A novel centrifugal impeller bioreactor. II. Oxygen transfer and power consumption

The effects of the impeller configuration, aeration rate, and agitation speed on oxygen transfer coefficient K(L)a were studied in a newly designed centrifugal impeller bioreactor (5-L). The oxygen transfer rates in the novel bioreactor were also compared with those in a cell-lift bioreactor with comparable dimensions. The cell-lift impeller produced much higher surface oxygen transfer rates than the centrifugal one at an agitation speed over 200 rpm. This result was in good agreement with our observation that the cell-lift impeller produced much higher unfavorable turbulence. In addition, the experiments using granulated agar particles as pseudo plant cells indicated that the K(L)a value decreased steadily with an increase in agar particle concentration, and the centrifugal impeller still demonstrated a larger K(L)a than the cell lift up to a high pseudo cell concentration of 19.5 g dry weight (DW)/L (under 150 rpm and 0.20 vvm) or 22.3 g DW/L (under 200 rpm and 0.20 vvm). Furthermore, the correlation between power number and impeller Reynolds number for both the centrifugal and the cell-lift impellers was successfully obtained, which could be used for predicting the power input required by each impeller. From the results obtained, the centrifugal impeller bioreactor is expected to have great potential in its application to shear-sensitive biological systems.     

Performance of mammalian cell culture bioreactor with a new impeller design

To improve the oxygen transfer in a mammalian cell bioreactor, a new type of impeller consisting of a double-screen concentric cylindrical cage impeller (annular cage impeller in short) was designed and its mass transfer rate evaluated. This new impeller design increases the specific screen area, and the convective mass transfer rate through the annular cage was significantly increased. The oxygen transfer rates with the new impeller and the commercially available cell-lift impeller (CelliGen by New Brunswick Scientific Co.) were evaluated and their performance compared at various rates of aeration and agitation. The results showed that with the new impeller, the oxygen transfer rate was increased by 19% in water and 21% in cell-free culture medium supplemented with 10% horse serum, the total hybridoma cell concentration was increased to 3.4 x 10(7) cells/mL, and the IgG(1) subtype monoclonal antibody (MAb) product concentration was also increased to 512 mg/L in perfusion culture of murine hybridoma cell line 62'D3. These improvements in oxygen transfer rate, cell concentration, and MAb product concentration are all very significant. The mass transfer resistance in the cell-lift impeller system was found to be mainly due to the surface area of the single-screen cage impeller. The new annular cage impeller not only provided the increased surface area for convective oxygen transfer but also protected cells from hydrodynamic shear damage, thereby achieving a significant bioprocess improvement in terms of higher viable cell concentration, higher product concentration, and higher oxygen transfer rate in the mammalian cell bioreactor system.     

搅拌式生物反应器悬浮培养水母雪莲细胞的研究

应用 2L通气搅拌式生物反应器一步批式培养水母雪莲细胞。采用倾斜式搅拌桨代替透平桨 ,研究了搅拌转速、通气量和接种量对细胞生长和黄酮合成的影响 ,发现在 75r min、70 0～1000L min和 4.0～ 5.0gDCW L接种量下细胞生长和黄酮合成比较好。经过 12d培养细胞干重达 13.8gDCW L ,黄酮产量 416mg L ,黄酮含量占细胞干重的 30%。水母雪莲细胞生长及黄酮合成的进程表明 ,黄酮积累与细胞生长呈正相关。对细胞聚集体分布的研究发现 ,流变压力使细胞聚集体分裂 ,使反应器中细胞生长受到影响 ,黄酮产量较摇瓶中降低     

