Accepted Manuscript

Pyrite-based autotrophic denitrification for remediation of nitrate contaminatedgroundwater

Jiaoyang Pu, Chuanping Feng, Ying Liu, Rui Li, Zhe Kong, Nan Chen, ShuangTong, Chunbo Hao, Ye Liu

PII: S0960-8524(14)01351-0DOI: http://dx.doi.org/10.1016/j.biortech.2014.09.092Reference: BITE 13982

To appear in: Bioresource Technology

Received Date: 7 August 2014Revised Date: 12 September 2014Accepted Date: 17 September 2014

Please cite this article as: Pu, J., Feng, C., Liu, Y., Li, R., Kong, Z., Chen, N., Tong, S., Hao, C., Liu, Y., Pyrite-based autotrophic denitrification for remediation of nitrate contaminated groundwater, Bioresource Technology(2014), doi: http://dx.doi.org/10.1016/j.biortech.2014.09.092

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Pyrite-based autotrophic denitrification for remediation of nitrate 1

contaminated groundwater 2

Jiaoyang Pua,b, Chuanping Fenga,b∗, Ying Liub, Rui Lib, Zhe Kongb, Nan Chenb, 3

Shuang Tongb, Chunbo Haob, Ye Liub 4

5

a Key Laboratory of Groundwater Circulation and Evolution (China University of 6

Geosciences, Beijing), Ministry of Education, No. 29 Xueyuan Road, Haidian District, 7

Beijing 100083, China 8

b School of Water Resources and Environment, China University of Geosciences 9

(Beijing), No. 29 Xueyuan Road, Haidian District, Beijing 100083, China 10

11

Abstract 12

In this study, pyrite-based denitrification using untreated pyrite (UP) and 13

acid-pretreated pyrite (AP) was evaluated as an alternative to elemental sulfur based 14

denitrification. Pyrite-based denitrification resulted in a favorable nitrate removal rate 15

constant (0.95 d-1)), sulfate production of 388.00 mg/L, and a stable pH. The 16

pretreatment of pyrite with acid led to a further increase in the nitrate removal rate 17

constant (1.03 d-1) and reduction in initial sulfate concentration (224.25±7.50 mg/L). 18

By analyzing the microbial community structure using Denaturing Gradient Gel 19

Electrophoresis, it was confirmed that Sulfurimonas denitrificans (S. denitrificans) 20

could utilize pyrite as an electron donor. A stable pH was observed over the entire 21

∗ Corresponding author: School of Water Resources and Environment, China University of Geosciences (Beijing),

No. 29 Xueyuan Road, Haidian District, Beijing 100083, China

Tel.: +86 010 8232 2281; Fax: +86 010 8232 1081

E-mail address: fengchuangping@gmail.com (C. Feng)

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experimental period, indicating that the use of a pH buffer reagent would not be 1

necessary for pyrite-based denitrification. Therefore, pyrite could effectively replace 2

elemental sulfur as an electron donor in autotrophic denitrification for 3

nitrate-contaminated groundwater remediation. 4

Keywords 5

Nitrate removal, Pyrite, Autotrophic denitrification, Groundwater, Acid-pretreatment 6

1. Introduction 7

Nitrate, as the most ubiquitous nonpoint-source (NPS) contaminant in groundwater, 8

severely hinders the usage of groundwater for drinking (Sun and Nemati, 2012). 9

Groundwater can be easily contaminated by nitrate, due to its high solubility and 10

mobility in both water and soil (Ghafari et al., 2009). The heavy use of nitrogen 11

fertilizer, discharge of untreated agricultural and industrial wastewater, and 12

atmospheric deposition of nitrogen oxide emissions, are just some of the human and 13

industrial actions leading to nitrate pollution (Yang and Lee, 2005; Ghafari et al., 14

2009). Elevated nitrate concentrations in drinking water represent the potential risks 15

for public health such as methemoglobinemia (Aslan and Cakici, 2007). Consequently, 16

the maximum contaminant level (MCL) for nitrate (NO3-, 10 mg-N/L) in drinking 17

water is stipulated by the World Health Organization (WHO, 2008) and China 18

(NHFPC, 2006). 19

Many physical-chemical technologies have been developed for nitrate 20

contaminated groundwater treatment, such as ion exchange (Samatya et al., 2006), 21

reverse osmosis (Schoeman and Steyn, 2003) and electrodialysis (Elmidaoui et al., 22

