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。     

利用甜菜糖蜜补料发酵生产丁醇

从土壤中分离出1株适合利用甜菜糖蜜发酵生产丁醇的丙酮丁醇梭菌(Clostridium acetobutylicum)2N,通过优化发酵条件,得到最适发酵温度为33℃,玉米浆最适添加量为15g/L,发现甜菜糖蜜中还原糖质量浓度高于50g/L时影响菌株的生长和溶剂生产。以补料分批发酵方式降低底物抑制,33℃发酵48h后,丁醇和总溶剂的质量浓度分别达到14.15g/L和19.65g/L,丁醇质量分数超过70%。     

化学改性甘蔗渣对固定化细胞发酵产丁醇的影响

旨在研究化学改性的甘蔗渣作为固定化载体对丙酮丁醇梭菌Clostridium acetobutylicum XY16发酵制备生物丁醇的影响。首先利用不同浓度的聚乙烯亚胺(PEI)和1 g/L戊二醛(GA)对甘蔗渣表面进行化学改性,增强甘蔗渣对Clostridium acetobutylicum XY16的附载能力。经4 g/L聚乙烯亚胺和1 g/L戊二醛改性的甘蔗渣(添加量10 g/L)应用到固定化批次发酵中,发酵36 h后丁醇和总溶剂浓度最高,分别达到了12.24 g/L和21.67 g/L,同时溶剂的生产速率达到0.60 g/(L·h),生产速率比游离细胞和未改性甘蔗渣固定化细胞分批发酵分别提高了130.8%和66.7%。在此基础上对改性甘蔗渣固定化的细胞进行6次重复批次发酵,丁醇和总溶剂的产量稳定,溶剂生产速率逐渐提高至0.83 g/(L·h),同时转化率也提高至0.42 g/g。     

丙酮丁醇梭菌丁醇耐受性

丁醇在发酵培养基中的积累所产生的毒性问题是限制丁醇产量的重要因素,然而对于Clostridium acetobutylicum是如何适应丁醇胁迫,进而调节菌体生长和代谢的,目前尚缺乏系统研究,不能全面揭示C.acetobutylicum的丁醇耐受性机制.对丙酮丁醇梭菌丁醇耐受性有关的研究成果进行了综述,旨在深入理解菌株丁醇耐受性发生改变的相关分子基础.希望为进行微生物丁醇耐受性分子机制的改造、提高菌株的丁醇耐受性提供新的研究思路.     

利用核糖体工程选育丙酮丁醇菌提高丁醇产量

利用核糖体工程技术对丙酮丁醇梭菌Clostridium acetobutylicum L7进行诱变筛选,以获得丁醇高产菌株。使用链霉素诱变C.acetobutylicum L7并结合设计的平板转接逐次提高链霉素浓度的筛选路线,获得丁醇产量较高的菌株S3。结果表明,S3丁醇产量为(12.48±0.03)g/L,乙醇产量为(1.70±0.07)g/L,相对于原始菌分别提高了11.2%及50%;丁醇/葡萄糖转化率由原始菌的0.19提高到0.22,丁醇生产率达到0.24 g/(L.h),相比提高30.5%;耐受丁醇浓度由原始菌的12 g/L提高到14 g/L;发酵液粘度下降到4 mPa/s,同比降低了60%,利于后续分离工作的进行,降低发酵成本。进一步研究工作表明,S3菌株遗传稳定性良好。因此,核糖体工程技术是一种选育丁醇高产菌株的有效方法。     

丙酮丁醇梭菌(Clostridium acetobutylicum)原生质体融合育种研究

为了提高丙酮丁醇梭菌(Clostridium acetobutylicum)的丁醇耐受能力和培养基总糖产丁醇的转化率,通过原生质体融合的方法,研究了溶菌酶浓度及其作用时间、再生培养基种类、55℃条件下菌体致死时间、不同PEG分子量以及作用时间、Ca^2+和Mg^2+不同的添加量对丙酮丁醇梭菌原生质体制备、融合、再生的影响,得到了一套比较系统的丙酮丁醇梭菌的原生质体融合条件,同时通过气相色谱检测了融合菌的产溶剂能力并计算总糖转化率。结果显示,最终得到的215I菌株的总糖转化率比原始菌株提高了34.7%,产丁醇能力比原始菌株提高了32.2%,并且发现1株融合菌能产生新物质。原生质体融合方法在丙酸丁醇梭菌育种方面有广泛的应用潜力,通过融合得到的菌株为丁醇生产奠定了基础。     

