Biotechnology of Microorganisms from Coal Environments: From Environmental Remediation to Energy Production

Simple Summary Despite the wide perception that coal environments are extreme habitats, they harbor resident microbial communities. Coal-associated habitats, such as coal mine areas/drainages, spoil heaps, and coalbeds, are defined as complex ecosystems with indigenous microbial groups and native microecological networks. Resident microorganisms possess rich functional potentials and profoundly shape a range of biotechnological processes in the coal industry, from production to remediation. Abstract It was generally believed that coal sources are not favorable as live-in habitats for microorganisms due to their recalcitrant chemical nature and negligible decomposition. However, accumulating evidence has revealed the presence of diverse microbial groups in coal environments and their significant metabolic role in coal biogeochemical dynamics and ecosystem functioning. The high oxygen content, organic fractions, and lignin-like structures of lower-rank coals may provide effective means for microbial attack, still representing a greatly unexplored frontier in microbiology. Coal degradation/conversion technology by native bacterial and fungal species has great potential in agricultural development, chemical industry production, and environmental rehabilitation. Furthermore, native microalgal species can offer a sustainable energy source and an excellent bioremediation strategy applicable to coal spill/seam waters. Additionally, the measures of the fate of the microbial community would serve as an indicator of restoration progress on post-coal-mining sites. This review puts forward a comprehensive vision of coal biodegradation and bioprocessing by microorganisms native to coal environments for determining their biotechnological potential and possible applications.


Introduction
Coal is a combustible fossil fuel utilized for different needs, including electricity generation, heating, steel manufacturing, as sources of liquid and gaseous fuels, and precursors in the production of various chemicals/materials [1]. Being a heterogeneous and complex geopolymer, coal and a diverse range of its extracts/derivatives may be subject to microbial attack. Ultimately, the various known microbial metabolic pathways are associated with the wide array of organic substrates, carbon and oxygen content, aromaticity, and relative moisture in coal. Moreover, some natural coals serve as a reservoir of microbial strains able to degrade lignin, having a molecular structure similar to those of coal components [2]. microbial attack. Ultimately, the various known microbial metabolic pathways are associated with the wide array of organic substrates, carbon and oxygen content, aromaticity, and relative moisture in coal. Moreover, some natural coals serve as a reservoir of microbial strains able to degrade lignin, having a molecular structure similar to those of coal components [2]. Coals are categorized into several ranks based on depositional, physicochemical, and coalification characteristics that eventually reflect coal microbiology. Coal extraction via surface or underground mining, coal processing/preparation, and energy generation may shape the microbial community structure and functional potential [3].
To date, the available literature body on coal microbiology has generally focused on investigating the physiology and ecology of various microbial community structures/diversity and their activities in coal biodesulfurization processes as well as biogenic coalbed methane production. However, coal environments appear to accommodate diverse microbial catalysis with rich functional potentials to convert coal substrates into value-added products and remediate post-mining sites and industrial deposits.
In order to evaluate current research trends and identify the most actively studied topics on coal microbiology, we performed a bibliometric analysis based on keyword cooccurrence. The results were visualized using the VOSviewer software, showing sophisticated interconnections of the topics related to coal microbiology ( Figure 1). As one can see from the figure, the major accents currently lay primarily on biotechnological (such as biodegradation, bioremediation, and reclamation) and somewhat less on ecological (microbial community, biofilm, etc.) aspects. One of our goals was to bring these two aspects closer to each other, or rather to emphasize their intrinsic community. Figure 1. Keyword co-occurrence network visualization map for publications involving "coal* OR low-rank coal* OR lignite*" and "microorganisms* OR microbes*". Only publications from 1980 to June 2022 were considered. After exporting the publications from the Scopus platform in RIS form, they were analyzed using the keyword co-occurrence function of the VOSviewer software. Here, the top 30 items in terms of the number of occurrences are shown; different circles in the figure represent keywords, and their size indicates the number of times the keywords appear. The lines between the circles indicate that two keywords have appeared together in an article, and the more times they appear, the thicker the line is. On this basis, the main aspects of coal microbiology involve substrates (coal, lignite, low-rank coal, soil, flue gas, and heavy metals), microorganisms (bacteria, microalgae, fungi, and microbial diversity), processes (biodegradation, bioremediation, and Figure 1. Keyword co-occurrence network visualization map for publications involving "coal* OR low-rank coal* OR lignite*" and "microorganisms* OR microbes*". Only publications from 1980 to June 2022 were considered. After exporting the publications from the Scopus platform in RIS form, they were analyzed using the keyword co-occurrence function of the VOSviewer software. Here, the top 30 items in terms of the number of occurrences are shown; different circles in the figure represent keywords, and their size indicates the number of times the keywords appear. The lines between the circles indicate that two keywords have appeared together in an article, and the more times they appear, the thicker the line is. On this basis, the main aspects of coal microbiology involve substrates (coal, lignite, low-rank coal, soil, flue gas, and heavy metals), microorganisms (bacteria, microalgae, fungi, and microbial diversity), processes (biodegradation, bioremediation, and biodesulfurization), environments (acid mine drainage, coal mining, wastewater, and sewage sludge), and products (methane, biofuel, and organic matter). By selecting the keyword "microalgae", as an example, we could observe the connection between this and other keywords, such as coal, flue gas, carbon dioxide, biodiesel, biofuel, etc.
The second aspect was the study of the general trend of research by indicating the change in the number of articles involving microbes (microorganisms) in coal (low-rank coal, lignite) over time ( Figure 2). Here, the yearly number of publications was counted, which was obtained from the Scopus platform from 1980 to 2021. As can be seen, there has been an upward trend in the number of publications over the years. Initially, many microbial strains were directly isolated from those environments, which may not be associated solely with coal sources; however, for achieving effective and sustained exploitation and remediation of coal, the relatedness of microbial isolates to the coal sources may be critical. It is intuitively clear that indigenous microbial communities are optimally adapted to their environment in the presence of coal, implying their higher coal degradation efficiency compared with that of exogenous microbial communities. Native strains of microorganisms are already widely documented and are able to grow in culture media with coal as the sole carbon source and solubilize this material, generating humified organic matter [4,5].
Investigation of the microbial communities and main functional genes in coal environments has provided strong evidence that coal sources could be a "seed bank" of various microorganisms with very different functional potentials [6].
In the present comprehensive review based on a systematic literature search, we aimed to take a deep look at the overall functional structure and metabolic potentials of key microorganisms (bacteria, fungi, and microalgae) native to coal environments in the production of value-added compounds and remediation of coal-impacted sites. However, the microbial potentials driving coal biodesulfurization and coalbed methane generation are beyond the scope of this review due to the numerous excellent works published recently [7][8][9].  1999 1998 1997 1996 1995 1994 1993 1992 1991 1990 1989 1988 1987 1986 1985 1984 1983 1982 1981 1980 Yearly publication number Initially, many microbial strains were directly isolated from those environments, which may not be associated solely with coal sources; however, for achieving effective and sustained exploitation and remediation of coal, the relatedness of microbial isolates to the coal sources may be critical. It is intuitively clear that indigenous microbial communities are optimally adapted to their environment in the presence of coal, implying their higher coal degradation efficiency compared with that of exogenous microbial communities. Native strains of microorganisms are already widely documented and are able to grow in culture media with coal as the sole carbon source and solubilize this material, generating humified organic matter [4,5].
Investigation of the microbial communities and main functional genes in coal environments has provided strong evidence that coal sources could be a "seed bank" of various microorganisms with very different functional potentials [6].
In the present comprehensive review based on a systematic literature search, we aimed to take a deep look at the overall functional structure and metabolic potentials of key microorganisms (bacteria, fungi, and microalgae) native to coal environments in the production of value-added compounds and remediation of coal-impacted sites. However, the microbial potentials driving coal biodesulfurization and coalbed methane generation are beyond the scope of this review due to the numerous excellent works published recently [7][8][9]. Relatively high content of viable biomass and spectrum of saprotrophic fungi and heterotrophic bacteria showed that the sediment was a microbially rich geological medium in which microorganisms could survive/thrive for a long time. [18] Brown coal mine deposit area Sokolov brown coal basin, Czech Republic Assessment of the development of bacterial communities throughout the succession in the mining area PLFA, microarray, and 16S rRNA gene-based analysis Bacterial community composition of the 6-year-old site with no vegetation cover greatly differed from those of the older sites, especially with higher contents of Gammaproteobacteria, Cyanobacteria, and some Alphaproteobacteria.
Bacterial communities were especially vital during primary succession in its initial and late phases, when they dominated over soil fungi. [19] Stockpiles of opencast coal mines Coal-rich Emalahleni area, South Africa Investigation of the microbial community and enzyme activities as soil quality indicators in stockpiles of coal mines PCR-DGGE analyses and enzyme activity determination The bacterial OTUs spanned two phyla (Firmicutes and Proteobacteria) and four genera viz Bacillus, Pseudomonas, Azomonas, and Lysinibacillus. All fungal OTUs belonged to Ascomycota. Overall, the microbial community from stockpiles was impaired compared with that of the unmined site Differences in microbial diversity and enzyme activities suggested that the soil's biological components were highly sensitive to soil disturbance [20]    Direct light and epifluorescence microscopy and "growth slide" method A total of 122 species of algae were found in both areas. Green algae prevailed in both areas, but in the brown coal area, cyanobacteria and diatoms were also quite diverse. The total abundance of algae ranged mostly from 10 4 -10 7 cells/g dry soil, and was one order higher in the brown coal area than in the lignite area Sludge and compost fertilization resulted in the rapid formation of visible algal crusts dominated by Klebsormidium crenulatum [46] Acid mine drainage Abandoned undergroundcoal mine, USA Identification of the dominant algae in photosynthetic assemblages observed in acid mine drainage

