Life Sciences Commons^™

Exploring The Function Of Gt2 In Physcomitrella Patens, Tess Scavuzzo-Duggan

Senior Honors Projects

Plant cell walls are composed of a variety of carbohydrates, among them cellulose, pectin and hemicellulose. Cellulose is deposited in the cell wall as microfibrils via cellulose synthesis complexes (CSCs). These complexes contain the cellulose synthase proteins (CESAs) and come in two different morphological forms: rosettes and linear complexes. Rosette shaped cellulose synthesis complexes occur in land plants, whilst linear complexes are commonly found in red algae. However, some land plants, notably bryophytes (mosses) and seedless vascular plants, contain genes that encode both CESAs of the type that form rosette CSCs and also genes similar to those found in red …

Combined Inactivation Of The Clostridium Cellulolyticum Lactate And Malate Dehydrogenase Genes Substantially Increases Ethanol Yield From Cellulose And Switchgrass Fermentations, Yongchao Li, Timothy J. Tschaplinski, Nancy L. Engle, Choo Y. Hamilton, Miquel Rodriguez, James C. Liao, Christopher W. Schadt, Adam M. Guss, Yunfeng Yang, David E. Graham

Microbiology Publications and Other Works

Background

The model bacterium Clostridium cellulolyticum 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, H₂ and 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 C. cellulolyticum, but targeted mutagenesis has not been reported for this organism, hindering genetic engineering.

Results …

Enhanced Microbial Utilization Of Recalcitrant Cellulose By An Ex Vivo Cellulosome-Microbe Complex, Chun You, Xiao-Zhou Zhang, Noppadon Sathitsuksanoh, Lee R. Lynd

Dartmouth Scholarship

A cellulosome-microbe complex was assembled ex vivo on the surface of Bacillus subtilis displaying a miniscaffoldin that can bind with three dockerin-containing cellulase components: the endoglucanase Cel5, the processive endoglucanase Cel9, and the cellobiohydrolase Cel48. The hydrolysis performances of the synthetic cellulosome bound to living cells, the synthetic cellulosome, a noncomplexed cellulase mixture with the same catalytic components, and a commercial fungal enzyme mixture were investigated on low-accessibility recalcitrant Avicel and high accessibility regenerated amorphous cellulose (RAC). The cellbound cellulosome exhibited 4.5- and 2.3-fold-higher hydrolysis ability than cell-free cellulosome on Avicel and RAC, respectively. The cellulosome-microbe synergy was not completely …

High Ethanol Titers From Cellulose By Using Metabolically Engineered Thermophilic, Anaerobic Microbes, D. Aaron Argyros, Shital A. Tripathi, Trisha F. Barrett, Stephen R. Rogers, Lawrence F. Feinberg, Daniel G. Olson, Justin M. Foden, Bethany B. Miller, Lee R. Lynd, David A. Hogsett, Nicky C. Caiazza

Dartmouth Scholarship

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 …

Single Molecule Analysis Of The Arabidopsis Fra1 Kinesin Shows That It Is A Functional Motor Protein With Unusually High Processivity, Chuanmei Zhu, Ram Dixit

Biology Faculty Publications & Presentations

The Arabidopsis FRA1 kinesin contributes to the organization of cellulose microfibrils through an unknown mechanism. The cortical localization of this kinesin during interphase raises the possibility that it transports cell wall-related cargoes along cortical microtubules that either directly or indirectly influence cellulose microfibril patterning. To determine whether FRA1 is an authentic motor protein, we combined bulk biochemical assays and single molecule fluorescence imaging to analyze the motor properties of recombinant, GFP-tagged FRA1 containing the motor and coiled-coil domains (designated as FRA1(707)–GFP). We found that FRA1(707)–GFP binds to microtubules in an ATP-dependent manner and that its ATPase activity is dramatically stimulated …

Determining The Composition Of The Dwelling Tubes Of Antarctic Pterobranchs, Lukasz J. Sewera

Honors Projects

Pterobranchs are a group of marine invertebrates within the Hemichordata, which share characteristics with both chordates and echinoderms. Pterobranchs live in colonies of secreted tubes, coenicia, which are composed of a gelatinous material of unknown composition. Visually, the tubes appear similar to the tunic of tunicates, a group of invertebrates within the Chordata. The nonproteinaceous tunic of tunicates is composed of cellulose, which is unusual. The goal of this study was to determine the composition of the pterobranch coenicium. Some aspects of pterobranch phylogeny are still unclear even after multiple molecular and morphological studies. Identification of any new shared characteristics …

