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

Life Cycle Inventory on the Production of OSB in the Northeast United States by Nathaniel Vacanti & Dr. Stephen Shaler

As part of the NSF-REU program here at the University of Maine I will be working with Dr. Stephen Shaler on a life cycle inventory (LCI) model on the manufacture of oriented strandboard (OSB).

Oriented strandboard is a material made out of wood strands and adhesive that can be substituted for plywood. Compared to plywood OSB is much less expensive which makes it a popular alternative in construction projects. However there are some drawbacks associated with OSB. OSB is more absorbent to water and consequently swells more in damp environments. This could present a problem if a constant thickness is absolutely necessary. Once swelled, OSB is much less permeable to water vapor which can cause a mold or fungus problem in confined areas.

A life cycle inventory is essentially a mass and energy balance on a process. Like any process, there are inputs and outputs to the manufacturing of OSB. The mass inputs include wood and adhesive while the energy inputs include the energy required to cut down the trees, to form wood strands, to transport the wood, to make the adhesive, to accomplish the high temperature and pressure necessary to manufacture the OSB, and to burn off volatile organic compounds. The mass outputs include the finished OSB, any unused raw materials, and any waste products (possibly toxic) from the process. The energy outputs take the form of lost heat and changes in internal energy. However, they are not as significant because the energy inputs are the dominant cost.

The software SimaPro LCA will be used to set up the mass and energy balances and create the LCI model. Once the model is created, parameters can be changed in specific processes and comparisons of environmental impacts can be made between productions with different compositions of raw materials; the goal being to find which parameters the negative environmental impacts (emissions, solid waste, energy use, etc.) are most sensitive to, and to propose ways to minimize these parameters in the actual industrial process.

These model outputs can also be inserted into a larger model, such as one for constructing a house. The effects of using OSB can then be compared to those for using plywood (if a model for the use of plywood is available). With the cost and environmental impacts of this substitution readily available, an educated decision between the two products can then be made.

REU Interview 07/17/07

http://efolio.umeedu.maine.edu/~tvassiliev/FBRI/2007FBRI/Nate.m4a

Role of Pore Size on Thermalconversion and Catalytic Product Distribution by Alex Canney, Clay Wheeler & Brian Frederick

Our project within the FBRI focuses on producing sustainable fuels and chemicals using catalytic thermochemical conversion. One of the ways this can be done is by pyrolysis. The woody biomass is heated in the absence of oxygen, producing oxygenated aromatic compounds. The oxygen must be removed to make fuels or other specialty chemicals. Development of metal catalysts for hydrodeoxygenation is a relatively new area of research.

One process option is to burn the woody biomass in the presence of a limited amount of oxygen, producing primarily a combination of carbon monoxide and hydrogen. This combination is commonly referred to as syngas. The syngas can then be combined with more hydrogen and polymerized to produce alkanes in a process called Fisher-Tropsch synthesis. These alkanes of various lengths can then be distilled to produce quantities of the desired compounds ranging from ethane to wax.

One strategy to narrow the product distribution is to prepare the FeCo metal catalyst in a nanoporous material. The first step in understanding the effect of pore size in catalysis is to study the conversion of a series of alcohols on porous tungsten oxide materials that have already been synthesized. A microreactor system will be used to measure the conversion of alcohols into alkenes and ethers. The inlet and outlet gas compositions will be measured using gas chromatography-mass spectrometry (GC-MS). The reaction rates on porous WO3 and on non-porous WO3 will be measured for a series of small-to-large alcohol molecules, and the ratio of porous to non-porous rates will be calculated. The goal is to show that the ratio decreases as the size of the molecule increases, therefore demonstrating that the active sites in the pores are not accessible to the larger molecules.

Before carrying out the alcohol/WO3 reactivity experiments the GC/MS microreactor system needs to be set up and calibrated. Tasks include calibrating mass flow controllers, maintaining and tuning the GC-MS, qualifying the performance of the reactant delivery cells, and measuring the reactivity of the inert SiO2 reactor packing material.

