An Operative Laboratory Investigation of Bioconversion Route From Waste Coal to Natural Energy

Purpose: In the present research, the potential of reactivated consortium for the methane production consuming waste coal as a carbon source (1% w/v) in the modied media at mesophilic temperature (37°C) was determined. Methods: Media modication was conducted for the enhancement of methane production by selecting three different components from the two media i.e. Methanosprillium sp producing media (MSP) and Methane producing bacteria media (MPB). From MSP medium; C 2 H 2 NaO 2 (sodium acetate), KH 2 PO 4 (potassium dihydrogen the phosphate) and NaHCO 3 (sodium bicarbonate) whereas from MPB medium; yeast extract, peptone, and NH 4 Cl (ammonium chloride) were selected in the range of 0.5-2.5 (g/l). Analytical assay i.e Fourier Transform Infrared Spectroscopy (FTIR) and Gas Chromatography Mass Spectrophotometry (GCMS) and Scanning electron microscopy (SEM) were conducted. Further, compatibility study and pathogenicity was performed. Results: In the present study reactivated consortia was used therefore key components of the media were modied. In case of MPB medium: 2g/l of yeast extract, 2g/l peptone and 1 g/l NH 4 Cl showed the promising results. Whereas, for MSP medium: 1 g/l of KH 2 PO 4 , 0.5 g/l of NaHCO 3 and 1.5 g/l of C 2 H 2 NaO 2 were noted to be the suitable range for methane production. Analytical studies conrmed the presences of -OH and aliphatic groups which majorly belongs to alkane, alkene, and phenol derivatives compounds whereas Scanning electron microscopy (SEM) studies delineated the active interaction of bacteria with coal particles. In addition, a compatibility study was performed and their successful results aid in the future approach of eld implementation. Further, pathogenicity data indicated the non-virulent and non-toxic nature of the consortia. Conclusions: were All the experiments were performed in triplicates, the inoculated inoculum) bottles were incubated at 37°C for 15–20 days.


Introduction
Low calori c value coal-generated from the coal mining industries is identi ed as waste coal or low-grade coal. Sometimes these are considered as a discarded coal; they generally form piles near the industries and appear as dark hills or unproductive small mountains. Waste coal usually creates metal leaching problems such as; iron, manganese, and aluminum in the water and further causes water pollution. It is also responsible for acid drainage. As these piles easily catch re they release toxic gases in the air and cause air pollution (www.energyjustice.net). Therefore, controlled production of methane from the waste coal is an economically valuable solution. Methane is one of the clean natural forms of energy which ful lls the need of many industries and households activity with less waste to the environment (Li et al., 2020). However, consistently expanding worldwide energy demands and limited fossil fuel sources has created enormous pressure for developing sustainable energy source for hydrocarbons (Gupta and Gupta 2014). Energy sources with low carbon emission, such as methane gas, are becoming important these days (Caposciutti et al., 2020).
Methane is usually trapped in the coal therefore its production becomes an alternative mode for the energy generation. Methane can be produced by thermogenic (abiogenic) and biological (biogenic) processes. Thermogenic, occurring in subsurface carbon deposits at early or late coali cation stages by the thermal cracking whereas biological occurs usually at or near the earth's surface using microorganisms (Chena et al., 2017;Wang et al., 2019). Biogenic methane is the result of complex biochemical reactions by groups of bacteria and archaea during the decomposition of organic matter in the anoxic environment. Due to the complexity in process of biogenic methane production, the procedure was poorly understood, but still, they are pervasive in nature (Wolfe 1996). In the past few years, numerous researchers have investigated the biodiversity of microbes residing in the coal seams and coal beds. The reported bacteria mainly belong to the three functional different trophic groups: hydrolytic fermentative, syntrophic acetogenic, and methanogenic bacteria (Boone 1991;Ritter et al., 2015). Hydrolytic fermentative and syntrophic acetogens, hydrolyse complex polymers (cellulose, polysaccharide, and protein) into monomers (fatty acids, sugars, amino acids, carbon dioxide, acetate, and hydrogen). These monomers are further utilized by methanogens to produce methane as depicted in Fig. 1 (Conrad et al., 1999;. Although, according to Enzmann et al.,2018, the universal mode of methane production is a hydrogen mediated reduction of carbon dioxide. Various, environmental (pH, salinity, temperature) and nutritional factors (inorganic and organic) can affect the process of methanogenesis (Boone 1991).
It has been reported that the rate of methane production depends on the maturity and functional microbial communities present in the coal. Con guration-wise, coal consists of condensed aromatic ring which makes it a complex and heterogeneous material. Lignin monolignols were considered as the main compound, whereas aromatic compounds considered as a derivative of coal which can further be substituted with hydroxyl, methoxy, and carboxyl groups. According to Mayumi et al., 2016 immature coals were commonly abundant in the methoxy groups. Since methanogenesis from coal tends to occur in immature coal rather than in mature coal, it was believed that coal-bed microorganisms may produce methane from methoxy groups (Rathi et al., 2015). Unlike coal mining, which required mechanical methods of extraction and processing, biogenic methane production is one of the conventional methods and found to be economically viable and environment-friendly.
In the present study, we proposed an approach for (1) the development and demonstration of the bioconversion process for the generation of methane from waste coal received from Tata Steel Jamshedpur, India. (2) Study the potential of developed consortia by modifying nutrient media (MSP and MPB) further, the analytical parameter of coal examination was conducted using FTIR, GCMS, followed by SEM techniques and, (3) pathogenicity assay and compatibility study was conducted. This study would help in proposing the suitable strategy for possible future in eld implementation.

