Skip to main content

An operative laboratory investigation of bioconversion route from waste coal to natural energy

Abstract

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 modified media at mesophilic temperature (37 °C) was determined.

Methods

Media modification 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, C2H2NaO2 (sodium acetate), KH2PO4 (potassium dihydrogen the phosphate), and NaHCO3 (sodium bicarbonate) whereas from MPB medium; yeast extract, peptone, and NH4Cl (ammonium chloride) were selected in the range of 0.5–2.5 (g/l). Analytical assay, i.e., Fourier transform infrared spectroscopy (FTIR), gas chromatography mass spectrophotometry (GCMS), scanning electron microscopy (SEM), and energy dispersive X-ray (EDX) 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 modified. In case of MPB medium, 2 g/l of yeast extract, 2 g/l peptone, and 1 g/l NH4Cl showed the promising results; whereas for MSP medium, 1 g/l of KH2PO4, 0.5 g/l of NaHCO3, and 1.5 g/l of C2H2NaO2 were noted to be the suitable range for methane production. Analytical studies confirmed the presences of -OH and aliphatic groups which majorly belongs to alkane, alkene, and phenol derivative compounds whereas SEM and EDX studies delineated the active interaction of bacteria with coal particles and presences of carbon (C) as a major peak in untreated coal and absence of C peak in microbial treated coal. In addition, a compatibility study was performed and their successful results aid in the future approach of field implementation. Further, pathogenicity data indicated the non-virulent and non-toxic nature of the consortia.

Conclusions

The production of waste coal is one of the most problematic and common activities of the mining industry. They release toxic substances into the environment (water, air, and soil) and damage the local biodiversity. Therefore, the generation of biogenic methane from waste coal is an environmentally friendly approach to overcome this problem.

Introduction

Low calorific value coal-generated from the coal mining industries is identified 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 fire, 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 fulfills 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 coalification 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 hydrolyze 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; Davis and Gerlach 2018). 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).

Fig. 1
figure 1

Pictorial representation of microorganism interactions in the bio-conversion process of coal to methane

It has been reported that the rate of methane production depends on the maturity and functional microbial communities present in the coal. Configuration-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) To study the potential of developed consortia by modifying nutrient media (MSP and MPB) further, the analytical parameter of coal examination was conducted using FTIR and GCMS, followed by SEM and EDX techniques, and (3) pathogenicity assay and compatibility study were conducted. This study would help in proposing the suitable strategy for methane generation and possible future approach (Fig. 2).

Fig. 2
figure 2

Schematic demonstrations of bio-conversion process of microorganism interactions with coal and generation of methane

Methods

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). The characterization of waste coal was conducted in terms of ash, moisture, volatile matter, and fixed carbon along with the specific carbon, hydrogen, nitrogen, sulfur, and oxygen (CHNSO). CHNSO analysis was determined using IS: 1350American Public Health Association guideline (Rathi et al., 2019).

Enrichment and modification of media

Initial optimization studies were based on one factor at a time analysis for enhanced methane production. In the modification studies, yeast & peptone (as a growth agent), KH2PO4 and NaHCO3 (as a buffering agent) and NH4Cl, CSL, and urea (as a nitrogen agent) were selected. And for the enhancement in methanation C2H2NaO2 was added to the study.

To study the potential of developed consortia at mesophilic condition, four enrichment cycles were performed in two different media specific for different species of methanogens. MSP (Methanosprillium sp.) and MPB (methanogen specific). Components like KH2PO4, C2H2NaO2, and NaHCO3 belongs to MSP medium and components peptone, yeast, and NH4Cl belongs to MPB medium. Detailed study based on urea and CSL was conducted (data presented in Supplementary). After obtaining the best range for the selected components optimized media was used for the further studies.

