高效液相色谱法测定必特螺旋霉素发酵液中有机酸

必特螺旋霉素是运用基因工程技术获得的工程茵产生的一组以异戊酰螺旋霉素为主要成分的多组分新型抗生素,其前体为乙酸、丙酸、丁酸和异戊酸等有机酸。本研究利用高效液相色谱法以0．01mol／L磷酸缓冲液（pH2．3）和甲醇为流动相,分别在反相C8（α-酮戊二酸、乙酸、柠檬酸、琥珀酸、丙酸）和C18（丁酸、异戊酸）柱上定量测定了必特螺旋霉素前体酸和三羧酸循环相关有机酸。所建立的测定方法的相对标准偏差为0．10％～0．42％,回收率为93．19％～102．08％。     

从发酵液中萃取L-乳酸的工艺

以L-乳酸发酵液为对象,以正丁醇为萃取剂,在pH为2、温度为25℃、n（乳酸）：n（正丁醇）=1：1、乳酸在发酵液中的质量分数为30％时,经3次萃取后,最终的萃取率可达到75．7％。萃取完成后,不需要将乳酸进行反萃,可将所得到的正丁醇-乳酸的混合体系作为底物进行酯化反应,生成乳酸丁酯,从而避开提取纯乳酸的高操作要求。     

HPLC-ELSD法测定发酵液中L-精氨酸含量

建立了一种快速、准确的高效液相色谱-蒸发光散射检测法(HPLC-ELSD)测定发酵液中L-精氨酸含量。采用Prevail C18色谱柱(C18 5μm,250×4.6 mm,Alltech),以5 mmol.L-1七氟丁酸(三氟乙酸调pH至1.0)和乙腈为流动相,采用梯度洗脱,总流速为1.0 mL/min。ELSD参数:漂移管温度为117.0℃,载气流速为3.2 L/min。L-精氨酸等氨基酸的回收率为96%~104%。能够快速、精确测定发酵样品中目的产物L-精氨酸与其它氨基酸含量。     

Effect of fermentation broth and biosurfactants on mass transfer during liquid-liquid extraction

Mass transfer rates in liquid-liquid extraction processes can be seriously affected by the presence of surface-active contaminants. This is especially true of applications of a biotechnological origin, where the microorganism used in the process may produce the surface-active contaminants. An investigation into the effects of soluble and insoluble fermentation broth components on mass transfer using chloramphenicol extraction into octanol as the model system was conducted. Soluble components produced during fermentation were found to adsorb to the interface, where they reduced the overall mass transfer coefficient by up to 70%. After fractionation it was found that components in the weight range from 10-30 kDa had the greatest effect on mass transfer. Protein and phospholipid compounds of similar size were found to reduce the overall mass transfer coefficient to a similar extent to the broth components at concentrations around 0.001mg/l. The biomass produced during the fermentation also reduced mass transfer substantially, and it is likely that this was due to physical blockage of the interface.     

应用HPLC一反相色谱法测定米根霉乳酸发酵液中的有机酸

提出了一种利用高效液相色谱法分析米根霉乳酸发酵液中有机酸的方法,应用反相Wakosil-II 5C18RS色谱柱,以0．01mol/L磷酸（pH2\5)作为流动相,发酵液经稀硫酸预处理后直接进样分离定量,在10min内把其中的乳酸,苹果酸,富马酸等完全分离定量,各种酸回收率大于97％。经多次实验结果证明：本方法是测定乳酸发酵液中各有机酸的快速,有效的定量测定方法。     

高效液相色谱法检测发酵液中木糖和木糖醇

建立高效液相色谱检测发酵液中木糖和木糖醇含量的分析方法。色谱柱为HypersilNH2 柱 (4 .6mmi.d.× 2 5 0mm ,5 μm) ,柱温 3 5℃ ,流动相为乙腈—水 (80∶2 0 ) ,流速 1 .0mL .min 1,示差折光检测器检测。木糖和木糖醇在 3 .0～ 60mg.mL 1范围内 ,峰面积与其浓度线性关系良好 (г=0 .9995 ) ;平均回收率分别为 96.0 7% (n =5 ,RSD =0 .5 1 % )和 97.47% (n =5 ,RSD =1 .1 3 % )。方法简便、快速、准确。     

