An Alternative to Traditional Methods of Chemical Fractionation of Woody Biomass through the use of an Enzyme Pretreatment focusing on the Lignin-Hemicellulose Bond

Morgan Urello
Mentor: Dr. Nancy Kravit

Woody biomass contains primarily three macromolecular components, cellulose, hemicellulose, and lignin.  Each has specific yet very different uses and optimization of the use of woody biomass as a renewable resource is directly correlated with the efficiency of the fractionation method used for separation.  Wood component separation is currently more inefficient and environmentally costly than ideal, largely because of chemical bonds between hemicellulose and lignin.  The most troublesome of these cross-links are ether bonds.  The Kravit lab in conjunction with a commercial partner Tethys Research LLC, is developing an alternative to traditional methods of chemical fractionation through the use of enzyme pretreatment.  The Kravit laboratory has created a substrate designed to mimic one of  the ether bonds between lignin and hemicellose (H-L bond) in soft woods.  The substrate is unique in that it fluoresces when the H-L bond is cleaved.  It has been used in bioprospecting experiments to discover an enzyme that may be capable of breaking lignin- hemicellulose bonds in softwoods.  The source of the enzyme is a newly discovered microorganism called B603.  It is now the goal to clone the gene for the enzyme from B603 and test the cloned enzyme on native lignin-hemicelluose complexes.

To test the capabilities of this enzyme, it must be obtained in high enough quantity and in sufficient purity  for testing, yet the cells found to produce this enzyme turn out very little of it.  For this reason, a more efficient and controlled method for optimizing expression has been developed using genetic engineering technology.  First a genetic library from Microbe B603 containing cDNA encoding the hemicellulose-lignin etherase (HLE) was obtained.  mRNA  was prepared from B603 cells that were expressing HLE.  For cost-efficiency, SeqWright, a specialty cloning laboratory, was contracted to actually construct the cDNA library.  The cloning strategy was to make the library in Lambda-ZAP II (Stratagene), a bacteriophage lambda vector which eventually lyses the host cell, allowing the recombinant proteins inside the host cells to access to the cell’s surroundings where the indicating substrate is located.  LambdaZap II’s cloning site is located in a phagemid to simplify subcloning.   The next step is to screen this amplified library using standard procedures.  To do this first XLIBlue, host cells, will be infected with the library in the presence of the indicating substrate and IPTG, a substance that induces the expression of the inserted gene.  The plates will be incubated until plaques appear. Then a hydrophobic membrane will be overlaid to bind and detect the 4MU (a fluorescent compound) that is released from the substrate as a result of any enzyme activity. Fluorescent spots on the membranes will be correlated back to the plaques, and the fluorescent plaques will then be picked and used as starting material for further rounds of subsequent infections until all plaques are fluorescent.  Exassist, a helper phage, will then be used to remove the phagemid from the lambda vector and the phagmid will be transformed into SOLR cells on ampicillin-containing  plates.  The desired phagemid is ampicillin resistant therefore colonies containing it will grow and all cells without it (the rest of the LambdaZapII sequence has no ampicillian resistance) will die. The surviving colonies will be selected, re-grown, and mini-prepped.  Because the enzyme produces auto-fluorescent proteins, it will be important to ensure the fluorescence was not caused by a fluorescent protein as opposed to the cleaved substrate.  To do this, cells will be grown on plates with and without indicating substrate.  If the desired enzyme is present, the first plate should not fluoresce and the second should.  If successful, more of this clone will be produced and purified.  Eventually, the recombinant enzyme will be tested on native lignin-hemicellulos complexes.

2009 REU Interview, July 23, 2009 – Morgan Urello

If you or your class has questions regarding this research or experience, please contact:

mau2107@columbia.edu

Acid hydrolysis of xylo-oligosaccharide extracts with sulfur dioxide (SO2): Effect of temperature and SO2 concentration on total pressure

By: Alex Haluska
Faculty Advisor: Adriaan van Heiningen
Graduate Advisor: Rory Jara

Hemicellulose extracts, wood pulp, have been identified as a byproduct from the production of higher value-added products such as ethanol and pulp in an Integrated Forest Bio-refinery (IFBR). In the IFBR process hemicelluloses are partially extracted via an aqueous solution at high pressure and temperature mainly as xylan-oligomers from wood material prior to application of further processes. These oligomers cannot be directly metabolized by microorganisms during the fermentation process used for the production of ethanol. The oligomers must be broken down in a cost effective way from oligomers to monomers. The study will examine if sulfur dioxide (SO2), a gas, is effective as a catalyst for the hydrolysis of hemicellulose oligomers. Since SO2 is a gas it is possible to recycle it through the process and this may reduce operational costs.

