Reduction of Fe(III)EDTA by Klebsiella sp. strain FD-3 in NOx scrubber solutions

Bioresource Technology 132 (2013) 210–216

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Reduction of Fe(III)EDTA by Klebsiella sp. strain FD-3 in NOx scrubber solutions Zuoming Zhou ⇑, Guohua Jing, Xiangjiao Zheng Department of Environmental Science & Engineering, Huaqiao University, Xiamen 361021, China

h i g h l i g h t s " A new bacterium FD-3 was isolated and identified as Klebsiella sp. strain. " FD-3 had a good performance on Fe(III)EDTA reduction. 



2

" Fe(II)EDTA–NO, NO3 , NO2 and SO3

inhibited the bio-reduction of Fe(III)EDTA. 



" FD-3 had a certain capability to reduce Fe(II)EDTA–NO, NO3 and NO2 . " The production of Fe(II)EDTA was associated with FD-3 growth.

a r t i c l e

i n f o

Article history: Received 27 September 2012 Received in revised form 1 January 2013 Accepted 5 January 2013 Available online 16 January 2013 Keywords: Nitrogen oxides Klebsiella sp. Fe(III)EDTA reduction Flue gas

a b s t r a c t Biological reduction of Fe(III) to Fe(II) is a key step in nitrogen oxides (NOx) removal by the integrated chemical absorption–biological reduction method, which determines the concentration of Fe(II) in the scrubbing liquid. A new Fe(III)EDTA reduction strain, named as FD-3, was isolated from mixed cultures used in the integrated NOx removal process and identified as Klebsiella sp. by 16S rDNA sequence analysis. The reduction abilities of FD-3 and the influence of nitrogen-containing compounds (Fe(II)EDTA–NO, NO 3 2 2 and NO 2 ) and sulfur-containing compounds (SO4 , SO3 ) on the Fe(III)EDTA reduction were investigated.   The results indicated that strain FD-3 could reduce Fe(III)EDTA efficiently. NO3 , NO2 and Fe(II)EDTA–NO inhibit the reduction of Fe(III)EDTA and could also serve as electron acceptor for strain FD-3. SO2 3 inhibited Fe(III)EDTA reduction while SO2 4 had no obviously effect on Fe(III)EDTA reduction. The relationship between cell growth and Fe(III)EDTA reduction could be described by the models based on Logistic equation. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Nitrogen oxides (NOx) are the main air pollutants causing acid rain, photochemical smog and other environmental problems, such as the elevation of ground-level ozone and the depletion of the ozone layer (Chien et al., 2009). The removal of NOx is important for both environment and human beings. Flue gases from power plants are considered to be one major source of NOx released into the atmosphere (Olivier et al., 1998). Methods to control and reduce NOx have been widely studied, in which an ammonia-based selective catalytic reduction is the best developed and worldwide used technology for the control of NOx emissions in fuel combustion from stationary sources (Nakahjima and Hamada, 1996). How⇑ Corresponding author. Postal address: College of Chemical Engineering, Huaqiao University, Xiamen, Fujian, 361021, China. Tel.: +86 592 6166216; fax: +86 592 6162345. E-mail address: [email protected] (Z. Zhou). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.01.022

ever, the high overall running cost and secondary pollutants have prompted a search for alternative methods for controlling NOx emissions (Jin et al., 2005; Mao et al., 2008). A promising technique for the removal of NOx from industrial flue gas is the BioDeNOx process. It is an integrated physico-chemical and biological technique based on wet absorption of NOx using Fe(II)EDTA as a chelating agent in a scrubber, followed by biological regeneration of this chelating agent in a bioreactor (Van der Maas et al., 2005a; Li et al., 2006). According to the research results of Chang et al. (1983), the absorption is fast and the equilibrium constant of Fe(II)EDTA–NO complex is about 107 M1 (25 °C). The nitrosyl complex formation can be written as:

FeðIIÞEDTA2 þ NO ! FeðIIÞEDTA  NO2

ð1Þ

When organic carbon source (e.g. ethanol, glucose) is used as electron donor, NO is reduced to N2 by the denitrifying bacteria according to Eq. (2) (Zhang et al., 2008):

Z. Zhou et al. / Bioresource Technology 132 (2013) 210–216

12FeðIIÞEDTA  NO2 þ C6 H12 O6 ! 12FeðIIÞEDTA

2

þ 6N2 þ 6H2 O þ 6CO2

ð2Þ

was also explored. Finally, a model for cell concentration as function of Fe(III)EDTA reduction based on a Logistic equation was evaluated.