Microscopic analyses
A diverse range of unicellular microalgae, such as Chlorella, Cylindrocystis, Botryococcus, and Navicula, and several filamentous forms identified as Microspora, Cladophora, and Binuclearia, were recorded The observed high algal diversity may be related to the long duration of acid mine drainage flow at the site, which has led to the development of adapted algal communities [47] * OTU, operational taxonomic units; PCR-DGGE, PCR-denaturing gradient gel electrophoresis; PLFA, phospholipid fatty acid analysis; RFLP, restriction fragment length polymorphism; TLFA, total fatty acid profiles.
Advanced next-generation sequencing methodologies based on metagenomic analyses and 16S or 18S rRNA genes have been used to survey coal mining-associated environments. Through the application of these multiple high-throughput technologies, microbial communities can be characterized in terms of function and structure, and these characteristic profiles can be monitored over time.

Coal Materials as Substrates for Microorganisms
Coal is a heterogeneous and carbonaceous material with mineral inclusions that microorganisms may be able to attack, degrade, or utilize several different constituents of. In principle, microorganisms can function either in the general breakdown of the coal molecule or in the selective removal of particular components [48,49]. The treatment of low-rank coals with aerobic coal-solubilizing microorganisms produces heterogeneous hydrocarbons and organics of different molecular weights and polarities, with a relatively high oxygen content [50]. Their structural and chemical nature renders low-rank coals more susceptible to microbial modifications, which can be attributed to [51]: (1) higher oxygen content compared with that of higher rank coals, thus providing "biological doorways" for degradation, (2) increased water solubility, which results in improved bioavailability, and (3) structural resemblance to lignin, which allows degradation by lignin-degrading microorganisms.
Several approaches for microbial coal modification have been proposed [52]. Microorganisms may attack coal by focusing on carbonaceous matter and/or interspersed inorganic materials. One approach is depolymerizing the coal polymer, breaking various key links-this could provide the basis for liquefaction. A second approach would be reducing the oxygen content through reducing C=O to CH 2 or decarboxylation to CO 2 -this could improve the calorific value. A third would be the removal of sulfur, nitrogen, or metals from the coal before firing, which would reduce unwanted emissions.
The pilot observations reported in the 1980s initiated intensive research, leading to a deeper understanding of the parameters and strategies of coal degradation [4]. To date, many studies have stated three principal mechanisms of coal biodegradation/bioconversion: solubilization, depolymerization, and utilization [53][54][55]. However, a number of other terms are often used interchangeably to describe different stages in coal biodegradation/bioconversion, such as solubilization, liquefaction, depolymerization, utilization, decolorization, etc.
Coal solubilization represents a nonenzymatic dissolution and occurs at an alkaline pH in the presence of alkaline substances, chelators, and surfactants, yielding black liquid. Coal depolymerization is mediated by enzymes that function at pH levels below 6. Lignindegrading oxidoreductases and certain hydrolases cleave linkages that maintain the 3D structure of coal and release substances with lower molecular masses [56]. Coal utilization consists of its biodegradation by various bacteria and fungi, which use components of the mobile part of lignite as carbon sources.
In general, microbial coal transformation is summarized and denoted as the ABCDE system (A = alkali, oxidative; B = biocatalysts; C = chelators; D = detergents; and E = esterases) [2,53]. In most bacteria and actinomycetes, alkaline action and chelation play the primary role, while enzymes play the most crucial role in fungi. Several microorganisms with distinctive physiological characteristics were found to exploit either one or a combination of these mechanisms.
A. alkaline substances (ammonia, biogenic amines, peptides, and their derivatives) and chelators are involved in the microbial solubilization (liquefaction) of coal. These non-enzymatic substances are produced by fungi and bacteria using the organic acids of the medium and increase oxidation by neutralizing the carboxylic acids present in coal, ultimately resulting in coal solubilization. B. coal depolymerization and solubilization can be achieved through catalytic metabolism, especially with lignin-degrading enzymes, because the structure of low-rank coal is very similar to that of lignin. These enzymes play a critical role in humic acid depolymerization by breaking the covalent bonds within the coal macromolecule. These enzymes can be divided into oxidative (lignin peroxidase, manganese peroxidase, and laccase) and non-oxidative (esterases). Numerous microorganisms (Penicillium sp., Trichoderma sp., Bacillus sp., Mycobacterium sp., Acinetobacter sp., Enterobacter sp., Rhodococcus sp.) have been documented to secrete ligninolytic enzymes on culture medium containing coal. Saprotrophic fungi and, in particular, ligninolytic microorganisms may act as biocatalysts for coal transformation [4]. C. chelating agents (e.g., oxalic acid, salicylic acid, and triethylamine) secreted by fungi can react with the metal ions (calcium, iron, and magnesium) in coal and depolymerize its molecular structure, resulting in the generation of small water-soluble molecules. D. detergents (surfactants) enhance coal solubilization/dissolution by promoting the absorption of biological enzymes on the coal's surface and by reducing surface tension. In addition, surfactants can also shift the reaction sites of certain enzymes, which may lead to higher coal biodegradation rates. E. like oxidases, non-oxidative esterases also play a large role in coal degradation. These enzymes are mainly produced by Gram-negative and Gram-positive soil bacteria and can hydrolyze coal polymers by the cleavage of ester or ether bonds.
Biological degradation/conversion of coal sources offers an alternative clean strategy for exploiting massive coal discards/dumps. Several non-fuel options for low-rank coal utilization have been prospected, such as the extraction of soil amendments/conditioning agents, organic moieties, and chemical feedstock for the subsequent generation of alternative fuels.

Peculiarities of Coal Degradation by Bacteria
A variety of bacterial species have been studied for their coal biosolubilization abilities, as they dissolve coal as an energy source for growth. Short-term cultivation, a faster conversion rate, and easier operation enable bacteria to achieve maximal coal degradation under standard temperature and pressure conditions [5,53,57]. Many recent studies suggest that indigenous bacterial isolates from coal environments (coal residues, coal-mining soils, and coal tailing water) have greater coal-solubilizing ability than exogenous microbial communities ( Table 2).      (1) and leonardite (2) Emalahleni coal fields (1) and No. 2 Seam (2), South Africa The biodegradation of coal may occur faster when the substrate is oxidized; therefore, many studies often employ either brown coal (lignite) or leonardite with different proximate/ultimate compositions. As mentioned above, depending on the type of mechanism involved in the studies, coal biodegradation can be described in many ways, such as biotransformation, biosolubilization, bioconversion, demineralization, decomposition, and bioliquefaction. The rates and extent of coal biodegradation depend upon several factors, including the rank and origin of the coal, type of microbial strains, mode of coal treatment/processing, etc.
Coal biodegradation by microbial isolates is being intensively studied to develop effective rehabilitation and revegetation strategies for coal mining areas and discard dump sites ( Figure 3). Such isolates, so-called "microbial cocktails" [51] native to coal environments, can presumably be the best tools for obtaining released humified organic matter from coal for subsequent agricultural applications.

