Search

Background: Conventional aqueous dilute sulfuric acid (DSA) pretreatment of lignocellulosic biomass facilitates hemicellulose solubilization and can improve subsequent enzymatic digestibility of cellulose to fermentable glucose. However, much of the lignin after DSA pretreatment either remains intact within the cell wall or readily redeposits back onto the biomass surface. This redeposited lignin has been shown to reduce enzyme activity and contribute to rapid enzyme deactivation, thus, necessitating significantly higher enzyme loadings than deemed economical for biofuel production from biomass., Results: In this study, we demonstrate how detrimental lignin redeposition on biomass surface after pretreatment can be prevented by employing Co-solvent Enhanced Lignocellulosic Fractionation (CELF) pretreatment that uses THF-water co-solvents with dilute sulfuric acid to solubilize lignin and overcome limitations of DSA pretreatment. We first find that enzymatic hydrolysis of CELF-pretreated switchgrass can sustain a high enzyme activity over incubation periods as long as 5 weeks with enzyme doses as low as 2 mg protein/g glucan to achieve 90% yield to glucose. A modified Ninhydrin-based protein assay revealed that the free-enzyme concentration in the hydrolysate liquor, related to enzyme activity, remained unchanged over long hydrolysis times. DSA-pretreated switchgrass, by contrast, had a 40% drop in free enzymes in solution during incubation, providing evidence of enzyme deactivation. Furthermore, measurements of enzyme adsorption per gram of lignin suggested that CELF prevented lignin redeposition onto the biomass surface, and the little lignin left in the solids was mostly integral to the original lignin-carbohydrate complex (LCC). Scanning electron micrographs and NMR characterization of lignin supported this observation., Conclusions: Enzymatic hydrolysis of solids from CELF pretreatment of switchgrass at low enzyme loadings was sustained for considerably longer times and reached higher conversions than for DSA solids. Analysis of solids following pretreatment and enzymatic hydrolysis showed that prolonged cellulase activity could be attributed to the limited lignin redeposition on the biomass surface making more enzymes available for hydrolysis of more accessible glucan.

Background: The amount of carbon dioxide in the atmosphere has been on the rise for more than a century. Bioenergy crops are seen by the Intergovernmental Panel on Climate Change as an essential part of the solution to addressing climate change. To understand the potential impact of shrub willow (Salix spp.) crop in the northeast United States, effective and transparent life cycle assessment of these systems needs to occur., Results: Here we show, ethanol produced from the fermentation of sugars from hot water extract of willow grown on cropland can sequester 0.012 ± 0.003 kg CO 2eq MJ -1 for a supply system incorporating summer harvest and storage. Despite decreases in soil organic carbon when willow is instead grown on grassland, the produced fuel still can provide significant climate benefits compared to gasoline., Conclusions: Shrub willow converted to ethanol can be a carbon negative source of transportation fuel when the electricity and heat required for the conversion process are generated from renewable biomass. The sequestration of carbon in the belowground portion of the plants is essential for the negative GHG balance for cropland and low GHG emissions in grassland.

Background: Lignin plays an important role in biochemical conversion of biomass to biofuels. A significant amount of lignin is precipitated on the surface of pretreated substrates after organosolv pretreatment. The effect of this residual lignin on enzymatic hydrolysis has been well understood, however, their effect on subsequent ABE fermentation is still unknown., Results: To determine the effect of residual extractable lignin on acetone-butanol-ethanol (ABE) fermentation in separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) processes, we compared ABE production from ethanol-washed and unwashed substrates. The ethanol organosolv pretreated loblolly pine (OPLP) was used as the substrate. It was observed that butanol production from OPLP-UW (unwashed) and OPLP-W (washed) reached 8.16 and 1.69 g/L, respectively, in SHF. The results showed that ABE production in SHF from OPLP-UW prevents an "acid crash" as compared the OPLP-W. In SSF process, the "acid crash" occurred for both OPLP-W and OPLP-UW. The inhibitory extractable lignin intensified the "acid crash" for OPLP-UW and resulted in less ABE production than OPLP-W. The addition of detoxified prehydrolysates in SSF processes shortened the fermentation time and could potentially prevent the "acid crash"., Conclusions: The results suggested that the residual extractable lignin in high sugar concentration could help ABE production by lowering the metabolic rate and preventing "acid crash" in SHF processes. However, it became unfavorable in SSF due to its inhibition of both enzymatic hydrolysis and ABE fermentation with low initial sugar concentration. It is essential to remove extractable lignin of substrates for ABE production in SSF processes. Also, a higher initial sugar concentration is needed to prevent the "acid crash" in SSF processes., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2020.)

