R E S EA RCH AR T I C L E

Ammonia transformations and abundance of ammoniaoxidizers in a clay soil underlying a manure pond

Yonatan Sher, Shahar Baram, Ofer Dahan, Zeev Ronen & Ali Nejidat

Department of Environmental Hydrology & Microbiology, Zuckerberg Institute for Water Research, Blaustein Institutes for Desert Research,

Ben-Gurion University of the Negev, Midreshet Sede Boqer, Israel

Correspondence: Ali Nejidat, Department of

Environmental Hydrology & Microbiology,

The Jacob Blaustein Institutes for Desert

Research, Ben-Gurion University of the

Negev, Sede Boqer Campus, Midreshet Sede

Boqer 84990, Israel. Tel.: +972 8 6596832;

fax: +972 8 6596831; e-mail: alineji@bgu.ac.il

Received 20 October 2011; revised 15

February 2012; accepted 21 February 2012.

DOI: 10.1111/j.1574-6941.2012.01347.x

Editor: Tillmann Lueders

Keywords

manure ponds; ammonia-oxidizing bacteria;

ammonia-oxidizing archaea; anammox

bacteria.

Abstract

Unlined manure ponds are constructed on clay soil worldwide to manage farm

waste. Seepage of ammonia-rich liquor into underlying soil layers contributes

to groundwater contamination by nitrate. To identify the possible processes

that lead to the production of nitrate from ammonia in this oxygen-limited

environment, we studied the diversity and abundance of ammonia-transform-

ing microorganisms under an unlined manure pond. The numbers of ammo-

nia-oxidizing bacteria and anammox bacteria were most abundant in the top

of the soil profile and decreased significantly with depth (0.5 m), correlating

with soil pore-water ammonia concentrations and soil ammonia concentra-

tions, respectively. On the other hand, the numbers of ammonia-oxidizing

archaea were relatively constant throughout the soil profile (107 amoA copies

per gsoil). Nitrite-oxidizing bacteria were detected mainly in the top 0.2 m. The

results suggest that nitrate accumulation in the vadose zone under the manure

pond could be the result of complete aerobic nitrification (ammonia oxidation

to nitrate) and could exist as a byproduct of anammox activity. While the

majority of the nitrogen was removed within the 0.5-m soil section, possibly

by combined anammox and heterotrophic denitrification, a fraction of the pro-

duced nitrate leached into the groundwater.

Introduction

Agricultural facilities known as concentrated animal feed-

ing operations extract large quantities of manure waste

(Burkholder et al., 2007), and different management

practices have been developed to control and mitigate

their impact on environmental quality (Day & Funk,

1998). Manure storage in anaerobic ponds is widely used

owing to the ponds’ low construction costs (Bernet &

Beline, 2009). However, the operation of these ponds

may involve severe environmental risks as a source of

contaminants to the air (Amon et al., 2006), as well as

to ground and surface water bodies (Arnon et al., 2008).

One of the major concerns of animal manure’s environ-

mental impact is its contamination of ground and

surface water bodies with nutrients such as phosphorus

and nitrogen (Mallin & Cahoon, 2003). Therefore, strict

regulations standardize the construction of manure

lagoons to control pollution through water seepage

(Sweeten, 1998).

The gradual accumulation of organic matter and the

development of microbial biofilms at the bottom of

earthen manure ponds and in the underlying soils (Tyner

& Lee, 2004) reduce hydraulic conductivity and inhibit

infiltration of manure liquor into the groundwater

(Maule et al., 2000). Nitrogen in manure ponds, originat-

ing from cattle feces and urine, appears mainly in the

form of ammonia and organic nitrogen (Safley et al.,

1986). The manure ponds are highly anoxic, and oxida-

tion of ammonia to nitrate can be achieved only by

intensive aeration (McGarvey et al., 2007). The construc-

tion of manure ponds on clay soil is based on the

assumption that its low hydraulic conductivity and high

cation exchange capacity would limit the downward

leaching of ammonia and its confinement to the anaero-

bic upper soil layers without further transformations

FEMS Microbiol Ecol && (2012) 1–11 ª 2012 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

MIC

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(DeSutter & Pierzynski, 2005; DeSutter et al., 2005).

However, nitrate has been detected in the vadose zone

and the groundwater under dairy manure ponds (Korom

& Jeppson, 1994; Parker et al., 1999).

