大孔树脂分离纯化蛹虫草深层发酵液中虫草素的工艺研究

通过比较10种不同大孔树脂吸附蛹虫草深层发酵液中虫草素的性能,筛选出合适的大孔树脂H103,并对大孔树脂H103的吸附和解吸条件进行了考察。实验结果表明,上样吸附前调节pH值为9,上样浓度为0．2mg·mL^-1,吸附流速为2BV·h^-1时,大孔树脂H103对蛹虫草深层发酵液中的虫草素有良好的吸附性能;解吸时先用去离子水洗去部分杂质,再用40％的乙醇对虫草素进行洗脱,可以得到较好的效果。     

大孔吸附树脂AB-8和十八烷基硅胶色谱分离纯化蛹虫草发酵液中虫草素工艺研究

主要研究了从蛹虫草发酵液中提取虫草素的工艺,确定了大孔吸附树脂AB-8及十八烷基键合硅胶反相层析柱对蛹虫草发酵液中虫草素的分离条件;吸附最佳工艺条件为上样液p H6,上样浓度0.4mg/m L,上样量5BV,吸附流速1.0BV/h;解吸最佳工艺条件包括解吸液20%乙醇,解吸体积15BV,解吸流速4BV/h。得到的解吸液进一步以十八烷基键合硅胶分离,条件为上样液p H6,梯度洗脱,流动相为p H6的水和p H6的乙醇。洗脱液通过结晶和重结晶得到精制虫草素。三步得到的虫草素纯度分别达到40%、90%和99%以上。     

Continuous acetone-butanol-ethanol (ABE) fermentation using immobilized cells of Clostridium acetobutylicum in a packed bed reactor and integration with product removal by pervaporation

An integrated solvent (ABE) fermentation and product removal process was investigated. A stable solvent productivity of 3.5 g/L h was achieved by using cells of Clostridium acetobutylicum immobilized onto a packed bed of bonechar, coupled with continuous product removal by pervaporation. Using a concentrated feed solution containing lactose at 130g/L, a lactose value of 97.9% was observed. The integrated fermentation and product removal system, with recycling of the treated fermentor effluent containing only low amount of solvents (/but lactose and acids), leads to only low acid losses. Therefore, most of the acids are converted to solvents, and this results in a high solvent yield of 0.39 g solvents/g lactose utilized. The pervaporation system provided a high product removal rate even at low solvent concentrations. A solvent membrane flux of 7.1 g/m(2) h with a selectivity of 5 was achieved during these investigations. The system proved to be very reliable.     

杀线虫芽孢杆菌发酵条件优化及大孔树脂筛选

芽孢杆菌 SMrs28分离自草原毒草瑞香狼毒根际土壤,其代谢产物有明显的线虫触杀活性。为了确定该菌株的最优发酵条件及初步纯化活性成分的树脂类型,采用单因素试验对发酵条件进行优化,通过静态吸附试验对大孔树脂类型进行筛选。结果表明: SMrs28菌株的最优发酵条件为:以葡萄糖和酵母粉作为最佳碳源和氮源,发酵时间48 h,接种量10%,温度28 ℃,转速180 r·min^-1,150 mL的三角瓶装液量30 mL,初始pH 7.2。静态吸附试验表明,大孔吸附树脂D101对发酵液中活性成分的吸附和解吸效果显著优于XAD-4、HP20和AB-8,其解吸液的杀线虫活性明显提高。发酵条件的优化和大孔吸附树脂的筛选,明显提高了发酵液的杀线虫活性,为进一步分离纯化该菌株的活性成分奠定了基础,为微生物杀线虫剂的开发和应用提供了理论依据。     

Energy-efficient recovery of butanol from model solutions and fermentation broth by adsorption

This article discusses the separation of butanol from aqueous solutions and/or fermentation broth by adsorption. Butanol fermentation is also known as acetone butanol ethanol (ABE) or solvent fermentation. Adsorbents such as silicalite, resins (XAD-2, XAD-4, XAD-7, XAD-8, XAD-16), bone charcoal, activated charcoal, bonopore, and polyvinylpyridine have been studied. Use of silicalite appears to be the more attractive as it can be used to concentrate butanol from dilute solutions (5 to 790–810 g L⁻¹) and results in complete desorption of butanol (or ABE). In addition, silicalite can be regenerated by heat treatment. The energy requirement for butanol recovery by adsorption–desorption processes has been calculated to be 1,948 kcal kg⁻¹ butanol as compared to 5,789 kcal kg⁻¹ butanol by steam stripping distillation. Other techniques such as gas stripping and pervaporation require 5,220 and 3,295 kcal kg⁻¹ butanol, respectively. Mention of trade names of commercial products in this article/publication is solely for the purpose of providing scientific information and does not imply recommendation or endorsement by the United States Department of Agriculture.     

