aspects are involved in the response of Saccharomyces cerevisiae cells to ultrasound.(3)The effect of ultrasonic treatment on the fermentation metabolites of Saccharomyces cerevisiae was explored through metabolomics analysis,and the effects of ultrasound on the activities of key enzymes in glycolysis were analyzed.Through LC-MS non-targeted metabonomics identification,498 differential metabolites were identified,of which 283 metabolites were significantly up-regulated and 215metabolites were significantly down-regulated,indicating that ultrasound treatment induces more metabolite synthesis than inhibition.The difference is conducive to the synthesis of ethanol.KEGG enrichment analysis showed that Saccharomyces cerevisiae cells could respond to the stress of ultrasound through multiple metabolic pathways such as amino acid and sugar metabolism,helping Saccharomyces cerevisiae cells to proliferate rapidly.The production of ethanol is affected by the efficiency of glycolysis.A gene-metabolism network was constructed using transcriptomics and metabolomics to analyze the gene expression jointly and metabolites of Saccharomyces cerevisiae.It was concluded that ultrasound has three main effects on Saccharomyces cerevisiae:pyruvate metabolism,amino acid metabolism,and carbohydrate metabolism.And clarified the change mechanism of glucose transport enzyme,cell membrane composition and reactive oxygen species clearance caused by ultrasonic stress of Saccharomyces cerevisiae cells.In order to explore the effect of ultrasonic treatment on glycolysis,the three key enzymes responsible,particularly hexokinase,phosphofructokinase,and pyruvate-kinase,were measured.Compared with the non-sonicated group,the activity of hexokinase,phosphofructokinase,and pyruvate-kinase increased by 59.02%,87.27%,and 109.05%,respectively.The results showed that the regulation of glycolysis efficiency by ultrasound was an important factor to promote ethanol fermentation.