Lignin Depletion Enhances the Digestibility of Cellulose in Cultured Xylem Cells

Plant lignocellulose constitutes an abundant and sustainable source of polysaccharides that can be converted into biofuels. However, the enzymatic digestion of native plant cell walls is inefficient, presenting a considerable barrier to cost-effective biofuel production. In addition to the insolubility of cellulose and hemicellulose, the tight association of lignin with these polysaccharides intensifies the problem of cell wall recalcitrance. To determine the extent to which lignin influences the enzymatic digestion of cellulose, specifically in secondary walls that contain the majority of cellulose and lignin in plants, we used a model system consisting of cultured xylem cells from Zinnia elegans . Rather than using purified cell wall substrates or plant tissue, we have applied this system to study cell wall degradation because it predominantly consists of homogeneous populations of single cells exhibiting large deposits of lignocellulose. We depleted lignin in these cells by treating with an oxidative chemical or by inhibiting lignin biosynthesis, and then examined the resulting cellulose digestibility and accessibility using a fluorescent cellulose-binding probe. Following cellulase digestion, we measured a significant decrease in relative cellulose content in lignin-depleted cells, whereas cells with intact lignin remained essentially unaltered. We also observed a significant increase in probe binding after lignin depletion, indicating that decreased lignin levels improve cellulose accessibility. These results indicate that lignin depletion considerably enhances the digestibility of cellulose in the cell wall by increasing the susceptibility of cellulose to enzymatic attack. Although other wall components are likely to contribute, our quantitative study exploits cultured Zinnia xylem cells to demonstrate the dominant influence of lignin on the enzymatic digestion of the cell wall. This system is simple enough for quantitative image analysis, but realistic enough to capture the natural complexity of lignocellulose in the plant cell wall. Consequently, these cells represent a suitable model for analyzing native lignocellulose degradation.     

Composition of cell wall phenolics and polysaccharides of the potential bioenergy crop –Miscanthus

The composition and concentrations of cell wall polysaccharides and phenolic compounds were analyzed in mature stems of several Miscanthus genotypes, in comparison with switchgrass and reed (Arundo donax), and biomass characteristics were correlated with cell wall saccharification efficiency. The highest cellulose content was found in cell walls of M. sinensis‘Grosse Fontaine’ (55%) and in A. donax (47%) and lowest (about 32%) in M. sinensis‘Adagio’. There was little variation in lignin contents across M. sinensis samples (all about 22–24% of cell wall), however, Miscanthus×giganteus (M × g) cell walls contained about 28% lignin, reed – 23% and switchgrass – 26%. The highest ratios of cellulose/lignin and cellulose/xylan were in M. sinensis‘Grosse Fontaine’ across all samples tested. About the same total content of ester‐bound phenolics was found in different Miscanthus genotypes (23–27 μg/mg cell wall), while reed cell walls contained 17 μg/mg cell wall and switchgrass contained a lower amount of ester‐bound phenolics, about 15 μg/mg cell wall. Coumaric acid was a major phenolic compound ester‐bound to cell walls in plants analyzed and the ratio of coumaric acid/ferulic acid varied from 2.1 to 4.3, with the highest ratio being in M × g samples. Concentration of ether‐bound hydroxycinnamic acids varied greatly (about two‐three‐fold) within Miscanthus genotypes and was also the highest in M × g cell walls, but at a concentration lower than ester‐bound hydroxycinnamic acids. We identified four different forms of diferulic acid esters bound to Miscanthus cell walls and their concentration and proportion varied in genotypes analyzed with the 5‐5‐coupled dimer being the predominant type of diferulate in most samples tested. The contents of lignin and ether‐bound phenolics in the cell wall were the major determinants of the biomass degradation caused by enzymatic hydrolysis.     

