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Switchgrass is a promising feedstock for cellulosic biorefineries, due to its ability to maintain comparatively high biomass yields across a wide range of soil and climatic conditions. However, there is an incomplete understanding of the economic and environmental tradeoffs associated with cultivating switchgrass on low-productivity land for conversion to biofuels. This study surveys prior literature and demonstrates a new integrated assessment framework, including agroecosystem, ecosystem services valuation, technoeconomic, and life-cycle assessment models, to quantify and contextualize the economic and environmental impacts of switchgrass cultivation on marginal land with downstream conversion to biofuels. Monetizing and incorporating the value of ecosystem services, such as improved water quality benefits from nitrate and sediment reductions, climate change mitigation benefits from CO2 emission reduction, and recreational and pollination benefits from increased biodiversity, the modeled multifunctional landscape reduces the ethanol production cost by 33.3–58.9 cents/L-gasoline-equivalent ($1.3–2.2/gge). Planting switchgrass in low productivity land improves soil health, resulting in the carbon footprint reduction credit of 12.8–20.2 gCO2e/MJ. For an improved switchgrass-to-ethanol conversion pathway with the maximum benefits from ecosystem services, the minimum ethanol selling price and carbon footprint of ethanol, respectively, could reach to 31 cents/L-gasoline-equivalent (47% reduction relative to average gasoline price) and 3 gCO2e/MJ (97% reduction relative to gasoline). This low carbon renewable ethanol leads to substantial State and/or Federal policy incentives (∼$1/L-gasoline-equivalent) providing a large benefit to biorefinery operators, farmers, and the public as a whole.

Production of 5-hydroxymethylfurfural (HMF) from biomass-derived glucose has great potential for synthesis of renewable fuels and chemicals. Selective glucose conversion to 5-hydroxymethylfurfural requires a balance between Lewis and Brønsted acids for the cascade of glucose isomerization followed by fructose dehydration. A dual Brønsted-Lewis acid, phosphotungstic acid encapsulated MIL-101(Al)–NH2 metal–organic frameworks (MOFs) was developed to catalyze the glucose dehydration reaction. The encapsulated catalysts had a high HMF selectivity of 58% at 44% glucose conversion at 120 °C in [C4C1im]Cl. Phosphotungstic acid was uniformly dispersed in the MOF pores, which provided both Brønsted and Lewis acid sites for this cascade reaction. The Brønsted acidic phosphotungstic acid-encapsulated MOF catalyst was stable and recyclable at least four times. These findings explain the effect of phosphotungstic acid location for maximizing the HMF selectivity and suggest a new approach for the design of bifunctional solid acid catalysts for selective HMF production from glucose. Moreover, the tunability of the acid properties of the encapsulated MOF catalysts provides opportunities for other biomass transformations.

Pretreatment of lignocellulosic biomass is essential for efficient conversion into biofuels and bioproducts. The present study develops a predictive toolset to computationally identify solvents that can efficiently dissolve lignin and therefore can be used to extract it from lignocellulose during pretreatment, a process known to reduce recalcitrance to enzymatic deconstruction and increase conversion efficiency. Two approaches were taken to examine the potential of eleven organic solvents to solubilize lignin, Hansen solubility parameters (HSP) and activity coefficients and excess enthalpies of solvent/lignin mixtures predicted by COSMO-RS (COnductor like Screening MOdel for Real Solvents). The screening revealed that diethylenetriamine was the most effective solvent, promoting the highest lignin removal (79.2%) and fermentable sugar yields (>72%). Therefore, a COSMO-RS-based predictive model for the lignin removal as a function of number and type of amines was developed. Among the fitted models, the non-linear regression model predicts the lignin solubility more accurately than the linear model. Experimental results demonstrated a >65% lignin removal and >70% of sugar yield from several amine-based solvents tested, which aligned very well with the model's prediction. Finally, to help understand the dissolution mechanism of lignin by these solvents, quantum theory of atoms in molecules (QTAIM) and quantum chemical calculations (interaction energies and natural bond orbital (NBO) analysis) was performed and suggest that amines exhibit strong electrostatic interactions and hydrogen bonding strengths with lignin leading to higher lignin removal. Together, these computational tools provide an effective approach for rapidly identifying solvents that are tailored for effective biomass pretreatment.

