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Effects of extractable lignin removal on enzymatic hydrolysis of a EP25, and b EP50. *p < 0.05, **p < 0.01 vs EP25 and EP50
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Background
The presence of lignin normally affects enzymatic saccharification of lignocellulose detrimentally. However, positive effects of lignin on enzymatic hydrolysis have been recently reported. Enzyme–lignin interactions could be the key to reveal the underlying mechanism of their discrepant behaviors. In this study, to elucidate the positive...
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Context 1
... evaluate the effects of EL removal on the enzymatic digestibility, enzymatic hydrolysis was performed on each of the four pretreated substrates (Fig. 2). The results Page 7 of 12 Lai et al. Biotechnol Biofuels (2019) 12:57 showed that EL removal decreased the 72-h hydrolysis yields from 43.6% (EP25) to 36.9% (EP25-EW), and from 50.0% (EP50) to 42.5% (EP50-EW). This suggests that EL removal decreased the enzymatic hydrolysis efficiency of EP25 and EP50. A similar result has been ...
Citations
... Additionally, the glucose yield of the ethanol-washed substrate was lower (12%-19%) than that of the water-washed substrate after 72 h of enzymatic hydrolysis. This is mainly caused by the fact that ethanol washing removes the extractable lignin deposited on the pretreated substrate surface, which in turn exposes the natural lignin that exhibits greater adsorption by the cellulase enzyme [37,38]. In conclusion, the proposed addition of PEG during GO pretreatment strategies holds promise for the development of economically viable and cost-effective enzyme-mediated biorefinery processes. ...
... Other studies explored the effects of alcohol hydroxyl, syringyl/ guaiacyl (S/G) ratio, β-β, β-5, etc. on the enzymatic hydrolysis of cellulose, but further investigations are needed to reach solid conclusions [101][102][103][104]. It should be noted that the effect of lignin structure on enzymatic hydrolysis is greatly affected by the content and distribution of lignin in substrates [24,26]. ...
Hydrolyzing lignocellulose, the most abundant biomass raw material in nature, into monosaccharides by enzymes and then transforming the sugars into chemicals and fuels by biological or chemical methods is one of the most important parts of biorefining. In lignocellulosic biomass, cellulose is often tightly wrapped by lignin along with hemicelluloses. Although some pretreatments can partially remove lignin, residual lignin in pretreated substrates still physically blocks the accessibility of cellulose, non-productively adsorbs cellulases, and sterically hinders the enzymatic hydrolysis of cellulose. So, lignin is generally considered to be an inhibitor to the enzy-matic hydrolysis of lignocellulosic substrates. However, new research shows that under the right conditions, lignin does not inhibit the enzymatic hydrolysis of lignocellulose, and can even increase the final sugar yield. For example, recently, some novel lignin-target pretreatments have been developed, they can reduce or overcome the inhibitory effect of lignin. And researchers also find that lignin can be functionalized and used as an activator/ promoter of enzymatic hydrolysis of lignocellulose, their effectiveness is comparable to common high-efficiency surfactants. What's more, different lignin-based carriers were also synthesized to carry and recycle cellulases during the hydrolysis, they can obviously reduce the load and cost of cellulases. These works indicate that lignin can play a positive role in the enzymatic hydrolysis of lignocellulose. In order for readers to dialectically view lignin and understand its multiplicity, this review is to report the progress in the studies of how researchers tame the "lignin dog" to facilitate enzymatic hydrolysis of lignocellulose.
... The former lignin was more depolymerized and less condensed, thus resulting in lower ineffective adsorption of the cellulase enzymes. Recently, the removal of these extractable lignin fragments from the substrate by ethanol washing was surprisingly observed to have an adverse effect on the enzymatic digestibility of organosolv-pretreated sweetgum [37]. In short, the extractable lignin fragments deposited on the substrate surface alleviated the occurrence of enzyme non-productive binding by sheltering the enzyme binding sites on the residual bulk lignin. ...
