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Five new sorbicillinoid derivatives, including (±)-aspersorbicillin A [(±)- 1 ], a pair of enantiomers at C-9, and aspersorbicillins B-D ( 2 - 4 ), together with two known analogs ( 5 and 6 ) were isolated from the endophytic fungus Aspergillus aculeatus TE-65L. Their structures including absolute configurations were determined by detailed spectroscopic analyses and electronic circular dichroism calculations. The herbicidal activity of sorbicillinoids on the germ and radicle elongation of various weed types was reported for the first time. Compound 1 displayed significant herbicidal activity against Eleusine indica germ elongation (IC 50 = 28.8 μg/mL), while compound 6 inhibited radicle elongation (IC 50 = 25.6 μg/mL). Both were stronger than those of glyphosate (66.2 and 30.9 μg/mL, respectively). Further transcriptomic and LC-MS/MS metabolomic analysis indicated that 6 induced the transcriptional expressions of genes related to the lignin biosynthetic pathway, resulting in lignin accumulation. Transmission electron microscopy confirmed the cell wall thickening of seeds treated with 6 , suggesting weed growth inhibition. This study reveals new lead compounds for fabricating natural herbicides and expands the agricultural use of sorbicillinoid analogs.

Understanding and recognizing the structural characteristics of lignin-carbohydrate complexes (LCCs) and lignin in different growth stages and tissue types of bamboo will facilitate industrial processes and practical applications of bamboo biomass. Herein, the LCC and lignin samples were sequentially isolated from fibers and parenchyma cells of bamboo with different growth ages. The diverse yields of sequential fractions not only reflect the different biomass recalcitrance between bamboo fibers and parenchyma cells but also uncover the structural heterogeneity of these tissues at different growth stages. The molecular structures and structural inhomogeneities of the isolated lignin and LCC samples were comprehensively investigated. The results showed that the structural features of lignin and LCC linkages in parenchyma cells were abundant in β- O -4 linkages but less with carbon-carbon linkages, suggesting that lignin and cross-linked LCC in parenchyma cells are simple in nature and easily to be tamed and tractable in the current biorefinery. Parallelly, the different ball-milled samples were directly characterized by high-resolution (800 M) solution-state 2D-HSQC NMR to analyze the whole lignocellulosic material. Overall, the scheme presented in this study will provide a comprehensive understanding of lignin and LCC linkages in fibers and parenchyma cells of bamboo and enable the utilization of bamboo biomass.

Brassica napus is currently the principal field crop for producing materials for primary, secondary and tertiary industries. B. napus shoots at stem elongation stage are rich in anthocyanins, vitamin C and mineral elements such as selenium, calcium and zinc, and represent a new type of green vegetable. However, the high crude fiber (CF) content of B. napus shoots affects their taste, and few studies have focused on the quality traits of these vegetables. In this study, we investigated five traits related to the CF components, including neutral detergent fiber (NDF), acid detergent fiber (ADF), acid detergent lignin (ADL), hemicellulose (Hem) and cellulose (Cel), of B. napus shoots. Whole-genome resequencing at a depth of ∼20× was utilized to genotype an association panel of 202 diverse accessions, which resulted in the identification of 6,093,649 single nucleotide polymorphisms (SNPs) and 996,252 indels, respectively. A genome-wide association study (GWAS) was performed for the five CF-related traits based on the phenotypic data observed in four environments. A total of 1,285 significant SNPs were detected at the threshold of -log 10 ( p ) = 5.16, and 97 significant association regions were obtained. In addition, seven candidate genes located on chromosomes A2 (one gene), A8 (three genes), A9 (two genes) and C9 (one gene) related to CF traits were identified, and ten lines containing low CF contents were selected as excellent germplasm resources for breeding. Our results contributed new insights into the genetic basis of CF traits and suggested germplasm resources for the quality improvement of B. napus shoots.

Monolignols and their derivatives exhibit various pharmaceutical and physiological characteristics, such as antioxidant and anti-inflammatory properties. However, they remain difficult to synthesize. In this study, we engineered several whole-cell bioconversion systems with carboxylate reductase (CAR)-mediated pathways for efficient synthesis of p -coumaryl, caffeyl, and coniferyl alcohols from l-tyrosine in Escherichia coli BL21 (DE3). By overexpressing the l-tyrosine ammonia lyase from Flavobacterium johnsoniae (FjTAL), carboxylate reductase from Segniliparus rugosus (SruCAR), alcohol dehydrogenase YqhD and hydroxylase HpaBC from E. coli , and caffeate 3-O-methyltransferase (COMT) from Arabidopsis thaliana , three enzyme cascades FjTAL-SruCAR-YqhD, FjTAL-SruCAR-YqhD-HpaBC, and FjTAL-SruCAR-YqhD-HpaBC-COMT were constructed to produce 1028.5 mg/L p -coumaryl alcohol, 1015.3 mg/L caffeyl alcohol, and 411.4 mg/L coniferyl alcohol from 1500, 1500, and 1000 mg/L l-tyrosine, with productivities of 257.1, 203.1, and 82.3 mg/L/h, respectively. This work provides an efficient strategy for the biosynthesis of p -coumaryl, caffeyl, and coniferyl alcohols from l-tyrosine.

Lignin provides structural support to plants; however, it reduces their utilization rate. According to our previous studies, selenium (Se) reduces lignin accumulation in alfalfa, but the specific mechanism involved remains unclear. Therefore, at the seedling stage, four root irrigation treatments using 2.5, 50, and 5 μmol/L sodium selenite (S-RI), selenomethionine (SS-RI), Se nanoparticles (SSS-RI), and deionized water (CK-RI) were performed. At the branching stage, four treatments of foliar spraying with the three Se fertilizers described above at a concentration of 0.5 mmol/L (S-FS, SS-FS, and SSS-FS) and deionized water (CK-FS) were administered. The results revealed that all Se treatments chiefly reduced the level of deposition of syringyl (S) lignin in the first internode of alfalfa stems. SS-FS and SSS-FS treatments mainly reduced the deposition of S and guaiacyl (G) lignins in the sixth internode of alfalfa stems, respectively, while S-FS treatment only slightly reduced the deposition of G lignin. S, SS, and SSS-RI treatments reduced the level of deposition of S and G lignins in the sixth internode of alfalfa stems. Se application increased plant height, stem diameter, epidermis (cortex) thickness, primary xylem vessel number (diameter), and pith diameter of alfalfa but decreased primary xylem area and pith parenchyma cell wall thickness of the first internode, and SS(SSS)-FS treatment reduced the mechanical strength of alfalfa stems. Therefore, Se application could decrease lignin accumulation by regulating the organizational structure parameters of alfalfa stems and the deposition pattern of the lignin monomers.