拜氏梭菌13-2发酵甘蔗渣水解液生产丁醇的研究

目的:蔗渣是一种重要的可再生生物质资源,蔗渣原料生产丁醇将大大降低丁醇的成本.方法:实验利用0.25 ～3.0%不同浓度稀H2SO4对蔗渣进行121℃的高温作用1h,以水解液为碳源,进行丁醇的发酵实验.结果:相对于8052菌株,13 -2菌株对甘蔗渣水解液具有更高的发酵效率,在0.5%硫酸用量条件下,13 -2菌株的丁醇发酵量最高,达到4.5g/L.而8052只有2.3g/L的丁醇发酵量.结论:在同等条件下,拜氏梭菌菌株13 -2比模式菌株8052具有更高的溶剂产量和抑制物耐受能力,最佳的蔗渣水解条件为1.5%硫酸用量,丁醇发酵量和总溶剂分别为4.57g/L和5.41 g/L.     

糖丁基梭菌产丁醇途径在大肠杆菌中的构建及发酵

摘要:【目的】通过克隆来源于糖丁基梭菌(Clostridium saccharobutylicum DSM13864)丁醇合成途径的关键酶基因(thlA,bcs-operon和adhE),构建产丁醇大肠杆菌。【方法】以Clostridium saccharobutylicum DSM13864的基因组为模板,分别扩增丁醇途径关键酶基因thlA,bcs-operon(crt-bcd1-etfB2-fixB2-hbd)和adhE,构建了两个重组质粒pETDuet-bcs和pRSFDuet-thlA-adhE,并成功转入E.coli JM109(DE3)实现异源表达,使大肠杆菌具备产丁醇能力。在半厌氧条件下进行重组菌的发酵,并研究不同培养基对产丁醇的影响。【结果】该重组菌在半厌氧条件下经摇瓶发酵丁醇产量达到25.4 mg/L,通过优化培养基后,在TB发酵培养基中丁醇产量可达到34.1 mg/L。【结论】通过构建重组共表达质粒,将糖丁基梭菌来源的丁醇途径关键酶基因在大肠杆菌中表达,成功构建产丁醇大肠杆菌。该研究提供了一株易于操作的丁醇发酵重组大肠杆菌,避免了传统梭菌发酵丁醇生产中苛刻的厌氧条件、易产孢子等限制问题。     

高抗逆高丁比拜氏梭菌的选育及其性能考察

以抗逆突变株Clostridium beijerinckii IB4为出发菌株,通过常压室温等离子体诱变(ARTP),刃天青平板初筛,摇瓶发酵复筛,筛选出1株高抗逆高丁比的突变菌株C.beijerinckii IT111。发酵结果表明:该突变菌株利用多种C源时均展现其高丁醇比的特性,以玉米芯酸解糖液为C源时,溶剂产量达到10.5 g/L,丁醇8.0 g/L,丁醇比高达76%。抑制物抗逆性测试结果显示:糠醛和酸类对C.beijerinckii发酵影响较小,酚类物质对C.beijerinckii抑制作用较强,其中以香草醛为最。综上所述,C.beijerinckii IT111是1株极具潜力的利用木质纤维原料制备丁醇的菌株。     

高抗逆高丁比拜氏梭菌的选育及其性能考察简

以抗逆突变株Clostridium beijerinckii IB4为出发菌株,通过常压室温等离子体诱变（ ARTP ）,刃天青平板初筛,摇瓶发酵复筛,筛选出1株高抗逆高丁比的突变菌株C.beijerinckii IT111。发酵结果表明：该突变菌株利用多种C源时均展现其高丁醇比的特性,以玉米芯酸解糖液为C源时,溶剂产量达到10.5 g/L,丁醇8.0 g/L,丁醇比高达76％。抑制物抗逆性测试结果显示：糠醛和酸类对C.beijerinckii发酵影响较小,酚类物质对C.beijerinckii抑制作用较强,其中以香草醛为最。综上所述,C.beijerinckii IT111是1株极具潜力的利用木质纤维原料制备丁醇的菌株。     

Transcriptional analysis of Clostridium beijerinckii NCIMB 8052 and the hyper-butanol-producing mutant BA101 during the shift from acidogenesis to solventogenesis

