基于多尺度参数相关分析的细胞培养过程优化与放大

细胞大规模培养过程是活体细胞代谢的过程,因此存在着基因、细胞、反应器多尺度相关关系。通过对生物反应过程中生理代谢特性参数检测,并分析参数的理化相关和生物相关,可以实现多尺度观察与调控。主要介绍了在生物过程优化与放大研究中,整合分析与细胞生理代谢特性相关的参数变化信息以及反应器流场特性参数变化信息,从而实现微观与宏观相结合的过程优化方法以及细胞生理和反应器流场特性相结合的过程放大策略。     

工业生物过程智能控制原理和方法进展

工业生物过程是一个复杂的系统过程,对活体细胞代谢过程的认识是实现高效工业生物制造的基础。文中首先综述了工业发酵过程多尺度优化控制原理和实践,包括多尺度理论与装备、细胞宏观代谢在线检测传感技术以及生理代谢参数相关分析。在此基础上,对工业生物过程智能控制——感知细胞内生理代谢特性新型传感技术、大数据库建立和数据深度计算以及生物过程智能决策进行了综述和展望。     

鸟苷发酵过程的多尺度问题研究

以鸟苷发酵为对象,在生物反应器中以多尺度问题的角度,对在线计算机数据进行相关分析,结合中间代谢物及有关酶活性的检测,建立了以代谢流为核心的多尺度调控方法;又通过与代谢流迁移有关的动态化学计量分析,实现了对批式发酵过程的时变系统估算,建立了主体代谢与产物形成的支路代谢间,包括能量与物质流的关系模型;最后,把具有不同生产能力菌株的关键基因与基因库数据进行了序列比较,发现了本文研究的菌株已具备高产能力,即编码sAMP合成酶的基因发生移码突变以及pur操纵子的多处突变,而中心代谢流迁移是过程优化的关键。对开展微生物功能基因组学的研究和工业过程优化的问题进行了探讨,作者提出有必要从生物反应器的系统生物学高度来认识和解决所面临的问题。     

大数据-模型混合驱动下生物过程优化与放大的新机遇与挑战

当前,生物制造技术和产业是世界关注的热点。然而,生物过程优化与放大过程中普遍面临以下几个难题,包括:过程检测手段缺乏,难以满足关键指标参数的监控;细胞代谢认知匮乏,无法理性实现过程最优化调控;反应器环境差异大,导致逐级放大效率低下。文中针对以上亟待解决的关键问题,通过案例分析介绍发酵过程实时检测-动态调控-理性放大全链条关键技术创新。在未来,生物过程设计将以集成细胞生理学(时空多尺度细胞代谢模型)和流体动力学(CFD模型)的全生命周期模型为指导,推进计算机辅助设计与开发,加速生物过程实现大规模智能化生产,开启绿色生物制造新时代。     

面向工业生物技术的系统生物学

工业生物技术是以微生物细胞工厂利用可再生的生物原料来生产能源、材料与化学品等的生物技术,在解决资源、能源与环境等问题方面起着越来越重要的作用。系统生物学是全面解析微生物细胞工厂及其发酵过程从"黑箱"到"白箱"的重要研究方法。系统生物学借助基因组、转录组、蛋白质组、代谢组以及代谢流组等多组学数据,可解析微生物细胞工厂在RNA、蛋白与代谢物等不同水平上的变化规律与调控机制。目前,系统生物学在微生物细胞工厂的设计创建与发酵工艺优化中起着越来越重要的指导作用,许多成功应用实例不断涌现,推动着工业生物技术的快速发展。文中重点综述基因组、转录组、蛋白质组、代谢组与代谢流组以及基因组规模的网络模型等各组学技术的最新发展及其在工业生物技术尤其是菌株改造与发酵优化中的应用,并就工业生物技术中系统生物学的未来发展方向进行展望。     

微生物发酵过程生理参数检测传感器技术与过程优化

工业生物发酵过程是一个复杂的代谢表达调控系统。阐述了开发和利用先进的发酵过程传感仪表技术,进行环境参数和细胞生理代谢特性参数的实时精确测定,是系统了解和分析工业微生物的宏观与微观代谢特性的基础。实现生物传感器与信息技术、过程多参数智能化过程分析的软件识别系统联合,是构建新型的智能化工业生物过程控制与生产模式、建立生产过程的绿色智能制造新技术的关键。     

基元模式分析在生物网络和途径分析中的应用

基元模式分析是应用最广泛的代谢途径分析方法。基元模式分析的研究对象从代谢网络发展到信号传导网络;研究尺度从细胞到生物反应器,甚至生态系统;数学描述从稳态分解到动态解析;研究领域从微生物代谢到人类疾病。以下综述了基元模式分析的算法和软件开发现状,以及其在代谢途径与鲁棒性、代谢通量分解、稳态代谢通量分析、动态模型与生物过程模拟、网络结构与调控、菌株设计和信号传导网络等方面的应用。开发新的算法解决组合爆炸问题,探索基元模式与代谢调控的关系以及提高菌株设计算法效率是今后基元模式的重要发展方向。     

