Evaluation of water quality and nitrogen removal bacteria community in Fuhe River

Abstract

The ecosystem of Fuhe River, which is the main inflow tributary of Baiyangdian Lake, is crucial to the ecological health and environmental safety of the lake. Understanding the variation of Fuhe River ecosystem and taking measures to reduce its pollutants can help relieve the eutrophication of Baiyangdian Lake. To investigate this, we monitored water quality and levels of nitrogen removal bacteria along the Fuhe River using comprehensive pollution indices and denaturing gradient gel electrophoresis (DGGE). Samples were collected in the low-flow, high-flow and mean-water periods of 2009 and 2010. The dissolved oxygen (DO) values were, on an average, lower than 4 mg/L suggesting that the river was in low oxygen conditions, mainly conducting anaerobic reactions. The chemical oxygen demand (CODCr) values decreased gradually along the river, with the highest value of 128.65 mg/L at the first sampling site. The concentrations of total nitrogen (TN) and total phosphorus (TP) increased to 56.21 mg/L and 4.96 mg/L respectively, with concentrations at the inlet of the wetland significantly higher than those in the lake. The comprehensive pollution indexes reached the highest in the low-flow period in 2009 and in 2010, up to 78.81 and 77.10, respectively. Overall, the high comprehensive pollution indices for TN and TP indicated that the Fuhe River was polluted by N and P nutrients. There were abundant denitrifying bacteria and anaerobic ammonium oxidation bacteria at the terrestrial-aquatic interface of the upstream region of the river according to the Shannon-Weiner index. (C) 2011 Published by Elsevier B. V. Selection and/or peer-review under responsibility of School of Environment, Beijing Normal University.

Full text

Procedia Environmental Sciences 13 (2012) 1809 – 1819
1878-0296 © 2011 Published by Elsevier B.V. Selection and/or peer-review under responsibility of School of Environment, Beijing Normal University.
doi:10.1016/j.proenv.2012.01.174
Available online at www.sciencedirect.com
Procedia
Environmental
Sciences
Procedia Environmental Sciences 8 (2011) 1835–1845
www.elsevier.com/locate/procedia
The 18th Biennial Conference of International Society for Ecological Modelling
Evaluation of water quality and nitrogen removal bacteria
community in Fuhe River
Yun Qi, Ziyuan Wang, Yuansheng Pei
MOE Key Laboratory of Water and Sediment Sciences, School of Environment, Beijing Normal
University, Beijing 100875, P.R. China
Abstract
The ecosystem of Fuhe River, which is the main inflow tributary of Baiyangdian Lake, is crucial to the ecological
health and environmental safety of the lake. Understanding the variation of Fuhe River ecosystem and taking
measures to reduce its pollutants can help relieve the eutrophication of Baiyangdian Lake. To investigate this, we
monitored water quality and levels of nitrogen removal bacteria along the Fuhe River using comprehensive pollution
indices and denaturing gradient gel electrophoresis (DGGE). Samples were collected in the low-flow, high-flow and
mean-water periods of 2009 and 2010. The dissolved oxygen (DO) values were, on an average, lower than 4 mg/L
suggesting that the river was in low oxygen conditions, mainly conducting anaerobic reactions. The chemical oxygen
demand (CODCr) values decreased gradually along the river, with the highest value of 128.65 mg/L at the first
sampling site. The concentrations of total nitrogen (TN) and total phosphorus (TP) increased to 56.21 mg/L and 4.96
mg/L respectively, with concentrations at the inlet of the wetland significantly higher than those in the lake. The
comprehensive pollution indexes reached the highest in the low-flow period in 2009 and in 2010, up to 78.81 and
77.10, respectively. Overall, the high comprehensive pollution indices for TN and TP indicated that the Fuhe River
was polluted by N and P nutrients. There were abundant denitrifying bacteria and anaerobic ammonium oxidation
bacteria at the terrestrial-aquatic interface of the upstream region of the river according to the Shannon-Weiner index.
© 2011 Published by Elsevier Ltd.
Keywords: Water quality; eutrophication; comprehensive pollution index; nitrogen removal bacteria
1. Introduction
 Corresponding author. Tel.: +86-5880-0160; fax: +86-5880-0160.
E-mail address: yspei@bnu.edu.cn
© 2011 Published by Elsevier B.V. Selection and/or peer-review under responsibility of School of
Environment, Beijing Normal University. Open access under CC BY-NC-ND license.
Open access under CC BY-NC-ND license.

