Abstract

This active learning exercise introduces students to the plant microbiome and the contributions that bacteria make to plant growth and food production. The Three Sisters are an ancient indigenous practice in which three plant crops and the associated microbiome contribute to each other’s growth. In this symbiotic relationship, the associated bacteria (Rhizobium leguminosarum biovar phaseoli) inhabit root nodules in bean plants, converting atmospheric nitrogen to ammonia through the process of nitrogen fixation. The plant takes up the ammonia as its nitrogen source, and provides the bacteria with organic carbon. The Lesson contains a pre-class reading, a 50-minute class session, and an after-class reading. In the first round of small group work during the class, students discuss and report back on the symbiosis between the three plants and between the bacteria and the plants, and the contributions to sustainable agriculture. In the second round of discussion and reporting, students discuss nitrogen fixation, emphasizing the nod genes for polypeptides involved in forming the root nodules and the nif genes that encode the nitrogenase enzyme complex that carries out nitrogen fixation. The after class reading provides students with an example of enhancing plant growth by adding nitrogen-fixing bacteria externally to beans. Altogether, this exercise provides students with a real life scenario relevant to sustainable agriculture.

Primary Image: Nitrogen fixation in bean root nodules. Nodule formation starts with the secretion of flavonoids from bean roots, leading to the induction of nodulation factors in the bacteria. After root curling and nodule formation, the enzyme nitrogenase catalyzes the reaction from atmospheric nitrogen to plant accessible ammonia.

Citation

Society Learning Goals

Microbiology

• Metabolism
• How are the interactions of microorganisms among themselves and with their environment determined by their metabolic abilities?
• Systems
• How do microorganisms, cellular and viral, interact with both human and non-human hosts in beneficial, neutral, or detrimental ways?
• Impact of Microorganisms
• How can humans utilize and harness microbes and their products?

Lesson Learning Goals

• “How do microorganisms, cellular and viral, interact with both human and non-human hosts in beneficial, neutral, or detrimental ways?”
• “How are the interactions of microorganisms among themselves and with their environment determined by their metabolic abilities?”
• “How can humans utilize and harness microbes and their products?”

Lesson Learning Objectives

• list the contributions of each of the Three Sisters plants to the symbiotic relationship.
• describe how bacterial nitrogen fixation in the bean root nodule is a driver behind nitrogen metabolism within the plant community.
• explain the process of nitrogen fixation, including how electrons flow.
• identify nutrients in each of the plants that are impacted by the symbiosis.
• expound on the potential contribution of nitrogen fixing bacteria to enhancing plant growth, disease resistance, and sustainable agriculture.

Introduction

The Three Sisters are an agricultural crop community or intercropping system containing corn, squash, and beans planted together by multiple North and South American indigenous tribes (1). The three plants are highly complementary, supporting each other’s growth, as well as providing a complete and balanced nutrition for humans. Most relevant for this activity, beans (Phaseolis vulgaris) contain bacteria in their root nodules that fix atmospheric nitrogen, which then gets converted to ammonia. Ammonia is more accessible to plants than atmospheric nitrogen. Corn provides a stalk for beans to climb up on, and squash keeps the soil from dehydrating. The increased crop productivity of the community relative to growing individual plants is due in part to the intertwined root architecture within the community (2). With respect to human nutrition (1), the ammonia produced by the bean root bacteria leads to the production of a highly satiating protein in the beans. Squash is high in vitamins, especially vitamin C, E, and A, and corn serves as a carbon and fiber source (1).

An essential part of the Three Sisters community is the bean root bacteria (Rhizobium leguminosarum biovar phaseoli) that fix nitrogen in this system (3). This bean-bacteria relationship is specific to this system; different bacteria fulfil the same roles with other legumes (e.g., Rhizobium leguminosarum bv viciae and peas). The positive effect of these bacteria on plant growth, nutritional value, plant health, crop yield, stress resistance, and disease resistance has been recognized for many years. Intriguingly, the bean-Rhizobium symbiosis has been used for many years to increase crop production by soaking the seeds with highly nitrogen fixing strains of Rhizobium phaseoli (5). This has even enabled corn roots to be colonized by Rhizobium phaseoli in an intercropping system similar to the Three Sisters (6).

Nitrogen fixation involves the transfer of 8 electrons from NADH + H⁺ to nitrogen (recently reviewed by Masson-Boivin and Sachs (4). The total reaction converts one molecule of atmospheric nitrogen (N₂, dinitrogen), 8 protons, 8 electrons, 16 molecules of ATP, and 16 molecules of H₂O to 2 molecules of ammonia (NH₃), hydrogen, 16 molecules of ADP, and 16 molecules of inorganic phosphate.

