木质纤维生物质资源高效利用分子技术的开发

木质纤维生物质是地球上最丰富的可再生生物质资源,可为造纸、化工、纺织和生物能源等工业提供重要的原材料。木质纤维生物质主要包括木质素、纤维素和半纤维素三种生物多聚物成分。如何利用分子手段改造这些生物聚合物,提高它们的工业利用率是目前高度关注的问题。综述了近年来木质纤维多聚物在生物合成与改造方面的研究进展,展望了利用分子技术改造植物木质纤维生物质实现其高效利用的前景。     

SO2‐catalyzed steam explosion: The effects of different severity on digestibility,accessibility, and crystallinity of lignocellulosic biomass

Lignocellulosic biomass is the most promising feedstock for biofuels production. To enhance the efficiency of enzymatic hydrolysis, lignocellulosics needs to be pretreated to lower their recalcitrance. SO₂‐catalyzed steam explosion is an efficient and relatively cost‐efficient pretreatment method for softwood. This work investigates the effects of steam explosion severity on the digestibility, accessibility, and crystallinity of Loblolly pine. Higher severity was found to increase the accessibility of the feedstock while also promoting nonselective degradation of carbohydrates. The adsorption behavior of Celluclast® enzymes on steam‐exploded Loblolly pine (SELP) can be described by a Langmuir isotherm. Cellulose crystallinity was found to first increase and then decrease with increasing pretreatment severity. A linear relationship between initial hydrolysis rates and crystallinity index (CrI) of pretreated Loblolly pine was found; moreover, a strong correlation between X‐ray diffraction intensities and initial rates was confirmed. The findings demonstrate the significance of CrI in enzymatic hydrolysis of pretreated lignocellulosic biomass. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:909–916, 2013     

Use of lignocellulose biomass for endoxylanase production by Streptomyces termitum

Actinobacteria isolates from Brazilian Cerrado soil were evaluated for their ability to produce enzymes of the cellulolytic and xylanolytic complex using lignocellulose residual biomass. Preliminary semiquantitative tests, made in Petri plates containing carboxymethylcellulose and beechwood xylan, indicated 11 potential species producing enzymes, all belonging to the genus Streptomyces. The species were subsequently grown in pure substrates in submerged fermentation and analyzed for the production of enzymes endoglucanase, β-glucosidase, endoxylanase, and β-xylosidase. The best results were obtained for endoxylanase enzyme production with Streptomyces termitum(UFLA CES 93). The strain was grown on lignocellulose biomass (bagasse, straw sugarcane, and cocoa pod husk) that was used in natura or acid pretreated. The medium containing sugarcane bagasse in natura favored the production of the endoxylanase that was subsequently optimized through an experimental model. The highest enzyme production 0.387?U?mL^?1, (25.8 times higher), compared to the lowest value obtained in one of the trials, was observed when combining 2.75% sugar cane bagasse and 1.0?g?L^?1 of yeast extract to the alkaline medium (pH 9.7). This is the first study using S. termitum as a producer of endoxylanase.     

Comparison of SHF and SSF processes for the bioconversion of steam-exploded wheat straw

Two processes for ethanol production from wheat straw have been evaluated — separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF). The study compares the ethanol yield for biomass subjected to varying steam explosion pretreatment conditions: temperature and time of pretreatment was 200°C or 217°C and at 3 or 10 min. A rinsing procedure with water and NaOH solutions was employed for removing lignin residues and the products of hemicellulose degradation from the biomass, resulting in a final structure that facilitated enzymatic hydrolysis. Biomass loading in the bioreactor ranged from 25 to 100 g l⁻¹ (dry weight). The enzyme-to-biomass mass ratio was 0.06. Ethanol yields close to 81% of theoretical were achieved in the two-step process (SHF) at hydrolysis and fermentation temperatures of 45°C and 37°C, respectively. The broth required addition of nutrients. Sterilisation of the biomass hydrolysate in SHF and of reaction medium in SSF can be avoided as can the use of different buffers in the two stages. The optimum temperature for the single-step process (SSF) was found to be 37°C and ethanol yields close to 68% of theoretical were achieved. The SSF process required a much shorter overall process time (≈30 h) than the SHF process (96 h) and resulted in a large increase in ethanol productivity (0.837 g l⁻¹ h⁻¹ for SSF compared to 0.313 g l⁻¹ h⁻¹ for SHF). Journal of Industrial Microbiology & Biotechnology (2000) 25, 184–192. Received 02 December 1999/ Accepted in revised form 20 July 2000     

