The Screening And Identifying Of Butanol Fermentation Strains

Objective:Based on the physiological and metabolic features of the bacteria producing butanol, an efficient and rapid screening method is established to screen the strains of high capacity of producing butanol from the soil and plant organs. Through making physiological and biochemical experiment,16S rDNA identification on the screening strains, preliminarily determining its taxonomy position.Through the investigation of fermentation characteristics of the screening strains, optimizing its fermentation conditions to further improve the butanol production.Method:The Known bacteria producing acetone and butanol are anaerobic bacteria who can produce spores resistant of high temperature,so kill the bacteria cells and enrich the spores by heat treatment, cultivate the bacteria in an anaerobic environment to eliminate the aerobic bacteria, and do separation and purification by picking single colony from cultivating flat. Through fermenting the single strains, testing the acetone content by the qualitative experiment of acetone, we eliminate the miscellaneous bacteria and the strains weak in solvent production. According to the characteristics of butanol fermentation process in which it produces acid in the prophase and late reduces the acid into acetone and butanol, we design the bromine cresol green plate to identify the ability of producing acid by its fade circle and meilan fading experiment to judge the reductive ability, to get the strains provided with both strong capacity of producing acid and the reductive ability. Through fermenting the small screening strains, and detecting the butanol content in the fermentation liquid by gas chromatography, we get the optimal strains named sat3.First of all, the physiological and biochemical experiment of the sat3,the amplification of its 27f to 1492r section in16S rDNA for sequencing the section is done. Comparison of the sequence to the sequence from the Blast in GeneBank, eight series are got that have the he highest similarity through similar matching. Finally, a system growth tree is constructed through MEGA4.1 software to preliminarily determine its taxonomy position and its fermentation conditions is optimized.Results:1. The strains producing butanol is screened from the soil samples and potato tuber. Through heat treatment enriching spores, acetone qualitative tests detecting acetone, bromine cresol green plate identifying the ability producing acid and meilan fading judging deoxidation ability, three strains are got. By fermenting the three strains and detecting the butanol content of their fermentation liquid, the strains named sat 3 is obtained, whose butanol production is 8.32 g/L.2. By morphology observation, staining reaction and physiological and biochemical experiment on sat3, preliminarily determine that the sat3 belongs to the clostridium. Finally through the amplification of 16SrDNA segment and the comparation of similarity and homology, confirm that the sat3 belongs to clostridium. 3. After optimizing the fermentation conditions of sat3, the sat3 can reach the better butanol yield 9.10 g/L in the initial pH 5.5, com medium concentration of 7% and temperature 37℃.Conclusion:1. Heat treatment method can effectively enrich heat resistant spores produced by acetone butanol bacteria, eliminate the miscellaneous bacteria that can't produce spores, and achieve the purpose of enriching spores.2, Through acetone qualitative test, we can detect whether the acetone exist in the fermentation liquid, and through the comparison of color depth, we can choose the strain that has strong ability of producing acetone.3.Through the fading situation of the bromine cresol green flat, we can identify the strains of strong ability to producing acid, and by the fading situation of the beautiful blue,we can judge the size of the reductive ability of strains.If a strain both has strong ability to produce acid and reductive ability, it has strong solvents production capacity.4. After the preliminary identification and the fermentation conditions optimization to the screening strain, we determined the species of the strains, and its optimal fermentation conditions.