培养基优化以促进超高浓度发酵生产乙醇(英文)

An optimal medium (300 g·L-1 initial glucose) comprising 6.3 mmol·L-1 Mg2+, 5.0 mmol·L-1 Ca2+, 15.0 g·L-1 peptone and 21.5 g·L-1 yeast extract was determined by uniform design to improve very high gravity (VHG) ethanol fermentation, showing over 30% increase in final ethanol (from 13.1% to 17.1%, by volume), 29% decrease in fermentation time (from 84 to 60 h), 80% increase in biomass formation and 26% increase in glucose utilization. Experiments also revealed physiological aspects linked to the fermentation enhancements. Compared to the control, trehalose in the cells grown in optimal fermentation medium increased 17.9-, 2.8-, 1.9-, 1.8- and 1.9-fold at the fermentation time of 12, 24, 36, 48 and 60 h, respectively. Its sharp rise at the early stage of fermentation when there was a considerable osmotic stress suggested that trehalose played an important role in promoting fermentation. Meanwhile, at the identical five fermentation time, the plasma membrane ATPase activity of the cells grown in optimal medium was 2.3, 1.8, 1.6, 1.5 and 1.3 times that of the control, respectively. Their disparities in enzymatic activity became wider when the glucose levels were dramatically changed for ethanol production, suggesting this enzyme also contributed to the fermentation improvements. Thus, medium optimization for VHG ethanol fermentation was found to trigger the increased yeast trehalose accumulation and plasma membrane ATPase activity.     

重组大肠杆菌流加发酵中短暂溶氧休克促进5-氨基乙酰丙酸生成(英文)

5-Aminolevulinic acid （ALA） is a common precursor for tetrapyrrole compounds in all kinds of organ isms and has wide applications in agriculture and medicines. In this study, a new strategy, i.e. short-term dissolved oxygen （DO） shock during aerobic fermentation, was introduced to produce 5-aminolevulinic acid with a recombi-nant E. coli. Effects of duration time of DO shock operation on plasmid concentration, intracellular ALA synthase （ALAS） activity and ALA production were investigated in Erlenmeyer shake flasks. The results indicated that both ALAS activity and ALA yield were enhanced in an anaerobic operation of 45 rain in the early exponential phase during fermentation, while they decreased when the anaerobic operation time was further increased to 60 rain. The DO shock protocol was confirmed with the fed-batch fermentation in a 15 L fermenter and the ALA production achieved 9.4 g.L-1 （72 mmol.L-1）, which is the highest yield in the fermentation broth reported up to now.     

氮源对利迪链菌素生产及相关次级代谢物分布的影响

The effects of nitrogen sources on streptolydigin production and distribution of secondary metabolites were investigated for flask cultured S.lydicus AS 4.2501.When peptone,asparamide,and glutamic acid were ex- amined as the nitrogen source,respectively,liquid chromatography-mass spectrometry（LC-MS）and photodiode array（PDA）analyses revealed the formation of two analogues of streptolydigin in the fermentation broth.When soybean meal was used as the source of nitrogen,three analogues of streptolydigin were detected.The use of am- monium sulfate as a source of nitrogen resulted in a lower pH value of the fermentation system,thus inhibiting streptolydigin biosynthesis and changing the metabolic profiling.Among the nitrogen sources that were made use of,glutamic acid was most favorable to the formation of streptolydigin.Simultaneously,this study also showed that the changing nitrogen sources resulted in altering the production and relative ratios of streptolydigin and its analogues.     

SBR好氧污泥颗粒的脱氮性能研究

The sequencing batch reactor （SBR） was started up by seeding the anaerobic granular sludge and the aerobic granular sludge was successfully cultivated. The performance characteristic of the aerobic granules for nitrogen removal was investigated in detail. The experimental results demonstrated the relationship between operational parameters [dissolved oxygen （DO） and pH] and variation of chemical oxygen demand （COD）, ammonium （NH4^＋-N） and total nitrogen （TN）. In continuous flow pattern, COD was too low in the reactor at the later stage of a cycle, which restrained denitrification and decreased the removal of nitrogen, while in discontinuous flow pattern, the carbon source could be supplemented in time, which improved denitrification and increased the removal of TN from 66% to 81%.     

