Simultaneous Sccharification And Ethanol Fermentation From Jerusalem Artichoke By Kluyveromyces Marxianus

Kluyveromyces marxianus is characterized by its thermal tolerant feature,and it is also fast growing and able to utilize broad range of substrates.Most importantly,it has the ability to produce inulinase,which enables direct ferementation of ethanol using inulin,the main carbohydrate in Jerusalem artichoke.In this study,a yeast strain of K.marxianus YX01 was selected for ethanol production using Jerusasalem artichoke.The extracellular inulinase production by K.marxianus YX01 was optimized and 57 U/mL inulinase was produced in shake flask culture,with the medium composition:inulin 40,yeast extract 4,peptone 4 and urea 1 g/L,under the culture conditions:pH 5.0,30℃and 150 rpm for 120 h.The optimal temperature and pH for the inulinase to hydrolysize inulin were 55℃and 5.0,but the enzyme was validated to be stable at the temperature as high as 60℃and the range of pH from 4.6 to 5.2.The activity ratio of the inulinase toward inulin and sucrose was 1.7 and 8%ethanol showed no significant effect on its activity.The growth and ethanol fermentation of K.marxianus YX01 were studied using inulin as substrate.The activity of inulinase,which attributes to the hydrolysis of inulin,was monitored in both shake flask culture and bioreactor.The optimum seed culture time is 24 h,and the pH of medium is 4.6.The optimum temperatures were 38℃for growth and inulinase production, and 35℃for ethanol fermentation.Aeration was not necessary for ethanol fermentation with the K.marxianus YX01 from inulin.Furthermore,the impact of aeration and substrate concentration was studied through batch fermentation in the 2.5 L bioreactor,and the results indicated that the average ethanol fermentation time was decreased at the aeration rates of 50 mL/min and 100 mL/min,but higher ethanol yield was achieved under non-aeration conditions with more substrate directed to ethanol production.The ethanol concentration of 92.2 g/L was obtained with the substrate containing 235 g/L inulin,and the ethanol yield was calculated to be 0.436,equivalent to 85.5%of its theoretical value.The fresh Jerusalem artichoke tubers grown in salina and irrigated with 25%and 50% seawater mixed with fresh water were further examined for ethanol fermentation with the K. marxianus YX01,and a higher ethanol yield was achieved for the Jerusalem artichoke tuber irrigated with 25%seawater.Furthermore,the dry meal of the Jerusalem artichoke tubers irrigated with 25%seawater was examined for ethanol fermentation at three solid concentrations of 200,225 and 250 g/L,and the highest ethanol yield of 0.439,or 86.1%of the theoretical value of 0.511,was achieved for the slurry with a solid concentration of 200 g/L.Finally,Jerusalem artichoke grown in salina and irrigated with seawater was fermented without sterilization treatment to further improve the economic competitiveness of the fermentation process,and 83.75 g/L ethanol was obtained with the substrate containing 280 g/L dry Jerusalem artichoke meal,with an ethanol yield of 0.432,indicating the Jerusalem artichoke could be an alternative feedstock for grain-based fuel ethanol production. An efficient high performance liquid chromatographic(HPLC) method for simultaneous determination of ethanol,sugar and organic acids in the K.marxianus YX01 culture broth was developed.Aminex HPX-87H organic acids analysis column was used and the mobile phage was 0.01 mol/L sulfic acid solution with the flow rate of 0.5 ml/min.The components of the culture broth containing glucose,fructose,ethanol,acetic acid,glycerol,lactic acid were detected with UV and RID dual detectors.The main by-products of the ethanol fermentation were determined to be lactic acid,glycerol and acetic acid.This method ensured rapid and accurate quantification of the metabolites of the K.marxianus YX01 in the ethanol fermentation from Jerusalem artichoke,and provided basis for the future metabolic flux analysis as well.Fragments of the lactic dehydrogenase gene(LDH) gene sequence from K.marxianus YX01 were cloned using degenerate primers and touch-down PCR.Kam gene conferring G418 resistance was inserted into the clone vector,and the LDH gene of the K.marxianus YX01was disrupted by homologous recombination.Cell growth and ethanol fermentation of the disruptant T₄ were investigated,and the results showed that the LDH activity of T₄ was 75%of that of the control under growth conditions and 54%of that of control in the fermentation process,and the concentrations of ethanol,glycerol,acetic acid and lactic acid in T4 were decreased,but the production of succinic acid production increased.The growth of T₄ was deficient comparing with the control,and the inulinase activity of T₄ was decreased 20-30%.However,due to its low biomass yield,the final specific ethanol productivity of T₄ was 11.25%higher that of the control strain(22.64 g/L/g(dcw) comparing to 20.35 g/L/g (dcw)).Improvement of the inulinase activity and stable expression of inulinase gene is beneficial to improve the ethanol productivity.To this end,inulinase gene INU with its native promoter was amplified from K.marxianus YX01 by PCR,and integrative vector with the HO flanking sequences was constructed.The integration vector was linerized by Not I and then transformed into K.marxianus YX01.The inulinase activity and ethanol fermentation performance of the transformant K/INU1 were investigated.The inulinase activity in K/INU1 doubled that of the control,and the fermentation time was shortened from 84 h in the control strain to 72 h in K/INU1,with similar final ethanol titer in the two strains.Using raw Jerusalem artichoke flour,fed batch culture was performed,and it was revealed that the fermentation rate of K/INU1 is faster than that of the wild strain,with similar final ethanol titer.Multicopy integration of INU will be an efficient way to further improve the ethanol fermentation performance of K.marxianus YX01.