3种木质素的主要理化性质分析

介绍了木质素、碱木质素和铵化木质素的制备方法,且对这3种木质素的比重、钠元素含量、X射线衍射、热重、溶解度等理化性质进行表征。研究结果表明,铵化木质素不含有碱金属钠,同时具有很好的水溶性,即铵化木质素解决了纯木质素难溶于水的问题,又解决了碱木质素与柴油乳化后对柴油发动机汽缸内的损坏和长期使用会存在积炭的风险。这说明铵化木质素与木质素和碱木质素相比更适于同柴油乳化混合,实现传统化石能源的添加剂,铵化木质素为我国林木废弃生物质资源化利用和替代能源开发提供了一条潜在的途径。     

木质素的生物合成及其调控研究进展

木质素是植物体中仅次于纤维素的一种重要大分子有机物质,具有重要生物学功能,其3种主要单体的生物合成途径已经基本清楚。从木质素生物合成及基因工程在调控木质素生物合成中的作用等方面的研究进展进行了综述,并提出了存在的问题及对策。     

Distribution and roles of p-hydroxycinnamate: CoA ligase in lignin biosynthesis

p-Hydroxycinnamate:CoA ligases were extracted from the xylems of angiosperms and gymnosperms, and the substrate specificities toward ferulate and sinapate were examined. Most of angiosperm and gymnosperm CoA ligases examined were active with ferulate but not with sinapate; however, the enzymes of Erythrina crista-galli, Robinia pseudoacacia and bamboo showed considerable activity with sinapate. The other enzymes, although inactive with sinapate, showed no inhibitory effect on the Erythrina CoA ligase reaction with sinapate. The K_ms for sinapate and ferulate of the Erythrina enzyme were 1.0 and 2.1 μM, respectively, and p-hydroxycinnamate was the best substrate among cinnamates examined. The MW of the CoA ligase was 40 000 and the pH optimum was between 7.2 and 7.6. The possible roles of p-hydroxycinnamate:CoA ligase in lignin biosynthesis are discussed.     

Downregulation of caffeoyl-CoA O-methyltransferase (CCoAOMT) by RNA interference leads to reduced lignin production in maize straw

Lignin is a major cell wall component of vascular plants that provides mechanical strength and hydrophobicity to vascular vessels. However, the presence of lignin limits the effective use of crop straw in many agroindustrial processes. Here, we generated transgenic maize plants in which the expression of a lignin biosynthetic gene encoding CCoAOMT, a key enzyme involved in the lignin biosynthesis pathway was downregulated by RNA interference (RNAi). RNAi of CCoAOMT led to significantly downregulated expression of this gene in transgenic maize compared with WT plants. These transgenic plants exhibited a 22.4% decrease in Klason lignin content and a 23.3% increase in cellulose content compared with WT plants, which may reflect compensatory regulation of lignin and cellulose deposition. We also measured the lignin monomer composition of the RNAi plants by GC-MS and determined that transgenic plants had a 57.08% higher S/G ratio than WT plants. In addition, histological staining of lignin with Wiesner reagent produced slightly more coloration in the xylem and sclerenchyma than WT plants. These results provide a foundation for breeding maize with low-lignin content and reveal novel insights about lignin regulation via genetic manipulation of CCoAOMT expression.     

木质素柴油制备工艺优化与理化性质评价

介绍了一种新型木质素柴油的制备工艺优化方法及其理化性质评价。根据添加木质素的比例、乳化剂的添加量和等影响因素进行正交试验设计,分别选择添加木质素的量为总质量2%、3%和4%的比例,以乳化剂的用量为4%、5%和6%,HLB为8.3、8.8和9.3以及0.2、0.3和0.4 g的助乳化剂用量的不同等条件为水平标准,配制了多种不同比例的乳化油,最终筛选乳化条件为：HLB值为9.3,乳化剂的用量为6.0%,助乳化剂的加入量为1.2%,乳化温度为室温,乳化时间2 min。同时还针对氧化安定性、含硫量、酸度、10%蒸余物残炭、铜片腐蚀、机械杂质、运动粘度、凝点、闪点（闭合）、十六烷值、馏程、密度十二项指标进行了国家标准检测均高于GB 252-2000轻柴油的国家标准。其中木质素柴油的十六烷值均到了46.1和48.8,远远超过了45的国家标准;这对开发柴油的替代燃料,缓解燃料供需紧张矛盾,降低燃料消耗率等都有重大意义。     

