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For Pichia pastoris fermentation process with multi-operating conditions, it is difficult to predict the cell concentration under the new operating conditions by the soft sensor model established under the specific operating conditions. Inspired by the idea of transfer learning, a method based on an improved balanced distribution adaptive regularization extreme learning machine (IBDA-RELM) was proposed to solve the problem. The domain adaptation (DA) method in transfer learning is developed to reduce distribution distance by transforming data. However, the joint distribution adaptation (JDA) and the balanced distribution adaptation (BDA) in DA cannot be directly applied to regression problems. The fuzzy sets (FSs) method was proposed to solve this issue. Finally, a soft sensor model of Pichia pastoris cell concentration was realized by inputting the converted data to the RELM model. Simulation verification was carried out with three operating conditions at the scene of fermentation. The transfer effects of three DA methods, including transfer component analysis (TCA), improved joint distribution adaptation (IJDA) as well as IBDA, were compared. The predicted results show that IBDA-RELM had a better performance in the soft sensor of Pichia pastoris cell concentration under multi-operating conditions.

Conventional sample preparation techniques require bulky and expensive instruments and are not compatible with next-generation point-of-care diagnostic testing. Here, we report a manually operated syringe-tip inertial microfluidic centrifuge (named i-centrifuge) for high-flow-rate (up to 16 mL/min) cell concentration and experimentally demonstrate its working mechanism and performance. Low-cost polymer films and double-sided tape were used through a rapid nonclean-room process of laser cutting and lamination bonding to construct the key components of the i-centrifuge, which consists of a syringe-tip flow stabilizer and a four-channel paralleled inertial microfluidic concentrator. The unstable liquid flow generated by the manual syringe was regulated and stabilized with the flow stabilizer to power inertial focusing in a four-channel paralleled concentrator. Finally, we successfully used our i-centrifuge for manually operated cell concentration. This i-centrifuge offers the advantages of low device cost, simple hand-powered operation, high-flow-rate processing, and portable device volume. Therefore, it holds potential as a low-cost, portable sample preparation tool for point-of-care diagnostic testing.

The seeding of cells into an organ is an important step in cell therapy because the final functional properties of the organ are related to the initial cell distribution throughout the organ. However, vessel occlusion is a serious problem that prevents uniform distribution of the cells in the entire organ. Understanding the mechanism of vessel occlusion can help optimize the seeding process. In this study, the vessel occlusion phenomenon under perfusion conditions during cell seeding was investigated. First, we applied a microfluidic system that enabled the observation of the occlusion events during injection. Second, we applied a multiphase numerical model that can describe the cell–cell interactions and cell–fluid interactions to investigate the vessel occlusion phenomenon during the seeding process. In particular, the effects of cell concentration and flow rate were investigated. The results indicate the importance of cell–cell interactions and cell–vessel interactions for the occurrence of vessel occlusion. In addition, it is found that the probability of occurrence of vessel occlusion increases with the increase in cell concentration and decrease in flow rate. The simulation model can help determine the optimum parameters to enhance cell seeding efficiency.

A careful selection of culture mediums and feeds has become necessary to maximize yields of recombinant proteins during bioprocesses of mammalian cells. Supplements contain a variety of concentrate nutrients, and their beneficial effects vary according to recombinant cell lines. In this study, the effects of PowerFeed A on growth kinetics, productivity and cellular metabolism were evaluated for two Chinese hamster ovary cell lines producing a monoclonal antibody in a batch culture. Supplemented cultures increased integral viable cell density of CRL-12444 and CRL-12445 cells by 2.4 and 1.6 times through extension of culture time at which viability was above 90% in 72 and 36 h, respectively, and increment of maximal cell concentration in 3.25 × 106 cells/ml (69%) for CRL-12445 cells. Product titer augmented 1.9 and 2.5 times for CRL-12444 and CRL-12445 cells, respectively, without changes in growth rate and specific productivity. Feed supplementation also stimulated full consumption of glucose and free glutamine and reduced 10 times lactate accumulation, while ammonium, sodium and potassium remained at similar concentrations at the end of the culture. About 44% of calcium, mainly provided by feed, was consumed by both cell lines. Maximization of cellular growth, viability and protein titer through feeding encourages extending its use to other cell lines and exploring novel combinations with other basal mediums or feeds. A thorough investigation of its impact on protein quality and the molecular mechanisms behind these effects will allow designing effective feeds and strategies to rationally optimize protein production in the biomanufacturing industry., Graphical Abstract

The objective of this study was to develop kinetic models to predict the growth of Vibrio parahaemolyticus in raw Pacific white shrimp as affected by storage temperature. Shrimp samples inoculated with a 2-strain cocktail of V. parahaemolyticus were stored between 8 and 35 °C to observe the growth during storage. The growth curves were analyzed with two primary models (Huang model and No-lag phase model) in combination with a suboptimal Huang square-root model using a one-step kinetic analysis method and the USDA-IPMP Global Fit software to estimate the kinetic parameters that minimized the global error. No growth of V. parahaemolyticus in samples was observed at storage temperatures below 10 °C. The minimum growth temperatures were 10.8 and 10.5 °C as estimated by the Huang and No-lag phase models, respectively. The maximum V. parahaemolyticus population in shrimp samples reached approximately 8.0 log CFU/g during storage. The estimated kinetic parameters, including the minimum growth temperature and maximum cell concentration, matched well with the experimental observations. The kinetic models were validated with two dynamic temperature profiles. The validation showed that both models were equally suitable for predicting the growth of V. parahaemolyticus in Pacific white shrimp. The standard error of prediction is about 1.0 log CFU/g. The models obtained from this study can be used to predict the dynamic growth of V. parahaemolyticus in Pacific white shrimp during post-harvest refrigerated storage and temperature abuse conditions.

