Chemical compositions of the PHL
The chemical compositions of the original PHL are listed in Table 1. As can be seen, xylosugars, lignin, acetic acid and acetyl groups were the main components of the PHL from hardwood dissolving pulp production processes. The concentration of XOS was 14.5 g/L, which was the major components of xylosugars (27.3 g/L). Also, it is generally considered that those XOS have the biological functions and are conductive to the growth of intestinal bifidobacterium [8, 28]. Hence, further enhancing the content of XOS with DP 2 ~ 6 from PHL will be of practical significance in this work. Additionally, acetic acid concentration was 3.1 g/L, while the content of acetyl groups was 3.7 g/L, implying that the dissolved xylan/XOS in PHL was still highly acetylated, as noted in the earlier reports [26].
Table 1
Main chemical compositions of original PHL (g/L)
Xylose | XOSa | Xylosugars | Lignin | Furfural | Acetic acid | Acetyl groupsb |
5.1 ± 0.22 | 14.5 ± 0.40 | 27.3 ± 0.43 | 7.1 ± 0.35 | 0.3 ± 0.02 | 3.1 ± 0.10 | 3.7 ± 0.12 |
a represents XOS with DP ranging from 2 to 6. |
b means the amount of acetyl groups linked to the dissolved xylan/XOS |
Process analysis
Effect of CH pretreatment on xylanase hydrolysis efficiency
The influence of CH dosages on the concentration of acetic acid, acetyl groups, XOS in CH treated PHL and subsequent xylanase hydrolysis PHL was investigated. As seen in Fig. 2a, with increasing the CH dosage, the concentration of acetyl groups bound to the dissolved XOS/xylan in PHL was obviously decreased. By contrast, the concentration of acetic acid in PHL was increased. This is due to the hydrolysis of acetyl groups under alkaline conditions and formation of acetic acid. When the CH dosage was 0.8%, around 80% of acetyl groups was released from backbone of XOS/xylan and acetic acid concentration became approximately doubled. Acetic acid can be successfully recovered in high purity to meet industrial needs [30, 31]. As can also be seen in Fig. 2b, when CH dosage was less than 0.8%, CH pretreatment had marginal effect on the XOS concentration before xylanase treatment, while further elevating CH dosage led to the obvious loss of XOS, as XOS were adsorbed on undissolved Ca(OH)2 particles [11].
Interesting, when xylanase treatment was performed after CH pretreatment, the concentration of XOS in CH-PHL increased rapidly by 23% with the increase of CH dosage (Fig. 2b). It indicated that CH pretreatment improved the efficiency of subsequent xylanase hydrolysis of CH-PHL. This behavior is related to the significant reduction of branched chains groups, such as the acetyl groups on XOS, which made xylan/XOS more linear and accessible to xylanase [10]. In this case, with increasing the CH dosage (above 0.8%), more xylosugars were adsorbed on undissolved Ca(OH)2 particles [11, 18], and it eventually reduced the concentrations of XOS at 1.2 wt..% dosage. In addition, results showed that 0.8 wt.% of CH could decrease the concentrations of lignin from 7.0 g/L to 4.5 g/L (Table 4). Based on these results, the CH pretreatment at 0.8 wt.% dosage was selected as the essential pretreatment step for purifying PHL, minimizing XOS loss and enhancing the efficiency of xylanase hydrolysis.
Single enzyme treatment of CH-PHL
The effect of laccase dosage and treatment time on the lignin removals of CH-PHL are shown in Fig. 3a. When the laccase dosage was increased from 0.5 U/g to 5 U/g, the lignin removal increased from 22.5–32.2%. The optimal lignin removal of 29.0% was achieved at 1 U/g of laccase dosage, indicating that a low usage of laccase could remove lignin efficiently, as noted in an earlier study [22]. The optimal treatment time for laccase treatment was 3 h, and further extension of the treatment time had a slight effect on the lignin removal. Laccase generally facilitate the polymerization of lignin via radical–radical coupling reaction of phenolic lignin [32]. The polymerized lignin has generally lower solubility than unpolymerized lignin, which promotes its removal from PHL [33].
