Welcome to the Zhang Group Webpage!

Driving Tomorrow by Biomass Sugars

through Imagination, Invention and Innovation

Select Honors

Biotechnology & Bioengineering Daniel IC Wang Award (2010)

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)

Major Achievements

Invented the most efficient lignocellulose fractionation technology (COSLIF) by using cellulose solvents (concnetrated phosphoric acid or ionic liquids). Patent filed in March 2006, and licensed in 2009.

Proposed cell-free synthetic pathway biotransformation (SyPaB) as the ultimate platform for low-cost production of biofuels, bioelectricity, and biochemicals.

Produced ~12 moles of hydrogen from per mole of glucose unit and water for the first time. Through this conversion, the energy output is more than the input based on useful chemical energy by absorbing useless ambient-temperature heat.

Proposed the Electricity-Carbohydrate-Hydrogen (ECHo) cycle, which would ultimately solve several important challenges for sustainability.

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 sustainability, such as (1) cost-efficient production of second generation biofuels -- cellulosic ethanol, alkanes, and third generation biofuels -- hydrogen and electricity; (2) cost-effective release fermentable sugars from non-food lignocellulosic biomass; (3) high efficient CO2 biological fixation; (4) electricity storage on large scales; (5) food production; (6) water conservation; and (7) constructing a sustainable ecosystem for space travel. The below figure shows our research topics associated with biofuels production.

Key Research Directions

1. Cell-free synthetic pathway biotransformation (SyPaB)

Cell-free synthetic pathway biotransformation (SyPaB) is assembly of a number of purified enzymes and coenzymes for implementing complicated biochemical reactions [1,2]. Due to its great advantages, such as high product yields, fast reaction rates, high product titers, and great engineering flexibility, SyPaB will become a disruptive technology as compared to microbial fermentation.

The successful examples are the production of high-yield hydrogen from starch or cellulosic materials and water [3,4].

Furthermore, we propose to use renewable low-cost carbohdyrates as a high-energy density hydrogen carrier (~8-14.8 H2 wt%). In the future, the sugar-powered vehicles will have a sugar container, on-board bioreforming, PEM fuel cells, rechargeable battery, and moter [5]. This hypothetic system will have numerous advantages over eletric vehicles [6]. Also, our life cycle analysis suggests that a small fraction of the USA biomass resource would meet 100% transportation fuel needs based on our new technology.

2. Cellulase engineering

Cellulase engineering includes directed evolution, rational design, and reconstitution of individual components [7]. The large challenge for cellulase engineering is how to evaluate their activties on pretreated heterogeneous cellulosic substrates [7,8]. Recently, we have improved beta-glucosidase thermostability and catalytic efficiency by directed evolution [9]. Endoglucanase performance has been improved by using directed evolution and cell-surface display (submitted).

3. Consolidated bioprocessing (CBP) microorganisms

Consolidated bioprocessing (CBP) is integration of cellulase production, cellulose hydrolysis and sugar fermentation in a single step [10-12]. Now we are working on a platform based on industrially-safe Bacillus subtilis. Now we have succeeded in expressing Clostridium phytofermentanas Cel48 cellobiohydrolase, Cel9 processive endoglucanase and several Cel5 endoglucanases in B. subtilis [13]. We have obtained the first celf-catalyzing recombinant cellulolytic microorganisms that can produce enough secretory cellulases, hydrolyze cellulose without any other organic nutrients, and support their growth and cellulase synthesis (in preparation for publication).

4. Cellulose solvent- and organic solvent-based lignocellulose fractionation (COSLIF)

The largest challenge of cellulosic ethanol biorefineries is to effectively break recalcitrant lignocellulose and release soluble fermentable sugars. We have invented a cellulose-solvent based lignocellulose fractionation technology [14], 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.

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 [15-18].

5. 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 [19]. 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 [20]. A new rapid method has been developed to determine the degree of polymerization of cellulose [21]; a new kind of non-substitution, high reactivity, homogeneous, regenerated amorphous cellulose (RAC) has been prepared for studying cellulose hydrolysis [22]. 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 [23].

See Our Vision about the Future Transportation and Energy Storage.

References

[1] Zhang Y-HP.* 2010.Production of biocommodities and bioelectricity by cell-free synthetic enzymatic pathway biotransformations (SyPaB): Challenges and opportunities. Biotechnology and Bioengineering (accepted).

[2] Zhang Y-HP*. 2009. Using extremophile enzymes to generate hydrogen for electricity. Microbe 4(12): In press .

[3] Zhang Y-HP., 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.

[4] 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).

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

[6] Zhang Y-HP*, Rollin JA. 2009. Technical comments on “Greater transportation energy and GHG offsets from bioelectricity than ethanol. Nature Precedings 2009.3725.1.

[7] Zhang Y-HP*, Himmel ME, Mielenz JR. 2006. Outlook for cellulase improvement: Screening and selection strategies. Biotechnology Advances 24(5): 452-481.

[8] Zhang Y-HP*, Hong J, Ye X. 2009. Cellulase Assays. Methods in Molecular Biology 581: 213- 231.

