The Performance Of Clostridium Phytofermentans For Biofuels Production From Lignocellulosic Biomass

Ethanol produced from lignocellulosic biomass is an alternative transportation fuel with the potential to lower greenhouse gas emissions and increase energy security. Source-separated organic waste (SSO) from the city of Toronto was investigated for feasibility as a lignocellulosic ethanol feedstock. Clostridium phytofermentans is a mesophilic, cellulolytic and ethanologenic species with potential application for ethanol production from lignocullulosic biomass. C. phytofermentans was considered for biofuels production through experiments on a variety of substrates including soluble sugars and pure celluloses. Results from this study found that C. phytofermentans produced 73% of the theoretical ethanol yield on cellobiose but grew poorly on glucose and xylose. In addition, C. phytofermentans grew marginally on microcrystalline and ball-milled cellulose, but with supplemental enzymes produced 55% of the theoretical ethanol yield.

The Performance Of Clostridium Phytofermentans For Biofuels Production From Lignocellulosic Biomass

Ethanol produced from lignocellulosic biomass is an alternative transportation fuel with the potential to lower greenhouse gas emissions and increase energy security. Source-separated organic waste (SSO) from the city of Toronto was investigated for feasibility as a lignocellulosic ethanol feedstock. Clostridium phytofermentans is a mesophilic, cellulolytic and ethanologenic species with potential application for ethanol production from lignocullulosic biomass. C. phytofermentans was considered for biofuels production through experiments on a variety of substrates including soluble sugars and pure celluloses. Results from this study found that C. phytofermentans produced 73% of the theoretical ethanol yield on cellobiose but grew poorly on glucose and xylose. In addition, C. phytofermentans grew marginally on microcrystalline and ball-milled cellulose, but with supplemental enzymes produced 55% of the theoretical ethanol yield.

A Targetron-Recombinase System for Large-Scale Genome Engineering of Clostridia

ABSTRACT Clostridia are a group of Gram-positive anaerobic bacteria of medical and industrial importance for which limited genetic methods are available. Here, we demonstrate an approach to make large genomic deletions and insertions in the model Clostridium phytofermentans by combining designed group II introns (targetrons) and Cre recombinase. We apply these methods to delete a 50-gene prophage island by programming targetrons to position markerless lox66 and lox71 sites, which mediate deletion of the intervening 39-kb DNA region using Cre recombinase. Gene expression and growth of the deletion strain showed that the prophage genes contribute to fitness on nonpreferred carbon sources. We also inserted an inducible fluorescent reporter gene into a neutral genomic site by recombination-mediated cassette exchange (RMCE) between genomic and plasmid-based tandem lox sites bearing heterospecific spacers to prevent intracassette recombination. These approaches generally enable facile markerless genome engineering in clostridia to study their genome structure and regulation. IMPORTANCE Clostridia are anaerobic bacteria with important roles in intestinal and soil microbiomes. The inability to experimentally modify the genomes of clostridia has limited their study and application in biotechnology. Here, we developed a targetron-recombinase system to efficiently make large targeted genomic deletions and insertions using the model Clostridium phytofermentans. We applied this approach to reveal the importance of a prophage to host fitness and introduce an inducible reporter by recombination-mediated cassette exchange.

