Optimization of culture conditions and scale-up to plant scales for teicoplanin production by <Emphasis Type="Italic">Actinoplanes teichomyceticus</Emphasis>

This report describes the optimization of culture conditions for teicoplanin production by Actinoplanes teichomyceticus KCCM-10601, an identified high-teicoplanin-producing strain (US 2006/0134757 A1). Among the conditions tested, temperature, pH, and the dissolved oxygen tension (DOT) were key factors affecting teicoplanin production. When the temperature, pH, and DOT were controlled at 34 degrees C, 7.0 and 20-30%, respectively, a dry-cell weight of 42.8 g l(-1) and a teicoplanin production of 2.9 g l(-1) were obtained after 120 h of batch culture, corresponding to a specific teicoplanin content of 67.8 mg g-DCW(-1). Teicoplanin production was scaled-up from a laboratory scale (7-l fermenter) to a pilot scale (300 l) and a plant scale (5,000 l) using the impeller tip velocity (V tip) as a scale-up parameter. Teicoplanin production at the laboratory scale was similar to those at the pilot and plant scales. This is the highest report of pilot- and plant-scale production of teicoplanin.     

Comparison of Cell Growth and Alkaloid Production of Catharanthus roseus Cells Cultured in Shake Flask and in Bioreactor

The cell growth and alkaloid production of Catharanthus roseus (L.) G. Don cells cultured in the shake flasks with different volumes and in the stirred tank bioreactor (10 L) were compared. Cell dry weight and alkaloid production showed no significant difference in the small volume scale-up shake flasks. When more broths were added to a certain volume in the shake flask, both cell weight and alkaloid production were decreased. The maximum cell dry weight was similar between the cell cultures in the shake flask and the bioreactor, but the alkaloid production of cells was much less in the bioreactor. Gas regime and shear stress were recognized to be the main factors contributing the important effect on alkaloid production during the scale-up processes.     

Comparative evaluation of compact photobioreactors for large-scale monoculture of microalgae

Engineering analyses combined with experimental observations in horizontal tubular photobioreactors and vertical bubble columns are used to demonstrate the potential of pneumatically mixed vertical devices for large-scale outdoor culture of photosynthetic microorganisms. Whereas the horizontal tubular systems have been extensively investigated, their scalability is limited. Horizontal tubular photobioreactors and vertical bubble column type units differ substantially in many ways, particularly with respect to the surface–to–volume ratio, the amount of gas in dispersion, the gas–liquid mass transfer characteristics, the nature of the fluid movement and the internal irradiance levels. As illustrated for eicosapentaenoic acid production from the microalga Phaeodactylum tricornutum, a realistic commercial process cannot rely on horizontal tubular photobioreactor technology. In bubble columns, presence of gas bubbles generally enhances internal irradiance when the Sun is low on the horizon. Near solar noon, the bubbles diminish the internal column irradiance relative to the ungassed state. The optimal dimensions of vertical column photobioreactors are about 0.2 m diameter and 4 m column height. Parallel east–west oriented rows of such columns located at 36.8°N latitude need an optimal inter-row spacing of about 3.5 m. In vertical columns the biomass productivity varies substantially during the year: the peak productivity during summer may be several times greater than in the winter. This seasonal variation occurs also in horizontal tubular units, but is much less pronounced. Under identical conditions, the volumetric biomass productivity in a bubble column is 60% of that in a 0.06 m diameter horizontal tubular loop, but there is substantial scope for raising this value.     

Metarhizium anisopliae conidia production in packed-bed bioreactor using rice as substrate in successive cultivations

This paper presents a study on scale-up and cost reduction of the production of spores of Metarhizium anisopliae IBCB 425, entomopathogenic fungus used in sugarcane crops. Rice was mixed with sugarcane bagasse (9:1 w/w) for substrate composition, assuring adequate physical structure for cultivation in packed-beds. Spores yield from only rice in bench-scale packed-bed bioreactor was 56 % of the one obtained from the mixture 9:1 w/w. In comparison to plastic packages used in bioindustries, equivalent spores yields per gram of substrate have been achieved in bench and pilot-scale bioreactors built by cylindrical jacketed modules, that provide better control of operational and environmental variables, attested by little variability among replicates. Although non-negligible temperature rise (5 °C above the ideal) occurred within the pilot-scale bioreactor, spores production was not harmed in comparison to bench-scale. By reusing rice up to three successive cultivations, a 2.5-fold increase of spores yields was achieved in comparison to single use. Temperatures and CO₂ profiles corroborates the fungus adapted differently to substrate at each usage. Such results are valuable for industrial producers of commercial formulations of the fungus spores, allowing process modernization by using packed-bed bioreactors and production costs and rice demand reduction by recycling the substrate.     

Efficient production of cordycepin by the Cordyceps militaris mutant G81-3 for practical use

Cordycepin is one of the most versatile metabolites of Cordyceps militaris. When C. militaris G81-3, the mutant obtained by a proton beam irradiation, was cultivated by liquid surface culture, cordycepin was found to crystallize in the medium due to high cordycepin concentration. Because the cordycepin crystals strongly attached to the mycelial mat, complete recovery of cordycepin was difficult. To prevent cordycepin crystallization, increase of the initial medium volume was examined to decrease the condensation rate by vaporization, in combination with decrease of the initial substrate level. Besides, addition of the water to the culture bottle was examined to cancel out the medium condensation. A 7.4 g/L of cordycepin was obtained without crystallization by the former method. The upper limit of cordycepin production, 14.3 g/L, was obtained by the latter method and this value was the highest level of all the previous reports. A 2.5-fold scale-up of the culture slightly declined the production rate, however, the amount of cordycepin production was properly 2.5 times higher than that of the small scale. In addition, differences in sensitivities to the substrates between the mutant and the wild-type strain are discussed.     

