Christine M. Ladisch

Pretreatment of yellow poplar sawdust by pressure cooking in water

The pretreatment of yellow poplar wood sawdust using liquid water at temperatures above 220°C enhances enzyme hydrolysis. This paper reviews our prior research and describes the laboratory reactor system currently in use for cooking wood sawdust at temperatures ranging from 220 to 260°C. The wood sawdust at a 6–6.6% solid/liquid slurry was treated in a 2 L, 304 SS, Parr reactor with three turbine propeller agitators and a proportional integral derivative (PID) controller, which controlled temperature within ±1°C. Heat-up times to the final temperatures of 220, 240, or 260°C were achieved in 60–70 min. Hold time at the final temperature was less than 1 min. A serpentine cooling coil, through which tap water was circulated at the completion of the run, cooled the reactor’s contents within 3 min after the maximum temperature was attained. A bottoms port, as well as ports in the reactor’s head plate, facilitated sampling of the slurry and measuring the pH, which changes from an initial value of 5 before cooking to a value of approx 3 after cooking. Enzyme hydrolysis gave 80–90% conversion of cellulose in the pretreated wood to glucose. Simultaneous saccharification and fermentation of washed, pretreated lignocellulose gave an ethanol yield that was 55% of theoretical. Untreated wood sawdust gave less than 5% hydrolysis under the same conditions.

Characterization of dicarboxylic acids for cellulose hydrolysis

In this paper, we show that dilute maleic acid, a dicarboxylic acid, hydrolyzes cellobiose, the repeat unit of cellulose, and the microcrystalline cellulose Avicel as effectively as dilute sulfuric acid but with minimal glucose degradation. Maleic acid, superior to other carboxylic acids reported in this paper, gives higher yields of glucose that is more easily fermented as a result of lower concentrations of degradation products. These results are especially significant because maleic acid, in the form of maleic anhydride, is widely available and produced in large quantities annually.

Pretreatment of corn fiber by pressure cooking in water

The pretreatment of corn fiber using liquid water at temperatures between 220 and 260°C enhances enzymatic hydrolysis. This paper describes the laboratory reactor system currently in use for cooking of corn fiber at temperatures ranging from 200 to 260°C. The corn fiber at approx 4.4% solid/liquid slurry was treated in a 2-L, 304 SS, Parr reactor with three turbine propeller agitators and a Proportional-Integral-Derivative (PID), controller that controlled temperature within ±1°C. Heat-up times to the final temperatures of 220, 240, or 260°C were achieved in 50 to 60 min. Hold time at the final temperature was less than 10 s. A serpentine cooling coil, through which tap water was circulated at the completion of the run, cooled the reactor’s contents to 180°C within 2 min after the maximum temperature was attained. Ports in the reactor’s head plate facilitated sampling of the slurry and monitoring the pH. A continuous pH monitoring system was developed to help observe trends in pH during pretreatment and to assist in the development of a base (2.0M KOH) addition profile to help keep the pH within the range of 5.0 to 7.0. Enzymatic hydrolysis gave 33 to 84% conversion of cellulose in the pretreated fiber to glucose compared to 17% for untreated fiber.

Cellulose to Sugars: New Path Gives Quantitative Yield

Cellulosic residues that had been treated with a small amount of chemical solvent under room conditions were quantitatively saccharified on enzyme hydrolysis. This treatment can be used to obtain simple sugars for the production of alcohol and other chemicals.

Reaction Kinetics, Molecular Action, and Mechanisms of Cellulolytic Proteins

Cellulolytic proteins form a complex of enzymes that work together to depolymerize cellulose to the soluble products cellobiose and glucose. Fundamental studies on their molecular mechanisms have been facilitated by advances in molecular biology. These studies have shown homology between cellulases from different microorganisms, and common mechanisms between enzymes whose modes of action have sometimes been viewed as being different, as suggested by the distribution of soluble products. A more complete picture of the cellulolytic action of these proteins has emerged and combines the physical and chemical characteristics of solid cellulose substrates with the specialized structure and function of the cellulases that break it down. This chapter combines the fundamentals of cellulose structure with enzyme function in a manner that relates the cellulose binding and biochemical kinetics at the catalytic site of the proteins to the macroscopic behavior of cellulase enzyme systems.