The bacterial nod genes and nif genes have distinct functions in the nitrogen fixation process: nod gene products permit nodule formation on the bean roots inhabited by Rhizobium phaseoli, and nif gene products catalyze the nitrogen fixation reaction. After Nod factor induction, root hairs curl and start forming the nodules harboring the R. phaseoli bacteria. These bacteria now convert atmospheric nitrogen to ammonia using the enzyme nitrogenase, encoded by nif genes. The positive regulator NifA is required for the transcription of all other nif genes. The nitrogenase enzyme itself contains two subunits; component I and component II. Component I is the MoFe protein dinitrogenase that is encoded by nifD and nifK. Component II is the homodimer of the Fe-containing dinitrogenase reductase and encoded by nifH.

This article describes an active learning exercise that builds on the traditional indigenous concept of the Three Sisters. Students will form groups and will use literature they find themselves, using Google and/or YouTube to familiarize themselves with the Three Sisters concept. They will identify the contributions each plant makes to the community, recognize nitrogen fixation as the driving force behind nitrogen metabolism in the community, and summarize the nutrients each of the plant contributes to human health. Each group will report their findings to the class. Since the exercise was developed for an upper level undergraduate class in Bacterial Physiology, students will spend the remaining time focusing on nitrogen fixation, especially electron flow from atmospheric nitrogen to ammonia. Students will be given a first review article to dissect nitrogen fixation (7). I recommend advising students to read the Introduction and the two sections on “Plant Available Nitrogen” and Nitrogen Fixation” of this article PRIOR to coming into class. Instructors may have to read the same sections to familiarize themselves with the topic. It may help both students and instructors to find the chemical reaction of nitrogen fixation within the chapter on Nitrogen Fixation on Wikipedia and read the paragraph on nodulation within the Root Nodule chapter of Wikipedia. After a short group discussion period, each group will report their findings to the class. Then at home, students will read the Abstract and the Introduction of a second article on the external addition of Rhizobium leguminosarum bv. phaseoli to beans to enhance agronomic performance (8). Finally, an online quiz containing four multiple choice questions will assess learning in this part.

Intended Audience

The targeted audience is microbiology majors or minors, as well as students from related disciplines, including but not limited to biology and biochemistry. The first exercise part can be taught at the freshman or sophomore level, but once students get to the scientific literature, the targeted level is junior and senior undergraduates, as well as graduate students. I developed the exercise for a 400/600 level Bacterial Physiology course as one of eight similarly structured exercises in the course. (CourseSource has already published one exercise as a Lesson Plan for face to face learning [9] and distance education [10].) The first exercise part helps students get used to other group members and start out with a culturally interesting and scientifically easy topic they can investigate with their commonly used online search skills. Reading the scientific articles will guide them down a path to knowledge acquisition using primary science tools, peer-reviewed journal articles.

Required Learning Time

Student learning time prior to class is approximately 45 minutes to read the first three sections of the article on nitrogen fixation (7) and to review the chemical reaction in the Nitrogen Fixation article on Wikipedia, as well as the information on nod genes and nodulation factors in the Root Nodules article of Wikipedia. This is followed by one class period of 50 min. Finally, the homework requires 30 minutes to read the Abstract and Introduction of the article on externally added Rhizobium (8) and 10 minutes to take the online quiz. For the instructor, time involvement is approximately the same; 1 hour and 15 minutes for reading the articles and Wikipedia sections, 50 minutes in class time, and 10 minutes to set up the online quiz that should import the points straight into the gradebook from the learning management system.

Prerequisite Student Knowledge

Students will need a general knowledge of bacteria and plants, microbiomes, and the concept of symbiotic and pathogenic relationships between bacteria and hosts. In addition to previous course work, this knowledge can be obtained from textbooks, as well as YouTube videos featuring Three Sisters companion planting and the nitrogen cycle in soil. See this link for an example featuring the nitrogen cycle.

Prerequisite Teacher Knowledge

Required content knowledge includes the bacterial metabolic pathways constituting the nitrogen cycle in soil (e.g., nitrogen fixation), as well as the general concept of plant/microbe symbiosis. This knowledge can be obtained from textbooks in microbiology and/or biochemistry. The inclusion of externally added bacteria in agricultural practices can be learned from the article provided to the students (8).

