Progress 09/15/01 to 09/30/06
Outputs
The research team has made steady and significant accomplishments in cellulase fermentation and foam fractionation. Robert Tanner's group at Vanderbilt University has (1) developed new analytical procedures for cellulase activity and its components in a micro or well-plate format to enable high-throughput sample analyses; (2) studied how synthetic surfactants affects the foaming behaviors and enzyme activity in cellulase (and lysozyme) solutions; and (3) developed a chaperone technique, using beta-cyclodextrin as the artificial chaperone, to renaturize the enzymes denatured by foaming. For example, with the addition of sodium dodecyl sulfate (surfactant) and cyclodextrin, 4-fold concentration of lysozyme has been achieved without losing any enzymatic activity. Patrick Lee's group at Tennessee Valley Authority has worked (with Lu-Kwang Ju's group at the University of Akron) towards comparison and optimization of pretreatment methods (including dilute acid method,
concentrated acid method, and non-acidic microwave-based methods) for producing hydrolysates containing higher contents of cellulo- and xylo-oligosaccharides for improved cellulase/xylanase production. The microwave-based hydrolysis involves two stages. In the first stage a delignin chemical is used to partially remove the lignin. The second-stage water (aqua) hydrolysis is performed to produce the xylooligosaccharides from the pretreated biomass. The final cellulose can be converted to glucose by additional acid-hydrolysis or enzymatic hydrolysis. The resulting sugar solution is suitable for bioethanol production. Ju's group has (1) evaluated the effects of various operation and culture (broth) factors on the foaming properties of the cellulase fermentation broths; (2) studied the cellulase productivity with different limiting nutrients and inducers in the medium and with different fermentation schemes (batch, fed-batch, and continuous, with various control mechanisms and feed
rates); (3) evaluated the productivity of cellulase in continuous cultures using lactose or mixtures of lactose and glycerol as the C-substrate; (4) worked towards developing a detailed kinetic model for cellulase expression, including descriptions on lactase synthesis, lactose hydrolysis (to glucose and galactose), glucose and galactose consumption by cells, cellulase induction (by lactose) and repression (by glucose and/or galactose), and cell growth on glucose, galactose and glycerol; and (5) demonstrated the feasibility of affinity foam fractionation to selectively concentrate/separate the enzymes from fermentation broth. A preliminary patent application has been filed for the affinity foam fractionation technique. Several groups of affinity foaming agents have been synthesized and/or studied. Rhamnolipids have been found to perform superbly in selective binding and enrichment of the cellulase component beta-glucosidase. The optimal agents for other components, endo- and
exo-glucanases, are yet to be finalized. Developing this simple and novel affinity foam fractionation technology will have significant impact on economical production of cellulase and other bioproducts.
Impacts
Economical cellulase production is of critical importance to the move of the nation towards increased use of renewable fuels. Developing process-level kinetic models helps our understanding of the complex interactions of multiple regulations in play and enables improved control and design for cellulase production. Foaming holds high potential as an economical and effective process for product (cellulase enzymes in this case) concentration and separation from fermentation broths. The development of affinity-foaming technology will prove valuable to the downstream processing of many products. The artificial chaperon approach to enhance product enrichment and minimize denaturation by foaming further facilitates the application of foam separation to a wide range of sensitive biological products.
Publications
- Burapatana, V., Prokop, A. and Tanner, R.D. 2005. Enhancing cellulase foam fractionation with surfactant addition. Applied Biochemistry and Biotechnology 121-124:541-552.
- Kirkland, R.A. and Tanner, R.D. 2005. Removing proteins from an aerated yeast fermentation by pulsing carbon dioxide: Replacing salting out as a method of protein precipitation. Applied Biochemistry and Biotechnology 121-124:685-694.
- Burapatana, V., Booth, E.A., Prokop, A. and Tanner, R.D. 2005. Effect of buffer and pH on detergent assisted foam fractionation of cellulase. Industrial and Engineering Chemistry Research 44:4968-4972.
