Isolation and Characterization of Pseudomonas

Geomicrobiology Journal

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Isolation and Characterization of Pseudomonas stutzeri Capable of Reducing Fe(III) and Nitrate from Skarn-type Copper Mine Tailings Guo-Wei Wang, Tian-Hu Chen, Zheng-Bo Yue, Yue-Fei Zhou & Jin Wang To cite this article: Guo-Wei Wang, Tian-Hu Chen, Zheng-Bo Yue, Yue-Fei Zhou & Jin Wang (2014) Isolation and Characterization of Pseudomonas stutzeri Capable of Reducing Fe(III) and Nitrate from Skarn-type Copper Mine Tailings, Geomicrobiology Journal, 31:6, 509-518, DOI: 10.1080/01490451.2013.847992 To link to this article: http://dx.doi.org/10.1080/01490451.2013.847992

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Date: 16 December 2016, At: 06:53

Geomicrobiology Journal (2014) 31, 509–518 C Taylor & Francis Group, LLC Copyright ISSN: 0149-0451 print / 1521-0529 online DOI: 10.1080/01490451.2013.847992

Isolation and Characterization of Pseudomonas stutzeri Capable of Reducing Fe(III) and Nitrate from Skarn-type Copper Mine Tailings GUO-WEI WANG, TIAN-HU CHEN∗, ZHENG-BO YUE, YUE-FEI ZHOU, and JIN WANG∗ School of Resources & Environmental Engineering, Hefei University of Technology, Hefei, China Received May 2013, Accepted September 2013

Nitrate and Fe(III) are two terminal electron acceptors in anaerobic respiration by microorganisms growing in the anoxic soil or sediment environment. In the current paper a facultative anaerobic dissimilatory Fe(III)- and nitrate-reducing bacterium was isolated from the Linchong tailings, a skarn-type copper mine tailings, located in Anhui. Skarn often formed at the contact zone between intrusions of granitic magma bodies into contact with carbonate sedimentary rocks. This made the tailings possessed strong acid neutralizing capacity and pH of the pore water was 6.8–8.6. The isolate, which was designated strain CW (CCTCC AB 2013114), was a gram-negative rod bacterium and belonged to the gamma subgroup p of the proteobacteria, closely (99.0%) related to Pseudomonas stutzeri. In defined medium, strain CW was shown to grow anaerobically with the acetate using the ferric iron or nitrate as the electron acceptors. Results also showed that strain CW could not grow in the presence of ferrous iron and nitrite. Keywords: copper tailing, dissimilatory Fe(III)-reducing, isolation, nitrate-reducing, Pseudomonas stutzeri

Introduction Many of the important geological elements are redox-active, and thus are prone to catalysis by microbes that exploited them as electron donors or electron acceptors. Fe(III) is often an abundant electron acceptor for microbial respiration in subsurface environments and aquatic sediments (Lovley 2000). Dissimilatory Fe(III)-reduction bacteria (DIRB) constituted an important metabolic group involved in environmental processes such as natural degradation of organic matter or bioremediation of organic contaminants (Becker and Seagren 2009). DIRB influenced not only the distribution of iron in the environment but also the fate of other metals, such as Mn(IV), Cr(VI), Se(V), U(VI), and Tc(VII), when their reduction was coupled to the oxidation of organic matter (Han and Gu 2009). This metabolism could lead to the complete mineralization of organic contaminants and the immobilization of metal contaminants under anaerobic conditions. Microbe dominated process using nitrate as the electron acceptors was another popular process happened under the anoxic conditions. A variety of nitrate-reducing bacteria had been found and reported (Chen et al. 2010). Microbe that could reduce both Fe(III) and nitrate had been isolated and identified from the nutrient rich environments (Table 1), ∗

Address correspondence to Dr. Tian-Hu Chen or Dr. Jin Wang, School of Resources & Environmental Engineering, Hefei University of Technology, Hefei 230009, China; E-mail: [email protected] or [email protected]

such as freshwater and marine sediments, hydrocarboncontaminated sediments, a mining-impacted lake, and the deep subsurface (Achtnich et al. 1995; Finneran 2003; Lovley 2000; Naganuma 2006; Obuekwe et al. 1981).This would improve their competition capacity for the substrate and might evolve as the dominant community. In the nutrient limited environment, for example ore tailings, these kinds of microbe would have a higher survival rate and play a significant role in the remediation of tailings in situ. Pseudomonas strains have been reported that could grow under the Fe(III) or nitrate reduction, but the strains were not isolated from the skarn-type copper tailings (Table 1). In the current paper a strain capable of reducing both nitrate and Fe(III), Pseudomonas stutzeri strain CW, was isolated from Linchong tailings, Tongling, China. The performance of strain CW was investigated using either Ferric iron or Nitrate as the only electron acceptor or both as the electron acceptors in the presence of acetate.

