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      The effect of switchgrass loadings on feedstock solubilization and biofuel production by Clostridium thermocellum

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          Abstract

          Background

          Efficient deconstruction and bioconversion of solids at high mass loadings is necessary to produce industrially relevant titers of biofuels from lignocellulosic biomass. To date, only a few studies have investigated the effect of solids loadings on microorganisms of interest for consolidated bioprocessing. Here, the effects that various switchgrass loadings have on Clostridium thermocellum solubilization and bioconversion are investigated.

          Results

          Clostridium thermocellum was grown for 10 days on 10, 25, or 50 g/L switchgrass or Avicel at equivalent glucan loadings. Avicel was completely consumed at all loadings, but total cellulose solubilization decreased from 63 to 37% as switchgrass loadings increased from 10 to 50 g/L. Washed, spent switchgrass could be additionally hydrolyzed and fermented in second-round fermentations suggesting that access to fermentable substrates was not the limiting factor at higher feedstock loadings. Results from fermentations on Avicel or cellobiose using culture medium supplemented with 50% spent fermentation broth demonstrated that compounds present in the supernatants from the 25 or 50 g/L switchgrass loadings were the most inhibitory to continued fermentation.

          Conclusions

          Recalcitrance alone cannot fully account for differences in solubilization and end-product formation between switchgrass and Avicel at increased substrate loadings. Experiments aimed at separating metabolic inhibition from inhibition of hydrolysis suggest that C. thermocellum’s hydrolytic machinery is more vulnerable to inhibition from switchgrass-derived compounds than its fermentative metabolism.

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          Most cited references46

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          Yield-determining factors in high-solids enzymatic hydrolysis of lignocellulose

          Background Working at high solids (substrate) concentrations is advantageous in enzymatic conversion of lignocellulosic biomass as it increases product concentrations and plant productivity while lowering energy and water input. However, for a number of lignocellulosic substrates it has been shown that at increasing substrate concentration, the corresponding yield decreases in a fashion which can not be explained by current models and knowledge of enzyme-substrate interactions. This decrease in yield is undesirable as it offsets the advantages of working at high solids levels. The cause of the 'solids effect' has so far remained unknown. Results The decreasing conversion at increasing solids concentrations was found to be a generic or intrinsic effect, describing a linear correlation from 5 to 30% initial total solids content (w/w). Insufficient mixing has previously been shown not to be involved in the effect. Hydrolysis experiments with filter paper showed that neither lignin content nor hemicellulose-derived inhibitors appear to be responsible for the decrease in yields. Product inhibition by glucose and in particular cellobiose (and ethanol in simultaneous saccharification and fermentation) at the increased concentrations at high solids loading plays a role but could not completely account for the decreasing conversion. Adsorption of cellulases was found to decrease at increasing solids concentrations. There was a strong correlation between the decreasing adsorption and conversion, indicating that the inhibition of cellulase adsorption to cellulose is causing the decrease in yield. Conclusion Inhibition of enzyme adsorption by hydrolysis products appear to be the main cause of the decreasing yields at increasing substrate concentrations in the enzymatic decomposition of cellulosic biomass. In order to facilitate high conversions at high solids concentrations, understanding of the mechanisms involved in high-solids product inhibition and adsorption inhibition must be improved.
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            Xylooligomers are strong inhibitors of cellulose hydrolysis by enzymes.

            Typically, the enzymatic hydrolysis rate of lignocellulosic biomass is fast initially but then slows down more rapidly than can be explained by just consumption of substrate. Although several factors including enzyme inhibition, enzyme deactivation, a drop in substrate reactivity, or nonproductive binding of enzyme to lignin could be responsible for this loss of effectiveness, we recently reported evidence that xylose, xylan, and xylooligomers dramatically decrease conversion rates and yields, but clarification was still needed for the magnitude of their effect. Therefore, in this study, xylan and various xylooligomers were added to Avicel hydrolysis at low enzyme loadings and found to have a greater effect than adding equal amounts of xylose derived from these materials or when added separately. Furthermore, xylooligomers were more inhibitory than xylan or xylose in terms of a decreased initial hydrolysis rate and a lower final glucose yield even for a low concentration of 1.67 mg/ml. At a higher concentration of 12.5mg/ml, xylooligomers lowered initial hydrolysis rates of Avicel by 82% and the final hydrolysis yield by 38%. Mixed DP xylooligomers showed strong inhibition on cellulase enzymes but not on beta-glucosidase enzymes. By tracking the profile change of xylooligomers, a large portion of the xylooligomers was found to be hydrolyzed by Spezyme CP enzyme preparations, indicating competitive inhibition by mixed xylooligomers. A comparison among glucose sugars and xylose sugars also showed that xylooligomers were more powerful inhibitors than well-established glucose and cellobiose. Copyright (c) 2010 Elsevier Ltd. All rights reserved.
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              High ethanol titers from cellulose by using metabolically engineered thermophilic, anaerobic microbes.

              This work describes novel genetic tools for use in Clostridium thermocellum that allow creation of unmarked mutations while using a replicating plasmid. The strategy employed counter-selections developed from the native C. thermocellum hpt gene and the Thermoanaerobacterium saccharolyticum tdk gene and was used to delete the genes for both lactate dehydrogenase (Ldh) and phosphotransacetylase (Pta). The Δldh Δpta mutant was evolved for 2,000 h, resulting in a stable strain with 40:1 ethanol selectivity and a 4.2-fold increase in ethanol yield over the wild-type strain. Ethanol production from cellulose was investigated with an engineered coculture of organic acid-deficient engineered strains of both C. thermocellum and T. saccharolyticum. Fermentation of 92 g/liter Avicel by this coculture resulted in 38 g/liter ethanol, with acetic and lactic acids below detection limits, in 146 h. These results demonstrate that ethanol production by thermophilic, cellulolytic microbes is amenable to substantial improvement by metabolic engineering.
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                Author and article information

                Contributors
                tobinjames.verbeke@ucalgary.ca
                Gabriela.Garcia@tufts.edu
                elkinsjg@ornl.gov
                Journal
                Biotechnol Biofuels
                Biotechnol Biofuels
                Biotechnology for Biofuels
                BioMed Central (London )
                1754-6834
                30 November 2017
                30 November 2017
                2017
                : 10
                : 233
                Affiliations
                [1 ]ISNI 0000 0004 0446 2659, GRID grid.135519.a, BioEnergy Science Center, , Oak Ridge National Laboratory, ; Oak Ridge, TN 37831-6038 USA
                [2 ]ISNI 0000 0004 0446 2659, GRID grid.135519.a, Biosciences Division, , Oak Ridge National Laboratory, ; Oak Ridge, TN 37831-6038 USA
                [3 ]ISNI 0000 0004 1936 7697, GRID grid.22072.35, Present Address: Department of Biological Sciences, , University of Calgary, ; Calgary, AB T2N 1N4 Canada
                Article
                917
                10.1186/s13068-017-0917-7
                5708108
                da448a9d-1530-4e3c-8e05-0e1d7c72cc71
                © The Author(s) 2017

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 13 June 2017
                : 8 August 2017
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/100006206, Biological and Environmental Research;
                Award ID: DE-PS02-06ER64304
                Categories
                Research
                Custom metadata
                © The Author(s) 2017

                Biotechnology
                clostridium thermocellum,consolidated bioprocessing,switchgrass,recalcitrance,inhibition,high-solid loading,ethanol

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