3.1. Raw Material and Autohydrolysis Treatment: Chemical Analysis Composition
The chemical composition of corn stover is shown in Table 1
, displaying similar values to those obtained by other authors [6
]. As seen, the sum of polysaccharides accounted for 56.8% of raw material, showing its potential for bioethanol production.
As indicated in Table 2
, corn stover was subjected to non-isothermal autohydrolysis at maximum temperatures of 200, 210, 220, 230, and 240 °C (corresponding with severities of 3.93, 4.20, 4.48, 4.75, and 5.03, respectively). The wide range of temperatures respond to the necessity of studying (i) milder conditions that aid the recovery of a great majority of hemicellulosic-derived compounds in the liquid fraction, and (ii) harsher conditions that facilitate the accessibility of enzymes to the cellulose of the solid fraction, making it suitable for bioethanol production. Table 2
shows the main results of the solid and liquid fractions after autohydrolysis pretreatment.
Firstly, solid yield (SY) decreased when the severity augmented, in a range of 64.5–55.5 g of autohydrolyzed corn stover/100 g of raw material. Similarly, nonvolatile compounds (NVCs) were reduced in harsher conditions from 32.3 g/100 g of raw material at 200 °C to 18.8 g/100 g of raw material at 240 °C. On the contrary, volatile compounds (VCs, calculated from the difference between SY and NVC) increased to 25.7 g/100 g of raw material for the highest severity evaluated (S0
= 5.03). This behavior can be explained due to the compounds in the extractives and to the sugar degradation reactions that provoke the transformation of saccharides into volatile compounds (such as acetic acid, hydroxymethylfurfural, or furfural) [39
Referring to the solid fraction, the glucan content stayed at similar values throughout the temperatures (55.4–62.6 g of glucan/100 g of autohydrolyzed corn stover) with an average recovery of 87.4% regarding the initial glucan. Klason lignin values varied between 28.5 and 39.7 g of lignin/100 g autohydrolyzed corn stover (with an average recovery of 87.0%), showing some degree of lignin solubilization at lower temperatures as reported for other lignocellulosic biomasses such as Eucalyptus globulus
] and higher recoveries in harsher conditions due to lignin repolimerization as explained for Paulownia wood [20
] and vine shoots [41
]. In addition, the relative percentage of acid-insoluble lignin extracted from hydrothermal pretreatment under high-severity conditions can be considered as pseudo-lignin [42
], which could be formed through reactions on lignin aromatic rings or acid-catalyzed condensation reactions on fragmented polysaccharides [43
]. On the other hand, hemicelluloses (xylan, arabinan, and acetyl groups) were mostly solubilized in all conditions (especially in the harshest ones). Hence, small amounts of hemicelluloses remained in the solid fraction, with values of about 6.55 g of hemicelluloses/100 g of autohydrolyzed corn stover in the mildest conditions, and an almost total solubilization in the harshest conditions (with recoveries of about 34% and 0.1% regarding initial hemicelluloses, respectively). Hence, glucan and lignin were recovered, almost quantitatively, in the solid phase, representing the almost total composition of this fraction with about a 93.4 g/100 g of autohydrolyzed corn stover (in average), increasing from 61.8 g/100 g regarding the raw corn stover. Similar results were obtained for autohydrolysis treatment of stover samples from several maize genotypes [36
Regarding the liquid fraction, the oligosaccharide content stands out in the milder conditions, especially the xylooligosaccharide form, with values up to 15.7 and 14.2 g oligomers/L (at temperatures of 200 and 210 °C, respectively), which represent 77.9% and 65.8% of compounds in the liquid fraction. In addition, xylooligosaccharides were released in the autohydrolysis liquor at values of 70.1% and 63.2% regarding initial xylan at temperatures of 200 and 210 °C. Similar operational conditions (205 °C, yielding 15.8 g oligosaccharides/L) were found by Buruiana et al. (2014) as optimal for the non-isothermal autohydrolysis of corn stover [35
]. In on, study, the hydrothermal processing of peanut shells [21
], performed at 210 °C (S0
= 4.09), resulted in concentrations of soluble oligomers compounds of 9.05 g/L, which are much lower than those obtained in this work. In another study, Gullón et al. (2018) reported high concentrations of oligosaccharides from chestnut shells (18.3 g/L) under milder autohydrolysis conditions (180 °C and S0
= 3.08) [44
]. Additionally, degradation products in the optimal temperature for oligosaccharide recovery (200 °C) accounted for almost 3 g/L, a very similar value to that reported by Rico et al. (2018) after autohydrolysis of peanut shell at 210 °C with about 3.7 g/L [21
In harsher conditions, the oligomer content was drastically reduced (values lower than 4 g/L) due to depolymerization phenomena into simple sugars (monomers). Acetic acid, hydroxymethylfurfural, and furfural had an increasing tendency with severity achieving values up to 5.18, 0.84, and 1.97 g/L, respectively.