Biosynthesis and Genetic Engineering of Lignin

Lignin, a complex heteropolymer of cinnamyl alcohols, is, second to cellulose, the most abundant biopolymer on Earth. Lignification has played a determining role in the adaptation of plants to terrestrial life. As all extracellular polymers, lignin confers rheological properties to plant tissues and participates probably in many other functions in cell and tissue physiology orin cell-to-cell communication. Economically, lignin is very important because it determines wood quality and it affects the pulp and paper-making processes as well as the digestibility of forage crops. For all these reasons the lignin biosynthesis pathway has been the subject of many studies. At present, most genes encoding the enzymes involved in the biosynthesis of lignin have been cloned and characterized. Various recent studies report on the alteration of the expression of these genes by genetic engineering, yielding plants with modified lignin. In addition, several mutants have been analyzed with changes in lignin content or lignin composition resulting in altered properties. Thanks to these studies, progress in the knowledge of the lignin biosynthesis pathway has been obtained. It is now clear that the pathway is more complex than initially thought and there is evidence for alternative pathways. A fine manipulation of the lignin content and/or composition in plants is now achievable and could have important economical and environmental benefits.     

The effect of fungal laccase on fractionated lignosulphonates (peritan Na)

Two fractions obtained after chromatography of lignosulphonates on Sephadex G-50, varying in M_rs, were treated with extracellular Trametes versicolor laccase. After incubation of the low M_r fraction, polymerization was observed, while in the case of the high M_r fraction the reverse process occurred. As a result of depolymerization, five new lower M_r fractions appeared. The reaction reached peak level after 2 hr of incubation and then the quantities of the products diminished, possibly due to their repolymerization. These studies indicate that laccase possesses both polymerization and depolymerization activity though the former was predominant.     

The influence of lignin migration and relocation during steam pretreatment on the enzymatic hydrolysis of softwood and corn stover biomass substrates

To be effective, steam pretreatment is typically carried out at temperatures/pressures above the glass transition point (Tg) of biomass lignin so that it can partly fluidize and relocate. The relocation of Douglas-fir and corn stover derived lignin was compared with the expectation that, with the corn stover lignin's lower hydrophobicity and molecular weight, it would be more readily fluidized. It was apparent that the Tg of lignin decreased as the moisture increased, with the easier access of steam to the corn stover lignin promoting its plasticization. Although the softwood lignin was more recalcitrant, when it was incorporated onto filter paper, it too could be plasticized, with its relocation enhancing enzymatic hydrolysis. When lignin recondensation was minimized, the increased hydrophobicity suppressed lignin relocation. It was apparent that differences in the accessibility of the lignin present in Douglas-fir and corn stover to steam significantly impacted lignin fluidization, relocation, and subsequent cellulose hydrolysis.     

Biodegradation of kraft lignin by a bacterial strain Comamonas sp. B-9 isolated from eroded bamboo slips

Aims: The aim was to obtain evidences for lignin degradation by unicellular bacterium Comamonas sp. B‐9. Methods and Results: Comamonas sp. B‐9 was inoculated into kraft lignin‐mineral salt medium (KL‐MSM) at pH 7·0 and 30°C for 7 days of incubation. The bacterial growth, chemical oxygen demand (COD) reduction, secretion of ligninolytic enzymes and productions of low‐molecular‐weight compounds revealed that Comamonas sp. B‐9 was able to degrade kraft lignin (KL). COD in KL‐MSM reduced by 32% after 7 days of incubation. The maximum activities of manganese peroxidase (MnP) of 2903·2 U l^?1 and laccase (Lac) of 1250 U l^?1 were observed at 4th and 6th day, respectively. The low‐molecular‐weight compounds such as ethanediol, 3, 5‐dimethyl‐benzaldehyde and phenethyl alcohol were formed in the degradation of KL by Comamonas sp. B‐9 based on GC‐MS analysis. Conclusions: This study confirmed that Comamonas sp. B‐9 could utilize KL as a sole carbon source and degrade KL to low‐molecular‐weight compounds. Significance and Impact of the Study: Comamonas sp. B‐9 may be useful in the utilization and bioconversion of lignin and lignin‐derived aromatic compounds in biotechnological applications. Meanwhile, using Comamonas sp. B‐9 in treatment of wastewater in pulp and paper industry is a meaningful work.     

