Effects of monocot and dicot types and species richness in mesocosm constructed wetlands on removal of pollutants from wastewater.
ABSTRACT The effects of planting type and species richness on removal of BOD5, COD, nitrogen and phosphorus were studied in mesocosms with monocot alone (M), dicot alone (D) and mixed planting of M+D, where each planting type had four species richness levels. Above- and below-ground plant biomasses increased with the M and M+D species richness as shown by one-way ANOVA. The M+D type had the highest above-ground biomass, whereas the M type had the highest below-ground biomass among planting types. Carbon, nitrogen and phosphorus in the microbial biomass increased with the richness of the M and M+D type. Removals of BOD5, COD, inorganic P and total P did not change with the richness, but removals of NH4-N, NO3-N increased. Planting type impacted only removal of inorganic P, with higher removal of inorganic P in the M type.
- SourceAvailable from: Baojing GuAtmospheric Environment 12/2012; · 3.06 Impact Factor
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ABSTRACT: Four horizontal subsurface flow constructed wetlands (HSFCWs), named HSFCW1 (three-stage, without step-feeding), HSFCW2 (three-stage, with step-feeding), HSFCW3 (five-stage, without step-feeding) and HSFCW4 (five-stage, with step-feeding) were designed to investigate the effects of dissolved oxygen (DO) and step-feeding on nitrogen removal. High removal of 90.9% COD, 99.1% ammonium nitrogen and 88.1% total nitrogen (TN) were obtained simultaneously in HSFCW4 compared with HSFCW1-3. The excellent TN removal of HSFCW4 was due to artificial aeration provided sufficient DO for nitrification and the favorable anoxic environment created for denitrification. Step-feeding was a crucial factor because it provided sufficient carbon source (high COD: nitrate ratio of 14.3) for the denitrification process. Microbial activities and microbial abundance in HSFCW4 was found to be influenced by DO distribution and step-feeding, and thus improve TN removal. These results suggest that artificial aeration combined with step-feeding could achieve high nitrogen removal in HSFCWs.Bioresource Technology 07/2014; 169C:395-402. · 5.04 Impact Factor
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ABSTRACT: This study investigated the effects of three planting patterns and related plant species richness on the structure and metabolic activities of microbial communities in vertical flow mesocosms fed with piggery wastewater. The planting pattern impacted the microbial community composition in the current mesocosm substrates, with 17, 12 and 10 fatty acids occurring in the substrates of monocot (M), dicot (D) and M + D planting pattern, respectively. In contrast, the planting pattern did not impact fatty acid abundance and the utilization intensity of six carbon guilds. Plant species richness from each planting pattern differently impacted fatty acid methyl ester (FAME) profiles, i.e. greater M richness enhanced the abundance of 18:3ω6c (fungi), D richness simultaneously enhanced the abundance of both i15:0 (Gram-positive bacteria) and 18:1ω9c (Gram-negative bacteria) and M + D richness increased the abundance of 18:0 (Gram-positive bacteria), 18:1ω9c and 18:2ω6c (fungi), respectively. Similarly, greater M, D and M + D richness enhanced the utilization of the amino acid, polymer and carbohydrate guilds, respectively. Therefore, the current study highlights the importance of plant species richness in distinguishing the microbial community in constructed wetland (CW) systems.Research in Microbiology 08/2013; · 2.83 Impact Factor
Effects of monocot and dicot types and species richness in mesocosm
constructed wetlands on removal of pollutants from wastewater
Chong-Bang Zhanga, Wen-Li Liua, Jiang Wanga, Yong Geb, Ying Gec, Scott X. Changd, Jie Changc,⇑
aSchool of Life Sciences, Taizhou University, Linhai 317000, PR China
bDepartment for Environmental Protection and Management, Huangyan Municipality, Zhejiang Province 3180220, PR China
cCollege of Life Sciences, Zhejiang University, Hangzhou 310058, PR China
dDepartment of Renewable Resources, University of Alberta, Edmonton, Alberta, Canada T6G 2E3
a r t i c l ei n f o
Received 7 June 2011
Received in revised form 15 August 2011
Accepted 18 August 2011
Available online 25 August 2011
Plant and microbial biomass
a b s t r a c t
The effects of planting type and species richness on removal of BOD5, COD, nitrogen and phosphorus were
studied in mesocosms with monocot alone (M), dicot alone (D) and mixed planting of M + D, where each
planting type had four species richness levels. Above- and below-ground plant biomasses increased with
the M and M + D species richness as shown by one-way ANOVA. The M + D type had the highest above-
ground biomass, whereas the M type had the highest below-ground biomass among planting types. Car-
bon, nitrogen and phosphorus in the microbial biomass increased with the richness of the M and M + D
type. Removals of BOD5, COD, inorganic P and total P did not change with the richness, but removals of
NH4-N, NO3-N increased. Planting type impacted only removal of inorganic P, with higher removal of
inorganic P in the M type.
