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Fe(III) reduction by Halomonas sp. TBZ9 and Maribobacter sp. TBZ23, isolated from Urmia Lake in Iran.

INTRODUCTION

Iron is an abundant element in the environment and therefore, iron cycling in different environmental processes has been widely studied. Environmental iron mobility and stability is partly under the control of its oxidation state [1]. Microbial reduction of Fe(III) to Fe(II) plays an important role in the iron cycle of aquatic ecosystems and affects the exchange of nutrients and trace elements between sediments and overlaying water body. Fe(III) reduction mediates phosphate and trace elements fluctuation, because these compounds are held by adsorption onto Fe(III) particles in the sediments and are released when Fe(III) is reduced [2, 3].

Dissimilatory reduction of ferric iron oxides plays an important role in mineralization of organic materials in anoxic soils and sediments [4, 5] and also makes up an important fraction of anaerobic carbon metabolism in special marine and freshwater environments. The extent of relationship between iron reduction and mineralization of organic materials depends partly on rates of organic material and ferric iron supply. This type of iron reduction plays an important key role in geochemistry of moderate- temperature (8-42[degrees]C) and hot (42121[degrees]C) environments [6, 7].

When iron acts as an electron acceptor in metabolic pathways leading to mineralization of organic matter, these pathways are more thermodynamically favorable than the mineralization of organic matter with methane production or sulfate reduction as the final step [2]. As well as importance in carbon cycling and metabolism, Fe(III)-reducing bacteria are notable environmental components in bioremediation of organic and metal contaminants at moderate temperature in freshwater and marine sediments and submerged soils [7, 8, 9].

Plant roots may be enhancing factors in Fe cycling, not only through the leakage of [O.sub.2] as an important mechanism of regenerating reactive Fe oxides in anoxic habitats, but also by excreting easily-available carbon substrates such as alcohols, sugars, and organic acids, which stimulate Fe(III) reduction as a carbon source and simultaneously, increase Fe(III) availability via acting as organic chelators maintaining Fe(III) in a soluble form and allowing a contact with microbial communities in surrounding anoxic micro- zones [10].

Some bacteria and archaea can grow anaerobically using Fe(III) as the sole electron acceptor. Fe(III) is an important electron acceptor in some environments such as minerotrophic fens, hydrothermal systems, and deep subsurface and may have also been an important electron acceptor on the early developmental stages of the Earth and other planets [11-14]. Numerous bacterial genera have been identified which are able to reduce ferric iron, such as Pseudomonas, Bacillus, Bacteroides, Desulfovibrio, Sulfolobus, Thiobacillus, Shewanella, Desulfomonas, Desulforomusa, Geobacter, and Pelobacter. Some fungi are also able to reduce Fe(III) to Fe(II) [4, 15, 16].

Numerous studies have reported microbial reduction of ferric Iron. David E. Cummings et al. (2000) studied microbial Fe(III) reduction in anoxic mining-impacted lake sediments. They concluded that the mean density of Fe-reducing bacteria was 8.3 x [10.sup.5] cells per gram of sediment (dry weight). They also isolated two new strains of dissimilatory Fe-reducing bacteria and supported the hypothesis that dissimilatory iron reduction is an important biogeochemical process in the environment [3]. Kristina L. Straub and coworkers (2001) isolated two strictly anaerobic dissimilatory ferric iron-reducing bacteria belonging to family Geobacteriaceae. In this study, electron donors such as hydrogen, acetate, pyruvate, formate, succinate, fumarate, and ethanol were used. The results showed that the closest known relative to these ferric iron-reducing isolates is Geobacter sulfurreducens with approximately 93% sequence identity. Phylogenetic and physiological data proved that these isolates were described as Geobacter bremensis and Geobacter pelophilus [4]. The microbial reduction of structural [Fe.sup.3+] in nontronite by thermophilic bacterium Thermoanaerobacter ethanolicus and its role in promoting the smectite to illite reaction was investigated by Gengxing Zhang et al (2009). Lactate was the sole electron donor and structural [Fe.sup.3+] in nontronite acted as the sole electron acceptor in their experiments. Their results showed that termophilic iron reducing bacteria have an important role in promoting the smectite to illite reaction under conditions common in sedimentary basins [17]. In another study, Jing Zhang and colleagues (2013) investigated the ability of thermophilic Methanothermobacter thermautotrophicus to reduce structural Fe(III) in iron-rich and iron-poor smectites. They came to the point that Fe(III) bioreduction and methanogenesis were mutually beneficial. The probable mechanism was that Fe(III) bioreduction lowered the reduction potential of the system and methanogenesis became favorable and in turn, methanogenesis stimulated the growth of methanogens, which enhanced Fe(III) bioreduction [18].

