Biogeochemistry (2017) 132:237–249
DOI 10.1007/s10533-017-0297-x
Potential roles of anaerobic ammonium oxidation
(anammox) in overlying water of rivers with suspended
sediments
Sibo Zhang . Xinghui Xia . Ting Liu . Lingzi Xia .
Liwei Zhang . Zhimei Jia . Yayuan Li
Received: 27 May 2016 / Accepted: 20 January 2017 / Published online: 9 February 2017
Ó Springer International Publishing Switzerland 2017
Abstract Anaerobic ammonium oxidation (anam-
mox) is believed to be an important sink for fixed
inorganic nitrogen in terrestrial and aquatic ecosys-
tems, and many studies have reported that macroscale
oxic–anoxic interfaces, such as riparian zones, were
hotspots of anammox reaction. However, no research
has linked microscale interfaces with the anammox
process in natural environments. This study provides
evidence for the presence of anammox bacteria and
potential anammox activity on the suspended sedi-
ment (SPS) in the oxic water of the Yellow River. The
anammox bacteria in the overlying water were mainly
attached to SPS. The abundance of anammox bacteria
in the overlying water was positively correlated with
SPS concentration (R2 = 0.97, P \ 0.01), with abun-
dance ranging from 9.5 9 102 to 1.5 9 104 hydrazine
synthase gene copies per g of SPS. Phylogenic
analysis of anammox bacteria revealed that the SPS
phase was dominated by Candidatus Brocadia.
Responsible Editor: Breck Bowden
Electronic supplementary material The online version of
this article (doi:10.1007/s10533-017-0297-x) contains supple-
mentary material, which is available to authorized users.
S. Zhang  X. Xia (&)  T. Liu  L. Xia 
L. Zhang  Z. Jia  Y. Li
School of Environment, Beijing Normal University/State
Key Joint Laboratory of Environmental Simulation and
Pollution Control, Beijing 100875, China
e-mail: xiaxh@bnu.edu.cn
Candidatus Scalindua genera was detected in this
study with a conductivity of 1100 lS cm-1. More-
over, 15 NHþ 4 -amended anaerobic incubation of the
overlying water showed that the average potential
anammox activity was 0.076 nmol-N L-1 day-1. The
15
N labeling simulation experiments demonstrated the
occurrence of anammox in the oxic water of the
Yellow River. This study suggests that the anammox
process at the SPS–water interface might be a non-
negligible pathway for the loss of fixed nitrogen in
natural freshwaters, but this remains to be determined
in further studies.
Keywords Anammox  Oxic–anoxic interface 
Suspended sediment  Freshwater  Nitrogen loss
Introduction
Anaerobic ammonium oxidation (anammox) is the
biological conversion of ammonium and nitrite into
dinitrogen gas (NHþ 
4 þ NO2 ! N2 þ 2H2 O)
(Kartal et al. 2011). So far, known anammox species
are divided into five genera, ‘Brocadia’, ‘Kuenenia’,
‘Anammoxoglobus’, ‘Jettenia’, and ‘Scalindua’. None
of these anammox bacteria have been obtained as
classical pure cultures, and so all have the taxonomical
status of ‘Candidatus’ (Kartal et al. 2013). At present,
the anammox process is identified as an important
pathway of global nitrogen loss, responsible for up to
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238
50% of N2 production in the oceans (Devol 2003;
Dalsgaard et al. 2005). Moreover, there has been
increasing evidence for the presence of anammox in
terrestrial and freshwater ecosystems (Hamersley et al.
2009; Hu et al. 2012; Moore et al. 2011; Naeher et al.
2015; Zhu et al. 2011).
Oxygen is a major regulatory factor influencing the
biogeographical distribution of anammox bacteria,
due to its strong inhibitory effect on the anammox
metabolism (Kalvelage et al. 2011). Reactor studies
have demonstrated that 1 lM O2 inhibited anammox
metabolism (Strous et al. 1997). The upper O2 limit for
anammox was estimated to be *20 lM in marine
oxygen minimum zones (OMZs) (Kalvelage et al.
2011). Additionally, anammox bacteria require the
concomitant existence of ammonium and nitrite.
Therefore, oxic–anoxic interfaces have been thought
to be hotspots of anammox reaction (Kuenen 2008;
Zhu et al. 2015). Most of the published studies on
anammox in natural systems have primarily focused
on macroscale potential ‘‘hot zones’’, such as the
water–sediment interface, the land–freshwater inter-
face, and the rhizosphere, especially in water-satu-
rated soils, etc. However, no studies have linked
microscale interfaces with the anammox process in
natural environments, especially for the suspended
particle–water interface. Only Woebken et al. (2007)
has reported the presence of anammox bacteria within
suspended particles in the marine environment, with
ambient oxygen of *25 lM.
Suspended particles, which are ubiquitous in
aquatic environments especially in river ecosystems,
play important roles in the transport and reactivity of
substances. Several studies have suggested the pres-
ence of anoxic microzones on the suspended particles
based on the theories of multispecies biofilm and
transport limitation (Michotey and Bonin 1997;
Stoodley et al. 2002). The significance of oxic–anoxic
interfaces between suspended particles and water to
the substance cycle in aquatic systems has always been
an area of active research (Falkowski et al. 2008; Jia
et al. 2016; Liu et al. 2013; Ochs et al. 2010). In
particular, our previous studies have demonstrated that
the denitrification process exists at the suspended
sediment (SPS)–water interface in oxic water of the
Yellow River due to the presence of suboxic/anoxic
microsites at the SPS (Jia et al. 2016; Liu et al. 2013).
Prior studies on wastewater treatment plants have
revealed the presence of the anammox process within
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Biogeochemistry (2017) 132:237–249
anoxic microsites in oxic bulk water. Anammox
bacteria were detected in aerobic zones with activated
sludge [dissolved oxygen (DO) [ 2 mg L-1] of
municipal wastewater treatment plants with an abun-
dance of 105–107 hydrazine synthase (hzs) gene copies
g-1 and with significant contributions to nitrogen loss
(Wang et al. 2015). Maximum anammox activity in a
biological rotating contactor was only slightly
decreased as the DO gradually increased up to
8.0 mg L-1, which presumedly benefited from the
close cooperation between aerobic and anaerobic
ammonium oxidation in the anammox consortium
(Liu et al. 2008). These anammox bacteria in the
bioreactor were enclosed by aerobic ammonia-oxidiz-
ing bacteria, which were distributed at the edge of the
anammox consortiums and were thought to consume
oxygen and provide protections for the anammox
bacteria. Similar to the sludge in wastewater treat-
ments, the SPS in oxic freshwaters might also provide
