Biodegradation of metal complex Naphthol Green B and formation of iron–sulfur nanoparticles by marine bacterium Pseudoalteromonas sp CF10-13

Accepted Manuscript Biodegradation of metal complex Naphthol Green B and formation of iron-sulfur nanoparticles by marine bacterium Pseudoalteromonas. sp CF10-13 Shuhua Cheng, Na Li, Li Jiang, Yating Li, Baiheng Xu, Weizhi Zhou PII: DOI: Reference:

S0960-8524(18)31514-1 https://doi.org/10.1016/j.biortech.2018.10.082 BITE 20646

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Bioresource Technology

Received Date: Revised Date: Accepted Date:

12 September 2018 28 October 2018 29 October 2018

Please cite this article as: Cheng, S., Li, N., Jiang, L., Li, Y., Xu, B., Zhou, W., Biodegradation of metal complex Naphthol Green B and formation of iron-sulfur nanoparticles by marine bacterium Pseudoalteromonas. sp CF10-13, Bioresource Technology (2018), doi: https://doi.org/10.1016/j.biortech.2018.10.082

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Biodegradation of metal complex Naphthol Green B and formation of iron-sulfur nanoparticles by marine bacterium Pseudoalteromonas. sp CF10-13 Shuhua Cheng, Na Li, Li Jiang, Yating Li, Baiheng Xu, Weizhi Zhou* School of Environmental Science and Engineering, Shandong University, Jinan, Shandong, China, 250100 *Corresponding author: Weizhi Zhou Postal address: School of Environmental Science and Engineering, Shandong University, 27 Shanda Nanlu, Jinan 250100, Shandong, China. Tel. /fax: +86 531 88361383. E-mail address: [email protected]

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Abstract Treatment of metal complex dye wastewater has attracted growing attention due to the degradation-resistant, high cost and potential hazards of current techniques. This study reported a marine bacterium (Pseudoalteromonas. sp CF10-13) with potential performance in decolorization and degradation of a metal complex dye—Naphthol Green B (NGB) at wide ranges of salinity, dye concentration and alkalinity under anaerobic conditions. It was inferred that the secretion of electron mediators in soluble extracellular metabolites by P. sp CF10-13 played important roles in NGB decolorization and degradation through extracellular electron transfer. Naphthalenesulfonate, the major structure in NGB molecule, was further degraded into low-toxic benzamide. Black stable iron-sulfur nanoparticles were formed endogenously avoiding H2S releasing, exogenous sulfur addition and metal sludge in accumulation. Accordingly, this study provided a cost-effective and eco-friendly biodegradation method to refractory NGB, further promoting the understanding of dye resources recovery. Keywords: Dye; Decolorization; Biodegradation; Redox mediator; Iron-sulfur nanoparticles.

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1. Introduction Metal-complex dyes (MCD) have been extensively used in textile and leather tanning industries due to the excellent light fastness, extensive adaptability to pH and temperature (Z & G, 2008). In reality, they are always persistent with high toxicity (Mallikarjuna et al., 2018). The refractory characters cause big troubles to the treatment of the wastewater containing MCD. When discharged into the natural water bodies, such as lakes or rivers, MCD wastewater will cause severe hazards to environment, plants and animals. Besides, complex metals such as chromium, cobalt, iron and copper etc. tend to release in the process of dye wastewater treatment, most likely causing secondary pollution and resource waste. Thus disposals of MCD in wastewater and the released metals become an urgent issue for wastewater restoration. A few treatment strategies about MCD wastewater have been reported, mainly physicochemical options such as advanced oxidation, adsorption and coagulation, etc. Naphthol green B (NGB), as a Fe (III) complex dye containing 1-nitroso-2-naphthol-6-sulfonate in the deprotonated state, is the most widely investigated MCD in wastewater treatment. Synthetic adsorbent magnetic halloysite-iron oxide nanocomposite (Riahi-Madvaar et al., 2017) could remove NGB fast, and photocatalysis technology (Devi & Ahmaruzzaman, 2017; Li et al., 2017a) has been also applied with outstanding degradation performance. But these methods may have some drawbacks such as high cost, complicated preparation technology and potential rerelease danger etc. Moreover, all technologies mentioned above ignored the disposal of iron ions, most likely causing an objectionable reddish-brown color to water and producing more sludge through nourishing to “iron bacteria” (Organization, 2004). 3