Recyclable biocatalysts and high-efficiency lignocellulose deconstruction are the crucial factors for cost-effective conversion of biomass into biofuels and bioproducts. Acetic acid-based catalytic hydrolysis of grassy lignocellulosic biomass presents a promising application because of its effectivity, recyclability, and other environmentally friendly features. However, this treatment is not as effective on woody biomass, such as poplar. One way to improve conversion performance of this process is to integrate it with other effective processes, such as pretreating biomass with protic ionic liquids (PILs) that have been shown to effectively solubilize lignin in reducing the recalcitrance of biomass to enzymatic deconstruction. In this work, an integrated acetic acid based one-pot ethanolamine acetate pretreatment (HAc-[EOA][OAc]) was developed for the efficient depolymerization of poplar polysaccharides. The configuration simultaneously removed ∼88% hemicellulose and selectively extracted up to ∼46% of the lignin from lignocellulosic biomass. HAc-[EOA][OAc] pretreated poplar yielded over 80% enzyme-hydrolyzed glucose that was attributed to an increase in the accessible surface area of cellulose to the hydrolytic enzymes. Analysis of the cellulose crystallinity and thermal decomposition profiles revealed that all pretreated samples have a higher cellulose crystallinity, indicating that amorphous cellulose had been removed during pretreatment. Conductor like screening model for real solvents (COSMO-RS) and Hansen solubility parameters (HSP) were used to provide insights into the mechanism of biomass pretreatment efficacy using both HAc and [EOA][OAc]. We found that a strong hydrogen-bonding and electrostatic misfit interaction between hemicellulose and HAc may explain the higher removal of hemicellulose during HAc pretreatment. Further, the close HSP values and COSMO-RS analysis indicate that [EOA][OAc] is a good lignin solvent, which leads to the higher delignification of biomass. This study demonstrates that the integration of IL with acid pretreatment is a promising strategy for conducting effective pretreatment on woody lignocellulose.

The molecular level details of dissolution of lignin in certain ionic liquids (ILs), such as cholinium-based ILs, are a relatively underexplored area and several key details to comprehend the dissolution mechanism are yet to be discovered. To understand, answer, and connect the missing links in the delignification mechanism during biomass pretreatment using cholinium-based ILs we employ COnductor like Screening MOdel for Real Solvents (COSMO-RS) and molecular dynamics (MD) simulations to evaluate the interactions between lignin-like model compounds and the anion and cation of several cholinium-based ILs. Initially, lignin dissolution was studied for cholinium-based ILs containing five different carboxylate anions ([For], [Ace], [But], [Hex], and [Oct]) and were compared with lysinate as the anion. The microscopic properties such as interaction energies, activity coefficient, radial and spatial distribution functions (RDF/SDF), and hydrogen bonds and their dynamics were assessed to characterize lignin dissolution in these ILs and were validated with experimental data. Among the anions studied, both octanoate and lysinate containing ILs demonstrated better lignin dissolution; lysinate being the best. The simulation data suggested that [Ch][Lys] has higher affinity for ether linkages of lignin (e.g., β-O-4) than for C-C linkages, which explains the higher delignification of hardwood and grassy biomasses (60-80% C-O-C linkages) in [Ch][Lys].

