pH-induced flocculation,indirect electrocoagulation,and hollow fiber filtration techniques for harvesting the saltwater microalga Dunaliella

This research assessed the efficacy of three harvesting methods on a strain of Dunaliella viridis. While there is strong potential to use lipids from microalgae as a feedstock for biofuels to replace petroleum-based fuel, at present microalgal harvesting for biofuel production is not yet economically feasible or energy efficient. pH-induced flocculation (by adjusting the pH of exponentially growing cells), indirect electrocoagulation (applying aluminum hydroxide coagulant to culture), and hollow fiber filtration (separating biomass from medium using tangential flow) were compared as potential harvesting mechanisms for small-scale (3–10 L) and large-scale (30–150 L) volumes of D. viridis. Both pH-induced flocculation and electrocoagulation yielded significant biomass recovery (>95 %), but both methods required removal of added chemicals and/or coagulant before the medium could be reused. In contrast, hollow fiber filtration did not require added chemicals or coagulant, and as another advantage, the filtrate was successfully reused as culture medium without apparent detrimental effects on cell size, cell shape, or cell production. When high salinity stress was imposed on the concentrate produced from the filtration method, total fatty acids (FAs) did not increase. However, total FAs did significantly increase after hollow fiber filtration (49 %) in comparison to FA content before filtration (36 %). This research indicates that hollow fiber filtration as a commercial harvesting mechanism offers attractive advantages as a harvesting mechanism for microalgae such as Dunaliella, relative to pH-induced flocculation and indirect electrocoagulation.     

Dissolvable Gelatin-Based Microcarriers Generated through Droplet Microfluidics for Expansion and Culture of Mesenchymal Stromal Cells

Microcarriers are synthetic particles used in bioreactor-based cell manufacturing of anchorage-dependent cells to promote proliferation at efficient physical volumes, mainly by increasing the surface area-to-volume ratio. Mesenchymal stromal cells (MSCs) are adherent cells that are used for numerous clinical trials of autologous and allogeneic cell therapy, thus requiring avenues for large-scale cell production at efficiently low volumes and cost. Here, a dissolvable gelatin-based microcarrier is developed for MSC expansion. This novel microcarrier shows comparable cell attachment efficiency and proliferation rate when compared to several commercial microcarriers, but with higher harvesting yield due to the direct dissolution of microcarrier particles and thus reduced cell loss at the cell harvesting step. Furthermore, gene expression and in vitro differentiation suggest that MSCs cultured on gelatin microcarriers maintain trilineage differentiation with similar adipogenic differentiation efficiency and higher chondrogenic and osteogenic differentiation efficiency when compared to MSCs cultured on 2D planar polystyrene tissue culture flask; on the contrary, MSCs cultured on conventional microcarriers appear to be bipotent along osteochondral lineages whereby adipogenic differentiation potential is impeded. These results suggest that these gelatin microcarriers are suitable for MSC culture and expansion, and can also potentially be extended for other types of anchorage-dependent cells.     

Periodic harvesting of embryonic stem cells from a hollow‐fiber membrane based four‐compartment bioreactor

Different types of stem cells have been investigated for applications in drug screening and toxicity testing. In order to provide sufficient numbers of cells for such in vitro applications a scale‐up of stem cell culture is necessary. Bioreactors for dynamic three‐dimensional (3D) culture of growing cells offer the option for culturing large amounts of stem cells at high densities in a closed system. We describe a method for periodic harvesting of pluripotent stem cells (PSC) during expansion in a perfused 3D hollow‐fiber membrane bioreactor, using mouse embryonic stem cells (mESC) as a model cell line. A number of 100 × 10⁶ mESC were seeded in bioreactors in the presence of mouse embryonic fibroblasts (MEF) as feeder cells. Over a cultivation interval of nine days cells were harvested by trypsin perfusion and mechanical agitation every second to third culture day. A mean of 380 × 10⁶ mESC could be removed with every harvest. Subsequent to harvesting, cells continued growing in the bioreactor, as determined by increasing glucose consumption and lactate production. Immunocytochemical staining and mRNA expression analysis of markers for pluripotency and the three germ layers showed a similar expression of most markers in the harvested cells and in mESC control cultures. In conclusion, successful expansion and harvesting of viable mESC from bioreactor cultures with preservation of sterility was shown. The present study is the first one showing the feasibility of periodic harvesting of adherent cells from a continuously perfused four‐compartment bioreactor including further cultivation of remaining cells. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:141–151, 2016     

