MICROSCOPIC AND LABORATORY SCALE CHARACTERIZATION METHODS TO EVALUATE BIOMASS DECONSTRUCTION

Renewable fuels from lignocellulosic biomass are an appealing option because they can seamlessly integrate into the existing fuel distribution infrastructure. Lignocellulosic biomass constitutes nonedible plant material obtained from plant cell walls. The natural recalcitrance of lignocellulosic biomass poses a challenge in accessing the cell wall carbohydrates during biochemical conversion. Despite various approaches, enzymatic hydrolysis of lignocellulosic biomass remains economically impractical due to incomplete knowledge about biomass recalcitrance and the influence of environmental factors on biomass quality. The first goal of this dissertation was to construct a microfluidic imaging reactor to better understand the tissue-specific deconstruction of plant materials. Confocal laser scanning microscopy was conducted on thin sections (60 mm thickness) of corn stems at different time points during dilute acid and dilute alkali pretreatment. The digestibility of the acid and alkali-pretreated biomass sections during enzymatic hydrolysis were evaluated using brightfield imaging. Corn stem parenchyma cells were more susceptible to deconstruction than vascular bundles in both pretreatment and enzymatic hydrolysis. After 48 hours of enzymatic hydrolysis, only the protoxylem remained undegraded. The second goal was to investigate how increasing stem solidness impacts enzymatic digestibility in wheat straw using the microfluidic imaging reactor. This was based on the rationale that the pith parenchyma cells are more digestible than the other vascular cell types. During the pretreatment and enzymatic hydrolysis, the solid stemmed samples showed considerably greater amenability to degradation than the hollow and semisolid cultivars, which based on the imaging was largely due to the greater digestibility of the pith parenchyma cells. The third goal was to develop a high-throughput, moderate-scale enzymatic hydrolysis method at high-solids loading to study the impact of drought and extreme weather conditions on biomass deconstruction. At the laboratory scale, high solids loading results in improper mixing and low saccharification due to low water availability. This was overcome using horizontal mixing on a laboratory scale roller to improve enzyme accessibility and obtain higher sugar yields. The saccharification for the roller bottle method was about 25-50% higher than the traditional shake flask method. This was evaluated for a variety of AFEX-pretreated feedstocks, including corn stover, sorghum, miscanthus, native prairie, and switchgrass

A high solids field-to-fuel research pipeline to identify interactions between feedstocks and biofuel production

Background: Environmental factors, such as weather extremes, have the potential to cause adverse effects on plant biomass quality and quantity. Beyond adversely affecting feedstock yield and composition, which have been extensively studied, environmental factors can have detrimental effects on saccharification and fermentation processes in biofuel production. Only a few studies have evaluated the effect of these factors on biomass deconstruction into biofuel and resulting fuel yields. This field-to-fuel evaluation of various feedstocks requires rigorous coordination of pretreatment, enzymatic hydrolysis, and fermentation experiments. A large number of biomass samples, often in limited quantity, are needed to thoroughly understand the effect of environmental conditions on biofuel production. This requires greater processing and analytical throughput of industrially relevant, high solids loading hydrolysates for fermentation, and led to the need for a laboratory-scale high solids experimentation platform. Results: A field-to-fuel platform was developed to provide sufficient volumes of high solids loading enzymatic hydrolysate for fermentation. AFEX pretreatment was conducted in custom pretreatment reactors, followed by high solids enzymatic hydrolysis. To accommodate enzymatic hydrolysis of multiple samples, roller bottles were used to overcome the bottlenecks of mixing and reduced sugar yields at high solids loading, while allowing greater sample throughput than possible in bioreactors. The roller bottle method provided 42–47% greater liquefaction compared to the batch shake flask method for the same solids loading. In fermentation experiments, hydrolysates from roller bottles were fermented more rapidly, with greater xylose consumption, but lower final ethanol yields and CO2 production than hydrolysates generated with shake flasks. The entire platform was tested and was able to replicate patterns of fermentation inhibition previously observed for experiments conducted in larger-scale reactors and bioreactors, showing divergent fermentation patterns for drought and normal year switchgrass hydrolysates. Conclusion: A pipeline of small-scale AFEX pretreatment and roller bottle enzymatic hydrolysis was able to provide adequate quantities of hydrolysate for respirometer fermentation experiments and was able to overcome hydrolysis bottlenecks at high solids loading by obtaining greater liquefaction compared to batch shake flask hydrolysis. Thus, the roller bottle method can be effectively utilized to compare divergent feedstocks and diverse process conditions

Microfluidic reactor designed for time-lapsed imaging of pretreatment and enzymatic hydrolysis of lignocellulosic biomass

The effect of tissue-specific biochemical heterogeneities of lignocellulosic biomass on biomass deconstruction is best understood through confocal laser scanning microscopy (CLSM) combined with immunohistochemistry. However, this process can be challenging, given the fragility of plant materials, and is generally not able to observe changes in the same section of biomass during both pretreatment and enzymatic hydrolysis. To overcome this challenge, a custom polydimethylsiloxane (PDMS) microfluidic imaging reactor was constructed using standard photolithographic techniques. As proof of concept, CLSM was performed on 60 μm-thick corn stem sections during pretreatment and enzymatic hydrolysis using the imaging reactor. Based on the fluorescence images, the less lignified parenchyma cell walls were more susceptible to pretreatment than the lignin-rich vascular bundles. During enzymatic hydrolysis, the highly lignified protoxylem cell wall was the most resistant, remaining unhydrolyzed even after 48 h. Therefore, imaging thin whole biomass sections was useful to obtain tissue-specific changes during biomass deconstruction