The MSP medium contained (g/l in de-ionized water): KH2PO4, 0.5 g; MgSO.7H2O, 0.4 g; NaCl, 0.4 g; CaCl2.2H2O, 0.05 g; FeSO4.7H2O, 0.002 g; yeast extract, 1 g; C2H2NaO2, 1 g; sodium formate, 2 g; NaHCO3, 4 g; resazurin, 0.001 g; and l-cysteine HCl, 0.5 g at pH 7.00 ± 0.2 (Lavania et al., 2014). The composition of MPB medium (g/l in de-ionized water) was K2HPO4, 0.3 g; KH2PO4, 0.3 g; NH4Cl, 0.5 g; MgSO4.6H2O, 0.2 g; NaCl, 1.0 g; yeast extract, 1.0 g; casein peptone, 1.0 g; resazurin, 0.001 g; and l-cysteine HCl, 0.5 g at pH 7.00 ± 0.2. The pH was adjusted with 1 M NaOH/1 M HCL. The medium was then boiled under a stream of oxygen-free nitrogen gas to remove all the dissolved oxygen. After cooling under continuous nitrogen flow, the medium was dispensed into 100 ml serum bottles containing 1% w/v of waste coal (used as a carbon source). The bottles were sealed with butyl rubber stoppers and sterilized at 121 °C for 20 min. All the experiments were performed in triplicates, the inoculated serum (10% inoculum) bottles were incubated at 37 °C for 15–20 days.

For maximum production of methane, media modification 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 NH4Cl and from MSP medium; sodium acetate, KH2PO4, and NaHCO3 were considered. Test range for ingredients was varied from 0.5, 1.0, 1.5, and 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. Modified medium contained (g/l in de-ionized water): KH2PO4, 1 g; NH4Cl, 1 g; MgSO4.6H2O, 0.2 g; NaCl, 1.0 g; yeast extract, 2 g; Peptone, 2; NaHCO3, 0.5 g; Sodium acetate, 1.5 g; resazurin, 0.001 g; and 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 1 M NaOH/1 M 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 efficiency of developed consortia, reactivation was conducted in the modified medium with 1% waste coal (w/v). To reactivate methanogens, an aliquot of developed consortia (5 ml) was added in 10 ml of the modified 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 purified using a PowerSoil DNA Isolation Kit (MoBio) as instructed in the manufacturer’s protocol. PCR amplification was done with universal bacterial primers 27F (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1492R (5′-ACG GCT TAC CTT GTT ACG CTT-3′) as well as archaeal primers 109f (5′-ACK GCT CAG TAA CAC GT-3′) Met 915R (3′GTG CTC CCC CGC CAA TTC CT-5′) (Lavania et al., 2014). For gene sequencing, PCR product was outsourced (AgriGenome Labs Pvt. Ltd). Following quality check of the FASTQ files using FastQC (v0.11.9), the sequencing data files were analyzed using DADA2 package (version 1.14.0) which included quality filtering, trimming of barcode/adaptors, dereplication, learn error rates, and chimera removal, merging of paired reads. The SILVA version 138 16S rRNA gene reference database was used to assign bacterial taxonomic classification. The phylogenetic tree was constructed using the neighbor-joining method in MEGA (version 6.06) package. The tree topologies were estimated with 1000 bootstrap data sets. The similarity value used for the identification of microbial population was of 97%, from the assessed microbial consortia (Fuertez et al., 2018).

Analytical analysis of sample

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 (mid-infrared 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 (2 m × 2 mm id NUCON, India) with a thermal conductivity detector (TCD), where argon acts as the carrier gas with flow 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 CH4 and CO2 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 and 325 °C. Initially, column temperature was set at 70 °C and further increased to 325 °C. Diluted sample (1/50 in methanol) of 0.1 μl was used. The components were identified 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. 0.1 M 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).

Energy dispersive X-ray (EDX)

The energy dispersive X-ray (EDX) is a known technique for detecting elemental present in the specimens. The X-ray revealed the true nature of the test sample. For the present study, the coal with or without treatment with bacteria was carried out in the Bruker X Flash 630 EDS detector using DX-700HS spectrometer (Shimadzu).

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 the National Toxicology Centre (APT Testing and Research Pvt. Ltd.), Pune. Twelve mice (6 males and 6 females) were designated to the dose groups: control and test (1 ml = 1.0*108 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 sacrificed 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 the 21st day of the experiment.

