Selecting a suitable incubation time for C. owensensis is important for assaying its enzymes. The growth and xylanase secretion lines were therefore analyzed at the beginning of this work. Figure 1 shows that regardless of xylose or corncob xylan as carbon source the cell quantity reached the highest after 24 h cultivation. The xylanase activities in the culture supernatant also increased to the peak after 24 h cultivation. The cell quantity by xylose was higher than that by xylan while the xylanase activity was reverse. It seems that xylose is more benefit for biomass accumulation while xylan can induce a higher xylanase secretion. The enzymes produced after 24 h cultivation were used in the following experiments.
Fig. 1Growth and xylanase secretion on corncob xylan and xylose
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The culture supernatants and cells were separated by centrifugation after 24 h cultivation. The extra-enzyme and intra-enzyme C. owensensis were respectively obtained after precipitation of the protein in supernatant by ammonia sulfate and breaking the cell wall by sonication. The hemicellulase (xylanase/Xyan, beta-xylosidase/pNPX, arabinofuranosidase/pNPAF, xylan esterase/pNPAC) and cellulase (endoglucanase/CMC, cellobiohydrolase/pNPC, beta-glucosidase/pNPG, filter paper activity/FPA) activities were measured.
Table 1 shows that no matter cultivated on xylose or corncob xylan both the extra-enzyme and intra-enzyme of C. owensensis had hemicellulase and cellulase activities. Hemicellulase and cellulase are not essential for cell growth and usually considered as the induced enzymes. However, the result of this experiment indicates that substrate inducing is not necessary for C. owensensis on hemicellulase and cellulase secretion. Maybe some genes for such enzymes share the promoters of the constitutive enzyme and are expressed during cell growth. On the whole, the hemicellulase activities cultivated on corncob xylan were higher than those on xylose. For example, the xylanase activities of extra-enzyme and intra-enzyme on corncob xylan were, respectively, 4.72 and 1.57 U/mg, while those on xylose were, respectively, 1.93 and 0.19 U/mg. However, the cellulase activities of the enzymes on corncob xylan and on xylose were varied slightly. This is possibly because both xylose and xylan are not the inducing substrates for cellulase secretion.
Table 1 Hemicellulase and cellulase activities (U/mg or mU/mg) of the crude proteins from C. owensensis and the thermophilic fungi reportedFull size table
Table 1 also shows that the activities of extra-enzyme and intra-enzyme were different. The extra-enzyme had higher xylanase and endoglucanase activities, while the intra-enzyme had higher β-d-xylosidase, β-d-glucosidase and arabinofuranosidase activities. Especially, the β-d-glucosidase activity of intra-enzyme on corncob xylan was about 125-fold higher than that of the extra-enzyme (532.4 mU/mg VS 4.2 mU/mg). This indicates that for degrading the lignocellulosic biomass by C. owensensis the main function of the extra-enzyme is to cleave the polysaccharides to oligosaccharides, while further hydrolysis of xylobiose and cellobiose takes place mainly in cell by intra-enzyme.
Comparing with the thermophilic fungi Thermoascus aurantiacus and Thielavia terrestris which were recently reported to be high cellulase producers [34], the hemicellulase activities of C. owensensis were higher than those of fungi (Table 1, the highest activities of each enzyme were used as the results for discussing). With regard to the cellulase, it showed that although the endoglucanase activity of C. owensensis was much lower than those of fungi, the cellobiohydrolase activity was almost the same as those of the fungi and the β-d-glucosidase activity was much higher than those of fungi. Three enzymes, endoglucanases, exoglucanases and β-d-glucosidases, compose the cellulase system functions in a coordinated manner for degradation of cellulose into glucose units [35]. Since most glucanases are inhibited by cellobiose and short cellooligosaccharides, β-d-glucosidases catalyze the rate limiting step of the cellulose hydrolysis process as a whole [36]. Filamentous fungi are the major source of commercial cellulases. Commercial cellulase preparations are mainly based on mutant strains of T. reesei which have usually been characterized by a low secretion of β-glucosidase [37]. Thus, T. reesei cellulase preparations had to be supplemented with added β-glucosidase to provide the more efficient saccharification of cellulosic substrates [32, 37]. The enzyme system of C. owensensis with high hemicellulases and β-d-glucosidase activities may complement with the fungi cellulase for deconstruction of native lignocellulose.