重组杆状病毒细小VP2蛋白40L生物反应器放大工艺研究

研究了Sf9细胞生产重组杆状病毒细小VP2蛋白在机械搅拌式生物反应器(STR)中从3L至40L的放大工艺。首先在3L反应器中,通过DO和搅拌转速的优化,使反应器中的VP2蛋白HA效价不低于摇瓶结果。在40L反应器放大时,温度、p H、DO保持不变,根据输入搅拌功率、体积溶氧系数和叶尖线速度等工程参数的计算,得到了该反应器的合理搅拌转速,最终HA效价测定结果与小罐一致。豚鼠免疫试验证实,反应器中表达的VP2蛋白制成疫苗,与HN2011灭活苗及商品化灭活疫苗相比,抗体水平上升较快且高于传统灭活苗。     

Optimization studies to develop a low-cost medium for production of the lipases of Rhizopus microsporus by solid-state fermentation and scale-up of the process to a pilot packed-bed bioreactor

A low-cost lipase preparation is required for enzymatic biodiesel synthesis. One possibility is to produce the lipase in solid-state fermentation (SSF) and then add the fermented solids (FS) directly to the reaction medium for biodiesel synthesis. In the current work, we scaled up the production of FS containing the lipases of Rhizopus microsporus. Initial experiments in flasks led to a low-cost medium containing wheat bran and sugarcane bagasse (50:50 w/w, dry basis), supplemented only with urea. We used this medium to scale-up production of FS, from 10 g in a laboratory column bioreactor to 15 kg in a pilot packed-bed bioreactor. This is the largest scale yet reported for lipase production in SSF. During scale-up, the hydrolytic activity of the FS decreased 57%: from 265 U g⁻¹ at 18 h in the laboratory bioreactor to 113 U g⁻¹ at 20 h in the pilot bioreactor. However, the esterification activity decreased by only 14%: from 12.1 U g⁻¹ to 10.4 U g⁻¹. When the FS produced in the laboratory and pilot bioreactors were dried and added directly to a solvent-free reaction medium to catalyze the esterification of oleic acid with ethanol, both gave the same ester content, 69% in 48 h.     

Production of adenovirus vector for gene therapy

The field of gene therapy is rapidly expanding with a major focus on the treatment of cancer. Replication-defective adenoviruses are vectors of choice for delivering corrective genes into human cells. Major efforts are directed to design new generations of adenoviral vectors that feature reduced immunogenicity and improved targeting ability. However, the production of adenoviral vectors for gene therapy applications faces a number of challenges that limit the availability of high quality material at the early stages of research and development in the gene therapy field. Moreover, very few papers have been published on the subject and information on large-scale production methods are only available through specialized conference proceedings. This review outlines the problems associated with mass production of adenovirus vectors and describes research efforts by a number of groups who have contributed to optimize production methods. Better understanding of the adenovirus infection and replication kinetics as well as better understanding of complementing cell line physiology and metabolism greatly contributed to improving vector titers and volumetric productivity at higher cell densities. Also, the critical aspect of viral vector quantitation is discussed.     

Multi-stage laccase extraction and separation using aqueous two-phase systems: Experiment and model

This work presents results of experimental and model investigation of continuous multi-stage enzyme extraction using aqueous two-phase systems for the first time. The aqueous two-phase system comprised polyethylene glycol 3000 and phosphate with additional sodium chloride buffered to pH 7. Two different laccases served as model enzymes. One of the laccases was directly taken from fungal culture supernatant, while the other laccase was solubilized lyophilisate. The modeling is based on an equilibrium stage approach. Equilibrium data were taken from single-stage experiments and approximated by different correlation equations. The model describes densities, phase equilibrium, enzyme activity partitioning between the phases. Moreover it allows to consider activity changes due to the aqueous two-phase system. Eight multi-stage mixer-settler experiments under varying operation conditions were performed to validate the proposed model; whereas the total throughput of all multi-stage extraction experiments was about 350 g h⁻¹. The average relative deviation of modeled activities from experimentally measured activities was 23%. Therefore, the model is able to calculate the behavior of the phases as well as the partitioning of the two enzymes between the two phases for a multi-stage process based on single-stage data.     

条斑紫菜藻红、藻蓝蛋白逐级放大的纯化工艺

采用“破碎-盐析-层析”的方法纯化条斑紫菜藻胆蛋白,并在提取规模上逐步放大。首先在综合比较凝胶层析去盐效率后,从Sephadex G-25、G-100、S-300和CL-6B中选择G-25作为实验流程中的去盐填料,其次将提取流程的初试原料条斑紫菜量逐步放大,选取了1g、20g和400g三个量,结果表明随着初试紫菜量逐步放大,最终所得藻胆蛋白中吸收光谱纯度>3.2的蛋白产率依次提高,其中400g冻干紫菜的藻红蛋白产率为0.323％,藻蓝蛋白产率为0.148％。由此认为该实验工艺流程具有规模放大的潜力,这为高纯度藻胆蛋白的规模生产提供了一条可行的方案。