Screening And Identification Of Lignin Degrading Bacteria And Whole Genome Sequencing Analysis Of Strain QL-Z3

Researchers are paying more and more attention to the development and utilization of biomass energy due to the unique advantages such as abundant,renewable and high cleanliness.Lignocellulose is the main component of biomass,and the complexity of lignin structure greatly limits the availability of natural fibrous materials protected by lignin from enzymatic hydrolysis.In addition,as a by-product of papermaking industry,lignin is difficult to be degraded so that it is easy lead to serious water ecological pollution.Therefore,research of isolating and domesticating high-efficiency lignin degradation strains and studying their degradation pathway have theoretical and practical meaning to utilize biomass energy and remediate contaminated water.In this study,the soil samples from deciduous soil in Qin Ling mountains of shaanxi province in order to obtain the efficient lignin-degrading bacteria.In the medium with alkali lignin as the sole growth carbon source,we studied the lignin degradation,depolymerization and enzyme secretion of Bacillus sp.QL-M1 and Erwinia sp.QL-Z3.On this basis,the lignin degradation gene of Erwinia sp.QL-Z3 was studied by whole genome sequencing.The main results are as follows:（1）Firstly,using the selected culture medium with alkali lignin as the sole carbon source,we obtained 10 strains with lignin degradation potential.Based on the cultural,morphological characteristics of the strains,and 16S r RNA gene sequence analysis,the bacteria belong to eight genera（Bacillus,Paenibcillus,Brevibacillus,Erwinia,Pseudomonas,Oerskscribe,Rhodococcus,Arthrobacter,respectively）of three classes（Bacilli,γ-Proteobacteria,Actinobacteria）.Then strains having ligini-degradation ability were screened by using the solid medium containing lignin structure analogues as sole carbon source.And the lignin-degrading enzyme activity was detected from the five strains by using plate culture with Azure-B and Remazol Brilliant Blue medium.we found the strain Bacillus sp.QL-M1and Erwinia sp.QL-Z3 showing remarkable lignin degrading performance.（2）Therefore the appropriate culture conditions of these two bacteria were determined.For Bacillus sp.QL-M1:lignin initial concentration of 0.5 g/L,initial p H value of9,（NH₄）₂SO₄ as nitrogen source and culture temperature is 25℃;The suitable culture conditions of Erwinia sp.QL-Z3 is:lignin initial concentration of 1.5 g/L,initial p H value of7,（NH₄）₂SO₄ as nitrogen source and culture temperature is 25℃.Under appropriate culture conditions,the lignin degradation related enzymatic activities of the strain QL-M1 reached to peak values at 48 h and 96 h:Mnp and Lip reached 298.76 U/L and 124.1U/L after 48 h incubation.Lac increased rapidly to 98.8U/L after 96 h incubation.The lignin degradation related enzymatic activities of strain QL-Z3 reached to peak values at 48 h and 60 h:Mnp reached 263.83 U/L after 60h incubation,Lip and Lac reached 104.11 U/L and 77 U/L respectively after 48 h incubation and then decreased slowly.（3）The scanning electron microscopy（SEM）was used to examine lignin samples to observe their morphological changes during degradation by Bacillus sp.QL-M1 and Erwinia sp.QL-Z3.The results showed that both strains could depolymerize the lignin samples,destroy the lignin structure and break it into small fragments.（4）The feasibility of biological pretreatment of Bacillus sp.QL-M1 and Erwinia sp.QL-Z3 strains in natural biomass raw materials（wheat straw,switchgrass and corn straw）was studied.The results showed that the fermentation broth of the two strains had certain biological pretreatment effect on the three substrates,and the effect of strain QL-M1 was slightly better than QL-Z3.（5）The whole genome of strain Erwinia sp.QL-Z3 was sequenced.The genome length of this strain’s chromosome is 4926645 bp,and the（G+C）%content is 55.1%.Contains a plasmid,plasmid genome length is 149,889 bp.（G+C）%content is 51.9%.The login number of strain QL-Z3 in Gen Bank was CP037949-CP037950.（6）Bioinformatic analysis shows that strains of QL-Z3 genome could contains abundant lignin-degrading genes encode several types of lignin-degrading enzymes,including the encoding genes of peroxidase（attack the main structures of lignin polymer）,β-etherase（cracking main connection lignin structure ofβ-Ο-4 linkage）,vanillic acid oxygenase、catechol 1,2–dioxygenase and protocatechuate 4,5–dioxygenase（cracking of benzene ring）and Fe-Mn superoxide dismutase and lactate dehydrogenase（provided H₂O₂ for other lignin peroxidase and manganese peroxidase）.The expression levels of these related to the degradation of lignin genes were higher when strain Erwinia sp.QL-Z3 was grown in a medium with lignin as the unique carbon source than in a medium with glucose as the unique carbon source.