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。     

Facile pretreatment of lignocellulosic biomass at high loadings in room temperature ionic liquids

Ionic liquids (ILs) have emerged as attractive solvents for lignocellulosic biomass pretreatment in the production of biofuels and chemical feedstocks. However, the high cost of ILs is a key deterrent to their practical application. Here, we show that acetate based ILs are effective in dramatically reducing the recalcitrance of corn stover toward enzymatic polysaccharide hydrolysis even at loadings of biomass as high as 50% by weight. Under these conditions, the IL serves more as a pretreatment additive rather than a true solvent. Pretreatment of corn stover with 1‐ethyl‐3‐methylimidizolium acetate ([Emim] [OAc]) at 125 ± 5°C for 1 h resulted in a dramatic reduction of cellulose crystallinity (up to 52%) and extraction of lignin (up to 44%). Enzymatic hydrolysis of the IL‐treated biomass was performed with a common commercial cellulase/xylanase from Trichoderma reesei and a commercial β‐glucosidase, and resulted in fermentable sugar yields of ～80% for glucose and ～50% for xylose at corn stover loadings up to 33% (w/w) and 55% and 34% for glucose and xylose, respectively, at 50% (w/w) biomass loading. Similar results were observed for the IL‐facilitated pretreatment of switchgrass, poplar, and the highly recalcitrant hardwood, maple. At 4.8% (w/w) corn stover, [Emim][OAc] can be readily reused up to 10 times without removal of extracted components, such as lignin, with no effect on subsequent fermentable sugar yields. A significant reduction in the amount of IL combined with facile recycling has the potential to enable ILs to be used in large‐scale biomass pretreatment. Biotechnol. Bioeng. 2011;108: 2865–2875. © 2011 Wiley Periodicals, Inc.     

Enzymatic hydrolysis of lignocellulosic biomass from low to high solids loading

Solid state enzymatic hydrolysis (SSEH) has many advantages, such as higher sugar concentration, lower operating costs, and less energy input. It should be a potential approach for the industrial application of lignocellulosic ethanol. The purpose of this work is to review the enzymatic hydrolysis of lignocellulosic biomass from low to high solids loading and introduce its both challenges and perspectives. The limitations of SSEH, including inhibition effects, water constraint, and rheology characteristic, are summarized firstly. Various strategies for overcoming these limitations are proposed correspondingly. Fed batch process and its feeding strategy to improve the SSEH efficiency are then discussed. Finally, several intensification methods, hydrolysis reactor, and pilot‐ and demonstration‐scale operations of SSEH are described. In‐depth analysis of main limitations and development of novel intensification methods and reactors should provide an effective way to achieve large‐scale implementation of SSEH.     

Comparative evaluation of acid and alkaline sulfite pretreatments for enzymatic saccharification of bagasses from three different sugarcane hybrids

Sugarcane bagasses from three experimental sugarcane hybrids and a mill‐reference sample were used to compare the efficiency and mode of action of acid and alkaline sulfite pretreatment processes. Varied chemical loads and reaction temperatures were used to prepare samples with distinguished characteristics regarding xylan and lignin removals, as well as sulfonation levels of residual lignins. The pretreatment with low sulfite loads (5%) under acidic conditions (pH 2) provided maximum glucose yield of 70% during enzymatic hydrolysis with cellulases (10 FPU/g) and β‐glucosidases (20 UI/g bagasse). In this case, glucan enzymatic conversion from pretreated materials was mostly associated with extensive xylan removal (70–100%) and partial delignification occurred during the pretreatment. The use of low sulfite loads under acidic conditions required pretreatment temperatures of 160°C. In contrast, at a lower pretreatment temperature (120°C), alkaline sulfite process achieved similar glucan digestibility, but required a higher sulfite load (7.5%). Residual xylans from acid pretreated materials were almost completely hydrolysed by commercial enzymes, contrasting with relatively lower xylan to xylose conversions observed in alkaline pretreated samples. Efficient xylan removal during acid sulfite pretreatment and during enzymatic digestion can be useful to enhance glucan accessibility and digestibility by cellulases. Alkaline sulfite process also provided substrates with high glucan digestibility, mainly associated with delignification and sulfonation of residual lignins. The results demonstrate that temperature, pH, and sulfite can be combined for reducing lignocellulose recalcitrance and achieve similar glucan conversion rates in the alkaline and acid sulfite pretreated bagasses. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:944–951, 2018     

Room temperature ionic liquids as emerging solvents for the pretreatment of lignocellulosic biomass

Room temperature ionic liquids (RTILs) are emerging as attractive and green solvents for lignocellulosic biomass pretreatment. The unique solvating properties of RTILs foster the disruption of the 3D network structure of lignin, cellulose, and hemicellulose, which allows high yields of fermentable sugars to be produced in subsequent enzymatic hydrolysis. In the current review, we summarize the physicochemical properties of RTILs that make them effective solvents for lignocellulose pretreatment including mechanisms of interaction between lignocellulosic biomass subcomponents and RTILs. We also highlight several recent strategies that exploit RTILs and generate high yields of fermentable sugars suitable for downstream biofuel production, and address new opportunities for use of lignocellulosic components, including lignin. Finally, we address some of the challenges that remain before large-scale use of RTILs may be achieved.     

