Electron transfer via the non-Mtr respiratory pathway from Shewanella putrefaciens CN-32 for methyl orange bioreduction
Graphical abstract
Introduction
Bioelectrochemical systems (BESs) are new routes to combat the over-reliance on fossil fuel based electricity and the promising remediation methods to the contaminated environment [1]. Exoelectrogens are defined as core genera in the BESs and play the core roles in energy recovery and environmental remediation [2]. One of the promising research issues on exoelectrogens is to gain a clearer understanding of their abilities for extracellular electron transfer to different electron acceptors, which is important for pushing forward the advancement of the development of BESs [[1], [2], [3]].
Azo dyes are recalcitrant pollutants commonly found in wastewater from the textile industry [[4], [5], [6]]. Discharge of these effluents into environment poses a serious threat to public health [5]. As a model azo dye, methyl orange (MO) is widely used in textiles, chemistry and paper industries. However, it is also known as water pollutant and a potent human carcinogen [5]. A wide variety of physico-chemical methods such as coagulation and chemical flocculation, filtration, adsorption, and advanced oxidation, have been adopted for MO reduction [7,8]. These methods are effective, but suffer from high costs. Bioremediation of wastewater containing MO has attracted much interest because of its eco-friendliness and low cost [[9], [10], [11], [12]]. The existence of electron withdrawing groups in MO makes it resistant to biodegradation. However, once MO is reduced by anaerobes, its reduction products are easily degraded by aerobes [[13], [14], [15], [16]]. Therefore, anaerobic bio-reduction is the basic prerequisite for complete degradation of MO [14].
A large diversity of bacteria and archaea such as lactic acid bacteria isolated from human intestine, Bacteroides sp., Clostridium sp., Eubacterium sp., Streptococcus faecalis, and Proteus vulgaris, were reported to reduce MO under anaerobic conditions [14]. Among these, exoelectrogens possessed high reduction efficiency due to their diverse respiratory capacities [[17], [18], [19], [20]]. Several Shewanella strains like Shewanella oneidensis MR-1, Shewanella oneidensis WL-7, Shewanella aquimarina, Shewanella algae and Shewanella decolorationis S-12, could couple the oxidation of electron donors to reduction of MO to sustain cellular growth [17,18,21]. Kinetic results showed that Geobacter sulfurreducens PCA had the highest reduction efficiency for MO among the currently known exoelectrogens [22]. Major advances had been made in our understanding of the anaerobic bioreduction mechanism of MO by exoelectrogens [23,24]. A gene encoding azoreductase was found in the genome of S. oneidensis MR-1. This purified azoreductase showed highest specific activity (153.16 U/mg) for MO at pH 6.5 and preferred for nicotinamide adenine dinucleotide (NADH) as electron donor [25]. The enzyme activities of NADH–dichloro phenol indophenol (NADH–DCIP) reductase were 2.67 times higher in the MO treatment as compared to the control treatment [26]. Moreover, the block of the Mtr (metal-reducing) respiratory pathway in S. oneidensis MR-1 resulted in a decrease of reduction rate by 80%, which demonstrated an extracellular process was mainly responsible for the anaerobic reduction of MO by S. oneidensis MR-1 [17,21]. Two outer membrane c-type cytochromes MtrC and OmcA were identified as the important reductive terminals for MO in S. decolorationis S-12 [27]. Our recent findings suggested that the bioreduction of MO by G. sulfurreducens PCA was an exclusive extracellular process [22]. Given the impressive diversity of physiology, genetics and genomics in exoelectrogens, reduction of MO by exoelectrogens might also occur via some unknown mechanisms.
