| There are many problems in fermentation industry,such as large water consumption,high energy consumption,heavy pollution and insufficient comprehensive utilization of resources.And as the fermentation scale expands,these problems will be increasingly prominent.In order to thoroughly solve the problem of energy and water saving and realize the comprehensive utilization of wastewater,this laboratory has put forward the theory of fermentation ecological engineering in recent years,and established the ethanol-methane fermentation process.The process can significantly shorten the time of ethanol fermentation,but the quality of reuse water is the key factor affecting the effect of ethanol fermentation.It was found that the low concentration of acetic acid in the recycled water could promote the ethanol fermentation,which was mainly manifested as increasing the yield of ethanol and decreasing the yield of glycerol.In addition,when urea,the non-preferred nitrogen source,was the only nitrogen source during the operation of the ethanol-methane fermentation process,Saccharomyces cerevisiae grew better than ammonium as the only nitrogen source,and the effect was good(high yield of ethanol and low yield of glycerol).Therefore,in this paper,the research topic was that the low-concentration of acetic acid and urea can promote ethanol fermentation,and the mechanism of improving ethanol fermentation can be studied by using microbial physiology,RNA-seq technology and metabolomics technology.The specific research results are as follows:1)The fermentation rate of mixed water is higher than that of tap water.The fermentation time in tap water was generally ended at 56 h,while the fermentation time in mixed water was generally ended at 32 h,which was 42.86%shorter than that of tap water.When the acetic acid concentration was 30 mM(1.8 g/L)and 60 mM(3.6 g/L),the fermentation speed was higher than that of tap water,but when the acetic acid concentration was 90 mM,the fermentation speed was lower than that of tap water.This indicated that when acetic acid concentration was greater than 60 mM,it had an obvious inhibitory effect on fermentation,while when the concentration was lower than 60 mM,it had a promoting effect.Ethanol fermentation with urea as nitrogen source can be completed within 42 hours,while with ammonium sulfate as nitrogen source,it takes 56 hours to complete it.Moreover,when urea was used as nitrogen source,the yield of ethanol was higher and the yield of glycerol was lower than that of ammonium sulfate.2)Although low concentration of acetic acid increased the pH difference between intracellular and extracellular,it did not affect the intracellular pH.The presence of low acetic acid concentration increased the intracellular ATP content and reduced the amount of NADH needed to be oxidized,thus reducing the production of glycerol.Compared with the control,intracellular trehalose increased by 32.27%when acetic acid concentration was 60 mM.When the acetic acid concentration was 90 mM,the intracellular trehalose concentration was increased by 51.72%compared with the control.The accumulation of trehalose may be to protect cellular proteins and cell membranes from the effects of acidification.When the acetic acid concentration reached 90 mM,the cells began to rupture and seriously affected the budding.When the concentration of acetic acid was 60 mM,the cell germination was affected and the germination rate was reduced.However,when the concentration of acetic acid was 30 mM,there was no significant difference in cell morphology compared with the control.In conclusion,low concentration of acetic acid would not cause cell membrane damage,but would stimulate S.cerevisiae to increase ethanol production per unit of cell.3)When ammonium sulfate was used as the nitrogen source,the pH in the fermentation process was lower than that in the same period when urea was used as the nitrogen source,and the pH of the fermentation broth remained below 3.0 after 4.5 h.The intracellular ATP(0.36 mg/g DCW)with ammonium was much higher than that with urea as nitrogen source(0.16 mg/g DCW).Meanwhile,at the logarithmic growth stage,the intracellular NADH(0.22 mg/g DCW)with urea was 69.23%higher than that with ammonium as the nitrogen source(0.13 mg/g DCW).Thereherefore,excess NADH inhibits ATP synthesis.The intracellular pH of the cell with urea as the nitrogen source was 7.0,which was higher than the intracellular pH of the cell with ammonium sulfate as the nitrogen source was 6.2,that is,the cells with ammonium sulfate as the nitrogen source showed acidification phenomenon.The enzyme activity of 3-phosphoglycerine dehydrogenase with ammonium as nitrogen source was higher than that with urea as nitrogen source,which was consistent with the results of fermentation with urea as nitrogen source producing less glycerol.Using ammonium sulfate as a nitrogen source increased the membrane potential of yeast cells,and the increase of membrane potential meant the increase of yeast mortality.4)RNA-seq technology was used to investigate the changes of global gene transcription level under the two conditions.Under the condition of low concentration acetic acid stimulation,there were 31 common significant differentially expressed genes at the three time points,among which 10 genes were up-regulated and 20 genes were down-regulated.Genes of oxidation-reduction with significant difference accounted for 75%,while down-regulated genes were mainly related to glycolysis.Ydr210w-d,ypr158w-b and ydr261c-d were up-regulated in different time periods,which was a manifestation of the stimulation of low concentration acetic acid.The up-regulation of REX4 gene is the key and prerequisite factor for S.cerevisiae to respond to the stimulation of low acetic acid concentration.The up-regulation of GRC3 gene means that the binding ability of GRC3 to RNA can be enhanced,The down-regulation of ENA1 transcription level changes the intracellular and extracellular salt ion concentration.5)Compared with urea,when ammonium sulfate was the only nitrogen source,the genes related to cell growth,such as ERR1,ERR2 and ERR3,were up-regulated.ERR2p and ERR3p convert 2-phosphoglyceric acid to phosphoenolpyruvate,so ammonium sulfate enhances the metabolic pathway for converting 2-phosphoglycerate to phosphoenolpyruvate.SSQ1 gene encoding molecular chaperone DnaK is closely related to the environment of cell growth.The significant up-regulation of this gene indicates that the growth environment of cell has changed a lot.The up-regulation of SPO20 gene may be a stress response of yeast cells to low pH growth environment.The SMP1 gene encodes a Hogl-dependent transcription factor,SMP1 interacts with Hogl,and SMP1 is phosphorylated in a Hogl-related manner under osmotic stress.It was speculated that the up-regulation of this gene was related to the increase of glycerol yield when ammonium sulfate was used as nitrogen source.6)According to the metabolomics analysis,most organic acids involved in the tricarboxylic acid cycle were significantly decreased in the logarithmic growth period under the stimulation of low concentration acetic acid.Except citric acid content slightly increased,the other organic acids all showed a decreasing trend,and it is worth noting that acetyl-coenzyme A showed a decreasing trend.The results indicate that the low concentration of acetic acid can weaken the tricarboxylic acid cycle.Meanwhile,the metabolic pathways of glycerol and glutamate were weakened.In particular,the down-regulation of glycerol triphosphate is the direct reason of glycerol decline induced by acetic acid stimulation at low concentration.7)Compared with urea,ammonium sulfate as a nitrogen source causes the content of most organic acids involved in the tricarboxylic acid cycle to significantly decrease in the logarithmic growth period.Except that the content of succinic acid increased slightly,the other organic acids all showed a downward trend.The results showed that the use of ammonium sulfate as a nitrogen source weakened the cycle of tricarboxylic acid.When ammonium sulfate was the nitrogen source,there was no significant change in aspartic acid and leucine.Compared with ammonium sulfate,using urea as a nitrogen source weakens the metabolic pathway of glycerol.Therefore,when ethanol fermentation was carried out with urea as nitrogen source,the by-product glycerol was less. |
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