Glycerol is an important platform compound with multidisciplinary applications, and glycerol production using low-cost sugar cane bagasse hydrolysate is promising. Candida glycerinogenes , an industrial yeast strain known for its high glycerol production capability, has been found to thrive in bagasse hydrolysate obtained through a simple treatment without detoxification. The engineered C. glycerinogenes exhibited significant resistance to furfural, acetic acid, and 3,4-dimethylbenzaldehyde within undetoxified hydrolysates. To further enhance glycerol production, genetic modifications were made to Candida glycerinogenes to enhance the utilization of xylose. Fermentation of undetoxified bagasse hydrolysate by CgS45 resulted in a glycerol titer of 40.3 g/L and a yield of 40.4%. This process required only 1 kg of bagasse to produce 93.5 g of glycerol. This is the first report of glycerol production using lignocellulose, which presents a new way for environmentally friendly industrial production of glycerol.

Sphingobium lignivorans SYK-6 can assimilate various lignin-derived aromatic compounds, including a β-5-type (phenylcoumaran-type) dimer, dehydrodiconiferyl alcohol (DCA). SYK-6 converts DCA to a stilbene-type intermediate via multiple reaction steps and then to vanillin and 5-formylferulic acid (FFA). Here, we first elucidated the catabolic pathway of FFA, which is the only unknown pathway in DCA catabolism. Then, we identified and characterized the enzyme-encoding genes responsible for this pathway. Analysis of the metabolites revealed that FFA was converted to 5-carboxyferulic acid (CFA) through oxidation of the formyl group, followed by conversion to ferulic acid by decarboxylation. A comprehensive analysis of the aldehyde dehydrogenase genes in SYK-6 indicated that NAD + -dependent FerD (SLG_12800) is crucial for the conversion of FFA to CFA. LigW and LigW2, which are 5-carboxyvanillic acid decarboxylases involved in the catabolism of a 5,5-type dimer, were found to be involved in the conversion of CFA to ferulic acid, and LigW2 played a significant role. The ligW2 gene forms an operon with ferD , and their transcription was induced during growth in DCA.

The cultivation of Agaricus bisporus was investigated with two commercial strains, A15 and W192. Nitrogen and lignocellulose were analyzed in absolute amounts based on mass balance to accurately compare the degradation efficiency of the compost, and the correlation between the degradation efficiency and extracellular enzyme activity of the mycelium was analyzed. Lignocellulose utilization efficiency positively correlated with mushroom yield. For the same strain, the compost with high lignocellulose content resulted in high utilization efficiency, which increased the yield of A. bisporus . For the same compost, the lignocellulose utilization efficiency of A15 was higher than that of W192. The activities of manganese-dependent peroxidase and β-glucosidase indicated that W192 may have a higher demand for lignin and cellulose. Therefore, a higher yield of W192 was obtained with high-lignocellulose compost. The metabolism of cellulose and hemicellulose in the mycelial growth stage seemed to be conducive to high mushroom yield.

There is a need for new protein sources to feed the world in a sustainable way. Converting non-food-grade "woody" side streams into food containing proteins will contribute to this mission. Mushroom forming fungi are unique in their capability to convert lignocellulosic substances into edible biomass containing protein. Especially if substrate mycelium can be used instead of mushrooms, this technology could be a serious contribution to addressing the protein challenge. In this Perspective, we discuss challenges toward production, purification, and market introduction of mushroom mycelium based foods.

Salicylic acid (SA) is an important plant hormone and signal required for establishing resistance to diverse pathogens and plant diseases. The abundant polyphenols in tea plants also defend plants from biotic and abiotic stresses. However, whether exogenous SA would increase the resistance of tea plants to adversity and the relationship between SA and polyphenols are still poorly understood. Here, we carried out SA treatment on tea seedlings and performed transcriptome sequencing. SA treatment inhibited the phenylpropanoid and flavonoid metabolic pathways but promoted the lignin metabolic pathways. The increased accumulation of lignin in tea leaves after treating with SA indicated that lignin might coordinate SA, enhance, and improve plant defense and disease resistance. Simultaneously, an SA-inducible flavonoid glucosyltransferase (CsUGT0554) specifically involved in 7-OH site glycosylation was characterized in vitro. These results provided valuable information about the effects of SA on tea seedlings and the molecular basis for SA-mediated immune responses.

Mycosporine-like amino acids (MAAs) have been used in cosmetics and pharmaceuticals. The purpose of this work was to develop yeast strains for sustainable and economical production of MAAs, especially shinorine. First, genes involved in MAA biosynthetic pathway from Actinosynnema mirum were introduced into Saccharomyces cerevisiae for heterologous shinorine production. Second, combinatorial expression of wild and mutant xylose reductase was adopted in the engineered S. cerevisiae to facilitate xylose utilization in the pentose phosphate pathway. Finally, the accumulation of sedoheptulose 7-phosphate (S7P) was attempted by deleting transaldolase-encoding TAL1 in the pentose phosphate pathway to increase carbon flux toward shinorine production. In fed-batch fermentation, the engineered strain (DXdT-M) produced 751 mg/L shinorine in 71 h. Ultimately, 54 mg/L MAAs was produced by DXdT-M from rice straw hydrolysate. The results suggest that shinorine production by S. cerevisiae might be a promising process for sustainable production and industrial applications.

