Ligninases and hemicellulases are crucial as accessory enzymes to increase the enzymatic hydrolysis of lignocellulose, boosting sugars production from which biofuels and bioproducts could be obtained. In order to find new sources of these accessory enzymes, this study evaluates the potential of laccase and mannanase enzymes from Streptomyces ipomoeae for improving the conventional hydrolysis with commercial cellulases of steam-pretreated softwood. For that, different laccase treatment and mannanase supplementation strategies were performed. S. ipomoeae laccase increased both glucose and xylose production (17.8% and 9.3%, respectively), which was attributed to a removal of phenols of 29%. Moreover, the combination of laccase and alkaline extraction produced a lignin reduction of 16.2%, improving the glucose and xylose production by almost 41.3% and 44.9%, respectively. On the other hand, the supplementation of S. ipomoeae mannanase to the hydrolysis 24 h before the addition of cellulases increased the glucose (18.4%), xylose (12.3%), and mannose (47.2%) production.
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Introduction
Lignocellulosic biomass represents a sustainable alternative in the gradual substitution of petroleum (Arevalo-Gallegos et al. 2017). It is a source of sugars from which biofuels and bioproducts could be produced, through chemical or biological processes, under the biorefinery concept. This represents a new way to face the serious consequences of climate change arising from the increasing accumulation of greenhouse gases in the atmosphere (Arevalo-Gallegos et al. 2017). Among different technologies assessed for the production of sugars from lignocellulosic biomass, enzymatic hydrolysis is one of the most realistic approaches (Alvira et al. 2013; Guo et al. 2023). However, the highly complex and recalcitrant structure of lignocellulose, mostly comprised of cellulose and hemicelluloses tightly bound to the lignin, restricts the accessibility of hydrolytic enzymes to the carbohydrates. To overcome this structural obstacle, a pretreatment step is required to modify the lignocellulose structure and, consequently to increase the carbohydrates accessibility to the hydrolytic enzymes (Moreno and Olsson 2017; Zou and Thian 2022). Among different pretreatment methodologies, the hydrothermal steam explosion is the most widely used and commercially applied pretreatment technology (Moreno and Olsson 2017; Zou and Thian 2022).
After pretreatment, hydrolytic enzymes are used to hydrolyze carbohydrates into monomeric sugars. Different enzymatic activities are implicated in this full catalytic conversion of carbohydrates contained in pretreated biomass. Cellulases, including endoglucanases, cellobiohydrolases and β-glucosidases, act synergistically to convert cellulose into glucose (Alvira et al. 2013; Guo et al. 2023). In addition to cellulases, other enzymatic activities have a relevant role to fully hydrolyze the complex structure of lignocellulose such as hemicellulases and ligninases, among others (Van Dyk and Pletschke 2012).
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Steam explosion bases its mode of action mainly on the extensive solubilization of the hemicellulosic fraction from lignocellulosic biomass (Moreno and Olsson 2017; Zou and Thian 2022). Nevertheless, some hemicelluloses may still remain in pretreated materials, mostly as insoluble hemicellulose and some solubilized oligomers, which prevent cellulases to hydrolyze cellulose efficiently (Alvira et al. 2013; Guo et al. 2023). The use of hemicellulases becomes essential to increase the yields of enzymatic hydrolysis and, therefore, reduce both the pretreatment intensity and cellulases dosages (Wang and Lü 2021). Considering the diversity of hemicellulosic sugars existing in the different lignocellulosic materials, multitude of hemicellulases (e.g. xylanases, mannanases, arabinofuranosidases, glucuronosidases, esterases, etc.,) can be employed (Alvira et al. 2013; Guo et al. 2023). In the case of softwoods, dominated by hexoses potentially fermentable into bioethanol, such as glucomanan, galactomannan, and galacto (gluco) mannan (Galbe and Zacchi 2002), endomannanases and β-mannosidases are the hemicellulases usually selected. Different fungal endomannanases, from Trichoderma reesei and Podospora anserina, have been described to increase both mannose and glucose production during the enzymatic hydrolysis of steam pretreated pine wood (von Freiesleben et al. 2018). A beneficial effect was also noticed on the enzymatic hydrolysis of ball milled pretreated Douglas fir wood when endomannanases and β-mannosidases from the fungus Aspergillus niger supplemented the cellulases cocktail (Inoue et al. 2015). Cameron et al. (2015) also reported a hydrolysis enhancement of steam-exploded Pinus radiata when a crude extract of Penicillium sp., with endomannanase activity, was added to a mixture of commercial hydrolytic enzymes.
In addition to hemicelluloses, the residual lignin embedded in pretreated materials also represents a significant limiting factor for the full hydrolysis of carbohydrates. Lignin constitutes a physical barrier that binds unspecifically different kinds of protein such as hydrolytic enzymes limiting its action during the enzymatic conversion of carbohydrates (Berlin et al. 2006). Moreover, lignin degradation during steam explosion releases different enzymatic inhibitors, i.e., phenolic compounds, which restrict the subsequent enzymatic hydrolysis and fermentation steps (Jönsson and Martín 2016; Panagiotou and Olsson 2007). Then, the usage of ligninases, including laccases and ligninolytic peroxidases, can transform lignin, reducing the non-productive binding of hydrolytic enzymes and, therefore enhancing the subsequent enzymatic hydrolysis (Fillat et al. 2017; Malhotra and Suman 2021). Moreover, these enzymes have also been largely evaluated to detoxify different pretreated materials (by removing phenolic compounds by selective oxidation of them) and, therefore, enhance the subsequent enzymatic hydrolysis and fermentation steps (Malhotra and Suman 2021; Roth and Spiess 2015). Different fungal laccases, from Sclerotium sp., have been employed on steam-exploded wheat straw, noticing a lignin oxidation that enhanced the subsequent cellulose hydrolysis (Qiu and Chen 2012). In the same way, Moreno et al. (2013) used a fungal laccase from Pycnoporus cinnabarinus to decrease the phenolic content of steam-exploded wheat straw, triggering its fermentation by Kluyveromyces marxianus CECT 10875 during simultaneous saccharification and fermentation processes.
