木质纤维素原料酶水解产乙醇工艺的研究进展

木质纤维素原料预处理后,经水解、发酵等过程,可生产乙醇作为清洁燃料,这大大提高了农业和林业废弃物的利用率,减轻了环境污染,并为经济的可持续发展提供了保证。目前木质纤维素酶水解因其具有明显优势而受到重视,被普遍研究和采用。综述了近年来木质纤维素原料的预处理方法、酶与水解技术、发酵工艺以及发酵耦合分离技术的最新研究成果。     

Potential and utilization of thermophiles and thermostable enzymes in biorefining

In today's world, there is an increasing trend towards the use of renewable, cheap and readily available biomass in the production of a wide variety of fine and bulk chemicals in different biorefineries. Biorefineries utilize the activities of microbial cells and their enzymes to convert biomass into target products. Many of these processes require enzymes which are operationally stable at high temperature thus allowing e.g. easy mixing, better substrate solubility, high mass transfer rate, and lowered risk of contamination. Thermophiles have often been proposed as sources of industrially relevant thermostable enzymes. Here we discuss existing and potential applications of thermophiles and thermostable enzymes with focus on conversion of carbohydrate containing raw materials. Their importance in biorefineries is explained using examples of lignocellulose and starch conversions to desired products. Strategies that enhance thermostablity of enzymes both in vivo and in vitro are also assessed. Moreover, this review deals with efforts made on developing vectors for expressing recombinant enzymes in thermophilic hosts.     

A short review on SSF – an interesting process option for ethanol production from lignocellulosic feedstocks

Simultaneous saccharification and fermentation (SSF) is one process option for production of ethanol from lignocellulose. The principal benefits of performing the enzymatic hydrolysis together with the fermentation, instead of in a separate step after the hydrolysis, are the reduced end-product inhibition of the enzymatic hydrolysis, and the reduced investment costs. The principal drawbacks, on the other hand, are the need to find favorable conditions (e.g. temperature and pH) for both the enzymatic hydrolysis and the fermentation and the difficulty to recycle the fermenting organism and the enzymes. To satisfy the first requirement, the temperature is normally kept below 37°C, whereas the difficulty to recycle the yeast makes it beneficial to operate with a low yeast concentration and at a high solid loading. In this review, we make a brief overview of recent experimental work and development of SSF using lignocellulosic feedstocks. Significant progress has been made with respect to increasing the substrate loading, decreasing the yeast concentration and co-fermentation of both hexoses and pentoses during SSF. Presently, an SSF process for e.g. wheat straw hydrolyzate can be expected to give final ethanol concentrations close to 40 g L^-1 with a yield based on total hexoses and pentoses higher than 70%.     

Direct production of commodity chemicals from lignocellulose using Myceliophthora thermophila

The production of fuels and chemicals from renewable plant biomass has been proposed as a feasible strategy for global sustainable development. However, the economic efficiency of biorefineries is low. Here, through metabolic engineering, Myceliophthora thermophila, a cellulolytic thermophilic fungus, was constructed into a platform that can efficiently convert lignocellulose into important bulk chemicals—four carbon 1, 4-diacids (malic and succinic acid), building blocks for biopolymers—without the need for extra hydrolytic enzymes. Titers of >200 g/L from crystalline cellulose and 110 g/L from plant biomass (corncob) were achieved during fed-batch fermentation. Our study represents a milestone in consolidated bioprocessing technology and offers a new and promising system for the cost-effective production of chemicals and fuels from biomass.     

A Weibull statistics‐based lignocellulose saccharification model and a built‐in parameter accurately predict lignocellulose hydrolysis performance

Renewable energy from lignocellulosic biomass has been deemed an alternative to depleting fossil fuels. In order to improve this technology, we aim to develop robust mathematical models for the enzymatic lignocellulose degradation process. By analyzing 96 groups of previously published and newly obtained lignocellulose saccharification results and fitting them to Weibull distribution, we discovered Weibull statistics can accurately predict lignocellulose saccharification data, regardless of the type of substrates, enzymes and saccharification conditions. A mathematical model for enzymatic lignocellulose degradation was subsequently constructed based on Weibull statistics. Further analysis of the mathematical structure of the model and experimental saccharification data showed the significance of the two parameters in this model. In particular, the λ value, defined the characteristic time, represents the overall performance of the saccharification system. This suggestion was further supported by statistical analysis of experimental saccharification data and analysis of the glucose production levels when λ and n values change. In conclusion, the constructed Weibull statistics‐based model can accurately predict lignocellulose hydrolysis behavior and we can use the λ parameter to assess the overall performance of enzymatic lignocellulose degradation. Advantages and potential applications of the model and the λ value in saccharification performance assessment were discussed.     

