Combined inactivation of the Clostridium cellulolyticum lactate and malate dehydrogenase genes substantially increases ethanol yield from cellulose and switchgrass fermentations

<p>Abstract</p> <p>Background</p> <p>The model bacterium <it>Clostridium cellulolyticum </it>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, H₂and 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 <it>C. cellulolyticum</it>, but targeted mutagenesis has not been reported for this organism, hindering genetic engineering.</p> <p>Results</p> <p>The first targeted gene inactivation system was developed for <it>C. cellulolyticum</it>, based on a mobile group II intron originating from the <it>Lactococcus lactis </it>L1.LtrB intron. This markerless mutagenesis system was used to disrupt both the paralogous <smcaps>L</smcaps>-lactate dehydrogenase (<it>Ccel_2485; ldh</it>) and <smcaps>L</smcaps>-malate dehydrogenase (<it>Ccel_0137; mdh</it>) 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.</p> <p>Conclusions</p> <p>The efficient intron-based gene inactivation system produced the first non-random, targeted mutations in <it>C. cellulolyticum</it>. As a key component of the genetic toolbox for this bacterium, markerless targeted mutagenesis enables functional genomic research in <it>C</it>. <it>cellulolyticum </it>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.</p

A formal model for output multimodal HCI - An Event-B formalization

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Hydrogen production by Clostridium cellulolyticum a cellulolytic and hydrogen-producing bacteria using sugarcane bagasse

Hydrogen (H2) production by Clostridium cellulolyticum was investigated. Anaerobic batch reactors were operated with cellobiose (2 g/L) and pretreated sugarcane bagasse (SCB) (2 g/L) using a hydrothermal system to observe the effects of carbon source on H2 production. Salts (NH4Cl, NaCl, MgCl2 and CaCl2) and vitamins (biotin, nicotinamide, p-aminobenzoic acid, thiamine, pantothenic acid, pyridoxamine, cyanocobalamin, riboflavin, folic and lipoic acid) were supplemented from stock solutions at different volumes percentages, ranging from 0 to 5%. The optimal concentration was 2.5% and the strain used both substrates and produced H2 which was higher for cellobiose (14.9±0.2 mmol/L) than for SCB (7.6±0.2 mmol/L), although the phase was much smaller when SCB (59.9 h) was used in relation to the assay with cellobiose (164 h). H2 was produced from SCB primarily through the fermentation of lactic and acetic acids.(undefined)info:eu-repo/semantics/publishedVersio