Autotrophic Vanadium(V) Bioreduction in Groundwater by Elemental

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Autotrophic Vanadium (V) Bio-reduction in Groundwater by Elemental Sulfur and Zerovalent Iron Baogang Zhang, Rui Qiu, Lu Lu, Xi Chen, Chao He, Jianping Lu, and Zhiyong Jason Ren Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b01317 • Publication Date (Web): 06 Jun 2018 Downloaded from http://pubs.acs.org on June 8, 2018

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Environmental Science & Technology

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Autotrophic Vanadium (V) Bio-reduction in Groundwater by

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Elemental Sulfur and Zerovalent Iron

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Baogang Zhanga,b*, Rui Qiua, Lu Lub, Xi Chenb, Chao Hea, Jianping Lua, Zhiyong Jason

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Renb,*

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a

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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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b

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Colorado Boulder, Boulder, Colorado 80309, United States

School of Water Resources and Environment, MOE Key Laboratory of Groundwater

Department of Civil, Environmental, and Architectural Engineering, University of

10 11 12 13 14 15 16

*

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[email protected], [email protected] (B. Zhang);

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Tel.: 303-492-4137; Fax: 303-492-7317. E-mail: [email protected] (Z. Ren).

Corresponding authors. Tel.: +86 10 8232 2281; Fax: +86 10 8232 1081. E-mail:

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ACS Paragon Plus Environment


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ABSTRACT

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Vanadium (V) is an emerging contaminant in groundwater that can adversely

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affect human health. Although bioremediation has been shown effective, little is

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known on autotrophic V(V) bio-reduction in the context of oligotrophic

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characteristics of groundwater. In this study we demonstrate that efficient V(V)

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bio-reductions can be coupled with bio-oxidation of elemental sulfur (S(0)) or

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zerovalent iron (Fe(0)), and the V(V) removal efficiencies reached 97.5 ± 1.2% and

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86.6 ± 2.5% within 120 h using S(0) and Fe(0), respectively. V(IV) is the main

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reduction product and precipitates naturally in near-neutral conditions. Microbial

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community, functional gene, and metabolites analyses reveal that synthetic

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metabolisms among autotrophs and heterotrophs played major roles in V(V) reduction

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using S(0) and Fe(0). These results demonstrate a new approach for V(V)

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contaminated groundwater remediation.

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TOC Art

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INTRODUCTION

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Vanadium contamination in groundwater is receiving increased attention due to

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rising public health concerns.1 Vanadium widely exists in the Earth's crust

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concomitant with minerals, crude oil and coal.2 Geological weathering of

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vanadium-containing minerals releases vanadium into aquifer naturally.3,4 The

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anthropogenic sources mainly include the combustion of vanadium-rich fossil fuels

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and wastewater discharge from mining and usage of vanadium catalysts.5,6 For

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example, high vanadium concentrations up to 0.77 mg/L were found in groundwater

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of a former vanadium and uranium ore processing facility in Rifle, Colorado, USA,7

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far exceeding a minimum reporting level of 0.2 µg/L proposed by the US

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Environmental

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microorganisms as a trace nutrient, but at high concentrations it becomes toxic to

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terrestrial organisms and humans in the same class as mercury, lead and arsenic.9,10

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The USEPA’s current reference concentration for vanadium indicates that ongoing

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exposure to vanadium at levels of more than 21 ppb per day may lead to negative

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health effects.11,12 Vanadium can exist in different oxidation states, with vanadium (V)

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(V(V)) considered as the most toxic and the most mobile form.13,14 Both physical and

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chemical treatments such as adsorption, precipitation and immobilization are used to

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remove V(V), however, the generation of large volumes of sludge and high

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operational costs restrict these applications.15 Vanadium in the form of V(IV) is less

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toxic and insoluble at near-neutral pH.16,17 Promoting the reduction of V(V) to V(IV)

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has been recognized as a promising remediation strategy for removing this

Protection

Agency

(USEPA).8

Vanadium

4


is

required

by

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contaminant from groundwater.8,18

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Bioremediation of V(V) contaminated groundwater under anaerobic condition is

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recently viewed as a feasible approach, and a variety of microorganisms including

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bacteria, archaea and eukaryotic strains were identified.2,19 Most of them are

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heterotrophic so organic substrates are supplemented during bioremediation.1,12