千烟洲针叶林的比叶面积及叶面积指数

根据实测数据计算了湿地松(Pinus elliotii)、马尾松(P. massoniana) 和杉木(Cunninghamia lanceolata)不同年龄、不同类型叶片的生物量和比叶面积,并结合样地调查数据和相对生长方程计算了中国科学院千烟洲试验站20年生湿地松林、马尾松林、杉木林和针叶混交林的叶面积指数。根据拟合结果,选择如下方程计算3个树种的叶生物量:湿地松W=12.074 1D^{2.151 5}、马尾松W=6.972 7D^{2.197 3}和杉木W=5.261 9D^{2.302 7}。湿地松林的叶生物量(0.822 kg·m^-2)最大,其次为针叶混交林(0.679 kg·m^-2),马尾松林和杉木林相差不大(分别为林0.528和0.572 kg·m^-2)。不同树种、不同年龄、不同类型叶片的比叶面积比较发现,新叶的比叶面积大于老叶,三针一束叶的比叶面积略大于两针一束叶,马尾松的平均半比表面积(8.62 m²·kg^-1)大于湿地松(6.04 m²·kg^-1)和杉木(7.91 m²·kg^-1)。胸径与单木叶片半表面积之间的经验方程为:湿地松LA=0.073D^{2.151 5}、马尾松LA=0.060D^{2.197 3}和杉木LA=0.042D^{2.302 7}。据此计算湿地松林的叶面积指数为5.03,马尾松林和杉木林为4.31,针叶混交林为4.77,该结果比利用CI-110植被冠层数字图像仪测得的结果偏大。     

冰雪灾害对中亚热带人工针叶林净初级生产力的影响

结合中亚热带江西千烟洲人工针叶林2005、2008和2011年3次树木清查数据以及树木相对生长方程,比较了2008年1月南方冰雪灾害前后的NPP,评价了森林生态系统灾后的恢复能力。结果表明:乡土树种马尾松(Pinus massoniana)与杉木(Cunninghamia lanceolata)比外来树种湿地松(Pinus elliottii)抗灾害能力强;在个体水平上,胸径(D)较大的树木抗灾害能力较差。灾后马尾松与杉木的D增长率降低,而湿地松增大。冰雪灾害导致大量碳(10.44 t C/hm2)从乔木层碳库转移到死生物量碳库,占乔木层碳储量的18.28%。灾前NPP和碳利用效率(CUE)分别为736.23 g C m-2a-1和0.41;灾后经过近4年的恢复,NPP和CUE分别为683.08 g C m-2a-1和0.38。     

Development of a helical-ribbon impeller bioreactor for high-density plant cell suspension culture

A double helical-ribbon impeller (HRI) bioreactor with a 11-L working volume was developed to grow high-density Catharanthus roseus cell suspensions. The rheological behavior of this suspension was found to be shear-thinning for concentrations higher than 12 to 15 g DW . L(-1). A granulated agar suspension of similar rheological properties was used as a model fluid for these suspensions. Mixing studies revealed that surface baffling and bottom profiling of the bioreactor and impeller speeds of 60 to 150 rpm ensured uniform mixing of suspensions. The HRI power requirement was found to increase singnificantly for agar suspensions higher than 13 g DW . L(-1), in conjunction with the effective viscosity increase. Oxygen transfer studies showed high apparent surface oxygen transfer coefficients (k(L)a approximately 4 to 45 h(-1)) from agar suspensions of 30 g DW . L(-1) to water and for mixing speeds ranging from 120 to 150 rpm. These high surface k(I)a values were ascribed to the flow pattern of this bioreactor configuration combined with surface bubble generation and entrainment in the liquid phase caused by the presence of the surface baffles. High-density C. roseus cell suspension cultures were successfully grown in this bioreactor without gas sparging. Up to 70% oxygen enrichment of the head space was required to ensure sufficient oxygen supply to the cultures so that dissolved oxygen concentration would remain above the critical level (>/=10% air saturation). The best mixing speed was 120 rpm. These cultures grew at the same rate ( approximately 0.4 d(-1)) and attained the same high biomass concentrations ( approximately 25 to 27 g DW . L(-1), 450 to 500 g filtered wet biomass . L(-1), and 92% to 100% settled wet biomass volume) as shake flask cultures. The scale-up potential of this bioreactor configuration is discussed.     

