(477a) Effects of Sequential Pretreatment of Bamboo on Fermentable Sugar Yields for the Production of Biofuels and Bioproducts

Alternative feedstock for biofuel production based on non-edible plants and agricultural wastes should be explored to diffuse the food versus fuel conflict in the use of food crops such as corn and sugarcane for biofuel production. This is especially relevant in low- and middle-income countries where food security must be balanced with efforts at decarbonization. Utilizing plants such as bamboo that grow well on marginal lands that are not economically viable for cultivating food crops is crucial to diffusing this conflict. Unfortunately, bamboo has been studied far less as a renewable carbon source, unlike other feedstock such as corn stover and poplar. Thus, in this work, we studied bamboo endemic to West Africa (Bambusa vulgaris) as a feedstock for the production of renewable fermentable sugars. We investigated the effects of sequential pretreatment of bamboo prior to enzymatic hydrolysis as a way of maximizing the fermentable sugar yield to make it a cost-effective source of renewable carbon for the production of biofuels and bioproducts.

Initially, the bamboo was pulverized using a hammer mill, ground with a coffee grinder, and sieved to a particle size range of 212 – 425 μm. Then, the bamboo particles were ball milled for 30 minutes (BM30) and 60 minutes (BM60). Bamboo was also subjected to a sequential pretreatment where delignification pretreatment was carried out before ball milling for 60 minutes (DL-BM60), or delignification pretreatment was done after the ball milling step (BM60-DL). Delignification was accomplished using sodium chlorite and acetic acid. Afterward, we carried out enzymatic hydrolysis of the pretreated bamboo samples using Cellic CTec2 cellulase enzymes (20 FPU/g biomass) in 50 mM citrate buffer (pH 5.0) at 50°C for 72 hours and analyzed the hydrolysates using HPLC.

XRD analysis showed that ball milling pretreatment decreased the crystallinity of the bamboo (amorphization), with the BM60 pretreated samples being more amorphous. This translated to a recovery of 62% of the available carbohydrates in bamboo as fermentable sugars, primarily in the form of glucose and xylose, for BM60 versus a 40% sugar recovery for BM30 after enzymatic hydrolysis. Further analysis with solid-state 13C NMR revealed that ball milling for 60 minutes resulted in a near-complete cellulose amorphization, leaving only ~ 10% in the crystalline state. In contrast, the hemicellulose and lignin fractions underwent minor (<5%) transformations during ball milling. Thus, the higher sugar yield for BM60 was attributed to increased accessible surfaces for enzyme reaction coupled with the increased cellulose amorphization, which is a key indicator of cellulose reactivity.

This was followed by an evaluation of sequential pretreatment (DL-BM60 or BM60-DL) to address the two critical factors affecting the enzymatic saccharification of lignocellulosic biomass – cellulose crystallinity and lignin content – to maximize the fermentable sugar yield obtainable from bamboo. We observed through HPLC analyses of the hydrolysates that up to 80 ± 5% of sugar yields is obtainable when delignification was done after ball milling (amorphization), i.e., BM60-DL, with the sugar yields increasing to 97 ± 4 % when a reversed order of sequential pretreatment (DL-BM60) is implemented. Solid-state NMR analysis of delignified bamboo indicated a >95% lignin removal, resulting in a 16 percentage point increase in sugar yield (versus BM60). BM60-DL increased the sugar yield by only 2 percentage points.

XRD and solid-state NMR analysis results revealed that cellulose crystallinity increased after delignification and that DL-BM60 was more amorphous than BM60-DL. This meant that ball milling after delignification pretreatment (DL-BM60) was necessary to realize the combined effects of lignin removal and cellulose amorphization to achieve near-quantitative sugar yields (97%) from bamboo.

Finally, techno-economic analysis for bamboo conversion was done using a model developed by NREL for corn stover conversion to sugars as a starting point. The scale of the biorefinery was adjusted for handling a circular area with a 15 km radius centered on the refinery, with all other parameters held constant from their set points. The pretreatment used for corn stover analysis was substituted with the sequential pretreatment processes for bamboo. The scale was re-adjusted to account for differences in productivity between bamboo and corn stover. All other parameters were held at their fixed point to allow a direct comparison. Costs and labor were based on the U.S. market. The projected net present value (NPV) of the bamboo biorefinery was positive ($190 MM, U.S.), whereas that of a corn stover biorefinery was negative NPV (-$430 MM, U.S.) as a result of the higher growth rate of bamboo and the effectiveness of the sequential pretreatment.

In conclusion, this study revealed that ball milling bamboo for 60 minutes results in near complete amorphization of cellulose and increases the accessible surface for enzyme reactivity but does not achieve quantitative sugar yields after enzymatic hydrolysis, with almost 40% of unrecovered sugars left in bamboo. Therefore, sequential ball milling (amorphization) and delignification (lignin removal) is necessary to increase the sugar yields substantially, but the order of sequential pretreatment is crucial for maximizing the sugar yield. There is a clear preference for delignification followed by amorphization, i.e., DL-BM60, which should be factored into any technological deployment. Techno-economic analysis for bamboo conversion to sugars indicated that a bamboo biorefinery based on the preferred sequential pretreatment (DL-BM60) was economically viable with a projected positive NPV. These findings support further investment in bamboo cultivation in marginal lands and conversion to fermentable sugars as a renewable carbon source to decarbonize transportation fuels and chemical production without competing with food, especially in low- and middle-income countries.