Introduction

Mercury (Hg), a heavy metal element, is a global pollutant that threatens the health of humans and ecosystems (Beckers and Rinklebe 2017; Driscoll et al. 2013). Methylmercury (MeHg) is one of the most biotoxic forms of Hg (Ha et al. 2017; Liu et al. 2021; Obrist et al. 2018; Stein et al. 1996). The transformation of divalent Hg (Hg2+) to MeHg is mainly undertaken by Hg-methylated microorganisms (Bravo et al. 2018b; Hudelson et al. 2020). Therefore, the mechanism of microbial Hg methylation has become a crucial research topic for understanding methylation in the environment and for improving the environmental risk management of Hg (Bravo and Cosio 2020; Ma et al. 2019). Numerous previous studies have shown the presence of microbial methylation of Hg in the environment, but the mechanism was unclear (Baldi 1997; Fischer et al. 1995; Ullrich et al. 2001). In 2013, the functional gene hgcAB of Hg methylation was reported, and the studies of Hg methylation entered the molecular level (Parks et al. 2013; Poulain and Barkay 2013).

The abundance and diversity of hgcAB are sensitive indicators of Hg methylation potential (Gilmour et al. 2013; Podar et al. 2015). Quantitative molecular probes of hgcAB genes have been developed to detect and quantify the potential of Hg-methylated microorganisms (Christensen et al. 2016). Various molecular biology techniques have also been applied to the detection and recognition of the gene hgcAB (Christensen et al. 2019). Molecular probes and molecular biological techniques developed were summarized and compared.

The current state of knowledge on globally isolated Hg microbial methylation strains and their associated methylation functions is reported. The potential of Hg methylation of these strains has been assessed using different indicators, which have not been uniformly standardized (Helmrich et al. 2022). Hg-methylated microorganisms reported in varied environments are also discussed here. Some strains have been reported in multiple environments, while others have been reported only in specific environments.

With regard to the biological community, we analyzed the driving factors affecting Hg methylation, including Hg2+ substrate, dissolved organic matter (DOM), sulfur, copper (Cu), anoxic environment, and selenium (Se) (Bravo et al. 2015; Bravo et al. 2017; Schartup et al. 2013; Tang et al. 2020). The effects of Hg bioavailability and microbial activity were analyzed and compared respectively. Microbial community interactions are also the driving factors of Hg methylation, and both intraspecific and interspecific interactions are considered and compared (Liu et al. 2019). To sum up, the driving factors and influencing mechanisms of microbial Hg methylation are urgent problems to be solved in further research.

The functional gene of microbial Hg methylation

Expression mechanism of gene hgcAB

The microbial Hg methylation process is carried out by methylators in the environment using the functional gene hgcAB to convert Hg2+ to MeHg (Fig. 1a). The model of the gene hgcAB was first proposed in 2013, in which the corrinoid protein encoded by gene hgcA converts Hg2+ to MeHg, and the corrinoid protein is reduced by the 2[4Fe-4S] ferredoxin encoded by gene hgcB (Parks et al. 2013). The expression mechanism of the gene hgcAB was further enriched in 2014, demonstrating that the corrinoid protein encoded by hgcA can transfer methyl groups to electrophilic substrates. Based on density functional theory, it is found that cysteine’s (Cys) thiolate coordination to corrinoid protein is conducive to the transfer of methyl radical and methyl carbanion to Hg2+ substrates (Zhou et al. 2013). A recent study has better described the protein structure of HgcAB through computational modeling and protein isolation. The model structure shows that there is no interaction between the two domains of HgcA; it is HgcB that forms a broad connection against the two domains, and the conserved Cys (Cys94 and Cys95) of HgcB obtain Hg2+ and pass it to corrinoid for methylation (Cooper et al. 2020). These studies have enriched the expression mechanism of the functional gene hgcAB through further verification of hgcAB theory and protein structure.

Fig. 1
figure 1

Expression mechanism (A) and diversity analysis (B, C) of gene hgcAB

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HgcAB is a sensitive indicator for the potential of microbial Hg methylation

Studies have demonstrated that the presence of hgcAB is a reliable predictor of the Hg methylation ability of microorganisms (Gilmour et al. 2013; Podar et al. 2015). The Hg methylation rate is limited by the expression of hgcAB (Capo et al. 2022a). Methylation of Hg can also occur at lower protein concentrations, requiring more sensitive genetic identification methods to detect hgcAB in samples (Date et al. 2019).

