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Guo, W., and Huang, J. (2023). "Environmental applications of immobilized and bio-resourced redox mediators: A Review," BioResources 18(1). Page numbers to be added.


Redox mediators (RMs), also known as electron shuttles, have been widely reported to promote both biotic and abiotic reductions of oxidized pollutants in water, soil, biogeochemical cycles, and wastewater treatment systems. However, the continuous addition of dissolved RMs is unaffordable and the potential environmental risks remain unknown because most applied RMs are synthetic chemicals. Immobilization technology enables RMs to be attached on non-dissolved supports, avoiding wash-out from the treatment systems. This realizes the reuse of RMs in scaled-up engineering applications and the in-situ remediation. Moreover, renewable natural biomass and their derivatives, such as biochar, have also aroused increased interest because they provide an economical and feasible way to solve the shortcomings of applying soluble RMs. This review presents different RM immobilization methods, which include entrapment, adsorption, and surface modification, as well as the use of bio-resourced RMs. The immobilization procedures and reaction mechanisms of the immobilized RMs and bio-resourced RMs in environmental applications are critically compared and summarized.

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Environmental Applications of Immobilized and Bio-Resourced Redox Mediators: A Review

Wenrui Guo,a and Jingang Huang b,c,*

Redox mediators (RMs), also known as electron shuttles, have been widely reported to promote both biotic and abiotic reductions of oxidized pollutants in water, soil, biogeochemical cycles, and wastewater treatment systems. However, the continuous addition of dissolved RMs is unaffordable and the potential environmental risks remain unknown because most applied RMs are synthetic chemicals. Immobilization technology enables RMs to be attached on non-dissolved supports, avoiding wash-out from the treatment systems. This realizes the reuse of RMs in scaled-up engineering applications and the in-situ remediation. Moreover, renewable natural biomass and their derivatives, such as biochar, have also aroused increased interest because they provide an economical and feasible way to solve the shortcomings of applying soluble RMs. This review presents different RM immobilization methods, which include entrapment, adsorption, and surface modification, as well as the use of bio-resourced RMs. The immobilization procedures and reaction mechanisms of the immobilized RMs and bio-resourced RMs in environmental applications are critically compared and summarized.

DOI: 10.15376/biores.18.1.Guo

Keywords: Immobilization; Redox mediator; Entrapment; Surface modification; Biomass

Contact information: a: PowerChina Huadong Engineering Corporation Limited, Hangzhou 311122, PR China; b: College of Materials and Environmental Science, Hangzhou Dianzi University, Hangzhou 310018, PR China; c: The Belt and Road Information Research Institute, Hangzhou Dianzi University, Hangzhou 310018, PR China; *Corresponding author:


In recent decades, the global environment has been challenged by serious pollution caused by synthetic chemicals, which are often recalcitrant, biorefractory, and carcinogenic. These contaminants include but are not limited to nitroaromatic compounds (NACs), azo dyes, nitrate, perchlorate, sulfate, halogenated compounds, polyhalogenated pollutants, hexavalent chromium (Cr(VI)), persistent organic pollutants, and so on. Because of the electron-withdrawing features of special groups associated with these oxidized pollutants, aerobic conditions usually fail to treat them (van der Zee and Cervantes 2009; Zhang et al. 2020). However, they can be moderately reduced under anaerobic conditions when electron donors are available, whereupon they can be readily removed aerobically. Thus, combined anaerobic-aerobic processes are typically applied to treat oxidized pollutants or concerned substances. In such approaches, the anaerobic reduction is the rate-limiting step and the bottleneck part of the process.

Redox mediators (RMs) are vital important compounds capable of accelerating redox reactions by shuttling electrons between their reduced and oxidized forms, as well as of lowering the activation energy for redox reactions. Within this catalytic process, the electrons from the primary electron donors would be quickly transferred to the final electron acceptors (Olivo-Alanis et al. 2018; Li et al. 2022a). They have been widely applied in the oxidation of H2, alcohols, biomass, cellulose, and so on (Anson and Stahl 2020; Chen et al. 2022). To promote the reductive transformation of oxidized pollutants, the use of RMs has also been extensively reported for both biotic and abiotic reduction of oxidized pollutants in water, soil, biogeochemical cycles, and wastewater treatment systems (Rau et al. 2002; O’Loughlin 2008; Uchimiya and Stone 2009; Song et al. 2021). Currently, the most frequently applied RMs consist of flavin-based compounds and quinone-based compounds. Flavin-based compounds are those such as flavin adenine dinucleotide, flavin mononucleotide, and riboflavin; quinone-based compounds include, among others, anthraquinone-2,6-disulfonate (AQDS), anthraquinone-2-sulfonate (AQS), juglone, lawsone, and natural organic matters (NOM ), such as humic acids (HA). Besides, 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) is another commercially available RM by the TEMPO/TEMPOH redox cycle (Zhan et al. 2019; Sun et al. 2022; Zhang et al. 2021). The chemical structure of three typical RMs and the conversion between their oxidized and reduced forms are shown in Fig. 1 (Buckel and Thauer 2018; Zhang et al. 2021).

