Multifrequency permittivity measurements enable on‐line monitoring of changes in intracellular conductivity due to nutrient limitations during batch cultivations of CHO cells

Lab and pilot scale batch cultivations of a CHO K1/dhfr^? host cell line were conducted to evaluate on‐line multifrequency permittivity measurements as a process monitoring tool. The β‐dispersion parameters such as the characteristic frequency (f_C) and the permittivity increment (Δε_max) were calculated on‐line from the permittivity spectra. The dual‐frequency permittivity signal correlated well with the off‐line measured biovolume and the viable cell density. A significant drop in permittivity was monitored at the transition from exponential growth to a phase with reduced growth rate. Although not reflected in off‐line biovolume measurements, this decrease coincided with a drop in OUR and was probably caused by the depletion of glutamine and a metabolic shift occurring at the same time. Sudden changes in cell density, cell size, viability, capacitance per membrane area (C_M), and effects caused by medium conductivity (σ_m) could be excluded as reasons for the decrease in permittivity. After analysis of the process data, a drop in f_C as a result of a fall in intracellular conductivity (σ_i) was identified as responsible for the observed changes in the dual‐frequency permittivity signal. It is hypothesized that the β‐dispersion parameter f_C is indicative of changes in nutrient availability that have an impact on intracellular conductivity σ_i. On‐line permittivity measurements consequently not only reflect the biovolume but also the physiological state of mammalian cell cultures. These findings should pave the way for a better understanding of the intracellular state of cells and render permittivity measurements an important tool in process development and control. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

On-line monitoring of responses to nutrient feed additions by multi-frequency permittivity measurements in fed-batch cultivations of CHO cells

Changes in the nutrient availability of mammalian cell cultures are reflected in the β-dispersion parameter characteristic frequency (f _C ) and the on-line dual frequency permittivity signal. Multi-frequency permittivity measurements were therefore evaluated in fed-batch cultivations of two different CHO cell lines. Similar responses to nutrient depletions and discontinuous feed additions were monitored in different cultivation phases and experimental setups. Sudden increases in permittivity and f _C occurred when feed additions were conducted. A constant or declining permittivity value in combination with a decrease in f _C indicated nutrient limitations. f _C correlated well with changes in oxygen uptake rate when cell diameter remained constant, indicating that metabolic activity is reflected in the value of f _C . When significant cell size changes occurred during the cultivations, the analysis of the β-dispersion parameters was rendered complex. For the application of our findings in other systems it will be hence required to conduct additional off-line measurements. Based on these results, it is hypothesized that multi-frequency permittivity measurements can give information on the intracellular or physiological state in fed-batch mode. Similar observations were made when using different cell lines and feeding strategies, indicating that the findings are transferable to other cell lines and systems. The results should lead to an improved understanding of routine fed-batch processes. Additional studies are, however, required to explore how these observations can be used for fed-batch process development and optimization.     

Analysis of long-term variation in phytoplankton biovolume in the northern basin of Lake Biwa

The long-term variation in phytoplankton biovolume in the northern basin of Lake Biwa was analyzed using periodic phytoplankton census data from January 1979 to December 2009. Population densities obtained from census data were transformed into biovolumes, and phytoplankton species were categorized into three size fractions: net phytoplankton (≥4,000 μm³ cell^?1, ≥ca. 20 μm in diameter), large nanophytoplankton (100–4,000 μm³ cell^?1, ca. 6–20 μm in diameter), and small nanophytoplankton (<100 μm³ cell^?1, <ca. 6 μm in diameter). Although the annual total biovolume gradually decreased over time, the total biovolumes in winter and spring were found to increase. Furthermore, a decrease in the biovolume of net phytoplankton and an increase in that of small nanophytoplankton were observed. Because of succession in the phytoplankton community, the average cell volume of the phytoplankton community decreased from 269 μm³ cell^?1 in the 1980s to 56 μm³ cell^?1 in the 2000s. Lake warming accompanied with the intensification of thermal stratification and the augmentation of wind speed were observed at Lake Biwa over the study period. Serial analysis correcting for autocorrelation revealed that oligotrophication in the epilimnion, induced by lake warming and limitation of light available for phytoplankton growth by wind-induced water mixing, was a potential factor in the succession of the phytoplankton community.     

