A process for the large-scale production of 4-hydroxyvalerate (4HV)-containing biopolyesters with a new monomer composition was developed by means of high-cell-density cultivation applying recombinant strains of Pseudomonas putida and Ralstonia eutropha, harboring the PHA-biosynthesis genes phaC and phaE of Thiocapsa pfennigii. Cell densities of about 20 g/L revealing a PHA content of 52% (w/w) and a molar fraction of 4HV of up to 15.4 mol % were obtained by a two-stage fed-batch cultivation process at a 25-L scale using octanoic acid during the growth phase and levulinic acid for the accumulation of 4HV-containing polyesters. Besides 4HV the polyester contained significant amounts of both 3-hydroxybutyric acid (3HB) and 3-hydroxyvaleric acid (3HV) and traces of 3-hydroxyhexanoic acid (3HHx) and 3-hydroxyoctanoic acid (3HO). With glucose or gluconic acid as the growth substrate, the components of the polyester could be reduced to mainly 3HV and 4HV with only a negligible fraction of 3HB, resulting in a polyester with a new composition. Scale-up of the cultivation process to a 500-L scale was successfully performed, resulting in the production of these polyesters at a pilot plant scale. Short-term shifts in temperature and pH resulted in the formation of cell agglomerates of about 50-100 microm by which the effectiveness of the semicontinuous centrifugation process was drastically increased. Washing of the freeze-dried cells with boiling methanol significantly shortened the extraction process and resulted in a polyester of higher purity. The physical and mechanical properties of these copolyesters were characterized by means of size exclusion chromatography, dynamic mechanical analysis, differential scanning calorimetry, stress-strain measurements, and measurements of the viscosity of the solution. The copolyesters were cast into films, spun to fibers, or processed into test bars by melt spinning and injection molding, respectively. They revealed an almost entirely amorphous structure and consequently were sticky and lacked strength. However they showed high thermal stability and an unusually high elongation at break of about 200%; the molecular weights (M(w)) were between 2.0 x 10(5) and 3.3 x 10(5) g/mol. It was shown that 4HV-containing polyesters belong to the class of thermoplastic elastomeres.

The fluorescence properties of one chemically and seven biologically produced polyhydroxyalkanoic acid were investigated as film castings and in living cells respectively after staining with Nile red. All these polyesters show a similar fluorescence behaviour, revealing a clear fluorescence maximum at an excitation wavelength between 540 nm and 560 nm and an emission wavelength between 570 nm and 605 nm. This could be shown by the use of two-dimensional fluorescence spectroscopy and flow cytometry. The examination of native poly(3-hydroxybutyric acid), poly(3HB), granules isolated from cells of Ralstonia eutropha H16 showed that the addition of 6.0 micrograms Nile red is necessary for total staining of 1.0 mg granules. The fluorescence intensity at an excitation wavelength of 550 nm and an emission wavelength of 600 nm showed high correlation to the poly(3HB) concentration of grana suspensions at different grana concentrations. These results and the staining of cell suspensions during cultivation experiments revealed that Nile red has a high potential for the quantitative determination of hydrophobic bacterial polyhydroxyalkanoic acids.

Production of monoclonal antibodies (MAb) for diagnostic or therapeutic applications has become an important task in the pharmaceutical industry. The efficiency of high-density reactor systems can be potentially increased by model-based design and control strategies. Therefore, a reliable kinetic model for cell metabolism is required. A systematic procedure based on metabolic modeling is used to model nutrient uptake and key product formation in a MAb bioprocess during both the growth and post-growth phases. The approach combines the key advantages of stoichiometric and kinetic models into a complete metabolic network while integrating the regulation and control of cellular activity. This modeling procedure can be easily applied to any cell line during both the cell growth and post-growth phases. Quadratic programming (QP) has been identified as a suitable method to solve the underdetermined constrained problem related to model parameter identification. The approach is illustrated for the case of murine hybridoma cells cultivated in stirred spinners.

