微生物降解秸秆木质素的研究进展

我国秸秆资源丰富，每年产生逾8亿t作物秸秆。通过秸秆直接还田或肥料化还田不仅可以减少化肥的施用量，缓解农业污染压力，还能实现农作物秸秆的循环利用。木质素结构复杂，且与纤维素和半纤维素相互缠绕，因此秸秆的自然腐解过程中，木质素是主要的限速因子，为了提高降解效率，木质素降解菌的发掘和降解机制也逐渐成为研究热点。本文综述了降解木质素的真菌和细菌的研究现状，对比其真菌和细菌降解特性的优缺点并分析复合降解菌群的优势。随后对木质素降解酶系的酶学性质、在不同微生物中的表达特性进行总结，对木质素降解机制及衍生芳烃代谢路径的研究进展进行综述。最后整理木质素降解微生物在秸秆肥料化技术中的应用进展，并探讨了微生物降解秸秆木质素的应用前景和未来的研究方向。     

木质素芳香族化合物降解菌Sphingobium sp. SYK-6的研究进展

木质素是木质的主要成分之一,在自然界中,高分子木质素被真菌的胞外酶分解成低分子芳香族化合物,然后土壤细菌将其完全降解为二氧化碳。由此可见,木质素的完全降解过程是真菌和细菌的共同作用。研究细菌的降解机制,一方面可以理解芳香族化合物在生态系中的碳素循环,另一方面可以为木质素的有效利用提供基因和酶工具,将可再生资源的木质素转化成高附加价值的工业产品。Sphingobium sp.SYK-6是1987年从造纸厂废水中以木质素中的联苯化合物(5,5’-脱氢联香草酸)作为唯一碳源分离出的木质素化合物降解菌。在长达25年以上的研究中我们阐明了一系列芳香族化合物的代谢途径,克隆了相关基因,2012年随着基因组测序的完成,整个降解功能的全貌展现出来。介绍内容:(1)基因组信息;(2)芳醚化合物代谢;(3)联苯化合物代谢;(4)阿魏酸代谢;(5)木质素化合物降解过程中四氢叶酸依赖型机制;(6)原儿茶酸4,5开环途径;(7)3-甲氧基没食子酸代谢的多样性;(8)应用研究。我们希望SYK-6菌株成为一个让人们理解木质素化合物降解的模式菌株。最后结合课题组现在的研究课题展望了木质素化合物的降解研究的发展方向。     

木质素生物炼制研究进展

木质素为天然的芳香族聚合物,是自然界第二大丰富的可再生碳源,占木质纤维素干重的15%~30%。因木质素富含芳香族结构,故其具有极高的应用价值。生物法转化利用木质素具有专一性强和环境友好等特点,使得木质素生物炼制成为研究热点。本文根据国内外研究进展,从木质素降解酶的研究现状、芳香族化合物胞内代谢途径及木质素生物基化学品研究进展等几个方面做了综述。     

木质素的微生物解聚与高值转化

木质纤维素是地球上最丰富的可再生资源。我国每年产生约9亿吨农业秸秆,因得不到有效利用,不仅造成资源浪费,也产生了诸多严峻的环境问题。缺少木质素的高效降解和资源化利用技术是限制木质纤维素产业化的主要瓶颈之一。虽然木质素的降解与转化多年来一直都受到关注,但是由于木质素结构的复杂性及异质性,使其高效利用受限。近年来,微生物具有的“生物漏斗”式转化特性为木质素的高值转化和利用提供了新方向。本文就生物质利用研究以来,微生物在木质素解聚与转化方面的研究历程与最新进展进行了简要的回顾与总结,并初步讨论了目前木质素高值转化面临的机遇与挑战。     

