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Self-generated peroxyacetic acid in phosphoric acid plus hydrogen peroxide pretreatment mediated lignocellulose deconstruction and delignification | Biotechnology for Biofuels | Full Text
Results and discussion . Synergistic removal of hemicellulose and lignin in PHP pretreatment . Both hemicellulose and lignin were generally considered as physical hindrances that prevented the access of cellulase from biomass surface into cellulose interior during enzymatic hydrolysis process [ 26 , 27 , 28 ]. It was found that PHP pretreatment could remove most of the hemicellulose and lignin by relieving the hindrance of the barrier components, therefore, enriching the cellulose component [ 29 ]. The influence of H 3 PO 4 to H 2 O 2 ratios on hemicellulose and lignin removal could reflect the functions of H 3 PO 4 and H 2 O 2 in the pretreatment process. Hemicellulose has a branched structure and a low degree of polymerization and is more sensitive to degrade compared to cellulose component. Removal of substrate hemicellulose from wheat straw and corn stalk was varied with pretreatment intensities (Fig.? 1 a, b). Complete removal of hemicellulose components could be achieved for both substrates when extending pretreatment time or increasing cooking temperature. The hemicellulose removal increased with the increase of H 3 PO 4 concentration for all the assessing time and temperature variables. It has been reported that the carbohydrates solubilization ability of concentrated H 3 PO 4 and the strong deconstruction ability of PHP pretreatment contributed to the removal of hemicellulose [ 5 , 30 ]. Therefore, hemicellulose removal effect was positively correlated with H 3 PO 4 concentration, indicating that higher acid concentration increased the acidity of the pretreatment system to cause more hemicellulose degradation. Fig. 1 Effect of H 3 PO 4 /H 2 O 2 concentrations on wheat straw and corn stalk hemicellulose removal and delignification. Pretreatment temperature, 30, 40, 50?°C, pretreatment time, 1, 3, 5?h. hemicellulose removal from wheat straw ( a ), hemicellulose removal from corn stalk ( b ), delignification from wheat straw ( c ), delignification from corn stalk ( d ). Correlations between delignification and hemicellulose removal of wheat straw ( e ) and corn stalk ( f ) Full size image In addition, cellulose digestibility could be enhanced by the removal of lignin fraction due to the reduction of non-productive cellulase adsorption and increased cellulose accessible surface [ 31 , 32 ]. As shown in Fig.? 1 c, d, after PHP pretreatment, all the assessing variables were influential in biomass delignification of these two substrates. When PHP pretreatment was conducted at mild conditions, i.e., short cooking time and low temperature, H 3 PO 4 concentration was dominant in biomass delignification. Furthering enhancing the pretreatment severity significantly contributed to delignification. It was interesting that the optimized delignification of 83.5% and 90.0% for wheat straw and corn stalk, respectively, was achieved at the H 3 PO 4 to H 2 O 2 ratio of 65–7.1% instead of 80–1.8%. Previous work has shown that acid-catalyzed lignin condensation would occur during prevalent single H 3 PO 4 pretreatment, which limited the lignin extraction/degradation from lignocellulose [ 33 , 34 ]. However, our recent work has shown that H 2 O 2 addition could significantly limit lignin condensation reactions among aromatic units [ 13 ]. In the work reported here, it appeared that the deconstruction and oxidation function of H 2 O 2 was obvious at rather severe pretreatment conditions. It was also shown that a better synergetic effect between H 3 PO 4 and H 2 O 2 occurred at higher temperatures. It could be interpreted that H 2 O 2 underwent more protonation reactions to produce a large number of hydroxyl cations (HO + ) under acidic conditions. The promoted electrophilic reactions likely eliminated the electron-rich active sites on the benzene rings to limit their condensation reactions with other electrophilic radicals involved in lignin depolymerization [ 19 ]. It was also likely that the severe pretreatment conditions significantly enhanced the overall oxidation ability of PHP pretreatment system thus facilitating hemicellulose and lignin removal through degradation reactions. It was also shown that H 3 PO 4 -catalyzed hemicellulose hydrolysis and H 2 O 2 -involved lignin oxidation were responsible for hemicellulose removal and delignification, respectively. When biomass delignification was plotted against its corresponding hemicellulose removal, a positive correlation of 0.74 and 0.85 was observed for wheat straw and corn stalk, respectively (Fig.? 1 e, f). This further supported the above analysis that a strong synergistic effect between H 2 O 2 and H 3 PO 4 existed during the PHP pretreatment process. Previous work has shown that either H 3 PO 4 or H 2 O 2 alone was far less effective in biomass delignification and hemicellulose removal than their combination [ 16 ]. To further determine the functional role of H 3 PO 4 and H 2 O 2 on wheat straw delignification, various ratios of H 3 PO 4 to H 2 O 2 were further assessed for their biomass deconstruction profile at the optimized conditions (50?