Efficient Production Of2,3-butanediol And Its Regulation In Bacillus Amyloliquefaciens

Interest in this bioprocess has increased remarkably because2,3-butanediol (2,3-BD) has a large number of industrial applications, and microbial production will alleviate the dependence on oil supply for the production of platform chemicals. Additionally,2,3-BD has potential applications in the manufacture of printing inks, perfumes, fumigants, moistening and softening agents, explosives, plasticizers, foods, and pharmaceuticals. So far, absolutely unbeatable in efficient production of2,3-BD are Klebsiella pneumoniae, K. oxytoca, Enterobacter aerogenes and Serratia marcescens. It is important to note that mainly class2(pathogenic) microorganisms are employed in the2,3-BD fermentation. Industrial-scale fermentation requires obeying safety regulations, which implies that class2microorganisms are unwanted in such applications. Therefore, an urgent need for class1microorganisms (safe) is pronounced. Such microorganisms have been reported as2,3-BD producers, however, the ef ficiency of the production was much too low for an economic process. In the current study, a GRAS (Generally Recognized As Safe) strain of Bacillus amyloliquefaciens producing2,3-BD designated as B10-127was isolated in our lab. The strain B10-127produced2,3-BD effectively under the condition of20%glucose (quality concentration), showed a high-glucose tolerance. In this current study,2,3-BD production by was B. amyloliquefaciens was studied by using traditional fermentation regulatory methods and modern metabolic engineering technique. The detailed work was introduced as following:1. Isolation and identification of2,3-BD high production and safe microorganismsGenerally, only a strain with the abilities of high-glucose tolerance and effective glucose utilization can be an excellent candidate for use in the production of2,3-BD at an industrial scale. Based on this perspective, we designed a screening culture containing300g/L of glucose. The enrichment process was to select the interested strains, which grown quickly to increase its proportion in the mixed culture using glucose as the carbon source. And it was also combined with voges-proskavr test. When action (precursor of2,3-BD) was detected in the enrichment broth, it might imply that2,3-BD producing microorganisms existed in the culture. After purified several times, several strains which could tolerate glucose up to300g/L and produce2,3-BD effectively were isolated from a soil sample collected from grassland. One isolate was further identified as B. amyloliquefaciens B10-127by its16S rRNA gene sequence and physiological biochemical analysis. Under a unoptimized condition, the titer of2,3-BD were52.2g/L with a2,3-BD productivity of0.68g/(L h).2. Optimization of medium and process parameters for the production of2,3-BDThe optimization of flask fermentation conditions and cultural medium composition on2,3-BD production have been studied. The results showed that the optimal culture conditions included initial pH of6.5, cultivation at37oC, inocultun size of6%(v/v) and shaking speed of150r/min. Corn steep liquor, soybean meal and ammonium citrate were found to be the key factors in the fermentation according to the results obtained from the Plackett–Burman experimental design. The optimal con-centration range of the three factors was examined by the steepest ascent path, and their optimal concentration were further optimized via response surface methodological approach and determined to be31.9,22.0and5.58g/L, respectively. Under optimized conditions, biomass increased by14.6%, the fermentation time was shorten from76to48h, the titer of2,3-BD increased by21.4%, the productivity of2,3-BD increased by91.3%, and acetoin decreased by34.4%, when compared with the results obtained under unoptimized conditions.3. Effects of corn steep liquor on2,3-BD and acetoin productionIt was found that the initial concentration of corn steep liquor (CSL) have remarkable effects on not only2,3-butanediol (2,3-BD) and acetoin production, but also on the ratio of2,3-BD to acetoin. Acetoin reductase catalyzes the conversion of acetoin to2,3-BD. Results obtained from tests performed under low CSL levels were compared to that obtained under high CSL levels. When a high concentration of CSL was supplemented, cell growth was improved, acetoin reductase (ACR) was stimulated, the concentration of2,3-BD increased by55.6%, acetoin decreased by69.0%, and the ratio of2,3-BD to acetoin increased by3.99-fold. Compared to the BD/AC ratio obtained in low CSL levels, the BD/AC ratio was much higher when CSL levels were high. This indicates that the NADH-oxidizing branch of acetoin to2,3-BD was enhanced, resulting in the decrease of the NADH/NAD+ratio.4. Enhanced2,3-BD production by overexpressing a NADH/NAD+regeneration systemIn the2,3-BD metabolic pathway, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the conversion of3-phosphate glyceraldehydes to1,3-bisphosphoglycerate with concomitant reduction of NAD+to NADH while acetoin reductase (ACR) catalyzes the conversion of acetoin to2,3-butanediol with concomitant oxidation of NADH to NAD+. So GAPDH and ACR are bound up with regeneration of cofactors. In this study, we firstly introduced extra copies of GAPDH/ACR enzymes into the GRAS strain B. amyloliquefaciens and studied their effect on2,3-BD fermentation. It was found that no difference of NADH and NAD+levels was observed between mutant and parent strain. While for mutant strain glucose fluxes were redistributed, in the NADH-dependent pathways, yield of2,3-BD of was16.7%higher and yields of by-products acetoin, lactate and succinate were separately60.9%,39.0%and25.9%lower than that of parent strain.5. The experiments in a5-L biorcaetorThe pH and oxygen supply are the very important variables in the2,3-BD fermentation. Batch fermentative production of2,3-BD by B. amyloliquefaciens was investigated using various oxygen supply methods though varying agitation speed. Based on the analysis of three kinetic parameters including specific cell growth rate (μ), specific glucose consumption rate (qs) and specific2,3-BD formation rate (qp), a multi-stage agitation speed control strategy, aimed at achieving high concentration, high yield and high productivity of2,3-BD, was proposed. At the first4h, agitation speed was controlled at350r/min, subsequently agitation speed was raised to400r/min until16h, and then, agitation speed was reduced to350r/min. Finally, the maximum titer of2,3-butanediol reached72.8g/L with the productivity of2.60g/(L h), which were8.2%and23.8%over the best results controlled by constant agitation speeds. In a pulse fed-batch fermentation, combining with a two-stage pH control strategy, the2,3-BD concentration and productivity were significantly improved to133.2g/L and2.78g/(L h), respectively.6. Enhancement of waste glycerol consumption with beet molasses co-fermentationThe production of2,3-BD from glycerol was inhibited when compared with the equivalent cultures performed on sugars. In the current study, Bacillus amyloliquefaciens was first reported to exhibit a remarkable producing potency of2,3-BD from biodiesel-derived glycerol, and the beet molasses was employed as co-substrate. Surprisedly, the molasses addition stimulated significant increases of2,3-BD production, and simultaneously reduced the duration of fermentation. At the beginning of fermentation the molasses addition enhanced the productivity of2,3-BD, and molasses fed during the fermentation increased the conversion rate of2,3-BD. In fed-batch fermentation,15g/L of molasses were added to the bioreactor before inoculation, and after6h, a solution containing80%glycerol and15%molasses was then fed into the bioreactor. The2,3-BD concentration, conversion, and productivity were improved significantly to83.3g/L,0.42g/g, and0.87g/(L h), respectively. To our knowledge, these results might hit a new record on2,3-BD fermentation from biodiesel-derived glycerol.