Figure 3b showed the effect of xylanase dosage and treatment time on the concentration of xylose and XOS in CH-PHL. As can be seen, the concentration of XOS increased with the extension of treating time while the xylose concentration changed slightly at the xylanase dosages of 2 U/g and 5 U/g. The major increases in the XOS concentration of CH-PHL were 40.0% and 39.7% at the xylanase dosage of 2 U/g for 3 h, and at xylanase dosage of 5 U/g for 1 h, respectively. XOS concentration also changed slightly when the treatment time prolonged from 3 h to 5 h, and the reason for such behavior was due to the lower xylanase dosage. With increasing the xylanase dosage, the maximum increase of XOS concentration in CH-PHL was 41.8% and 44.2% at the xylanase dosages of 10 U/g and 20 U/g for 1 h, respectively. XOS concentration continued to decrease with prolonging the treatment time from 1 h to 5 h resulting in further degradation of XOS to xylose (i.e., a xylose concentration increase). In brief, the optimal conditions of xylanase treatment were the combination of high xylanase dosage and short treating time, or low xylanase dosage and long treating time. Thus, the long treatment time can be considered as a pathway for reducing the usage of xylanase and thus the cost of enzyme treatment. It can be concluded that the optimal xylanase dosage was 2 U/g at the treating time was 3 h (Fig. 3). Thus, the similarity in the optimal treatment time in both laccase and xylanase treatments can contribute to the treatment of laccase and xylanase (LX) to efficiently and simultaneously purify and prepare XOS from CH-PHL.
Laccase and xylanase treatment (LX) of CH-PHL
The simultaneous use of xylanase and laccase in CH-PHL at different pH and temperatures were evaluated for enhancing lignin removal and elevating XOS concentration. The lignin removal in single laccase treated PHL (La-PHL) and XOS concentration in single xylanase treated PHL (Xy-PHL) were also analyzed for comparison.
Influence of temperature on lignin removal and XOS concentration in different enzyme treatment processes was presented in Fig. 4. It can be seen that the lignin concentration of LX-PHL was similar to that of the single laccase treatment process. Both laccase and LX treatments for the lignin removal were effective in the temperature range of 45 to 55 °C (Fig. 4a). The lignin removal decreased rapidly with further elevating temperature, which was attributed to laccase deactivation at a high temperature (60 °C) [11]. In opposition to lignin, the XOS concentration of CH-PHL treated by xylanase and LX treatment gradually increased when the temperature increased from 25 °C to 55 °C. Moreover, with subsequently increasing the temperature to 60 °C, the XOS concentration decreased owing to xylanase deactivation [34].
The effect of pH value on lignin removal and concentration of XOS in the LX-PHL are shown in Fig. 4b. The optimal pH range for the lignin removal in laccase and LX treating processes were 3.5 ~ 5.5. Additionally, the concentration of XOS in xylanase and LX treated PHL were similar, and the maximum increase in the XOS concentration of 40.0% and 41.4% was obtained at pH 5.5, respectively. The XOS concentration decreased when the pH value was further increased to 6.5. The optimal pH value and temperature were 5.5 and 55 °C, respectively, based on lignin removal and XOS concentration increase. Compared with laccase and xylanase treatment, LX treatment can simultaneously remove the lignin and concentrate XOS in PHL.
AC adsorption
Figure 5 shows the effect of activated carbon treatment on the concentrations of dissolved organics in the LX-PHL. It was observed that the residual lignin of LX-PHL can be removed more extensively than sugars, when AC dosage was lower than 0.6%. As compared to xylose, the loss of XOS was significant with concentrating AC in the system. As also shown in Fig. 5, the optimal dosage of AC was 0.6%, under which the concentration of lignin in LX-PHL could effectively decreased by 59.4 wt.%, while XOS loss was minimal. In the present study, the relatively high selectivity of lignin removal is very meaningful, and it can promote the downstream processing and utilization of the dissolved XOS.