Materials and Methods Source of Microorganism Soil samples were collected from the LinChong tailings, Tongling, China. The tailings, which contain significant amounts of calcite, possess strong acid neutralizing capacity. pH of the pore water of the tailings was in the range of 6.8–8.6. In the past 50 years, the surface sulfides had been weathered and oxidized to sulfate, iron oxides and hydroxides.

510

For/lac

Ac/H2 /lac/mal/pept/ pyr/suc etc.

For/eth/lac/pyr/ac

Deferribacter thermophilus

Geobacter strain JW-3

Fe(III)/Fe(II), nitrate/ND


Fe(III)/Fe(II), nitrate/ammonia Fe(III)/Fe(II), nitrate/nitrite Fe(III)/Fe(II), nitrate/nitrite

Desulfuromusa Ac/prop/citr etc. kysingii Ferrimonas balearica Lac

Bacillus infernus

Fe(III)/Fe(II), nitrate/ammonia

Ac/buty/pyr etc.

Fe(III)/Fe(II), nitrate/N2 Fe(III)/Fe(II), nitrate/nitrite Fe(III)/Fe(II), nitrate/ND

H2

Ac

Electron acceptors/ corresponding productsb

Yeast extract /pept etc.

Pseudomonas sp. strains KNA-6-3 and KNA-6-5 Geobacter metallireducens

Pseudomonas sp. strain CW Pseudomonas sp.

Microorganisms

Electron donors oxidized with Fe(III)a

Table 1. Microorganisms capable of reducing nitrate/ Fe(III)

AQDS/So/Mn(IV)

Mn(IV)

Mn(IV)/TMAO

So/Fumarate/ malate etc. Mn(IV)

Mn(IV)/U(VI)/ AQDS etc.

O2 /U(VI)

O2

ND

Other electron acceptors

Marine sediment at the coast of Mallorca (Spain) Deep subsurface ca. 2, 700 m below the land surface in the Taylorsville Triassic Basin in Virginia Sea oil field in the British sector of the North Sea near the coast of Scotland at a depth of 2, 058 m Freshwater sediments collected from a shallow wetland in Fairfax, Va., that appeared to be contaminated with hydrocarbons

Aquatic sediment in the Potmac river, Maryland. Freshwater

Groundwater of the Tono uranium mine

Skarn-type copper mine tailings Swampy soil

Resources

Lovley (2000)

Lovley (2000) Lovley (2000) Lovley (2000)

Lovley (2000)

Lovley (2000)

30◦ C/pH 7.0 37◦ C/pH 6.0–9.0 60◦ C/pH 7.3–7.8

60◦ C/pH 7.1

30◦ C/pH 6.7

Balashova and Zavarzin (1979) Naganuma et al. (2006)

This article

Reference

33◦ C/pH 6.7

5–40◦ C/pH 5.0–10.0

30–40◦ C/pH 6.7–8.0 30◦ C/pH 7.0

Growth conditions

511

Lac/pyr/for/ buty/suc etc.

Carboxydocella manganica

Fe(III)/Fe(II), nitrate/ammonia

Fe(III)/Fe(II), nitrate/ammonia Mn(IV)

Fumarate/malate/U(VI)/ Mn(IV)/So/PCE/TCE

Lovley (2000) Lovley (2000)

Lovley (2000)

Lovley (2000) Francis et al. (2000)

Finneran et al. (2003)

Sung et al. (2006) Slobodkina et al. (2012)

37◦ C/pH 7.0 65◦ C/pH 6.5–7.0

30◦ C/pH 7.0

25◦ C/pH 7.0 5–40◦ C/ pH 6.0–8.5 4–30◦ C/ pH 6.7–7.1

35◦ C/pH 6.5–7.2 26–70◦ C/ pH 5.5–8.0

for electron donors and acceptors: Acetate (Ac), Butyrate (Buty), Benzoate (Benz), Citrate (Citr), Ethanol (Eth), Formate(For), Glycerol (Glyc), Lactate (Lac), Malate (Mal), Pyruvate (Pyr), Propionate (Prop), Peptone (Pept), Succinate (Suc); Trimetlylamine oxide (TMAO), 2,6-anthraquinone disulfonate (AQDS), Tetrachloroethene (PCE) and trichloroethene (TCE). bND = not determined.