Ecosystem respiration derived from 222Rn measurements on Rishiri Island, Japan

We evaluated the nighttime CO₂ flux (ecosystem respiration) on Rishiri Island, located at the northern tip of Hokkaido, Japan, from 2009 to 2011, by using the relationship between atmospheric ²²²Rn and CO₂ concentrations. The annual mean CO₂ flux was 1.8 μmol m^?2 s^?1, with a maximum monthly mean in July (4.6 ± 2.6 μmol m^?2 s^?1) and a broad minimum from December to March (0.33 ± 0.29 μmol m^?2 s^?1). The annual mean was comparable to fluxes at the JapanFlux sites in northern Japan. During the season of snow cover (mid-December to early April), the CO₂ flux was low (0.45 ± 0.43 μmol m^?2 s^?1). Total annual respiration was estimated at 679 ± 174 g cm^?2, about 8 % of which occurred during the season of snow cover.     

Recovery of avocado (<Emphasis Type="Italic">Persea americana</Emphasis> Mill.) plants transformed with the antifungal plant defensin gene PDF1.2

Embryogenic avocado cultures derived from ‘Hass’ protoplasts were genetically transformed with the plant defensin gene (pdf1.2) driven by the CaMV 35S promoter in pGPTV with uidA as a reporter gene and bar, the gene for resistance to phosphinothricin, the active ingredient of the herbicide Finale® (Basta) (Bayer Environmental Science, Research Triangle Park, Durham, NC ). Transformation was mediated by Agrobacterium tumefaciens strain EHA105. Transformed cultures were selected in the presence of 3.0 mg l^?1 phosphinothricin in liquid maintenance medium for 3–4 mo. Liquid maintenance medium consisted of modified MS medium containing (per liter) 12 mg NH₄NO₃ and 30.3 mg KNO₃ and supplemented with 0.1 mg l^?1 thiamine HCl, 100 mg l^?1 myo-inositol, 30 g l^?1 sucrose, 3.0 mg l^?1 phosphinothricin, and 0.41 μM picloram. Somatic embryo development from transformed cultures was initiated on MS medium supplemented with 45 g l^?1 sucrose, 4 mg l^?1 thiamine HCl, 100 mg l^?1 myo-inositol, 10% (v/v) filter-sterilized coconut water, 3.0 mg l^?1 phosphinothricin, and 6.0 g l^?1 gellan gum. Limited plant recovery occurred from somatic embryos on semi-solid MS medium supplemented with 3.0 mg l^?1 phosphinothricin, 4.44 μM 6-benzylaminopurine (BA), and 2.89 μM GA₃; transformed shoots were micrografted on in vitro-grown seedling rootstocks. Approximately 1 yr after acclimatization in the greenhouse, transformed shoots were air-layered to recover transformed roots. Genetic transformation of embryogenic cultures, somatic embryos, and regenerated plants was confirmed by polymerase chain reaction (PCR), Southern blot hybridization, the XGLUC reaction for uidA, and application of the herbicide Finale® to regenerated plants.     

Oxygen transfer as a tool for fine-tuning recombinant protein production by <Emphasis Type="Italic">Pichia pastoris</Emphasis> under glyceraldehyde-3-phosphate dehydrogenase promoter

Effects of oxygen transfer on recombinant protein production by Pichia pastoris under glyceraldehyde-3-phosphate dehydrogenase promoter were investigated. Recombinant glucose isomerase was chosen as the model protein. Two groups of oxygen transfer strategies were applied, one of which was based on constant oxygen transfer rate where aeration rate was Q _O/V = 3 and 10 vvm, and agitation rate was N = 900 min^?1; while the other one was based on constant dissolved oxygen concentrations, C _DO = 5, 10, 15, 20 and 40 % in the fermentation broth, by using predetermined exponential glucose feeding with μ _o = 0.15 h^?1. The highest cell concentration was obtained as 44 g L^?1 at t = 9 h of the glucose fed-batch phase at C _DO = 20 % operation while the highest volumetric and specific enzyme activities were obtained as 4440 U L^?1 and 126 U g^?1 cell, respectively at C _DO = 15 % operation. Investigation of specific enzyme activities revealed that keeping C _DO at 15 % was more advantageous with an expense of relatively higher by-product formation and lower specific cell growth rate. For this strategy, the highest oxygen transfer coefficient and oxygen uptake rate were K _L a = 0.045 s^?1 and OUR = 8.91 mmol m^?3 s^?1, respectively.     

