Biodiversity,Physiological And Ecological Characteristics Of Small Bacteria In Freshwater Environments

As a basic component on the earth,aquatic bacteria not only motivate the material and energy flow within the food web,but also play important roles in the global biochemical processes.For the inland freshwater environments,indigenous bacteria have been proven to be closely related to the migration and transformation of material and energy within specific ecosystems and their dynamnic responses to global and regional environmental changes.Thus,it is essential to have an in-depth understanding of the importance and roles of bacteria in the ecological functions of freshwater environments.However,most of the aquatic bacteria can not be effectively detected and isolated through the conventional culture-based method(e.g.,heterotrophic plating),leading to a lack of complete understanding of aquatic bacteria to some extent.With the development of bacterial detection techniques,it has been indicated that these“un-cultured”bacteria generally had characterastics of small cell size,high flexibility,oligotrophic growth,and low nucleic acid-content.Hence,different terminologies,including“ultromicrobacteria,”“oligotrophic bacteria,”“filterable bacteria,”and“low nucleic acid-content bacteria”,have been used to define these bacteria.In this study,we used small bacterial communities and pure culture strains from different freshwater habitats to analyze their biodiversity,physiological characteristics,and ecological functions.Furthermore,we investigated the growth and survival mechanisms of small bacteria under ologotrophic conditions,and then revealed their important roles in the global material and energy cycles.The main research subjects and conclusions are summarized as follows:1.The study in Chapter 2 was to investigate the biodiversity,distribution,metabolic and ecological characteristics of small bacteria in a lake ecosystem.Three bacterial groups in the lake water were clearly identified in terms of cell size and nucleic acid-content through flow cytometric fingerprint combined with fluorescent staining.0.45?m-filtration was used to separate and enrich small bacteria from the total microbial community.Furthermore,their distribution,potential metabolic and ecological functions were investigated by amplicon sequencing,correlation analysis,and bioinformation tools.We found that small bacteria were the dominant bacterial group within the lake water,whereas they were phylogenetically less diverse than other bacteria.The external environmental factors have significant influence on the distribution of low-abundance small bacteria,among which the nutrient concentrations(e.g.,total nitrogen and phosphate)were the key factors that determined the distribution and growth of small bacteria.Additionally,several small bacterial taxa had potential pathogenicity and ability in degrading organic materials.2.The study in Chapter 3 was to investigate the dynamic changes of the groundwater microbial community and related nitrogen-cycling functions during the environmental disturbances.A bioreactor was used to mimic the environmental disturbances(i.e.,temperature and p H)that affected the small bacteria and nitrogen-cycling functions in groundwater ecosystems.The small bacteria accounted for a large proportion of the groundwater microbial community,among which most of them were symbiotic and parasitic.Upon the sharp changes of temperature and p H over a short period,the cell membrane structure of small bacteria was more tolerant than that of other bacteria to external disturbances.During the long-term mild changes,we found that small bacteria could not adapt well to the temperature increase,whereas they were resistant to the external p H decrease.Furthermore,the environmental disturbances led to the changes in groundwater bacterial structure,and further elevated the denitrification potential.Conversely,the nitrification potential was weakened.3.The study in Chapter 4 was to isolate and characterize pure small bacterial cultures,and then to investigate their genomic characteristics.In this study,a pure small bacterial strain,namely,“strain SW”,which belongs to the genus Brevundimonas,was isolated by the extinction dilution method from an aquatic environment.This strain was able to grow at the 1 mg/L dissolved organic carbon level with a maximum yield of10⁷cells/m L at the stationary phase.The scanning electron microscopic figure showed that this strain was short rod-shaped with a cell size range of 0.25-1.2?m.By comparing the bacterial genome with a model strain(eutrophic bacteria)of Brevundimonas in terms of Kyoto Encyclopedia of Genes and Genomes pathway,it could be found that strain SW had more integrated metabolic pathways than the model strain,indicating that strain SW could simultaneously use diverse nutrient sources for its basic growth and survival under oligotrophic conditions.4.The study in Chapter 5 was to investigate the ability of representative small bacteria in utilization of different phosphrous sources under oligotrophic conditions.PO₄^3--P and ATP-P were used as different P-sources.The results showed that PO₄^3--P could clearly enhance the bacterial growth.In contrast,the bacterial strain reached the stationary phase faster when ATP-P was provided as the P-source and maintained a constant cell concentration over a long period.The results of the polyphosphate concentration changes and the expression of relevant regulatory genes suggested hat ATP could not only be used as an energy source,but also be reserved in bacterial cells in the form of polyphosphate.During the stationary phase,the polyphosphate could gradually be hydrolyzed to support the long-term bacterial growth and survival.5.The study in Chapter 6 was to investigate the resistance and resilience of small bacterial strains isolated from drinking water sources to free chlorine exposure.A bioreactor was used to set up the chlorine disinfection and drinking water distribution processes.Three stages,namely,“inactivation stage,”“regrowth stage,”and“resistant stage”were set to mimic different chlorine exposure under the low carbon condition.Two small bacterial strains isolated from drinking water sources,Sphingopyxis sp.strain 15Y-HN and Polynucleobacter sp.strain CB,and two microbial indicators of drinking water quality were used for the comparison of resistant abilities to free chlorine exposure.The results indicated that the small bacterial strains were more tolerant than microbial indicators under the same conditions(i.e.,same carbon concentration,temperature,and p H).Futhermore,the strain 15Y-HN was more resistant to chlorine during the second chlorination stage.The results of the flow cytometric fingerprint and fluorescent staining on the basis of nucleic acid and cell membrane permeability analysis suggested that small bacterial strains and microbial indicators had different cell membrane component.The overall results indicated that it is difficult to oxidize the cell membrane of small bacterial strains by free chlorine,leading to the strong chlorine-resistant abilities of small bacteria.