Letter pubs.acs.org/journal/estlcu
Rapid Release of Arsenite from Roxarsone Bioreduction by Exoelectrogenic Bacteria Jun-Cheng Han, Feng Zhang, Lei Cheng, Yang Mu, Dong-Feng Liu,* Wen-Wei Li, and Han-Qing Yu CAS Key Laboratory of Urban Pollutant Conversion, Department of Chemistry, University of Science and Technology of China, Hefei 230026, China S Supporting Information *
ABSTRACT: The extensive use of roxarsone in the poultry and livestock industry has led to increasing arsenic contamination of soil and aquatic environments. Microbial activity, especially exoelectrogenic bacterium (EEB)-mediated roxarsone bioreduction, plays important roles in such a bioconversion. However, the biomolecular-level mechanism behind this process and the reduction pathways remain largely unclear. Herein, the rapid anaerobic reduction of roxarsone by several EEB was explored, and the degradation pathways were clarified by using Shewanella putrefaciens CN32 as the model. The knockout of undA/mtrC led to a 70% loss of the roxarsone bioreduction ability within the initial 48 h. Both extracellular and intracellular reductions occurred simultaneously, resulting in the production of As(III) as the main inorganic arsenic species. Adding anthraquinone 2,6-disulfonate as a mediator considerably increased the roxarsone reduction rate by 119%. Given the wide distribution of EEB in environments, our findings facilitate a better understanding of the transformation behaviors of arsenic compounds in natural environments and highlight the necessity of re-evaluating the environmental risks of roxarsone.
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present in subsurface environments11 and critically involved in the geochemical cycling of many elements.12 Shewanella oneidensis MR-1, as a model EEB, can anaerobically degrade roxarsone to a single product, HAPA(V),10 but the specific biotransformation pathway is still unclear. In addition, genomic analysis showed that many types of EEB such as Geobacter sulf urreducens, Aeromonas hydrophila, and several other Shewanella species all possess the genes for encoding arsenic detoxification proteins (ars operon) and arsenic respiration proteins (arr operon),13 and their ability to reduce As(V) into As(III) under anaerobic conditions has been demonstrated.14−16 Nevertheless, direct evidence of roxarsone bioreduction by these EEB is still lacking. In light of the mobility and toxicity of As(III) being much higher than those of As(V),17,18 understanding the ultimate products of reduction of roxarsone by EEB, if any, is also of great significance for mitigating its environmental impacts. Herein, we explore the roxarsone reduction ability of EEB by using Shewanella putrefaciens CN32 and several other representative EEB as the models. The reaction kinetics and products of reduction of roxarsone by S. putrefaciens CN32 were examined in detail, and the relevant electron transfer pathways were elucidated by comparing the performances of the wide-type and several mutant strains with a modified metal
INTRODUCTION Roxarsone (3-nitro-4-hydroxyphenylarsonic acid) is widely used in the poultry and livestock industry as an anticoccidial agent and growth factor.1 The unmetabolized roxarsone is ultimately released into the environment with the feces.2,3 The annual release of roxarsone into the environment is approximately 900 t in the United States alone4 and can reach thousands of tons globally.5 The released roxarsone is further transformed into more toxic and mobile inorganic arsenic (As), severely threatening aquatic ecology and water security. Arsenic pollution of surface water and groundwater is today a global environmental concern.1,2,6,7 The biodegradation of roxarsone in chicken feces and sewage sludge into 3-amino-4-hydroxybenzene arsonic acid [HAPA(V)] and arsenate has been previously reported,4,8 wherein Clostridium species were identified as the main roxarsone degrader under anaerobic conditions. A roxarsone-respiring Clostridium bacterium OhILAs has been isolated.8 Anaerobic sludge could also effectively degrade roxarsone under methanogenic and sulfate-reducing conditions, and up to 99% of the reduction intermediates [HAPA(V) and 4-aminophenylarsonic acid] could be further mineralized into inorganic arsenic, i.e., As(III) and As(V).4 In addition, roxarsone biodegradation activities of soil microorganisms and exoelectrogenic bacteria (EEB) have also been demonstrated.9,10 The growth rate of the microorganisms in soil was 1.4-fold higher in the presence of roxarsone, and 81% of the roxarsone was transformed after incubation for 7 days.9 EEB are widely © XXXX American Chemical Society
Received: June 7, 2017 Accepted: June 27, 2017 Published: June 27, 2017 A
DOI: 10.1021/acs.estlett.7b00227 Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX
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Environmental Science & Technology Letters reducing and respiratory pathway (Mtr respiratory pathway) or mediator generation. Our work may facilitate a better understanding on the microbially mediated transformation behaviors of arsenic compounds in natural environments.
