PUBLICATIONS
Journal of Geophysical Research: Oceans
RESEARCH ARTICLE
10.1002/2014JC010554
Bingbing Fu and Jiwen Liu contributed
equally to this work.
Shift of anammox bacterial community structure along the
Pearl Estuary and the impact of environmental factors
Bingbing Fu1, Jiwen Liu1, Hongmei Yang2, Ting Chang Hsu3, Biyan He4, Minhan Dai3, Shuh Ji Kao3,
Meixun Zhao2, and Xiao-Hua Zhang1
1
Special Section:
Pacific-Asian Marginal Seas
Key Points:
Shift in anammox bacterial
community was observed along the
Pearl Estuary
Four novel genera of anammox
bacteria were identiﬁed
Anammox bacterial abundance
related positively with NO2
2 and
inversely with DO
Supporting Information:
Supporting Information S1
Figure S1
Figure S2
Table S1
Table S2
Correspondence to:
X.-H. Zhang,
[email protected]
Citation:
Fu, B., J. Liu, H. Yang, T. C. Hsu, B. He,
M. Dai, S. J. Kao, M. Zhao, and
X.-H. Zhang (2015), Shift of anammox
bacterial community structure along
the Pearl Estuary and the impact of
environmental factors, J. Geophys. Res.
Oceans, 120, 2869–2883, doi:10.1002/
2014JC010554.
Received 30 OCT 2014
Accepted 16 MAR 2015
Accepted article online 23 MAR 2015
Published online 15 APR 2015
C 2015. American Geophysical Union.
V
All Rights Reserved.
FU ET AL.
College of Marine Life Sciences, Ocean University of China, Qingdao, China, 2Key Laboratory of Marine Chemistry Theory
and Technology, Ministry of Education, Ocean University of China, Qingdao, China, 3State Key Laboratory of Marine
Environmental Science, Xiamen University, Xiamen, China, 4School of Bioengineering, Jimei University, Xiamen, China
Abstract Anaerobic ammonium oxidation (anammox) plays an important role in the marine nitrogen
cycle. The Pearl Estuary, a typical subtropical estuary characterized by hypoxia upstream and high loads of
organic matter and inorganic nutrients caused by anthropogenic activities, has received extensive attention.
In this study, anammox bacterial community structures in surface sediments along the Pearl Estuary were
investigated using 16S rRNA and hydrazine oxidoreductase (HZO) genes. In addition, abundance of anammox bacteria in both water and surface sediments was investigated by quantitative PCR. Obvious anammox
bacterial community structure shift was observed in surface sediments, in which the dominant genus
changed from ‘‘Candidatus Brocadia’’ or ‘‘Candidatus Anammoxoglobus’’ to ‘‘Candidatus Scalindua’’ along the
salinity gradient from freshwater to the open ocean based on 16S rRNA gene and HZO amino acid phylotypes. This distribution pattern was associated with salinity, temperature, pH of overlying water, and particularly C/N ratio. Phylogenetic analysis unraveled a rich diversity of anammox bacteria including four novel
clusters provisionally named ‘‘Candidatus Jugangensis,’’ ‘‘Candidatus Oceanicum,’’ ‘‘Candidatus Anammoxidans,’’ and ‘‘Candidatus Aestuarianus.’’ The abundance of anammox bacteria in surface sediments, bottom
and surface waters ranged from 4.22 3 105 to 2.55 3 106 copies g21, 1.24 3 104 to 1.013105 copies L21,
and 8.073103 to 8.863105 copies L21, respectively. The abundance of anammox bacteria in the water col2
umn was positively correlated with NO2
2 and NO3 , and negatively correlated with dissolved oxygen,
although an autochthonous source might contribute to the observed abundance of anammox bacteria.
1. Introduction
The anammox reaction was discovered in bioreactor sludge of a wastewater treatment plant in the Netherlands two decades ago [Mulder et al., 1995]. The signiﬁcance of anammox bacteria has been increasingly
recognized ever since because of their wide presence in oxygen-depleted estuarine and marine sediments [Thamdrup and Dalsgaard, 2002; Risgaard-Petersen et al., 2004; Penton et al., 2006; Zhang et al.,
2007], wastewater [Schmid et al., 2003; Kartal et al., 2008; Quan et al., 2008], marine oxygen minimum
zones [Dalsgaard et al., 2003; Kuypers et al., 2003, 2005; Lam et al., 2007], multiyear sea ice [Rysgaard and
Glud, 2004], and even in geothermal subterranean oil reservoirs [H. Li et al., 2010] and black smokers
2
[Byrne et al., 2009]. Anammox removes bioavailable nitrogen through NH1
4 oxidation coupled with NO2
reduction and likely accounts for up to 50% of the total N2 production in marine environments [Arrigo,
2005]. Anammox bacteria belong to the phylum Planctomycetes [Strous et al., 1999] and have a unique
compartmentalized cell consisting of anammoxosome, riboplasm, and paryphoplasm [van Niftrik et al.,
2004]. Recently, a protein surface layer (a crystalline array of protein subunits) was described as the outermost layer of the anammox cell, which was the latest addition to the cell structure [van Teeseling et al.,
2014]. Nevertheless, due to the limitation of traditional microbiological isolation and cultivation techniques, anammox bacteria are not yet in pure culture, which hampers further understanding about anammox bacteria in various perspectives. At present, anammox bacteria belong to ﬁve recognized genera and
16 species [Sonthiphand et al., 2014]; and ‘‘Candidatus Scalindua zhenhei’’ I, II, III, and ‘‘Candidatus Scalindua sinooilﬁeld,’’ which were retrieved from the South China Sea and petroleum reservoirs, respectively
[H. Li et al., 2010; Hong et al., 2011], were the four new species afﬁliated to the ‘‘Candidatus Scalindua’’
genus.
ANAMMOX BACTERIA ALONG THE PEARL ESTUARY
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Journal of Geophysical Research: Oceans
Figure 1. Location map of sampling sites in the Pearl Estuary, modiﬁed from Liu et al.
[2014b].
10.1002/2014JC010554
Fluorescent in situ hybridization
(FISH) and PCR ampliﬁcation targeting the 16S rRNA genes of
anammox bacteria have been
main methods for anammox bacteria detection and community
analyses [Schmid et al., 2005].
However, due to the highly conservative nature of the 16S rRNA
genes, and biased coverage and
speciﬁcity of most PCR primers,
anammox bacterial diversity may
not be fully characterized [Amano
et al., 2007; Byrne et al., 2009].
