Search

Modern biotechnology has the potential to substantially advance the feasibility, structure, and efficiency of future biofuel supply chains. Advances might be direct or indirect. A direct advance would be improving the efficiency of biochemical conversion processes and feedstock production. Direct advances in processing may involve developing improved enzymes and bacteria to convert lignocellulosic feedstocks to ethanol. Progress in feedstock production could include enhancing crop yields via genetic modification or the selection of specific natural variants and breeds. Other direct results of biotechnology might increase the production of fungible biofuels and bioproducts, which would impact the supply chain. Indirect advances might include modifications to dedicated bioenergy crops that enable them to grow on marginal lands rather than land needed for food production. This study assesses the feasibility and advantages of near-future (10-year) biotechnological developments for a US biomass-based supply chain for bioenergy production. We assume a simplified supply chain of feedstock, logistics and land use, conversion, and products and utilization. The primary focus is how likely developments in feedstock production and conversion technologies will impact bioenergy and biofuels in the USA; a secondary focus is other innovative uses of biotechnologies in the energy arenas. The assessment addresses near-term biofuels based on starch, sugar, and cellulosic feedstocks and considers some longer-term options, such as oil-crop and algal technologies. Published 2015. This article is a U.S. Government work and is in the public domain in the USA. Biofuels, Bioproducts and Biorefining published by Society of Industrial Chemistry and John Wiley & Sons Ltd [ABSTRACT FROM AUTHOR]

Potential economic and environmental benefits of increasing nitrogen-use efficiency (NUE) are widely recognized but scarcely quantified. This study quantifies the effects of increased NUE on 1) the national agricultural economy using a simulation model of US agriculture and 2) regional water quality effects using a biogeochemical model for the Arkansas-White-Red river basin. National economic effects are reported for NUE improvement scenarios of 10%, 20%, 50%, and 100%, whereas regional water quality effects are estimated for a 20% NUE improvement scenario in the Arkansas-White-Red river basin. Simulating a 20% increase in NUE in row crops is shown to reduce N requirements by 1.4 million tonnes y−1 and increase farmer net profits by 1.6% ($743 million) per year by 2026 over the baseline simulation for the same period. For each 10% increase in NUE, annual farm revenues for commodity crops increased over the baseline by approximately $350 million per year by 2026. Changes in crop prices and land-use relative to the baseline were less than 2%. This suggests a net benefit even though fertilizer cost savings can result in increased cultivation of land, i.e., 'Jevon's paradox'. Results from the biogeochemical model of the Arkansas-White-Red river basin suggest that a 20% increase in NUE corresponds to a 5.72% reduction in nitrate loadings to freshwaters, with higher reductions in agricultural watersheds. The value of these reductions was estimated as $43 ha−1, for a total of $15.3 to 136.7 million yr−1 in avoided water treatment costs. After estimating the social value of increased NUE, we conclude with a discussion of potential strategies to increase efficiency and the research needed to achieve this goal. These include perennialization of the agricultural landscape, genetic crop improvement, targeted fertilizer application, and manipulation of the plant-root microbiome. Unlabelled Image • Increasing nitrogen use efficiency (NUE) by 20% can save $743 m yr−1 in the US. • Increasing (NUE) by 20% can reduce N loadings in freshwaters by 5.7% in a case study. • The N reductions in the case study are estimated to be worth $15.3 to 136.7 million yr−1. [ABSTRACT FROM AUTHOR]

Ethanol to middle distillates (ETMD) is a promising pathway to produce sustainable liquid fuels to decarbonize the hard-to-electrify transportation sectors due to (1) the abundant sugar/starch and lignocellulosic biomass, (2) the existing deployment scale of fuel ethanol production (∼29 billion gallons per year globally), and (3) emerging opportunities in C2+ alcohol synthesis from CO2. Here we report a conceptual market-responsive biorefinery centered around a new ETMD pathway based on one-step ethanol to butene-rich olefins (ETO) over a Cu–Zn–Y/Beta catalyst. Specifically, this ethanol conversion pathway comprises one-step ETO, oligomerization, and hydrotreating. This ETO is distinct from that in the conventional ethanol-to-jet process which is based on two-step ethanol to ethylene and ethylene oligomerization to butenes. Butene-rich olefins can be shifted to butadiene-rich products by simply changing the reaction atmosphere from hydrogen to inert gas over the same ETO catalyst. Leveraging the experimental results, baseline techno-economic analysis (TEA) and sensitivity analysis indicate that the ethanol conversion cost is $0.60 per gallon gasoline equivalent (GGE), with opportunities for further cost reduction via improving the liquid hydrocarbon yield and space velocities, and process optimization on balancing dewatering of ethanol feed prior to the ETO step. The minimum fuel selling price (MFSP) of liquid hydrocarbons derived from corn starch ethanol with butadiene as coproduct is $1.64 per GGE, in the range that is cost competitive with petroleum kerosene-type jet fuel. Projected MFSP for cellulosic ethanol (corn stover) derived hydrocarbons is below $3.00 per GGE and co-production of butadiene further reduces the MFSP to $1.70 per GGE. The Well-to-Wake life-cycle analysis indicates that 85% greenhouse gas emission reduction can be achieved when using corn stover compared to petroleum reference and the associated carbon credits will provide significant economic incentives to favor the cellulosic ethanol-derived hydrocarbon fuels. This study demonstrates a low-cost pathway to middle distillate fuels leveraging existing ethanol infrastructure, where catalysis innovation drives the reduction of process complexity and flexible coproduction of a value-added chemical product. [ABSTRACT FROM AUTHOR]

Biomass-derived solvents have been proposed as a novel pathway in biorefining for the realization of biofuels and bioproducts derived from lignocellulosic biomass. Cyrene derived from cellulose has recently been shown to have a high potential as a green organic solvent for pretreating poplar biomass. However, due to its high dynamic viscosity nature, high Cyrene concentration could cause negative effects on the sugar release of the pretreated biomass as well as driving up the operational cost of the lignin recovery. In this study, we combine experimental and computational approaches to examine the impact of Cyrene pretreatment with reduced Cyrene concentration under mild conditions on switchgrass lignin. Our experimental studies indicated correlation between pretreatment condition and recovery and structure modification of lignin. Switchgrass lignin extracted by Cyrene pretreatment possessed high preservation of β-O-4 ether inter-unit linkage, which could provide versatility in the integration of downstream lignin valorization into the modern biorefinery industries. Molecular modeling examining the solvation of switchgrass lignin polymer and the disaggregation of low-molecular weight lignin aggregates under pretreatment conditions indicated that a preferential interaction exists between Cyrene and lignin, which likely drives lignin release, and that the disruption of inter-lignin contacts can be modulated as a non-monotonic function of Cyrene : water ratio. Further, while Cyrene–lignin interactions permit the solubilization of lignin, simulations with proxy reactive-species reveal that changes to the diffusion of these reactive proxies and their localization near linkage sites under Cyrene conditions may inhibit chemical processes. The results indicated that loss of pretreatment efficacy caused by low Cyrene concentration could be compensated by prolonged pretreatment time and high catalyst dosage. [ABSTRACT FROM AUTHOR]