2001). These technologies are used for nitrate enrichment and separation, and require 23

secondary treatments (Ghafari et al., 2009). Presently, biological denitrification 24

represents a promising approach for nitrate contaminated groundwater remediation 25

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(Sánchez et al., 2008). Heterotrophic denitrifiers utilize organic substances as electron 1

donors to convert nitrate into nitrogen, under anoxic conditions (Sierra-Alvarez et al., 2

2007). Organic carbon availability is a critical factor for heterotrophic denitrification 3

(Zhang et al., 2012). Liquid carbon sources such as methanol (Tong et al., 2013) and 4

hydrolyzed molasses (Quan et al., 2005), as well as solid carbon sources, including 5

biodegradable plastic and wheat straw (Zhang et al., 2012), have been utilized for 6

heterotrophic denitrification. However, the supplementation of organic compounds 7

can lead to additional costs and secondary pollution resulting from heterotrophic 8

denitrification (Moon et al., 2004). 9

Due to its low cost and stable denitrification performance, autotrophic 10

denitrification using elemental sulfur as an electron donor, for groundwater treatment, 11

has been extensively studied in recent years. Soares (2002) used a laboratory column 12

packed with elemental sulfur to treat synthetic groundwater containing 100 mg/L of 13

nitrate, and denitrification rates of up to 0.20kg N/(m3·d) were obtained and sulfate 14

concentrations increased from 50-80 mg/L up to 320 mg/L. High average nitrate 15

removal efficiencies (98.8%) for synthetic groundwater contaminated by nitrate (7.3 16

mM) were maintained in the bioreactor supplied with elemental sulfur and limestone 17

granules (1:1,v:v) (Sierra-Alvarez et al., 2007). By using a sulfur-based permeable 18

reactive barrier for nitrate-contaminated groundwater treatment, 60 mg-N/L of nitrate 19

was reduced to nitrogen gas with sulfate production of 250 mg-S/L (Moon et al., 20

2008). In the batch experiment, the removal of 50 mg-N/L of nitrate in 500 mL of 21

synthetic groundwater resulted in the production of 792.3 mg/L sulfate and a decrease 22

in pH from 7.5 to less than 5.0 with addition of elemental sulfur (Qambrani and 23

Oh ,2013). High sulfate production and sharp pH decrease are primary factors limiting 24

elemental sulfur based autotrophic denitrification; limestone is, therefore, frequently 25

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used as a pH-buffering reagent (Soares, 2002). 1

Autotrophic denitrification, based on ferrous sulfide (FeS), was shown to be an 2

efficient method for nitrate removal in freshwater systems by Haaijer et al. (2007). Li 3

et al. (2013) also used this approach and treated real wastewater with FeS, resulting in 4

nitrate reduction from 14.9 mg-N/L to 1.1 mg-N/L in 18 hours. 5

Microbial oxidation of pyrite by Thiobacillus denitrificans (T. denitrificans), as a 6

significant natural attenuation process, has been reported to play a role in natural 7

attenuation of nitrate contaminated groundwater. Juncher Jørgensen et al. (2009) 8

observed nitrate reduction when pyrite particles were added to the sediments from an 9

adjacent aquifer. Torrentó et al. (2010) showed that pyrite minerals could be utilized 10

by T. denitrificans as the single electron donor for denitrification. Pyrite is the most 11

abundant sulfide mineral in the earth’s crust, constituting a major reservoir in global 12

cycles of sulfur and iron (Bosch et al., 2012). However, few studies have been 13

conducted concerning pyrite utilization for treatment of nitrate contaminated 14

groundwater. 15

The objectives of this study were (1) to confirm the feasibility of pyrite-based 16

autotrophic denitrification by analyzing the nitrate removal rate, sulfate production, 17

and pH variation, (2) to investigate pyrite-based denitrification mechanisms through 18

analysis of the bacterial community structures, and (3) to compare the denitrification 19

performances of UP (untreated pyrite) and AP (acid-pretreated pyrite) as electron 20

donors. 21

2. Materials and methods 22

2.1 Preparation of materials 23

Natural pyrite crystals were obtained from Qujing, Yunnan Province, China, and 24

ground by a pulverizer (ZN-04, KINGSLH, China) to obtain 0.15-0.25 mm particle 25

