Analysis of process integration and intensification of enzymatic cellulose hydrolysis in a membrane bioreactor

Enzymatic cellulose hydrolysis has been studied for many years, generating rich literatures and knowledge in respect to the underlying reaction mechanism, reaction kinetics, and bioreactor systems. This paper attempts to offer some additional information and new understanding of how reaction kinetics and reactor productivity can be improved in a process involving simultaneous reaction and product separation using a purpose‐built membrane reactor with a single combined reaction zone and separation zone. Different operating strategies of batch, fed batch and continuous cellulose hydrolysis were investigated with intermittent or simultaneous removal of products (reducing sugars) to reduce enzyme inhibition and improve reactor productivity. The effect of continuous and selective product removal, reduced enzyme inhibition and higher enzyme concentration in retention were examined for the potential benefit in process integration and intensification in order to lower the high process cost of the enzymatic hydrolysis process, mainly due to slow reaction kinetics and expensive enzymes. A mathematical model was offered to account for the effect of selective product (reducing sugars) separation, permeate flux, reduced cellulase inhibition, dynamic structural change of the solid substrate and possible shear deactivation of the enzyme. Computer analysis was also carried out to analyse the quasi‐steady state of the reaction intermediates in order to gain an insight into the reaction mechanism in simultaneous reaction and separation systems. Some original analysis and simulation of the effect of membrane separation parameters on the overall reactor performance is offered, including the effect of membrane selectivity (rejection coefficient) and flux. Copyright © 2005 Society of Chemical Industry     

Effect of Pectinase Immobilization in a Polymeric Membrane on Ultrafiltration of Fluid Foods

The aim of this research was to obtain an enzymatic membrane reactor for use to clarify a model system and apple juice with a 12% soluble solid content and 1 mg/mL of pectin by water-soluble pectinolytic enzymes which were immobilized on polysulfone membranes. These membranes were synthesized in the laboratory by immersion precipitation (phase inversion) from polysulfone with and without active carbon. To ensure the cut-off separation of the membrane in the ultrafiltration process a commercial flat membrane with an 8-kDa molecular weight cut-off was used. The influence of different parameters (viscosity, density, reducing sugars, color, pH, and presence of pectin) on the initial sample and the volumetric permeate flux was also evaluated. The obtained membranes were characterized physically using a scanning electron microscopy SEM and analyses were performed with IFME® software.

The results indicated that immobilization of the enzyme in the membrane allowed an interaction between the recirculating sample and the membrane, the one obtained with activated carbon and immobilized enzymes showed higher degradation of pectin, improving the performance of the volumetric permeate flux.     

Use of membrane separation in enzymatic hydrolysis of waste paper

A three-stage process containing phosphoric acid pretreatment, enzymatic hydrolysis, and membrane filtration was performed on waste paper as a lignocellulosic material. In the first two stages, the effect of phosphoric acid concentration, enzyme loading, hydrolysis time, and substrate concentration on the amount of products was investigated. At the third stage using a proper membrane, the effect of substrate concentration and transmembrane pressure (TMP) on yield of the reducing sugars was studied. The novelty of the present study was to demonstrate the application of ultrafiltration membrane on the enzymatic hydrolysis process of waste paper. The reducing sugars concentration was determined by using the 3,5-dinitrosalicylic acid (DNS) reagent method. According to the results, a value of 0.5% was determined as the optimum concentration for phosphoric acid in the pretreatment stage. The reducing sugars yield was obtained as 67.4% in this concentration. Moreover, for the enzymatic hydrolysis of waste paper, the suitable amounts of cellulase enzyme loading and hydrolysis time were determined as 50 mg/g substrate and 48 h, respectively. In the filtration stage, increase of substrate concentration and decrease of TMP resulted in higher rejection of the reducing sugars. The experimental results revealed that the highest rejection was 19.2% at TMP of 3 bar and substrate concentration of 100 g/L.     