Shewanella putrefaciens inhabited a wide range of terrestrial and marine environments [28,29]. S. putrefaciens strain AS96 could decolorize 100 mg/L azo dyes at salt concentrations up to 60 g/L under static and low oxygen conditions [30]. S. putrefaciens strain CCT1967 showed a high efficiency in reduction of seven azo dyes at pH 8.5 [31]. The resting cells of S. putrefaciens strain B-3-1 could reduce Direct red 81 with lactic acid as the electron donor under anaerobic conditions and c-type cytochromes involved in such a reduction process [32]. As a model strain of S. putrefaciens, S. putrefaciens CN-32 showed a considerable potential for the remediation of environments contaminated by organoarsenical or nitrobenzene [[33], [34], [35]]. S. putrefaciens CN-32 could also decolorize azo dyes under anaerobic conditions [36]. Dimethyl sulphoxide, nitrate, nitrite, and oxygen could act as competitive electron acceptors to inhibit the reduction efficiency of azo dyes. Genome analysis showed the azoreductase gene (Sputcn32_0423), the laccase gene (Sputcn32_1023), and a locus that encoded the mtr-like gene cluster (mtrA, Sputcn32_1477; mtrB, Sputcn32_1476; mtrC, Sputcn32_1478; undA, Sputcn32_1479) existed in the genome of S. putrefaciens CN-32. Moreover, S. putrefaciens CN-32 also secreted flavins acting as electron mediators to accelerate electron transfer between cells surfaces to extracellular electron acceptors such as lepidocrocite, electrodes, organoarsenical, and nitrobenzene [37]. Nevertheless, so far, the exact mechanism of reduction of azo dyes such as MO by S. putrefaciens CN-32 is not clearly understood yet.
Therefore, this work aims to explore the reduction mechanism of MO by S. putrefaciens CN-32 at molecular level. For this purpose, the reduction products were first identified. Next, the effects of the initial cell density and mediators on MO reduction by S. putrefaciens CN-32 were also investigated. Furthermore, the kinetic parameters of MO reduction by different Shewanella mutants were calculated and the roles of azoreductase, laccase, and the Mtr respiratory pathway in the MO reduction were evaluated. Finally, the reduction mechanism of MO by S. putrefaciens CN-32 was further analyzed via transposon mutagenesis.
Section snippets
Bacteria strains and MO reduction tests
Escherichia coli WM3064 was cultured in Luria-Broth (LB) medium supplemented with 50 μg/mL diaminopimelic acid at 37 °C (Table 1). S. putrefaciens CN32 and its mutants were inoculated from frozen stocks and activated in LB at 30 °C (Table 1). MO reduction experiments were conducted as follows: Shewanella strains were cultivated in an anaerobic minimal salt (MS) medium (pH 7.2), which contained 11.91 g/L 4-(2-hydroxyerhyl) piperazine-1-erhanesulfonic acid, 0.46 g/L NaCl, 0.225 g/L NH4Cl,
Reduction products of MO by S. putrefaciens CN-32
MO could absorb visible light and its absorption spectrum showed a peak at 465 nm (azo-bond) [17]. When S. putrefaciens CN-32 was added into the anoxic reaction mixture of serum vials with lactic acid and MO as the sole electron donor and acceptor, respectively, we observed a dramatic decrease in intensity of 465 nm, concomitant with the increase in the intensity of 245 nm (Fig. 1a). This result suggested the conjugated azo-bond in the MO was cleaved and at least one of the reduction products
Conclusions
S. putrefaciens CN-32 could efficiently decolorize MO under anaerobic conditions. MO reduction by S. putrefaciens CN-32 occurred via the cleavage of azo bond and the major products of MO reduction by all strains were identified as 4-aminobenzenesulfonic acid and N,N-dimethylbenzene-1,4-diamine. The initial cell density and mediators affected MO reduction kinetics by S. putrefaciens CN-32. We unexpectedly found that S. putrefaciens CN-32 adopted a distinctive electron transfer mechanism for
CRediT authorship contribution statement
Di Min: Conceptualization, Formal analysis, Writing - original draft. Lei Cheng: Methodology, Formal analysis. Dong-Feng Liu: Conceptualization, Methodology, Writing - review & editing. Wen-Wei Li: Formal analysis. Han-Qing Yu: Writing - review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
The authors wish to thank the National Key Research and Development Program of China (2019YFA0905503, 2018YFA0901301, and 2018YFC0406303), the National Natural Science Foundation of China (51878638, 21607146, 21590812 and 51821006), the Scientific Research Grant of Hefei National Synchrotron Radiation Laboratory (UN2017LHJJ), and the Program for Changjiang Scholars and Innovative Research Team in University of the Ministry of Education of China for supporting this work.
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