Background: Cellulase adsorbed on cellulose is productive and helpful to produce reducing sugars in enzymatic hydrolysis of lignocellulose; however, cellulase adsorbed on lignin is non-productive. Increasing productive adsorption of cellulase on cellulose would be beneficial in improving enzymatic hydrolysis. Adding lignin that was more hydrophilic in hydrolysis system could increase productive adsorption and promote hydrolysis. However, the effect mechanism is still worth exploring further. In this study, lignosulfonate (LS), a type of hydrophilic lignin, was used to study its effect on cellulosic hydrolysis., Results: The effect of LS on the enzymatic hydrolysis of pure cellulose (Avicel) and lignocellulose [dilute acid (DA) treated sugarcane bagasse (SCB)] was investigated by analyzing enzymatic hydrolysis efficiency, productive and non-productive cellulase adsorptions, zeta potential and particle size distribution of substrates. The result showed that after adding LS, the productive cellulase adsorption on Avicel reduced. Adding LS to Avicel suspension could form the Avicel-LS complexes. The particles were charged more negatively and the average particle size was smaller than Avicel before adding LS. In addition, adding LS to cellulase solution formed the LS-cellulase complexes. For DA-SCB, adding LS decreased the non-productive cellulase adsorption on DA-SCB from 3.92 to 2.99 mg/g lignin and increased the productive adsorption of cellulase on DA-SCB from 2.00 to 3.44 mg/g cellulose. Besides, the addition of LS promoted the formation of LS-lignin complexes and LS-cellulase complexes, and the complexes had more negative charges and smaller average sizes than DA-SCB lignin and cellulase particles before adding LS., Conclusions: In this study, LS inhibited Avicel's hydrolysis, but enhanced DA-SCB's hydrolysis. This stemmed from the fact that LS could bind cellulase and Avicel, and occupied the binding sites of cellulase and Avicel. Thus, a decreased productive adsorption of cellulase on Avicel arose. Regarding DA-SCB, adding LS, which enhanced hydrolysis efficiency of DA-SCB, increased the electrostatic repulsion between DA-SCB lignin and cellulase, and therefore, decreased non-productive adsorption of cellulase on DA-SCB lignin and enhanced productive adsorption of cellulase on DA-SCB cellulose., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2020.)

Background: Lignocellulosic biomass, which is composed of cellulose, hemicellulose and lignin, represents the most abundant renewable carbon source with significant potential for the production of sustainable chemicals and fuels. Current biorefineries focus on cellulose and hemicellulose valorization, whereas lignin is treated as a waste product and is burned to supply energy to the biorefineries. The depolymerization of lignin into well-defined mono-aromatic chemicals suitable for downstream processing is recognized increasingly as an important starting point for lignin valorization. In this study, conversion of all three components of Eucalyptus grandis into the corresponding monomeric chemicals was investigated using solid and acidic catalyst in sequence., Results: Lignin was depolymerized into well-defined monomeric phenols in the first step using a Pd/C catalyst. The maximum phenolic monomers yield of 49.8 wt% was achieved at 240 °C for 4 h under 30 atm H 2 . In the monomers, 4-propanol guaiacol (12.9 wt%) and 4-propanol syringol (31.9 wt%) were identified as the two major phenolic products with 90% selectivity. High retention of cellulose and hemicellulose pulp was also obtained, which was treated with FeCl 3 catalyst to attain 5-hydroxymethylfurfural, levulinic acid and furfural simultaneously. The optimal reaction condition for the co-conversion of hemicellulose and cellulose was established as 190 °C and 100 min, from which furfural and levulinic acid were obtained in 55.9% and 73.6% yields, respectively. Ultimately, 54% of Eucalyptus sawdust can be converted into well-defined chemicals under such an integrated biorefinery method., Conclusions: A two-step process (reductive catalytic fractionation followed by FeCl 3 catalysis) allows the fractionation of all the three biopolymers (cellulose, hemicellulose, and lignin) in Eucalyptus biomass, which provides a promising strategy to make high-value chemicals from sustainable biomass., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2020.)

Background: Biomass fuel has been used to supply heat or crude materials in industry to replace the traditional fossil fuel which was one of the chief causes of climate warming. However, the large-scale utilization of biomass fuel was restricted due to the low density and high hydrophilicity of biomass, which causes the problem of transportation and storage. Therefore, pelletization of biomass was used to improve its fuel density. At present, the biomass pellet was widely used to supply heat, gas or electricity generation via gasification, which supplied clean and sustainable energy for industry. However, the energy consumption during pelletization and high hydrophilicity of pellets were still the problem for the large-scale application of biomass pellet. In this study, hydrothermal carbonization and surfactant played the role of permeation, adsorption and wetting in the solution, which was expected to improve the fuel properties and pelletization effectivity of corn stover., Results: In the article, surfactant (PEG400, Span80, SDBS) was chosen to be combined with wet torrefaction to overcome the drawbacks and improve the pelletization and combustion properties of Corn stover (CS). Especially, hydrothermal carbonization (HTC) combined with surfactant improves the yield of solid products and reduces the ash content of solid product, which was beneficial for reducing the ashes of furnace during gasification. Meanwhile, surfactant promotes the formation of pseudo-lignin and the absorption for oil with low O and high C during HTC, which improves the energy density of solid product. Furthermore, the oil in solid product plays the role of lubricant and binder, which reduces the negative effect of high energy consumption, low bulk density and weak pellets strength caused by HTC during pelletization. HTC combined with surfactant improved the hydrophobicity of pellet as well as grindability due to the modification of solid product. Moreover, surfactant combined with HTC improved the combustion characteristic of solid product such as ignition and burning temperature as well as kinetic parameters due to the bio-oil absorbed and the improvement of surface and porosity., Conclusions: The study supplied a new, less-energy intensive and effective method to improve the pelletization and combustion properties of corn stover via hydrothermal carbonization combined with surfactant, and provided a promising alternative fuel from corn stover ., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2019.)

