固定化热带假丝酵母发酵氨浸稻秸水解液生产木糖醇

采用海藻酸钙固定化热带假丝酵母细胞发酵氨水浸泡稻秸半纤维素水解液生产木糖醇。为了提高木糖醇的转化率,对发酵条件进行了研究。发酵在250 mL锥形瓶中进行。向水解液中补充适量氮源和营养盐等营养物质提高了木糖醇的生产速率,但木糖醇转化率没有因此而提高。适宜的初始pH和细胞干浓度分别为4-5和1.22 g/L。在这些条件下,进行了固定化细胞重复法较高浓缩度水解液的试验。结果发现,固定化细胞能在初始木糖浓度为104.2 g/L的水解液中重复批式发酵5次,木糖醇平均得率和生产速率分别为0.737 g/g和0.533 g/(L.h)。     

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.     

一株热带假丝酵母木糖醇高产新菌株的筛选

自木糖厂环境样品中分离出一株编号为1-18的产木糖醇分离物。在初始木糖浓度200g/L的条件下重复循环利用1-18细胞摇瓶发酵,木糖醇转化率高达0.91(木糖醇g/木糖g),接近理论极限值;木糖醇生成速率达6.7g/L·h。无论产物浓度,产物生成速率与产物转化率都达到了令人满意的高水平。1-18发酵液以40ml/kg·d的最大剂量饲喂小白鼠一周,受试动物未出现中毒反应。分离物1-18在木糖醇生产中显然具有重要的生物工程应用潜力。根据常规形态鉴别,生理生化实验测定,以及26S rDNA D1/D2区域核酸序列同源性比较分析,证实分离物1-18属于Candida tropicalis(热带假丝酵母)。分离物1-18已保存于中国典型培养物保藏中心,保藏编号CCTCC M 205067,其26S rDNA D1/D2区域核酸序列在GenBank的核酸序列登记号为DQ176428。     

Fed‐batch l‐(+)‐lactic acid fermentation of brewer's spent grain hydrolysate

Brewer's spent grain (BSG) hydrolysates were used for l ‐(+)‐lactic acid (LA) fermentation by Lactobacillus rhamnosus ATCC 7469. The aim of this study was to evaluate fed‐batch LA fermentation of BSG hydrolysate with the addition of glucose, glucose and yeast extract, and wort during LA fermentation and its effect on fermentation parameters such as LA concentration, its volumetric productivity and yield, and L. rhamnosus cell viability. The highest LA yield, volumetric productivity and concentration of 93.3%, 2.0 g/L/h, and 116.1 g/L, respectively, were achieved with glucose and yeast extract addition during fermentation. In fed‐batch fermentation with glucose and yeast extract addition significantly higher LA concentration, yield and volumetric productivity (by 194.8; 2.2, and 20.7%, respectively) were achieved compared with batch fermentation. The results indicated that fed‐batch fermentation could be used to increase LA fermentation efficiency. Copyright © 2017 The Institute of Brewing & Distilling     

Ethanol production by a new pentose-fermenting yeast strain, Scheffersomyces stipitis UFMG-IMH 43.2, isolated from the Brazilian forest

The ability of a recently isolated Scheffersomyces stipitis strain (UFMG-IMH 43.2) to produce ethanol from xylose was evaluated. For the assays, a hemicellulosic hydrolysate produced by dilute acid hydrolysis of sugarcane bagasse was used as the fermentation medium. Initially, the necessity of adding nutrients (MgSO(4)·7H(2)O, yeast extract and/or urea) to this medium was verified, and the yeast extract supplementation favoured ethanol production by the yeast. Then, in a second stage, assays under different initial xylose and cell concentrations, supplemented or not with yeast extract, were performed. All these three variables showed significant (p < 0.05) influence on ethanol production. The best results (ethanol yield and productivity of 0.19 g/g and 0.13 g/l/h, respectively) were obtained using the hydrolysate containing an initial xylose concentration of 30 g/l, supplemented with 5.0 g/l yeast extract and inoculated with an initial cell concentration of 2.0 g/l. S. stipitis UFMG-IMH 43.2 was demonstrated to be a yeast strain with potential for use in xylose conversion to ethanol. The establishment of the best fermentation conditions was also proved to be of great importance to increasing the product formation by this yeast strain. These findings open up new perspectives for the establishment of a feasible technology for ethanol production from hemicellulosic hydrolysates.     

