Sugar release and growth of biofuel crops are improved by downregulation of pectin biosynthesis.

Cell walls in crops and trees have been engineered for production of biofuels and commodity chemicals, but engineered varieties often fail multi-year field trials and are not commercialized. We engineered reduced expression of a pectin biosynthesis gene (Galacturonosyltransferase 4, GAUT4) in switchgrass and poplar, and find that this improves biomass yields and sugar release from biomass processing. Both traits were maintained in a 3-year field trial of GAUT4-knockdown switchgrass, with up to sevenfold increased saccharification and ethanol production and sixfold increased biomass yield compared with control plants.

Integrated omics analyses reveal the details of metabolic adaptation of to lignocellulose-derived growth inhibitors released during the deconstruction of switchgrass.

Background: is capable of solubilizing and converting lignocellulosic biomass into ethanol. Although much of the work-to-date has centered on characterizing this microbe's growth on model cellulosic substrates, such as cellobiose, Avicel, or filter paper, it is vitally important to understand its metabolism on more complex, lignocellulosic substrates to identify relevant industrial bottlenecks that could undermine efficient biofuel production. To this end, we have examined a time course progression of grown on switchgrass to assess the metabolic and protein changes that occur during the conversion of plant biomass to ethanol.

Transgenic switchgrass (Panicum virgatum L.) targeted for reduced recalcitrance to bioconversion: a 2-year comparative analysis of field-grown lines modified for target gene or genetic element expression.

Transgenic Panicum virgatum L. silencing (KD) or overexpressing (OE) specific genes or a small RNA (GAUT4-KD, miRNA156-OE, MYB4-OE, COMT-KD and FPGS-KD) was grown in the field and aerial tissue analysed for biofuel production traits. Clones representing independent transgenic lines were established and senesced tissue was sampled after year 1 and 2 growth cycles.

Ethanol and High-Value Terpene Co-Production from Lignocellulosic Biomass of Cymbopogon flexuosus and Cymbopogon martinii.

Cymbopogon flexuosus, lemongrass, and C. martinii, palmarosa, are perennial grasses grown to produce essential oils for the fragrance industry. The objectives of this study were (1) to evaluate biomass and oil yields as a function of nitrogen and sulfur fertilization, and (2) to characterize their utility for lignocellulosic ethanol compared to Panicum virgatum (switchgrass).

Genome-scale resources for Thermoanaerobacterium saccharolyticum.

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.

Development of Agave as a dedicated biomass source: production of biofuels from whole plants.

Background: Agave species can grow well in semi-arid marginal agricultural lands around the world. Selected Agave species are used largely for alcoholic beverage production in Mexico. There are expanding research efforts to use the plentiful residues (bagasse) for ethanol production as the beverage manufacturing process only uses the juice from the central core of mature plants.

Consolidated bioprocessing of transgenic switchgrass by an engineered and evolved Clostridium thermocellum strain.

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.

Two-year field analysis of reduced recalcitrance transgenic switchgrass.

Switchgrass (Panicum virgatum L.) is a leading candidate for a dedicated lignocellulosic biofuel feedstock owing to its high biomass production, wide adaptation and low agronomic input requirements. Lignin in cell walls of switchgrass, and other lignocellulosic feedstocks, severely limits the accessibility of cell wall carbohydrates to enzymatic breakdown into fermentable sugars and subsequently biofuels.

Simultaneous saccharification and fermentation and a consolidated bioprocessing for Hinoki cypress and Eucalyptus after fibrillation by steam and subsequent wet-disk milling.

An advanced pretreatment method that combines steam treatment (ST) with wet disk milling (WDM) was evaluated using two different species of woods, viz., Hinoki cypress (softwood) and Eucalyptus (hardwood). Bioconversion of the pretreated products was performed using enzymatic saccharification via a commercial cellulase mixture and two types of fermentation processing, i.

Global transcriptome analysis of Clostridium thermocellum ATCC 27405 during growth on dilute acid pretreated Populus and switchgrass.

Background: The thermophilic anaerobe Clostridium thermocellum is a candidate consolidated bioprocessing (CBP) biocatalyst for cellulosic ethanol production. The aim of this study was to investigate C. thermocellum genes required to ferment biomass substrates and to conduct a robust comparison of DNA microarray and RNA sequencing (RNA-seq) analytical platforms.

Industrial robustness: understanding the mechanism of tolerance for the Populus hydrolysate-tolerant mutant strain of Clostridium thermocellum.

Background: An industrially robust microorganism that can efficiently degrade and convert lignocellulosic biomass into ethanol and next-generation fuels is required to economically produce future sustainable liquid transportation fuels. The anaerobic, thermophilic, cellulolytic bacterium Clostridium thermocellum is a candidate microorganism for such conversions but it, like many bacteria, is sensitive to potential toxic inhibitors developed in the liquid hydrolysate produced during biomass processing. Microbial processes leading to tolerance of these inhibitory compounds found in the pretreated biomass hydrolysate are likely complex and involve multiple genes.

Kinetic modeling of batch fermentation for Populus hydrolysate tolerant mutant and wild type strains of Clostridium thermocellum.

The extent of inhibition of two strains of Clostridium thermocellum by a Populus hydrolysate was investigated. A Monod-based model of wild type (WT) and Populus hydrolysate tolerant mutant (PM) strains of the cellulolytic bacterium C. thermocellum was developed to quantify growth kinetics in standard media and the extent of inhibition to a Populus hydrolysate.

Enhanced characteristics of genetically modified switchgrass (Panicum virgatum L.) for high biofuel production.