The study focuses on the effect of temperature and SO2 concentration on total pressure during the acid hydrolysis process. The study used an oil batch reactor system operation, wireless pressure sensor operation, and bio-refinery concept to study the effect on the total pressure. The study will carry out multiple experiments changing temperature, SO2 concentration and using different extracts. The two techniques to be used are the hot water extract (HWE) and the near neutral extract (NNE). The HWE uses hot water to increase reaction rate. NNE is a technique using sodium acetate to keep the ph level near 7 to reduce the amount of substance needed to neutralize it before the fermentation process.  The temperatures to be examined will be: 100°C, 130°C, 140°C, 150°C, 160°C.  The SO2 concentration to be examined in this study were: 1%, 2% 5% 10%. As an end result, this information will be used to develop new equipment for the acid hydrolysis of xylan to xylose during the fermentation process in the ethanol production process of an IFBR.

2009 REU Interview, July 21, 2009 – Alex Haluska

If you or your class has questions regarding this research or experience, please contact:

aahalusk@syr.edu

Utilizing Ultrafiltration for the Concentration of Hemicellulose Extracts from the Kraft Pulping Process

Rob Jonson
Mentor: Peter can Walsum

Currently the kraft pulping process burns hemicelluloses to recover energy.  However, the hemicellulose can be extracted from pulping process and utilized to produce fuels and other chemicals.  Unfortunately the green liquor extraction process only produces dilute hemicellulose extract that must be concentrated before it can be converted economically into fuels or other chemicals.  Simple evaporation is not suitable for concentration because the resulting concentrations of sodium and acetic acid inhibit the fermentation process.  This research examines the use of tangential flow ultrafiltration for concentrating hemicellulose extracts.  Tangential flow membrane ultrafiltration will allow for the concentration of the hemicellulose without concentrating the salts or acids.  Experiments will be performed on both the extraction and filtration conditions.  The molecular weight distributions of the extracts can then be compared with the filtration system’s performances and modeled for the design of a concentration system.

2009 REU Interview, July 21, 2009 – Rob Jonson

If you or your class has questions regarding this research or experience, please contact:

jonson@ksu.edu

Extraction of Shikimic Acid from the Picea Species

Alex Shaffer
Advisors: Drs. Barbara Cole and Raymond Fort

Foliage from the Picea species will be investigated as a potential macro-scale source of shikimic acid.    Shikimic acid is an important reagent in the synthesis of oseltamivir phosphate, or Tamiflu, which is used in the treatment of the H5N1 strain (bird flu) and H1N1 strain (swine flu).  Specific focus will be initially placed upon white pine needles because of the simplicity, low cost, and industrial practicality of the extraction process.  Ultimately, focus will expand to include all sub-species of the Picea species in an effort to simulate a real forest management situation. The overall goal of the project will be to minimize tree waste in an optimized industrial ready process that maximizes yield of shikimic acid at a low cost while maintaining other industrial operations at their current capacity.
Techniques will be based upon micro-scale methods and research previously conducted at the University of Maine.  Modifications will also be enacted to ensure thata safe product results from the extraction process.  Samples will be freshly collected from various species in the surrounding areas of Orono, Maineand then finely ground for investigation.  Whole needles will also be tested to determine the necessity of the grinding process.  Micro-scale extractions will be performed via an accelerated solvent extractor (ASE), and quantification will be analyzed via a liquid chromatographer combined with a mass spectrophotometer (HPLC-MS).  Additionally, UV light and liquid-liquid extractions will be used to isolate specific compounds of interest within thesamples.  Macro-scale techniques will first include soxhlet extraction with water as the solvent.  Other solvents may be tested to optimize the extraction of shikimic acid.  Experiments will then expand to a high temperature circular digester.  A rocking digester may also be tested to compareresults.  Polarity of the compounds in each sample will play a major role in the choice of extraction solvent.