Pretreatment of lignocellulosic biomass using ultrasound aiming at obtaining fermentable sugar

This study aims to evaluate the activity of the cellulase enzyme forward the use of ultrasound technology in different conditions of temperature, pH and exposure time, as well, to match the steps of pretreatment and enzymatic hydrolysis in one step. A central composite design (CCRD) and response surface analysis were used to evaluate the effect of ultrasound power, temperature and pH on enzyme activity. Optimum condition in the studied range was 30% for ultrasound power, pH 4.6 and 50?°C, yielding an enzyme activity of 15.5 UPF/mL. From this, we carried out kinetics of enzymatic hydrolysis on filter paper and bagasse malt, in optimized conditions. Total reducing sugars (TRS) were 3.85 and 0.46?mg/mL when the filter paper and bagasse malt were used as substrate, respectively. Ultrasound showed to be a good technology to increase the enzyme activity aiming to intensify enzymatic processes.     

Saccharification of biomass using whole solid‐state fermentation medium to avoid additional separation steps

The enzymatic hydrolysis of steam‐exploded sugarcane bagasse (SESB) was investigated using enzymatic extracts (EE) and whole fermentation media (WM), produced in‐house, from Aspergillus niger 3T5B8 and Trichoderma reesei Rut‐C30 cultivated on wheat bran under solid‐state fermentation (SSF). A detailed and quantitative comparison of the different hydrolysis conditions tested was carried out using the Chrastil approach for modeling enzymatic reactions by fitting the experimental data of total reducing sugar (TRS) released according to hydrolysis time. Conversion of SESB using A. niger enzymatic complex were up to 3.2‐fold higher (in terms of TRS) than T. reesei at similar enzyme loadings, which could be correlated to the higher β‐glucosidase levels (up to 35‐fold higher) of A. niger enzymatic complex. Conversion yields after 72 h exceeded 40% in terms of TRS when the WM was supplemented with a low dosage of a commercial enzyme preparation. When the combination of WM (from either T. reesei or A. niger) and commercial cellulase was used, the dosage of the commercial enzyme could be reduced by half, while still providing a hydrolysis that was up to 36% more efficient. Furthermore, SESB hydrolysis using either EE or WM resulted in similar yields, indicating that the enzyme extraction/filtration steps could be eliminated from the overall process. This procedure is highly advantageous in terms of reduced enzyme and process costs, and also avoids the generation of unnecessary effluent streams. Thus, the enzymatic conversion of SESB using the WM from SSF is cost‐effective and compatible with the biorefinery concept. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1430–1440, 2013     

Quantitative proteomic analysis of lignocellulolytic enzymes by Phanerochaete chrysosporium on different lignocellulosic biomass

Lignocellulosic biomass from agricultural crop residues and forest waste represents an abundant renewable resource for bioenergy and future biofuel. The current bottleneck of lignocellulosic biofuel production is the hydrolysis of biomass to sugar. To understand the enzymatic hydrolysis of complex biomasses, in this report, lignocellulolytic enzymes secretion by Phanerochaete chrysosporium cultivated in different natural lignocellulosic biomass such as corn stover, hay, sawdust, sugarcane baggase, wheat bran and wood chips were quantitatively analyzed with the iTRAQ technique using LC-MS/MS. A diverse groups of enzymes, including cellulases, glycoside hydrolases, hemicellulases, lignin degrading enzymes, peroxidases, esterases, lipases, chitinases, peptidases, protein translocating transporter and hypothetical proteins were quantified, of which several were novel lignocellulosic biomass hydrolyzing enzymes. The quantitative expression and regulation of lignocellulolytic enzymes by P. chrysosporium were dependent on the nature and complexity of lignocellulosic biomass as well as physical size of the biomass. The iTRAQ data revealed oxidative and hydrolytic lignin degrading mechanism of P. chrysosporium. Numerous proteins presumed to be involved in natural lignocellulosic biomass transformation and degradation were expressed and produced in variable quantities in response to different agricultural and forest wastes.     