REU Interview Notes 07/10/07

Alex Canney REU Interview Notes (pfd file)

Optimization of Soil Block Assay Techniques and the Analysis of the Effect of Fenton Chemistry on Cellulose Crystallinity by Stewart Gramlich & Dr. Jody Jellison

This research will investigate how various fungal species are able to colonize and chemically modify lignocellulose. Fungi best grow in dark wet environments and can be found anywhere organic material is present. Yeasts, mushrooms, and molds are examples of fungi. Fungi are usually aerobic, but some yeasts are able to anaerobically obtain energy via fermentation. Fungi, other than yeasts, are composed of many threadlike structures called hypha, which make up mycelium. This mycelium makes up molds and the nutrient gathering portion of mushrooms, which break down complex organic materials into their usable constituents.

Fungi have potential utilization in the processes of bioconversion because of their ability to produce enzymes and other metabolites that can break down lignocellulose into simple organic compounds and inorganic molecules, a process called biodegration. Simple organic compounds such as sugars can then go through the process of fermentation to produce ethanol. By looking at more aggressive fungal species and optimizing soil block assay techniques, more effective ways of breaking down lignocellulose can be identified. My work will focus on exploring the effects of soil characteristics, incubation times and water content of soil block assays on biodegradation rates. Irpex lacteus, Gloeophyllum trabeum, and Pictomyces sanguineus will be used to test decay rates in white pine blocks. I will also be involved in laboratory experiments looking at composite biodegradation and the effect of microbial colonization on cellulose crystallinity.

REU Interview Notes 07/10/07

Stewart Gramlich Interview Notes 07/10/07 (pdf file)

The Separation of Forest Bio-Product Components through Near-Infrared Spectroscopy and Chemometrics by Abby Hamilton, Andy Mishou, Dr. Darrell W. Donahue & Amy St. Peter

The use of near-infrared spectroscopy (NIRS) could maximize the productivity of the forest bio-products process by aiding the separation of output woody biomass. NIRS and chemometric techniques could eventually be used online as a process control tool for the forest bio-product process. But before this technology is applied, research must be performed off-line at first. Hardwood and softwood mixtures of wood chips were scanned before and after treatment. NIRS was also used for the extracted liquid product removed from the wood chips after the extraction process. Two separate modules of the spectrometer were used to measure the reflectance of the extracts and wood chip samples.

Partial least squares calibration method (PLS), a multivariate calibration method, was used as well as selected pretreatments to create a mathematical model of the spectra. After calibrating the spectra of the solutions created in the laboratory, the calibration was tested by scanning the extracts that were removed from wood chips after the extraction process.

Differences in physical appearance of the wood chip samples, such as the surface color, grain size and thickness must be taken into account. A variation between the treated and untreated wood chips mostly occurred within the 1000-1350nm range. Effects of the viscosity, the amount of liquid scanned and the type of solvent used for the solution were investigated on the liquid extracts prepared in the laboratory. Generally there was a difference in the liquid extract spectra between 1000-1400nm.

Interview 07/11/07

http://efolio.umeedu.maine.edu/~tvassiliev/FBRI/2007FBRI/Abby&Andy.m4a

The Investigation of Value Added Applications of Paper Products in Areas of Bio-separations and Bio-detections by Nimesh Patel & Dr. Michael Mason

One rapidly growing area of research involves the investigation of value added applications of paper products in areas of bio-separations and bio-detections. These novel systems are now being referred to as “bio-active paper”. One potential future application of this paper is in Biological Warfare Agents (BWA) and infectious diseases (ID) detection. At this point, there are two ways in which to do this. One is by using Polymerase Chain Reaction to amplify the sample in a laboratory. The advantage of this is reliability and specificity. However, it has drawbacks of being time consuming and expensive. The other method of detection includes the types used in the field, which work similar in a manner to pregnancy tests in the sense of an almost immediate response. The drawbacks to these is that they are not able to distinguish between similar pathogens that exhibit large variance in toxicity (not specific) nor are they able to detect certain BWA/ID in trace, yet harmful, concentrations. These drawbacks can lead to false positives and false negatives. Thus, the overall long-term goal of the research is to use the production of bio-active paper along with chemistry to develop a field detection system that is cost-effective, fast, and accurate.

