瘤胃中木质纤维素降解菌及降解酶基因的研究进展

摘要：反刍动物瘤胃是公认的木质纤维素高效降解的天然反应器,对瘤胃微生物的研究成为开发生物能源的热点领域之一。其研究手段已经从传统的依赖分离培养从瘤胃中获得木质纤维素降解菌,并对降解菌中的木质纤维素降解酶逐一分析,发展到通过基因组/元基因组技术,直接从瘤胃中发现获得大量新的木质纤维素降解酶基因/基因簇,进而探讨其降解的分子机理。已有的研究结果表明,瘤胃微生物降解木质纤维素的过程非常复杂,其中涉及到大量不同种类的微生物、酶及基因/基因簇,随着新分析技术的建立和完善,对这些微生物、酶和基因的研究已取得了诸多进展。本论文综述报道了近期有关该方向的研究进展。     

瘤胃木质纤维素降解菌及降解酶基因的研究进展

反刍动物瘤胃是公认的木质纤维素高效降解的天然反应器,对瘤胃微生物的研究已成为开发生物能源的热点领域之一。目前的研究手段已经从传统的依赖分离培养从瘤胃中获得木质纤维素降解菌,并对降解菌中的木质纤维素降解酶逐一分析,发展到通过基因组/元基因组学技术,直接从瘤胃中发现并获得大量新的木质纤维素降解酶基因/基因簇,进而探讨其降解的分子机理。已有的研究结果表明,瘤胃微生物降解木质纤维素的过程非常复杂,涉及大量不同种类的微生物及基因/基因簇,随着新分析技术的建立和完善,对这些微生物和基因的研究已取得了诸多进展。本论文综述了近期有关该方向的研究进展。     

瘤胃纤维素降解细菌的研究进展

反刍动物瘤胃是自然界中最有效的纤维素降解系统，其纤维素降解能力主要源于寄居于其中的纤维素降解细菌、真菌和原虫。其中，瘤胃纤维素降解细菌因数量庞大、种类繁多以及代谢途径丰富，在木质纤维素降解及利用方面发挥着重要作用。本文综述了国内外瘤胃纤维素降解细菌的种类，分析了瘤胃纤维素降解细菌的特性；阐述了瘤胃纤维素降解细菌通过纤维小体对纤维素的降解过程，以及瘤胃微生物之间的相互作用和相互制约关系；简述宏组学技术在开发新纤维素降解菌和新纤维素酶方面的应用，旨在为进一步研究纤维素降解细菌的降解机理，开发新的纤维素菌种和酶资源提供新的思路。     

筛选微生物降解木质纤维素的研究进展

木质纤维素资源是自然界中含量丰富的可再生资源,利用微生物降解木质纤维素是一种重要的策略。在综合国内外对木质纤维素降解微生物的筛选方法和研究策略的基础上,从单一菌株、复合微生物菌系和组学技术三个方面对筛选微生物降解木质纤维素进行了总结和分析,阐述了各个策略的优势特点和应用价值,即单一菌株易于培养但降解能力较低,复合菌系降解能力强但传代稳定性较差,组学技术能够更好的解释微生物降解木质纤维素的机理,为筛选木质纤维素降解微生物提供一定的指导。同时提出使用合成生物学的策略进行相应微生物的筛选,旨在为筛选高效降解木质纤维素的微生物提供一定的参考。     

木质纤维素的微生物降解

木质纤维素广泛存在于自然界中,因结构复杂,其高效降解需要多种微生物的协同互作,由于参与木质纤维素降解的微生物种类繁多,其协同降解机理尚不完全明确。随着微生物分子生物学和组学技术的快速发展,将为微生物协同降解木质纤维素机制的研究提供新的方法和思路。笔者前期研究发现,细菌复合菌系在50℃下表现出强大的木质纤维素降解能力,菌系由可分离培养和暂时不可分离培养细菌组成,但是可分离培养细菌没有降解能力。通过宏基因组和宏转录组研究表明,与木质纤维素降解相关的某些基因表达量发生显著变化,通过组学方法有可能更加深入解释微生物协同降解木质纤维素的微生物学和酶学机理。文中从酶、纯培养菌株和复合菌群三个方面综述了木质纤维素微生物降解研究进展,着重介绍了组学技术在解析复合菌群作用机理方面的现状和应用前景,以期为探索微生物群落协同降解木质纤维素的机理提供借鉴。     

降解纤维素的“超分子机器”研究进展

综述了目前关于纤维小体组装模式、纤维小体结构多样性及人工设计纤维小体等方面的研究进展.纤维小体是某些厌氧菌产生的由多个亚基共同组装而成的大分子机器,是致力于组织、协调多种酶组分协同高效催化降解木质纤维素的胞外蛋白质复合体.纤维小体是厌氧微生物水解纤维素的主体,具有非常高效的打破结晶纤维素的结晶结构和降解纤维素链的作用.纤维小体对木质纤维素降解的高效性来自于其自发组装而成的复杂的高级结构,其结构的复杂性因不同的厌氧微生物而有所不同.     

