Microbial Continuous Culture Research Articles

Omics integration for in-depth understanding of the low-carbon co-culture platform system of Chlorella vulgaris-Escherichia coli

Many fuels and chemicals can be commercially produced by microbial processes to substitute the petrochemical processes. However, the microbial processes suffer from the instability, low efficiency or discontinuous cultures. The continuous culture can greatly increase the productivity and reduce costs for the microbial production. The challenges for the application of continuous culture in industry are high contamination risk and strain degeneration. The co-culture system of photoautotrophs and heterotrophs is probably able to solve bottlenecks of microbial culture. The micro-ecological balance can be achieved for the continuous microbial culture by the co-culture of photoautotrophs and heterotrophs. Here, a system for the co-cultivation of Chlorella vulgaris and Escherichia coli was established for the biosynthesis of isoprene. Compared with axenic culture, the isoprene production in the co-culture process was improved 10-fold to 0.6 g/L and the fermentation was prolonged from 100 h to 350 h. C. vulgaris promoted the isoprene synthesis and E. coli growth, while E. coli restricted C. vulgaris growth. The interactions between E. coli and C. vulgaris were closely associated with oxidative pressure from photomixotrophic metabolism. The consumption of exogenous glucose resulted in excess photomixotrophic electrons and subsequently resultant toxic reactive oxygen species (ROS). The oxidative pressure was reflected by the high activity of intracellular antioxidative (CysK, CysE, SerA, AhpC, AhpF) and repair (YtfE, NfuA, YebG) systems. C. vulgaris might protect E. coli against the oxidative pressure and improve the growth of E. coli through the inter-species cross-feeding. The biosynthesis of cysteine was greatly up-regulated in C. vulgaris to reduce ROS, and the cysteine necessary for antioxidation in E. coli might be provided by C. vulgaris. This study on the co-culture of C. vulgaris and E. coli is significant for revealing the common interactions between photoautotrophs and heterotrophs for the continuous culture.

A MICROBIAL CONTINUOUS CULTURE SYSTEM WITH DIFFUSION AND DIVERSIFIED GROWTH

A reaction-diffusion model is presented to describe the microbial continuous culture with diversified growth. The existence of nonnegative solutions and attractors for the system is obtained, the stability of steady states and the steady state bifurcation are studied under three growth conditions. In the case of no growth inhibition or only product inhibition, the system admits one positive constant steady state which is stable; in the case of growth inhibition only by substrate, the system can have two positive constant steady states, explicit conditions of the stability and the steady state bifurcation are also determined. In addition, numerical simulations are given to exhibit the theoretical results.

Identification and robustness analysis of nonlinear hybrid dynamical system of genetic regulation in continuous culture

In this paper, we present a framework to infer the possible transmembrane transport of intracellular substances. Considering four key enzymes, a modified fourteen-dimensional nonlinear hybrid dynamic system is established to describe the microbial continuous culture with enzyme-catalytic and genetic regulation. A novel quantitative definition of biological robustness is proposed to characterize the system's resilience when system parameters were perturbed. It not only considers the expectation of system output data after parameter disturbance but also considers the influence of the variance of these data. In this way, the definition can be used as an objective function of the system identification model due to the lack of data on the concentration of intracellular substances. Then, we design a parallel computing method to solve the system identification model. Numerical results indicate that the most likely transmembrane mode of transport is active transport coupling with passive diffusion for glycerol and 1, 3-propanediol.

Thermoelectric heat exchange and growth regulation in a continuous yeast culture.

We have designed a thermoelectric heat exchanger (TEHE) for microbial fermentations that is able to produce electric power from a microbial continuous culture using the intrinsic heat generated by microbial growth. While the TEHE was connected, the system proved able to stably self‐maintain both the temperature and the optical density of the culture. This paves the way toward a more sustainable operation of microbial fermentations, in which energy could be saved by converting part of the metabolic heat into usable electric power.

A class of dynamic models describing microbial flocculant with nutrient competition and metabolic products in wastewater treatment

In this paper, based on the related theories of microbial continuous culture, fermentation dynamics, and microbial flocculant, a class of dynamic models which describe microbial flocculant with resource competition and metabolic products in wastewater treatment is proposed. By analyzing the global dynamic properties of the model, the feasibility of employing microbial metabolites as flocculant to remove harmful microorganisms is considered.