Biological methods could be alternative for dyes removal, owing to the superiorities including inexpensive operation, eco-friendly approach, and extensive adaptability (Vikrant et al., 2018). With excellent performance and lower expense, anaerobic biotreatment is frequently utilized to remedy dye wastewater (Victral et al., 2017). Generally, it could be attributed to non-specific redox reaction, in which dyes act as electron acceptors supplied by organic carbon sources through electron transport chain (Singh et al., 2007). It is noteworthy that, a unique microorganisms’ metabolism mechanism—extracellular electron transfer (EET) referred to the anaerobic electron transfer between cell and extracellular substrate to provide energy for metabolic activity (Richter et al., 2017). Many organic pollutants (Saratale et al., 2011a) and metal ions (Liu et al., 2016a) were disposed separately involving EET. Actually, relying on electricigens with the cytochrome or pilus, EET could be divided into two strategies: direct electron transfer (DET) and mediated electron transfer (MET) (Huang et al., 2017). Unlike the DET which needs direct contact between cells and electron acceptors, MET pathway usually shuttles electrons through redox mediators (Schröder, 2007). Several extracellular redox mediators have been reported so far, including flavin, melanin and quinone etc. Flavin , a redox mediator produced by Shewanella species could accelerate the reduction of poorly crystalline Fe (III) oxides (Edwards et al., 2015). And melanin, a redox-active molecule secreted by Shewanella. oneidensis, enhanced the reduction rate of dye (Brigé et al., 2010). Reducing pollutants with no direct contact between cells and pollutants, MET pathway seemed to circumvent or attenuate the adverse effect of hazardous pollutants on cells which could benefit the

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biodegradation. However, due to the high biotoxicity, referenced microbes for MCD degradation are still very limited. To our knowledge, only one bacterial strain (Shewanella oneidensis MR-1) has been reported about the decolorization of NGB and the immobilization of iron in the form of ferrous sulfide nanoparticles with sulfur addition (Xiao et al., 2012; Xiao et al., 2016). Since there are three sulfonic acid groups existing in NGB molecule, it was indicated that NGB decolorization by Shewanella oneidensis MR-1 was a one-step reduction with a large amount of intermediates accumulate. Sulfur addition for the nanoparticles synthesis might increase operation cost and aggravate sulfur emissions. It was also implied that it would be more favorable to release sulfur from sulfonic acid group on naphthalene ring through further bioregradation of NGB and to immobilize iron. This work investigated the NGB decoloration and degradation under anaerobic condition by a reductive marine bacterium Pseudoalteromonas. sp CF10-13 (P. sp CF10-13). The effects of electron donors, inorganic salts, pH, oxygen, salinity and initial NGB concentration were studied. The characterization methods including UV-Vis spectra, cyclic voltammetry (CV) and high performance liquid chromatography-mass spectrometry (HPLC-MS) were used to investigate the decolorization mechanisms of NGB. In addition, the microscopic characteristics of biosynthetic nanoparticles were observed through Transmission Electron Microscope (TEM) and Scanning Electron Microscope (SEM). X-Ray Diffraction (XRD) was also applied to reveal the possible component of the synthetic particles.