Effect of culture container and carbohydrate content on in vitro slow growth storage of the cherry rootstock ‘Gisela<Superscript>®</Superscript>5’

The present study investigated the effects of gas-tight and gas-permeable culture containers and different sucrose concentrations, as well as sucrose and mannitol combinations on the development of an effective in vitro slow growth storage protocol (at 4 °C, in darkness) for ‘Gisela^®5’ shoot cultures. ‘Gisela^®5’ is the most widely used cherry rootstock in Europe. This dwarf triploid hybrid has many advantages over the conventional cherry rootstocks. Optimizations for the cold storage of ‘Gisela^®5’ in vitro shoot cultures included use of storage medium supplemented with 10, 20, 30, 45, and 60 g L^?1 sucrose and sucrose (15, 30 g L^?1) and mannitol (15 g L^?1) combinations, contained in gas-tight glass jars and gas-permeable ‘Star Pac?’ bags. Cold storage was prolonged to 12 months, during which in every 3 months, cultures were evaluated. Possibility of 16 month-cold storage in gas-tight glass jars was also explored, during which gas chromatographic analysis was performed for the detection of CO₂ and ethylene accumulation for the first 5 months of cold storage. Our results showed that both the 12- and 16-month conservations were possible, especially when 45 or 60 g L^?1 sucrose was supplemented to storage medium, contained in glass jars. Mannitol inclusion to the storage medium was also effective to reduce the metabolic activity of the shoot cultures during storage; however, it did not have a significant positive influence on shoot quality in post-conservation.     

The paths of Andrew A. Benson: a radio-autobiography

Among marine phytoplankton groups, diatoms span the widest range of cell size, with resulting effects upon their nitrogen uptake, photosynthesis and growth responses to light. We grew two strains of marine centric diatoms differing by ~4 orders of magnitude in cell biovolume in high (enriched artificial seawater with ~500 µmol L^?1 µmol L^?1 NO₃ ^?) and lower-nitrogen (enriched artificial seawater with <10 µmol L^?1 NO₃ ^?) media, across a range of growth light levels. Nitrogen and total protein per cell decreased with increasing growth light in both species when grown under the lower-nitrogen media. Cells growing under lower-nitrogen media increased their cellular allocation to RUBISCO and their rate of electron transport away from PSII, for the smaller diatom under low growth light and for the larger diatom across the range of growth lights. The smaller coastal diatom Thalassiosira pseudonana is able to exploit high nitrogen in growth media by up-regulating growth rate, but the same high-nitrogen growth media inhibits growth of the larger diatom species.     

Impact of microcarrier coverage on using permittivity for on-line monitoring high adherent Vero cell densities in perfusion bioreactors

The paper presents the interest of on-line permittivity monitoring to estimate the density of Vero cells growing on microcarriers (MCs), even when high cell densities were reached in perfusion bioreactors (4.5 × 10⁶ cells ml⁻¹). Cultures were performed with various MCs concentrations in a reactor equipped with a settling tube. A linear correlation between on-line permittivity and off-line volumetric cell concentration was observed provided that MCs are not fully covered by cells. High permittivities such as 250 pF cm⁻¹ could be measured without signal saturation of the Fogale Biomass system^®. The correlation was no longer linear when cell density per carrier exceeded 100% cell confluency corresponding to 150 cells MC⁻¹ (0.15 × 10⁶ cells cm⁻²). This behaviour was attributed to the decrease of cell volume when cells saturated MCs surface. It mainly happened when low MCs concentration and continuous medium renewing were used. Therefore, permittivity sensor can be considered as a reliable tool to monitor on-line adherent cell densities not exceeding total cell confluency. Moreover, it could be useful to detect when cell confluency occurs.     

