Strain Screening And Development For Industrial Acetone-butanol-ethanol (ABE) Production And Its Fermentation Process

Acetone-butanol fermentation is a traditional fermentation, once is fermentation of the world’s second largest, after alcoholic fermentation. In the1950s, the acetone-butanol fermentation is gradually being replaced by the rapid development petrochemical industry. With the increasingly tense of the oil resources, that microbial fermentation with the use of biomass as feedstock can replace petroleum fuels has become a research focus. Acetone-butanol fermentation as an important means of converting biomass into chemical raw materials and fuel is re-attracted wide attention. The traditional Acetone-butanol fermentation is grain fermentation, raw material costs are too high, in order to reduce the cost of raw materials, People a lot further study on the fermentation raw material including crop straw, bran and industrial production waste,etc. These rich resources of raw materials are low prices, and fermentation used of these resources is in favor of protecting the environment. In this paper, acetone-butanol fermentation produces ABE from industrial waste xylose mother liquor and corn steep liquor, but acetone-butanol fermentation yields low solvent and extraction costs are too high, which seriously hampered the development of the industry, so by breeding and fermentation process optimization and other means to improve solvent production is necessary. The main contents of this paper:(1Establishing the method of the product detectionA fast and effective way to detect Acetone-butanol fermentation products was established, fermentation products were detected by using GC1690gas chromatograph, AC-20capillary column and FID (flame ionization) detectors. Detecting conditions were optimized, so the ABE detecting time was shorten to be4.2min, and if the byproduct acetic acid and butyric are detected, the detecting time of individual sample needs8.5min.Isobutanol as the internal standard, we carried on the quantitative analysis of fermentation products by the peak area internal standard method. Drawing the regression curve for each solvent, the five kinds of solvents regression correlation coefficient R2are greater than0.99, which indicates that the As/Ai is good linear correlation with the concentration of the solvent. Repeatability and stability experiments indicate that the relative standard deviation (RSD) of test results is very small. So the measuring method and instrument conditions can quickly and accurately detect fermentation samples and provide protection for screening high-yield strain and the detection of the large-scale fermentation.(2)Saccharification and fermentation of corncob residuesWe pretreated corncob residues with dilute sulfuric acid, dilute NaOH solution or tap water. Corncob residues pretreated with dilute sulfuric acid released the most。 While that pretreated with dilute sulfuric acid or dilute NaOH solution released less sugars after saccharification. Temperature had a great influence on the reaction of the cellulase enzyme. Saccharification was performed at37℃,45℃or55℃conditions, and the results showed that the optimum saccharification temperature was45℃. Laccase added in Saccharification system can inhibit the reaction of the cellulase enzyme and the conversion of cellulose was reduced. Compared with separate hydrolysis and fermentation, simultaneous saccharification and fermentation had higher solvent production.(3) Breeding of high-yielding strainsClostridium beijerinckii ATCC55025was carried on natural breeding, and then the strains L-lwas selected. By the carbon and nitrogen experiments, we determined that the fermentation medium was10%xylose mother liquor and3.5%corn steep liquor without any other ingredients. This reduced the cost of the media and simplified the composition of the medium. L-1as the original strain was mutationed by UV and Low Energy N+Implantation. With10%xylose mother liquor and3.5%corn steep liquor as medium for fermentation in anaerobic bottles, we screened out high-yielding strains. After mutagenesis, solvent production has increased significantly. Butanol is very toxic to cells, and Strains can tolerate up to11g/L butanol.(4) Optimization of the fermentation process After inoculation quantities and feeding experiments in anaerobic flasks, we determined that the optimum inoculation quantities was5%, feeding time was36h, and complementary feeding amount was15g/L. with continuous expansion in liquid culture,bacteria negative mutation was quite quickly, and solvent yields plummeted. Strains passaged four times in a solid medium had no effect on fermentation yield, therefore, The mutant was genetically stable in the solid passaged.Fermenting in5L tank, with solvent production as the main index, the fermentation conditions were optimized:initial pH6.0, temperature37℃, stirring speed100rpm. To verify the above optimization results, the total solvent yield reached21.138g/L under the optimized conditions.