The pattern of cell wall deterioration in lignocellulose fibers throughout enzymatic cellulose hydrolysis

Cell wall deterioration throughout enzymatic hydrolysis of cellulosic biomass is greatly affected by the chemical composition and the ultrastructure of the fiber cell wall. The resulting pattern of cell wall deterioration will reveal information on cellulose activity throughout enzymatic hydrolysis. This study investigates the progression and morphological changes in lignocellulose fibers throughout enzymatic hydrolysis, using (transmission electron microscopy) TEM and field emission scanning electron microscopy (FE‐SEM). Softwood thermo‐mechanical pulp (STMP) and softwood bleached kraft pulp (SBKP), lignocellulose substrates containing almost all the original fiber composition, and with lignin and some hemicellulose removed, respectively, was compared for morphology changes throughout hydrolysis. The difference of conversion between STMP and SBKP after 48 h of enzymatic hydrolysis is 11 and 88%, respectively. TEM images revealed an even fiber cell wall cross section density, with uneven middle lamella coverage in STMP fibers. SKBP fibers exhibited some spaces between cell wall and lamella layers due to the removal of lignin and some hemicellulose. After 1 h hydrolysis in SBKP fibers, there were more changes in the fiber cross‐sectional area than after 10 h hydrolysis in STMP fibers. Cell wall degradation was uneven, and originated in accessible cellulose throughout the fiber cell wall. FE‐SEM images illustrated more morphology changes in SBKP fibers than STMP fibers. Enzymatic action of STMP fiber resulted in a smoother fiber surface, along with fiber peeling and the formation of ribbon‐disjunction layers. SBKP fibers exhibited structural changes such as fiber erosion, fiber cutting, and fiber splitting throughout enzymatic hydrolysis. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Biomass Enzymatic Saccharification Is Determined by the Non-KOH-Extractable Wall Polymer Features That Predominately Affect Cellulose Crystallinity in Corn

Corn is a major food crop with enormous biomass residues for biofuel production. Due to cell wall recalcitrance, it becomes essential to identify the key factors of lignocellulose on biomass saccharification. In this study, we examined total 40 corn accessions that displayed a diverse cell wall composition. Correlation analysis showed that cellulose and lignin levels negatively affected biomass digestibility after NaOH pretreatments at p<0.05 & 0.01, but hemicelluloses did not show any significant impact on hexoses yields. Comparative analysis of five standard pairs of corn samples indicated that cellulose and lignin should not be the major factors on biomass saccharification after pretreatments with NaOH and H₂SO₄ at three concentrations. Notably, despite that the non-KOH-extractable residues covered 12%–23% hemicelluloses and lignin of total biomass, their wall polymer features exhibited the predominant effects on biomass enzymatic hydrolysis including Ara substitution degree of xylan (reverse Xyl/Ara) and S/G ratio of lignin. Furthermore, the non-KOH-extractable polymer features could significantly affect lignocellulose crystallinity at p<0.05, leading to a high biomass digestibility. Hence, this study could suggest an optimal approach for genetic modification of plant cell walls in bioenergy corn.     

Biomass yield in a genetically diverse Miscanthus sinensis germplasm panel evaluated at five locations revealed individuals with exceptional potential

To breed improved biomass cultivars of Miscanthus ×giganteus, it will be necessary to select the highest‐yielding and best‐adapted genotypes of its parental species, Miscanthus sinensis and Miscanthus sacchariflorus. We phenotyped a diverse clonally propagated panel of 569 M. sinensis and nine natural diploid M. ×giganteus at one subtropical (Zhuji, China) and five temperate locations (Sapporo, Japan; Leamington, Ontario, Canada; Fort Collins, CO; Urbana, IL; and Chuncheon, Korea) for dry biomass yield and 14 yield‐component traits, in trials grown for 3 years. Notably, dry biomass yield of four Miscanthus accessions exceeded 80 Mg/ha in Zhuji, China, approaching the highest observed for any land plant. Additionally, six M. sinensis in Sapporo, Japan and one in Leamington, Canada also yielded more than the triploid M. ×giganteus ‘1993‐1780’ control, with values exceeding 20 Mg/ha. Diploid M. ×giganteus was the best‐yielding group at the northern sites. Genotype‐by‐environment interactions were modest among the five northern trial sites but large between Zhuji, and the northern sites. M. sinensis accessions typically yielded best at trial sites with latitudes similar to collection sites, although broad adaptation was observed for accessions from southern Japan. Genotypic heritabilities for third year yields ranged from 0.71 to 0.88 within locations. Compressed circumference was the best predictor of yield. These results establish a baseline of data for initiating selection to improve biomass yield of M. sinensis and M. ×giganteus in a diverse set of relevant geographies.     

