Production Of 2,3-butanediol From Inulin By Klebsiella Pneumoniae DL-H3

With the depletion of fossil fuels,the preparation of 2,3-butanediol by biological methods has received much attention.The use of non-food crops of Jerusalem artichoke instead of glucose as a fermentation carbon source for biosynthesis 2,3-butanediol is an important research direction.However,the inulin in the tubers of Jerusalem artichoke is difficult to be used directly by microorganisms,resulting in low yield and low production intensity.Therefore,it is important for screening suitable strains and rationally optimizing the fermentation process for biosynthesis of 2,3-butanediol by using inulin as the main carbon source.Firstly,a strain was screened which can directly convert inulin to 2,3-butanediol.In solid culture,the colony was fully developed in short time with smooth and hemispherical surface,and was sticky and easy to form lace.It was identified as Klebsiella pneumoniae by 16S rRNA identification and named DL-H3.Secondly,the catalytic characteristics and influencing factors of inulinase were explored.The inulinase produced by strain DL-H3 was an exo-inulinase mainly located in the cell.The optimal pH and temperature for inulinase was pH 6.0 and 30?C,respectively.In a reaction buffer containing 2%inulin,it was found metal ion K⁺and Mn²⁺can promote the catalytic efficiency of inulinase,while the metabolites of succinic acid and acetic acid can inhibit the catalytic activity of inulinase.The addition of ammonium sulfate to 10 common nitrogen sources maximized the total sugar utilization of the strain DL-H3 to 77.68%.Then,the phenomenon that inulin could not be fully utilized was studied.Under the optimization conditions?pH 6.5,stirring rate 200 r/min,aeration rate 0.2 vvm,addition of16.53 g/L ammonium sulfate to Jerusalem artichoke tuber extract?,the total sugar utilization rate reached 86.98%which was about 20%higher than that at the initial stage.During the fermentation process,the average degree of polymerization of inulin increased from 2.82 to8.08,whereas the inulinase maintained a high activity and reached 1.143 U/ml in the end.The short-chain inulin was preferentially ingested and utilized during fermentation,while long-chain inulin could not be ingested into cells and accumulated gradually,thus the enzymatic hydrolysis reaction was blocked.After that,the full utilization of inulin was realized through the strategy of pre-treatment.Partial degradation of inulin was achieved by a pretreatment method composed of adjusting the initial pH of the fermentation broth to 3.00 and autoclaving.The strain DL-H3 showed tolerance to high concentrations of substrate and consumed most of the pretreated inulin.By optimizing the oxygen supply strategy in batch fermentation,the target product?2,3-butanediol and acetoin?reached 80.83 g/L with a yield of 0.426 g/g sugar and productivity of 2.23 g/?L*h?under the conditions of pH 6.0,stirring rate 250 r/min,aeration rate 0.2 vvm and substrate concentration 202.55 g/L.Theses results indicate the industrial potential of K.pneumoniae DL-H3.Finally,the separation of 2,3-butanediol and acetoin was studied using the salting-out extraction system of ethyl acetate/K₂HPO₄.The results showed that the system was more suitable for the separation of acetoin.When the system was used for salting-out extraction of2,3-butanediol in the fermentation broth produced by the strain DL-H3,the impurities in the fermentation broth caused the emulsification phenomenon to be severe,resulting in poor extraction effect.