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2. Materials and methods 2.1. Dye, strain and culture medium The dye (Naphthol Green B, NGB) used in this study was purchased from Tianjin Kemiou Chemical Reagent Co., Ltd. And the bacterial strain P. sp CF10-13 used in this study was isolated from abyssal sediments in South China Sea. Species of the strain has been identified using 16S rDNA gene sequencing in early report (Li et al., 2016) and included in NCBI databases (Gene Bank: KP294525). The cells were cultured in Luria-Bertani (LB) medium on a rotary shaker (200 rpm) at 25°C. The LB medium included (g/L): peptone, 10.0; yeast extract, 35.0; NaCl, 30.0. Besides, all the chemicals used in this study were analytical-grade purity or above. 2.2. Decolorization experiments Decolorization experiments were performed in 200 mL serum bottles. In advance, P. sp CF10-13 was cultivated in 250 mL shaking erlenmeyer flask under aerobic conditions for 24 h, and then collected by centrifugation at 10,000 RCF for 10 min. After discarding the supernatant, cells were re-suspended by sterile water for three times and then formed 50 ml concentrated solution. Eventually, the concentrate was added in 100 mL sterilized serum bottle system containing 20 mM organics as electron donor and 100 mg/L NGB. To eliminate oxygen, the serum bottle medium was filled with high purity of N2 for 10 min, and sealed with butyl rubber stoppers immediately. All reagents were prepared in sterile water under aseptic conditions. Same as the above operations, effects of different parameters on decolorizing were investigated, including different electron donors (acetate, lactate, glucose, glycerinum, citrate and succinate; 20 mM) and extra inorganic salts (nitrate, nitrite, thiosulfate; 20 mM), pH (5.5-9.0), salinity (0 6

g/L-50 g/L) and initial NGB concentration (25 mg/L-1000 mg/L). In order to adjust pH, 30 mM 4-Morpholineethanesulfonic acid (MES) and 50 mM Tris-HCl were added to buffer the solution pH in the range of 5.5-6.5 and 7.0-9.0. In this study, all the experiments were carried out at 25°C, and each experiment was conducted in triplicate. The cell suspension of P. sp CF10-13 after 24 h cultivation was centrifuged and then filtered with a 0.22 μm filter membrane. The collected filtrate containing the extracellular metabolites of P. sp CF10-13 is called supernatant. Experiments to verify the effect of extracellular metabolites upon NGB decolorization were undertaken by adding 50 mL supernatant instead of cell concentrate in serum bottle system with adding the same volume of fresh LB as the control. Also, other cell departments (cytomembrane and cytoplasm) were extracted (Li et al., 2017b) to prove the color removal performance of NGB, respectively. Besides, different riboflavin dosagse (0 uM-8 uM) were added to the intact cell-NGB system separately in order to explore the biological decolorization mechanism of P. sp CF10-13. 2.3. Chemical analyses 2.3.1. UV-Vis spectrophotometry Samples were extracted regularly by a sterile injector to maintain the oxygen-free system. Residual NGB was evaluated by an UV-Vis scanning spectrophotometer (SHIMADZU, Japan) at 714 nm. The decolorization efficiency (DE) was calculated using the following equation: DE (%) = (A0―A1) /A0 ×100, Where A0 represents the initial absorbance at 714 nm and A1 refers to the absorbance at some sampling time. Moreover, the samples withdrawn at 0h, 72h and 120h were scanned (300 nm-900 nm)

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by UV-Visible spectrophotometer to detect the generation and disappearance of substances in the NGB decolorization system. 2.3.2. Cyclic voltammetry Direct electrode reactions of P. sp CF10-13 cells suspension, supernatant and fresh LB were examined by cyclic voltammetry using a CHI 660D Electrochemical Workstation (Shanghai, China) at a scan rate range of 100 mVs−1. A glassy car-bon working electrode, a platinum counter electrode and a silver/silver chloride reference electrode were used in the electrochemical system cycled between -1.0 and 1.0 V. Tests were carried out at 25°C under anaerobic conditions. 2.3.3. HPLC-MS analysis In order to identify the biodegradation products of NGB by P. sp CF10-13, HPLC-MS was performed by an Agilent 1260 Infinity Bio-inert Quaternay LC System coupled to an Agilent 6130B Single Quadrupole LC/MS System, which equipped with ESI source in positive ion mode (Agilent Technologies, USA). The eluents A (ultrapure water containing 0.1% methanoic acid) and B (acetonitrile) served as mobile phase in a gradient mode (5% B at 0-15 min, 10-25% B at 15-55 min, 100% B at 55-65 min). 2.3.4. TEM, SEM and XRD The micromorphology of black synthesized particles appearing at the terminal stage of decolorizing was observed by Transmission electron microscope (TEM) and Scanning electron microscope (SEM). The pretreatments of samples referred to early report (Xiao et al., 2016). Besides, for the component analysis, the black precipitations were handled by centrifugation and freeze-drying in advance. Large amounts of biomass adhered to the black particles surface and glue them together, which was hard to clean 8

