Progress 12/01/02 to 09/30/08
Outputs
OUTPUTS: The purification of proteins from various sources is an important aspect of bioprocessing of pharmaceuticals, enzymes, antibodies, etc. Traditional separation and recovery techniques can be quite expensive. Hence, targeting better protein separation and purification techniques may result in a significant reduction in downstream processing costs. Foam fractionation has been shown to be a feasible technique for the separation and concentration of a variety of proteins and enzymes. This project focuses on the feasibility of using foam fractionation as a means to separate/concentrate industrial enzymes and other valuable proteins. Over the course of the project we have investigated foam fractionation as a possible strategy to i) concentrate whey proteins from dilute and concentrated whey solutions, ii) recover an industrial enzymes, cellulose, from a crude protein solution, and iii) recover a recombinant histidine-tagged protein from a tobacco extract solution. In addition, we have investigated the use of light scattering to characterize the foam structure and the performance of the foam fractionation column. This overall project represents a wide-range of application of foam fractionation as a recovery strategy. Foam fractionation is expected to be a low-cost alternative for the recovery and concentration of various proteins. PARTICIPANTS: Kelly Gillette (technician): helped to set up the system and the lab, worked on the BSA paper. Melanie Loiselle (post-doc): worked on the recovery from tobacco, Indu Maiti (collaborator): helped with the tobacco project, Paul Bummer (collaborator): helped with the tobacco project, Mike Jay (collaborator): helped with the tobacco project, Mustafa Aslan (collaborator): helped with the foam sensor project, Daniel Tao (collaborator): helped with the foam sensor project, Pinar Menguc (collaborator): helped with the foam sensor project, JN Swamy (PhD student): worked on the foam sensor project, Aubrey Shae (MS student): worked on the foam sensor project, Fred Payne (collaborator): heled with the foam sensor project, Over a dozen undergraduate students also worked on the various projects. TARGET AUDIENCES: The target audience for this project is bioprocessing companies. PROJECT MODIFICATIONS: On the most part, the project proceeded as planned. One objective, recovery of industrial enzymes, was abandoned after initial experiments. However, a new objective, development of a foam sensor, was included and proved successful.
Impacts
Recovering a Recombinant Protein: Tobacco plants can be used for the production of proteins for pharmaceutical applications. One of the most difficult and expensive tasks associated with this technology is isolating the product of interest from the hundreds of other chemicals found in tobacco. This work describes a new recovery strategy where the protein of interest is tagged with a histidine structure, which forms a complex with metal ions and a surfactant that will accumulate in the foamate of a foam fractionation step. His-gus, a histidine tagged enzyme, was selectively recovered in the presence of two different surfactants and two different metal ions. The foam fractionation with DCL surfactant and Ni2+ ions resulted in an average His-gus activity recovery value of 88% and an activity enrichment of 2.27. The performance of the recovery strategy without tobacco extract resulted in an average activity recovery value of 63.32% and an average activity enrichment value of 5.16, utilizing LED3A surfactant and Ni2+ ions. It was shown, that even though a majority of the native tobacco proteins are removed during the prefoaming step, the presence of tobacco extract does affect the recovery of His-gus. Recovering Industrial Enzymes: We were able to concentrate cellulose and xylanase samples, where the final product is up to 1.4 times more concentrated than the original sample. However, on an industrial scale the enrichment needs to be much higher. For this reason, this aspect of the project was abandoned. Recovering a Waste Product: Whey, a byproduct of cheese production, is often considered a waste stream and typically transported out of dairy plant for a per volume charge. One possible method to reduce the waste volume and disposal costs is to concentrate whey by foam fractionation and potentially produce a valuable co-product, a concentrated whey protein solution. We have been able to recovery as much as 90% of the original whey proteins and have been able to achieve a nine-fold increase in the whey protein concentration on a laboratory scale. We have also investigated the use of surfactants, heat, and gas type as a means to increase performance. Development of a Foam Sensor: Liquid foams being dynamic systems with a wide variety of industrial applications require continuous and non-intrusive diagnostics for monitoring of their bulk properties. The utility of polarized light scattering for characterizing liquid foams has been investigated with a vision for developing in-line diagnostic systems that can be used to monitor the foam properties and provide a basis for process control. A theoretical understanding of the polarized light propagation in foams was obtained through Monte Carlo ray tracing simulations by coupling a statistical model based on foam physics with a vector Monte Carlo scheme. The effects of the average bubble size and polydispersity on the depolarization of incident polarized laser beam were investigated. The results showed that depolarization ratios were sensitive to these parameters.
