Studying undisturbed autotrophic biofilms: still a technical challenge

Biofilms in surface waters are characterized by their spatial structure and the heterogeneous distribution of the microorganisms that interact in a complex and dynamic way. Therefore, the assessment of phototrophic and heterotrophic biofilms' metabolic activity should be done without disturbing their structure. Several optical methods that allow the study of undisturbed living biofilms have been developed and are claimed to have high potential in the analysis of biofilms. However, natural biofilms can develop into thick packages of cells that may limit light penetration into the biofilm matrix, hindering the use of optical methods. In this paper we evaluate the use of nondestructive optical and destructive methods for the study of natural algal-bacterial biofilms. Pulse amplitude modulation (PAM) fluorimetry (with both single and multiple wavelength excitation) and confocal laser scanning microscopy (CLSM) are used as optical methods and compared to chemical extraction of plant pigments and exopolysaccharides. We demonstrate that the attenuation of the light intensity by the biofilm matrix represents a limitation to optical methods that is difficult to overcome in mature natural biofilms; but nevertheless, optical methods are very reliable for the study of thin or young biofilms. Apart from the biofilm thickness, the degree of compactness should also be taken into account. The density of some natural biofilms could be a limitation of CLSM especially if high molecular weight probes are used for specific biofilm components. In conclusion, a combination of both approaches still appears to be necessary in order to follow the complete developmental period of biofilms.