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sizes. In order to investigate the effects of acid-pretreatment on denitrification 1

performance, a portion of the ground pyrite particles were immersed in 10% (v/v) HCl 2

solutions for 30 min, rinsed by deionized water until the pH of the rinsing solution 3

was 7.0, and dried at 105 for 4 hours. 4

Synthetic groundwater was prepared by adding KNO3 to deionized water, 5

containing (g/L) 0.40 KNO3, 0.40 KH2PO4 and 0.50 NaHCO3. The initial nitrate 6

concentration in the synthetic groundwater was 55 mg-N/L. 7

All chemical reagents used in the experiments were analytical-grade. 8

2.2 Characterization of pyrite 9

The pyrite crystals were confirmed to be pure pyrite, with no evidence of other 10

mineral phases, by X-ray diffraction analysis (D8 FOCUS, Bruker, Germany). 11

The elemental compositions of the prepared pyrite samples (natural pyrite crystals, 12

UP, and AP) were analyzed by electron microprobe analysis (EPMA-1600, Shimadzu, 13

Japan). The natural pyrite crystals contained 52.81% (w/w) Fe, 46.41% S, 0.46% Si, 14

and 0.32% Al. The elemental composition of UP was 50.10% Fe, 49.42% S, 0.29% Si, 15

and 0.19% Al. The elemental composition of the ground sample was similar to the 16

natural crystals. For the acid-pretreatment, the elemental composition was 17.11% S, 17

75.13% Fe, 3.66% Si, and 4.10% Al. During the acid-pretreatment, pyrite could be 18

chemically oxidized according to the following equation: 19

FeS2+2HCl→H2S+FeCl2+S (1) 20

Hydrogen sulfide was produced and released into the air during the acid-pretreatment, 21

therefore, a lower mass percentage of S was detected in the acid-pretreated pyrite 22

sample. 23

2.3 Cultivation of microorganisms 24

Anaerobic sludge was obtained from Tsinghe Sewage Treatment Plant (Beijing, 25

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China) and cultivated in a liquid nutrient medium (2L) at 30 for 50 days. The 1

nutrient medium consisted of Na2S2O3·5H2O (20 mM), NaHCO3 (30 mM), KNO3 (20 2

mM), KH2PO4 (14.7 mM), NH4Cl (18.7 mM), MgSO4·7H2O (3.25 mM), FeSO4·7H2O 3

(0.08 mM), and CaCl2·2H2O (0.05 mM) (Beller, 2005). The nutrient medium was 4

replaced every two days. Before inoculation, the cultures were centrifuged for 10 min 5

(4500 rpm, 30 ) by a centrifuge (GT10-1，SHKIC，China) and suspended in 6

sterilized physiological saline solutions to remove nitrate and sulfate residues in 7

cultures. 8

2.4 Experimental procedure 9

All the denitrification experiments using UP and AP as electron donors were carried 10

out in 500 mL flasks, in duplicate. 11

The respective flasks were filled with 50 g of UP or AP and 300 mL of the synthetic 12

groundwater, and sterilized at 115 for 30 min. The flasks were then inoculated 13

with 20 mL of the prepared cultures, described in 2.3, on a Clean Bench (SW-GJ-IFD, 14

AIRTECH, China). The flasks were sparged by nitrogen gas for 5 min prior to sealing 15

with rubber plugs. Un-inoculated flasks were used as controls. All flasks were 16

cultivated at 100 rpm and 30 in a constant temperature incubator (DDHZ-300, 17

TCSSYSBC, China) for 6 days. 18

After 6 days, the inoculated UP flask was still standing for 30 min, microorganisms 19

grown on the UP surface and suspended in the supernatant were taken for 16S 20

rRNA-based microbial analysis. 21

2.5 Analysis 22

A 10 mL sample was collected from each flask, every 18 h, and the pH of the 23

sample was measured immediately with a pH meter (Seven Multi S40, Mettler Toledo, 24

