Biomass Enzymatic Digestibility Research Articles

Determination of Lignin Monomer Contents in Rice Straw Using Visible and Near-infrared Reflectance Spectroscopy

Genetic modification of plant lignin composition is an important strategy for improving biomass enzymatic digestibility without sacrificing the normal growth of the plant. However, the absence of fast and convenient methods for rapid determination of lignin composition has impeded corresponding research. Near-infrared reflectance spectroscopy (NIRS) analysis has potential as a solution for this dilemma, while the NIRS measurement for expediently assaying lignin composition in rice straws is still lacking. In this study, visible and near-infrared reflectance spectroscopy (VIS/NIRS) and modified partial least squares (MPLS) method were combined to develop calibration models for predicting the lignin monomer contents in a diverse rice population. Four optimal equations for predicting the content of p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) lignin units and their total amount (H + S + G) were generated with acceptable determination coefficients for calibration (0.85 to 0.93), cross-validation (0.75 to 0.88), and external validation (0.82 to 0.88), and the ratio performance deviation (RPD, 2 to 3.01). This study was the first to demonstrate that VIS/NIRS could give a sufficiently accurate prediction of lignin monomer contents in rice and could be applied for rapid assessments of large-scale rice straw samples.

Genetic loci simultaneously controlling lignin monomers and biomass digestibility of rice straw

Lignin content and composition are crucial factors affecting biomass digestibility. Exploring the genetic loci simultaneously affecting lignin-relevant traits and biomass digestibility is a precondition for lignin genetic manipulation towards energy crop breeding. In this study, a high-throughput platform was employed to assay the lignin content, lignin composition and biomass enzymatic digestibility of a rice recombinant inbred line population. Correlation analysis indicated that the absolute content of lignin monomers rather than lignin content had negative effects on biomass saccharification, whereas the relative content of p-hydroxyphenyl unit and the molar ratio of p-hydroxyphenyl unit to guaiacyl unit exhibited positive roles. Eight QTL clusters were identified and four of them affecting both lignin composition and biomass digestibility. The additive effects of clustered QTL revealed consistent relationships between lignin-relevant traits and biomass digestibility. Pyramiding rice lines containing the above four positive alleles for increasing biomass digestibility were selected and showed comparable lignin content, decreased syringyl or guaiacyl unit and increased molar percentage of p-hydroxyphenyl unit, the molar ratio of p-hydroxyphenyl unit to guaiacyl unit and sugar releases. More importantly, the lodging resistance and eating/cooking quality of pyramiding lines were not sacrificed, indicating the QTL information could be applied to select desirable energy rice lines.

A precise and consistent assay for major wall polymer features that distinctively determine biomass saccharification in transgenic rice by near-infrared spectroscopy

BackgroundThe genetic modification of plant cell walls has been considered to reduce lignocellulose recalcitrance in bioenergy crops. As a result, it is important to develop a precise and rapid assay for the major wall polymer features that affect biomass saccharification in a large population of transgenic plants. In this study, we collected a total of 246 transgenic rice plants that, respectively, over-expressed and RNAi silenced 12 genes of the OsGH9 and OsGH10 family that are closely associated with cellulose and hemicellulose modification. We examined the wall polymer features and biomass saccharification among 246 transgenic plants and one wild-type plant. The samples presented a normal distribution applicable for statistical analysis and NIRS modeling.ResultsAmong the 246 transgenic rice plants, we determined largely varied wall polymer features and the biomass enzymatic saccharification after alkali pretreatment in rice straws, particularly for the fermentable hexoses, ranging from 52.8 to 95.9%. Correlation analysis indicated that crystalline cellulose and lignin levels negatively affected the hexose and total sugar yields released from pretreatment and enzymatic hydrolysis in the transgenic rice plants, whereas the arabinose levels and arabinose substitution degree (reverse xylose/arabinose ratio) exhibited positive impacts on the hexose and total sugars yields. Notably, near-infrared spectroscopy (NIRS) was applied to obtain ten equations for predicting biomass enzymatic saccharification and seven equations for distinguishing major wall polymer features. Most of the equations exhibited high R2/R2cv/R2ev and RPD values for a perfect prediction capacity.ConclusionsDue to large generated populations of transgenic rice lines, this study has not only examined the key wall polymer features that distinctively affect biomass enzymatic saccharification in rice but has also established optimal NIRS models for a rapid and precise screening of major wall polymer features and lignocellulose saccharification in biomass samples. Importantly, this study has briefly explored the potential roles of a total of 12 OsGH9 and OsGH10 genes in cellulose and hemicellulose modification and cell wall remodeling in transgenic rice lines. Hence, it provides a strategy for genetic modification of plant cell walls by expressing the desired OsGH9 and OsGH10 genes that could greatly improve biomass enzymatic digestibility in rice.