The level of glucose-6-phosphate dehydrogenase activity strongly influences xylose fermentation and inhibitor sensitivity in recombinant Saccharomyces cerevisiae strains

Disruption of the ZWF1 gene encoding glucose-6-phosphate dehydrogenase (G6PDH) has been shown to reduce the xylitol yield and the xylose consumption in the xylose-utilizing recombinant Saccharomyces cerevisiae strain TMB3255. In the present investigation we have studied the influence of different production levels of G6PDH on xylose fermentation. We used a synthetic promoter library and the copper-regulated CUP1 promoter to generate G6PDH-activities between 0% and 179% of the wild-type level. G6PDH-activities of 1% and 6% of the wild-type level resulted in 2.8- and 5.1-fold increase in specific xylose consumption, respectively, compared with the ZWF1-disrupted strain. Both strains exhibited decreased xylitol yields (0.13 and 0.19 g/g xylose) and enhanced ethanol yields (0.36 and 0.34 g/g xylose) compared with the control strain TMB3001 (0.29 g xylitol/g xylose, 0.31 g ethanol/g xylose). Cytoplasmic transhydrogenase (TH) from Azotobacter vinelandii has previously been shown to transfer NADPH and NAD(+) into NADP(+) and NADH, and TH-overproduction resulted in lower xylitol yield and enhanced glycerol yield during xylose utilization. Strains with low G6PDH-activity grew slower in a lignocellulose hydrolysate than the strain with wild-type G6PDH-activity, which suggested that the availability of intracellular NADPH correlated with tolerance towards lignocellulose-derived inhibitors. Low G6PDH-activity strains were also more sensitive to H(2)O(2) than the control strain TMB3001.     

不依赖IPTG诱导产木糖醇大肠杆菌工程菌的构建

该研究采用分子生物学技术构建不依赖异丙基-β-D-硫代半乳糖苷（IPTG）诱导产木糖醇的大肠杆菌（Escherichia coli）工程菌,并研究启动子、质粒拷贝数、诱导剂IPTG的添加、基因xylA和xylB的敲除以及木糖含量对工程菌发酵产木糖醇的影响。结果表明,当转速为200 r/min时,E. coli AI07/pWYZ-1（启动子为lacP）的木糖醇产量是E. coli AI07/pAGI02（启动子为pflB-p6）的1.8倍;E. coli AI07/pWYZ-2（中拷贝质粒pBR322）的木糖醇产量高于E. coli AI07/pWYZ-1（高拷贝质粒pUC19）,分别为19.56 g/L、7.90 g/L;是否添加诱导剂IPTG对E. coli AI07/pWYZ-2产木糖醇影响不大,且在发酵96 h时,木糖醇产量极显著高于E. coli AI05/pWYZ-2（P＜0.01）,其在不添加IPTG的条件下,当木糖初始质量浓度为80 g/L,发酵时间为108 h时,木糖醇产量达48.7 g/L。     

Brewers' spent grain and thin stillage as raw materials in l‐(+)‐lactic acid fermentation

Brewer's spent grain (BSG) hydrolysates were used for l ‐(+)‐lactic acid (LA) fermentation by Lactobacillus rhamnosus ATCC 7469. In this study the effect of the addition of various amounts of thin stillage (TS) in BSG hydrolysate on LA fermentation parameters were evaluated. TS addition significantly increased utilization of glucose by up to 43.0%. In batch fermentation the highest LA concentration and volumetric productivity of 31.0 g/L, and 0.93 g/L/h, respectively, were obtained with the addition of 50% TS. L. rhamnosus cell viability also increased with the addition of 50% TS (by 2.4%). TS addition significantly increased free amino nitrogen concentration (by up to 209%) which is important for bacterial growth. A strong positive correlation between free amino nitrogen and LA concentration was noted. Compared with the results obtained in the batch fermentation (50% TS), significantly higher LA concentration, yield and volumetric productivity (54.8, 1.9 and 4.0%, respectively) were achieved in fed‐batch fermentation with glucose and TS addition. The results suggest that the combination of the by‐products of brewing and bioethanol industries could be suitable for LA production. Copyright © 2017 The Institute of Brewing & Distilling     

