International Biodeterioration & Biodegradation xxx (2014) 1e9

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International Biodeterioration & Biodegradation

journal homepage: www.elsevier.com/locate/ibiod

Bioaugmentation in lab scale constructed wetland microcosms for

treating polluted river water and domestic wastewater in northern
China
Yuanyuan Shao a, c, Haiyan Pei a, b, *, Wenrong Hu a, b, Christopher Peter Chanway c, d,
Panpan Meng a, Yan Ji a, Zheng Li a
a

School of Environmental Science and Engineering, Shandong University, 250061 Jinan, Shandong Province, People's Republic of China
Shandong Provincial Engineering Centre on Environmental Science and Technology, 250061 Jinan, Shandong Province, People's Republic of China
Department of Forest Sciences, Faculty of Forestry, University of British Columbia, 3041-2424 Main Mall, Vancouver, BC V6T 1Z4, Canada
d
Faculty of Land and Food Systems, University of British Columbia, 248-2357 Main Mall, Vancouver, BC V6T 1Z4, Canada
b
c

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 14 February 2014
Received in revised form
26 May 2014
Accepted 30 May 2014
Available online xxx

Nutrient removal in constructed wetland (CW) microcosms planted with Phragmites and inoculated with
a consortium of six denitrifying bacteria was evaluated as a possible treatment for polluted river water
(RW) and domestic wastewater (DW) in northern China. Experiments were conducted using the batchloaded method over a 15 day period. Bio1 (RW with Phragmites inoculated with the bacterial consortium)
and Abio1 (DW with Phragmites and bacterial inoculation) showed signicant decreases in chemical
oxygen demand (CODcr), and increases in total nitrogen (TN), ammonia (NH3eN) and total phosphorus
(TP) removal compared with Bio2 and Abio2, i.e., Phragmites without bacteria inoculation. On the 7th day,
the removal efciencies of Bio1 were found to be 75.7% for CODcr, 96.7% for TN, 96.8% for NH3eN and
90.4% for TP in RW microcosms, and 85.7% for CODcr, 75% for TN, 88.6% for NH3eN and 88% for TP in
Abio1 DW microcosms. Optimal hydraulic retention time (HRT) was 7 days. We conclude that bioaugmentation in CWs with inoculation repeated every 14 days is a cost-effective measure for nutrient
removal of polluted river water.
2014 Elsevier Ltd. All rights reserved.

Keywords:
Constructed wetlands
River water
Domestic wastewater
Denitrifying bacteria
Hydraulic retention time

1. Introduction
Rural pollution involving contaminated river water (RW) and
domestic wastewater (DW) has attracted increasing attention
over the past decade in northern China (Chen et al., 2003; Wu
et al., 2011). Currently, the water quality of many rivers in
China is inferior according to level V standards. Since chemical

Abbreviations: CW, constructed wetland; RCW, reed constructed wetland;

CWM, constructed wetland microcosms; RW, river water; DW, domestic wastewater; HRT, hydraulic retention time; DO, dissolved oxygen; COD, chemical oxygen


demand; N, nitrogen; P, phosphorus; NH3eN, ammonia; NO

2 eN, nitrite; NO3 eN,
nitrate.
* Corresponding author. School of Environmental Science and Engineering,
Shandong University, 250061 Jinan, Shandong Province, People's Republic of China.
Tel./fax: 86 543 88392983.
E-mail addresses: yuanyuan4872@126.com (Y. Shao), haiyanhup@126.com
(H. Pei), wenrongh@sdu.edu.cn (W. Hu), chris.chanway@ubc.ca (C.P. Chanway),
mpp0701@126.com (P. Meng), jiyan0523@163.com (Y. Ji), lizheng0330@126.com
(Z. Li).

oxygen demand (COD) is controlled by sewage treatment plants,

nitrogen (N), particularly ammonia-N (NH3eN), has become the
main pollutant responsible for this poor water quality. High N
and phosphorus (P) levels cause problems including eutrophication and the generation of undesirable odors, and ultimately
threaten surface and ground water quality and human health
(Camargo and Alonso, 2006; Ding et al., 2012). Early treatment
measures for polluted RW simply depended on the self-purifying
mechanisms of natural water bodies (Yuan et al., 2012). However,
the concentration and volume of wastewater discharged into
rivers is now too great to be treated by natural processes alone.
Furthermore, centralized wastewater treatment plants based on
activated sludge or bacterial bed processes often utilized in cities
are not suitable for DW treatment in rural areas (Ye and Li,
2009).
The constructed wetland system (CW) is a viable option for
treating wastewater because it is low cost and maintenance as
well as ecologically friendly. This system has been used successfully to treat municipal and industrial wastewater, as well as

http://dx.doi.org/10.1016/j.ibiod.2014.05.027
0964-8305/ 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Shao, Y., et al., Bioaugmentation in lab scale constructed wetland microcosms for treating polluted river water
and domestic wastewater in northern China, International Biodeterioration & Biodegradation (2014), http://dx.doi.org/10.1016/
j.ibiod.2014.05.027