Background: As a renewable and clean energy carrier, the production of biohydrogen from low-value feedstock such as lignocellulose has increasingly garnered interest. The NADH-dependent reduced ferredoxin:NADP + oxidoreductase (NfnAB) complex catalyzes electron transfer between reduced ferredoxin and NAD(P) + , which is critical for production of NAD(P)H-dependent products such as hydrogen and ethanol. In this study, the effects on end-product formation of deletion of nfnAB from Thermoanaerobacterium aotearoense SCUT27 were investigated., Results: Compared with the parental strain, the NADH/NAD + ratio in the ∆nfnAB mutant was increased. The concentration of hydrogen and ethanol produced increased by (41.1 ± 2.37)% ( p  < 0.01) and (13.24 ± 1.12)% ( p  < 0.01), respectively, while the lactic acid concentration decreased by (11.88 ± 0.96)% ( p  < 0.01) when the ∆nfnAB mutant used glucose as sole carbon source. No obvious inhibition effect was observed for either SCUT27 or SCUT27/ ∆nfnAB when six types of lignocellulose hydrolysate pretreated with dilute acid were used for hydrogen production. Notably, the SCUT27/ ∆nfnAB mutant produced 190.63-209.31 mmol/L hydrogen, with a yield of 1.66-1.77 mol/mol and productivity of 12.71-13.95 mmol/L h from nonsterilized rice straw and corn cob hydrolysates pretreated with dilute acid., Conclusions: The T. aotearoense SCUT27/ ∆nfnAB mutant showed higher hydrogen yield and productivity compared with those of the parental strain. Hence, we demonstrate that deletion of nfnAB from T. aotearoense SCUT27 is an effective approach to improve hydrogen production by redirecting the electron flux, and SCUT27/ ∆nfnAB is a promising candidate strain for efficient biohydrogen production from lignocellulosic hydrolysates., Competing Interests: Competing interestsThe authors declare that they have no competing interests.

Background: Butanol is an important biofuel and chemical. The development of butanol-tolerant strains and the identification of functional butanol-tolerant genes is essential for high-yield bio-butanol production due to the toxicity of butanol., Results: Escherichia coli BW25113 was subjected for the first time to error-prone PCR-based whole-genome shuffling. The resulting mutants BW1847 and BW1857 were found to tolerate 2% (v/v) butanol and short-chain alcohols, including ethanol, isobutanol, and 1-pentanol. The mutants exhibited good stability under butanol stress, indicating that they are potential host strains for the construction of butanol pathways. BW1847 had better butanol tolerance than BW1857 under 0-0.75% (v/v) butanol stress, but showed a lower tolerance than BW1857 under 1.25-2% (v/v) butanol stress. Genome resequencing and PCR confirmation revealed that BW1847 and BW1857 had nine and seven single nucleotide polymorphisms, respectively, and a common 14-kb deletion. Functional complementation experiments of the SNPs and deleted genes demonstrated that the mutations of acrB and rob gene and the deletion of TqsA increased the tolerance of the two mutants to butanol. Genome-wide site-specific mutated strains DT385 ( acrB C 1198 T) and DT900 ( rob AT 686-7 ) also showed significant tolerance to butanol and had higher butanol efflux ability than the control, further demonstrating that their mutations yield an inactive protein that enhances butanol resistance characteristics., Conclusions: Stable E. coli mutants with enhanced short alcohols and high concentrations of butanol tolerance were obtained through a rapid and effective method. The key genes of butanol tolerance in the two mutants were identified by comparative functional genomic analysis., Competing Interests: The authors declare that they have no competing interests.

