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Remediation and Control Technologies
Microbial Chromate Reduction Coupled to Anaerobic Oxidation of Elemental Sulfur or Zerovalent Iron Jiaxin Shi, Baogang Zhang, Rui Qiu, Chunyu Lai, Yufeng Jiang, Chao He, and Jianhua Guo Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b05053 • Publication Date (Web): 18 Feb 2019 Downloaded from http://pubs.acs.org on February 18, 2019
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Microbial Chromate Reduction Coupled to Anaerobic Oxidation of
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Elemental Sulfur or Zerovalent Iron
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Jiaxin Shia, Baogang Zhanga,*, Rui Qiua, Chunyu Laib, Yufeng Jianga, Chao Hea,
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Jianhua Guob,*
5
a School
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Circulation and Environmental Evolution, China University of Geosciences (Beijing),
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Beijing 100083, P. R. China
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of Water Resources and Environment, MOE Key Laboratory of Groundwater
b Advanced
Water Management Centre, The University of Queensland, St Lucia, Queensland 4072, Australia
9 10 11 12 13 14 15
*Corresponding
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Baogang Zhang: Tel.: +86 10 8232 2281; Fax: +86 10 8232 1081. E-mail:
17
[email protected].
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Jianhua Guo: Tel.: + 61 7 3346 3222; Fax: + 61 7 3365 4726; E-mail:
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[email protected].
authors.
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ABSTRACT: Chromate (Cr(VI)), as one of ubiquitous contaminants in groundwater,
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has posed a major threat to public health and ecological environment. Although
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various electron donors (e.g., organic carbon, hydrogen and methane) have been
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proposed to drive chromate removal from contaminated water, little is known for
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microbial chromate reduction coupled to elemental sulfur (S(0)) or zerovalent iron
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(Fe(0)) oxidation. This study demonstrated chromate could be biologically reduced by
27
using S(0) or Fe(0) as inorganic electron donor. After 60-day cultivation, the sludge
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achieved a high Cr(VI) removal efficiency of 92.9 ± 1.1% and 98.1 ± 1.2% in two
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independent systems with S(0) or Fe(0) as the sole electron donor, respectively. The
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deposited Cr(III) was identified as the main reduction product based on X-ray
31
photoelectron spectroscopy. High-throughput 16S rRNA gene sequencing indicated
32
that Cr(VI) reduction coupled to S(0) or Fe(0) oxidation was mediated synergically by
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a microbial consortia. In such the consortia, S(0)- or Fe(0)-oxidizing bacteria (e.g.,
34
Thiobacillus or Ferrovibrio) could generate volatile fatty acids as metabolites, which
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were further utilized by chromate-reducing bacteria (e.g., Geobacter or Desulfovibrio)
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to reduce chromate. Our findings advance our understanding on microbial chromate
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reduction supported by solid electron donors and also offer a promising process for
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groundwater remediation.
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TOC Art
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INTRODUCTION
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Although chromium (Cr) is required by human and other organisms as an essential
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micronutrient element, it becomes toxic and carcinogenic in the form of chromate
45
(Cr(VI)).1 Cr(VI) is one of widely detected contaminants in groundwater.2 The natural
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concentration of Cr in groundwater is usually low (5 μg/L).3 However, serious Cr(VI)
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pollutions occurred in groundwater at numerous places over the past few decades,
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mainly due to Cr-containing wastewater released from tannery, metallurgical and
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nuclear industrial sectors.4,5 According to a survey conducted by Environmental
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Protection Agency (EPA) in the US, the concentration of Cr(VI) is above 10 μg/L in
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4% of groundwater systems, with the maximum concentration up to 97 μg/L.6 The
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exposure of Cr(VI) can cause severe adverse effects on human health, e.g., respiratory
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irritation, nasal or lung cancer, and damage to the gastrointestinal tract.5 In terms of
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its toxicity, the Cr(VI) concentration in drinking water is limited to 10 μg/L in
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California US.7
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Removing excessive Cr(VI) from groundwater has become an urgent
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environmental problem to be solved.5 Conventional remediation methods include
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physical adsorption, chemical reduction, ion exchange, and phytoremediation.8,9
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However, both physical and chemical processes are usually costly and may cause a
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secondary pollution (e.g., residual metal sludge), while the cycle time for
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phytoremediation is generally long (more than 4 months).5,10,11 Biological chromate
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removal methods provide a promising pathway for Cr(VI)-contaminated groundwater
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remediation.12 Recently, some strains such as Pseudomonas dechromaticans, 4
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Desulfovibrio vulgaris and Leucobacter sp. have been reported to possess potential
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capacities for microbial Cr(VI) reduction.13-15 Cr(VI) can be microbially reduced to
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Cr(III) when it is utilized as an electron acceptor by these microorganisms with
