Compatible ionic liquid-cellulases system for hydrolysis of lignocellulosic biomass

Ionic liquids (ILs) have been increasingly recognized as novel solvents for dissolution and pretreatment of cellulose. However, cellulases are inactivated in the presence of ILs, even when present at low concentrations. To more fully exploit the benefits of ILs it is critical to develop a compatible IL‐cellulases system in which the IL is able to effectively solubilize and activate the lignocellulosic biomass, and the cellulases possess high stability and activity. In this study, we investigated the stability and activity of a commercially available cellulases mixture in the presence of different concentrations of 1‐ethyl‐3‐methylimidazolium acetate ([Emim][OAc]). A mixture of cellulases and β‐glucosidase (Celluclast1.5L, from Trichoderma reesei, and Novozyme188, from Aspergillus niger, respectively) retained 77% and 65% of its original activity after being pre‐incubated in 15% and 20% (w/v) IL solutions, respectively, at 50°C for 3 h. The cellulases mixture also retained high activity in 15% [Emim][OAc] to hydrolyze Avicel, a model substrate for cellulose analysis, with conversion efficiency of approximately 91%. Notably, the presence of different amounts of yellow poplar lignin did not interfere significantly with the enzymatic hydrolysis of Avicel. Using this IL‐cellulase system (15% [Emim][OAc]), the saccharification of yellow poplar biomass was also significantly improved (33%) compared to the untreated control (3%) during the first hour of enzymatic hydrolysis. Together, these findings provide compelling evidence that [Emim][OAc] was compatible with the cellulase mixture, and this compatible IL‐cellulases system is promising for efficient activation and hydrolysis of native biomass to produce biofuels and co‐products from the individual biomass components. Bioeng. 2011; 108:1042–1048. © 2010 Wiley Periodicals, Inc.     

纤维素酶与木质纤维素生物降解转化的研究进展

利用纤维素酶将预处理后的秸秆降解成可发酵性单糖,然后发酵生产所需的液体燃料及化工产品的技术,对于我国解决能源、环境、人口就业等难题有着巨大的积极影响。在木质纤维素生物降解转化工艺中,减少纤维素酶用量及提高酶解效率是降低木质纤维素降解成本的关键。纤维素酶系和木质纤维素酶水解技术的改进需要深入了解纤维素酶系统的组成及其协同作用、纤维素酶的结构与功能以及纤维素酶的生产技术。将就以上几个方面的研究进展进行讨论,并深入探讨了纤维素酶糖化能力的评价方法。     

High‐yield lipid production from lignocellulosic biomass using engineered xylose‐utilizing Yarrowia lipolytica

Lignocellulosic biomass shows high potential as a renewable feedstock for use in biodiesel production via microbial fermentation. Yarrowia lipolytica, an emerging oleaginous yeast, has been engineered to efficiently convert xylose, the second most abundant sugar in lignocellulosic biomass, into lipids for lignocellulosic biodiesel production. Yet, the lipid yield from xylose or lignocellulosic biomass remains far lower than that from glucose. Here we developed an efficient xylose‐utilizing Y. lipolytica strain, expressing an isomerase‐based pathway, to achieve high‐yield lipid production from lignocellulosic biomass. The newly developed xylose‐utilizing Y. lipolytica, YSXID, produced 12.01 g/L lipids with a maximum yield of 0.16 g/g, the highest ever reported, from lignocellulosic hydrolysates. Consequently, this study shows the potential of isomerase‐based xylose‐utilizing Y. lipolytica for economical and sustainable production of biodiesel and oleochemicals from lignocellulosic biomass.     

