High Substrate Loading Research Articles

Fusion of Hydrophobic Anchor Peptides Promotes the Hydrolytic Activity of PETase but not the Extent of PET Depolymerization

Enzymatic recycling of polyethylene terephthalate (PET) has attracted significant attention in recent years. While the fusion of anchor peptides to PET hydrolases is believed to enhance PET hydrolytic activity, a quantitative analysis is yet lacking. Here, we construct four fusion enzymes by fusing anchor peptides (including hydrophobic LCI, LCIM1 and TA2, and hydrophilic EK4) to the C terminus of HotPETase, one of the most active PET hydrolases for high‐crystallinity PET (HC‐PET). Single‐molecule force spectroscopy (SMFS) demonstrates that hydrophobic anchor peptides promote adhesive interactions between the fusion enzymes and the PET surface. This is also validated by the adsorption kinetics and isotherms, and the saturated adsorption capacity remains unaltered compare to HotPETase. At low substrate loadings, the apparent hydrolytic activity of these fusion enzymes is positively related to the hydrophobicity of the anchor peptides. Among them, HotPETase‐LCI stands out as the most effective enzyme for HC‐PET degradation, demonstrating a 1.5‐fold increase in hydrolytic activity. At high substrate loadings, the advantages of fusion with anchor peptides diminish. We conclude that fusion enzymes only facilitate the hydrolytic rates of HC‐PET but have little effect on the final conversion extent.

Improving the catalytic performance of carbonyl reductase based on the functional loops engineering.

Vibegron functions as a potent and selective β3-adrenergic receptor agonist, with its chiral precursor (2S,3R)-aminohydroxy ester (1b) being crucial to its synthesis. In this study, loop engineering was applied to the carbonyl reductase (EaSDR6) from Exiguobacterium algae to achieve an asymmetric reduction of the (rac)-aminoketone ester 1a. The variant M5 (A138L/A190V/S193A/Y201F/N204A) was obtained and demonstrated an 868-fold increase in catalytic efficiency (kcat/Km = 260.3 s-1 mM-1) and a desirable stereoselectivity (>99% enantiomeric excess, e.e.; >99% diastereomeric excess, d.e.) for the target product 1b in contrast to the wild-type EaSDR6 (WT). Structural alignment with WT indicated that loops 137-154 and 182-210 potentially play vital roles in facilitating catalysis and substrate binding. Moreover, molecular dynamics (MD) simulations of WT-1a and M5-1a complex illustrated that M5-1a exhibits a more effective nucleophilic attack distance and more readily adopts a pre-reaction state. The interaction analysis unveiled that M5 enhanced hydrophobic interactions with substrate 1a on cavities A and B while diminishing unfavorable hydrophilic interactions on cavity C. Computational analysis of binding free energies indicated that M5 displayed heightened affinity towards substrate 1a compared to the WT, aligning with its decreased Km value. Under organic-aqueous biphasic conditions, the M5 mutant showed >99% conversion within 12 h with 300 g/L substrate 1a (highest substrate loading as reported). This study enhanced the catalytic performance of carbonyl reductase through functional loops engineering and established a robust framework for the large-scale biosynthesis of the vibegron intermediate.

High-solids saccharification of non-pretreated citrus peels through tailored cellulase

Citrus peels, characterized by their low lignin and high sugar content, have been drawing increasing attention as a valuable lignocellulosic biomass with significant potential in biorefinery. Notably, in this study, the citrus waste was found to be enzymatically accessible without any pretreatment. Moreover, to promote the high-solids saccharification of the citrus peels, a tailored cellulase cocktail was formulated by response surface methodology (RSM), along with a fed-batch strategy aiming to obtain a high substrate loading. The study resulted in an optimized cellulase cocktail (7.08 U/g DM of β-glucosidase, 164.17 U/g DM of hemicellulase, 47.38 mg/g DM of sophorolipid, and 64.68 mg/g DM of Tween 80) and achieved solids loading of 22 % with a total sugar concentration of 123.84 g/L, corresponding to a yield of 93.12 % (65.28 % in batch operation). These findings provided essential validation for the efficient utilization of citrus waste, ensuring them promising potential as feedstock for sugar platforms.

