Environmental Science and Pollution Research (2023) 30:45231–45243 https://doi.org/10.1007/s11356-023-25376-z RESEARCH ARTICLE Combining nanoscale zero‑valent iron and anaerobic dechlorinating bacteria to degrade chlorinated methanes and 1,2‑dichloroethane Dani Salom1 · David Fernández‑Verdejo1 · Javier Moral‑Vico1 · Xavier Font1 · Ernest Marco‑Urrea1 Received: 16 August 2022 / Accepted: 13 January 2023 / Published online: 27 January 2023 © The Author(s) 2023 Abstract Nanoscale zero-valent iron (nZVI) has the potential to degrade a diversity of chlorinated compounds, and it is widely used for remediation of contaminated groundwaters. However, some frequently detected contaminants such as dichloromethane (DCM) and 1,2-dichloroethane (1,2-DCA) have shown nearly no reactivity with nZVI. Here, we tested the feasibility of com- bining anaerobic dechlorinating bacteria, Dehalobacterium and Dehalogenimonas, and nZVI as a treatment train to detoxify chlorinated methanes (i.e., chloroform-CF- and DCM), and 1,2-DCA. First, we showed that CF (500 μM) was fully degraded by 1 g/L nZVI to DCM as a major by-product, which was susceptible to fermentation by Dehalobacterium to innocuous products. Our results indicate that soluble compounds released by nZVI might cause an inhibitory impact on Dehalobacte- rium activity, avoiding DCM depletion. The DCM dechlorination activity was recovered when transferred to a fresh medium without nZVI. The increase in H ­ 2 production and pH was discarded as potential inhibitors. Similarly, a Dehalogenimonas- containing culture was unable to dichloroeliminate 1,2-DCA when exposed to 1 g/L nZVI, but dechlorinating activity was also recovered when transferred to nZVI-free media. The recovery of the dechlorinating activity of Dehalobacterium and Dehalogenimonas suggests that combination of nZVI and bioremediation techniques can be feasible under field conditions where dilution processes can alleviate the impact of the potential inhibitory soluble compounds. Keywords Nanoscale zero-valent iron · Organohalide respiring bacteria · Dehalobacterium · Dehalogenimonas · Chlorinated organic pollutants Introduction The use of nZVI has been encouraged due to its greater specific surface area and higher degradation rates in compar- Nanoscale zero-valent iron (nZVI) is an emerging technol- ison with conventional iron powder (Wang and Zhang 1997). ogy to remediate groundwaters polluted with chlorinated In addition, nZVI allows in situ groundwater treatments by organic compounds, which are persistent and toxic pollut- direct injection under pressure or by gravity into groundwa- ants that threaten the environment and health (Koenig et al. ter, which is preferred to ex situ treatments (i.e., pump and 2015; Wang et al. 2016; Zhang 2003; El-Shahawi et al. treat) because of the lower cost and energy consumption, 2010). Reductive dehalogenation carried out by nZVI is a reduced worker exposure, and limited site disruption (Elliott surface-mediated reaction in which electrons are transferred and Zhang 2001; Wang et al. 2016). from nZVI to organochlorines and produce lesser chlorin- Despite the reactivity of nZVI toward a broad spec- ated compounds and various oxides on the surface with the trum of chlorinated compounds, dichloromethane (DCM) ­ l− and H release of C ­ 2 (Wang and Zhang 1997; Zhang 2003). and 1,2-dichloroethane (1,2-DCA) have shown nearly no reactivity with nZVI (Koenig et al. 2016; Song and Car- raway 2006; Song and Carraway 2005; Tobiszewski and Responsible Editor: Gerald Thouand Namiesnik 2012). This presents a problem for the remedia- tion of groundwater contaminated with complex chemical * Ernest Marco‑Urrea ernest.marco@uab.cat mixtures containing DCM and 1,2-DCA as primary pol- lutants. Interestingly, another abiotic source of DCM in 1 Departament d’Enginyeria Química, Biològica i Ambiental, groundwaters is the hydrogenolysis of chloroform (CF) by Universitat Autònoma de Barcelona (UAB), 08193 nZVI, which accumulates in the medium with no further Bellaterra, Barcelona, Spain 13 Vol.:(0123456789) 45232 Environmental Science and Pollution Research (2023) 30:45231–45243 degradation (Song and Carraway 2005). In this scenario, interfere with respiration due to their reducing power, and the approach of coupling nZVI treatments with functional extensive DNA and protein damage by the generation of anaerobic dechlorinating bacteria is promising to fully reactive oxygen species (ROS) if nanoparticles enter into the detoxify such chlorinated compounds (Chen et al. 2014; Liu cytoplasm (Koenig et al. 2015; Rangan et al. 2020; Saccà et al. 2022; Kocur et al. 2015; Kocur et al. 2016; Wang et al. et al. 2014; Wang et al. 2016; Xie et al. 2017). 