Precipitate analysis
Molecular Weight analysis of lignin samples
The molecular weight and polydispersity (PDI, Mw/Mn) of lignin samples was determined using GPC and the results are listed in Table 2. The Mw, Mn and PDI of the F-lignin was similar to those reported in other studies [35, 36], suggesting that a substantial lignin with a low molecular weight was generated in the pre-hydrolysis process. Furthermore, the molecular weight of LX-lignin generated in the LX-PHL treated at 45 °C was higher than that at 25 °C, which confirmed that the higher temperature was more favorable for LX polymerization of lignin and thereby accelerated lignin removal (Fig. 4a). In addition, the similar Mw and Mn of LX-lignin at the pH range of 3.5–5.5 in the LX treatment suggested that laccase was reactive in this pH range. As compared to alkali lignin with a higher molecular weight (Mw = 3135, Mn = 1886), the PHL lignin may be suitable for production of platform chemicals such as epoxies, acrylates, polyurethanes and polymer blends [37].
Table 2
Molecular weight of lignin samples
Lignin Sample | Condition | Mw (g/mol) | Mn (g/mol) | Mw/Mn |
F-lignin | - | 2320 | 1152 | 2.01 |
LX-Lignina | T = 25 °C | 1768 | 890 | 1.98 |
LX-Lignina | T = 45 °C | 2532 | 1325 | 1.91 |
LX-Ligninb | pH = 3.5 | 2280 | 1142 | 2.00 |
LX-Ligninb | pH = 5.5 | 2465 | 1180 | 2.04 |
PHL lignin [35] | - | 2975 | 794 | 3.75 |
PHL lignin [36] | - | 2200 | 1294 | 1.70 |
Note: F-lignin obtained via filtrating the original PHL; (a): the lignin in CH-PHL obtained by LX treatment under the conditions of laccase dosage 1 U/g, xylanase dosage 2 U/g, 3 h, pH 5.5; (b): the lignin in CH-PHL obtained by LX treatment under the conditions of laccase dosage 1 U/g, xylanase dosage 2 U/g, 3 h, 55 °C. |
DSC analysis of lignin samples
Glass transition has an important effect on the processing and mixing of lignin with other polymers [38]. To explore the glass transition temperature (Tg) of PHL-induced lignin, heat flow peaks were measured and shown in Fig. S1, and the Tg values are listed in Table 3. At different LX treatment temperatures of 25 and 45 oC, the Tg was different (63 and 114 oC, respectively), which was possibly attributed to the molecular weight of lignin (Table 2). However, Tg seems not to be affected by pH. In addition, the Tg of LX-lignin was similar to that of F-lignin and commercial alkaline lignin in Table 3. Overall, the Tg of lignin was not significantly changed when extracted via different methods from PHL [39, 40].
Table 3
Glass transition temperature (Tg) of lignin derivatives of PHL.
Lignin sample | Condition | Tg (°C) |
F-lignin | - | 92 |
LX-Lignina | T = 25 °C | 63 |
LX-Lignina | T = 45 °C | 114 |
LX-Ligninb | pH = 3.5 | 96 |
LX-Ligninb | pH = 5.5 | 102 |
Lignin [40] | - | 109 |
Note: F-lignin: lignin obtained via filtrating from original PHL; LX-lignin: lignin obtained via LX treatment. Other experimental conditions: (a) laccase dosage of 1 U/g, xylanase dosage of 2 U/g, 3 h, pH 5.5; (b) laccase dosage of 1 U/g, xylanase dosage of 2 U/g, 3 h, 55 °C. |
FTIR analysis of XOS
The FT-IR spectrum of PHL samples and commercial XOS are shown in Fig. 6. Evidently, the commercial XOS and PHL-induced XOS in the all PHL samples had similar absorption peaks at 800 to 1200 cm− 1, implying that the hemicellulose structures were not obviously changed in successive purification process. Specifically, the similar absorption peak at 1045 cm− 1 assigned to the C–O–C stretching of glycosidic linkages is typical characteristic peak of xylan [15], the peak at 894 cm− 1 is ascribed to the vibrations of the dominant b-glycosidic linkages between the sugars’ units [6]. The presence of two low intensity shoulders at 1161 cm− 1 and 987 cm− 1 are the typical characteristic peaks of arabinosyl side chains [41, 42]. In addition, the band at 3420 cm− 1 was ascribed to the O − H stretching vibration of OH groups, and the band at 2926 cm− 1 originated from the methylene groups. However, compared to the peak intensity of lignin in F-PHL, the relative peak intensity of lignin in S-CH, S-LX, S-AC was weak or disappeared, indicating the high efficiency of the present purifying methods in removing lignin from XOS. For instance, the typical characteristic peaks of lignin appeared at 1600 cm− 1and 1515 cm− 1 (aromatic ring), 1336 cm− 1 (syringyl (S) ring), 1250 cm− 1 (guaiacyl (G) ring breathing with C − O stretching) [43]. In addition, the major absence of absorption at 1730 cm− 1 of PHL samples including S-CH, S-LX, S-AC, was due to the low abundance of acetyl groups, which was consistent with remarkable removal of acetyl groups of xylan in PHL (Fig. 2a).
