Optimization Conditions Of Fermentation And Biocontrol Effect Of Bacillus Amyloliquefaciens H1 Research

According to the Food and Agriculture Organization of the United Nations（FAO）information,the world population has been growing every day,hence increasing food supply is important.In the purpose to increase crop yield,controlling plant disease is crucial.The five major crops such as wheat,rice,maize,potatoes are damaged by diseases or pests.The global range of yield loss ranges from 17 to 30%.Many researchers have been focusing on the potential of bio controlling microorganisms as viable alternatives for plant disease.Biocontrol is a technique of using organism controlling pests by organisms including insects,mites,weed and plant diseases by means of certain other living organisms or their products.It is factors of using pest management access environmentally safe and high potential for decreasing pests using natural enemies.It is an environmentally effective means of reducing or mitigating pests and pest effects using natural enemies.Microorganisms as viable alternatives for plant disease.Biological control was taking advantage agents were producing from predators,parasite insects and microorganisms（bacteria,virus,and fungi）.Microbial biological control agents（MBCAs）are applied to crops for biological control of plant pathogens where they through a range of modes of action,some microbial biological control agents interact with plants by inducing immunology of priming plants without any direct interaction with the targeted pathogen.Thus,these new solutions to plant disease problems provide control while minimizing negative consequences for human health and environment.The aim of research would like to study optimal condition and optimization of medium components for biocontrol application.Among endophytic bacteria Bacillus species,have obtained much attention because it has biocontrol ability and capable to be plant growth promote.Bacillus genus can induce plant resistant plant pathogen and they has broad spectrum,which can eliminate various fungal species.They capable to produced abundant non-ribosomal peptides,through directed synthesis by enormous enzyme complexes,that have modular structured organization and orderly link the amino acid residues of the final peptide the product from process is lipopeptide,inturin,and surfactin.These substances were used in use of biosurfactant for applications in foods,environmental management,biomedical fields,and cosmetics.they have shown their potential for use as major immunomodulatory molecules and antiadhesive agents.They are also being used for gene transfection,as ligands for binding immunoglobulins.They proved to be useful in eradicating nosocomial infections caused by different microorganisms by inhibiting their biofilms,and Biosurfactants can be used in production of green electricity as biosurfactants increase the surface area and emulsify hydrophobic compounds like oil also.This research using biocontrol potential of Bacillus amyloiquefaciens H1 to studied optimal condition and optimal composition of medium to enhance biocontrol activity which relate with lipocyclic compound production.The cells of this strain often appear as long chains,unlike many other Bacillus species that form as single cells.That in this research aim to find sustainable of biocontrol for replace chemical fungicide and it has good properties to use in crops and improve immune in plants.Among microorganisms Bacillus species,have obtained so much attention.Bacillus amyloiquefaciens H1 has a good biocontrol efficiency.It has a broad spectrum of antifungal activity with co culture method by incubating microorganisms at 28℃for 5days.The result has clear-zone around mycelial of fungus,that shows inhibition activity,because it could produce antifungal substance.This bacterium was used to control 6pathogens being Fusarium solani,Alternaria solani.Sauer,Fusa Hum graminearum Sehw,Fusarium oxysporum,Sclerotinia sclerotiorum de Bary,and Alternaria tenuissima.These pathogens mainly cause plant diseases and represents a problem in agricultural crops,which lead to yield loss.This research was using Bacillus amyloiquefaciens H1.It was isolated from the forest at Dailing District,Yichun City,Heilongjiang Province,after observed its phenotype by using optical microscope and gram stain method found H1 strains has rod shape and gram positive and H1 strains is the one that has a good biocontrol efficiency.The phenotype of H1 strain was observed by light microscope and Gram staining.H1strain was rod shaped and gram positive.H1 strain and pathogen were cultured at 28℃for 5 days by duo culture method.The results showed that H1 strain had clear zone surround plant pathogen mycelium,that shown antifungal substance producing.Before optimization,must observe the growth curve of strain H1 to find the optimal to prepare seed inoculation,at of 8h ferment duration.The seeds were inoculated in BPY medium to observe the inhibitory activity.The inhibition activity of antibiotics substance was studied by extract supernatant of H1 strain by using centrifuge at 4℃ and 15000 rpm,25min,after that filter the supernatant through 0.22μm diameter of membrane,which this supernatant will be used in inhibition activity.In this study,strain H1 was separated from F.solani,A.solani