【作者】 杨玉锁；

【导师】 周集体；

【作者基本信息】 大连理工大学 ， 环境科学， 2012， 博士

【摘要】 木质素是仅次于纤维素的第二大植物生物质,也是生物圈中最丰富的芳香化合物。木质素生物降解的研究对于了解全球碳循环、发展环境友好生产技术(如造纸过程中选择性去除木质素)有重要意义。同时有效转化木质素,充分利用它作为新材料或能源也是很重要的。有关木质素转化和降解的研究主要集中于木腐菌,尤其是白腐菌。然而,由于白腐菌生长慢、抵抗其它微生物污染能力差、酶类易失活及培养成本等问题,在非灭菌状态下,单独白腐菌的应用面临很多困难。鉴于此,本文基于微生物微生态学原理,在筛选具有降解、转化木质素的功能微生物基础上,建立了高效降解和/或转化木质素微生物群落体系,构建了可在非灭菌状态下持续运行的生物反应器。在侧耳属6株真菌中筛选出一株白腐菌--糙皮侧耳(P. ostreatus) G5,该菌生长快,在限氮液体培养基中降解木质素活性较高、稳定,可耐受较高浓度的木质素。与两个模式白腐菌株--黄孢原毛平革菌(P.chrysosprium)5.776、杂色栓菌(T. versicolor)5.161进行了比较,结果表明,本实验室的糙皮侧耳(P. ostreatus) G5是一株很好的木质素降解真菌。从木质素降解反应器中分离出一株有降解木质素活性的细菌,经过形态观察和16SrRNA基因序列同源性分析,初步鉴定为微杆菌属菌株Microbacterium sp.GE1017.16S rRNA基因序列已提交GenBank,登录号FJ405359。Microbacterium sp.GE1017具有漆酶活性、很高的锰过氧化物酶活性(MnP2493.3U·L-1)和较强木质素降解能力(4d,去除率50%以上),单株菌纯培养可使木质素去除率达近60%。从大连森林土中新分离到两株放线菌。根据形态特征、培养特征和16S rDNA序列分析,将它们鉴定为链霉菌Streptomyces spp. strains F-6and F-7。rRNA基因序列已提交GenBank,登录号F-6FJ405358, F-7FJ405357。两株菌都具有高的木质素降解酶活性。漆酶活性(U·g-1细胞干重)：F-6935.4；F-7191.7。MnP活性：F-6198.8；F-7324.3。两株菌纯培养物具有强的碱木素降解能力(12d碱木素去除率：F-752%；F-649%)。两株菌可以与白腐真菌—糙皮侧耳G5共存,并在真菌-链霉菌共培养条件下有效降解碱木素(12d碱木素去除率：F-7+G557%;F-6+G552%)。通过GC-MS分析由链霉菌F-6菌株降解碱木素后的产物,我们了解了主要的小分子产物。分析结果表明,F-6菌株可以有效地降解碱木素并将其转化为苯或开环化合物(C2--C5)。从大连森林土中新分离出一株霉菌。经过形态观察和rRNA基因ITS区序列分析,该菌属曲霉属,即Aspergillus sp. strain F-3。rRNA基因ITS区序列已提交GenBank,登录号GQ149340。该菌株具有独特的菌丝膨大或厚壁节点结构。曲霉菌株F-3具有木质素降解酶的活性,能够单独降解碱木素,碱木素去除率64.8%、色度去除率100%(8d)。降解最适pH7.0,添加酒石酸胺和葡萄糖会延迟或抑制降解活性表达。在降解过程中,可检测到MnP(28.2U·L-1)、Lac(3.5U·L-1)活性。通过GC-GS分析被菌株F-3降解的小分子产物,表明碱木素可以被降解为小分子或者转换成可利用的产物。在曲霉F-3--糙皮侧耳G5共同培养的条件下能有效地降解碱木素,12d碱木素去除率56.6%。以球形陶瓷珠及不规则陶瓷块为填料,由曲霉F-3、链霉菌F-6,F-7和糙皮侧耳G5四株菌共固定生物膜反应器,一个月可完成挂膜,基本进入稳定运行。在非灭菌状态下,连续运行18个月,在降解液含碱木素2000mg L-1,葡萄糖10g L-1,HRT12h,室温(16-26℃)条件下,碱木素去除率平均>70%,色度去除率>90%,COD去除率>90%。碱木素降解的小分子产物GC-MS分析显示,它们为芳香醇类、芳香酸酯类、酚类、及小分子的醇、羧酸类化合物,大部分都是芳香族化合物。结果说明,共固定生物膜反应器具有稳定、持续降解/转化碱木素活性和很强的抗污染能力。更多还原