Cellulose- And Xylan-Degrading Thermophilic Anaerobic Bacteria From Biocompost, M. V. Sizova, J. A. Izquierdo, N. S. Panikov, L. R. Lynd

Dartmouth Scholarship

Nine thermophilic cellulolytic clostridial isolates and four other noncellulolytic bacterial isolates were isolated from self-heated biocompost via preliminary enrichment culture on microcrystalline cellulose. All cellulolytic isolates grew vigorously on cellulose, with the formation of either ethanol and acetate or acetate and formate as principal fermentation products as well as lactate and glycerol as minor products. In addition, two out of nine cellulolytic strains were able to utilize xylan and pretreated wood with roughly the same efficiency as for cellulose. The major products of xylan fermentation were acetate and formate, with minor contributions of lactate and ethanol. Phylogenetic analyses of 16S …

Quantitative Analysis Of Lignocellulosic Components Of Non-Treated And Steam Exploded Barley, Canola, Oat And Wheat Straw Using Fourier Transform Infrared Spectroscopy, P. K. Adapa, L. G. Schonenau, Thomas Canam, T. Dumonceaux

Thomas Canam

Rapid and cost effective quantification of lignocellulosic components (cellulose, hemicelluloses and lignin) of agricultural biomass (barley, canola, oat and wheat) is essential to determine the effect of various pre-treatments (such as steam explosion) on biomass used as feedstock for the biofuel industry. Fourier Transformed Infrared (FTIR) spectroscopy was considered as an option to achieve this objective. Regression equations having R2 values of 0.89, 0.99 and 0.98 were developed to predict the cellulose, hemicelluloses and lignin compounds of biomass, respectively. The average absolute difference in predicted and measured cellulose, hemicellulose and lignin in agricultural biomass was 7.5%, 2.5%, and 3.8%, respectively.

Quantitative Analysis Of Lignocellulosic Components Of Non-Treated And Steam Exploded Barley, Canola, Oat And Wheat Straw Using Fourier Transform Infrared Spectroscopy, P. Adapa, L. Schonenau, Thomas Canam, T. Dumonceaux

Faculty Research & Creative Activity

Rapid and cost effective quantification of lignocellulosic components (cellulose, hemicelluloses and lignin) of agricultural biomass (barley, canola, oat and wheat) is essential to determine the effect of various pre-treatments (such as steam explosion) on biomass used as feedstock for the biofuel industry. Fourier Transformed Infrared (FTIR) spectroscopy was considered as an option to achieve this objective. Regression equations having R2 values of 0.89, 0.99 and 0.98 were developed to predict the cellulose, hemicelluloses and lignin compounds of biomass, respectively. The average absolute difference in predicted and measured cellulose, hemicellulose and lignin in agricultural biomass was 7.5%, 2.5%, and 3.8%, respectively.

Quantitative Analysis Of Lignocellulosic Components Of Non-Treated And Steam Exploded Barley, Canola, Oat And Wheat Straw Using Fourier Transform Infrared Spectroscopy, P. K. Adapa, L. G. Schonenau, Thomas Canam, T. Dumonceaux

Faculty Research & Creative Activity

Rapid and cost effective quantification of lignocellulosic components (cellulose, hemicelluloses and lignin) of agricultural biomass (barley, canola, oat and wheat) is essential to determine the effect of various pre-treatments (such as steam explosion) on biomass used as feedstock for the biofuel industry. Fourier Transformed Infrared (FTIR) spectroscopy was considered as an option to achieve this objective. Regression equations having R2 values of 0.89, 0.99 and 0.98 were developed to predict the cellulose, hemicelluloses and lignin compounds of biomass, respectively. The average absolute difference in predicted and measured cellulose, hemicellulose and lignin in agricultural biomass was 7.5%, 2.5%, and 3.8%, respectively.

Diversity Of Bacteria And Glycosyl Hydrolase Family 48 Genes In Cellulolytic Consortia Enriched From Thermophilic Biocompost, Javier A. Izquierdo, Maria V. Sizova, Lee R. Lynd

Dartmouth Scholarship

The enrichment from nature of novel microbial communities with high cellulolytic activity is useful in the identification of novel organisms and novel functions that enhance the fundamental understanding of microbial cellulose degradation. In this work we identify predominant organisms in three cellulolytic enrichment cultures with thermophilic compost as an inoculum. Community structure based on 16S rRNA gene clone libraries featured extensive representation of clostridia from cluster III, with one cluster sharing as little as 73% sequence similarity with the closest known relative. In all, 14 new GHF48 gene sequences were added to the known diversity of 35 genes from cultured …

