Slight acid pre-hydrolysis for pectin extraction
H2SO4, as the most commonly used inorganic acid, was used for pectin extraction owing to its low cost and ability to highly efficiently extract pectin . In this study, pectin is extracted by sulfuric acid at pH 2.0 with conventional heating techniques (95 °C) for 0.5-4.0 hours [17, 18]; the pectin solids was then collected by ethanol precipitation and washing process. The results showed that pectin with a wide range of yield of 6.5-14.8% could be obtained from the dry powder of SSP. Figure 1 indicates that the yield of pectin increased linearly with the increase in duration of extraction over the range of 0.5-2.0 h. However, the yield of pectin did not increase when it was extracted for more than 2 h, and it even decreased when the duration of extraction was excessive. A similar phenomenon was obtained by Yapo et al. (2007) that the overlong extraction time for pectin from sugar beet resulted in large molecules were almost completely degraded into smaller sized ones . It was deduced that the pectin obtained in the extraction medium had been destroyed and disintegrated . Overall, the extraction time was fixed at 2 h for the pectin extraction experiments to minimize the cost.
Since the presence of high content galacturonic acids, therefore, pectic materials all have a characteristic fingerprint region in FTIR spectroscopy , the distinctive peaks shown for galacturonic acids in Fig. 2 are at 1150, 1100 and 1020 cm−1, which confirmed that its identities was pectin [5, 16]. Moreover, the HPLC analysis showed that the galacturonic acid content was approximately 67% from the SSP, indicating that the extracted solids were pectic materials. The average molecular weight calculated from the GPC analysis was 33.2 kDa. Although the average Mw of SSP-pectin was lower than that the values of commercial pectin from apple or citrus, it was still consistent with previous studies that show that the average Mw of pectic materials from various sources, which typically in the range of 10-100 kDa, and can be used as value-added food additives .
Reinforced acid pretreatment for improving the efficiency of enzymatic hydrolysis
After the pectin had been extracted with H2SO4 at pH 2.0, approximately 95.8% of the glucan was retained in PE-SSP residues, and the relative content increased from 32.3% to 51.5%. The retained glucan in pretreated solids can be enzymatically hydrolyzed into glucose; subsequently, glucose is easily bioconverted into ethanol or other biochemicals. It is well known that pectin is the major component of the primary cell walls and the main of pectin is hydration and adhesion of wall cell. Thus, the presence of pectin can influence the porosity of cell wall and morphogenesis of plant. To a certain extent, pectin, as a physical barrier, restrict the access of enzymes to the cellulosic part of cell wall , . Namely, pectin can affect the accessibility of cellulases. Thus, to verify that the removal of pectin improves the saccharification of cellulose, the raw SSP and solids after pectin-extracted SSP (PE-SSP) were all offered to enzymatic hydrolysis process that 4% (w/v) of the solids were loaded.
It can be observed that in Fig. 3, the glucose yield from SSP was 44.2% at 72 h when 20 FPU/g of cellulose was loaded, indicating that the raw SSP was poorly digested by the enzymes. Correspondingly, a glucose yield of 68.1% was obtained by enzymatic hydrolysis of the 4% PE-SSP solids, indicating that the slight acid pretreatment to remove the pectin with H2SO4 could improve the efficiency of saccharification. While the pectin-extracted strategy improved the yield of enzymatic hydrolysis, the results have not been entirely satisfactory for generating the maximum quantity of profit-generating products.
The crystallinity of cellulose is known to significantly affect the digestibility of cellulases. The XRD analysis (Fig. 4) showed that the characteristic peak of the SSP and the PE-SSP were at 2θ=22.1°, which was cellulose I. The CrI of the PE-SSP solids sample (46.3%) increased merely 10% compared with that of the raw SSP material (36.7%). The results suggested that the PE-SSP required additional pretreatment to increase the CrI and improve the enzymatic accessibility for cellulose. Dilute acid pretreatment is the most frequently studied process for agricultural biomass and has been considered to be a suitable technology for bioethanol production at an industrial scale . Thus, a second step, aimed at improving the efficiency of enzymatic hydrolysis, was performed with 0.75% (w/v) H2SO4 at 150 ℃ for 30 min .
An analysis of the reinforced pretreated PE-SSP (RP-PE-SSP) showed that the relative content of glucan increased significantly from 51.5 % to 71.1 % with a yield of 94.1%, and the xylan content decreased from 7.2% to 3.1%. Moreover, the peak of step-2 pretreatment was sharper, and the CrI increased substantially to 62.4%. In addition, the physical structure of the SSP raw material and RP-PE-SSP residues were studied using SEM. As shown in Fig. 5a, the surface morphologies of SSP were smooth and highly ordered. Observation of Fig. 5b showed that the surface morphologies of PE-SSP changed slightly, and only a small part of the cellulose was exposed, while RP-PE-SSP (Fig. 5c) depicted a highly unstructured rough surface with a substantial amount of cracks.