Factors indicating culture status during cultivation of Spirulina (Arthrospira) platensis

Factors indicating culture status of two Spirulina platensis strains were monitored in a batch mode cultivation for 36 days. Changing mode in all factors showed a common turning point, indicating shift of cell or culture status. Mean biomass productivity was highly sustained until day 22, chlorophyll a concentration peaked on day 22, pH value was >12 on day 22, coil number was abruptly shortened on day 22, and floating activity was sustained at greater than 79% after day 22, indicating that day 22 is a criterion reflecting phase-transfer in cell physiology in a batch culture system. Many of these changes may have been caused by increased pH, suggesting that pH control is essential for mass production of S. platensis. Fluctuations in floating activity were likely induced by the number of cellular gas vacuoles. Consequently, coil number per trichome and floating activity of S. platensis could readily act as simple indicators for determination of culture status or harvesting time of cells.     

Regulation of prostaglandin production and ectoenzyme activities in cultured aortic endothelial cells

Prostaglandin production, angiotensin-converting enzyme, and 5'-nucleotidase were measured in porcine aortic endothelial cells in situ (with a multi-well template on an opened aorta), in primary culture and in subcultures. Changes during culture were monitored and the effects of culture conditions were investigated by growing cells on a biological matrix or on plastic, by adding different sera to the growth medium, and by harvesting cells enzymically or mechanically. Prostacyclin production by endothelium in primary culture is highest immediately after cell isolation and subsequently declines; this pattern is repeated each time the cells are subcultured. The level at which production stabilises is approximately 200 pg X 10(6) cells-1 X h-1. Detaching cells by physical means stimulates production much more than enzymic dispersion; the type of serum or the presence of a biological matrix does not alter prostaglandin production. The relative amount of prostaglandin E produced increases with time, from approximately 20% of the prostacyclin production shortly after isolation to greater than 100% in subcultured cells. None of the culture conditions that we tested altered this trend. Angiotensin-converting enzyme activity decreases during primary culture, but activity can be sustained by including homologous serum (from whole blood or from platelet-free plasma) in the culture medium. The method of harvesting cells, or the presence of a matrix, did not affect enzyme activity. 5'-Nucleotidase also declines during culture, with a progressive decrease in both Km and Vmax from template to primary culture to subcultures. None of the variations in culture conditions prevented this change. Ecto-adenosine-deaminase activity, not detectable in cultured cells, can be measured in the template. Part of this activity was released by the vascular wall and could be due to plasma diffusing from the interstitial space.     

Bioreactor-Based Online Recovery of Human Progenitor Cells with Uncompromised Regenerative Potential: A Bone Tissue Engineering Perspective

