Sulfur-based mixotrophic bio-reduction for efficient removal of chromium (VI) in groundwater

Journal Pre-proofs Sulfur-based mixotrophic bio-reduction for efficient removal of chromium (VI) in groundwater Baogang Zhang, Zhongli Wang, Jiaxin Shi, Hailiang Dong PII: DOI: Reference:

S0016-7037(19)30654-4 https://doi.org/10.1016/j.gca.2019.10.011 GCA 11479

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Geochimica et Cosmochimica Acta

Received Date: Revised Date: Accepted Date:

12 June 2019 3 October 2019 5 October 2019

Please cite this article as: Zhang, B., Wang, Z., Shi, J., Dong, H., Sulfur-based mixotrophic bio-reduction for efficient removal of chromium (VI) in groundwater, Geochimica et Cosmochimica Acta (2019), doi: https:// doi.org/10.1016/j.gca.2019.10.011

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Sulfur-based mixotrophic bio-reduction for efficient removal of chromium (VI) in groundwater

Baogang Zhanga, Zhongli Wanga, Jiaxin Shia, and Hailiang Dongb*

a School

of Water Resources and Environment, MOE Key Laboratory of Groundwater

Circulation and Environmental Evolution, China University of Geosciences (Beijing), Beijing 100083, P. R. China

b State

Key Laboratory of Biogeology and Environmental Geology, China University of Geosciences, Beijing 100083, China

*Corresponding

author. Tel.: + 010-82320969

E-mail: [email protected] (H. Dong)

Revised for Geochimica et Cosmochimica Acta

October 3, 2019

1

Abstract Organic matter and reduced sulfur compounds commonly coexist in groundwater aquifers and their respective roles in Cr(VI) bio-reduction have been well established, but Cr(VI) bio-reduction under mixotrophic condition, where organics and elemental sulfur simultaneously occur as co-donors of electrons, remains largely unknown. Herein a sulfur-based mixotrophic bio-reduction process is demonstrated to be effective to detoxify Cr(VI), with a removal efficiency of 95.5 ± 0.74% within 48 h at an initial concentration of 50 mg/L. In addition to direct reduction by heterotrophic Cr(VI) reducers such as Desulfovibrio and Desulfuromonas, volatile fatty acids (VFAs) produced from autotrophic sulfur oxidation served as electron donors for heterotrophic Cr(VI) reducers. Part of VFAs was also assimilated and accumulated as glycogen within cells, which enhanced their Cr(VI) removal capacity. Metabolic pathway analysis suggested that Cr(VI) was reduced to insoluble Cr(III) both extracellularly by cytochrome c and intracellularly by nicotinamide adenine dinucleotide in the presence of upregulated chrA gene. Constituents of extracellular polymeric substances (EPS) also contributed to Cr(VI) reduction enzymatically, through binding of toxic Cr(VI) by carboxyl and hydroxyl groups. Results from this study have important implications for understanding the biogeochemical behavior and environmental remediation of Cr(VI) in groundwater aquifers and sediments/soils. Keywords:

Cr(VI);

Elemental

sulfur;

bio-reduction 2

Metabolic

analysis;

Mixotrophic

1. INTRODUCTION Chromium is a heavy metal that naturally occurs in the Earth's crust with an average concentration of about 100 mg/kg (Zhang et al., 2012a; Kazakis et al., 2015). It is one of the most abundant and toxic metals in groundwater worldwide (Dimitroula et al., 2015; Němeček et al., 2016; Huang et al., 2017). In many locations, such as California of the United States, concentrations of geogenic chromium in groundwater aquifers can exceed 50 μg/L, the limit of chromium in drinking water established by the World Health Organization (Mills et al., 2011). Chromium naturally exists in two stable oxidation states: hexavalent chromium [Cr(VI)] and trivalent chromium [Cr(III)] (Zhong et al., 2017; Goring-Harford et al., 2018; Wang et al., 2018a). Cr(VI) is highly toxic to all forms of living organisms, with a high solubility, mobility, and bioavailability, while Cr(III) is less toxic and tends to form insoluble minerals at neutral pH (Kim et al., 2015; Lu et al., 2018; Bishop et al., 2019). Hence, the most commonly used approach to detoxify Cr(VI) in groundwater is the reduction of Cr(VI) to Cr(III) (Wang et al., 2015; Sun et al., 2015; Singh et al., 2017; Shao et al., 2018). Microbial reduction of Cr(VI) to Cr(III) takes place in groundwater aquifer, soils, and sediments, which is also the most common and promising route for in situ remediation of Cr(VI)-contaminated groundwater (Daulton et al., 2007; Lai et al., 2016; Zhao et al., 2017a). In this biogeochemical process, electron donor bioavailability plays a vital role (Sikora et al., 2008; Long et al., 2017). Heterotrophic bio-reduction of Cr(VI), where organic carbon serves as electron donor, is an efficient and reliable process for Cr(VI) removal, as many Cr(VI)-reducing microorganisms 3

