Promoting mechanism of electronic shuttle for bioavailability of Fe(III) oxide and its environmental significance

Iron is the fourth most abundant element in the earth's crust. From the perspective of geochemistry, iron is mostly present in sediments in the form of iron oxide, especially insoluble hydrated iron oxide (Liu et al. 2007). Bioreduction reactions with Fe(III) oxide as the electron acceptor can degrade macromolecular organic matter in groundwater (Botton & Parsons 2007; Liang et al. 2009) and landfill leachate (Li et al. 2018), oxidize ammonia nitrogen in soil and water (Park et al. 2009; Yang et al. 2012), and decompose organic matter in deep-water lakes and deep seas (contribution rates of 44% and 75%) (Roden & Wetzel 2002; Thomsen et al. 2004). The Fe(III) bioreduction process not only strongly affects the cyclical transformation of key elements such as C, N, O and P in nature, but also plays an important role in the transformation and degradation of organic and inorganic pollutants (Lovley et al. 1991). The main Fe(III) oxides include goethite (α-FeOOH), hematite (α-Fe₂O₃), lepidocrocite (γ-FeOOH), maghemite (γ-Fe₂O₃) and Fe(OH)₃ (ferrihydrite, also known as amorphous iron). These forms of iron oxide are basically insoluble, and their biological activity in decreasing order is: Fe(OH)₃, γ-Fe₂O₃, γ-FeOOH, α-Fe₂O₃, and α-FeOOH. The higher thermodynamic stability and stronger crystal structure of Fe(III) oxide result in lower bioavailability and lower pollutant removal efficiency. In recent years, the promotion of the bioavailability of Fe(III) oxide by electronic shuttle has gradually become a research hotspot.

By adding a redox active material to form an electron shuttle between Fe(III) oxide and microorganisms, the rate of electron transfer between the microorganism and the extracellular electron acceptor can be accelerated. In addition to the common humic substances (HS) (Lovley & Woodward 1996), a variety of redox active substances have been discovered that accelerate the reduction of Fe(III), such as quinonoids (O'Loughlin 2008), biochar (Zhou et al. 2017), activated carbon (Wu et al. 2017), sulfur species, and microbial autocrine substances. By virtue of a unique phenolic hydroxyl group and a quinone group structure, these redox active substances can form an electron shuttle cycle of mutual conversion of oxidized and reduced states between microorganisms and Fe(III) oxides. The electron shuttle cycle acts as a bridge for electron transfer, improves electron transport efficiency (Dunnivant et al. 1992; Scott et al. 1998), and promotes the transition of crystalline Fe(III) to an amorphous state, thereby improving the bioavailability of Fe(III) (Li et al. 2018). Although domestic and foreign scholars have carried out a large amount of research on the promotion mechanism of Fe(III) bioavailability, the effects, restrictive factors and environmental applications of this process have not yet been systematically summarized and analyzed. In this paper, the research progress of related frontiers in recent years is reviewed, and the promotion mechanism of Fe(III) bioavailability, the Fe(III) reduction effect and restrictive factors of different redox active substances are discussed. From promoting the removal of contaminants such as heavy metals, polycyclic aromatic hydrocarbons, and nitrogen dyes, the environmental significance of the electron shuttle to promote the bioavailability of Fe(III) is evaluated, which provides reference and consideration for the selection and application of redox active substances.

There are many possible mechanisms for the bioreduction of Fe(III) oxide: (1) direct contact: this involves the direct transfer of electrons by contact between insoluble Fe(III) oxide and the extracellular membrane protein, (I); (2) chelation and dissolution: the small molecule organic matter interacts with the insoluble Fe(III) oxide to form a complex, which promotes the contact between the iron-reducing bacteria and Fe(III) oxide after dissolution (II); (3) mediated reduction: the microorganism first transmits the electrons to the redox active substance and reduces it; then the reduced substance transfers electrons to Fe(III) oxide, and also returns to an oxidized state itself to continue to participate in a new round of electron transfer (III); (4) Electronic transition: electrons are transferred multiple times between microorganisms and finally transferred to Fe(III) oxide (IV) (Chi et al. 2007) (Figure 1). Among these mechanisms, the mediated reduction has the best promoting effect on Fe(III) bioavailability because it does not require direct contact of microorganisms with Fe(III) oxide.

