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bit25022-sm-0001-SuppFig-S1.JPG3704KFigure S1. Comparison of unpelletized (“loose”) and pelletized AFEX™ treated corn stover. Each 2.8 L Erlenmeyer flask contains 180 g corn stover. The bulk density of pellets is approximately 550 kg/m3 dry weight compared to 60 kg/m3 for the loose biomass.
bit25022-sm-0001-SuppFig-S2.JPG3603KFigure S2. Water, citrate buffer, antibiotics, and enzymes were added to each flask to bring the total weight up to 1,000 g in order to perform hydrolysis at 18% solid loading. All pellets are completely submerged in the solution, and there is a significant amount of free water present to allow dispersion of biomass and even mixing. In contrast, approximately half of the loose biomass is above the water line, and there is very little free liquid. If this approach is used for hydrolysis, it takes approximately 5 h for the slurry to liquefy enough that all biomass becomes submerged in the solution, and another hour until it becomes an easily mixable slurry. Because of this slow liquefaction, a fed batch approach is used for loose biomass, in which half of the biomass is added immediately and half after 3 h. Fed batch addition is not required for pellets.
bit25022-sm-0001-SuppFig-S3.JPG3615KFigure S3. This figure shows a close-up of high solids hydrolysis of pellets 1 h after enzyme addition. Pellets have partially dispersed into free particulates, which are fully susceptible to enzymatic attack. Although the slurry has thickened due to absorption of water by the loose biomass particulates, the relatively slow rate of pellet dispersion maintains an easily mixable slurry.
bit25022-sm-0001-SuppFig-S4.JPG3609KFigure S4. After 3 h, the thickness of the slurry has decreased dramatically. Very few if any intact pellets can be observed, and the solids are easily suspended. After this point, the opaque brown mixture will not change appearance throughout the rest of the hydrolysis. There is little difference in performance between pellets and loose biomass once both are fully liquefied.
bit25022-sm-0001-SuppFig-S5.JPG699KFigure S5. Pellets were added to a 5-L stirred tank reactor equipped with a single marine impeller. Working volume is 3 L. The solid loading of pellets was 18% by weight. The vertical placement of the impeller is at approximately the mid-point of the total slurry. This image was taken immediately after all pellets were added. As seen in this image, the pellets only account for less than one-third of the total volume, allowing for adequate mixing.
bit25022-sm-0001-SuppFig-S6.JPG2259KFigure S6. After 20 min of enzymatic hydrolysis in the stirred tank reactor, the impeller was turned off and biomass allowed to settle to take this image. Most pellets were disrupted by this time, although some pellets remained visible. After starting the impeller, the biomass that was built up along the bottom of the reactor went into suspension. This required a high speed (600 rpm), but it is expected that the speed would decrease with a different impeller configuration.
bit25022-sm-0001-SuppFig-S7.JPG2125KFigure S7. After 2 h of enzymatic hydrolysis in the stirred tank reactor, the impeller was turned off and biomass allowed to settle. No pellets were visible and the biomass particulates were very fine. At this point, an impeller speed of less than 150 rpm was sufficient to maintain all biomass in suspension. This impeller speed was sufficient throughout the remaining residence time.
bit25022-sm-0001-SuppVideo-S1.wmv19100KVideo S1. Video showing pelletized biomass that had settled out after 20 min of hydrolysis (see Supplemental Fig. S6). The impeller speed was gradually increased until the biomass was fully suspended.
bit25022-sm-0001-SuppVideo-S2.wmv9294KVideo S2. Video showing loose biomass 20 min after all biomass is loaded for loose AFEX biomass at 18% solid loading. Biomass was added in a fed batch manner. Only the bottom portion of the hydrolysate is well mixed, while a solid cake of biomass is present at the top. Regardless of the impeller configuration, a significant mass of solids would be immobile during the liquefaction phase. It can take up to 24 h before all biomass is suspended with a marine impeller.

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