Purification during crossflow electromicrofiltration of fermentation broth

The present study was to investigate the purification of a fermentation broth by an electromicrofiltration membrane. Microfiltration runs with a crude and a centrifuged broth, with solution of particles recovered from centrifugation and with permeates from microfiltration experiments were thus compared.Microfiltration performances were governed by colloids and small particles that induced sharp initial flux declines. For these results, the evolution of the overall membrane resistance was increased by 80% in comparison with the electromicrofiltration membrane. The main focus of this study was set on the enhancement of the filtrate flux by an electric field. This pressure electrofiltration leads to a drastic improvement of the filtration by 100% and the filtration time was thereby reduced. Pressure electrofiltration serves as an interesting alternative to the cross-flow filtration and it effectively separates advantageous constituents such as amino acids and biopolymers from a fermentation broth. They were equally maintained during the microelectrofiltration, although they were significantly reduced by 45% by the microfiltration without the application of an electric field. Accordingly, since the electrofiltration membrane was provided more permeability, this study experimentally demonstrates that the permeability inside a membrane can be controlled using an electric field.     

Accelerated solvent extraction of monacolin K from red yeast rice and purification by high-speed counter-current chromatography

Monacolin K from red yeast rice was extracted by accelerated solvent extraction (ASE). The effects of various extraction parameters including extraction temperature, static extraction time and cycle index on yield were investigated using a DIONEX ASE 300 system to select the optimal conditions by an orthogonal test design L₉ (3)³. The optimum extraction conditions were determined as follows: extraction temperature 120 °C, static extraction time 7 min, and cycle index 3. Under the optimal conditions, the yield of ASE extract and monacolin K was 5.35% and 9.26 mg/g of dry red yeast rice, respectively. A separation and purification method of monacolin K was then established using high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane–ethyl acetate–methanol–water (8:2:5:5, v/v/v/v). From 300 mg of crude extract, 51.2 mg of monacolin K was obtained with the purity of 98.7%. The chemical structure of isolated compound was identified by UV, ESI-MS and ¹H NMR.     

青霉素酶发酵液的预处理和酶学特性

研究了在发酵液中添加絮凝剂对发酵液进行预处理,对蜡状芽孢杆菌（Bacillus cereus）产生青霉素酶的影响与酶学特性。实验结果表明,发酵液膜处理的难易程度与芽孢释放程度成正相关,在发酵液中添加0．02g／mL自制絮凝剂进行预处理,过滤效率提高10倍,而酶活损失仅5％;酶学特性研究结果表明,该酶最佳反应温度55℃时,酶的最适反应pH值7．0,金属离子锌、锰、镁对酶有激活作用,其浓度为0．25moL／L;酶热稳定性研究结果表明,在75℃下保温30min时,酶活损失85％。该酶在过量青霉素底物下,酶促反应为0级反应。     

Production of ethanol by coupling fermentation and solvent extraction

Summary A new technology of fermentation is proposed. The inhibitor product is removed continuously by coupling fermentation and solvent extraction. Applied to ethanol fermentation this technology is suitable to any case where the terminal product is inhibitory.The proposed technology uses both plug flow reactor and liquid-liquid extraction to achieve continuously the extractive fermentation of ethanol. The solvent used for liquid-liquid extraction is dodecanol. A new reactor was used. It is a column packed with a porous material . The fermentation broth is pulsed (a) to increase the interfacial area between the liquid medium and the dodecanol, and (b) to: decrease the gas hold up.Alcoholic fermentations were performed on glucose syrup at 35°C using Saccharomyces cerevisiae, with adsorbed cells as reference, with adsorbed cells and extractive fermentation. The results show that the fermentation is substantially improved. By this new method the ethanol productivity was multiplied by 5 and a solution of 407 g/l of glucose was totally fermented with a yeast which cannot normally transform more than 200 g/l glucose.     