anoxic conditions for the anammox process.
Therefore, we hypothesize that the microscale
oxic–anoxic interface between SPS and water is a
potential zone of the anammox process in natural
freshwaters even under oxic conditions. To test this
hypothesis, the Yellow River was selected as our study
area. The average annual sediment load of the Yellow
River varies from 0.26 to 0.53 Gt (Yellow River
Sediment Bulletin 2015), making it the most sediment-
laden river in the world (Wang et al. 2015). The
anammox bacterial abundance in the water, the SPS,
and the surface sediment phases were determined with
the most probable number (MPN)-PCR method, and
their correlations with environmental parameters were
analyzed. The community structure of the anammox
bacteria in the SPS phase was investigated. Moreover,
the potential anammox activities of the overlying
water containing SPS were estimated in anaerobic
conditions, and laboratory simulation experiments
with the 15N-labeling technique were conducted to test
whether the anammox could occur in the oxic
overlying water with SPS.
Materials and methods
Site description and sampling
The Yellow River derives from the Bayan Har
Mountains and flows into the Bohai Bay, with the
Biogeochemistry (2017) 132:237–249
basin area of 7.95 9 105 km2. As the largest turbid
river in the world, the average annual SPS concentra-
tion along the Yellow River ranges from 0.57 to
22.1 g L-1 (from 1987 to 2015; Yellow River Sedi-
ment Bulletin). The water quality of the Yellow River,
a drinking water source in northern and northwestern
China, has been threatened by nitrogen pollution
(Wang et al. 2008). The average annual ammonium
concentration at most of the hydrological stations
along the Yellow River was appropriately 1 mg L-1,
with the maximum of 31 mg L-1 (Xia et al. 2004).
In this study, nine sampling sites in the middle and
lower reaches of the Yellow River were selected
(Fig. 1). The surface sediment (0–5 cm depth) and
water samples (0.2 m below the surface) were col-
lected at each site in May 2015. The sediment samples
were placed in sterile plastic bags and, together with
the water samples, were transported to the laboratory
on ice as soon as possible. Concurrent with sample
collection, the temperature, velocity, DO, pH, and
conductivity were measured. For each water sample,
one part was sequentially filtered through polycar-
bonate filters with pore sizes of 0.45 and 0.20 lm
Fig. 1 Locations (red circles) of the nine sampling sites in the
Yellow River, China. Four sites were sampled on the middle
reaches: Wubu (WB) (37°260 51.900 N, 110°420 54.900 E), Longmen
(LM) (35°390 33.900 N, 110°360 06.400 E), Tongguan (TG)
(34°360 27.300 N, 110°160 55.400 E), and Sanmenxia (SMX)
(34°480 02.80 ’N, 111°120 49.60 ’E). Another five sampling sites
239
(Millipore, USA). The filters were frozen (-20 °C) for
DNA extraction and molecular analysis of the anam-
mox bacteria in the SPS phase (0.45 lm) and water
phase (0.20 lm), respectively; another part of the
water samples were used for the determination of
physicochemical properties. Subsamples from the HK
and AS stations were incubated to estimate the
potential anammox activities. Regarding the sediment
samples, one part was stored at -20 °C for DNA
extraction and anammox bacteria quantification, and
another part was used for analysis of physicochemical
parameters. Additionally, subsamples from the KF
station were used to conduct simulation experiments
to explore whether the anammox process could occur
at the SPS–water interface under oxic conditions.
Physicochemical analysis
One part of each water sample was filtered through
0.45-lm pore-size filters for physicochemical analy-
sis. The filters were dried in a freeze drier to constant
weight and then weighed to determine the SPS
concentrations. The concentrations of ammonium
were located at Huayuankou (HYK) (34°540 16.800 N,
113°410 07.700 E), Kaifeng (KF) (34°550 26.500 N,
114°350 22.100 E), Aishan (AS) (36°130 44.400 N, 116°160 39.700 E),
Lijin (LJ) (37°290 20.800 N, 118°160 21.600 E) and Hekou (HK)
(37°500 1.3700 N, 119°020 17.3100 E) in the lower reaches of the
Yellow River. (Color figure online)
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240
and NO 
2 þ NO3 in each water sample were mea-
sured colorimetrically with an Autoanalyser-3 (Bran
& Luebbe, France). The DOC concentration of the
water samples was determined using the TOC analyser
(Shimadzu, Japan). Salinity values of the sampling
sites in the Yellow River were obtained from conduc-
tivities based on transformation equations (Fofonoff
and Millard 1983). As for the sediment samples,
ammonium and NO 
2 þ NO3 were extracted with
2 M KCl and analyzed with the Autoanalyser-3.
Particle size distribution was measured with the
Microtrac S3500 Laser Particles Size Analyzer (Mon-
togmeryville, PA, USA). For the TOC content, the
sediment samples were first treated with HCl (1:1, v/v)
to remove the inorganic carbon and dried in an oven at
60 °C, and were then determined with a Costech ECS
4010 element analyzer.
Molecular analysis
Genomic DNA was extracted from the filters (both
0.20 and 0.45 lm) and 500 mg of each sediment
sample using the FastDNA SPIN Kit for Soil (QBIO-
gene, Carlsbad, CA, USA), according to the manu-
facturer’s instructions. In this study, the anammox
bacterial abundance in the SPS, water and surface
sediment phases was determined by the MPN-PCR
method. The abundance of anammox bacteria in the
overlying water was calculated as the sum of the
anammox bacterial abundance in the water and SPS
phases. The primer set hzsA 526f/hzsA 1857r
(Harhangi et al. 2012), targeting the subunit of the
hydrazine synthase genes of the anammox bacteria,
was used in this study. Detailed information on the
quantification procedures is shown in supporting
information (SI).
Furthermore, a nested PCR method was applied to
retrieve the anammox 16S rRNA genes from the SPS
phase from the HYK and HK stations with the
following PCR primer sets: Pla46f 50 -GGATTAGG-
CATGCAAGTC-30 , 630r 50 -CAKAAAGGAGGT-
GATCC-30 ; Amx368f 50 -TTCGCAATGCCCGAA
AGG-30 , Amx820r 50 -AAAACCCCTCTACT-TAGT
GCCC-30 . Detailed information on the reaction mix-
tures and thermal profiles was reported previously by
Zhu et al. (2013). Then, the PCR products derived
from the HK and HYK stations were purified and
ligated into a pMD18-T vector (TaKaRa, Dalian,
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Biogeochemistry (2017) 132:237–249
China). The inserts with the correct patterns were used
for sequencing analysis with an ABI3730 DNA
Sequencer (USA). All the sequences and their closest