Although Fe(II)EDTA can chelate NO with a high absorption rate, it is easily oxidized to Fe(III)EDTA by O2 present in the flue gas (Zhu et al., 2011; Wubs and Beenackers, 1993).

2. Methods

4FeðIIÞEDTA2 þ O2 þ 4Hþ ! 4FeðIIIÞEDTA þ 2H2 O

2.1. Bacterial strain, media and identification

ð3Þ

Since only Fe(II)EDTA can absorb NO, it is important to reduce Fe(III)EDTA timely, as it determines Fe(II)EDTA concentration in the scrubber liquor and thus influences the removal efficiency of NO (Kumaraswamy et al., 2006). The biological Fe(III)EDTA reduction can be expressed by Eq. (4) (Zhang et al., 2008):

24FeðIIIÞEDTA þ C6 H12 O6 þ 24OH ! 24FeðIIÞEDTA2 þ 6CO2 þ 18H2 O

ð4Þ

In most of the reported literatures, mixed cultures, which are cultivated with Fe(III)EDTA as the sole electron acceptor, has been found to reduce Fe(III)EDTA efficiently (Jing et al., 2012; Li et al., 2006; Van der Maas et al., 2005b). However, the low density of the dedicated iron-reducing bacteria makes the operation unstable, and under unfavorable conditions, some others (for instance, sulfate reducing bacteria) may become predominant bacteria.  Fe(II)EDTA–NO, NO 3 , and NO2 , which are formed during the absorption of NOx, can inhibit the biological reduction of Fe(III)EDTA (Zhang et al., 2009; Van der Maas et al., 2009). To abate the inhibition of these components on Fe(III)EDTA reduction, a new approach of two-stage reduction with immobilized denitrifying bacteria and iron-reducing bacteria has been proposed by our group(Jing et al., 2012; Zhou et al., 2012). Nitrogen containing  compounds (Fe(II)EDTA–NO, NO 3 , and NO2 ) are reduced at the first stage and Fe(III)EDTA is reduced at the second stage, by which, the inhibition effect will be reduced, and thus the NOx removal efficiency can be improved obviously. However, it is necessary to know and isolate the predominant bacteria which will be used in this technology. Kumaraswamy et al. (2005) applied denaturing gradient gel electrophoresis to analysis the species composition of the lab- and pilot-scale BioDeNOx reactors. They found the predominant microorganisms to reduce Fe(III)EDTA closely affiliated with members of the phylum Deferribacteres, an iron-reducing group of bacteria, but failed to isolate the bacteria from the BioDeNOx reactors. Van der Maas et al. (2005b) reported that Escherichia coli NCTC 9002, a nondissimilatory iron-reducing bacterium unable to reduce poorly crystalline Fe(III) oxides, was capable of reducing Fe(III)EDTA at 37 °C during the oxidation of glucose. In the study of Li et al. (2007), they isolated a strain E. coli FR-2 (GenBank DQ411026) from the cultivated mixed culture and found it was a key player in Fe(III)EDTA reduction. More recently, Dong et al. (2012) demonstrated that a denitrifying bacterium Paracoccus denitrificans ZGL1 could reduce Fe(III)EDTA efficiently. From the above statements, several bacteria had been reported to be able to reduce Fe(III)EDTA, but only a very few has been isolated from the mixed culture used in the integrated running process for NOx removal. To better understand the predominant strains used in the two-stage bio-reduction system, in this study, a new Fe(III)EDTA reduction bacterial strain, named as FD-3, was isolated from mixed culture used in the integrated process and identified as Klebsiella sp. The strain showed a good performance in Fe(III)EDTA reduction. Furthermore, FD-3 could use Fe(II)ED TA–NO, NO 3 and NO2 as the electron acceptor. Therefore, the  reduction of Fe(II)EDTA–NO, NO 3 and NO2 by the strain FD-3 as well as the effect of those nitrogen-containing compounds on Fe(III)EDTA reduction were investigated. Since SO2 were always 2 present in typical flue gas, SO2 3 and SO4 were also formed in the liquid. The effect of these constituents on Fe(III)EDTA reduction

