# Analysis of Sequential Pretreatments to Enhance the Early-Stage Biorefinery Designs
## 3 Results  
### Pretreatment Screening  
The review of the different pretreatments contemplated sequencing at least two pretreatment schemes to maximize the lignocellulosic fraction of interest. It was observed that single-step processes cannot completely fractionate the biomass with maximum accessibility for platform products [17]. Consequently, the performance of further processes could be reduced, as in saccharification, where some studies report the lignin inhibition on enzyme activity due to steric hindrance, the non-accessibility of the enzyme on cellulose, and the non-performance of the enzyme on lignin, leading to its deactivation [24]. Therefore, proposing different sequential schemes to remove unwanted lignocellulosic fractions is needed. Table 2 summarizes the sequential schemes proposed for isolating lignocellulosic compounds. The selection of the schemes was particular to each fraction. Considering the partial removals after each pretreatment, the sequence must have a specific order guided by the technological readiness level (TRL) or the possibility of removing unwanted fractions. For example, sequences were proposed for cellulose isolation that maximized hemicellulose hydrolysis and lignin solubilization through minimal loss of hexoses. It was expected that DA and kraft should be present in cellulose isolation since they are the main pretreatments for biofuel production and pulping of lignocellulosic materials, respectively. Both schemes have high TRLs; however, kraft processes are widely industrialized, enabling their use in the first sequencing pretreatment for the kraft and SE scheme. On the other hand, DA is characterized by high hemicellulose fractionation compared to lignin. Thus, the use of DA as a first step, and a pretreatment with high lignin removal as a second step can be proposed to facilitate the total biomass fractionation in biorefinery schemes, such as the sequence of DA and wet air oxidation (WAO). However, it has been reported that the hydrolysate may contain glucose according to the pretreatment severity and the structural biomass configuration. [PERSON] and co-authors showed that the content of hydrolyzed pentoses could vary between 42% and 66% of the quantified monosaccharides based on the feedstock type [25]. Concerning hemicellulose-based schemes, the sequential order relates the acidic character of the pretreatments in an upward pH to avoid the formation of inhibitory compounds toward the bioconversion processes [26]. Therefore, DA was never the first-step pretreatment for hemicellulose hydrolysis. Although ammonia-recycled percolation (ARP) could be an option in these schemes, there are large fractionations of cellulose [27]. Finally, pretreatments were first performed for the lignin sequences with greater biomass cleaning by removing hemicellulose or those schemes where lignin solubilization is minimal. Table 2 also shows the results of the operational indicators (Indicators (i)-(v)). It was identified that, for the cellulose sequences, the overall removals or losses were less than 19%. Likewise, the accessibility of cellulose in the WIS was more than 90%, except for the DA and alkali sequential pretreatment, indicating a remaining lignin composition that has not been solubilized. However, proposing a third pretreatment step for this sequence leads to higher capital and operating costs, as well as a possible increase in cellulose removal. Accessibility is directly related to I\({}_{\text{CR}}\), demonstrating a higher potential for processes requiring cleaner cellulose, as for SE and WAO sequential pretreatment. However, this sequence is the second most severe, followed by DA and WAO, since they are the most energy-intensive heating schemes. In addition, this sequence implements high-pressure processes using saturated steam.