Coal Degradation by Fungi
Primarily, coal degradation by fungal species differs from that by bacteria in terms of the nature of the released organics. Indeed, while the bacterial degradation of coal results in the generation of a mixed pool of organics, including aromatics and aliphatics, the fungal degradation machinery involves mainly polyaromatic hydrocarbons, single-ring aromatics, aromatic nitrogen compounds, and a minor fraction of aliphatics [74]. The elucidated operative mechanisms behind coal solubilization and depolymerization by fungi include both enzymatic (hydrolases; peroxidases, viz., manganese peroxidase and lignin peroxidase; and phenol oxidases, viz., laccases) and non-enzymatic (alkaline metabolites, surfactants, and chelators) agents [75].
A variety of coal-native fungal species have been reported, which efficiently modify the coal matrix and liberate fractions with different molecular weights ( Table 3). The indigenous fungal species are preferable to exogenous species because they are more likely to fit into complex coal environments and are well-adapted climatically. In most studies, the fungal strains playing a crucial role in effective coal degradation were isolated from decaying wood around coal mines, coal-soil mixtures, coal mining sites, and purely coal environments.
One of the prospects that can be derived from the data summarized in Table 3 is the use of fungal strains in coal degradation for agricultural sustainability and environmental safety. The application of coal treated with fungal strains has been extensively reported by Gokcay et al. and in other studies devoted to chemical feedstock processing and the production of humic substances for soil conditioning [76].

Bioremediation of Contaminated Sites by Native Microorganisms
Coal remains the largest fuel source for power generation worldwide, comprising around 40% of global energy production [96]. Mining activities produce a high volume of mine discards and tailings, causing soil erosion, heavy metal contamination, and acid mine drainage. Furthermore, land degradation due to coal mining alters biogeochemical and hydrological cycles, posing severe environmental and health risks in vast mining operations areas [97].
Land devastation in coal mining sites is well documented and emphasizes the need to invest focused effort in developing sound rehabilitation technologies [98,99]. Various traditional chemical approaches have been proposed to restore contaminated mining soils. However, their effectiveness and cost-efficiency are still debatable [100][101][102]. Bioremediation and bioreclamation are increasingly being considered as the primary choice for contaminated site recovery worldwide due to economic, environmental, and safety reasons [4,103].
While the exact biochemical and molecular mechanisms involved in coal biodegradation under ambient conditions remain to be better elucidated, there certainly appears to be a contribution of microbial solubilization [104], oxidation [105], and liquefaction [5,93] that demonstrate the potential to use microorganisms in effective rehabilitation strategies.

Bioremediation of Coal Mining Areas by Microorganisms
Several microbial cultures have been isolated from coal sources (slurries and disposal of coal tailings), screened for coal biodegradation competence, and characterized. Hamidović et al. [97] isolated and identified autochthonous lignite mine spoil bacteria and evaluated their potential in the bioremediation of mine-overburdened soil. The findings of that study also illustrated the soil fertility potential of recovered native species Bacillus simplex and Bacillus cereus. In a study by David et al. [5], 45 bacterial strains were isolated from the coal sludge, and four strains belonging to Cupriavidus sp., Pseudomonas sp., and Alcaligenes sp., were further evaluated for their coal-degrading activity. The observed ability of these strains in coal depolymerization may be attributed to the aggressive degradation of aromatic compounds (phenol, toluene, benzaldehyde, benzoic acid, and indole), since these compounds have been widely used as models for coal degradation.
The colonization and oxidative metabolism of discarded coal by fungal strains (Fusarium oxysporum, Paecilomyces farinosus, Lentinula edodes Trametes versicolor, and Phanerochaete chrysosporium) isolated from coal environments are also well-documented [106,107]. Another fungal organism, Neosartorya fischeri, could rapidly colonize and use various complex organics, highlighting its biotechnological potential for application in rehabilitating recalcitrant substrates [95,108].
Apart from using individual microbial isolates, consortia-based remediation could be an attractive alternative for enhancing the rehabilitation of mining sites. Like crude oil and petroleum, coal is a complex hydrocarbon material that usually requires cooperation between species or assemblages of microbial populations to be degraded [109,110]. Specific microbial taxa within a complex consortium demonstrate a different level of substrate specificity proliferating on particular coal fractions. Detman et al. reported that the formation of microbial consortia and their synergistic interactions dramatically enhance lignite degradation [64]. According to Maka et al., mixed cultures of Bacillus strains sufficiently degraded crude lignite within two weeks [111]. In a study by Olawale et al. [57], the most successful coal-degrading consortia contained either Serratia sp. ECCN 24b or Exiguobacterium sp. ECCN 21b, or both, and reduced the coal substrates' masses bỹ 10% and~30%, respectively. Indeed, in another study by Mohanty et al., species of the Exiguobacterium were able to actively utilize n-alkanes (C9-C26) [112], while Serratia spp. were identified by Benedek et al. as hydrocarbon degraders [113]. The biocatalytic efficacy of microbial consortia has been especially noticeable in studies on the bioconversion of coal to methane [114].

Bioremediation Potential of Arbuscular Mycorrhizal Fungi
Bioremediation strategies for soils affected by coal mining activities primarily aim to establish pioneer vegetation, because plant root systems stabilize degraded soils by controlling soil erosion and restoring soil fertility. The introduction of plant cover to mining areas can be facilitated by beneficial soil microorganisms, including plant-growth-promoting (PGP) bacteria and arbuscular mycorrhizal fungi (AMF). These essential microbial "biocatalysts" provide mutualistic support to sustain plant growth in degraded lands by enhancing the ability of plants to resist predominant conditions and by increasing plant performance through the biotransformation of pollutants to less toxic forms [49,115]. AMF, due to their vast mycelial network, can colonize plants and effectively extract nutrients and water from soil [116]. In addition, AMF enhance the root microbial community structure and contribute to the capture of macro-and micronutrients by plants [117]. Very recent studies by Sekhohola-Dlamini et al. and Widhayasa et al. showed that the period of post-coal-mining reclamation, vegetation, and soil physicochemical properties are profoundly determined by the soil AMF [117,118].
Another beneficial aspect of AMF in ecosystems is the facilitation of carbon conservation in coalfield soils. For example, in a study by Wang et al. [119], increased AMF inoculation significantly enhanced carbon sequestration, respiration, and photosynthesis in many plant species (wild cherry, cerasus humilis, shiny leaf yellow horn, and apricot). In the same study, the AMF promoted plant growth, significantly increasing the leaf area, chlorophyll content, and Q 10 value.
Taheri et al. [120] tested the potential for AMF to mediate plant adaptation to mine soil conditions and found that the plants (common grass and forb) with fungal communities (dominated by Paraglomus occultum, but also harbored an undescribed Entropospora species and Glomus mosseae derived from mine soil) grew larger, regardless of the soil type in which they were grown. The authors suggest that microbial communities collected from harsh environments can be the best group to draw upon for the fungal inoculation of nursery plants destined to be planted in reclaimed areas. A field experiment was conducted by Bi et al. [121] to study the ecological effects of AMF (Funneliformis mosseae and Rhizophagus intraradices) on the growth of Amygdalus pedunculata Pall. and their root development in coal-mine-subsided areas. The results showed that AMF increased the quantity of microorganisms in the rhizosphere as well as soil quality compared with the non-inoculated treatment.
Salim et al. observed that the AMF population shows a tendency to increase along with the increasing revegetation age classes. In one of their studies, the eight-year revegetation age classes had the highest average number of spores [122]. Another study by the same research group [123] showed that the increase in revegetation age led to an increase in the number of AMF populations, with Glomus sp. and Acaulospora sp. being the dominant AMF representatives in every land revegetation age.
The combined inoculation of AMF (Glomus mosseae) and phosphate solubilizing bacteria (Pantoesstewarti) significantly enhanced the soil quality and ecological efficiency of the coal mining waste, and also improved the plant biomass of Medicago sativa L. [124]. These results imply that bacteria and AMF together play an essential role in phytate mineralization and subsequent transfer to the host plant.
Of particular interest are the approaches involving biofertilizers (Rhizobium sp., Azotobacter sp.), effluent treatment plant sludge, and mycorrhizal fungi (Glomus sp. and Gigaspora sp. isolated from plants growing near mine spoil dumpsites) along with suitable plant species. Inoculating such biofertilizers allowed Juwarkar et al. to reduce heavy metals and improve the rhizosphere microbiological characteristics for plant growth [125].
Fungcoal, exploiting fungi-plant mutualism, was developed in South Africa as a viable and alternative strategy for rehabilitating coal discard dumps and opencast spoils [126]. In short, Fungcoal is composed of mutualistic networks between (a) C4 grasses (Eragrostis tef , Cynodon dactylon, and Pennisetum clandestinum, (b) AMF inoculates (Paraglomus occultum, Glomus clarum, Glomus mossea, and Gigaspora gigantea), and (c) coal-degrading fungi (Neosartorya fischeri ECCN 84 and/or Aspergillus ECCN 84). Fungcoal was created as a strategy to address numerous issues, including (1) biodegradation of carbon pollutants, (2) biogeneration of technosol with humified organic matter, (3) promotion of mutualism between plants and microbes, and (4) activation of relevant rhizosphere microbes, etc. [117]. Based on in situ studies, the authors postulated that soil fertility can be improved de novo through a microbial consortium, so-called 'humifiers', and that combinations of specific biocatalysts act synergistically in maintaining soil dynamics.
Mutualistic interaction between plant roots and nonmycorrhizal fungi can also aid in the growth of plants. For example, in a study conducted by Igbinigie et al. [127], a phyto-bioconversion of hard coal involving plants and free-living fungi (Aspergillus spp., Ulocladium sp., Alternaria sp., and Penicillium sp.) occurring in the rhizosphere facilitated the growth of Cynodon dactylon (Bermuda grass) in the coal dump.

