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。     

甘蔗渣和糖蜜混合发酵制备燃料丁醇

以抗逆突变株Clostridium beijerinckii IB4为研究对象,葡萄糖为C源,对其进行补料分批发酵过程的优化,同时将该优化工艺应用于甘蔗渣和糖蜜混合发酵制备燃料丁醇。结果表明:在5 L发酵罐中,先加入作为还原糖的甘蔗渣酸解糖液10 g/L,16 h后补加甘蔗糖蜜30 g/L,于35℃、100 r/min发酵50 h,丁醇和总溶剂产量分别达到11.1和15.3 g/L,丁醇比例高达72.5%。     

丙酮丁醇梭菌XY16丁醇发酵特性

以诱变选育的1株突变菌株丙酮丁醇梭菌XY16为对象,对影响该菌发酵特性的相关因素(N源、生长因子、热激)进行研究。结果显示:无机N源乙酸铵比其他N源更有利于丙酮丁醇的发酵,玉米浆或玉米蛋白可以直接替代生长因子进行丙酮丁醇发酵,热激可以提高总溶剂产量,最高可以达到21.28 g/L。该菌还可以同时利用葡萄糖和木糖,当葡萄糖利用完后,木糖才能被有效利用。     

响应面法优化甘蔗糖蜜发酵产丁醇

以甘蔗糖蜜为底物,用响应面法对高丁醇比突变菌株拜氏梭菌(Clostridium beijerinckii)ART124发酵生产丁醇的培养条件进行优化.首先利用Plackett - Burman试验设计筛选出影响丁醇生产的3个重要因素CaCO3和NH4 HCO3和K2HPO4的用量,再通过最陡爬坡路径逼近最大向应区域,最后根据响应面中心组合设计理论,确定主要影响因素的最佳条件:CaCO3、NH4HCO3和K2HPO4的质量浓度分别为2.65、2.16和0.43 g/L.利用数学模型分析预测得甘蔗糖蜜质量浓度为30 g/L时,最佳的丁醇产量为8.10 g/L,比优化前提高了53.14％.在最佳工艺条件下得到的实验结果与模型预测值很吻合,说明所建立的模型是有效的.     

筛选具有高α-淀粉酶酶活的丙酮丁醇梭菌及C源对其酶活的影响

利用淀粉平板筛选到1株α-淀粉酶比酶活为94.67 U/mL的丙酮丁醇梭菌(Clostridium acetobutylicum)菌株D3 1 1,比酶活比原菌株提高了58.21%,丁醇产量达11.88 g/L,比原菌株提高了25.45%。考察不同C源对丙酮丁醇梭菌D3 1 1α-淀粉酶活的影响,其中葡萄糖要比其他C源对α淀粉酶的抑制作用更强,且随着葡萄糖浓度的加大,抑制作用也越强。     

一株拜氏梭菌利用甘蔗废糖蜜发酵生产丙酮丁醇

以甘蔗废糖蜜作为原料,利用Clostridium beijerinckii DSM 6422菌株进行丙酮丁醇发酵的初步研究.结果表明:采用H2SO4预处理糖蜜,初糖质量浓度60 g/L,(NH4)2SO4 2g/L,CaCO3 10 g/L,温度30℃,pH 5.5～7.0,接种量6％(体积分数),在5L发酵罐中发酵培养96 h,总溶剂产量为16.17 g/L,其中丁醇质量浓度为10.07 g/L,总溶剂产率为30.2％,糖利用率为89.3％.     

常压室温等离子体诱变高效利用木糖产丁二酸菌株

大肠杆菌Escherichia coli AFP111是E. coli NZN111 (△pflAB△ldhA) 的ptsG自发突变株,其转化1 mol的木糖合成丁二酸的过程中净产生1.67 mol ATP,但是转化1 mol的木糖合成丁二酸的过程中实际需要2.67 mol ATP,因此在厌氧条件下,ATP的供给不足导致E. coli AFP111不能代谢木糖。采用常压室温等离子体射流诱变产丁二酸大肠杆菌菌株,在厌氧条件下,利用以木糖为碳源的M9培养基,筛选得到一株可以代谢木糖并积累丁二酸的突变株DC111。该突变菌株在发酵培养基中,72 h内可以消耗10.52 g/L木糖产6.46 g/L的丁二酸,丁二酸的得率达到了0.78 mol/mol。而且突变株中伴有ATP产生的磷酸烯醇式丙酮酸羧激酶 (PCK) 途径得到加强,PCK的比酶活相对于出发菌株提高了19.33倍,使得其在厌氧条件下能够有足够的ATP供给来代谢木糖发酵产丁二酸。     

C．shehatae TZ8耐乙醇菌株的筛选及发酵性能研究

以C.shehataeTZ8为出发茵株,利用1％溶壁酶和1％蜗牛酶酶解1．5h,制备成C.shehataeTZ8原生质体,并对原生质体进行紫外诱变,以含不同浓度乙醇的木糖液体培养基培养进行初筛和复筛,获得一株遗传性能稳定、耐乙醇能力达5．5％（v／v）的蕾株C．shehataeTZ8-4,比初始菌株耐乙醇能力提高了2％。对突变株C．shehataeTZ8-4发酵性能的研究结果表明：C．shehataeTZ8-4发酵糖能力从80g/L（葡糖糖和木糖比为2：1）提高到120g／L,最大乙醇产量从27．41g／L提高到43．12g／L。     

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%。     

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.     

