Bio-conversion of coal to methane can be considered as a healthy and feasible approach for the environment. As studies reported by Ge H et al., 2016; Ribeiro et al., 2012; Hao et al., 2016 suggested the toxic nature of waste coal piles generated near the industries during coal mining. Therefore, through this study recovery of significant methane was observed using waste coal.
In the present research, microbes from the developed consortia were reactivated and further used as a source for biogenic methane production. Figure 2 showed the production of methane gas (29.2 % in MBP and 27.4 % in MSP) along with the carbon-dioxide (5.2 % in MBP and 6.6 % in MSP). Therefore, to maintain the composition of gas (majorly methane) modification studies were conducted by selecting two specific media (MPB and MSP). In MPB medium concentration of yeast extract, peptone and NH4Cl whereas in MSP medium concentration of C2H2NaO2, KH2PO4 and NaHCO3 were altered. Modification provided promising results for methane generation in the scale-up analysis.
Each selected component plays a vital role in the methanation process as depicted in Fig. 3 and Fig. 4. Selected components from the MPB medium were; yeast extract, peptone, and NH4Cl which behaves like a common complex and defined nitrogen source in the medium. Previous studies have been examined for their potential to enhance coal-to-methane conversion (Verstraete et al., 1984; Wagner et al., 2012; Davis et al., 2018). Preceding researches also investigated urea and CLS (corn steep liquor) compounds as a respectable nitrogen source (Yang et al., 2014; Tan et al., 2016). But in this investigation yeast extract, peptone and NH4Cl showed promising results (Fig. 3) whereas urea and CLS were not found to be that effective (Figure S1). In MSP medium; C2H2NaO2, KH2PO4, and NaHCO3 were elected. According to Ulrich & Bower 2008 study, C2H2NaO2 was considered as an essential ingredient for methanogenesis. Furthermore, pH also plays an important role in the methanation process. And with the proper buffering system optimized pH can be achieved (Gupta and Gupta 2014; Yang et al., 2018). KH2PO4 and NaHCO3 were considered as the chief components in maintaining the pH of the medium (Eduok et al., 2018). As the selected components of MPB and MSP media had a significant role in the methane generation process, they were varied in a certain range (0.5-2 g/l) for the modifying study. The reactivated consortium showed the highest methane production at 37°C in the modified medium when waste coal was used as a carbon source. By comparing Fig. 2, Figs. 3 and 4 noteworthy differences in methane generation were noted. In the case of MBP and MSP media, methane production was observed to be 29.2 % and 27.4 % respectively (Fig. 2), whereas in modified medium methane generation was in the range of 40–50%. These results prove that the nutrient amendment was a successful strategy for methane production.
Figure 3 and Fig. 4 data also illustrate the importance of coal in the medium. By observing with coal (Fig. 3A, 3B, 3C, 4A, 4B, 4C) and without coal (Fig. 3D, 4D) data sets, maximum production of methane after the 10th day of incubation was noticed in sets having coal. This study emphasizes the importance of methane production in a low incubation period as previous literature, on waste coal showed more than a month of the incubation period (Opara et al., 2012; Gupta and Gupta 2014).
The microbial community present in the reactivated culture showed a 95% similarity with developed consortia (Fig. 5). Both bacterial and archaeal domain was observed. The bacterial domain was comprised of firmicutes (Clostridium beijerinckii and Clostridium sp.) and Proteobacteria (Pseudomonas sp. and Comamonas sp.) Similar species was reported by many scientists for methane generation (Bi et al., 2017). Further, methanation by similar species at 23°C was also observed (Fuerteza et al., 2018). The archaeal domain includes majorly Methanoculleus sp. which are responsible for methane production was also noted. According to Zellner et al., 1998; Zhu et al., 2011 research on methanation similar archeal species were reported. The genera Methanoculleus were related to the family Methanomicrobiaceae, this family contains methanogens of highly irregular coccoid shape with optimal growth temperature 25–60°C (Spring et al., 2005). By looking into the mechanism of methane production by the microbial community; it was reported by previous researchers that acetogenic microorganisms oxidize organic compounds partially into acetate which was further consumed by methanogens for methane production (Kushkevych et al., 2017) (Fig. 1). Clostridium sp. is a well-known acetogenic species; it utilized the organic component from the environment and produces acetate (Schmidt et al., 1985). Further, the byproduct of Clostridium sp. (acetate) is consumed by methanogens for methane production.
In analytical studies, FTIR provided the details of functional groups present in the coal sample (Fig. 6A). As reported by Reddy and vinu 2016; Sonibare et al., 2012 organic part of coal contains aromatic, aliphatic, and oxygen groups. The spectrum obtained from FTIR of coal sample attributed the presence of -OH and C = C groups. The presence of aromatic C = C stretch demonstrated that the carbon content was more in the sample. The CHNS data also proved the same, the possible reason for high carbon content could be the reduction of oxygen due to the conversion of C = O to CH2 or decarboxylation (Manoj et al., 2009). FTIR spectra reported by Li et al., 2018; Zhang et al., 2018 showed similar trends. Further, extending the analysis in identifying the chemical groups of coal sample GC-MS was considered as a powerful tool (Fig. 6B). Aliphatic compounds present in the sample contained various range of hydrocarbons, alkene, and cyclic or acyclic compounds (Table S2). By observing the fragmentation pattern, the peak at RT 24.4 corresponds to 2,4-Di-tert-butyl which has a role in bacterial metabolites (National Center for Biotechnology Information NIH). Damin et al., 2010; Shi et al., 2013 demonstrated the presence of alkenes, cyclic, and acyclic organic species in the coal sample. FTIR and GCMS data revealed that bacteria can utilize the components from coal for the production of methane. Further, an SEM micrograph depicted the interaction between coal and bacteria (Fig. 7). Stephen et al., 2014 study, explained the interactions between bacteria (rod-shaped, anaerobic) and coal. According to Wang et al., 2017 SEM images illustrated the growth of microflora on the surface of coal and their effects on the coal surface in terms of morphological change.
Further, pathogenicity data of consortia revealed that consortium was safe for the large scale analysis or the field trial (Table S3-S6). In the experiment of compatibility (Fig. 8) potential of waste coal for methane production was noted significantly. The nitrogen sparged sets; set 1 and set 2 demonstrated the maximum production of methane (51.6% and 49.91% respectively) this study also proved that an anaerobic environment is a vital factor for methanogenesis. Whereas, sets without sparged (set 3 and set 4) showed considerably low and negligible methanation (13.2% and 0.54% respectively). This research provides an idea for establishing a feasible way for creating a pollution-free environment from waste coal to clean and natural energy.