Background: Dilute oxalic acid pretreatment has drawn much attention because it could selectively hydrolyse the hemicellulose fraction during lignocellulose pretreatment. However, there are few studies focusing on the recovery of oxalic acid. Here, we reported a new approach to recycle oxalic acid used in pretreatment via ethanol extraction., Results: The highest xylose content in hydrolysate was 266.70 mg xylose per 1 g corncob (85.0% yield), which was achieved using 150 mmol/L oxalic acid under the optimized treatment condition (140 °C, 2.5 h). These pretreatment conditions were employed to the subsequent pretreatment using recycled oxalic acid. Oxalic acid in the hydrolysate could be recycled according to the following steps: (1) water was removed via evaporation and vacuum drying, (2) ethanol was used to extract oxalic acid in the remaining mixture, and (3) oxalic acid and ethanol were separated by reduced pressure evaporation. The total xylose yields could be stabilized by intermittent adding oxalic acid, and the yields were in range of 46.7-64.3% in this experiment., Conclusions: This sustainable approach of recycling and reuse of oxalic acid has a significant potential application for replacing traditional dilute mineral acid pretreatment of lignocellulose, which could contribute to reduce CO 2 emissions and the cost of the pretreatment.

Background: The recalcitrant structure of lignocellulosic biomass made it challenging for their bioconversion into biofuels and biochemicals. Pretreatment was required to deconstruct the intact structure by the removal of hemicellulose/lignin, improving the cellulose accessibility of enzyme. Combinatorial pretreatments with liquid hot water/H 2 SO 4 and ethanol/NaOH of sugarcane bagasse were developed to improve enzymatic hydrolysis under mild conditions., Results: After one-step 60% ethanol containing 0.5% NaOH pretreatment with solid to liquid ratio of 1/10, the glucose yield after hydrolysis for 72 h with enzyme dosage of 20 FPU/g substrate was enhanced by 41% and 205% compared to that of NaOH or 60% ethanol pretreated solids, respectively. This improvement was correlated with the removal of hemicellulose and lignin. However, using combinatorial pretreatments with 1% H 2 SO 4 followed by 60% ethanol containing 0.5% NaOH, the highest glucose yield with Tween 80 reached 76%, representing 84.5% of theoretical glucose in pretreated substrate. While retaining similar glucose yield, the addition of Tween 80 capacitated either a reduction of enzyme loading by 50% or shortening hydrolysis time to 24 h. However, the enhancement with the addition of Tween 80 decreased as hydrolysis time was extended., Conclusions: This study demonstrated that a combinatorial pretreatment with 1% H 2 SO 4 followed by 60% ethanol containing 0.5% NaOH had significant effects on improving the enzymatic hydrolysis of sugarcane bagasse. The addition of Tween 80 enabled reducing the enzyme loading or shortening the hydrolysis time. This study provided an economically feasible and mild process for the generation of glucose, which will be subsequently converted to bioethanol and biochemicals.

Background: Cellulase adsorption to lignin is considered a cost barrier for bioethanol production; however, its detailed association mechanism is still not fully understood. In this study, two natural poplar variants with high and low sugar release performance were selected as the low and high recalcitrant raw materials (named L and H , respectively). Three different lignin fractions were extracted using ethanol, followed by p -dioxane and then cellulase treatment from the dilute acid pretreated poplar solids (fraction 1, 2, and 3, respectively)., Results: Each lignin fraction had different physicochemical properties. Ethanol-extracted lignin had the lowest weight average molecular weight, while the molecular weights for the other two lignin fractions were similar. 31 P NMR analysis revealed that lignin fraction with higher molecular weight contained more aliphatic hydroxyl groups and less phenolic hydroxyl groups. Semi-quantitative analysis by 2D HSQC NMR indicated that the lignin fractions isolated from the natural variants had different contents of syringyl (S), guaiacyl (G) and interunit linkages. Lignin extracted by ethanol contained the largest amount of S units, the smallest amounts of G and p -hydroxybenzoate (PB) subunits, while the contents of these lignin subunits in the other two lignin fractions were similar. The lignin fraction obtained after cellulase treatment was primarily comprised of β- O -4 linkages with small amounts of β-5 and β-β linkages. The binding strength of these three lignin fractions obtained by Langmuir equations were in the order of L 1  >  L 3  >  L 2 for the low recalcitrance poplar and H 1  >  H 2  >  H 3 for the high recalcitrance poplar., Conclusions: Overall, adsorption ability of lignin was correlated with the sugar release of poplar. Structural features of lignin were associated with its binding to CBH. For natural poplar variants, lignin fractions with lower molecular weight and polydispersity index (PDI) exhibited more CBH adsorption ability. Lignins with more phenolic hydroxyl groups had higher CBH binding strength. It was also found that lignin fractions with more condensed aromatics adsorbed more CBH likely attributed to stronger hydrophobic interactions.