2009 REU Interview, July 21,2009 – Alex Shaffer

If you or your class has questions regarding this research or experience, please contact:

atshaffer@gmail.com

Acid springing and extraction using trioctylamine

Audrey Polifka
Mentor:

One avenue to the conversion of cellulosic biomass into ethanol is to extract a by-product stream from the wood chips entering a kraft pulp mill. It has been shown that under optimized conditions, aqueous extraction can yield a dilute stream of fermentable wood-derived sugars while still maintaining the pulp yield and quality. In addition to sugars, the extract also contains furfural and acetic acid, which have been shown to inhibit microbial fermentation and can also be sold as commodity chemicals if separated from the extract at sufficient purity.

The aim of this project is to remove the acetic acid from the aqueous wood extract. Triocytylphosphine oxide (TOPO) in a water insoluble solvent (undecane) has already been tested to remove the acid via liquid-liquid extraction. In this work, trioctylamine (TOA) will be used with a co-solvent (octanol). Key investigations include: developing extraction equilibrium data for water/acetic acid versus an organic-amine system, comparing extraction performance on real extracts versus a pure component system, developing distillation equilibrium data for real extracts versus pure components, and recycling amines in both systems.

These characterizations of the extraction and solvent recovery operations lead to the final objective of the project. TOPO costs nearly ten times as much as TOA per gram; therefore, the recyclability and effectiveness of both TOPO and TOA will be compared to determine the preferable extraction technology.  Looking at the economics of the extraction/distillation system will complete the project and provide substantial insight for pulp mills wanting to also extract and market acetic acid.

2009 REU Interview, July 20, 2009 – Audrey Polifka

If you or your class has questions regarding this research or experience, please contact:

apolifka@ksu.edu

Evaluation of Nanocellulose Fibrils On Mechanical Properties in Amorphous Inorganic Composites

John Attonito
Advisors: Yousoo Han, Douglas J. Gardner, Yucheng Peng

Cellulose is an increasingly important part of the materials industry.  It is the most abundant material on Earth and its natural ability for self adhesion has long been recognized. On the nano-scale, microcrystalline and cellulose nanofibrils have been shown to have similar tensile strengths and stiffness as glass fibers.  The introduction of microcrystalline cellulose into polymers such as polypropylene has produced stronger and more thermostable materials than neatplastics.  Fillers such as cellulose nanofibrils are also environmentally and economically favorable in terms of plastic products because they are natural “green” products.

Cellulose nanofibrils are expected to go even further in applications due to their long aspect ratio and relative strength.  Expected uses may include surface strengthening, bio-nanocomposites, food, cosmetics, medical/pharmaceutical, absorbents, emulsion/dispersion and oil recovery applications (Mikael Ankerfors et al, 2007).  Cellulose nanofibrils are currently being used in composites with polylactic acid as microwave safe food packaging.  However, the nano sized fibrils are difficult to analyze due to agglomeration via hydrogen bonding in aqueous solutions.  Agglomeration results in different sized clumps of fibrils in a polymer matrix leading to a loss in the enhancing properties of the cellulose nanofibrils, rendering them less effective as polymer additives.  Therefore we have chosen sodium silicate or “water glass,” as an alternative matrix material, for preparing cellulose nanofibril composites.  Use of an aqueous-based matrix is expected to keep the fibrils separate at lower concentrations due to strong ionic bonding between the fibrils and the sodium silicate.  In the past, water glass has had applications in automotive and metal repair, food preservation, water treatment, fire protection and fireproofing and insulation.  Although water glass is used in many solutions that act as coatings, it is not itself an ideal coating as it becomes very brittle upon the removalof water.  It is known that treating wood with silicates preserves wood from insects, decay and offers some flame-retardant properties as well as some dimensional stability.  However, because of the high hygroscopicity, high pH values, and increased moisture absorption, strength loss of water glass treated wood isfrequently observed.  It is our goal to improve the properties of water glass as a structural material and/or coating by addition of cellulose nanofibrils.
To make a water glass-cellulose nanofibril composite, the sodium silicate matrix may be cured by mild heating to initiate dehydrolysis and condensation reactions between the silicatemolecules.  The cellulose nanofibril suspension will then be added and dispersed into the matrix using a Speed Mixer®.  Other additives are added to increase the strength of the composite.  The composite mixture will be poured into a mold and cured until a testable solid is produced.  The new composite will be tested and analyzed for improvements upon the properties of solidified water glass alone.  Flexural, tensile and impact strength measurements will be made as well as thermo stability measurements using thermogravimetric analysis (TGA).

