Crystallinity Of Lignocellulose Research Articles

Engineering Trichoderma reesei for the hyperproduction of cellulose induced protein 1 (Cip1) on a sophorose-containing inducer to efficiently saccharify alkali-pretreated corn stover

Trichoderma reesei was induced to produce cellulase by a combination of glucose and β-disaccharide; however, lower levels of auxiliary proteins for degrading lignocellulosic biomass were detected by iTRAQ analysis compared with cellulose as an inducer, especially cellulose induced protein 1 (CIP1). In this study, A pdc1 promoter-driven overexpression of the endogenous Trcip1 gene was observed in T. reesei Rut C30, and the Trcip1 transcription levels of the two transformants, T. reesei OE-cip1-1 and OE-cip1-2, demonstrated 31.2- and 164.6-fold increases, respectively, but there was no significant change in cellobiohydrolase, endoglucanase and filter paper activity at 48 h. The crude enzyme was then used to hydrolyze corn stover. For T. reesei OE-cip1-1 and OE-cip1-2, the hydrolysis efficiency increased by 25.0 and 28.6% with a solid loading of 5% at 2 h, respectively. Simultaneously, 85.5 and 85.2 g/L glucose were released using a cellulase cocktail at high solid loading (20%), and these glucose release rates were significantly greater than that of T. reesei Rut C30 cellulase (77.4 g/L) at 120 h. Furthermore, scanning electron microscopy (SEM) and X-ray diffraction (XRD) showed that the enhanced hydrolysis efficiency was primarily triggered by the decrease in the crystallinity of lignocellulose, and the fiber structure had varying degrees of loosening and disintegration.

Effect of Different Pretreatment of Birch Sawdust on the Production of Active Polysaccharides by Inonotus obliquus Under Submerged Fermentation and Its Structural Mechanism.

This study examined the effects of different pretreatments of birch sawdust on the production and activity of polysaccharides by Inonotus obliquus, and in order to explore the mechanism, structural characterization and analysis were carried out. The result clearly indicated that alkali treatment, ozone treatment, and alkali combined with ozone treatment of birch sawdust could be all helpful for the production of active polysaccharide by I. obliquus. Among four pretreatment groups, birch sawdust treated with alkali showed the highest increase in the exo-polysaccharide content (39.90%) and the inhibition rate of α-glucosidase (80.78%) within 11 days by the mycelium of I. obliquus through deep fermentation, in comparison to water-washed birch sawdust. Through a single-factor analysis and orthogonal experimental design, the optimum alkali treatment condition was as follows: NaOH concentration 1%, temperature 60 °C, and time 3 h. Moreover, the structural characteristics of pretreated birch sawdust with the optimum alkali treatment condition before and after fermentation by the mycelium of I. obliquus was performed by Fourier transform infrared spectroscopy, X-ray diffraction, and scanning electronic microscopy. The results showed that alkali treatment destroyed the lignin structure of birch sawdust, exposed the cellulose in the amorphous area, reduced the crystallinity of lignocellulose, and damaged the surface structure of birch sawdust, which had a further damage and a greater degradation degree of birch sawdust after fermentation, indicating that alkali pretreatment was beneficial for utilization of birch sawdust by I. obliquus.

Study of delignification process and crystallinity index on lignocellulose components of corn cob in different pretreatments: a combination of pretreatment (ionic choline acetate and NaOH) and NaOH pretreatment

This study studied the effect of combination pretreatment (ionic liquid choline acetate and NaOH) and pretreatment of NaOH in the process of delignification and lignocellulose crystallinity index. This process was carried out to obtain a good lignocellulose biomass, which has a low lignin content and a high crystallinity index to be prepared as a raw material for making bioethanol. Low lignin content and high index of crystallinity can influence the success of the enzymatic hydrolysis process and of course bioethanol production. In the combination of pretreatment, the highest lignin removal was found from process with 5% of NaOH as much as 41.63% but the cristalinity index was found from proces with 3% of NaOH as much as 46.33. While, In the NaOH pretreatment, the highest lignin removal and cristalinity index was found from process with 5% of NaOH as much as 7.63% and 3.87, respectively.