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organic carbons as electron donors.16 Compared to Cr(VI), Cr(III) is less toxic and
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shows limited mobility due to its low solubility in groundwater.17
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Due to the low bioavailability of organic carbons in groundwater, additional
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supplies of electron donors are usually required to support heterotrophic Cr(V)
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reduction. However, the extra addition of organic carbons may cause a secondary
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pollution.18 In addition, the formation of organic-Cr(III) complexes increases the
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solubility of Cr(III).19 Recently, microbial reduction of Cr(VI) has also been proposed
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by using gaseous substrates as electron donors, such as hydrogen or methane in terms
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of low operation cost and substantial availability.20 Although hydrogen- or methane-
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based methods are efficient to reduce Cr(VI) to Cr(III) and satisfactory results have
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been achieved,21,22 the safety issues resulting from the usage and storage of these
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explosive gases require precautionary measures before their wide applications in
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practice. Very recently, batch and continuous flow bioreactors utilizing S(0) or Fe(0)
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as electron donor have been evaluated to achieve removals of nutrients and toxic
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metals in groundwater.23-25 Since particulate S(0) is non-toxic and insoluble in water,
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it can be persistently fixed in biosystems, then release electrons slowly for microbes
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to remove contaminants. The application of S(0) as electron donor have advantages,
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including less biomass production and enhanced biological stability. Meanwhile, S(0)
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can be obtained from the waste byproduct of oil refining.26 Similarly, Fe(0) enables to
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react with water through iron corrosion process under anaerobic conditions, thus
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releasing hydrogen to drive microbial reactions.25 For example, Geets et al.27 added
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Fe(0) to permeable reactive barriers to support sulfate-reducing bacteria (SRB) to
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improve removal efficiencies of heavy metals (e.g., Cu and Zn). For in situ
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application of Fe(0) for groundwater remediation, it can reduce biomass clogging due
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to a lower biomass yield compared to using organic carbons as electron donors.28
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Nevertheless, the feasibility of S(0) or Fe(0) as the sole electron donor for microbial
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Cr(VI) reduction has not been well explored so far.
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The objective of this study is to investigate the potential of microbial Cr(VI)
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reduction coupled with S(0) or Fe(0) as the sole electron donor. Six lab-scale
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bioreactors were set up to test the feasibility of chromate removal after seeding
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anaerobic digestion sludge as inoculums. Firstly, we monitored chromate removal
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efficiency and identified the intermediates and final products of different systems with
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S(0) or Fe(0) alone as electron donors. Then, potential influencing factors (including
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initial Cr(VI) and bicarbonate concentrations) during the Cr(VI) reduction were
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assessed. Finally, the potential players for microbial reduction of Cr(VI) were
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identified based on 16S rRNA gene sequencing. In this study, microbial chromate
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reduction coupled to S(0) or Fe(0) oxidation was stably achieved after 60-day
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cultivation. The findings of this study shed a light on microbial reduction of Cr(VI)
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and could guide a process design to achieve groundwater remediation with Cr(VI)
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contamination.
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MATERIALS AND METHODS
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Bioreactor setting-up and the inoculum
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Six identical serum bottles with a working volume of 250 mL were set up as
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bioreactors to test chromate removal in this study. Each of bottle was sealed with
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butyl rubber stoppers and aluminum crimp caps to maintain a strict anaerobic
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condition. Initially, 50 mL anaerobic sludge obtained from an up-flow anaerobic
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sludge blanket reactor (Beijing YanJing Brewery Co., Ltd., China) were inoculated
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into each bioreactor. These six bioreactors were divided into three groups equally
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(each group with duplicate reactors). The first group (B-S) was operated with 5 g S(0)
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supplement as electron donors, while the second group (B-Fe) with 5 g Fe(0) addition.
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Neither S(0) nor Fe(0) was added into the third group (B-I) in order to confirm
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whether chromate reduction was available under the absence of electron donor. In
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addition, extra two reactors filled with the same amount of S(0) and Fe(0) but without
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inoculum were named as “C-S” and “C-Fe” as abiotic controls, respectively.