Organosolv pretreatment of lignocellulosic biomass for enzymatic hydrolysis

Production of ethanol by bioconversion of lignocellulosic biomass has attracted much interest in recent years. However, the pretreatment process for increasing the enzymatic digestibility of cellulose has become a key step in commercialized production of cellulosic ethanol. During the last decades, many pretreatment processes have been developed for decreasing the biomass recalcitrance, but only a few of them seem to be promising. From the point of view for integrated utilization of lignocellulosic biomass, organosolv pretreatment provides a pathway for biorefining of biomass. This review presents the progress of organosolv pretreatment of lignocellulosic biomass in recent decades, especially on alcohol, organic acid, organic peracid and acetone pretreatments, and corresponding action mechanisms. Evaluation and prospect of organosolv pretreatment were performed. Finally, some recommendations for future investigation of this pretreatment method were given.     

Microwave-assisted conversion of lignocellulosic biomass into furans in ionic liquid

Production of 5-hydroxymethylfurfural (HMF) and furfural from lignocellulosic biomass was studied in ionic liquid in the presence of CrCl₃ under microwave irradiation. Corn stalk, rice straw and pine wood treated under typical reaction conditions produced HMF and furfural in yields of 45–52% and 23–31%, respectively, within 3 min. This method should be valuable to facilitate energy-efficient and cost-effective conversion of biomass into biofuels and platform chemicals.     

Catalytic hydrolysis of lignocellulosic biomass into 5-hydroxymethylfurfural in ionic liquid

Production of 5-hydroxymethylfurfural (HMF) from cellulose catalyzed by solid acids and metal chlorides was studied in the 1-butyl-3-methylimidazolium chloride ([BMIM]Cl) under microwave irradiation. Among the applied catalysts, the use of CrCl₃/LiCl resulted in the highest yield of HMF. The effects of catalyst dosage (mole ratio of catalyst to glucose units in the feedstock) and reaction temperature on HMF yields were investigated to obtain optimal process conditions. With the 1:1 mol ratio of catalyst to glucose unit, the HMF yield reached 62.3% at 160 °C for 10 min. Untreated wheat straw was also investigated as feedstock to produce HMF for the practical use of raw biomass, in which the HMF yield was comparable to that from pure cellulose. After the extraction of HMF, [BMIM]Cl and CrCl₃/LiCl could be reused and exhibited no activity loss after three successive runs.     

Carboxymethyl cellulase production optimization from Glutamicibacter arilaitensis strain ALA4 and its application in lignocellulosic waste biomass saccharification

Abstract

In this context, carboxymethyl cellulase (CMCase) production from Glutamicibacter arilaitensis strain ALA4 was initially optimized by one factor at a time (OFAT) method using goat dung as proficient feedstock. Two-level full factorial design (2⁵ factorial matrix) using first-order polynomial model revealed the significant (p?<?0.05) influence of pH, moisture, and peptone on CMCase activity. Central composite design at N?=?20 was further taken into account using a second-order polynomial equation, and thereby liberated maximum CMCase activity of 4925.56?±?31.61?U/g in the goat dung medium of pH 8.0 and 100% moisture containing 1% (w/w) peptone, which was approximately two fold increment with respect to OFAT method. Furthermore, the partially purified CMCase exhibited stability not only at high pH and temperature but also in the presence of varied metal ions, organic solvents, surfactants, and inhibitors with pronounced residual activities. The enzymatic hydrolysis using partially purified CMCase depicted the maximum liberation of fermentable sugars from alkali pretreated lignocellulosic wastes biomass in the order of paddy straw (13.8?±?0.15?mg/g)?>?pomegranate peel (9.1?±?0.18?mg/g)?>?sweet lime peel (8.37?±?0.16?mg/g), with saccharification efficiency of 62.1?±?0.8, 40.95?±?0.4, and 37.66?±?0.4%, respectively after 72?hr of treatment.     