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However, natural organic availability decreases with the increase of depth, and the

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external injection of substrates becomes costly and energy intensive with the

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possibility of secondary contamination.20 Considering the oligotrophic nature of

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groundwater, autotrophic V(V) reduction is more feasible and cost effective without

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excess accumulation of biomass.21 Though H2 based V(V) reduction was reported,22

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low solubility and difficulty in transportation and storage makes the implementation

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difficult. In this context, redox-active minerals such as elemental sulfur (S(0)) and

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zerovalent iron (Fe(0)) may serve as ideal electron donors. S(0) is a waste byproduct

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of oil refining, which is inexpensive, non-toxic, water insoluble, and stable under

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normal conditions.23 Fe(0) can supply hydrogen in situ via the iron corrosion

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process.24 S(0) and Fe(0) have been successfully used for biological denitrification

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and perchlorate reduction.25,26 However, little is known about feasibility of V(V)

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bio-reduction using S(0) and Fe(0) as sole electron donors.

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To fill this knowledge gap, we investigated for the first time the possibility of

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bio-reducing V(V) using S(0) and Fe(0) as electron donors. Naturally available

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bicarbonate served as the carbon source. Reaction products were analyzed, and

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operational factors were systematically examined. In addition, microbial communities

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involved in the process were analyzed and functional genes were identified. The

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findings from this study will assist in the development of viable solutions for

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bioremediation of V(V) contaminated aquifers.

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MATERIAL AND METHODS

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Bioreactor setup and operation. Six plexiglass bottles with a total volume of

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250 mL were employed as reactors. Each reactor was covered with aluminum foil and

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sealed with a rubber stopper to maintain the anaerobic condition. Each reactor was

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filled with 200 mL synthetic groundwater containing the following ingredients (per L):

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0.504 g NaHCO3, 0.2464 g CaCl2, 0.035 g NH4Cl, 1.0572 g MaCl2·6H2O,0.4459 g

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NaCl, 0.0283 g KCl, 0.0299 g KH2PO4.12 V(V) was supplied in the form of

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NaVO3·2H2O with a given concentration described below. Four reactors were

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inoculated with 50 mL anaerobic sludge obtained from an up-flow anaerobic sludge

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blanket reactor (Yanjing Brewery, Beijing, China). Then they were divided into 2

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groups with 2 reactors fed with 5 g S(0) (B-S) and the other 2 reactors fed with 5 g

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Fe(0) (B-Fe) with a similar range of particle size (0.8-4.0 mm). Two reactors each

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served as a control by adding the same amount of S(0) (C-S) or Fe(0) (C-Fe) but

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without inoculum.

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The bioreactors were operated in batch mode and took almost 2 months for

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microbial cultivation before formal data collection. Hardly any V(V) was removed

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after the organics in the sludge were depleted with S(0) or Fe(0) absent during this

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process (Figure S1). Average concentration of total organic carbon (TOC) for the

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cultivated inocula was as low as 4.49 ± 0.21 mg/L (p < 0.01). Since then, V(V)

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reduction with S(0) or Fe(0) as the sole electron donor was individually assessed in

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three consecutive operating cycles. The initial V(V) concentration was 50 mg/L. For

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each cycle, B-S and B-Fe reactors were supplemented with 5 g S(0) and 5 g Fe(0),

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respectively. Reaction products as well as solution conditions were analyzed, and the

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impacts of operating factors were evaluated, including initial V(V) concentration (25

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mg/L, 50 mg/L, 75 mg/L, 100 mg/L) with fixed bicarbonate concentration (360 mg/L),

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as well as the effects of initial bicarbonate concentration (0 mg/L, 180 mg/L, 360

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mg/L, 540 mg/L) with specified V(V) concentration (50 mg/L). After another 2

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months of operation, high-throughput 16S rRNA gene sequencing was performed to

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identify the distribution of microbial community. Preliminary functional gene groups

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were also identified to elucidate the degradation pathways. All experiments were

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conducted at room temperature (22 ± 2 ºC) with duplicate reactors, and the mean

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values of experimental data were reported.

