螺带桨搅拌在木质纤维素稀酸预处理反应器中的应用

在干式稀酸预处理的反应器中采用螺带桨搅拌器,对秸秆预处理体系进行混合。在带有螺带式搅拌的预处理过程中,在质量分数2.0%和2.5%的H2SO4用量条件下,预处理后72 h秸秆的酶解糖化得率分别为77.55%和87.11%,比静态预处理得到的得率分别增长了7.6%和2.4%,抑制物的生成显著降低。通过计算流体力学方法验证,螺带桨搅拌器可以有效地改善玉米秸秆在稀酸预处理过程中的蒸汽和秸秆两相的混合情况。     

极端嗜热厌氧菌 Caldicellulosiruptor 木质纤维素降解研究简

随着能源危机的加剧,木质纤维素作为生产生物能源的重要原料得到人们的广泛关注。目前,极端嗜热厌氧菌Caldicellulosiruptor属已发现8个种,具有高效的木质纤维素降解能力,甚至可以作用于未经预处理的木质纤维素。自从20世纪80年代以来,人们在Caldicellulosiruptor属的菌株生理生化性质、木质纤维素降解机制及转化能力、基因组、转录组及蛋白质组、遗传转化体系等方面,都取得了一系列研究成果。笔者对嗜热厌氧菌Caldicellulosiruptor属木质纤维素降解的研究现状及前景进行综述及展望。     

粗糙脉孢菌木质纤维素降解利用研究进展简

粗糙脉孢菌作为木质纤维素降解真菌,不仅具有完整的木质纤维素降解酶系,而且还拥有全基因组基因敲除突变体库,是研究丝状真菌纤维素酶表达分泌和木质纤维素降解机制的优秀体系。近年来,国内外利用粗糙脉孢菌系统,在木质纤维素降解机制方面取得了显著进展,包括纤维素酶信号传导、调控以及生物质降解后糖的转运利用等。笔者就相关方面的进展进行综述,并对利用粗糙脉孢菌研究木质纤维素降解利用进行展望,总结和分析木质纤维素降解机制研究的国际前沿动态,有助于加深本领域研究人员对真菌体系纤维素降解机制的理解。     

木质纤维素酶基因资源挖掘及真菌酶系改造简

简述了木质纤维素酶基因资源挖掘的策略和方法及其在丝状真菌酶系改造中的应用。从候选基因的获取（木质纤维素酶基因资源的挖掘和高效利用）、外源基因的表达、酶系的复配和重构等方面综述了丝状真菌酶系改造的最新进展,并提出了丝状真菌酶系改造中亟须解决的关键问题。     

球毛壳菌降解天然木质纤维素能力差异及酶系基因分析简

目的：以不同植物中分离到的4株内生球毛壳菌NK102、NK103、NK104和NK105为对象,研究不同生态来源球毛壳菌降解木质素和纤维素的能力。方法：首先采用羧甲基纤维素和纤维素刚果红平板检测各菌株的纤维素降解能力,并利用Bavendamm平板反应检测各菌株的木质素降解能力;将4株菌分别培养在以微晶纤维素、杨树叶和木屑为惟一碳源的液体培养基中,通过检测培养液中纤维素酶和漆酶的酶活力,比较各菌株分解利用天然木质纤维素材料的能力,连续培养12d后检测培养液中次级代谢产物的合成情况;利用已测序的球毛壳菌CBS148．51的基因组信息,寻找编码木质纤维素降解酶类的基因,为球毛壳菌分解利用木质纤维素提供分子生物学依据。结果：NK102、NK103、NK104和NK105在羧甲基纤维素培养基和纤维素刚果红培养基上都能够生长并形成水解圈;Bavendamm平板反应显示4株菌降解木质素的能力由强到弱依次是NK103、NK102、NK105和NK104。4株菌都能分解利用微晶纤维素、杨树叶和木屑,分泌纤维素酶和漆酶,其中NK102在以木屑为碳源的培养基上纤维素酶活力最强,达到0．76U/mL发酵液,NK103在以杨树叶为碳源的培养基上漆酶活力最强。与此同时,4株菌在发酵培养过程中都能够稳定地合成球毛壳甲素（ChA）,ChA产量受到碳源影响,在以杨树叶为碳源的培养基上,NK104的ChA产量最高,可达到14．88mg/L发酵液。利用已测序的球毛壳菌CBS148．51的基因组信息,寻找到119个编码纤维素半纤维素酶的基因、8个编码漆酶的基因和2个编码锰过氧化物酶的基因,球毛壳菌具有完整的降解纤维素半纤维素的酶体系,在木质纤维素降解真菌的开发过程中具有重要的研究价值。结论：本研究为球毛壳菌木质纤维素降解过程的研究及该菌种的开发利用奠定了基础。     

