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Sulfur-driven iron reduction coupled to anaerobic ammonium oxidation Peng Bao, and Guo-Xiang Li Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 30 May 2017 Downloaded from http://pubs.acs.org on May 31, 2017

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Environmental Science & Technology

TOC 426x249mm (300 x 300 DPI)

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Figure 1. Maximum-likelihood taxonomic diversity of HJ-4 consortium, and affiliation of closely related species. Bootstrap values based on 1,000 replicates (over 50%) are indicated at each node. Phylotypes identified in the consortium are in color with OTU numbers. The OTUs (green text) were Anaerospora hongkongensis, the related species are shown in green box; the OTUs (violet text) were unclassified Comamonadaceae phylotypes, the related species are shown in violet box. The photo shown morphology of Anaerospora hongkongensis (OTU_1, 2, 5) (a) and Comamonadaceae (OTU_3, 4) (b). 300x142mm (300 x 300 DPI)


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Figure 2. HCl-extractable Fe2+ production from ferrihydrite (a), and ammonium oxidation (b) in incubations of HJ-4 consortium under the condition of with/without ammonium, ferrihydrite, or sulfide, respectively. 450x181mm (300 x 300 DPI)



Figure 3. HCl-extractable Fe2+ production from ferrihydrite (a) coupling with ammonium oxidation (b) in incubations of HJ-4 consortium driven by different sulfur species. Medium with sulfide, but without HJ-4 consortium inoculation was as a control. 233x316mm (300 x 300 DPI)


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Figure 4. Coupling transformation of sulfur, iron and ammonium by HJ-4 consortium. (a), 29N2 (filled squares) and 30N2 (open squares) production from 15NH4+ (control: filled triangles; HJ-4 treatment: open triangles) by HJ-4 consortium. (b), Nitrate (filled symbols) and nitrite (open symbols) production and consumption in the absence (diamond) and presence (circle) of HJ-4. In (c) and (d), sulfide concentration is indicated in orange lines. Ferrous are indicated in the column. Symbols in all plots represent averages of three individual experiments. Error bars represent standard deviations of three biological replicates. 478x302mm (300 x 300 DPI)



Figure 5. Nitrite/nitrate dependent iron oxidation of HJ-4 consortium under anaerobic condition. The main products are nitrate (b) and ammonium (c) and nitrite (d). 456x303mm (300 x 300 DPI)


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Figure 6. Conceptual model of sulfur-driven iron reduction coupled to anaerobic ammonium oxidation based on the relative abundance of representative abundant OTUs and known physiologies of Anaerospora hongkongesis and Comamonadaceae. Yellow cycle represents sulfur redox cycling driven by Anaerospora hongkongesis. Green cycle represents iron redox cycling. Blue cycle represents an ammonium oxidation pathway via simultaneous nitrification and denitrification driven by Comamonadaceae. 416x217mm (300 x 300 DPI)



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Sulfur-driven iron reduction coupled to anaerobic ammonium oxidation

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Peng Bao1, 2, * and Guo-Xiang Li1, 2, 3

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Academy of Sciences, Xiamen, 361021, P. R. China;

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2

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Environment Observation and Research Station, Chinese Academy of Sciences,

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Ningbo, 315800, China;

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3

Key Lab of Urban Environment and Health, Institute of Urban Environment, Chinese

Key Lab of Urban Environmental Processes and Pollution Control, Ningbo Urban

University of Chinese Academy of Sciences, Beijing, China;

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Address correspondence to Professor Peng Bao, Institute of Urban Environment,

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Chinese Academy of Sciences, Xiamen, 361021, P. R. China. E-mail: [email protected]

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Phone: +86-574-86085998; Fax: +86-574-86784810

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ABSTRACT

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A new biogeochemical pathway has been suggested to be present in terrestrial

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ecosystems, linking the nitrogen and iron cycles via ferric iron reduction coupled to

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anaerobic ammonium oxidation. However, the underlying microbiological process has

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not been demonstrated to date. Here we report a stable consortium, HJ-4, composed of

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Anaerospora hongkongensis (85%) and facultative anaerobe, Comamonadaceae

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(15%), which can process ferrihydrite reduction coupled to anaerobic ammonium

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oxidation driven by sulfur redox cycling. In this process, A. hongkongensis reduces

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elemental sulfur, sulfite, and polysulfides to sulfide, which fuels ferrihydrite reduction.