Switzerland). 25

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Samples were filtered through a 0.45 μm membrane filter. Two mL of each 1

sample were treated with 0.5 mL of H2O2 (3 %, v/v) in order to oxidize sulfite and 2

thiosulfate in the samples to sulfate. Sulfate and total sulfate were measured with an 3

ion chromatograph (ICS900 Dionex IonPac, Thermo Fisher Scientific, US) with 4

detection limit of 0.09 mg/L. 5

According to the Water and Wastewater Monitoring Analysis Method (SEPA, 2002), 6

concentrations of nitrate, nitrite, ammonium, total iron and ferrous ion (Fe (II)) were 7

measured with a spectrophotometer (DR6000, HACH, US) and the detection limits 8

were 0.08 mg/L for nitrate, 0.003 mg/L for nitrite, 0.025 mg/L for ammonium, 0.03 9

mg/L for total iron and 0.03 mg/L for ferrous ion. ATPs in the cultures were measured 10

with an ATP fluorescence detector (AF-100, TOADKK, Japan) with the detection 11

limit of 0.2 fM ATP. 12

The standard deviations were analyzed at a confidence level of 90%, and Origin 9.0 13

(OriginLab, trial version) was used to compute the nitrate removal rate constants in 14

the inoculated UP and AP flasks. 15

For 16S rRNA-based microbial analysis, genomic DNA was extracted and 16

amplified with 968F GC and 1401R primers, using the PCR system (initial 17

denaturation, 95 for 5 min; subsequent denaturation, 95 for 0.5 min; annealing, 18

54 for 0.5 min; extension, 72 for 45 s and final extension, 72 for 10 min). 19

Another round of PCR was performed with amplified 16S rRNA Genes. Denaturing 20

Gradient Gel Electrophoresis (DGGE) analysis was performed using the Bio-Rad 21

C-1000 system (Bio-Rad, USA). The PCR products were loaded in a 1 mm-thick gel 22

containing, 9 % (w/v) polyacrylamide and a denaturant gradient of 40-60 % (100% 23

denaturant was 7 M urea and 40 % formamide). The electrophoresis was run in 1 × 24

TAE (20 mM Tris, 10mM acetate, 0.5mM EDTA, at pH 8.0) at 60 for 12 h at 100 25

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V. The gel was then stained with dye solution and placed on a UV illuminator 1

(Bio-Rad, USA). The excised gel from each target band was placed into a 1.5 mL 2

sterile tube containing 30 µL of sterile ultrapure water and crushed. One µL of eluted 3

DNA was amplified with primer 968F/1401R. DNA was sequenced and the sequences 4

were determined by Shanghai Sangon Co., Ltd., Shanghai, China. 5

2.6 Nucleotide Sequence Accession numbers 6

The nucleotide sequences reported in this paper have been submitted to GenBank 7

with accession numbers: KM220911-KM220924. 8

3. Results and discussion 9

3.1 Nitrate reduction 10

As shown in Fig. 1a, nitrate sharply decreased from 56.69±0.22 mg-N/L to 11

5.21±1.33 mg-N/L during the first 3 days, and then decreased slowly to 0.08±0.06 12

mg-N/L in the inoculated UP flasks. Nitrate concentrations ranged from 53.31±0.28 13

mg-N/L to 55.87±0.14 mg-N/L in the un-inoculated UP flasks during the experimental 14

period. Similarly, the nitrate concentration decreased from 55.57±0.21 mg-N/L to 15

2.62±0.36 mg-N/L during first 3.75 days, and then nitrate concentration declined 16

steadily to 0.05±0.00 mg-N/L in the inoculated AP flasks. In the un-inoculated AP 17

flasks, the nitrate concentration was nearly stable at 55 mg-N/L (Fig. 1b). Nitrate 18

reduction efficiencies in the inoculated UP and AP flasks both exceeded 99%. In 19

contrast, no nitrate reduction was observed in un-inoculated flasks, indicating that the 20

pyrite could not directly reduce nitrate. 21

According to the first-order kinetics model, nitrate removal rates in the inoculated 22