To do this we will employ a bio-chemical amplification technique using enzymes. The process begins with a molecular beacon that will be engineered down to a specific base on the DNA of the sample to ensure selectivity. If there is an exact match, the hairpin shaped DNA that is attached to the inhibitor of the enzyme will uncoil. This will release the inhibitor and allow the enzyme to start a chemical reaction. We will use a lipase to produce H+ ions. These H+ ions will be used to activate an indicator molecule or dye that will show the presence of the specific pathogen.

The current specific research will deal with maximizing the efficiency of detection not just in solution but on bio-active paper as well. The preliminary unit of this efficiency is: Signal photons (dye) / (time x [enzyme]).

To do this we will have to choose the proper dye that will show the best on the paper in the correct pH of the reaction. In addition, since no two enzyme molecules are alike, there is the variable of different enzyme molecules within an ensemble of similar enzymes that can sometimes produce different results. Consequently, we must find what aspects of the single enzyme molecule optimize the amount of signal photons.

Once the enzyme has been selected and characterized at the sub-ensemble level and also the dye indicator system selected, then device geometries that involve bio-active paper loading will be investigated. Specifically, the “on paper” enzyme activity and dye sensitivity will be compared to data obtained from the solution and ensemble measurements.

Finally, after the first two stages, there is further research to be done on creating an H+ permeable membrane to remove the excess H+ from the system so it does not slow down the reaction according to Le Chatelier’s principle by fueling the reverse reaction.

REU Interview 07/11/07

http://efolio.umeedu.maine.edu/~tvassiliev/FBRI/2007FBRI/nimesh.m4a

Acetic Acid Inhibition of E. coli K011 during Fermentation by Brittany Oetter & Sara Walton

Research being done on forest bioproducts includes researching the efficiency and feasibility of using wood to produce ethanol. One of the three components of wood, hemicellulose, can be separated from the other two components and can then be fermented using bacteria such as Escherichia coli (E. coli). During fermentation, the bacteria convert the sugars of the hemicellulose into ethanol. However, this process may not be an effective way to produce fuel ethanol if it is an inefficient process. One problem is that certain compounds produced during the pretreatment of the hemicellulose before fermentation can inhibit the bacteria that are used in fermentation. One such degradation product is acetic acid, which may inhibit E. coli by penetrating the cell walls of the bacteria and making the cell cytoplasm acidic. This would make the process of using wood to produce ethanol by fermentation less efficient. One area for research involves investigating the use of genetically engineered strains of bacteria that might be more resistant to these degradation products.

This research will focus specifically on analyzing the effect of acetic acid on the fermentation of hemicellulose by E. coli K011, a recombinant strain of E. coli that is thought to be more resistant to the adverse effects of acetic acid. After growing this recombinant strain of E. coli it will be used to ferment sugars that have been extracted from wood chips. At first, a few fermentations will be done using pure sugars, and later the actual hemicellulose extracts will be used in the fermentations. This research will focus on finding out if fermentation done by this strain of recombinant E. coli is affected by the acetic acid in the same way that naturally occurring E. coli is. The goal will be to determine the extent to which acetic acid inhibits this strain of E. coli. The samples will be analyzed using HPLC (high performance liquid chromatography) to determine the amount of ethanol produced by the E. coli during fermentation and to examine how much of the sugars from the hemicellulose are converted into ethanol. The knowledge gained from this research could help determine the efficiency of using this fermentation process to produce ethanol from hemicellulose.

The inhibition of bacteria by degradation products of hemicellulose poses a problem to the idea of using biomass to produce ethanol. This particular research could help clarify this problem and help determine whether or not wood can become a practical resource for fuel ethanol production.

Brittany Oetter REU Interview 07/10/07

http://efolio.umeedu.maine.edu/~tvassiliev/FBRI/2007FBRI/Brittany.m4a

Imaging of Nanofibrils by Atomic Force Microscopy by Ryan Lena & Dr. Doug Bousfield

The process of making nanofibrils is well-established today. Many groups around the world have found methods, but unfortunately none of these are very high-yield processes, and they are expensive. The challenge, then, is finding a way to get a high yield of nanofibrils, and doing so in such a way as to reduce the time it takes to produce the fibrils, as well as reducing the cost and the energy consumption.