Compatibility studies

Before field 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 significance p values are ≤0.05.

Results

Coal characterization

The detailed analysis of collected waste coal samples in terms of ash, moisture, volatile matter, and fixed carbon along with the specific 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 fixed carbon. The calorific value of waste coal was 4092 kcal/kg. Obtained data of waste coal indicated the significant 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.

Enrichment studies

Figure 3 illustrated the gas production (methane and carbon dioxide) in all four successive enrichment cycles in both the specific 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 to carbon-dioxide), the ratio was ranging from 1.8 to 5.5. Similarly, in the case of MPB medium, rises in methane generation were 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).

Fig. 3
figure 3

Explained the percent of gas generation by developed consortia in MSP and MPB medium respectively where waste coal was used as carbon source in various enrichment cycles. Data recorded after 20 days of incubation

Modification of nutrient media

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 4 data demonstrated the methane production in selected range of components form MPB medium in with and without coal sets. Selected components were peptone (Fig. 4A), yeast extract (Fig. 4B), and NH4Cl (Fig. 4C) in a range between 0.5, 1.0, 1.5, 2.0, and 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, 2 g/l was obtained to be preeminent concentration for methane production (42.3% and 35.23%, respectively), whereas in NH4Cl, 1 g/l was showed the respectable result with 38.1% of methane after the 20th day of incubation. Figure 4D depicted methane generation in set without coal (control). Methane was observed at the 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.

Fig. 4
figure 4

AC The methane gas production in peptone, yeast extract, and NH4Cl selected components from MPB medium in range of 0.5–2.5 g/L with coal (1.0% w/v), whereas D depicted the methane production without coal

Further in MSP medium (Fig. 5), 1 g/l of KH2PO4 (Fig. 5A), 0.5 g/l of NaHCO3 (Fig. 5B), and 1.5 g/l of C2H2NaO2 (Fig. 5C) indicated the suitable range for methane production with 23.34%, 23.45%, and 34.22%, respectively, after the 20th day of incubation. Figure 5D showed the methane production without coal (control) after the 20th day. As the range increased for selected components, the methane production was increased up to particular range beyond which it seems to be not favorable. Data obtained in with and without coal sets confirmed that a significant amount of coal converted into energy which was not in the case of set without coal. With coal, methane generation was observed to be more. Further, in modification study, impact of different nitrogen source on methane generation was studied using CSL and Urea components. Obtained data was not favorable for methanogenesis as represented in Fig. S1.

Fig. 5
figure 5

AC 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

Identification of microbial community present in reactivated consortia

The phylogenetic studies of reactivated CBM65 were studied, and it was observed that it shows the resemblance with published manuscript (Lavania et al. 2014). Both bacterial and archaeal domain were noted, and the tree was constructed (Fig. 6). The phylogenetic profile indicated that the consortium contained mixed phyla of anaerobic archaeal and firmicutes. These are available under the accession numbers OK175643, OK175645, and OK175646.

Fig. 6
figure 6

The phylogenetic tree of the consortium was constructed using neighbor-joining. The tree topologies were estimated with 1000 bootstrap data sets. The tree shows the relationship within the archaeal and bacterial domain. GenBank accession numbers are provided in brackets. The scale bar indicates sequence divergence

CBM65 shows maximum similarity with archaeal phylum containing Methanoculleus thermophiles and Methanoculleus receptaculi and minimum with Methanoculleus sediminis, Methanoculleus chikugoensis, and Methanoculleus marisnigri. Furthermore, bacterial domain was also noted in reactivated consortia which were comprised of fermicutes (Clostridium sp. and Clostridium beijerinckii). The obtained microbial community was able to generate methane at mesophilic condition.

Analytical analysis of sample (FT-IR and GC-MS)

In order to understand the functional groups present in the coal sample, FITR analysis was performed as shown in Fig. 7A. Spectra fragment lies between 1600 and 1800 cm− 1 was attributed to aromatic group (C=C). Further the stretching of 3000–2800 cm− 1 corresponded to -OH and -NH groups. The presence of aliphatic groups (methyl and methylene) illustrated by spectra range from 2400 to 2000 cm− 1. Smaller peaks beyond 1600 cm− 1 were represented the presence of mineral compositions in the sample (Damin et al., 2010; Zhou et al., 2014; Singh and Zondlo, 2017).

