纤维素乙醇生产重组酿酒酵母菌株的构建与优化研究进展

寻找化石能源的替代品以及开发和利用生物能源已引起国内外研究者的广泛关注。提高酿酒酵母利用来源广泛、贮存丰富的农林废弃物等木质纤维素原料生产燃料乙醇的效率是生物能源的重要研究内容,但是,重组酿酒酵母木糖发酵性能低是限制纤维素乙醇经济性的关键问题。本文总结了酿酒酵母中木糖代谢途径的构建和优化以及木糖转运对木糖利用的影响,分析了重组酵母利用纤维素水解液进行乙醇发酵的研究现状,并对进一步提高重组酿酒酵母纤维素乙醇生产效率的研究趋势进行了展望。目前国内外已经构建了可有效利用木糖产乙醇的重组酵母,但对其木糖代谢机制的研究还尚未深入,限制了重组菌株的定向改造。此外,目前缺少在纤维素生物质水解液发酵实际应用过程中对重组菌株的评价。因此,加强重组酵母菌株对木糖利用相关代谢调控机理的分析,注重多种抑制物对菌株发酵性能的影响,结合真实底物纤维素乙醇发酵过程进行重组菌株的构建和优化,从而进一步提高纤维素乙醇生产的经济性,是未来菌株构建的重要研究方向。     

Formation of formic acid from glycerine using a hydrothermal reaction

BACKGROUND: Glycerine, a main by‐product of the biodiesel manufacturing process, has potential to be an important biorefinery feedstock with the rapid increase in biodiesel production all over the world. Hydrothermal experiments with glycerine were carried out at 250 °C using H₂O₂ as an oxidant. RESULTS: Glycerine was converted into formic acid with a yield of 31.0% based on the starting mass of carbon in glycerine. A possible oxidation pathway for the formation of formic acid from glycerine is proposed. In the proposed pathway, glycerine may first be oxidised and then decomposed into formic acid and oxalic acid. Oxalic acid was indirectly attributed to the increase of formic acid production from glycerine, but it instead acts as a retardant to prevent further oxidation of formic acid. However, when an alkali was added to the experimental conditions, the yield of formic acid was not greatly improved, reaching only 34.7%. CONCLUSION: The present work should help to facilitate further studies to develop a new green process for the production of formic acid from renewable biomass. © 2012 Society of Chemical Industry     

Options analysis for long-term capacity design and operation of a lignocellulosic biomass refinery

The growth of the lignocellulosic fuels has been hindered by technological and market uncertainty. This paper optimizes strategic investment decisions by prospective biobased fuel and chemical enterprises. A real options-based stochastic integer programming model is developed in this paper. We model a hypothetical, vertically integrated lignocellulosic enterprise that produces cellulosic ethanol and biosuccinic acid. Uncertainty is represented in bioproduct demands and prices. Strategic options including investment in research and development, investments in a flexible production platform and deferral of project investment are modeled. A hypothetical market model is also developed to correlate crude oil prices with the evolution of bioproduct markets. The discounted value of equity free cash flows is optimized. The optimal results include multiple capacity design plans based on the long term evolution of bioproduct markets. Monte Carlo simulations are also conducted to quantify the risk adjusted NPV's and returns on investment for the optimal capacity design trajectories.     

高温液态水中葡萄糖无催化降解反应动力学

The decomposition kinetics of glucose was studied in high-temperature liquid water （HTLW） from 180 to 220℃ under a pressure of 10 MPa. It was found the main products from glucose decomposition were 5-hydroxymethylfurfural （5-HMF） and levulinic acid （LA）. The decomposition kinetics of 5-HMF and stability of LA in HTLW were further investigated. A kinetic model for glucose decomposition was proposed accordingly. In the model, a series of first-order reactions with the consideration of parallel by-reactions were used to illustrate the decomposition of glucose. The decomposition activation energies of glucose, 5-HMF, and LA were evaluated as 118.85, 95.40, and 31.29 kJ·mol^-1, respectively.     