aAbbreviations

H2 /pyr

Geobacter lovleyi

Mn(IV)/O2 /fumarate


Ac/lac/mal/ prop/ pyr/suc/benz

Mn(IV)/Cr(VI)/AQDS/So


Ac/H2

Rhodoferax ferrireducens

Fumarate


Ac

Rock and groundwater samples collected from the Witwatersrand Supergroup at a 3.2-km depth in a South African gold mine Sediments of petroleum-contaminated aquifer at the Defense Fuel Supply Center in Hanahan, SC, USA. Mining-impacted lake sediments Surficial sediments collected from Salt Pond, a coastal pond near Woods Hole, Mass. Subsurface environments, aquifer material from a Department of Energy subsurface study site in Oyster Bay Su-Zi Creek sediment with no reported contamination A terrestrial hot spring on the Kamchatka peninsula

O2 /So etc.

Fumarate/Mn(IV)/AQDS

Freshwater sediments

Fumarate/Co(III)/Se(VI)

Ferribacter limneticum Pantoea agglomerans


Fe(III)/Fe(II), nitrate/nitrite Fe(III)/Fe(II), nitrate/nitrite

Ac/prop/lac

H2 /lac

Glyc/lac/suc

Geothrix fermentans

Aeromonas hydrophila genus Thermus

512 The abandoned ore on the upper layer of tailings was covered with arable soil to the thickness of 40 cm. The samples used for our bacterial enrichments were taken from the soil over the tailings at a depth of 1 m and then stored at −20◦ C. Enrichment and Isolation The liquid culture medium contained (grams per liter of deionized water): NaCl 5.0, MgCl2 •6H2 O 0.2, CaCl2 •2H2 O 0.1, KCl 3.0, KH2 PO4 1.0, NH4 Cl 1.25, Fe(III)-Citrate 6.0. Several supplemental solutions, sterilized by passing through 0.22 μm filters, were added to the sterilized medium. The supplemental solutions that were added included trace mineral solution (1 mL), vitamin solution (1 mL) (Dong and Schnell 2001), ascorbic acid solution (5 g ascorbic acid in 100 mL sterilized oxygen-free deionized water) (2 mL), a solution of Na acetate and NaHCO3 (6.7 g Na acetate and 8.5 g NaHCO3 in 100 mL sterile oxygen-free deionized water) (30 mL). The final concentration of acetate in the culture medium was 23.7 mM. The pH was adjusted to 7.0. Using sterile conditions, soil samples were serially diluted in distilled water and immediately thereafter, 0.1 mL was spread over the medium. All transfers and samplings of the cultures were performed with sterile syringes. The nitrate liquid medium was prepared by replacing the ferric citrate with sodium nitrate. All incubations were cultured in anaerobic tubes or serum bottles capped with thick butyl rubber stoppers in the dark at 35◦ C unless otherwise noted. The medium was extensively bubbled with N2 -CO2 (80 : 20) before inoculations in an anaerobic glove box. Medium constituents were sterilized by autoclaving (121◦ C, 20 min). One of the isolates, strain CW (CCTCC AB 2013114, China Center for Type Culture Collection), was used in the subsequent study.

Wang et al. identified by agarose gel electrophoresis (Figure 1) were used for the determination of 16S rDNA gene sequence (Sangon Biotech CO., Ltd., China). The sequences amplified from this strain were compared with those in the GenBank nucleotide database by using BLAST program packages. Analysis of the 16S rDNA gene sequence data was performed by using the software package MEGA 4.0 after multiple sequence alignment of the data by CLUSTAL X. A phylogenetic tree was constructed using the neighbor-joining methods. Bootstrap analysis based on 1,000 replications was undertaken to test the robustness of the phylogenetic tree. Reduction Tests The performance of strain CW in the reduction of nitrate and Fe(III) was also investigated with three batch tests. In Test 1, 23.5 mM of nitrate was used as electron acceptor. In Test 2, 22.5 mM of ferric citrate, 45 mM of goethite (FeOOH) and 45 mM of hematite (Fe2 O3 ) was used as electron acceptors, respectively. For the study of reduction of ferric citrate, the experiments were divided to four groups: G1 with no cell and no acetate in the medium as Control 1, G2 with killed cells and acetate in the medium as Control 2, G3 with active cells but no acetate in the medium to investigate whether the cells can survive with the citrate, and G4 with both active cells and acetate in the medium. In Test 3, 23.5 mM of nitrate coexisting with 22.5 mM of ferric citrate was used as electron acceptors. 23.7 mM of acetate was served as electron donor and carbon source in all of three tests. All systems were extensively bubbled with