Sample Collection and Characterization
In the present study, waste coal was received from Tata Steel, Jamshedpur, India. Sampling was performed in sterilized bottles and stored at ambient temperature and further transported to the laboratory (The Energy and resources of Institute, New Delhi). Characterization of waste coal was conducted in terms of ash, moisture, volatile matter, and xed carbon along with the speci c carbon, hydrogen, nitrogen, sulfur, and oxygen (CHNSO). CHNSO analysis was determined using IS: 1350/ American Public Health Association guideline (Rathi et al., 2015).
For maximum production of methane, media modi cation studies were performed in which three components from two media (MSP and MPB) were selected in a range of 0.5-2.5 g/l. Ingredients from MPB medium were; yeast extract, peptone, and NH 4 Cl and from MSP medium; Sodium acetate, KH 2 PO 4 , and NaHCO 3 were considered. Test range for ingredients was varied from 0.5, 1.0, 1.5, 2.0 to 2.5 g/l. The medium was boiled under the inert environment (using nitrogen gas). Inoculated coal bottles were kept at 37°C and gas was monitored in 5th, 10th, 15th, and 20th day. Further, to study the effect of different nitrogen source on methane production CSL (corn steep liquor) and urea was also used. Modi ed medium contained (g/l in de-ionized water): KH 2 PO 4 , 1 g; NH 4 Cl, 1 g; MgSO 4 .6H 2 O, 0.2 g; NaCl, 1.0 g; yeast extract, 2 g; Peptone, 2; NaHCO 3 , 0.5 g; Sodium acetate, 1.5 g; resazurin, 0.001 g; L-cysteine HCl, 0.5 g at pH 7.00 ± 0.2. Resazurin was added as an oxygen indicator (resazurin has a pink color at redox potentials of about150 mV). The pH was adjusted with 1M NaOH/ 1M HCL. The media was prepared anaerobically through nitrogen sparging. The medium was used for bacterial reactivation and scale-up analysis.

Reactivation of developed Consortia (Reactivated Consortia)
To study the e ciency of developed consortia, reactivation was conducted in the modi ed medium with 1% waste coal (w/v). To reactivate methanogens, an aliquot of developed consortia (5 ml) was added in 10 ml of the modi ed medium. Further, after obtaining 0.5 MacFarland standard turbidity of bacterial growth which was equivalent to 1.5 × 106 CFU/ml, subculturing was performed for inoculum preparation which was considered as reactivated consortium (Wayne 2003). All the inoculated serum bottles were incubated at 37°C for 15-20 days.

Microbial Community present in Reactivated Consortia
To identify the enriched/isolated microbial community from developed consortia, total genomic DNA was extracted and puri ed using a PowerSoil DNA Isolation Kit (MoBio,) as instructed in the manufacturer's protocol. PCR ampli cation was done with universal bacterial primers 27F

Fourier Transform Infrared Spectroscopy (FTIR)
FT-IR was carried out to identify the functional groups present in the bacterially degraded coal sample. Functional group was characterized by using Fourier Transform Infrared Spectroscopy (Perkin Elmer). All spectra were recorded in an absorbance scale with a mid-measuring region of 400-4000 cm − 1 (midinfrared range). The resolution was set at 4 cm − 1 with 64 scans per spectrum.