Investigating the role of mechanics in lignocellulosic biomass degradation during hydrolysis: Part II

Lignocellulose breakdown in biorefineries is facilitated by enzymes and physical forces. Enzymes degrade and solubilize accessible lignocellulosic polymers, primarily on fiber surfaces, and make fibers physically weaker. Meanwhile physical forces acting during mechanical agitation induce tearing and cause rupture and attrition of the fibers, leading to liquefaction, that is, a less viscous hydrolysate that can be further processed in industrial settings. This study aims at understanding how mechanical agitation during enzymatic saccharification can be used to promote fiber attrition. The effects of reaction conditions, such as substrate and enzyme concentration on fiber attrition rate and hydrolysis yield were investigated. To gain insight into the fiber attrition mechanism, enzymatic hydrolysis was compared to hydrolysis by use of hydrochloric acid. Results show that fiber attrition depends on several factors concerning reactor design and operation including drum diameter, rotational speed, mixing schedule, and concentrations of fibers and enzymes. Surprisingly, different fiber attrition patterns during enzymatic and acid hydrolysis were found for similar mixing schedules. Specifically, for tumbling mixing, slow continuous mixing appears to function better than faster, intermittent mixing even for the same total number of drum revolutions. The findings indicate that reactor design and operation as well as hydrolysis conditions are key to process optimization and that detailed insights are needed to obtain fast liquefaction without sacrificing saccharification yields.     

Suitability of aspenwood biologically delignified with Pheblia tremellosus for fermentation to ethanol or butanediol

Summary Enzymatic conversion of lignocellulosic material to fuels and chemicals depends on a initial pretreatment to render the cellulose more susceptible to enzymatic attack. Biological delignification of aspenwood with the fungus Phlebia tremellosus was compared to steaming as a pretreatment method.The biologically delignified aspenwood (BDA) had a high pentosan content and did not contain inhibitors of enzymatic hydrolysis or subsequent fermentation. In contrast, the steamed aspenwood required a water extraction step to remove the inhibitory material and this step also removed most of the pentosan. The yield of treated material was 90% from biological delignification and 70% from steaming.The cellulose in the BDA was less accessible to the cellulase enzymes than the steamed aspenwood. Combined hydrolysis and fermentation with Saccharomyces cerevisiae gave a lower yield of ethanol from BDA than from the steamed aspenwood, but the yields based on the weight of substrate before pretreatment were comparable. Combined hydrolysis and fermentation with Klebsiella pneumoniae gave higher yields of butanediol from BDA than from steamed aspenwood, because Klebsiella can ferment the xylose which was present in the biologically treated aspenwood. Trichoderma harzianum produced lower levels of cellulase enzymes when grown on BDA than when grown on steamed aspenwood and this was related to the xylan found in the biologically treated material.Abbreviations BDA biologically delignified aspenwood - SEA-WI steam-exploded, water-extracted aspenwood - AI-SEA-WI acid-impregnated, steam-exploded, water-extracted aspenwood - CHF combined hydrolysis and fermentation - FP filter paper     

Fractionating recalcitrant lignocellulose at modest reaction conditions

Effectively releasing the locked polysaccharides from recalcitrant lignocellulose to fermentable sugars is among the greatest technical and economic barriers to the realization of lignocellulose biorefineries because leading lignocellulose pre-treatment technologies suffer from low sugar yields, and/or severe reaction conditions, and/or high cellulase use, narrow substrate applicability, and high capital investment, etc. A new lignocellulose pre-treatment featuring modest reaction conditions (50 degrees C and atmospheric pressure) was demonstrated to fractionate lignocellulose to amorphous cellulose, hemicellulose, lignin, and acetic acid by using a non-volatile cellulose solvent (concentrated phosphoric acid), a highly volatile organic solvent (acetone), and water. The highest sugar yields after enzymatic hydrolysis were attributed to no sugar degradation during the fractionation and the highest enzymatic cellulose digestibility ( approximately 97% in 24 h) during the hydrolysis step at the enzyme loading of 15 filter paper units of cellulase and 60 IU of beta-glucosidase per gram of glucan. Isolation of high-value lignocellulose components (lignin, acetic acid, and hemicellulose) would greatly increase potential revenues of a lignocellulose biorefinery.     

