Showing papers on "Chemostat published in 2010"

Effect of biodiesel-derived waste glycerol impurities on biomass and 1,3-propanediol production of Clostridium butyricum VPI 1718

Abstract: Aim of the present study was to assess and evaluate the impact of various kinds of impurities of biodiesel-derived raw glycerol feedstock, upon microbial growth and 1,3-propanediol (1,3-PDO) production by Clostridium butyricum. Preliminary trials in 200-mL anaerobic bottles revealed that the presence of NaCl at a concentration of 4.5% (w/w of glycerol) in growth medium imposed an evident inhibitory effect, in contrast with phosphoric salts. However, the application of NaCl at elevated quantities during batch bioreactor experiments [up to 30% (w/w of glycerol)], did neither affect the microbial growth, nor the 1,3-PDO production. Moreover, when oleic acid was added into the growth medium at 2% (w/w of glycerol), a total preclusion of the strain was observed. In order to further investigate whether the nature of oleic acid itself or the presence of the double bond induced the inhibitory phenomenon, stearic acid was added into the medium at the same concentration (2%, w/w, of glycerol). Indeed, no inhibitory effect was observed in the fermentor, suggesting that the presence of the double bond may play a key role in the growth behavior of the microorganism. Finally, methanol effect was tested in batch and continuous bioreactor operations. Interestingly enough, the alcohol addition did not affect the microbial bioconversion of glycerol into 1,3-PDO, even when imposed at relatively high concentrations (10%, w/w, of glycerol) in batch-bioreactor operations. In continuous experiments, methanol was added when steady state had been achieved, and although in one case high concentration was added into the chemostat (5 g/L), the system re-obtained a steady state without indications of negative effect upon biomass production due to the alcohol.

Genome-derived minimal metabolic models for Escherichia coli MG1655 with estimated in vivo respiratory ATP stoichiometry.

Abstract: Metabolic network models describing growth of Escherichia coli on glucose, glycerol and acetate were derived from a genome scale model of E. coli. One of the uncertainties in the metabolic networks is the exact stoichiometry of energy generating and consuming processes. Accurate estimation of biomass and product yields requires correct information on the ATP stoichiometry. The unknown ATP stoichiometry parameters of the constructed E. coli network were estimated from experimental data of eight different aerobic chemostat experiments carried out with E. coli MG1655, grown at different dilution rates (0.025, 0.05, 0.1, and 0.3 h(-1)) and on different carbon substrates (glucose, glycerol, and acetate). Proper estimation of the ATP stoichiometry requires proper information on the biomass composition of the organism as well as accurate assessment of net conversion rates under well-defined conditions. For this purpose a growth rate dependent biomass composition was derived, based on measurements and literature data. After incorporation of the growth rate dependent biomass composition in a metabolic network model, an effective P/O ratio of 1.49 +/- 0.26 mol of ATP/mol of O, K(X) (growth dependent maintenance) of 0.46 +/- 0.27 mol of ATP/C-mol of biomass and m(ATP) (growth independent maintenance) of 0.075 +/- 0.015 mol of ATP/C-mol of biomass/h were estimated using a newly developed Comprehensive Data Reconciliation (CDR) method, assuming that the three energetic parameters were independent of the growth rate and the used substrate. The resulting metabolic network model only requires the specific rate of growth, micro, as an input in order to accurately predict all other fluxes and yields.

A proteomic and transcriptional view of acidogenic and solventogenic steady-state cells of Clostridium acetobutylicum in a chemostat culture

Abstract: The complex changes in the life cycle of Clostridium acetobutylicum, a promising biofuel producer, are not well understood. During exponential growth, sugars are fermented to acetate and butyrate, and in the transition phase, the metabolism switches to the production of the solvents acetone and butanol accompanied by the initiation of endospore formation. Using phosphate-limited chemostat cultures at pH 5.7, C. acetobutylicum was kept at a steady state of acidogenic metabolism, whereas at pH 4.5, the cells showed stable solvent production without sporulation. Novel proteome reference maps of cytosolic proteins from both acidogenesis and solventogenesis with a high degree of reproducibility were generated. Yielding a 21% coverage, 15 protein spots were specifically assigned to the acidogenic phase, and 29 protein spots exhibited a significantly higher abundance in the solventogenic phase. Besides well-known metabolic proteins, unexpected proteins were also identified. Among these, the two proteins CAP0036 and CAP0037 of unknown function were found as major striking indicator proteins in acidogenic cells. Proteome data were confirmed by genome-wide DNA microarray analyses of the identical cultures. Thus, a first systematic study of acidogenic and solventogenic chemostat cultures is presented, and similarities as well as differences to previous studies of batch cultures are discussed.

