Synergistic Biotransformation of Low‐Rank Coal Into Chemical Feedstocks by Indigenous Fungi and Chemical Pretreatment

ABSTRACT

Fungal biotransformation of subbituminous coal into chemical feedstocks provides an eco‐friendly and cost‐effective approach for sustainable coal utilization. In this study, the fungal isolates AD‐3 (Neurospora crassa) and AD‐5 (Penicillium simplicissimum) from a Pakistani subbituminous coal, optimization, and enhancing agents play a valuable role in the enhanced biodegradation processes. Optimal conditions yielded substantial organic release with 1% and 1.5% (w/v) glucose, 0.5% pulverized coal within 7 and 17 days of incubation. NaOH and sodium acetate enhanced fungal coal degradation by modifying coal structure and promoting organic compound release. The GC–MS analysis of the solubilized black liquid showed a more diverse variety of organic compounds in chemically treated coal than the virgin coal. The analyses showed 31.29% ± 0.23% (AD‐3) and 44.9% ± 0.26% (AD‐5) solubilization of virgin coal, increasing to 50.9% ± 0.23% (AD‐3) and 68.4% ± 0.30% (AD‐5) after chemical pretreatment. The solubilized fraction and residual coal were analyzed with FTIR to explore the functional group aroused by the conjugative effect of the chemical and fungal attacks. This analysis showed that the combination of chemical treatment and biotransformation by indigenous fungi from coal environments has the potential to exploit the low‐rank coal for chemical feedstock production.

Indigenous fungi Neurospora crassa and Penicillium simplicissimum, isolated from Pakistani subbituminous coal, are combined with chemical enhancers—NaOH and sodium acetate—to boost biosolubilization from 31%–45% to 51%–68% within 5 days. GC–MS and FT‐IR analyses reveal a broader diversity of organic compounds in treated coal, highlighting fungal–chemical synergy as a green route to convert low‐rank coal into valuable chemical feedstocks.

1. Introduction

The transformation technology of any rank of coal and coal wastes into value‐added products and alternative energy is inevitable, as the combustion of coal emits pollutants into the environment. As a promising technology, biotransformation of coal has been widely investigated for the sustainable generation of clean fuels and value‐added products. Moreover, it is now well acknowledged and practicable that the technology has substantial benefits over the traditional physicochemical methods [1]. The huge indigenous coal assets of Pakistan (∼185.5 billion tons) could be utilized biologically for the production of alternative fuels and non‐fuel chemical stock in a cost‐effective manner [2]. This could possibly open future opportunities for the manufacturing of coal‐derived products.

Coal is a three‐dimensional, diverse carbonaceous rock that comprises concentrated carbon and aromatic clusters linked by aliphatic and hetero‐aliphatic bonds acting as a shield against direct biodegradation [3, 4, 5]. Fortunately, a multifarious microbial system can readily depolymerize the complex polymers into monomers [6, 7, 8, 9, 10, 11, 12]. Numerous studies have investigated the biotransformation of coal, but the exact process is not well understood due to the complexity of such systems [12, 13]. However, it had been reported that during depolymerization, coal can be converted into lower and higher molecular masses of brownish and yellowish humic and fulvic acid‐like components along with many other compounds [14, 15, 16]. However, refractoriness of coal is the major hurdle in such applications [17]. In general, coal biosolubilization can be facilitated by a number of agents to enhance the solubilization efficacy and hence, the bioavailability of coal carbons. Several studies using chemical agents as the enhancers could encourage the activity of biological entities for liberating a huge variety of beneficial organic moieties through a sequential breakdown of coal particles [15, 16, 18, 19, 20].

Biotransformation of Pakistani subbituminous coal for alternative compound extraction has been rarely investigated. In this research, indigenous Neurospora crassa (AD‐3) and Penicillium sp. (AD‐5) fungal strains were isolated from a subbituminous coal sample and optimized for enhancing the release of organic fractions from the coal matrix. In addition, different enhancing agents were used to stimulate the activity of these biological entities on the subbituminous coal. The structural changes aroused by the biological attack on the coal were investigated by UV–Visible spectroscopy and Fourier transform infrared spectroscopy (FT‐IR). The chemical composition of the released fractions was characterized by gas chromatography‐mass spectrometry (GC–MS). For clarity, we use the following terms as operationally defined in this study: biosolubilization refers to the fungal‐mediated conversion of solid coal into soluble, liquid‐phase organic products, quantified gravimetrically and UV‐vis absorbance; depolymerization refers specifically to the breakdown of high‐molecular‐weight coal macromolecular structures into lower‐molecular‐weight fragments, inferred here from FT‐IR spectral changes and the detection of low‐molecular‐weight compounds by GC–MS; and biotransformation is used as the broader umbrella term encompassing both biosolubilization and depolymerization as components of the overall fungal‐mediated conversion of coal into chemical products. While fungal biosolubilization, chemical pretreatment, and GC–MS‐based product characterization have each been applied to coal biotransformation previously [7, 12, 14, 16, 19], this study is, to our knowledge, the first to (i) isolate and molecularly identify indigenous coal‐associated fungi from a Pakistani subbituminous coal source and (ii) directly compare, under individually optimized conditions, the biosolubilization of virgin versus NaOH‐pretreated coal by the same isolates, quantifying the relative contribution of chemical pretreatment to overall solubilization yield. This comparative framework, together with the compositional inventory of the released products, extends the empirical basis for evaluating chemically enhanced fungal treatment as a route to chemical feedstocks from previously uncharacterized low‐rank coal resources.

2. Materials and Methods

2.1. Coal

Subbituminous coal was freshly mined at various depths ranging from 312 to 321 m below the ground surface from the Duki region, Baluchistan coalfield of Pakistan [20]. The coal was sampled at the coordinates 30°08'36.5''N, 68°34'41.0''E of the coal mine. All the samples were securely sealed in airtight zipper polyethylene bags. They were transferred to the Environmental Microbiology Lab, Quaid‐I‐Azam University, Islamabad, Pakistan, and stored at 4°C until being used. All mined samples were mixed homogenously to make a representative sample for all experiments. To isolate native microflora, the coal samples were finely ground with a sterile pestle and mortar in a laminar flow hood. The particle size of the coal was less than 650 µm. The selected coal characterization, including proximate analysis, calorific values, and vitrinite reflectance was determined following the ASTM methods. The coal vitrinite reflectance and calorific gross value were measured by a LECO AC500 Isoperibol Calorimeter (Saint Joseph, Michigan, USA) and a Leitz Orthoplan microscope (Solms, Germany) with a photometer. For vitrinite reflectance and incident light composition analyses, a peak detector and a Leica DMRX (Solms, Germany) with a mechanical stage were used simultaneously with a xenon and a halogen discharge light source. The CHNS analyzer (LECO TruMac Series, Saint Joseph, Michigan) was used for the ultimate analysis of the selected coal samples. The results of these coal analyses were summarized and presented in Tables 1 and 2.

TABLE 1.

Proximate analysis, heating value, and vitrinite reflectance of coal.

Proximate analysis (mean ± SD)
Moisture	Ash	Volatile matter	Fixed carbon	Calorific value (m, mmf) a	Vitrinite reflectance
10.12 ± 0.09	24.41 ± 0.12	25.65 ± 0.21	39.82 ± 0.11	9995	0.44

^a Moisture, mineral‐matter‐free.

