A Genome-Scale Metabolic Model for Methylococcus capsulatus (Bath) Suggests Reduced Efficiency Electron Transfer to the Particulate Methane Monooxygenase.

Background: Genome-scale metabolic models allow researchers to calculate yields, to predict consumption and production rates, and to study the effect of genetic modifications in silico, without running resource-intensive experiments. While these models have become an invaluable tool for optimizing industrial production hosts like Escherichia coli and S. cerevisiae, few such models exist for one-carbon (C1) metabolizers. Results: Here, we present a genome-scale metabolic model for Methylococcus capsulatus (Bath), a well-studied obligate methanotroph, which has been used as a production strain of single cell protein (SCP). The model was manually curated, and spans a total of 879 metabolites connected via 913 reactions. The inclusion of 730 genes and comprehensive annotations, make this model not only a useful tool for modeling metabolic physiology, but also a centralized knowledge base for M. capsulatus (Bath). With it, we determined that oxidation of methane by the particulate methane monooxygenase could be driven both through direct coupling or uphill electron transfer, both operating at reduced efficiency, as either scenario matches well with experimental data and observations from literature. Conclusion: The metabolic model will serve the ongoing fundamental research of C1 metabolism, and pave the way for rational strain design strategies toward improved SCP production processes in M. capsulatus.

Reflections on the aerobic fermentation stoichiometry of Crabtree positive yeasts.

In this communication a stoichiometric steady state model for Crabtree positive yeasts is proposed. The model is sufficiently simple to be corroborated by experimental data on the key metabolic events around Dcrit. The key feature of the model is that the bottleneck aperture for biomass production in the model of Sonnleitner and Käppeli, 1986 shrinks abruptly at Dcrit and continues to decrease with increasing dilution rate. A black box stoichiometric analysis of experiments reported in literature indicates that production of acetaldehyde might account for the abrupt shrinkage through a severe poisoning effect on the respiratory system.

Dynamic effects related to steady-state multiplicity in continuous Saccharomyces cerevisiae cultivations.

The behavioral differences between chemostat and productostat cultivation of aerobic glucose-limited Saccharomyces cerevisiae were investigated. Three types of experiments were conducted: a chemostat, where the dilution rate was shifted up or down in stepwise manner; and a productostat, with either stepwise changed or a rampwise increased ethanol setpoint, i.e., an accelero-productostat. The transient responses from chemostat and productostat experiments were interpreted using a simple metabolic flux model. In a productostat it was possible to obtain oxido-reductive steady states at dilution rates far below Dcrit due to a strong repression of the respiratory system. However, these steady states could not be obtained in a chemostat, since a dilution rate shift-down from an oxido-reductive steady state led to a derepression of the respiratory system. It can therefore be concluded that the range of dilution rates where steady-state multiplicity can be obtained differs depending on the operation mode and that this dilution rate multiplicity range may appear larger in a productostat than in a chemostat. A more narrow multiplicity range, however, was obtained when the productostat was operated as an accelero-productostat.

Conservation principles suspended solids distribution modelling to support ATS introduction on a recirculating WWTP.

A model for the description of the SS distribution in a full-scale recirculating activated sludge WWTP was developed. The model, based on conservation principles, uses on-line plant data as model inputs, and provides a prediction of the SS load in the inlet to the secondary clarifiers and the SS distribution in the WWTP as outputs. The calibrated model produces excellent predictions of the SS load to the secondary clarifiers, an essential variable for the operation of the aeration tank settling (ATS) process. A case study illustrated how the calibrated SS distribution model can be used to evaluate the potential benefit of ATS implementation on a full-scale recirculating WWTP. A reduction of the maximum SS peak load to the secondary clarifiers with 24.9% was obtained with ATS, whereas the cumulative SS load to the clarifiers is foreseen to be reduced with 22.5% for short rain events (4 hours duration) and with 16.6% for long rain events (24 hours duration). The SS distribution model is a useful tool for off-line studies of the potential benefits to be obtained by introducing ATS on a recirculating WWTP. Finally, the successful operation of the ATS process on the full-scale plant is illustrated with data.

Experimental investigations of multiple steady states in aerobic continuous cultivations of Saccharomyces cerevisiae.

The steady-state behavior of a glucose-limited, aerobic, continuous cultivation of Saccharomyces cerevisiae CEN.PK113-7D was investigated around the critical dilution rate. Oxido-reductive steady states were obtained at dilution rates up to 0.09 h(-1) lower than the critical dilution rate by operating the bioreactor as a productostat, where the dilution rate was controlled on the basis of an ethanol measurement. Thus, the experimental investigations revealed that multiple steady states exist in a region of dilution rates below the critical dilution rate. The existence of multiple steady states was attributed to two distinct physiological effects occurring when growth changed from oxidative to oxido-reductive: (i) a decrease in the efficiency of ATP production and utilization (at ethanol concentrations below 3 g/L) and (ii) repression of the oxidative metabolism (at higher ethanol concentrations). The first effect was best observed at low ethanol concentrations, where multiple steady states were observed even when no repression of the oxidative metabolism was evident, i.e., the oxidative capacity was constant. However, at higher ethanol concentrations repression of the oxidative metabolism was observed (the oxidative capacity decreased), and this resulted in a broader range of dilution rates where multiple steady states could be found.