A fed-batch strategy is proposed to produce microalgae biomass under non-axenic heterotrophic conditions. The strategy induces the alternation of N-deplete (Glucose-replete) and N-replete (Glucose-deplete) cultivation phases by the periodic and uncoupled supply of glucose and NO3− to the culture. Cultivation of the microalga T. obliquus with this strategy reduced the ratio of the bacteria to microalgae cell concentration from 1.6, attained by conventional photoautotrophic cultivation, to 0.03. During the N-deplete phase, microalgae duplication stopped and biomass concentration increased 1.9 times, while during the N-replete phase, microalgae duplicated halving their average size and losing about 25% of their weight. The process proved to be effective under several consecutive cycles. Biomass productivity until 6.1 g/Ld and biomass concentration until 26 g/L were achieved. The results demonstrate that the proposed strategy can effectively prevent bacterial contamination, paving the way to the large scale production of microalgae biomass under non-axenic heterotrophic conditions.

Cryopreservation is a common methodology for long-term microalgae storage. Current cryopreservation methods are based on using diverse cryoprotectants and two-step cooling protocols, followed by sample storage at the temperature of liquid nitrogen (− 196 °C). However, the use of this methodology requires a continuous liquid N2 supply as well as facilities with dedicated equipment, which is not affordable for every laboratory. In our work, we report on the successful development of a simple and cost-effective method for the long-term cryogenic storage of Tetradesmus obliquus at temperatures (− 80 °C) used in commonly available deep freezers that are more readily accessible to laboratories. Two procedures were evaluated that were originally devised for other microalgae; this was followed by the optimization of critical parameters such as the sample’s microalgal concentration and the cryoprotectant reagent’s incubation time. Cell viability was monitored using the survival rates obtained by direct agar plating and the growth recovery times in liquid cultures. Viability-related variables were recorded following different storage times of up to 3 years. The main operational factors involved in the process (cell concentration, incubation time, and storage time) were statistically analyzed with regard to their influence on the survival rate. The statistical analysis showed interdependence (a two-factor interaction) between the cellular concentration and the cryoprotectant’s incubation time, on the one hand, and between the incubation time and the storage time on the other. Survival rates above 70% were obtained under optimized conditions after 3 months of storage, along with 20–35% viabilities after 3 years. These results open up the possibility of extending this method to other Scenedesmaceae, or even other microalgal species, and for its use in resource-limited laboratories.

Cell counting has become an essential method for monitoring the viability and proliferation of cells. A hemacytometer is the standard device used to measure cell numbers in most laboratories which are typically automated to increase throughput. The principle of both manual and automated hemacytometers is to calculate cell numbers with a fixed volume within a set measurement range (105 ~ 106 cells/ml). If the cell concentration of the unknown sample is outside the range of the hemacytometer, the sample must be prepared again by increasing or decreasing the cell concentration. We have developed a new hemacytometer that has a multi-volume chamber with 4 different depths containing different volumes (0.1, 0.2, 0.4, 0.8 µl respectively). A multi-volume hemacytometer can measure cell concentration with a maximum of 106 cells/ml to a minimum of 5 × 103 cells/ml. Compared to a typical hemacytometer with a fixed volume of 0.1 µl, the minimum measurable cell concentration of 5 × 103 cells/ml on the multi-volume hemacytometer is twenty times lower. Additionally, the Multi-Volume Cell Counting model (cell concentration calculation with the slope value of cell number in multi-chambers) showed a wide measurement range (5 × 103 ~ 1 × 106 cells/ml) while reducing total cell counting numbers by 62.5% compared to a large volume (0.8 µl-chamber) hemacytometer.

The effect of light on biomass and fucoxanthin (Fx) productivities was studied in two microalgae, Tisochrysis lutea and Phaeodactylum tricornutum. High and low biomass concentrations (1.1 and 0.4 g L−1) were tested in outdoor pilot‐scale flat‐panel photobioreactors at semi‐continuous cultivation mode. Fluorescence spectroscopy coupled with chemometric modeling was used to develop prediction models for Fx content and for biomass concentration to be applied for both microalgae species. Prediction models showed high R 2 for cell concentration (.93) and Fx content (.77). Biomass productivity was lower for high biomass concentration than low biomass concentration, for both microalgae (1.1 g L−1: 75.66 and 98.14 mg L−1 d−1, for T. lutea and P. tricornutum, respectively; 0.4 g L−1: 129.9 and 158.47 mg L−1 d−1, T. lutea and P. tricornutum). The same trend was observed in Fx productivity (1.1 g L−1: 1.14 and 1.41 mg L−1 d−1, T. lutea and P. tricornutum; 0.4 g L−1: 2.09 and 1.73 mg L−1 d−1, T. lutea and P. tricornutum). These results show that biomass and Fx productivities can be set by controlling biomass concentration under outdoor conditions and can be predicted using fluorescence spectroscopy. This monitoring tool opens new possibilities for online process control and optimization., Graphical abstract Biomass and fucoxanthin outdoor production and monitoring with brown microalgae