Development Of A Ligno-Cellulosic Polymeric And Reinforced Sheet Molding Compound (Smc), Ryan Harris Mills

Electronic Theses and Dissertations

The overall objective of this dissertation was to study the surface energy and acid-base characteristics of natural fibers, glass, a wood extract, and a sheet molding compound prepreg to facilitate the fabrication of totally synthetic and partially renewable sheet molding compounds (SMCs). The water absorption and micro-mechanical performance of the totally synthetic and partially renewable SMC composites were compared through accelerated aging experiments. Reinforcing glass sized for polyester, bast kenaf fibers, hot water extract from Acer rubrum, and a dicyclopentadiene modified polyester prepreg were analyzed by inverse gas chromatography to evaluate and help predict how the various components may interact …

Decomposition Of Bt And Non-Bt Corn Hybrid Residues In The Field, David D. Tarkalson, Stephen D. Kachman, Johannes M. H. Knops, Janice E. Thies, Charles S. Wortmann

Department of Agronomy and Horticulture: Faculty Publications

Results of a previous laboratory study indicated that six transgenic crops expressing the Cry1Ab insecticidal protein from Bacillus thuringiensis (Bt) decomposed at a slower rate than their respective non-Bt isolines. Consequently, litter decomposition rates, nitrogen cycling, and carbon pools may change in agricultural systems as the result of the widespread use of Bt crops. In this study, we assessed the decomposition rates and chemical composition of commonly grown hybrids of Bt and non-Bt isolines of corn (Zea mays L.) in the field. Leaves, stalks, and cobs from two Bt corn hybrids (Pioneer 34N44 Bt and NC+ 4990 Bt) and their …

Over-Expression Of Udp-Glucose Pyrophosphorylase In Hybrid Poplar Affects Carbon Allocation, Heather D. Coleman, Thomas Canam, Kyu-Young Kang, David D. Ellis, Shawn D. Mansfield

Thomas Canam

The effects of the over-expression of the Acetobacter xylinum UDP-glucose pyrophosphorylase (UGPase) under the control of the tandem repeat Cauliflower Mosaic Virus promoter (2335S) on plant metabolism and growth were investigated in hybrid poplar (Populus alba3grandidentata). Transcript levels, enzyme activity, growth parameters, leaf morphology, structural and soluble carbohydrates, and soluble metabolite levels were quantified in both transgenic and wild-type trees. Transgenic 2335S::UGPase poplar showed impaired growth rates, displaying reduced height growth and stem diameter. Morphologically, 2335S::UGPase trees had elongated axial shoots, and leaves that were substantially smaller in size when compared with wild-type trees at equivalent developmental stages. Biochemical analysis …

Over-Expression Of Udp-Glucose Pyrophosphorylase In Hybrid Poplar Affects Carbon Allocation, Heather Coleman, Thomas Canam, Kyu-Young Kang, David Ellis, Shawn Mansfield

Faculty Research & Creative Activity

The effects of the over-expression of the Acetobacter xylinum UDP-glucose pyrophosphorylase (UGPase) under the control of the tandem repeat Cauliflower Mosaic Virus promoter (2335S) on plant metabolism and growth were investigated in hybrid poplar (Populus alba3grandidentata). Transcript levels, enzyme activity, growth parameters, leaf morphology, structural and soluble carbohydrates, and soluble metabolite levels were quantified in both transgenic and wild-type trees. Transgenic 2335S::UGPase poplar showed impaired growth rates, displaying reduced height growth and stem diameter. Morphologically, 2335S::UGPase trees had elongated axial shoots, and leaves that were substantially smaller in size when compared with wild-type trees at equivalent developmental stages. Biochemical analysis …

Over-Expression Of Udp-Glucose Pyrophosphorylase In Hybrid Poplar Affects Carbon Allocation, Heather D. Coleman, Thomas Canam, Kyu-Young Kang, David D. Ellis, Shawn D. Mansfield

Faculty Research & Creative Activity

The effects of the over-expression of the Acetobacter xylinum UDP-glucose pyrophosphorylase (UGPase) under the control of the tandem repeat Cauliflower Mosaic Virus promoter (2335S) on plant metabolism and growth were investigated in hybrid poplar (Populus alba3grandidentata). Transcript levels, enzyme activity, growth parameters, leaf morphology, structural and soluble carbohydrates, and soluble metabolite levels were quantified in both transgenic and wild-type trees. Transgenic 2335S::UGPase poplar showed impaired growth rates, displaying reduced height growth and stem diameter. Morphologically, 2335S::UGPase trees had elongated axial shoots, and leaves that were substantially smaller in size when compared with wild-type trees at equivalent developmental stages. Biochemical analysis …

