pH-controlled cell release and biomass distribution of alginate-immobilized Lactococcus lactis subsp. lactis.

AIMS: To investigate the growth and release of Lactococcus lactis subsp. lactis in gel beads and to affect rates of cell release by changing the growth conditions. METHODS AND RESULTS: The rate of release and the distribution of immobilized L. lactis subsp. lactis in alginate beads were studied in continuous fermentations for 48 h. A change in operating pH from 6.5 to 9.25 initially reduced the ratio of the rates of cell release to lactate production by almost a factor of 105. Compared with fermentations at pH 6.5, growth at pH 9.25 also increased the final internal bead biomass concentration by a factor of 5 and increased the final rate of lactate production by 25%. After 48 h, the ratio of the rates of cell release to lactate production was still 10 times lower than in fermentations at pH 6.5. CONCLUSIONS: A change in the operating pH from 6.5 to 9.25 reduced rates of cell release throughout 48 h of fermentation and increased the final rates of lactate production and internal bead biomass concentration. SIGNIFICANCE AND IMPACT OF THE STUDY: These data illustrate that diffusional limitations and corresponding pH gradients can be exploited in affecting the distribution of immobilized growing cells and their concomitant release.

Cell release from alginate immobilized Lactococcus lactis ssp. lactis in chitosan and alginate coated beads.

The effects of chitosan and alginate coatings of alginate beads with entrapped Lactococcus lactis ssp. lactis were studied in batch and continuous fermentations. Chitosan coating reduced the final concentrations of free cells, the initial release of free cells and the rate of lactate production in milk fermented batch-wise to a final pH of 4.7 in five consecutive batch fermentations. An alternative experimental system based on continuous fermentation with controlled pH and a high dilution rate was developed to better study the phenomenon of cell release. To estimate the effects of different bead coatings on cell release, alginate beads were coated with chitosan or alginate, or sequentially with chitosan/alginate or chitosan/alginate/chitosan. Chitosan coating alone seemed to reduce the rate of cell release only in the early stages of the fermentation, while sequential coatings with chitosan and alginate showed significant reduction throughout the whole test period. To examine whether the observed effects of bead coating could be explained only by a decrease in cell activity, the ratios between the rate of cell release and the rate of lactate production were examined during the fermentations for the different beads. This ratio showed qualitatively the same behavior as direct results of volumetric cell release.

Transport of osmoprotective compounds in hybridoma cells exposed to hyperosmotic stress.

Addition of osmoprotective compounds has a positive effect on growth and monoclonal antibody production in hyperosmotic hybridoma cell cultures. In order to better understand the processes involved in the osmoprotective response, uptake of the osmoprotective compounds glycine betaine, proline, sarcosine and glycine in mouse hybridoma cell line 6H11 during exposure to hyperosmotic stress was studied. Hyperosmotic stress (510 mOsmol/kg) was introduced through the addition of NaCl (100 mM) to the growth medium, and amino acid transport activity was measured immediately after transfer of the cells to the hyperosmotic medium. The osmoprotective capability of the four osmoprotectants tested was negatively affected if methylaminosobutyric acid (MeAiB), a specific substrate for amino acid transport system A, was simultaneously included in the hyperosmotic medium in equimolar amounts with one of the osmoprotective compounds. This was due to accumulation of MeAiB in the stressed cells, giving a significant reduction in the concentration of the osmoprotective compound inside the cells. Furthermore, addition of excess meAiB gave approx. 905 reduction in the initial rate of uptake of glycine betaine, while 40-50% reduction in the initial rate of uptake of proline, glycine and sarcosine. Similarly, addition of proline, glycine or sarcosine also gave a significant reduction in the initial rate of glycine betaine uptake. These results suggest that the four osmoprotective compounds share, at least in part, a common, MeAiB inhibitable carrier for transport into osmotically stressed hybridoma cells. This carrier is probably equal to amino acid transport system A.

Hyperosmotic hbridoma cell cultures: Increased monoclonal antibody production with addition of glycine betaine.

When mouse hybridoma cells were grown in culture media which were made hyperosmotic through the addition of NaCl or sucrose, the specific rate of antibody production increased with medium osmolality, reaching approx. 1.9 times the level obtained at physiological osmolality. However, due to a simultaneous reduction of the maximal cell density in the hyperosmotic media, the effect of the increased production rate did not give significant increases in the maximum antibody titer obtained in the cultures. When the osmoprotective compound, glycine betaine, was included in the NaCl- or sucrose-stressed cultures, the specific antibody production rate wasincreased up to 2.6-fold and maximum antibody titer up to twofold over that obtained in the control culture (physiological osmolality). A similar pattern of response was observed when other osmoprotective compounds (sarcosine, proline, glycine) were added to NaCl-stressed hybridoma cell cultures. For the present experiments, the results suggest that medium osmolality, rather than growth rate, will determine the specific antibody production rate by hybridoma cell line 6H11 growing in hyperosmotic culture media. (c) 1994 John Wiley & Sons, Inc.