- Burapatana, V., Prokop, A. and Tanner, R.D. 2005. A comparison of the activity reduction occurring in two detergent-assisted protein (cellulase and lysozyme) foam fractionation processes. Separation Science and Technology 40:2445-2461.
- Lo, C.-M., Zhang, Q., Lee, P. and Ju, L.-K. 2005. Cellulase production by Trichoderma reesei using sawdust hydrolysate. Applied Biochemistry and Biotechnology 121-124:561-573.
- Jeong, G.-T., Park, E.-S., Wahlig, V.L., Burapatana, V., Park, D.-H. and Tanner, R.D. 2004. Effect of pH on the foam fractionation of Mimosa putida L. seed proteins. Industrial & Engineering Chemistry Research 43:422-427.
- Burapatana, V., Prokop, A. and Tanner, R.D. 2004. Degradation of beta-glucosidase activity in a foam fractionation process. Applied Biochemistry and Biotechnology 113-116:619-625.
- Lambert, W.D., Du, L.P., Ma, Y., Loha, V., Burapatana, V., Prokop, A., Tanner, R.D. and Pamment, N.B. 2003. The effect of pH on the foam fractionation of beta-glucosidase and cellulase. Bioresource Technology 87:247-253.
- Zhang, Q., Lo, C.-M. and Ju, L.-K. 2007. Factors affecting foaming behavior in cellulase fermentation by Trichoderma reesei Rut C-30. Bioresource Technol. 98:753-760.
- Lee, K.-C.P. and Ju, L.-K. 2006. Hydrolysis of lignocellulosic materials by microwave irradiation. Presented at the 232nd American Chemical Society National Meeting in San Francisco. September 9-14, 2006.
- Zhang, Q., Lo, C.-M. and Ju, L.-K. 2006. Affinity Foam Fractionation of Trichoderma Cellulase. Applied Biochemistry and Biotechnology 129-132:1051-1065.
- Burapatana, V., Prokop, A. and Tanner, R.D. 2006. Effect of beta-cyclodextrin in artificial chaperones assisted foam fractionation of cellulase. Applied Biochemistry and Biotechnology 129-132:247-255.
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Progress 10/01/04 to 09/30/05
Outputs
Over the past four years, we have made steady and significant progresses. Briefly, Dr. Robert Tanner's group at Vanderbilt University has (1) studied how the addition of synthetic surfactants affects the foaming behaviors and enzyme activity (against denaturation) in cellulase solutions, and (2) developed a 'chaperone' technique for renaturizing the enzymes denatured by foaming. Dr. Patrick Lee's group at Tennessee Valley Authority has (1) improved the hardwood acid hydrolysis to produce hydrolysate with less/insignificant inhibition to cells and (2) developed a unique process for producing xylan hydrolysate. Dr. Lu-Kwang Ju's group at The University of Akron has (1) evaluated the effects of various operation and culture (broth) factors on the foaming properties of the cellulase fermentation broths, (2) studied the cellulase productivity with different limiting nutrients and inducers in the medium and with different fermentation schemes (batch, fed-batch, and
continuous, with various control mechanisms and feed rates), and (3) demonstrated the feasibility of affinity foam fractionation to selectively concentrate/separate the enzymes from fermentation broth. A preliminary patent application has been filed for the affinity foam fractionation technique. Unfortunately, despite the above progresses, a directly coupled foaming-fermentation process has not been successfully developed. The difficulty comes primarily from the cell removal occurring during the in-situ foaming. We have spent much time trying various foam column arrangements and backwash rates, and various cell immobilization techniques. None have succeeded well enough. In studies with cell immobilization, we have used media designed with certain limiting nutrients (C, N or P), to prevent growth of free cells outside of the immobilization matrix (e.g. Celite particles and polyurethane foam particles). With C-limited media, the mass transfer limitation caused the cells to sporulate in
the inner core. When switched to N- or P-limited media, the cell physiology changed much, and the cells had even higher tendency to sporulate. In the final phase of the project, we will pursue the following studies: (1) Develop a two-stage Aqua process for biomass pretreatment using microwave heating. The first objective is to optimize the two-stage aqua pretreatment process in a 2-gal Parr stainless steel agitated reactor while providing data to understand the new process. The second objective is to carry out similar reactions using a microwave heated reaction system. (2) Evaluate the productivity and composition of cellulase (and xylanase, if induced at elevated levels) using lactose vs. the enzyme hydrolysate of Aqua-pretreated biomass as the inducing substrates. (3) Develop the optimal affinity foaming agent for selective collection of cellulase from fermentation broths. We have had some promising results using simple substrate analogs. We have designed some much better agents for
the purpose. We believe this affinity foam fractionation technique will be much more economical than other conventional enzyme/protein separation/purification methods.