Slab models are simple and useful structural descriptions which have long been used to describe lyotropic lamellar phases, such as lipid bilayers. Typically, slab models assume a midline symmetry and break a bilayer structure into three pieces, a central solvent‐free core and two symmetric outer layers composed of the soluble portion of the amphiphile and associated solvent. This breakdown matches reasonably well to the distribution of neutron scattering length density and therefore is a convenient and common approach for the treatment of small‐angle scattering data. Here, an implementation of this model within the SasView software suite is reported. The implementation is intended to provide physical consistency through the area per amphiphile molecule and number of solvent molecules included within the solvent‐exposed outer layer. The proper use of this model requires knowledge of (or good estimates for) the amphiphile and solvent molecule volume and atomic composition, ultimately providing a self‐consistent data treatment with only two free parameters: the lateral area per amphiphile molecule and the number of solvent molecules included in the outer region per amphiphile molecule. The use of this code is demonstrated in the fitting of standard lipid bilayer data sets, obtaining structural parameters consistent with prior literature and illustrating the typical and ideal cases of fitting for neutron scattering data obtained using single or multiple contrast conditions. While demonstrated here for lipid bilayers, this model is intended for general application to block copolymers, surfactants, and other lyotropic lamellar phase structures for which a slab model is able to reasonably estimate the neutron scattering length density/electron‐density profile of inner and outer layers of the lamellae. [ABSTRACT FROM AUTHOR]

Background: Lignin dehydrogenation polymers (DHPs) are polymers generated from phenolic precursors for the purpose of studying lignin structure and polymerization processes Methods: Here, DHPs were synthesized using a Zutropfverfahren method with horseradish peroxidase and three lignin monomers, sinapyl (S), coumaryl (H), and coniferyl (G) alcohols, in the presence of hydrogen peroxide. The H monomer was reacted with G and a 1:1 molar mixture of S:G monomers at H molar compositions of 0, 5, 10, and 20 mol% to study how the presence of the H monomer affected the structure and composition of the recovered polymers. Results: At low H concentrations, solid-state NMR spectra suggest that the H and G monomers interact to form G:H polymers that have a lower average molecular weight than the solely G-based polymer or the G:H polymer produced at higher H concentrations. Solid-state NMR and pyrolysis-MBMS analyses suggest that at higher H concentrations, the H monomer primarily self-polymerizes to produce clusters of H-based polymer that are segregated from clusters of G- or S:G-based polymers. Thioacidolysis generally showed higher recoveries of thioethylated products from S:G or S:G:H polymers made with higher H content, indicating an increase in the linear ether linkages. Conclusions: Overall, the experimental results support theoretical predictions for the reactivity and structural influences of the H monomer on the formation of lignin-like polymers. [ABSTRACT FROM AUTHOR]

Amphiphilic co‐solvents can have a significant impact on the structure, organization and physical properties of lipid bilayers. Describing the mutual impact of partitioning and induced structure changes is therefore a crucial consideration for a range of topics such as anesthesia and other pharmacokinetic effects, as well as microbial solvent tolerance in the production of biofuels and other fermentation products, where molecules such as ethanol, butanol or acetic acid might be generated. Small‐angle neutron scattering (SANS) is a key method for studying lipid and polymer bilayer structures, with many models for extracting bilayer structure (thickness, area per lipid etc.) from scattering data in use today. However, the molecular details of co‐solvent partitioning are conflated with induced changes to bilayer structure, making interpretation and modeling of the scattering curves a challenge with the existing set of models. To address this, a model of a bilayer structure is presented which invokes a two‐term partition constant accounting for the localization of the co‐solvent within the bilayer. This model was validated using a series of SANS measurements of lipid vesicles in the presence of the co‐solvent tetrahydrofuran (THF), showing several strategies of how to deploy the two‐parameter partition constant model to describe scattering data and extract both structure and partitioning information from the data. Molecular dynamics simulations are then used to evaluate assumptions of the model, provide additional molecular scale details and illustrate its complementary nature to the data fitting procedure. This approach results in estimates of the partition coefficient for THF in 1,2‐dimyristoyl‐sn‐glycero‐3‐phosphocholine at 35°C, along with an estimate of the fraction of THF residing in the hydrophobic core of the membrane. The authors envision that this model will be applicable to a wide range of other bilayer/amphiphile interactions and provide the associated code needed to implement this model as a fitting algorithm for scattering data in the SasView suite. [ABSTRACT FROM AUTHOR]

A response by Brian H. Davison, Arthur J. Ragauskas, Richard Templer, Timothy J. Tschaplinski, and Jonathan R. Mielenz to a letter to the editor about their article "The Path Forward for Biofuels and Biomaterials," in the January 27, 2006 issue is presented.

Biomass represents an abundant carbon-neutral renewable resource for the production of bioenergy and biomaterials, and its enhanced use would address several societal needs. Advances in genetics, biotechnology, process chemistry, and engineering are leading to a new manufacturing concept for converting renewable biomass to valuable fuels and products, generally referred to as the biorefinery. The integration of agroenergy crops and biorefinery manufacturing technologies offers the potential for the development of sustainable biopower and biomaterials that will lead to a new manufacturing paradigm. [ABSTRACT FROM AUTHOR]

To achieve a bio‐based economy, it is necessary to consider variability within a feedstock population. We must understand the range of key phenotypic characteristics when selecting economically advantageous genotypes for domestication in an optimized supply chain. In this analysis we measured cell‐wall composition traits in a large natural variant population of Populus trichocarpa. The results were combined with agronomic growth data from the matching genotype to conduct various techno‐economic analyses, evaluating the impacts of physical and compositional variability and determining the ultimate phenotypic drivers for yield and economic metrics. Here we show that, although ethanol yield per land area per year and minimum fuel selling price were most strongly impacted by tree size, when considering the largest 25% of trees, size and carbohydrate content were nearly identical influences on minimal fuel selling price, highlighting the need to focus on both size and carbohydrate content in selecting economically optimal feedstocks. © 2020 The Authors. Biofuels, Bioproducts, and Biorefining published by Society of Chemical Industry and John Wiley & Sons, Ltd [ABSTRACT FROM AUTHOR]

The disruptive effect of organic solvents on microbial membranes represents a significant challenge to the economical production of green fuels and value-added chemicals from lignocellulosic feedstocks. One route to overcoming this challenge is to engineer microbes with membranes capable of resisting organic solvent stresses. In this regard, it is useful to understand the mechanisms by which organic solvents disrupt typical biomembranes. Here, molecular dynamics (MD) simulation, complemented by small-angle X-ray and neutron scattering (SANS/SAXS), provide a molecular-scale view of the disruption of a microbial model membrane by 1-butanol and tetrahydrofuran (THF), two common water–organic cosolvent mixtures of importance in biofuel production. Solvent interactions at the interface between the head-group and fatty acid tail regions lead to more dramatic membrane changes than interactions solely at the head-groups or tails. Although both organic solvents are found to partition into the membrane, the depth of solvent penetration into the membrane is quite different. Specifically, 1-butanol localizes near the interface between the lipid heads and tails at low concentrations, but partitions into both the head and tail regions at high concentrations. In contrast, THF, overall, partitions less than 1-butanol and prefers the lipid tail regions. Importantly, the presence of 1-butanol near the head/tail interface introduces drastic membrane changes not seen with THF. The organic solvent interactions with the lipids lead to membrane thinning and fluidization, but more so for 1-butanol than for THF. These results suggest that an aim for the future engineering of robust membranes could be to design lipid head groups that reduce the accumulation of organic solvents at the head–tail interface and that rational designs need also be cognizant of the different solvent-specific mechanisms responsible for membrane disruption. [ABSTRACT FROM AUTHOR]