2D-HSQC NMR Spectra analysis
To further understand the precise structural features of the associated carbohydrates and lignin fractions (LCC, side-chain regions and aromatic regions) in the PHL, 2D-HSQC NMR spectra of the S-F and the purified PHL solids (including S-CH, S-LX and S-AC) were analyzed. The obtained spectra and assigned cross-signals of these samples were identified according to published literatures [7, 9, 28, 44] and showed in Fig. 7 and Table S1, respectively. The main substructures of carbohydrates and lignin fractions in 2D-HSQC spectra were also illustrated in Fig. S2 in Supporting Information.
As shown in Fig. 7a, the main substructures of carbohydrates and lignin fractions can be verified by their correlation signals in the 2D-HSQC spectra at δC/δH 51–109/2.6–5.6. In general, the signals of carbohydrates and lignin fractions in spectrum of the S-F were stronger and more complete than that in purified PHL solids, and the spectra of the purified PHL were considerably similar. Firstly, the signals for the carbohydrates were interpreted as follows: (a) β-(1→4)-D-xylopyranoside of internal xylan (X, δC/δH i.e., X2 (73.0/3.03)); (b) 2-O-acetyl-β-D-xylopyranoside (X22, δC/δH 73.9/4.50), 3-O-acetyl-β-D-xylopyranoside (X33, δC/δH 75.4/4.80); (c) a-(1→4)-D-xylopyranoside with reducing end (aXR1, δC/δH 92.8/4.86); (d) β-(1→4)-D-xylopyranoside with reducing end (XR1, δC/δH 98.1/4.22); (e) β-(1→4)-D-xylopyranoside with non-reducing end (XNR, δC/δH i.e., XNR1 (102.3/4.26) and XNR3 (77.0/3.07)); (f) a-(1→4)-L-arabinofuranoside (Ara, δC/δH i.e., Ara5 (61.1/3.52)); (g) β-(1→4)-D-mannopyranoside (Man3, δC/δH 71.4/3.51); (h) β-(1→4)-D-glucopyranoside (Glc6, δC/δH 62.0/3.52)) and 4-O-methyl-a-D-glucuronic acid (UA, δC/δH i.e., UA4 (82.1/3.11), UA-OMe (59.8/3.35)) and X-UA-OMe4 (77.2/3.62). It can be noted that the cross-signals of xylosugars (xylan/XOS) in the S-F and purified S-CH, S-LX and S-AC were clearly observed through the C2-H2, C3-H3, C4-H4, and C5-H5 correlations from β-(1→4)-D-xylopyranoside of internal xylan (X2, X3, X4, and X5). Obviously, these signals associated with xylan were the prime cross-signals in all spectra, suggesting that the degraded carbohydrate in the PHL was mainly β-(1→4)-D-xylan. In addition, the presence of other sugars (Ara, Man, Gal, Glc and UA) with intensive cross-signals, such as Ara5, Man3, Glc6 and UA, were detected in both non-purified and purified PHL, which suggested that there were little structure changes for those sugars. Also, the correlation signal of X-UA-OMe4 unit (δC/δH 77.2/3.62) is assigned to the C4-H4 of 4-O-methyl-α-D-glucuronic acid linked to O-2 position of xylan backbone. However, the intensive cross-signals of O-acetyl-β-D-xylopranoside in the CH-PHL, such as the cross-signals of C3-H3 from 3-O-acetyl-β-D-xylopranoside (X33), respectively, cannot be detected after CH pretreatment because of their low frequency. It suggest that CH-PHL contained XOS/xylan with less acetyl groups due to its alkaline hydrolysis, which was consistent with the analysis of acetyl groups in Fig. 2a and 6. In brief, the above results suggested that the polysaccharides in PHL were mainly made up of linear XOS/xylan decorated with a few side chains, such as 4-O-methyl-D-glucuronic acids, and acetyl groups.