The behavior of organophosphate esters (OPEs) in plants has drawn considerable attention because of their adverse effects to biota. However, the root uptake pathways and cell wall accumulation mechanisms of OPEs in plants are still unclear. In this study, the uptake pathways, subcellular distribution, and accumulation mechanisms of OPEs in wheat roots were elucidated. The results demonstrated that the symplast is the major pathway for uptake of both tris(2-chloroethyl) phosphate (TCEP) and triphenyl phosphate (TPHP) in wheat roots. Inhibitor experiments showed that the transmembrane transport of OPEs is a passive uptake process, and aquaporins and carrier proteins contribute to the uptake of OPEs. More than 69% of TCEP was accumulated in cell sap due to its high hydrophilicity, while the hydrophobic TPHP was mainly stored in the root cell wall. The sorption affinity of TPHP decreased gradually following the sequential fractionation of wheat roots, which confirmed the significant contribution to TPHP sorption on wheat roots. A significant positive correlation between the sorption affinity values and the percentage of aromatic carbon was observed ( r 2 = 0.856, p < 0.01), indicating that the accumulation of hydrophobic OPEs in roots does not just depend on lipids alone, but the aromatic moieties of lignin in the cell wall also contribute to OPE accumulation.

A diet with a high dietary fiber content is often recommended in today's nutrition due to several beneficial health effects related to its intake. Lignin as a part of dietary fiber is the second most abundant natural polymer and considered to be stable during digestion. However, some studies indicate a partial degradation during the intestinal metabolism. To further elucidate this hypothesis, the aim of this study was to investigate whether lignin is metabolized by the gut microbiota using the ex vivo pig cecum model. As potential lignin-derived metabolites might already naturally occur in the pig cecal matrix, an approach using isotopically labeled 13 C lignin was chosen for this study. Ten small phenolic lignin degradation products and their time-dependent metabolism were identified via an untargeted HPLC-HRMS approach, and the quantity of the metabolites was estimated. From the results, we conclude that lignin is partially degraded releasing small phenolic metabolites.

Maize stalks support leaves and reproductive structures and functionally support water and nutrient transport; besides, their anatomical and biochemical characteristics have been described as a plant defense against stress, also impacting economically important applications. In this study, we evaluated agronomical and stem description traits in a subset of maize inbred lines that showed variability for cell wall composition in the internodes. Overall, a great proportion of lignin subunit G and a low concentration of p -coumaric acid and lignin subunit S are beneficial for greater rind puncture resistance and taller plants, with a greater biomass yield. Also, the greater the proportions of subunit H, the longer the internode. Finally, the lower the total hemicellulose content, the greater the rind puncture resistance. Our results confirmed the effect of the cell wall on agronomic and stalk traits, which would be useful in applied breeding programs focused on biomass yield improvement.

To improve the reactivity and enrich the functionality of lignin for valorization, kraft lignin was depolymerized and demethylated via cleaving aryl and alkyl ether bonds in acidic lithium bromide trihydrate (∼60% LiBr aqueous solution). It was found that the cleavage of the ether bonds followed the order of β-O-4 ether > aryl alkyl ether in phenylcoumaran > dialkyl ether in resinol > methoxyl (MeO). The depolymerization via β-O-4 cleavage occurred under mild conditions (e.g., <0.5 M HCl at 110 °C), while sufficient demethylation of the lignin needed harsher conditions (>1.5 M HCl). Both depolymerization and demethylation generated new aromatic hydroxyl (ArOH). With 2.4 M HCl, MeO content dropped from 4.85 to 0.95 mmol/g lignin, and ArOH content increased from 2.78 to 5.09 mmol/g lignin. The depolymerized and demethylated kraft lignin showed excellent antioxidant activity and Cr(VI)-scavenging capacity, compared with original kraft lignin and tannins.

Lignin-carbohydrate complexes (LCCs) have recently emerged as natural products with pharmaceutical and nutraceutical potential. Here, we compared the structure of LCCs from ginkgo (GK, gymnosperms), wheat straw (WST, monocotyledons), and aspen white poplar (AW, dicotyledons). We also investigated the biotransformation of LCCs by intestinal microbiota in vitro. We found that human intestinal microbiota could use LCCs as a carbon source for growth, breaking resistant cross-linkages in LCCs to generate a plethora of short-chain fatty acids (SCFAs) and aromatic compounds with putative beneficial effects on human health. The yield of SCFAs reached 1837.8 ± 44.1 μmol/g using AW LCCs as a carbon source. The biomass of intestinal microbiota increased the fastest using GK LCCs. The greatest amounts of phenolics were present at 4 h in a WST LCCs fermentation system. Many phenolic acids with potential bioactivity were obtained after 24 h fermentation using each LCCs, such as ferulic acid.

It is generally considered that lignin is a three-dimensional amorphous polymer consisting of methoxylated phenylpropane structures. However, high yields of monomer structural units of lignin cannot be obtained through various ways, which inspired us to gain insights into the structures of lignin. Herein, enzymatic lignin (EL) was directly characterized by a solid-state 13 C nuclear magnetic resonance spectrometer and Fourier transform infrared spectrometer and then subjected to ruthenium ion-catalyzed oxidation. According to the spectral characterization, it can be inferred that multi-ring aromatic clusters exist in EL because of the aromatic bridgehead carbon ratio of 0.136. Based on the results of ruthenium ion-catalyzed oxidation of the EL, it can be deduced that (1) double- and triple-aromatic ring clusters exist in the EL besides the traditional phenylpropane single-aromatic ring clusters, and (2) some aromatic rings with long-alkyl chain substituents exist in the EL, which is quite different from the traditional cognition of lignin. This investigation provides a new insight into the structure of EL.

Many studies have shown that phenolic compounds such as lignin and flavonoids enhance plant resistance. Tea plants are rich in flavonoid compounds. Whether these compounds are related to tea plant resistance is unclear. In this study, an interesting conclusion was drawn on the basis of experimental results: in response to abiotic stress (except for sucrose treatment), gene expression was increased in the phenylpropanoid and lignin pathways and was reduced in the flavonoid pathway in tea plants. CsHCT s, the genes located at the branch point of the lignin and flavonoid pathways, are most suitable for regulating the ratio of carbon flow in the lignin pathway and flavonoid synthesis. Enzymatic and genetic modification experiments proved that CsHCT s encode hydroxycinnamoyl-coenzyme A:shikimate/quinate hydroxycinnamoyl transferase in vitro and in vivo . Furthermore, the genetic modification results showed that the contents of phenolic acids and lignin were increased in tobacco and Arabidopsis plants overexpressing CsHCT s, whereas the content of flavonol glycosides was decreased. Both types of transgenic plants showed resistance to many abiotic stresses and bacterial infections. We speculate that CsHCT s participate in regulation of the metabolic flow of carbon from the flavonoid pathway to the chlorogenic acid, caffeoylshikimic acid, and lignin pathways to increase resistance to biotic and abiotic stresses.