? 2011 Elsevier Ltd. All rights reserved.
Constructed wetland (CW) represents an alternative to con-
ventional wastewater treatment for a wide range of pollutants
(Maltais-Landry et al., 2007). When wastewater is treated using
CWs, numerous factors influence the removal efficiency of pollu-
tants. These factors include the filling material such as sand,
gravel, furnace slag, zeolite and apatite (Scholz and Xu, 2002),
substrate redox condition (Wießner et al., 2005), wastewater
loading rate (Maltais-Landry et al., 2007), flow rate (Lavrova
and Koumanova, 2010), retention time, carbon source availabil-
ity, electron acceptor concentrations, temperature in the envi-
ronment (Krauter et al., 2005) and plant species (Maine et al.,
In CWs, plants play both direct and indirect roles in the removal
of pollutants from wastewater (Kyambadde et al., 2004; Marchand
et al., 2010; Lizama et al., 2011). Direct roles include the uptake,
sorption and filtering of pollutants by plants, while indirect roles
include enhancement of oxygen availability, and release of organic
compounds (such as sugars and organic acids) into filter matrix to
stimulate microbial activities (Ryan and Delhaize, 2001; Chaudhry
et al., 2005; Maine et al., 2007; Maltais-Landry et al., 2007). Each
plant species may have a different nutrient uptake rate and specific
growth pattern in natural and CWs, thus different plants have been
applied to CWs on different scales for testing the effects on the re-
moval efficiency of pollutants from wastewater. It was demon-
strated that macrophyte diversity enhanced the functioning of
wetland ecosystems such as removal of phosphorus and nitrate
(Engelhardt and Ritchie, 2001; Sirianuntapiboon and Jitvimolnimit,
2007) and increased diversity and activity of microbial communi-
ties (Zhang et al., 2010a,b).
The majority of studies concerning the removal of pollutants in
the CWs have been focused on monocots (M) such as the species of
Gramineae, since these plants are dominant in many wetland sys-
tems (Visser et al., 2000). Nevertheless, dicot (D) species often oc-
cur in waterlogged or wetland environments as well (Snowden and
Wheeler, 1995; Visser et al., 2000). Both types of plants have differ-
ent anatomical and physiological properties such as root morphol-
ogy, aerenchyma, photosynthetic rate and radial oxygen loss
(Visser et al., 2000; Mogami et al., 2006; Marchand et al., 2010).
We hypothesized that monocot and dicotous plant types as well
as their species richness would impact differently the removal effi-
ciencies of pollutants in CWs. To test our hypothesis, we collected
eight M and D species from subtropical wetlands between March
and April of 2009, and established three planting types: M plants
alone, D plants alone and mixed planting of M + D in mesocosms.
Within each plant type, each of mesocosms planted with two, four
and eight plant species were replicated three times, respectively.
Unplanted mesocosms were used as controls. The experiment
was designed to answer the questions of how planting type
impacts removal efficiency of pollutants in wastewater and if
0960-8524/$ - see front matter ? 2011 Elsevier Ltd. All rights reserved.
⇑Corresponding author. Tel.: +86 571 88206466; fax: +86 571 88206465.
E-mail address: firstname.lastname@example.org (J. Chang).
