Accepted Manuscript Pyrite-based autotrophic denitrification for remediation of nitrate contaminated groundwater Jiaoyang Pu, Chuanping Feng, Ying Liu, Rui Li, Zhe Kong, Nan Chen, Shuang Tong, Chunbo Hao, Ye Liu PII: DOI: Reference:
S0960-8524(14)01351-0 http://dx.doi.org/10.1016/j.biortech.2014.09.092 BITE 13982
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
7 August 2014 12 September 2014 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., Pyritebased 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
2
contaminated groundwater
3
Jiaoyang Pua,b, Chuanping Fenga,b∗, Ying Liub, Rui Lib, Zhe Kongb, Nan Chenb,
4
Shuang Tongb, Chunbo Haob, Ye Liub
5 6
a
7
Geosciences, Beijing), Ministry of Education, No. 29 Xueyuan Road, Haidian District,
8
Beijing 100083, China
9
b
10
Key Laboratory of Groundwater Circulation and Evolution (China University of
School of Water Resources and Environment, China University of Geosciences
(Beijing), No. 29 Xueyuan Road, Haidian District, Beijing 100083, China
11 12
Abstract In this study, pyrite-based denitrification using untreated pyrite (UP) and
13 14
acid-pretreated pyrite (AP) was evaluated as an alternative to elemental sulfur based
15
denitrification. Pyrite-based denitrification resulted in a favorable nitrate removal rate
16
constant (0.95 d-1)), sulfate production of 388.00 mg/L, and a stable pH. The
17
pretreatment of pyrite with acid led to a further increase in the nitrate removal rate
18
constant (1.03 d-1) and reduction in initial sulfate concentration (224.25±7.50 mg/L).
19
By analyzing the microbial community structure using Denaturing Gradient Gel
20
Electrophoresis, it was confirmed that Sulfurimonas denitrificans (S. denitrificans)
21
could utilize pyrite as an electron donor. A stable pH was observed over the entire
∗
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: [email protected] (C. Feng)
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experimental period, indicating that the use of a pH buffer reagent would not be
2
necessary for pyrite-based denitrification. Therefore, pyrite could effectively replace
3
elemental sulfur as an electron donor in autotrophic denitrification for
4
nitrate-contaminated groundwater remediation.
5
Keywords
6
Nitrate removal, Pyrite, Autotrophic denitrification, Groundwater, Acid-pretreatment
7
1. Introduction
8
Nitrate, as the most ubiquitous nonpoint-source (NPS) contaminant in groundwater,
9
severely hinders the usage of groundwater for drinking (Sun and Nemati, 2012).
10
Groundwater can be easily contaminated by nitrate, due to its high solubility and
11
mobility in both water and soil (Ghafari et al., 2009). The heavy use of nitrogen
12
fertilizer, discharge of untreated agricultural and industrial wastewater, and
13
atmospheric deposition of nitrogen oxide emissions, are just some of the human and
14
industrial actions leading to nitrate pollution (Yang and Lee, 2005; Ghafari et al.,
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2009). Elevated nitrate concentrations in drinking water represent the potential risks
16
for public health such as methemoglobinemia (Aslan and Cakici, 2007). Consequently,
17
the maximum contaminant level (MCL) for nitrate (NO3-, 10 mg-N/L) in drinking
18
water is stipulated by the World Health Organization (WHO, 2008) and China
19
(NHFPC, 2006).
20
Many physical-chemical technologies have been developed for nitrate
21
contaminated groundwater treatment, such as ion exchange (Samatya et al., 2006),
22
reverse osmosis (Schoeman and Steyn, 2003) and electrodialysis (Elmidaoui et al.,
23
2001). These technologies are used for nitrate enrichment and separation, and require
24
secondary treatments (Ghafari et al., 2009). Presently, biological denitrification
25
represents a promising approach for nitrate contaminated groundwater remediation
2
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(Sánchez et al., 2008). Heterotrophic denitrifiers utilize organic substances as electron
2
donors to convert nitrate into nitrogen, under anoxic conditions (Sierra-Alvarez et al.,
3
2007). Organic carbon availability is a critical factor for heterotrophic denitrification
4
(Zhang et al., 2012). Liquid carbon sources such as methanol (Tong et al., 2013) and
5
hydrolyzed molasses (Quan et al., 2005), as well as solid carbon sources, including
6
biodegradable plastic and wheat straw (Zhang et al., 2012), have been utilized for
7
heterotrophic denitrification. However, the supplementation of organic compounds
8
can lead to additional costs and secondary pollution resulting from heterotrophic
9
denitrification (Moon et al., 2004).
10
Due to its low cost and stable denitrification performance, autotrophic
11
denitrification using elemental sulfur as an electron donor, for groundwater treatment,
12
has been extensively studied in recent years. Soares (2002) used a laboratory column
13
packed with elemental sulfur to treat synthetic groundwater containing 100 mg/L of
14
nitrate, and denitrification rates of up to 0.20kg N/(m3·d) were obtained and sulfate
15
concentrations increased from 50-80 mg/L up to 320 mg/L. High average nitrate
16
removal efficiencies (98.8%) for synthetic groundwater contaminated by nitrate (7.3
17
mM) were maintained in the bioreactor supplied with elemental sulfur and limestone
18
granules (1:1,v:v) (Sierra-Alvarez et al., 2007). By using a sulfur-based permeable
19
reactive barrier for nitrate-contaminated groundwater treatment, 60 mg-N/L of nitrate
20
was reduced to nitrogen gas with sulfate production of 250 mg-S/L (Moon et al.,
21
2008). In the batch experiment, the removal of 50 mg-N/L of nitrate in 500 mL of
22
synthetic groundwater resulted in the production of 792.3 mg/L sulfate and a decrease
23
in pH from 7.5 to less than 5.0 with addition of elemental sulfur (Qambrani and
24
Oh ,2013). High sulfate production and sharp pH decrease are primary factors limiting
25
elemental sulfur based autotrophic denitrification; limestone is, therefore, frequently
3
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used as a pH-buffering reagent (Soares, 2002).
2
Autotrophic denitrification, based on ferrous sulfide (FeS), was shown to be an
3
efficient method for nitrate removal in freshwater systems by Haaijer et al. (2007). Li
4
et al. (2013) also used this approach and treated real wastewater with FeS, resulting in
5
nitrate reduction from 14.9 mg-N/L to 1.1 mg-N/L in 18 hours.
6
Microbial oxidation of pyrite by Thiobacillus denitrificans (T. denitrificans), as a
7
significant natural attenuation process, has been reported to play a role in natural
8
attenuation of nitrate contaminated groundwater. Juncher Jørgensen et al. (2009)
9
observed nitrate reduction when pyrite particles were added to the sediments from an
10
adjacent aquifer. Torrentó et al. (2010) showed that pyrite minerals could be utilized
11
by T. denitrificans as the single electron donor for denitrification. Pyrite is the most
12
abundant sulfide mineral in the earth’s crust, constituting a major reservoir in global
13
cycles of sulfur and iron (Bosch et al., 2012). However, few studies have been
14
conducted concerning pyrite utilization for treatment of nitrate contaminated
15
groundwater.
16
The objectives of this study were (1) to confirm the feasibility of pyrite-based
17
autotrophic denitrification by analyzing the nitrate removal rate, sulfate production,
18
and pH variation, (2) to investigate pyrite-based denitrification mechanisms through
19
analysis of the bacterial community structures, and (3) to compare the denitrification
20
performances of UP (untreated pyrite) and AP (acid-pretreated pyrite) as electron
21
donors.
22
2. Materials and methods
23
2.1 Preparation of materials
24
Natural pyrite crystals were obtained from Qujing, Yunnan Province, China, and
25
ground by a pulverizer (ZN-04, KINGSLH, China) to obtain 0.15-0.25 mm particle
4
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sizes. In order to investigate the effects of acid-pretreatment on denitrification
2
performance, a portion of the ground pyrite particles were immersed in 10% (v/v) HCl
3
solutions for 30 min, rinsed by deionized water until the pH of the rinsing solution
4
was 7.0, and dried at 105 ℃ for 4 hours.
5
Synthetic groundwater was prepared by adding KNO3 to deionized water,
6
containing (g/L) 0.40 KNO3, 0.40 KH2PO4 and 0.50 NaHCO3. The initial nitrate
7
concentration in the synthetic groundwater was 55 mg-N/L.