To identify the protein components in extra- and intra-enzymes of C. owensensis cultivated on corncob xylan, the proteins were analyzed with HPLC/MS. More than 100 and 150 kinds of proteins were identified in the extra- and intra-enzymes respectively. Enzymes related to polysaccharide degradation were shown in Table 2. The extra-enzymes include β-xylanase (Calow_, Calow_), β-galactosidase (Calow_), α-N-arabinofuranosidase (Calow_), polysaccharide deacetylase (Calow_), pectate disaccharide-lyase (Calow_), esterase (Calow_), α-l-fucosidase (Calow_), α-amylase (Calow_ and Calow_), pullulanase, type I (Calow_) and a glycoside hydrolase belong to family 28 (Calow_). The intra-enzymes include the glycoside hydrolases belong to family 18 (Calow _), family 31 (Calow_), family 43 (Calow_, Calow_), family 4 (Calow_) and family 20 (Calow_), pullulanase, type I (Calow_, Calow_), arabinogalactan endo-β-1,4-galactanase (Calow_) and α-l-fucosidase (Calow_). Although the cellulase were not identified, the enzymes belong to different GH families might have multi-activity, including cellulolytic activity. Besides these enzymes related to polysaccharide degradation, the extra-enzymes related to carbohydrate metabolism were also identified and shown in Additional file 1, including glycosyltransferase, extracellular solute-binding protein, ATP-binding cassette (ABC) transporter-related protein, and S-layer domain-containing protein. They gave useful information for further research on carbohydrate hydrolysis and metabolism of C. owensensis.
Table 2 Detected enzymes relation to polysaccharide degradation by HPLC/MSFull size table
The enzymes of C. owensensis were used to hydrolyze native corn stover, native corncob and steam-exploded corn stover with the loading rate of 15 mg enzyme per gram dry substrate at 70 °C for 48 h. The experiment was performed in three groups respectively using extra-enzyme, intra-enzyme and extra-enzyme mixed with intra-enzyme at the ratio of 1:1. The data in Table 3 show that the enzyme of C. owensensis had high ability of degrading hemicellulose. The highest conversion rates of xylan on native corn stover, native corncob and steam-exploded corn stover were respectively 14.7, 16.8 and 59.1 %. Moreover, the conversion rates of araban on native corn stover and native corncob reached 53.5 and 60.0 %, respectively. However, the enzyme of C. owensensis was not such perfect at degrading cellulose. As Table 3 shows the glucose can not be detected after hydrolysis of both native corncob and steam-exploded corn stover for 48 h. This may because the endoglucanase in the enzyme of C. owensensis was weak (shown in Table 1). Blumer&#;Schuette et al. [19] analyzed the core genomes, pangenomes, and individual genomes and predicted that the ancestral Caldicellulosiruptor was likely cellulolytic and evolved, in some cases, into species that lost the ability to degrade crystalline cellulose while maintaining the capacity to hydrolyze amorphous cellulose and hemicelluloses. The results in this experiment were in accord with the prediction.
Table 3 Hydrolysis rates of lignocellulosic biomass by enzymes of C. owensensisFull size table
As described in the section of characteristic of cellulase and hemicellulase, the xylanase was mainly existed in the extra-enzyme of C. owensensis while the β-d-xylosidase and arabinofuranosidase were mainly existed in the intra-enzyme of C. owensensis. The extra-enzyme and the intra-enzyme may have synergetic function for hemicellulose hydrolysis. For each substrate, extra-enzyme mixed with intra-enzyme contributed higher levels of xylose releasing than those by extra-enzyme and intra-enzyme respective hydrolysis. However, the extra-enzyme led to the highest reducing sugar releasing, indicating that the xylanase, with the function of cleaving the xylan to xylo-oligosaccharides and xylose, is the most important enzyme for xylan degradation.
The morphology changes induced by hydrolysis with the extra-enzyme of C. owensensis were examined by SEM to provide direct insight into the structure modification in the native corn stover. Before hydrolysis, the vascular bundle and the epidermis (with stoma and epidermal hair, Fig. 2a, b) of the samples were intact. After hydrolysis, the residual corn stover was changed dramatically (Fig. 2c, d); the initial structure was destroyed and replaced by a collapsed and distorted cell wall structure. The cuticle waxy layer appeared to be almost desquamated, and the microfibrils were exposed to the surface. Clearly, the structure of the native corn stover was greatly changed in appearance after hydrolysis. Figure 2e, f shows the images of the corn stover after incubation in the acetate buffer at 70 °C for 48 h as control. When comparing with the initial corn stover (Fig. 2a, b), the acetate-buffer-incubated corn stover was slightly changed with some fissures in the sample. The acetate-buffer incubation cannot make much change for the biomass structure was proved by the hydrolysis experiment: The acetate-buffer-incubated corn stover and corncob and the samples without incubation were hydrolyzed by CTec2 (Novoyzmes) at 50 °C for 72 h. As a result, the sugar yields of the buffer-incubated samples and the corresponding samples without incubation were almost the same. The glucan conversion rates (%) were as follows: incubated corn stover 18.1 ± 1.7, corn stover without incubation 17.9 ± 1.5, incubated corncob 20.1 ± 1.6, corncob without incubation 20.4 ± 1.9.