The use of a 3D perfusion culture environment for stem cell expansion has been shown to be beneficial for maintenance of the original cell functionality but due to several system inherent characteristics such as the presence of extracellular matrix, the continued development and implementation of 3D perfusion bioreactor technologies is hampered. Therefore, this study developed a methodology for harvesting a progenitor cell population from a 3D open porous culture surface after expansion in a perfusion bioreactor and performed a functional characterization of the expanded cells. An initial screening showed collagenase to be the most interesting reagent to release the cells from the 3D culture surface as it resulted in high yields without compromising cell viability. Subsequently a Design of Experiment approach was used to obtain optimized 3D harvest conditions by assessing the interplay of flow rate, collagenase concentration and incubation time on the harvest efficiency, viability and single cell fraction. Cells that were recovered with the optimized harvest protocol, by perfusing a 880 U/ml collagenase solution for 7 hours at a flow rate of 4 ml/min, were thereafter functionally analyzed for their characteristics as expanded progenitor cell population. As both the in vitro tri-lineage differentiation capacity and the in vivo bone forming potential were maintained after 3D perfusion bioreactor expansion we concluded that the developed seeding, culture and harvest processes did not significantly compromise the viability and potency of the cells and can contribute to the future development of integrated bioprocesses for stem cell expansion.     

Cultivation, photobioreactor design and harvesting of microalgae for biodiesel production: A critical review

Microalgae have the ability to mitigate CO₂ emission and produce oil with a high productivity, thereby having the potential for applications in producing the third-generation of biofuels. The key technologies for producing microalgal biofuels include identification of preferable culture conditions for high oil productivity, development of effective and economical microalgae cultivation systems, as well as separation and harvesting of microalgal biomass and oil. This review presents recent advances in microalgal cultivation, photobioreactor design, and harvesting technologies with a focus on microalgal oil (mainly triglycerides) production. The effects of different microalgal metabolisms (i.e., phototrophic, heterotrophic, mixotrophic, and photoheterotrophic growth), cultivation systems (emphasizing the effect of light sources), and biomass harvesting methods (chemical/physical methods) on microalgal biomass and oil production are compared and critically discussed. This review aims to provide useful information to help future development of efficient and commercially viable technology for microalgae-based biodiesel production.     

Biodiesel production with microalgae as feedstock: from strains to biodiesel

Due to negative environmental influence and limited availability, petroleum-derived fuels need to be replaced by renewable biofuels. Biodiesel has attracted intensive attention as an important biofuel. Microalgae have numerous advantages for biodiesel production over many terrestrial plants. There are a series of consecutive processes for biodiesel production with microalgae as feedstock, including selection of adequate microalgal strains, mass culture, cell harvesting, oil extraction and transesterification. To reduce the overall production cost, technology development and process optimization are necessary. Genetic engineering also plays an important role in manipulating lipid biosynthesis in microalgae. Many approaches, such as sequestering carbon dioxide from industrial plants for the carbon source, using wastewater for the nutrient supply, and maximizing the values of by-products, have shown a potential for cost reduction. This review provides a brief overview of the process of biodiesel production with microalgae as feedstock. The methods associated with this process (e.g. lipid determination, mass culture, oil extraction) are also compared and discussed.     

Kinetics of biphasic growth of yeast in continuous and fed-batch cultures

The influence of dilution rate on the production of biomass, ethanol, and invertase in an aerobic culture of Saccharomyces carlsbergensis was studied in a glucose-limited chemostat culture. A kinetic model was developed to analyze the biphasic growth of yeast on both the glucose remaining and the ethanol produced in the culture. The model assumes a double effect where glucose regulates the flux of glucose catabolism (respiration and aerobic fermentation) and the ethanol utilization in yeast cells. The model could successfully demonstrate the experimental results of a chemostat culture featuring the monotonic decrease of biomass concentration with an increase of dilution rate higher than 0.2 hr^?1 as well as the maximum ethanol concentration at a particular dilution rate around 0.5 hr^?1. Some supplementary data were collected from an ethanol-limited aerobic chemostat culture and a glucose-limited anaerobic chemostat culture to use in the model calculation. Some parametric constants of cell growth, ethanol production, and invertase formation were determined in batch cultures under aerobic and anaerobic states as summarized in a table in comparison with the chemostat data. Using the constants, a prediction of the optimal control of a glucose fed-batch yeast culture was conducted in connection with an experiment for harvesting a high yield of yeast cells with high invertase activity.     