8 9 10 11 12
All chemical reagents used in the experiments were analytical-grade. 2.2 Characterization of pyrite The pyrite crystals were confirmed to be pure pyrite, with no evidence of other mineral phases, by X-ray diffraction analysis (D8 FOCUS, Bruker, Germany). The elemental compositions of the prepared pyrite samples (natural pyrite crystals,
13
UP, and AP) were analyzed by electron microprobe analysis (EPMA-1600, Shimadzu,
14
Japan). The natural pyrite crystals contained 52.81% (w/w) Fe, 46.41% S, 0.46% Si,
15
and 0.32% Al. The elemental composition of UP was 50.10% Fe, 49.42% S, 0.29% Si,
16
and 0.19% Al. The elemental composition of the ground sample was similar to the
17
natural crystals. For the acid-pretreatment, the elemental composition was 17.11% S,
18
75.13% Fe, 3.66% Si, and 4.10% Al. During the acid-pretreatment, pyrite could be
19
chemically oxidized according to the following equation:
20
FeS2 +2HCl→H2 S+FeCl2 +S
21
Hydrogen sulfide was produced and released into the air during the acid-pretreatment,
22
therefore, a lower mass percentage of S was detected in the acid-pretreated pyrite
23
sample.
24
2.3 Cultivation of microorganisms
25
(1)
Anaerobic sludge was obtained from Tsinghe Sewage Treatment Plant (Beijing, 5
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China) and cultivated in a liquid nutrient medium (2L) at 30 ℃ for 50 days. The
2
nutrient medium consisted of Na2S2O3·5H2O (20 mM), NaHCO3 (30 mM), KNO3 (20
3
mM), KH2PO4 (14.7 mM), NH4Cl (18.7 mM), MgSO4·7H2O (3.25 mM), FeSO4·7H2O
4
(0.08 mM), and CaCl2·2H2O (0.05 mM) (Beller, 2005). The nutrient medium was
5
replaced every two days. Before inoculation, the cultures were centrifuged for 10 min
6
(4500 rpm, 30 ℃) by a centrifuge (GT10-1，SHKIC，China) and suspended in
7
sterilized physiological saline solutions to remove nitrate and sulfate residues in
8
cultures.
9
2.4 Experimental procedure
10 11 12
All the denitrification experiments using UP and AP as electron donors were carried out in 500 mL flasks, in duplicate. The respective flasks were filled with 50 g of UP or AP and 300 mL of the synthetic
13
groundwater, and sterilized at 115 ℃ for 30 min. The flasks were then inoculated
14
with 20 mL of the prepared cultures, described in 2.3, on a Clean Bench (SW-GJ-IFD,
15
AIRTECH, China). The flasks were sparged by nitrogen gas for 5 min prior to sealing
16
with rubber plugs. Un-inoculated flasks were used as controls. All flasks were
17
cultivated at 100 rpm and 30 ℃ in a constant temperature incubator (DDHZ-300,
18
TCSSYSBC, China) for 6 days.
19
After 6 days, the inoculated UP flask was still standing for 30 min, microorganisms
20
grown on the UP surface and suspended in the supernatant were taken for 16S
21
rRNA-based microbial analysis.
22
2.5 Analysis
23
A 10 mL sample was collected from each flask, every 18 h, and the pH of the
24
sample was measured immediately with a pH meter (Seven Multi S40, Mettler Toledo,
25
Switzerland). 6
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Samples were filtered through a 0.45 μm membrane filter. Two mL of each
2
sample were treated with 0.5 mL of H2O2 (3 %, v/v) in order to oxidize sulfite and
3
thiosulfate in the samples to sulfate. Sulfate and total sulfate were measured with an
4
ion chromatograph (ICS900 Dionex IonPac, Thermo Fisher Scientific, US) with
5
detection limit of 0.09 mg/L.
6
According to the Water and Wastewater Monitoring Analysis Method (SEPA, 2002),
7
concentrations of nitrate, nitrite, ammonium, total iron and ferrous ion (Fe (II)) were
8
measured with a spectrophotometer (DR6000, HACH, US) and the detection limits
9
were 0.08 mg/L for nitrate, 0.003 mg/L for nitrite, 0.025 mg/L for ammonium, 0.03
10
mg/L for total iron and 0.03 mg/L for ferrous ion. ATPs in the cultures were measured
11
with an ATP fluorescence detector (AF-100, TOADKK, Japan) with the detection
12
limit of 0.2 fM ATP.
13
The standard deviations were analyzed at a confidence level of 90%, and Origin 9.0
14
(OriginLab, trial version) was used to compute the nitrate removal rate constants in
15
the inoculated UP and AP flasks.
16
For 16S rRNA-based microbial analysis, genomic DNA was extracted and
17
amplified with 968F GC and 1401R primers, using the PCR system (initial
18
denaturation, 95 ℃ for 5 min; subsequent denaturation, 95℃ for 0.5 min; annealing,
19
54 ℃ for 0.5 min; extension, 72 ℃ for 45 s and final extension, 72 ℃ for 10 min).
20
Another round of PCR was performed with amplified 16S rRNA Genes. Denaturing
21
Gradient Gel Electrophoresis (DGGE) analysis was performed using the Bio-Rad
22
C-1000 system (Bio-Rad, USA). The PCR products were loaded in a 1 mm-thick gel
23
containing, 9 % (w/v) polyacrylamide and a denaturant gradient of 40-60 % (100%
24
denaturant was 7 M urea and 40 % formamide). The electrophoresis was run in 1 ×
25
TAE (20 mM Tris, 10mM acetate, 0.5mM EDTA, at pH 8.0) at 60 ℃ for 12 h at 100 7
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V. The gel was then stained with dye solution and placed on a UV illuminator
2
(Bio-Rad, USA). The excised gel from each target band was placed into a 1.5 mL
3
sterile tube containing 30 µL of sterile ultrapure water and crushed. One µL of eluted
4
DNA was amplified with primer 968F/1401R. DNA was sequenced and the sequences
5
were determined by Shanghai Sangon Co., Ltd., Shanghai, China.
6
2.6 Nucleotide Sequence Accession numbers
7
The nucleotide sequences reported in this paper have been submitted to GenBank
8
with accession numbers: KM220911-KM220924.
9
3. Results and discussion
10 11
3.1 Nitrate reduction As shown in Fig. 1a, nitrate sharply decreased from 56.69±0.22 mg-N/L to
12
5.21±1.33 mg-N/L during the first 3 days, and then decreased slowly to 0.08±0.06
13
mg-N/L in the inoculated UP flasks. Nitrate concentrations ranged from 53.31±0.28
14
mg-N/L to 55.87±0.14 mg-N/L in the un-inoculated UP flasks during the experimental
15
period. Similarly, the nitrate concentration decreased from 55.57±0.21 mg-N/L to
16
2.62±0.36 mg-N/L during first 3.75 days, and then nitrate concentration declined
17
steadily to 0.05±0.00 mg-N/L in the inoculated AP flasks. In the un-inoculated AP
18
flasks, the nitrate concentration was nearly stable at 55 mg-N/L (Fig. 1b). Nitrate
19
reduction efficiencies in the inoculated UP and AP flasks both exceeded 99%. In
20
contrast, no nitrate reduction was observed in un-inoculated flasks, indicating that the
21
pyrite could not directly reduce nitrate.
22
According to the first-order kinetics model, nitrate removal rates in the inoculated
23
UP and AP flasks were computed (determination coefficients [R2]> 0.9). The nitrate
24
removal rate constant in the inoculated AP flasks was 1.03 d-1, slightly higher than
25
that of the inoculated UP flasks (0.95 d-1). This difference was potentially due to the
8
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acid-pretreatment process, removing micro-particles such as iron and sulfur impurities
2
from the pyrite surface, and roughening the pyrite surface to allow microorganisms to
3
more easily attach to and utilize the pyrite.
4
The initial nitrite concentration was less than 0.02 mg-N/L in the inoculated UP
5
flasks. The nitrite concentration increased rapidly, reaching 0.55±0.04 mg-N/L during
6
first 3 days, then decreased steadily to less than 0.02 mg-N/L (Fig. 1c). In the
7
inoculated AP flasks, the nitrite concentration increased from less than 0.02 mg-N/L
8
to 0.47±0.05 mg-N/L during the first 2.25 days, then decreased to 0.03±0.00 mg-N/L
9
(Fig. 1d). Pyrite-based denitrification followed the typical pattern of basic microbial
10
denitrification, expressed as NO3-→NO2- →NO→N2O→N2 (Torrentó et al., 2010).