TABLE 2.

The ultimate analysis and atomic ratio of coal.

Ultimate analysis (mean ± SD)					Atomic ratio
% C	% H	% N	% S	% O	H/C	O/C	N/O
70.29 ± 0.23	5.99 ± 0.12	2.06 ± 0.15	4.87 ± 0.28	16.79 ± 0.11	1.02	0.17	0.14

2.2. Isolation and Screening of Coal Solubilizing Fungi

Native coal solubilizing fungal strains were isolated from the coal samples. Briefly, a suspension solution was made by mixing 100 mL sterilized distilled water with 1 g ground coal. 1 mL of the suspension was inoculated into a 50 mL minimal salts medium (MSM) at pH 5.5. The culture was incubated in a shaker at 120 rpm at 25°C. The constituents of the MSM were (per L) 1 g K₂HPO₄, 0.2 g KH₂PO₄, 1 g NaCl, 0.002 g CaCl₂·2H₂O, 0.005 g Boric Acid, 1 g (NH₄)₂SO₄, 0.5 g MgSO₄·7H₂O, 0.001 g CuSO₄, 0.01 g ZnSO₄·7H₂O, 0.001 g MnSO₄, 0.01 g FeSO₄·7H₂O, and 3 g glucose supplement [21]. After 2 days, the culture solution was diluted 50 times and inoculated on the surface of malt extract agar plates (MEA) containing (per L) 10 g glucose, 5 g malt extract, 1 g peptone, and 10 g agar at pH 5.5 [14]. The plates were incubated in a fungal incubator at 30°C for 4 days. The fungal mycelia were further purified on MEA plates and stored on MEA slants at 4°C. A total of six fungal strains were isolated from the coal sample. A qualitative plate assay was used to screen all six isolated prior to selection. Each fungal strain was grown on MEA plates for 4 days at 30°C to form a complete mycelial mat, after which 0.5 g of sterilized, pulverized virgin coal was spread evenly over the mat surface. Plates were incubated for 5 days at 30°C. Coal‐solubilizing activity was evaluated qualitatively by (i) the appearance of dark brown‐to‐black exudate/guttation droplets on the coal‐fungus interface and (ii) visible darkening or dispersal of the coal particles on the mycelial mat relative to uninoculated coal controls. The two isolates (designated as AD‐3 and AD‐5) exhibiting the best coal solubilizing capability were selected for downstream molecular identification and further optimization.

2.3. Molecular Typing

The total genomic DNA was extracted by a modified method [22]. Briefly, 0.5 mL cetyltrimethyl ammonium bromide (CTAB) extraction buffer and 0.1 g of freshly grown mycelia were mixed and ground in a sterile mortar and pestle. The mixture was transferred to a microcentrifuge tube and further lysed by homogenization with glass beads (4–6 mm). The tube was placed in a water bath at 65°C for 30 min. An equal volume of chloroform isoamyl alcohol (24:1) was added to the tube and centrifuged at 14 000 rpm at room temperature for 15 min. The procedure was repeated once. The supernatant was transferred into a new tube with an equal volume of isopropanol and stored at −4°C overnight. Then, the DNA was thawed and centrifuged for 10 min at 14 000 rpm. The supernatant was discarded. The obtained pellet was rinsed with 200 µL of 70% ethanol and centrifuged for 10 min at 14 000 rpm. The pellet was resuspended in a 50 µL TE buffer and stored at −4°C until further use.

For the identification of AD‐3 and AD‐5 strains, the ITS internal region amplification was done with ITS1 (TCC GTA GGT GAA CCT GCG G) and ITS4 (TCC TCC GCT TAT TGA TAT GC) universal primers through PCR [11]. The amplifying conditions were as follows: 94°C for 3 min, 94°C for 30 s (35 cycles), 56°C for 60 s, 72°C for 60 s, followed by 72°C for 10 min. The PCR band confirmation was done through agarose gel electrophoresis. The DNA was sequenced by Macrogen Inc. (South Korea). The data were searched and analyzed in the GenBank database for sequence similarities by using NCBI BLASTn.

2.4. Chemical Pretreatment

Coal samples for the chemical pretreatment experiment were dried, ground, and sieved to particle sizes of 0.1–0.2 mm in diameter. The coal was treated with 8 M sodium hydroxide (NaOH) at a ratio of 1:3 (w/v) for 24 h at room temperature. After that, the sample was transferred into microcentrifuge tubes and centrifuged at 10 000 rpm, 25°C, for 20 min. The supernatant was separated from the coal residues. The coal residues were suspended in distilled water and centrifuged under the same conditions 10 times to wash any chemical residues. Ten sequential washing cycles were applied as a conservative measure intended to minimize residual NaOH. The treated coal samples were stored in airtight containers for further experimentation [7, 16, 19].

2.5. Plate Biosolubilization Assay

The quantitative estimation of biosolubilized release fraction from coal was performed with a Petri plate assay. The AD‐3 and AD‐5 fungal isolates were grown on MEA medium plates. The plates were incubated for 4 days at 30°C in order to develop complete mycelia mats. 0.5 g virgin and chemically pretreated coal particles (0.1–0.2 mm) were spread on the mats. Two control references were run along with this assay, that is, MEA agar plates having AD‐3 and AD‐5 without coal and MEA plates having coal without fungi. The plates were incubated for 5 days at 30^°C. The guttation droplets produced by the fungi were collected by sterile microsyringes and stored at 4°C for further analysis. After incubation, the coal particles were collected and weighed. Biosolubilization efficacy was determined by a gravitational method with Equation (1) [12]. The initial weight (W ₀) of the coal was measured before spreading on the plate. After the incubation period, the residual particles were collected and weighed (W ₁):

$$P=\left(1-\frac{W_1}{W_o}\right)\times 100$$ (1)

2.6. Biosolubilization Optimization

The fungal isolates were optimized to enhance the release of organic fraction from subbituminous coal matrix. Major parametric conditions, that is, glucose concentrations (0.1%, 0.5%, 1.0%, and 1.5% (w/v)), incubation period (7, 12, 17, and 21 days), and coal loading ratio (0.1%, 0.5%, 1.0%, and 1.5% (w/v)), were optimized for releasing organics. The resultants were determined by a SPECORD 200 Analytik UV–Vis Spectrophotometer (Jena, Germany). The size of the dry coal particles was reduced to 0.1‐0.2 mm in diameter by properly sieving. After optimization of major conditions, 0.25 g sodium acetate (C₂H₃NaO₂) was added as an enhancing agent in the MSM medium for both fungal isolates. Triplicate experiments were performed to check variations. The medium having sodium acetate as the sole carbon source was also prepared as a control [23, 24]. Initially, the fungal isolates were grown in a MSM having 1% w/v glucose supplement for 4 days at 25°C and 120 rpm. After 4 days, the mycelia were suspended in distilled water, ground by homogenization, and adjusted to an absorbance of 0.4 at 600 nm. A 2 mL aliquot of the homogenate and autoclaved coal were used for solubilization optimization. All experiments were conducted in pre‐autoclaved, aerobic, sterile glass flasks. Two controls, that is, MSM having coal without fungal inoculation and MSM having fungal inoculation without coal, were used for all studies. After a specified incubation period, the samples were filtered with 0.45 µm nylon syringe membrane filters (CNW Technologies, Düsseldorf, Germany) for further analysis.

