Studies On The Algicidal Characteristics And Mechanisms Of Bacillus Licheniformis Sp34 And Its Algicidal Substances

The water eutrophication and outbreak of harmful cyanobacteria blooms are one of the major environmental problems in the world.The outbreak frequency and intensity of cyanobacterial blooms are increasing worldwide,which could cause severe problems,e.g.,damage the structure and function of aquatic ecosystems,affect the healthy development of tourism,aquaculture and other industries,and directly threaten human life and health.Besides,bacteria,an important component in the structure and function of aquatic ecosystems,play an important role in maintaining the balance of algae biomass.Therefore,the utilization of algicidal bacteria in the biocontrol of blooms have received great attention.Dianchi Lake,the largest plateau lake in China,is of great important economic and ecological significance.However,it has been colonized by cyanobacteria blooms for the last two decades.Therefore,the algicidal characterizations and mechanisms of algicidal bacteria and the algicidal substances released from it were studied to provide biocontrol agent resources and theoretical guidance for cyanobacteria bloom controlling in Dianchi Lake.The major consequences are listed as follows:1.Isolation and identification of algicidal bacteria.A strain,Sp34 with strong algicidal activity against Microcystis aeruginosa was isolated from Dianchi Lake.Sp34was identified as Bacillus licheniformics according to their cell morphology and the analysis of 16S rDNA gene sequences.2.Studies on algicidal characterizations of Sp34 against M.aeruginosa.Sp34could kill M.aeruginosa DCM4 with an initial Chl-a concentration as high as almost2.0 mg/L with a cell density of no less than 1.35×10⁵ CFU/mL.Besides,the algicidal efficiency against M.aeruginosa NIES843 can reach 97%on the 10^th day,indicating that Sp34 could kill M.aeruginosa efficiently.Sp34 can also inhibit the growth of some other harmful algae species,including M.wesenbergii and Phormidium sp.Among them,M.aeruginosa is one of the most common bloom-causing species,which can produce microcystins,a kind of poisonous metabolite,thus posing a serious threat to environmental safety and human health.Those results indicate the great potential of Sp34 in cyanobacterial bloom control.Sp34 killed M.aeruginosa DCM4 through the release of algicidal substances,which have good heat and acid-base resistance,and it can provide guidance and support for the isolation and purification of the algicidal substances.3.Studies on influence factors of algicidal activity of algicidal substances produced by Sp34.The algicidal efficiency of Sp34 supernatant against M.aeruginosa under 24 h light and 14:10 h light-dark cycle were much higher than that of dark conditions,indicating that its algicidal activity was relatively light-dependent.In addition,the algicidal activity under 28°C was significantly higher than that of 15°C,while the growth of algae was promoted at 28°C and inhibited at 15°C.Combined with the significantly positive correlation?p<0.001?between algal growth of control group and algicidal ratio of treatment group in both illumination and temperature experiments,it was confirmed that the higher algicidal efficiency of algicidal substances released from Sp34 depended on faster growth of M.aeruginosa.So,the application of strain Sp34 or its algicidal substances in the controlling of fast developing M.aeruginosa bloom is promising.4.Studies on the algicidal mechanisms of algicidal substances produced by Sp34.After treated with Sp34 supernatant,the integrity of M.aeruginosa cells was severely damaged.The activity of catalase?CAT?and the content of malonaldehyde?MDA?in M.aeruginosa cells were significantly increased,and the content of reduced glutathione?GSH?was first increased and then significantly decreased for 28 h.Sp34 supernatant may cause oxidative stress and then lipid peroxidation in algal cells.This study confirmed that the algicidal substances produced by Sp34 seriously inhibited the transcriptional expression of photosynthesis-related genes?psaB,psbD1 and rbcL?,DNA repair gene?recA?and microcystin synthesis gene?mcyB?in M.aeruginosa cells.The transcription of psaB,psbD1,rbcL,recA and mcyB were down regulated?0.02,0.03,0.23,0.31 and 0.07 fold relative to control group,respectively?after treated for 24 h.Therefore,it is speculated that the algicidal mechanisms of Sp34 against M.aeruginosa was as follows:the algicidal substances released from Sp34 caused oxidative stress and triggered the antioxidant mechanism in algae cells,which leads to the increase of CAT activity and the decrease of GSH content;then the oxidative stress further caused lipid peroxidation reaction in algae cell membrane,which leads to the increase of lipid peroxidation product?MDA?.Next the algicidal substances damaged the integrity of algae cells.And caused DNA damage and dysfunction of DNA repair function,weakened photosynthesis system and inhibited microcystin synthesis of M.aeruginosa cells,finally lead to the lysis and death of algal cells.5.Identification of tryptamine and studies on its algicidal characteristics against M.aeruginosa.Sp34 could use tryptophan to produce tryptamine.And Sp34 can release tryptamine when grown in 1/8 LB medium.Tryptamine is a natural algicide,with good algicidal activity against M.aeruginosa DCM4.Sp34 killed DCM4 through the release of extracellular algicidal substances.Therefore it is presumed that tryptamine is one of algicidal substances produced by Sp34.The algicidal efficiency of 6ppm of tryptamine against M.aeruginosa DCM4 was 80.0%on the 5th day.Similarly,the algicidal activity of tryptamine was also relatively light-dependent and temperature-dependent.Besides,combined with illumination and temperature experiments,the algal growth of control group were also significantly positive correlated with the algicidal ratio of treatment group?p<0.001?,so the algicidal activity of tryptamine against M.aeruginosa also depends on the well growth of alage.6.Preliminary study on the algicidal mechanisms of tryptamine against M.aeruginosa.Tryptamine can stimulate the accumulation of ROS in M.aeruginosa cells,and increase the level of intracellular oxidation significantly,which may cause oxidative damage to M.aeruginosa cells.The concentrations of total protein and sugar in M.aeruginosa cells decreased significantly,but the protein and sugar content of per cell increased,which indicated that tryptamine may inhibit the cell division of M.aeruginosa,and seriously affected the M.aeruginosa growth.In conclusion,the algicidal characteristics and mechanism of an algicidal bacteria isolated from Dianchi Lake and the algicidal substances produced by it against M.aeruginosa were investigated.This research will provide biocontrol agent resources and theoretical guidance and support for the biological control of cyanobacteria bloom.