Y. Shao et al. / International Biodeterioration & Biodegradation xxx (2014) 1e9

nutrient-rich agricultural land runoff (Svika and Mrkved, 2008;

Morari and Giardini, 2009; Nyquist and Nyquist, 2009; Papadia
et al., 2011). CWs have been used to treat more than 20 different
types of wastewater and have also been used in ecological engineering for more than two decades in China (Zhang et al., 2012).
This system has also been adapted as an ecologically benign tool
for pollution treatment and conservation of the Ganga River (Rai
et al., 2013). Results from a pilot-scale CW used to purify highly
polluted Erh-Ren River water in Taiwan suggested that macrophytes had a direct positive inuence on the water treatment (Jing
et al., 2001). A wide range of studies of nutrient removal in CWs
has been reported to optimize the long hydraulic retention time
(HRT) and low removal efciency. Jia et al. (2010) found that
intermittent operation increased oxidizing conditions in the
microcosm wetlands and greatly enhanced the removal of NH3eN,
COD and TP. Waste rubber tire chips were introduced as a novel
medium and biolm carrier of a subsurface ow CW (Chyan et al.,
2013). However, many wetlands failed to meet environmental
standards, particularly for N and P in polluted water bodies (Fraser
et al., 2004; Greenway, 2005).
Various pollutants are removed from wastewater by microbial,
physical and chemical mechanisms in CWs containing plants and
gravel or soil (Hamilton et al., 1997; Reed, 2000; Garca et al., 2004;
Voeks and Rahmatian, 2004). N removal in particular, is associated
with specic microbial functional groups (Faulwetter et al., 2009).
Biological N removal involves nitrication of NH3eN to nitrite


(NO

2 eN) and nitrate (NO3 eN), followed by denitrication of these
products to volatile oxides of N and N2 gas (Sun et al., 2010). Microbial activity is crucial to N removal from wetlands by contributing to the processes of sedimentation, ltration, precipitation,
volatilization, adsorption and plant uptake (Kadlec and Knight,
1996).
Pollutant-degrading microorganisms are often introduced
into a contaminated environment to accelerate bioremediation
and meet efuent standards. Bioaugmentation is the introduction of microorganisms with specic catabolic abilities into
contaminated environments to enable or accelerate degradation
of targeted pollutants (Perelo, 2010). It is considered a relatively
cost-effective and environmentally friendly technology for
pollutant elimination from affected environments such as
wastewater treatment systems, in which overall microbiological
activity is enhanced (Wakako et al., 2013). Bioaugmentation
processes have been designed to remediate environments
polluted with pesticides (Lima et al., 2009; Barreiros et al., 2012)
and aromatic compounds, and wastewaters contaminated with
phenol, phenanthrene, nitrobenzene (Wang and Li, 2007;
Angeles and Refugio, 2011; Wang et al., 2011; Lin et al., 2012)
and phenol dye (Khandare et al., 2013). A new strain of denitrifying bacteria, Pseudomonas mendocina LR, isolated from
rhizosphere soil of aquatic plants living in a river contaminated
with industrial wastewater and domestic sewage, exhibited a
maximal N reduction rate of 97.3% in natural wastewater (Feng
et al., 2012a, b).
The usefulness of CW in northern China cannot be predicted due
to the large diurnal and seasonal temperature variations that
characterize the region. In addition, removal of N and P from
polluted RW and DW in CWs in northern China through bioaugmentation remains largely unexplored. Therefore, the objectives of this study are to: (i) investigate the rate and amount of N
and P removal from polluted RW and DW by inoculating CW with
denitrifying bacteria; (ii) evaluate the length of time bioaugmentation takes to reduce the chemical composition of CW
efuent to acceptable environmental standards; and (iii) compare
the efcacy of pollutant removal by microbial augmentation in RW
and DW in northern China.