A particularly promising approach to deconstructing and fractionating lignocellulosic biomass to produce green renewable fuels and high-value chemicals pretreats the biomass with organic solvents in aqueous solution. Here, neutron scattering and moleculardynamics simulations reveal the temperature-dependent morphological changes in poplar wood biomass during tetrahydrofuran (THF):water pretreatment and provide a mechanism by which the solvent components drive efficient biomass breakdown. Whereas lignin dissociates over a wide temperature range (>25 °C) cellulose disruption occurs only above 150 °C. Neutron scattering with contrast variation provides direct evidence for the formation of THF-rich nanoclusters (Rg ~ 0.5 nm) on the nonpolar cellulose surfaces and on hydrophobic lignin, and equivalent waterrich nanoclusters on polar cellulose surfaces. The disassembly of the amphiphilic biomass is thus enabled through the local demixing of highly functional cosolvents, THF and water, which preferentially solvate specific biomass surfaces so as to match the local solute polarity. A multiscale description of the efficiency of THF:water pretreatment is provided: matching polarity at the atomic scale prevents lignin aggregation and disrupts cellulose, leading to improvements in deconstruction at the macroscopic scale. [ABSTRACT FROM AUTHOR]

Technoeconomic and life-cycle analyses are presented for catalytic conversion of ethanol to fungible hydrocarbon fuel blendstocks, informed by advances in catalyst and process development. Whereas prior work toward this end focused on 3-step processes featuring dehydration, oligomerization, and hydrogenation, the consolidated alcohol dehydration and oligomerization (CADO) approach described here results in 1-step conversion of wet ethanol vapor (40 wt% in water) to hydrocarbons and water over a metal-modified zeolite catalyst. A development project increased liquid hydrocarbon yields from 36% of theoretical to >80%, reduced catalyst cost by an order of magnitude, scaled up the process by 300-fold, and reduced projected costs of ethanol conversion 12-fold. Current CADO products conform most closely to gasoline blendstocks, but can be blended with jet fuel at low levels today, and could potentially be blended at higher levels in the future. Operating plus annualized capital costs for conversion of wet ethanol to fungible blendstocks are estimated at $2.00/GJ for CADO today and $1.44/GJ in the future, similar to the unit energy cost of producing anhydrous ethanol from wet ethanol ($1.46/GJ). Including the cost of ethanol from either corn or future cellulosic biomass but not production incentives, projected minimum selling prices for fungible blendstocks produced via CADO are competitive with conventional jet fuel when oil is $100 per barrel but not at $60 per barrel. However, with existing production incentives, the projected minimum blendstock selling price is competitive with oil at $60 per barrel. Life-cycle greenhouse gas emission reductions for CADO-derived hydrocarbon blendstocks closely follow those for the ethanol feedstock. [ABSTRACT FROM AUTHOR]

Background: Caldicellulosiruptor bescii, a promising biocatalyst being developed for use in consolidated bioprocessing of lignocellulosic materials to ethanol, grows poorly and has reduced conversion at elevated medium osmolarities. Increasing tolerance to elevated fermentation osmolarities is desired to enable performance necessary of a consolidated bioprocessing (CBP) biocatalyst. Results: Two strains of C. bescii showing growth phenotypes in elevated osmolarity conditions were identified. The first strain, ORCB001, carried a deletion of the FapR fatty acid biosynthesis and malonyl-CoA metabolism repressor and had a severe growth defect when grown in high-osmolarity conditions—introduced as the addition of either ethanol, NaCl, glycerol, or glucose to growth media. The second strain, ORCB002, displayed a growth rate over three times higher than its genetic parent when grown in high-osmolarity medium. Unexpectedly, a genetic complement ORCB002 exhibited improved growth, failing to revert the observed phenotype, and suggesting that mutations other than the deleted transcription factor (the fruR/cra gene) are responsible for the growth phenotype observed in ORCB002. Genome resequencing identified several other genomic alterations (three deleted regions, three substitution mutations, one silent mutation, and one frameshift mutation), which may be responsible for the observed increase in osmolarity tolerance in the fruR/cra-deficient strain, including a substitution mutation in dnaK, a gene previously implicated in osmoresistance in bacteria. Differential expression analysis and transcription factor binding site inference indicates that FapR negatively regulates malonyl-CoA and fatty acid biosynthesis, as it does in many other bacteria. FruR/Cra regulates neighboring fructose metabolism genes, as well as other genes in global manner. Conclusions: Two systems able to effect tolerance to elevated osmolarities in C. bescii are identified. The first is fatty acid biosynthesis. The other is likely the result of one or more unintended, secondary mutations present in another transcription factor deletion strain. Though the locus/loci and mechanism(s) responsible remain unknown, candidate mutations are identified, including a mutation in the dnaK chaperone coding sequence. These results illustrate both the promise of targeted regulatory manipulation for osmotolerance (in the case of fapR) and the challenges (in the case of fruR/cra). [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Deuterated and protiated switchgrass hemicelluloses had a similar structure. • Hemicelluloses had similar arabinose to xylose ratio. • Deuterated switchgrass hemicellulose can be used in SANS or pharmaceutical studies. Abstract This work describes the structural characterization of hemicellulose isolated from hydroponically grown switchgrass in H 2 O medium (protiated) or 50% D 2 O medium (deuterated) through compositional analysis, GPC, FTIR, 13C and 1H/13C HSQC NMR. 4-O-methyl glucuronoarabinoxylan (GAX), the major hemicellulose in switchgrass isolated from deuterated switchgrass, had structural properties similar to hemicellulose isolated from protiated switchgrass. Both had comparable arabinose to xylose ratio (0.25) and molecular weight (47–50 kDa). Structural similarities show that deuterated switchgrass hemicellulose can be used as a model carbohydrate polymer in neutron scattering, or pharmaceutical studies due to their immunomodulatory activity and gastroprotective effects. [ABSTRACT FROM AUTHOR]

Neutron scattering of deuterated plants can provide fundamental insight into the structure of lignocellulosics in plant cell walls and its deconstruction by pretreatment and enzymes. Such plants need to be characterized for any alterations to lignocellulosic structure caused by growth in deuterated media. Here we show that glucose yields from enzymatic hydrolysis at lower enzyme loading were 35% and 30% for untreated deuterated and protiated switchgrass, respectively. Lignin content was 4% higher in deuterated switchgrass but there were no significant lignin structural differences. Transmission electron microscopy showed differences in lignin distribution and packing of fibers in the cell walls that apparently increased surface area of cellulose in deuterated switchgrass, increasing cellulose accessibility and lowering its recalcitrance. These differences in lignification were likely caused by abiotic stress due to growth in deuterated media. [ABSTRACT FROM AUTHOR]