Biochemical Tests and Identification by 16S rDNA Sequence Analysis Strain CW was first cultured anaerobically in the nitrate liquid medium. As it grew to the late logarithmic phase, the mixture solution was centrifuged at 3000 rpm for 10 min. The pellets were suspended in the buffer solution and centrifuged again. Strain identification with gram staining and conventional biochemical tests followed the methods developed by Godkar (1994) and Prescott and Harley (2002), respectively. All tests were conducted in 100 mL glass serum bottles with a medium of 50 mL. Without other note, each test had 10% (v/v) inoculums. The growth of strain CW under different temperature (30–40◦ C) and pH (5.0–8.0) was monitored. Results showed that the optimum temperature and pH were 35◦ C and 7.0, respectively. Confirmation of the taxonomical status of the selected strains was done by molecular methods. DNA from 20 mL of samples was isolated and purified using a UNIQ-10 column bacteria genomic DNA reagent kit (Sangon Biotech Co., Ltd, China). The 16S rDNA gene was amplified by polymerase chain reaction (PCR) using bacterial universal primers 50F (5’-AACACATGCAAGTCGAACG-3’) and 1492R (5’GGTTACCTTGTTACGACTT–3’), and the PCR products

Fig. 1. 16S rRNA gene agarose gel electrophoresis.

Isolation and Characterization of Pseudomonas stutzeri

513

argon before inoculations. Without other note, each test had a 10% (v/v) inoculum and was run in triplicate. Controls consisted of uninoculated medium. All tests were conducted in an anaerobic glove box at 35◦ C except where noted. Gas and liquid samples were collected regularly with sterile syringes. The precipitates formed in the bottles were stored anaerobically at -4◦ C for X-ray diffraction (XRD) analysis. Aseptic and strict anaerobic techniques were used throughout this study.

The pure culture was identified on the basis of morphology and biochemical characters. Only one of the four isolated bacterial showed the ability to reduce both Fe(III) and nitrate. This strain was designated as strain CW and was selected for further investigation. Strain CW was Gram-negative, short rod-shaped bacterium. Cells were around 2.0 μm in length and 0.6 μm in diameter, and lacked flagella as determined by SEM (Figure 2). Comparative analysis of the sequences showed that the strain CW was phylogenetically similar to Pseudomonas sp (AJ387903) and Pseudomonas stutzeri (AB088754) with a sequence homology of 99.0%. Braker et al. (2010) reported that Pseudomonas sp (AJ387903), which they isolated from an anaerobic, trichlorobenzene transforming consortium, could reduce nitrate to nitrogen gas. Pseudomonas stutzeri (AB088754), which could degrade carbazole, was isolated from activated sludge (Shintani et al. 2003). A detailed phylogenetic analysis of the isolate is shown in Figure 3. Another Gamma-proteobacteria capable of iron reduction, such as Shewanella sp (X81623), serve as the outgroup and some Pseudomonas sp. that can reduce Fe(III) (AB095005) or oxidize Fe(II) coupled to nitrate reduction (U26420) also list in the phylogenetic tree (Naganuma et al. 2006; Rossello-Mora et al. 1995; Straub et al. 1996). DNA similarity between strains CW and the reference Pseudomonas putida was 25%, but the Pseudomonas stutzeri showed more than 89% DNA homology. Accordingly, strain CW was affiliated with Pseudomonas stutzeri with an accession number of KC244183, based on the genetic and phenotypic analyses.