Fig. 1. The chemical structure and conversion cycle of flavin-based (a), quinone-based (b) and TEMPO (c) RMs

The RMs have been extensively reported to accelerate biological/chemical reduction of oxidized pollutants in various bioreactors and further prevent the toxic inhibition of them to the biological processes (van der Zee et al. 2001; Liu et al. 2009; Costa et al. 2010; Saratale et al. 2011; Mani et al. 2018). In these processes, RMs could speed up the electron transferring from the electron donors – carbohydrate, protein, volatile fatty acids, alcohol, hydrogen, (biogenic)sulfide, zero valent iron and ferrous iron, nicotinamide adenine dinucleotide phosphate hydrate, and electrical power – to the acceptors of oxidized contaminants, increasing the reduction rates by several folds or even dozens. Mixed cultures of microorganisms (such as activated sludge) as well as pure strains have been reported to contribute to the above mediated processes (Borch et al. 2005; dos Santos et al. 2005; Bhushan et al. 2006; Kwon and Finneran 2006, 2008; Bibi et al. 2019; Liu et al. 2021; Ren et al. 2022). Moreover, TEMPO-mediated decolorization, cellulose oxidation, photocatalysis and electroreduction have also been reported in the literature, implying the electron shuttling role of TEMPO/TEMPOH couple (Gharehkhani et al. 2019; Wen et al. 2019; Lukyanov et al. 2022).

In practical applications, the continuous addition of soluble RMs has been unaffordable because of their washout from the treatment systems. Moreover, most applied RMs are synthetic chemicals, for which the potential environmental and health risks are still unknown. Thus, urgent ways are needed for avoiding or mitigating the loss of soluble RMs (Zhou et al. 2012; Zhang et al. 2020). These methods include decreasing the soluble RM dosage, immobilizing RMs on special materials, and/or utilizing economical and naturally available alternatives. The RM immobilization enables soluble RMs to be attached to non-dissolved and inert carriers that fulfill the electron shuttling role in the redox process. This technology has aroused the most interest because it provides an economical and feasible way to solve the shortcomings of applying soluble RMs in continuously operating reactors. The abundance of target pollutants followed the order: azo compounds (52%) > polyhalogenated pollutants (15%) > NACs (13%) > nitrate and/or nitrite (12%) > Fe (4%) and Cr(VI) (3%) (Dai et al. 2016). Furthermore, some natural biomass or their derivatives, such as activated carbon, biochar, and natural humic substance (HS) are also capable of shuttling electrons during the redox reactions, providing cost-saving alternatives in the future. However, the direct use of natural biomass as RM would bring problems such as the release of unwanted organic compounds, which might increase secondary pollution.

The immobilization of soluble RMs is a complex process, depending on the physical-chemical features of both RMs and solid carriers. Thus, the selection of immobilization methods for specific chemical RMs has always been challenging. To guide the future applications of RMs in the environmental field, it is necessary to summarize different RM immobilization methods, focusing on immobilization procedures, and further prospects; in addition, natural and bio-resourced RMs are also proposed because of their economical and eco-friendly advantages. This review paper attempts to (1) summarize different RM immobilization methods (procedures and mechanisms) such as entrapment, adsorption, and surface modification, (2) investigate the catalytic effect and bottlenecks of various immobilized RMs in environmental applications, and (3) select bio-resourced RMs to speed up the bioredox processes for pollutants the removal.


Entrapment-type Immobilization

In biological systems, entrapment has been traditionally considered for the physical retention or fixation of microorganisms and/or enzymes by polymeric carriers such as alginate, nanomaterials, silica gel, polyacrylamide, gelatin, polyion complex, and so on (Moyo et al. 2012; Romero-Soto et al. 2021). This method keeps the suspended cells and/or enzymes trapped in a porous solid matrix and thus condenses them, preventing their wash-out or loss from environmental systems or developing enzyme electrodes/biosensors for bioelectrochemical systems (Sakurada et al. 2017). Therefore, immobilization using polymeric materials as carriers of bioactive components can improve the efficiency and stability of microorganisms compared to that with free cells and/or enzymes, thus providing a promising technology to mitigate the pollutants in the environment (Fernández-Fernández et al. 2013; Somu et al. 2022).

