Amylose-like polysaccharide accumulation and hyphal cell-surface structure in relation to citric acid production by Aspergillus niger in shake culture

When 120 mg glucose/ml was used as a carbon source, in shake culture Aspergillus niger Yang no. 2 maximally produced only 15.4 mg citric acid/ml but accumulated 3.0 mg extracellular polysaccharide/ml. The polysaccharide secreted by mycelia of Yang no. 2 in shake culture was confirmed to be an amylose-like alpha-1,4-glucan by hydrolysis analysis with acid, amylase and glucoamylase. However, in static cultures, such as semisolid and surface cultures free from physical stresses caused by shaking damage, Yang no. 2 produced more citric acid but did not accumulate the polysaccharide. With cultivation time in shake culture, the amount of extracellular polysaccharide and the viscosity of the culture broth increased. The increase of shaking speed caused a remarkable increase in the accumulation of extracellular polysaccharide, e.g. 11.2 mg extracellular polysaccharide/ml was accumulated in the medium at a shaking speed of 200 rpm. The addition of 2.0 mg carboxymethylcellulose (CMC)/ml as a viscous additive to the medium reduced drastically the amount of extracellular polysaccharide accumulated to 1.5 mg/ml, but increased the citric acid produced to 52.0 mg/ml. However, intracellular polysaccharide accumulation kept up a steady rate of 0.26 microgram/mg dried mycelium through the entire period of cultivation. The addition of 3.0 mg polysaccharide/ml purified from the culture broth to the medium at the start of a culture resulted in a decrease of extracellular polysaccharide accumulation but an increase of citric acid accumulation. From electronmicroscopic observation, cell surfaces of hyphae cultivated with CMC were smooth, while hyphae cultivated without CMC had fibrous and granular polysaccharide on the cell surface. These results suggested that Yang no. 2 secreted the polysaccharide on the cell surface as a viscous substance and/or a shock absorber to protect itself from physical stresses caused by shaking damage in shake culture.     

樟叶越桔细胞悬浮培养条件的优化

为了提高樟叶越桔(Vacciniumdunalianum)悬浮培养细胞的生物量,以樟叶越桔叶片愈伤组织为试材,通过单因素试验探究不同蔗糖浓度、培养基pH值、培养基体积、初始接种量和摇床转速对悬浮培养细胞生长的影响,并根据响应面法Box-Behnken试验设计原理进行组合试验以优化培养条件。结果显示,以改良WPM培养基为基础培养基,樟叶越桔细胞悬浮培养的最优条件为40 g·L^–1蔗糖、培养基pH5.2、培养基体积45 mL、初始接种量2.64 g和摇床转速为149 r·min^–1,其细胞生物量干重为0.184 4 g,与理论预测值0.184 5 g较为接近,且细胞的生长曲线呈S型。研究结果为樟叶越桔悬浮培养细胞次生代谢产物的生产调控奠定了技术基础。     

Assessment of Cultivation Factors that Affect Biomass and Geraniol Production in Transgenic Tobacco Cell Suspension Cultures

A large-scale statistical experimental design was used to determine essential cultivation parameters that affect biomass accumulation and geraniol production in transgenic tobacco (Nicotiana tabacum cv. Samsun NN) cell suspension cultures. The carbohydrate source played a major role in determining the geraniol yield and factors such as filling volume, inoculum size and light were less important. Sucrose, filling volume and inoculum size had a positive effect on geraniol yield by boosting growth of plant cell cultures whereas illumination of the cultures stimulated the geraniol biosynthesis. We also found that the carbohydrates sucrose and mannitol showed polarizing effects on biomass and geraniol accumulation. Factors such as shaking frequency, the presence of conditioned medium and solubilizers had minor influence on both plant cell growth and geraniol content. When cells were cultivated under the screened conditions for all the investigated factors, the cultures produced ∼5.2 mg/l geraniol after 12 days of cultivation in shaking flasks which is comparable to the yield obtained in microbial expression systems. Our data suggest that industrial experimental designs based on orthogonal arrays are suitable for the selection of initial cultivation parameters prior to the essential medium optimization steps. Such designs are particularly beneficial in the early optimization steps when many factors must be screened, increasing the statistical power of the experiments without increasing the demand on time and resources.     