Because of the important role of biological iron reduction in iron and organic material cycling [4, 6, 7], we were interested in evaluating Fe(III) reduction potential of bacteria isolated from ecosystems of our own homeland. The objective of this study was to survey the ability of two bacterial isolates, namely TBZ9 and TBZ23, isolated from Urmia Lake in Iran [19], to reduce Fe(III) to Fe(II) by the aid of differential pulse polarography. Both isolates were able to reduce ferric iron, but in comparison with the other isolate, the isolate TBZ9 showed higher bioreduction potential.

MATERIALS AND METHODS

Culture Media:

Bacterial isolates were first sub-cultured from stock cultures on Marine Agar Medium containing (per liter): 5.0 g peptone, 1.0 g yeast extract, 0.1 g ferric citrate, 19.45 g NaCl, 8.8 g magnesium chloride, 3.24 g sodium sulfate, 1.8 g calcium chloride, 0.55 g potassium chloride, 0.16 g sodium bicarbonate, 0.08 g potassium bromide, 34.0 mg strontium chloride, 22.0 mg boric acid, 4.0 mg sodium silicate, 2.4 mg sodium fluoride, 1.6 mg ammonium nitrate, 8.0 mg disodium phosphate, 15.0 g agar and pH 7.0. After incubation at 30[degrees]C for 3 days, the isolates were transferred to Marine Broth Medium, containing (per liter): 5.0 g peptone, 1.0 g yeast extract, 0.1 g ferric citrate, 19.45 g sodium chloride, 5.9 g magnesium chloride, 3.24 g magnesium sulfate, 1.8 g calcium chloride, 0.55 g potassium chloride, 0.16 g sodium bicarbonate, 0.08 g potassium bromide, 34.0 mg strontium chloride, 22.0 mg boric acid, 4.0 mg sodium silicate, 2.4 mg sodium fluoride, 1.6 mg ammonium nitrate, 8.0 mg disodium phosphate and pH 7.0. [20]. After achieving proper turbidity within 4 days, 500 pL of standard 0.5 McFarland turbidity was used for Fe(III) reduction experiment in a defined culture medium containing (per liter): sodium hydrogen carbonate, 2.5 g; calcium chloride, 0.08 g; ammonium chloride, 1.5 g; magnesium chloride, 0.2 g; and sodium chloride, 70.0 g and 5.0 g for TBZ23 and TBZ9, respectively. 2 mM ferric citrate was used as Fe(III) source with 50 mM HEPES buffer (Sigma-Aldrich, USA) and pH was adjusted on 7.0. This culture medium was purged with nitrogen gas (99.9999%) before autoclaving to remove the oxygen and after autoclaving at 121[degrees]C and 1.5 psi for 20 minutes, 5-ml aliquots were quickly dispensed into capped test tubes and supplemented with 50 mM sterile HEPES buffer [21, 22]. The cultures were incubated in anaerobic jar at 30[degrees]C for 10 days. The gaseous atmosphere in the jar was altered using gas packs to minimize the effect of oxygen on the reductive conditions.

