Mass spectrometry imaging reveals different allocation of the starting steps of benzalkonium biotransformation in wastewater biofilms.

In urban water engineering, biofilm reactors are often used to remove organic micropollutants from water both in drinking water production and wastewater treatment. However, the functioning of multispecies biofilms is largely unknown. We used mass spectrometry imaging (MSI) to reveal spatially localized reactions of the pollutant benzalkonium chloride to test whether the degradation reactions are evenly distributed over the biomass or localized in reactive centers. Here we show, that several reactions can be localized. It can thus be concluded that the few microorganisms that can metabolize these pollutants are not evenly (homogeneously) distributed over the biofilm. We also show that the different biotransformations have different reaction centers, especially i) the oxidation of the terminal carbon atom by ω-oxidation, followed by both α- and β-oxidation, and ii) the second hydroxylation to form bis-hydroxy derivatives are located differently. Thus, these two different reactions are conducted by different microbial complexes or microcolonies. These differed further in their lipid patterns, which were measured in the same MSI analysis. These findings could have vast impact on designing biofilms for wastewater engineering as they provide a new analytical tool that can tell which domains in a multispecies biofilm need to be promoted for dedicated tasks.