Journal of Environmental Sciences 21(2009) 1036–1043
Effect of carbon source on the denitrification in constructed wetlands
LU Songliu, HU Hongying∗, SUN Yingxue, YANG Jia
ESPC State Key Joint Laboratory, Department of Environmental Science and Engineering, Tsinghua University, Beijing 100084, China.
Received 25 October 2008; revised 08 December 2008; accepted 10 December 2008
The ability of constructed wetlands with different plants in nitrate removal were investigated. The factors promoting the rates
of denitrification including organic carbon, nitrate load, plants in wetlands, pH and water temperature in field were systematically
investigated. The results showed that the additional carbon source (glucose) can remarkably improve the nitrate removal ability of the
constructed wetland. It demonstrated that the nitrate removal rate can increase from 20% to more than 50% in summer and from 10%
to 30% in winter, when the nitrate concentration was 30–40 mg/L, the retention time was 24 h and 25 mg/L dissolved organic carbon
(DOC) was ploughed into the constructed wetland. However, the nitrite in the constructed wetland accumulated a little with the supply
of the additional carbon source in summer and winter, and it increased from 0.15 to 2 mg/L in the effluent. It was also found that the
abilities of plant in adjusting pH and temperature can result in an increase of denitrification in wetlands. The seasonal change may also
impact the denitrification.
Key words: constructed wetland; carbon source; denitrification; nitrate; nitrite
Constructed wetlands have become increasingly favored
in the remediation of agricultural runoff and municipal
wastewatertreatment due to their lowcapital cost, very low
operating cost, and environmental friendliness (Kadlec,
1995; Bezbaruah and Zhang, 2003; Huett et al., 2005).
of nitrate which makes up the majority of the nitrogen in
wastewater (Lin et al., 2003; Huett et al., 2005). Treated
municipal wastewaters still contain relatively high level
of nitrate, which would enter water environments such
as rivers, lakes, and sea. The high nitrate load in water
stimulates algal blooms which could degrade water quality
and reduce biological diversity. This eutrophic condition
is the major cause of the hypoxia in water, which has an
adverse effect on its ecosystem and economy (Sirivedhin
and Gray, 2006). Furthermore, it has been reported that
high nitrate concentration in drinking water can cause
2001). Therefore, nitrate removal from agricultural runoffs
and effluent of treated wastewater has become more and
more important (Bezbaruah and Zhang, 2003).
Nitrogen in wetlands is removed from the water mainly
by plant uptake, soil adsorption, and microbe catabolism,
and permanent and dominating nitrogen removal occurs
via denitrification by denitrifying bacteria (Clement et
* Corresponding author. E-mail: firstname.lastname@example.org
al., 2002; Poe et al., 2003). As the main mechanism of
removing nitrate in constructed wetlands, denitrification
is an anaerobic dissimilative pathway in which nitrate is
used as an electron acceptor for anaerobic respiration to
generate energy (IWA, 2000). Denitrification is affected by
many factors including oxygen availability, temperature,
concentration of nitrite, organic carbon supply, and the
species of the wetlands plants (Beauchamp et al., 1989;
Ingersoll and Baker, 1998; Sirivedhin and Gray, 2006).
Because denitrification is an anaerobic dissimilative path-
way in which the synthesis of the enzyme involved in each
denitrification step and the corresponding denitrification
rates are greatly repressed by the presence of O2(IWA,
2000; Sirivedhin and Gray, 2006). The process of deni-
trification is strictly anoxic process and is sensitive to the
oxygen levels presented in constructed wetlands systems.
However, denitrification activities have been observed in
wetland systems that have measurable dissolved oxygen
concentrations in their surface waters (Phipps and Crump-
ton, 1994; Sirivedhin and Gray, 2006). The concentration
of nitrate also has great impacts on the denitrification in
constructed wetlands. Many researchers have found that
in wetlands (Willems et al., 1997; Sartoris et al., 2000).