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The original cultivation was done in six bioreactors with 200 mL synthetic
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groundwater containing the following components per liter: 0.504 g NaHCO3, 0.2464
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g CaCl2, 0.035 g NH4Cl, 1.0572 g MaCl2·6H2O, 0.4459 g NaCl, 0.0283 g KCl, 0.029
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g KH2PO4. In the medium, bicarbonate was supplemented as inorganic carbon sources
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and K2CrO4 with a given concentration was added to the bioreactors as the source of
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Cr(VI). Both S(0) and Fe(0) (Analytical reagent) were purchased from Sinopharm
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Chemical Reagent Co., Ltd. (Beijing, China) and used directly without further
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processing. The whole experiments were performed in an anaerobic chamber, which 7
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avoided S(0) and Fe(0) oxidation. According to the manufacture information, both
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S(0) and Fe(0) have a similar range of particle size (i.e., 0.8-4.0 mm).
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Experimental procedure
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Long-term incubation was conducted in all six bioreactors by feeding 200 mL
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synthetic groundwater with 50 mg/L Cr(VI) at the beginning of each cycle (120 h),
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refreshing the aqueous solution every 120 h. In each bioreactor, 5 g S(0) or 5 g Fe(0)
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was supplemented initially at the incubation stage. After 60-d incubation, each
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bioreactor achieved a steady state at room temperature (22 ± 2 ºC). Soluble organics
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existing in the original inoculum were exhausted after 60-d cultivation in terms of
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total organic of less than 5 mg/L. Then three consecutive cycle studies were
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conducted to evaluate the performance of microbial reduction of Cr(VI) with S(0) and
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Fe(0) as the sole electron donor, respectively. For B-S and B-Fe systems, 5 g S(0) and
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5 g Fe(0) were freshly supplied to B-S and B-Fe at the beginning of these three
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consecutive cycles, respectively. In addition, both Cr(VI) and bicarbonate were
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provided to keep initial Cr(VI) concentration of 50 mg/L and initial bicarbonate
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concentration of 360 mg/L. During the cycle study, 5 mL supernatants in bioreactors
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were collected every 12 h and filtered through 0.22 μm membrane filter to determine
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the Cr(VI) concentrations and identify reaction metabolites. Furthermore, in order to
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investigate the effect of solution conditions on the microbial Cr(VI) reduction, batch
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experiments with different initial Cr(VI) concentrations (25, 50, 75, and 100 mg/L)
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and various initial bicarbonate concentrations (0, 180, 360, and 540 mg/L) were
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carried out in triplicates. 8
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Analytical methods
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The concentration of Cr(VI) was measured by the colorimetric method by using an
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UV-visible spectrophotometer (DR 6000, HACH, USA) at 540 nm.29 The
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determination of dissolved total Cr was performed by an Inductively Coupled Plasma
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Mass Spectrometry (ICP-MS, X Series II, Thermo Fisher Scientific, USA). The pH
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level was determined using a pH-201 meter (Hanna, Italy). The sulfate, sulfite and
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thiosulfate concentrations in B-S were measured using an ion chromatography (ICS-
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1100, Thermo Fisher Scientific, USA ). Gas chromatograph (Agilent 4890, J&W
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Scientific, USA) equipped with a flame ionization detector was used to identify the
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potential reaction intermediates (mainly volatile fatty acids, VFAs) produced in
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bioreactors. The morphology of solid S(0) and Fe(0) were observed through scanning
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electron microscope (SEM) with energy dispersive X-ray (EDS) operated at 20 kV
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(JEOL JAX-840, Hitachi Limited, Japan). The precipitates adhered to the bioreactors
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were collected and characterized by X-ray photoelectron spectroscopy (XPS)
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(XSAM-800, Kratos, UK).
