| Biological lignin valorization has attracted extensive attention due to its potential to increase the cost-effectiveness of lignocellulosic biorefineries.However,the complex and heterogeneous structure of lignin hinders its value-added utilization process.In this study,the effect of lignin fractionation on its bioconversion with Pseudomonas putida KT2440 was investigated systematically.The gel permeation chromatography(GPC),two-dimensional heteronuclear single quantum coherence(2D HSQC)NMR,and FTIR spectroscopy were applied to characterize lignin fractions before and after fermentation.Combined with the fermentation results including cell amount,PHA production,and lignin degradation,the ideal lignin substrate properties for bioconversion were discussed.Based on lignin fractionation,laccase and mediator were added to further facilitate the bioconversion.The main results are as follows:1.Lignin fractionation results in lignin with uniform molecular weight,which promotes lignin bioconversion effectively.The parent lignin was obtained by combinatorial pretreatment,the molecular weight is 17440 g/mol.Three lignin fractions were obtained by gradient acid precipitation,the molecular weight is 21202g/mol,4189 g/mol,1631 g/mol respectively.Compared with the parent lignin,the fermentation performance utilizing fractionated lignin was significantly improved.Especially,the fermentation performance of low-molecular-weight lignin fraction was better than that of high-molecular-weight fractions.By utilizing the low-molecular-weight lignin fraction,the highest cell amount was 1.45×10~8 CFU/m L;the highest PHA concentration was 0.350 g/L;and the highest lignin degradation rate was 13.24%.Therefore,low-molecular-weight lignin can be utilized by microbes effectively and is suitable for lignin bioconversion.2.Laccase facilitates lignin depolymerization effectively.The molecular weight of lignin was reduced through breaking the linkages(i.e.,β-O-4)in lignin,which effectively improved the properties of lignin substrate,thereby improving the lignin fermentation performance and promoting PHA production.Compared with those without laccase addition,the cell amount after 48 h fermentation within the four fractions were increased by 84.21%,56.04%,61.96%,and 7.44%,respectively.The PHA production of F3 was the highest(0.400 g/L),which increased by 1.14 times than that without laccase addition.Lignin degradation of four fractions increased by13.51%,18.23%,19.87%and 9.22%,respectively,than those without laccase addition.3.Laccase-HBT can effectively facilitate enzymatic lignin depolymerization and improve lignin fermentation performance.Compared with those without laccase addition,the cell amount after 48 h fermentation within four lignin fractions increased by 62.79%,57.56%,24.30%,and 12.81%,respectively.The highest PHA production reached 0.413 g/L when using F3 as substrate,which was 3.25%higher than that with laccase treatment alone.The degradation of four fractions increased by8.94%,21.84%,16.98%,and 7.38%respectively,compared with those without laccase addition.4.Statistic analysis showed that the structure of lignin affected the fermentation performance significantly.According to correlation analysis and linear regression analysis results,lignin molecular weight has the most significant influence on fermentation performance,which provides a direction for promoting lignin bioconversion by reducing the lignin molecular weight.In summary,fractionation improves lignin properties and can obtain low-molecular-weight lignin for fermentation.The addition of laccase and mediator enhances lignin depolymerization,resulting in a higher proportion of lower-molecular-weight lignin that can be utilized by microbes.Therefore,the lignin degradation is facilitated and thus the PHA yield is increased,which promotes lignin bioconversion effectively. |