Along with fungal strains, actinomycetes also have the ability to secrete a wide array of accessory enzymes active against the major components of lignocellulose (Saini et al. 2015). Among them, Streptomyces spp. is actually considered one of the main groups of lignocellulolytic microorganisms, producing high levels of mannanase and laccase activities (Molina-Guijarro et al. 2009; Montiel et al. 1999). These enzymes have been already evaluated in lignocellulosic industry for bio-bleaching softwood Kraft pulps (Arias et al. 2003; Montiel et al. 2002), but not as accessory enzymes for improving the production of monomeric sugars. Therefore, considering the importance of evaluating new enzymes for boosting enzymatic hydrolysis of lignocellulose biomass, this work aims to study for the first time the potential of mannanase and also of laccase enzymes secreted by S. ipomoeae as accessory enzymes to improve the enzymatic hydrolysis of steam-exploded softwood, the dominant raw material in the Northern hemisphere.
Materials and methods
Raw material and steam explosion pretreatment
Pine wood (Pinus spp.), available at INIA-CSIC (Madrid, Spain), was used as raw material. Firstly, it was ground and sieved to obtain chips (size of 0.5–1.5 cm length and 1–2 mm width). After that, pine chips were pre-moistened at 25 °C for 16 h with H2SO4 (1% w/w) and steam exploded in a 10L reactor at 210 °C for 5 min. Then, one portion of whole slurry generated was filtered to provide a liquid fraction and a solid fraction. The solid fraction was further washed with water to obtain the water insoluble solids (WIS) fraction.
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Chemical composition of both raw and WIS fraction was determined using the National Renewable Energies Laboratory procedures (NREL) for biomass analysis (NREL 2011) (Sect. “Analytical methods”). Sugars and degradation products contained in the liquid fraction were also analyzed (NREL 2011) (Sect. “Analytical methods”).
Enzymes
The laccase from S. ipomoeae CECT 3341 was overproduced and purified according to Molina-Guijarro et al. (2009), checking the activity by oxidation of 5 mM 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) to its cation radical (ε436 = 29 300 M−1 cm−1) (De La Torre et al. 2017). This enzyme shows a low redox potential (+ 450 mV) and is active in a range of pH 5–9 and at high temperature (Molina-Guijarro et al. 2009).
The mannanase (endo-β-mannanase) from S. ipomoeae CECT 3341 was produced and purified according to Montiel et al. (2002), checking the activity by the release of reducing sugars (Ghose 1987) using locust bean gum (0.5%, w/v in 70 mM phosphate buffer, pH 7.5) as substrate. This enzyme shows an optimal pH of 7.5 and temperature reaction at 55 °C (Montiel et al. 2002).
Celluclast 1.5L (cellulase) and Novozyme 188 (β-glucosidase) from Novozymes (Denmark) were used for enzymatic hydrolysis assays. Activities were determined using filter paper (Whatman No. 1 filter paper strips) and cellobiose, respectively, by the release of reducing sugars (Ghose 1987).
Laccase treatment and enzymatic hydrolysis
The WIS fraction (2.5 g dry weight (DW)) was subjected to different laccase treatment strategies according to De La Torre et al. (2017).
Laccase followed by enzymatic hydrolysis (L): the laccase treatment of WIS fraction (2.5 IU/g DW substrate of laccase) was carried out at 5% consistency (w/v) in 50 mM phosphate buffer (pH 8) for 24 h at 50 °C and 150 rpm. After that, samples were filtered and washed with 1 L of water. In a subsequent step, the laccase-treated WIS fraction was subjected to enzymatic hydrolysis at 5% consistency (w/v) in 50 mM citrate buffer (pH 5.5) for 96 h at 50 °C and 150 rpm. 2.5 FPU Celluclast 1.5L/g DW substrate and 2.5 IU Novozyme 188/g DW substrate were used.
Laccase followed by alkaline extraction and subsequent enzymatic hydrolysis (L + AE): the laccase treatment of WIS fraction was carried out as explained above. After filtration and washing, the resulting laccase-treated WIS fraction was extracted with alkali under mild conditions [2.5% NaOH for 1 h at 60 °C and 5% consistency (w/v)] followed by filtration and water washing. Then, the alkali-extracted sample was again subjected to enzymatic hydrolysis as explained above.
Alkaline extraction followed by laccase and subsequent enzymatic hydrolysis (AE + L): WIS fraction was extracted with alkali as described above, followed by filtration and water washing. Then, the alkali-extracted sample was treated with laccase followed by enzymatic hydrolysis as explained above.
The effects of laccase treatment strategies on WIS fraction were analyzed in terms of chemical composition according to the NREL (2011) (Sect. “Analytical methods”), and enzymatic hydrolysis yields. For that, the enzymatic hydrolysates obtained from laccase-treated WIS samples were centrifuged, and the liquid fractions were analyzed to quantify glucose and hemicellulosic sugars (xylose and mannose) concentrations. For a better evaluation between assays, relative sugars recoveries (glucose, RGR; hemicellulosic, RHR) were calculated using the following equation (Moreno et al. 2016a):
For RHR (%) an identical equation was used but taking into account both xylose and mannose concentrations instead.
Control assays were performed under the same conditions described for the different strategies without the addition of laccase. All the experiments were carried out by triplicate and the average and standard deviation values are shown. When appropriate, one‐way analysis of variance (ANOVA) was performed to evaluate the effect of the laccase treatment strategies. The level of statistical significance was set at p < 0.05 or p < 0.01.
