Dynamic investigation and modeling of the nitrogen cometabolism in Methylococcus capsulatus ( Bath).

The methanotrophic bacterium Methylococcus capsulatus is capable of assimilating methane and oxygen into protein-rich biomass, however, the diverse metabolism of the microorganism also allows for several undesired cometabolic side-reactions to occur. In this study, the ammonia cometabolism in Methylococcus capsulatus is investigated using pulse experiments. Surprisingly Methylococcus capsulatus oxidizes ammonia to nitrate through a yet unknown mechanism and fixes molecular nitrogen even at a high dissolved oxygen tension. The observed phenomena can be modeled using 14 ordinary differential equations and 18 kinetic parameters, of which 6 were revealed by Morris screening to be identifiable from the experimental data. Monte Carlo simulations showed that the model was robust and accurate even with uncertainty in the parameter values as confirmed by statistical error analysis.

Mixing and mass transfer in a pilot scale U-loop bioreactor.

A system capable of handling a large volumetric gas fraction while providing a high gas to liquid mass transfer is a necessity if the metanotrophic bacterium Methylococcus capsulatus is to be used in single cell protein (SCP) production. In this study, mixing time and mass transfer coefficients were determined in a 0.15 m3 forced flow U-loop fermenter of a novel construction. The effect on the impeller drawn power when a gas was introduced into the system was also studied. Mixing time decreased and mass transfer increased with increasing volumetric liquid flow rate and specific power input. This happened also for a large volume fraction of the gas, which was shown to have only minor effect on the power drawn from the pump impeller. Very large mass transfer coefficients, considerably higher than those obtainable in an STR and previous tubular loop reactors, could be achieved in the U-loop fermenter equipped with static mixers at modest volumetric liquid and gas flow rates. Biotechnol. Bioeng. 2017;114: 344-354. © 2016 Wiley Periodicals, Inc.

Oxidative phosphorylation revisited.

The fundamentals of oxidative phosphorylation and photophosphorylation are revisited. New experimental data on the involvement of succinate and malate anions respectively in oxidative phosphorylation and photophosphorylation are presented. These new data offer a novel molecular mechanistic explanation for the energy coupling and ATP synthesis carried out in mitochondria and chloroplast thylakoids. The mechanism does not suffer from the flaws in Mitchell's chemiosmotic theory that have been pointed out in many studies since its first appearance 50 years ago, when it was hailed as a ground-breaking mechanistic explanation of what is perhaps the most important process in cellular energetics. The new findings fit very well with the predictions of Nath's torsional mechanism of energy transduction and ATP synthesis. It is argued that this mechanism, based on at least 15 years of experimental and theoretical work by Sunil Nath, constitutes a fundamentally different theory of the energy conversion process that eliminates all the inconsistencies in Mitchell's chemiosmotic theory pointed out by other authors. It is concluded that the energy-transducing complexes in oxidative phosphorylation and photosynthesis are proton-dicarboxylic acid anion cotransporters and not simply electrogenic proton translocators. These results necessitate revision of previous theories of biological energy transduction, coupling, and ATP synthesis. The novel molecular mechanism is extended to cover ATP synthesis in prokaryotes, in particular to alkaliphilic and haloalkaliphilic bacteria, essentially making it a complete theory addressing mechanistic, kinetic, and thermodynamic details. Finally, based on the new interpretation of oxidative phosphorylation, quantitative values for the P/O ratio, the amount of ATP generated per redox package of the reduced substrates, are calculated and compared with experimental values for fermentation on different substrates. It is our hope that the presentation of oxidative phosphorylation and photophosphorylation from a wholly new perspective will rekindle scientific discussion of a key process in bioenergetics and catalyze new avenues of research in a truly interdisciplinary field.

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.

Use of high-gradient magnetic fishing for reducing proteolysis during fermentation.

Proteolysis during fermentation may have a severe impact on the yield and quality of a secreted product. In the current study, we demonstrate the use of high-gradient magnetic fishing (HGMF) as an efficient alternative to the more conventional methods of preventing proteolytic degradation. Bacitracin-linked magnetic affinity adsorbents were employed directly in a fermenter during Bacillus licheniformis cultivation to remove trace amounts of unwanted proteases. The constructed magnetic adsorbents had excellent, highly specific binding characteristics in the fermentation broth (K(d) = 1.94 micromolar; Q(max) = 222.8 mg/g), which obeyed the Langmuir isotherm and had rapid binding kinetics (equilibrium in <300 s). When applied directly in shake-flask cultures or in a 1-L fermenter and then removed by HGMF, the degradation of the model protein bovine serum albumin was stopped. The adsorbents could be recycled and reused during the same fermentation to remove freshly produced proteases, extending the life of the model protein in the fermenter. HGMF may provide an efficient method of stabilizing heterologous proteins produced in cultivation processes.