In an anaerobic microenvironment, iron-reducing bacteria with strong metabolism can significantly affect the degradation of various organic pollutants (saccharides, organic acids, aromatic hydrocarbons, etc.) during the bioreduction of Fe(III) oxide. Therefore, this reaction is critical in the repair and treatment of contaminated environments. The addition of a redox active material capable of forming an electron shuttle cycle can effectively promote this bioreduction reaction. Iron-reducing bacteria can transfer electrons from saccharide compounds to substances with redox activity, thereby promoting the formation of electron shuttles, which can oxidize carbohydrates (glucose) to CO₂ (Manfred et al. 2009). Under anaerobic conditions, iron-reducing bacteria can also use Fe(III) oxide as an electron acceptor and organic acid as an electron donor (Champine & Goodwin 1991). The quinone and hydroquinone functional groups in specific organic compounds can continuously lose electrons through the redox reaction to form an electron shuttle cycle, and oxidize formic acid, lactic acid and pyruvate to CH₃COO⁻ and CO₂. In addition, the metal reduction bacterium is the first microorganism that can oxidize aromatic compounds with Fe(III) as an electron acceptor. This electron shuttle can catalyze the degradation of various aromatic compounds (such as benzene, toluene, phenol, cresol, p-cresol, phenol, aniline, benzoic acid, p-hydroxybenzoic acid, benzaldehyde, p-hydroxybenzaldehyde, phenylethyl alcohol, p-hydroxyphenylethanol, etc.) (Lovley et al. 1989; Lovley & Lonergan 1990; Kazumi et al. 1995). The specific reaction equations are shown in Table 1.

HS is a complex polymer compound formed by long-term physical, chemical and biological reactions of animals, plants and microbial residues. It is generally black or dark brown and is a major component (50%–65%) of natural organic matter. HS is widely found in soil, sediments and aquatic environments, and it can be divided into humic acid (HA), fulvic acid (FA) and humin (Pettit 2004). HA contains many functional groups, such as carboxyl, hydroxyl, and sulfhydryl groups.

HA promotes the reduction efficiency of hematite higher than glucose, and can also synergize with glucose to improve the bioavailability of Fe(III). Unlike glucose, HA can form an electron shuttle to promote the biological reaction process (Stern et al. 2018). Lovley (Lovley & Woodward 1996) proposed that HA promotes the bioavailability of Fe(III) oxides in two stages: first, iron-reducing bacteria oxidize acetate, and HA is reduced as an electron acceptor; second, the reduced HA transfers electrons to Fe(III). Jiang et al. (Jiang et al. 2018) firstly confirmed that HA can promote the bioavailability of Fe(III) oxide. Their results showed that the first reaction rate of Fe(III) oxide bioreduction was increased at least 27 times compared with no HA addition, and the second reaction rate was increased 7 times.

HS concentration is a key factor in promoting Fe(III) bioavailability. Jiang et al. (Jiang et al. 2018) investigated the effects of various HS standards on reducing ferrihydrite by Geobacter sulfureducens, and concluded that the minimum concentration of HS was 5–10 mg C/L to promote the electron shuttle process. In another study, Wolf et al. (Wolf et al. 2009) reported that HA promoted the reduction of ferrihydrite by G. metallireducens. When the HA concentration was as low as 0.59 mg C/L, it still promoted the reduction of ferrihydrite. This may be due to the different abilities of various HS and microbes to transfer electrons. The study of HS promoting the bioavailability of Fe(III) has been carried out around soluble iron oxides, but the promotion of solid humus is still unclear. It is also important to thoroughly explore the optimal initial concentration of HS.

Carbonaceous materials include biochar, soot, activated carbon (AC), graphite, carbon nanotubes, and graphene oxide (Langley & Fairbrother 2007; Fu et al. 2015; Saquing et al. 2016). They have a highly porous structure, high alkalinity and abundant surface functional groups, which have great potential for carbon sequestration, soil fertility and quality (Woolf et al. 2010; Mohan et al. 2014; Inyang et al. 2016). The carbonaceous material contains many redox functional groups with high conductivity, and it has the same electron shuttle capability as HS. However, unlike the mechanism of HS in promoting the bioavailability of Fe(III) oxide, the carbonaceous material can transmit electrons through multiple oxidation reduction reactions (Kappler et al. 2014).

The added concentration of carbonaceous material is a key factor affecting the bioavailability of Fe(III) oxide. Kappler et al. (Kappler et al. 2014) studied the effects of different concentrations of biochar prepared with wood chips on the bioreduction process of Fe(III) oxide, and found that higher concentrations (5 g/L, 10 g/L) of biochar can improve the bioavailability of Fe(III) oxide. The reduction efficiency of ferrihydrite, anthraquinone-2,6-disulfonic acid (AQDS) and biochar synergistically promoted the bioavailability of Fe(III). When the biochar concentration was lower than (0.5 g/L, 1 g/L), the Fe(III) biological reaction was inhibited. Moreover, Wu et al. studied the maximum reduction rate of ferrihydrite promoted by activated carbon, and found that it was as high as 0.127–0.434 mM/h, which was similar to the Fe(III) reduction rate promoted by biochar (0.12–1.49 mM/h). Also, they found that activated carbon (AC-N2, AC-N4) was more potent after pretreatment with nitric acid (Wu et al. 2017). However, the mechanism for the improvement in bioavailability of Fe(III) oxides by solid-phase carbon materials and the influencing factors need to be further explored.