Protein chromatography using a continuous stationary phase

Abstract A continuous stationary phase consisting of yarns woven into a fabric is rolled and packed into mechanically stable liquid chromatography columns. This work utilized yarns having a characteristic width of 200–400 μm, made from 10–20-μm fibers consisting of 95% poly(m-phenylene isophthalamide) and 5% poly(p-phenylene terephthalamide). Although loadings on this stationary phase were low at 4 mg/g for bovine serum albumin and 6 mg/g for β-galactosidase, this material shows the interesting characteristic of a leveling off of plate height at mobile phase velocities of 30–80 cm/min. This phenomenon is explained on the basis of a coupling argument whereby a fraction of the mobile phase flows through the intramatrix pore space, and convective transport through the pore space dominates transport by diffusion. A modified Van Deemter expression is derived and shown to fit plate height data for polyethylene glycol standards having molecular weights of 200 and 20 000. The characteristics of this continuous stationary phase at high eluent velocities are discussed conditions which give separation of immunoglobulin G, bovine serum albumin, insulin and β-galactosidase in 12 min are described.

Chapter 1 - Fermentation Substrates from Cellulosic Materials: Production of Fermentable Sugars from Cellulosic Materials

Publisher Summary This chapter discusses the production of fermentable sugars from cellulosic materials. The chapter presents a comparison of the availability and the economy of utilization of cellulosic wastes as an alternative natural resource to those of petroleum crude oil, which is the major raw material source of current chemical industries. The chapter discusses the use of selective solvent extraction to fractionate cellulosic wastes into three individual components: (1) cellulose, (2) hemicellulose, and (3) lignin. Once cellulose is dissolved in a solution, it is neither protected by a crystalline structure nor lignin seal. The composition of cellulosic materials varies widely from plant species to species. It also varies because of different harvesting and storage conditions.

Chromatography for Rapid Buffer Exchange and Refolding of Secretory Leukocyte Protease Inhibitor

A DEAE‐cellulose stationary phase in a rolled configuration was used to separate recombinant secretory leukocyte protease inhibitor (rSLPI) from denaturants and reducing agents (3 M guanidine‐HCl and 5 mM DTT) in less than 5 min to promote refolding of the protein to an active form. The mobile phase consisted of buffer and 500 mM NaCl, where NaCl suppressed binding of protein to this stationary phase. Separation of an initial concentration of 2 mg/mL protein from the other constituents resulted in 96% recovery of the rSLPI at an average concentration of 1.28 mg/mL. When incubated for 4 h at 20 °C, the fractionated rSLPI gave a 46% yield of properly refolded protein. The protein concentration was 6.4 times higher than that reported in a previously published method, where refolding was carried out by diluting the mixture of protein, denaturants, and reducing agents by a factor of 10. The results show that a combination of rapid chromatographic separation over a cellulosic stationary phase followed by protein refolding will significantly enhance process throughput by minimizing tankage, water requirements, and process time.

Transport properties of rolled, continuous stationary phase columns

Continuous stationary phase columns consist of woven textile matrixes of fibers rolled into a cylindrical configuration and inserted into a liquid chromatography column. This configuration allows separations to be carried out at interstitial mobile phase velocities in excess of 100 cm/min and pressures of up to 700 psig for stationary phases based on cellulose. Ordinarily, these conditions would cause compaction of a cellulosic stationary phase to the point where flow is no longer possible. The packing of the column with cellulose as a continuous stationary phase enables these linear velocities to be achieved. Most importantly, this type of column allows the study of momentum transport and mass transfer in a media in which the mobile phase explores almost all of the void volumes in the column. The analysis of flow patterns in these columns has been modeled using elution patterns of both retained and unretained components, and plate height has been correlated as a function of velocities in the range of 1–100 cm/min. Engineering analysis of this type of chromatography column based on visual representation of the packed fibers by scanning electron microscopy, analysis of porosities using unretained (nonadsorbing) molecular probes, and application of momentum and mass transport equations is discussed.

Optimal Packing Characteristics of Rolled, Continuous Stationary-Phase Columns

Rolled, continuous stationary phases were constructed by tightly rolling and inserting a whole textile fabric into a chromatography column. This work reports the column performance, in terms of plate height, void fraction, and resolution, of 10 cellulose‐based fabrics. The relation between fabric structural properties of yarn diameter, fabric count, fabric compressibility, and column performance are quantitated. General requirements, including reproducibility of packing, for choosing fabrics to make a good SEC column are identified. This research showed that the packed columns have an optimal mass of fabric that minimizes plate height and maximizes resolution, in a manner that is consistent with chromatography theory. Mass of material packed is then an important column parameter to consider when optimizing columns for the rapid desalting of proteins. Proteins were completely separated from salt and glucose in less than 8 min at a pressure drop less than 500 psi on the rolled, continuous stationary‐phase columns. These results, together with stability and reproducibility, suggest potential industrial applications for cellulose‐based rolled, continuous stationary‐phase columns where speed is a key parameter in the production process.