Based on the generation of nitrate from ammonia in

these soil layers, it can be hypothesized that ammonia-

oxidizing microorganisms do colonize this oxygen-limited

environment. Nitrate can be generated by two processes:

(1) complete nitrification (oxidation of ammonia to

nitrate) and (2) as a byproduct of anammox bacteria

activity that consumes ammonia and nitrite (Mulder

et al., 1995). Partial nitrification (oxidation of ammonia

to nitrite) can provide nitrite for the activity of anammox

bacteria (Sliekers et al., 2002). However, to the best of

our knowledge, the microbial groups that may contribute

to nitrate generation in the soils underlying anaerobic

unlined manure ponds have not been studied before. In

this study, we report upon the diversity and abundance

of ammonia-transforming microorganisms [ammonia-

oxidizing bacteria (AOB), ammonia-oxidizing archaea

(AOA), and anammox bacteria] in a soil profile below an

unlined anaerobic manure pond.

Materials and methods

Study site

Samples analyzed in this study were collected from a

dairy farm, located in the lowlands (Shfelat Yehuda) of

southern central Israel, containing 60 dairy cows and 30

heifers and calves. It discharges ~ 7 m3 day�1 of liquid

waste (manure, feces, water from washing shades, and

cooling water) into an unlined earthen manure pond,

with dimensions of ~ 16 9 12 m and depth ~ 0.8 m.

The manure pond is constructed in clayey soil (51% clay,

85% of which consists of illite/smectite minerals), and the

sediment in its bed consists of organic sludge and coarse

sand derived from the wear of the dairy farm’s concrete

structures.

Sampling of soil and pore water under the

manure pond

To sample the sediment underlying the pond, a metal

ring (1 m high and 0.6 m diameter) was placed in the

manure pond to allow slurry removal and exposure of

the bottom of the pond’s top sediments. Sediment and

soil samples were then collected from the bottom of the

pond using two types of core barrels: a 0.5-m long steel

corer with a diameter of 0.15 m to collect a large undis-

turbed 0.5-m long sediment sample, and a 0.1-m long

sterilized PVC cylinder with diameter of 0.075 m for

higher sampling resolution of the topsoil. In the field, the

core samples were immediately covered with aluminum

foil and kept on ice until they reached the laboratory

(< 12 h). The 0.5- and 0.1-m cylinders were cut into sec-

tions every 10 and 2 cm, respectively. Samples were taken

from each section of the core for further analyses, after

removing the layers that had been in contact with either

the core barrel walls or the cutting tools. All samples were

kept at 4 °C until use. Core sections are reported as: (1)

sediment – representing the sediment in the manure

pond bed, (2) interface – representing the interface layer

between the pond sediment and the underlying soil, and

(3) soil profile – representing soil samples at different

depths below the interface.

To continuously sample the propagating pore water in

the unsaturated zone, a custom-made suction cup (6 cm

long and 2 cm diameter) was installed in the soil under-

lying the manure pond, at a vertical depth of 0.5 m.

Chemical analyses

Water content was determined after 72 h of air-drying at

105 °C, and total organic matter was determined by the

combustion of these air-dried soil samples at 450 °C(Nelson & Sommers, 1996). Total ammonia nitrogen was

determined following extraction with 1 M KCl. Nitrate,

nitrite, and pore-water ammonia were extracted (~ 910

dilution) with double distilled water (DDW). Ammonia

was determined by the phenate method, nitrite by the

sulfanilamide colorimetric method, and nitrate by cad-

mium reduction of nitrate and subsequent analysis as for

the nitrite (Clesceri et al., 1998). Pore-water ammonia

concentrations were assessed with the assumption of no

desorption from the solids during the DDW extractions;

hence, the concentrations in the DDW extractions repre-

sented solely the dilution of the pore water. Oxidation–reduction potentials (ORPs) of the manure pond slurry

and propagating pore water in the unsaturated zone were

monitored continuously for 4 months using an ORP elec-

trode (Cole-Parmer KH27300-19, Vernon Hills, IL).

