Lee R. Lynd

Dartmouth College

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Daniel G. Olson

Oak Ridge National Laboratory

117

Evert K. Holwerda

Dartmouth College

Liang Tian

Nanjing Agricultural University

Adam M. Guss

Oak Ridge National Laboratory

Shuen Hon

Dartmouth College

Marybeth Maloney

Dartmouth College

Mark Laser

Dartmouth College

Robert L. Hettich

Oak Ridge National Laboratory

Richard J. Giannone

Oak Ridge National Laboratory

Xiongjun Shao

Dartmouth College

Cooperative Institutions

Oak Ridge National Laboratory

198

Dartmouth College

148

Bioenergy Life Science (United States)

University of Tennessee at Knoxville

Lawrence Berkeley National Laboratory

National Renewable Energy Laboratory

Joint Genome Institute

United States Department of Energy

Universidade Estadual de Campinas (UNICAMP)

Massachusetts Institute of Technology

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Form and Function of Clostridium thermocellum Biofilms

Applied and Environmental Microbiology (2012)

Alexandru Dumitrache Gideon Wolfaardt Grant D. Allen Steven N. Liss Lee R. Lynd

The importance of bacterial adherence has been acknowledged in microbial lignocellulose conversion studies; however, few reports have described the function and structure of biofilms supported by cellulosic substrates. We investigated the organization, dynamic formation, and carbon flow associated with biofilms of the obligately anaerobic cellulolytic bacterium Clostridium thermocellum 27405. Using noninvasive, in situ fluorescence imaging, we showed biofilms capable of near complete substrate conversion with a characteristic monolayered cell structure without an extracellular polymeric matrix typically seen in biofilms. Cell division at the interface and terminal endospores appeared throughout all stages of biofilm growth. Using continuous-flow reactors with a rate of dilution (2 h(-1)) 12-fold higher than the bacterium's maximum growth rate, we compared biofilm activity under low (44 g/liter) and high (202 g/liter) initial cellulose loading. The average hydrolysis rate was over 3-fold higher in the latter case, while the proportions of oligomeric cellulose hydrolysis products lost from the biofilm were 13.7% and 29.1% of the total substrate carbon hydrolyzed, respectively. Fermentative catabolism was comparable between the two cellulose loadings, with ca. 4% of metabolized sugar carbon being utilized for cell production, while 75.4% and 66.7% of the two cellulose loadings, respectively, were converted to primary carbon metabolites (ethanol, acetic acid, lactic acid, carbon dioxide). However, there was a notable difference in the ethanol-to-acetic acid ratio (g/g), measured to be 0.91 for the low cellulose loading and 0.41 for the high cellulose loading. The results suggest that substrate availability for cell attachment rather than biofilm colonization rates govern the efficiency of cellulose conversion.

10.1128/aem.02563-12

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Citations (56)

Consolidated bioprocessing of cellulosic biomass: an update

Current Opinion in Biotechnology (2005)

Lee R. Lynd Willem H. van Zyl John E. McBride Mark Laser

Cellulosic ethanol

Lignocellulosic Biomass

Metabolic Engineering

10.1016/j.copbio.2005.08.009

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Citations (1,408)

Protein feeds coproduction in biomass conversion to fuels and chemicals

Biofuels Bioproducts and Biorefining (2009)

Bruce E. Dale M.S. Allen Mark Laser Lee R. Lynd

Abstract Agriculture has changed greatly in the past in response to changing human needs. Now agriculture is being called on to provide raw materials for very large‐scale fuel and chemical production. Agriculture will change again in response to this demand and all producers and users of agricultural feedstocks will be affected by this change. For example, livestock feeding practices have already changed in response to the availability of distillers' grains from corn ethanol production. A fuels industry based on herbaceous biomass energy crops will be many‐fold larger than the existing corn ethanol industry and will produce its own set of impacts on livestock feeding. We explore here one of these impacts: the availability of large new sources of feed protein from biomass energy crops. In addition to structural carbohydrates, such as cellulose and hemicellulose, herbaceous biomass energy crops can easily be produced with approximately 10% protein, called ‘leaf protein’. This leaf protein, as exemplified by alfalfa leaf protein, is superior to soybean meal (SBM) protein in its biological value. Leaf protein recovery and processing fit well into many process flow diagrams for biomass fuels. When leaf protein is properly processed to concentrate it and remove antinutritional factors, as we have learned over the years to do with soybean meal protein, protein in leaf protein concentrate (LPC) will probably be at least as valuable in livestock diets as SBM protein. If LPC is used to meet 20% of total animal protein requirements (i.e., market penetration of 20%) then the potential utilization of leaf protein concentrate could reach as much as 24 million metric tons annually. This leaf protein will replace protein from SBM and other sources. This much leaf protein will reduce by approximately 16 million hectares the amount of land required to provide protein for livestock. Likewise the amount of land required to meet fuel needs will effectively be reduced by 8 million hectares because this land will effectively do ‘double duty’ by producing needed animal protein as well as feedstocks for fuel production. © 2009 Society of Chemical Industry and John Wiley & Sons, Ltd

