In present study, the combined hydrothermal-chemical pretreatments were used for increasing lignin removal and accelerating the process of sugars digestion and the biogas production which are done by microorganisms. The chemical solutions used were SH, PA, Ph, Ph + SH and Ph + PA solutions that their effects on chemical structure of spruce wood were investigated.
Effect Of Pretreatments On Composition Of Spruce Wood
The amounts of lignin content for treated and untreated spruce woods are summarized in Table 1. As observed, Acid Insoluble Lignin (AIL) with 30.366% is the main part of lignin in spruce wood. The amount of AIL increased after using all of applied pretreatments, with the lowest and highest values for PA (31.756%) and Ph (48.878%) solutions, respectively. Acid Soluble Lignin (ASL) decreased by SH, Ph and SH + Ph pretreatments and increased by PA and PA + Ph pretreatments (Table 1). Forasmuch as reduction in lignin content is a key outcome of an efficient pretreatment (Hendriks and Zeeman 2009), lignin removal was calculated for each solution in order to more clarify (Table 1).
Table 1
The amounts of lignin content for treated and untreated spruce woods
Solution type | ASL (%) | AIL (%) | Lignin removal (%) |
Ph | 0.475 | 48.878 | 42.362 |
SH | 0.493 | 35.364 | 12.487 |
Ph + SH | 0.480 | 34.645 | 10.186 |
PA | 0.559 | 31.756 | 1.580 |
Ph + PA | 0.551 | 33.345 | 6.112 |
Untreated | 0.530 | 30.366 | - |
According Table 1, all of the pretreatments positively affected the lignin removal and their variation range was between 1.580 (PA) and 42.362% (Ph). As can be deduced, the effect of PA solution on lignin removal alone was not too much (1.580%). But, combining two solutions of PA and Ph, caused to enhance their effect on lignin removal up to 6.112%. This demonstrates that adding Ph solution improves the performance of PA in terms of lignin removal. However, addition of Ph to SH lead to the opposite results so that the lignin removal slightly decreased. As observed, among the studied pretreatments, the effect of Ph on lignin removal was more than the others.
It should be mentioned that most studies performed on lignocellulose materials attempted to focus on the pretreatments which could accelerate the hydrolysis of cellulosic parts and break the bonds between the sugars. Reason for ignoring lignin removal is that lignin content in lignocellulose materials generally could not be effectively removed in those studies. The results obtained from present study indicate the good effect of the selected pretreatments on lignin removal of spruce wood. In a research studied on spruce wood by Mirahmadi et al. (2010), it was approximately observed no destruction of lignin content using SH pretreatment in mild condition, while SH pretreatment in present study could remove lignin up to12.487%.
The closely research with present study performed by Mohsenzadeh et al. (2012) who investigated the effect of alkali pretreatment on spruce woods. Their lignin removals were between 11.1 and 23.4% in all of used pretreatments. In their research, the highest value of lignin removal was obtained for mixing SH with Polyethylene glycol at temperature of 22 °C. While the highest lignin removal of 42.362% was obtained using Ph pretreatment in present research.
Compositional And Morphological Analysis
Using FTIR spectroscopic technique, the changes in hemicellulose and cellulose structures before and after the pretreatments were investigated. Table 2 shows the FTIR peaks for the following samples: untreated spruce wood, Ph, SH, SH + Ph, PA, and PA + Ph. FTIR spectra of untreated wood shows a band at 897 cm− 1, which represent β-(1–4) glycosidic linkages of cellulose and it is attributed to amorphous cellulose. For all treated spruce woods compared to the untreated, absorbance of amorphous cellulose has increased, except Ph. However, the absorbance is higher for SH and PA + Ph pretreatments compared to SH + Ph and PA. This data proposes maximum structure changed and enhancement of cellulose content for SH and PA + Ph treatments (Khedkar et al. 2018; Pal et al. 2016). According to the previous study by Li et al. (2016) on M. lutarioriparius samples, steam explosion increased peak intensity at 897 cm− 1 and also they suggested the augmentation of cellulose content after pretreatment. This is consistent with results of the current study.