3.3. Simultaneous Saccharification and Fermentation (SSF) of the Whole Slurries after Autohydrolysis
After the autohydrolysis process, a suspension of autohydrolyzed corn stover (mainly composed of glucan and lignin, as shown in Table 2
) and an autohydrolysis liquor (mainly composed of hemicellulosic-derived compounds, as shown in Table 2
) was obtained, echibiting three different possibilities for their use in the production of bioethanol (as seen in Figure 1
): (i) employing the whole slurry in the SSF process, (ii) partially separating both liquid and solid fractions for different liquor percentages in the SSF, and (iii) totally separating both fractions, with a consequent washing of the solid phase, employing water as the liquid phase in the SSF assays. The former option is the easiest and most economic one, using a one-pot process, even allowing the possibility of fermenting the hemicellulosic-derived compounds, which increases the ethanol concentration [24
]. However, the degradation products present in the liquor (namely, furan and phenolic compounds) may inhibit the growth of the yeast and reduce the enzyme activity [49
]. On the other hand, the latter option permits the separation of the hemicellulosic-derived compounds in a different stream to be used in other applications for the food industry (e.g., prebiotics and sweeteners) or the chemical industry (e.g., furfural) [50
], albeit implying a higher economic cost due to the solid–liquid separation and the washing of the solid fraction to avoid technical issues due to the presence of inhibitory compounds.
Bearing this in mind, the use of liquor in different proportions (100%, 75%, 50%, 25%) and the use of water (0% of liquor) as the liquid phase for the SSF processes was proposed. Furthermore, taking into account the results from previous enzymatic hydrolysis, substrates from the temperature of 200 °C were discarded for the subsequent step of fermentation due to their low enzymatic susceptibility, compared with the others. Thus, solid fractions from autohydrolysis of 210, 220, 230, and 240 °C were subjected to SSF at industrial-level conditions of high-solid loading (LSR = 6 g/g, CSR = 15 FPU/g, and CCR = 5 UI/FPU), which allow the production of ethanol at a high concentration reducing evaporation costs [25
]. In addition, a commitment temperature of 35 °C, between that optimal for the enzyme (48.5 °C) and the yeast (32 °C), was selected. Figure 3
displays the time course of ethanol concentration reached during SSF of autohydrolyzed corn stover using different liquor percentages. Table 3
summarize the maximum ethanol concentration, maximum ethanol conversion, ethanol concentration at 72 h, and volumetric productivity at 72 h.
In general, concentrations between 32 and 42 g of ethanol/L were achieved in all of the experiments, with the exception of the SSF performed using the solid pretreated at 240 °C and with 100% and 75% of liquor, which did not lead to ethanol production, probably owing to the high quantity of inhibitory compounds in the autohydrolysis liquor such as acetic acid (5.18 g/L), hydroxymethylfurfural (0.65 g/L), or furfural (1.63 g/L) that inhibit the yeast growth [52
Firstly, SSF of the solid pretreated at 210 °C (Figure 3
a) showed faster and higher ethanol productions when using 50% or 25% liquor as liquid phase. Ethanol concentrations at 72 h displayed significant differences (p
< 0.05) for SSF assays with those liquor percentages when compared to the other experiments. This may be as a consequence of the glucose and glucooligosaccharides in the medium (that can be easily and rapidly employed by the yeast or attacked by the enzymes) and the lower amounts of xylooligosaccharides in the diluted liquor that may act as inhibitors to enzymes and yeasts in a whole-slurry fermentation [16
]. Meanwhile using water as liquid phase resulted in similar ethanol concentrations to those obtained with 25% (about 35–36 g/L).
SSF from autohydrolyzed solid at 220 and 230 °C (Figure 3
b,c, respectively) resulted in similar values of ethanol titer regardless of the liquor percentage employed (between 37.7 and 41.9 g/L), reaching the highest value with the SSF using the solid hydrothermally treated at 230 °C and 100% liquor (close to 80% ethanol conversion). However, the first 24 h in this SSF reflected a delay in the delivery of ethanol, possibly because the yeast stayed longer in the lag phase, accustomed to the higher amounts of degradation products [53
], which favored an increase in the concentration and a faster assimilation of the glucose. In this case, SSF carried out with solid and liquor from hydrothermally pretreated corn stover at 230 °C did not show significant differences (p
> 0.05) at maximum ethanol concentrations, whereas SSF assays with solid and liquors from 220 °C were significantly different (p
< 0.05) when using water (0% of liquor) and using any other percentage of liquor.
