Adsorption of Microbial β-Amylase on Starch

The effect of lime pretreatment of brown midrib sorghums on enzymatic saccharification was investigated. Under most of the pretreatment conditions, the saccharification yields of bmrs were higher than those of the normal counterparts. This result suggests that bmr is useful to reduce pretreatment costs, because the amount of lime necessary for the pretreatment of biomass can reduced by using bmr mutants.     

RT-CaCCO process: an improved CaCCO process for rice straw by its incorporation with a step of lime pretreatment at room temperature

We improved the CaCCO process for rice straw by its incorporation with a step of lime pretreatment at room temperature (RT). We firstly optimized the RT-lime pretreatment for the lignocellulosic part. When the ratio of lime/dry-biomass was 0.2 (w/w), the RT lime-pretreatment for 7-d resulted in an effect on the enzymatic saccharification of cellulose and xylan equivalent to that of the pretreatment at 120°C for 1h. Sucrose, starch and β-1,3-1,4-glucan, which could be often detected in rice straw, were mostly stable under the RT-lime pretreatment condition. Then, the pretreatment condition in the conventional CaCCO process was modified by the adaptation of the optimized RT lime-pretreatment, resulting in significantly better carbohydrate recoveries via enzymatic saccharification than those of the CaCCO process (120°C for 1 h). Thus, the improved CaCCO process (the RT-CaCCO process) could preserve/pretreat the feedstock at RT in a wet form with minimum loss of carbohydrates.     

Characterization of novel Brown midrib 6 mutations affecting lignin biosynthesis in sorghum

The presence of lignin reduces the quality of lignocellulosic biomass for forage materials and feedstock for biofuels. In C4 grasses,the brown midrib phenotype has been linked to mutations to genes in the monolignol biosynthesis pathway. For example,the Bmr6 gene in sorghum(Sorghum bicolor) has been previously shown to encode cinnamyl alcohol dehydrogenase(CAD),which catalyzes the final step of the monolignol biosynthesis pathway. Mutations in this gene have been shown to reduce the abundance of lignin,enhance digestibility,and improve saccharification efficiencies and ethanol yields. Nine sorghum lines harboring five different bmr6 alleles were identified in an EMS-mutagenized TILLING population. DNA sequencing of Bmr6 revealed that the majority of the mutations impacted evolutionarily conserved amino acids while three-dimensional structural modeling predicted that all of these alleles interfered with the enzyme's ability to bind with its NADPH cofactor. All of the new alleles reduced in vitro CAD activity levels and enhanced glucose yields following saccharification. Further,many of these lines were associated with higher reductions in acid detergent lignin compared to lines harboring the previously characterized bmr6-ref allele. These bmr6 lines represent new breeding tools for manipulating biomass composition to enhance forage and feedstock quality.     

Identification and Characterization of Four Missense Mutations in Brown midrib 12 (Bmr12), the Caffeic O-Methyltranferase (COMT) of Sorghum

Modifying lignin content and composition are targets to improve bioenergy crops for cellulosic conversion to biofuels. In sorghum and other C4 grasses, the brown midrib mutants have been shown to reduce lignin content and alter its composition. Bmr12 encodes the sorghum caffeic O-methyltransferase, which catalyzes the penultimate step in monolignol biosynthesis. From an EMS-mutagenized TILLING population, four bmr12 mutants were isolated. DNA sequencing identified the four missense mutations in the Bmr12 coding region, which changed evolutionarily conserved amino acids Ala71Val, Pro150Leu, Gly225Asp, and Gly325Ser. The previously characterized bmr12 mutants all contain premature stop codons. These newly identified mutants, along with the previously characterized bmr12-ref, represent the first allelic series of bmr12 mutants available in the same genetic background. The impacts of these newly identified mutations on protein accumulation, enzyme activity, Klason lignin content, lignin subunit composition, and saccharification yield were determined. Gly225Asp mutant greatly reduced protein accumulation, and Pro150Leu and Gly325Ser greatly impaired enzyme activity compared to wild type (WT). All four mutants significantly reduced Klason lignin content and altered lignin composition resulting in a significantly reduced S/G ratio relative to WT, but the overall impact of these mutations was less severe than bmr12-ref. Except for Gly325Ser, which is a hypomorphic mutant, all mutants increased the saccharification yield relative to WT. These mutants represent new tools to decrease lignin content and S/G ratio, possibly leading toward the ability to tailor lignin content and composition in the bioenergy grass sorghum.     