In accordance with the State Pharmacopoeia (SPh) requirements for live plague vaccine, a reference standard has to be used when testing the specific activity and thermal stability of plague vaccine commercial batches in order to assess the consistency and acceptability of the test results. Since there is no international reference standard for plague vaccine, the certification of a new batch of the industry reference standard (IRS) of live plague vaccine in terms of the above-mentioned quality parameters is an urgent challenge. Therefore, a certification programme for the industry reference standard was developed that establishes the design and scope of testing required to obtain statistically significant results. A candidate IRS was represented by a commercial batch of the product meeting the specification requirements for live plague vaccine. The certification parameters were: «Specific activity: microbial cell concentration», «Specific activity: live microbial cell percentage» and «Thermal stability». The article presents the results of the certification of a new batch of the live plague vaccine IRS, detailed evaluation of the candidate IRS in terms of: «Average weight and uniformity of weight», «Loss on drying», and statistical interpretation of the test results. It also summarises the results of the product testing in terms of «Specific activity: immunogenicity». The results of application of the previous batch of the live plague vaccine IRS (OSO 42-28-392-2013) and the results of monitoring the stability of its certification parameters demonstrated that the IRS shelf life could be extended by 6 months relative to the established period (from 2 to 2.5 years). All the certified and additional characteristics are reflected in the official documents for the scientific/technological product — live plague vaccine IRS, OSO 42-28-392-2017: passport, labelling and patient information leaflet.

The objective of this study was to investigate the growth of Escherichia coli O157:H7 in raw ground beef under competition from background flora. The growth of E. coli O157:H7 was observed in sterile irradiated and non-irradiated raw ground beef under dynamically changing temperature conditions. A one-step dynamic analysis method was used to directly construct tertiary models for describing bacterial growth with and without competition and to estimate the kinetic parameters from dynamic growth curves to prove the hypothesis that the growth of E. coli O157:H7 was significantly affected by competition from background flora in raw ground beef. The one-step dynamic method successfully modelled the growth of E. coli O157:H7 and background flora in ground beef and the competition between the two. The estimated minimum growth temperature for E. coli O157:H7 was 7.7 °C, and the maximum cell concentration was 9.0 log CFU/g in irradiated ground beef. Under competition, the specific growth rate of E. coli O157:H7 was reduced by approximately 18% in raw ground beef. The resulting dynamic models and kinetic parameters were validated with separate dynamic growth data, showing that the Root Mean Square Error (RMSE) of prediction was The results of this study demonstrated that the one-step dynamic analysis is a useful and efficient method for investigating bacterial growth with and without competition under dynamic conditions and for developing growth kinetic models. Since the dynamic models have been validated, they can be used to predict the shelf-life of ground beef (background flora) and conduct risk assessment of E. coli O157:H7 and non-O157 STEC.

Wine fermentations using rice media containing either Monascus koji or rice nuruk were performed and fermentative characteristics based on the koji type were investigated. Cultivations were performed in a 20 °C room in a 20 L bottle with the rice media that included Monascus rice koji at both 20 and 30%, or rice nuruk at 20%. After 22 days of cultivation, the ethanol yield reached 14.2–14.6% (v/v) for M. koji and 16.5% (v/v) for rice nuruk. This lower yield with use of M. koji was thought to be due to rapid cell concentration decreases in the later stage. Total amounts of organic acids and volatile compounds in fermentations using M. koji were 166–172 and 1779–1874 mg/L, respectively, being 8.7–12.9% and 46.3–54.1% higher than with use of rice nuruk. With M. koji, a high quality rice wine was produced with high levels of volatile compounds and monacolin K.

Since the infection strategy in the baculovirus-insect cell system mostly affects production of the vector itself or the target product, and given that individual infection parameters interact with each other, the optimal combination must be established for each such specific system. In this work an artificial neural network was used to model infection strategy, including the cell concentration at infection, the multiplicity of infection, the medium recycle, and agitation intensity, and to evaluate the relative importance of each factor in the baculovirus production obtained. The results demonstrate that this model can be used to select an optimal infection strategy. For the baculovirus-insect cell system used in this study, this includes low multiplicity of infection and agitation intensity, along with high cell concentration at infection and medium recycle. Our model is superior to regression methods and predicts baculovirus production more precisely, thus meaning that it could be useful for the development of feasible processes, thereby improving process performance and economy.

Tagatose is a novel low-calorie sweetener which addition changes food product properties, e.g., aw, and formula, that may influence food-microbial growth dynamics in both liquid and solid products. The aim of this work was to study growth dynamics of Salmonella Typhimurium and Listeria monocytogenes in liquid and solid media enriched with tagatose (1.5, 4.5 and 7.5% (w/v)), at 4, 8 and 20 °C. More specifically, liquid media were chemically defined minimal medium, general medium and UHT skimmed milk; gelatine and a gelatine-dextran mixture were the gelling agents used, leading respectively a homogeneous and heterogeneous solid system. At regular intervals, cell concentration was determined and the Baranyi and Roberts (1994) model was fitted for growth parameter estimation. Results show a reduced and even no growth of S. Typhimurium with increasing tagatose concentrations, especially at low temperatures and when increasing media complexity. The behaviour of L. monocytogenes is not affected by tagatose, except in liquid at 4 °C, where tagatose facilitates the growth in general culture media. Varying responses of the studied bacteria to changes in media formulation should be considered for future product design.

