Inline characterization of cell concentration and cell volume in agitated bioreactors using in situ microscopy: application to volume variation induced by osmotic stress

A new in situ microscope (ISM) was developed and tested to perform in-line monitoring of average cell volume and cell concentration in agitated cultures subjected to osmotic stress. The ISM is directly immersed into the agitated broth in a bioreactor and generates still images of cells by using pulsed luminescent diode illumination and a virtual probe volume defined by depth of focus. This technique allows the acquisition of microscopic still images without mechanical sampling techniques. The front end of the sensor fits into a standard 25-mm port and it can be steam sterilized together with the bioreactor. The automatic image evaluation generates signals of the cell concentration and the average cell volume with a time resolution of a few minutes per data point (if a 200 MHz PC is used). Without the need for evaluation, the images can be acquired and stored at a rate of one image per 0.6 s. Hansenula anomala was cultivated as batch fermentation and monitored inline with the ISM. The ISM signal of the cell concentration agreed well with referential growth curves that were obtained from counting with a hemocytometer. The ISM signal of the average cell volume shows a gradual volume reduction as a result of the aging of the culture, and it monitors an abrupt and strong cell contraction if osmotic shocks are generated in the bioreactor. Systematic in vitro studies of osmotic shocks were performed by applying the ISM to agitated culture samples of H. anomala. The volume signal of H. anomala during osmotic shocks showed a very fast cell contraction within less than a second. Within half an hour after the shocks, no signal drifts were observed, which would indicate volume restoration. These findings suggest that the ISM volume signal can be used as an inline indicator of osmotic stress in cell cultures.     

The viability of animal cell cultures in bioreactors: Can it be estimated online by using in situ microscopy?

This work aims at checking the possibility of estimating mammalian cell viability from images provided by an in situ microscope (ISM). It was found that images of cells in bioreactors obtained by a high-resolution ISM contain a certain part of cells which exhibits strong morphological similarity with images of dying cells or dead cells obtained by epifluorescence. Cell images of this fraction have less homogeneous texture and less smooth borders as compared to regular cells. Modifications of intracellular organelles and irregularities of the plasma membrane can explain such visual features. Therefore, by only using the texture effect, a criterion is proposed in order to distinguish living cells from the other ones. It is based on the variability of the inside part of the image of the cell. A quantitative estimate of viability (e_v) is then calculated from a set of images obtained for each sample. The viabilities obtained from the conventional flow cytometry method are inside the 5% confidence interval of these estimations.     

In situ microscopy for on-line characterization of cell-populations in bioreactors, including cell-concentration measurements by depth from focus

A new technique is presented which allows the use of a front-end sensor head for in situ and on-line characterization of cell concentration and cell size during fermentation. An epifluorescence microscope is mounted in a port of a bioreactor viewing directly into the agitated broth. Still images from cells are generated using pulsed illumination. They are directly visualized on a monitor and used for automatic image analysis. The cell concentration and morphological information are determined by counting and evaluating the cell images with respect to their depth from focus characteristic. An in situ microscope was successfully tested during yeast fermentations and yielded results which correlated well with results from a hemocytometer. (c) 1995 John Wiley & Sons, Inc.     

Toward a control of lignin and manganese peroxidases hypersecretion by Phanerochaete chrysosporium in agitated vessels: Evidence of the superiority of pneumatic bioreactors on mechanically agitated bioreactors

Phanerochaete chrysosporium and cultivated both mechanically agitated and pneumatic bioreactors. In the pneumatic devices, the yields of lignin and manganese peroxidases as well as extracellular protein, were considerably increased as compared with mechanically agitated bioreactors. Lignin peroxidase and manganese peroxidase activities as high as 4500 U . L(-1) and 1812 U . L(-1) respectively, were produced in an airlift bioreactor. By using enzyme markers, the secretion pathway and the respiration were shown to be dramatically activated in pneumatic bioreactors. The general metabolism of the fungus, when cultivated in the conventional fermentors, is oriented toward the synthesis of biomass at the expense of the synthesis of peroxidases. The use of pneumatic devices for the production of extracellular peroxidases by P. chrysosporium, avoids shear effects due to turbine agitator in the conventional fermentors, and provides a good example for the production of shear-sensitive metabolites. (c) 1993 John Wiley & Sons, Inc.     