【Abstract】 Lignin, next to cellulose,is the second most abundant compound in plant biomass and by far the most abundant aromatic substance present in the biosphere.Studies on the biodegradation of lignin have significant implications for our understanding of the global carbon cycle and development of an environmentally friendly technique for selective removal of lignin in paper-making.Also the effective bioconversion of lignin is important to fully utilize it for new materials or energy.Most lignin research on bioconversion and biodegradation has been on wood-rot fungi,particularly on white-rot fungi.and not on other lignolytic organisms.However, because of slow growth rates;easily counteracted by other lower microorganisms;damageable enzymes and production costs for them,etc.,the direct use of single white rot fungus and their enzymes in industrial processes is still very difficult now.For that reason, according to micro-ecology principle,the community combined system of actinomyces—fungi was set up. And further a coimmobolized bed reactor was structured, which can be running continuously under non sterilization.One strain of white rot fungus--P. ostreatus G5was selected from6strains of Genus Pleurotus with its high growth rate, degrading alkali lignin and tolerance to lignin. Furthermore, P. ostreatus G5was compared to the typical white rot fungi--P.chrysosprium5.776and T. versicolor5.161. The results show that P. ostreatus G5is a better strain for lignin decomposing.One strain of bacteria was isolated from the lignin degrading reactor. It was identified as one Microbacterium sp.GE1017with its morphologic and high homology to the genus of rDNA sequence. The sequence was registered in the NCBI GenBank Data Library under the accession number FJ405359.Strain GE1017possesses high activity of MnP(2493.3U·L-1) and ability of decomposing lignin (degrading rate about60%).Through screening from forest soil of Dalian, two novel isolated actinomycete strains were identified as Streptomyces spp.strains F-6and F-7by their morphology, cultural characteristics and high homology to the16S rRNA gene.The sequences were registered in the NCBI GenBank Data Library under the accession numbers F-6FJ405358,F-7FJ405357.Both strains possessed laccase and manganese peroxidase activities.Laccase activity produced by strain F-6was up to935.4U·g-1dry cell weights.More than50%of alkali lignin was removed by strains F-6and F-7in12days of incubation. GC-MS analysis of the biodegraded products showed strain F-6converted lignin into phenol and broken phenol compounds.The two strains could co-culture with white rot fungus, and the combined actinonycete-fungus system decomposed alkali lignin effectively.A fungus strain F-3was selected from fungal strains isolated from forest soil in Dalian of China. It was identified as one Aspergillus sp.stain F-3with its morphologic,cultural characteristics and high homology to the genus of ITS rDNA sequence. The sequence was registered in the NCBI GenBank Data Library under the accession number GQ149340.The budges or thickened node-like structures are peculiar structures of hyphae of the strain.The fungus degraded64.8%of alkali lignin (2000mg L-1) after day8of incubation at30℃at pH7. The removal of colority was up to100%at8days. The biodegradation of lignin by Aspergillus sp. F-3favored initial pH7.0. Excess acid or alkali conditions were not propitious to lignin decomposing.Addition of Ammonium L-tartrate or glucose delayed or repressed biodegradation activities.During lignin degradation, manganese peroxidase (28.2U·L-1) and laccase (3.5U·L-1) activities were detected after day7of incubation.GC-MS analysis of biodegraded products showed strain F-3could convert alkali lignin into small molecules or other utilizable products.Strain F-3may co-culture with white rot fungus and decompose alkali lignin effectively(removal ratio56.6%)Four strains of Aspergillus F-3,Streptomyces spp.strains F-6and F-7, P. ostreatus G5were used to deal with coimmobilized biofilm formation on the ceramic ball-packed bed reactor. The start-up was finished in one month. Under non sterilization and the operation conditions of degradation liquor with alkali lignin2000mg L-1,glucose10g L-1,HRT12h, at room temperature,the reactor was continuously running for18months.During the running period, the removal ratios were>70%of alkali lignin,>90%of colourity,>90%of COD. GC-GS analysis of products degraded by the reactor shows lignin can be decompose and convert into aromatic alcohols, aromatic acid esters,phenols,and small molecule alcohols, carboxylic acid compounds.Most of them are aromatic compounds.The reactor can maintain the degrading functions and antimicrobial pollution ability, which implies its potential in industrial use.更多还原