Effect of thermal pretreatment on equilibrium moisture content of lignocellulosic biomass

The equilibrium moisture content (EMC) of raw lignocellulosic biomass, along with four samples subjected to thermal pretreatment, was measured at relative humidities ranging from 11% to 97% at a constant temperature of 30 °C. Three samples were prepared by treatment in hot compressed water by a process known as wet torrefaction, at temperatures of 200, 230, and 260 °C. An additional sample was prepared by dry torrefaction at 300 °C. Pretreated biomass shows EMC below that of raw biomass. This indicates that pretreated biomass, both dry and wet torrefied, is more hydrophobic than raw biomass. The EMC results were correlated with a recent model that takes into account additional non-adsorption interactions of water, such as mixing and swelling. The model offers physical insight into the water activity in lignocellulosic biomass.     

An effective chemical pretreatment method for lignocellulosic biomass with substituted imidazoles

Lignocellulosic biomass is the most abundant naturally renewable organic resource for biofuel production. Because of its recalcitrance to enzymatic degradation, pretreatment is a crucial step before hydrolysis of the feedstock. A variety of pretreatment methods have been developed and intensively studied to achieve optimal yield without imposing significant adverse impact on the environment. Herein, we present a novel chemical pretreatment method using substituted heterocycles with low temperature and short residence time requirements. 1‐Methylimidazole (MI) is a precursor to some imidazolium‐based ionic liquids. In this study, its potential utilization as a biomass pretreatment agent is being investigated for the first time. At mild conditions, such as 25°C for 5 min at ambient pressure, a substantial increase in the hydrolysis rate throughout the entire course of conversion for cellulose substrate was obtained. Furthermore, the pretreatment effectiveness of MI on both untreated and steam‐exploded lignocellulosic biomass including loblolly pine, switchgrass, and sugarcane bagasse has been studied and MI was found to be an efficient delignifier. Remarkable rate enhancement was also observed for the non‐woody lignocellulosic substrates after a short period of MI pretreatment at ambient conditions. The mechanism of MI pretreatment is explored through analysis of cellulose physical properties including crystallinity index, degree of polymerization, accessibility, and lignin dissolution quantification. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:25–34, 2015     

Optimal configuration and combination of multiple lignocellulosic biomass feedstocks delivery to a biorefinery

Biomass availability and transportation are major challenges in establishing a large-scale biorefinery. The objective of this study was to assess the delivery cost of different combinations of multiple forms of lignocellulosic feedstocks including agricultural and woody biomass. Three types of biomass i.e., wheat straw, corn stover and forest biomass were considered in different forms such as loose biomass, bales/bundles, chopped/chipped and pellets. It was found that the delivery cost of a combination of woody and agricultural biomass feedstocks is lower than that for a single type of biomass. The delivery of agricultural residues as bales and woody biomass as chips is an economically attractive option with optimal combination of 30% bales and 70% wood chips to a biorefinery of capacity 5000 dry tonnes per day. The anticipated traffic congestions resulting from biomass supply to a large facility could be significantly reduced by increasing the density of biomass.     