Allelic Association, Chemical Characterization and Saccharification Properties of brown midrib Mutants of Sorghum (Sorghum bicolor (L.) Moench)

Genetic improvement of biomass crops can significantly reduce the overall cost of biomass-to-ethanol conversion. The conversion of cellulose to monomeric sugar units is affected by lignin content and composition. Sorghum has attracted the attention of the scientific and industrial community as a promising source of biomass for bioenergy due to its great yield potential and tolerance to stresses. The brown midrib (bmr) mutants of sorghum are characterized by brown vascular tissue associated with altered lignin content. Twenty-eight bmr mutants have been identified since the late 1970s, but the allelic relationships have not been fully established, and the function of only one of the Bmr loci has been unequivocally established. In this study, we combined genetic and chemical approaches to establish that there are mutations at least four independent bmr loci, represented by the bmr2, bmr6, bmr12 and bmr19 groups. Since each allelic group presents unique staining characteristics, rapid classification of emerging bmr lines into the existing groups can be achieved using phloroglucinol-HCl as a histochemical stain. In addition, pyrolysis-gas chromatography-mass spectrometry, enabled the characterization of changes in subunit lignin composition in each of the allelic groups, to help predict the genes underlying the mutations. Enzymatic saccharification of stover from plants representing each allelic bmr group demonstrated that lignin changes in lines belonging to the bmr2, bmr6 and bmr12 groups can increase glucose yields, up to 25% compared to wild-type isolines. In order to expedite the selection of the bmr mutant alleles in breeding populations, we have developed molecular markers specific for bmr7 and bmr25, two novel mutant alleles of the gene encoding caffeic acid O-methyl transferase. Based on the results from this study, we propose to rename the bmr mutants in a manner that reflects the number of independent loci.     

Comparative study on enzymatic digestibility of switchgrass varieties and harvests processed by leading pretreatment technologies

Feedstock quality of switchgrass for biofuel production depends on many factors such as morphological types, geographic origins, maturity, environmental and cultivation parameters, and storage. We report variability in compositions and enzymatic digestion efficiencies for three cultivars of switchgrass (Alamo, Dacotah and Shawnee), grown and harvested at different locations and seasons. Saccharification yields of switchgrass processed by different pretreatment technologies (AFEX, dilute sulfuric acid, liquid hot water, lime, and soaking in aqueous ammonia) are compared in regards to switchgrass genotypes and harvest seasons. Despite its higher cellulose content per dry mass, Dacotah switchgrass harvested after wintering consistently gave a lower saccharification yield than the other two varieties harvested in the fall. The recalcitrance of upland cultivars and over-wintered switchgrass may require more severe pretreatment conditions. We discuss the key features of different pretreatment technologies and differences in switchgrass cultivars and harvest seasons on hydrolysis performance for the applied pretreatment methods.     

Molecular characterization of allelic variation in spontaneous brown midrib mutants of sorghum (Sorghum bicolor (L.) Moench)