are heterotrophic, however insufficient amounts of organic carbon in subsurface environments often limit this process (Pradhan et al., 2017; Ni et al., 2018). Presence of organic carbon can accelerate this process, but can also result in pore clogging from excess biomass growth (McLeod et al., 2018). Sulfur-based autotrophic bio-reduction of Cr(VI), where reduced inorganic substances such as sulfur compounds serve as electron donor, may be important to Cr(VI) removal as well. Elemental sulfur is one of important sulfur species present in natural environments (Knöller and Schubert, 2010; Graham and Bouwer, 2012; Findlay et al., 2014) and is able to serve as electron donor to couple with reduction of nitrate and heavy metals (Juang et al., 2015; Peng et al., 2016; Shi et al., 2019). This process can be considered as an alternative for contaminant removal without requirement of organic carbon (Peng et al., 2016; Zhang et al., 2018). Nonetheless, the generations of acid and sulfate from oxidation of elemental sulfur are some of the main disadvantages of this process, which may cause the acidification of groundwater and result in diarrhea when water with a large amount of sulfate is drunk (Zhang et al., 2018; Runtti et al., 2018). In many anoxic environments, organic matter and elemental sulfur can co-occur (Graham et al., 2009; Zhang et al., 2012b; Knabe et al., 2018; Phan et al., 2019), which may work together to impact Cr(VI) biotransformation. Therefore, a mixotrophic process, which utilizes organic carbon and elemental sulfur as co-donors of electrons to combine autotrophic and heterotrophic activities, is an effective 4

strategy to overcome their respective shortcomings. In such process, autotrophy and heterotrophy may occur simultaneously due to different microbial populations in a community. Heterotrophic activity can remove sulfate generated in autotrophic activity and the alkalinity generated in heterotrophic activity can neutralize the acidity generated in autotrophic activity, thus forming a synergistic interaction between these two processes to further improve the removal efficiencies of nitrate and metals (Sahinkaya et al., 2012; Zhang et al., 2015). A sulfur-based mixotrophic denitrification process has been observed for efficient nitrate removal (Juang et al., 2015). In the Juang et al. study, Cr(VI) was used as a competing electron acceptor, thus the mechanisms involved in Cr(VI) removal and Cr(VI) reduction products are not known. Although the geochemistry of Cr(VI) has been investigated in this denitrification process (Sahinkaya et al., 2013; 2017), little is known about Cr(VI) bio-reduction kinetics. In studies with Cr(VI) as the sole electron acceptor, Cr(VI) reduction is typically coupled with oxidation of either organic carbon (heterotrophy, Singh et al., 2015, 2017; Bishop et al., 2019) or elemental sulfur (autotrophy, Shi et al., 2019), a synergistic interaction between these two processes, i.e., mixotrophy, has not been studied, despite potential importance of such interaction in natural environments (Frohne et al., 2015). Furthermore, both heterotrophs and autotrophs co-exist in this mixotrophic process, and thus examining the microbial community structure is vital to reveal the complex microbial interactions occurring in this process and to optimize this strategy for potential application. However, a mechanistic understanding of microbial functions in sulfur-based mixotrophic Cr(VI) reduction is 5

still lacking. Herein, we studied sulfur-based mixotrophic bio-reduction with Cr(VI) as the sole electron acceptor to reveal the mechanisms of Cr(VI) biotransformation and the biogeochemical fate of reduced chromium in groundwater. In this process, sulfate generation decreased, and alkalinity and acidity were completely eliminated. The objectives of this study were to: (1) evaluate the rate and extent of Cr(VI) reduction in a sulfur-based mixotrophic process in comparison with individual autotrophic and heterotrophic processes; (2) investigate the effects of key environmental conditions on the Cr(VI) reduction kinetics and the Cr(VI) reduction products under sulfur-based mixotrophic condition; and (3) provide insights into the mechanisms of Cr(VI) reduction under sulfur-based mixotrophic condition by analyzing the microbial community composition and metabolic pathways (functional genes, pertinent compounds, and possible metabolites). This study provides new insights into the biogeochemical cycling of Cr in those environments where elevated Cr, organic carbon and reduced sulfur compounds co-exist (such as contaminated estuary sediments, groundwater aquifers, and soils) with important implications for developing an efficient Cr(VI) remediation strategy. 2. MATERIALS AND METHODS 2.1. Chemicals K2Cr2O7 (99.8%), sodium acetate, and elemental sulfur (99.0%) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). Deionized water (18.2 6

MΩ·cm) produced by a Milli-Q Water System (Heal Force NW) was used in the experiment. Other chemicals were of analytical grade and used directly without further purification. 2.2. Bioreactor setup All experiments were conducted in 280-mL plexiglass bioreactors which were sealed by air-tight rubber stoppers to maintain an anaerobic condition and were covered with aluminum foil to avoid light exposure. An anaerobic consortium of 50 mL in volume, obtained from a reactor in YanJing Brewery (Beijing, China), was added to each bioreactor for initial inoculation. The microbial community structure of the inoculum was similar to the one in a chromium contaminated aquifer (Zhang et al., 2019b). Every reactor was filled with 200 mL synthetic groundwater, which was prepared according to the following components (per L): 0.504 g NaHCO3, 1.0572 g MgCl2·6H2O, 0.2464 g CaCl2, 0.035 g NH4Cl, 0.0299 g KH2PO4, 0.4459 g NaCl, and 0.0283 g KCl (Zhang et al., 2015; Liu et al., 2017). Aqueous Cr(VI) was provided in the form of K2Cr2O7 with a concentration of 50 mg/L unless indicated otherwise. A heterotrophic bioreactor, designated as HB, was amended with acetate, with the amount equivalent to 60 mg/L chemical oxygen demand (COD), while 5 g elemental sulfur (instead of acetate) was fed into an autotrophic bioreactor (AB). Acetate concentration equivalent to 60 mg/L was chosen in consideration of typical concentration (25 mg/L) of natural organic matter in aquifer (Moser et al., 2003). Mixotrophic bioreactor was labeled as MB with supplements of both COD (60 mg/L) in the form of acetate and elemental sulfur (5 g). Abiotic reactor without inoculation 7

