【作者】 石岩；

【导师】 柴立元；

【作者基本信息】 中南大学 ， 冶金工程， 2013， 博士

【摘要】 摘要：木质素是地球上最丰富的苯化合物。自然界中由木质素构成的生物量仅次于纤维素和半纤维素位于地球生物总量的第二位。木质素结构复杂,相对分子量高,难溶于水,这些特点使其很难被降解。木质素的微生物降解已经成为国内外研究的热点,而且微生物主要集中于真菌。近年来越来越多的研究表明,由于具有极强的环境适应性和丰富的生化多样性,细菌将在木质素生物降解中起到越来越重要的作用。论文以碱木质素及木质素模型苯化合物为对象,利用从被微生物腐蚀的三国吴简的浸泡液中分离的Pandoraea sp.B-6和Cupriavidus basilensis B-8对木质素及其模式苯化合物降解的特性和机制进行了比较系统的研究,获得如下研究成果：系统研究了Pandoraea sp.B-6和Cupriavidus basilensis B-8的木质素降解特性,首次发现Pandoraea属和Cupriavidus属的菌株具有木质素降解能力。两株细菌降解碱木质素的最适pH和温度分别为10,30℃和7,30℃；两株细菌在初始浓度为1-6g/L的碱木质素培养基中COD和色度分别降低38.2%,41.6%和31.3%,37.5%以上。两株细菌分泌胞外过氧化物酶降解木质素,其中Pandoraea sp.B-6中锰过氧化物酶和漆酶的最大活性分别为2249.2U/L和1120.6U/L, Cupriavidus basilegnsis B-8中锰过氧化物酶和漆酶的最大活性分别为1685.3U/L和815.6U/L。通过GC-MS分析两株细菌降解碱木质素过程中产生的中间代谢物,发现有多种化合物(如阿魏酸、肉桂酸等),由此推测两株细菌首先将木质素解聚成低聚物,然后进一步降解成木质素单体苯化合物及其他小分子化合物直至进入三羧酸循环被矿化。Pandoraea sp.B-6和Cupriavidus basilensis B-8在以阿魏酸和对香豆酸为唯一碳源的培养基中正常生长的同时还能够高效的降解这两种化合物。除了Pandoraea sp.B-6降解对香豆酸的最适pH为8以外,两株细菌降解这两种化合物的最适pH和温度均分别为7和30℃,完全降解这两种苯化合物的最大浓度分别为1000mg/L,800mg/L和1000mg/L,1000mg/L.通过两株细菌降解苯化合物的液相色谱分析,在Pandoraea sp.B-6和Cupriavidus basilemsis B-8降解对香豆酸的过程中只发现对羟基苯甲酸这一种中间产物(最大浓度分别为169mg/L和253mg/L)；在Pandoraea sp.B-6降解阿魏酸的过程中发现了香草酸和香草醛,最大浓度分别为426mg/L和193mg/L；在Cupriavidus basilensis B-8降解阿魏酸的过程中发现了4-乙烯基愈创木酚,香草酸和香草醛,最大浓度为别为548mg/L,238mg/L和低于30mg/L。通过基因RT-PCR(逆转录PCR)分析发现两株细菌都是通过β-酮中心代谢途径对两种苯化合物进行降解,而Cupriavidus basilensis B-8中还存在另外一条降解阿魏酸的途径—还原转化途径。准确测定了Pandoraea sp.B-6和Cupriavidus basilensis B-8的全基因组序列。结果发现,这两株细菌的基因组均具有单染色体,大小分别为5037162bp(136contigs)和8705938bp(1458contigs).平均GC含量分别为63.56%和65.40%。两株细菌基因组编码区的GC含量(分别为64.02%和65.62%)均比非编码区的GC含量(分别为60.08%和64.29%)高。Pandoraea sp.B-6基因组中共有4803个编码序列,总长度为4312731bp,其中包括4692或4695个蛋白编码基因,55个代表20种氨基酸的tRNA编码基因,5个核糖体RNA编码基因,3个或5个rRNA编码基因,31个sRNA编码基因以及15个miRNA编码基因。Cupriavidus basilensis B-8基因组中共有8448个编码序列,总长度为6740730bp,其中包括8371个蛋白编码基因,65个代表20种氨基酸的tRNA编码基因,5个核糖体RNA编码基因,12个rRNA编码基因。对两株细菌的基因组及特定的表达蛋白进行系统的生物信息学分析和RT-PCR分析。结果发现：Pandoraea sp.B-6和Cupriavidus basilenisis B-8均含有多种降解木质素及苯化合物的基因和降解途径。Pandoraea sp.B-6共发现119个木质素及苯化合物降解相关基因；四种木质素降解相关途径：β-酮中心代谢途径降,龙胆酸代谢途径,2,3-二羟基苯基丙酸间位裂解途径和Box途径；以及5种其他苯化合物降解途径：3,4-二羟基苯乙酸甲酯间位裂解途径,苯基乙酰辅酶A开环途径,2,5-二羟基苯乙酸途径,2-氨基苯甲酰基-辅酶A途径和3-羟基邻氨基苯甲酸途径。Cuprjavidus basilensis B-8共发现165个木质素基本化合物降解相关基因；5种木质素降解相关途径：β-酮中心代谢途径,苯酚代谢途径,龙胆酸代谢途径,2,3-二羟基苯基丙酸间位裂解途径和Box途径；以及5种其他苯化合物降解途径：3,4-二羟基苯乙酸甲酯间位裂解途径,苯基乙酰辅酶A开环途径,2,5-二羟基苯乙酸途径,2-氨基苯甲酰基-辅酶A途径和3-羟基邻氨基苯甲酸途径。研究结果证实了Pandoraea sp.B-6和Cupriavidus basilensis B-8具有降解木质素及其模型苯化合物的能力。全基因组的测序和分析确定了这两株细菌降解木质素和苯化合物的步骤和降解产物。研究对木质素及苯化合物降解机制及木质素降解应用研究具有重要意义。更多还原

【Abstract】 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 ρ-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 ρ-coumaric acid by Pandoraea sp. B-6was8. The maximum concentration of complete degradation for these two compounds degradation were all1000mg/L, except that for ρ-coumaric acid by Pandoraea sp. B-6was800mg/L. The HPLC analysis of aromatic compounds degradation by these two strains showed that ρ-hydroxybenzoic acid was the only compounds detected during the process of ρ-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 β-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 β-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 β-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.更多还原