Influence of non-dechlorinating microbes on trichloroethene reduction based on vitamin B12 synthesis in anaerobic cultures

Environmental Pollution 259 (2020) 113947

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Influence of non-dechlorinating microbes on trichloroethene reduction based on vitamin B12 synthesis in anaerobic cultures Li-Lian Wen a, b, 1, Yaru Li b, 1, Lizhong Zhu b, He-Ping Zhao b, * a

College of Resource and Environmental Science, Hubei University, Wuhan, 430062, China MOE Key Lab of Environmental Remediation and Ecosystem Health, College of Environmental and Resource Science, Zhejiang University, Hangzhou, 310058, China

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 31 July 2019 Received in revised form 28 September 2019 Accepted 7 January 2020 Available online 8 January 2020

In this study, the YH consortium, an ethene-producing culture, was used to evaluate the effect of vitamin B12 (VB12) on trichloroethene (TCE) dechlorination by transferring the original TCE-reducing culture with or without adding exogenous VB12. Ultra-high performance liquid chromatography - tandem mass spectrometry (UPLC-MS/MS) was applied to detect the concentrations of VB12 and its lower ligand 5,6dimethylbenzimidazole (DMB) in the cultures. After three successive VB12 starvation cycles, the dechlorination of TCE stopped mostly at cis-dichloroethene (cDCE), and no ethene was found; methane production increased significantly, and no VB12 was detected. Results suggest that the co-cultured microbes may not be able to provide enough VB12 as a cofactor for the growth of Dehalococcoides in the YH culture, possibly due to the competition for corrinoids between Dehalococcoides and methanogens. The relative abundances of 16 S rRNA gene of Dehalococcoides and reductive dehalogenase genes tceA or vcrA were lower in the cultures without VB12 compared with the cultures with VB12. VB12 limitation changed the microbial community structures of the consortia. In the absence of VB12, the microbial community shifted from dominance of Chloroflexi to Proteobacteria after three consecutive VB12 starvation cycles, and the dechlorinating genus Dehalococcoides declined from 42.9% to 13.5%. In addition, Geobacter, Clostridium, and Desulfovibrio were also present in the cultures without VB12. Furthermore, the abundance of archaea increased under VB12 limited conditions. Methanobacterium and Methanosarcina were the predominant archaea in the culture without VB12. © 2020 Elsevier Ltd. All rights reserved.

Keywords: Trichloroethene Reductive dechlorination Dehalococcoides Methanogens Vitamin B12

1. Introduction Trichloroethene (TCE) is a common contaminant in groundwater, mainly originating as discharges from metal degreasing sites and other factories (USEPA, 2001). It has been listed as a priority pollutant due to the risks it poses to human health, such as immune disorders and cancer (ASTDR, 2015; Ordaz et al., 2017). The maximum contaminant level of TCE in drinking water is 5 mg/L (USEPA, 2007). Anaerobic microbial reductive dechlorination (AMRD) is a viable and cost-effective method for TCE removal (Stroo et al., 2012; Zhao et al., 2010). Microbes capable of coupling the reductive dechlorination of TCE to energy conservation and biomass growth are called

* Corresponding author. E-mail address: [email protected] (H.-P. Zhao). 1 Share the first author. https://doi.org/10.1016/j.envpol.2020.113947 0269-7491/© 2020 Elsevier Ltd. All rights reserved.

organohalide-respiring bacteria (OHRB), and are widely present in the various environments including soils, sediments, and aquifers (Maphosa et al., 2010). OHRB belonging to the genera Anaeromyxobacter, Desulfitobacterium, Sulfurospirillum and Geobacter are metabolically versatile because of their broad range of electron donors and acceptors, but these microbes can only partially dechlorinate TCE to cis-dichloroethene (cDCE) (Maphosa et al., 2010). Dehalococcoides and Dehalogenimonas are the only bacteria able to achieve further reduction of VC to ethene (Yang et al., 2017). The dechlorinating capability of different microbes depends on their reductive dehalogenases (RDases). Several TCE RDases have been purified and characterized, including multiple PceAs, MbrA, TceA, VcrA, and BvcA (Hug et al., 2013). The PceAs, which were isolated from Desulfitobacterium, Sulfurospirillum, Dehalobacter, and Dehalococcoides, can catalyze TCE to cDCE (Neumann et al., 1996; Magnuson et al., 1998; Krasotkina et al., 2001). MbrA from Dehalococcoides dechlorinates TCE to trans-DCE (Chow et al., 2010). While TceA, VcrA and BvcA, which were exclusively identified from