and bioreactor without any electron donor served as controls under otherwise identical conditions. 2.3. Batch reactor experimental design Aqueous solutions in all bioreactors were replenished once every 3 days during a 2-month incubation period to ensure that the bioreactors achieved a stable performance. The old solution was removed with a syringe and replaced with a new solution under anaerobic atmosphere. Cr(VI) detoxification was subsequently evaluated in three consecutive cycles, with 48 h for each cycle, which was sufficient to remove most Cr(VI). Variations of dissolved organic carbon (DOC) and pH were monitored through all three cycles. Products of Cr(VI) bio-reduction and elemental sulfur oxidation were analyzed. Microbial community was studied with high-throughput 16S rRNA gene sequencing. Potential metabolic pathways were investigated by examining functional genes, pertinent compounds, and metabolites. Subsequently, the effects of key performance parameters in sulfur-based mixotrophic Cr(VI) bio-reduction were investigated: 1) initial Cr(VI) concentrations (25 mg/L, 50 mg/L, 75 mg/L, 100 mg/L) with a constant COD of 60 mg/L; 2) initial COD concentrations (30 mg/L, 60 mg/L, 90 mg/L, 120 mg/L) with a constant Cr(VI) concentration of 50 mg/L. All experiments were performed in triplicate at ambient temperature (22 ± 2 ºC). Mean values of experimental data were presented. 2.4. Aqueous speciation analysis All aqueous samples were filtered through a 0.22 μm pore size syringe. Aqueous 8

Cr(VI) concentration was monitored by a spectrophotometric method (Zhang et al., 2012a). Dissolved total Cr was measured with inductively coupled plasma mass spectrometry (ICP-MS, Thermo Fisher X series, Germany). Sulfate, sulfite and thiosulfate were analyzed with ion chromatography (Basic IC 792, Metrohm, Switzerland). COD was measured with a potassium dichromate method based on digestion in concentrated sulfuric acid (Zhang et al., 2009). DOC was measured with a MultiN/C 3000 TOC analyzer (Analytik Jena AG, Germany). pH was determined by using a pH-201 meter (Hanna, Italy). 2.5. Solid characterization To further characterize the solid precipitates produced in MB, a suite of characterization methods was employed. The morphology of biomass was observed with scanning electron microscope (SEM) operating at 20 kV (JEOL JAX-840, Hitachi, Japan), which was equipped with energy dispersive X-ray spectroscopy (EDS). X-ray diffraction (XRD) (D/MAX-PC 2500, Rigaku, Japan) and X-ray photoelectron spectroscopy (XPS) (XSAM-800, Kratos, UK) analyses were performed to examine the Cr(VI) reduction precipitates. The obtained XRD and XPS data were analyzed using Jade 6.0 and XPS Peak Fit software, respectively. To visualize intracellular Cr precipitation, ultrathin sections (50-60 nm) of microbial cells were prepared as previously described (Wang et al., 2017). Briefly, the washed cells were fixed overnight in a glutaraldehyde solution (2.5%, w/w). After rinsing three times with phosphate buffer, biomass was fixed with osmium tetroxide

9

(1%, w/w) for2 h. Biomass was then gradually dehydrated with acetone and polymerized. Ultrathin sections were made by using a ultramicrotome (EM UC6, Leica Germany). The intracellular distribution of reduced Cr products was visualized under scanning transmission electron microscopy (STEM)/EDS (JEM-2100 LaB6, JEOL, Japan) with a 200-kV accelerating voltage. 2.6. Microbial analysis Biomass in initial inoculum and bioreactors was collected using an inoculating loop and pre-treated ultrasonically (Zhang et al., 2018). FastPrep®-24 kit (MP Biomedicals, the USA) and FastDNA® SPIN Kit for Soil (Qiagen, CA, the USA) were used together to extract and purify genomic DNA from collected biomass samples. The obtained DNA was amplified with PCR with bacterial primer pairs 338F/806R, by following reported procedures (Liu et al., 2017). A previous study (Wang et al., 2018a) revealed that biomass in initial inoculum and bioreactors was predominated by bacteria, and thus, only bacterial community was analyzed. Briefly, a 2-min denaturation at 94 oC was performed initially, and subsequently 25 cycles were conducted. Each cycle lasted 90 s, including 30 s at 94oC, 30 s at 57 oC, and 30 s at 72 oC. Finally, a 5-min extension at 72 oC was performed. The obtained amplicons were exercised from 2% agarose gels followed by purification and quantification with the AxyPrep DNA GelExtraction Kit (Axygen Biosciences, Union City, CA, U.S.) and QuantiFluor™ -ST (Promega, the USA), respectively. After pooling of the purified amplicons in equimolar concentrations according to the standard protocols (Zhang et al., 2018), they were sent to Shanghai Majorbio Technology Co., 10