Glycerol organosolv (GO) pretreatment is revealed to be potent in selectively deconstructing the lignocellulosic biomass and effectively enhancing its enzymatic hydrolysis, but the conventional solid washing and GO lignin extraction processes frequently consume large amounts of water, resulting additionally in the difficulty of recycling the glycerol. In this study, an anhydrous two-step organosolv pretreatment process was explored, followed by the membrane ultrafiltration of glycerol lignin. Results showed that the solid washing of the residual glycerol after the atmospheric glycerol organosolv (AGO) pretreatment was necessary for the subsequent operation of high-solid enzymatic hydrolysis. Washing with ethanol was desired as an alternative to water as only a low glycerol content of 5.2% resided in the substrate. Membrane ultrafiltration was helpful in extracting the AGO lignin from the pretreatment liquor, in which a high lignin extraction of 81.5% was made with a regenerated cellulose membrane (cut-off for 1 kDa) under selected ultrafiltration conditions. With the characterization of membrane-extracted lignin, it was observed for the first time that the AGO lignin had a well-preserved structure of G/S type. Moreover, the lignin was enriched with reactive groups, i.e., β-O-4´ linkages and aliphatic hydroxyl groups, which was very likely owing to the glycerol grafting onto the lignin via α-etherification reaction. The two-step organosolv pretreatment process allowed 86% of glycerol and 92% of the ethanol recovery with ~78% of distillation energy-savings, which was applicable for extraction of organosolv lignin and recycling use of organic solvents.
... Such reduction of enzymatic hydrolysis upon post lignin extraction after pretreatment was also observed for organosolv pretreatment (Lai et al., 2015(Lai et al., , 2014 and dilute acid pretreatment (Lin et al., 2021;Jia et al., 2021) The extractable lignin from the pretreated substrate was also reported to promote the enzyme digestion of Avicel (Lai et al., 2015;Lin et at, 2021;Jia et al., 2020) and pretreated biomass (Lai et al., 2015(Lai et al., , 2014Lai et al., 2018). The hypothesis was that the presence of extractable lignin in pretreated substrate blocks the non-productive binding between the enzyme and residual bulk lignin (Lai et al., 2019). However, this hypothesis could not explain the positive effect that extractable lignin had on the enzymatic hydrolysis of Avicel, as Avicel is purified microcrystalline cellulose bearing no lignin. ...
The role of solvent extractable lignin in enzymatic hydrolysis of hydrothermally pretreated sweetgum was investigated. Lignin extraction with acetone, methanol or acetone/methanol removed 27-33% of lignin in hy-drothermally pretreated sweetgum pulp, resulting in total sugar yield reduction. Comprehensive characterization indicated that the extractable lignin is highly degraded with low molecular weight, high phenolic hydroxyls, and low native lignin interunit linkages and exhibited neither stimulation nor inhibition effects on enzymatic hy-drolysis of filter paper, Avicel, and bleached eucalyptus kraft pulp. The extractable lignin was mainly deposited on the surface of the fibers and pores, which removal caused significant morphological change and collapse of the mesopores and macropores. Thus, the reduction in enzymatic digestion efficiency was more likely caused by the pore collapse in fiber and decreased accessible area, rather than the stimulative action of the extractable lignin.
... As the mechanisms behind the beneficial effect of prior, non-catalytic proteins adsorption onto biomass substrates has yet to be fully elucidated, a softwood derived Kraft lignin and a hardwood derived organosolv lignin were assessed for their ability to preferentially bind several non-catalytic proteins and cellulases. As quartz Crystal Microbalance with Dissipation monitoring (QCM-D) has been successfully used to monitor the adsorption of cellulase and hemicellulase enzymes onto lignin films that were spin-coated onto a QCM sensor (Kellock et al., 2017;Lai et al., 2019;Rahikainen et al., 2013b;Song et al., 2017) it was used to assess the preferential binding of various "blocking" proteins and cellulases onto the different lignin films which had been spin-coated onto the QCM sensor. This helped capture any changes in frequency (Δf) that resulted from any mass changes at the surface of the sensor, reflecting the relative adsorption of non-catalytic proteins and/or cellulase enzymes. ...
... Hydroxyl group characterization based on quantitative 31 P NMR analysis (mmol/g) Molecular weight analyzed by GPC Dissipation monitoring (QCM-D) was used to monitor the adsorption and desorption of non-catalytic (blocking) proteins and enzymes onto lignin films. Previous work had shown that QCM-D could provide realtime monitoring of enzyme adsorption onto lignin films that were spin-coated onto a QCM sensor (Kellock et al., 2017;Lai et al., 2019). When the non-catalytic BSA, lysozyme and ovalbumin proteins were added to the lignin films, followed by enzyme addition, two stages were observed, a non-catalytic (blocking) protein adsorption stage and an enzyme adsorption stage, facilitated by buffer rinsing (Fig. 3). ...
The pre-adsorption of non-catalytic/blocking proteins onto the lignin component of pretreated biomass has been shown to significantly increase the effectiveness of subsequent enzyme-mediated hydrolysis of the cellulose by limiting non-productive enzyme adsorption. Layer-by-layer adsorption of non-catalytic proteins and enzymes onto lignin was monitored using Quartz Crystal Micro balancing combined with Dissipation monitoring (QCM-D) and conventional protein adsorption. These methods were used to assess the interaction between soft/hardwood lignins, cellulases and the three non-catalytic proteins BSA, lysozyme and ovalbumin. The QCM-D analysis showed higher adsorption rates for all of the non-catalytic proteins onto the lignin films as compared to cellulases. This suggested that the “blocking” proteins would preferentially adsorb to the lignin rather than the enzymes. Pre-incubation of the lignin films with blocking proteins resulted in reduced adsorption of cellulases onto the lignin, significantly enhancing cellulose hydrolysis.