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Baiyangdian Lake, called the “Pearl of North China” due to its unique landscape [1], is the largest
natural freshwater body and wetland ecological system in northern China. It is also an important storage
pool of the south-to-north water diversion project. This lake maintains the ecological system equilibrium
of the plains of northern China and regulates climate change. The total area of Baiyangdian Lake is 366
km2, and the depth of the lake varies between 2-3 m according to the hydrologic conditions [2]. It has 9
tributaries of which the Fuhe River is the only inflow river with perennial flow. During the non-flood
season, this river becomes the largest inflow river of Baiyangdian Lake.
The Fuhe River receives urban sewage and effluents of wastewater treatment plants from Baoding City
in northern China. More than 105 m3 of sewage and treated wastewater with high nutrients flow into the
lake every day, and accounts for 45.24% of the flow of Fuhe River. This has resulted in eutrophication of
Baiyangdian Lake and has become a threat to the health of the ecosystem. The biodiversity of the area
around the lake has decreased significantly and the biological population structure destroyed, which has
caused the degradation of the Baiyangdian ecosystem. To investigate the variation of Fuhe River
ecosystem and its effect on Baiyangdian wetland, we monitored water quality of the river and the
terrestrial–aquatic interface of the riverbank from 2009 to 2010.
2. Materials and Methods
2.1. Study area
Six monitoring sites, F1-F6, were chosen along the Fuhe River (Fig. 1), and one sampling site F7 was
located in the northern part of the lake; F7 represents the wetland environment and was treated as the
reference site. Sites F1 and F2 were located in the upstream portion of the river, sites F3 and F4 were in
the midstream portion, and sites F5 and F6 were in the downstream portion. F1, F3 and F5 were the
historical monitoring sites approved by the Baoding Environmental Protection Bureau. Urban sewage
from Baoding City is discharged into the river at site F1, while effluents from the secondary wastewater
treatment plants enter the river at site F2. The monitoring sites were chosen based on factors such as
drainage outlet, distance between sites and compatibility with historically monitored sections of the river
[3]. The sites cover regions typically found along the Fuhe River and fully reflect the water and
ecological environments along the river.
Based on temperature and rainfall, the Fuhe River can be divided into the low-flow period (March to
May), high-flow period (June to August) and mean-water period (September and October); the Fuhe
River and Baiyangdian Lake are frozen for the rest of the year. Samples were collected at each of the sites
once every month from March to October.

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Y. Qi et al / Procedia Environmental Sciences 8 (2011) 1835–1845 1837
Figure 1. Schematic graph of the Fuhe River and sampling sites.
2.2. Sampling
Water samples were collected from the river at a depth of 30 cm in the vertical direction and stored in
acid-washed polyethylene bottles for further analysis. Prior to measuring the parameters of each sample,
the water was filtered through a 0.45 μm acetate fiber film.
Surface sediments (the top 10 cm layer) on the riverbank were collected and homogenized. The
homogenized samples were sealed and labeled. Small parts were stored at 4 ℃ for immediate analysis
while the residuals were stored at -20℃ for subsequent analysis.
2.3. Laboratory analysis
Water temperature and dissolved oxygen (DO) were measured on site (Thermo Orion, America). Total
nitrogen (TN), ammonia nitrogen (NH3-N), total phosphorus (TP) and chemical oxygen demand (CODCr)
values of each water sample were analyzed in accordance with standard methods [4].
Sediment samples were dried at 105 ℃ for 24 hours, and passed through a 100 mesh sieve. TP and TN
concentrations of the sediments were determined using standard methods [5].
2.4. Statistical analysis of data
Comprehensive pollution index, P, which was calculated by the algebra superposition method, was
used to quantify water quality changes during different periods [6]. The following formula describes P:

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74
0
j1 1
,/
ij i i
i
P PP C C
 
 