The RMs, like microorganisms and enzymes, also can be non-covalently entrapped in different porous polymeric substances. The reported entrapmental immobilization methods of RMs and their applications in the reductive biotransformation of oxidized pollutants and bioelectrocatalysis capability are listed in Table 1. It indicates that the entrapmental immobilization is quite simple and the operating conditions are relatively moderate, meaning that high temperature and/or high pressure are not required during the redox reaction. Calcium alginate (CA), polyvinyl alcohol (PVA), and agar gel are the popularly used polymer supports for RMs entrapment because of their relatively easy procedures and low costs (Guo et al. 2007). The RMs entrapped on CA/PVA beads have been used in the bio-decolorization and denitrification processes. In these RM-assisted systems, the adsorption of pollutants by CA and PVA polymer beads only accounted for 0.2% to 2.0% of the total removal, indicating that the main removal pathways were via bio-reduction. Under the electron-shuttling assistance of polymer-trapped RMs, the removal of azo dyes and nitrate would be promoted to some extent with yeast extract, peptone, acetate, and glucose as electron donors (Liu et al. 2012b). Additionally, silica-entrapped TEMPO is an efficient heterogeneous catalyst and is more environmental-friendly and less tedious during immobilization, attracting attentions on the selective oxidation of cellulose, glucoside, and alcohols (Palmisano et al. 2006; Chen et al. 2022).

Table 1. Entrapment-type Immobilization of RMs and Their Applications

Although presents entrapment has the advantages in immobilizing RMs, it would wrap the selected RM groups into the the polymer matrix. This means that the substrates, including electron donors and acceptors, must pass through the porous matrix to reach active RM groups and react with them, thereby limiting the electron-transferal during the bio-reduction process. Previous studies indicated that the RMs immobilized by entrapment are only capable of increasing the bio-decolorization/bio-denitrification rates up to two-fold compared to rates in the control systems (Guo et al. 2007, 2010; Liu et al. 2012b). In contrast, the RM polymer matrix might be disrupted because of the weakening mechanical strength of CA/PVA/Sol-gel beads after the long operation, resulting in the blockage of the matrix pores. To maintain the effective entrapment of soluble RMs on a more durable polymeric matrix than CA and PVA, hydrophobic RMs, such as anthraquinone, AQS, and 1,5-dichloroanthraquinone are preferred. Feng et al. (2017) developed a “foldable” but strengthened RM, i.e., AQSA-Na (anthraquinone-2-sulfonic acid sodium salt)-doped PVA-H2SO4 robust gel film electrolyte, and suggested a great contribution of loaded RMs to the outstanding electron-transferring capability. This advance enabled the fabrication of smarter bio-electrochemical systems for the continuous removal of oxidized pollutants.

For the future application of RM entrapment as a technique to enhance pollutants removal, the mechanical strength of the polymer matrix and the diffusion rates of soluble substrates must be improved. To solve these problems, the co-entrapment of RMs and microorganism/enzymes seems promising, as this approach provides a better electron/mass transferring condition between microbial cells and entrapped RMs, thereby stably accelerating the reductive transformation of oxidized pollutants. Su et al. (2009) reported that the co-entrapment of RMs and microorganisms in a polymer matrix achieved better decolorization performance than was achieved with mono-immobilization of only RMs or microorganisms. Repeated decolorization batch experiments using co-entrapment systems showed that the RMs were reusable after several operating batch cycles (Su et al. 2009; Sharma et al. 2016).

Adsorptive Immobilization

Adsorption is another frequently employed RM immobilization method (Dai et al. 2016). After adsorption of soluble RMs into the surface of solid adsorbents, the electron-shuttling groups can be concentrated, enabling their reutilization during the redox reaction of oxidized pollutants. The adsorptive types by which soluble RMs are incorporated into special adsorbents include physical and chemical pathways. Physical adsorption is governed by the van der Waals intermolecular force, which weakly binds the soluble RMs with the adsorbent’s surface and the adsorption was even reversible (Wang et al. 2010). Thus, physically adsorped RMs can be easily detached from their carrier. For a wastewater treatment system as an example, a fluctuating influent can easily wash off physically immobilized RMs from the adsorbents. However, chemically adsorbed RMs are co-joined by chemical bonds, such as covalent bonds and hydrogen bonds, which are much stronger and more stable than physical force. Consequently, chemical-adsorptive RM immobilization has been widely used, depending on the linked functional groups (hydrophilicity/hydrophobicity) and/or charges (positive/negative) between adsorbents and RMs (Cervantes et al. 2010). As indicated in recent reports, ceramsite-based products, anion exchange resin, cellulose acetate, activated carbon (AC), polyurethane foam, porous silica beads, and metal-oxides nanoparticles are typically used as the potential adsorbents to immobilize different soluble RMs (Table 2).

