Study On Lactic Acid Fermentation By Bacillus Coagulans With Mixed Carbon Source Based On Quantitative Metaboletome And Transcriptome

Bacillus coagulans is of great interest in the lactic acid fermentation industry because of its high culture temperature and low pH tolerance,open fermentation and high optical purity L-lactic acid production.Using different neutralizers may lead to variations in the metal ions present in medium during lactic acid fermentation,which may impact the physiological metabolism of the bacteria.The industrial lactic acid fermentation by B.coagulans mainly uses corn starch hydrolysate as raw material.Due to the carbon catabolite repressive effect(CCR),residual sugars(isomaltose and trehalose,etc.)after glucose depletion lead to lower lactic acid yield,longer fermentation cycle and greater cost of post-purification.Therefore,studying the effect of neutralizers on metabolism and the metabolic characteristics and mechanisms of mixed carbon sources during lactic acid fermentation of B.coagulans can help optimize the lactic acid fermentation process and improve the efficiency of carbon source utilization.In this paper,we first established an analytical procedure applicable to quantitative metabolome of B.coagulans.We compared the quenching and extracting methods of the bacterium by calculating the leakage rate and recovery rate,and established a method to obtain intracellular metabolites by quenching with 60%cold methanol and then extracting with boiling ethanol.According to 13C-labeled metabolites isotope dilution mass spectrometry principle,the standard curves for the quantitative analysis of 19 amino acids,6 organic acids and 11 phosphate sugars were established.We further investigated the effects of different neutralizers on the autolysis of B.coagulans ATCC7050 during lactic acid fermentation with mixed carbon sources in the switch of carbon source substrate.(1)Compared the effects of glucose with 10 other carbon sources on lactic acid fermentation,the results showed that glucose+trehalose displayed the most obvious hierarchical utilization effect,and the experimental condition were determined for the further studies.(2)By analyzing the differences in fermentation profiles between shake flasks and 5 L bioreactor,it was experimentally verified that the neutralizer was the key factor causing the difference in cell autolysis after glucose depletion during the fermentation process.NaOH as neutralizer caused massive cell autolysis after glucose depletion,and there was obvious secondary growth in the late stage of fermentation.Using Ca(OH)2 as neutralizer could significantly reduce the cell autolysis,and cell density(OD620)was always maintained at a high level,which significantly improved the trehalose utilization and lactate generation rate in the late fermentation period.(3)Investigating the effects of metal ions and osmotic pressure on the cell autolysis of B.coagulans under starvation,it was found that Ca2+and Mg2+ alleviated the cell autolysis.(4)Finally,combined with quantitative metabolome and transcriptome analysis,it was found that Ca(OH)2 as neutralizer alleviated cell autolysis by up-regulating genes related to cell division(ftsA、ftsE、ftsX、ftsZ and sepF)and central carbon metabolism and down-regulating genes related to cell wall hydrolysis(walK and cwlJ)after glucose depletion when the bacterium was in a starved state.Finally,we studied the lactic acid fermentation characteristics of B.coagulans ATCC7050 with mixed carbon source.(1)Multi-parameter collection and analysis of the fermentation process of glucose+trehalose was carried out on a 5 L bioreactor,and the experimental results showed that the fermentation process could be divided into glucose consumption phase(stage Ⅰ),organic acids consumption phase(stage Ⅱ)and trehalose consumption phase(stage Ⅲ).At stage Ⅰ lactic acid production rate was 2.04 g/L/h,sugar-acid conversion rate was 0.89 g/g,and very little by-products(acetic acid and acetoin)was producted.At stage Ⅱ organic acids were consumed as carbon source.At stage Ⅲ lactic acid production rate was 0.19 g/L/h,sugar-acid conversion rate was 0.71 g/g,and more by-products(acetic acid and acetoin)were producted.(2)The combined analysis of the metabolome and transcriptome data showed that there was a clear metabolic shift in the cell before and after glucose depletion.At stage Ⅰ the metabolism of cell demonstrated to be homolactic fermentation with EMP as the main metabolic pathway,while at stage Ⅲ to be heterolactic fermentation with more carbon source allocating to the PKP pathway.(3)Meanwhile,ccpA and hprK,key genes of inducer rejection effect were upregulated at stage I,indicating that B.coagulans triggered the CCR effect in the presence of mixed carbon source.