(1)
where P stands for comprehensive pollution index; ∑Pi for comprehensive pollution subindex; Ci for
measured values of each parameter; C0 for standards of each parameter; ∑Pj for pollution subindex of
each parameter.
The annual average concentration of each nutrient is the arithmetic mean of eight samples (three
samples each for the low-flow and high-flow periods and two samples for the mean-flow period).
2.5. Nitrogen removal bacteria diversity analysis
Bacterial DNA was extracted from the sediment samples according to the instruction of the fast DNA
spin kit for soil. DNA was amplified by polymerase chain reaction (PCR) using primers specific to the for
16S rRNA of anammox bacteria and nosZ gene of denitrifying bacteria. The primer pairs are
PLA46~AMX820 and nosZ-F~nosZ-R, respectively [7-9]. PCR products were analyzed by DGGE. The
photographs were analyzed using the Quantity One software (Quantity One; Bio-Rad). Based on to the
DGGE profile of each sample, the Shannon-Wiener index (H) was calculated to evaluate biological
diversity. The Shannon-Wiener index is a measure of the evenness and abundance of species within a
given microbial community. The H value is determined using:
s
1
ln
ii
i
H pp

 

(2)
where
ii
p nN
,
i
n
is the number of individuals of the
i
th species,
N
the total number of
individuals;
s
the total number of species and
i
p
the relative abundance of species
i
in a sample of
s
species.
3. Results and discussion
3.1. Spatiotemporal distribution characteristics of DO and CODCr
The DO value of a water body directly reflects the growth situation for aquatic organisms and pollution
conditions. Low DO values, especially lower than 5 mg/L, are harmful to aquatic life. As shown in Fig. 2,
the DO values of the six sampling sites along the Fuhe River showed the same variation trend in 2009 and
2010. For example, the DO value at site F1, the entrance to Fuhe River, was low in both years possibly
due to the industrial and municipal wastewater discharged into the river from Baoding city. DO values at
site F2 were 1.46 mg/L and 1.73 mg/L in 2009 and 2010, respectively; these were the lowest amongst all
six sites. This was probably caused by the sewage discharged from the wastewater treatment plant located
at this site. Interestingly, DO values showed an increasing trend along the Fuhe River with the highest
values at site F6, which is the inlet to Baiyangdian Lake, and site F7, the reference site. This suggests that
the Fuhe River retains a self-purification capability and indicates that the water quality of the lake is
better than that of the Fuhe River. Overall, given that the DO values of sites F1, F2 and F3 were below
3mg/L, while the DO values of site F4, F5 and F6 ranged from 3-5 mg/L, it can be concluded that the
Fuhe River is under low DO or anaerobic conditions.

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1
2
3
4
5
6
F1 F2 F3 F4 F5 F6 F7
2
4
6
8
10
Mar. Apr. May. Jun.
Jul. Agu. Sep. Oct.
2009
DO (mg/L)
Sample sites
Annual average
1
2
3
4
5
6
7
F1 F2 F3 F4 F5 F6 F7
2
4
6
8
10
12
14
DO (mg/L)
Sample sites
Mar. Apr. May. Jun.
Jul. Agu. Sep. Oct.
2010
Annual average
Figure 2. Spatiotemporal distribution of DO values of river water at sites F1 through F7 from 2009 (left panel) and 2010 (right
panel).
CODCr is a comprehensive index used to characterize water pollution caused by reducing substances
and organic matters. It was observed that the CODCr values decreased from upstream regions of the Fihe
River to downstream regions (Fig. 3) in both 2009 and 2010. Moreover, the estimated CODCr values of
the sampling sites F1 to F4 were lower in 2010 as compared to 2009 (Fig. 3). This was probably due to
the pollution control measures taken by the environmental protection bureau of Baoding City. Finally,
CODCr values did not vary at site F7 from 2009 to 2010 suggesting that the water quality of Baiyangdian
Lake changed little in these two years.
F1 F2 F3 F4 F5 F6 F7
40
60
80
100
120
140
COD
Cr
(mg/L)
Sample sites
2009
2010
Figure 3. The trends of CODCr of river water in 2009 and 2010.
3.2. Spatiotemporal distribution characteristics of nutrients
3.2.1. Spatiotemporal distribution characteristics of TN