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Dehalococcoides, can dechlorinate TCE, all DCE isomers, and vinyl chloride (VC) to ethene (Magnuson et al., 2000; Müller et al., 2004; Krajmalnik-Brown et al., 2004). Recently, new RDase genes involved in TCE dechlorination from Dehalogenimonas have been found and a putative VC RDase was identified (Yang et al., 2017). Corrinoid is an essential part of RDases. A complete corrinoid, namely cobamide, is composed of three parts: an upper ligand, a cobalt-containing corrin ring, and a lower base. Cobamides are categorized in three groups, benzimidazole, purine, and phenolic, based on the structure of lower base (Men et al., 2015). Yi et al. (2012) showed that Dehalococcoides mccartyi strain 195, which contains the RDase TceA, could only utilize benzimidazole cobamides and remodel other corrinoids with a functional benzimidazole base. Researchers further found that the dechlorinating rates and extents of D. mccartyi strains harboring the RDases VcrA and BvcA were affected by the lower bases of corrinoids such that the dechlorinating activity was highest in the presence of 5,6dimethylbenzimidazolyl-cobamide. However, due to the rigorous growth conditions, the prosthetic cofactor of RDases from D. mccartyi remains elusive. Vitamin B12 (VB12), one of benzimidazole cobamides, carries the cyano group as the upper base and 5,6-dimethylbenzimidazole (DMB) as the lower base (Roth et al., 1996). VB12 is an indispensable growth cofactor for D. mccartyi (Maymo-Gatell et al., 1995; He et al., 2007). Dehalococcoides have no ability to de novo produce corrinoids, as confirmed by the genome sequence of the D. mccartyi strain 195 (Seshadri et al., 2005). However, they can utilize the VB12 exogenously added into cultures. He et al. (2007) indicated that the biomass and reductive rates of D. mccartyi strain 195 increased along with increasing concentrations of VB12 with an optimum VB12 concentration of 25 mg/L. In addition, Dehalococcoides can take up VB12 produced by other microbes in co-culture or salvage and remodel other corrinoids with an appropriate free lower ligand (Yi et al., 2012). Many microorganisms are capable of de novo synthesizing corrinoids. Clostridium, Desulfovibrio, Acetobacterium, and Geobacter can generate VB12 and other types of corrinoids, eg. 50 -methylbenzimidazolyl cobamide, in specific circumstances (Stupperich et al., 1988; Yan et al., 2012; Men et al., 2012). He et al. (2007) found that the dechlorination efficiency of D. mccartyi strain 195 was significantly promoted when cocultured with Desulfovibrio and Acetobacterium. Yan et al. (2012) found that Geobacter lovleyi facilitated the growth of D. mccartyi strains BAV1 and FL2, and Geobacter sulfurreducens benefited Dehalococcoides growth only if the cultures were supplemented with DMB. In addition, the archaea Methanobacterium and Methanococcus can also synthesize 50 -hydroxybenzimidazolyl cobamide and pseudo-VB12 (Stupperich et al.,1990). However, most TCE dechlorinating cultures are complex mixtures including fermenters, acetogens, methanogens, and dechlorinators. Previous studies showed that the co-cultured microorganisms affected the dechlorination efficiencies due to electron competition (Wen et al., 2015). However, whether the VB12 synthesized by other microbes is enough for the growth of Dehalococcoides remains unclear, as VB12 also plays an important role in the metabolism of folate, methionine, and ubiquinone (Romine et al., 2017). Therefore, the objective of this study is to evaluate if TCE dechlorination remains steady in the YH culture without exogenous VB12 amendment, and further to understand the metabolic interactions within the microbial community.