Assessment of nitrification potential

Aerobic ammonia oxidation potential was assessed in 0.5-

L flasks containing a 200 mL medium of 25 mM

K2HPO4 (pH 7.8) and 2.86 mM (NH4)2SO4, covered with

air-permeable paper stoppers. Following amendment of

5 g soil, flasks were incubated for 24 h in the dark with

continuous shaking (200 r.p.m.). Samples were with-

drawn and frozen at �80 °C. Nitrogen species were ana-

lyzed as described in the previous section. Nitrification

rates were calculated based on the linear regression

(R2 = 0.76–0.94, P-value < 0.006) of nitrate accumulation

vs. time.

ª 2012 Federation of European Microbiological Societies FEMS Microbiol Ecol && (2012) 1–11Published by Blackwell Publishing Ltd. All rights reserved

2 Y. Sher et al.

DNA extraction and PCR amplification

Genomic DNA was extracted with a PowerSoilTM DNA

Isolation kit (MO BIO Laboratory Inc., Solana Beach,

CA). The column of the kit was rinsed twice to ensure

maximal DNA extraction. The abundance of AOA, AOB,

nitrite-oxidizing bacteria (NOB), and anammox was esti-

mated via a SYBR green chemistry quantitative PCR

(qPCR) of the following marker genes: putative archaeal

amoA gene, bacterial amoA, 16S rRNA gene, and anam-

mox 16S rRNA gene, respectively. The primers and PCR

conditions are given in Table 1. qPCR contained 12.5 µlreaction mix (DyNAmoTM Flash SYBR® Green qPCR

kit; Finnzymes, Espoo, Finland), 2.5 µl of each of the rel-

evant primers, 5 µl of DNA template or standard, and

2.5 µl DDW in a total volume of 25 µl. Melting curves

(72–95 °C) showed only one peak for all qPCR reactions.

Calibration curves were created according to a 10-fold

dilution series (103–109 copies) of plasmids containing

environmental copies of the relevant genes. Calibration

curves had R2 > 0.975, and the slope was between �3.0

and �3.9, corresponding to PCR efficiencies of 90–111%.

Amplification reactions were carried out in a Rotor-

GeneTM 6000 (Corbett Life Science, Concorde, NSW,

Australia).

For denaturing gradient gel electrophoresis (DGGE)

analysis, the following genes were amplified: putative

archaeal amoA gene and 16S rRNA gene fragments of the

AOB (Table 1). The latter were amplified by CTO prim-

ers using a nested PCR approach, with initial amplifica-

tion using 27f-1492r primers, as indicated in Table 1

(Mahmood et al., 2006). PCRs were carried out in a vol-

ume of 50 µl, containing 5 µl of 109 PCR buffer

(Sigma), 250 lM of each deoxynucleoside triphosphate,

2.5 mM MgCl2, 0.1 mg mL�1 BSA, 0.5 µM of each of

the relevant primers, and 2 µl of DNA template. Amplifi-

cation reactions were carried out in a TGradient thermo-

cycler (Biometra, Gottingen, Germany).

DGGE analysis

DGGE analysis was performed with a DcodeTM Universal

Mutation Detection System (Bio-Rad, Hercules, CA) in a

1-mm thick 8% (w/v) polyacrylamide gel at 60 °C. PCRproducts of the putative archaeal amoA gene and 16S

rRNA gene fragments were analyzed with denaturing gra-

dients of 15–50% and 35–50% (Nejidat, 2005; Nicol

et al., 2008) urea/formamide, for 1160 min at 80 V and

980 min at 70 V for AOA and AOB, respectively. Poly-

acrylamide gels and all DGGE solutions were prepared

according to the manufacturer’s instructions (Bio-Rad).

Ethidium bromide-stained gels were visualized on a Gel

Doc XR gel-imaging system (Bio-Rad), and DNA bands

were excised on a UV transilluminator table using a scal-

pel. The DNA was eluted from the gel and used as a tem-

plate for reamplification using the same sets of PCR

primers (Table 1) except CTO primers without the

GC-clamp.

