Addition of Shewanella oneidensis MR-1 to the Dehalococcoides-containing culture enhances the trichloroethene dechlorination

Environment International 133 (2019) 105245

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Addition of Shewanella oneidensis MR-1 to the Dehalococcoides-containing culture enhances the trichloroethene dechlorination Yaru Lia,b, Li-Lian Wena,c, He-Ping Zhaoa, Lizhong Zhua,b,

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a

College of Environmental and Resource Science, Zhejiang University, Hangzhou 310058, China Key Laboratory of Organic Pollution Process and Control, Zhejiang Province, Zhejiang University, Hangzhou 310058, China c College of Resource and Environmental Science, Hubei University, Wuhan 430062, China b

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ABSTRACT

Handling Editor: Da Chen

Dehalococcoides is able to completely dehalogenate tetrachloroethene (PCE) and trichloroethene (TCE) to ethene (ETH). However, the dechlorination efficiency of Dehalococcoides is low and result in the accumulation of toxic intermediates. In this study, Shewanella oneidensis MR-1 (S. oneidensis MR-1) was added to the Dehalococcoidescontaining culture and the complete TCE to ETH dechlorination was shortened from 24 days to 16 days. Dehalococcoides-targeted 16S rRNA gene and two model reductive dehalogenase (RDase) genes (tceA and vcrA), responsible for dechlorinating TCE to vinyl chloride (VC) and VC to ETH respectively, were characterized. Results showed that S. oneidensis MR-1 has no effect on the cell growth while the RDase genes expression was upregulated and the RDase activity of Dehalococcoides was elevated. The mRNA abundance of vcrA increased approximately tenfold along with the increased concentration of vitamin B12 (cyanocobalamin). Interestingly, the addition of S. oneidensis MR-1 increased the concentration of vitamin B12 by affecting the microbial community structure. Therefore, the addition of S. oneidensis MR-1 might have a positive effect on regulating the activity of RDase of functional microorganisms and uptake of vitamin B12, and further provided a practical vision of chloroethene dechlorination by the Dehalococcoides–containing culture.

Keywords: Dehalococcoides Shewanella oneidensis MR-1 Trichloroethene Dehalogenase Gene expression Vitamin B12

1. Introduction As a result of improper handling, storage and disposal, trichloroethene (TCE) has contaminated groundwater and sediments at many industrial sites. Due to its potential carcinogenicity, the drinkingwater maximum contaminant level (MCL) of TCE was regulated at 5 μg/ L (Guha et al., 2012; USEPA, 2007). A number of remediation technologies for TCE pollution have been developed including abiotic nanoscale zero-valent iron (nZVI) and organohalide-respiring bacteria degradation (Shih et al., 2010). Organohalide-respiring bacteria, utilizing H2 as electron donor, obtain energy through metabolic dechlorination. Dehalococcoides and Dehalogenimonas are the microorganisms ever reported that can reduce TCE to the nontoxic benign product ethene or ethane, while other identified microbes only partially metabolize TCE to dichloroethene (DCE) or vinyl chloride (VC) (Amos et al., 2007; Atashgahi et al., 2016; Jugder et al., 2016; Weatherill et al., 2018; Yang et al., 2017). Stepwise dechlorination results in the accumulation of VC in the environment, which is of particular concern (MCL in drinking-water was 2 μg/L) (Abelson, 1990; He et al., 2003). Furthermore, the low abundance of Dehalococcoides at bioremediation sites

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results in low dechlorination rate and long dechlorination period. The low abundance of Dehalococcoides and the likely accumulation of toxic intermediates are the challenges for in-situ bioremediation (Wang et al., 2016). The redox conditions, carbon source, and electron donor also critical to support reductive dechlorination by Dehalococcoides (DeAngelis et al., 2010; Lee et al., 2016). In addition, Dehalococcoides has specific metabolic requirements for hydrogen, cobamides, acetate, biotin, and thiamine, which are provided by other anaerobic microbes through a complex network (He et al., 2007; Mao et al., 2017; Maymó-Gatell et al., 1997; Men et al., 2014; Türkowsky et al., 2018; Wang et al., 2019; Ziv-El et al., 2012). For instance, nZVI can promote microbial reductive dechlorination by reducing the redox potential and providing H2 (Rosenthal et al., 2004; Shi et al., 2011). However, nZVI is also cytotoxic to Dehalococcoides, causing cell membrane disruption and DNA/protein damage (Chaithawiwat et al., 2016; Xiu et al., 2010b). Desulfovibrio vulgaris Hildenborough and Pelosinus fermentans R7 can provide hydrogen and cobamides, which facilitate the growth of Dehalococcoides mccartyi 195 and maintain sustainable dechlorination (Men et al., 2014). Sulfurospirillum can also produce hydrogen, acetate