α-Pinene, an important biologically active natural monoterpene, has been widely used in fragrances, medicines, and fine chemicals, especially, in high-density renewable fuels such as jet fuel. The development of an α-pinene production platform in a highly modifiable microbe from renewable substitute feedstocks could lead to a green, economical avenue, and sustainable biotechnological process for the biosynthesis of α-pinene. Here, we report engineering of an orthogonal biosynthetic pathway for efficient production of α-pinene in oleaginous yeast Yarrowia lipolytica that resulted in an α-pinene titer of 19.6 mg/L when using glucose as the sole carbon source, a significant 218-fold improvement than the initial titer. In addition, the potential of using waste cooking oil and lignocellulosic hydrolysate as carbon sources for α-pinene production from the engineered Y. lipolytica strains was analyzed. The results indicated that α-pinene titers of 33.8 and 36.1 mg/L were successfully obtained in waste cooking oil and lignocellulosic hydrolysate medium, thereby representing the highest titer reported to date in yeast. To our knowledge, this is also the first report related to microbial production of α-pinene from waste cooking oil and lignocellulosic hydrolysate.

Understanding the contribution of stem cell wall components to lodging is important in developing breeding programs aimed at reducing lodging in cereal crops. This study is one of the first to investigate the correlation between the amounts of cell wall-bound ferulic acid, p -coumaric acid, and lignin in the nodes and internodes of cereals (oat, wheat, and barley) and their lodging susceptibility during grain fill. All samples, except two-row barley, were susceptible to lodging and expressed a significantly lower stalk strength. Lignin and phenolic contents between nodes and internodes of all samples were significantly different, with internodes having higher amounts (5.5-7.0 and 10.9-16.2 μg/g p- coumaric acid, and 2.5-3.2 and 3.9-7.1 μg/g ferulic acid in nodes and internodes, respectively). The acid-soluble lignin content was different between nodes and internodes but not between crops. This data set did not correlate with lodging classification, possibly due to sample size and type.

A 6.2 m high immature bamboo ( Phyllostachys nigra ) was divided into seven fractions. The bamboo cell walls and lignin samples from young to old were characterized by 1 H- 13 C correlation heteronuclear single-quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectroscopy both qualitatively and semiquantitatively. Mature bamboo and bamboo shoot samples were used as comparison references. HSQC-NMR analysis proved that cellulose and arabinoxylan have already deposited in bamboo shoot, and cellulose amount increased during growth. Lignin side chain linkage formation started from β-ether (β- O -4), then phenylcoumaran (β-5), and finally resinol (β-β). Ferulic acid and p -coumaric acid ( p CA) were formed at the earlier stages in the immature bamboo, and the p CA proportion decreased throughout the lignification process. We propose that the bamboo lignification process is distinct from both woody and other herbaceous plants, where syringyl units deposited at the early stage and polymerized with the β- O -4 linkage. Then guaiacyl units formed gradually, and finally, p -hydroxyphenyl units formed.

The production of acetone-butanol-ethanol by solventogenic Clostridium using lignocellulosic biomass can be a potential alternative to petroleum-based butanol. However, previous studies on nondetoxified lignocellulose hydrolysate could not provide better results when compared to those in synthetic medium. In this study, we engineered the pentose pathway of Clostridium beijerinckii NCIMB 8052, which was then subjected to adaptive laboratory evolution in the gradient mixture of synthetic medium and pretreated corn stover enzymatic hydrolysate (CSH) prepared according to the National Renewable Energy Laboratory (NREL) standard. The final resultant strain CIBTS1274A produced 20.7 g/L of total solvents in NREL CSH diluted to 6% initial total sugars, supplemented with ammonium acetate. This performance was comparable with that of corn-based butanol. In addition, this strain was successfully used in the scale-up operation using nondetoxified corn stover and corncob hydrolysate at Lignicell Refining Biotechnologies Ltd., which once was the only commercial biobutanol industry in the world.

Farnesene is an important chemical platform for many industrial products, such as biofuels and polymers. We performed high-efficiency utilization of corncobs for β-farnesene production by separate hydrolysis and fermentation with an optimized Escherichia coli strain. First, we developed a recycling strategy for both corncob pretreatment and cellulose hydrolysis, which saved great amounts of pretreatment reagents and presented a 96.83% cellulose conversion rate into glucose. However, the corncob hydrolysate strongly repressed cell growth and β-farnesene production, being caused by high-concentrated citrate. Through expressing a heterologous ATP citrate lyase and screening for a suitable expression host, an optimized strain was constructed that produced β-farnesene at 4.06 g/L after 48 h in a 5 L fermenter, representing an approximately 2.3-fold increase over the initial strain. Therefore, the proposed strategy about the recycling process and repression elimination was successful and suitable for the production of lignocellulosic-based β-farnesene, which can be further studied to scale up for industrialization.

p-Quinone methide (QM) is formed as an intermediate during lignin biosynthesis. The aromatization of the QM by the attack of a nucleophile at the α-position of its side chain generates a phenolic hydroxy group in a growing polymer and creates stereoisomeric forms in the side chain. A series of β-O-4-aryl ether QMs was reacted with water at 25 °C to replicate the formation of p-hydroxyphenyl (H) and guaiacyl (G) β-O-4 structures in plant cell walls. Water addition occurred in 3-methoxy-substituted QMs (G-type QMs) with half-lives ( t 1/2 ) between 13 and 15 min, at pH 7, in 50% water solution (dioxane-water, 1:1). The rate increased as the water concentration increased to 99% ( t 1/2 , 1.2-1.4 min). Similar solvent effects were observed for more reactive nonsubstituted QMs (H-type QMs with t 1/2 of <1 min). Consequently, t 1/2 of the H-type QMs was shorter than that of the G-type QMs under every solvent condition. Upon increasing the water concentration, the variation in the erythro/ threo ratios of the four dimeric β-O-4 products increased. Interestingly, the effect of pH on the stereopreference, which was observed in 50% water solution, was small and became imperceptible as the water concentration increased to 99%, suggesting that the effect of the solvent, as well as the effect of the pH, plays an important role in understanding the reaction conditions in cell walls during lignin biosynthesis. The threo isomer was preferentially formed in the four dimeric β-O-4 structures, which is inconsistent with the structural features of compression wood lignin rich in H-units. However, the erythro-selective formation was attained in an H-type QM at every pH studied (pH 3.5-7) by introducing a biphenyl structure into the β-etherified ring moiety.