3D bioprinted vascular constructs have gained increased interest due to their significant potential for creating customizable alternatives to autologous vessel grafts. In this study, we developed a new approach for biofabricating fibrin-based vascular constructs using a novel rotary 3D bioprinter developed in our lab. We formulated a new bioink by incorporating fibrinogen with gelatin to achieve a desired shear-thinning property for rotary bioprinting. The blending of heat-treated gelatin with fibrinogen turned unprintable fibrinogen into a printable biomaterial for vessel bioprinting by leveraging the favorable rheological properties of gelatin. We discovered that the heat-treatment of gelatin remarkably affects the rheological properties of a gelatin-fibrinogen blended bioink, which in turn influences the printability of the ink. Further characterizations revealed that not only concentration of the gelatin but the heat treatment also affects cell viability during printing. Notably, the density of cells included in the bioinks also influenced printability and tissue volumetric changes of the printed vessel constructs during cultures. We observed increased collagen deposition and construct mechanical strength during two months of the cultures. The burst pressure of the vessel constructs reached 1110 mmHg, which is about 52% of the value of the human saphenous vein. An analysis of the tensile mechanical properties of the printed vessel constructs unveiled an increase in both the circumferential and axial elastic moduli during cultures. This study highlights important considerations for bioink formulation when bioprinting vessel constructs. Statement of Significance There has been an increased demand for small-diameter tissue-engineered vascular grafts. Vascular 3D bioprinting holds the potential to create equivalent vascular grafts but with the ability to tailor them to meet patient’s needs. Here, we presented a new and innovative 3D rotary bioprinter and a new bioink formulation for printing vascular constructs using fibrinogen, a favorable biomaterial for vascular tissue engineering. The bioink was formulated by blending fibrinogen with a more printable biomaterial, gelatin. The systematic characterization of the effects of heat treatment and gelatin concentration as well as bioink cell concentration on the printability of the bioink offers new insight into the development of printable biomaterials for tissue biofabrication.

Despite its simplicity, which makes it the most commonly used bioprinting method today, extrusion-based bioprinting suffers from its inability to reproduce the complex tissue architecture found in organs. Generally, this printing method allows for the dispensing of solutions of a predefined cell concentration through a rudimentary needle. Moreover, to avoid cell lysis in the dispensing needle, which is detrimental to the viability of the printed tissue, as well as cell loss in dead volumes of tubing, thereby increasing the cost of printing tissue, a common strategy has been to print with cell concentrations much lower in comparison to the concentrations found in living tissues. As a result, cell-to-cell distance is increased in the dispensed samples impairing communication through cytokines. Here, we present a microfluidic-based print head capable of modulating the printed cell concentration in real-time. This device allows bioprinting at high cell concentrations by concentrating and dispensing fibroblasts at concentrations up to 10 million cells∙mL−1. We also demonstrate that this device can be used to print bladder organoids. As the cell seeding concentration is of major importance for organogenesis in 3D culture, organoid printing allows the user to standardize the process of organoid formation and achieve more reliable and reproducible results.

Considering its high content of hemicellulose, oil palm empty fruit bunch (EFB) lignocellulosic biomass waste from palm oil processing has the potential to be utilized as the raw material for the production of xylitol, a low calorie, low GI, and anti cariogenic alternative sugar with similar sweetness to sucrose. This research explored the possibility of converting EFB to xylitol via green microbial fermentation, in particular the effects of inoculum and initial pH on the fermentation performance. It was observed that the cell concentration in the inoculum and the initial pH affect cell growth and xylitol production. pH 5 was observed to give the best fermentation performance. Further, the fermentation tended to yield more xylitol at higher initial cell concentration. It was also observed that no growth or fermentation inhibitory compounds were found in the EFB hydrolysate obtained from enzymatic hydrolysis of EFB. Thus it can be used directly as substrate for xylitol fermentation.

In situ Microscopy (ISM) is an optical non-invasive technique to monitor cells in bioprocesses in real-time. Pichia pastoris is one of the most promising protein expression systems. This yeast combines fast growth on simple media and important eukaryotic features such as glycosylation. In this work, the ISM technology was applied to Pichia pastoris cultivations for online monitoring of the cell concentration during cultivation. Different ISM settings were tested. The acquired images were analyzed with two image processing algorithms. In seven cultivations the cell concentration was monitored by the applied algorithms and offline samples were taken to determine optical density (OD) and dry cell mass (DCM). Cell concentrations up to 74 g/L dry cell mass could be analyzed via the ISM. Depending on the algorithm and the ISM settings, an accuracy between 0.3 % and 12 % was achieved. The overall results show that for a robust measurement a combination of the two described algorithms is required.

Cut surfaces of fresh produce, which directly expose plant cell walls to the environment, are particularly susceptible to contamination by pathogens, including Salmonella enterica . The effect of Salmonella cell concentration in liquids (such as rinses or washes) on their attachment to plant cell walls was investigated using bacterial cellulose-based plant cell wall models. Numbers of Salmonella cells attaching to the plant cell wall models increased linearly with cell concentration. A simple linear model (y = 0.916x) was constructed to predict the number of Salmonella cells that will attach to per unit surface area of plant cell wall (CFU/cm 2 ), assuming their initial inoculum concentration lies within the range of 3 log to 8 log CFU/mL. The linear regression model generated from the model surfaces was validated for a range of variables (different plant tissues, S. enterica subspecies and other foodborne bacteria). The validation supported the use of the linear regression model in predicting Salmonella attachment to plant cell walls regardless of the Salmonella subspecies. The use of the model can also be extended to other bacteria within the cell concentration range. This work may contribute to generating risk-assessment tools for control of Salmonella associated with the processing of fresh produce.

To ensure cell-based assays are performed properly, both cell concentration and viability have to be determined so that the data can be normalized to generate meaningful and comparable results. Cell-based assays performed in immuno-oncology, toxicology, or bioprocessing research often require measuring of multiple samples and conditions, thus the current automated cell counter that uses single disposable counting slides is not practical for high-throughput screening assays. In the recent years, a plate-based image cytometry system has been developed for high-throughput biomolecular screening assays. In this work, we demonstrate a high-throughput AO/PI-based cell concentration and viability method using the Celigo image cytometer. First, we validate the method by comparing directly to Cellometer automated cell counter. Next, cell concentration dynamic range, viability dynamic range, and consistency are determined. The high-throughput AO/PI method described here allows for 96-well to 384-well plate samples to be analyzed in less than 7 min, which greatly reduces the time required for the single sample-based automated cell counter. In addition, this method can improve the efficiency for high-throughput screening assays, where multiple cell counts and viability measurements are needed prior to performing assays such as flow cytometry, ELISA, or simply plating cells for cell culture.

