提高木质纤维素酶解糖化效率的研究进展

木质纤维素高效水解为可发酵糖是其在生物燃料及高附加值化学品转化过程中至关重要的环节。从环保的角度出发,水解方法中的酶解是木质纤维素被彻底降解而无环境污染的有效途径,并且酶水解反应糖损耗低、副产物少、条件温和,因此受到广泛关注。但木质纤维素的组成与结构极为复杂,加之纤维素酶存在稳定性差、寿命短、活性低等缺陷,致使酶解效率较低,酶解糖化成本过高,为此国内外学者对如何提高木质纤维素酶解效率开展诸多方面的研究,综述了近年来提高木质纤维素酶解效率研究取得的最新理论研究及工艺进展,并就木质纤维素的预处理、产酶菌株/技术、酶复配/重组、酶解助剂、酶固定、外场作用、酶回收重利用及酶解反应器多方面的研究情况进行了总结,进一步展望了木质纤维酶解糖化的发展方向。     

表面活性剂对木质纤维素酶解产糖的影响研究进展

木质纤维素是生产生物燃料乙醇的主要原料,其含量丰富、绿色环保以及可再生性,因此有效地利用木质纤维素有望解决能源短缺问题。表面活性剂能够有效地促进木质纤维素的酶解反应,通过探讨不同表面活性剂对酶解反应的影响及机理,为实际的酶解过程找到合适表面活性剂提供一定的理论指导。     

稀酸和稀碱预处理对四种不同生物质资源制备还原糖的影响

本论文探讨了不同浓度的稀H_2SO_4和稀NaOH预处理对大豆秸秆、水稻秸秆、象草和狼尾草四种不同生物质酶解制备还原糖的影响。结果表明,大豆秸秆、水稻秸秆、象草和狼尾草具有较高的纤维素和半纤维素含量,是制备还原糖的理想原料。与稀H_2SO_4预处理相比,经稀NaOH预处理后的样品表现出较好的酶解性能。通过使用4%的NaOH对大豆秸秆和狼尾草进行预处理,还原糖产量分别为145.8 mg/mL和319.2 mg/mL。此外,以1%NaOH预处理后的水稻秸秆和象草为原料,可以分别获得385.2 mg/mL和231.6 mg/mL还原糖产量。     

评价木质纤维素预处理效果的新方法

以玉米秸秆为代表性纤维质原料,尝试建立一种评估预处理效果的新方法——持水率测定法,即:将试样在室温下浸泡1 h,在分离因数1 000下离心5 min后测定持水率。结果表明:在机理上木质纤维的持水率与可消化性具有一致性,在某种程度上具有正相关性;持水率作为一种简单快捷的新型测定方法,能够用来评估木质纤维素类生物质的预处理效果,不同预处理方法通过打破木质纤维的复杂致密结构,破坏氢键和酯键作用增加其孔径和孔穴,同时使其暴露出更多的游离羟基等亲水性基团,最终增加了木质纤维的持水率。     

乙醇预处理麦秆的酶解性能研究

采用H2SO4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:H2SO4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H2SO4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)。     

解糖热解纤维素菌F32降解未预处理秸秆及产纤维素酶特性分析

【目的】明确极端嗜热厌氧木质纤维素降解菌解糖热解纤维素菌F32代谢特征,并分析其产酶特性。【方法】使用细胞计数法绘制菌株的生长曲线,使用离子色谱及气相色谱进行产物和残糖量分析,以DNS法及对硝基苯酚法检测菌株胞外蛋白的酶活性。【结果】解糖热解纤维素菌F32在以葡萄糖、微晶纤维素和未经预处理小麦秸秆为碳源时生长状况优于解糖热解纤维素菌DSM 8903。在以葡萄糖为碳源进行培养时,与菌株DSM 8903相比,菌株F32具有产乳酸较多,而产氢气较少的特点。在以微晶纤维素和未经预处理小麦秸秆为碳源进行培养时,与菌株DSM 8903相比,菌株F32胞外蛋白具有较高的内切纤维素酶活性和木聚糖酶活性。【结论】解糖热解纤维素菌F32表现出较强的木质纤维素降解能力,其与DSM 8903的产物组成及胞外蛋白的酶活性具有明显差异。     

碱液湿磨预处理稻草的酶解性能

预处理是提高酶法木质纤维素糖化效率的重要加工过程.本论文对稻草进行碱液湿磨预处理和酶解,探讨了预处理碱液浓度对稻草的成分、结构和酶解的影响,同时利用米氏方程对稻草酶解反应进行动力学分析,求出了米氏常数Km 和最大反应速率rmax.实验结果表明,碱液湿磨预处理明显改善了稻草的酶解性能.未处理稻草酶解的还原糖收率为13.4%、Km 为66.8 mg/mL、rmax 为312.5μmol/(min · mL).采用1%NaOH 溶液对稻草进行预处理1h 后,还原糖收率提高到41.4%,Km 减小到15.9 mg/mL, rmax 提高到666.7μmol/(min · mL).预处理过程中木质素去除、纤维素晶体结构消除、底物可及度增加是酶解中还原糖收率和反应速率上升的主要原因.     