Gas Chromatography (GC)
In the present analysis, concentration of gas produced in the headspace (methane and carbon-dioxide in %) of media bottles were analyzed with GC 7890A Agilent Ltd. USA equipped with a packed stainless steel column (2m×2mm id NUCON, INDIA) with a thermal conductivity detector (TCD). Where, argon acts as the carrier gas with ow rate of 1.0 ml/min. The operating temperatures of the injection port, oven and the detector were 100, 50 and 150°C respectively (Rathi et al., 2015). The incubated cultures were tested for CH 4 and CO 2 production after 15-20 days by taking 0.5 ml of headspace gas samples from the anaerobic serum bottles using gas-tight syringe.

Gas Chromatography Mass Spectrophotometry (GCMS)
The sample was analyzed using GCMS (model GC-7890A, Agilent Ltd., United States) equipped with DB-WAX capillary column. Helium was used as the carrier gas. Temperature ranges between 230-325°C.
Initially, column temperature was set at 70°C and further increased to 325°C. Diluted sample (1/50 in methanol) of 0.1µl was used. The components were identi ed on the basis of their mass spectra using NIST (National Institute for Standards and Technology) library data base.

Scanning Electron Microscopy (SEM)
Interactions between bacterial species and coal were studied by Scanning Electron Microscopy (Carl Zeiss) (Hayat 2000). Under aseptic conditions, sample was absorbed for 2 to 4 h in 2.5% glutaraldehyde solution. 0.1M phosphate buffer was used for primary washing where pH maintained up to 7.2 further sample was dehydrated with ethanol solution in a series of 10-100% followed by acetone. Samples were air dried overnight and coated with thin layer of metal (gold and palladium).

Pathogenicity Test
The pathogenicity test of reactivated consortia was examined by acute oral toxicity under EPA 712-C-96-322 OPPTS 885.3550 guidelines at National Toxicology Centre (APT Testing and Research Pvt. Ltd.), Pune. Twelve mice (6 male and 6 female) were designated to the dose groups: control and test (1 ml = 1.0*10 8 CFU) were administrated by the gauge to six mice per sex. The mice were fasted overnight and 2 h after administration of the test material.
The mice were observed for 21 days after dosing. At the end of the inspection period, the surviving experimental animals were sacri ced for testing. Gross necropsy was performed and all animals were carefully examined for the presence of anaerobic bacteria. The body weight was recorded. All animals were observed for mortality throughout the observation period. RBC (red blood cell), WBC (white blood cell), Hemoglobin, Packed cell Volume, Glucose, BUN (blood urine nitrogen), Total Proteins and Albumin were studied on 21st day of the experiment.

Compatibility Studies
Before eld implementation test, compatibility studies were conducted in the lab. In this analysis obtained tube well water was used for media preparation (available near the washeries in Jharia). Experiment was conducted in four sets; in set 1: anaerobic condition was maintained without autoclaving (referred as S-A), in set 2: anaerobic condition was maintained with proper sterilization (referred as S + A), in set 3: aerobic condition without autoclaving (referred as US-A) and in set 4: aerobic condition with autoclaving (referred as US + A). While preparing the media no precipitation was observed with commercial grade of chemicals in tube well water. In all sets waste coal (1% w/v) was used. After inoculating with inoculum (10%) all sets were incubated at 37°C for 15-20 days.

Statistical Analysis
All the experiments were performed in triplicates. The data points are average of the triplicate ± standard deviation (less than5% of average) and calculated signi cance p values are ≤ 0.05.

