Cellulosic ethanol stillage for methane production by integrating single-chamber anaerobic digestion and microbial electrolysis cell system.

Anaerobic digestion provides a solution to the inefficient use of carbon resources caused by improper disposal of corn stover-based ethanol stillage (CES). In this regard, we developed a single-chamber anaerobic digestion integrated microbial electrolysis cells system (AD-MEC) to convert CES into biogas while simultaneously upgrading biogas in-situ by employing voltages ranging from 0 to 2.5 V.

Sulphite addition during steam pretreatment enhanced both enzyme-mediated cellulose hydrolysis and ethanol production.

Sulphite addition during steam pretreatment of softwoods under acidic, neutral and alkaline conditions was assessed to try to minimize lignin condensation. Although pretreatment under neutral/alkaline conditions resulted in effective lignin sulphonation, non-uniform size reduction was observed. In contrast, acidic sulphite steam treatment at 210 °C for 10 min resulted in homogenous particle size reduction and water-insoluble component that was 62% carbohydrate and 33% lignin.

The production of lactic acid from chemi-thermomechanical pulps using a chemo-catalytic approach.

Previous work has shown that sulfonation and oxidation of chemi-thermomechanical pulps (CTMPs) significantly enhanced enzyme accessibility to cellulose while recovering the majority of carbohydrates in the water-insoluble component. In the work reported here, modified (sulfonated and oxidized) CTMPs derived from hard-and-softwoods were used to produce a DL-mix of lactic acid via a chemo-catalytic approach using lanthanide triflate (Ln (OTf)) catalysts (Ln = La, Nd, Er, and Yb). It was apparent that sulfonation and oxidation of chemi-thermomechanical pulps (CTMPs) also enhanced Ln(OTf) catalyst accessibility to the carbohydrate components of the pulps, with the Er(OTf) catalysts resulting in significant lactic acid production.

Enhancing Enzyme-Mediated Cellulose Hydrolysis by Incorporating Acid Groups Onto the Lignin During Biomass Pretreatment.

Lignin is known to limit the enzyme-mediated hydrolysis of biomass by both restricting substrate swelling and binding to the enzymes. Pretreated mechanical pulp (MP) made from Aspen wood chips was incubated with either 16% sodium sulfite or 32% sodium percarbonate to incorporate similar amounts of sulfonic and carboxylic acid groups onto the lignin (60 mmol/kg substrate) present in the pulp without resulting in significant delignification. When Simon's stain was used to assess potential enzyme accessibility to the cellulose, it was apparent that both post-treatments enhanced accessibility and cellulose hydrolysis.

Non-productive celluase binding onto deep eutectic solvent (DES) extracted lignin from willow and corn stover with inhibitory effects on enzymatic hydrolysis of cellulose.

In this work, deep eutectic solvent (DES) was prepared by mixing choline chloride (ChCl) with lactic acid (LA), and effects of cellulase non-productive binding onto DES-extracted lignin from willow and corn stover on enzymatic hydrolysis of cellulose was investigated. The correlation between hydrolysis yield of cellulose and chemical features of lignin was evaluated, and a potential inhibitory mechanism was proposed. Condensation of lignin was observed during DES treatment, and these condensed aromatic structures had an increased tendency to adsorb enzymes through hydrophobic interactions.

Enzyme-Mediated Lignocellulose Liquefaction Is Highly Substrate-Specific and Influenced by the Substrate Concentration or Rheological Regime.

The high viscosities/yield stresses of lignocellulose slurries makes their industrial processing a significant challenge. However, little is known regarding the degree to which liquefaction and its enzymatic requirements are specific to a substrate's physicochemical and rheological properties. In the work reported here, the substrate- and rheological regime-specificities of liquefaction of various substrates were assessed using real-time in-rheometer viscometry and offline oscillatory rheometry when hydrolyzed by combinations of cellobiohydrolase ( Cel7A), endoglucanase ( Cel45A), glycoside hydrolase (GH) family 10 xylanase, and GH family 11 xylanase.

Alkaline sulfonation and thermomechanical pulping pretreatment of softwood chips and pellets to enhance enzymatic hydrolysis.

To assess the impact of alkalinity on sulfonation and the enzyme-mediated hydrolysis of softwood cellulose, Lodgepole pine chips were impregnated with 8% sodium sulfite and increasing loadings of sodium carbonate before thermomechanical pulping. It was apparent that alkali addition enhanced lignin sulfonation with an additional 4% loading of sodium carbonate proving optimal. TEM indicated that sulfonation predominantly occurred within the secondary-cell-wall lignin, increasing cellulose accessibility to the cellulase enzymes.

The influence of lignin on the effectiveness of using a chemithermomechanical pulping based process to pretreat softwood chips and pellets prior to enzymatic hydrolysis.

Over the last century the pulp and paper sector has assessed various technologies to fractionate woody biomass to produce strong, bright fibers. Several of these processes have also been assessed for their potential to pretreat and fractionate biomass to enhance the subsequent enzymatic hydrolysis of the cellulosic component. Although many of these pretreatments are effective on agricultural residues, softwoods have proven more recalcitrant, primarily due to their high lignin content and structure.