Rhizosphere Microbial Community as a Bioindicator of Soil Restoration in Coal Mining Sites
The community structure and composition of soil microorganisms undergo considerable taxonomic and functional changes during remediation and restoration in disturbed coal mining ecosystems. In the last two decades, the interaction between the rhizosphere microbial communities and their host plants in mining-affected environments and their responses to various factors have received considerable scientific attention. The microbiological nature of the rhizosphere presents essential information regarding the screening and management of plant species for degraded land revegetation. The studies summarized in Table 4 illustrate the idea that assessing microbial diversity in coal mine-affected soils may be a sensitive indicator of ecological stress and the restoration processes. The accompanying comprehensive analyses conducted by the authors reveal the dynamic nature and complexity of the soil microbial composition in the field of vegetation restoration in mining areas (Table 4).    Roots affected the microbial community and had a larger size and higher growth than the control [144] * NLFA, neutral lipid fraction analysis; PLFA, phospholipid fatty acid analysis.

Bioremediation of Heavy Metals and Selenium in Coal Mining Areas
Heavy metal pollution by industrial sources and anthropogenic activities poses severe threats to the environment and public health because of its high toxicity, nonbiodegradability, and bioaccumulation. Coal mining is a prominent global geogenic and anthropogenic source of heavy metal pollution through acid mine drainages (AMD) [145,146]. The AMD from abandoned coal mines is a serious problem faced by many countries and may remain hazardous for decades or even centuries after mine closure, persistently shaping unfavorable environmental scenarios. To date, research on the impact, assessment, and management of AMD has received extensive attention worldwide, focusing on sustainable remediation strategies. Significant efforts have been devoted to reducing heavy metal loads and enhancing microbial activities [147]. Among the biotic approaches, autochthonous microorganisms are one of the best options for the decommissioning of AMD stressors [148,149].
Sulfate-reducing bacteria (SRB), including Desulfosporosinus, Desulfitobacterium, and some members of Firmicutes and Actinobacteria, attenuating toxic metal/metalloid concentrations, have been repeatedly isolated from mining sites [150,151]. AMD treatment by SRB reduces sulfates to hydrogen sulfide, which binds with metals, thus removing them from the solution. In addition, SRB generate alkalinity, contributing to the neutralization of AMD acidity [152]. In a study by Luptakova et al., a mixed SRB culture of Desulfovibrio and Desulfotomaculum effectively removed Cu 2+ from model solutions after 5-6 days using one batch reactor [153]. Dong et al. showed that SRB combined with coal gangue achieved high treatment efficiency in repairing AMD, i.e., it achieved the highest removal percentages of chemical oxygen, SO 4 2− , Fe 2+ , and Mn 2+ [154]. SRB isolated by Ma et al. [155] from loess polluted by coal AMD exhibited almost 100% immobilization of Fe 2+ , Cd 2+ , and Zn 2+ by hydrogen sulfide precipitation after 18 days.
The bioremediation potential of indigenous heavy metal-tolerant bacteria isolated from a rat-hole coal mine environment was well documented by Shylla et al. [156]. In their study, three isolates (Serratia marcescens KH-CC, Bacillus siamensis KH-12A, and Bacillus altitudinis KH-16F) out of twelve exhibited a high maximum tolerable concentration (MTC) against Pb (1400 ppm), Mn (830 ppm), and Fe (500 ppm). Serratia marcescens exhibited the highest Mn and Pb remediation, with 72.5 and 83% removal capacities, respectively. Kaot et al. [157] discovered that two bacterial isolates, Bacillus subtilis sub sp. inaquosorum SK22 and Bacillus cereus SK44, isolated from rat-hole coal mines showed resistance to 100 mg/L of Fe and 1 mg/L of both Cd and Cr compared with the control strains of Bacillus subtilis MTCC 441 and Bacillus cereus MTCC 430. The same authors, in another study [158], aimed to profile the native bacterial isolates from a rat-hole coal mine for their bioprospection as bioremediating agents, and found that the minimum inhibitory concentration and maximum bactericidal concentration of Cd 2+ , Fe 2+ , and Cr 6+ against Enterobacter huaxiensis KHED8 were 4000, 4096, and 256 mg/L, respectively. Furthermore, E. huaxiensis KHED8 was able to remove 89%, 90%, and 82.45% of Fe 2+ , Cd 2+ , and Cr 6+ , respectively. Previously, Zheng et al. [159] identified a total of 23 highly sensitive genera (Actinobacteria, Acidobacteria, Candidate division WS3, Chloroflexi, Gemmatimonadetes, Proteobacteria, and Thermotogae) and 16 highly resistant genera (Bacteroidetes and Proteobacteria) to Cd 2+ and Hg 2+ in coal-mine-affected agricultural soil.
Some bacterial populations can thrive under coal-overburdened strata's mineraldepleted and highly toxic heavy metal conditions. Singh et al. [160] enumerated the bacterial diversity of active opencast coal mining sites stratum-wise and found that the bacterial isolates belonging to Firmicutes, Actinobacteria, and Proteobacteria exhibited high tolerance (5 to 12 mM) to heavy metals (Ni 2+ , Cu 2+ , Cr 6+ , As 3+ , and Cd 2+ ) and could be promising agents for the bioremediation of contaminated sites. Enterobacter spp., Klebsiella spp., and Acinetobacter gyllenbergii were selected by Gandhi et al. [161] based on their high level of heavy metal resistance (Cd 2+ , Pb 2+ , Fe 2+ , Mn 2+ , and Cu 2+ ) and their biochemical characterization. Interestingly, a high degree of metal resistance was associated with multiple-antibiotic resistance of these isolates.
Micromycetes can also precipitate metals as insoluble oxalates, participating in metal removal from geochemical cycling. Thermophilic/thermotolerant micromycete cultures of Aspergillus spp. Isolated from coal seam spoil are capable of binding Cu 2+ as lowsolubility crystalline moolooite [162]. The Ni 2+ and Cd 2+ resistance of fungi from coal mining environments has also been described elsewhere [163].
As mentioned, microbial consortia can exhibit excellent sequestration of various multicomponent toxic heavy metal mixtures in AMD. Such consortia of bacteria indigenous to coal AMD that tolerate elevated concentrations of heavy metal mixtures can be specifically designed in order to develop targeted bioremediation strategies for alleviating heavy metal toxicity in situ [164]. For example, the study by Oyetibo et al. revealed that a consortium of seven autochthonous bacterial taxa (Υ-Proteobacteria: groups of Acinetobacter pittii, Enterobacteriaceae, Pseudomonas citronellolis, and unclassified FWNZ species, and Bacilli: groups of Sporosarcina koreensis, Bacillus cereus group, and Exiguobacterium aurantiacum) exhibited excellent urease activities (≥253 µmol urea min −1 ) with subsequent stemming of acidic pH to >8.2 and sequestration of toxic metals (~100% efficiency) as precipitates (15.6 ± 0.92 mg ml −1 ). Bacterial ureases hydrolyze urea into ammonia and carbamate, which subsequently release ammonia and carbonic acid that can stem acidic and toxic metal impacts [165]. Naghavi et al. reported that Acidithiobacillus ferrooxidans isolated from coal mining, when added to Cu 2+ coal mining samples, showed synergistic effects toward natural ferrous oxidizing microorganisms, manifested as up to a 46.7% increase in Cu 2+ extraction [166].
A number of bacterial communities with good heavy metal tolerance and bioremediation potential in extreme environments have been discovered and characterized through high-throughput sequencing technology. According to Liu et al., metal-tolerant Proteobacteria and Actinobacteria are predominant at the phylum level in mining area soils [167]. The study by Ma et al. [168] indicated that acidic coal gangue was relatively rich with SRB, containing six genera: Desulfosporosinus, Desulfovibrio, Desulfotomaculum, Desulfobulbus, Desulfitobacterium, and Desulfurella.
Oxidation states influence the solubility and bioavailability of another important pollutant, selenium. One Se 6+ -reducing bacterium, Enterobacter hormaechei, and four Se 4+ -reducing bacteria, Klebsiella pneumoniae, Pseudomonas fluorescens, Stenotrophomonas maltophilia, and Enterobacter amnigenus, were isolated by Siddique et al. [169] from coal mine tailings pond sediment. The results suggested that E. hormaechei could remove up to 96% of the added Se 6+ (0.92 mg L −1 ) from the effluents, with potential application in removing Se from industrial effluents.
Bacillus paramycoides SP3, a native strain to the leachate of coal-mine-overburden rocks, was isolated by Borah et al. [170] and investigated for its potential to produce Se nanoparticles by the biogenic reduction of selenite, one of the most toxic forms of selenium. B. paramycoides SP3 exhibited extremely high selenite tolerance (1000 mM). It reduced 10 mM selenite under 72 h to produce spherical monodisperse Se nanoparticles with an average size of 149.1 ± 29 nm, indicating that this strain could be utilized for the ecofriendly removal of selenite from contaminated sites with the concomitant biosynthesis of Se nanoparticles.