Enzyme–Microbe Synergy During Cellulose Hydrolysis By Clostridium Thermocellum, Yanpin Lu, Yi-Heng P. Zhang, Lee R. Lynd

Dartmouth Scholarship

Specific cellulose hydrolysis rates (g of cellulose/g of cellulase per h) were shown to be substantially higher (2.7- to 4.7-fold) for growing cultures of Clostridium thermocellum as compared with purified cellulase preparations from this organism in controlled experiments involving both batch and continuous cultures. This “enzyme–microbe synergy” requires the presence of metabolically active cellulolytic microbes, is not explained by removal of hydrolysis products from the bulk fermentation broth, and appears due to surface phenomena involving adherent cellulolytic microorganisms. Results support the desirability of biotechnological processes featuring microbial conversion of cellulosic biomass to ethanol (or other products) in the absence of …

Cellulose Utilization By Clostridium Thermocellum: Bioenergetics And Hydrolysis Product Assimilation, Yi-Heng P. Zhang, Lee R. Lynd

Dartmouth Scholarship

The bioenergetics of cellulose utilization by Clostridium thermocellum was investigated. Cell yield and maintenance parameters, Y(X/ATP)True = 16.44 g cell/mol ATP and m = 3.27 mmol ATP/g cell per hour, were obtained from cellobiose-grown chemostats, and it was shown that one ATP is required per glucan transported. Experimentally determined values for G(ATP)P-T (ATP from phosphorolytic beta-glucan cleavage minus ATP for substrate transport, mol ATP/mol hexose) from chemostats fed beta-glucans with degree of polymerization (DP) 2-6 agreed well with the predicted value of (n-2)/n [corrected] (n = mean cellodextrin DP assimilated). A mean G(ATP)(P-T) value of 0.52 +/- 0.06 was calculated …

Towards A Systems Approach To Understanding Plant Cell Walls, Chris R. Somerville, Stefan Bauer, Ginger Brininstool, Michelle Facette, Thorsten Hamann, Jennifer Milne, Erin Osborne, Alex Paradez, Staffan Persson, Ted K. Raab, Sonja Vorwerk, Heather Youngs

Ted K. Raab

One of the defining features of plants is a body plan based on the physical properties of cell walls. Structural analyses of the polysaccharide components, combined with highresolution imaging, have provided the basis for much of the current understanding of cell walls. The application of genetic methods has begun to provide new insights into how walls are made, how they are controlled, and how they function. However, progress in integrating biophysical, developmental, and genetic information into a useful model will require a system-based approach.

Microbial Cellulose Utilization: Fundamentals And Biotechnology, Lee R. Lynd, Paul J. Weimer, Willem H. Van Zyl, Isak S. Pretorius

Dartmouth Scholarship

Fundamental features of microbial cellulose utilization are examined at successively higher levels of aggregation encompassing the structure and composition of cellulosic biomass, taxonomic diversity, cellulase enzyme systems, molecular biology of cellulase enzymes, physiology of cellulolytic microorganisms, ecological aspects of cellulase-degrading communities, and rate-limiting factors in nature. The methodological basis for studying microbial cellulose utilization is considered relative to quantification of cells and enzymes in the presence of solid substrates as well as apparatus and analysis for cellulose-grown continuous cultures. Quantitative description of cellulose hydrolysis is addressed with respect to adsorption of cellulase enzymes, rates of enzymatic hydrolysis, bioenergetics of microbial …

Crystalline Structure Properties Of Bleached And Unbleached Wheat Straw (Triticum Aestivum L.) Soda-Oxygen Pulp, Esat Gümüşkaya, Mustafa Usta

Turkish Journal of Agriculture and Forestry

In this study, the crystallinity index and crystallite size of wheat straw powder, soda-oxygen pulp and soda oxygen pulp bleached with hypochlorite (H) and hypochlorite and peroxide (HP) sequences were determined using an x-ray diffractometer method. The crystallinity indexes of these pulp samples were found to be 45.61%, 52.00%, 52.60% and 54.11%, respectively. The crystallite sizes of these pulp samples were also determined and were 6.4 nm, 3.4 nm, 4.3 nm and 4.6 nm, respectively. On the other hand, the crystallinity index and crystallite size of holocellulose, cellulose and alpha-cellulose in wheat straw were then found to be 65.00%, 55.20% …

Fermentation Of Cellulosic Substrates In Batch And Continuous Culture By Clostridium Thermocellum, Lee R. Lynd, Hans E. Grethlein, Richard H. Wolkin