Hydrazine oxidoreductase (HZO), a
key player in the anammox biochemical process [Klotz and Stein,
2008], can oxidize hydrazine to N2
[Schalk et al., 2000]. The hzo gene
has been used as an alternative
functional
and
phylogenetic
marker for anammox bacteria
[Schmid et al., 2008; Li et al., 2009,
M. Li et al., 2010], and can verify
and improve the 16S rRNA genebased methods.
Estuaries, the ecotones between riverine and marine ecosystems, are characterized by complex natural and
anthropogenic environmental gradients, which make them ideal candidates for investigating the microbial
response to environmental changes. Signiﬁcant anthropogenic loads into estuaries strongly stimulate
microbial respiration, resulting in excessive O2 consumption and subsequent hypoxia [Dai et al., 2006]. As a
consequence, many anaerobic processes such as anammox and denitriﬁcation involved in N-nutrients
removal become prevalent, upgrading the role of estuaries in the global N budget [Capone et al., 2008].
Although both anammox and denitriﬁcation reactions play important roles in removal of the anthropogenic
excess nitrogen in estuarine environments, anammox reduces the production of greenhouse gases (N2O
and CO2) compared to denitriﬁcation [Ward et al., 2013], making their ecological consequences and environmental responses entirely different. The Pearl River, which ranks 2nd in terms of discharge volume in China,
ﬂows through large catchment areas into the Pearl Estuary (Figure 1) and ﬁnally reaches the South China
Sea, the largest marginal sea on the western boundary of the Paciﬁc Ocean [Dai et al., 2013]. The Pearl Estuary is located in one of the densely populated and industrialized areas in China [Ding et al., 2004; Dai et al.,
2006, 2008]. As receiving increasing wastewater discharge from anthropogenic activities (ﬁsheries, agriculture, industry, etc.), the Pearl Estuary carries a high load of dissolved inorganic nitrogen (DIN) and phosphate (PO32
4 ), and eutrophication was the most outstanding environmental problem in this area [Huang
et al., 2003; Cai et al., 2004; Dai et al., 2008]. The wide spectrum of environmental conditions in the Pearl
Estuary as documented previously [Liu et al., 2014a] may support a diverse community of anammox bacteria. Our study therefore aims to examine the abundance, diversity, and spatial distribution of anammox bacteria along a subtropical estuary through constructing 16S rRNA and hzo genes clone libraries and
quantitative PCR (Q-PCR) technology. This study may provide new insights into the ecology of anammox
bacteria in estuarine ecosystems.
2. Materials and Methods
2.1. Site Description, Sampling, and DNA Extraction
Sediment and water samples were collected during a cruise in summer 2012 from nine sites along the salinity gradient of the Pearl Estuary (Figure 1). Surface sediments (0–2 cm) were collected individually using a
FU ET AL.
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10.1002/2014JC010554
box corer. At each site, near surface (1 m below surface) and bottom (1–2 m above bottom) waters were collected and processed as reported previously [Liu et al., 2014a]. All the samples were stored at 280 C immediately until laboratory analysis. The nine stations were divided into three groups: freshwater (P01, P03, and
P07), low-salinity (A04, A08, C2, and C3), and seawater sites (F412 and F414) (Figure 1).
2
32
1
Nutrients (NO2
3 , NO2 , NH4 , and PO4 ), chlorophyll a (Chl-a), salinity, and dissolved oxygen (DO) of the
water samples were measured according to Dai et al. [2008]. Depth, temperature, pH, and turbidity of the
water samples were obtained by CTD (SBE 25 Sealogger, Sea-Bird Electronic, Inc., Washington, USA) [Liu
et al., 2014a]. The sediment pore water was extracted using the Rhizon Soil Moisture Samplers (19.21.23F
2
1
Rhizon CSS, Netherlands) [Song et al., 2013; Seeberg-Elverfeldt et al., 2005] and nutrients (NO2
3 , NO2 , NH4 ,
32
42
PO4 , and SiO4 ) therein were measured using an AutoAnalyzer3 (Seal Analytical, Mequon, WI). Total
organic carbon (TOC) and total nitrogen (TN) were measured using a Thermo Flash2000 OEA Analyzer
(Thermo Fisher Scientiﬁc, Waltham, MA, USA) as described previously [Ge et al., 2013; Liu et al., 2014b].
Sediment DNA was extracted using the PowerSoil DNA Kit (MoBio Laboratories, Inc., Carlsbad, CA) according
to the manufacturer’s manual. Water DNA was extracted following the method described by Yin et al. [2013]
with several modiﬁcations. Brieﬂy, the sample membranes were put into sterile 1.5 mL tubes, and 600 mL of
STE buffer and 30 mL of lysozyme (10 mg mL21) were added before incubation at 37 C for 30 min. Then, 60
mL of 10% (w/v) sodium dodecyl sulfate (SDS) and 6 mL of proteinase K (10 mg mL21) were added into the
tubes, and the mixture was incubated at 65 C for 20 min. The concentration of DNA extracted from sediments and waters were measured with a NanoDrop spectrometer (Thermo Scientiﬁc, Wilmington, DE, USA).
2.2. PCR Amplification
A nested PCR assay was performed to amplify anammox bacterial 16S rRNA genes as described previously
[Dale et al., 2009]. The initial ampliﬁcation was conducted with the primers Pla46F and 1307R under the
thermal proﬁle: 94 C for 4 min, followed by 30 cycles of 95 C for 45 s, 59 C for 50 s, and 72 C for 3 min. The
nested PCR was performed by using the primers Amx 368F and Amx 820R with the condition: 94 C for 4
min followed by 30 cycles of 45 s at 95 C, 50 s at 59 C, and 1 min at 72 C. The 50 mL PCR reaction included
10 mL of 5 3 Go Taq Flexi buffer (Promega, USA), 4 mL of dNTPs (2 mM, TOYOBO Co., Japan), 4 mL of MgCl2
(25 mM, Promega), 3 mL of DNA (15–25 ng mL21), 1 mL of bovine serum albumin (BSA, 100 mg mL21, Takara),
0.5 mL of each primer (50 mM), 0.5 mL of Go Taq DNA Polymerase (5 U mL21, Promega) and 26.5 mL of distilled water.
The hzo gene fragments were ampliﬁed using a nested PCR approach with the primers hzoAB1F/hzoAB1R
and hzoAB4F/hzoAB4R [Hirsch et al., 2011]. The PCR mixture contained a ﬁnal volume of 50 mL: 5 mL of
53Go Taq Flexi buffer, 5 mL of dNTPs (2 mM), 4 mL of MgCl2 (25 mM), 4 mL of DNA (15–25 ng mL21), 2 mL of
BSA (100 mg mL21), 0.5 mL of each primer (50 mM), 0.5 mL of Go Taq DNA Polymerase (5 U mL21), and
28.5mL of distilled water.