Pretreatment is an essential step to enable the efficient conversion of lignocellulosic biomass to biofuels and bioproducts. The most effective pretreatment methods currently in use are based on severe thermochemical approaches, which are costly and energy-intensive. Here we explored whether the common practice of ensiling grassy biomass, such as sorghum, could be used as a pre-processing step to increase the conversion efficiency under milder pretreatment conditions. We determined the impact of replacing dry sorghum biomass with ensiled sorghum biomass on the deconstruction efficiency, process economics, and carbon footprint of a lignocellulosic biorefinery that employs a separation-free ionic liquid pretreatment coupled to enzymatic saccharification and microbial conversion. Our results indicate that the use of ensiled biomass allowed for a 50% reduction in both the amount of ionic liquid (from 5 to 2.5% (w/w) as measured by initial pretreatment loading) and the time required for enzymatic saccharification (from 72 h to 24 h) without sacrificing efficiency. We show that the resulting hydrolysate can be used to cultivate an engineered strain ofRhodosporidium toruloidesto convert >90% of the monomeric sugars into bisabolene, a promising intermediate to biofuels and bioproducts. Overall, we estimate that the replacement of field-dried biomass sorghum with ensiled sorghum in combination with an ionic liquid-based deconstruction process could reduce the minimum selling price and carbon footprint of biofuel production in a biorefinery by at least 13% and 8.2%, respectively.

Over the last few decades, efforts to transition the global production of fuels and chemicals toward renewable carbon feedstocks have accelerated. A large portion of these efforts have focused on valorization of one of the most abundant renewable carbon sources, lignocellulose. Pretreatment of lignocellulose is the first critical step in this process. In this study, novel ionic liquid (IL) systems consisting of multiple ions known to be effective at biomass pretreatment were tested on woody and grassy biomass. Molecular simulations and experimental results established the synergistic advantages of combining specific individual components in these systems. For pine (woody) biomass, pretreatment with the combination of imidazolium, cholinium, acetate, and lysinate ions achieved 80% glucose and 70% xylose yields at high biomass loading. For sorghum biomass, an IL system comprising cholinium, lysinate, and palmitate ions not only enabled a 98% glucose yield but was also found to be biocompatible in a one-pot configuration, producing the biofuel precursor bisabolene using an engineered strain of the yeast Rhodosporidium toruloides.

BackgroundIn an effort to ensure future energy security, reduce greenhouse gas emissions and create domestic jobs, the US has invested in technologies to develop sustainable biofuels and bioproducts from renewable carbon sources such as lignocellulosic biomass. Bio-derived jet fuel is of particular interest as aviation is less amenable to electrification compared to other modes of transportation and synthetic biology provides the ability to tailor fuel properties to enhance performance. Specific energy and energy density are important properties in determining the attractiveness of potential bio-derived jet fuels. For example, increased energy content can give the industry options such as longer range, higher load or reduced takeoff weight. Energy-dense sesquiterpenes have been identified as potential next-generation jet fuels that can be renewably produced from lignocellulosic biomass.ResultsWe developed a biomass deconstruction and conversion process that enabled the production of two tricyclic sesquiterpenes, epi-isozizaene and prespatane, from the woody biomass poplar using the versatile basidiomycete Rhodosporidium toruloides. We demonstrated terpene production at both bench and bioreactor scales, with prespatane titers reaching 1173.6 mg/L when grown in poplar hydrolysate in a 2 L bioreactor. Additionally, we examined the theoretical fuel properties of prespatane and epi-isozizaene in their hydrogenated states as blending options for jet fuel, and compared them to aviation fuel, Jet A.ConclusionOur findings indicate that prespatane and epi-isozizaene in their hydrogenated states would be attractive blending options in Jet A or other lower density renewable jet fuels as they would improve viscosity and increase their energy density. Saturated epi-isozizaene and saturated prespatane have energy densities that are 16.6 and 18.8% higher than Jet A, respectively. These results highlight the potential of R. toruloides as a production host for the sustainable and scalable production of bio-derived jet fuel blends, and this is the first report of prespatane as an alternative jet fuel.