11
After nitrate was partially reduced to nitrite, nitrate and nitrite co-existed in the
12
inoculated UP and AP flasks. The synthesis and activity of nitrite reductase could be
13
inhibited by high concentrations of remaining nitrate (Peng and Zhu, 2006).
14
Furthermore, nitrate was the preferred electron acceptor, over nitrite (Glass and
15
Silverstein, 1998); therefore, competition between nitrate and nitrite was stronger
16
when pyrite was utilized as the sole electron donor.
17
The ammonium concentration increased rapidly from 0.28±0.02 to 5.47±0.29
18
mg-N/L during the first 4.5 days, then increased slowly to 5.88±0.28 mg-N/L in the
19
inoculated UP flasks (Fig. 1e). In the inoculated AP flasks, ammonium continued
20
increasing to 6.82±0.74 mg-N/L over the trial period (Fig. 1f). The dissimilatory
21
nitrate reduction to ammonium (DNRA) process was considered to be the primary
22
cause of ammonium accumulation (Kelso et al., 1997; Zhang et al., 2012). The
23
inoculated cultures derived from anaerobic sludge and were cultivated in a specific
24
liquid culture medium, resulting in more complex microbial community. Clostridium
25
sp. was identified in the inoculated UP and AP flasks before and after the experiment
9
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1
(Table 1). Specific sulfur-oxidizing chemolithotrophic bacteria such as Clostridium sp.
2
could reduce nitrate to ammonium using sulfur or sulfide compounds as electron
3
donors (Brunet and Garcia-Gil, 1996).
4
3.2 Sulfate accumulation
5
In the inoculated UP and AP flasks, sulfate concentrations increased from
6
261.75±5.50 and 224.25±7.50 mg/L to 649.75±7.00 and 661.25±10.00 mg/L,
7
respectively (Fig. 2). No sulfide or sulfur was added to the flasks, so pyrite could be
8
used as the sole electron donor for autotrophic denitrification. In the un-inoculated UP
9
and AP flasks, sulfate concentrations increased from 252.00±8.00 and 195.50±12.00
10
mg/L to 348.00±2.00 and 270.50± 10.50 mg/L, respectively (Fig. 2). Significantly
11
higher sulfate accumulation occurred in the inoculated UP and AP flasks due to
12
autotrophic denitrification with pyrite as the electron donor.
13
To evaluate their dissolvability, UP or AP was immersed in flasks containing
14
sterilized deionized water. In the UP and AP flasks of sterilized deionized water,
15
sulfate productions were 22.00 and 15.00 mg/L, respectively (Fig. 2). These results
16
indicated that pyrite dissolution led to minimal sulfate production under anoxic
17
conditions.
18
According to Torrentó et al. (2010), if the biomass generation was ignored, basic
19
equation of pyrite-based denitrification could be given as follows:
20
NO3 + 3 FeS2 + 3 H2 O→ 2 N2 + 3 SO4 + 3 Fe(OH)3 + 3 H+
21
-
1
2
1
2
2-
1
1
(2)
Theoretical mass ratio of produced sulfate to removed nitrate (S/N) based on Eq. (2)
22
was 1.03. As shown in Table 2, the actual mass ratios of S/N were always higher than
23
theoretical ratios, due to (1) S. denitrificans (Sulfurimonas denitrificans), which is
24
detected in the experiments (Table 1), being the facultative anaerobic bacteria, could
25
utilize oxygen to produce sulfate (Sievert et al., 2008); (2) small amounts of oxygen 10
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1
were inevitably introduced during sampling, and pyrite could be chemically oxidized
2
by oxygen in flasks (Akcil and Koldas, 2006). Therefore, sulfate also rose slightly in
3
the un-inoculated UP and AP flasks. Furthermore, a small amount of sulfate could be
4
produced by microorganisms, given a small amount of oxygen in the inoculated UP
5
and AP flasks. However, sulfate production by S. denitrificans was considered to be
6
significant. Hence, it is reasonable to assume that the actual S/N was higher.
7
The S/N ranged from 1.11 to 2.23 in the inoculated UP flasks during the first 4 days
8
(Table 2), which indicated that microbial denitrification was the primary
9
sulfate-production process when nitrate and carbon sources were sufficient. During
10
the last 4 days, the S/N was much higher than 1.03 (Table 1), indicating that chemical
11
oxidation of pyrite played a dominant role with the consumption of the nitrate and
12
carbon sources. Torrentó et al. (2010) also reported similar phenomenon in batch
13
experiments, i.e., the S/N of 3.3 (nitrate initial concentration was 0.69 mM with 20 g
14
pyrite particles and 1 mL pure T. denitrificans supplied in the flask) was attributed to
15
the additional oxidation of pyrite by trace levels of dissolved oxygen.
16
Sulfate concentrations in the un-inoculated UP flasks were always higher than in
17
the un-inoculated AP flasks (Fig. 2). Elemental sulfur could be produced during
18
acid-pretreatment (Eq. (1)). Elemental sulfur could not be chemically oxidized under
19
anoxic conditions. The chemical oxidization capability of AP was weaker because a
20
part of the pyrite samples was transformed to elemental sulfur. Accordingly, this may
21
partially explain why sulfate concentrations in the inoculated UP flasks were always
22
higher than in the AP flasks. However, the S/N mass ratios in the inoculated UP flasks
23
were always lower than in the AP flasks, except on Day 5 (Table 2). The equation of
24
denitrification based on elemental sulfur was as follows (Sierra-Alvarez et al., 2007):
25
NO3 +1.10S+0.40CO2 +0.76H2 O+0.08NH+4 →0.50N2 +1.10SO4 +1.28H+ +0.08C5 H7 O2 N
2-
-
11
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1
(3)
2
According to Eq. (2) and (3), when 1.00 mol of nitrate was reduced, sulfate
3
production by elemental sulfur based denitrification (1.10 mol) was higher than that
4
by pyrite-based denitrification (0.67 mol). Therefore, the S/N ratio was higher in the
5
inoculated AP flasks.
6
3.3 Microbial community analysis
7
Table 1 showed that S. denitrificans grew in both the UP and AP flasks after 6 days
8
incubation. S. denitrificans is a type of mesophilic, neutrophilic,
9
facultatively-anaerobic, and denitrifying chemolithoautotrophic bacteria, which used
10
sulfide or thiosulfate as an electron donor (Takai et al., 2006). S. denitrificans and T.
11
denitrificans were the only species that can perform sulfo-oxidizing denitrification
12
under neutral and freshwater conditions, both utilizing sulfide and thiosulfate as
13
electron donors (Sievert et al., 2008). It has been proven that pyrite could be used by T.
14
denitrificans as an electron donor (Torrentó et al., 2010). However, until now, only
15
Kelly and Wood (2006) reported pyrite utilization by S. denitrificans. According to
16
the microbial community illustrated in Table 1, pyrite was most likely to be utilized
17
by S. denitrifican as an electron donor for denitrification. The activity and viable
18
count of microorganisms can be characterized by ATP concentrations. The ATP
19
concentrations in the inoculated UP and AP flasks increased from 4.12±0.63 and
20
4.13±0.73 nM to 9.12±2.71 and 9.84±1.87 nM, respectively, indicating that
21
microorganisms continued growing with the nitrate consumption in the inoculated
22
flasks, and viable counts of microorganisms were higher in the AP flasks. Therefore,
23
cost-effective pretreatment may be necessary for the future utilization of pyrite for
24
autotrophic denitrification.
25
According to the light band representing S. denitrificans in Line A, S. denitrificans
12
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
1
was identified on pyrite surfaces (Fig. 3). However, the band representing S.
2
denitrificans in Line B was not bright, indicating that S. denitrificans was not
3
prevalent in the solutions (Fig. 3). S. denitrificans could oxidize electron donors using
4
a multi-enzyme complex catalyzing in vitro (Sievert et al., 2008). Sulfur-binding
5
proteins of some microorganisms such as Thiobacillus ferrooxidans have been found
6
in denitrification using elemental sulfur as an electron donor (Soares, 2002). It is
7
speculated that S. denitrificans might also attach to the pyrite surface using some
8
binding proteins, oxidizing pyrite to reduce nitrate out of cells. It is necessary to
9
further study the binding proteins of S. denitrificans and the enzyme systems of
10 11
pyrite-based denitrification in the future. Bands that represented Dechlorosoma suillum were detected only in Line A and B,
12
which indicated Dechlorosoma suillum grew continuously during incubation.