These above immobilization procedures are relatively easy, but in some cases they are costly due to the usage of some advanced adsorptive materials such as nanoparticles or metal-organic frameworks (Li et al. 2017; Lou et al. 2023). The adsorption performance of an adsorbent to immobilize RMs is usually determined by adsorption isotherms modeling, the result of which provides proper guidance to maximize the electron-transferring capacity. Functional groups of chemical-adsorptive immobilized RMs are tightly attached to the adsorbent’s surface. This leads to much better mass/electron transferal capability as compared with entrapmental immobilized RMs, which was inside the polymer matrix (Dai et al. 2016). Thus, the pollutant removal rates achieved using adsorptive immobilized RMs (maximally 10.4-fold) are much higher than that achieved using entrapped immobilization RMs (maximally 2.1-fold). Furthermore, benefitted by immobilized RMs, an anaerobic consortium can even utilize toxic substrates (such as phenol) as the sole energy source (electron donor) (Martínez et al. 2013).

Surface modification could potentially improve the adsorption capacity, enabling RMs to be immobilized as designed. Yuan et al. (2012) produced organic–inorganic hybrid materials using adsorption/covalence coupling methods. With OH-ceramsite and NH2-ceramsite as a base, the sulfonic acid group associated in AQS can covalently bind with the -OH and -NH2 bonds to form AQS-ceramsite. The grafted AQS on ceramsite has been shown to effectively catalyze the bio-decolorization process in a high salt environment. Furthermore, Fe3O4-quinone/TEMPO nanocomposites have also been identified as a promising RM complex because they can be easily and quickly separated from the mixed liquor with the assistance of a magnetic field; once separated, they can then be reused. Zhang and Hu (2017) introduced nano-scale Fe3O4 as an adsorbent to immobilize quinone-based RMs, achieving efficient and stable bio-reduction of NACs during multiple use cycles. Gao et al. (2018) used amino-functionalized magnetic Fe3O4 nanoparticles as a support to develop a co-immobilize TEMPO/laccase complex to remove acid fuchsin, and 50% residual activity was retained after eight cycles of operation.

Table 2. Adsorptive Immobilization of RMs and Their Applications

Solid Carbon Materials and their Modification

Solid carbon-based materials can be derived from carbon-enriched substances including coal, petcoke, resin, and agricultural/forestry residues. The commercially available carbon materials include powdered activated carbon, activated carbon felt, carbon paper, carbon nanotubes, graphene oxide (GO), graphite electrodes, and so on. These carbon materials were associated with functional groups of carboxyl, quinone, carbonyl, lactone, hydroxyl, and carboxylic anhydride in their surface, enhancing the reductive biotransformation of various oxidized pollutants by improving electron shuttling ability (van der Zee et al. 2003; Pereira et al. 2014; Colunga et al. 2015; Amezquita-Garcia et al. 2016). However, the density of these active groups of solid carbon materials is at a relatively low level, limiting the electron-shuttling rates during the pollutant removal processes. Thus, carbon materials can slightly enhance pollutant removal (maximally 3.7-fold over the control) in chemical and/or biological decolorization, denitrification, anammox, and NAC reduction processes. Although GO and reduced graphene oxide (rGO), with proper size distribution, unique spatial structure, and better redox potential, lead to a higher redox conversion of oxidized pollutant (up to 10-fold), they are costly for full-scale applications (Yin et al. 2015; Colunga et al. 2015; Li et al. 2016b).

In consideration of the above drawbacks, researchers have attempted to selectively modify the surface chemistry of carbon materials to promote the richness of desired groups and thereby develop the electron-transferal capacity for contaminants removal (Li et al. 2016a). Thermal (heat), chemical (acidic and alkaline/basic treatments), electrochemical, and biological (bio-adsorption) modification methods are typically used to change the surface characteristics of carbon materials, depending on their group features (acidic, basic, and/or neutral) (Yin et al. 2007). Research studies regarding different modification methods to fix RMs are briefly summarized in Table 3. For example, Pereira et al. (2010) compared chemical oxidation (with HNO3 and O2) and thermal treatments (in an H2/N2 atmosphere) in modifying the surface chemistry of commercial AC, suggesting that chemical modification introduced more quinone groups than did thermal treatments. In this case, the first-order reduction rate constants of azo dyes increased by 9.0-fold compared with the control treatment. Further, a two-step procedure consisting of amination and the Buchwald–Hartwig reaction was also developed to immobilize quinone-based RMs, efficiently mediating the reductive removal of azo dyes, nitrate, and NACs (Zhang et al. 2014b; Xu et al. 2015).