Nutrient control of eukaryote cell growth: a systems biology study in yeast

Abstract: To elucidate the biological processes affected by changes in growth rate and nutrient availability, we have performed a comprehensive analysis of the transcriptome, proteome and metabolome responses of chemostat cultures of the yeast, Saccharomyces cerevisiae, growing at a range of growth rates and in four different nutrient-limiting conditions. We find significant changes in expression for many genes in each of the four nutrient-limited conditions tested. We also observe several processes that respond differently to changes in growth rate and are specific to each nutrient-limiting condition. These include carbohydrate storage, mitochondrial function, ribosome synthesis, and phosphate transport. Integrating transcriptome data with proteome measurements allows us to identify previously unrecognized examples of post-transcriptional regulation in response to both nutrient and growth-rate signals. Our results emphasize the unique properties of carbon metabolism and the carbon substrate, the limitation of which induces significant changes in gene regulation at the transcriptional and post-transcriptional level, as well as altering how many genes respond to growth rate. By comparison, the responses to growth limitation by other nutrients involve a smaller set of genes that participate in specific pathways. See associated commentary http://www.biomedcentral.com/1741-7007/8/62

Dynamic analysis of Michaelis-Menten chemostat-type competition models with time delay and pulse in a polluted environment

Abstract: In this paper, a new Michaelis–Menten type chemostat model with time delay and pulsed input nutrient concentration in a polluted environment is considered. We obtain a ‘microorganism-extinction’ semi-trivial periodic solution and establish the sufficient conditions for the global attractivity of the semi-trivial periodic solution. By use of new computational techniques for impulsive differential equations with delay, we prove and support with numerical calculations that the system is permanent. Our results show that time delays and the polluted environment can lead the microorganism species to be extinct.

Dynamic gene expression regulation model for growth and penicillin production in Penicillium chrysogenum

Abstract: As is often the case for microbial product formation, the penicillin production rate of Penicillium chrysogenum has been observed to be a function of the growth rate of the organism. The relation between the biomass specific rate of penicillin formation (q(p)) and growth rate (mu) has been measured under steady state conditions in carbon limited chemostats resulting in a steady state q(p)(mu) relation. Direct application of such a relation to predict the rate of product formation during dynamic conditions, as they occur, for example, in fed-batch experiments, leads to errors in the prediction, because q(p) is not an instantaneous function of the growth rate but rather lags behind because of adaptational and regulatory processes. In this paper a dynamic gene regulation model is presented, in which the specific rate of penicillin production is assumed to be a linear function of the amount of a rate-limiting enzyme in the penicillin production pathway. Enzyme activity assays were performed and strongly indicated that isopenicillin-N synthase (IPNS) was the main rate-limiting enzyme for penicillin-G biosynthesis in our strain. The developed gene regulation model predicts the expression of this rate limiting enzyme based on glucose repression, fast decay of the mRNA encoding for the enzyme as well as the decay of the enzyme itself. The gene regulation model was combined with a stoichiometric model and appeared to accurately describe the biomass and penicillin concentrations for both chemostat steady-state as well as the dynamics during chemostat start-up and fed-batch cultivation.

The effects of delayed growth response on the dynamic behaviors of the Monod type chemostat model with impulsive input nutrient concentration

Abstract: This paper investigates the effects of delayed growth response on the dynamic behaviors of the Monod type chemostat model with impulsive input nutrient concentration. By the use of the discrete dynamical system determined by the stroboscopic map, we obtain a ‘microorganism-extinction’ periodic solution, further, prove that the ‘microorganism-extinction’ periodic solution is globally attractive if the impulsive period satisfies some conditions. Using the theory on delay functional and impulsive differential equation, we obtain sufficient condition with time delay for the permanence of the system, and prove that time delays, impulsive input nutrient can bring obvious effects on the dynamic behaviors of the model.

Insufficient uracil supply in fully aerobic chemostat cultures of Saccharomyces cerevisiae leads to respiro-fermentative metabolism and double nutrient-limitation.

Abstract: A combination of chemostat cultivation and a defined medium was used to demonstrate that uracil limitation leads to a drastic alteration in the physiology of auxotrophic cells of Saccharomyces cerevisiae. Under this condition, the carbon source is dissimilated mainly to ethanol and acetate, even in fully aerobic cultures grown at 0.1 h(-1), which is far below the critical dilution rate. Differently from nitrogen-, sulphur-, or phosphate-limited cultures, uracil limitation leads to residual sugar (either glucose or sucrose) concentrations below 2 mM, which characterizes a situation of double-limitation: by the carbon source and by uracil. Furthermore, the specific rates of CO2 production and O2 consumption are increased when compared to the corresponding prototrophic strain. We conclude that when auxotrophic strains are to be used for quantitative physiological studies, special attention must be paid to the cultivation conditions, mainly regarding medium formulation, in order to avoid limitation of growth by the auxotrophic nutrient.