2009 REU Interview, July 20, 2009 – John Attonito

If you or your class has questions regarding this research or experience, please contact:

jdatt2@gmail.com

Cellulose Nanofiber Coated Paper

Jacqueline Beckvermit
Mentor: Dr. Doug Bousfield

Abstract: The increase in public awareness and pressure to discover and use a renewable resource while having an ecologicallyfriendly process is reaching the paper industry.  Many publication grade papers are coated with pigments that need petroleum based binders to obtain a high quality print.  Other uncoated papers are treated with additional chemicals to help improve printing.   My research is focused on using cellulose nanofibers to coat paper.  The process utilizes a draw down coater which evenly distributes the nanofibers.   The nanofibers will be mixed with other materials that add pigment and/or act as a binder. Pigments such as calcium carbonate or kaolin and binders such as starch and polyvinyl alcohol will be investigated. The nanofiber coated sheets will be examined and compared with industrial grade products for print resolution, ink distribution, and other product characteristics.  The nanofibers have a unique ability to capture ink pigments at the surface of the paper allowing for little ink penetration.  Papers will be tested and compared based on the inks absorption or penetration rate and the inks density. The latter is performed using a reflection densitometer, a Bristow wheel and a microscopic video camera used to measure the contact angles to characterize the surface energy. My research is focused on finding an affordable green method to upgrade paper that should allow companies to reduce their use of petroleum products.

2009 REU Interview, July 20, 2009 – Jacqueline Beckvermit

If you or your class has questions regarding this research or experience, please contact:

jcbeckvermi@fortlewis.edu

Life Cycle Assessment of Wood Hemicellulose Derived Bio-Ethanol

Rachel Bowman
Mentor: Anthony Halog

Life cycle assessment (LCA) is a commonly accepted technique for determining the environmental sustainability of a product or process. The goal of this research is to complete a cradle to gate LCA of wood hemicellulose derived bio-ethanol from a modified Kraft mill that produces pulp and paper using the LCA modeling software Eco-LCA, Open LCA, and commercial SimaPro. The system boundaries of this study may change depending on data availability. An LCA of wood based bio-ethanol has already been completed using SimaPro, but such LCA technique only accounts for green house gas emissions and non-renewable resources. Other LCA programs consider land usage in their evaluation, and end-point impact assessments also exist that incorporate multiple factors.  Eco-LCA and Open LCA are newly developed models that offer a more complete approach to LCA.  Eco-LCA, a free LCA model available to the public, considers ecosystem goods and services, referred to as natural capital, which accounts for the environmental impact of a process on natural goods and services such as water, soil, wood, and grass. Eco-LCA uses an input-output model to assess a system, resulting in a more comprehensive outlook that requires only simple resource input data, rather than specific information about the emissions from individual processes. SimaPro requires setting a boundary for the types of factors that will be included in the LCA and specific emissions data from each individual process, therefore potentially yielding different results than the Eco-LCA evaluation.  The results of Eco-LCA and Open LCA will be compared to the results of SimaPro LCA, particularly in green house gas emissions and net energy consumption. The discrepancies among the three models will be reported to determine the model that reflects better representation of the environmental impacts of bio-ethanol.

2009 REU Interview, July 17, 2009- Rachel Bowman

If you or your class has questions regarding this research or experience, please contact:

rachel.bowman708@wku.edu

Turning lignin into carbon nano-materials

Alden Earle
Mentored by David Neivandt

Wood chips utilized in a kraft pulp mill are chemically broken down to remove lignin from cellulose and hemicellulose.  Though numerous applications have been found for the later, lignin (approximately 30% of the dry mass of wood) has proven to be more difficult to utilize and for the most part is burnt for fuel to power the mill.  Because it is so underutilized, inexpensive, plentiful and renewable, lignin is attractive as a feedstock for product development.  A proprietary method of converting lignin into nano structured carbon materials has been developed in the Neivandt laboratory.  However, significant room exists for optimization of the process.  Consequently, the present work aims to further the fundamental understanding of the process and to gain greater control over product specifications through modification of process variables.

