通气量和菊粉浓度对克鲁维酵母乙醇发酵的影响

马克斯克鲁维酵母能够利用集成生物加工技术发酵菊芋生产乙醇,具有非粮燃料乙醇生产潜力.文中研究了该技术中的两个关键因素(通气量和底物浓度)对于K.marxinaus YX01乙醇发酵过程和菊粉酶活性的影响.研究结果表明,底物浓度对乙醇得率影响不大,底物浓度为250 g/L时,发酵终点乙醇浓度为84.74 g/L,但乙醇得率由低浓度50 g/L的86.4％(理论值),降为84.7％.通气能够加速K.marxinaus YX01的乙醇发酵过程,但降低了乙醇得率,当底物浓度为250 g/L时,乙醇得率由不通气的84.7％降为1.0 vvm时的73.3％.随底物浓度的升高及通气量的降低,K.marxinaus YX01分泌的菊粉酶活力表现出降低的趋势.在不通气及底物浓度为250 g/L时,菊粉酶的活性为6.59 U/mL,而底物浓度50 g/L,通气量1.0 vvm时的酶活力为21.54 U/mL.乙醇发酵过程中的副产物甘油随通气量的降低及底物浓度的升高而增大,而乙酸的浓度随通气量的增大及底物浓度的升高而升高.     

酶水解菊芋糖浆发酵生产琥珀酸的初步研究

用产菊粉酶的一株黑曲霉菌株进行产酶发酵条件和水解条件研究,在30℃,pH 6.0,摇床转速200 r/min,发酵时间为3 d的最适产酶条件下,酶活可以达到45.9 U/mL.以总糖含量为85.2 g/L的菊芋粉为初始底物,最适酶水解条件为温度50℃,加黑曲霉培养液的量为10%(v/v),水解12 h后,水解率达到99.6%.用此酶解液在5 L搅拌发酵罐中进行琥珀酸发酵,初始还原糖浓度53.5 g/L,36 h发酵产琥珀酸43.8 g/L,琥珀酸产率0.83 g/g,糖利用率99.0%,琥珀酸生产强度1.22 g/(L·h).     

酿酒酵母L610利用菊糖生产乙醇发酵条件的研究

以1株能够直接利用菊糖产乙醇的酿酒酵母L610为出发菌株,对其利用菊糖生产乙醇的发酵条件进行了一系列研究。结果表明,L610最适乙醇发酵温度为37℃,且40℃高温发酵对其产乙醇能力无显著影响;L610对酸性发酵环境有良好的耐受性,当发酵液p H值降至3.5时,其糖醇转化率及乙醇产量仍保持较高水平;以0.025~0.10 vvm的通气量通气12 h有利于L610发酵菊糖产乙醇;L610对350 g/L的高浓度菊糖有良好的转化率,乙醇浓度和生产强度分别达到129 g/L和1.35 g/(L·h);当直接以300 g/L菊芋粗粉为唯一底物进行发酵时,L610发酵产乙醇浓度达到89.6 g/L,为理论产量的78.1%。本研究所取得的成果为酿酒酵母一步法发酵菊芋生产乙醇的工业化发展提供参考。     

一步法发酵菊芋产乙醇菌种的筛选及产酶条件、酶学性质的研究

从腐烂的菊芋及实验室保存的菌种中,选育到一株发酵菊芋产乙醇的菌株克鲁维酵母Kluyveromyces marxianus Y1。利用正交实验法对克鲁维酵母产菊粉酶的培养基组成及培养条件进行优化,确定培养基组成（g/L）为：菊粉40,酵母粉4,蛋白胨4,尿素1;初始pH5．0,温度30℃,150r／min条件下培养达到最佳产酶效果（57U／mL）。该菌株所产菊粉酶的性质测定结果表明：以菊粉为底物,该菊粉酶最适反应温度为55℃,在60℃以下稳定性很好,高于60℃时酶迅速失活;最适pH为5．0,pH4．6—5．2范围内酶稳定性很好;该酶属于外切型菊粉酶,体积分数为8％的乙醇对酶活力基本没有影响。     