2016), but the effect of nZVI on bacterial degraders has been In this work, we investigated the impact of nZVI on two poorly explored yet. bacterial models capable of detoxifying DCM and 1,2-DCA. Due to the frequent anoxic conditions of contaminated First, we intend to evaluate the feasibility of a treatment groundwaters, organohalide-respiring bacteria (ORB) train to transform CF to DCM via nZVI and the subsequent are commonly used for the detoxification of chlorinated fermentation of DCM to acetate via a Dehalobacterium-con- compounds. At the heart of this process are membrane- taining culture. For this, the effect of different concentrations bound reductive dehalogenases, which use chlorinated of nZVI on the chemical reduction of CF and the inhibi- compounds as electron acceptors to generate cellular tion of Dehalobacterium was studied. Second, 1,2-DCA has energy while using ­H2 as electron donor (Koenig et al. 2015; shown no reactivity with nZVI, but it can be used as electron Richardson 2013; Wang et al. 2016). As for nZVI, enhanced acceptor by the ORB belonging to the Dehalogenimonas biological reductive dechlorination has been proven to genus. Therefore, we studied the inhibitory effect of nZVI on be an effective in situ treatment to remove chlorinated a Dehalogenimonas-containing culture that transformed 1,2- compounds in anoxic groundwaters with a relatively DCA to ethene. This study provides valuable information to low cost compared to other physicochemical techniques delineate remediation strategies that combine both anaerobic (Chen et al. 2015; Blázquez-Pallí et al. 2019). To date, dechlorinating bacteria and nZVI at contaminated sites. dichloroelimination of 1,2-DCA to ethene under anaerobic conditions has been demonstrated for several ORB, including Dehalococcoides mccartyi, Dehalogenimonas, Materials and methods Desulfitobacterium, Sulfurospirillum, and Dehalobacter (De Wildeman et al. 2003; Maymó-Gatell et al. 1999; Moe nZVI synthesis and bacterial cultures et al. 2009; Grostern and Edwards 2006; van der Zaan et al. 2009). On the other hand, anaerobic DCM degradation is a Particles of nZVI were synthesized as described elsewhere relatively novel pathway for which only three bacteria, all (Wang and Zhang 1997). Briefly, nZVI was synthesized belonging to Peptococcaceae family, have been identified: using a 3.6 M sodium borohydride ­(NaBH4, ≥ 98%, Sigma- “Candidatus Formimonas warabiya” (formerly referred Aldrich) aqueous solution and a 0.9 M ferrous chloride (III) as strain DCMF), Dehalobacterium formicoaceticum, and ­(FeCl3, ≥ 98%, Sigma-Aldrich) aqueous solution. Both solu- “Candidatus Dichloromethanomonas elyuquensis” (Mägli tions were prepared using ultrapure water (Milli-Q). Water et al. 1998; Chen et al. 2017a; Kleindienst et al. 2019; used to prepare the ­FeCl3 solution was previously deoxygen- Holland et al. 2021; Trueba-Santiso et al. 2020). ated by bubbling with N ­ 2. Then, the N ­ aBH4 solution was To adequately detoxify sites with complex mixtures added dropwise to the ­FeCl3 solution while stirring at 200 containing CF and 1,2-DCA, a combined strategy includ- rpm and bubbling with N ­ 2. Then, nZVI was washed three ing both nZVI and anaerobic dechlorinating bacteria can times using anaerobic ultrapure water, and it was finally be of interest. For instance, CF has been reported to inhibit stored in a serum bottle and sealed with Teflon-coated butyl microbial reductive dechlorination of chloroethenes, chlo- rubber aluminum crimp caps until use to keep anaerobic roethanes, chlorophenols, and DCM (Bagley et al. 2000; conditions. Trueba-Santiso et al. 2020; Futagami et al. 2013), but nZVI The cultures containing Dehalogenimonas and Dehalo- might alleviate such inhibition by decreasing CF concen- bacterium derived from the Besòs River (Barcelona, Spain) tration yielding DCM. On the other hand, the use of nZVI estuary sediments and slurry samples collected from a can benefit ORB by creating the strong reducing conditions membrane bioreactor operating in a centralized indus- required for their growth and generating H ­ 2 as electron donor trial wastewater treatment plant, respectively. Both cul- for organohalide respiration (Kocur et al. 2015; Kocur et al. tures were cultivated for more than 5 years in an anaerobic 2016; Koenig et al. 2016; Xiu et al. 2010). At the same time, defined medium described elsewhere (Trueba-Santiso et al. anaerobic dechlorinating bacteria can detoxify 1,2-DCA and 2017; Martín-González et al. 2015). Briefly, both media DCM, which showed almost no reactivity with nZVI. How- contained vitamins and trace elements ­(KH2PO4, ­NH4Cl, ever, the presence of nZVI can be detrimental to microbial ­CaCl2, ­MgCl2·6H2O, KCl, ­CaCl2·2H2O, NTA, ­FeCl2·4H2O, reductive dechlorination. Reasons for this nZVI toxicity are ­ZnCl2, ­MnCl2·2H2O, ­CoCl2·6H2O, ­H3BO3, ­NiCl2·6H2O, not still clear. nZVI particles can interact with cell mem- and ­NaMoO4·H2O) and N ­ a2S·9H2O and L-cysteine (0.2 branes causing disturbances in cell membrane integrity and mM each) as reducing agents. Dehalobacterium cultures 13 Environmental Science and Pollution Research (2023) 30:45231–45243 45233 also contained tungsten (22.8 μM) and selenium (24.2 μM). SE963 operated