Pretreatment strategies for microbial valorization of bio‐oil fractions produced by fast pyrolysis of ash‐rich lignocellulosic biomass

This work evaluates a biorefinery approach for microbial valorization of bio‐oil fractions produced by fast pyrolysis of ash‐rich lignocellulosic biomass. Different methods are presented for the pretreatment of the low‐sugar complex bio‐oil consisting of organic condensate (OC) and aqueous condensate (AC) to overcome their strong inhibitory effects and unsuitability for common analytical methods. Growth of Pseudomonas putida KT2440, which was chosen as a reference system, on untreated bio‐oil fractions was only detectable using solid medium with OC as sole carbon source. Utilization of a pretreated OC which was filtered, autoclaved, neutralized and centrifuged enabled growth in liquid medium with significant remaining optical instability. By subjecting the pretreated fractions to solid phase extraction, more stable and less inhibitory bio‐oil fractions could be obtained enabling the appliance of common analytical methods. Furthermore, this pretreatment facilitated growth of the applied reference organism Pseudomonas putida KT2440. As there is currently no convincing strategy for reliable application of bio‐oil as a sole source of carbon in industrial biotechnology, the presented work depicts a first step toward establishing bio‐oil as a future sustainable feedstock for a bio‐based economy.     

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.     

High-throughput microplate technique for enzymatic hydrolysis of lignocellulosic biomass

Several factors will influence the viability of a biochemical platform for manufacturing lignocellulosic based fuels and chemicals, for example, genetically engineering energy crops, reducing pre-treatment severity, and minimizing enzyme loading. Past research on biomass conversion has focused largely on acid based pre-treatment technologies that fractionate lignin and hemicellulose from cellulose. However, for alkaline based (e.g., AFEX) and other lower severity pre-treatments it becomes critical to co-hydrolyze cellulose and hemicellulose using an optimized enzyme cocktail. Lignocellulosics are appropriate substrates to assess hydrolytic activity of enzyme mixtures compared to conventional unrealistic substrates (e.g., filter paper, chromogenic, and fluorigenic compounds) for studying synergistic hydrolysis. However, there are few, if any, high-throughput lignocellulosic digestibility analytical platforms for optimizing biomass conversion. The 96-well Biomass Conversion Research Lab (BCRL) microplate method is a high-throughput assay to study digestibility of lignocellulosic biomass as a function of biomass composition, pre-treatment severity, and enzyme composition. The most suitable method for delivering milled biomass to the microplate was through multi-pipetting slurry suspensions. A rapid bio-enzymatic, spectrophotometric assay was used to determine fermentable sugars. The entire procedure was automated using a robotic pipetting workstation. Several parameters that affect hydrolysis in the microplate were studied and optimized (i.e., particle size reduction, slurry solids concentration, glucan loading, mass transfer issues, and time period for hydrolysis). The microplate method was optimized for crystalline cellulose (Avicel) and ammonia fiber expansion (AFEX) pre-treated corn stover.     

High‐solids biphasic CO2–H2O pretreatment of lignocellulosic biomass

A high pressure (200 bar) CO₂–H₂O process was developed for pretreating lignocellulosic biomass at high‐solid contents, while minimizing chemical inputs. Hardwood was pretreated at 20 and 40 (wt.%) solids. Switchgrass, corn stover, big bluestem, and mixed perennial grasses (a co‐culture of big bluestem and switchgrass) were pretreated at 40 (wt.%) solids. Operating temperatures ranged from 150 to 250°C, and residence times from 20 s to 60 min. At these conditions a biphasic mixture of an H₂O‐rich liquid (hydrothermal) phase and a CO₂‐rich supercritical phase coexist. Following pretreatment, samples were then enzymatically hydrolyzed. Total yields, defined as the fraction of the theoretical maximum, were determined for glucose, hemicellulose sugars, and two degradation products: furfural and 5‐hydroxymethylfurfural. Response surfaces of yield as a function of temperature and residence time were compared for different moisture contents and biomass species. Pretreatment at 170°C for 60 min gave glucose yields of 77%, 73%, and 68% for 20 and 40 (wt.%) solids mixed hardwood and mixed perennial grasses, respectively. Pretreatment at 160°C for 60 min gave glucan to glucose yields of 81% for switchgrass and 85% for corn stover. Biotechnol. Bioeng. 2010;107: 451–460. © 2010 Wiley Periodicals, Inc.     

