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The cultivation of Bacillus licheniformis and the production of subtilisin Carlsberg serine protease were investigated in complex medium with starch and glucose, respectively, and Na-caseinate as substrates to maximize the protease concentration. The turbidity and culture fluorescence were monitored in situ, the optical density on-line and the (dry) sediment and (dry) cell mass concentrations as well as the cell count and the DNA content were monitored off-line. These values are closely interrelated and were quantified by particular relationships. By means of the six-channel flow injection analyser (FIA) system, the following medium components were monitored on-line: glucose, maltose, starch, ammonium, urea, phosphate and protease activity. The same components as well as protein, intracellular phosphate and-α-amylase activity were evaluated off-line. The off-gas composition was analysed on-line as well. Various control strategies were tested in order to maximize the protease concentration: On one hand, starch in various concentrations was used as substrate. These runs were performed at non-controlled starch decomposition, at controlled and non-controlled pH-values, respectively, and non-controlled Po2-values. On the other hand, glucose was used as substrate in fed-batch mode. These runs were performed with closed loop controlled pH- and Po2-values and open-loop and closed-loop controlled glucose concentrations, respectively. The latter strategy yielded a higher protease concentration than the former. With complex medium and closed-loop controlled process, extremely high protease activities (24 000 EPE ml −1) were obtained.

SummaryWe developed a very sensitive assay for functional factor XII. This assay is based on the property of glass-bound factor XII to activate prekallikrein (PK) into kallikrein in factor XH-deficient plasma, which is assessed by measuring the formation of kallikrein- C1 -inhibitor complexes in this plasma by radioimmunoassay.Incubation of varying amounts of factor XII in glass tubes led to a dose-dependent increase in kallikrein-C1-inhibitor complexes in factor XH-deficient plasma with a lower limit of detection of ± 20 pg of factor XII. The specificity of the assay for factor XII was demonstrated by experiments with plasmas deficient for factor XII, PK or high molecular weight kininogen (HK) and by incubation of factor XII with Polybrene or with a monoclonal antibody that inhibits the amidolytic and procoagulant activity of factor XII. The high sensitivity of the assay appeared to be due to the inability of Cl-inhibitor to inhibit factor XII bound to glass, which resulted in a molar ratio of generated kallikrein to glass-bound factor XII of at least 100:1.This assay for factor XII may be a feasible tool in studies on structural and functional aspects of (recombinant) factor XII species synthesized by cultured cells.

The influence of stirrer speed in the third preculture on the performance of penicillin V production by Penicillium chrysogenum in complex medium in a 100-l air-lift tower loop reactor was investigated. The process performance in the main culture was improved by increasing the stirrer speed from 500 to 750 rpm: the pellet size was reduced to half, the cell growth was influenced only slightly, but the production phase was extended considerably, and the final penicillin concentration was increased from 5.1 g 1-1 to 10.4 g 1-1.