木质纤维原料生物转化燃料乙醇的研究进展

木质纤维生物量能够用来生产一种可替代有限的石油产品的能源——乙醇。木质纤维的转化主要分两个步骤木质纤维生物量中纤维素水解生成还原糖;糖发酵成乙醇。基于目前的技术,木质纤维原料生产乙醇的主要问题是得率低、水解成本高。促进木质纤维水解的方法包括木质纤维原料预处理脱除木素和半纤维素;纤维素酶的优化;同步糖化发酵法(SSF)。     

氨预处理对大豆秸秆纤维素酶解产糖影响的研究

为了提高大豆秸秆酶解产糖能力, 以利于从大豆秸秆中提取生物降解性塑料的原料 ?? 乳酸, 对大豆秸秆纤维素预处理过程的影响因素进行了探索,对预处理前后大豆秸秆的物理结构变化、化学成分变化及预处理条件对大豆秸秆酶水解产糖的影响进行了研究。研究结果表明,粉碎结合氨处理对大豆秸秆酶水解影响较大,较适宜的预处理条件为大豆秸秆粉碎至 140 目,10%氨水处理 24h。经过预处理后大豆秸秆纤维素含量提高 70.27%, 半纤维素含量下降 41.45%, 木质素含量下降 30.16%, 有利于大豆秸秆酶解产糖。     

木质纤维素燃料乙醇生物转化预处理技术

由丰富的木质纤维素资源制备乙醇有利于缓解能源紧缺、减少环境污染、实现可持续发展.然而某些物理、化学因素阻碍了木质纤维素中纤维素和半纤维素的转化和利用.预处理引起物理和/或化学上的变化,主要目的是改变或去除各种结构和(或)化学障碍,增加纤维素酶解率和转化效果,是一系列纤维素乙醇转化技术中的关键和核心.本文就纤维素乙醇生物...     

Synergistic effect of pretreatment and hydrolysis enzymes on the production of fermentable sugars from date palm lignocellulosic waste

The sequential addition of the enzymes, laccase for lignin degrading, followed by xylanase for hemicelluloses hydrolysis, then cellulase for cellulose hydrolysis, was compared to the synergistic action of using the three enzymes together. It was shown that the reducing sugars yield increased from 5.6% using cellulase only to 45.6% by pretreatment with laccase and xylanase, prior to the enzymatic hydrolysis. A higher conversion of 60% was achieved by using the three enzymes together for the same incubation period. The proposed synergistic enzymes approach is a simpler and less energy intensive alternative compared to the conventional lignocelluloses pretreatment techniques.     

Green Liquor Pretreatment of Mixed Hardwood for Ethanol Production in a Repurposed Kraft Pulp Mill

Abstract

The development of a new, relatively simple process, which uses green liquor (sodium carbonate and sodium sulfide) as a pretreatment for the production of ethanol is described in this article. The pulps produced by this process can be enzymatically hydrolyzed to monomeric sugars with a high overall sugar recovery. The use of green liquor for pretreatment ensures that the chemicals used during pretreatment can be recovered efficiently using proven technology and can be easily implemented in a repurposed kraft pulp mill. The yield of pulps produced by the green liquor pretreatment process is about 80% with nearly 100% cellulose and 75% xylan in retention in mixed southern hardwood. The low pH prevents the random hydrolysis of polysaccharide and secondary peeling reactions from occurring during the pretreatment, resulting in higher retention of the polysaccharides in pulp. About 35% of the lignin is removed during the green liquor pretreatment process, which is sufficient for efficient enzymatic hydrolysis. The amount of sugar produced in enzymatic hydrolysis increased with both the green liquor and enzyme charge. The increase in enzymatic hydrolysis efficiency was small as the total titrateable alkali was increased beyond 12–16%. With green liquor pretreatment at 16% Total Titrateable Alkali (TTA), the overall sugar recovery for hardwood was shown to be around 77% at a cellulase charge of 20 FPU/gm of substrate. A sugar recovery of 80% could be achieved at higher enzyme charges. These levels of sugar recovery are competitive with other pretreatments for hardwood. This novel pretreatment process can be used to repurpose kraft mills, which are being closed due to a decrease in the demand for paper in North America, for production of ethanol.     

皇竹草稀硫酸预处理研究

研究了中低温稀酸预处理对皇竹草化学组成变化、纤维素酶水解得率与总糖得率的影响,并采用扫描电镜(SEM)对皇竹草纤维结构变化进行了分析.结果表明,随着硫酸浓度的增大、温度的升高和时间的延长,半纤维素含量大幅度降低,且预处理后纤维素酶水解得率也逐渐增大.较好的预处理条件为100 g皇竹草原料,在固液比1:5(g:mL)条件下,用质量分数4.0%硫酸在温度110 ℃下,经过8 h预处理后,纤维素保留率为87.48%,半纤维素水解率为93.68%,所得固体渣经纤维素酶水解72 h后得率为86.3%(纤维素酶用量40 FPIU/g,以纤维素质量计),100 g原料可得到总糖量为54.53 g.预处理后皇竹草纤维表面和细胞壁受到破坏,表面积增大,有利于纤维素酶水解作用的进行.     