at 200 kV to obtain energy electron loss Yeast extract (200 mg/L) and acetate (5 mM) were added spectroscopy (EELS) analysis. This analysis defines an L32 to the Dehalobacterium and Dehalogenimonas cultures, ratio, which corresponds to a defined oxidation state of iron respectively. Microcosms containing Dehalobacterium according to Tan et al. (2012). According to it, a value of were gassed with ­N2 (0.4 bar overpressure), and Dehalo- 2.99 of this ratio corresponds to zero-valent iron, while oxi- genimonas cultures were gassed with ­N2/CO2 (4:1, v/v, 0.2 dation states of + 2 and + 3 correspond to L32 values of 3.99 bar overpressure) and ­H2 (added to an overpressure of 0.4 and 4.55, respectively. bar) unless otherwise stated. Both media were buffered with bicarbonate solution (0.01 M). When indicated, either Tris- Analytical methods HCl (25 mM, pH 7.5) or HEPES (60 mM) was added as buffer solutions. Volatile halogenated compounds were analyzed by injecting 0.5 mL from the headspace samples into an Agilent 6890N Establishment of microcosms gas chromatography (GC) fitted with an Agilent DB-624 column (30 m × 0.32 mm with 0.25 μm film thickness) and Sixty-five mL of the anaerobic medium described above a flame ionization detector. Helium was used as the carrier was added into 100-mL serum bottles and sealed with gas (0.9 mL/min), and the injector and detector temperatures Teflon-coated butyl rubber septa aluminum crimp caps. were set at 250 and 300 °C, respectively. To analyze CF, pro- When indicated, some of the medium components were pene, cis-1,2-dichloroethene, vinyl chloride, chloroethane, omitted. At least three treatments were included for each methane, ethene, and 1,2-DCA, the initial oven temperature experiment: (i) abiotic controls containing the anaerobic was 35 °C; it was held for 3 min and then ramped at 10 °C/ defined medium and nZVI, (ii) biotic controls containing the min to 240 °C, which was held for 4 min (Trueba-Santiso anaerobic defined medium and the corresponding bacterium, et al. 2017). To analyze DCM, the initial temperature was and (iii) biotic-abiotic treatments containing the anaerobic 60 °C, and it was raised to 140 °C in 4 min. Then, the tem- defined medium, nZVI, and the corresponding bacterium. perature was maintained at 140 °C for 1 min. Hydrogen was Each microcosm was amended with either 1,2-DCA, CF or analyzed on 0.1-mL headspace samples using an Agilent DCM (1,2-DCA from acetone stock solutions). In the biotic 7820A GC equipped with a thermal conductivity detector. treatments, Dehalobacterium and Dehalogenimonas were Columns used for separation were MolSieve 5A 60/80 SS inoculated by transferring 3 mL and 5 mL from cultures (1.82 m × 2 mm, Agilent) and Porapak Q 60/80 UM (1.82 m during the exponential degradation phase of DCM and 1,2- × 2 mm, Agilent). The carrier gas was nitrogen at 138 kPa, DCA, respectively. The microcosms containing nZVI were and the oven temperature was held isothermal at 40 °C, the not gassed with H ­ 2 but with N ­ 2/CO2 (4:1, v/v, 0.2 bar over- injector temperature at 200 °C, and the detector temperature pressure). Biotic controls were cultivated under static con- at 250 °C. The pH was measured by taking 0.5 mL of liquid ditions; biotic-abiotic treatments and abiotic controls were samples and using a pH probe HACH. incubated under shaking conditions (150 rpm) with the aim of maintaining the nanoparticles in suspension. All treat- ments were maintained at 25 °C in the dark. Results and discussion Characterization of nZVI particles Degradation of CF, DCM, and 1,2‑DCA by nZVI The morphological features of nZVI and the interaction We first studied the abiotic dechlorination of CF, DCM, and between cells and nZVI particles were analyzed with a 1,2-DCA by nZVI and the effect of omitting vitamins (V−) Zeiss Merlin Field-Emission Scanning Electron Microscope and sulfur-cysteine (S−) from the complete medium (CM) (SEM). Samples were prepared as follows: a drop from the described in the “nZVI synthesis and bacterial cultures” sample was placed in silicon chips, where it was then fixed section. with 2.5% glutaraldehyde in phosphate buffer 0.1 M for 2 Based on previous reports, CF reacted with nZVI produc- h at 4 °C, post-fixed with 1% osmium tetroxide with 0.8% ing mainly DCM (Song and Carraway 2006). Therefore, we potassium ferrocyanide for 2 h, and dehydrated in increas- studied the optimal concentration of nZVI to transform 500 ing concentrations of ethanol (50, 70, 90, 96, and 100%). μM CF, which will be later used to determine the inhibitory Finally, it was chemically dried with hexamethyldisilazane. effect of nZVI on the DCM-fermenting culture. The con- Samples were also analyzed by transmission elec- centrations of nZVI tested were in the order of those typi- tron microscopy (TEM) to measure the oxidation state of cally applied in field studies: 0.05, 0.1, 0.5, and 1 g nZVI/L nZVI. A FEI Tecnai G2 F20 HR(S) was used. This TEM (Koenig et al. 2016; Wang et