Molecular biology methods for measuring gene abundance include metagenomic shotgun sequencing, 16S rRNA gene pyrosequencing, quantitative PCR amplification, and metaproteomics. Current common methods used to quantify hgcAB gene abundance include hgcAB PCR amplification, 16S rRNA sequencing and metagenomic sequencing. The detection depth of hgcAB PCR amplification is based on the development of specific primers for the hgcAB gene (Fig. 1b). In 2014, a variety of hgcA primers were designed to study hgcA diversity in different environments (wetland soils, paddy soils, and swamp sediments) (Bae et al. 2014; Liu et al. 2014a; Schaefer et al. 2014). The development of wide-range degenerate primers for the hgcAB gene was achieved in 2016, along with the development of branch-specific degenerate qPCR primers targeting three major clades (Deltaproteobacteria, Firmicutes, and Archaea) (Christensen et al. 2016). In addition, a new broad-range primer set for hgcAB and an expanded hgcAB reference library have been used to improve hgcAB amplification efficiency (Gionfriddo et al. 2020). Another way to quantify hgcAB is to detect, identify, and quantify hgcAB genes from metagenomics (Fig. 1c). According to the protocol reported by Capo et al., gene hgcAB in the metagenomic genome can be detected, identified, and counted through the latest hgc gene catalog, Hg-MATE database v1, and the marky-coco bioinformatics pipeline (Capo et al. 2022b). Comparing these methods of hgcAB gene detection reported above (16S rRNA sequencing, hgcAB PCR amplification, and metagenomic sequencing), it was found that (1) 16S rRNA gene pyrosequencing could not identify enough hgcAB+ species; (2) hgcAB clone library estimates a deeper diversity of Hg-methylators than 16S rRNA sequencing; and (3) the results from metagenomic screening showed the same diversity of hgcAB+ microorganisms’ recognition (Christensen et al. 2019). Thus, developing new techniques that fulfill the requirements of both the diversity and specificity of hgcAB is urgent. Prior to the technology being updated, it is recommended that combining hgcAB amplification diversity and metagenomic data accurately identifies Hg-methylated microbial communities and their Hg methylation potential in the environment.

Functional strains of Hg methylation

Classification of Hg methylation functional strains

The discovery of the functional gene hgcAB in 2013 was an essential improvement in Hg methylation mechanism research (Parks et al. 2013; Poulain and Barkay 2013). The gene cluster hgcAB has been used to estimate whether a strain has the potential for methylation (Christensen et al. 2016; Jones et al. 2019). In the phylogenetic distribution of genes containing the clusters hgcAB, methylators were reported among sulfate-reducing bacteria (SRB), iron-reducing bacteria (IRB), methanogens, and a small number of other unclassified microorganisms (Baldi 1997; Fischer et al. 1995). Although all species containing the gene hgcAB were distributed in these clades, not every strain in these clades has the potential for Hg methylation (Bravo et al. 2018b; Isaure et al. 2020; Liu et al. 2014b). Sixty-two strains, which had been isolated and experimentally demonstrated to have Hg methylation potential, were classified, and their methylation potential was compared (Fig. 2, Table S1). These strains included thirty-six SRBs, nine IRBs, eight methanogens, and nine other methylators. SRBs with the most significant proportion in quantity (> 60% of 62 hgcAB+ strains) dominate microbial Hg methylation. In addition, SRB strains are primarily distributed in family Desulfovibironaceae, with no significant difference in the proportion of strains in the remaining families. The number of IRBs and methanogens is much smaller than that of SRBs, but the Hg methylation potential of individual strains is comparable to that of SRBs. IRB strains were mainly distributed in family Geobacteraceae, while the proportion of methanogens in each family was evenly distributed (Fig. 2).

