膜生物反应器封闭循环发酵系统对野生酵母的驯化

自然界中选育的野生酵母菌S3在PDMS膜生物反应器长期封闭循环系统中进行驯化,在经过3轮连续的封闭循环发酵适应性驯化实验后,对第四轮实验和第一轮实验的酵母发酵性能进行比较,分析野生酵母的适应性进化行为,乙醇-细胞平均比产率、葡萄糖转化率分别提高42.6%、2.1%,细胞平均死亡率减少11.04%;与商用的安琪酵母（ADY）相比,发酵性能也更优秀。结果表明在膜生物反应器封闭循环发酵系统中可以使野生酵母菌得到驯化。      

野生酵母菌在膜生物反应器中的适应性培养与发酵动力学研究

进行了野生酵母菌在硅橡胶膜生物反应器长期封闭循环连续发酵环境中的多轮发酵-优势菌株筛选-转移培养的适应性进化实验,研究了菌株的发酵性能变化和发酵动力学.发酵实验共进行了5轮,每轮发酵持续时间500h,且采用前一轮发酵残液中筛选出的优势菌株作为发酵菌种.第4轮、第5轮发酵实验在乙醇产率、乙醇生成比速率、细胞比生长率等发酵性能上较第一轮有显著提高.多轮适应性培养有效增强了酵母细胞对乙醇的耐受力以及缩短了发酵适应期.     

利用硅橡胶膜生物反应器适应性进化培育耐持久力酵母菌株

酵母是影响乙醇发酵的关键因素之一。本实验设计了一种通过适应性进化改进酵母菌株性能的方法:将工业酵母菌株接种到硅橡胶膜生物反应器中进行长期封闭循环连续发酵,通过递进转移的方式进行适应性培养并筛选出优势酵母菌株。硅橡胶膜渗透汽化同步分离乙醇使发酵过程长期连续运行,发酵代谢产物中多种非透过性物质连续累积,发酵液不断恶化的环境为酵母适应性进化提供了选择压力。通过实验发现,从每轮500 h连续发酵终止时分离繁衍约200代的菌株,在残液培养基中具有更高的生长速率和乙醇产率,表现出明显的适应性进化特征。研究表明,采用这种方法选育的酵母菌株,延长封闭循环发酵的时间并维持较高的乙醇得率是可行的,对推进封闭循环乙醇发酵生产工艺的发展有利。     

膜生物反应器用于乙醇连续发酵菌株的适应性培养

将一种耐高温酿酒干酵母在体积为5L、膜而积为0.08m2的硅橡胶膜生物反应器乙醇连续发酵系统中进行适应性培养,选育出适应该环境的优势菌种.三轮乙醇连续发酵实验的结果表明,糖的转化率差别不大,但单位酵母细胞代谢产生乙醇的能力有了提高,说明酵母细胞在长期封闭循环发酵环境条件下,原种菌经过几百代繁殖后其后代遗传特征发生了改变,能逐渐适应所处的不利环境.表明通过膜生物反应器长期封闭循环发酵对酵母细胞进行适应性培养,可以获得具有某种遗传优势的新菌株.     

用硅橡胶膜生物反应器系统选育高性能酿酒酵母

应用硅橡胶膜生物反应器的发酵-渗透汽化耦合功能,进行长期封闭循环发酵,使酵母在这种特定环境下完成适应性自然进化,从而获得优势的突变株。以工业安琪酵母ADY为原始菌株,进行每一轮500h的封闭循环发酵,在发酵终点取菌悬液做平板筛菌,分离出遗传约200代的相对强势的新菌株,再转移新菌株进行同样的发酵和终点筛选实验。如此的发酵-筛选-转移实验共进行了3轮。试验结果表明:ADY遗传超过600代的菌株S33,在500h封闭循环发酵残液配制的培养基条件下,表现出了明显的环境适应性遗传优势,其细胞生长速率、产物乙醇的比生成速率和糖转化率都相对原种菌ADY分别提高了约71.8%、53.6%和52.7%。     

细胞稀释强化硅橡胶膜生物反应器连续乙醇发酵

通过原位重力沉降分离酵母和渗透汽化分离乙醇构建了细胞稀释连续乙醇发酵的硅橡胶膜生物反应器。采用酿酒酵母,以0.05/h的细胞稀释率在膜生物反应器中实现了170 h的连续稳定乙醇发酵。重力沉降分离酵母对硅橡胶膜生物反应器产生的细胞稀释作用可以通过反应器内酵母自身生长得到平衡,发酵液细胞浓度稳定在5g/L。渗透汽化原位分离使发酵液内乙醇浓度维持在50 g/L。细胞稀释膜生物反应器连续发酵的乙醇体积产率达到1.63 g/(L.h),相对于同等工艺参数的细胞封闭循环膜生物反应器连续乙醇发酵细胞比产率提高了31%。     