Corresponding author at: College of Environmental and Resource Science, Zhejiang University, Hangzhou 310058, China. E-mail address: [email protected] (L. Zhu).

https://doi.org/10.1016/j.envint.2019.105245 Received 26 July 2019; Received in revised form 28 September 2019; Accepted 4 October 2019 Available online 01 November 2019 0160-4120/ © 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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and the reductive dehalogenase (RDase) cobamide cofactor to support Dehalococcoides (Kruse et al., 2019). The growth of Dehalococcoides could also be optimized with high concentration of vitamin B12 and the possibly high availability of vitamin B12 (He et al., 2007). While the aforementioned studies have identified numerous factors that contribute to enhanced dechlorination, the expression level of RDase genes and its relationship to dechlorination rate were mostly undiscussed. RDase can facilitate the final step of the electron transfer in reductive dechlorination at the outer side of the membrane of Dehalococcoides (Schubert et al., 2018; Wang et al., 2018). The expression of RDase genes tceA and vcrA is often used as a biomarker of Dehalococcoides’s activity (Kranzioch et al., 2015; Lee et al., 2006, 2008; Türkowsky et al., 2018). The expression of RDase genes could be affected by the concentration of residual chloroethenes, redox conditions, and dissolved organic carbon etc. For example, nZVI has been found to inhibit the expression of tceA and vcrA possibly by direct interaction (Xiu et al., 2010a). A little of information about the effect of biotic factors on the expression of RDase genes are valuable. Previous studies have found that RDase requires cobalamin as a cofactor, which contains a cobalt atom at the center (Bommer et al., 2014; Yan et al., 2018). Payne et al. (2015) proposed a vitamin B12-dependent dechlorination mechanism via electron transfer on the basis of the structure of RDase, which depends on the valence variation of vitamin B12. Reductive cleavage of chloroethene requires vitamin B12(I) as the catalyst in respiratory RDase (Hug et al., 2012; Schubert et al., 2016). However, the effect of biotic factors on the valence variation of vitamin B12, which further affects the activity of RDase, was studied less. Shewanella oneidensis MR-1 (S. oneidensis MR-1) has attracted wide attention for its potential in biodegradation of pollutants such as nitrobenzene, NO3−, or as anodes of mediator-less microbial fuel cells (Cai et al., 2012). Contrary to nZVI, S. oneidensis MR-1 does not exert cytotoxicity to Dehalococcoides, and possess a special MtrABC electron transport pathway (Brutinel et al., 2012; Gralnick et al., 2007; Hernandez et al., 2001). Although S. oneidensis MR-1 cannot degrade chloroethenes independently, it has great potential to optimize Dehalococcoides-based systems. For example, S. oneidensis MR-1 may modulate the chemical forms of various compounds such as cobamides, metal elements and electron donors. Under anoxic conditions, acetate is a known end product in S. oneidensis MR-1 (Scott et al., 1994). Considering the interspecies electron transfer from Geobacter sulfurreducens to Thiobacillus denitrificans facilitates acetate oxidation coupled to nitrate reduction (Kato et al., 2012), similar phenomena may occur that oxidation of lactate by S. oneidensis MR-1 could provide electron to Dehalococcoides through special nanowire, which ultimately support TCE dechlorination (Brutinel et al., 2012). These effects produced by S. oneidensis MR-1 play an important role in the optimization of Dehalococcoides-based communities. Therefore, the objectives of this study are: (1) to investigate the effect of S. oneidensis MR-1 on TCE dechlorination in the Dehalococcoides-containing culture; (2) to explain the interaction mechanism between S. oneidensis MR-1 and Dehalococcoides by exploring the effect of S. oneidensis MR1 on the genes expression and the activity of RDase in Dehalococcoides. The knowledge gained from this study provides us with a deeper understanding of Dehalococcoides-based dechlorinating communities and will help solve the main challenges faced by in-situ bioremediation.