瘤胃微生物对纤维素的降解及其应用

瘤胃微生物主要包括细菌、真菌和原生动物。其中,瘤胃细菌和瘤胃真菌能分泌纤维素酶,对纤维素有较强的降解能力,主要介绍了瘤胃微生物对纤维素的降解作用及其广阔的应用前景。     

高效降解木质纤维素的白蚁肠道微生物组

木食性白蚁是自然界木质纤维素的高效降解者,在长期进化过程中白蚁与其肠道微生物组协同作用发展出不同的纤维素降解机制。木食性白蚁具有分别来源于白蚁和共生微生物的两套纤维素酶系统。在低等白蚁中,木质颗粒经过白蚁前、中肠分泌的内源性酶初步消化后,在后肠共生鞭毛虫中被降解为乙酸、二氧化碳和氢。高等木食性白蚁在进化中丢失了鞭毛虫,木质颗粒经白蚁自身分泌的酶初步消化后,在后肠大量共生细菌的帮助下被有效降解。培菌类白蚁利用其菌圃中的蚁巢伞菌和肠道微生物协同作用降解木质纤维素。共生微生物在白蚁的氮素固定与循环、中间产物代谢及纤维素降解等过程中发挥了重要作用。学习和模拟白蚁高效降解木质纤维素的体系,对生物质能源的产业化发展具有积极的意义。     

白蚁-共生微生物系统降解木质纤维素研究进展

开发利用木质纤维素材料能显著增加地球上可再生资源的储备量。白蚁分布广泛,常见于热带和亚热带地区,它们借助细菌、古细菌、真菌等肠道微生物和原生动物协同降解食物中的木质纤维素,在生态系统的碳、氮循环中发挥着十分重要的作用。本文概括了近年来白蚁肠道微生物研究的进展,特别是近年来已被证明的肠道微生物在木质纤维素降解方面的作用,以期为后续研究木质纤维素的降解提供参考信息。     

真菌降解木质纤维素的功能基因组学研究进展

木质纤维素利用的核心问题之一是生物质的降解,即如何将生物质由高聚大分子降解为可发酵的小分子糖,又称为糖化。自然界中向胞外大量分泌降解生物质酶类的微生物主要是真菌,研究真菌木质纤维素降解途径的分子机理对生物质的综合利用意义重大,是木质纤维素能否实现全面生物炼制的关键之一。以下将针对真菌降解木质纤维素的研究进展,特别是对利用功能基因组学所取得的进展进行评述。     

iTRAQ-based quantitative secretome analysis of Phanerochaete chrysosporium

The basidiomycete fungi such as Phanerochaete chrysosporium secrete large amount of hydrolytic and oxidative enzymes and degrade lignocellulosic biomass. The lignin depolymerizing proteins were extensively studied, but cellulose, hemicellulose and pectin hydrolyzing enzymes were poorly explored. In this study P. chrysosporium was grown in cellulose, lignin and mixture of cellulose and lignin, and secretory proteins were quantified by isobaric tag for relative and absolute quantitation (iTRAQ)-based quantitative proteomics using liquid chromatography tandem mass spectrometry (LC-MS/MS). An iTRAQ quantified 117 enzymes comprising cellulose hydrolyzing endoglucanases, exoglucanases, beta-glucosidases; hemicelluloses hydrolyzing xylanases, acetylxylan esterases, mannosidases, mannanases; pectin-degrading enzymes polygalacturonase, rhamnogalacturonase, arabinose and lignin degrading protein belonging to oxidoreductase family. Under cellulose and cellulose with lignin culture conditions, enzymes such as endoglucanases, exoglucanases, β-glucosidases and cellobiose dehydrogenase were significantly upregulated and iTRAQ data suggested hydrolytic and oxidative cellulose degradation. When lignin was used as a major carbon source, enzymes such as copper radical oxidase, isoamyl oxidase, glutathione S-transferase, thioredoxin peroxidase, quinone oxidoreductase, aryl alcohol oxidase, pyranose 2-oxidase, aldehyde dehydrogenase, and alcohol dehydrogenase were expressed and significantly regulated. This study explored cellulose, hemicellulose, pectin and lignin degrading enzymes of P. chrysosporium that are valuable for lignocellulosic bioenergy.     