A comprehensive study of the loss of enzyme activity in a continuous membrane reactor – application to starch hydrolysis

Loss of enzyme activity is a problem associated with enzymatic reactions in continuous recycled membrane reactors (CRMR). It may result from catalyst leakage and also enzyme denaturation due to the effects of pH, temperature, shear effects or adsorption/deposit on membrane. In this study, the relative importance of these various factors has been assessed in order to reduce their adverse effects on starch hydrolysis in a CRMR. The effects of temperature and denaturation by adsorption/deposit on membrane were the most limiting phenomena. Reducing the temperature to overcome thermal denaturation was not a practical solution since this increases viscosity and thereby decreases permeate flux and reactor performance. Insofar that adsorption/deposit of enzymes on the membrane is directly linked to membrane fouling, back‐flushing or regularly purging retentate should reduce this phenomenon by lowering accumulation of high molecular weight products. © 2001 Society of Chemical Industry     

Enzymatic saccharification of lignocellulosic materials after treatment with supercritical carbon dioxide

Lignocellulosic materials, such as agricultural residues, are abundant renewable resources for bioconversion to sugars. The sugar cane bagasse was studied here to obtain simple sugars for the production of alcohols and other chemicals. The crystalline structure of cellulose and the lignin that physically seals the surrounding cellulose fibers makes enzymatic hydrolysis difficult by preventing the contact between the cellulose and the enzyme. Two different samples of sugar cane (bagasse pulp and skin) were used and compared with microcrystalline cellulose (Avicel). The investigated samples were pretreated with SC-CO₂ explosion before hydrolysis. The experiments were conducted at 12, 14 and 16 MPa at a temperature of 60 °C. In this process, particles of celluloses within the size range from 0.25 to 0.42 mm were placed in defined amounts inside the experimental vessel, CO₂ was injected and let stand for 5 and 60 min. The explosion pretreatment of cellulosic materials by SC-CO₂ was performed in an apparatus of a static type with 300 ml of volume. The hydrolysis reaction using cellulose enzyme was carried at 55 °C for 8 h. After the pretreatment, the glucose yield increased in 72% to the bagasse sample. The SC-CO₂ pretreatment together with alkali increased the glucose yield in 20% as compared with alkali only. X-ray, microscopy and thermal analysis were used to investigate the effect of the pretreatment.     

A study on enzymatic hydrolysis of cellulose in an attrition bioreactor

In the biological conversion of cellulose, a physical or chemical pretrealment proceeds the hydrolysis by an enzyme. The hydrolysis rate however, is slowed down as the active sites in the pretreated substrates are reduced. In this study, attempts were made to use an attrition bioreactor in which the pretreatment and the hydrolysis were carried out at the same time, where higher hydrolysis rales were achieved. Glass beads of 0.3-cm-diameter and sand were used as the milling media in a batch reactor where pure cellulose, Solka Floc BW200 was hydrolyzed by cellulase secreted from a fungus mutant,Trichoderma reesei. The higher rates observed are believed due to the synergistic effects of the size reduction and the conversion of the crystalline to the amorphous form of cellulose which was observed by comparing the X-ray diffractorgrams of the cellulose hydrolyzed in the reactors with and without milling medium. A simple kinetic model was found satisfactory in depicting the hydrolysis mechanism, and the kinetic parameters were estimated. Higher power consumption as compared to a reqular stirred reactor was observed and a quantitative expression was derived for its estimation.     