In addition, carbon source is a controlling factor in the
process of denitrification. The carbon source in the system
of constructed wetlands usually comes from wastewater,
soil, and the root exudates of plants. The addition of
various carbon sources such as glucose, sodium acetate,
No. 8Effect of carbon source on the denitrification in constructed wetlands1037
methanol, starch, cellulose, plant materials, and wheat
straw (Ragab et al., 1994) can also enhance the denitrifica-
and Stahl, 2000; Robins et al., 2000; Sirivedhin and Gray,
2006). Temperature can not only influence the activity of
denitrifying bacteria but also impact the growth of plants
in wetland. Herkowitz (1986) found denitrifying bacteria
in wetland sediments to be more abundant in spring and
summer compared to fall and winter. Subsequently, Stober
et al. (1997) demonstrated that the overall nitrate removal
rate was significantly higher in summer than in winter.
Plant is the most important composition in constructed
wetlands. It can not only take up the nitrogen or phosphate
as nutrition, but also provide a convenient condition for
nitrification and denitrification. Wetland plants can simul-
taneously provide strictly anaerobic and aerobic habitats
in the rhizosphere, which provides convenient conditions
for nitrogen removal (Nikolausz et al., 2008). Wetland
systems supporting with plant communities tend to remove
nitrate more effectively than non-vegetated systems (Zhu
and Sikora, 1995). Belmont and Metcalfe (2003) reported
that plants influenced nitrogen removal rates significantly.
Nitrate can be effectively removed without the addition of
carbon source as long as macrophytes present in wetlands
(Lin et al., 2003; Bastviken et al., 2005).
Several studies have investigated how these factors
affect denitrification in riparian buffer zones (Willems
et al., 1997; Martin et al., 1999; Clement et al., 2002;
Matheson et al., 2003; Rotkin-Ellman et al., 2004), and
some have investigated the factors affecting the denitri-
fication in creating wetlands receiving non-point source
pollution or river flood water (Poe et al., 2003; Sidle and
Goodrich, 2003; Srivedhin and Gray, 2006). To date, there
is still very limited study aiming at the performance of the
constructed wetland in nitrate removal and denitrification
when receiving wastewater with high concentration of
The purpose of this research was to investigate system-
atically the ability of constructed wetlands with different
plants in nitrate removal with the artificial influent contain-
ing high level of nitrate, to determine the effect of carbon
source on the denitrification, and to evaluate the nitrite
accumulation and the factors impacting the nitrate removal
in the constructed wetland.
1 Materials and methods
1.1 Constructed wetlands
The constructed wetlands studied were operated during
February 2006 to December 2006 in Tsinghua University,
Beijing, China (39.92◦N, 116.46◦E). Three parallel con-
structed wetlands were undercurrent constructed wetlands,
and area of each wetland was 3 m2(with length of 4 m,
width 0.75 m, depth 0.7 m, and gradient of 1%). The
wetlands were built with concrete and contained a 50-
cm layer of slag at bottom and a 10-cm of local soil
above the soil layer. The wetlands included 30 cm of river
gravel (nominal diameter 10–20 mm) in the entrance of
influent, following 40 cm of subsurface water flow within
the gravel layer. Three wetlands were planted with Canna,
Zizania caduciflora and Lythrum salicari, respectively,
with a space of 30 cm between each plant. A lateral
perforated pipe was installed at the inlet of each wetland
for the distribution of inflow. To control the water depth, a
lateral trough-shaped collector for drainage was installed
at the upper wall of the distal end of the wetlands, and
a lateral perforated pipe served as a collection drain was
installed at the bottom of distal end of the wetlands.
1.2 Materials and water samples
From the early February 2006 to the middle July 2006,
the sodium nitrate concentration in wetlands influent was
30–40 mg/L NO3−-N and without supplement of organic
carbon. From the middle July 2006 to the November 2006,
the influent has sodium nitrate concentration of 30–40
mg/L NO3−-N with glucose as additional carbon source,
and the proportion of glucose to NO3−-N was 3:1. The
influent was prepared according to water consumption,
stored in a tank and can flow continuously via gate valves
and distribution pipes into the wetlands by a peristaltic
pump. During this study, the inflow rates of the wetlands
were maintained constant by adjusting the gate.