Production of ethanol from liquefied cassava starch using co-immobilized cells of Zymomonas mobilis and Saccharomyces diastaticus

Co-immobilized cells of Saccharomyces diastaticus and Zymomonas mobilis produced a high ethanol concentration compared to immobilized cells of S. diastaticus during batch fermentation of liquefied cassava starch. The co-immobilized cells produced 46.7 g/l ethanol from 150 g/l liquefied cassava starch, while immobilized cells of yeast S. diastaticus produced 37.5 g/l ethanol. The concentration of ethanol produced by immobilized cells was higher than that by free cells of S. diastaticus and Z. mobilis in mixed-culture fermentation. In repeated-batch fermentation using co-immobilized cells, the ethanol concentration increased to 53.5 g/l. The co-immobilized gel beads were stable up to seven successive batches. Continuous fermentation using co-immobilized cells in a packed bed column reactor operated at a flow rate of 15 ml/h (residence time, 4 h) exhibited a maximum ethanol productivity of 8.9 g/l/h.     

木糖浓度及补料发酵对树干毕赤酵母乙醇发酵的影响

探究不同浓度木糖及补料对树干毕赤酵母（Pichia stipitis）菌株1K-9发酵木糖产乙醇的影响,提高木糖产乙醇的发酵水平,为扩大规模发酵木糖产乙醇打下基础。结果表明,菌株1K-9先采用10%木糖进行乙醇发酵,36 h补加与10%木糖培养基等体积的20%木糖培养基继续发酵,发酵至108 h时菌数也达到了（12.16±0.07）×108个/mL,较未补料发酵时有所提高;发酵108 h时醪液中残留的木糖含量为（1.03±0.02）g/L,较未补料发酵时有所降低;乙醇含量达到了6.56%vol,较未补料时提高了1.85%vol。因此补料发酵是有效的。     

Continuous production of pectinase by immobilized yeast cells on spent grains

A yeast strain secreting endopolygalacturonase was used in this work to study the possibility of continuous production of this enzyme. It is a feasible and interesting alternative to fungal batch production essentially due to the specificity of the type of pectinase excreted by Kluyveromyces marxianus CCT 3172, to the lower broth viscosity and to the easier downstream operations. In order to increase the reactors' productivity, a cellulosic carrier obtained from barley spent grains was tested as an immobilization support. Two types of reactors were studied for pectinase production using glucose as a carbon and energy source--a continuous stirred tank reactor (CSTR) and a packed bed reactor (PBR) with recycled flow. The highest value for pectinase volumetric productivity (P(V)=0.98 U ml(-1) h(-1)) was achieved in the PBR for D=0.40 h(-1), a glucose concentration on the inlet of S(in)=20 g l(-1), and a biomass load in the support of X(i)=0.225 g g(-1). The results demonstrate the attractiveness of the packed bed system for pectinase production.     

工业酿酒酵母木糖代谢途径的建立及酒精发酵初步研究

以酵母AS2.1190为出发菌株,把含有木糖还原酶基因(XYL1)、木糖醇脱氢酶基因(XYL2)以及木酮糖激酶基因(XKS1)的质粒载体pYMIKP-xy127线性化后多拷贝整合进入其基因组,筛选得到基因工程菌株GZ4-127,并对此工程菌株进行葡萄糖、木糖共发酵试验.结果显示GZ4-127比出发菌株的菌体密度提高5%,木糖消耗提高2倍,酒精产率提高12%,说明工程菌已能够有效地利用木糖生产乙醇.     

Effective onion vinegar production by a two-step fermentation system

A two-step fermentation system combining a repeated batch process using a flocculating yeast with a charcoal pellet bioreactor was developed for onion vinegar production. Juice from the red onion R-3, which contained 67.3 g/l total sugar, was smoothly converted to onion alcohol containing 30.6 g/l ethanol by repeated batch operation using the flocculating yeast Saccharomyces cerevisiae strain IR-2. Stable operation was possible and the maximum productivity was about 8.0 g/l/h. A packed bed bioreactor containing charcoal pellets produced from waste mushroom medium was then applied to continuous onion vinegar production from the onion alcohol. Onion vinegar was successfully produced, with a maximum productivity and acetic acid concentration of about 3.3 g/l/h and 37.9 g/l, respectively. The total acetic acid yield calculated from the amount of sugar consumed was 0.86. The two-step system was operated for 50 d and proved to be competitive with other systems in terms of its high productivity, high acetic acid yield, operational stability and low production costs.     

