Characterization of a lignin-specific O-Methyltransferase in aspen wood

O-Methyltransferases were extracted from the differentiating xylem of 10-yr-old Populus euramericana. The enzymes were partially purified by ammonium sulfate precipitation, and column chromatography on DEAE-cellulose, Sephadex G200 and hydroxyapatite. The enzymes were resolved into two peaks by DEAE-cellulose chromatography, and the MWs of the respective enzymes were estimated to be 72 000 and 75 000 by gel filtration chromatography. The enzyme corresponding to the latter peak was unstable and thus only the former peak enzyme was characterized completely. Magnesium ions had no effect, EDTA moderately stimulated and heavy metals and SH group inhibitors strongly inhibited enzyme activity. K_mm values for caffeate and 5-hydroxyferulate were estimated to be 3.8 x 10⁻⁴ and 3.1 x 10⁻⁴ M, respectively. The ratio of V_max/K_m for 5-hydroxyferulate was 5.4 times greater than that for caffeate. The enzyme(s) catalysing the formation of ferulate from caffeate and of sinapate from 5-hydroxyferulate were not separated during the purification or by the disc electrophoresis using polyacrylamide gel. Quercetin, cyanin and catechin were not methylated by the enzyme preparation. The O-methyltransferase of aspen wood, where the phenolic metabolism is almost exclusively directed to lignin biosynthesis, catalyses the methylation of both guaiacyl and syringyl lignin precursors, with preferential utilization of the latter substrate. These findings lead to the conclusion that the enzyme is a typical angiosperm-type O-methyltransferase related to guaiacyl and syringyl lignin biosynthesis in aspen wood.     

Lignin depolymerization/repolymerization and its critical role for delignification of aspen wood by steam explosion

Steam explosion is an important process for the fractionation of biomass components. In order to understand the behaviour of lignin under the conditions encountered in the steam explosion process, as well as in other types of steam treatment, aspen wood and isolated lignin from aspen were subjected to steam treatment under various conditions. The lignin portion was analyzed using NMR and size exclusion chromatography as major analytical techniques. Thereby, the competition between lignin depolymerization and repolymerization was revealed and the conditions required for these two types of reaction identified. Addition of a reactive phenol, 2-naphthol, was shown to inhibit the repolymerization reaction strongly, resulting in a highly improved delignification by subsequent solvent extraction and an extracted lignin of uniform structure.     

Solid state degradation of paddy straw by Phlebia floridensis in the presence of different supplements for improving its nutritive status

Effect of different supplements on the degradation of paddy (rice) straw by Phlebia floridensis was studied and the conditions for best ligninolysis, lower loss in total organic matter (TOM) and enhancement in in vitro digestibility (IVD) were established. Effect of different supplements on lignocellulolytic enzymes production, degradation of cell wall fibres of paddy straw and their resultant effect on its nutritional quality was studied. Ammonium chloride, soya bean meal and moisture content were selected for response surface study on the basis of their important role in degradation. Finally, the process was successfully scaled up from 5 g to 200 g under optimized sold state conditions and the straw quality was upgraded in terms of increased IVD (40%) with a moderate loss (6%) in TOM during 20 days of incubation. Protein content, amino acid, total phenolics and antioxidant properties of the paddy straw improved with its fermentation.     

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.     

Novel renewable ionic liquids as highly effective solvents for pretreatment of rice straw biomass by selective removal of lignin

Cholinium amino acids ionic liquids ([Ch][AA] ILs), a novel type of bio‐ILs that can easily be prepared from renewable biomaterials, were investigated for pretreatment of rice straw by selective extraction of lignin from this abundant lignocellulosic biomass material. Of the eight ILs examined, most were demonstrated to be excellent pretreatment solvents. Upon pretreatment using these ILs, the initial saccharification rates of rice straw residues were substantially improved as well as the extent to which polysaccharides could be digested (>90% for cellulose and >60% for xylan). Enzymatic hydrolysis of pretreated rice straw by Trichoderma reesei cellulase/xylanase furnished glucose and xylose with the yields in excess of 80% and 30%, respectively. Detailed spectroscopic characterization showed that the enhancement of polysaccharides degestibility derived mainly from delignification rather than changes in cellulose crystallinity. The yields of fermentable reducing sugars were significantly improved after individual optimization of pretreatment temperature and duration. With [Ch][Lys] as the solvent, the sugar yields of 84.0% for glucose and 42.1% for xylose were achieved after pretreatment at 90°C for 5 h. The IL [Ch][Lys] showed excellent reusability across five successive batches in pretreatment of rice straw. These bio‐ILs performed as well as or better than previously investigated non‐renewable ILs, and thus present a new and environmentally friendly way to pretreat lignocellulose for production of fermentable sugars and total utilization of the biomass. Biotechnol. Bioeng. 2012; 109: 2484–2493. © 2012 Wiley Periodicals, Inc.     