Purification of monoclonal antibodies from whole hybridoma fermentation broth by fluidized bed adsorption

To achive the coarse purification of a monoclonal antibody from whole hybridoma fermentation broth a fluidized bed cation exchange process was used. The procedure consisted of application of the crude sample and washing of the bed in a fluidized mode and elution in a fixed bed mode. A completely clarified eluate was obtained with purification factors between 4 and 8 and a concentration of the desired product (monoclonal antibody) by a factor of more than 3 was achived. Thus, a combination of the three early steps of the downstream process clarification, concentration and coarse purification was possible. Two different materials were tested: a commercially available agarose-based matrix (Stream-line-SP), and a self-derivatized material based on controlled-pore glass (Bioran). Initial experiments were performed to describe the fluidization of the glass material. Comparison with the agarose material showed several differences, the agarose matrix allowing liquid flow closer to plug flow than the glass material. Increased backmixing in the liquid phase was detected when fluidizing the glass adsorbent compared with the agarose-based matrix. Despite this fact, comparison of the two materials with respect to antibody binding and elution demonstrated a similar performance. (c) 1995 John Wiley & Sons, Inc.     

非水介质大孔树脂分离纯化虾壳中虾青素

通过7种大孔树脂对虾青素的静态吸附容量和解析率等指标的考察,筛选出AB-8大孔吸附树脂,用于分离虾壳中虾青素,同时利用高效液相色谱（HPLC）法测量虾青素的含量。结果表明,AB-8树脂对虾青素的吸附量为（24．17±0．5）mg／g,解吸率为95．2％,最大上样量（每g干树脂）以虾青素计为（23．07±0．2）mg,并确定用8倍量柱床体积的乙酸乙酯为洗脱剂,纯化所得虾青素的纯度为14．73％。     

Selective extraction of acetic acid from the fermentation broth produced by Mannheimia succiniciproducens

Acetic acid is by-product from fermentation processes for producing succinic acid using Mannheimia succiniciproducens . To obtain pure succinic acid from the final fermentation broth, acetic acid was selectively removed based on the different extractability of succinic acid and acetic acid with pH using tri-n-octylamine (TOA) as extractant. When successive batch extractions were performed using 0.25 mol TOA kg(-1) dissolved in 1-octanol at pH 5, the mol ratio of succinic acid to acetic acid before extraction was 4.9 and the final ratio after the fourth batch was 9.4.     

Enhanced production of bioethanol from waste of beer fermentation broth at high temperature through consecutive batch strategy by simultaneous saccharification and fermentation

Malt hydrolyzing enzymes and yeast glycolytic and fermentation enzymes in the waste from beer fermentation broth (WBFB) were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A new ‘one-pot consecutive batch strategy’ was developed for efficient bio-ethanol production by simultaneous saccharification and fermentation (SSF) using WBFB without additional enzymes, microbial cells, or carbohydrates. Bio-ethanol production was conducted in batches using WBFB supernatant in the first phase at 25–67 °C and 50 rpm, followed by the addition of 3% WBFB solid residue to the existing culture broth in the second phase at 67 °C. The ethanol production increased from 50 to 102.5 g/L when bare supernatant was used in the first phase, and then to 219 g ethanol/L in the second phase. The amount of ethanol obtained using this strategy was almost equal to that obtained using the original WBFB containing 25% solid residue at 33 °C, and more than double that obtained when bare supernatant was used. Microscopic and gel electrophoresis studies revealed yeast cell wall degradation and secretion of cellular material into the surrounding medium. Scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) supported the existence of enzymes in WBFB involved in bioethanol production at elevated temperatures. The results of this study will provide insight for the development of new strategies for biofuel production.     

余甘原汁中多酚的大孔树脂提取分离及其抗氧化活性

为研究余甘子多酚的分离提取方法,本文以余甘原汁为原料,采用NKA-Ⅱ大孔吸附树脂,对上样量、洗脱剂、洗脱体积及洗脱速度等条件进行了考察,并对提取物的抗氧化活性进行了对比分析。通过试验确定了余甘原汁多酚的大孔树脂分离提取条件为:上样速度2BV/h,上样量0.8BV,洗脱剂为70%乙醇溶液,洗脱体积3BV,洗脱速度8BV/h,在此条件下NKA-Ⅱ大孔树脂对余甘原汁中多酚的吸附率可达到88.42%,洗脱率为90.93%,提取率为80.39%;对提取物的抗氧化活性分析显示,与分离前的余甘原汁干燥物相比,提取物的多酚和维生素C含量均提高了0.53倍,类超氧化物歧化酶活性(SODL)提高了1.4倍;铁离子还原能力(FRAP)明显高于分离前,对DPPH自由基、ABTS自由基及脂质氧化的半抑制浓度(IC50)均显著低于分离前的原汁干燥物,表明通过大孔吸附树脂分离,使余甘原汁中的多酚等抗氧化活性成分得到了有效的分离纯化。     