2.3. Clone Libraries Construction, Sequencing, and Phylogenetic Analyses
PCR products were electrophoresed on 1% agarose gels and then puriﬁed using the Agarose Gel Puriﬁcation Recovery Kit (Biomed Co., Beijing, China), following the manufacturer’s instructions. Later, the puriﬁed
fragments were inserted into pUCm-T vectors (Sangon Biotech Co., Shanghai, China) and Escherichia coli
TOP10 competent cells were used as transformers. Transformants were selected using Xgal-IPTG LB plates
with 100 mg mL21 ampicillin. Approximately 50 positive clones were randomly selected from each library
and the correct insertions were checked by PCR ampliﬁcation with primers M13F and M13R before ﬁnally
sequencing.
The 16S rRNA gene sequences and the deduced amino acid sequences of hzo genes were aligned with the
ClustalX program [Thompson et al., 1997; Larkin et al., 2007]. Operational taxonomic units (OTUs) of 16S
rRNA gene and HZO amino acid sequences were deﬁned at a 3% distance level, using the DOTUR program
[Schloss and Handelsman, 2005]. Rarefaction, Chao I and Shannon indices were calculated for each library.
Before constructing the 16S rRNA gene and the HZO amino acid phylogenetic trees, the sequences with
97% nucleotide similarity and 97% amino acid similarity were grouped into one representative sequence,
respectively. Phylogenetic trees were constructed by the neighbor-joining algorithm [Saitou and Nei, 1987]
with Kimura-2 parameters and P-distance methods, respectively [Kimura, 1980] and followed by 1000 bootstrap replicates using the MEGA software (version 4.0) [Tamura et al., 2007].
FU ET AL.
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Journal of Geophysical Research: Oceans
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2.4. Q-PCR Assay
Total bacterial and anammox bacterial 16S rRNA gene copy numbers of surface sediments, surface, and bottom waters at each site were quantiﬁed with an ABI 7500 sequence detection system (Applied Biosystems,
Foster City, CA, USA) using a SYBR green method [Hamersley et al., 2007; Dang et al., 2010a, M. Li et al.,
2010]. Q-PCR was performed in triplicate with the primers 341F and 518R [Shinkai and Kobayashi, 2007] for
total bacterial 16S rRNA genes and the primers AMX-808-F and AMX-1040-R [Dale et al., 2009] for anammox
bacterial 16S rRNA genes. The 20 mL Q-PCR mixture contained 10 mL of 23SYBR Premix II (TaKaRa, Tokyo,
Japan), 6 mL of H2O, 0.8 mL of each primer (10 mM), 0.4 mL of Rox reference dye II, and 2 mL of template DNA
(0.015–0.025 ng mL21 and 15–25 ng mL21 of bacterial DNA and anammox bacterial DNA, respectively). For
total bacterial 16S rRNA genes, the Q-PCR cycle was started with 95 C for 2 min, followed by 40 cycles of
94 C for 5 s, annealing for 20 s at 60 C, and extension for 45 s at 72 C [Dang et al., 2010b]. For anammox
bacterial 16S rRNA genes, the Q-PCR protocols were performed as follows: 95 C for 3 min, followed by 45
cycles of 95 C for 30 s, 55 C for 30 s, and 72 C for 30 s. Standard plasmids containing the total bacterial and
anammox bacterial 16S rRNA genes were generated by cloning with the same primers and condition of QPCR. Standard curves were obtained with tenfold serial dilutions of standard plasmids. The speciﬁcity of the
Q-PCR ampliﬁcation was conﬁrmed with melting curve and gel electrophoresis analyses. In all experiments,
negative controls containing no template DNA were performed under the same Q-PCR procedure to detect
and exclude any possible contamination or carryover.
2.5. Statistical Analyses
Comparison of anammox bacterial assemblages was determined with principal coordinates analysis (PCoA)
embedded in UniFrac [Lozupone et al., 2006, 2007]. Correlations between anammox bacterial community
structures in surface sediments and environmental parameters of the bottom water were explored by
canonical correspondence analysis (CCA) using the software CANOCO version 4.5 [Dang et al., 2010a,
2010c]. The Pearson correlation between the abundance of total bacterial and anammox bacterial 16S rRNA
genes and environmental factors was determined using the R base package [Ihaka and Gentleman, 1996].
2.6. Nucleotide Sequence Accession Numbers
The 16S rRNA and hzo gene sequences have been deposited in the GenBank database under the accession
numbers KF897027 to KF897508 and KF934768 to KF935222, respectively.
3. Results
3.1. Environmental Characteristics
2
2
32
Signiﬁcantly higher (about 3–30 times) concentrations of nutrients (NH1
4 , NO2 , NO3 , and PO4 ) and Chl-a,
and lower DO and pH values were observed in the freshwater sites compared to the seawater sites (Table 1)
[Liu et al., 2014a] consistent with previous reports [Dai et al., 2008]. The water salinity varied from 0 to 33.6
across the nine sites. The water depth of the sampling sites ranged from a few meters to 27 m at the deepest. In the downstream estuary, the surface water had a lower salinity resulting in a vertical stratiﬁcation
(typical salt wedge phenomenon), but a higher NO2
3 concentration than the bottom water. Severe hypoxia
was found at freshwater sites (DO level <1 mg L21) except for P03 (3.14–3.47 mg L21). At the seawater sites,
bottom water had a lower DO content compared to surface water but did not reach hypoxia. Although
2
2
32
42
there were no signiﬁcant differences in nutrient (NH1
4 , NO2 , NO3 , PO4 , and SiO4 ) concentrations
between the freshwater and seawater sediments, the concentrations of nutrient at site P01 was signiﬁcantly
higher than at all other sites (Table 2). The sedimentary TOC and C/N ratio showed decreased trends along
the gradient from freshwater to the open ocean, despite of similar TN values in all sites (Table 2) [Liu et al.,
2014b].
3.2. Diversity of Anammox Bacterial 16S rRNA Gene and HZO Amino Acid Sequences in Surface
Sediments
The 16S rRNA gene and the deduced HZO amino acid sequences of anammox bacteria retrieved from sediment samples were analyzed by Blast search, which conﬁrmed that all the clones represented anammoxlike sequences. Nine anammox bacterial 16S rRNA gene clone libraries of the Pearl Estuary sediment samples were constructed and in total 482 sequences were obtained, representing 426 unique sequences and
42 OTUs at a 3% distance level. The rarefaction analysis of all libraries indicated that anammox bacteria
FU ET AL.