related matches obtained from NCBI-BLAST were
aligned using the Clustal X1.83 program (Thompson
et al. 1997). The sequences sharing 97% similarity
were grouped into one operational taxonomic unit
(OTU) with Mothur software (http://www.mothur.org/
wiki/Main_Page) by the furthest neighbor approach.
The phylogenetic trees were constructed using the
Mega 5 package (Tamura et al. 2011).
Measurement of potential anammox activity
15
N-labeling experiments were performed upon the
arrival of water samples from the HK and AS stations
to determine potential anammox activities. The incu-
bation protocol was slightly modified from the method
described by Dalsgaard et al. (2003). A number of
100-mL unfiltered water samples from each site were
transferred into a series of 120-mL serum vials. Then,
the vials were capped by silicone rubber septa and
crimp-sealed with an aluminum closure (AGI
182,463; Agilent). Each vial was flushed with He for
10 min to remove the background N2 and O2. He-
purged 15NH4Cl (99.0 atm% 15N; Shanghai Research
Institute of Chemical Industry, China) was injected
through the septa into the vials to a final concentration
of 2 mg-N L-1. Another set of assays were sterilized
to serve as a control. Throughout the incubation
period, the vials were shaken at 150 rpm and placed in
the dark at 20 ± 1 °C. These vials were destructively
sampled at 0, 1, 3, and 5 days by injecting 1 mL
saturated solution of mercuric chloride (HgCl2). The
14 15
N N:14N14N ratio of headspace gas was determined
by Delta V isotope ratio mass spectrometry (Thermo
Fisher Scientific, Germany). The potential anammox
activities were estimated from the 29N2 production
during the experiment period using the expressions
described by Holtappels et al. (2011).
Laboratory simulation experiment of anammox
The simulation experiments of anammox were con-
ducted in incubation chambers sealed by rubber
stoppers; the chamber was composed of a polymethyl
methacrylate column and combined with mechanical
agitators to stir the water. Detailed information can be
found in our previous research (Liu et al. 2013). A total
Biogeochemistry (2017) 132:237–249
of 6.4 g (dry weight) homogenized sediment from KF
and sterile pure water were loaded into a series of
incubation chambers with a final SPS concentration of
8 g L-1. Glucose (5 mg-C L-1) was added to simu-
late the DOC concentration in river water (Liu et al.
2013), followed by the addition of 150 mg L-1
allylthiourea (ATU), which can completely inhibit
nitrification activity while having no significant influ-
ence on the anammox in these chambers under oxic
conditions (detailed information for the determination
of optimum ATU concentration can be seen in the SI).
Six hours later, 5 mL stock solution of isotopic
mixture, 14NH4?(99.0 atm% 15N) plus 15 NO 2
(99.0 atm% 15N) and 15NO3- (99.0 atm% 15N), were
mixed into each incubation chamber, leading to a final
concentration of 2 mg-N L-1, 1 mg-N L-1, and
2 mg-N L-1 in the final 800-mL solution, respec-
tively. Experiments with sterile sediment served as
controls, and the microbial activities in the controls
were also inhibited by the addition of HgCl2. Each set
of experiments was performed in triplicate. The
agitation rates were set at 180 rpm to suspend the
sediment, and the incubation temperature was constant
at 20 °C. The incubation period lasted for 20 days
based on our previous studies in the Yellow River (Jia
et al. 2016; Liu et al. 2013). At 10 and 20 days, the
headspace gas of the chambers was collected by a gas-
tight syringe to determine the ratios of 14N15N:14N14N
and 15N15N:14N14N, and subsequently the incubation
chambers were aerated for 10 min to purge the 15N2 of
previous incubation periods and to maintain oxygen
saturation. DO concentrations in the chambers were
determined at the start and end of each incubation
interval.
Results and discussion
Physicochemical properties of water and sediment
samples
The physicochemical properties of water and sediment
samples of the Yellow River are shown in Table 1.
Salinity values of the Yellow River ranged from 0.53
to 1.03 psu (practical salinity units), with the highest
at the HK station, which were relatively higher than
the global mean salinity of river waters (*0.12 psu)
(Wetzel 2001). Dissolved oxygen concentrations of
241
the sampling sites ranged from 7.85 to 10.36 mg L-1.
The DOC concentration of the LM water samples was
about 4- to 6-fold higher than that of other sampling
sites partly resulting from the wastewater input from a
neighboring temporary sewage outlet. The SPS con-
centrations of the sampling sites ranged from 5 to
1440 mg L-1, which were relatively lower than the
average annual average SPS concentrations recorded
in the Yellow River Sediment Bulletin (2015). This
was mainly due to the fact that the water samples were
collected during the dry period of the Yellow River. In
addition, the SPS concentrations in the lower reaches
of the Yellow River (from HYK to HK) were
dramatically higher than those of the middle reaches
(from WB to SMX).
Spatial distribution and abundance of anammox
bacteria in the Yellow River
Distribution of anammox bacteria in the water
and SPS phases
The abundance of anammox bacteria in the water
phase was below the detected limit (Fig. 2), which was
primarily attributable to the inhibitory effect of
oxygen on the anammox bacteria. The anammox
metabolism was believed to be inhibited by oxygen
with a concentration of *20 lM in the marine OMZ
(Kalvelage et al. 2011), which is much lower than the
DO concentrations of all the sampling sites in the
Yellow River (Table 1). Moreover, the anammox
bacteria of the SPS and water phases were obtained in
this study by filtering the overlying water samples
sequentially through 0.45- and 0.20-lm pore-size
filters. However, it was observed that the anammox
bacteria preferentially existed as clusters with diam-
eters of up to 17 lm in a rotating disk contactor (Egli
et al. 2001). This might also be responsible for the
failure to detect the anammox bacteria in the water
phase.
Unlike the water phase, anammox bacteria were
detected in the SPS phase of all sampling sites, with
the abundance ranging from 9.5 9 102 to 1.5 9 104
copies g-1. Mutual effects of aerobic degradation of
organic compounds and diffusion retardation to oxy-
gen on the suspended particle could largely lower the
DO concentration (Stoodley et al. 2002). Our previous
research has revealed net oxygen influx around the
SPS in the oxic water of the Yellow River (Jia et al.
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242 Biogeochemistry (2017) 132:237–249