Enhanced shear protection and increased production of an anti-tumor polysaccharide by Agaricus blazei in xanthan-supplemented cultures

Xanthan supplementation provided shear protection and stimulated polysaccharide production by Agaricus blazei. In xanthan-free cultures, the optimal cell yield, 0.63 g biomass g^–1 glucose, and product yield, 0.19 g polysaccharide g^–1 glucose, were, respectively, when the critical impeller tip speed was 50.3 cm s^–1 and 100.5 cm s^–1. Furthermore, the critical impeller tip speed of cell yield shifted from 50.3 cm s^–1 to 100.5 cm s^–1 with the supplementation of 1 g xanthan l^–1. Maximum specific product yield, namely 0.74 g polysaccharide g^–1 biomass, was achieved with inlet air supply of 3% O₂ and impeller tip speed of 100.5 cm s^–1.     

The disruption ofSaccharomyces cerevisiae cells and release of glucose 6-phosphate dehydrogenase (G6PDH) in a horizontal dyno bead mill operated in continuous recycling mode

Baker’s yeast was disrupted in a 1.4-L stainless steel horizontal bead mill under a continuous recycle mode using 0.3 mm diameter zirconia beads as abrasive. A single pass in continuous mode bead mill operation liberates half of the maximally released protein. The maximum total protein release can only be achieved after passaging the cells 5 times through the disruption chamber. The degree of cell disruption was increased with the increase in feeding rate, but the total protein release was highest at the middle range of feeding rate (45 L/h). The total protein release was increased with an increase in biomass concentration from 10 to 50% (w/v). However, higher heat dissipation as a result of high viscosity of concentrated biomass led to the denaturation of labile protein such as glucose 6-phosphate dehydrogenase (G6PDH). As a result the highest specific activity of G6PDH was achieved at biomass concentration of 20% (ww/v). Generally, the degree of cell disruption and total protein released were increased with an increase in impeller tip speed, but the specific activity of G6PDH was decreased substantially at higher impeller tip speed (14 m/s). Both the degree of cell disruption and total protein release increased, as the bead loading increased from 75 to 85% (v/v). Hence, in order to obtain a higher yield of labile protein such as G6PDH, the yeast cell should not be disrupted at biomass concentration and impeller tip speed higher than 20% (w/v) and 10 m/s, respectively.     

Enhancing polyhydroxybutyrate production from high cell density fed-batch fermentation of Bacillus megaterium BA-019

This study demonstrated the improved polyhydroxybutyrate (PHB) production via high cell density cultivation of Bacillus megaterium BA-019 with balanced initial total sugar concentration and carbon to nitrogen (C/N) weight ratio. In the 10 L stirred fermentor operated at 30 °C, pH 7.0, 600 rpm, and 1.0 vvm air, with the initial total sugar concentration of 60 g/L and urea at the C/N weight ratio of 10:1, 32.48 g/L cell biomass with the corresponding PHB weight content of 26.94 % and volumetric productivity of 0.73 g/L h were obtained from batch cultivation. Continuing cultivation by intermittent feeding of the sugarcane molasses along with urea at the C/N weight ratio of 12.5:1 gave much improved biomass and PHB production (90.71 g/L biomass with 45.84 % PHB content and 1.73 g/L h PHB productivity). Similar biomass and PHB yields were obtained in the 90 L stirred fermentor when using the impeller tip speed as the scale-up criterion.     

High-density spore production of Piriformospora indica, a plant growth-promoting endophyte, by optimization of nutritional and cultural parameters

Piriformospora indica is an axenically cultivable root endophytic fungus which exerts plant growth promoting effects on its host plants. To enable commercial production of its spores, the medium composition and culture conditions have been optimized in a 14 L bioreactor such that they result in maximum biomass during growth phase and in maximum spore yield during subsequent sporulation phase. Maximum spore yields were obtained with modified Kaefer medium using a glucose deprivation strategy. An enhancement of 100% in overall biomass productivity (0.18 g L(-1) h(-1)) and reduction of about 70% in the time (60 h) required to achieve the maximum spore yield (9.25×10(7) spores/mL) was achieved in comparison to the original Kaefer medium. The high spore yield obtained in the present study seems to be economical for commercial production of P. indica.     