Characterization of Miscanthus cell wall polymers

Efficient utilization of lignocellulosic Miscanthus biomass for the production of biochemicals, such as ethanol, is challenging due to its recalcitrance, which is influenced by the individual plant cell wall polymers and their interactions. Lignocellulosic biomass composition differs depending on several factors, such as plant age, harvest date, organ type, and genotype. Here, four selected Miscanthus genotypes (Miscanthus sinensis, Miscanthus sacchariflorus, Miscanthus × giganteus, Miscanthus sinensis × Miscanthus sacchariflorus hybrid) were grown and harvested, separated into stems and leaves, and characterized for their non‐starch polysaccharide composition and structures, lignin contents and structures, and hydroxycinnamate profiles (monomers and ferulic acid dehydrodimers). Polysaccharides of all genotypes are mainly composed of cellulose and low‐substituted arabinoxylans. Ratios of hemicelluloses to cellulose were comparable, with the exception of Miscanthus sinensis that showed a higher hemicellulose/cellulose ratio. Lignin contents of Miscanthus stems were higher than those of Miscanthus leaves. Considering the same organs, the four genotypes did not differ in their Klason lignin contents, but Miscanthus × giganteus showed the highest acetylbromide soluble lignin content. Lignin polymers isolated from stems varied in their S/G ratios and linkage type distributions across genotypes. p‐Coumaric acid was the most abundant ester‐bound hydroxycinnamte monomer in all samples. Ferulic acid dehydrodimers were analyzed as cell wall cross‐links, with 8‐5‐coupled diferulic acid being the main dimer, followed by 8‐O‐4‐, and 5‐5‐diferulic acid. Contents of p‐coumaric acid, ferulic acid, and ferulic acid dimers varied depending on genotype and organ type. The largest amount of cell wall cross‐links was analyzed for Miscanthus sinensis.     

Characterization of lignocellulose particles during lignocellulose solubilization by Clostridium thermocellum

Clostridium thermocellum is a candidate bacterium for lignocellulose utilization due to its efficient lignocellulose solubilization ability. It has been reported that C. thermocellum efficiently degrades purified cellulose substrates, but cannot completely degrade milled lignocellulose powders. Evaluation of cellulose and hemicellulose contents in a lignocellulose residue after the cultivation of C. thermocellum indicated that C. thermocellum degraded cellulose and hemicellulose equally. Microscopic observations demonstrated that C. thermocellum significantly degraded small-sized lignocellulose particles, but it only partially degraded the larger sized particles. The lignin content of the large-sized particles was higher than that of the small particles. The remained large-sized particles included vascular tissues. These results suggest that the lignified structures such as vascular tissues in milled lignocellulose were less susceptible to bacterial lignocellulose solubilization.     

Lignin solubilisation by Thermomonospora mesophila

Summary Thermomonospora mesophila degraded [¹⁴C]lignin-labelled wheat lignocellulose to yield high molecular weight water-soluble products and a small amount of ¹⁴CO₂. Solubilisation of [¹⁴C]lignin was found to be extracellular and inducible by growth on lignocellulose (straw) and hemicellulose (xylan), but was not correlated with xylanase or cellulase production.The acid-precipitable product of straw degradation by T. mesophila was found to be a complex of lignin, pentose-rich carbohydrate and protein with some similarity to humic acids. Solid-state ¹³C-NMR spectra of the dried product were generally similar to those of chemically extracted milled straw lignin but showed an increased content of carbonyl groups.The relationship between degradation and solubilisation of lignin is discussed and a role suggested for actinomycetes in humification and the exploitation of lignocellulose bioconversion.     