Not only must the process be cheap and efficient, but the fibrils produced must be able to be utilized. To be useful in industry, these fibrils must have high aspect ratios (the ratio of fibril length to diameter). The problem with measuring the fibril’s aspect ratio is that the fibrils often entangle in the pulp-water suspension due to hydrogen bonding between the fibers. Thus, the technique for creating the fibrils must also have a method of separation that is not deleterious to the fibrils themselves. Furthermore, only fibrils of a certain diameter are useful—ideal nanofibrils have diameters less than 100 nanometers.

Many groups have researched this process in a water-based medium, without pre-treating the sample with enzymes. The aim of this project is to determine which enzymes have the greatest effect on nanofibril production, as well as the most effective method of production. The two machines that are used are the Kady mill, which is an industrial mixer and applies an extremely high shear force to the sample in a short period of time, and the homogenizer, which forces the sample through a series of tubes at 45,000 psi and breaks the sample down into smaller pieces, making the sample more uniform. The Kady mill and homogenizer are used in this experiment to break down the larger microfibrils (which have diameters in the micron range) into smaller nanofibrils. The experiment varies how many passes through the homogenizer produce the greatest number of nanofibrils, how long the sample should be put in a Kady mill and how it should be filtered afterward, and so on. There are many different combinations of methods that involve high-shearing forces that can be tested.

My focus in this project will pertain to the use of Atomic Force Microscopy in this research project. I will gain expertise in using the AFM, and analyze different samples given to me. Different methods of sample preparation will be used, i.e. scanning a dry sample vs. scanning the sample in an aqueous solution. My findings will provide support on the nanoscale for observations made through optical and electron microscopes. I will determine which method of production gives the best nanofibrils.

The results of this project could have many different applications. Many people have thought of uses for nanofibrils were they to become readily available. Nanofibrils can be used to strengthen other materials, such as polymers, without altering the overall appearance. They also have potential to be used in the medical field, such as in dressings or drug delivery systems. These are just a few examples of the applications, but there are many more out there, and certainly more to come.

REU Interview 07/11/07

http://efolio.umeedu.maine.edu/~tvassiliev/FBRI/2007FBRI/ryan.m4a

Pre Extraction Research by Gracson Andrews & Dr. Weiping Ban

Dr. Van Heiningen’s research group is working on a project called pre-extraction of modified kraft pulping to increase overall pulp yield and decrease the amount of necessary chemicals for the pulping process. Some background information regarding softwoods and hardwoods is important for understanding the pre-extraction process. Softwoods include woods such as pine, fir, cedar, spruce, redwood, and hemlock. Hardwoods include woods such as birch, beech, aspen, ash, and cottonwood. With standard pulping processes, a percent yield of 40%-45% is obtained with softwoods while hardwoods usually obtain a percent yield of around 50%. However, hardwoods have shorter fibers than softwoods and therefore make a more uniform and consistent product. Because of the longer fibers in softwood, the paper made from softwood is usually stronger than that from hardwood. Any method which increases pulp yield and reduces chemical consumption is beneficial to paper companies because it increases profit and decreases waste. Currently, the group has attained a 5% overall increase in pulp yield, and has also attained a 3% decrease in overall chemical consumption.

The pre-extraction modified pulping process has three main steps. First, the hemicellulose is extracted from the raw wood chips. This step is the extraction step, which is what allows an overall increase in yield. The hemicellulose is set aside for later use. Second, the remaining post-extracted wood chips are made into pulp by cooking with chemicals such as NaOH and NaS (white liquor) at high temperatures around 170 degrees C. The third and final step is adsorption, in which the hemicellulose is redeposited into the pulp mixture. By extracting the hemicellulose before cooking the woods chips to make pulp, a higher overall percent yield is obtained.

