Activation and regeneration of whole cell biocatalysts: initial and periodic induction behavior in starved Escherichia coli after immobilization in thin synthetic films

Activation and regeneration of whole cell biocatalytic activity via initial and subsequent induction of the lacZ gene was investigated in starved Escherichia coli using a novel synthetic biofilm. Stationary-phase bacteria were entrapped in 10-80 mum thick multi-layer films, where a copolymer of acrylic and vinyl acetate was the immobilization matrix. The E. coli were placed in a defined starvation medium containing essentially no nitrogen or carbon source and induced initially using lactose or isopropylthiogalactoside (IPTG). Subsequent inductions were performed with IPTG. Comparison studies with suspended bacteria showed that when IPTG was the initial inducing agent, induction kinetics are linear for both immobilized and suspended cells. After induction with lactose, however, a lag time is noted for suspended cells, but not for E. coli in the biofilm. Biocatalytic activity was successfully regenerated by re-inducing starved suspended cells 1-3 days after an initial induction with lactose. This regeneration was demonstrated in the synthesis of additional active beta-galactosidase. However, immobilized cells could be re-induced for at least 17 days after the initial induction, and viability in the synthetic biofilms remained greater than 90%, demonstrating that periodic induction is a valuable method for extending the life of whole cell biocatalysts. (c) 1996 John Wiley & Sons, Inc.     

Low multiplicity infection of insect cells with a recombinant baculovirus: The cell yield concept

In vitro infection of insect cells with baculoviruses is increasingly considered a viable means for the production of biopesticides, recombinant veterinary vaccines, and other recombinant products. Batch fermentation processes traditionally employ intermediate to high multiplicities of infection necessitating two parallel scale-up processes-one for cells and one for virus. In this study, we consider the use of multiplicities of infection as low as 0.0001 plaque-forming units per cell, a virus level low enough to enable infection of even large reactors (e.g., 10 m(3)) directly from a frozen stock. Using low multiplicities in the Sf9/beta-gal-AcNPV system, recombinant protein titers comparable with the maximum titer observed in high multiplicity infections were achieved. Cultures yielding the maximum titer were characterized by reaching a maximum cell density between 3 and 4 x 10(9) cell L(-1). This optimal cell yield did not depend on the multiplicity of infection, supporting the existing view that batch cultures are limited by availability of substrate. Up to a certain cell density, product titer will increase almost linearly with availability of biocatalyst, that is, cells. Beyond this point any further cell formation comes at the expense of final product titer. Low multiplicity infections were found not to cause any significant dispersion of the protein production process. Hence, product stability is not a major issue of concern using low multiplicities of infection. The sensitivity to initial conditions and disturbances, however, remains an issue of concern for the commercial use of low multiplicity infections. (c) 1996 John Wiley & Sons, Inc.     

Growth kinetics of Lactobacillus acidophilus under ohmic heating

Lactobacillus acidophilus OSU133 was inoculated into MRS broth in a fermenter vessel and incubated at 30, 35, or 45 degrees C with agitation. Incubation temperatures were attained by conventional or ohmic heating. An electrical current at low (15 V) or high (40 V) voltage was used to heat the culture directly during fermentations under ohmic heating. The growth parameters (lag period, minimum generation time, and maximum growth) and changes in pH were determined during fermentation. Metabolic activities (consumption of glucose and production of lactic acid and bacteriocin) were determined during fermentation at 35 degrees C under both heating methods. Lag period for L. acidophilus was affected appreciably by the method of heating, but the magnitude of these changes depended on the fermentation temperature. When fermentation was done at 30 degrees C, lag period decreased by 94% under low-voltage ohmic, compared with conventional, heating methods. Ohmic heating did not change the generation time significantly and caused slight, but significant (p < 0.01) decrease in maximum growth. Therefore, the electric current enhances the early stages, but it inhibits the late stages of growth. Ohmic, compared with conventional, heating resulted in higher final pH and lower bacteriocin activity in the fermented medium. However, ohmic heating at 35 degrees C had minimal effect on glucose utilization and lactic acid production by L. acidophilus. Results show that measurement of the electric current when ohmic heating is done at a constant voltage may be used in monitoring such fermentations. In conclusion, ohmic heating is potentially useful in certain applications related to fermented foods. (c) 1996 John Wiley & Sons, Inc.     