Hemicellulose

Distillers grains

Animal Feed

Energy crop

10.1002/bbb.132

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Citations (106)

Contribution of Scaffoldins to Biomass Degradation by Clostridium Thermocellum: The Effect of Scaffoldin-Deletions on Expression of Other Genes

36th Symposium on Biotechnology for Fuels and Chemicals (April 28-May 1, 2014) (2014)

Qi Xu Kara Podkaminer Michael G. Resch Bryon S. Donohoe Daniel G. Olson

The cellulosome system contributes greatly to the extreme efficiency of C. thermocellum cellulose degradation. In order to further understand the cellulosome working mechanism, we have knocked out C. thermocellum scaffoldin genes to generate a variety of deletion mutants. The knockout most detrimental to enzymatic hydrolysis by the secretome is that of the primary scaffoldin CipA. Deletion of multiple secondary scaffoldins results in secretome activities intermediate between those of the parent strain and the CipA-knockout mutants. The order of relative secretome activities is the same, whether the cellulosic substrate is microcrystalline cellulose (Avicel) or deacetylated acid-pretreated corn stover (DACS), but the relative magnitudes of the deletion effects are strongly substrate-dependent. Similar trends are observed in fermentation studies of the abilities of the parent and knockout strains themselves to utilize Avicel and DACS. Data from transcriptomic and proteomic studies of these strains when grown on both substrates are used to relate the activity and growth effects of the deletions to their effects on the overall expression of lignocellulose-degrading enzymes by C. thermocellum.

Cellulosome

Cellulosic ethanol

Source

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Genome-scale resources for Thermoanaerobacterium saccharolyticum

BMC Systems Biology (2015)

Devin H. Currie Babu Raman Christopher M. Gowen Timothy J. Tschaplinski Miriam Land

Thermoanaerobacterium saccharolyticum is a hemicellulose-degrading thermophilic anaerobe that was previously engineered to produce ethanol at high yield. A major project was undertaken to develop this organism into an industrial biocatalyst, but the lack of genome information and resources were recognized early on as a key limitation.Here we present a set of genome-scale resources to enable the systems level investigation and development of this potentially important industrial organism. Resources include a complete genome sequence for strain JW/SL-YS485, a genome-scale reconstruction of metabolism, tiled microarray data showing transcription units, mRNA expression data from 71 different growth conditions or timepoints and GC/MS-based metabolite analysis data from 42 different conditions or timepoints. Growth conditions include hemicellulose hydrolysate, the inhibitors HMF, furfural, diamide, and ethanol, as well as high levels of cellulose, xylose, cellobiose or maltodextrin. The genome consists of a 2.7 Mbp chromosome and a 110 Kbp megaplasmid. An active prophage was also detected, and the expression levels of CRISPR genes were observed to increase in association with those of the phage. Hemicellulose hydrolysate elicited a response of carbohydrate transport and catabolism genes, as well as poorly characterized genes suggesting a redox challenge. In some conditions, a time series of combined transcription and metabolite measurements were made to allow careful study of microbial physiology under process conditions. As a demonstration of the potential utility of the metabolic reconstruction, the OptKnock algorithm was used to predict a set of gene knockouts that maximize growth-coupled ethanol production. The predictions validated intuitive strain designs and matched previous experimental results.These data will be a useful asset for efforts to develop T. saccharolyticum for efficient industrial production of biofuels. The resources presented herein may also be useful on a comparative basis for development of other lignocellulose degrading microbes, such as Clostridium thermocellum.

