In most natural and engineered environments, microorganisms are commonly found closely associated with surfaces or interfaces in the form of multicellular aggregates known as biofilms. A number of studies have suggested that biofilm mode of life provides a range of advantages to the cells, especially under unfavourable conditions such as exposure to toxic chemicals. Hence, biofilm-mediated environmental bioprocess provides a promising alternative to conventional bioprocesses relying on planktonic cells. However, loss of biofilm structure and biomass caused by the toxicity exerted by contaminants is one of the main challenges for biofilm-based bioprocesses. For example, in our recent work, we have shown that the exposure of Comamonas testosteroni biofilms to 3-chloroaniline (3-CA) induced cell detachment from the biofilms. Hence, novel approaches capable of enhancing biofilm stability and improving an overall performance of biofilm bioprocesses are required.
In this study, we report for the first time that nitrate dosing significantly improves viability of cells in C. testosteroni biofilms (from ~69% to ~90%) and greatly enhances structure stability of the biofilms in the presence of 3-CA. Genome sequence suggests that this organism may be capable of using nitrate as a sole electron acceptor for anaerobic respiration. We showed that, when nitrate was dosed, anaerobic nitrate respiration did occurr in the C. testosteroni biofilms. We further demonstrated that nitrate respiration was the key to the observed enhancement of the biofilm structure stability. To elucidate the underlying mechanisms, we employed a RNA-sequencing method and compared the gene expression profiles of C. testosteroni cells grown with oxygen and nitrate as an electron acceptor. Intriguingly, a number of genes involved in the synthesis of bis-(3’ -5’)-cyclic dimeric guanosine monophosphate (c-di-GMP) were found to be upregulated in the cultures with nitrate as the electron acceptor, suggesting that nitrate respiration may increase the intracellular c-di-GMP level. We quantified the intracellular c-di-GMP level using LC-MS/MS and found that the c-di-GMP level in the cells respiring on nitrate was significantly higher than those grown with oxygen as the electron acceptor (134.0 vs. 7.9 fmol per 109 cells). The c-di-GMP is a ubiquitous second messenger that determines bacterial lifestyle between the planktonic and biofilm mode of life. Previous reports have shown that elevated c-di-GMP levels stimulate the synthesis of EPS components and inhibit various forms of motility, favouring biofilm mode of life. Hence, the elevated level of c-di-GMP in cells respiring on nitrate underlies the enhancement of the biofilm structure stability by nitrate dosing.