Formation and Ecological Dynamics of Synthetic Biofilms Derived from Microbial Components of the Cladonia arbuscula Thallus

Abstract

Synthetic multi-species consortia provide valuable insights into the ecological and structural dynamics of complex microbial biofilms. However, the specialized functional contributions of individual components under severe nutrient limitations remain poorly understood. This study investigated the population dynamics, matrix biogenesis and metabolic potential of a synthetic ‘protolichen biofilm’ model comprising Asterochloris microalgae, Gordonia bacteria, Thelebolus filamentous fungi and Occultifur yeast. The biofilms were cultivated under strict carbohydrate-deficient conditions for 30 days. Population changes, extracellular polymeric substance (EPS) matrix formation, and the concentrations of extracellular DNA (exDNA) and proteins (exProt), as well as potential dehydrogenase activity (via iodonitrotetrazolium reduction), were evaluated across monocultures, binary, ternary and quaternary consortia. Under carbon starvation, the photoautotrophic microalgae dominated the consortium, driving an 11-fold increase in population size in the four-component system and serving as the primary source of exDNA, which increased by up to three orders of magnitude by day 30. The Gordonia sp. exhibited a tenfold expansion by actively localizing to fungal hyphae and microalgal cell walls. This was directly correlated with a sharp increase in metabolic activity. By contrast, Thelebolus sp. initially provided the structural framework via EPS production, but exhibited limited metabolic activity over time. Meanwhile, the Occultifur sp. yeast population was severely suppressed, adopting a sit-and-wait ecological strategy. Spearman correlation analysis revealed that multi-species integration stabilized the community and triggered significant emergent effects in exDNA accumulation and metabolic potential, but only when microalgae were present. These findings demonstrate that microalgae and bacteria primarily drive metabolism and regulation within the protolichen consortia investigated, while fungi and yeast play structural or opportunistic roles. This provides a robust framework for understanding complex symbiotic interactions.