Mannanase supplementation of enzymatic hydrolysis
Effect of mannanase at different supplementation times
The WIS fraction (2.5 g DW) was subjected to enzymatic hydrolysis with different mannanase supplementation strategies according to Alvira et al. (2011). For that, WIS fraction was subjected to enzymatic hydrolysis at 5% consistency (w/v) in 50 mM citrate buffer (pH 5.5) for 96 h at 50 °C and 150 rpm, using 2.5 FPU Celluclast 1.5L/g DW substrate and 2.5 IU Novozyme 188/g DW substrate. Then, enzymatic hydrolysis was carried out by adding mannanase enzyme (2.5 IU /g DW substrate) in different ways: (i) mannanase (M) and hydrolytic enzymes (HE) were added simultaneously at 0 h (M0HE0); (ii) mannanase was added 24 h before the addition of hydrolytic enzymes (M0HE24); and (iii) mannanase was added after 24 h starting of enzymatic hydrolysis (M24HE0). RGR and RHR were calculated as described above.
Control assays were performed under same conditions without addition of mannanase. All the experiments were carried out in triplicate and the average and standard deviation values are shown. When appropriate, one‐way analysis of variance (ANOVA) was performed to evaluate the effect of the mannanase supplementation strategies. The level of statistical significance was set at p < 0.05 or p < 0.01.
Effect of pH on mannanases performance
In a second set of experiments, the same mannanase supplementation strategies were carried out using higher enzymes dosages (15 FPU Celluclast 1.5L/g DW substrate and 15 IU Novozyme 188/g DW substrate). Finally, M0HE24 strategy was carried out at the optimal conditions for mannanase activity. Thus, mannanase treatment of WIS fraction (15 IU/g DW substrate) was carried out at 5% consistency (w/v) in 50 mM citrate buffer (pH 7.5) at 50 °C for 24 h in a rotatory shaker (150 rpm). Then, the pH was adjusted at 5.5 and hydrolytic enzymes were added (15 FPU Celluclast 1.5L/g DW substrate, 15 IU Novozyme 188/g DW substrate) for another 72 h. In these last two sets of experiments samples were taken at 24, 48, 72 and 96 h of enzymatic hydrolysis, centrifuged and the liquid fractions analyzed for glucose, xylose and mannose production. RGR and RHR were calculated as described above, being distinguished RHRX and RHRM for xylose and mannose, respectively, in the case of RHR.
Control assays were performed under same conditions without addition of mannanase. All the experiments were carried out in triplicate and the average and standard deviation values are shown. When appropriate, one‐way analysis of variance (ANOVA) was performed to evaluate the effect of the mannanase supplementation strategies. The level of statistical significance was set at p < 0.05 or p < 0.01.
Analytical methods
Chemical composition of raw, pretreated material (WIS fraction) and the WIS samples resulting from the different laccase treatment strategies was determined according to NREL (2011), being the NREL/TP-510-42618 protocol used. Carbohydrate content was quantified by High Performance Liquid Chromatography (HPLC) equipment using an Agilent Technologies 1260 HPLC with a refractive index detector G1362A (Agilent, Waldbronn, Germany), and an Agilent Hi-PlexH column at 65 °C with a mobile phase (0.6 mL/min) of sulfuric acid (5 mmol/L). The solid residue from acid hydrolysis is referred to as acid-insoluble lignin. The composition (monosaccharides, acetic acid, furfural and 5-hydroxymethylfurfural (5-HMF)) of the liquid fractions generated during steam explosion pretreatment was also analyzed by HPLC (using Hi-PlexH column) according to NREL (2011), being the NREL/TP-510-42623 protocol used. Furthermore, liquid fraction was also post-hydrolyzed (4% H2SO4, at 120 °C, for 60 min) before HPLC analysis. Increment in the concentration of monosaccharides caused by post-hydrolysis was used to measure the concentrations of oligomers.
Glucose, xylose and mannose concentrations obtained from enzymatic hydrolysis resulting from laccase and mannanase strategies were determined by HPLC using an Agilent Hi-PlexPb column at 70 °C using a mobile phase (0.6 mL/min) of ultrapure water.
The total phenols content of WIS samples resulting from laccase treatment was determined according to Folin–Ciocalteau method (Santos et al. 2014).
Results and discussion
Chemical composition of raw and pretreated materials
Table 1 displays the chemical composition of pine wood: cellulose, 43.6%; hemicelluloses, 23.1%, which corresponds to xylan (9.9%), mannan (11.4%), and arabinan (1.7%); and lignin 25.7%. The potential of pine wood as a source of sugars is evidenced, showing high amount of glucan, mannan and xylan. Interesting, the mannan content is higher than xylan, a typical characteristic of softwoods compared to hardwoods (Galbe and Zacchi 2002). Mannose is a hexose than can be fermented together with glucose by normal baker’s yeast, which leads to increase the bioethanol production (Galbe and Zacchi 2002). In order to increase the accessibility of the hydrolytic enzymes to these carbohydrates, steam explosion was used as pretreatment of pine wood pre-moistened with sulfuric acid. Steam explosion has been successfully applied to different lignocellulosic materials, including non-woody materials such as wheat straw, barley straw, etc. (Díaz et al. 2022; Ilanidis et al. 2021), and hardwoods such as eucalypt, poplar, etc. (Ballesteros et al. 2004; He et al. 2022). However, this hydrothermal pretreatment is less effective on softwoods as this type of woody material comprises more lignin and fewer acetyl groups compared to both non-woody and hardwood materials, which decreases the autohydrolysis effect of steam explosion (Zou and Thian 2022). In these cases, the addition of an external acid catalyst is often suitable to complement steam explosion pretreatment (Alvira et al. 2010; Zou and Thian 2022). Table 1 shows the effects produced by steam explosion on pine wood pre-moistened with sulfuric acid, displaying WIS and liquid fractions composition. Steam explosion pretreatment produced a relative increase of the cellulose proportion in the resulting WIS fraction (from 43.6% of untreated pine to 51.9% for WIS). This increment is related to a broad solubilization of hemicelluloses as pointed out by the reduction of hemicelluloses in WIS sample (from 23.1% of untreated pine to 11.2% of WIS) together with the high hemicellulose content in the liquid fraction (16.6 g/L (6.6 g/100 g DW material). A significant proportion of degradation products derived from hemicelluloses was also observed in the liquid fraction, including furfural, 5-HMF, formic acid and acetic acid. Furfural and 5‐HMF are formed from pentoses and hexoses decomposition, respectively, and their subsequent degradation leads to the production of formic acid. Whereas, acetic acid is released from the acetyl groups of xylose. Similar observations have been previously described during steam explosion with sulfuric acid of different softwood materials (Janzon et al. 2014; Stoffel et al. 2017). Regarding lignin, it was also evident a relative increment in WIS fraction (from 25.7% of untreated pine to 35.7% for WIS) because of hemicellulose removal. Nevertheless, this lignin content can also be increased by pseudo-lignin formation, due to repolymerization of degradation products (such as furfural) and/or polymerization with lignin, during acid pretreatment (Hu et al. 2012). Moreover, phenols from lignin degradation were observed in the liquid fraction. Some of them have been identified (4-hydroxybenzoic acid, vanillic acid, syringic acid, guaiacol, cathecol, vanillin, etc.) in other studies during steam explosion of softwoods impregnated with acid (Jönsson et al. 1998).
