| Increase in fossil fuel depletion and increase in environmental pollution forced the researchers to find an alternate source to fossil fuel.Among all the renewable sources,lignocellulose is the only resource that can be directly converted to fuels or high value products.However,lignin is an obstacle in lignocellulosic biomass utilization due to its recalcitrance.Lignin is natural aromatic polymer comprosed phenylpropanoid units linked through various types of chemical bonds,and is the second most abundant biopolymer.In sipte of its abundance,the utilization of lignin has been particularly challenging due to its recalcitrant structure.However,many biomass conversion systems,including white rotten fungi,bacteria,and termites,are indeed capable of utilizing lignin through the secretion of various lignolytic enzymes.In recent years,bacteria have in recent attracted a growing attention due to their unique advantages,such as diversity,rapid growth,wide range of environmental adaptability,and a relatively easy-going genetic manipulation.Termites and their intestinal microbial symbiosis system have a unique and efficient degrading ability to transform lignocellulose,which has aroused widespread concerns in various fields of scientific research.But the diversity and lignolytic functions of their symbiotic bacteria still remain unknown.In this study,four termite species,Odontotermes hainanensis,Macrotermes annandalei,Odontotermes formosanus,Microtermes pakistanicus collected from Xishuangbanna were chosen as research subjects.Some symbiotic bacteria with lignin degradation potential have been successfully isolated and identified from these termite guts.A bacterial strain,which exhibited the highest growth rate on lignin substrate,was further selected to evaluate its degradation ability on lignin.The functions of enzyme genes related to lignin degradation were also investigated via transcriptomic analysis.The main results of this study are summarized as follows:(1)With alkali lignin as the sole carbon source,a total of 96 bacterial strains were isolated from the gut of a wood-feeding termite.The identification results showed that the bacterial strains belonged to four bacterial phyla: Proteobacteria,Actinobacteria,Firmicutes and Bacteriodetes,where they are classified into 21 genus and 33 species.Among those strains,Enterobacteriaceae were the dominant bacterial community.The genus of Kosakonia and Pseudocit robacter were isolated and recorded from the gut of termtie for the first time.Uniquely,R.ornithinolytica MP-132 exhibited the highest growth rate on lignin and lignin derivatives among all identified bacterial symbionts from termite guts.(2)After P.anthropi MP-4 and R.ornithinolytica MP-132 were inoculated into alkali lignin mineral salt medium for 7 d,two strains have been recorded a significant reduction for the chemical oxygen demand(COD)at > 50%,which indeed indicated a degradation processing on lignin components.The greatest enzymatic activities were recorded as 84.5 U/L for lignin peroxidase,36.67 U/L for laccase from P.anthropi MP-4,105.6 U/L for lignin peroxidase and 32 U/L for laccase from R.ornithinolytica MP-132.The results of SEM,FTIR,Py-GC/MS and TGA analyses showed that two strains had the ability to degrade lignin substrates.(3)The various lignin substrate samples were evaluated and characterized for a unique bacterial strain,R.ornithinolytica MP-132,on its lignin degradation processing.The results showed that the maximum degradation rate of lignin by this bacterial strain was recorded as high as 25.3% at an optimum pH of 8.0.In addition,the FTIR results clearly indicated that R.ornithinolytica MP-132 not only caused side chain oxidation and demethylation modifications but also modified and further attacked the aromatic skeletal backbone during the lignin degradation process.TGA results showed that the control and treated lignin samples indicated a significant difference in their thermal decomposition behaviours,suggesting that the strain MP-132 would indeed modify the lignin structures.(4)The pine sawdust degradation by R.ornithinolytica MP-132 was also systematically evaluated.After R.ornithinolytica MP-132 were inoculated into pine sawdust mineral salt medium for 15 days,the activities of laccase,lignin peroxidase,cellulose and xylanase were recorded as 142.6 U/L,55.97 U/L,2.92 U/mL and 2.08 U/mL,respectively.Moreover,the degradation rate of cellulose,hemicelluloses and lignin were also observed as high as 28.78%,32.9% and 30%,respectively.The XRD and FTIR analyses revealed that the bacterial strain effectively degraded the pine sawdust crystalline cellulose and lignin.As a further evidence,the SEM pictures showed the structural changes in pine sawdust substrates.At last,the TGA and Py-GC/MS also confirmed that the milled wood lignin of pine sawdust had a significant lignin structural change made by this bacterial strain.(5)In order to reveal a potential metablic mechanism involved in lignin degradation,transcriptomic analyses were carried out for R.ornithinolytica MP-132 that was grown both glucose medium and lignin medium,respectively.Compared with glucose medium,a total of 2644 differentially expressed genes including 1360 up-regulated genes and 1284 down-regulated genes were recorded.With a analysis by differentially expressed genes functional categories defined by the Gene Ontology(GO),there are 1277 genes classified as biological process,837 genes classified as molecular function and 277 genes classified as cellular.Pathways enrichment analysis of differentially expressed genes was also carried out based on Kyoto Encyclopedia of Genes and Genomes(KEGG).The majority of the genes were enriched in the metabolism/degradation pathway.A large number genes involved in lignin degradation were found in the metabolism pathway.According to the KEGG analysis,it was found that R.ornithinolytica MP-132 may be followed by the central reaction of the benzoate metabolic pathways for subsequent metabolism.Moreover,a large number of genes related to the ?-ketoadipate pathway were found in the up-regulated differential genes.With these analyses and evidences,the lignin degradation mechanism by this bacterial strain can be proposed as the followings: Initially cracking down the lignin structure by this bacterial strain was the first designed action through various oxidation reactions to form some of lower molecular weight aromatic compounds;then these aromatic compounds were further degraded through benzoate metabolic pathways;at last,the benzoate metabolic pathway converted aromatic compounds into either catechol or protocatechuate,for subsequent degradation by the ?-ketoadipate pathway. |
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