Study On The Selecting Of High Acetone And Butanol Producing Strains And Its New Fermentation Technology

A rapid and efficient method of detecting acetone,butanol and ethanol was acquired in this paper.Its detecting conditions were showed as below: AT-FFAP capillary column(30 m×0.25 mm×0.33μm);carrier gas flow rate: N₂ 35 mL/min,H₂ 35 mL/min,air 350 mL/min;column temperature:180℃;injection temperature:180℃;detector temperature:200℃,injection volume:1μL.Under these conditions,detecting time needs 5 min.A high acetone and butanol producing strains,named B513,was obtained through repeated mutation of microwave,UV and ion beam. ABE yield reached 18.27 g/L,in which butanol concentration arrived 11.43 g/L.The production of ABE was increased 100.77%in comparison with that of the parent strain.Protoplast preparation and regeneration conditions were studied on the osmotic pressure stabilizer,sucrose concentration,enzyme concentration, enzyme hydrolysis time.The most appropriate conditions were as follow: sucrose,as a stabilizer,concentration was 0.3 M;cell culture time was 16-18 h;lysozyme concentration was 1 mg/mL;enzymatic hydrolysis temperature was 37℃;enzymatic hydrolysis time was 40 min.Under these conditions,the rates of protoplast preparation and regeneration were 70% and 99%respectively.Strains B513,D01 were inactivated by UV and heat treatment separately.80 fusants were gained by biparental inactivation. ABE yield was lower and maximum solvent production was 10.31 g/L. There was also not shortening on fermentation time.In addition,ABE fermentation conditions were studied on fermentation temperature,heat treatment time,inoculation amount, fermentation broth pH,oscillations,and fermentation time.The optimum fermentation conditions were as follow:fermentation temperature was 38℃;heat treatment time was 1.5 min in boiling water;inoculation amount reached 3%;corn mash maintained the original pH;every 10 h shaked once when mash cap appeared;fermentation time was 70 h.Under these conditions,ABE yield can reach 18 g/L.Research on cell immobilization using orthogonal test has obtained the best immobilization conditions.The concentrations of PVA,SA,CaCl₂ were 9%,1.1%,4%,individually;immobilized time was 4 h;solvent yield reached 10.82 g/L.Study on immobilized cell fermentation coupled extraction shows that 70-80 h was the first suitable feed time in fed-batch fermentation.Author tried to enhance butanol production through knocking-out ptb gene,but results showed that the PCR amplified fragment containing Kan gene couldn't be successfully transformed into Clostridium acetobutylicum. The result of enzyme activity in ABE metabolic pathway influenced by different pH showed that butanol synthesis enzymes can not be completely induced by low pH.So cutting off butyrate branch metabolic pathway of ABE fermentation through gene knockout technology is unfavorable to butanol production,which could not produce solvent.