2.1 Structure characterizations of PSS
PSS was characterized by FT-IR, 1H-NMR and viscosity molecular weight and the results were presented in Fig. 1 as 1a, 1b and 1c, respectively. In Fig. 1a, the O-H stretching vibration was observed at band centered at 3436 cm-1, and sulfonic acid group and the S=O characteristic absorption peaks in sulfonate were observed at 1182 cm-1 and 1035 cm-1 [20]. In Fig. 1b, the proton peak assignment from different carbon was presented in the chart. The resonances from 6.4 to 7.9 ppm were due to the para-substitution on the benzene ring [21]. From the 1H-NMR spectra it can be seen that the PSS sample was quite pure. The molecular weight of PSS was assessed by intrinsic viscosity approach, since the intrinsic viscosity has close relationship with polymer molecular weight and fluid temperature [22]. The intrinsic viscosity of PSS was determined as shown in Fig. 1c, and the value was determined to be 0.2256 g/100mL. Mƞ of PSS was calculated based on Eq. (1) to be 61080, which agreed with the value (~70000) claimed by the supplier very well, since the viscosity averaged molecular weight is usually less than the weight averaged molecular weight. In sum, the structure of PSS used in this investigation was well-defined.
2.3 Chemical composition of poplar variants with green liquor pretreatment and enzymatic hydrolysis efficiency
As a mild alkali pretreatment method to remove some lignin and a small portion of hemicellulose, GL pretreatment mainly used two chemical reagents, Na2S and Na2CO3, which highly enhanced the enzymatic hydrolysis productivity of hardwood [23]. As shown in Table 1, the solid recovery was 84%, while most glucan (95%) in the poplar was reserved in the substrate.
Table 1 The main components of poplar and GL-P
Materials
|
Solid recovery (%)
|
Carbohydrate (%)
|
Lignin (%)
|
Ash
(%)
|
Glucan
|
Xylan
|
KLb
|
ASLc
|
Poplar
|
/
|
56.0±0.03
|
8.7±0.2
|
17.4±0.9
|
2.4±0.1
|
0.69±0.01
|
GL-Pa
|
84.00
|
53.5±0.07
|
6.9±0.2
|
19.7±0.1
|
2.5±0.1
|
1.97±0.04
|
a Green liquor pretreated poplar.
b Klason lignin.
c Acid-soluble lignin.
We have known that LS inhibits the enzymatic hydrolysis conversion of pure cellulose, but promotes the enzymatic hydrolysis conversion of lignocellulose [11, 16]. To verify whether the PSS has the similar function as LS or not, different loadings of PSS were added in enzymatic hydrolysis system to evaluate its performance. In enzymatic hydrolysis of pure cellulose (i.e., Whatman filter paper) at the enzyme loading of 10 FPU/g-glucan, the SED or glucose yield was 32.0% without PSS or LS addition (Fig. 2a). When adding 0.05 g/g-substrate LS, the glucose yield dropped to 29.3%. The enzymatic digestibility was decreased with the further increase of LS addition amount, and reduced to 23.5% and 23.2% for the LS dosages of 0.2 and 0.4 g/g-substrate, respectively. From the work by Wang [11], their enzymatic hydrolysis efficiency after 72h decreased from 74.0% to 66.6% with adding 0.25 g/g-substrate commercial LS at enzyme loading of 15 FPU/g-glucan. Therefore, our results were consistent with theirs quite well in trend. Different from LS, PSS, even at the dosage as low as 0.025 g/g-substrate, lowered the glucose yield dramatically to 9.5%. With the PSS addition increased to 0.2 g/g-substrate, the glucose yield was gradually reduced to 7%. Despite this experiment adopted a lower enzyme loading (i.e., 10 FPU/g-glucan, PSS showed much stronger inhibition on enzymatic hydrolysis of pure cellulose than LS.
