Isolation And Identification Of Bacillus Amyloliquefaciens And Its Degradation Effect

The African ostrich,a member of the ostrich family,is native to Africa and the Arabian Desert and is the largest surviving herbivorous bird in the world.The herbivorous habits of the African ostrich are closely linked to its gut microbiology.The African ostrich is a herbivorous bird with a more developed colon and rectum than other birds,long and large and knobby,with a dense and highly diverse anaerobic flora that is thought to be beneficial for nutritional contributions and the host’s immune and defence systems.The microorganisms in the gut of herbivorous birds are predominantly Bacillus mimicus and thick-walled bacteria.Cellulolytic bacteria are very important in the gut system of the African ostrich,producing cellulolytic enzymes that degrade the less decomposable parts of the feed,improving feed utilisation,increasing the absorption of nutrient content,promoting animal growth and reducing the probability of intestinal disease,which is of great importance in terms of animal husbandry and feed production.In this study,African ostrich manure was collected and proliferated in enrichment medium,firstly,the strain was initially screened by carboxymethyl cellulose plate method,then re-screened by shake flask fermentation method,morphological observation and biochemical test were used for preliminary identification,r RNA was extracted by Trizol method,c DNA was obtained by reverse transcription,16 S r DNA gene was used for amplification,16 S rDNA sequence of the strain was determined,sequencing results were used The results were analysed by BLAST on the NCBI website and a phylogenetic tree was constructed using MEGA 11 software;the growth and enzymatic characteristics of the screened strains were then investigated and finally the fermentation and degradation effect of the strains on maize straw was further investigated.The results and conclusions of this study are as follows:1.15 samples of African ostrich faeces were collected aseptically and initially screened by sodium carboxymethyl cellulose plate method to obtain eight sample colonies,then Congo red staining and determination of cellulase(CMCase)activity,and one dominant strain H1 with an enzyme activity of 0.61 U/m L was screened for subsequent tests.2.The cellulolytic bacteria were identified by morphological identification,physiological and biochemical identification and 16 S r DNA analysis,and it was concluded that strain H1 belongs to the genus Bacillus and is the closest relative to Bacillus aryabhattai strain 5-Sj-2-6-2-M(KJ009541.1J),with 98% homology,and is a starch-dissolving bacterium.The growth curve was further determined and it was concluded that from 4 to 28 h,strain H1 entered the logarithmic growth phase;from 28 to 52 h of incubation it was in the basic stability phase and lasted for about 24 h.After52 h,it entered the decline phase.3.The biological properties and stability of Bacillus amyloliquefaciens H1 strain were investigated using the 3,5-dinitrosalicylic acid colour development method:(1)The effect of p H on enzyme activity and stability.p H 3-4.6,cellulase activity increased with p H;p H 4.6-7,cellulase activity was negatively correlated with p H.The optimum p H for cellulase secretion by strain H1 was 4.6.(2)The effect of temperature on enzyme activity and stability.Cellulase activity increases with the rise of temperature,when the temperature reaches 60℃the enzyme activity decreases with the rise of temperature,the optimum temperature for cellulase to react is 60℃,its enzyme activity is 0.8 U/m L;when the temperature is 40～50℃,the enzyme activity is around 83%,the stability is strong;after the temperature is higher than 50℃the enzyme activity starts to weaken rapidly.(3)The effect of reaction time on enzyme activity and stability.The enzyme activity peaked when the time was 72 h.When the reaction time exceeded 72 h,the enzyme activity decreased as the reaction time was extended.4、The enzyme production conditions of Bacillus amyloliquefaciens H1 strain were investigated,and the content of reducing sugars and soluble proteins of maize straw before and after fermentation were also measured.The optimum feed to water ratio was1:1.5 at 60 mesh(0.25 mm)sieve and the optimum bacterial solution inoculum was 3%.The highest values of reducing sugars and soluble protein in the maize straw fermentation broth were 2.134 mg and 41.5 mg on the third day after fermentation.In summary,this study successfully isolated a superior cellulolytic strain of Bacillus amyloliquefaciens H1 from African ostrich manure,which has good enzymatic activity and stability,and can effectively degrade maize straw,and explored the optimal feed addition,optimal maize straw crushing degree,optimal material to water ratio and optimal inoculum for the strain to be used in fermentation and degradation,which has certain reference significance for the further development and application of this strain..