Impact of Rhizobium Tropici-Produced EPS on Bermuda Grass Root Growth

Erosion control is imperative to soils vulnerable to flooding, such as watersheds in the Mississippi River Basin. A potential solution is provided by extracellular polymeric substances (EPS): hydrated microbial biopolymers whose high water-holding capacity, entrapment of nutrients, and resistance to environmental stressors can elevate soil fertility. EPS also promotes aggregate formation, which helps maintain a soil’s structural integrity. A more indirect benefit of EPS in soil is their ability to enhance subterranean root growth. This corresponding increase in root mass or length can stabilize topsoil and aid in erosion control. However, the specific mechanism by which EPS benefits root growth was studied by different research groups with partial success. This project aimed to further investigate the influence of EPS produced by Rhizobium tropici bacteria (RT-EPS) on Bermuda grass, particularly on its root growth. By gaining a deeper understanding of this impact, a more effective approach for the fabrication and deployment of RT-EPS can be developed, which will be used on large scale applications to minimize the soil damage caused by extreme weather conditions.<br/>EPS produced from the Rhizobium tropici bacteria was used to generate an ethanol precipitate material (EPM). After cell-free biofilms of the bacteria were generated, the cells were removed by centrifuge and the crude EPM collected through precipitation with ethanol, thus producing two forms of EPM: non-dialyzed and dialyzed (where an additional dialysis step was applied). For the plant study, Bermuda grass seeds were germinated in sand treated with non-dialyzed- and dialyzed- EPM (and water as a control). The plants were grown in one of three conditions in the greenhouse: a controlled chamber, inside the greenhouse, or outdoors. After a 60-day period, gravimetric and length measurements were taken of both the roots and shoots, indicating that EPM treatment increased relative root mass growth. The structure, density, and elemental composition of roots from Bermuda grass specimens grown in sand were analyzed by a Keyence VHX-7000 Microscope. After retrieving images, qualitative and quantitative analyses (nodule counting) found higher root density and greater branching in EPM-treated samples, for both dialyzed and non-dialyzed polymers. Additionally, X-ray fluorescence (XRF) spectroscopy was performed to determine the concentration of biologically important ions in roots with EPM, revealing higher amounts of Ca, K, and P. X-ray diffraction (XRD) was then performed to determine the presence of crystalline structures on top of the roots.<br/>In conclusion, it was observed that the Bermuda grass samples treated with non-dialyzed EPM had the highest root-to-shoot mass ratios and coverage from tertiary branching. These samples also contained higher concentrations of ions that promote root growth, demonstrating enrichment of the nutrient profile. Prospective analysis includes in-depth research on the protein fraction EPM contains, with a focus on their influence on root stem cell differentiation. Further experiments will be conducted to compare the impact of different EPM dialysis procedures (by increasing the dialysis pores) on Bermuda grass root growth. The knowledge obtained from these studies will be utilized to conduct open-field experiments to examine the effect of EPM on large-scale growth, including subsequent soil analysis.<br/>The authors would like to thank the US Army Corps of Engineers (ERDC) for their support (W912HZ-20-2-0054) in this research.