内循环气升式生物反应器培养甘草细胞

自行设计研制了７Ｌ,９Ｌ及２５Ｌ内循环气升式生物反应器,并应用于甘草细胞的放大培养研究。在接种量为８％（Ｗ／Ｖ）通气率为０．２—０．２５ｖｖｍ的条件下,甘草细胞在反应器中生长迅速。其中在９Ｌ反应器中生长最好,最高生物量达１６．２５ｇ／Ｌ,生长速率达０．９ｇ／Ｌ．ｄ,均高于摇瓶培养。培养过程中ｐＨ值、溶氧状况通过电极自动显示记录,说明设计的气升式生物反应器适合于甘草细胞的大规模培养。     

甘草细胞在搅拌式生物反应器中的放大培养

在建立了稳定的甘草细胞搅拌式生物反应器放大培养体系的基础上,本文研究了甘草细胞在搅拌式反应器中悬浮培养的生长特性,包括细胞生长、细胞膜的透性、培养体系的p H变化及甘草黄酮合成情况等,并与摇瓶培养作比较。结果发现,同等条件下,反应器中培养细胞生物量的积累低于摇瓶培养,整个培养周期较摇瓶培养缩短。培养过程中同一时间段反应器中的p H值略低于摇瓶中的p H,细胞中H2O2的浓度是摇瓶中的1.8倍,甘草黄酮的产量是摇瓶培养的1.5倍,表明反应器中机械搅拌与流体剪切的培养环境对细胞生长起到一定程度的抑制作用,但刺激了细胞次生代谢产物甘草黄酮较高水平的合成。     

胀果甘草悬浮培养细胞合成甘草总黄酮

比较了胀果甘草(Glycyrrhiza inflata)悬浮细胞在逐级放大摇瓶中的生长、黄酮产量以及营养消耗过程,以便了解其放大规律。结果表明,在250和500mL摇瓶中,细胞的最大生物量、黄酮产量以及最大比生长速率没有显著性差异,但是在1L的摇瓶中,这三种参数都显著地降低,分别比250mL摇瓶中降低了27%,30%和27%。在逐级放大的摇瓶中,氮、磷、铵浓度都随着培养时间延长而逐渐降低,尽管在1L的摇瓶中磷消耗得最慢,但三种摇瓶中磷在细胞生长对数期基本都被消耗尽了。此外,硝态氮在第18天时基本被消耗完,而铵态氮在细胞收获时仍能维持在100mg/L。因此在反应器中培养时,主要的培养条件还需进一步优化。     

产油微藻自养培养条件调控

作为新兴生物燃料的生物柴油近年来发展迅速,以微藻为代表的第二代生物能源是解决能源危机的长远之计,但如何提高其产量仍是研究的热点问题。以提高产油自养微藻生物量和油脂含量为目的,在气升式光反应器中运用均匀设计实验方法进行了条件优化试验。分别得出了氮原子浓度、通气速率、二氧化碳体积浓度和光照强度4个因素对小球藻C2生物量积累和油脂含量影响的显著回归方程和反应器优化培养条件。以生物量为指标的优化培养条件是:氮原子浓度0.178 g/L,通气速率5 L/min,二氧化碳体积浓度3%(V/V),光照强度6000 lx。该优化条件下,生物量为2.11 g/L,即生产速率为0.352 g/(L.d),比测试实验中检测到的最高生物量[1.88 g/L,即生产速率为0.313 g/(L.d)]提高了12.2%;以油脂含量为指标的优化培养条件是:进气速率0.400 L/min,二氧化碳体积浓度1.94%(V/V),得到油脂含量为22.4%,比测试实验中检测到的最高油脂量(20.7%)提高7.7%。     

小球藻高密度培养及油脂提取条件的优化

【目的】高密度培养小球藻及优化油脂提取条件。【方法】通过进行单因素实验研究不同培养基组成及环境因子对其细胞生长影响,并采用超声波提取法进行正交实验对藻粉油脂提取条件进行研究。【结果】对椭圆小球藻Y4进行异养培养,最适培养条件为:葡萄糖50 g/L,硝酸钾2 g/L,适宜的培养温度、摇床转速和接种量分别为29°C、180 r/min和20%。在此基础上,进行了1 L发酵罐培养实验,获得了干重18.25 g/L的生物量。通过对油脂提取条件进行优化,Y4的油脂提取率由优化前的25.0%提高到60.2%,提高了35.2%。【结论】优化了小球藻的培养条件及油脂提取条件,促进了小球藻的开发和利用。     