Bioremediation of Salt-Affected Soils
Soil salinization is one of the immense environmental stresses and global issues, deleteriously affecting the growth and yield of crops and thereby threatening food security. High salt concentrations in soil trigger extensive alterations in the physiology of agriculturally important plants, ultimately leading to their death. The rehabilitation and remediation of salt-affected soils have already been addressed with different technologies, including salt leaching and applying various amendments such, as gypsum and sulfuric acid [171,172].
Soil enrichment with different sources of humified organic matter and humic substances could help to alleviate the negative effect of salt accumulation on salt-sensitive crops [173]. Low-rank coal has been considered to be a superior amendment for soil quality and productivity. Low-rank coals can themselves serve as a nutritional medium for different soil microorganisms, stimulating their growth and development, and, through various metabolic mechanisms, result in a high yield of humic substances [174]. Among the microorganisms that can transform/solubilize coal to generate humified organic matter are different species of bacteria isolated from coal samples, including Pseudomonas sp., Streptomyces sp., Rhodococcus sp., Bacillus sp., and Escherichia sp. [175,176]. Cubillos-Hinojosa et al. reported that soil amendment with 1% coal and coal-solubilizing bacteria promoted short-term biological activity (increase in soil respiration and hydrolytic enzyme activity) associated with coal biotransformation and increased the soil cation exchange capacity [177]. Incorporating coal as a source of humic substances together with coal-solubilizing bacteria in saline-sodic soils under field conditions significantly and positively affected the chemical and biological soil properties [178]. This was reflected in decreases in the electrical conductivity, sodium adsorption ratio, and exchangeable sodium percentage, as well as in increases in microbiological activity and soil respiration [177,179]. For this reason, coal discards containing native microorganisms could be well used as an organic amendment for managing disturbed lands with the presence of salt-affected soils. However, evidence supporting the inherent chemical heterogeneity and functional diversity of coal as an amendment for salt-affected soils is scarce, and there are still uncertainties regarding the chemical mechanisms of coal as a slow-release fertilizer. Furthermore, depending on its origin and rank, coal may itself contain elevated levels of organically bound chlorides and inorganic constituents, implying a substantial risk of soil contamination [180].

Agricultural Applications of Microorganisms Native to Coal Environments
Green agriculture requires applying effective organo-mineral amendments that contain macro-and microelements and plant-growth biostimulants, which are a source of biologically active compounds. Today, low-rank coals (lignite and leonardite), as a source of humic acid, have attracted considerable interest in agriculture and environment conservation. Humic acids, as a key source of soil organic amendment, can be easily extracted and mobilized from soils through native strains of microorganisms [181]. Many of them are known as plant-growth-promoting bacteria (bio-inoculants) and represent a vital part of the healthy soil microbiome [182].

Production of Humic Substances through Coal Biodegradation
Humic substances are the most complex and biologically active organic matter compounds in the soil/sediments. They improve the microbial community structure and activity, regulate the availability of higher macro-and micronutrients for plant growth, and help maintain soil physicochemical properties. Furthermore, humic substances can play a considerable role in increasing plant resistance against common diseases and hostile environmental conditions. Humic substances of low-rank coals or run-of-mine coals have properties very similar to those of soils' humic substances. Typically, such coals contain between 25% and 85% humic acids, as compared with 1-5% in many soils/sediments [183]. This implies a novel and robust way to produce humic acids products from coal discards. It has long been observed that coal is susceptible to microbial attack, and a great number of studies have been published on this subject (Tables 2 and 3). Among these studies, many investigated humified organics as products of coal biodegradation. However, limited evidence exists concerning the targeted production of humic acid and fulvic acid-like substances.
Microbial consortia from coal-and diesel-contaminated soil slurries appear to be more efficient as biocatalysts in degrading coal than individual strains. Such associations of bacterial strains can grow on humic acid and fulvic acid as the sole carbon source [57]. In the study by Olawale et al., the most effective coal-degrading consortia contained native strains, either Serratia ECCN 24b or Exiguobacterium ECCN 21b, or both, and these decreased the coal substrate mass by~30% and~10%, respectively.
Commercial coal-derived humic substances used for crop cultivation could be considered as suitable substrates for isolating microbial strains capable of stimulating plant growth. For example, bacterial and fungal genera (i.e., Bacillus and Aspergillus) isolated from raw humic substances resulted in a significant increase in lettuce biomass in hydroponic cultivations and enhanced resistance to NaCl-related abiotic stresses [184].
Coal-degrading fungi from coal-polluted sites possess the potential for bioconversion of coal to value-added products, including humic substances. Based on the accumulation of humic acid, which is a marker of successful biosolubilization, Mucor circinelloides (118.9 mg/L) and Aspergillus tubingensis (43.9 mg/L) were the most active fungi in a study reported by Nsa et al. [185]. On the other hand, Cunninghamella bertholletiae (67.03 mg/L), Simplicillium subtropicum (45.95 mg/L), Penicillium daleae (42.70 mg/L), and Trichoderma koningiopsis (42.43 mg/L) produced high amounts of fulvic acid, indicating the occurrence of depolymerization. Penicillium ortum MJ51 was isolated by Li et al. from lignite, and they used its cell-free filtrates to extract HA from lignite [186].
The results of the recent study by Valero et al. [187] provided further evidence that lowrank coals, alone or inoculated with native bacteria (Bacillus mycoides, Acinetobacter baumannii, and Microbacterium sp.), serve as a valuable amendment to improve soil reclamation processes after mining activities.