The full utilization of plant biomass for the production of energy and novel materials often involves high temperature treatment. Examples include melt spinning of lignin for manufacturing low-cost carbon fiber and the relocalization of lignin to increase the accessibility of cellulose for production of biofuels. These temperature-induced effects arise from poorly understood changes in lignin flexibility. Here, we combine molecular dynamics simulations with neutron scattering and dielectric spectroscopy experiments to probe the dependence of lignin dynamics on hydration and thermal history. We find a dynamical and structural hysteresis: at a given temperature, the lignin molecules are more expanded and their dynamics faster when the lignin is cooled than when heated. The structural hysteresis is more pronounced for dry lignin. The difference in dynamics, however, follows a different trend, it is found to be more significant at high temperatures and high hydration levels. The simulations also reveal syringyl units to be more dynamic than guiacyl. The results provide an atomic-detailed description of lignin dynamics, important for understanding lignin role in plant cell wall mechanics and for rationally improving lignin processing. The lignin glass transition, at which the polymer softens, is lower when lignin is cooled than when heated; therefore extending the cooling phase of processing and shortening the heating phase may offer ways to lower processing costs. [ABSTRACT FROM AUTHOR]

Summary: Sustainable utilization of lignocellulosic perennial grass feedstocks will be enabled by high biomass production and optimized cell wall chemistry for efficient conversion into biofuels. MicroRNAs are regulatory elements that modulate the expression of genes involved in various biological functions in plants, including growth and development. In greenhouse studies, overexpressing a microRNA (miR156) gene in switchgrass had dramatic effects on plant architecture and flowering, which appeared to be driven by transgene expression levels. High expressing lines were extremely dwarfed, whereas low and moderate‐expressing lines had higher biomass yields, improved sugar release and delayed flowering. Four lines with moderate or low miR156 overexpression from the prior greenhouse study were selected for a field experiment to assess the relationship between miR156 expression and biomass production over three years. We also analysed important bioenergy feedstock traits such as flowering, disease resistance, cell wall chemistry and biofuel production. Phenotypes of the transgenic lines were inconsistent between the greenhouse and the field as well as among different field growing seasons. One low expressing transgenic line consistently produced more biomass (25%–56%) than the control across all three seasons, which translated to the production of 30% more biofuel per plant during the final season. The other three transgenic lines produced less biomass than the control by the final season, and the two lines with moderate expression levels also exhibited altered disease susceptibilities. Results of this study emphasize the importance of performing multiyear field studies for plants with altered regulatory transgenes that target plant growth and development. [ABSTRACT FROM AUTHOR]

The DOE BioEnergy Science Center has operated as a virtual center with multiple partners for a decade targeting overcoming biomass recalcitrance. BESC has redefined biomass recalcitrance from an observable phenotype to a better understood and manipulatable fundamental and operational property. These manipulations are the result of deeper biological understanding and can be combined with other advanced biotechnology improvements in biomass conversion to improve bioenergy processes and markets. This article provides an overview of key accomplishments in overcoming recalcitrance via better plants, better microbes, and better tools and combinations. A perspective on the aspects of successful center operation is presented. [ABSTRACT FROM AUTHOR]

Selective conversion of bio-derived ethanol to benzene, toluene and xylenes (BTX) is desirable for producing renewable BTX. In this work, we show that addition of Ga to H-ZSM-5 leads to a two-fold increase in the BTX yield as compared with H-ZSM-5 when ethanol is converted over these zeolites at 450 °C and ambient pressure. Besides promoting BTX formation, Ga also plays an important role in enhancing molecular hydrogen production and suppressing hydrogen transfer reactions for light alkane formation. The ion exchange synthesis of Ga-ZSM-5 results in the majority of Ga at the outer surface of zeolite crystals as extra-zeolitic Ga2O3 particles and only a small fraction of Ga exchanging with the Brønsted acid sites which appears to be responsible for higher ethanol conversion to BTX. The interface between H-ZSM-5 and Ga2O3 particles is not active since H-ZSM-5 and the physical mixture of β-Ga2O3/H-ZSM-5 furnish an almost identical product distribution. Hydrogen reduction of the physical mixtures facilitates movement of Ga to ion exchange locations and dramatically increases the BTX yield becoming comparable to those obtained over ion-exchanged Ga-ZSM-5, suggesting that exchanged Ga(iii) cations are responsible for the increased BTX production. A linear correlation between the BTX site time yield and exchanged Ga sites further confirms that Ga occupying cationic sites are active sites for enhancing BTX formation. Reduction of physical mixtures (β-Ga2O3/H-ZSM-5) also provides an economical and environmentally friendly non-aqueous method for large scale catalyst synthesis without sacrificing catalyst performance for ethanol conversion application. [ABSTRACT FROM AUTHOR]

Improving access to the carbohydrate content of lignocellulose is key to reducing recalcitrance for microbial deconstruction and conversion to fuels and chemicals. Caldicellulosiruptor bescii completely solubilizes naked microcrystalline cellulose, yet this transformation is impeded within the context of the plant cell wall by a network of lignin and hemicellulose. Here, the bioavailability of carbohydrates to C. bescii at 70°C was examined for reduced lignin transgenic switchgrass lines COMT3(+) and MYB Trans, their corresponding parental lines (cultivar Alamo) COMT3(-) and MYB wild type (WT), and the natural variant cultivar Cave-in-Rock (CR). Transgenic modification improved carbohydrate solubilization by C. bescii to 15% (2.3- fold) for MYB and to 36% (1.5-fold) for COMT, comparable to the levels achieved for the natural variant, CR (36%). Carbohydrate solubilization was nearly doubled after two consecutive microbial fermentations compared to one microbial step, but it never exceeded 50% overall. Hydrothermal treatment (180°C) prior to microbial steps improved solubilization 3.7-fold for the most recalcitrant line (MYB WT) and increased carbohydrate recovery to nearly 50% for the least recalcitrant lines [COMT3(+) and CR]. Alternating microbial and hydrothermal steps (T→M→T→M) further increased bioavailability, achieving carbohydrate solubilization ranging from 50% for MYB WT to above 70% for COMT3(+) and CR. Incomplete carbohydrate solubilization suggests that cellulose in the highly lignified residue was inaccessible; indeed, residue from the T→M→T→M treatment was primarily glucan and inert materials (lignin and ash). While C. bescii could significantly solubilize the transgenic switchgrass lines and natural variant tested here, additional or alternative strategies (physical, chemical, enzymatic, and/or genetic) are needed to eliminate recalcitrance. IMPORTANCE Key to a microbial process for solubilization of plant biomass is the organism's access to the carbohydrate content of lignocellulose. Economically viable routes will characteristically minimize physical, chemical, and biological pretreatment such that microbial steps contribute to the greatest extent possible. Recently, transgenic versions of plants and trees have been developed with the intention of lowering the barrier to lignocellulose conversion, with particular focus on lignin content and composition. Here, the extremely thermophilic bacterium Caldicellulosiruptor bescii was used to solubilize natural and genetically modified switchgrass lines, with and without the aid of hydrothermal treatment. For lignocellulose conversion, it is clear that the microorganism, plant biomass substrate, and processing steps must all be considered simultaneously to achieve optimal results. Whether switchgrass lines engineered for low lignin or natural variants with desirable properties are used, conversion will depend on microbial access to crystalline cellulose in the plant cell wall. [ABSTRACT FROM AUTHOR]