The combined enzymatic hydrolysis and fermentation of hemicellulose to 2,3-butanediol

Summary Hemicellulose-rich fractions from several agricultural residues were converted to 2,3-butanediol by a combined enzymatic hydrolysis and fermentation process. Culture filtrates from Trichoderma harzianum E58 were used to hydrolyze the substrates while Klebsiella pneumoniae fermented the liberated sugars to 2,3-butanediol. Approximately 50–60% of a 5% (w/v) xylan preparation could be hydrolyzed and quantitatively converted to 2,3-butanediol using this procedure. Although enzymatic hydrolysis was optimal at pH 5.0 and 50° C, the combined hydrolysis and fermentation was most efficient at pH 6.5 and 30° C. Combined hydrolysis and fermentation resulted in butanediol levels that were 20–40% higher than could be obtained with a separate hydrolysis and fermentation process. The hemicellulose-rich water-soluble fractions obtained from a variety of steam-exploded agricultural residues could be readily used by the combined hydrolysis and fermentation approach resulting in butanediol yields of 0.4–0.5 g/g of reducing sugar utilized.     

添加纤维素酶的青贮研究进展

青贮是一种传统的青绿饲料作物的保存方法,在青贮中添加纤维素酶可以增强青贮的效果,促进植物细胞壁的酶解,提高青贮料的消化率,添加纤维素酶的青贮也可用于从植物细胞中提取有价值的天然产物,不仅条件温和,且提取率大大提高,应用在线生产纤维素与青贮发酵耦合能大大降低该工艺的成本,对添加纤维素酶青贮的研究可望促进纤维素资源的转化利用。     

Characterization of the interaction between surfactants and enzymes by fluorescence probe

In order to investigate the mechanism of the different stimulatory effects of the biosurfactant rhamnolipid and the chemical surfactant Tween 80 on enzymatic hydrolysis of lignocellulose, the interaction between surfactants and enzymes was analyzed by the fluorescence probe method using pyrene as probe. Based on the evolution law of pyrene fluorescence spectroscopy in the “surfactants-enzymes” systems, the interaction relationship between surfactants and enzymes was analyzed and discussed in this paper. The results show that enzyme molecules bind with rhamnolipid molecules, participate in the formation of rhamnolipid micelles, and increase the inner hydrophobic polarity of micelles, but do not change the properties of rhamnolipid micelles above the CMC (Critical Micelle Concentration). Nevertheless, for Tween 80, enzyme molecules also participate in the forming of micelles, however, they exhibit a stronger interaction with enzymes above the CMC. Both rhamnolipid and Tween 80 bind more strongly with xylanase than cellulase. Considering also previous experimental results, it can be concluded that the interaction between surfactants and enzymes improve enzyme stability and activity, and, therefore, the efficiency of enzymatic hydrolysis of lignocellulose is enhanced. The findings further provide theoretical knowledge about the mechanism of the stimulative effects of surfactants on enzymatic hydrolysis of lignocellulose.     

Biodelignification of lignocellulose substrates: An intrinsic and sustainable pretreatment strategy for clean energy production

Lignocellulosic biomass (LB) is a promising sugar feedstock for biofuels and other high-value chemical commodities. The recalcitrance of LB, however, impedes carbohydrate accessibility and its conversion into commercially significant products. Two important factors for the overall economization of biofuel production is LB pretreatment to liberate fermentable sugars followed by conversion into ethanol. Sustainable biofuel production must overcome issues such as minimizing water and energy usage, reducing chemical usage and process intensification. Amongst available pretreatment methods, microorganism-mediated pretreatments are the safest, green, and sustainable. Native biodelignifying agents such as Phanerochaete chrysosporium, Pycnoporous cinnabarinus, Ceriporiopsis subvermispora and Cyathus stercoreus can remove lignin, making the remaining substrates amenable for saccharification. The development of a robust, integrated bioprocessing (IBP) approach for economic ethanol production would incorporate all essential steps including pretreatment, cellulase production, enzyme hydrolysis and fermentation of the released sugars into ethanol. IBP represents an inexpensive, environmentally friendly, low energy and low capital approach for second-generation ethanol production. This paper reviews the advancements in microbial-assisted pretreatment for the delignification of lignocellulosic substrates, system metabolic engineering for biorefineries and highlights the possibilities of process integration for sustainable and economic ethanol production.     

