Bamboo pretreated with SAA exhibited substantial delignification in comparison with unpretreated bamboo (Figure 2.5). Furthermore, there was a significant increase in the level of delignification with higher aqueous ammonia concentrations (ANOVA, p-value>0.05). The amount of mass loss during SAA pretreatment ranged from 23.1% to 29.8% of the initial biomass, and the majority of this was due to delignification. Despite this high level of delignification during SAA pretreatments, the majority of sugars were preserved within the bamboo material at all conditions. At the most severe pretreatment condition of 24 hours with 25% NH4OH, glucan was only reduced from 38.4% to 37.2% and xylan from 20.4% to
18.3%; they were thus retained at 97% and 89% of their original contents, respectively. Under this condition, lignin was reduced from its initial level of 20.8% to 10.8% of DM, representing a 48.4% reduction. With the exception of lignin, there was relatively little variation in the biomass composition despite the range of pretreatment concentrations (10- 25% w/w NH4OH) and times (6-24 hours) tested.
Figure 2.5 Complete mass closures of bamboo pretreated with soaking in aqueous
ammonia expressed as a percentage of dry matter.
38.4 37.0 37.6 37.7 37.5 37.0 35.4 36.0 37.1 37.2 37.4 37.7 37.2 20.5 18.9 20.2 19.8 19.3 18.7 17.7 17.3 17.7 18.4 18.5 18.5 18.3 3.6 2.0 3.2 3.2 2.8 3.2 2.9 2.7 20.8 16.5 14.5 13.5 16.7 15.3 14.5 14.4 12.5 13.7 13.4 11.9 10.8 23.1 23.4 26.2 22.5 25.5 26.6 24.0 24.2 26.0 25.9 27.1 29.8 13.5 0 20 40 60 80 100 % o f DM
Glucan Xylan Galactan Arabinan Lignin
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Levels of delignification reported in the literature vary significantly depending on the pretreatment conditions and feedstocks used, demonstrating that the optimal conditions have yet to be identified and/or these are linked to differences in biomass composition and structure. While almost half of the original lignin content was solubilised during the most severe SAA pretreatment, this amount is on the lower end of the range of 47% to 74% reported for other herbaceous feedstocks (Kim and Lee, 2005, Kim et al., 2008, Isci et al., 2008, Ko et al., 2009), and is substantially lower than the delignification levels achieved in corn stover (up to 80% of initial lignin content) (Cao et al., 1996, Kim et al., 2008). Although the amount of delignification achieved in this study is comparable to that reported for rice straw (47% delignification) (Ko et al., 2009), those results were achieved after 10 days of soaking compared to 24 hours used in this study. Despite these discrepancies, studies have been fairly consistent in demonstrating that after pretreatment, nearly 100% of glucan and around 85% of xylan are retained in the material – this post-pretreatment sugar-rich, low lignin composition was expected to be a major contributing factor towards increasing sugar release during enzymatic saccharification.
2.3.3.2
Effect of soaking in aqueous ammonia pretreatment on sugar yields
The sugar release from pretreatment and enzymatic saccharification in this section are reported in the same way as in Section 2.3.2.2. Following SAA pretreatment the total sugar release ranged from 21.8% to 30.6% of DM (equivalent to 34.0% to 47.7% of the theoretical maximum) (Figure 2.6). In terms of the predicted ethanol yield per dry tonne of biomass, this is equivalent to a range of 120 to 166 litres of bioethanol. At each aqueous ammonia concentration, there was an increase in total sugar release when time was raised from 6 to 14 to 24 hours. However, the difference between concentrations was not significant (ANOVA, p-value>0.05) with the exception of pretreatment with 15% NH4OH for 24 hours,
where total sugar release was maximised amongst the conditions tested. In contrast to LHW pretreatment, there was a greater proportion of sugar released from saccharification compared with pretreatment, such that this comprised approximately 67-82% of the total release. However, it was found that the pretreatment condition with the highest sugar release (15% NH4OH for 24 hours) also had the greatest level of sugar solubilisation during
pretreatment (16% compared with 6-11% of the theoretical maximum). At this condition, sugar release during saccharification (approximately 32% of the theoretical maximum) was exceeded by two other pretreatment conditions which reached 33% of the theoretical maximum.
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Figure 2.6 Effect of soaking in aqueous ammonia pretreatment (PT) and enzymatic
saccharification (ES) on total sugar release. Sugar release is expressed as a percentage of dry matter under a standardised enzyme loading of 60 FPU/g glucan. Other sugars refer to the sum of galactose and arabinose solubilised during pretreatment. Theoretical maximum sugars indicated by the red dashed line and is equal to 64.2% of DM. *Optimal SAA pretreatment condition.
Delignification as a result of alkali pretreatment is viewed as an alternative approach to hemicellulose solubilisation for improving enzyme accessibility to cellulose. In general, although harsher pretreatment conditions led to enhanced delignification, this did not consistently produce the substrate most amenable to enzymatic digestion, as shown by sugar yields in saccharification (Figure 2.6). The highest saccharification yields amongst these conditions were achieved during pretreatments with 25% and 20% NH4OH for 24
hours, which both released approximately 33% of the theoretical maximum. This was statistically higher (ANOVA, p-value<0.05) than the next best condition of pretreatment with 15% NH4OH for 24 hours, which released 32% of the theoretical maximum. Between these
conditions however, a substantial difference in the level of delignification was found, whereby pretreatment with 25% NH4OH and 20% NH4OH lost 48% and 31% from initial
lignin contents, respectively. Although increased delignification was observed with higher aqueous ammonia concentrations and longer pretreatment times, these did not necessarily correspond to an enhanced sugar yield during saccharification when delignification exceeded 31% of initial lignin. Furthermore, there were other conditions which also achieved around 30% delignification during pretreatment that released significantly lower levels of sugar during saccharification. These variable results seem to suggest that there
0 20 40 60 80 100 S u g ar r ele as e (% o f DM )
PT other sugars PT Glucose PT Xylose ES Glucose ES Xylose *
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does not seem to be a strong correlation between cell wall accessibility and the absolute level of lignin, but possibly stronger associations could be found between accessibility and other factors such as lignin chemistry and/or its physical or chemical linkage to other cell wall components.
On the one hand, numerous studies on herbaceous feedstocks have successfully demonstrated that progressive SAA-catalysed delignification enhances sugar release via greater enzyme accessibility to its substrate and reduced non-productive enzyme adsorption to lignin (Kim and Lee, 2007, Kim et al., 2008, Isci et al., 2008, Ko et al., 2009). However, other studies (Fan et al., 1981, Berlin et al., 2006, Zhu et al., 2008) have also contradicted this by showing that above a certain level of delignification, there is no further improvement in biomass digestibility. Two possible explanations have been proposed for the second argument: 1) extensive delignification results in a collapse of the cell wall structure, therefore reducing the available surface area for enzyme adsorption and preventing potential sugar release; and 2) the chemical composition of lignin is more important than absolute lignin content in reducing recalcitrance and thus enzymatic digestibility (Fan et al., 1981, Zhu et al., 2008, Moxley et al., 2012). Additionally, a recent finding on switchgrass revealed that while SAA pretreatment caused mild improvements in accessibility due to a lower lignin content, the remaining lignin was more evenly distributed (as opposed to being clustered) with a higher surface area to volume ratio, which actually resulted in hindering enzymes from effectively releasing cell wall sugars (Rollin et al., 2011). Though our results could possibly support this theory, further ultrastructural studies outside the scope of this investigation area would be required to establish whether bamboo biomass exhibits the same response during SAA-catalysed delignification.