固定化细胞应用进展

固定化细胞技术是酶工程的核心技术之一,它将酶工程提高到一个新水平。该技术简化了工业分离纯化的步骤,并使酶反应的连续生产成为现实。目前,该技术已经广泛应用于食品、发酵、三废处理等行业,经济效益显著。首先分析了固定化细胞的优缺点,介绍了近年来在食品、发酵和三废处理行业的应用,最后对其应用进行了展望。     

病毒逃逸补体的机制

补体系统是宿主免疫防御外来病原体的第1道防线,包括对病毒的防御.补体系统可通过黏附在病原体表面利于宿主细胞吞噬、形成膜攻击复合体导致病原体溶解、释放过敏毒素引起炎症反应等多条途径清除外来病原体.然而,在与宿主一起进化的过程中,某些病毒已经建立了逃逸补体系统的策略,这些策略包括编码补体调控蛋白、从宿主获得膜调控蛋白以及利用宿主膜补体受体进入宿主细胞.本文就病毒逃逸补体系统作用的策略的研究进展作一综述.     

L-色氨酸发酵过程偶联膜分离提取工艺研究

目的:将发酵过程与膜分离提取过程相偶联,解决现有产酸周期短、乙酸积累较为严重等问题,以提高目的产物的产量,降低代谢抑制物质的积累量。方法:将发酵罐与陶瓷膜分离装置相偶联,在发酵时长21 h、乙酸积累量达到5 g/L时开始膜过滤浓缩菌液,结束后补加透析培养基继续发酵。结果:采用膜偶联发酵技术,与传统发酵工艺相比,菌体生物量提高3.4%,谷氨酸积累量下降54.34%,乙酸积累量下降55.36%。整个发酵过程产酸周期延长了12 h,发酵过程中糖酸转化率提高6.9%。结论:采用膜偶联发酵技术发酵生产色氨酸,可以显著提高色氨酸产量,降低代谢副产物的积累量,延长产酸周期,提高糖酸转化率。     

甾体微生物转化在制药工业中的应用

对几种重要的甾体微生物转化反应如甾体边链降解、甾体羟基化反应的机理及其发展与应用作了概述;同时也介绍了固定化微生物细胞、非水溶液中酶催化反应及混合发酵等微生物转化技术在制药工业中的应用。     

固定化微生物细胞在发酵工业上的应用

近年来,固定化微生物细胞用于单一酶反应的工业化生产,在发酵工程方面越来越引起人们的兴趣。特别是固定化微生物细胞,用于多步生化反应中合成产品以及在发酵工业中,必将有广泛的前途。这个新技术的应用范围也越来越广。我们以乙醇、乳酸、柠檬酸和葡萄糖酸的生产为例子,来阐明厌气和好气性微生物细胞固定化后的发酵过程。     

电容法在线测定发酵过程中毕赤酵母浓度的研究

活细胞量是与细胞生长、代谢、生产力相关的重要生理参数。在线活细胞测定仪能够利用电容的原理检测发酵液中的活细胞量。本文详细介绍了国产在线活细胞测定仪的检测原理,并说明了其在毕赤酵母发酵过程中的应用情况。结果表明,我们设计的在线活细胞测定仪能够直接、实时、在线获得发酵过程中可靠的活细胞量,且有效避免采样操作的干扰。这一技术符合过程分析技术(PAT)的要求,并可以与其他参数相结合进行分析,作为发酵过程开发、控制和优化的有利工具。     

典型工业发酵过程环境变化下的细胞自适应行为与系统优化

工业发酵过程中,当细胞遭受到极端环境胁迫或剧烈环境变化时,细胞必须要作出必要的举动来适应环境的剧烈变化。细胞的自适应行为有可能不能应对剧烈的环境变化,导致发酵失败;但也有可能产生意想不到的效果,改善发酵性能。文中以毕赤酵母Pichia pastoris生产异源蛋白和丁醇发酵过程为例,阐述了环境变化条件下的细胞自适应行为及其基于自适应行为的发酵过程优化方法和策略,为利用基于细胞自适应行为的发酵过程优化提供参考。     

疏水网格滤膜技术检测食源性致病菌的研究进展

疏水网格滤膜技术利用膜的疏水性黏附细菌细胞,通过膜的过滤作用截留细菌细胞,然后将膜进行选择性培养,达到检测鉴定待测菌的目的。主要概述了疏水网格滤膜技术的原理和特点,以及该技术与分子生物学技术、免疫学技术偶联在食源性致病菌检测中的应用。     