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Spatiotemporal variation of TN in Fuhe River is shown in Fig. 4. In 2009, at each site except F7, TN
concentrations were highest in March, i.e., the start of the low-flow period, while the concentrations
reached the lowest value in July and August, i.e., the mean-flow period. A similar trend was observed in
2010 as well. Moreover, in 2009, the highest TN concentration was observed at site F1 at 43.19 mg/L,
while the TN concentration at site F1 was 14.65 mg/L in 2010. The TN concentration peaked at site F2 in
2010 at 40.72 mg/L. This was probably because the sewage discharged from Baoding city reduced in
2010. On the contrary, the effluents from secondary treatment plants increased close to site F2, thus
increasing TN concentration at this site. Although TN concentrations decreased gradually along the river
due to the effect of self purification, the TN concentration at site F6, which is the entrance of Baiyangdian
Lake, was much higher than that at site F7. This suggests that exogenous nitrogen brought by the Fuhe
River contributed a lot to the eutrophication of water in Baiyangdian Lake.
0
10
20
30
40
50
F1 F2 F3 F4 F5 F6 F7
0
10
20
30
40
50
Sample sites
Mar. Apr. May. Jun.
Jul. Agu. Sep. O ct.
TN (mg/L)
Annual average
2009
0
10
20
30
40
F1 F2 F3 F4 F5 F6 F7
0
10
20
30
40
50
60
70
Sample sites
Mar. Apr. May. Jun.
Jul. Agu. Sep. Oct.
2010
Annual average
TN (mg/L)
Figure 4. Spatiotemporal distribution of TN in Fuhe River water from 2009 to 2010.
3.2.2. Spatiotemporal distribution characteristics of TP
Spatiotemporal variation of TP in the Fuhe River is shown in Fig. 5. The distribution of TP
concentrations in the river showed the same variation trend as that of TN (Fig. 4). High concentrations
were mainly observed in March of 2009 and 2010, while TP concentrations in July and August were low.
This was probably due to increased rainfall, strengthened plant uptake and biotransformation by
microorganisms. In 2009, the highest TP concentration was observed at site F1, i.e., 3.15 mg/L, whereas
in 2010, the TP concentration at F1 was only 0.68 mg/L. In contrast, the TP concentration at site F2
changed from 3 mg/L in 2009 to 1.6 mg/L in 2010 making it the highest value in 2010. This can be
attributed to the decrease in urban sewage discharged from Baoding city at site F1 that contains several
phosphorous pollutants. Overall, TP concentrations in 2010 were lower than those in 2009 and can be
chiefly correlated to the pollution control measures taken by Baoding city. A comparison of the TP
concentrations at sites F6 and F7 suggested the contribution of the Fuhe River to Baiyangdian Lake
pollution. The local government should continue to take effective measures to relieve the pollution of
Fuhe River to protect the Baiyangdian Lake.

