Studies On The Diversity Of Epiphytic Bacteria And The Functional Microorganisms Of Nitrogen Cycling Associated With Gracilariopsis Lemaneiformis

Economic macroalgae are major primary producers in coastal marine ecosystems as well as important builders and shapers in nearshore habitats.Algal epiphytic bacteria are very important for normal development and growth of the algal body,play a crucial role in the material circulation and energy flow of the marine ecosystem and a unique role in the biogeochemical cycle of marine carbon and nitrogen elements.Therefore,it is very important to understand the epiphytic bacterial diversity and community structure,functional microorganisms in epiphytic bacterial communities（EBC）,and ecological function of functional microorganisms to explore the interaction between algal host and its epiphytic bacteria.In recent years,most studies on algal epiphytic bacteria are only rest on the diversity of microbial communities,and there are few reports on the function of nitrogen cycling in algal epiphytic bacteria.Gracilariopsis lemaneiformis is a very important red macroalgae to produce agar,which has been cultivated on a large-scale along the southeast coast of China.The artificial algal farm formed by G.lemaneiformis has an important role in regulating the circulation of marine nutrient elements.Hence,it is of great significance for us to understand the ecological function of macroalgae to study the diversity and nitrogen cycling function of epiphytic bacteria associated with G.lemaneiformis.Thus,in this study:（1）The diversity and community composition of epiphytic bacteria on G.lemaneiformis were analyzed by using 16S r RNA gene amplicon sequencing technology,and the environmental factors affecting the EBC composition were discussed.（2）Urease-producing bacteria（UPB）were isolated from the cultivation environment of G.lemaneiformis.The effects of UPB on the physiological parameters of G.lemaneiformis were analyzed by using stable ¹⁵N isotope labeling technique,and the effects of UPB on urea absorption and utilization of G.lemaneiformis were further explored.（3）Nitrogenase activity,nif H gene abundance of epiphytic bacteria on G.lemaneiformis,and the diazotrophs community diversity associated with G.lemaneiformis and seawater in different cultivation periods were analyzed by using acetylene reduction,q PCR,and nif H gene high-throughput sequencing technology,and the nitrogen fixations of diazotrophs in different environmental conditions were further investigated.This study aims to explore the mechanism of epiphytic bacteria associated with macroalgae G.lemaneiformis influence on the algal host.The research results are of great significance for insights into the interaction between G.lemaneiformis and its epiphytic bacteria,and lay a foundation for further studying the action mechanism of nitrogen cycling functional microorganisms associated with macroalgae in the future.Main research results in this thesis are as follows:1.G.lemaneiformis and seawater samples were collected from different geographical locations（Nan’ao Island,NA,117°6′40″E,23°29′9″N;Nanri Island,NR,119°33′13″E,25°13′29″N;Lianjiang County,LJ,119°43′35″E,26°23′31″N）.G.lemaneiformis samples were used for extracting the total DNA of epiphytic bacteria,and the 16S r RNA gene Hiseq sequencing was performed after DNA purification.Seawater samples were used for the determination of various nitrogen and phosphorus nutrients.The data of environmental factors showed that there were significant（p<0.05）differences in nitrogen and phosphorus concentrations between different geographical locations.A total of 1,234,507 valid sequences were obtained from 23epiphytic bacteria samples,with an average of 45,722 sequences per sample.6,238OTUs were generated at 97%sequence identity classified into 11 phyla,21 classes,52orders,94 families,and 196 genera.There were considerable differences in the EBC composition associated with G.lemaneiformis in different geographical locations.For example,Bacteroidetes was the most predominant phylum on the surface of G.lemaneiformis in NR,with a relative abundance of 44.982%.While the most predominant phylum on the surface of G.lemaneiformis in NA and LJ was Proteobacteria,with relative abundance of 61.727%and 64.437%,respectively,which were significantly（p<0.05）higher than that of NR.RDA analysis showed that the relative abundance of Bacteroidetes,Firmicutes,Verrucomicrobia,and Epsilonbacteraeota in the EBC of G.lemaneiformis at NR were strongly positively correlated with nitrate nitrogen（NO₃-N）,total inorganic nitrogen（DIN）,total nitrogen（TN）,and total phosphorus（TP）.The abundance of Cyanobacteria in the EBC G.lemaneiformis at NA and LJ was markedly positively correlated with nitrite nitrogen（NO₂-N）.Moreover,the