title: Combination of synchrotron radiation-based Fourier transforms infrared microspectroscopy and confocal laser scanning microscopy to understand spatial heterogeneity in aquatic multispecies biofilms.
abstract: Understanding the spatial heterogeneity within environmental biofilms can provide an insight into compartmentalization of different functions in biofilm communities. We used a non-destructive and label-free method by combining Synchrotron Radiation-based Fourier Transform Infrared Microspectroscopy (SR-FTIR) with Confocal Laser Scanning Microscopy (CLSM) to distinguish the spatial chemical changes within multispecies biofilms grown from natural storm waters in flow cells. Among the different surfaces tested for biofilm growth and optimal imaging, mylar membranes were most suited and it enabled successful spatial infrared imaging of natural biofilms for obtaining reliable and interpretable FTIR spectra. Time series analysis of biofilm growth showed that influx of water during biofilm growth, results in significant changes in biofilm formation. Early biofilms showed active nutrient acquisition and desiccation tolerance mechanisms corresponding with accumulation of secreted proteins. Statistical approach used for the evaluation of chemical spectra allowed for clustering and classification of various regions of the biofilm. Microheterogeneity was observed in the polymeric components of the biofilm matrix, including cellulose, glycocalyx and dextran-like molecules. Fructan and glycan-rich regions were distinguishable and glycocalyx was abundant in the strongly adhering peripheral regions of biofilms. Inner core showed coexistence of oxygen dimers and ferrihydrite that will likely support growth of Fe (II)-oxidising bacteria. The combined SR-FTIR microspectroscopy and CSLM approach for complex natural biofilms described here will be useful both in understanding heterogeneity of matrix components and in correlating functions of juxtaposed microbial species in complex natural biofilms with physicochemical microenvironment to which they are exposed.
authors.affiliation: Singapore Delft Water Alliance (SDWA), National University of Singapore, 2 Engineering Drive 2, Engineering Workshop 1, #02-05 Singapore 117577, Singapore.
authors.forename: Sheela
authors.initials: S
authors.lastname: Reuben
authors.affiliation: Singapore Synchrotron Light Source (SSLS), National University of Singapore, 5 Research Link, Singapore 117603, Singapore.
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Banas
authors.affiliation: Singapore Synchrotron Light Source (SSLS), National University of Singapore, 5 Research Link, Singapore 117603, Singapore.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Banas
authors.affiliation: Metabolites Biology Laboratory, Department of Biological Sciences, National University of Singapore, Singapore 117543, Singapore; NUS Environmental Research Institute (NERI), T-Lab Building, 5A Engineering Drive 1, Singapore 117411, Singapore; Singapore Center for Environmental Life Sciences Engineering (SCELSE), Nanyang Technological University, 60 Nanyang Drive, SBS-01N-27, Singapore 637551, Singapore. Electronic address: sanjay@nus.edu.sg.
authors.forename: Sanjay
authors.initials: S
authors.lastname: Swarup
mesh_terms: D001419:Bacteria / Q000254:growth & development* / Q000378:metabolism; D001426:Bacterial Proteins / Q000032:analysis; D018441:Biofilms / Q000145:classification / Q000254:growth & development*; D002482:Cellulose / Q000032:analysis; D003911:Dextrans / Q000032:analysis; D019276:Glycocalyx / Q000737:chemistry; D064307:Microbiota; D018613:Microscopy, Confocal; D011108:Polymers / Q000032:analysis; D012846:Singapore; D017550:Spectroscopy, Fourier Transform Infrared; D017356:Synchrotrons; D013997:Time Factors; D014871:Water Microbiology
chemical_list: D001426:Bacterial Proteins; D003911:Dextrans; D011108:Polymers; D002482:Cellulose
keywords: Aquatic biofilms; Biofilm heterogeneity; Synchrotron; Water