Steam-exploded biomass saccharification is predominately affected by lignocellulose porosity and largely enhanced by Tween-80 in Miscanthus

In this study, total ten Miscanthus accessions exhibited diverse cell wall compositions, leading to largely varied hexoses yields at 17%–40% (% cellulose) released from direct enzymatic hydrolysis of steam-exploded (SE) residues. Further supplied with 2% Tween-80 into the enzymatic digestion, the Mis7 accession showed the higher hexose yield by 14.8-fold than that of raw material, whereas the Mis10 had the highest hexoses yield at 77% among ten Miscanthus accessions. Significantly, this study identified four wall polymer features that negatively affect biomass saccharification as p<0.05 or 0.01 in the SE residues, including cellulose DP, Xyl and Ara of hemicellulose, and S-monomer of lignin. Based on Simons’ stain, the SE porosity (defined by DY/DB) was examined to be the unique positive factor on biomass enzymatic digestion. Hence, this study provides the potential strategy to enhance biomass saccharification using optimal biomass process technology and related genetic breeding in Miscanthus and beyond.

Gene Profiling of Plant Cell Wall Biosynthesis for Genetic Enhancing Biomass Enzymatic Saccharification in Cereal Crops

Cereal crops, including rice, wheat, corn, and sweet sorghum, produce major food grains for humans and generate huge amount biomass for biofuels production. Due to lignocellulose recalcitrance, however, the most biomass residues of cereal crops are not well used for biofuel application, and genetic modification of plant cell walls has thus proposed as a promising solution for enhancing biomass enzymatic saccharification. In the present review, we described plant cell wall compositions and wall polymer features in four cereal crops, and presented their major wall factors that significantly affect biomass enzymatic digestibility under various pretreatments including cellulose crystallinity, arabinose substitution degree of hemicellulose and three monomers proportions of lignin. Furthermore, using globe genomic sequence database and gene expression information, we compared all candidate genes that are involved in cell wall biosynthesis in the four cereal crops, and discussed the potential cell wall modifications for increasing both biomass yields and lignocellulose enzymatic saccharification in the cereal crops by using desire genes and selecting appropriate genetic manipulation approaches.

Tween-80 is effective for enhancing steam-exploded biomass enzymatic saccharification and ethanol production by specifically lessening cellulase absorption with lignin in common reed

In this study, eight physical and chemical pretreatments were compared in terms of their enhancements on biomass enzymatic saccharification in reed. Despite 8% NaOH pretreatment could result in 100% biomass enzymatic digestion while co-supplied with 1% Tween-80, it only produced bioethanol at 10% (% dry matter). By comparison, 10% CaO pretreatment with Tween-80 is a relatively low-cost biomass conversion with ethanol yield at 12%. Notably, the steam-explosion pretreatment with 1% Tween-80 could cause a complete biomass enzymatic hydrolysis with bioethanol yield at 17%. The sequential 5% CaO pretreatment with the steam-exploded residues could lead to the highest ethanol yield at 19% with an almost complete sugar–ethanol conversion rate. Due to much low-DP cellulose and less noncellulosic polymers (lignin, hemicelluloses) that increase biomass surfaces, the steam-exploded residues were specifically effective for Tween-80 either to block lignin absorbing with cellulases or to disassociate hemicelluloses, leading to an efficient lignocellulose enzymatic digestion. Compared with previously reported pretreatments in other C4-grasses (Miscanthus, corn, sweet sorghum, switchgrass), to our knowledge, this study has therefore provided three more applicable approaches for high ethanol production with relatively low cost, less contaminate release and efficient biomass conversion rates in reed.

G-lignin and hemicellulosic monosaccharides distinctively affect biomass digestibility in rapeseed

In this study, total 19 straw samples from four Brassica species were determined with a diverse cell wall composition and varied biomass enzymatic digestibility under sulfuric acid or lime pretreatment. Correlation analysis was then performed to detect effects of cell wall compositions and wall polymer features (cellulose crystallinity, hemicellulosic monosaccharides and lignin monomers) on rapeseeds biomass digestibility. As a result, coniferyl alcohol (G-lignin) showed a strongly negative effect on biomass saccharification, whereas hemicellulosic monosaccharides (fucose, galactose, arabinose and rhamnose) were positive factors on lignocellulose digestions. Notably, chemical analyses of four typical pairs of samples indicated that hemicellulosic monosaccharides and G-lignin may coordinately influence biomass digestibility in rapeseeds. In addition, Brassica napus with lower lignin content exhibited more efficiency on both biomass enzymatic saccharification and ethanol production, compared with Brassica junjea. Hence, this study has at first time provided a genetic strategy on cell wall modification towards bioenergy rapeseed breeding.