2.7. In Vivo Degradation

The comparative analysis for the enhanced release of organics from the virgin and chemically pretreated coal was carried out in vivo. The AD‐3 and AD‐5 fungal inocula were prepared by adding loops full of pure mycelia from an active growth zone of the plates into distilled water. The suspensions were homogenized and UV‐Vis absorbance was adjusted to 0.2 at 600 nm. Two milliliters of inocula were added to the MSM medium supplemented with optimized glucose concentrations of 1% w/v for the AD‐3 strain and 1.5% w/v for the AD‐5 fungal isolate, at pH 5.5, and incubated at 25°C and 120 rpm for 4 days. Autoclaved virgin and chemically pretreated dried coal (0.1–0.2 mm) was added at 0.5%. Triplicate experiments were run along with positive and negative controls. After the optimized time of incubation, the samples were syringe filtered for further analysis.

2.8. Fourier Transform‐Infrared Spectroscopy (FT‐IR)

The structural changes in the coal‐derived biosolubilized products and residues were investigated by FT‐IR. The analysis was conducted in the range of 3500–500 cm⁻¹ with a spectrum‐65 FT‐IR spectrometer (Perkin Elmer, USA). The coal particles were washed with distilled water to remove all the fungal mycelia. The water content of the biosolubilized liquid products was reduced with a vacuum rotary evaporator at 70°C. The biologically treated coal residues, released organic fractions, and raw coal were dried at ambient temperature for 24 h. The potassium bromide (KBr) pellets were prepared by uniformly mixing 2 mg of each sample with 200 mg dried KBr powder. The acquisition of the IR spectra can be referred to in the previous literature [14, 15].

2.9. Gas Chromatography–Mass Spectrometry (GC–MS) of Biosolubilized Products

GC–MS was used for component separation and identification according to [12, 14, 19]. A vacuum rotary evaporator was used to remove water content from the biosolubilized liquid products at 70°C. Ethyl acetate was used to extract organic constituents from the biosolubilized liquid in a ratio of 1 to 10 (viscous liquid: organic solvent) by volume for 6 h. An Agilent GC–MS (6890 model number) equipped with a DB‐5 MS fused silica packed capillary column (5% phenyl methyl polysiloxane, 30 m × 0.25 mm, 0.25 µm film thickness, Agilent JW Scientific) was used for characterization of the coal‐derived organics. The GC–MS was operated in a spitless mode with a constant helium flow rate of1.5 mL/min as the carrier gas. The capillary column was heated from 120°C to 280°C with an increment of 10°C/min and held at 280°C for 10 min. The inlet temperature was set at 280°C. 2 µL of the sample was injected manually. For MS, 50–1000 a.m.u. was selected for the molecular weight range. The running mode was electron impact ionization with a 70 eV ionization electron beam for producing 0–315 µA ionization current. Peaks were analyzed by using the NIST Ver. 02 library with a minimum match quality of 80% for compound identification [9, 12, 14, 19].

All experiments were conducted in triplicate, with results expressed as mean ± standard deviation (SD). Statistical significance between treatment groups was assessed using one‐way analysis of variance (ANOVA) followed by Tukey's Honest Significant Difference (HSD) post‐hoc test. Differences were considered statistically significant at p < 0.05. For UV‐Vis spectroscopic analysis, each sample was scanned three times, and the average absorbance values were reported.

3. Results and Discussion

3.1. Coal Characterization

Proximate analysis, calorific value, vitrinite reflectance, and ultimate analysis were summarized in Tables 1 and 2. The elemental composition of the coal was 70.29% ± 0.23 C, 5.99% ± 0.12 H, 2.06% ± 0.15 N, 4.87% ± 0.28 S, and 16.79% ± 0.11 O on a dry, ash‐free basis. The heating value of the selected coal was 23,248 kJ/kg (9995 Btu/lb), and vitrinite reflectance index was 0.44%. The coal rank can be classified by vitrinite reflectance and gross calorific values on a moist, mineral‐matter‐free basis for low rank coal while for high rank coal, it is determined on a dry fixed carbon basis. The selected coal was determined as subbituminous B coal based on the heating value and vitrinite reflectance index. Low rank coal is more susceptible to biological attack than high rank coal as it has high volatile matter and low carbon content [25]. However, some studies also reported biodegradation of high rank coal [26, 27, 28].

3.2. Isolation of AD‐3 and AD‐5 Fungal Isolates

The amplicon sequences of AD‐3 and AD‐5 fungal isolates by BLASTn search depicted 99% similarity with Neurospora crassa (MG669239) and Penicillium simplicissimum (MH855126), respectively. Several studies on Penicillium sp. have been documented for their effective role in coal transformation [29, 30, 31, 32, 33, 34, 35, 36]. Penicillium simplicissimum has been utilized for the conversion of long‐chain plastics and coal polymers into monomers, remediation of metal contaminations, and production of stable enzymes for degradation [37, 38, 39, 40]. Neurospora crassa was also studied for coal solubilization. Under specific environments, Neurospora crassa could significantly release black liquified products from coal [41]. However, this is the first study to analyze the native Neurospora crassa and Penicillium simplicissimum isolates for enhanced solubilization of Pakistani subbituminous coal. The AD‐3 and AD‐5 isolates have shown high solubilization capabilities, suggesting their acclimatization in the coal environment.

3.3. Plate Solubilization Extent

The AD‐3 and AD‐5 fungal isolates liberated about 1∼2 mL black liquid from 1 g of virgin and chemically pretreated coal respectively within 5 days of incubation. Chemical treatment and fungal attacks conjugatively facilitated the solubilization process. The chemically pretreated coal showed 50.9% ± 0.23% and 68.4% ± 0.30% solubilization by AD‐3 and AD‐5 while 31.29% ± 0.23% and 44.9% ± 0.26% solubilization were observed in the virgin coal in 5 days. The solubilization percentages are given in Table 3 (p < 0.05). The results show that the indigenously isolated fungal strains minimized the solubilizing time to a comparable extent as those documented in the literature, many of which used coal ranks lower than this study [11, 42, 43]. Yin et al. reported a wood‐decaying fungus was able to solubilize 31.83% of nitric acid pretreated lignite within 10.5 days [12]. Pseudomonas putida showed 25% solubilization of chemically oxidized low rank coal within 14 days [8]. Jiang et al. reported 36.77% solubilization of Chinese lignite by Bacillus sp. Y7 within 2 weeks of incubation [44]. Gokcay et al. reported 60% solubilization of Elbistan lignite by Phanerochaete chrysosporium [23]. This suggests the high efficacy of indigenously isolated AD‐3 and AD‐5 fungal strains in terms of both time and solubilization extents of Pakistani subbituminous coal. The biotransformation of coal is directly proportional to the pore size in coal residues. The rate of coal dissolution is intensified by reducing coal particle sizes and increasing concentrations of pores [23]. Recent studies have shown that surface area is also an important factor for enhanced coal biosolubilization. The biosolubilization time and extent were enhanced four times by reducing particle size, that is, 600–850 µm to 150–300 µm [23, 24]. In submerged cultures, the size of coal particles up to 0.2–0.5 mm have remarkable effects on accelerating solubilization activity than those between 600 and 650 nm [12, 24]. Therefore, the chances for biosolubilization on pulverized (0.1–0.2 mm) coal particles were increased. It can be deduced that the biosolubilizing ratio of coal in terms of time and percentage highly depends on its particle size, porosity, and the ability of microbes for this purpose. The indigenous community may have developed such enzyme systems that can effectively degrade coal.