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Microbial analysis
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In order to identify potential players for microbial chromate reduction, sludge samples
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(0.5 g) were collected from B-S and B-Fe systems after the three consecutive cycles
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for high-throughput 16S rRNA gene sequencing. Microbial DNA was extracted by
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using FastDNA® SPIN Kit for Soil (Qiagen, CA, the USA) according to
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manufacturer’s protocols. We used primers 338F (ACTCCTACGGGAGGC
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AGCAG) and 806R (GGACTACHVGGGTWTCTAAT) to amplify the V4-V5
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regions of the bacteria 16S ribosomal RNA gene (GeneAmp® 9700, ABI, the
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USA).30 The primers 524F10ext (TGYCAGCCGCCGCGGTAA) and Arch958R
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(YCCGGCGTTGAVTCCAATT) were used to amplify archaea. The PCR
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amplification and high-throughput 16S rRNA gene sequencing were performed by
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Majorbio Technology (Shanghai, China) on an Illumina HiSeq4000 platform
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(Illumina, Inc., San Diego, CA, USA). Based on 16S rRNA gene amplicon
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sequencing data, the microbial community structure was analyzed by following the
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pipeline described previously by Zhang et al.23
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RESULTS AND DISCUSSION
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Microbial Cr(VI) reduction performance
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With an initial Cr(VI) concentration of 50 mg/L, Figure 1 shows the variations of
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Cr(VI) and total Cr concentrations over time in three consecutive cycles for both of B-
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S and B-Fe systems after 60-d cultivation. When S(0) or Fe(0) was supplied as the
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sole electron donor, both B-S and B-Fe systems exhibited a high Cr(VI) removal
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efficiency, reaching 92.9 ± 1.1% and 98.1 ± 1.2%, respectively. The Cr(VI) removal
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efficiencies in both B-S and B-Fe were comparable with the previous results by using
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organic electron donors (e.g. acetate).31 The profile of total Cr concentration in the
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supernatant of B-S and B-Fe followed the similar trend with the profile of Cr(VI)
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(Figure 1). The obtained removal efficiency of total Cr in B-S and B-Fe was 88.9 ±
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1.2% and 97.4 ± 0.9%, respectively. Compared to Cr(VI) reduction rate ( 0.39 ±
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0.04 mg/L·h in B-S and 0.42 ± 0.02 mg/L·h in B-Fe), the obtained total Cr reduction
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rate was slower (0.38 ± 0.07 mg/L·h in B-Fe and 0.41 ± 0.03 mg/L·h). In contrast,
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only 3.9 ± 0.9% of Cr(VI) was removed in B-I without any supply of S(0) or Fe(0)
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(Figure S1), confirming that Cr(VI) reduction is associated with the presence of S(0)
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or Fe(0) in the proposed bioprocess. In addition, abiotic experiments without biomass
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(C-S and C-Fe) were conducted to verify whether chemical reduction also made
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contributions for chromate removal. Results showed that the C-S system without
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biomass did not exhibit any Cr(VI) removal capacity (Figure S1), ruling out the
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contribution of chemical reaction in the B-S system. Differently, Cr(VI) removal
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efficiency reached 14.8 ± 0.7% in the first cycle of C-Fe, while this percentage
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decreased to 7.9 ± 0.5% after 3-cycle operation (Figure S1a). Eventually, only 1.9 ±
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0.3% of Cr(VI) was removed after 60-day operation (Figure S1b). Therefore, a higher
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removal efficiency of Cr(VI) in B-Fe (98.1 ± 1.2%) might be partially associated with
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the combined microbial reduction and chemical reaction. Chemical reduction of
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chromate might be driven by electrons released from Fe(0) (Reaction 1).32 Iron
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corrosion could also occur and the corresponding products (e.g., H2 and Fe2+) would
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further contribute to Cr(VI) reduction (Reactions 2-4).33
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CrO42- + Fe(0) + 4H2O → Cr3+ + Fe3+ + 8OH-
(1)
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Fe(0) + 2H2O → H2 ↑ + Fe2+ + 2OH-
(2)
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CrO42- + 1.5H2 + H2O → Cr3+ + 5OH-
(3)
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CrO42- + 3Fe2+ + 4H2O → Cr3+ + 3Fe3+ + 8OH-
(4)
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It should be noted that a distinct decrease of chemical chromate reduction from
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Day 1 (14.8 ± 0.7%) to Day 60 (1.9 ± 0.3%) was due to the Fe(0) passivation, which
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was evidenced by iron oxides coating on the surface according to EDS spectrums and
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SEM images. The EDS results showed that the initial S(0) and Fe(0) are pure (Figure
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S2a), while iron oxides were observed in C-Fe after the three operating cycles (Figure
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S2b) and 60-day operation (Figure S2c). The SEM images showed that the surface of
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S(0) in C-S appeared to be uniform and smooth (Figure S3a). In C-Fe, botryoidal
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clusters depositions on the surface of Fe(0) were found (Figure S3b), which might be
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precipitates of iron oxides derived from chemical oxidation of Fe(0) by Cr(VI).34 This
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Fe(0) passivation could inhibit chemical reactions in which Fe(0) acted as electron
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donor for Cr(VI) reduction. Obvious morphology variations of S(0) and Fe(0) in
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bioreactors were also observed. Surfaces of S(0) and Fe(0) in B-S and B-Fe were
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relatively rough, with microbes attaching on the surface (Figure S3c and d). Microbes
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used S(0) and Fe(0) as electron donors to accelerate the Cr(VI) reduction.26, 35 The
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EDS spectrums also indicated solid Cr component was formed on the surface of S(0)
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and Fe(0) in both B-S and B-Fe systems (Figure S2d).