微生物法降解木质素的研究进展

生物质是代替石化资源生产能源和化学品的关键资源,木质素作为植物细胞壁的主要成分已经在很多行业中得到了广泛的应用。然而,由于木质素结构复杂且难以降解,成为生物质资源利用的最大障碍,因此,去除或者降解木质素是利用细胞壁中其他成分的关键步骤。许多行业使用有害化学物质降解木质素,严重危害了生态环境,自然界中木质素经常被包括真菌和细菌在内的微生物降解,因此,研究微生物降解木质素的机制为解决这一问题提供了可能性。本文讨论了木质素的化学组成成分,重点讨论了自然界降解木质素的微生物种类及其降解机制,包括各种真菌和细菌的木质素降解活性,描述了由各种微生物特别是白腐真菌、褐腐真菌和细菌产生的木质素降解酶,并展望了今后木质素生物降解的研究和应用的可能方向。     

白蚁及共生微生物木质纤维素水解酶的种类

白蚁是热带生态系统重要的木质纤维素降解者。白蚁种类丰富,可分成高等白蚁和低等白蚁,食性也具有各自特点。白蚁自身可以产生纤维素酶,主要是GHF9的内切葡聚糖酶(EG),也有β-葡萄糖苷酶(GB)。低等白蚁共生的原虫中已发现丰富的纤维素酶基因,属于GHF5,7和45。同时还有其他相关功能基因,如木聚糖酶和果胶类物质水解酶。高等白蚁肠道中没有共生原虫。高等培菌白蚁可以利用共生蚁巢伞属真菌促进木质纤维素降解,真菌可以产生纤维素酶,果胶质水解酶类、木聚糖酶,同时还产生可能与木质素分解相关的一种漆酶,但是从分子水平,关于共生真菌纤维素水解酶的研究还较少。白蚁肠道已分离出许多具有木质纤维素降解能力的菌株,最近的研究也发现了大量细菌纤维素酶基因。白蚁-共生系统丰富的木质纤维素水解酶类为发展生物方法开发纤维素乙醇这一思路提供有价值的资源。     

环境微生物介导的木质素代谢及其资源化利用研究进展

木质素是一种丰富的芳烃生物大分子聚合物,其分解代谢与地球元素循环和生物资源利用密切相关。但由于木质素结构的复杂性和无规则性导致其难以降解,使得木质素降解的研究成为全球碳循环和生物质资源利用研究的难点。近年来,来自不同环境的微生物陆续被发现具有木质素降解能力,并解析出参与木质素分解代谢的多种氧化还原酶。然而对木质素详细的代谢过程仍不十分清楚,因此,探究木质素降解酶系、作用机理和代谢网络是研究微生物代谢木质素机理的关键。本文综述环境中参与木质素降解的微生物,重点解析其木质素解聚酶系组成、分泌机制和木质素的代谢途径,并在此基础上阐明近年来木质素生物转化的最新研究进展,以期为今后环境微生物代谢木质素机理及其资源化利用的研究提供参考。     

烟草废弃物中的难降解有机物的微生物降解研究进展

烟草废弃物的资源化利用及无害处理过程,需要利用微生物高效降解其中的难降解物质,如木质素与尼古丁。本文主要综述烟草废弃物中难降解物质的生物降解研究进展。迄今,已经发现了不少木质素和尼古丁的微生物降解菌株,对其降解机理及应用已有不少研究报道,但其在烟草废弃物处理中的应用方面报道较少。木质素和尼古丁降解菌可以用于废次烟叶（烟梗）木质素的消减和尼古丁去除,但同时也需要考察菌株的降解能力和应用环境的适用性。具备降解木质素和尼古丁双重功能的菌株更有应用前景,但迄今发现较少。基于全基因组分析和微生物组学技术的复合菌群的研究也是重要的研究方向,将推动含木质素和尼古丁等多种难降解物质的废次烟叶的处置技术发展和实际应用。     

基因工程在能源植物改良中的应用

利用基因工程技术改良能源植物,对降低能源植物向生物燃料(生物乙醇、生物柴油)的转化成本、提高能源转化效率有着非常重要的意义。目前,基因工程技术已被广泛应用于提高植物总的生物产量、降低或改变植物木质素的含量与成分、在植物体中大量表达纤维素降解酶、提高油料植物的产油量以及改变植物油酯的组成成分等方面的研究。概述了利用基因工程技术在以上方面对能源植物进行改良已取得的进展,讨论了现存问题及未来的发展前景。     