13
Dechlorosoma suillum could utilize Fe (II) as an electron donor under anaerobic
14
conditions (Lack et al., 2002). It was proven that Fe (II) was an intermediate product
15
in pyrite based denitrification (Torrentó et al., 2010), which provided a suitable
16
condition for Dechlorosoma suillum. The band representing Prosthecobacter
17
fluviatilis was only detected in Line B, indicating that Prosthecobacter fluviatilis was
18
cultivated during the experiment. As mentioned above, ammonium was produced by
19
DNRA, thus, ammonium could be utilized as a nitrogen source by Prosthecobacter
20
fluviatilis (Takeda et al., 2008). Consequently, Prosthecobacter fluviatilis was able to
21
grow in the solutions.
22
According to Fig. 2, total sulfate concentrations were always higher than sulfate
23
concentrations in inoculated UP and AP flasks, indicating that sulfite, thiosulfate and
24
other sulfur compounds were formed during the denitrification process. Sulfite in the
25
inoculated UP and AP flasks was qualitatively detected using the Basic Fuchsin
13
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
1
solution. In addition, Sievert et al. (2008) have proved that sulfite dehydrogenase
2
could be produced by S. denitrificans, so sulfite which could be utilized by S.
3
denitrificans, might be an ideal intermediate product during denitrification. It is
4
speculated that a process exists involving a series of intermediate products, such as
5
sulfite, during sulfate production in pyrite-based denitrification.
6
Concentrations of total iron and Fe (II) were always undetectable in both the
7
inoculated UP and AP flasks. The pH of all flasks was higher than 6.30 during the
8
experiment (Fig. 4). Fe (II) was produced by microbial and chemical pyrite oxidation,
9
and then oxidized to Fe (III) by dissolved oxygen. Fe (III) was precipitated under the
10
pH levels mentioned above. Torrentó et al. (2010) reported that iron concentrations in
11
all batch experiments were below the detection limit, as the pH in the flasks ranged
12
between 6.50 and 7.50.
13
3.4 pH variation
14
The pH in the inoculated UP flasks decreased slightly from 6.68 to 6.51 during first
15
3.75 days, and then remained stable at approximately 6.55 (Fig. 4). In the inoculated
16
AP flasks, the pH decreased from 6.92±0.07 to 6.40±0.11 during the experimental
17
period (Fig. 4). The pH in the un-inoculated UP flasks ranged between 6.80±0.03 and
18
7.15±0.14, and remained stable during the 6 days (Fig. 4). Similarly, pH in the
19
un-inoculated AP flasks remained fairly constant at 6.40. The pH remained at
20
approximately 7.00 and 6.70 in the un-inoculated UP and AP flasks containing
21
sterilized deionized water, respectively (Fig. 4). According to Eq. (2), 0.33 mol of H+
22
was produced by reducing 1.00 mol of nitrate. Although the pH in the inoculated
23
flasks decreased, the pH variation was insignificant, in particular when compared with
24
the H+ production during denitrification based on elemental sulfur. According to Eq.
25
(3), 1.00 mol of nitrate reduction would produce 1.28 mol of H+. A pH buffer reagent,
26
such as limestone, was not necessary for pyrite-based denitrification. To its advantage, 14
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
1
pyrite-based denitrification is a pH-buffering reaction.
2
4. Conclusions
3
Nitrate in synthetic groundwater could be effectively reduced by pyrite-based
4
denitrification with a nitrate reduction efficiency of more than 99%. S. denitrificans
5
could utilize pyrite as electron donor for denitrification. Pyrite-based denitrification
6
exhibited a considerable nitrate removal rate, low sulfate production, and more stable
7
pH. The nitrate removal rate constant was higher, and the initial concentration of
8
sulfate was lower, with the acid-pretreatment. Pyrite is a promising candidate as an
9
electron donor for autotrophic denitrification.
10
Acknowledgements
11
This research work was supported by the Foundation for the Advisor of Beijing
12
Excellent Doctoral Dissertation (No. 20121141501; No. 20131141502), the National
13
Natural Science Foundation of China (NSFC) (No. 31140082) and the Fundamental
14
Research Funds for the Central Universities (No. 2652014100).
15
References
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[15] Moon, H.S., Ahn, K.H., Lee, S., Nam, K., Kim, J.Y., 2004. Use of autotrophic
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sulfur-oxidizers to remove nitrate from bank filtrate in a permeable reactive
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[16] Moon, H.S., Shin, D.Y., Nam, K., Kim, J.Y., 2008. A long-term performance test
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on an autotrophic denitrification column for application as a permeable reactive
11
barrier. Chemosphere, 73, 723–728.
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[17] NHFPC, 2006, Standards for Drinking Water Quality, 1st edition, National Health and Family Planning Commission of the PRC, Beijng.
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[18] Peng, Y.Z., Zhu, G.B., 2006. Biological nitrogen removal with nitrification and
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denitrification via nitrite pathway. Appl. Microbiol. Biotechnol., 73, 15-26.
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[19] Qambrani, N. A., Oh, S. E., 2013. Effect of dissolved oxygen tension and
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agitation rates on sulfur-utilizing autotrophic denitrification: batch tests. Appl.
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[20] Quan, Z.X., Jin, Y.S., Yin, C.R., Lee, J.J., Lee, S.T., 2005. Hydrolyzed molasses
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96, 1690-1695.
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addition of Thiobacillus denitrificans and Thiomicrospira denitrificans to
24
chemolithoautotrophic denitrifying bioflasks. Int. Microbiol., 11, 179-184.
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[22] Samatya, S., Kabay, N., Yüksel, Ü., Arda, M., Yüksel, M., 2006., Removal of
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nitrate from aqueous solution by nitrate selective ion exchange resins. React.
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Funct. Polym., 66, 1206-1214.
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[23] Schoeman, J.J., Steyn, A., 2003. Nitrate removal with reverse osmosis in a rural area in South Africa. Desalination, 155, 15-26. [24] SEPA, 2002. Water and Wastewater Monitoring Analysis Method, 4th edition. China Environmental Science Press, Beijing. [25] Sierra-Alvarez, R., Beristain-Cardoso, R., Salazar, M., Gómez, J., Razo-Flores, E.,
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Field, J.A., 2007. Chemolithotrophic denitrification with elemental sulfur for
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groundwater treatment. Water Res., 41, 1253-1262.
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[26] Sievert, S.M., Scott, K.M., Klotz, M.G., Chain, P.S., Hauser, L.J., Hemp, J.,
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Hügler, M., Land, M., Lapidus, A., Larimer, F.W., Lucas, S., Malfatti, S.A.,
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Meyer, F., Paulsen, I.T., Ren, Q., Simon, J., 2008. Genome of the
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Epsilonproteobacterial Chemolithoautotroph Sulfurimonas denitrificans. Appl.
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Environ. Microbiol., 74, 1145-1156.
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[27] Soares, M.I.M., 2002. Denitrification of groundwater with elemental sulfur. Water Res., 36, 1392-1395. [28] Sun, Y.M., Nemati, M., 2012. Evaluation of sulfur-based autotrophic
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denitrification and denitritation for biological removal of nitrate and nitrite from
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contaminated waters. Bioresour. Technol., 114, 207-216.
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[29] Takai, K., Suzuki, M., Nakagawa, S., Miyazaki, M., Suzuki, Y., Inagaki, F.,
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Horikoshi, K., 2006. Sulfurimonas paralvinellae sp. nov., a novel mesophilic,
22
hydrogen- and sulfur-oxidizing chemolithoautotroph within the
23
Epsilonproteobacteria isolated from a deep-sea hydrothermal vent polychaete
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nest, reclassification of Thiomicrospira denitrificans as Sulfurimonas
25
denitrificans comb. nov. and emended description of the genus Sulfurimonas, Int.
18
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I. Koizumi, J., 2008. Prosthecobacter fluviatilis sp. nov., which lacks the bacterial
4
tubulin btubA and btubB genes, Int. J. Syst. Evol. Microbiol, 58,1561-1565.