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Identification of final products
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It was observed that gray-blue precipitates appeared in the solution of B-S and B-Fe
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(Figure S4). The produced precipitates were identified by XPS. As shown in Figure
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2a, two distinct peaks appeared between 576.0-578.0 eV and 586.0-588.0 eV,
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corresponding to Cr 2p3/2 and Cr 2p1/2, respectively. Such the spectrum indicated
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that the precipitates were Cr(III) species.36 The XPS results further indicated that Cr3+
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from biotransformation of Cr(VI) formed Cr(III) precipitates immediately after its 12
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production in bioreactors, which was consistent with the main components of
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Cr(OH)3 and Cr2O3 reported previously.36 In addition, the produced Cr(III) could also
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form complexes with extracellular polymeric substances (EPS) secreted by
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microorganisms.37 Considering Cr(III) is less toxic and insoluble under near neutral
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pH condition,17 the remediation process based on S(0) or Fe(0) could be practically
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useful to treat Cr(VI) contaminated groundwater.
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In order to investigate whether electron donors, including both Fe(0) and S(0),
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were oxidized or not, both sulfur and iron species were monitored during microbial
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chromate reduction. The sulfate concentration gradually increased in a cycle in B-S
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(Figure 2b), indicating the transformation of S(0) to sulfate. This also confirmed that
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microbial Cr(VI) reduction was coupled with S(0) oxidation, similar to a previous
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study.24 Other sulfur-related intermediates such as sulfite and thiosulfate were
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undetected in B-S. Regarding the B-Fe system, soluble iron species in the supernatant
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were hardly found, while brown precipitates adhering to the bottle were observed
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(Figure S4). The XPS analysis showed these precipitates had two typical peaks
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assigned to Fe 2p1/2 (725.0-727 eV) and Fe 2p3/2 (710.0-712.5 eV) (Figure 2c),
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which are similar to the peaks of Fe3O4 oxides obtained in a previous study.38 This
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result suggested that Fe(0) might have been oxidized into Fe3O4, which could release
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energy to synthesize intermediates for microbial Cr(VI) reduction.
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Accumulations of VFAs were observed within a cycle for 120 h (Figure 2d), in
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which 8.63 ± 0.15 mg/L and 18.09 ± 0.44 mg/L VFAs were produced in B-S and B-
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Fe, respectively. Isobutyrate was found as the main form of residual VFAs in both B-
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S and B-Fe, which is consistent with our previous study in S(0)- or Fe(0)-based
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biological vanadate reduction.23 Differently, acetate was observed as the dominant
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component of VFAs in methane-based bromate or nitrate removals.39,40 The total
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concentration of residual VFAs in B-Fe was higher than that in B-S, which might lead
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to a faster Cr(VI) removal rate. In autotrophic biosystems, VFAs could be derived
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from bicarbonate reduction or carbon dioxide fixation.41,42 In this bioprocess,
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autotrophs employed bicarbonate or carbon dioxide to synthesize VFAs via the Calvin
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cycle.43,44 These synthesized VFAs could be utilized as electron donors to achieve
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Cr(VI) reduction.
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The pH decreased gradually in B-S within a typical operating cycle (120 h),
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which is due to the generation of hydrogen ion during the S(0) bio-oxidation
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process24. In contrast, the pH profile showed an ascending trend in B-Fe (Figure S5),
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as hydroxyl ion can be produced from both chemical corrosion and biological
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oxidation of Fe(0).25 The decreasing bicarbonate concentration in both biosystems
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indicated that bicarbonate acted as an inorganic carbon source for autotrophic
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microbes to synthesize VFAs, which supported microbial Cr(VI) reduction with S(0)
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and Fe(0) as electron donors, respectively.