A new method for the preparation of crystalline L-arabinose from arabinoxylan by enzymatic hydrolysis and selective fermentation with yeast

Arabinoxylan (`Cellace') corn fiber, containing 28.1% (w/w) as l-arabinose and 32.8% (w/w) as d-xylose, was hydrolyzed by a crude enzyme containing -xylanase, -xylosidase and -l-arabinofuranosidase originating from the extracellular culture broth of Penicillium funiculosum. The resultant hydrolysate contained l-arabinose, d-xylose and small amounts of other mono- and oligosaccharides. The l-arabinose and d-xylose were 21.3% (w/w) and 18.7% (w/w), respectively, based on the initial arabinoxylan. Williopsis saturnus var. saturnus, which metabolizes d-xylose without using l-arabinose, was aerobically cultured in the hydrolysate at 30 °C for 96 h. The sugar solution after removal of yeast cells contained l-arabinose and d-xylose which were 20.3% (w/w) and 0.002% (w/w), respectively, of the initial arabinoxylan. The solution was decolorized with activated carbon, and deionized with cation- and anion-exchange resins. The clear sugar solution thus obtained was composed of l-arabinose and d-xylose which were 19.3% (w/w) and 0.001% (w/w), respectively, of the initial arabinoxylan. After concentration of the sugar solution under reduced pressure, followed by crystallization of l-arabinose, 16% (w/w) l-arabinose (based on the initial arabinoxylan) was obtained as crude crystals. No d-xylose was detected in the final preparation.     

Optimized delignification of wood‐derived lignocellulosics for improved enzymatic hydrolysis

One of the major bottlenecks in the bioconversion of lignocelluosic feedstocks to liquid ethanol is the recalcitrance of residue following pretreatment, specifically softwood derived residues. Peroxide delignification has previously been shown to effectively aid in the removal of condensed lignaceous moieties from substrates following pretreatment, and thereby improve the hydrolyzability of the polymeric carbohydrates to their monomeric constituents. Despite the effectiveness of peroxide, drawbacks in this system still remain, as the concentration of peroxide required for adequate hydrolysis performance is currently uneconomical. In an attempt to improve the efficacy of the delignification process, we evaluated other post‐treatment operations and concurrently attempted to limit the decomposition of peroxide loading; with the over arching aim to improve the efficiency of the bioconversion process. By employing several peroxide stabilizers and pre‐chelating the steam exploded recalcitrant substrates, we were able to substantially improve the delignification treatment of Douglas‐fir wood chips, and to reduce peroxide loading by more than 50% without negative effects on the hydrolysis rates and yield. Biotechnol. Bioeng. 2010;106: 884–893. © 2010 Wiley Periodicals, Inc.     

Selectivity and delignification kinetics for oxidative and nonoxidative lime pretreatment of poplar wood, part III: long-term

Lime pretreatment is an effective method for improving lignocellulose digestibility by removing lignin. For several weeks, mixtures of poplar wood, water, and calcium hydroxide (lime) were submitted to temperatures from 25 to 65°C, with and without aeration. Kinetic models for lignin and carbohydrate degradation were obtained as functions of temperature, time, and aeration using first-order kinetics in lignin and carbohydrates. Model 1 considered two reacting moieties (slow and fast), and Model 2 considered three (slow, medium, and fast). Model 1 was statistically better and was employed to determine differential and integral selectivities, which measure the ability of pretreatment to retain carbohydrates while removing lignin. During the first 2 weeks, when lignin content ≥ 0.80 g/g lignin in raw biomass, both glucan and xylan differential and integral selectivities decreased rapidly. Afterwards, selectivities were nearly constant ranging between 0 and 3 g lignin removed/g carbohydrate degraded.     