Decomposition of plant residues at low temperatures separates turnover of nitrogen and energy rich tissue components in time

Carbon and nitrogen loss patterns from stems and leaves from Elephant grass (Miscanthus × ogiformis Honda cv. Giganteus), and five commonly used cover crop species: Hairy vetch (Vicia villosa Roth), Italian ryegrass (Lolium multiflorum L.), Crimson clover (Trifolium incarnatum L.), Rye (Secale cereale L.), and Radish (Raphanus sativus L.) were examined at 3 and 9 °C. The stratified incubation system allowed `dry' recovery of the decomposing plant residues with minimal soil contamination and without loss of soluble substances. The recovered materials were characterized biochemically and by light and scanning electron microscopy. When the data was analysed across all treatments and sampling dates, there was no significant effect of temperature on N loss, whereas C loss was significantly affected (P<0.0001) by temperature. Decomposition at 3 °C led to wider C-to-N ratios in the plant residues. At 3 °C there was no net immobilization of N, whereas at 9 °C net immobilization was strong in the L. multiflorum and M. × ogiformis treatments. The biochemical and microscopic evidence supports that microbial growth and macro-polymer utilization was reduced at 3 °C. It was apparent that the dicot materials leaked substantially more carbon during the early phase of decomposition, whereas in the monocot materials and especially in the M. × ogiformis treatment the microbial growth and substrate utilization must have been contained within the decomposing tissues. Based on this evidence, we propose that the decomposition of intracellular low molecular substances and proteins can be viewed as a process separate from the decomposition of macro-polymers in cell walls. At higher temperatures these processes coincide and thus the distinctiveness is blurred, whereas at low temperatures they may occur more separated in time as well as space due to leaking.     

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.     

Regulation of cytoplasmic pH in rat sublingual mucous acini at rest and during muscarinic stimulation

The regulation of intracellular pH (pHi) in rat sublingual mucous acini was monitored using dual-wavelength microfluorometry of the pH-sensitive dye BCECF (2',7'-biscarboxyethyl-5(6)-carboxyfluorescein). Acini attached to coverslips and continuously superfused with HCO3(-)-containing medium (25 mM NaHCO3/5% CO2; pH 7.4) have a steady-state pHi of 7.25 +/- 0.02. Acid loading of acinar cells using the NH4+/NH3 prepulse technique resulted in a Na(+)-dependent, MIBA-inhibitable (5-(N-methyl-N-isobutyl) amiloride, Ki approximately 0.42 microM) pHi recovery, the kinetics of which were not influenced by the absence of extracellular Cl-. The rate and magnitude of the pHi recovery were dependent on the extracellular Na+ concentration, indicating that Na+/H+ exchange plays a critical role in maintaining pHi above the pH predicted for electrochemical equilibrium. When the NH4+/NH3 concentration was varied, the rate of pHi recovery was enhanced as the extent of the intracellular acidification increased, demonstrating that the activity of the Na+/H+ exchanger is regulated by the concentration of intracellular protons. Switching BCECF-loaded acini to a Cl(-)-free medium did not significantly alter resting pHi, suggesting the absence of Cl-/HCO3- exchange activity. Muscarinic stimulation resulted in a rapid and sustained cytosolic acidification (t 1/2 < 30 sec; 0.16 +/- 0.02 pH unit), the magnitude of which was amplified greater than two-fold in the presence of MIBA (0.37 +/- 0.05 pH unit) or in the absence of extracellular Na+ (0.34 +/- 0.03 pH unit). The agonist-induced intracellular acidification was blunted in HCO3(-)-free media and was inhibited by DPC (diphenylamine-2-carboxylate), an anion channel blocker. In contrast, the acidification was not influenced by removal of extracellular Cl-. The Ca2+ ionophore, ionomycin, mimicked the effects of stimulation, whereas preloading acini with BAPTA (bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid) to chelate intracellular Ca2+ blocked the agonist-induced cytoplasmic acidification. The above results indicate that during muscarinic stimulation an intracellular acidification occurs which: (i) is partially buffered by increased Na+/H+ exchange activity; (ii) is most likely mediated by HCO3- efflux via an anion channel; and (iii) requires an increase in cytosolic free [Ca2+].     

Decomposition of sugarcane bagasse with lignocellulose-derived thermotolerant and thermoresistant Penicillia and Aspergilli

To promote the decomposition of sugarcane bagasse (SCB) for conversion into value-added products and to reduce waste, the capability of fungal mixes (FMs) to degrade SCB was examined. A total of 169 isolates from SCB and non-SCB were categorized as thermotolerant and thermoresistant. Thirty-six fungal candidates were screened for the presence of polyphenol oxidase, endoglucanase (EDN) and xylanase (XLN) activities, and EDN and XLN activities were quantitated. Five identified isolates (Aspergillus flavus AG10; Aspergillus niger AG68 & NB23; and Penicillium citrinum AG93 & AG140) were selected as the best enzyme producers, and 15 moderately to highly xylolytic, cellulolytic and ligninolytic isolates were added to construct FMs. Using a Taguchi design, the top ten reducing sugar-producing FMs (no. 12 showed the maximum amount of reducing sugar, at 2.11 mg g⁻¹, followed by no. 7, 15, 2, 16, 11, 13, 6, 4, & 8) were selected as potential agents for decomposition durations of 1, 2 and 3 months. The maximum decrease in SCB materials compared with the control was generated by FM 6 (9.08% cellulose reduction); FM 13 (21.03% hemicellulose reduction); and FM 16 (9.21% lignin reduction). These results indicate the potential use of SCB as a substrate for synergistic FMs. These FMs could be applied to the large-scale composting of SCB and other related agricultural residues, thus improving the biological pretreatment of lignocellulose.