冻干腮腺炎活疫苗细胞培养生产工艺研究

报告了冻干腮腺炎活疫苗细胞培养生产工艺研究结果,通过对鸡胚疫苗株的适应培养及对细胞培养生产工艺的试验优化,建立了连续细胞培养多次收获疫苗生产工艺并制备出了冻干细胞培养腮腺炎活疫苗制剂。本生产工艺切实可行,生产成本低、投入产出率高,所用原材料规范、质量易于控制,具有明显的技术优势。生产的冻干疫苗制剂质量稳定可靠,符合中国生物制品规程要求,有利于预防腮腺炎的规模化推广使用     

Gene targeting in C57BL/6 ES cells. Successful germ line transmission using recipient BALB/c blastocysts developmentally matured in vitro.

There are significant advantages to the production of gene-knockout mice directly in mouse strains other than 129. The availability now of ES cells derived from the C57BL/6 mouse strain presents workers with a valuable alternative. A major difficulty, however, is the requirement for BALB/c blastocysts as recipients for ES cell injection. Using standard procedures, few BALB/c blastocysts can be obtained. This limitation has now been resolved by harvesting BALB/c embryos at the early morula stage and maturing these to blastocysts by in vitro culture. Of early morulae harvested and cultured, over 70% were recovered as fully expanded and injectable blastocysts. C57BL/6 ES cell injection of these blastocysts has enabled the production of a number of gene-knockout mice with a success rate similar to that reported for ES cells derived from the 129 mouse strains.     

Glucose and amino acid metabolism in neonatal rat skeletal muscle in tissue culture

The characteristics of glucose and amino acid metabolism over a 98-hour incubation period were studied in a primary culture of neonatal rat skeletal muscle cells. The cells formed large myotubes in culture, were spontaneously highly contractile, and had cell phosphocreatine levels exceeding ATP concentrations. Medium glucose fell from 7.2±0.2 to 1.5±0.1 mM between 0 and 98 hours; intracellular glucose was readily detectable, indicating glycolysis was limited by phosphorylation, not glucose transport. Alanine levels in the medium increased from 0.06±0.01 to 0.82±0.04 mM between 0 and 48 hours and decreased to 0.72±0.04 mM by 98 hours. The period of net alanine production correlated with the rise in the cell mass action ratio of the alanine aminotransferase reaction. Cell aspartate, glutamate, and calculated oxalacetate levels were inversely related to the cell NADH/NAD⁺ ratio, as represented by the intracellular lactate/pyruvate ratio (r=0.78–0.88). The branched chain amino acids (leucine, isoleucine, valine) were actively utilized, e.g., medium leucine fell from 0.70±0.01 to 0.30±0.06 mM between 0 and 98 hours. In addition, arginine and serine consumption was observed in conjunction with ornithine, proline, and glycine production. Conclusions: (1) A major driving force for the high rates of alanine production by skeletal muscle cells in tissue culture is the active utilization of branched chain amino acids. (2) Intracellular aspartate and glutamate pools are linked, probably via the malate-aspartate shuttle, to the cell NADH/NAD⁺ redox state. (3) Muscle cells in tissue culture metabolize significant amounts of arginine and serine in association with the production of ornithine and proline, and these pathways may possibly be related to creatine production.     

Increasing GPI-anchored protein harvest concentrations from suspension and porous microcarrier CHO cell cultures