Cloning, sequencing, and phylogenetic analysis

Reamplified DGGE bands were cloned in a pTZ57R

plasmid using an InsTAcloneTM PCR cloning kit (MBI

Fermentas, Hanover, MD). Cloned DNA was then sent

Table 1. PCR and qPCR primers and reaction conditions applied in this study

Target gene Application Primers Conditions Size (bp) Reference

AOA (amoA) DGGE and

qPCR

Arch amoAF-5′-TTATGGTCTGGCTTAGACG-3′

Arch amoAR-5′-GCGGCCATCCATCTGTATG

T-3′

95 °C 5 min; 35 cycles: 95 °C 35 s,

54 °C 45 s, 72 °C 50 s

635 Francis et al.

(2005)

AOB (amoA) qPCR amoAF-5′-GGGGHTTYTACTGGTGGT-3′

amoA2R-5′-CCCCTCKGSAAAGCCTT

CTTC-3′

95 °C 5 min; 35 cycles: 95 °C 35 s,

54 °C 45 s, 72 °C 50 s

491 Rotthauwe

et al. (1997)

AOB

(16S rRNA

gene)

DGGE CTO189F-5′-(GC clamp)-GAGRAAAGCA

GGGATC G-3′

CTO649R-5′-CTAGCTTGTAGTTTCAAA

CGC-3′

95 °C 5 min; 30 cycles: 94 °C 45 s,

58 °C 45 s,72 °C 1 min; 10 min at

72 °C

460 Kowalchuk

et al. (1997)

Bacteria

(16S rRNA

gene)

Nested PCR 27F-5′-AGAGTTTGATCCTGGCTCAG-3′

1492R-5′-GGTTACCTTGTTACGACTT-3′

95 °C 5 min; 30 cycles: 94 °C 45 s,

55 °C 45 s, 72 °C 1 min; 10 min at

72 °C

1465 Lane (1991)

NOB

(16S rRNA

gene)

qPCR FGPS 872f-5′-CTAAAACTCAAAGGAAT

TGA-3′

FGPS 1269r-5′-TTTTTTGAGATTTGCTAG-3′

95 °C 15 min; 35 cycles: 95 °C 60 s,

50 °C 60 s, 72 °C 60 s

397 Degrange &

Bardin (1995)

Anammox

(16S rRNA

gene)

qPCR AMX 368F-5′-TTCGCAATGCCCGAAAGG-3′

AMX 820F-5′-AAAACCCCTCTACTTAGTGC

CC-3′

95 °C 15 min; 35 cycles: 95 °C 45 s,

59 °C 50 s, 72 °C 60 s

452 Schmid et al.

(2003)

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Ammonia transformations under a manure pond 3

for sequencing (Macrogen Inc., Seoul, Korea). To iden-

tify the anammox bacteria in the soil, corresponding

16S rRNA gene fragments were PCR-amplified (Table 1)

and a cloned library was constructed. Ten clones were

randomly selected and sequenced. Phylogenetic trees,

based on 16S rRNA gene sequences of AOB and anam-

mox bacteria, were constructed using the neighbor-

joining method with evolutionary distances computed

using the maximum composite likelihood method. Phy-

logenetic analyses were conducted with MEGA4 software

(Tamura et al., 2007). Sequences obtained from this

study were deposited in GenBank and assigned accession

numbers HQ652082–HQ652103, HQ652105–HQ652107,

HQ407496, and JF313147.

Results

Chemical parameters along a soil profile under

the manure pond

Soil chemical parameters under the manure pond under-

went notable changes throughout the soil profile (Fig. 1).

The gravimetric water content and the organic matter

content in the soil decreased with depth from values of

38% and 5%, respectively, at the interface between the

pond sediments and the underlying clay, to the values of

24% and 3%, respectively, at a depth of 0.5 m (Fig. 1a

and b). Ammonia concentrations followed the same trend

(Fig. 1c and d). Soil ammonia concentration (KCl

extracts) decreased from 3442 mg-N per kgsoil at the

interface to 2 mg-N per kgsoil at a depth of 0.5 m under

the manure pond. In the first 10 cm of the profile,

ammonia concentration remained relatively constant and

then decreased below this depth. Pore-water ammonia

concentrations, extracted with DDW (concentration cal-

culated per liter of pore water), also decreased from

1098 mg-N per Lpore water at the interface to 32 mg-N per

Lpore water at a depth of 0.5 m under the manure pond.

The decrease in pore-water ammonia concentrations

showed the same trend as the KCl-extracted ammonia

concentrations. However, in the sediment of the manure

pond bed, the latter (442 mg-N per kgsoil) was signifi-

cantly lower than its concentrations in the underlying soil

profile. Nitrate was not detected in the sediment or in the

top 0.1 m of the soil profile, whereas detectable nitrate

concentrations were measured at a depth of about 0.3 m,

reaching up to 6 mg-N per kgsoil at 0.5 m (Fig. 1e).