The aim of this article is to review a number of variables that may affect the cryopreservation of minimally manipulated products containing allogeneic or autologous hemopoietic progenitor cells (HPC) used for transplantation, with particular reference to processing, type and addition of cryoprotectant, cell concentration, volume, freezing procedure, cooling rate, storage, thawing, and quality management. After defining final product's requirements in compliance with norms, laws and regulations, it is crucial to define the critical control points of the process. New approaches of processing were developed in the last few years such as automatic devices for volume reduction and high cell concentration in the frozen product. DMSO at 10% final concentration is still the most used cryoprotectant for HPC cryopreservation. Although controlled rate freezing is the recommended method for HPC cryopreservation, alternative methods may be used. Last generation vapor storage vessels ensure temperature stability better than older tanks. Their use may reduce risks of cross-contamination. Finally we review advantages and disadvantages of thawing procedures that may be carried out in the laboratory or at the patient's bedside.

The effects of important production variables on the biotransformation of sucrose by immobilized cells of Phaffia rhodozyma were investigated. Cell concentration had negative effect on the ...

Purpose To investigate the potential of two flow imaging microscopy (FIM) techniques (Micro-Flow Imaging (MFI) and FlowCAM) to determine total cell concentration and cell viability. Methods B-lineage acute lymphoblastic leukemia (B-ALL) cells of 2 different donors were exposed to ambient conditions. Samples were taken at different days and measured with MFI, FlowCAM, hemocytometry and automated cell counting. Dead and live cells from a fresh B-ALL cell suspension were fractionated by flow cytometry in order to derive software filters based on morphological parameters of separate cell populations with MFI and FlowCAM. The filter sets were used to assess cell viability in the measured samples. Results All techniques gave fairly similar cell concentration values over the whole incubation period. MFI showed to be superior with respect to precision, whereas FlowCAM provided particle images with a higher resolution. Moreover, both FIM methods were able to provide similar results for cell viability as the conventional methods (hemocytometry and automated cell counting). Conclusion FIM-based methods may be advantageous over conventional cell methods for determining total cell concentration and cell viability, as FIM measures much larger sample volumes, does not require labeling, is less laborious and provides images of individual cells. Electronic supplementary material The online version of this article (10.1007/s11095-018-2422-5) contains supplementary material, which is available to authorized users.

Continuous culture for the production of biopharmaceutical proteins offers the possibility of steady state operations and thus more consistent product quality and increased productivity. Under some conditions, multiplicity of steady states has been observed in continuous cultures of mammalian cells, wherein with the same dilution rate and feed nutrient composition, steady states with very different cell and product concentrations may be reached. At those different steady states, cells may exhibit a high glycolysis flux with high lactate production and low cell concentration, or a low glycolysis flux with low lactate and high cell concentration. These different steady states, with different cell concentration, also have different productivity. Developing a mechanistic understanding of the occurrence of steady state multiplicity and devising a strategy to steer the culture toward the desired steady state is critical. We establish a multi-scale kinetic model that integrates a mechanistic intracellular metabolic model and cell growth model in a continuous bioreactor. We show that steady state multiplicity exists in a range of dilution rate in continuous culture as a result of the bistable behavior in glycolysis. The insights from the model were used to devise strategies to guide the culture to the desired steady state in the multiple steady state region. The model provides a guideline principle in the design of continuous culture processes of mammalian cells. Biotechnol. Bioeng. 2015;112: 1437–1445. © 2015 Wiley Periodicals, Inc.

In situ Microscopy (ISM) is an optical non-invasive technique to monitor cells in bioprocesses in real-time. Escherichia coli is the most studied and used organism in biotechnology. In this article the cell density in Escherichia coli cultivations was monitored by applying ISM in these cultivations. The acquired images were analyzed with an image processing algorithm to determine the turbidity of the cultivation medium. In three cultivations the cell density was monitored with the algorithm and offline samples were taken to determine the dry cell mass (DCM). Both results were correlated and concentrations up to 70 g/L DCM could be measured via ISM. For higher cell densities a saturation was recognized. The deviation of the calibration lines within three cultivations was 8%.

To protect the enzymes during fed-batch cellulase production by means of partial enzyme recovery at regular intervals. Extracellular enzymes were partially recovered at the intervals of 1, 2, or 3 days. Mycelia were also removed to avoid contamination. Increases in the total harvested cellulase (24–62%) and β-glucosidase (22–76%) were achieved. In fermentor cultivation when the enzymes were recovered every day with 15% culture broth. The total harvested cellulase and β-glucosidase activity increased by 43 and 58%, respectively, with fungal cell concentration maintained at 3.5–4.5 g l−1. Enzyme recovery at regular intervals during fed-batch cellulase cultivation could protect the enzyme in the culture broth and enhance the enzyme production when the fungal cell concentration is maintained in a reasonable range.