Chinese hamster ovary (CHO) cells expressing the human melanoma tumour antigen, p97, were used to develop a controlled release process for the production of recombinant glycosyl-phosphatidylinositol (GPI) anchored proteins. The cells were cultured either in suspension or immobilized on porous microcarriers and p97 was selectively cleaved from the cell surface by the bacterial enzyme, phosphatidylinositol-phospholipase C (PI-PLC). The kinetics of p97 cleavage from the cell surface by PI-PLC was shown to be approximated by Michaelis-Menten kinetics. The recovered p97 concentrations were increased by reusing the PI-PLC enzyme solution to harvest multiple batches of cells. A convenient PI-PLC assay was developed to monitor the harvesting process and to determine the stability of PI-PLC under harvesting conditions. Although the Pl-PLC was stable under harvesting conditions, it rapidly adsorbed to the cell surface and was depleted from the reused enzyme solution. In order to maintain PI-PLC activity, it was necessary to add fresh PI-PLC to the reused enzyme solution before harvesting a fresh batch of cells. The maximum p97 concentration that could be obtained from harvesting CHO cells cultured on porous microcarriers was limited by the dilution effects of sample removal, adding fresh PI-PLC and liquid associated with settled microcarriers. A model was developed that adequately predicted the p97 concentration after each harvest and the maximum p97 concentration that could be achieved by this harvesting method. The dilution effects were minimized by harvesting from centrifuged suspension culture cells and the harvested p97 concentration was increased by over sixfold to 0.64 mg/mL. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 136-147, 1997.     

微藻两阶段培养技术研究进展

微藻细胞富含油脂、淀粉及其他高值代谢物,可用于食品、饲料、化学品和能源的生产。但在规模化培养中,微藻的高生长速率和高产物含量难以兼得,制约了其商业化应用。通过微藻的两阶段培养技术可以将生长和产物积累的时期分离,从而同时获得较高的微藻生物量和产物含量。该技术具有产品得率高、节能减排、适用范围广的优点,是推进微藻商业化的关键之一。本综述总结了现有微藻两阶段培养技术的优势和产品类型,解析了目前微藻两阶段培养技术的限制因素及发展前景,并提出微藻两阶段培养中存在阶段转换时间尚不明确、中间采收步骤成本高这两个限制该技术应用的关键瓶颈,从而为未来微藻两阶段培养技术规模化生产方案的科学决策与实施提供参考。     

Flotation characteristics of cyanobacterium Anabaena flos-aquae for gas vesicle production

Cyanobacterium Anabaena flos-aquae was cultivated in photobioreactors for production of intracellular gas vesicles (GVs), as potential oxygen microcarriers. Natural flotation of the buoyant culture was investigated as a potential means of cell harvesting, because filtration and centrifugation tended to destroy the vesicles. Best flotation was found with actively growing culture and when conducted in the dark. The flotation-related cell properties, including the specific GV content, vesicle-collapsed filament density, and intracellular carbohydrate content, were measured to understand the phenomena. During the batch culture, the specific GV content remained relatively constant at 370 microL/(g dry cells) but the filament density (ranging 1.02 to 1.08 g/cm3) showed a decrease-then-increase profile. The increase began when the growth slowed down because of the reduced light availability at high cell concentrations. The dark flotation was studied with both actively growing (mu approximately 0.2 day-1) and stationary-phase cultures. The specific GV content of the stationary-phase culture remained relatively constant while that of the growing culture increased slightly. The intracellular carbohydrate content of the growing culture decreased much faster and more significantly, from 57 to 10 mg/(g dry cells) in 相似文献   

Spontaneous level of sister chromosome exchanges and their distribution in human cells]

