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).
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.
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.
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.