and interfered with the intensity of diffraction peak. To precisely assess the particle component, the samples analyzed by XRD were ignited for 4 h and 6 h at 600 °C by muffle roaster (SX2-4-10, China), respectively. After cooling to room temperature, powders were analyzed using a high resolution XRD with Cu Kα irradiation (D8-Advance, Germany). The samples were scanned with 2θ from 10o to 80o. 3. Results and discussion In the NGB decolorization system, it was obvious that the solution color changed from green to colorless, and then gradually formed black turbid liquid with lots of small particles which of them even adhered to the bottle wall. UV-Visible spectrum was employed to determine the component changes of decolorization system at different stages. After 72 h of decolorizing, the absorption peak of NGB at 714 nm almost disappeared, indicating NGB was removed completely. At 120 h, two new peaks at 415 nm and 549 nm came out visibly along with mass black particles formation, suggesting that biodegradation existed in the decolorization process of NGB. In addition, the above biological decolorization of P. sp CF10-13 was independent of biosorption because the cell color was nearly invariable. 3.1. Effects of parameters on NGB decolorization Most microbes cannot utilize dyes as sole carbon sources, due to the high molecular weight and complicated structure of dyes didn’t allow them to across the cell membrane into cytoplasm (Anjaneya et al., 2011). Early reports showed that dyes tend to be biodegraded through co-metabolism with adequate available organics, such as glucose, lactate, sucrose et.al (Tan et al., 2016). Fig. 1a represented the effects of different electron donors on the decolorization of NGB under anaerobic conditions. As 9

shown, P. sp CF10-13 could use several organics to act as electron donors for decolorizing. Acetate, lactate, glucose, citrate and succinate as electron donors all achieved the decolorization efficiency (DE) above 50.00%. Among them, acetate and lactate could reach to 94.90% and 92.64% respectively. Thus acetate was selected as the optimal electron donor for NGB decolorization in the subsequent experiments. Generally, glucose as a common substrate was more easily to be taken up and metabolize for many strains such as Pichia sp. TCL (Qu et al., 2012) which decolorized Acid Red B with the maximum DE of 90.00% by using glucose as carbon source. However, in this study, glucose as electron donor had a poor performance with DE of 54.99%, which may be explained that sulfonic acid group on dye molecule obstructed the transport of certain organics into the cytoplasm (Seesuriyachan et al., 2007). It is known that various inorganic salts are added into dyeing process as accessory ingredients to improve color quality, causing lots of inorganic salts remained in wastewater (Anjaneya et al., 2011). However, some inorganic salts residues could restrain dyes decolorization (Chen et al., 2008). The influences of three common inorganic salts on NGB decolorization were evaluated respectively. As shown in Fig. 1b, nitrate deeply suppressed the NGB removal with only 19.46% of DE, which corresponded to previous researches (Liu et al., 2016b; Xiao et al., 2012). The decolorization depression might result from the electron competition between nitrate and dyes (Liu et al., 2016b). Besides, nitrite also revealed slight inhibition in the preliminary stage of NGB decolorizaton, but the DE could achieve 96.38% ultimately. This phenomenon was distinct from early reports such as 18 uM of nitrite drastically restrained NGB decolorizing by S. oneidensis MR-1 (Xiao et al., 2012), and 10 uM 10