Table 1 Physicochemical properties of water and sediment samples of the Yellow River
Parametera Values for the sampling sites
WB LM TG SMX HYK KF AS LJ HK

Water sample
NH4-N (mg L-1) 0.25 0.24 0.34 0.76 0.40 0.77 0.36 0.42 0.32
-1
(NO2 þ NO3 )–N (mg L ) 2.82 5.45 6.25 4.60 4.50 4.51 4.45 4.46 4.48
DOC (mg L-1) 3.17 14.05 3.58 3.18 3.32 2.82 2.87 2.35 2.34
Temp (°C) 22.1 22.9 24.7 24.0 18.8 17.6 20.8 22.4 21.9
pH 8.61 8.49 8.16 8.32 8.30 8.32 8.27 8.40 8.33
DO (mg L-1) 8.53 9.38 7.85 8.90 10.36 10.0 9.67 9.22 8.56
Conductivity (lS cm-1) 1370 1250 1060 1070 1100 1090 1090 1890 1890
b
Salinity (psu) 0.73 0.65 0.53 0.54 0.64 0.63 0.59 1.01 1.03
SPS concentration (mg L-1) 100 24 177 5 327 693 1440 454 1230
Flow velocity (ms-1) 0.1 \0.1 \0.1 \0.1 0.3 0.1 0.1 0.3 0.6
Sediment sample
Clay (%) (\0.002 mm) 0 0 0 0 0 0 0 0.04 0.05
Silt (%) (0.002–0.02 mm) 7 1 1 6 6 1 10 26 31
Sand (%) (0.02–2 mm) 93 99 99 94 94 99 99 74 69
NH4-N (mg Kg-1) 44.4 59.45 44.35 44.37 35.92 46.27 34.43 43.75 50.21
(NO  -1
2 þ NO3 )–N (mg L ) 2.02 2.11 2.03 2.45 1.44 2.83 1.97 2.80 2.11
Organic carbon (g Kg-1) 3.95 1.16 0.64 2.88 5.39 0.84 0.89 2.04 3.30
a
Values are means for triplicate water and sediment samples
b
The salinity values are obtained from the conductivity value according to transformation equations (Fofonoff and Millard 1983)

