Rheological and Toxicological Properties of the Extracellular Polymeric Material (EPM) Produced by Rhizobium Tropici ATCC 49672

<br/>Soil erosion is a significant issue costing the U.S. economy forty-four billion dollars per year and increasing water pollution (Sulaeman & Westhoff, 2020). Typical erosion treatments use toxic, non-biodegradable petrochemicals that pose threats to surrounding ecosystems and agricultural efforts. An alternative may come from symbiotic Rhizobium tropici bacteria which secrete gel-like biopolymers known as EPS that can then be ethanol precipitated into EPM (non-dialyzed EPS) (Maróti & Kondorosi, 2014). EPS soil treatments have been tested by the ERDC and results indicate that the soil was strengthened substantially, reducing erosion. REF Analysis of the vegetation showed that plant root systems were larger, which was postulated to be a contributing factor to the soil stability increase. The role of bacterial infection and nitrogen sequestration in root development has been extensively studied, but less is known about the role of bacterial EPS in strengthening the soil via gelation or other interactions with the soil particles. In order to study the possible mechanisms through which the EPS can strengthen soil, we performed a series of experiments where the biopolymer was precipitated in alcohol and subsequently rehydrated. In this manner no bacteria was present and only the material properties of the EPS are probed.<br/>The rehydrated EPM’s rheology behavior with various concentrations was tested on DHR-2 rheometer oscillation-amplitude and oscillation-frequency mode. The results show that upon rehydration the polymer formed a gel at concentrations of 2% with a modulus G’=20Pa. The modulus increases approx. linearly with concentration to a value of G’=300Pa at a concentration of 20%. In each case a distinct yield point was observed, at 20-170Pa between the lowest and highest concentrations measured, where G’ approached zero. The systems were rested for 30 min without an applied force and the amplitude scan was repeated. The gels completely recovered in each case after at least three cycles, indicating that in each case physical gels were formed and then reformed when the forces were removed. Measurement of the gels in the frequency mode indicated shear thinning behavior in all cases at room temperature. The chemical structure of the gels was investigated using Raman spectroscopy and TGA. RAMAN spectroscopy indicated that the EPM was composed of polysaccharides with a high content of potassium dihydrogen phosphate. TGA showed that ~50% of the gel was removed at T&lt;100C, and another 10% was removed at T&lt;257C. This is consistent of a gel with large water content, while the additional 20% indicated strongly adsorbed water and a high degree of water retention. A surprisingly high fraction, ~30% of the gel was stable to temperatures of at least 800C. This temperature is higher than organic polymers, and was attributed to the dihydrogen phosphate salts. RAMAN spectroscopy of this residue confirmed this where one large and narrow peak was observed consistent with a crystalline compound formation. Auxiliary testing for the antibacterial, antiviral, and cytotoxic effects of the EPM were performed to determine the safety of EPM application into soil. Results indicated that EPM is neither antiviral nor antibacterial. Application of EPM into human fibroblast cultures indicated no adverse effects on the doubling time nor any other function of the tissue.<br/>Work supported by the ERDC (W912HZ-20-2-0054) and the Morin Charitable Trust.<br/>E. M. G. K. (2014, June). Nitrogen-fixing rhizobium-legume symbiosis: Are polyploidy and host peptide-governed symbiont differentiation general principles of endosymbiosis? Frontiers in Microbiology.<br/>Larson, S., Nijak Jr, G., Corcoran, M., Lord, E., & Nestler, C. (2016, June). Evaluation of Rhizobium tropici-derived Biopolymer for Erosion Control of Protective Berms. US ERDC.<br/>Sulaeman, D., & Westhoff, T. (2020, Feb.). The causes and effects of soil erosion, and how to prevent it. World Resources Institute.