nitrite decreased the DE of Congo Red only with 18.00% (Li et al., 2015). Hence, P. sp CF10-13 possessed of a better adaptability to nitrite than some other functional strain. Notably, the addition of thiosulfate dropped DE to 89.70% but accelerated the generation of black precipitate. Reportedly, the final reduction product of thiosulfate was identified as hydrogen sulfide (Xiao et al., 2012) which may react with some NGB degradation product to form black particles. Overall, compared with the control, all the three treatments with inorganic salts inhibited the NGB decolorization. In fact, microbes tend to choose the more easily accessible electron acceptors which were at upstream of the electron transport chain or easier to be utilized when several electron acceptors coexisted(Coby & Picardal, 2005; Feinberg & Holden, 2006). With the high redox potential, oxygen was superior to dyes on receiving electrons, resulting into a lower DE under aerobic conditions (Gupta et al., 2015; Saratale et al., 2011b). Thus it also accounted for the excellent performance of anaerobic decolorization (Fig. 1d). Normally, pH has a considerable impact on biological decolorization of dyes (Tan et al., 2016). The maximum DE was obtained at the group of pH 7.5 after 70 h (Fig. 1c), suggesting that neutral condition was appropriate for P. sp CF10-13 to NGB decolorization. The results were consistent to many existing literatures of anaerobic decolorization bacteria such as Mutant Bacillus sp. VUS (Dawkar et al., 2009) and Citrobacter sp. CK3 (Wang et al., 2009). While at pH of 5.5-6.5, biological decolorization declined sharply and the maximal DE was only 50.55%. It might be because acidic condition greatly inhibited the metabolic activity of P. sp CF10-13 (Li et al., 2016). When at pH of 9.0, the decolorization rate was suppressed in the first 70h but eventually exceeded 90.00%, indicating that P. sp CF10-13 had a strong tolerance to 11

alkalinity although requiring an adaptive phase. The alkali-resistance of P. sp CF10-13 might result from the secretion of large amount of extracellular polymeric substances (EPS) which was capable of adjusting alkalinity to neutral (Zhou et al., 2016). In conclusion, P. sp CF10-13 expressed a favorable decolorizing ability with wide ranges of pH, encouraging a broader application to the treatment of dye wastewater. A large number of salts are frequently used as dyeing assistant during the tincture of textile products, leading to higher salinity in printing and dyeing wastewater (Xu et al., 2016). The metabolic activities of most microorganisms in activated sludge were impaired when the wastewater salinity is higher than 3% (Hessel et al., 2007). Hence, salinity of wastewater could be a significant limitation for the application of biological decolorization. Fig. 1e reflected the impacts of different salinities on NGB decolorization by P. sp CF10-13. When salinity was 0-10 g/L, DEs showed an upward trend with the increased salt content. And salinity at 20 g/L, DE was more than 90%. P. sp CF10-13, as a deep-sea bacterium, possessed more resistance to salinity and even required certain salts for growth and metabolism (Li et al., 2016). Whereas salinity ascended to 30 g/l and 50 g/l, DEs were diminished apparently only with 38.10% and 24.43%. Thus there was a tolerance limitation for P. sp CF10-13 to salinity, and biological metabolic activities would be restrained once salinity surpassed the optimum dosage. All the same, P. sp CF10-13 owned tremendous preponderance over conventional activated sludge in the treatment of hyperhaline dyeing wastewater. Various initial dye concentrations of wastewater may have different shocks to biological treatment technology due to the interaction between dye molecules and microorganisms (Khataee et al., 2010). In this study, the effects of initial NGB 12

concentrations on decolorization were investigated. As shown in Fig. 1f, DEs decreased visibly with the increasing initial dye concentration, due to the higher concentration of dye was more toxic to bacteria. At 80 h, all groups achieved more than 95% decolorization, except for the group of 1000 mg/L NGB. It eventually reached to 97.15% DE although requiring a little more time for bacteria to adapt the stress of high NGB concentration (Xiao et al., 2012). These phenomena fully demonstrated the strong tolerance and decolorization performance of P. sp CF10-13 to NGB, which supported the biological treatment of dye wastewater to some extent. 3.2. NGB biodegradation mechanism Apparently, the formation of new products revealed that the anaerobic decolorization of NGB by P. sp CF10-13 must refer to biodegradation. Different parts of P. sp CF10-13 replacing entire cell were employed to get rid of NGB, respectively. As a result, only the cell free supernatant had excellent ability to remove NGB with 90% DE in 70 h (Fig. 2a). Compared to the useless fresh LB, the above result indicated that there were some functional substances in the extracellular metabolites to degrade NGB effectively. Nevertheless, the supernatant-NGB system didn’t generate black particles like the intact cell-NGB system, thus there may be distinct between these two systems. As mentioned above, Shewanella oneidensis MR-1, the most typical bacteria with EET pathway, could secrete flavin to accelerate electron transport inside and outside the cell (Lin et al., 2018). By report, adding exogenous redox mediators could significantly promote the organics anaerobic degradation relying on EET (Rau et al., 2002). Therefore, NGB degradation tests with additional riboflavin were implemented to verify whether EET mechanism worked. Results revealed that NGB decolorization rates 13