PDMS膜生物反应器中ABE发酵的实验研究

采用PDMS膜生物反应器和丙酮丁醇梭菌进行了丙酮-丁醇-乙醇(ABE)发酵实验研究.进行了2轮封闭循环连续发酵实验,发酵时间分别为264h和300h.2轮实验的发酵-膜分离耦合操作模式有区别:第1轮实验仅在白天(每天8:00～20:00)运行膜渗透汽化同步分离提取ABE产物(间断耦合);第2轮实验则在前192 h发酵阶段全程运行膜同步分离(连续耦合),之后采用和第1轮相同的间断耦合发酵模式.结果表明,第2轮发酵细胞的生长状况和发酵能力好于第1轮;2轮实验发酵液平均浓度分别为1.781g/L和1.884g/L;第2轮实验丁醇体积产率、得率系数、丁醇总产量分别比第1轮提高了95.2％、14.2％、121.2％.对发酵动力学行为进行了初步分析.     

补充营养物对硅橡胶膜生物反应器长期封闭循环发酵性能的影响

通过向发酵液中补充营养物质,研究了补充营养物对膜生物反应器长期封闭循环发酵行为的影响;实验得到的细胞浓度为7.10 g/L,细胞存活率为0.90,发酵液内的平均乙醇浓度为68.6 g/L,乙醇产率为1.85 g/(L·h),乙醇得率为0.411 g/g,乙醇比产率为0.286 g/(g·h),与不补充营养物质的实验相比,发酵性能有所提高;实验发现补充营养物质,没有提高细胞浓度和细胞存活率,而是增强了细胞的生命活性,提高了细胞产乙醇的能力。     

固定化酵母-膜生物反应器连续发酵生产乙醇的研究

对固定化酿酒活性干酵母-硅橡胶膜生物反应器连续发酵生产乙醇进行了实验研究。对固定化酵母和游离酵母的发酵能力进行了比较,实验研究了系统在长期运行过程中的发酵反应动力学参数和膜传质动力学参数等基本性能。结果表明,酵母细胞固定化后,其发酵速度有明显提高;膜渗透汽化分离产物乙醇使发酵反应能够长时间连续稳定地进行,葡萄糖消耗率和乙醇体积产率都较间歇发酵有明显的提高;在连续运行中,发酵液中葡萄糖浓度控制在20～50g/L的范围内,乙醇体积产率为4.0g/（L·h）,罐内乙醇浓度基本保持在45g/L左右,固定化酵母浓度维持在1.05×10^10～2.15×10^10个/克胶,发酵液中游离酵母浓度维持在0.02×10^10～0.098×10^10个/mL,膜的渗透通量和分离因子分别为300～500g/（m^2·h）和3.6～7.2;实验系统连续运行了294h,收得渗透冷凝液4841.7g,平均质量分数为21.3%。     

猕猴桃发酵果酒的研制

通过对猕猴桃发酵果酒的发酵工艺以及筛选的野生猕猴桃酵母菌种的发酵性能的研究,结果表明:被筛选的猕猴桃酵母菌株具有良好的发酵性能;在主发酵期间,酵母用量确定为7.0%～9.0%,发酵温度为27℃～30℃。     

Characterization of membrane bioreactor for dry wine production

Characteristics of yeast growth and ethanol fermentation were examined in membrane bioreactor using a grape juice. After inoculation, batch fermentation was carried out for 24 h. When yeast growth reached the stationary phase, continuous fermentation was initiated. In continuous fermentation, a linear relationship was observed between cell concentration and dilution rate. In single-vessel membrane bioreactor, the cell concentrations of 18.7 g/l and 76.9 g/l (15 and 60 times higher than that of the batch fermentation, respectively) were observed at dilution rates of 0.1 h(-1) and 0.3 h(-1), respectively. The residual sugar concentration was higher than 10 g/l at the dilution rate of 0.1 h(-1), 0.2 h(-1) or 0.3 h(-1), therefore the single-vessel membrane bioreactor was not suitable for producing dry wine (sugar concentration: 4 g/l or less). In the double-vessel membrane bioreactor, it is most suitable to set the recycle ratio at 0.15 for keeping the sugar concentration below 4 g/l. The productivity of dry wine in the double-vessel membrane bioreactor was 28 times higher than that in the batch fermentation.     