Structure‐Guided Engineering of a Short‐Chain Dehydrogenase LfSDR1 for Efficient Biosynthesis of (<i>R</i>)‐9‐(2‐Hydroxypropyl)adenine, the Key Intermediate of Tenofovir

(<i>R</i>)‐9‐(2‐hydroxypropyl) adenine ((<i>R</i>)‐HPA) is an important intermediate for the synthesis of tenofovir and its prodrugs. Herein, structure‐guided rational design of short‐chain dehydrogenase LfSDR1 was adopted to improve the catalytic performance for enantioselective synthesis of (<i>R</i>)‐HPA at high substrate loading. The crystal structures of LfSDR1 in its apo form as well as in complex with NADPH were solved, which were used for mutagenesis studies and illustration mechanism. Three residues (G92, E141 and V186) were identified as hotspots by structural analysis, and variants V186A/G92V and V186A/G92V/E141L with remarkably improved activity were obtained. By molecular dynamics (MD) simulation of WT and variants, G92V plays a key role in enzyme‐substrate interaction in the binding pocket. Whole cells expressing the mutant LfSDR1‐V186A/G92V and glucose dehydrogenase BsGDH from <i>Bacillus subtilis</i> were used as the catalyst, and up to 200 g L‐1 substrate without cosolvent was completely converted to (R)‐HPA with 99.9% ee and a high space‐time yield (STY) of 800 g L‐1 day‐1. This study improves the understanding of the catalytic mechanism of LfSDR and provides a potential biocatalytic strategy for industrial synthesis of (<i>R</i>)‐HPA.

Toward an environmentally friendly synthesis of chiral γ-lactones: A biocatalytically oxidative desymmetrization pathway

The direct use of oxygen for the enantioselective Baeyer–Villiger oxidation of cyclobutanone represents a promising, environmentally benign method of generating substituted chiral γ-lactones, a recurrent motif in natural product skeletons. We herein demonstrate a highly efficient process for the desymmetrization of cyclobutanones by a Baeyer–Villiger monooxygenase that originated from Acinetobacter radioresistens (ArBVMO). By coupling a formate dehydrogenase to the monooxygenase, the reaction can readily be performed at high substrate loading (200 mM) in buffer/toluene biphasic systems. Scaling up the reaction led to bioconversion with a space-time yield of up to 115 g L−1 day−1, and perfect enantioselectivity (> 99% ee). The discovery of ArBVMO provides a practical synthetic route to γ-lactones, overcoming the problems of poor accessibility and the high cost of waste disposal incurred by previous methodologies.

One-Pot Two-Stage Biocatalytic Cascade to Produce l-Phosphinothricin by Two Enantioselective Complementary Aminotransferases at High Substrate Loading via a Deracemization Process.

The bioderacemization of racemic phosphinothricin (D, L-PPT) is a promising route for the synthesis of l-phosphinothricin (L-PPT). However, the low activity and tolerance of wild-type enzymes restrict their industrial applications. Two stereocomplementary aminotransferases with high activity and substrate tolerance were identified in a metagenomic library, and a one-pot, two-stage artificial cascade biocatalytic system was developed to produce L-PPT through kinetic resolution and asymmetric amination. We observed that 500 mM D, L-PPT (100 g/L) could be converted into L-PPT with 94% final conversion and >99.9% enantiomeric excess (e.e.) within 24 h, with only 0.02 eq amino acceptor pyruvate and 1.2 eq amino donor l-aspartate required. The process could be scaled up to 10 L under sufficient oxygen and stirring. The superior catalytic performance of this system provides an eco-friendly and sustainable approach to the industrial deracemization of D, L-PPT to L-PPT.