Scientific Teaching Themes

Active Learning

This active learning exercise is one of eight similarly structured exercises used for a Bacterial Physiology class. Active learning has been described as a tool to enhance student engagement and equity in the classroom (11, 12). Strategies to promote active learning include (i) “giving students time to think and talk” and (ii) “actively managing the participation of all students” (11).

The exercise involves no lecturing by the instructor. Instead, students have to perform individual tasks, such as reading, and group exercises, such as retrieving and combining information. When reading the literature at home, students can take all the time they need to process the information. During class, time is more limited. However, there are multiple opportunities to digest information, either in the group discussion or while another student reports group results to the class. Also, the exercise concludes with another at home reading, where students can spend more time processing information. All students have the opportunity to talk during the group discussions, with the instructor drawing less engaged students into the discussion and calling upon different students to report the group activity back to the class.

Assessment

The instructor will assess learning primarily in a formative way, by listening to student reports and responding to questions. The construction of a table and figure on the whiteboard will also give the instructor feedback on student understanding. At the same time, students can assess their own understanding and compare it with class knowledge and instructor expectations. It is anticipated that students will enhance each other’s understanding during discussion and reporting. The online quiz is summative to assess the depth of each student’s comprehension of the content.

Inclusive Teaching

The 400/600 level Bacterial Physiology course is required for Microbiology majors but also enrolls students from other disciplines, sometimes even including those in engineering or medical programs. The academic level is most often junior/senior undergraduate level or first/second year graduate level students that are also diverse in other respects, including but not limited to gender, race, and nationality. Many use English as a second (possibly third) language. To ensure that all students are engaged and participate in learning, this STEM learning activity has an intrinsic cultural component, the Three Sisters concept, developed by indigenous populations. The book Braiding Sweetgrass by R.W. Kimmerer, a member of the Citizen Potawatomi Nation, includes this and other indigenous lectures on plants for foods and medicine (13). The inclusion of an indigenous concept into a STEM science course communicates to students from underrepresented groups that different cultures are valued. This is especially important since evidence is growing that the achievement gap between underrepresented students and white students is due to a sense of belonging (or lack thereof) and not an understanding of the learning material (14).

Along with this deliberate ‘pedagogical’ choice to include diverse cultures into a STEM science classroom, another strategy promoted by Dewsbury and Brame for inclusive teaching is ‘empathy’ (15). I have always found it helpful to connect to students early in the course by showing up before class and engaging them in discussion on course and other topics.

Lesson Plan

Lesson: Overview

The Three Sisters active learning exercise contains a pre-class reading for individual students to do at home, two sections of group work in class totaling 50 minutes, a second reading at home, and an online quiz. Table 1 guides instructors through these activities.

Table 1. Lesson Plan Timeline

Pre-class Preparation

Students will have to read the first three chapters of the review article on nitrogen fixation at home (7), as well as the Nitrogen Fixation article and the Root Nodule article on Wikipedia. It is recommended that the instructor advise the students to take notes on the nif genes from the review article, the chemical reaction of nitrogen fixation from the Wikipedia chapter on nitrogen fixation, and the nod genes from the Wikipedia chapter on root nodules. It is anticipated that this will take approximately 45 minutes. Remind students that because in class discussion of this paper is only 15 minutes, they must read the recommended literature prior to class.

Class Session (50 min)

The class session contains two rounds of student group discussion and student reporting with the instructor using this information to compile the information on the board. The first discussion/reporting round covers general information on the concept of Three Sisters intercropping. Each group of four to five students has 10 minutes to use Google and YouTube to retrieve information on the symbiosis between the three plants, the bacteria/plant symbiosis on the beans, and the nutritional benefits of each plant to humans. One student per group reports the group’s finding to the class. The instructor guides this process by asking each group a sub-question from Table 2, while summarizing the information from all groups on the board, constructing a table similar to Table 2.

Table 2. Assessment Table-Symbiosis

Note that the answers are examples. Students may find more information under the questions.

The second discussion/reporting round focuses on nitrogen fixation. This process has been reviewed by Soumare (7) in an article provided to the students. Students use this article and the Wikipedia articles on Nitrogen Fixation and Rood Nodules to search for specific information pieces. Student groups are the same as for the first round of discussion/reporting. The instructor gives one half of the groups the assignment to investigate the nod genes (group 1, 3, 5, if needed) and the other half of the groups the task to investigate the nif genes (groups 2, 4, 6, if needed). I highly recommend the instructor guide students towards the genes from Figure 1 to help the students focus on the central parts of the process. Once student groups start reporting, the instructor draws a figure on the board similar to Figure 1, starting with the plant, and asking the questions to the groups in an order that permits the drawing of the figure in an orderly manner (e.g., plant first, then nod genes, then nif genes). Ideally, students will volunteer to report to the class. I recommend that groups select a student reporter different from the one who reported in the first round of discussion/reporting.