Analyses The samples were treated firstly with Na2 CO3 aqueous solution (3.6 mmol/L) and centrifuged at 8000 rpm for 10 min. The pellets was acidified and used to measure the Fe3+/Fe2+ using Ferrozine method (Lovley and Phillips, 1987). The liquid was passed the 0.45-μm membrane and was used to measure nitrate and nitrite using an ion chromatograph (WYIC 6100A, China) equipped with a conductivity detector and a Shodex SI-52 4E analytical column. The mobile phase for the chromatograph was Na2 CO3 aqueous solution (3.6 mmol/L) with a flow rate of 0.8 mL/min. The temperature of the column and conductivity detector cell was 45 ◦ C and 50 ◦ C, respectively. The sample injection volume was 50 μL. N2 concentrations in the headspace were analyzed with a gas chromatograph (TianMei GC-7890T; Shanghai, China) with argon as the carrier gas and equipped with a GDX502 packed column and TCD detector. The temperatures of injector, detector, and column were 120◦ C, 100◦ C, and 70◦ C, respectively. Acetate was quantified by gas chromatography (GC2010, Shimadzu) on a capillary column (RTX-1, 25 m × 0.25 mm) with a flame ionization detector. The column temperature was 110◦ C, and the injector and flame ionization detector temperature was 300◦ C. The morphology of isolate was determined by the Scanning Electron Microscope (Sirion 200 FE-SEM, USA). The mineral characteristic of the precipitates were analyzed using X-ray powder diffraction method (Japan, Rigaku D/max2500pc). Mineral residue from the reduction experiments was smeared on a glass slide in anaerobic glove box, and dried at 40◦ C for X-ray diffraction analysis. The slides were maintained under anoxic atmosphere in the anaerobic bags until it was analyzed.

Results Enrichment, Isolation and Identification of Strain CW After 5 days of incubation, the color of the culture changed from yellow-brown to colorless and the primary enrichment was transferred to fresh basal medium (10% inoculum). After four successive 10% (vol/vol) transfers, the culture of the target bacteria was serially diluted and plated anaerobically on LB solid medium amended with ferric citrate. Visible colonies on the plates ranged from 0.5 to 2 mm in diameter and were white and yellow in color. The isolated distinct colonies on the selective medium were subcultured repeatedly on the same medium for purification.

Nitrate as the Electron Acceptor The nitrate concentration decreased significantly during the initial 24 h after inoculation, resulting in transitory accumulation of nitrite and production of nitrogen gas (Figure 4). After 24 h, nitrite accumulation ceased since nitrate was completely consumed and accumulated nitrite was converted to nitrogen gas. No ammonium was detected in the process. The high conversion rate of nitrate to nitrogen gas (94% based on molar concentration) indicated the high denitrification ability of the strain CW. The other nitrogen might be converted to other gaseous end-products such as NO and/or N2 O or be consumed for the microbial growth. The maximum rates of nitrogen gas production and nitrate reduction were 0.38 mM/h (R2 = 0.99) and 0.14 mM/h (R2 = 0.98), respectively, which were calculated by performing a linear regression on the plots for nitrogen gas production and nitrate consumption during the logarithmic growth phase (8–24 h).

Fe(III) as the Electron Acceptor Figure 5a showed the variation of soluble Fe(II) concentration when ferric citrate was used as electron acceptor. No Fe(II) was generated in the control tests of G1-G3. This indicated that citrate cannot serve as the carbon resource or electron donors for the growth of strain CW. On the contrary, a high

514

Wang et al.

Fig. 2. SEM images of the Pseudomonas stutzeri strain CW (a × 40000, b × 80000).

increment of Fe(II) concentration in the solution of G4 was observed. A maximum 10.88 mM of Fe(II) [equivalent to approximately 49.34% of the initial Fe(III)] was obtained finally. A large number of high density small particles were found in the bottom of serum bottles, which indicated that a part of the Fe(II) produced may have precipitated in the medium. Fe(II) might react with phosphate to generate the vivianite. This was verified by the XRD analysis results (Figure 5b). Since no soluble phosphate was detected, it was speculated that 11.03 mM Fe(II) was precipitated. Based on the initial Fe dosages, the undetected 0.62 mM Fe(II) might be assimilated by the cells and or converted to other state or otherwise like absorbed to the precipitate.