211

The basal media for the bacterial growth included the following components (mg L1): glucose 1500, NH4Cl 1000, KH2PO4 625, K2HPO43H2O 1000, CaCl2 2, Na2SO3 70, MgSO4 100, MnSO4 0.5, Na2MoO4 0.1, CuSO45H2O 0.1. The bacterial strain used in this study was isolated from the cultivated mixed culture used for NOx removal in our laboratory, purified and named as FD-3. The detailed cultivation was seen in Jing et al. (2012). Isolation of pure culture was carried out anaerobically on agar plates at 40 °C with 50 ll of enriched mixed culture preliminarily diluted 108 times in sterile 200 mg L1 phosphate buffer. Colonies were picked and then purified twice to obtain pure cultures. Selection of strain FD-3 was based on its highest efficiency of Fe(III)EDTA reduction among the six strains isolated. Taxonomical identification was performed by 16S ribosomal DNA amplification and sequencing at Shanghai Sangon Biological Engineering & Technology and Service Co., Ltd. (Shanghai, China). After purification, FD-3 was cultivated in 250 ml conical flasks with 100 ml basal medium under anaerobic condition by replacing the air above the solution surface with nitrogen gas. The flasks were located in a rotary shaker with a temperature of 40 °C and a rotation rate of 140 rpm. Cells in the medium were harvested by centrifugation at 8000 rpm for 10 min and washed twice with distilled water, and then suspended in the phosphate buffer at certain concentrations for use. 2.2. Fe(III)EDTA reduction experiments The reduction experiment was performed in 100 ml batch-bottles filled with 50 ml of the sterilized medium (autoclaving for 20 min at 121 °C) and 10 mM Fe(III)EDTA. Fe(III)EDTA solution was prepared according to Jing et al. (2012). The pH was adjusted to 6.8 ± 0.1 with 0.1 M HCl or NaOH. The bottles were sealed with Teflon-coated rubber septa in a gyrating shaker at 140 rpm and 40 °C. The headspace of the solution was flushed with oxygen-free nitrogen gas. Samples were taken regularly to measure the concentration of Fe(II)EDTA and cells. And the control experiments were simultaneously studied. To evaluate the influence of Fe(II)EDTA–  2 NO, NO and SO2 on Fe(III)EDTA reduction, different 3 , NO2 , SO3 4 concentrations of Fe(II)EDTA–NO, NaNO3, NaNO2, NaSO4 and Na2SO3 were added respectively to the serum bottles. The Fe(II)EDTA–NO solution was prepared by bubbling NO into the ferrous EDTA solution (Li et al., 2007). The FD-3 inoculum concentration was 0.15 g DCW L1 in the experiments to evaluate the inhibition of Fe(II)EDTA–NO and NO 2 on Fe(III)EDTA reduction. In other experiments, the FD-3 inoculum concentration was 0.10 g DCW L1. 2.3. Analytical methods The concentration of Fe(II)EDTA–NO was determined from a standard curve with linear correlation between absorbance and Fe(II)EDTA–NO concentration. The absorbance was directly measured by a model 723A spectrophotometer at 438 nm. The concentration of NO 3 was measured by an ultraviolet spectrophotometric method (The National Geological Mineral Industry Standards of China, 1996). The concentration of NO 2 was measured by N-(1naphthyl)ethylene diamine dihydrochloride spectrophotometric

Z. Zhou et al. / Bioresource Technology 132 (2013) 210–216

7.0

9

6.8

8

6.6

7

6.4 6.2

6 Fe(III)EDTA pH cells

5

0

2

4

6

8

10

0.25

0.20

0.15

6.0 5.8

4

3. Results and discussion

0.30

Cell concentration (g/L)

10

pH

method (The National Environmental Protection Standards of China, 2009). The concentration of ferrous irons and total irons in solution was determined colorimetrically using 1,10-phenanthroline at 510 nm (Jing et al., 2012). The Fe(III)EDTA concentration was calculated by the difference between the total Fe and Fe(II). Cell concentration in the samples was analyzed by measuring the optical density at 610 nm(OD610) using an UV–visible spectrophotometer with the culture medium, then calculated according a linear relationship between OD610 and dry cell weight (DCW) (Jing et al., 2012). All the data shown in this study were the mean values of the duplicate or triplicate experiments.

Fe(III)EDTA concentration (mM)

212

0.10

12

Time (h)

3.1. Characterization of strain FD-3 Strain FD-3 was a Gram-negative coccus that formed white rounded opaque colonies on agar plates. The nearly complete 16S rDNA gene of strain FD-3 was amplified and sequenced. Based on the BLAST DNA sequence analysis, the 16S rDNA gene sequence of strain FD-3 exhibited 99% identity with those of the genus Klebsiella. The sequence of stain FD-3 was submitted to GenBank and gained a number GU167258. The whole sequence alignment was carried out by using clustarx1.8 software and the phylogenetic tree was made by Phylip3.69 package with maximum likelihood. Phylogenetic relationships were shown in Fig. 1. Strain FD-3 could grow at 35–55 °C with an optimal temperature of 40–45 °C.