High-efficiency anaerobic digestion of soybean curb residue with simultaneous phosphorus recovery by Mg[sbnd]Fe layered double hydroxides addition

Anaerobic digestion of food waste faces several technical problems, such as process failure caused by over-acidification, low heating value of biogas, and high treatment cost of liquid digestate. Layered double hydroxides (LDH) are expected to simultaneously enhance biogas production and quality and immobilize soluble phosphorus in AD due to their excellent anion exchange capability. This study first verified the above hypothesis, indicated by a dramatically enhanced methane yield of 17 folds under a high substrate load and a high quality of generated biogas with 89 % methane content and 90 % lowered hydrogen sulfide. Meanwhile, the soluble phosphorus was efficiently reduced to 3 mg/L after AD. Moreover, the calcined product of LDH showed comparable performance, indicating the possibility of reusing LDH in the AD process. The above results implied the great potential of LDH as a multifunctional material for enhancing the efficiency of waste treatment and energy/resource conversion in the AD process.

Genome and secretome insights: unravelling the lignocellulolytic potential of Myceliophthora verrucosa for enhanced hydrolysis of lignocellulosic biomass.

Lignocellulolytic enzymes from a novel Myceliophthora verrucosa (5DR) strain was found to potentiate the efficacy of benchmark cellulase during saccharification of acid/alkali treated bagasse by ~ 2.24 fold, indicating it to be an important source of auxiliary enzymes. The De-novo sequencing and analysis of M. verrucosa genome (31.7Mb) revealed to encode for 7989 putative genes, representing a wide array of CAZymes (366) with a high proportions of auxiliary activity (AA) genes (76). The LC/MS QTOF based secretome analysis of M. verrucosa showed high abundance of glycosyl hydrolases and AA proteins with cellobiose dehydrogenase (CDH) (AA8), being the most prominent auxiliary protein. A gene coding for lytic polysaccharide monooxygenase (LPMO) was expressed in Pichia pastoris and CDH produced by M. verrucosa culture on rice straw based solidified medium were purified and characterized. The mass spectrometry of LPMO catalyzed hydrolytic products of avicel showed the release of both C1/C4 oxidized products, indicating it to be type-3. The lignocellulolytic cocktail comprising of in-house cellulase produced by Aspergillus allahabadii strain spiked with LPMO & CDH exhibited enhanced and better hydrolysis of mild alkali deacetylated (MAD) and unwashed acid pretreated rice straw slurry (UWAP), when compared to Cellic CTec3 at high substrate loading rate.

Integrated preservation of water activity as key to intensified chemoenzymatic synthesis of bio-based styrene derivatives

The valorization of lignin-derived feedstocks by catalytic means enables their defunctionalization and upgrading to valuable products. However, the development of productive, safe, and low-waste processes remains challenging. This paper explores the industrial potential of a chemoenzymatic reaction performing the decarboxylation of bio-based phenolic acids in wet cyclopentyl methyl ether (CPME) by immobilized phenolic acid decarboxylase from Bacillus subtilis, followed by a base-catalyzed acylation. Key-to-success is the continuous control of water activity, which fluctuates along the reaction progress, particularly at high substrate loadings (triggered by different hydrophilicities of substrate and product). A combination of experimentation, thermodynamic equilibrium calculations, and MD simulations revealed the change in water activity which guided the integration of water reservoirs and allowed process intensification of the previously limiting enzymatic step. With this, the highly concentrated sequential two-step cascade (400 g·L–1) achieves full conversions and affords products in less than 3 h. The chemical step is versatile, accepting different acyl donors, leading to a range of industrially sound products. Importantly, the finding that water activity changes in intensified processes is an academic insight that might explain other deactivations of enzymes when used in non-conventional media.