Impacts
The use of cellulose hydrolysate for cellulase production and for production of other value-added products is utterly important to the nation's move to increased use of renewable fuels. The development of the 2-stage Aqua pretreatment process has several potential advantages over the traditional acid hydrolysis process. It minimizes sugar degradation and inhibitor generation, eliminates the need of special reactor material for the acid hydrolysis process, removes most of the hemicellulose and some of the lignin, and produces the residues that retain the original biomass texture instead of the mud-like lignin material from the traditional dilute-acid process. The residues from this Aqua process can be effectively hydrolyzed with cellulase enzymes. Fermentation yields and rates from the enzyme hydrolysate should be higher because of the lower concentrations of inhibitors present. Foaming holds high potential as an economical and effective process for product (cellulase
enzymes in this case) concentration and separation from fermentation broths. The development of affinity-foaming technology will prove valuable to the downstream processing of many products. The artificial chaperon approach to enhance product enrichment and minimize denaturation by foaming further facilitates the application of foam separation to a wide range of sensitive biological products.
Publications
- Enhancing Cellulase Foam Fractionation with Surfactant Addition, V. Burapatana, A. Prokop and R. D. Tanner, Applied Biochemistry and Biotechnology, 121-124: 541-552 (2005).
- Removing Proteins from an Aerated Yeast Fermentation By Pulsing Carbon Dioxide: Replacing Salting Out as a Method of Protein Precipitation, R. A. Kirkland and R. D. Tanner, Applied Biochemistry and Biotechnology, 121-124: 685-694 (2005).
- Effect of Buffer and pH on Detergent Assisted Foam Fractionation of Cellulase, V. Burapatana, E. A. Booth, A. Prokop and R. D. Tanner, Industrial and Engineering Chemistry Research, 44: 4968-4972 (2005).
- Modeling the Dynamics of an Activated Sludge Process Using Yield and Growth Functions, Q. Deng and R. D. Tanner, Journal of Chemical Engineering of Japan, (2005).
- A Comparison of the Activity Reduction Occuring in Two Detergent-Assisted Protein (Cellulase and Lysozyme) Foam Fractionation Processes, V. Burapatana, A. Prokop and R. D. Tanner, Separation Science and Technology, 40: 2445-2461 (2005).
- Effect of Beta-Cyclodextrin in Artificial Chaperones Assisted Foam Fractionation of Cellulase, A. Prokop and R. D. Tanner, Applied Biochemistry and Biotechnology, (2006, in press).
- Affinity Foam Fractionation of Trichoderma Cellulase. Q. Zhang, C.-M. Lo and L.-K. Ju, Applied Biochemistry and Biotechnology (in press, 2006).
- Cellulase production by Trichoderma reesei using sawdust hydrolysate. C.-M. Lo, Q. Zhang, P. Lee, and L.-K. Ju, Applied Biochemistry and Biotechnology 121-124: 561-573 (2005).