Conversion of rye straw into fuel and xylitol: a technical and economical assessment based on experimental data

A contribution to the development of economically sustainable processes to obtain lignocellulosic bioethanol is presented.The simultaneous production of bioethanol and xylitol from rye straw is investigated, as this compound can become a realistic alternative to the transformation of xylose into ethanol. A complete process from the raw material to the end products is developed and simulated with respect to all major operations: hot liquid water pretreatment, fermentation of glucose to ethanol, fermentation of xylose to xylitol, separation and purification of both products. Calculations are based on experimental as well as on literature data. In particular, experimental data about the pretreatment and enzymatic hydrolysis steps previously obtained (Ingram et al., 2009) are used in order to achieve a realistic model that was then implemented using the software Aspen Plus™. The optimization of the process energy duties is carried out by means of the pinch technology analysis. Mass balances from the simulation are used in order to size the equipments and calculate the capital investment. Finally production costs and some financial indexes are estimated by an economical analysis.     

Fast pyrolysis of glucose‐based carbohydrates with added NaCl part 1: Experiments and development of a mechanistic model

Sodium ions, one of the natural inorganic constituents in lignocellulosic biomass, significantly alter pyrolysis behavior and resulting chemical speciation. Here, experiments were conducted using a micropyrolyzer to investigate the catalytic effects of NaCl on fast pyrolysis of glucose‐based carbohydrates (glucose, cellobiose, maltohexaose, and cellulose), and on a major product of cellulose pyrolysis, levoglucosan (LVG). A mechanistic model that addressed the significant catalytic effects of NaCl on the product distribution was developed. The model incorporated interactions of Na⁺ with cellulosic chains and low molecular weight species, reactions mediated by Na⁺ including dehydration, cyclic/Grob fragmentation, ring‐opening/closing, isomerization, and char formation, and a degradation network of LVG in the presence of Na⁺. Rate coefficients of elementary steps were specified based on Arrhenius parameters. The mechanistic model for cellulose included 768 reactions of 222 species, which included 252 reactions of 150 species comprising the mechanistic model of glucose decomposition in the presence of NaCl. © 2015 American Institute of Chemical Engineers AIChE J, 62: 766–777, 2016     

Low‐temperature and highly efficient conversion of saccharides into formic acid under hydrothermal conditions

Production of formic acid, which has been regarded as an important H₂ carrier, from biomass can be a highly potential way to provide human societies with renewable energy source. To attain economically viable production of formic acid from biomass on an industrial scale, the system operation at low reaction temperature is crucially important. In this work, a low‐temperature hydrothermal conversion of carbohydrates such as monosaccharides and disaccharides into formic acid is reported. A good formic acid yield of 80–85% was obtained at a lower temperature of 423 K for only 15–20 min in the presence of NaOH without any other catalyst. The alkali was found to act as two roles in enhancing the production of formic acid. One was inhibition of the formic acid decomposition; another was favorable for the oxidation selectively at C‐1 for aldoses, which leads to the formation of formic acid via the rupture of the C1–C2 bond. © 2016 American Institute of Chemical Engineers AIChE J, 62: 3657–3663, 2016     

Enzymatic saccharification of dissolution pretreated waste cellulosic fabrics for bacterial cellulose production by Gluconacetobacter xylinus