TABLE 3.

Biosolubilization extent of virgin and chemically pretreated coal.

Test	Virgin coal (mean ± SD)						Chemically pretreated coal (mean ± SD)
	AD‐3			AD‐5			AD‐3			AD‐5
Initial mass	0.50 ± 0.2	0.40 ± 0.3	0.51 ± 0.1	0.50 ± 0.2	0.52 ± 0.2	0.51 ± 0.4	0.50 ± 0.3	0.47 ± 0.2	0.49 ± 0.3	0.50 ± 0.1	0.47 ± 0.3	0.49 ± 0.3
Final mass	0.34 ± 0.1	0.34 ± 0.3	0.34 ± 0.2	0.27 ± 0.2	0.29 ± 0.1	0.29 ± 0.1	0.23 ± 0.3	0.24 ± 0.1	0.24 ± 0.1	0.15 ± 0.2	0.16 ± 0.1	0.15 ± 0.2
Solubilization %	31.8 ± 0.2	29.5 ± 0.3	32.5 ± 0.2	45.6 ± 0.3	45.6 ± 0.2	43.5 ± 0.3	52.4 ± 0.3	48.7 ± 0.2	51.6 ± 0.2	71.0 ± 0.3	65.1 ± 0.3	69.1 ± 0.3

Hard coal is considered appropriate for thermal power generation. Its bioconversion is limited. Previous biological benefaction of hard coal was reported with less promising results [27, 28, 45, 46, 47, 48]. However, more encouraging results of low rank coal have been reported due to high fraction of pores and oxygen contents [6, 11, 12, 15, 23, 29, 49, 50, 51, 52, 53, 54]. In Pakistan, microbial conversions of soft coal have been studied extensively, but limited data is available on hard coal conversions [14, 30, 31, 32, 42, 55]. A unique and multifarious biochemical system could be applied to subbituminous coal for the generation of fuels and nonfuel constituents. These findings may enable the development of sustainable fuel production that also reduces environmental pollution. Several studies reported pretreatment strategies to alter coal structure up to some extent for enhanced microbial attack and elution of various organic assets [6, 7, 8, 12, 18, 19, 23, 27, 49, 52, 56, 57, 58]. Chemically pretreated subbituminous coal by NaOH before biological benefaction may have great potential to incorporate oxygen, reduce carbon content, enhance pore size and make it more vulnerable for biological attacks. AD‐3 and AD‐5 fungal isolates might be very efficacious in solubilization of these partially distorted coal particles and release multiple compounds.

3.4. In Vivo Experiment Optimization

The AD‐3 and AD‐5 fungal isolated were optimized to accelerate the liberation of organic moieties from coal particles. These variables including glucose concentration, coal loading ratio, and incubation period have been reported to act as keys to significantly enhance solubilization of coal [11, 12, 14, 23, 30]. Tao et al. suggested that the adsorption of the biosolubilized organics by fungal pretreatments occurs at 240 nm in the range of 200–600 nm UV‐Vis spectral scan [53]. To determine the released organic moieties by the fungal attack, 240 nm of UV‐VIS was used [12, 14, 53]. The adsorption with optimizing variables at 240 nm was listed in Table 4. The intensity of the NaOH‐treated samples was much higher than the other samples. This suggests that the chemical treatment has played a critical role in the biosolubilization process. The unsaturated chemicals and aromatic compounds have been reported the components of the released products from coal [12, 59]. In the enhanced degradation experiment, (Acetate + Glucose) has been shown to significantly enhance the biosolubilization. The intensity was much higher than acetate or glucose alone. The intensity of the two controls was higher than some samples with enhancers, for example, low glucose concentrations and acetate, suggesting not all the setups had positive impacts on the coal biosolubilization. The aromatics [12, 60] and humic moieties [30, 58] liberation indicating at 220–320 nm suggests that the fungal strains could break down the complex crosslinkers connecting the aromatic rings in coal.

TABLE 4.

Absorption at 240 nm UV‐Visible with variables for optimization of fungal treatments.

Glucose b	Intensity a		Coal loading b	Intensity a		Incubation (days)	Intensity a		Enhance degradation	Intensity a
	AD‐3	AD‐5		AD‐3	AD‐5		AD‐3	AD‐5		AD‐3	AD‐5
0.1	0.3 ± 0.02	0.82 ± 0.23	0.1	1.15 ± 0.09	0.94 ± 0.19	7	1.80 ± 0.45	1.25 ± 065	Acetate	0.79 ± 0.45	0.96 ± 0.10
0.5	0.79 ± 0.02	0.97 ± 0.11	0.5	2.27 ± 0.57	1.16 ± 0.23	12	1.79 ± 0.08	1.44 ± 0.01	Acetate + Glucose	3.22 ± 0.33	3.33 ± 0.46
1.0	2.41 ± 0.31	1.62 ± 0.07	1.0	1.41 ± 0.27	0.97 ± 0.09	17	1.66 ± 0.26	1.97 ± 0.89	Coal reference	0.94 ± 0.54	0.94 ± 0.23
1.5	2.21 ± 0.42	2.25 ± 0.32	1.5	1.62 ± 0.09	0.99 ± 0.05	21	1.38 ± 0.23	1.66 ± 0.49	Fungal reference	1.09 ± 0.09	1.08 ± 0.06
—	—	—	—	—	—	—	—	—	Virgin coal	0.02 ± 0.51	2.23 ± 0.51
—	—	—	—	—	—	—	—	—	NaOH treated coal	4.10 ± 0.11	4.83 ± 0.71

^a at 240 nm.

^b %.

3.4.1. Glucose Effect

Glucose (0.1%, 0.5%, 1.0%, and 1.5% (w/v)) as an auxiliary carbon source was tested to determine the released organics from coal (1% w/v coal loading ratio). One percent for AD‐3 (Figure 1A) and 1.5% for AD‐5 fungal isolate (Figure 1D) were determined the ratios for maximum release of organics. Without glucose supplement, coal‐fungal solubilization extent was found very low, signifying its role in enhanced solubilization. Glucose supplements were found valuable in maturation and fungal biomass that enhanced the coal solubilization activity. Enhanced release of organics from coal has been documented by utilizing specific concentrations of glucose [11, 14, 23, 30]. Glucose supplements may encourage coal depolymerization processes by activating the growth of fungi and the production of fungal extracellular enzymes in coal‐rich environments [10, 12, 23, 61]. Sabar et al. reported a 1.5% optimal glucose concentration for enhancing coal solubilization by Rhizopus oryzae [14]. On the contrary, a study demonstrated that high glucose concentrations could stimulate the growth of T. atroviride ES11 but suppress the solubilization [11]. The high fungal mass by excessive glucose may limit the dissolved oxygen inaccessible enzymatic attack and hence, low depolymerization. Therefore, it can be concluded that 1.0% (w/v) and 1.5% (w/v) glucose concentration was optimal for AD‐3 and AD‐5 fungal isolates for enhancing solubilization of the subbituminous coal.