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。     

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。     

Simultaneous solid–liquid separation and primary purification of clavulanic acid from fermentation broth of Streptomyces clavuligerus using salting‐out extraction system

Clavulanic acid (CA) is usually used together with other β‐lactam antibiotics as combination drugs to inhibit bacterial β‐lactamases, which is mainly produced from the fermentation of microorganism such as Streptomyces clavuligerus. Recently, it is still a challenge for downstream processing of low concentration and unstable CA from fermentation broth with high solid content, high viscosity, and small cell size. In this study, an integrated process was developed for simultaneous solid–liquid separation and primary purification of CA from real fermentation broth of S. clavuligerus using salting‐out extraction system (SOES). First, different SOESs were investigated, and a suitable SOES composed of ethanol/phosphate was chosen and further optimized using the pretreated fermentation broth. Then, the optimal system composed of 20% ethanol/15% K₂HPO₄ and 10% KH₂PO₄ w/w was used to direct separation of CA from untreated fermentation broth. The result showed that the partition coefficient (K) and recovery yield (Y) of CA from untreated fermentation broth were 29.13 and 96.8%, respectively. Simultaneously, the removal rates of the cells and proteins were 99.8% and 63.3%, respectively. Compared with the traditional method of membrane filtration or liquid–liquid extraction system, this developed SOES showed the advantages of simple operation, shorter operation time, lower process cost and higher recovery yield of CA. These results demonstrated that the developed SOES could be used as an attractive alternative for the downstream processing of CA from real fermentation broth.     

Improvement in bioconversion efficiency and reduction of ammonia emission by introduction of fruit fermentation broth in a black soldier fly larvae and kitchen waste conversion system

The black soldier fly (BSF), Hermetia illucens (Diptera: Stratiomyidae), is an insect commonly used for the bioconversion of various organic wastes. Not only can the BSF convert organic waste into macromolecular organic substances, such as insect proteins, but it can also lessen the pollution associated with these waste products by reducing ammonia emissions, for example. In this study, we measured the effects of adding fruit fermentation broth (Fer) and commercial lactic acid bacteria fermentation broth (Em) to kitchen waste (KW), as deodorizing auxiliary substances, on the growth performance of black soldier fly larvae (BSFL), the intestinal flora structure of BSFL, the ammonia emission from the KW substrate, and the microbial community structure of the KW substrate. We found that the addition of Fer or Em increased the body weight of BSFL after 6 d of culture, increasing the growth rate by 9.96% and 7.96%, respectively. The addition of Fer not only reduced the pH of the KW substrate but also increased the relative abundance of probiotics, such as Lactobacillus, Lysinibacillus, and Vagococcus, which inhibited the growth of ammonifiers such as Bacillus, Oligella, Paenalcaligenes, Paenibacillus, Pseudogracilibacillus, and Pseudomonas, resulting in the reduction of ammonia emission in the KW substrate. Moreover, the addition of Fer or Em significantly increased the relative abundances of Bacteroides, Campylobacter, Dysgonomonas, Enterococcus, and Ignatzschineria in the gut of BSFL and increased the species diversity and richness in the KW substrate. Our findings provide a novel way to improve the conversion rate of organic waste and reduce the environmental pollution caused by BSF.     

树干毕赤酵母酒精发酵碳元素代谢流向分析

实验对树干毕赤酵母（Pichia stipitis）进行了4个阶段共400 h连续恒化培养,在不同阶段以30.0 g/L葡萄糖作为基本碳源,添加30.0或15.0 g/L的木糖,通过控制温度（35±1）℃,进气量100～150mL/min,搅拌转速250～300 r/min。4个阶段共建立4个连续培养的"稳态"。对碳元素进行物料衡算发现,四个阶段碳元素回收率分别为118.0 %、105.6 %、113.5 %和94.7 %。对4个近似"稳态"的碳元素的代谢流向进行分析发现：将近50.0 %左右碳元素流向产物酒精,其次是CO2和酵母细胞;木糖醇浓度与流入底物中木糖浓度有直接关系,在相同发酵条件下流入的木糖浓度越大代谢生成木糖醇浓度也越高;实验所采用的通气条件更适合底物为30.0 g/L葡萄糖和30.0 g/L木糖混合液的连续发酵。     

基于 AOPAN 纳米纤维膜的粘杆菌素发酵液后处理研究

使用静电纺丝再经胺肟化改性制备AOPAN纳米纤维膜,并基于AOPAN纳米纤维膜构建膜分离装置,用于粘杆菌素发酵液的后处理。研究中,对不同厚度的纳米纤维膜的渗透通量及分离性能进行比较,最终确定双层叠合的纳米纤维膜为分离膜,最适操作压力为0.14 MPa。在此条件下,分离膜的渗透通量为2.61 L/m2．min,蛋白质的截留率达到90％,色素等其他杂质也得到有效去除。     

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.     