BACKGROUND: Waste textiles, such as dyed cellulosic and/or polyester blended fabrics have the potential to serve as an alternative feedstock for the production of biological products via microbial fermentation. Dissolution pretreatment was employed to enhance the enzymatic saccharification of dyed and synthetic fiber blended cellulosic fabrics. The fermentable reducing sugars obtained from waste cellulosic fabrics were used to culture Gluconobacter xylinus for value‐added bacterial cellulose (BC) production. RESULTS: Concentrated phosphoric acid was the ultimate cellulose solvent for dissolution pretreatment since 5% w/w cellulosic fabric can be completed dissolved at 50 °C. After regeneration in water, the cellulosic precipitate was subjected to cellulase hydrolysis, resulting in at least 4‐fold enhancement of saccharification rate and reducing sugars yield. The colored saccharification products can be utilized by G. xylinus to produce BC, approximately 1.8 g L^?1 BC pellicle was obtained after 7 days static cultivation. CONCLUSION: Dyed and blended waste fabric can be pretreated effectively by dissolution to produce fermentable sugars by cellulase hydrolysis. Dissolution pretreatment can expose the dyed or polyester fiber covered digestible cellulosic fibers to cellulase and leads to a significant enhancement of saccharification yield. The colored saccharification products have no significant inhibiting effect on the fermentation activity of G. xylinus for BC production. Copyright © 2010 Society of Chemical Industry     

发酵抑制物对葡萄糖发酵产乙醇的影响

在纤维素乙醇研究中,木质纤维原料在酸性预处理过程中会产生甲酸、乙酸、乙酰丙酸、糠醛和5-羟甲基糠醛等发酵抑制物,这些发酵抑制物会影响葡萄糖发酵生产乙醇的收率。本文考察了发酵液中各种发酵抑制物含量对高温超级酿酒酵母发酵乙醇收率的影响。研究结果表明,多种发酵抑制物的协同作用对乙醇发酵的影响要高于单一种类发酵抑制物对乙醇发酵的影响。发酵液中发酵抑制物总量一般控制在3.0g/L以内时,对葡萄糖发酵生产乙醇的抑制作用不明显。     

高温液态水中木糖无催化分解反应动力学

The kinetics of non-catalyzed decompositions of xylose and its decomposition product furfural in high temperature liquid water （HTLW） was studied for temperature from 180 to 220℃ and under pressure of 10MPa. The main products of xylose decomposition were furfural and formic acid, and furfural further degraded to formic acid under HTLW condition. With the assumption of first order kinetics e.quation, the evaluated activation energy of xylose and furfural decomposition was 123.27kJ·mol^-1 and 58.84kJ·mol^-1, respectively.     

Reaction Rates for the Partial Dehydration of Glucose to Organic Acids in Solid-Acid,Molecular-Sieving Catalyst Powders

Molecular-sieving catalysts have the potential to promote the production of oxygenated hydrocarbons from glucose. A kinetic model for the partial dehydration of glucose to organic acids by micro- and mesoporous aluminosilicate catalysts was developed. Kinetic parameters were estimated from glucose conversion and product yield versus time data at 150°C for HY-zeolite, aluminum-pillared montmorillonite, MCM-20 and MCM-41 catalyst powders of 0·5 mmol H⁺ g⁻¹ solid-acid activity. Rate constants for the partial dehydration of glucose to 5-hydroxymethylfurfural (HMF) and the rehydration and cleavage of HMF to formic acid and 4-oxopentanoic acid were maximized at catalyst pore diameters of 10–30 Å. The final organic acid product yields were low, less than 60% of theoretical for formic acid and 10% of theoretical for 4-oxopentanoic acid, due to significant coke formation. © 1997 SCI.     

高温液态水中果糖无催化分解反应动力学

研究了压力10 MPa下、温度453.15～493.15 K范围内果糖在高温液态水中的无催化分解反应动力学.实验结果表明,在无任何催化剂条件下,果糖能顺利进行分解反应,且随着温度的升高,分解反应速率常数显著地增大.在本实验范围内,果糖分解反应的活化能为126.16 kJ•mol^-1.另外,对不同温度下反应时间对果糖分解的主要产物——5-羟甲基糠醛收率的影响进行了考察.通过控制反应条件可以有选择性地调节产物分布.     