丙酮丁醇梭菌发酵菊芋汁生产丁醇

对丙酮丁醇梭菌Clostridium acetobutylicum L7发酵菊芋汁酸水解液生产丁醇进行了初步研究。实验结果表明,以该水解液为底物生产丁醇,不需要添加氮源和生长因子。当水解液初始糖浓度为48.36 g/L时,其发酵性能与以果糖为碳源的对照组基本相同,发酵终点丁醇浓度为8.67 g/L,丁醇、丙酮和乙醇的比例为0.58∶0.36∶0.06,但与以葡萄糖为碳源的对照组相比,发酵时间明显延长,表明该菌株葡萄糖转运能力强于果糖。当水解液初始糖浓度提高到62.87 g/L时,发酵终点残糖浓度从3.09 g/L增加到3.26 g/L,但丁醇浓度却提高到11.21 g/L,丁醇、丙酮和乙醇的比例相应为0.64∶0.29∶0.05,表明适量糖过剩有助于C.acetobutylicum L7胞内代谢从丙酮合成向丁醇合成途径调节;继续提高水解液初始糖浓度,发酵终点残糖浓度迅速升高,丁醇生产的技术经济指标受到明显影响。     

Kluyveromyces marxianus发酵己糖和戊糖生产乙醇

对利用底物广泛的乙醇发酵菌株马克斯克鲁维(Kluyveromyces marxianus)DL1菌株与工业用乙醇发酵菌株酿酒酵母(Saccharomyces cerevisiae)6525利用己糖(葡萄糖、甘露糖、半乳糖)和戊糖(木糖、阿拉伯糖)的情况进行对比研究。结果发现:以己糖为底物时,K.marxianus DL1均表现出细胞生长快、乙醇得率高的特点;在不通气、糖20 g/L条件下,K.marxianus DL1的最大乙醇质量浓度均比S.cerevisiae 6525高出10%左右,细胞量及乙醇生产强度分别是S.cerevisiae 6525的近2和1.7倍。当以戊糖为底物时,K.marxianus DL1可以利用木糖和阿拉伯糖;在不通气、糖20g/L条件下,K.marxianus DL1利用木糖产木糖醇和乙醇,乙醇终质量浓度可达7.68 g/L,木糖醇质量浓度为9.12 g/L;以阿拉伯糖为发酵底物时,阿拉伯糖醇的产量可达6 g/L左右;而S.cerevisiae 6525不能利用戊糖。马克斯克鲁维酵母比酿酒酵母更适合纤维乙醇生产。     

亚硫酸盐甘蔗渣浆酶解液发酵制备乙醇

以亚硫酸盐甘蔗渣浆酶解液作为原料,利用C. shehatae发酵制取燃料乙醇。结果表明：还原糖最适初始质量浓度为葡萄糖140 g/L、木糖60 g/L、酶解液总糖80 g/L。利用初始葡萄糖55.06 g/L、木糖11.18 g/L、纤维二糖4.51 g/L的亚硫酸盐甘蔗渣浆酶解液发酵,经18 h获得乙醇22.98 g/L。乙醇得率为67.23%,葡萄糖利用率为99.27%,木糖利用率为32.96%,C. shehatae适合作为蔗渣为原料的乙醇发酵菌株。     

混合糖发酵重组酿酒酵母的菌株构建和菊芋秸秆同步糖化发酵研究

【目的】构建可用于纤维素乙醇高效生产的混合糖发酵重组酿酒酵母菌株,并利用菊芋秸秆为原料进行乙醇发酵。【方法】筛选在木糖中生长较好的酿酒酵母YB-2625作为宿主菌,构建木糖共代谢菌株YB-2625 CCX。进一步通过r DNA位点多拷贝整合的方式,以YB-2625 CCX为出发菌株构建木糖脱氢酶过表达菌株,并筛选得到优势菌株YB-73。采用同步糖化发酵策略研究YB-73的菊芋秸秆发酵性能。【结果】YB-73菌株以90 g/L葡萄糖和30 g/L木糖为碳源进行混合糖发酵,乙醇产量比出发菌株YB-2625 CCX提高了13.9%,副产物木糖醇产率由0.89 g/g降低至0.31 g/g,下降了64.6%。利用重组菌YB-73对菊芋秸秆进行同步糖化发酵,48 h最高乙醇浓度达到6.10%(体积比)。【结论】通过转入木糖代谢途径以及r DNA位点多拷贝整合过表达木糖脱氢酶基因可有效提高菌株木糖发酵性能,并用于菊芋秸秆的纤维素乙醇生产。这是首次报道利用重组酿酒酵母进行菊芋秸秆原料的纤维素乙醇发酵。     