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.

Silica distinctively affects cell wall features and lignocellulosic saccharification with large enhancement on biomass production in rice

Rice is a typical silicon-accumulating crop with enormous biomass residues for biofuels. Silica is a cell wall component, but its effect on the plant cell wall and biomass production remains largely unknown. In this study, a systems biology approach was performed using 42 distinct rice cell wall mutants. We found that silica levels are significantly positively correlated with three major wall polymers, indicating that silica is associated with the cell wall network. Silicon-supplied hydroculture analysis demonstrated that silica distinctively affects cell wall composition and major wall polymer features, including cellulose crystallinity (CrI), arabinose substitution degree (reverse Xyl/Ara) of xylans, and sinapyl alcohol (S) proportion in three typical rice mutants. Notably, the silicon supplement exhibited dual effects on biomass enzymatic digestibility in the mutant and wild type (NPB) after pre-treatments with 1% NaOH and 1% H2SO4. In addition, silicon supply largely enhanced plant height, mechanical strength and straw biomass production, suggesting that silica rescues mutant growth defects. Hence, this study provides potential approaches for silicon applications in biomass process and bioenergy rice breeding.

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.

The minor wall-networks between monolignols and interlinked-phenolics predominantly affect biomass enzymatic digestibility in Miscanthus.

Plant lignin is one of the major wall components that greatly contribute to biomass recalcitrance for biofuel production. In this study, total 79 representative Miscanthus germplasms were determined with wide biomass digestibility and diverse monolignol composition. Integrative analyses indicated that three major monolignols (S, G, H) and S/G ratio could account for lignin negative influence on biomass digestibility upon NaOH and H2SO4 pretreatments. Notably, the biomass enzymatic digestions were predominately affected by the non-KOH-extractable lignin and interlinked-phenolics, other than the KOH-extractable ones that cover 80% of total lignin. Furthermore, a positive correlation was found between the monolignols and phenolics at p<0.05 level in the non-KOH-extractable only, suggesting their tight association to form the minor wall-networks against cellulases accessibility. The results indicated that the non-KOH-extractable lignin-complex should be the target either for cost-effective biomass pretreatments or for relatively simply genetic modification of plant cell walls in Miscanthus.

Comparison of lab, pilot, and industrial scale low consistency mechanical refining for improvements in enzymatic digestibility of pretreated hardwood

Mechanical refining has been shown to improve biomass enzymatic digestibility. In this study industrial high-yield sodium carbonate hardwood pulp was subjected to lab, pilot and industrial refining to determine if the mechanical refining improves the enzymatic hydrolysis sugar conversion efficiency differently at different refining scales. Lab, pilot and industrial refining increased the biomass digestibility for lignocellulosic biomass relative to the unrefined material. The sugar conversion was increased from 36% to 65% at 5FPU/g of biomass with industrial refining at 67.0kWh/t, which was more energy efficient than lab and pilot scale refining. There is a maximum in the sugar conversion with respect to the amount of refining energy. Water retention value is a good predictor of improvements in sugar conversion for a given fiber source and composition. Improvements in biomass digestibility with refining due to lab, pilot plant and industrial refining were similar with respect to water retention value.

Biomass digestibility is predominantly affected by three factors of wall polymer features distinctive in wheat accessions and rice mutants

BackgroundWheat and rice are important food crops with enormous biomass residues for biofuels. However, lignocellulosic recalcitrance becomes a crucial factor on biomass process. Plant cell walls greatly determine biomass recalcitrance, thus it is essential to identify their key factors on lignocellulose saccharification. Despite it has been reported about cell wall factors on biomass digestions, little is known in wheat and rice. In this study, we analyzed nine typical pairs of wheat and rice samples that exhibited distinct cell wall compositions, and identified three major factors of wall polymer features that affected biomass digestibility.ResultsBased on cell wall compositions, ten wheat accessions and three rice mutants were classified into three distinct groups each with three typical pairs. In terms of group I that displayed single wall polymer alternations in wheat, we found that three wall polymer levels (cellulose, hemicelluloses and lignin) each had a negative effect on biomass digestibility at similar rates under pretreatments of NaOH and H2SO4 with three concentrations. However, analysis of six pairs of wheat and rice samples in groups II and III that each exhibited a similar cell wall composition, indicated that three wall polymer levels were not the major factors on biomass saccharification. Furthermore, in-depth detection of the wall polymer features distinctive in rice mutants, demonstrated that biomass digestibility was remarkably affected either negatively by cellulose crystallinity (CrI) of raw biomass materials, or positively by both Ara substitution degree of non-KOH-extractable hemicelluloses (reverse Xyl/Ara) and p-coumaryl alcohol relative proportion of KOH-extractable lignin (H/G). Correlation analysis indicated that Ara substitution degree and H/G ratio negatively affected cellulose crystallinity for high biomass enzymatic digestion. It was also suggested to determine whether Ara and H monomer have an interlinking with cellulose chains in the future.ConclusionsUsing nine typical pairs of wheat and rice samples having distinct cell wall compositions and wide biomass saccharification, Ara substitution degree and monolignin H proportion have been revealed to be the dominant factors positively determining biomass digestibility upon various chemical pretreatments. The results demonstrated the potential of genetic modification of plant cell walls for high biomass saccharification in bioenergy crops.