Differential proteomic analysis highlights metabolic strategies associated with balhimycin production in Amycolatopsis balhimycina chemostat cultivations

Abstract: Background Proteomics was recently used to reveal enzymes whose expression is associated with the production of the glycopeptide antibiotic balhimycin in Amycolatopsis balhimycina batch cultivations. Combining chemostat fermentation technology, where cells proliferate with constant parameters in a highly reproducible steady-state, and differential proteomics, the relationships between physiological status and metabolic pathways during antibiotic producing and non-producing conditions could be highlighted.

Nonlinear Modelling and Qualitative Analysis of a Real Chemostat with Pulse Feeding

Abstract: The control of substrate concentration in the bioreactor medium should be due to the substrate inhibition phenomenon Moreover, the oxygen demand in a bioreactor should be lower than the dissolved oxygen content The biomass concentration is one of the most important factors which affect the oxygen demand In order to maintain the dissolved oxygen content in an appropriate range, the biomass concentration should not exceed a critical level Based on the design ideas, a mathematical model of a chemostat with Monod-type kinetics and impulsive state feedback control for microorganisms of any biomass yield is proposed in this paper By the existence criteria of periodic solution of a general planar impulsive autonomous system, the conditions for the existence of period-1 solution of the system are obtained The results simplify the choice of suitable operating conditions for continuous culture systems It also points out that the system is not chaotic according to the analysis on the existence of period-2 solution The results and numerical simulations show that the chemostat system with state impulsive control tends to a stable state or a period solution

Isolation of NH₄+-tolerant mutants of Actinobacillus succinogenes for succinic acid production by continuous selection.

Abstract: Actinobacillus succinogenes, a typical succinic acid producing microorganism, was inhibited seriously by ammonium ion, which hampered industrialization of A. succinogenes with ammonium ion based material as the pH controller. In this study, we have isolated an ammonium ion-tolerant mutant of A. succinogenes by continuous-culture technique in which all environmental factors beside the stress (ammonium ion) were maintained constant. In this technique, the mutant-generating system was not operated as a nutrient-limited chemostat, but as a nutrient-unlimited system where cells were continuous cultured at the maximum specific growth rate. Mutants were isolated on agar plates containing a acid-base indicator bromothymol blue and high level of ammonium ion which gives 100% killing of the parent strain. When cultured in anaerobic bottles at ammonium ion concentration of 354 mmol/L, the mutant YZ0819 could produce succinic acid 40.21 g/L with yield 80.4%, while the parent strain NJ113 did not grow. With NH4OH being used to buffer the culture pH in 3.0 liter stirred bioreactor, YZ0819 produced 35.15 g/L succinic acid with yield 70.3%, 155% higher than that obtained by NJ113. In addition, the morphology of YZ0819 in fermentation broths was changed. Cells of YZ0819 were aggregated from the beginning to the end of fermentation. These results indicate that YZ0819 would be used to be the potential strain produced efficiently succinic acid with NH4OH as the pH controller and the formation of aggregates may be useful as an aid in the transferring of cells from a cultivation medium for various industrial applications.

Global behaviors of Monod type chemostat model with nutrient recycling and impulsive input

Abstract: In this paper, we consider the global behaviors of Monod type chemostat model with nutrient recycling and impulsive input. By introducing a new study method, the sufficient and necessary conditions on the permanence and extinction of the microorganisms are obtained. Furthermore, by using the Liapunov function method, the sufficient condition on the global attractivity of the system is established. Lastly, an example is given, the numerical simulation shows that if only the system is permanent, then it also is globally attractive.