Optimization of enzymatic hydrolysis and fermentation conditions for improved bioethanol production from potato peel residues

The aim of this work was the optimization of the enzyme hydrolysis of potato peel residues (PPR) for bioethanol production. The process included a pretreatment step followed by an enzyme hydrolysis using crude enzyme system composed of cellulase, amylase and hemicellulase, produced by a mixed culture of Aspergillus niger and Trichoderma reesei. Hydrothermal, alkali and acid pretreatments were considered with regards to the enhancement of enzyme hydrolysis of potato peel residues. The obtained results showed that hydrothermal pretreatment lead to a higher enzyme hydrolysis yield compared to both acid and alkali pretreatments. Enzyme hydrolysis was also optimized for parameters such as temperature, pH, substrate loading and surfactant loading using a response surface methodology. Under optimized conditions, 77 g L^?1 of reducing sugars were obtained. Yeast fermentation of the released reducing sugars led to an ethanol titer of 30 g L^?1 after supplementation of the culture medium with ammonium sulfate. Moreover, a comparative study between acid and enzyme hydrolysis of potato peel residues was investigated. Results showed that enzyme hydrolysis offers higher yield of bioethanol production than acid hydrolysis. These results highlight the potential of second generation bioethanol production from potato peel residues treated with onsite produced hydrolytic enzymes. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:397–406, 2017     

Sporulation of Metarhizium anisopliae in solid-state fermentation with forced aeration

Metarhizium anisopliae was grown by solid-state fermentation using a medium based on a mixture of rice bran and rice husk (1:1 on a dry weight basis) where the initial water content was fixed at a value of 47%. The experiments were performed in glass column bioreactors of three different sizes. Moist saturated air was continuously passed through the bottom of each column at a flow rate of 0.34 l h⁻¹ g⁻¹ initial dry matter, and cultivation was performed for two weeks at 27 ± 1°C. The effect of medium packing density was studied in the columns of small size. For initial apparent densities of 0.270 and 0.357 g ml⁻¹, no significant differences were observed either in total biomass production or spore yields; however, when the initial apparent density was increased up to a value of 0.496 g ml⁻¹, both growth and sporulation were strongly affected.

Thermal gradients in the medium and larger columns packed at an apparent density of 0.357 g ml⁻¹ were observed both in the radial and axial directions. Heat build-up effects were analyzed in the larger column. In this case, there were significant differences between different segments of the column both in the water content of the fermented matter and the sporulation pattern.     

Selectivity and delignification kinetics for oxidative short‐term lime pretreatment of poplar wood,part I: Constant‐pressure

Kinetic models applied to oxygen bleaching of paper pulp focus on the degradation of polymers, either lignin or carbohydrates. Traditionally, they separately model different moieties that degrade at three different rates: rapid, medium, and slow. These models were successfully applied to lignin and carbohydrate degradation of poplar wood submitted to oxidative pretreatment with lime at the following conditions: temperature 110–180°C, total pressure 7.9–21.7 bar, and excess lime loading of 0.5 g Ca(OH)₂ per gram dry biomass. These conditions were held constant for 1–6 h. The models properly fit experimental data and were used to determine pretreatment selectivity in two fashions: differential and integral. By assessing selectivity, the detrimental effect of pretreatment on carbohydrates at high temperatures and at low lignin content was determined. The models can be used to identify pretreatment conditions that selectively remove lignin while preserving carbohydrates. Lignin removal ≥50% with glucan preservation ≥90% was observed for differential glucan selectivities between ～10 and ～30 g lignin degraded per gram glucan degraded. Pretreatment conditions complying with these reference values were preferably observed at 140°C, total pressure ≥14.7 bars, and for pretreatment times between 2 and 6 h depending on the total pressure (the higher the pressure, the less time). They were also observed at 160°C, total pressure of 14.7 and 21.7 bars, and pretreatment time of 2 h. Generally, at 110°C lignin removal is insufficient and at 180°C carbohydrates do not preserve well. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Simultaneous saccharification and fermentation of cellulose for lactic acid production

Lactic acid production from α-cellulose by simultaneous saccharification and fermentation (SSF) was studied. The cellulose was converted in a batch SSF using cellulase enzyme Cytolase CL to produce glucose sugar andLactobacillus delbrueckii to ferment the glucose to lactic acid. The effects of temperature, pH, yeast extract loading, and lactic acid inhibition were studied to determine the optimum conditions for the batch processing. Cellulose was converted efficiently to lactic acid, and enzymatic hydrolysis was the rate controlling step in the SSF. The highest conversion rate was obtained at 46°C and pH 5.0. The observed yield of lactic acid from α-cellulose was 0.90 at 72 hours. The optimum pH of the SSF was coincident with that of enzymatic hydrolysis. The optimum temperature of the SSF was chosen as the highest temperature the microorganism could withstand. The optimum yeast extract loading was found to be 2.5 g/L. Lactic acid was observed to be inhibitory to the microorganisms’ activity.     