Description of Bacterial Isolates:

Isolate TBZ23:

Cells were short rods, oxidase- and catalase-positive. Gram-staining reaction was negative. Colonies produced after 72 h at 32[degrees]C on Marine Agar medium were convex, smooth, and orange in color. It was incapable of growing in the absence of NaCl. It did not produce acid from D- glucose, D-mannose, D-fructose, maltose, D-mannitol, rhamnose, sucrose, D-trehalose, D-galactose, cellobiose, D- raffinose, xylose, lactose, ribose, arabinose, D-melezitose, D-salicin and aesculin. Indole and [H.sub.2]S production were negative. Urease activity was positive. Phenylalanine-deaminase was not produced. Tween 20 was hydrolyzed, but tyrosine, starch, casein, gelatin and Tween 80 were not hydrolyzed. Nitrate was not reduced to nitrite. Argenine, lysine, and ornithine were not deaminated. According to the 16S rRNA gene sequence, this isolate belongs to genus Marinobacter [19].

Isolate TBZ9:

Cells were Gram-negative rods with light cream colonies. Colonies were convex and smooth. Oxidase test was negative. It was incapable of growing in the absence of NaCl and did not produce acid from D-glucose, Dmannose, D-fructose, maltose, D-mannitol, rhamnose, sucrose, D-trehalose, D-galactose, cellobiose, Draffinose, xylose, lactose, ribose, arabinose, D-melezitose, D-salicin and aesculin. Urease activity was positive. Indole and [H.sub.2]S production were negative, and phenylalanine deaminase production was not observed. Hydrolysis of Z-tyrosine was negative, but Tween 20 and Tween 80 were hydrolyzed. Starch, casein, and gelatin were not hydrolyzed. Nitrate was reduced to nitrite. 16S rRNA gene sequencing showed that this isolate is a member of genus Halomonas [19].

Evaluation of Ferric Iron Reduction:

Differential pulse polarography (DPP) with Computrace VA 757 (Metrohm, Switzerland) polarograph equipped with dropping mercury electrode as working electrode, Pt rod as auxiliary electrode, and Ag/AgCl reference electrode was exploited to evaluate iron reduction capability of bacterial isolates. After 10 days of incubation, the samples were transferred to titration vessel and after 180 seconds of purging with nitrogen (99.9999%) to remove the residual oxygen, analysis was done with 50-mV pulse amplitude, pulse time of 40 ms, scanning pace of 10mV/s, drop lifetime of 1 second, and potential scanning between -0.1 V to -0.8 V. Standard addition was used to identify peaks related to different forms of Fe. 100, 200, and 300[micro]L aliquots of standard 50 mM ferric citrate (Merck, Germany) solution were added to each sample and differential pulse polarography experiment was done for them exactly with the conditions mentioned above. The data gathered this way was plotted in Microsoft Excel 2007 software. All of the experiments were done at least in triplicates [23, 24].

RESULRS AND DISCUSSION

Evaluation of isolates TBZ9 and TBZ23 capability to reduce ferric iron was accomplished via differential pulse polarography method. The concentrations of ferric, ferrous and metallic iron were evaluated in treated and untreated samples. In differential pulse polarogram of control sample with 2 mM ferric iron (in the form of ferric citrate), two peaks appeared respectively at -0.34 V and -0.51 V in comparison with reference electrode. The intensity of second peak is two folds of the first; therefore, these peaks can be related to the reductive reactions below on the surface of mercury electrode (equations 1 and 2):

First Peak (-0.34 V/ref): [Fe.sup.3+] + [e.sup.-] [right arrow] [Fe.sup.2+] (1)

Second Peak (-0.51 V/ref): [Fe.sup.2+] + 2[e.sup.-] [right arrow] Fe (2)

Differential pulse polarograms of control (untreated) samples are shown in Fig. 1.

In the sample treated with TBZ9 for 10 days, two peaks were observed again at the same potentials, but the intensity of peaks was much lower than control (untreated) samples, showing reduction of ferric iron to ferrous iron by isolate TBZ9. If TBZ9 only reduced Fe(III) to Fe(II), it had been necessary for the intensity of the second peak, which is related to reduction of Fe(II) to metallic Fe, to be same as the relevant peak of untreated sample. However, in differential pulse polarograms of TBZ9-treated samples, again the intensity of the second peak is twice the intensity of the first peak, showing that TBZ9 reduces Fe(III) to metallic Fe. Therefore, isolate TBZ9 shows a high potential of reducing Fe(III) to metallic Fe. The results of iron reduction by isolate TBZ9 are summarized in Fig. 2.