Enhancing enzymatic saccharification yields of cellulose at high solid loadings by combining different LPMO activities

BackgroundThe polysaccharides in lignocellulosic biomass hold potential for production of biofuels and biochemicals. However, achieving efficient conversion of this resource into fermentable sugars faces challenges, especially when operating at industrially relevant high solid loadings. While it is clear that combining classical hydrolytic enzymes and lytic polysaccharide monooxygenases (LPMOs) is necessary to achieve high saccharification yields, exactly how these enzymes synergize at high solid loadings remains unclear.ResultsAn LPMO-poor cellulase cocktail, Celluclast 1.5 L, was spiked with one or both of two fungal LPMOs from Thermothielavioides terrestris and Thermoascus aurantiacus, TtAA9E and TaAA9A, respectively, to assess their impact on cellulose saccharification efficiency at high dry matter loading, using Avicel and steam-exploded wheat straw as substrates. The results demonstrate that LPMOs can mitigate the reduction in saccharification efficiency associated with high dry matter contents. The positive effect of LPMO inclusion depends on the type of feedstock and the type of LPMO and increases with the increasing dry matter content and reaction time. Furthermore, our results show that chelating free copper, which may leak out of the active site of inactivated LPMOs during saccharification, with EDTA prevents side reactions with in situ generated H2O2 and the reductant (ascorbic acid).ConclusionsThis study shows that sustaining LPMO activity is vital for efficient cellulose solubilization at high substrate loadings. LPMO cleavage of cellulose at high dry matter loadings results in new chain ends and thus increased water accessibility leading to decrystallization of the substrate, all factors making the substrate more accessible to cellulase action. Additionally, this work highlights the importance of preventing LPMO inactivation and its potential detrimental impact on all enzymes in the reaction.

Discovery of Latent Cannabichromene Cyclase Activity in Marine Bacterial Flavoenzymes.

Production of phytocannabinoids remains an area of active scientific interest due to the growing use of cannabis by the public and the underexplored therapeutic potential of the over 100 minor cannabinoids. While phytocannabinoids are biosynthesized by Cannabis sativa and other select plants and fungi, structural analogs and stereoisomers can only be accessed synthetically or through heterologous expression. To date, the bioproduction of cannabinoids has required eukaryotic hosts like yeast since key, native oxidative cyclization enzymes do not express well in bacterial hosts. Here, we report that two marine bacterial flavoenzymes, Clz9 and Tcz9, perform oxidative cyclization reactions on phytocannabinoid precursors to efficiently generate cannabichromene scaffolds. Furthermore, Clz9 and Tcz9 express robustly in bacteria and display significant tolerance to organic solvent and high substrate loading, thereby enabling fermentative production of cannabichromenic acid in Escherichia coli and indicating their potential for biocatalyst development.

Effects of organic loading rate on hydrogen and methane production in a novel two-stage reactor system: performance, enzyme activity and microbial structure

Two-stage anaerobic digestion (AD) system is an efficient technology; however, organic loading rate (OLR), a critical design and process parameter, influences the performance and capacity of digesters. This study investigates the impact of various OLRs on biogas production during co-digestion of food waste, chicken manure and corn straw in an innovatively designed two-stage reactor system, including variations in the enzyme and electron transfer system (ETS) activities and microbial community. The production of hydrogen and methane increased gradually with increasing OLR, with maximum production at 8 g VS/L d (690 mLH2/L d and 690 mLCH4/L d), which is 30–97 % higher than OLR of 1.5 to 6 g VS/L d. However, the maximum methane yield of 535 mL/g VSadded was attained at OLR of 4 g VS/L d consistent with the highest coenzyme F420 activity. Although no substantial ETS activity was seen during the acidogenic stage, the ETS activity decreased with increasing OLR in the methanogenesis stage, indicating that high substrate loading results in low syntrophic methanogenesis. Microbial analysis revealed a more diverse enrichment of acidogens and acetogens at high OLR with a wider range of substrate consumption, resulting in balanced acetoclastic and hydrogenotrophic methanogens (Methanosarcina and Methanothermobacter) in the biomethane process. These findings suggest the strengthening of syntrophic methanogenesis in high substrate-loading AD systems.

