Recent advances in second generation ethanol production by thermophilic bacteria

There is an increased interest in using thermophilic bacteria for the production of bioethanol from complex lignocellulosic biomass due to their higher operating temperatures and broad substrate range. This review focuses upon the main genera of thermophilic anaerobes known to produce ethanol, their physiology, and the relevance of various environmental factors on ethanol yields including the partial pressure of hydrogen, ethanol tolerance, pH and substrate inhibition. Additionally, recent development in evolutionary adaptation and genetic engineering of thermophilic bacteria is highlighted. Recent developments in advanced process techniques used for ethanol production are reviewed with an emphasis on the advantages of using thermophilic bacteria in process strategies including separate saccharification and fermentation, simultaneous saccharification and fermentation (SSF), and consolidated bioprocessing (CBP).Peer Reviewe

Genome-scale metabolic analysis of Clostridium thermocellum for bioethanol production

<p>Abstract</p> <p>Background</p> <p>Microorganisms possess diverse metabolic capabilities that can potentially be leveraged for efficient production of biofuels. <it>Clostridium thermocellum </it>(ATCC 27405) is a thermophilic anaerobe that is both cellulolytic and ethanologenic, meaning that it can directly use the plant sugar, cellulose, and biochemically convert it to ethanol. A major challenge in using microorganisms for chemical production is the need to modify the organism to increase production efficiency. The process of properly engineering an organism is typically arduous.</p> <p>Results</p> <p>Here we present a genome-scale model of <it>C. thermocellum </it>metabolism, <it>i</it>SR432, for the purpose of establishing a computational tool to study the metabolic network of <it>C. thermocellum </it>and facilitate efforts to engineer <it>C. thermocellum </it>for biofuel production. The model consists of 577 reactions involving 525 intracellular metabolites, 432 genes, and a proteomic-based representation of a cellulosome. The process of constructing this metabolic model led to suggested annotation refinements for 27 genes and identification of areas of metabolism requiring further study. The accuracy of the <it>i</it>SR432 model was tested using experimental growth and by-product secretion data for growth on cellobiose and fructose. Analysis using this model captures the relationship between the reduction-oxidation state of the cell and ethanol secretion and allowed for prediction of gene deletions and environmental conditions that would increase ethanol production.</p> <p>Conclusions</p> <p>By incorporating genomic sequence data, network topology, and experimental measurements of enzyme activities and metabolite fluxes, we have generated a model that is reasonably accurate at predicting the cellular phenotype of <it>C. thermocellum </it>and establish a strong foundation for rational strain design. In addition, we are able to draw some important conclusions regarding the underlying metabolic mechanisms for observed behaviors of <it>C. thermocellum </it>and highlight remaining gaps in the existing genome annotations.</p

Progress in Second Generation Ethanol Production with Thermophilic Bacteria

Thermophilic bacteria have gained increased attention as prospective organisms for bioethanol production from lignocellulosic biomass due to their broad substrate spectra including many of the hexoses pentoses, and disaccharides found in biomass and biomass hydrolysates, fast growth rates, and high tolerance for extreme cultivation conditions. Apart from optimizing the ethanol production by varying physiological parameters, genetic engineering methods have been applied. This review focuses upon those thermophilic anaerobes recognized as being highly ethanologenic, their metabolism, and the importance of various culture parameters affecting ethanol yields, such as the partial pressure of hydrogen, pH, substrate inhibition, and ethanol tolerance. Also, recent developments in evolutionary adaptation and genetic engineering of thermophilic anaerobes are addressed

Exploring complex cellular phenotypes and model-guided strain design with a novel genome-scale metabolic model of Clostridium thermocellum DSM 1313 implementing an adjustable cellulosome

Additional file 1. Model structure and biomass term calculations

Lignocellulosic Biomass Utilization Toward Biorefinery Using Meshophilic Clostridial Species

Lignocellulosic biomass such as agricultural, industrial, and forestry residues as well asdedicated crops constitute renewable and abundant resources with great potential for a lowcostand uniquely sustainable bioconversion to value-added bioproducts. Thus, manyorganic fuels and chemicals that can be obtained from lignocellulosic biomass can reducegreenhouse gas emissions, enhance energy security, improve the economy, dispose ofproblematic solid wastes, and improve air quality. In particular, liquid biofuels are attractivecandidates, since little or no change is needed to the current petroleum-based fueltechnologies. However, the biorefining process remains economically unfeasible due to alack of biocatalysts that can overcome costly hurdles such as cooling from high temperature,pumping of oxygen/stirring, and, neutralization from acidic or basic pH. Therefore,bioconversion of the lignocellulosic components into fermentable sugars is an essential stepin the biorefinery

Characterization and Metabolic Engineering of Transcription Factors and Redox Dynamics in Candidate Consolidated Bioprocessing Biocatalysts