Bioresource Technology 102 (2011) 10260–10265
Contents lists available at SciVerse ScienceDirect
journal homepage: www.elsevier.com/locate/biortech
species richness or planting type are more important in enhancing
2.1. Mesocosm design
Similar to the design of a previously described full-scale wet-
land system (Zhang et al., 2010a), 30 mesocosms were established
at Taizhou University, Zhejiang Province, in Eastern China from Au-
gust to October of 2009. The mesocosm containers were made
from polyethylene, and 1.1 (height) ? 1.0 (length) ? 1.0 (width)
m in size. Each mesocosm was filled with fine river sand (1–
2 mm) in the top layer (40 cm deep), coarse sand (4–6 mm) in
the middle layer (30 cm), and gravel (50–85 mm) in the bottom
layer (30 cm). To ensure that each mesocosm received the same
wastewater, all mesocosms were connected to the wastewater-
storage tank that supplied the wastewater. The wastewater used
for this study was collected from the effluent of a pig breeding farm
BOD5= 37.56 mg L?1, COD = 48.46 mg L?1,
NO3-N = 0.79 mg L?1, total nitrogen (N) = 22.97 mg L?1, inorganic
P = 0.36 mg L?1, and total P = 1.47 mg L?1. All mesocosms were
fed by pulse-irrigation of wastewater, and operated with the same
hydraulic loading rate (0.67 m3day?1) and retention time (8 days).
Between March and April of 2010, eight M plants (Scirpus tri-
queter, Phragmites australis, Juncus effuses, Plantago asiatica, Acorus
calamus, Ophiopogon japonicus, Cyperus rotundus, Canna indica)
and eight D plants (Penthorum chinense, Houttuynia cordata,
Kalimeris indica, Alternanthera philoxeroides, Oenanthe javanica, Des-
modium podocarpum, Ranunculus asiaticus, Artemisia lavandulaefoli-
a) were collected from Shanjiang National City Swamp near Linhai
City (28?500N, 121?060E), Zhejiang Province. The plants were grown
in a greenhouse using healthy rhizomatous and stolon cuttings, or
seedlings with the same size rather than seeds, since the percent of
seed germination is generally low in perennial aquatic plants
(Engelhardt and Ritchie, 2002). After one month of growth in the
greenhouse, the plants were transplanted into the mesocosms.
The planting patterns were: M plants alone, D plants alone and
mixed planting of M + D. Within each planting type, two, four
and eight plant species were planted in triplicate. Unplanted mes-
ocosms were used as controls. The special species composition for
each type of mesocosm was showed in Table 1. The mixtures of
two, four and eight species were assembled by constraint random
selection from the species pool, i.e., selecting the same species
twice was not allowed in this procedure (van Ruijven and Ber-
endse, 2009). The planting density in each mesocosm was 16 viable
seedlings. Seedlings that died were replanted and other invasive
species were removed throughout the experiment. Because the dif-
ferences between the mono-cultured and unplanted treatments in
terms of nutrient retention, and the structure and function of
microbial communities in our previous vertical flow CW system
were small (Zhang et al., 2010a,b), the current study did not con-
sider a treatment planted with one species. Based on the present
design, our experiment included a total of 30 mesocosms.
NH4-N = 1.30 mg L?1,
2.2. Sampling methods
After the mesocosms were established for 60 days, wastewater
samples including the influent and effluent in each mesocosm
were taken biweekly in July, August and October 2010. Each sam-
pling was completed within one day. Three influent and effluent
water samples were collected from each mesocosm and stored in
polyethylene bottles (1 L) for water quality parameter analysis. Ex-
cept for the water samples using immediately for BOD5analysis,
other samples were temporarily stored in a refrigerator of ?20 ?C
for at least one week.
Plant and substrate samples were collected only at the end of
October 2010 using the harvesting and spade-sampling methods
(Zhang et al., 2010a), and were completed within one day. Because
unit were difficult to separate, above- and below-ground samples
were not separated by species to keep the consistent scale. Newly
produced above-ground plant tissue (stems, shoots and leaves)
was clipped at the stem base above the substrate surface for net
(roots plus rhizomes) were collected, washed carefully the samples
through a sieve (3 mm openings) for biomass analysis.
Once plant sampling was completed, five substrate samples (to
20 cm depth) were immediately collected in each mesocosm unit
using a sampling spade (the sampling date referred to the plant
sampling), and were mixed into a composite sample. After roots
and macrofauna were removed by hand (Zhang et al., 2010a), the
moist substrate samples were sieved (2 mm) and immediately col-
lected in Ziploc™ bags, which were stored temporarily in a refrig-
erator at 4 ?C for microbial biomass analysis.
