Isolation, Screening And Optimization Of Fermentation Conditions For The Multi-functional And Salt Tolerant Bacteria In Saline Lake Of Yuncheng

Saline of Yuncheng is a special water system that rich of mirabilite and salt,it salinizes the soil seriously.This experiment was conducted to isolate and identify the salt tolerant bacteria which could produce functional enzymes in the soil of Saline Lake in Yuncheng,understanding the characteristics of salt tolerant bacteria in this region.Combining some physiological and biochemical characteristics of salt tolerant functional bacteria,selecting different functional enzyme screening medium,optimizing the experimental conditions during the process of strains screening,by adding the enzyme specific substrate in the culture medium so as to targeted manner to screening the strains that produced different functional enzymes,this is more conducive to the enrichment and isolation of bacteria which produce different functional enzymes.At the same time,the fermentation conditions of the strains with high enzyme activity were optimized,which provided theoretical basis for the study of the enzyme production of moderately thermophilic bacteria.The main results are as follows:(1)A total of 20 strains of enzyme producing bacterial strain were isolated from the experiment,including 9 strains of protease producing strains,7 strains of amylase producing strains and 4 strains of lipase producing strains.The bacterial strains producing high protease activity were YH-7,YH-8,YH6-1.The strains were inoculated into the amylase screening medium and the lipase screening medium to detected,we found that the strain YH6-1 had the ability to produce amylase and lipase,it is a strain with more functions.The 3 producing high enzyme activity strains were identified by morphological indexes,physiological and biochemical indexes and molecular sequence identification.16 SrDNA sequence analysis results showed: the similarity of strain YH-7(accession number: KU363164)and Bacillus sonorensis NB RC101234T(accession number: AYTN010000)was to 99.85%.T strain YH-7 belongs to the genus bacillus,named the bacteria as Bacillus sp.YH-7.Strain YH-8(accession number: KU363162)is the highest similarity with halotolerans YIM 70185 T Zhihengliuella(accession number: DQ372937),reaching 99.91%,named the bacteria as Zhihengliuella sp.YH-8.The strain YH6-1 and flexus IFO 15715 T Bacillus(accession number: AB021185)the highest degree of similarity,reaching 99.93%,named the bacteria as Bacillus sp.YH6-1;(2)Through the research on the multi function of bacteria YH6-1,we found that bacteria YH6-1 not only produces protease,but also has a transparent circle on the screening medium of amylase and lipase,it showed that strain YH6-1 was a multifunctional enzyme producing strain,the strain could still be grown on the medium with 13% concentration of sodium chloride.The protease activity was highest at 0.5%.YH6-1 producing protease activity(1471.765 U/ml was 39.5% of that produced on the the medium with sodium chloride close to 0.5%(3726.732 U/ml)).(3)The amylase producing ability of strain sp.YH6-1 Bacillus was analyzed by single factor analysis.The results showed that when the inoculation concentration reached 8%,the strain YH6-1 producing amylase activity reached the highest(29.133 U/ml).When temperature was 30?,the enzyme activity reached the maximum(28.05 U/ml);when p H reached 8.5,the bacteria amylase producing ability was the highest;(4)Optimization of fermentation conditions for the production of lipase by strain YH6-1,results showed when the inoculation concentration reached 8%,the strain YH6-1 producing amylase activity reached the highest(4.09 U/ml).When temperature was 35?,the enzyme activity reached the maximum(4.56 U /ml);when p H reached 7.5,the bacteria lipase producing ability was the highest(3.83 U/ml);(5)Optimization of fermentation conditions for the production of protease by strain YH6-1,results showed when the inoculation concentration reached 10%,the strain YH6-1 producing protease activity reached the highest(3330.624 U/ml).When temperature was 35?,the enzyme activity reached the maximum(3413.354 U/ml);when p H reached 8.5,the bacteria protease producing ability was the highest(3996.49 U/ml);(6)The effects of carbon source,nitrogen source and inorganic salt ions on the fermentation of strain YH6-1 producing protease activity were studied,results showed that protease activity was highest when using beef extract as carbon source,reached 3469.21 Um/L;when yeast extract was used as nitrogen source,the protease activity reached the highest 4624.86 U/ml;the enzyme activity reached 4444.56 U/ml when the added ion was Mg2+.(7)By response surface software analysis: When p H was 8.63,the concentration of beef extract was 1.16%,and the amount of magnesium ion was 0.42%,the protease activity of strain YH6-1 was reached 4428.75 U/m L.