Fig. 2
figure 2

Classification and number of strains of SRBs, IRBs, methanogens, and other Hg-methylated microorganisms at phylum and family levels

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Sulfate-reducing bacteria is an obligate anaerobe that uses sulfate as an electron acceptor for energy generation and uses acetic acid, lactic acid, and pyruvate as electron donors. In recent decades, SRB has been shown to contribute significantly to microbial Hg methylation in terms of quantity and Hg methylation potential. With our knowledge, 36 Hg-methylated SRB strains have been reported to date. These strains have been distributed in phylum Proteobacteria (33 strains) and phylum Firmicutes (3 strains). SRB Hg-methylators are distributed in eight families, Desulfovibrionaceae (20 strains), Desulfobacteraceae (6 strains), Desulfomicrobiaceae (4 strains), Peptococcaceae (3 strains), Desulfobulbaceae (1 strain), Desulfohalobiaceae (1 strain), and Syntrophobacteraceae (1 strain) (Benoit et al. 2001; Bridou et al. 2011; Brown et al. 2011; Compeau and Bartha 1985; Ekstrom et al. 2003; Feng et al. 2022; Gilmour et al. 2011; Gilmour et al. 2013; Goni-Urriza et al. 2020; Graham et al. 2012; Kerin et al. 2006; King et al. 2001; Limper et al. 2008; Lin and Jay 2007; Liu et al. 2018c; Malcolm et al. 2010; Moreau et al. 2015; Ranchou-Peyruse et al. 2009; Xiang et al. 2020; Yu et al. 2018).

Iron-reducing bacteria are members of Deltaproteobacteria, which use ferric iron as an electron acceptor. The role of IRBs in Hg methylation has also attracted more attention (Fleming et al. 2006). Nine IRBs have been reported to have the potential to methylate Hg, distributed in phylum Proteobacteria (8 strains) and phylum Firmicutes (1 strain). IRB Hg-methylators are distributed in three families: Geobacteraceae (6 strains), Desulfuromonadaceae (2 strains), and Peptococcaceae (1 strain) (Bravo et al. 2018a; Fleming et al. 2006; Graham et al. 2012; Guo et al. 2021; Warner et al. 2003).

Methanogens were the first microorganisms found to have Hg methylation potential in the early 1960s (Bravo et al. 2015; Wood et al. 1968). Nevertheless, Pak and Bartha failed to replicate the Hg methylation of methanogens in pure culture experiments in 1998, and the study of Hg methylation in methanogens has been neglected for half a century (Ma et al. 2019; Pak and Bartha 1998). Until the 2010s, inhibitory culture experiments with field samples (lake, peatlands, and sediments) suggested that methanogens might be principal methylators (Bravo et al. 2018a; Hamelin et al. 2011; Zhang et al. 2015). Subsequent studies demonstrated the Hg methylation potential of methanogens in pure culture experiments (Gilmour et al. 2018). Eight methanogens have been reported to have Hg methylation potential. They are all distributed in phylum Euryarchaeota. These strains are distributed in seven families, Methanosarcinaceae (2 strains), Methanomicrobiaceae (1 strain), Methanoregulaceae (1 strain), Methanomassiliicoccus (1 strain), Methanocellaceae (1 strain), Methanocorpusculaceae (1 strain), and Methanospirillaceae (1 strain) (Gilmour et al. 2018; Pak and Bartha 1998; Wood et al. 1968; Yu et al. 2012; Yu et al. 2013).

In addition to the microorganisms mentioned above, methylators include fermentative, acetogenic, cellulolytic, and other unclassified microorganisms (Gilmour et al. 2018; Gilmour et al. 2013; Jones et al. 2019). These strains were isolated from wastewaters, sediments, rice paddies, and animal guts. Nine Hg-methylated strains were reported but not attributed to SRB, IRB, or methanogens, all of which have been confirmed to carry the gene hgcAB. It is important to note that these strains are not all strictly anaerobic and also include facultative aerobic bacteria and aerobic bacteria (Cao et al. 2021; Feng et al. 2022). This conflicts with the current belief that Hg-methylated strains are strictly anaerobic. In the future, the role of non-anaerobic bacteria in Hg methylation is calling for more attention. Predictions of methylation potential based on the functional gene hgcAB expanded the number of Hg-methylators. More strains with strong Hg methylation potential are expected to be found in other species.

