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Isolation and Characterization of Bacterial Cellulase Producers for Biomass Deconstruction: A Microbiology Laboratory Course

    Authors: Jesus F. Barajas1,2, Maren Wehrs2,3, Milton To2,3, Lauchlin Cruickshanks4, Rochelle Urban2,3,5, Adrienne McKee2,3,6, John Gladden7, Ee-Been Goh2,3,8, Margaret E. Brown2,3,9, Diane Pierotti2,3, James M. Carothers10, Aindrila Mukhopadhyay2,3, Jay D. Keasling2,3,10,11,12,13,14,15, Jeffrey L. Fortman2,3,15, Steven W. Singer2,3,*, Constance B. Bailey2,3,11,#,*
    VIEW AFFILIATIONS HIDE AFFILIATIONS
    Affiliations: 1: Agile BioFoundry, Emeryville, CA 94608; 2: Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720; 3: Joint BioEnergy Institute, Emeryville, CA 94608; 4: Oakland Technical High School, Oakland, CA 94611; 5: University of Southern California Viterbi School of Engineering, Los Angeles, CA 90089; 6: Helix OpCo, San Carlos, CA 94070; 7: Sandia National Laboratories, Livermore CA 94551; 8: Lygos Inc., Berkeley, CA 94710; 9: MicroByre, Berkeley, CA 94720; 10: Department of Chemical Engineering, University of Washington, Seattle, WA 98195; 11: QB3 Institute, University of California-Berkeley, Emeryville, CA 94608; 12: University of California, Berkeley, Department of Chemical & Biomolecular Engineering, Berkeley, CA 94720; 13: University of California, Berkeley, Department of Bioengineering, Berkeley, CA 94720; 14: Novo Nordisk Foundation Center for Biosustainability, Technical University Denmark, DK2970-Horsholm, Denmark; 15: Synthetic Biochemistry Center, Institute for Synthetic Biology, Shenzhen Institutes for Advanced Technologies, Shenzhen, China; 16: Center for Health Security, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205
    AUTHOR AND ARTICLE INFORMATION AUTHOR AND ARTICLE INFORMATION
    • Received 11 December 2018 Accepted 22 February 2019 Published 26 July 2019
    • ©2019 Author(s). Published by the American Society for Microbiology
    • [open-access] This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial-NoDerivatives 4.0 International license (https://creativecommons.org/licenses/by-nc-nd/4.0/ and https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode), which grants the public the nonexclusive right to copy, distribute, or display the published work.

    • Supplemental materials available at http://asmscience.org/jmbe
    • *Corresponding author. Mailing address: Joint BioEnergy Institute, 5885 Hollis St, 4th Floor, Emeryville, CA 94608. Phone: 510-486-5556. E-mail: [email protected].
    • # Current address: University of Tennessee-Knoxville, Knoxville, 1420 Circle Drive, Knoxville, TN 37996. Phone 865-974-8378. E-mail: [email protected].
    Source: J. Microbiol. Biol. Educ. July 2019 vol. 20 no. 2 doi:10.1128/jmbe.v20i2.1723
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    Abstract:

    The conversion of biomass to biofuels presents a solution to one of the largest global challenges of our era, climate change. A critical part of this pipeline is the process of breaking down cellulosic sugars from plant matter to be used by microbes containing biosynthetic pathways that produce biofuels or bioproducts. In this inquiry-based course, students complete a research project that isolates cellulase-producing bacteria from samples collected from the environment. After obtaining isolates, the students characterize the production of cellulases. Students then amplify and sequence the 16S rRNA genes of confirmed cellulase producers and use bioinformatic methods to identify the bacterial isolates. Throughout the course, students learn about the process of generating biofuels and bioproducts through the deconstruction of cellulosic biomass to form monosaccharides from the biopolymers in plant matter. The program relies heavily on active learning and enables students to connect microbiology with issues of sustainability. In addition, it provides exposure to basic microbiology, molecular biology, and biotechnology laboratory techniques and concepts. The described activity was initially developed for the Introductory College Level Experience in Microbiology (iCLEM) program, a research-based immersive laboratory course at the US Department of Energy Joint BioEnergy Institute. Originally designed as an accelerated program for high-potential, low-income, high school students (11th–12th grade), this curriculum could also be implemented for undergraduate coursework in a research-intensive laboratory course at a two- or four-year college or university.