UP and AP flasks were computed (determination coefficients [R2]> 0.9). The nitrate 23

removal rate constant in the inoculated AP flasks was 1.03 d-1, slightly higher than 24

that of the inoculated UP flasks (0.95 d-1). This difference was potentially due to the 25

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acid-pretreatment process, removing micro-particles such as iron and sulfur impurities 1

from the pyrite surface, and roughening the pyrite surface to allow microorganisms to 2

more easily attach to and utilize the pyrite. 3

The initial nitrite concentration was less than 0.02 mg-N/L in the inoculated UP 4

flasks. The nitrite concentration increased rapidly, reaching 0.55±0.04 mg-N/L during 5

first 3 days, then decreased steadily to less than 0.02 mg-N/L (Fig. 1c). In the 6

inoculated AP flasks, the nitrite concentration increased from less than 0.02 mg-N/L 7

to 0.47±0.05 mg-N/L during the first 2.25 days, then decreased to 0.03±0.00 mg-N/L 8

(Fig. 1d). Pyrite-based denitrification followed the typical pattern of basic microbial 9

denitrification, expressed as NO3-→NO2

- →NO→N2O→N2 (Torrentó et al., 2010). 10

After nitrate was partially reduced to nitrite, nitrate and nitrite co-existed in the 11

inoculated UP and AP flasks. The synthesis and activity of nitrite reductase could be 12

inhibited by high concentrations of remaining nitrate (Peng and Zhu, 2006). 13

Furthermore, nitrate was the preferred electron acceptor, over nitrite (Glass and 14

Silverstein, 1998); therefore, competition between nitrate and nitrite was stronger 15

when pyrite was utilized as the sole electron donor. 16

The ammonium concentration increased rapidly from 0.28±0.02 to 5.47±0.29 17

mg-N/L during the first 4.5 days, then increased slowly to 5.88±0.28 mg-N/L in the 18

inoculated UP flasks (Fig. 1e). In the inoculated AP flasks, ammonium continued 19

increasing to 6.82±0.74 mg-N/L over the trial period (Fig. 1f). The dissimilatory 20

nitrate reduction to ammonium (DNRA) process was considered to be the primary 21

cause of ammonium accumulation (Kelso et al., 1997; Zhang et al., 2012). The 22

inoculated cultures derived from anaerobic sludge and were cultivated in a specific 23

liquid culture medium, resulting in more complex microbial community. Clostridium 24

sp. was identified in the inoculated UP and AP flasks before and after the experiment 25

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(Table 1). Specific sulfur-oxidizing chemolithotrophic bacteria such as Clostridium sp. 1

could reduce nitrate to ammonium using sulfur or sulfide compounds as electron 2

donors (Brunet and Garcia-Gil, 1996). 3

3.2 Sulfate accumulation 4

In the inoculated UP and AP flasks, sulfate concentrations increased from 5

261.75±5.50 and 224.25±7.50 mg/L to 649.75±7.00 and 661.25±10.00 mg/L, 6

respectively (Fig. 2). No sulfide or sulfur was added to the flasks, so pyrite could be 7

used as the sole electron donor for autotrophic denitrification. In the un-inoculated UP 8

and AP flasks, sulfate concentrations increased from 252.00±8.00 and 195.50±12.00 9

mg/L to 348.00±2.00 and 270.50± 10.50 mg/L, respectively (Fig. 2). Significantly 10

higher sulfate accumulation occurred in the inoculated UP and AP flasks due to 11

autotrophic denitrification with pyrite as the electron donor. 12

To evaluate their dissolvability, UP or AP was immersed in flasks containing 13

sterilized deionized water. In the UP and AP flasks of sterilized deionized water, 14

sulfate productions were 22.00 and 15.00 mg/L, respectively (Fig. 2). These results 15

indicated that pyrite dissolution led to minimal sulfate production under anoxic 16

conditions. 17

According to Torrentó et al. (2010), if the biomass generation was ignored, basic 18

equation of pyrite-based denitrification could be given as follows: 19

NO3-+1

3FeS2+

2

3H2O→

1

2N2+

2

3SO4

2-+1

3Fe(OH)