Clostridium beijerinckii is an anaerobic bacterium used for the fermentative production of acetone and butanol. The recent availability of genomic sequence information for C. beijerinckii NCIMB 8052 has allowed for an examination of gene expression during the shift from acidogenesis to solventogenesis over the time course of a batch fermentation using a ca. 500-gene set DNA microarray. The microarray was constructed using a collection of genes which are orthologs of members of gene families previously found to be important to the physiology of C. acetobutylicum ATCC 824. Similar to the onset of solventogenesis in C. acetobutylicum 824, the onset of solventogenesis in C. beijerinckii 8052 was concurrent with the initiation of sporulation. However, forespores and endospores developed more rapidly in C. beijerinckii 8052 than in C. acetobutylicum 824, consistent with the accelerated expression of the sigE- and sigG-regulated genes in C. beijerinckii 8052. The comparison of gene expression patterns and morphological changes in C. beijerinckii 8052 and the hyper-butanol-producing C. beijerinckii strain BA101 indicated that BA101 was less efficient in sporulation and phosphotransferase system-mediated sugar transport than 8052 but that it exhibited elevated expression of several primary metabolic genes and chemotaxis/motility genes.     

Optimization of butanol production from tropical maize stalk juice by fermentation with Clostridium beijerinckii NCIMB 8052

Mixed sugars from tropical maize stalk juice were used to carry out butanol fermentation with Clostridium beijerinckii NCIMB 8052. Batch experiments employing central composite design (CCD) and response surface methodology (RSM) optimization were performed to evaluate effects of three factors, i.e. pH, initial total sugar concentration, and agitation rate on butanol production. Optimum conditions of pH 6.7, sugar concentration 42.2 g/L and agitation rate 48 rpm were predicted, under which a maximum butanol yield of 0.27 g/g-sugar was estimated. Further experiments demonstrated that higher agitation facilitated acetone production, leading to lower butanol selectivity in total acetone–butanol–ethanol (ABE). While glucose and fructose are more preferable by C. beijerinckii, sucrose can also be easily degraded by the microorganism. This study indicated that RSM is a useful approach for optimizing operational conditions for butanol production, and demonstrated that tropical maize, with high yield of biomass and stalk sugars, is a promising biofuel crop.     

The acetone butanol fermentation on glucose and xylose. II. Regulation and kinetics in fed-batch cultures

The kinetics in fed-batch cultures of acetone butanol fermentation by Clostridium acetobutylicum is compared on glucose, xylose, and mixtures of both sugars. The final conversion yield of sugars into solvents always increases with the sugar feeding rate. At low feeding rates, the sugar concentration in the medium becomes limiting, which results in a slower cellular growth, a slower metabolic transition from an acid to a solvent fermentation and, thus, a higher accumulation of acids. It is only at sufficiently high feeding rates that fed-batch fermentations yield kinetic results comparable to those of batch fermentations. With mixtures of glucose and xylose, because of a maintained low glucose level, both sugars are taken up at the same rate during a first fermentation period. An earlier accumulation of xylose when the fermentation becomes inhibited suggest that xylose utilization is inhibited when the catabolic flux of glucose alone can satisfy the metabolic activity of the cell. Kinetic results with batch and fed-batch fermentations indicate several important features of the regulation of C. acetobutylicum metabolism: an early inhibition by the produced acids; an initiation of solvent formation between 4 and 6 g/L acetic and butyric acid depending on the metabolic activity of the cell; a metabolic transition from acids to solvents production at a rate closely related to the rate of sugar uptake; during solvent production, a reassimilation of acids above a minimal rate of sugar consumption of 0.2 h(-1); a final inhibition of the fermentation at a total butanol and acids concentration of ca. 20 g/L.     

Studies on the stability of solvent production by Clostridium acetobutylicum in continuous culture

Various methods of continuous flow culture of Clostridium acetobutylicum NCIB 8052 were investigated, with the aim of obtaining prolonged production of acetone and butanol. In ammonia-limited chemostat culture, maximal concentrations of solvents were obtained at pH 5–5 at a relatively high biomass concentration of 1.3–2.0 g/1 dry weight maintained at a dilution rate of 0.06/h. Similar dependence of solvent production on the sustenance of a relatively high cell density was observed in magnesium- or phosphate-limited chemostat cultures. Solvent production was always transient, however, with a shift to production of only acetic and butyric acids being observed after 4–16 volume changes. Longer term solvent production was obtainable under conditions of glucose limitation but the solvent yield was low. Cultivation in a pH-auxostat permitted solvent production in reasonably high yield over at least 70 volume changes with no signs of culture degeneration. Although none of the continuous flow cultures achieved a true steady state, we conclude that turbidostat or pH-auxostat culture are the methods of choice for continuous solvent production by Cl. acetobutylicum NCIB 8052.     