Fig. 7
figure 7

A The FTIR spectra of coal sample. B The fragmentation pattern of coal sample

Previous reports stated the presence of low molecular weight hydrocarbons in coal sample, where GCMS plays the important role in identifying those compounds (Fan et al., 2013). The organic moiety present in coal was observed to be cyclic and acyclic hydrocarbons mainly alkanes, alkenes, and derivatives of phenols (Table S2). Figure 7B illustrated the fragmentation pattern of compounds present in the sample. Major peaks were recorded at RT 27.39, 37.74, 44.32, 47.05, and 61.00 which corresponds to 2,4-Di-tert-butyl phenol, 5-Octadecene, E-15 Heptadecenal, 1-Nonadecanol, and Cyclotetracosane, respectively (https://pubchem.ncbi.nlm.nih.gov/compound/2_4-Di-tert-butylphenol).

Scanning electron microscopy (SEM) and energy disruptive X-ray (EDX)

SEM micrographs depicted the coal particles were destructed and reduced in sized after the bacterial intervention (Fig. 8A, B). Figure 8A shows the intact coal piece without any bacterial treatment whereas the 8B has bacteria with coal particles. 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. 8b, where red arrows indicated the presence of bacteria, white arrows showed the coal particles and yellow circle illustrated the bacterial interaction with coal. Mixed natures of anaerobic bacteria were noted, where rod and coccus shaped were observed and bacteria in clusters were also recorded. By observing EDX graphs of coal and bacterial treated coal, major peaks were found to be missing. In coal samples, peaks for carbon (C), oxygen (O), silicon (Si), iron (Fe), titanium (Ti), magnesium (Mg), sodium (Na), sulfur (S), potassium (K), phosphorus (P), calcium (Ca), and aluminum (Al) were noted whereas in case of after bacterial treatment peaks of C and O were not observed (Fig. 8C, D).

Fig. 8
figure 8

A The untreated (before bacterial treatment) coal particles (scale bar 2 μm). B The treated (after bacterial treatment) coal particles (scale bar 2 μm)

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 significant 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 the 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 sacrificed 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 field implementation (Table S3–S6).

Compatibility studies

Compatibility study of waste coal in modified media with reactivated consortia was executed as represented in Fig. 9. Methane and carbon-dioxide were observed in all experimental sets with different percentage. In set 1 and set 2, significantly high amount of methane and low amount of carbon-dioxide was noticed with 51.6%, 49.91 methane, and 6.25%, 7.61% CO2, respectively. In set 3, methane was found to be significantly 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.

Fig. 9
figure 9

The compatibility test in modified media with waste coal and tube well water (Set 1: S-A, where S signifies nitrogen sparge media (anaerobic) and -A represents without autoclave media; Set 2: S + A, where S signifies nitrogen sparge media and + A represents Autoclave media; Set 3: US-A, where US signifies without sparging media (aerobic) and -A represents without autoclave media; Set 3: US+A, where US signifies without sparging media (aerobic) and + A represents autoclave media

Discussion

Bio-conversion of coal to methane can be considered as a healthy and feasible approach for the environment (Fig. 2), as studies reported by Ge et al. (2016), (Ribeiro et al. 2012), and 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 3 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 and 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 Figs. 4 and 5. Selected components from the MPB medium were yeast extract, peptone, and NH4Cl which behaves like a common complex and defined growth and nitrogen source for the microorganism. 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). The 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. 4) whereas urea and CLS were not found to be that effective (Fig. 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 Figs. 3, 4, and 5, noteworthy differences in methane generation were noted. In the case of MBP and MSP media, the methane production was observed to be 29.2% and 27.4%, respectively (Fig. 3), 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.