3+1

3H+ (2) 20

Theoretical mass ratio of produced sulfate to removed nitrate (S/N) based on Eq. (2) 21

was 1.03. As shown in Table 2, the actual mass ratios of S/N were always higher than 22

theoretical ratios, due to (1) S. denitrificans (Sulfurimonas denitrificans), which is 23

detected in the experiments (Table 1), being the facultative anaerobic bacteria, could 24

utilize oxygen to produce sulfate (Sievert et al., 2008); (2) small amounts of oxygen 25

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were inevitably introduced during sampling, and pyrite could be chemically oxidized 1

by oxygen in flasks (Akcil and Koldas, 2006). Therefore, sulfate also rose slightly in 2

the un-inoculated UP and AP flasks. Furthermore, a small amount of sulfate could be 3

produced by microorganisms, given a small amount of oxygen in the inoculated UP 4

and AP flasks. However, sulfate production by S. denitrificans was considered to be 5

significant. Hence, it is reasonable to assume that the actual S/N was higher. 6

The S/N ranged from 1.11 to 2.23 in the inoculated UP flasks during the first 4 days 7

(Table 2), which indicated that microbial denitrification was the primary 8

sulfate-production process when nitrate and carbon sources were sufficient. During 9

the last 4 days, the S/N was much higher than 1.03 (Table 1), indicating that chemical 10

oxidation of pyrite played a dominant role with the consumption of the nitrate and 11

carbon sources. Torrentó et al. (2010) also reported similar phenomenon in batch 12

experiments, i.e., the S/N of 3.3 (nitrate initial concentration was 0.69 mM with 20 g 13

pyrite particles and 1 mL pure T. denitrificans supplied in the flask) was attributed to 14

the additional oxidation of pyrite by trace levels of dissolved oxygen. 15

Sulfate concentrations in the un-inoculated UP flasks were always higher than in 16

the un-inoculated AP flasks (Fig. 2). Elemental sulfur could be produced during 17

acid-pretreatment (Eq. (1)). Elemental sulfur could not be chemically oxidized under 18

anoxic conditions. The chemical oxidization capability of AP was weaker because a 19

part of the pyrite samples was transformed to elemental sulfur. Accordingly, this may 20

partially explain why sulfate concentrations in the inoculated UP flasks were always 21

higher than in the AP flasks. However, the S/N mass ratios in the inoculated UP flasks 22

were always lower than in the AP flasks, except on Day 5 (Table 2). The equation of 23

denitrification based on elemental sulfur was as follows (Sierra-Alvarez et al., 2007): 24

NO3-+1.10S+0.40CO2+0.76H2O+0.08NH4

+→0.50N2+1.10SO42-+1.28H++0.08C5H7O2N 25

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(3) 1

According to Eq. (2) and (3), when 1.00 mol of nitrate was reduced, sulfate 2

production by elemental sulfur based denitrification (1.10 mol) was higher than that 3

by pyrite-based denitrification (0.67 mol). Therefore, the S/N ratio was higher in the 4

inoculated AP flasks. 5

3.3 Microbial community analysis 6

Table 1 showed that S. denitrificans grew in both the UP and AP flasks after 6 days 7

incubation. S. denitrificans is a type of mesophilic, neutrophilic, 8

facultatively-anaerobic, and denitrifying chemolithoautotrophic bacteria, which used 9

sulfide or thiosulfate as an electron donor (Takai et al., 2006). S. denitrificans and T. 10

denitrificans were the only species that can perform sulfo-oxidizing denitrification 11

under neutral and freshwater conditions, both utilizing sulfide and thiosulfate as 12

electron donors (Sievert et al., 2008). It has been proven that pyrite could be used by T. 13

denitrificans as an electron donor (Torrentó et al., 2010). However, until now, only 14

Kelly and Wood (2006) reported pyrite utilization by S. denitrificans. According to 15

the microbial community illustrated in Table 1, pyrite was most likely to be utilized 16

by S. denitrifican as an electron donor for denitrification. The activity and viable 17

count of microorganisms can be characterized by ATP concentrations. The ATP 18

concentrations in the inoculated UP and AP flasks increased from 4.12±0.63 and 19