Production of somatic embryos in a helical ribbon impeller bioreactor

Embryogenic cultures of a transformed Eschscholtzia californica cell line were carried out in a 11-L helical ribbon impeller bioreactor operated under various conditions to evaluate the performance of this equipment for somatic embryo (SE) production. All bioreactor cultures produced SE suspensions with maximum concentrations at least comparable to those obtained from flask control cultures ( approximately 8-13 SE . mL(-;1)). However, an increase of the mixingspeed, from 60 to 100 rpm, and low sparging rate ( approximately 0.05 VVM, k(L) a approximately 6.1 h(-;1)) for dissolved oxygen concentration (DO) control yielded poorer quality embryogenic cultures. The negative effects on SE production were attributed mainly to the low but excessive shear experienced by the embryogenic cells and/or embryoforming aggregates. High DO ( approximately 60% of air saturation) conditions favored undifferentrated biomass production and high nutrient uptake rates at the expense of the slower SE differentiation process in both flask and bioreactor cultures. Too low DO (-5-10%) inhibited biomass and SE production. The best production of SE ( approximately 44 SE . mL(-1) or approximately 757 SE . g dw(-1) . d(-1)) was achieved by operating the bioreactor at 60 rpm while controlling DO at approximately 20%by surface oxygenation only (0.05 VVM, k(L) a approximately 1.4 h(-;1)). This production was found to be a biomass production/growth-associated process and was mainly limited by the availability of extracellular phosphate, magnesium, nitrogen salts, and carbohydrates. (c) 1994 John Wiley & Sons, Inc.     

Focussed beam reflectance measurement (FBRM) monitoring of particle size and morphology in suspension cultures of Morinda citrifolia and Centaurea calcitrapa

Laser light scattering technology, as applied in the Lasentec focussed beam reflectance measurement (FBRM) system, was used to characterise two morphologically dissimilar plant cell suspension cultures, Morinda citrifolia and Centaurea calcitrapa. Shake-flask suspensions were analysed in terms of biomass concentration and aggregate size/shape over the course of typical batch growth cycles. For the heavily aggregated C. calcitrapa, biomass levels [from 10-160 g fresh weight (fw) l(-1))] were linearly correlated with FBRM counts. For M. citrifolia, which grows in unbranched chains of 2-10 elongated cells, linear correlation of biomass concentration with FBRM counts was applicable in the range 0-100 g fw l(-1); at higher levels (100-300 g fw l(-1)), biomass was non-linearly correlated with FBRM counts and length-weighted average FBRM chord length. For both cell systems, particle morphology (size/shape) was quantified using semi-automated digital image analysis. The average aggregate equivalent diameter (C. calcitrapa) and average chain length (M. citrifolia), determined using image analysis, closely tracked the FBRM average chord length. The data clearly demonstrate the potential for applying the FBRM technique for rapid characterisation of plant cell suspension cultures.     

The influence of bakers’ yeast cells on protein adsorption in anion exchange expanded bed chromatography

Baker’s yeast was disrupted in a 1.4-L stainless steel horizontal bead mill under a continuous recycle mode using 0.3 mm diameter zirconia beads as abrasive. A single pass in continuous mode bead mill operation liberates half of the maximally released protein. The maximum total protein release can only be achieved after passaging the cells 5 times through the disruption chamber. The degree of cell disruption was increased with the increase in feeding rate, but the total protein release was highest at the middle range of feeding rate (45 L/h). The total protein release was increased with an increase in biomass concentration from 10 to 50% (w/v). However, higher heat dissipation as a result of high viscosity of concentrated biomass led to the denaturation of labile protein such as glucose 6-phosphate dehydrogenase (G6PDH). As a result the highest specific activity of G6PDH was achieved at biomass concentration of 20% (ww/v). Generally, the degree of cell disruption and total protein released were increased with an increase in impeller tip speed, but the specific activity of G6PDH was decreased substantially at higher impeller tip speed (14 m/s). Both the degree of cell disruption and total protein release increased, as the bead loading increased from 75 to 85% (v/v). Hence, in order to obtain a higher yield of labile protein such as G6PDH, the yeast cell should not be disrupted at biomass concentration and impeller tip speed higher than 20% (w/v) and 10 m/s, respectively.     