Ltd.(Shanghai, China) for paired-endhigh-throughput sequencing (2 × 250) on an Illumina MiSeq platform (the USA). Accession number of SPR21440 was assigned to the raw sequence data deposited in NCBI Sequence Read Archive Database. QIIME (version 1.17) was used to demultiplex and to quality-filter the raw fastq files with universal criteria (Jiang et al., 2018). UPARSE (version 7.1) and UCHIME were employed to cluster operational taxonomic units (OTUs) at a 97% similarity and to remove chimeric sequences. Rarefaction curves and alpha-diversity were evaluated by using Mothur (version v.1.30.1). RDP Classifier was used to analyze the taxonomy by referring to the silva (SSU115) 16S rRNA database at a 70% confidence threshold. Real-time quantitative PCR (qPCR) was performed (ABI 7500, Applied Biosystems, the USA) to quantify functional genes (chrA and soxB) with previously recommended primers (Table EA-1) (He et al., 2011; Meyer et al., 2010). The chrA gene encodes a putative Cr(VI) transporter, which confers Cr(VI) resistance. The soxB gene encodes enzyme complexes that are widespread among sulfur-oxidizing bacteria and responsible for oxidizing elemental sulfur to sulfate (Meyer et al., 2010). Certain compounds, such as cytochrome c on cell surface, intracellular nicotinamide adenine dinucleotide (NADH), and extracellular polymeric substances (EPS) [extracellular protein (PN), polysaccharide (PS), humic-like substances (HS)], may be involved in Cr(VI) reduction and detoxification. The variation patterns of these compounds may be related to Cr(VI) reduction activity. Therefore, these compounds were extracted and measured by following published procedures (Zhao et al., 2017b; Kang et al., 2018; Laiet al., 2018). Cytochrome c is a heme protein that is localized in 11

the inner and outer membranes, and plays an important role in Cr(VI) reduction through electron transfer in the respiratory chain (Zhao et al., 2017b). NADH is a coenzyme that functions as a hydride donor for chromate reductase to detoxify Cr(VI) (He et al., 2011). EPS ingredients, especially PN and HS, are involved in the thermodynamically favorable process of binding with Cr(VI) during Cr(VI) reduction process by membrane-associated chromate reductase (Jin et al., 2017). The abundance data of these compounds were normalized to volatile suspended solids (VSS) reflecting the amount of viable cells (Lai et al., 2018). Fourier-transform infrared (FTIR) spectrometry (Nicolet 5700, Thermo, the USA) was employed to analyze functional groups of collected EPS. Possible metabolites, including extracellular volatile fatty acids (VFAs) in aqueous solution and intracellular organic carbon polymers, i.e.glycogen, were determined. VFAs were measured with gas chromatography (Agilent 4890, J&W Scientific, USA) that was equipped with a flame ionization detector (Zhang et al., 2018). Glycogen was measured with the phenol method (Cai et al., 2013). A phenol reagent was used to break up cells to release glycogen and its concentration was measured using a spectrophotometer at 470 nm, with glucose as a standard (Randall et al., 2002). 3. RESULTS 3.1. Cr(VI) removal in the bioreactors Cr(VI) was progressively removed in all bioreactors, with faster and more complete removal in MB than those in individual HB and AB (Fig. 1A). Cr(VI)

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removal extents and rates were similar in three consecutive cycles, suggesting that their performance was steady and reproducible. Cr(VI) removal efficiency in a typical cycle (48 h) were 95.5 ± 0.74%, 45.9 ± 0.81%, and 40.1 ± 0.52% in MB, HB, and AB, respectively. The calculated Cr(VI) removal rate in these three bioreactors was 3.22 ± 0.10 mg L-1 h-1, 1.58 ± 0.09 mg L-1 h-1 and 0.77 ± 0.13 mg L-1 h-1, respectively. Cr(VI) concentration in abiotic control remained stable (Fig. EA-1). Without addition of any electron donor, Cr(VI) concentration decreased only in the first spike, likely due to the presence of some residual organic carbon from the inoculum. Once this carbon source was exhausted, Cr(VI) reduction ceased. These data suggest that Cr(VI) removals in bioreactors were microbially-mediated and electron donors were essential. Aqueous total Cr concentration was nearly identical to aqueous Cr(VI), indicating that reduced Cr(VI) product, e.g., likely Cr(III), was not soluble. Similar to the patterns of Cr(VI) removal, total Cr displayed a faster removal in MB than those in HB and AB (Fig. 1B). As expected, pH slightly increased in HB but decreased in AB (Fig. 2A). In contrast, pH in MB remained stable, likely due to neutralization between the alkalinity generated in HB and the acidity generated in AB. DOC was consumed in a typical Cr(VI) reduction cycle, with a lower extent of consumption in MB (89.6 ± 0.5%) than in HB (95.0 ± 0.4%) (Fig. 2B). Likewise, DOC consumption rate was slightly slower in MB (0.45 ± 0.01 mg L-1 h-1) than in HB (0.49 ± 0.02 mg L-1 h-1). Sulfate was continuously produced from microbial oxidation of elemental sulfur in both AB and MB (Fig. 2C), while neither sulfite nor thiosulfate was detected in both 13

