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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 ([less-than or equal to]180 °C), enzymatic, and microbial recalcitrance. Down-regulation of the switchgrass caffeic acid O-methyltransferase gene decreases lignin content modestly, reduces the syringyl:guaiacyl lignin monomer ratio, improves forage quality, and, most importantly, increases the ethanol yield by up to 38% using conventional biomass fermentation processes. The down-regulated lines require less severe pretreatment and 300-400% lower cellulase dosages for equivalent product yields using simultaneous saccharification and fermentation with yeast. Furthermore, fermentation of diluted acid-pretreated transgenic switchgrass using Clostridium thermocellum with no added enzymes showed better product yields than obtained with unmodified switchgrass. Therefore, this apparent reduction in the recalcitrance of transgenic switchgrass has the potential to lower processing costs for biomass fermentation-derived fuels and chemicals significantly. Alternatively, such modified transgenic switchgrass lines should yield significantly more fermentation chemicals per hectare under identical process conditions.

Abstract Background: The model bacterium Clostridium cellulolyticum efficiently degrades crystalline cellulose and hemicellulose, using cellulosomes to degrade lignocellulosic biomass. Although it imports and ferments both pentose and hexose sugars to produce a mixture of ethanol, acetate, lactate, H2 and CO2 , the proportion of ethanol is low, which impedes its use in consolidated bioprocessing for biofuels production. Therefore genetic engineering will likely be required to improve the ethanol yield. Plasmid transformation, random mutagenesis and heterologous expression systems have previously been developed for C. cellulolyticum , but targeted mutagenesis has not been reported for this organism, hindering genetic engineering. Results: The first targeted gene inactivation system was developed for C. cellulolyticum , based on a mobile group II intron originating from the Lactococcus lactis L1.LtrB intron. This markerless mutagenesis system was used to disrupt both the paralogous L-lactate dehydrogenase (Ccel_2485; ldh ) and L-malate dehydrogenase (Ccel_0137; mdh ) genes, distinguishing the overlapping substrate specificities of these enzymes. Both mutations were then combined in a single strain, resulting in a substantial shift in fermentation toward ethanol production. This double mutant produced 8.5-times more ethanol than wild-type cells growing on crystalline cellulose. Ethanol constituted 93% of the major fermentation products, corresponding to a molar ratio of ethanol to organic acids of 15, versus 0.18 in wild-type cells. During growth on acid-pretreated switchgrass, the double mutant also produced four times as much ethanol as wild-type cells. Detailed metabolomic analyses identified increased flux through the oxidative branch of the mutant's tricarboxylic acid pathway. Conclusions: The efficient intron-based gene inactivation system produced the first non-random, targeted mutations in C. cellulolyticum . As a key component of the genetic toolbox for this bacterium, markerless targeted mutagenesis enables functional genomic research in C . cellulolyticum and rapid genetic engineering to significantly alter the mixture of fermentation products. The initial application of this system successfully engineered a strain with high ethanol productivity from cellobiose, cellulose and switchgrass.

Doc number: 71 Abstract 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 its glucoside 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.

Impedance changes of the anode, cathode and solution were examined for an air-cathode microbial fuel cell (MFC) under varying conditions. An MFC inoculated with a pre-enriched microbial culture resulted in a startup time of less than ten days. Over this period, the anode impedance decreased below the cathode impedance, suggesting a cathode-limited power output. Increasing the anode flow rate did not impact the anode impedance significantly, but it decreased the cathode impedance by 65%. Increasing the anode-medium ionic strength also decreased the cathode impedance. These impedance results provide insight into electron and proton transport mechanisms and can be used to improve MFC performance.

Switchgrass ( Panicum virgatum ) is an important biofuel crop candidate thought to have low disease susceptibility. As switchgrass production becomes more prevalent, monoculture and production fields in close proximity to one another may increase the spread and severity of diseases such as switchgrass rust caused by the pathogen Puccinia emaculata . The objective of this research was to examine the impact of rust on ethanol yield in switchgrass. In 2010 and 2012, naturally infected leaves from field-grown ‘Alamo’ and ‘Kanlow’ in Knoxville, TN (2010, 2012) and Crossville, TN (2012) were visually categorized as exhibiting low, medium, or high disease based on the degree of chlorosis and sporulation. P. emaculata was isolated from each disease range to confirm infection. Samples from 2010 were acid/heat pretreated and subjected to two runs of simultaneous saccharification and fermentation (SSF) with Saccharomyces cerevisiae D 5 A to measure ethanol yield. Near-infrared spectroscopy (NIRS) was used to estimate ethanol yield for 2012 samples. SSF and NIRS data were analyzed separately using ANOVA. Disease level effects were significant within both models ( P  < 0.05) and both models explained a large amount of variation in ETOH (SSF: R 2  = 0.99, NIRS: R 2  = 0.99). In the SSF dataset, ethanol was reduced by 35 % in samples exhibiting medium disease symptoms and by 55 % in samples exhibiting high disease symptoms. In the NIRS dataset, estimated ethanol was reduced by 10 % in samples exhibiting medium disease symptoms and by 21 % in samples exhibiting high disease symptoms. Results indicate that switchgrass rust will likely have a negative impact on ethanol yield in switchgrass grown as a biofuel crop.

