Demonstrating Microbial Growth and Metabolic Kinetics with Methanotrophic Bacteria: A Classroom Laboratory Experiment

Andrew Ross Pfluger; Weimin Wu

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Conference: 2011 ASEE Annual Conference & Exposition
Location: Vancouver, BC
Publication Date: June 26, 2011
Start Date: June 26, 2011
End Date: June 29, 2011
ISSN: 2153-5965
Conference Session: Techniques to Enhance Environmental Engineering Courses
Tagged Division: Environmental Engineering
Page Count: 10
Page Numbers: 22.421.1 - 22.421.10
DOI: 10.18260/1-2--17702
Permanent URL: https://peer.asee.org/17702
Download Count: 518

Paper Authors

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Andrew Ross Pfluger U.S. Military Academy orcid.org/0000-0001-6960-2075

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Major Andrew Pfluger is an officer in the U.S. Army and an Instructor at the U.S. Military Academy (USMA) in the Department of Geography and Environmental Engineering. He holds two graduate degrees from the Department of Civil and Environmental Engineering at Stanford University. He currently teaches Environmental Science and Air Pollution Engineering at USMA.

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biography

Weimin Wu Stanford University

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Wei-Min Wu, Senior Research Engineer of Department of Civil & Environmental Engineering, Department of Civil & Environmental Engineering, Stanford University, Stanford, CA, USA since 2001; Program Manager of Center for Sustainable Development & Global Competitiveness, Stanford University; and Part-time Ph.D. adviser of Harbin Institute of Technology, Harbin, China. He graduated from Harbin Architectural & Civil Engineering Institute, Harbin, China (1976), received a Masters of Engineering degree from Tsinghua University, Beijing, China (1984) and a Ph.D. degree in Environmental Engineering from Michigan State University, East Lansing, MI (1991).

Dr. Wu was Engineer/Project Leader of Michigan Biotechnology Institute (1992 - 2001) and the Manager for Asia-Pacific Business Development (1998 - 2001). In the 1980s, he was an Assistant Engineer and Assistant Administrator of the Chief Engineer in the 2nd Division of the China National Construction Engineering Corp. Dr. Wu’s research interests are in the area of environmental biotechnology including the bioremediation of organic toxicants and heavy metals in contaminated groundwater and sediments, the anaerobic digestion of solid wastes and wastewaters for energy recovery, microbial ecology, and biodegradable plastics. He has more than 90 publications and holds two U.S. patents.

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Abstract

Demonstrating Microbial Growth and Metabolic Kinetics with Methanotrophic Bacteria: A Classroom Laboratory ExperimentWith the field of biotechnology rapidly expanding, it is increasingly important to providerelevant, hands-on education to environmental engineering students in the area of microbialgrowth and metabolic kinetics. Students enrolled in introductory microbiology courses can havedifficultly visualizing the physical growth of bacteria, the stoichiometric ratios of substrateconsumption, and the results of metabolic processes that produce end products. Methanotrophicbacteria can be used as a model, allowing students in an experimental laboratory class tounderstand microbiological fundamentals. Methanotrophs have been well studied and manystrains are available for purchase through American Type Culture Collection (ATCC) and otherculture collection services. Methanotrophs are also ubiquitous in nature and can be isolated frommany different environments, including soils and aqueous sediments. Additionally,methanotrophs possess a relatively simple and easily understandable metabolic pathway,consuming methane (electron donor) and oxygen (electron acceptor) as their primary substrates,and producing carbon dioxide and water as end products. The objective of this study was todevelop a simple experiment that clearly shows students the concepts of microbial growth, andthe stoichiometry associated with substrate consumption, using simple techniques with a readilyavailable source of bacteria, i.e., methanotrophs. The laboratory study is conducted as follows:in sterilized serum bottles, methanotrophic bacteria in the exponential growth phase areinoculated in a Milli-Q water-based medium containing nitrate, other key nutrients (P, K, Na,Mg, Ca, etc.), and selected trace metals. The primary substrates, methane and oxygen, are thenintroduced and each bottle is placed on a shaker-table for incubation. Gas samples areperiodically withdrawn from the headspace of each bottle and manually injected into a gaschromatograph to measure the concentrations of methane, oxygen, and carbon dioxide. Thisallows students to monitor the amount of substrates consumed and the amount of end productproduced at different time points. Students can also track microbial growth by measuring theincrease in turbidity of the medium via changes in optical density (OD). Results of the substrateconsumption and carbon dioxide production are then plotted in a spreadsheet program (e.g.,Excel). Typical results clearly depict the different rates of substrate consumption for oxygen andmethane, and the rate of carbon dioxide production. This unique approach allows students to seefirsthand the stoichiometry of the chemical reactions associated with the metabolism ofmetanotrophs. The results also clearly depict the microbial growth concepts of lag, exponentialgrowth, and, if the experiment is continued long enough, microbial death. This experiment hasbeen successfully conducted by both graduate and undergraduate students with several differentgenuses of methanotrophic bacteria including Methylobacter, Methylonsinus, and Methylocystis,indicating that any genus of the methanotroph family can be successfully grown under theseconditions. After using this approach, students will have a better understanding of microbialgrowth kinetics and the stoichiometry associated with the chemical reactions in themethanotrophic metabolic pathway, while gaining additional relevant lab experience.

