Welcome to the Zhang Group Webpage!

BSE 5984 Enzyme Engineering (fall 2008, 2010)
BSE 3524 Unit Operation (spring 2009)

Research Statement

1. Cellulose solvent- and organic solvent-based lignocellulose fractionation (COSLIF) Technology
The largest challenge of cellulosic ethanol biorefineries is to effectively break recalcitrant lignocellulose and release soluble fermentable sugars. In response to this challenge, we have invented a novel Version 1 cellulose-solvent based lignocellulose fractionation technology [1], which can separate lignocellulose to four parts: amorphous cellulose, hemicellulose sugars, lignin, and acetic acid in the below flowchart. It features modest reaction condition (~50^oC, atmospheric pressure), amorphous cellulose generation, and efficient chemical recycling as shown below. As compared to (dilute acid) steam explosion, this technology can produce at least 20% more sugar yields and increase 20% revenues from acetic acid.

In addition, we have developed Version 2 and 3 lignocellulose fractionation, which have solved two main shortcomings of Version 1 -- reducing solvent use volume by several fold and avoiding dilute sugar reconcentration. This technology has been successfully applied to numerous feedstocks, such as corn stover, switchgrass, poplar, industrial hemp hurds, bamboo, common reed, etc. Furthermore, this technology can drastically decrease costly cellulase use for efficient glucan hydrolysis [2-4].

The improvement of specific cellulase activities can be implemented through cellulase engineering -- rational design and directed evolution for each cellulase component as well as the reconstitution of cellulase components [5]. We are applying the combinatorial technologies involving DNA mutagenesis techniques, enzyme cell surface display techniques, and evolutionary engineering for improving cellulase activity on insoluble substrate cellulose. Recently, we have improved beta-glucosidase thermostability and catalytic efficiency by directed evolution [6].

3. Cellulose hydrolysis mechanisms mediated by cellulase, cellulosome, and cellulolytic microbes

Cellulose biodegradation requires three types of cellulases (endoglucanase, exoglucanases, and beta-glucosidase) to work together [7]. The unidentified characteristics of heterogeneous cellulose, the complicated interaction between solid cellulose and cellulase components, and the synergic/competitive relationship among various cellulase components limit our understanding of this de-polymerization process and also lags behind the developments in lignocellulose pretreatment and cellulase improvement.

We have developed a generic functionally based mathematical model to simulate this complicated process [8]. A new rapid method has been developed to determine the degree of polymerization of cellulose [9]; a new kind of non-substitution, high reactivity, homogeneous, regenerated amorphous cellulose (RAC) has been prepared for studying cellulose hydrolysis [10]. Now we are developing several new technologies which will help us to characterize key substrate characteristics, and elucidate in-depth mechanisms that will provide new insights into lignocellulose pretreatment and cellulase improvement.

In addition, we have discovered a new cellulose hydrolysis mechanism for microbial cellulose hydrolysis mediated by a complexed cellulase system -- the Clostridium thermocellum cellulosome [11,12].

4. Enzymatic hydrogen production by cell-free synthetic enzymatic biotransformation[13,14]
The overall reaction is C₆H₁₀O₅ + 7 H₂O --> 12 H₂ + 6 CO₂. Our idea is to utilize the energy stored in sugars to break up water and produce energy in the form of hydrogen or oxidate sugars using water rather than oxygen. This technology would be perfect for mobile applications because of mild reaction conditions (~20-80 degree C and high hydrogen yields), complete conversion, after the technology improvement. We first propose SUGAR-powered CARS which stores sugar, convert sugar to hydrogen on board, generate electricity via PEM fuel cells, and drive motor [15]. This technology would solve the technical challenges associated with hydrogen storage, distribution, production, and safety simultaneously. The synthetic enzymatic pathway is shown below,

What is more important, the integration of this technology with PEM fuel cell would achieve the highest energy efficiency from chemical energy to mechanical energy. The details of life cycle analysis pertaining to the carbohydrate hydrogen economy vs other hypocretical future economies, such as methanol economy, electron economy, are being conducted.

References

[1] Zhang Y.-H.P., Ding S.-Y., Mielenz J.R., Cui J.-.B., Elander R.T., Laser M, Himmel M.E., McMillan, J.D., Lynd, L.R. 2007. Fractionating recalcitrant lignocellulose at modest reaction conditions. Biotechnology and Bioengineering 97(2): 214-223.

