Screening And Identification Of Alginate Degrading Bacteria And Optimization Of Fermentation Conditions

Apostichopus japonicus is a marine organism with a high nutritional and economic value.The body wall of Apostichopus japonicus is nutritious with biologically active substances.With the rapid development of the sea cucumber breeding industry,large-scale and intensive aquiculture is becoming the new trend for sea cucumber production.However,the cost of feeding and seeding of the intensive aquaculture accounts for about 54%of the total cost,and there will be a huge economic loss when the feed is not fully used.This study aimed to degrade alginate in kelp with the biologically method,in an effort to improve the efficiency of feed utilization and increase the additional value of feeding products yield by kelp.The main work of this thesis was as follows:1.A total of 15 strains with alginate-degrading ability were screened from rotting kelp and laboratory preserved samples of terrestrial origin,and identified by 16S r RNA sequencing.Most of the strains were identified as Pseudoalteromonas.To further screen the ideal strain,the selected strains were subjected to the determination of alginate lyase enzyme activity.Considering the safety of the strain and the enzyme activity of alginate lyase,the strain E1 was selected for the following experiments.2.Through the methods of molecular biology,physiological and biochemical analysis,strain E1 can be confirmed as Paenibacillus pabuli and it can use a variety of carbohydrates as energy materials for growth.The strain E1 showed a high ability to produce specific mannuronic acid lyases,which can form an 18-20 mm transparent circle in the solid selective medium with alginate as the only carbon source.3.In the experiment of kelp fermentation with different ratio of material to solvent,it was found that when the ratio was 1:3,the alginate lyase activity and the amount of alginate degradation reached the highest at 96 h.Through the investigation of fermentation time,fermentation temperature and inoculum amount,it was found that when the fermentation time was 96 h,the fermentation temperature was 37°C and the inoculum amount was 10¹⁰ CFU/m L,the activity of alginate lyase and the degradation of alginate reached the highest level.4.Box-Behnken Design experiment with reference to the optimal fermentation conditions obtained by single factor.The interaction between fermentation time X₁,fermentation temperature X₂,and inoculum amount X₃ was investigated by response surface analysis.The optimized fermentation condition was obtained through regression analysis:the fermentation time was 102.28 h,the fermentation temperature was 34.15℃,and the inoculum was 2.4×10¹⁰CFU/m L.Under these conditions,the maximum alginate lyase activity predicted by the regression model was 53.04 U/m L.Experiments verified that the actual fermentation enzyme activity could reach to 50.21 U/m L,and the amount of alginate degradation was 61.2%.In conclusion,Paenibacillus pabuli E1 can produce alginate lyases with a high alginate degradation ability,and the strain can effectively degrade alginate in kelp.The study provided evidence that Paenibacillus pabuli E1 has a certain application potential in the preparation of kelp feedstuff with low alginate content.