Determination of intracellular osmotic volume and sodium concentration in dunaliella

A new method to measure intracellular volume in Dunaliella was developed, where lithium ions are used as monitors of the extracellular volume. Li⁺ is shown to be impenetrable to the intracellular volume, insignificantly absorbed to the algae, and is rapidly and evenly distributed within the extracellular volume. The method is suggested to be free of several limitations and consistent errors present in several previously employed techniques.

Using the new technique it is shown that both Dunaliella salina and Dunaliella bardawil adjust to a constant cellular volume when grown in a medium containing salt concentrations ranging from 0.5 molar to 4 molar NaCl. That volume is 90 femtoliter per cell for D. salina and 600 femtoliter per cell for D. bardawil. Nonosmotic volume accounts for about 10% of the total cell volume.

The intracellular sodium concentration, as determined with the new technique, was under all experimental conditions tested below 100 millimolar. This was true both for cells grown on 0.5 to 4 molar NaCl, and during the osmoregulatory process. It is thus concluded that intracellular NaCl is a minor contributor to the overall intracellular osmotic pressure in Dunaliella.

     

The relationship between cell injury and osmotic volume reduction: II. Red cell lysis correlates with cell volume rather than intracellular salt concentration

It is possible to simulate freezing by suspending cells in progressively hyperosmotic solutions. It is not generally possible, however, to discriminate between cell volume reduction and solute concentration as the cause of injury since, in a normal cell behaving as an osmometer, volume is an obligate function of solution osmolality. The paper describes experiments in which osmolality and volume were disassociated by loading red cells with additional KCl by making them slowly permeable to potassium through treatment with valinomycin. It is shown that cell hemolysis is associated with the reduction of cell volume beyond some minimum volume regardless of the concentration of intracellular or extracellular electrolyte. Similarly, it is shown that hemolysis from thermal shock is related to a decrease in cell volume rather than to an increase in solute concentration.     

Inhibition of photosynthetic processes in foliose lichens induced by temperature and osmotic stress

Negative effects of osmotically-induced dehydration of two foliose lichen species, Lasallia pustulata and Umbilicaria hirsuta, was studied at physiological (22 °C), low (5 °C) and freezing temperature (−10 °C), using chlorophyll (Chl) fluorescence. In both species, exposure to increasing sucrose concentrations led to a pronounced decrease in potential (F_V/F_M), and actual (Φ₂) quantum yields of photochemical processes in photosystem 2. L. pustulata was more sensitive to osmotic stress, because comparable osmotic dehydration inhibited F_V/F_M and Φ₂ more than in U. hirsuta. Critical concentration of sucrose that fully inhibited photochemical processes of photosynthesis was 2.5 M, which represented water potential (Ψ_w) of −18.8 MPa. Decrease in background Chl fluorescence (F₀) and increase in non-photochemical quenching (qN) revealed two phases of osmotic stress in lichens: phase I with no change (Ψ_w 0 to −6.6 MPa) and phase II (Ψ_w −11.3 to −18.8 MPa) typical by substantial change in Chl fluorescence parameters. Effects of thallus anatomy on species-specific response to osmotic dehydration is discussed and attributed to the results obtained by optical microscopy and Chl fluorescence imaging technique.     

Membrane fouling by cell-protein mixtures: in situ characterisation using multi-photon microscopy

Fouling of the membrane by cell and protein mixtures can result in severe flux declines, leading to the eventual need to clean or replace the membrane. In this study multi-photon microscopy, a fluorescence-based technique is used to 3-D image in situ the fouling of microfiltration membranes by suspensions containing combinations of washed yeast, bovine serum albumin (BSA) and ovalbumin. Appropriate fluorescent labelling allows the three foulant species to be clearly identified. Images correlate well with filtration data and clearly show the cake of yeast cells capturing protein aggregates. The proteins exhibited very different filtration behaviour. When filtering washed yeast together with ovalbumin and/or a 50:50 mixture by mass of BSA and ovalbumin, the ovalbumin fouling dominates the system. Capture of aggregates by the cake did not reduce fouling of the membrane by the protein and increased the resistance of the cake. For mixtures of BSA and washed yeast, the presence of a cake of yeast cells did reduce fouling of the membrane by the protein, however, the extra resistance due to the cake resulted in a flux lower than that when filtering BSA alone.     