[2] Moxley G, Zhu Z, Zhang Y-HP*. 2008. Efficient sugar release by the cellulose solvent-based lignocellulose fractionation technology and enzymatic cellulose hydrolysis. Journal of Agriculture and Food Chemistry 56: 7885–7890 (PDF)

[3] Zhu Z, Sathitsuksanoh N, Vinzant T, Schell DJ, McMillan JD, Zhang Y-HP*. 2009. Comparative study of corn stover pretreated by dilute acid and cellulose solvent-based lignocellulose fractionation: Enzymatic hydrolysis, supramolecular structure, and substrate accessibility. Biotechnology and Bioengineering (Epub, PDF).

[4] Sathitsuksanoh N, Zhu Z, Templeton N, Rollin J, Harvey S, Zhang Y-HP*. 2009. Saccharification of a potential bioenergy crop, Phragmites australis (common reed), by lignocellulose fractionation followed by enzymatic hydrolysis at decreased cellulase loadings. Industrial & Engineering Chemistry Research (accepted).

[5] Zhang Y.-H.P., Himmel E. M., Mielenz J.R. 2006. Outlook for cellulase improvement: Screening and selection strategies. Biotechnology Advances, 22(5): 452-481.

[6] Liu W, Hong J, Bevan DR, Zhang Y-HP*. 2009. Fast identification of thermostable beta-glucosidase mutants on cellobiose by a novel combinatorial selection/screening approach. Biotechnology and Bioengineering (Epub, PDF).

[7] Zhang Y.-H.P., and Lynd L.R. 2004. Toward an aggregated understanding of enzymatic hydrolysis of cellulose: non-complexed cellulase systems. Biotechnology and Bioengineering 88:797-824.

[8] Zhang Y.-H.P., Lynd L.R. 2006. A functionally-based model for hydrolysis of solid cellulose by fungal cellulase. Biotechnology and Bioengineering, 94(5): 888-898.

[9] Zhang Y.-H.P., and Lynd L.R. 2005. Determination of the number average degree of polymerization of cellodextrins and cellulose with application to enzymatic cellulose hydrolysis Biomacromolecules 6: 1510-1515.

[10] Zhang Y.-H.P., Cui X.B., Lynd L.R., and Huang L. 2006. A transition from cellulose swelling to cellulose dissolution by o-phosphoric acid: Evidences from supramolecular structures and enzymatic hydrolysis. Biomacromolecules 7(2): 644-648.

[11] Zhang Y.-H.P., and Lynd L.R. 2005. Cellulose utilization by Clostridium thermocellum: bioenergetics and hydrolysis product assimilation. Proceedings of the National Academy of Sciences of USA 102:7321-7325.

[12] Lu Y.P., Zhang Y.-H.P., Lynd L.R. 2006. Evidence for enzyme-microbe synergy in cellulose utilization by Clostridium thermocellum. Proceedings of the National Academy of Sciences of USA 103(44): 16165-16169.

[13] Zhang Y.-H.P., Mielenz J.R., Evans B.R., Hopkins R.C., Adams M.W.W. 2007. High-yield hydrogen production from starch and water by synthetic enzymatic pathway. PLOS ONE 2(5): e456.

[14] Ye X, Wang Y, Hopkins RC, Adams MWW, Evans BR, Mielenz JR, Zhang Y-HP*. 2009. Spontaneous high-yield hydrogen generation from cellulosic materials catalyzed by enzyme cocktails. ChemSusChem 2:149-152. (PDF).

[15] Zhang Y-HP*. 2009. A sweet out-of-the-box solution for the hydrogen economy: Is sugar-powered car science fiction? Energy Environmental Science 2: 272-282 (PDF).