2. Materials and methods

2.1. Experimental wetland system
2.1.1. Experimental setup and design
Constructed wetland microcosms (CWM) were designed according to a surface ow style and established under a rain shelter
outdoors in March, 2012 in Shandong province, northern China
(36.650 N, 117.00 E). The climate is cold and dry during spring and
winter, with wetter and warmer summers (annual precipitation of
670.7 mm, and an average temperature of 14.3  C). Eighteen
wetlands were constructed using circular barrel-type polyethylene
containers, each of which had a surface area of 0.19 m2 and a
depth of 65 cm, with an outlet at the bottom (Fig. 1). The microcosm wetland lter beds consisted of three successive layers from
top to bottom with soil to a height of 10 cm, washed river sand
(particle size <2 mm, mainly Si2O3, Al2O3, Fe2O3) to a height of
5 cm, detritus, which was used as the supporting layer, to a height
of 35 cm. Water depth was kept at 15 cm above the soil surface.
One vertical, perforated PVC pipe (65 cm in length and 3 cm in
diameter) was inserted into the substrate in the middle of each
wetland system to enable measurements of various physical and
chemical parameters. Each CWM system held 15 L water when
lled.
2.1.2. Composition of polluted RW and DW
Two kinds of simulated water were used to minimize variability:
polluted RW and DW (Table 1). Their composition was based on the
systematic analysis of the on-line data of water quality monitoring
sections from the environmental protection authorities and the
Grade I treatment standard of municipal sewage treatment plants
in China. The key pollutants were organic matter, NH3eN, P and N.
The simulated water was prepared from tap water containing
glucose, (NH4)2SO4, KH2PO4 and KNO3. Micronutrients (mg/L):
21Ca, 10 Mg, 14S, 0.8P, 0.3Fe, 0.03Zn, 0.01Cu, 0.03Mn, 0.03B, and
0.002Mo were kept at the same level in all the treatments by
adding CaCl2, MgSO4, Fe-EDTA, ZnSO4, CuSO4, H3BO3, H2MoO4, and
MnCl2 (Huetta et al., 2005). Average pollutant concentrations of the
RW and DW inuents are depicted in Table 1.

Fig. 1. Schematic diagram of a constructed wetland microcosm (CWM) (a: water; b:

soil; c: washed sand; d: detritus; e: perforated PVC pipe; f: inuent tap; g: water
outlet; h: Phragmites australis).

Please cite this article in press as: Shao, Y., et al., Bioaugmentation in lab scale constructed wetland microcosms for treating polluted river water
and domestic wastewater in northern China, International Biodeterioration & Biodegradation (2014), http://dx.doi.org/10.1016/
j.ibiod.2014.05.027

Y. Shao et al. / International Biodeterioration & Biodegradation xxx (2014) 1e9

Table 1
pH, total phosphorus (TP), chemical oxygen demand (CODcr), ammonia-N (NH3eN),


nitrate-N (NO

3 eN), nitrite-N (NO2 eN) and total N (TN) concentrations of RW and
DW.

RW
DW

pH

TP
(mg/L)

CODcr
(mg/L)

NH3eN
(mg/L)

NO

3 eN
(mg/L)

NO

2 eN
(mg/L)

TN
(mg/L)

7.58
7.5

1.53
3.4e3.6

66.2
175e180

8.3
45e47

10.2
0.03e0.1

0.08
ND

19.2
48e50

2.1.3. Preparation of bacterial ora

Six bacteria with known denitrifying and phosphorus-removal
capabilities were isolated from the rhizosphere soil of wetlands
plants at the School of Environmental Science and Engineering in
Shandong University and preserved in the China Center Type Culture Collection (CCTCC) (Table 2). Five strains belonged to the
Pseudomonas family according to 16S rDNA sequence analysis and
one strain (XP-1) showed 100% similarity with Paenibacillus sp. (DO
272469) (Fig. 2).
Enrichment medium (pH 7.2e7.4) was prepared by dissolving
10 g Tryptone, 5 g Yeast extract, 10 g NaCl in 1 L of distilled water.
This medium (100 mL) was autoclaved for 30 min at 121  C, inoculated with 2 mL of bacterial solution and incubated at 35  C for
24 h. Bacteria were harvested by centrifugation (5000 rpm for
15 min at 4  C) and re-suspended in sterile water to achieve 1.5 g
(wet weight) bacteria L
1. The bacterial solution comprised the six
strains described above and was measured by Most-ProbableNumber (MPN) analysis (Rowe et al., 1977).
2.1.4. Plant culture in constructed wetlands
Young Phragmites australis were collected from Nansihu Lake in
Shandong Province and cultured in the local plant nursery in
March, 2012. Roots were washed in running tap water to remove
soil and dead plant tissue. Plants were then placed in 10% modied
Hoagland's solution (Hoagland and Arnon, 1950) to grow before
use. After one week, healthy Phragmites australis of similar size
(approximately 0.3e0.5 m in height) were transplanted by hand
into twelve of the constructed wetland microcosm units (RCW)
with 8 plants per microcosm, while six others were left unplanted.
After planting, microcosms were kept ooded for about one and a
half months until plants were well established.
2.1.5. Operation of the microcosm wetland systems
Six RCWs and three unplanted units were used for the RW
treatment and remaining units were used for the DW treatment.
The following treatment designations were used: polluted river
water (Bio) and domestic wastewater (Abio) with (1) or without (2)
bacteria or without plants (3), which resulted in the following
specic treatments: Bio1 RW with Phragmites inoculated with
the bacterial consortium, Abio1 DW with Phragmites and bacterial inoculation, Bio2 RW with Phragmites but no bacterial inoculation, Abio2 DW with Phragmites and no bacterial inoculation,