Quinonoids are a class of substances with a quinoid structure, which are simple, diverse and widely found in the natural environment. They can be mainly classified into four types: benzoquinone, naphthoquinone, phenanthrenequinone and anthracene. Many researchers have used quinonoids as analogs or substitutes for humus to promote the bioavailability of Fe(III) oxides (Lovley et al. 1998; Wolf et al. 2009). Unlike HS, quinonoids do not exhibit properties such as complexation and adsorption of Fe(III) oxides (Royer et al. 2002; Piepenbrock et al. 2011; Masayuki et al. 2013; Eusterhues et al. 2014).

Under the action of sodium 2-sulfonate (AQS), the bioreduction efficiencies of goethite and hematite were significantly improved, and the content of Fe(II) in the reaction product was increased by 0.118 and 0.149 mg/L, respectively (Yu et al. 2016). In addition, AQS extended the service life of ferrihydrite, and the Fe(III) reduction rate was increased by 43.9% (Zang 2011). Low concentrations (10 μM) of quinonoids also promoted the efficiency of Fe(III) bioreduction. Due to the structural diversity of quinonoids, the current research is limited to several common quinonoids, whose results do not represent all quinonoids. Therefore, further studies on the reaction mechanisms are still needed.

Sulfur compounds are widely present in the environment and usually exist in the form of sulfides, sulfates or simple substances, which promote the bioavailability of Fe(III) oxides. At present, there are two explanations for the mechanism of action of sulfur compounds: (1) Iron-reducing bacteria reduce S₀ to sulfide. Then, sulfide reduces Fe(III) oxide and is simultaneously reoxidized to S₀ (Figure 2(I)) (Nevin & Lovley 2000); (2) Acidophilic bacteria can simultaneously produce S₀ oxidation and Fe(III) bioreduction under anaerobic conditions. Then, S₀ forms H₂S and SO₄²⁻ by disproportionation reaction, and one part of Fe(III) is reduced by acidophilic bacteria, while another part is indirectly reduced by H₂S (Figure 2(II)) (Osorio et al. 2013). In both mechanisms, Fe(III) oxide is not required to be in direct contact with the microorganism. Thereby, the rate of redox reaction can be increased.

Flynn et al. (Flynn et al. 2014) found that S₀ enhanced the bioavailability of Fe(III) oxide by simulating alkaline groundwater, and the Fe(III) reduction rate was increased by three times. Wang et al. (Wang et al. 2015) added S₀ to the cultures of S. oneidensis and G. sulfurreducens under alkaline conditions and found that the reduction rate of ferrihydrite was increased by 1.7 and 1.8 times, respectively. In addition, Wang et al. (Wang et al. 2015) found that the ability of AQDS to promote Fe(III) bioavailability was much higher than that of S₀. Therefore, in most natural environments, except for those with high sulfur content, HS and quinonoids are more capable of promoting Fe(III) reduction than sulfur species.

Wang et al. (Wang et al. 2017) used underground sediments as research objects and concluded that low-concentration sulfates have limited promotion effect, while high-concentration sulfates can significantly improve Fe(III) reduction efficiency. However, Hansel (Hansel et al. 2015) found that low concentrations of sulfate (200 μM) can also promote the bioavailability of goethite and hematite. The difference between the two results may be due to the different experimental conditions in the two studies. Eitel (Eitel & Taillefert 2017) used electrochemical methods and found that low molecular weight organic sulfur compounds such as cysteamine, cysteine, homocysteine and glutathione can improve the bioavailability of ferrihydrite. The degree of reduction was found to be proportional to the complexity of the sulfur species. In summary, both pH and concentration can affect the bioavailability of Fe(III) by sulfur compounds, and the promotion effect of low concentration sulfur compounds is limited, which is not conducive to their applications. At present, the mechanism of action is still unclear.