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Sulfide, elemental sulfur, sulfite, and polysulfides serve as electron shuttles,

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completing the sulfur cycle between A. hongkongensis and ferrihydrite. In addition,

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Comamonadaceae shows ammonium oxidation potential under aerobic conditions

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with nitrite as the main product. We inferred that Comamonadaceae mediates

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simultaneous nitrification–denitrification coupled to iron redox cycling through

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nitrate/nitrite-dependent ferrous oxidation under anaerobic conditions. Hence, we

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discovered a novel pathway for ferric iron reduction coupled to ammonium oxidation,

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highlighting the key role of electron shuttles and nitrate/nitrite-dependent ferrous

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oxidation in this process. The biogeochemical cycling of sulfur, iron, and nitrogen

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could be coupled in aquatic and terrestrial ecosystems.

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INTRODUCTION Anaerobic ammonium (NH4+) oxidation can be coupled to the reduction of ferric

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iron [Fe (III)] to produce N2, NO3−, or NO2−

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microorganisms in many environments, including wetland soils 3-5, upland soils 6, and

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continuous flow membrane reactors 7. Environments rich in poorly crystalline Fe

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minerals such as ferrihydrite have the potential to support anaerobic ammonium

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oxidation coupled to ferric iron reduction (AAOFe). This implies that AAOFe plays

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an important role in nitrogen loss, except in three known microbial processes,

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denitrification, codenitrification, and anammox, which involve nitrogen loss from

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terrestrial ecosystems via the generation of nitrous oxide (N2O) or N2. In soils rich in

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poorly crystalline Fe minerals, the generation of N2 (equation 1) by AAOFe is

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energetically more favorable than that of NO2− (equation 2) or NO3− (equation 3); it is

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also favorable over a wider range of conditions 5, 6.

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3Fe(OH )3 + 5H + + NH 4 + → 3Fe 2+ + 9 H 2O + 0.5 N 2

(−245 kJ mol−1)

(1)

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6 Fe(OH )3 + 10 H + + NH 4 + → 6 Fe2+ + 16 H 2O + NO2 −

(−164 kJ mol−1)

(2)

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8Fe(OH )3 + 14 H + + NH 4 + → 8Fe 2+ + 21H 2O + NO3−

(−207 kJ mol−1)

(3)

1-4

. This process can be mediated by

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Two possible mechanisms exist for AAOFe: (1) surface Fe reduction coupled to

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NH4+ oxidation, or (2) use of O2 liberated from Fe oxides for intra-aerobic NH4+

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oxidation coupled to ferric iron reduction

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whether the AAOFe process overlaps with denitrification, co-denitrification, or

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anammox. Hence, requirement for information about the microorganism responsible

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for AAOFe process is gradually increased.

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. We have not completely understood


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We realized that an electron transfer mediator is crucial for AAOFe because iron

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reduction during this pathway is an extracellular electron transfer process. Various

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sulfur species are widespread but often neglected electron transfer mediators for iron

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reduction

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sulfide to iron, with little or no precipitation of black iron sulfides. Instead, ferrous

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iron and oxidized sulfur species are produced, and the latter can again serve as an

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electron acceptor. Further, oxidized products such as elemental sulfur or sulfite and/or

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polysulfides can complete iron reduction mediated by the sulfur redox cycle.

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Simultaneous nitrification–denitrification (SND) that converts ammonium to N2 via a

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nitrite pathway

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involved in biotic and/or abiotic nitrate/nitrite-dependent ferrous oxidation at the

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meeting point of ammonium oxidation and iron reduction.

