| In recent years,with the frequent occurrence and duration of water eutrophication and global warming,cyanobacterial blooms have broken out frequently around the world.It has already seriously destroyed the stability of aquatic ecosystem,caused enormous economic losses and posed a great threat to human survival and health.Microcystis aeruginosa is the dominant species of cyanobacteria bloom in China and even all over the world.It could produce and secret microcystins to environments,which has a serious impact on the growth of organisms in water and human health.Therefore,how to timely and scientifically prevent cyanobacterial bloom and eliminate the effects quickly and effectively has become one of the important environmental problems that need to solve.When cyanobacterial blooms have broken out in a large area,the main method to control is taking short-term effective emergency remedial measures.In contrast,biological methods have attracted more and more attention because of their simple operation,low pollution and strong specificity.Among them,microorganisms or microbial agents have become an important research topic in this field,which has great application potential for the removal of cyanobacteria blooms.In this research,microbial control and mitigation of M.aeruginosa was studied.Firstly,Functional bacteria was isolated and screened from mangrove wetland or phycosphere,then phylogenetic analysis,extraction and purification of active substances and functional studies of these microorganisms were preformd.The study mainly include one algicidal bacteria(strain SZ-14)and one flocculation bacteria(strain H9).Main results of this study are shown as follows:1.A total of 74 strains of bacteria were isolated from mangrove wetland environmental samples and phycosphere of M.aeruginosa.Among them,7 strains showed algicidal activity to M.aeruginosa cells,2 strains showed inhibitory activity and 1 strain showed flocculation activity.A total of 6 strains were potential new species.Three strains of GS-14(algicidal activity and potential new species),SZ-14(higher algicidal activity)and H9(unique flocculation strain)were studied in this research.2.New genus reclassification was performed for the algicidal bacterium GS-14.It had the highest 16S rRNA gene sequence similarity(96.63%)to Aestuariibacter aggregatus CGMCC 1.8995T.Values of ANI and dDDH between two strains were 79.48%and 21.9%.Together with the differences in morphological characteristics,physiological and biochemical characteristics and chemical composition,we propose that strain GS-14T represents a novel species of a new genus,for which the name Marisediminitalea mangrovi gen.nov.,sp.nov.is proposed.In addition,it is proposed that A.aggregatus CGMCC 1.8995T is reclassified as a member of the genus Marisediminitalea.3.Zooshikella sp.SZ-14 showed algicidal activity by means of prodiginines(PGs),including five linear and three cyclic types,and it is an unusual bacterium which could produce cyclic PGs.Two main PGs were obtained by means of silica-gel column chromatography and preparative HPLC.One was identified as prodigiosin(m/z=324)and the other was identified as cycloprodigiosin(m/z=322)by NMR.The other six PGs were identified as homologs of prodigiosin and cycloprodigiosin by LC-MS/MS.Among them,four other linear types of PGs were 2-methyl-3-propyl prodiginine(m/z=296),2-methyl-3-butyl prodiginine(m/z=310),2-methyl-3-hexyl prodiginine(m/z=338)and 2-methyl-3-heptyl prodiginine(m/z=352),respectively.Two other cyclic types of PGs were cyclohexylprodigiosin(m/z=336)and Cycloheptylprodigiosin(m/z=350).The method to extract and purify prodigiosin(m/z=324)was optimized and the method to extract and purify cycloprodigiosin(m/z=322)was established.4.PGs were mainly synthesized at the logarithmic phase in strain SZ-14,and the proportion of prodigiosin was the same as cycloprodigiosin,while only prodigiosin was synthesized at the stationary phase.Compared with two other PGs-produced srains(Z.ganghwensis KCTC 12044T and Hahella sp.KA22),strain SZ-14 had a higher PGs production and the ability to produce cycloprodigiosin.Analysis of genomes showed that there were a complete prodigiosin biosynthesis gene cluster,namely Pig cluster,occurring in the genome of strain SZ-14 and KCTC 12044.We found a hypothetical PGs cyclizing gene SZ14GL004460 in strain SZ-14 and B9G39 RS21880 in strain KCTC 12044,which might encode a homologous protein to PRUB680 of strain Pseudoalteromonas rubra DSM 6842.Results of gene quantification showed that the genes related to PGs synthesis and cyclization were normally expressed in strain SZ14,so it could produce linear and cyclic types of PGs during cell growth.However,no cyclic types of PGs produced because this cyclizing gene was unexpressed in strain KCTC 12044.Moreover,there was no PGs cyclizing gene detected in strain KA22.5.According to the results of proteome,we found a potential metabolic pathway of the biosynthetic substrates of PGs in strain SZ-14.Both proteins in the prodigiosin biosynthesic pathway and cyclizing proteins SZ14GL004460 were normally expressed and had the highest expressions at 15 h,in which the PGs synthesis rate reached a peak.During the growth of SZ-14,all proteins in the fatty acid synthesis pathway were downregulated,while all proteins in the fatty acid degradation pathway were up-regulated,indicating that 2-octenal,the precursor of prodigiosin synthesis,might come from the degradation of fatty acids.Moreover,the expression of ornithine cyclodeaminase(Ocd)and proline iminopeptidase(Pip)were significantly up-regulated,indicating that proline,another precursor of prodigiosin synthesis,might come from ornithine metabolism and degradation of polypeptide.In addition,a large number of serine and methionine also contributed to the biosynthesis of prodigiosin.6.Algicidal species and algicidal activity to M.aeruginosa were firstly compared for prodigiosin and cycloprodigiosin.Both prodigiosin and cycloprodigiosin showed obvious algicidal activity against a variety of harmful algae.Compared to prodigiosin,cycloprodigiosin had a poor photostability and a stronger inhibitory effect on the photosynthetic system(Fv/Fm and rRTR)of M.aeruginosa in the early stage of stress.While prodigiosin had a relatively strong photostability and the algicidal activity was higher in the late stage of stress.They could cause the rupture and death of algal cells,leading to the release and efflux of microcystins(MCs).However,both of them could inhibit the synthesis of MCs and reduce the total MCs in the environment compared to the natural growth.7.Under stress of prodigiosin,the transient respiration rate of M.aeruginosa was increased.Nucleoid became diffuse.Cell membrane permeability was enhanced and the integrity was damaged.Thylakoid was dispersed,the content of chlorophyll a was decreased and the photosynthetic capacity was inhibited.It was also found that the content of phycobiliprotein was increased in a short time(3 h)under the stress,and the related genes of phycobiliprotein synthesis,including phycocyanin β subunit(CpcB),allophycocyanin β subunit(ApcB)and the ferredoxin related gene(PcyA)were significantly up-regulated.It was speculated that prodigiosin hindered the absorption and utilization of red light,as a result,more phycobiliproteins need to be synthesized to capture light energy to maintain the cell growth.8.It was the first time to find that a bacterium(strain H9)belonged to Halobacillus could remove M aeruginosa quickly by bioflocculation.The flocculation mode of strain H9 to M aeruginosa was indirect-flocculation,and flocculation active substances were existed in the supernatant.Flocculation is in a dose-dependent manner,and when the algal cell density was not higher than 3×107 cells/ml,5%dose of supernatant could flocculate more than 90%of algal cells within 2 h.Strain H9 supernatant had excellent thermal stability and strong acid-base stability,which could meet the environmental conditions.Zeta potential of M aeruginosa cells was increased significantly after flocculation,indicating that charge neutralization was occurred during the flocculation process.Compared to the algicidal methods,H9 induced flocculation without visual cell damage.Hence,bioflocculation would not cause the release of MCs and had little impact on the environment. |
PDF Full Text Request