Biorefining of plant feedstocks into fuels and specialty chemicals, using biological conversion, requires the solubilization of lignocellulosics into simpler oligomeric compounds. However, non-pretreated woody biomass has shown high resistance to hydrolysis by cellulolytic microbes or purified cellulases. We investigate the limited solubilization of Populus deltoides by the cellulolytic thermophile Clostridium thermocellum in the absence of solute inhibitors. Compared to control samples, fermented poplar revealed that the hydrolysis of carbohydrates in secondary cell walls ceased prematurely as the presence of lignin increased at the surface. In quantitative fluorescence colocalization analysis by confocal laser scanning microscopy, the Manders’ coefficient of the fractional overlap between lignin and cellulose signals increased from an average of 0.67 to a near-maximum of 0.92 in fermented tissue. Chemical imaging by time-of-flight secondary ion mass spectrometry revealed a 49% decline in surface cellulose and a compensatory 30% and 11% increase in surface S- and G-lignin, respectively. Although 72% of the initial glucan was still present in the lignocellulose matrix of this feedstock, subsequent treatments with cell-free purified cellulases did not significantly restore hydrolysis. This confirmed that biomass surfaces had become non-productive for the C. thermocellum hydrolytic exoproteome. This study provides direct evidence for an explicit definition of feedstock recalcitrance, whereby depletion of surface carbohydrate increases lignin exposure which leads to inhibition of enzyme activity, while the bulk residual biomass retains significant undigested carbohydrate content. The analysis presented here establishes a novel method for the quantitation of lignocellulose recalcitrance. [ABSTRACT FROM AUTHOR]

[Display omitted] • Biomass-derived solvents of current interest for pretreatment were identified. • Solubility parameters theory is an effective tool for pretreatment solvent screen. • Biomass pretreatment using biosolvents were summarized. • Existing challenges and prospects of biosolvents are emphasized for future research. Biomass pretreatment is considered a key step in the 2nd generation biofuel production from lignocellulosic biomass. Research on conventional biomass pretreatment solvents has mainly been focused on carbohydrate conversion efficiency, while their hazardousness and/or carbon intensity were not comprehensively considered. Recent sustainability issues request further consideration for eco-friendly and sustainable alternatives like biomass-derived solvents. Carbohydrate and lignin-derived solvents have been proposed and investigated as green alternatives in many biomass processes. In this review, the applications of different types of biomass pretreatment solvents, including organic, ionic liquid, and deep eutectic solvents, are thoroughly discussed. The role of water as a co-solvent in these pretreatment processes is also reviewed. Finally, current research challenges and prospects of utilizing biomass-derived pretreatment solvents for pretreatment are discussed. Given bioethanol's market potential and increasing public awareness about environmental concerns, it will be a priority adopting sustainable and green biomass pretreatment solvents in biorefinery. [ABSTRACT FROM AUTHOR]

Background: The thermophilic, anaerobic bacterium Thermoanaerobacterium saccharolyticum digests hemicellulose and utilizes the major sugars present in biomass. It was previously engineered to produce ethanol at yields equivalent to yeast. While saccharolytic anaerobes have been long studied as potential biomass-fermenting organisms, development efforts for commercial ethanol production have not been reported. Results: Here, we describe the highest ethanol titers achieved from T. saccharolyticum during a 4-year project to develop it for industrial production of ethanol from pre-treated hardwood at 51–55 °C. We describe organism and bioprocess development efforts undertaken to improve ethanol production. The final strain M2886 was generated by removing genes for exopolysaccharide synthesis, the regulator perR, and re-introduction of phosphotransacetylase and acetate kinase into the methyglyoxal synthase gene. It was also subject to multiple rounds of adaptation and selection, resulting in mutations later identified by resequencing. The highest ethanol titer achieved was 70 g/L in batch culture with a mixture of cellobiose and maltodextrin. In a "mock hydrolysate" Simultaneous Saccharification and Fermentation (SSF) with Sigmacell-20, glucose, xylose, and acetic acid, an ethanol titer of 61 g/L was achieved, at 92 % of theoretical yield. Fungal cellulases were rapidly inactivated under these conditions and had to be supplemented with cellulosomes from C. thermocellum. Ethanol titers of 31 g/L were reached in a 100 L SSF of pre-treated hardwood and 26 g/L in a fermentation of a hardwood hemicellulose extract. Conclusions: This study demonstrates that thermophilic anaerobes are capable of producing ethanol at high yield and at titers greater than 60 g/L from purified substrates, but additional work is needed to produce the same ethanol titers from pre-treated hardwood. [ABSTRACT FROM AUTHOR]

Background: Higher ratios of syringyl-to-guaiacyl (S/G) lignin components of Populus were shown to improve sugar release by enzymatic hydrolysis using commercial blends. Cellulolytic microbes are often robust biomass hydrolyzers and may offer cost advantages; however, it is unknown whether their activity can also be significantly influenced by the ratio of different monolignol types in Populus biomass. Hydrolysis and fermentation of autoclaved, but otherwise not pretreated Populus trichocarpa by Clostridium thermocellum ATCC 27405 was compared using feedstocks that had similar carbohydrate and total lignin contents but differed in S/G ratios. Results: Populus with an S/G ratio of 2.1 was converted more rapidly and to a greater extent compared to similar biomass that had a ratio of 1.2. For either microbes or commercial enzymes, an approximate 50 % relative difference in total solids solubilization was measured for both biomasses, which suggests that the differences and limitations in the microbial breakdown of lignocellulose may be largely from the enzymatic hydrolytic process. Surprisingly, the reduction in glucan content per gram solid in the residual microbially processed biomass was similar (17-18 %) irrespective of S/G ratio, pointing to a similar mechanism of solubilization that proceeded at different rates. Fermentation metabolome testing did not reveal the release of known biomass-derived alcohol and aldehyde inhibitors that could explain observed differences in microbial hydrolytic activity. Biomass-derived p-hydroxybenzoic acid was up to ninefold higher in low S/G ratio biomass fermentations, but was not found to be inhibitory in subsequent test fermentations. Cellulose crystallinity and degree of polymerization did not vary between Populus lines and had minor changes after fermentation. However, lignin molecular weights and cellulose accessibility determined by Simons' staining were positively correlated to the S/G content. Conclusions: Higher S/G ratios in Populus biomass lead to longer and more linear lignin chains and greater access to surface cellulosic content by microbe-bound enzymatic complexes. Substrate access limitation is suggested as a primary bottleneck in solubilization of minimally processed Populus, which has important implications for microbial deconstruction of lignocellulose biomass. Our findings will allow others to examine different Populus lines and to test if similar observations are possible for other plant species. [ABSTRACT FROM AUTHOR]