Background: As a natural renewable biomass, the tea oil fruit hull (TOFH) mainly consists of lignocellulose, together with some bioactive substances. Our earlier work constructed a two-stage solvent-based process, including one aqueous ethanol organosolv extraction and an atmospheric glycerol organosolv (AGO) pretreatment, for bioprocessing of the TOFH into diverse bioproducts. However, the AGO pretreatment is not as selective as expected in removing the lignin from TOFH, resulting in the limited delignification and simultaneously high cellulose loss., Results: In this study, acetic acid organosolv (AAO) pretreatment was optimized with experimental design to fractionate the TOFH selectively. Alkaline hydrogen peroxide (AHP) pretreatment was used for further delignification. Results indicate that the AAO-AHP pretreatment had an extremely good selectivity at component fractionation, resulting in 92% delignification and 88% hemicellulose removal, with 87% cellulose retention. The pretreated substrate presented a remarkable enzymatic hydrolysis of 85% for 48 h at a low cellulase loading of 3 FPU/g dry mass. The hydrolyzability was correlated with the composition and structure of substrates by using scanning electron microscopy, confocal laser scanning microscopy, and X-ray diffraction., Conclusion: The mild AAO-AHP pretreatment is an environmentally benign and advantageous scheme for biorefinery of the agroforestry biomass into value-added bioproducts.

Background: Bamboo is a highly abundant source of biomass which is underutilized despite having a chemical composition and fiber structure similar as wood. The main challenge for the industrial processing of bamboo is the high level of silica, which forms water-insoluble precipitates negetively affecting the process systems. A cost-competitive and eco-friendly scheme for the production of high-purity dissolving grade pulp from bamboo not only requires a process for silica removal, but also needs to fully utilize all of the materials dissolved in the process which includes lignin, and cellulosic and hemicellulosic sugars as well as the silica. Many investigations have been carried out to resolve the silica issue, but none of them has led to a commercial process. In this work, alkaline pretreatment of bamboo was conducted to extract silica prior to pulping process. The silica-free substrate was used to produce high-grade dissolving pulp. The dissolved silica, lignin, hemicellulosic sugars, and degraded cellulose in the spent liquors obtained from alkaline pretreatment and pulping process were recovered for providing high-value bio-based chemicals and fuel., Results: An integrated process which combines dissolving pulp production with the recovery of excellent sustainable biofuel and biochemical feedstocks is presented in this work. Pretreatment at 95 °C with 12% NaOH charge for 150 min extracted all the silica and about 30% of the hemicellulose from bamboo. After kraft pulping, xylanase treatment and cold caustic extraction, pulp with hemicellulose content of about 3.5% was obtained. This pulp, after bleaching, provided a cellulose acetate grade dissolving pulp with α-cellulose content higher than 97% and hemicellulose content less than 2%. The amount of silica and lignin that could be recovered from the process corresponded to 95 and 77.86% of the two components in the original chips, respectively. Enzymatic hydrolysis and fermentation of the concentrated and detoxified sugar mixture liquor showed that an ethanol recovery of 0.46 g/g sugar was achieved with 93.2% of hydrolyzed sugars being consumed. A mass balance of the overall process showed that 76.59 g of solids was recovered from 100 g (o.d.) of green bamboo., Conclusions: The present work proposes an integrated biorefinery process that contains alkaline pre-extraction, kraft pulping, enzyme treatment and cold caustic extraction for the production of high-grade dissolving pulp and recovery of silica, lignin, and hemicellulose from bamboo. This process could alleviate the silica-associated challenges and provide feedstocks for bio-based products, thereby allowing the improvement and expansion of bamboo utilization in industrial processes.

Background: The biomass recalcitrance resulting from its chemical compositions and physical structures impedes the conversion of biomass into fermentable sugars. Pretreatment is a necessary procedure to increase the cellulase accessibility for bioconversion of lignocelluloses into bioethanol. Alternatively, ionic liquids, a series of promising solvents, provide unique opportunities for pretreating a wide range of lignocellulosic materials. In this study, a two-step treatment including ionic liquids pretreatment and successive alkali fractionations was performed on Eucalyptus to achieve a high enzymatic digestibility. The compositional and structural changes of Eucalyptus cell walls and their possible effect on saccharification ratio were comprehensively investigated., Results: After the ionic liquids pretreatment, the cell walls became loose and even swelled, accompanying with the decrease of cellulose crystallinity. As compared to the simplex ionic liquids pretreatment, the integrated process resulted in the significant removal of hemicelluloses and lignin, enhancing the disruption of the cell walls and increasing the exposure of cellulose, which led to a higher conversion of cellulose to glucose. The glucose yield of Eucalyptus underwent the combination of [Bmim]OAc and alkali treatments reached the maximum (90.53 %), which was 6.6 times higher than that of the untreated Eucalyptus. The combination of chemical compositions and physical structure of Eucalyptus affected the efficiency of cellulose enzymatic hydrolysis. Especially, the changes of cellulose crystallinity played a major role in enhancing the enzymatic digestibility of Eucalyptus in this study., Conclusions: The two-step treatment with ionic liquids pretreatment and successive alkali fractionation can be considered as a promising method to improve the conversion of cellulose to glucose. The detailed information obtained about chemical and anatomical changes was helpful to understand the underlying mechanism of the integrated treatment process acting on Eucalyptus for enhancing enzymatic digestibility.