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Progress 10/01/03 to 09/30/04
Outputs
Continuing on developing the combined foam fractionation and cellulase fermentation process, our effort has been focused on the following: 1. Enhance Selective Foaming of Cellulase from Fermentation Broth - When applying foam fractionation to cellulase separation, we found that cellulase is surface-active but not the most hydrophobic in the fermentation broth. To enhance their selectivity and enrichment, we have developed a new technology, affinity foam fractionation, and have filed a patent disclosure on the technology. More detailed descriptions and results will be reported later when the patent application is formally filed. 2. Evaluate Effects of Surfactant Addition - Along a similar line to the above affinity foam fractionation technology, we have evaluated the effects of addition of surfactant (cetyltrimethylammonium bromide, CTAB) and artificial chaperones (b-cyclodextrin, b-CD) on foam fractionation. These included the effects of CTAB and b-CD concentrations,
and the dynamics and temperature effects on cellulase refolding in b-CD solutions. Similar technique has also been applied to lysozyme. Like the multi-component cellulase, lysozyme could only be concentrated by foam fractionation with the addition of a surfactant. The surfactant, CTAB, did not work well with lysozyme, but sodium dodecyl sulfate (SDS) performed very well. With the addition of SDS, it was possible to concentrate lysozyme 4-fold without losing any enzymatic activity. We can now calibrate cellulase with the much better studied lysozyme (single protein) enzyme, each in a surfactant-assisted foam fractionation process. 3. Improve Cell Retention in Fermentation with In-Situ Foaming - a. Foam Column Design with Backwash - We have studied several foam column designs to improve the effectiveness of backwash on cell retention. Coupling the foam column with fermentor still needs more study. b. Cell Immobilization - We have also studied the possibility of immobilizing the cells in
celite particles for improving cell retention due to their larger sizes and higher densities. The study is ongoing, partly to address the problem of attrition of celite particles in the agitated fermentor. 4. Maintain Culture at Optimal Metabolic State for Cellulase Synthesis by Online Process Control - Cellulase synthesis peaks in the late growth/early stationary phase, which is extremely brief in batch culture. Fed-batch processes have therefore been suggested in the literature. We are developing an online control scheme, based on the rate of culture pH change, to guide the addition of inducing substrate(s) for continuously maintaining the optimal metabolic stage for cellulase synthesis. 5. Expand Foaming to Ethanol Production from Fermentation of Biomass Hydrolysate - Ethanol fermentation is known to be inhibited by accumulating product (ethanol) and hydrolysis compounds. The volatile ethanol and inhibitory compounds such as acetic acid and furfurals can be continuously removed
from the fermentation broth by foaming and collected by condensation. A model simulation has been developed to estimate the scope of applicability.
Impacts
The use of hydrolysate as soluble inducing substrate has potential for significantly improving the productivity and controllability of cellulase production processes, not limited to the application in the combined foam fractionation and fermentation process under development in this project. The continuing study on cell partition to the foam phase advances our understanding of the low cost, environment-friendly separation technology and enhances its future application to other bioprocesses. The artificial chaperon approach to enhance protein enrichment and, particularly, the potential renaturation of denatured proteins by addition of its native forms is fundamentally significant and, once developed, has broad applications to foam purification of various proteins.
Publications
- "Effect of pH on the Foam Fractionation of Mimosa putida L. Seed Proteins,"(with G.-T. Jeong, E.-S.Park, V.L.Wahlig,V.Burapatana and D.-H. Park), Industrial & Engineering Chemistry Research, Vol.43, No.2 (January 21, 2004), 422-427.
- "Degradation of Beta-Glucosidase Activity in a Foam Fractionation Process,"(with V. Burapatana and A. Prokop), Proceedings of the Twenty-Fifth Symposium on Biotechnology for Fuels and Chemicals, M. Finkelstein, J.D. McMillan, B.H. Davison and B. Evans, eds: Applied Biochemistry and Biotechnology, Vols.113-116 (Spring, 2004), 619-625.
- "Enhancing Cellulase Foam Fractionation with Surfactant Addition," (with V. Burapatana and A. Prokop), Proceedings of the Twenty-Sixth Symposium on Biotechnology for Fuels and Chemicals, B. H. Davison, J.D. McMillan and B.Evans, eds: Applied Biochemistry and Biotechnology , Vols. 121-124 (Spring, 2005).