Coal Characterization
The detailed analysis of collected waste coal samples in terms of ash, moisture, volatile matter and xed carbon along with the speci c carbon, hydrogen, nitrogen, sulfur and oxygen (CHNSO) was determined as per the guidelines of ASTM standard (Table S1) (Rathi et al., 2015). Proximate analysis data showed that waste coal contains 0.49% of moisture, 14.85% of volatile matter along with high 42.52% of ash and 42.14% of xed carbon. The calori c value of waste coal was 4092 kcal/kg. Obtained data of waste coal indicated the signi cant potential in the bioconversion process of methane. The ultimate analysis of the waste coal samples showed 44.71% of carbon, 2.55% of hydrogen, 0.04% of nitrogen, 0.28% of sulfur, and 9.41% of oxygen. Figure 2 illustrated the gas production (methane and carbon dioxide) in all four successive enrichment cycles in both the speci c media (MSP and MPB) at 37°C. However, in the MSP medium, an increase in methane generation was observed from 9.99 % to 27.4 % in 1st and 4th enrichment cycle respectively whereas; carbon-dioxide was decreased from 5.2 % to 4.9 %. Further, CH4: CO2 (methane: carbon-dioxide) the ratio was ranging from 1.8 to 5.5. Similarly, in the case of MPB medium, rises in methane generation was noted from 12 % to 29.2%, and reduction in carbon-dioxide from 6.6 % to 4.5% in the 1st and 4th cycles respectively. Also, CH4:CO2 the ratio was ranging from 2.0 to 6.4. In the developed consortium, both media showed almost the similar trends in methane production (29.2 % in MPB and 27.4% in MSP)

Modi cation of Nutrient Media
The modi cation study was performed to enhance the biogenic methane content. Methane production by consortia was tested with different sets of MPB and MSP media. Gas was monitored at interval of 5 days during incubation period. Figure 3 data demonstrated the methane production in selected range of components form MPB medium in with and without coal sets. Selected components were peptone (Fig. 3A), yeast extract (Fig. 3B), and NH 4 Cl (Fig. 3C) in a range between 0.5, 1.0, 1.5, 2.0 to 2.5 g/l with waste coal (1% w/v). In this experiment, it was observed that methane was increased up to a concentration after that production was not supportive. In case of yeast extract and peptone, 2g/l was obtained to be preeminent concentration for methane production (42.3% and 35.23% respectively) whereas, in NH 4 Cl; 1 g/l was showed the respectable result with 38.1% of methane after 20th day of incubation. Figure 3D depicted the methane generation in without coal set. Methane was observed at 20th day of incubation. By comparing the data of with and without coal, it was noted that methane generation was more in case of set containing coal.
Further in MSP medium (Fig. 4), 1 g/l of KH 2 PO 4 (Fig. 4A), 0.5 g/l of NaHCO 3 (Fig. 4B) and 1.5 g/l of C 2 H 2 NaO 2 (Fig. 4C) indicated the suitable range for methane production with 23.34%, 23.45% and 34.22% respectively after 20th day of incubation. Figure 4D showed the methane production without coal after 20th day. As the range increased for selected components, methane production was increased up to particular range beyond which it seems to be not favorable. Data obtained in with and without coal sets, con rmed that coal was participating actively in methane generation. With coal, methane generation was observed to be more. Further, in modi cation study with different nitrogen source on methane generation was studied with CSL and Urea components. Obtained data was not favorable for methanogenesis as represented in Figure S1.

Identi cation of Microbial Community Present in Reactivated Consortia
As shown in Fig. 5, reactivated consortia from developed consortia contained majority of similar community as described in published manuscript (Lavania et al., 2014). Both bacterial and archaeal domain was noted. Methanoculleus thermophiles was observed as the major archaeal species in Methanoculleus sp. Apart from Methanoculleus sp., Comamonas sp. was also obtained. In reactivated consortia majority of species were similar (Methanoculleus sp. and Comamonas sp.). Along with archaeal domain, bacterial domain was also noted in reactivated consortia which were comprised of fermicutes (Clostridium sp.) and proteobacteria (Pseudomonas sp.). The obtained microbial community was able to generate methane at mesophilic condition.

Scanning Electron Microscopy (SEM)
SEM images depicted the coal particles were destructed and reduced in sized after the bacterial intervention ( Fig. 7A and 7B). The reduced sized coal provided the large surface area which makes adherence easy for the bacteria. Morphology of bacteria and bacterial interaction with coal particles were clearly visible in Fig. 7C, where red arrows indicated the presence of bacteria and black arrows showed the coal particles. Mixed natures of anaerobic bacteria were noted, where rod and coccus shaped were observed and presences of bacteria were recorded in the clusters form.