1.3 Water quality analysis
Water samples of influent and effluent were collected
once a week from the three wetlands. Because the three
wetlands received the same contaminated groundwater,
their influent samples were taken from the influent port
identically. The effluent samples of each wetland were
taken from the effluent port separately. Water samples were
analyzed for NO2−-N, and NO3−-N concentration by ultra-
violet spectrophotometer (UV-2401 PC, Shimadzu, Japan)
according to standard method (American Public Health
Association, 1995). Dissolved organic carbon (DOC) was
determined using a total organic carbon (TOC) analyzer
(TOC 5000A, Shimadzu, Japan). Dissolved oxygen con-
tent (DO), pH and oxidation-reduction potential (ORP)
were measured in situ when sampling the water sample.
2 Results and discussion
2.1 Dissolved organic carbon removal ability of the
The results in Fig. 1 show that DOC in influent and ef-
fluent of the three constructed wetlands was about 2 mg/L
without additional carbon source from February to July.
Three constructed wetlands with different plants showed
no obvious difference during long time performance. It
is widely recognized that root-deposited photosynthate
serves as an important carbon source for microorganisms
in the vicinity of growing roots, and previous studies
showed that 28%–59% of the photosynthate was trans-
ferred to the underground, 4%–70% of which entered into
the soil (Lynch and Whipps, 1990). In turn, plants rely
on the microbially mediated decomposition of organic
materials for their supply of available nutrients. Several
1038LU Songliu et al.Vol. 21
Dissolved organic carbon (DOC) concentrations of influent and effluent in the three constructed wetlands.
studies have examined the partitioning of photosynthate
throughout the plant-soil system and few of them have
monitored the incorporation of this photosynthate into the
soil microbial biomass (Kuzyakov et al., 2002; Butler et
al., 2003). The results indicated that all root-deposited
photosynthate and decomposition of organic materials in
the soil can be utilized by the microorganisms in the
Most of the carbon required to fuel denitrification comes
from the plants growing in the wetlands (Bachand and
Horne, 2000) and additional carbon source (Huett et al.,
2005). In this study, 25 mg/L glucose were added to the
influent since the end of July, however, DOC in the effluent
of three constructed wetlands were still low (3 mg/L)
(Fig. 1). The results indicated that the microorganisms
in wetlands can consume the carbon inner the wetlands
and additional carbon simultaneously. The carbon source
was a controlling factor in the process of denitrification
in wetlands, which was in agreement with the previous
research (Starr and Gillham, 1993; Davidsson and Stahl,
2.2 Nitrate removal ability of the three constructed
The nitrate concentrations in influent and effluent and
nitrate removal efficiencies of the three constructed wet-
lands are presented in Fig. 2. As shown in Fig. 2a,
there was a little difference in nitrate removal efficiency
among the three constructed wetlands during the perfor-
mance period. The nitrate removal efficiencies of the three
wetlands were varied along with seasonal change and
plants growth. Nitrate removal efficiencies were slightly
increased from Feb. to July, which was in accordance with
growth periods of wetlands plants. The possible reason
for this phenomenon may be that a part of nitrate was
assimilated by plants in wetlands and the wetland plants
can provide increasing organic carbon source with plants
growth, which can improve the ability of denitrification.
However, the removal do not perform excellent even in
July when plants in exuberant growth, which can only
attain 20% in three wetlands.
Various additional carbon sources, including methanol
(Huett et al., 2005), glucose (Davidsson and Stahl, 2000),
and starch (Robins et al., 2000), have been tentatively
added into carbon-limited wastewaters to enhance the
heterotrophic denitrification rate in constructed wetlands.
source and the proportion of DOC and nitrate was about
1:1 in influent. When glucose was added as organic carbon
source from day 150 to 160, nitrate removal efficiency
was improved rapidly from 20% to 40% and the following
largest removal efficiency was almost 60%, which indi-
cated that organic carbon source improved the removal
of nitrate in the wetlands. Additional carbon source con-
tributed to only 10 mg/L of nitrate removal comparing the
removal efficiency between day 140 to 170 (Fig. 2). Less
than 40% of additional glucose was used by denitrification
process and a majority of additional carbon source was
assimilated by other microorganism in wetlands, which
was similar to previous study which employed C:nitrate-
N ratios of 1.13:1 (Skrinde and Bhagat, 1982).