Homogenisation and oxygen transfer rates in large agitated and sparged animal cell bioreactors: Some implications for growth and production

Because of concern for cell damage, very low agitation energy inputs have been used in industrial animal cell bioreactors, typical values being two orders of magnitude less than those found in bacterial fermentations. Aeration rates are also very small. As a result, such bioreactors might be both poorly mixed and also unable to provide the higher oxygen up-take rates demanded by more intensive operation. This paper reports experimental studies both of K _L a and of mixing (via pH measurements) in bioreactors up to 8 m³ at Wellcome and of scaled down models of such reactors at Birmingham. Alongside these physical measurements, sensitivity of certain cell lines to continuously controlled dO₂ has been studied and the oxygen up-take rates measured in representative growth conditions. An analysis of characteristic times and mixing theory, together with other recent work showing that more vigorous agitation and aeration can be used especially in the presence of Pluronic F-68, indicates ways of improving their performance. pH gradients offer a special challenge.     

Improvement of Panax notoginseng cell culture for production of ginseng saponin and polysaccharide by high density cultivation in pneumatically agitated bioreactors

A Panax notoginseng cell culture was successfully scaled up from shake flask to 1.0-L bubble column reactor and concentric-tube airlift reactor. High-density bioreactor batch cultivation was carried out using a modified MS medium. The maximum cell density in batch cultures reached 20.1, 21.0 and 24.1 g/L in the shake flask, bubble column and airlift reactors, respectively, and their corresponding biomass productivity was 950, 1140 and 1350 mg/(L x d) for each. The productivity of ginseng saponin was 70, 96 and 99 mg/(L x d) in the flask, bubble column and airlift reactors, respectively; and the polysaccharide productivity reached 104, 119 and 151 mg/(L x d) for each. Furthermore, a fed-batch cultivation strategy was developed on the basis of specific oxygen uptake rate (SOUR), i.e., sucrose feeding before a sharp decrease of SOUR, and the highest cell density of 29.7 g/L was successfully achieved in the airlift bioreactor on day 17 with a very high biomass productivity of 1520 mg/(L x d). The concentrations of ginseng saponin and polysaccharide reached about 2.1 and 3.0 g/L, respectively, and their productivity was 106 (saponin) and 158 mg/(L x d) (polysaccharide). This work successfully demonstrated the high-density bioreactor cultivation of P. notoginseng cells in pneumatically agitated bioreactors and the reproduction of the shake flask culture results in bioreactors. The cell density, biomass productivity, production titer and productivity of both ginseng saponin and polysaccharide obtained here were the highest that have been reported on a reactor scale for all the ginseng species.     

Proteomics characterization of cell model with renal fibrosis phenotype: osmotic stress as fibrosis triggering factor

Renal fibroblasts are thought to play a major role in the development of renal fibrosis (RF). The mechanisms leading to this renal alteration remain poorly understood. We performed differential proteomic analyses with two established fibroblast cell lines with RF phenotype to identify new molecular pathways associated with RF. Differential 2-DE combined with mass spectrometry analysis revealed the alteration of more than 30 proteins in fibrotic kidney fibroblasts (TK188) compared to normal kidney fibroblast (TK173). Among these proteins, markers of the endoplasmic reticulum (ER) stress- and the unfolded protein response (UPR) pathway (GRP78, GRP94, ERP57, ERP72, and CALR) and the oxidative stress pathway proteins (PRDX1, PRDX2, PRDX6, HSP70, HYOU1) were highly up-regulated in fibrotic cells. Activation of these stress pathways through long time exposition of TK173, to high NaCl or glucose concentrations resulted in TK188 like phenotype. Parallel to an increase in reactive oxygen species, the stressed cells showed significant alteration of fibrosis markers, ER-stress and oxidative stress proteins. Similar effects of osmotic stress could be also observed on renal proximal tubule cells. Our data suggest an important role of the ER-stress proteins in fibrosis and highlights the pro-fibrotic effect of osmotic stress through activation of oxidative stress and ER-stress pathways.     