Optimal fed-batch cultivation when mass transfer becomes limiting.

In the design of an aerobic fed-batch process to produce, for example, a pharmaceutical protein, the volumetric production rate will eventually become limited by mass transfer when the biomass concentration exceeds a certain upper limit x*. It appears to be common practice to switch from exponential feed of substrate to a constant feed rate when x* is reached. This is done to avoid oxygen starvation with a potential risk of undesired stress responses. But with a constant feed rate the carbon source (glucose) concentration may decrease to a low level with a resulting loss of viability and an undesired production of endotoxins. It is shown that an exponential feeding strategy may be continued, but with a smaller exponent than the one used before oxygen limitation occurs. This will diminish the potential detrimental effects on the culture due to low glucose concentration, and the total time to reach a given final biomass concentration will be reduced.

Scale-up of enzymatic production of lactobionic acid using the rotary jet head system.

Enzymatic oxidation of lactose to lactobionic acid (LBA) by a carbohydrate oxidase from Microdochium nivale was studied in a pilot-scale batch reactor of 600 L working volume using a rotary jet head (RJH) for mixing and mass transfer (Nordkvist et al., 2003, Chem Eng Sci 58:3877-3890). Both lactose and whey permeate were used as substrate, air was used as oxygen source, and catalase was added to eliminate the byproduct hydrogen peroxide. More than 98% conversion to LBA was achieved. Neither enzyme deactivation nor enzyme inhibition was observed under the experimental conditions. The dissolved oxygen tension (DOT) was constant throughout the tank for a given set of operating conditions, indicating that liquid mixing was sufficiently good to avoid oxygen gradients in the tank. However, at a given oxygen tension measured in the tank, the specific rate of reaction found in the RJH system was somewhat higher than previously obtained in a 1 L mechanically stirred tank reactor (Nordkvist et al., 2007, in this issue, pp. 694-707). This can be ascribed to a higher pressure in the recirculation loop which is part of the RJH system. Compared to mechanically stirred systems, high values of the volumetric mass transfer coefficient, k(L)a, were obtained when lactose was used as substrate, especially at low values of the specific power input and the superficial gas velocity. k(L)a was lower for experiments with whey permeate than with lactose due to addition of antifoam. The importance of mass transfer and of the saturation concentration of oxygen on the volumetric rate of reaction was demonstrated by simulations.

Oxidation of lactose to lactobionic acid by a Microdochium nivale carbohydrate oxidase: kinetics and operational stability.

Oxidation of lactose to lactobionic acid by a Microdochium nivale carbohydrate oxidase was studied. The K(m)-value for lactose, obtained by a traditional enzymatic assay, was 0.066 mM at pH 6.4 and 38 degrees C. The effect of oxygen on the enzymatic rate of reaction as well as the operational stability of the enzyme was studied by performing reactions at constant pH and temperature in a stirred tank reactor. Catalase was included in all reactions to avoid inhibition and deactivation of the oxidase by hydrogen peroxide. At pH 6.4 and 38 degrees C, K(m) for oxygen was 0.97 mM, while the catalytical rate constant, k(cat), was 94 s(-1). Furthermore, we found that the operational stability of the oxidase was dependent on the type of base used for neutralization of the acid produced. Thus, when 2 M NaOH was used for neutralization of a reaction medium containing 50 mM phosphate buffer, significant deactivation of the oxidase was observed. Also, we found that the oxidase was protected against deactivation by base at high lactose concentrations. A simple model is proposed to explain the obtained results.

Glucose metabolism in Lactococcus lactis MG1363 under different aeration conditions: requirement of acetate to sustain growth under microaerobic conditions.

Lactococcus lactis subsp. lactis MG1363 was grown in batch cultures on a defined medium with glucose as the energy source under different aeration conditions, namely, anaerobic conditions, aerobic conditions, and microaerobic conditions with a dissolved oxygen tension of 5% (when saturation with air was used as the reference). The maximum specific growth rate was high (0.78 to 0.91 h(-1)) under all aeration conditions but decreased with increasing aeration, and more than 90% of the glucose was converted to lactate. However, a shift in by-product formation was observed. Increasing aeration resulted in acetate, CO(2), and acetoin replacing formate and ethanol as end products. Under microaerobic conditions, growth came to a gradual halt, although more than 60% of the glucose was still left. A decline in growth was not observed during microaerobic cultivation when acetate was added to the medium. We hypothesize that the decline in growth was due to a lack of acetyl coenzyme A (acetyl-CoA) needed for fatty acid synthesis since acetyl-CoA can be synthesized from acetate by means of acetate kinase and phosphotransacetylase activities.

The simultaneous biosynthesis and uptake of amino acids by Lactococcus lactis studied by (13)C-labeling experiments.