Evaluation of iron reduction by isolate TBZ23 showed slight reduction rate. In the case of this isolate, it is also interpreted that reductive reaction progresses until production of metallic Fe. Fig. 3 summarizes iron

Based on extrapolation of peaks of standard addition diagrams at -0.34 V and - 0.51 V, it seems that TBZ23 reduces almost 14.11% of available Fe(III) to Fe(II) and this reduction rate is about 72.12% for TBZ9 during 10 days of incubation. The rate of reducing Fe(II) to metallic Fe is seemingly 21.14% for TBZ23 and 64.49% for TBZ9 during 10 days of incubation. The extrapolation results for control, TBZ9- and TBZ23-treated samples are shown in Fig. 5-7 respectively.

In some studies, biological iron reduction is assessed using complexometric methods [12, 25]. It is while in this study, differential pulse polarography technique utilized for measuring different reduced forms of iron. In comparison with complexometry, differential pulse polarography is a technique providing higher accuracy and sensitivity and can produce more precise results [21, 22]. As a result, the iron reduction potential of both isolates TBZ9 and TBZ23 can be more reliably stated.

It is also notable that both TBZ9 and TBZ23 play the role of electron transport intermediates. In other words, iron reduction is dependent on simultaneous oxidation of another compound in the culture medium. Under experimental conditions, it seems that this compound is the citrate ion available in the culture medium. This is clear that the limited concentration of oxidizable compound in the culture medium affects the ability of bacteria to reduce Fe(III) completely.

Conclusion:

Analysis of different reduced forms of iron was done by the aid of differential pulse polarography technique and the results show that both isolates are able to reduce ferric iron until production of metallic Fe. Two peaks were observed at -0.34 V and -0.51 V, which were respectively related to reduction of Fe(III) to Fe(II), and reduction of Fe(II) to metallic Fe. The intensity of these two peaks shows that almost 14.11% of available Fe(III) was reduced to Fe(II) by isolate TBZ23, while this reduction rate is about 72.12% for TBZ9 during 10 days of incubation. The rate of reducing Fe(II) to metallic Fe is seemingly 21.14% for TBZ23 and 64.79% for TBZ9 during 10 days of incubation.

Based on our results, it is concluded that both bacterial isolates are able to reduce Fe(III) to Fe(II), and Fe(II) to Fe, but the reduction rate is higher for TBZ9. Therefore, in comparison with isolate TBZ23,

isolate TBZ9 is an environmental bacterium with higher potential to reduce ferric iron to ferrous iron and also to reduce ferrous iron to metallic iron. Consequently, this isolate can play an important role in environmental cycling of iron, carbon, and other elements.

ARTICLE INFO

Article history:

Received 11 October 2014

Received in revised form 21 November 2014

Accepted 25 December 2014

Available online 16 January 2015

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(1,2) Youssof Sefidi Heris, (1) Nader Hajizadeh, (1,3) Sepideh Zununi Vahed, (4) Javad Vallipour, (5) Mohammad Amin Hejazi, (4) Sayyed Mahdi Golabi, (4) Karim Asadpour-Zeynali, (1) Mohammad Saeid Hejazi

(1) Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz 51664, Iran.

(2) Department of Microbiology, Zanjan Branch, Islamic Azad University, Zanjan, Iran.

(3) Department of Medical Biotechnology, School of Advanced Biomedical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran.

(4) Department of Analytical Chemistry, Faculty of Chemistry, University of Tabriz, Tabriz, Iran.

(5) Branch for the Northwest and West Region, Agriculture Biotechnology Research Institute of Iran (ABRII), Tabriz, Iran.

Corresponding Author: Nader Hajizadeh, Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz 51664, Iran.

Tel: +98 (41)34786646 Fax: +98(41)35531009, E-mail: Hajizadeh.nader@yahoo.com
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Author:Heris, Youssof Sefidi; Hajizadeh, Nader; Vahed, Sepideh Zununi; Vallipour, Javad; Hejazi, Mohammad A
Publication:Advances in Environmental Biology
Article Type:Report
Geographic Code:7IRAN
Date:Dec 1, 2014
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