Background: Previous work has demonstrated that glycerol organosolv pretreatment can effectively improve the hydrolyzability of various lignocellulosic substrates. This pretreatment process strategy is ideal to integrate a commercially successful lignocellulosic and vegetable oil biorefinery industry. However, industrially relevant high-solid-loading hydrolyzability and fermentability of the pretreated substrates have yet to be considered for enzyme-based lignocellulosic biorefineries., Results: In this study, an AGO pretreatment of sugarcane bagasse was evaluated with regard to the component selectivity, structural modification, hydrolyzability, and fermentation of pretreated substrates. The results showed that the AGO pretreatment presented good component selectivity, removing approximately 70 % lignin and hemicellulose, respectively, from sugarcane bagasse with a near-intact preservation (94 %) of the overall cellulose. The pretreatment deconstructed the recalcitrant architecture of natural lignocellulosic biomass, thereby modifying the structure at the macro-/micrometer level (fiber size, surface area, average size, roughness) and supermolecular level (key chemical bond dissociation) of lignocellulosic substrates towards good hydrolyzability. Notably, extraordinarily few fermentation inhibitors (<0.2 g furfural and 5-hydromethyl furfural/kg feedstock) were generated from the AGO pretreatment process, which was apparently due to the prominent role of glycerol organic solvent in protecting monosaccharides against further degradation. The 72-h enzymatic hydrolysis of pretreated substrates at 15 % solid content achieved 90 % completion with Cellic CTec2 at 10 FPU/g dried substrate. With a simple nutrition (only 10 g/L (NH4)2SO4) addition, the fed-batch semi-SSF of AGO-pretreated substrates (30 % solid content) almost reached 50 g/L ethanol with cellulase preparation at 10 FPU/g dried substrate. These results have revealed that the pretreated substrate is susceptible and accessible to cellulase enzymes, thereafter exhibiting remarkable hydrolyzability and fermentability., Conclusion: The AGO pretreatment is a promising candidate for the current pretreatment process towards industrially relevant enzyme-based lignocellulosic biorefineries.

Background: Lignocellulosic biomass from softwood represents a valuable resource for the production of biofuels and bio-based materials as alternatives to traditional pulp and paper products. Hemicelluloses constitute an extremely heterogeneous fraction of the plant cell wall, as their molecular structures involve multiple monosaccharide components, glycosidic linkages, and decoration patterns. The complete enzymatic hydrolysis of wood hemicelluloses into monosaccharides is therefore a complex biochemical process that requires the activities of multiple degradative enzymes with complementary activities tailored to the structural features of a particular substrate. Glucuronoarabinoxylan (GAX) is a major hemicellulose component in softwood, and its structural complexity requires more enzyme specificities to achieve complete hydrolysis compared to glucuronoxylans from hardwood and arabinoxylans from grasses., Results: We report the characterisation of a recombinant α-glucuronidase (Agu115) from Schizophyllum commune capable of removing (4-O-methyl)-glucuronic acid ((Me)GlcA) residues from polymeric and oligomeric xylan. The enzyme is required for the complete deconstruction of spruce glucuronoarabinoxylan (GAX) and acts synergistically with other xylan-degrading enzymes, specifically a xylanase (Xyn10C), an α-l-arabinofuranosidase (AbfA), and a β-xylosidase (XynB). Each enzyme in this mixture showed varying degrees of potentiation by the other activities, likely due to increased physical access to their respective target monosaccharides. The exo-acting Agu115 and AbfA were unable to remove all of their respective target side chain decorations from GAX, but their specific activity was significantly boosted by the addition of the endo-Xyn10C xylanase. We demonstrate that the proposed enzymatic cocktail (Agu115 with AbfA, Xyn10C and XynB) achieved almost complete conversion of GAX to arabinofuranose (Araf), xylopyranose (Xylp), and MeGlcA monosaccharides. Addition of Agu115 to the enzymatic cocktail contributes specifically to 25 % of the conversion. However, traces of residual oligosaccharides resistant to this combination of enzymes were still present after deconstruction, due to steric hindrances to enzyme access to the substrate., Conclusions: Our GH115 α-glucuronidase is capable of finely tailoring the molecular structure of softwood GAX, and contributes to the almost complete saccharification of GAX in synergy with other exo- and endo-xylan-acting enzymes. This has great relevance for the cost-efficient production of biofuels from softwood lignocellulose.