Bifurcations of a periodically forced microbial continuous culture model with restrained growth rate.

A three dimensional microbial continuous culture model with a restrained microbial growth rate is studied in this paper. Two types of dilution rates are considered to investigate the dynamic behaviors of the model. For the unforced system, fold bifurcation and Hopf bifurcation are detected, and numerical simulations reveal that the system undergoes degenerate Hopf bifurcation. When the system is periodically forced, bifurcation diagrams for periodic solutions of period-one and period-two are given by researching the Poincaré map, corresponding to different bifurcation cases in the unforced system. Stable and unstable quasiperiodic solutions are obtained by Neimark-Sacker bifurcation with different parameter values. Periodic solutions of various periods can occur or disappear and even change their stability, when the Poincaré map of the forced system undergoes Neimark-Sacker bifurcation, flip bifurcation, and fold bifurcation. Chaotic attractors generated by a cascade of period doublings and some phase portraits are given at last.

Global dynamics of a delayed chemostat model with harvest by impulsive flocculant input

A mathematical model describing continuous microbial culture and harvest in a chemostat, incorporating a control strategy and defined by impulsive differential equations, is presented and investigated. Theoretical results indicate that the model has a microbe-extinction periodic solution, which is globally attractive if the threshold R_{1} is less than unity, and the model is permanent if the threshold R_{2} is greater than unity. Further, we consider the control strategy under time delay and periodical impulsive effect. Analysis shows that continuous microbial culture and harvest process can be implemented by adjusting time delay, impulsive period or input amount of flocculant. Finally, we give an example with numerical simulations to illustrate the control strategy.

Robust State Estimation in Presence of Parametric Uncertainty by NL-PI observers. An Application to Continuous Microbial Cultures

A state observer with integral action, robust and applicable to uniformly observable systems,is presented in this paper. Asymptotic stability of these observers is demonstrated applying Lyapunov convergence functions. In addition, it is shown that high-gain observers with integral action (NL-PI) provide robust estimation, despite parametric variabilities normally observed in bioreactors. The advantages of the proposed design are compared with a classical high gain observerthrough simulations and real-time cultivations of Saccharomyces cerevisiae.

DYNAMICAL ANALYSIS OF A CONTINUOUS-CULTURE AND HARVEST CHEMOSTAT MODEL WITH IMPULSIVE EFFECT

In this paper, we aim to propose a new chemostat model with continuous microbial culture and harvest, and to investigate the dynamics of the model. Different to the conventional ones, our model includes a constant periodic flocculant transmission. For the proposed system, by using theory of impulsive differential equations, we show that the microbe-extinction periodic solution is globally asymptotically stable when a threshold value is less than 1, and system is permanent when a certain threshold value is greater than 1. Then, according to the threshold associated with microbial extinction or existence, the control strategy for microbial continuous cultivation and harvest is discussed. Under such control strategy, continuous microbial culture and harvest can be achieved by adjusting input time, input amount or concentration of the flocculant. Finally, an example with numerical simulations is given to illustrate our theoretical conclusions.

Noteworthy Facts about a Methane-Producing Microbial Community Processing Acidic Effluent from Sugar Beet Molasses Fermentation

Anaerobic digestion is a complex process involving hydrolysis, acidogenesis, acetogenesis and methanogenesis. The separation of the hydrogen-yielding (dark fermentation) and methane-yielding steps under controlled conditions permits the production of hydrogen and methane from biomass. The characterization of microbial communities developed in bioreactors is crucial for the understanding and optimization of fermentation processes. Previously we developed an effective system for hydrogen production based on long-term continuous microbial cultures grown on sugar beet molasses. Here, the acidic effluent from molasses fermentation was used as the substrate for methanogenesis in an upflow anaerobic sludge blanket bioreactor. This study focused on the molecular analysis of the methane-yielding community processing the non-gaseous products of molasses fermentation. The substrate for methanogenesis produces conditions that favor the hydrogenotrophic pathway of methane synthesis. Methane production results from syntrophic metabolism whose key process is hydrogen transfer between bacteria and methanogenic Archaea. High-throughput 454 pyrosequencing of total DNA isolated from the methanogenic microbial community and bioinformatic sequence analysis revealed that the domain Bacteria was dominated by Firmicutes (mainly Clostridia), Bacteroidetes, δ- and γ-Proteobacteria, Cloacimonetes and Spirochaetes. In the domain Archaea, the order Methanomicrobiales was predominant, with Methanoculleus as the most abundant genus. The second and third most abundant members of the Archaeal community were representatives of the Methanomassiliicoccales and the Methanosarcinales. Analysis of the methanogenic sludge by scanning electron microscopy with Energy Dispersive X-ray Spectroscopy and X-ray diffraction showed that it was composed of small highly heterogeneous mineral-rich granules. Mineral components of methanogenic granules probably modulate syntrophic metabolism and methanogenic pathways. A rough functional analysis from shotgun data of the metagenome demonstrated that our knowledge of methanogenesis is poor and/or the enzymes responsible for methane production are highly effective, since despite reasonably good sequencing coverage, the details of the functional potential of the microbial community appeared to be incomplete.