FIGURE 1.

UV‐Vis absorbance scans (200–600) of released organics from AD‐3 (panels a–c) and AD‐5 (panels d–f) under varying optimization parameters. (a) AD‐3 glucose concentration effect; (b) AD‐3 coal loading effect; (c) AD‐3 incubation time effect; (d) AD‐5 glucose concentration effect; (e) AD‐5 coal loading effect; (f) AD‐5 incubation time effect. X‐axis: wavelength (nm); y‐axis: absorbance intensity.

3.4.2. Coal Loading Effect

Different concentrations of coal (0.1%, 0.5%, 1.0%, and 1.5% (w/v)) were treated with AD‐3 (Figure 1B) and AD‐5 (Figure 1E) fungal isolates with respective optimized glucose supplements. 0.5% coal loading ratio for both isolates was found to be optimal. High coal ratio could be toxic to the fungi by lysing the mycelia [44, 62, 63]. Consequently, minimum organics were detected at 220 and 300 nm for higher coal loading ratios because increasing coal concentrations may cause inhibitory effects on fungal growth and suppress the peak intensity. Haider et al. reported the same effect [30]. The rate of coal dissolution becomes constant after incorporating a certain amount of coal residues. One possible reason for this could be the limited surface area of residues as a result of blockage by microbial metabolites and dead cell debris. Sabar et al. also reported a 0.5% coal loading ratio in solubilization of low rank coal by Rhizopus oryzae [14]. However, a diverse range of optimal coal loading ratios was investigated in a number of studies. A 1.0% low rank coal was used for solubilization and humic acid extraction [30, 36, 64]. Bacillus sp. Y7 showed maximal liberation of organics by using 1% coal particles after 12 days of incubation [44]. G. alkanivorans S7 and B. mycoides NS1020 sp. were reported to solubilize 5% (w/v) of low rank coal [52]. Trichoderma sp. was optimized with 1.5% (w/v) coal for enhanced coal solubilization activity [53]. These have shown that the optimal coal loading ratio depends on specific fungi and other conditions.

3.4.3. Incubation Effect

The extent of released fractions from coal matrix by fungal attacks could be optimized with incubation periods. Optimized coal loading ratio and glucose concentrations were used to optimize incubation periods for 7, 12, 17, and 21 days. The AD‐3 (Figure 1C) fungal isolate was shown to have maximal solubilization on Day 7 of incubation while the AD‐5 was on Day 17 (Figure 1F). The stationary phase may facilitate coal dissolution by accelerating enzymatic and metabolites production for coal attack. However, it is speculated that the enzymatic activity was limited in the longer incubation period as the absorbance reduces at 240‐280 nm. This agrees with the results reported elsewhere [14, 30]. Previous studies had revealed that 7 to 9 days incubation during fungal pretreatment was beneficial to amplify the solubilization extent of organic moieties [30, 53]. These released moieties were continuously detected by a UV‐Visible spectrophotometer in the range of 215–300 nm. Therefore, a substantial increase in absorption intensity was also observed at 240 nm. Whereas a longer incubation period representing the reduction in absorbance intensity indicates the decline in released fractions. This might be attributed to the subsequent utilization of soluble organics by the fungal strains for growth and hence lower their concentrations in the supernatant. Moreover, Sabar et al. reported that 11 days incubation is optimal for the AD‐1 fungal strain in terms of biosolubilization while it is 7 days for the MW‐1 fungal strain [14, 30]. Tao et al. also reported a significant reduction of absorption intensity for longer incubations from 9 to 15 and 19 days [53]. The majority of coal biosolubilization occurred within 12 days incubation period though [44].

3.4.4. Sodium Acetate Effect

Besides major optimization parameters, sodium acetate (C₂H₃NaO₂) has been reported to stabilize microbial growth and enhance the solubilization of organic moieties from coal [23]. As an enhancing agent, 0.25 g sodium acetate was added in the MSM medium with optimized parameters for both fungal isolates. The activity of AD‐3 (Figure 2A) and AD‐5 (Figure 2B) fungal isolates was significantly enhanced by the acetate amendments along with glucose supplements and an optimized incubation period. However, the setup without any glucose supplement has shown low intensity. These results are contrary to the findings of [23]. These authors reported that acetate as the primary carbon source could enhance lignite solubilization. In't Zandt et al. also revealed that acetate and nutrients alterations can significantly increase the number of microbial cells [65]. Excessive nutrients in the growth medium may lead to a fungal flock‐like structure by dense growth. It depletes coal dissolution by lowering fungal enzymatic action due to the cloaked coal surface by the fungus and other reactants produced in the reaction vessels. Manganese peroxidase observed as the dominant enzyme released in the presence of alkali lignin and responsible for the mineralization of wide range of coal aromatics [66].

FIGURE 2.

UV‐Vis absorbance scans (200–600 nm) of released organics under sodium acetate amendment and optimized fungal treatment conditions. (a) Effect of sodium acetate in AD‐3 bio‐solubilization efficiency; (b) Effect of sodium acetate in AD‐5 bio‐solubilization efficiency; (c) Optimized release of organics by AD‐3 strain; (d) Optimized release of organics by AD‐5 strain.

The synergistic mechanism between NaOH pretreatment and fungal degradation operates through complementary molecular pathways. NaOH treatment facilitates the cleavage of ether and ester linkages within the coal matrix, increasing porosity and exposing aromatic clusters to enzymatic attack. This initial chemical modification reduces steric hindrance that typically limits fungal enzyme accessibility. The indigenous fungal isolates, particularly AD‐5 (Penicillium simplicissimum), subsequently employ extracellular oxidative enzymes to cleave C‐C bonds and oxidize aromatic rings. This two‐step approach overcomes the recalcitrance of the subbituminous coal structure, as evidenced by the significantly enhanced solubilization percentages (50.9% ± 0.23%–68.4% ± 0.30%) compared to either treatment alone. The identification of diverse oxygen‐containing functional groups (hydroxyl, carboxyl, carbonyl) in the biosolubilized products further confirms that fungal enzymes target the partially oxidized linkages created by NaOH pretreatment, resulting in the release of aromatic and aliphatic compounds through a sequential breakdown mechanism.

Statistical analysis revealed significant differences between virgin and chemically pretreated coal biosolubilization for both fungal strains (p < 0.01 for AD‐3; p < 0.001 for AD‐5). The enhancement effect of sodium acetate was also statistically significant (p < 0.05) for both isolates under optimized conditions.