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MATERIALS AND METHODS
Bacterial Cultures and Medium. Mutant construction and the culture details of bacteria are provided in the Supporting Information. The preparation of the medium for roxarsone bioreduction is described in detail in the Supporting Information. Roxarsone Bioreduction Tests. The roxarsone bioreduction experiments were conducted under environment-mimicking motility-restricted conditions by statically culturing EEB. All tested EEB strains except G. sulfurreducens PCA in stationary phase were harvested by centrifugation (7000g for 5 min), washed with phosphate-buffered saline (PBS), and added to mineral medium (Supporting Information). For G. sulf urreducens PCA, the modified DSMZ medium (Supporting Information) with 5 mM acetate as the electron donor and 0.2 mM roxarsone as the electron acceptor was used for roxarsone reduction. The strains were cultivated in serum vials at 30 °C with an initial concentration of 1.0 in optical density (OD600). The culture medium was sparged with a N2/CO2 (80:20) gas mixture to ensure an anaerobic atmosphere. The reduction of roxarsone by different cell fractions of S. putrefaciens CN32 was analyzed following the methods reported previously.19 To evaluate the roles of mediators in the biotransformation of roxarsone by S. putrefaciens CN32, different concentrations of mediators, i.e., riboflavin and anthraquinone-2,6-disulfonate (AQDS), were also spiked into the 50 mL sterile serum vials. All experiments were conducted in the dark to avoid the photoreduction of roxarsone. For sample collection, 1 mL of bacterial culture was withdrawn from each serum at given time intervals and immediately centrifuged at 7000 rpm for 120 s. The supernatant was used for the subsequent analysis. The details for analyzing roxarsone and the degradation products are provided in the Supporting Information.
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Figure 1. Transformation of roxarsone by S. putrefaciens CN32. (A) Roxarsone, HAPA, lactate, and acetate concentration profiles for the Shewanella-mediated roxarsone reduction. (B) Anaerobic reduction of roxarsone at 0.2 mM by S. putrefaciens CN32 wild-type and derivative strains (ΔmtrA, ΔmtrB, ΔundA/mtrC, and ΔcymA). The batch test with the heat-inactivated strain was conducted as the negative control.
exhibited the highest roxarsone degradation activity (Figure 1B and Figure S1). The reduction activities of ΔmtrA and ΔmtrB were lower than that of the wide type in the initial 48 h but became much higher later, resulting in a roxarsone reduction efficiency comparable with that of the wide type at the end of the 84 h incubation. The ΔundA/mtrC mutant showed a 70% loss of the roxarsone bioreduction ability within the initial 48 h, and only ∼60% roxarsone reduction was achieved at the end of the test. UndA and MtrC of S. putrefaciens W3-18-1 were reported to be involved in iron reduction.22 Such a loss of roxarsone degradation ability for the ΔundA/mtrC mutant indicates that extracellular respiration is an important pathway of roxarsone bioreduction by S. putrefaciens CN32. Nevertheless, the partially remaining degradation activity of ΔundA/ mtrC implies that some intracellular proteins or other alternative extracellular proteins might also be involved in the roxarsone reduction. CymA is a key inner membrane protein in the Mtr respiratory chain of Shewanella, principally responsible for transferring cytoplasmic electrons to the periplasmic space for various reductive reactions.23,24 We found that the knockout of CymA led to an only 30% loss of roxarsone bioreduction ability (Figure 1B). Such a sustained high degradation activity might be attributed to the presence of some alternative cytoplasmic membrane-bound c-type cytochrome proteins25,26 in the electron transfer chain or intracellular degradation process, which warrants further investigation. To better understand the roxarsone reduction process in S. putrefaciens CN32, we further investigated the reduction of roxarsone by different cell fractions. Both the membrane fraction and the cytoplasmic fractions exhibited high roxarsone reduction activities (Table S2). Taken together, these results indicate that both extracellular and intracellular reductions of roxarsone occurred simultaneously. Role of Mediators in Roxarsone Bioreduction. Many natural materials, such as humic substances, and microbially excreted small molecules, such as flavins (riboflavin and flavin
RESULTS AND DISCUSSION