Figures 4 and 5 data also illustrate the importance of coal in the medium. By observing with coal (Figs. 4A–C and 5A–C) and without coal (Figs. 4D and 5D) data sets, maximum production of methane after the 10th day of incubation was noticed in sets having coal. This study emphasized the importance of the methane production in a low incubation period in comparison to the 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 CBM65 has maximum similarity with those species which was obtained in the developed consortia (Fig. 6). Both bacterial and archaeal domain were observed. The bacterial domain was comprised of firmicutes (Clostridium beijerinckii and Clostridium 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 (Fuertez 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) and Zhu et al. (2011), research on methanation similar archeal species was 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 and Cooney, 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. 7A). As reported by Reddy and Vinu (2016) and Sonibare et al. (2012), the 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) and 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. 7B). 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) and 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. 8). 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.

By analyzing the data set of EDX graph, it was confirmed that untreated coal contain majorly C, O, Si, F, Ti, Mg, Na, S, K, P, Ca, and Al (Fig. 8C). Long C peak is clearly visible in the graph with other element. Similar data was reported by Sellaro et al. (2015), according to their studies, the EDX of coal dust from mining area contains 85% carbon and 15% oxygen due to the coal particles in the mine dust. The coal dust was obtained after pulverizing the sample; further, it was collected in the polycarbonate (PC) filter and EDX was conducted the results showed the presence of C, O, Na, Mg, Al, Si, K, Ca, Ti, Fe, and Cu peaks. In another research conducted by Essex et al. (2017) in coal mine dust, their studies illustrated the presence of C because of coal particles. A study in India coal using SEM/EDX was conducted by Manoj (2016); in his investigation, he revealed that a coal contains C, N, O, and H in significant amount and Al and Si show their presence.

Figure 8D depicted the case of a treated coal. The peak that corresponds to C and O was missing; the possible reason for this could be the behavior of microorganism towards the coal particle as it can be said that microbes were actively participated in utilizing coal and generating methane as a byproduct. The presence of other peaks corresponds to the residual of media and coal.

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. 9), the 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.

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 and the compositional analysis was studied by EDX. 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.

Availability of data and materials

Not applicable.

References

  • Bi Z, Zhang J, Park S, Harpalani S, Liang Y (2017) A formation water-based nutrient recipe for potentially increasing methane release from coal in situ. Fuel 209:498–508

    Article  CAS  Google Scholar 

  • Boone DR (1991) Ecology of methanogenesis. In: Rogers JE, Whitman WB (eds) Microbial production and consumption of greenhouse gases: methane, nitrogen oxides and halomethanes. American Society for Microbiology, Washington, DC, pp 57–70

    Google Scholar 

  • Caposciutti G, Baccioli A, Ferrari L, Desideri U (2020) Biogas from anaerobic digestion: power generation or biomethane production. Energies. 13:743

  • Chena T, Zheng H, Hamilton S, Rodrigues S, Golding DS, Rudolpha V (2017) Characterisation of bioavailability of Surat Basin Walloon coals for biogenic methane production using environmental microbial consortia. Int J Coal Geol 179:92–112

    Article  Google Scholar 

  • Conrad R (1999) Contribution of hydrogen to methane production and control of hydrogen concentrations in methanogenic soils and sediments. FEMS Microbiol Ecol 28:193–202

    Article  CAS  Google Scholar 

  • Damin T, Xiangyum L, Mingjie D (2010) CS2 extarction and FTIR and GC/MS analysis of a Chinese brown coal. Min Sci Technol 20:0562–0565

    Google Scholar 

  • Davis JK, Gerlach R (2018) Transition of biogenic coal-to-methane conversion from the laboratory to the field: a review of important parameters and studies. Int J Coal Geol 185:33–43

    Article  CAS  Google Scholar 

  • Davis KJ, Lu S, Barnhart EP, Parker AE, Fields MW, Gerlach R (2018) Type and amount of organic amendments affect enhanced biogenic methane production from coal and microbial community structure. Fuel 211:600–608

    Article  CAS  Google Scholar 

  • Eduok S, John O, Ita B, Inyang E, Coulon F (2018) Enhanced biogas production from anaerobic co-digestion of lignocellulosic biomass and poultry feces using source separated human urine as buffering agent. Front Environ Sci 6:67