Biotechnological strategies to overcome inhibitors in lignocellulose hydrolysates for ethanol production: review

One of the major challenges faced in commercial production of lignocellulosic bioethanol is the inhibitory compounds generated during the thermo-chemical pre-treatment step of biomass. These inhibitory compounds are toxic to fermenting micro-organisms. The ethanol yield and productivity obtained during fermentation of lignocellulosic hydrolysates is decreased due to the presence of inhibiting compounds, such as weak acids, furans and phenolic compounds formed or released during thermo-chemical pre-treatment step such as acid and steam explosion. This review describes the application and/or effect of biological detoxification (removal of inhibitors before fermentation) or use of bioreduction capability of fermenting yeasts on the fermentability of the hydrolysates. Inhibition of yeast fermentation by the inhibitor compounds in the lignocellulosic hydrolysates can be reduced by treatment with enzymes such as the lignolytic enzymes, for example, laccase and micro-organisms such as Trichoderma reesei, Coniochaeta ligniaria NRRL30616, Trametes versicolor, Pseudomonas putida Fu1, Candida guilliermondii, and Ureibacillus thermosphaericus. Microbial and enzymatic detoxifications of lignocellulosic hydrolysate are mild and more specific in their action. The efficiency of enzymatic process is quite comparable to other physical and chemical methods. Adaptation of the fermentation yeasts to the lignocellulosic hydrolysate prior to fermentation is suggested as an alternative approach to detoxification. Increases in fermentation rate and ethanol yield by adapted micro-organisms to acid pre-treated lignocellulosic hydrolysates have been reported in some studies. Another approach to alleviate the inhibition problem is to use genetic engineering to introduce increased tolerance by Saccharomyces cerevisiae, for example, by overexpressing genes encoding enzymes for resistance against specific inhibitors and altering co-factor balance. Cloning of the laccase gene followed by heterologous expression in yeasts was shown to provide higher enzyme yields and permit production of laccases with desired properties for detoxification of lignocellulose hydrolysates. A combination of more inhibitor-tolerant yeast strains with efficient feed strategies such as fed-batch will likely improve lignocellulose-to-ethanol process robustness.     

Detoxification of Lignocellulose Hydrolysates: Biochemical and Metabolic Engineering Toward White Biotechnology

Chemical hydrolysis of lignocellulosic biomass (LB) produces a number of inhibitors in addition to sugars. These inhibitors include lignin-derived phenolics, carbohydrate-derived furans, and weak acids that have shown a marked effect on the productivities of various metabolites and the growth of biocatalysts in the fermentative reaction. In the past, a number of physicochemical and biological approaches have been proposed to overcome these fermentation inhibitors, including modified fermentative strategies. Additionally, the timely intervention of genetic engineering has provided an impetus to develop suitable technologies for the detoxification of lignocellulosics in biorefineries. However, the improvements in detoxification strategies for lignocellulose hydrolysates have resulted in significant loss of sugars after detoxification. Hydrolysis of myco-LB (LB after fungal pretreatment) has been recognized as a promising approach to avoid fermentation inhibitors and improve total sugar recovery. Biotechnological inventions have also made it possible to widen the range of suitable biocatalysts for biorefineries by microbial-routed induction of enzymatic expression for the elimination of inhibitors, eventually improving ethanol production from acid hydrolysates. This article aims to highlight the strategies that have been adopted to detoxify lignocellulosic hydrolysates and their effects on the chemical composition of the hydrolysates to improve the fermentability of lignocellulosics. In addition, genetic manipulation could widen the availability and variety of substrates and modify the metabolic routes to produce bioethanol or other value-added compounds in an efficient manner.     