enhanced as riboflavin concentration increasing. By comparison to the control, the group with 8 uM riboflavin decolorized NGB faster at 2.7 times with 96.70% DE in 46 h (Fig. 2b). According to these phenomena, the NGB biodegradation by P. sp CF10-3 should depend on EET mechanism rather than enzymatic activity. Based on the excellent decolorization ability of cell free supernatant, the hypothesis could be made that some redox mediators might be produce by P. sp CF10-13 in the extracellular metabolites and play a crucial role in the MET pathway. Research showed that, a strain Sphingomonas facilitated a 20-fold increase on the reduction of sulfonated azo dye through excreting 1, 2-dihydroxynaphthalene (Keck et al., 1997). To further verify the existence of redox mediator, cell suspensions of P. sp CF10-13 were prepared under anaerobic conditions to determine their electrochemical activities via cyclic voltammetry. Fig. 3 expressed the cyclic voltammograms (CVs) of cell suspensions, cell free supernatant and fresh LB medium. The supernatant reflected the strongest electrochemical activity with a redox potential of around -0.20 V against the Ag/AgCl reference electrode, and the cell suspensions revealed a little behind at -0.15 V. But the cell suspensions had a higher peak current, possibly since electrochemical system stimulated the excretion of more redox mediators by entire cells to cause higher electron transfer rate. This result indicated that the interfacial electron transfer rates between cells and electrodes were significantly accelerated (Lin et al., 2018) by the support of redox mediator in the extracellular metabolites of P. sp CF10-13. In consideration of strong electrochemical activity of the redox mediator, P. sp CF10-13 could be expected to be further researched and applied in microbial fuel

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cells (Rossi, 2017), a booming bioelectrochemical device, for organic compounds degradation and electrical production. 3.3. Characterization of biodegradation products Disodium 4-amino-3-hydroxy-2, 7-naphthalenedisulfonate was monitored in the biodegradation products of NGB. Obviously, the chromophoric group N=O (Mohammadi & Ashkarran, 2016) was transformed into NH2, which explained the NGB decolorization in the biological reduction process. Besides, complex iron was released from NGB to form naphthalene hydroxyl, which was consistent with previous research that most MCD tend to release metals in sewage treatment (Blánquez et al., 2004). The compound with the base peak of 119.0494 was identified as benzamide. The appearance of the harmfulless and stable degradation product should be due to the breakage of naphthalene ring which with delocalization-conjugated bond had pernicious mutagenic effect on microorganisms, fish, and human (Clemente & Fedorak, 2005). Besides, sulfonic acid groups, frequently introduced into dye molecule to improve water solubility, were eliminated from NGB structure. Based on above results of HPLC-MS, possible pathways of NGB biodegradation by P. sp CF10-13 under anaerobic condition were proposed, that coordinate bond Fe—O and chromophore groups N=O were firstly broken, and then the sulfonic acid groups were removed, finally benzamide was generated though the cleavage of the benzene ring. Hence P. sp CF10-13 economically achieved the degradation and toxicity weakening of NGB, contributing to the future complete mineralization. After complete decolorization, small black synthesized particles appeared and increased gradually. To observe the black synthesized particles on the microscopic scale, 15