2. Materials and methods 2.1. Description of the ethene-producing enrichment culture The ethene-producing enrichment culture YH has been

maintained in the laboratory for 4 years with lactate as the sole electron donor and ATCC vitamin supplement and VB12 as growth factors (Kranzioch et al., 2013; Wen et al., 2017a). The culture is dominated by Dehalococcoides, the main dechlorinating bacteria, and non-dechlorinating bacteria including Geobacter, Clostridium, Petrimonas, Syntrophobacter, and Desulfovibrio, which probably provide necessary cofactors (eg. VB12) for the growth of Dehalococcoides. 2.2. The effect of VB12 on the ethene-producing culture We prepared an anaerobic medium for the experiment as described by Wen et al. (2015). The mineral salts medium contained the following reagents (per liter): 3.17 g KH2PO4, 14.33 g Na2HPO412H2O, 0.45 g (NH4)2HPO4, 0.04 g MgHPO43H2O, 1 mL of trace element solution A, and 1 mL of trace element solution B (Kranzioch et al., 2013). The medium was boiled and flushed with argon (Ar) gas for 15 min to remove oxygen. We added 0.2 mM Lcysteine, 0.2 mM Na2S9H2O and 0.5 mM DL-dithiothreitol (DTT) into the medium to create reducing conditions. In addition, we added 10 mM NaHCO3 and 10 mM tris-ethanesulfonic acid (TES) as buffering agents (He et al., 2007), and 0.025% (vol/vol) resazurin as a redox indicator (Wen et al., 2016). Then, we transferred 75 mL of medium into 120-mL glass serum bottles under a stream of Ar gas and sealed the bottles with butyl rubber stoppers and aluminum crimps. Finally, the medium was sterilized at 121  C for 30 min and stored in an anaerobic chamber (Britain, AW200SG). We added 200 mL lactate (1 M in a stock solution, and final concentration was 2.5 mM) as the sole electron donor and carbon source; 2.4 mL TCE (99.9% in purity, and final concentration was 0.33 mM) as the electron acceptor. No ATCC vitamin supplement (ATCC MD-VS, U.S.A.) or VB12 was added. To investigate the effect of VB12 on the ethene producing culture, we performed sub-culturing three times. First, we transferred 5 mL YH culture into bottles amended with 0 mg/L VB12 (O1) or 100 mg/L VB12 (V1); second, we transferred 5 mL from O1 subculture bottle into bottles amended with 0 mg/L VB12 (O2) or 100 mg/L VB12 (V2); third, we transferred 5 mL from O2 subculture bottle into bottles amended with 0 mg/L VB12 (O3) or 100 mg/L VB12 (V3). Each bottle was sampled periodically for TCE analysis (every 12 h during TCE reduction; every 2 days after TCE was completely reduced). All experiments were performed with duplicate bottles. The results are presented as the average values from the duplicates. 2.3. Chemical analysis Chlorinated ethenes (TCE, cDCE, VC), ethene, and methane were measured by injecting 100 mL of headspace samples with a gastight syringe into a gas chromatograph (Agilent Technologies GC system, model 6890N, Agilent Technologies Inc., U.S.A.) equipped with a flame-ionization detector (FID), and a packed column (30 m long, 0.32 mm i.d., 0.5 mm thickness, cross-linked polydimethysiloxane film, J&W scientific, U.S.A.) (Ziv-El et al., 2011; Wen et al., 2017b). N2 was the carrier gas fed at a constant flow rate of 0.065 m3/d, and the temperature conditions for injector and detector were 200 and 250  C, respectively. The program was as follows: holding at 60  C for 1 min, heating gradually to 200  C  (20 C/min), and holding at 200  C for 2 min. Analytical grade chloroethenes, ethene, and methane were added into 80 mL of water in 120 mL bottles to make standards for calibration curves, which were linear (R2  0.996). We calculated the concentrations of ethene and methane in the liquid according to their Henry’s constants (KH): ½Compoundliq ¼ ½Compoundgas =KH The calculated dimensionless Henry’s constants (mMgas/mMliq,