Laccase pretreatment of wheat straw: effects of the physicochemical characteristics and the kinetics of enzymatic hydrolysis

BackgroundWheat straw, the most abundant lignocellulosic biomass in China, is rich in cellulose that can be hydrolyzed and then converted into biofuels, such as bioethanol and biohydrogen. However, the accessibility of cellulose and the enzyme activity are greatly reduced in the presence of lignin. This significantly increases the enzyme cost in the saccharification, which hampers industrial production of lignocellulosic biofuels. In this study, a laccase treatment system mediated by 1-hydroxybenzotriazole was employed to modify and degrade lignin to enhance subsequent enzymatic saccharification of wheat straw. A kinetic model considering enzyme adsorption on lignin was proposed to estimate the saccharification performance.ResultsFourier transform infrared spectroscopy (FTIR) analyses showed that the peak intensity of lignin structure characteristics significantly changed after laccase-mediated system (LMS) treatment. 2D-nuclear magnetic resonance (NMR) analyses indicated that the aromatic ether bonds were cleaved and that guaiacyl (G) was oxidized after LMS treatment. X-ray diffraction (XRD) analyses suggested that the crystallinity of lignocellulose increased due to the partial degradation of lignin. As a result, the nonproductive adsorption of the enzyme on lignin was reduced by 28%, while the reducing sugar yield increased by 26%. A semi-empirical kinetic model was used to estimate the reducing sugar yield, the initial hydrolysis rate (KM) and deactivation rate coefficient (α) of LMS-pretreated wheat straw were 0.157 (h−1) and 0.214 (h−1), respectively. The model showed high accuracy (predicting error < 10%) for describing the behavior of laccase-treated wheat straw hydrolysis when the solid loading is < 5%.ConclusionsThe adsorption ability of the enzyme to lignin was reduced after LMS pretreatment. Physicochemical analyses showed that the chemical groups of lignin and lignocellulose were changed, with the crystallinity of the lignocellulose increasing after LMS treatment. A semi-empirical kinetic model was proposed to estimate the reducing sugar yield, which showed high accuracy for predicting the hydrolysis performance of laccase-treated wheat straw.

Valorization of Lignocellulosic Waste (Crotalaria juncea) Using Alkaline Peroxide Pretreatment under Different Process Conditions: An Optimization Study on Separation of Lignin, Cellulose, and Hemicellulose

ABSTRACTLignocellulose materials, essentially consisting of lignin, cellulose and hemicelluloses, are abundant sources of fermentable sugars. The bast fiber of Crotalaria juncea (Sunn hemp), a native cover crop of India, was used as feedstock for this study. The primary objective of this study was to add value to the waste C. juncea bast fiber. The same was achieved by pretreating the waste fiber using alkaline peroxide solution at various process conditions. The optimal process condition for alkaline peroxide pretreatment was identified for the fiber to pretreatment solution (S/S) ratio of 1/40 at 50°C for 5h with respect to all response variables (lignin removal, hemicellulose recovery, recovery of solid pretreated material, and crystallinity of lignocellulose). Three-way ANOVA results revealed that S/S ratio had no significant effect; whereas, both temperature and time, and the combination of parameters, exhibited significant effect on response variables. The characteristic peaks associated with lignin and cellulose demonstrated a higher amount of lignin removal and increased cellulose content with elevated treatment time. Autoclave assisted pretreatment proved to be inefficient due to removal of lower amount of lignin in addition to higher hemicellulose degradation. On the other hand, pretreatment using ultrasound was found to be most effective in removing lignin, liberating hemicelluloses along with diminition in cellulose crystallinity.

Ammonium oxalate-extractable uronic acids positively affect biomass enzymatic digestibility by reducing lignocellulose crystallinity in Miscanthus

Based on systems biology analyses of total 179 representative Miscanthus accessions, ammonium oxalate (AO)-extractable uronic acids could either positively affect biomass digestibility or negatively alter lignocellulose crystallinity at p<0.01 or 0.05. Comparative analysis of four typical pairs of Miscanthus samples indicated that the AO-extractable uronic acids, other than hexoses and pentoses, play a predominant role in biomass enzymatic saccharification upon various chemical pretreatments, consistent with observations of strong cell tissue destruction in situ and rough biomass residue surface in vitro in the unique Msa24 sample rich in uronic acids. Notably, AO-extraction of uronic acids could significantly increase lignocellulose CrI at p<0.05, indicating that uronic acids-rich polymers may have the interactions with β-1,4-glucan chains that reduce cellulose crystallinity. It has also suggested that increasing of uronic acids should be a useful approach for enhancing biomass enzymatic digestibility in Miscanthus and beyond.