The nitrate was removed most effectively during the
last ten days, in August (around 190 d, Fig. 2b), which
accorded with the growth cycle of plants. Plants grow best
during August and September, and become contabescence
from October. Seasonal changes affect the assimilation of
Although efficient nitrate removal in constructed wetlands,
e.g., 68% or higher, has been frequently reported by
other researchers, comparative studies also revealed a poor
nitrate removal performance, e.g., < 25%, or even an
increase in nitrate concentration from influent to effluent
No. 8Effect of carbon source on the denitrification in constructed wetlands 1039
Fig. 2 Nitrate concentrations of the influent and effluent and nitrate removal efficiencies of the three constructed wetlands from Feb to July (a), from
July to Dec (b).
in constructed wetlands (Yang et al., 2008). The difference
available carbon source. The additional carbon source can
obviously improve the ability of nitrate removal.
The difference of nitrate removal efficiency between
August and December was about 25%–30% (Fig. 2b)
when plants grow best and almost cease in wetlands.
Comparing the results in Fig. 2, the difference of nitrate
removalefficiencybetween day 160and day 170wasabout
20%, which was contributed by the additional glucose
to denitrification. In conclusion, additional carbon source
caused about 10% nitrate removal, which was according
to nitrate removal efficiency during February and March.
Moreover, for these three wetlands, nitrate was removed
35% in summer and 10% in winter without additional
carbon source. The addition of plants will increase the abil-
ity of buffer action and the denitrification in the wetland
is slightly impacted by temperature and pH because the
nitrate removal efficiency is relatively high in the winter
when additional carbon source was added (Fig. 2b).
Organic carbon sources for denitrification include addi-
tional carbon source, root-deposited of plants, and organic
materials from soil (Butler et al., 2003). Plants assimi-
lation and plant roots decomposition accorded with the
plant growth cycle. The contribution of additional carbon
change without considering the activity of microorganism
impacted by temperature. Thereby, it is important to con-
sider carbon sources in designing a wetland to treat nursery
runoff, which has a low DOC concentration. Plants may
be required to enhance nutrient removal. The addition of
plants will increase water loss through evapotranspiration
and then less water will be available for recycling (Huett et
al., 2005). The additional carbon source will be added to
the influent and substance with rich organic carbon source
will be filled into the soil of constructed wetlands.
2.3 Nitrite accumulation in the three constructed wet-
Nitrite concentrations of influent and effluent and nitrate
removal efficiencies of the constructed wetlands during
Feb. to Dec. are presented in Fig. 3. Nitrite would be
accumulated during denitrification. Nitrite have a sig-
nificant impact on ecosystem functioning and microbial
populations, since it is toxic to both plants and soil
microorganisms (Gelfand and Yakir, 2008).
It was obvious that the nitrite concentration was below
0.15 mg/L and the peak concentration was less than 0.25
1040 LU Songliu et al.Vol. 21
Fig. 3 Nitrite concentrations of influent and effluent and nitrate removal efficiencies of three constructed wetlands from Feb. to July (a) and from Aug.
to Dec. (b).
When nitrate concentration in influent was more than 30
mg/L and nitrate removal efficiency was above 10%, the
nitrite produced during the removal process in wetlands
accounted for less than 5% of the removed nitrate. These
results illuminated that there was no nitrite accumulation
during denitrification in the wetlands without additional
organic carbon source. The results are in agreement with
previous research by Rittmann and McCarty (2001), who
indicated that nitrite accumulation was rarely observed
in the environment due to a low substrate concentration
capable of supporting biomass.