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and may support the AAOFe process. Electrons are transferred from

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may occur during the AAOFe process, while nitrite may be

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In view that there are ubiquitous sulfur reduction process occur in AAOFe

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environment, including wetland soils, upland soils and continuous flow membrane

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reactors

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ammonium oxidation process may be widely distributed in aquatic and terrestrial

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ecosystems. Therefore, we concluded that the study of microorganisms involved in

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this coupling process is feasible and significant.

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, we proposed that sulfur-driven iron reduction coupled to the anaerobic

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EXPERIMENTAL SECTION

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Enrichment of AAOFe consortium



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Paddy soil sediments (0–20 cm depth) were collected from the upper section of a

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paddy field in the Guangxi Dahuanjiang region (24°53ʹ52ʺ N, 108°17ʹ43ʺ E) in the

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Southwest of China for enrichment of the cultures involved in AAOFe. These soil

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samples were immediately transferred into a plastic bottle, filled with water to

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eliminate any air, and tightly sealed. The samples were then air-dried, ground with a

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mortar, and crushed to pass through a 2.0-mm sieve for chemical analyses. Soil

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organic carbon (19.81 g kg−1) was determined using potassium dichromate oxidation

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titration, and soil total Fe (31.9 g kg−1) was determined by ICP-OES. Plant available

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sulfur (29.1 mg kg−1) was determined by turbidimetry. The pH value (3.99) of soil

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slurry was measured using a pH meter. The concentrations of other elements in the

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soil slurry, determined by ICP-OES, were as follows: Zn, 2473.3 mg kg−1; Pb, 1053.1

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mg kg−1; As, 2742.9 mg kg−1; and Cu, 147.7 mg kg−1.

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For enrichment, approximately 0.5 g of paddy soil was transferred to 100 ml of

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double-distilled water. After shaking at 100 rpm for 2 min, 5 ml of the suspension was

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inoculated into 100 ml of anaerobic freshwater medium

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Ferrihydrite (1.6 mM) and ammonium (15NH4Cl 99.08%, 1.0 mM) were then added,

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and the medium was incubated under static conditions at 30°C in dark for 1 week.

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The basal anaerobic freshwater medium (pH 7.0) contained the following (g l−1):

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NaCl (1.0), KH2PO4 (0.2), MgCl·6H2O (0.4), CaCl2·2H2O (0.1), and KCl (0.5). The

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trace elemental mixture contained the following (g l−1): distilled water (987 ml), 25%

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HCl (12.5 ml, 100 mM), FeSO4·7H2O (2100 mg, 7.5 mM), H3BO3 (30 mg, 0.5 mM),

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MnCl2·4H2O (100 mg, 0.5 mM), CoCl2·6H2O (190 mg, 0.8 mM), NiCl·6H2O (24 mg,


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flushed with He gas.

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0.1 mM), CuCl2·2H2O (2 mg, 0.01 mM), ZnSO4·7H2O (144 mg, 0.5 mM), and

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Na2MoO4·2H2O (36 mg, 0.15 mM). Selenite–tungstate solution contained the

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following (l−1): NaOH (0.4 g), Na2SeO3·5H2O (6 mg), and Na2WO4·2H2O (8 mg).

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Bicarbonate solution contained NaHCO3 (84 g l−1, 30 ml). In total, 100 ml of vitamin

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mixture was prepared in sodium phosphate buffer (10 mM, pH 7.1) using the

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following vitamins: 4-aminobenzoic acid (4 mg), D-biotin (1 mg), nicotinic acid (10

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mg), calcium D-pantothenate (5 mg), and pyridoxine dihydrochloride (15 mg).

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Thiamine solution was prepared by dissolving 10 mg thiamine chloride

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dihydrochloride in 100 ml sodium phosphate buffer (25 mM, pH 3.4). Vitamin B12

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solution was prepared by dissolving 5 mg cyanocobalamin in 100 ml sodium

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phosphate buffer (25 mM, pH 3.4). Other solutions included sulfide solution (0.48 g

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in 100 ml, 0.2 M) and yeast extract (0.5 g in 100 ml). The above mentioned stock

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solutions or aliquots were aseptically added to the basal medium in the following

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quantities (l−1): trace element solution (1.0 ml), selenite–tungstate solution (0.1 ml),