Background: Lignocellulosic biomass is one of the most promising renewable and clean energy resources to reduce greenhouse gas emissions and dependence on fossil fuels. However, the resistance to accessibility of sugars embedded in plant cell walls (so-called recalcitrance) is a major barrier to economically viable cellulosic ethanol production. A recent report from the US National Academy of Sciences indicated that, "absent technological breakthroughs", it was unlikely that the US would meet the congressionally mandated renewable fuel standard of 35 billion gallons of ethanol-equivalent biofuels plus 1 billion gallons of biodiesel by 2022.

Evaluation of the bioconversion of genetically modified switchgrass using simultaneous saccharification and fermentation and a consolidated bioprocessing approach.

Background: The inherent recalcitrance of lignocellulosic biomass is one of the major economic hurdles for the production of fuels and chemicals from biomass. Additionally, lignin is recognized as having a negative impact on enzymatic hydrolysis of biomass, and as a result much interest has been placed on modifying the lignin pathway to improve bioconversion of lignocellulosic feedstocks.

Results: Down-regulation of the caffeic acid 3-O-methyltransferase (COMT) gene in the lignin pathway yielded switchgrass (Panicum virgatum) that was more susceptible to bioconversion after dilute acid pretreatment.

Down-regulation of the caffeic acid O-methyltransferase gene in switchgrass reveals a novel monolignol analog.

Background: Down-regulation of the caffeic acid 3-O-methyltransferase EC 2.1.1.

Anaerobic high-throughput cultivation method for isolation of thermophiles using biomass-derived substrates.

Flow cytometry (FCM) techniques have been developed for sorting mesophilic organisms, but the difficulty increases if the target microbes are thermophilic anaerobes. We demonstrate a reliable, high-throughput method of screening thermophilic anaerobic organisms using FCM and 96-well plates for growth on biomass-relevant substrates. The method was tested using the cellulolytic thermophiles Clostridium thermocellum (T(opt) = 55 °C), Caldicellulosiruptor obsidiansis (T(opt) = 78 °C) and the fermentative hyperthermophiles, Pyrococcus furiosus (T(opt) = 100 °C) and Thermotoga maritima (T(opt) = 80 °C).

Mutant selection and phenotypic and genetic characterization of ethanol-tolerant strains of Clostridium thermocellum.

Clostridium thermocellum is a model microorganism for converting cellulosic biomass into fuels and chemicals via consolidated bioprocessing. One of the challenges for industrial application of this organism is its low ethanol tolerance, typically 1-2% (w/v) in wild-type strains. In this study, we report the development and characterization of mutant C.

Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum.

Clostridium thermocellum is a thermophilic, obligately anaerobic, gram-positive bacterium that is a candidate microorganism for converting cellulosic biomass into ethanol through consolidated bioprocessing. Ethanol intolerance is an important metric in terms of process economics, and tolerance has often been described as a complex and likely multigenic trait for which complex gene interactions come into play. Here, we resequence the genome of an ethanol-tolerant mutant, show that the tolerant phenotype is primarily due to a mutated bifunctional acetaldehyde-CoA/alcohol dehydrogenase gene (adhE), hypothesize based on structural analysis that cofactor specificity may be affected, and confirm this hypothesis using enzyme assays.

Transcriptomic analysis of Clostridium thermocellum ATCC 27405 cellulose fermentation.

Background: The ability of Clostridium thermocellum ATCC 27405 wild-type strain to hydrolyze cellulose and ferment the degradation products directly to ethanol and other metabolic byproducts makes it an attractive candidate for consolidated bioprocessing of cellulosic biomass to biofuels. In this study, whole-genome microarrays were used to investigate the expression of C. thermocellum mRNA during growth on crystalline cellulose in controlled replicate batch fermentations.

Use of label-free quantitative proteomics to distinguish the secreted cellulolytic systems of Caldicellulosiruptor bescii and Caldicellulosiruptor obsidiansis.

The extremely thermophilic, Gram-positive bacteria Caldicellulosiruptor bescii and Caldicellulosiruptor obsidiansis efficiently degrade both cellulose and hemicellulose, which makes them relevant models for lignocellulosic biomass deconstruction to produce sustainable biofuels. To identify the shared and unique features of secreted cellulolytic apparatuses from C. bescii and C.

Genetic manipulation of lignin reduces recalcitrance and improves ethanol production from switchgrass.

Switchgrass is a leading dedicated bioenergy feedstock in the United States because it is a native, high-yielding, perennial prairie grass with a broad cultivation range and low agronomic input requirements. Biomass conversion research has developed processes for production of ethanol and other biofuels, but they remain costly primarily because of the intrinsic recalcitrance of biomass. We show here that genetic modification of switchgrass can produce phenotypically normal plants that have reduced thermal-chemical (≤180 °C), enzymatic, and microbial recalcitrance.

Sequencing of multiple clostridial genomes related to biomass conversion and biofuel production.

Modern methods to develop microbe-based biomass conversion processes require a system-level understanding of the microbes involved. Clostridium species have long been recognized as ideal candidates for processes involving biomass conversion and production of various biofuels and other industrial products. To expand the knowledge base for clostridial species relevant to current biofuel production efforts, we have sequenced the genomes of 20 species spanning multiple genera.

Impact of pretreated Switchgrass and biomass carbohydrates on Clostridium thermocellum ATCC 27405 cellulosome composition: a quantitative proteomic analysis.

Background: Economic feasibility and sustainability of lignocellulosic ethanol production requires the development of robust microorganisms that can efficiently degrade and convert plant biomass to ethanol. The anaerobic thermophilic bacterium Clostridium thermocellum is a candidate microorganism as it is capable of hydrolyzing cellulose and fermenting the hydrolysis products to ethanol and other metabolites. C.