Charles R. Wilke

Enzymatic catalysis in a supercritical fluid

The enzyme alkaline phosphatase, EC 3. 1. 3. 1, was found to be active in a supercritical carbo dioxide solvent system. A batch reaction of disodium p-nitrophenyl phosphate with a 0.1 vol. % water solution in supercritical CO2 at 100 atm and 35°C produced p-nitrophenol when catalyzed by alkaline phosphatase.

The transient responses of hybridoma cells to nutrient additions in continuous culture: II. Glutamine pulse and step changes

The transient and steady‐state responses of hybridoma growth and metabolism to glutamine pulse and step changes have been examined. Metabolic quotients are reported for oxygen, glucose, lactate, ammonia, glutamine, alanine, and other amino acids. The specific glutamine consumption rate increased rapidly after all glutamine additions, but the responses of the glucose and oxygen consumption rates and the cell concentration were found to depend on the intial feed glutamine concentration. The glucose consumption rate was 1.4‐10.9 times that of glutamine, and serine and branched‐chain amino acids were consumed in larger amounts at the higher glucose: glutamine uptake ratios. It was estimated that maintenance accounted for ca. 60% of the cellular ATP requirements at specific growth rates ranging from 0.57 to 0.68 day−1.

Transient responses of hybridoma cells to lactate and ammonia pulse and step changes in continuous culture

Ammonia and lactate are the major byproducts from mammalian cells grown in medium containing glutamine and glucose. Both can be toxic to cells, and may limit the productivity of commercial bioreactors. The transient and steady-state responses of hybridoma growth and metabolism to lactate and ammonia pulse and step changes in continuous suspension culture have been examined. No inhibition was observed at 40 mM lactate. Cell growth was inhibited by 5 mM ammonia, but the cells were able to adapt to ammonia concentrations as high as 8.2 mM. Ammonia production decreased and alanine production increased in response to higher ammonia concentrations. Increased ammonia concentrations also inhibited glutamine and oxygen consumption. The specific oxygen consumption rate decreased by an order of magnitude after an ammonia pulse to 18 mM. Under these conditions, over 90% of the estimated ATP production was due to glycolysis and a large fraction of glutamine was converted to lactate.

Analysis of affinity separations: I: Predicting the performance of affinity adsorbers

Abstract The analysis of affinity separations has lagged behind the rapid increase in new applications. The purpose of this work is to outline an approach to analysis that will aid the design and optimization of large-scale affinity separations. This paper is an introduction to the theory of fixed bed adsorption as it applies to affinity chromatography. This theory provides analytical tools for evaluating and predicting industrial-scale affinity column performance, based on data from laboratory-scale experiments. Design equations are included for continuous separations on a rotating annular chromatograph and for batch operation of the adsorption and wash steps. The design equations are derived from a general model by introducing appropriate simplifying assumptions for rate-limiting mass transfer and sorption steps.

In-situ recovery of butanol during fermentation

End product inhibition can be reduced by the in situ removal of inhibitory fermentation products as they form. Extractive fermentation, in which an immiscible organic solvent is added to the fermentor in order to extract inhibitory products, was applied to the acetone-butanol fermentation. Six solvents or solvent mixtures were tested in batch extractive fermentations: kerosene, 30 wt% tetradecanol in kerosene, 50 wt% dodecanol in kerosene, oleyl alcohol, 50 wt% oleyl alcohol in a decane fraction and 50 wt% oleyl alcohol in benzyl benzoate. The best results were obtained with oleyl alcohol or a mixture of oleyl alcohol and benzyl benzoate. In normal batch fermentation of Clostridium acetobutylicum, glucose consumption is limited to about 80 kg/m3 due to the accumulation of butanol in the broth. In extractive fermentation using oleyl alcohol or a mixture of oleyl alcohol and benzyl benzoate, over 100 kg/m3 of glucose can be fermented. Removal of butanol from the broth as it formed also increased the rate of butanol production. Maximum volumetric butanol productivity was increased by as much as 60% in extractive fermentation compared to batch fermentation. Butanol productivities obtained in extractive fermentation compare favorably with other in situ product removal fermentations.

In situ recovery of fermentation products

Abstract In situ recovery of fermentation products can increase the rate of product inhibited fermentations, reduce costs of waste-water treatment and minimize product degradation. Some methods of in situ recovery show more potential than others for the production of chemicals and pharmaceuticals by fermentation.

Lactic acid production by Lactobacillus delbreuckii in a hollow fiber fermenter

SummaryLactic acid was produced by viable Lactobacillus delbreuckii NRRL-B445 in a hollow fiber fermenter. Final cell densities in the fluid surrounding the fibers in the fermenter were apparently as high as 480 gms DW/L, and volumetric productivities reached 100 gms/L-hr lactic acid. The observed cell yields were appreciably lower than batch cell yields.

Enhanced cellulase production in fed-batch culture of Trichoderma reesei C30

Abstract The use of a fed-batch cultivation of the fungus Trichoderma reesei (C30) allows cellulase [see 1,4-(1,3;1,4)-β- d -glucan 4-glucanohydrolase, EC 3.2.1.4] production to occur under optimum conditions, and results in extremely high enzyme titres and productivities. Enzyme levels of 26 U ml−1 at productivities >130 U l−1 h−1 have been achieved. These results are compared with the values obtained in two-stage continuous cultivation of the organism at optimum pH and temperature.

The application of cell recycle to continuous fermentative lactic acid production

SummaryA continuous stirred tank reactor (CSTR) with cell recycle was used to produce lactic acid from glucose usingLactobacillus delbreuckii NRRLB445. A volumetric productivity of 76 gm/1-h was obtained with an effluent concentration of 35 gm/1 lactic acid and a residual glucose concentration of less than 0.02 gm/l.

Continuous production of lactic acid in a cell recycle reactor

The production of lactic acid from glucose has been demonstrated using a CSTR (continuous stirred-tank reactor) with cell recycle. Studies were conducted withLactobacillus delbrueckii at a fermentation temperature of 42°C and a pH of 6.25. A cell density of 140 g dry weight/L and a volumetric productivity of 150 g/L.h, with complete glucose consumption, were obtained. It was not possible to obtain a lactic acid concentration above 60 g/L because of product inhibition. A cell purge was not necessary to maintain high viability bacteria culture or to obtain a steady state. At steady state the net cell growth appeared to be negligible. The specific glucose consumption for cell maintenance was 0.33 g glucose/g cells-h.