TEM and SEM as effective tools were utilized. As expected, some aggregated black particles existed in medium, and some were densely deposited on the surface of P. sp CF10-13 with the average diameter approximate 200 nm. Early literatures indicated that some marine microbes tend to produce a lot of EPS under the presence of toxic substances (Sheng et al., 2005). EPS could serve as template for assembling nanomaterials (Xiao et al., 2016). As mentioned above, during the biodegradation of NGB, iron released and the black nanomaterials appeared. It could be inferred that the unknown biosynthetic nanomaterials might contain iron. The XRD results of unburned sample showed a peak at 2θ = 20.03o in approached to FeS, but it had a low fitting degree resulting from lots of biomass adherence. And the samples burned for 2 h and 4 h presented peaks at 2θ = 31.74o, 35.63o and 45.52o, confirming the presences of Na2SO4, Fe2O3 and Fe3S4. Evidently, iron sulfides were contained in the black synthesized nanomaterials. In NGB degradation system, the only sulfur came from the sulfonic acid group on the molecular structure of NGB. Previous research has reported that linear alkylbenzenesulfonate surfactants and other similar structures could be utilized by Pseudomonas putida S-3 as sulfur source for growth and achieve desulfonation simultaneously (Kertesz et al., 1994). Thus P. sp CF10-13 might be competent for metabolizing the sulfonic acid groups on naphthalene ring. In this study, at the end of NGB experiment, some stink egg gas was released when opening the stopper of serum bottle. The gas turned out to be H2S because it could blacken the lead acetate test paper (not shown in this paper).During the anaerobic sulfate reduction, sulfate or sulfur was always as terminal electron acceptor with H2S as the end-product (Rasool et al., 2016). Hence, P. sp CF10-13 most likely 16

transported sulfonic acid groups to H2S, which was in accordance with the proposed pathways of NGB biodegradation to some extent. At the same time, NGB system consisted of a large number of free iron ions. Therefore, the primary synthesis mechanism of black nanomaterials could be as follows: 2Fe3+ + 3H2 S = 2FeS ↓ +S ↓ +6H + Moreover, there were some other iron-sulfur compounds which may involve other side reactions. These inferences also explained the phenomena that no black nanomaterials generated in the supernatant-NGB system and the addition of thiosulfate accelerated the formation of black nanomaterials, because the sulfur of iron-sulfur nanoparticles could be provided by the biological metabolism of P. sp CF10-13 or exogenous addition. In conclusion, this study was the first report about the biological synthesis of the iron-sulfur nanoparticles without additional sulfur supply. 4. Conclusions P. sp CF10-13 effectively achieved anaerobic decolorization and biodegradation of NGB with wide adaptability, low-cost and little pollution, owing to EET and the redox mediators secreted by P. sp CF10-13. The ultimate product of NGB indicated the stability and attenuation effect of biodegradation, promoting the next dye permineralization. Besides, redox mediators would require to be explored and future applied in MFC for pollutant degradation and electrical production. The synthesis of iron-sulfur nanoparticles avoided the dangers of H2S and ferric iron, and realized dye resources recovery. This study applied an alternative, environmentally friendly and economical technique for NGB degradation. Acknowledgements 17