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0.5
1.0
1.5
2.0
2.5
3.0
3.5
F1 F2 F3 F4 F5 F6 F7
0
1
2
3
4
5
6
TP (mg/L)
Mar. Apr. May. Jun.
Jul. Agu. Sep. Oct.
Sample sites
2009
Annual average
0.5
1.0
1.5
F1 F2 F3 F4 F5 F6 F7
1
2
3
4
5
TP (mg/L)
Sample sites
Mar. Apr. M ay. Jun.
Jul. Agu. Sep. Oct.
2010
Annual average
Figure 5. Spatiotemporal distribution of TP of river water from 2009 to 2010.
3.2.3. Individual indexes and comprehensive pollution index of water quality
Comprehensive pollution indices (P) of water quality of the Fuhe River are listed in Table 1. In 2009,
the values of P were highest in the low-flow period, followed by the mean-flow and high-flow periods.
With a total P value of 78.81, the worst water quality was observed during the low-flow period. Amongst
the four parameters tested, the annual P value of TN was the highest, reaching 82.23, while the annual P
value of CODCr was the lowest, at 12.05. In contrast, the 2010 P values were as follows: low-flow
period > high-flow period > mean-flow period. This can be explained by the fact that the rainfall in 2010
was mainly in August and September as compared to June, July and August in 2009. However, similar to
2009, the worst water quality was observed during the low-flow period of 2010, with a P value of 77.19.
The four parameters also followed the same trend in 2010 with the pollution index of TN being the
highest at 77.19 and the pollution index of CODCr being the lowest at 7.18. Overall, the comprehensive
pollution indices were lower in 2010 as compared to 2009, except during the high-flow period, indicating
a general improvement in the water quality of the Fuhe River.
Table 1. Individual indexes and comprehensive pollution index of water quality in Fuhe River in 2009 and 2010.
Year Period P ∑Pi
TN TP NH3-N CODCr
2009 low-flow period 30.59 16.42 28.46 3.34 78.81
high-flow period 23.33 7.11 13.33 4.66 48.43
mean-flow period 28.31 12.1 19.36 4.05 63.83
∑Pj 82.23 35.63 61.15 12.05
2010 low-flow period 33.95 8.11 32.05 2.99 77.1
high-flow period 22.17 3.33 21.58 2.78 49.87
mean-flow period
21.06
3.43
20.24
1.41
46.15

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∑Pj 77.19 14.87 73.87 7.18
3.3. Spatiotemporal distribution of nutrients at the terrestrial-aquatic interface
The riparian zone is the important accumulation library for nutrients in the water. The pollutants in
water are easy to be transferred into sediment and contaminate the sediment, and the contaminated
sediment can enrich N and P nutrients much more [10, 11]. Under certain conditions, N and P nutrients in
the sediment can diffuse into the overlying water by physical, chemical and biological actions, which can
aggravate the eutrophication of the water body [12]. Thus measures should be taken to remove nutrients
from the water as well as from the sediments.
Spatiotemporal distribution of TN and TP at the terrestrial-aquatic interface, i.e., the riparian zone, is
shown in Fig. 6. In 2009, the TN content decreased gradually along the river, with site F6 having the
lowest average at 1.02 g/kg. In 2010, the content of TN at sites F1 and F2 were significantly lower than
those in 2009, while the content of TN at sites F5 and F6 became higher. The content of TN in sediments
was positively correlated to the TN concentration in the water expect for site F7 (compare Figs. 4 and 6).
The content of TN in sediments at site F7 was much higher than TN concentration in water. This might be
because the N nutrients in the sediments could not diffuse to the overlying water because of the stillness
of the lake. In contrast to TN concentrations, the distribution of TP in the riparian zone in 2009 was
similar to that in 2010, i.e., a decreasing trend along the river. While the TP concentrations in the river
water decreased from 2009 to 2010 (Fig. 5), the TP content of the sediment did not change significantly
from 2009 to 2010. Finally, at site F7, the content of N and P nutrients in the sediment remained the same
from 2009 to 2010 indicating that nutrient loading to the Baiyangdian Lake sediment did not increase in
these two years.
F1 F2 F3 F4 F5 F6 F7
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
TN (g/kg)
Sample sites
2009
2010
F1 F2 F3 F4 F5 F6 F7
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
TP (g/kg)
Sample sites
2009
2010
Figure 6. Spatiotemporal distribution of TN and TP concentrations at the terrestrial-aquatic interface.
3.4. Diversity characteristics of nitrogen removal bacteria
Because of the intensive water exchange between the riparian zone and ground water in small rivers,
they exhibit a higher number of active denitrifying reactions as compared to large rivers [13]. This is most
likely true in the case of the Fuhe River whose water depth is variable according to the hydrologic