Vitamins are important nutrients for many fermentations, but they are generally costly. Agricultural lignocellulose biomass contains considerable amounts of vitamin B compounds, but these water-soluble vitamins are easily lost into wastewater discharge during pretreatment or detoxification of lignocellulose in biorefinery processes. Here, we showed that the dry acid pretreatment and biodetoxification process allowed the preservation of significant amounts of vitamin B, which promoted l-lactic acid fermentation efficiency significantly. Supplementation with specific vitamin B compounds, VB 3 and VB 5 , into corn stover hydrolysate led to further increases of cellulosic l-lactic acid yield and fermentation rates. This study provided a new solution for the enhancement of biorefinery fermentation efficiency by using vitamin B compounds in lignocellulose biomass.

A lignin amphoteric surfactant and betaine could enhance the enzymatic hydrolysis of lignocellulose and recover cellulase. The effects of lignosulfonate quaternary ammonium salt (SLQA) and dodecyl dimethyl betaine (BS12) on enzymatic hydrolysis digestibility, ethanol yield, yeast cell viability, and other properties of high-solid enzymatic hydrolysis and fermentation of a corncob residue were studied in this research. The results suggested that SLQA and 1 g/L BS12 effectively improved the ethanol yield through enhancing enzymatic hydrolysis. SLQA had no significant effect on the yeast cell membrane and glucose fermentation. However, 5 g/L BS12 reduced the ethanol yield as a result of the fact that 5 g/L BS12 damaged the yeast cell membrane and inhibited the conversion of glucose to ethanol. Our research also suggested that 1 g/L BS12 enhanced the ethanol yield of corncob residue fermentation, which was attributed to the fact that lignin in the corncob adsorbed BS12 and decreased its concentration in solution to a safe level for the yeast.

Our previous research showed that Pleurotus eryngii and Pleurotus ostreatus were effective fungi for pretreatment of industrial hemp stalks to improve enzymatic saccharification. The secretomes of these two fungi were analyzed to search for the effective enzyme cocktails degrading hemp lignin during the pretreatment process. In total, 169 and 155 proteins were identified in Pleurotus eryngii and Pleurotus ostreatus, respectively, and 50% of the proteins involved in lignocellulose degradation were CAZymes. Because most of the extracellular proteins secreted by fungi are glycosylated proteins, the N-linked glycosylation of enzymes could be mapped. In total, 27 and 24 N-glycosylated peptides were detected in Pleurotus eryngii and Pleurotus ostreatus secretomes, respectively. N-Glycosylated peptides of laccase, GH92, exoglucanase, phenol oxidase, α-galactosidase, carboxylic ester hydrolase, and pectin lyase were identified. Deglycosylation could decrease enzymatic saccharification of hemp stalks. The activities of laccase, α-galactosidase, and phenol oxidase and the thermal stability of laccase were reduced after deglycosylation.

Insoluble residue (INS) is a lignin-rich fraction of brewer's spent grain (BSG) that also contains β-glucan and arabinoxylan, the major constituents of dietary fiber. We investigated the effects of INS in diet-induced obese mice in terms of lipid metabolism and metabolic diseases. Male mice (C57bl6) were fed a high-fat diet (HFD), a HFD + 20% INS, a HFD + 20% cellulose (CEL), a HFD with a combination of 20% INS-CEL (1:1), or a control diet for 14 weeks. Insulin and glucose tolerance tests were performed after 12 weeks. Fasting plasma lipids, bile acid, and fecal bile acid were measured after 14 weeks of feeding, and tissues were collected for gene expression analysis. Body weight gain was significantly reduced with all fibers, but only INS and INS-CEL decreased fasting plasma low-density lipoprotein cholesterol and total cholesterol compared to HFD. CEL and INS-CEL significantly improved insulin resistance. Fecal bile acids were significantly increased by all fibers, but there was no change in plasma bile acid. Clostridium leptum was increased with all fibers, but universal bacterial diversity was only with INS and INS-CEL. In addition, INS significantly increased the abundance of Bacteriodes, while CEL decreased Atopobium and Lactobacillus. INS feeding significantly upregulated various genes of cholesterol and bile acid metabolism, such as Srebp2, Hmgcr, Ldlr, Cyp7a1, Pparα, Fxr, and Pxr, in the liver. INS, INS-CEL, and CEL significantly attenuated liver steatosis. Our results suggest that INS from BSG induced beneficial systemic changes in mice via gut microbiota, bile acids, and gene expression in the liver.

Kluyveromyces marxianus IIPE453 can utilize biomass-derived fermentable sugars for xylitol and ethanol fermentation. In this study, the xylitol production in the native strain was improved by overexpression of endogenous d-xylose reductase gene. A suitable expression cassette harboring the gene of interest was constructed and incorporated in the native yeast. qPCR analysis demonstrated the 2.1-fold enhancement in d-xylose reductase transcript levels in the modified strain with 1.62-fold enhancement in overall xylitol yield without affecting its ethanol fermenting capacity. Material balance analysis on 2 kg of sugar cane bagasse-derived fermentable sugars illustrated an excess of 58.62 ± 0.15 g of xylitol production by transformed strain in comparison to the wild variety with similar ethanol yield. The modified strain can be suitably used as a single biocatalyst for multiproduct biorefinery application.