黑曲霉产菊粉酶菌株的选育及培养基优化

为获得高产菊粉酶的黑曲霉菌株,以Aspergillus niger YH-1为出发菌株,经过亚硝基胍(NTG)诱变,以高温高菊芋粉相结合的方式进行梯度驯化,选育出一株产菊粉酶菌株YH-3,并运用响应面实验方法对该菌株的培养基进行优化。确定了最佳培养基组成:菊芋粉25.2 g/L、豆饼粉40 g/L、蔗糖酯4.9 g/L、NaCl 5.5 g/L。发现内切菊粉酶活力(I)由60.9 U/mL提高到165.0 U/mL,比出发菌株提高了1.7倍。研究证明蔗糖酯对于黑曲霉YH-3发酵产菊粉酶是一种有效的促进剂。     

鹰嘴豆孢克鲁维酵母利用菊芋原料同步糖化与发酵生产乙醇

菊芋含有大量的菊粉多糖,且种植简单、产量高,是极具开发价值的替代玉米等粮食作物生产燃料乙醇的原料。文中研究了鹰嘴豆孢克鲁维酵母Y179利用菊芋原料同步糖化与发酵生产乙醇。鹰嘴豆孢克鲁维酵母Y179具有高效分泌菊粉酶的能力,摇瓶试验显示Y179酵母能够利用完全由菊芋原料配制而成的培养基良好生长并发酵产生乙醇。通气及温度对乙醇产量影响明显,相对厌氧环境对Y179酵母发酵产乙醇具有促进作用,30℃发酵温度相对37℃和42℃更有利于乙醇产量提高。种子液培养时间及接种量对乙醇产量影响较小。在5 L发酵罐中以10%(V/V)量接入预培养36 h的Y179种子液,发酵液完全由菊芋干粉配制而成,总糖含量22%(W/V),30℃不通气,300 r/min搅拌,发酵144 h时,乙醇浓度达到12.3%(V/V),糖醇转化效率86.9%,糖利用率大于93.6%。初步研究结果显示鹰嘴豆孢克鲁维酵母Y179在利用菊芋原料生产乙醇方面具有良好应用前景。     

Improved ethanol production in Jerusalem artichoke tubers by overexpression of inulinase gene in Kluyveromyces marxianus

Ethanol production from Jerusalem artichoke tubers through a consolidated bioprocessing (CBP) strategy using the inulinase-producing yeast Kluyveromyces marxianus is an economical and competitive than that from a grainbased feedstock. However, poor inulinase production under ethanol fermentation conditions significantly prolongs the fermentation time and compromises ethanol productivity. Improvement of inulinase activity appears to be promising for increasing ethanol production from Jerusalem artichoke tubers by CBP. In the present study, expression of the inulinase gene INU with its own promoter in K. marxianus (K/INU2) was explored using the integrative cassette. Overexpression of INU was explored using chromosome integration via the HO locus of the yeast. Inulinase activity and ethanol were determined from inulin and Jerusalem artichoke tubers under fed-batch operation. Inulinase activity was 114.9 U/mL under aerobic conditions for K/INU2, compared with 52.3 U/mL produced by the wild type strain. Importantly, inulinase production was enhanced in K/INU2 under ethanol fermentation conditions. When using 230 g/L inulin and 220 g/L Jerusalem artichoke tubers as substrates, inulinase activities of 3.7 and 6.8 U/mL, respectively, were measured using K/INU2, comparing favorably with 2.4 and 3.1 U/mL, respectively, using the wide type strain. Ethanol concentration and productivity for inulin were improved by the recombinant yeast to 96.2 g/L and 1.34 g/L/h, respectively, vs 93.7 g/L and 1.12 g/L/h, respectively, by the wild type strain. Ethanol concentration and productivity improvements for Jerusalem artichoke tubers were 69 g/L and 1.44 g/L/h, respectively, from the recombinant strain vs 62 g/L and 1.29 g/L/h, respectively, from the wild type strain.     