Coal Microorganisms with Plant-Growth-Promoting Characteristics
Plant-growth-promoting (PGP) bacteria have already gained worldwide recognition within sustainable agriculture and soil remediation: they are responsible for a broad scope of biotic activities as they positively influence plant growth, hormone balance, immunity pathways, and plant stress resistance, and improve soil nutrient availability. PGP bacteria solubilize inorganic phosphorus compounds by producing organic acids and acid phosphatases and, in return, gain root-borne carbon compounds essential for bacterial growth [188]. Therefore, coal gangue and coal discard contain phosphorus and other essential macronutrients in a form available for plant growth and development [189].
Coal-solubilizing/degrading bacteria isolated from coal slurry from discard dumps often display characteristics typical of PGP bacteria. To bring more clarity to this issue, Titilawo et al. [190] sought to establish the genetic relatedness of coal-degrading rhizosphere bacteria (Bacillus, Escherichia, Citrobacter, Serratia, Exiguobacterium, and Microbacterium) from coal discard dumps to sequences of PGP bacteria from the NCBI GenBank database.
Analyses of indole and ammonium production revealed that these bacteria may have PGP characteristics.
The strains Pseudomonas sp. NU36 and Acinetobacter sp. NU25 from dump soils were characterized by Upadhyay et al. using the BIOLOG identification system that showed their high plant growth promotion effect (arising from indole-3-acetic acid (IAA) production, siderophore production, and the potential to solubilize inorganic phosphate) combined with excellent stress tolerance characteristics (high temperatures, drought, salt stress, pH, and heavy metal toxicity) [191]. Several other studies also indicated the stress tolerance characteristics of PGP bacteria. For example, Barman et al., in their recent study [192], isolated and characterized 10 bacterial strains from the coal dumping area; among them, Bacillus toyonensis DD1, B. mycoides DD2, B. velezensis DD9, and B. flexus DD10 strains demonstrated tolerance to two or more heavy metals. Additionally, these PGP bacteria could solubilize phosphate, produce IAA, produce siderophore, and show ACC deaminase activity.
An alternative to the recovery of degraded coal mining areas is revegetation with fast-growing species of legumes, which promote nutrient cycling, increase incorporation of carbon in the soil, and minimize erosion. In addition, legumes associate with symbiotic rhizobia (N-fixing bacteria), who promote increased deposition of nitrogen, reduce the soil C/N ratio, and increase soil organic matter (humus), favoring the mineralization and cycling of nutrients [193]. In a study conducted by Moura et al. [194] to evaluate the effectiveness of indigenous rhizobia isolated from coal mining areas in nodulation and their capacity to promote the growth of leguminous trees, the isolates were able to nodulate bracatinga (Mimosa) plants, ensuring shoot dry matter increases of 165%, and also favored nodulation and the growth of Marica.
Xia et al. determined whether culturable PGP isolates could be isolated from the surface of switchgrass (Panicum virgatum L.) from coal fields and investigated the subsequent effects of these isolates on switchgrass growth and development. A total of 307 bacterial isolates were cultured and identified into 76 strains, 36 species, and 5 phyla. Approximately 58% of bacterial strains, when reintroduced into surface-sterilized switchgrass seeds, were observed to increase the lamina length relative to the uninoculated controls [195].
The genus Delftia has been widely accepted as a group of PGP bacteria, although originally isolated as a free-living bacterium [196]. A new facultative chemolithoautotrophic heavy-metal-resistant Delftia sp. strain SR4 was isolated by Roy and Roy [197] from an open cast coal mine. It exhibited many PGP characteristics upon 48 h of incubation, including the production of IAA (23 µg mL −1 ), siderophore (55% siderophore units), ammonia (6 µmol mL −1 ), and HCN (30 ppm). Furthermore, this strain showed encouraging results on the growth of Brassica juncea, designating Delftia sp. as a versatile strain for multiple ecosystem functions.
Actinomycete strains (Streptomyces sp. and Amycolatopsis sp.) isolated from the rhizosphere of birch (Betula pendula) inhabiting a coal mine dump seemed to be effective in producing siderophores and antibacterial compounds, and displayed somewhat increased survival in the presence of heavy metals [198].
Plants may harbor endophytic fungi that are functionally important for their health. In a study performed by Xia et al. [199], the diversity and specificity of culturable endophytic fungal communities (the most abundant class, order, and species were Sordariomycetes, Hypocreales, and Fusarium spp., respectively) were explored in switchgrass (Panicum virgatum L.) growing on a reclaimed coal-mining site for around 20 years. The isolated fungi were able to enhance the heights of the shoots by about 86%, the fresh shoot weights by 69%, and the dry shoot weights by 62% after being recultivated back into the plants, demonstrating their functional features.
As already described in Chapter 4.2, arbuscular mycorrhizal fungi (AMF) are common endophytic fungi that exhibit potentially symbiotic associations with terrestrial plants. For example, inoculation with AMF Funneliformis mosseae significantly promoted the survival rate of sea buckthorn over 50 months while also increasing plant height after 14 (53.9%), 26 (24.2%), and 50 (16.2%) months compared with the uninoculated treatment [200]. The application of AMF (inocula of Glomus intraradices BEG140, G. claroideum BEG96, and G. mosseae BEG95 adapted to adverse soil conditions) together with PGP bacteria (Sinorhizobium spp. And Azotobacter spp.) to coal mine spoil banks could increase the growth of reed canary grass and high-biomass hemp, and compensate for reduced doses of organic amendments [201].
In spite of various technological advancements and extensive experience in employing different PGP bacteria, it is still challenging to establish an integrated bioprocess using native coal PGP bacteria on a commercial scale. Indigenous microorganisms with plant-growth-promoting characteristics should be recognized as key "bioengineers" when developing the rhizosphere in disturbed soils, as they are better adapted to local conditions and contribute to the growth of selective plants.

Coal Microalgae Hold Great Biotechnological Potential in Coal Utilization and Processing
Microalgae are known as bioremediation and feedstock production agents: they offer several advantages related to their high productivity, cost-efficiency, simple cultivation conditions, high stress resistance, and easy product recovery [202]. The isolation of indigenous microalgae strains and communities adapted to the coal environment is essential in both research and commercial applications for environmental sustainability and economic feasibility ( Figure 4). Aquatic environments in post-coal-mining sites possess ideal conditions for the growth and productivity of microalgae. Abandoned coal mines are usually vast barren land with unlimited sunlight and carbon dioxide (CO 2 ), which are the ultimate requirements for microalgal cultivation to yield great biomass [203]. Different microalgae strains could be isolated from various local coal environments, ranging from freshwater bodies to effluents generated from mining activities [204].
The main benefit of employing microalgae is associated with the bioremediation of coal-contaminated wastewater [205]. Coal mining/processing is a water-intensive industry that requires a systematic approach to treating and recycling coal effluents/drainages. For example, a typical 1000 MW coal-fired power station produces half a billion liters of metal-contaminated effluent yearly [206]. Successful wastewater treatment for the removal/biotransformation of different pollutants from coal mining areas remains challenging. However, it is worth noting that indigenous microalgae can be used as a convenient bioindicator to assess aquatic ecosystems affected by coal mining activities [207][208][209].

Phycoremediation
Phycoremediation is the process of employing macro and microalgae for the remediation of wastewater and effluents. This type of treatment has many advantages (selfrenewing capacity, low cost, and sustainable nature) over conventional ones, which are energy-consuming, very costly, and generate a high amount of sludge [210]. Over the last few decades, algae-based systems have shown promising prospects for removing various heavy metals from AMD. Algae function as "hyper-accumulators" (when an active mechanism is involved) and "hyper-adsorbents" (through passive mechanisms), with a pronounced selectivity for different elements [211,212]. Microalgal species such as Spirulina sp., Chlorella, Chlamydomonas, Scenedesmus, Cladophora, and Oscillatoria are widely employed for heavy metal removal [212][213][214][215]. A cyanobacterium, Nostoc sp. KX814344, isolated by Warjri et al. [216] from a coal mine water sample showed the ability to grow at 15 ppm Cr 4+ , which is the highest Cr concentration observed in the area. In their study, Cr biosorption by Nostoc sp. was optimum at pH 6.0, and the biomass reached 3 µg mL −1 . In a study by Goswami et al. [217], a cyanobacterium Nostoc muscorum isolated from a coal mining pit exhibited the ability to remove 66% of Zn 2+ and 71% of Cu 2+ within a 24 h contact time; metal binding on the cell surface was found to be the primary mode of uptake, followed by internalization.
coal-contaminated wastewater [205]. Coal mining/processing is a water-intensive industry that requires a systematic approach to treating and recycling coal effluents/drainages. For example, a typical 1000 MW coal-fired power station produces half a billion liters of metal-contaminated effluent yearly [206]. Successful wastewater treatment for the removal/biotransformation of different pollutants from coal mining areas remains challenging. However, it is worth noting that indigenous microalgae can be used as a convenient bioindicator to assess aquatic ecosystems affected by coal mining activities [207][208][209]. Figure 4. Native microalgae from the aquatic coal environment possess enormous biotechnological potential: they capture and sequester carbon to offset flue gas emissions from power plants, and the easily obtained microalgal biomass can be used for lipid/biofuel production. Coal mine drainage and power plant effluents can be decontaminated with microalgae. Finally, coal discards/fines can be burned with microalgae as a heat-efficient coal-microalgae composite. Microalgal bioremediation becomes even more attractive when the biomass cultivated in wastewater treatment systems is used as a feedstock.