Particle concentration and yield stress of biomass slurries during enzymatic hydrolysis at high‐solids loadings

Effective and efficient breakdown of lignocellulosic biomass remains a primary barrier for its use as a feedstock for renewable transportation fuels. A more detailed understanding of the material properties of biomass slurries during conversion is needed to design cost‐effective conversion processes. A series of enzymatic saccharification experiments were performed with dilute acid pretreated corn stover at initial insoluble solids loadings of 20% by mass, during which the concentration of particulate solids and the rheological property yield stress (τ_y) of the slurries were measured. The saccharified stover liquefies to the point of being pourable (τ_y ≤ 10 Pa) at a total biomass conversion of about 40%, after roughly 2 days of saccharification for a moderate loading of enzyme. Mass balance and semi‐empirical relationships are developed to connect the progress of enzymatic hydrolysis with particle concentration and yield stress. The experimental data show good agreement with the proposed relationships. The predictive models developed here are based on established physical principles and should be applicable to the saccharification of other biomass systems. The concepts presented, especially the ability to predict yield stress from extent of conversion, will be helpful in the design and optimization of enzymatic hydrolysis processes that operate at high‐solids loadings. Biotechnol. Bioeng. 2009; 104: 290–300 © 2009 Wiley Periodicals, Inc.     

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。     

d‐lactic acid production from renewable lignocellulosic biomass via genetically modified Lactobacillus plantarum

d ‐lactic acid is of great interest because of increasing demand for biobased poly‐lactic acid (PLA). Blending poly‐l ‐lactic acid with poly‐d ‐lactic acid greatly improves PLA's mechanical and physical properties. Corn stover and sorghum stalks treated with 1% sodium hydroxide were investigated as possible substrates for d ‐lactic acid production by both sequential saccharification and fermentation and simultaneous saccharification and cofermentation (SSCF). A commercial cellulase (Cellic CTec2) was used for hydrolysis of lignocellulosic biomass and an l ‐lactate‐deficient mutant strain Lactobacillus plantarum NCIMB 8826 ldhL1 and its derivative harboring a xylose assimilation plasmid (ΔldhL1‐pCU‐PxylAB) were used for fermentation. The SSCF process demonstrated the advantage of avoiding feedback inhibition of released sugars from lignocellulosic biomass, thus significantly improving d ‐lactic acid yield and productivity. d ‐lactic acid (27.3 g L^?1) and productivity (0.75 g L^?1 h^?1) was obtained from corn stover and d ‐lactic acid (22.0 g L^?1) and productivity (0.65 g L^?1 h^?1) was obtained from sorghum stalks using ΔldhL1‐pCU‐PxylAB via the SSCF process. The recombinant strain produced a higher concentration of d ‐lactic acid than the mutant strain by using the xylose present in lignocellulosic biomass. Our findings demonstrate the potential of using renewable lignocellulosic biomass as an alternative to conventional feedstocks with metabolically engineered lactic acid bacteria to produce d ‐lactic acid. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:271–278, 2016     

Hydrodynamic cavitation as a strategy to enhance the efficiency of lignocellulosic biomass pretreatment

Hydrodynamic cavitation (HC) is a process technology with potential for application in different areas including environmental, food processing, and biofuels production. Although HC is an undesirable phenomenon for hydraulic equipment, the net energy released during this process is enough to accelerate certain chemical reactions. The application of cavitation energy to enhance the efficiency of lignocellulosic biomass pretreatment is an interesting strategy proposed for integration in biorefineries for the production of bio-based products. Moreover, the use of an HC-assisted process was demonstrated as an attractive alternative when compared to other conventional pretreatment technologies. This is not only due to high pretreatment efficiency resulting in high enzymatic digestibility of carbohydrate fraction, but also, by its high energy efficiency, simple configuration, and construction of systems, besides the possibility of using on the large scale. This paper gives an overview regarding HC technology and its potential for application on the pretreatment of lignocellulosic biomass. The parameters affecting this process and the perspectives for future developments in this area are also presented and discussed.     