Secondly, the LCC linkages of phenyl glycoside (PhGlc) was found in all PHL spectra in Fig. 7a, indicating that lignin of the PHL was linked with different carbohydrates by phenyl glycoside linkages to form LCC structures [28, 45]. It was revealed that LCC can be released into PHL under mild pre-hydrolysis conditions [9]. However, the correlation signal of PhGlc2 (δC/δH 99.4/4.70) was disappeared in S-CH spectrum, which was probably due to the removal of lignin in CH pretreatment process. It demonstrated that the removed lignin fractions containing LCC linkages were mainly PhGlc2. On the contrary, the LCC linkages of PhGlc3 (δC/δH 102.3/4.92) was present in all PHL spectra, implying that the soluble lignin fractions existed in the form of LCC structures before and after purifying PHL. These results are in accordance with those of a previous report stating that purified-XOS contained LCC structures [28].
Thirdly, the side-chain regions of all PHL solids spectra were also shown in Fig. 7a. Considering the basic substructures of lignin fractions, β-O-4 (A), β-β (B), and β-5 (C) were observed by their interunit correlation signals, such as β-O-4 (A): Cα-Hα (Aα) at δC/δH 71.6/4.62, Cβ − Hβ (Aβ(G)) at δC/δH 83.8/4.30 and Cγ − Hγ (Aγ) at δC/δH 59.8/3.35; β-β (B): Cγ − Hγ (Bγ) at δC/δH 71.2/4.16, 3.80; β-5 (C): Cγ − Hγ (Cγ) at δC/δH 63.5/3.46. The above results indicated that the common lignin substructures were preserved during the hot water pre-hydrolysis process [46, 47]. Also, these signals became weak or absent with continuous purification steps. Interestingly, only the side-chain signals: Aγ at δC/δH 59.4/3.54 and Cγ at δC/δH 63.5/3.48 were found in the purified PHL samples (S-CH, S-LX and S-AC) spectra. The results revealed that a part of residual lignin in the purified PHL, especially in the AC-treated PHL, possessing at least a dimeric form, might be also linked with carbohydrates by the β-O-4 and β-5 linkages.
As shown in Fig. 7b, prominent correlation signals for syringyl units (S), guaiacyl units (G) and p-hydroxyphenyl (H) lignin in the PHL samples can be observed in the aromatic region (δC/δH 100–131/6.0-7.8). Overall, the signals about lignin fractions in spectrum of the S-F were stronger than those in the purified PHL solids (S-CH, S-LX and S-AC).
In the S lignin units, the correlation signals at δC/δH 104.3/6.60 was attributed to the C2,6-H2,6, and the signals of oxidized S lignin units occurred at δC/δH 106.4/7.30 (C2,6-H2,6). In addition, the signals at δC/δH 111.3/6.89, 115.2/6.74, and 119.3/6.78 were attributed to the C2-H2, C5-H5, and C6-H6 in G lignin units. The signals of these units in S-AC spectrum were much weaker than those in the S-F spectrum, indicating only a small quantity of lignin in purified PHL. Noticeably, the lignin in F-PHL appeared to have the typical condensed S units, which was also reported by others in the extraction of lignin from Eucalyptus via γ-valerolactone/water/acid system [43].