VERO细胞生物反应器放大培养初探

目的:研究用生物反应器放大进行Vero细胞微载体培养,实现生物反应器之间Veto细胞放大培养.方法:5L微载体生物反应器以10g/L微载体浓度培养Vero细胞,96h时经漂洗、消化、接种于30L微载体生物反应器,实现放大后的30L微载体生物反应器细胞怏速增殖,期间对不同时期的微载体细胞进行细胞计数、细胞代谢分析和形态观察.结果:5L生物反应器细胞经过96h灌注培养,平均细胞密度达到7.81×10~6cells/mL.5L微载体细胞放大到30L微载体生物反应器,平均细胞收获率为32.3%;放大到30L生物反应器后经过144h培养,细胞密度达到9.19×10~6cells/mL;放大后的细胞代谢途径依然以葡萄糖氧化代谢乳酸为主.结论:生物反应器由5L到30L进行Veto细胞放大培养是可行的.     

红色诺卡菌固体培养基配方的优化

应用响应面优化设计法优化固体培养基配方,增大红色诺卡菌的固体培养细胞生物量。首先用Plackett-Burman法从现有培养基组分中找到影响红色诺卡菌细胞生物量的关键因素,再通过最陡爬坡法确定细胞生物量最大的配方,用作中心组合设计(Central Composite Design, CCD)实验的基础起始值,拟合数学模型方程,最后找到最优组分的组合。优化的配方转移至企业实施放大实验,对结果进行验证和比较。试验结果表明,培养基各组分中影响红色诺卡菌细胞生物量的关键因素为蛋白胨、NaCl、牛肉膏;最优固体培养基配方:蛋白胨42 g/L、牛肉膏8 g/L、NaCl 1.2 g/L、甘油10 mL/L、Na_2HPO_4·12H_2O 0.3 g/L、琼脂20 g/L。在细胞生物量方面最优固体培养基配方比原配方高104%。响应面优化设计可用于提高红色诺卡菌细胞生物量固体培养基的优化,也为红色诺卡菌培养条件、液体发酵的优化研究提供参考。     

光生物反应器中螺旋藻培养条件的优化

利用正交实验对搅拌式光生物反应器中钝顶螺旋藻(Spirulina platensis Geitl)的培养条件即搅拌速度、通气量和光照强度进行优化.实验结果表明:当培养温度为30℃时,通过正交实验所获得的最佳培养条件为搅拌转速120 r·min-1,通气量80 L·h-1,光照强度5000lx.在最佳培养条件下,收获时螺旋藻的干重为1.922 g·L-1.根据回归模型得到相应的优化条件为:光照强度5000lx,通气量150L·h-1,搅拌转速111.70r·min-1,收获量(干重)的预测值为2.293 g·L-1.另外,10%的接种量有利于螺旋藻的生长.     

高生物量富硒酵母的选育及培养条件初步优化

通过筛选、单倍体分离、诱变和原生质体融合,从融合子中选育了一株高生物量富硒酵母菌株（编号为ZFF-28）,其细胞硒总含量分别是原始亲株ZY-67和ZY-198的2.8倍和2.0倍。通过单因素实验和正交试验设计,确定了优化培养条件：6%糖浓度的蔗糖糖蜜,添加0.5% (NH4)2SO4、0.1% H3PO4、60μg/mL Se,pH60～6.5,装液量50mL/250mL三角瓶,接种量10%,培养时间25h。在优化培养条件下,菌株ZFF_28的生物量可达8.2g/L,细胞中硒的含量达2050μg/g,硒总含量达到了16810μg/L,是培养条件优化前的1.3倍。细胞硒含量的91%为有机硒。     

均匀设计法优化发菜细胞悬浮培养条件

通过摇瓶发酵实验研究了培养温度、光照强度等培养条件对发菜细胞悬浮培养生物量和代谢产物发菜多糖累积的影响,通过均匀设计试验对培养条件进行了优化。结果表明:在培养温度24℃、培养基初始pH8.0、光照强度60μmol/(m2.s)、转速150r/min的条件下培养20d,发菜细胞生物量(细胞质量浓度)达到1.34g/L,胞外多糖产量达到208.32mg/L;与优化前相比,发菜细胞生物量和胞外多糖产量分别提高27.3%、111.17%。     

A genetic algorithm for the optimization of the thermoinduction protocol for high-level production of recombinant human-like collagen from Escherichia coli

Production of recombinant human-like collagen (RHLC) by thermoinduction of recombinant Escherichia coli BL 21 during high cell density cultivation was investigated in a 30 L bioreactor. The effects of induction temperature (T), pH, and carbon-to-nitrogen molar ratio of the nutrient medium (C/N) were examined. The optimal thermoinduction protocol for RHLC production was determined by using a model coupling genetic algorithm and artificial neural networks. The optimal operating conditions were as follows: maintenance of induction temperature at 42°C for 3 H and then at 39.4°C until the end, induction pH at 7.03, and C/N at 4.8 (mol/mol). The theoretical maximum concentration of RHLC was 12.5 g/L, whereas the experimental value was 12.1 g/L under the optimal induction conditions.     