重组大肠杆菌高密度发酵生产类人胶原蛋白的过程控制研究

Recombinant E. coli BL 21 was cultivated in high cell density to produce human-like collagen. The effects of the feeding of nitrogen source, controlled by an auto on/off-feeding mode with two different cycles of 0.5min and 4min intervals, oxygen-enrichment methods and inducement strength on the cell yield and human-like collagen production were investigated. The studies showed that nitrogen source feeding in fast cycle could result in higher human-like collagen production than that in slow cycle; and the feedback regulation of glucose, increase of the pressure of fermentation bioreactor, and supply of oxygen-enriched air could all increase cell yield and human-like collagen production. The effects of inducement strength on protein expression were found important. When OD600 reached 90-100, the cultivation temperature was increased to 42℃ to begin induction for 2-3 h, and then shifted to 39℃ for 5-6h induction, the cell density and human-like collagen production could reach 96g·L-1 [DCW (dry cell mass)     

Enhanced production of glycyrrhetic acid 3-O-mono-β-D-glucuronide by fed-batch fermentation using p H and dissolved oxygen as feedback parameters

Glycyrrhetic acid 3-O-mono-β-D-glucuronide(GAMG), the major functional ingredient in licorice, has widespread applications in food, pharmacy and cosmetics industry. The production of GAMG through Penicillium purpurogenum Li-3 cultivation was for the first time performed through both batch and fed-batch processes in bioreactors. In batch process, under optimal conditions(p H 5.0, temperature 32 °C, agitation speed 100 r·min-1), 3.55 g·L-1GAMG was obtained in a 2.5 L fermentor. To further enhance GAMG production, a fine fed-batch process was developed by using p H and DO as feedback parameters. Starting from 48 h, 100 ml 90 g·L-1substrate Glycyrrhizin(GL) was fed each time when p H increased to above 5.0 and DO was increased to above 80%. This strategy can significantly enhance GAMG production: the achieved GL conversion was 95.34% with GAMG yield of 95.15%, and GAMG concentration was 16.62 g·L-1which was 5 times higher than that of batch. Then, a two-step separation strategy was established to separate GAMG from fermentation broth by crude extraction of 15 ml column packed with D101 resin followed by fine purification with preparative C18 chromatography. The obtained GAMG purity was 95.79%. This study provides a new insight into the industrial bioprocess of high-level GAMG production.     

钌基氨合成催化剂氢氮吸附性能的研究

The effects of promoters K, Ba, Sm on the chemisorption and desorption of hydrogen and nitrogen, dispersion of metallic Ru and catalytic activity of active carbon (AC) supported ruthenium catalyst for ammonia synthesis have been studied by means of pulse chromatography, temperature-programmed desorption, and activity test. Promoters K, Ba and Sm increased the activity of Ru/AC catalysts for ammonia synthesis significantly, and particularly, potassium exhibited the best promotion on the activity because of the strong electronic donation to metallic Ru. Much higher activity can be obtained for Ru/AC catalyst with binary or triple promoters. The activity of Ru/AC catalyst is dependent on the adsorption of hydrogen and nitrogen. The high activity of catalyst could be ascribed to strong dissociation of nitrogen on the catalyst surface. Strong adsorption of hydrogen would inhibit the adsorption of nitrogen, resulted in decrease of the catalytic activity. Ru/AC catalyst promoted by Sm2O3 shows the best dispers     

Anaerobic hydrogen production of molasses from mixed microbial communities immobilized by activated granular carbon