    Article  Google Scholar 

  • Enzmann F, Mayer F, Rother M, Holtmann D (2018) Methanogens: biochemical background and biotechnological applications. AMB Expr 8:1–22

    Article  CAS  Google Scholar 

  • Essex VJ, Cigdem K, Sarver E (2017) A computer-controlled SEM-EDX routine for characterizing respirable coal mine dust. Minerals 7:15

    Article  Google Scholar 

  • Fan X, Wei XY, Zong ZM (2013) Application of gas chromatography/mass spectrometry in studies on separation and identification of organic species in coals. Fuel 109:28–32

    Article  CAS  Google Scholar 

  • Fuertez J, Córdoba G, McLennan JD, Adams DJ, Sparks TD (2018) Potential application of developed methanogenic microbial consortia for coal bio-gasification. Int J Coal Geol 188:165–180

    Article  CAS  Google Scholar 

  • Ge H, Feng Y, Li Y, Yang W, Gong N (2016) Heavy metal pollution diagnosis and ecological risk assessment ofthe surrounding soils of coal waste pile at Naluo coal MineLiupanshui, Guizhou. Int J Min Reclam Env 30:312–318

    Article  CAS  Google Scholar 

  • Gupta P, Gupta A (2014) Biogas production from coal via anaerobic fermentation. Fuel 118:238–242

    Article  CAS  Google Scholar 

  • Hao G, Yun F, Fangfang L, Yang L, WenLia Y, Yin Y (2016) Soil diagnosis and land suitability assessment for vegetation restoration on coal waste piles in Liupanshui, Guizhou, China. Int J Min Reclam Env 30:209–216 https://pubchem.ncbi.nlm.nih.gov/compound/2_4-Di-tert-butylphenol

    Article  CAS  Google Scholar 

  • Hayat, MA (ed.) (2000) Principles and techniques of electron microscopy: Biological applications. Cambridge University press, 4th ed.: 1-80, 400-431

  • Kushkevych I, Vítězová M, Vítěz T, Bartoš M (2017) Production of biogas: relationship between methanogenic and sulfate-reducing microorganisms. Open Life Sci 12:82–91

    Article  CAS  Google Scholar 

  • Lavania M, Cheema S, Sarma PM, Ganapathi R, Lal B (2014) Methanogenic potential of a thermophilic consortium enriched from coal mine. Int Biodeterior Biodegradation 93:177–185

    Article  CAS  Google Scholar 

  • Li J, Li Z, Yang Y, King B, Wang C (2018) Laboratory study on the inhibitory effect of free radical scavenger on coal spontaneous combustion. Fuel Process Technol 171:350–360

    Article  CAS  Google Scholar 

  • Li H, Zuo J, Wang L, Li P, Xu X (2020) Mechanism of structural damage in low permeability coal material of coalbed methane reservoir under cyclic cold loading. Energies 13:519

    Article  Google Scholar 

  • Manoj B (2016) A comprehensive analysis of various structural parameters of Indian coals with the aid of advanced analytical tools. Int J Coal Sci Technol 3:123–132

    Article  CAS  Google Scholar 

  • Manoj B, Kunjomana AG, Chandrasekharan (2009) Chemical leaching of low rank coal and its characterization using SEM/EDAX and FTIR. JMMCE 8:821–832

    Article  Google Scholar 

  • Mayumi D, Mochimaru H, Tamaki H, Yamamoto K, Yoshioka H et al (2016) Methane production from coal by a single methanogen. Science. 354:222–225

    Article  CAS  PubMed  Google Scholar 

  • Opara A, Adams DJ, Free ML, McLennan J, Hamilton J (2012) Microbial production of methane and carbon dioxide from lignite, bituminous coal, and coal waste materials. Int J Coal Geol 96(97):1–8

    Article  Google Scholar 

  • Rathi R, Priya A, Vohra M, Lavania M, Lal B, Sarma PM (2015) Development of a microbial process for methane generation from bituminous coal at thermophilic conditions. Int J Coal Geol 148:25–34