野生蓝莓保健果酒的工艺优化

以实验室保藏的产酯酵母T1及耐酸酵母菌株YM1-1为试验菌株,野生蓝莓为原料,通过单因素及正交试验研究了初始pH值、菌种复配比例、接种量、发酵温度、SO2添加量等对蓝莓保健酒品质的影响,确定了最佳的主发酵工艺。结果显示,蓝莓果酒的最佳主发酵工艺为SO2添加量60 mg/L,产酯酵母∶耐酸酵母接种比例1∶2（V∶V）,接种量6.0%,果胶酶添加量30 mg/L,发酵初始pH值3.8,发酵温度26 ℃。在此条件下发酵得到果香浓郁、口感醇厚的低酒精度蓝莓酒,其酒精度为4.2%vol,残糖量为5.0 °Bx,感官评分为87分,花青素含量为368.5 mg/L,糖醇转化率为33.6%。     

响应面法优化野生火棘果保健果酒发酵工艺

以实验室保藏的产酯酵母菌株T6-1和耐酸酵母菌株Y2-1为试验菌株,野生火棘果和枸杞干果为原料制作保健果酒。在单因素试验基础上,选取接种量、初始pH值、发酵温度、发酵时间为影响因子,以野生火棘果保健果酒感官评分为响应值,利用Box- Behnken试验设计,建立发酵工艺回归模型并进行响应面分析。结果表明,野生火棘果保健果酒发酵的最佳条件为产酯酵母菌株T6-1和耐酸酵母菌株Y2-1的接种比例1∶4（V/V）,接种量9%,初始pH值5.5,发酵温度22 ℃,发酵时间8 d。在此优化条件下,野生火棘果保健果酒的色泽鲜艳、果香浓郁、口感醇厚、整体饱满协调,酒精度为11.5%vol～12.0%vol,感官评分达到94.61分。     

Optimization of ethanol production from starch by an amylolytic nuclear petite Saccharomyces cerevisiae strain

Ethanol fermentation characteristics of the 100% respiratory-deficient nuclear petite amylolytic Saccharomyces cerevisiae NPB-G strain was investigated in both shake-flask and controlled bioreactor cultivation conditions, and comparison with the earlier reported results revealed 54.6% increase in ethanol yield. Efforts to improve the starch utilization rate by increasing the selection pressure or supplying the fermentation medium with glucose did not prevent the observed decrease in time-dependent amylolytic activity. Response surface methodology (RSM) was then used as a statistical tool to optimize the initial yeast extract and starch contents of the medium, which resulted in a substantial increase in the stability of the expression plasmid in both the respiratory-deficient NPB-G and the parental respiratory-sufficient WTPB-G strains, with concomitant improvement in their amylolytic potentials. High ethanol yields on substrate values of the bioreactor cultures, which were very close to the theoretical yield, indicated that the amylolytic respiratory-deficient NPB-G strain was effective in the direct fermentation of starch into ethanol.     

Reduced acetaldehyde production by genome shuffling of an industrial brewing yeast strain

High levels of acetaldehyde produced by yeast during fermentation can be of concern to product quality. A novel approach, based on genome shuffling, was applied to reduce the production of acetaldehyde by industrial brewing strain YS86. Four isolates with different impacts of acetaldehyde concentration were obtained from populations generated by ultraviolet irradiation and nitrosoguanidine mutagenesis. These yeast strains were then subjected to recursive pool‐wise protoplast fusion. A strain library that was likely to yield positive colonies was created by fusing the lethal protoplasts obtained from both UV irradiation and heat treatments. After two rounds of genome shuffling, a recombinant YSF2–9 strain produced less acetaldehyde than wild‐type strain YS86, by 64.5 and 66.2% in laboratory and pilot plant fermentations, respectively. The shuffled yeast strain YSF2–9 was genetically stable and may have a potential application in brewing industry for managing acetaldehyde in beer. Copyright © 2017 The Institute of Brewing & Distilling     

Increase of xylitol productivity by cell-recycle fermentation of Candida tropicalis using submerged membrane bioreactor

Candida tropicalis, an osmophilic strain isolated from honeycomb, produced xylitol at a maximal volumetric productivity of 3.5 g l(-1) h(-1) from an initial xylose concentration of 200 g l(-1). Even at a very high xylose concentration, e.g., 350 g l(-1), this strain produced xylitol at a moderate rate of 2.07 g l(-1) h(-1). In a fed-batch fermentation of xylose and glucose, 260 g l(-1) xylose was added, and the xylitol production was 234 g l(-1) for 48 h, corresponding to a rate of 4.88 g l(-1) h(-1). To increase xylitol productivity, cells were recycled in a submerged membrane bioreactor with suction pressure and air sparging. For each recycle round in cell-recycle fermentation, the average concentration of xylitol produced, fermentation time, volumetric productivity, and product yield were 180 g l(-1), 19.5 h, 8.5 g l(-1) h(-1), and 85%, respectively. When cell-recycle fermentation was started with the cell mass concentrated twofold after batch fermentation and performed for 10 recycle rounds, we achieved a very high productivity of 12 g l(-1) h(-1). The productivity and total amount of xylitol in cell-recycle fermentation were 3.4- and 11.0-fold higher than those in batch fermentation, respectively.