Sorauer,F.graminearum,F.oxysporum,S.sclerotiorum,and,A.tenuissima 6 kinds of pathogenic microorganisms,such as,which are the main causes of crop diseases,were tested in the antagonistic activity.After cultured at 37℃for 5 days,there were obvious antibacterial spots around the mycelium,and strain H1 showed inhibitory activity.The results showed that strain H1 had the ability to produce antifungal substances.The inhibition rates of strain H1 to different pathogens were F.solani 23.56%,A.solani Sorauer 30.18%,F.graminearum 27.06%,F.oxysporum 29.26%,S.sclerotiorum 26.97%,A.tenuissima 32.10%.In the purpose to enhance the biocontrol ability of B.amyloiquefaciens H1,this research has improved the composition of the fermentation medium by substituting carbon sources,nitrogen sources,and inorganic salts,because these nutrients were composition of antibiotics production.They have roles in electron exchange in the reaction.BPY medium was used for optimized medium.Carbon source is an energy source and has an important role in the growth the same as in the production of primary and secondary metabolites.The rate at which the carbon source is metabolized and can influence the formation of biomass and the production of primary or secondary metabolites,nitrogen source has a critical effect on metabolite production.The microorganism can utilize both inorganic and organic sources of nitrogen.The carbon source of the BPY medium was changed from glucose into mannitol,maltose,soluble starch,dextrin,and sucrose Among carbon sources used.mannitol had the highest inhibition rate.The peptone was used as a nitrogen source,but after substituting nitrogen sources,it was found that NH₄Cl had the highest potential to inhibit plant pathogens.Inorganic salt in the BPY medium which is NaCl was changed by MgCl₂,CaCl₂,KCl and,LiCl,where KCl had the most antifungal rate.Afterwards,using single factor and orthogonal method,the result has showed the optimal composition of medium（g/L）being ammonium chloride 9 g,mannitol 9 g,potassium chloride 5 g,yeast extract 5 g,beef extract 5 g,in 1000 ml of water,this composition increased antagonistic spectrum to up to 37.03%.The optimization of fermentation conditions were carried out using this formula of medium composition:NH₄Cl 0.9 g,mannitol 0.9 g,KCl 0.5 g,yeast extract 0.5 g,beef extract 0.5 g in 100 ml.Optimizing condition were conducted by varying inoculum size1%,2%,3%,4%,5%,6%,and 7%,in shaker at 37℃and rotation rate of 180 rpm for 48h.Results showed that 2%of inoculum size where it had 47.89%of antifungal rate.The initial pH of the medium varying from 5,6,7,8,and 9 respectively in a shaker condition at 37℃under rotation rate of 180 rpm for 48 h.It was found that at pH 6 was optimal because it had 49.57%of inhibition rate.The temperature of fermentation was varied from 25℃,28℃,31℃,34℃,37℃,and 40℃ in shaker with rotation rate of 180 rpm for48 h.The temperature of 28℃,was optimal for the microorganism to secrete antifungal substance with an inhibition rate of 49.86%.The fermentation time was varied from 12 h,24 h,36 h,48 h,60 h,and 72 h and cultured in shaker at 37℃and rotation rate of 180rpm where best performances of fermentation was found at 36 h and highest inhibition rate reached 51.20%.Therefore,optimal conditions of fermentation with H1 strain were:2%of inoculum size,initial pH of the medium at pH of 6,temperature of 28℃ and fermentation time of 36 h,and under these conditions,inhibition rate reached 48.48%,which was higher than before optimized medium.The secondary metabolite producing H1 strain must study stability further,because it contains a lot of secondary metabolites compounds,and each compound can stable in different condition.The activity of lipopeptides depend on pH value,because lipopeptide has activate groups such as ionic groups,carboxyl group,amino group,thiol group,imidazole group and,phenolic hydroxyl group,and so on.which these structures have active side to affinity to other substrates,in environment has variety of pH,because in root rhizosphere can lead induced pH change all the time.The stability of the antibiotics was studied by adjusting the pH of supernatant adding HCl and Na OH into 3,4,5,6,7,8,9,10 and 11 and mixed it with PDA medium at 55℃,after that,mycelial of pathogenic fungal were placed in the center of the plate,cultured for 5 days.At pH of 5-8,a highest antibiotic activity was still present in 75%.Studying the effect of metal ions on antibiotics activity,the same culture method as the pH effect study was carried out.Ca²⁺can improve antibiotic activity.It has inhibition rate of 41.82%.which was higher than the control（24.27%）.And this means Ca²⁺can increase antibiotics ability.Also,another ion influenced antibiotic activity.Cu²⁺had 30.02%of inhibition rate.While Mg²⁺,Na⁺and K⁺had a little effect on antagonistic activity,because when compared with control it still has stability to control plant pathogen,at the same time K⁺has 34.80%of antifungal rate,but Na⁺has no affect on biocontrol activity,and Mg²⁺had 20.41%of biocontrol rate,which according to control it is a small effect with lipopeptide activity.At the same time Li⁺and Zn²⁺reduce antibiotics activity when added to the supernatant of H1 strain.It only has antifungal rate of 11.89%and 10.69%respectively.It decreases antibiotics activity because of its partial denaturation or the decrease in ion exchange in antibiotics.