Whereas, nitrite concentration was more than 2 mg/L
in the effluent when additional carbon source was added
(Fig. 3b). The nitrite produced during removal processes
in the wetlands occupied more than 15% of the removed
nitrate with additional carbon source. It was determinated
that nitrite was accumulated under this condition. The
nitrite concentration in effluent varies randomly (Fig. 3b),
which may be due to the variation of nitrate and DOC
concentration in influent. Comparing DOC concentration
in the influent, DOC may be the leading factor for nitrite
in the effluent. The phenomena of nitrite accumulation
were also observed in other studies, and there were many
special conditions that can conduce to produce nitrite,
such as at low DO concentrations, high temperatures and
inhibitory nitrite-oxidization environment (Alleman, 1985;
Kim et al., 2008). The results are in agreement with the
hypothesis that the oxidation rate of nitrate is faster than
that of nitrite, leading to the conversion of nitrate to nitrite
phenomena were also observed in the flask experiments of
denitrification. As shown in Fig. 3, nitrite accumulation
became more evident with the increasing temperature,
which is in agreement with the faster increase of bacteria
activity converting nitrate to nitrite. This result is in line
with that of Kim et al. (2008).
No. 8Effect of carbon source on the denitrification in constructed wetlands 1041
2.4 Factors affecting the ability of nitrate removal in
Constructed wetland is a complex system and there are
many factors would influence nitrate removal. In addition
to such controlling factors as readily available carbon
source, temperature, and pH, high rates of denitrification
depend upon anaerobic circumstance in denitrification
constructed wetlands. The rhizosphere theory indicated
that there are intersectional aerobic and anaerobic re-
actions. The denitrification constructed wetlands are in
favor of the plants which have slightly ability of oxygen
transmission. The influences of pH and temperature in
constructed wetlands were also investigated in this study.
As shown in Fig. 4a, the pH in the influent and efflu-
ent were in the range of 7–8, mostly above 7.5, which
was propitious to denitrification. Although denitrification
process would produce alkalinity, the root secretion and
putrefaction of dead plants can consume alkalinity. As a
result, the constructed wetlands system has a strong ability
of maintaining suitable pH for denitrification.
It is well known that most suitable temperature range
for denitrifying bacteria was from 20 to 40°C. Sirivedhin
and Gray (2006) indicated that denitrification slow down
below 15°C and almost ceases below 5°C. As shown in
Fig. 4b, temperatures in influent were little higher than
in effluent, and were above 15°C except November and
December. As a result, the temperature may impact the
denitrification in winter. Sirivedhin and Gray (2006) also
found that the overall nitrate removal rate was significantly
temperature on denitrification in constructed wetland was
little compare with that of carbon source.
Nutrient removal and transformation processes in
constructed wetlands include microbial conversion, de-
composition, plant uptake, sedimentation, volatilization
and adsorption-fixation reactions (Tchobanoglous, 1993).
Plants uptake and denitrification are the main processes
Variation curves of pH (a) and temperature (b) in the constructed wetlands.
1042LU Songliu et al. Vol. 21
of nitrate removal in constructed wetlands considering
nitrogen conservation. The factors impacting plants uptake
and denitrification significantly affect the nitrate removal
in wetlands. Wetland plants enhance nutrient removal
through biomass accumulation, fixation of inorganic and
organic particulates and the creation of an oxidized and
anaerobic rhizosphere which are controlling factors for
nitrification and denitrification (Burgoon et al., 1995). The
contribution of plants in removing nitrate varies with the
species of plants and the characteristics of the effluent.
structed wetlands planted with Canna, Zizania caduciflora
or Lythrum salicari during almost one year performance.
In conclusion, the constructed wetland can not pro-
vide enough available organic carbon sources for nitrate
removal when influent are rich in nitrate and poor in
organic chemistry contamination. The additional carbon
source (glucose) can remarkably improve the nitrate re-
move ability of the constructed wetland. However, nitrite
in the constructed wetland accumulated a little when the
additional carbon source was supplied. The wetland plants
will increase the ability of buffer action which will result
in that the denitrification in wetland is slightly impacted
by temperature and pH. Three constructed wetlands plant-
ed with Canna, Zizania caduciflora or Lythrum salicari
showed no obvious difference during almost one year
performance because those three plants have similar ability
of nitrogen assimilation. In the design of constructed
wetland to treat wastewater with high nitrate and low
carbon source, the plants, carbon source and substance
should all be considered. Further studies about how to
enhancetheremovalofnitrateby plants,carbonsource and
filling material are recommended.
This work was supported by the National Key Technolo-
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