Driving Tomorrow by Biomass Sugars

Select Honors

Courses Taught

Research Statement

Research Support

The Zhang Lab
Home Biography Research Members Publications Activities News Awards Contact	Driving Tomorrow by Biomass Sugars through Imagination, Invention and Innovation Select Honors ASABE Sunkist Young Designer Award (2009) British Petroleum Young Scientists Award (IBS2008) DuPont Young Professor Award (2008) Outstanding New Assistant Professor Award from the COE of Virginia Tech (2008) Air Force Young Investigator Award (AFOSR) (2007) Best and Brightest of Esquire Magazine (2006) Ralph E. Powe Junior Faculty Enhancement Award (2006) Research Interests Biofuels, biomass conversion, and biorefinery Biomass pretreatement/fractionation -- Cellulose solvent- and organic solvent-based lignocellulose fractionation (COSLIF) Cell-free synthetic biology (Synthetic Pathway Biotransformation -- SyPaB) Cellulase engineering Cellulose hydrolysis mechanism and modeling (Bio)Chemical Process Integration, Optimization and Modeling Life Cycle Analysis Courses Taught BSE 5984 Enzyme Engineering (fall 2008, 2010) BSE 3524 Unit Operation (spring 2009) Research Statement Our research integrates chemical engineering design principles with protein biochemistry, microbiology, and modern biotechnology to solve several most important challenges for biofuels (transportation fuels, second generation biofuels -- cellulosic ethanol, butanol, microdiesel, and alkanes; third generation biofuels -- hydrogen and electricity) production from non-food renewable lignocellulose as below. Four main directions are: (1) cellulose solvent-based lignocellulose fractionation featuring modest reaction conditions, (2) cellulase engineering and cellulose hydrolysis mechanisms mediated by free cellulase, cellulosome, and cellulolytic microbe; (3) consolidated bioprocessing microorganisms from advanced biofuels and biobased products, and (4) hydrogen and electricity production by cell-free synthetic biology approaches. 1. Cellulose solvent- and organic solvent-based lignocellulose fractionation (COSLIF) Technology The largest challenge of cellulosic ethanol biorefineries is to effectively break recalcitrant lignocellulose and release soluble fermentable sugars. In response to this challenge, we have invented a novel Version 1 cellulose-solvent based lignocellulose fractionation technology [1], which can separate lignocellulose to four parts: amorphous cellulose, hemicellulose sugars, lignin, and acetic acid in the below flowchart. It features modest reaction condition (~50^oC, atmospheric pressure), amorphous cellulose generation, and efficient chemical recycling as shown below. As compared to (dilute acid) steam explosion, this technology can produce at least 20% more sugar yields and increase 20% revenues from acetic acid. In addition, we have developed Version 2 and 3 lignocellulose fractionation, which have solved two main shortcomings of Version 1 -- reducing solvent use volume by several fold and avoiding dilute sugar reconcentration. This technology has been successfully applied to numerous feedstocks, such as corn stover, switchgrass, poplar, industrial hemp hurds, bamboo, common reed, etc. Furthermore, this technology can drastically decrease costly cellulase use for efficient glucan hydrolysis [2-4]. *2. Cellulase Engineering via* Biomolecular Engineering The improvement of specific cellulase activities can be implemented through cellulase engineering -- rational design and directed evolution for each cellulase component as well as the reconstitution of cellulase components [5]. We are applying the combinatorial technologies involving DNA mutagenesis techniques, enzyme cell surface display techniques, and evolutionary engineering for improving cellulase activity on insoluble substrate cellulose. Recently, we have improved beta-glucosidase thermostability and catalytic efficiency by directed evolution [6]. 3. Cellulose hydrolysis mechanisms mediated by cellulase, cellulosome, and cellulolytic microbes** Cellulose biodegradation requires three types of cellulases (endoglucanase, exoglucanases, and beta-glucosidase) to work together [7]. The unidentified characteristics of heterogeneous cellulose, the complicated interaction between solid cellulose and cellulase components, and the synergic/competitive relationship among various cellulase components limit our understanding of this de-polymerization process and also lags behind the developments in lignocellulose pretreatment and cellulase improvement. We have developed a generic functionally based mathematical model to simulate this complicated process [8]. A new rapid method has been developed to determine the degree of polymerization of cellulose [9]; a new kind of non-substitution, high reactivity, homogeneous, regenerated amorphous cellulose (RAC) has been prepared for studying cellulose hydrolysis [10]. Now we are developing several new technologies which will help us to characterize key substrate characteristics, and elucidate in-depth mechanisms that will provide new insights into lignocellulose pretreatment and cellulase improvement. In addition, we have discovered a new cellulose hydrolysis mechanism for microbial cellulose hydrolysis mediated by a complexed cellulase system -- the Clostridium thermocellum cellulosome [11,12]. 