Publications
- Crofcheck, C., Gillette, K. 2003. Evaluation of Foam Fractionation Column Scale-Up for Recovering a Model Protein. Trans. ASAE, 46(6):1759-1764.
- Crofcheck, C. L., Loiselle, M., Weekley, J., Maiti, I., Pattanaik, S., Bummer, P. M., Jay, M.. 2003. Histidine Tagged Protein Recovery from Tobacco Extract by Foam Fractionation. Biotech. Progress, 19(2):680-682.
- Crofcheck, C., Maiti, I., Pattanaik, S. and Jay, M. 2004. Effect of Ion and Surfactant Choice on the Recovery of a Histidine Tagged Protein from Tobacco Extract Using Foam Fractionation. Applied Biochemistry and Biotechnology, 119(1): 79-92.
- Aslan, M. M., C. Crofcheck, D. Tao, M. P. Menguc. 2006. Evaluation of Micro-bubble Size and Gas Hold-up in Two-Phase Gas Liquid Columns via Scattered Light Measurements. J. Quant. Spectro. Radiative Transfer. 101, 527-539.
- Swamy, J. N., C. Crofcheck, M. P. Menguc. 2007. A Monte Carlo Ray Tracing study of Polarized Light Propagation in Liquid Foams. Journal of Quantitative Spectroscopy and Radiative Transfer, 104, 277-287.
- Shea, A. P., C. L. Crofcheck, F. A. Payne, and Y. L. Xiong. 2008. Foam Fractionation of alpha-Lactalbumin and beta-Lactoglobulin from a Whey Solution, Asia-Pacific Journal of Chemical Engineering (in press).
- Swamy, J. N., C. Crofcheck, M. P. Menguc. 2008. Time Dependent Scattering Properties of Slow Decaying Liquid Foams, Colloids and Surfaces A: Physicochemical and Engineering Aspects (in press).
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Progress 01/01/07 to 12/31/07
Outputs
The purification of proteins from various sources is an important aspect of bioprocessing of pharmaceuticals, enzymes, antibodies, etc. Traditional separation and recovery techniques can be quite expensive. Hence, targeting better protein separation and purification techniques may result in a significant reduction in downstream processing costs. Foam fractionation has been shown to be a feasible technique for the separation and concentration of a variety of proteins and enzymes. This project focuses on the feasibility of using foam fractionation as a means to separate/concentrate industrial enzymes and other valuable proteins. We are currently continuing work on optimizing the concentration of whey proteins from dilute and concentrated whey solutions and the development of a light-based sensor to monitor the foam fractionation process. We have been able to recovery as much as 90% of the original whey proteins and have been able to achieve a nine-fold increase in the
whey protein concentration on a laboratory scale. We have also investigated the use of surfactants, heat, and gas type as a means ti increase performance. We have been able to show that a light scattering technique has the potential to monitor the bubble size and the liquid hold up in liquid foams; however it doesn't appear that the inclusion of the polarized state of the light contributes to the system. This overall project represents a wide-range of application of foam fractionation as a recovery strategy. Foam fractionation is expected to be a low-cost alternative for the recovery and concentration of various proteins.
Impacts
This project focuses on the development of a separation/concentration process that could result in significant decreases in the cost of producing industrial enzymes and pharmaceutical proteins, as well as waste disposal.
Publications
- Shea, A. P., C. L. Crofcheck, F. A. Payne, and Y. L. Xiong. 2008. Foam Fractionation of α-Lactalbumin and β-Lactoglobulin from a Whey Solution, Asia-Pacific Journal of Chemical Engineering (submitted).