纤维素乙醇高温发酵的研究进展与展望

利用高温细菌发酵,纤维素乙醇生产有望实现“生物质降解-乙醇发酵-乙醇蒸馏”过程的同步化,从而最大限度地降低纤维素乙醇的生产成本;这是一个目标更高、道路更远、科学性更强的可再生能源发展策略.纤维素乙醇高温发酵研究已经取得了重要进展,目前面临的主要挑战包括发酵乙醇的高温细菌的遗传转化系统不够稳定、缺少内源的高活性和耐热性纤维素酶,以及乙醇代谢调控机理有待进一步解析.这些科技难题将会在DNA生物合成和进化技术、细胞生物学技术,以及合成生物学技术的发展中得到解决.     

微生物菌群发酵生产化学品的研究进展

廉价生物质资源的利用是工业生物技术领域研究的热点,复杂的成分和较多的杂质使传统的单菌发酵方式难以应对,成为产业化的关键问题。文中从微生物菌群的工业应用、微生物菌群发酵与纯种发酵的比较、微生物细胞间的相互作用等方面综述了微生物菌群发酵技术的最新研究进展,并对微生物菌群的设计和应用进行了展望。微生物菌群发酵可以充分利用廉价生物质基质、生产多个产品或减少副产物的生成,在生物基化学品和燃料的生产中将是一种有前景的发酵技术。     

Dialysis cultures

Dialysis techniques are discussed as a means for effective removal of low-molecular-mass components from fermentation broth to reach high cell density. Reactor systems and process strategies, the relevant properties of membranes and examples for high-density fermentation with dialysis, and problems related to scale-up are addressed. The dialysis technique has turned out to be very efficient and reliable for obtaining high cell densities. As in dialysis processes the membranes are not perfused, membrane clogging is not a problem as it is for micro- and ultrafiltration. By applying a “nutrient-split” feeding strategy, the loss of nutrients can be avoided and the medium is used very efficiently. The potential of dialysis cultures is demonstrated on the laboratory scale in a membrane dialysis reactor with an integrated membrane and in reactor systems with an external dialysis loop. In dialysis cultures with different microorganisms (Staphylococci, Escherichia coli, extremophilic microorganisms, Lactobacilli) the cell densities achieved were up to 30 times higher than those of other fermentation methods. The technique enables high cell densities to be attained without time-consuming medium optimization. For animal cell cultures the concept of a fixed bed coupled with dialysis proved to be very effective. Received: 24 March 1998 / Received revision: 18 June 1998 / Accepted: 19 June 1998     

Nutrient-split feeding strategy for dialysis cultivation of Escherichia coli

Reduction in nutrient loss during dialysis cultivation of Escherichia coli on a glycerol medium was investigated. A dialysis reactor with an inner fermentation and an outer dialysis chamber was used. Aerobic condition was maintained by limiting the glycerol feed rate to an optimum value which was estimated from the oxygen requirements for glycerol oxidation and oxygen transfer capacity of the reactor. High reduction in nutrient loss was achieved by using water as the dialyzing fluid. However, osmotic movement of water from the dialysis to the fermentation chamber was observed, and the final cell concentration was low. With a nutrient-split feeding strategy (feeding glycerol directly to the fermentation chamber and dialyzing with salt solution), glycerol loss was small, there was no osmotic flux of water to the fermentation chamber, and the cell concentration was high. Both glycerol and salt loss could be avoided, and a cell concentration of 170 g/L was obtained when the dialysis process was substituted by addition of XAD adsorbents to the dialysis chamber. Application of this nutrient-split feeding strategy to cell cultivation in a stirred tank reactor, coupled with dialysis in external dialyzer modules, resulted in low cell concentrations. (c) 1993 Wiley & Sons, Inc.     

Scale-up of dialysis fermentation for high cell density cultivation of Escherichia coli.