Evolution of a Biomass-Fermenting Bacterium To Resist Lignin Phenolics

ABSTRACT Increasing the resistance of plant-fermenting bacteria to lignocellulosic inhibitors is useful to understand microbial adaptation and to develop candidate strains for consolidated bioprocessing. Here, we study and improve inhibitor resistance in Clostridium phytofermentans (also called Lachnoclostridium phytofermentans), a model anaerobe that ferments lignocellulosic biomass. We survey the resistance of this bacterium to a panel of biomass inhibitors and then evolve strains that grow in increasing concentrations of the lignin phenolic, ferulic acid, by automated, long-term growth selection in an anaerobic GM3 automat. Ultimately, strains resist multiple inhibitors and grow robustly at the solubility limit of ferulate while retaining the ability to ferment cellulose. We analyze genome-wide transcription patterns during ferulate stress and genomic variants that arose along the ferulate growth selection, revealing how cells adapt to inhibitors through changes in gene dosage and regulation, membrane fatty acid structure, and the surface layer. Collectively, this study demonstrates an automated framework for in vivo directed evolution of anaerobes and gives insight into the genetic mechanisms by which bacteria survive exposure to chemical inhibitors. IMPORTANCE Fermentation of plant biomass is a key part of carbon cycling in diverse ecosystems. Further, industrial biomass fermentation may provide a renewable alternative to fossil fuels. Plants are primarily composed of lignocellulose, a matrix of polysaccharides and polyphenolic lignin. Thus, when microorganisms degrade lignocellulose to access sugars, they also release phenolic and acidic inhibitors. Here, we study how the plant-fermenting bacterium Clostridium phytofermentans resists plant inhibitors using the lignin phenolic, ferulic acid. We examine how the cell responds to abrupt ferulate stress by measuring changes in gene expression. We evolve increasingly resistant strains by automated, long-term cultivation at progressively higher ferulate concentrations and sequence their genomes to identify mutations associated with acquired ferulate resistance. Our study develops an inhibitor-resistant bacterium that ferments cellulose and provides insights into genomic evolution to resist chemical inhibitors.

Direct Image-Based Enumeration of Clostridium phytofermentans Cells on Insoluble Plant Biomass Growth Substrates

ABSTRACTA dual-fluorescent-dye protocol to visualize and quantifyClostridium phytofermentansISDg (ATCC 700394) cells growing on insoluble cellulosic substrates was developed by combining calcofluor white staining of the growth substrate with cell staining using the nucleic acid dye Syto 9. Cell growth, cell substrate attachment, and fermentation product formation were investigated in cultures containing either Whatman no. 1 filter paper, wild-typeSorghum bicolor, or a reduced-ligninS. bicolordouble mutant (bmr-6 bmr-12double mutant) as the growth substrate. After 3 days of growth, cell numbers in cultures grown on filter paper as the substrate were 6.0- and 2.2-fold higher than cell numbers in cultures with wild-type sorghum and double mutant sorghum, respectively. However, cells produced more ethanol per cell when grown with either sorghum substrate than with filter paper as the substrate. Ethanol yields of cultures were significantly higher with double mutant sorghum than with wild-type sorghum or filter paper as the substrate. Moreover, ethanol production correlated with cell attachment in sorghum cultures: 90% of cells were directly attached to the double mutant sorghum substrate, while only 76% of cells were attached to wild-type sorghum substrate. With filter paper as the growth substrate, ethanol production was correlated with cell number; however, with either wild-type or mutant sorghum, ethanol production did not correlate with cell number, suggesting that only a portion of the microbial cell population was active during growth on sorghum. The dual-staining procedure described here may be used to visualize and enumerate cells directly on insoluble cellulosic substrates, enabling in-depth studies of interactions of microbes with plant biomass.

Physiology, Genomics, and Pathway Engineering of an Ethanol-Tolerant Strain of Clostridium phytofermentans

ABSTRACTNovel processing strategies for hydrolysis and fermentation of lignocellulosic biomass in a single reactor offer large potential cost savings for production of biocommodities and biofuels. One critical challenge is retaining high enzyme production in the presence of elevated product titers. Toward this goal, the cellulolytic, ethanol-producing bacteriumClostridium phytofermentanswas adapted to increased ethanol concentrations. The resulting ethanol-tolerant (ET) strain has nearly doubled ethanol tolerance relative to the wild-type level but also reduced ethanol yield and growth at low ethanol concentrations. The genome of the ET strain has coding changes in proteins involved in membrane biosynthesis, the Rnf complex, cation homeostasis, gene regulation, and ethanol production. In particular, purification of the mutant bifunctional acetaldehyde coenzyme A (CoA)/alcohol dehydrogenase showed that a G609D variant abolished its activities, including ethanol formation. Heterologous expression ofZymomonas mobilispyruvate decarboxylase and alcohol dehydrogenase in the ET strain increased cellulose consumption and restored ethanol production, demonstrating how metabolic engineering can be used to overcome disadvantageous mutations incurred during adaptation to ethanol. We discuss how genetic changes in the ET strain reveal novel potential strategies for improving microbial solvent tolerance.