Background: Woody biomass is highly recalcitrant to enzymatic sugar release and often requires significant size reduction and severe pretreatments to achieve economically viable sugar yields in biological production of sustainable fuels and chemicals. However, because mechanical size reduction of woody biomass can consume significant amounts of energy, it is desirable to minimize size reduction and instead pretreat larger wood chips prior to biological conversion. To date, however, most laboratory research has been performed on materials that are significantly smaller than applicable in a commercial setting. As a result, there is a limited understanding of the effects that larger biomass particle size has on the effectiveness of steam explosion pretreatment and subsequent enzymatic hydrolysis of wood chips., Results: To address these concerns, novel downscaled analysis and high throughput pretreatment and hydrolysis (HTPH) were applied to examine whether differences exist in the composition and digestibility within a single pretreated wood chip due to heterogeneous pretreatment across its thickness. Heat transfer modeling, Simons' stain testing, magnetic resonance imaging (MRI), and scanning electron microscopy (SEM) were applied to probe the effects of pretreatment within and between pretreated wood samples to shed light on potential causes of variation, pointing to enzyme accessibility (i.e., pore size) distribution being a key factor dictating enzyme digestibility in these samples. Application of these techniques demonstrated that the effectiveness of pretreatment of Populus tremuloides can vary substantially over the chip thickness at short pretreatment times, resulting in spatial digestibility effects and overall lower sugar yields in subsequent enzymatic hydrolysis., Conclusions: These results indicate that rapid decompression pretreatments (e.g., steam explosion) that specifically alter accessibility at lower temperature conditions are well suited for larger wood chips due to the non-uniformity in temperature and digestibility profiles that can result from high temperature and short pretreatment times. Furthermore, this study also demonstrated that wood chips were hydrated primarily through the natural pore structure during pretreatment, suggesting that preserving the natural grain and transport systems in wood during storage and chipping processes could likely promote pretreatment efficacy and uniformity.

Background: Corncob as one of the most suitable feedstock for the production of a variety of high-value-added chemicals is receiving increasing attention worldwide because of the characteristics of high carbohydrate (cellulose and hemicelluloses) contents and high energy densities. Furfural produced from hemicelluloses is a highly versatile and key feedstock used in the manufacture of a wide range of biofuel and important chemicals in different fields. Achieving high furfural yields from corncob combining green approaches and efficient equipment has the promising potential for biomass-to-biofuel technologies. To understand the dissolving mechanism of corncob sugars and reveal the relationship between the hydrolysate composition and furfural yields, a two-step approach was proposed using microwave-assisted hydrothermal pretreatment and subsequently heterogeneous catalytic process., Results: Released hemicelluloses in the first stage were mainly in forms of monosaccharide, oligosaccharides, and water-soluble polysaccharide. Hydrolysates with the maximum xylose content (99.94 mg g(-1), 160 °C, 90 min), the maximum xylobiose content (20.89 mg g(-1), 180 °C, 15 min), and the maximum total xylose content in monosaccharide and oligosaccharides (DP ≤ 6) (272.06 mg g(-1), 160 °C, 60 min) were further converted to furfural using tin-loaded montmorillonite as the catalyst in a biphasic system. The highest furfural yield (57.80 %) was obtained at 190 °C for 10 min from hydrolysates with the maximum xylose content. Moreover, controlled experiments showed that furfural yields from corncob hydrolysates were higher than those from the pure xylose solutions, and lower initial xylose concentration may be in favor of the furfural production., Conclusions: This work provides an efficient approach to produce furfural by a two-step process for the biomass-to-biofuel industry. Results indicated that the production of furfural from biomass raw materials can be controlled by the depolymerization degree of hemicelluloses.

Background: Microbial lipid is a potential raw material for large-scale biodiesel production and lignocellulosic hydrolysate has been considered as promising low-cost substrate for lipid fermentation. Lignocellulosic biomass needs to be pretreated before enzymatic hydrolysis, and biocompatible cholinium ionic liquids (ILs) have been demonstrated to be highly efficient for pretreatment. However, the impact of these ILs residues in hydrolysates on downstream biotransformation remains unknown. Therefore, the influence of three typical cholinium ILs on the lipid production by Trichosporon fermentans was first investigated., Results: The cell growth of T. fermentans was stimulated in the presence of cholinium lysine ([Ch][Lys]) and cholinium serine ([Ch][Ser]), while the lipid accumulation was inhibited by [Ch][Lys]) and [Ch][Ser]. Both cell growth and lipid accumulation of T. fermentans were inhibited in the presence of cholinium acetate ([Ch][OAc]). Despite the reduction in lipid content, the lipid production by T. fermentans was improved in the presence of low concentrations of [Ch][Lys] (≤30 mM) and [Ch][Ser] (≤20 mM) due to the remarkable increase of biomass. It was found that cholinium cation had minor influence on lipid production. However, the anions of [Ch][Lys] and [Ch][Ser] could be assimilated as nitrogen source by T. fermentans and the reduced C/N ratio accounts for the inhibition of lipid accumulation, which could be alleviated by improving C/N ratio of medium. In addition, the anion of [Ch][OAc] could be metabolized by T. fermentans, leading to a rapid alkaline-pH shift and strong inhibition of lipid production. And this inhibitory effect on lipid production could be significantly reduced by controlling culture pH., Conclusions: The anions of [Ch][Lys], [Ch][Ser] and [Ch][OAc] play an important role in affecting the cell growth and lipid accumulation of T. fermentans, and the inhibition of these three ILs on lipid production can be alleviated by careful fermentation condition control. Hence, T. fermentans is a promising strain for microbial lipid production from cholinium ILs-pretreated lignocellulosic hydrolysates.