Electrophysical monitoring of culture process of recombinant Escherichia coli strains

A novel method for monitoring the cell culture process has been developed. The method is based on the measurements of electro-optical characteristics of cell suspension, calculation of cell structure parameters, and the relationship between accumulation of proteins and change of these parameters' employment. Application of the method for the monitoring of a culture process of a recombinant strain is considered. The process of growth of recombinant strains cannot be sufficiently predicted and the direct measurement of cell culture parameters is unlikely to be the most efficient way of solving the problem.Escherichia coli plasmid-free and recombinant strains synthesizing the fusion protein consisting of tumor necrosis factor-alpha (TNF) and thymosin-alpha(1) (T) were studied. It was found that cytoplasmic electroconductivity of the strains investigated increased during the culture process. The accumulation of insoluble recombinant pThy-315-encoded hybrid protein TNF(SINGLEBOND)T in cells resulted in a decrease of the membrane dielectric permeability. To determine variations of membrane dielectric permeability the amount of insoluble recombinant protein TNF(SINGLEBOND)T in the bacterial cells should be calculated.     

Localized delivery of epidermal growth factor improves the survival of transplanted hepatocytes

Hepatocyte transplantation may provide a new approach for treating a variety of liver diseases if a sufficient number of the transplanted cells survive over an extended time period. In this report, we describe a technique to deliver growth factors to transplanted hepatocytes to improve their engraftment. Epidermal growth factor (EGF) was incorporated (0.11%) into microspheres (19 +/- 12 mum) fabricated from a copolymer of lactic and glycolic acid using a double emulsion technique. The incorporated EGF was steadily released over 1 month in vitro, and it remained biologically active, as determined by its ability to stimulate DNA synthesis, cell division, and long-term survival of cultured hepatocytes. EGF-containing microspheres were mixed with a suspension of hepatocytes, seeded onto porous sponges, and implanted into the mesentery of two groups of Lewis rats. The first group of animals had their portal vein shunted to the inferior vena cava prior to cell transplantation (portal-caval shunt = PCS), and the second group of animals did not (non-PCS). This surgical procedure improves the survival of transplanted hepatocytes. The engraftment of transplanted hepatocytes in PCS animals was increased two-fold by adding EGF microspheres, as compared to adding control microspheres that contained no growth factors. Devices implanted into non-PCS animals had fewer engrafted hepatocytes than devices implanted into PCS animals, regardless of whether blank or EGF-containing microspheres were added. These results first indicate that it is possible to design systems which can alter the microenvironment of transplanted hepatocytes to improve their engraftment. They also suggest that hepatocyte engraftment is not improved by providing single growth factors unless the correct environment (PCS) is provided for the transplanted cells. (c) 1996 John Wiley & Sons, Inc.     

Modulation of metabolism of Clostridium acetobutylicum grown in chemostat culture in a three-electrode potentiostatic system with methyl viologen as electron carrier

The metabolism of Clostridium acetobutylicum was manipulated in chemostat culture at pH 5 and 6.5 in a three-electrode potentiostatic system with methyl viologen (MV) as the electron carrier. When a constant potential was applied at pH 5, the broth redox potential continuously decreased and, simultaneously, a high increase in the reduced MV concentration (MV(+.)) and the specific rate of butanol production was observed while butyric acid was taken up. A linear relationship was reported between the specific rate of NAD(P)(+) reduction by ferredoxin-NAD(P)(+) reductase and the broth redox potential, as long as the growth rate was not affected. To reach a steady state in glucose limited culture, a control system of the redox potential was required. However, it seems that C. acetobutylicum is able to adapt its metabolism when the broth redox potential was regulated at low value. On the other hand, at pH 6.5, the current generated by the electrochemical device had no effect either on broth redox potential and MV(+.) concentration or on the metabolism. (c) 1996 John Wiley & Sons, Inc.     

Poly(fumaric-co-sebacic) microspheres as oral drug delivery systems

The current study focuses on the development of bioadhesive oral delivery systems based on bioerodible polyanhydrides. The polymers were studied and characterized using a novel tensiometer based on a very sensitive electrobalance. The system was designed to mimic in vivo interactions, thus all experiments were conducted with freshly excised tissue immersed in physiological saline at 37 degrees C. Poly(fumaric-co-sebacic) [P(FA:SA)] was found to be the most bioadhesive polymer from a series of different thermoplastic materials evaluated. Correlation with in vivo performance was investigated by determining gastrointestinal (GI) residence time of barium-loaded microspheres. Residence times of 24 to 36 h provided a strong indication that these microspheres were good candidates for bioadhesive drug delivery systems. To evaluate the effect of these materials on bioavailability, the anticoagulant drug, dicumarol, was encapsulated. Systemic blood levels demonstrated increased bioavailability for the encapsulated dicumarol formulation as compared with unencapsulated drug. (c) 1996 John Wiley & Sons, Inc.     