The authors acknowledge the Major Program of Shandong Province Natural Science Foundation (ZR2018ZB0211); the Major Program of Shandong Province Technological Innovation Project (2018CXGC0307) and the Natural Science Foundation of Shandong Province (ZR2017MEE024). Appendix A. Supplementary data E-supplementary data of this work can be found in online version of the paper. References 1. Anjaneya, O., Souche, S.Y., Santoshkumar, M., Karegoudar, T.B. 2011. Decolorization of sulfonated azo dye Metanil Yellow by newly isolated bacterial strains: Bacillus sp strain AK1 and Lysinibacillus sp strain AK2. Journal of Hazardous Materials, 190(1-3), 351-358. 2. Blánquez, P., Casas, N., Font, X., Gabarrell, X., Sarrà, M., Caminal, G., Vicent, T. 2004. Mechanism of textile metal dye biotransformation by Trametes versicolor. Water Research, 38(8), 2166-2172. 3. Brigé, A., Motte, B., Borloo, J., Buysschaert, G., Devreese, B., Van Beeumen, J.J. 2010. Bacterial decolorization of textile dyes is an extracellular process requiring a multicomponent electron transfer pathway. Microbial Biotechnology, 1(1), 40-52. 4. Chen, C.-H., Chang, C.-F., Ho, C.-H., Tsai, T.-L., Liu, S.-M. 2008. Biodegradation of crystal violet by a Shewanella sp NTOU1. Chemosphere, 72(11), 1712-1720. 5. Clemente, J.S., Fedorak, P.M. 2005. A review of the occurrence, analyses, toxicity, and biodegradation of naphthenic acids. Chemosphere, 60(5), 585-600. 6. Coby, A.J., Picardal, F.W. 2005. Inhibition of NO3− and NO2− Reduction by Microbial Fe(III) Reduction: Evidence of a Reaction between NO2− and Cell Surface-Bound Fe2+. Applied and Environmental Microbiology, 71(9), 5267-5274. 7. Dawkar, V.V., Jadhav, U.U., Ghodake, G.S., Govindwar, S.P. 2009. Effect of inducers on the decolorization and biodegradation of textile azo dye Navy blue 2GL by Bacillus sp VUS. Biodegradation, 20(6), 777-787. 8. Devi, T.B., Ahmaruzzaman, M. 2017. AgNPs‐AC Composite for Effective Removal (Degradation) of Napthol Green B Dye from Aqueous Solution. ChemistrySelect, 2(28), 9201-9210. 9. Edwards, M.J., White, G.F., Norman, M., Tome-Fernandez, A., Ainsworth, E., Shi, L., Fredrickson, J.K., Zachara, J.M., Butt, J.N., Richardson, D.J. 2015. Redox linked flavin sites in extracellular decaheme proteins involved in microbe-mineral electron transfer. Scientific reports, 5, 11677. 10. Feinberg, L.F., Holden, J.F. 2006. Characterization of Dissimilatory Fe(III) versus NO3− Reduction in the Hyperthermophilic Archaeon Pyrobaculum aerophilum. Journal of Bacteriology, 188(2), 525-531. 11. Gupta, V.K., Khamparia, S., Tyagi, I., Jaspal, D., Malviya, A. 2015. Decolorization of mixture of dyes: A critical review. Global Journal of Environmental Science and Management, 1(1), 71-94. 12. Hessel, C., Allegre, C., Maisseu, M., Charbit, F., Moulin, P. 2007. Guidelines and legislation for dye house effluents. Journal of Environmental Management, 83(2), 171-180. 13. Huang, B., Gao, S., Xu, Z., He, H., Pan, X. 2017. The Functional Mechanisms and Application of 18

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Figures captions Fig. 1. Effects of different parameters on decolorization: (a) electron donors; (b) inorganic salts; (c) pH; (d) dissolved oxygen; (e) salinity; (f) initial dye concentration. (If not mentioned, at pH of 7.5, initial NGB concentration of 100 mg/L and anaerobic conditions, with acetate as electron donors, without addition of salts. This also applied for the following figures.) Fig. 2. Effects of (a) parts from P. sp CF10-13 culture (Fresh LB as a contrast to supernatant) and (b) doses of riboflavin on NGB decolorization. Fig.3. Cyclic voltammetry curves of fresh LB, supernatant and cell suspension of P. sp CF10-13.

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Fig. 1. Effects of different parameters on decolorization: (a) electron donors; (b) inorganic salts; (c) pH; (d) dissolved oxygen; (e) salinity; (f) initial dye concentration. (If not mentioned, at pH of 7.5, initial NGB concentration of 100 mg/L and anaerobic conditions, with acetate as electron donors, without addition of salts. This also applied for the following figures.)

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Fig.3. Cyclic voltammetry curves of fresh LB, supernatant and cell suspension of P. sp CF10-13.

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Biodegradation of metal complex Naphthol Green B and formation of iron-sulfur nanoparticles by marine bacterium Pseudoalteromonas. sp CF10-13

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Highlights  P. sp CF10-13 discolored NGB at wide conditions.  NGB biodegradation achieved by extracellular electron transfer.  Redox mediator secreted by P. sp CF10-13 accelerated the reduction of NGB.  P. sp CF10-13 synthetized iron-sulfur nanoparticles without extra sulfur.

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