BackgroundDue to their high energy density and compatible physical properties, several monoterpenes have been investigated as potential renewable transportation fuels, either as blendstocks with petroleum or as drop-in replacements for use in vehicles (both heavy and light-weight) or in aviation. Sustainable microbial production of these biofuels requires the ability to utilize cheap and readily available feedstocks such as lignocellulosic biomass, which can be depolymerized into fermentable carbon sources such as glucose and xylose. However, common microbial production platforms such as the yeast Saccharomyces cerevisiae are not naturally capable of utilizing xylose, hence requiring extensive strain engineering and optimization to efficiently utilize lignocellulosic feedstocks. In contrast, the oleaginous red yeast Rhodosporidium toruloides is capable of efficiently metabolizing both xylose and glucose, suggesting that it may be a suitable host for the production of lignocellulosic bioproducts. In addition, R. toruloides naturally produces several carotenoids (C40 terpenoids), indicating that it may have a naturally high carbon flux through its mevalonate (MVA) pathway, providing pools of intermediates for the production of a wide range of heterologous terpene-based biofuels and bioproducts from lignocellulose.ResultsSixteen terpene synthases (TS) originating from plants, bacteria and fungi were evaluated for their ability to produce a total of nine different monoterpenes in R. toruloides. Eight of these TS were functional and produced several different monoterpenes, either as individual compounds or as mixtures, with 1,8-cineole, sabinene, ocimene, pinene, limonene, and carene being produced at the highest levels. The 1,8-cineole synthase HYP3 from Hypoxylon sp. E74060B produced the highest titer of 14.94 ± 1.84 mg/L 1,8-cineole in YPD medium and was selected for further optimization and fuel properties study. Production of 1,8-cineole from lignocellulose was also demonstrated in a 2L batch fermentation, and cineole production titers reached 34.6 mg/L in DMR-EH (Deacetylated, Mechanically Refined, Enzymatically Hydorlized) hydrolysate. Finally, the fuel properties of 1,8-cineole were examined, and indicate that it may be a suitable petroleum blend stock or drop-in replacement fuel for spark ignition engines.ConclusionOur results demonstrate that Rhodosporidium toruloides is a suitable microbial platform for the production of non-native monoterpenes with biofuel applications from lignocellulosic biomass.

Microbial production of fuels and commodity chemicals has been performed primarily using natural or slightly modified enzymes, which inherently limits the types of molecules that can be produced. Type I modular polyketide synthases (PKSs) are multi-domain enzymes that can produce unique and diverse molecular structures by combining particular types of catalytic domains in a specific order. This catalytic mechanism offers a wealth of engineering opportunities. Here we report engineered microbes that produce various short-chain (C5-C7) ketones using hybrid PKSs. Introduction of the genes into the chromosome of Streptomyces albus enables it to produce >1 g · l-1 of C6 and C7 ethyl ketones and several hundred mg · l-1 of C5 and C6 methyl ketones from plant biomass hydrolysates. Engine tests indicate these short-chain ketones can be added to gasoline as oxygenates to increase the octane of gasoline. Together, it demonstrates the efficient and renewable microbial production of biogasolines by hybrid enzymes.

Lignin undergoes catalytic depolymerization in the presence of a variety of transfer hydrogenation agents, however the mechanisms for non-catalytic depolymerization of lignin via transfer hydrogenation are not well understood; this makes process optimization difficult. Herein, for the first time a mechanism for this process is proposed. For the purposes of understanding the mechanisms involved in these non-catalytic lignin depolymerization processes, this study investigates the equilibrium system of formic acid, methyl formate and carbon monoxide, as agents for the depolymerization of lignin, in the presence of either water or methanol as solvents. In the methyl formate/water (at 300 °C) system, 73 wt% oil was produced which contained a significant amount of low molecular weight alkylphenols, with less than 1 wt% char produced. In aqueous media, the results showed that methyl formate maintains an equilibrium with formic acid which is itself in equilibrium with carbon monoxide. It was found that using either formic acid or methyl formate for non-catalytic transfer hydrogenation of lignin can produce high amounts of oil, and can be described as a two-stage mechanism. After 10 min of reaction at 300 °C, around a quarter of the formic acid is consumed via hydride transfer of the formate proton, preventing the condensation of lignin fragments. At the same time, approximately three quarters of the formic acid decomposes to carbon dioxide and carbon monoxide. Once the formic acid is consumed, the carbon monoxide was identified as the precursor to a reactive reductive reagent and was able to activate the proton of the water molecule preventing further condensation of the lignin fragments. It has been previously thought that transfer hydrogenation in lignin using formic acid occurs via the production of molecular hydrogen. Here it is demonstrated that formic acid reacts directly with the lignin, without this hydrogen formation. Therefore the key parameters for efficient transfer hydrogenation of the lignin to maximize bio-oil yield appear to involve controlling the reactions between lignin and formic acid, methyl formate or carbon monoxide under aqueous conditions, thereby reducing the reagent cost and loading while maintaining efficient lignin conversion.