2009 REU Interview, July 21, 2009 – Alden Earle

If you or your class has questions regarding this research or experience, please contact:

drivenformore@gmail.com

Characterization of Hydrodeoxygenation Catalysts in the Upgrading of the Model Compound Guaiacol in Pyrolysis Oil

Author:  Nick Dunn
Advisor: Dr. Brian Frederick

Methods are being developed to convert wood into fuels like heating oil and gasoline that will reduce the dependence of the United States on foreign oil. Wood is a renewable resource and fuels derived from it can reduce the effects of global warming because growth of new wood reabsorbs the carbon emissions produced by burning the fuel.  When wood is heated without air, it forms an oily mixture of chemicals, called “pyrolysis oil”, which is composed of approximately half carbon and half oxygen. The oxygen reduces the energy content of the oil.  To convert pyrolysis oil into high quality fuel it is necessary to design catalysts which will facilitate the efficient removal of oxygen at lower temperatures, thereby upgrading the oil’s energy content.  The precise chemical composition of the raw oil depends on the wood source and pyrolysis processing conditions, while the product quality depends on the upgrading process. The goal of this project is to characterize catalysts in the deoxygenation of the model compound guaiacol over catalysts such as cobalt/molybdenum sulfides in addition to nitrides on carbon and oxide supports, as well as pure oxides.  Although it comprises only a tiny fraction of the mass of pyrolysis oil, guaiacol is representative of many of the lignin-derived compounds.  Comparison of the efficiency and specificity of different catalysts will be accomplished by performing reactions of guaiacol in decalin solvent in a Parr continuous stirred tank batch reactor at hydrogen pressures of 50 bar. Reaction mixture samples will be removed from the reactor at regular intervals and chemical composition will be determined quantitatively with gas chromatography (GC).  Specifically, conversion is the rate of consumption of guaiacol.  Selectivity will be defined as the amount of deoxygenated products produced compared to the less desirable hydrogenation products.  Products will be identified by comparison with standards using GC, and with GC/mass spectrometry or nuclear magnetic resonance as necessary.  The catalysts will be compared based on guaiacol conversion. They will also be compared based on their selectivity toward deoxygenated products such as benzene, and the minimization of hydrogenated products such as cyclohexane.  Also of interest is the composition of the catalyst itself before and after the reaction, which will be determined using X-ray photoelectron spectroscopy.

REU Interview, July 21, 2009 – Nick Dunn

If you or your class has questions regarding this research or experience, please contact:

njhdunn@gmail.com

Fluorescent enzymes

Rosie Ochoa
Advisor: Dr. Nancy Kravit

The purpose of this project is to use woody biomass efficiently by discovering enzymes that break ether bonds between lignin and hemicellulose. Wood has three major macromolecular components: cellulose, hemicelluloses and lignin. It is believed ether bonds between lignin and hemicelluloses are a primary reason for the strength of both hardwoods and softwoods and for the difficulty of fractionating wood into separate streams of cellulose, hemicelluloses and lignin.

There are several advantages having separate streams of cellulose, hemicelluloses and lignin. Cellulose is used for papermaking. Hemicellulose can be depolymerized into its component sugars and the sugars then used in fermentations to produce building blocks for polymers, fine chemicals, chiral chemicals, or biofuels. The remaining lignin stream can be burned for heat and energy or sold for synthesis of aromatic fine chemicals.

2009 REU Interview, July 20, 2009 – Rosie Ochoa

If you or your class has questions regarding this research or experience, please contact:

compgik@yahoo.com

An investigation into the existance of high value compounds found in the bark and foliage of trees

Annemarie Nauert
Advisors: Dr. Fort & Dr. Cole

As natural resources have become increasingly valued, there has been a movement in the pulp and paper industry towards a more holistic model of resource use and production.  This includes interest in potentially high value compounds found in the bark and foliage of trees, which is otherwise discarded as waste.  Particular polyphenolic compounds are noted for their role as antioxidants and their ability to mimic the effects of calorie restriction.  Studies have shown it may protect against age-related ailments including Alzheimer’s disease and cancer.  A group of phytosterols—campestrol, stigmasterol, and sitosterol—have also been identified as important in preventing cardiovascular diseases and high cholesterol.