Mechanism of Dilute Acid Hydrolysis of Cellulose Accounting for its Degradation in the Solid State

It is known that dilute acid hydrolysis of cellulose at high temperature results in yields not exceeding 60–65% of the potential glucose. All the models presented in the literature are based on the sequence: cellulose #x00BB; glucose #x00BB; degradation products. The underlying assumption of this sequence is that the unconverted cellulose retains its chemical integrity during the course of the hydrolysis reaction. Experimental evidence based on physico-chemical characterization of cellulosic residues obtained from dilute acid hydrolysis processes allows us to propose a new mechanism for cellulose hydrolysis accounting for its degradation in the solid state. Such degradation results in the formation of a non- carbohydrate residue.     

Developing Ways of Obtaining Quality Hydrolyzates Based on Integrating Catalytic Peroxide Delignification and the Acid Hydrolysis of Birch Wood

Traditional processes of acid-catalyzed hydrolysis of wood are ineffective due to the low quality of formed glucose solutions contaminated with impurities that inhibit fermentation of glucose to ethanol. This problem grows during the hydrolysis of birch wood containing large amounts of hemicellulose. This work proposes producing quality glucose solutions using sulfuric acid (H₂SO₄, 80%) catalyzed hydrolysis at 25°C the cellulosic products formed during the catalytic peroxide delignification of birch wood. It is established that the composition of cellulosic products strongly affects the contents of glucose, xylose, and impurities inhibiting the enzymatic synthesis of bioethanol: furfural, 5-hydroxymethyl furfural, and levulinic acid. High yields (80.4–83.5 wt %) of glucose are achieved using cellulosic products produced by integrating the processes of sulfuric acid hydrolysis of hemicelluloses from birch wood and peroxide delignification of prehydrolyzed wood in the presence of catalysts: 2% H₂SO₄ and 1% TiO₂. Concentration of inhibitors of enzymatic processes in these hydrolyzates is below the allowable limits. Hydrolyzates with maximum glucose content (86.4–88.5 wt %) and minimum concentration of inhibiting impurities produced by acid hydrolysis of cellulosic products treated with an 18% solution of NaOH. Gas chromatography, HPLC, and chromato-mass spectrometry are used to analyze the composition of hydrolyzates. Cellulosic products are examined by SEM, XRD, and chemical analysis.     

高温液态水中甜高粱渣半纤维素水解及其机理

为了回收甜高粱渣中的半纤维素衍生糖并了解其水解机理,在自行设计的Flowthrough反应器中对甜高粱渣进行了高温液态水水解,分别考察了不同反应温度和反应液流量下水解液中产物的生成情况。研究表明,相对葡萄糖和阿拉伯糖而言,木糖的生成受反应温度和反应液流量影响更大。温度高于195 ℃时糖降解加剧,总木糖浓度不断降低;低流量（5 ml?min-1）条件下生成的木糖不能被及时排出而进一步降解。通过产物分析可知,甜高粱渣半纤维素中含有典型的O-乙酰基-4-O-甲基葡萄糖醛酸基阿拉伯糖基木聚糖结构,木糖、葡萄糖、阿拉伯糖、各种低聚糖、乙酸和葡萄糖醛酸等是半纤维素水解的直接产物,糠醛和5-羟甲基糠醛等是糖类的降解产物, 甲酸等小分子酸是它们的进一步降解产物。     

过表达谷氧还蛋白基因GRX5提高酿酒酵母乙酸耐性

利用可再生的纤维素原料生产燃料乙醇是国内外研究的热点。但纤维素原料一些预处理过程产生的乙酸对酿酒酵母细胞生长和乙醇发酵产生强烈抑制,因此,提高酿酒酵母细胞的乙酸耐受性是提高纤维素乙醇发酵效率的重要手段。本文研究了谷氧还蛋白家族中GRX5p的编码基因的过表达对酿酒酵母在乙酸胁迫条件下细胞生长和发酵性能的影响。结果表明,过表达GRX5的重组菌株在含有5 g·L^-1乙酸的平板中生长优于对照菌株;在含有5 g·L^-1乙酸的培养基中进行乙醇发酵,过表达GRX5的重组菌株可在48 h基本消耗培养基中所有的葡萄糖,发酵周期比对照菌株缩短了12 h。过表达GRX5菌株的乙醇生产强度为0.897 g·L^-1·h^-1,比对照提高了28.5%。代谢物分析结果表明,过表达GRX5的重组菌株可产生更多的保护性物质海藻糖和甘油,有利于增强菌株胁迫耐受性。     