3.4.5. Comparative In Vivo Assessment

To establish a comparative assessment, in vivo experiments of AD‐3 (Figure 2C) and AD‐5 (Figure 2D) on virgin and pretreated coal were set up. The experiment was run in triplicates along with positive and negative controls. The UV‐Vis spectral scans of both controls were conducted in the range of 200–600 nm. The solubilization extent of organics in the negative controls was minimal. However, peaks were observed in the positive controls that might suggest fungal metabolic actions. The release fraction from virgin coal showed peaks in the region of 200–250 nm. Strong absorbance at 240 nm was observed that may represent alkyl substitutes of unsaturated aldehydes and ketones [53]. However, the decrease of absorbance intensity above this range was also reported by [67]. Conjugative effects of fungal attacks and chemical pretreatment of coal were effective in liberating aromatics and humic substances. The AD‐5 fungal isolate showed prominent peaks in the region of 220‐400 nm while peaks at 200–380 nm for AD‐3 strain were observed from chemically pretreated coal. The absorbance at 210–300 nm showed the existence of conjugated double bonds and aromatic compounds [12, 59]. Similar results were reported by Shi K. Y et al. Moreover, Huang et al. and Jaffrennou et al. also reported one‐ and two‐ring aromatics at 300–350 nm and PAHs at 370–480 nm [16, 68]. The fungal attack on the chemically pretreated coal may cause breakage at major linkages that lead to the enhanced liberation of humic and aromatic moieties in the liquid culture system. Yin et al. detected simple aromatics in the supernatant at 260–300 nm [12]. Jiang et al. reported aromatics at 260–450 nm. The Rhizopus oryzae AD‐1 strain showed maximal absorbance at 220–330 nm, indicating the presence of humic and aromatic compounds [14]. However, the main difficulty regarding the hard coal biosolubilization profiling is the heterogenicity nature of the coal and the yields of a wide variety of degraded products.

The consistently higher solubilization achieved by AD‐5 (Penicillium simplicissimum) relative to AD‐3 (Neurospora crassa) both in virgin and chemically pretreated coal is consistent with several reported characteristics of Penicillium species relevant to coal biotransformation. Penicillium species has been reported to produce a broad extracellular enzyme repertoire including esterase, lipases, and lignin‐modifying peroxidases [35, 36, 39], and to degrade structurally recalcitrant polymers such as polyethylene [40], suggesting a more versatile extracellular catabolic system than typically reported for Neurospora species. This is also reflected in our optimization results: AD‐5 required a higher optimal glucose concentration (1.5% vs. 1.0% w/v) and a longer optimal incubation period (17 days vs. 7 days) than AD‐3, consistent with sustained biomass and extracellular enzyme production over a longer growth phase, whereas AD‐3 reached peak activity earlier and its solubilization declined at extended incubation times, suggesting a more transient or growth‐phase‐restricted enzymatic response. Together, these observations offer a plausible explanation for AD‐5 superior performance, however direct enzyme activity assays would be needed to confirm this mechanistically.

3.5. FT‐IR Analysis

FT‐IR analyses of the reference, bio‐oxidized residues, and biosolubilized extracts were conducted. The fungal‐treated virgin coal residues and the biosolubilized extracts exhibited different spectral peaks in terms of band intensity and absorbance frequency. This might be due to the nature of fungal attacks on coal. A broad band of ‐OH stretching was observed in the AD‐3‐treated coal at 3498.67–3405.91 cm⁻¹ (Figure 3A). A short stretching of sp² C‐H was observed at 2923.31 cm⁻¹ in the AD‐5‐treated coal, suggesting fewer hydroxyl and aliphatic stretching in the residual coal. These were not observed in the control samples. A prominent CH₃ stretching was observed at 1392.09 cm⁻¹ in the AD‐3‐treated coal, signifying vibrational changes of the aromatic rings. A similar observation was reported by [58, 69]. The AD‐5‐treated coal also exhibits intense peaks of C═O, carbonyl C═C stretching, and –C‐H bending at 1693.07, 1496.07, and 1031.24 cm⁻¹, indicating vibrational changes in ether bonds. These were not observed in the controls and the biosolubilized extract. A weak sp³ ‐CH stretching at 2971.37 cm⁻¹ indicated asymmetric aliphatics in the AD‐3 biosolubilized extract. The aliphatic ether band at 1099.10–1032.26 cm⁻¹ was observed in the controls and both fungal mediated biosolubilized extracts, indicating that these indigenous fungal strains have an ability to degrade complex linkages in coal. Moreover, the peak intensity decreased in the 900–600 cm⁻¹ region in the AD‐3‐treated coal, suggesting the release of aromatics in the biosolubilized fraction. However, the AD‐5‐treated coal showed bands with higher absorbance than the biosolubilized extracts. This has suggested that the AD‐3 fungal strain has a greater ability to degrade the side chains of aromatics than the AD‐5 fungal strain.

FIGURE 3.

FT‐IR spectra of (a) AD‐3 fungal strain treatments; (b) AD‐5 fungal strain treatments. A: Virgin coal reference; B: Released extract of virgin coal; C: Virgin residual coal; D: Treated coal reference; E: Released extract of treated coal; F: Treated coal residue.

In the case of chemically pretreated coal, the AD‐5‐treated coal showed enhanced spectra in terms of band intensity and absorbance frequency while AD‐3‐treated coal and biosolubilized extracts exhibited spectra with minor differences (Figure 3B). In the spectra of the AD‐5‐treated coal, two peaks were observed at 3498.67 and 2923.18 cm⁻¹, owing to –OH and sp²–C–H stretches. These indicated enhanced hydroxyl or humic acid functionalities. However, only a weak stretching of sp² –C‐H was observed in the AD‐3‐treated coal at 2923.18cm⁻¹. In addition, C═C, C═O stretch, and CH₃ bending in the residual coal and biosolubilized extracts at 1635.34, 1601.93, and 1450.07 cm⁻¹ were observed, representing carboxylic, methylene, and ester group stretching. The spectra of AD‐5‐treated coal exhibited more intense C═C stretching and CH₂ bending at 1683.56 and 1470.43 cm⁻¹. This indicates the presence of carboxylic, carbonyl ester, and methylene groups. They were not found in the biosolubilized extracts and controls. Both fungal‐treated coals showed a sharp edge loop of ether at 1032.39 cm⁻¹ and C–O–C asymmetric stretching in the biosolubilized extract at 1161.02–1152.82 cm⁻¹. However, the sp²–C–H stretching almost disappeared in both biosolubilized extracts, consistent with the results reported in [12]. These fungal isolates efficiently solubilize multifaceted coal particles and enhance the release of functional groups. Higher intensity representing aromatic, oxygen‐ and nitrogen‐containing groups in the residual coal and the biosolubilized extract by the AD‐5 than the AD‐3. Nitric acid pretreated lignite particles also showed improved solubilization of nitrogen‐ and oxygen‐containing functional groups [12].

The FT‐IR spectral changes provide mechanistic insights into the fungal degradation process. The disappearance of sp³ C–H stretching bands (2971–2923 cm^{− 1}) in treated coal residues indicates cleavage of aliphatic crosslinks. The appearance and intensification of C═O stretching bands (1693–1635 cm^{− 1}) is consistent with oxidation of aromatic rings where fungal laccases and peroxidases are known to catalyze this type of transformation in other lignin‐ and coal‐degrading fungi [70, 71, 72, 73], though no enzyme activity assays were performed in the present study to confirm their involvement. The reduction in ether band intensity (1099–1032 cm^{− 1}) indicates cleavage of ether linkages that connect aromatic clusters. These spectral changes, combined with the increased O/C ratio from the ultimate analysis, are consistent with fungal enzymatic activity preferentially targeting aliphatic bridges and ether linkages, though this was not directly assayed.