Biological sulfate reduction using synthesis gas as energy and carbon source

Biological sulfate reduction was studied in laboratory-scale gas-lift reactors. Synthesis gas (gas mixtures of H(2)/CO/CO(2)) was used as energy and carbon source. The required biomass retention was obtained by aggregation and immobilization on pumice particles. Special attention was paid to the effect of CO addition on the sulfate conversion rate, aggregation, and aggregate composition.Addition of 5% CO negatively affected the overall sulfate conversion rate; i.e., it dropped from 12-14 to 6-8 g SO(2-) (4)/L day. However, a further increase of CO to 10 and 20% did not further deteriorate the process. With external biomass recycling the sulfate conversion rate could be improved to 10 g SO(2-) (4)/L day. Therefore biomass retention clearly could be regarded as the rate-limiting step. Furthermore, CO affected the aggregate shape and diameter. Scanning electron microscopy (SEM) photographs showed that rough aggregates pregrown on H(2)/CO(2) changed into smooth aggregates upon addition of CO. Addition of CO also changed the aggregate Sauter mean diameter (d(32)) from 1.7 mm at 5% CO to 2.1 mm at 20% CO. After addition of CO, a layered biomass structure developed. Acetobacterium sp. were mainly located at the outside of the aggregates, whereas Desulfovibrio sp. were located inside the aggregates. (c) 1996 John Wiley & Sons, Inc.     

A novel centrifugal impeller bioreactor. I. Fluid circulation, mixing, and liquid velocity profiles

A novel centrifugal impeller bioreactor for shear-sensitive biological systems was designed by installing a centrifugal-pumplike impeller in a stirred vessel. The fluid circulation, mixing, and liquid velocity profiles in the new bioreactor (5-L) were assessed as functions of the principal impeller designing and bioreactor operating parameters. The performances of the centrifugal impeller bioreactor were compared with those of a widely used cell-lift bioreactor. The newly developed bioreactor showed higher liquid lift capacity and shorter mixing time than the cell lift with comparable dimensions. Furthermore, the experiments of the liquid velocity profiles around an impeller region indicated that the centrifugal impeller bioreactor produced lower shear stress than the cell lift. This conclusion was also supported by evaluating the changes in size distributions of granulated agar particles that were sheared with those two types of impeller.     

Influence of pH and impeller tip speed on the cultivation of<Emphasis Type="Italic">Bifidobacterium pseudocatenulatum</Emphasis> G4 in a milk-based medium

Biomass production ofBifidobacterium pseudocatenulatum G4 in a milk-based medium was carried out in a 2- and 10-L stirred tank fermenters. The effects of impeller tip speed (0.28, 0.56, and 0.83 m/s) and pH control (6.0, 6.5, and 7.0) on the biomass production were investigated. The growth performance in the 2-L fermenter was significantly improved when the impeller tip speed was held constant at 0.56 m/s and the pH was controlled at 6.5. These conditions yielded a maximum biomass of 1.687×10⁹ cfu/mL, a maximum specific growth rate of 0.504 h⁻¹, a biomass productivity of 9.240×10⁷ cfu/mL·h, and a biomass yield of 9.791×10¹⁰ cfu/g lactose. The consumption of milk lactose resulted in the accumulation of 7.353 g/L acetic acid and 6.515 g/L lactic acid, with an acetic:lactic ratio of 1.129. Scale-up of the fermentation process to a 10-L fermenter based on a constant impeller tip speed of 0.56 m/s yielded reproducible results with respect to biomass production and cell viability.     