Table 2
Species identication, denitrication products and China Center Type Culture
Collection (CCTCC) number of the six bacterial strains used in constructed wetlands.
Strain
number

Bacterial species

Dentrication
products

CCTCC
number

LZ-4
LZ-14
XP-1
XP-2
CL-1
CL-3

Pseudomonas stutzeri
Pseudomonas stutzeri
Paenibacillus sp.
Pseudomonas stutzeri
Pseudomonas pseudoalcaligenes
Pseudomonas oleovorans

N2
N2
N2
N2
N2
N2

M2011430
M2011119
M2011120
M2011431
M2012225
M2012226

Fig. 2. Phylogenetic analysis of the bacterial strains used in this experiment based on
16S rDNA gene sequences.

and Bio3 RW no Phragmites and no bacterial inoculation, and

Abio3 no Phragmites and no bacterial inoculation.
Each group consisted of three microcosm units and three replicates. Before experiments were performed, plants were watered
every 2 days by simulated RW/DW to maintain the water level at
the soil surface. The water level was gradually increased to 15 cm
and held at that level for 2 weeks to acclimate before the experiment started. A ll-and-draw batch mode was applied to inuent
from Sep. 20 to Oct. 5, 2012. The units were manually lled with
test solution from a tank containing simulated RW/DW. The water
depth above the soil surface was 15 cm with 15 L test solution in
total. Units were re-lled with fresh test solution immediately after
drainage. A 0.15 L aliquot of a suspension containing 1% bacterial
solution (described above): 99% polluted river water (v/v) with a
total bacterial population of 3.8  108 cfu/ml was inoculated into
Bio1 and Abio1 microcosms. Bio2, Bio3, Abio2 and Abio3 were not
treated in the rst phase. An injector (Chinese patent number ZL
20112485865.3) was used to add an equivalent volume of inoculum
into the units at three different depths below the water surface
(0.20 m, 0.35 m and 0.45 m).
Water samples were collected using a syringe 10 cm below the
water surface of each unit every day until Bio1 water quality
achieved the GB3838-2002 standard (China) for Grade guideline
and Abio1 reached the GB18918-2002 standard (China) for Class IB criteria. The units were then manually drained through a valve at
the bottom and immediately re-lled with fresh test solution. In
the second phase, Bio1 and Abio1 treatment units were not
inoculated with bacteria. Water samples from the three experimental systems were examined until the end of the same HRT in
the rst phrase. Estimated evapotranspiration was compensated
for with distilled water for each wetland unit during the
experiment.

Please cite this article in press as: Shao, Y., et al., Bioaugmentation in lab scale constructed wetland microcosms for treating polluted river water
and domestic wastewater in northern China, International Biodeterioration & Biodegradation (2014), http://dx.doi.org/10.1016/
j.ibiod.2014.05.027

Y. Shao et al. / International Biodeterioration & Biodegradation xxx (2014) 1e9

3. Results and discussion

Nitrogen and P removal are affected by temperature, indirectly

through effects on oxygen availability which then inuences redox
levels (Bachand and Horne, 2000). Akratos and Tsihrintzi (2007)
found that in the range of 2.0e26.0  C, water temperature did not
have a signicant effect on COD. However, higher temperatures
signicantly impact nitrication activity, with an optimum temperature range for nitrication at 30e40  C in CW soils. When
temperatures fall below 20  C, a slower rate of nitrication is found,
but the process continues to temperatures of 10  C. The rate decreases rapidly to zero below 6  C and does not resume until the
temperature increases to well over 10  C (Herskowitz et al., 1987;
Bachand and Horne, 2000; Vymazal, 2007). During the two tests
the average water temperature ranged from 20.3  C to 23.5  C and
the lowest water temperature was 16.5  C. The maximum temperature was 28.3  C (Table 3). The average temperature favors both
nitrication and denitrication. Both RW and DW tests were conducted under similar environmental conditions.

3.1. Water temperature, dissolved oxygen and pH

3.2. Chemical Oxygen Demand (CODcr)

The physical characteristics and chemical composition of wetlands affect all biological processes. Three of the most important
factors affecting the performance of CW are pH, DO, and water
temperature (Kadlec and Wallace, 2008). Most bacteria are unable
to exist outside the range of 4.0 < pH < 9.5 (Tchbanoglous, 1991).
Nitrifying bacteria prefer pH > 7.2 and they are inhibited at pH < 6.
Denitriers operate optimally in a range 6.5 < pH < 7.5 (Kadlec and
Knight, 1996). Average pH values during the RW treatment period
ranged from 7.0 to 7.5 and did not vary signicantly (Fig. 3). Similar
trends for pH were observed in the DW treatment (Table 3). These
data suggest that the pH in CMWs was conducive for nitrication
and denitrication.
DO, which induces different bioreactions, plays a major role in
determining whether the CW is aerobic or anaerobic (Chyan et al.,
2013) and should be considered a key factor in achieving successful
bioaugmentation (Quan et al., 2012). Low DO concentration (about
0.5 mg/L) favored the enrichment of denitriers and the coexistence of algae and bacteria (Feng et al., 2012a, b). In addition, Ruiz
et al. (2003) found that below 0.5 mg/L DO, NH3eN accumulated
and over a DO of 1.7 mg/L complete nitrication to nitrate was
achieved. In all the CMW systems the average DO ranged from
0.82 mg/L to 0.85 mg/L in both tests (Table 3). Based on these results, we conclude that both nitrication and denitrication can
occur in the presence of DO.