多噬伯克霍尔德氏菌WS-FJ9对林木病原菌物的拮抗作用及代谢产物的稳定性

【背景】伯克霍尔德氏菌(Burkholderia)是一类重要的植物根际促生细菌,许多菌株具有抑制植物病原菌生长和促进植物生长等功能。【目的】探究高效解磷促生细菌多噬伯克霍尔德氏菌(B. multivorans) WS-FJ9对不同林木病原菌物的抑菌作用。【方法】采用平板对峙法检测菌株WS-FJ9对5株林木病原真菌和卵菌的抑制效果;基于比色法检测经菌株WS-FJ9处理后病原菌菌丝细胞内含物的变化;使用antiSMASH 5.0在线预测网站对其次生代谢物质进行预测;通过菌丝生长抑制速率法对其无菌发酵滤液的抑菌活性和稳定性进行研究。【结果】菌株WS-FJ9对5种林木病原菌均具有不同程度的抑制作用,其中菌悬液对樟疫霉(Phytophthora cinnamomi)的抑制作用最好,抑菌带宽度为14.82±0.20mm,无菌发酵滤液对真菌拟茎点霉(Phomopsismacrospore)和松杉球壳孢(Sphaeropsis sapinea)的抑制效果显著,抑菌率分别为62.22%和62.78%;经无菌发酵滤液处理后的病原菌菌丝内的丙二醛含量增高,还原糖和可溶性蛋白含量显著降低。WS-FJ9菌株的基因组中含27个不同的次级代谢产物编码基因簇,其中包含编码嗜铁素、细菌素和抗生素等抑菌基因簇;该菌株发酵液在高温、紫外照射和强酸强碱环境条件下及经蛋白酶处理后,其抑菌活性均未受到影响。【结论】多噬伯克霍尔德氏菌WS-FJ9对林木病原菌物具有很好的生防潜力。     

Emulsion stability and demulsification during the direct extraction of penicillin broth

In this article, we present the factors which influence the stability of the emulsion forming under the circumstances of the direct extraction of penicillin fermentation broth and the conditions of demulsification. A laboratory method has been developed for testing demulsifiers and a quantitative evaluation of the properties determining the separation of phases has been worked out as well. It has been found that an emulsion of oil in water (o/w) forms and that this is very stable; it can be broken only by the presence of demulsifier and by using a field of centrifugal force. We have discovered that the most effective demulsifiers are cation-active surfactants for 1-4 g/L broth concentration. Furthermore, it is advantageous if the demulsifier contains a nonionic surfactant and wetting agent as well. From the 19 surface-active compounds studied in the laboratory, we found that the NADAR 107 A and ARMOGARD D-5306 demulsifiers contained the best properties for the extraction of the penicillin broth.     

Stress-related challenges in pentose fermentation to ethanol by the yeast Saccharomyces cerevisiae

Conversion of agricultural residues, energy crops and forest residues into bioethanol requires hydrolysis of the biomass and fermentation of the released sugars. During the hydrolysis of the hemicellulose fraction, substantial amounts of pentose sugars, in particular xylose, are released. Fermentation of these pentose sugars to ethanol by engineered Saccharomyces cerevisiae under industrial process conditions is the subject of this review. First, fermentation challenges originating from the main steps of ethanol production from lignocellulosic feedstocks are discussed, followed by genetic modifications that have been implemented in S. cerevisiae to obtain xylose and arabinose fermenting capacity per se. Finally, the fermentation of a real lignocellulosic medium is discussed in terms of inhibitory effects of furaldehydes, phenolics and weak acids and the presence of contaminating microbiota.