Fig. 2SEM of native corn stover before (a, c) and after (b, d) 48 h hydrolysis by the extra-enzyme of C. owensensis and the native corn stover after incubated in the acetate buffer (pH 6.0) at 70 °C for 48 h (e, f)
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Fig. 3Sugar conversion rates from synergetic hydrolysis by extra-enzyme of C. owensensis and CTec2 on native corn stover (a) and native corncob (b). c, d are the HPLC lines of the hydrolysate at the end of hydrolysis (72 h) on native corn stover and native corncob respectively. SH (sequential hydrolysis): hydrolyzed by the enzyme of C. owensensis at 70 °C for 48 h (the xylan and glucan conversion rates after this hydrolysis were shown at 0 h in a, b) then adding CTec2 and incubating at 50 °C for 72 h. CH (co-hydrolysis): co-hydrolyzed by the enzyme of C. owensensis and CTec2 at 50 °C for 72 h. CTec2: hydrolyzed by CTec2 only as control. The loading rates of CTec2 for synergetic hydrolysis were 30 mg/g glucan (High loading). The loading rates of enzyme of C. owensensis for synergetic hydrolysis were 15 mg/g dry substrate. The amounts of released glucose and xylose were used for calculating glucan and xylan conversions, respectively
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Two trials were performed for synergetic hydrolysis by the enzymes of C. owensensis cultivated on corncob xylan and the commercial enzyme cocktail Cellic CTec2 (Novoyzmes). One was that the native corn stover and corncob were sequentially hydrolyzed (SH) by the enzymes of C. owensensis at 70 °C for 48 h then added CTec2 and incubated at 50 °C for 72 h. The other was that these lignocellulosic biomasses were co-hydrolyzed (CH) by the enzyme of C. owensensis and CTec2 at 50 °C for 72 h. The loading rates of CTec2 (http://www.bioenergy.novozymes.com/) for synergetic hydrolysis were 30 mg/g glucan (High loading). These lignocellulosic biomasses were hydrolyzed by CTec2 only at 50 °C for 72 h as controls.
Figure 3 shows that after sequential hydrolysis (SH) on native corn stover and native corncob by extra-enzyme and CTec2, the conversion rates of glucan were 31.2 and 37.9 %, which respectively were 1.7- and 1.9-fold of each control (hydrolyzed by CTec2 only). Using the same loading (high loading, 30 mg enzyme/g glucan) of CTec2 for hydrolysis of the steam-exploded (SE) corn stover and SE corncob the glucan conversion rates were 38.2 and 39.6 %, respectively (Fig. 4a), which were not much higher than glucan conversion rates of the native corn stover and corncob sequentially hydrolyzed (SH) by the enzyme of C. owensensis and CTec2. The glucan conversion rates of native corn stover and corncob by SH were respectively 81.7 % and 95.7 % of those of the SE corn stover and SE corncob hydrolyzed by CTec2 (Fig. 4b). It seems that in this experiment, the hydrolysis by the extra-enzyme of C. owensensis made almost the same contribution as steam-exploded pretreatment for glucan degradation from native lignocellulosic biomass. Sequential hydrolysis by the extra-enzyme of C. owensensis and CTec2 could greatly increase the hydrolysis rate for native lignocellulosic biomass possibly due to the hemicelluloses degraded by the hemicellulase in extra-enzyme of C. owensensis hence increasing the accessibility of cellulose to CTec2. The cellulases, especially the endoglucanase in the extra-enzyme (shown in Tables 1, 2) would also contribute to improve the cellulose hydrolysis. This is made sure by what Brunecky et al. [38] have recently reported that pre-digestion of biomass with the cellulases (CelA and endocellulase E1) from extremely thermophilic bacterium C. becsii, and Acidothermus cellulolyticus at elevated temperatures prior to addition of the commercial cellulase formulation increased conversion rates and yields when compared to commercial cellulase formulation alone.
Fig. 4Comparison of glucan conversion rates on native corn stover and corncob by sequential hydrolysis (SH) and on steam-exploded (SE) corn stover and corncob by CTec2 alone at High loading (30 mg/g glucan). a Time course of glucan conversion. b Relative glucan conversion rate at the end of hydrolysis
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Co-hydrolyzed (CH) by the extra-enzyme of C. owensensis and CTec2 the conversion rates of glucan from native corn stover and corncob were 21.4 and 23.1 % which were respectively 1.19 and 1.13 times of each control (Fig. 3). The increased extents of glucan conversion were not high as those of SH. This is because the optimum temperature for the enzymes of C. owensensis was 70&#;80 °C [33]. When co-hydrolyzed with CTec2 at 50 °C the enzyme activity was decreased.