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Y. Qi et al / Procedia Environmental Sciences 8 (2011) 1835–1845 1843
conditions between 0.8 and 2.7m. The anoxic and anaerobic areas were formed in the riparian zone as
organic matters were consumed gradually, which accelerated the denitrifying process. To understand this
further, the microbial diversity of the Fuhe River was analyzed. Based on the observations that effluents
from the secondary wastewater treatment plant enter the river at site F2 and the water at this site has low
DO levels and high concentrations of N and P, it was presumed that this site harbors several nitrogen
removal bacteria. Therefore, F2 was chosen as the sampling site for the analysis of bacterial composition
and distribution. Total DNA was prepared from sediment samples collected from this site in April, June,
July, August and October of 2010 and analyzed for the presence of denitrifying bacteria (using primers
specific to the nosZ gene) and anaerobic ammonium oxidation bacteria (using primers specific to the 16s
rDNA gene).
3.4.1 Diversity characteristics of denitrifying bacteria
The DGGE profiles of denitrifying bacteria at site F2 are illustrated in Fig.7. As expected, the results
showed that there were abundant denitrifying bacteria at site F2. Each sample was variable in number,
size, intensity and brightness indicating the presence of different bacterial species during different months
of the year. This could be because the changing water environment influenced bacterial diversity. The
sample collected in October had the highest Shannon-Weiner index of 1.53, which suggested a high
diversity of denitrifying bacteria in this month. Shannon-Weiner index in other months were apparently
different. So bacteria diversity in different months fluctuated significantly due to the changing water
environment.
Figure 7. DGGE fingerprints of nosZ genes of denitrifying bacteria collected from the water-soil interface (at site F2). DGGE
profile is shown in the left panel, and the schematic diagram processed by Quantity One is shown in the right panel, using lane 5 as
the reference lane.
3.4.2 Diversity characteristics of anammox bacteria
Oct. Aug. Jun. Jul. Apr.

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As shown in Fig. 8, the PCR-based DGGE analysis identified several anaerobic ammonium oxidation
bacteria in different months at site F2. In contrast to the denitrifying bacteria, the differences between the
five samples were fewer. Although the April sample had the largest number of bands followed by the
October sample, the latter had the highest Shannon-Weiner index at 2.07. This suggested that the highest
anaerobic ammonium oxidation bacterial diversity is present in the month of October. Interestingly,
samples collected in the summer months had low Shannon-Weiner indices for anaerobic ammonium
oxidation bacteria. This is possibly because, in the summer, river flow fluctuated significantly thus
increasing the redox potential of the water, which in turn was harmful for the growth of these bacteria.
Taken together, this research demonstrated for the first time that the diversity of anaerobic ammonium
oxidation bacteria changed significantly with seasons and was closely related to temperature and water
quality.
Figure 8. DGGE fingerprints of 16S rDNA of anaerobic ammonium oxidation bacteria collected from the water-soil interface (at site
F2). DGGE profile is shown in the left panel, and the schematic diagram processed by Quantity One is shown in the right panel,
using lane 5 as the reference lane.
4. Conclusion
The parameters measured in this study provide a comprehensive view of the water quality of the Fuhe
River and its impact on the eutrophication of the Baiyangdian Lake. The DO values exhibited an
increasing trend and the CODCr a decreasing trend along the Fuhe River suggesting that the river retains
its self-purification capacity. Also, the water quality in Baiyangdian Lake was superior to that in the Fuhe
River. The CODCr values in 2010 were lower than those in 2009, probably due to the preventive measures
taken by the Baoding city environmental protection bureau. TN and TP concentrations decreased
gradually along the river, with the concentrations at the inlet to Baiyangdian Lake considerably higher
than those in the lake. This meant that the nutrients brought by Fuhe River contributed a lot to the
eutrophication of Baiyangdian Lake. The poorest water quality with the highest comprehensive pollution
Oct. Aug. Jun. Jul. Apr.

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index was observed during the low-flow period. There were abundant denitrifying bacteria and anaerobic
ammonium oxidation bacteria at the terrestrial-aquatic interface of site F2. In conclusion, the Fuhe River
was highly polluted and the effluent nutrients brought by Fuhe River significantly contributed to the
eutrophication of Baiyangdian Lake. Effective measures are urgently needed to control Fuhe River
pollution and to relieve the eutrophication status of Baiyangdian Lake.
Acknowledgements
This study was supported by the National Natural Science Foundation of China (Grant No. 50979007
and Grant No. 51179008) and the National Water Environment Management Projects of China
Foundation (Grant No. 2008ZX07209-007). The authors would like to thank Wang Jun, Gao Gao, and
Wang Changhui for assistance with sample collection and laboratory procedures.
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