Uniformly (13)C labeled glucose was fed to a lactic acid bacterium growing on a defined medium supplemented with all proteinogenic amino acids except glutamate. Aspartate stemming from the protein pool and from the extracellular medium was enriched with (13)C disclosing a substantial de novo biosynthesis of this amino acid simultaneous to its uptake from the growth medium and a rapid exchange flux of aspartate over the cellular membrane. Phenylalanine, alanine, and threonine were also synthesized de novo in spite of their presence in the growth medium.

Production of teicoplanin by Actinoplanes teichomyceticus in continuous fermentation.

Production of the potent antibiotic teicoplanin by Actinoplanes teichomyceticus was studied in batch and in chemostat cultures. It is found that the producing strain deactivates to a non-producing strain named NP-12. This strain is used to find the growth kinetics of the A. teichomyceticus without interference from the product teicoplanin. In batch experiments with NP-12 grown on glucose at different initial concentrations and with different added amounts of teicoplanin, the strong inhibitory effect of teicoplanin was determined. These results obtained on NP-12 were validated in a series of chemostat experiments with the processing strain. All experiments in batch and in chemostat cultures were well represented by Monod kinetics with respect to the carbon and energy source (glucose) and with a substantial inhibitory effect of teicoplanin. Further experiments were made with the producing strain in a continuous reactor coupled to a microfilter that delivers a cell-free permeate. It was found that the derived kinetics almost exactly simulated the behavior of the cell recirculation reactor in addition to when the cell concentration in the reactor was more than four times higher than in the chemostat. For industrial production of teicoplanin, a continuous reactor with cell recirculation and working with a low effluent glucose concentration was by far the best mode of operation. Finally, the deactivation of the producing strain to NP-12 was modeled by a two-step deactivation mechanism. Deactivation was independent of dilution rate but dependent on the inoculum preparation and on the previous history of the inoculum.

High exogenous concentrations of phenoxyacetic acid are crucial for a high penicillin V productivity in Penicillium chrysogenum.

A high-penicillin-yielding strain of Penicillium chrysogenum was grown in continuous culture on a chemically defined medium with glucose as the growth-limiting component. The cultivations were operated at a constant dilution rate of 0.05 h-1 and the feed concentration of the penicillin V sidechain precursor phenoxyacetic acid was varied between 0 and 6.5 g l-1. Subsequent formation of penicillin V and by-products related to the penicillin biosynthetic pathway was monitored at steady state. It was established that the concentration of phenoxyacetic acid in the growth medium had to be kept high to obtain a high productivity of penicillin V. The specific production rate of penicillin V as a function of the phenoxyacetic acid concentration followed Michaelis--Menten-type kinetics, from which an overall apparent Km value of 42 mM for the incorporation of intracellular phenoxyacetic acid into penicillin V could be obtained. High phenoxyacetic acid concentrations tended to lower the formation of the by-products 6-aminopenicillanic acid and 8-hydroxypenillic acid. Furthermore the undesirable loss of the pathway intermediate isopenicillin N into the extracellular medium was lowered, whereas the opposite effect was observed for the pathway intermediate δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine and the by-product 6-oxo-piperidine-2-carboxylic acid, the δ-lactam form of α-aminoadipic acid.

Flux distributions in anaerobic, glucose-limited continuous cultures of Saccharomyces cerevisiae.

A stoichiometric model describing the anaerobic metabolism of Saccharomyces cerevisiae during growth on a defined medium was derived. The model was used to calculate intracellular fluxes based on measurements of the uptake of substrates from the medium, the secretion of products from the cells, and of the rate of biomass formation. Furthermore, measurements of the biomass composition and of the activity of key enzymes were used in the calculations. The stoichiometric network consists of 37 pathway reactions involving 43 compounds of which 13 were measured (acetate, CO2, ethanol, glucose, glycerol, NH4+, pyruvate, succinate, carbohydrates, DNA, lipids, proteins and RNA). The model was used to calculate the production rates of malate and fumarate and the ethanol measurement was used to validate the model. All rate measurements were performed on glucose-limited continuous cultures in a high-performance bioreactor. Carbon balances closed within 98%. The calculations comprised flux distributions at specific growth rates of 0.10 and 0.30 h-1. The fluxes through reactions located around important branch points of the metabolism were compared, i.e. the split between the pentose phosphate and the Embden-Meyerhoff-Parnas pathways. Also the model was used to show the probable existence of a redox shunt across the inner mitochondrial membrane consisting of the reactions catalysed by the mitochondrial and the cytosolic alcohol dehydrogenase. Finally it was concluded that cytosolic isocitrate dehydrogenase is probably not present during growth on glucose. The importance of basing the flux analysis on accurate measurements was demonstrated through a sensitivity analysis. It was found that the accuracy of the measurements of CO2, ethanol, glucose, glycerol and protein was critical for the correct calculation of the flux distribution.