Fig. 2. Characterization of Klebsiella sp. FD-3 growth and Fe(III)EDTA reduction. (Klebsiella sp. FD-3, T = 40 °C, pH = 6.71, [Fe(III)EDTA]0 = 10 mM, [Cell]0 = 0.10 DCW L1, data were the mean values of triplicate experiments).

4.14 mmol h1 per g DCW) with an initial Fe(III)EDTA concentration of 12 mM. Zhang et al. (2009) reported that a bacterial strain of E. coli had a reduction rate about 0.77 mmol h1 per g DCW within the cultivation of 12 h. Compared with these strains, FD-3 had a better performance for Fe(III)EDTA reduction. During Fe(III)EDTA reduction, OH was consumed and the pH gradually declined, from 7.0 to 6.0, which could be explained by the reaction shown in Eq. (4).

3.2. FD-3 growth and Fe(III)EDTA reduction The results of the Fe(III)EDTA reduction and cell growth experiments were shown in Fig. 2. Reduction of Fe(III)EDTA by the strain FD-3 was fast and effective. During the first 6 h lag phase, Fe(III)EDTA decreased slowly. While during the next 8 h, Fe(III)EDTA declined from 9.20 to 4.76 mM, and the cell concentration increased from 0.10 to 0.23 g (DCW/L). After 12 h, Fe(III)EDTA reduction efficiency reached 52.4%, and the average reduction rate was about 4.30 mmol h1 per g DCW. Li et al. (2005) reported that a Klebsiella Trevisan sp with an inoculation of approximately 0.15 g DCW/L, could reduce about 70.0% Fe(III)EDTA in 13.5 h (about

0.802

0.514

0.1

 3.3. Effects of Fe(II)EDTA–NO, NO 3 and NO2 on Fe(III)EDTA reduction

Fig. 3 illustrated the effects of different initial concentrations of  Fe(II)EDTA–NO (0–8 mM), NO 3 (0–8 mM), and NO2 (0–4 mM) on Fe(III)EDTA reduction. Fig. 3(a) showed that Fe(II)EDTA–NO inhibited the reduction of Fe(III)EDTA, and the inhibition increased with the increase of Fe(II)EDTA–NO concentration. After 24 h, the reduction efficiency reached 95.1%, 60.0%, 18.7% and 10.6% when 0, 2, 4 and 8 mM Fe(II)EDTA–NO were added, respectively. According to the previous researches (Van der Maas et al., 2005b; Zhang et al.,

0.1 Plasmodium falciparum X07455.1 Plasmodium falciparum X07455.1 Bacillus sporothermodurans U49078.1 0.085 Bacillus sporothermodurans U49078.1 Bacillus azotoformans strain D783.9.1 Bifidobacterium HGAT10 HM245216.1 BBifidobacterium sp. HGAT10 sp. HM245216.1 0.156 Bifidobacterium sp. HGAT9 HM245212.1 0.107 Cellulophaga lytica M62796.1 0.184 0.125 D ysgonomonas CDC Group DF-3 U41355.1 capnocytophagoides DF-3 U41355.1 Desulfovibrio dechloracetivorans AB546253.1 dechloracetivorans AB546253.1 0.126 Desulfovibrio 0.032 dechloracetivorans Desulfovibrio Desulfovibrio dechloracetivorans strain SF3 NR_025078.1 strain SF3 NR-025078.1 0.193 ferrooxidans Leptosp Leptospirillumirillum ferrooxidans strain Z-15 HQ405790.1 strain Z-15 HQ405790.1 0.046 ferrooxidans Acidithiobacillus ferrooxidans strain af1 EU652051.1 strain af1 EU652051.1 0.076 Acidithiobacillus A ferrooxidans cidithiobacillus Acidithiobacillus ferrooxidans strain IESL32 HQ902071.1strain IESL32 HQ902071.1 0.023 Acidithiobacillus ferrooxidans Acidithiobacillus ferrooxidans strain SRDSM2 GQ292748.1strain SRDSM2 GQ292748.1 Burkholderia sp. GG4 HQ728437.1 Burkholderia sp. GG4 HQ728437.1 0.068 0.064 Gallionella PN013 HQ117915.1 Gallionella sp. PNO13sp. HQ117915.1 Gallionellaceae Gallionellaceae bacterium HDDbacterium HQ263247.1 HDD HQ263247.1 Siderox ydans spp. PN022 HQ117918.1 Sideroxydans sp. PNO22 HQ117918.1 0.030 ydans Siderox sp. PN032 HQ117920.1 Sideroxydans sp. PNO32 HQ117920.1 seudomonas strain cT180 JF303029.1 Pseudomonas stutzeri strainstutzeri cT180 JF303029.1 0.101 P P seudomonas strain cT224 JF303038.1 Pseudomonas stutzeri strainstutzeri cT224 JF303038.1 0.052 E scherichia coliHQ286917.1 strain skg007 HQ286917.1 Escherichia coli strain skq007 0.088 Klebsiella Klebsiella sp. SRC-DSAS sp. GU373992.1 SRC-DSAS GU373992.1 FD3 GU167258.1 FD3 GU167258.1 Klebsiella sp. ANctcri2 Klebsiella sp.HQ286642.1 ANctcri2 HQ286642.1