Salvia suspension cultures as production systems for oleanolic and ursolic acid

Oleanolic acid (OA) and ursolic acid (UA) are triterpenic acids with diverse biological activities that are of interest to the pharmaceutical industry. To investigate the scope for producing these compound using cell suspension cultures of Salvia species, calli from Salvia officinalis, S. virgata and S. fruticosa were induced using several plant growth regulator combinations. Eleven lines were selected for suspension induction from a pool of calli. Six suspension cultures were established successfully and cultivated in the respiration activity monitoring system (RAMOS^®) to obtain online data on their growth kinetics and to establish appropriate sampling schedules for the determination of their OA and UA production. Based on their observed growth behaviour, OA and UA contents, and aggregation properties, one suspension culture from each studied Salvia species was selected for further optimisation. The µ_max values for these suspension cultures ranged from 0.20 to 0.37 day^?1, their OA and UA contents were greater than 1.3 and 1.2 mg g^?1, respectively, and they afforded maximum volumetric yields of 21.0 mg l^?1 for OA and 32.8 mg l^?1 for UA. These results will be useful in the development of a refined Salvia suspension-based process for OA and UA production.     

A dual near-infrared and dielectric spectroscopies strategy to monitor populations of Chinese hamster ovary cells in bioreactor

Objective

to develop a new strategy combining near-infrared (NIR) and dielectric spectroscopies for real-time monitoring and in-depth characterizing populations of Chinese hamster ovary cells throughout cultures performed in bioreactors.

Results

Spectral data processing was based on off-line analyses of the cells, including trypan blue exclusion method, and lactate dehydrogenase activity (LDH). Viable cell density showed a linear correlation with permittivity up to 6 × 10⁶ cells ml^?1, while a logarithmic correlation was found between non-lysed dead cell density and conductivity up to 10⁷ cells ml^?1. Additionally, partial least square technique was used to develop a calibration model of the supernatant LDH activity based on online NIR spectra with a RMSEC of 55 U l^?1. Considering the LDH content of viable cells measured to be 110 U per 10⁹ cells, the lysed dead cell density could be then estimated. These calibration models provided real-time prediction accuracy (R² ≥ 0.95) for the three types of cell populations.

Conclusion

The high potential of a dual spectroscopy strategy to enhance the online bioprocesses characterization is demonstrated since it allows the simultaneous determination of viable, dead and lysed cell populations in real time.

     

Establishment and characterization of Stevia rebaudiana (Bertoni) cell suspension culture: an in vitro approach for production of stevioside

A protocol has been standardized for establishment and characterization of cell suspension cultures of Stevia rebaudiana in shake flasks, as a strategy to obtain an in vitro stevioside producing cell line. The effect of growth regulators, inoculum density and various concentrations of macro salts have been analyzed, to optimize the biomass growth. Dynamics of stevioside production has been investigated with culture growth in liquid suspensions. The callus used for this purpose was obtained from leaves of 15-day-old in vitro propagated plantlets, on MS medium fortified with benzyl aminopurine (8.9 μM) and naphthalene acetic acid (10.7 μM). The optimal conditions for biomass growth in suspension cultures were found to be 10 g l^?1 of inoculum density on fresh weight basis in full strength MS liquid basal medium of initial pH 5.8, augmented with 2,4-dichlorophenoxy acetic acid (0.27 μM), benzyl aminopurine (0.27 μM) and ascorbic acid (0.06 μM), 1.0× NH₄NO₃ (24.7 mM), 3.0× KNO₃ (56.4 mM), 3.0× MgSO₄ (4.5 mM) and 3.0× KH₂PO₄ (3.75 mM), in 150 ml Erlenmeyer flask with 50 ml media and incubated in dark at 110 rpm. The growth kinetics of the cell suspension culture has shown a maximum specific cell growth rate of 3.26 day^?1, doubling time of 26.35 h and cell viability of 75 %, respectively. Stevioside content in cell suspension was high during exponential growth phase and decreased subsequently at the stationary phase. The results of present study are useful to scale-up process and augment the S. rebaudiana biological research.     