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Progress 01/01/06 to 12/31/06
Outputs
The purification of proteins from various sources is an important aspect of bioprocessing of pharmaceuticals, enzymes, antibodies, etc. Traditional separation and recovery techniques can be quite expensive. Hence, targeting better protein separation and purification techniques may result in a significant reduction in downstream processing costs. Foam fractionation has been shown to be a feasible technique for the separation and concentration of a variety of proteins and enzymes. This project focuses on the feasibility of using foam fractionation as a means to separate/concentrate industrial enzymes and other valuable proteins. We are currently working on optimizing the concentration of whey proteins from dilute and concentrated whey solutions and the development of a light-based sensor to monitor the foam fractionation process. We have been able to recovery as much as 90% of the original whey proteins and have been able to achieve a nine-fold increase in the whey
protein concentration on a laboratory scale. We have been able to show that a light scattering technique has the potential to monitor the bubble size and the liquid hold up in liquid foams; however it doesn't appear that the inclusion of the polarized state of the light contributes to the system. This overall project represents a wide-range of application of foam fractionation as a recovery strategy. Foam fractionation is expected to be a low-cost alternative for the recovery and concentration of various proteins.
Impacts
This project focuses on the development of a separation/concentration process that could result in significant decreases in the cost of producing industrial enzymes and pharmaceutical proteins, as well as waste disposal.
Publications
- Swamy, J. N., C. Crofcheck, M. P. Menguc. 2007. A Monte Carlo Ray Tracing study of Polarized Light Propagation in Liquid Foams. Journal of Quantitative Spectroscopy and Radiative Transfer, 104, 277-287.
- Aslan, M. M., C. Crofcheck, D. Tao, M. P. Menguc. 2006. Evaluation of Micro-bubble Size and Gas Hold-up in Two-Phase Gas Liquid Columns via Scattered Light Measurements. J. Quant. Spectro. Radiative Transfer. 101, 527-539.
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Progress 01/01/05 to 12/31/05
Outputs
We are currently working on optimizing the concentration of whey proteins from dilute and concentrated whey solutions. We are investigating the use of a continuous system with and without the addition of surfactant to increase foaming and protein recovery. Preliminary results indicate that the enrichment is better for more dilute solutions. Strategies are being tested to increase the enrichment and recovery at protein levels similar to those found in industry. In a related project, we're looking at the protein recovery of histidine tagged protein from tobacco extract facilitating the use of nickel sub-micron particles. While this project doesn't involve foam fractionation, the foam fractionation work has lead directly to these additional studies. We have developed and assembled the experimental system and confirmed that the presence of nickel particles doesn't harm the histidine-tagged protein. Preliminary experiments indicate that the strategy does work, where the
histidine tagged proteins complex with the surface of the nickel particles and then the nickel particles are collected using a magnetic field. Future work will focus on quantifying and optimizing recovery performance.