al. 2016; Dong et al. 2019). was equipped with a Gatan Image Filter (GIF) Quantum 13 45234 Environmental Science and Pollution Research (2023) 30:45231–45243 CF did not significantly react with nZVI when added at of this, the following microcosms were performed in com- 0.05 and 0.1 g nZVI/L in any treatment (CM, V−, S−), and plete culture media. DCM was detected as a byproduct at very low concentra- In addition to DCM, some other volatile compounds tions (≤ 10 μM) after 115 days (Fig. 1A, B). In microcosms derived from CF degradation were detected at lower con- amended with 0.5 g nZVI/L (Fig. 1C), degradation of CF centrations, including methane, ethene, propene, vinyl occurred in all treatments during the first 15 days, reaching chloride, chloroethane, cis-1,2-dichloroethene, and 1,2- a plateau since then. At this time point, the degradation of DCA (Table S1). These byproducts were also detected in CF was ~ 80% in the CM and S− treatments and ~ 45% in previous studies employing nZVI to degrade chlorinated the V− treatment. When the amended concentration was methanes (Song and Carraway 2006; Lee et al. 2015). 1 g nZVI/L, full CF removal was achieved in 7 days in the Alternative byproducts described from CF degradation by CM and V- treatments, with a degradation rate of 80 ± 8 nZVI that were not detected in this study include short-chain μM/d (Fig. 1D). When sulfur-cysteine lacked in the medium hydrocarbons (C1–C5) (Lee et al. 2015). The mass balance (S−), the degradation rate decreased at 20 ± 1 μM/d. The of the detected compounds accounted for ~ 30% of the CF amount of DCM produced after the depletion of CF was 70 supplied. In addition to the unidentified compounds, a closed ± 30 μM DCM in CM and 50 ± 5 μM DCM in V−, which mass balance is difficult because some of the generated prod- accounted for ~ 15 and 10% of the CF supplied, respec- ucts can be adsorbed on the surface of the nanoparticles (Lee tively. According to previous studies, solutes such as ­Cl− or et al. 2015). bicarbonate (present in our culture medium) can deactivate Based on the above results, the abiotic degradation of reaction sites in the nZVI surface (Tang et al. 2012; Han DCM (2000 μM) and 1,2-DCA (500 μM) was assessed using and Yan 2014). However, vitamins and sulfur-cysteine can 1 g nZVI/L. Consistent with previous reports (Gillham and act as electron shuttling compounds, hence enhancing elec- O’Hannesin 1994; Song and Carraway 2005; Koenig et al. tron transfer from nZVI to CF (Watanabe et al. 2009). This 2016; Lee et al. 2015), there was no significant reactivity of fact can explain the moderately slower reaction rate when nZVI toward both 1,2-DCA and DCM (Fig. S1). Thus, these sulfur-cysteine was missing in the culture medium. Because pollutants remained in the medium and needed alternative Fig. 1  CF abiotic degradation and DCM production by nZVI at four different concentra- tions: A 0.05 g nZVI/L, B 0.1 g nZVI/L, C 0.5 g nZVI/L, and D 1 g nZVI/L. Three different culture media were used: com- plete medium (CM), vitamins lacking medium (V−), and sulfur-cysteine lacking medium (S−). Values plotted are means ± standard deviations for dupli- cate cultures 13 Environmental Science and Pollution Research (2023) 30:45231–45243 45235 remediation treatments, which opens the window to their microcosms fully fermented DCM in 4 days (Fig. 2A). It biodegradation using anaerobic dechlorinating bacteria. indicates that nZVI exerts an inhibitory effect on Dehalo- bacterium. Then, the next step was exploring the factor Effect of nZVI on Dehalobacterium activity that provokes the inhibitory effect. Impact of nZVI Adsorption of nZVI on bacterial cells As stated before, biodegradation of DCM under anoxic It has been reported that nZVI can provoke physical disrup- conditions has been reported solely in three bacterial gen- tion of cell membranes. Although this mechanism is not era. Since DCM is the by-product from CF degradation fully understood, it has been hypothesized that it is primar- by nZVI but it is not degraded further by this treatment, ily due to the chemical interaction between cells and the we studied whether nZVI has a toxic effect on microbial reactive surface of nZVI (e.g., iron is a strong reductant), transformation of DCM (2000 μM) by a Dehalobacterium- which could favor the decomposition of functional groups containing culture. For this purpose, we used the two nZVI in proteins and lipopolysaccharides from cell membranes concentrations, which were found to fully remove CF in (Lee et al. 2008; Li et al. 2010). SEM analysis revealed previous tests: 0.5 and 1 g nZVI/L (Fig. 1). a deformed cell structure with adsorption of nZVI on the DCM fermentation by Dehalobacterium was not cell surface with 1 g nZVI/L (Fig. 3). To study whether observed with either 0.5 or 1 g nZVI/L, while biotic nZVI adsorbed on cell membranes caused the inhibition, Fig. 2  Time course of DCM degradation by a Dehalobacterium- under static conditions. Biotic