Mercury methylation potential of strains

Mercury methylation potential can be characterized by the MeHg production (%MeHg), Hg methylation rate constants (Km), and normalized to protein content (picomoles of MeHg/mg protein) (Helmrich et al. 2022). In field experiments, MeHg/THg was used to represent the in situ potential for Hg methylation (Drott et al. 2008). In the laboratory studies, the variations of MeHg/THg, Km, and picomoles of MeHg/mg protein with culture time were obtained through culturing field samples to verify the methylation potential better. In addition, isotopic tracers (i.e., 196Hg, 198Hg, 199Hg, 200Hg, 201Hg, 202Hg, and 204Hg) were added to characterize the potential for methylation and demethylation clearly (Tang et al. 2020). Isotope labeling can allow for more accurate estimates of Km and Hg demethylation rate constants (Kd) by removing the effect of Hg compound morphology on Km. At the same time, marker elements’ localization can indicate the active transport mechanism during Hg methylation (Pedrero et al. 2012), whereas it is worth noting that the above three methods for assessing the Hg methylation potential of strains do not have a uniform standard to date.

The Hg methylation potential of Hg-methylated strains in the above classification, which has been reported in pure culture experiments and field studies, was analyzed. The Hg methylation potential of Hg-methylated strains in the above classification categories was analyzed. In SRB, strains Desulfovibrio desulfuricans ND132 and Desulfovibrio caledoniensis BerOc1 are often used as model strains to explore the mechanism of Hg methylation (Gilmour et al. 2011; Goni-Urriza et al. 2015). Studies of pure cultures showed that the methylation potential in %MeHg of strains Desulfovibrio sp. X2, D. desulfuricans ND132, and Desulfomicrobium baculatum X are 62.0%, 53.0%, and 34.1%, respectively (Gilmour et al. 2011; Gilmour et al. 2013) (Table S1). When picomoles of MeHg/mg protein were used to characterize Hg methylation potential, strains D. desulfuricans ND132 and D. baculatum X also have high methylation potential in picomoles of MeHg/mg protein up to 22.9 pmoles MeHg/mg protein and 26.6 pmoles MeHg/mg protein (Table S1). In IRB, strain Geobacter sulfureducens PCA is the model strain to study the potential of Hg methylation. The methylation potential in %MeHg of G. sulfureducens PCA in an iron medium reached 14.0% (Kerin et al. 2006). A laboratory study of pure cultures showed that Geobacter daltonii FRC-32 and Geobacter bemidjensis Bem were strong methylators with the methylation potential in %MeHg of 30.0% and 74.9% (Gilmour et al. 2013) (Table S1). In methanogens, Methanospirillum hungatei JF-1 cultured in DSM 864 has a strong potential for Hg methylation (Yu et al. 2012; Yu et al. 2013), and the methylation potential in %MeHg reached 64.2% (Gilmour et al. 2018). Methanomassiliicoccus luminyensis B10, Methanosphaerula palustris E1-9c, and Methanocella paludicola SANAE were confirmed to be Hg-methylators with methylation potential in %MeHg of 53.4%, 15.0%, and 8.6%, respectively (Gilmour et al. 2013; Podar et al. 2015) (Table S1). In addition, small amounts of Hg methylation strains in other classes have also shown noteworthy Hg methylation potential (Cao et al. 2021; Feng et al. 2022; Gilmour et al. 2013).

Hg-methylated microorganisms in the environment

Hg-methylated microorganisms in various environments are identified by analyzing the composition of microbial communities in field studies. Proteobacteria and methanogens have been widely reported in field research settings of Hg methylation. Proteobacteria have been reported as dominant Hg-methylated microorganisms in numerous studies on paddy soils, sediments, oceans, glaciers, and other environments (Azaroff et al. 2020; Capo et al. 2020; Lin et al. 2021; Liu et al. 2018b; Zhang et al. 2020a) (Fig. 3). In addition, when the Proteobacteria were further divided, SRB and IRB in proteobacteria were found to dominate microbial Hg methylation in different environmental samples. In studies on paddy soil in Guizhou, China, and sediments from 10 lakes in Spain, Proteobacteria SRB was found to be the largest Hg-methylated microbial community (Bravo et al. 2018a; Vishnivetskaya et al. 2018), whereas it was found that the leading Hg-methylated microorganisms belong to Proteobacteria IRB in two studies with sediments (Bravo et al. 2018b; Du et al. 2017). The contribution of methanogens to Hg methylation has been reported in a variety of settings, including landfills, paddy soils, and lake sediments (An et al. 2022; Jones et al. 2020; Liu et al. 2018c) (Fig. 3). In the above studies, other microbial communities (Firmicutes, Chloroflexi, Spirochaetes, etc.) are also involved in Hg methylation in various environments, whereas their abundance is much lower than the abundance of Proteobacteria and methanogens. Specific strains have also been found in certain environments. Nitrospina (microaerophilic nitrite-oxidizing bacteria) is currently the only Hg-methylated microorganism reported to be present in the ocean (Tada et al. 2021; Villar et al. 2020). More environmentally representative Hg-methylated communities remain to be discovered.

