Effect of flavin compounds on uranium(VI) reduction- kinetic study using electrochemical methods with UV-vis spectroscopy

Highlights

• The rate constant for the U(VI) reduction by flavin analogues was determined.

• The flavins showed a mediator effect on the U(VI) reduction.

• The logarithmic rate constants for the U(VI) reduction was proportional to redox potential of the mediator.

• The presence of the mediator increased about 0.2 V apparent redox potential of U(VI) to U(IV).

Abstract

The reduction of uranium hexavalent (U(VI)) to tetravalent (U(IV)) is an important reaction because of the change in its mobility in the natural environment. Although the flavin mononucleotide (FMN) has acted as an electron shuttle for the U(VI) reduction in vivo system, which is called an electron mediator, only the rate constant for the electron transfer from FMN to U(VI) has been determined. This study examined the rate constant for the U(VI) reduction process by three flavin analogues (riboflavin, flavin mononucleotide, flavin adenine dinucleotide) to elucidate their substituent group effect on the U(VI) reduction rate by electrochemical methods. The formation of the U(IV) was monitored by UV-vis spectrometry at 660 nm during the constant potential electrolysis of the U(VI) solution in the presence of the mediator. The cyclic voltammograms indicated that the three flavin analogues behaved as electron mediator to reduce U(VI). The logarithmic rate constant for the U(VI) reduction was related to the standard redox potential of the mediators. This linear relationship indicated that the redox-active group of the mediator and the substituent group of the mediator dominate capability of the U(VI) reduction and its rate, respectively. The apparent reduction potential of U(VI) increased about 0.2 V in the presence of the mediators, which strongly suggests that the biological electron mediator makes the U(VI) reduction possible even under more oxidative conditions.

Introduction

The bioreduction of U(VI) has been studied in terms of radioactive waste management and a better understanding of the migration process of uranium in anaerobic subsurface environments (Lovley et al., 1991, Lovley and Phillips, 1992). In the absence of a complexing agent, such as organic acids, the solubility change from the soluble U(VI) to the insoluble U(IV), UO₂, causes a significant impact on its mobility in the environment. Bioreduction has been considered a potent process of the U(VI) immobilization in addition to the physicochemical adsorption on the cell surface (biosorption (Suzuki and Banfield, 1999)), the formation of insoluble precipitates, such as phosphate (biomineralization (Macaskie et al., 1992, Ohnuki et al., 2005, Mondani et al., 2011)) and transport of some minerals into the cell body, which has a chemical behavior similar to essential elements in the cell (bioaccumulation (Suzuki and Banfield, 2004)). Most living organisms obtain energy by decomposing hydrocarbons during respiration. Because this energy formation process is an oxidation reaction, an electron acceptor is required to allow this reaction. For example, aerobic microorganisms use oxygen as an electron acceptor, whereas anaerobic microorganisms use ferric ions, sulfate ions (Plugge et al., 2011), etc. In the absence of oxygen, that is, an anaerobic condition, U(VI) can also be an electron acceptor. Some shewanella species, which are some of the iron reducing bacteria, have displayed the U(VI) reduction (Cherkouk et al., 2016, Sheng and Fein, 2014). The c-type cytochromes are the redox-active membrane protein immobilized in the membrane. It has been reported that several c-type cytochromes (cytochrome c, cytochrome c₃ and cytochrome c₅₅₃) show a fast enzymatic redox reaction (Bianco and Haradjian, 1994). Because of their fast electrochemical reaction, the c-type cytochromes are able to act as an electron shuttle from microorganisms to U(VI). According to the literature (Lojou and Bianco, 1999), the kinetics of the electron transfer reaction from cytochrome c₃ to U(VI) has also been elucidated using an electrochemical in vitro system technique, and the rate constant for the U(VI) reduction by cytochrome c₃ was determined to be 9000 M⁻¹ s⁻¹. The c-type cytochromes are observed in sulfate reducing bacteria (Lojou and Bianco, 1999) and act as electron transfer chains, in addition, their kinetics was very fast. These results of previous studies indicated that the c-type cytochromes play an important role in the biological U(VI) reduction process. However, the c-type cytochrome reduction is limited to the membrane surface.