Consolidated bioprocessing performance of Thermoanaerobacterium thermosaccharolyticum M18 on fungal pretreated cornstalk for enhanced hydrogen production.

BackgroundBiological hydrogen production from lignocellulosic biomass shows great potential as a promising alternative to conventional hydrogen production methods, such as electrolysis of water and coal gasification. Currently, most researches on biohydrogen production from lignocellulose concentrate on consolidated bioprocessing, which has the advantages of simpler operation and lower cost over processes featuring dedicated cellulase production. However, the recalcitrance of the lignin structure induces a low cellulase activity, making the carbohydrates in the hetero-matrix more unapproachable. Pretreatment of lignocellulosic biomass is consequently an extremely important step in the commercialization of biohydrogen, and for massive realization of lignocellulosic biomass as alternative fuel feedstock. Thus, development of a pretreatment method which is cost efficient, environmentally benign, and highly efficient for enhanced consolidated bioprocessing of lignocellulosic biomass to hydrogen is essential.ResultsIn this research, fungal pretreatment was adopted for enhanced hydrogen production by consolidated bioprocessing performance. To confirm the fungal pretreatment efficiency, two typical thermochemical pretreatments were also compared side by side. Results showed that the fungal pretreatment was superior to the other pretreatments in terms of high lignin reduction of up to 35.3% with least holocellulose loss (the value was only 9.5%). Microscopic structure observation combined with Fourier transform infrared spectroscopy (FTIR) analysis further demonstrated that the lignin and crystallinity of lignocellulose were decreased with better holocellulose reservation. Upon fungal pretreatment, the hydrogen yield and hydrogen production rate were 6.8 mmol H2 g-1 pretreated substrate and 0.89 mmol L-1 h-1, respectively, which were 2.9 and 4 times higher than the values obtained for the untreated sample.ConclusionsResults revealed that although all pretreatments could contribute to the enhancement of hydrogen production from cornstalk, fungal pretreatment proved to be the optimal method. It is apparent that besides high hydrogen production efficiency, fungal pretreatment also offered several advantages over other pretreatments such as being environmentally benign and energy efficient. This pretreatment method thus has great potential for application in consolidated bioprocessing performance of hydrogen production.

High-level hemicellulosic arabinose predominately affects lignocellulose crystallinity for genetically enhancing both plant lodging resistance and biomass enzymatic digestibility in rice mutants.

Rice is a major food crop with enormous biomass residue for biofuels. As plant cell wall recalcitrance basically decides a costly biomass process, genetic modification of plant cell walls has been regarded as a promising solution. However, due to structural complexity and functional diversity of plant cell walls, it becomes essential to identify the key factors of cell wall modifications that could not much alter plant growth, but cause an enhancement in biomass enzymatic digestibility. To address this issue, we performed systems biology analyses of a total of 36 distinct cell wall mutants of rice. As a result, cellulose crystallinity (CrI) was examined to be the key factor that negatively determines either the biomass enzymatic saccharification upon various chemical pretreatments or the plant lodging resistance, an integrated agronomic trait in plant growth and grain production. Notably, hemicellulosic arabinose (Ara) was detected to be the major factor that negatively affects cellulose CrI probably through its interlinking with β-1,4-glucans. In addition, lignin and G monomer also exhibited the positive impact on biomass digestion and lodging resistance. Further characterization of two elite mutants, Osfc17 and Osfc30, showing normal plant growth and high biomass enzymatic digestion in situ and in vitro, revealed the multiple GH9B candidate genes for reducing cellulose CrI and XAT genes for increasing hemicellulosic Ara level. Hence, the results have suggested the potential cell wall modifications for enhancing both biomass enzymatic digestibility and plant lodging resistance by synchronically overexpressing GH9B and XAT genes in rice.

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.

