Stablity And Dynamic Changes Of Community Composition Of Bacteria Attached To Cyanobacterial Colony

Cyanobacteria blooms caused by eutrophication are the focus of ecological management of lakes at present.Taihu and Chaohu are two typical eutrophic freshwater lakes in China.The mechanism of cyanobacteria blooms,including the causes of cyanobacteria blooms from the perspectives of water temperature,nutrients,hydraulic engineering,has been studied by many scholars,but it has not been fully clarified yet.Attached bacteria of cyanobacteria are essential material recyclers,energy converters and information transmitters in the occurrence and development of cyanobacteria blooms,and there are closely relationships between becteria and cyanobacteria.Therefore,it is of great significance to understand the relationship between cyanobacteria and bacteria and to reveal the development of cyanobacteria blooms by in-depth understanding of the stability and dynamic changes of community composition of bacteria attached to cyanobacterial colony.Combined with field investigation and laboratory microcosm experiments,this paper used 16S r DNA amplicon sequencing technology and bioinformatic analysis methods to explore the community composition and core species of bacteria attached to cyanobacteria of different species and different colony sizes in field.The key environmental driving factors affecting the bacterial community structure were analyzed.On the other hand,a colonial Microcystis aeruginosa isolated from Taihu was used to analyze the stability and dynamic changes of bacterial community composition in the culture system under the influence of temperature and concentration of nitrogen and phosphorus.1.Community composition of bacteria attached to different cyanobacterial species and the key driving environmental factors.In Taihu and Chaohu,colonies of different cyanobacterial species were identified and collected for 16S r DNA ampland sequencing and analysis.（1）NMDS analysis showed that there were significant differences in the composition of attached bacterial community among different cyanobacterial species in the two lakes（PERMANOVA,P<0.05）.The diversity index and richness of bacterial community of attached with Microcystis flos-aquae in Taihu was significantly higher than those attached with Dolichospermum flos-aquae（Wilcoxon rank-sum test,P<0.05）.The diversity and richness of attached bacterial community of D.flos-aquae in Chaohu was higher than those of Aphanizomenon flos-aquae（Wilcoxon rank-sum test,P<0.05）.The diversity of attached bacterial community in Microcystis wesenbergii was significantly lower than that in M.aeruginosa and M.flos-aquae（Wilcoxon rank-sum test,P<0.05）.（2）Burkholderiales and Rhizobiales were all dominant order in the attached community of D.flos-aquae collected from both lakes.Rhodospirillales and Rhodobacterales were the dominant order of attached bacterial community of M.flos-aquae.Burkholderiales and Rhodospirillales are the dominant order in babacterial community associated with colonial M.aeruginosa.（3）In Taihu,the relative abundance of the core dominant bacterial species,such as Cytophagaceae in the attached bacteria of D.flos-aquae,Ralstonia and Porphyrobacter in the attached bacteria of M.flos-aquae,Rhodobacter,Roseomonas and Cytophagaceae in the attached bacteria of M.aeruginosa,remained relatively stable.In Chaohu,the relative abundance of these core dominant bacterial species,including Rhodospirillales in the attached bacteria of D.flos-aquae,Phenylobacterium in the attached bacteria of M.aeruginosa,Rhodobacter and Porphyrobacter in the attached bacteria of M.flos-aquae,Alcaligenaceae and Cytophagaceae in the attached bacteria of M.wesenbergii,remained relatively stable.（4）Network analysis revealed that the interaction among bacterial communities attached with Microcystis spp.was relatively more intense compared with that of D.flos-aquae in Taihu Lake.The interaction between attached bacterial communities of M.flos-aquae in Taihu and Chaohu was strongest.（5）In Taihu,temperature and total phosphorus（TP）were the major driving environmental factors on distribution pattern of the attached bacterial community of M.aeruginosa.Nitrate（NO₃^-）was the major driving factor on distribution pattern of attached bacterial community of D.flos-aquae.In Chaohu,the total dissolved nitrogen（DTN）and ammonium salt（NH₄⁺）affected the distribution pattern of the attached bacterial community of D.flos-aquae and M.flos-aquae,while the total dissolved phosphorus（DTP）and temperature affected the attached bacterial community of the other three Microcystis spp.2.Community composition of bacteria attached to different particle sizes of cyanobacterial colony and its key driving factors.Colonies of different cyanobacterial particle sizes（>120μm,36-130μm and 3-36μm）were collected monthly in Taihu and Chaohu for 16S r DNA ampland sequencing and analysis.（1）NMDS analysis showed that there were significant differences in the composition of attached bacterial community among different cyanobacterial particle sizes between Taihu and Chaohu（PERMANOVA,P<0.05）.The diversity index and richness of bacterial community of attached with cyanobacterial colony at 3-36μm was higher than those of>120μm and 36-120μm in both lakes.（2）Burkholderiales is dominant order in bacterial community of cyanobacterial colony at>120μm and 36-120μm in both lakes.（3）The relative abundance of Rhodobacter,Roseomonas and Alcaligenaceae in bacterial community of attached with cyanobacterial colony at36-120μm and 3-36μm varied less than that of other core dominant bacterial species in Taihu.The relative abundance of Cytophagaceae and Ralstonia were relatively stable in bacterial community of attached with cyanobacterial colony at 36-120μm and 3-36μm in Chaohu,while the relative abundance of Comamonadaceae was also relatively stable in bacterial community of attached with cyanobacterial colony at>120μm and 36-120μm.（4）The bacterial community of cyanobacterial colony at36-120μm in Taihu was the most complex,while the bacterial community of cyanobacterial colony at 36-120μm in Chaohu was the least interrelated.