Ethanol fermentation with Kluyveromyces marxianus from Jerusalem artichoke grown in salina and irrigated with a mixture of seawater and freshwater

Aims: To study fuel ethanol fermentation with Kluyveromyces marxianus ATCC8554 from Jerusalem artichoke (Helianthus tuberosus) grown in salina and irrigated with a mixture of seawater and freshwater. Methods and Results: The growth and ethanol fermentation of K. marxianus ATCC8554 were studied using inulin as substrate. The activity of inulinase, which attributes to the hydrolysis of inulin, the main carbohydrate in Jerusalem artichoke, was monitored. The optimum temperatures were 38°C for growth and inulinase production, and 35°C for ethanol fermentation. Aeration was not necessary for ethanol fermentation with the K. marxianus from inulin. Then, the fresh Jerusalem artichoke tubers grown in salina and irrigated with 25% and 50% seawater were further examined for ethanol fermentation with the K. marxianus, 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^?1, and the highest ethanol yield of 0·467, or 91·5% of the theoretical value of 0·511, was achieved for the slurry with a solid concentration of 200 g l^?1. Conclusions: Halophilic Jerusalem artichoke can be used for fuel ethanol production. Significance and Impact of the Study: Halophilic Jerusalem artichoke, not competing with grain crops for arable land, is a sustainable feedstock for fuel ethanol production.     

Simultaneous saccharification of inulin and ethanol fermentation by recombinantSaccharomyces cerevisiae secreting inulinase

VariousSaccharomyces cerevisiae strains were transformed with a 2 μ-based multicopy expression plasmid, pYIGP, carryingKluyveromyces marxianus inulinase gene under the control ofGAPDH promoter. Among them two strains, SEY2102 and 2805, showed high levels of cell growth and inulinase expression, and were selected to study their fermentation properties on inulin. Jerusalem artichoke inulin was more effective for cell growth (10∼11 g-dry wt./L at 48 hr) and inulinase expression (1.0 units/mL with SEY2102/pYIGP and 2.5 units/mL with 2805/pYIGP) than other inulin sources such as dahlia and chicory. It was also found that maximal ethanol production of 9 g/L was obtained from Jerusalem artichoke inulin at the early stationary phase (around 30 hr), indicating that recombinantS. cerevisiae cells secreting exoinulinase could be used for the simultaneous saccharification of inulin and ethanol fermentation.     

Consolidated bioprocessing strategy for ethanol production from Jerusalem artichoke tubers by Kluyveromyces marxianus under high gravity conditions

Aims: Developing an innovative process for ethanol fermentation from Jerusalem artichoke tubers under very high gravity (VHG) conditions. Methods and Results: A consolidated bioprocessing (CBP) strategy that integrated inulinase production, saccharification of inulin contained in Jerusalem artichoke tubers and ethanol production from sugars released from inulin by the enzyme was developed with the inulinase‐producing yeast Kluyveromyces marxianus Y179 and fed‐batch operation. The impact of inoculum age, aeration, the supplementation of pectinase and nutrients on the ethanol fermentation performance of the CBP system was studied. Although inulinase activities increased with the extension of the seed incubation time, its contribution to ethanol production was negligible because vigorously growing yeast cells harvested earlier carried out ethanol fermentation more efficiently. Thus, the overnight incubation that has been practised in ethanol production from starch‐based feedstocks is recommended. Aeration facilitated the fermentation process, but compromised ethanol yield because of the negative Crabtree effect of the species, and increases the risk of contamination under industrial conditions. Therefore, nonaeration conditions are preferred for the CBP system. Pectinase supplementation reduced viscosity of the fermentation broth and improved ethanol production performance, particularly under high gravity conditions, but the enzyme cost should be carefully balanced. Medium optimization was performed, and ethanol concentration as high as 94·2 g l^?1 was achieved when 0·15 g l^?1 K₂HPO₄ was supplemented, which presents a significant progress in ethanol production from Jerusalem artichoke tubers. Conclusions: A CBP system using K. marxianus is suitable for efficient ethanol production from Jerusalem artichoke tubers under VHG conditions. Significance and Impact of the Study: Jerusalem artichoke tubers are an alternative to grain‐based feedstocks for ethanol production. The high ethanol concentration achieved using K. marxianus with the CBP system not only saves energy consumption for ethanol distillation, but also significantly reduces the amount of waste distillage discharged from the distillation system.     