Assessing student learning during the class session is formative and uses Table 2 and Figure 1. The instructor will first notice when students are floundering by visiting with groups during their discussions, particularly during the longer group discussion on nitrogen fixation. While collecting information from the students, the instructor will notice when individual students or student groups struggle with a particular information piece. If time permits, students can be asked to go back to the paper and retrieve the information. If time is short, the instructor can tell students the correct answer. At the end of the 50 minute class time, all students should know the symbiosis between the three plants of the Three Sisters community, the key aspects of the symbiosis between the Rhizobium bacteria in the bean nodules and the beans, and how nodulation factors and nitrogenase work together during nitrogen fixation.

Post-class Assignment

The post class assignment permits students to study a societal aspect of nitrogen fixation, the external addition of R. phaseoli to beans. The assigned review article (8) seeks to overcome the fact that most strains of Rhizobium that are currently in use were selected for their superior ability to perform nitrogen fixation, but are often not successful at competing with the natural microflora. Using a naturally evolved and locally sourced strain of R. leguminosarum bv. phaseoli, the authors aimed to obtain grain yields at least equal to current fertilization techniques. The outcome of the study is that their LCS0306 strain outperformed the two type strains of Rhizobium etli and R. phaseoli (8).

I advise instructors to guide students towards the Abstract and the Introduction of the article and give them the questions from Table 1 to guide them through the reading. For the online quiz, the instructor will enter all correct answers included in Assessment Quiz Table 3 or different correct answers as identified from the paper. Instructors will enter a selection of the incorrect answers from Table 3. It will help students to let them know whether they are expected to mark only one correct answer or several. If the instructor uses a learning management system, the grades can be integrated straight into the grade book, which will involve no additional grading time for the instructor.

Table 3. Assessment Table-Quiz

Teaching Discussion

The main point behind this exercise is to keep students engaged by providing an active learning environment. All students are expected to contribute to the group discussions, and by visiting with different groups during the discussions, the instructor can identify and help individual students who may be struggling with some aspects of the learning material. The 400/600 level Bacterial Physiology class this exercise was developed for is part of the College of Agriculture, Food Systems, and Natural Resources. The plant microbiome fits in with the North Dakota Agribiome Initiative, and the bacterial impact on crop production represents the influence bacteria have on our society. The real-life scenario makes the exercise interesting for the students. In my class, all students always participated in the group discussions. To enhance engagement, I ask different students from each group to report the group’s findings from the different parts of the exercise.

The learning objectives focus on symbiosis: between different plants, and between bacteria and their plant hosts. This includes the contributions of the metabolic capabilities of bacteria to plant growth and food production. The exercise covers all aspects of this symbiosis with a focus on bacterial metabolic pathways. Students usually have no difficulty performing the first round of group discussion and student reporting during the 50 minute class period. The nitrogen fixation part can be more difficult. Since repetition can enhance student learning, I have a lecture on nitrogen fixation following the exercise. This lecture repeats much of the learning material in a tightly organized manner. It is up to the individual instructor whether to devote the additional time in their course for this, but the follow up lecture is one adaptation of the Lesson Plan I recommend.

Of course, adaptations always have to be made for class sizes. I chose group sizes of 4 to 5 students, which gives me 5 or 6 student groups. If the class is larger, the instructor will either have to increase the student number per group or the number of groups. Increasing the student number per group will increase the opportunity for some students to not contribute; increasing the number of groups makes it more difficult to visit with all groups during the group discussions. However, I believe both these adaptation methods should work for classes up to 50 students.

I developed the exercise for in person teaching, but it required minimal adaptation to the online or HyFlex learning environment. The only two differences were (i) the group discussions took place in breakout rooms that Zoom or Microsoft Teams offer and (ii) an online whiteboard (Zoom or Teams) was used instead of the physical one in the classroom.

Obviously, the first round of group discussion and student reporting is much easier than the second. This enables instructors who teach lower level undergraduate classes to use the exercise by omitting the advanced, second part and giving students more time for the easier, first part.

Acknowledgments

I thank the NDSU Gateways ND program for introducing me to the concept of active learning and the College of Agriculture, Food Systems, and Natural Resources for awarding me with the H. Roald and Janet Lund Excellence in Teaching Award. This encouraged me to publish a second active learning exercise.

References

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