Enhancement of butanol tolerance and butanol yield in Clostridium acetobutylicum mutant NT642 obtained by nitrogen ion beam implantation

As a promising alternative biofuel, biobutanol can be produced through acetone/butanol/ethanol (ABE) fermentation. Currently, ABE fermentation is still a small-scale industry due to its low production and high input cost. Moreover, butanol toxicity to the Clostridium fermentation host limits the accumulation of butanol in the fermentation broth. The wild-type Clostridium acetobutylicum D64 can only produce about 13 g butanol/L and tolerates less than 2% (v/v) butanol. To improve the tolerance of C. acetobutylicum D64 for enhancing the production of butanol, nitrogen ion beam implantation was employed and finally five mutants with enhanced butanol tolerance were obtained. Among these, the most butanol tolerant mutant C. acetobutylicum NT642 can tolerate above 3% (v/v) butanol while the wide-type strain can only withstand 2% (v/v). In batch fermentation, the production of butanol and ABE yield of C. acetobutylicum NT642 was 15.4 g/L and 22.3 g/L, respectively, which were both higher than those of its parental strain and the other mutants using corn or cassava as substrate. Enhancing butanol tolerance is a great precondition for obtaining a hyper-yield producer. Nitrogen ion beam implantation could be a promising biotechnology to improve butanol tolerance and production of the host strain C. acetobutylicum.     

Bioproduction of butanol in bioreactors: new insights from simultaneous in situ butanol recovery to eliminate product toxicity

Simultaneous acetone butanol ethanol (ABE) fermentation by Clostridium beijerinckii P260 and in situ product recovery was investigated using a vacuum process operated in two modes: continuous and intermittent. Integrated batch fermentations and ABE recovery were conducted at 37 °C using a 14-L bioreactor (7.0 L fermentation volume) containing initial substrate (glucose) concentration of 60 g/L. The bioreactor was connected in series with a condensation system and vacuum pump. Vacuum was applied continuously or intermittently with 1.5 h vacuum sessions separated by 4, 6, and 8 h intervals. A control ABE fermentation experiment was characterized by incomplete glucose utilization due to butanol toxicity to C. beijerinckii P260, while fermentation coupled with in situ recovery by both continuous and intermittent vacuum modes resulted in complete utilization of glucose, greater productivity, improved cell growth, and concentrated recovered ABE stream. These results demonstrate that vacuum technology can be applied to integrated ABE fermentation and recovery even though the boiling point of butanol is greater than that of water.     

Efficient butanol production under aerobic conditions by coculture of Clostridium acetobutylicum and Nesterenkonia sp. strain F

Clostridium acetobutylicum is widely used for the microbial production of butanol in a process known as acetone–butanol–ethanol (ABE) fermentation. However, this process suffers from several disadvantages including high oxygen sensitivity of the bacterium which makes the process complicated and necessitate oxygen elimination in the culture medium. Nesterenkonia sp. strain F has attracted interests as the only known non-Clostridia microorganism with inherent capability of butanol production even in the presence of oxygen. This bacterium is not delimited by oxygen sensitivity, a challenge in butanol biosynthesis, but the butanol titer was far below Clostridia. In this study, Nesterenkonia sp. strain F was cocultivated with C. acetobutylicum to form a powerful “coculture” for butanol production thereby eliminating the need for oxygen removal before fermentation. The response surface method was used for obtaining optimal inoculation amount/time and media formulation. The highest yield, 0.31 g/g ABE (13.6 g/L butanol), was obtained by a coculture initiated with 1.5 mg/L Nesterenkonia sp. strain F and inoculated with 15 mg/L C. acetobutylicum after 1.5 hr in a medium containing 67 g/L glucose, 2.2 g/L yeast extract, 4 g/L peptone, and 1.4% (vol/vol) P2 solution. After butanol toxicity assessment, where Nesterenkonia sp. strain F showed no butanol toxicity, the coculture was implemented in a 2 L fermenter with continual aeration leading to 20 g/L ABE.     