bioreactors. However, the accumulated amount of sulfate in MB (25.7 ± 1.02 mg L-1) was lower than that in AB (31.2 ± 1.03 mg L-1), suggesting that there was a removal mechanism of sulfate by heterotrophs in MB. 3.2. Fate of Cr(VI) reduction product At the end of the third cycle, dark green precipitates accumulated at the bottom of all three bioreactors (Fig. EA-2). SEM image in MB indicated that large amounts of mineral precipitates accumulated (Fig. 3A). EDS results showed that the main elemental composition of the precipitates consisted of Ca, Cr, S, O, and Cl (Fig. 3B). The XRD patterns identified these precipitates as Cr2(SO4)3, CrO(OH), and Cr(OH)3·3H2O, along with some salts likely precipitated from the medium (Fig. 3C). XPS results showed two distinct peaks at 586.3 eV (Cr 2p1/2) and 576.6 eV (Cr 2p3/2), (Fig. 3D), suggesting that chromium in the precipitates was mostly Cr(III) (Wang et al., 2018b) from microbial reduction of Cr(VI) to Cr(III). Intracellular solid deposits were clearly observed under STEM (Fig. 3E). An elemental line scan revealed high signal intensity of chromium inside and around a bacterial cell (Fig. 3F). 3.3. Effects of initial Cr(VI) concentration and COD on Cr(VI) removal The effects of initial Cr(VI) concentration and COD on Cr(VI) removal in MB were investigated in order to understand optimal conditions for Cr(VI) removal. As expected, Cr(VI) removal efficiency decreased with the increasing Cr(VI) concentration (Fig. 4A). Cr(VI) was completely removed in 48 h at the lowest concentration of 25 mg/L, but only 53.8 ± 1.7% Cr(VI) was removed at the highest 14

concentration of 100 mg/L. However, the rate of Cr(VI) removal increased from 2.46 ± 0.14 mg L-1 h-1 at Cr(VI) concentration of 25 mg/L to 3.70 ± 0.29 mg L-1 h-1at Cr(VI) concentration of 100 mg/L. The extent and rate of Cr(VI) removal were positively correlated with initial COD concentration (Fig. 4B). When the initial COD concentration was 30 mg/L, 82.8 ± 1.7% Cr(VI) was removed, while a complete removal of Cr(VI) was achieved when the initial COD concentration increased to 120 mg/L. The Cr(VI) removal rate increased by 2.4 folds when the initial COD concentration increased from 30 mg/L (1.86 ± 0.19mg L-1 h-1) to 120 mg/L (4.54 ± 0.47mg L-1 h-1). 3.4. Microbial community dynamics Both microbial diversity and community structure changed after incubation with Cr(VI). By the end of the third cycle, the number of OTUs decreased in incubated bioreactors relative to that in the initial inoculum, especially in AB (Fig. 5, Table 1). Among the three bioreactors, the number of OTUs in MB was higher than those in HB and AB, suggesting that more organisms could tolerate and detoxify Cr(VI) under the mixotrophic condition. Moreover, microbial community in MB displayed the maximum Shannon index and minimum Simpson index among all three bioreactors and the initial inoculum, implying that microbial diversity under the mixotrophic condition may have actually increased after incubation with Cr(VI). After two-month incubation with three cycles of Cr(VI), microbial community structure showed significant changes at class level (Fig. 6A). Relative to the inoculum, the relative abundance of Betaproteobacteria increased to 13.3% and 14.3% in HB 15

and AB, respectively. However, the abundance of Gammaproteobacteria decreased to 15.3% and 9.24%, respectively. Furthermore, Bacteroidia and Spirochaetes were enriched in HB and further enriched in AB, while Anaerolineae and Deltaproteobacteria accumulated in AB. However, these classes were not particularly enriched in MB, but among all three bioreactors and the inoculum the abundances of Bacteroidetes vadin HA17 and Thermotogae were the highest in MB. At the genus level, specific organisms either diminished or accumulated under different conditions (Fig. 6B). Limnobacter was enriched in HB (0.27%) and AB (0.25%), but diminished in MB. Abundances of Proteiniphilum and Desulfovibrio increased greatly in MB relative to the inoculum and the other two bioreactors. Desulfuromonas emerged in AB and HB and was further enriched in MB (1.77%). Geobacter was enriched more in HB than in AB (2.98%). Relative to the inoculum (3.07%), Mesotoga diminished in HB (0.97%), but became enriched in AB (4.10%) and in MB (8.03%). 3.4. Quantitation of functional genes, compounds and metabolites The chrA gene was present in all bioreactors, with the highest amount in MB (Fig. 7A). The soxB gene was also found in MB but was expectedly lower than in AB. Both cytochrome c and NADH were detected in all bioreactors (Fig. 7B). The content of cytochrome c in HB was higher than in AB, but NADH was higher in AB than in HB. In MB, the concentration of cytochrome c was the lowest, while the NADH level was the highest.

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Concentration of EPS in HB, AB, and MB was 3.54 ± 0.42 mg/g VSS, 6.77 ± 0.59 mg/g VSS, and 5.79 ± 0.51 mg/g VSS, respectively. Extracellular protein (PN) accounted for the largest fraction of EPS in all bioreactors, followed by humic-like substances (HS) and a negligible amount of polysaccharide (PS) (Fig. 7C). FTIR spectra revealed major functional groups in EPS, such as carboxyl (COO-), and hydroxyl (-OH) (Fig. EA-3). VFAs accumulated at the end of a typical cycle (48 h). The total amount of VFAs was the highest in AB (7.06 ± 0.27 mg/L) followed by those in HB (5.08 ± 0.19 mg/L) and MB (3.26 ± 0.14 mg/L) (Fig. 8). Valerate and isovalerate were the major forms of VFAs in all bioreactors. The accumulation of intracellular glycogen was also found, with the highest concentration (4.64 mg/g VSS) in MB. 4. DISCUSSION 4.1. Advantages of sulfur-based mixotrophic Cr(VI) bio-reduction Heterotrophic Cr(VI) reduction has been intensively investigated in groundwater systems (Panousi et al., 2017), but lack of sufficient organics would limit Cr(VI) reduction and detoxification via this mechanism. A previous study has shown that around 65% of 52 mg/L Cr(VI) could be biologically removed with 10 mM acetate (640 mg/L COD) (Wang et al., 2018a). In our experiment, because 60 mg/L COD was used, which was apparently not adequate to reduce 50 mg/L Cr(VI) in a single cycle (Fig. 1). Likewise, sulfur-based autotrophic Cr(VI) removal has been studied, however, low solubility of elemental sulfur and acid accumulation may render this 17

