The Research On Whey Fermentation And Ethanol Tolerance Of Thermoanaerobacterium Calidifontis Rx1

Thermophilic anaerobic bacterium has a wide range of substrates for its use,and it has the ability to use polysaccharides,oligosaccharides and a variety of monosaccharides(pentaose,six carbon sugar)for the production of ethanol fermentation,and is currently a research hotspot in the field of bioethanol.In this study,Thermoanaerobacterium calidifontis Rx1 and its mutant strain T.calidifontis Rx1 △ack were studied.The characteristics of ethanol production from whey were studied,and the optimal ethanol fermentation conditions were optimized.At the same time,the mechanism of ethanol tolerance was studied by the distribution of metabolites and the activity of key enzyme in carbon center metabolism.1.Ethanol metabolism in whey using Rx1 strain and optimization of its fermentation conditions Thermoanaerobacterium calidifontis Rx1 and T.calidifontis Rx1 △ack whey fermentation analysis.Whey is mostly not used as a by-product of cheese production.In this study,the production process was studied by using Rx1 and Rx1 △ack fermented whey to produce ethanol.Firstly,the single factor experiment preliminary confirmed that the main factors affecting the ethanol fermentation process are: inoculation amount,lactose concentration,fermentation pH and fermentation temperature.Through the orthogonal experiment design,the end point of fermentation pH,substrate utilization,ethanol yield and ethanol yield were used as indicators.Finally,the optimal conditions for ethanol fermentation were determined as substrate concentration 15 g/L,pH value 8.5,and fermentation temperature.It is 60°C and the inoculation amount is 8%.Under optimized fermentation conditions,the yield of ethanol was 4.633 g/L,which was 0.318 g/g,which was increased by 229% compared to the unoptimized 1.407 g/L;the yield of lactic acid was reduced from 1.520 g/L to 0.505,which was not optimized.g/L;Acetic acid production changes little,from 0.743g/L under unoptimized conditions to 0.603g/L;substrate utilization increased from 86% to 97%.2.Effect of Acetyl kinase Knockout on Ethanol Tolerance of Rx1 This study found that the ethanol tolerance of the mutant Rx1 Δack strain was increased by 2% compared to the wild-type strain.Analysis of different ethanol volume fraction conditions,Rx1 and Rx1 △ack product distribution and carbon center metabolism key enzyme activity,provide a theoretical basis for the biochemical mechanism of thermophilic anaerobic bacterium ethanol tolerance.Adding exogenous ethanol had a great influence on the yields of wild-type lactic acid,acetic acid,and ethanol.The ethanol-free volume fractions were 2.011 g/L,1.078 g/L,and 1.796 g/L,respectively,and the production of exogenous ethanol decreased.27%,36%,and 58%;mutant strains due to the knockout of the acetate kinase gene,the addition of exogenous ethanol had little effect on it,but lactic acid is slightly increased,not much,compared to 1.124 g without ethanol volume fraction /L,increased by 14%,47%,62%,and 63%,respectively;the ethanol yield of the mutant strain was 1.884g/L without ethanol concentration,and the ethanol yield was 1%-3% ethanol volume fraction.The increase was increased by 1%,18%,and 16%,respectively,and decreased by 6% under the condition of 4% ethanol fraction.Compared to the condition without adding exogenous ethanol,the wild type Rx1 significantly increased the LDH enzyme activity under the condition of adding exogenous ethanol.The wild type had the highest PK enzyme activity at the 3% ethanol volume fraction,compared with the ethanol-free condition.Under the conditions,the activity of LDH,ADH,and ALDH increased by 150%,130%,and 91%,respectively.There was no significant change in ACK enzyme activity.The PK enzyme activity of the mutant strain was the highest under the condition of exogenous ethanol-free,and the highest enzyme activity was 7.798 U,1.023 U,0.840 U,and 1.043 U,respectively.There was also no significant change in ACK enzyme activity.3.Exploration of the construction of Rx1 strain gene knockout genetics operating system.At present,it is difficult to find stable selection markers under high temperature conditions to construct a suitable targeting vector.Even for high temperature resistant kanamycin,its effective resistance can only be maintained at 48-50°C for about 48 hours,and it can’t be effectively screened only higher.It is necessary to develop a screening marker for an auxotrophic marker gene for positive recombinants that grow at temperature.Due to the need to transform multiple genes,a reusable marker gene needs to be established.The auxotrophic marker gene pyrF can be reused and the method of operation is simple and mature.Orotidine 5’-phosphate decarboxylase is orotidine phosphoribosyltransferase,which affects the synthesis of uracil and can be screened with 5-fluoroorotic acid(5-FOA).We constructed the knockout plasmid PMU1101 of the pyrf gene,expecting to obtain an engineered strain,unfortunately failed to successfully screen positive transformants.We speculate that the main reason may be that the special cell membrane structure of the high-temperature anaerobic bacteria and the quality of T.calidifontis Rx1 complicates the transformation of foreign genes.