白蚁肠道木质素降解菌分离鉴定及其降解特性

以白蚁肠道为菌株来源,富集分离木质素降解细菌,并进一步解析木质素降解过程。从白蚁肠道中筛选木质素降解菌,对其进行16S rRNA基因序列鉴定,通过FTIR和TGA等手段解析了该菌对木质素的降解特性。结果显示,菌株MP-132经鉴定为解鸟氨酸拉乌尔菌,以碱木质素为唯一碳源时,培养7 d后,木质素降解率可达53.2%,漆酶和木质过氧化物酶活力最大分别为32 U/L和105.6 U/L。该菌能够使苯胺蓝和天青B脱色。此外,FTIR和TGA分析结果表明细菌生物处理改变了木质素结构。较为系统揭示了菌株MP-132的木质素降解特性,为木质素资源化利用方面提供优良菌株资源。     

The emerging role for bacteria in lignin degradation and bio-product formation

The microbial degradation of lignin has been well studied in white-rot and brown-rot fungi, but is much less well studied in bacteria. Recent published work suggests that a range of soil bacteria, often aromatic-degrading bacteria, are able to break down lignin. The enzymology of bacterial lignin breakdown is currently not well understood, but extracellular peroxidase and laccase enzymes appear to be involved. There are also reports of aromatic-degrading bacteria isolated from termite guts, though there are conflicting reports on the ability of termite gut micro-organisms to break down lignin. If biocatalytic routes for lignin breakdown could be developed, then lignin represents a potentially rich source of renewable aromatic chemicals.     

Microbial degradation of lignin

The transformations of lignin that occur during its biodegradation are complex and incompletely understood. Certain fungi of the white-rot group, and possibly other fungi and bacteria, completely decompose lignin to carbon dioxide and water. Other fungi and bacteria apparently degrade lignin incompletely. Differences in lignin-degrading abilities observed for different organisms may result from differences in the completeness of their ligninolytic enzyme systems. Not all lignin components may be attacked by a particular organism. Alternatively, different organisms may differ in their basic mechanisms of attack on lignin. The basic pathways of lignin degradation have been elucidated only for certain representatives of the white-and brown-rot fungi. Although it is known that each of the principal structural components of lignin is attacked by other fungi and bacteria, the biochemistry of that attack has not been elucidated. Work with low molecular weight lignin models has provided only limited information on possible pathways of lignin degradation by microorganisms. There is little evidence to suggest a correlation between abilities to degrade single-ring aromatic or lignin model compounds and the ability to degrade polymeric lignin. More evidence has come from analysis of spent culture media for lignin breakdown products and from comparative chemical analyses of sound lignins versus decayed lignin residues. Accumulated evidence with the most thoroughly studied white-rot fungi suggests that with these fungi lignin degradation proceeds by way of extracellular mixed-function oxygenases and dioxygenases, which catalyse demethylations, hydroxylations and ring-fission reactions within a largely intact polymer, concomitant with some release of low molecular weight lignin fragments. There are also apparent relationships between lignin, carbohydrate and nitrogen metabolism for some organisms, but the relationships may vary from one organism to another. Although research is now mostly at a basic level, industrial applications may result from lignin degradation research. Considerable potential exists for the development of bioconversions which might produce low molecular weight chemicals from waste lignins, and thereby reduce our dependence on petroleum as a source of these chemicals. Alternatively, such bioconversions might produce chemically altered forms of polymeric lignin that may be valuable industrially.     

On the bacterial degradation of lignin

Summary Several bacteria which can degrade numerous phenols with structural relationships to lignin were tested for their ability to degrade lignin. The biodegradation with all the tested bacteria was poor. The method of lignin extraction, presence of glucose as cosubstrate and changes in the nitrogen source of the medium did not affect the extent of lignin degradation. The poor degradation does not seem to be influenced by medium composition and culture condition but is more probably due to the inability of the tested bacteria to degrade lignin to any considerable extent.     