°C and 3?h). It was shown when H 3 PO 4 concentration was 65%, increasing H 2 O 2 concentration from 3.0 to 7.0% facilitated the delignification from 27.6 to 74.9% (Fig.? 2 ). Similarly, when H 2 O 2 concentration was 1.8%, increasing H 3 PO 4 concentration from 50 to 80% also significantly enhanced wheat straw delignification from 0.3 to 64.8%. This indicated that the oxidative function of H 2 O 2 needed the activation by H 3 PO 4 . It was clearly showed that the synergy between H 2 O 2 and H 3 PO 4 in biomass pretreatment. It appeared that H 3 PO 4 could effectively disrupt biomass recalcitrant structure thus allow the H 2 O 2 to access the inner of the substrate, while H 2 O 2 was activated to generate strong oxidant to further facilitate biomass cell wall deconstruction [ 35 , 36 ]. Fig. 2 Influence of H 3 PO 4 and H 2 O 2 concentration on wheat straw delignification at 50?°C for 3?h. Blue line, when H 3 PO 4 concentration was 65%, H 2 O 2 concentration was designed as 3%, 5% and 7%. Red line, when H 2 O 2 concentration was 1.8%, H 3 PO 4 concentration was designed as 50%, 65%, and 80% Full size image Peroxyacetic acid-involved delignification profile of PHP pretreatment . To find the key factor that mediated the overall biomass deconstruction, the degradation products in the liquid fraction were analyzed using Gas chromatography–mass spectrometry (GC–MS). Six representative products, i.e., acetic acid, furfural, formic acid, furan, acrylic acid and benzoic acid were detected from the residue of PHP pretreatment (Additional file 1 : Table S1). This result further demonstrated the strong oxidation ability of the PHP system thus various organic acids products were obtained after oxidation. Amongst these, the appearance of benzoic acid indicated that lignin could be further fragmented and degraded after acid-catalyzed β-O-4′ cleavage [ 37 ]. The presence of saturated and unsaturated aliphatic carboxylic acids, such as formic acid, acetic acid, and acrylic acid, showed more pieces of evidence for the deconstruction and oxidation of lignocellulose [ 38 ]. The appearance of furfural and furan suggested that pentose and hexose monosaccharides resulted from cellulose and hemicellulose hydrolysis underwent further degradation [ 39 , 40 ]. To get more information about the degradation products of PHP pretreatment, the gas fraction generated during the cooking was simultaneously collected and analyzed using GC–MS (Additional file 1 : Fig. S1). The spectrum results showed that a large peak correlated to carbon dioxide appeared, indicating that the benzene ring structure was oxidized to form quinone compounds, and even mineralized into carbon dioxide and water [ 41 ]. This clearly demonstrated the high extent of lignin oxidative degradation was corresponded to high delignification performance of PHP pretreatment (Fig.? 1 ). Previous work has shown that when lignocellulose was pretreated under rather acidic conditions, much acetic acid and formic acid would be released through the deacetylation and oxidation process [ 5 ]. However, the GC–MS analysis showed that only a limited amount of acetic acid was detected in the volatile gas compounds (Additional file 1 : Fig. S1, retention time, 6.38?min). The possible reason was that most of the generated acetic acid was consumed during the pretreatment or the concentration of acetic acid in the gas was close to detection limit. To quantify these degradation products, the gas fraction was enriched in water and analyzed using high performance liquid chromatography (HPLC). Interestingly, both formic acid and peroxyacetic acid were detected (Fig.? 3 a). Although the concentration of peroxyacetic acid was quite low even after enrichment, this result clearly demonstrated the existence of peroxyacetic acid during PHP pretreatment. This exciting result encouraged us to further look at the liquid fraction of PHP pretreatment. As shown in Fig.? 3 b, when the liquid fraction was detected after dilution using HPLC, representative products including H 2 O 2 , formic acid, acetic acid and peroxyacetic acid were clearly detected. Earlier work has shown that the acetic acid extraction was 0.21?