Molasses wastewater was evaluated as substrate for biohydrogen production by anaerobic fermentation in a novel continuous mixed attached growth reactor ( CMAGR ) with aeration pretreated sludge attached onto granular activated carbon under continuous flow condition.It was indicated that the CMAGR system was operated at the conditions of influent COD of 2000～6000mg / L , hydraulic retention time ( HRT ) of 6hand temperature of 35 ℃ , when the pH value and oxidation-reduction potential ( ORP ) ranged from 4.16and-434 mV respectively , stable ethanol-type fermentation was formed with the sum of ethanol and acetate concentration ratio of 89.3%to the total liquid products after 40days operation.The H 2 content in biogas and chemical oxygen demand ( COD ) removal were estimated to be 46.6% and 13% , respectively.It was also investigated that the effects of organic loading rates ( OLRs ) on CMAGR hydrogen production system.It was found that hydrogen production yield increased from 3.72 mmol / hL to 12.51 mmol / hL as OLRs increased from 8 kg / m 3 d to 32 kg / m 3 d.The maximum hydrogen production rate of 12.51mmol / hL at a OLR of 32kg / m 3 d and the maximum hydrogen yield by substrate consumed was 130.57 mmol / mol happened at OLR of 16 kg / m 3 d.Greater pHs appeared to be favour to butyrate production and the maximum of 0.51mol / mol was obtained at pH of 4.14.However , ethanol / acetate ratio was greater than 1.1at pH fluctuated between 3.4 - 3.6and 4.1 - 4.4which indicated that these pHs were favour to ethanol type fermentation.Therefore , the continuous mixed attached growth reactor ( CMAGR ) could be a promising attached growth system for biohydrogen fermentation.     

短乳杆菌CGMCC1306pH和温度两阶段控制结合底物补加方式生产γ-氨基丁酸(英文)

Methods to optimize the production of gamma-aminobutyric acid （GABA） by Lactobacillus brevis CGMCC 1306 were investigated. Results indicated that cell growth was maximal at pH 5.0, while pH 4.5 was pref-erable to GABA formation. The optimal temperature for cell growth （35 °C） was lower than that for GABA forma-tion （40 °C）. In a two-stage pH and temperature control fermentation, cultures were maintained at pH 5.0 and 35 °C for 32 h, then adjusted to pH 4.5 and 40 °C, GABA production increased remarkably and reached 474.79 mmol·L-1 at 72 h, while it was 398.63 mmol·L-1 with one stage pH and temperature control process, in which cultivation con-ditions were constantly controlled at pH 5.0 and 35 °C. In order to avoid the inhibition of cell growth at higher L-monosodium glutamate （L-MSG） concentrations, the two-stage control fermentation with substrate feeding strat-egy was applied to GABA production, with 106.87 mmol （20 g） L-MSG supplemented into the shaking-flask at 32 h and 56 h post-inoculation separately. The GABA concentration reached 526.33 mmol·L-1 at 72 h with the fer-mentation volume increased by 38%. These results will provide primary data to realize large-scale production of GABA by L. brevis CGMCC 1306.     

黑曲霉Aspergillus niger P-6021浆态发酵常山胡柚皮渣产果胶霉

The peel of Citrus changshan-huyou, coupled with wheat bran, could be utilized by Aspergillus niger P-6021 in slurry-state fermentation to produce pectinase with suitable enzyme composition for application in apple juice processing. The production of pectinase is improved by additional nitrogen source substances and mineral supplements. The ratio of carbon source substances to nitrogen source substances in the medium also has significant effect on the pectinase production by A. niger P-6021 in slurry-state fermentation. In the optimized medium composition, the maximal enzyme activity could reach 42 U.L^- 1 （polymethylgalacturonase）, 6.7 U.L^- 1 （polymethygalacturatesterase）, and 4.3 U.L^-1 （polymethylgalacturonate lyase）, respectively, after 3 days at 180 r.min^- 1 and 30℃. The crude pectinase shows significant effect to improve the yield and clarification of apple juice. Keywords Aspergillus niger, slurry-state fermentation, pectinase, Citrus changshan-huyou, apple juice     