2.2. Culture A mixed culture containing Dehalococcoides, stored at Zhejiang University, was originally enriched from Yangtze Three Gorges Reservoir sediment and grown with lactate as carbon source and electron donor. The consortium YH was sub-culture from the YCQ1 culture maintained under anaerobic conditions and incubated in the dark at 30 °C (Wen et al., 2015, 2017). The anaerobic medium contained the following reagents per liter: 10 mL of salts solution, 1 mL of Se/W solution, 1 mL of trace element solution, 0.25 mL of 0.1% (w/v) resazurin stock solution, as described by Löffler et al. (2005). Then 2.292 g N-[Tris(hydroxymethyl) methyl]2-aminoethanesulfonic acid (TES) was added as buffer and 1.87 g sodium lactate was added as carbon source and electron donor. The medium was flushed with N2, boiled 10 min to remove oxygen and was autoclaved. Then 0.048 g Na2S·9H2O, and 0.242 g L-cysteine were added as the reducing agents, and 2.52 g NaHCO3 was added as buffer. 0.0771 g DL-dithiothreitol was added. Then 45 mL medium and 0.2 mL vitamin supplement solution with or without vitamin B12 were transferred into glass serum bottles sealed with butyl rubber stoppers and aluminum crimps. The initial concentration of TCE was 450 μmol/L. Finally, 5 mL inoculum culture was transferred into every bottle, half of which were also amended with S. oneidensis MR-1 (OD600 = 0.1). S. oneidensis MR-1 was originally grown in autoclaved Luria-Bertani (LB) medium at 30 °C and washed three times with the above-mentioned anaerobic medium before added to the Dehalococcoides-containing culture. The volume ratio between the added S. oneidensis MR-1 and the final reaction volume was 1:250. All experiments were performed at 30 °C in the dark. Results are presented as the average values from triplicates. Assays with sterile medium and TCE only, as well with sterile medium, TCE and S. oneidensis MR-1, were run as negative controls. 2.3. Analytical methods 2.3.1. Chemical analysis Chloroethenes and ethene (ETH) were measured using a GC-FID (Agilent Technologies GC system, model 6890 N, Agilent Technologies Inc., USA) equipped with a packed column (30 m long, 0.32 mm i.d., 0.5 mm thickness, cross-linked polydimethysiloxane film, J&W scientific, USA) by injecting 100 μL headspace samples (Kranzioch et al., 2013; Zhao et al., 2010). The carrier gas was N2 with a constant flow rate of 0.065 m3/day, and the temperature conditions for the injector and detector were 180 °C and 220 °C, respectively. A temperature program was kept at 60 °C for 2 min, heated gradually to 200 °C (20 °C/ min), and kept at 200 °C for 5 min (Wen et al., 2015). Standard curves were described with every five-point for all chloroethenes and ETH. The concentration of each compound was calculated based on gas-liquid equilibrium using Henry’s law constants. The simple acids (lactate and acetate) were measured by filtering 1 mL of sample through a 0.22 μm membrane filter using an Agilent 1100 high performance liquid chromatography with an Aminex HPX87H (Bio-Rad) column. The temperature maintained at 50 °C and 2.5 mM of sulfuric acid as the eluent at a flow rate of 0.6 mL/min. The extraction and measurement of vitamin B12 in the different cultures were fulfilled in the Institute of Applied Ecology, Chinese Academy of Sciences. 1.5 mL culture was incubated in a sonication water bath to achieve homogeneous suspensions and transferred to sterile 50 mL plastic tubes. Total corrinoids were extracted in the cyano form and purified using the potassium cyanide extraction method (Yan et al., 2013). Liquid chromatography–mass spectrometry analysis was performed using a Dionex Ultimate 3000 system (Thermo Fisher Scientific) fitted to an Exactive Plus Orbitrap Mass Spectrometer with an electrospray ionization source (Thermo Fisher Scientific). The detail information of detection was reported in previous study (Yan et al., 2016). The concentration of vitamin B12 reduced by S. oneidensis MR-1