±?0.05% (w/w) at composition from 10?g dry biomass in pre-pulping hemicellulosic extraction hydrolysate [ 42 ]. However, due to the compositional complexity and structural unstability of these degraded products in the PHP pretreatment system, quantification of peroxyacetic acid has been technically challenging using HPLC detection. Since the peroxyacetic acid in the liquid fraction was responsible for the overall lignocellulose deconstruction performance, we roughly detected the content ratio of peroxyacetic acid in the liquid and gas fraction through chemical titration. The concentration of peroxyacetic acid in the liquid fraction was 5.4% (w/w), which was 100-fold higher than that in the gas fraction. The peroxyacetic acid concentration corresponded to 54?g/kg dried wheat straw substrate in optimized PHP pretreatment condition (50?°C, 3.0?h, and H 3 PO 4 /H 2 O 2 loading of 65%/7.1%). It appeared that under such acidic conditions, acetic acid was oxidized by H 2 O 2 thus a large amount of peroxyacetic acid was generated. It was also deduced the generated peroxyacetic acid with much higher oxidation ability acted as the key intermediate that influenced the overall lignocellulose deconstruction. Fig. 3 Determination of the main degradation products of PHP pretreatment through HPLC–UV, gas fraction of pretreatment after enrichment in water ( a ), the liquid fraction of the pretreatment after dilution in water ( b ) Full size image Previous work has shown that peroxyacetic acid would produce hydronium ion (HO + ) in the reaction process to selectively oxidate lignin by replacing its side chains [ 19 ]. The guaiacyl and syringyl units were converted into quinone methide, and the aldehyde intermediate was converted into low molecular weight carboxylic acid by Baeyer–Villiger oxidation [ 43 ]. Therefore, it was speculated that the peroxyacetic acid formed during the PHP pretreatment reaction has a strong degrading effect on lignin. To explore whether there was a certain relationship between the delignification obtained by PHP pretreatment and the concentration of peroxyacetic acid formed by the pretreatment system, the content of peroxyacetic acid in the representative pretreatment conditions process was tested (Fig.? 4 ). It was shown a positive non-linear correlation between delignification and peroxyacetic acid concentration was obtained. It appeared that the generated peroxyacetic acid might facilitate biomass delignification. However, it was also apparent that more peroxyacetic acid could be generated at severe pretreatment conditions. Both earlier work and the discussion above showed that increasing the pretreatment severity could enhance the delignification ability of PHP pretreatment. It was still conflicting that whether the generated peroxyacetic acid could boost biomass delignification. Fig. 4 Positive non-linear correlation between delignification and peroxyacetic acid concentration Full size image To verify the boosting effect of peroxyacetic acid, as well as gain insights into the function of peroxyacetic acid in PHP pretreatment, an increasing amount of extra peroxyacetic acid with 0.2%, 0.5% and 1.0% concentration were artificially added to the PHP pretreatment system, respectively, to check whether the overall biomass deconstruction was enhanced (Fig.? 5 ). Interestingly, compared to original PHP pretreatment systems, all these modified pretreatment systems gave increased biomass delignification. The extent of boosting effect was highly dependent on the additional amount of peroxyacetic acid and pretreatment temperature. It was shown the delignification boosting effect was obvious at mild conditions, corresponding to the maximum delignification enhancement of 14.4 and 43.81% at 30?°C and 40?°C, respectively, with 1% peroxyacetic acid addition. Despite the already rather high delignification of 67.8% at 50?