定点突变的谷氨酰胺合成酶的表达条件和在酶法合成谷氨酰胺中的应用

针对酶法生产谷氨酰胺中高浓度铵盐条件下的谷氨酰胺合成酶（GS）腺苷酰化问题,从谷氨酸棒杆菌Corynebacterium glutamicum ATCC 14067调取编码GS的基因glnA,将GS的腺苷酰化位点Tyr405定点突变为Phe405,并在大肠杆菌中表达突变后的GS,优化产酶条件。用突变的GS在摇瓶中进行酶催化过程,通过补加酶催化底物谷氨酸钠和氯化铵,可以提高定点突变的GS生产谷氨酰胺的能力,谷氨酰胺产量达到16.8 g·L^-1;在5 L反应器规模的酶催化生产谷氨酰胺过程中,通过调控底物补加方案和反应条件,谷氨酰胺的产量达到34.2 g·L^-1,谷氨酰胺对谷氨酸的摩尔转化率为96.3%。     

杂交瘤细胞流加培养中葡萄糖和谷氨酰胺的代谢控制

对底物限制的流加培养中杂交瘤细胞HB 58的生长、代谢和单抗生成进行了研究。葡萄糖或 谷氨酰胺限制引起比生长速率下降。葡萄糖限制时,乳酸生成减少,细胞对葡萄糖的得率增 加,乳酸对葡萄糖的得率降低,说明葡萄糖更多地参与三羧酸循环。谷氨酰胺限制时,氨和丙氨酸生成减少,细胞和氨对谷氨酰胺的得率增加,丙氨酸对谷氨酰胺的得率减小,说明谷氨酰胺更多地参与脱氢反应,其有效利用率得到提高。     

谷氨酰胺合成酶腺苷酰化位点的定点突变和产胺的初步研究

为解决谷氨酰胺合成酶腺苷酰化修饰失活的问题,利用基因定点突变的方法将谷氨酸棒杆菌的谷氨酰胺合成酶(Glutamine Synthetase, GS)腺苷酰化位点由Tyr405突变为Phe405,并在大肠杆菌中获得突变后GS的表达. 对比腺苷酰化位点突变前后的重组大肠杆菌pET-3a/GSI和pET-3a/GSIM在高氨环境下的GS活性和谷氨酰胺产量,发现重组菌pET-3a/GSIM在高氨环境下的最大酶活是150 U/L,产谷氨酰胺浓度为17.5 g/L,分别是pET-3a/GSI酶活(30 U/L)的5.0倍和产谷氨酰胺水平(3.4 g/L)的5.1倍,GS定点突变使谷氨酸转化为谷氨酰胺的途径得到强化.     

周期变温和后期通氮气或二氧化碳发酵对提高甘油产量的影响

引　言甘油是一种重要的工业原料 ,广泛用于化工、食品、医药等领域 .生产甘油的方法主要有化学合成、油脂水解和肥皂液提取以及糖类原料经微生物作用的发酵法 .由于化学合成甘油的产量有限 ,肥皂行业受到洗涤剂行业的冲击 ,肥皂生产减少 ,甘油产量也随之下降 ,市场供不应求 .因此 ,发酵法以其原料来源丰富、设备简单等得到人们的青睐[1～ 3 ] .发酵法是以糖类为原料 ,利用某些微生物在一定条件下通过代谢作用而产生甘油 .用耐高渗酵母发酵生产甘油的研究已有许多文献进行了报道[4～ 7] ,我国在这方面也做了大量的研究工作 .但大规模工业化…     

陶瓷球吸附污水中低含量氨氮性能研究

针对污水中低含量的氨氮,考察在陶瓷生产过程中产生的陶瓷球的吸附性能.用静态吸附法测定氨离子在陶瓷球上的吸附等温线、了解动力学特性,并探讨pH、改性等方法对陶瓷球吸附氨氮的影响规律.结果表明,随着溶液中吸附质含量的上升,吸附剂的吸附容量明显上升,达到动态平衡的时间为7h;陶瓷球吸附氨氮的最佳pH在6～9,吸附方式有离子交换作用和物理吸附作用;使用NaCl溶液浸泡振荡的改性方法可明显提高陶瓷球对氨氮的吸附性能,成为一种有效且廉价的改性方法.证明陶瓷厂排出的废料陶瓷球可以应用于污水中低含量氨氮的吸附去除.     

