Spyros A. Svoronos

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(ΔldhA ΔadhE ΔackA ΔfocA‐pflB ΔmgsA ΔpoxB). 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 (ΔldhA ΔadhE ΔfocA‐pflB ΔmgsA ΔpoxB ΔtdcDE ΔcitF ΔaspC ΔsfcA Δpta‐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. Biotechnol. Bioeng.

Effect of temperature and ph on the effective maximum specific growth rate of nitrifying bacteria

For modeling nitrification in wastewater treatment processes it is necessary to determine the dependence of the maximum specific growth rate (μA) of nitrifying bacteria on temperature and pH. A functional relationship for the simultaneous dependence of the effective maximum specific growth rate (μA) minus the decay coefficient, bA) on temperature and pH, obtained from theoretical arguments, was verified via batch experiments with sludge from a local wastewater treatment plant. The parameters for the functional relationship were determined from the experimental data using a nonlinear regression scheme. An optimum pH of approx. 7.8 was determined and the effective maximum specific growth rate was found to be a monotonically increasing function of temperature in the range of 15–25°C.

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.

One-dimensional modeling of secondary clarifiers using a concentration and feed velocity-dependent dispersion coefficient

A one-dimensional model of activated sludge secondary clarifiers with a dispersion term dependent on concentration and feed velocity was developed. Motivation for this model was derived from an analysis of a model employing a constraint on the gravity flux which has been shown to give excellent fits to a previously published data set. It was noted that the gravity flux constraint disappears as the level of discretization is increased, a difficulty which our model overcomes. Data collected from a full-scale clarifier at the Kanapaha Water Reclamation Facility were used to evaluate our model. Better matches to observed concentration profiles were achieved with our model than with the gravity-flux-constraining model. In addition, our model, when calibrated using data from experiments in which the sludge blanket reached steady levels, successfully predicted the outcomes of five experiments which exhibited continuously rising blankets. These failures to reach steady blanket levels were not predicted by either limiting total solids flux theory or the gravity-flux-constraining model.

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.

Discretization of nonlinear control systems via the Carleman linearization

Abstract A new explicit method for the discretization of nonlinear continuous-time systems is presented. A nonlinear discrete-time model is obtained by integrating a Carleman approximation of the original continuous-time system. In contrast to conventional explicit discretization methods, the proposed Carleman discretization does not deteriorate rapidly as the sampling period increases. Examples of open-loop and deadbeat closed-loop responses of an isothermal continuous stirred tank reactor reveal the advantages of the proposed discretization scheme.

Optimization of a periodic biological process for nitrogen removal from wastewater

Abstract The Bio-Denipho® process, a “phased isolation ditch” technology, varies both aeration pattern and flow path in a continuous flow multi-reactor system to force oscillation of organic and nutrient concentrations in process reactors. Using a six-phase cycle, desired biochemical transformations (e.g. nitrification, denitrification), are accomplished at different times in the same reactor. We used an industry-standard biokinetic model (IAWPRC) to develop and test three control strategies of increasing sophistication: (a) fixed phase lengths, (b) use of constant set points to switch between phases thus resulting in variable phase lengths, and (c) use of switching set points which are a function of on-line measurements (criteria functions). These strategies were optimized for nominal diurnal operating conditions, with the objective of minimizing effluent soluble nitrogen, subject to an upper bound on the oxygen transfer coefficient. In addition, performance of the strategies was simulated against 1-, 2-, and 5-day sustained peak loads. Under nominal diurnal conditions, the performance obtained with each strategy was comparable. Under peak loading conditions, both of the switching set point strategies gave substantially better performance than the fixed phase length strategy. Criteria functions were marginally better than constant switch points.

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.

Optimizing cellulase usage for improved mixing and rheological properties of acid-pretreated sugarcane bagasse

Consolidation of bioprocessing steps with lignocellulose is limited by hydrolysate toxicity, the fibrous nature of suspensions, and low activity of cellulase enzymes. Combinations of enzyme dose and treatment conditions improved the flow properties and pumping of acid-pretreated sugarcane bagasse slurries (10% dry weight). Low levels of cellulase enzyme (0.1 and 0.5 FPU/g dry weight acid-pretreated bagasse) were found to reduce viscosities by 77-95% after 6 h, solubilizing 3.5% of the bagasse dry weight. Flow of slurries through small funnels was a useful predictor of success with centrifugal and diaphragm pumps. Equations were derived that describe viscosity and solubilized carbohydrates as a function of time and cellulase dosage. Blending of acid-pretreated bagasse (10% dry weight) with suspensions of acid-pretreated bagasse (10% dry weight) that had been previously digested with cellulase enzymes (low viscosity) did not increase viscosity in a linear fashion. Viscosity of these mixtures remained relatively constant until a threshold level of new fiber was reached, followed by a rapid increase with further additions. Up to 35% fresh acid-pretreated bagasse could be blended with enzyme-digested fiber (5.0 FPU/g dry weight acid-pretreated fiber; 6 h) with only a modest increase in viscosity. The smooth surfaces of enzyme-treated fiber are proposed to hinder the frequency and extent of interactions between fibrils of fresh fiber particles (acid-pretreated) until a threshold concentration is achieved, after which fiber interactions and viscosity increase dramatically. These results were used to model the viscosity in an ideal continuous stirred tank reactor (liquefaction) as a function of residence time and enzyme dosage.

Collector-frother interaction in column flotation of Florida phosphate

Abstract This study examines main and interaction effects of frother type, collector and pH on the efficiency of column flotation of Florida phosphate. Two types of frothers are used including sodium alkyl ether sulfate, as an anionic frother, and mixed polyglycols as a nonionic frother. A mixture of fatty acid and fuel oil is used as a collector. The results show that column flotation, depending on the operating conditions, can give phosphate concentrates of high grade (∼31% P 2 O 5 ) at a recovery as high as (∼99%). However, interaction between frothers and the collector and consequently the flotation efficiency were found to be dependent on frother type. Specifically, anionic frother was found to lose its ability to stabilize the froth in the presence of low dosages of collector. An anionic frother used in the presence of conditioning water containing residual collector (i.e., supernatant water after conditioning solids with collector) resulted in a significant reduction in recovery. This phenomenon does not occur with the polyglycol (nonionic) frother. Flotation results of the three-phase system have been confirmed by results obtained in two-phase experiments where a low dosage of collector is added while measuring air hold-up in the presence of different frothers. The residual collector reduces air hold-up and increases bubble size when added with the alkyl ether sulfate (anionic) frother. On the other hand, a synergistic effect was obtained in the presence of the nonionic frother.