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T ¼ 25  C) used in this study were 8.35 for ethene and 28.99 for methane. The volatile fatty acids (VFAs) lactate, acetate, and propionate were analyzed using liquid chromatography (LC, Waters) equipped with a 1525 Binary Pump, a 717 plus Autosampler, a 2487 Dual l Absorbance Detector and an organic acid column (Acclaim™ OA 5 mm, 4  250 mm) (Wen et al., 2017b). The monitored parameters were as follows: the mobile phase was 100 mM Na2SO4, the pH was adjusted to 2.65 with methylsulphonic acid (MSA), the flow rate was 0.6 mL/min, the column temperature was set at 30  C, the absorbance wavelength was 210 nm, and the injection volume was 10 mL. Liquid samples (1 mL) were filtered through a 0.22-mm polyvinylidene fluoride membrane syringe filter (Shanghai Xingya Purifying Materials Company, China) into 1 mL glass vials for subsequent analysis. Calibration curves were generated for all VFAs during every HPLC run. The detection limits for VFAs on the HPLC were 0.1 mg/L. VB12 and DMB were detected using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assembled with an UPLC (Acquity I-Class, Waters), a liquid chromatograph-triple quadrupole mass spectrometer (AB SCIEX QTRAP® 5500, USA) and a column (ACQUITY UPLC® BEH C18, 1.7 mm, 2.1  50 mm). LC was performed at 0.3 mL/min with initial mobile phase conditions of 90% miliQ water with 0.1% formic acid (A) and 10% methanol with 0.1% formic acid (B) held for 2 min,

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increased to 95% B over 2 min and held for 2 min, and decreased back to 10% B over 0.1 min and held for 1.9 min. The column temperature was set at 40  C, and the injection volume was 1 mL. The declustering potential was set at 120 V for the MS2 scan, and the collision energy was set at 42 V for product ion scanning. Liquid samples (1 mL) were filtered through a 0.22-mm polyvinylidene fluoride membrane syringe filter (Shanghai Xingya Purifying Materials Company, China), and final samples for subsequent analysis contained 10% methanol. Calibration curves were generated for VB12 and DMB during every run. The detection limits for VB12 and DMB on the UPLC-MS/MS were 25 and 10 ng/L respectively.

2.4. Molecular analysis At the end of experiment, 30 mL of liquid samples were transferred into 50-mL centrifuge tubes and then centrifuged for 1 h at 8000 rpm (5900 g) at 4  C (Eppendorf 5415R, Germany). We collected the pellets for DNA extraction as described by Wen et al. (2017b). We used SYBR Premix Ex Taq Kits (Takara Bio Inc., Japan) and performed qPCR amplification to target Dhc (for Dehalococcoides), mcrA (for methanogens), FTHFS (for acetogens) and the functional reductive dehalogenase genes tceA, vcrA and bvcA (Wen et al., 2015). The slopes of the plasmid standard curves and efficiency values for quantification by qPCR are listed in Table S1. We

Fig. 1. The time course of TCE reductive dechlorination in mixed consortia amended without VB12 (A, B, C) and with VB12 (A1, B1, C1). Transfer 1 cultures were originated from the YH culture, transfer 2 cultures were developed from the transfer 1 culture without VB12, and transfer 3 cultures were derived from the transfer 2 culture without VB12 (Fig. S1). The left Y-axis means the concentrations of chlorinated ethenes and ethene.