Biotransformation of lignin: Mechanisms,applications and future work

As one of the most abundant polymers in biosphere, lignin has attracted extensive attention as a kind of promising feedstock for biofuel and bio-based products. However, the utilization of lignin presents various challenges in that its complex composition and structure and high resistance to degradation. Lignin conversion through biological platform harnesses the catalytic power of microorganisms to decompose complex lignin molecules and obtain value-added products through biosynthesis. Given the heterogeneity of lignin, various microbial metabolic pathways are involved in lignin bioconversion processes, which has been characterized in extensive research work. With different types of lignin substrates (e.g., model compounds, technical lignin, and lignocellulosic biomass), several bacterial and fungal species have been proved to own lignin-degrading abilities and accumulate microbial products (e.g., lipid and polyhydroxyalkanoates), while the lignin conversion efficiencies are still relatively low. Genetic and metabolic strategies have been developed to enhance lignin biodegradation by reprogramming microbial metabolism, and diverse products, such as vanillin and dicarboxylic acids were also produced from lignin. This article aims at presenting a comprehensive review on lignin bioconversion including lignin degradation mechanisms, metabolic pathways, and applications for the production of value-added bioproducts. Advanced techniques on genetic and metabolic engineering are also covered in the recent development of biological platforms for lignin utilization. To conclude this article, the existing challenges for efficient lignin bioprocessing are analyzed and possible directions for future work are proposed.     

Characterization of soluble lignin released from alfalfa by sheep digestion

A faecal soluble lignin fraction (FSL) extracted with 90% dioxane from the faeces of sheep fed on alfalfa hay was characterized by chemical analysis, nitrobenzene oxidation, fourier transform infrared spectroscopy and gel permeation chromatography, and compared with milled wood lignin (MWL) isolated from the alfalfa hay. The amount of FSL in the faeces was low, accounting for only 1% of the lignin present in the alfalfa hay. FSL and MWL consisted mostly of lignin components and contained a small amount of carbohydrate. FSL had a much higher proportion of syringylpropane units than MWL and showed a wide molecular size distribution. The results indicate the selective and limited solubilization of syringyl-rich lignin from alfalfa by sheep digestion.     

Expression of a bacterial 3‐dehydroshikimate dehydratase reduces lignin content and improves biomass saccharification efficiency

Lignin confers recalcitrance to plant biomass used as feedstocks in agro‐processing industries or as source of renewable sugars for the production of bioproducts. The metabolic steps for the synthesis of lignin building blocks belong to the shikimate and phenylpropanoid pathways. Genetic engineering efforts to reduce lignin content typically employ gene knockout or gene silencing techniques to constitutively repress one of these metabolic pathways. Recently, new strategies have emerged offering better spatiotemporal control of lignin deposition, including the expression of enzymes that interfere with the normal process for cell wall lignification. In this study, we report that expression of a 3‐dehydroshikimate dehydratase (QsuB from Corynebacterium glutamicum) reduces lignin deposition in Arabidopsis cell walls. QsuB was targeted to the plastids to convert 3‐dehydroshikimate – an intermediate of the shikimate pathway – into protocatechuate. Compared to wild‐type plants, lines expressing QsuB contain higher amounts of protocatechuate, p‐coumarate, p‐coumaraldehyde and p‐coumaryl alcohol, and lower amounts of coniferaldehyde, coniferyl alcohol, sinapaldehyde and sinapyl alcohol. 2D‐NMR spectroscopy and pyrolysis‐gas chromatography/mass spectrometry (pyro‐GC/MS) reveal an increase of p‐hydroxyphenyl units and a reduction of guaiacyl units in the lignin of QsuB lines. Size‐exclusion chromatography indicates a lower degree of lignin polymerization in the transgenic lines. Therefore, our data show that the expression of QsuB primarily affects the lignin biosynthetic pathway. Finally, biomass from these lines exhibits more than a twofold improvement in saccharification efficiency. We conclude that the expression of QsuB in plants, in combination with specific promoters, is a promising gain‐of‐function strategy for spatiotemporal reduction of lignin in plant biomass.     

Extraction of lignins from aqueous-ionic liquid mixtures by organic solvents

The commercial development of ionic liquids (ILs) to pretreat lignocellulose by dissolution of whole biomass and cellulose precipitation by addition of water is hindered by the absence of an effective technique to recover the lignin content of the biomass from the IL. Three organic solvents [ethyl acetate, 1,4-dioxane, and tetrahydrofuran (THF)] were studied for their ability to form a two-liquid-phase system with water and 1-ethyl-3-methylimidazolium acetate ([C(2)mim][OAc]), and for partitioning model lignins and lignin monomers between the two liquid phases. Ternary diagrams were obtained for three [C(2)mim][OAc]/organic solvent/water systems at 22°C. Partition coefficients were measured for several types of lignin in these three systems. Partition coefficients increase with rising water content in the IL phase, and depend strongly on the type of lignin and on the organic solvent. Partition coefficients rise as the pH of the ionic-liquid-rich phase falls. Small molecule model lignin monomer compounds (guaiacol, syringaldehyde) are also readily extracted from the IL/water system by THF.     

