Characterization of corn stover before and after pretreatments
The effects of different pretreatments on the glucan, xylan, and lignin content of the pretreated corn stover are shown in Table 1 and suggest that both pretreatments can drastically change the cell wall composition of corn stover.
Table 1
Composition of corn stover expressed as percentage of dry matter. Different letters indicate significant differences (P < 0.05) among different pretreatments. Different letters indicate significant differences (P < 0.05) among different pretreatments.
Pretreatment
|
Component (%)
|
Glucan
|
Xylan
|
Acid insoluble lignin
|
Non-pretreated
|
27.43±0.50c
|
18.91±1.13a
|
21.13±1.45b
|
AHP
|
41.03±4.87b
|
20.67±0.33a
|
15.47±0.92c
|
H₂SO₄
|
53.67±3.26a
|
16.69±0.76b
|
27.93±0.61a
|
The raw material used in this investigation contained 46.34% total carbohydrate (27.43% glucan and 18.91% xylan) and 21.13% acid-insoluble lignin, which is consistent with that of the corn stover analyzed by Liu et al., 2013 [37]. After the treatment of AHP, the content of lignin was 15.47%, which showed a significant reduction compared to the raw material (P < 0.05), while the content of glucan significantly increased by 49.58% with a slight rise of xylan (Table 1). In this study, 1% H2SO4 pretreatment resulted in a significant increase of glucan content (53.67%, P < 0.05) compared to that of the raw material, while the xylan content significantly decreased to 16.69% (P < 0.05) (Table 1). In a previous study, the alkali pretreatment removed most of the lignin by breaking hydrogen and other covalent bonds, leaving behind a highly porous cell wall for better penetration of enzymes [38]. In contrast, acid pretreatment such as H2SO4 mainly induces the chemical conversion of xylan to xylose [39] which is soluble in acidic liquor and hence removed from the solids after pretreatment. Thus, the acid-pretreated corn stover had a reduced xylan content compared to non-pretreated corn stover (Table 1).
Production of CMCase and xylanase by A0, AS2B, and CH20S1
All three bacterial strains, A0, AS2B, and CH20S1, showed excellent CMCase and xylanase activity when corn stover was used as the sole carbon source, and with a similar variation tendency (Fig. 1). After 12 h of incubation, all tested strains secreted the majority of CMCase and xylanase into the medium. The maximum CMCase activities for A0, AS2B, and CH20S1 were 2.11, 2.58, and 1.96 U ml-1, respectively (Fig. 1A), while the highest corresponding xylanase activities were 20.26, 26.65, and 29.44 U ml-1, respectively (Fig. 1B).
Both CMCase and xylanase activity decreased rapidly after reaching peak values, possibly because the CMCase and xylanase were degraded by proteolytic enzymes [40]. Previous studies have proven that several Bacillus sp. strains can produce CMCase and xylanase and degrade cellulose and hemicellulose [41, 42]. In addition, the maximum CMCase activity of the three strains studied here (1.96-2.58 U ml-1) were all remarkably higher than the typical cellulase-producing Bacillus sp. strain (0.81 U ml-1) previously reported [43]. The highest increase in xylanase activity (29.44 U ml-1) detected in this study was significantly more than other Bacillus sp. strains (1.8-4.03 U ml -1) and the fungal strain Aspergillus wentii (8.1 U ml-1) [44, 45]. The results indicate that corn stover containing rich cellulose and hemicellulose [46] could effectively induce the production of cellulolytic enzymes in bacteria.
Optimal temperature and pH for enzymatic activities
The best hydrolytic performance of enzymes produced by the three strains was determined by the influence of temperature and pH on enzyme activity. The optimal temperature for CMCase activity was 55°C for A0, AS2B, and CH20S1, however, the optimal temperatures for xylanase activity were 60°C, 50°C, and 60°C, respectively (Fig. 2).
The optimal pH of CMCase and xylanase activity was 5.5 in A0, while for both AS2B and CH20S1 the optimal pH was 5.0 (Fig. 3). These optimal temperature and pH conditions were consistent with the CMCase and xylanase produced by some fungi and bacteria, with the optimal values ranging from 40°C to 70°C and pH 4.0 to 6.5, respectively [47-49].
Saccharification of corn stover using bacterial crude enzyme extracts
The crude enzymes extracted from the three strains in the current study exhibited different abilities to release reducing sugars from pretreated and non-pretreated corn stover in the saccharification process (Fig. 4). After 72 h of incubation, the DCE from the A0 strains hydrolyzed AHP and H2SO4 pretreated and non-pretreated corn stovers, and produced 63.27, 53.16, and 48.23 mg g-1 reducing sugars, respectively. Similarly, strain AS2B released 70.36, 53.89, and 52.27 mg g-1 reducing sugars, respectively, and strain CH20S1 produced 71.69, 63.89, and 55.24 mg g-1of reducing sugars, respectively (Fig. 4). When comparing these three strains, CH20S1 demonstrated an advantage of hydrolytic ability on AHP and H2SO4 pretreated and non-pretreated corn stovers and released the highest amount of reducing sugars.