斑茅酶解转化可发酵单糖的液氨预处理及参数优化

斑茅(Saccharum arundinaceum Retz.)的生物产量高,对土壤条件要求低,可作为纤维素乙醇生产的原料作物在我国南方地区广泛种植.实验以斑茅为原料,采用液氨预处理法克服其水解顽抗性,并添加纤维素酶进行酶解,运用高效液相色谱(HPLC)测定了酶解液中的单糖含量.实验结果表明在纤维素酶添加量为15FPU/(g当量葡聚糖)、预处理原料含水率为80％、预处理温度为130℃、预处理驻留时间为10 min、液氨与生物质的质量比例为2∶1时,葡聚糖和木聚糖的总转化率分别为69.34％和82.60％,相比于未作预处理的原料分别提高了573％和1 056％,单糖产量提高8倍.实验结果表明液氨预处理对斑茅是一种有效的预处理方式,并优于稀酸或湿爆法预处理,与酸预处理和氨爆法(AFEX)处理效果接近.     

超临界CO2流体对纤维素酶催化反应的影响

超临界二氧化碳流体预处理对纤维素超分子结构及纤维素酶催化反应有重要影响。一定含水量的微晶纤维素用SC-CO₂ 在 10MPa,50℃处理 30min ,其结构发生了有利于进一步被酶解的变化。上述超临界条件单独作用于纤维素酶时 ,并未造成酶催化活力的降低 ;但与纤维素共同进行SC-CO₂ 处理时 ,纤维素酶则失去催化活性 ,但这种处理却能提高纤维素进一步被酶解的效率。一定范围内处理时的酶用量与酶解效率的增加正相关。纤维素的含水量对SC-CO₂ 处理后的酶解效率有显著影响.     

Enhancement of the enzymatic digestibility of sugarcane bagasse by steam pretreatment impregnated with hydrogen peroxide

Sugarcane bagasse was subjected to steam pretreatment impregnated with hydrogen peroxide. Analyses were performed using 2³ factorial designs and enzymatic hydrolysis was performed at two different solid concentrations and with washed and unwashed material to evaluate the importance of this step for obtaining high cellulose conversion. Similar cellulose conversion were obtained at different conditions of pretreatment and hydrolysis. When the cellulose was hydrolyzed using the pretreated material in the most severe conditions of the experimental design (210°C, 15 min and 1.0% hydrogen peroxide), and using 2% (w/w) water‐insoluble solids (WIS), and 15 FPU/g WIS, the cellulose conversion was 86.9%. In contrast, at a milder pretreatment condition (190°C, 15 min and 0.2% hydrogen peroxide) and industrially more realistic conditions of hydrolysis (10% WIS and 10 FPU/g WIS), the cellulose conversion reached 82.2%. The step of washing the pretreated material was very important to obtain high concentrations of fermentable sugars. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Ionic liquid‐mediated selective extraction of lignin from wood leading to enhanced enzymatic cellulose hydrolysis

Lignocellulose represents a key sustainable source of biomass for transformation into biofuels and bio‐based products. Unfortunately, lignocellulosic biomass is highly recalcitrant to biotransformation, both microbial and enzymatic, which limits its use and prevents economically viable conversion into value‐added products. As a result, effective pretreatment strategies are necessary, which invariably involves high energy processing or results in the degradation of key components of lignocellulose. In this work, the ionic liquid, 1‐ethyl‐3‐methylimidazolium acetate ([Emim][CH₃COO]), was used as a pretreatment solvent to extract lignin from wood flour. The cellulose in the pretreated wood flour becomes far less crystalline without undergoing solubilization. When 40% of the lignin was removed, the cellulose crystallinity index dropped below 45, resulting in >90% of the cellulose in wood flour to be hydrolyzed by Trichoderma viride cellulase. [Emim] [CH₃COO] was easily reused, thereby resulting in a highly concentrated solution of chemically unmodified lignin, which may serve as a valuable source of a polyaromatic material as a value‐added product. Biotechnol. Bioeng. 2009;102: 1368–1376. © 2008 Wiley Periodicals, Inc.     