Figure 5c showed the changes over time in Fe(II) concentration in the inoculated medium with natural goethite and natural hematite as well as those in the uninoculated controls. Reduction of the iron of the natural goethite and natural hematite resulted in an obvious color change in the mineral from gold-yellow to Gray-yellowish. Fe(II) concentration in the inoculated medium with natural goethite was higher than that with natural hematite. This may be due to the difference in structure and surface area and a consequent difference in efficiency of microbial utilization of the two minerals (Roden and Zachara 1996). It was worth noting that the specific rate of Fe(II) production sharply increased from 0.003 mM/d to 0.089 mM/d and 0.028 mM/d to 0.128 mM/d after 14 days of

47 Pseudomonas sp (AJ387903) 100

Pseudomonas stutzeri strain CW (KC244183)

79

Proteobacterium WJQ (HM142822)

33 59 41

Pseudomonas stutzeri (AB088754)

Gamma proteobacterium (GU594655) Pseudomonas stutzeri strain ZoBell (U26420) Azotobacter chroococcum (AB681887)

61 24

Pseudomonas alcaligenes (Z76653) Pseudomonas sp. KNA6-5 (AB095005) Pseudomonas nitroreducens (AF494091) Shewanella.putrefaciens (X81623)

0.01

Fig. 3. Phylogenetic tree of strain CW based on 16S rRNA gene sequences. The tree was constructed using the neighbor-joining method. GenBank sequence accession numbers were given in parentheses.

25

15

20

12 NO3--N

9

10

NO2--N N2

6

-

15

5

3

0

0 60

N2 (mM)

NOX -N (mM)

Isolation and Characterization of Pseudomonas stutzeri

515 and earlier in terms of time, and the maximum rates of nitrogen gas production and nitrate reduction were 0.44 mM/h (R2 = 0.99, 0–16 h) and 2.1 mM/h (R2 = 0.91, 0–16 h), respectively, both of which were higher than that in Test 1 as described previously. This indicated that the addition of Fe(III) had promoted the conversion of nitrite to nitrogen gas.

Discussion and Conclusions Characteristics of Strain CW

0

10

20

30

40

50

t (h) Fig. 4. Changes over time in NOx −-N concentration and nitrogen gas in Test 1.

cultivation. Additionally, the specific rate of Fe(II) production (Figure 5c) was orders of magnitude lower than that shown in Figure 5a, which indicates that the release and reduction of Fe(III) from iron oxide minerals which was much slower compared to the soluble Fe(III).

Strain CW was consistent with its classification within the genus Pseudomonas and was related to Pseudomonas stutzeri. Usually Pseudomonas sp can grow with the nitrate as the electron acceptor under anaerobic condition (Braker et al. 2010; Su et al. 2012). As illustrated in Table 1, some of Pseudomonas species were reported to reduce Fe(III) and convert nitrate to nitrite or nitrogen oxides (Balashova and Zavarzin 1979; Naganuma 2006), but there was less information about Pseudomonas sp., which can reduce Fe(III) and transfer nitrate to nitrogen gas as well. Therefore, strain CW isolated from a 12 (a)

10

2+

Fe

8

G1 G2 G3 G4

6 4 2 0

0

40

80

120

160

200

t (h) (b) v v

v

V:vivianite v

v

v

v v v

vv

10 15 20 25 30 35 40 45 50 55 60

2θ (o) (c)

2+

(mM)

1.6

Fe

To study nitrate reduction, Fe(III) reduction, and likely nitrate-dependent Fe(II)-oxidation by strain CW, a liquid medium containing 23.5 mM Na-nitrate, 23.7 mM acetate, and 22.5 mM Fe(III)-citrate was prepared. A same medium without nitrate was also prepared for a control culture of strain CW. Although nitrate and Fe(III) coexisted under anaerobic conditions, nitrate reduction and N2 production preceded Fe(III) reduction as the main respiratory process (Figure 6). Nitrate was almost completely depleted before significant Fe(III) reduction process started. The concentration of Fe(II) increased gradually in both groups (Figure 6a). Compared with the control, it showed a negative effect of nitrate on the Fe(II) production rate in the period of nitrate reduction. After that, the Fe(II) generation rate was pretty similar. The maximum cell numbers in the group with nitrate addition reached 8.1 × 108 cells/mL, which was much more than that in the control culture (2.9 × 108 cells/mL).This was might due to that ferric bio-reduction was over competed by nitrate reduction while nitrate and Fe(III) coexisted under anaerobic conditions. As the nitrate and nitrite was depleted totally (after 48 h), the cell number in the culture with nitrate also declined. Strain CW can grow with acetate as the electron donor. Fe(III) or nitrate reduction was accompanied by an increment in cell number and a loss of acetate (Figure 6a). The acetate consumption in culture with nitrate (ca. 19.8 mM) was much more than no nitrate culture (ca. 3.9 mM), which was consistent with the electronic exchange and the cell growth. After 112 h, the cell number and Fe(III) reduction rate begin to drop, which can be attributed to the shortage of the electron acceptors. On the other hand, in contrast to the nitrate reduction in Test 1 (Figure 4), the accumulation of nitrite was much lower