Optimal dilution strategy for a microbial continuous culture based on the biological robustness

A robust optimal parameter selection model is proposed to formulate the microbial continuous culture process of glycerol bio-dissimilation to 1,3-propanediol (1,3-PD), in which the dilution rate and the glycerol concentration in feed medium are taken as the optimization variables. In consideration of the uncertain factors that some system parameters may be changed with the change of the optimization variables, we establish a novelty mathematical model which is represented by an eight-dimensional nonlinear dynamical system with the unknown parameters. On the basis of biological robustness, we give a quantitative definition of robustness index. The concentration of 1,3-PD at the approximately steady-state time together with the robustness index are taken as the cost functional in consideration of uncertain metabolic mechanisms. A parallel particle swarm optimization -- optimal parameter selection algorithm (PPSO-OPSA) is constructed to find the optimal dilution rate and the feeding glycerol concentration. Numerical results show that, by employing the obtained optimal input strategy, not only the concentration of 1,3-PD at the approximately steady-state time can be increased considerably compared with the previous experimental results, but also the obtained optimal parameters are robust for the dynamical system.

Modelling of a nonlinear switching system in microbial continuous culture and its parameter optimization via sensitivity functions

To describe the continuous bioconversion of glycerol to 1,3-propanediol concerning the intracellular and extracellular dynamics and the transports across cell membrane, we establish a nonlinear switching system and discuss its some basic properties. For this over parameterized dynamical system, we propose a two-phase method to determine its parameters. The first phase carries out parametric sensitivity analysis for the purpose of reducing the number of the unknown parameters and the second phase covers the optimization of those parameters with higher sensitivity index. Numerical calculations on the basis of multi-groups experiments indicate that the proposed switching system is appropriate and the developed methods are valid.

Sensitivity analysis and parameter identification of nonlinear hybrid systems for glycerol transport mechanisms in continuous culture

In this paper, we establish a modified fourteen-dimensional nonlinear hybrid dynamic system with genetic regulation to describe the microbial continuous culture, in which we consider that there are three possible ways for glycerol to pass the cell's membrane and one way for 1,3-PD (passive diffusion and active transport). Then we discuss the existence, uniqueness, continuous dependence of solutions and the compactness of the solution set. We construct a global sensitivity analysis approach to reduce the number of kinetic parameters. In order to infer the most reasonable transport mechanism of glycerol, we propose a parameter identification model aiming at identifying the parameter with higher sensitivity and transport of glycerol, which takes the robustness index of the intracellular substance together with the relative error between the experimental data and the computational values of the extracellular substance as a performance index. Finally, a parallel algorithm is applied to find the optimal transport of glycerol and parameters.

μ-Synthesis of dissimilation process of glycerol to 1,3-propanediol in microbial continuous culture

In this paper, robust control problem using μ-synthesis in microbial continuous culture is studied. The dissimilation process of glycerol to 1,3-propanediol cannot avoid the disturbances caused by uncertain factors. Based on the biodynamical model, a control system with the initial glycerol concentration as input control is proposed to simplify the controller design. μ-synthesis method is applied to find a feedback controller to assure both of robust stability and robust performance of the closed-loop system simultaneously. To solve the corresponding structured singular value optimization problem, a converged result is obtained through D-K iteration method. The μ-synthesis system is also compared with the corresponding H(∞) system. The simulation results indicate that the μ-controller might be more feasible for the continuous bioprocess controlling.