内生解淀粉芽孢杆菌CC09产Iturin A摇瓶发酵条件优化

【目的】提高内生解淀粉芽孢杆菌CC09发酵产抗菌脂肽Iturin A的产量。【方法】首先采用单因子实验研究了碳源、氮源、NaCl浓度、pH、温度、转速和装液量等因子对CC09产Iturin A能力的影响,然后对其中显著性因子:氮源浓度、pH、温度及装液量4个因素进行正交实验,进一步优化发酵条件。【结果】优化培养基组成及发酵条件可以提高CC09菌株的生长速度及产Iturin A的量,其中可溶性淀粉以及一定比例的蛋白胨和酵母粉是CC09菌株产Iturin A的良好碳源和氮源;培养温度、装液量、培养液pH等也对CC09菌株产Iturin A有显著影响。优化后的培养基成分:可溶性淀粉(碳源)5 g/L、比例为3:1的胰蛋白胨酵母粉混合氮源15 g/L、NaCl 1 g/L;最佳培养条件:pH 6.0、28°C、摇床转速120 r/min、培养瓶装液量20%。【结论】在此条件下,Iturin A的产量可达到690 mg/L,较优化前的138 mg/L提高了4倍。     

Trypanosoma congolense: mechanical removal of the surface coat in vitro

By shaking suspensions of Trypanosoma congolense in isotonic buffer the surface coat could be separated from the cell body. The release of radioactivity from trypanosomes, selectively labeled in the surface coat by diazoniobenzenesulfonate, was used to follow the kinetics of coat detachment. The proteins in the supernatants of shaken trypanosomes were analyzed by sodium dodecyl sulfate—polyacrylamide gel electrophoresis. The shaking conditions had to be carefully controlled to avoid complete rupture of trypanosomes. Otherwise the coat protein was rapidly degraded by endogenous proteases. The influence of several parameters on the yield of coat release and the degree of degradation of the coat protein was investigated, including the ratio of trypanosome suspension volume to shaking vessel volume, vessel surface, temperature, shaking frequency, and preincubation of the trypanosomes at 0 C. By combining these parameters an optimal scheme was developed which allowed the separation of more than 90% of the coat protein from T. congolense, the detached protein showing no degradation at all. These results could be confirmed by electron microscopy of shaken and unshaken trypanosomes.     

Physical stress for overproduction of biomass and flavonoids in cell suspension cultures of Boesenbergia rotunda

Flavonoid, a bioactive compound isolated from the rhizomes of Boesenbergia rotunda, exhibited antibiotic and anti-inflammatory properties and has also shown high inhibitory activity of dengue-2 virus protease. Several factors are responsible for the production of flavonoid in cell cultures. In the present study, the effects of initial inoculation volume, temperature and speed of agitation on cell growth, total and selected flavonoid in suspension cultures of B. rotunda were determined. High performance liquid chromatography analysis showed that a 2 % inoculation volume induced a significantly high accumulation of biomass and of flavonoid in the cells. The cells cultured at 25 °C showed significantly high biomass and selected flavonoid accumulation while differences in medium agitation significantly affected the yield of selected flavonoid.     

Enhancement of oleandrin production in suspension cultures of Nerium oleander by combined optimization of medium composition and substrate feeding

Abstract

Oleandrin has been identified as the most potent antitumor ingredient of the Mediterranean herb Nerium oleander L. A strategy for optimization of medium compositions and conditions was developed for enhanced oleandrin production in suspension cultures from leaf-origin explants of Nerium oleander. The cell suspension cultures were grown in various modifications of MS medium as a basal medium. The effects of different natural extracts, plant growth substances, carbon and nitrogen sources and phosphate on the growth and oleandrin accumulation were investigated as well as effect of light, pH, shaking speed and substrate feeding. The highest oleandrin yield was obtained when the nitrogen concentration was lowered to two-thirds and the phosphate concentration increased by two-thirds of that specified in the MS medium in the presence of 3% sucrose, coconut milk, indolebutyric acid and benzyladenine in concentrations of 1 and 2 mg l^?1, respectively. Lower pH and faster shaking speed favored oleandrin accumulation. Chemical feeding of progesterone and cholesterol boosted the oleandrin concentration to higher levels reaching 8.23±0.05 mg g^?1 dry weight. This was about 10-fold higher than that detected in field-grown plants using the same extraction and analytic conditions, and about 24-fold higher than that determined in control cultures with regular MS medium and without precursor feeding.     