Clostridial strain degeneration

Abstract: Strain degeneration, the loss of the capacity to produce solvents and form spores, typically occurs when Clostridium acetobutylicum and related clostridia are repeatedly subcultured in batch culture or grown in continuous culture, as opposed to being grown from germinated, heat-treated spores. Several mechanisms for degeneration have been identified thus far. (i) Degeneration can be caused by excessive acidification of the culture during exponential growth. We present data interpreted to mean that C. beijerinckii (formerly C. acetobutylicum ) NCIMB 8052 cells ferment glucose to acetic and butyric acids at an uncontrolled rate, so that, during rapid growth, the rate of acid production can exceed the rate of induction of the solventogenic pathway enzymes. As a result, the medium pH drops to bactericical levels, and the cells cannot switch to solventogenesis and sporulation. The clostridia seem to be poised either to produce excess acids, or to initiate solventogenesis, depending on small differences in the rates of growth. (ii) We have isolated transposon-insertion mutants of C. beijerinckii NCIMB 8052 that are resistant to degeneration, suggesting the involvement of a regulatory region of the clostridial chromosome. (iii) Involvement of a global regulatory gene has been inferred in C. beijerinckii NCIMB 8052 which degenerates irreversibly in chemostat culture. (iv) Impairment of butanol formation due to a defect in NADH generation has been reported in an oligosporogenous strain which can revert to the non-degenerate phenotype. (v) In continuous culture, degenerate cells may be selected because they continue to divide, while the non-degenerate cells stop dividing and start differentiating.     

拜氏梭菌Clostridium beijerinckii NCIMB 8052发酵玉米皮生产丁醇

玉米皮作为玉米淀粉加工的副产物,是一种可用于生产液体燃料的潜在廉价优质的生物质资源。本文以玉米皮为原料,对拜氏梭菌发酵生产丁醇进行了研究。实验结果表明,玉米皮首先在最优的预处理温度140℃下使用0.5%硫酸水溶液以固液比1∶8处理20 min,再添加200 IU/g底物糖化酶、1.0 IU/g底物木聚糖酶进行酶解,可以使原料中的淀粉和半纤维素转化为可发酵糖,此时水解液中的总糖浓度为50.46 g/L。然后使用1.0%的活性炭对水解液进行脱毒处理以去除发酵抑制物,再进行丁醇发酵,丁醇产量为9.72 g/L,总溶剂产量可达14.09 g/L,糖醇转化率为35.1%。上述研究结果证明玉米皮作为一种粮食加工废弃物用于液体燃料丁醇的生产在技术上是完全可行的。     

Molecular genetics and the initiation of solventogenesis in Clostridium beijerinckii (formerly Clostridium acetobutylicum) NCIMB 8052

Abstract: A physical map of the Clostridium beijerinckii (formerly Clostridium acetobutylicum ) NCIMB 8052 chromosome has been constructed, encompassing about 90 rare restriction sites. The 14 rrn operons together with 40 genes have been assigned positions on the map. Genetic analysis and gene transfer have been developed in this organism to enable in vivo analysis of the roles of cloned genes using marker replacement technology. Experiments using the available genetic tools have shown that spo0A plays a cardinal role in controlling several aspects of the transition from exponential growth to stationary phase in C. beijerinckii . These include initiation of sporulation, accumulation of the storage polysaccharide, granulose, and production of acetone and butanol. Several C. beijerinckii and C. acetobutylicum genes concerned with fermentative metabolism, whose expression is modulated at the onset of solventogenesis, contain sequence motifs resembling 0A boxes in their 5' regulatory regions. This invites the speculation that they are under direct control of Spo0A, and additional data are now required to test this prediction.     

Transfer of Tn1545 and Tn916 to Clostridium acetobutylicum

Tn1545, a conjugative transposon originally discovered in Streptococcus pneumoniae, has been transferred from Enterococcus faecalis and Bacillus subtilis to Clostridium acetobutylicum NCIB 8052. Transfer between different strains of C. acetobutylicum has also been observed. Insertion of Tn1545 into the C. acetobutylicum chromosome occurred at multiple sites, as shown by Southern hybridization. Although ermAM (erythromycin-resistance) was the most satisfactory marker for primary selection of transconjugants, all three Tn1545-encoded antibiotic resistance genes (aphA-3, ermAM, and tetM) were apparently expressed in C. acetobutylicum. Our results indicate that Tn1545 is potentially useful for undertaking mutagenesis and mutational cloning in this industrially important organism. Transfer of another conjugative transposon, Tn916, from E. faecalis to C. acetobutylicum NCIB 8052 was also apparently detected. Circumstantial evidence suggests that there may be a hot spot for Tn916 insertion in the C. acetobutylicum NCIB 8052 chromosome.