Microorganisms frequently co-exist in matrix-embedded microbial communities with high species diversity in natural environments. Member species usually exhibit non-random spatial organization and occupy their own favorite microenvironment, mainly driven by nutrient availability, microbial physiological properties and interspecies inter-actions. Individual species residing in multispecies biofilms often receive growth ad-vantages that their single-species biofilm counterparts cannot offer, such as enhanced bi-omass production, resistance to antimicrobials and capability utilizing complex com-pounds. Spatial organization of member species is believed to play important roles in shaping the development, structure and function of multispecies communities, leading to the increased growth fitness mentioned above. Therefore, exploring the dynamic spatial organization can indispensably increase our understanding of interspecies interactions and molecular mechanisms behind these activities of complex communities in combina-tion with omics technologies. Manuscript I demonstrates the apparent and predictable correlation between interspecific interactions and spatial organization of microbes in mul-tispecies biofilms, which provides theoretical and practical arguments for further ad-vancement of this field.
Here, a reproducible four-species biofilm, composed of Stenotrophomonas rhizophila, Xan-thomonas retroflexus, Microbacterium oxydans and Paenibacillus amylolyticus, was established to study the effect of member species’ spatial organization on biofilm formation and community assembly. The observations from Manuscript II suggest that low abundance key species can significantly impact the spatial organization and hereby stabilize the func-tion and composition of complex microbiomes. Manuscript III represents a novel ap-proach performing three-dimensional pair-species cross correlation analyses on the tem-poral development of the four-species community. The results show that interspecies in-teractions mediate local intermixing of bacterial species and have a positive effect on bio-film formation and community assembly. Manuscript IV presents a comparative gene expression analysis of individual species, indicating that local interspecies interactions are indeed an important factor driving species’ spatial organization, leading to the enhanced biomass production of the whole community. Manuscript V focuses on investigating a simpler dual-species biofilm composed of X. retroflexus and P. amylolyticus, in order to explore how X. retroflexus evolves to stabilize the coexistence with P. amylolyticus, hereby leading to the increased biofilm formation by changing spatial organization within the biofilm structure.