Study on Interactive Effects of Different Levels of Lead and Mercury on Nitrogen Fixation of Some Diazotrophs
Authors: Aroh Kenechukwu U. , Udensi Justina U. 
Published: May 2021

Researchers have studied the effects of addition of metal elements in combination with nitrogen fixing organisms as inoculants on the plants (growth) predominantly in legumes, however there is a major gap because responses and effects of these proposed micronutrients on the nitrogen fixation activity of these microbes both free living and symbiotic remains sketchy at best. Therefore, the effect of supplementation of lead and Mercury (bioaugmentation) on the nitrogen fixation potential of two (2) diazotrophs was evaluated in this study.
Aims: To evaluate the interactive effects of different levels of Lead and Mercury on Nitrogen fixation of both Rhizobium and Xanthobactersppin-vitro.
Place and Duration of Study: Sample organisms where collected from Groundnut rhizospheric soil of a farm in Cross River state, Nigeria. The microorganism isolation and nitrogen fixation analysis was further carried out at MacCliff General services Laboratory, Owerri, Nigeria for a duration of 3 months.
Study Design: The interactive plots serve to show the effect of one variable (lead) on the value of mercury (the other) and is derived by selecting high and low values for lead (Pb) and entering them into the equation along with the range of values for Mercury (Hg). The values of independent variables (lead and mercury levels) used in the plots were selected by observing the highest concentration (+1) and lowest concentration (-1) values which are able to support nitrogen fixation independently in Rhizobium and Xanthobacter.
Methodology: The soil samples were collected from groundnut rhizosphere at a 20 cm depth using sterile soil corer (sterilized with 95% ethanol) and matured Groundnut plants were uprooted with care. From these samples, both Rhizobium and Xanthobacterspp were isolated. The isolated organisms were re-vitalized in Jensen’s nitrogen free broth and standardized to 0.5 McFarland standards. To determine nitrogen fixation, the broth cultures were examined for nitrate nitrogen (NO3-N) and amino nitrogen (Amino-N) levels after ten days of the experiment under continuous airflow using the Jensen’s nitrogen free broth containing the metal salts, Mercury (II) chloride HgCl2 and Lead (II) acetate trihydratePb (CH3COO)2.3H2O). Nitrate nitrogen and amino Nitrogen was obtained using cataldo and ninhydrins methods respectively. The data obtained was made in triplicates and reported as mean values. Interactive effect plots and statistical analysis were done using Minitab 17 software at 5% level of significance (p<0.05).
Results: The main effect plots illustrate that to maximize nitrogen fixation in Xanthobacterspthrough the utilization of the selected metals as micronutrient, we should use lead at 6.25 mg/L and mercury at 25mg/L yielding 0.508 mg/L for nitrogen fixation response. The plot also suggests that if lead metals are used at a higher concentration than stated nitrogen fixation will decline. On the interaction plots, the slopes indicate that an interference or antagonistic interaction effect (crossed lines) exist between lead and mercury in the nitrogen fixation activity of Xanthobacter. The R-squared adjusted value suggests that 70.87% of the variations in nitrogen fixation response is explained by the interaction of lead and mercury, hence the model likely fits the data. However, the P-value was not significant at 0.102. For Rhizobium sp. mercury also has a higher fixation magnitude than Lead but relatively at 0.554 mg/L. However, the interaction plot showed parallel lines indicating that there was no interaction effect. Therefore, one can say that the relationship between lead and nitrogen fixation does not depend on the concentration of mercury and vice versa. The model was also statistically insignificant at 0.981.
Conclusion: Interactive effect only occurred in the nitrogen fixation of Xanthobactersp. This raises a need for further study combination of metal elements which could be utilized to stimulate nitrogen, phosphorus and potassium production in Diazotrophs both in the field and in-vitro.