In our study, isobaric tags for relative and absolute quantification (iTRAQ) was conducted to determine the significantly changed proteins in the fleshy roots of carrots under different carbon dioxide (CO 2 ) treatments. A total of 1523 proteins were identified, of which 257 were differentially expressed proteins (DEPs). On the basis of annotation analysis, the DEPs were identified to be involved in energy metabolism, carbohydrate metabolism, and some other metabolic processes. DcC4H and DcPER, two lignin-related proteins, were identified from the DEPs. Under elevated CO 2 stress, both carrot lignin content and the expression profiles of lignin biosynthesis genes changed significantly. The protein-protein interactions among lignin-related enzymes proved the importance of DcC4H and DcPER. The results of our study provided potential new insights into the molecular mechanism of lignin content changes in carrot roots under elevated CO 2 stress.

Water supply and valorization are two urgent issues in the utilization of maize biomass in the context of climate change and replacement of fossil resources. Maximizing maize biomass valorization is of interest to make biofuel conversion competitive, and to increase forage energetic value for animal fodder. One way to estimate biomass valorization is to quantify cell wall degradability. In this study, we evaluated the impact of water supply on cell wall degradability, cell wall contents and structure, and distribution of lignified cell types in maize internodes using dedicated high-throughput tools to effectively phenotype maize internodes from 11 inbred lines under two contrasting irrigation scenarios in field trials over three years. Overall, our results clearly showed that water deficit induced significant changes in lignin content and distribution along with a reduction in lignin p-coumaroylation, thereby impacting cell wall degradability. Additionally, we also observed that responses to a water deficit varied between the lines examined, underscoring biochemical and histological target traits for plant breeding.

A homemade microcurrent reactor was used to treat the fermentation of Trichoderma reesei. Results indicated that the yield of saccharides for T. reesei RUT-C30 cultivated in pumpkin lignocellulose broth reaches 38.86% (w/w) when a microcurrent treatment (20 mA, at the 48th hour for 60 min) was carried out, which is significantly higher than the control group (p < 0.05). Additionally, activities of endoglucanase, cellobiohydrolase, xylanase, and pectinase were significantly increased in days 3-7. Furthermore, the fungal growth was facilitated by microelectric treatment, showing a 0.57-fold increase of spore numbers at the sixth day of cultivation. Besides, the monosaccharide composition, including glucose (1.03 mg/mL), xylose (0.12 mg/mL), arabinose (0.31 mg/mL), and fructose (0.13 mg/mL), extracted from the reactor was higher than that without the current treatment. In this work, we improved the biodegradation of lignocellulosic wastes by applying a microcurrent to lignocellulose-degrading fungal cultures and provided a new idea for the lignocellulose material pretreatment and bioconversion.

Brewer's spent grain (BSG) is the major side-stream from brewing. As BSG is rich in dietary fiber and protein, it could be used in more valuable applications, such as nutritional additives for foods. Our aim was to elucidate whether an insoluble lignin-rich fraction (INS) from BSG is metabolized by mice gut microbiota and how it affects the microbiota. Our results indicated that lignin was partially degraded by the gut microbiota, degradation products were absorbed, and finally excreted in urine. Therefore, they contribute to the phenolic pool circulating in the mammalian body, and may have systemic effects on health. In addition, the effects of the test diets on the microbiota were significant. Most interestingly, diversities of predominant cecal and fecal bacteria were higher after the intervention diet containing INS than after the intervention diet containing cellulose. Since low fecal bacterial diversity has been linked with numerous diseases and disorders, the diversity increasing ability opens very interesting perspectives for the future.

To clarify the mechanisms of selenium (Se) tolerance in peanut seedlings, we grew peanut seedlings with sodium selenite (0, 3, and 6 mg/L), and investigated the phenylpropanoids metabolism in seedling roots. The results showed that selenite up-regulated the expression of genes and related enzyme activities involving in the phenylpropanoids biosynthesis cascade, such as phenylalanine ammonia-lyase, trans-cinnamate-4-hydroxylase, chalcone synthase, chalcone isomerase, and cinnamyl-alcohol dehydrogenase. Selenite significantly increased phenolic acids and flavonoids, which contributed to the alleviation of selenite-induced stress. Moreover, selenite enhanced the formation of endodermis in roots, which may be attributed to the up-regulation of lignin biosynthesis mediated by the selenite-induced changes of H2O2 and NO, which probably regulated the selenite uptake from an external medium. Accumulation of polyphenolic compounds via the phenylpropanoid pathway may be one of the mechanisms of the increasing selenite tolerance in plants, by which peanut seedlings survived in seleniferous soil, accompanied by accumulation of Se.

Lignin is part of dietary fiber, but its conversion in the gastrointestinal tract is not well understood. The aim of this work was to obtain structural information on brewer's spent grain (BSG) lignin and to understand the behavior of the polymeric part of lignin exposed to fecal microbiota. The original BSG and different lignin fractions were characterized by pyrolysis-GC/MS with and without methylation. Methylation pyrolysis proved that the ratio between guaiacyl and syringyl units was similar in all lignin samples, but the ratio between p-coumaric and ferulic acids varied by the isolation method. Combined pyrolysis results indicated higher acylation of γ-OH groups in syringyl than in guaiacyl lignin units. The polymeric lignin structure in the alkali-soluble fraction after enzymatic hydrolysis was slightly altered in the in vitro colon fermentation, whereas lignin in the insoluble residue after enzymatic treatments remained intact.

Six lignocellulosic hydrolysates produced through acid pretreatment were analyzed for the occurrence of formaldehyde, acetaldehyde, and glycolaldehyde. Acetaldehyde was found in all six (0.3-1.6 mM) and formaldehyde in four (≤ 4.4 mM), whereas glycolaldehyde was not detected. To assess the relevance of these findings, fermentations with yeast and formaldehyde or acetaldehyde were performed in the concentration interval 0.5-10 mM. Formaldehyde already inhibited at 1.0 mM, whereas 5.0 mM acetaldehyde was needed to obtain a clear inhibitory effect. After 24 h of fermentation, 1.5 mM formaldehyde reduced the glucose consumption by 85%, the balanced ethanol yield by 92%, and the volumetric productivity by 91%. The results show that formaldehyde and acetaldehyde are prevalent in pretreated lignocellulose and that formaldehyde in some cases could explain a large part of the inhibitory effects on yeast by lignocellulosic hydrolysates, as three of six hydrolysates contained ≥ 1.9 mM formaldehyde, which was shown to be strongly inhibitory.