Methods for Intense Aeration, Growth, Storage, and Replication of Bacterial Strains in Microtiter Plates

Miniaturized growth systems for heterogeneous culture collections are not only attractive in reducing demands for incubation space and medium but also in making the parallel handling of large numbers of strains more practicable. We report here on the optimization of oxygen transfer rates in deep-well microtiter plates and the development of a replication system allowing the simultaneous and reproducible sampling of 96 frozen glycerol stock cultures while the remaining culture volume remains frozen. Oxygen transfer rates were derived from growth curves of Pseudomonas putida and from rates of oxygen disappearance due to the cobalt-catalyzed oxidation of sulfite. Maximum oxygen transfer rates (38 mmol liter⁻¹ h⁻¹, corresponding to a mass transfer coefficient of 188 h⁻¹) were measured during orbital shaking at 300 rpm at a shaking diameter of 5 cm and a culture volume of 0.5 ml. A shaking diameter of 2.5 cm resulted in threefold-lower values. These high oxygen transfer rates allowed P. putida to reach a cell density of approximately 9 g (dry weight) liter⁻¹ during growth on a glucose mineral medium at culture volumes of up to 1 ml. The growth-and-replication system was evaluated for a culture collection consisting of aerobic strains, mainly from the genera Pseudomonas, Rhodococcus, and Alcaligenes, using mineral media and rich media. Cross-contamination and excessive evaporation during vigorous aeration were adequately prevented by the use of a sandwich cover of spongy silicone and cotton wool on top of the microtiter plates.     

Optimization of cultivation conditions in spin tubes for Chinese hamster ovary cells producing erythropoietin and the comparison of glycosylation patterns in different cultivation vessels

This article describes the optimization of cultivation factor settings, that is the shaking rate and working volume in 50 mL spin tubes for a Chinese hamster ovary cell line expressing recombinant human α‐erythropoietin, using a response D‐optimal surface method. The main objectives of the research were, firstly, to determine a setting in which the product titer and product quality attributes in spin tubes are equivalent to those in 250 mL shake flasks in a seven day batch and, secondly, to find a setting in which the product titer is maximal. The model for product titer prediction as a function of shaking rate and working volume in the defined design space was successfully applied to the optimization of cultivation conditions in spin tubes for the tested cell line. Subsequently, validation experiments were carried out simultaneously in spin tubes, shake flasks and bench scale bioreactors to compare cell culture performance parameters such as growth, productivity and product quality attributes in the form of isoform profiles and glycan antennarity structures. The results of the experiments showed that similar cell culture performance and product quality could be achieved in spin tubes when compared to shake flasks. Additionally, bioreactor titers could be reproduced in spin tubes at high shaking rates and low working volumes, but with differing product quality. Cultivation at lower shaking rates in spin tubes and shake flasks produced a glycoprotein with a product quality slightly comparable to that from bioreactors, but with titers being only two thirds. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

A Retinoblastoma Orthologue Is Required for the Sensing of a Chalone in Dictyostelium discoideum