Research will focus on developing an economically and environmentally viable isolation method for obtaining polyphenolic compounds and phytosterols from Piceaspecies and other trees commonly harvested for paper and fiber production.  First of all, species in which polyphenolic compounds and phytosterols have been identified in high concentrations must be further validated by another round of experimental testing.  Because the compounds produced by trees vary according to species, location in the tree,and season, it is critical to take a variety of samples.  Analytical methods to be used for the further study of samples include Gas Chromatography-Mass Spectrometry for qualitative identification of certain polyphenolic compounds and a quantitative measure of phytosterols and several tests of antioxidant ability.

Second, the refinement processes being used in the research setting (accelerated solvent extraction, liquid-liquid extraction) must be scaled-up and adjusted for industrial and commercial applications.  This will include identifying environmentally benign, inexpensive, and simpler extraction methods by choosing solvents appropriately or finding alternative refinement processes.

July 16, 2009 REU Interview – Annemarie Nauert

If you or your class has questions regarding this research or experience, please contact:

aennt7@gmail.com

Novel nanocellulose polymer composites as green materials

Jacob Schual-Berke
Advisor: Dr. Doug Bousfield

Cellulose is the most abundant biological compound on the planet, with exceptional physical and chemical properties. Cellulose nanofibers (several nanometers in diameter) possess superior properties, such as increased tensile strength, elasticity and toughness because of their web-like structure. Modern technologies have made it possible to mechanically produce cellulose nanofibers in large quantities from common cellulose feedstocks. With its enhanced quality, natural abundance and non-toxicity, cellulose nanofibers may be the key to manufacturing inexpensive, sustainable, durable and environmentally unobtrusive materials. With packaging materials accounting for approximately 40% of U.S. municipal landfills, such a material would represent a significant advance.

Previous research has shown that incorporating cellulose nanofibers into other polymers improves the physical qualities of the material. In this research project, cellulose nanofibers will be combined with polymers such as polyvinyl alcohol (PVA) and latex to produce a composite whose characteristics will be analyzed. The liquid materials will be mechanically mixed for one hour to ensure thorough dispersion and then sonicated for a further hour to remove any air bubbles. The composites will be made into test strips by heating in a silicone mold and tested for tensile strength and elasticity with an Instron (5562). The effects of adding cross-linking compounds will also be investigated by incorporating them into the materials in various quantities. It is hoped that the addition of these compounds with cellulose nanofibers to known polymers will produce a low-cost material with increased strength, durability and environmental benefits.

REU Interview July 17, 2009 – Jacob Schual-Berke

If you or your class has questions regarding this research or experience, please contact:

jakeschualberke@gmail.com

Analysis of the Hemicellulose Pre-Extraction from Red Maple Wood.

Diego Rosso
Advisors: Adriaan van Heiningen and Rory Jara
Aqueous hemicellulose extracts from wood are considered a viable resource for the production of higher value products such as ethanol. The proposed process extracts hemicelluloses mainly as xylan-oligomers from wood material. Red maple ( Acer rubrum L.) wood will be  used for the pre-extraction of hemicellulose using  hot water extractions. The extractions will be made in both batch reactor and continuous flow reactor processes. The hemicelluloses extraction yields and extraction rates of both processes will be compared. Temperature and time of exposure for the batch reactor process will vary in the ranges of 140oC-170oC and 45-90 minutes, respectively.  The severity factor Ro, a function of time of exposure and temperature, will be used to measure the intensity of the reaction conditions in the batch reactor process. High Performance Liquid Chromatography and High Performance Anion Exchange Chromatography will be used to quantify the amount of oligomeric sugars, acetic acid, formic acid, and furfural in the extracted liquor. A UV-VIS detector will be used to determine the amount of lignin in the continuous flow reactor process. The sugar analysis will focus on the quantification of xylan, arabinan, galactan, glucan, and mannan. The extraction rate of red maple wood strands, wood chips, and wood meal will be analyzed and compared.

July 16, 2009 REU Interview: Diego Rosso

If you or your class has questions regarding this research or experience, please contact:

diego.rosso@upr.edu