Flocculation Mechanism Of Streptomyces Sp. Hsn06 On Chlorella Biomass

Chlorella is an unicellular microalgae of the genus Chlorophyta,which widely distributed in natural environment.It possesses the ability of fast growth and high application value,and is commonly used in biofuels,medical raw materials,high-quality food sources and livestock husbandry.In recent years,improvements in the production of Chlorella have been achieved through transformation of photobioreactors,screening of high-yield strains and genetic modification of metabolic pathways.However,research and innovation in the downstream processes of microalgae treatment have been insufficient,and especially the acquisition of microalgae biomass has become the bottleneck of its industrial development.Microorganisms can secrete a large number of extracellular metabolites such as chitosan,polysaccharides,proteins,and glycoproteins during the growth process.The particulates are flocculated by charge neutralization or ion bridging,and the extracellular metabolites of microorganisms belong to the biological class.Therefore,these substances will not cause damage to environment.Therefore,microorganisms are always used to flocculate various types of particulate matter.Due to the extensive flocculation capacity of microorganisms,many researchers have used microbial flocculation technology to harvest microalgal biomass and obtain a large amount of flocculating microbial resources.However,due to the complexity of microbial metabolites and the specificity of "relationship between bacteria and algae",the flocculation mechanism of microbial flocculation on microalgae remains unclear.Therefore,this paper studies the flocculation mechanism of a flocculanting microorganism,Streptomyces,which lays a theoretical foundation for the collection of energy producing microalgae.Finally,the transcriptome sequencing was combined to determine the flocculation related genes.The main experimental conclusions of this paper are as follows:(1)According to the morphological characteristics,the 16 S r RNA gene sequence and phylogenetic analysis,strain hsn06 was confirmed as a species of the genus Streptomyces,for which name Streptomyces sp.hsn06 is proposed.(2)In this study,the bioflocculant from an actinomycete Streptomyces sp.hsn06 was used to harvest Chlorella vulgaris biomass.Consecutive treatment with 20 mg·L-1 bioflocculant and 5 m M Ca Cl2 for 5 min showed the highest flocculating activity.The bioflocculant was a nonprotein substance with thermal stability and p H stability,which can be used in comprehensive applications.Chemical analysis of the bioflocculant indicated that it is a small molecule substance of moderate polarity with containing triple bond and cumulated double bonds.Algal temperature,p H,and metal ions showed enormous effects on the flocculation efficiency of the bioflocculant.(3)Streptomyces sp.hsn06 exhibited flocculation activity on algal cells through mycelial pellets with adding calcium.Calcium was determined to promote flocculation activity of mycelial pellets as a bridge binding with mycelial pellets and algal cells,which implied that calcium bridging is the main flocculation mechanism for mycelial pellets.Characteristics of flocculation activity confirmed proteins in mycelial pellets involved in flocculation procedure.The morphology and structure of mycelial pellets also caused dramatic effects on flocculation activity of mycelial pellets.According to the results,Streptomyces sp.hsn06 can be used as a novel flocculating microbial resource for high-efficiency harvesting of microalgae biomass.(4)Streptomyces sp.hsn06 exhibited different flocculation ability after cultured with glucose(G)and starch(S)as sole carbon source,respectively.Samples G1-1,G2-1 and G3-1 which showed flocculation effect on Chlorella and samples S1-1,S2-1 and S3-1 which showed no flocculation effect on Chlorella were used to sequence by the Illumina Hiseq sequencing platform,and resulted in 18,031,906,200 reads,averaging 3 billion reads per sample.Through the statistical analysis of the data,2482 GO annotation sequences were obtained,and 5582 sequences with COG / KOGG annotation were obtained,811 differential genes were obtained,including 403 up-regulated genes and 408 down-regulated genes.Based on the G and S transcriptome databases,five significantly up-regulated genes were screened,which proved to be associated with protein.The q RT-PCR technique was used to verify the relative expression level of five proteins relatedgenes in actinomycete hsn06.The results showed that the gene BC342_RS16555,BC342_RS27795,BC342_RS16560,BC342_RS15410,BC342_RS01575 was significantly up-regulated in G vs S.The results of the validation were consistent with the results of the transcriptome sequencing.