Utilization of osmoprotective compounds by hybridoma cells exposed to hyperosmotic stress.

A search was undertaken for osmoprotective compounds for mouse hybridoma cell line 6H11 grown in culture. When the osmolality of the growth medium was increased above the normal osmolality of 330 mOsmol/kg, growth rates were decreased in a dose-dependent fashion, reaching zero when the osmolality of the medium reached approx. 435 mOsmol/kg through the addition of KCl (60 mM), or 510 mOsmol/kg through the addition of NaCl (100 mM), or sucrose (175 mM). For NaCl or sucrose-stressed cultures, the inclusion of glycine betaine, sarcosine, proline, glycine, or asparagine in the growth medium gave a moderate to strong osmoprotective effect, measured as the ability of these compounds to enhance cell growth rates under hyperosmotic conditions. Inclusion of dimethylglycine may also give a strong osmoprotective effect under these stress conditions.In KCl-stressed cell cultures, addition of glycine betaine, sarcosine, or dimethylglycine gave strong osmoprotective effects. Of 38 compounds tested during NaCl stress, 7 gave weak osmoprotective effects and 25 gave no osmoprotective effect. The osmoprotective compounds accumulated inside the stressed cells. Accumulation was completed after 4 to 8 h, reaching intracellular concentrations of approx. 0.27 pmol/cell, or 0.15 M, in NaCl stressed cells (100 mM NaCl added).Glycine betaine, dimethylglycine, and sarcosine accumulation was observed only when these protectants were included in the medium. For all osmoprotectants, a growth medium concentration between 5 and 30 mM gave the maximal protective effect, with the exception of dimethylglycine, for which the optimum concentration was approx. 65 mM. Osmoprotective effects obtained with glycine, sarcosine, dimethylglycine, and glycine betaine, indicate that the more methylated compounds are the most effective protectants.The cellular content of glycine betaine and the glycine betaine uptake rate increased with medium osmolality in a linear fashion. Glycine betaine uptake was described by a model comprising a saturable component obeying Michaelis-Menten kinetics and a nonsaturable component. K(m) and V(max) for glycine betaine uptake were determined at 420 mOsmol/kg (50 mM NaCl added) and 510 mOsmol/kg (100 mM NaCl added). A K(m) value of approx. 2.5 mM was obtained at both medium osmolalities, while V(max) increased from 0.010 pmol/cell . h to 0.018 pmol/cell . h as the osmolality of the growth medium was increased, indicating an effect of medium osmolality on the maximal rate of transport rather than on the affinity of the transporters for glycine betaine. Hybridoma cells were not able to utilize the glycine betaine precursors choline or glycine betaine aldehyde for osmoprotection, suggesting that the cells lack part, or all, of the choline-glycine betaine pathway or the appropriate uptake mechanism.The uptake rate for glycine in NaCl-stressed hybridoma cells was approx. four times higher than the uptake rate for glycine betaine. Furthermore, if equimolar amounts of glycine betaine, glycine, sarcosine, and proline were simultaneously added to NaCl-stressed cell cultures, the intracellular concentrations of glycine, proline, and sarcosine were significantly higher than the concentration of glycine betaine.A 40% increase in hybridoma cell volume was observed when the growth medium osmolality was increased from 300 to 520 mOsmol/kg. (c) 1994 John Wiley & Sons, Inc.

Production and characterization of guluronate lyase from Klebsiella pneumoniae for applications in seaweed biotechnology.

Cultures of Klebsiella pneumoniae fermenting sodium alginate produce an extracellular guluronate-specific alginate lyase. This enzyme production was studied in stirred-tank fermentors. Different alginate substrates gave moderate differences in growth and enzyme yield. Alginates with low guluronic content gave reduced biomass but favored enzyme production. Low molecular weight (down to DPn approximately 270) also favored enzyme production. Excessive depolymerization of substrates occurred during heat sterilization of culture media. The enzyme was characterized by its specificity and sensitivity to pH, salt, and calcium. Improved yields of viable protoplasts were documented for Laminaria digitata (Huds.) Lamour.