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Table 1. Environmental Parameters of the 18 Water Samplesa
Samples
Depth
(m)
T
( C)
S
pH
Turb.
(FTU)
DO
(mg L21)
Chla
(lg L21)
PO32
4
(lmol L21)
NO2
3
(lmol L21)
NO2
2
(lmol L21)
NH1
4
(lmol L21)
P01_0b
P01_2b
P03_0b
P03_2b
P07_0b
P07_2b
A04_0
A04_2
A08_0b
A08_2b
C2_0b
C2_2b
C3_0
C3_2
F412_0b
F412_2b
F414_0
F414_2
1
7
1
3
1
19
1
7
1
13
1
6
1
6
1
14
1
27
30.14
30.10
29.61
29.61
29.35
29.32
29.72
28.81
28.34
26.45
28.31
27.22
28.54
26.47
27.50
26.39
28.61
25.36
0.00
0.00
0.00
0.00
0.00
0.10
1.15
6.74
16.50
31.50
18.66
28.34
7.84
25.44
23.50
31.41
23.10
33.60
7.01
7.01
7.24
7.21
6.99
6.98
7.65
7.46
8.04
8.09
8.01
8.03
7.99
7.98
8.11
8.13
8.24
8.07
24.10
24.11
24.10
24.08
22.14
24.11
24.12
24.11
5.26
23.42
9.08
24.11
NA
16.23
3.90
7.82
3.42
4.24
0.22
0.51
3.47
3.14
0.72
0.62
6.05
4.68
6.45
4.68
5.48
4.09
6.82
4.89
6.95
5.39
7.59
3.85
16.45
17.36
5.16
6.45
11.54
9.73
4.90
3.37
4.81
0.91
1.55
1.13
1.10
0.16
4.29
NA
4.46
NA
4.8
4.7
2.0
2.2
2.0
2.0
1.2
1.5
1.0
0.5
1.0
0.8
1.5
1.0
0.9
0.4
0.2
0.4
78.6
96.1
102.5
103.0
105.0
106.4
92.6
101.7
61.5
4.4
54.3
18.5
92.2
22.6
36.9
4.4
35.6
3.1
14.8
14.7
24.6
26.4
43.1
41.9
8.4
14.1
8.5
4.6
6.5
4.9
7.3
3.3
4.2
1.8
4.5
1.0
139.7
140.1
21.4
30.6
34.4
31.1
2.2
8.3
0.8
ND
1.1
ND
8.2
ND
ND
ND
ND
0.9
a
Abbreviation: T, Temperature; S, salinity; Turb, Turbidity; NA, no analysis; ND, nondetectable.
Data reported by Liu et al. [2014a].
b
were mostly represented in these clone libraries (supporting information Figure S1a). Based on Shannon
and Chao I diversity indices, site C3 exhibited the highest OTU diversity and the lowest diversity was found
at sites A08 and F412. In addition, anammox bacteria showed high diversity in freshwater and low-salinity
sites (except for site A08) and low diversity in seawater sites (Table 3).
In total, 455 sequences were obtained from the nine anammox bacterial hzo gene clone libraries of the
Pearl Estuary sediment samples. The deduced amino acid sequences of the clones comprised 455 unique
sequences distributed over 66 OTUs at a 3% cutoff value. The rarefaction analysis of all libraries indicated
that the majority of phylotypes were detected (supporting information Figure S1b). Chao I estimator and
Shannon index calculated based on HZO amino acid sequences exhibited similar variations in anammox
bacterial diversity compared with that calculated using 16S rRNA gene sequences. Sites A08 and C2 had the
lowest and highest diversity, respectively. However, sites P01, P07, and F412 showed higher diversity in
HZO diversity calculation (Table 3). Thus, the diversity indices based on 16S rRNA gene and HZO amino acid
sequences both indicated differential diversity distribution of anammox bacteria across all the sites.
3.3. Phylogeny of Anammox Bacterial 16S rRNA Gene Sequences
The phylogenetic analysis based on 16S rRNA genes indicated three known and four potential new anammox bacterial genera in the Pearl Estuary. Most of the sequences recovered from the freshwater sites were
identiﬁed as ‘‘Ca. Brocadia’’ organisms, which accounted for 74.4% of all the clones (Figure 2). The closestTable 2. Environmental Parameters of the Nine Sediment Samplesa
Samples
P01
P03
P07
A04
A08
C2
C3
F412
F414
T
( C)
S
pH
SiO42
4
(lmol L21)
PO32
4
(lmol L21)
NO2
3
(lmol L21)
NO2
2
(lmol L21)
NH1
4
(lmol L21)
TONb
(%)
TOCb
(%)
C/Nb
Porosity
30.10
29.61
29.32
28.81
26.45
27.22
26.47
26.39
25.36
0.00
0.00
0.10
6.74
31.50
28.34
25.44
31.41
33.60
7.01
7.21
6.98
7.46
8.09
8.03
7.98
8.13
8.07
175.3
113.6
83.5
85.3
98.3
86.3
108.7
83.2
NA
8.74
2.40
0.81
5.37
2.17
2.44
2.97
1.50
NA
10.90
0.20
1.76
0.18
4.05
1.72
1.63
2.40
NA
1.24
0.26
0.36
0.26
0.16
0.64
0.48
0.43
NA
278.0
105.2
104.9
105.0
73.4
117.6
107.6
43.8
NA
0.16
0.12
0.10
0.09
0.07
0.12
0.11
0.05
0.14
2.15
1.44
1.02
0.89
0.59
1.07
0.91
0.31
0.89
13.16
11.54
9.88
9.93
8.88
8.94
8.50
6.43
6.24
NA
0.3993
0.5325
0.3233
0.5834
NA
NA
0.4487
NA
a
Abbreviation: T, temperature; S, salinity; TN, total nitrogen; TOC, total organic carbon; C/N, organic carbon/organic nitrogen. NA, no
analysis.
b
Data reported by Liu et al. [2014b].