(mM)

Nitrate and Fe(III) as the Electron Acceptors

Goethite without cell Goethite with cell Hematite without cell Hematite with cell

1.2 0.8 0.4 0.0 0

5

10

15

20

25

t (d) Fig. 5. Variation of Fe(II) concentration respectively using (a) Fe(III)-citrate and (b) the produced minerals in Fe(III)-citrate culture analyzed by XRD, and (c) iron oxides as electron acceptors in Test 2. The experiments for the reduction of ferric citrate were divided to four groups: G1 (no cell and no acetate), G2 (acetate and killed cells), G3 (active cells an no acetate), and G4 (active cells and acetate).

516

Wang et al.

10

With nitrate Without nitrate

8

Fe 2+ (mM)

8

6

6

With nitrate Without nitrate

4 2

With nitrate Without nitrate 0

40

80

120

160

4 2

20

15

10

5

0 200

Acetate concentration (mM)

10

Cell number (× 108 cells/mL)

(a)

0

25

12

12

0

t (h) 24 12

(b) 20

12

NO2--N NO3--N

8

N2

6

8

4

4

2

0 0

10

20

30

40

50

60

N2 (mM)

NOX--N (mM)

10 16

0 70

t (h)

Fig. 6. Changes in (a) Fe(II) concentration and cells number and acetate concentration, and (b) NOx −-N concentration and total N2 production in Test 3, in which nitrate and Fe(III) coexisted in the medium.

skarn-type copper mine tailing improves our understanding of the Pseudomonas sp. Moreover, our observations further confirm the presence of functional microorganisms in older tailings (Diaby et al. 2007; Kock and Schippers 2008; Mendez et al. 2008) and broaden our understanding about the habitats of Fe(III) and nitrate reducers. A few microbes that could reduce nitrate and Fe(III) have been isolated as follows: Pseudomonas (Balashova and Zavarzin 1979; Naganuma 2006), Geobacter sp. (Sung et al. 2006), Bacillus infernus (Lovley 2000), Geothrix fermentans (Lovley 2000), Pantoea agglomerans (Francis et al. 2000), Carboxydocella manganica (Slobodkina et al. 2012), et (Table 1). However, there was less information available about an isolate previously obtained from skarn-type copper tailings. Ore tailings, especially those containing sulfide, are characterized by elevated concentrations of arsenic and heavy metals such as lead, zinc, etc. The microbial communities from these sources are limited in species richness and in ability to use diverse carbon sources compared to microbial communities in uncontaminated soil. Molecular biology techniques were used to study the vertical distribution of biomes in the older tailings pond. A few anaerobic sulfate-, iron-, arsenic- and nitrate-reducing bacteria were found in these special environments (Diaby et al. 2007; Kock and Schippers 2008; Mendez et al. 2008). To our knowledge, it is rarely report about an