This study presents a novel cell docking system based on microwells integratded with microvalves. Conventional cell docking device based on micro-well suffers from generation of dead volume and shear stress within micro-wells resulting in low efficiency of cell docking, limitation of nutrient, and low cell viability. Our approach to solve the problems adopts integration of microvalve controlled by pressure with microwells for provinding guided flow stream of cells and nutrients into microwell. We have optimized the efficiency of cell docking by varying several experimental parameters including flow rate, cell concentration, microvalve pressure, and size of microvalve. Under the optimized flow rate (1 µL/sec) and valve pressure (0.2 MPa), we obtain high efficiency of cell docking as 14.1 cells/microwell. In this study, we confirm that the perfusion culture of cells in microfluidic chip provides suitable environmental condition for cell culture at small scale and demonstrate the feasibility of universal cell culture platform.

Monitoring cell growth and measuring physical features of food-borne pathogenic bacteria are important for better understanding the conditions under which these organisms survive and proliferate. To address this challenge, buoyant masses of live and dead Escherichia coli O157:H7 and Listeria innocua were measured using Archimedes, a commercially available suspended microchannel resonator (SMR). Cell growth was monitored with Archimedes by observing increased cell concentration and buoyant mass values of live growing bacteria. These growth data were compared to optical density measurements obtained with a Bioscreen system. We observed buoyant mass measurements with Archimedes at cell concentrations between 10 5 and 10 8 cells/ml, while growth was not observed with optical density measurements until the concentration was 10 7 cells/ml. Buoyant mass measurements of live and dead cells with and without exposure to hydrogen peroxide stress were also compared; live cells generally had a larger buoyant mass than dead cells. Additionally, buoyant mass measurements were used to determine cell density and total mass for both live and dead cells. Dead E. coli cells were found to have a larger density and smaller total mass than live E. coli cells. In contrast, density was the same for both live and dead L. innocua cells, while the total mass was greater for live than for dead cells. These results contribute to the ongoing challenge to further develop existing technologies used to observe cell populations at low concentrations and to measure unique physical features of cells that may be useful for developing future diagnostics.

In this work, we introduced an integrated microfluidic system for fast and efficient circulating tumor cell (CTC) isolation and capture. In this microfluidic platform, a combination of microfluidic deterministic lateral displacement array and affinity-based cell capture architecture, allows for the high efficiency cancer cell enrichment and continuous high throughput and purity cancer cell capture. Using this device to isolate breast cancer cells from spiked blood samples, we achieved an enrichment factor of 1500×, and a high processing throughput of 9.6mL/min with 90% capture yield and more than 50% capture purity at cell concentration 10(2)cells/mL. This integrated platform offers a promising approach for CTC capture with high recovery rates, purity and stability, and exhibits potential capability in cancer cell culture and drug screening.

We present a method for retrieval of cells encapsulated in alginate-polyethersulfone (AP) microcapsules. AP microcapsules consist of alginate hydrogel core and a semi-permeable polymer membrane. Proposed method is based on chemical destruction of microcapsule structure that is, polymer membranes are removed by dissolution in 1-methyl-2-pyrrolidone subsequently the cells immobilized in the cores are released by liquefaction of alginate gel. The viability of Saccharomyces cerevisiae cells retrieved by this technique exceeds 98%, while the concentration of recovered cells reaches 98%-102% of cell concentration in suspension used for microencapsulation, which proves that proposed method allows to effectively and quantitatively retrieve encapsulated cells without compromising their viability.

A liquid marble has a porous and superhydrophobic shell, which prevents the liquid core within the marble from making any contact with outside surfaces, but at the same time allows gases to transport freely across the shell. In this study we demonstrated the use of this unique property of liquid marbles to build respirable micro-biological reactors to cultivate microorganisms. Liquid marbles loaded with two kinds of microorganism cultures with different preference to oxygen were studied. Due to the presence of oxygen in the cultivation environment, significant differences in cell proliferation between the two microorganisms were observed within the liquid marbles. Furthermore, we found that this respirable bioreactor provides a more suitable environment for the growth of an aerobe than that in McCartney bottles with shaking incubation; cell concentration increased more rapidly in liquid marbles.

Background: Nowadays, Leaching-ore bacteria, especially Acidithiobacillus ferrooxidans is widely used to retrieve heavy metals, many researches reflected that extra adding microorganism could promote bioleaching efficiency by different mechanisms, but few of them discussed the interaction between microorganisms and based on growth model. This study aimed to provide theoretical support for the collaborative bioleaching of multiple microorganisms by using the Lotka-Volterra (L-V) model. Results: This study investigated the interaction of Acidithiobacillus ferrooxidans , Rhizobium phaseoli , and Rhodotorula sp . Results showed that the individual growth of the three microorganisms fit the logistic curves. The environmental capacities of A. ferrooxidans , R. phaseoli , and Rhodotorula sp. were 1.88x10 9 , 3.26x10 8 , and 2.66x10 8 cells/mL, respectively. Co-bioleaching showed mutualism between A. ferrooxidans and R. phaseoli with mutualism coefficients of α = 1.19 and β = 0.31, respectively. The relationship between A. ferrooxidans and Rhodotorula sp. could be considered as commensalism. The commensalism coefficient γ of the effect of Rhodotorula sp. on A. ferrooxidans was 2.45. The concentrations of A. ferrooxidans and R. phaseoli were 3.59x10 9 and 1.44x10 9 cells/mL in group E, respectively, as predicted by the model. The concentrations of A. ferrooxidans and Rhodotorula sp. were 2.38x10 9 and 2.66x10 8 cells/mL, respectively. The experimental peak values of the concentrations in microorganism groups E and F were detected on different days, but were quite close to the predicted values. Conclusion: The relationship among microorganisms during leaching could be described appropriately by Lotka-Volterra model between the initial and peak values. The relationship of A. ferrooxidans and R. phaseoli could be considered as mutualism, whereas, the relationship of A. ferrooxidans and R. phaseoli could be considered as commensalism. Normal 0 21 false false false EN-US ZH-CN X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Tabla normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:"Times New Roman","serif"; mso-ansi-language:EN-US; mso-fareast-language:ZH-CN;}

The objective of this work was to study the production of GSH by Saccharomyces cerevisiae ATCC 7754 in a fermentor (5 L) using a cell recycle system with magnets. The fermentation conditions were 20°C, 500 rpm, 5% (v/v) of inoculum, pHinitial 5, 1.1 vvm aeration and total fermentation time of 72 h. The time of application of MF ranged from 24, 48 or 72 h. In comparison to the control experiment, the best results were obtained with 72 h of application of MF. The cell concentration reached 19.5 g/L and GSH concentration was 271.9 mg/L that corresponded to an increase of 2.63 and 32.1% compared to the control experiment, respectively.