Table 2
Assignments of FTIR spectrum with relative band intensity absorption for studied pretreatments
Band no. | Band Region in Wave number (cm− 1) | Assignments | Relative absorbance |
Untreated | Ph | SH | SH + Ph | PA | PA + Ph |
1 | ~835 | C-H out of plane vibration in lignin | 0.010 | 0.008 | 0.018 | 0.015 | 0.014 | 0.020 |
2 | ~ 897 | C-H deformation in cellulose | 0.011 | 0.009 | 0.023 | 0.017 | 0.018 | 0.021 |
3 | 1040–1060 | C-O stretch in cellulose and hemicellulose | 0.037 | 0.031 | 0.066 | 0.039 | 0.060 | 0.055 |
4 | 1160–1170 | C-O-C vibration in cellulose and hemicellulose | 0.013 | 0.011 | 0.026 | 0.018 | 0.022 | 0.026 |
5 | 1240–1260 | Guaiacyl ring breathing, C-O stretch in lignin | 0.014 | 0.011 | 0.016 | 0.014 | 0.020 | 0.024 |
6 | 1320–1330 | Syringyl ring breathing in lignin | 0.009 | 0.007 | 0.019 | 0.015 | 0.015 | 0.020 |
7 | 1370–1380 | C-H deformation in cellulose and hemicellulose | 0.008 | 0.006 | 0.015 | 0.012 | 0.013 | 0.020 |
8 | 1420–1430 | Aromatic skeleton vibration (methyl) in lignin combined with C-H plane deformation in carbohydrates | 0.007 | 0.006 | 0.016 | 0.011 | 0.011 | 0.015 |
9 | 1450–1460 | Aromatic C-H deformation; asymmetric in CH3, and CH2 | 0.006 | 0.005 | 0.014 | 0.010 | 0.010 | 0.013 |
10 | 1510–1520 | Aromatic C = C stretch from aromatic lignin | 0.004 | 0.003 | 0.010 | 0.004 | 0.006 | 0.009 |
11 | 1600–1610 | Aromatic skeletal vibration plus C = O stretch | 0.006 | 0.004 | 0.010 | 0.009 | 0.006 | 0.012 |
12 | 1630–1640 | Absorbed O-H, Conjugate C = O, ketone | 0.005 | 0.002 | 0.007 | 0.005 | 0.003 | 0.008 |
13 | ~ 1705 | C = O stretch unconjugated ketone, esters in xylan | 0.002 | 0.001 | 0.003 | 0.001 | 0.002 | 0.005 |
14 | 2900–2910 | C-H stretching, from methyl, methylene groups | 0.004 | 0.002 | 0.008 | 0.010 | 0.006 | 0.011 |
15 | 3300–3400 | O-H vibration from aromatic and aliphatic groups | 0.009 | 0.005 | 0.017 | 0.014 | 0.013 | 0.017 |
The peak intensities of 1000–1250 cm− 1 can be recognized to contributions of cellulose and hemicellulose having maxima at 1040 cm− 1, due to C-O stretching and 1165 cm− 1 due to the asymmetrical C-O-C stretching. The band absorption at 1247 cm− 1 is due to C-O stretching and this absorption region show feature of hemicellulose and lignin (Fig. 1). The band at 1247 cm− 1 indicates elimination of hemicellulose as compared to untreated wood. The band intensity for Ph pretreatment had the lowest value. FTIR analysis indicates that maximum solubilization of hemicellulose and lignin is allocated to Ph pretreatment.
The region of 1400–1460 cm− 1 reveals aromatic skeleton C-H plane deformations in lignin. For, 1500–1650 cm− 1 is similarly included aromatic skeletal vibrations. The lowest absorbance values are reported for Ph pretreatment. In the wavenumber of 1500 cm− 1, the lowest value allocated to Ph pretreatment as compared to the others. According the results, it is suggested that addition of Ph to spruce wood causes more lignin changes as compared to the other pretreatments. This results are consistent to the results obtained from acid hydrolysis (Table 1) in which the lignin removal had the highest value for Ph pretreatment.
The crystallinity index is one of the key parameters to be considered during enzymatic hydrolysis. Hence, the crystallinity index was evaluated based on CLL, LOI, and HBI. Generally, increased CLL, LOI and HBI values represent the highest degree of crystallinity and a more ordered cellulose structure. While decreasing these values designate the amorphous structure of cellulose (Khedkar et al. 2018). Table 3 summarizes the values of different crystallinity ratios (CLL, LOI and HBI).