Evaluation of the effects of operational parameters in the pretreatment of sugarcane bagasse with diluted sulfuric acid using analysis of variance

The pretreatment of lignocellulosic residues has been extensively studied as a method to disrupt the cellulose–hemicelluloses–lignin complex in biomass to access the sugars in their respective components. In this work, we carried out a study using sulfuric acid pretreatment of sugarcane bagasse by varying the following operational parameters: solid loading (10–30% of bagasse relative to the volume of the sulfuric acid solution), sulfuric acid concentration (0.5–2.5% relative to the dry mass of bagasse), reaction time (5–25?min), and temperature (135–195°C). The obtained solids from each pretreatment condition were submitted to enzymatic hydrolysis under the same process conditions: 0.232?g of Celluclast 1.5?L and 0.052?g of Novozym 188 per g of pretreated sugarcane bagasse, 72?h of hydrolysis, and 200?rpm of agitation at 50°C. Using central composite rotational design configuration in the experiments and analysis of variance, the results indicate that the conditions that produced larger quantities of glucose by enzymatic hydrolysis (0.35?g glucose/g pulp) with minimum amounts of degradation products were as follows: 20% solids loading, 15?min of reaction time, 1.5% sulfuric acid, and a minimum temperature of reaction of 170°C.     

Kinetic studies of alkyl formate hydrolysis using formic acid as a catalyst

BACKGROUND: The hydrolysis of methyl formate is the major industrial process for the production of formic acid. The aim of the work is to determine the reaction kinetics quantitatively in the presence of formic acid catalyst, develop a mathematical model for the reaction system and estimate the kinetic parameters for the purpose of optimization. RESULTS: Liquid phase hydrolysis kinetics of alkyl formates (ethyl and methyl formate) was studied in an isothermal batch reactor at 80–110 °C and 20 bar nitrogen pressure. The catalyst of choice was formic acid. The reaction rate was enhanced but the formic acid product yield was slightly suppressed relative to the uncatalysed system. A kinetic model comprising mass balances and rate equations was developed and the kinetic and equilibrium parameters included in the rate equations were estimated from the experimental data with non‐linear regression analysis. CONCLUSION: The model was able to predict the experimental results successfully. The results obtained were compared quantitatively with an earlier model involving alkyl formate hydrolysis in a neutral aqueous solution. Copyright © 2011 Society of Chemical Industry     

Formation of polyelectrolyte complexes between cellulose derivatives and their blood compatibility

Formation of polyelectrolyte complexes (PECs) between cellulose derivatives in aqueous solution and their blood compatibility were examined. To this end, two types of quaternary ammonium cellulose derivatives, Q-Cell and Q-HEC, were prepared by treating cellulose and hydroxyethyl cellulose (HEC), respectively, with glycidyl trimethylammonium chloride. Anionic derivatives were carboxymethyl cellulose (CMC) with different degrees of substitution (DS) and cellulose sulfate (CS). In vivo blood tests were made by a method of peripheral vein indwelling suture. The results showed that the complex formation of Q-Cell did not follow a stoichiometric reaction, but Q-HEC reacted stoichiometrically with CMC and CS. It was also found that water-insoluble cellulosic PECs are soluble in formic acid, showing that the cellulosic PEC films can be prepared from formic acid solutions. The blood tests revealed that by the criteria of the test method employed in this work, cellulosic PECs had a good blood compatibility.