Based on our findings and documented metabolic capabilities of these fungal species [35, 36, 39, 40], we propose the following degradation pathway: (1) initial attack on aliphatic side chains by cytochrome P450 monooxygenases; (2) oxidative cleavage of aromatic rings by laccases and peroxidases; (3) cleavage of ether and ester crosslinks by esterases and etherases; (4) release of lower molecular weight aromatic and aliphatic compounds. The superior performance of AD‐5 (P. simplicissimum) likely reflects its well‐characterized ability to produce a diverse array of extracellular enzymes, including lignin peroxidases and esterases [35, 36].

3.6. Gas Chromatography‐Mass Spectrometry (GC–MS) of Solubilization Products

The compounds in the biosolubilized extracts from the plate assay were extracted and analyzed by GC–MS and listed in Tables 5 and 6. In total, 12 compounds from the solubilized virgin coal were detected, compared to 18 from the chemically pretreated coal by AD‐3. Most of the identified compounds from the biosolubilization of virgin coal were saturated fatty acids, polycyclic aromatic hydrocarbons (PAHs), benzene, and phenol derivatives. The biosolubilization of the chemically pretreated coal contained PAHs, ketones, aldehydes, fatty acids, nitrogen‐ and oxygen‐containing compounds. The lengths of the carbon chain of the identified compounds ranged between C7‐C35. Various studies reported that low rank coal yields more aliphatic compounds while hard coal yields mainly classes of aromatics [14, 30, 74, 75]. Several studies reported that microbial solubilization of chemically treated coal produces compounds of ethers, alcohols, hydrocarbons, aromatic acids, carboxylic acids, phenols, and benzene derivatives in the solubilized products [7, 11, 58, 76, 77].

TABLE 5.

GC/MS detectable compounds in biosolubilized extract by AD‐3.

Virgin coal			Chemically oxidized coal
RT (Liu et al.)	Compound name	Formula	RT	Compound name	Formula
6.61	Phenol, 3,5‐bis (1,1‐dimethylethyl)‐	C14H22O	6.62	Phenanthrene, 4, 5‐dimethyl‐	C16H14
9.63	9‐Octadecene, (Machnikowska et al.) ‐	C18H36	7.41	Bifenox	C14H9Cl2NO5
9.95	Isopropyl Myristate	C17H34O2	8.61	2, 4‐Dimethyl‐1‐hexene	C8H16
11.01	7,9‐Di‐tert‐butyl‐1‐oxaspiro (4,5) deca‐6,9‐diene‐2,8‐dione	C17H24O3	8.95	Methanone, (2‐methylphenyl) phenyl‐	C14H12O
11.47	1,2‐Benzenedicarboxylic acid, butyl octyl ester	C20H30O4	9.62	Pentafluoropropionic acid, pentadecyl ester	C18H31F5O2
11.67	1 –Octadecene	C18H36	9.94	Isopropyl Myristate	C17H34O2
13.55	1‐Heptadecene	C17H34	11.47	Dibutyl phthalate	C16H22O4
15.27	1‐Octadecene	C18H36	11.67	Cyclopentane, 2‐isopropyl‐1, 3‐dimethyl‐	C10H20
15.62	Phenol, 2,2’‐methylenebis [6‐(1,1‐dimethylethyl)‐4‐methyl‐	C23H32O2	13.55	Ethanone, 1‐cyclohexyl‐	C8H140
16.52	Di‐n‐octyl phthalate	C24H38O4	14.18	2‐Methyl‐d‐glucose	C7H14O6
16.95	Pentatriacontane	C35H72	15.27	2‐Aziridinone, 1‐tert‐butyl‐3‐(1‐methylcycloheptyl)‐	C14H25NO
19.25	Disulfide dihexadecyl	C32H66S2	15.53	Androstane‐3,12,17‐trione, (5. beta.)‐	C19H28O2
—	—	—	15.62	Phenol, 2,2’‐methylenebis[6‐(1,1‐dimethylethyl)‐4‐methyl‐	C23H32O2
—	—	—	16.51	1,2‐Benzenedicarboxylic acid, dicyclohexyl ester	C20H26O4
—	—	—	16.95	2‐Nonanol	C9H20O
—	—	—	19.35	Lanostan‐3. beta. ‐ol,11. beta.,18‐epoxy‐, acetate	C30H54O
—	—	—	28.31	Copper, bis (2,4‐pentanedionato‐O, O’)‐	C10H14CuO4
—	—	—	33.94	n‐Heptyl‐2‐{3‐[(heptyl‐methyl‐carbamoyl)‐methyl]‐quinoxalin‐2‐yloxy}‐ N‐ methyl‐acetamide	C8H9ClN2O

TABLE 6.

GC/MS detectable compounds in biosolubilized extract by AD‐5.

Virgin coal			Chemically oxidized coal
RT (Liu et al.)	Compound name	Formula	RT	Compound name	Formula
6.62	Phenol, 2,4‐bis (1,1‐dimethylethyl) ‐	C14H22O	3.54	2H‐1‐Benzopyran, 3,4‐dihydro‐6,7‐dimethoxy‐3‐(2,3,4,5‐tetramethoxyphenyl (S)‐	C11H13NO2
7.42	Trichloroacetic acid, tetradecyl ester	C16H29C13O2	5.48	Cresyl diphenyl phosphate	C19H17O4P
9.62	Cis‐2‐Nonene	C9H18	7.41	4‐(4‐Hydroxyphenyl) pyrimidine	C12H12N2O4
11.16	Pyrazol‐4‐amine,3,5‐dimethyl‐1‐(3‐phenoxybenzyl)‐	C18H19N3O	8.61	2,2’‐Bipyridine,1,1’–dioxide	C10H8N2O2
11.65	Dioxathion	C12H26O6P2S4	8.97	3,4‐Dimethoxyphenyl isothiocyanate	C9H9NO2S
13.52	Pseudosolasodine o, N‐diacetate	C31H47NO4	9.63	5,18:9,14‐Dietheno‐6H‐dibenzo [d, k] thiacyclotetradecin,8,15,16,17‐tetrahydro	C25H22S
15.23	4‐trifluoroacetoxytetradecane	C16H29F3O2	9.95	Isopropyl Myristate	C17H34O2
16.89	Phenol,2‐[4‐(2‐hydroxyethylamino)‐2‐quinazolinyl]‐	C16H15N3O2	11.00	Butanal, (2.4‐dinitrophenyl) hydrazine	C10H12N4O4
19.29	Phthalocyanine	C32H18N8	11.47	Terephthalanilide,4’,4’’‐dinitro‐	C20H14N4O6
—	—	—	11.66	Benzene‐1,3‐dicarboxamide,5‐nitro‐N, N’‐dicyclohexyl‐N, N’‐dimethyl‐	C22H31N3O4
—	—	—	13.55	N, N’‐Dicyclohexylformamidine	C13H24N2
—	—	—	13.69	3‐(1,3‐Dihydroxyisopropyl)‐1.5,8,11,14‐pentaoxacyclohexadecane
—	—	—	15.27	Z‐11‐Tetradecen‐1‐ol trifluoroacetate	C14H28O
—	—	—	15.62	Phenol,2,2’‐methylenebis[6‐methoxy‐3‐(2‐propenyl)‐	C21H24O4
—	—	—	16.10	4‐Methyl‐E‐9‐octadecene	C19H38
—	—	—	16.51	1,2‐ Benzenedicarboxylic acid, mono (2‐ethylhexyl) ester	C16H22O4
—	—	—	16.96	19‐Norpregn‐4‐ene‐3,20‐dione	C20H28O2
—	—	—	17.47	Thionazin	C8H13N2O3PS
—	—	—	17.75	Trans‐Zearalen‐. beta. ol, tri‐TMS
—	—	—	19.26	4‐Oxocyclopentane‐1,2‐dicarboxylic acid	C7H8O5
—	—	—	21.92	C(14a)‐Homo‐27‐norgammacer‐14‐ene,3. beta. ‐methoxy‐	C32H52O3
—	—	—	25.28	3, 5‐Dimethoxyphenethylamine	C10H15NO2