Background: Feedstock recalcitrance is the most important barrier impeding cost-efective production of cellulosic biofuels. Pioneer commercial cellulosic ethanol facilities employ thermochemical pretreatment and addition of fungal cellulase, relecting the main research emphasis in the ield. However, it has been suggested that it may be possible to process cellulosic biomass without thermochemical pretreatment using thermophilic, cellulolytic bacteria. To further explore this idea, we examine the ability of various biocatalysts to solubilize autoclaved but otherwise unpretreated cellulosic biomass under controlled but not industrial conditions. Results: Carbohydrate solubilization of mid-season harvested switchgrass after 5 days ranged from 24 % for Caldicellulosiruptor bescii to 65 % for Clostridium thermocellum, with intermediate values for a thermophilic horse manure enrichment, Clostridium clarilavum, Clostridium cellulolyticum, and simultaneous sacchariication and fermentation (SSF) featuring a fungal cellulase cocktail and yeast. Under a variety of conditions, solubilization yields were about twice as high for C. thermocellum compared to fungal cellulase. Solubilization of mid-season harvested switchgrass was about twice that of senescent switchgrass. Lower yields and greater dependence on particle size were observed for Populus as compared to switchgrass. Trends observed from data drawn from six conversion systems and three substrates, including both time course and end-point data, were (1) equal fractional solubilization of glucan and xylan, (2) no biological solubilization of the non-carbohydrate fraction of biomass, and (3) higher solubilization for three of the four bacterial cultures tested as compared to the fungal cellulase system. Brief (5 min) ball milling of solids remaining after fermentation of senescent switchgrass by C. thermocellum nearly doubled carbohydrate solubilization upon reinnoculation as compared to a control without milling. Greater particle size reduction and solubilization were observed for milling of partially fermented solids than for unfermented solids. Physical disruption of cellulosic feedstocks after initiation of fermentation, termed cotreatment, warrants further study. Conclusions: While the ability to achieve signiicant solubilization of minimally pretreated switchgrass is widespread, a ivefold diference between the most and least efective biocatalyst--feedstock combinations was observed. Starting with nature's best biomass-solubilizing systems may enable a reduction in the amount of non-biological processing required, and in particular substitution of cotreatment for pretreatment. [ABSTRACT FROM AUTHOR]

Background: Clostridium thermocellum is a promising consolidated bioprocessing candidate organism capable of directly converting lignocellulosic biomass to ethanol. Current ethanol yields, productivities, and growth inhibitions are industrial deployment impediments for commodity fuel production by this bacterium. Redox imbalance under certain conditions and in engineered strains may contribute to incomplete substrate utilization and may direct fermentation products to undesirable overflow metabolites. Towards a better understanding of redox metabolism in C. thermocellum, we established continuous growth conditions and analyzed global gene expression during addition of two stress chemicals (methyl viologen and hydrogen peroxide) which changed the fermentation redox potential. Results: The addition of methyl viologen to C. thermocellum DSM 1313 chemostat cultures caused an increase in ethanol and lactate yields. A lower fermenter redox potential was observed in response to methyl viologen exposure, which correlated with a decrease in cell yield and significant differential expression of 123 genes (log2 > 1.5 or log2 < -1.5, with a 5 % false discovery rate). Expression levels decreased in four main redox-active systems during methyl viologen exposure; the [NiFe] hydrogenase, sulfate transport and metabolism, ammonia assimilation (GSGOGAT), and porphyrin/siroheme biosynthesis. Genes encoding sulfate transport and reduction and porphyrin/ siroheme biosynthesis are co-located immediately downstream of a putative iscR regulatory gene, which may be a cis-regulatory element controlling expression of these genes. Other genes showing differential expression during methyl viologen exposure included transporters and transposases. Conclusions: The differential expression results from this study support a role for C. thermocellum genes for sulfate transport/reduction, glutamate synthase-glutamine synthetase (the GS-GOGAT system), and porphyrin biosynthesis being involved in redox metabolism and homeostasis. This global profiling study provides gene targets for future studies to elucidate the relative contributions of prospective pathways for co-factor pool re-oxidation and C. thermocellum redox homeostasis. [ABSTRACT FROM AUTHOR]

Background: Thermoanaerobacterium saccharolyticum is a hemicellulose-degrading thermophilic anaerobe that was previously engineered to produce ethanol at high yield. A major project was undertaken to develop this organism into an industrial biocatalyst, but the lack of genome information and resources were recognized early on as a key limitation. Results: Here we present a set of genome-scale resources to enable the systems level investigation and development of this potentially important industrial organism. Resources include a complete genome sequence for strain JW/SL-YS485, a genome-scale reconstruction of metabolism, tiled microarray data showing transcription units, mRNA expression data from 71 different growth conditions or timepoints and GC/MS-based metabolite analysis data from 42 different conditions or timepoints. Growth conditions include hemicellulose hydrolysate, the inhibitors HMF, furfural, diamide, and ethanol, as well as high levels of cellulose, xylose, cellobiose or maltodextrin. The genome consists of a 2.7 Mbp chromosome and a 110 Kbp megaplasmid. An active prophage was also detected, and the expression levels of CRISPR genes were observed to increase in association with those of the phage. Hemicellulose hydrolysate elicited a response of carbohydrate transport and catabolism genes, as well as poorly characterized genes suggesting a redox challenge. In some conditions, a time series of combined transcription and metabolite measurements were made to allow careful study of microbial physiology under process conditions. As a demonstration of the potential utility of the metabolic reconstruction, the OptKnock algorithm was used to predict a set of gene knockouts that maximize growth-coupled ethanol production. The predictions validated intuitive strain designs and matched previous experimental results. Conclusion: These data will be a useful asset for efforts to develop T. saccharolyticum for efficient industrial production of biofuels. The resources presented herein may also be useful on a comparative basis for development of other lignocellulose degrading microbes, such as Clostridium thermocellum. [ABSTRACT FROM AUTHOR]

Fine-tuning plant cell wall properties to render plant biomass more amenable to biofuel conversion is a colossal challenge. A deep knowledge of the biosynthesis and regulation of plant cell wall and a high-precision genome engineering toolset are the two essential pillars of efforts to alter plant cell walls and reduce biomass recalcitrance. The past decade has seen a meteoric rise in use of transcriptomics and high-resolution imaging methods resulting in fresh insights into composition, structure, formation and deconstruction of plant cell walls. Subsequent gene manipulation approaches, however, commonly include ubiquitous mis-expression of a single candidate gene in a host that carries an intact copy of the native gene. The challenges posed by pleiotropic and unintended changes resulting from such an approach are moving the field towards synthetic biology approaches. Synthetic biology builds on a systems biology knowledge base and leverages high-precision tools for high-throughput assembly of multigene constructs and pathways, precision genome editing and site-specific gene stacking, silencing and/or removal. Here, we summarize the recent breakthroughs in biosynthesis and remodelling of major secondary cell wall components, assess the impediments in obtaining a systems-level understanding and explore the potential opportunities in leveraging synthetic biology approaches to reduce biomass recalcitrance. [ABSTRACT FROM AUTHOR]