On the other hand, the extracellar electron transfer pathway from the biological electron mediator, which is a redox-active molecule, to U(VI) has recently been suggested (Brutinel and Gralnick, 2012). The organic molecules acted as electron shuttles between the bacteria and U(VI). For example, the mutant Desulfovibrio desulfuricans, which lacks the cytochrome c₃, can reduce U(VI) in the presence of lactate as an electron donor (Payne et al., 2002). The effect of riboflavin on the U(VI) reduction by Shewanella oneidensis MR-1 has also been investigated in a previous study (Cherkouk et al., 2016). In addition to the flavin analogues, 9,10-anthraquinone 2,6-disulfonate (2,6-AQDS), which has been used as a humic substance analogue, also showed acceleration of the U(VI) reduction in the presence of microorganisms (Gu et al., 2005). When Fe(III) exists in the system, the U(VI) reduction resulting from the 2,6-AQDS electron shuttle, was not observed because the kinetics of the U(VI) reduction was slower than that of the Fe(III) reduction (Finneran et al., 2002). Marsili et al. (2008) reported that the flavin derivatives, which can be secreted by Shewanella, mediate the electron transfer by using a biofilm. This examination showed the possibility of an electron shuttle in the natural environment. These reports have indicated that the extracellular soluble redox-active molecules and the molecules secreted from bacteria are potential mediators of the U(VI) reduction process in some cases. Therefore, understanding of the kinetics of the U(VI) reduction is required in order to estimate the uranium migration process in the natural environment. However, the details about the electron transfer reaction from a biological mediator to U(VI) have not been fully understood. A direct electron transfer pathway for the U(VI) reduction mediated by the flavin mononucleotide (FMN), which is secreted by Shewanella putrifaciens, has been reported by Suzuki et al. (2010). They showed that FMN may act as a mediator and accelerate the reduction of U(VI) to U(IV). To date, an electrochemical approach to understanding the electron transfer from the mediator to U(VI) has been limited to the FMN in vitro system.

As already described, some microbial species can potentially release redox-active compounds, such as FMN (Canstein et al., 2008, Marsili et al., 2008). These compounds can be used as electron shuttles. The experiment using microorganisms is very complex because there are many biological effects such as a release from phosphate to form a precipitate of uranyl phosphate (Murakami et al., 2005). Therefore, an electrochemical method is proposed to quantitatively handle the amount consumed of the reduced mediator by reducing the U(VI) by an electric current compared to the in vivo system. Fig. 1 shows a schematic illustration of this study. As shown in Fig. 1, the formation of the reduced mediator inside the microorganisms (Fig. 1(a)), which results from respiration, was simulated by the reduction of the mediator on the electrode surface (Fig. 1(b)). The more the U(VI) concentration increases, the more reduced mediator is consumed by the electron transfer reaction as shown in Fig. 1(b). The enhancement of the oxidized mediator generation by the U(VI) reduction results in amplification of the cathodic current. This relationship was used for the determination of the rate constant of the U(VI) reduction. The theoretical background of this analysis is described in section 3.2 of the results and discussion.

In the present study, the electrochemically controlled U(VI) reduction by biological mediators was investigated in order to examine the effect of the FMN analogues riboflavin (RF), which is vitamin B2, flavin adenine dinucleotide (FAD), which is a coenzyme to control the biological redox reaction and flavin mononucleotide (FMN), which is also a coenzyme, on the U(VI) reduction kinetics and to investigate the substituent effect on the U(VI) reduction rate constant. The molecular structures of the compounds are shown in the supplement (Fig. S1). Cyclic voltammetry was used to determine the rate constant for the U(VI) reduction by the mediators, and UV-vis spectrometry together with constant potential electrolysis was performed for the direct observation of the U(IV) formation. The basic concept of cyclic voltammetry is described in the supplemental information, and the literature should be investigated for a better understanding of the electrochemistry (Bard and Faulkner, 2000). Our objective is to determine other electron mediators for the U(VI) reduction and to examine the substituent effect of flavins on the U(VI) reduction kinetics.