The glucose yield of enzymatic hydrolysis of GL-P is depicted in Fig. 2b. As can be seen, the yield was only 39.8% in the absence of any PSS or LS, but increased significantly to 48.3% even at a very low dosage of PSS (i.e., 0.0125 g/g-substrate). Increasing PSS dosage led to the higher glucose yield, and reached the highest (60.3%) at the PSS dosage of 0.1 g/g-substrate. However, when more PSS was added, the glucose yield started to decline, as observed in the cases of PSS dosages at 0.2 and 0.4 g/g-substrate. The promotion effect induced by PSS appeared to be similar to LS reported by Wang [24], and their data are plotted in Fig 2b for comparison. For commercial LS at the enzyme loading of 20 FPU/g-glucan, the highest glucose yield of GL-P reached to 88.0% with addition 0.2 g/g-substrate. Though the enzymatic hydrolysis rate induced by LS was higher than those obtained by PSS, the enzyme loading in the current PSS system was only half of that applied in LS system. Under the same enzyme loading (i.e., 10 FPU/g-glucan), LS at its optimal dosage of 0.2 g/g-substrate led to the glucose yield of GL-P at 55.7%, which was close to the yield resulted from PSS at the same dosage (Fig. 2c). From this point of view, the promoting effect of PSS was better than that of LS. What’s more, the glucose yield of GL-P with PSS addition was slightly higher than that (52.5%) of poplar after SPORL pretreatment with SPORL hydrolysate addition [25].
To compare the effect of PSS and LS on enzymatic hydrolysis process for lignocellulose, the glucose yield of GL-P after PSS/LS addition was plotted again enzymatic hydrolysis time. The dynamic results shown in Fig. 2c. Indicated that the optimal dosage of LS and PSS occurred at 0.2 g/g-substrate and 0.1 g/g-substrate, respectively. Since PSS processed a stronger inhibition to the pure cellulose enzymatic hydrolysis, a lower PSS dosage at 0.05 g/g-substrate was included as well. Clearly, the glucose yield of GL-P was obviously increased from 39.8% to 55.7% and 60.3% with optimal LS and PSS dosage applied, respectively. Even for the low PSS dosage (half of the optimal dosage), its glucose yield after 72 h also reached to 56.7%, which was slightly higher than that of LS at optimal dosage. After fully enzymatic hydrolysis for 72 h, the enzymatic digestibility of GL pretreated substrate in the presence of additives followed the order of PSS 0.1 > PSS 0.05 > LS 0.2 > Control. Lou et al. [15] reported a much higher glucose yield when applied LS as additive, compared with the results in our case addressed above, but the substrate they used was the mixture of Whatman paper and enzymatic hydrolysis lignin. Even though both substrates contained similar chemical compositions, however, the lignin chemistry and its distribution in the fiber should be rather different from the lignocellulose used in our work.
Interestingly, when the enzymatic digestibility at less than 48 h was examined, the different order was observed. For example, for the enzymatic digestibility of 24 h, the order followed LS 0.2 > PSS 0.05 > PSS 0.1 > control, which was opposite with the order of 72 h. The results indicated both additives could promote the enzymatic efficiency if compared with the control. However, if comparing enzymatic digestibility among LS and PSS additives, it was found that the enzyme cocktails of cellulase and PSS showed a lower enzyme activity in a short processing period but with a prolonged processive time. This unique and interesting phenomenon was noticed for the first time in the current, which has not been reported yet in previous work. However, it still remains unclear why PSS lowers the enzyme activity but with a longer processive time than LS, which is worth further investing.
2.4 Interactions between PSS and cellulase
To roughly investigate the effect of PSS on cellulase, the zeta potentials of the cellulase and the mixtures of cellulase with various loading of PSS were measured and the results are given in Table 2. The cellulase used in this investigation had a slightly positive charge of 1.89±0.05 mV. When PSS molecules were added into the system and bound to cellulase, all the mixtures of cellulase with various loading of PSS exhibited negatively charged due to the strongly charged sulfonate group of PSS, which rendered the complexes of cellulase and PSS with negative charges. With the increment of PSS addition from 0.05 to 0.10 g/g-substrate, the zeta potential of the mixtures dropped from -28.95 to -31.37 mV. Very interestingly, with further increment of PSS to 0.15 and 0.20 g/g-substrate, the zeta potential of cellulase-PSS complex started to recover some extent to -25.55 and -21.53 mV, respectively. The most negatively charged mixture was achieved for the PSS dosage of 0.10 g/g-substrate. This agreed with the previous SED results.