Chemical oxygen demand in RW inuent uctuated signicantly (Fig. 4a). The average CODCr was 68 mg/L in phase of the RW
test, but it tripled in phase of the DW test to approximately
165e175 mg/L (Fig. 4b). The CODCr removal efciency of Bio1
exceeded 75.7% after bioaugmentation in CMW, while the CODCr
removal efciency of the Bio2 CMW by the autochthonal microbial
effect was about 68% and Bio3 CMW was 41.8%. These results are in
agreement with Wu et al. (2011) who found that plant uptake is an
important component of nutrient removal from wastewater. The
CODCr was within Chinese government standards (GB3838-2002
CODCr <20 mg/L) by day 7. HRT of Bio1 CMW was also effectively
reduced. In contrast, CODCr in the efuent in phase was not
reduced signicantly but still met the GB3838-2002 standard in
phase . In phase , CODCr removal efciency was 72.8% at the end
of 7 days. Differences between Bio2 and Bio3 CMWs on CODCr
removal efciency in the two phases were not apparent.
The CODcr removal efciency of Abio1 in phase of the DW test
exceeded 85.7% after bioaugmentation in CWM, while the CODcr
removal efciency of the Abio2 CWM by the autochthonal microbial effect was about 63.6% and 58.8% of Abio3 CWM (Fig. 4b). The
CODcr of Abio1 CMW had already met local and national Chinese
government standards, i.e, Class I-B criteria of GB18918-2002
(CODcr <60 mg/L) by the 4th day of phase . HRT of Abio1 CWM
was also shortened compared with Abio2 and Abio3 CWMs.
However, CODcr of the Abio1 CWM inuent was not reduced
signicantly in phase , with the removal efciency of 69.1%, but it
did meet the GB18918-2002 standard after 7 days. In addition,
CODcr of Abio2 and Abio3 CWMs in both phases did not attain Class
I-B criteria of GB18918-2002 by the end of the test. These results
suggest that the addition of bacteria enhanced CODCr removal from
simulated RW water as well as DW water.

2.2. Water quality monitoring

The following laboratory analyses were performed on water
samples: CODcr, NH3eN (ammonia-N), TN (total nitrogen), TP (total


phosphorus), NO

3 eN (nitrate-N) and NO2 eN (nitrite). Water
temperature and DO (dissolved oxygen) were monitored using a
DO Meter (DO-200, Lovibond, Germany) in situ. The pH of the
efuent was measured using a pH meter (Shanghai Leici Instrument Co. Ltd., PHS-3C, China). All parameters were determined
according to standard methods (APHA, 1998). TN, NH3eN and
NO

3 eN were evaluated by a UVeVis spectrophotometer (Shimadzu Instrument Co. Ltd., UV-2450, Japan). Treatment efciency
was calculated as the percentage of removal for N and P as follows:
Removal efciency (%) (Ci
Ce)/Ci  100.where Ci and Ce are the
inuent and efuent concentrations in mg/L.

3.3. Removal of TP

Fig. 3. pH of RW microcosms during phases I and II (: CWM s were inoculated with

bacteria; : CWM s were drained and then lled with simulated RW without bacterial
inoculation).

Strains CL-1 and CL-3 were previously found to be effective

denitrifying and phosphorus-removing bacteria with high TP
removal efciency (Shao et al., 2013). The TP concentration decreased
signicantly in Bio1 CWM compared with Bio2 and Bio3 groups in
both phase and (Fig. 5a). Inuent TP uctuated with an average of
1.56 mg/L in phase and met local and national Chinese government
standards by 7th day, i.e., Class criteria of GB3838-2002 (<0.2 mg/
L), with a concentration of 0.15 mg/L and a removal rate of 90.4%.
However, TP in the inuent in phase with an 86% removal rate did
not meet the GB3838-2002 standard after 7 days, likely because the
added microbial consortium was not well immobilized. TP removal

Please cite this article in press as: Shao, Y., et al., Bioaugmentation in lab scale constructed wetland microcosms for treating polluted river water
and domestic wastewater in northern China, International Biodeterioration & Biodegradation (2014), http://dx.doi.org/10.1016/
j.ibiod.2014.05.027