三株高效秸秆纤维素降解真菌的筛选及其降解效果

【目的】利用多种筛选方法,获得高效秸秆纤维素降解真菌,并研究其秸秆纤维素的降解能力。【方法】采用滤纸片孔洞法、滤纸条降解法、羧甲基纤维素钠(CMC-Na)水解圈测定法、秸秆失重法、纤维素分解率测定法、胞外酶活测定法等常规秸秆纤维素降解菌的筛选方法。【结果】筛选到3株具有较强纤维素降解能力的真菌菌株,经初步鉴定菌株98MJ为草酸青霉(Penicillium oxalicum)、菌株W3为木霉(Trichoderma sp.)、菌株W4为扩张青霉(Penicillium expansum)。菌株W4具有非常强的秸秆纤维素降解能力,10d内对秸秆的降解率可达56.3%,对纤维素、半纤维素和木质素的分解率分别为59.06%、78.75%和33.79%。菌株W4的胞外纤维素酶活力在14.25-49.75U/mL之间。【结论】筛选获得3株高效秸秆纤维素降解真菌菌株,其中菌株W4的纤维素酶活高于已报道的菌株,是一株十分具有研究开发潜力的纤维素酶生产菌株。     

Investigation of lignin-carbohydrate complexes in kraft pulps by selective enzymatic treatments

The occurrence of covalent bonds between residual lignin and polysaccharides in birch and pine kraft pulps was investigated by specific enzymatic treatments. Pure enzymes degrading cellulose, xylan and mannan were used both separately and in combination. Comparison of the molar masses of polysaccharides and lignin in the orginal pulps and in the residual pulps after enzymatic treatments showed that residual lignin in birch kraft pulp is linked at least to xylan. A minor portion may also be linked to cellulose. In pine kraft pulp some of the residual lignin appears to be linked to cellulose, glucomannan and xylan. The linkages between lignin and cellulose and hemicelluloses may be either native or formed during pulp processing. The results also provided new information on the synergistic action of cellulose- and hemicellulose-degrading enzymes on pulp fibres. The synergism appears to be mainly due to the structure of the pulp fibres, with different layers of cellulose sheets, hemicelluloses and lignin. On the other hand the results also provided information about fibre structure. The degradation of xylan clearly enhanced the action of enzymes on cellulose, suggesting that xylan partially covers the cellulose. A similar phenomenon was not observed in the simultaneous hydrolysis of glucomannan and cellulose. However, the results suggest that glucomannan does interact with cellulose, possibly by non-covalent linkages. Received: 8 July 1998 / Received revision: 7 October 1998 / Accepted: 11 October 1998     

油菜秸秆混合发酵降解菌的筛选

油菜秸秆含有大量木质纤维素,该类物质结构稳定,不易降解,限制了其工业化应用.通过对10株包括细菌、酵母菌和白腐真菌的菌株产酶能力和特性进行比较,并进行共同培养试验,筛选出5株可共同生长的木质纤维素降解菌BS09、BL、PC、TS和KS.通过对这5个菌株单独发酵降解油菜秸秆的能力考察,结果表明:PC对木质纤维素的降解能力...     

Biosoftening of coir fiber using selected microorganisms

Coir fiber belongs to the group of hard structural fibers obtained from coconut husk. As lignin is the main constituent of coir responsible for its stiffness, microbes that selectively remove lignin without loss of appreciable amounts of cellulose are extremely attractive in biosoftening. Five isolated strains were compared with known strains of bacteria and fungi. The raw fiber treated with Pseudomonas putida and Phanerocheate chrysosporium produced better softened fiber at 30±2 °C and neutral pH. FeSO₄ and humic acid were found to be the best inducers for P. chrysosporium and P. putida, respectively, while sucrose and dextrose were the best C-sources for both. Biosoftening of unretted coir fibers was more advantageous than the retted fibers. Unlike the weak chemically softened fiber, microbial treatment produced soft, whiter fibers having better tensile strength and elongation (44.6–44.8%) properties. Scanning electron microscopy photos showed the mycelia penetrating the pores of the fiber, removing the tylose plug and degrading lignin.     