Specific light uptake rates can enhance astaxanthin productivity in <Emphasis Type="Italic">Haematococcus lacustris</Emphasis>

Lumostatic operation was applied for efficient astaxanthin production in autotrophic Haematococcus lacustris cultures using 0.4-L bubble column photobioreactors. The lumostatic operation in this study was performed with three different specific light uptake rates (q _e) based on cell concentration, cell projection area, and fresh weight as one-, two- and three-dimensional characteristics values, respectively. The q _e value from the cell concentration (q _e1D) obtained was 13.5 × 10^?8 μE cell^?1 s^?1, and the maximum astaxanthin concentration was increased to 150 % compared to that of a control with constant light intensity. The other optimum q _e values by cell projection area (q _e2D) and fresh weight (q _e3D) were determined to be 195 μE m^?2 s^?1 and 10.5 μE g^?1 s^?1 for astaxanthin production, respectively. The maximum astaxanthin production from the lumostatic cultures using the parameters controlled by cell projection area (2D) and fresh weight (3D) also increased by 36 and 22 % over that of the controls, respectively. When comparing the optimal q _e values among the three different types, the lumostatic cultures using q _e based on fresh weight showed the highest astaxanthin productivity (22.8 mg L^?1 day^?1), which was a higher level than previously reported. The lumostatic operations reported here demonstrated that more efficient and effective astaxanthin production was obtained by H. lacustris than providing a constant light intensity, regardless of which parameter is used to calculate the specific light uptake rate.     

Abscisic acid,gibberellins and brassinosteroids in Kelpak®, a commercial seaweed extract made from Ecklonia maxima

The seaweed extract Kelpak^® made from the kelp Ecklonia maxima is registered as a biostimulant for use in agriculture. It elicits many beneficial responses including improved root and shoot growth, higher yields and greater resistance to abiotic and biotic stresses. Previously, cytokinins, auxins and polyamines were identified in Kelpak^®. The aim of the present study was to quantify other groups of plant growth regulators (PGRs)—abscisic acid (ABA), gibberellins (GAs) and brassinosteroids—that may be present in E. maxima and Kelpak^®. Kelpak^® samples harvested between 2008 and 2010 and stored for up to 26 months were analysed using ultra performance liquid chromatography tandem mass spectrometry. ABA levels were below the limits of detection in E. maxima but were detected in low concentrations in Kelpak^®, ranging from 0.31 to 20.70 pg mL^?1 Kelpak^®. Eighteen GAs were found in E. maxima and Kelpak^® with concentrations from 187.54 to 565.96 pg mL^?1 Kelpak^®. The biologically active GAs (GA₁, GA₃, GA₄, GA₅, GA₆ and GA₇) comprised less than 3 % in Kelpak^®. Although GA₁₃ (a final product in the metabolic pathway) was present in low concentrations in E. maxima, very high concentrations were present in Kelpak^®. The brassinosteroids brassinolide (BL) and castasterone (CS) were identified in E. maxima and Kelpak^®. Concentrations varied with harvest and storage time, ranging from 384.72 to 793.23 pg BL mL^?1 Kelpak^® and 62.84 to 567.51 pg CS mL^?1 Kelpak^®. It is likely that this cocktail of natural PGRs present in Kelpak^® may act individually or in concert and thus contribute to the numerous favourable physiological responses elicited by Kelpak^® application to plants.     

In vitro plantlet regeneration and assessment of alkaloid contents from callus cultures of Ephedra foliata (Unth phog), a source of anti-asthmatic drugs

Ephedra foliata, (Gymnosperm) is a pharmaceutically important plant known for the last 5,000 years and has a number of medicinal properties. We describe here for the first time, a method for plant regeneration from callus established from axillary buds as explant, with the aim of optimizing alkaloids production in vitro. The tissue cultures initiated are being maintained for the last 3 years on Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium containing 0.5 mg l^?1 each of 2, 4-D and Kin. Maintained callus cultures exhibited regeneration potential and maximum number (23.5 ± 0.44 shoots per culture vessel) of shoots with an average height (4.94 ± 0.23 cm) was achieved on MS medium containing combination of 0.25 mg l^?1 each of Kin, BA and 0.1 mg l^?1 of NAA. About 84.9 % regenerated shoots were rooted under ex vitro conditions on Soilrite^®, if their base was treated with 500 mg l^?1 of IBA for 5 min. The rooted plantlets were successfully acclimatized under greenhouse conditions with ≈80 % survival rate. We analyzed alkaloid contents of tissue culture raised plants/callus as affected by the different concentrations and combination of two additives, i.e., l-phenylalanine and IBA. The alkaloid production was higher in the in vitro grown cultures than field-grown plants. Highest alkaloid content was recorded in callus culture on M₅ medium having 0.5 mg l^?1 each of 2, 4-D and Kin, 100 mg l^?1 l-phenylalanine and 5 mg l^?1 IBA. The present protocol may be applicable for the large-scale cultivation of E. foliata and selection of cell line having higher secondary metabolite contents of this pharmaceutically important threatened plant species.     