2. Materials and methods 2.1. Reagents Trichloroethene (TCE; 99.5%), cis-dichloroethene (cis-DCE; 98%), trans- dichloroethene (trans-DCE; 98%), 1,1-dichloroethene (1,1-DCE; 99%) and vinyl chloride (VC; 99%) were obtained from J&K Chemicals. All other chemicals were of analytical reagent or guaranteed reagent. 2

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was measured using a Shimadzu UV-1800 spectrophotometer. Scanning range was from 200 nm to 600 nm using a fast scan speed. The peak wavelengths were 361 nm for vitamin B12(III) and 312 nm for vitamin B12(II). Standard curves generated for vitamin B12(III) and vitamin B12(II) were linear (R2 ≥ 0.997). Samples were kept away from air exposure to avoid rapid oxidation (Workman et al., 1997).

anaerobic condition, the sample was centrifuged at 4 °C and 8000 g for 30 min (Hitachi CF16RXII, Japan). The fresh pellets were re-suspended in 250 μL of anoxic 1× sample buffer containing 1% digitonin provided by the NativePAGE sample Prep Kit (Invitrogen, California, USA) (Molenda et al., 2016; Tang et al., 2013). Suspended cell pellets were transferred to 1.5 mL Eppendorf tubes and combined with 250 mg of 75-μm-diameter glass beads. The sealed cells were bead beaten using a Vortex Shaker QL-861 (Haimen Kylin-Bell lab Instruments Co., Ltd, China) for 10 min, during which they were placed on ice for 1 min every 2 min. The lysed cells were centrifuged at 13,000g for 10 min at 4 °C to separate dissolved proteins. The supernatant containing dissolved protein was transferred into a new 1.5 mL Eppendorf tube. Protein concentrations were determined by the Bradford assay (Bradford et al., 1976) with bovine serum albumin as the standard. All the experiments were repeated triplicates. Dechlorination activity assays Dechlorination activity assays were performed in an anaerobic workstation using 20 μL crude protein extract with S. oneidensis MR-1 as a variable. The assays were conducted in 2 mL glass vials containing 1 mL anaerobic medium (vitamin B12 supplemented) with 450 μmol/L TCE or 100 μmol/L VC as substrate. Each mixture was incubated in a glove box for 24 h at 30 °C. Dechlorination products were analyzed by GC-FID as described previously. Assays with substrate and medium only, as well with substrate, medium, and denatured protein (10 min at 99 °C), were run as negative controls. The results are presented as the average values from triplicates.

2.3.2. DNA and RNA extractions Chromosomal DNA was collected from 1.5 mL liquid culture without concentrating the cells and extracted using an Ezup Column Bacteria Genomic DNA Purification Kit (Sangon Biotech, Shanghai, China). mRNA was extracted from 1.5 mL liquid culture using an RNAprep Pure Cell/Bacteria Kit (TianGen Biotech, Beijing, China) according to the manufacturer’s protocol. The mRNA was treated with DNase to remove contaminating DNA during the extraction process. Master mix for reverse transcription containing 10 μL FastKing gDNA Dispelling RT SuperMix (TianGen Biotech, Beijing, China), 4 μL mRNA and 6 μL double distilled water was prepared. The FastKing gDNA Dispelling RT SuperMix included FastKing RT Enzyme, RNase inhibitor, random primers, oligo dT primers, dNTP Mixture, reaction buffer and gDNase. Reverse transcription was performed in a DNA Engine Peltier Thermal Cycler (Bio-Rad, California, USA) with an incubation time of 15 min at 42 °C followed by 3 min at 95 °C so as to obtain cDNA. The synthetic cDNA was stored at −80 °C until subsequent analyses (DoğanSubaşı et al., 2014). 2.3.3. Real-time PCR Bacteria, S. oneidensis MR-1, Dehalococcoides and two RDase genes were quantified with the Bac16S (bacteria 16S rRNA gene), cymA (encoding a c-type cytochrome in the electron transfer chain of S. oneidensis MR-1), Dhc16S (Dehalococcoides-targeted 16S rRNA gene), tceA and vcrA (RDase genes of Dehalococcoides), respectively. For Bac16S and cymA genes, real-time PCR (RT-PCR) amplification was performed in 15 μL reactions, containing 7.5 μL SYBR Green mix (DBI Bestar, Ludwigshafen, Germany), 0.5 μL Rox, 0.5 μL of each primer, 2 μL DNA template and 4 μL double distilled water. TaqMan probes were used for Dhc16S, tceA and vcrA (Ritalahti et al., 2006; Wen et al., 2016). Each reaction contained 7.5 μL 2 × TaqMan Fast qPCR Master Mix (Sangon Biotech, Shanghai, China), 0.5 μL of each primer, 0.5 μL probe, 2 μL DNA template and 4 μL double distilled water. The RT-PCR for cymA was run for 10 min at 95 °C for Taq activation, followed by 40 cycles of 15 s at 95 °C, 30 s at 55 °C, 1 min at 72 °C and a melting curve stage from 55 °C to 95 °C. For Dhc16S, tceA and vcrA, the cycle parameters consisted of 15 s at 95 °C and 1 min at 58 °C without melting curves. In this experiment, the method limit of quantitation was 103 copies/mL. Primers and probes were synthesized by Sangon Biotech; sequences and reaction conditions are showed in Table S1.