Fig. 3
figure 3

Hg-methylated microorganisms in the environments

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Driving factors of Hg methylation

The bioavailability of Hg

Substrates that can be used for microbial Hg methylation include Hg2+, Hg complexes with natural organic matter (Hg2+-NOM), cinnabar (α-HgS), metacinnabar (β-HgS), Hg2+-complexes, and other small molecule compounds (Schaefer et al. 2011; Xiang et al. 2022; Zhang et al. 2012a). The thermodynamic stability of Hg2+-complexes is the main controlling factor methylation, and unstable complexes (mixed-ligation complexes containing low-molecular-mass thiol (LMM-RSH), OH–, and Cl–) have higher methylation rates than stable complexes (Hg(LMM-RS)2) (Adediran et al. 2019). Factors affecting the bioavailability of Hg2+ include DOM, sulfides, and selenium.

The concentration of DOM and the combination of DOM to Hg2+ affect simultaneously on Hg bioavailability (Leclerc et al. 2015; Luo et al. 2017; Van et al. 2021). The concentration of DOM and the bioavailability of Hg2+ are inversely correlated, with low concentrations of DOM increasing the bioavailability of Hg2+, and high concentrations decreasing the availability of Hg2+ (Fig. 4a). Studies have shown that DOM with 0–0.01 mg·g−1 increases the bioavailability of Hg2+ by tenfold, compared to DOM with 0.01–0.05 mg·g−1 which reduces the bioavailability of Hg2+ (Chiasson-Gould et al. 2014). The combination of DOM and Hg2+ promoted the bioavailability of Hg. Most studies have focused on the binding of thiols to Hg and its affection on the bioavailability of Hg2+ (Bouchet et al. 2018; Thomas et al. 2020). As a special DOM molecule, thiols are the most affinity for Hg2+ cell ligands; thus, increasing the concentration of total thiols could promote methylation (Leclerc et al. 2015). For example, low molecular weight thiols (LMW-Thiols, such as cysteine) form Hg-thiol complexes (Hg-thiol), directly leading to the increase of MeHg production (Cardiano et al. 2011; Leclerc et al. 2015). Studies have explored the binding strength and binding law between DOM and Hg2+; the results show that the relative binding strength of Hg2+ is dimercaptopropanesulfonic (DMPS) > glutathione (GSH) > penicillamine (PEN) > cysteine > ethylenediaminetetraacetic (EDTA) > citric, acetic, and glycine, at a molar ratio of ligand-Hg < 2 (Liang et al. 2019). These results provide a new theoretical basis for studying the influence of multi-component DOM on Hg2+ transformation and bioavailability. In addition, DOM can slow down the precipitation of nano Hg sulfide (HgSnp), thereby improving microbial Hg methylation (Gilmour et al. 2018).

Fig. 4
figure 4

Driving factors of mercury methylation

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The form and size of the combination of sulfide and Hg affect its bioavailability (Fig. 4a). The increase of sulfide could inhibit microbial Hg methylation (Benoit et al. 1998; Liu et al. 2018a). Sulfides, including iron sulfide (FeS), iron disulfide (FeS2), carboxymethyl cellulose (CMC-FeS), and other forms of sulfides, will combine with dissolved Hg to form Hg sulfide (HgS) or Hg-complexes, and these combinations are stable (Pierce et al. 2022; Wang et al. 2020a). In addition, the morphology and binding characteristics of HgS also affect the bioavailability of Hg. HgS small molecules can be used by methylators, so the increase in HgS small molecule content has a promoting effect on Hg methylation (Li et al. 2022; Xu et al. 2021). In addition, HgS-EPS binding also increases the bioavailability of Hg, which is influenced by cell-nanoparticle interface reactions (Zhang et al. 2020b).

Recent studies have also found that antagonism between Se and Hg has an inhibitory effect on Hg methylation, mainly due to the formation of Hg-Se complexes reducing the bioavailability of Hg2+ (Cai et al. 2020; Truong et al. 2014; Wang et al. 2016) (Fig. 4a).