Fed-batch coculture of Lactobacillus kefiranofaciens with Saccharomyces cerevisiae for effective production of kefiran

In a batch coculture of kefiran-producing lactic acid bacteria Lactobacillus kefiranofaciens and lactate-assimilating yeast Saccharomyces cerevisiae, lactate accumulation in the medium was observed, which inhibited kefiran production. To enhance kefiran productivity by preventing lactate accumulation, we conducted lactose-feeding batch operation with feedforward/feedback control during the coculture, so that the lactate production rate of L. kefiranofaciens was balanced with the lactate consumption rate of S. cerevisiae. The lactate concentration was maintained at less than 6 g l(-1) throughout the fed-batch coculture using a 5 l jar fermentor, although the concentration reached 33 g l(-1) in the batch coculture. Kefiran production was increased to 6.3 g in 102 h in the fed-batch coculture, whereas 4.5 g kefiran was produced in 97 h in the batch coculture. The kefiran yield on lactose basis was increased up to 0.033 g g(-1) in the fed-batch coculture, whereas that in the batch coculture was 0.027 g g(-1).     

Xylooligosaccharide fermentation with Leuconostoc lactis

Strains of Leuconostoc lactis SHO-47 and Le. lactis SHO-54, producing the clinically useful enzyme NAD-specific 6-phosphoglucanate dehydrogenase, were cultivated with a hydrolyzed birch wood xylan as the unique carbon source to produce D-lactic acid for poly(D-lactic acid). In addition to the strains SHO-47 and SHO-54, Lactococcus lactis IO-1, well known as a good xylose-utilizing lactic acid bacterium, was used as a control to confirm the extent of hemicellulose hydrolysis. The fermentation time for lactic acid of strains SHO-47 and SHO-54 was 12 h, and produced respectively 2.3 and 2.2 g/l lactic acid from 8.5 g/l hydrolyzed xylan, whereas the fermentation time of strain IO-1 was 21 h, and produced 1.3 g/l lactic acid. Xylooligosaccharides from xylobiose to xylohexose were utilized more rapidly than xylose in the cultures of strains SHO-47 and SHO-54. However, xylose concentration increased temporarily and then decreased in the culture of strain IO-1. On the other hand, xylooligosaccharides larger than xyloheptaose were not utilized by these three strains. The xylosidase activities of SHO-47, SHO-54, and IO-1 were induced by xylose or a mixture of xylobiose and xylotriose. The xylosidases of these three strains were localized in their cytoplasm.     

THE ENHANCEMENT OF AMMONIA PRETREATMENT ON THE FERMENTATION OF RICE STRAW HYDROLYSATE TO XYLITOL

In xylitol fermentation from rice straw hydrolysate by Candida tropicalis As2.1776, ammonia steeping was employed to pretreat the rice straw. Experimental results showed that the content of toxic compounds created in the hydrolysis process, such as acetic acid and phenolic compounds, was greatly reduced and the fermentation of the hydrolysate was enhanced. Xylitol fermentation was investigated in flasks and a 2‐L bioreactor, respectively. The xylitol yield factor and volumetric productivity were 0.746 g/g and 0.686 g/(L·h) in the lab‐flask fermentation, respectively. The corresponding results conducted in bioreactor fermentation were 0.689 g/g and 0.697 g/(L·h), respectively.     

Continuous production of lactic acid from molasses by perfusion culture of Lactococcus lactis using a stirred ceramic membrane reactor