Rapid Reduction of Roxarsone by S. putrefaciens CN32. We first tested whether roxarsone could be anaerobically degraded by S. putrefaciens CN32 with lactate as the carbon source. Figure 1A shows that roxarsone was rapidly degraded at a rate of 2.4 × 10−3 mM/h and was completely eliminated after 84 h. Meanwhile, lactate was oxidized to acetate with a lactate consumption rate of 14.2 × 10−3 mM/h and acetate production rate of 11.5 × 10−3 mM/h (Figure 1A). No obvious degradation of roxarsone was observed in the control with the heat-inactivated bacteria, suggesting that the roxarsone degradation was a biological process. The Mtr respiratory pathway is an important anaerobic respiratory pathway of Shewanella cells to transfer electrons from the cytoplasmic membrane to the extracellular electron acceptor.20,21 To determine whether the roxarsone bioreduction by S. putrefaciens CN32 also depends on the Mtr respiratory pathway, we compared the bioreduction performances of S. putrefaciens CN32 and several of its mutants lacking the specific genes for encoding the proteins in the Mtr respiratory pathway. These mutants included ΔmtrA, ΔmtrB, ΔundA/mtrC, and ΔcymA (Table S1). The wild-type strain B
DOI: 10.1021/acs.estlett.7b00227 Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX
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Environmental Science & Technology Letters mononucleotide), can function as mediators to carry electrons from EEB cells to extracellular electron acceptors, thereby accelerating extracellular reduction.27−29 To determine whether a mediated bioreduction of roxarsone occurs in S. putrefaciens CN32, riboflavin or AQDS (the humic acid analog) was added as the mediator, and its impacts on the roxarsone degradation performance of wild-type CN32 were evaluated. The first-order rate constants (k) for all the tests were calculated to evaluate their roxarsone transformation kinetics (Table S3). Within the initial 60 h, no obvious increase in the roxarsone reduction rate was observed upon addition of 1 μM AQDS (0.0087 ± 0.0005 h−1) or riboflavin (0.0078 ± 0.0003 h−1) compared to that of the mediator-free control (0.0075 ± 0.0004 h−1). However, a 119% increase in the roxarsone reduction rate was observed when 5 μM AQDS (0.016 ± 0.0006 h−1) was added, while 5 μM riboflavin (0.0095 ± 0.0008 h−1) supplementation led to a slight increase in the roxarsone reduction rate. AQDS enhanced roxarsone reduction to a degree greater than that seen with riboflavin, suggesting that it reacted more quickly with roxarsone. This observation is in contrast to the behavior of these electron shuttles in iron(III) oxide reduction.21,30 Roxarsone Bioreduction Products. To elucidate the roxarsone bioreduction mechanism by S. putrefaciens CN32, its bioreduction metabolites were detected by high-performance liquid chromatography (HPLC), HPLC−hydride generation− atomic fluorescence spectrometry (HPLC−HG−AFS), HPLC−electrospray ionization tandem mass spectrometry (HPLC−EIS-MS), and high-resolution mass spectrometry. The HPLC analysis showed that HAPA(V) was the main product of roxarsone bioreduction, which agrees with a previous study.8 The mass balance of the roxarsone reduction was calculated by measuring the concentrations of the roxarsone and HAPA(V) (Figure 1A). The stoichiometric ratio between the produced HAPA(V) and the removed roxarsone was 0.85 at 84 h, indicating that HAPA(V) was the main reduction product. In addition, analysis of the arsenic species by HPLC−HG−AFS showed the presence of a considerable amount of As(III) and a relatively smaller amount of As(V) at 84 h, indicating that HAPA(V) was further mineralized to inorganic arsenic (Figure S2). The HPLC−EISMS analysis (Figure S3) shows that HAPA(III) was a reduction product of HAPA(V). The high-resolution mass spectrometric analysis demonstrates that HAPA(III) was further transformed into O-aminophenol and As(III) through cleavage of the C−As bond (Figure S4). The putative pathways of roxarsone degradation and biotransformation by S. putrefaciens strain CN32 are illustrated in Figure S5. The exact mechanism of the release of inorganic arsenic from the roxarsone reduction by S. putrefaciens CN32 warrants further investigation. Reduction of Roxarsone by Other EEB. The potential roxarsone reduction abilities of several other model EEB were also evaluated at identical bacterial concentrations [OD600 = 1 (Figure 2A)]. The roxarsone reduction rates of all these strains except for that of A. hydrophila ATCC7966 were higher than that of S. putrefaciens CN32. Specifically, roxarsone was completely degraded by S. oneidensis MR-1 and Shewanella decolorationis S12 within 24 h. The G. sulfurreducens PCA showed relatively slow reduction despite its powerful extracellular electron transfer ability. These results indicate that roxarsone reduction ability is a common ability possessed by EEB. Compared to those of the EEB strains listed in Table S4, the mixed cultures had weaker roxarsone reduction ability. In microcosms of groundwater with nutrient supplementation,
Figure 2. (A) Roxarsone reduction and (B) inorganic arsenic release by the tested EEB. Abbreviations: CN32, S. putrefaciens CN32; S12, S. decolorationis S12; MR-1, S. oneidensis MR-1; ATCC, A. hydrophila ATCC7966; PCA, G. sulfurreducens PCA.