(2007) reported that anammox bacteria existed in the

Namibian upwelling regions with DO up to 30 lM,
60000
and they attributed this to the presence of suspended
particles.
hzs A gene (copies g-1)

56000
SPS
Sediment In addition, a positive correlation was found
52000 between the anammox bacterial abundance of SPS
16000 (copies g-1) and the SPS concentration (P \ 0.01)
12000 (Table S1). It is believed that higher extents of SPS
8000 flocculation would occur with the increase of SPS
4000 concentration (Droppo and Ongley 1994), which
0
would cause a higher depletion ratio of oxygen in
WB LM TG SMX HYK KF AS LJ HK the suspended particles (Alldredge and Cohen 1987).
Fig. 2 Distribution pattern of anammox bacteria in the Yellow This suggests that the anoxic microzones per SPS
River, indicating the abundance in the SPS phase (white) and the would increase with SPS concentration, leading to the
sediment phase (grey). The abundance of anammox bacteria in positive correlation between the anammox bacterial
the water phase of all sampling sites is not shown because the abundance of SPS and the SPS concentration in the
value was below the detection limit (18 copies L-1). Data
represent mean values of three replicates ±SD. In this study, Yellow River.
Figs. 2 and 4 have been made with the Arcgis 10.2 and Mega 5 The anammox bacterial abundance of the overlying
packages, respectively; other figures were created by OriginPro 8 water in the sampling sites ranged from 10 to
1.9 9 104 copies L-1, which are much lower than
2016). This will lead to the formation of anoxic those of the anoxic water column of freshwater lakes
microsites on the SPS, creating a suitable environment (Hamersley et al. 2009; Schubert et al. 2006). This was
for the growth of anammox bacteria. Woebken et al. mainly due to the inhibitory effect of oxygen on the

123
Biogeochemistry (2017) 132:237–249 243

anammox bacteria, although the SPS could provide dissimilatory nitrate reduction to ammonium
shelter for the anammox bacteria in oxic environ- (DNRA), and anammox] were thought to be predom-
ments. However, the abundance of anammox bacteria inantly responsible for such tight correlations when
in the overlying water (copies L-1) (y) increased either ammonium or nitrite was insufficient for the
significantly with SPS concentration (g L-1) (x) as a anammox bacteria (Hu et al. 2012).
power function (y = 8.7237x0.2769) (P \ 0.01)
(Fig. 3). The SPS concentrations during the wet Distribution of anammox bacteria in the sediment
period of the Yellow River are much higher than phase
those of the dry period (Xu 2002). This suggests that
the abundance of anammox bacteria in the overlying The distribution of anammox bacteria in the surface
water during the wet periods would be greater than that sediment phase had distinctive spatial heterogeneity
found in this study (dry period). along the Yellow River (Fig. 2). The anammox
Compared with the significant influence of SPS bacterial abundance of the sampling sites ranged from
concentration on the anammox bacterial abundance 3.2 9 103 to 5.8 9 104 copies g-1 sediment in the
(copies L-1), there was no significant correlation lower reaches (from KF to HK), which were relatively
between NHþ  
4 or NO2 þ NO3 of the overlying water higher than those in the middle reaches. The WB
and the anammox bacterial abundance in the overlying stations harbored the highest anammox bacterial
water (Table S1). This indicates that the NHþ 4 and
abundance in the middle reaches (from WB to
 
NO2 þ NO3 were not the significant factors influ- SMX), which was 9.0 9 102 copies g-1. Correlation
encing the distribution of anammox bacteria. This was analysis revealed that anammox bacterial abundance
mainly because the variation of SPS concentration (5– was positively correlated with the NO 2 þ NO3


1440 mg L-1) was significant in the river water concentration of the sediment (P \ 0.05). This may
(Table 1) and the SPS concentration dominated the indicate that NO 
2 þ NO3 , the potential substrates
distribution of anammox bacteria in the overlying for anammox bacteria, influence the distribution of
water. However, the sediment and OMZ water column anammox bacteria in the sediment. Moreover, positive
are the sites where the anammox bacterial abundance, correlations were found between the anammox bacte-
community structure or potential activity tend to rial abundance and the fine particle (clay and silt)
correlate with NHþ  
4 and/or NO2 þ NO3 (Hou et al.
content of the sediment (P \ 0.01) (Table S2). This
2013; Lam et al. 2009; Minjeaud et al. 2008; Rich et al. makes sense because oxygen renewal should be
2008; Zhu et al. 2013). Interactions of various nitrogen greater in coarser sediments and should therefore
metabolism processes [nitrification, denitrification, reduce anammox activity and abundance.
The surface sediment in the Yellow River had
relatively lower abundance of anammox bacteria,
Ln(Anammox bacteria abundance)

10
compared with that of the Qiantang River sediment of
China (4.9 9 106 to 3.7 9 107copies g-1) (Hu et al.
8
y=8.7237x0.2769 2012). This discrepancy is explainable, especially
R2=0.9698
considering that all samples were collected during the
(copies L-1 )

6
dry season of the Yellow River. During such periods,
4 the water depth is relatively low and the diffusion
capacity of oxygen into the surface sediment would be
2 greatly enhanced. This was supported by the fact that
higher anammox bacterial abundance was observed in
0
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
the lower reaches with a greater water depth. Further-
SPS concentration (g L-1) more, as mentioned above, enhanced oxygen diffusion
into the sediment would occur in coarse sediments.
Fig. 3 Correlation between the anammox bacterial abundance The sediment particle size of the Yellow River was
and SPS concentration of the sampling sites in the middle and
larger than that of the Qiantang River (Jiyu et al.
lower reaches of the Yellow River. Data represent the mean
values of three replicates 1990). This might be another cause for the lower