原位显微镜在细胞生物量在线监测中的发展与应用

随着现代生物技术的快速发展,生物发酵过程在工业生产中的重要性日益增加。为获得质量稳定的发酵产品,通常需要对发酵过程进行监测与调控。生物量可以直接反映生物反应器中细胞代谢的主体——细胞的生长状况,因此实现生物量的在线监测对发酵过程的调控具有重要意义。原位显微镜是一项非侵入式的、基于图像分析的技术,可以实时监测生物过程中的细胞量。文中就原位显微镜的发展及其在细胞生物量实时监测中的应用进行了综述。     

生物热对传统固态发酵菌群演替及其代谢影响的研究进展

解析传统固态发酵中产生的生物热对微生物菌群代谢的影响,是认识发酵机制、调控发酵过程、保证发酵效率的关键之一。固态发酵过程中,微生物菌群代谢活动所产生的生物热及传热效率低等问题引起微环境温度升高,进而影响微生物的生长与代谢。然而,关于传统固态发酵微生物受生物热的影响及其适应机制仍不明晰。因此,本文以传统固态发酵体系为研究对象,阐述持续生物热介导的高温对固态发酵过程中微生物群落演替和代谢功能的影响,并提出复杂群落中具有多层次调控微生物代谢以适应高温环境的方式,主要从微生物群体与个体层面介绍可能存在的耐热机制。了解生物热对传统固态发酵微生物的影响及潜在的耐热机制,有助于靶向调控发酵过程、强化高温发酵等,以满足未来的工业化需求。     

生物反应器流场特性研究及其在生物过程优化与放大中的应用研究

生物反应器是实现生物过程的核心设备,其内进行的生物反应过程受不同反应器内流场结构的影响会产生差异显著的结果,这也是导致生物过程放大困难的关键问题之一。为研究这一问题,必须对不同规模反应器内流场结构有充分的认识,必须对反应器内的混合、传质、剪切等影响生物过程的几个关键方面进行深入研究。首先简单介绍了反应器流场研究的实验及数值模拟方法,在此基础上详细论述了流场混合与传质对生物反应过程的影响,流场剪切对丝状菌形态及其生物过程的影响,最后从反应器流场与细胞生理代谢特性整合的角度,介绍了反应器流场与细胞生理之间的相互作用关系,并以三个实例简单介绍了基于Euler-Lagrange模拟框架的整合流场和细胞动力学模型的模拟方法及模拟结果。旨在通过介绍反应器流场研究及其在生物过程优化放大中的应用,提出基于反应器流场特性与细胞动力学响应的生物过程放大研究方法,为更高效、理性地生物过程放大提供一些理论参考。     

Propionic acid fermentation by Propionibacterium freudenreichii CCTCC M207015 in a multi-point fibrous-bed bioreactor

Propionic acid was produced in a multi-point fibrous-bed (MFB) bioreactor by Propionibacterium freudenreichii CCTCC M207015. The MFB bioreactor, comprising spiral cotton fiber packed in a modified 7.5-l bioreactor, was effective for cell-immobilized propionic acid production compared with conventional free cell fermentation. Batch fermentations at various glucose concentrations were investigated in the MFB bioreactor. Based on analysis of the time course of production, a fed-batch strategy was applied for propionic acid production. The maximum propionic acid concentration was 67.05 g l⁻¹ after 496 h of fermentation, and the proportion of propionic acid to total organic acids was approximately 78.28% (w/w). The MFB bioreactor exhibited excellent production stability during batch fermentation and the propionic acid productivity remained high after 78 days of fermentation.     

Comparison of fluidized bed and ultrasonic cell-retention systems for high cell density mammalian cell culture

Economically viable biopharmaceutical production is to a high degree dependent on high product yields and stable fermentation systems that are easy to handle. In the current study we have compared two different fermentation systems for the production of recombinant protein from CHO cells. Both systems are fully scaleable and can be used for industrial high cell density bioprocesses. As a model cell line we have used a recombinant CHO cell line producing the enzyme arylsulfatase B (ASB). CHO cells were cultivated as adherent cell culture attached on Cytoline macroporous microcarrier (Amersham Biosciences, Sweden) using a Cytopilot Mini fluidized bed bioreactor (FBR, Vogelbusch-Amersham Biosciences, Austria) and as suspension culture using a stirred tank bioreactor equipped with a BioSep ultrasonic resonator based cell separation device (Applikon, The Netherlands). Both systems are equally well-suited for stable, long-term high cell density perfusion cell culture and provide industrial scalability and high yields. For products such as the recombinant ASB, high perfusion rates and therefore short product bioreactor residence times may be of additional benefit.     

