Studies of biofilm development by advanced microscopic techniques and high-throughput sequencing

Abstract

This study was conducted to investigate the biofilm formation by using advanced microscopic and high-throughput sequencing techniques. The major tasks were (1) to quantitatively evaluate the initial bacterial attachment processes by Atomic Force Microscopy (AFM); (2) to characterize the chemical variation during biofilm formation by Raman microscopy; (3) to analyze the microbial structure and functions in the wastewater and drinking water biofilms by metagenomic analysis.

To determine the lateral detachment force for bacteria, a quantitative method using contact mode of AFM was developed. The established method had good repeatability and sensitivity to various bacteria and substrata, and was applied to evaluate the roles of bacterial surface polymers in Phase I and II attachment, i.e. lipopolysaccharides, type 1 fimbria and capsular colanic acid. The results indicated lipopolysaccharides largely enhanced Phases I and II attachment. Fimbriae increased Phase I attachment but not significantly influence the adhesion strength in Phase II. Moreover, colanic acid had negative effect on attachment in both of Phases I and II.

Surface-enhanced Raman scattering was applied to evaluate the chemical components in the biofilm matrix at different growth phases, including initial attached bacteria, colonies and mature biofilm. Three model bacteria, including Escherichia coli, Pseudomonas putida, and Bacillus subtilis, were used to cultivate biofilms. The results showed that the content of carbohydrates, proteins, and nucleic acids in biofilm matrix increased significantly along with the biofilm growth of three bacteria judging from the intensities and appearance probabilities of related marker peaks in the spectra. The content of lipids, however, only increased in the Gram-negative biofilms.

Moreover, metagenomic data, coupled with PCR-based 454 pyrosequencing reads, were generated for activated sludge and biofilm from a full-scale hybrid reactor to study the microbial taxonomic and functional differences/connections between activated sludge and biofilm. The results showed that the dominant bacteria co-existed in two samples. Global functions in activated sludge and biofilm metagenomes showed quite similar pattern, revealing the limited differences of overall functions existed in two samples. For nitrogen removal, the diversity and abundance of nitrifiers and denitrifiers in biofilm did not surpass that in activated sludge. Whilst, higher abundances of nitrification and denitrification genes were indeed found in biofilm, suggesting the increased nitrogen removal by applying biofilm might be attributed to removal efficiency rather than biomass accumulation of nitrogen removal bacteria.

To investigate the bacterial structure and functions of drinking water biofilm, PCR-based 454 pyrosequencing of 16S rRNA gene and Illumina metagenomic data were generated and analyzed. Significant differences of bacterial diversity and taxonomic structure were found between biofilms formed on stainless steel and plastics. Moreover, ecological succession could be obviously observed during biofilm formation. The metabolic network analysis for drinking water biofilm constructed for the first time. Moreover, the occurrence and abundance of specific genes involving in the bacterial pathway of glutathione metabolism and production/degradation of extracellular polymeric substances were also evaluated.