... These lignin structural changes during pretreatment exacerbate the lignin heterogeneity. Our previous studies have observed that the residual lignins from acid pretreated or ethanol organosolv pretreated materials could be fractionated into two distinct fractions [16]. One of lignin fractions was mainly generated during lignin depolymerization, which had a relatively small molecular weight, and was prone to mitigate to the fiber surface as the pretreatment temperature rose beyond the lignin glass transition temperature. ...
... One of lignin fractions was mainly generated during lignin depolymerization, which had a relatively small molecular weight, and was prone to mitigate to the fiber surface as the pretreatment temperature rose beyond the lignin glass transition temperature. These lignins tended to redeposit on the surface of pretreated biomass when the pretreatment temperature cooled down [16,17]. Moreover, a more pronounced lignin redeposition was observed on surfaces of organosolv pretreated biomass, because of the precipitation of dissolved lignins [16,18]. ...
... These lignins tended to redeposit on the surface of pretreated biomass when the pretreatment temperature cooled down [16,17]. Moreover, a more pronounced lignin redeposition was observed on surfaces of organosolv pretreated biomass, because of the precipitation of dissolved lignins [16,18]. While, another had a dramatically higher molecular weight, potentially due to the less depolymerization and stronger lignin repolymerization, and preferred to be embedded in the substrates [16]. ...
It has been reported that the lignins deposited on surfaces of pretreated biomass directly affected the enzyme adsorption, thus impeding enzymatic hydrolysis of lignocellulose. Alleviating the surface lignin inhibition is urgently needed for establishing an efficient lignocellulose biorefinery process. In this study, ethanol organosolv lignins (EOLs) from masson pine and poplar were employed as representatives of surface lignins. Aiming to mitigate the EOLs inhibition on enzymatic hydrolysis, 2-naphthol as a carbocation scavenger was added in the organosolv pretreatment. The function of 2-naphthol for suppressing lignin condensation was verified by investigating the lignin structural changes with NMR analysis. Moreover, it was verified that 2-naphthol modification remarkably eliminated the negative effects of EOLs from both two wood species, leading to a 6.8–34.4% increase in hydrolysis yields. The effects of 2-napthol modified lignins were even reversed to slightly promote the enzymatic hydrolysis of pure cellulose. The mechanism of 2-naphthol modification for relieving lignin inhibition was revealed by evaluating the relationship between lignin structural features and hydrolysis performance with Pearson's correlation heatmap. Results enrich our understanding of surface lignin inhibition, thereby providing ideas for promoting enzymatic hydrolysis by alleviating the surface lignin inhibition.
... Some lignin with low molecular weight obtained from pretreatment of lignocellulosic materials could also promote enzymatic hydrolysis [30,138]. These types of lignin could be easily obtained by a simple organic solvent extraction, which is more depolymerized and typically less condensed [139]. For example, Lai et al. found that the promoting mechanism of ethanol organosolv lignin for improving cellulase hydrolysis efficiency was related to pH, and higher pH could intensify the suppression of interaction between residual lignins and enzymes [140]. ...
Even traditional pretreatments can partially remove or degrade lignin and hemicellulose from lignocellulosic biomass for enhancing its enzymatic digestibility, the remaining lignin in pretreated biomass still restricts its enzymatic hydrolysis by limiting cellulose accessibility and lignin-enzyme nonproductive interaction. Therefore, many pretreatments that can modify lignin structure in a unique way and approaches to block the lignin’s adverse impact have been proposed to directly improve the enzymatic digestibility of pretreated biomass. In this review, recent development in sulfite pretreatment that can transform the native lignin into lignosulfonate and subsequently enhance saccharification of pretreated biomass under certain conditions was summarized. In addition, we also reviewed the approaches of the addition of reactive agents to block the lignin’s reactive sites and limit the cellulase-enzyme adsorption during hydrolysis. It is our hope that this summary can provide a guideline for workers engaged in biorefining for the goal of reaching high enzymatic digestibility of lignocellulose.
... Enzymatic hydrolysis experiments were performed in duplicate. To calculate the glucose yields, sugars released in the hydrolysis supernatants were determined by HPLC (Agilent 1260, Palo Alta, CA, United States) with an Aminex Bio-Rad HPX-87H column according to the previous work (Lai et al., 2019). To evaluate the effects of surfactant on enzyme adsorption during hydrolysis process, the free enzyme contents in supernatants were detected according to Bradford assay. ...