In dialysis fermentations inhibiting metabolites can be removed from cell suspensions resulting in a prolonged exponential growth phase and higher production yields. Because of successful high cell density cultivations of Escherichia coli in a laboratory dialysis reactor, a scale-up of the process was investigated. To provide sufficient membrane area for dialysis in a technical scale fermenter, an external membrane module was used, that was also applied for oxygen supply to the culture in the external loop. Cultivations with recombinant E. coli K12, with and without induction, in 2- and 300-l reactors were carried out using external modules. Cell densities exceeding 190 g l(-1), previously obtained in laboratory dialysis fermentation, were also produced with external dialysis modules. Protein concentration in a 300-l reactor was increased to the 3.8-fold of industrial fed-batch-fermentations.     

Specific cephalosporin C production of Acremonium chrysogenum is independent of the culture density

Specific cephalosporin C production of Acremonium chrysogenum grown on a glucose-based minimal medium using conventional batch and dialysis membrane reactor systems was independent of the cell density in the range of 0.4 to 40 g biomass l^–1.     

Fixed-bed dialysis culture of a transfectoma cell line producing chimeric Fab-fragments with “nutrient-split”-feeding strategy

The very shear sensitive transfectoma cell line, FAB 10, producing a chimeric Fab-antibody fragment specific for the human carcinoembryonic antigen (CEA), was cultivated in fixed bed reactors where the cells were immobilized in macroporous carriers. As the cell line had a very low productivity, cultures with process integrated product enrichment were performed in a membrane dialysis bioreactor with integrated fixed bed, where low molecular weight metabolites are removed over the membrane and high molecular weight products are enriched. In a new nutrient-split feeding strategy for dialysis cultures concentrated medium was supplied directly to the fixed bed unit, whereas a buffer solution was used as dialysis fluid. With the dialysis technique up to 5800 g Fab l–1 could be obtained, more than 10 times higher compared to fixed bed cultures without dialysis or batch cultures with suspended cells. The nutrient-split-feeding strategy reduced the amount of medium required per mg of antibody significantly. © Rapid Science Ltd. 1998     

Performance of a membrane-dialysis bioreactor with a radial-flow fixed bed for the cultivation of a hybridoma cell line

A bioreactor system for the continuous cultivation of animal cells with a high potential for scale-up is presented. This reactor system consists of radial-flow fixed-bed units coupled with a dialysis module. The dialysis membrane enables the supply of low-molecular-weight nutrients and removal of toxic metabolites, while high-molecular-weight nutrients and products (e.g. monoclonal antibodies) are retained and accumulated. This concept was investigated on the laboratory scale in a bioreactor with an integrated dialysis membrane. The efficiency of the reactor system and the reproducibility of the cell activity (hybridoma cells) under certain process conditions could be demonstrated in fermentations up to 77 days. Based on model calculations, an optimized fermentation strategy was formulated and experimentally confirmed. Compared to chemostat cultures with suspended cells, a ten-times higher mAb concentration (383 mgl⁻¹) could be obtained. The highest volumetric specific mAb production rate determined was 6.1 mg mAb (1 fixed bed)⁻¹ h⁻¹.     

Growth kinetics and nitrogen-nutrition of the marine diatom Phaeodactylum tricornutum in continuous dialysis culture

The growth kinetics and nitrogen (N)-nutrition of the marine pennate diatom Phaeodactylum tricornutum Bohlin were determined in continuous dialysis culture at different cell densities. Inflow nutrient medium was supplied as natural unenriched estuarine seawater to a dialysis culture system with a high ratio of membrane surface area/culture volume (Am/Vc). Under the experimental conditions, the supply of inorganic macronutrients (NO ₃ ^? + NO ₄ ^? and PO ₄ ^?3 ) by diffusion (Nd) was markedly greater than that provided by the dilution (FfCN) of the culture (Nd ? FfCN), thereby establishing an inverse relationship between the cell density and the dilution rate (D). This continuous dialysis system allows for the maintenance of prolonged growth (> two weeks) at various cell densities (1.4 to 27.2 × 10⁹ cells 1^?1) within a range of dilution rates between 0.30 to 1.08 d^?1. In high cell density cultures, where the extracellular medium was characterized as nutrient deficient, a lower growth rate (μ_e) was exhibited than in cultures with lower cell densities. The growth rate (μ_e) remained equivalent to the dilution rate (D) throughout the culture cycle, indicating that equilibrated growth was achieved. High cell density cultures yielded higher productivity (P), relative to that of cultures grown at lower cell densities, in terms of cell-N and ?C produced per unit time. However, cell quotas of both N and C declined with increasing cell concentrations. Denser cultures were characterized by an enhanced N-conversion efficiency (Y_N) and a higher cellular N/C atomic ratio. The nutritional response of this diatom in dense cultures reveals an efficient use of N-nutrients, presumably as a result of cellular nutrient adaptation to oligotrophic conditions.     