Y. Shao et al. / International Biodeterioration & Biodegradation xxx (2014) 1e9

Table 3
Maximum, minimum and average pH, temperature (T) and dissolved oxygen concentration (DO) of RW and DW microcosms during phases I and II.
Parameters

RW

DW

pH
T ( C)
DO (mg/L)
pH
T ( C)
DO (mg/L)

Phase

Phase

Maximum

Minimum

Average

Maximum

Minimum

Average

7.5
28.6
1.01
7.7
28.3
1.05

7.0
18.5
0.6
7.1
18.0
0.63

7.32
23.5
0.82
7.4
23.2
0.84

7.5
25.6
1.10
7.6
25.8
1.02

7.36
16.5
0.56
7.1
17.5
0.66

7.42
20.3
0.85
7.32
20.3
0.85

efciency of Bio2 CWMs did not differ from Bio3 CMWs in the two
phases. The average inuent TP was 3.5 mg/L in phase in DW
(Fig. 5b) and met the Class I-B criteria of GB18918-2002 (<0.5 mg/L)
by the 7th day with a concentration of 0.4 mg/L and removal rate of
88.6%. However, efuent TP in phase had a 62% removal rate and
did not meet the GB18918-2002. There were no apparent differences
in TP removal by Abio2 and Abio3 CWMs in either phase I or II.
Similarly, the TP concentration in RW was reduced more in Abio1
CWM compared with Abio2 and Abio3 in both phases. Even in
autumn when plants had withered and absorbed little TP, inoculated
bacteria had a noticeable effect on TP removal.
3.4. Removal of N

implementation, efciency and cost effectiveness (Chen et al.,

2003; Kim et al., 2005; Khardenavis et al., 2007) and has been
assumed to result from common nitrication and denitrication
processes. Nitrication is thought to result in the oxidation of


NH3eN to NO

3 eN via NO2 eN by autotrophic bacteria under aerobic conditions, while denitrication results in the conversion of


NO

2 eN and NO3 eN to volatile N oxides and N2 gas by denitrifying
bacteria mostly under anaerobic conditions (Sundberg et al., 2007;
Daniel et al., 2009; Ji and Chen, 2010; Chang et al., 2011).
Nitrication is dependent on the NH3eN concentration in
wastewater (Paredes et al., 2007), while denitrication is promoted
by higher NO

3 eN concentrations (Sirivedhin and Grey, 2006). Bio1,
Bio2 and Bio3 treatments in our work evaluated removal

Nitrogen removal by biological means is a widely adopted

approach for wastewater treatment due to its ease of

Fig. 4. Mean chemical oxygen demand (CODcr) in the outow of constructed wetland
microcosms (CWMs) (a: RW; b: DW; : CWMs were inoculated with bacteria; : CWMs
were drained and then lled with simulated water without bacterial inoculation).

Fig. 5. Mean total phosphorus (TP) concentration in the outow of constructed

wetland microcosms (CWMs) (a: RW; b: DW; : CWMs were inoculated with bacteria;
: CWMs were drained and then lled with simulated river water without bacterial
inoculation).

Please cite this article in press as: Shao, Y., et al., Bioaugmentation in lab scale constructed wetland microcosms for treating polluted river water
and domestic wastewater in northern China, International Biodeterioration & Biodegradation (2014), http://dx.doi.org/10.1016/
j.ibiod.2014.05.027

Y. Shao et al. / International Biodeterioration & Biodegradation xxx (2014) 1e9

efciencies of NH3eN, NO

3 eN and TN. The inuent NH3eN concentrations varied signicantly with an average of 8.3 mg/L in RW
(Fig. 6a) and 48 mg/L in DW (Fig. 6b). In the RW CWM, the concentration of NH3eN decreased faster in Bio1 than Bio2 and Bio3
with 96.8% of NH3eN removed after 7 days in phase . This trend
continued in phase , with a 91.7% NH3eN removal rate from the
efuent by day 7. A similar trend was observed in DW, where
NH3eN concentration decreased more signicantly in Abio1 CWM
than Abio2 and Abio3 in both phases. NH3eN met the Class I-B
criteria of GB18918-2002 (<8 mg/L) by day 7 in phase , with a
concentration of 6.1 mg/L and a removal rate of 88%, while removal

rates of Abio2 and Abio3 were 52.6% and 44.5%, respectively.

NH3eN in the efuent did not reach the Class I-B criteria of
GB18918-2002 in phase II, though it still presented a high removal
rate of 81% on day 7. Our data suggest that adding denitrifying
bacteria indirectly increases nitrication in both phases of RW and
DW CWM's.
Biological denitrication appears to be an efcient, economical,
and environmentally friendly approach, which can simultaneously
remove organics and nitrogen (Vidali, 2001; Lynch and Moffat,
2005; Farhadian et al., 2008). Different denitrier species have
been placed in the natural environment, where they have exhibited

Fig. 6. Mean ammonia-N (NH3eN), nitrate-N (NO

3 eN) and total nitrogen (TN) concentrations in the outow of constructed wetland microcosms (CWMs) (a: RW; b: DW; : CWMs
were inoculated with bacteria; : CMWs were drained and then lled with simulated river water without bacterial inoculation).