In addition to artificially added HS and quinonoids, microorganisms in the natural environment can secrete a variety of redox active substances in their own metabolic processes, which can form electron shuttles involved in extracellular electron transport to enhance Fe(III) oxide bioavailability. Wang and Newman (Wang & Newman 2008) considered that microbial-derived flavin mononucleotides (FMN) may play multiple roles in the reduction of Fe(III) oxides: (1) As a cofactor for cytochrome C (MtrC), FMN promotes the bioavailability of lepidocrocite, hematite and goethite; (2) FMN forms an electron shuttle system to promote Fe(III) bioreduction when reducing lepidocrocite; (3) FMN acts as an unreactive electron in the reduction of hematite and goethite.

The phenazine compound secreted by microorganisms has a lower redox potential than the weakly crystalline Fe(III) oxide and can reduce Fe(III) oxide to promote its bioavailability. Hernandez et al. (Hernandez et al. 2004) found that Pseudomonas chlororaphis PCL1391 isolated from the rhizosphere of plants can produce phenazine-1-carboxamide, which can form an electron shuttle to promote the bioavailability of weakly crystalline iron oxides. S. oneidensis secretes sulfhydryl compounds such as cysteine, homocysteine, glutathione and cysteamine, participates in the electron shuttle cycle, and promotes the bioavailability of Fe(III) oxide (Cooper 2016). Until now, there is no consensus on the mechanism by which microbial autocrine promotes the bioavailability of Fe(III) oxide and this area needs further study.

The promotion of Fe(III) bioavailability by electronic shuttle can accelerate the redox removal of environmental pollutants. At present, this strategy has been widely used in the treatment of trace metals, polycyclic aromatic hydrocarbons, azo dyes and other pollutants.

The promotion of Fe(III) bioavailability by electronic shuttles can affect the geochemical cycle of heavy metal elements. Han Rui (Han 2016) studied six different electron transport proteins as research objects, and found that cytochrome C enhanced the bioavailability of Fe(III). Cytochrome C improved the reduction rate of Cr(VI) by 66.7%, and played a significant role in controlling Cr(VI) in the environment.

The electron shuttle promotes the Fe(III) bioreaction process, which can degrade pollutants such as polycyclic aromatic hydrocarbons (Xu 2012; Zhou 2018). It was found that HS- and AQS-enhanced the Fe(III) bioreaction process increased the degradation rate of nitrobenzene by 10.3% and 1.2% (Xu 2012). Moreover, the AQS-enhanced Fe(III) bioreduction process promoted the degradation of 4-nitroacetophenone, and the removal rate of 4-nitroacetophenone was increased by 34% when AQS was added (Zang 2011). Another study found that the biochar prepared at 600 °C had the best effect on the degradation of pentachlorophenol (PCP) and could degrade 97.6% of PCP (Zhou 2018).

Azo dyes are widely used in the printing and dyeing industry. These dyes are difficult to biodegrade and cause significant pollution to the environment. The electron shuttle can promote the Fe(III) bioreaction to accelerate the decolorization of azo dye wastewater. The newly formed Fe(II) also has a flocculation effect to reduce the color of wastewater. The Fe(III) bioreduction process enhanced by exogenous riboflavin can promote the decolorization of cationic red X-GRL dye (Cationic Red x-GRL, X-GRL). When the concentration of riboflavin reached 5 mM, the reaction efficiency was increased by two times (Xiao et al. 2017). The Fe(III) bioreduction process enhanced by riboflavin also decolorized methyl orange (MO) and naphthol green B (NGB) dyes.

Table 2 details the impact of several electronic shuttles on the removal of environmental pollutants. It can be concluded that the effect of different electronic shuttles is quite different through comparison. When the pollutant is a heavy metal, the activated carbon will play a certain role in inhibition; when the pollutant is organic, the promotion effect of HA is better.

The introduction of redox active substances to construct an electronic shuttle between Fe(III) oxide and microorganisms not only enhances Fe(III) bioavailability and accelerates electron transfer, but also improves pollutant removal efficiency. However, the promoting effect and mechanism of action of redox active substances for enhancing the bioavailability of Fe(III) need to be studied further in terms of the following aspects: (1) analysis of the diversity of microbial species that can utilize electron shuttles; (2) exploration of the decisive factors for bioavailability of different forms of Fe(III) oxide under different environmental conditions, and studying each redox active material-mediated electronic shuttle cycle; (3) analyze the electron shuttle from the perspectives of thermodynamics, kinetics, chemistry, materials science, etc., and study the mechanism of action of the shuttle on improving the bioavailability of Fe(III) oxide; (4) Application of this strategy to actual contaminated water and soil samples.

This study was supported by the National Major Science and Technology Program for Water Pollution Control and Treatment, China (2018ZX07109-003) and Beijing Municipal Science and Technology Project, China (Z191100006919001).