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calculated gene copy numbers for biomass samples using the standard curves. DNA samples were sent to Novogene (Beijing, China) to perform Illumina MiSeq sequencing with standard protocols including amplicon generation, which used primers 341F (50 CCTAYGGGRBGCASCAG - 30 ) and 806R (50 - GGACTACNNGGGTATCTAAT - 30 ) to target the conserved V3 to V4 regions of the bacterial 16 S rRNA gene (Caporaso et al., 2010; Wen et al., 2017b; Lv et al., 2019). The data were processed using the QIIME (version 1.7.0) pipeline (Caporaso et al., 2010). 3. Results and discussion 3.1. TCE reductive dechlorination under VB12 starvation We performed three consecutive subculture experiments to explore the effects of VB12 on TCE reduction (Fig. 1, Fig. S1). TCE was degraded to cDCE within 2 days in all the bottles tested. Reduction of DCE and VC was severely inhibited in the cultures without exogenous VB12 compared to the cultures with VB12, where DCE and VC were completely converted to ethene at Day 16. Transfer 1 cultures (Fig. 1 A &A-1) derived from the YH microcosms maintained TCE-to-ethene dechlorination activity without added VB12, which may be due to certain cofactors (eg. VB12) being carried over from the YH inoculum. Transfer 2 cultures (Fig. 1 B & B-1) developed from O1 microcosms (Transfer 1 cultures without VB12, see Fig. S1) showed distinct TCE dechlorination patterns. TCE was completely reduced to ethene at Day 12 in cultures with added VB12, but only ~39 mmol/L ethene was produced. VC was the main product accumulated in the cultures without VB12 (O2 bottles). In the transfer 3 cultures (Fig. 1 C & C-1) originating from O2 microcosms (Transfer 2 cultures without VB12), TCE dechlorination was severely inhibited. In O3 bottles, DCE accumulated and was partially converted to VC, and no ethene was detected in the cultures. After two successive VB12 starvation cycles, the TCE-toethene dechlorination activity was restored in cultures again amended with VB12, indicating that VB12 was important for reductive dechlorination of DCE and VC. Men et al. (2013) showed that dechlorination of TCE to VC and ethene occurred in cultures without VB12 present. However in this study, the cultures without exogenous VB12 lost the capability of producing ethene, suggesting that other organisms could not provide enough cofactors (eg. VB12) for Dehalococcoides in the tested cultures. During our experiment, we also measured the concentration of methane (Fig. 1). In the cultures with added VB12, the concentration of methane was lower, maintained at the level of 0e50 mmol/L. In the cultures without VB12, the concentration of methane increased as the sub-culturing generation increased (up to ~240 mmol/L). In typical TCE dechlorinating consortia, methanogens are usually considered as the main competitors for electron donors, causing the dechlorination of TCE to lag when the electron donors are limiting (Wen et al., 2015). Moreover, methanogens also require corrinoids as growth cofactors (Kenealy and Zeikus, 1981), hence

there might be competition between Dehalococcoides and methanogens for cofactors under VB12 limited condition, resulting in a lower dechlorination activity of the culture without exogenous VB12. 3.2. The concentrations of VB12 in mixed consortia Table 1 shows the concentrations of VB12 and DMB at the beginning and end of the tests in all the cultures. In the cultures with added VB12, the initial concentrations of VB12 for transfer 1, 2, and 3 were 12.42, 2.39, and 1.81 mg/L respectively, and all were consumed at the end of the experiment (the level of VB12 in all the cultures was negligible at the end). He et al. (2007) found that 1 mg/ L VB12 was enough to support Dehalococcoides growth. In the cultures without added VB12, VB12 was never detectable, but DMB (the lower ligand of VB12) was detected. The initial concentration of DMB ranged from 2.16 to 6.69 mg/L. Nevertheless, the conversion of TCE to ethene only occurred in the cultures with 6.69 mg/L DMB, which possibly suggested that some microorganisms along with Dehalococcoides could salvage DMB and remodel it into necessary cofactors (eg. VB12). Many microbes observed in the dechlorinating enrichments can biosynthesize corrinoids, including Geobacter, Desulfovibrio, Clostridium, and Methanobacterium (Stupperich et al., 1988, 1990; Yan et al., 2012; Men et al., 2012). Hazra et al. (2015) reported that some anaerobic microorganisms synthesize DMB. 3.3. Functional gene abundance Fig. 2 shows the relative abundance of functional genes of transferred consortia with and without VB12. Overall, the copies of 16s rRNA genes of Dehalococcoides (Dhc) and its functional genes tceA and vcrA in cultures amended with VB12 were higher than the cultures without VB12, which is consistent with the time courses of TCE dechlorination shown in Fig. 1. In subsequent cultures, Dehalococcoides genes increased in cultures added with VB12 (V1O2>O3). The TceA RDase is responsible for the conversion of TCE to VC, and the transformation of TCE to ethene is mainly driven by the VcrA RDase. Thus in cultures without added VB12, reductive dechlorination mediated by TceA or VcrA was inhibited due to VB12 limitation. However, when there was enough VB12, ethene production recovered, indicating that the organisms containing TceA or VcrA were resistant to VB12 starvation. In all the cultures, the copy number of bvcA was relatively low, a possible consequence of a small portion of Dehalococcoides strains carrying bvcA in YH culture maintained with TCE as electron acceptor. The BvcA RDase catalyzes all DCE isomers and VC to ethene, and bvcA was detected in some VC-respiring cultures. However, when cultures were enriched with TCE as the electron acceptor, eg. BDI and PRTB cultures, bvcA was maintained at a low abundance (Krajmalnik-Brown et al., 2004; Amos et al., 2008; Wen et al., 2017a). The abundance of the bvcA gene in the V2 culture was higher than in the V1 culture, but lower in V3 culture, indicating that after two successive VB12