Genetic variations of cell wall digestibility related traits in floral stems of Arabidopsis thaliana accessions as a basis for the improvement of the feeding value in maize and forage plants

Floral stems of Arabidopsis thaliana accessions were used as a model system relative to forage plant stems in genetic variation studies of lignin content and cell wall digestibility related traits. Successive investigations were developed in a core collection of 24 Arabidopsis accessions and in a larger collection of 280 accessions. Significant genetic variation for lignin content in the cell wall, and for the two in vitro cell wall digestibility investigated traits, were found both in the core collection and in the large collection. Genotype × environment interactions, investigated in the core collection, were significant with a few genotypes contributing greatly to interactions, based on ecovalence value estimates. In the core collection, genotypes 42AV, 224AV, and 8AV had low cell wall digestibility values, whatever be the environmental conditions. Genotype 157AV, observed only in one environment, also appeared to have a low cell wall digestibility. Conversely, genotypes 236AV, 162AV, 70AV, 101AV, 83AV had high cell wall digestibility values, genotype 83AV having a slightly greater instability across differing environments than others. The well-known accession Col-0 (186AV) appeared with a medium level of cell wall digestibility and a weak to medium level of interaction between environments. The ranges of variation in cell wall digestibility traits were higher in the large collection than in the core collection of 24 accessions, these results needing confirmation due to the lower number of replicates. Accessions 295AV, 148AV, and 309AV could be models for low stem cell wall digestibility values, with variable lignin content. Similarly, accessions 83AV and 162AV, already identified from the study of the core collection, and five accessions (6AV, 20AV, 91AV, 114AV, and 223AV) could be models for high stem cell wall digestibility values. The large variations observed between Arabidopsis accessions for both lignin content and cell wall digestibility in floral stems have strengthened the use this species as a powerful tool for discovering genes involved in cell wall biosynthesis and lignification of dicotyledons forage plants. Investigations of this kind might also be applicable to monocotyledons forage plants due to the basic similarity of the genes involved in the lignin pathway of Angiosperms and the partial homology of the cell wall composition and organization of the mature vascular system in grasses and Arabidopsis.     

Kinetic behavior of liquefaction of Japanese beech in subcritical phenol

Non-catalytic liquefaction of Japanese beech (Fagus crenata) wood in subcritical phenol was investigated using a batch-type reaction vessel. After samples were treated at 160 °C/0.9 MPa–350 °C/4.2 MPa for 3–30 min, they were fractionated into a phenol-soluble portion and phenol-insoluble residues. These residues were then analyzed for their chemical composition. Based on the obtained data, the kinetics for liquefaction was modeled using first-order reaction rate law. Subsequently, the liquefaction rate constants of the major cell wall components including cellulose, hemicellulose, and lignin were determined. The different kinetic mechanisms were found to exist for lignin and cellulose at two different temperature ranges, lower 160–290 °C and higher 310–350 °C, whereas for hemicellulose, it was only liquefied in the lower temperature range. Thus, the liquefaction behaviors of these major cell wall components highlighted hemicellulose to be the most susceptible to liquefaction, followed by lignin and cellulose.     

Potential of selected fungal species to degrade wheat straw,the most abundant plant raw material in Europe

Background

Structural component of plant biomass, lignocellulose, is the most abundant renewable resource in nature. Lignin is the most recalcitrant natural aromatic polymer and its degradation presents great challenge. Nowadays, the special attention is given to biological delignification, the process where white-rot fungi take the crucial place owing to strong ligninolytic enzyme system. However, fungal species, even strains, differ in potential to produce high active ligninolytic enzymes and consequently to delignify plant biomass. Therefore, the goals of the study were characterization of Mn-oxidizing peroxidases and laccases of numerous mushrooms as well as determination of their potential to delignify wheat straw, the plant raw material that, according to annual yield, takes the first place in Europe and the second one in the world.