Lignin, a phenylpropanoid polymer present in secondary cell walls, has a negative impact on feed digestibility. TT8 and HB12 genes were shown to have low expression levels in low-lignin tissues of alfalfa, but to date, there has been no study on the effect of down-regulation of these two genes in alfalfa on nutrient chemical profiles and availability in ruminant livestock systems. The objectives of this study were to investigate the effect of transformation of alfalfa with TT8 and HB12 RNAi constructs on carbohydrate (CHO) structure and CHO nutritive value in ruminant livestock systems. The results showed that transformation with TT8 and HB12 RNAi constructs reduced rumen, rapidly degraded CHO fractions (RDCA4, P = 0.06; RDCB1, P < 0.01) and totally degraded CHO fraction (TRDCHO, P = 0.08). Both HB12 and TT8 populations had significantly higher in vitro digestibility of neutral detergent fiber (NDF) at 30 h of incubation (ivNDF30) compared to the control (P < 0.01). The TT8 populations had highest ivDM30 and ivNDF240. Transformation of alfalfa with TT8 and HB12 RNAi constructs induced molecular structure changes. Different CHO functional groups had different sensitivities and different responses to the transformation. The CHO molecular structure changes induced by the transformation were associated with predicted CHO availability. Compared with HB12 RNAi, transformation with TT8 RNAi could improve forage quality by increasing the availability of both NDF and DM. Further study is needed on the relationship between the transformation-induced structure changes at a molecular level and nutrient utilization in ruminant livestock systems when lignification is much higher.

Wineries and olive oil industries are dominant agro-industrial activities in southern European regions. Olive pomace, exhausted grape marc, and vine shoot trimmings are lignocellulosic residues generated by these industries, which could be valued biotechnologically. In the present work these residues were used as substrate to produce cellulases and xylanases through solid-state fermentation using Aspergillus uvarum MUM 08.01. For that, two factorial designs (3(2)) were first planned to optimize substrate composition, temperature, and initial moisture level. Subsequently, the kinectics of cellulolytic enzyme production, fungal growth, and fermented solid were characterized. Finally, the process was performed in a packed-bed bioreactor. The results showed that cellulase activity improved with the optimization processes, reaching 33.56 U/g, and with the packed-bed bioreactor aeration of 0.2 L/min, reaching 38.51 U/g. The composition of fermented solids indicated their potential use for animal feed because cellulose, hemicellulose, lignin, and phenolic compounds were partially degraded 28.08, 10.78, 13.3, and 28.32%, respectively, crude protein was increased from 8.47 to 17.08%, and the mineral contents meet the requirements of main livestock.

The lignin deposition in the stem of two sugarcane genotypes was assessed on exposure to water stress. The lignin content and the morphoanatomical characterization of the stem indicated that IACSP94-2094 plants are more lignified than those of IACSP95-5000 genotype, under normal water supply conditions, which was especially associated with higher lignin contents in the rind of mature internodes. Water deficit had negative impact on the biomass production, mostly with IACSP94-2094 plants, possibly due to stress severity or higher susceptibility of that genotype during the stem-lengthening phase. Water deficit led to significant alterations in the expression levels of lignin biosynthesis genes and led to an approximate 60% increase of lignin content in the rind of young internodes in both genotypes. It is concluded that the young rind region was more directly affected by water stress and, depending on the genotype, a higher lignin accumulation may occur in the stem, thus implying lower quality biomass for bioethanol production.

A process based on a steam explosion pretreatment and alkali solution post-treatment was applied to fractionate olive stones (whole and fragmented, without seeds) and olive cake into their main constitutive polymers of cellulose (C), hemicelluloses (H), and lignin (L) under optimal conditions for each fraction according to earlier works. The chemical characterization (chromatographic method and UV and IR spectroscopy) and the functional properties (water- and oil-holding capacities, bile acid binding, and glucose retardation index) of each fraction were analyzed. The in vitro studies showed a substantial bile acid binding activity in the fraction containing lignin from olive stones (L) and the alkaline extractable fraction from olive cake (Lp). Lignin bound significantly more bile acid than any other fraction and an amount similar to that bound by cholestyramine (a cholesterol-lowering, bile acid-binding drug), especially when cholic acid (CA) was tested. These results highlight the health-promoting potential of lignin from olive stones and olive cake extracted from olive byproducts.

We have investigated the impact of the brown-midrib bm5 mutation on lignins and on p-coumaric acid and ferulic acid ester-linked to maize (Zea mays L.) cell walls. Lignified stalks or plant aerial parts (without ears) collected at grain maturity were studied in three genetic backgrounds. Relative to the control, bm5 mutants displayed lower levels of lignins and of p-coumarate esters but increased levels of ferulate esters. Thioacidolysis revealed that bm5 lignins display an increased frequency of free-phenolic guaiacyl units. More importantly, thioacidolysis provided unusual amounts of 1,2,2-trithioethyl ethylguaiacol, a marker compound diagnostic for the incorporation of free ferulic acid into lignins by bis 8-O-4 cross-coupling. As the resulting acetal bonding pattern is a chemically labile branch point introduced in maize lignins by the bm5 mutation, this alteration is prone to facilitate the delignification pretreatments used in the cellulose-to-ethanol process.

Brewer's spent grain (BSG), the major side-stream from brewing, is rich in protein, lignin, and nonstarch polysaccharides. Lignin is a polyphenolic macromolecule considered resilient toward breakdown and utilization by colon microbiota, although some indications of release of small phenolic components from lignin in animals have been shown. The aim of this study was to investigate if the human intestinal microbiota can release lignans and small phenolic compounds from whole BSG, a lignin-enriched insoluble fraction from BSG and a deferuloylated fraction, in a metabolic in vitro colon model. The formation of short-chain fatty acid (SCFA) was also investigated. More lignin-related monomers and dilignols were detected from the lignin-enriched fraction than from BSG or deferuloylated BSG. SCFA formation was not suppressed by any of the fractions. It was shown that small lignin-like compounds were released from these samples in the in vitro colon model, originating most likely from lignin.