Kinetics, Microbial Growth

Abstract: The article summarizes basic principles, methods, and the most essential results of kinetic studies in microbiology. Microbial kinetics is based on experimental studies of all dynamic manifestations of microbial life (growth, survival, death, product formation, mutations, cell cycles, environmental effects) and analysis of underlying mechanisms using mathematical models. The article starts from introduction to basic principles of growth stoichiometry (quantitative relationships between substrates and products of microbial processes) and enzyme kinetics and then, step-by-step surveys simple and structured models of microbial growth, cell cycle, mutation, and population dynamics. Special emphasis is given to similarity and difference in kinetic terms as applied to chemical reactions, enzyme-catalyzed processes, and microbial growth. The last part of the chapter is devoted to characterization of diversity of cultivation methods including various continuous methods (chemostat, turbidostat, pH-auxostat, viscostat, bistat), batch, fed-batch, phased and dialysis culture, plug-flow, columns packed, colonies and biofilms. Keywords: growth kinetic and stoichiometry; maintenance; wasteful catabolic reactions; cell size distribution; growth models; catabolic and anabolic substrates; physiological state; structured models; autoselection; biofilms; bistat; chemostat; fed-batch; dialysis culture; biomass recycle; sensitivity analysis; inhibitors and activators; colonies

New Approach to the Nonlinear Analysis of a Chemostat with Impulsive State Feedback Control

Abstract: Bioreactor control is an important area of research concerning continuous microorganism cultivation. The possible occurrence of the substrate inhibition phenomenon or substrate deficiency engenders the necessity to the control the substrate concentration. Moreover in order to maintain the dissolved oxygen content in an appropriate range, the biomass concentration should not exceed the set out level. To maintain the substrate and biomass concentration in certain desired ranges, a proposal of a new chemostat with pulse state feedback control is presented in the work. Then, a universal mathematical model of the chemostat is formulated, and the dynamic properties of the model including the existence and stability of the periodic solution are discussed. After this, in order to optimize the biomass production, two objective functions are introduced and the optimization is performed. The work shows that the proposed chemostat extends functionality of a chemostat providing in easy way the control of biomass and substrate concentration. The analytical results presented in the work are validated by numerical simulations.

Complex responses to culture conditions in Pseudomonas syringae pv. tomato DC3000 continuous cultures: the role of iron in cell growth and virulence factor induction.

Abstract: The growth of a model plant pathogen, Pseudomonas syringae pv. tomato DC3000, was investigated using a chemostat culture system to examine environmentally regulated responses. Using minimal medium with iron as the limiting nutrient, four different types of responses were obtained in a customized continuous culture system: (1) stable steady state, (2) damped oscillation, (3) normal washout due to high dilution rates exceeding the maximum growth rate, and (4) washout at low dilution rates due to negative growth rates. The type of response was determined by a combination of initial cell mass and dilution rate. Stable steady states were obtained with dilution rates ranging from 0.059 to 0.086 h(-1) with an initial cell mass of less than 0.6 OD(600). Damped oscillations and negative growth rates are unusual observations for bacterial systems. We have observed these responses at values of initial cell mass of 0.9 OD(600) or higher, or at low dilution rates (<0.05 h(-1)) irrespectively of initial cell mass. This response suggests complex dynamics including the possibility of multiple steady states.Iron, which was reported earlier as a growth limiting nutrient in a widely used minimal medium, enhances both growth and virulence factor induction in iron-supplemented cultures compared to unsupplemented controls. Intracellular iron concentration is correlated to the early induction (6 h) of virulence factors in both batch and chemostat cultures. A reduction in aconitase activity (a TCA cycle enzyme) and ATP levels in iron-limited chemostat cultures was observed compared to iron-supplemented chemostat cultures, indicating that iron affects central metabolic pathways. We conclude that DC3000 cultures are particularly dependent on the environment and iron is likely a key nutrient in determining physiology.

Competition of two microbial species in a turbidostat

Abstract: The competition between two microbial species in a chemostat definitely leads to the disappearance of one of them (Smith and Waltman, 1995) In this paper, a turbidostat is taken into account, ie, a continuous bioreactor in which the microbial concentration is controlled by manipulating the feed rate, and the competition between two microbial species is modelled by means of Mathematica® and AUTO97 simulation codes The growth of the two populations is supposed to be described by a simple unstructured model, the growth rate being a function of the limiting substrate concentration for both species It is shown that there is a range of the set point parameters for which the continuous bioreactor has a steady state in which the microbial species can coexist The stability of such solutions depends on both the growth rate and the microbial yield If the growth kinetics are monotonic with substrate concentration (such as the Monod-like one), no periodic solutions can occur On the contrary, if the growth rates are not monotonic, the bioreactor can show periodic regimes, too Such regimes can involve either oscillations of the concentration of a single species (while the other is constantly equal to zero) or oscillations of the concentrations of both the populations

The chemostat with lateral gene transfer

Abstract: We investigate the standard chemostat model when lateral gene transfer is taken into account. We will show that when the different genotypes have growth rate functions that are sufficiently close to a common growth rate function, and when the yields of the genotypes are sufficiently close to a common value, then the population evolves to a globally stable steady state, at which all genotypes coexist. These results can explain why the antibiotic-resistant strains persist in the pathogen population.