Table 1 The concentrations of VB12 and DMB at the stages of initial and end of the experiments in mixed consortia with VB12 and without VB12 (ND means not detectable). Cultures without VB12

Treatments

Cultures with VB12

Concentrations mg/L

VB12

DMB

VB12

DMB

12.42 ± 0.36 ND 2.39 ± 0.42 ND 1.81 ± 0.05 0.01 ± 0.00

1.09 ± 0.08 ND 0.09 ± 0.01 0.05 ± 0.00 0.05 ± 0.00 0.03 ± 0.00

ND ND ND ND ND ND

6.69 0.87 1.91 0.20 2.16 0.06

Transfer 1 Transfer 2 Transfer 3

0 day 18 day 0 day 16 day 0 day 16 day

± ± ± ± ± ±

1.22 0.01 0.56 0.05 0.23 0.01

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Fig. 2. The abundance of genes in mixed consortia. O1- Transfer 1 without VB12; O2Transfer 2 without VB12; O3-Transfer 3 without VB12; V1- Transfer 1 with VB12; V2Transfer 2 with VB12; V3-Transfer 3 with VB12.

starvation cycles, the BvcA enzyme activity of isolates could not recovered.

3.4. Shifts in microbial community structure Fig. 3 shows the microbial community structures in the tested cultures at phylum level while Fig. 4 is at genus level. The different patterns of dealing with VB12 reshaped the microbial community structures of YH culture. In the presence of VB12, the dominant bacteria were Chloroflexi, Spirochaetes, and Firmicutes, accounting for more than 30%, 20%, and 18% respectively in the V1 culture. After two consecutive VB12 starvation cycles, once VB12 was again added, Chloroflexi, Spirochaetes, and Bacteroidetes dominated in the V3 culture. In the absence of VB12, the microbial community shifted from dominance of Chloroflexi in the O1 and O2 cultures to Proteobacteria in the O3 culture. Consistent with this taxonomic shift, the dechlorinating genus Dehalococcoides was abundant in the O1 and O2 cultures (~40%), but decreased to ~13% in the O3 culture. The population change of Dehalococcoides among O1, O2, and O3 is significant (P value is 0.004 calculated by one-way

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Fig. 4. The microbial community structure of tested consortia at the end of experiments at genus level. O1- Transfer 1 without VB12; O2- Transfer 2 without VB12; O3Transfer 3 without VB12; V1- Transfer 1 with VB12; V2- Transfer 2 with VB12; V3Transfer 3 with VB12; YH- Inoculum culture.

analysis of variance (ANOVA) using SPSS). The decrease in Dehalococcoides might hinder the complete dechlorination of TCE, and we previously reported that an absolute high abundance of active Dehalococcides was vital for TCE dechlorination (Wen et al., 2017a). Geobacter was present in the subcultures without VB12, accounting for 4.3%e11.9%. Geobacter can partly dechlorinate TCE to cDCE with acetate as an electron donor and provides corrinoids for the growth of Dehalococcoides (Yan et al., 2012). Clostridium and Desulfovibrio were also present in the tested cultures; these organisms possibly fermented lactate to acetate and hydrogen (Zhao et al., 2008; Men et al., 2012), which then could be used as carbon source and electron donor for Dehalococcoides. In addition to bacteria, the cultures contained Euryarchaeota, which increased from 0.8% in the O1 culture to 4.8% in the O2 culture and 5.4% in the O3 culture. Methanobacterium and Methanosarcina were the predominant archaea in the O3 culture, accounting for 4.0%, and 1.3% respectively. Methanobacterium can generate corrinoids, specifically those involved in methanogenesis (Kenealy and Zeikus, 1981; Stupperich et al., 1990).

3.5. The mechanism of VB12 effect on TCE dechlorinaiton

Fig. 3. The microbial community structure of tested consortia at the end of experiments at phylum level. O1- Transfer 1 without VB12; O2- Transfer 2 without VB12; O3Transfer 3 without VB12; V1- Transfer 1 with VB12; V2- Transfer 2 with VB12; V3Transfer 3 with VB12; YH- Inoculum culture.