Methodological analysis for determination of enzymatic digestibility of cellulosic materials

Accurate measurement of enzymatic cellulose digestibility (X) is important in evaluating the efficiency of lignocellulose pretreatment technologies, assessing the performance of reconstituted cellulase mixtures, and conducting economic analysis for biorefinery processes. We analyzed the effect of sugars contained in enzymes solutions, usually added as a preservative, and random measurement errors on the accuracy of X calculated by various methods. The analysis suggests that exogenous sugars at levels measured in several commercial enzyme preparations significantly bias the results and that this error should be minimized by accounting for these sugars in the calculation of X. Additionally, a method of calculating X equating the ratio of the soluble glucose equivalent in the liquid phase after hydrolysis to the sum of the soluble glucose equivalent in the liquid phase and the insoluble glucose equivalent in the residual solid after hydrolysis was found to be the most accurate, particularly at high conversion levels (>ca. 50%).     

Effect of xylan and lignin removal by batch and flowthrough pretreatment on the enzymatic digestibility of corn stover cellulose

Compared with batch systems, flowthrough and countercurrent reactors have important potential advantages for pretreating cellulosic biomass, including higher hemicellulose sugar yields, enhanced cellulose digestibility, and reduced chemical additions. Unfortunately, they suffer from high water and energy use. To better understand these trade-offs, comparative data are reported on xylan and lignin removal and enzymatic digestibility of cellulose for corn stover pretreated in batch and flowthrough reactors over a range of flow rates between 160 degrees and 220 degrees C, with water only and also with 0.1 wt% sulfuric acid. Increasing flow with just water enhanced the xylan dissolution rate, more than doubled total lignin removal, and increased cellulose digestibility. Furthermore, adding dilute sulfuric acid increased the rate of xylan removal for both batch and flowthrough systems. Interestingly, adding acid also increased the lignin removal rate with flow, but less lignin was left in solution when acid was added in batch. Although the enzymatic hydrolysis of pretreated cellulose was related to xylan removal, as others have shown, the digestibility was much better for flowthrough compared with batch systems, for the same degree of xylan removal. Cellulose digestibility for flowthrough reactors was related to lignin removal as well. These results suggest that altering lignin also affects the enzymatic digestibility of corn stover.     

Enhancing the enzymatic digestibility of sugarcane bagasse through the application of an ionic liquid in combination with an acid catalyst

Various ionic liquids have been identified as effective pretreatment solvents that can enhance the cellulose digestibility of lignocellulose by removing lignin, one of the main factors contributing to the recalcitrant nature of lignocellulose. 1-Butyl-3-methylimidazolium methylsulfate ([BMiM]MeSO(4)) is a potential delignification reagent, hence its application as a pretreatment solvent for sugarcane bagasse (SB) was investigated. The study also evaluated the benefit of an acid catalyst (i.e., H(2) SO(4)) and the effect of pretreatment conditions, which varied within a time and temperature range of 0-240 min and 50-150°C, respectively. The use of an acid catalyst contributed to a more digestible solid and a higher degree of delignification. However, the [BMiM]MeSO(4)-H(2) SO(4) combination failed to produce a fully digestible solid, as a maximum cellulose digestibility of 77% (w/w) was obtained at the optimum pretreatment condition of 125°C for 120 min. Furthermore, up to half of the lignin content could be extracted during pretreatment, while simultaneously extensive, sometimes complete, removal of xylan, the presence of which, also hampers cellulose digestibility. Hence, [BMiM]MeSO(4) has been identified an effective pretreatment solvent for SB as the application thereof both significantly improved digestibility, and simultaneously removed two of the main factors contributing to the recalcitrant nature of lignocellulose. As xylan and lignin have potential value as precursor chemicals, the existing process may in future be extended toward substrate fractionation, a biorefinery concept where value is added to all feedstock constituents.     

Extraction and characterization of wax from sugarcane bagasse and the enzymatic hydrolysis of dewaxed sugarcane bagasse

Extraction of high-value products from agricultural wastes is an important component for sustainable bioeconomy development. In this study, wax extraction from sugarcane bagasse was performed and the beneficial effect of dewaxing pretreatment on the enzymatic hydrolysis was investigated. About 1.2% (w/w) of crude sugarcane wax was obtained from the sugarcane bagasse using the mixture of petroleum ether and ethanol (mass ratio of 1:1) as the extraction agent. Results of Fourier-transform infrared characterization and gas chromatography–mass spectrometry qualitative analysis showed that the crude sugarcane wax consisted of fatty fractions (fatty acids, fatty aldehydes, hydrocarbons, and esters) and small amount of lignin derivatives. In addition, the effect of dewaxing pretreatment on the enzymatic hydrolysis of sugarcane bagasse was also investigated. The digestibilities of cellulose and xylan in dewaxed sugarcane bagasse were 18.7 and 10.3%, respectively, compared with those of 13.1 and 8.9% obtained from native sugarcane bagasse. The dewaxed sugarcane bagasse became more accessible to enzyme due to the disruption of the outermost layer of the waxy materials.     