Blood of practically healthy donors of both sexes (27 females and 23 males) was cultured under the standard conditions during 96 hours. Bromodeoxyuridine (BUdR) was added at the final concentration of 10 mkg/ml 28 hours before harvesting. The slides were stained with acridine orange and Giemsa for differential staining of chromatids. In each culture sister chromatid exchanges (SCE) were analysed in 50 cells, and the part of cells undergoing the first, second and third mitoses at the time of harvesting, was calculated. According to the mean number of SCE per cell, the distribution of individuals was consistent with the normal law, the mean being 6.525 and standard deviation--0.956. A significant heterogeneity in the speed of cell cycle of cultures was observed. The coefficient of variation for the part of cells undergoing the first mitosis was 50%, for the cells in the second mitosis--15%, and for the cells in the third mitosis--154%. Correlation analysis showed a positive dependence of the mean level of SCF upon the age of a donor and upon the part of cells in the second mitosis in this individual. No reliable correlation of the SCE level with the donor's sex was observed. The distribution of cells, obtained from the culture of one individual, was best approximated by beta-distribution, and the distribution of cells obtained from the cultures of different individuals--by gamma-distribution. In both there was obtained a satisfactory approximation by Pearson's distribution of the 1 type, and significant deviations were found from the normal, Poison's and the negative binomial distribution. The conditions were found of similarity of empirical distribution of SCE in cells to the normal one. For that, it is not the value of SCE for a separate cell that should be used as a unit of measurement, but the mean from the values of frequencies for 5-10 cells. Hence, it was shown that for the evaluation of the mean frequency of SCE with the precision of 1 exchange in separate individuals it is necessary to analyse 40 cells, and to observe the 15% increase of spontaneous SCE level under the action of deleterious factors--8 individuals are enough to analyse.     

Additive-free harvesting of oleaginous phagotrophic microalga by oil and air flotation

A unique oleaginous phagotrophic microalga Ochromonas danica is poised for effective lipid production from waste. Cell harvesting and dewatering are major costs in making algae-based products. In this work an effective additive-free harvesting method was developed, taking advantage of O. danica’s comparatively more hydrophobic surface and larger size. The algal cells’ partitioning to oil/water interface was evaluated. Recovery by flotation with waste cooking oil was optimized using an L-9 Taguchi orthogonal-array design. Further, additive-free cell collection and concentrating by air flotation was studied for the effects of both physical factors (column dimension, air–stone pore size, sample-to-column volume ratio) and culture properties (pH, culture growth stage, cell concentration, and pure versus impure cultures). The optimized process consistently achieved >90 % recovery in a single stage. 98+ % recovery could be achieved when starting concentrations were >10⁸ cells/ml, or potentially using a two- or multi-stage process for diluter cultures.     

Synthesis of rotavirus-like particles in insect cells: comparative and quantitative analysis

When the three major structural proteins, VP2, VP6, and VP7, of rotavirus are co-expressed in insect cells infected with recombinant baculoviruses, they self-assemble into triple-layered virus-like particles (VLPs) that are similar in morphology to native rotavirus. In order to establish the most favorable conditions for the synthesis of rotavirus VLPs, we have compared the kinetics of 2/6/7-VLP synthesis in two different insect cell lines: High Five cells propagated in Excell 405 medium and Spodoptera frugiperda 9 cells in Excell 400 medium. The majority of VLPs produced in both cell lines were released into the culture medium, and these released VLPs were predominantly triple-layered and were found to be stable for the period of six or seven days examined. The optimal synthesis of VLPs depended upon the cell line and the culture medium used as well as the time of harvesting infected cell cultures. The highest yield of VLPs was obtained from High Five cultures in the late phase of infection when the yield was at least 5-fold higher than that from S. frugiperda 9 cultures on a per cell basis. Our results demonstrate the usefulness of High Five cells for the production of VLPs as potential rotavirus subunit vaccines.     

Modern aspects of mushroom culture technology

The production and culture of new species of mushrooms is increasing. The breeding of new strains has significantly improved, allowing the use of strains with high yield and resistance to diseases, increasing productivity and diminishing the use of chemicals for pest control. The improvement and development of modern technologies, such as computerized control, automated mushroom harvesting, preparation of compost, production of mushrooms in a non-composted substrate, and new methods of substrate sterilization and spawn preparation, will increase the productivity of mushroom culture. All these aspects are crucial for the production of mushrooms with better flavor, appearance, texture, nutritional qualities, and medicinal properties at low cost. Mushroom culture is a biotechnological process that recycles ligninocellulosic wastes, since mushrooms are food for human consumption and the spent substrate can be used in different ways.