Enzymatic Hydrolysis of Cassava Starch into Maltose Syrup in a Continuous Membrane Reactor

This study deals with the use of a membrane reactor for the enzymatic conversion of cassava starch to maltose. The enzymes used were Maltogenase and Promozyme (Novo Nordisk). Maltogenase activity was unaffected after a 5 h incubation period at 65°C, but Promozyme was markedly heat-unstable even at 37°C. Batch hydrolysis of liquefied cassava starch (30% w/w) by Maltogenase and Promozyme resulted in a maximum degree of starch conversion to maltose of 72% (≈254 g dm⁻³ maltose). The conversion degree fell by 11% when no debranching enzyme was used. The residence time distribution of the ultrafiltration reactor (UFR) was that of an ideal continuously stirred tank reactor. Rejection of Maltogenase by Carbosep M4 membranes (MWCO: 50 kDa) was not total. The overall enzyme activity loss after a 5 h diafiltration period was 28%, however about half this loss appeared to be due to enzyme denaturation inside the reactor. During saccharification trials conducted in the UFR at a starch concentration of 30% (w/w), severe membrane fouling occurred. The average permeate fluxes obtained were 14 and 23 dm³ h⁻¹ m⁻² at constant transmembrane pressures of 100 and 200 kPa respectively. When the reactor was operated at a space-time of 4·2 h, the degree of starch conversion to maltose in the permeate rapidly stabilized around 55–56%. © 1997 SCI.     

利用乳液酶膜反应器拆分萘普生甲酯实验研究

利用乳液酶膜反应器进行外消旋萘普生甲酯的水解反应,以制备光学纯对映体(S)-萘普生,反应同时从膜透过侧收集产物,实现反应分离一体化。实验研究了固定酶前后膜传质阻力和反应过程的水相跨膜通量,考察了透过侧的产物浓度、反应器的转化率、产量和对映体选择性。结果表明:固定化酶引起的传质阻力远大于膜本身的阻力;透过侧的产物浓度与水相渗透通量密切相关,通量较低时,产物浓度较高;固定化酶的初始反应速率为3.660μmol/(h.g),为自由酶的20倍以上,固定化酶的对映体过剩值为99%—100%,远高于自由酶的选择性,表明该反应体系为脂肪酶催化拆分反应提供了良好环境。     

Simultaneous hydrolysis and fermentation of pulp mill primary clarifier sludge

Bioconversion of sludge from the primary clarifier of a sulphite pulping operation to ethanol offers a number of advantages over conventional disposal options. The amount of material which must be disposed of is reduced while, at the same time, salable and environmentally friendly fuel-ethanol is produced. In this study, primary clarifier sludge (PCS) was shown to be hydrolysed to produce fermentable sugars at a rate proportional to enzyme loading. Initial (1 hour) hydrolysis rates as high as 12.6 g reducing sugar/L · h were observed at an initial enzyme loading of 10 filter paper units (FPU)/g. Hydrolysis was inhibited by spent sulphite liquor (SSL), an inhibition which could be completely overcome by fermenting the SSL to remove sugars. Surfactants were found to only marginally improve the production of sugars. To reduce the deleterious effects of end product inhibition, single stage simultaneous hydrolysis and fermentation (SHF) was carried out using cellulase enzymes and Saccharomyces cerevisiae.     

Application of a microassay method to study enzymatic hydrolysis of pretreated wheat straw

BACKGROUND: The conversion of lignocellulosic biomass to ethanol includes a disruptive pretreatment process followed by enzyme‐catalyzed hydrolysis of the cellulose and hemicellulose components to fermentable sugars. As the cost and hydrolytic efficiency of enzymes are major factors that restrict the commercialization of biomass conversion processes, significant efforts are made nowadays to improve the enzymatic mixtures and make the process cost‐effective. RESULTS: In this work, enzymatic microassays have been developed and validated to test new different enzymatic formulations on real lignocellulosic substrates. Homogeneous handsheets from steam pretreated wheat straw were elaborated to be used as substrate. The microassay was adapted to test both water‐insoluble solids and the whole slurry as substrates. Results in hydrolysis microassays were comparable with those obtained in standard flask assays using pretreated wheat straw. Moreover, using the enzymatic microassays, two novel preparations have been evaluated, demonstrating the ability of microassays to discriminate between different enzymatic mixtures. CONCLUSIONS: This enzymatic microassay represents a rapid method to test the performance of new selected cellulase enzymes on real pretreated lignocellulosic substrates. This microassay will enable evaluation of enzyme components separately, or optimized mixtures. Copyright © 2010 Society of Chemical Industry     

汽爆秸秆膜循环酶解耦合丙酮丁醇发酵

利用新型的汽爆玉米秸秆膜循环酶解耦合发酵系统进行了丙酮丁醇发酵的研究,并对使用该系统所导致的丙酮丁醇梭菌(Clostridium acetobutylicum AS1.132)代谢的变化进行了讨论. 在稀释率为0.075 h-1的条件下,丁醇的产量为0.14 g/g (纤维素+半纤维素),最大丁醇产率达到0.31 g/(L×h),溶剂组成为丁醇:丙酮:乙醇65.3:24.3:10.4(体积比),纤维素和半纤维素的转化率分别为72%和80%,使用单位纤维素酶所产生的丁醇量为3.9 mg/IU,是分步水解批次发酵的1.5倍. 利用该系统使酶解和发酵分别在各自最适的条件下同时连续进行,减少了纤维素酶的用量,有效地解除了酶解产物对纤维素酶的抑制作用,并减轻了溶剂产物尤其是丁醇对微生物活性的影响,延长了发酵周期.     