Comparisons of SPORL and dilute acid pretreatments for sugar and ethanol productions from aspen

This study reports comparative evaluations of sugar and ethanol production from a native aspen (Populus tremuloides) between sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL) and dilute acid (DA) pretreatments. All aqueous pretreatments were carried out in a laboratory wood pulping digester using wood chips at 170°C with a liquid to oven dry (od) wood ratio (L/W) of 3:1 at two levels of acid charge on wood of 0.56 and 1.11%. Sodium bisulfite charge on od wood was 0 for DA and 1.5 or 3.0% for SPORL. All substrates produced by both pretreatments (except DA with pretreatment duration of 0) had good enzymatic digestibility of over 80%. However, SPORL produced higher enzymatic digestibility than its corresponding DA pretreatment for all the experiments conducted. As a result, SPORL produced higher ethanol yield from simultaneous saccharification and fermentation of cellulosic substrate than its corresponding DA pretreatment. SPORL was more effective than its corresponding DA pretreatment in reducing energy consumption for postpretreatment wood chip size-reduction. SPORL, with lower energy input and higher sugar and ethanol yield, produced higher sugar and ethanol production energy efficiencies than the corresponding DA pretreatment.     

Renewable bio ionic liquids‐water mixtures‐mediated selective removal of lignin from rice straw: Visualization of changes in composition and cell wall structure

Pretreatment of rice straw by using renewable cholinium amino acids ionic liquids ([Ch][AA] ILs)‐water mixtures and the subsequent enzymatic hydrolysis of the residues were conducted in the present work. Of the eight mixtures composed of ILs and water, most were found to be effective for rice straw pretreatment. After pretreatment with 50% ILs‐water mixtures, the enzymatic digestion of the lignocellulosic biomass was enhanced significantly, thus leading to satisfactory sugar yields of >80% for glucose and approximately 50% for xylose. To better understand the ILs pretreatment mechanism, confocal laser scanning microscopy combined with immunolabeling and transmission electron microscopy were used to visualize changes in the contents and distribution of two major components—lignin and xylan. The results coupled with changes in chemical structures (infrared spectra) of the substrates indicated occurrence of extensive delignification, especially in cell corner and compound middle lumen of cell walls, which made polysaccharides more accessible to enzymes. This pretreatment process is promising for large‐scale application because of the high sugar yields, easy handling, being environmentally benign and highly tolerant to moisture, and significantly reduced cost and energy consumption. Biotechnol. Bioeng. 2013; 110: 1895–1902. © 2013 Wiley Periodicals, Inc.     

固态发酵下酵母自溶的工艺优化

固态下酵母自溶可以有效促进菌体内多种活性物质的释放,进而提高酵母类产品的品质。通过优化自溶温度、自溶时间及自溶促进剂锌离子浓度以获得固态发酵下酵母自溶的最佳工艺,对固态发酵物料中游离氨基酸、可溶性蛋白、α-氨基氮含量和A260/A280等指标的分析来确定固态酵母自溶工艺条件,在此基础上以自溶温度40 ℃、50 ℃、55 ℃;作用时间12、18、24 h;锌离子添加浓度2、4、8 mg/kg设置L9(33)正交试验,进一步优化固态酵母自溶的工艺参数。结果表明酵母自溶的最佳工艺条件为：自溶温度55 ℃、作用时间18 h、锌离子浓度2 mg/kg,此时其可溶性蛋白含量可达9.31 mg/g、游离氨基酸14.36 mg/g、α-氨基氮10.16 μg/g、A260/A280为1.73。经工艺优化后,可显著提高酵母自溶产物可溶性蛋白、游离氨基酸和α-氨基氮的含量,从而明显提高了复合菌培养物的品质。     