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Chemical and biological analyses. Before the analysis, water samples taken

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from the reactors were immediately filtered through a 0.22 µm membrane. Soluble

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V(V) concentration was measured using a spectrophotometric method through

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forming

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(5-Br-PADAP),13 and the total V in aqueous solution was analyzed by ICP-MS

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(Thermo Fisher X series, Germany). pH was determined by using a pH-201 meter

complexes

with

2-(5-bromo-2-pyridylazo)-5-diethylaminophenol

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(Hanna, Italy). Analyses of sulfate, sulfite and thiosulfate ions were carried out by ion

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chromatography (Basic IC 792, Metrohm, Switzerland) using standard solutions. TOC

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was analyzed by Multi N/C 3000 TOC analyzer (Analytik Jena AG, Germany).

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Volatile fatty acids (VFAs) were monitored by a gas chromatograph (Agilent 4890,

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J&W Scientific, USA) equipped with a flame ionization detector. Solid S(0) and Fe(0)

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were examined by scanning electron microscope (SEM) with energy dispersive X-ray

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(EDS) operated at 20 kV (JEOL JAX-840, Hitachi Limited, Japan). Generated

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precipitates were analyzed by X-ray photoelectron spectroscopy (XPS) (XSAM-800,

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Kratos, UK).

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Microbial samples were collected from the inoculum as well as different stages

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of operation. Samples were pretreated with ultrasonic method,27 and the genomic

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DNA was extracted using the FastDNA® SPIN Kit for Soil (Qiagen, CA, the USA)

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according to the manufacturer’s instructions. The extracted DNA was pooled,

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amplified by PCR before sending to Majorbio Technology (Shanghai, China) for

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high-throughput Illumina MiSeq sequencing. Raw sequencing data were submitted to

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the NCBI Sequence Read Archive Database with the accession number of SPR071706

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and SPR096247. Phylogenetic affiliations and metagenomic results were analyzed as

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previously described,28 with reference to Kyoto Encyclopedia of Genes and Genomes

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(KEGG) database and Clusters of Orthologous Groups of proteins (COG) database.

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RESULTS AND DISCUSSION

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Performance of autotrophic V(V) bio-reduction. For each batch, an initial 50

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mg/L of V(V) was applied in each reactor, and Figure 1a shows gradual V(V) removal

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in both B-S and B-Fe reactors in three consecutive operating cycles, implying that

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microbially-mediated V(V) reduction took place under autotrophic condition by using

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S(0) or Fe(0) as the sole electron donor. By the end of the 120 h batch, the V(V) was

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reduced by 97.5 ± 1.2% when using S(0) as the sole electron donor (B-S), higher than

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the results observed in B-Fe reactor (86.6 ± 2.5%) (p < 0.05). The total V also

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decreased progressively, indicating the precipitation of reduction product. The

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removal of the total V was lower than V(V), because it not only includes V(V) but

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also V(IV), which is a reduction product of V(V).2,18 The difference between total V

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and V(V) could indicate the dissolved V(IV). For example, an average amount of 44.6

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± 1.9 mg/L V(V) was converted to V(IV) in each batch cycle in B-S reactor. In

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comparison, approximately 5.7 ± 0.8 mg/L dissolved V(IV) existed in aqueous

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solution after batch reaction.

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In a typical operating cycle (120 h), V(V) removal rates were 0.41 ± 0.06 mg/L·h

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in B-S and 0.36 ± 0.04 mg/L·h in B-Fe, respectively (p < 0.05) (Figure 1b). The

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Pseudo first-order kinetics rate constants were calculated to be 0.0299 h-1 for B-S and

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0.0139 h-1 for B-Fe, correspondingly (Table S1). In abiotic control reactors, hardly any

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V(V) was removed in C-S, further confirming the functions of microbes in V(V)

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reduction while slight V(V) removal was obtained in C-Fe through chemical reduction

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(Figure S2).29 SEM images show that the surface of S(0) and Fe(0) became rougher 9



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after interactions with microbes in the bioreactors, which is an apparent contradiction

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with the smooth surfaces shown in abiotic reactors (Figure S3). This observation

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supports the removal data that microbial activities facilitated the V(V) reduction by

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using S(0) or Fe(0) as the electron donor. The detection of an oxygen peak in B-Fe

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and C-Fe (Figure S4) suggests Fe(0) passivation to iron oxides maybe induced,30

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which can partially explain the slower V(V) reduction compared to S(0) (Figure 1a).

