The Escherichia coli K-12 strain TG1 was grown at 28 6C in aerobic glucose-limited continuous cultures at dilution rates ranging from 0?044 to 0?415 h "1 . The rates of biomass formation, the specific rates of glucose, ammonium and oxygen uptake and the specific carbon dioxide evolution rate increased linearly with the dilution rate up to 0?3 h "1 . At dilution rates between 0?3 h "1 and 0?4 h "1 , a strong deviation from the linear increase to lower specific oxygen uptake and carbon dioxide evolution rates occurred. The biomass formation rate and the specific glucose and ammonium uptake rates did not deviate that strongly from the linear increase up to dilution rates of 0?4 h "1 . An increasing percentage of glucose carbon flow towards biomass determined by a reactor mass balance and a decreasing specific ATP production rate concomitant with a decreasing adenylate energy charge indicated higher energetic efficiency of carbon substrate utilization at higher dilution rates. Estimation of metabolic fluxes by a stoichiometric model revealed an increasing activity of the pentose phosphate pathway and a decreasing tricarboxylic acid cycle activity with increasing dilution rates, indicative of the increased NADPH and precursor demand for anabolic purposes at the expense of ATP formation through catabolic activities. Thus, increasing growth rates first result in a more energy-efficient use of the carbon substrate for biomass production, i.e. a lower portion of the carbon substrate is channelled into the respiratory, energy-generating pathway. At dilution rates above 0?4 h "1 , close to the wash-out point, respiration rates dropped sharply and accumulation of glucose and acetic acid was observed. Energy generation through acetate formation yields less ATP compared with complete oxidation of the sugar carbon substrate, but is the result of maximized energy generation under conditions of restrictions in the tricarboxylic acid cycle or in respiratory NADH turnover. Thus, the data strongly support the conclusion that, in aerobic glucose-limited continuous cultures of E. coli TG1, two different carbon limitations occur: at low dilution rates, cell growth is limited by cell-carbon supply and, at high dilution rates, by energy-carbon supply.

Pseudomonas sp. strain MT1 is capable of degrading 4-and 5-chlorosalicylates via 4-chlorocatechol, 3-chloromuconate, and maleylacetate by a novel pathway. 3-Chloromuconate is transformed by muconate cycloisomerase of MT1 into protoanemonin, a dominant reaction product, as previously shown for other muconate cycloisomerases. However, kinetic data indicate that the muconate cycloisomerase of MT1 is specialized for 3-chloromuconate conversion and is not able to form cis-dienelactone. Protoanemonin is obviously a dead-end product of the pathway. A trans-dienelactone hydrolase (trans-DLH) was induced during growth on chlorosalicylates. Even though the purified enzyme did not act on either 3-chloromuconate or protoanemonin, the presence of muconate cylcoisomerase and trans-DLH together resulted in considerably lower protoanemonin concentrations but larger amounts of maleylacetate formed from 3-chloromuconate than the presence of muconate cycloisomerase alone resulted in. As trans-DLH also acts on 4-fluoromuconolactone, forming maleylacetate, we suggest that this enzyme acts on 4-chloromuconolactone as an intermediate in the muconate cycloisomerase-catalyzed transformation of 3-chloromuconate, thus preventing protoanemonin formation and favoring maleylacetate formation. The maleylacetate formed in this way is reduced by maleylacetate reductase. Chlorosalicylate degradation in MT1 thus occurs by a new pathway consisting of a patchwork of reactions catalyzed by enzymes from the 3-oxoadipate pathway (catechol 1,2-dioxygenase, muconate cycloisomerase) and the chlorocatechol pathway (maleylacetate reductase) and a trans-DLH.

Multiplicity of steady states of a continuous culture with an inhibitory substrate was used to estimate kinetic parameters under steady‐state conditions. A continuous culture of Pseudomonas cepacia G4, using phenol as the sole source of carbon and energy, was overloaded by increasing the dilution rate above the critical dilution rate. The culture was then stabilized in the inhibitory branch by a proportional controller using the carbon dioxide concentration in the reactor exhaust gas as the controlled variable and the dilution rate as the manipulated variable. By variation of the set point, several unstable steady states in the inhibitory branch were investigated and the specific phenol conversion rates calculated. In addition, phenol degradation was investigated under substrate limitation (chemostat operation). The results show that the phenol degradation by P. cepacia can be described by the same set of inhibition parameters under substrate limitation and under high substrate concentrations in the inhibitory branch. Biomass yield and maintenance coefficients were identical. Fitting of the data to various inhibition models resulted in the best fit for the Yano and Koga equation. The well‐known Haldane model, which is most often used to describe substrate inhibition by phenol, gave the poorest fit. The described method allows a precise data estimation under steady‐state conditions from the maximum of the biological reaction rate up to high substrate concentrations in the inhibitory branch. Inhibition parameter estimation by controlling unstable steady states may thus be useful in avoiding discrepancies between data generated by batch runs and their application to continuous cultures which have been often described in the literature. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 567–576, 1997.

The microbial degradation of quinoline by Comamonas acidovorans was studied in a laboratory scale stirred tank reactor. In continuous culture experiments using quinoline as a sole source of carbon and nitrogen, it was shown by means of mass balances that quinoline was converted completely to biomass, carbon dioxide, and ammonia. Degradation rates up to 0.7 g/L h were obtained. Measured yield coefficients Y(x/s) for quinoline were about 0.7 g/g, which is in agreement with the theoretical value for complete mineralization. Kinetic constants based on Haldane substrate inhibition were evaluated. The values were micro(max) = 0.48 h(-1), K(i) = 69 mg/L, and K(s) < 1.45 mg/L.

Analysis of the growth of Pseudomonas cepacia G4 on phenol in continuous culture has been carried out. The data were checked for consistency using both available electron and carbon balances. Coupled with the covariate adjustment estimation technique, the best estimates for true biomass energetic yield, ηmax and maintenance, me, were obtained when the carbon dioxide measurements were excluded. However, upon making corrections to the gas measurements, the best estimates were the maximum likelihood estimates (MLE) based on the complete data. The method therefore allows discrimination to be made between data. Also, similar estimates were obtained using Pirt's model based on the Monod approach and a modified form based on substrate uptake rate being the limiting factor. For the aerobic growth of P. cepacia G4 on phenol, ηmax = 0·417 and me = 0·0513 h−1 were obtained when the CO2 data were excluded. When corrections were made to the gas measurements to take into account the dissolved CO2 and the effect of operating temperature, ηmax = 0·432 and me = 0·0684 h−1 were obtained. From the 95% confidence intervals, a maximum of about 38–47·5% of the energy contained in phenol is incorporated into the biomass while the balance (52·5–62%) is evolved as heat with only a little energy needed for the maintenance of the organism.