Nitrite concentrations throughout the soil profile were in

the range of zero to 0.2 mg-N per kgsoil. Sampling of the

vadose zone pore water under the manure pond, at a

depth of 0.5 m, showed high nitrate concentrations of

513 mg-N per Lpore water, coinciding with low ammonia

concentrations of 0.3 mg-N per Lpore water. Manure pond

slurry exhibited a highly reduced ORP of �0.44 V, while

the underlying pore water, collected from a depth of

0.5 m below the pond bed, showed an ORP of 0.18 V.

Nitrification activity throughout the soil

profile

The decrease in ammonia concentrations and the accu-

mulation of nitrate throughout the soil profile indicated

nitrification activity. Nitrification potential was, therefore,

assessed throughout the soil profile (Fig. 2). The highest

nitrification activity (30 mg-N (NO�3 ) per kgsoil per h)

was measured at a depth of 0.1 m in the soil layer and

decreased steadily with depth, reaching 0.7 mg-N (NO�3 )

per kgsoil per h at 0.5 m.

Abundance of ammonia-transforming

microorganisms

The abundance of the aerobic ammonia oxidizers

throughout the soil profile was estimated based on the

copy number of the amoA genes of the bacterial and

archaeal ammonia oxidizers using qPCR (Fig. 3). Copy

Fig. 1. Physiochemical properties of the soil-depth profile under the manure pond: (a) water content, (b) organic matter, (c) soil ammonia, (d)

pore-water ammonia, and (e) nitrate. Dashed line indicates the interface between the manure pond sediment and the underlying soil. Water

content and organic matter values are the averages of two measurements – one from each core. Soil ammonia, pore-water ammonia, and

nitrate values are averages of four measurements – two from each core.

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4 Y. Sher et al.

numbers of the putative archaeal amoA were, in most

cases, higher than those of the AOB, with an average

copy number of 5 9 106 per gsoil, with no significant

change with depth and with no correlation to ammonia

concentration (Fig. 1) or nitrification potential (Fig. 2).

On the other hand, the highest copy number of the

bacterial amoA (1 9 107 copies per gsoil) was recorded

in the top 10 cm, followed by a steep decrease in three

orders of magnitude at a depth of 0.5 m (Fig. 3),

correlated with pore-water ammonia concentration

(R2 = 0.83, P = 0.0001) and nitrification potential (R2 =0.80, P = 0.042).

The anammox 16S rRNA gene showed an increase in

copy number from 9 9 104 copies per gsoil in the sedi-

ment of the manure pond to 2 9 107 copies per gsoil at

the interface, followed by a sharp decrease with depth in

the soil profile, to a concentration of 2 9 103 copies per

gsoil at 0.5 m (Fig. 3). The abundance pattern of anam-

mox 16S rRNA gene copies correlated with soil ammonia

concentration (R2 = 0.74, P = 0.0006), largely due to the

low soil ammonia concentration and anammox abun-

dance in the manure pond sediment (Figs 1c and 3).

Nitrobacter species were detected in 7 out of 18 soil samples

tested and spanning the 0.5 m soil profile. The positive

samples were mainly in the upper parts of the soil profile

(top 0.2 m), and their16S rRNA gene copy numbers were

in the range of 2 9 106 to 8 9 106 copies per gsoil.

Community structure of ammonia oxidizers

The dominant species of AOB along the soil profile were

identified by DGGE (Fig. 4a). Higher numbers of DNA

bands were found in the samples from the top 0.1 m of

the soil profile than in those from its lower parts. The

major DNA bands revealed by the DGGE analysis

(Fig. 4a) were sequenced, and the DNA sequences were

used to construct a phylogenetic tree (Fig. 5). AOB

sequences obtained from soil samples at a depth of 0.1–0.5 m were related to both Nitrosomonas and Nitrosospira

lineages. Within the latter, manure pond soil sequences

clustered mainly near Nitrosospira briensis and Nitrosospir-

a multiformis sequences within cluster 3 of the Nitroso-

spira genus (Purkhold et al., 2000). Nitrosomonas species

were distributed in several clusters, including those of

Nitrosomonas europea and Nitrosomonas communis

(Fig. 5). DGGE analysis of AOA amoA gene fragments

throughout the soil profile did not show major changes

(Fig. 4b), and a dominant DNA band (4; Fig. 4b) was

evident throughout the soil profile. Its sequence

(JF313147) showed 95% similarity to corresponding

sequences (DQ148902, DQ148904, and DQ148891) of

AOA retrieved from saline estuaries (Francis et al., 2005).