Pretreatment of Siam weed stem by several chemical methods for increasing the enzymatic digestibility

Siam weed [Chromolaena odorata (L.) King & Robinson], an invasive exotic weed in China, was proposed as a feedstock for bioethanol production. This would be a promising way of using for an invasive weed that needs management and control. It was found that the glucan content of the weed stem was similar to that of sugarcane bagasse, but higher than those of corn stover and wheat straw. Several chemical pretreatment methods were applied to the weed stem to increase its enzymatic digestibility. Mild sulfuric acid (<120°C) or alkali pretreatment did not markedly increase the enzymatic digestibility. However, peracetic acid (PAA) pretreatment dramatically enhanced the enzymatic hydrolysis of the weed stem. Compared to some other common agricultural residues, the weed stem was more difficult to pretreat and digest by cellulase. Fourier transform infrared (FTIR) spectra analysis indicated that the cellulose-related bands became more intensive after pretreatment, especially for PAA-pretreated samples. According to X-ray diffraction spectra, the biomass solids had higher crystallinity indices after pretreatment, although these indices were similar for all of the pretreated samples.     

Enhancement of the enzymatic digestibility of sugarcane bagasse by alkali–peracetic acid pretreatment

The enzymatic digestibility of sugarcane bagasse was greatly increased by alkali (NaOH)–peracetic acid (PAA) pretreatment under mild conditions. The effects of several factors affecting the pretreatment were investigated. It was found that when bagasse was pre-pretreated by 10% (based on initial dry materials) NaOH with 3:1 liquid-to-solid ratio at 90 °C for 1.5 h and further delignified by 10% peracetic acid (based on initial dry materials) at 75 °C for 2.5 h, the yield of reducing sugars reached 92.04% by enzymatic hydrolysis for 120 h with cellulase loading of 15 FPU/g solid. Compared with acid and alkali pretreatment, alkali–PAA pretreatment could be conducted under milder conditions and was more effective for delignification with less carbohydrates being degraded in the pretreatment process. Alkaline stage played an important role for partial delignification, swelling fibers and subsequently reducing PAA loading. No loss of cellulase activity (FPA) was observed in the liquid phase for alkali–PAA pretreated bagasse after enzymatic hydrolysis for 120 h.     

Liquid hot water and alkaline pretreatment of soybean straw for improving cellulose digestibility

Soybean straw was pretreated with either liquid hot water (LHW) (170-210 °C for 3-10 min) or alkaline soaking (4-40 g NaOH/100 g dry straw) at room temperature to evaluate the effects on cellulose digestibility. Nearly 100% cellulose was recovered in pretreated solids for both pretreatment methods. For LHW pretreatment, xylan dissolution from the raw material increased with pretreatment temperature and time. Cellulose digestibility was correlated with xylan dissolution. A maximal glucose yield of 70.76%, corresponding to 80% xylan removal, was obtained with soybean straw pretreated at 210 °C for 10 min. NaOH soaking at ambient conditions removed xylan up to 46.37% and the subsequent glucose yield of pretreated solids reached up to 64.55%. Our results indicated LHW pretreatment was more effective than NaOH soaking for improving cellulose digestibility of soybean straw.     

Enzymatic digestibility and pretreatment degradation products of AFEX‐treated hardwoods (Populus nigra)

There is a growing need to find alternatives to crude oil as the primary feed stock for the chemicals and fuel industry and ethanol has been demonstrated to be a viable alternative. Among the various feed stocks for producing ethanol, poplar (Populus nigra × Populus maximowiczii) is considered to have great potential as a biorefinery feedstock in the United States, due to their widespread availability and good productivity in several parts of the country. We have optimized AFEX pretreatment conditions (180°C, 2:1 ammonia to biomass loading, 233% moisture, 30 minutes residence time) and by using various combinations of enzymes (commercical celluloses and xylanases) to achieve high glucan and xylan conversion (93 and 65%, respectively). We have also identified and quantified several important degradation products formed during AFEX using liquid chromatography followed by mass spectrometry (LC‐MS/MS). As a part of degradation product analysis, we have also quantified oligosaccharides in the AFEX water wash extracts by acid hydrolysis. It is interesting to note that corn stover (C4 grass) can be pretreated effectively using mild AFEX pretreatment conditions, while on the other hand hardwood poplar requires much harsher AFEX conditions to obtain equivalent sugar yields upon enzymatic hydrolysis. Comparing corn stover and poplar, we conclude that pretreatment severity and enzymatic hydrolysis efficiency are dictated to a large extent by lignin carbohydrate complexes and arabinoxylan cross‐linkages for AFEX. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009