2.3. Water quality analysis
Water quality parameters (the five-day biological oxygen de-
mand (BOD5), chemical oxygen demand (COD), total P, NH4-N,
Species composition across each species richness series within three planting types.
Species richnessMonocots DicotsM + D
C.-B. Zhang et al./Bioresource Technology 102 (2011) 10260–10265
NO3-N and inorganic P) were analyzed using standard methods
established by the National Environment Protection Agency of Chi-
na (2002). The removal efficiencies of pollutants were computed
based on mass balance (Maltais-Landry et al., 2007):
R ð%Þ ¼ ½1 ? ðEv? Ec=Iv? IcÞ? ? 100
where R is the removal efficiency, Evis the treated effluent volume,
Ecis the treated effluent concentration; Ivis the influent volume, Ic
is the concentration in the influent.
2.4. Plant biomass analysis
Plant samples were dried until constant weight at 60 ?C. The to-
tal above- and below-ground biomasses were converted into dry
weight per square meter according to the surface area of the mes-
ocosm (1 m2).
2.5. Microbial biomass measurement
Microbial biomass C (MBC), N (MBN) and P (MBP) were deter-
mined by the chloroform fumigation–extraction technique (Amato
and Ladd, 1988; Brookes et al., 1982; Sparling and West, 1988). Ten
grams of fresh substrate was fumigated with chloroform at 25 ?C
for 24 h. Following fumigation, the extractable organic C was ex-
tracted with 0.5 mol L?1K2SO4and was determined using a heated
K2Cr2O7–H2SO4 digestion. The KEC-factor used was 0.35 which
means that 35% of microbial C was extractable as organic C after
the fumigation (Sparling and West, 1988). The extractable organic
N was extracted with 2 mol L?1KCl and was measured photomet-
rically (UV2102-PC photoelectric photometer, UNICO) at 570 nm
after the ninhydrin reaction. The KEN-factor used was 3.1 repre-
senting that 1 lg biomass N per g dry substrate is equivalent to
3.1 lg ninhydrin-reactive N per g dry substrate (Amato and Ladd,
1988). The P in samples was extracted with 0.5 mol L?1NaHCO3
and measured photometrically at 882 nm. The biomass P was
determined from the difference in extractable inorganic P between
fumigated and non-fumigated samples. A KEP-factor of 0.4 was
used representing that 40% P in the biomass is rendered extract-
able as inorganic P by chloroform (Brookes et al., 1982).
2.6. Data analysis
The mean removals of pollutants over three months were calcu-
lated and, along with plant and microbial data (measured at end of
October), are presented as means of triplicates of mesocosms with
standard deviations. All data were log-transformed to maintain
homogeneity of variances (Reinhart and Callaway, 2004). Signifi-
cant difference between treatments was analyzed using two one-
way ANOVAs (analysis of variance), i.e., one used plant species
richness as fixed factor for identifying the difference of variables
among richness levels within each of M, D and M + D types, while
another used plant type as fixed factor for identifying the differ-
ence of planted variables among M, D and M + D types. Multiple
comparisons of means were conducted using Tukey test at
P = 0.05. All statistical analyses were performed with the SPSS soft-
ware for Windows version 11.5 (2002).
3. Results and discussion
3.1. Plant biomass production in mesocosms
Studies conducted on the relationship between plant diversity
and productivity in mesocosms have yielded inconsistent results.
