Degree Of Oxygen Limitation Research Articles

Model‐based derivation, analysis and control of unstable microaerobic steady‐states—Considering Rhodospirillum rubrum as an example

Microaerobic (oxygen-limited) conditions are critical for inducing many important microbial processes in industrial or environmental applications. At very low oxygen concentrations, however, the process performance often suffers from technical limitations. Available dissolved oxygen measurement techniques are not sensitive enough and thus control techniques, that can reliable handle these conditions, are lacking. Recently, we proposed a microaerobic process control strategy, which overcomes these restrictions and allows to assess different degrees of oxygen limitation in bioreactor batch cultivations. Here, we focus on the design of a control strategy for the automation of oxygen-limited continuous cultures using the microaerobic formation of photosynthetic membranes (PM) in Rhodospirillum rubrum as model phenomenon. We draw upon R. rubrum since the considered phenomenon depends on the optimal availability of mixed-carbon sources, hence on boundary conditions which make the process performance challenging. Empirically assessing these specific microaerobic conditions is scarcely practicable as such a process reacts highly sensitive to changes in the substrate composition and the oxygen availability in the culture broth. Therefore, we propose a model-based process control strategy which allows to stabilize steady-states of cultures grown under these conditions. As designing the appropriate strategy requires a detailed knowledge of the system behavior, we begin by deriving and validating an unstructured process model. This model is used to optimize the experimental conditions, and identify properties of the system which are critical for process performance. The derived model facilitates the good process performance via the proposed optimal control strategy. In summary the presented model-based control strategy allows to access and maintain microaerobic steady-states of interest and to precisely and efficiently transfer the culture from one stable microaerobic steady-state into another. Therefore, the presented approach is a valuable tool to study regulatory mechanisms of microaerobic phenomena in response to oxygen limitation alone. Biotechnol. Bioeng. 2014;111: 734-747. © 2013 Wiley Periodicals, Inc.

Stepwise reduction of the culture redox potential allows the analysis of microaerobic metabolism and photosynthetic membrane synthesis in Rhodospirillum rubrum

Bacterial growth under oxygen-limited (microaerobic) conditions is often accompanied by phenomena of great interest for fundamental research and industrial application. The microaerobic lifestyle of anoxygenic photosynthetic bacteria like Rhodospirillum rubrum harbors such a phenomenon, as it allows the formation of photosynthetic membranes and related interesting products without light. However, due to the technical difficulties in process control of microaerobic cultivations and the limited sensitivity of available oxygen sensors, the analysis of microaerobic growth and physiology is still underrepresented in current research. The main focus of the present study was to establish an experimental set-up for the systematic study of physiological processes, associated with the growth of R. rubrum under microaerobic conditions in the dark. For this purpose, we introduce a robust and reliable microaerobic process control strategy, which applies the culture redox potential (CRP) for assessing different degrees of oxygen limitation in bioreactor cultivations. To describe the microaerobic growth behavior of R. rubrum cultures for each of these defined CRP reduction steps, basic growth parameters were experimentally determined. Flux variability analysis provided an insight into the metabolic activity of the TCA cycle and implied its connection to the respiratory capacity of the cells. In this context, our results suggest that microaerobic growth of R. rubrum can be described as an oxygen-activated cooperative mechanism. The present study thus contributes to the investigation of metabolic and regulatory events responsible for the redox-sensitive formation of photosynthetic membranes in facultative photosynthetic bacteria. Furthermore, the introduced microaerobic cultivation setup should be generally applicable for any microbial system of interest which can be cultivated in common stirred-tank bioreactors.

Fundamental Escherichia coli biochemical pathways for biomass and energy production: creation of overall flux states

We have previously shown that the metabolism for most efficient cell growth can be realized by a combination of two types of elementary modes. One mode produces biomass while the second mode generates only energy. The identity of the four most efficient biomass and energy pathway pairs changes, depending on the degree of oxygen limitation. The identification of such pathway pairs for different growth conditions offers a pathway-based explanation of maintenance energy generation. For a given growth rate, experimental aerobic glucose consumption rates can be used to estimate the contribution of each pathway type to the overall metabolic flux pattern. All metabolic fluxes are then completely determined by the stoichiometries of involved pathways defining all nutrient consumption and metabolite secretion rates. We present here equations that permit computation of network fluxes on the basis of unique pathways for the case of optimal, glucose-limited Escherichia coli growth under varying levels of oxygen stress. Predicted glucose and oxygen uptake rates and some metabolite secretion rates are in remarkable agreement with experimental observations supporting the validity of the presented approach. The entire most efficient, steady-state, metabolic rate structure is explicitly defined by the developed equations without need for additional computer simulations. The approach should be generally useful for analyzing and interpreting genomic data by predicting concise, pathway-based metabolic rate structures.

Metabolism of 2,4-dinitrotoluene (2,4-DNT) by Alcaligenes sp. JS867 under oxygen limited conditions.