Secretome analysis of Trichoderma reesei and Aspergillus niger cultivated by submerged and sequential fermentation processes: Enzyme production for sugarcane bagasse hydrolysis

Cellulases and hemicellulases from Trichoderma reesei and Aspergillus niger have been shown to be powerful enzymes for biomass conversion to sugars, but the production costs are still relatively high for commercial application. The choice of an effective microbial cultivation process employed for enzyme production is important, since it may affect titers and the profile of protein secretion. We used proteomic analysis to characterize the secretome of T. reesei and A. niger cultivated in submerged and sequential fermentation processes. The information gained was key to understand differences in hydrolysis of steam exploded sugarcane bagasse for enzyme cocktails obtained from two different cultivation processes. The sequential process for cultivating A. niger gave xylanase and β-glucosidase activities 3- and 8-fold higher, respectively, than corresponding activities from the submerged process. A greater protein diversity of critical cellulolytic and hemicellulolytic enzymes were also observed through secretome analyses. These results helped to explain the 3-fold higher yield for hydrolysis of non-washed pretreated bagasse when combined T. reesei and A. niger enzyme extracts from sequential fermentation were used in place of enzymes obtained from submerged fermentation. An enzyme loading of 0.7 FPU cellulase activity/g glucan was surprisingly effective when compared to the 5–15 times more enzyme loadings commonly reported for other cellulose hydrolysis studies. Analyses showed that more than 80% consisted of proteins other than cellulases whose role is important to the hydrolysis of a lignocellulose substrate. Our work combined proteomic analyses and enzymology studies to show that sequential and submerged cultivation methods differently influence both titers and secretion profile of key enzymes required for the hydrolysis of sugarcane bagasse. The higher diversity of feruloyl esterases, xylanases and other auxiliary hemicellulolytic enzymes observed in the enzyme mixtures from the sequential fermentation could be one major reason for the more efficient enzyme hydrolysis that results when using the combined secretomes from A. niger and T. reesei.     

同步分泌高效纤维素酶和木聚糖酶菌株YB的鉴定及其酶学性质研究

筛选和鉴定可降解木质纤维素的真菌,并研究其产酶特征。采用刚果红平板涂布法,从荔枝腐叶中筛选具有木质纤维素降解能力的真菌,结合ITS-rDNA序列分析进行鉴定,初步测定其产酶条件,然后采用DEAE Sepharose Fast Flow阴离子交换层析与Sephadex G-100凝胶层析对硫酸铵沉淀的粗酶液进行分离纯化,对其开展酶学性质研究。结果显示,筛选出一株可降解木质纤维素降解的菌株YB,鉴定为绿木霉(Trichoderma virens)。在发酵过程中,纤维素酶和木聚糖酶的最大活力分别为313.53±26.78 U/mL和18 120.87±500.37 U/mL。分离纯化得到纤维素酶(CMC酶)Ⅰb、Ⅳ和木聚糖酶Ⅰa;通过SDS-PAGE检测,其分子量分别为58.5 kD、22.8 kD和44.5 kD。3种酶的最适酶促反应条件均为:50℃,pH 5.0。其中,木聚糖酶能有效降解玉米芯木聚糖为木糖和多种木寡糖。菌株Trichoderma virens YB可分泌高效木质纤维素降解酶,具有应用于木聚糖酶和木寡糖生产的潜力。     

Precipitation of Trichoderma reesei commercial cellulase preparations under standard enzymatic hydrolysis conditions for lignocelluloses

Comparative studies between commercial Trichoderma reesei cellulase preparations show that, depending on the preparation and loading, total protein precipitation can be as high as 30 % under standard hydrolysis conditions used for lignocellulosic materials. ATR-IR and SDS-PAGE data verify precipitates are protein-based and contain key cell wall hydrolyzing enzymes. Precipitation increased considerably with incubation temperature; roughly 50-150 % increase from 40 to 50 °C and 800 % greater at 60 °C. All of the reported protein losses translated into significant, and often drastic, losses in activity on related 4-nitrophenyl substrates. In addition, supplementation with the non-ionic surfactant PEG 6,000 decreased precipitation up to 80 % in 24 h precipitation levels. Protein precipitation is potentially substantial during enzymatic hydrolysis of lignocelluloses and should be accounted for during lignocellulose conversion process design, particularly when enzyme recycling is considered.     

Enzymatic synthesis of ATP from RNA and adenine

Summary The title compound was prepared by a three-stage enzymatic procedure consisting of (i) RNA hydrolysis to a mixture of ribonucleosides using intact mycelium of Spicaria violacea, (ii) transribosylation of exogenous adenine employing whole cells of Escherichia coli as a biocatalyst, and (iii) conversion of formed adenosine into ATP by the enzymes of alcohol fermentation and the kinases extracted from baker's yeast.