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Bioseparations Presentations

Liquid Hot Water Pretreatment Inhibitors

E. Ximenes, Y. Kim, C. Farinas, M. R. Ladisch, 251st National ACS Meeting, Biofuel & Biobased Chemical Production: Biomass Pretreatment and Hydrolysis, San Diego, CA, March 14, 2016, , 


Abstract: Liquid hot water pretreatment enhances the rates and extents of cellulose hydrolysis for corn stover, sugar cane bagasse, switchgrass, hardwood, and other lignocellulosic materials as long as there is sufficient enzyme present to catalyze the reaction. The rationale that drives the use of pretreatment is the reduction in cost of enzyme and feedstock by increasing yields of fermentable sugars, principally glucose and xylose. Compared to untreated lignocellulose, pretreated feedstocks result in enhanced hydrolysis since pretreatment opens up the cell wall structure of the substrate, thereby enabling access of enzyme to the cellulose and disrupting the tightly packed cellulose structure. However, pretreatments also release inhibitors. More severe pretreatments are not always better since they can release greater amounts of inhibitors and deactivators which significantly reduce enzyme activity. Inhibitors include xylo-oligosaccharides, acetic acid, tannic acid, and phenolics. This effect is particularly noticeable as enzyme loading is decreased and the ratio of biomass derived inhibitors to added enzyme protein increases. Higher severity pretreatment may also expose more lignin as well as more cellulose in the cell wall structure. The lignin may unproductively adsorb proteins, including enzymes. Hence pretreatment can both help and hinder the enzyme hydrolysis of cellulose. This paper describes interactions between multiple enzyme components, inhibitors, and pretreated lignocellulosic substrates. Mitigation strategies are presented that reduce the amount of enzymes required to overcome inhibition due to pretreatment and achieve high conversion of lignocellulosic feedstocks to fermentable monosaccharides.

Research Area: Bioseparations

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Bacterial Transport in Rolled Stationary Phase Monoliths

Bwatwa, J., N. S. Mosier, T. Huang, X. Liu, A. Stewart, C. M. Ladisch, and M. R. Ladisch,  Division of Analytical Chemistry, Paper 349, 229th National ACS Meeting of the American Chemical Society, San Diego, CA, March 15, 2005


Abstract: Rolled cotton monoliths enable rapid desalting of proteins in 1 to 10 minutes, and constitute an excelent hydrophilic chromatography support. The monoliths display rigidity and robustness at mobile phase linear velocities of 100 cm/min, unlike beds of cellulose particles which collapse at these conditions. This stationary phase is able to pass microorganisms without plugging. This has led to investigation of rolled stationary phase for rapid preprocessing of homogenized meat broths to separate and recover microbial cells. We report results fro the fractionation of microorganisms from a broth of lipids, protein and other colloidal particles and innoculated with GFP expressing E. coli. The location of GFP expressing E. coli during their passage through the monlith is readily monitored using fluorescence microscopy. The overall characteristics of the rolled monolith having a 2.5 cm diameter ar emodeled, and the probable trajectory of microbial cells, based on work with particle flow over single fibers, is estimated. The passage of the bacteria entails both tangential and radial flow. Application of rolled stationary phase monoliths to rapid filtering, fractionation, and detection of both proteins and bacteria using microfluidic devices is presented with examples.

Research Area: Bioseparations

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Microscopic Examination of Changes of Plant Cell Structure in Corn Stover Due to Cellulase Activity and Hot Water Pretreatment

Zeng, M., N. S. Mosier, C. Huang, D. Sherman, J. Goetz, and M. R. Ladisch, Division of Biochemical Technology, Poster 335, 229th National ACS Meeting of the American Chemical Society, San Diego, CA, March 16, 2005


Abstract: Particle size has a significant impact on the saccharification of plant cell walls by cellulolytic enzymes. It is believed that small particle sizes of a cellulosic substrate are more readily hydrolyzed than large ones and that pretreatment enlarges accessible and susceptible surface area. These hypotheses are being tested using ground corn stover (stalks and leaves) in the size range of 425 to 710 um and 53 to 75 um. Scanning electron microscopy shows that enzyme treatment induces pore formation in the surface of the corn stover. Corn stover pretreated at 190 C for 15 min generates a few pores on the surface. When followed by enzyme hydrolysis, pretreated stover exhibits greater porosity than the enzyme hydrolyzed stover that has not been pretreated. Comparison of the microscopic changes to macroscopic features of hydrolysis suggests that mechanism of enzyme action is more complex than would be suggested by particle size or surface area. This paper correlates microscopic change in structure to the activity of enzyme hydrolysis before and after pretreatment. The objective is to understand the changes that occur at cellular level, compared to a particulate or macroscopic level. In this manner, a specific understanding of enzyme activity on a cellular level can be developed and ultimately translated to pretreatment processes that impove hydrolysis.

Research Area: Bioseparations

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Rapid Prototyping of Purification Platforms

Huang, T., N. S. Mosier, and M. R. Ladisch, Division of Biochemical Technology, Paper 63, 229th National ACS Meeting of the American Chemical Society, San Diego, CA, March 14, 2005


Abstract: The development of protein products, particularly monoclonal antibodies, for pharmaceutical applicatins requires rapid development of purification methods. Previously small analytical columns, and advance scale systems have been used to evaluate different types of stationary phase, and to quickly evaluation whether or not its separation characteristics are compatible with the molecules to be fractionated. This particular paper presents an approach which utilizes rapid prototyping of microchips in order to rapidly evaluate different types of stationary phases. These chips are based on fibers to which particles of different ion exchange groups or antibodies are anchored. The labeled proteins are then microscopically observed with respect to the retention behavior. This work describes the rapid assembly of different types of stationary phases required for separation, and methodologies for the rapid evaluation of the observed fractionation. Examples are based on lgG class antibody interactions with affinity base stationary phases such as Protein A. The methods show how the observed properties can be used to quickly define the most appropriate stationary phase, and then begin rapid evaluation with respect to scale parameters. Since the methodology is based on path lengths that are less than 10 microns from the liquid to the surface of the stationary phase, difficusion control is the limiting factor. Consequently, close observation of separation chracteristics can be quickly conceived, reduced to practice, and be evaluated.

Research Area: Bioseparations

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Bioseparations for Biochips

Chen, W.-T. (Speaker), T. Geng, R. Hendrickson, A. K. Bhunia, and M. R. Ladisch, Paper 328A, AIChE Annual Meeting, Indianapolis, IN, November 7, 2002


Abstract: Biochip technology has opened the door to rapid detection of pathogens compared to conventional methods. Detection of some pathogens, notably Listeria monocytogenes, takes up to a week. It is costly both economically and in terms of food safety. Biochips offer detection times in hours, not in days. However, sample procesisng is required in order to remove the interferring substances, ideally leaving only the microorganisms. Biological samples, especially food products are complex substances containing crabohydrates, proteins, lipids, and salts, which interfere with the selectivity of the binding sites on the chips. This paper describes preparation for food samples by various chromatographic resins, including cationic, anionic ion exchangers, hydrophobic, bifunctional and reverse-phase resins. Among these, Amberlite 35, which is a strong cationic ion exchanger, gave the highest adsorption of proteins.

Research Area: Bioseparations

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