- "Removing Proteins from an Aerated Yeast Fermentation By Pulsing Carbon Dioxide: Replacing Salting Out as a Method of Protein Precipitation," (with R. A. Kirkland), Proceedings of the Twenty-Sixth Symposium on Biotechnology for Fuels and Chemicals, B. H. Davison, J.D. McMillan and B. Evans, eds: Applied Biochemistry and Biotechnology , Vols. 121-124 (Spring, 2005).
- "Effect of Buffer and pH on Detergent Assisted Foam Fractionation of Cellulase," (with V. Burapatana, E. A. Booth and A. Prokop), Industrial and Engineering Chemistry Research, (August, 2005).
- "Factors affecting foaming behavior in cellulase fermentation by Trichoderma reesei Rut C-30." (Q. Zhang, C.-M. Lo, and L.-K. Ju) submitted to Bioresource Technology (2004).
- "Cellulase production by Trichoderma reesei using sawdust hydrolysate." (Q. Zhang, C.-M. Lo, P. Lee, and L.-K. Ju) Applied Biochemistry and Biotechnology (in press, 2005).
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Progress 10/01/02 to 09/30/03
Outputs
Continuing on developing the combined foam fractionation and cellulase fermentation process, our effort has been focused on the following: 1. Replace solid cellulose with a soluble inducing substrate, to minimize the substrate loss by being foamed out. This has been accomplished by using a sawdust hydrolysate prepared by a process developed by Tennessee Valley Authority. The hydrolysate has minor inhibition to cell growth but was demonstrated as an effective cellulase inducer, probably more effective than the pure cellulose. The soluble hydrolysate can be continuously and accurately added to the fermentation for optimal productivity. We are further developing the hydrolysis process and optimizing the hydrolysate utilization for cellulase production. 2. Evaluate cell retention with genetically engineered strain. The foaming would remove Trichoderma reesei RUT C-30 cells from fermentor. Making cell wall more hydrophilic may minimize this. T. reesei incorporates
hydrophobic proteins, particularly hydrophobin I, in the cell wall. We obtained a genetically engineered strain that does not synthesize hydrophobin I. We are evaluating the cell retention behavior of this strain during the foaming process. 3. Study cellulase enrichment in foam fractionation. After studying the foaming of fermentation broth, we extended the study to solutions of relatively pure cellulase. At pH 3-8, pure cellulase barely foamed. At pH 10 and above, it foamed well, but most of its activity was destroyed. Addition of organic surfactants can promote foaming to an appropriate level. Nine surfactants (nonionic, anionic, and cationic) were studied. SDS yielded the highest enrichment, but caused the most activity reduction. Pluronic F-68 and Tetronic 1107 yielded very good mass recoveries but did not lead to cellulase enrichment. The activity of the cellulase components (exocellulase, endocellulase and beta-glucosidase) changed in similar patterns as the whole cellulase
activity (FPU) did. The assays used were developed for a 96 well-plate reader under this USDA grant. We are working on the artificial chaperone method to help recover lost activity of foam-fractionated cellulase. One method uses cetyltrimethylammonium bromide (CTAB) and beta-cyclodextrin to reorient the protein into the enzymatically active position. CTAB is added before foaming and then washed off with beta-cyclodextrin after foaming. Another method is the pulse renaturation, which involves dilution of the denatured cellulase with active cellulase. Initial results of this latter method are promising.
Impacts
The use of hydrolysate as soluble inducing substrate has potential for significantly improving the productivity and controllability of cellulase production processes, not limited to the application in the combined foam fractionation and fermentation process under development in this project. The continuing study on cell partition to the foam phase advances our understanding of the low cost, environment-friendly separation technology and enhances its future application to other bioprocesses. The artificial chaperon approach to enhance protein enrichment and, particularly, the potential renaturation of denatured proteins by addition of its native forms is fundamentally significant and, once developed, has broad applications to foam purification of various proteins.