8-(6-氨基己烷)-氨基-NADH的制备和表征

采用1,6-己二胺对烟酰胺辅酶(NADH)的腺嘌呤C8位点进行定点修饰,通过DEAE-Sepharose层析分离得到产物8-(6-氨基己烷)-氨基-NADH. 经优化制备过程和分离条件,其收率可达60%. 在pH 2.2~8.0内系统考察了修饰产物的pH稳定性,结果表明在不同种类的缓冲液中,修饰后辅酶的稳定性均有显著提高,在pH 6.5的柠檬酸盐和pH 8.0的磷酸盐缓冲液中,半衰期分别延长11和5倍. 通过利用乳酸脱氢酶(LDH)和谷氨酸脱氢酶(GDH)测定修饰前后辅酶的活性,发现相同反应条件下修饰后辅酶的反应速率分别是天然NADH的3.0~3.8倍(LDH)和1.3~1.4倍(GDH).     

Electrochemical sensor array for bioprocess monitoring

A silicon-based biosensor chip consisting of an array of amperometric enzyme sensors has been developed for monitoring the concentration of glutamate and glutamine in cell-culture fermentation processes. The glutamate sensor was constructed by immobilising glutamate oxidase, while the glutamine sensor by sequential coupling of glutaminase and glutamate oxidase. The enzymes were immobilised by means of cross-linking with glutaraldehyde on the surface of the patterned platinum thin-film electrodes. The developed biosensors have been electrochemically characterised in solutions with different concentrations of glutamate and glutamine in terms of sensitivity, response time, linear working range and lifetime. A high sensitivity of 96 nA/mM and 100 nA/mM was registered for the glutamate and glutamine sensors, respectively. The preliminary experiments in cell-culture medium have shown a good correlation between the glutamine and glutamate concentrations measured with the biosensor chip and the commercially available biochemistry analyser. The obtained results demonstrate the feasibility of the realised biosensor chip for monitoring the glutamine and glutamate concentrations in fermentation processes.     

一起制氮设备的故障分析

在浮法玻璃生产过程中,氮气流量保持恒定,是保持锡槽工况稳定的重要条件,是生产优质浮法玻璃的重要前提条件之一.只有制氮设备的稳定运行,才能保证氮气供应.本文就制氮空压机运行中出现的故障现象、原因进行了分析,并提出了改进措施.     

表达重组谷氨酸脱羧酶工程菌发酵条件的优化

目的对表达谷氨酸脱羧酶(Glutamate decarboxylase,EC4.1.1.15,简称GAD或GDC)的重组大肠杆菌的发酵条件进行优化。方法通过单因素试验优化表达GAD的重组大肠杆菌B3C1的诱导剂IPTG添加时间、IPTG浓度、诱导时间、诱导温度,再经响应面分析法优化工程菌的发酵条件(氯化钠、胰化蛋白胨、酵母粉、摇床转速、初始pH值、培养时间、接种量、装液量)。结果工程菌的最佳诱导条件为:IPTG添加时间为工程菌接种后4 h,IPTG浓度为0.8 mmol/L,诱导时间为4 h,诱导温度为35℃;工程菌的最佳发酵条件为:酵母粉1.01%,氯化钠0.6%,胰化蛋白胨1.5%,培养时间11.25 h,摇床转速250 r/min,初始pH值6.5,接种量3.0%,装液量11.70 ml。工程菌在该条件下发酵培养,GAD酶活达1 227.33 U,比优化前提高了12.29%。结论已成功对表达GAD的重组大肠杆菌的发酵条件进行了优化,为其工业化生产奠定了基础。