A biofilm is a complex aggregate of microorganisms that embed din extracellular polymeric substances (EPS) and can develop on almost all the surface. Biofilm studies using model species in laboratory have been extensively performed. However, biofilm communities in natural environments are much complex and the molecular mechanisms governing these biofilm development remain largely unexplored. Herein we applied artificial surface-based biofilm as an ideal model to study biofilm development in marine environments. The integration of ecogenomic techniques, including metagenome, metatranscriptome, metaproteome and single genome binning, facilitates understanding of mechanisms governing biofilm development in intertidal zone and deep-sea cold seep system.

In the first part of the th...[ Read more ]

A biofilm is a complex aggregate of microorganisms that embed din extracellular polymeric substances (EPS) and can develop on almost all the surface. Biofilm studies using model species in laboratory have been extensively performed. However, biofilm communities in natural environments are much complex and the molecular mechanisms governing these biofilm development remain largely unexplored. Herein we applied artificial surface-based biofilm as an ideal model to study biofilm development in marine environments. The integration of ecogenomic techniques, including metagenome, metatranscriptome, metaproteome and single genome binning, facilitates understanding of mechanisms governing biofilm development in intertidal zone and deep-sea cold seep system.

In the first part of the thesis, biofilms were developed on artificial surfaces deployed in intertidal and subtidal zones near Hong Kong Water. Using comparative metagenomics, mechanisms governing biofilm development in the intertidal zone were illuminated. The genes responsible for resistance to metal ion and oxidative stresses were enriched in both 6-day and 12-day intertidal biofilms, compared to the subtidal biofilms. We proposed that a complex signaling network was used for stress tolerance and a model illustrating the relationships between these functions and environmental metal ion concentrations and oxidative stresses. This was the first successful use of metagenomic analysis on artificial surface-based biofilm model.

The second part of the thesis work focuses on biofild development under extreme environment. In the Thuwal cold seeps II, which comprises a brine pool and the adjacent normal bottom water (NBW). Biofilms were developed on the surface of different materials, iucluding aluminium (Al), polyether ether ketone (PEEK), polyvinyl chloride (PVC), polytetrafluoroethene (PTFE), stainless steel (SS) and titanium (Ti). The comparative metagenomic results demonstrated that biofilm developed on different surfaces used similar mechanisms. We presented different mechanisms of biofilm development from previous biofilm models. The role of polysaccharides in biofilm development was more complex and fundamental than previously reported (as a component of the matrix). The replacement of oxygen by nitrate as an electron acceptor was an important process in brine biofilm development. Remarkably, exploiting and organizing niche-specific functional features through inter-species interaction and generation of special micro-environments would be important mechanisms of biofilm-dependent adaptation.

We also used the artificial surface-based biofilm in the cold seep system as a model to test ecological sheories, such as species sorting, that is, the filtering by local environmental conditions is important in assembly of bacterial communities. We designed an experiment to investigate the effects of environmental switching between the brine pool and the NBW on biofilm assembly, which could reflect environmental filtering effects during bacterial immigration to new environments. Analyses of 16S rRNA genes of 71 biofilm samples suggested that the microbial composition of biofilms established in new environments was determined by both the source community and the incubation conditions. The results shed new light on microbial community assembly in special habitats and bridges the gaps in species sorting theory.

As a direct extension of the cold seep biofilm work, we performed a large scale genome binning work to study the environment-microbe interaction using cold seep biofilms as a model. Genome analyses of 66 biofilm members spanning seven phyla exemplified by Proteobacteria, Marinimicrobia and Cloacimonetes reveal genes coding for fermentative metabolism of recalcitrant carbohydrates and energy generation. Polysaccharide metabolism is further evidenced by metatranscriptomic reads mapping and in vitro characterization of enzymes catalyzing deconstruction of both α- and β-D-glucosides. The results suggested that polysaccharides affect both genomic content and post-genomic activity; simultaneously expression of diverse fermentative respiratory-related elements, together with enzymatic flexibility, underwrite microbial polysaccharide utilization.

To complement the naural biofilm study, we also performed study on the model single species, Pseudomonas aeruginosa biofilm. We applied iTRAQ-based quantitative proteomics to quantitify matrix-associated proteins isolated from different phases of Pseudomonas aeruginosa ATCC27853 biofilms. The increased abundance of stress resistance and nutrient metabolism-related proteins during biofilm development was consistent with the hypothesis that biofilm matrix forms micro-environments in which cells are optimally organized to resist stress and use available nutrients. Further analysis revealed complex interactions among these modulated proteins, and the mutation of selected proteins attenuated biofilm development. Interestingly, there was a good correlation between the abundance changes of matrix-associated proteins and their expression. Collectively, this work presents the first dynamic picture of matrix-associated proteins during biofilm development, and provides evidences that the matrix-associated proteins may form an integral and well regulated system that contributes to stress resistance, nutrient acquisition, pathogenesis and the stability of biofilm.

In summary, the main contribution of thesis work lies in establishment of a biofilm model to address ecologigical questions from multiply aspects. It integrates omics, molecular and biochemical methods to study biofilms. It contributes to understanding of classical ecological theories, microbial roles in biogeochemical cycles, microbial adaptation straties to extrem envrionments, as well as controlling strategy of biofilm development in the biofouling and bioinfection frameworks.