Pathogenicity Assessment
Reactivated consortia (1 ml of dose) did not show any case of mortality in the treated mice (both male and female). All the mice were appeared to be normal and showed no clinical signs of intoxication after dosing till the end of the study. No statistically signi cant difference in the hematological and blood chemistry parameters (Red Blood Cells, White Blood Cells, Hemoglobin, Packed Cell Volume, BUN, Albumin, Total protein and Glucose) was observed in the test group. After evaluating the test groups with control group, there were no numerically decreased in body weight was observed. The results from the necropsy revealed no abnormalities in the test group when compared with the control group animals. The consortia did not induce any gross pathological alterations, in experimental models during their necropsy. The sacri ced mice's were thoroughly examined and were found to be completely free from any live anaerobic bacteria. After analyzing the data, reactivated consortia were considered to be non-toxic and non-virulent. Hence, it is safe for eld implementation (Table S3-S6).

Compatibility Studies
Compatibility study of waste coal in modi ed media with reactivated consortia was executed as represented in Fig. 8. Methane and carbon-dioxide was observed in all experimental sets with different percentage. In set 1 and set 2, signi cantly high amount of methane and low amount of carbon-dioxide was noticed with 51.6%, 49.91 methane and 6.25%, 7.61% CO 2 respectively. In set 3, methane was found to be signi cantly reduced which was 13.2% with 7.21% of carbon-dioxide. Whereas, in set 4; negligible amount of methane (0.54%) was observed with 7.56% of carbon-dioxide.

Discussion
Bio-conversion of coal to methane can be considered as a healthy and feasible approach for the 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) modi cation studies were conducted by selecting two speci c media (MPB and MSP). In MPB medium concentration of yeast extract, peptone and NH 4 Cl whereas in MSP medium concentration of C 2 H 2 NaO 2 , KH 2 PO 4 and NaHCO 3 were altered. Modi cation 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 NH 4 Cl which behaves like a common complex and de ned 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;. 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 NH 4 Cl showed promising results (Fig. 3) whereas urea and CLS were not found to be that effective ( Figure S1). In MSP medium; C 2 H 2 NaO 2 , KH 2 PO 4 , and NaHCO 3 were elected. According to Ulrich & Bower 2008 study, C 2 H 2 NaO 2 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). KH 2 PO 4 and NaHCO 3 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 signi cant 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 modi ed 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 modi ed 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 rmicutes (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 CH 2 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) Further, pathogenicity data of consortia revealed that consortium was safe for the large scale analysis or the eld trial (Table S3-S6). In the experiment of compatibility (Fig. 8) potential of waste coal for methane production was noted signi cantly. 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.

Conclusions
Our study emphasizes the production of renewable energy (methane) from the waste piles of coal present near the coal mining area. This study proves the enhancement of methane generation in the presence of coal containing medium. The data of FTIR and GCMS illustrated the complex nature of the coal sample. Moreover, active interactions of bacteria with coal particles were detected in the SEM micrograph. Further, pathogenicity assay explained the non-pathogenic nature of the consortium. The results highlighted the potential of the bioconversion process from waste to renewable energy generation. This study can be seen as a promising alternative method for energy generation through coal waste piles. 4 . Zhou, C., Liu, G., Cheng, S., Fang, T., and Lam, P. K. S., (2014). Thermochemical and trace element behavior of coal gangue, agricultural biomass and their blends during co-combustion. Bioresources  Figure 1 Schematic demonstration of microorganism interactions in the bio-conversion process of coal to methane.

Figures
Page 17/24 Figure 2 Explained the percent of gas generation by developed consortia in MSP and MPB medium where waste coal was used as carbon source in various enrichment cycles. Data recorded after 20 days of incubation.  A, B and C; demonstrated the methane gas production in KH2PO4, NaHCO3 and C2H2NaO2 selected components from MSP medium in range 0.5-2.5 g/L with coal (1.0% w/v). Whereas D; depicted the methane production without coal.   A, Depicted the untreated (before bacterial treatment) coal particles (scale bar 2 µm). B; represented the treated (after bacterial treatment) coal particles (scale bar 2 µm). C; illustrated the interaction between bacteria and coal particle (scale bar 2 µm).

Figure 8
Depicted the compatibility test in modi ed medium with waste coal and tube well water (Set 1: S-A, where S signify Nitrogen sparge media (anaerobic) and -A represent without autoclave media; Set 2: S+A,where S signify nitrogen sparge media and +A represent Autoclave media; Set 3: US-A,where US signify without sparging media (aerobic) and -A represent without Autoclave media; Set 3: US+A, where US signify without sparging media (aerobic) and +A represent autoclave media.