Analytical strategies for studying multi-domain biofilms: challenges and emerging approaches

Multi-domain biofilms (MDBs), composed of bacteria, fungi, algae, and archaea, dominate natural, clinical, and engineered environments, yet remain poorly characterized relative to single-domain biofilms. This disparity arises from the added complexity of MDBs and from emergent behaviors that do not occur in, and cannot be predicted from, single-domain systems. Most existing methodologies developed for single-domain biofilms do not readily translate to MDBs, where interactions, organization, and collective properties span multiple microbial domains. Here, we review current analytical approaches and methods with emerging potential for MDB research, with a focus on quantitative microbial identification across taxonomic levels from kingdom to species, characterization of functional and metabolic interactions among multiple domains, and spatially resolved analyses in complex MDB structures. We highlight how amplicon- and biomarker-based profiling, multi-omics techniques, advanced microscopy, spectroscopy, isotopic probing, and mechanobiological measurements have been applied to MDB research. We further emphasize how analytical and practical limitations along with uneven adoption across microbial domains hinder accurate characterization of MDBs. By identifying critical technical gaps and emerging opportunities, this review outlines the analytical foundations needed to improve MDB characterization and model development, an essential step toward informing effective mitigation and control strategies for environmentally and clinically relevant biofilms.