Background: Forest residue is one of the most cost-effective feedstock for biofuel production. It has relatively high bulk density and can be harvested year round, advantageous for reducing transportation cost and eliminating onsite storage. However, forest residues, especially those from softwood species, are highly recalcitrant to biochemical conversion. A severe pretreatment for removing this recalcitrance can result in increased sugar degradation to inhibitors and hence cause difficulties in fermentation at high solid loadings. Here, we presented high titer ethanol production from Douglas-fir forest residue without detoxification. The strong recalcitrance of the Douglas-fir residue was removed by sulfite pretreatment to overcome the recalcitrance of lignocelluloses (SPORL). Sugar degradation to inhibitors was substantially reduced using a novel approach of "pH profiling" by delaying acid application in pretreatment, which facilitated the simultaneous enzymatic saccharification and fermentation of undetoxified whole slurry at a solid loading of 21%., Results: "pH profiling" reduced furan production by approximately 70% in using SPORL pretreating Douglas-fir forest residue (FS-10) comparing with the control run while without sacrificing enzymatic saccharification of the resultant substrate. pH profiling also reduced carbohydrate degradation. The improved carbohydrate yield in pretreated solids and reduced fermentation inhibitors with pH profiling resulted in a terminal ethanol titer of 48.9 ± 1.4 g/L and yield of 297 ± 9 L/tonne FS-10, which are substantially higher, i.e., by 27% in titer and by 38% in yield, than those of a control SPORL run without pH profiling., Conclusions: Economical and large-volume production of commodity biofuels requires the utilization of feedstocks with low value (therefore low cost) and sustainably producible in large quantities, such as forest residues. However, most existing pretreatment technologies cannot remove the strong recalcitrance of forest residues to produce practically fermentable high titer sugars. Here, we demonstrated a commercially scalable and efficient technology capable of removing the strong recalcitrant nature of forest residues using "pH profiling" together with "low temperature SPORL". The resultant pretreated whole slurry of a Douglas-fir forest residue using this technology can be easily processed at high solids of 21% without detoxification to achieve a high ethanol yield of 297 L/tonne at 48.9 g/L. Graphical AbstractGraphic table of content.

Background: The recalcitrance of lignocellulosic biomass is a major limitation for its conversion into biofuels by enzymatic hydrolysis. The use of a pretreatment technology is an essential step to diminish biomass recalcitrance for bioethanol production. In this study, a two-step pretreatment using hydrothermal pretreatment at various temperatures and alkali fractionation was performed on eucalyptus fiber. The detailed chemical composition, physicochemical characteristics, and morphology of the pretreated fibers in each of the fractions were evaluated to advance the performance of eucalyptus fiber in enzymatic digestibility., Results: The hydrothermal pretreatment (100 to 220°C) significantly degraded hemicelluloses, resulting in an increased crystallinity of the pretreated fibers. However, as the pretreatment temperature reached 240°C, partial cellulose was degraded, resulting in a reduced crystallinity of cellulose. As compared to the hydrothermal pretreatment alone, a combination of hydrothermal and alkali treatments significantly removed hemicelluloses and lignin, resulting in an improved enzymatic hydrolysis of the cellulose-rich fractions. As compared with the raw fiber, the enzymatic hydrolysis rate increased 1.1 to 8.5 times as the hydrothermal pretreatment temperature increased from 100 to 240°C. Interestingly, after a combination of hydrothermal pretreatment and alkali fractionation, the enzymatic hydrolysis rate increased 3.7 to 9.2 times. Taking into consideration the consumption of energy and the production of xylo-oligosaccharides and lignin, an optimum pretreatment condition was found to be hydrothermal pretreatment at 180°C for 30 min and alkali fractionation with 2% NaOH at 90°C for 2.5 h, in which 66.3% cellulose was converted into glucose by enzymatic hydrolysis., Conclusions: The combination of hydrothermal pretreatment and alkali fractionation was a promising method to remove hemicelluloses and lignin as well as overcome the biomass recalcitrance for enzymatic hydrolysis from eucalyptus fiber. In addition, the various techniques applied in this work constituted an efficient approach to understand the underlying chemical and morphological changes of the cellulose-rich fractions.