For example, some studies showed that plant shoot biomass was
not affected by species richness due to sampling effects, i.e., the
presence of a competitively dominant but less productive macro-
phyte species (Engelhardt and Ritchie, 2001, 2002), whereas other
studies demonstrated a higher plant biomass with greater species
richness (Sirianuntapiboon and Jitvimolnimit, 2007; Zhang et al.,
2010a). In the current study, total above- and below-ground plant
biomasses of the D type did not respond significantly to the species
richness (2, 4 and 8 species) regardless of the unplanted treatment,
as shown by the one-way ANOVA (Fig. 1A). This observation sup-
ports results of Engelhardt and Ritchie (2001, 2002). Differently,
the M and M + D types had significantly higher above- and be-
low-ground plant biomass productions in the treatment planted
with 8 species than in the treatment planted with two species, thus
confirming the results of Sirianuntapiboon and Jitvimolnimit
(2007) and Zhang et al. (2010a). Among different planting types,
significant changes in the above-ground plant biomass were ob-
served, i.e., both D and M + D types had significantly higher bio-
mass productions than the M type (Fig. 1A). This was on the one
hand related to higher competition of dicots for nutrients (Call-
away et al., 2004), and on the other hand indicated that positive
complementary effects occurred between the D and M types. The
below-ground biomasses of the D type did not change significantly
across the richness levels (2, 4 and 8 species, Fig. 1B), as shown by
one-way ANOVA, but for the M and M + D types, treatments
planted with 8 species had significantly higher biomasses than
the treatments planted with 2 and 4 species. For the plant type ef-
fects, the M type had higher below-ground biomass than the D
type. This was most likely attributed to efficient utilization of
nutrients through the rhizomes and dense fibrous roots of the M
3.2. Microbial biomass in mesocosms
Microorganisms and their activities have been regarded as the
cornerstone of the CW treatment technology for removal of most
pollutants in wastewater (Faulwetter et al., 2009). In the current
study, MBC increased significantly with species richness of three
planting types, while MBN and MBP increased differently with spe-
cies richness of the M and M + D type, as shown by one-way ANO-
VA (Fig. 2A–C). MBC did not match the changes in above- and
below-ground plant biomass, while the MBN and MBP matched
those of the plant biomass parameters of the M and M + D types,
indicating that the plant biomass for the M and M + D was an
important factor mediating the substrate microbial community
size in mesocosms, thus supporting previous findings with a full-
scale CW (Zhang et al., 2010a). Studies on grassland ecosystems
also provided evidence that soil microbial biomass significantly in-
creased with plant species richness (Zak et al., 2003; Liu et al.,
2008). Among three planting types, when not considering the un-
planted treatment, the M + D type had slightly or significantly
higher MBC and MBN compared with the M or D type alone, thus
indicating complementary effects of the M and D types on the sub-
strate microbial biomass.
3.3. Pollutant removal efficiencies in mesocosms
Plant species with different rooting depth and root productivity
specifically influence removal of pollutants in wastewater (Kyamb-
adde et al., 2004; Calheiros et al., 2007). In the present vertical flow
plantedor plantedtreatment, andwere not affected byspeciesrich-
ness or planting type, as shown by one-way ANOVA (Fig. 3A and B).
Our monitoring was conducted from July to October, a period when
pollutant removal is most efficient (Werker et al., 2002), thus we
speculated that the temperature was possibly a more important
factor in controlling both BOD5and COD removals than plants. Our
findings supported previous results that the cultivated–plant types
C.-B. Zhang et al./Bioresource Technology 102 (2011) 10260–10265
ensis, Sirianuntapiboon and Jitvimolnimit, 2007) or plant species
solid (SS), BOD5and COD in a subsurface flow CW. This outcome
Fig. 1. Effects of species richness and planting type on the above- (A) and below-ground (B) plant biomass production in vertical flow mesocosms. Results from one-way
ANOVA are presented in each diagram. Bars denote standard deviations of the means of plant biomass. M + D indicates a mixed planting type of monocot and dicot species.
Small letters above the bars indicate significance of mean biomass variance, where bars with the different letters indicate significant difference of plant biomass at P = 0.05,
but bars with the same letters indicate no significant difference of plant biomass among species richness levels. Bars, letters and abbreviations were the same below.
Fig. 2. Effects of species richness and planting type on production of microbial biomass carbon (A) and ninhydrin-reactive nitrogen (B) and phosphorus (C) in vertical flow
mesocosms. Results from one-way ANOVA are presented in the diagrams.
Fig. 3. Effects of species richness and planting type on removal efficiency of BOD5(A), COD (B), NH4-N (C), NO3-N (D), inorganic phosphorus (E) and total phosphorus (F) in
vertical flow mesocosms. Results from one-way ANOVA are presented in the diagrams.
C.-B. Zhang et al./Bioresource Technology 102 (2011) 10260–10265
could be attributed to the dominant role played by microbial degra-
dation (Sirianuntapiboon and Jitvimolnimit, 2007; Faulwetter et al.,
2007) or since plant roots and rhizomes can ‘support’ growth of cer-
tion, release of oxygen from the root tissues is possibly another
important cause for stimulating microbial growth (Kantawanichkul
et al., 2009).