... Prior to QCM analysis, lignin films were prepared by coating the QCM gold sensors (QSX301, Västra Frölunda, Sweden) with isolated lignins as described previously (Lai et al., 2019). Then QCM analysis was applied to investigate the binding of enzyme protein or Tween 80 on lignin film. ...
... The corresponding QCM frequency changes were also monitored. All the QCM analysis for each condition was run in triplicate, and frequency changes (Δf, Hz) were processed using Lagergren kinetic Eq. (1) according to the previous study (Turon et al., 2008;Lai et al., 2019). ...
In this study, lignin blockers including non-catalytic protein and surfactants were employed to promote enzymatic digestibility of pretreated poplars. Among them, Tween 80 exhibited the most pronounced facilitation, improving the glucose yield from 26.6% to 99.6% at a low enzyme loading (10 FPU/g glucan), and readily reduced the required cellulase loading by 75%. The underlying mechanism for this remarkable improvement on glucose yields by Tween 80 was elucidated. The impacts of Tween 80 on the enzyme-lignin interaction were explored by quartz crystal microbalance analysis, revealing that the binding rate of Tween 80 on lignin surfaces was 3-fold higher than that of enzyme. More importantly, Tween 80 remarkably decreased the binding capacity and binding rate of enzyme on lignins. Furthermore, the substrate properties dominating the increase in glucose yields with Tween 80 were explored. The results facilitate to understand the underlying mechanism of the promotion of surfactants on enzymatic hydrolysis.
... Generally, the lignin in softwood is mostly composed of the guaiacyl (G), which is the precursor for condensed lignin. Hence, it is speculated that the residual lignin contains some level of abundance of condensed lignin embodying new carbon-carbon bonds at lignin's formerly free aromatic C-5 positions [34]. The condensed subunit contents were more inhibitory for the enzymatic hydrolysis by enzyme non-productive binding [35]. ...
The high degree of lignification in larch shows strong recalcitrance for its enzymatic digestibility to produce fermentation sugars for the further bio-energy production. Hence, pretreatment with excellent delignification ability should be carried out for larch. In this work, a high delignification degree of 77.3% could be achieved for larch after kraft pretreatment, while it still had a low enzymatic digestibility (30.3%), which was due to the remained stout cell wall structure revealed by confocal laser scanning microscopy. To elucidate the reason for low enzymatic digestibility, the addition of bovine serum albumin (BSA) and ball-milling were performed to investigate the substrate-related inhibiting factors of lignin and cellulose, respectively. It is found that the enzymatic digestibility of pretreated larch was enhanced to 58.8% by the addition of BSA, which is due to the reduction of the non-productive binding between residual lignin and cellulases. In addition, ball-milled could disrupt the cellulose's crystalline structures, which enhance the enzymatic hydrolysis of BSA-absorbed larch to 77.7%. This result indicated that cellulose structure is another inhibition bastion towards enzymatic digestion. This work revealed the effect of substrate-related factors that restrict the enzymatic digestibility of larch.
... Alkaline-based pretreatments, such as using sodium hydroxide or Kraft pulping, are typical delignification methods that could solubilize, redistribute or extract lignin from the biomass (Mittal et al., 2017). In addition, pretreatments using organic solvent (Lai et al., 2019), DES (Ling et al., 2020), and ionic liquid (Caporgno et al., 2016) were also heavily investigated with an aim to remove the lignin as well as preserve its structural integrity for the downstream valorization. Nevertheless, these technologies are performed under severe conditions, which is impractical for large-scale applications due to the aforementioned high capital cost. ...
A delignification saturation point (DSP) was observed for bamboo alkaline hydrogen peroxide pretreatment (AHP). The lignin removal was increased from 52.23% to ∼70% when increasing H2O2 dosage from 0% to 2% at the optimum pH, but it cannot be further reinforced as increasing the H2O2. With partial lignin preserved, the glucan hydrolysis yield was found to have a ceiling of ∼80%. Following study indicated a strong association between enzymatic digestibility and lignin removal. Anatomical analysis by fluorescence microscope and confocal Raman microscope revealed that the undegradable lignin was mainly existed in the cell corner of sclerenchyma fibers, causing the DSP in the bamboo AHP. Finally, the residual lignin in pretreated bamboo was characterized with GPC, HSQC NMR, and 31P NMR, which revealed the nature of DSP. This study could help to understand the lignin modification during the AHP and further contribute to the establishment of a chemical-saving biorefinery.