Table 2 Zeta potential (ζ) of cellulase and the mixtures of cellulase and PSS
Samples
|
Cellulase
|
Cellulase+0.05g/g-substrate PSS
|
Cellulase+0.10g/g-substrate PSS
|
Cellulase+0.15g/g-substrate PSS
|
Cellulase+0.20g/g-substrate PSS
|
ζ (mV)
|
1.89±0.05
|
-28.95±1.88
|
-31.37±0.17
|
-25.55±0.55
|
-21.53±1.37
|
To profoundly study the interaction between cellulase and PSS, a QCM E4 was employed. Initially, the cellulase was immobilized on the gold surface of QCM sensors and then the interaction between PSS and cellulase were monitored in situ and in real time. The overtone data of frequency and energy dissipation changes during cellulase immobilization and complexes of cellulase-PSS formation monitored are presented in Fig. 3a. The cellulase solution was injected into the chamber at 119 min when the frequency (Δf) and the dissipation (ΔD) became smooth or steady. Once the PSS solution flowing into the chamber at 145.3 min, the Δf3 was dropped from +116.3 Hz to +100 Hz, and it recovered 5 Hz to +105 Hz after buffer rinse. In the meanwhile, the ΔD3 increased mildly by about 1.25×10−6. This adsorption process proceeded relatively fast, taking approximately 17.5 min. Regarding the interaction between cellulase and LS, the Δf3 dropped by ca. 7.8 Hz and the ΔD3 raise ca. 1.43×10−6 in the same medium [17]. It demonstrated there exists a stronger interaction between cellulase and PSS than that between cellulase and LS. In other words, PSS can form the complexes with cellulase like LS does, but with a more efficient manner. The schematic of the interaction between cellulase and PSS was shown in Fig. 3b.
Based on the QCM-D overtone data, the thickness, viscositic and shear moduli of cellulase and PSS films were fitted with sauerbrey and viscoelastic model by Dfind and the results were depicted in Figs. 3c and 3d. Since Sauerbery always underestimates the film thickness when the film is viscoelastic and cellulase layer and the formed complex layer were indeed viscoelastic, thereafter the thickness was referred as viscoelastic thickness. The thickness of cellulase film was 11.56 nm, which was quite close to the value (11.7 nm) reported previously by Wang et al. [17]. This demonstrated the cellulase immobilization on QCM sensors was repeatable and the protocol was reliable. When the stable complexes of cellulase and PSS were formed and subjected to buffer rinsing, the thickness of PSS layer was 4.22 nm, which was the half of the thickness of LS film adsorbed on the cellulase film [17]. The Fig. 3d demonstrated the viscositic and shear moduli real-time change with the formation of the cellulase and PSS films. The viscositic and shear moduli of cellulase adlayer was 0.00165 kg/ms and 9.7 KPa, respectively. When the PSS solution was loaded, the viscositic moduli dropped slightly to 0.00157 kg/ms and the shear moduli increased significantly to 28.7 KPa. The viscositic moduli of PSS adlayer recovered to 0.00162 kg/ms and the shear moduli dropped to 12.66 KPa after buffer rinse. Compared with the values obtained for cellulase and LS, the viscositic moduli of both were quite close whereas the shear moduli of the complex of celluase and PSS was much lower than that of the complex of cellulase and LS. This may attribute to the soft nature of PSS, since it exists as linear and random coils in solution, whereas LS is a relative rigid polymer with 3D network. Overall, PSS was much flexible and it might change its conformation when forming complexes with cellulase. That was the underlying reason why PSS was more efficient to promote lignocellulose saccharification than LS.
Since the same roles of the cellulase-PSS as played by the complexes of cellulase-LS, the improved performance of as-formed cellulase-PSS complexes should be attributed to their promotion of lignocellulose enzymatic saccharification due to as the reduction of the non-productive binding to residual lignin remained in the matrix of lignocellulose. The promoting mechanism lies in the increasing processive action of cellulase with the addition of PSS, along with a trade-off of reduced enzymatic hydrolysis. Therefore, the promoting effect became more profound and desired further investigation.