| Lignin is a three-dimensional heteropolymer synthesized from phenylpropane units in the process of photosynthesis, which exists widely in plant cell walls and contains various types of complex chemical bonds (C-C and C-O-C). It is generally accepted that lignin is one of the most refractory biodegradable aromatic compounds. Lignin biodegradation is of great significance in the global carbon cycle, because most renewable carbon is either in lignin or in compounds protected by lignin from enzymatic degradation (cellulose and hemicellulose).Six bacteria which were stored in our laboratory were screened based on their ability to utilize the seven lignin model monomers including guaiacol, vanillic acid, veratric acid, cinnamic acid, p-coumaric acid, ferulic acid and sinapic acid as the sole carbon source. The strain B-8was found to degrade all of these lignin model monomers; and quantitative experiments indicated that the growth condition of B-8on the medium of the seven lignin model monomers was much better than that of control, while the growth on sinapic acid was poorer than others. Based on morphological, biophysical and biochemical characteristics, and homology identification of16S rDNA sequence, the strain B-8was identified as Cupriavidus sp. B-8.Biodegradation and degradation mechanisms of three typical lignin monomers, namely p-coumaric acid, ferulic acid and sinapic acid, by Cupriavidus sp. B-8were studied,. Higher concentration of p-coumaric acid, ferulic acid and sinapic acid had an inhibitory effect on growth of Cupriavidus sp. B-8. Under1mM of the three substrates, Cupriavidus sp. B-8grew better. Under optimum concentration of substrtes, Cupriavidus sp. B-8was highly efficient in degradation ofp-coumaric acid and ferulic acid.99.44%and99.84%of p-coumaric acid, ferulic acid was degraded within12hour, respectively. However,98.13%degradation of sinapic acid was observed after6days. The biodegradation mechanism of ferulic acid was further investigated. UV and FTIR analysis revealed that the four-hour period between8h and12h was an important time for ferulic acid degradation by Cupriavidus sp. B-8. Based on GC-MS analysis,4-vinylguaiacol and vanillic acid were identified as the main intermediates of ferulic acid degradation. To further confirm the two biodegradation intermediates, degradations of4-vinylguaiacol and vanillic acid by Cupriavidus sp. B-8were examined. Both4-vinylguaiacol and vanillic acid could support the growth of B-8as the sole carbon and energy source. But degradation of4-vinylguaiacol by Cupriavidus sp. B-8was a little slower, compared with the fact that93.21%of vanillic acid could be degraded by Cupriavidus sp. B-8within24hour. With vanillic acid as substrate, protocatechuic acid was detected as the major product formed in the culture by GC-MS analysis. Protocatechuic acid could also be utilized as the sole carbon and energy source by Cupriavidus sp. B-8. Based on above results, the biodegradation pathway of ferulic acid degradation by Cupriavidus sp. B-8was proposed as following:ferulic acid was initially converted to4-vinylguaiacol, and then4-vinylguaiacol was further oxidized to vanillic acid and finally to protocatechuic acid.Biodegradation and degradation mechanisms of kraft lignin by Cupriavidus sp. B-8were studied using kraft lignin as the sole carbon source. Higher concentration of kraft lignin had no inhibitory effect on growth of Cupriavidus sp. B-8and degradation rate of kraft lignin can all reach above32%under different initial concentrations of kraft lignin. Microbial growth and kraft lignin degradation by Cupriavidus sp. B-8were determined when the initial concentration of kraft lignin was2g/L.37.98%of COD removal efficiency was observed after2day’s incubation and then remained a stable level. During the degradation of kraft lignin, B-8grew fast initially indicated by OD600values. Within24h of incubation, OD600values had increased from0.026to0.348and then decreased slowly. In addition, three ligninolytic enzymes during the degradation process were also investigated. A low enzyme activity of laccase was observed during the initial two days followed by a gradual increase till the sixth day with the maximum value of345U/L. Enzyme activity of manganese peroxidase increased slowly till the third day with the maximum of787U/L and then it declined rapidly e after the fourth day. However, nearly no Lignin peroxidase activity was detected throughout the course of degradation. According to GC-MS analysis, cinnamic acid, two kinds of ketone-type compounds whose structure was similar to ferulic acid and sinapic acid (ferulic acid, sinapic acid, and cinnamic acid were all precursors of lignin synthesis), and other low molecular weight aromatic compounds with carbonyl group in a position was observed. FTIR and SPM analysis have also provided evidences of kraft lignin degradation by Cupriavidus sp. B-8. In conclusion, in the process of kraft lignin degradation, cleavage and oxidation of side-chain, depolymerization, and aromatic ring cleavage were occurred. These results favored the idea that kraft lignin was degradaed and depolymerized into low molecular weight aromatic compounds. |
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