There were 9 compounds identified in the biosolubilized extract of the virgin coal by AD‐5, compared to 22 compounds in the chemically pretreated extract (Table 6). The compounds in the virgin coal extract exhibit mostly phenolic derivatives, monocyclic aromatics, nitrogenous compounds, and hydrocarbons. In the extract of the chemically pretreated coal, PAHs, fatty acids, ketones, and oxygen‐containing compounds were the main products. The length of the carbon chain ranged between C7–C32, which is comparable to the AD‐3 treatment. Hofrichter et al. reported the liberation of benzene derivatives and polycyclic aromatic hydrocarbons (PAHs) from powdered hard coal by Coprinus sclerotigenis, similar to many other studies [12, 19, 30, 46, 76]. These organics are enriched with carboxyl groups that can be utilized as potential substrates for methane generation.

The GC–MS analysis of the solubilized fractions was consistent with the UV Visible and FT‐IR analysis. The UV scans showed aromatic and aliphatic substitutions in the biosolubilization products. Concomitantly, aliphatic and aromatic derivatives were detected by the GC–MS. Furthermore, it is evident that fungal mediated degradation of the chemically pretreated coal produced more organics than the virgin coal in terms of variety. A greater number and diversity of aromatic compounds classes than aliphatic compound classes were identified in the solubilized fraction (Tables 5 and 6), consistent with effective depolymerization of the aromatic‐rich crosslinked coal structure. The aromatics and derivatives containing hydroxyl, carboxyl, and ether groups were also confirmed by FT‐IR analyses. In addition, both GC–MS and FT‐IR analyses showed low molecular weight compounds in the biosolubilized products. The chemically pretreated coal produced more diverse compounds than the virgin coal, suggesting the treatment open more sites accessible for fungal attacks.

Across both isolates, chemical pretreatment shifted the compound profile toward a broader representation of fatty acids, ketones, and nitrogen/oxygen‐containing heterocycles, in addition to the PAHs and phenolics already present in extracts from virgin coal. This represents a more diverse array than previously reported for subbituminous coal biosolubilization. Shi et al. reported primarily aromatic compounds from nitric acid‐pretreated coal [58]. The practical implications of this compound diversity are significant. The identified fatty acids (C7–C35) can serve as precursors for biodiesel production or as industrial lubricants. The aromatic hydrocarbons, particularly PAHs and phenolic derivatives, have potential applications in the production of resins, adhesives, and specialty chemicals. The nitrogen‐containing compounds (e.g., pyrazol‐4‐amine, thionazin) may find applications in pharmaceutical and agricultural chemistry.

From a process‐design perspective, this biological conversion approach offers potential advantages over conventional thermochemical methods, chiefly because it operates at ambient temperature and pressure, avoiding the substantial energy input required by pyrolysis or gasification (typically > 500°C). The NaOH pretreatment step measurably improved conversion efficiency at a comparatively low reagent cost. However, we emphasize that no formal techno‐economic analysis (capital costs, reagent and energy costs at scale, downstream separation/purification costs) has been performed in this study, and the cost‐effectiveness claim should therefore be read as a process‐level observation rather than a validated economic conclusion. Given Pakistan's substantial subbituminous coal reserves (over 185.5 billion tons [2]), we view this system as a promising candidate for further scale‐up and economic evaluation, which we identify explicitly as necessary future work. Furthermore, the environmental benefits are equally compelling. This process avoids the CO₂ and SO_x emissions associated with coal combustion, instead converting coal into valuable chemical products. The use of indigenous fungal isolates eliminates the need for introduced microorganisms, reducing both cost and potential ecological risks.

The reproducibility of our findings was confirmed through triplicate experiments with consistent results (variation < 5% for key parameters). Control experiments confirmed that neither fungal metabolites alone nor chemical treatment alone accounted for the observed solubilization, establishing the synergistic nature of the process. While the current study provides comprehensive characterization, future studies should focus on: (1) identification and purification of the specific fungal enzymes responsible for coal degradation; (2) optimization of the NaOH pretreatment conditions (concentration, temperature, duration), including direct pH monitoring of the wash effluent to confirm complete removal of residual alkalinity, to maximize efficiency; (3) corroboration of gravimetric solubilization estimates with an independent, particle‐loss‐insensitive measure such as total organic carbon released into the liquid fraction, given that fine particle loss during sample handling and residual fungal biomass on the coal surface represent potential opposing sources of bias in the gravimetric method, (4) scale‐up studies to assess process feasibility at larger volumes; and (5) detailed economic assessment of the process at pilot scale.

4. Conclusion

The present study showed that indigenous fungal isolates AD‐3 and AD‐5 are potentially effective to oxidatively solubilize subbituminous coal. The results clearly show that chemical pretreatments and nutrient amendments enhance the biosolubilization of hard coal. The biosolubilized products are chemically heterogeneous and complex. This suggests that coal can be treated by indigenous fungi to produce alternative chemical stock/fuels like humic and felvic acids, amides, aromatic and aliphatic in an environment‐friendly and cost‐effective manner. The analysis has shown that the AD‐3 and AD‐5 fungal isolates can depolymerize subbituminous coal whose efficiency can be enhanced by chemical pretreatment to coal. This demonstrates that by combining chemical and biological processes, there could be an effective approach for the cleaner and sustainable alternative utilization of coal.

Author Contributions

Samia Anjum: conceptualization, validation, methodology, investigation, writing – original draft. Muhammad Adnan Sabar: investigation, validation, visualization, methodology, supervision. Javed Khan: methodology, validation, formal analysis, resources. Aneela Younas Malik: investigation, validation, formal analysis, data curation. Asif Jamal: investigation, visualization, methodology, resources, formal analysis. Zaixing Huang: investigation, formal analysis, visualization, validation. Ahsan Habib: writing – original draft, formal analysis, supervision, investigation, visualization. Muhammad Ishtiaq Ali: investigation, visualization, project administration, resources, data curation. Yusuf Tutar: investigation, visualization, validation, supervision. All authors contributed equally.

Funding

This research received no specific grant from funding agencies in the public, commercial, or not‐for‐profit sectors.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The datasets generated or analyzed are available from the corresponding author on request.