铁改性沸石对Clostridium acetobutylicum XY16固定效率及发酵性能的影响

考察4种无机铁盐改性沸石对丁醇生产菌Clostridium acetobutylicum XY16的固定效率及其发酵产丁醇性能的影响。结果表明:铁改性沸石对菌体的固定效率均优于未改性沸石,而Fe3+改性效果优于Fe2+,经FeCl3改性的沸石对菌体具有良好的吸附作用,当Fe3+-zeolite用量为180 g/L时,细胞的固定效率达到87%。在此基础上,比较了沸石负载的铁离子量对丁醇发酵性能的影响,沸石负载的铁离子量为6.0 mg/g时可显著提高丁醇发酵性能,当葡萄糖质量浓度为60 g/L时进行发酵,丁醇产量为13.5 g/L,总溶剂可达20 g/L,总溶剂的生产速率为0.385g/(L.h),比游离细胞发酵分别提高了9.5%、10.3%和40%。     

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%。     

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%。     

聚乙烯亚胺/戊二醛交联法固定化重组酯酶大肠杆菌细胞

利用聚乙烯亚胺/戊二醛交联法对重组酯酶大肠杆菌E.coli BL21细胞进行固定化研究,并对交联工艺条件进行优化。结果表明:在大肠杆菌细胞质量浓度200 g/L、硅藻土质量浓度2 g/L、聚乙烯亚胺(PEI)体积分数3%、交联时间1.5 h、戊二醛(GA)体积分数0.5%以及交联时间0.5 h时,固定化细胞的酯酶活力最高。固定化细胞的最适反应温度和pH分别为45℃和8.0,且温度稳定性和pH稳定性均高于游离细胞。当底物浓度为300mmol/L时,固定化细胞重复使用15批次后,其相对酶活仍能保留在80%以上。因此,该固定化细胞具有良好的操作稳定性。     

膜反应器固定化米根霉发酵产富马酸的工艺研究

以膜反应器固定化米根霉发酵产富马酸为研究对象,以Na2CO3为中和剂,考察固定化米根霉在5L搅拌式发酵罐中的发酵特征,采用智能可视化软件(IVOS)优化发酵工艺条件。结果表明,在80g/L初始糖浓及最优工艺下,富马酸产量、生产速率及转化率分别为21.1g/L、0.25g/(L·h)和28%;采用40g/L初始糖浓及连续批次发酵工艺时,富马酸产量、生产速率及转化率最高分别为10.8 g/L、0.36g/(L·h)和27%。搅拌式反应器中,固定化米根霉的膜反应器比表面积有限,以及菌膜的空间阻隔效应对传质传氧的限制作用,显著影响了富马酸的生产强度和转化率。因此,亟需发掘新的固定化方法及反应器形式,达到既解决米根霉形态控制问题,又有助于生产性状提升的目标。     

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。     

可发酵糖为底物不同菌种丁醇生产能力的研究

随着新一代生物质能源的研发,利用梭菌的发酵生产丁醇已成为热点。选用能生产丁醇的Clostridium acetobutylicum AS1.7,Clostridium acetobutylicum AS1.132,Clostridium acetobutylicumAS1.134和Clostridium beijerinckii NCMIB 8052,在多种糖源下进行发酵培养,通过比较其在不同糖源条件下的生长情况、糖利用率、丁醇及副产物产量、对丁醇、木糖耐受能力等,综合筛选出了最适用于发酵生产丁醇的备选菌种。NCMIB8052因具有最高产量、相对优良的耐受性及可利用多种糖源的特点,而被确定为发酵能力最强的菌种。     

低成本绿色循环工艺发酵生产丙酸联产维生素B12

对提取维生素B12后的费氏丙酸杆菌废菌体进行水解处理,考察以菌体水解液作为N源用于丙酸发酵的可行性.利用正交设计得到了提取维生素B12后的废菌体水解优化条件.基于此,构建利用植物纤维床反应器固定化生产丙酸联产维生素B12的低成本绿色循环工艺.结果表明:在4.5L的发酵体系中,单批次总糖质量浓度为200 g/L,发酵进行了5批次共1192h,丙酸生成总量为2 328.75 g,单批次丙酸质量浓度103.50 g/L,丙酸生产效率达0.43 g/(L·h),干菌质量浓度达到19.52 g/L.将菌体注入微好氧发酵罐中发酵获得112.8 mg/L维生素B12.     

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.     

Production of succinic acid and lactic acid by Corynebacterium crenatum under anaerobic conditions

A new succinic acid and lactic acid production bioprocess by Corynebacterium crenatum was investigated in mineral medium under anaerobic conditions. Corynebacterium crenatum cells with sustained acid production ability and high acid volumetric productivity harvested from the glutamic acid fermentation broth were used to produce succinic acid and lactic acid. Compared with the first cycle, succinic acid production in the third cycle increased 120% and reached 43.4 g/L in 10 h during cell-recycling repeated fermentations. The volumetric productivities of succinic acid and lactic acid could maintain above 4.2 g/(L·h) and 3.1 g/(L·h), respectively, for at least 100 h. Moreover, wheat bran hydrolysates could be used for succinic acid and lactic acid production by the recycled C. crenatum cells. The final succinic acid concentration reached 43.6 g/L with a volumetric productivity of 4.36 g/(L·h); at the same time, 32 g/L lactic acid was produced.     