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Process elucidation and evaluation
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The processes of reduction of Cr(VI) coupled with S(0) and Fe(0) oxidations
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(Reactions 5 and 6, respectively) were proposed in terms of determined reaction
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products, and stoichiometric relationship:
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CrO42- + 10.5S0 + 15HCO3- + 3NH4+ → 3C5H7NO2 + Cr3+ + 10.5SO42- + H2O + 4H+ 14
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CrO42- + 1.5Fe0 +0.375HCO3- + 0.075NH4+ + 4.825H2O → 0.075C5H7NO2 + Cr3+ +
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1.5Fe(OH)3↓ + 5.3OH(6)
284 285
Both mass and electron balances were conducted to elucidate these two
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processes. In B-S, the measured sulfate concentration (93.7 ± 1.6 mg/L) within in a
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typical operating cycle (120 h), indicating that solid phase S(0) could be oxidized to
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SO42-.45,46 The released electrons resulting from S(0) oxidation to sulfate was 1.46 ±
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0.03 mmol, which is higher than 0.69 ± 0.06 mmol electrons required for transfering
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Cr(VI) to Cr(III). It is assumed that approximately 47.3% of released electrons were
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transferred from S(0) to Cr(VI) in one cycle, while the remaining electrons might
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support microbial growth and intermediates synthesis.23 According to Reaction 5, the
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theoritical required bicarbonate were 839.5 ± 73.2 mg/L in B-S based on the
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measured reduction of Cr(VI), while the measured bicarbonate consumption was
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353.2 ± 4.1 mg/L. The insufficient bicarbonate consumption in B-S might be
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compensated by carbon dioxide production from anaerobic respiration of accumulated
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organic intermediates.47
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It is too difficult to perform the electron balance in B-Fe, because no soluble iron
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species was availably detected. The consumption of bicarbonate was monitored in B-
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Fe. It is found that a larger amount of bicarbonate (177.1 ± 0.9 mg/L) was reduced
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than the theoretical value (21.9 ± 1.8 mg/L) in B-Fe, likely due to the growth of
302
autotrophic bacteria and the transformation of bicarbonate to carbonate along with
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increasing of pH. Furthermore, the biomass production was stoichiometrically 0.16 g
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to remove 1 g Cr(VI) supported by Fe(0) (Reaction 6), which was much lower than
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that in bioreactors using lactose and cheese whey as electron donors for Cr(VI)
306
reduction.48 The low biomass production of this reaction was also comparable to the
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cell yield of the autotrophic microorganisms (ranging 0.073 to 0.280 g cell dry weight
308
per gram chemical oxygen demand equivalent of hydrogen).23 Such a low biomass
309
yield might be a significant advantage of our proposed biosystems using Fe(0) as an
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electron donor, since aquifer clogging in organic carbon-based systems could be
311
alleviated.49
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Influencing factors examination
313
The effects of initial Cr(VI) and bicarbonate concentration on microbial Cr(VI)
314
reduction performance were further investigated. The initial Cr(VI) concentrations
315
would determine the reaction duration to achieve a complete Cr(VI) reduction to the
316
final Cr(VI) concentration below the detection limit. It was observed that the
317
complete reduction of Cr(VI) with concentration of 25 mg/L required approximately
318
72 h for both B-S and B-Fe, whereas the time needed for a complete removal of 100
319
mg/L Cr(VI) took more than 120 h in both systems (Figure 3a and 3b). However, the
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maximum reduction rate increased from 0.70 ± 0.11 mg/L·h to 1.59 ± 0.17 mg/L·h for
321
B-S, and the rate increased from 0.69 ± 0.02 mg/L·h to 2.73 ± 0.07 mg/L·h for B-Fe.
322
These results demonstrated that microbial chromate reduction processes with S(0) and
323
Fe(0) as electron donors were very robust to treat a wide range of Cr(VI)
324
concentrations (25 to 100 mg/L). In addition, B-Fe seems to be more robust towards 16
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higher Cr(VI) concentration than B-S. Increasing bicarbonate concentration could provide sufficient carbon source for
327
microbes to promote Cr(VI) reduction in both B-S and B-Fe.50 The removal efficiency
328
of Cr(VI) was 70.1 ± 1.5% and 80.4 ± 1.2% in B-S and B-Fe, respectively, without
329
the addition of bicarbonate. With the increase of initial bicarbonate concentration, the
330
reduction rate and the removal efficiency of Cr(VI) in B-S and B-Fe were enhanced.
331
When the bicarbonate concentration increased from 0 mg/L to 540 mg/L, the
332
percentage of Cr(VI) removal in B-S and B-Fe increased by 26.4 ± 1.1% and 19.0 ±
333
1.3% respectively (Figure 3c and 3d). Meanwhile, the removal rates increased from
334
0.29 ± 0.01 to 0.40 ± 0.02 mg/L·h and from 0.34 ± 0.01 to 0.42 ± 0.01 mg/L·h with
335
the same bicarbonate concentration change, respectively (Figure 3c and 3d).