Transformation of 2,4-dinitrotoluene (2,4-DNT) by Alcaligenes JS867 under varying degrees of oxygen limitation was examined. Complete 2,4-DNT removal was observed under oxygen excess with near stoichiometric release (83%) of nitrite. Average kinetic parameters were estimated based on a dual-Monod biokinetic model with 2,4-DNT and O2 as growth limiting substrates. The negative impact of nitrite accumulation on the reaction rate was adequately described by inclusion of a noncompetitive inhibition term for NO2-. Under aerobic conditions, mumax, KsDNT, and KiNO were 0.058 (0.004)hr(-1), 3.3(+/-1.3) mg 2,4-DNT/L, and 1.2(+/-0.2)hr(-1), respectively. At increasing oxygen limitation, rates of 2,4-DNT disappearance and nitrite production decreased and incomplete removal of 2,4-DNT commenced. JS867 was able to use NO2- as a terminal electron acceptor when grown on glucose or succinate under anaerobic conditions. However, during growth on 2,4-DNT and under O2-limited conditions, JS867 did not use released nitrite as electron acceptor. The nearly constant molar ratios of DNT removed over NO2- released under various degrees of oxygen limitation suggested that oxygenolytic denitration pathways continued. No evidence of nitroreduction was obtained under the examined oligotrophic conditions. JS867 displayed a high affinity for oxygen consumption with K(SO2) value of 0.285(+/-0.198) mg O2/L. Our results indicate that under oligotrophic conditions with 2,4-DNT as dominant carbon source, oxygen availability and nitrite accumulation may limit 2,4-DNT biomineralization, but the accumulation of reduced 2,4-DNT transformation products will be small.

Xylitol formation by Candida boidinii in oxygen limited chemostat culture

Production of xylitol by Candida boidinii NRRL Y-17213 occurs under conditions of an oxygen limitation. The extent to which substrate is converted to xylitol and its coproducts (ethanol, other polyols, acetic acid), and the relative flow rates of substrate to energetic and biosynthetic pathways is controlled by the degree of oxygen limitation. With decrease in oxygen concentration in the inlet gas, for a constant dilution rate of 0.05 1/h. the specific oxygen uptake rate decreased from 1.30 to 0.36 mmol/gh Xylitol was not produced at specific oxygen uptake rates above 0.91 mmol/gh. Upon shift to lower oxygen rates, specific xylitol production rate increased more rapidly than specific ethanol production rate:

An energetic model for oxygen‐limited metabolism

Microbial production of 2,3-butanediol by Klebsiella oxytoca occurs under conditions of an oxygen limitation. The extent to which substrate is oxidized to 2,3-butanediol and its coproducts, (acetic acid, acetoin, and ethanol) and the relative flow rates of substrate to energetic and biosynthetic pathways are controlled by the degree of oxygen limitation. Two energetic relationships which describe the response to an oxygen limitation have been derived. The first relationship describes the coupling between growth and energy production observed under oxygen-limited conditions. This allows calculation of energetic parameters and modeling of the cell mass and substrate profiles in terms of the degree of oxygen limitation only. The second relationship describes the average degree of oxidation and the rate of the end-product flow. The model has been tested with both batch and continuous culture. During these kinetic studies, two phases of growth have been observed: energy-coupled growth, which was described above; and, energy-uncoupled growth, which arises when the degree of oxygen limitation reaches a critical value. Optimal culture performance with respect to 2,3-butanediol productivity occurs during energy-coupled growth.

Intermediary metabolite concentrations in xylose fermentingCandida tropicalis at varying oxygen limitations

The perchloric acid extraction method for measuring concentrations of intracellular intermediary metabolites was adapted and optimized forCandida tropicalis. Cells were harvested by filtration and rapidly frozen in liquid nitrogen in order to image anin vivo situation. Intracellular metabolites were determined with NAD/NADH linked enzymatic assays and detected spectrofluorimetrically, which allowed determinations in the mM range. The assays allow measurements of 50–60 samples at the same time. Six different metabolites (fructose 1,6-diphosphate, pyruvate, phosphoenolpyruvate, citrate, malate and fumarate) could be determined in the same sample. The method was applied to study the relation of product formation and the intracellular intermediary metabolite concentrations inCandida tropicalis at varying degrees of oxygen limitation in xylose fermenting cells. Maximum metabolite concentrations as well as maximum ratio of Krebs cycle to glycolysis metabolites were found at 5 mM/h OTR, at which point maximum ethanol production was observed.

Activity of nitrogenase and glutamine synthetase in relation to availability of oxygen in continuous cultures of a strain of cowpea Rhizobium sp. supplied with excess ammonium

In samples from nitrogen-fixing continuous cultures of strain CB756 of the cowpea type rhizobia ( Rhizobium sp.), newly fixed NH 4 + is in equilibrium with the medium, from where it is assimilated by the glutamine synthetase/glutamate synthase pathway. In samples from steady state cultures with different degrees of oxygen-limitation, nitrogenase activity was positively correlated with the biosynthetic activity of glutamine synthetase in cell free extracts. Also, activities in biosynthetic assays were positively correlated with activities in γ-glutamyl transferase assays containing 60 mM Mg 2+. Relative adenylylation of glutamine synthetase was conveniently measured in cell free extracts as the ratio of γ-glutamyl transferase activities without and with addition of 60 mM Mg 2+. Automatic control of oxygen supply was used to facilitate the study of transitions between steady-state continuous cultures with high and low nitrogenase activities. Adenylylation of glutamine synthetase and repression of nitrogenase activity in the presence of excess NH 4 +, were masked when oxygen strongly limited culture yield. Partial relief of the limitation in cultures supplied with 10 mM NH 4 + produced early decline in nitrogenase activity and increase in relative adenylylation of glutamine synthetase. Decreased oxygen supply produced a rapid decline in relative adenylylation, followed by increased nitrogenase activity, supporting the concept that control of nitrogenase synthesis is modulated by glutamine synthetase adenylylation in these bacteria.