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Table 3. The Diversity Indices and Richness Estimators of the Anammox Bacterial 16S rRNA Gene and HZO Amino Acid Sequences
Number of
Sequences
Number of OTUsa
Shannon
ChaoI
Station Number
16S
hzo
16S
hzo
16S
hzo
16S
hzo
P01
P03
P07
A04
A08
C2
C3
F412
F414
46
60
41
46
40
56
76
44
73
49
50
57
39
60
48
53
51
48
5
10
5
8
4
12
16
4
5
11
12
12
13
4
15
13
13
6
1.17
1.86
1.26
1.60
1.36
2.11
2.50
1.12
1.07
1.92
2.17
2.10
2.27
1.20
2.34
2.19
2.18
1.28
5
10.33
5
11
4
12.17
21
4
6
16
15
17
20.5
4
29
28
20.5
6.5
a
OTUs were determined at a 3% distance cut-off for 16S rRNA gene and HZO amino acid sequences using the DOTUR program.
match GenBank sequences (98–99% similarity) of ‘‘Ca. Brocadia’’-like sequences found in our study were
retrieved from wastewater, groundwater, agricultural soil, river, lake, and wetland sediments [Shen et al.,
2013]. Three known anammox genera: ‘‘Ca. Brocadia,’’ ‘‘Ca. Kuenenia,’’ and ‘‘Ca. Scalindua’’ coexisted at the
four middle sites where freshwater mixed with seawater, and accounted for 5.85%, 14.63%, and 64.88%,
respectively, of the total middle sites clones. The rest, 14.64% of sequences retrieved from the middle sites,
were identiﬁed as new genera of anammox bacteria. The results indicated that ‘‘Ca. Kuenenia’’ and ‘‘Ca. Scalindua’’ thrived at low salinity. The sequences afﬁliated with ‘‘Ca. Kuenenia’’ were close (with 98–99% similarity) to those retrieved from paddy soil, tidal, and estuarine sediments [Hu et al., 2012, 2013; Zheng et al.,
2012]. The sequences from seawater sites were afﬁliated solely with the genus ‘‘Ca. Scalindua’’ except for
sequence F414-16S-29 which belonged to a new anammox bacterial genus. The closest-match GenBank
sequences were originally retrieved from the Cape Fear River Estuary, Mai Po Nature Reserve, South China
Sea, mangrove, and arctic sediments [Dale et al., 2009; Li et al., 2011a, 2011b] and the similarities were more
than 98%.
Additionally, four clusters of sequences were not afﬁliated with any known anammox bacterial genera, and
they may represent new anammox genera (Figure 2). Sequences of cluster I were less related to the 16S
rRNA genes of any known anammox bacterial genera with 87–89% identity. The closest-match sequences
of this novel cluster were recovered from the North Carolina groundwater with 98% identity [Hirsch et al.,
2011]. We propose to provisionally name this anammox genus and species as ‘‘Candidatus Jugangensis sediminum.’’ Cluster II was distantly related to the ﬁve known genera with 82–84% sequence identity, and it
had a low similarity (89%) with the closest-match sequence found in the Taihu Lake sediment. We suggest
to provisionally name ‘‘Candidatus Oceanicum marisluti’’ as a new genus and species. Cluster III was also distant to the known genera with less than 80% identity. The closest relatives were retrieved from the Mai Po
Nature Reserve and Zhoushan Island marine sediment. We suggest to provisionally name ‘‘Candidatus
Anammoxidans sediminis,’’ ‘‘Ca. Anammoxidans luti,’’ and ‘‘Ca. Aanmmoxidans orientalis’’ (corresponding to
16S rRNA gene sequences C2–16S-4, C2–16S-28, and A08–16S-10, respectively). Sequences of cluster IV
were distantly related (< 80% similarity) to the ﬁve known candidate anammox genera and the closest relatives were retrieved from the Shimokita Peninsula. For cluster IV, we propose to provisionally name it as a
new genus ‘‘Candidatus Aestuarianus.’’ This genus included three new species: ‘‘Ca. Aestuarianus humenicus,’’ ‘‘Ca. Aestuarianus asiatica,’’ and ‘‘Ca. Aestuarianus orientis’’ (corresponding to 16S rRNA sequences
P07–16S-20, A08–16S-41, and P07–16S-43, respectively). Comparing the sequences of the above four novel
genera with previously proposed new phylotypes of anammox bacteria, i.e., ‘‘Ca. Scalindua zhenhei’’ I, II, III
in the South China Sea [Hong et al., 2011], novel ‘‘Ca. Scalindua’’ clade in the Jiaozhou Bay [Dang et al.,
2010b], and ‘‘Ca. Scalindua sinooilﬁeld’’ in petroleum reservoirs [H. Li et al., 2010], low sequence identity
(<90%) was exhibited.
3.4. Phylogeny of Anammox Bacterial HZO Amino Acid Sequences
The phylogenetic analysis of HZO amino acid sequences showed that ‘‘Ca. Anammoxoglobus’’ dominated
the anammox bacteria in the freshwater sites accounting for 72.1% of all the freshwater sites clones, which
was different from that observed with 16S rRNA genes (Figures 2 and 3). Similar to the phylogenetic analysis
results of 16S rRNA genes, the seawater sites only had sequences related to ‘‘Ca. Scalindua.’’ The four middle
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Figure 2. Phylogenetic tree of the anammox bacterial 16S rRNA gene sequences recovered from surface sediment of the Pearl Estuary,
constructed using neighbor-joining method of Phylip 3.66. The clone sequences were named by retrieved site and randomly picked clone
series number. The numbers in parentheses mean the number of 16S rRNA sequences assigned to the representative phylotype sequence.
Planctomyces maris (AJ231184), Planctomyces limnophilus (X62911), and Nitrosomonas europaea (AF353160) were used as out-groups.
sites had a diverse composition of anammox genera: ‘‘Ca. Anammoxoglobus,’’ ‘‘Ca. Jettenia,’’ and ‘‘Ca. Scalindua,’’ which accounted for 0.57%, 44.57%, and 54.86%, respectively, of all the sequences of the middle sites,
indicating the prevalence of ‘‘Ca. Jettenia’’ and ‘‘Ca. Scalindua’’ in the low-salinity environment.
The representative amino acid sequences of the 66 OTUs were 93%–99% identical to the closest-match
GenBank sequences retrieved from the Cape Fear River Estuary, Dongjiang River, Black River, South China
Sea, deep sea tephra deposits, deep sea hydrothermal vent, mangrove sediments, oil reservoir, waste water
treatment plant, anoxic groundwater, and paddy soil [Dale et al., 2009; Hirsch et al., 2011; Hu et al., 2013; H.
Li et al., 2010, 2011a, 2011b; Wang and Gu, 2013; Sun et al., 2014] (Figure 3).