4.13±0.73 nM to 9.12±2.71 and 9.84±1.87 nM, respectively, indicating that 20

microorganisms continued growing with the nitrate consumption in the inoculated 21

flasks, and viable counts of microorganisms were higher in the AP flasks. Therefore, 22

cost-effective pretreatment may be necessary for the future utilization of pyrite for 23

autotrophic denitrification. 24

According to the light band representing S. denitrificans in Line A, S. denitrificans 25

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13

was identified on pyrite surfaces (Fig. 3). However, the band representing S. 1

denitrificans in Line B was not bright, indicating that S. denitrificans was not 2

prevalent in the solutions (Fig. 3). S. denitrificans could oxidize electron donors using 3

a multi-enzyme complex catalyzing in vitro (Sievert et al., 2008). Sulfur-binding 4

proteins of some microorganisms such as Thiobacillus ferrooxidans have been found 5

in denitrification using elemental sulfur as an electron donor (Soares, 2002). It is 6

speculated that S. denitrificans might also attach to the pyrite surface using some 7

binding proteins, oxidizing pyrite to reduce nitrate out of cells. It is necessary to 8

further study the binding proteins of S. denitrificans and the enzyme systems of 9

pyrite-based denitrification in the future. 10

Bands that represented Dechlorosoma suillum were detected only in Line A and B, 11

which indicated Dechlorosoma suillum grew continuously during incubation. 12

Dechlorosoma suillum could utilize Fe (II) as an electron donor under anaerobic 13

conditions (Lack et al., 2002). It was proven that Fe (II) was an intermediate product 14

in pyrite based denitrification (Torrentó et al., 2010), which provided a suitable 15

condition for Dechlorosoma suillum. The band representing Prosthecobacter 16

fluviatilis was only detected in Line B, indicating that Prosthecobacter fluviatilis was 17

cultivated during the experiment. As mentioned above, ammonium was produced by 18

DNRA, thus, ammonium could be utilized as a nitrogen source by Prosthecobacter 19

fluviatilis (Takeda et al., 2008). Consequently, Prosthecobacter fluviatilis was able to 20

grow in the solutions. 21

According to Fig. 2, total sulfate concentrations were always higher than sulfate 22

concentrations in inoculated UP and AP flasks, indicating that sulfite, thiosulfate and 23

other sulfur compounds were formed during the denitrification process. Sulfite in the 24

inoculated UP and AP flasks was qualitatively detected using the Basic Fuchsin 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

14

solution. In addition, Sievert et al. (2008) have proved that sulfite dehydrogenase 1

could be produced by S. denitrificans, so sulfite which could be utilized by S. 2

denitrificans, might be an ideal intermediate product during denitrification. It is 3

speculated that a process exists involving a series of intermediate products, such as 4

sulfite, during sulfate production in pyrite-based denitrification. 5

Concentrations of total iron and Fe (II) were always undetectable in both the 6

inoculated UP and AP flasks. The pH of all flasks was higher than 6.30 during the 7

experiment (Fig. 4). Fe (II) was produced by microbial and chemical pyrite oxidation, 8

and then oxidized to Fe (III) by dissolved oxygen. Fe (III) was precipitated under the 9

pH levels mentioned above. Torrentó et al. (2010) reported that iron concentrations in 10

all batch experiments were below the detection limit, as the pH in the flasks ranged 11

between 6.50 and 7.50. 12

3.4 pH variation 13

The pH in the inoculated UP flasks decreased slightly from 6.68 to 6.51 during first 14

3.75 days, and then remained stable at approximately 6.55 (Fig. 4). In the inoculated 15

AP flasks, the pH decreased from 6.92±0.07 to 6.40±0.11 during the experimental 16

period (Fig. 4). The pH in the un-inoculated UP flasks ranged between 6.80±0.03 and 17