    Article  Google Scholar 

  • Rathi R, Lavania M, Singh N, Sarma PM, Kishore P et al (2019) Evaluating indigenous diversity and its potential for microbial methane generation from thermogenic coal bed methane reservoir. Fuel 250:362–372

    Article  CAS  Google Scholar 

  • Reddy BR, Vinu R (2016) Microwave assisted pyrolysis of Indian and Indonesian coals and product characterization. Fuel Process Technol 154:96–103

    Article  CAS  Google Scholar 

  • Ribeiro J, Silva T, Filho JGM, Flores D (2012) Polycyclic aromatic hydrocarbons (PAHs) in burning and non-burning coal waste piles. J Hazard Mater 200:105–110

    Article  Google Scholar 

  • Ritter D, Vinson D, Barnhart E, Akob DM, Fields MW, Cunningham AS et al (2015) Enhanced microbial coalbed methane generation: a review of research, commercial activity and remaining challenges. Int J Coal Geol 146:28–41

    Article  CAS  Google Scholar 

  • Schmidt RL, Cooney CL (1985) Production of acetic acid form hydrogen and carbon dioxide by Clostridium species ATCC 29792. Chem Eng Commun 45:61–73

    Article  Google Scholar 

  • Sellaro R, Sarver E, Baxter D (2015) A standard characterization methodology for respirable coal mine dust using SEM-EDX. Resources 4:939–957

    Article  Google Scholar 

  • Shi DL, Wei XY, Fan X, Zong ZM, Chen B et al (2013) Characterizations of the extracts from geting bituminous coal by spectrometries. Energy Fuel 27:3709–3717

    Article  CAS  Google Scholar 

  • Singh K, Zondlo J (2017) Characterization of fuel properties for coal and torrefied biomass mixtures. J Energy Inst 90:505–512

    Article  CAS  Google Scholar 

  • Sonibare OO, Haeger T, Foley SF (2012) Structural characterization of Nigerian coals by X-ray diffraction, Raman and FTIR spectroscopy. Energy 35:5347–5353

    Article  Google Scholar 

  • Spring S, Schumann P, Sproer (2005) Methanogenium frittonii Harris et al. 1996 is a later synonym of Methanoculleus thermophilus (Rivard and smith 1982) Maestrojua’n et al. 1990. Int. J. Syst. Evol Microbiol; 55,1097–1099

  • Stephen A, Adebusuyi A, Baldygin A, Shuster J, Southam et al (2014) Bioconversion of coal: new insights from a core flooding study. RSC Adv 4:22779

    Article  CAS  Google Scholar 

  • Tan JP, Jahim JM, Wu TY, Harun S, Mumtaz T (2016) Use of corn steep liquor as an economical nitrogen source for biosuccinic acid production by Actinobacillus succinogenes. International Conference on Chemical Engineering and Bioprocess Engineering 36,012058

  • Ulrich G, Bower S (2008) Active methanogenesis and acetate utilization in Powder River Basin coals, United States. Int J Coal Geol 76:25–33

    Article  CAS  Google Scholar 

  • Verstraete W, Assche PV, Devocht M, Baere LAD (1984) Influence of high NaCl and high NH4Cl salt levels on methanogenic associations. Water Res 18:543–548

    Article  Google Scholar 

  • Wagner O, Hohlbrugger P, Lins P, Illmer P (2012) Effects of different nitrogen sources on the biogas production – a lab-scale investigation Andreas. Micro Biol Res 167:630–636

    Article  CAS  Google Scholar 

  • Wang B, Tai C, Wu L, Chen L, Liu J et al (2017) Methane production from lignite through the combined effects of exogenous aerobic and anaerobic microflora. Int J Coal Geol 173:84–93

    Article  CAS  Google Scholar 

  • Wang Q, Guo H, Wang H, Urynowicz MA, Hu A et al (2019) Enhanced production of secondary biogenic coalbed natural gas from a subbituminous coal treated by hydrogen peroxide and its geochemical and microbiological analyses. Fuel. 236:1345–1355

    Article  CAS  Google Scholar 

  • Wayne PA (2003) National Committee for clinical laboratory standards (NCCLS), methods for dilution antimicrobial susceptibility tests for bacterial that grow aerobically, 6thedn. Approved standard M7-A6