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Figure 3. Phylogenetic tree of the deduced anammox bacterial HZO amino acid sequences recovered from surface sediment of the Pearl
Estuary, constructed using neighbor-joining method of Phylip 3.66. The clone sequences were named by retrieved site and randomly
picked clone series number. The numbers in parentheses mean the number of HZO amino acid sequences assigned to the representative
phylotype sequence. Planctomycete KSU-1 (BAF98478) was used as out-group.
3.5. Anammox Bacterial Community Structure and Spatial Distribution
Obvious geographically speciﬁc distribution of anammox bacteria in the Pearl Estuary surface sediments
were revealed by PCoA. Based on 16S rRNA gene sequences (Figure 4a), the ﬁrst two principal coordinates
(P1 and P2) explained 49.33% of the anammox bacterial community variability among all the sampling sites.
The results indicated anammox bacterial assemblages fell into three groups. Sites P01, P03, and P07 were
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Figure 4. PCoA analysis of the Pearl Estuary sediment anammox bacterial assemblages calculated with 16S rRNA gene sequences (a) and HZO amino acid sequences (b). The plots of
the ﬁrst two principal coordinate axis (P1 and P2) for PCoA and the distributions of the anammox bacterial assemblages (designated with the sampling site names) in response to these
axis are shown.
grouped together and the sequences retrieved from them fell into group I, which shared the freshwater
characteristics. Sites F412 and F414 possessed obvious marine features, and the sequences recovered from
them belonged to group III. The remaining sequences belonging to group II were recovered from middle
sites where freshwater and seawater mixed and had low salinity. The PCoA analysis of anammox bacterial
assemblages based on 16S rRNA gene and HZO amino acid sequences showed a slightly different classiﬁcation (Figure 4b): P07 was slightly away from the other sites in group I in the latter analysis compared to the
former one. In all, PCoA analyses of both 16S rRNA gene and HZO amino acid sequences evidently revealed
a niche segregation of anammox bacteria in the Pearl Estuary.
The correlations between anammox bacterial communities and environmental factors in surface sediments
of the Pearl Estuary were examined by CCA analysis (Figure 5). The ﬁrst two CCA axis (CCA1 and CCA2) in
16S rRNA gene and HZO amino acid sequences CCA analyses explained 71.0% and 54.8% of the total
Figure 5. CCA ordination plots for the ﬁrst two principal dimensions of the correlation between the distribution of the sediment anammox
bacterial assemblages and environmental parameters in the Pearl Estuary as viewed by assessing the anammox 16S rRNA gene OTUs (a)
and the anammox bacterial HZO OTUs (b). Correlations between environmental variables and CCA axis are represented by the length and
angle of arrows (environmental factor vectors).
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Table 4. Abundance of Total Bacterial and Anammox Bacterial 16S rRNA Genes in Sediment, Surface Water, and Bottom Water of the
Nine Sampling Sites of the Pearl Estuarya
No. of Total Bacterial 16S Copies
No. of Anammox Bacterial 16S Copies
Station Number
Sediment_0
(copies g21)
Water_0
(copies L21)
Water_2
(copies L21)
Sediment_0
(copies g21)
Water_0
(copies L21)
Water_2
(copies L21)
P01
P03
P07
A04
A08
C2
C3
F412
F414
5.22 3 1011
5.99 3 1011
2.82 3 1011
5.39 3 1011
2.66 3 1011
6.68 3 1011
5.59 3 1011
3.29 3 1011
4.49 3 1011
2.04 3 1011
3.20 3 1011
1.91 3 1011
2.88 3 1011
1.18 3 1011
3.85 3 1011
4.73 3 1011
1.36 3 1011
1.20 3 1011
1.05 3 1011
2.55 3 1011
5.45 3 1011
8.64 3 1010
2.89 3 1011
9.43 3 1010
9.99 3 1010
2.18 3 1011
1.53 3 1011
1.12 3 106
1.08 3 106
5.76 3 105
1.02 3 106
4.22 3 105
2.55 3 106
2.03 3 106
6.53 3 105
2.27 3 106
1.18 3 105
1.03 3 105
8.86 3 105
2.54 3 104
1.84 3 104
4.16 3 104
8.45 3 104
1.35 3 104
8.07 3 103
9.31 3 104
9.84 3 104
1.01 3 105
1.24 3 104
2.02 3 104
1.78 3 104
2.91 3 104
2.51 3 104
1.26 3 104
a
Surface and bottom samples were designated _0 and _2, respectively.
anammox bacterial variation, respectively (Figure 5). Of all the environmental factors analyzed, salinity
(p 5 0.0112), temperature (p 5 0.0019), pH (p 5 0.0145), and C/N (p 5 0.0015) were shown to be signiﬁcant
in driving the composition and distribution of anammox bacteria, and these four environmental factors
together provided 57.7% of the total CCA explanatory power. In the CCA analysis of HZO amino acid
sequences, p values of the above four environmental factors were 0.0004, 0.0028, 0.0009, and 0.0056,
respectively. These four environmental factors together provided 51.8% of the total CCA explanatory power.
Moreover, the geospatial heterogeneity was also revealed by CCA analysis in which distinguishable anammox bacterial communities occurred in habitats with different environmental factors. The anammox bacterial communities of freshwater sites (P01, P03, P07) and seawater sites (F414, F412) were signiﬁcantly
inﬂuenced by nutrients and salinity, respectively. The communities of middle sites (A04, A08, C2, C3) with
salinity ranging from 6.74 to 31.50 seemed to be inﬂuenced synergetically by various environmental factors.
TN had a signiﬁcant inﬂuence on the communities of sites A04 and C2 based on the 16S rRNA genes CCA
analysis (Figure 5).
3.6. Quantification of Anammox Bacteria and Environmental Controls
Melting-curve analysis of the total bacterial and anammox bacterial 16S rRNA genes conﬁrmed that ﬂuorescent signals were derived from speciﬁc PCR products in our Q-PCR quantiﬁcations. The ampliﬁcation efﬁciency of total bacterial and anammox bacterial 16S rRNA genes was 102% and 95%, respectively. Standard
curves were generated with the threshold cycle (Ct) and the log value of gene copy numbers. The high linearity (R2 5 0.9843 and R2 5 0.9996, respectively) obtained over six orders of magnitude of the standard plasmid DNA concentration indicated a high primer hybridization and extension efﬁciency.