Table 1
Composition of pretreated samples (acid pre-moistened pine followed by steam explosion at 210 °C for 5 min)
Raw material composition (% dry weight, w/w)
Cellulose
43.6
Hemicelluloses
23.1
Lignin
25.7
210 °C, 5 min
WIS composition (% dry weight, w/w)
Cellulose
51.9
Hemicelluloses
11.2
Lignin
35.7
Prehydrolyzate composition (g/L)
Monosaccharides
Glucose
2.3 (1.9)*
Hemicelluloses
Xylose/Mannose
16.6 (13.1)*
Arabinose
–
Galactose
–
Degradation products
Formic acid
1.6
Acetic acid
2.4
5-HMF
0.7
Furfural
1.3
Phenols
3.3
*Oligomeric form
Evaluation of the laccase enzyme effect
Laccases have been extensively reported as a valuable tool for delignification and detoxification of pretreated lignocellulose, boosting subsequent enzymatic hydrolysis and fermentation processes (Fillat et al. 2017; Malhotra and Suman 2021). The laccase action on steam-pretreated materials results in the oxidation of residual lignin as well as of soluble phenols generated during steam explosion. Lignin oxidation leads to the generation of phenoxyl radicals resulting in alkyl-aryl ether and carbon–carbon bonds cleavage as well as aromatic backbone breakdown, giving rise to lignin depolymerization (Roth and Spiess 2015). In addition, formation of phenoxyl radicals from soluble phenols could lead to their polymerization, yielding oligomers with lower toxicity (Roth and Spiess 2015). Fungal laccases have been extensively evaluated for delignification and detoxification of different pretreated materials (Ibarra et al. 2023; Moilanen et al. 2011; Moreno et al. 2013, 2016a; Palonen and Viikari 2004; Qiu and Chen 2012; Schneider et al. 2020; Suman et al. 2022). Lastly, bacterial laccases have also become of great interest for these purposes (De la Torre et al. 2017; Moreno et al. 2016b), showing a great ability to retain activity at extreme pH and temperature (Rezaie et al. 2017). In this study, a bacterial laccase isolated from S. ipomoeae is evaluated on the WIS fraction obtained after steam explosion of pine pre-moistened with sulfuric acid. For that, different strategies without or with alkaline extraction, after or before laccase treatment (L; L + AE and; AE + L) were performed.
Laccase treatment: delignification effect
The chemical composition of WIS fractions subjected to the different laccase treatment strategies is shown in Table 2. Laccase treatment without alkaline extraction (L) did not show significant evidence of delignification compared to control. Similar observation was previously reported when the same enzyme was assayed on steam-exploded wheat straw (De La Torre et al. 2017). In the same way, Moreno et al. (2016b) did not detect any lignin content variation when Metzyme® bacterial laccase was also evaluated on steam-exploded wheat straw. Even, several studies have described an increment in lignin content using different fungal laccases. For instance, Moilanen et al. (2011) described a lignin content increment in steam-pretreated spruce treated with a fungal laccase from Cerrena unicolor. Similarly, Moreno et al. (2016a) also reported a slight increment in the lignin content of steam-exploded wheat straw after treatment with a fungal laccase from P. cinnabarinus. The higher redox potential of fungal laccases (around + 730 mV to + 790 mV) compared to that of the bacterial laccases (+ 450 mV) increases their ability to act toward a wider range of soluble phenolic compounds of steam-pretreated materials (De la Torre et al. 2017). Among them, the phenoxyl radicals generated by fungal laccase, with low EHOMO (eV) values, show a major tendency to undergo grafting reactions oxidation (Barneto et al. 2012), describing the lignin increment. Contrary, the generation of phenoxy radicals by bacterial laccases, with high EHOMO (eV) values, shows a higher tendency to undergo polymerization (Barneto et al. 2012), explaining the unaltered lignin content of the substrate treated with S. ipomoeae laccase. Regarding alkaline extraction, it is known for its ability to eliminate lignin from lignocellulosic materials (Alvira et al. 2010; Zou and Thian 2022). In this study, the alkaline extraction under mild conditions produced a delignification of steam-exploded pine around 10.0–11.2% (Table 2). This delignification range increased up to 16.2% when S. ipomoeae laccase treatment was carried out prior to alkaline extraction (L + AE). In this sense, Schneider et al. (2020) reported a lignin degradation of 31% when milled wood of Eucalyptus globulus was treated with the whole enzymatic mixture, rich in laccase, of the white-rot basidiomycete Marasmiellus palmivorus followed by alkaline peroxide extraction. However, Suman et al. (2022) observed a rather low percentage reduction in lignin content (2–3%) when jute sticks biomass was treated with a fungal laccase from Trametes maxima followed by peroxide alkaline extraction. When alkaline extraction was applied prior to S. ipomoeae laccase treatment (AE + L) a slightly lower delignification (14.6%) was observed compared to L + AE. The use of this enzyme on alkali-extracted WIS sample from steam-exploded wheat straw achieved a delignification range around to 22% (De La Torre et al. 2017). Moreno et al. (2016b) reported a delignification of 11% when Metzyme® bacterial laccase was applied to the alkali-extracted WIS from steam-exploded wheat straw.