Comparative ligninolytic and polysaccharolytic potentials of an alkaliphilic basidiomycete on native ligninocellulose

A comparison of the ligninolytic, cellulolytic and hemicellulolytic abilities of an alkaliphilic white-rot fungus. Coprinus fimetarius, on wheat straw under varying conditions of solid-substrate fermentation is presented. The extent of fractional degradation (percentage of the original dry weight of the fraction) of straw under an optimized set of cultural conditions (pH 9·0, moisture 65%, temperature 37°C, period 21 days) was in the following order: lignin (45%), cellulose (42%), hemicellulose (27%). Urea nitrogen favoured the degradation of lignin as well as cellulose and hemicellulose up to a certain level (1·5% sterile urea or 3% unsterile urea on a dry weight basis) beyond which the degradation of lignin was relatively more adversely affected than cellulose. The addition of phosphorus and sulphur was found essential for selective lignin removal. Increasing the C:N ratio by addition of free carbohydrates resulted in an overall decrease in the degradation wherein cellulose utilization was the most affected event. The pre-treatment (physical or chemical) of the substrate caused a general increase in biodegradation of lignin, cellulose and hemicellulose. The degrading activity of the fungus declined with the scaling-up of the fermentation particularly under non-sterile conditions.     

Microbial population genomes from the Amazon River reveal possible modulation of the organic matter degradation process in tropical freshwaters

Rivers connect the carbon cycle in land with that in aquatic ecosystems by transporting and transforming terrestrial organic matter (TeOM). The Amazon River receives huge loads of TeOM from the surrounding rainforest, promoting a substantial microbial heterotrophic activity and consequently, CO₂ outgassing. In the Amazon River, microbes degrade up to 55% of the lignin present in the TeOM. Yet, the main microbial genomes involved in TeOM degradation were unknown. Here, we characterize 51 population genomes (PGs) representing some of the most abundant microbes in the Amazon River deriving from 106 metagenomes. The 51 reconstructed PGs are among the most abundant microbes in the Amazon River, and 53% of them are not able to degrade TeOM. Among the PGs capable of degrading TeOM, 20% were exclusively cellulolytic, while the others could also oxidize lignin. The transport and consumption of lignin oxidation byproducts seemed to be decoupled from the oxidation process, being apparently performed by different groups of microorganisms. By connecting the genomic features of abundant microbes in the Amazon River with the degradation machinery of TeOM, we suggest that a complex microbial consortium could explain the quick turnover of TeOM previously observed in this ecosystem.     

Infra-red spectroscopic analyses of banana waste degraded by oyster mushroom

Carbon, hydrogen and nitrogen analyses of banana leaf and pseudostem biomass revealed their potentiality as substrates for microorganisms. Infra-red (IR) spectra of both biomass show presence of cellulose, xylan and lignin. IR spectra of leaf and pseudostem biomass degraded in solid state fermentation (SSF) by two Pleurotus species (P. sajor-caju and P. ostreatus) for 40 days showed the utilization of cellulose, xylan and lignin by these microbes. Dynamics of various lignocellulolytic enzymes of Pleurotus species and analyses of carbon, hydrogen and nitrogen contents of degraded biomass supported the same. Both the Pleurotus species exhibited lignin consumption ability on both the substrates.     

Bio-softening of mature coconut husk for facile coir recovery

Bio-softening of the mature coconut husk using Basidiomyceteous fungi was attempted to recover the soft and whiter fibers. The process was faster and more efficient in degrading lignin and toxic phenolics. Phanerochaete chrysosporium, Pleurotus eryngii and Ceriporiopsis subvermispora were found to degrade lignin efficiently without any appreciable loss of cellulose, yielding good quality fiber ideal for dyeing.     

Influence of particle size and pH on anaerobic degradation of cellulose by ruminal microbes

Batch experiments were performed to investigate the influence of cellulose particle size and pH on the anaerobic degradation of crystalline cellulose by ruminal microbes. At a particle size of 50 μm there was a higher hydrolysis and acidogenesis rate, and a reduced degradation time, than for 100-μm particles. Reduction in cellulose particle size resulted in decreased methane production, but an increase of soluble products. Cellulose degradation increased with pH from pH 6.0 to 7.5, whereas at pH⩽5.5 there was no degradation. The inhibitory effect of low pH (⩽5.5) on ruminal microbes was not completely remedied even when the pH of the medium was adjusted to a neutral range. In an anaerobic cellulosic waste degrading system inoculated with ruminal microbes the fermentation system should therefore be maintained above pH 6.0. In all cases, volatile fatty acids were the major water-soluble products of cellulose degradation; acetate and propionate accounted for more than 90% of the volatile fatty acid total.