The Q-PCR results suggested a geographically heterogeneous distribution of total bacterial and anammox
bacterial 16S rRNA genes abundance across all the sampling sites of the Pearl Estuary (Table 4 and supporting information Figure S2). The copy numbers of anammox bacterial 16S rRNA genes were between 4.22 3
105 and 2.55 3 106 g21 in sediments, and site C2 had the highest abundance whereas site A08 had the lowest abundance. In bottom and surface waters, the copy numbers of anammox bacterial 16S rRNA genes
ranged from 1.24 3 104 to 1.01 3 105 and 8.07 3 103 to 8.86 3 105 L21, respectively. In general, the abundance of anammox 16S rRNA genes was higher in freshwater sites than seawater sites among all the water
samples. In the sediment, the ratio of anammox bacterial 16S rRNA gene copies to total bacterial 16S rRNA
gene copies was about 1:5 3 105. However, the ratio was low in water samples: from 1:5 3 106 to 1:1 3
107.
Pearson correlation analysis indicated that the abundance of total bacterial 16S rRNA genes in both sediment and water samples did not show signiﬁcant correlations with any environmental factors measured
(supporting information Tables S1 and S2) except for the bottom water samples that showed a positive correlation with NO2
2 concentration. By contrast, the abundance of anammox bacteria in the water column,
particularly the bottom water, revealed signiﬁcantly positive correlations with temperature, Chl-a, PO32
4 ,
2
NO2
,
NO
,
and
negative
correlations
with
salinity,
pH,
and
DO.
In
surface
sediments,
a
positive
correlation
3
2
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Figure 6. Community structure of anammox bacteria reads from the 16S rRNA genes clone libraries of the surface sediment of the Pearl
Estuary.
of TN to anammox bacterial abundance was observed with a correlation coefﬁcient of 0.59 (supporting
information Table S2).
4. Discussion
4.1. Anammox Bacterial Diversity and Distribution
High anammox bacterial diversity (‘‘Ca. Brocadia,’’ ‘‘Ca. Kuenenia,’’ ‘‘Ca. Anammoxoglobus,’’ ‘‘Ca. Jettenia,’’
and ‘‘Ca. Scalindua’’) in surface sediments of the Pearl Estuary was evidenced by phylogenetic analyses of
16S rRNA gene and HZO amino acid sequences (Figures 2, 3, and 6).
The shift in anammox bacterial community, i.e., the dominant genus changed from ‘‘Ca. Brocadia’’ or ‘‘Ca.
Anammoxoglobus’’ to ‘‘Ca. Scalindua,’’ along the salinity gradient from freshwater to the open ocean in our
study was consistent with previous reports [Dale et al., 2009; Hou et al., 2013], which may be related to the
different salinity tolerance of different anammox genera. A previous study on riparian sediments of the
Pearl Estuary indicated that ‘‘Ca. Brocadia’’ accounted for 53.6% of total sequences, with the highest abundance in winter [Wang et al., 2012]. The percentage was lower than in our study where the proportion of
‘‘Ca. Brocadia’’ was 74.4% at the freshwater sites (in summer). Another study in the Pearl Estuary [Li et al.,
2013a] observed ‘‘Ca. Kuenenia’’ but not ‘‘Ca. Brocadia’’ in the estuary where salinity at the sites was 3.52–
7.05 (in summer). The ability of ‘‘Ca. Brocadia’’ to adapt to the freshwater environment may explain these
differences. The salinity in riparian sediments [Wang et al., 2012] was 0.09–0.18, while it was almost zero in
our investigation. The extremely low salinity provided a suitable environment for the growth of ‘‘Ca. Brocadia.’’ The decreased abundance of ‘‘Ca. Brocadia’’ with increased salinity in our investigation is in agreement
with the low salinity tolerance of this genus. The relatively high proportion (14.6%) of ‘‘Ca. Kuenenia’’ in the
middle sites (salinity ranged from 6.74 to 28.34) and the positive correlation of the proportion of ‘‘Ca. Kuenenia’’ with salinity both indicated its adaptive capability to low-saline environments. The presence of ‘‘Ca.
Kuenenia’’ was also observed in hot springs, deep-sea hydrothermal vents, petroleum reservoirs, wastewater
treatment plant, underground aquifers, estuaries, and many other environments [Dale et al., 2009; Woebken
et al., 2008; Byrne et al., 2009; Jaeschke et al., 2009a; Li et al., 2009, H. Li et al., 2010; Hirsch et al., 2011; Hu
et al., 2012; Wang et al., 2012], underscoring the importance to further elucidate its speciﬁc survival strategies especially under extreme conditions. The high tolerance of ‘‘Ca. Scalindua’’ to salinity was reconﬁrmed
in this study as sequences retrieved from the seawater sites of the Pearl Estuary were all afﬁliated with ‘‘Ca.
Scalindua’’ except for one sequence representing a novel anammox assemblage (Figure 2).
Additionally, four novel anammox bacterial genera were observed based on phylogenetic analysis of 16S
rRNA genes. The sequence identities between the four novel genera in our study and the ‘‘Ca. Scalindua
zhenhei’’ I, II, III in previous report of the Pearl Estuary [Li et al., 2013b] were low (90%). It was notable that
the novel genera mainly appeared in middle sites (A04, A08, C2, C3), where tidally induced interaction
between freshwater and seawater is intensive. Moreover, the concentration of N-nutrients and the content
of trace metals were elevated over the last two decades due to the increasing use of nitrogen-based
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fertilizer and discharge of municipal wastewaters, agricultural runoff, and particularly metal industries in the
Pearl Estuary [Yu et al., 2010]. It thus could be inferred that a kind of adaptation mechanism exists to help
them to cope with the dynamic environmental conditions. Additionally, the closest-match sequences
(98% identity) of the three novel genera (‘‘Ca. Jugangensis,’’ ‘‘Ca. Anammoxidans,’’ ‘‘Ca. Aestuarianus’’)
were retrieved from the North Carolina groundwater [Hirsch et al., 2011], Mai Po Nature Reserve mangrove
sediment, Zhoushan Island marine sediment, and Shimokita Peninsula sediment, respectively. This indicates
a global presence of the above three novel genera in aquatic environments. However, the low identity
(89%) between ‘‘Ca. Oceanicum’’ and the closest relatives (found in the Taihu Lake sediment) indicates the
habitat-speciﬁc characteristics of this genus in the Pearl Estuary.
The phylogenetic analysis based on HZO amino acid sequences revealed sequences afﬁliated with ‘‘Ca.
Anammoxoglobus’’ and ‘‘Ca. Jettenia’’ which were not found by phylogenetic analysis of 16S rRNA genes. A
similar observation was also reported in petroleum reservoirs [H. Li et al., 2010]. Differences in genera
observed may be related to the different coverage and speciﬁcity of the PCR primers. This indicates the
complementary role of hzo marker gene in anammox bacterial phylogenetic analysis. Other gene markers
such as nirS, hzs, and cytochrome c genes could also be employed to evaluate this possibility and provide
more information on anammox bacteria in further studies.
