## 4 Discussion  
The majority of the influences of earthworms on nutrient cycling and biogeochemical processes of the ecosystem is due to the joint activities of earthworms and the microorganisms selectively activated within their functional domains [2; 30]. The gut of the earthworm is dominated by high amounts of mucus, plant derived saccharides, organic acids, several denitrification-derived gases such asnitrous oxide (N\({}_{2}\)O), molecular hydrogen (H\({}_{2}\)) and molecular nitrogen (N\({}_{2}\)). The earthworm gut also experiences increased total organic carbon, nitrogen, and moisture contents which provide an optimal environment for the activation of dormant microbes and support the germination of ingested endospores [3; 31]. The earthworm gut environment can influence EPS producing microorganisms [4]. The isolation of an EPS producing bacterial strain _Bacillus licheniformis_ with accession number KX65784 from the earthworm gut (_Metaplire posthumua_) has been reported earlier [4; 20]. The isolated strain was unique in producing EPS only in the presence of L-asparagine monohydrate and maximum EPS yield was observed when sucrose was the carbon substrate [4]. The phenotypic and genotypic characterization, optimal conditions for growth and EPS production of the strain isolated from the earthworm gut were also reported earlier [4]. The current study focuses on the application aspect of the EPS produced by the above bacterial strain. The FTIR and NMR analyses act as powerful tools for studying and speculating several functional groups [32], carbohydrate compositions and structure of complex substances in biomolecules such as EPS. The \({}^{1}\)H NMR and \({}^{13}\)C NMR spectra (Figure 1a,b) confirmed that the isolated EPS possessed electronegative substituents or groups, unsaturated carbon, CH\({}_{2}\)-chain and carbonyl groups. The signals from \(\delta\)-1.9 ppm and \(\delta\)-2.3 ppm resembling -CH\({}_{2}\) protons, and signals from \(\delta\)-3.5-3.8 ppm indicating the presence of electronegative group deshielding were in accordance to earlier report [26]. Previous authors [26] similarly reported a strong signal at \(\delta\)-1.87 ppm indicating the presence of alkyl -CH\({}_{2}\) protons, while the \(\delta\)-3.5-3.8 ppm signals indicated the presence of electronegative group. The FTIR analysis showed that majority of the functional groups in the isolated EPS belonged to alkanes, alkynes, carboxylic and alcoholic groups. The results of FTIR and NMR analyses obtained by previous studies [33; 34; 35] were in conformity with our results. The EPS composition consisted of carbohydrates with some amounts of proteins and sugar acids, and also traces of nucleic acids. Mass spectra (Figure 2) revealed the mass of the extracted EPS. The anionic nature of the EPS extracted from the gut of the earthworm was confirmed from the zeta potential value [4]. The biological functioning of EPS and EPS producing bacteria are determined by the occurrence of the charged (positive or negative) functional groups. The presence of a significant quantity of negatively charged functional groups, such as carboxyls and hydroxyls, in the EPS evident from the NMR and IR studies indicated a high carbohydrate to protein ratio in the biomolecules. This feature of the EPS might offer a vast promise in the removal and remediation of heavy metals from polluted environments by this EPS [23; 28; 36]. medium [38]. In the present study, the bioflecculant showed a flocculation efficiency of 74% at 4 mg L\({}^{-1}\) concentration of EPS (Figure 3a). Different microbial strains have different flocculation efficiency at different concentrations. The ability of _Bacillus licheniformis_ strains to produce biofleculant has been reported [39; 40]. Studies showed that bioflecculant from the strain _Bacillus licheniformis_ produced 700 U mL\({}^{-1}\) flocculation activities after cultivation at 37 \({}^{\circ}\)C for 48 h [40]. Another study on _Bacillus_ sp.