Effect of media composition on yield values of bacteria growing on binary and ternary substrate mixtures in continuous culture

Abstract

Pseudomonas aeruginosa 142 and a presumed variant were grown axenically in chemostats on salicylate/benzoate or salicylate/glucose binary feeds. Each substrate was supplied at 2, 10, 50, 90, 98, or 100% of the total energy flux. Two experiments were also run with ternary mixtures using the same substrates. Aliquots were transferred to fed-batch reactors receiving the same substrates at the same specific rates as the chemostat, but with one substrate radiolabeled with 14C. Radiolabel incorporated into biomass, 14CO2, and soluble microbial products over a period of 8 minutes was used to establish the biomass yield, CO2 yield, and product yield, respectively, associated with a given substrate. The effect of the percent substrate in the feed on the yields depended on the pair of substrates supplied. When benzoate comprised 50% or more of the applied substrate in salicylate/benzoate feeds, the fraction of benzoate in the feed had a small effect on the yield values associated with benzoate. However, when benzoate constituted 2% or 10% of the feed, CO2 yields were lower, biomass yields were slightly lower, and product yields were higher. In contrast, the percent of salicylate in the feed had little effect on any of the salicylate yields for cells growing on the salicylate/benzoate feeds. When salicylate was mixed with glucose, the yields associated with salicylate behaved quite differently. Biomass and CO2 yields were lower and product yields higher when salicylate was 2% or 10% of the feed than when it was higher. In the same substrate mixtures, glucose-based biomass yields were higher and CO2 yields were lower when glucose constituted 2% or 10% of the feed but were constant for higher percentages. The results suggest that the fate of a substrate is relatively independent of the feed composition as long as the substrate in question constitutes a significant percentage of the mixture. Thus, in those situations the assumption of a constant biomass yield in multicomponent substrate modeling is justified. However, when a given substrate constitutes a small percentage of the feed, significant changes in yield may occur.

Catabolic enzyme levels in bacteria grown on binary and ternary substrate mixtures in continuous culture

“…As seen in Table I, the in vitro enzyme activities in all experiments were much higher than the in vivo speci®c uptake rates required to handle the salicylate entering the chemostat, suggesting a well developed capability to metabolize the substrate over the entire substrate range, even when it contributed only 2% of the energy¯ux. This was consistent with the fact that there was little change in the fate of the carbon in the salicylate over the range of energy¯uxes studied (Rudolph and Grady, 2001). In addition, no salicylate was detected in the euents, with a detection limit of 100 lg/L (Rudolph, 1999).…”

“…Tests to establish the fate of the benzoate carbon showed that the percentages going to cell mass, carbon dioxide, and soluble The speci®c uptake rate required to remove all of the applied substrate from the chemostat. microbial products were no dierent from those in experiments 10Sa/90Be and 100Be (Rudolph and Grady, 2001), suggesting that the amount of enzyme expressed in experiment 2Sa/98Be was sucient to handle all of the benzoate entering the chemostat. Con®rmation of this can be obtained in Table I, where it can be seen that the activity of catechol 1,2-dioxygenase measured in vitro in experiment 2Sa/98Be was more than 10 times the amount needed in vivo to metabolize all of the benzoate entering the chemostat.…”

“…Culture B metabolized glucose more rapidly than culture A, with a maximum OUR of 1.38 0.16 mg O 2 /(Lámináabs) versus 0.51 0.04 mg O 2 /(Lámináabs) for culture A. Both cultures exhibited similar biomass yields on glucose, but more of the glucose carbon was released as soluble microbial products by culture A, whereas more was respired by culture B (Rudolph and Grady, 2001). Figure 1 presents the relationships between each of the key enzymes and the ability of the culture to respire the substrate degraded by the pathway containing the enzyme.…”

“…Alternatively, benzoate might have been acted upon by the enzymes responsible for salicylate degradation, even though salicylate 5-hydroxylase, the ®rst enzyme in the pathway (Grund et al, 1992), is thought to be fairly speci®c (Buswell et al, 1980). Whatever the cause, benzoate utilization was less ecient in experiments 98Sa/2Be and 90Sa/10Be because approximately 25% of the ring-carbon in the benzoate was released as soluble metabolic products, whereas less than 10% was released as products in the other experiments (Rudolph and Grady, 2001). Thus, metabolism of benzoate was impaired when it contributed 10% or less of the energy¯ux through the chemostats.…”

“…Culture B, on the other hand, used benzoate at a rate similar to that of culture A, with a maximum OUR of 4.87 0.07 mg O 2 / (Lámináabs), but used salicylate more slowly, with a maximum OUR value of 2.25 0.22 mg O 2 /(Lámi-náabs). Furthermore, culture A exhibited a slightly higher biomass yield than culture B on both of the aromatic substrates (Rudolph and Grady, 2001). Culture B metabolized glucose more rapidly than culture A, with a maximum OUR of 1.38 0.16 mg O 2 /(Lámináabs) versus 0.51 0.04 mg O 2 /(Lámináabs) for culture A.…”