Presupuesto

1. INTRODUCTION

2. MATERIAL AND METHODS
2.1. Organism and culture conditions
C. acetobutylicum ATCC 824 was obtained from the American Type Culture Collection. Cultures were grown anaerobically at 37°C in the synthetic medium containing the following components per liter of distilled water: (components y cantidades de sustacias)
2.2. Software
Para la solución del modeloestequiométrico planteado en este trabajo, se utilizó GAMS (General Algebraic Modeling System) versión 23.9.1. Este último es un software desarrollado por A. Brooke, D. Kendrick y A. Meeraus. [19].

El servidor utilizado para la ejecución de la optimización es NEOS versión 5.0, el cual es un “solver” desarrollado por el centro de tecnología y optimización de la Universidad de Wisconsin [20].

3.METHODOLOGY
Performing a detailed description of the 29 individual reactions, including the glycolytic pathway and 1:2 molar ratio are obtained from glucose and pyruvic acid respectively (Campbell and Farrell, 2004). Since the formation of pyruvate is not certain of the reactants and amounts of metabolites that are formed in the following consecutive reactions, therefore assigned letters (a, b, c,d, f, g, l, h, i, j, k, q) to these amounts (mol), and also takes advantage of the conservation of the stoichiometric ratios for propose the model.
In the metabolic pathway is the production of some important intermediary metabolites such as pyruvate, acetyl-CoA, butyryl-CoA, NADH and Fd-Rd (ferredoxin), which have a major role in the synthesis of extracellular products and biomass. The deductionof these reactions is shown in detail in Appendix A.

3.2. Stoichiometric model
Various techniques have been developed to try to understand cell metabolism. The initial efforts resulted in the development of metabolic control theory (MCA), Unfortunately, the lack of sufficient kinetic information renders MCA impractical for most biological systems. (Papoutsakis, Et al, 1999).

To address thisproblem was proposed an alternative approach that is based on metabolic flux analysis (MFA) using metabolic pathway balances to develop a model of cellular metabolism was reported by Papoutsakis (1984).

The development of this model is based on excess production of ATP, and the biochemical formation of fermentation products [15].
The stoichiometric model takes the form of a system ofequations obtained to perform mass balances of intracellular metabolites.
The system of equations is typically underdetermined and has singularities so it is necessary to make some approximations on intermediary metabolites to reduce underdetermined nature of the system [16.13].
The literature reports that the metabolic pathway of C. acetobutilycum has the presence of some singuliarities that haveprevented the calculation of some critical flows by the general method posed Papoutzakis (1984). It has been blamed formation of acetic acid and consumption, as well as production and consumption of butyric acid by the presence of these singularities [16].
To solve the problem of singularities in the model is necessary to introduce principles of global optimization using linear programmingtechniques [16]. And so calculate the theoretical maximum yield of butanol. The general method described above was used in this work to calculate the general equation of the metabolic pathway of C. acetobutilycum ATCC 824.
3.3. Reaction System
The stoichiometric balance for the 12 reactions considered for the approach of the model can be written as follow:C6H12O6+2NAD++2HPO4-2+2ADP→2C3H4O3+2NADH+2ATP+2H2O

C3H4O3+CoASH+OxFd→C2H3OSCoA+RdFd+CO2
RdFd→OxFd+H2
RdFd+NAD+→OxFd+NADH+H+
C2H3OSCoA+ HPO4-2+ADP+H+→C2H4O2+ATP+CoASH
C2H3OSCoA+2NADH+2H+→C2H8O+CoASH+2NAD+
C4H7OSCoA+HPO4-2+ADP+H+↔ C4H8O2+ATP+CoASH
C4H7CoA+2NADH+2H+→C4H10O+CoASH+2NAD+
2C2H3OSCoA+2NADH+2H+↔ C4H7OSCoA+2NAD++H2O+CoASH
2C3H3OSCoA→ C3H6O+CO2+ATP+CoASH
C3H4O3+NADH+H+→C3H6O3+NAD+

Formation of...