Efficient production of l-lactic acid from hydrolysate of Jerusalem artichoke with immobilized cells of Lactococcus lactis in fibrous bed bioreactors

Hydrolysate of Jerusalem artichoke was applied for the production of l-lactic acid by immobilized Lactococcus lactis cells in a fibrous bed bioreactor system. Preliminary experiments had indicated that the high quality hydrolysate, which was derived from the 40min acid treatment at 95°C and pH 1.8, was sufficient to support the cell growth and synthesis of l-lactic acid. With the addition of 5g/l yeast extract, the fermentative performance of free cell system was evidently improved. After the basal settlement of hydrolysate based fermentation, the batch mode and the fed-batch mode fermentation were carried out in the free cell system and the fibrous bed bioreactor system, respectively. In all cases the immobilized cells presented the superior ability to produce l-lactic acid. The comparison of batch mode and fed-batch mode also indicated that the growth-limiting feeding strategy could reduce the lag phase of fermentation process and enhance the production of l-lactic acid. The achieved maximum concentration of l-lactic acid was 142g/l in the fed-batch mode. Subsequent repeated-batch fermentation of the fibrous bed bioreactor system had further exhibited the persistence and stability of this system for the high production of l-lactic acid in a long term. Our work suggested the great potential of the fibrous bed bioreactor system and hydrolysate of J. artichoke in the economical production of l-lactic acid at industrial scale.     

Continuous propionate production from whey permeate using a novel fibrous bed bioreactor

Continuous production of propionate from whey lactose by Propionibacterium acidipropionici immobilized in a novel fibrous bed bioreactor was studied. In conventional batch propionic acid fermentation, whey permeate without nutrient supplementation was unable to support cell growth and failed to give satisfactory fermentation results for over 7 days. However, with the fibrous bed bioreactor, a high fermentation rate and high conversion were obtained with plain whey permeate and de-lactose whey permeate. About 2% (wt/vol) propionic acid was obtained from a 4.2% lactose feed at a retention time of 35 to 45 h. The propionic acid yield was approximately 46% (wt/vol) from lactose. The optimal pH for fementation was 6.5, and lower fermentation rates and yields were obtained at lower pH values. The optimal temperature was 30 degrees C, but the temperature effect was not dramatic in the range of 25 to 35 degrees C. Addition of yeast extract and trypticase to whey permeate hastened reactor startup and increased the fermentation rate and product yields, but the addition was not required for long-term reactor performance. The improved fermentation results with the immobilized cell bioreactor can be attributed to the high cell density, approximately 50 g/L, attained in the bioreactor, Cells were immobilized by loose attachement to fiber surfaces and entrapment in the void spaces within the fibrous matrix, thus allowing constant renewal of cells. Consequently, this bioreactor was able to operate continuously for 6 months without encountering any clogging, degeneration, or contamination problems. Compared to conventional batch fermentors, the new bioreactor offers many advantages for industrial fermentation, including a more than 10-fold increase in productivity, acceptance of low-nutrient feedstocks such as whey permeate, and resistance to contamination. (c) 1994 John Wiley & Sons, Inc.     

Enhanced production of L-(+)-lactic acid in chemostat by Lactobacillus casei DSM 20011 using ion-exchange resins and cross-flow filtration in a fully automated pilot plant controlled via NIR

Due to the lack of suitable in-process sensors, on-line monitoring of fermentation processes is restricted almost exclusively to the measurement of physical parameters only indirectly related to key process variables, i.e., substrate, product, and biomass concentration. This obstacle can be overcome by near infrared (NIR) spectroscopy, which allows not only real-time process monitoring, but also automated process control, provided that NIR-generated information is fed to a suitable computerized bioreactor control system. Once the relevant calibrations have been obtained, substrate, biomass and product concentration can be evaluated on-line and used by the bioreactor control system to manage the fermentation. In this work, an NIR-based control system allowed the full automation of a small-scale pilot plant for lactic acid production and provided an excellent tool for process optimization. The growth-inhibiting effect of lactic acid present in the culture broth is enhanced when the growth-limiting substrate, glucose, is also present at relatively high concentrations. Both combined factors can result in a severe reduction of the performance of the lactate production process. A dedicated software enabling on-line NIR data acquisition and reduction, and automated process management through feed addition, culture removal and/or product recovery by microfiltration was developed in order to allow the implementation of continuous fermentation processes with recycling of culture medium and cell recycling. Both operation modes were tested at different dilution rates and the respective cultivation parameters observed were compared with those obtained in a conventional continuous fermentation. Steady states were obtained in both modes with high performance on lactate production. The highest lactate volumetric productivity, 138 g L(-1) h(-1), was obtained in continuous fermentation with cell recycling.     