Xylose metabolism

Hemicellulose

10.1186/s12918-015-0159-x

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Citations (26)

Simultaneous saccharification and co‐fermentation of paper sludge to ethanol by Saccharomyces cerevisiae RWB222. Part II: Investigation of discrepancies between predicted and observed performance at high solids concentration

Biotechnology and Bioengineering (2009)

Jiayi Zhang Xiongjun Shao Lee R. Lynd

The simultaneous saccharification and co-fermentation (SSCF) kinetic model described in the companion paper can predict batch and fed batch fermentations well at solids concentrations up to 62.4 g/L cellulose paper sludge but not in batch fermentation at 82.0 g/L cellulose paper sludge. Four hypotheses for the discrepancy between observation and model prediction at high solids concentration were examined: ethanol inhibition, enzyme deactivation, inhibition by non-metabolizable compounds present in paper sludge, and mass transfer limitation. The results show that mass transfer limitation was responsible for the discrepancy between model and experimental data. The model can predict the value of high paper sludge SSCF in the fermentation period with no mass transfer limitation. The model predicted that maximum ethanol production of fed-batch fermentation was achieved when it was run as close to batch mode as possible with the initial solids loading below the mass transfer limitation threshold. A method for measuring final enzyme activity at the end of fermentation was also developed in this study.

Volatile suspended solids

Enzymatic Hydrolysis

Ethanol Fermentation

10.1002/bit.22465

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Citations (31)

MOESM9 of Enhanced ethanol formation by Clostridium thermocellum via pyruvate decarboxylase

Figshare (2017)

Liang Tian Skyler Perot Shuen Hon Jilai Zhou Xiaoyu Liang

Additional file 9. Assay of plasmid loss by CFU count.

Pyruvate decarboxylase

10.6084/m9.figshare.c.3897049_d12

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Citations (0)

Kinetics and Relative Importance of Phosphorolytic and Hydrolytic Cleavage of Cellodextrins and Cellobiose in Cell Extracts of Clostridium thermocellum

Applied and Environmental Microbiology (2004)

Y.‐H. Percival Zhang Lee R. Lynd

ABSTRACT Rates of phosphorolytic cleavage of β-glucan substrates were determined for cell extracts from Clostridium thermocellum ATCC 27405 and were compared to rates of hydrolytic cleavage. Reactions with cellopentaose and cellobiose were evaluated for both cellulose (Avicel)- and cellobiose-grown cultures, with more limited data also obtained for cellotetraose. To measure the reaction rate in the chain-shortening direction at elevated temperatures, an assay protocol was developed featuring discrete sampling at 60°C followed by subsequent analysis of reaction products (glucose and glucose-1-phosphate) at 35°C. Calculated rates of phosphorolytic cleavage for cell extract from Avicel-grown cells exceeded rates of hydrolytic cleavage by ≥20-fold for both cellobiose and cellopentaose over a 10-fold range of β-glucan concentrations (0.5 to 5 mM) and for cellotetraose at a single concentration (2 mM). Rates of phosphorolytic cleavage of β-glucosidic bonds measured in cell extracts were similar to rates observed in growing cultures. Comparisons of V max values indicated that cellobiose- and cellodextrin-phosphorylating activities are synthesized during growth on both cellobiose and Avicel but are subject to some degree of metabolic control. The apparent K m for phosphorolytic cleavage was lower for cellopentaose (mean value for Avicel- and cellobiose-grown cells, 0.61 mM) than for cellobiose (mean value, 3.3 mM).

Cleavage (geology)

10.1128/aem.70.3.1563-1569.2004

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Citations (98)

Analysis of conversion of particulate biomass to ethanol in continuous solids retaining and cascade bioreactors

Applied Biochemistry and Biotechnology (1994)

Colin R. South Lee R. Lynd

Dilution

Residence time distribution

Batch reactor

Continuous reactor

10.1007/bf02941822

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Citations (22)

Characterization of sugarcane bagasse solubilization and utilization by thermophilic cellulolytic and saccharolytic bacteria at increasing solid loadings

Bioresource Technology (2024)

Isabela U. Zambello Evert K. Holwerda Lee R. Lynd

In Brazil the main feedstock used for ethanol production is sugarcane juice, resulting in large amounts of bagasse. Bagasse has high potential for cellulosic ethanol production, and consolidated bioprocessing (CBP) has potential for lowering costs. However, economic feasibility requires bioprocessing at high solids loadings, entailing engineering and biological challenges. This study aims to document and characterize carbohydrate solubilization and utilization by defined cocultures of Clostridium thermocellum and Thermoanaerobacterium thermosaccharolyticum at increasing loadings of sugarcane bagasse. Results show that fractional carbohydrate solubilization decreases as solids loading increases from 10 g/L to 80 g/L. Cocultures enhance solubilization and carbohydrate utilization compared to monocultures, irrespective of initial solids loading. Rinsing bagasse before fermentation slightly decreases solubilization. Experiments studying inhibitory effects using spent media and dilution of broth show that negative effects are temporary or reversible. These findings highlight the potential of converting sugarcane bagasse via CBP, pointing out performance limitations that must be addressed.

Bagasse

Cellulosic ethanol

10.1016/j.biortech.2024.130973

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Citations (1)