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Removal of Water-Soluble Extractives Improves the Enzymatic Digestibility of Steam-Pretreated Softwood Barks.

ABSTRACT: Softwood bark contains a large amounts of extractives-i.e., soluble lipophilic (such as resin acids) and hydrophilic components (phenolic compounds, stilbenes). The effects of the partial removal of water-soluble extractives before acid-catalyzed steam pretreatment on enzymatic digestibility were assessed for two softwood barks-Norway spruce and Scots pine. A simple hot water extraction step removed more than half of the water-soluble extractives from the barks, which improved the enzymatic digestibility of both steam-pretreated materials. This effect was more pronounced for the spruce than the pine bark, as evidenced by the 30 and 11% glucose yield improvement, respectively, in the enzymatic digestibility. Furthermore, analysis of the chemical composition showed that the acid-insoluble lignin content of the pretreated materials decreased when water-soluble extractives were removed prior to steam pretreatment. This can be explained by a decreased formation of water-insoluble "pseudo-lignin" from water-soluble bark phenolics during the acid-catalyzed pretreatment, which otherwise results in distorted lignin analysis and may also contribute to the impaired enzymatic digestibility of the barks. Thus, this study advocates the removal of extractives as the first step in the processing of bark or bark-rich materials in a sugar platform biorefinery.

SUBMITTER: Franko B

PROVIDER: S-EPMC5767193 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Removal of Water-Soluble Extractives Improves the Enzymatic Digestibility of Steam-Pretreated Softwood Barks.

Frankó Balázs B Carlqvist Karin K Galbe Mats M Lidén Gunnar G Wallberg Ola O

Applied biochemistry and biotechnology 20170814 2

Softwood bark contains a large amounts of extractives-i.e., soluble lipophilic (such as resin acids) and hydrophilic components (phenolic compounds, stilbenes). The effects of the partial removal of water-soluble extractives before acid-catalyzed steam pretreatment on enzymatic digestibility were assessed for two softwood barks-Norway spruce and Scots pine. A simple hot water extraction step removed more than half of the water-soluble extractives from the barks, which improved the enzymatic dige ...[more]

PMID: 28808883

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Project description:BackgroundTo realize the full potential of softwood-based forest biorefineries, the bottlenecks of enzymatic saccharification of softwood need to be better understood. Here, we investigated the potential of lytic polysaccharide monooxygenases (LPMO9s) in softwood saccharification. Norway spruce was steam-pretreated at three different severities, leading to varying hemicellulose retention, lignin condensation, and cellulose ultrastructure. Hydrolyzability of the three substrates was assessed after pretreatment and after an additional knife-milling step, comparing the efficiency of cellulolytic Celluclast + Novozym 188 and LPMO-containing Cellic CTec2 cocktails. The role of Thermoascus aurantiacus TaLPMO9 in saccharification was assessed through time-course analysis of sugar release and accumulation of oxidized sugars, as well as wide-angle X-ray scattering analysis of cellulose ultrastructural changes.ResultsGlucose yield was 6% (w/w) with the mildest pretreatment (steam pretreatment at 210 °C without catalyst) and 66% (w/w) with the harshest (steam pretreatment at 210 °C with 3%(w/w) SO2) when using Celluclast + Novozym 188. Surprisingly, the yield was lower with all substrates when Cellic CTec2 was used. Therefore, the conditions for optimal LPMO activity were tested and it was found that enough O2 was present over the headspace and that the reducing power of the lignin of all three substrates was sufficient for the LPMOs in Cellic CTec2 to be active. Supplementation of Celluclast + Novozym 188 with TaLPMO9 increased the conversion of glucan by 1.6-fold and xylan by 1.5-fold, which was evident primarily in the later stages of saccharification (24-72 h). Improved glucan conversion could be explained by drastically reduced cellulose crystallinity of spruce substrates upon TaLPMO9 supplementation.ConclusionOur study demonstrated that LPMO addition to hydrolytic enzymes improves the release of glucose and xylose from steam-pretreated softwood substrates. Furthermore, softwood lignin provides enough reducing power for LPMOs, irrespective of pretreatment severity. These results provided new insights into the potential role of LPMOs in saccharification of industrially relevant softwood substrates.