In addition, the sequence (HQ407496) of the DNA bands

Fig. 2. Nitrification potential, measured by the accumulation of

nitrate along a large-scale soil core (0.5 m). Error bars represent

standard error of the slope, calculated by linear regression of four to

six time points.

Fig. 3. Abundance of ammonia-transforming microorganisms

throughout the soil profile under the manure pond, represented as

copy numbers of marker genes per gram dry soil. Abundance of AOB

(●), AOA (○), and anammox [AMX (▼)]. Each point represents

average of four qPCR runs – two from each core, and error bars

indicate standard deviation.

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Ammonia transformations under a manure pond 5

(6–1; Fig. 4b), which showed a significant reduction in its

intensity along the soil profile, has a 99% similarity to

sequences (AB542171 and AB542169) of AOA retrieved

from composted cattle manure (Yamamoto et al., 2011).

Ten randomly selected anammox 16S rRNA gene

clones that form a library were sequenced, and a phyloge-

netic tree was constructed. The sequences were highly

similar and clustered with the sequence of Candidatus

Jettenia asiatica (Fig. 6), suggesting selection for limited

diversity in the manure pond soil environment.

Discussion

Nitrate, in contrast to ammonia that adsorbs to clay par-

ticles, is easily leached and contaminates groundwater.

Therefore, this research aimed at identifying the ammo-

nia-consuming microbial communities that are involved

in the generation of nitrate from ammonia in a reduced

environment of clay soil that underlies anaerobic manure

ponds. We have detected and studied the abundance and

diversity of AOB, AOA, and anammox bacteria in a soil

profile beneath a dairy farm manure pond. The detection

of these microbial groups in the reduced vadose zone

suggested the subsistence of heterogeneous environmental

conditions that allowed aerobic (AOA and AOB) and

anaerobic (anammox) microbial activity. This can be

attributed to the complexity of the soil environment in

which aerobic and anaerobic niches can subsist in very

close proximity (Tiedje et al., 1984; Holden & Fierer,

2005).

Abundance of ammonia-transforming

microorganisms

The generation of nitrate from ammonia is an indicator

of aerobic nitrification. Aerobic AOB and AOA, which

oxidize ammonia to nitrite, were highly abundant at the

interface between the manure pond sediment and the

underlying soil (Fig. 3). The AOB 16S rRNA gene

sequences belonged to both the Nitrospira and Nitroso-

monas genera (Fig. 5). All Nitrospira-like 16S rRNA gene

sequences were associated with cluster 3 (Fig.5). Nitroso-

monas (Koops et al., 2003) and Nitrosospira cluster 3

(Kowalchuk et al., 2000; Webster et al., 2005) species

may have been selected for by the high ammonia concen-

tration throughout the soil profile (Fig. 1).

The number of AOB amoA gene copies (Fig. 3)

decreased with soil depth (Fig. 4a), possibly driven by

variations in ammonia concentration (Princic et al., 1998;

Avrahami et al., 2002) and heterogeneity of the environ-

mental conditions within the soil matrix (Brune et al.,

2000). In contrast, the gene copy number of the putative

archaeal amoA and AOA diversity did not change signifi-

cantly with soil depth (Figs 3 and 4b, respectively), in

accordance with the suggestion that they can also grow at

low levels of ammonia (Erguder et al., 2009; Martens-

Habbena et al., 2009), as found at the lower soil layers.

In addition, the distribution patterns of the AOB and the

AOA were possibly affected by the availability of oxygen

throughout the soil profile as AOA were reported to be

able to occupy environments of very low dissolved oxygen

concentrations (Coolen et al., 2007; Erguder et al., 2009).

