Biofilm Formation And Community Assembly In Porous Media

The vast majority of microorganisms on the earth exist in the form of biofilms,which are attached to the surface of solid particles,plant rhizosphere,marine sediments and so on.The abundance of microorganisms in soil is high,which is a hot spot for biofilm formation.Soil biofilms show great potential in crop production,soil improvement and environmental protection.However,the complex composition and highly heterogeneity of natural soil hampered the soil biofilms research.In this study,a microfluidic chip was designed to simulate the physical porous structure of soil,micropillars with diameter of 50 ?m were used to represent soil particles.With the help of laser confocal microscope(CLSM),fluorescent staining technology,and digital image analysis,we studied the dynamic processes of the morphology and extracellular polymer(EPS)of soil biofilms;What's more,combined with 16 S r RNA sequencing and bioinformatics,get the information about the changes of microbial communities in soil biofilms during the cultivation times.The main findings are as follows:The results of laser confocal microscopy showed that the processes of soil biofilms formation can be divided into four stages: initial adsorption stage(0-36 h),initial formation stage(36-48 h),development stage(48-96 h),and mature stage(96-108 h).Quantitative results showed that the thickness and roughness coefficient of soil biofilms increased with the cultivation time.At four observation times,the thickness of soil biofilms were 3.35 ± 0.34 ?m?3.68 ± 0.36 ?m?5.43 ± 0.68 ?m?12.00 ± 1.97 ?m,respectively.The variation of biofilm roughness coefficient is consistent with the variation of biofilm thickness.At 108 h,it has the largest roughness coefficient value.Different EPS components have different contributions during biofilms formation.In the early stage,polysaccharides and e DNA acted more closely,while proteins and polysaccharides acted more closely in the late times.The above results indicate that the structure and matrix components of soil biofilms can change dynamically during the formation process to adapt to the porous environment.In order to characterize the changes in microbial communities during biofilms formation under the porous environment,16 S r RNA sequencing was carried out.Thesequencing results showed that the composition of soil biofilms in the porous media had changed significantly.The community had the highest Simpson index,Shannon index and evenness index at 12 h,with the value: 0.84 ± 0.08?5.91 ± 1.74?0.55 ± 0.10,respectively;However,at 72 h,the above three indexes were at the lowest level,with the value: 0.17 ±0.09?1.10 ± 0.65?0.12 ± 0.06,respectively.The biofilm community structure fluctuated greatly in the early stage,but tended to be stable in the later stage.There is no obvious correlation between biofilm morphology and community diversity.Proteobacteria,Actinobacteria,Firmicutes,Bacteroidetes are the dominant categories of biofilm communities.The proportion of Proteobacteria in the early stage was less than that in the later time,after cultivation,it's relative abundance has increased by 31.9%;The proportion of Actinobacteria in the initial inoculation was 24.62% ± 1.84%,and it's ratio dropped to 5.59% ± 3.22% when cultured to 108 h.The ratio has decreased by 77.29%within cultivation periods.Pseudomonas is the most abundant genus of the community,it's number changes greatly in the early stage.It's relative abundance reached 95.76% ±1.89% at 72 h,which is the maximum value during cultivation times.Ochrobactrum,Herbaspirillum,Vibrionimonas,Dyella,Pseudarthrobacter,and Terrabacter were the remaining abundant genera.The relative abundance of Ochrobactrum,Vibrionimonas,Herbaspirillum,and Pseudarthrobacter have been changing during the cultivation periods.The relative abundance of Dyella had little difference before 36 h,and it's abundance continued to increase since then;The relative abundance of Terrabacter has decreased with the cultivation times,at the end of sampling,it's proportion decreased by nearly double compared with the initial inoculation.