process non-sustainable (Sahinkaya et al., 2014). Indeed, such weaknesses were observed in our AB bioreactor (Fig. 2) and the achieved Cr removal efficiencies were comparable to those previously reported under the same condition (Shi et al., 2019). These weaknesses inherent within heterotrophic and autotrophic Cr(VI) reduction can be overcome by synergistically combining these two processes. Although previous studies have demonstrated efficient Cr(VI) removal by the sulfur-based mixotrophic process (Sahinkaya et al., 2013; Peng et al., 2016; Sahinkaya et al., 2017), other electron acceptors such as nitrate often co-existed in those studies, which complicated the determination of the kinetics and mechanisms of Cr(VI) removal by this novel process. Sulfur-based mixotrophic Cr(VI) bio-reduction, with Cr(VI) as the sole electron acceptor, has never been reported before. In this study, by combining the heterotrophic and autotrophic processes into a single mixotrophic process, we demonstrated that both the rate and extent of Cr(VI) removal in MB were significantly enhanced (Fig. 1A). The Cr(VI) removal rate in MB (3.22 ± 0.10 mg L-1 h-1) was even higher than the sum of those in HB (1.58 ± 0.09 mg L-1 h-1) and AB (0.77 ± 0.13 mg L-1 h-1). The Cr(VI) removal extent exhibited a similar pattern. These results suggest a synergetic collaboration between heterotrophic and autotrophic organisms. Thus, mixotrophic Cr(VI) reduction performed better than the combination of autotrophic and heterotrophic reductions, as consistent with a previous study (Chojnacka et al., 2012). Similar enhancement was also found in biodiesel production by microalgae, where the productivity of fatty acid methyl ester in the mixotrophic culture was 1.53 times higher than the combination of 18

productivities in heterotrophic and autotrophic cultures (Shen et al., 2018). With acetate as electron donor, heterotrophic Cr(VI) reduction to Cr(III) should proceed as Reaction (1). Proton was consumed and alkalinity was produced, which should have been responsible for the observed increase in pH (Fig. 2A). Cr(VI) bio-reduction in AB could be expressed as Reaction (2), with the oxidation of elemental sulfur to sulfate, consumption of bicarbonate, and production of protons, resulting in a decrease in pH (Fig. 2A). By synergistically linking the heterotrophic and autotrophic processes, the mixotrophic Cr(VI) reduction process can be presented as Reaction (3). CrO42- + 0.5CH3COO- + 0.05NH4++ 5H+ → Cr3+ + 0.05C5H7NO2 + 0.45HCO3-+ 0.3CO2 + 2.95H2O(1) CrO42- + 10.5S0 + 15HCO3- + 3NH4+→ Cr3+ + 3C5H7NO2 + 10.5SO42- + H2O + 4H+ (2) CrO42- + 0.25CH3COO-+ 5.25S0+ 7.275HCO3-+ 1.525NH4++ 0.5H+ → Cr3+ + 1.525C5H7NO2+ 5.25SO42-+ 0.15CO2 + 1.975H2O(3) Advantages of this mixotrophic process are multiple folds. First, in the heterotrophic process, insufficient supply of organics could limit the bio-reduction process, but addition of excess organic carbon can result in blooming of fermentative bacteria, causing pore clogging (McLeod et al., 2018). Mixotrophic Cr(VI) reduction can overcome this limitation, because organic carbon can be synthesized by autotrophs from sulfur oxidation (Feng et al., 2010). Therefore, the problem of

19

biomass growth and subsequent pore clogging would have been avoided in the mixotrophic process. Second, less sulfate accumulated in MB. The amount of sulfate generation in the mixotrophic process was only about a half of that generated in the autotrophic process, when the same amount of Cr(VI) was removed [Reactions (1) and (3)]. Third, alkalinity produced from heterotrophic activity neutralizes the protons produced from the autotrophic reaction, thus maintaining a steady pH condition in MB, which would sustain Cr(VI) reduction to Cr(III). 4.2. Characterization of functional microbes and metabolic pathways Functional species responsible for oxidation of organics/elemental sulfur were detected. Genus Limnobacter of class Betaproteobacteria that was enriched in AB possesses genes involved in sulfur oxidization (Chen et al., 2016). Because this genus can grow both autotrophically and heterotrophically, its accumulation was also observed in HB (Nguyen and Kim, 2017). The growth of Mesotoga in Thermotogae could be stimulated by elemental sulfur under strictly anaerobic condition (Nesbø et al., 2012). Potential Cr(VI) reducers were identified in both the inoculum and all bioreactors, such as genus Geobacter of Deltaproteobacteria that are capable of reducing Cr(VI) through the function of c-type cytochromes and menaquinone (Lovley

et

al.,

1993).