Phycoremediation
Phycoremediation is the process of employing macro and microalgae for the remediation of wastewater and effluents. This type of treatment has many advantages (self-renewing capacity, low cost, and sustainable nature) over conventional ones, which are energy-consuming, very costly, and generate a high amount of sludge [210]. Over the last few decades, algae-based systems have shown promising prospects for removing various heavy metals from AMD. Algae function as "hyper-accumulators" (when an active mechanism is involved) and "hyper-adsorbents" (through passive mechanisms), with a pronounced selectivity for different elements [211,212]. Microalgal species such as Spirulina sp., Chlorella, Chlamydomonas, Scenedesmus, Cladophora, and Oscillatoria are widely employed for heavy metal removal [212][213][214][215]. A cyanobacterium, Nostoc sp. KX814344, isolated by Warjri et al. [216] from a coal mine water sample showed the ability to grow at 15 ppm Cr 4+ , which is the highest Cr concentration observed in the area. In their study, Cr Figure 4. Native microalgae from the aquatic coal environment possess enormous biotechnological potential: they capture and sequester carbon to offset flue gas emissions from power plants, and the easily obtained microalgal biomass can be used for lipid/biofuel production. Coal mine drainage and power plant effluents can be decontaminated with microalgae. Finally, coal discards/fines can be burned with microalgae as a heat-efficient coal-microalgae composite. Microalgal bioremediation becomes even more attractive when the biomass cultivated in wastewater treatment systems is used as a feedstock.
Extremophilic indigenous coal microalgae, such as Klebsormidium, Euglena, Mougeotia, and Chlamydomonas, were repeatedly isolated from coal-generated AMD, where they were continuously exposed to very harsh chemical or physical conditions, including low pH and the presence of heavy metals [218][219][220]. In addition, the data reported by Freitas et al. [221] showed that the aquatic environment impacted by AMD could be also inhabited by Microspora, Eunotia, Euglena, Mougeotia, and Frustulia, accumulating huge concentrations of metals in their biomass.
Different technologies may be applied for AMD detoxification using native algae communities. For example, the oxidation pond systems containing cyanobacterial mats employed by Phillips et al. [222] showed a removal rate of up to 2.59 g Mn day −1 m −2 . Bench-scale biological treatment test cells (blue-green algae, predominantly Oscillatoria spp./microbial mat consortium) utilized by Sheoran and Bhandari [223] were revealed to be a cost-effective treatment technique for removing SO 4 and precipitating metals from AMD. Microcosm systems developed by Sheoran et al. using Eunotia exigua and Chlamydomonas sp. efficiently reduced the Fe content and SO 4 level from 14 mg L −1 to 0.2 mg L −1 and from 344 mg L −1 to 124 mg L −1 , respectively [45]. Finally, Microspora quadrata, a green filamentous algae from streams heavily contaminated by coal-generated AMD, was shown to simultaneously accumulate Pb 2+ and Fe 2+ ions [224].
Regarding the mechanisms of bioaccumulation, there are interesting studies conducted by Molwantwa et al. and later by Boshoff et al., where they described the pivotal role of extracellular polysaccharides. [225,226].

Cultivation of Microalgae with Flue Gas from Coal-Fired Power Plants
Global warming has profound implications for all aspects of ecosystems and human life. The trend of atmospheric carbon dioxide (CO 2 ) emissions has been increasing over the years, and its concentration reached an average of 400 ppm, while the safe level is considered to be 350 ppm [227]. The energy power sectors are responsible for ca. 42% of CO 2 emissions; thus, a transition toward renewable energy and alternative fuels is among the contemporary approaches to reducing CO 2 emissions [228].
The photosynthetic process is an attractive, sustainable pathway for the biological fixation of CO 2 by converting it into biomass as a low-carbon emission source, thus contributing to greenhouse gas reduction. Consequently, microalgae, as a globally dominant photosynthetic group, have received growing attention regarding their rapid conversion rate of CO 2 , high biomass productivity, and flexibility in the cultivation environment. It was estimated that 1 kg of dry algal biomass utilizes~1.83 kg of CO 2 , meaning that microalgae fix waste CO 2 ten times more efficiently than terrestrial plants [229,230]. The algal biomass can be converted into many valuable products, such as biofuel, nutritional food, and fertilizers [231].
Intensive studies focused on microalgae have been primarily conducted to develop an effective system for CO 2 mitigation on the one hand and to increase downstream production at laboratory and industrial scales on the other. However, considerable importance should be also attached to the upstream process, such as employing and optimizing native microalgae species to expedite the acclimatization period and alleviate the in situ biological CO 2 fixation. A significant effort toward this goal was recently made by Yahya et al. [232], who screened native microalgae species in a coal-fired power plant's surroundings for carbon fixation ability and identified three dominant species (Nannochloropsis sp., Tetraselmis sp., and Isochrysis sp.). Among them, Isochrysis sp. was elected as the superior carbon fixer, with a fixation rate of 0.35 g CO 2 L −1 day −1 under actual coal-fired flue gas exposure using a customized lab-scale photobioreactor. This finding was of great importance in exploring the biotechnological potential of microalgae for carbon emission mitigation from coal-based power plants.
In 2007, De Morais et al. [233] isolated Scenedesmus obliquus and Chlorella kessleri from the wastewater of a power plant and investigated their growth characteristics under different concentrations of CO 2 . The results demonstrated a high growth rate (µ max ) of 0.267 day for C. kessleri, with a maximum biomass productivity (P max ) of 0.087 g L −1 day −1 when cultivated with 6% and 12% CO 2 and the highest maximum dry weight biomass value of 1.14 g L −1 with 12% CO 2 for S. obliquus. Later, in a study by Radmann et al. [234], Synechococcus nidulans and Chlorella vulgaris were isolated from waste treatment ponds and compared with Scenedesmus obliquus and Spirulina sp. for CO 2 biofixation: the results indicated that C. vulgaris possessed similar fixation performance to Spirulina sp., with a maximum daily fixation of 13.43%, when growing in reservoirs for CO 2 biofixation from coal combustion gas.
Direct exposure to unfiltered flue gas from coal combustion is apparently challenging for microalgal communities, since they can be subjected to very high amounts of SO x and NO x compounds as well as heavy metals. However, mixed ("biodiverse") microalgal communities, containing different algal genera each preadapted to high carbonate contents, can be designed and adapted to tolerate growth in as much as 100% flue gas [235]. In some cases, coal combustion wastes could even provide microalgae with minerals, which substitute the nutrients needed for their growth. Vaz et al. [236] found that Chlorella fusca LEB 111 and Spirulina sp. LEB 18 did not show significant differences in their maximum biomass concentration (ranged between 0.64 g L −1 and 0.58 g L −1 ) when cultivated in a natural lagoon or in a waste pond at a thermoelectric power plant, implying no significant difference in the growth support capacity between a "natural" growth medium and an "industrial" one.
Carefully designed and scaled photobioreactors for growing microalgae can possess high economic value. For instance, a novel photobioreactor (total volume of 30 m 3 ) filled with Spirulina platensis developed by Chen et al. [237] allowed CO 2 utilization at an annual rate of 2234 kg of CO 2 .