Acoustic cell retention devices have provided a practical alternative for up to 50 L/day perfusion cultures but further scale-up has been limited. A novel temperature-controlled and larger-scale acoustic separator was evaluated at up to 400 L/day for a 10(7) CHO cell/mL perfusion culture using a 100-L bioreactor that produced up to 34 g/day recombinant protein. The increased active volume of this scaled-up separator was divided into four parallel compartments for improved fluid dynamics. Operational settings of the acoustic separator were optimized and the limits of robust operations explored. The performance was not influenced over wide ranges of duty cycle stop and run times. The maximum performance of 96% separation efficiency at 200 L/day was obtained by setting the separator temperature to 35.1 degrees C, the recirculation rate to three times the harvest rate, and the power to 90 W. While there was no detectable effect on culture viability, viable cells were selectively retained, especially at 50 L/day, where there was a 5-fold higher nonviable washout efficiency. Overall, the new temperature-controlled and scaled-up separator design performed reliably in a way similar to smaller-scale acoustic separators. These results provide strong support for the feasibility of much greater scale-up of acoustic separations.

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A low-cost closed tubular glass photobioreactor allowing axenic cultivation of phototrophic microorganisms was constructed. Standard glass tubes were arranged in a helical array providing a working volume of 80 1. The glass tubes were connected with a degassing chamber, which also provided ports for measuring and regulating oxygen supply, pH, foam, and optical density and for adding substrates and antifoam agents as well as disposing of vent gas. A pump module allowed agitation of the medium in the bioreactor at a laminar flow rate of 1.5 m/s. Upstream of the pump module a gas inlet was located, allowing efficient mixing of the used gases with the medium. The temperature of the medium was controlled by a Pt-100 sensor and by a heat exchanger with an effective surface of 0.12 m2 connected to an external thermostat. Irradiation was provided by three light panels each consisting of ten fluorescent tubes. The entire photobioreactor - apart from the light panels and motor - could be sterilized at 121 degrees C in an autoclave. In addition to a detailed description of this photobioreactor, we report on first experiments to cultivate the anoxygenic phototrophic bacteria Rhodobacter sphaeroides and Rhodospirillum rubrum, the oxygenic phototrophic cyanobacterium Synechocystis sp. strain PCC6803, and the microalga Chlorella sp. in this photobioreactor.

Increasing worldwide demand for mammalian cell production capacity will likely be partially satisfied by a greater use of higher volumetric productivity perfusion processes. An important additional component of any perfusion system is the cell retention device that can be based on filtration, sedimentation, and/or acoustic technologies. A common concern with these systems is that pumping and transient exposure to suboptimal medium conditions may damage the cells or influence the product quality. A novel air-backflush mode of operating an acoustic cell separator was developed in which an injection of bioreactor air downstream of the separator periodically returned the captured cells to the reactor, allowing separation to resume within 20 s. This mode of operation eliminated the need to pump the cells and allows the selection of a residence time in the separator depending on the sensitivity of the cell line. The air-backflush mode of operating a 10L acoustic separator was systematically tested at 10(7) cells/mL to define reliable ranges of operation. Consistent separation performance was obtained for wide ranges of cooling airflow rates from 0 to 15 L/min and for backflush frequencies between 10 and 40 h(-1). The separator performance was optimized at a perfusion rate of 10 L/day to obtain a maximum separation efficiency of 92 +/- 0.3%. This was achieved by increasing the power setting to 8 W and using duty cycle stop and run times of 4.5 and 45 s, respectively. Acoustic cell separation with air backflush was successfully applied over a 110 day CHO cell perfusion culture at 10(7) cells/mL and 95% viability.

Acoustic cell filters operate at high separation efficiencies with minimal fouling and have provided a practical alternative for up to 200 L/d perfusion cultures. However, the operation of cell retention systems depends on several settings that should be adjusted depending on the cell concentration and perfusion rate. The impact of operating variables on the separation efficiency performance of a 10-L acoustic separator was characterized using a factorial design of experiments. For the recirculation mode of separator operation, bioreactor cell concentration, perfusion rate, power input, stop time and recirculation ratio were studied using a fractional factorial 2(5-1) design, augmented with axial and center point runs. One complete replicate of the experiment was carried out, consisting of 32 more runs, at 8 runs per day. Separation efficiency was the primary response and it was fitted by a second-order model using restricted maximum likelihood estimation. By backward elimination, the model equation for both experiments was reduced to 14 significant terms. The response surface model for the separation efficiency was tested using additional independent data to check the accuracy of its predictions, to explore robust operation ranges and to optimize separator performance. A recirculation ratio of 1.5 and a stop time of 2 s improved the separator performance over a wide range of separator operation. At power input of 5 W the broad range of robust high SE performance (95% or higher) was raised to over 8 L/d. The reproducible model testing results over a total period of 3 months illustrate both the stable separator performance and the applicability of the model developed to long-term perfusion cultures.