3. Results and discussion 3.1. Dechlorination of TCE in the presence of S. Oneidensis MR-1 The dechlorination of TCE to ETH by the Dehalococcoides-containing culture with or without S. oneidensis MR-1 are shown in Fig. 1 (the concentration of vitamin B12 was 4.00 µg/L). The culture with the same amount of Dehalococcoides had the best dechlorination efficiency when

2.3.4. Microbial community structure At the beginning and the end of operation, 15 mL of inoculum samples were centrifuged at 8000g for 30 min (Hitachi CF16RXII, Japan) in Falcon tubes. The pellets were collected for DNA extraction according to the Ezup Column Bacteria Genomic DNA Purification Kit (Sangon Biotech, Shanghai, China). The DNA samples were sent to Sangon Biotech (Shanghai, China) to perform high-throughput 16S rRNA gene Illumina MiSeq sequencing. To target the conserved V3 to V4 regions of the bacterial 16S rRNA gene, primers 341F (5′-CCTACGGGNGGCWGCAG-3′) and 805R (5′-GACTACHVGGGTATCTAATCC-3′) were used. PCR amplification, PCR product purification and quantification, sequencing using the Illumina MiSeq platform. The detail procedures and data analysis were processed as described by Cui et al. (2016). Fig. 1. Dechlorination of TCE by different cultures. (a) Dehalococcoides-containing culture (with vitamin B12 supplemented), (b) Dehalococcoides-containing culture and S. oneidensis MR-1 (with vitamin B12 supplemented).

2.3.5. Dehalogenase analysis Crude protein extracts 60 mL of culture was flushed with N2-CO2 (80/ 20, vol/vol) to dislodge residual chlorinated compounds. Under 3

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Fig. 2. The microbial community structure of the two different cultures at genera level of 0 and 24 day (D represents the Dehalococcoides-containing culture and M represents S. oneidensis MR-1).