Table 2
Composition of WIS samples treated by S. ipomoeae laccase using different strategies without or with alkaline extraction
Strategies
Composition (% dry weight, w/w)a
Cellulose
Hemicelluloses
Lignin
C
51.9 ± 0.5
11.2 ± 0.1
35.7 ± 0.3
L
51.2 ± 0.6
11.5 ± 0.1
35.9 ± 0.3
C + AE
55.1 ± 0.5
11.0 ± 0.2
32.1 ± 0.2
L + AE
56.3 ± 0.6
11.7 ± 0.3
29.9 ± 0.3
AE + C
55.0 ± 0.3
11.3 ± 0.2
31.7 ± 0.5
AE + L
56.6 ± 0.3
11.4 ± 0.2
30.5 ± 0.4
aThe remaining percent (of the whole 100%) for pine composition is represented by other components such as ashes and acid soluble lignin. C, control without alkaline extraction; C + AE, control followed by alkaline extraction; AE + C, alkaline extraction prior to control; L, laccase treatment; L + AE, laccase treatment followed by alkaline extraction; AE + L, alkaline extraction prior to laccase treatment. Differences in means are not statistically significant
Laccase treatment: enzymatic hydrolysis of WIS
The relative glucose (RGR) and hemicelluloses (RHR) recoveries obtained at 96 h of enzymatic hydrolysis of the WIS fraction treated with S. ipomoeae laccase without alkali extraction (L) are shown in Fig. 1, whereas the enzymatic hydrolysis yields (%) are shown in Table S1. RGR value of laccase-treated WIS fraction was increased by almost 17.8% (3.71 ± 0.01 g/L) compared to control (3.15 ± 0.02 g/L), whereas an increase in RHR (9.3%), which mainly corresponds to xylose, was also observed (from 0.75 ± 0.04 g/L for control samples to 0.82 ± 0.05 g/L for laccase-treated sample). However, no improvements of glucose and xylose recoveries were observed when this bacterial laccase was previously assayed on steam-exploded wheat straw (De La Torre et al. 2017). Contrary, glucose and xylose recoveries increased by almost 5% and 3%, respectively, when Metzyme® bacterial laccase was also applied to steam-exploded wheat straw (Moreno et al. 2016b).
Fig. 1
Relative glucose (RGR) (a) and hemicelluloses (RHR) (b) recovery values (%) at 96 h of enzymatic hydrolysis of WIS samples resulting from the different laccase treatment and enzymatic hydrolysis strategies. Strategy 1, laccase followed by enzymatic hydrolysis (C, control sample; L, laccase sample). Strategy 2, laccase followed by alkaline extraction and subsequent enzymatic hydrolysis (C + AE, control sample followed by alkaline extraction; L + AE, laccase sample followed by alkaline extraction). Strategy 3, alkaline extraction followed by laccase and subsequent enzymatic hydrolysis (AE + C, alkaline extraction of control sample; AE + L, alkaline extraction followed by laccase sample). Control strategies (dark gray bars). Laccase strategies (light gray bars). Mean values and standard deviations were calculated from the triplicates. Dosages: Celluclast 1.5L, 2.5 FPU/g DW substrate and Novozyme 188, 2.5 IU/g DW substrate. The mean difference is significant at the (*) 0.05 or (**) 0.01
×
Although no lignin content variation was detected after treatment with S. ipomoeae laccase (Sect. “Laccase treatment: delignification effect”), the slightly higher enzymatic hydrolysis yield obtained herein for laccase-treated WIS fraction could be due to the lignin structure alteration, which could affect the interaction of hydrolytic enzymes with the laccase-treated material. In this context, the oxidation of phenolic lignin units by laccase alters the hydrophobicity of lignin and, therefore, diminishes the non-productive binding of hydrolytic enzymes (Fillat et al. 2017; Malhotra and Suman 2021). In this sense, Palonen and Viikari (2004) reported a lignin from steam-pretreated spruce with a high content of carboxyl groups after treatment with a fungal laccase from T. hirsute. Consequently, the hydrophobicity of lignin was decreased and the surface charge increased, which reduced the non-specific adsorption of hydrolytic enzymes on lignin, enhancing saccharification yields by almost 13% compared to control. Similar effects were also described by Moilanen et al. (2011) when steam-pretreated spruce was treated with a fungal laccase from C. unicolor, improving the enzymatic hydrolysis by 12%.
Together with lignin modifications by laccase treatment, the removal of soluble phenols contained in WIS fraction could also result in an improvement of enzymatic hydrolysis. The phenols content after S. ipomoeae laccase treatment of WIS fraction was determined, observing a reduction around 29% (from 0.7 ± 0.3 mg/L for control sample to 0.5 ± 0.1 mg/L for laccase sample) in the liquid fraction generated after laccase treatment. Similar phenols reduction was reported when this bacterial enzyme was also used on steam-exploded wheat straw (De la Torre et al. 2017) as well as with the bacterial laccase Metzyme® (Moreno et al. 2016b), increasing the enzymatic hydrolysis by 5%. Nazar et al. (2022) have also described a removal of phenols (44.8%) from milled rice straw treated with a bacterial laccase from Bacilus ligniniphilus, enhancing the enzymatic hydrolysis by 28%. Nevertheless, as previously described, greater ranges of phenols removal have been reported with fungal laccases of high redox potential, as opposed to bacterial laccases, such as that of S. ipomoeae, which has a low redox potential and, therefore, a lower action spectrum on different phenolic compounds (De La Torre et al. 2017). Kalyani et al. (2012) described a huge reduction in the phenols content (76% of the total phenols) from steam-exploded rice straw when it was subjected to a treatment with a fungal laccase from Coltricia perennis, increasing enzymatic hydrolysis yield by 48%.