As compared to S-F, S-CH had limited condensed S units as seen from the low frequency of condensed S signals, demonstrating that CH pretreatment was effective in removing lignin with condensed S unit structure. In addition, the monomeric lignin-degraded fractions, such as p-coumaric acid (PCA) and ferulic acid (FA) structures, were identified in the F-PHL spectrum. While PCA structures (PCA8 and PCA2,6) could not be found, and the signals of FA structures (FA2 and FA6) was obviously lower in all purified PHL spectra.
Thermal stability analysis
To better understand the thermal behavior of the final purified XOS, the mass loss and mass loss rate of S-AC were analyzed, and the results are compared with those of commercial XOS in Fig. 8. When the temperature increased to 600 °C, approximately 80 wt.% of the XOS was decomposed. In this decomposition process, the mass loss process could be divided into three stages. The temperature of the first stage was lower than 200 °C. During this stage, a small weight loss of XOS appeared, which was mainly caused by the removal of moisture and the generation of noncombustible gases (e.g., formic acid and acetic acid) [41]. It is noted that the major weight loss of XOS sample was occurred in the second stage (temperature of 200 to 450 °C), which was due to the degradation reactions forming the combustible gases when heating (e.g., depolymerization, hydrolysis) [41, 48]. The third stage of weight loss ranging from 450 to 600 °C had no significant changes with the combustion of few volatile components [49]. Furthermore, the DTG curve of XOS (Fig. 8) showed a wider decomposition temperature range for the commercial XOS, which may show that the decomposition performance of XOS samples were different.
Overall performance
Table 4 shows the concentration of xylose, XOS, xylosugars, lignin and furfural at each purification step and the overall changes in the compositions of the chemicals in the process. Firstly, F-lignin was obtained as insoluble lignin by filtrating the original PHL which had non-obvious influence on the removal of other components as the concentration of lignin and furfural in F-PHL was basically similar to original PHL. Next, CH pretreatment led to 35.7 wt.% lignin and 66.7 wt.% furfural removals with an insignificant sugar loss, which showed the high selectivity in impurity removals. Additionally, LX treatment was effective in eliminating lignin and producing XOS, and it was noted that the XOS concentration remarkably increased by 40.7 wt.%. The recovered lignin including F-lignin and LX-lignin possessed relative lower molecular wight and Tg. Overall, the concentration of lignin, furfural and xylosugars decreased by 81.7 wt.% 100 wt.% and 5.1 wt.%, respectively, but XOS concentration was increased by 36.6 wt.%. In addition, lignin carbohydrate complexes (LCC) existed in the finial purified XOS. In previous reports, the application of poly ethylene imine led to 20.2% xylosugars loss and 58.4% lignin removal [50]. In another work, the treatment of CH and AC resulted in 6.8% xylosugars loss and 66.9% lignin removal [18]. Comparatively, the combination of CH, LX and AC treatment process not only acquires a satisfying purification effect, but also enables a substantial increase in XOS content simultaneously.
Table 4
Effect of different treating steps on the concentration of PHL components and the final increase or removal of each component after purification
PHL (g/L) | Xylose | XOS | Xylosugars | lignin | Furfural |
Original PHL | 5.1 ± 0.22 | 14.5 ± 0.40 | 27.3 ± 0.43 | 7.1 ± 0.35 | 0.3 ± 0.02 |
F-PHL | 5.1 ± 0.20 | 14.5 ± 0.35 | 27.2 ± 0.41 | 7.0 ± 0.28 | 0.3 ± 0.02 |
CH-PHL | 5.0 ± 0.24 | 14.4 ± 0.28 | 26.9 ± 0.36 | 4.5 ± 0.33 | 0.1 ± 0.01 |
LX-PHL | 5.3 ± 0.29 | 20.4 ± 0.38 | 26.8 ± 0.32 | 3.2 ± 0.18 | 0.1 ± 0.01 |
AC-PHL | 5.2 ± 0.16 | 19.8 ± 0.25 | 25.9 ± 0.29 | 1.3 ± 0.20 | 0 |
Change (wt.%) | Xylose | XOS | Xylosugars | lignin | Furfural |
Present work | + 2.0 | + 36.6 | -5.1 | -81.7 | -100 |
[18] | -4.0 | - | -6.8 | -66.9 | -70.1 |
[50] | - | - | -20.2 | -58.4 | - |