Consolidated bioprocessing (CBP), which merges enzyme production, biomass hydrolysis, and fermentation into a single step, has the potential to become an efficient and economic strategy for the bioconversion of lignocellulosic feedstocks to transportation fuels or chemicals. In this study, we evaluated wild-type Clostridium thermocellum, Caldicellulosiruptor bescii, and Caldicellulosiruptor obsidiansis, three thermophilic, cellulolytic, mixed-acid fermenting candidate CBP microorganisms, for their fermentation capabilities using dilute acid pretreated Populus as a model biomass feedstock. Under pH-controlled anaerobic fermentation conditions, each candidate successfully digested a minimum of 75 % of the cellulose from dilute acid pretreated Populus, as indicated by an increase in planktonic cells and end-product metabolites and a concurrent decrease in glucan content. C. thermocellum, which employs a cellulosomal approach to biomass degradation, required approximately 50 h to achieve 75 % cellulose utilization. In contrast, the noncellulosomal, secreted hydrolytic enzyme system of the Caldicellulosiruptor sp. required about 100 h after a significant lag phase to achieve similar results. End-point fermentation conversions for C. thermocellum, C. bescii, and C. obsidiansis were determined to be 0.29, 0.34, and 0.38 g of total metabolites per gram of loaded glucan, respectively. These data provide a starting point for future strain engineering efforts that can serve to improve the biomass fermentation capabilities of these three promising candidate CBP platforms.

A biological process for removal of mercury from coal is under investigation. Iron and sulfur oxidizing bacteria have previously been used for desulfurization of coal and for mineral mining. We have shown that removal of mercury from coal is also possible via the same principles. Two pure cultures, Leptospirillum ferrooxidans and Acidithiobacillus ferrooxidans and four environmental consortium samples obtained from an acid mine drainage site were studied for mercury removal from coal. Four different coal samples were included in the study and the preliminary results have shown that up to 20% of the mercury can be removed in batch cultures compared to control. Additional parameters such as media composition and inoculum size were also studied. This is the first report demonstrating successful leaching of mercury from coal using biological treatment.

Switchgrass (Panicum virgatum) is an important biofuel crop candidate thought to have low disease susceptibility. As switchgrass production becomes more prevalent, monoculture and production fields in close proximity to one another may increase the spread and severity of diseases such as switchgrass rust caused by the pathogen Puccinia emaculata. The objective of this research was to examine the impact of rust on ethanol yield in switchgrass. In 2010 and 2012, naturally infected leaves from field-grown 'Alamo' and 'Kanlow' in Knoxville, TN (2010, 2012) and Crossville, TN (2012) were visually categorized as exhibiting low, medium, or high disease based on the degree of chlorosis and sporulation. P. emaculata was isolated from each disease range to confirm infection. Samples from 2010 were acid/heat pretreated and subjected to two runs of simultaneous saccharification and fermentation (SSF) with Saccharomyces cerevisiae D5A to measure ethanol yield. Near-infrared spectroscopy (NIRS) was used to estimate ethanol yield for 2012 samples. SSF and NIRS data were analyzed separately using ANOVA. Disease level effects were significant within both models (P < 0.05) and both models explained a large amount of variation in ETOH (SSF: R2 = 0.99, NIRS: R2 = 0.99). In the SSF dataset, ethanol was reduced by 35 % in samples exhibiting medium disease symptoms and by 55 % in samples exhibiting high disease symptoms. In the NIRS dataset, estimated ethanol was reduced by 10 % in samples exhibiting medium disease symptoms and by 21 % in samples exhibiting high disease symptoms. Lastly, results indicate that switchgrass rust will likely have a negative impact on ethanol yield in switchgrass grown as a biofuel crop.

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. Down-regulation of the switchgrass caffeic acid O-methyltransferase gene decreases lignin content modestly, reduces the syringyl:guaiacyl lignin monomer ratio, improves forage quality, and, most importantly, increases the ethanol yield by up to 38% using conventional biomass fermentation processes. The down-regulated lines require less severe pretreatment and 300–400% lower cellulase dosages for equivalent product yields using simultaneous saccharification and fermentation with yeast. Furthermore, fermentation of diluted acid-pretreated transgenic switchgrass using Clostridium thermocellum with no added enzymes showed better product yields than obtained with unmodified switchgrass. Therefore, this apparent reduction in the recalcitrance of transgenic switchgrass has the potential to lower processing costs for biomass fermentation-derived fuels and chemicals significantly. Alternatively, such modified transgenic switchgrass lines should yield significantly more fermentation chemicals per hectare under identical process conditions.

A biological process for removal of mercury from coal is under investigation. Iron and sulfur oxidizing bacteria have previously been used for desulfurization of coal and for mineral mining. We have shown that removal of mercury from coal is also possible via the same principles. Two pure cultures, Leptospirillum ferrooxidans and Acidithiobacillus ferrooxidans and four environmental consortium samples obtained from an acid mine drainage site were studied for mercury removal from coal. Four different coal samples were included in the study and the preliminary results have shown that up to 20% of the mercury can be removed in batch cultures compared to control. Additional parameters such as media composition and inoculum size were also studied. This is the first report demonstrating successful leaching of mercury from coal using biological treatment.