Lastly, SSF from solid pretreated at 240 °C (Figure 3
d) at higher percentages of liquor (100% and 75%) was unable to produce ethanol. This was due to the elevated concentration of inhibitors, such as acetic acid, furans, or even humic compounds (that were not identified in this work) that disabled the yeast in the fermentation process [55
]. In spite of this, the enzymes aided the production of glucose, as can be seen in the time courses, obtaining cellulose-to-glucose conversions of 58% and 62% for 100% and 75% liquor, respectively, which can be compared to results obtained for enzymatic hydrolysis of autohydrolyzed corn stover with water as the liquid medium. Thus, under this condition, enzyme inhibition was observed, probably as a consequence of the higher concentration of phenolic compounds in the liquor and high-solid loading in the fermentation assay, causing precipitation of the enzymes [25
]. On the other hand, SSF experiments with liquor percentages of 0%, 25%, and 50% reached maximum ethanol concentrations between 36.8 and 37.5 g/L, with significant differences (p
< 0.05) between them and those from assays with liquor percentages of 75% and 100%.
Summing up, ethanol conversions of around 70% were achieved in the best conditions of SSF from autohydrolysis at 210, 220, and 240 °C, whereas a conversion of about 80% was accomplished in the SSF from autohydrolysis at 230 °C while employing a liquor percentage of 100%. In addition, 40 g/L of ethanol is the suggested ethanol concentration goal for a commercial manufacture for second-generation bioethanol [56
], and the SSF assays from autohydrolyzed corn stover achieved values close to and above this recommendation. Moreover, if employing the xylose and xylooligosaccharides for biofuel production, the concentration may increase to 7.6 g/L from hemicellulosic ethanol of autohydrolyzed liquor of 210 and 220 °C.
In Table 3
, the volumetric productivity of ethanol was calculated at 72 h owing to the fact that, at that time, the great majority of ethanol was already produced. In this case, values ranging from 0.254–0.571 g ethanol/(L·h) were achieved, with the exception of the SSF at 240 °C with 100% and 75% of liquor with 0.012 and 0.017, respectively. Higher values of QP
, above 0.500 g/(L·h), were obtained with the solid and liquor of autohydrolysis at 230 °C. Volumetric productivities close or above 0.400 g/(L·h) were achieved for SFF of 210 and 240 °C. On the contrary, SSF assays at 220 °C resulted in a QP
around 0.300 g ethanol/(L·h). These results demonstrate faster and higher ethanol production when employing the solid and liquid phase from autohydrolysis at 230 °C.
SSF assays carried out with the autohydrolyzed corn stover at 210 °C with liquor percentages of 100% and 25% seem interesting due to the possibility of using the compounds in the remaining liquor for other purposes (such as in the food or the chemical industries or even for the production of ethanol when employing xylose-metabolizing microorganisms [48
]), while exhibiting ethanol concentrations of 32.6–36.3 g/L and ethanol conversions between 59% and 68%. On the other hand, SSF from autohydrolyzed corn stover at 230 °C with liquor percentages of 100% and 75% achieved very positive results regarding the maximum ethanol concentration (40.7–41.9 g/L), maximum ethanol conversion (77–79%), and volumetric productivity (0.383–0.522 g/(L·h)).
3.4. Overall Mass Balance
In order to summarize and get a better perception of the results, an overall mass balance was calculated using the data from autohydrolysis and SSF. Figure 4
shows the schemes leading to higher ethanol titers for each temperature configuration (keeping in mind the amount of liquor employed in the SSF assays).
Beginning with 100 kg of corn stover o.d.b., the autohydrolysis process resulted in different amounts of oligomers in the liquid fraction, depending on the severity of treatment: 13.0, 3.66, 0.97, and 0.48 kg respectively. Conversely, furans and acetic acid increased from values of 3.85 kg at 210 °C to 7.30 kg at 230 °C. However, the autohydrolyzed solid fraction maintained values of glucan and lignin of 32.235.6 kg and 17.2–22.1 kg, while hemicelluloses decreased from 3.26 to 0.02 kg.
After the SSF process at optimal conditions of percentage of liquor for each temperature, 13.7–15.6 kg of ethanol was obtained. On the one hand, the scheme with autohydrolysis at 210 °C and 25% of liquor as the liquid medium could achieve 14.0 kg of ethanol, while liquor rich in hemicellulose-derived compounds could be employed for other applications. Conversely, autohydrolysis at 230 °C seemed optimal for the employment of a highly susceptible solid fraction toward enzymes and a percentage of liquor–water of 100% (15.6 kg of ethanol), which implies a whole-slurry SSF scheme that may be the base for a one-pot process for the production of bioethanol from autohydrolyzed corn stover.