Fig. 1. Phylogenetic tree of 16S rDNA for Klebsiella sp. strain FD-3. The numbers on the branches show the genetic distances.

213

(a)

Fe(III)EDTA concentration (mM)

Z. Zhou et al. / Bioresource Technology 132 (2013) 210–216

reached 8 mM. After 16 h, the reduction efficiency reached 49.5%, 55.2%, 57.4%, 58.5% and 34.9% when 0, 0.5, 1, 2, 4 and 8 mM NO 3 were added, respectively. The presence of 8 mM NO 3 inhibited Fe(III)EDTA reduction. Zhang et al. (2009) reported that more than 3 mM of NO 3 was added, the reduction of Fe(III)EDTA was inhibited and the inhibition effect increased with the increase of NO 3 concentration. From Fig. 3(c), it indicated that with the addition of 0.5–2 mM NO 2 , the reduction rate of Fe(III)EDTA was not inhibited but promoted a little. When more NO 2 (4, 8 mM) was added, the Fe(III)EDTA reduction rate decreased. After 26 h, Fe(III)EDTA reduction efficiency was 97.8%, 99.6%, 97.4%, 97.2%, 90.3%, 86.4% and 49.7% with 0, 0.5, 1, 2, 4, 6 and 8 mM NO 2 , respectively. However, with the increase of the reaction time, Fe(III)EDTA could be reduced completely. Fe(III)EDTA reduction efficiency could reach 92.8% and 90.4% after 36 h, with 4 and 6 mM NO 2 respectively, and reached 93.2% after 50 h with 8 mM NO 2 . Meanwhile, the cell concentration increased with the addition of NO 2 , however, at high concentration of NO , the cell concentration declined (data not 2 shown). This observation agreed with the previous researches that prolonged exposure to elevated concentrations of NO 2 may be toxic (Huang et al., 2000). The results from Fig. 3(b) and (c) suggested that the reduction of Fe(III)EDTA was accelerated at low concentrations and inhibited at  high concentrations of NO 3 and NO2 . The reason was that, when  NO and NO served as electron acceptors, the microorganisms 2 3 could obtain more energy from the dissimilatory reduction of  NO 2 and NO3 than from the dissimilatory reduction of Fe(III), which was beneficial to shorten the adaptation period of the bacteria and then increased the reduction efficiency. At high concentrations, due to the higher standard redox potential of NO 2 /NO  (+0.99 V) and NO /NO (+0.433 V) than that of Fe(III)/Fe(II) 3 2  (+0.34 V), electrons could transferred to NO 2 and NO3 (Zhang  et al., 2009). The inhibition of NO and NO on Fe(III)EDTA reduc2 3 tion might be caused by the differences in the electron transport to  NO 2 , NO3 and Fe(III)EDTA, that had evolved to allow preferential use of the most favorable oxidant (Cooper et al., 2003; Li et al., 2011; Jing et al., 2012). Moreover, the oxidation of Fe(II)EDTA was accelerated by the following chemical reaction with nitrite (Van der Maas et al., 2004):

10 8 0 mM 2 mM 4 mM 8 mM control

6 4 2 0

0

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Time (h) Fe(III)EDTA concentration (mM)

(b)

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8 0 mM 0.5 mM 1 mM 2 mM 4 mM 8 mM control

