The accessibility of almost complete genome sequences of uncultivable microbial species from metagenomes necessitates computational methods predicting microbial phenotypes solely based on genomic data. Here we investigate how comparative genomics can be utilized for the prediction of microbial phenotypes. The PICA framework facilitates application and comparison of different machine learning techniques for phenotypic trait prediction. We have improved and extended PICAs support vector machine plug-in and suggest its applicability to large-scale genome databases and incomplete genome sequences. We have demonstrated the stability of the predictive power for phenotypic traits, not perturbed by the rapid growth of genome databases. A new software tool facilitates the in-depth analysis of phenotype models, which associate expected and unexpected protein functions with particular traits. Most of the traits can be reliably predicted in only 60-70% complete genomes. We have established a new phenotypic model that predicts intracellular microorganisms. Thereby we could demonstrate that also independently evolved phenotypic traits, characterized by genome reduction, can be reliably predicted based on comparative genomics. Our results suggest that the extended PICA framework can be used to automatically annotate phenotypes in near-complete microbial genome sequences, as generated in large numbers in current metagenomics studies.