the OD600 = 0.1 of S. oneidensis MR-1 (Fig. S1). In the absence of S. oneidensis MR-1, TCE was completely reduced to ETH in 24 days, while DCE and VC accumulated at the peak values on days 2 and 10, respectively. In the presence of S. oneidensis MR-1, TCE was completely reduced to ETH in only 16 days; DCE and VC reached their peak values at days 2 and 4, respectively. The dechlorination experiment with VC as substrate was also conducted in the same condition. In the presence of S. oneidensis MR-1, the complete VC to ETH dechlorination was shortened from 8 days to 6 days (Fig. S2). The addition of S. oneidensis MR-1 obviously increased the dechlorination rate. Fig. 2 showed the microbial community structure at genus level of the Dehalococcoides-containing culture with or without S. oneidensis MR-1 at the beginning and end of the experiment. With S. oneidensis MR-1 addition, the relative abundance of S. oneidensis MR-1 decreased from 70.48% to 10.32% while the Dehalococcoides increased from 2.76% to 7.70% at the end of experiment. When S. oneidensis MR-1 was absent, the Dehalococcoides remained stable throughout the experiment. The rapid production and subsequent dechlorination of DCE and VC in the culture might be due to the increased abundance of Dehalococcoides. Previous studies have reported that Pelobacter SFB93 and Syntrophomonas wolfei could keep a long-term sustainable syntrophic association with Dehalococcoides in co-culture and they significantly improved the cell growth of Dehalococcoides by providing hydrogen (Mao et al., 2015, 2017). To investigate whether this effect was a result of cell growth or enhanced bioactivity of Dehalococcoides, Dhc16S was quantified and demonstrated similar trends with or without S. oneidensis MR1 (2.1 × 105 to 1.2 × 107 copies/mL from day 0 to day 24, Fig. S3; standard curves and amplification efficiency of different genes are displayed in Table S2). These results suggested that S. oneidensis MR-1 had no effect on the cell growth of Dehalococcoides. Therefore, the higher dechlorination rate upon addition of S. oneidensis MR-1 was a result of higher bioactivity of Dehalococcoides. However, the abundance of S. oneidensis MR-1 decreased by approximately 90% in 24 days, possibly due to the accumulation of intermediates DCE and VC that have been shown toxicity (Giri et al., 1995). The copy number of cymA gradually decreased to 6.1 × 106 copies/mL from the beginning of the production of VC, and the reduced rate was likely proportional to the concentration of VC. When VC was depleted, the copy number of cymA reached a stable level (Fig. S4). Various studies have also observed that chloroethenes are toxic to microorganisms, such as Pseudomonas putida F1, Acetobacterium, sulfate reducers (García-Solares et al., 2013; Singh et al., 2010; Ziv-El et al., 2012). Organisms other than Dehalococcoides may also contribute to the dechlorination of chloroethenes. For example, Geobacter lovleyi strain

SZ was described to dechlorinate PCE to DCE (Sung et al., 2006). Though Geobacters were also identified in the Dehalococcoides-containing culture, considering that very few Geobacters dechlorinate chloroethenes (Türkowsky et al., 2018) and the low abundance in the culture, the likelihood that these Geobacters were not main contributors for dechlorination. 3.2. Dechlorination activity of Dehalococcoides To investigate changes in the bioactivity of Dehalococcoides, the reverse transcript abundances of RDase genes were measured, results were shown in Fig. 3. There was no obvious difference for the abundance of tceA transcripts with or without S. oneidensis MR-1, which is consistent with the DCE accumulation curves. Along with DCE depletion, the tceA transcripts decayed exponentially with a half-life of approximately 2 days. However, the abundance of vcrA transcripts was approximately 1 order of magnitude higher in the presence of S. oneidensis MR-1 than in the culture without S. oneidensis MR-1 at day 12 (6.93 transcripts/gene vs. 0.59 transcripts/gene). In addition to the expression of RDase genes, the activity of RDase was also examined and used as biological marker of Dehalococcoides. In the presence of S. oneidensis MR-1, the dechlorination of TCE and VC by the same amount of crude protein extract was slightly higher (Table S3), confirmed the positive effect of S. oneidensis MR-1 on the activity of RDase.

Fig. 3. The expression of reductive dehalogenases genes in Dehalococcoidescontaining culture with or without S. oneidensis MR-1 (with vitamin B12 supplemented). 4