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4 0

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Fe(III)EDTA

(c)

concentration (mM)

Time (h) 10 0 mM 0.5 mM 1 mM 2 mM 4 mM 6 mM 8 mM control

8 6 4 2 0

2FeðIIÞEDTA2 þ NO2 þ 2Hþ ! FeðIIIÞEDTA þ FeðIIÞEDTA 0

10

20

30

40

 NO þ H2 O

50

Time (h)  Fig. 3. Effect of different concentration of Fe(II)EDTANO (a), NO 3 (b) and NO2 (c) on Fe(III)EDTA reduction. (Klebsiella sp. FD-3, T = 40 °C, pH = 6.98, [Fe(III)EDTA]0 = 10 mM, (a), (c): [Cell]0 = 0.15 DCW L1, (b): [Cell]0 = 0.10 DCW L1, data were the mean values of triplicate experiments).

ð6Þ

Additionally, the inhibition of Fe(II)EDTA–NO on Fe(III)EDTA reduction was demonstrated, and Fe(II)EDTA–NO might be accumulated in the liquid phase with the increase of the NO 2 concentration, which would affect the reduction of Fe(III)EDTA. 3.4. Effects of Na2SO4 and Na2SO3 on Fe(III)EDTA reduction

2008), during the bio-reduction of Fe(II)EDTA–NO, Fe(II)EDTA can be oxidized to Fe(III)EDTA according to Eq. (5).

2FeðIIÞEDTA  NO2 þ 2FeðIIÞEDTA2 þ 4Hþ ! 4FeðIIIÞEDTA þ N2 þ H2 O

ð5Þ

Li et al. (2007) found that when Fe(II)EDTA–NO concentration was up to 3.7 mM, about 1 mM Fe(III)EDTA was increased after the cultivation. Thus, the reduction efficiency decreased accordingly due to the oxidation of Fe(II). Similar findings were observed as for the strain E. coli FR-2 (Zhang et al., 2008). When the concentration of Fe(II)EDTA–NO reached 3.7 mM, the FR-2 cell growth and Fe(III)EDTA reduction almost stopped. Fig. 3(b) showed that there was no significant influence in Fe(III)EDTA reduction in response to NO 3 until the concentration

The addition of Na2SO4 (0–8.0 mM) in the medium had no influence on Fe(III)EDTA reduction (data not shown), which was in coincidence with the results using mixed cultures (Jing et al., 2012). Fig. 4 showed the effect of the addition of Na2SO3 (0.5, 1, 2, 4 mM) on the maximum reduction rate of 10 mM Fe(III)EDTA observed in batch conical flasks inoculated with 0.1 g L1 FD-3 cells. According to Fig. 4, addition of Na2SO3 resulted in lower Fe(III)EDTA reduction rates than that of the control group without Na2SO3. When 0.5, 1, 2, 4 mM Na2SO3 were added, after 24 h, the Fe(III)EDTA reduction rate declined from 60.0% to 49.8%, 47.4%, 44.4% and 33.0% respectively. The strong inhibition of Fe(III)EDTA reduction by sulfite might be caused by the combined results of a direct toxic effect of sulfite itself on the bacterial population and a competition for electrons

(a)

8

0 mM 0.5 mM 1 mM 2 mM 4 mM control

4

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4

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Time (h)

 3.5. Reduction of Fe(II)EDTA–NO, NO 3 , and NO2 by FD-3

Fig. 5(a) showed the results of Fe(II)EDTA–NO reduction by FD3. When 4, 6 and 8 mM Fe(II)EDTA–NO were added, during the first 23 h, the specific reduction capacity as measured by the reduction rate of Fe(II)EDTA–NO were up to 74.1%, 75.4%, 19.4%, respectively, while the depletion of Fe(II)EDTA–NO was not found in the control samples without FD-3 cells. Though each complex, e.g., Fe(II)EDTA–NO or Fe(III)EDTA, could be reduced by its own dedicated bacterial strain, strain FD-3 capable of reducing Fe(II)EDTA–NO would enlarge NO elimination capacity. As shown in Fig. 5(b), during cultivation, NO 3 decreased with corresponding increase in cell concentration and NO 2 concentration. NO 3 could also serve as the sole electron acceptor, and the concentration of bacterial cells reached the highest value after 4 h cultivation, about 0.11 and 0.13 g DCW L1 with the addition of 4 and 8 mM NO 3 (data did not show), respectively. The amount of NO 3 in the culture medium decreased rapidly, companied with the growth of cells. After 8 h cultivation, 4 and 8 mM NO 3 were reduced to 0 and 0.15 mM, respectively. Depletion of NO 3 was not found in the control samples without FD-3 cells. During the reduc tion of NO 3 , it was interesting to note that an increase of NO2 concentration was observed with NO was reduced. Approximately 3 2.27 mM and 7.42 mM NO 2 were produced respectively in the 8 h of cultivation, almost equal to the amount of NO 3 reduced. It  was indicated that FD-3 could reduce NO to NO . 3 2 Fig. 5(c) showed the results of NO 2 reduction by FD-3 without adding Fe(III)EDTA. When 1, 2, 4, 6 and 8 mM NO 2 were added, after 8 h, the reduction rate reached 94.8%, 67.1%, 48.1%, 29.2%

