Inoculating Clover Seeds for Nitrogen Fixation

In many developing nations, populations are growing faster than food production can support. In order to get nutrition efficiently, farmers in these nations grow plants with high protein content, which inadvertently drains their soil of vital nutrients, such as nitrogen. As soil becomes nutrient deficient, agriculture becomes impossible, as is natural in many developing arid climates. This desertification will continue to be an issue as land that can support agriculture grows in demand. A solution to the lack of nitrogen in soil lies in inoculation (the binding of bacteria with seeds) and nitrogen fixation (the product of this symbiosis between bacteria and plants, wherein colonies form on the roots and produce nitrogen for the plant in return for carbohydrates). Although inoculation is a viable solution across the world’s many climates, subsistence farmers in developing nations do not have the funds to use the method; modern inoculation typically requires the use of expensive industrial treatment materials marketed towards commercial farmers. However, more simple solutions for inoculating seeds exist—materials that cost less and are more available to farmers than designated treatments.

Our research involves testing the efficacy of different cheap inoculation treatments for Rhizobia on Trifolium repens seeds. Using Coca Cola, Kroger orange juice, glue, and corn syrup, we will attempt to bind the Rhizobia bacteria to the clover seeds to see which solution encourages the most success in nitrogen fixation. After a three week period of growth, we will record the number of nodules that form along the taproot of the plants and the amount of leghemoglobin (a protein that is integral to fixing nitrogen in the plant) in the nodules to determine the validity of fixation. A compound light microscope, a centrifuge, and a mass spectrophotometer will be used to quantify this data.

Due to the purpose of this research—to simulate the procedures a subsistence farmer would use—most of our study is straight-forward and requires only basic supplies. Treatment solutions like Coca Cola will need to be purchased at a local grocery store (King Soopers). The actual bacteria, R. trifolii, can be purchased in powder form through an online agro-supplier. The most need lies in the chemicals required to separate the leghemoglobin from the plant material for analysis. These include sodium phosphates, pyridine, and sodium dithionite.

The help of our mentors has been invaluable, from the preliminary research to the present day. Dr. Jim Ippolito, Associate Professor of Soil Fertility and Environmental Soil Quality in the College of Agricultural Sciences at Colorado State University has provided us with critical expertise on a variety of aspects of this study. We thank him for his time, generosity, and insight as we conduct our research. Also, we thank Franklin Harris, a graduate student in the Plant Science Department of Colorado State University, for donating materials to make this research possible and providing resources that allowed us to further explore this topic.