The closest-match GenBank sequences of 16S rRNA gene and HZO amino acids both had sequences originally retrieved from paddy soil and agricultural soil [Hu et al., 2013; Shen et al., 2013], indicating that these
anammox bacteria were introduced from land. In all, anammox bacteria have a wide distribution and great
adaptive capability to varietal environments.
4.2. Anammox Bacterial Abundance in Surface Sediments
Anammox bacterial 16S rRNA gene copies in sediments of the Pearl Estuary ranged from 4.22 3 105 to 2.55 3
106 g21, similar with that observed in the Cape Fear River Estuary (1.3 3 10528.4 3 106 16S rRNA gene copies
g21) [Dale et al., 2009] and Jiaozhou Bay (3.5 3 10525.9 3 106 hzo gene copies g21) [Dang et al., 2010b]. The
ratio of anammox bacterial 16S rRNA genes to total bacterial 16S rRNA genes in sediments of our study was
about 1:5 3 105, similar to the ratio observed in the Jiaozhou Bay [Dang et al., 2010b]. These results may indicate a generally high anammox bacterial abundance in eutrophic estuaries. However, a previous study performed in riparian sediments of the Pearl Estuary observed a much higher abundance of anammox bacteria
with copy numbers of anammox bacterial hzsB genes ranging from 1.3 3 106 to 1.2 3 107 g21 in summer
and even two orders of magnitude higher in winter, which was the highest abundance in natural environments recorded so far [Wang et al., 2012]. This discrepancy may be related to different copy numbers of 16S
rRNA genes and hzsB operons contained in anammox bacteria. Moreover, the difference between sampling
sites may also inﬂuence the result because of the different impacts from anthropogenic activities.
4.3. Anammox Bacterial Abundance in the Water Column
It was noteworthy that the anammox bacterial abundance at most sites of the freshwater and low-salinity
areas was higher in surface water than in bottom water. At site P07, the anammox bacterial abundance in
the surface water was even higher than in the sediment. This may be explained by major anthropogenic
inputs since these sites are located in areas either with dense population (freshwater and low-salinity sites)
or close to industry (site P07, near an industrial park in Dongguan city). Moreover, a higher anammox bacterial abundance was observed in upstream sites than further downstream. The input of anammox bacteria
from soil and wastewater could contribute to this high anammox bacterial abundance in upstream sites.
Moreover, this horizontal difference in anammox bacterial abundance of water samples was positively correlated with N-nutrients and negatively correlated with DO and pH. The signiﬁcant role of NO2
2 in anammox
process was reported in other estuaries [Trimmer et al., 2003; Dang et al., 2010b; Hou et al., 2013; Sun et al.,
2
2014]. As anammox activity requires the simultaneous presence of NH1
4 and NO2 , the observed low con2
1
centration of NO2 and excess NH4 in the Pearl Estuary (Table 1) might shed light on the importance of
2
NO2
2 availability in governing the anammox bacterial abundance. NO3 was positively correlated with anammox bacterial abundance in the bottom water (supporting information Table S1). NO2
3 might act as an electron acceptor of anammox-mediated denitriﬁcation since anammox bacteria have been conﬁrmed to
perform denitriﬁcation themselves in natural ecosystems [Kartal et al., 2007]. It was notable that anammox
bacteria appeared in the water column which did not reach hypoxia (Table 1). The tolerance of anammox
bacteria to elevated oxygen concentration was previously reported [Kuypers et al., 2005; Trimmer et al.,
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2005; Jaeschke et al., 2009b]. The high abundance of anammox bacteria in water samples of the Pearl Estuary where DO ranged from 0.22 to 7.59 mg L21 might imply that anammox bacteria have a high tolerance
to oxygen or they are in a dormant stage, which needs further research. However, river runoff from
upstream of the estuary especially in the wet season can result in a strong dilution which might also result
in the declining trend of anammox bacteria abundance from the upstream to the further downstream. Furthermore, the observed high abundance of anammox bacteria might be originated from sediments and the
real factors relevant to the anammox bacteria abundance should be further evaluated.
4.4. Biogeography of Anammox Bacteria in Surface Sediments in Response to Changes in Estuarine
Ecosystems
Environmental factors may be the driving force to shape the observed spatial distribution pattern of anammox bacteria from freshwater to seawater of the Pearl Estuary. Salinity was evident to be a signiﬁcant contributor (p 5 0.0112 and p 5 0.0004 in 16S rRNA gene and HZO amino acid sequences CCA analysis,
respectively). The important role of salinity was related to the different salinity tolerance of anammox bacteria. The signiﬁcance of salinity to the anammox bacterial distribution was also observed in other estuaries
[Dale et al., 2009; Hou et al., 2013], deep-sea tephra, and deep-sea hydrothermal vent [Hirsch et al., 2011].
Temperature and C/N were also the important determinants in our study area. Similarly, the inﬂuence of
temperature on anammox community structure was also observed in the Yangtze Estuary [Hou et al., 2013].
The temperature in our study was relatively stable and ranged from 26.39oC at site F412 to 30.10 C at site
P01 (Table 2). However, it has been conﬁrmed that anammox bacteria appear at a broader temperature
range, i.e., deep-sea hydrothermal vent (4–100 C) [Byrne et al., 2009], petroleum reservoirs (55–75oC) [H. Li
et al., 2010], hot springs (24–65 C) [Jaeschke et al., 2009a], and arctic sea ice (22.5 to 20.3 C) [Rysgaard and
Glud, 2004]. High C/N was usually related to the terrestrial input, and the strong positive correlation
(p 5 0.0015 and p 5 0.0056 in 16S rRNA gene and HZO amino acid sequences CCA analysis, respectively)
between anammox bacterial distribution and C/N in sediment may indicate the signiﬁcant inﬂuence of
anthropogenic activities on anammox bacterial communities in the Pearl Estuary.
Acknowledgments
The sequence data are available from
the GenBank database (http://www.
ncbi.nlm.nih.gov/nucleotide/) as
mentioned in section 2.6 of the current
study. We thank all the scientists and
members on the R/V Tian Long for
their great assistance in sample
collection during expedition. We also
thank Sumei Liu of Ocean University of
China for providing nutrients data of
sediment, and. Jianping Gan of Hong
Kong University of Science and
Technology for providing CTD records.
This work was supported by the
National Natural Science Foundation
of China through grants of 41476112,
41276141, 41106038, 41221004, and
41130857.
FU ET AL.
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