产1,3-丙二醇菌株的诱变和筛选

为提高克雷伯氏肺炎杆菌产1,3-丙二醇的能力,以离子束、紫外线和氯化锂为复合诱变法,建立了产酸圈和产物耐受相结合的平板筛选方法,获得可耐受高浓度1,3-丙二醇并且副产物中乙醇含量较少的优良突变菌株2株。与出发菌株相比,两株高产突变菌株Klebsiella pneumoniae LM 03和Klebsiella pneumoniae LM05的1,3-丙二醇产量分别提高了33% 和30% ,达到66.74 g/L和65.12 g/L;乙醇产量分别降低了38% 和24% ,降低为6.59 g/L和8.05 g/L。同时测定了诱变前后还原途径中甘油脱水酶（GDHt）和1,3-丙二醇氧化还原酶（PDOR）的酶活变化,研究表明诱变对GDHt有明显的促进作用,而对PDOR的影响不明显。该诱变和筛选方法目标明确、易操作、效率高,在1,3-PD工业规模的生物法生产中将具有良好的应用价值,而且对于其他具有工业应用价值的菌株筛选工作也具有一定的借鉴意义。     

1,3-丙二醇产生菌的诱变育种及培养基优化

以实验室自然筛选的克雷伯氏杆菌(Klebsiella sp.)为出发株,采用紫外诱变及亚硝基胍和超声波协同处理获得一株1,3-丙二醇高产突变株。在摇瓶发酵中,其产1,3-丙二醇产量由17.39 g/L提高到24.11 g/L,提高38.64%。变异株经10次传代培养,发酵能力稳定。对发酵培养基成分进行了优化,优化后1,3-丙二醇产量为30.05g/L,为优化前的1.25倍。     

新型复合诱变法筛选高产1,3-丙二醇菌株

为提高肺炎克雷伯氏茵生产l,3-丙二醇的能力,使用一种新型的以氮气为工作气体的等离子体复合紫外的诱变系统(MPMS-UV)对菌种进行诱变.用含有90g/L～100g/L1,3-丙二醇的选择培养基进行筛选驯化,最终获得一株高产PDO且甘油利用率高的优良菌株k2.2L批式流加发酵结果表明,该菌株PDO产量为69.71 g/L,摩尔产率为0.57 mol/mol,比原始菌株分别提高了44.7％和21％.     

丁酸梭杆菌发酵甘油制备1,3-丙二醇的研究

本研究采用丁酸梭芽孢杆菌(Clostridium butyricum)从甘油发酵制备1,3—丙二醇。该发酵需厌氧培养,发酵培养基为：甘油6％,葡萄糖1％,玉米浆2％,(NH4)2SO40．2％。发酵温度为34℃,pH为6．5～7。在最佳条件下,发酵50h可产1,3—丙二醇40．7g／L,甘油摩尔转化率达68％。     

丁酸梭菌1，3-丙二醇氧化还原酶基因dhaT的克隆及在大肠杆菌中的表达

以丁酸梭菌（Clostridium butyricum）基因组DNA为模板,利用PCR技术扩增得到1,3-丙二醇氧化还原酶基因dhaT,将它连接到pMD18一T载体上,得到重组质粒pMD—dhaT,对此重组质粒进行序列测定,对其DNA序列分析表明,dhaT基因全长为1 158bp。将dhaT基因插入表达载体pSE-380中,构建成重组子pSE—dhaT,并在大肠杆菌JMl09中进行诱导表达。研究表明,以1,3-丙二醇为底物时,基因工程菌在37℃下,以1．0mmol／L IPTG诱导14h,酶活力达到16．28U／mL,比原始菌株提高5、6倍。     

腐乳毛霉蛋白酶菌株紫外线诱变育种

目的：选育酶活力高、耐热性好的毛霉蛋白酶菌株,用于腐乳发酵实验或生产。方法：从腐乳中分离毛霉蛋白酶菌株,对其出发菌株进行紫外线诱变,并经控温培养,选育优良变株。结果：获得一株酶活力高、耐热性强的优良变株SCLG—毛霉16,该菌株在32℃条件下,生长茂盛,其HE值为1.39、蛋白酶活力为180.375u/g,分别较出发菌株高出7.8%和51.4%。结论：将此优良变株用于腐乳发酵实验或生产,在缩短发酵时间,提升腐乳质量,延长生产季节等方面具有一定的应用价值。     

产1,3-丙二醇基因工程菌发酵条件的研究

利用途径工程的方法,将来源于克雷伯氏菌(Klebsiella pneumoniae)的甘油脱水酶基因dhaB和1,3-丙二醇氧化还原酶基因dhaT构建成多顺反子重组质粒pSE-dhaB-dhaT并在大肠杆菌JM 109中进行表达,在大肠杆菌中构建一条新的产1,3-丙二醇代谢途径。研究表明,重组菌株JM 109/pSE-dhaB-dhaT在微好氧条件下,尝试用廉价的乳糖为诱导物、维生素B12为辅酶,可以将甘油转化为1,3-丙二醇,产量达15.34 g/L,甘油转化率为35.7%,对低成本生产1,3-丙二醇作了有益的探索。     