Oxidation and amidation of salicylate by Streptomyces species

Seven streptomycete strains were tested for biotransformation of salicylate. The products were identified by nuclear magnetic resonance spectroscopy and three types of conversion were found. Streptomyces cinnamonensis and Streptomyces spectabilis formed gentisate and salicylamide concurrently. Streptomyces rimosus transformed salicylate to salicylamide. Streptomyces lividans, Streptomyces coelicolor, Streptomyces griseus and Streptomyces avermitilis produced only gentisate. Time course studies of salicylate conversion by thin-layer chromatography and high pressure liquid chromatography showed that salicylamide was accumulated in the culture broth, whereas gentisate was further metabolized.Key words: salicylate, gentisate, salicylamide, biotransformation, Streptomyces spp.     

Xylitol production by immobilized recombinant Saccharomyces cerevisiae in a continuous packed-bed bioreactor

Continuous xylitol production with two different immobilized recombinant Saccharomyces cerevisiae strains (H475 and S641), expressing low and high xylose reductase (XR) activities, was investigated in a lab-scale packed-bed bioreactor. The effect of hydraulic residence time (HRT; 1.3-11.3 h), substrate/cosubstrate ratio (0.5 and 1), recycling ratio (0, 5, and 10), and aeration (anaerobic and oxygen limited conditions) were studied. The cells were immobilized by gel entrapment using Ca-alginate as support and the beads were treated with Al(3+) to improve their mechanical strength. Xylose was converted to xylitol using glucose as cosubstrate for regeneration of NAD(P)H required in xylitol formation and for generation of maintenance energy. The stability of the recombinant strains after 15 days of continuous operation was evaluated by XR activity and plasmid retention analyses. Under anaerobic conditions the volumetric xylitol productivity increased with decreasing HRT with both strains. With a recycling ratio of 10, volumetric productivities as high as 3.44 and 5.80 g/L . h were obtained with the low XR strain at HRT 1.3 h and with the high XR strain at HRT 2.6 h, respectively. However, the highest overall xylitol yields on xylose and on cosubstrate were reached at higher HRTs. Lowering the xylose/cosubstrate ratio from 1 to 0.5 increased the overall yield of xylitol on xylose, but the productivity and the xylitol yield on cosubstrate decreased. Under oxygen limited conditions the effect of the recycling ratio on production parameters was masked by other factors, such as an accumulation of free cells in the bioreactor and severe genetic instability of the high XR strain. Under anaerobic conditions the instability was less severe, causing a decrease in XR activity from 0.15 to 0.10 and from 3.18 to 1.49 U/mg with the low and high XR strains, respectively. At the end of the fermentation, the fraction of plasmid bearing cells in the beads was close to 100% for the low XR strain; however, it was significantly lower for the high XR strain, particularly for cells from the interior of the beads. (c) 1996 John Wiley & Sons, Inc.     

On-line monitoring of intracellular ATP concentration in Escherichia coli fermentations

A method was developed to provide a real-time measurement of intracellular adenosine 5'-triphosophate (ATP) concentrations in growing Escherichia coli. The bacteria to be monitored must first be modified by inserting the cDNA for firefly luciferase expressed from a constitutive promoter. Such a construct leads to constant specific activity of firefly luciferase during both the lag phase and exponential growth. When the luciferase substrate, D-luciferin, is added to the medium, ATP within the cells is utilized in the luciferase-catalyzed reaction that produces light. The light is carried from the bioreactor to a computer-based detector by an optical fiber. The detected per cell light emission varies during exponential growth. Analysis of cytoplasm extracts shows that this variance is related to changes in the ATP concentration, which ranges from 1 to 6 times the literature value for K(M). Experimental analyses demonstrated that inner filter effects are not a significant factor affecting the use of this system. The method was tested in a benchtop fermentor at cell densities above 13 g/L dry cell weight. A correction factor based on the accumulated light data is calculated and used in real time to account for consumption of luciferin from the culture broth by the light producing reaction. Dissolved oxygen concentrations must be kept above 15% of air saturation to ensure constant light output, but no detectable increase in oxygen demand is seen. The method does not significantly affect growth or production rates. (c) 1996 John Wiley & Sons, Inc.