Optimization of culture conditions and bench-scale production of L-asparaginase by submerged fermentation of Aspergillus terreus MTCC 1782

Optimization of culture conditions for L-asparaginase production by submerged fermentation of Aspergillus terreus MTCC 1782 was studied using a 3-level central composite design of response surface methodology and artificial neural network linked genetic algorithm. The artificial neural network linked genetic algorithm was found to be more efficient than response surface methodology. The experimental L-asparaginase activity of 43.29 IU/ml was obtained at the optimum culture conditions of temperature 35 degrees C, initial pH 6.3, inoculum size 1% (v/v), agitation rate 140 rpm, and incubation time 58.5 h of the artificial neural network linked genetic algorithm, which was close to the predicted activity of 44.38 IU/ml. Characteristics of L-asparaginase production by A. terreus MTCC 1782 were studied in a 3 L bench-scale bioreactor.     

Modeling <Emphasis Type="Italic">Euglena</Emphasis> sp. growth under different conditions using an artificial neural network

Microalgae are considered as the future source of biofuels because of their high biomass productivity and neutral lipid content as triacylglycerides (TAG). Microalgae have high photosynthetic efficiency and the possibility of being cultivated in different wastewaters. The isolation of potential microalgae followed by the optimization of cultivation conditions is prerequisite for successful cultivation and accumulation of high lipid content. In the present work, a three-layer artificial neural network (ANN) model is developed to predict the essential parameters (such as pH, temperature, light intensity, photoperiod, and medium composition) based on 156 sets of laboratory experiments for achieving maximum biomass from Euglena sp. The independent parameters (viz., temperature, light intensity, photoperiod and number of days at fixed pH, and media composition) were fed as input to the ANN, and biomass yield was investigated. The comparison of the simulated environmental conditions using the ANN model and experimental results are found to have an excellent correlation coefficient of about 0.97 for the model variables used in this study. The model results established that artificial neural network design may be judiciously employed for optimization of different environmental conditions for this isolated microalga.     

Online filtering of CO<Subscript>2</Subscript> signals from a bioreactor gas outflow using a committee of constructive neural networks

This work proposes a committee of Cascade Correlation neural networks as an online smoother for random measurement noise. The goals of this paper are twofold: first it intends to explore the possibilities of using a constructive neural network algorithm to learn how to filter typical noisy data from a bioreactor, CO₂ mol fractions of the effluent gas during the aerobic cultivation of Bacillus megaterium to produce the enzyme penicillin G acylase. Second, to propose a committee of such networks for achieving more realistic results, capturing the inherent trend of the process. In order to do that this paper discusses the advantages of using a constructive neural network algorithm, describes how the committee of NNs operates and evaluates its performance using real CO₂ online data obtained in laboratorial experiments. The paper also presents results obtained with classical filtering algorithms, for comparison.     

Optimization of fed-batch bioreactor using neural network model

An algorithm using feedforward neural network model for determining optimal substrate feeding policies for fed-batch fermentation process is presented in this work. The algorithm involves developing the neural network model of the process using the sampled data. The trained neural network model in turn is used for optimization purposes. The advantages of this technique is that optimization can be achieved without detailed kinetic model of the process and the computation of gradient of objective function with respect to control variables is straightforward. The application of the technique is demonstrated with two examples, namely, production of secreted protein and invertase. The simulation results show that the discrete-time dynamics of fed-batch bioreactor can be satisfactorily approximated using a feedforward sigmoidal neural network. The optimal policies obtained with the neural network model agree reasonably well with the previously reported results.     

Effect of aggregate size in cell cultures of<Emphasis Type="Italic"> Saussurea medusa</Emphasis> on cell growth and jaceosidin production

Cell suspension cultures of Saussurea medusa were grown in shake flasks and a 5-l stirred tank bioreactor. Biomass and jaceosidin distribution in cell aggregates of different sizes were investigated during the cultivation period. The results showed that on day 10, jaceosidin accumulation showed an increase with increasing size of the cell aggregate to 4 mm in diameter, with the highest jaceosidin accumulation being 12.2 mg/g. An inverse tendency was observed with cell aggregates larger than 4 mm in diameter, with the lowest accumulation being 3.1 mg/g. However, all of the cell aggregates, despite their size, synthesized almost the same amount of jaceosidin at day 12. Oxygen diffusion limitation and cell-cell contact may explain this behavior. In comparison with cells cultivated in shake flasks, decreased biomass and decreased jaceosidin concentration were observed when the cells were cultivated in a stirred tank bioreactor. The sublytic effects caused by the hydrodynamic stress in combination with insufficient nutrients in the bioreactor may cause cell damage.     