Thermotolerant Kluyveromyces marxianus and Saccharomyces cerevisiae strains representing potentials for bioethanol production from Jerusalem artichoke by consolidated bioprocessing

Thermotolerant inulin-utilizing yeast strains are desirable for ethanol production from Jerusalem artichoke tubers by consolidated bioprocessing (CBP). To obtain such strains, 21 naturally occurring yeast strains isolated by using an enrichment method and 65 previously isolated Saccharomyces cerevisiae strains were investigated in inulin utilization, extracellular inulinase activity, and ethanol fermentation from inulin and Jerusalem artichoke tuber flour at 40?°C. The strains Kluyveromyces marxianus PT-1 (CGMCC AS2.4515) and S. cerevisiae JZ1C (CGMCC AS2.3878) presented the highest extracellular inulinase activity and ethanol yield in this study. The highest ethanol concentration in Jerusalem artichoke tuber flour fermentation (200?g?L(-1)) at 40?°C achieved by K. marxianus PT-1 and S. cerevisiae JZ1C was 73.6 and 65.2?g?L(-1), which corresponded to the theoretical ethanol yield of 90.0 and 79.7?%, respectively. In the range of 30 to 40?°C, temperature did not have a significant effect on ethanol production for both strains. This study displayed the distinctive superiority of K. marxianus PT-1 and S. cerevisiae JZ1C in the thermotolerance and utilization of inulin-type oligosaccharides reserved in Jerusalem artichoke tubers. It is proposed that both K. marxianus and S. cerevisiae have considerable potential in ethanol production from Jerusalem artichoke tubers by a high temperature CBP.     

Consolidated ethanol production from Jerusalem artichoke tubers at elevated temperature by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> engineered with inulinase expression through cell surface display

Ethanol fermentation from Jerusalem artichoke tubers was performed at elevated temperatures by the consolidated bioprocessing strategy using Saccharomyces cerevisiae MK01 expressing inulinase through cell surface display. No significant difference was observed in yeast growth when temperature was controlled at 38 and 40 °C, respectively, but inulinase activity with yeast cells was substantially enhanced at 40 °C. As a result, enzymatic hydrolysis of inulin was facilitated and ethanol production was improved with 89.3 g/L ethanol produced within 72 h from 198.2 g/L total inulin sugars consumed. Similar results were also observed in ethanol production from Jerusalem artichoke tubers with 85.2 g/L ethanol produced within 72 h from 185.7 g/L total sugars consumed. On the other hand, capital investment on cooling facilities and energy consumption for running the facilities would be saved, since regular cooling water instead of chill water could be used to cool down the fermentation system.     

Efficient production of butyric acid from Jerusalem artichoke by immobilized Clostridium tyrobutyricum in a fibrous-bed bioreactor

Butyric acid is an important specialty chemical with wide industrial applications. The feasible large-scale fermentation for the economical production of butyric acid requires low-cost substrate and efficient process. In the present study, butyric acid production by immobilized Clostridium tyrobutyricum was successfully performed in a fibrous-bed bioreactor using Jerusalem artichoke as the substrate. Repeated-batch fermentation was carried out to produce butyric acid with a high butyrate yield (0.44 g/g), high productivity (2.75 g/L/h) and a butyrate concentration of 27.5 g/L. Furthermore, fed-batch fermentation using sulfuric acid pretreated Jerusalem artichoke hydrolysate resulted in a high butyric acid concentration of 60.4 g/L, with the yield of 0.38 g/g and the selectivity of ∼85.1 (85.1 g butyric acid/g acetic acid). Thus, the production of butyric acid from Jerusalem artichoke on a commercial scale could be achieved based on the system developed in this work.     

菊粉酶基因在酿酒酵母中的表达及乙醇发酵

以乙醇耐受力较强的酿酒酵母为受体菌,构建了能够分泌菊粉酶的基因工程菌并进行了菊芋粉的生料发酵。首先,以马克斯克鲁维酵母Kluyveromyces marxianus中的基因组DNA为模板,PCR扩增菊粉酶编码基因inu,分别使用菊粉酶自身启动子和酵母磷酸甘油激酶 (Phosphoglycerate kinase,pgk) 启动子,构建重组表达质粒HO/p-inu和HO/pgk-inu。经NotⅠ线性化后,采用电击法转化酿酒酵母工业菌株Saccharomyces cerevisiae 6525,分别得到含菊