Results

During wheat straw fermentation, Lentinus edodes HAI 858 produced the most active Mn-dependent and Mn-independent peroxidases (1443.2 U L⁻¹ and 1045.5 U L⁻¹, respectively), while Pleurotus eryngii HAI 711 was the best laccase producer (7804.3 U L⁻¹). Visualized bends on zymogram confirmed these activities and demonstrated that laccases were the dominant ligninolytic enzymes in the studied species. Ganoderma lucidum BEOFB 435 showed considerable ability to degrade lignin (58.5%) and especially hemicellulose (74.8%), while the cellulose remained almost intact (0.7%). Remarkable selectivity in lignocellulose degradation was also noted in Pleurotus pulmonarius HAI 573 where degraded amounts of lignin, hemicellulose and cellulose were in ratio of 50.4%:15.3%:3.8%.

Conclusions

According to the presented results, it can be concluded that white-rot fungi, due to ligninolytic enzymes features and degradation potential, could be important participants in various biotechnological processes including biotransformation of lignocellulose residues/wastes in food, feed, paper and biofuels.

     

In vitro Studies on lignocellulose degradation by microbial strains isolated from composting processes

An in vitro study of different strains isolated from composting piles in relation to their capacity to biodegrade lignocellulose was achieved. Thirteen microorganisms (five bacteria, one actinomycete, and seven fungi) isolated from compost windrows were grown on agricultural wastes and analyzed for cellulose, hemicellulose, and lignin degradation. Hemicellulose fraction was degraded to a lesser extent because only two of the isolates, B122 and B541, identified as Bacillus licheniformis and Brevibacillus parabrevis, respectively, were able to decrease the concentration of this polymer. On the contrary, most of the isolates were capable of reducing cellulose and lignin concentrations; strain B541 was the most active cellulose degrader (51%), while isolate B122 showed higher lignin degradation activity (68%). Consequently, an increase in humification indices was detected, especially with respect to humification index (HI) for both bacteria and CAH/AF in the case of strain B122. According to these data, the use of microbial inoculants as a tool to improve organic matter biodegradation processes (i.e., composting) may become important if microorganisms’ capabilities are in accordance with the final characteristics required in the product (high humic content, lignin content decrease, cellulose concentration decrease, etc.).     

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.     

Genetic Diversity and Population Structure of Miscanthus sinensis Germplasm in China

Miscanthus is a perennial rhizomatous C4 grass native to East Asia. Endowed with great biomass yield, high ligno-cellulose composition, efficient use of radiation, nutrient and water, as well as tolerance to stress, Miscanthus has great potential as an excellent bioenergy crop. Despite of the high potential for biomass production of the allotriploid hybrid M. ×giganteus, derived from M. sacchariflorus and M. sinensis, other options need to be explored to improve the narrow genetic base of M. ×giganteus, and also to exploit other Miscanthus species, including M. sinensis (2n = 2x = 38), as bioenergy crops. In the present study, a large number of 459 M. sinensis accessions, collected from the wide geographical distribution regions in China, were genotyped using 23 SSR markers transferable from Brachypodium distachyon. Genetic diversity and population structure were assessed. High genetic diversity and differentiation of the germplasm were observed, with 115 alleles in total, a polymorphic rate of 0.77, Nei’s genetic diversity index (He) of 0.32 and polymorphism information content (PIC) of 0.26. Clustering of germplasm accessions was primarily in agreement with the natural geographic distribution. AMOVA and genetic distance analyses confirmed the genetic differentiation in the M. sinensis germplasm and it was grouped into five clusters or subpopulations. Significant genetic variation among subpopulations indicated obvious genetic differentiation in the collections, but within-subpopulation variation (83%) was substantially greater than the between-subpopulation variation (17%). Considerable phenotypic variation was observed for multiple traits among 300 M. sinensis accessions. Nine SSR markers were found to be associated with heading date and biomass yield. The diverse Chinese M. sinensis germplasm and newly identified SSR markers were proved to be valuable for breeding Miscanthus varieties with desired bioenergy traits.     