Stilbenes are diphenyl ethene compounds produced naturally in a wide variety of plant species and some bacteria. Stilbenes are also derived from lignin during kraft pulping. Stilbene cleavage oxygenases (SCOs) cleave the central double bond of stilbenes, forming two phenolic aldehydes. Here, we report the structure of an SCO. The X-ray structure of NOV1 from Novosphingobium aromaticivorans was determined in complex with its substrate resveratrol (1.89 Å), its product vanillin (1.75 Å), and without any bound ligand (1.61 Å). The enzyme is a seven-bladed β-propeller with an iron cofactor coordinated by four histidines. In all three structures, dioxygen is observed bound to the iron in a side-on fashion. These structures, along with EPR analysis, allow us to propose a mechanism in which a ferric-superoxide reacts with substrate activated by deprotonation of a phenol group at position 4 of the substrate, which allows movement of electron density toward the central double bond and thus facilitates reaction with the ferric superoxide electrophile. Correspondingly, NOV1 cleaves a wide range of other stilbene-like compounds with a 4'-OH group, offering potential in processing some solubilized fragments of lignin into monomer aromatic compounds.

Screening a large number of biologically derived molecules for potential fuel compounds without recourse to experimental testing is important in identifying understudied yet valuable molecules. Experimental testing, although a valuable standard for measuring fuel properties, has several major limitations, including the requirement of testably high quantities, considerable expense, and a large amount of time. This paper discusses the development of a general-purpose fuel property tool, using machine learning, whose outcome is to screen molecules for desirable fuel properties. BioCompoundML adopts a general methodology, requiring as input only a list of training compounds (with identifiers and measured values) and a list of testing compounds (with identifiers). For the training data, BioCompoundML collects open data from the National Center for Biotechnology Information, incorporates user-provided features, imputes missing values, performs feature reduction, builds a classifier, and clusters compounds. BioCompoundML then collects data for the testing compounds, predicts class membership, and determines whether compounds are found in the range of variability of the training data set. This tool is demonstrated using three different fuel properties: research octane number (RON), threshold soot index (TSI), and melting point (MP). We provide measures of its success with these properties using randomized train/test measurements: average accuracy is 88% in RON, 85% in TSI, and 94% in MP; average precision is 88% in RON, 88% in TSI, and 95% in MP; and average recall is 88% in RON, 82% in TSI, and 97% in MP. The receiver operator characteristics (area under the curve) were estimated at 0.88 in RON, 0.86 in TSI, and 0.87 in MP. We also measured the success of BioCompoundML by sending 16 compounds for direct RON determination. Finally, we provide a screen of 1977 hydrocarbons/oxygenates within the 8696 compounds in MetaCyc, identifying compounds with high predictive strength for high or low RON.