Three lignocellulose features that distinctively affect biomass enzymatic digestibility under NaOH and H2SO4 pretreatments in Miscanthus

In this study, total 80 typical Miscanthus accessions were examined with diverse lignocellulose features, including cellulose crystallinity (CrI), degree of polymerization (DP), and mole number (MN). Correlation analysis revealed that the crude cellulose CrI and MN, as well as crystalline cellulose DP, displayed significantly negative influence on biomass enzymatic digestibility under pretreatments with NaOH or H2SO4 at three concentrations. By contrast, the comparative analysis of two Miscanthus samples with similar cellulose contents showed that crude cellulose DP and crystalline cellulose MN were positive factors on biomass saccharification, indicating cross effects among the cellulose levels and the three cellulose features. The results can provide insights into mechanism of the lignocellulose enzymatic digestion, and also suggest potential approaches for genetic engineering of bioenergy crops.

A rapid and consistent near infrared spectroscopic assay for biomass enzymatic digestibility upon various physical and chemical pretreatments in Miscanthus

Near infrared spectroscopy (NIRS) has been broadly applied as a quick assay for biological component and property analysis. However, NIRS remains unavailable for in-depth analysis of biomass digestibility in plants. In this study, NIRS was used to determine biomass enzymatic digestibility using 199 Miscanthus samples, which represents a rich germplasm resource and provides for a stable calibration model. The intensive evaluation indicates that the calibration and validation sets are comparable. Using the modified partial least squares method, seven optimal equations were generated with high determination coefficient on calibration (R2) at 0.75–0.89, cross-validation (R2cv) at 0.69–0.87, and the ratio performance deviation (RPD) at 1.80–2.74, which provide multiple options for NIRS prediction of biomass digestibility under different pretreatments. As biomass digestibility is a crucial parameter for biofuel processing, NIRS is a powerful tool for the high-throughput screening of biomass samples in plants.

Correlation between anatomical characteristics of ethanol organosolv pretreated Buddleja davidii and its enzymatic conversion to glucose

Buddleja davidii is a unique biomass that has many attractive agroenergy features, especially its wide range of growth habitat. The anatomical characteristics of B. davidii were investigated before and after ethanol organosolv pretreatment (one of the leading pretreatment technologies) in order to further understand the alterations that occur to the cellular structure of the biomass which can then be correlated with its enzymatic digestibility. Results showed that the ethanol organosolv pretreatment of B. davidii selectively removes lignin from the middle lamella (ML), which does not significantly disrupt the crystalline structure of cellulose. The removal of ML lignin is a major factor in enhancing enzymatic cellulose-to-glucose hydrolysis. The pretreatment also causes cell deformation, resulting in cracks and breaks in the cell wall. These observations, together with characterization analysis of the cell wall polymer material, lend support to the hypothesis that the physical distribution of lignin in the biomass matrix is an important structural feature affecting biomass enzymatic digestibility.

Structural features affecting biomass enzymatic digestibility

The rate and extent of enzymatic hydrolysis of lignocellulosic biomass highly depend on enzyme loadings, hydrolysis periods, and structural features resulting from pretreatments. Furthermore, the influence of one structural feature on biomass digestibility varies with the changes in enzyme loading, hydrolysis period and other structural features as well. In this paper, the effects of lignin content, acetyl content, and biomass crystallinity on the 1-, 6-, and 72-h digestibilities with various enzyme loadings were investigated. To eliminate the cross effects among structural features, selective pretreatment techniques were employed to vary one particular structural feature during a pretreatment, while the other two structural features remained unchanged. The digestibility results showed that lignin content and biomass crystallinity dominated digestibility whereas acetyl content had a lesser effect. Lignin removal greatly enhanced the ultimate hydrolysis extent. Crystallinity reduction, however, tremendously increased the initial hydrolysis rate and reduced the hydrolysis time or the amount of enzyme required to attain high digestibility. To some extent, the effects of structural features on digestibility were interrelated. At short hydrolysis periods, lignin content was not important to digestibility when crystallinity was low. Similarly, at long hydrolysis periods, crystallinity was not important to digestibility when lignin content was low.