Non-invasive methods for determination of cellular growth inPodophyllum hexandrum suspension cultures

Culture conductivity and on-line NADH fluorescence were used to measure cellular growth in plant cell suspension cultures ofPodophyllum hexandrum. An inverse correlation between dry cell weight and medium conductivity was observed during shake flask cultivation. A linear relationship between dry cell weight and culture NADH fluorescence was obtained during the exponential phase of batch cultivation in a bioreactor under the pH stat (pH 6) conditions. It was observed that conductivity measurement were suitable for biomass characterisation under highly dynamic uncontrolled shake flask cultivation conditions. However, if the acid/alkali feeding is done for pH control the conductivity measurement could not be applied. On the other hand the NADH fluorescence measurement allowed online-in situ biomass monitoring of rather heterogenous plant cell suspension cultures in bioreactor even under the most desirable pH stat conditions.     

Fluorescence-based real time monitoring and diagnostics of recombinant Pichia pastoris cultivations in a bioreactor

This study describes the application of the multivariate curve resolution (MCR) analysis technique for real-time analysis of culture fluorescence during recombinant Pichia pastoris cultivation in a bioreactor. Fluorescence spectra were acquired with an on-line dual excitation wavelength fluorometer and then used to develop a real time MCR-based bioprocess monitoring and diagnostics tool. Initial bioreactor experiments using two similar recombinant antibody secreting P. pastoris cell lines showed significant differences in protein production. To distinguish between the contributions of operating conditions and the specific cell line's genetic composition to the observed differences in protein production, the bioreactor experiments were repeated and accompanied by real time MCR analysis. The tests demonstrated high sensitivity of MCR-derived “pure concentration” profiles to growth as well as to initial conditions, thus enabling real-time cultivation process trend diagnostics and fault detection. © 2018 Her Majesty the Queen in Right of Canada © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2761, 2019.     

Submerged cultivation of Stephania glabra (Roxb.) Miers cells in different systems: Specific features of growth and accumulation of alkaloid stepharine

Strains of a Stephania glabra suspension culture grown in flasks and two types of bioreactors (laboratory-scale bubble and pilot-scale stirred reactors) have been compared according to their growth characteristics and accumulation of the alkaloid stepharine. The best characteristics have been recorded for strains 113 and 261. In the case of batch cultivation in flasks, the maximal accumulation of dry biomass by these strains reaches 19–21 g/l; that of the alkaloid stepharine, 0.30–0.35% of dry biomass. The used strains differ in their response to cultivation scale-up from flasks to bioreactors, strain 254 displaying the lowest adaptation to such changes. A bubble reactor is the most beneficial system for submerged cultivation of S. glabra. The absence of detectable stepharine synthesis on the background of a considerable decrease in all growth characteristics of the cultures has been observed when using a pilot stirred bioreactor. The batch cultures of strains 113 and 261 in a bubble bioreactor accumulate 11–16 g/l of dry biomass containing 0.05–0.16% of the alkaloid. It has been shown that strains 113 and 261 retain satisfactory physiological characteristics in a semi-flow regime of a bubble bioreactor. This scale-up scheme can be used for further industrial cultivation.     

Enhanced production of podophyllotoxin by Podophyllum hexandrum using in situ cell retention bioreactor

The rhizomes of the rare plant Podophyllum hexandrum contain podophyllotoxin, which is a precursor of the anticancer drugs etoposide and teniposide. Batch cultivation of Podophyllum hexandrum was conducted using optimized medium in a 3 L bioreactor, which resulted in biomass and podophyllotoxin concentrations of 21.4 g/L and 13.8 mg/L in 24 and 26 days, respectively. The batch kinetics was used to identify the mathematical model. The model was extrapolated to identify the nutrient feeding rate (150 mL/d) and substrate concentration (105 g/L) in the incoming feed for nonlimiting and noninhibitory glucose concentration in the cell retention bioreactor. An improvement in cell growth to 53 g/L and intracellular podophyllotoxin accumulation of 48.8 mg/L was achieved in 60 days, when the bioreactor was operated in continuous cell retention cultivation mode.