Modification of lignin composition and content are important to enhance the saccharification potential of lignocellulosic biomass. Brown midrib (bmr) mutants with altered lignin and enhanced glucose yields are a valuable resource for modification of the lignin biosynthetic pathway in sorghum (Sorghum bicolor (L.) Moench). Of the 38 bmr mutants reported in sorghum, some have been classified into four independent groups, namely bmr2, bmr6, bmr12 and bmr19, based on the allelic test, and a few have been characterized at the molecular level. The bmr2, bmr6 and bmr12 groups have mutations that impair 4-coumarate:coenzyme A ligase (4CL), cinnamyl alcohol dehydrogenase (CAD2) and caffeic O-methyltransferase (COMT), respectively. The molecular basis of bmr19 is unknown. In the present study, four spontaneous bmr mutants of sorghum were analyzed for allelic variation at two candidate gene loci. cDNAs of CAD2 and COMT genes were cloned and sequenced from these mutants. Sequence analysis revealed that two of these mutants, IS23789 and IS23253, share a new allele of CAD2. These mutants have a G-to-C transversion at position 3699 of the genomic sequence that leads to glycine-to-arginine (G191R) substitution in the CAD2 protein sequence. This mutation lies in the highly conserved glycine-rich motif ¹⁸⁸G(X)GGV(L)G¹⁹³ that participates in the binding of the pyrophosphate group of NADP⁺ cofactor and hence might impair the activity of CAD2. Phloroglucinol staining of midribs of these mutants also showed a dark wine-red color that is characteristic of the bmr6 group. These two mutants can be distinguished by an intron length polymorphic marker developed based on the COMT gene sequence in this study. Mutant IS23549, which has also been assigned to the bmr6 group, was found to have another new allele with alanine-to-valine (A164V) substitution in CAD2. Alanine-164 is highly conserved among MDR proteins in plants and hence may be necessary for the activity of the enzyme. In mutant IS11861, there was no mutation that led to a change in amino acid in CAD2, while a threonine-to-serine (T302S) substitution was found in COMT. This single nucleotide polymorphism (SNP) at position 2645 in the COMT gene was converted into a cleaved amplified polymorphic sequence marker that can be used for its identification. In addition, additional SNP- and/or indel-based markers were developed, which can be used for exploiting these alleles in the molecular breeding of sorghum for dedicated bioenergy feedstock.     

Enhanced saccharification kinetics of sugarcane bagasse pretreated in 1-butyl-3-methylimidazolium chloride at high temperature and without complete dissolution

Dissolution of bagasse with 1-butyl-3-methylimidazolium chloride at high temperatures (110-160 °C) is investigated as a pretreatment process for saccharification and fermentation based biofuel production. Material balances are reported and used along with enzymatic saccharification data to identify optimum pretreatment conditions (150 °C for 90 min). At all pretreatment temperatures, dissolved and reprecipitated material is enriched in cellulose, has a low crystallinity and the cellulose component is easily and quantitatively hydrolysed (100%, 3h, 15 FPU). At pretreatment temperatures ≤ 150 °C, the undissolved material has only slightly lower crystallinity than the starting. At pretreatment temperatures ≥ 150 °C, the undissolved material has low crystallinity and when combined with the dissolved material has a saccharification rate and extent similar to completely dissolved material. Complete dissolution is not necessary to maximise saccharification efficiency at temperatures ≥ 150 °C.     

Simultaneous saccharification and fermentation of lime-treated biomass

Simultaneous saccharification and fermentation (SSF) was performed on lime-treated switchgrass and corn stover, and oxidatively lime-treated poplar wood to determine their compatibility with Saccharomyces cerevisiae. Cellulose-derived glucose was extensively utilized by the yeast during SSF. The ethanol yields from pretreated switchgrass, pretreated corn stover, and pretreated-and-washed poplar wood were 72%, 62% and 73% of theoretical, respectively, whereas those from -cellulose were 67 to 91% of theoretical. The lower ethanol yields from treated biomass resulted from lower cellulose digestibilities rather than inhibitors produced by the pretreatment. Oxidative lime pretreatment of poplar wood increased the ethanol yield by a factor of 5.6, from 13% (untreated) to 73% (pretreated-and-washed).     

Comparison of Dilute Acid and Sulfite Pretreatment for Enzymatic Saccharification of Earlywood and Latewood of Douglas fir

This study applied dilute acid (DA) and sulfite pretreatment to overcome the recalcitrance of lignocelluloses (SPORL) to deconstruct earlywood and latewood cell walls of Douglas fir for fermentable sugars production through subsequent enzymatic hydrolysis. DA pretreatment removed almost all the hemicelluloses, while SPORL at initial pH?=?4.5 (SP-B) removed significant amount of lignin between 20 and 25 %. But both are not sufficient for effective enzymatic saccharification. SPORL at low initial pH?=?2 (SP-AB) combines the advantage of both DA and SPORL-B to achieve approximately 90 % hemicellulose removal and delignification of 10–20 %. As a result, SP-AB effectively removed recalcitrance and thereby significantly improved enzymatic saccharification compared with DA and SP-B. Results also showed that earlywood with significantly lower density produced less saccharification after DA pretreatment, suggesting that wood density does not contribute to recalcitrance. The thick cell wall of latewood did not limit chemical penetration in pretreatments. The high lignin content of earlywood limited the effectiveness of DA pretreatment for enzymatic saccharification, while hemicellulose limits the effectiveness of high pH pretreatment of SP-B. The higher hemicellulose content in the earlywood and latewood of heartwood reduced saccharification relative to the corresponding earlywood and latewood in the sapwood using DA and SP-AB.     

