Optimization of fermentation media and processes is a difficult task due to the potential for high dimensionality and nonlinearity. Here we develop and evaluate variations on two novel and highly efficient methods for experimental fermentation optimization. The first approach is based on using a truncated genetic algorithm with a developing neural network model to choose the best experiments to run. The second approach uses information theory, along with Bayesian regularized neural network models, for experiment selection. To evaluate these methods experimentally, we used them to develop a new chemically defined medium for Lactococcus lactis ILI403, along with an optimal temperature and initial pH. to achieve maximum cell growth. The media consisted of 19 defined components or groups of components. The optimization results shou, that the maximum cell growth from the optimal process of each novel method is generally comparable to or higher than that achieved using a traditional statistical experimental design method, but these optima are reached in about half of the experiments (73-94 vs. 161, depending on the variants of methods). The optimal chemically defined media developed in this work are rich media that can support high cell density growth 3.5-4 times higher than the best reported synthetic medium and 72% higher than a commonly used complex medium (M17) at optimization scale. The best chemically defined medium found using the method was evaluated and compared with other defined or complex media at flask- and fermentor-scales. (C) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 25: 1587-1597, 2009