Retinoblastoma-like proteins regulate cell differentiation and inhibit cell proliferation. The Dictyostelium discoideum retinoblastoma orthologue RblA affects the differentiation of cells during multicellular development, but it is unclear whether RblA has a significant effect on Dictyostelium cell proliferation, which is inhibited by the secreted proteins AprA and CfaD. We found that rblA⁻ cells in shaking culture proliferate to a higher density, die faster after reaching stationary density, and, after starvation, have a lower spore viability than wild-type cells, possibly because in shaking culture, rblA⁻ cells have both increased cytokinesis and lower extracellular accumulation of CfaD. However, rblA⁻ cells have abnormally slow proliferation on bacterial lawns. Recombinant AprA inhibits the proliferation of wild-type cells but not that of rblA⁻ cells, whereas CfaD inhibits the proliferation of both wild-type cells and rblA⁻ cells. Similar to aprA⁻ cells, rblA⁻ cells have a normal mass and protein accumulation rate on a per-nucleus basis, indicating that RblA affects cell proliferation but not cell growth. AprA also functions as a chemorepellent, and RblA is required for proper AprA chemorepellent activity despite the fact that RblA does not affect cell speed. Together, our data indicate that an autocrine proliferation-inhibiting factor acts through RblA to regulate cell density in Dictyostelium, suggesting that such factors may signal through retinoblastoma-like proteins to control the sizes of structures such as developing organs or tumors.     

Large‐scale culture of a megakaryocytic progenitor cell line with a single‐use bioreactor system

The increasing application of regenerative medicine has generated a growing demand for stem cells and their derivatives. Single‐use bioreactors offer an attractive platform for stem cell expansion owing to their scalability for large‐scale production and feasibility of meeting clinical‐grade standards. The current work evaluated the capacity of a single‐use bioreactor system (1 L working volume) for expanding Meg01 cells, a megakaryocytic (MK) progenitor cell line. Oxygen supply was provided by surface aeration to minimize foaming and orbital shaking was used to promote oxygen transfer. Oxygen transfer rates (k_La) of shaking speeds 50, 100, and 125 rpm were estimated to be 0.39, 1.12, and 10.45 h^?1, respectively. Shaking speed was a critical factor for optimizing cell growth. At 50 rpm, Meg01 cells exhibited restricted growth due to insufficient mixing. A negative effect occurred when the shaking speed was increased to 125 rpm, likely caused by high hydrodynamic shear stress. The bioreactor culture achieved the highest growth profile when shaken at 100 rpm, achieving a total expansion rate up to 5.7‐fold with a total cell number of 1.2 ± 0.2 × 10⁹ cells L^?1. In addition, cells expanded using the bioreactor system could maintain their potency to differentiate following the MK lineage, as analyzed from specific surface protein and morphological similarity with the cells grown in the conventional culturing system. Our study reports the impact of operational variables such as shaking speed for growth profile and MK differentiation potential of a progenitor cell line in a single‐use bioreactor. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:362–369, 2018     

Tonicity-volume relations in partially hemolyzed hypotonic systems

The linear relation between the red cell volume V and the reciprocal of the tonicity T of a hypotonic medium is not the linear one expected on the basis of the van't Hoff-Mariotte law, particularly when a fraction p of the cells are hemolyzed and the volume V/(1 – p) of the (1 – p) cells which remain intact is considered. In systems of relatively high tonicity in which p is zero, the relation is linear but its slope is usually too small; at lower tonicities in which p has a value between zero and 0.35, the volumes are larger than those expected on the basis of the van't Hoff-Mariotte law, while at still smaller tonicities they are much smaller than expected. The volume measurements referred to are made with a high speed hematocrit; the results obtained in systems containing relatively high and relatively low volume concentrations of cells are contrasted with each other, and allowance is made in the calculations for the volume of the hypotonic medium surrounding the cells being limited. Attempts are made at an explanation of the anomalous results in terms of incomplete packing, of a stepwise as opposed to an all-or-none loss of Hb from the cells, of a heterogeneity in the swelling properties of the cells of the population, of a loss of osmotically active substances from the intact cells, and of the red cell ghost having some degree of rigidity. These explanations are not satisfying, although some of them in combination may account for the phenomena observed. It seems likely that the structures involved in the hemolytic process (cells and ghosts) have different properties in different tonicity ranges and even when the same tonicity is established in systems which are dense, as opposed to dilute, with respect to cell concentration. The form of the tonicity-volume relation can be changed substantially, although not in the direction of greater linearity, by treating the cells with resorcinol, colloidal silicic acid, iodoacetate, or sodium oxalate. Small changes in pH, exposure to temperatures as high as 37°C., allowing the cells to stand for periods up to 24 hours, or the substitution of hypotonic plasma for hypotonic NaCl-buffer, produce, on the other hand, only minor changes in the tonicity-volume relation.     