Evaluation of Mut+ and MutS Pichia pastoris phenotypes for high level extracellular scFv expression under feedback control of the methanol concentration

Extracellular secretion of over 4 g x L(-1) of the A33 scFv antibody fragment was achieved in Pichia pastoris at the 10 L bioreactor scale using minimal medium and feedback control of the methanol concentration. Since methanol acts as both inducer and carbon source, its close regulation is a crucial factor in achieving optimal fermentation conditions. The antibody fragment production levels of both Mut+ and MutS phenotypes were compared in a bioreactor under closed-loop PID control of the methanol level. As expected, the MutS phenotype has a growth rate lower than that of the Mut+ (0.37 vs 1.05 d(-1)) when growing under methanol. However, protein productivity and cell yield on substrate are almost double that of the Mut+ (18.2 vs 9.3 mg A33 sc per gram of methanol). Induction at wet cell weight of 350 g x L(-1) for the MutS also has a positive effect on the final product concentration. Both Mut+ and MutS phenotypes reach a maximum biomass density around 450 g x L(-1) wet cell weight, independent of methanol concentration, reactor scale, or induction density. This reactor configuration allows for reproducible fermentation schemes with different Pichia pastoris phenotypes with AOX promoters, without prior knowledge of the culture growth parameters.     

乳酸钠促进法夫酵母虾青素合成代谢流作用分析

【目的】研究乳酸钠(一种糖代谢产物)的加入对法夫酵母JMU-VDL668发酵过程中细胞生长和虾青素合成的影响。【方法】分别在摇瓶和7 L发酵罐实验基础上,采用代谢通量分析的方法分析添加乳酸钠对法夫酵母菌株JMU-VDL668合成虾青素代谢流的影响。【结果】在7 L发酵罐实验中添加乳酸钠,虾青素产量最高可达17.70 mg/L,与对照组相比提高26%。代谢通量分析表明,乳酸钠可以调节丙酮酸、乙酰辅酶A节点处的代谢通量分布,乳酸在乳酸脱氢酶的作用下可以直接进入代谢网络的后半程,乙酰辅酶A的通量和进入TCA循环的通量得到了显著加强。【结论】乳酸钠的加入提供了更多的乙酰辅酶A等前体物质和能量供给,因此促进了虾青素的合成。     

High cell density induces spontaneous bifurcations of dissolved oxygen controllers during CHO cell fermentations

High cell density cultures of CHO cells growing in a bioreactor under dissolved oxygen control were found to undergo spontaneous bifurcations and a subsequent loss of stability some time into the fermentation. This loss of stability was manifested by sustained and amplified oscillations in the bioreactor dissolved oxygen concentration and in the oxygen gas flow rate to the reactor. To identify potential biological and operational causes for the phenomenon, linear stability analysis was applied in a neighborhood of the experimentally observed bifurcation point. The analysis revealed that two steady state process gains, K(P1) and K(P2), regulated k(l)a and gas phase oxygen concentration inputs, respectively, and the magnitude of K(P1) was found to determine system stability about the bifurcation point. The magnitude of K(P1), and hence the corresponding open-loop steady state gain K(OL1), scaled linearly with the bioreactor cell density, increasing with increasing cell density. These results allowed the generation of a fermentation stability diagram, which partitioned K(C)-N operating space into stable and unstable regions separated by the loci of predicted critically stable controller constants, K(C,critical), as a function of bioreactor cell density. This consistency of this operating diagram with experimentally observed changes in system stability was demonstrated. We conclude that time-dependent increases in cell density are the cause of the observed instabilities and that cell density is the critical bifurcation parameter. The results of this study should be readily applicable to the design of a more robust controller.     

Investigation of the bacitracin biosynthesis in an airlift bioreactor

Results of pilot plant studies using an external-loop airlift bioreactor (170 l fermentation volume, liquid height-to-riser diameter: 27, loop-to-tower cross-section-area: 0.1225) have proven the relative merits of such a system in the bacitracin biosynthesis produced by the Bacillus licheniformis submerged aerobic cultivation. The results were compared to those obtained in a pilot-scale stirred-tank bioreactor with the same values of k_La. Excepting the aeration rate of 0.2 vvm, the fermentation process performed at 0.5 vvm and 1/0 vvm, respectively, unfolded similarly in the two fermentation devices with respect to the cell mass production, substrate utilization and bacitracin production during the fermentation process. In the riser section of the airlift bioreactor, the dissolved oxygen levels were higher, while in the downcomer section they were lower than those realized in the stirred tank bioreactor. Power requirements of the airlift fermenter were by 17–64% lower than those for a mechanically agitated system, depending on the aeration rates, which led to an important energy saving. Moreover, the lack of mechanical devices in the airlift system provides safety and a more gentle environment for the cultivation of microorganisms.