Eliminating side products and increasing succinate yields in engineered strains of Escherichia coli C.

Derivatives of Escherichia coli C were previously described for succinate production by combining the deletion of genes that disrupt fermentation pathways for alternative products (ldhA::FRT, adhE::FRT, ackA::FRT, focA-pflB::FRT, mgsA, poxB) with growth-based selection for increased ATP production. The resulting strain, KJ073, produced 1.2 mol of succinate per mol glucose in mineral salts medium with acetate, malate, and pyruvate as significant co-products. KJ073 has been further improved by removing residual recombinase sites (FRT sites) from the chromosomal regions of gene deletion to create a strain devoid of foreign DNA, strain KJ091(DeltaldhA DeltaadhE DeltaackA DeltafocA-pflB DeltamgsA DeltapoxB). KJ091 was further engineered for improvements in succinate production. Deletion of the threonine decarboxylase (tdcD; acetate kinase homologue) and 2-ketobutyrate formate-lyase (tdcE; pyruvate formate-lyase homologue) reduced the acetate level by 50% and increased succinate yield (1.3 mol mol(-1) glucose) by almost 10% as compared to KJ091 and KJ073. Deletion of two genes involved in oxaloacetate metabolism, aspartate aminotransferase (aspC) and the NAD(+)-linked malic enzyme (sfcA) (KJ122) significantly increased succinate yield (1.5 mol mol(-1) glucose), succinate titer (700 mM), and average volumetric productivity (0.9 g L(-1) h(-1)). Residual pyruvate and acetate were substantially reduced by further deletion of pta encoding phosphotransacetylase to produce KJ134 (DeltaldhA DeltaadhE DeltafocA-pflB DeltamgsA DeltapoxB DeltatdcDE DeltacitF DeltaaspC DeltasfcA Deltapta-ackA). Strains KJ122 and KJ134 produced near theoretical yields of succinate during simple, anaerobic, batch fermentations using mineral salts medium. Both may be useful as biocatalysts for the commercial production of succinate.

Effect of nitrate exposure history on the oxygen/nitrate diauxic growth of Pseudomonas denitrificans.

Pseudomonas denitrificans pre-cultured (revived from agar plates) under varying conditions (nitrate absent or present; dissolved oxygen absent or present) was subsequently grown aerobically with or without nitrate present, and finally exposed to anoxic conditions (i.e., aeration stopped and dissolved oxygen stripped from solution). The occurrence and length of diauxic lags following transition from aerobic to anoxic conditions were affected strongly by the nitrate and oxygen exposure history of the biomass.

Stable performance of anaerobic digestion in the presence of a high concentration of propionic acid.

An automatically controlled, glucose-fed, anaerobic digester was deliberately inhibited by addition of phenol. To overcome the phenol inhibition the feed dilution rate was lowered in such a way that the methane yield from glucose was kept the same as that under normal conditions. The concentrations of acetic and butyric acids remained below 100 mg/l, however, propionic acid accumulated to 2,750 mg/l. Phenol apparently inhibited all tropic groups of organisms and it was shown that the propionic acid was formed from the metabolism of phenol. From the nature of the operating strategy, it was deduced that the digester continued to convert all the glucose that was supplied to methane showing that propionic acid accumulation did not inhibit conversion of glucose to methane. Therefore, propionic acid accumulation may be an effect and not a cause of inhibition of the anaerobic digestion process.

Expert system for control of anaerobic digesters.

Continuous anaerobic digesters are systems that present challenging control problems including the possibility that an unmeasured disturbance can change the sign of the steady-state process gain. An expert system is developed that recognizes changes in the sign of process gain and implements appropriate control laws. The sole on-line measured variable is the methane production rate, and the manipulated input is the dilution rate. The expert system changes the dilution rate according to one of four possible strategies: a constrained conventional set-point control law, a constant yield control law (CYCL) that is nearly optimal for the most common cause of change in the sign of the process gain, batch operation, or constant dilution rate. The algorithm uses a t test for determining when to switch to the CYCL and returns to the conventional set-point control law with bumpless transfer. The expert system has proved successful in several experimental tests: severe overload; mild, moderate, and severe underload; and addition of phenol in low and high levels. Phenol is an inhibitor that in high concentrations changes the sign of the process gain.

Experimental and modeling study of diauxic lag of Pseudomonas denitrificans switching from oxic to anoxic conditions.

We have shown that Pseudomonas denitrificans undergo a diauxie when switching from dissolved oxygen to nitrate as terminal electron acceptor. The length of time under aeration significantly affected the length of the diauxic lag, whereas the presence or absence of nitrate in the culture under aeration had a marginal effect. Nitrate consumption was very low during the lag period and then increased rapidly, coinciding with exponentially increasing biomass concentrations. Biochemical rate expressions that account for enzyme synthesis and activity in response to culture conditions and enzyme specific levels were developed. The new model successfully predicts the different lengths of diauxic lags observed in the experiments as well as the growth pattern and nitrate uptake.

Avoiding digester imbalance through real-time expert system control of dilution rate.

Process control of anaerobic digesters is a particularly challenging problem because of the diversity of possible causes that can lead to digester imbalance. Conventional control schemes can fail in consequence of a reversal in the sign of the steady-state gain caused by some type of disturbance. In this work we present an expert system approach that takes into account the particularity of this process. The developed algorithm is demonstrated to compensate successfully for changes in the digester feed medium when simulated against a model for a continuous anaerobic digester.

A simple model to describe transient differences between cell number and biomass growth rates of Escherichia coli.

Information on the response of a microbial culture to dynamic environmental conditions is necessary for the design of transient operation processes. However, most attempts at modelling culture response have been directed at describing the steady-state behavior. Thus, there is a need for adequate dynamic models for process design. Simulations of nutrient shifts were completed using a "single-cell" model for Escherichia coli. It was discovered that the specific mass growth rate and the specific number of cells growth rate were different under transient conditions, whereas at steady state (balanced growth) these rates are equivalent. Using these observations, a simple delay model to describe the transient behavior of the two growth rates is formulated and tested. The model contains as state variables only the readily measurable macroscopic quantities (biomass, cell number, and limiting nutrient). This model agreed well with the predictions of the single-cell model.

A new method for the adaptive determination of optimum pH and temperature.

An adaptive control algorithm for the on-line determination of optimal temperature or pH for biomass production in a continuous fermentor is presented. The algorithm requires no prior information and uses a dynamic Hammerstein model to identify parameters and to estimate an optimal steady-state control value. A check of the estimated performance measure second derivative is included to ensure that the target extremum is an optimum. The process is driven towards this optimum with a variable step size that depends on the quality of the on-line identified model. Numerical simulations are performed on a dynamic chemostat model that incorporates a metabolic time delay. The algorithm successfully finds the optimum temperature or pH values and maintains the reactor at the optimum steady state.

Adaptive steady-state optimization of biomass productivity in continuous fermentors.

An adaptive steady-state optimization algorithm is presented and applied to the problem of optimizing the production of biomass in continuous fermentation processes. The algorithm requires no modeling information but is based on an on-line identified linear model, locates the optimum dilution rate, and maintains the chemostat at its optimum operating condition at all times. The behavior of the algorithm is tested against a dynamic model of a chemostat that incorporates metabolic time delay, and it is shown that large disturbances in the subtrate feed concentration and the specific growth rate, causing a shift in the optimum, are handled well. The developed algorithm is also used to drive a methylotroph single-cell production process to its optimum.