The group has successfully created lab size batches of pulp using the new pre-extraction method, but further testing is necessary in regards to the products physical properties. Dr. Ban will be testing the physical properties of the paper made using the pre-extraction process. Because the physical tests will be run on sheets of paper, preparation of 100 grams of product is necessary. Hopefully, the group will be successful in producing 10 to 12 10-gram samples and then concentrating and combining these samples to obtain a larger batch size for use in the physical properties tests. The tests that will be performed include tensile strength, tear strength, burst strength, and fold strength compared against a control sample. Beating, which is blending the pulp to obtain shorter fibers and increase bonding surface area, will also be tested. Tests using different refining times will be performed to determine which beating yields the highest strength paper. A bleaching comparison will also be performed to make sure the bleaching process is not affected by the pre-extraction process.

The experimental analysis of the physical properties testing will allow Dr. Van Heiningen’s group to determine whether this method of pre-extraction can be used to obtain equally viable final products while increasing yield and decreasing chemical consumption in industrial applications.

Interview with Gracson 07/03/07

http://efolio.umeedu.maine.edu/~tvassiliev/FBRI/2007FBRI/gracson.m4a

Xylanase Enzyme by Andru O’Farrill, Dr. Ray Fort & Dr. Barbara Cole

The work conducted will consist of two interconnected types of research based on the same topic. The research will include both chemistry lab work, as well as in the chemistry computer lab classroom. The experiments will be done with the enzyme, xylanase, and the substrate xylobiose. Xylobiose is a product that is formed from the hemicelluloses in trees. Currently there is research being conducted in the chemistry lab to use the xylanase enzyme to cleave hemicelluloses into desired lengths. One of the molecules that should theoretically be detected is the xylobiose molecule. Using the xylanase enzyme the linkage between two xylose units in xylobiose should be cleaved. There will be two aspects of this that will be studied.
First, a way to identify the xylobiose molecule must be found using gas chromatography/ mass-spectrometry (gc/ms), and liquid chromatography/ mass-spectrometry (lc/ms), most likely lc-ms. These trials should theoretically find a way to determine if xylobiose is present in a mixture, and to see if the particular strand of xylanase being used is an efficient enzyme. Secondly, the research will consist of altering the reaction that is obtained when xylan is hydrolyzed by xylanase enzymes. Temperature and time will be altered to find the optimum conditions to drive the reaction.

In the computer lab, the computer simulation program Autodock will be used to examine how the xylobiose binds to the enzyme. By setting up a 3-D grid across the enzyme, and analyzing the interactions at different points on the enzyme, the location on the enzyme where xylobiose binds, and the best conformation of the xylobiose for this reaction to occur can be found. Additionally, different conformations will be analyzed to see the effects on the enzyme’s ability to catalyze the reaction. This will in turn give a good idea of what must be done in the lab to accomplish the selective cleavage of the hemicellulose bonds., and will reduce the amount of different xylanases that will have to be bought for lab experiments, and lab work done. This research will also help understand the same problems that are being found under the larger scale hemicellulose molecules.

Interview with Andru July 5, 2007

http://efolio.umeedu.maine.edu/~tvassiliev/FBRI/2007FBRI/andru.m4a

Biomass Availability in Maine by Jacob Kavkewitz & Jonathan Rubin

The specific goal of the research project is to use data mapping to find the best possible locations for biofuel plants in Maine, and the best methods to achieve the economically optimal supply side results of the Forest Products Research Initiative. The project goals will be accomplished by the following methods: 1) The collection and reconciliation of data on the location of biomass availability with the potential for biofuel in Maine. Two major sources of data have currently been identified: data provided by the Billion Tons Report and data from the Maine Department of Conservation. , Other data sources are also used in the project. 2) Mapping these data using a Geographic Information System (GIS). Part of this task will be to identify relevant GIS mapping that may already be available. 3) Identify relevant roadways, rivers and barriers to access and transport of biomass. 4) Identify flows of biomass into and out of Maine that affect the biomass availability, and 5) Identify and, if feasible, estimate price relationships that affect the physical biomass supply.

Interview July 5, 2007

http://efolio.umeedu.maine.edu/~tvassiliev/FBRI/2007FBRI/jacob.m4a

Research Summeries

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RET

Tracy Vassiliev – tracy.nason@umit.maine.edu