controls (-nZVI) consisted of Dehalo- containing consortium in a complete anaerobic culture medium with bacterium-containing cultures amended with DCM without nZVI. 0.5 and 1 g nZVI/L. This test presents two phases: A cultures under Values plotted are means ± standard deviations for triplicate cultures shaken conditions (150 rpm) and B cultures with nZVI precipitated Fig. 3  SEM micrographs from Dehalobacterium-containing cultures with 1 g nZVI/L 13 45236 Environmental Science and Pollution Research (2023) 30:45231–45243 on the fifteenth day, we stopped shaking the cultures and precipitated nZVI at the bottom of the serum bottles using a magnet, while microorganisms remained in suspension, thus avoiding contact between nZVI and Dehalobacte- rium (Fig. 2B). In the following 55 days, no degradation of DCM was observed in the bottles with 0.5 and 1 g nZVI/L (Fig. 2B). Also, on the fifteenth day, 3 mL of both cultures containing 0.5 and 1 g nZVI/L were transferred to fresh complete culture media without nZVI and amended with DCM (1000 μM). DCM was completely degraded after 20–30 days, with a lag phase of 10 days (Fig. 4). These results suggested that inhibition of DCM fermentation by nZVI was reversible, and that nZVI adsorption to cells was not the main cause of the inhibition. pH The increase in pH due to corrosion of nZVI can nega- tively impact Dehalobacterium activity. pH on day 65 increased in cultures amended with nZVI (8.8 ± 0.1 with 1 g nZVI/L and 8.1 ± 0.1 with 0.5 g nZVI/L) com- pared with cultures without nZVI (4.9 ± 0.1) (Fig. 2). To determine whether DCM fermentation was inhib- ited in a basic medium, we established new Dehalo- bacterium cultures at pH 7 (control), 8.5, and 9 without nZVI. Degradation of DCM in cultures at pH 7 and 8.5 occurred at similar degradation rates after a lag phase of 6 days, but cultures at pH 9 exhibited an extended lag phase of 18 days before DCM was fully consumed (Fig. 5). Interestingly, cultures at pH 9 (9.4 ± 0.1) did not start degrading DCM until pH reached a value of 8.7 Fig. 5  A Time course of DCM degradation by a Dehalobacte- rium-containing consortium in complete anaerobic culture media at different pH values (7, 8.5, and 9). B Changes in pH during DCM degradation. Values plotted are means ± standard deviations for triplicate cultures ± 0.8 due to the slow degradation of DCM that acidified the medium because the production of acetate and ­H + (Trueba-Santiso et al. 2020). To avoid the negative impact of high pH values on Dehalobacterium activity, we buffered the Dehalobac- terium-containing cultures with 25 mM Tris-HCl (pH = 7.5) and amended 1 g nZVI/L, but degradation of DCM was not observed for 12 days although pH was 8.2 ± 0.3 (Fig. 6). Cultures that were not buffered with Tris-HCl reached pH = 9.4 ± 0.4, whereas controls that did not contain nZVI degraded DCM in 7 days (Fig. 6). These results confirm that although degradation of DCM is Fig. 4  Time course of DCM degradation by a Dehalobacterium- delayed at pH values up to 9, other factors contributed containing consortium after being transferred to fresh complete medium from microcosms containing nZVI. Values plotted are to the cytotoxicity of Dehalobacterium when exposed means ± standard deviations for triplicate cultures to nZVI. 13 Environmental Science and Pollution Research (2023) 30:45231–45243 45237 with 23 ± 3 μM H ­ 2 obtained in the biotic microcosms (with- out nZVI). It provides evidence that ­H2 accumulated because of water hydrolysis by nZVI even though hydrogenotrophs were present in the bacterial consortium (Trueba-Santiso et al. 2020). To study whether ­H2 exerted an inhibitory effect on Dehalobacterium activity in the cultures buffered with Tris- HCl and amended with 1 g nZVI/L, we periodically removed ­H2 after day 12 by purging with nitrogen (Fig. 7). DCM was then reamended (2000 μM). In addition, a second treatment in parallel cultures consisted of introducing air (1 mL) after purging with nitrogen to decrease the reductant power of nZVI and reduce the ­H2 production. Although the produc- tion of ­H2 decreased in cultures amended with air, ­H2 was rapidly generated after each purge (Fig. 7). After 70 days, ­H2 produced exceeded the concentration of 3000 ppmv (126 Fig. 6  A Time course of DCM degradation by a Dehalobacterium- containing consortium in complete anaerobic culture media with 1 g nZVI/L and buffered and non-buffered with 25 mM Tris-HCl. B Changes jn pH during DCM degradation. Values plotted are means ± standard deviations for duplicate cultures. Controls consisted of Dehalobacterium-containing cultures amended with DCM without nZVI Effect of hydrogen and soluble compounds Elevated ­H2 partial pressures have been found to inhibit the fermentation of DCM (Chen et al. 2017b; Lee et al. 2012; Trueba-Santiso et al. 2020). The reason for this inhibitory effect is still unclear. According to Chen et al. (2017b), this fact could be due to the hindrance of formate oxidation at such hydrogen