°C, further adding peroxyacetic acid could still boost the delignification with the maximum enhancement to 73.8%. Moreover, biomass delignification was also enhanced with the increasing addition of peroxyacetic acid at the same temperature. These results clearly showed that peroxyacetic acid could facilitate biomass delignification during PHP pretreatment. Earlier work has shown peroxide-involved pretreatment showed a delignification fashion through strong ring-opening of guaiacyl and syringyl units [ 13 ]. The work reported here showed that the addition of peroxyacetic acid in PHP solvent system showed an obvious marginal effect on biomass delignification, likely due to decreasing content of guaiacyl and syringyl units in biomass. Fig. 5 Influence of artificially added peroxyacetic acid (0.2, 0.5 and 1.0%) on wheat straw delignification. To minimize the interference of peroxyacetic acid self-generated in PHP solvent system, a short reaction time of 1?h was selected Full size image Formation pathway of peroxyacetic acid in PHP pretreatment system . Since peroxyacetic acid could be generated by an oxidation reaction between acetic acid and H 2 O 2 under acidic conditions. It was also widely accepted that large amounts of acetic acid could be released during various chemical pretreatments [ 44 , 45 ]. Therefore, it was proposed the peroxyacetic acid was generated through the oxidation of the released acetic acid and introduced H 2 O 2 of PHP pretreatment. To verify this hypothesis, various amounts of acetic acid with 0.20, 0.36, 0.70, 1.00% loading was artificially added into the PHP solvent system without biomass involvement to check whether there was a correlation between peroxyacetic acid generation and acetic acid release. Since the pretreatment condition of H 3 PO 4 /H 2 O 2 ratio, 65%/7.1%, with cooking temperature and time of 50?°C, 3?h showed good compromise of biomass delignification and polysaccharide yield, we selected it for the subsequent demonstration experiment. Earlier work has shown that about 1% acetic acid would be harvested in a typical pretreatment, thus we selected it as the maximum acetic acid addition accordingly. Results have shown that no peroxyacetic acid was detected in the original PHP solvent system (Fig.? 6 a), indicating PHP itself couldn’t produce peroxyacetic acid. However, increasing acetic acid addition significantly contributed to peroxyacetic acid generation (Fig.? 6 b). When the peroxyacetic acid generation was plotted against the acetic acid addition, an obvious positive linear correlation was obtained, which means that 1% (w/w) acetic acid addition could generate 0.35% (w/w) peroxyacetic acid with the excessive H 2 O 2 in PHP liquor system. This analysis clearly supported the above hypothesis that the involvement of lignocellulose to release acetic acid was essential for peroxyacetic acid formation. Nonetheless, the specific source of acetic acid during PHP pretreatment, the formation route of peroxyacetic acid and the deconstruction pathway of lignocellulose components need to be further studied. Fig. 6 HPLC detection of peroxyacetic acid resulted from acetic acid addition in PHP system with an increasing concentration ( a ). The positive correlation between the concentration of peroxyacetic acid formed in the PHP system and the concentration of acetic acid artificially added, 0.00%, 0.20%, 0.36%, 0.70% and 1.00% ( b ) Full size image To address the above concerns, the structural changes of representative biomass model compounds during PHP pretreatment at the same conditions were traced. Microcrystalline cellulose was selected as cellulose model in this case due to its high purity and small particle size. After pretreatment, both acetic acid and peroxyacetic acid were not detected in the liquid fraction. This indicated that cellulose or its hydrolyzed products hardly participated in the formation of acetic acid. In PHP pretreatment, cellulose was deconstructed or swelled by concentrated H 3 PO 4 and then regenerated after water dilution. The above analysis showed rather high cellulose yield was obtained after PHP pretreatment, which was in line with the results here that only a few parts of cellulose were degraded. The major composition of the entire hemicellulose presented in agricultural residues was dominantly represented by xylan. When xylan was used as the hemicellulose model compound for the assessment, a variety of deconstruction products in both gas and liquid fraction were detected by GC–MS and HPLC–UV, respectively. It was shown acetic acid was detected in the gas fraction, while peroxyacetic acid was also traced in the liquid fraction (Fig.? 7 ). To further verify the existence of acetic acid in the liquid fraction, HPLC-RI with higher resolution was complementally conducted (Additional file 1 : Figs. S2, S3,?S4). Results showed a trace amount of acetic acid was detected. These results clearly showed that acetic acid could be released under PHP condition, which was rapidly in situ converted into peroxyacetic acid with the oxidation of excessive H 2 O 2 thus limited acetic acid was detected in the liquid fraction. In nature, the C-2 and C-3 positions of linear xylan are replaced by arabinose and acetyl groups, which could be hydrolyzed at acid environment to release acetic acid. It appeared