Mantel tests showed that there were significant correlation between the EBC composition associated with G.lemaneiformis and environmental factors,and Spearman correlation analysis also confirmed this.Thus,environmental factors such as NO₃-N and DIN play a pivotal role in the EBC composition associated with G.lemaneiformis.2.To study the diversity of UPB in the cultivation environment of G.lemaneiformis and the ecological effects of UPB on the algal host,different algal samples（G.lemaneiformis,Porphyra haitanensis）and seawater samples were collected from the Shen’ao Bay artificial seaweed farm（117°6′40″E,23°29′9″N）on Nan’ao Island,Guangdong Province.Eight UPB species containing ure C gene were obtained from the cultivation environment of G.lemaneiformis by isolation,screening,and PCR identification.There were some differences in the urease activity of different UPB measured by Berthelot reaction colorimetry.For instance,the urease activity of UPB associated with G.lemaneiformis was generally higher than that of UPB associated with P.haitanensis.The co-culture of UPB and the sterilized G.lemaneiformis was performed under different single nitrogen sources(CH₄¹⁵N₂O、¹⁵NH₄Cl)by constructing the macroalgae-bacteria co-culture system.Physiological analysis showed that urea consumption in the cultivation system increased significantly（p<0.05）after the addition of UPB or mixed epiphytic bacteria compared with axenic cultivation,and urea consumption in mixed epiphytic bacteria group was significant（p<0.05）higher than UPB group.In addition,elemental analysis showed that total nitrogen content in tissues of G.lemaneiformis increased dramatically（p<0.05）under the action of UPB compared with axenic cultivation.Stable ¹⁵N isotope analysis showed thatδ¹⁵N isotope values in tissues of G.lemaneiformis increased observably（p<0.01）under the condition of adding UPB or mixed epiphytic bacteria,δ¹⁵N isotope values in mixed epiphytic bacteria group was significant（p<0.01）higher than UPB group.The above results indicated that urea could be absorbed and utilized by G.lemaneiformis.UPB or mixed epiphytic bacteria significantly promoted urea absorption and utilization in G.lemaneiformis,and mixed epiphytic bacteria had a better promotion effect on urea absorption and utilization in G.lemaneiformis.3.To investigate nitrogenase activity,nif H gene abundance of epiphytic bacteria on G.lemaneiformis,and the variations of diazotrophs community diversity associated with G.lemaneiformis and seawater in different cultivation periods（cultivation Jan.,CJ;cultivation Feb.,CF;cultivation Mar.,CM;cultivation Apr.,CA）,G.lemaneiformis and seawater samples were collected termly from the Zoumapu Village artificial seaweed farm（117°2′39″E,23°28′32″N）on Nan’ao Island,Guangdong Province.Thirteen nitrogen-fixing bacteria species containing nif H gene were obtained from the surface of G.lemaneiformis and seawater by isolation,screening,and PCR identification.There were significant（p<0.05）differences in nitrogenase activity and nif H gene abundance of epiphytic bacteria on G.lemaneiformis in different cultivation periods by acetylene reduction and q PCR.Among them,the nitrogenase activity and nif H gene abundance of epiphytic bacteria on G.lemaneiformis in CM was significantly（p<0.05）higher than those in CJ,CF,and CA,indicating that the nitrogen fixation of eiphytic bacteria on G.lemaneiformis was significant enhanced.Combined with the data of environmental factors,it was found that the low concentration of nitrogen and phosphorus in CM might significantly enhance the nitrogen fixation of epiphytic bacteria on G.lemaneiformis.Total DNA of epiphytic bacteria on G.lemaneiformis and seawater bacteria were extracted and purified,and nif H gene Novaseq sequencing was performed after library construction.The results showed that theα-diversity of diazotrophs communities associated with G.lemaneiformis and seawater was higher than that in other cultivation periods.The results of community composition analysis showed that there were significant differences in diazotrophs communities on the surface of G.lemaneiformis in different cultivation periods,which were significantly diverse from the diazotrophs communities of seawater,but the diazotrophs communities of seawater were relatively similar in different cultivation periods.LEf Se analysis showed that Rhodobacterales,Hyphomonadaceae,Robiginitomaculum,and Robiginitomaculum antarcticum had a great influence on the differences between groups（GL.CJ vs GL.CF vs GL.CM vs GL.CA）.The above results suggested that Rhodobacterales,Hyphomonadaceae,Robiginitomaculum,and Robiginitomaculum antarcticum under Alphaproteobacteria played a significant（p<0.05）role in biological nitrogen fixation in response to nutrient deficiency.