| With the increase of energy demand,the exhaustion of fossil energy and the aggravation of environmental pollution,developing safe,clean,convenient and sustainable energy(such as solar,wind and biomass energy)has become an urgent problem that needs to be solved.At present,the vast majority of ethanol is produced from the Saccharomyces cerevisiae and Zymomonas mobilis using simple biomass,but they can only utilize fermentable sugars typically released from the hydrolysis of sugar/starchy biomass(such as beets,sugarcane corn and sorghum).By contrast,Thermoanaerobacterium aotearoense has the characteristics of efficient conversion of substrates at high temperature,wide range of substrate utilization,high catabolic activity,short fermentation time and easy recovery of ethanol.It is the dominant species that can use complex lignocellulose to produce bioethanol,but the low ethanol tolerance and various by-products restrict its application.Therefore,it is of great significance toincreasetheethanoltoleranceandthefermentationperformanceof Thermoanaerobacterium aotearoense.Based on the transcriptome data of T.aotearoense SCUT27 under different ethanol concentrations,the pyruvate formate lyase-activating protein gene(pflA),a key enzyme in the formic acid synthesis pathway which was significantly down-regulated under high ethanol stimulation,was selected as the research object.Knocking out of pflA could block the the synthetic pathway of formic acid and distribute more carbon flux to ethanol production.Previously,the pyruvate formate lyase-activating protein gene(pflA)was knocked out and an engineered strain T.aotearoense SCUT27/?ldh/?pflA was obtained in our lab.In this study,the deletion of pflA was confirmed by Southern blot.Although the inhibited growth and reduced acetic acid production of the engineered strain was observed,the yield of ethanol did not increase when modified liquid medium of T.aotearoense(MTC_m)was used as the fermentation medium.However,in the nutrient rich medium(MTC_n),the ethanol concentration and conversion rate of T.aotearoense SCUT27/?ldh/?pflA were increased by12.36%and 20%,respectively.In addition,the yield of acetic acid decreased by 28.81%.T.aotearoense SCUT27/?ldh/?pflA exhibited higher ethanol tolerance than T.aotearoense SCUT27/?ldh and maintained higher activity in the condition of high ethanol concentration in the MTC_n.Free cell fed-batch fermentation showed that the final ethanol concentration and the ethanol productivity of T.aotearoense SCUT27/?ldh/?pflA reached 38.82 g/L and 0.35g/L·h,increased by 49.02%and 9.38%compared with T.aotearoense SCUT27/?ldh.In addition,the acetic acid concentration of the engineered strain was 6.12 g/L,which was 36.77%lower than that of T.aotearoense SCUT27/?ldh.In order to further increase the fermentation performance of T.aotearoense SCUT27/?ldh/?pflA,fed-batch fermentation was carried out with cells immobilized in the fibrous bed bioreactor(FBB).Under this condition,the ethanol concentration reached to45.01 g/L,which was 26.39%higher than that of T.aotearoense SCUT27/?ldh.At the same time,the ethanol productivity of the engineered strain was also increased by 32.35%compared with the parent strain.For the purpose of reducing the toxicity of ethanol on T.aotearoense,ethanol was removed in situ using gas stripping.The key factors in the gas stripping process are ethanol concentration in fermentation broth,feed temperature,gas flow rate,and condensing temperature,and the optimized values we obtained are:25 g/L,55?,1.7 L/min,and 0?,respectively.In order to achieve continuous thermophilic ethanol fermentation,a coupling bioprocess for in situ ethanol recovery coupled with immobilized fed-batch fermentation was established.The ethanol concentration after recovery achieved120 g/L,which would greatly reduce the subsequent purification cost.The combination of genetic engineering and process engineering not only increased the production capacity of ethanol but also reduced the toxicity of ethanol to cells,achieving multi-batch fermentation.In addition,a preliminary study on the use of lignocellulosic hydrolysates for ethanol production by T.aotearoense SCUT27/?ldh/?pflA was conducted.First,the optimum conditions for peanut shells pretreatment were determined by response surface analysis:treatment temperature(121?),the concentration of dilute sulfuric acid(0.03 mol/L),pretreatment time(48 min),and ratio of liquid to material(10.18).The total sugar concentration after pretreatment and enzymatic hydrolysis was 22.57 g/L.The hydrolysis of the corn cob was performed using an optimized method according to the literature,and the total sugar concentration was 28.95 g/L.Then corn cob hydrolyzate(CCH)and peanut shell hydrolyzate(PSH)with a total sugar about 25 g/L were used as the substrates for ethanol production.The ethanol concentration of T.aotearoense SCUT27/?ldh/?pflA were 4.84 g/L and 5.82 g/L,respectively,which were increased by 9.19%and 9.25%compared with T.aotearoense SCUT27/?ldh.In summary,knocking out of pflA can significantly enhance the ethanol production of Thermoanaerobacterium aotearoense at the expense of the reduced acetic acid production.Furthermore,by coupling gas stripping with immobilized fed-batch fermentations,the ethanol productivity of T.aotearoense SCUT27/?ldh/?pflA could be further increased,which provides a new idea for the development of efficient ethanol production by microorganisms. |
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