Biogeosciences, 12, 769–779, 2015 www.biogeosciences.net/12/769/2015/ doi:10.5194/bg-12-769-2015 © Author(s) 2015. CC Attribution 3.0 License. Characterization of incubation experiments and development of an enrichment culture capable of ammonium oxidation under iron-reducing conditions S. Huang and P. R. Jaffé Department of Civil and Environmental Engineering, Princeton University, Princeton, NJ 08544, USA Correspondence to: P. R. Jaffé ([email protected]) Received: 14 July 2014 – Published in Biogeosciences Discuss.: 14 August 2014 Revised: 28 November 2014 – Accepted: 1 December 2014 – Published: 10 February 2015 C Abstract. Incubation experiments were conducted using soil bacteria/archaea (AOB/AOA). A novel anaerobic NH4 ox- samples from a forested riparian wetland where we have pre- idation process coupled to iron reduction was first noted in viously observed anaerobic ammonium oxidation coupled to a forested riparian wetland in New Jersey (Clement et al., C iron reduction. Production of both nitrite and ferrous iron was 2005). In this reaction, NH4 is the electron donor, which is measured repeatedly during incubations when the soil slurry − oxidized to nitrite (NO2 ), and ferric iron – Fe(III) – is the was supplied with either ferrihydrite or goethite and ammo- electron acceptor, which is reduced to ferrous iron – Fe(II). nium chloride. Significant changes in the microbial commu- The stoichiometry and change in free energy when ferrihy- nity were observed after 180 days of incubation as well as drite is the Fe(III) source is: in a continuous flow membrane reactor, using 16S rRNA • C C C C ! 2C gene PCR-denaturing gradient gel electrophoresis, 454 py- 3Fe2O3 0:5H2O 10H NH4 6Fe (1) rosequencing, and real-time quantitative PCR analysis. We C C − ≤ − −1 believe that one of the dominant microbial species in our 8:5H2O NO2 1Gr 145:08 kJ mol ; Supplement 1:1 system (an uncultured Acidimicrobiaceae bacterium A6), be- C − longing to the Acidimicrobiaceae family, whose closest cul- No proven pathway for the oxidation of NH4 to NO2 in tivated relative is Ferrimicrobium acidiphilum (with 92 % anaerobic environments has been described in the literature 15 C identity) and Acidimicrobium ferrooxidans (with 90 % iden- before this process was reported. Using labeled NH4 in 15 tity), might play a key role in this anaerobic biological pro- a microcosm experiment resulted in the production of N2, cess that uses ferric iron as an electron acceptor while oxi- which conclusively showed that ammonium-N was converted dizing ammonium to nitrite. After ammonium was oxidized to nitrogen gas (N2) in these sediments under iron-reducing to nitrite, nitrogen loss proceeded via denitrification and/or conditions (Shrestha et al., 2009). Either this same path- C anammox. way for NH4 oxidation, or a very similar one, was also ob- served in a biological reactor (Sawayama, 2006) and a trop- ical rainforest soil (Yang et al., 2012), and coined Feammox (Sawayama, 2006). These pathways have been reported to C − 1 Introduction oxidize NH4 to NO2 (Clement et al., 2005; Shrestha et al., − 2009), to NO3 (Sawayama, 2006), or directly to N2 (Yang et The most common removal of nitrogen from soil environ- al., 2012), using Fe(III) as an electron acceptor. ments is mineralization (for organic nitrogen), followed by Our understanding of the Feammox process is still incom- nitrification and then denitrification (Canfield et al., 2010). plete; particularly, information about the microorganism(s) In water-saturated and organic carbon rich sediments or soils, responsible for it is lacking. This makes further study into such as wetland sediments, there is little oxygen for signif- the mechanism of the Feammox process difficult. Here we C icant nitrification by aerobic ammonium (NH4 ) oxidation focus on a series of incubations and establishing a Feammox Published by Copernicus Publications on behalf of the European Geosciences Union. 770 S. Huang and P. R. Jaffé: Characterization of incubation experiments and development enrichment culture to identify the microbial community re- treatment). pH was adjusted to 4.5 in the ferrihydrite/goethite sponsible for the process described previously (Clement et augmented samples, and to between 3.5 and ∼ 4.0 in the al., 2005; Shrestha et al., 2009). Soil samples were collected ferric chloride/citrate augmented samples. Soil-slurry sam- from the same location and used for laboratory incubation ples, which were prepared to have an initial concentration −1 −1 C experiments as well as to set up an enrichment system for of 12.0 mmol L Fe(III) and/or 2.00 mmol L NH4 , were Feammox in a continuous flow membrane reactor. Various incubated in a series of 50 mL vials with an oxygen-free incubation conditions (Fe(III) sources, inorganic