Lipid Production by Native Microalgae
Apart from the bioremediation aspects, microalgal biomass is advantageous for highvalue-added product generation. Lipids extracted from the algae biomass can act as precursors to produce biodiesel. Ikenaga et al. examined the co-liquefaction of Chlorella, Spirulina, and Littorale with brown coal in 1-methylnaphthalene under a hydrogen atmosphere at 300-400 • C [238]. They used Fe(CO) 5 -S (at a high S/Fe ratio) and Ru 3 (CO) 12 as catalysts and observed a high oil yield. In another study, biofuel production by Chlamydomonas PW95 (isolated from coal-bed methane production water) was assessed by Corredor et al. [239] using an optimal combination of culture conditions. The combination of 30 • C and 0.5 mM nitrate resulted in maximum daily biomass accumulation reaching 5.30 × 10 6 cells/mL, high biofuel productivity (16 mg/L/d), and desirable fatty acid profiles, represented by saturated and unsaturated C16 and C18 chains. Their study may serve as a model to elicit physiological responses of microalgae to diverse culture conditions mimicking those of outdoor biofuel production.
An interesting autoflocculating microalgal strain, Scenedesmus sp. NC1, was isolated by Kumar et al. [240] from coal mine effluent wastewater. Its lipid characterization exhibited a complex profile (18.55% monounsaturated, 22.74% polyunsaturated, and 35.15% saturated fatty acids), designating this strain as a prospective candidate for biodiesel production. Moreover, due to its significant bioflocculation potential, Scenedesmus sp. NC1 can be used for better harvesting other non-flocculating microorganisms.
Abandoned coal areas can be an alternative place for the cultivation of microalgae for lipid production. Such algae samples were collected by Kumar et al. from wastewater accumulated in different coal mining areas and identified as Spirogyra sp. and Oscillatoria sp. [203]. Lipid content estimation revealed that the lipid content from algae grown in mine water was 16.3% higher than that of algae grown in river and pond water. In some cases, a double benefit could be achieved using microalgae, in which valuable biomass is produced while remediating residues for heavy metals. This was demonstrated by Chlamydomonas acidophila LAFIC-004 (from coal mining drainage), which is capable of growing in acidic and heavy-metal-rich mining residues [241].

Co-Firing of Microalgae with Coal for Power Generation
Environmental and health concerns regarding coal combustion have recently facilitated biomass utilization as a partial substitute for fossil fuels to yield high-quality coal with the desired characteristics [242]. Here, the term biomass refers to organic matter (wood, herbaceous, and aquatic biomass) generated as a result of photosynthesis and organic wastes originating from industrial, municipal, and animal materials [243]. The combustion of microalgae with fossil fuels positively affects the environment and economics of power generation. In addition, microalgae have a number of benefits, including a higher growth rate, elevated level of photosynthesis, high CO 2 fixation efficiency, and lower requirements for environmental conditions [244].
Several studies have been conducted on the co-firing of microalgae and coal, where microalgal species have been obtained/isolated from different sources. Scenedesmus sp. has been reported as a promising feedstock for carbon-neutral solutions, as it offsets the CO 2 emitted through combustion [245]. Moreover, this microalgae strain has been observed to offer higher values, i.e., 77.5 wt.% of volatile matter, 21.4 wt.% of calorific value, and low ash content (7.3 wt.%) [246]. Scenedesmus biomass blended with discarded ultra-fine coal has shown a prominent synergistic effect, upgrading the ignition temperature and the rate of combustion [247].
Coal-Scenedesmus blends, under the commercial name Coalgae ® 5-20% (coal and microalgae ratio at mass basis) composite, have exhibited improved combustion behavior and evolved greenhouse gases [248]. In addition, a decrease in the emissions of CO 2 , NOx, and SO 2 from coal to Coalgae ® 5-20% was observed.
The following possible synergistic effects explained the co-pyrolysis of coal and Scenedesmus sp.: the results revealed the occurrence of three pyrolysis stages with temperature ranges of 200-400 • C, 430-650 • C, and >750 • C, and activation energies of 131-138, 72-78, and 864.5-1235 kJ/mol, respectively [249]. According to the coal pyrolysis models, three main components in microalgae (glycine, medium-chain triglyceride, and starch) were studied by Wu et al. [250]. Glycine demonstrated positive synergistic effects under a 25% mass ratio, with a higher volatile yield than the calculated value.
An open pond microalgal culture system integrated with a coal-fired power plant seems to be a prospective setup in which the produced biomass is co-fired in the coal plant's boiler. Geostri et al. [228] investigated the smart integration of a 500 ha microalgae Tetraselmis suecica-culturing facility with a large-scale coal power plant (758.6 MWe), though the produced algal biomass contributed to only around 1% of the boiler's heat input. A fraction of the CO 2 contained in the coal plant flue gases was used for the algal cultivation and a fraction of the low-temperature flue gas heat available was used for the biomass drying; finally, the target biomass was co-combusted in the coal plant.

Limitations
All of the above-referenced studies should facilitate an understanding of the basic concepts of coal microbiology and the applications of native microorganisms in enhancing agricultural production and environmental protection through sustainable approaches. However, there are a number of important issues that should be borne in mind when considering the various microbial groups in coal ecosystems:

1.
Since every coal environment is unique in terms of nature and geology, it is difficult to formulate a set of general principles that could enhance the bio-utilization of coal universally. Furthermore, every coal source, which behaves as a part of a geomicrobial reactor, may have unique characteristics; therefore, the selection criteria of respective microbial species should be considered carefully; 2.
More efforts should be made to promote better characterization of the native microorganisms, their metabolic capacities, and/or exact metabolic pathways. An understanding of the detailed mechanisms of coal biodegradation/bioconversion and their exploitation at the molecular level may be required for sustainable agricultural and environmental systems. Studies examining the metabolic and physiological characteristics of microorganisms associated with coal environments have the potential to address fundamental questions about the primary functional drivers, a key area of investigation in coal biotechnology; 3.
Exploiting indigenous "microbial cocktails" native to coal may help to achieve optimized coal bioprocessing/utilization; however, this may be quite selective to a given coal environment; 4.
The traditional culture-dependent techniques, despite their advantages, might have limitations in capturing and studying large variates/amounts of microorganisms from coal environments; it is thus imperative to evolve more advanced techniques to discover novel microorganisms possessing unique metabolic characteristics;

5.
The underlying mechanisms of the functional roles of bacteria, fungi, and plants in coal-associated sites (abandoned mines, surface coal mines, and post-coal mining activities) and affecting environmental factors are yet to be fully explored; 6.
Although recent evidence has implicated the vast potential of microalgae in coal environments, a further understanding of their ecology, adaptation mechanisms, and efficient application of these organisms could be crucial for successful bioenergy production and environmental protection.

1.
Until now, fundamental research on coal biodegradation and bioutilization has focused mainly on the laboratory-scale screening of various methodologies and exogenous microorganisms; however, the implementation and optimization of these processes using indigenous microorganisms in full-scale outdoor systems remain attractive; 2.
Many research outcomes are too preliminary to predict the details of a commercialized process. Some consideration must be given to upgrading the technology to bring these processes to regulatory issues and policy that may exert a strong influence in the future; 3.
Because coal bioutilization is a complex and intricate process, efficient organisms and processes will be critical for economic competitiveness. The modern techniques of recombinant DNA technology and protein science may serve as enhanced tools for the manipulation of indigenous microorganisms; 4.
Furthermore, exogenous coal-solubilizing bacteria may become part of the indigenous microbial consortia that colonize coal environments, though they may be able to naturally thrive and subsequently solubilize coal in this ecological niche.

Conclusions
The relationships of the microbial species with the coal environment are one of the critical factors for achieving increased coal degradation and utilization, as they appear to be relatively favorable microbial substrates. The heterogeneous and aromatically condensed structure of coal cannot be regarded as completely understood in terms of its bioavailability and bio-efficiency due to the complexity of coal-microbial interactions. The subsequent application of various microorganisms to benefit coal largely depends on their metabolic and functional characteristics. Therefore, establishing highly adaptive and selective microbial groups may help to determine rate-limiting steps and enhance the prospects of coal bioutilization. Furthermore, despite the lengthy research history and promising reports regarding the efficiency of indigenous microorganisms, there is still no precise prescription for successful technological innovation in a practical large-scale application.

Conflicts of Interest:
The authors declare no conflict of interest.