Project description:Background Lignocellulosic biomass is considered as a potential source for sustainable biofuels. In the conversion process, a pretreatment step is necessary in order to overcome the biomass recalcitrance and allow for sufficient fermentable sugar yields in enzymatic hydrolysis. Steam explosion is a well known pretreatment method working without additional chemicals and allowing for efficient particle size reduction. However, it is not effective for the pretreatment of softwood and the harsh conditions necessary to achieve a highly digestible cellulose fraction lead to the partial degradation of the hemicellulosic sugars. Previous studies showed that the autohydrolysis pretreatreatment of softwood can benefit from the addition of 2-naphthol. This carbocation scavenger prevents lignin repolymerisation leading to an enhanced glucose yield in the subsequent enzymatic hydrolysis.Results In order to prevent the degradation of the hemicellulose, we investigated in this study a two-stage 2-naphthol steam explosion pretreatment. In the first stage, spruce wood is pretreated at a severity which is optimal for the autocatalytic hydrolysis of the hemicellulose. The hydrolyzate containing the solubilized sugars is withdrawn from the reactor and the remaining solids are pretreated with different amounts of 2-naphthol in a second stage at a severity that allows for high glucose yields in enzymatic hydrolysis. The pretreated spruce was subjected to enzymatic hydrolysis and to simultaneous saccharification and fermentation (SSF). In the first stage, the maximal yield of hemicellulosic sugars was 47.5% at a pretreatment severity of log R0 = 3.75 at 180 °C. In the second stage, a 2-naphthol dosage of 0.205 mol/mol lignin C9-unit increased the ethanol yield in SSF with a cellulose loading of 1% using the whole second stage pretreatment slurry by 17% from 73.6% for the control without 2-naphthol to 90.4%. At a higher solid loading corresponding to 5% w/w cellulose, the yields decreased due to higher concentrations of residual 2-naphthol in the biomass and the pretreatment liquor, but also due to higher concentrations of potential inhibitors like HMF, furfural and acetic acid. Experiments with washed solids, vacuum filtered solids and the whole slurry showed that residual 2-naphthol can inhibit the fermentation as a single inhibitor but also synergistically together with HMF, furfural and acetic acid.Conclusions This work shows that a two-stage pretreatment greatly enhances the recovery of hemicellulosic sugars from spruce wood. The presence of 2-naphthol in the second pretreatment stage can enhance the ethanol yield in SSF of steam explosion pretreated softwood at low cellulose concentrations of 1% w/w. However, with higher solid loadings of 5% w/w cellulose, the ethanol yields were in general lower due to the solid effect and a synergistic inhibition of HMF, furfural, acetic acid with residual 2-naphthol. The concentration of residual 2-naphthol tolerated by the yeast decreased with increasing concentrations of HMF, furfural, and acetic acid.

Project description:BackgroundSoftwood is a promising feedstock for lignocellulosic biorefineries, but as it contains galactoglucomannan efficient mannan-degrading enzymes are required to unlock its full potential.ResultsBoosting of the saccharification of pretreated softwood (Canadian lodgepole pine) was investigated for 10 fungal endo-β(1→4)-mannanases (endomannanases) from GH5 and GH26, including 6 novel GH26 enzymes. The endomannanases from Trichoderma reesei (TresMan5A) and Podospora anserina (PansMan26) were investigated with and without their carbohydrate-binding module (CBM). The pH optimum and initial rates of enzyme catalysed hydrolysis were determined on pure β-mannans, including acetylated and deacetylated spruce galactoglucomannan. Melting temperature (Tm) and stability of the endomannanases during prolonged incubations were also assessed. The highest initial rates on the pure mannans were attained by GH26 endomannanases. Acetylation tended to decrease the enzymatic rates to different extents depending on the enzyme. Despite exhibiting low rates on the pure mannan substrates, TresMan5A with CBM1 catalysed highest release among the endomannanases of both mannose and glucose during softwood saccharification. The presence of the CBM1 as well as the catalytic capability of the TresMan5A core module itself seemed to allow fast and more profound degradation of portions of the mannan that led to better cellulose degradation. In contrast, the presence of the CBM35 did not change the performance of PansMan26 in softwood saccharification.ConclusionsThis study identified TresMan5A as the best endomannanase for increasing cellulase catalysed glucose release from softwood. Except for the superior performance of TresMan5A, the fungal GH5 and GH26 endomannanases generally performed on par on the lignocellulosic matrix. The work also illustrated the importance of using genuine lignocellulosic substrates rather than simple model substrates when selecting enzymes for industrial biomass applications.

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Removal of Water-Soluble Extractives Improves the Enzymatic Digestibility of Steam-Pretreated Softwood Barks.

Publications

Removal of Water-Soluble Extractives Improves the Enzymatic Digestibility of Steam-Pretreated Softwood Barks.

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