Sugar-rich sweet sorghum is distinctively affected by wall polymer features for biomass digestibility and ethanol fermentation in bagasse

Sweet sorghum has been regarded as a typical species for rich soluble-sugar and high lignocellulose residues, but their effects on biomass digestibility remain unclear. In this study, we examined total 63 representative sweet sorghum accessions that displayed a varied sugar level at stalk and diverse cell wall composition at bagasse. Correlative analysis showed that both soluble-sugar and dry-bagasse could not significantly affect lignocellulose saccharification under chemical pretreatments. Comparative analyses of five typical pairs of samples indicated that DP of crystalline cellulose and arabinose substitution degree of non-KOH-extractable hemicelluloses distinctively affected lignocellulose crystallinity for high biomass digestibility. By comparison, lignin could not alter lignocellulose crystallinity, but the KOH-extractable G-monomer predominately determined lignin negative impacts on biomass digestions, and the G-levels released from pretreatments significantly inhibited yeast fermentation. The results also suggested potential genetic approaches for enhancing soluble-sugar level and lignocellulose digestibility and reducing ethanol conversion inhibition in sweet sorghum.

Exogenously applied 24-epi brassinolide reduces lignification and alters cell wall carbohydrate biosynthesis in the secondary xylem of Liriodendron tulipifera

The roles of brassinosteroids (BRs) in vasculature development have been implicated based on an analysis of Arabidopsis BR mutants and suspension cells of Zinnia elegans. However, the effects of BRs in vascular development of a woody species have not been demonstrated. In this study, 24-epi brassinolide (BL) was applied to the vascular cambium of a vertical stem of a 2-year-old Liriodendron, and the resulting chemical and anatomical phenotypes were characterized to uncover the roles of BRs in secondary xylem formation of a woody species. The growth in xylary cells was clearly promoted when treated with BL. Statistical analysis indicated that the length of both types of xylary cells (fiber and vessel elements) increased significantly after BL application. Histochemical analysis demonstrated that BL-induced growth promotion involved the acceleration of cell division and cell elongation. Histochemical and expression analysis of several lignin biosynthetic genes indicated that most genes in the phenylpropanoid pathway were significantly down-regulated in BL-treated stems compared to that in control stems. Chemical analysis of secondary xylem demonstrated that BL treatment induced significant modification in the cell wall carbohydrates, including biosynthesis of hemicellulose and cellulose. Lignocellulose crystallinity decreased significantly, and the hemicellulose composition changed with significant increases in galactan and arabinan. Thus, BL has regulatory roles in the biosynthesis and modification of secondary cell wall components and cell wall assembly during secondary xylem development in woody plants.

Global Identification of Multiple OsGH9 Family Members and Their Involvement in Cellulose Crystallinity Modification in Rice

Plant glycoside hydrolase family 9 (GH9) comprises typical endo-β-1,4-glucanase (EGases, EC3.2.1.4). Although GH9A (KORRIGAN) family genes have been reported to be involved in cellulose biosynthesis in plants, much remains unknown about other GH9 subclasses. In this study, we observed a global gene co-expression profiling and conducted a correlation analysis between OsGH9 and OsCESA among 66 tissues covering most periods of life cycles in 2 rice varieties. Our results showed that OsGH9A3 and B5 possessed an extremely high co-expression with OsCESA1, 3, and 8 typical for cellulose biosynthesis in rice. Using two distinct rice non-GH9 mutants and wild type, we performed integrative analysis of gene expression level by qRT-PCR, cellulase activities in situ and in vitro, and lignocellulose crystallinity index (CrI) in four internodes of stem tissues. For the first time, OsGH9B1, 3, and 16 were characterized with the potential role in lignocellulose crystallinity alteration in rice, whereas OsGH9A3 and B5 were suggested for cellulose biosynthesis. In addition, phylogenetic analysis and gene co-expression comparison revealed GH9 function similarity in Arabidopsis and rice. Hence, the data can provide insights into GH9 function in plants and offer the potential strategy for genetic manipulation of plant cell wall using the five aforementioned novel OsGH9 genes.

