In the Laboratory
Build Your Own Second-Generation Bioethanol Plant in the Classroom! Janneke R. van Seters,* Jeroen P. J. Sijbers, Misha Denis, and Johannes Tramper Bioprocess Engineering, Wageningen University and Research Centre, The Netherlands *
[email protected] Bioethanol is produced widely today for use as fuel, particularly in the United States and Brazil (1). The experiment described here illustrates the possible solution to the problem of using primarily agricultural feedstocks (such as corn or cane sugar) for bioethanol production. This so-called first generation of bioethanol manufacturing competes with the world's food supply and is thus one of the factors giving rise to higher food prices (2). The production of bioethanol from cellulosic waste (so-called second-generation production) is a possible solution for this problem (3). Chemists, biochemists, and chemical engineers play an important role in finding ways to solve this problem by optimizing pretreatment procedures (4) and identifying enzyme mixtures that help to degrade the cellulose (5). The goal of this experiment is the production of bioethanol from cellulosic waste. This experiment is suitable for execution by students in high school classroom settings and should lead to bioethanol in a concentration high enough to burn the liquid. In this experiment, bioethanol is produced from a material used and thrown away by the students on a daily basis: facial tissues. Another example of cellulosic waste is grass. Grass, however, is much more difficult to process into bioethanol because it contains lignocellulose, which is difficult to break down. Although learning that experiments do not always work is a useful experience for students, we aimed to develop an experiment with a high probability of success. Furthermore, materials were chosen that most high schools would easily be able to obtain. Learning more about biotechnology might encourage high school students to choose a university degree in chemistry or engineering, thus, ensuring sufficient numbers of scientists for the future. We also believe that it is important for individuals in our society to form a fact-based opinion on subjects such as bioethanol that are covered extensively in the media. This experiment provides a unique opportunity to educate students in the multidisciplinary and interdisciplinary field of biotechnology. Overview of the Experiment This experiment requires about 6 h of activity by the students, but involves a lot of waiting, so it takes 5 days from start to finish. The production of bioethanol involves several steps. For the experiment, this process is simplified to three steps: (i) the cellulose of the waste material is converted to glucose by cellulase enzymes, (ii) the resulting glucose solution is fermented using baker's yeast to a solution of at least 5% ethanol, and (iii) the solution is distilled to yield an ethanol solution of at least 40%. The use of primarily agricultural feedstocks for bioethanol production is not preferable, so waste materials are used in this experiment. We chose facial tissues, as this material is used by students in daily life. Facial tissues belong to the group of cellulosic waste. About 70%
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of all domestic waste consists of cellulosic compounds (6) making it a good example for students. Of the various cellulosic waste compounds, paper-like materials have several advantages and have therefore been chosen for this experiment: they do not contain lignocelloluse, which is difficult to break down, and the structure of the cellulose in facial tissues is largely amorphous in nature, making the material easily accessible for cellulases. Enzyme Step The cellulose in the tissues is converted to glucose by placing the tissues in a cellulase solution for 2-3 days at 50 °C. Several methods were tried to shorten the time needed for this step; the best method was to place the folded tissues in a plastic container with a lid and pour the enzyme solution over it. Agitation appeared not to be necessary, and taking into account the minimization of equipment, this action was not included in the protocol. The glucose concentration was measured with glucose strips after 1 h and repeated after 24, 48, and 72 h. This enzyme step aims to obtain a solution containing about 100 g of glucose/L of solution. Fermentation Step Baker's yeast, available from supermarkets and bakeries, can convert glucose to ethanol under anaerobic conditions. The aim of this step is to obtain a solution with 5% ethanol. Several conditions were tested and most gave similar results, so the easiest method was chosen. In this method, the entire solution, including the remaining tissues, from the enzyme step is decanted into a flask with a water lock. Yeast is then added and the solution is incubated at 33 °C for 1 day while stirring. Distillation Step The distillation step should yield an ethanol solution of at least 40%. It can be difficult to obtain enough ethanol after fermentation, but collecting different fractions in the distillation helps to obtain a sample that will burn. In this step, a standard distillation setup with vigreux column is used. The first fraction of the distillation will burn; however, the second fraction sometimes needs heating first. Other fractions will probably not burn due to too much water still being present after distillation. Materials Most materials used are readily available at high schools (see the supporting information for a list of the materials). Glucose strips are commercially available (7). The enzymes used in this experiment are Multifect CX 10L (Genencor International). Water locks can be ordered from home brewery suppliers (8).
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r 2010 American Chemical Society and Division of Chemical Education, Inc. pubs.acs.org/jchemeduc Vol. 88 No. 2 February 2011 10.1021/ed100791w Published on Web 11/23/2010
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In the Laboratory
Hazards The enzymes may cause sensitization by inhalation. However, because the enzyme is used in solution and made up by the instructor, this hazard is unlikely to cause problems in classrooms. Heating the reaction mixture in the distillation step should not be done with the use of flames, because it is flammable.
(www.b-basic.nl) through B-Basic, a public-private NWO-ACTS program (ACTS = Advanced Chemical Technologies for Sustainability). Literature Cited
The development of an experiment for use in high schools to illustrate the second generation of bioethanol production was successful. Students enjoy carrying out the experiment since it gives them real experience of how bioethanol can be produced.
1. Balat, M.; Balat, H. Appl. Energy 2009, 86, 2273-2282. http://dx. doi.org/10.1016/j.apenergy.2009.03.015 (accessed Nov 2010). 2. Cassman, K. G.; Liska, A. J. Biofuels, Bioprod. Biorefin. 2007, 1, 18-23. http://dx.doi.org/10.1002/bbb.3 (accessed Nov 2010). 3. Naik, S. N.; Goud, V. V.; Rout, P. K.; Dalai, A. K. Renewable Sustainable Energy Rev. 2010, 14, 578-597. http://dx.doi.org/ 10.1016/j.rser.2009.10.003 (accessed Nov 2010). 4. Alvira, P.; Tomas-Pejo, E.; Ballesteros, M.; Negro, M. J. Bioresour. Technol. 2010, 101, 4851-4861. http://dx.doi.org/10.1016/j.biortech.2009.11.093 (accessed Nov 2010). 5. Sun, Y.; Cheng, J. Bioresour. Technol. 2002, 83, 1-11. http://dx.doi. org/10.1016/S0960-8524(01)00212-7 (accessed Nov 2010). 6. van Wyk, J. P. H.; Mohulatsi, M. J. Polym. Environ. 2003, 11, 23-28. http://dx.doi.org/10.1023/A:1023883428359 (accessed Nov 2010). 7. Fisher Scientific Home Page. Glucose strips, product number 530702. http://www.emergolab.com (accessed Nov 2010). 8. Brouwland Home Page. http://www.brouwland.com (accessed Nov 2010).
Acknowledgment
Supporting Information Available
This project is supported financially by the Netherlands Ministry of Economic Affairs and the B-Basic partner organizations
Student handout; notes for the instructor. This material is available via the Internet at http://pubs.acs.org.
Results The experiment was executed in classroom setting at a high school that was not involved in the development of the experiment. The experiment was part of a larger module about bioethanol production. According to responses to a questionnaire and interviews held by the teacher with a number of students, the students liked the practical nature of the experiment (“this is how you really learn things”). Eleven groups of two students executed the experiment of which 10 obtained flammable end products (the group that did not have a flammable product could not use a vigreux column for distillation). One group even obtained three flammable fractions of 5 mL. Conclusion
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