[9] 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 103:1087-1094.

[10] Lu Y, Zhang Y-HP, Lynd LR*. 2006. Evidence for enzyme-microbe synergy in cellulose utilization by Clostridium thermocellum. Proceedings of the National Academy of Sciences of the USA 103(44): 16165-16169.

[11] Zhang Y-HP, Lynd LR*. 2005. Cellulose utilization by Clostridium thermocellum: Bioenergetics and hydrolysis product assimilation. Proceedings of the National Academy of Sciences of the USA 102: 7321-7325.

[12] Zhang Y-HP*, Lynd LR. 2008. New generation biomass conversion: Consolidated bioprocessing. Biomass Recalcitrance: Deconstructing the Plant Cell Wall for Bioenergy (ed. by Himmel, M.E.) Blackwell Publishing. ISBN: 9781405163606. pp 480-493.

[13] Zhang XZ, Zhang ZM, Sathitsuksanoh N, Yang D, Zhang Y-HP*. 2009. The non-cellulosomal family 48 cellobiohydrolase from Clostridium phytofermentans ISDg: Heterologous expression, characterization, and processivity. Applied Microbiology and Biotechnology [Epub].

[14] Zhang Y-HP*, Ding S-Y, Mielenz JR, Cui J, Elander RT, Laser M, Himmel ME, McMillan JD, Lynd LR. 2007. Fractionating recalcitrant lignocellulose at modest reaction conditions. Biotechnology and Bioengineering 97(2): 214-223. Accelerated publication.

[15] 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 Agricultural and Food Chemistry 56 (17), 7885–7890.

[16] 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 103: 715-724.

[17] 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 48: 6441-6447.

[18] Sathitsuksanoh N, Zhu ZG, Ho T-J, Bai M-D, Zhang Y-HP*. 2010. Bamboo saccharification through cellulose solvent-based biomass pretreatment followed by enzymatic hydrolysis at ultra-low cellulase loadings. Bioresource Technology (Epub)

[19] 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.

[20] 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.

[21] 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.

[22] 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.

[23] Hong J, Ye X, Zhang Y-HP*. 2007. Quantitative determination of cellulose accessibility to cellulase based on adsorption of a non-hydrolytic fusion protein containing CBM and GFP with its applications. Langmuir 23 (25): 12535-12540.

Selected Research Supports

DOE BioEnergy Science Center (BESC)

AFOSR Young Investigator Award and Muri Program

USDA

DoD -- U.S. Army Research

DuPont Young Professor Award

VT ICTAS

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)