336
Microbial community structure
337
In order to compare microbial community structure at different conditions, 16S rRNA
338
gene sequencing was performed for both the inoculum and two biomass samples (one
339
from B-S and another from B-Fe) after 75-day cultivation. Compared to the inoculum,
340
microbial richness was decreased in both B-S and B-Fe systems in terms of
341
rarefaction curves (Figure S6). In particular, the B-S system harbored less diverse
342
microbial communities in terms of OTU numbers, Simpson and Shannon indexes,
343
suggesting microbial communities tended to be highly selected (Table S1). However,
344
microbial diversity increased in B-Fe, implying more species existed due to Fe(0) as
345
well as its corrosion products.51
346
Compared to the inoculum, microbial communities changed significantly at the 17
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class level after feeding solid electron donors (Figure 4a). The predominant classes in
348
B-S were Betaproteobacteria, Gammaproteobacteria and Bacteroidia, with a relative
349
abundance of 31.1%, 22.8% and 18.3%, respectively. These three representative
350
classes accounted for around 72.1% of the total bacterial population in B-S, while
351
accounted for only about 14.1% in the inoculum. Meanwhile, a new class Bacilli with
352
a relative abundance of 4.17% appeared in B-S, which was not detected in either
353
inoculum or B-Fe. In B-Fe, Alphaproteobacteria, Spirochaetes and
354
Betaproteobacteria were enriched by 2.5%, 5.3% and 10.9% compared to the
355
inoculum, with the proportions of Anaeroline and Gammaproteobacteria remained
356
basically constant.
357
Potential bacteria associated with Cr(VI) reduction and oxidation of S(0) and
358
Fe(0) were further analyzed at the genus level (Figure 4b). The relative abundance of
359
Geobacter was increased both in B-S and B-Fe, which had been reported performed
360
well in situ bioremediation performance under high concentration of Cr(VI)
361
contamination.52 Desulfovibrio was enriched in B-Fe and its ability of reducing
362
Cr(VI) had been previously confirmed.53 In B-Fe, an iron-oxidizing bacteria
363
Ferrovibrio was accumulated, which used Fe(0) as direct electron donor.54 The
364
increased Syntrophus (0.86%) might be involved in the production and consumption
365
of VFAs.55 In B-S, chemoautotrophic Thiobacillus increased significantly from 0.25%
366
in the inoculum to 17.1% after 75-day cultivation. It has been reported that
367
Thiobacillus could oxidize S(0) to release electrons and gain energy to synthesize
368
metabolites to reduce Cr(VI).56,57 The accumulated Blvii28 (17.9%) might play an
18
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important role in autotrophic S(0) oxidation in B-S, which has been reported to be
370
able to generate hydrogen for S(0) autotrophic microbes.58
371
In both B-S and B-Fe, archaeal communities were also observed to shift
372
significantly. Methanobacteria became the only dominating archaea at the class level
373
in B-S and B-Fe, compared to the diverse archaeal species in the inoculum (Figure
374
S7a). The genus Methanobacterium was less in the inoculum (7.6%) but
375
predominated in B-S (91.0%) and B-Fe (81.0%) (Figure S7b). It was reported that
376
Methanobacterium have the potential of detoxifying Cr(VI) through bio-reduction.59
377
Potential microbial C(VI) reduction mechanism and environmental implications
378
In the light of the performances of batch tests, intermediate identification and microbial
379
community structure, a synergistic mechanism for the reduction of Cr(VI) coupled with
380
oxidation of S(0) and Fe(0) was proposed (Figure 5). Firstly, autotrophic microbes such
381
as Thiobacillus and Ferrovibrio might have synthesized VFAs as byproducts through
382
bicarbonate reduction with energy from oxidation of S(0) to sulfate or Fe(0) to
383
Fe(II)/Fe(III), respectively.54,56,57 Secondly, heterotrophic Cr(VI)-reducing microbes
384
such as Geobacter in B-S and Desulfovibrio in B-Fe would reduce Cr(VI) by using
385
VFAs as electron donors and carbon sources. Both extracellular and intracellular
386
reduction of Cr(VI) could occur, since mixed anaerobic culture was employed.11 Fox
387
example, Geobacter could reduce Cr(VI) extracellularly with the aid of c type
388
cytochromes on out membrane.60 Finally, the reduction products, Cr(III) would be
389
precipitated naturally in a near-neutral pH environment. Similar synergistic
390
mechanisms had also been reported in the bio-reduction of bromate driven by 19
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391
methane.39 However, it should be noted that more studies are needed to elucidate the
392
pathways involved and reveal the functions of the key members of a complex microbial
393
community in our S(0)- and Fe(0)-based systems through meta-omics (e.g.,
394
metagenomics and metatranscriptomics).