In

AB

and

MB,

accumulated

Desulfovibrio

in

Deltaproteobacteria could reduce Cr(VI) through c3-type cytochrome as a Cr(VI) reductase (Lovley et al., 1994). Other potential Cr(VI) reducers included

20

Desulfuromonas, which emerged only after incubation with Cr(VI). This genus possesses polyheme a-type cytochromes on its membrane, which may be involved in enzymatic Cr(VI) reduction (Assfalg et al., 2002). Interestingly, the Proteiniphilum of Bacteroidia was present in the inoculum, HB, and MB, and it is a fermentative bacterium, producing acetic acid, hydrogen, and carbon dioxide by fermenting proteins (Niu et al., 2008), thus it could potentially provide organic carbon and electrons to heterotrophic Cr(VI) reducers. Cr(VI) reduction can occur both extracellularly and intracellularly. Extracellular Cr(VI) reduction can take place by compounds located on cell membranes, for instance, membrane-bound cytochrome c can transfer electrons to Cr(VI) (Middleton et al., 2003). The protein component in EPS could be involved in Cr(VI) reduction, while COO- and -OH groups (Fig. EA-3) could bind Cr(VI) to protect cells against Cr(VI) toxicity (Lu et al., 2018). In addition, Cr(VI) ion can enter cells, and cytoplasmic fractions with soluble proteins can reduce it as a way of detoxification (Wang et al., 2017). For example, intracellular NADH can serve as electron donor to reduce Cr(VI) (He et al., 2011). The detection of cytochrome c and NADH in all three bioreactors illustrated that both extracellular and intracellular Cr(VI) reduction pathways may have occurred, likely because of a mixed culture of high diversity used in the bioreactor (Focardi et al., 2012). Compared to HB and AB, the decreased cytochrome c and increased NADH under mixotrophic condition imply decreased extracellular Cr(VI) reduction but increased intracellular Cr(VI) detoxification. Such enhanced intracellular Cr(VI) reduction was evidenced by intracellular Cr 21

accumulation (Figs. 3E-F). Furthermore, the higher chrA gene abundance in MB than in HB and AB (Fig. 7A) suggests a high resistance to Cr(VI) toxicity (He et al., 2018) under mixotrophic condition, because this gene encodes Cr(VI) ion transport protein that is responsible for intracellular Cr(VI) efflux. The accumulation of organic metabolites was found after incubation with Cr(VI). In AB, the measured VFAs (except acetate) should have been derived from bicarbonate reduction, with the energy possibly from elemental sulfur oxidation (Zhang et al., 2018), with the upregulation of the soxB gene. The soxB gene encodes enzymes involved in catalytic oxidation of sulfur to sulfate (Meyer et al., 2010). In HB, the metabolic activities of fermentative bacteria, such as Proteiniphilum, could induce the accumulation of VFAs through fermenting microbially synthesized proteins (Niu et al., 2008). In MB, both pathways for VFAs accumulation, e.g., bicarbonate reduction and fermentation of proteins, may have occurred through the activities of Mesotoga (autotrophic sulfur oxidizer) (Nesbø et al., 2012) and Proteiniphilum (protein fermenter) (Niu et al., 2008). Part of VFAs could have been transferred into cell to synthesize glycogen that can provide energy for intracellular Cr(VI) reduction (Ji et al., 2017). Indeed, the amount of glycogen appeared to be correlated with the level of Cr(VI) reduction, e.g., in MB, both the amount of glycogen and extent of Cr(VI) reduction were the highest, but in AB both were the lowest (compare Figs. 1 and 8).

22

4.3. Possible mechanisms and environmental implications On the basis of product identification, microbial analysis, and metabolite determination, possible mechanisms for the sulfur-based mixotrophic Cr(VI) reduction process can be proposed (Fig. 9). In addition to heterotrophic Cr(VI) reducers such as Desulfovibrio and Desulfuromonas using acetate as carbon source and electron donor to reduce Cr(VI), synergistic interaction was recognized as a pivotal characteristic in mixotrophic Cr(VI) reduction (Zhang et al., 2018). In such synergy, autotrophic sulfur-oxidizing bacteria partnered with heterotrophic Cr(VI) reducers to jointly achieve this goal, with their produced organic metabolites (VFAs) serving as links (Lai et al., 2016). Autotrophic sulfur-oxidizing bacteria Limnobacter and Mesotoga oxidized elemental sulfur to sulfate through the upregulated soxB gene, accompanied by the release of energy (Nesbø et al., 2012; Chen et al., 2016). With this energy, bicarbonate was reduced to synthesize VFAs. A portion of VFAs was consumed by heterotrophic Cr(VI) reducers to reduce Cr(VI) extracellularly, catalyzed by cellular compounds including membrane-bound cytochrome c (Middleton et al., 2003). Some VFAs were assimilated by these heterotrophic Cr(VI) reducers and transformed to glycogen. The lowest level of residual VFAs and the highest glycogen in MB suggested that a large fraction of Cr(VI) was reduced inside cells (Fig. 3E-F). Eventually, Cr(III) was identified as the main product of Cr(VI) reduction, and it precipitated both on and within cells (Fig. 3). Considering the common co-occurrence of organic matter and reduced sulfur compounds in groundwater aquifer (Zhang et al., 2009; Zhang et al., 2012b; Knabe et 23

