This lecture explores the application of thermodynamics in metabolic pathways, focusing on the use of Flux Balance Analysis and the formulation of the problem as a Mixed Integer Linear Programming. It discusses the constraints imposed by thermodynamics, the role of bi-directional reactions, and the group contribution method for estimating Gibbs free energy. The uncertainty in linear regression models and the impact of experimental errors are also covered. Additionally, it delves into the influence of factors like pH and ionic strength on free energy calculations, as well as the effect of crowding conditions on metabolic pathway feasibility.