The Response of Duck Erythrocytes to Hypertonic Media : Further evidence for a volume-controlling mechanism

The addition of a hypertonic bathing medium to duck erythrocytes results in an initial instantaneous phase of osmotic shrinkage and, when the [K]_o of the hypertonic solution is larger than "normal," in a second, more prolonged phase, the volume regulatory phase. During the latter, which also requires extracellular Na, the cells swell until they approach their initial isotonic volume. The increase in cell volume during the volume regulatory phase is accomplished by a gain in the cell content of K, Cl, and H₂O. There is also a smaller increase in the Na content of the cell. Potassium is accumulated against an electrochemical gradient and is therefore actively transported into the cell. This accumulation is associated with an increase, although dissimilar, in both K influx and efflux. Changes in cell size during the volume regulatory phase are not altered by 10^-4 M ouabain, although this concentration of ouabain does change the cellular cation content. The response is independent of any effect of norepinephrine. The changes in cell size during the volume regulatory phase are discussed as the product of a volume controlling mechanism identical in principle to the one reported in the previous paper which controls cell volume in hypotonic media. Similarly, this mechanism can regulate cell size, when the Na-K exchange, ouabain-inhibitable pump mechanism is blocked.     

蜡质芽孢杆菌AR156发酵培养基及发酵条件的优化

对前期筛选得到的在田间试验中防治根结线虫效果较好的蜡质芽孢杆菌(Bacillus cereus)AR156,通过单因素筛选及正交试验的方法进行了发酵培养基优化,得到的最佳配比为:麦芽糖0.25%,玉米粉0.5%,黄豆粉0.5%,胰蛋白胨0.5%,CaCl2·2H2O0.05%,MnSO4·H2O0.05%,K2HPO40.1%。同时对实验室摇瓶条件下液体发酵的主要影响因子温度、转速、初始pH值等进行实验探讨,确定了最佳培养条件:初始pH值7.0,装液量200mL/L,接种量5%,发酵温度28℃,转速200r/min,发酵时间48h。优化后芽孢产量为1.03×109CFU/mL,芽孢生成率在97%以上,明显高于初始发酵培养基发酵结果。     

Effectiveness of orbital shaking for the aeration of suspended bacterial cultures in square-deepwell microtiter plates

Growth of heterogeneous culture collections in microtiter plates is advantageous for logistic reasons and also in enabling significant savings in medium costs, labor input and use of equipment during large screening projects. The main hurdles to overcome for aerobic microbial strains are the prevention of cross-contamination and excessive evaporation while assuring sufficient aeration rates. For this purpose we developed a sandwich spongy silicone/cotton wool cover to close the wells of square-deepwell microtiter plates. Oxygen transfer rates were derived from growth curves of Pseudomonas putida and were shown to be threefold higher during orbital shaking at a shaking diameter of 5cm at 300rpm (24mmolO(2)l(-1)h(-1) at a culture volume of 0.75ml) in comparison to a shaking diameter of 2.5cm. Photographic analysis showed a clear influence of the shaking diameter on the hydrodynamic behavior in the wells; during shaking at a 2.5cm amplitude, out-of-phase conditions occurred resulting in poor vertical mixing, while a 5cm shaking amplitude led to an optimal surface to volume ratio and a turbulent flow.     

Production and release of pigments by culture of transformed hairy root of red beet

Adventitious roots were induced from red beet (Beta vulgaris L. cv. Detroit dark red) by infecting the plant with a soil bacterium, Agrobacterium rhizogenes. Based on analysis of opines which are uniquely produced in transformed hairy roots, the established clone was proved to be a transformed hairy root. In a shake culture of the beet hairy root clone with a liquid medium, it was found that significant amounts of pigments, mainly betanin and vulgaxanthin-I, were released into the medium by the cessation of culture shaking (temporary limitation of oxygen supply). The hairy root cells were capable of propagation even after the cells were subjected to shaking cessation. Repeated-batch culture of the beet hairy root was performed with the cell growth phases for 9 or 10 d and with pigment leakage phases during shaking cessation for 2 d, and more than 20% of the total intracellular pigments were recovered from the culture broth at a culture time of 35 d. The released pigments were confirmed to be substantially identical to those extracted from the hairy root and original plant cells of red beet.     