al., 2018; Phan et al., 2019), sediments (Graham et al, 2009), and soils (Frohne et al., 2015), a mixotrophic condition may be expected in such environments. This study provides new knowledge on the biogeochemical transformation process of Cr(VI) when Cr(VI) and other metals are present in such aquifers/sediments/soils (Mollema et al., 2015). For example, when organic matter, reduced sulfur compounds, and Cr co-exist in soils (Frohne et al., 2015), a mixotrophic process may be important and even dominant in controlling biogeochemical cycling of Cr. In such contaminated soils, enhanced Cr(VI) bio-detoxification under mixotrophic condition can potentially be employed for efficient Cr(VI) removal. In practical application, the proposed mixotrophic process can be embedded in a variety of remediation techniques. For example, biostimulation with amendment of proposed substances (e.g., elemental sulfur and acetate) can be implemented to successfully remediate Cr(VI)-contaminated aquifer (Wang et al., 2014). In addition, solid organics, such as sawdust and straw, are also efficient electron donors for microbes (Lepine et al., 2016). They can act as the reactive materials and can be packed in a biological permeable reactive barrier (bio-PRB), which is recognized as a promising technology for in situ groundwater remediation (Gibert et al., 2011). The bio-PRB can be placed in the migration pathway of contaminated plumes so that the reactive materials can react with Cr(VI) in groundwater, and this reaction can be catalyzed by microbes in groundwater flows (Gibert et al., 2019). In practical application, other co-contaminants, such as nitrate and other metal ions, should be taken into account (Jiang et al., 2018; Wang et al., 2018a) and conditions can be 24

further optimized. 5. CONCLUSIONS Fast Cr(VI) bio-reduction in sulfur-based mixotrophic process with organics and elemental sulfur as co-donors of electrons was achieved in this study, with removal efficiency as high as 95.5 ± 0.74% within 48 h, which was higher than the combined autotrophic and heterotrophic reductions. Environmental conditions, such as initial Cr(VI) concentration and COD concentration, affected the efficiency of this process. Heterotrophic Cr(VI) reducers such as Desulfovibrio and Desulfuromonas could directly reduce Cr(VI) with acetate. In mixotrophic system, these heterotrophs partnered with autotrophic sulfur-oxidizing bacteria to jointly accomplish Cr(VI) reduction, with VFAs as serving links between them. Both extracellular and intracellular reduction Cr(VI) to insoluble Cr(III) occurred as inferred from metabolite analysis and solid analysis. This study is helpful to reveal the biogeochemical behavior of Cr(VI) in aquifers with co-presence of organics and

reduced sulfur

compounds. The proposed process can also be potentially implemented for in-situ bioremediation of Cr(VI) contaminated groundwater. ACKNOWLEDGMENTS This research was supported by Beijing Nova Program (No. Z171100001117082), the National Natural Science Foundation of China (NSFC) (No. 41672237 and 41630103) and the Fundamental Research Funds for the Central Universities (No. 2652018176). We are grateful to two anonymous reviewers, the Associate Editor, and 25

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38

Table 1. The bacterial richness and diversity indices for the initial inoculum and the communities by the end of the third cycle in all bioreactors. Systems

Reads

OTU

Ace

Chao1

Shannon Simpson

Coverage

Inoculum

36576

508

545

565

4.11

0.050

0.998

HB

35110

495

483

496

3.81

0.058

0.998

AB

34155

389

456

459

4.18

0.032

0.998

MB

35967

434

471

479

4.20

0.027

0.998

39

Figure captions Fig. 1. Time-course decrease of (A) aqueous Cr(VI) concentration during three consecutive cycles and (B) total dissolved chromium under heterotrophic, autotrophic and mixotrophic conditions. Grey arrows indicate the times of replacement of the reaction products by a new spike of Cr(VI) in synthetic groundwater. Fig. 2. Time-course variations of pH, DOC and sulfate in employed bioreactors. (A) pH in all three types of bioreactors; (B) DOC in MB and HB; (C) sulfate in MB and AB. Fig. 3. Morphology, composition, mineral phase, and valence state of the produced precipitates in MB. (A) SEM; (B) EDS; (C) XRD; (D) XPS; (E) A TEM thin section of a bacterial cell showing precipitation around and within the cell; (F) A SEM line scan showing Cr distribution across the thin section of the cell. In Fig. 3A, the surface of two bacterial cells appeared to be coated by Cr precipitates. In Fig. 3B, the composition of Cr precipitates shows Cr, S, and O as major elements. Ca and Cl peaks are likely from synthetic groundwater medium. In Fig. 3C, three Cr(III) minerals are identified, Cr2(SO4)3, CrO(OH), and Cr(OH)3 ·3H2O. In Fig. 3D, the blue and green curves represent the peaks of Cr2p3/2 and Cr2p1/2, respectively. The orange curve is the baseline. The red curve is fitted from the experimental curve (grey one) by XPS Peak Fit software. In Fig. 3E and F, chromium distribution across a cell is detected. Fig. 4. The effects of initial Cr(VI) concentration (A) and COD (B) on Cr(VI) removal.

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Fig. 5. Rarefaction curves based on the sequencing results of initial inoculated consortium and bacterial communities in the bioreactors at the end of three Cr(VI) cycles (2-month). Fig. 6. Relative microbial abundance in the initial inoculum and three bioreactors. (A) class-level; (B) genus-level. Fig. 7. Quantitation of functional genes, pertinent compounds and EPS components. (A) functional genes chrA and soxB; (B) pertinent compounds cytochrome and NADH normalized to volatile suspended solids (VSS); (C) Abundances of constituents of EPS; PN: extracellular protein; PS: polysaccharides; HS: humic-like substances. Fig. 8. Quantitation of possible organic metabolites including residual volatile fatty acids (VFAs) in aqueous solution and intracellular glycogen in all bioreactors. Fig. 9. A Proposed mechanism of sulfur-based mixotrophic Cr(VI) bio-reduction. VFAs: volatile fatty acids; EPS: extracellular polymeric substances; NADH: nicotinamide adenine dinucleotide.

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Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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