This work presents a systematic investigation of the toxic effects of acrylic acid on the growth of Clostridium propionicum and the isolation of acrylic acid-tolerant mutants. The results suggest that addition of acrylic acid prolonged the lag phase of the fermentation and reduced the initial-specific growth rate, as well as the final cell concentration. Moreover, the toxic effect of acrylic acid was sensitive to the pH value. The minimal inhibition concentration of acrylic acid increased from 1.11 to 31.25 mM when the pH value rose from 5.8 to 7.4. In addition, the molar concentration ratio of products (acetic acid:propionic acid) was enhanced with the supplementation of acrylic acid. The highest ratio was 0.7:1 when acrylic acid was 20.83 mM at pH 7.4. Two acrylic acid-tolerant mutants were isolated, which could still grow at a high concentration (43.06 mM) of acrylic acid. These strains could be instrumental for improved bioproduction of acrylic acid.

Aim: The investigation of the different culture systems in order to get high yield of baby hamster kidney (BHK) cell line. Materials and Methods: Different cultivation systems, vibrofermentor and 5-L stirred tank bioreactors, have been used for the production of BHK cell line. The production systems were evaluated with respect to pH, temperature, viable cell number, aeration rate and stirring speed. Results: BHK cells were successfully cultivated in vibrofermentor and stirred tank bioreactor (STR) systems. In addition, higher cell concentration was obtained with low aeration rate (0.005 vvm) comparing to those of 0.02 and 0.05 vvm. The cultivation in STR can be operated with low aeration rate due to decrease the expenditure and improve the cell productivity. Conclusion: In order to improve the cell yield of the anchorage semi-dependent cell line in pilot scale, it should be preferred that the cells are firstly adapted to suspension culture by vibrational stirring and then be cultivated in stirred tank bioreactor which is operated at low aeration rate. These results showed consistency with the previous studies.

Adhesion behavior of Escherichia coli from its suspension onto stainless steel (type 304) surface was studied, focusing on the effects of initial cell concentration of suspension, suspending medium, and roughness of stainless steel surface. The results demonstrated that the adhesion of E. coli was significantly enhanced by the cell concentration of contaminants. The adhesion was observed even in short contact periods of time, but it increased and reached a plateau within 3–4 h. The plateau value of surface cell density was roughly proportional to the cell concentration of the suspension. Rinsing experiments indicated that a relatively high percentage of cells were found to be irreversibly attached on the surface when peptone saline was used as a suspending medium. The adherent cells were however shown to be quite vulnerable to removal with water as a rinsing medium. Moreover surface roughness was found to affect the removability of adherent cells.

Application of an automated colony counter for evaluation of the viability of microbial cultures was investigated with yeast cultures as a model. Statistical comparison of the results of automated and visual (“manual”) colony counting is presented, as well as the results of the application of the bundled software to digital images obtained by light microscopy for determination of the cell concentration in suspensions. Automated counting is concluded to significantly accelerate the evaluation of culture viability by colony-forming capacity, provided that a certain requirements of sample preparation and analysis are observed.

The asymmetric reduction of o-chloroacetophenone 1 with Candida pseudotropicalis 104 produced the corresponding (S)-l-(2-chloro-phenyl)-ethanol 2 with the enantiomeric excess (ee >99%) without addition of any cosolvent. The cell could tolerate high ketone 1 concentration of 233.8 mmol/L (i.e., 36 g/L) with considerable reduction activity in this method. The product 2 concentration achieved 38.9 and 58.4 mmol/L with cells of 40 and 60 g DCW (dry cell weight)/ L, respectively, in 24 h. The optimum reaction time, the effect of substrate concentration, cosubstrate type and concentration, and cell concentration in the reaction were investigated in this paper.

Conditions were evaluated for optimum cryopreservation of primary chicken embryo kidney (CEK) cells. The recovery of viable CEK cells was best (50.8% viability) when the concentration of dimethyl sulfoxide (DMSO) in the freezing medium was 20% (v/v). The viability of primary CEK cells was not influenced by the concentration of calf serum in the freezing medium, the duration of storage at -70 degrees C before storage in liquid nitrogen, cell concentration, or the method of addition or dilution of DMSO. Thawed cells recovered and grew in complete growth medium similarly to cells freshly isolated from kidney, and influenza viruses produced plaques in the monolayer. The cryopreservation procedures described here may facilitate maintenance of a standard stock of primary CEK cells for laboratories where preparation of primary CEK cells is not an option.