395
Considering Cr(VI) contamination in aquifer becomes serious, bioremediation
396
has been proposed as a promising pathway to alleviate its negative environmental
397
impacts.61 This study demonstrates for the first time that Cr(VI) could be biologically
398
reduced to Cr(III) coupled with oxidation of S(0) or Fe(0) with an efficient removal
399
rate. We further compared chromate removal performance with different electron
400
donors in literature. Cr(VI) reduction rate in both B-S (0.39 ± 0.04 mg/L·h) and B-Fe
401
(0.42 ± 0.02 mg/L·h) was much higher than that obtained with gaseous electron
402
donors (0.003 mg/L·h for hydrogen and 0.01~0.06 mg/L·h for methane),19, 21 and
403
comparable to the rate obtained using organics, such as acetate (0.62 mg/L·h) and
404
glucose (0.60 mg/L·h) (Table S2).31, 62 Moreover, employing solid inorganic electron
405
donors such as S(0) and Fe(0) can minimize possible secondary contamination and
406
aquifer clogging induced by organic substrates injection. The proposed bioprocesses
407
can be implemented as bio-permeable reactive barriers (bio-PRB) for efficient
408
bioremediation of Cr(V) contaminated groundwater. Bio-PRB is an in-situ
409
remediation project based on a wall of porous material cross the path of contaminated
410
groundwater flow which mainly removes contaminants by biodegradation combined
411
with adsorption, oxidation/reduction and other processes. 63 In such the bio-PRB
412
system, indigenous microorganisms can be accumulated and solid inorganic electron
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donors can be periodically replaced to maintain a high-efficiency. In addition, nitrate,
414
as a frequent co-contaminant in groundwater, might compete with chromate to utilize
415
electron donor, thus decreasing Cr(VI) removal efficiency,64,65 which should be
416
further investigated in the future.
417
Supporting Information
418
The bacterial richness and diversity of inoculated sludge and bioreactors;
419
comparison of common bioprocesses for Cr(VI) removal with different electron
420
donors; changes of Cr(VI) in C-S, C-Fe and B-I at different stages; EDS patterns
421
of S(0) and Fe(0) at initial states and after use at different states in employed
422
reactors; corresponding SEM images of solid electron donor in reactors; images
423
of the precipitates generated in employed bioreactors during operation; changes
424
of pH and bicarbonate concentration in employed bioreactors within a typical
425
operating cycle; rarefaction curves for species abundance in the inoculum,
426
biomass in B-S and B-Fe; archaeal community compositions.
427
ACKNOWLEDGEMENTS
428
This research work was supported by the National Natural Science Foundation of
429
China (NSFC) (No. 41672237, No. 91647115), Beijing Nova Program (No.
430
Z171100001117082) and the Fundamental Research Funds for the Central
431
Universities (No. 2652018193).
432
NOTES
433
The authors declare no competing financial interest.
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Figure 1. Variations of Cr(VI) and total Cr in B-S and B-Fe during three consecutive operating cycles. Red arrows represent the replacement of synthetic groundwater.
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Figure 2. Final products during Cr(VI) reduction in B-S and B-Fe. (a) XPS spectra of Cr 2p for the generated precipitates; (b) Variations of products from S(0) oxidation with time; (c) Fe 2p XPS spectra for precipitates from Fe(0) oxidation; (d) Average concentration of residual VFAs in a typical operating cycle.
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Figure 3. Effects of different operating factors on microbial reduction of Cr(VI) in proposed bioreactors. Initial Cr(VI) concentration in (a) B-S and (b) B-Fe; initial bicarbonate concentration in (c) B-S and (d) B-Fe.
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Figure 4. Relative abundances of microbial communities revealed by high-throughput sequences for the inoculum, biomass in the B-S and B-Fe after 75 d cultivation at levels of (a) class and (b) genus.
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Figure 5. Mechanistic pathways in microbial Cr(VI) reduction processes coupled to S(0) oxidation (a) and Fe(0) oxidation (b) under anaerobic condition. In B-Fe, chemical reduction also made approximately 1.9% of chromate removal efficiency, as indicated by a dash line.
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