| Abstract:Lignin is the most abundant aromatic compound on earth and is second only to cellulose in its contribution to living terrestrial biomass. The structural complexity of lignin, its high molecular weight and its insolubility make its degradation very difficult. Microbio-degradation of lignin has become a research hotspot, mainly focused on the fungi. In recent years, a variety of literature has shown that given the immense environmental adaptability and biochemical versatility, bacterial could play a potential role in lignin degradation.In present study, two bacterial strains Pandoraea sp. B-6and Cupriavidus basilensis B-8were isolated from the steeping fluid of the eroding bamboo slips. Characterization and biochemical and molecular mechanisms of lignin and aromatic compounds degradation were studied. Through a series of studies, the research finding and conclusions of this paper are as follows:Kraft Lignin (KL) degradation capability of Pandoraea sp. B-6and Cupriavidus basilensis B-8were firstly investigated. The optimum pH and temperature for KL degradation were10and30?, respectively, for Pandoraea sp. B-6, and7and30?, respectively, for Cupriavidus basilensis B-8. At least38.2%of COD and41.6%of color for Pandoraea sp. B-6, and31.3%COD and37.5%color for Cupriavidus basilensis B-8could be removed in7days under the initial concentrations from1g/L to6g/L. The extracellular peroxidase enzymes were detected during KL degradation by these two strains. The greatest activities of2249.2U/L for manganese peroxidase and1120.6U/L for laccase from Pandoraea sp. B-6, and1120.6U/L for manganese peroxidase and815.6U/L for laccase from Cupriavidus basilensis B-8were observed. Many intermediates such as ferulic acid, cinnamic acid and so on, were formed during the period of KL degradation base on GC-MS analysis. Thus we predicated that these two strains were able to depolymerize lignin polymer into oligomers and fuither degrade into lignin aromatic monomer compounds and other small molecule compounds and finally enter into tricarboxylic acid cycle.Pandoraea sp. B-6and Cupriavidus basilensis B-8grew well and displayed great degradation capability in the medium using ferulic acid and ?-coumaric acid as the sole carbon source, respectively. The optimum pH and temperature for these two compounds degradation were all10and30?, respectively, except that the optimum pH for ?-coumaric acid by Pandoraea sp. B-6was8. The maximum concentration of complete degradation for these two compounds degradation were all1000mg/L, except that for ?-coumaric acid by Pandoraea sp. B-6was800mg/L. The HPLC analysis of aromatic compounds degradation by these two strains showed that ?-hydroxybenzoic acid was the only compounds detected during the process of ?-coumaric acid degradation by Pandoraea sp. B-6and Cupriavidus basilensis B-8, and the maximum cumulative concentration were169mg/L and253mg/L, respectively. The vanillic acid and vanillin were detected duri4ng the process of ferulic acid degradation, the maximum cumulative concentration were426mg/L and193mg/L, respectively, by Pandoraea sp. B-6, and238mg/L and less than30mg/L, respectively, by Cupriavidus basilensis B-8. In addition,4-vinylguaiacol was detected during the process of ferulic acid degradation, the maximum cumulative concentration were548mg/L. RT-PCR analysis showed that these two compounds were all degraded through ?-ketoadipate central pathway, furthermore, another ferulic acid degradation pathway, reductive transformation pathways, were found.The whole genomes of Pandoraea sp. B-6and Cupriavidus basilensis B-8were accurately sequenced. The Pandoraea sp. B-6genome consists of a single circular chromosome of5,037,162bp (136contigs) with an average G+C content of63.56%. The G+C content of the coding region (64.02%) is higher than the G+C content of the noncoding region (60.82%).4803putative coding sequences (CDSs) with the length of4,312,731bp were identified. It contained4692or4695protein-coding genes,55tRNA genes representing all20amino acids,5scattered ribosomal RNA genes,3or5rRNA genes,31sRNA genes and15miRMA genes. Cupriavidus basilensis B-8genome consists of a single circular chromosome of8,705,938bp (1458contigs) with an average G+C content of65.40%(Fig.1). The G+C content of the coding region (65.62%) is higher than the G+C content of the noncoding region (64.29%).8,448putative coding sequences (CDSs) with the length of6,740,730bp were identified. It contained8371protein-coding genes,65tRNA genes representing all20amino acids,12rRNA genes.Many lignin and aromatic compounds degradation related genes and metabolic pathways were founds through bioinformatics analysis combined with RT-PCR. In Pandoraea sp. B-6,119related genes were identified. Four metabolic pathways for lignin degradation included ?-ketoadipate central pathway, gentisate pathway, the2,3-dihydroxyphenylpropionate meta ring-cleavage pathway and box pathway, five metabolic pathways for other aromatic compounds degradation included3,4-dihydroxyphenylacetate meta-cleavage pathway, the phenylacetyl-CoA ring-cleavage pathway, homogentisate pathway,2-aminobenzoyl-CoA pathway and3-hydroxyanthranilate pathway were confirmed. In Pandoraea sp. B-6,165related genes were identified. Five metabolic pathways for lignin degradation included ?-ketoadipate central pathway, phenol degradation pathway, gentisate pathway, the2,3-dihydroxyphenylpropionate meta ring-cleavage pathway and box pathway, five metabolic pathways for other aromatic compounds degradation included3,4-dihydroxyphenylacetate meta-cleavage pathway, the phenylacetyl-CoA ring-cleavage pathway, homogentisate pathway,2-aminobenzoyl-CoA pathway and3-hydroxyanthranilate pathway were confirmed.These results confirmed the capability of Pandoraea sp. B-6and Cupriavidus basilensis B-8to promote lingnin and aromatic compounds degradation. Whole genomic sequencing and systematic analysis of the Pandoraea sp. B-6and Cupriavidus basilensis B-8genome identified degradation steps and intermediates. Our findings provide a theoretical basis for research into the mechanisms of lignin and aromatic compounds degradation as well as a practical basis for biofuel production using lignin materials. |
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