A perfusion culture system was used for continuous production of lactic acid by retaining cells at a high density of Lactococcus lactis in a stirred ceramic membrane reactor (SCMR). After the cell concentration increased to 248 g/l, half of the culture broth volume was replaced with the fermentation medium. Subsequently, a substrate solution containing glucose (run 1) or molasses (run 2) was continuously supplied to the cells retained in the SCMR. Simultaneously, the culture supernatant was extracted using a ceramic filter with a pore size of 0.2 mum. The dilution rate was initially set at 0.4 h(-1) and gradually decreased to 0.2 h(-1) due to reduction in the permeability of the filter. The concentration of glucose in the substrate solution was adjusted to 60 g/l for the transition and the first period until 240 h, 90 g/l for the second period from 240 h to 440 h, and 70 g/l for the third period from 440 h to 643 h. The average concentration of lactic acid in the filtrate reached 46 g/l in the first period, 43 g/l in the second period, and 33 g/l for the third period. The productivity obtained for the first period reached 15.8 g.l(-1).h(-1), twice as much as that achieved in repeated batch fermentations. Based on the results obtained in run 1, the substrate solution containing 120 g/l of molasses was continuously supplied for 240 h in run 2. The concentration and productivity of lactic acid reached 40 g/l and 10.6 g.l(-1).h(-1), respectively, by continuously replenishing the culture medium at a dilution rate of 0.26 h(-1). These results demonstrated that the filtration capacity of the SCMR was sufficient for a continuous and rapid replenishment of molasses solution from the dense cell culture and, therefore, the perfusion culture system is considered to provide a low-cost process for continuous production of lactic acid from cheap resources.     

转木糖还原酶基因XYL1酿酒酵母的构建及产木糖醇能力研究

将人工合成的树干毕赤酵母（Pichia stipitis）的木糖还原酶基因XYL1插入酿酒酵母（Saccharomyces cerevisiae）表达载体pYES2中,然后将重组质粒pYES2-XYL1导入酿酒酵母INVSc1中,构建转木糖还原酶基因XYL1酿酒酵母菌株INVSc1/pYES2-XYL1,最后采用营养缺陷培养基筛选转木糖还原酶基因酿酒酵母并对其产木糖醇的能力进行检测。结果表明,成功获得2株转木糖还原酶基因XYL1酿酒酵母菌株INVSc1/pYES2-XYL1-01、INVSc1/pYES2-XYL1-02,当两菌株以50 g/L木糖及10 g/L半乳糖为碳源发酵5 d后,木糖醇产量分别高达（13.68±2.37）g/L、（12.09±1.45）g/L,显著高于非转基因酿酒酵母INVSc1的木糖醇产量（1.08±0.37）g/L（P＜0.05）,说明XYL1基因的导入显著提高了酿酒酵母INVSc1生产木糖醇的能力（P＜0.05）。为采用基因工程酿酒酵母制备食用木糖醇提供了理论及技术基础。     

NADPH-dependent D-aldose reductases and xylose fermentation in Fusarium oxysporum

Two aldose (xylose) reductases (ARI and ARII) from Fusarium oxysporum were purified and characterized. The native ARI was a monomer with M(r) 41000, pI 5.2 and showed a 52-fold preference for NADPH over NADH, while ARII was homodimeric with a subunit of M(r) 37000, pI 3.6 and a 60-fold preference for NADPH over NADH. In this study, the influence of aeration and the response to the addition of electron acceptors on xylose fermentation by F. oxysporum were also studied. The batch cultivation of F. oxysporum on xylose was performed under aerobic, anaerobic and oxygen-limited conditions in stirred tank reactors. Oxygen limitation had considerable influence on xylose metabolism. Under anaerobic conditions (0 vvm), xylitol was the main product with a maximum yield of 0.34 mole of xylitol/mole of xylose while the maximum ethanol yield (1.02 moles of ethanol/mole of xylose) was obtained under aerobic conditions (0.3 vvm). When the artificial electron acceptor acetoin was added to an anaerobic batch fermentation of xylose by F. oxysporum, the ethanol yield increased while xylitol excretion was also decreased.     

响应面法优化不同型态热带假丝酵母发酵生产木糖醇的工艺

利用Design Expert软件对菌丝型和酵母型热带假丝酵母发酵生产木糖醇实验进行设计及结果分析,建立木糖和木糖醇浓度与4个关键因子(菌型、发酵温度、pH、初始木糖浓度)的二次多项式回归模型,并对模型进行解析。结果表明:菌丝型热带假丝酵母转化木糖为木糖醇的能力高于酵母型;升高发酵温度,有利于木糖转化为木糖醇,而pH升高对转化过程并没有明显促进;发酵液中初始木糖浓度与木糖转化率呈正相关关系;获得最佳发酵工艺条件为菌种采用菌丝型酵母,发酵温度37℃,pH8,初始木糖浓度60mg/mL,此时木糖醇浓度达到17.21mg/mL。