Critical evaluation of biochar effects on methane production and process stability in anaerobic digestion

Biochar is an emerging biomaterial for managing residual biomass while simultaneously sequestering carbon. To extend the biochar value chain, applying biochar to enhance anaerobic digestion (AD) processes is gaining attention in the context of a circular economy and cascading use of biomass. However, the comparative effects of various biochar dosages under normal and severe AD conditions are still unclear. To further our understanding of its potential application, this work investigated the impact of adding various biochar dosages on AD processes under normal and high substrate loadings. Three inoculum-to-substrate ratios (ISRs): one representing normal substrate loading (ISR 2) and two representing substrate overloading (ISR 1 and 0.5) were investigated. Each substrate loading rate was tested with a biochar dosage of 0% (control), 10%, and 25% based on substrate volatile solids. The results revealed that under the severe condition of high substrate overload (ISR 0.5), a high biochar dosage of 25% significantly increased cumulative methane production by 5.6% (p = 0.06) when compared to the control. Under the same condition (ISR 0.5, 25%), the time required to achieve a particular extent of ultimate methane potential was significantly reduced (p = 0.04), indicating that the methane production rate was increased. At ISR 0.5, the increase of process stability was also significant with 25% biochar addition, while the control (0%) and 10% biochar addition exhibited high variance among replicates. However, biochar did not affect AD processes under normal substrate loading (ISR 2) and mild substrate overload (ISR 1). Thus, a positive effect of biochar on the AD process was only observed under severe conditions with the highest biochar dosage. Future works should consider optimising substrate loadings and biochar dosages under real conditions when testing the practical application of biochar addition in AD processes.

Efficient asymmetric synthesis of ethyl (R)-3-hydroxybutyrate by recombinant Escherichia coli cells under high substrate loading using eco-friendly ionic liquids as cosolvent.

Ionic liquids (ILs) which synthesized from bio-renewable materials have recently attracted much attention for their applications in biocatalysis. Ethyl (R)-3-hydroxybutyrate ((R)-EHB) as a versatile chiral intermediate is of great interest in pharmaceutical synthesis. This study focuses on evaluating the performances of choline chloride (ChCl)-based and tetramethylammonium (TMA)-based neoteric ILs in the efficient synthesis of (R)-EHB via the bioreduction of ethyl acetoacetate (EAA) at high substrate loading by recombinant Escherichia coli cells. It was found that choline chloride/glutathione (ChCl/GSH, molar ratio 1:1) and tetramethylammonium/cysteine ([TMA][Cys], molar ratio 1:1) as eco-friendly ILs not only enhanced the solubility of water-insoluble EAA in the aqueous buffer system, but also appropriately improved the membrane permeability of recombinant E. coli cells, thus boosting catalytic reduction efficiency of EAA to (R)-EHB. In the developed ChCl/GSH- or [TMA][Cys]-buffer systems, the space-time yields of (R)-EHB achieved 754.9g/L/d and 726.3g/L/d, respectively, which are much higher than neat aqueous buffer system (537.2g/L/d space-time yield). Meanwhile, positive results have also been demonstrated in the bioreduction of other prochiral ketones in the established IL-buffer systems. This work exhibits an efficient bioprocess for (R)-EHB synthesis under 325g/L (2.5M) substrate loading, and provides promising ChCl/GSH- and [TMA][Cys]-buffer systems employed in the biocatalysis for hydrophobic substrate.

Optimisation of β-Glucosidase Production in a Crude Aspergillus japonicus VIT-SB1 Cellulase Cocktail Using One Variable at a Time and Statistical Methods and its Application in Cellulose Hydrolysis.