4. Enzymatic hydrogen production by cell-free synthetic enzymatic biotransformation[13,14] The overall reaction is C₆H₁₀O₅ + 7 H₂O --> 12 H₂ + 6 CO₂. Our idea is to utilize the energy stored in sugars to break up water and produce energy in the form of hydrogen or oxidate sugars using water rather than oxygen. This technology would be perfect for mobile applications because of mild reaction conditions (~20-80 degree C and high hydrogen yields), complete conversion, after the technology improvement. We first propose SUGAR-powered CARS which stores sugar, convert sugar to hydrogen on board, generate electricity via PEM fuel cells, and drive motor [15]. This technology would solve the technical challenges associated with hydrogen storage, distribution, production, and safety simultaneously. The synthetic enzymatic pathway is shown below, What is more important, the integration of this technology with PEM fuel cell would achieve the highest energy efficiency from chemical energy to mechanical energy. The details of life cycle analysis pertaining to the carbohydrate hydrogen economy vs other hypocretical future economies, such as methanol economy, electron economy, are being conducted. See Our Vision about the Future Transportation and Energy Storage . References [1] Zhang Y.-H.P., Ding S.-Y., Mielenz J.R., Cui J.-.B., Elander R.T., Laser M, Himmel M.E., McMillan, J.D., Lynd, L.R. 2007. Fractionating recalcitrant lignocellulose at modest reaction conditions. *Biotechnology and Bioengineering* 97(2): 214-223. [2] Moxley G, Zhu Z, *Zhang Y-HP. 2008. Efficient sugar release by the cellulose solvent-based lignocellulose fractionation technology and enzymatic cellulose hydrolysis. Journal of Agriculture and Food Chemistry 56: 7885–7890 (PDF) [3] Zhu Z, Sathitsuksanoh N, Vinzant T, Schell DJ, McMillan JD, Zhang Y-HP. 2009. Comparative study of corn stover pretreated by dilute acid and cellulose solvent-based lignocellulose fractionation: Enzymatic hydrolysis, supramolecular structure, and substrate accessibility. Biotechnology and Bioengineering* (Epub, PDF). [4] Sathitsuksanoh N, Zhu Z, Templeton N, Rollin J, Harvey S, Zhang Y-HP. 2009. Saccharification of a potential bioenergy crop, Phragmites australis* (common reed), by lignocellulose fractionation followed by enzymatic hydrolysis at decreased cellulase loadings. *Industrial & Engineering Chemistry Research* (accepted). [5] Zhang Y.-H.P., Himmel E. M., Mielenz J.R. 2006. Outlook for cellulase improvement: Screening and selection strategies. *Biotechnology Advances, 22(5): 452-481. [6] Liu W, Hong J, Bevan DR, Zhang Y-HP. 2009. Fast identification of thermostable beta-glucosidase mutants on cellobiose by a novel combinatorial selection/screening approach. Biotechnology and Bioengineering (Epub, PDF). [7] Zhang Y.-H.P., and Lynd L.R. 2004. Toward an aggregated understanding of enzymatic hydrolysis of cellulose: non-complexed cellulase systems. Biotechnology and Bioengineering 88:797-824. [8] Zhang Y.-H.P., Lynd L.R. 2006. A functionally-based model for hydrolysis of solid cellulose by fungal cellulase. Biotechnology and Bioengineering, 94(5): 888-898. [9] Zhang Y.-H.P., and Lynd L.R. 2005. Determination of the number average degree of polymerization of cellodextrins and cellulose with application to enzymatic cellulose hydrolysis Biomacromolecules 6: 1510-1515. [10] Zhang Y.-H.P., Cui X.B., Lynd L.R., and Huang L. 2006*. A transition from cellulose swelling to cellulose dissolution by o-phosphoric acid: Evidences from supramolecular structures and enzymatic hydrolysis. Biomacromolecules* 7(2): 644-648. [11] Zhang Y.-H.P., and Lynd L.R. 2005. Cellulose utilization by Clostridium thermocellum: bioenergetics and hydrolysis product assimilation. *Proceedings of the National Academy of Sciences of USA* 102:7321-7325. [12] Lu Y.P., Zhang Y.-H.P., Lynd L.R. 2006. Evidence for enzyme-microbe synergy in cellulose utilization by Clostridium thermocellum. *Proceedings of the National Academy of Sciences of USA* 103(44): 16165-16169. [13] Zhang Y.-H.P., Mielenz J.R., Evans B.R., Hopkins R.C., Adams M.W.W. 2007. High-yield hydrogen production from starch and water by synthetic enzymatic pathway. *PLOS ONE* 2(5): e456. [14] Ye X, Wang Y, Hopkins RC, Adams MWW, Evans BR, Mielenz JR, *Zhang Y-HP. 2009.** Spontaneous high-yield hydrogen generation from cellulosic materials catalyzed by enzyme cocktails. ChemSusChem 2:149-152. (PDF). [15] *Zhang Y-HP. 2009. A sweet out-of-the-box solution for the hydrogen economy: Is sugar-powered car science fiction? Energy Environmental Science 2: 272-282 (PDF). Research Support DOE BioEnergy Science Center (BESC) AFOSR Young Investigator Award and Muri Program USDA ACS PRF DoD -- U.S. Army Research DuPont Young Professor Award VT ICTAS more If you are interested in our technology transfer (lignocellulose fractionation and enzymatic hydrogen production), please feel free to contact me or VTIP. Imagination, Innovation, Implementation (3I Biofuels Lab)**