20

25

8

6

6 -

-

NO3

NO2 4 mM 8 mM control

4

4 mM 8 mM control

4

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NO3 concentration (mM)

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with sulfite, which has been described in our previous study using mixed cultures (Jing et al., 2012). As reported by Huang et al. (2000), the high concentration of sulfite was toxic to the microbial populations present in the bio-assays. For instance, 0.9 mM sodium sulfite completely inhibited the activity of Methanobacterium fornicicum (Balderston and Payne, 1976). Van der Maas et al. (2009) also suggested that the strong inhibition of Fe(III)EDTA reduction by calcium sulfite might be due to a direct toxic effect of sulfite on the bacterial population. In the study of Li et al. (2011), they pointed out that sulfite inhibited cell growth and Fe(III)Cit reduction whereas sulfate had no influence on Fe(III)Cit reduction. On 2 the other hand, the standard redox potential of SO2 3 /S (+0.342 V) was close to that of Fe(III)/Fe(II) (+0.34 V). Therefore, electrons could transport to sulfite and Fe(III), which resulted in the inhibition of Fe(III) reduction. Sulfate had no toxicity and cannot compete for electrons since the standard redox potential of 2 SO2 4 /SO3 was 0.04 V, lower than that of Fe(III)/Fe(II) (+0.34 V).

(b)

NO2 concentration (mM)

Fig. 4. Effect of Na2SO3 on Fe(III)EDTA reduction. (Klebsiella sp. FD-3, T = 40 °C, pH = 6.86, [Fe(III)EDTA]0 = 10 mM, [Cell]0 = 0.10 DCW L1).

15

Time (h)

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NO2 concentration (mM)

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Fe(II)EDTA-NO concentration (mM)

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Fe(III)EDTA concentration (mM)

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6

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2

0mM 2mM 6mM control

1mM 4mM 8mM

50

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0 0

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Time (h)  Fig. 5. The reduction of Fe(II)EDTA–NO (a), NO 3 (b), NO2 (c) by FD-3 without Fe(III)EDTA in the solution. (Klebsiella sp. FD-3, T = 40 °C, pH = 6.92, [Fe(III)EDTA]0 = 10 mM, [Cell]0 = 0.10 DCW L1, data were the mean values of duplicate experiments).

and 15.7% respectively. After 32 h, the reduction rate reached 81.5% with the addition of 4 mM NO 2 , while the reduction rates were 32.8% and 28.2% with 6 and 8 mM NO 2 added respectively, and the prolonging of reaction time had no obvious increase in the reduction rate. Even after 56 h, the reduction rate was only increased to 34.4% and 31.1% when 6 and 8 mM NO 2 were added respectively. These results further demonstrated the conclusions drawn from Fig. 3(b) and (c), that Fe(III)EDTA reduction was promoted from the dissimilatory reduction of low concentrations of     NO 2 and NO3 . At high concentrations of NO2 and NO3 , NO2 would accumulate, which was toxic to FD-3 and thus inhibited the Fe(III)EDTA reduction.