Large specific surface area and total pore volume can offer high adsorption capacity, therefore, the specific surface area and pore volume of lignocellulosic materials are considered to be key factors impacting the enzymatic hydrolysis. The specific surface area of raw SSP, PE-SSP and RP-PE-SSP were 1.529, 2.803, and 3.591 m2/g, respectively, and the corresponding total pore volume was 0.0026, 0.0051, and 0.0064 cm3/g, respectively. The result depicted that the specific surface area and total pore volume significantly increased by enhancement of pretreatment intensity. Such an increase can be attributed to the partial breakdown of the raw material microstructure, resulting in the formation of more cracks and larger pore size, as evident from the micrographs of SEM . Enzymatic hydrolysis is known to be strongly affected by porosity, including specific surface area and total pore volume, since the cellulase can directly contact the cellulose structure through the pores. Overall, incremented specific surface area and pore volume percentage allow better access of the cellulase to inner portion of lignocellulosic solids. Visual observations show that reinforced pretreatment efficiently dissected the physical structure after reinforced pretreatment with 0.75% H2SO4 at 150 °C for 0.5 h, which resulted in an enzymatic hydrolysis yield of up to 92.3% at 72 h with a loading dosage of 4% RP-PE-SSP solids. Obviously, reinforced pretreated can enhance interfacial interactions between the solids and cellulases, resulting in improvement of saccharification efficiency [27, 28].
High-solids enzymatic hydrolysis with batch and fed-batch modes
The fermentable sugars should be at levels as high as possible in the industrial scale utilization of lignocellulosic materials due to high final sugars is benefit to improve utilization rate of equipment, and low the consumption of water and energy. Using bioethanol generation as an example, obtaining fermentation broths of at least 4% (w/v) of ethanol is essential for economical large-scale production owing to the costs of ethanol purification, which dramatically increase when the ethanol titer is lower . Thus, in this framework, at least more than 80 g/L of total reducing sugars obtained from the biomass is required. Correspondingly, enzymatic hydrolysis must be conducted with a pretreated solid loading of more than 10~20% (w/v) on the basis of the cellulose proportion. Moreover, high-solids enzymatic hydrolysis is preferred for economic reasons for biorefinery processes on an industrial scale. Thus, enzymatic hydrolysis assays with batch mode were first performed with 4, 8, 12, and 16% RP-PE-SSP solids loading to produce a high titer of glucose.
Figure 6 shows that a glucose titer of 28.9, 51.6, 63.5, and 54.4 g/L final glucose accumulated with a solid loading of 4, 8, 12, and 16%, respectively. It was apparent that 4% solid in the system could be liquefied within 8 h. However, the mixing became fouled with a batch operation of more than 8% (w/v) solids loading, and in the case of 12% (w/v) RP-PE-SSP solids loading, the reaction medium was not mixed at the beginning of the experiments, even with a significant increase in the rate of agitation. In addition, the system with high solids loading had difficulty becoming liquefied even when treated for more than 24 h, which speculate that this due to the reduction of crystallinity and the limited catalytic sites for cellulases [30, 31]. It is apparent that a high consistency of hydrolysis may result in difficulties mass transfer owing to high viscosity as a result of high solids loading and the lack of free water in the enzymatic system. Thus, glucose yields from the loading of 8, 12, and 16% solids declined linearly. The enzymatic hydrolysis yield from loading of 16% solids was only 43.4%.
Although increasing the solids loading was the simplest and most direct way to enable a high concentration of sugars, this technique resulted in high viscosity, poor mixing, heat transfer and enzyme distribution problems, which reduced the efficiency of enzymatic hydrolysis [30, 32]. The fed-batch process is regarded as an effective way to minimize these negative effects. Thus, we conducted a fed-batch enzymatic hydrolysis of RP-PE-SSP material to produce glucose. A fed-batch operation of 4%-4%-4%-4% (every 12 h) solids dosage was conducted for 72 h for enzymatic hydrolysis. When the enzymatic hydrolysis was successfully performed with a solid content as high as 16% (w/v), the glucose titer obtained and yield reached 103.1 g/L and 83.6 %, respectively. Overall, the fed-batch strategy enables an easy dynamical load and the production of a solution with a high glucose titer. The mass balance of the whole process for releasing pectin and glucose showed that a total of 120 g pectin and 260 g glucose were recovered from 1,000 g oven-dried raw material using a two-step pretreatment technology and the fed-batch enzymatic hydrolysis mode. The entirety of the results clearly suggests that the two-step acid-treatment with H2SO4 is a profitable option for the further exploitation of sunflower residue.