123
244
anammox bacterial abundance observed in the Yellow
River.
Biodiversity and community structure
of anammox bacteria on the SPS
A total of 100 gene sequences were produced from the
SPS phase of the HYK (50) and HK (50) stations based
on the nested PCR amplification, and a total of 13
OTUs were obtained based on the cutoff of 0.97.
Phylogenetic analysis of 16S rRNA genes showed that
the SPS of HYK station had relatively low anammox
bacteria diversity. Most of the sequences (48 of 50)
were closely affiliated to the Brocadia genera (Fig. 4),
when combined with related sequences deposited in
the Genbank. One sequence shared high similarity
(99%) with Scalindua sp. retrieved from the sediment
of Randers Fjord, Denmark (FM992881). Previous
studies have demonstrated that the Scalindua group
are the most halotolerant of the anammox genera and
primarily exist in the marine environments (Kartal
et al. 2013). However, increasing evidence for the
presence of Scalindua genera in freshwater environ-
ments has been found in recent years (Hamersley et al.
2009; Hu et al. 2012; Moore et al. 2011; Zhao et al.
2013). Among all the freshwater environments
reported for the presence of Scalindua genera, accord-
ing to our knowledge, the conductivity/salinity value
of the HYK station in the present study was the lowest.
The salinity of the anammox zone in Lake Rassnitzer
ranged from ca. 6 to 13 psu and in Lake Tanganyika
was greater than 2 psu (Hamersley et al. 2009;
Schubert et al. 2006), both of which were far higher
than that of the HYK station with the estimated value
of 0.64 psu. Comparable salinity values were found in
the contaminated groundwater of Canada, where the
Scalindua genera were detected at the Z103 and Z106
sites with conductivities of 1675 and 1440 lS cm-1,
respectively. However, these conductivity values are
also relatively higher than that of the HYK station
(1100 lS cm-1). Contrasting with their dominant
distribution in marine environments, the Scalindua
genera existed in these freshwater environments with
relatively low abundance, except for Rassnitzer Lake
with the highest salinity value among these freshwater
environments. Therefore, the salinity is assumed to be
the controlling factor influencing the distribution of
Scalindua genera in freshwater environments. More-
over, some unknown variables might also play
123
Biogeochemistry (2017) 132:237–249
important roles in their distribution, especially con-
sidering the failure to detect the Scalindua genera in
groundwater environments with conductivity values
ranging from 2140 to 2220 lS cm-1 (Moore et al.
2011), which were higher than that of the HYK station
in this study. Systematic studies involved in marine,
freshwater and transition environments (such as
estuary) could provide some insights.
High biodiversity of the anammox bacteria was
discovered in the SPS of the HK station, with all
sequences grouped into 11 OTUs based on the 97%
cutoff. Most of the sequences were also closely related to
the Brocadia genera (40 of 50). A total of eight sequences
showed high similarity (97–99%) to 16S rRNA genes of
the Scalindua genera. The community structure of the
anammox bacteria at the HK station provided implica-
tions of land–sea interactions at this place, especially
considering that the highest conductivity value existed at
the HK station (Table 1). High anammox bacterial
diversity is always observed in estuarine environments
(Dale et al. 2009; Hou et al. 2013), which might result
from the larger variety of suitable niches for anammox
bacteria in this transition zone.
The potential anammox activity of the overlying
water
The potential anammox activities of the overlying
water at the HK and HYK stations were measured
under anaerobic conditions with 15 NHþ 4 (2 mg-
N L-1). There was no significant production of 29N2
in the sterile assay throughout the incubation period,
and the formations of 29N2 were observed without a
lag phase in the incubation experiments (Fig. 5). The
calculated results showed that the potential anammox
activities at the HK and HYK stations were 0.095 and
0.057 nmol-N L-1 day-1, respectively, with an aver-
age of 0.076 nmol-N L-1 day-1. These potential rates
were much lower than those of freshwater lakes
(Tanganyika and Rassnitzer) (Hamersley et al. 2009;
Schubert et al. 2006), especially Lake Tanganyika
with anammox rates up to 10 nM h-1. This difference
might be primarily attributable to larger anammox
bacterial abundance in these lakes. Based on the
results of fluorescence in situ hybridization analysis,
600–13,000 and 2.7–5.2 9 104 cells mL-1 were,
respectively, identified as anammox bacteria in the
water columns of the Tanganyika and Rassnitzer
Biogeochemistry (2017) 132:237–249 245

99 OTU01|HK(22/50);HYK(34/50)
57 Ca.Brocadia fulgida (KU217748.1)
OTU05|HK(4/50);HYK(1/50)
82
98 paddy soil (KJ508639.1)
OTU02|HK(6/50);HYK(8/50)
36 99 Lvshuiwan wetland soils (KT162139.1)
53 OTU03|HK(5/50);HYK(4/50)
OTU06|HK(2/50);HYK(1/50)
30
55 OTU09|HK(1/50);HYK(0/50)
100 Unvegetated soil (KM095318.1)
OTU07|HK(2/50);HYK(0/50)
77 73 KU217839.1 (Yangtze Estuary sediment)
99 OTU04|HK(5/50);HYK(0/50)
60 Ca.Scalindua brodea (KM925748.1)
OTU11|HK(1/50);HYK(0/50)
83 OTU08|HK(1/50);HYK(0/50)
47
Yangtze Estuary sediment (KU218214.1)
99 OTU12|HK(1/50);HYK(0/50)
56
Ca.Scalindua wagneri (KU218283.1)
99 OTU13|HK(0/50);HYK(1/50)
91 Ca. Scalindua sp. (FM992881.1)
OTU10|HK(0/50);HYK(1/50)
87 hypersaline groundwater (JF747636.1)
Planctomyces (EU360295)