Accelerating the degradation of green plant waste with chemical decomposition agents

Degradation of green plant waste is often difficult, and excess maturity times are typically required. In this study, we used lignin, cellulose and hemicellulose assays; scanning electron microscopy; infrared spectrum analysis and X-ray diffraction analysis to investigate the effects of chemical decomposition agents on the lignocellulose content of green plant waste, its structure and major functional groups and the mechanism of accelerated degradation. Our results showed that adding chemical decomposition agents to Ficus microcarpa var. pusillifolia sawdust reduced the contents of lignin by 0.53%-11.48% and the contents of cellulose by 2.86%-7.71%, and increased the contents of hemicellulose by 2.92%-33.63% after 24 h. With increasing quantities of alkaline residue and sodium lignosulphonate, the lignin content decreased. Scanning electron microscopy showed that, after F. microcarpa var. pusillifolia sawdust was treated with chemical decomposition agents, lignocellulose tube wall thickness increased significantlyIncreases of 29.41%, 3.53% and 34.71% were observed after treatment with NaOH, alkaline residue and sodium lignosulphonate, respectively. Infrared spectroscopy showed that CO and aromatic skeleton stretching absorption peaks were weakened and the C-H vibrational absorption peak from out-of-plane in positions 2 and 6 (S units) (890-900 cm(-1)) was strengthened after F. microcarpa var. pusillifolia sawdust was treated with chemical decomposition agents, indicating a reduction in lignin content. Several absorption peaks [i.e., C-H deformations (asymmetry in methyl groups, -CH(3)- and -CH(2)-) (1450-1460 cm(-1)); Aliphatic C-H stretching in methyl and phenol OH (1370-1380 cm(-1)); CO stretching (cellulose and hemicellulose) (1040-1060 cm(-1))] that indicate the presence of a chemical bond between lignin and cellulose was reduced, indicating that the chemical bond between lignin and cellulose had been partially broken. X-ray diffraction analysis showed that NaOH, alkaline residue and sodium lignosulphonate can reduce the relative crystallinity of lignocellulose in F. microcarpa var. pusillifolia by 2.64%, 13.24%, 12.44%, respectively. The C-H vibrational absorption peak from out-of-plane in positions 2 and 6 (S units) comes from the vibration of the sugar anomeric carbon. Because lignin is a phenolic, not carbohydrate polymer, the relative absorption intensity of this peak should be stronger at lower lignin contents. Compared to CK, the peak intensities increased in treatments T1, T5 and T9, indicating reduced lignin contents and increased sugar contents after CDA treatment.

Enzymatic hydrolysis of pretreated sugar cane bagasse using Penicillium funiculosum and Trichoderma harzianum cellulases

In this study, we investigated the enzymatic hydrolysis of pretreated sugarcane bagasse using eight different enzymatic blends obtained from concentrated crude enzyme extracts produced by Penicillium funiculosum and Trichoderma harzianum as well as from the extracts in combination with a commercial enzymatic cocktail. The influence of different levels of biomass delignification, degree of crystallinity of lignicellulose, composition of enzymatic activities and BSA on enzymatic hydrolysis yields (HYs) was evaluated. Our X-ray diffraction studies showed that crystallinity of lignocellulose is not a key determinant of its recalcitrance toward enzymatic hydrolysis. In fact, under the experimental conditions of our study, an increase in crystallinity of lignocellulosic samples resulted in increased glucose release by enzymatic hydrolysis. Furthermore, under the same conditions, the addition of BSA had no significant effect on enzymatic hydrolysis. The most efficient enzyme blends were obtained by mixing a commercial enzymatic cocktail with P. funiculosum or T. harzianum cellulase preparations (HYs above 97%) followed by the concentrated extract of P. funiculosum alone (HY=88.5%). Increased hydrolytic efficiencies appeared to correlate with having an adequate level of both β-glucosidase and xylanase activities in the blends.

Recrystallization behavior of cellulose and lignocellulose from Pinus massoniana

In an X-ray diffraction investigation, the recrystallization behavior of ball-milled Chinese southern pine wood was found to differ from that of cellulose. The crystallinity of lignocellulose milled for a very long time and recrystallized by wetting in water did not decrease with increasing milling time as was the case with milled cellulose. In addition, it appeared that the presence of lignin tended to restrict the amorphous cellulose produced by milling from recrystallizing into Cellulose II, whereas it had no influence on the recrystallization into Cellulose I. These results were discussed in the context of the mechanism of the conversion of Cellulose I to Cellulose II.