Enhanced enzymatic hydrolysis of lignocellulosic biomass pretreated by low-pressure steam autohydrolysis

Summary The use of low-pressure steam autohydrolysis in the pretreatment of corn stover and hybrid poplar has been assessed. In terms of yield of prehydrolyzed solids, minimal by-product formation and extent of subsequent enzymatic saccharification, the results of low-pressure steam pretreatment were found to be as good as or better than those reported for more severe pretreatment processes. Almost complete saccharification of the cellulose in the prehydrolyzed biomass solids was obtained within 24h with a commercial cellulase preparation — Celluclast. The presence of grinding elements (glass beads) during the enzymatic hydrolysis was found to increase the extent of saccharification by 40% to 50% over controls without any grinding elements.     

超声波对木质纤维素糖化过程影响的研究

将超声波应用在木质纤维素预处理及其酶解糖化过程中,通过SEM、FTIR研究了处理前后纤维素的形态结构和结晶性能,并考察了不同预处理方式对原料 成分的影响和超声波对酶解糖化率的影响。结果表明,超声波作用能有效的破坏纤维素分子中的氢键,降低其结晶程度,而且能有效地提高木质素的脱除率和酶解糖化率。对超声波作用于酶解过程中的机理进行了初步探讨     

Monitoring and Analyzing Process Streams Towards Understanding Ionic Liquid Pretreatment of Switchgrass (Panicum virgatum L.)

Fundamental understanding of biomass pretreatment and its influence on saccharification kinetics, total sugar yield, and inhibitor formation is essential to develop efficient next-generation biofuel strategies, capable of displacing fossil fuels at a commercial level. In this study, we investigated the effect of residence time and temperature during ionic liquid (IL) pretreatment of switchgrass using 1-ethyl-3-methyl imidazolium acetate. The primary metrics of pretreatment performance are biomass delignification, xylan and glucan depolymerization, porosity, surface area, cellulase kinetics, and sugar yields. Compositional analysis and quantification of process streams of saccharides and lignin demonstrate that delignification increases as a function of pretreatment temperature and is hypothesized to be correlated with the apparent glass transition temperature of lignin. IL pretreatment did not generate monosaccharides from hemicellulose. Compared to untreated switchgrass, Brunauer–Emmett–Teller surface area of pretreated switchgrass increased by a factor of ～30, with a corresponding increase in saccharification kinetics of a factor of ～40. There is an observed dependence of cellulase kinetics with delignification efficiency. Although complete biomass dissolution is observed after 3 h of IL pretreatment, the pattern of sugar release, saccharification kinetics, and total sugar yields are strongly correlated with temperature.     

A candidate-gene approach to clone the sorghum <Emphasis Type="Italic">Brown midrib </Emphasis>gene encoding caffeic acid <Emphasis Type="Italic">O</Emphasis>-methyltransferase

The brown midrib (bmr) mutants of sorghum have brown vascular tissue in the leaves and stem as a result of changes in lignin composition. The bmr mutants were generated via chemical mutagenesis with diethyl sulfate (DES) and resemble the brown midrib (bm) mutants of maize. The maize and sorghum brown midrib mutants are of particular value for the comparison of lignin biosynthesis across different, yet evolutionarily related, species. Although the sorghum brown midrib mutants were first described in 1978, none of the Brown midrib genes have been cloned. We have used a candidate-gene approach to clone the first Brown midrib gene from sorghum. Based on chemical analyses of the allelic mutants bmr12, bmr18 and bmr26, we hypothesized that these mutants had reduced activity of the lignin biosynthetic enzyme caffeic acid O-methyltransferase (COMT). After a northern analysis revealed strongly reduced expression of the COMT gene, the gene was cloned from the mutants and the corresponding wild types using PCR. In all three mutants, point mutations resulting in premature stop codons were identified: bmr12, bmr18 and bmr26 are therefore mutant alleles of the gene encoding COMT. RT-PCR indicated that all three mutants express the mutant allele, but at much lower levels relative to the wild-type controls. Molecular markers were developed for each of the three mutant alleles to facilitate the use of these mutant alleles in genetic studies and breeding programs.     