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The addition of S. oneidensis MR-1 may stimulate the expression of RDase genes by affecting the specific metabolites of Dehalococcoides and reducing conditions. For example, previous studies have reported the connection between RDase genes expression and the substrate and reducing conditions (Kranzioch et al., 2015; Xiu et al., 2010a). Firstly, S. oneidensis MR-1 could oxidize lactate to acetate, which can be used as a carbon source of Dehalococcoides. The concentration of acetate was slightly increased with the addition of S. oneidensis MR-1 (Fig. S5), and may contribute to the higher bioactivity of Dehalococcoides. More importantly, even though S. oneidensis MR-1 cannot synthesize vitamin B12 alone, it may facilitate other microbes in the Dehalococcoides-containing culture to produce and secrete vitamin B12 (Zhang et al., 2009). For example, a number of methanogens could produce vitamin B12 and possibly other cofactors necessary for Dehalococcoides, even if it competed hydrogen with Dehalococcoides (He et al., 2007). With the addition of S. oneidensis MR-1, the production of methane was significantly increased, and may increase the concentration of vitamin B12. On the 24th day, the concentration of vitamin B12 increased from 3.08 μg/L to 6.01 μg/L with the addition of S. oneidensis MR-1. The increased concentration of vitamin B12 also boost the genes expression and the activity of RDase. 3.3. Influence of co-factors on bioactivity 3.3.1. Dechlorination of TCE with different concentration of vitamin B12 To further explore the effect of vitamin B12 on the RDase genes expression, we set up cultures with different initial concentration of vitamin B12. Fig. 4 showed that the dechlorination rate increased along with the increased initial concentration of vitamin B12. Researches showed that the TCE dechlorination rate of Dehalococcoides ethenogenes 195 would double when the vitamin B12 concentration increased from 1 to 25 μg/L (He et al., 2007). The production of methane was gradually suppressed in pace with the higher initial concentration of vitamin B12. Combined the result of S. oneidensis MR-1, we concluded that S. oneidensis MR-1 increased the concentration of vitamin B12 by affecting the microbial community structure. Meanwhile, the expression of RDase genes was up-regulated due to the increased the initial concentration of vitamin B12 (Fig. S6). To investigate the effect of S. oneidensis MR-1 on the activity of RDase, we set up cultures without extrinsic vitamin B12. Before conducting this experiment, we transferred the inoculum several generations by adding TCE and other nutrients but without providing vitamin B12 to reach a culture without extrinsic vitamin B12. The vitamin B12 concentration of the last transferred culture was under detection limit. Compared with the culture having initial vitamin B12 concentration at 4 μg/L (Fig. 5a), the dechlorination rate of the culture without extrinsic vitamin B12 decreased dramatically (Fig. 5b), while VC accumulated as the main intermediate with no ETH detected at day 28. It was reported that VC accumulated significantly when the concentration of vitamin B12 was limited to 1 μg/L (Yan et al., 2013). Interestingly, in the no extrinsic vitamin B12 cultures in the presence of S. oneidensis MR-1, the dechlorination rates were slightly higher (Fig. 5c) than those without adding S. oneidensis MR-1 (Fig. 5b). VC was produced faster and then reduced at day 23, after which ETH was detected on the following day. Based on quantification of functional genes, the cell growth of Dehalococcoides was inhibited due to the lack of vitamin B12 (Fig. S7). In the no extrinsic vitamin B12 culture with the addition of S. oneidensis MR-1, Dhc16S and tceA increased slower and recovered to the same level as in the vitamin B12-amended culture while vcrA didn’t increase. In order to explain these results, we measured the concentration of vitamin B12 in different cultures. In the cultures without extrinsic vitamin B12, the concentration of vitamin B12 were 1.40 μg/L and 2.79 μg/L in cultures without and with S. oneidensis MR-1 at day 28. It indicated that the higher concentration of vitamin B12 stimulated by S. oneidensis MR-1 was responsible for the increased number of Dhc16S and tceA. However, the lower concentration of vitamin B12 couldn’t

Fig. 4. Dechlorination of TCE by Dehalococcoides-containing cultures with different extrinsic concentration of vitamin B12: (a) 1 µg/L; (b) 4 µg/L; (c) 8 µg/L; (d) 25 µg/L.

sustain the growth of Dehalococcoides which has vcrA, which might due to the different cobalamin requirement of tceA and vcrA. The similar phenomenon was found in the expression of RDase genes (Fig. S8). According to these results, S. oneidensis MR-1 likely affected the dechlorination of VC by mechanisms other than improved growth of Dehalococcoides or up-regulation vcrA gene in the no extrinsic vitamin B12 culture. Future study is needed to examine if S. oneidensis MR-1 as an electricigen can influence the redox state of vitamin B12. 3.3.2. Reduction of vitamin B12 by S. oneidensis MR-1 Fig. 6 showed that S. oneidensis MR-1 reduced approximately 50% of vitamin B12(III) in 7 days using lactate as the electron donor, generating vitamin B12(II). The concentration of TCE and vitamin B12 were 450 μmol/L and 50 μmol/L, respectively. The abundance of S. oneidensis MR-1 increased approximately tenfold over 7 days when vitamin B12 and TCE were present (Fig. S9). Liu reported that the cell number of S. oneidensis MR-1 increased as long as appropriate electron acceptors existed (Liu et al., 2017). However, when TCE was absent, the quantity of S. oneidensis MR-1 with vitamin B12(III) as electron acceptor 5