carbon con- headspace, created by purging with a CO2 :N2 (80 V 20) mix- C 15 C tent, NH4 concentration, NH4 , and acetylene gas (C2H2) ture. Triplicate samples were collected destructively every 2 as a selected inhibitor) were used to study the Feammox days to analyze iron and nitrogen species. mechanism. Molecular biology methods, such as denaturing The second incubation was conducted to extend the gradient gel electrophoresis (DGGE), 454 pyrosequencing, anoxic incubation with ferrihydrite to 180 days, with re- C and real-time quantitative PCR (qPCR) analysis were used peated NH4Cl additions after the NH4 in solution was ex- to investigate the bacterial community change during incu- hausted. The initial concentration of Fe(III) was 25.0 and −1 C bations. 1.00 mmol L NH4 was added on days 4, 24, and 60; fur- −1 thermore, 0.20 mmol L NaHCO3 was added on day 50 and day 90 of the incubation. On day 125, incubation vials 2 Methods were divided into two sets to study the effect of differ- ent inorganic carbon contents on Feammox. Either 1.20 or −1 −1 2.1 Sample collection and processing 0.20 mmol L of NaHCO3 plus 2 mmol L of NH4Cl were added to each set. NaHCO3 was then added every 10 days, Soils for all the experiments described in this study were which increased the soil pH to ∼ 5 in the samples amended −1 taken from a temperate forested riparian wetland at the As- with 1.20 mmol L or NaHCO3. For this incubation, sam- sunpink Wildlife Management Area, New Jersey. Ten soil ples were collected every 4 days. Finally, soil samples col- cores were collected from 10 cm below the surface with lected on day 180 of the incubations were used to enrich polyethylene column containers (8 cm diameter and 30 cm the Feammox bacteria in a membrane reactor. To study how long) and transported to the laboratory within 2 h. The soil the organic carbon content affects the Feammox bacteria, pH was between 3.5 and 4.5, and no manganese oxides 1.00 mmol L−1 sodium citrate was also supplied on day 125 −1 were detected. The detailed physicochemical characteris- to four of the 1.20 mmol L NaHCO3 amended samples. tics of these wetland soils have been described elsewhere In the third experiment, inorganic nitrogen species were (Clement et al., 2005). Prior to all incubation experiments, quantified through incubations in the presence of C2H2. Soil soil slurry from the field site was aerated for a month to de- slurries were first incubated for 90 days in eighty 50 mL grade much of the labile organic carbon. After 30 days of aer- vials. The Fe(III) concentration at the beginning of the −1 −1 ation, the dissolved organic carbon (DOC) content was sta- incubations was 25 mmol L . One mmol L NH4Cl and ± −1 −1 ble at 2.06 0.20 mg g . Following the aeration treatment, 0.20 mmol L NaHCO3 were added on days 24, 60, and 90. × the soil was divided into 400 10 g (air-dry equivalent) sub- After this incubation, 5 mL of pure C2H2 gas were added samples, and added to 50 mL serum vials, with 30 mL deion- to 40 vials, which resulted in a final C2H2 concentration of −1 ized water. The soil slurries were purged thoroughly with a 100 µmol L . Samples with and without C2H2 were then in- CO2 :N2 (80 V 20) mixture, resulting in a final pH of ∼ 4 to cubated anaerobically for 20 days. The headspace gas was 4.5. The vials were sealed tightly with rubber stoppers and sampled every 24 h for N2O analysis, and soil samples were were stored in an anaerobic glove box for 30 days at ambi- analyzed every 2 days for Fe and N species. ent temperature to allow for stabilization before starting the incubations. 2.3 Continuous flow membrane Feammox reactor 2.2 Batch incubation experiments Soil samples collected on day 180 from the incubation with −1 ferrihydrite, NH4Cl, and 1.20 mmol L NaHCO3 additions All incubations, addition of reagents, and sampling were con- were inoculated into a continuous flow membrane reactor ducted in an anaerobic glove box with a solution of resazurin (Abbassi et al., 2014), which was operated under anaero- as the redox indicator. Soil samples were first incubated with bic conditions by constantly purging N2 through the reac- different Fe(III) sources to determine which source would tor’s headspace at room temperature (25◦), and with a 48 h yield a more active Feammox process: 6-line ferrihydrite hydraulic retention time. (Fe2O3C · 0.5H2O) or goethite (FeO(OH); prepared accord- The enrichment medium contained the following com- ing to Cornell and Schwertmann, 2003) C NHC addition; 4 ponents per liter: 177 mg NH4Cl, 77.9 mg (NH4/2SO4, C C C C ferric chloride NH4 addition; ferric citrate NH4 ad- 19.8 mg NaHCO3, 71.0 mg KHCO3, 9.00 mg KH2PO4, C dition; either only ferrihydrite or NH4 addition; and auto- 100 mg MgSO4 × 7H2O, and 60.0 mg CaCl2 × 2H2O.