Nondestructive 3D imaging and quantification of hydrated biofilm matrix by confocal Raman microscopy coupled with non-negative matrix factorization

Biofilms are ubiquitous in natural and engineered environments and of great importance in drinking water distribution and biological wastewater treatment systems. Simultaneously acquiring the chemical and structural information of the hydrated biofilm matrix is essential for the cognition and regulation of biofilms in the environmental field. However, the complexity of samples and the limited approaches prevent a holistic understanding of the biofilm matrix. In this work, an approach based on the confocal Raman mapping technique integrated with non-negative matrix factorization (NMF) analysis was developed to probe the hydrated biofilm matrix in situ. The flexibility of the NMF analysis was utilized to subtract the undesired water background signal and resolve the meaningful biological components from Raman spectra of the hydrated biofilms. Diverse chemical components such as proteins, bacterial cells, glycolipids and polyhydroxyalkanoates (PHA) were unraveled within the distinct Pseudomonas spp. biofilm matrices, and the corresponding 3-dimensional spatial organization was visualized and quantified. Of these components, glycolipids and PHA were unique to the P. aeruginosa and P. putida biofilm matrix, respectively. Furthermore, their high abundances in the lower region of the biofilm matrix were found to be related to the specific physiological functions and surrounding microenvironments. Overall, the results demonstrate that our NMF Raman mapping method could serve as a powerful tool complementary to the conventional approaches for identifying and visualizing the chemical components in the biofilm matrix. This work may facilitate the online characterization of the biofilm matrix widely present in the environment and advance the fundamental understanding of biofilm.