that under acidic PHP environment, xylan was deconstructed and hydrolyzed rapidly to release acetic acid. In addition to acetic acid, formic acid was also simultaneously detected (Fig.? 7 ). The appearance of formic acid indicated that xylan underwent both acid hydrolysis and further deconstruction reactions. It appeared that hexose in xylan was dehydrated to form 5-methyl furfural, and further completely deconstructed to levulinic acid and formic acid under severe conditions [ 46 ]. Fig. 7 Determination of the main compounds in liquid fraction after PHP pretreatment of xylan through HPLC–UV Full size image When alkali, dealkali and cellulytic enzyme lignin were selected as lignin model compounds, the deconstruction products in the liquid fraction were assessed using HPLC–UV (Fig.? 8 ). It was shown peroxyacetic acid was quite dominant among these three liquid fractions. These results also demonstrated that peroxyacetic acid could be produced from the degradation and oxidation of lignin, even though lignin types were varied. When these gas fractions were further identified by GC–MS, various degradation products including acetic acid were detected (Additional file 1 :Table S2). It appeared that alkali lignin tended to give more kinds of degradation products compared to dealkali and cellulytic enzyme lignin. It was also shown that apart from the acetic acid generation that was involved for the subsequent peroxyacetic acid formation, small molecular formic acid, saturated fatty acids with longer molecular chains, such as n -undecanoic acid and n -dodecanoic acid, unsaturated fatty acids, such as acrylic acid and crotonic acid, aromatic acids, and a large amount of low molecular esters were also produced. These results clearly showed the degradation route of lignin in PHP pretreatment. Peroxyacetic acid exhibited an aggressive oxidative degradation effect on biomass lignin in the PHP pretreatment system. After the lignin was degraded into various small molecular compounds, most of the degradation products were removed from the system through the subsequent washing stage. Therefore, high delignification was achieved with the mediation of peroxyacetic acid. Fig. 8 Determination of the main compounds in the liquid fraction of alkali, dealkali and cellulytic enzyme lignin after PHP pretreatment through HPLC–UV Full size image According to the discussion above, the profile of PHP pretreatment, route of peroxyacetic acid generation (Fig.? 9 ) and delignification mechanism (Fig.? 10 ) were proposed. In the initial stage of PHP pretreatment, lignocellulose was penetrated by the concentrated H 3 PO 4 , while hemicellulose underwent acid-catalyzed hydrolysis and dehydration due to its branched and amorphous chemical structure [ 47 ]. The acetyl groups on the branches of hemicellulose structure released and formed acetic acid. Then H 2 O 2 in PHP solvent system reacted with acetic acid in acidic environment to form peroxyacetic acid, which initiated the selective lignin oxidation and degradation. Peroxyacetic acid then oxidized xylose and lignin to produce more acetic acid [ 48 ], which consequently boosted the peroxyacetic acid formation and biomass deconstruction. Due to the continuous production of peroxyacetic acid, the cyclic synergistic effect of PHP pretreatment was continuously increasing. The lignin component with rather high structural recalcitrance underwent both fragmentation and oxidation-induced ring-opening reactions. These products contained low-molecular-weight monocarboxylic acids, saturated long-chain aliphatic carboxylic acids, unsaturated short-chain fatty acids, and some aromatic compounds. These small molecular compounds were removed from the substrate along with the washing process after the pretreatment. These acidic and oxidative conditions also showed a rather high ability to deconstruct cellulose, corresponding to about 87.0% recovery with high digestibility [ 49 ]. The formation of peroxyacetic acid significantly boosted lignin degradation to enhance the overall deconstruction effect of lignocellulose. The boosting effect of the self-generated peroxyacetic acid thus could mediate the synergy between hemicellulose removal and biomass delignification to achieve a better deconstruction effect. Fig. 9 Formation pathway of peroxyacetic acid in PHP pretreatment system. The yellow arrow indicates the continuous production of peroxyacetic acid enhanced the synergistic effect of PHP pretreatment Full size image Fig. 10 Mechanism of self-generated peroxyacetic acid in PHP pretreatment mediated lignocellulose deconstruction Full size image .
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