循环利用重组大肠杆菌细胞转化合成丁二酸

研究了回收丁二酸发酵液中的大肠杆菌进行细胞转化的可行性,以转化率和生产效率为指标,考察了不同菌体浓度、底物浓度、pH调节剂对细胞转化的影响。发酵结果表明大肠杆菌可以在仅含有葡萄糖和pH调节剂的水环境中转化生产丁二酸,并确定了最佳的转化条件为:细胞浓度(OD600)50,底物浓度40g/L,缓冲盐为MgCO3。基于优化好的条件,在7L发酵罐中进行重复批次转化,第1次转化的转化率和生产效率分别达到91%和3.22g/(L·h),第2次转化的生产效率和转化率达到了86%和2.04g/(L·h),第3次转化的转化率和生产效率分别达到了83%和1.82g/(L·h)。     

大肠杆菌AFP111菌体回收连续转化生产丁二酸

在利用大肠杆菌AFP111厌氧发酵生产丁二酸过程中,随着产物丁二酸的不断积累,菌体活力和产酸能力逐渐降低,而通过回收菌体在新鲜培养基中重复发酵,可延长厌氧发酵时间,但是丁二酸生产效率较低。为了提高菌体回收丁二酸的转化效率,通过在回收菌体时有氧诱导 3 h,以纯水为培养基,进行丁二酸转化发酵。在连续进行 3 批次的发酵后,丁二酸的总产量和最终收率分别为 56.50 g/L和90%,生产速率达到了 0.81 g/(L·h),比未诱导情况下的生产速率提高了13%。     

Microbial production of xylitol from D-xylose and sugarcane bagasse hemicellulose using newly isolated thermotolerant yeast Debaryomyces hansenii

A thermotolerant yeast capable of fermenting xylose to xylitol at 40°C was isolated and identified as a strain of Debaryomyces hansenii by ITS sequencing. This paper reports the production of xylitol from D-xylose and sugarcane bagasse hemicellulose by free and Ca-alginate immobilized cells of D. hansenii. The efficiency of free and immobilized cells were compared for xylitol production from D-xylose and hemicellulose in batch culture at 40°C. The maximum xylitol produced by free cells was 68.6 g/L from 100 g/L of xylose, with a yield of 0.76 g/g and volumetric productivity 0.44 g/L/h. The yield of xylitol and volumetric productivity were 0.69 g/g and 0.28 g/L/h respectively from hemicellulosic hydrolysate of sugarcane bagasse after detoxification with activated charcoal and ion exchange resins. The Ca-alginate immobilized D. hansenii cells produced 73.8 g of xylitol from 100 g/L of xylose with a yield of 0.82 g/g and volumetric productivity of 0.46 g/L/h and were reused for five batches with steady bioconversion rates and yields.     

Anaerobic mixed‐culture fermentation of aqueous ammonia‐treated sugarcane bagasse in consolidated bioprocessing

The MixAlco process is an example of consolidated bioprocessing (CBP) in which anaerobic mixed‐culture fermentation biochemically converts any biodegradable feedstock into carboxylate salts. Downstream processing thermochemically transforms the resulting salts into mixed alcohol fuels or gasoline. To enhance digestibility, sugarcane bagasse was treated under mild conditions (55°C, 24 h, and 30% aqueous ammonia solution with a loading of 10 mL/g dry biomass). Using NH₄HCO₃ buffer, the feedstock (80% ammonia‐treated sugarcane bagasse/20% chicken manure) was anaerobically fermented by a mixed culture of marine microorganisms at 55°C. Four‐stage countercurrent fermentations were performed at various volatile solids loading rates (VSLRs) and liquid residence times (LRTs). The highest acid productivity (1.14 g/(L day)) occurred at a total acid concentration of 29.8 g/L. The highest conversion (65%) occurred at a total acid concentration of 27.6 g/L. The continuum particle distribution model (CPDM) predicted the experimental total acid concentrations and conversions within 4.98% and 10.41%, respectively. When using NH₄HCO₃ buffer, ammonia pretreatment is an attractive option. The CPDM “map” shows that both high volatile solid conversions (78.8%) and high acid concentrations (32.6 g/L) are possible with 300 g/(L liquid) substrate concentration, 30 days LRT, 2 g/(L day) solid loading rate and NH₄HCO₃ buffer. Biotechnol. Bioeng. 2010;106: 216–227. © 2010 Wiley Periodicals, Inc.     

Biobutanol production in a Clostridium acetobutylicum biofilm reactor integrated with simultaneous product recovery by adsorption

Background

Clostridium acetobutylicum can propagate on fibrous matrices and form biofilms that have improved butanol tolerance and a high fermentation rate and can be repeatedly used. Previously, a novel macroporous resin, KA-I, was synthesized in our laboratory and was demonstrated to be a good adsorbent with high selectivity and capacity for butanol recovery from a model solution. Based on these results, we aimed to develop a process integrating a biofilm reactor with simultaneous product recovery using the KA-I resin to maximize the production efficiency of biobutanol.