Background Dielectric spectroscopy (DS) has been used for over two decades to measure the live cell concentration of animal cells in bioreactors online. The technology measures the build up of charge around live cell membranes under the influence of an induced electric field. Dead cells, debris, air bubbles and solid media particles do not have the capability to hold charge around themselves and are hence, invisible to the technology. The capacitance thus measured is proportional to the live bio-volume of the culture. DS has been used extensively to monitor and control live cell concentrations in bioreactors from process development through to large scale production. For DS to be employed as a PAT tool, it is essential that it successfully measures cell concentration over a wide range of culture volumes. Utilising the same cell measurement technique through the entire scale up protocol can also be a distinct advantage. This paper describes the latest developments in dielectric spectroscopy, especially the scaling down of the technology for measurement of lower volume cultures (up to 1 ml) in real time. The paper will also illustrate the application of dielectric spectroscopy to single use bioreactors.

Yeast identification and characterization is of significant importance for yeast strain quality and purity, and hence for the final product. This chapter describes the different brewing yeast species and identification as well as differentiation methods. Yeast strains are described from the perspective of fermentation technology. Universal yeast quality specifications are being defined. Diverse methods to determine cell concentration, viability, vitality, main fermentation parameters, fermentation by-products, online measured parameters, and microbiological values are described and discussed. Information from this chapter can be applied to optimize the use of specific brewing yeast strains, as well as their quality and process control.

The development of fermentation conditions for the production of C595 diabody fragment (dbFv) inE. coli HB2151 clone has been explored. Investigations were carried out to study the effect of carbon supplements over the expression period, the comparison of C595 dbfv production in synthetic and complex media, the influence of acetic acid upon antibody production, and comparison of one-stage and two-stage processes operated at batch or fed-batch modes in bioreactor. Yeast extract supplied during expression yielded more antibody fragment than any other carbon supply. The synthetic medium presented higher specific productivity (0.066 mg dbFv g−1 dry cell weight) when compared to the complex medium (0.044 mg dbFv g−1 DCW). The comparison of fermentation strategies demonstrated that (1) one-stage fed-batch fermentation performed higher C595 dbFv production than that operated in batch mode which was significantly affected by acetate concentration; (2) a two-stage batch operation could enhance C595 dbFv production. It was found that a concentration of 12.3 mg L−1 broth of C595 dbFv and a cell concentration of 10.8 g L−1 broth were achieved at the end of two-stage operation in 5-L fermentation.

Sinorhizobium sp., which can convert d-fructose into d-psicose, was isolated from soil. The optimal pH, temperature, and cell concentration for d-psicose production with the isolated strain were 8.5, 40°C, and 60 mg/ml, respectively. The toluene-treated cells showed 2.5- and 4.8-fold increases in the d-psicose concentration and productivity compared with untreated washed cells. Under the optimal conditions, the toluene-treated cells produced 37 g d-psicose/l from 70% (w/v) (3.9 M) d-fructose after 15 h.

In order to achieve an effective decolorization with Funalia trogii, fungal immobilization and repeated batch experiment were investigated. The activities of three enzymes, namely laccase, lignin peroxidase (LiP) and manganese peroxidase (MnP), were reported during decolorization of acid black 52 under the condition of immobilized and suspended fungal mycelia. When F. trogii were immobilized in Na-alginate, the cell viability increased as alginate concentration was increased, although the cell concentration decreased. Na-alginate of 4% (w/v) was an optimum concentration. Decolorization rate by fungal immobilization was higher than that by suspended cultivation. Decolorization could be stably maintained with the repeated batch experiments during long period (at least for 300 h).

Lyophilized non-proliferating microbial cells of Pseudomonas sp. 42A2 showed their ability to catalyze the bioconversion of oleic acid into (E)-10-hydroxy-8-octadecenoic acid (MHOD) and (E)- 7,10-dihydroxy-8-octadecenoic acid (DHOD). The reaction conditions were optimized in order to produce the maximum amount of MHOD. At 30°C and using phosphate buffer (0.1 M and pH=8), an initial substrate concentration of 10 mg mL� 1 and a cell concentration of 8 mg mL� 1 , practically all the oleic acid in the medium was biotransformed within 42 h, with hardly any DHOD produced (1.4 mg mL� 1 ).

We investigated rapid and simple colorimetric microplate assays with tryptone–yeast–glucose broth (TYG) containing oxidation–reduction (redox) indicators (alamarBlue, MTS and WST-8), AquaTest (selective medium for coliforms) and PYP broth containing pH indicator (phenol-red) to assess cell counts using Escherichia coli, Staphylococcus aureus and Bacillus cereus strains as test organisms. In all redox systems, the estimation of aerobic cell concentration was possible by incubating at 35 °C for several hours. Although optical densities for MTS or WST were higher than that of alamarBlue, the time to obtain results (which may be correlated with doubling time) was longer. Color changes in AquaTest were 200–300 min later than for TYG containing alamarBlue and PYP.

This study proposes lumostatic operations using a specific light uptake rate ( q e ) as a control parameter in order to maintain a desirable light condition. As a model system, bubble column photobioreactors have been operated lumostatically for photoautotrophic cultivations of Haematococcus pluvialis . Lumostatic operations were conducted at three levels of specific light uptake rates ( q e =1.5×10 −8 , 3×10 −8 , and 4.5×10 −8 μE cell −1 s −1 ) and compared with constant light energy supply ( I 0 =40, 90, and 140 μE m −2 s −1 ). In the case of the lumostatic operation at 4.5×10 −8 μE cell −1 s −1 , the cell concentration was enhanced over 250% than that obtained under constant light energy, without inducing astaxanthin accumulation. For the lumostat at q e of 3.0×10 −8 μE cell −1 s −1 , the cell concentration increased almost 200% than that of the control experiments. These results indicate that a specific light uptake rate can be a useful parameter for lumostatic operation of photobioreactors and that the productivity of vegetative stage can be greatly improved by regulating the light condition based on the specific light uptake rates.