Variability and Vulnerability of Coastal ‘Blue Carbon’ Stocks: A Case Study from Southeast Australia

‘Blue carbon’ ecosystems—seagrasses, tidal marshes, and mangroves—serve as dense carbon sinks important for reducing atmospheric greenhouse gas concentrations, yet only recently have stock estimates emerged. We sampled 96 blue carbon ecosystems across the Victorian coastline (southeast Australia) to quantify total sediment stocks, variability across spatial scales, and estimate emissions associated with historical ecosystem loss. Mean sediment organic carbon (C_org) stock (±SE) to a depth of 30 cm was not significantly different between tidal marshes (87.1 ± 4.90 Mg C_org ha^?1) and mangroves (65.6 ± 4.17 Mg C_org ha^?1), but was significantly lower in seagrasses (24.3 ± 1.82 Mg C_org ha^?1). Location (defined as an individual meadow, marsh, or forest) had a stronger relationship with C_org stock than catchment region, suggesting local-scale conditions drive variability of stocks more than regional-scale processes. We estimate over 2.90 million ± 199,000 Mg C_org in the top 30 cm of blue carbon sediments in Victoria (53% in tidal marshes, 36% in seagrasses, and 11% in mangroves) and sequestration rates of 22,700 ± 5510 Mg C_org year^?1 (valued at over $AUD1 million ± 245,000 year^?1 based on the average price of $AUD12.14 Mg CO₂ eq^?1 at Australian Emissions Reduction Fund auctions). We estimate ecosystem loss since European settlement may equate to emissions as high as 4.83 million ± 358,000 Mg CO₂ equivalents (assuming 90% remineralization of stocks), 98% of which was associated with tidal marsh loss, and what would have been sequestering 9360 ± 2500 Mg C_org year^?1. This study is among the first to present a comprehensive comparison of sediment stocks across and within coastal blue carbon ecosystems. We estimate substantial and valuable carbon stocks associated with these ecosystems that have suffered considerable losses in the past and need protection into the future to maintain their role as carbon sinks.     

Effect of elicitation and precursor feeding on accumulation of 20-hydroxyecdysone in <Emphasis Type="Italic">Achyranthes aspera</Emphasis> Linn. cell suspension cultures

20-Hydroxyecdysone is one of the most common ecdysteroids in plants with potential therapeutic applications. In this study, cell suspension cultures of Achyranthes aspera were raised in shake flasks to investigate the production of 20-hydroxyecdysone. The quantification and characterization of 20-hydroxyecdysone in the cultures were done by High performance liquid chromatography (HPLC) and Liquid Chromatography-quadrupole time-of- flight mass spectrometry (LC-Q-TOF) analyses. For raising the suspension, calli initiated from in vitro grown leaf explants were cultured in liquid Murashige and Skoog (MS) medium augmented with combinations of 2, 4-dichlorophenoxyacetic acid (1 mg L^?1) and α-naphthaleneacetic acid (1 mg L^?1). Maximum growth index of the cell suspension was 9.9, which was achieved during 20th day of culture (final phase of exponential growth). At this stage, the biomass accumulated was 1.09 ± 0.09 g dry weight (DW) and the 20-hydroxyecdysone concentration was 0.24 mg g^?1 DW. Eliciting the cultures with 0.6 mM Methyl jasmonate for 6 days; enhanced the production of 20-hydroxyecdysone production to 0.35 mg g^?1 DW. By augmenting the cultures with the precursors namely cholesterol (10 mg L^?1) and 7-dehydrocholesterol (10 mg L^?1), production of 20-hydroxyecdysone was boosted to 0.31 mg g^?1 DW and 0.28 mg g^?1 DW respectively.     