产1,3-丙二醇新型重组大肠杆菌的构建

利用PCR技术从大肠杆菌(Escherichia coli )中扩增出1.16 kb的编码1,3-丙二醇氧化还原酶同工酶的基因yqhD,将其连接到表达载体pEtac,得到重组载体pEtac-yqhD,重组载体在大肠杆菌JM109中得到高效表达。SDS_PAGE分析显示融合表达产物的分子量均为43 kD,同核酸序列测定所推导的值相符。对含有yqh-D的基因工程菌进行表达研究表明：37 ℃,以1.0 mmol /L IPTG诱导4 h,1,3-丙二醇氧化还原酶同工酶的酶活力达到120 u/mg蛋白,而对照菌株的酶活力为0.5 u/mg蛋白。再将含甘油脱水酶基因dhaB和含1,3-丙二醇氧化还原酶同工酶基因yqhD的重组质粒共转化大肠杆菌JM109得到重组大肠杆菌JM109（pUCtac-dhaB, pEtac-yqhD）,该菌株在好氧条件下,以1.0mmol/L IPTG诱导可将50 g/L甘油转化为38.0 g/L 1,3-丙二醇。首次发现1,3-丙二醇氧化还原酶同工酶在好氧条件下表现出较高的活性。     

原生质体紫外诱变技术选育1,3-丙二醇高产菌株

以肺炎克雷伯氏杆菌(Klebsiella pneumoniae)为研究对象,应用原生质体紫外诱变技术提高其对甘油及1,3-丙二醇的耐受性,获得1,3-丙二醇高产菌.在原生质体制备过程中,运用滤膜去除酶解后细胞悬液中的正常菌体,简化菌体酶解过程,提高再生率及形成率.经过原生质体诱变后,以耐受高浓度甘油和1,3-丙二醇及高产酸能力为筛选方向,最终筛选到了3株高产菌株(Kp-1、Kp-4和Kp-5).在补料发酵实验中,上述诱变菌产1,3-丙二醇能力分别为70.24 、65.21和75.51 g/L,比野生菌株WT(55.78 g/L)分别提高了25.92%、16.91%和35.37%.     

耐高浓度甘油的1,3-丙二醇产生菌诱变选育与固定化培养初步研究

紫外诱变筛选耐高浓度甘油的1,3-PD克雷伯杆菌,诱变条件为30 W紫外灯,距离34 cm,照射6 m in,平板涂布稀释度为10-5,经六轮诱变筛选出耐90 g/L甘油的变异菌株,甘油转化率稳定在45%左右,1,3-PD生成量稳定在40 g/L左右,比原始出发菌株提高了近30%,诱变菌株经考察具有一定的遗传稳定性。在此基础上应用溶胶-凝胶法进行固定化细胞实验并与游离细胞进行了四个批次的对比连续发酵,结果显示溶胶-凝胶法具有一定的稳定性。     

A robust soft sensor to monitor 1,3-propanediol fermentation process by Clostridium butyricum based on artificial neural network

With the aggravation of environmental pollution and energy crisis, the sustainable microbial fermentation process of converting glycerol to 1,3-propanediol (1,3-PDO) has become an attractive alternative. However, the difficulty in the online measurement of glycerol and 1,3-PDO creates a barrier to the fermentation process and then leads to the residual glycerol and therefore, its wastage. Thus, in the present study, the four-input artificial neural network (ANN) model was developed successfully to predict the concentration of glycerol, 1,3-PDO, and biomass with high accuracy. Moreover, an ANN model combined with a kinetic model was also successfully developed to simulate the fed-batch fermentation process accurately. Hence, a soft sensor from the ANN model based on NaOH-related parameters has been successfully developed which cannot only be applied in software to solve the difficulty of glycerol and 1,3-PDO online measurement during the industrialization process, but also offer insight and reference for similar fermentation processes.     

Production of 1,3-Propanediol by Clostridium butyricum VPI 3266 in continuous cultures with high yield and productivity

The effects of dilution rate and substrate feed concentration on continuous glycerol fermentation by Clostridium butyricum VPI 3266, a natural 1,3-propanediol producer, were evaluated in this work. A high and constant 1,3-propanediol yield (around 0.65 mol/mol), close to the theoretical value, was obtained irrespective of substrate feed concentration or dilution rate. Improvement of 1,3-propanediol volumetric productivity was achieved by increasing the dilution rate, at a fixed feed substrate concentration of 30, 60 or 70 g l⁻¹. Higher 1,3-propanediol final concentrations and volumetric productivities were also obtained when glycerol feed concentration was increased from 30 to 60 g l⁻¹, at D=0.05–0.3 h⁻¹, and from 60–70 g l⁻¹, at D=0.05 and 0.1 h⁻¹·30 g l⁻¹ of 1,3-propanediol and the highest reported value of productivity, 10.3 g l⁻¹ h⁻¹, was achieved at D=0.30 h⁻¹ and 60 g l⁻¹ of feed glycerol. A switch to an acetate/butyrate ratio higher than one was observed for 60 g l⁻¹ of feed glycerol and a dilution rate higher than 0.10 h⁻¹; moreover, at D=0.30 h⁻¹ 3-hydroxypropionaldehyde accumulation was observed for the first time in the fermentation broth of C. butyricum.     