Enzymatic hydrolysis from carbohydrates of barley straw pretreated by ionic liquids

BACKGROUND: Lignocellulosic biomass offers many potential advantages in comparison with the traditionally used sugars or starchy biomass since it is very widely available and does not compete with food and feed production. The abundance and high carbohydrates content of barley straw make it a good candidate for bioethanol production in Europe. Since biomass must be pretreated before enzymatic hydrolysis to improve the digestibility of both the cellulose and the hemicellulose biomass, the use of ionic liquids (IL) has been proposed as an environment‐friendly pretreatment of biomass. RESULTS: Different pretreatment conditions were investigated to determine the effects of the experimental conditions (temperature and time) on the enzymatic digestibility of pretreated material. The pretreatment of barley straw with 1‐ethyl‐3‐methyl imidazolium acetate treatment resulted in up to a 9‐fold increase in the cellulose conversion and a 13‐fold increase in the xylan conversion when compared with the untreated barley straw. CONCLUSION: Ionic liquid pretreatment of barley straw at 110°C for 30 min, followed by enzymatic hydrolysis, leads to a sugar yield of 53.5 g per 100 g raw material. It is then ready available for conversion into ethanol and is equivalent to more than 86% from potential sugars. The increase in saccharification was possible due to rupture of the lignin–hemicellulose linkages by treatment with 1‐ethyl‐3‐methyl imidazolium acetate. © 2012 Society of Chemical Industry     

Comparing the efficiency of plant material bioconversion processes using biocatalysts based on Trichoderma and Penicillium verruculosum enzyme preparations

A comparative study of the effectiveness of six commercial biocatalysts based on enzyme preparations derived from a fungus of the genus Trichoderma as a producer (Cellic Ctec-1, Cellic Ctec-2, Accelerase 1000, Accelerase 1500, Accelerase XY, Accelerase DUET), and of laboratory biocatalysts based on enzyme preparations derived from the fungus Penicillium verruculosum for the hydrolysis of the four types of plant cellulose material (steam explosion pretreated corn stalks and bagasse, ground pine and aspen), and of microcrystalline cellulose is performed. Biocatalysts activities toward various substrates, and the dependence of the depth of exhaustive hydrolysis of plant material on the dosage of these biocatalysts are determined. It is shown that biocatalysts based on the strains of P. verruculosum are competitive with widely used commercial biocatalysts based on the Trichoderma strain if we scale the biotechnological processes of bioconversion of renewable plant raw materials.     

木薯酒精渣的预处理及补料同步糖化发酵制取乙醇

木薯酒精渣的处置是制约木薯燃料乙醇大规模产业化的问题之一。本文立足于探索木薯酒精渣利用途径,分析了木薯酒精渣的主要成分,对比了氨水、氢氧化钠、氨水组合稀硫酸3种预处理方式对于木薯酒精渣纤维素和木素含量及纤维素酶水解效率的影响,分析了处理前后木薯酒精渣的表面结构及纤维素结晶度,并以氨水处理后的木薯酒精渣为底物,进行了同步糖化发酵。结果表明,3种预处理方法中组合预处理能更好地增加纤维素含量和提高纤维素酶水解效率,与未处理原料相比,组合预处理后纤维素含量增加了111.26%,木素下降了35.05%,酶水解72h纤维素转化率从42.10%增加到61.71%。氨水预处理后,原料的木素含量降低,处理后木薯酒精渣的表面变得更加粗糙,纤维素结晶度有所增加,以氨水处理后的木薯酒精渣为底物进行分批补料同步糖化发酵,当初始底物浓度为100.0g/L,分别在20h、40h、60h进行补料至最终底物浓度为400.0g/L时,发酵120h乙醇浓度达到51.0g/L。