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However, microbial activities in B-Fe could alleviate this inhibition through reductive

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dissolution by employing these iron oxides as alternative electron acceptors.31

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Microbial autotrophic V(V) reduction in groundwater has only been reported by

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using H2 as the electron donor,22 but the reported V(V) reduction rate (0.005 mg/L·h)

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was orders of magnitude lower than what this study observed, presumably due to the

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slow mass transfer and less efficient microbial activities. This study demonstrates that

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S(0) and Fe(0) may have good advantages for autotropic V(V) reduction, as they

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provide abundant electron donor sources in the subsurface. It should be noted that H2

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from iron corrosion may contribute as an alternative electron donor in B-Fe (Equation

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(1)),32 but no gas bubbles were observed during the study so such contribution was

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deemed not significant. Though externally amended organic electron donors such as

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acetate showed higher V(V) reduction,27 the high cost, difficulty in application, and

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potential to cause secondary contamination make it still a challenging approach as

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compared with this in situ inorganic donor method.

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Fe(0) + 2H2O → H2 + Fe2+ + 2OH-

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Identification of reaction products. Blue precipitates were observed in the

(1)

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reactors along with V(V) reduction (Figure S5). XPS results showed the peak located

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at 515.8 eV and was confirmed as V(IV) with the main components of VO(OH)2

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and/or mineral sincosite [CaV2(PO4)2(OH)4·3H2O], similar to the products reported

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before (Fig. 2a).33,34 This finding confirmed that V(V) was bio-reduced to less mobile

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V(IV) using S(0) and Fe(0) as the sole electron donor. Peaks corresponding to V(V)

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were also discovered,12 probably resulting from the re-oxidation of V(IV) in the

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processes of sampling and testing as V(IV) is easily oxidized in air.18

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In addition to reduction products, oxidation products of S(0) and Fe(0) were also

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investigated. In B-S, the concentration of sulfate increased with time, while sulfite and

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thiosulfate were hardly detected (Figure 2b). This indicates that S(0) was oxidized

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mainly to sulfate by releasing electrons to V(V) during biological utilization, which

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had also been observed in S(0) based autotrophic denitrification processes for

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simultaneous nitrate and Cr(VI) reduction.35 Regarding B-Fe, dissolved iron species

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were low in the solution, but brown precipitates were found accumulated. Analytic

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results of XPS of Fe 2p for these precipitates suggested that Fe(III) was the main state

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of the oxidation products of Fe(0) with the difference between Fe 2p1/2 at 24.9 eV

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and Fe 2p3/2 at 711.3 eV in peak splitting value of 13.6 eV (Figure 2c), which is

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consistent with the literature value for Fe(III) oxides.36 The Fe(III) product has more

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negative effects on V(V) bio-reduction than SO42-, as the standard reduction potential

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for Fe(III)/Fe(II) is 0.771 V, higher than that for SO42-/S0 (0.621 V). This leads to

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lower energy gain during V(V) reduction to V(IV) (0.991 V), which correlates with

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the slower kinetics observed in B-Fe (Figure 1a). Similar findings and explanations

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were also used in explaining acetate-supported V(V) bio-reductions and

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hydrogen-based microbial reduction of uranium (VI).37,38 Furthermore, sulfate and

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Fe(III) could also be produced from oxygen-induced consumption of S(0) and Fe(0).

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Both of them were competitive electron acceptors with V(V), which would adversely

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affect the treatment efficiency of V(V).37

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VFAs were also monitored as possible metabolic intermediates. In a typical

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operating cycle (120 h), concentrations of residual VFAs were 11.64 ± 2.89 mg/L for

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B-S and 4.55 ± 1.74 mg/L for B-Fe, respectively (p < 0.05). These numbers are

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comparable with data obtained from methane mediated biological bromate

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reduction.39,40 Higher VFAs could also induce faster V(V) removal in B-S with

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ongoing consumption by heterotrophic V(V) reducing microorganisms, which were

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identified in microbial community analysis. Residual VFAs were dominated by valeric

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species in this study (Figure 2d), differing from acetate as the main form under

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quasi-anaerobic conditions,39 probably due to the priority for V(V) reducers to

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consume acetate.27

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The solution pH decreased gradually from 8.02 ± 0.02 to 7.38 ± 0.07 in B-S (p

Autotrophic Vanadium(V) Bioreduction in Groundwater by Elemental

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