Please cite this article in press as: Shao, Y., et al., Bioaugmentation in lab scale constructed wetland microcosms for treating polluted river water
and domestic wastewater in northern China, International Biodeterioration & Biodegradation (2014), http://dx.doi.org/10.1016/
j.ibiod.2014.05.027

Y. Shao et al. / International Biodeterioration & Biodegradation xxx (2014) 1e9

Table 4
Chemical oxygen demand (CODcr), ammonia-N (NH3eN), total N (TN), total phosphorus (TP), removal efciency of bioaugmentation treatments and optimum hydraulic
retention time (HRT) from different studies.
Tests

Inuent (mg/L)

Met Class criteria

of GB3838-2002 (d)

Met Class I-B criteria

of GB18918-2002 (d)
CODcr

TN

TP

CODcr

NH3eN

TN

TP

CODcr

TN

TP

19.2

1.56

32

143

4.7
NO

3 eN
17.2
NO

3 eN

90.4
26.3
24.2
88.6
64.9
60.2
e

7
N
N

3.5

96.7
33.9
31.4
79
44.8
38.8
83

7
N
N

50

96.8
46.9
38.7
88
52.6
44.5
e

7
N
N

48

75.7
68
41
85.7
63.6
58.8
48.7

82.5

95

CODcr
Bio1
Bio2
Bio3
Abio1
Abio2
Abio3
Ji et al., 2013a

68

170

Pei et al.

NH3eN

Removal efciency in
efuent (%)

8.3

NH3eN

Optimum HRT (d)

CODcr

TP

4
6
N

7
N
N

TP

7
N
N

N did not meet the criteria e not evaluated.

dissimilar denitrication proles in response to changes in the

environment (Feng et al., 2012a, b). Some denitrifying bacteria
cannot survive or perform denitrication due to the suboptimal
environment (Song et al., 2011). The inuent NO

3 -N concentrations
uctuated with an average of 10.2 mg/L (Fig. 6a). The NO

3 eN
concentration demonstrated the same changes with NH3eN in the
Bio1 CWM treatment. The NO

3 eN removal efciency of Bio1 was
much higher than Bio2 and Bio3 in both phases. In the Bio1 CWM,
the concentration of NO

3 eN reached removal efciency of 94.1% in
phase . However, in phase , NO

3 eN in the inuent of Bio CMW
presented a slightly lower rate of 91.7% on the 7th day. The inuent
NO

3 eN concentrations of 0.06 mg/L in DW was much lower than
RW (Fig. 6b). In phase , the concentration of NO

3 eN in Abio1 CWM
increased to 18.7 mg/L within 3 days, and then decreased to 2.5 mg/
L during the last 4 days. Presumably, part of NO

3 eN was transformed to NO

eN
under
anoxic
conditions
and
strains
preferen2
tially utilized NO

2 eN as an electron donor and nally N2 was
returned to the atmosphere during denitrication. The NO

3 eN
removal efciency of Abio1 was much higher and Abio1 CWM
shows much more obvious denitrication than Abio2 and Abio3 in
the two phases due to the effect of inoculated bacteria.
The pattern and efciency of TN removal by bioaugmentation
were partially affected by different N species (Fig. 6). NH3eN and
NO

3 eN are the dominant types of N with 96%e97% in the RW
inuent. For DW inuent, NH3eN concentration is almost the
same as the total N. As shown in Fig. 6a, the TN concentration
decreased signicantly in Bio1 CWM compared with the Bio2 and
Bio3 groups in both phases and . TN in the inuent uctuated
with an average value of 19.2 mg/L in phase . TN had already met
the Class III criteria of GB3838-2002 (<1 mg/L) on the 7th day,
with a concentration of 0.6 mg/L, and a removal rate of 96.7%. Long
term N removal from polluted water of the Liu River basin CW was
tested and an average TN removal efciency of 58.7% was attained
(Tang et al., 2013). This shows that the bioaugmentation may be an
effective means of increasing N removal efciency. However, in
phase , TN in the inuent, with an 81.6% removal rate, did not
meet the GB3838-2002 standard after 7 days. TN removal efciency of Bio2 CWMs did not differ greatly from Bio 3 in the two
phases. The removal efciency of TN ranged from 33.9% to 38.5%.
TN concentration decreased more signicantly in Abio1 CWMs
than Abio2 and Abio3 groups in both phases (Fig. 6b). The removal
efciency of TN in the Abio1 CMW was similar to that of NH3eN,
with a removal efciency of 73%e79%, while of Abio2 and Abio3
showed 44.6%e44.8% and 38.8%e40.7% removal rates, respectively
in the two phases. These results suggest that establishment and
growth of the added bacteria are very important factors for
successful CWs.