Effect of dilute acid pretreatment on the saccharification and fermentation of rye straw

This research shows the effect of dilute acid pretreatment with various sulfuric acid concentrations (0.5–2.0% [wt/vol]) on enzymatic saccharification and fermentation yield of rye straw. After pretreatment, solids of rye straw were suspended in Na citrate buffer or post-pretreatment liquids (prehydrolysates) containing sugars liberated after hemicellulose hydrolysis. Saccharification was conducted using enzymes dosage of 15 or 25 FPU/g cellulose. Cellulose saccharification rate after rye straw pretreatment was enhanced by performing enzymatic hydrolysis in sodium citrate buffer in comparison with hemicellulose prehydrolysate. The maximum cellulose saccharification rate (69%) was reached in sodium citrate buffer (biomass pretreated with 2.0% [wt/vol] H₂SO₄). Lignocellulosic complex of rye straw after pretreatment was subjected to separate hydrolysis and fermentation (SHF) or separate hydrolysis and co-fermentation (SHCF). The SHF processes conducted in the sodium citrate buffer using monoculture of Saccharomyces cerevisiae (Ethanol Red) were more efficient compared to hemicellulose prehydrolysate in respect with ethanol yields. Maximum fermentation efficiency of SHF processes obtained after rye straw pretreatment at 1.5% [wt/vol] H₂SO₄ and saccharification using enzymes dosage of 25 FPU/g in sodium citrate buffer, achieving 40.6% of theoretical yield. However, SHCF process using cocultures of pentose-fermenting yeast, after pretreatment of raw material at 1.5% [wt/vol] H₂SO₄ and hydrolysis using enzymes dosage of 25 FPU/g, resulted in the highest ethanol yield among studied methods, achieving 9.4 g/L of ethanol, corresponding to 55% of theoretical yield.     

Artificial neural network and response surface methodology: a comparative analysis for optimizing rice straw pretreatment and saccharification

Abstract

The present study demonstrates a comparative analysis between the artificial neural network (ANN) and response surface methodology (RSM) as optimization tools for pretreatment and enzymatic hydrolysis of lignocellulosic rice straw. The efficacy for both the processes, that is, pretreatment and enzymatic hydrolysis was evaluated using correlation coefficient (R²) & mean squared error (MSE). The values of R² obtained by ANN after training, validation, and testing were 1, 0.9005, and 0.997 for pretreatment and 0.962, 0.923, and 0.9941 for enzymatic saccharification, respectively. On the other hand, the R² values obtained with RSM were 0.9965 for cellulose recovery and 0.9994 for saccharification efficiency. Thus, ANN and RSM together successfully identify the substantial process conditions for rice straw pretreatment and enzymatic saccharification. The percentage of error for ANN and RSM were 0.009 and 0.01 for cellulose recovery and for 0.004 and 0.005 for saccharification efficiency, respectively, which showed the authority of ANN in exemplifying the non-linear behavior of the system.     

Reducing agents improve enzymatic hydrolysis of cellulosic substrates in the presence of pretreatment liquid

Enzymatic hydrolysis of pretreated lignocellulosic substrates has emerged as an interesting option to produce sugars that can be converted to liquid biofuels and other commodities using microbial biocatalysts. Lignocellulosic substrates are pretreated to make them more accessible to cellulolytic enzymes, but the pretreatment liquid partially inhibits subsequent enzymatic hydrolysis. The presence of pretreatment liquid from Norway spruce resulted in a 63% decrease in the enzymatic saccharification of Avicel compared to when the reaction was performed in a buffered aqueous solution. The addition of 15 mM of a reducing agent (hydrogen sulfite, dithionite, or dithiothreitol) to reaction mixtures with the pretreatment liquid resulted in up to 54% improvement of the saccharification efficiency. When the reducing agents were added to reaction mixtures without pretreatment liquid, there was a 13-39% decrease in saccharification efficiency. In the presence of pretreatment liquid, the addition of 15 mM dithionite to Avicel, α-cellulose or filter cake of pretreated spruce wood resulted in improvements between 25 and 33%. Positive effects (6-17%) of reducing agents were also observed in experiments with carboxymethyl cellulose and 2-hydroxyethyl cellulose. The approach to add reducing agents appears useful for facilitating the utilization of enzymes to convert cellulosic substrates in industrial processes.     