Publications
- Lambert W.D., Du L.P., Ma Y, Loha V., Burapatana V, Prokop A., Tanner R.D., Pamment NB (2003) The effect of pH on the foam fractionation of beta-glucosidase and cellulase. Bioresource Technology 87(3): 247-253.
- Burapatana V., Prokop A., Tanner R.D. Minimizing degradation of beta-glucosidase in foam fractionation column. Applied Biochemistry and Biotechnology (Accepted for publication, 2004)
- Zhang Q., Lo C.-M., Lee P., Ju L.-K. (2004) Cellulase Production by Trichoderma reesei Using Sawdust Hydrolysate. Submitted for publication.
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Progress 10/01/01 to 09/30/02
Outputs
A detailed study has been conducted for evaluating the effects of various fermentation parameters on foaming behaviors of cellulase fermentation. Samples were taken for evaluation at different stages of the batch cellulase fermentation. The factors evaluated included cellulose concentration, cell concentration, extracellular protein concentration, cellulase concentration, pH, and aeration rate. The broth foamability was confirmed to increase with increasing cellulase (FPU) activity. More importantly, the solids (both cellulose and cells) were found to come out with the foam. A detailed study confirmed that the fungal cells have hydrophobic surface and, thus, tend to partition to the foam. The cell hydrophobicity appears to result from the presence of a hydrophibic surface protein. We are currently working on ways of solving the cell removal problem, including the use of wash stream in the foam column and the use of mutant cells lacking the ability to synthesize the
surface hydrophobic protein. We have also collected several commercially available samples of cellulase (complex) as well as some pure components, and are evaluating their foaming capacity and determining what activity (out of the complex mixture) is preferentially adsorbed onto the foam. Separately, we have developed several new procedures for determining cellulase activity and its components. These standard methods were converted into a micro or well-plate format to enable processing of a large number of samples at one time. Methods included involved beta-glucosidase (BG, cellobiose as a substrate) (an exoglucanase enzyme) assay, filter paper activity (FP, a total cellulase activity), MUC assay (EXG, exoglucanase, based on methylumbelliferyl-beta-D-cellobioside as a substrate, an exocellulase assay) and CMC assay (Cx, ENG, an endoclucanase assay). The MUC methods, since it can be run in large format, allow us to study the effect of BG inhibitor, in order to distinguish between
different activities present in a sample. In another effort, the surface tension-pH profile of beta-glucosidase was established to determine its relationship to the corresponding profile of cellulase and to the foam fractionation of that cellulase. The goal of this work was to determine the optimal foaming points for both cellulase and cellobiase. These data may prove useful in the separation of certain components of cellulase, since the non-foaming hydrophilic beta-glucosidase does not foam as well as the hydrophobic components of cellulase at low concentrations. A key finding from these experiments was that there are two local minima in the surface tension-pH trajectory for Trichoderma reesei cellulase, as contrasted to the usual single minimum. The lower of these minimum points corresponds to the cellulase isoelectric point. The double minimum surface tension-pH profile was also observed for cellobiase alone. The optimal foaming pH for cellobiase alone was determined to be around
10.5, while for cellulase it was between 6 and 9.
Impacts
Understanding the contribution of various components to the foaming behavior of cellulase fermentation is crucial to the development of effective cellulase production process based on an environment-friendly foam fractionation technique. Economical cellulase production is important to the conversion of the abundant renewable cellulosic resources to useful products and energy.
Publications
- Zhang, Q., Lo, K. and Ju, L.-K. 2003. Factors affecting foaming behaviors of cellulase fermentation by Trichoderma reesei RutC-30. Enz. Microb. Technol. (submitted for publication).
- Lambert, W.D., Du, L., Ma, Y., Loha, V., Burapatana, V., Prokop, A., Tanner, R.D. and Pamment, N.B. 2003. The effect of pH on the foam fractionation of beta-glucosidase and cellulase. Bioresource Technol. 87 (3): 247-253.
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