Blake A. Simmons

CHAPTER 3:Ionic Liquid Pretreatment of Lignocellulosic Biomass for Biofuels and Chemicals

This chapter focuses on the recent development of biomass pre-treatment using ionic liquids (ILs). Lignocellulosic biomass, such as wood, grass, and agricultural residues, are a promising alternative source for the production of renewable fuels and chemicals. Because of the inherent recalcitrance, pre-treatment of lignocellulosic biomass is essential to improve the bioconversion process. Different physical, chemical and physicochemical pre-treatments are reported in literature and each process has its own advantages and disadvantages. Ionic liquids (ILs) are promising solvents for biomass pre-treatment as some ILs are capable of completely solubilizing lignocellulosic biomass and the biomass can then be regenerated upon addition of a suitable anti-solvent. Dissolution in IL drives structural changes in the regenerated biomass, making it easier to hydrolyse. The chapter starts with an overview of biomass and different emerging pre-treatment technologies, with an emphasis on IL-based pre-treatment technology. This section covers the history along with a concise literature review of different IL-based pre-treatment processes. The second section is devoted to IL pre-treatment chemistry and cellulose–IL interaction, with a short discussion of lignin and its prospects for the production of renewable platform chemicals. The last part of this chapter outlines the challenges and opportunities of the emerging IL pre-treatment technology in a biorefinery concept and provides concluding remarks on the outlook for the biorefinery.

Development of a High Throughput Platform for Screening Glycoside Hydrolases Based on Oxime-NIMS

Cost-effective hydrolysis of biomass into sugars for biofuel production requires high-performance low-cost glycoside hydrolase (GH) cocktails that are active under demanding process conditions. Improving the performance of GH cocktails depends on knowledge of many critical parameters, including individual enzyme stabilities, optimal reaction conditions, kinetics, and specificity of reaction. With this information, rate- and/or yield-limiting reactions can be potentially improved through substitution, synergistic complementation, or protein engineering. Given the wide range of substrates and methods used for GH characterization, it is difficult to compare results across a myriad of approaches to identify high performance and synergistic combinations of enzymes. Here, we describe a platform for systematic screening of GH activities using automatic biomass handling, bioconjugate chemistry, robotic liquid handling, and nanostructure-initiator mass spectrometry (NIMS). Twelve well-characterized substrates spanning the types of glycosidic linkages found in plant cell walls are included in the experimental workflow. To test the application of this platform and substrate panel, we studied the reactivity of three engineered cellulases and their synergy of combination across a range of reaction conditions and enzyme concentrations. We anticipate that large-scale screening using the standardized platform and substrates will generate critical datasets to enable direct comparison of enzyme activities for cocktail design.

Investigation of inter- and intraspecies variation through genome sequencing of Aspergillus section Nigri

Aspergillus section Nigri comprises filamentous fungi relevant to biomedicine, bioenergy, health, and biotechnology. To learn more about what genetically sets these species apart, as well as about potential applications in biotechnology and biomedicine, we sequenced 23 genomes de novo, forming a full genome compendium for the section (26 species), as well as 6 Aspergillus niger isolates. This allowed us to quantify both inter- and intraspecies genomic variation. We further predicted 17,903 carbohydrate-active enzymes and 2,717 secondary metabolite gene clusters, which we condensed into 455 distinct families corresponding to compound classes, 49% of which are only found in single species. We performed metabolomics and genetic engineering to correlate genotypes to phenotypes, as demonstrated for the metabolite aurasperone, and by heterologous transfer of citrate production to Aspergillus nidulans. Experimental and computational analyses showed that both secondary metabolism and regulation are key factors that are significant in the delineation of Aspergillus species.De novo assembly of 23 Aspergillus section Nigri and 6 Aspergillus niger genome sequences allows for inter- and intraspecies comparisons and prediction of secondary metabolite gene clusters.