响应面分析法优化黑根霉胞外多糖发酵工艺

实验以商品化的马铃薯葡萄糖液体培养基为基础培养基,以胞外粗多糖产量为考察指标,运用响应面分析法考察玉米浆浓度、KH₂PO₄浓度和发酵时间3个因素对胞外多糖发酵产量的影响,以获得黑根霉胞外多糖发酵最优工艺,建立高产、稳定、可控的胞外多糖发酵生产工艺技术方案。经响应面分析,各因素按照对响应值的影响顺序为：玉米浆浓度>发酵时间> KH₂PO₄浓度,且玉米浆浓度、发酵时间对胞外多糖产量的影响极显著,KH₂PO₄浓度对胞外多糖产量的影响不显著。胞外多糖发酵最优工艺为：玉米浆3.2mg/mL、KH₂PO₄ 1.5mg/mL和发酵时间132h,在此条件下胞外多糖的最大预测产量为0.824mg/mL。实验重复性好,是一个高产、稳定、可控的胞外多糖发酵生产工艺技术方案,可以指导黑根霉胞外多糖发酵。     

离子交换法分离发酵液中鸟苷和肌苷

乌苷发酵液中乌苷与副产物肌苷的分离对乌苷工业有重要的意义。对乌苷和肌苷的分离条件进行研究,得到了最佳分离条件：采用Hd-8树脂,树脂床高度为4cm,前120mL采用0．05mol／L盐酸洗脱收集肌苷,120mL至240mL采用0．74mol／L pH5．0醋酸钠缓冲液洗脱收集乌苷,可以获得很好的效果。     

青海湖M1菌发酵液体外抗菌活性及稳定性的初步研究

从青海湖中分离得到一株霉菌M1,采用平板抑菌法进行体外抗菌作用以及pH值、温度因素对其抗菌活性的影响。结果表明：该菌株发酵液对大肠杆菌、枯草芽孢杆菌及金黄色葡萄球菌都有明显的抑菌效果。M1菌株发酵液的抑菌活性对热稳定性较差,在pH值3～7的条件下抑菌效果最佳,遗传稳定性也不高。     

乳腺生物反应器表达产物层析分离过程中介质的污染机理及其再生策略

考察了磺酸基离子交换层析介质 (SP Sepharose FF) 在分离表达人乳铁蛋白的重组牛乳过程中的污染机理及其再生策略。通过层析原料及流分中各组分含量的检测分析,发现牛乳中的脂类通过堵塞效应或疏水相互作用残留在层析柱上,造成层析运行压力升高;部分酪蛋白通过静电相互作用占据介质的配基位点,导致介质的交换容量降低;乳糖与介质之间无直接相互作用。连续层析运行次数的增加以及层析-再生时间间隔的延长,均能导致残留组分和介质之间的相互作用逐渐增强,最终影响介质的再生效率。使用NaOH进行及时清洗,可以有效地清除柱上残留的脂类和蛋白,恢复离子交换介质的层析性能和微观形态。     

响应面法优化富硒荷叶离褶伞菌丝体胞内粗多糖发酵条件

为了优化富硒荷叶离褶伞菌丝体胞内粗多糖发酵条件,在单因素试验基础上,考察灭菌条件、培养时间、硒添加量和硒添加时间对富硒荷叶离褶伞菌丝体胞内粗多糖的影响,并通过Box-Benhnken实验设计和响应面分析法确定最优发酵条件。结果表明,在121℃、20min条件下对培养基和Na₂SeO₃溶液分别灭菌后混合,在发酵至第129h,发酵液中添加4µg/mL Na₂SeO₃,继续发酵至第244h,发酵产生的富硒荷叶离褶伞菌丝体胞内粗多糖含量为349.8mg/100mL。     

Determination of total silver and silver species in coastal seawater by inductively-coupled plasma mass spectrometry after batch sorption experiments with Chelex-100 resin

Abstract

Sorption experiments in batch mode with Chelex-100 resin have been carried out for the determination of total Ag, labile and inert fractions in seawater from Ria de Vigo (NE Atlantic ocean) by inductively coupled plasma-mass spectrometry (ICP-MS). The method allows an accurate determination of the total Ag thus avoiding the effect of the saline matrix when direct determination by ICP-MS is performed. Similarly, the distribution patterns of Ag among species with different lability can be established in seawater as a function of pH. Species distribution and total Ag contents have been obtained in CRM CASS-4 (near shore seawater) and four coastal seawater samples. While the free silver ion concentration accounts for a significant fraction at low pH, the labile inorganic fraction is most important at natural pH (≈8) in non-polluted seawater. The inert and labile organic fractions were the most relevant in polluted coastal seawater. Complexation mechanisms of silver were also investigated from the sorption curves of silver on Chelex-100 according to the Gibbs-Donnan model. Two complexes were formed, AgL and AgL₂ with the following intrinsic complexation constants log β_(1np) = ?2.11 and ?10.02, respectively. The formation of the complex AgL predominates up to pH 4 but is negligible due to chloride ions effect, while the complex AgL₂ predominates from pH 4.     