酿酒酵母 L-阿拉伯糖转化乙醇代谢工程的研究进展简

L-阿拉伯糖是木质纤维素原料中一种重要的五碳糖组分,但传统的乙醇生产菌株酿酒酵母（ Saccharomyces cerevisiae）不能利用L 阿拉伯糖。通过代谢途径工程手段,在酿酒酵母中引入L 阿拉伯糖初始代谢途径可以获得能利用L 阿拉伯糖乙醇发酵的重组菌株。并且,通过代谢途径的疏通以及吸收系统的优化可以强化重组菌株代谢L 阿拉伯糖的能力。笔者从以上角度综述了近年来酿酒酵母转化L 阿拉伯糖生产乙醇的研究进展。     

黄翅大白蚁后肠降解滤纸微生物群落的分析简

黄翅大白蚁（ Macrotermes barneyi）具有高效降解木质纤维素的能力,其后肠内存在着丰富的共生微生物。采用活性电泳和变形梯度凝胶电泳的方法对黄翅大白蚁后肠降解滤纸微生物群落进行分析。活性电泳实验证实了此微生物群落纤维素酶的存在（内切葡聚糖酶、β葡萄糖苷酶和木聚糖酶）,变形梯度凝胶电泳实验鉴定出微生物组的群落结构,即7种细菌和3种真菌。本研究初步阐明了黄翅大白蚁后肠内与滤纸降解相关的微生物种类,为进一步了解黄翅大白蚁纤维素的降解机制以及生物质资源的高效利用提供了理论基础。     

高抗逆高丁比拜氏梭菌的选育及其性能考察简

以抗逆突变株Clostridium beijerinckii IB4为出发菌株,通过常压室温等离子体诱变（ ARTP ）,刃天青平板初筛,摇瓶发酵复筛,筛选出1株高抗逆高丁比的突变菌株C.beijerinckii IT111。发酵结果表明：该突变菌株利用多种C源时均展现其高丁醇比的特性,以玉米芯酸解糖液为C源时,溶剂产量达到10.5 g/L,丁醇8.0 g/L,丁醇比高达76％。抑制物抗逆性测试结果显示：糠醛和酸类对C.beijerinckii发酵影响较小,酚类物质对C.beijerinckii抑制作用较强,其中以香草醛为最。综上所述,C.beijerinckii IT111是1株极具潜力的利用木质纤维原料制备丁醇的菌株。     

乙醇预处理麦秆的酶解性能研究简

采用H2 SO4催化和自催化乙醇法对麦秆进行预处理,比较预处理后麦秆的主要化学组成、纤维素酶解性能和半同步糖化发酵生产乙醇特性,并进行物料衡算。结果表明：H2 SO4催化和自催化乙醇预处理过程中纤维素固体回收率大于90％。添加非离子表面活性剂吐温20和吐温80没有显著提高H2 SO4催化乙醇预处理后纤维素的酶解葡萄糖得率及半同步糖化发酵过程中乙醇的产量,而对自催化乙醇处理后麦秆的酶解和半同步糖化发酵过程有一定程度的促进作用,相应的酶解葡聚糖转化率由72.7％提高到85.0％,而半同步糖化发酵过程中乙醇质量浓度提高了11.4％。物料衡算结果表明：酸催化和自催化乙醇预处理后葡聚糖回收率分别为91.0％和95.4％;半同步糖化发酵生产乙醇的得率分别为10.4和11.6 g（按100 g原料计）。     