L-丝氨酸高产菌株的选育和摇瓶发酵条件优化

采用B revibacterium flavmC-11A为出发菌株,经紫外线照射和亚硝基胍诱变处理,选育出一株L-丝氨酸高产菌株C32为目的突变株,使摇瓶产酸率由12.1 g.L-1增加到16.4 g.L-1,然后对其进行摇瓶发酵条件优化,使菌株C32的L-丝氨酸产率提高到30.1 g.L-1。     

酪酸菌对动物肠道致病菌体外拮抗作用的研究

探讨了酪酸菌对三种畜禽肠道致病菌的体外拮抗作用。将酪酸菌分别和猪大肠杆菌、鸡大肠杆菌、鸡白痢沙门氏菌三种畜禽肠道致病菌按不同的比例混合接种于CL液体培养基中进行厌氧培养,培养过程中三种致病菌的活菌数急剧下降,30h后的菌落数都逐渐降为零。说明酪酸菌在体外实验中对上述三种畜禽肠道致病菌都有较强的拮抗作用,其中酪酸菌对猪大肠杆菌、鸡大肠杆菌和鸡白痢沙门氏菌的最佳接种比例分别为：10：1～100：1、10：1～100：1和5：1～10：1。     

丁酸梭菌在医学领域中的研究进展

肠道益生菌是定植于人体肠道内,能产生确切健康功效从而改善宿主微生态平衡,对宿主发挥有益作用的一类活性微生物总称。最近越来越多的研究表明肥胖、糖尿病、肠易激综合征、骨质疏松等与肠道菌群密切相关,而丁酸梭菌由于能够促进其他有益菌（乳杆菌、双歧杆菌）生长,具有其他有益生菌没有的特性,受到越来越多研究者的重视。本文通过综述近几年有关丁酸梭菌与神经、内分泌、消化等系统中部分疾病的关系,总结了丁酸梭菌可能具有的治疗机制,并将其与益生菌在治疗骨质疏松症时所发挥的机制进行对比,以展望丁酸梭菌可能具有治疗骨质疏松的潜力。     

丁醇产生菌育种研究进展

生物丁醇产业因发酵法的产量、产率和比例低等原因受到限制。菌种改良是解决问题的一个重要和根本的策略。诱变育种仍然是工业育种的主要方法,复合诱变和理性化筛选更有效。基因工程育种对丙酮丁醇梭菌和大肠杆菌丁醇合成途径进行改造和构建优化,还可改造途径外影响合成的其它基因,以获得高产菌株,发展最为迅猛,但效果仍低于诱变育种。今后的菌种改良方向仍为提高产量和比例。     

Fermentation of raw glycerol to 1,3-propanediol by new strains ofClostridium butyricum

Industrial glycerol obtained through the transesterification process using rapeseed oil did not support growth of several strains ofClostridium butyricum obtained from bacterial culture collections. Ten new strains ofC. butyricum were obtained from mud samples from a river, a stagnant pond, and a dry canal. These new isolates fermented the commercial glycerol and produced 1,3-propanediol as a major fermentation product with concomitant production of acetic and butyric acids. Four of the ten isolates were able to grow on industrial glycerol obtained from rapeseed oil. One strain,C. butyricum E5, was very resistant to high levels of glycerol and 1,3-propanediol. Using fed-batch fermentation, 109 g L^–1 of industrial glycerol were converted into 58 g of 1,3-propanediol, 2.2 g of acetate and 6.1 g of butyrate per liter.     

酪酸梭菌活菌散预防新生儿高胆红素血症临床观察

目的观察酪酸梭菌活菌散(商品名:宝乐安)预防新生儿高胆红素血症的临床疗效。方法将产科分娩的正常足月新生儿212例,随机分为2组,预防组106例,对照组106例,2组均在生后半小时内开始喂奶。预防组喂服酪酸梭菌活菌散500 mg/次,3次/d,用至黄疸消退,观察黄疸变化情况及不良反应。结果出生后48 h内胎便转黄率预防组为56.6%,对照组为26.4%;生后7 d内皮肤巩膜黄染达高峰预防组为58.5%,对照组为43.3%;高胆红素血症发生率预防组为7.5%,对照组为22.6%;出生后7 d内血清胆红素达高峰率预防组为62.3%,对照组为47.1%。以上各指标2组间差异均具有统计学意义(P0.01或0.05)。结论酪酸梭菌活菌散预防新生儿高胆红素血症安全有效,值得临床推广应用。