Short‐term lime pretreatment of poplar wood

Short‐term lime pretreatment uses lime and high‐pressure oxygen to significantly increase the digestibility of poplar wood. When the treated poplar wood was enzymatically hydrolyzed, glucan and xylan were converted to glucose and xylose, respectively. To calculate product yields from raw biomass, these sugars were expressed as equivalent glucan and xylan. To recommend pretreatment conditions, the single criterion was the maximum overall glucan and xylan yields using a cellulase loading of 15 FPU/g glucan in raw biomass. On this basis, the recommended conditions for short‐term lime pretreatment of poplar wood follow: (1) 2 h, 140°C, 21.7 bar absolute and (2) 2 h, 160°C, and 14.8 bar absolute. In these two cases, the reactivity was nearly identical, thus the selected condition depends on the economic trade off between pressure and temperature. Considering glucose and xylose and their oligomers produced during 72 h of enzymatic hydrolysis, the overall yields attained under these recommended conditions follow: (1) 95.5 g glucan/100 g of glucan in raw biomass and 73.1 g xylan/100 g xylan in raw biomass and (2) 94.2 g glucan/100 g glucan in raw biomass and 73.2 g xylan/100 g xylan in raw biomass. The yields improved by increasing the enzyme loading. An optimal enzyme cocktail was identified as 67% cellulase, 12% β‐glucosidase, and 24% xylanase (mass of protein basis) with cellulase activity of 15 FPU/g glucan in raw biomass and total enzyme loading of 51 mg protein/g glucan in raw biomass. Ball milling the lime‐treated poplar wood allowed for 100% conversion of glucan in 120 h with a cellulase loading of only 10 FPU/g glucan in raw biomass. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

六种白腐菌预处理对毛白杨木材酶水解效果的影响

通过化学分析和酶水解试验,研究了不同的白腐菌对毛白杨的预处理效果及不同组分的降解对酶水解的影响。毛白杨木片经6种白腐菌预处理30d后,各组分都发生了降解,其中半纤维素的损失最为显著,Trametes ochracea C6888引起半纤维素降解率高达47.19%,其次是纤维素和酸不溶木素的降解。在后续酶水解过程中,6种白腐菌处理后的样品显示出不同的水解模式,菌株Trametes ochracea C6888、T. pubescens C7571和T. versicolor C6915预处理效果最为显著,还原糖得率在整个酶水解过程中一直高于对照,其中T. ochracea C6888在水解96h后还原糖得率达到15.93%,比未处理样品提高了25%。分析酸不溶木素降解率及半纤维素降解率与还原糖得率的关系发现,不同菌株在作用同一种基质时,预处理效果差异显著,木质素和半纤维素的脱除都会影响木质纤维素的酶水解。     

两株内生真菌菌株固态发酵培养基优化

【目的】研究内生真菌菌株YCEF005、YCEF053固态发酵培养基。【方法】通过单因素试验和正交试验设计优化固态发酵培养基基质组成;通过单因素试验和均匀试验设计及神经网络结合遗传算法优化基质外加组分和接种量。【结果】优化的YCEF005培养基基质组成(质量比)为麸皮50%、豆饼粉10%、米糠20%、玉米碎粒20%;YCEF053培养基基质组成为麸皮60%、豆饼粉10%、米糠10%、玉米碎粒20%。YCEF005基质外加组分和接种量(/kg基质)为蔗糖12.96 g、蛋白胨12.70 g、NH4NO3 4.00 g、KH2PO4 1.50 g、Ca SO4 10.00 g、Mg SO4 0.48 g、含水量500 g、接种量24 m L。YCEF053基质外加组分和接种量(/kg基质)为蔗糖13.37 g、蛋白胨14.02 g、Na NO33.85 g、KH2PO4 1.23 g、Ca SO4 10.89 g、Mg SO4 0.52 g、含水量480 g、接种量20 m L。在优化培养基上发酵7-9 d获得最大发酵生物产量。【结论】通过对固态发酵培养基的优化提高了发酵的生物产量。     

Effect of surfactants on separate hydrolysis fermentation and simultaneous saccharification fermentation of pretreated lodgepole pine

The effects of surfactants addition on enzymatic hydrolysis and subsequent fermentation of steam exploded lodgepole pine (SELP) and ethanol pretreated lodgepole pine (EPLP) were investigated in this study. Supplementing Tween 80 during cellulase hydrolysis of SELP resulted in a 32% increase in the cellulose‐to‐glucose yield. However, little improvement was obtained from hydrolyzing EPLP in the presence of the same amount of surfactant. The positive effect of surfactants on SELP hydrolysis led to an increase in final ethanol yield after the fermentation. It was found that the addition of surfactant led to a substantial increase in the amount of free enzymes in the 48 h hydrolysates derived from both substrates. The effect of surfactant addition on final ethanol yield of simultaneous saccharification and fermentation (SSF) was also investigated by using SELP in the presence of additional furfural and hydroxymethylfurfural (HMF). The results showed that the surfactants slightly increased the conversion rates of furfural and HMF during SSF process by Saccharomyces cerevisiae. The presence of furfural and HMF at the experimental concentrations did not affect the final ethanol concentration either. The strategy of applying surfactants in cellulase recycling to reduce enzyme cost is presented. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009