Comparison of different options for harvest of a therapeutic protein product from high cell density yeast fermentation broth

Recovery of therapeutic protein from high cell density yeast fermentations at commercial scale is a challenging task. In this study, we investigate and compare three different harvest approaches, namely centrifugation followed by depth filtration, centrifugation followed by filter-aid enhanced depth filtration, and microfiltration. This is achieved by presenting a case study involving recovery of a therapeutic protein from Pichia pastoris fermentation broth. The focus of this study is on performance of the depth filtration and the microfiltration steps. The experimental data has been fitted to the conventional models for cake filtration to evaluate specific cake resistance and cake compressibility. In the case of microfiltration, the experimental data agrees well with flux predicted by shear induced diffusion model. It is shown that, under optimal conditions, all three options can deliver the desired product recovery ( >80%), harvest time ( <15 h including sequential concentration/diafiltration step), and clarification ( <6 NTU). However, the three options differ in terms of process development time required, capital cost, consumable cost, ease of scale-ability and process robustness. It is recommended that these be kept under consideration when making a final decision on a harvesting approach.     

High-yield production of L-valine in engineered Escherichia coli by a novel two-stage fermentation

L-valine is an essential amino acid and an important amino acid in the food and feed industry. The relatively low titer and low fermentation yield currently limit the large-scale application of L-valine. Here, we constructed a chromosomally engineered Escherichia coli to efficiently produce L-valine. First, the synthetic pathway of L-valine was enhanced by heterologous introduction of a feedback-resistant acetolactate acid synthase from Bacillus subtilis and overexpression of other two enzymes in the L-valine synthetic pathway. For efficient efflux of L-valine, an exporter from Corynebacterium glutamicum was subsequently introduced. Next, the precursor pyruvate pool was increased by knockout of GTP pyrophosphokinase and introduction of a ppGpp 3′-pyrophosphohydrolase mutant to facilitate the glucose uptake process. Finally, in order to improve the redox cofactor balance, acetohydroxy acid isomeroreductase was replaced by a NADH-preferring mutant, and branched-chain amino acid aminotransferase was replaced by leucine dehydrogenase from Bacillus subtilis. Redox cofactor balance enabled the strain to synthesize L-valine under oxygen-limiting condition, significantly increasing the yield in the presence of glucose. Two-stage fed-batch fermentation of the final strain in a 5 L bioreactor produced 84 g/L L-valine with a yield and productivity of 0.41 g/g glucose and 2.33 g/L/h, respectively. To the best of our knowledge, this is the highest L-valine titer and yield ever reported in E. coli. The systems metabolic engineering strategy described here will be useful for future engineering of E. coli strains for the industrial production of L-valine and related products.     

Fermentation and recovery of glutamic acid from palm waste hydrolysate by Ion-exchange resin column

Glutamic acid produced from palm waste hydrolysate by fermentation with Brevibacterium lactofermentum ATCC 13869 is produced with a remarkably high yield compared with that produced from pure glucose as a carbon source. The produce yield is 70 g/L with glucose, wherease, when palm waste hydrolysate is the fermentation medium in the same bioreactor under same conditions, it is 88 g/L. The higher yield may be attributed to the fact that this organism has the ability to convert sugars other than only glucose present in the hydrolysate. Bioreactor conditions most conducive for maximum production are pH 7.5, temperature of 30 degrees rmentation period of 48 h, inoculum size 6%, substrate concentration of 10 g per 100 mL, yeast extract 0.5 g per 100 mL as a suitable N source, and biotin at a concentration of 10 pg/L. Palm waste hydrolysate used in this study was prepared by enzymic saccharification of treated palm press fiber under conditions that yielded a maximum of 30 g/L total reducing sugars. Glutamic acid from fermentation broth was recovered by using a chromatographic column (5cm x 60 cm) packed with a strong ion-exchange resin. The filtered broth containing glutamic acid and other inorganic ions was fed to the fully charged column. The broth was continuously recycled at a flow rate of 50 mL/min (retention time of 55 min) until glutamic acid was fully adsorbed on the column leaving other ions in the effluent. Recovery was done by eluting with urea and sodium hydroxide for total displacement of glutamic acid from the resin. The eluent containing 88 g/L of glutamic acid was concentrated by evaporation to obtain solid crystals of the product. (c) 1995 John Wiley & Sons, Inc.     

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.