Screening And The Transcriptomic Studies Of Lignin Degrading Functional Genes Of Sporosarcina Sp.N2

As an important grain production base in China,Northeast China produces a large amount of agricultural waste such as crop straw every year.Straw is rich in lignocellulose,and lignin is difficult to be biodegraded due to its complex composition.In addition,the special temperature conditions in Northeast China result in slow biodegradation of straw under natural conditions.The breeding of efficient lignin degrading microbial resources,artificial inoculation to enhance straw biodegradation,and the promotion of straw return in cold regions have become research hotspots in this field.This study attempts to screen low temperature resistant and efficient lignin-degrading bacteria,optimize the conditions of lignin degradation by bacteria,and study the functional genes related to lignin degradation by the strains.The main results are as follows:Four strains of bacteria N2,K1,S2 and J5 capable of degrading lignin at low temperature（15℃）were screened from soil samples.Under the culture temperature of 15℃and the rotation speed of 150 rpm,their lignin degradation rates were 20.8%,19.48%,19.19%and 18.23%,respectively.By observing the decolorization ability of aniline blue,the strain N2 with the highest lignin degradation rate was finally selected for subsequent experiments.The strain was identified as Sporosarcina by 16S r RNA molecular biological identification and physiological and biochemical reaction test,and named Sporosarcina sp.N2.InLB liquid medium,Entering logarithmic growth phase at 1-3 days,the strain was in the stable phase at 3-9 d,After 9 days,the strain enters the decay phase.The strain increased from initial p H 5.5 to 8.4 on the 7th day.The maximum enzyme activity of Mn P was 152.26 U·m L^-1on the 2nd day,the maximum enzyme activity of Li P was 25.08 U·m L^-1on the 3rd day,and the maximum enzyme activity of Lac was 32.47 U·m L^-1on the 4th day.Optimize the degradation conditions of lignin by strain N2,selecting five conditions:inoculation amount,initial p H,lignin addition amount,loading amount,and nitrogen source addition ratio for single factor experiments.Through Plackett-Burman screening single factor test analysis,it was determined that inoculation amount,initial p H and nitrogen source addition ratio were finally determined as significant influencing factors.Subsequent response surface optimization experiments were carried out to determine the optimal culture conditions:dealkali lignin 0.5 g,yeast extract 5.0 g,tryptone 2.5 g,NaCl 5.0 g,p H6.5,liquid volume 50 m L/100 m L,temperature 15℃.Shaker speed 150 rpm.After optimization,the lignin degradation rate was 31.70%,which was 10.90%higher than that before optimization,and the lignin degradation effect was 52.4%higher than that before optimization.In this study,samples from the initial stage（1 day）,peak stage（3 days）and final stage（5 days）of N2 degradation were taken as research objects.Through transcriptome sequencing（RNA-seq）analysis,The enzyme genes related to lignin degradation were excavated.Through COG database and KEGG database annotation,the functional genes of strain N2 are mainly annotated to amino acid metabolism and carbohydrate metabolism,indicating that strain N2 has a large number of enzymes related to carbohydrate metabolism.A large number of lignin degradation pathways were enriched in functional enrichment analysis,such as phenylpropanoid metabolism,β-alanine metabolism,tyrosine metabolism,benzoic acid degradation,glycerolipid metabolism,fatty acid metabolism,etc.Among them,a large number of differential genes encoding degradation of aromatic compounds were significantly up-regulated,such as dehydrogenase,oxidoreductase,decarboxylase,and dioxygenase.It shows that Sporosarcina sp.N2 has the ability to degrade lignin.In summary,this study screened a strain Sporosarcina sp.N2 that can efficiently degrade lignin under low temperature conditions.The degradation ability of lignin was significantly improved by optimizing the culture conditions.Differential expression genes at different times were identified through transcriptome sequencing analysis,and lignin degradation related functional enzyme genes were analyzed through functional gene annotation.The research has enriched the microbial resources of low-temperature lignin degradation and provided data support for the subsequent low-temperature bio enhanced degradation of straw.