木质素降解菌BYL-7的筛选及降解条件优化

【背景】微生物降解木质素因其具有降解效率高和环保等特点而备受关注。【目的】筛选高效木质素降解真菌,并对其降解条件进行优化。【方法】通过愈创木酚-马铃薯葡萄糖琼脂(potato dextrose agar,PDA)和苯胺蓝平板法筛选高效木质素降解菌株,利用单因素筛选及响应面试验对培养条件进行优化。【结果】筛选到一株高效木质素降解菌BYL-7,经形态和多序列分析初步确定为Trametes versicolor。单因素试验证明初始pH、温度和接种量为降解木质素显著影响因子,响应面试验确定降解木质素最优条件为：初始pH 6.7,温度25 °C,接种量8%。在此条件下,碱性木质素降解率为36.5%,比未优化前提高54.0%;水稻秸秆木质素、半纤维素和纤维素降解率分别为32.8%、21.5%、13.2%,其中木质素降解率比未优化前提高36.1%;漆酶活性在第6天达到峰值120.0 U/L,比未优化前提高25.0%;木质素过氧化物酶活性在第6天达到峰值1 343.8 U/L,比未优化前提高36.0%;锰过氧化物酶活性在第5天达到峰值463.8 U/L,比未优化前提高31.7%。【结论】研究结果为木质素的降解提供了良好的菌种资源,同时也为后续木质素的研究积累了相关数据。     

Lignin degradation by microorganisms: A review

Lignin is an abundant plant-based biopolymer that has found applications in a variety of industries from construction to bioethanol production. This recalcitrant branched polymer is naturally degraded by many different species of microorganisms, including fungi and bacteria. These microbial lignin degradation mechanisms provide a host of possibilities to overcome the challenges of using harmful chemicals to degrade lignin biowaste in many industries. The classes and mechanisms of different microbial lignin degradation options available in nature form the primary focus of the present review. This review first discusses the chemical building blocks of lignin and the industrial sources and applications of this multifaceted polymer. The review further places emphasis on the degradation of lignin by natural means, discussing in detail the lignin degradation activities of various fungal and bacterial species. The lignin-degrading enzymes produced by various microbial species, specifically white-rot fungi, brown-rot fungi, and bacteria, are described. In the end, possible directions for future lignin biodegradation applications and research investigations have been provided.     

耐高温木质素降解菌筛选及降解效果研究

为提高育苗基质中废弃物木质素降解速率,在废弃物堆腐生产育苗基质高温阶段取样,筛选耐高温木质素降解菌,并对菌种进行鉴定,同时测定其对秸秆木质素和菌糠木质素的降解效果。获得了1株较好的木质素高温降解菌HZ11,鉴定为解淀粉芽胞杆菌（Bacillus amyloliquefaciens）,结果显示,该菌株对秸秆木质素和菌糠木质素降解效果较好,50 ℃条件下,20 d木质素降解率分别为46.7%和42.4%。菌株HZ11在降解秸秆和菌糠方面具有很好的应用潜力,为利用农业废弃物生产育苗基质提供更加丰富的菌种资源,具有重要的参考价值。     

