| Lignin is a complex natural heteropolymer comprising phenyl-propanoid aryl-C3units linked via a variety of ether and carbon-carbon linkages. Lignin is resistant to biodegradation due to its complex structure and stable linkages. However, the lignin degrading microorganisms still can be found in nature, and the white-rot fungi, especially Phanerochaete chrysosporium, were considered as the most efficient lignin decomposer. Despite the study of fungal lignin degradation since the1980s, there is as yet no commercial biocatalytic process for lignin degradation, in part due to the practical challenges of fungal protein expression and genetic manipulation. On the other hand, more and more rencent investigations indicated that bacterial lignin degradation showed more advantages and broader practical application prospects in liquid media than the fungi.Two bacterial strains Novosphingobium sp. B-7and Comamonas sp. B-9were isolated from the steeping fluid of the eroded bamboo slips in our study. The two bacterial strains showed potentially efficient lignin degradation due to their vigorous growth on the medium containing lignin monomer compounds/kraft lignin/lignosulfonate as sole carbon source. To further determine the ability and practical application prospects in lignin biodegradation process, a systematic study on lignin degradation and related degradation genes of Novosphingobium sp. B-7and Comamonas sp. B-9were carried out. Through a series of studies, the research findings and conclusions of this paper are as follows:The lignin degradation capacity of genus of Novosphingobium and Comamonas were first found by a systematic test in this paper. By culturing in the liquid medium containing kraft lignin as sole carbon source for7days, Novosphingobium sp. B-7could reduce the lignin content, total carbon, chemical oxygen demand and color by38.84%,50.11%,34.7%,51.94%, respectively, as well as45.06%,47.23%,32%,53.97%by Comamonas sp. B-9. Simultaneously, the extracellular lignolytic enzymes were secreted by Novosphingobium sp. B-7and Comamonas sp. B-9to degrade lignin. The activity of manganese peroxidase (MnP) of Novosphingobium sp. B-7and Comamonas sp. B-9 were3229.8U·L-1and2903U·L-1, respectively, as well as1275U·L-1and1250U·L-1of Laccase (Lac). The results revealed that Novosphingobium sp. B-7and Comamonas sp. B-9could degrade lignin efficiently by using lignin as sole carbon source.The depolymerization pathways and decolorization mechanisms of Novosphingobium sp. B-7and Comamonas sp. B-9were demonstrated. The results of gel permeation chromatography and GC-MS showed that Novosphingobium sp. B-7and Comamonas sp. B-9were able to depolymerize lignin polymer into oligomer, further degrading into lignin monomer compounds and finally entering into the tricarboxylic acid cycle. There were a variety of valuable chemical products and alcohols produced by Novosphingobium sp. B-7and Comamonas sp. B-9via lignin bioconversion, such as vanillic acid and ethanediol. If the biocatalytic degradation of lignin by Novosphingobium sp. B-7and Comamonas sp. B-9could be harnessed and controlled, the practical application on lignin bioconversion to chemicals could be expected. In addition, from the UV and FT-IR spectra, the kraft lignin was decolorized by Novosphingobium sp. B-7and Comamonas sp. B-9via cleavage of the benzene rings, conjugated ethylenic linkages and conjugated carbonyl groups.The whole genomes of Novosphingobium sp. B-7and Comamonas sp. B-9were accurately sequenced, and the lignin-degrading genes were cloned, identified and analysed, respectively. Base on the whole genome sequences and bioinformatic analysis, the genome size and GC content of Novosphingobium sp. B-7were4.06Mb and65.11%as well as4.57Mb and63.62%of Comamonas sp. B-9, respectively.4639genes from Novosphingobium sp. B-7and4639genes from Comamonas sp. B-9were predicted by using Glimmer3.0.60lignin-degrading genes were annotated in Novosphingobium sp. B-7as well as45in Comamonas sp. B-9by comparing with the database of KEGG and COG. These genes encode several types of lignin-degrading enzymes, including the encoding genes of laccase-like phenol oxidases (attacking the main structures of lignin polymer), peroxidase,(3-etherase (cracking of β-0-4linkage), protocatechuate4,5-dioxygenase and catechol1,2-dioxygenase and (cracking of benzene ring). The sequence specific primers were used to clone22lignin-degrading genes in Novosphingobium sp. B-7and Comamonas sp. B-9by PCR and RT-PCR, and partial fragments of the corresponding genes were obtained. The22genes were identified and analyzed by resequencing and nr/nt database BLAST, and the results indicated that the whole genome sequencing and annotation of Novosphingobium sp. B-7and Comamonas sp. B-9were convinced.Due to their ability of degrading lignin into valuable chemicals, Novosphingobium sp. B-7and Comamonas sp. B-9could be used to develop the biotechnology on dealing with the environmental pollution caused by lignin, and could also be applied to produce valuable chemicals on lignin degradation. Moreover, according to bioinformatics analysis, Novosphingobium sp. B-7and Comamonas sp. B-9encoded the enzymes involving in the process of lignin polymer→oligomer→monomer benzene compounds→benzene ring-opening products→tricarboxylic acid cycle. These results convinced at gene level that Novosphingobium sp. B-7and Comamonas sp. B-9could grow and catabolize lignin as sole carbon source. Besides, this research provides a valuable data for using Novosphingobium sp. B-7and Comamonas sp. B-9to build genetically engineered bacteria for the production of lignin-degrading enzymes. In conclusion, it is a considerably potential application on lignin bio-conversion by Novosphingobium sp. B-7and Comamonas sp. B-9. |
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