Asymptotic Behavior of a Chemostat Model with Watt Type Functional Response and Variable Yield

To make the theoretical analysis of the microbial continuous culture more close to the experimental results, we consider a chemostat model with Watt type functional response and variable yield. The existence of limit cycles and Hopf bifurcation is investigated, which is useful in the further study of the oscillatory behaviors of the microbial growth in the vessel. The conditions for the global asymptotical stability of the model are obtained by Dulac criterion.

Stability analysis and controller design in microbial continuous culture with discrete time delay

In this paper, stability analysis and controller design in microbial continuous culture with discrete time delay are studied. The dissimilation process of glycerol to 1,3-propanediol cannot avoid the disturbances caused by time delay. Time delay can limit and degrade the achievable performance of controlled systems, and even induce instability. Based on the biodynamic model, some properties of its solutions are discussed. In addition, we investigate how the time-delay affects the stability of the system. A linear matrix inequalities method is applied to find a feedback controller to ensure the stability of the closed-loop system. The simulation results indicate that this controller might be feasible for continuous bioprocess control.

Hopf bifurcation of a chemostat model

A chemostat model with discrete time delay is proposed to describe oscillatory behavior in microbial continuous culture. Stability and Hopf bifurcation are discussed, including the changing regularity of bifurcation value. Explicit algorithm for determining the direction of Hopf bifurcation and the stability of periodic solution is derived, using the theory of normal form and center manifold. Finally, some numerical simulations are performed to illustrate the analytical results found.

Parameter identification and optimization algorithm in microbial continuous culture

In this study, a novelty mathematical model is established to formulate the continuous culture of glycerol to 1,3-Propanediol (1,3-PD) by Klebsiella pneumoniae, in which the inhibition of 3-hydroxypropionaldehyde (3-HPA) to cells growth and activity of some enzymes (such as glycerol dehydratase (GDHt) and 1,3-PD oxidoreductase (PDOR)), and the passive diffusion and active transport of glycerol and 1,3-PD across cell membrane are all taken into consideration. Taking the mean relative error between the experimental data and calculated values as the performance index, a parameter identification model involving multiple nonlinear dynamic systems is presented. The identifiability of the parameter identification model is also proved. Finally, an improved particle swarm optimization (PSO) algorithm is constructed to find the optimal parameters for the systems under substrate limitation and excess conditions, respectively. Numerical results not only show that the established model can be used to describe the continuous fermentation reasonably, but also the improved PSO algorithm is valid.

Pathway identification using parallel optimization for a complex metabolic system in microbial continuous culture

The bio-dissimilation of glycerol to 1,3-propanediol (1,3-PD) by Klebsiella pneumoniae ( K. pneumoniae) is a complex bioprocess due to the multiple inhibitions of substrate and products onto the cell growth. In consideration of the fact that both the inhibition mechanisms of 3-hydroxypropionaldehyde (3-HPA) onto the cell growth and the transport systems of glycerol and 1,3-PD across the cell membrane are still unclear, we consider 72 possible metabolic pathways, and establish a novel mathematical model which is represented by an eight-dimensional nonlinear dynamical system. The existence, uniqueness, continuous dependence of solutions to the system and the compactness of the solution set are explored. On the basis of biological robustness, we give a quantitative definition of robustness index of the intracellular substances. Taking the robustness index of the intracellular substances together with the relative error between the experimental data and the computational values of the extracellular substances as a performance index, a parameter identification model is proposed for the nonlinear dynamical system, in which 43848 continuous variables and 1152 discrete variables are involved. A parallel particle swarm optimization — pathways identification algorithm (PPSO-PIA) is constructed to find the optimal pathway and parameters under various experiments conditions. Numerical results show that the optimal pathway and the corresponding dynamical system can describe the continuous fermentation reasonably.

Hopf bifurcation of a five-dimensional delay differential system

A five-dimensional delay differential system is proposed to describe oscillatory behaviour in microbial continuous culture. Stability and Hopf bifurcation are discussed, including the changing regularity of the bifurcation value. An explicit algorithm for determining the direction of Hopf bifurcation and the stability of periodic solution is derived, using the theory of normal form and centre manifold. Finally, some numerical simulations are performed to illustrate the analytical results found.