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bicarbonate solution (10.0 ml), vitamin mixture (1.0 ml), thiamine solution (1.0 ml),

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vitamin B12 solution (1.0 ml), sulfide solution (1.0 ml), and yeast extract (5.0 ml). A

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total of 0.5 ml of positive culture, in which iron reduction coupled to anaerobic

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ammonium oxidation occurred with the aid of sulfide, was sub–inoculated into 5.0 ml

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of freshwater agar medium in Hungate tubes. Positive samples were continuously

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subcultured using the anaerobic freshwater medium to form a stable AAOFe

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consortium, HJ-4. It was enriched after sub-cultivation for five times, and was

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periodically subcultivated during a four-year period.



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Experimental design This study was performed using a series of experiments that coupled transformations of sulfur, iron and ammonium by HJ-4 including the following:

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(I) 0.12 mM sulfide + 1.6 mM ferrihydrite + 1.0 mM 15NH4Cl

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(II) HJ-4 + 0.12 mM sulfide + 1.6 mM ferrihydrite + 1.0 mM 15NH4Cl.

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Samples were collected on days 0, 3, 7, 11, 17, 23, 29, and 34.

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Anaerobic nitrate/nitrite-dependent ferrous oxidation by HJ-4 was studied using

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two sets of experiments conducted after 34 days of culture:

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(I) 0.12 mM sulfide + 1.6 mM ferrihydrite + 1.0 mM 15NH4Cl

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(II) HJ-4 + 0.12 mM sulfide + 1.6 mM ferrihydrite + 1.0 mM 15NH4Cl

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Further, 0.25 mM NaNO3 was added to both the treatment groups, and sampling

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was performed at days 0, 3, 7, 11, and 15. Experiments were performed anaerobically

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in 100-ml serum bottles in the dark at 30oC. Each batch of experiments was

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performed in triplicates, and inoculation was performed with 1.0 ml of HJ-4 in the

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exponential phase, if necessary. For experiments performed under aerobic conditions,

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1.0 ml of HJ-4 in the exponential phase was inoculated in a 100-ml flask containing

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freshwater medium with 10 mM acetate as an additional carbon source. Samples were

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incubated in the dark at 30°C.

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Analytical methods for sampling

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(i) Cell growth. Cell growth was measured using a microplate reader (Infinite

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200PRO; TECAN). Samples were collected on days 0, 3, 7, 11, 17, 23, 29, and 34 and

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analyzed for cell growth (OD at 600 nm).


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(ii) Electron microscopy. The morphology of the consortium HJ-4 was examined

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using a field emission scanning electron microscope (FESEM) (Hitachi S-4800,

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Hitachi, Ltd, Tokyo, Japan). Cells and iron material from the cultures were collected

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by centrifugation, fixed with 2.5% glutaraldehyde solution (pH 7.5), critical

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point-dried using an ethanol series and mounted on aluminium stumps. The specimens

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were coated with gold in a sputter coater (EMS150 T, ES, Quorum, UK) prior to

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microscopic examination. The material was examined immediately at an accelerating

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voltage of 15 KV.

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(iii) Genomic DNA extraction and Illumina sequencing. Approximately 100 ml of

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HJ-4 culture was used for genomic DNA extraction using the FastDNA™ SPIN Kit

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for microorganisms (Sangon Biotech, China), and the extraction was performed

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according to the protocols provided by the manufacturer. Microbial sequencing was

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performed using the Illumina HiSeq platform at Novogene Biotechnology Company

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(Beijing, China). In brief, the V4 region of the 16S bacterial rRNA gene was

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amplified

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(5ʹ-GTGCCAGCMGCCGCGGTAA-3ʹ)

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(5ʹ-GGACTACHVGGGTWTCTAAT-3ʹ) to generate an amplicon size of 291 bp. A

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paired-end library with an insert size of 500 bp was sequenced using an Illumina

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HiSeq2500 by PE125 mode. Illumina PCR adapter reads and low-quality reads

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(containing ambiguous bases or an average sequencing quality score of

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