concentrations, leading to the accumulation of formate into the cells of “Candidatus Dichloromethano- Fig. 7  A Effect of purging hydrogen with nitrogen (­H2 purged) and monas elyuquensis.” Hydrogen reached concentrations of nitrogen plus air ­(H2 purged + air) on DCM degradation and B ­H2 generation in Dehalobacterium-containing cultures buffered with 1600 ± 100 μM and 15000 ± 3000 μM when nZVI was Tris-HCl and amended with 1 g nZVI/L. Arrows indicate when ­H2 amended at 0.5 and 1 g nZVI/L on day 15 in the experiment was purged. Values plotted are means ± standard deviations for dupli- depicted in Fig. 2, and were dramatically higher compared cate cultures 13 45238 Environmental Science and Pollution Research (2023) 30:45231–45243 μM) found inhibitory for DCM fermentation in Dehalobac- terium (Chen et al. 2017b). Due to the continued production of H ­ 2 by nZVI, it was difficult to establish whether the high partial pressure of ­H2 was the only factor responsible for the inhibition of DCM fermentation. Therefore, we assessed whether hydrogen or a soluble inhibitory compound from nZVI were inhibitory factors in the Dehalobacterium activity. We prepared nine abiotic-biotic microcosms containing Dehalobacterium and 1 g nZVI/L in a complete medium buffered with 25 mM Tris-HCl and 500 μM CF. When CF was depleted by nZVI on day 7 (Fig. 8), three different treat- ments were established in triplicate: i) particles of nZVI were removed from microcosms using a magnet, but cul- tures were gassed with ­H2 at the concentration reached in the microcosms before ZVI removal (–nZVI, ­H2), ii) parti- cles of nZVI were removed from microcosms with no addi- tion of ­H2 (–nZVI, –H2), and iii) particles of nZVI were not removed (control). All treatments were amended with 2000 μM DCM after CF depletion. On day 7, H­ 2 concentration was 4 ± 1 mM (Fig. 8), and pH was 7.8 ± 0.1 in all treatments. No degradation of DCM was observed in the following 53 days for any of the three treatments. Hydrogen was steadily consumed in cultures that did not contain nZVI to levels below the inhibition con- centration (Fig. 8), but DCM was not degraded, providing additional supporting evidence that dissolved compounds derived from nZVI could contribute to the inhibition of Dehalobacterium. It has been described that soluble sub- stances released or produced by nZVI particles that can cause inhibition to bacterial cells include radical species (i.e., hydroxyl radicals) (Anang et al. 2021; Xie et al. 2017), soluble iron (Fajardo et al. 2013; Koenig et al. 2016; Lee Fig. 8  A Time course of abiotic-biotic degradation of CF by 1 g et al. 2008; Rangan et al. 2020) and soluble metals (Xie et al. nZVI/L in Dehalobacterium-containing cultures (1). Time-course degradation of DCM in Dehalobacterium-containing cultures after 2017). Soluble iron can enter into the cells and generate ROS the removal of nZVI with (-nZVI, ­H2) and without (–nZVI, –H2) species by Fenton reactions, which could lead to damage by amended ­H2 (2). B Hydrogen produced by nZVI. Values plotted are oxidative stress (Fajardo et al. 2013; Lee et al. 2008; Saccà means ± standard deviations for triplicate cultures et al. 2014). On day 17, 3 mL of each microcosm was transferred to fresh culture media plus DCM (2000 μM), and complete the Dehalogenimonas population after the injection of 1 g degradation of DCM was observed after a lag phase of 14 ± nZVI/L stabilized with carboxymethyl cellulose in a con- 3 days in microcosms derived from treatment “–nZVI, –H2” taminated aquifer. A similar phenomenon was observed for (Fig. S2), indicating that inhibition was reversible. the ORB Dehalococcoides mccartyi (Xiu et al. 2010; Kocur Further research is needed to identify the inhibitory et al. 2015). compound or compounds released or catalyzed by nZVI to overcome the inhibition of DCM degradation in Dehalobac- nZVI characterization terium. Some strategies to attenuate such inhibitory effect include the surface modification of nanoparticles with sur- The morphological characterization of nZVI from two dif- face stabilizers such as surfactants or polyelectrolytes, which ferent treatments (CF + nZVI, shown in Fig. 9A–C and slow down nZVI reaction rates, the production of H ­ 2, the DCM + Dehalobacterium-containing culture + nZVI, release of ROS and iron ions, and avoid contact between shown in Fig. 9D–F) was performed with SEM. Sam- nZVI particles and cells (Xie et al. 2017; Wang et al. 2016). ples were taken in three different stages: beginning (t = For instance, Kocur et al. (2016) detected an increase in 0, Fig. 9A and D), mid-term (Fig. 9B and E), and final 13 Environmental Science and Pollution Research (2023) 30:45231–45243 45239 Fig. 9  SEM micrographs of nZVI from abiotic treatments (CF + nZVI) (A to C) and abiotic-biotic treatments (DCM + Dehalobacterium+ nZVI) (D to F) at different stages: initial time (A and D), half time (B and E), and final time (C and F) time (when the experiments were