Early stage of biofilm assembly on microplastics is structured by substrate size and bacterial motility

Abstract

The taxonomic structure of biofilms on 0.3-mm microplastics differed significantly from that on 3-mm microplastics or glass particles. Compared with the 3-mm microplastics, biofilms on 0.3-mm microplastics were enriched for genes involved in flagellar-based motility and chemotaxis, pointing to a more 'mobile' community. The association between motility and bacterial colonization of 0.3-mm microplastics was observed through laboratory experiments using isolated strains.

Figure 1

The experimental workflow and taxonomic composition of biofilm and seawater samples. (A) The experimental workflow. Biofilms were developed on two sizes (3 and 0.3 mm) of microplastic and glass particles, in the subtidal zone, for 10 and 20 days. Then they were transferred to the laboratory and subjected to metagenomic (three biological replicates) and metatranscriptomic sequencing and analyses. Experiments with an isolated bacterial strain were used to confirm the results of the in silico analyses. (B and C) Taxonomic composition analyses on miTags derived from the 27 metagenomes. The 30 most abundant phyla (B) or genera (C) in terms of maximum relative abundance are shown, and the remaining were merged into “Others.” Proteobacteria were classified down to the class level. The 10‐day biofilms on 3‐mm and 0.3‐mm glass particles, 3‐mm and 0.3‐mm microplastic particles were termed 3‐GP‐10, 0.3‐GP‐10, 3‐MP‐10, and 0.3‐MP‐10, respectively. The 20‐day biofilms were termed 3‐GP‐20, 0.3‐GP‐20, 3‐MP‐20, and 0.3‐MP‐20.

Figure 2

Metagenomics to reveal differentially presented genes of the 10‐day biofilms and metatranscriptomics to reveal expression levels of cell motility‐related genes in the 3‐MP‐10 and 0.3‐MP10 biofilms. (A) Annotation and abundance distribution of the top 7 (indicated by F‐value in one‐way analysis of variance [ANOVA]) significantly differentially presented motility‐related genes by metagenomics. The genes were annotated by the Kyoto Encyclopedia of Genes and Genomes (KEGG) and those in the 0.3‐MP‐10 biofilms were compared with those in the other four microbiota to identify significantly enriched genes (p‐value < 0.001 in one‐way ANOVA). (B) Relative abundance distribution of the total genes and cell motility‐related genes by metatranscriptomics. The relative abundance of genes was converted into an exponential form. (C) Expression‐level ranks of all cell motility‐related genes in the 0.3‐MP‐10 and 3‐MP10 biofilms by metatranscriptomics. The relative abundance of genes was also shown as an exponential form. (D) Functional profiles of the cell motility‐related genes. The genes are listed based on the fold change (FC = 0.3‐MP‐10/3‐MP‐10) of expression levels between the two biofilms revealed by metatranscriptomics.

Figure 3

Biofilm formation of Alteromonas sp009811495 PMMA93 treated with different concentrations of carbonyl cyanide 3‐chlorophenylhydrazone (CCCP). PMMA93 cells were incubated with 3‐MP or 0.3‐MP together with the addition of 0, 12.5, and 25 μm CCCP for 12 h, followed by scanning electron microscope evaluation and imaging (A), cell density measurement (B), and colony counting (C). The CCCP treatment was found to exert a dose‐dependent impact on PMMA93 colonization on the 0.3‐MP while having no significant effect on its colonization on the 3‐MP. The error bars represent the standard deviation of three biological replicates. ** indicates p‐value < 0.01, *** indicates p‐value < 0.001, while NS indicates nonsignificant.