  • Wolfe RS (1996) 1776-1996: Alessandro Volta’s combustible air. ASM news 62,529-534 www.energyjustice.net “burning waste coal is much more polluting that burning coal”

  • Yang JI, Gang LIU, Jing MA, Bin ZG, Hua X (2014) Effects of urea and controlled release urea fertilizers on methane emission from paddy fields: a multi-year field study. Pedosphere. 24:662–673

    Article  Google Scholar 

  • Yang F, Li W, Sun M, Li Q, Wang M, Sun Y (2018) Improved buffering capacity and methane production by anaerobic co-digestion of corn stalk and straw deploymerization waste water. Energies. 11:1751

    Article  Google Scholar 

  • Zellner G, Messner P, Stackebrandt E (1998) Methanoculleuspalmolei sp. nov., an irregularly coccoid methanogen from an anaerobic digester treating wastewater of a palm oil plant in North-Sumatra, Indonesia. Int J Syst Bacteriol 48, 1111–1117

  • Zhang X, Zhang S, Li P, Ding Z, Hao Z (2018) Investigation on solubility of multi-components from semi-anthracite coal and its effect on coal structure by fourier transform infrared spectroscopy and x ray diffraction. Fuel Process Technol 174:123–131

    Article  CAS  Google Scholar 

  • Zhou C, Liu G, Cheng S, Fang T, Lam PKS (2014) Thermochemical and trace element behavior of coal gangue, agricultural biomass and their blends during co-combustion. Bioresour Technol 166:243–251

    Article  CAS  PubMed  Google Scholar 

  • Zhu C, Zhang J, Tang Y, Xu Z, Song R (2011) Diversity of methanogenicarchaea in a biogas reactor fed with swine feces as the mono-substrate by mcrA analysis. Microbiol Res 166:27–35

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

Authors are thankful to TERI for providing the infrastructural facilities for executing the present study. The authors would also like to acknowledge the facility of TDNBC and TERI for GC-MS, FTIR, and SEM analysis. The funder had the involvement in technical guidance in waste coal washery selection and physico-chemical property data interpretations of waste coal.

Funding

The authors declare that this study received funding from TATA Steel Ltd. (IN-DL13508522907519P) Tisco General Office, Bistupur, and Jamshedpur.

Author information

Authors and Affiliations

Authors

Contributions

PB and ML designed the experiments. PB conducted the experiments as per design and data generation and further wrote the manuscript. ML critically reviewed the manuscript. OS and SKS involved in the technical guidance in waste coal washeries and their selection and characterization of waste coal. BL provided the resources for performing the experiments. The authors read and approved the final manuscript.

Corresponding author

Correspondence to Meeta Lavania.

Ethics declarations

Completing interests

The authors declare that they have no competing interests.

Ethics approval and consent to participate

The pathogenicity was studied by acute oral toxicity under EPA 712-C-96-322 OPPTS 885.3550 guidelines. The study was performed at the National Toxicology Centre (APT Testing and Research Pvt. Ltd.), Pune.

Consent for publication

Not applicable.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

ESM 1

Table S1: Proximate and ultimate analysis of collected waste coal sample. Fig. S1 A and B, illustrated the percent of gas generation by reactivated consortia with variable nitrogen sources (Urea and CLS). Data recoded after 15-20th days of incubation. Table S2: Chemical moiety present in coal containing cultured bottles, identified through GCMS. Table S3: Group mean clinical signs data of male and female mice. Table S4: Group mean hematology data of male (M) and female (F). Table S5: Group mean body weight data of male (M) and female (F). Table S6: Group mean mortality data.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Basera, P., Lavania, M., Shinde, O. et al. An operative laboratory investigation of bioconversion route from waste coal to natural energy. Ann Microbiol 72, 13 (2022). https://doi.org/10.1186/s13213-021-01659-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s13213-021-01659-z

Keywords

  • Waste coal
  • Modified medium
  • Reactivated microbial consortia
  • Methane
  • Analytical methods