Removal of NH4-N and NO3-N may be attributed to two major
mechanisms, i.e., plant uptake and microbial transformation in
the CW (Faulwetter et al., 2009). In the current study, removal effi-
ciencies of NH4-N and NO3-N increased significantly with M spe-
cies richness regardless of the unplanted treatment (Fig. 3C and
D), and is thus related to the increases in plant and microbial bio-
masses of the M type. However, removal of NH4-N and NO3-N did
not change significantly with D and D + M richness (Fig. 3C and D).
These changes matched those in the plant biomass, MBN and MBP
of the D type, but did not match the changes in both plant and
microbial biomasses of the M + D type. This results for the M + D
type can perhaps be attributed to other removal ways such as sub-
strate sorption or uptake by green filamentous algae during the
wastewater retention in CWs, since increased retention of both
NH4-N and NO3-N in substrate with species richness was observed
in our previous study (Zhang et al., 2010b). In contrast, the study
by Engelhardt and Ritchie (2001) showed that the green filamen-
tous algae attached to plants played an important role in removing
pollutants such as phosphorus. Kyambadde et al. (2004) claimed
also that the epiphytic nitrifiers appeared more important in
removing NH4-N in wastewater than those in substrate or sus-
pended in water. Our original expectation was that the mixed
planting treatment would removal more NH4-N and NO3-N
through complementary uptake by two plant types than the M or
D type alone. However, one-way ANOVA showed that no signifi-
cant removal differences of NH4-N and NO3-N were observed
among three planting types (Fig. 3C and D), indicating that plant
type was perhaps not an important factor in enhancing removal
of NH4-N and NO3-N in the present mesocosms. This case was
not consistent with the differences in the determined plant and
microbial biomasses among planting types. Since less information
on nutrient usage for planting types is available, this observation
cannot be currently explained.
Removal of P in CWs depends on uptake by plants and microbes,
as well as sorption on the substrate (Hunter et al., 2001; Braskerud,
2002; Kelderman et al., 2007). Vegetation has been reported to im-
prove P removal in some CW systems (Hunter et al., 2001; Kyamb-
adde et al., 2004), but no significant difference between the
vegetated and un-vegetated treatments has been reported in other
systems (Burgoon et al., 1989; Calheiros et al., 2007). Discrepancies
among studies may be caused by differences in vegetation type and
density, growth media type, retention time, loading rate, tempera-
ture, and size of treatment systems (Hunter et al., 2001). In the cur-
rent study, inorganic P removal was not enhanced significantly by
species richness regardless of the unplanted treatment (Fig. 3E),
indicating that plant uptake and complementary usage among
plant species was not perhaps an important mechanism on inor-
ganic P removal, since phosphorus accumulated in plant tissues
or microbial cells is rapidly returned into soil or substrate through
degradation (Richardson, 1985). However, the removal of inorganic
P was affected by planting type, and was unexpectedly higher in
the M type than in the D and M + D planting types. This result cor-
related with the highest below-ground plant biomass and MBP
across M species richness, indicating that planting type was per-
haps a more important factor for inorganic P removal than the spe-
cies richness in the CW system (Hunter et al., 2001, p. 302; Khan
and Shah, 2010). Total P removal in the current study did not
increase significantly with species richness, and was not impacted
by the planting type, as shown by one-way ANOVA (Fig. 3F). This
case was most possibly attributed to the small difference in MBP
among planting types.
Effects of three planting types using monocots and dicots and
their species richness levels on removal of pollutants were studied
in mesocosms receiving wastewater from a pig breeding farm.
Monocot species richness was important for removals of NH4-N
and NO3-N in the piggery wastewater, while monocot planting
type enhanced removal of inorganic P more than other planting
types such as dicot alone and mixture of monocots + dicots. There-
fore, a monocot planting pattern and its species richness were the
best choices for removing inorganic nitrogen and phosphorus from
wastewater in the current mesocosm treatment system.
We are grateful for the funding provided by the Natural Science
Foundation from Zhejiang Province, China (No. Y5100017) and Na-
tional Science Foundation of China (Nos. 30870235 and 31000256).
We wish to thank Professor Ke Shisheng and Engineer Yuan Qingq-
ing from School of Life Sciences of Taizhou University for their help
in the analysis of wastewater quality.
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