Citation
Format

Pfluger, A. R., & Wu, W. (2011, June), Demonstrating Microbial Growth and Metabolic Kinetics with Methanotrophic Bacteria: A Classroom Laboratory Experiment Paper presented at 2011 ASEE Annual Conference & Exposition, Vancouver, BC. 10.18260/1-2--17702

TY  - CPAPER
AB  -         Demonstrating Microbial Growth and Metabolic Kinetics with        Methanotrophic Bacteria: A Classroom Laboratory ExperimentWith the field of biotechnology rapidly expanding, it is increasingly important to providerelevant, hands-on education to environmental engineering students in the area of microbialgrowth and metabolic kinetics. Students enrolled in introductory microbiology courses can havedifficultly visualizing the physical growth of bacteria, the stoichiometric ratios of substrateconsumption, and the results of metabolic processes that produce end products. Methanotrophicbacteria can be used as a model, allowing students in an experimental laboratory class tounderstand microbiological fundamentals. Methanotrophs have been well studied and manystrains are available for purchase through American Type Culture Collection (ATCC) and otherculture collection services. Methanotrophs are also ubiquitous in nature and can be isolated frommany different environments, including soils and aqueous sediments. Additionally,methanotrophs possess a relatively simple and easily understandable metabolic pathway,consuming methane (electron donor) and oxygen (electron acceptor) as their primary substrates,and producing carbon dioxide and water as end products. The objective of this study was todevelop a simple experiment that clearly shows students the concepts of microbial growth, andthe stoichiometry associated with substrate consumption, using simple techniques with a readilyavailable source of bacteria, i.e., methanotrophs. The laboratory study is conducted as follows:in sterilized serum bottles, methanotrophic bacteria in the exponential growth phase areinoculated in a Milli-Q water-based medium containing nitrate, other key nutrients (P, K, Na,Mg, Ca, etc.), and selected trace metals. The primary substrates, methane and oxygen, are thenintroduced and each bottle is placed on a shaker-table for incubation. Gas samples areperiodically withdrawn from the headspace of each bottle and manually injected into a gaschromatograph to measure the concentrations of methane, oxygen, and carbon dioxide. Thisallows students to monitor the amount of substrates consumed and the amount of end productproduced at different time points. Students can also track microbial growth by measuring theincrease in turbidity of the medium via changes in optical density (OD). Results of the substrateconsumption and carbon dioxide production are then plotted in a spreadsheet program (e.g.,Excel). Typical results clearly depict the different rates of substrate consumption for oxygen andmethane, and the rate of carbon dioxide production. This unique approach allows students to seefirsthand the stoichiometry of the chemical reactions associated with the metabolism ofmetanotrophs. The results also clearly depict the microbial growth concepts of lag, exponentialgrowth, and, if the experiment is continued long enough, microbial death. This experiment hasbeen successfully conducted by both graduate and undergraduate students with several differentgenuses of methanotrophic bacteria including Methylobacter, Methylonsinus, and Methylocystis,indicating that any genus of the methanotroph family can be successfully grown under theseconditions. After using this approach, students will have a better understanding of microbialgrowth kinetics and the stoichiometry associated with the chemical reactions in themethanotrophic metabolic pathway, while gaining additional relevant lab experience.
AU  - Andrew Ross Pfluger
AU  - Weimin Wu
CY  - Vancouver, BC
DA  - 2011/06/26
PB  - ASEE Conferences
TI  - Demonstrating Microbial Growth and Metabolic Kinetics with Methanotrophic Bacteria: A Classroom Laboratory Experiment
UR  - https://peer.asee.org/17702
DO  - 10.18260/1-2--17702
ER  -