finished, Fig. 9C and F). Table 1  L32 index of samples from experimental microcosms con- Mid-term was the third day for microcosms containing CF taining nZVI. Sample 1 derived from the experiment depicted in + nZVI and the eighth day for the microcosms contain- Fig. 1C on day 7; sample 2 derived from the experiment depicted in Fig. 8 on day 7; sample 3 derived from the experiment depicted in ing Dehalobacterium. At time zero, nZVI were spherical Fig. 2 on day 15; sample 4 derived from the experiment depicted in or near spherical in shape (50–100 nm) (Fig. 9A and D) Fig. 6 on day 12 and eventually formed aggregates in the mid-term stage L32 index values (Fig. 9B and E). Aggregation of nZVI and the subsequent formation of a particle network (gelation) have been pointed 1) CF + 0.5 g nZVI/L 4.9 to limit transport of nZVI in water-saturated porous media 2) CF + Dehalobacterium + 1 g nZVI/L+ Tris-HCl 5.77 (Phenrat et al. 2007). At the end of abiotic and biotic-abi- 3) DCM + Dehalobacterium + 1 g nZVI/L 5.34 otic experiments, nZVI showed a rough surface topography, 4) DCM + Dehalobacterium + 1 g nZVI/L + Tris- 4.56 which is consistent with the formation of iron oxides and HCl hydroxides on the surface of nZVI (Fig. 9C and F). The oxidation state of the nZVI particles used in some of the treatments carried out in this study was determined Effect of nZVI on Dehalogenimonas activity by EELS analysis. Each treatment showed an oxidation state ≥ + 3 at the end of the experiment according to the As shown in Fig. S1, 1,2-DCA was not removed by 1 g L32 ratio values (Table 1), indicating oxidation of nZVI nZVI/L. Because of this, we aimed to test the capability through the experiments is in accordance with micro- of a Dehalogenimonas-containing culture to dechlorinate graphs depicted in Fig. 9. 1,2-DCA in the presence of nZVI using a HEPES buffered medium. Four different treatments were tested: i) biotic 13 45240 Environmental Science and Pollution Research (2023) 30:45231–45243 microcosms (control + ­H2), ii) biotic microcosms with- Despite the inhibition of Dehalogenimonas activity by out ­H2 (control -H2), iii) abiotic-biotic microcosms con- nZVI, our results are not necessarily transferable to field taining 0.5 g nZVI/L, and iv) abiotic-biotic microcosms studies. The high complexity of the soil and groundwater containing 1 g nZVI/L. Both control treatments removed composition could avoid the direct contact between nano- 1,2-DCA at the same rate with the concomitant production particles and cells while providing optimum conditions for of ethene, while those microcosms containing nZVI did ORB growth (low redox potential, ­H2 production). not show 1,2-DCA depletion despite the circumneutral pH and the high production of ­H2 that could be used as elec- tron donor for Dehalogenimonas (Fig. 10). Therefore, we Conclusions transferred 5 mL of the microcosms to fresh culture media with 1,2-DCA (50 μM) to test whether the inhibitory effect Our laboratory tests demonstrated that dechlorinating was reversible, and full removal of 1,2-DCA was observed activity of Dehalobacterium and Dehalogenimonas in 50 days after a lag phase of ~ 20 days (Fig. S3). This is was inhibited at 1 g nZVI/L, which was the optimal in line with the tests performed by Koenig et al. (2016), concentration of nZVI found to fully remove CF (500 μM). indicating that concentrations of nZVI supplied above 0.5 Further experiments discarded that changes in both pH and g/L inhibited the dechlorination activity of groundwater partial pressure of ­H2 provoked by nZVI exerted inhibition containing Dehalogenimonas likely due to ROS species). on Dehalobacterium. The fact that both Dehalogenimonas and Dehalobacterium recovered the dechlorinating Fig. 10  A Time course of 1,2- DCA degradation by 0.5 and 1 g nZVI/L in Dehalogenimonas- containing cultures. Ethene production is also shown. B ­H2 concentrations in microcosms with Dehalogenimonas-contain- ing cultures. Values plotted are means ± standard deviations for triplicate cultures 13 Environmental Science and Pollution Research (2023) 30:45231–45243 45241 capability after being transferred to nZVI-free medium Bagley DM, Lalonde M, Kaseros V, Stasiuk KE, Sleep BE (2000) pointed to soluble compounds released by particles as Acclimation of anaerobic systems to biodegrade tetrachloroethene in the presence of carbon tetrachloride and chloroform. Water Res potential inhibitors. These findings have implication for 34(1):171–178. https://​doi.​org/​10.​1016/​S0043-​1354(99)​00121-9 remediation strategies aiming to combine both abiotic and Blázquez-Pallí N, Rosell M, Varias J, Bosch M, Soler A, Vicent T, Marco- biotic treatments to decontaminate aquifers with mixtures Urrea E (2019) Integrative isotopic and molecular approach for the of chlorinated compounds. In particular, our study suggests diagnostic and implementation of an efficient in-situ enhanced bio- logical reductive dechlorination of chlorinated ethenes. Water Res that factors such as the buffering capacity of groundwaters 167:115106. https://​doi.