References & Citations

1. Blanch HW, Adams PD, Andrews-Cramer KM, Frommer WB, Simmons BA, Keasling JD 2008 Addressing the need for alternative transportation fuels: the Joint BioEnergy Institute ACS Chem Biol 3 17 20 10.1021/cb700267s 18205287 http://dx.doi.org/10.1021/cb700267s
2. Reardon KF 2014 Lignocellulosic biorefineries: concepts and possibilities 255 265 McCann MC, Buckeridge MS, Carpita NC Plants and BioEnergy Springer New York, New York, NY 10.1007/978-1-4614-9329-7_15 http://dx.doi.org/10.1007/978-1-4614-9329-7_15
3. Bhutto AW, Qureshi K, Abro R, Harijan K, Zhao Z, Bazmi AA, Abbas T, Yu G 2016 Progress in the production of biomass-to-liquid biofuels to decarbonize the transport sector—prospects and challenges RSC Adv 6 32140 32170 10.1039/C5RA26459F http://dx.doi.org/10.1039/C5RA26459F
4. Chundawat SPS, Beckham GT, Himmel ME, Dale BE 2011 Deconstruction of lignocellulosic biomass to fuels and chemicals Annu Rev Chem Biomol Eng 2 121 145 10.1146/annurev-chembioeng-061010-114205 22432613 http://dx.doi.org/10.1146/annurev-chembioeng-061010-114205
5. De S, Luque R 2015 Integrated enzymatic catalysis for biomass deconstruction: a partnership for a sustainable future Sustain Chem Process 3 4 10.1186/s40508-015-0030-9 http://dx.doi.org/10.1186/s40508-015-0030-9
6. Blanch HW, Simmons BA, Klein-Marcuschamer D 2011 Biomass deconstruction to sugars Biotechnol J 6 1086 1102 10.1002/biot.201000180 21834132 http://dx.doi.org/10.1002/biot.201000180
7. Zhang F, Rodriguez S, Keasling JD 2011 Metabolic engineering of microbial pathways for advanced biofuels production Curr Opin Biotechnol 22 775 783 10.1016/j.copbio.2011.04.024 21620688 http://dx.doi.org/10.1016/j.copbio.2011.04.024
8. Zargar A, Bailey CB, Haushalter RW, Eiben CB, Katz L, Keasling JD 2017 Leveraging microbial biosynthetic pathways for the generation of “drop-in” biofuels Curr Opin Biotechnol 45 156 163 10.1016/j.copbio.2017.03.004 28427010 6283405 http://dx.doi.org/10.1016/j.copbio.2017.03.004
9. Henrissat B 1994 Cellulases and their interaction with cellulose Cellulose 1 169 196 10.1007/BF00813506 http://dx.doi.org/10.1007/BF00813506
10. Sukharnikov LO, Alahuhta M, Brunecky R, Upadhyay A, Himmel ME, Lunin VV, Zhulin IB 2012 Sequence, structure, and evolution of cellulases in glycoside hydrolase family 48 J Biol Chem 287 41068 41077 10.1074/jbc.M112.405720 23055526 3510808 http://dx.doi.org/10.1074/jbc.M112.405720
11. Wang F, Li F, Chen G, Liu W 2009 Isolation and characterization of novel cellulase genes from uncultured microorganisms in different environmental niches Microbiol Res 164 650 657 10.1016/j.micres.2008.12.002 19230636 http://dx.doi.org/10.1016/j.micres.2008.12.002
12. Park JI, Steen EJ, Burd H, Evans SS, Redding-Johnson AM, Batth T, Benke PI, D’haeseleer P, Sun N, Sale KL, Keasling JD, Lee TS, Petzold CJ, Mukhopadhyay A, Singer SW, Simmons BA, Gladden JM 2012 A thermophilic ionic liquid-tolerant cellulase cocktail for the production of cellulosic biofuels PLOS One 7 e37010 10.1371/journal.pone.0037010 22649505 3359315 http://dx.doi.org/10.1371/journal.pone.0037010
13. Schuster SM 2007 Commentary: a “biofuels” teaching moment Biochem Mol Biol Educ 35 221 10.1002/bmb.57 http://dx.doi.org/10.1002/bmb.57
14. Pedwell RK, Fraser JA, Wang JTH, Clegg JK, Chartres JD, Rowland SL 2018 The beer and biofuels laboratory: a report on implementing and supporting a large, interdisciplinary, yeast-focused course-based undergraduate research experience Biochem Mol Biol Educ 46 213 222 10.1002/bmb.21111 29383870 http://dx.doi.org/10.1002/bmb.21111
15. Emmert EAB ASM Task Committee on Laboratory Biosafety 2013 Biosafety guidelines for handling microorganisms in the teaching laboratory: development and rationale J Microbiol Biol Educ 14 78 83 10.1128/jmbe.v14i1.531 23858356 3706168 http://dx.doi.org/10.1128/jmbe.v14i1.531

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2019-07-26
2019-08-24

Abstract:

The conversion of biomass to biofuels presents a solution to one of the largest global challenges of our era, climate change. A critical part of this pipeline is the process of breaking down cellulosic sugars from plant matter to be used by microbes containing biosynthetic pathways that produce biofuels or bioproducts. In this inquiry-based course, students complete a research project that isolates cellulase-producing bacteria from samples collected from the environment. After obtaining isolates, the students characterize the production of cellulases. Students then amplify and sequence the 16S rRNA genes of confirmed cellulase producers and use bioinformatic methods to identify the bacterial isolates. Throughout the course, students learn about the process of generating biofuels and bioproducts through the deconstruction of cellulosic biomass to form monosaccharides from the biopolymers in plant matter. The program relies heavily on active learning and enables students to connect microbiology with issues of sustainability. In addition, it provides exposure to basic microbiology, molecular biology, and biotechnology laboratory techniques and concepts. The described activity was initially developed for the Introductory College Level Experience in Microbiology (iCLEM) program, a research-based immersive laboratory course at the US Department of Energy Joint BioEnergy Institute. Originally designed as an accelerated program for high-potential, low-income, high school students (11th–12th grade), this curriculum could also be implemented for undergraduate coursework in a research-intensive laboratory course at a two- or four-year college or university.

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Figures

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FIGURE 1

Biomass to biofuels and bioproducts pipeline. The activity described falls under deconstruction, the process of discovering enzymes that break down polysaccharides to form sugars and lignin-derived intermediates (indicated by orange text and box) that can be metabolized by bacteria to generate petrochemical replacements.

Source: J. Microbiol. Biol. Educ. July 2019 vol. 20 no. 2 doi:10.1128/jmbe.v20i2.1723
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Image of FIGURE 2

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FIGURE 2

Student confidence assessment for learning objective 4 (see Table 1 ). Survey was on a scale of 1 to 10, with 1 being the least confident (Very Low) and 10 being the most confident (Very High).

Source: J. Microbiol. Biol. Educ. July 2019 vol. 20 no. 2 doi:10.1128/jmbe.v20i2.1723
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