5
[31] Tong, S., Zhang, B.G., Feng, C.P., Zhao, Y.X., Chen, N., Hao, C.B., Pu, J.Y.,
6
Zhao, L.W, 2013. Characteristics of heterotrophic/biofilm-electrode autotrophic
7
denitrification for nitrate removal from groundwater. Bioresour. Technol., 148,
8
121-127.
9
[32] Torrentó, C., Cama, J., Urmeneta, J., Otero, N., Soler, A., 2010. Denitrification of
10
groundwater with pyrite and Thiobacillus denitrificans. Chemi. Geol., 278, 80-91.
11
[33] WHO, 2008. Guidelines for drinking-water quality, incorporating first and second
12
addenda, Volume 1, Recommendations, 3rd edition. World Health Organization,
13
Geneva.
14 15
[34] Yang, G.C.C., Lee, H.L., 2005. Chemical reduction of nitrate by nanosized iron: kinetics and pathways. Water Res., 39, 884-894.
16
[35] Zhang, J.M, Feng, C.P., Hong, S.Q., Hao, H.L., Yang, Y.N., 2012. Behavior of
17
solid carbon sources for biological denitrification in groundwater remediation.
18
Water Sci. Technol., 65, 1696-1704.
19
Table 1 Bacterial species based on DGGE profile before and after experiment. Sample location
Cultivated cultures before experiment
Cultures from pyrite surface after experiment
Cultures from the solution after experiment
Most closely related sequence
% Similarity
Phylogenetic group
Sulfurovum sp.
97
Epsilonproteobacteria
Sulfurimonas denitrificans
94
Epsilonproteobacteria
Clostridium sp.
92
Firmicutes
Sulfurovum sp.
97
Epsilonproteobacteria
Thermomonas sp.
90
Gammaproteobacteria
Clostridium sp
92
Firmicutes
Dechlorosoma suillum
92
Betaproteobacteria
Sulfurimonas denitrificans
90
Epsilonproteobacteria
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.
S0960-8524(14)01351-0 http://dx.doi.org/10.1016/j.biortech.2014.09.092 BITE 13982
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
7 August 2014 12 September 2014 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., Pyritebased 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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1
Pyrite-based autotrophic denitrification for remediation of nitrate
2
contaminated groundwater
3
Jiaoyang Pua,b, Chuanping Fenga,b∗, Ying Liub, Rui Lib, Zhe Kongb, Nan Chenb,
4
Shuang Tongb, Chunbo Haob, Ye Liub
5 6
a
7
Geosciences, Beijing), Ministry of Education, No. 29 Xueyuan Road, Haidian District,
8
Beijing 100083, China
9
b
10
Key Laboratory of Groundwater Circulation and Evolution (China University of
School of Water Resources and Environment, China University of Geosciences
(Beijing), No. 29 Xueyuan Road, Haidian District, Beijing 100083, China
11 12
Abstract In this study, pyrite-based denitrification using untreated pyrite (UP) and
13 14
acid-pretreated pyrite (AP) was evaluated as an alternative to elemental sulfur based
15
denitrification. Pyrite-based denitrification resulted in a favorable nitrate removal rate
16
constant (0.95 d-1)), sulfate production of 388.00 mg/L, and a stable pH. The
17
pretreatment of pyrite with acid led to a further increase in the nitrate removal rate
18
constant (1.03 d-1) and reduction in initial sulfate concentration (224.25±7.50 mg/L).
19
By analyzing the microbial community structure using Denaturing Gradient Gel
20
Electrophoresis, it was confirmed that Sulfurimonas denitrificans (S. denitrificans)
21
could utilize pyrite as an electron donor. A stable pH was observed over the entire
∗
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: [email protected] (C. Feng)
1
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1
experimental period, indicating that the use of a pH buffer reagent would not be
2
necessary for pyrite-based denitrification. Therefore, pyrite could effectively replace
3
elemental sulfur as an electron donor in autotrophic denitrification for
4
nitrate-contaminated groundwater remediation.
5
Keywords
6
Nitrate removal, Pyrite, Autotrophic denitrification, Groundwater, Acid-pretreatment
7
1. Introduction
8
Nitrate, as the most ubiquitous nonpoint-source (NPS) contaminant in groundwater,
9
severely hinders the usage of groundwater for drinking (Sun and Nemati, 2012).
10
Groundwater can be easily contaminated by nitrate, due to its high solubility and
11
mobility in both water and soil (Ghafari et al., 2009). The heavy use of nitrogen
12
fertilizer, discharge of untreated agricultural and industrial wastewater, and
13
atmospheric deposition of nitrogen oxide emissions, are just some of the human and
14
industrial actions leading to nitrate pollution (Yang and Lee, 2005; Ghafari et al.,
15
2009). Elevated nitrate concentrations in drinking water represent the potential risks
16
for public health such as methemoglobinemia (Aslan and Cakici, 2007). Consequently,
17
the maximum contaminant level (MCL) for nitrate (NO3-, 10 mg-N/L) in drinking
18
water is stipulated by the World Health Organization (WHO, 2008) and China
19
(NHFPC, 2006).
20
Many physical-chemical technologies have been developed for nitrate
21
contaminated groundwater treatment, such as ion exchange (Samatya et al., 2006),
22
reverse osmosis (Schoeman and Steyn, 2003) and electrodialysis (Elmidaoui et al.,
23
2001). These technologies are used for nitrate enrichment and separation, and require
24
secondary treatments (Ghafari et al., 2009). Presently, biological denitrification
25
represents a promising approach for nitrate contaminated groundwater remediation
2
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1
(Sánchez et al., 2008). Heterotrophic denitrifiers utilize organic substances as electron
2
donors to convert nitrate into nitrogen, under anoxic conditions (Sierra-Alvarez et al.,
3
2007). Organic carbon availability is a critical factor for heterotrophic denitrification
4
(Zhang et al., 2012). Liquid carbon sources such as methanol (Tong et al., 2013) and
5
hydrolyzed molasses (Quan et al., 2005), as well as solid carbon sources, including
6
biodegradable plastic and wheat straw (Zhang et al., 2012), have been utilized for
7
heterotrophic denitrification. However, the supplementation of organic compounds
8
can lead to additional costs and secondary pollution resulting from heterotrophic
9
denitrification (Moon et al., 2004).
10
Due to its low cost and stable denitrification performance, autotrophic
11
denitrification using elemental sulfur as an electron donor, for groundwater treatment,
12
has been extensively studied in recent years. Soares (2002) used a laboratory column
13
packed with elemental sulfur to treat synthetic groundwater containing 100 mg/L of
14
nitrate, and denitrification rates of up to 0.20kg N/(m3·d) were obtained and sulfate
15
concentrations increased from 50-80 mg/L up to 320 mg/L. High average nitrate
16
removal efficiencies (98.8%) for synthetic groundwater contaminated by nitrate (7.3
17
mM) were maintained in the bioreactor supplied with elemental sulfur and limestone
18
granules (1:1,v:v) (Sierra-Alvarez et al., 2007). By using a sulfur-based permeable
19
reactive barrier for nitrate-contaminated groundwater treatment, 60 mg-N/L of nitrate
20
was reduced to nitrogen gas with sulfate production of 250 mg-S/L (Moon et al.,
21
2008). In the batch experiment, the removal of 50 mg-N/L of nitrate in 500 mL of
22
synthetic groundwater resulted in the production of 792.3 mg/L sulfate and a decrease
23
in pH from 7.5 to less than 5.0 with addition of elemental sulfur (Qambrani and
24
Oh ,2013). High sulfate production and sharp pH decrease are primary factors limiting
25
elemental sulfur based autotrophic denitrification; limestone is, therefore, frequently
3
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1
used as a pH-buffering reagent (Soares, 2002).
2
Autotrophic denitrification, based on ferrous sulfide (FeS), was shown to be an
3
efficient method for nitrate removal in freshwater systems by Haaijer et al. (2007). Li
4
et al. (2013) also used this approach and treated real wastewater with FeS, resulting in
5
nitrate reduction from 14.9 mg-N/L to 1.1 mg-N/L in 18 hours.