The Zhang Lab
Home Biography Research Members Publications Activities News Awards Contact	Driving Tomorrow by Biomass Sugars through Imagination, Invention and Innovation Select Honors Biotechnology & Bioengineering Daniel IC Wang Award (2010) 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) Short CV Major Achievements Invented the most efficient lignocellulose fractionation technology (COSLIF) by using cellulose solvents (concnetrated phosphoric acid or ionic liquids). Patent filed in March 2006, and licensed in 2009. Proposed cell-free synthetic pathway biotransformation (SyPaB) as the ultimate platform for low-cost production of biofuels, bioelectricity, and biochemicals. Produced ~12 moles of hydrogen from per mole of glucose unit and water for the first time. Through this conversion, the energy output is more than the input based on useful chemical energy by absorbing useless ambient-temperature heat. Proposed the Electricity-Carbohydrate-Hydrogen (ECHo) cycle, which would ultimately solve several important challenges for sustainability. 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 sustainability, such as (1) cost-efficient production of second generation biofuels -- cellulosic ethanol, alkanes, and third generation biofuels -- hydrogen and electricity; (2) cost-effective release fermentable sugars from non-food lignocellulosic biomass; (3) high efficient CO2 biological fixation; (4) electricity storage on large scales; (5) food production; (6) water conservation; and (7) constructing a sustainable ecosystem for space travel. The below figure shows our research topics associated with biofuels production. Key Research Directions 1. Cell-free synthetic pathway biotransformation (SyPaB) Cell-free synthetic pathway biotransformation (SyPaB) is assembly of a number of purified enzymes and coenzymes for implementing complicated biochemical reactions [1,2]. Due to its great advantages, such as high product yields, fast reaction rates, high product titers, and great engineering flexibility, SyPaB will become a disruptive technology as compared to microbial fermentation. The successful examples are the production of high-yield hydrogen from starch or cellulosic materials and water [3,4]. Furthermore, we propose to use renewable low-cost carbohdyrates as a high-energy density hydrogen carrier (~8-14.8 H2 wt%). In the future, the sugar-powered vehicles will have a sugar container, on-board bioreforming, PEM fuel cells, rechargeable battery, and moter [5]. This hypothetic system will have numerous advantages over eletric vehicles [6]. Also, our life cycle analysis suggests that a small fraction of the USA biomass resource would meet 100% transportation fuel needs based on our new technology. 2. Cellulase engineering Cellulase engineering includes directed evolution, rational design, and reconstitution of individual components [7]. The large challenge for cellulase engineering is how to evaluate their activties on pretreated heterogeneous cellulosic substrates [7,8]. Recently, we have improved beta-glucosidase thermostability and catalytic efficiency by directed evolution [9]. Endoglucanase performance has been improved by using directed evolution and cell-surface display (submitted). 3. Consolidated bioprocessing (CBP) microorganisms Consolidated bioprocessing (CBP) is integration of cellulase production, cellulose hydrolysis and sugar fermentation in a single step [10-12]. Now we are working on a platform based on industrially-safe Bacillus subtilis. Now we have succeeded in expressing Clostridium phytofermentanas Cel48 cellobiohydrolase, Cel9 processive endoglucanase and several Cel5 endoglucanases in B. subtilis [13]. We have obtained the first celf-catalyzing recombinant cellulolytic microorganisms that can produce enough secretory cellulases, hydrolyze cellulose without any other organic nutrients, and support their growth and cellulase synthesis (in preparation for publication). 4. Cellulose solvent- and organic solvent-based lignocellulose fractionation (COSLIF) The largest challenge of cellulosic ethanol biorefineries is to effectively break recalcitrant lignocellulose and release soluble fermentable sugars. We have invented a cellulose-solvent based lignocellulose fractionation technology [14], 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. 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 [15-18]. 5. 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 [19]. 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 [20]. A new rapid method has been developed to determine the degree of polymerization of cellulose [21]; a new kind of non-substitution, high reactivity, homogeneous, regenerated amorphous cellulose (RAC) has been prepared for studying cellulose hydrolysis [22]. 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 [23]. See Our Vision about the Future Transportation and Energy Storage. References *[1] Zhang Y-HP. 2010.Production of biocommodities and bioelectricity by cell-free synthetic enzymatic pathway biotransformations (SyPaB): Challenges and opportunities. Biotechnology and Bioengineering (accepted). [2] Zhang Y-HP. 2009.* Using extremophile enzymes to generate hydrogen for electricity. *Microbe* 4(12): In press . [3] Zhang Y-HP., 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. [4] 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). *[5] Zhang Y-HP. 2009. A sweet out-of-the-box solution to the hydrogen economy: Is sugar-powered car science fiction? Energy and Environmental Science 2: 272-282 (PDF). [6]** Zhang Y-HP, Rollin JA. 2009. Technical comments on “Greater transportation energy and GHG offsets from bioelectricity than ethanol. Nature Precedings* 2009.3725.1. [7] Zhang Y-HP, Himmel ME, Mielenz JR. 2006. Outlook for cellulase improvement: Screening and selection strategies. Biotechnology Advances* 24(5): 452-481. [8] Zhang Y-HP, Hong J, Ye X. 2009. Cellulase Assays. Methods in Molecular Biology* 581: 213- 231. [9] 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 103:1087-1094. [10] Lu Y, Zhang Y-HP*, Lynd LR. 2006. Evidence for enzyme-microbe synergy in cellulose utilization by Clostridium thermocellum. Proceedings of the National Academy of Sciences of the USA 103(44): 16165-16169. [11] Zhang Y-HP, Lynd LR. 2005. Cellulose utilization by Clostridium thermocellum: Bioenergetics and hydrolysis product assimilation. Proceedings of the National Academy of Sciences of the USA* 102: 7321-7325. [12] Zhang Y-HP, Lynd LR. 2008. New generation biomass conversion: Consolidated bioprocessing. Biomass Recalcitrance: Deconstructing the Plant Cell Wall for Bioenergy* (ed. by Himmel, M.E.) Blackwell Publishing. ISBN: 9781405163606. pp 480-493. [13] Zhang XZ, Zhang ZM, Sathitsuksanoh N, Yang D, Zhang Y-HP. 2009.* The non-cellulosomal family 48 cellobiohydrolase from Clostridium phytofermentans ISDg: Heterologous expression, characterization, and processivity. *Applied Microbiology and Biotechnology* [Epub]. [14] Zhang Y-HP, Ding S-Y, Mielenz JR, Cui J, Elander RT, Laser M, Himmel ME, McMillan JD, Lynd LR. 2007.* Fractionating recalcitrant lignocellulose at modest reaction conditions. *Biotechnology and Bioengineering* 97(2): 214-223. Accelerated publication. [15] 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 Agricultural and Food Chemistry 56 (17), 7885–7890. [16] 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* 103: 715-724. [17] 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* 48: 6441-6447. [18] Sathitsuksanoh N, Zhu ZG, Ho T-J, Bai M-D, Zhang Y-HP. 2010.* Bamboo saccharification through cellulose solvent-based biomass pretreatment followed by enzymatic hydrolysis at ultra-low cellulase loadings. *Bioresource Technology* (Epub) [19] 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. [20] 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. [21]* 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. [22] 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. [23] Hong J, Ye X, *Zhang Y-HP. 2007. Quantitative determination of cellulose accessibility to cellulase based on adsorption of a non-hydrolytic fusion protein containing CBM and GFP with its applications. Langmuir 23 (25): 12535-12540. Selected Research Supports DOE BioEnergy Science Center (BESC) AFOSR Young Investigator Award and Muri Program USDA 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)**