Bioaugmentation showed signicant effects on pollutant

removal regardless of the CODcr in both RW and DW as government
standards were met by day 7 in both cases (Table 4).
In a study using Pseudomonas stutzeri LZ-14 to treat wastewater
in CWs (Table 4; Ji et al., 2013a), TP removal was not detected and
the main form of N in wastewater was NO

3 eN, the electron
acceptor in denitrication and an easily oxidized nutrient for plants
and microorganisms. These studies suggest microorganisms may
be useful for treating water containing a large range of pollutants
from industrial, agricultural and domestic sources.
The inuent Carbon/Nitrogen (C/N) ratio is thought to uctuate
naturally and affect aerobic and anaerobic conditions in CWs (Wu
et al., 2009), however it did not change signicantly during bioaugmentation in RW and DW (Fig. 7).
The C/N ratios of 2e3 for RW, and 3e4 for DW in our study were
suitable for the inoculated bacteria. The bacterial strain XP1 was
most effective at denitrication when C/N was 7 (Hou et al., 2011), 3
for strain XP2 (Pei et al., 2013) and 5 for strains LZ-4 (Ji et al., 2013b)
and LZ-14 (Chen et al., 2009). The C/N ratios in the CWMs in our
study were suitable for the inoculated bacteria which suggests that
they did not adversely affect microbial activity; a fact emphasized
by the reduction of TN in microcosms. The positive effect of
Phragmites and bacterial inoculation suggests that certain
plantemicrobe combinations may constitute an effective treatment
for wastewater. For example, Bacillus subtilis strain FY99-01
signicantly enhanced microbial NO

3 eN removal in an articial
riparian wetland in southern China (Pei et al., 2010). Results from
that study suggested that NO

3 eN was removed more efciently in
summer (>35  C) than in winter (3.8  Ce8.6  C) and the maximal

Fig. 7. Relationship between N removal (TN removal %) and C/N ratio in river water
(RW) and DW.

Please cite this article in press as: Shao, Y., et al., Bioaugmentation in lab scale constructed wetland microcosms for treating polluted river water
and domestic wastewater in northern China, International Biodeterioration & Biodegradation (2014), http://dx.doi.org/10.1016/
j.ibiod.2014.05.027

Y. Shao et al. / International Biodeterioration & Biodegradation xxx (2014) 1e9

removal efciency of NO

3 eN was 36.1%. In order to develop more
effective applications for bioaugmentation, selection of low temperature tolerant, plant colonizing bacteria may be an effective
strategy to enhance microbial growth, survival and detoxifying
activity in CW.
4. Conclusions
The study shows that an introduced microbial consortium
designed for bioaugmentation promoted N and P removal from
polluted RW and DW in Northern China. The bacterial consortium
comprised six allochthonous denitrifying strains previously isolated from rhizosphere soil of wetlands plants and selected for
pollutant removal from RW and DW. Microbial activity was
enhanced when plants were present.
RCW inoculated with bacterial ora (Bio1 and Abio1) in phase
showed signicant improvements in COD, TN and TP removal efciency compared with phase treatments without bacteria. The
removal efciency of Bio1 on the 7th day was for CODcr, 96.7% for
TN, 96.8% for NH3eN and 90.4% for TP. The optimal HRT for TN, TP
and CODcr achieving the GB3838-2002 standard (China) for Grade
III guideline of 1.0 mg/L, 0.2 mg/L and 20 mg/L was 7 days, but Bio2
and Bio3 did not meet these criteria even by the end of phase II.
Similar trends in removal efciency were observed in Abio1, i.e.,
85.7% for CODcr, 75% for TN, 88.6% for NH3eN and 88% for TP on the
7th day. The optimal HRT for CODcr, NH3eN and TP achieving the
GB18918-2002 standard (China) for Class I-B criteria of 60 mg/L,
8.0 mg/L and 0.5 mg/L was 7 days, and Abio2 and Abio3 also did not
meet these criteria during the experimental period. Water ow
eroded microorganism population sizes during the experiments
resulting in a reduced positive effect on pollutant removal in the
articial wetland system. In order to maintain the positive effect of
micro ora, bacterial inoculations were repeated every 14 days in
the wetland system. Future research should focus on the use of
psychrophilic bacteria immobilized on a suitable solid substrate
such as a sponge to enhance microbial persistence and pollutant
removal in constructed wetlands.
Acknowledgements
This research was nancially supported by the National Natural
Science Foundation of China (51378300), (50978156); Nature Science Foundation of Shandong Province (ZR2009BZ007); Key
Research Foundation of Shandong Provincial Environmental protection bureau (hcyf0602) and Technology Development Projects of
Shandong Province (2009GG2GC06002). The authors thank Tim A.
Sheedy for revising the English in the manuscript.
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j.ibiod.2014.05.027