7.15±0.14, and remained stable during the 6 days (Fig. 4). Similarly, pH in the 18

un-inoculated AP flasks remained fairly constant at 6.40. The pH remained at 19

approximately 7.00 and 6.70 in the un-inoculated UP and AP flasks containing 20

sterilized deionized water, respectively (Fig. 4). According to Eq. (2), 0.33 mol of H+ 21

was produced by reducing 1.00 mol of nitrate. Although the pH in the inoculated 22

flasks decreased, the pH variation was insignificant, in particular when compared with 23

the H+ production during denitrification based on elemental sulfur. According to Eq. 24

(3), 1.00 mol of nitrate reduction would produce 1.28 mol of H+. A pH buffer reagent, 25

such as limestone, was not necessary for pyrite-based denitrification. To its advantage, 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

15

pyrite-based denitrification is a pH-buffering reaction. 1

4. Conclusions 2

Nitrate in synthetic groundwater could be effectively reduced by pyrite-based 3

denitrification with a nitrate reduction efficiency of more than 99%. S. denitrificans 4

could utilize pyrite as electron donor for denitrification. Pyrite-based denitrification 5

exhibited a considerable nitrate removal rate, low sulfate production, and more stable 6

pH. The nitrate removal rate constant was higher, and the initial concentration of 7

sulfate was lower, with the acid-pretreatment. Pyrite is a promising candidate as an 8

electron donor for autotrophic denitrification. 9

Acknowledgements 10

This research work was supported by the Foundation for the Advisor of Beijing 11

Excellent Doctoral Dissertation (No. 20121141501; No. 20131141502), the National 12

Natural Science Foundation of China (NSFC) (No. 31140082) and the Fundamental 13

Research Funds for the Central Universities (No. 2652014100). 14

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Table 1

Bacterial species based on DGGE profile before and after experiment.

Sample location Most closely related sequence %

Similarity Phylogenetic group

Cultivated cultures before experiment

Sulfurovum sp. 97 Epsilonproteobacteria

Sulfurimonas denitrificans 94 Epsilonproteobacteria

Clostridium sp. 92 Firmicutes

Cultures from pyrite surface after

experiment

Sulfurovum sp. 97 Epsilonproteobacteria

Thermomonas sp. 90 Gammaproteobacteria

Clostridium sp 92 Firmicutes

Dechlorosoma suillum 92 Betaproteobacteria

Sulfurimonas denitrificans 90 Epsilonproteobacteria

Cultures from the solution after

experiment

Sulfurovum sp. 97 Epsilonproteobacteria

Thermomonas sp. 90 Gammaproteobacteria

Clostridium sp. 92 Firmicutes

Dechlorosoma suillum 92 Betaproteobacteria

Sulfurimonas denitrificans 90 Epsilonproteobacteria

Prosthecobacter fluviatilis 99 Verrucomicrobia

Table 2

The mass ratio of produced sulfate to nitrate removal in flasks with UP and AP.

Period (d) 1 2 3 4 5 6 7 8

UP 1.11 1.27 1.16 2.23 6.58 2.36 4.41 5.03

AP 1.27 1.58 2.86 3.07 2.00 4.60 4.48 6.84

Figure Caption

Fig. 1 Concentrations of nitrate, nitrite and ammonium over time in UP and AP flasks.

Fig. 2 Concentrations of sulfate over time in UP and AP flasks.

Fig. 3 DGGE profile of microbial community before and after experiment in

inoculated UP flask: Line A was sampled from pyrite surface; Line B was sampled

from solution and Line C was sampled before experiment.

Fig. 4 Variations of pH over time in UP and AP flasks.

Fig. 1 Concentrations of nitrate, nitrite and ammonium over time in UP and AP flasks.

Fig. 2 Concentrations of sulfate over time in UP and AP flasks.

Fig. 3 DGGE profile of microbial community before and after experiment in inoculated UP flask: Line A was sampled from pyrite surface; Line

B was sampled from solution and Line C was sampled before experiment.

Fig. 4 Variations of pH over time in UP and AP flasks.

Highlights

Nitrate could be effectively removed by pyrite-based denitrification.

Sulfurimonas denitrificans was confirmed by microbial community structure

analysis.

A stable pH was kept and a pH buffer reagent was unnecessary for denitrification.