The relative contribution of the two ammonia-oxidizing

groups to the nitrification activity that is measured in

environmental samples is still under debate with conclu-

sions being based mainly on the relative abundance of the

respective amoA gene copy numbers (Prosser & Nicol,

2008). Whereas in some environments, AOB have been

found to be the dominant ammonia oxidizer, in others,

AOA numbers surpass those of AOB (e.g., Nicol et al.,

2008; De Corte et al., 2009; and Jia & Conrad, 2009). It

is difficult to determine the relative contribution of the

two groups in the studied system. The correlation of

ammonia concentrations in the soil layers (Fig. 1) and

ammonia oxidation activity in batch experiments (Fig. 2)

with AOB abundance (Fig. 3) suggests their significant

role. However, it should be mentioned that the measured

activity in the batch experiments do not necessarily repre-

sent the in situ activity because the used medium can be

preferable to one of the ammonia-oxidizing groups (AOB

vs. AOA). Although ammonia concentration in the nitri-

fication medium was relatively high (5.6 mM), it was still

lower than its concentration in the pore water in most

sections of the soil profile (Fig. 1), which further supports

Fig. 4. DGGE profiles of two soil cores sampled under the manure

pond: (a) AOB and (b) AOA communities. Arrows indicate DNA bands

that were sequenced; AOB identification numbers correspond to

those in the phylogenetic tree in Fig. 5.

ª 2012 Federation of European Microbiological Societies FEMS Microbiol Ecol && (2012) 1–11Published by Blackwell Publishing Ltd. All rights reserved

6 Y. Sher et al.

the role of AOB. However, the effects of other factors,

such as oxygen concentrations (Lam et al., 2007) that

might differentiate between the in situ and the batch

experiments’ measured activities cannot be ruled out. In

addition, the measured ammonia concentrations can be,

in part, owing to transport processes and organic matter

contents. Nevertheless, the high copy number of the

amoA gene of the AOB and AOA, in particular, in the

upper soil samples, indicates an active mixture of ammo-

nia-oxidizing population.

Fig. 5. AOB phylogenetic tree based on 16S rRNA gene fragments (440 bp) and inferred using the neighbor-joining method. Sequences

obtained in this study are indicated in bold. The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary

history of the taxa analyzed. Branches corresponding to partitions reproduced in < 50% bootstrap replicates are collapsed. The percentage of

replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown to the left of the branches. The

tree is drawn to scale; evolutionary distances were computed using the maximum composite likelihood method and are in the units of the

number of base substitutions per site.

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Ammonia transformations under a manure pond 7

Possible routes for oxygen supply

Although measurements showed that the environment is

reduced, the generation of nitrate from ammonia indi-

cates that considerable amounts of oxygen were reaching

the environment and supporting aerobic nitrification. The

oxygen supply for aerobic microbial activity can be facili-

tated by the sediment and soil structure under the man-

ure pond. Deposition of fine organic particles on the bed

of the manure pond reduces its hydraulic conductivity

(Parker et al., 1999; Cihan et al., 2006). In return, the

underlying clay layer becomes unsaturated (Fig. 1a) and

susceptible to the formation of desiccation cracks (Chert-

kov & Ravina, 1999; Chertkov, 2002). Indeed, desiccation

cracks with average aperture of 5.5 ± 1.7 cm and average

depth of 65 ± 19 cm (with average aperture larger than

6 mm) were observed over the entire land surface at our

site, including the margins of the manure pond (Baram

et al., 2012). Cracks to the depths of 12 m were also mea-

sured. The cracks network was found to remain opened

and serves as a preferential flow pathway year-round

(Baram et al., 2012). It is highly possible that desiccation

cracks from the margins create a network of horizontal

connectivity under the pond, as has been observed in

large-scale lysimeter experiments and modeled by numeri-

cal models (Chertkov, 2002; Greve et al., 2010). It is sug-

gested that the formation of desiccation cracks around

and under the manure pond can accelerate the aeration

of the unsaturated vadose zone, via a variety of mecha-

nisms: (1) thermal-induced air convection in the cracks

(Nachshon et al., 2008), (2) barometric pressure fluctua-

tions in the vadose zone caused by daily atmospheric

pressure fluctuations (Rimon et al., 2011), and (3) wind

gusts (Auer et al., 1996; Neeper, 2001).