Strategies for the production of cell wall‐deconstructing enzymes in lignocellulosic biomass and their utilization for biofuel production

Microbial cell wall‐deconstructing enzymes are widely used in the food, wine, pulp and paper, textile, and detergent industries and will be heavily utilized by cellulosic biorefineries in the production of fuels and chemicals. Due to their ability to use freely available solar energy, genetically engineered bioenergy crops provide an attractive alternative to microbial bioreactors for the production of cell wall‐deconstructing enzymes. This review article summarizes the efforts made within the last decade on the production of cell wall‐deconstructing enzymes in planta for use in the deconstruction of lignocellulosic biomass. A number of strategies have been employed to increase enzyme yields and limit negative impacts on plant growth and development including targeting heterologous enzymes into specific subcellular compartments using signal peptides, using tissue‐specific or inducible promoters to limit the expression of enzymes to certain portions of the plant or certain times, and fusion of amplification sequences upstream of the coding region to enhance expression. We also summarize methods that have been used to access and maintain activity of plant‐generated enzymes when used in conjunction with thermochemical pretreatments for the production of lignocellulosic biofuels.     

Comparison of aromatic monomers in lignocellulosic biomass prehydrolysates

Differences in the relative toxicity of xylose-rich prehydrolysates derived from woody and herbaceous feedstocks are likely due to the relative abundance of a variety of inhibitory compounds. Acetate, as well as several aromatic monomers, has been shown to be an inhibitor of the xylose-fermenting yeast, Pichia stipitis. Comparative information on the concentration of known and likely inhibitors, other than acetate, is lacking. The present study provides data on the aromatic monomer composition of representative herbaceous and woody prehydrolysates. Dilute-acid prehydrolysates were prepared from three feedstocks; two herbaceous, corn stover and switchgrass (Panicum virgatum L.), and one woody (poplar). The prehydrolysates were neutralized with Ca(OH)₂ extracted with ethyl acetate, trimethylsilylated, and analyzed by GC-MS. Fourteen aromatic monomers were tentatively identified by comparison with published mass spectra. The concentrations of the aromatic monomers totalled 112, 141 and 247 mg L⁻¹ for corn stover, switchgrass and poplar prehydrolysates, respectively. This is also the order of increasing inhibition of growth and ethanol productivity observed for Pichia fermentations. The woody prehydrolysate contained approximately four-fold more syringyl-based monomers than did the herbaceous prehydrolysates, while guaiacyl-containing compounds were more evenly distributed. Received 24 April 1998/ Accepted in revised form 8 June 1998     

木质纤维素生物转化产氢技术现状与发展趋势

氢能是一种清洁能源,利用木质纤维素类生物质生产氢气,在生产可再生绿色能源的同时,避免了木质纤维素资源未被充分利用而造成的环境污染和资源浪费,它的开发与应用对人类未来能源与经济发展具有十分重要意义。以下综述了木质纤维素生物转化产氢技术的研究现状,提出了木质纤维素生物转化产氢的总体构想与对产业发展方向的建议。     

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     

RNAi suppression of lignin biosynthesis in sugarcane reduces recalcitrance for biofuel production from lignocellulosic biomass

Sugarcane is a prime bioethanol feedstock. Currently, sugarcane ethanol is produced through fermentation of the sucrose, which can easily be extracted from stem internodes. Processes for production of biofuels from the abundant lignocellulosic sugarcane residues will boost the ethanol output from sugarcane per land area. However, unlocking the vast amount of chemical energy stored in plant cell walls remains expensive primarily because of the intrinsic recalcitrance of lignocellulosic biomass. We report here the successful reduction in lignification in sugarcane by RNA interference, despite the complex and highly polyploid genome of this interspecific hybrid. Down‐regulation of the sugarcane caffeic acid O‐methyltransferase (COMT) gene by 67% to 97% reduced the lignin content by 3.9% to 13.7%, respectively. The syringyl/guaiacyl ratio in the lignin was reduced from 1.47 in the wild type to values ranging between 1.27 and 0.79. The yields of directly fermentable glucose from lignocellulosic biomass increased up to 29% without pretreatment. After dilute acid pretreatment, the fermentable glucose yield increased up to 34%. These observations demonstrate that a moderate reduction in lignin (3.9% to 8.4%) can reduce the recalcitrance of sugarcane biomass without compromising plant performance under controlled environmental conditions.