Table 3
Infrared crystallinity ratio of untreated and treated samples of spruce wood.
Index | Relative absorbance |
Untreated | Ph | SH | SH + Ph | PA | PA + Ph |
CLL | 0.72 | 0.98 | 1.09 | 0.56 | 1.12 | 0.81 |
LOI | 0.64 | 0.58 | 0.67 | 0.65 | 0.62 | 0.70 |
HBI | 0.85 | 0.53 | 0.73 | 0.85 | 0.72 | 0.78 |
CLL value of the sample treated with SH + Ph decreased as compared to untreated sample. In reverse, for other pretreatments, CLL values were higher than untreated sample. This reflects an abundance in lignin with condensed and cross-linked G-type lignin structures. One explanation could be that lignin is solubilized during the pretreatment and then repolymerized/recondensed (Auxenfans et al. 2017). Auxenfans et al. (2017) used steam explosion pretreatment combined with dilute sulfuric acid on spruce wood. Their results showed that CLL values of the treated spruce samples were not statistically different compared to the untreated ones. According Table 3, the lowest and highest LOI values were obtained for Ph and PA + Ph, respectively. HBI of the all pretreated samples were lower than untreated sample (except SH + Ph which was equal to it). This indicates decreased crystallinity and increased amorphous cellulose structure of spruce wood which increases the enzymatic efficiency.
SEM Analysis
The morphological changes of the untreated and treated spruce woods in two magnitudes of 200 and 20 µm were evaluated using SEM analysis (Fig. 2). The SEM image of untreated sample indicated an intact surface with well-arranged structure of cellulose, hemicellulose, and lignin (Fig. 2a). After treating with Ph, SH and SH + Ph, the surface layer of spruce wood was destructed (Fig. 2b, c and d). That’s reason is that the pretreatment removes the amorphous cellulose and hemicellulose from inner part. According SEM image, SH + Ph pretreatment caused the lignin re-deposition on the biomass surface. This created a corrugated surface for the wood and could result to accelerate in the enzymatic hydrolysis process. The result of CLL concurs with SEM analysis for SH + Ph so that CLL value for this pretreatment had the lowest value (0.560). Moreover, in Fig. 2e, when using PA pretreatment, the morphology of wood did not change. But, using PA + Ph, the morphology of wood demonstrated tiny destruction (Fig. 2f). This result is also consistence with conformational analysis (Table 1) and CLL. As given in Table 1, the lignin removal for Ph and PA were 42.362 and 1.580%, respectively. When Ph is mixed with PA, caused to arise lignin removal from 1.580 to 6.112% for spruce sample. In addition, from Table 3 is inferred that with mixing Ph (CLL = 0.976) and PA (CLL = 1.118), the CLL of the sample reduces to 0.813.
Summarizing the results of performed analysis in this research demonstrate that among five different pretreatments, Ph represented the best effect on restructuring the spruce wood. Generally, for restructuring lignocellulose materials, in previous researches were focused on the effect of alkali, acid and oxidizer chemicals as pretreatments. Ph used as an oxidizer pretreatment in those studies has environmental hazards because of its toxicity. In this study, in order to reducing Ph toxicity, the effect of the combinations of Ph with alkali and acidic pretreatments on structural change of spruce wood compared to single pretreatments was investigated. The results obtained from LOI and CLL showed that mixing Ph to SH caused to increase the structural change of spruce wood as compared to individually use of SH. It can be make easier for enzymes to attack and diffuse into the cellulosic structure of spruce wood. The results of SEM analysis confirmed these results. In mixing Ph with acidic pretreatment was obtained similar results. Combining Ph with PA led to an increase in PA power in changing structure of spruce wood. This is also well inferred from the results of SEM analysis. After Ph, comparing the obtained results indicates that the combination of Ph with SH is more effective compared to the mix of Ph and PA in terms of changing structure of spruce wood. In addition, between two alkali (SH) and acidic (PA) pretreatments studied in this research, SH pretreatment represents more structural change in spruce wood.