​org/​10.​1016/j.​watres.​2019.​115106 and dilution processes can play crucial roles in the viability Chen G, Kleindienst S, Griffiths DR, Mack EE, Seger ES, Löffler FE of this combined strategy in bioremediation treatments. (2017b) Mutualistic interaction between dichloromethane-and chloromethane-degrading bacteria in an anaerobic mixed cul- Supplementary Information The online version contains supplemen- ture. Environ Microbiol 19(11):4784–4796. https://​doi.​org/​10.​ tary material available at https://d​ oi.​org/​10.1​ 007/​s11356-​023-​25376-z. 1111/​1462-​2920.​13945 Chen G, Murdoch RW, Mack EE, Seger ES, Löffler FE (2017a) Com- Author contribution Conceptualization and funding acquisition: EMU. plete genome sequence of Dehalobacterium formicoaceticum Investigation: DS and DFV. Methodology: XF, JMV, DFV, DS, and strain DMC, a strictly anaerobic dichloromethane-degrading EMU. Data collection and analysis: DS and DFV. Supervision and bacterium. Genome Announc 5(37):e00897–e00817. https://​ validation: XF, JMV, and EMU. Writing (original draft): DS. Writing doi.​org/​10.​1128/​genom​eA.​00897-​17 (review and editing): XF, JMV, DFV, DS, and EMU. XF, JMV, DFV, Chen M, Xu P, Zeng G, Yang C, Huang D, Zhang J (2015) Bioreme- DS, and EMU read and approved the final version of the manuscript. diation of soils contaminated with polycyclic aromatic hydro- All authors that appear in the submission made substantial contribu- carbons, petroleum, pesticides, chlorophenols and heavy met- tions to the conception or design of the work; drafted the work or als by composting: applications, microbes and future research revised it critically for important intellectual content; approved the needs. Biotechnol Adv 33(6):745–755. https://​d oi.​o rg/​1 0.​ version to be published; and agree to be accountable for all aspects of 1016/j.​biote​chadv.​2015.​05.​003 the work in ensuring that questions related to the accuracy or integrity Chen X, Chen G, Qiu M, Sun G, Guo J, Xu M (2014) Synergistic of any part of the work are appropriately investigated and resolved. degradation of deca-BDE by an enrichment culture and zero- valent iron. Environ Sci Pollut Res 21:7856–7862. https://​doi.​ Funding Open Access Funding provided by Universitat Autonoma de org/​10.​1007/​s11356-​014-​2734-x Barcelona. This work was supported by the I+D+i project PID2019- De Wildeman S, Diekert G, Van Langenhove H, Verstraete W (2003) 103989RB-100 funded by the Ministerio de Ciencia e Innovación y Stereoselective microbial dehalorespiration with vicinal dichlo- Agencia Estatal de Investigación (CIN/AEI/10.13039/501100011033). rinated alkanes. Appl Environ Microbiol 69(9):5643–5647. https://​doi.​org/​10.​1128/​AEM.​69.9.​5643-​5647.​2003 Declarations Dong H, Li L, Lu Y, Cheng Y, Wang Y, Ning Q, Wang B, Zhang L, Zeng G (2019) Integration of nanoscale zero-valent iron and functional Consent for publication All authors agree with the content of this anaerobic bacteria for groundwater remediation: a review. Environ manuscript, and all give explicit consent to submit in Environmental Int 124:265–277. https://​doi.​org/​10.​1016/j.​envint.​2019.​01.​030 Science and Pollution Research. This work is new and original, and it Elliott DW, Zhang WX (2001) Field assessment of nanoscale bime- is not under consideration elsewhere. tallic particles for groundwater treatment. Environ Sci Technol 35(24):4922–4926. https://​doi.​org/​10.​1021/​es010​8584 Competing interests The authors declare no competing interests. El-Shahawi MS, Hamza A, Bashammakh AS, Al-Saggaf WT (2010) An overview on the accumulation, distribution, transformations, toxicity and analytical methods for the monitoring of persistent Open Access This article is licensed under a Creative Commons Attri- organic pollutants. Talanta 80(5):1587–1597. https://​doi.​org/​10.​ bution 4.0 International License, which permits use, sharing, adapta- 1016/j.​talan​ta.​2009.​09.​055 tion, distribution and reproduction in any medium or format, as long Fajardo C, Saccà ML, Martinez-Gomariz M, Costa G, Nande M, as you give appropriate credit to the original author(s) and the source, Martin M (2013) Transcriptional and proteomic stress responses provide a link to the Creative Commons licence, and indicate if changes of a soil bacterium Bacillus cereus to nanosized zero-valent iron were made. The images or other third party material in this article are (nZVI) particles. Chemosphere 93(6):1077–1083. https://​doi.​ included in the article's Creative Commons licence, unless indicated org/​10.​1016/j.​chemo​sphere.​2013.​05.​082 otherwise in a credit line to the material. 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Bioresour Technol 101(4):1141– Publisher’s note Springer Nature remains neutral with regard to 1146. https://​doi.​org/​10.​1016/j.​biort​ech.​2009.​09.​057 jurisdictional claims in published maps and institutional affiliations. 13