Monoterpenoid oxindole alkaloid production by Uncaria tomentosa (Willd) D.C. cell suspension cultures in a stirred tank bioreactor

Cell growth, monoterpenoid oxindole alkaloid (MOA) production, and morphological properties of Uncaria tomentosa cell suspension cultures in a 2-L stirred tank bioreactor were investigated. U. tomentosa (cell line green Uth-3) was able to grow in a stirred tank at an impeller tip speed of 95 cm/s (agitation speed of 400 rpm), showing a maximum biomass yield of 11.9 +/- 0.6 g DW/L and a specific growth rate of 0.102 d(-1). U. tomentosa cells growing in a stirred tank achieved maximum volumetric and specific MOA concentration (467.7 +/- 40.0 microg/L, 44.6 +/- 5.2 microg/g DW) at 16 days of culture. MOA chemical profile of cell suspension cultures growing in a stirred tank resembled that of the plant. Depending on culture time, from the total MOA produced, 37-100% was found in the medium in the bioreactor culture. MOA concentration achieved in a stirred tank was up to 10-fold higher than that obtained in Erlenmeyer flasks (agitated at 110 rpm). In a stirred tank, average area of the single cells of U. tomentosa increased up to 4-fold, and elliptical form factor increased from 1.40 to 2.55, indicating enlargement of U. tomentosa single cells. This work presents the first report of U. tomentosa green cell suspension cultures that grow and produce MOA in a stirred tank bioreactor.     

湿地松体细胞胚胎发生和植株再生

以湿地松的未成熟合子胚为外植体,在附加８ｍｇ／Ｌ,２,４－Ｄ和４ｍｇ／Ｌ ＢＡ的ＬＰ培养基上诱导出胚性愈伤组织。在含１ｍｇ／Ｌ,２,４－Ｄ和０．５ｍｇ／Ｌ ＢＡ的培养基上保持并增殖。提高培养基的渗透压后,愈伤组织内大量的胚性胚柄细胞团和早期原胚。     

Kinetics of ethanol fermentations in membrane cell recycle fermentors

Ethanol fermentation by yeast was carried out in a cell filtration recycle system with a hollow-fiber membrane filter. Maximum biomass concentrations up to 210 g dry wt/L were obtained, but in normal operation concentrations they were between 100 and 150 g/L. The ethanol productivity using 14% glucose feed was 85 g/L h, with an ethanol concentration of 65 g/L and an ethanol yield of over 90%. The ethanol productivity and yeast growth rate decreased as the cell concentration increased beyond a certain level. The cell mass in the reactor was maintained by a proper manipulation of diluticn rate and bleed ratio depending on the growth rate.     

雨雪冰冻灾害对中亚热带人工林的影响——以江西省千烟洲为例

为深入研究2008年初低温雨雪冰冻灾害对我国南方森林生态系统的影响, 实地调查了中国科学院千烟洲红壤丘陵综合开发试验站(简称千烟洲站)人工林受损状况, 并结合气象资料与空间数据进行了分析。在千烟洲站内主要的5种林型中, 受损林分多为湿地松(Pinus elliottii)林, 倒伏区面积为7.72 hm², 占森林面积的6.12%, 地上植被碳储量损失1 462 g·m^-2(范围在655-5 230 g·m^-2之间)。持续低温、阴雨潮湿等气象条件是导致湿地松林受损的直接原因, 特别是2008年2月1日的低温、强降水和大风, 加剧了灾害程度。在吉泰盆地低山丘陵区(海拔60-140 m), 高程与局地地形、气象条件密切相关, 因而对冰雪灾害具有较强的指示作用。丘陵顶部土壤贫瘠、受风影响强烈, 林木易受损倒伏。阳坡的林木易偏冠, 这可能是其受损较为严重的原因之一。湿地松林受损的内因主要有冠型、材质、根系分布、叶片特征和年龄等;此外, 人为割脂也可能是造成湿地松大量倒伏的重要因素。