草鱼肠道纤维素降解细菌的分离与鉴定简

为更好地弄清草鱼(Ctenopharyngodon idella)肠道纤维素降解细菌的种类,采用羧甲基纤维素(CMC)作为唯一碳源的选择性培养基,分别从草鱼肠道内容物和肠道黏膜中分离到了40株产纤维素酶细菌。16S rRNA基因序列的分析结果显示,大多数产纤维素酶细菌为气单胞菌属(Aeromonas)的种类,其次为肠杆菌属(Enterobacter)的细菌以及未经分离纯培养的细菌(Uncultured bacterium)。进一步研究细菌产纤维素酶能力发现,纤维素酶活性显著性高于其他菌株的分别是A.veronii MC2、A.veronii BC6、肠杆菌科(Enterobacteriaceae)中一种未经分离纯培养的细菌BM3(Uncultured bacterium BM3)和A.jandaei HC9。草鱼肠道中简答气单胞菌(A.jandaei)、类志贺邻单胞菌(Plesiomonas shigelloides)、阴沟肠杆菌(E.cloacae)以及产气肠杆菌(E.aerogenes)是被作为产纤维素酶细菌的首次报道。     

Differences in glucose yield of residues from among varieties of rice,wheat, and sorghum after dilute acid pretreatment

Bio-refinery processes require use of the most suitable lignocellulosic biomass for enzymatic saccharification and microbial fermentation. Glucose yield from biomass solid fractions obtained after dilute sulfuric acid (1%) pretreatment (at 180 °C) was investigated using 14, 8, and 16 varieties of rice, wheat, and sorghum, respectively. Biomass solid fractions of each crop showed similar cellulose content. However, glucose yield after enzymatic hydrolysis (cellulase loading at 6.6 filter paper unit/g-biomass) was different among the varieties of each crop, indicating genotypic differences for rice, wheat, and sorghum. Nuclear magnetic resonance method revealed that the high residual level of lignin aromatic regions decreased glucose yield from solid fraction of sorghum.     

木质纤维素预处理抑制物产生及脱除方法的研究进展

利用纤维素酶将木质纤维素降解成可发酵性糖,然后发酵生产氢气、乙醇、丁醇等生物燃料及高附加值产品,是当今全球研究的热点。预处理是生物质转化过程中至关重要的步骤,而预处理过程中产生的抑制物对木质纤维素后续的酶解和发酵微生物有负面影响。因此了解预处理方法及其过程中产生的抑制物及脱除方法是能否高效转化生物质的基础。文中首先介绍了木质纤维素常用的两类预处理方法即化学法和物理化学法。随后阐述了不同抑制物的产生及其抑制机制,并重点介绍了多种脱毒方法。最后展望了脱除木质纤维素预处理抑制物的研究趋势:应用交联聚乙烯亚胺和金属有机骨架化合物等新型材料脱除抑制物或通过基因工程、代谢工程技术等构建抑制物耐受性菌株等。     

Effect of dilute acid pretreatment on the saccharification and fermentation of rye straw

This research shows the effect of dilute acid pretreatment with various sulfuric acid concentrations (0.5–2.0% [wt/vol]) on enzymatic saccharification and fermentation yield of rye straw. After pretreatment, solids of rye straw were suspended in Na citrate buffer or post-pretreatment liquids (prehydrolysates) containing sugars liberated after hemicellulose hydrolysis. Saccharification was conducted using enzymes dosage of 15 or 25 FPU/g cellulose. Cellulose saccharification rate after rye straw pretreatment was enhanced by performing enzymatic hydrolysis in sodium citrate buffer in comparison with hemicellulose prehydrolysate. The maximum cellulose saccharification rate (69%) was reached in sodium citrate buffer (biomass pretreated with 2.0% [wt/vol] H₂SO₄). Lignocellulosic complex of rye straw after pretreatment was subjected to separate hydrolysis and fermentation (SHF) or separate hydrolysis and co-fermentation (SHCF). The SHF processes conducted in the sodium citrate buffer using monoculture of Saccharomyces cerevisiae (Ethanol Red) were more efficient compared to hemicellulose prehydrolysate in respect with ethanol yields. Maximum fermentation efficiency of SHF processes obtained after rye straw pretreatment at 1.5% [wt/vol] H₂SO₄ and saccharification using enzymes dosage of 25 FPU/g in sodium citrate buffer, achieving 40.6% of theoretical yield. However, SHCF process using cocultures of pentose-fermenting yeast, after pretreatment of raw material at 1.5% [wt/vol] H₂SO₄ and hydrolysis using enzymes dosage of 25 FPU/g, resulted in the highest ethanol yield among studied methods, achieving 9.4 g/L of ethanol, corresponding to 55% of theoretical yield.     