90% roxarsone degradation was achieved within 15 days under anaerobic conditions.31 Under methanogenic and sulfatereducing conditions, 95% of roxarsone was removed by anaerobic sludge within 12 days.4 A poised potential of 0.5 V had been used to electrochemically stimulate the anaerobic degradation of roxarsone.32 Although it still took 2 days to reduce 90% of roxarsone, the work implies an activity of EEB much higher than those of other anaerobic bacteria in roxarsone degradation. Previous studies suggest that S. oneidensis MR-1 could anaerobically convert roxarsone to HAPA(V) as the sole product.10 The biotransformation of roxarsone to As(V) as the main inorganic arsenic species by a non-EEB Clostridium sp. strain OhILAs has also been reported.8 Unlike these earlier findings, here we found an abundant presence of As(III) in all the tested EEB cultures (Figure 2B). In addition, a trace amount of As(V) was detected in the cultures of S. putrefaciens CN32, S. oneidensis MR-1, and S. decolorationis S12. The involvement of several enzymes in Clostridium sp. strain OhILAs in the oxidation of the benzene ring of roxarsone has been reported.33 However, our findings show that the release of inorganic arsenic in Shewanella originated from the further reduction of HAPA(V) and direct cleavage of the C−As bond in HAPA(III) instead of benzene ring oxidation. Thus, As(III) was the main inorganic arsenic species in EEB cultures. Arsenate reductase (ArsC), the Mtr respiratory pathway, and intracellular C·As lyase might be involved in this process. Given the high toxicity and mobility of As(III), our results imply a high risk of roxarsone bioreduction by EEB in the environment, which should be examined further. C
DOI: 10.1021/acs.estlett.7b00227 Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX
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Environmental Science & Technology Letters
Figure 3. Proposed anaerobic degradation mechanisms of roxarsone by S. putrefaciens CN32. The red arrows represent the pathway of electron flow. The question marks indicate the unknown proteins or enzymes involved in the roxarsone bioreduction by S. putrefaciens CN32.
conditions.34,35 The abundant minerals and humic substances in the environment could further accelerate the transformation of roxarsone by EEB. In summary, we demonstrated that EEB could effectively use roxarsone as an electron acceptor for anaerobic respiration. For S. putrefaciens CN32, MtrC and UndA were the key cytochromes involved in the extracellular reduction of roxarsone. Both extracellular and intracellular reductions occurred simultaneously, resulting in the production of As(III) as the main inorganic arsenic species. Such a manner of respiration of EEB might expand the scope of electron acceptors to other organic arsenic compounds. Our findings facilitate a better understanding of the fates of organic arsenic compounds in natural environments and imply the necessity of re-evaluating the environmental risks of roxarsone.
Mechanism of Roxarsone Reduction. On the basis of the results described above, we propose the following mechanism of roxarsone anaerobic reduction by S. putrefaciens CN32 (Figure 3). The electrons generated from lactate oxidation are transported from the quinone pool to the inner membraneanchored CymA, through MtrA and MtrB, and reach the outer membrane-anchored c-type cytochromes MtrC and UndA. When roxarsone reaches the bacterial outer membrane, a portion of roxarsone receives electrons from the Mtr respiratory pathway and is initially transformed into HAPA(V). Meanwhile, a portion of roxarsone crosses the inner membrane and is transformed into HAPA(V) via catalysis by some unknown enzymes or proteins. The further reduction of HAPA(V) and a direct cleavage of the C−As bond in HAPA(III) occur intracellularly and lead to the release of As(III). The reduction of roxarsone can be significantly accelerated by the presence of extracellular redox mediators (AQDS or riboflavin), aggravating the release of toxic As(III) to the environment. The potential environmental risks of such reduction of roxarsone by EEB should not be ignored for several reasons. First, EEB could transform roxarsone rapidly and release inorganic arsenic into the environment. Inorganic arsenic is more toxic and mobile than its organic arsenic counterparts and poses a greater environmental threat.1 Second, EEB are widely present in anoxic environments11 and could survive in roxarsone-rich environments because of their high roxarsone toleration levels. Roxarsone (water solubility of