Results

KA-I showed great affinity for butanol and butyrate and could selectively enhance acetoin production at the expense of acetone during the fermentation. The biofilm reactor exhibited high productivity with considerably low broth turbidity during repeated batch fermentations. By maintaining the butanol level above 6.5 g/L in the biofilm reactor, butyrate adsorption by the KA-I resin was effectively reduced. Co-adsorption of acetone by the resin improved the fermentation performance. By redox modulation with methyl viologen (MV), the butanol-acetone ratio and the total product yield increased. An equivalent solvent titer of 96.5 to 130.7 g/L was achieved with a productivity of 1.0 to 1.5 g?·?L^-1?·?h^-1. The solvent concentration and productivity increased by 4 to 6-fold and 3 to 5-fold, respectively, compared to traditional batch fermentation using planktonic culture.

Conclusions

Compared to the conventional process, the integrated process dramatically improved the productivity and reduced the energy consumption as well as water usage in biobutanol production. While genetic engineering focuses on strain improvement to enhance butanol production, process development can fully exploit the productivity of a strain and maximize the production efficiency.     

An novel immobilization method of Saccharomyces cerevisiae to sorghum bagasse for ethanol production

Natural sorghum bagasse without any treatment was used to immobilize Saccharomyces cerevisiae at 0.6+/-0.2g dry cell weight (DCW)/g dry sorghum bagasse weight (DSW) through solid-state or semi-solid state incubation. The scanning electron microscopy (SEM) of the carriers revealed the friendship between yeast cells and sorghum bagasse are adsorption and embedding. The ethanol productivity of the immobilized cells was 2.24 times higher than the free cells. In repeated batch fermentation with an initial sugar concentration of 200g/L, nearly 100% total sugar was consumed after 16 h. The ethanol yield and productivity were 4.9 g/g consumed sugar on average and 5.72 g/(Lh), respectively. The immobilized cell reactor was operated over a period of 20 days without breakage of the carriers, while the free cell concentration in the effluent remained less than 5 g/L thoughout the fermentation. The maximum ethanol productivity of 16.68 g/(Lh) appeared at the dilution rate of 0.3h(-1).     

电子载体对丁醇发酵的影响

研究在培养基中加入不同电子载体对丁醇发酵的影响。结果表明:添加微量的苄基紫精可以促进丁醇的产生,同时可强烈抑制丙酮的合成,丁醇体积分数由66.92%提高到82.35%。苄基紫精可促进菌株快速进入产溶剂期,发酵周期明显缩短,丁醇生产强度显著提高。7%玉米培养基中加入40 mg/L苄基紫精,丁醇产量最高达16.10 g/L,生产强度为0.37 g/(L.h),分别较对照提高10.96%和60.87%。在初始丁醇体积分数较低的条件下,苄基紫精对丁醇合成的促进作用更明显。     

分阶段pH调控提高2-酮基-L-古龙酸生产

为了提高酮古龙酸菌Ketogulonicigenium vulgare和巨大芽胞杆菌Bacillus megaterium生产2-酮基-L-古龙酸(2-KLG)的生产效率,分析了pH对K.vulgare和B.megaterium生长和产酸的影响,发现K.vulgare和B.megaterium的最适生长pH值分别为6.0和8.0,但是K.vulgare的糖酸转化活力在pH7.0时达到最大值,因此提出了三阶段pH控制策略(第一阶段:0～8h,pH8.0;第二阶段:8～20h,pH6.0;第三阶段:20h至发酵结束,pH7.0)以促进K.vulgare生长和2-KLG生产。结果表明,三阶段pH控制策略的实施进一步提高了2-KLG的产量(77.3g/L)、生产强度(1.38g/(L·h))和L-山梨糖消耗速率(1.42g/(L·h)),分别比恒定pH7.0时提高了9.7%、33.2%和25.7%。     

发酵产丁二酸过程中废弃细胞的循环利用

对厌氧发酵产丁二酸后的废弃细胞进行破壁处理,考察了以细胞水解液作为有机氮源重新用于丁二酸发酵的可行性。比较了超声破碎、盐溶、酶解3种方法破碎细胞获得的水解液作为氮源发酵产丁二酸的效果,结果表明酶解制得的细胞水解液效果最佳。以总氮含量为1.11g/L的酶解液(相当于10g/L酵母膏)作为氮源发酵,丁二酸产量可达42.0g/L,继续增大酶解液用量对耗糖、产酸能力没有显著提高。将细胞酶解液与5g/L酵母膏联用发酵36h后,丁二酸产量达75.5g/L,且丁二酸生产强度为2.10g/(L·h),比使用10g/L酵母膏时提高了66.7%。因此,厌氧发酵产丁二酸结束后的废弃细胞酶解液可以替代原培养基中50%的酵母膏用于发酵。