## 2 Mechanism of Action
### Impact of Amphiphiles as Biomass Modifier  
The presence of lignin in pretreated biomass solutions is an important factor, since processes that use organic solvents to extract lignin are currently too expensive to make the cellulosic ethanol economically justifiable [18]. Evidence shows that lignin is released into solution during pretreatment with acid or just water/steam, and then reacts to form compounds with limited solubility. These compounds precipitate onto the surface of substrate [58,59], and the deposited lignin irreversibly adsorbs enzymes [60]. When enzymatic hydrolysis of mixtures of cellulose and lignin was conducted in the presence of tri-block-polymer of L64, cellulose was observed to settle at 50 \({}^{\circ}\)C in a water bath; however, lignin and L64 remained suspended in solution. An even further separation of lignin into the L64-rich phase occurred at 70 \({}^{\circ}\)C. Other surfactants such as PEG 4000 and Triton X-100 also demonstrated similar behaviors [28].  
According to [PERSON] _et al._[18], when pretreated biomass is treated with surfactants, a significant amount of the lignin released into the liquid phase could be captured by surfactant-water emulsions, thereby reducing the amount of lignin re-deposited on biomass. [PERSON] _et al._[22] showed that application of 3.33 wt% of the non-ionic surfactant of Tween 20 during pretreatment of baggase at 170-190 \({}^{\circ}\)C enhanced subsequent enzymatic hydrolysis and diminished the amount of residual lignin deposited in/on treated substrate by 22%-27% compared to those treated with water (control). Recently it was demonstrated that soaking corn stover in Tween 80 followed by dilute acid pretreatment (140 \({}^{\circ}\)C, 1% H\({}_{2}\)SO\({}_{4}\)) increased lignin removal by 52% [18]. A similar Tween 80 soaked-corn stover, hydrothermally pre-treated at 220 \({}^{\circ}\)C, resulted in 114% increase in lignin removal [18]. Lignin removal in these samples was calculated based on actual compositional analysis of biomass with the Klasson-lignin method according to NREL Laboratory Analytical Procedure #003 (NREL, 1996). It is probable that the surfactant emulsions isolated the released lignin, and prevented its re-deposition on the surface of biomass (_via_ hydrophobic interaction) that would non-productively adsorb or block enzyme during enzymatic hydrolysis (See Figure 1 for clarification).  
**Figure 1.** A schematic of enzymatic hydrolysis of lignocellulosic biomass pretreated with dilute acid technique in which part of the released lignin materials are re-deposited back onto the biomass and irreversibly adsorbed the enzyme **(I)**; Treatment of dilute acid pretreated biomass with surfactants isolated part of the lignin with monomers of surfactants. This prevents irreversible adsorption of enzymes to non-productive sites of the biomass (e.g., lignin) **(II)**. Brown lines: lignin, grey lines: crystalline and amorphous cellulose, red dots: enzymes, blue objects: surfactants. For simplicity, hemicellulose was not shown in this image.  
Hydrophobicity tests conducted by [PERSON] _et al._[18] showed that the biomass surface became more hydrophilic following surfactant treatment. It was postulated that this could have been due to the surfactant-assisted removal of lignin, which is hydrophobic in nature. Furthermore, [PERSON] _et al._[54] demonstrated a significant increase in water retention in biomass treated with Tween 20; similar to that of NaOH. They hypothesized that Tween 20 can affect the swelling of lignocellulosic as much as NaOH. These results were in agreement with [PERSON] _et al._[19] who demonstrated (based on SEM analysis) that application of Tween 80 caused cellulose filter paper to swell, and enhanced surface cracks and filaments caused by native CBH I but not by core CBH I [19].