A Preliminary Study On The Multiple Species Interaction Among Chlamydomonas Reinhardtii And Bacteria

Algae are important primary producers in aquatic ecosystems.Algae can provide nutrients for heterotrophic bacteria,at the same time,bacteria can produce beneficial or harmful substances which may affect algae,resulting in different types of bacteria-algae interactions.These interactions not only affect the viability and physiological state of them,but also have an important impact on the balance of the water ecological environment.Thus,the form and mechanism of interactions among algae and bacteria are worthy of in-depth study.Researchers normally focus on exploring the interaction between algae and a kind of bacteria in the past decades.However,there are multiple species with complex interactions under natural conditions.We still don't know much about the complex relationship among various species of bacteria and algae.Therefore,it is of great significance to establish a research system for studying multi-species interaction among bacteria and algae to deepen the understanding of complex interspecies relationships.In this study,we used the eukaryotic model algae Chlamydomonas reinhardtii,to co-culture with two different species of bacteria,and we explored the relationship among algae and bacteria in response to different temperatures and carbon sources through the analysis of the heat resistance of algae.This study has initially established a artificial system for studying the multi-species interaction between bacteria and algae,which provides a new foundation for the in-depth exploration of the role and mechanism of multi-species interactions of microorganisms.The main contents of this study are as follows:1:Enrichment and screening of Chlamydomonas reinhardtii interacting bacteria.The bacteria,separated and purified from the cultures prepared by co-cultivating the environmental sample with 21gr,were co-inoculated with 21gr under normal temperature and high temperature conditions,respectively.The types of bacteria that accelerate the death of 21gr(K type)and promote the heat resistance of 21gr at high temperature(P type)are obtained by phenotypic observation of 21gr.The 16S rRNA gene sequencing and comparison results of these strains show that the most strains were Proteobacteria with only two Bacteroidetes strains(K type).2:Interaction between Chlamydomonas reinhardtii and single bacteria.As algae are autotrophic organisms,and the isolated bacteria are mostly heterotrophic microorganisms,we explored whether different carbon source conditions can affect the interactions between Chlamydomonas reinhardtii and single bacteria.The results show that the acetic acid-containing medium lead to the consistent results with minimal medium,while the other three carbon sources have different influences on bacteria-21 gr interactions,indicating that certain carbon sources play a role in the interaction between 21gr and single bacteria.3:Interaction between Chlamydomonas reinhardtii and two different bacteria.21gr was co-cultured with the combination of two different bacteria(K or P type)to test how its growth is affected by the coexisting multiple bacterial species.The results show that the simultaneous presence of two bacteria in the minimal medium has no significant effect on the growth tendency of 21 gr at room temperature;at high temperature,while about 75%of the co-cultivation combinations enhanced the heat resistance of 21gr(P type),about 25%weakened the heat resistance of 21gr(K type).Compared with minimal medium,acetic acid containing medium will reverse the heat resistance of 21gr in the co-culture of a few combinations(21gr+K9+P9,etc.).The above results indicated that the culture system of the two bacterial co-cultures is more beneficial to promote the heat resistance of 21gr than single bacterial co-cultures.4:Research on the three-species co-cultivation system affected by carbon sources.To investigate whether the change in the heat resistance of Chlamydomonas caused by acetic acid in the few co-cultivation combinations mentioned above is due to the change in the relative number of bacteria,we selected 21gr+K9+P9 combination for the downstream analysis.The results show that the relative abundance of live bacteria under different carbon source conditions is not related to its actual effect,suggesting that there may be other cell number-independent mechanisms involved in this interaction;In addition,both K9 and P9 strains are sensitive to high temperature,and P9 can promote the heat resistance of K9 in the presence of acetic acid;acetic acid significantly promoted the growth of K9 and P9 at room temperature.5:Genomic analysis of strains K9 and P9.These two strains were selected for physiological and biochemical determination and genome sequencing analysis.The results showed that the 16S rRNA gene sequences of K9 and P9 had the highest sequence homology with Blastomonas natatoria and Hydrogenophaga sp.,respectively.The three-generation genome sequencing results of K9 and P9 provided genetic information to further clarify the mechanism of the multiple species interaction.In summary,we screened the bacteria with different effect on the heat resistance of 21gr,constructed a three partner co-culture system of algae and two bacteria,and performed a preliminary study of the multi-species interaction between bacteria and algae under different temperatures and carbon sources.Our study provides a new scientific basis for exploring the relationship between multi-species and its role in the shaping of microbial communities.