Pulp and paper mill sludge (PPMS) is currently disposed of into landfills which are reaching their maximum capacity. Valorisation of PPMS by enzymatic hydrolysis using cellulases is an alternative strategy. Existing commercial cellulases are expensive and contain low titres of β-glucosidases. In this study, β-glucosidase production was optimised by Aspergillus japonicus VIT-SB1 to obtain higher β-glucosidase titres using the One Variable at a Time (OVAT), Plackett Burman (PBD), and Box Behnken design (BBD)of experiments and the efficiency of the optimised cellulase cocktail to hydrolyse cellulose was tested. β-Glucosidase production was enhanced from 0.4 to 10.13 U/mL, representing a 25.3-fold increase in production levels after optimisation. The optimal BBD production conditions were 6 days of fermentation at 20 °C, 125 rpm, 1.75% soy peptone, and 1.25% wheat bran in (pH 6.0) buffer. The optimal pH for β-glucosidase activity in the crude cellulase cocktail was (pH 5.0) at 50 °C. Optimal cellulose hydrolysis using the crude cellulase cocktail occurred at longer incubation times, and higher substrate loads and enzyme doses. Cellulose hydrolysis with the A. japonicus VIT-SB1 cellulase cocktail and commercial cellulase cocktails resulted in glucose yields of 15.12 and 12.33 µmol/mL glucose, respectively. Supplementation of the commercial cellulase cocktail with 0.25 U/mg of β-glucosidase resulted in a 19.8% increase in glucose yield.

Green solvents and biocatalysis: A bigger picture

Biocatalysis has matured to be used in an ample array of reaction media, from pure aqueous solutions to (water-free) non-conventional media. In most of the applications solvents are needed as reaction media and/or during the downstream unit to extract and purify the product. Aligning processes with the Green Chemistry principles, some green solvents have been introduced. This article discusses critically the environmental impact that solvents may have in a biocatalytic reaction, from the raw material extraction to the solvent synthesis and to its use in biocatalysis and ultimate disposal. Some of the observed impacts – quantified as kg CO2·kg product−1 – are surely unavoidable, as they depend on the solvent synthesis or on the raw material used. However, the intensification of the biocatalytic reaction (e.g. using higher substrate loadings) and investing efforts in solvent recycling may have a clear impact in the final ecological footprint of the enzymatic process. Instead of qualitatively stating that green solvents are used, experiments in these directions should be performed to provide reaction conditions with minimized environmental metrics for sustainable chemistry.

A detailed genome-scale metabolic model of Clostridium thermocellum investigates sources of pyrophosphate for driving glycolysis

Lignocellulosic biomass is an abundant and renewable source of carbon for chemical manufacturing, yet it is cumbersome in conventional processes. A promising, and increasingly studied, candidate for lignocellulose bioprocessing is the thermophilic anaerobe Clostridium thermocellum given its potential to produce ethanol, organic acids, and hydrogen gas from lignocellulosic biomass under high substrate loading. Possessing an atypical glycolytic pathway which substitutes GTP or pyrophosphate (PPi) for ATP in some steps, including in the energy-investment phase, identification, and manipulation of PPi sources are key to engineering its metabolism. Previous efforts to identify the primary pyrophosphate have been unsuccessful. Here, we explore pyrophosphate metabolism through reconstructing, updating, and analyzing a new genome-scale stoichiometric model for C. thermocellum, iCTH669. Hundreds of changes to the former GEM, iCBI655, including correcting cofactor usages, addressing charge and elemental balance, standardizing biomass composition, and incorporating the latest experimental evidence led to a MEMOTE score improvement to 94%. We found agreement of iCTH669 model predictions across all available fermentation and biomass yield datasets. The feasibility of hundreds of PPi synthesis routes, newly identified and previously proposed, were assessed through the lens of the iCTH669 model including biomass synthesis, tRNA synthesis, newly identified sources, and previously proposed PPi-generating cycles. In all cases, the metabolic cost of PPi synthesis is at best equivalent to investment of one ATP suggesting no direct energetic advantage for the cofactor substitution in C. thermocellum. Even though no unique source of PPi could be gleaned by the model, by combining with gene expression data two most likely scenarios emerge. First, previously investigated PPi sources likely account for most PPi production in wild-type strains. Second, alternate metabolic routes as encoded by iCTH669 can collectively maintain PPi levels even when previously investigated synthesis cycles are disrupted. Model iCTH669 is available at github.com/maranasgroup/iCTH669.