一株乳杆菌对蒸汽爆破处理纤维物料的乳酸发酵研究

用筛选得到的一株乳酸杆菌ZJU-1(Lactobacillus sp．)对蒸汽爆破预处理纤维物料的乳酸发酵进行了研究,结果表明该菌株能对蒸汽爆破处理的纤维物料酶解液进行乳酸发酵,在还原糖质量浓度为52．1mg/mL时,乳酸量为42．1mg/mL;与纤维素酶的酶解过程相耦合,对蒸汽爆破处理的物料进行同时糖化乳酸发酵。其发酵周期比分步糖化发酵的过程周期短,在底物质量分数为80mg/mL,接种量为10％,温度为45％时,乳酸量为45．3mg/mL。     

Comparative study of corn stover pretreated by dilute acid and cellulose solvent‐based lignocellulose fractionation: Enzymatic hydrolysis,supramolecular structure,and substrate accessibility

Liberation of fermentable sugars from recalcitrant biomass is among the most costly steps for emerging cellulosic ethanol production. Here we compared two pretreatment methods (dilute acid, DA, and cellulose solvent and organic solvent lignocellulose fractionation, COSLIF) for corn stover. At a high cellulase loading [15 filter paper units (FPUs) or 12.3 mg cellulase per gram of glucan], glucan digestibilities of the corn stover pretreated by DA and COSLIF were 84% at hour 72 and 97% at hour 24, respectively. At a low cellulase loading (5 FPUs per gram of glucan), digestibility remained as high as 93% at hour 24 for the COSLIF‐pretreated corn stover but reached only ～60% for the DA‐pretreated biomass. Quantitative determinations of total substrate accessibility to cellulase (TSAC), cellulose accessibility to cellulase (CAC), and non‐cellulose accessibility to cellulase (NCAC) based on adsorption of a non‐hydrolytic recombinant protein TGC were measured for the first time. The COSLIF‐pretreated corn stover had a CAC of 11.57 m²/g, nearly twice that of the DA‐pretreated biomass (5.89 m²/g). These results, along with scanning electron microscopy images showing dramatic structural differences between the DA‐ and COSLIF‐pretreated samples, suggest that COSLIF treatment disrupts microfibrillar structures within biomass while DA treatment mainly removes hemicellulose. Under the tested conditions COSLIF treatment breaks down lignocellulose structure more extensively than DA treatment, producing a more enzymatically reactive material with a higher CAC accompanied by faster hydrolysis rates and higher enzymatic digestibility. Biotechnol. Bioeng. 2009;103: 715–724. © 2009 Wiley Periodicals, Inc.     

Culture of insect cells in helical ribbon impeller bioreactor

An 11-L helical ribbon impeller (HRI) bioreactor was tested for the culture of Spodoptera frugiperda (Sf-9) cells. This impeller and surface baffling ensured homogeneous mixing and high oxygen transfer through surface aeration and surface-induced babble generation. Serum-supplemented and serum-free cultures, using TNMFH and IPL/41 media, respectively, grew a similar specific growth rates(0.031 and 0.028 h(-1)) to maximum cell densities of 5.5 x 10(6)-6.0 x 10(6) cells. mL(-1) with viability exceeding 98% during exponential growth phase. Growth limitation coincided with glucose and glutamine depletion and production of significant amounts of alanine. The bioreactor was further tested under more stringent conditions by infecting a serum-free medium culture with a recombinant baculovirus. Heterologous protein production of approximately 35 mug per 10(6) cells was comparable to yields obtained in serum-free cultures grown in spinner flasks and petri dishes. Average specific oxygen up-take and carbon dioxide production rates of the serum-free culture prior to infection as measured by on-line mass spectroscopy were 0.20 mumol O(2)mu.(10(6) cells)(-1) h(-1) and 0.22 mumol CO(2) . (10(6) cells)(-1)h(-1) and increased by 30-40% during infection. Therefore, the mixing and oxygenation conditions of this bioreactor were suitable for insect cell culture and recombinant protein production, with limitation being mainly attributed to nutrient depletion and toxic by-product generation.     