0.05

Fig. 4 Phylogenetic trees showing the affiliations of the
anammox bacterial 16S rRNA gene sequences retrieved from
the SPS phase of the HK and HYK stations of the Yellow River.
The tree was constructed based on the neighbor-joining method
using the Kimura two-parameter distance. Bootstrap values
were 1000 replicates. The clones of the HK and HYK stations in
each operational taxonomic unit are indicated. The clone names
are labeled with the sample name followed by the number of
times a sequence was detected among all tested clones of a
sample. The sequences obtained in this study are accessible in
Genbank under Accession Numbers KX244502–KX244601. In
this study, Figs. 2 and 4 have been made with the Arcgis 10.2
and Mega 5 packages, respectively; other figures were created
by OriginPro 8

2.5 1.5
HK
N2 (nmol vial -1)

Concentration of 29 N2 (nmol vial-1)

AS
2.0 Incubation with 15NH4+ 1.2 Incubation with 15 NH 4+
Control
Control
1.5 0.9
29
Concentration of

1.0 0.6

0.5 0.3

0.0 0.0
0 1 2 3 4 5 0 1 2 3 4 5

Time (d) Time (d)

Fig. 5 Production of 14N15N in the incubation of overlying water samples collected from the HK and AS stations in the Yellow River.
Control represents the sterile experiments. Data represent the mean values of three replicates ±SD

123
246
lakes. Moreover, the possible formation of 28N2
through co-occurrence of micro-aerobic nitrification
and micro-aerotolerant anammox (Holtappels et al.
2011) was not considered in the calculation of the
anammox rates. Therefore, there would be an under-
estimate of the potential anammox activities of the
overlying water in the Yellow River.
However, the cell-specific activities showed active
metabolism of anammox bacteria in the overlying
water of the Yellow River. Based on the assumption
that the hzsA gene is specific to anammox bacteria and
each genome only contains one gene copy (Harhangi
et al. 2012), the specific cellular anammox activity was
calculated based on the anammox bacterial abundance
and activity. The cell-specific activities at the HK and
HYK stations were 5.06 and 5.60 fmol L-1 day-1,
respectively, which were comparable with that of the
Benguela upwelling system (4.5 fmol L-1 day-1)
(Kuypers et al. 2005). In addition, the cell-specific
activities of HYK and HK stations were approximately
nine times higher than that of Lake Rassnitzer
(Hamersley et al. 2009). Such a discrepancy might
be due to the fact that the temperature in Lake
Rassnitzer (8 °C) was lower than that at the HYK and
HK stations (19 and 22 °C, respectively). In addition,
substrate concentration might be another reason for
the difference in cell-specific anammox rates between
the Yellow River and Lake Rassnitzer. NO  NO3 ) and ATU, the DO concentration ranged from
2 þ NO3
concentrations in the overlying water of the HYK and
HK stations were *4.5 mg-N L-1 (321 lM), while it
was only 8.3 or 20.8 lM in Lake Rassnitzer, and
15
NHþ 4 2 mg-N L
-1
(143 lM) in this study, which was
4.3- to 17.2-fold higher than that in Lake Rassnitzer.
To compare the potential anammox rates in the
overlying water with that in the sediment of the
Yellow River, it was assumed here that the cell-
specific anammox activity in the sediment of the
Yellow River was similar to that of the Yangtze
estuary (5.18 fmol N day-1) (Hou et al. 2013) and that
the hot zones of anammox in the sediment was
0–10 cm (Zhu et al. 2013). For the middle and lower
reaches of the Yellow River, the dry sediment density
was 1.1 g cm-3 (Li et al. 1998), with an average water
area of 996 km2 (average river width = 500 m,
length = 1992 km) and average water depth of 3 m.
Average anammox bacterial abundance in the sedi-
ment of the middle and lower reaches of the Yellow
River was 9.7 9 103 copies/g. The annual nitrogen
123
Biogeochemistry (2017) 132:237–249
loss from anammox in the sediment was estimated to be
28.13 t N (5.18 fmol N day-1 9 9.7 9 103 copies/
g 9 1.1 g cm-3 9 14 g mol-1 9 996 km2 9 10 cm)
in the middle and lower reaches of the Yellow River,
and 1.16 t N (0.076 nmol-1 L-1 day-1 9 14 g
mol-1 9 996 km2 9 3 m) could be removed from
the anammox in the overlying water on an annual basis.
The ratio of overlying water to sediment in N2
production through anammox was estimated to be
1:24.24, which means the overlying water can cover 4%
of N2 production through anammox in the middle and
lower reaches of the Yellow River. Because the SPS
distribution, water and active bed-sediment depth in the
Yellow River spatiotemporally varied, leading to the
variation of anammox rates, our current knowledge of
anammox in turbid rivers is far too preliminary to put
much confidence in this estimate. However, this
estimate does indicate that the anammox in the
overlying water might be an important pathway of
nitrogen loss in the Yellow River.
Occurrence of anammox at SPS-water interfaces
in oxic water
During the simulation experiments conducted with
15
N-labeled nitrogen compounds (14 NHþ 15 
4 , NO2 and
15 
7.32 to 7.47 mg L-1 at the end of the incubation
intervals. In the incubation chambers, the anammox
and denitrification processes were the only pathways
of 29N2 production. Sterile experiments were con-
ducted to examine the effect of abiotic processes on
the production of 29N2. Anammox bacteria would
convert 14 NHþ 4 with
15
NO
2 into
29
N2 (14 NHþ 4 þ
15  29
NO2 ! N2 ). With the addition of ATU, the
production of 29N2 through coupled nitrification and
denitrification could be avoided. For the denitrification
process, it would mainly reduce 15 NO 15
2 and NO3 to