6
Microbial oxidation of pyrite by Thiobacillus denitrificans (T. denitrificans), as a
7
significant natural attenuation process, has been reported to play a role in natural
8
attenuation of nitrate contaminated groundwater. Juncher Jørgensen et al. (2009)
9
observed nitrate reduction when pyrite particles were added to the sediments from an
10
adjacent aquifer. Torrentó et al. (2010) showed that pyrite minerals could be utilized
11
by T. denitrificans as the single electron donor for denitrification. Pyrite is the most
12
abundant sulfide mineral in the earth’s crust, constituting a major reservoir in global
13
cycles of sulfur and iron (Bosch et al., 2012). However, few studies have been
14
conducted concerning pyrite utilization for treatment of nitrate contaminated
15
groundwater.
16
The objectives of this study were (1) to confirm the feasibility of pyrite-based
17
autotrophic denitrification by analyzing the nitrate removal rate, sulfate production,
18
and pH variation, (2) to investigate pyrite-based denitrification mechanisms through
19
analysis of the bacterial community structures, and (3) to compare the denitrification
20
performances of UP (untreated pyrite) and AP (acid-pretreated pyrite) as electron
21
donors.
22
2. Materials and methods
23
2.1 Preparation of materials
24
Natural pyrite crystals were obtained from Qujing, Yunnan Province, China, and
25
ground by a pulverizer (ZN-04, KINGSLH, China) to obtain 0.15-0.25 mm particle
4
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
1
sizes. In order to investigate the effects of acid-pretreatment on denitrification
2
performance, a portion of the ground pyrite particles were immersed in 10% (v/v) HCl
3
solutions for 30 min, rinsed by deionized water until the pH of the rinsing solution
4
was 7.0, and dried at 105 ℃ for 4 hours.
5
Synthetic groundwater was prepared by adding KNO3 to deionized water,
6
containing (g/L) 0.40 KNO3, 0.40 KH2PO4 and 0.50 NaHCO3. The initial nitrate
7
concentration in the synthetic groundwater was 55 mg-N/L.
8 9 10 11 12
All chemical reagents used in the experiments were analytical-grade. 2.2 Characterization of pyrite The pyrite crystals were confirmed to be pure pyrite, with no evidence of other mineral phases, by X-ray diffraction analysis (D8 FOCUS, Bruker, Germany). The elemental compositions of the prepared pyrite samples (natural pyrite crystals,
13
UP, and AP) were analyzed by electron microprobe analysis (EPMA-1600, Shimadzu,
14
Japan). The natural pyrite crystals contained 52.81% (w/w) Fe, 46.41% S, 0.46% Si,
15
and 0.32% Al. The elemental composition of UP was 50.10% Fe, 49.42% S, 0.29% Si,
16
and 0.19% Al. The elemental composition of the ground sample was similar to the
17
natural crystals. For the acid-pretreatment, the elemental composition was 17.11% S,
18
75.13% Fe, 3.66% Si, and 4.10% Al. During the acid-pretreatment, pyrite could be
19
chemically oxidized according to the following equation:
20
FeS2 +2HCl→H2 S+FeCl2 +S
21
Hydrogen sulfide was produced and released into the air during the acid-pretreatment,
22
therefore, a lower mass percentage of S was detected in the acid-pretreated pyrite
23
sample.
24
2.3 Cultivation of microorganisms
25
(1)
Anaerobic sludge was obtained from Tsinghe Sewage Treatment Plant (Beijing, 5
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
1
China) and cultivated in a liquid nutrient medium (2L) at 30 ℃ for 50 days. The
2
nutrient medium consisted of Na2S2O3·5H2O (20 mM), NaHCO3 (30 mM), KNO3 (20
3
mM), KH2PO4 (14.7 mM), NH4Cl (18.7 mM), MgSO4·7H2O (3.25 mM), FeSO4·7H2O
4
(0.08 mM), and CaCl2·2H2O (0.05 mM) (Beller, 2005). The nutrient medium was
5
replaced every two days. Before inoculation, the cultures were centrifuged for 10 min
6
(4500 rpm, 30 ℃) by a centrifuge (GT10-1，SHKIC，China) and suspended in
7
sterilized physiological saline solutions to remove nitrate and sulfate residues in
8
cultures.
9
2.4 Experimental procedure
10 11 12
All the denitrification experiments using UP and AP as electron donors were carried out in 500 mL flasks, in duplicate. The respective flasks were filled with 50 g of UP or AP and 300 mL of the synthetic
13
groundwater, and sterilized at 115 ℃ for 30 min. The flasks were then inoculated
14
with 20 mL of the prepared cultures, described in 2.3, on a Clean Bench (SW-GJ-IFD,
15
AIRTECH, China). The flasks were sparged by nitrogen gas for 5 min prior to sealing
16
with rubber plugs. Un-inoculated flasks were used as controls. All flasks were
17
cultivated at 100 rpm and 30 ℃ in a constant temperature incubator (DDHZ-300,
18
TCSSYSBC, China) for 6 days.
19
After 6 days, the inoculated UP flask was still standing for 30 min, microorganisms
20
grown on the UP surface and suspended in the supernatant were taken for 16S
21
rRNA-based microbial analysis.
22
2.5 Analysis
23
A 10 mL sample was collected from each flask, every 18 h, and the pH of the
24
sample was measured immediately with a pH meter (Seven Multi S40, Mettler Toledo,
25
Switzerland). 6
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
1
Samples were filtered through a 0.45 μm membrane filter. Two mL of each
2
sample were treated with 0.5 mL of H2O2 (3 %, v/v) in order to oxidize sulfite and
3
thiosulfate in the samples to sulfate. Sulfate and total sulfate were measured with an
4
ion chromatograph (ICS900 Dionex IonPac, Thermo Fisher Scientific, US) with
5
detection limit of 0.09 mg/L.
6
According to the Water and Wastewater Monitoring Analysis Method (SEPA, 2002),
7
concentrations of nitrate, nitrite, ammonium, total iron and ferrous ion (Fe (II)) were
8
measured with a spectrophotometer (DR6000, HACH, US) and the detection limits
9
were 0.08 mg/L for nitrate, 0.003 mg/L for nitrite, 0.025 mg/L for ammonium, 0.03
10
mg/L for total iron and 0.03 mg/L for ferrous ion. ATPs in the cultures were measured
11
with an ATP fluorescence detector (AF-100, TOADKK, Japan) with the detection
12
limit of 0.2 fM ATP.
13
The standard deviations were analyzed at a confidence level of 90%, and Origin 9.0
14
(OriginLab, trial version) was used to compute the nitrate removal rate constants in
15
the inoculated UP and AP flasks.
16
For 16S rRNA-based microbial analysis, genomic DNA was extracted and
17
amplified with 968F GC and 1401R primers, using the PCR system (initial
18
denaturation, 95 ℃ for 5 min; subsequent denaturation, 95℃ for 0.5 min; annealing,
19
54 ℃ for 0.5 min; extension, 72 ℃ for 45 s and final extension, 72 ℃ for 10 min).
20
Another round of PCR was performed with amplified 16S rRNA Genes. Denaturing
21
Gradient Gel Electrophoresis (DGGE) analysis was performed using the Bio-Rad
22
C-1000 system (Bio-Rad, USA). The PCR products were loaded in a 1 mm-thick gel
23
containing, 9 % (w/v) polyacrylamide and a denaturant gradient of 40-60 % (100%
24
denaturant was 7 M urea and 40 % formamide). The electrophoresis was run in 1 ×
25
TAE (20 mM Tris, 10mM acetate, 0.5mM EDTA, at pH 8.0) at 60 ℃ for 12 h at 100 7
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
1
V. The gel was then stained with dye solution and placed on a UV illuminator
2
(Bio-Rad, USA). The excised gel from each target band was placed into a 1.5 mL
3
sterile tube containing 30 µL of sterile ultrapure water and crushed. One µL of eluted
4
DNA was amplified with primer 968F/1401R. DNA was sequenced and the sequences
5
were determined by Shanghai Sangon Co., Ltd., Shanghai, China.
6
2.6 Nucleotide Sequence Accession numbers
7
The nucleotide sequences reported in this paper have been submitted to GenBank
8
with accession numbers: KM220911-KM220924.