Monoclonal antibody production in dialyzed continuous suspension culture

Hybridoma cell growth and monoclonal antibody production in dialyzed continuous suspension culture were investigated using a 1.5-L Celligen bioreactor. Medium supplemented with 1.5% fetal bovine serum was fed directly into the reactor at a dilution rate of 0.45 d(-1). Dailysis tubing with a molecular weight cut-off (MWCO) of 1000 was coiled inside the bioreactor. Fresh medium containing no serum or serum substitues passed through the dialysis tubing at flow rates of 2 to 5 L/d. The objective was to remove low molecular weight inhibitors, such as lactic acid and ammonia, by diffusion through the tubing, while continuoulsy replenishing essential nutrients by the same mechanism. Due to the low MWCO of the dialysis tubing high molecular weight components such as growth factors and antibody were not removed by the dialyzing stream. In the batch start-up phase, the monoclonal antibody (MAb) titer was almost 3 times that achieved in typical batch cultures (i.e., 170 to 180 mg/L). During dialyzed continuous operation, a substantial increase (up to 40%) in cell density, monoclonal antibody (MAb) titer, and reactor MAb productivity was observed, as compared with a conventional continuous suspension culture. The cell viability and the specific MAb productivity remained practically constant at different dialysis rates. This finding suggests that the steady state growth and death rate in continuous suspension hybridoma cultures are not direct functions of the nutrient or inhibitor concentrations.     

Production of the siderophore enterobactin: use of four different fermentation systems and identification of the compound by HPLC

The article describes four different fermentation procedures for Escherichia coli AN311, a producer of enterobactin. A regular rotary shaker culture with a biphasic system consisting of an agar layer (as a reservoir for feeding processes) and a layer of liquid medium, 2.4 L and 10 L batch cultures, and a novel dialysis membrane fermentor were used. With the use of this latter fermentor type, the production of enterobactin could be increased by a factor of about 9.5, while growth increased by a factor of 12 compared to the other systems. For the rapid and reliable quantification of the concentration and purity of enterobactin an analytical and preparative high-performance liquid chromatography (HPLC) method was established. The degradation compounds of this siderophore were detected by diodearray and bioassays. A comparison of total catechol production as well as the distribution between enterobactin and its degradation compounds is given. (c) 1993 John Wiley & Sons, Inc.     

Dialysis cultures with immobilized hybridoma cells for effective production of monoclonal antibodies

An industrial scale reactor concept for continuous cultivation of immobilized animal cells (e.g. hybridoma cells) in a radial-flow fixed bed is presented, where low molecular weight metabolites are removed via dialysis membrane and high molecular products (e.g. monoclonal antibodies) are enriched. In a new nutrient-split feeding strategy concentrated medium is fed directly to the fixed bed unit, whereas a buffer solution is used as dialysis fluid. This feeding strategy was investigated in a laboratory scale reactor with hybridoma cells for production of monoclonal antibodies. A steady state monoclonal antibody concentration of 478 mg l^-1 was reached, appr. 15 times more compared to the concentration reached in chemostat cultures with suspended cells. Glucose and glutamine were used up to 98%. The experiments were described successfully with a kinetic model for immobilized growing cells. Conclusions were drawn for scale-up and design of the large scale system.Abbreviations: c_Glc – glucose concentration, mmol l^-1; c_Gln – glutamine concentration, mmol l^-1; c_Amm – ammonia concentration, mmol l^-1; c_Lac – lactate concentration, mmol l^-1; c_MAb – MAb concentration, mg l^-1; D – dilution rate, d^-1; D_i – dilution rate in the inner chamber of the membrane dialysis reactor, d^-1; D₀ – dilution rate in the outer chamber of the membrane dialysis reactor, d^-1; q*_FB,Glc – volume specific glucose uptake rate related to the fixed bed volume, mmol l_FB ^-1 h^-1; q*_FB,Gln – volume specific glutamine uptake rate related to the fixed bed volume, mmol l_FB ^-1 h^-1.