Biological synthesis of ursodeoxycholic acid.

Ursodeoxycholic acid (UDCA) is a fundamental treatment drug for numerous hepatobiliary diseases that also has adjuvant therapeutic effects on certain cancers and neurological diseases. Chemical UDCA synthesis is environmentally unfriendly with low yields. Biological UDCA synthesis by free-enzyme catalysis or whole-cell synthesis using inexpensive and readily available chenodeoxycholic acid (CDCA), cholic acid (CA), or lithocholic acid (LCA) as substrates is being developed. The free enzyme-catalyzed one-pot, one-step/two-step method uses hydroxysteroid dehydrogenase (HSDH); whole-cell synthesis, mainly uses engineered bacteria (mainly Escherichia coli) expressing the relevant HSDHs. To further develop these methods, HSDHs with specific coenzyme dependence, high enzyme activity, good stability, and high substrate loading concentration, P450 monooxygenase with C-7 hydroxylation activity and engineered strain harboring HSDHs must be exploited.

Orientated inhibition of humin formation in efficient production of levulinic acid from cellulose with high substrate loading: Synergistic role of additives

The main bottleneck in the direct conversion of cellulose to levulinic acid (LA), a promising bio-based platform chemical, lies in the severe formation of humins, especially at high substrate loading (>10 wt%). Herein, we report an efficient catalytic system consisting of a 2-methyltetrahydrofuran/water (MTHF/H2O) biphasic solvent with NaCl and cetyltrimethylammonium bromide (CTAB) as additives for converting cellulose (15 wt%) to LA in the presence of a benzenesulfonic acid catalyst. We show that both NaCl and CTAB accelerated the depolymerization of cellulose and formation of LA. However, NaCl favored the humin formation via degradative condensations, whereas CTAB inhibited humin formation by restraining the routes of both degradative and dehydrated condensations. A synergistic role of NaCl and CTAB on suppressing humin formations is illustrated. The combined use of NaCl and CTAB led to an increased LA yield (60.8 mol%) from microcrystalline cellulose in MTHF/H2O (VMTHF/VH2O = 2/1) at 453 K for 2 h. Moreover, it was efficient for converting cellulose fractioned from several kinds of lignocellulosic biomass, wherein a high LA yield of 81.0 mol% was achieved from wheat straw cellulose. This work presents a new strategy for advancing LA biorefinery by synergistically promoting cellulose depolymerization with orientated inhibition of undesired humin formation.

Enhancing methane recovery by intermittent substrate feeding and microbial community response in anaerobic digestion of glycerol

Accumulation of intermediate volatile fatty acids, especially propionic acid, is a limiting factor in anaerobic digestion of glycerol. To mitigate this problem, intermittent substrate feeding was investigated and evaluated. The results showed that intermittent substrate feeding could decrease the effects of high concentrations of propionic acid and improve the performance of CH4 production at high substrate loading rates. CH4 production rate with intermittent substrate feeding (1.60 NL/L/day) was higher than single substrate feeding (1.47 NL/L/day) at 5.0 g COD/L/day of glycerol loading rate. NGS and PICRUSt were applied to monitor the changes in the microbial community. The proportion of dominant bacteria changed with alterations in glycerol loading rate and substrate feeding technique. NGS results elucidated three main bacterial groups that degraded and competed for glycerol, namely: Delftia, Trichococcus, and Desulfuromonas. Trichococcus and Desulfuromonas tended to withstand a higher rate of glycerol loading than Delftia. PICRUSt results demonstrated that Trichococcus is paramount in the glycerol degradation. Delftia and Desulfuromonas might compete with methanogens by utilizing acetate and/or H2. This study suggested the role of microbial communities in CH4 production from glycerol. The intermittent feeding, which balanced organic acid production and its consumption, was clarified to improve methane production from glycerol.