（5）Temperature,NO₃^-,NH₄⁺,TP,DTP and phosphate(PO₄^3-)were the main environmental factors affecting the the attached community composition of cyanobacterial colony at three particle size in Taihu and Chaohu.3.Effects of temperature on composition and stability of bacterial community in colonial M.aeruginosa culture systems.A colonial M.aeruginosa strain was cultivated at different temperatures（15,20,25 and 30℃）to analyse the similarities and differences in the composition of different particle sizes（>20μm,3-20μm and 0.2-3μm）in each culture system.（1）Temperature significantly affected bacterial community compositions in all the three different particle size fractions including Microcystis colony（>20μm）attached bacteria,single cell-small colony（3-20μm）attached bacteria and free-living（0.2-3μm）bacteria（PERMANOVA,P<0.01）.（2）Sphingomonadales,Pseudomonadales and Cytophagales were the most dominant order in the three groups,accounting for 21.35%,19.74%and 33.44%of the total relative abundance,respectively.（3）There were some core dominant bacterial species in the three groups of bacterial communities,which were relatively stable in abundance and insensitive to temperature changes.The relative abundance of Brevundimonas and OPB56,the dominant genus among the core bacterial community attached with Microcystis colony,were relatively stable when temperature changed from 20 to 30℃.The relative abundance of Mariniradius,the dominant genus among the core bacteria attached with single cell-small colony,was also relatively stable when temperature changed from 20 to 30℃,and the relative abundance of Gemmobacter,the another dominant genus among the core bacteria attached with single cell-small colony,was relatively stable in response to temperature changes.The relative abundance of Porphyrobacter,the dominant genus among the core bacterial community in free living bacteria,were also relatively stable when temperature changed from 20 to 30℃.（4）Compared with the situations at other three higher temperatures,the complexity of species correlation network at 15℃was the highest but the cooperative correlation was the weakest.4.Effects of nitrogen and phosphorus concentrations on composition and stability of bacterial community in colonial M.aeruginosa culture systems.The colonial M.aeruginosa was cultured in nitrogen deficiency,oligotropher,mesotropher,eutropher and BG11,and the similarities and differences in bacterial community attached to different particle sizes in each microcosm culture system were analyzed.（1）NMDS analysis showed that the concentration of nitrogen and phosphorus significantly affected the bacterial community compositions in all the three different particle size fractions including Microcystis colony attached bacteria,single cell-small colony attached bacteria and free-living bacteria.The attached bacterial community composition was more similar under nitrogen deficiency,oligotropher,mesotropher conditions,while the difference of bacterial community composition increased under eutropher and BG11 conditions.Alpha diversity analysis showed that the community composition richness and diversity of the attached and free-living bacterial communities of M.aeruginosa decreased when the concentration of nitrogen and phosphorus was higher.（2）Rhizobiales is the most dominant order of Microcystis colony attached bacterial and single cell-small colony attached bacterial community,with relative abundance of 27.59%and 24.19%,respectively.Pseudomonadales was the most dominant order of free-living bacterial community,accounting for 27.62%.（3）The relative abundance of Brevundimonas,the core dominant genus of Microcystis colony attached bacterial community,and Pseudomonas,the core dominant genus of single cell-small colony attached bacterial community,were relatively stable at high nitrogen and phosphorus concentrations.The relative abundance of Rhizobium,the core dominant species of single cell-small colony attached bacterial community,fluctuated relatively little under five diffreent nitrogen and phosphorus concentrations.The relative abundance of Sphingobacteriales,the core dominant genera of the free-living bacterial community,was also relatively stable except in the BG11conditions.（4）With the increase of nitrogen and phosphorus concentrations,the complexity of species correlation networks increased gradually,and the complexity of species correlation network was still high under nitrogen deficiency conditions.The cooperative interaction between bacteria decreased with the increase of nitrogen and phosphorus concentrations.In conclusion,the composition and diversity of attached bacterial communities of cyanobacterial colony in field were significantly different at both species and particle sizes levels,but there were some relatively stable core bacterial species in each species and particle sizes.The dominant order and the stable core bacterial species of community of bacteria attached to the same cyanobacterial colony were different in Taihu and Chaohu.The bacterial community of cyanobacterial colony at36-120μm in Taihu was the most complex,while bacterial community of cyanobacterial colony at 36-120μm in Chaohu was the least interrelated.Water temperature and concentration of nitrogen and phosphorus were the main environmental factors affecting the composition of bacterial community of attached with cyanobacterial colony at different species and particle sizes in field.Laboratory microcosm experiments on colonial M.aeruginosa strain further showed that temperature and concentration of nitrogen and phosphorus significantly affected the bacterial community compositions.Under low temperature and low nitrogen and phosphorus concentrations,the diversity of free-living bacterial community increased,but under low temperature and high nitrogen and phosphorus concentrations,the complexity of species correlation network increased.There were also some bacterial species that were relatively insensitive to temperature and concentration of nitrogen and phosphorus changes in these particle sizes communities.No matter the bacterial community attached to cyanobacterial colony with multiple genera and multiple particle sizes in the field,or the bacterial community of a single colonial Microcystis in the laboratory culture system,the cooperation among bacteria was always dominant.