Rational assembly of synthetic marine biofilm community with chitinase production

Highly diverse multispecies biofilms are ubiquitous in microbial ecosystems; however, our current understanding of biofilm dynamics is limited to single species or low richness studies. We aimed to design a multispecies biofilm with a targeted function, chitinase production, using natural marine bacteria. We present a top-down assembly approach to design functional biofilm communities. Using our method, we found that final community membership was established within 24 hours, regardless of nutrient availability. However, cultivation in nutrient-rich media enabled rapid identification of the competitive dominant taxon, Pseudoalteromonas , among the 17 initial isolates used in the assembly. By repeating community assembly in a low-nutrient medium without these highly competitive taxa, we achieved the highest species diversity in the biofilm. The resulting multispecies biofilm exhibited chitinase production and maintained ~ 50% persistence during peak invasion. By comparison, a single species chitinase-producing biofilm formed lower biomass and suffered higher displacement during invasion. Importantly, one member that withstood invasion challenge in the multispecies community was completely undetectable at seven days post-invasion as a single species biofilm, indicating collective invasion resilience in the multispecies community. Further evidence of cooperation for coexistence is supported by increased β-N-acetylglucosaminidase, enzyme that hydrolyzes chitin oligomers, in the 14-member community at later timepoints, while the detected exochitinase activity remained stable. Our findings present a streamlined strategy to assemble diverse and functional biofilm communities for targeted biofilm engineering in marine and applied microbiome contexts, and our achievement of engineered function using natural bacteria offers a powerful complement to synthetic biology.