Identification of DypB from Rhodococcus jostii RHA1 as a lignin peroxidase

Rhodococcus jostii RHA1, a polychlorinated biphenyl-degrading soil bacterium whose genome has been sequenced, shows lignin degrading activity in two recently developed spectrophotometric assays. Bioinformatic analysis reveals two unannotated peroxidase genes present in the genome of R. jostii RHA1 with sequence similarity to open reading frames in other lignin-degrading microbes. They are members of the Dyp peroxidase family and were annotated as DypA and DypB, on the basis of bioinformatic analysis. Assay of gene deletion mutants using a colorimetric lignin degradation assay reveals that a ΔdypB mutant shows greatly reduced lignin degradation activity, consistent with a role in lignin breakdown. Recombinant DypB protein shows activity in the colorimetric assay and shows Michaelis-Menten kinetic behavior using Kraft lignin as a substrate. DypB is activated by Mn(2+) by 5-23-fold using a range of assay substrates, and breakdown of wheat straw lignocellulose by recombinant DypB is observed over 24-48 h in the presence of 1 mM MnCl(2). Incubation of recombinant DypB with a β-aryl ether lignin model compound shows time-dependent turnover, giving vanillin as a product, indicating that C(α)-C(β) bond cleavage has taken place. This reaction is inhibited by addition of diaphorase, consistent with a radical mechanism for C-C bond cleavage. Stopped-flow kinetic analysis of the DypB-catalyzed reaction shows reaction between the intermediate compound I (397 nm) and either Mn(II) (k(obs) = 2.35 s(-1)) or the β-aryl ether (k(obs) = 3.10 s(-1)), in the latter case also showing a transient at 417 nm, consistent with a compound II intermediate. These results indicate that DypB has a significant role in lignin degradation in R. jostii RHA1, is able to oxidize both polymeric lignin and a lignin model compound, and appears to have both Mn(II) and lignin oxidation sites. This is the first detailed characterization of a recombinant bacterial lignin peroxidase.     

Lignin valorization meets synthetic biology

Lignin, an abundant renewable resource in nature, is a highly heterogeneous biopolymer consisting of phenylpropanoid units. It is essential for sustainable utilization of biomass to convert lignin to value‐added products. However, there are technical obstacles for lignin valorization due to intrinsic heterogeneity. The emerging of synthetic biology technologies brings new opportunities for lignin breakdown and utilization. In this review, we discussed the applications of synthetic biology on lignin conversion, especially the production of value‐added products, such as aromatic chemicals, ring‐cleaved chemicals from lignin‐derived aromatics and bio‐active substances. Synthetic biology will offer new potential strategies for lignin valorization by optimizing lignin degradation enzymes, building novel artificial converting pathways, and improving the chassis of model microorganisms.     

Biotechnology in the degradation and utilization of lignocellulose

Lignocellulose is the predominant renewable resource. It uses include fuel, as the feedstock for the pulp and paper industry, and for animal nutrition. It also constitutes a large proportion of agricultural and urban waste. Biotechnology has roles in its efficient production and utilisation. The types of lignin substrates available for study of lignin biodegradation are described. The white rot fungus Phanerochaete chrysosporium is the archetypal system for the study of lignocellulose degradation, since it mineralises lignin and degrades both cellulose and hemicellulose. The salient features of the P. chrysosporium system are described. The lignin peroxidases are a family of proteins, and it is shown that expression of their genes is differential. P. chrysosporium is heterokaryotic with two gene equivalents that have abundant RFLPs. A set of basidiospore-derived strains with genetic compositions defined by such RFLPs provided the potential basis for a strain improvement programme for lignin degradation. However, analysis of this system using radiolabelled synthetic lignin (DHP) as the substrate confirmed previous evidence that both the substrate and the fungal cultures displayed much variation, so that it was difficult to quantify performance for this property. The cellobiohydrolase I enzymes are also coded for by a family of genes, and evidence is also presented for allelic variants, for differential expression and for differential splicing. In contrast, the cellobiohydrolase II function is encoded at a unique genetic locus. Approaches to an homologous integrative transformation system are discussed. Some actinomycete bacteria represent an alternative system for lignin solubilisation in which strains differ in their spectra of activities on lignocellulose substrates. The xylanase system of Streptomyces cyaneus is shown to include three enzymes, two of which are inducible by xylan. A novel assay method was developed and used to demonstrate that the third is constitutive and also non-repressible by glucose. It is proposed that this acts as a sensor for xylans in the environment that can yield breakdown products that are taken up and can then act as inducers of the other two enzymes. The studies on microbial lignocellulose degradation from different laboratories have allowed the formulation of specific biotechnological goals, and some of the problems and opportunities in this area are identified.