Combining the fact that TCE dechlorination was inhibited severely due to VB12 limitation, and the observation that relative abundances of Clostridium, Geobacter, Desulfovibrio, Methanobacterium, Methanosarcina, and Dehalococcoides changed along with the different concentrations of VB12, we propose a possible working mechanism of the effect of VB12 on dechlorination by the YH culture (Fig. 5). Dehalococcoides is a VB12 auxotrophic bacterium and requires VB12 as a necessary cofactor. VB12 can be supplied by exogenous amendment or other organisms in the culture. Clostridium, Geobacter and Desulfovibrio were the major corrinoidproducers in the YH culture without exogenous VB12 amendment. Clostridium and Desulfovibrio produced acetate, hydrogen, and corrinoids, which provide Dehalococcoides with a carbon source, an electron donor, and cofactors. Stupperich et al. (1988) reported that Clostridium formicoaceticum produces 5-methoxy-6methylbenzimidazolyl cobamide and VB12. Geobacter possesses a near-complete de novo corrinoid biosynthesis pathway, and can generate VB12 or other forms of corrinoids (Yan et al., 2012). In addition, it was the major bacterium to convert TCE to cDCE under the VB12-limiting conditions. In the presence of specific corrinoids

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isolates containing TceA or VcrA were more resistant to VB12 starvation. VB12 limitation changed the microbial community structures of the consortia. In the absence of VB12, the microbial community shifted from dominance of Chloroflexi in the O1 culture to Proteobacteria in the O3 culture. The abundance of archaea increased under VB12-limiting conditions. Methanobacterium and Methanosarcina were the predominant archaea in the O3 culture, accounting for 4.0%, and 1.3% respectively. Acknowledgments Authors greatly thank the “National Natural Science Foundation of China (Grant No. 21377109, and 21577123)”, the “National Key Technology R&D Program (2018YEC1802203)” and “National Key Research and Development Program of China (2017TFA0207002)” for their financial support. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.envpol.2020.113947. Fig. 5. The possible mechanism of VB12 effect on the dechlorination of TCE in the tested cultures.

like benzimidazole cobamide and 5-methoxybenzimidazole, Dehalococcoides could only dechlorinate TCE to VC, but when the appropriate lower base such as DMB was present in the culture, Dehalococcoides remodeled corrinoids into VB12 and achieved complete dechlorination of TCE. DMB was detected but at low levels in the cultures without exogenous VB12 amendment, which indicated that some microorganisms could produce DMB anaerobically (Men et al., 2015). However, other microbes also require corrinoids, including fermenters and methanogens. In the all cultures tested, lactate was fermented to propionate in 2 days (Fig. S2), which indicated that fermenters might preferentially utilize corrinoids. The activity of methanogens increased in the cultures without VB12 compared with the cultures with VB12. Methanogens possibly took up corrinoids synthesized by themselves or other microbes. Meanwhile, the biosynthesis of folate, methionine, and ubiquinone also requires VB12 (Romine et al., 2017). These microbes possibly inhibited TCE dechlorination by competing for corrinoids, and as a result, there were not enough VB12 for Dehalococcoides to further dechlorinate the DCE and VC to ethene.

4Conclusion The effect of VB12 on TCE reduction was evaluated by conducting three sub-culturing events in mixed consortia without exogenous VB12 amendment. Compared to the cultures with added VB12, TCE dechlorination was severely inhibited and cDCE accumulated as the main product, with only partial to VC; no ethene was detected in the cultures after three successive VB12 starvation cycles. Methane production increased as the sub-culturing generation increased in the cultures without VB12. Therefore, we conclude that other microbes could not provide enough cofactors (eg. VB12) to support the growth of Dehalococcoides in the YH culture, possibly due to competition for corrinoids between Dehalococcoides and methanogens. In the cultures without added VB12, VB12 was not detectable, but DMB was present, and the conversion of TCE to ethene only occurred in the cultures with concentrations of DMB above a certain threshold, suggesting that Dehalococcoides could salvage other types of corrinoids in the cultures and remodel them into necessary cofactors (eg. VB12). The qPCR results indicated that the

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