Cloning and characterization of a putative 12-oxophytodienoic acid reductase cDNA induced by osmotic stress in roots of foxtail millet.

Foxtail millet is a gramineous crop with low water requirement. Cloning of osmotic responses-related genes from foxtail millet is a key step for understanding the mechanism of its tolerance to drought. Here we reported the cloning and characterization of a cDNA (SiOPR1) encoding a putative 12-oxophytodienoic acid reductase 1 from foxtail millet by using RACE methods. Sequence analysis showed that SiOPR1 encoded a polypeptide of 374 amino acids with a predicted molecular mass of 41.9 kDa and pI of 5.14. Multiple alignment result showed that OPR1 protein was very conservative among gramineous crops. RNA gel blot analysis results indicated that SiOPR1 was up-regulated by osmotic stress, and its expression was limited in the roots of foxtail millet. However, SiOPR1 expression was not affected by ABA, NaCl and MeJA treatments both in roots and shoots. Therefore, it is suggested that SiOPR1 gene play an important role in response to drought stress.     

Yeast cell permeabilization by osmotic shock allows determination of enzymatic activities in situ

Yeast cells were permeabilized by incubation in 0.8 M sorbitol followed by suspension in dilute buffer. A preincubation with 2-mercaptoethanol was also included for optimal permeabilization. More than 90% of the treated cells were stainable with methylene blue. Determinations of cell wall-synthesizing enzymes (beta(1 --> 3)glucan and chitin synthases) and cytosolic enzymes in permeabilized cells yielded similar or higher activities than those in cell extracts. With chitin synthase III, the activity obtained with cells was 4- to 6-fold higher than in membrane preparations. Little protein leaks from the cells during permeabilization; yet the cells appear to be readily permeable to substrates and even proteins. Thus, these preparations may be of wide use for the study of enzymes and of biological processes in situ.     

Influence of nitric oxide in protection of tomato seedling against oxidative stress induced by osmotic stress

Osmotic stress associated with drought and salinity is a serious problem that inhibits the growth of plants mainly due to disturbance of the balance between production of ROS and antioxidant defense and causes oxidative stress. In this research, sodium nitroprusside (SNP) was used as NO donor in control and drought-stressed plants, and the role of NO in reduction of oxidative damages were investigated. In this study, we observed that SNP pretreatment prevented drought-induced decrease in RWC and membrane stability index, increase in lipid peroxidation and lipoxygenase activity and increase in hydrogen peroxide content. However, pretreatment of plants with SNP and phenyl 4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (a NO scavenger) reversed the protective effects of SNP suggesting that protective effect by SNP is attributable to NO release. In addition, the relationship between these defense mechanisms and activity of antioxidant enzymes were checked. Results showed that in drought-stressed plants ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and catalase activities were elevated over the controls, while GR decreased under drought condition. Activity of GPX was inhibited under SNP pretreatment in drought-stressed plants specially, while the activity of APX and GR increased under SNP pretreatment and it seems that under this condition APX had a key role of detoxification of ROS in tomato plants. This result corresponded well with ASA and total acid-soluble thiols content. Therefore, reduction of drought-induced oxidative damages by NO in tomato leaves is most likely mediated through either NO ability to scavenge active oxygen species or stimulation of antioxidant enzyme such as APX.     

Osmotic adjustment by intact isolated chloroplasts in response to osmotic stress and its effect on photosynthesis and chloroplast volume

Spinach leaf chloroplasts isolated in isotonic media (330 millimolar sorbitol, −1.0 megapascals osmotic potential) had optimum rates of photosynthesis when assayed at −1.0 megapascals. When chloroplasts were isolated in hypertonic media (720 millimolar sorbitol, −2.0 megapascals osmotic potential) the optimum osmotic potential for photosynthesis was shifted to −1.8 megapascals and the chloroplasts had higher rates of CO₂-dependent O₂ evolution than chloroplasts isolated in 330 millimolar sorbitol when both were assayed at high solute concentrations.