Fungal pretreatment of lignocellulosic biomass

Pretreatment is a crucial step in the conversion of lignocellulosic biomass to fermentable sugars and biofuels. Compared to thermal/chemical pretreatment, fungal pretreatment reduces the recalcitrance of lignocellulosic biomass by lignin-degrading microorganisms and thus potentially provides an environmentally-friendly and energy-efficient pretreatment technology for biofuel production. This paper provides an overview of the current state of fungal pretreatment by white rot fungi for biofuel production. The specific topics discussed are: 1) enzymes involved in biodegradation during the fungal pretreatment; 2) operating parameters governing performance of the fungal pretreatment; 3) the effect of fungal pretreatment on enzymatic hydrolysis and ethanol production; 4) efforts for improving enzymatic hydrolysis and ethanol production through combinations of fungal pretreatment and physical/chemical pretreatment; 5) the treatment of lignocellulosic biomass with lignin-degrading enzymes isolated from fungal pretreatment, with a comparison to fungal pretreatment; 6) modeling, reactor design, and scale-up of solid state fungal pretreatment; and 7) the limitations and future perspective of this technology.     

树干毕赤酵母和酿酒酵母混合糖发酵产乙醇

以树干毕赤酵母和酿酒酵母为发酵菌株,酸性蒸汽爆破玉米秸秆预水解液和纯糖模拟液为C源,采用固定化酵母细胞的方法,研究了酸爆玉米秸秆预水解液初始pH、N源种类及其浓度、3种发酵模式对树干毕赤酵母戊糖发酵的影响。结果表明:玉米秸秆预水解液适合发酵的初始pH范围为6.0～7.0;1.0 g/L的(NH4)2SO4作为N源,在40 g/L葡萄糖和25 g/L木糖培养基中发酵24 h,糖利用率达到99.47%,乙醇质量浓度为24.72 g/L,优于尿素和蛋白胨作为N源;3种模式的发酵体系中,以游离树干毕赤酵母和固定化酿酒酵母发酵性能最好,糖利用率和乙醇得率分别为99.43%和96.39%。     

树干毕赤酵母木糖还原酶基因的克隆及在大肠杆菌中的表达

根据NCBI中的木糖还原酶基因序列设计引物,利用高保真聚合酶克隆树干毕赤酵母木糖还原酶基因,加A后克隆到质粒pGM-T中,测序验证.然后将目的基因克隆到舍有强启动子的穿梭表达载体p424GPD中,构建含有XYL1基因的重组质粒p424GPD-XYL1.将p424GPD-XYL1转化到大肠杆菌中,提取总蛋白,聚丙烯酰胺凝胶电泳分析.酶活测定确定木糖还原酶基因XYL1在大肠杆菌中得到活性表达,表明表达载体构建成功.表达载体的成功构建为后续构建重组酿酒酵母利用木糖发酵奠定基础.     

Label free quantitative proteomic analysis of secretome by Thermobifida fusca on different lignocellulosic biomass

Solid state fermentation of lignocellulosic biomass by filamentous microorganisms to induced enzyme production has been recognized as an attractive and cost effective technology. The secretion profile of lignocellulolytic enzymes by thermostable filamentous Thermobifida fusca (T. fusca) in solid state fermentation of different lignocellulosic biomasses, such as corn stover, hay; saw dust; sugarcane bagasse; wood chips; and un-dried green plant were explored using label-free exponentially modified protein abundance index (emPAI) based quantitative proteomics. Comparative analyses of T. fusca secretion profiles between cellulose and the various lignocellulosic biomasses showed induced expression of cellulolytic proteins by cellulose, and expression of hemicellulose, pectin and lignin degrading enzymes were induced by lignocellulosic biomasses. The solid state fermentation by T. fusca on lignocellulosic biomasses also revealed increased expressions of various transport proteins and hypothetical proteins. The Bray-Curtis similarity indices, clustering, and multidimensional scaling plot explicated differential protein expressions by T. fusca on different lignocellulosic biomasses, indicating that protein secretion by T. fusca is reliant on substrate complexity.     