Guiding optimal biofuels

In the current study, processes to produce either ethanol or a representative fatty acid ethyl ester (FAEE) via the fermentation of sugars liberated from lignocellulosic materials pretreated in acid or alkaline environments are analyzed in terms of economic and environmental metrics. Simplified process models are introduced and employed to estimate process performance, and Monte Carlo analyses were carried out to identify key sources of uncertainty and variability. We find that the near-term performance of processes to produce FAEE is significantly worse than that of ethanol production processes for all metrics considered, primarily due to poor fermentation yields and higher electricity demands for aerobic fermentation. In the longer term, the reduced cost and energy requirements of FAEE separation processes will be at least partially offset by inherent limitations in the relevant metabolic pathways that constrain the maximum yield potential of FAEE from biomass-derived sugars.

Trojan Horse Strategy for Deconstruction of Biomass for Biofuels Production

Production of renewable biofuels to displace fossil fuels currently consumed in the transportation sector is a pressing multi-agency national priority. Currently, nearly all fuel ethanol is produced from corn-derived starch. Dedicated energy crops and agricultural waste are preferred long-term solutions for renewable, cheap, and globally available biofuels as they avoid some of the market pressures and secondary greenhouse gas emission challenges currently facing corn ethanol. These sources of lignocellulosic biomass are converted to fermentable sugars using a variety of chemical and thermochemical pretreatments, which disrupt cellulose and lignin cross-links, allowing exogenously added recombinant microbial enzymes to more efficiently hydrolyze the cellulose for deconstruction into glucose. This process is plagued with inefficiencies, primarily due to the recalcitrance of cellulosic biomass, mass transfer issues during deconstruction, and low activity of recombinant deconstruction enzymes. Costs are also high due to the requirement for enzymes and reagents, and energy-intensive and cumbersome pretreatment steps. One potential solution to these problems is found in synthetic biology; they propose to engineer plants that self-produce a suite of cellulase enzymes targeted to the apoplast for cleaving the linkages between lignin and cellulosic fibers; the genes encoding the degradation enzymes, also known as cellulases, are obtained from extremophilic organismsmorexa0» that grow at high temperatures (60-100 C) and acidic pH levels (<5). These enzymes will remain inactive during the life cycle of the plant but become active during hydrothermal pretreatment i.e., elevated temperatures. Deconstruction can be integrated into a one-step process, thereby increasing efficiency (cellulose-cellulase mass-transfer rates) and reducing costs. The proposed disruptive technologies address biomass deconstruction processes by developing transgenic plants encoding a suite of enzymes used in cellulosic deconstruction. The unique aspects of this technology are the rationally engineered, highly productive extremophilic enzymes, targeted to specific cellular locations (apoplast) and their dormancy during normal plant proliferation, which become Trojan horses during pretreatment conditions. They have been leveraging established Sandias enzyme-engineering and imaging capabilities. Their technical approach not only targets the recalcitrance and mass-transfer problem during biomass degradation but also eliminates the costs associated with industrial-scale production of microbial enzymes added during processing.«xa0less

Understanding and engineering enzymes for enhanced biofuel production.

Today, carbon-rich fossil fuels, primarily oil, coal and natural gas, provide 85% of the energy consumed in the United States. The release of greenhouse gases from these fuels has spurred research into alternative, non-fossil energy sources. Lignocellulosic biomass is renewable resource that is carbon-neutral, and can provide a raw material for alternative transportation fuels. Plant-derived biomass contains cellulose, which is difficult to convert to monomeric sugars for production of fuels. The development of cost-effective and energy-efficient processes to transform the cellulosic content of biomass into fuels is hampered by significant roadblocks, including the lack of specifically developed energy crops, the difficulty in separating biomass components, the high costs of enzymatic deconstruction of biomass, and the inhibitory effect of fuels and processing byproducts on organisms responsible for producing fuels from biomass monomers. One of the main impediments to more widespread utilization of this important resource is the recalcitrance of cellulosic biomass and techniques that can be utilized to deconstruct cellulosic biomass.