Table 3. Immobilization of RMs by Surface Modification and Their Applications

In (bio)electrochemical systems, electrodeposition, adsorption, and crossing-linking have been successfully applied to prepare RM-modified electrodes for use in MFC and microbial electrolysis cell (MEC) systems; which in turn benefit the electron-transferal during redox reactions, enhancing the bioelectricity generation, pollutant removal, and energy storage (Table 3). In addition, electropolymerization has been used to increase the required density of electron shuttling groups on the surface of carbon materials or functional composites. This method is usually a coating procedure to enable a conducting polymer to be fixed or deposited into a conducting material from a solution, which was previously used in the enzyme immobilization (such as encapsulating glucose oxidase electrochemical biosensors) by choosing a defined electrical potential and current (Chiorcea-Paquim et al. 2008). When applying the electropolymerization to immobilize RMs on an electrode, attempts have been made to coat polymer materials, such as polypyrrole and pyrrole, on a carbon electrode by doping designed RMs. This polymer/RMs composite film has a vast surface area and pseudo-capacitance, which has aroused great interest in its application in electricity-assisted or electricity-produced systems (Lang et al. 2010; Anwer et al. 2021). Regarding the interest in RMs to enhance pollutant removal, this composite film can also promote the functional group density and prevent the wash-out of RMs from systems. Compared to the entrapped AQDS/PVA particles, AQDS/electropolymerization-modified anode in an MFC system shows better performance in both electricity generation and pollutants removal (Martinez et al. 2017).

Natural and Bio-resourced Redox Mediator

The previously mentioned RM immobilization technology has enabled the separation of RM retention time from hydraulic retention time in environmental applications. However, the complex procedures of these immobilization processes, the time-consuming lab work to select suitable process conditions and unaffordable carriers always limited their wide applications (Rocha-Martín et al. 2021; Wang et al. 2021). To overcome these raised issues, data-driven approaches such as machine learning algorithms have been attempted to predict and optimize those complex procedures and to obtain feasible immobilization conditions (Shi et al. 2022). Moreover, some natural biomass and/or their derivatives such as biochar would be expected to be good and low-cost alternatives because of the renewable raw materials. The reported uses of these materials, i.e., natural biomass and bio-resourced derivates in facilitating pollutant removal are shown in Tables 4 and 5, respectively.

Henna plant (Lawsonia inermis) biomass (HPB) has been reported to be a natural source of lawsone (an effective RM), with lawsone comprising up to 1.8% of the plant’s dry weight (Almeida et al. 2012). Previous studies indicated that natural HPB, including stem, leaf, flower, and seed, could serve as both an electron donor and RM source to improve the bio-reduction of azo dyes and Cr(VI) in batch and continuous bioreactors, all showing positive effects of HPB on pollutants removal (Huang et al. 2014, 2015, 2016a 2021; Tang et al. 2016; Rau et al. 2002). Moreover, HPB could be co-fermented with waste sludge in municipal wastewater treatment plants and enhance the production of VFAs (Huang et al. 2016b). As a protein-rich biomass, waste sludge could be firstly hydrolyzed to amino acid, and then converted to carboxylic acid and NH4+-N by the Stickland redox reaction (Huang et al. 2019). The associated RM of lawsone in HPB could speed up the transfer of electron from one amino acid to another, resulting in higher VFAs production. Moreover, another waste biomass of Punica granatum peal was also approved as a natural RM to efficiently degrade orange G dye (Bibi et al. 2019). In contrast, some extractions from plant biomass or algae, such as chlorophyll, laccase, syringaldehyde, and acetosyringone, were also reported to serve as natural RMs for enhanced denitrification, decolorization, and electricity generation (Ma et al. 2017; Mani et al. 2018; Lu et al. 2020; Song et al. 2021). Furthermore, a novel biomass-derived lignin with electron transfer 3D networks was developed to act as cathode interlayer, achieving high-efficient performance of solar cells (Hu et al. 2020). However, raw plant biomass or its extractions without proper treatment can cause problems such as secondary pollutants (e.g., organic substances lost from biomass as well as wash-out of associated RMs). To address these problems in engineering applications, researchers have suggested that natural biomass could be prepared as biochar to retain or modify most of the active electron transferral groups.

Table 4. Natural Biomass Acting as Redox Mediator