## 1 Introduction
### Impact of Amphiphiles on Enzyme Recycling  
Due to the high cost of cellulolytic enzymes and their high dosage rates (e.g., 15 FPU/g glucan) for biomass conversion, enzyme recycling has been evaluated in several studies. One study noted an 85% reduction in enzyme activity (Ctec\({}_{2}\)-15 FPU/g glucan) after 24 h hydrolysis of corn stover that contained \(\sim\)20.2% lignin [36]. The author's hypothesized that the reason for the reduced enzyme activity was deformation and/or de-solubilization of cellulase caused by the high concentration of lignin in pretreated biomass. For enzymes to be efficiently recycled, it is important for them to remain free in solution or be washed off the biomass with sodium chloride solution to some extent [1]. The ability of amphiphiles to adhere to non-productive sites of the biomass and prevent irreversible enzyme adsorption has been clearly demonstrated before [27, 33, 36, 48]. According to [PERSON] _et al._[27], the adsorption of cellobiohydrolase decreased by 60%-70% onto steam exploded lodgepole pine (SELP) using Tween 20. Likewise, desorption of cellulases (cellobiohydrolase and endoglucanase) into the liquid phase was improved from 46% to73% during the hydrolysis of SELP when Tween 80 was used as an enzyme stabilizer [31]. This property of amphiphiles can be exploited for recycling of cellulase if the revenue made from the additional amount of sugar and ethanol is higher than the cost paid for the amphiphiles.  
For enzyme recycling, a re-adsorption technique was first proposed by [PERSON] _et al._[49] as an alternative to costly ultra-filtration techniques. In this process, the slurry of hydrolysate or fermentation broth is centrifuged or filtered, and the supernatant containing soluble enzymes is incubated with fresh biomass for 2 h to allow the adsorption of free enzymes onto fresh biomass. Then after a second separation step, the biomass residues are re-solubilized in fresh buffer and additional \(\beta\)-glucosidase is added for another cycle of hydrolysis or fermentation [31, 46]. It has been suggested that the re-adsorption technique prevents the accumulation of lignin degradation by-products to toxic levels [31,50,51]. Due to the advantage of the SSF process that assimilates the inhibitors and sugars as they are generated, we believe that a re-adsorption process might not be necessary considering that it adds additional costs to the process through the addition of an extra separation step, water and \(\beta\)-glucosidase.  
When surfactant was applied to ethanol pretreated lodgepole pine (EPLP), it increased the free enzyme levels of celluclast and spezyme cp from 71% of the original amount to 96%. Similarly, in another study it was found that the efficiency of enzyme recycling (using re-adsorption technique) was significantly higher in lower lignin content substrates, compared to feedstocks such as SELP that contained higher lignin levels [31]. Differences in enzyme recycling also appear to be related to the source of enzymes. For instance, under similar experimental conditions (similar substrate and surfactant), enzymes from _Trichoderma ressei_ were successfully recycled for 4 cycles, while _Penicillium_-derived enzyme was only recycled once successfully [31].  
According to [PERSON] _et al._[52], after two steps recycling of fermentation liquor containing enzymes, the ethanol yield was improved by 80% and 130% with the aid of Tween and liquid casein micelles, respectively. Polymeric micelles (PMs) of PEG-Tween and PEG-Casein improved enzyme recycling further, such that the ethanol yield was improved by 50% and 108% beyond that obtained with only Tween and casein, respectively. Amphiphiles of acid casein were also found to improve the sugar recovery and fermentability of dilute acid, lime, alkali, extrusion pretreated corn stover by up to 31% and 33%, respectively. Neither of Tween 20, nor the accumulated sugars showed toxicity to microbial or enzyme activity [42].