Environmental factors affect microorganisms and microbial Hg methylation

Environmental factors drive Hg methylation by affecting the biological activity or metabolic processes of microorganisms. For substrates of microbial Hg methylation, the morphology and bioavailability of extracellular Hg2+ control Hg methylation by microorganisms rather than intracellular Hg2+ (Wang et al. 2020b). DOM promotes microbial Hg methylation because DOM provides carbon sources or nutrients for microorganisms (Bravo et al. 2017; Lei et al. 2019) (Fig. 4b). Low cysteine concentrations (0–0.06 mg·g−1) enhanced cell growth thus promoting methylation, whereas the growth was inhibited when the cysteine concentration reached 0.6 mg·g−1(Gilmour et al. 2018). The mechanism of sulfate that promotes microbial Hg methylation is that sulfate provides sufficient electron acceptors for SRB, thereby promoting SRB activity and leading to an increase in the production of MeHg (Pierce et al. 2022; Wang et al. 2018) (Fig. 4b). Moderate sulfate concentrations (0.1 mg·g−1) promoted the methylation significantly higher than low (0 mg·g−1) and high sulfate concentrations (0.5 mg·g−1) (Lei et al. 2021; Shao et al. 2012). In addition, copper ions (Cu2+) could promote methylation (Fig. 4b). The absorption of Cu2+ has a synergistic effect on the absorption and methylation of Hg2+, which is due to copper transporters or metal binding (Lu et al. 2018). Anoxic environment is more conducive to the increase and accumulation of MeHg (Yang et al. 2019). Earlier study suggested that higher MeHg production was observed in anaerobic sediments than in aerobic sediments (Olson and Cooper 1976). Since then, more studies have confirmed that microbial Hg methylation has been observed in anoxic environments (Liu et al. 2009; Mehrotra and Sedlak 2005; Warner et al. 2003). Studies indicated that the deep brine layer prevents oxygen from contacting with the DOM in the sediment and inhibits demethylation, allowing MeHg to be produced and accumulated (Valdes et al. 2017). Hg-Se antagonism could inhibit microbial Hg methylation in the environment (Dang et al. 2019). The mechanism by which selenium inhibits methylation may include (1) the formation of HgSe nanoparticle and (2) the effect of selenium on methylators or demethylators (Wright et al. 2020; Zhang et al. 2012).

The Hg methylation potential is affected by the metabolic functions of the methylators

The classification of Hg-methylated strains includes SRB, IRB, and methanogenic bacteria (Luo et al. 2023; Ma et al. 2019). The methylation potential of microorganisms varies with their metabolic functions. There is a synergy between sulfate reduction and Hg methylation of SRB (Fig. 4c). When the sulfate reduction process was inhibited by molybdate, Hg methylation is greatly or even completely inhibited, such as 70.0% and 87.7% in peatlands and sediments, respectively (Correia and Guimaraes 2017). In addition, increasing sulfate content leads to the enhancement of SRB activity and thus promotes Hg methylation (the “Environmental factors affect microorganisms and microbial Hg methylation” section). This also demonstrated that sulfate reduction and Hg methylation are positively correlated. Many studies focus on the correlation between methanogenesis and Hg methylation of methanogens; however, there is not a clear understanding yet (Fig. 4c). Some studies suggested that the methanogenic pathway is synergistic with the Hg methylation pathway in which methane production was inhibited by 2-bromoethanesulfonate (BES) and Hg methylation is inhibited by 100% and 90% (Hamelin et al. 2011; Wang et al. 2020c), whereas a fierce competition for carbon sources and electron donors between the methanogenic and Hg methylation has also been reported, in which Hg methylation was significantly promoted in paddy soil (16.6-fold) and sediment (2-fold) when the methanogenesis was inhibited (Roth et al. 2021; Wu et al. 2020). The relationship between iron reduction and Hg methylation of IRB has been less reported. The mechanism by which iron reduction processes promote or inhibit Hg methylation remains unclear. Studies have shown that Hg methylation of IRB is not promoted when electron acceptors (FeOOH) enhance the iron reduction process (Wu et al. 2020), whereas iron reduction rates (FeRR) have a negative correlation with demethylation rates in the sediments (Avramescu et al. 2011).