Kinetic Study and Mathematical Modeling of Chromium(VI) Reduction and Microorganism Growth Under Mixed Culture

The kinetics of chromium(VI) reduction by Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E. coli) was studied under both pure and mixed cultures. Initially, the study of kinetics was performed in pure culture. It was observed that the growth of the two bacteria was both inhibited in the presence of chromium(VI). The maximum specific growth rate (μ _m ) of P. aeruginosa decreased from 2.3942 h^?1 (without Cr(VI)) to 1.8551 h^?1 (with Cr(VI)). Under the mixed culture, the growth of E. coli was inhibited by P. aeruginosa. The maximum specific growth rate (μ _m ) of E. coli decreased form 0.871 h^?1 (in pure culture) to 0.153 h^?1 (in mixed culture). When the concentration of each bacterium was 4.5 × 10⁸ cells ml^?1, the half-velocity reduction rate constant (K _C) and the maximum specific reduction rate constant (v _max) of chromium(VI) were 80.05 mg chromium(VI) l^?1 and 3.674 mg chromium(VI) cells^?1 h^?1, respectively. The results showed that the simulation appeared in good agreement with the experimental data, supporting the series of mathematical models represented the bacteria growth and chromium(VI) reduction in both pure and mixed cultures usefully.     

On-line monitoring of physiological parameters of insect cell cultures during the growth and infection process

On-line monitoring of insect cell cultures used for the production of recombinant proteins with the baculovirus expression vector system (BEVS) provides valuable tools for the optimization, operation, and control of the production process. The relative permittivity (epsilon') and CO(2) evolution rates (CER) were measured on-line using the biomass monitor and the infrared CO(2) analyzer, respectively. The growth and infection phases of two different cell lines, Spodoptera frugiperda (Sf-9) and Trichoplusia ni(High-5), were monitored using the above measurements. These in turn were correlated to the progress of the culture by using the off-line measurements of protein produced, virus titer, and biovolume, which is the product of viable cell density and mean cell volume. The epsilon', CER, and the biovolume profiles were closely matched during the growth phase of cells when grown in a batch or fed batch culture. The relationship became more complex when the cultures were either in stationary phase or in the postinfection phase. The epsilon' profile was found to be a good indicator of the process of synchronous baculoviral infection, showing a plateau between 18 and 24 h postinfection (hpi), the period during which budded virus is produced, and a peak at approximately 48 hpi correlated to the onset of accelerated cell lysis. The CER profile continues to increase after the growth period with a peak around the 24 hpi period, after which there is a decline in the profile corresponding to release of virus as seen from virus titer determinations. This was examined for Sf-9 cultures under conditions of cell densities from 3 to 50 x 10(6) cells/mL and MOI values ranging from 0.001 to 1000. The profiles were found to be similar also in the case of the High-5 cells. Thus both measurements give reliable information regarding the physiological status of the cells as seen from their correlation to virus and protein production. A further combination of these with the off-line measured parameters such as the biovolume and metabolite concentrations can give a more detailed understanding of the process and help in the better design and automation of these processes.     

Quantitative modeling of viable cell density,cell size,intracellular conductivity,and membrane capacitance in batch and fed‐batch CHO processes using dielectric spectroscopy

Dielectric spectroscopy was used to analyze typical batch and fed‐batch CHO cell culture processes. Three methods of analysis (linear modeling, Cole–Cole modeling, and partial least squares regression), were used to correlate the spectroscopic data with routine biomass measurements [viable packed cell volume, viable cell concentration (VCC), cell size, and oxygen uptake rate (OUR)]. All three models predicted offline biomass measurements accurately during the growth phase of the cultures. However, during the stationary and decline phases of the cultures, the models decreased in accuracy to varying degrees. Offline cell radius measurements were unsuccessfully used to correct for the deviations from the linear model, indicating that physiological changes affecting permittivity were occurring. The β‐dispersion was analyzed using the Cole–Cole distribution parameters Δε (magnitude of the permittivity drop), f_c (critical frequency), and α (Cole–Cole parameter). Furthermore, the dielectric parameters static internal conductivity (σ_i) and membrane capacitance per area (C_m) were calculated for the cultures. Finally, the relationship between permittivity, OUR, and VCC was examined, demonstrating how the definition of viability is critical when analyzing biomass online. The results indicate that the common assumptions of constant size and dielectric properties used in dielectric analysis are not always valid during later phases of cell culture processes. The findings also demonstrate that dielectric spectroscopy, while not a substitute for VCC, is a complementary measurement of viable biomass, providing useful auxiliary information about the physiological state of a culture. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010