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。     

Screening and characteristics of a butanol-tolerant strain and butanol production from enzymatic hydrolysate of NaOH-pretreated corn stover

As a gasoline substitute, butanol has advantages over traditional fuel ethanol in terms of energy density and hydroscopicity. However, solvent production appeared limited by butanol toxicity. The strain of Clostridium acetobutylicum was subjected to mutation by mutagen of N-methyl-N'-nitro-N-nitrosoguanidine for 0.5?h. Screening of mutants was done according to the individual resistance to butanol. A selected butanol-resistant mutant, strain 206, produced 50?% higher solvent concentrations than the wild-type strain when 60?g glucose/l was employed as substrate. The strain was also able to produce solvents of 23.47?g/l in 80?g/l glucose P2 medium after 70?h fermentation, including 5.41?g acetone/l, 15.05?g butanol/l and 3.02?g ethanol/l, resulting in an ABE yield and productivity of 0.32?g/g and 0.34?g/(l?h). Subsequently, Acetone-butanol-ethanol (ABE) production from enzymatic hydrolysate of NaOH-pretreated corn stover was investigated in this study. An ABE yield of 0.41 and a productivity of 0.21?g/(l?h) was obtained, compared to the yield of 0.33 and the productivity of 0.20?g/(l?h) in the control medium containing 52.47 mixed sugars. However, it is important to note that although strain 206 was able to utilize all the glucose rapidly in the hydrolysate, only 32.9?% xylose in the hydrolysate was used after fermentation stopped compared to 91.4?% xylose in the control medium. Strain 206 was shown to be a robust strain for ABE production from lignocellulosic materials and has a great potential for industrial application.     

Butanol production from agricultural residues: Impact of degradation products on Clostridium beijerinckii growth and butanol fermentation

During pretreatment and hydrolysis of fiber-rich agricultural biomass, compounds such as salts, furfural, hydroxymethyl furfural (HMF), acetic, ferulic, glucuronic, rho-coumaric acids, and phenolic compounds are produced. Clostridium beijerinckii BA101 can utilize the individual sugars present in lignocellulosic [e.g., corn fiber, distillers dry grain solubles (DDGS), etc] hydrolysates such as cellobiose, glucose, mannose, arabinose, and xylose. In these studies we investigated the effect of some of the lignocellulosic hydrolysate inhibitors associated with C. beijerinckii BA101 growth and acetone-butanol-ethanol (ABE) production. When 0.3 g/L rho-coumaric and ferulic acids were introduced into the fermentation medium, growth and ABE production by C. beijerinckii BA101 decreased significantly. Furfural and HMF are not inhibitory to C. beijerinckii BA101; rather they have stimulatory effect on the growth of the microorganism and ABE production.     

Production of acetone butanol ethanol (ABE) by a hyper-producing mutant strain of Clostridium beijerinckii BA101 and recovery by pervaporation.

A silicone membrane was used to study butanol separation from model butanol solutions and fermentation broth. Depending upon the butanol feed concentration in the model solution and pervaporation conditions, butanol selectivities of 20.88-68.32 and flux values of 158.7-215.4 g m(-)(2) h(-)(1) were achieved. Higher flux values (400 g m(-)(2) h(-)(1)) were obtained at higher butanol concentrations using air as sweep gas. In an integrated process of butanol fermentation-recovery, solvent productivities were improved to 200% of the control batch fermentation productivities. In a batch reactor the hyper-butanol-producing mutant strain C. beijerinckii BA101 utilized 57.3 g/L glucose and produced 24.2 g/L total solvents, while in the integrated process it produced 51.5 g/L (culture volume) total solvents. Concentrated glucose medium was also fermented. The C. beijerinckii BA101 mutant strain was not negatively affected by the pervaporative conditions. In the integrated experiment, acids were not produced. With the active fermentation broth, butanol selectivity was reduced by a factor of 2-3. However, the membrane flux was not affected by the active fermentation broth. The butanol permeate concentration ranged from 26.4 to 95.4 g/L, depending upon butanol concentration in the fermentation broth. Since the permeate of most membranes contains acetone, butanol, and ethanol (and small concentrations of acids), it is suggested that distillation be used for further purification.     

Production of butanol from glucose and xylose with immobilized cells of Clostridium acetobutylicum

Pretreated cotton towels were used as carriers to immobilize Clostridium acetobutylicum CGMCC 5234 cells for butanol or ABE production from glucose and xylose. Results showed that cell immobilization was a promising method to increase butanol concentration, yield and productivity regardless of the sugar sources compared with cell suspension. In this study, a high butanol concentration of 10.02 g/L with a yield of 0.20 g/g was obtained from 60 g/L xylose with 9.9 g/L residual xylose using immobilized cells compared with 8.48 g/L butanol and a yield of 0.141 g/g with 20.2 g/L residual xylose from 60 g/L xylose using suspended cells. In mixed-sugar fermentation (30 g/L glucose plus 30 g/L xylose), the immobilized cultures produced 11.1 g/L butanol with a yield of 0.190 g/g, which were 28.3% higher than with suspended cells (8.65 g/L) during which 30 g/L glucose was utilized completely using both immobilized and suspended cells while 3.46 and 13.1 g/L xylose maintained untilized for immobilized and suspended cells, respectively. Based on the results, we speculated that immobilized cells showed enhanced tolerance to butanol toxicity and the cultures preferred glucose to xylose during ABE fermentation. Moreover, the cultures showed obvious difference when grown between high initial concentrations of glucose and those of xylose. Repeated-batch fermentations from glucose with immobilized cells showed better long-term stability than from xylose. At last, the morphologies of free and immobilized cells adsorbed on pretreated cotton towels during the growth cycle were examined by SEM.     

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。