Transfer of chloroplasts isolated in 330 millimolar sorbitol to 720 millimolar sorbitol resulted in decreased chloroplast volume but this shrinkage was only transient and the chloroplasts subsequently swelled so that within 2 to 3 minutes at 20°C the chloroplast volume had returned to near the original value. Thus, actual steady state chloroplast volume was not decreased in hypertonic media. In isotonic media, there was a slow but significant uptake of sorbitol by chloroplasts (10 to 20 micromoles per milligram chlorophyll per hour at 20°C). Transfer of chloroplasts from 330 millimolar sorbitol to 720 millimolar sorbitol resulted in rapid uptake of sorbitol (up to 280 micromoles per milligram chlorophyll per hour at 20°C) and after 5 minutes the concentration of sorbitol inside the chloroplasts exceeded 500 millimolar. This uptake of sorbitol resulted in a significant underestimation of chloroplast volume unless [¹⁴C]sorbitol was added just prior to centrifuging the chloroplasts through silicone oil. Sudden exposure to osmotic stress apparently induced a transient change in the permeability of the chloroplast envelope since addition of [¹⁴C]sorbitol 3 minutes after transfer to hypertonic media (when chloroplast volume had returned to normal) did not result in rapid uptake of labeled sorbitol.

It is concluded that chloroplasts can osmotically adjust in vitro by uptake of solutes which do not normally penetrate the chloroplast envelope, resulting in a restoration of normal chloroplast volume and partially preventing the inhibition of photosynthesis by high solute concentrations. The results indicate the importance of matching the osmotic potential of isolation media to that of the tissue, particularly in studies of stress physiology.

     

Quantitative NMR microscopy of osmotic stress responses in maize and pearl millet.

The effect of osmotic stress (-0.35 MPa) on the cell water balance and apical growth was studied non-invasively for maize (Zea mays L., cv. LG 11) and pearl millet (Pennisetum americanum L., cv. MH 179) by (1)H NMR microscopy in combination with water uptake measurements. Single parameter images of the water content and the transverse relaxation time (T(2)) were used to discriminate between the different tissues and to follow the water status of the apical region during osmotic stress. The T(2) values of non-stressed stem tissue turned out to be correlated to the cell dimensions as determined by optical microscopy. Growth was found to be strongly inhibited by mild stress in both species, whereas the water uptake was far less affected. During the experiment hardly any changes in water content or T(2) in the stem region of maize were observed. In contrast, the apical tissue of pearl millet showed a decrease in T(2) within 48 h of stress. This decrease in T(2) is interpreted as an increase in the membrane permeability for water.     

Nonideality of volume flows and phase transitions of F-actin solutions in response to osmotic stress.

Ovalbumin and G-actin solutions decreased their volume in a concentration-dependent manner in response to an osmotic stress, arising from an osmotic pressure gradient of 5-20 cm H2O at 25 degrees C, at protein concentrations as high as 20 mg/ml. In contrast, solutions of F-actin exhibited a concentration-dependent decrease in their rate of volume change in response to the osmotic stress. Shortening of F-actin by gelsolin did not affect this decrease, suggesting that the elastic response of the filaments underlies the osmotically nonideal behavior. However, above a critical actin concentration of approximately 7 mg/ml, no volume change occurred in response to osmotic gradients as high as 20 cm H2O. The concentration at which this critical phenomenon occurred and its abolition by shortening of F-actin by gelsolin suggest that a transition of diffusible rods to a glassy state is the cause of this critical phenomenon. Above the critical concentration, an increase in the osmotic pressure applied to an F-actin solution to greater than 20 cm H2O produced a transient increase in flow rate to that expected for a solution containing no polymer. This finding may represent a transition from an isotropic glassy state to an anisotropic and heterogeneous one wherein regions of pure solvent coexist with domains of pure polymer.