isolate, which was obtained from skarn-type copper tailings, capable of reducing both nitrate and Fe(III). Competing Electron Acceptors Theoretically, the degradation of organic matter is performed by the sequential reduction of O2 , nitrate, Mn4 +, Fe(III), SO4 2−, and CO2. Electron acceptor with higher redox potential will be reduced first and will promote the sequential dominance of the related group of microorganisms (Bethke et al. 2011). A competition between nitrate and Fe(III) iron reducing bacteria for electron donors was known (Achtnich et al. 1995; Guo et al. 2010; Obuekwe et al. 1981). As shown in Figure 6, nitrate reduction preceded Fe(III) reduction as the main respiratory process when both of nitrate and Fe(III) were as electron acceptors. Addition of sufficient quantity of acetate as electron donor promoted bioreduction of both Fe(III) and nitrate (Guo et al. 2010). Furthermore, the negative effect of nitrate on Fe(III) reduction (Figure 6a) was consistent with previous studies in pure and mixed cultures of Fe(III)-reducing bacteria (Achtnich et al. 1995; Cooper et al. 2003; Guo et al. 2010). Reduction of 1 mol Fe(III) needs 1 mol electrons while reduction equimolar amount of nitrate needs 5 mol electrons. However, strain CW can reduce nitrate at a maximum rate of 1.14 mM NO3 −-N/h, which was significantly higher than Fe(III) reduction rate of 0.14 mM Fe(III)/h (24–48 h). The negative effect of nitrate on the Fe(II) production rate and total Fe(II) concentration in Figure 6a may be due to the reasons as follows: (i) Nitrate/nitrite, nitrate/N2 and Fe(OH)3 /Fe(II) redox potential were 0.431 V, 0.713 V, and 0.014 V at pH 7.0, respectively (Weber et al. 2006). The bio-thermodynamic principle has been proposed that the process obtaining more energy will happen prior to be reduced in the functional microbial communities (Achtnich et al. 1995; Bethke et al. 2011). Accordingly, when strain CW grew with acetate as the electron donor, the nitrate reduction process, which had the thermodynamic advantages of obtaining more energy, got more electrons and consequently over competed the Fe(III) reduction process. (ii) The retention time of nitrite in the culture containing Fe(II) was shorter than the culture without Fe(II) (Figures 4 and 6b), which indicated that addition of Fe(III) promoted the conversion of nitrite to nitrogen gas. As the intermediate product of nitrate reduction, a part of nitrite might be reduced by reductase related to the bio-produced Fe(II) (Coby and Picardal 2005; Cooper et al. 2003; Obuekwe et al. 1981). What’s more, the ligand, such as citrate, in the culture perhaps promoted the negative effect of nitrate on the Fe(II) production (Kopf et al. 2013). (iii) Nitrate-dependent Fe(II) oxidation (Carlson et al. 2012; Straub et al. 1996), that is the produced Fe(II) was directly bio-oxidized by nitrate. Additionally, Fe(III)-citrate solution tend to form Fe(OH)3 colloids or small granular, which restrain the dissolution and reduction of Fe(III) in neutral pH. Moreover, it is mentioning that the formation of secondary minerals in this study led to the decrease of Fe(II) content monitored and sampling error as well.

Isolation and Characterization of Pseudomonas stutzeri References

Environmental Significance and Bioremediative Potential The soils for strain isolation were collected from the Linchong tailing, a skarn-type copper mine tailings which was closed at the end of 1980. Tailings consisted of large amount of calcite which possessed strong acid neutralizing capacity. Thus, the pH value of its pore water was at 6.8–8.6, which was appropriate for the growth of strain CW. Metal sulfides on the surface of tailings had been weathered and oxidized to form a few of sulfate, iron oxides and hydroxides. Nitrogen compounds, released from ammonia mineral decomposition, lighting or plants fixed, were partly transformed into nitrate by aerobic bacteria. On the other hand, secretion and debris of the plants in the tailings provided organic matters. These iron hydroxides or oxides, nitrate and organic matters from the surface layer penetrated into the deeper anaerobic regions with rain water, which provided suitable growing conditions for anaerobic nitrate-, iron- and sulfate-reducers (Diaby et al. 2007). Strain CW was isolated from the above environment, in which it can reduce Fe(III) and nitrate accompanied with the dissimilation of acetate at neutral or weakly alkaline conditions. The isolate can use acetate (or other organic matters) as carbon source to convert nitrate or nitrite into nitrogen gas (Equation 1). As Fe(III) oxides or hydroxides exist, it can bioreduce Fe(III) in the mineral crystals to free Fe(II) (Equation 2). Moreover, as described in Equation 3, the bio-produced Fe(II) can be combined with phosphate in the solution to form Fe3 (PO4 )2 (Ksp = 1.0 × 10−36) precipitate, which is readily removed from water. mi cr obi al

NO3− /NO2− + C H3 COO− −→ N2 + CO2 − mi cr obi al

Fe + C H3 COO −→ Fe + CO2 Fe2+ + PO43+ → Fe3 (PO4 )2 ↓ 3+

2+

517

(1) (2) (3)

The chemical composition of vivianite is Fe3 (PO4 )2 ·8H2 O. The theoretical molar ratio of Fe2+ produced/PO4 3− precipitated for Fe3 (PO4 )2 ·8H2 O is 1.5. Based on this, Fe(II) produced from iron ore was used for the removal of phosphate from reject water by the chemical precipitation (Guo et al. 2010). It suggested that strain CW can be used to remove nitrate and phosphate in the wastewater simply by casting iron oxides or ore. Microorganisms with a function similar to that of strain CW have been proposed for use for the treatment of nitrogen and phosphorus simultaneously (Guo et al. 2010).

Funding This study was financially supported by the National Natural Science Foundation of China (41130206, 40902019), National Basic Research Program of China (2011CB411904) and Ph.D. Programs Foundation of Ministry of Education of China (20110111110003).

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