Cooperation between nitrogen-transforming

microorganisms

The lack of nitrite accumulation (only residual levels were

detected) in the soil samples may stem from either effi-

cient activity of the NOB, nitrite consumption by the

anammox bacteria and heterotrophic denitrifiers, or as a

result of their combined activity. Detection of autotrophic

anammox bacteria (Fig. 3) indicates the existence of

anaerobic niches in the soil layers because of the con-

sumption of the limited amounts of oxygen by aerobic

microbial activity. Carbon dioxide originated from the

crack-perfused air or was generated by the heterotrophic

microbial activity that can support the autotrophic

growth of both the ammonia oxidizers and the anammox

bacteria. Anammox bacteria have mostly been detected in

aquatic environments and wastewater treatment plants

(Dalsgaard et al., 2005; Kuenen, 2008). However, anam-

mox bacteria were also recently detected in terrestrial eco-

systems (Humbert et al., 2010; Hu et al., 2011), and the

anammox 16S rRNA gene sequences obtained from the

soil profile were related to C. Jettenia asiatica (Fig. 6),

which has been detected in terrestrial ecosystems (Quan

et al., 2008; Humbert et al., 2010). The number of the

anammox bacteria was relatively low in the manure pond

sediment, and the highest numbers were measured in the

interface between the sediment and the soil (Fig. 3). The

Fig. 6. Phylogenetic tree, based on 16S rRNA gene fragments (462 bp) that are unique to anammox bacteria, inferred using the neighbor-

joining method. Sequences obtained in this study are indicated in bold. The bootstrap consensus tree inferred from 1000 replicates is taken to

represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates

are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown

to the left of the branches. The tree is drawn to scale; evolutionary distances were computed using the maximum composite likelihood method

and are in the units of the number of base substitutions per site.

ª 2012 Federation of European Microbiological Societies FEMS Microbiol Ecol && (2012) 1–11Published by Blackwell Publishing Ltd. All rights reserved

8 Y. Sher et al.

low anammox numbers in the sediment may be related

to their specific ecological requirements of nitrite concen-

tration and C/N ratio and their preference to colonize

microscopic particles, such as clay particles, in the soil

profile, rather than sand particles (Woebken et al., 2007;

Dang et al., 2010). On the other hand, the decreasing

number of anammox bacteria throughout soil profile

(Fig. 3) can be attributed to the decreasing concentration

of ammonia (Fig. 1).

Nitrifiers have been found to interact with anammox

bacteria under extremely low oxygen systems, such as the

water column of oxygen minimum zones in marine eco-

systems (Lam et al., 2007; Yan et al., 2010). The activity of

anammox bacteria consumes ammonia and nitrite while

producing mainly gaseous N2 and nitrate as a byproduct

(Kuenen, 2008). In the studied system, nitrite can be pro-

duced as an intermediate of nitrification (ammonia oxida-

tion to nitrite) and/or denitrification (reduction of nitrate

to nitrite), as well. Nitrate was found to be the dominant

nitrogen form (166 ± 100 mg L�1 NO�3 -N) in the propa-

gating pore water at the depth of 0.5 below the pond bed,

which is significantly lower than the expected concentra-

tion that may result from the oxidation of the ammonia

pore water in the upper soil layers (Fig. 1), indicating

nitrogen removal processes. Nitrogen removal from the

vadose zone underlying the manure pond can be the result

of anammox activity (consuming ammonia and nitrite

resulting from partial nitrification), although the occur-

rence of canonical denitrification cannot ruled out. The

relative contribution of each pathway to nitrogen removal

in the studied system is not yet clear. However, the resid-

ual nitrate can leach down into the deeper soil layers (Paul

& Zebarth, 1997) and can potentially contaminate the

groundwater.

Conclusions

This study showed that aerobic and anaerobic ammonia-

transforming microbial species inhabit the soil layers that

underlie an anaerobic pond receiving ammonia-rich cow

manure. Therefore, leaked ammonia from manure ponds

can be readily transformed, and nitrate is produced.

While most of the nitrogen is removed within a 0.5-m

soil layer, significant levels of nitrate were detected in the

vadose zone. Therefore, the results indicate that manure

ponds that are underlined with clay soil should be con-

sidered as a potential point source for the contamination

of groundwater with nitrogenous compounds.

Acknowledgements

We thank the dairy farm owner for allowing us to con-

duct the research at his farm. The work was funded by

Israel’s Water Authority and by a grant from the Israel

Science Foundation (734/05).

Authors’ contribution

Yonatan Sher and Shahar Baram contributed equally to

this work.

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Ammonia transformations under a manure pond 11