Biofilm reactors are often mass transfer limited due to excessive biofilm growth, impeding reactor performance. Fluidic conditions play a key role for biofilm structural development and subsequently for overall reactor performance. Continuous interfacial forces generated by aqueous-air segmented flow are controlling biofilm structure and diminish mass transfer limitations in biofilm micro-reactors. A simple three step method allows the formation of robust biofilms under aqueous-air segmented flow conditions: a first-generation biofilm is developing during single phase flow, followed by the introduction of air segments discarding most of the established biofilm. Finally, a second-generation, mature biofilm is formed in the presence of aqueous-air segments. Confocal laser scanning microscopy experiments revealed that the segmented flow supports the development of a robust biofilm. This mature biofilm is characterized by a three to fourfold increase in growth rate, calculated from an increase in thickness, a faster spatial distribution (95% surface coverage in 24 h), and a significantly more compact structure (roughness coefficient <1), as compared to biofilms grown under single phase flow conditions. The applicability of the concept in a segmented flow biofilm microreactor was demonstrated using the epoxidation of styrene to (S)-styrene oxide (ee>99.8%) catalyzed by Pseudomonas sp. strain VLB120 Delta C cells in the mono-species biofilm. The limiting factor affecting reactor performance was oxygen transfer as the volumetric productivity rose from 11 to 46 g L-tube(-1) day(-1) after increasing the air flow rate. In summary, different interfacial forces can be applied for separating cell attachment and adaptation resulting in the development of a robust catalytic biofilm in continuous microreactors. (c) 2014 Wiley Periodicals, Inc.