Background Switchgrass is an abundant and dedicated bioenergy feedstock, however its inherent recalcitrance is one of the economic hurdles for producing biofuels. The downregulation of the caffeic acid O-methyl transferase (COMT) gene in the lignin pathway of switchgrass reduced lignin content and S/G ratio, and the transgenic lines showed improved fermentation yield with Saccharomyces cerevisiae and wild-type Clostridium thermocellum (ATCC 27405) in comparison to the wild-type switchgrass. Results Here we examine the conversion and yield of the COMT transgenic and wild-type switchgrass lines with an engineered and evolved C. thermocellum (M1570) strain. The fermentation of the transgenic switchgrass by M1570 had superior conversion relative to the wild-type control switchgrass line with an increase in conversion of approximately 20% and ethanol being the primary product accounting for 90% of the total metabolites measured by HPLC analysis. Conclusions The engineered and evolved C. thermocellum M1570 was found to respond to the apparent reduced recalcitrance of the COMT switchgrass with no substrate inhibition, producing more ethanol on the transgenic feedstock than the wild-type substrate. Since ethanol was the main fermentation metabolite produced by an engineered and evolved C. thermocellum strain, its ethanol yield on a transgenic switchgrass substrate (gram/gram (g/g) glucan liberated) is the highest produced thus far. This result indicates that the advantages of a modified feedstock can be combined with a modified consolidated bioprocessing microorganism as anticipated. [ABSTRACT FROM AUTHOR]

Lignocellulosic biomass, a potentially important renewable organic source of energy and chemical feedstock, resists degradation to glucose in industrial hydrolysis processes and thus requires expensive thermochemical pretreatments. Understanding the mechanism of biomass breakdown during these pretreatments will lead to more efficient use of biomass. By combining multiple probes of structure, sensitive to different length scales, with molecular dynamics simulations, we reveal two fundamental processes responsible for the morphological changes in biomass during steam explosion pretreatment: cellulose dehydration and ligninhemicellulose phase separation. We further show that the basic driving forces are the same in other leading thermochemical pretreatments, such as dilute acid pretreatment and ammonia fiber expansion. [ABSTRACT FROM AUTHOR]

Background: Zymomonas mobilis ZM4 is a capable ethanologenic bacterium with high ethanol productivity and ethanol tolerance. Previous studies indicated that several stress-related proteins and changes in the ZM4 membrane lipid composition may contribute to ethanol tolerance. However, the molecular mechanisms of its ethanol stress response have not been elucidated fully. Methodology/Principal Findings: In this study, ethanol stress responses were investigated using systems biology approaches. Medium supplementation with an initial 47 g/L (6% v/v) ethanol reduced Z. mobilis ZM4 glucose consumption, growth rate and ethanol productivity compared to that of untreated controls. A proteomic analysis of early exponential growth identified about one thousand proteins, or approximately 55% of the predicted ZM4 proteome. Proteins related to metabolism and stress response such as chaperones and key regulators were more abundant in the early ethanol stress condition. Transcriptomic studies indicated that the response of ZM4 to ethanol is dynamic, complex and involves many genes from all the different functional categories. Most down-regulated genes were related to translation and ribosome biogenesis, while the ethanol-upregulated genes were mostly related to cellular processes and metabolism. Transcriptomic data were used to update Z. mobilis ZM4 operon models. Furthermore, correlations among the transcriptomic, proteomic and metabolic data were examined. Among significantly expressed genes or proteins, we observe higher correlation coefficients when fold-change values are higher. Conclusions: Our study has provided insights into the responses of Z. mobilis to ethanol stress through an integrated “omics” approach for the first time. This systems biology study elucidated key Z. mobilis ZM4 metabolites, genes and proteins that form the foundation of its distinctive physiology and its multifaceted response to ethanol stress. [ABSTRACT FROM AUTHOR]

The production of cellulosic ethanol from biomass is considered a promising alternative to reliance on diminishing supplies of fossil fuels, providing a sustainable option for fuels production in an environmentally compatible manner. The conversion of lignocellulosic biomass to biofuels through a biological route usually suffers from the intrinsic recalcitrance of biomass owing to the complicated structure of plant cell walls. Currently, a pretreatment step that can effectively reduce biomass recalcitrance is generally required to make the polysaccharide fractions locked in the intricacy of plant cell walls to become more accessible and amenable to enzymatic hydrolysis. Dilute acid and hydrothermal pretreatments are attractive and among the most promising pretreatment technologies that enhance sugar release performance. This review highlights our recent understanding on molecular structure basis for recalcitrance, with emphasis on structural transformation of major biomass biopolymers (i.e., cellulose, hemicellulose, and lignin) related to the reduction of recalcitrance during dilute acid and hydrothermal pretreatments. The effects of these two pretreatments on biomass porosity as well as its contribution on reduced recalcitrance are also discussed. [ABSTRACT FROM AUTHOR]

Background: Down-regulation of the caffeic acid 3-O-methyltransferase EC 2.1.1.68 (COMT) gene in the lignin biosynthetic pathway of switchgrass (Panicum virgatum) resulted in cell walls of transgenic plants releasing more constituent sugars after pretreatment by dilute acid and treatment with glycosyl hydrolases from an added enzyme preparation and from Clostridium thermocellum. Fermentation of both wild-type and transgenic switchgrass after milder hot water pretreatment with no water washing showed that only the transgenic switchgrass inhibited C. thermocellum. Gas chromatography-mass spectrometry (GCMS)-based metabolomics were undertaken on cell wall aqueous extracts to determine the nature of the microbial inhibitors. Results: GCMS confirmed the increased concentration of a number of phenolic acids and aldehydes that are known inhibitors of microbial fermentation. Metabolomic analyses of the transgenic biomass additionally revealed the presence of a novel monolignol-like metabolite, identified as trans-3, 4-dimethoxy-5-hydroxycinnamyl alcohol (iso-sinapyl alcohol) in both non-pretreated, as well as hot water pretreated samples. iso-Sinapyl alcohol and itsglucoside were subsequently generated by organic synthesis and the identity of natural and synthetic materials were confirmed by mass spectrometric and NMR analyses. The additional novel presence of iso-sinapic acid, iso-sinapyl aldehyde, and iso-syringin suggest the increased activity of a para-methyltransferase, concomitant with the reduced COMT activity, a strict meta-methyltransferase. Quantum chemical calculations were used to predict the most likely homodimeric lignans generated from dehydration reactions, but these products were not evident in plant samples. Conclusions: Down-regulation of COMT activity in switchgrass resulted in the accumulation of previously undetected metabolites resembling sinapyl alcohol and its related metabolites, but that are derived from para-methylation of 5-hydroxyconiferyl alcohol, and related precursors and products; the accumulation of which suggests altered metabolism of 5-hydroxyconiferyl alcohol in switchgrass. Given that there was no indication that iso-sinapyl alcohol was integrated in cell walls, it is considered a monolignol analog. Diversion of substrates from sinapyl alcohol to free iso-sinapyl alcohol, its glucoside, and associated upstream lignin pathway changes, including increased phenolic aldehydes and acids, are together associated with more facile cell wall deconstruction, and to the observed inhibitory effect on microbial growth. However, iso-sinapyl alcohol and iso-sinapic acid, added separately to media, were not inhibitory to C. thermocellum cultures. [ABSTRACT FROM AUTHOR]