Figure 1 also shows the relative glucose (RGR) and hemicelluloses (RHR) recoveries obtained at 96 h of enzymatic hydrolysis of the WIS fraction treated with S. ipomoeae laccase and alkali extraction (L + AE and AE + L). Enzymatic hydrolysis yields (%) are also shown in Table S1. As previously observed, alkaline extraction produced a lignin removal, which leads to a lignocellulose structure with a major porosity and available surface area and, therefore a reduction in the non-productive binding of hydrolytic enzymes (Alvira et al. 2010; Zou and Thian 2022). These effects improve the accessibility of hydrolytic enzymes and, consequently the enzymatic hydrolysis (Ibarra et al. 2021; Yang et al. 2002). Therefore, alkaline extraction of steam-pretreated pine produced increments in RGR and RHR recoveries of 21.7% (3.83 ± 0.06 g/L) and 29.3% (0.98 ± 0.02 g/L), respectively, in the case of C + AE; and 16.5% (3.67 ± 0.07 g/L) and 13.3% (0.85 ± 0.01 g/L), respectively, in the case of AE + C. These increments were improved when S. ipomoeae laccase was combined with alkaline extraction, according to the higher delignification ranges produced (Sect. “Laccase treatment: delignification effect”). Then, in the case of L + AE strategy, the RGR and RHR recoveries increased by almost 41.3% (4.45 ± 0.05 g/L) and 44.9% (1.10 ± 0.13 g/L), respectively compared to the enzymatic hydrolysis of steam-pretreated pine without laccase and alkaline extraction. In this sense, Schneider et al. (2020) also reported a 10% increase in the glucose yield, as well as a 15% increase in xylose yield, after hydrolysis of milled wood of E. globulus treated with the whole enzymatic mixture of M. palmivorus followed by alkaline peroxide extraction. Similarly, Suman et al. (2022) also performed a delignification of jute sticks biomass with a fungal laccase from T. maxima followed by peroxide alkaline extraction, reaching a 13.3% increase in glucose production. With AE + L strategy, the increments of RGR and RHR recoveries were around 37.4% (4.33 ± 0.19 g/L) and 19.7% (0.90 ± 0.00 g/L). S. ipomoeae laccase was already used after alkaline extraction of steam-exploded wheat straw, increasing the RGR and RHR recoveries around 66.1% and 13.2% respectively, compared to enzymatic hydrolysis of steam-exploded wheat straw without laccase and alkaline extraction (De La Torre et al. 2017). Similarly, Yang et al. (2011) reported how the enzymatic hydrolysis of Brassica campestris straw reached 48.8% after applying alkaline extraction followed by laccase treatment with a fungal enzyme from Trametes hirsuta.
According to the RGR and RHR recoveries obtained herein by the different strategies assayed with the S. ipomoeae laccase, it can be suggested that the removal of the laccase-modified lignin from steam-pretreated softwood by alkaline extraction (L + AE strategy) has a major positive effect on the enzymatic hydrolysis than the increment of laccase accessibility to lignin when steam-pretreated softwood is previously extracted with alkali (AE + L strategy).
Evaluation of the mannanase enzyme effect
The use of hemicellulolytic enzymes also becomes critical to increase enzymatic hydrolysis yields and, consequently, to improve different biofuels and bioproducts yields (Guo et al. 2023; Wang and Lü 2021). When softwoods are used as feedstock, endomannanases and β-mannosidases are the hemicellulases generally used as they hydrolyze the main chains of glucomanan, galactomannan, and galacto (gluco) mannan. Different fungal endomannanases have been reported to increase both mannose and glucose production during the enzymatic hydrolysis of steam-pretreated softwood (Cameron et al. 2015; von Freiesleben et al. 2018; Várnai et al. 2011). However, a few studies have shown the use of bacterial mannanases for improving enzymatic hydrolysis of pretreated biomass (Malgas and Pletschke 2020). In this study, a mannanase (endo-β-mannanase) from S. ipomoeae is evaluated using different supplementation strategies (M0HE0; M0HE24; M24HE0) to improve the enzymatic hydrolysis of WIS fraction obtained after steam explosion of pine pre-moistened with sulfuric acid.
Effects of mannanase supplementation at different times on enzymatic hydrolysis
In a first set of mannanase supplementation experiments, the enzymatic hydrolysis of steam-exploded pine was carried out using 2.5 FPU Celluclast 1.5L/g DW substrate and 2.5 IU Novozyme 188/g DW substrate supplemented with mannanase enzyme (2.5 IU/g DW substrate). Figure 2 shows RGR and RHR recovery values at 96 h of enzymatic hydrolysis, whereas enzymatic hydrolysis yields (%) are also shown in Table S2. In general, mannanase addition resulted in higher increments of RGR and RHR recoveries in comparison to the control assay without mannanase addition (Fig. 2). When hydrolytic enzymes and mannanase were added simultaneously at 0 h (M0HE0), RGR and RHR values were increased by almost 10.7% (from 3.13 ± 0.05 g/L for control to 3.46 ± 0.03 g/L for M0HE0) and 19.2% (from 0.77 ± 0.03 g/L for control to 0.92 ± 0.01 g/L for M0HE0), respectively. With M0HE24, in which manannase was added at 24 h before hydrolytic enzymes, the enzymatic hydrolysis was also enhanced (increments of RGR and RHR values around 15.7% (3.62 ± 0.05 g/L) and 12.3% (0.86 ± 0.03 g/L), respectively). Finally, when mannanase was supplemented at 24 h of enzymatic hydrolysis (M24HE0), RGR and RHR values were increased by almost 6.8% (3.34 ± 0.03 g/L) and 13.6% (0.87 ± 0.01 g/L), respectively.