Application of high throughput pretreatment and co‐hydrolysis system to thermochemical pretreatment. Part 1: Dilute acid

Because conventional approaches for evaluating sugar release from the coupled operations of pretreatment and enzymatic hydrolysis are extremely time and material intensive, high throughput (HT) pretreatment and enzymatic hydrolysis systems have become vital for screening large numbers of lignocellulosic biomass samples to identify feedstocks and/or processing conditions that significantly improve performance and lower costs. Because dilute acid pretreatment offers many important advantages in rendering biomass highly susceptible to subsequent enzymatic hydrolysis, a high throughput pretreatment and co‐hydrolysis (HTPH) approach was extended to employ dilute acid as a tool to screen for enhanced performance. First, a single‐step neutralization and buffering method was developed to allow effective enzymatic hydrolysis of the whole pretreated slurry. Switchgrass and poplar were then pretreated with 0.5% and 1% acid loadings at a 5% solids concentration, the resulting slurry conditioned with the buffering approach, and the entire mixture enzymatically hydrolyzed. The resulting sugar yields demonstrated that single‐step neutralizing and buffering was capable of adjusting the pH as needed for enzymatic saccharification, as well as overcoming enzyme inhibition by compounds released in pretreatment. In addition, the effects of pretreatment conditions and biomass types on susceptibility of pretreated substrates to enzymatic conversion were clearly discernible, demonstrating the method to be a useful extension of HTPH systems. Biotechnol. Bioeng. 2013; 110: 754–762. © 2012 Wiley Periodicals, Inc.     

Morphology and clavulanic acid production of Streptomyces clavuligerus: Effect of stirrer speed in batch fermentations

Streptomyces clavuligerus ATCC 20764 was grown from spore-inocuia on a glycerol, malt extract, bacteriological peptone medium in 5-L batch fermentations at 490, 990, and 1300 rpm. Dry cell weights, clavulanic acid production, and the morphological parameters main hyphal length, total hyphal length, number of tips, and hyphal growth unit were measured. Growth and productivity were hardly dependent on stirrer speed. After early growth fragmentation of long, highly branched mycelia to shorter, less branched fragments occurred. This was followed by regrowth and, at 1300 rpm, a second fragmentation phase. The effect of increasing stirrer speed was to accelerate the initial fragmentation phase. It was clearly possible to obtain the same biomass concentration and clavulanic acid liter, with different morphologies depending on stirrer speed. This shows that for this fermentation at least there is no direct link between morphology and productivity and, hence, that it might be possible to manipulate them independently to improve fermentor performance.     

Effect of physical pretreatment on dilute acid hydrolysis of water hyacinth (Eichhornia crassipes)

Effects of different physical pretreatments on water hyacinth for dilute acid hydrolysis process (121 ± 3 °C, 5% H₂SO₄, 60 min) were comparatively investigated. Untreated sample had produced 24.69 mg sugar/g dry matter. Steaming (121 ± 3 °C) and boiling (100 ± 3 °C) for 30 min had provided 35.9% and 52.4% higher sugar yield than untreated sample, respectively. The highest sugar yield (132.96 mg sugar/g dry matter) in ultrasonication was obtained at 20 min irradiation using 100% power. The highest sugar production (155.13 mg sugar/g dry matter) was obtained from pulverized samples. Hydrolysis time was reduced when using samples pretreated by drying, mechanical comminution and ultrasonication. In most methods, prolonging the pretreatment period was ineffective and led to sugar degradations. Morphology inspection and thermal analysis had provided evidences of structure disruption that led to higher sugar recovery in hydrolysis process.     

Redistribution of xylan in maize cell walls during dilute acid pretreatment

Developing processes for the conversion of biomass for use in transportation fuels production is becoming a critically important economic and engineering challenge. Dilute acid pretreatment is a promising technology for increasing the enzymatic digestibility of lignocellulosic biomass. However, a deeper understanding of the pretreatability of biomass is needed so that the rate of formation and yields of sugars can be increased. Xylan is an important hemicellulosic component of the plant cell wall and acts as a barrier to cellulose, essentially blocking cellulase action. To better understand xylan hydrolysis in corn stover, we have studied changes in the distribution of xylan caused by dilute acid pretreatment using correlative microscopy. A dramatic loss of xylan antibody signal from the center of the cell wall and an increase or retention of xylan at the plasma membrane interface and middle lamella of the cell were observed by confocal laser scanning microscopy (CLSM). We also observed a reduction in xylan fluorescence signal by CLSM that is generally consistent with the decrease in xylan content measured experimentally in the bulk sample, however, the compartmentalization of this xylan retention was not anticipated. Biotechnol. Bioeng. 2009;102: 1537–1543. © 2008 Wiley Periodicals, Inc.