Lignin is a constituent of plant cell walls and thus is classified as part of dietary fiber. However, little is known about the role of lignin in gastrointestinal fermentation. In this work, a lignin-rich fraction was prepared from brewer's spent grain and subjected to an in vitro colon model to study its potential bioconversions and interactions with fecal microbiota. No suppression of microbial conversion by the fraction was observed in the colon model, as measured as short-chain fatty acid production. Furthermore, no inhibition on the growth was observed when the fraction was incubated with strains of lactobacilli and bifidobacteria. In fact, the lignin-rich fraction enabled bifidobacteria to survive longer than with glucose. Several transiently appearing phenolic compounds, very likely originating from lignin, were observed during the fermentation. This would indicate that the gut microbiota was able to partially degrade lignin and metabolize the released compounds.

This paper presents a prefermentation treatment method involving fungi to improve flavonoid extraction from the leaves of Ginkgo biloba . The fungi employed for this treatment were screened from the soil present under an ancient ginkgo tree. Seventy-six strains belonging to 23 genera were isolated and identified by a molecular identification method employing 18S rDNA sequences. Thirty-three strains grew well using ginkgo leaves as the growth medium. One strain, Gyx086, with higher extracted yield of flavonoids and more similar to the control, was finally selected for prefermentation processing. The major fermentation factors were optimized by response surface methodology. The optimal conditions for the highest total falvonoid yield were 27.8 °C for temperature, 64.2% for moisture content, and 61 h for fermentation time. Under the optimal condition, a actual total flavonoid yield of 27.59 ± 0.52 mg/g dry weight culture sample was obtained, which was about 70% higher than that of unfermented gingko leaf samples.

Ozonolysis is potentially an effective method for pretreating lignocellulosic biomass to improve the production of fermentable sugars via enzymatic hydrolysis. Further understanding of the ozonolysis process and identifying specific lignin structural changes are crucial for improving the pretreatment process. Investigation into pretreatment of wheat straw using ozonolysisis is reported in this paper, with special emphasis on selective modification/degradation of lignin subunits. The ozonolysis was performed for 2 h with less than 60 mesh particles in order to achieve maximum lignin oxidation. The results showed that the lignin structure was significantly modified under these conditions, leading to higher sugar recovery of more than 50% which increased from 13.11% to 63.17% corresponding to the control and ozone treated samples, respectively. Moisture content was found to be an important parameter for improving sugar recovery. Ninety percent (w/w) moisture produced the highest sugar recovery. The concentration of acid soluble lignin in the ozone treated sample increased from 4% to 11% after 2 h treatment. NMR analysis revealed that the S2/6 and G2 lignin units in the wheat straw were most prone to oxidation by ozone as the concentration of aromatic units decreased while the carboxylic acids became more abundant. The experimental data suggest the degradation of β-O-4 moieties and aromatic ring opening in lignin subunits. The pyrolysis-gas chromatography/mass spectrometry results revealed that the rate of lignin unit degradation was in the following order: syringyl > guaiacyl > p-hydroxyphenyl. Long ozone exposure resulted in few condensed lignin structure formation. In addition, the formation of condensed units during this process increased the activation energy from ASTM-E, 259.74 kJ/mol; Friedman-E, 270.08 kJ/mol to ASTM-E, 509.29 kJ/mol; Friedman-E, 462.17 kJ/mol. The results provide new information in overcoming lignin barrier for lignocellulose utilization.

Termites are highly effective in lignocellulose degradation; however, the process of lignin deconstruction along the alimentary canal is not well understood. In this study, the wood metabolites in each gut segment were tentatively analyzed using pyrolysis-gas chromatography-mass spectrometry in the presence of tetramethylammonium hydroxide. Collectively, the significant differences in the pyrolysate distribution among each sample established (1) conservation of the major β-O-4' bonds of lignin during termite digestion, although a selective lignin substructure modification was observed across the whole gut; (2) initiation of lignin-polysaccharide dissociation, aliphatic oxidation/carboxylation, phenolic dehydroxylation in the foregut, and linkage modification of the 5-5', β-5', and β-1' substructures; (3) the continuation of foregut reactions into the midgut with further phenolic carboxylation/demethoxylation/carbonylation; and (4) phenolic/aliphatic esterifications in the hindgut. Overall, elucidation of the stepwise lignin unlocking mechanism in termites provides a valuable insight for understanding plant cell wall structure and its recalcitrance.

The methane yields and conversion of pentoses (xylose) and hexoses (cellulose) in hemp, maize, and white lupin were studied over 30 days of anaerobic digestion. Preservation of hemp increased the methane yield by 23% compared with the fresh hemp. The increased methane yield of hemp was verified by the enhanced conversion of C6 sugars, increasing from 48% to about 70%, whereas the conversion of C5 sugars increased from only 9% to nearly 50%. The consumption of all carbohydrates in fresh maize was almost complete in the 30 days of anaerobic digestion. Hence, there was no major difference in carbohydrate consumption between fresh and preserved maize during biogas production. Fresh white lupin produced the highest methane yield (343 ± 33 dm(3) kg(-1) TS) in this work, mainly due to its highest amount of proteins. Conversion of C6 sugars was 80%, but that of C5 sugars was notably less at 46%.

Apoplastic targeting of secondary metabolites compatible with monolignol polymerization may provide new avenues for designing lignins that are less inhibitory toward fiber fermentation. To identify suitable monolignol substitutes, primary maize cell walls were artificially lignified with normal monolignols plus various epicatechin, quercetin glycoside, and gallate derivatives added as 0 or 45% by weight of the precursor mixture. The flavonoids and gallates had variable effects on peroxidase activity, but all dropped lignification pH. Epigallocatechin gallate, epicatechin gallate, epicatechin vanillate, epigallocatechin, galloylhyperin, and pentagalloylglucose formed wall-bound lignin at moderate to high concentrations, and their incorporation increased 48 h in vitro ruminal fiber fermentability by 20-33% relative to lignified controls. By contrast, ethyl gallate and corilagin severely depressed lignification and increased 48 h fermentability by about 50%. The results suggest several flavonoid and gallate derivatives are promising lignin bioengineering targets for improving the inherent fermentability of nonpretreated cell walls.