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reduction of vitamin B12(III) by S. oneidensis MR-1, coupled to the oxidation of lactate was of benefit to the dechlorination of chloroethenes. And the reduced vitamin B12(II) might be used more effectively by Dehalococcoides. Many studies reported the microbial reductive dechlorination via RDase, which contains vitamin B12, cooperated with iron sulfur clusters. The detailed enzymatic mechanism of dechlorination is still unclear. Payne proposed that RDase achieve reduction of the organohalide substrate via halogen–cobalt bond formation (Payne et al., 2015). However, these structures and mechanistic explanations are studied in RDase not stemming from Dehalococcoides. In this study, the intracellular and extracellular experiments proved that the increased concentration and the possible reduction of vitamin B12 by S. oneidensis MR-1 improved dechlorination. Reinhold also showed that low concentration of extrinsic vitamin B12 inhibit the expression of RDase genes and further affect the activity of RDase of Desulfitobacterium, which was similar to the low dechlorination rate of the culture without extrinsic vitamin B12 in this study (Reinhold et al., 2012). By oxidizing lactate to release electrons, S. oneidensis MR-1 reduced the high-valent vitamin B12, which might have a positive effect on the uptake of vitamin B12 by Dehalococcoides and improve the dechlorination efficiency of the culture. 4. Conclusion We studied the effect of S. oneidensis MR-1 on TCE dechlorination in a Dehalococcoides-containing culture and the interaction mechanism between S. oneidensis MR-1 and Dehalococcoides (Fig. S10). The complete TCE to ethene dechlorination was shortened from 24 days to 16 days with the addition of S. oneidensis MR-1. Based on absolute quantification of Dhc16S, tceA and vcrA, the cell growth of Dehalococcoides was not promoted with the addition of S. oneidensis MR-1. The expression of RDase genes and the activity of RDase was elevated, which might be caused by the increased concentration of vitamin B12. The mRNA abundance of vcrA was almost tenfold greater. In the culture without extrinsic vitamin B12, the S. oneidensis MR-1 might have a positive effect on the the RDase activity of Dehalococcoides and further improved its TCE dechlorination efficiency via generating vitamin B12. Overall, the elevated expression of RDase genes and activity of RDase may help solve the problems caused by the low abundance of Dehalococcoides and the likely accumulation of toxic intermediates. Meanwhile, the addition of S. oneidensis MR-1 had an effect on the growing environment of Dehalococcoides, which was benefit to dechlorination. These results further provided a practical vision of chloroethenes dehalogenation by the Dehalococcoides–containing culture.

Fig. 5. Dechlorination of TCE by different cultures. (a) Dehalococcoides-containing culture (with extrinsic vitamin B12), (b) Dehalococcoides-containing culture (without extrinsic vitamin B12), (c) Dehalococcoides-containing culture and S. oneidensis MR-1 (without extrinsic vitamin B12).

increased by only 58%, suggesting that the reduction of vitamin B12(III) by S. oneidensis MR-1 and oxidation of vitamin B12(II) played an essential role in reducing TCE (Table S4). Shewanella alga Strain BrY was reported to reduce vitamin B12(III) by oxidizing lactate or hydrogen, which further allowed the abiotic transformation of carbon tetrachloride, chloroform, and dichloromethane (Workman et al., 1997). Amir and Huang also reported that dechlorination could be performed by vitamin B12(III) with low-valent metal as an electron donor (Amir and Lee, 2012; Huang et al., 2013). Thus, it’s plausible that the

Fig. 6. Reduction of vitamin B12(III) to vitamin B12(II) by S. oneidensis MR-1 (V(III) represents vitamin B12(III) and V(II) represents vitamin B12(II); TP represents TCE present and TA represents TCE absent).

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Acknowledgments

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