Pretreatment of lignocellulosic materials with hydrogen peroxide in presence of manganese compounds

Pretreatment of lignocellulosic materials such as newspaper, rice straw, pulp waste, and municipal solid waste with hydrogen peroxide in the presence of manganese compounds greatly enhances their susceptibility to enzymatic saccharification. This pretreatment can be achieved using rather mild conditions with only a minimal decrease in the recovery and little change in composition. Manganese salts in this hydrogen peroxide pretreatment works effectively in particular when the concentration of hydrogen peroxide is relatively low. The susceptibility of hydrogen-peroxide-pretreated substrate to enzymatic saccharification increases with increasing the molar ratio of manganes to hydrogen peroxide up to 1 : 100.     

Evaluation of the biocompatibile ionic liquid 1-methyl-3-methylimidazolium dimethylphosphite pretreatment of corn cob for improved saccharification

Ionic liquid (IL) pretreatment of lignocellulose materials is a promising process in biomass conversion to renewable biofuel. More in-depth research involving environment-friendly IL is much needed to explore pretreatment green route. In our case, IL 1-methyl-3-methylimidazolium dimethylphosphite ([Mmim]DMP) was chosen as an environment-friendly solvent to pretreat corn cob in view of its biocompatibility with both lignocellulose solubility and cellulase activity. The pretreatment/saccharification process and in situ saccharification process involving [Mmim]DMP were efficiently performed in bioconversion of corn cob to sugars, and more than 70% saccharification rates were obtained. Furthermore, the fermentability of reducing sugars obtained from the hydrolyzates was evaluated using Rhodococcus opacus strain ACCC41043 (R. opacus). High lipid production 41–43% of cell dry matter was obtained after 30 h of cultivation. GC/MS analysis indicated that lipids from R. opacus contained mainly long-chain fatty acids with four major constituent/oleic acid, stearic acid, palmitic acid, palmitoleic acid which are good candidates for biodiesel. These elucidated that corn cob pretreated by IL [Mmim]DMP did not bring negative effects on saccharification, cell growth, and accumulation of lipid of R. opacus. In conclusion, the IL [Mmim]DMP shows promise as green pretreatment solvent for cellulosic materials.     

Substrate-Related Factors Affecting Enzymatic Saccharification of Lignocelluloses: Our Recent Understanding

Enzymatic saccharification of cellulose is a key step in conversion of plant biomass to advanced biofuel and chemicals. Many substrate-related factors affect saccharification. Rather than examining the role of each individual factor on overall saccharification efficiency, this study examined how each factor affects the three basic processes of a heterogeneous biochemistry reaction: (1) substrate accessibility to cellulose—the roles of component removal and size reduction by pretreatments, (2) substrate and cellulase reactivity limited by component inhibition, and (3) reaction conditions—substrate-specific optimization. Our in-depth analysis of published literature work, especially those published in the last 5 years, explained and reconciled some of the conflicting results in literature, especially the relative importance of hemicellulose vs. lignin removal and substrate size reduction on enzymatic saccharification of lignocelluloses. We concluded that hemicellulose removal is more important than lignin removal for creating cellulase accessible pores. Lignin removal is important when alkaline-based pretreatment is used with limited hemicellulose removal. Partial delignification is needed to achieve satisfactory saccharification of lignocelluloses with high lignin content, such as softwood species. Rather than using passive approaches, such as washing and additives, controlling pretreatment or hydrolysis conditions, such as pH, to modify lignin surface properties can be more efficient for reducing or eliminating lignin inhibition to cellulase, leading to improved lignocellulose saccharification.