produce N2 (15 NO
30
3 !
15
NO2 !
30
N2 ). How-
14  14 
ever, NO2 and NO3 could be introduced into the
chambers by the addition of sediment and 15N-labeled
nitrogen compounds (15N at 99%). This means that
there would be 29N2 produced from denitrification in
our chambers, and that this part of 29N2 could be
calculated based on the assumption that the 30N2 was
completely produced from denitrification (detailed
information is shown in the SI). Therefore, the
Biogeochemistry (2017) 132:237–249
anammox rates could be corrected by subtracting the
29
N2 produced by denitrification from the total 29N2
produced in this study. In addition, the DNRA process
might occur on the SPS in the chambers, leading to the
conversion of 15 NO 2 and
15
NO3 into
15
NHþ4 , and
subsequently the anammox would combine 15 NHþ
and 15 NO 2 into
30
N2. Therefore, there might be an
overestimate of the production of 29N2 from denitri-
fication, and consequently an underestimate of that
from the anammox.
Furthermore, as mentioned in ‘‘Laboratory simula-
tion experiment of anammox’’, the incubation cham-
bers were purged with ambient air before each
experiment interval. The 28N2 concentration of the
headspace gas was assumed to be constant. So any
difference in the ratios of 29N2 to 28N2 between the
headspace gases of non-sterile and sterile chambers
was caused by the anammox and denitrification
processes during the incubation period, and the
anammox was further corrected by subtracting the
ratio of 29N2 to 28N2 produced from denitrification.
As shown in Fig. 6, the ratios of 29N2 to 28N2 in the
non-sterile chambers caused by the anammox were
higher than those of the sterile chambers on the 10th
day, but there was no significant difference through a
one-tailed t test (P [ 0.1). This was predictable be-
cause the SPS was simulated by the surface sediment
collected from KF in suboxic/anoxic conditions, but
the simulation system was under oxic conditions
through incubation periods. The lag phase periods
would be required for microbes to adapt themselves to
new conditions. However, the ratios of 29N2 to 28N2 of
the non-sterile chambers were higher than those of the
sterile chambers with a significance level of 0.1
(P = 0.077) on the 20th day. This indicated the
occurrence of anammox at the SPS–water interface
under oxic conditions.
The results obtained in this research suggest that the
SPS could provide suitable micro-environments for the
anammox even in oxic conditions. The anoxic zones on
the SPS mainly result from the active aerobic degra-
dation of organic compounds. It has been reported that
microzones of nutrient enrichment on suspended
particles would attract microorganisms and support
high metabolic activity, leading to the formation of
oxygen-depleted microzones around the SPS (All-
dredge and Cohen 1987). This provides microenviron-
ments for the anammox as well as denitrification. Our
247
Sterile
0.00730 non-Sterile
0.00725
28
N2
0.00720
Ratios of 29N2 to
4 0.00715
0.00710
0.00705
0.00700
10 20
Time (d)
Fig. 6 Average ratios of 29N2 to 28N2 of the headspace gas in
the incubation chambers on the 10th and 20th days. Data
represent the mean values of three replicates ±SD; the data
were corrected by the ratios of 29N2 to 28N2 caused by
denitrification in the non-sterile chambers
previous study has shown that the denitrifier abun-
dance and denitrification rate were positively corre-
lated with the TOC content of SPS (Jia et al. 2016).
Compared with the Yellow River, most of the inland
rivers in the world have lower concentrations of SPS,
but higher TOC contents in the SPS (Ludwig et al.
1996; Bianchi et al. 2002; Tesi et al. 2013; Xia et al.
2016). This may indicate that there would be more
anoxic microzones in the SPS in other rivers to support
the occurrence of the anammox process. However, it
remains to be determined if anammox on the SPS is an
important nitrogen loss route in the Yellow River as
well as in other rivers around the world.
Conclusions
The Yellow River was selected to investigate the
potential roles of anammox at the SPS–water interface
in natural rivers. The results show that the anammox
bacteria in the overlying water of the Yellow River
were attached to the SPS, and the anammox bacterial
abundance in the overlying water increased with the
SPS concentration (P \ 0.01). The abundance of
anammox bacteria in the SPS phase (copies g-1)
increased with SPS concentration (P \ 0.01). Phylo-
genic analysis of anammox bacteria revealed that the
SPS phase was dominated by Brocadia. Scalindua
genera were detected in the Yellow River with a
conductivity of 1100 lS cm-1, which is the lowest
salinity/conductivity value reported so far for the
123
248
presence of the Scalindua genera. Moreover, 15 NHþ 4-
amended incubations of the unfiltered overlying water
from the Yellow River under anoxic conditions
showed that the average potential anammox activity
was 0.076 nmol-N L-1 day-1, and that the cell-
specific anammox activities at the HK and HYK
stations were 5.06 and 5.60 fmol L-1 day-1, respec-
tively. Furthermore, 15N-labeling simulation experi-
ments demonstrated the occurrence of anammox in the
oxic water containing SPS. This study suggests that
anammox could occur at the SPS–water interface in
natural oxic freshwaters, but it remains to be deter-
mined whether anammox in the SPS is an important
nitrogen loss route in the Yellow River as well as
in other rivers around the world.
Acknowledgements This study is financially supported by the
National Science Foundation of China (91547207), the National
Science Foundation for Distinguished Young Scholars
(51325902), and the Fund for Innovative Research Group of
the National Natural Science Foundation of China (51421065).
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