This thesis studies the metabolic engineering of candidate consolidated bioprocessing biocatalyst microorganisms through targeting regulatory genes, with an emphasis on redox metabolism. Consolidated bioprocessing is the single-step hydrolysis and conversion of lignocellulosic material to biofuels. The biocatalysts considered are Clostridium thermocellum and Caldicellulosiruptor bescii, and the primary product of interest is ethanol. Both organisms are thermophilic anaerobic bacteria which encode and express genes that facilitate the deconstruction and solubilization of lignocellulose into fermentable carbohydrates. Furthermore, these organisms ferment these carbohydrates into ethanol, organic acids, as well as other fermentation products. We seek to improve redox metabolism and osmotolerance in these organsisms toward a biorefining objective goal of engineering a biocatalyst capable of facilitating economically viable consolidated bioprocessing.Expression profiling, transcription factor regulon mapping, genetic engineering, and analytical fermentation were approaches employed to assay and understand which specific traits can be beneficially altered. The traits sought to be altered are characteristically complex, co-opting many cellular sub-processes to enable a molecular mechanism resulting in an observable trait. Such traits are notoriously difficult not only to understand, but to alter through classical metabolic engineering. Instead, the possibility of making system-wide changes through a minimal number of genetic alterations to methodically selected and/or screened regulatory genes was investigated.Active redox-dependent systems were characterized in both bacteria, many of which are controlled by the global redox-state sensing transcription factor Rex. Eliminating Rex control over gene expression in C. bescii resulted in a more reduced intracellular redox state, and ultimately drives increased ethanol synthesis. A method for quantifying important redox metabolites intracellularly is also adopted and validated for use with C. thermocellum. This approach was extended to less characterized gene targets and, arguably, even more complex traits. Screening of single-gene deletion mutants identified two strains of C. bescii showing phenotypic growth differences in elevated osmolarity conditions. One strain housed a deletion of the fapR gene, while the other a deletion of the fruR/cra gene. Characterizing these transcription factors and their regulons elucidates mechanisms which this organism uses to facilitate survival at elevated osmolarities. We are also able to construct genetic variants in C. bescii which are substantially more osmotolerant than native strains, highlighting the usefulness of these genes as targets and the applicability, and important considerations, of our metabolic engineering approach

Ethanol production by novel thermophilic anaerobe isolate/S via consolidated bioprocessing

The research work summarizes a very significant, industrially and globally important topic. Particularly the isolation of microorganisms efficient in fermentation at higher than normal temperature and utilizing renewable biomass, is of economic importance. The work presented in this thesis with future perspective has a potential for larger scale consolidated bioprocessing for ethanol production.<br /

Engineered Saccharomyces cerevisiae for lignocellulosic valorization: a review and perspectives on bioethanol production

The biorefinery concept, consisting in using renewable biomass with economical and energy goals, appeared in response to the ongoing exhaustion of fossil reserves. Bioethanol is the most prominent biofuel and has been considered one of the top chemicals to be obtained from biomass. Saccharomyces cerevisiae, the preferred microorganism for ethanol production, has been the target of extensive genetic modifications to improve the production of this alcohol from renewable biomasses. Additionally, S. cerevisiae strains from harsh industrial environments have been exploited due to their robust traits and improved fermentative capacity. Nevertheless, there is still not an optimized strain capable of turning second generation bioprocesses economically viable. Considering this, and aiming to facilitate and guide the future development of effective S. cerevisiae strains, this work reviews genetic engineering strategies envisioning improvements in 2nd generation bioethanol production, with special focus in process-related traits, xylose consumption, and consolidated bioprocessing. Altogether, the genetic toolbox described proves S. cerevisiae to be a key microorganism for the establishment of a bioeconomy, not only for the production of lignocellulosic bioethanol, but also having potential as a cell factory platform for overall valorization of renewable biomasses.This work was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020, the PhD grants [SFRH/BD/ 130739/2017 to CEC; SFRH/BD/146367/2019 to POS; SFRH/ BD/132717/2017 to SLB], the MIT-Portugal Program [PhD Grant PD/BD/128247/2016 to JTC], BioTecNorte operation [NORTE-01-0145-FEDER-000004] and Biomass and Bioenergy Research Infrastructure (BBRI)- LISBOA-01-0145-FEDER- 022059] funded by the European Regional Development Fund (ERDF) under the scope of Norte2020 - Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

Transcriptomic analysis of Clostridium thermocellum ATCC 27405 cellulose fermentation

<p>Abstract</p> <p>Background</p> <p>The ability of C<it>lostridium thermocellum </it>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 <it>C. thermocellum </it>mRNA during growth on crystalline cellulose in controlled replicate batch fermentations.</p> <p>Results</p> <p>A time-series analysis of gene expression revealed changes in transcript levels of ~40% of genes (~1300 out of 3198 ORFs encoded in the genome) during transition from early-exponential to late-stationary phase. K-means clustering of genes with statistically significant changes in transcript levels identified six distinct clusters of temporal expression. Broadly, genes involved in energy production, translation, glycolysis and amino acid, nucleotide and coenzyme metabolism displayed a decreasing trend in gene expression as cells entered stationary phase. In comparison, genes involved in cell structure and motility, chemotaxis, signal transduction and transcription showed an increasing trend in gene expression. Hierarchical clustering of cellulosome-related genes highlighted temporal changes in composition of this multi-enzyme complex during batch growth on crystalline cellulose, with increased expression of several genes encoding hydrolytic enzymes involved in degradation of non-cellulosic substrates in stationary phase.</p> <p>Conclusions</p> <p>Overall, the results suggest that under low substrate availability, growth slows due to decreased metabolic potential and <it>C. thermocellum </it>alters its gene expression to (i) modulate the composition of cellulosomes that are released into the environment with an increased proportion of enzymes than can efficiently degrade plant polysaccharides other than cellulose, (ii) enhance signal transduction and chemotaxis mechanisms perhaps to sense the oligosaccharide hydrolysis products, and nutrient gradients generated through the action of cell-free cellulosomes and, (iii) increase cellular motility for potentially orienting the cells' movement towards positive environmental signals leading to nutrient sources. Such a coordinated cellular strategy would increase its chances of survival in natural ecosystems where feast and famine conditions are frequently encountered.</p