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(a)

The specific growth rate of cells in a batch system was defined as (Blanch and Clark, 1997):

  1 dX X  ¼ lmax 1  X dt X max

ð7Þ

where X was the cell concentration, g DCW L1; lmax was the maximum specific growth rate of the microorganisms at the exponential phase of the growth curve, h1; dX/dt was the cell growth rate; and t was the cultivation time, h. Assuming that Fe(II)EDTA production obeyed the LuedekingPiret equation (Luedeking and Piret, 1959), which was the universal equation to describe the product formation rate:

dP dX ¼a þ bX dt dt

0.28

Cell concentration (g/L)

3.6. Model for cell growth and Fe(III)EDTA reduction

0.24

0.20

0.16

0.12

0

dS dX  ¼a þ bX dt dt

dX ¼ lmax  X  ð1 

ð9Þ

X Þ  dt X max

ð10Þ

dS ¼ adX þ bXdt

dS ¼ a  lmax  X  ð1 

X Þ  dt þ bXdt X max

ð12Þ

According to initial condition, when t = 0, X = X0 and S = S0. Integrating Eqs. (10) and (12), the following expressions were obtained (Li et al., 2011):

X 0 X max elmax t X¼ X max  X 0 þ X 0 elmax t  ln

6

8

10

25

Fe(III)EDTA concentration (mM)

(b)

5 mM 7.5 mM 10 mM 12.5 mM

20

15

25 mM

10

5

0 0

2

4

6

8

10

Time (h) Fig. 6. Comparison of the experimental and modeled data for FD-3 growth (a) and Fe(III)EDTA reduction (b) under different initial concentrations of Fe(III)EDTA. (Klebsiella sp. FD-3, T = 40 °C, 140 rpm, pH = 6.70, [Cell]0 = 0.10 DCW L1, data were the mean values of triplicate experiments).

ð11Þ

Substituting dX by Eq. (10) into Eq. (11), Eq. (12) could be described as follows:

lmax

4

Time (h)

where dS/dt was the Fe(III)EDTA consumption rate. Eqs. (7) and (9) could be rewritten as follows:

b  X max

2

ð8Þ

where dP/dt was Fe(II)EDTA formation rate; a was the product formation coefficient; b was a non-growth correlation coefficient. Gaden (1959) classified the mode of product formation in terms of the relationship with microorganism growth as follows: for a – 0, b = 0, product formation was associated to microorganism growth; for a = 0, b – 0, product formation was unrelated to microorganism growth; for a – 0, b – 0, product formation was partially associated to microorganism growth. Since Fe(III)EDTA only served as electron acceptor, the consumption of Fe(III)EDTA was equal to the production of Fe(II)EDTA, i.e. dS/dt = dP/dt. As such, Eq. (8) could be described as follows:

S¼

5 mM 7.5 mM 10 mM 12.5 mM 25 mM

ð13Þ

X  X max þ a  ðX 0  XÞ þ S0 X 0  X max

ð14Þ

Eq. (13) described the relation between cell growth and time. Eq. (14) described the relation between cell growth and Fe(III) EDTA reduction. Table 1 listed the values of the parameters used

Table 1 Values of parameters used in the model.

in the above mathematical model. The simulation data for FD-3 growth and Fe(III)EDTA reduction were compared with the experimental data as shown in Fig. 6(a) and (b), respectively. The simulation results showed that the model predictions matched the experimental data satisfactorily, which suggested that the production of Fe(II)EDTA was associated to cell growth. 4. Conclusions A new strain, named FD-3, was isolated and identified as the genus Klebsiella. FD-3 had a good performance in Fe(III)EDTA reduction. Fe(III)EDTA reduction would be affected by those coexisting component in the NOx absorption solution (e.g. Fe(II)ED 2 2 TA–NO, NO had no 3 , NO2 and SO3 ), while the presence of SO4 influence on Fe(III)EDTA reduction. In addition, FD-3 had a certain capability to reduce those nitrogen-containing compounds, which was beneficial to Fe(III)EDTA reduction. Furthermore, the developed model suggested that the production of Fe(II)EDTA was associated with FD-3 growth. Acknowledgements

S0 (mM)

Xmax

lmax

a

b

5 7.5 10 12.5 25

0.307 ± 0.153 0.269 ± 0.158 0.298 ± 0.191 0.216 ± 0.097 0.338 ± 0.067

1.050 ± 0.243 1.217 ± 0.429 0.772 ± 0.188 1.676 ± 0.543 2.676 ± 0.408

3.177 ± 4.296 19.059 ± 7.181 50.655 ± 9.726 56.252 ± 6.321 32.723 ± 6.854

1.253 ± 0.404 2.800 ± 0.735 0.235 ± 0.653 6.887 ± 1.606 7.773 ± 1.158

This work was sponsored by the National Natural Science Foundation of China (No. 21077035), the Program for New Century Excellent Talents in the University of China (NCET-11-0851) and Fujian Province (11FJRC02), and the Fundamental Research Funds for the Central Universities ((No.JB-GJ1004).

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