Isolation of Bacterial Strain from Sediment Core of Pulp and Paper Mill Industries for Production and Purification of Lignin Peroxidase (LiP) Enzyme

Five bacterial strains were isolated and purified (CSA101 to CSA105) from the sediment core of the effluent released from the Century Pulp and Paper Mill Ltd., India. These strains were grown in minimal salt medium (MSM) containing pulp (10% as a carbon source). The production of lignin peroxidase, CMCase, Fpase, and xylanase together with protein and reducing sugar by all bacterial strains was observed. All of the bacterial isolates responded differently with respect to growth and ligninocellulolytic enzyme production. The maximum lignin peroxidase (LiP) was obtained from the cell extract of Bacillus sp. (CSA105) strain, which was used for purification, fractionation and characterization. The culture filtrate from Bacillus sp. (CSA105) was purified with ammonium sulfate precipitation. Crude protein was desalted by dialyzing with Tris buffer. The lignolytic enzyme produced in the liquid medium was fractionated by gel filtration on Sephadex G-100. In the present study, 12.4-fold purification of LiP enzyme was obtained and 35.85% yield of lignin peroxidase was achieved in the cell extract of Bacillus sp. (CSA105). Lignin peroxidase enzyme plays an important role in lignin degradation process. The ligninolytic enzymes were produced by all of the bacterial strains but maximum lignin peroxidase activity was found in cell extract of CSA105. On the basis of the results obtained, the bacterial strain (CSA105) was found most suitable for the purification of the LiP enzyme.     

Characterization and biosynthesis of mistletoe lignin

Mistletoe lignin was a typical angiosperm one based on the spectral (UV, IR, ¹³C-NMR) and functional group analyses, and on degradation products (nitrobenzene oxidation and acidolysis), the analytical results of which were compared with those of the host lignin. l-Phenylalanine-[U-¹⁴C] was efficiently incorporated into mistletoe lignin. Phenylalanine ammonia-lyase and cinnamate-4-hydroxylase were detected by incubation of the tissue slices under illumination. It was also found that O-methyltransferase activity of the crude homogenate catalysed the methylation of 5-hydroxyferulic but not the methylation of caffeic acid. However, the latter methylation activity could be recovered by purification. These results indicate that mistletoe lignin is synthesized independently from that of its host.     

Non-degradative dissolution and acetylation of ball-milled plant cell walls: high-resolution solution-state NMR

Two solvent systems for fully dissolving, and optionally derivatizing, finely ground plant cell wall material at room temperature are described: dimethylsulfoxide (DMSO) and tetrabutylammonium fluoride (TBAF) or N-methylimidazole (NMI). In situ acetylation produces acetylated cell walls (Ac-CWs) that are fully soluble in chloroform. Lignin structures tested remain fully intact. The dispersion of 13C-1H correlations afforded by two-dimensional (2D) nuclear magnetic resonance (NMR) experiments reveals the major lignin units, allowing the whole lignin fraction to be analyzed by high-resolution solution-state NMR methods for the first time. Non-degradative cell wall dissolution offers the potential to analyze polysaccharide components, and improve current cell wall analytical methods by using standard homogeneous solution-state chemistry.     

Tailoring lignin biosynthesis for efficient and sustainable biofuel production

Increased global interest in a bio‐based economy has reinvigorated the research on the cell wall structure and composition in plants. In particular, the study of plant lignification has become a central focus, with respect to its intractability and negative impact on the utilization of the cell wall biomass for producing biofuels and bio‐based chemicals. Striking progress has been achieved in the last few years both on our fundamental understanding of lignin biosynthesis, deposition and assembly, and on the interplay of lignin synthesis with the plant growth and development. With the knowledge gleaned from basic studies, researchers are now able to invent and develop elegant biotechnological strategies to sophisticatedly manipulate the quantity and structure of lignin and thus to create economically viable bioenergy feedstocks. These concerted efforts open an avenue for the commercial production of cost‐competitive biofuel to meet our energy needs.