The Hg methylation potential of methylators is affected by non-methylators

Due to the complex structure of microbial communities in the environment, other strains also affect Hg methylation strains. There is a great correlation between non-Hg methylators and Hg-methylators, and studies have shown that non-Hg methylated communities play an important role in predicting Hg methylation in paddy soil (Liu et al. 2019). In addition, there are interactions between different functional strains. For example, there is a synergistic relationship between SRB and methanogens, and studies have shown that syntrophic microbial interactions dominate microbial Hg methylation (Roth et al. 2021; Yu et al. 2018).

Perspective

Better use of molecular biological techniques in the evaluation of methylation

The hgcAB+ microorganisms and community characteristics have been used to estimate the potential of Hg methylation (Bravo and Cosio 2020). Molecular biological techniques used in field studies for environmental samples include 16S rRNA, real-time PCR, high-throughput sequencing, and metagenomics (Christensen et al. 2016; Lin et al. 2023; Puglisi et al. 2019; Regnell and Watras 2019). Through statistical analyses, correlation analyses, and phylogenetic analyses of microbial community structure and hgcAB abundance, the dominant microbial community of Hg methylation in the environment was identified, and the mechanism of methylation by microorganisms was explored (Christensen et al. 2016). However, due to the small proportion of methylators in the total microbial community, identifying all Hg-methylated microbial communities is difficult. Therefore, molecular sequencing technology should be developed to consider both diversity and accuracy in future research. The data of microorganic diversity and metagenomic are supposed to be combined to avoid that a single index could not well represent the real situation of Hg-methylated microorganisms. In addition, the phylogenetic information for microbiota should be given more attention. First, the three-generation sequencing technology should be better used to identify Hg-methylated microorganisms (Utturkar et al. 2015). Second, newly developed high-throughput, single-microbe genomics techniques are compelling tools for characterizing the genomics information of Hg methylation strains (Zheng et al. 2022).

Understanding methylation from microbial communities view

Field studies about the community abundance and diversity of Hg methylation microorganisms based on the functional genes hgcAB have been conducted. And many studies have isolated functional strains, then explored the process and mechanism of Hg methylation in the laboratory. However, to clearly understand the Hg methylation process in different environments, it is not enough to focus on the functional flora, but also to treat the environment as a micro-ecosystem and study them from a holistic perspective (Liu et al. 2019). The recommended reasons are as follows: (1) non-functional strains and functional strains may compete for limited energy sources (carbon sources, nitrogen sources, etc.) (Song et al. 2023a); (2) the metabolic of non-functional bacteria may be interrelated with the Hg methylation; (3) the abundance and proportion of functional bacteria in the ecological structure varied with environmental characteristics and non-functional bacteria. The complexity of microbial communities and interactions between communities (synergy, competition, etc.) need to be considered (Yang et al. 2021). From the perspective of microbial ecological structure, it is possible to understand the real environmental behavior mechanism of Hg more comprehensively. In addition, as with other pollutants, identifying the migration and transformation pathways of Hg and MeHg in the microbial community is also crucial for better risk management of MeHg in the environment (Song et al. 2023b; Zhang et al. 2022).

More precise and efficient inhibitors are needed

Current research uses gene hgcAB as molecular markers to identify methylators in environmental samples, whereas the role of microbial communities in Hg methylation remains unclear. Inhibition culture was used to verify the contribution of microbial communities to Hg methylation (Roth et al. 2021; Wang et al. 2020c). For example, molybdate or BES was used to inhibit the sulfate-reducing pathway of SRB or the methanogenic pathway of methanogens to identify primary Hg-methylated microorganisms. However, the interpretation of inhibition culture results can be influenced by (1) complex environmental factors and (2) microbial synergies (Lei et al. 2023). Therefore, it is necessary to design a more reasonable inhibition culture experiment and consider the appropriate inhibitor gradient as well as the background concentration of substrate and electron acceptor. In addition, a more accurate interpretation of inhibitor results is needed to avoid uncertainty in evaluating the relative contributions of different microbial groups.

The complexity of the environment, the diversity and interactions of microbial communities, and the pending development of molecular biotechnology pose limitations on the study of microbial Hg methylation. Further attention needs to be paid to the simultaneous contribution of the microbial community to Hg methylation, as well as the recent application of pioneer molecular biotechnology techniques, which are important for the clear understanding of microbial Hg methylation mechanisms in the environment.