Laccase-mediated hydrophilization of lignin decreases unproductive enzyme binding but limits subsequent enzymatic hydrolysis at high substrate concentrations.

One of the predominant mechanisms by which lignin restricts effective enzymatic deconstruction of lignocellulosic materials is the unproductive adsorption of enzymes. Although this inhibition can be partially mitigated through hydrophilization of lignin during thermochemical pretreatment, these types of treatments could potentially worsen slurry rheology, consequently making it more difficult to process the material at high substrate concentrations. In the work reported here, laccases were used to specifically modify lignin hydrophilicity within steam-pretreated substrate via in situ phenolic compound grafting.

The influence of lignin migration and relocation during steam pretreatment on the enzymatic hydrolysis of softwood and corn stover biomass substrates.

To be effective, steam pretreatment is typically carried out at temperatures/pressures above the glass transition point (Tg) of biomass lignin so that it can partly fluidize and relocate. The relocation of Douglas-fir and corn stover derived lignin was compared with the expectation that, with the corn stover lignin's lower hydrophobicity and molecular weight, it would be more readily fluidized. It was apparent that the Tg of lignin decreased as the moisture increased, with the easier access of steam to the corn stover lignin promoting its plasticization.

Lignin valorization: lignin nanoparticles as high-value bio-additive for multifunctional nanocomposites.

Background: Although conversion of low value but high-volume lignin by-product to its usable form is one of the determinant factors for building an economically feasible integrated lignocellulose biorefinery, it has been challenged by its structural complexity and inhomogeneity. We and others have shown that uniform lignin nanoparticles can be produced from a wide range of technical lignins, despite the varied lignocellulosic biomass and the pretreatment methods/conditions applied. This value-added nanostructure lignin enriched with multifunctional groups can be a promising versatile material platform for various downstream utilizations especially in the emerging nanocomposite fields.

A comparison of various lignin-extraction methods to enhance the accessibility and ease of enzymatic hydrolysis of the cellulosic component of steam-pretreated poplar.

Background: Current single-stage delignification-pretreatment technologies to overcome lignocellulosic biomass recalcitrance are usually achieved at the expense of compromising the recovery of the polysaccharide components, particularly the hemicellulose fraction. One way to enhance overall sugar recovery is to tailor an efficient two-stage pretreatment that can pre-extract the more labile hemicellulose component before subjecting the cellulose-rich residual material to a second-stage delignification process. Previous work had shown that a mild steam pretreatment could recover >65% of the hemicellulose from poplar while limiting the acid-catalysed condensation of lignin.

The influence of lignin on steam pretreatment and mechanical pulping of poplar to achieve high sugar recovery and ease of enzymatic hydrolysis.

With the goal of enhancing overall carbohydrate recovery and reducing enzyme loading refiner mechanical pulping and steam pretreatment (210°C, 5 min) were used to pretreat poplar wood chips. Neutral sulphonation post-treatment indicated that, although the lignin present in the steam pretreated substrate was less reactive, the cellulose-rich, water insoluble component was more accessible to cellulases and Simons stain. This was likely due to lignin relocation as the relative surface lignin measured by X-ray photoelectron spectroscopy increased from 0.

The addition of accessory enzymes enhances the hydrolytic performance of cellulase enzymes at high solid loadings.

The pretreatment process used and the nature of the biomass feedstock will influence the role that accessory enzymes can play in synergistically interacting with cellulases to effectively deconstruct the substrate. The work reported here assessed the possible boosting effects of the xylanase and lytic polysaccharide monooxygenase (AA9, formerly known as GH61) on the hydrolytic potential of cellulase enzyme mixtures during hydrolysis of steam pretreated poplar and corn stover at high (10-20% w/v) substrate concentrations. A higher proportion of xylanase was required when the substrate had a relatively high xylan content and at high substrate concentrations.

Steam pretreatment of agricultural residues facilitates hemicellulose recovery while enhancing enzyme accessibility to cellulose.

The origins of lignocellulosic biomass and the pretreatment used to enhance enzyme accessibility to the cellulosic component are known to be strongly influenced by various substrate characteristics. To assess the impact that fibre properties might have on enzymatic hydrolysis, seven agricultural residues were characterised before and after steam pretreatment using a single pretreatment condition (190°C, 5min, 3% SO2) previously shown to enhance fractionation and hydrolysis of the cellulosic component of corn stover. When the fibre length, width and coarseness, viscosity, water retention value and cellulose crystallinity were monitored, no clear correlation was observed between any single substrate characteristic and the substrate's ease of enzymatic hydrolysis.

Lignin valorization: improving lignin processing in the biorefinery.

Research and development activities directed toward commercial production of cellulosic ethanol have created the opportunity to dramatically increase the transformation of lignin to value-added products. Here, we highlight recent advances in this lignin valorization effort. Discovery of genetic variants in native populations of bioenergy crops and direct manipulation of biosynthesis pathways have produced lignin feedstocks with favorable properties for recovery and downstream conversion.

How effective are traditional methods of compositional analysis in providing an accurate material balance for a range of softwood derived residues?