9
3. Results and discussion
10 11
3.1 Nitrate reduction As shown in Fig. 1a, nitrate sharply decreased from 56.69±0.22 mg-N/L to
12
5.21±1.33 mg-N/L during the first 3 days, and then decreased slowly to 0.08±0.06
13
mg-N/L in the inoculated UP flasks. Nitrate concentrations ranged from 53.31±0.28
14
mg-N/L to 55.87±0.14 mg-N/L in the un-inoculated UP flasks during the experimental
15
period. Similarly, the nitrate concentration decreased from 55.57±0.21 mg-N/L to
16
2.62±0.36 mg-N/L during first 3.75 days, and then nitrate concentration declined
17
steadily to 0.05±0.00 mg-N/L in the inoculated AP flasks. In the un-inoculated AP
18
flasks, the nitrate concentration was nearly stable at 55 mg-N/L (Fig. 1b). Nitrate
19
reduction efficiencies in the inoculated UP and AP flasks both exceeded 99%. In
20
contrast, no nitrate reduction was observed in un-inoculated flasks, indicating that the
21
pyrite could not directly reduce nitrate.
22
According to the first-order kinetics model, nitrate removal rates in the inoculated
23
UP and AP flasks were computed (determination coefficients [R2]> 0.9). The nitrate
24
removal rate constant in the inoculated AP flasks was 1.03 d-1, slightly higher than
25
that of the inoculated UP flasks (0.95 d-1). This difference was potentially due to the
8
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
1
acid-pretreatment process, removing micro-particles such as iron and sulfur impurities
2
from the pyrite surface, and roughening the pyrite surface to allow microorganisms to
3
more easily attach to and utilize the pyrite.
4
The initial nitrite concentration was less than 0.02 mg-N/L in the inoculated UP
5
flasks. The nitrite concentration increased rapidly, reaching 0.55±0.04 mg-N/L during
6
first 3 days, then decreased steadily to less than 0.02 mg-N/L (Fig. 1c). In the
7
inoculated AP flasks, the nitrite concentration increased from less than 0.02 mg-N/L
8
to 0.47±0.05 mg-N/L during the first 2.25 days, then decreased to 0.03±0.00 mg-N/L
9
(Fig. 1d). Pyrite-based denitrification followed the typical pattern of basic microbial
10
denitrification, expressed as NO3-→NO2- →NO→N2O→N2 (Torrentó et al., 2010).
11
After nitrate was partially reduced to nitrite, nitrate and nitrite co-existed in the
12
inoculated UP and AP flasks. The synthesis and activity of nitrite reductase could be
13
inhibited by high concentrations of remaining nitrate (Peng and Zhu, 2006).
14
Furthermore, nitrate was the preferred electron acceptor, over nitrite (Glass and
15
Silverstein, 1998); therefore, competition between nitrate and nitrite was stronger
16
when pyrite was utilized as the sole electron donor.
17
The ammonium concentration increased rapidly from 0.28±0.02 to 5.47±0.29
18
mg-N/L during the first 4.5 days, then increased slowly to 5.88±0.28 mg-N/L in the
19
inoculated UP flasks (Fig. 1e). In the inoculated AP flasks, ammonium continued
20
increasing to 6.82±0.74 mg-N/L over the trial period (Fig. 1f). The dissimilatory
21
nitrate reduction to ammonium (DNRA) process was considered to be the primary
22
cause of ammonium accumulation (Kelso et al., 1997; Zhang et al., 2012). The
23
inoculated cultures derived from anaerobic sludge and were cultivated in a specific
24
liquid culture medium, resulting in more complex microbial community. Clostridium
25
sp. was identified in the inoculated UP and AP flasks before and after the experiment
9
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
1
(Table 1). Specific sulfur-oxidizing chemolithotrophic bacteria such as Clostridium sp.
2
could reduce nitrate to ammonium using sulfur or sulfide compounds as electron
3
donors (Brunet and Garcia-Gil, 1996).
4
3.2 Sulfate accumulation
5
In the inoculated UP and AP flasks, sulfate concentrations increased from
6
261.75±5.50 and 224.25±7.50 mg/L to 649.75±7.00 and 661.25±10.00 mg/L,
7
respectively (Fig. 2). No sulfide or sulfur was added to the flasks, so pyrite could be
8
used as the sole electron donor for autotrophic denitrification. In the un-inoculated UP
9
and AP flasks, sulfate concentrations increased from 252.00±8.00 and 195.50±12.00
10
mg/L to 348.00±2.00 and 270.50± 10.50 mg/L, respectively (Fig. 2). Significantly
11
higher sulfate accumulation occurred in the inoculated UP and AP flasks due to
12
autotrophic denitrification with pyrite as the electron donor.
13
To evaluate their dissolvability, UP or AP was immersed in flasks containing
14
sterilized deionized water. In the UP and AP flasks of sterilized deionized water,
15
sulfate productions were 22.00 and 15.00 mg/L, respectively (Fig. 2). These results
16
indicated that pyrite dissolution led to minimal sulfate production under anoxic
17
conditions.
18
According to Torrentó et al. (2010), if the biomass generation was ignored, basic
19
equation of pyrite-based denitrification could be given as follows:
20
NO3 + 3 FeS2 + 3 H2 O→ 2 N2 + 3 SO4 + 3 Fe(OH)3 + 3 H+
21
-
1
2
1
2
2-
1
1
(2)
Theoretical mass ratio of produced sulfate to removed nitrate (S/N) based on Eq. (2)
22
was 1.03. As shown in Table 2, the actual mass ratios of S/N were always higher than
23
theoretical ratios, due to (1) S. denitrificans (Sulfurimonas denitrificans), which is
24
detected in the experiments (Table 1), being the facultative anaerobic bacteria, could
25
utilize oxygen to produce sulfate (Sievert et al., 2008); (2) small amounts of oxygen 10
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
1
were inevitably introduced during sampling, and pyrite could be chemically oxidized
2
by oxygen in flasks (Akcil and Koldas, 2006). Therefore, sulfate also rose slightly in
3
the un-inoculated UP and AP flasks. Furthermore, a small amount of sulfate could be
4
produced by microorganisms, given a small amount of oxygen in the inoculated UP
5
and AP flasks. However, sulfate production by S. denitrificans was considered to be
6
significant. Hence, it is reasonable to assume that the actual S/N was higher.
7
The S/N ranged from 1.11 to 2.23 in the inoculated UP flasks during the first 4 days
8
(Table 2), which indicated that microbial denitrification was the primary
9
sulfate-production process when nitrate and carbon sources were sufficient. During
10
the last 4 days, the S/N was much higher than 1.03 (Table 1), indicating that chemical
11
oxidation of pyrite played a dominant role with the consumption of the nitrate and
12
carbon sources. Torrentó et al. (2010) also reported similar phenomenon in batch
13
experiments, i.e., the S/N of 3.3 (nitrate initial concentration was 0.69 mM with 20 g
14
pyrite particles and 1 mL pure T. denitrificans supplied in the flask) was attributed to
15
the additional oxidation of pyrite by trace levels of dissolved oxygen.
16
Sulfate concentrations in the un-inoculated UP flasks were always higher than in
17
the un-inoculated AP flasks (Fig. 2). Elemental sulfur could be produced during
18
acid-pretreatment (Eq. (1)). Elemental sulfur could not be chemically oxidized under
19
anoxic conditions. The chemical oxidization capability of AP was weaker because a
20
part of the pyrite samples was transformed to elemental sulfur. Accordingly, this may
21
partially explain why sulfate concentrations in the inoculated UP flasks were always
22
higher than in the AP flasks. However, the S/N mass ratios in the inoculated UP flasks
23
were always lower than in the AP flasks, except on Day 5 (Table 2). The equation of
24
denitrification based on elemental sulfur was as follows (Sierra-Alvarez et al., 2007):
25
NO3 +1.10S+0.40CO2 +0.76H2 O+0.08NH+4 →0.50N2 +1.10SO4 +1.28H+ +0.08C5 H7 O2 N
2-
-
11
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
1
(3)
2
According to Eq. (2) and (3), when 1.00 mol of nitrate was reduced, sulfate
3
production by elemental sulfur based denitrification (1.10 mol) was higher than that
4
by pyrite-based denitrification (0.67 mol). Therefore, the S/N ratio was higher in the
5
inoculated AP flasks.
6
3.3 Microbial community analysis
7
Table 1 showed that S. denitrificans grew in both the UP and AP flasks after 6 days
8
incubation. S. denitrificans is a type of mesophilic, neutrophilic,
9
facultatively-anaerobic, and denitrifying chemolithoautotrophic bacteria, which used
10
sulfide or thiosulfate as an electron donor (Takai et al., 2006). S. denitrificans and T.