氨基磺酸应用于甘蔗渣预处理的研究

本研究尝试将氨基磺酸应用于甘蔗渣预处理,探究其作为酸预处理试剂对甘蔗渣成分和酶解的影响。氨基磺酸预处理最优条件为浓度3%,温度121℃,预处理1 h。在该条件下,甘蔗渣的固体回收率为64.45%,半纤维素和木质素去除率分别为70.81%和25.10%,纤维素损失率仅7.56%。与硫酸、盐酸预处理相比,氨基磺酸的半纤维素和木质素去除率不如硫酸、盐酸预处理,但固体回收率更高,纤维素损失率低,能保留更多纤维素有效成分。进一步酶解显示,氨基磺酸预处理的纤维素转化率高于硫酸、盐酸预处理。氨基磺酸作为一种新的酸预处理试剂,在木质纤维素降解上有良好应用前景。     

Selection strategies for developing smooth bromegrass cell wall ideotypes

Summary A random sample of 80 families of the B8HD smooth bromegrass (Bromus inermis Leyss.) population were tested in three environments for forage yield and cell wall constituents. Expected progress from one cycle of family selection was computed for single-trait selection and multiple-trait restricted selection. Expected gains were compared to desired goals and actual results from one cycle of phenotypic selection. Desired goals were: Model I = reduced lignin and cellulose, with increased hemicellulose, resulting in no change in cell wall content; Model II = reduced lignin and cellulose with no change in hemicellulose; or Model III = reduced lignin, cellulose, and hemicellulose. Single-trait selection for high hemicellulose in first harvest or low cellulose in second harvest had the best expected responses, of any single trait, for Model I. Possible undesirable effects of selection for low cellulose would be a reduction in forage yield potential. Multiple-trait restricted selection was judged to be more effective, with responses all in the desired direction, by specifying increased hemicellulose in index development. Selection in second harvest was expected to have similar responses as first harvest, except for a greater increase in forage yield. Development of Models II or III is expected to be difficult due to a negative correlation estimate between first and second harvest cell wall concentration.     

Investigating the potential of Andean lupin as a lignocellulosic feedstock for Europe: First genome-wide association study on Lupinus mutabilis biomass quality

The development of multipurpose crops will drive the transformation of agricultural “waste” into added-value products, helping to meet biomass demands without competing with food production or increasing environmental pressure. Lupinus mutabilis, has been proposed not only as a valid source of protein and oil for Europe but also as a possible source of lignocellulosic feedstock for the biorefinery industry. In this study, the quality of L. mutabilis lignocellulosic biomass and its genetic architecture are investigated for the first time, using a panel of 223 accessions planted across three locations in two different European cropping conditions. Biomass quality was evaluated based on the estimation of neutral detergent fiber, cellulose, hemicellulose and acid detergent lignin fractions, and on the basis of the monosaccharide composition of cell wall polysaccharides. The broad variation in yield and composition of biomass encountered in the panel confirms the potential of L. mutabilis as lignocellulosic feedstock and points out the value of this panel as a breeding tool for the improvement of biomass quality. A genome-wide association study was conducted to identify single-nucleotide polymorphisms (SNPs) associated with biomass quality, both across locations and per specific location. Scanning of 16,781 SNPs across the whole genome identified 46 unique quantitative trait loci for biomass quality, 4 of which were detected as common either among traits or GWAS models. For each of the traits analyzed, between 3 and 10 SNPs were detected, explaining 2.7%–15.9% of the phenotypic variation. Underlying these loci, 28 genes were proposed as candidate genes for biomass quality. Important genes involved in cellulose and sucrose synthesis (CESA4, SPP1,WRKY33, GONST2), monolignol biosynthesis (SKIP31, WAT1, CCR-SNL6) and pectin degradation (RAV1, PE) were identified and will require validation to confirm their value for application in L. mutabilis breeding.