Figure 3 also shows the xylan conversion rates from native corn stover and corncob synergetically hydrolyzed by the extra-enzyme of C. owensensis and CTec2. Totally, the xylan conversion rates of native corncob were higher than those of native corn stover. Especially, the xylan conversion rate of native corncob by sequential hydrolysis (SH) reached 34.8 %. The possible reason is that the enzyme of C. owensensis used in this experiment was produced using corncob as inducing substrate; hence, the ratio of the compositions in this enzyme was more fitted for corncob hydrolysis. It is believed that deconstructing of hemicellulose in lignocellulose will benefit cellulose degradation. This was proved by the results in Fig. 3. Namely, the higher xylose releasing (34.8 % from native corncob vs 11.8 % from native corn stover) led to a higher glucose releasing (37.9 % from native corncob vs 31.2 % from native corn stover).
Figure 5 shows the sugar conversion rates from native corn stover and corncob by synergetic hydrolysis using intra-enzyme of C. owensensis and CTec2. It can be see that the conversion rates of glucan and xylan were lower than those counterparts of synergetic hydrolysis with extra-enzyme of C. owensensis and CTec2 (Fig. 3). This is possibly due to the higher xylanase and endoglucanase activities in the extra-enzyme.
Fig. 5Sugar conversion rates from synergetic hydrolysis by intra-enzyme of C. owensensis and CTec2 on native corn stover (a) and native corncob (b). c, d are the HPLC lines of the hydrolysate at the end of hydrolysis (72 h) on native corn stover and native corncob respectively. SH (sequential hydrolysis), CH (co-hydrolysis), CTec2: hydrolyzed by CTec2 only as control. The loading rates of CTec2 and enzyme of C. owensensis were the same as described in Fig. 3. The amounts of released glucose and xylose were used for calculating glucan and xylan conversions, respectively
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The furfural and 5-hydroxymethyl furfural (HMF) were not detected in the hydrolysates from the native corn stover and corncob by sequential hydrolysis (SH). It is not surprising since at the temperature of 70 and 50 °C in the hydrolysis buffer the sugars are stable.
Kataeva et al. [39] found that C. bescii could solubilize all components of switchgrass, including lignin. Therefore, the phenolics may be released from lignin during hydrolysis by the extra-enzyme of C. owensensis. Really, the phenolics concentrations of the hydrolysates form native corn stover and corncob by SH were respectively 35.8 ± 3.2 and 34.3 ± 2.7 mg/l, which were slightly higher than these of the hydrolysates form native corn stover and corncob by CTec2 with 24.4 ± 1.8 and 25.1 ± 2.3 mg/l respectively. The phenolics concentrations of the hydrolysis buffer soaked (at 70 °C for 48 h) with native corn stover and corncob were, respectively, 4.2 ± 0.3 and 3.7 ± 0.3 mg/l. While the phenolics concentrations of the hydrolysates form the stream exploded corn stover and corncob by CTec2 were much high as 232 ± 17.6 and 219 ± 20.5 mg/l, respectively. These results show that only few phenolics from lignin can be released by the extra-enzyme of C. owensensis and CTec2. Among the biofuel-production microorganisms, Clostridium is very sensitive to phenolics which are lethal to Clostridium even at low concentrations [40, 41]. Even so, the research by Lee et al. [42] showed that the cell growth and metabolite production of Clostridium tyrobutyricum and Clostridium beijerinckii were not or slight inhibited when the phenolics concentrations were less than 100 mg/l. Wang and Chen [43] used the detoxified hydrolysate from steam-exploded rice straw to produce butanol by Clostridium acetobutylicum ATCC 824, and found that fermentation was improved when the phenolics concentration of the hydrolysate was less than 890 mg/l. The phenolics concentration of the hydrolysate by SH in this work was below 40 mg/l. This suggests that the hydrolysate by SH may be used for biofuels production without detoxification.
Hemicelluloses are polysaccharides in plant cell walls that have beta-(1-->4)-linked backbones with an equatorial configuration. Hemicelluloses include xyloglucans, xylans, mannans and glucomannans, and beta-(1-->3,1-->4)-glucans. These types of hemicelluloses are present in the cell walls of all terrestrial plants, except for beta-(1-->3,1-->4)-glucans, which are restricted to Poales and a few other groups. The detailed structure of the hemicelluloses and their abundance vary widely between different species and cell types. The most important biological role of hemicelluloses is their contribution to strengthening the cell wall by interaction with cellulose and, in some walls, with lignin. These features are discussed in relation to widely accepted models of the primary wall. Hemicelluloses are synthesized by glycosyltransferases located in the Golgi membranes. Many glycosyltransferases needed for biosynthesis of xyloglucans and mannans are known. In contrast, the biosynthesis of xylans and beta-(1-->3,1-->4)-glucans remains very elusive, and recent studies have led to more questions than answers.
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