11
denitrificans were the only species that can perform sulfo-oxidizing denitrification
12
under neutral and freshwater conditions, both utilizing sulfide and thiosulfate as
13
electron donors (Sievert et al., 2008). It has been proven that pyrite could be used by T.
14
denitrificans as an electron donor (Torrentó et al., 2010). However, until now, only
15
Kelly and Wood (2006) reported pyrite utilization by S. denitrificans. According to
16
the microbial community illustrated in Table 1, pyrite was most likely to be utilized
17
by S. denitrifican as an electron donor for denitrification. The activity and viable
18
count of microorganisms can be characterized by ATP concentrations. The ATP
19
concentrations in the inoculated UP and AP flasks increased from 4.12±0.63 and
20
4.13±0.73 nM to 9.12±2.71 and 9.84±1.87 nM, respectively, indicating that
21
microorganisms continued growing with the nitrate consumption in the inoculated
22
flasks, and viable counts of microorganisms were higher in the AP flasks. Therefore,
23
cost-effective pretreatment may be necessary for the future utilization of pyrite for
24
autotrophic denitrification.
25
According to the light band representing S. denitrificans in Line A, S. denitrificans
12
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
1
was identified on pyrite surfaces (Fig. 3). However, the band representing S.
2
denitrificans in Line B was not bright, indicating that S. denitrificans was not
3
prevalent in the solutions (Fig. 3). S. denitrificans could oxidize electron donors using
4
a multi-enzyme complex catalyzing in vitro (Sievert et al., 2008). Sulfur-binding
5
proteins of some microorganisms such as Thiobacillus ferrooxidans have been found
6
in denitrification using elemental sulfur as an electron donor (Soares, 2002). It is
7
speculated that S. denitrificans might also attach to the pyrite surface using some
8
binding proteins, oxidizing pyrite to reduce nitrate out of cells. It is necessary to
9
further study the binding proteins of S. denitrificans and the enzyme systems of
10 11
pyrite-based denitrification in the future. Bands that represented Dechlorosoma suillum were detected only in Line A and B,
12
which indicated Dechlorosoma suillum grew continuously during incubation.
13
Dechlorosoma suillum could utilize Fe (II) as an electron donor under anaerobic
14
conditions (Lack et al., 2002). It was proven that Fe (II) was an intermediate product
15
in pyrite based denitrification (Torrentó et al., 2010), which provided a suitable
16
condition for Dechlorosoma suillum. The band representing Prosthecobacter
17
fluviatilis was only detected in Line B, indicating that Prosthecobacter fluviatilis was
18
cultivated during the experiment. As mentioned above, ammonium was produced by
19
DNRA, thus, ammonium could be utilized as a nitrogen source by Prosthecobacter
20
fluviatilis (Takeda et al., 2008). Consequently, Prosthecobacter fluviatilis was able to
21
grow in the solutions.
22
According to Fig. 2, total sulfate concentrations were always higher than sulfate
23
concentrations in inoculated UP and AP flasks, indicating that sulfite, thiosulfate and
24
other sulfur compounds were formed during the denitrification process. Sulfite in the
25
inoculated UP and AP flasks was qualitatively detected using the Basic Fuchsin
13
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
1
solution. In addition, Sievert et al. (2008) have proved that sulfite dehydrogenase
2
could be produced by S. denitrificans, so sulfite which could be utilized by S.
3
denitrificans, might be an ideal intermediate product during denitrification. It is
4
speculated that a process exists involving a series of intermediate products, such as
5
sulfite, during sulfate production in pyrite-based denitrification.
6
Concentrations of total iron and Fe (II) were always undetectable in both the
7
inoculated UP and AP flasks. The pH of all flasks was higher than 6.30 during the
8
experiment (Fig. 4). Fe (II) was produced by microbial and chemical pyrite oxidation,
9
and then oxidized to Fe (III) by dissolved oxygen. Fe (III) was precipitated under the
10
pH levels mentioned above. Torrentó et al. (2010) reported that iron concentrations in
11
all batch experiments were below the detection limit, as the pH in the flasks ranged
12
between 6.50 and 7.50.
13
3.4 pH variation
14
The pH in the inoculated UP flasks decreased slightly from 6.68 to 6.51 during first
15
3.75 days, and then remained stable at approximately 6.55 (Fig. 4). In the inoculated
16
AP flasks, the pH decreased from 6.92±0.07 to 6.40±0.11 during the experimental
17
period (Fig. 4). The pH in the un-inoculated UP flasks ranged between 6.80±0.03 and
18
7.15±0.14, and remained stable during the 6 days (Fig. 4). Similarly, pH in the
19
un-inoculated AP flasks remained fairly constant at 6.40. The pH remained at
20
approximately 7.00 and 6.70 in the un-inoculated UP and AP flasks containing
21
sterilized deionized water, respectively (Fig. 4). According to Eq. (2), 0.33 mol of H+
22
was produced by reducing 1.00 mol of nitrate. Although the pH in the inoculated
23
flasks decreased, the pH variation was insignificant, in particular when compared with
24
the H+ production during denitrification based on elemental sulfur. According to Eq.
25
(3), 1.00 mol of nitrate reduction would produce 1.28 mol of H+. A pH buffer reagent,
26
such as limestone, was not necessary for pyrite-based denitrification. To its advantage, 14
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
1
pyrite-based denitrification is a pH-buffering reaction.
2
4. Conclusions
3
Nitrate in synthetic groundwater could be effectively reduced by pyrite-based
4
denitrification with a nitrate reduction efficiency of more than 99%. S. denitrificans
5
could utilize pyrite as electron donor for denitrification. Pyrite-based denitrification
6
exhibited a considerable nitrate removal rate, low sulfate production, and more stable
7
pH. The nitrate removal rate constant was higher, and the initial concentration of
8
sulfate was lower, with the acid-pretreatment. Pyrite is a promising candidate as an
9
electron donor for autotrophic denitrification.
10
Acknowledgements
11
This research work was supported by the Foundation for the Advisor of Beijing
12
Excellent Doctoral Dissertation (No. 20121141501; No. 20131141502), the National
13
Natural Science Foundation of China (NSFC) (No. 31140082) and the Fundamental
14
Research Funds for the Central Universities (No. 2652014100).
15
References
16
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21
minerals by the chemolithoautotrophic bacterium Thiobacillus denitrificans. Appl.
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Environ. Microbiol., 71, 2170-2174.
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[4] Bosch, J., Lee, K.Y., Jordan, G., Kim, K.W., Meckenstock, R.U., 2012. Anaerobic,
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Environ. Sci. Technol., 46, 2095-2101.
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[6] Elmidaoui, A., Elhannouni, F., Menkouchi Sahli, M.A., Chay, L., Elabbassi, H.,
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[9] Haaijer, S. C. M., Lamers, L. P. M., Smolders, A. J. P., Jetten, M. S. M., Op den
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Camp, H. J. M., 2007. Iron sulfide and pyrite as potential electron donors for
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[10] Juncher Jørgensen, C., Jacobsen, O.S., Elberling, B., Aamand, J., 2009. Microbial
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[11] Kelly, D.P., Wood, A.P., 2006. The chemolithotrophic prokaryotes, in: Dworkin,
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[12] Kelso, B., Smith, R.V., Laughlin, R.J., Lennox, S.D., 1997. Dissimilatory nitrate
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[13] Lack, J.G., Chaudhuri, S.K., Chakraborty, R., Achenbach, L.A., Coates, J.D.,
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Table 1 Bacterial species based on DGGE profile before and after experiment. Sample location
Cultivated cultures before experiment
Cultures from pyrite surface after experiment
Cultures from the solution after experiment
Most closely related sequence
% Similarity
Phylogenetic group
Sulfurovum sp.
97
Epsilonproteobacteria
Sulfurimonas denitrificans
94
Epsilonproteobacteria
Clostridium sp.
92
Firmicutes
Sulfurovum sp.
97
Epsilonproteobacteria
Thermomonas sp.
90
Gammaproteobacteria
Clostridium sp
92
Firmicutes
Dechlorosoma suillum
92
Betaproteobacteria
Sulfurimonas denitrificans
90
Epsilonproteobacteria
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.