The hydrolysis effects on pretreated materials were superior to non-pretreated materials, mainly because of the fiber exposure of the materials after pretreatments [50]. Moreover, the yields of reducing sugars released by DCE were 3.3–7.0 fold higher than that of the buffer solution alone (Fig. 4), demonstrating that the addition of bacterial enzymes can significantly improve the hydrolysis of corn stover. However, according to previous research [51], the contents of reducing sugars released from non-pretreated/pretreated corn stover by commercial cellulase ranged from 100 to 400 mg g-1, which is higher than the results of this study.
Several enzymes, such as endoxylanases, β-xylosidases, and various accessory enzymes, are required in abscission of bonded side groups to the main chain of substituted xylan [52]. Cellulolytic enzymes from bacteria can hydrolyze CMC and xylan into reducing sugars, and the hydrolysis efficiency is limited to the various lignocellulosic biomasses used as substrates [12, 53]. Furthermore, the types and activities of enzymes induced by different biomasses differ greatly [53]. So, we can perhaps conclude that the remarkably different hydrolysis effects between the crude bacterial enzymes and commercial cellulase studied here could be due to the absence of some necessary hydrolytic enzymes for efficient hydrolysis of lignocellulosic biomass in the bacterial crude enzyme extracts.
Comparison of effects of crude enzymes and commercial cellulase on saccharification
To further explore the possibility of the practical application of bacterial enzymes in the industrial production of bioethanol, more enzymatic saccharification experiments were performed by partially replacing commercial cellulase with bacterial enzyme extracts. Strain CH20S1 was selected from the three strains previously studied for its ability to hydrolyze corn stover, and 5.0 ml of DCE was added to each treatment. The reducing sugar yields of each treatment group were markedly different (Fig. 5).
After 72 h of incubation, the contents of reducing sugars released from non-pretreated, AHP pretreated, and H2SO4 pretreated corn stovers by using 20 FPU g-1 of commercial cellulase were 162.25, 260.08, and 317.60 mg g-1, respectively. The reducing sugar yield from other treatments are depicted in Fig. 5. The maximum amount of reducing sugars obtained from non-pretreated raw materials was almost the same as that of the pretreated corn stovers, which demonstrates these treatments were efficient in the hydrolysis of corn stovers with or without pretreatments. Furthermore, these amounts were significantly higher than the reducing sugar produced by using the crude enzymes from bacteria (Fig. 4) and confirms our previous hypothesis that the crude enzyme extracts lack the necessary hydrolytic enzymes, which might be supplemented by commercial cellulase.
More importantly, for AHP pretreated corn stover, the treatments using commercial cellulase with crude enzymes (260.08–320.65 mg g-1) generally exhibited superior hydrolysis effects compared to the treatment using 20 FPU g-1 of commercial cellulase only. In addition, the content of reducing sugar released by 4, 8, and 12 FPU g-1 of commercial cellulase added to DCE declined with values of 320.65, 302.57, and 260.08 mg g-1, respectively (Fig. 5B). These results illuminate a synergistic effect in the enzymatic hydrolysis process of AHP pretreated corn stover when bacterial enzyme extracts were used to partially replace the commercial cellulase. Furthermore, the synergistic effect increased with the proportion of crude enzymes used in this study. According to previous research, the non-hydrolytic protein isolated from fresh corn stover substantially increased both the hydrolysis rate of cellulose and the yield of reducing sugar [54]. The addition of non-hydrolytic proteins was considered not only for enzyme deactivation but also to loosen the tightly packed and highly ordered regions of the cellulose resulting in more access for the cellulolytic enzymes to the cellulose [55, 56]. The water-soluble extracts of wheat straw were recently proven to contain non-hydrolytic proteins and a promotional effect on the enzymatic hydrolysis of pretreated wheat straw. The synergistic effect found in this study could therefore possibly have been caused by the non-hydrolytic proteins contained in the crude enzyme extracts produced by strain CH20S1 when corn stover was used as a carbon source. A similar synergistic effect appeared in the enzymatic hydrolysis of non-pretreated corn stover, while the synergistic effect increased with the rise in the proportion of commercial cellulase (Fig. 5A). Specifically, after 72 h of incubation, the yields of reducing sugar from non-pretreated corn stover using 4, 8, and 12 FPU g-1 of commercial cellulase with DCE, were 181.73, 183.58, and 315.90 mg g-1, respectively (Fig. 5A). The main reason for this increase could be that the cell wall structure of non-pretreated corn stover was not damaged, thus requiring more hydrolytic enzymes than pretreated corn stover [29]. In addition, the bacterial enzymes showed no synergistic effect when commercial cellulase and H2SO4 pretreated corn stover was used as the substrate. The H2SO4 pretreatment might generate inhibitors of the non-hydrolytic proteins in the reaction mixture, while partial by-products of acidic pretreatment have been reported to promote deactivation of proteins [57, 58]. Despite all this, our results suggest that partially replacing costly commercial enzymes has the potential to be an economically friendly and efficient saccharification process in biofuel production.