The fast pyrolysis behaviour of pretreated banagrass was examined at four temperatures (between 400 and 600 C) and four residence times (between ~1.2 and 12 s). The pretreatment used water washing/leaching to reduce the inorganic content of the banagrass. Yields of bio-oil, permanent gases and char were determined at each reaction condition and compared to previously published results from untreated banagrass. Comparing the bio-oil yields from the untreated and pretreated banagrass shows that the yields were greater from the pretreated banagrass by 4 to 11 wt% (absolute) at all reaction conditions. The effect of pretreatment (i.e. reducing the amount of ash, and alkali and alkali earth metals) on pyrolysis products is: 1) to increase the dry bio-oil yield, 2) to decrease the amount of undetected material, 3) to produce a slight increase in CO yield or no change, 4) to slightly decrease CO2 yield or no change, and 5) to produce a more stable bio-oil (less aging). Char yield and total gas yield were unaffected by feedstock pretreatment. Four other tropical biomass species were also pyrolyzed under one condition (450°C and 1.4 s residence time) for comparison to the banagrass results. The samples include two hardwoods: leucaena and eucalyptus, and two grasses: sugarcane bagasse and energy-cane. A sample of pretreated energy-cane was also pyrolyzed. Of the materials tested, the best feedstocks for fast pyrolysis were sugarcane bagasse, pretreated energy cane and eucalyptus based on the yields of 'dry bio-oil', CO and CO2. On the same basis, the least productive feedstocks are untreated banagrass followed by pretreated banagrass and leucaena.

Lignin confers recalcitrance to plant biomass used as feedstocks in agro-processing industries or as source of renewable sugars for the production of bioproducts. The metabolic steps for the synthesis of lignin building blocks belong to the shikimate and phenylpropanoid pathways. Genetic engineering efforts to reduce lignin content typically employ gene knockout or gene silencing techniques to constitutively repress one of these metabolic pathways. Recently, new strategies have emerged offering better spatiotemporal control of lignin deposition, including the expression of enzymes that interfere with the normal process for cell wall lignification. In this study, we report that expression of a 3-dehydroshikimate dehydratase (QsuB from Corynebacterium glutamicum) reduces lignin deposition in Arabidopsis cell walls. QsuB was targeted to the plastids to convert 3-dehydroshikimate - an intermediate of the shikimate pathway - into protocatechuate. Compared to wild-type plants, lines expressing QsuB contain higher amounts of protocatechuate, p-coumarate, p-coumaraldehyde and p-coumaryl alcohol, and lower amounts of coniferaldehyde, coniferyl alcohol, sinapaldehyde and sinapyl alcohol. 2D-NMR spectroscopy and pyrolysis-gas chromatography/mass spectrometry (pyro-GC/MS) reveal an increase of p-hydroxyphenyl units and a reduction of guaiacyl units in the lignin of QsuB lines. Size-exclusion chromatography indicates a lower degree of lignin polymerization in the transgenic lines. Therefore, our data show that the expression of QsuB primarily affects the lignin biosynthetic pathway. Finally, biomass from these lines exhibits more than a twofold improvement in saccharification efficiency. We conclude that the expression of QsuB in plants, in combination with specific promoters, is a promising gain-of-function strategy for spatiotemporal reduction of lignin in plant biomass.

Eucalypt species are a group of flowering trees widely used in pulp production for paper manufacture. For several decades, the wood pulp industry has focused research and development efforts on improving yields, growth rates and pulp quality through breeding and the genetic improvement of key tree species. Recently, this focus has shifted from the production of high quality pulps to the investigation of the use of eucalypts as feedstocks for biofuel production. Here the structure and chemical composition of the heartwood and sapwood of Eucalyptus dunnii, E. globulus, E. pillularis, E. urophylla, an E. urophylla-E. grandis cross, Corymbia citriodora ssp. variegata, and Acacia mangium were compared using nuclear magnetic resonance spectroscopy (NMR), X-ray diffraction (XRD) and biochemical composition analysis. Some trends relating to these compositions were also identified by Fourier transform near infrared (FT-NIR) spectroscopy. These results will serve as a foundation for a more comprehensive database of wood properties that will help develop criteria for the selection of tree species for use as biorefinery feedstocks.