Impacts
This project focuses on the development of a separation/concentration process that could result in significant decreases in the cost of producing industrial enzymes and pharmaceutical proteins, as well as waste disposal.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs
We are currently investigating the use of foam fractionation with several different protein systems. In one system, recombinant proteins are recovered from tobacco extract via foam fractionation. In another, a dilute solution of xylanase, which is an industrial enzyme used in the conversion of hemicellulases into sugars, was concentrated. In the final system, whey proteins are recovered from dilute and concentrated solutions, in an attempt to recover proteins that could be used again, instead of needing to be disposed of. Tobacco plants can be used for the production of proteins for pharmaceutical applications. One of the most difficult and expensive tasks associated with this technology is isolating the product of interest from the hundreds of other chemicals found in tobacco. This work describes a new recovery strategy where the protein of interest is tagged with a histidine structure, which forms a complex with metal ions and a surfactant that will accumulate in
the foamate of a foam fractionation step. His-gus, a histidine tagged enzyme, was selectively recovered in the presence of two different surfactants and two different metal ions. The foam fractionation with DCL surfactant and Ni2+ ions resulted in an average His-gus activity recovery value of 88% and an activity enrichment of 2.27. The performance of the recovery strategy without tobacco extract resulted in an average activity recovery value of 63.32% and an average activity enrichment value of 5.16, utilizing LED3A surfactant and Ni2+ ions. It was shown, that even though a majority of the native tobacco proteins are removed during the prefoaming step, the presence of tobacco extract does affect the recovery of His-gus. We have been able to concentrate xylanase samples, where the final product is up to 1.4 times more concentrated than the original sample. The conditions need to be further investigated to test the robustness of the procedure. For every pound of cheese manufactured,
nine pounds of whey are produced. This large volume of whey produced is typically considered a waste stream and must be transported out of the plant for a per volume charge. If the waste volume could be reduced by reducing the volume to be transported, costs could be significantly reduced. One way to reduce the amount of whey leaving a cheese making process could be foam fractionation to concentrate the waste stream. The recovery and enrichment values for whey vary with pH and initial concentration. There are several different types of whey waste streams with drastically different proteins levels. For these results, it appears that foam fractionation would be better suited to remove proteins from the dilute processing waste streams. In order to maintain foam stability, the column will most likely be thin and tall or will require the addition of a surfactant. The pH may need to be adjusted, depending on the initial pH of the stream.
Impacts
This project focuses on the development of a separation/concentration process that could result in significant decreases in the cost of producing industrial enzymes and pharmaceutical proteins, as well as waste disposal.
Publications
- Crofcheck, C., Maiti, I., Pattanaik, S. and Jay, M. 2004. Effect of Ion and Surfactant Choice on the Recovery of a Histidine Tagged Protein from Tobacco Extract Using Foam Fractionation. Applied Biochemistry and Biotechnology, 119(1): 79-92.
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Progress 01/01/03 to 12/31/03
Outputs
The purification of proteins from various sources is an important aspect of bioprocessing of pharmaceuticals, enzymes, antibodies, etc. Traditional separation and recovery techniques can be quite expensive. Hence, targeting better protein separation and purification techniques may result in a significant reduction in downstream processing costs. Foam fractionation has been shown to be a feasible technique for the separation and concentration of a variety of proteins and enzymes. This project focuses on the feasibility of using foam fractionation as a means to separate/concentrate industrial enzymes and other valuable proteins. We are currently working on optimizing the i) concentration of whey proteins from dilute and concentrated whey solutions, ii) recovery of the industrial enzyme cellulase from a crude protein solution, and iii) the recovery of a recombinant histidine-tagged protein from a tobacco extract solution. We have been able to recovery as much as 90% of
the original whey proteins and have been able to achieve a nine-fold increase in the whey protein concentration on a laboratory scale. For the cellulase work, we have been able to recover as much as 50% of the original protein, while concentrating the solution by four-fold. This represents a significant accomplishment and further work is necessary to further verify and improve this recovery. For the recovery of a histidine-tagged protein from a tobacco extract, we have been able to recover as much as 90% of the original protein with a 2.5-fold increase in the protein concentration from a yeast extract solution. The results are not as promising for the recovery experiments from a tobacco extract solution. This suggests that further work is need to improve the recovery from a tobacco extract. This overall project represents a wide-range of application of foam fractionation as a recovery strategy. Foam fractionation is expected to be a low-cost alternative for the recovery and
concentration of various proteins. It is our hope to start looking into the scale up challenges for each of these applications within the next year.
Impacts
This project focuses on the development of a separation/concentration process that could result in significant decreases in the cost of producing industrial enzymes and pharmaceutical proteins, as well as waste disposal.
Publications
- Crofcheck, C. , Gillette, K. 2003. Evaluation of Foam Fractionation Column Scale-Up for Recovering a Model Protein. Trans. ASAE, 46(6):1759-1764.
- Crofcheck, C. L., Loiselle, M., Weekley, J., Maiti, I., Pattanaik, S., Bummer, P. M., Jay, M.. 2003. Histidine Tagged Protein Recovery from Tobacco Extract by Foam Fractionation. Biotech. Progress, 19(2):680-682.
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