Fig. 2
Relative glucose (RGR) (a) and hemicelluloses (RHR) (b) recovery values (%) at 96 h of enzymatic hydrolysis of WIS samples with different mannanase supplementation strategies. Strategy 1, mannanase (M) and hydrolytic enzymes (HE) added simultaneously at 0 h (M0HE0). Strategy 2, mannanase added at 0 h before hydrolytic enzymes addition at 24 h (M0HE24). Strategy 3, mannanase supplemented at 24 h of the enzymatic hydrolysis (M24HE0). Control strategies without mannanase supplementation (C). Mean values and standard deviations were calculated from the triplicates. Dosages: Celluclast 1.5L, 2.5 FPU/g DW substrate and Novozyme 188, 2.5 IU/g DW substrate. The mean difference is significant at the (*) 0.05
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In a second set of mannanase supplementation assays, the enzymatic hydrolysis of WIS samples from steam-exploded pine was carried out using 15 FPU Celluclast 1.5L/g DW substrate and 15 IU Novozyme 188/g DW substrate supplemented with mannanase enzyme (15 IU/g DW substrate). Figure 3 shows the glucose, xylose and mannose production during the enzymatic hydrolysis using the different mannanase supplementation strategies (M0HE0; M0HE24; M24HE0), whereas Fig. 4 displays RGR, RHRX, and RHRM recovery values. Enzymatic hydrolysis yields (%) are also shown in Table S3. Control assays without mannanase supplementation showed the typical batch hydrolysis pattern for glucose production, with a rapid glucose release at the beginning of the process (during the first 48 h), and then a slowing down of the glucose production rate (Fig. 3a). Contrary, the xylose production rate was continuous during the whole enzymatic hydrolysis (Fig. 3b), whereas no mannose release was observed (Fig. 3c). This is to be expected since the commercial enzyme Celluclast 1.5L contains α-L-arabinofuranosidase, endo-xylanase and β-xylosidase activities, but lacks mannan-hydrolytic enzymes (Cameron et al. 2015). The higher hydrolytic enzymes dosages used in this control assay, compared to the control of the first set of experiments, increased the glucose production until 16.2 ± 0.11 g/L after 96 h of enzymatic hydrolysis. In the same way, a xylose production (2.41 ± 0.1 g/L) was also observed.
Fig. 3
Time course for glucose (a), xylose (b), and mannose (c) production during enzymatic hydrolysis of WIS samples with different mannanase supplementation strategies. Strategy 1, mannanase (M) and hydrolytic enzymes (HE) added simultaneously at 0 h (M0HE0). Strategy 2, mannanase added at 0 h before hydrolytic enzymes addition at 24 h (M0HE24). Strategy 3, mannanase supplemented at 24 h of the enzymatic hydrolysis (M24HE0). Control strategies without mannanase supplementation. Mean values and standard deviations were calculated from the triplicates. Dosages: Celluclast 1.5L, 15 FPU/g DW substrate and Novozyme 188, 15 IU/g DW substrate
Fig. 4
Relative glucose (RGR) (a) and hemicelluloses (RHR) (b) recovery values (%) at 96 h of enzymatic hydrolysis of WIS samples with different mannanase supplementation strategies. Strategy 1, mannanase (M) and hydrolytic enzymes (HE) added simultaneously at 0 h (M0HE0). Strategy 2, mannanase added at 0 h before hydrolytic enzymes addition at 24 h (M0HE24). Strategy 3, mannanase supplemented at 24 h of the enzymatic hydrolysis (M24HE0). RGR, samples from control strategies without mannanase supplementation (C). RHR, samples from control strategies without mannanase supplementation (C, xylose and mannose), and samples from strategies with mannanase supplementation (xylose, light gray bars; mannose, white bars). Mean values and standard deviations were calculated from the triplicates. Dosages: Celluclast 1.5L, 15 FPU/g DW substrate and Novozyme 188, 15 IU/g DW substrate. The mean difference is significant at the (*) 0.05 or (**) 0.01
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The addition of mannanase enzyme from S. ipomoeae to enzymatic hydrolysis also increased the sugars production in all strategies assayed in comparison to the control experiment without mannanase supplementation. When hydrolytic enzymes and mannanase were added simultaneously at 0 h (M0HE0), the glucose production rate followed the pattern of the control hydrolysis (Fig. 3a), obtaining 18.66 ± 0.02 g/L of glucose at the end of enzymatic hydrolysis. Xylose and mannose release rates were continuous during the whole enzymatic hydrolysis (Figs. 3b and c, respectively), producing 2.59 ± 0.03 g/L and 0.99 ± 0.03 g/L after 96 h of enzymatic hydrolysis. These results show the synergistic action of hydrolytic enzymes with mannanase by modifying the hemicellulosic matrix and, therefore augmenting the porosity and accessibility of hydrolytic enzymes. Mannanase supplementation at 24 h (M24HE0) also enhanced the glucose (17.67 ± 0.30 g/L), xylose (2.54 ± 0.03 g/L), and mannose production (0.92 ± 0.02 g/L) at the end of enzymatic hydrolysis. In this case, glucose and xylose production rates displayed similar hydrolysis patterns to control and M0HE0 experiments (Fig. 3a and b, respectively), whereas the mannose production was delayed 24 h after beginning of enzymatic hydrolysis (Fig. 3c). In this strategy, the positive effect of the addition of mannanase several hours after beginning of enzymatic hydrolysis could be due to an improvement hydrolysis of the most recalcitrant cellulose by the hydrolytic enzymes. Interesting, the highest sugars production was achieved when mannanase supplementation was carried out 24 h before the addition of hydrolytic enzymes (M0HE24). Enzymatic hydrolysis rate for glucose and xylose increased after hydrolytic enzymes addition at 24 h (Fig. 3a and b, respectively), whereas mannose production rate was continuous during the whole enzymatic hydrolysis (Fig. 3c). Then, the production of glucose (19.85 ± 0.32 g/L), xylose (2.70 ± 0.10 g/L) and mannose (1.09 ± 0.03 g/L) at the end of the process was improved with respect to the other strategies (M0HE0 and M24HE0). Compared to the control experiment without mannanase supplementation, M0HE24 increased RGR by 22.7% (Fig. 4a), RHRX by 12.3% and RHRM by 47.2% (Fig. 4b).