Effector cells which mediate antibody-dependent cell-mediated cytotoxicity. II. Ontogeny of effector activity in murine spleen.

The specificity of T cells for syngeneic target cells is directed to both antigens and products of the major histocompatibility complex (MHC) on the target cell surface. This dual requirement is best accounted for by the altered-self hypothesis, which implies that the MHC products on a cell's surface are able to form complexes with many other proteins on the surface of the same cell. To account for the ability of MHC products to bind so many different cell surface antigens we propose that interactions in general among macromolecules on the surface of a membrane may be dramatically enhanced by a purely physical effect. This effect derives from the confinement of membrane macromolecules to an effective volume which is the product of membrane surface area times d, the distance over which the center of mass of the molecules can move in a vertical direction (perpendicular to the membrane surface). Because d is very small the effective concentrations of surface molecules are extremely high and their interactions are correspondingly enhanced.     

Alternate pathways of secretion of simian immunodeficiency virus envelope glycoproteins.

A biotinylation assay was used to detect the envelope glycoprotein of the simian immunodeficiency virus (SIV) envelope glycoprotein expressed by a recombinant vaccinia virus on the surface of HeLa T4 cells. The relationship between the detection of the envelope glycoprotein on the cell surface and its secretion from the cell was examined. It was found that much more gp120 was released into the culture medium than could be accounted for by shedding of the biotinylated SIV envelope protein from the cell surface. Treatment with the ionophore monensin showed that this drug did not block the secretion of gp120 into the culture medium even though the expression of gp120 on the cell surface was strongly downregulated. Similar results were observed for the secretion of gp120 in HUT78 cells infected with SIVmac251 virus. Brefeldin A, on the other hand, inhibited both the detection of gp120 on the cell surface and its secretion into the culture medium. On the basis of these results, we propose that gp120 can be secreted into the culture medium via at least two pathways. One pathway involves the dissociation of gp120 from membrane-associated gp41-gp120 complexes on the cell surface. However, the major pathway involves the secretion of gp120 without its transitory appearance on the cell surface as part of a gp41-gp120 complex.     

Freezing-Out Technique Applied to the Concentration of Biologically Active Materials

When applied to a dilute solution of folic acid and glucose, a freezing-out (with agitation) technique was shown to be an effective method of achieving a 20-fold reduction in volume with a loss of 10% of the active material being concentrated. Concentration of a stimulatory factor for Lactobacillus casei produced by Candida albicans in a complex medium was limited by the total solute concentration. Salts in the medium were concentrated to levels inhibitory for L. casei. The process is not selective and all solutes are concentrated.     

Water Transfer and Cell Structure in Isolated Crayfish Muscle Fibers

Changes in volume of crayfish single muscle fibers in response to changes in ionic or electrical conditions have been studied in conjunction with electrophysiological measurements and electron microscopic examinations. The occurrence of at least three mechanisms of water movements is revealed, two being processes which are superimposed on the normal osmotic water movement that results from a change in the concentration of solute in the medium. Differences between the time courses of the changes in volume and potential on changing K_i/K_o indicate that water may be distributed unequally for a time within compartments of the fiber. Electron micrographs reveal a selective accumulation of water at the periphery of the fiber under certain conditions. A correlation of H₂O transfer with a change in membrane potential is apparent in crayfish muscle fibers and is probably due to electroosmotic effects. Electrokinetic water movements are produced whenever the membrane potential is changed to a considerable degree by changing the level of K and/or Cl in the medium, or by applying currents with an intracellular microelectrode. Depolarizations cause shrinkage. Hyperpolarizations or repolarizations cause swelling. The volume changes are independent of the occurrence or absence of swelling in the anion-permselective transverse tubular system. They indicate that the fiber membrane along the surface is heterogeneous, not only with respect to the signs of its fixed charge sites, but also with respect to the sizes and relative permselectivities of these charged channels.