Ethanol production from hardwood spent sulfite liquor using an adapted strain of Pichia stipitis

Conditions have been optimized for fermentation of pretreated hardwood spent sulfite liquor (HSSL) using an adapted strain of Pichia stipitis. The pretreatments, consisting of boiling and overliming with Ca(OH)₂ of HSSL, to partially remove inhibitors, and adaptation of the yeast strain to HSSL, were both critical for a successful fermentation. Ethanol concentration was increased from 6.7 to 20.2 g l⁻¹ using adapted P. stipitis (A) and pretreated HSSL. The maximum ethanol yield (Y _p/s) and productivity (Q _p) were 0.41 g g⁻¹ and 0.44 g l⁻¹ h⁻¹, respectively, at an oxygen transfer rate of 2.0 mmol O₂ l⁻¹ h⁻¹. The optimized results with this strain were compared to those of other xylose-fermenting yeasts and Saccharomyces cerevisiae (SSL-acclimatized) currently used at an industrial plant for the fermentation of spent sulfite liquor. Journal of Industrial Microbiology & Biotechnology (2001) 26, 145–150. Received 23 June 2000/ Accepted in revised form 21 October 2000     

木质纤维素资源生物精炼技术研究进展

开发利用可再生性的木质纤维素资源来生产液体燃料和大宗化学品,对于解决人类发展面临的资源与环境危机具有重要的意义。然而,作为其代表性工艺的纤维素乙醇生产却因为经济上无法过关而迟迟不能真正实现产业化。采用生物精炼技术,充分利用木质纤维素材料中各种组分,生产包括部分高值产品的多种产品,是克服其转化技术产业化经济可行性问题的有效措施。综述了木质纤维素原料生物精炼技术的研究发展现状,着重阐述了玉米芯的生物精炼技术产业化进展,并对木质纤维素的生物精炼前景进行了展望。     

纤维素乙醇发酵液中复杂组分对乙醇渗透汽化传质过程的影响

考察了木质纤维素乙醇发酵液中各组分对乙醇透过聚二甲基硅氧烷(PDMS)-silicalite-1渗透汽化膜传质性能的影响。结果表明:酵母细胞、玉米秸秆残渣和发酵用无机盐可增加乙醇通过膜的通量和选择性;而葡萄糖和甘油的存在会对乙醇的透膜传质产生负面影响;木质纤维素水解后的产物如糠醛和羟基丙酮,表现出对膜分离乙醇轻微的抑制作用。本文建立了渗透汽化优先透醇与纤维素乙醇发酵集成过程,批次发酵20 h后乙醇产率从最初的12.95下降到10.22 g/(L·h),60 h后乙醇产率下降为0,葡萄糖消耗速率与乙醇消耗产率呈同样趋势;连续发酵过程中,乙醇产率较稳定地维持在13.30 g/(L·h)。实验证明,集成过程可及时地将产物乙醇分离出去,能够有效地消除产物抑制,提高乙醇生产速率和葡萄糖转化率。     

Yeast strains for ethanol production from lignocellulosic hydrolysates during in situ detoxification

Yeast strains Y1, Y4 and Y7 demonstrated high conversion efficiencies for sugars and high abilities to tolerate or metabolize inhibitors in dilute-acid lignocellulosic hydrolysates. Strains Y1 and Y4 completely consumed the glucose within 24 h in dilute-acid lignocellulosic hydrolysate during in situ detoxification, and the maximum ethanol yields reached 0.49 g and 0.45 g ethanol/g glucose, equivalent to maximum theoretical values of 96% and 88.2%, respectively. Strain Y1 could metabolize xylose to xylitol with a yield of 0.64 g/g xylose, whereas Y4 was unable to utilize xylose as a substrate. Strain Y7 was able to consume sugars (glucose and xylose) within 72 h during hydrolysate in situ detoxification, producing a high ethanol yield (equivalent to 93.6% of the maximum theoretical value). Y1 and Y7 are the most efficient yeast strains yet reported for producing ethanol from non-detoxified dilute-acid lignocellulosic hydrolysates. These findings offer huge potential for improving the economics of bio-ethanol production from lignocellulosic hydrolysates.