Comparable observations have been described by Alvira et al. (2011) using similar xylanase supplementation strategies to improve the enzymatic hydrolysis of slurry from steam-exploded wheat straw, reporting the best results when xylanase enzyme was added 24 h before hydrolytic enzymes. These authors attributed the enzymatic hydrolysis improvement by xylanase supplementation to the degradation of hemicellulosic matrix, increasing the porosity and the accessibility of hydrolytic enzymes. Moreover, they also explained this enhancement by the action of xylanase enzyme on the xylooligomers solubilized in the pretreated material. It has been described that xylooligomers are strong inhibitors of cellulase activity (Qing et al. 2010). In our case, although the WIS fraction was used, some soluble hemicellulosic oligomers from mannose might be still present in the materials. Consequently, the mannanase supplementation 24 h before the addition of commercial hydrolytic enzymes could act on these soluble oligomers from mannose obtaining better results compared to the other mannanase supplementation strategies.
As previously commented, different studies have already described the improvement of enzymatic hydrolysis of pretreated materials when mannanase enzymes, especially from fungal origin, were used as accessory enzymes to other hydrolytic enzymes. Concerning this, von Freiesleben et al. (2018) supplemented a commercial hydrolytic preparation (Cellic® CTec3) with different fungal endomannanases, from T. reesei and P. anserina, to hydrolyze pretreated pine wood. After 24 h of enzymatic hydrolysis, the release of glucose and mannose produced with mannanase supplementation was enhanced. Yields of glucose and mannose obtained with the mannanase from T. reesei (TresMan5A) were 30% and 15% higher than those obtained with control. Várnai et al. (2011) also described the improvement of enzymatic hydrolysis of steam pretreated spruce when the process was supplemented with xylanases and mananases from T. reseei, increasing the sugars production yield by 4%. In the same way, Katsimpouras et al. (2016) used a thermostable endo-β-mannanase from Myceliophtora thermophila as a supplement to the commercial enzymes Celluclast 1.5L and Novozyme 188 during enzymatic hydrolysis of pretreated beech wood sawdust, improving the production of total reducing sugars and glucose by 13% and 12%, respectively. Cameron et al. (2015) also described a saccharification improvement of steam-exploded P. radiata when a crude extract of Penicillium sp., containing endomannanase activity, was added to a mixture of the commercial enzymes Celluclast 1.5L and Novozyme 188. Regarding mannanases from bacterial origin, very few studies have been published. To our knowledge, only Malgas and Pletschke (2020) studied the supplementation effect of different endomannanases from Bacillus sp. in combination with the commercial cellulase Cellic CTec2 for enzymatic hydrolysis of sugarcane bagasse and pineapple pulps, reporting a 1.3-fold increase in reducing sugars production in comparison when just CTec2 was used.
Effect of pH on mannanases performance
Finally, the M0HE24 strategy was carried out at the optimal pH of mannanase activity (pH 7.5). After 24 h of mannanase treatment, the pH was adjusted at 5.5 and commercial hydrolytic enzymes were added. Figure 5 shows the glucose, xylose and mannose production during the enzymatic hydrolysis using the different mannanase supplementation strategies (M0HE0; M0HE24; M24HE0), whereas enzymatic hydrolysis yields (%) are also shown in Table S4. No differences were observed between the hydrolysis patterns with mannanase enzyme supplementation at pH 5.5 or 7.5. Then, the enzymatic hydrolysis rate for glucose and xylose increased after hydrolytic enzymes addition at 24 h (Fig. 5a and b, respectively) in both cases, whereas mannose production rate was continuous during the whole enzymatic hydrolysis (Fig. 5c). Nevertheless, it is remarkable that, a higher sugars production was obtained when mannanase activity was used at its optimal pH (7.5). Thus, a higher glucose (21.40 ± 0.32 g/L), xylose (2.82 ± 0.08 g/L) and mannonse (1.30 ± 0.04 g/L) production was obtained at the end of enzymatic hydrolysis, which involves increments of RGR, RHRX and RHRM of 7.7%, 4.5% and 19.3%, respectively, compared to M0HE24 strategy with mannanase supplementation at pH 5.5. This result allows to emphasize the suitability of mannanases from S. ipomoeae to increase the hexose yields from residual substrates. This fact, together with the positive results achieved by S. ipomoeae laccase enzyme, makes consider combining both enzymes in next experiments to further increase the sugars production.
Fig. 5
Time course for glucose (a), xylose (b), and mannose (c) production during enzymatic hydrolysis of WIS samples with mannanase added at 0 h before hydrolytic enzymes addition at 24 h (M0HE24). Mannanase supplementation is performed at pH 5.5 and 7.5. Control strategies without mannanase supplementation are also performed at pH 5.5 and 7.5. Mean values and standard deviations were calculated from the triplicates. Dosages: Celluclast 1.5L, 15 FPU/g DW substrate and Novozyme 188, 15 IU/g DW substrate
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Conclusion
The suitability of laccase and mannanase enzymes from S. ipomoeae for improving enzymatic hydrolysis of steam-pretreated softwood has been demonstrated. Laccase reduced the phenols content of WIS, increasing both glucose and xylose production. Moreover, lignin reduction was observed when laccase was combined with alkaline extraction, detecting a higher sugars release. The addition of the mannanase to the enzymatic hydrolysis of WIS resulted in higher increments of glucose, xylose and mannose production in all supplementation strategies evaluated. The highest sugars production was achieved when mannanase supplementation was carried out at its optimal pH 24 h before the addition of hydrolytic enzymes.
Acknowledgements
This research was funded by Comunidad de Madrid via Project SUSTEC‐CM S2018/EMT‐ 4348; Spanish MCINN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe” via Project RTI2018‐096080‐B‐C22; and Spanish MCINN via Projects TED2021‐132122B‐C21 (LIGNUCELL) and PID2022-141965OB-C21 (SUS-BIO-FILM). María E. Eugenio and David Ibarra are grateful for the support of the Interdisciplinary Platform for Sustainable Plastics toward a Circular Economy (SusPlast-CSIC), Madrid, Spain and Interdisciplinary Platform for Sustainability and Circular Economy (SosEcoCir-CSIC), Madrid, Spain; and Interdisciplinary Platform Horizonte Verde (CSIC), Madrid, Spain.
Declarations
Competing interests
The authors declare no competing interests.
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