Background: Forest residues represent an abundant and sustainable source of biomass which could be used as a biorefinery feedstock. Due to the heterogeneity of forest residues, such as hog fuel and bark, one of the expected challenges is to obtain an accurate material balance of these feedstocks. Current compositional analytical methods have been standardised for more homogenous feedstocks such as white wood and agricultural residues.

Fibre size does not appear to influence the ease of enzymatic hydrolysis of organosolv-pretreated softwoods.

To determine the effect of fibre size on enzymatic hydrolysis, organosolv-pretreated lodgepole pine was size-fractionated into six substrates ranging in average size from 0.20 to 3.4mm.

The lignin present in steam pretreated softwood binds enzymes and limits cellulose accessibility.

The influence of cellulose accessibility and protein loading on the efficiency of enzymatic hydrolysis of steam pretreated Douglas-fir was assessed. It was apparent that the lignin component significantly influences the swelling/accessibility of cellulose as at low protein loadings (5FPU/g cellulose), only 16% of the cellulose present in the steam pretreated softwood was hydrolyzed while almost complete hydrolysis was achieved with the delignified substrate. When lignin (isolated from steam pretreated Douglas-fir) was added back in the same proportions it was originally found to the highly accessible and swollen, delignified steam pretreated softwood and to a cellulose control such as Avicel, the hydrolysis yields decreased by 9 and 46%, respectively.

Influence of steam pretreatment severity on post-treatments used to enhance the enzymatic hydrolysis of pretreated softwoods at low enzyme loadings.

It is recognized that some form of post-treatment will usually be required if reasonable hydrolysis yields (>60%) of steam pretreated softwood are to be achieved when using low enzyme loadings (5 FPU/g cellulose). In the work reported here we modified/removed lignin from steam pretreated softwood while investigating the influence that the severity of pretreatment might have on the effectiveness of subsequent post-treatments. Although treatment at a lower severity could provide better overall hemicellulose recovery, post-treatment was not as effective on the cellulosic component.

The effects of increasing swelling and anionic charges on the enzymatic hydrolysis of organosolv-pretreated softwoods at low enzyme loadings.

Organosolv-pretreated Lodgepole pine substrates were physically and chemically treated to increase their hydrophilicity and swelling as these are two substrate attributes which have been shown to improve cellulolytic hydrolysis. Surprisingly, mechanical treatment of the organosolv-treated substrates by PFI-mill refining did not significantly increase hydrolysis yields despite decreases in particle size and crystallinity and increases in swelling. However, sulfonation of the substrate did, significantly, increase enzymatic hydrolysis at loadings of both 5 and 2.

The influence of pretreatment and enzyme loading on the effectiveness of batch and fed-batch hydrolysis of corn stover.

To try to improve hydrolysis yields at elevated solids loadings, a comparison was made between batch and fed-batch addition of fresh substrate at the initial and later phases of hydrolysis. Both ethanol (EPCS) and steam-pretreated corn stover (SPCS) substrates were tested at low (5 FPU) and high (60 FPU) loadings of cellulase per gram of cellulose. The fed-batch addition of fresh substrate resulted in a slight decrease in hydrolysis yields when compared with the corresponding batch reactions.

The isolation, characterization and effect of lignin isolated from steam pretreated Douglas-fir on the enzymatic hydrolysis of cellulose.

Douglas-fir was SO(2)-steam pretreated at different severities (190, 200, and 210°C) to assess the possible negative effect of the residual and isolated lignins on the enzymatic hydrolysis of the steam pretreated substrates. When various isolated lignins were added to the Avicel hydrolysis reactions, the decrease in glucose yields ranged from 15.2% to 29.

Enhancing the enzymatic hydrolysis of lignocellulosic biomass by increasing the carboxylic acid content of the associated lignin.

To assess the effects that the physical and chemical properties of lignin might have on the enzymatic hydrolysis of pretreated lignocellulosic substrates, protease treated lignin (PTL) and cellulolytic enzyme lignin (CEL) fractions, isolated from steam and organosolv pretreated corn stover, poplar, and lodgepole pine, were prepared and characterized. The adsorption of cellulases to the isolated lignin preparations corresponded to a Langmuir adsorption isotherm. It was apparent that, rather than the physical properties of the isolated lignin, the carboxylic acid functionality of the isolated lignin, as determined by FTIR and NMR spectroscopy, had much more of an influence when lignin was added to typical hydrolysis of pure cellulose (Avicel).

Can the same steam pretreatment conditions be used for most softwoods to achieve good, enzymatic hydrolysis and sugar yields?

Wood chips from six different Douglas-fir trees and a representative Lodgepole pine were steam pretreated at a single pretreatment condition (200 °C 4% SO₂ 5 min) which had previously been shown to be effective for Spruce and Lodgepole pine chips. All of the softwood samples responded in a similar fashion with more than 60% of the cellulose hydrolysed after 72 h, at an enzyme loading of 20 FPU/g cellulose. However, when the enzyme loading was reduced to 5FPU, less than 27% of the cellulose was hydrolysed.