Dartmouth Scholarship

Fermentation of dilute-acid-pretreated mixed hardwood and Avicel by Clostridium thermocellum was comparedinbatchandcontinuouscultures.Maximumspecificgrowthratesper hourobtainedon cellulosic substrateswere 0.1inbatchcultureand>0.13incontinuousculture.Cellyields(gramsofcellsper gram of substrate)inbatchculturewere 0.17forpretreatedwoodand0.15forAvicel.Ethanolandacetatewere the mainproductsobservedunderalconditions.Ethanol:acetateratios(ingrams)were approximately1.8:1in batchcultureand generallyslightlylessthan 1:1incontinuousculture.Utilizationofcellulosicsubstrateswas essentially complete in batch culture. A prolonged lag phase was initialy observed in batch culture on pretreated wood; the length of the lag phase could be shortened by addition of cell-free spent medium. In continuousculturewith-5g ofglucoseequivalentper literinthefeed,substrateconversionrelativeto theoreticalrangedfrom0.86ata dilutionrate(D)of0.05/hto0.48ata D of0.167/hforAvicelandfrom0.75 ata D of0.05/hto0.43ata D of0.11/hforpretreatedwood.Atfeedconcentrationsof<4.5g ofglucose equivalentperliter,conversionofpretreatedwoodwas80to90%atD= 0.083/h.Lowerconversionwas obtainedathigherfeedsubstrateconcentrations,consistentwitha limitingfactorotherthancellulose.Free Avicelaseactivitiesof12to84mU/mlwere observed,withactivityincreasinginthisorder:batchceliobiose, batchpretreatedwood< batchAvicel,continuouspretreatedwood< continuousAvicel.Freecellulaseactivity was higheratincreasingextentsofsubstrateutilizationforbothpretreatedwoodandAvicelunderal conditions tested. The results indicate that fermentation parameters, with the exception of free cellulase activity,are essentiallythesame forpretreatedmixedhardwoodandAvicelundera varietyofconditions. HydrolysisyieldsobtainedwithC.thermocellumcellulaseactingeitherinvitroor invivowere comparableto thosepreviouslyreportedforTrichodermareeseion thesame substrates.

Fermentation of dilute-acid-pretreated mixed hardwood and Avicel by Clostridium thermocellum was comparedinbatchandcontinuouscultures.Maximumspecificgrowthratesper hourobtainedon cellulosic substrateswere 0.1inbatchcultureand>0.13incontinuousculture.Cellyields(gramsofcellsper gram of substrate)inbatchculturewere 0.17forpretreatedwoodand0.15forAvicel.Ethanolandacetatewere the mainproductsobservedunderalconditions.Ethanol:acetateratios(ingrams)were approximately1.8:1in batchcultureand generallyslightlylessthan 1:1incontinuousculture.Utilizationofcellulosicsubstrateswas …

Effects Of Lignification, Cellulose Crystallinity And Enzyme Accessible Space On The Digestibility Of Plant Cell Wall Carbohydrates By The Ruminant, M. S. Kerley, G. C. Fahey Jr., J. M. Gould, E. L. Iannotti

Food Structure

Intrinsic characteristics of plant cell walls limiting susceptibility of structural carbohydrates to microbial attack in the ruminant's gastrointestinal tract are lignification of the cell wall, covalent bonding of phenolic acids to cell wall polysaccharides, the crystalline structure of cellulose and limited fibrolytic enzyme accessible space. The exact mechanism by which or degree to which each of these characteristics affect rate and/or extent of cell wall polysaccharide hydrolysis by gastrointestinal tract microbes is not well understood. Lignification and limited enzyme accessible space probably affect the extent of cell wall degradation by preventing contact between microbial enzymes and cell wall polysaccharides. Phenolic …

The Isolation Of Ecdysterone Inducible Genes By Hybridization Subtraction Chromatography, Michael P. Vitek, Susan G. Kreissman, Robert H. Gross

Dartmouth Scholarship

We have developed a procedure for selectively enriching a mRNA population for inducible sequences. Other than the induced mRNA species, the population of mRNA in control cells is approximately the same as the mRNA population in induced cells. Cytoplasmic mRNA from control cells is bound to oligo (dT)-cellulose and used as a template for reverse transcriptase, the oligo (dT) serving as a primer. After removing the template mRNAs, the cDNA-cellulose column is used to hybridize a population of mRNAs from induced cells. The non-hybridized poly A ⁺ RNAs are greatly enriched in the inducible sequences. We have used this technique …