We present a lab-on-a-chip device, the Envirostat 2.0, which allows for the first time contactless cultivation of a single bacterial cell by negative dielectrophoresis (nDEP) in a precisely controllable microenvironment. Stable trapping in perfusing growth medium was achieved by a miniaturization of octupole electrode geometries, matching the dimensions of bacteria. Temperature sensitive fluorescent measurements showed that these reductions of microelectrode distances led to reduced Joule heating during cell manipulation. The presented miniaturization is not possible with conventional manufacturing processes. Therefore, we present a novel bonding technology, which permits miniaturization of 3D octupole electrode geometry with biocompatible materials. To exclude the influence of other cells and to enable sampling of perfusion medium from the isolated living bacterium under study, computer aided flow simulations were used to develop a microfluidic nDEP isolation procedure. The developed microchannel and microelectrode design integrates for the first time well characterized nDEP cell sorting mechanisms and time-resolved contactless single bacterial cell cultivation in a 1.7 picoliter bioreactor system. The cell type independent trapping is demonstrated with singularized Bacillus subtilis, Escherichia coli, Corynebacterium glutamicum and other industrially relevant microbes. The static and precisely controlled microenvironment resulted in a consistent and significant faster growth compared to maximal growth rates observed on population level. Preventing the influence of surfaces and cell–cell interactions, the Envirostat 2.0 chip permits total microenvironmental control by the experimenter and therefore provides major opportunities for microfluidic based cell analysis of bacteria and small eukaryotes.