Background: Zymomonas mobilis ZM4 (ZM4) produces near theoretical yields of ethanol with high specific productivity and recombinant strains are able to ferment both C-5 and C-6 sugars. Z. mobilis performs best under anaerobic conditions, but is an aerotolerant organism. However, the genetic and physiological basis of ZM4's response to various stresses is understood poorly. Results: In this study, transcriptomic and metabolomic profiles for ZM4 aerobic and anaerobic fermentations were elucidated by microarray analysis and by high-performance liquid chromatography (HPLC), gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) analyses. In the absence of oxygen, ZM4 consumed glucose more rapidly, had a higher growth rate, and ethanol was the major end-product. Greater amounts of other end-products such as acetate, lactate, and acetoin were detected under aerobic conditions and at 26 h there was only 1.7% of the amount of ethanol present aerobically as there was anaerobically. In the early exponential growth phase, significant differences in gene expression were not observed between aerobic and anaerobic conditions via microarray analysis. HPLC and GC analyses revealed minor differences in extracellular metabolite profiles at the corresponding early exponential phase time point. Differences in extracellular metabolite profiles between conditions became greater as the fermentations progressed. GC-MS analysis of stationary phase intracellular metabolites indicated that ZM4 contained lower levels of amino acids such as alanine, valine and lysine, and other metabolites like lactate, ribitol, and 4-hydroxybutanoate under anaerobic conditions relative to aerobic conditions. Stationary phase microarray analysis revealed that 166 genes were significantly differentially expressed by more than two-fold. Transcripts for Entner-Doudoroff (ED) pathway genes (glk, zwf, pgl, pgk, and eno) and gene pdc, encoding a key enzyme leading to ethanol production, were at least 30-fold more abundant under anaerobic conditions in the stationary phase based on quantitative-PCR results. We also identified differentially expressed ZM4 genes predicted by The Institute for Genomic Research (TIGR) that were not predicted in the primary annotation. Conclusion: High oxygen concentrations present during Z. mobilis fermentations negatively influence fermentation performance. The maximum specific growth rates were not dramatically different between aerobic and anaerobic conditions, yet oxygen did affect the physiology of the cells leading to the buildup of metabolic byproducts that ultimately led to greater differences in transcriptomic profiles in stationary phase. [ABSTRACT FROM AUTHOR]

We report measurements of solubility limits for benzene, toluene, and TCE in systems that contain varying levels of biomass up to 0.13gmL−1 for TCE and 0.25gmL−1 for benzene and toluene. The solubility limit increased from 21 to 48mM when biomass (in the form of yeast) was added to aqueous batch systems containing benzene. The toluene solubility limit increased from 4.9 to greater than 20mM. For TCE, the solubility increased from 8mM to more than 1000mM. Solubility for TCE (trichloroethylene) was most heavily impacted by biomass levels, changing by two orders of magnitude as the microbial concentrations approach those in biofilms. [Copyright &y& Elsevier]

Following publication of the original article [1], the authors reported that the omission of author name. [ABSTRACT FROM AUTHOR]

Background: The recalcitrance of cellulosic biomass is widely recognized as a key barrier to cost-effective biological processing to fuels and chemicals, but the relative impacts of physical, chemical and genetic interventions to improve biomass processing singly and in combination have yet to be evaluated systematically. Solubilization of plant cell walls can be enhanced by non-biological augmentation including physical cotreatment and thermochemical pretreatment, the choice of biocatalyst, the choice of plant feedstock, genetic engineering of plants, and choosing feedstocks that are less recalcitrant natural variants. A two-tiered combinatoric investigation of lignocellulosic biomass deconstruction was undertaken with three biocatalysts (Clostridium thermocellum, Caldicellulosiruptor bescii, Novozymes Cellic® Ctec2 and Htec2), three transgenic switchgrass plant lines (COMT, MYB4, GAUT4) and their respective nontransgenic controls, two Populus natural variants, and augmentation of biological attack using either mechanical cotreatment or cosolvent-enhanced lignocellulosic fractionation (CELF) pretreatment. Results: In the absence of augmentation and under the conditions tested, increased total carbohydrate solubilization (TCS) was observed for 8 of the 9 combinations of switchgrass modifications and biocatalysts tested, and statistically significant for five of the combinations. Our results indicate that recalcitrance is not a trait determined by the feedstock only, but instead is coequally determined by the choice of biocatalyst. TCS with C. thermocellum was significantly higher than with the other two biocatalysts. Both CELF pretreatment and cotreatment via continuous ball milling enabled TCS in excess of 90%. Conclusion: Based on our results as well as literature studies, it appears that some form of non-biological augmentation will likely be necessary for the foreseeable future to achieve high TCS for most cellulosic feedstocks. However, our results show that this need not necessarily involve thermochemical processing, and need not necessarily occur prior to biological conversion. Under the conditions tested, the relative magnitude of TCS increase was augmentation > biocatalyst choice > plant choice > plant modification > plant natural variants. In the presence of augmentation, plant modification, plant natural variation, and plant choice exhibited a small, statistically non-significant impact on TCS. [ABSTRACT FROM AUTHOR]

Clostridium (Ruminiclostridium) thermocellum is a model organism for its ability to deconstruct plant biomass and convert the cellulose into ethanol. The bacterium forms biofilms adherent to lignocellulosic feedstocks in a continuous cell-monolayer in order to efficiently break down and uptake cellulose hydrolysates. We developed a novel bioreactor design to generate separate sessile and planktonic cell populations for omics studies. Sessile cells had significantly greater expression of genes involved in catabolism of carbohydrates by glycolysis and pyruvate fermentation, ATP generation by proton gradient, the anabolism of proteins and lipids and cellular functions critical for cell division consistent with substrate replete conditions. Planktonic cells had notably higher gene expression for flagellar motility and chemotaxis, cellulosomal cellulases and anchoring scaffoldins, and a range of stress induced homeostasis mechanisms such as oxidative stress protection by antioxidants and flavoprotein co-factors, methionine repair, Fe-S cluster assembly and repair in redox proteins, cell growth control through tRNA thiolation, recovery of damaged DNA by nucleotide excision repair and removal of terminal proteins by proteases. This study demonstrates that microbial attachment to cellulose substrate produces widespread gene expression changes for critical functions of this organism and provides physiological insights for two cells populations relevant for engineering of industrially-ready phenotypes. [ABSTRACT FROM AUTHOR]

Direct catalytic conversion of ethanol to hydrocarbon blend-stock can increase biofuels use in current vehicles beyond the ethanol blend-wall of 10-15%. Literature reports describe quantitative conversion of ethanol over zeolite catalysts but high C2 hydrocarbon formation renders this approach unsuitable for commercialization. Furthermore, the prior mechanistic studies suggested that ethanol conversion involves endothermic dehydration step. Here, we report the complete conversion of ethanol to hydrocarbons over InV-ZSM-5 without added hydrogen and which produces lower C2 (<13%) as compared to that over H-ZSM-5. Experiments with C2H5OD and in situ DRIFT suggest that most of the products come from the hydrocarbon pool type mechanism and dehydration step is not necessary. Thus, our method of direct conversion of ethanol offers a pathway to produce suitable hydrocarbon blend-stock that may be blended at a refinery to produce fuels such as gasoline, diesel, JP-8, and jet fuel, or produce commodity chemicals such as BTX. [ABSTRACT FROM AUTHOR]

A summary is presented of the authors' research into improving biorefinery processing of the aromatic polymer lignin for power generation. Topics include advances in bioengineering the structure or components of lignin, research into extraction efficiency, and potential lignin products to replace petroleum-based versions such as plastics and fuels.