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Bioresource Technology 102 (2011) 94629470

Contents lists available at ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Enhancement of rural domestic sewage treatment performance, and assessment

of microbial community diversity and structure using tower vermiltration
Longmian Wang a, Feihong Guo a, Zheng Zheng b,, Xingzhang Luo b,, Jibiao Zhang b
a
State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing 210046, PR China
b
Environmental Science & Engineering Department, Fudan University, No. 220, Handan Road, Shanghai 200433, PR China

a r t i c l e i n f o a b s t r a c t

Article history: The performance of a novel three-stage vermiltration (VF) system using the earthworm, Eisenia fetida,
Received 21 March 2011 for rural domestic wastewater treatment was studied during a 131-day period. The average removal ef-
Received in revised form 21 July 2011 ciencies of the tower VF planted with Penstemon campanulatus were as follows: chemical oxygen demand,
Accepted 23 July 2011
81.3%; ammonium, 98%; total nitrogen, 60.2%; total phosphorus, 98.4%; total nitrogen, mainly in the form
Available online 28 July 2011
of nitrate. Soils played an important role in removing the organic matter. The three-sectional design with
increasing oxygen demand concentration in the efuents, and the distribution of certain oxides in the
Keywords:
padding were likely benecial for ammonium and phosphorus removal, respectively. The microbial com-
Domestic wastewater
Nutrient removal
munity proles revealed that band patterns varied more or less in various matrices of each stage at dif-
Polymerase chain reaction-denaturing ferent sampling times, while the presence of earthworms intensied the bacterial diversity in soils.
gradient gel electrophoresis (PCR-DGGE) Retrieved sequences recovered from the media in VF primarily belonged to unknown bacterium and
Vermilter Bacilli of Firmicutes.
2011 Elsevier Ltd. All rights reserved.

1. Introduction wastewater must be developed to protect these ecosystems from

Non-point source pollution of enclosed watersheds (e.g., lakes,
reservoirs, and parts of rivers) is a global concern. The current dete-
rioration of water quality in Lake Taihu in China is primarily due to
pollution from domestic wastewater produced in rural areas (Li
et al., 2009a). The essential issue regarding the increased incidence
of eutrophication is the input of nitrogen (N) and phosphorus (P).
Excessive amounts of these nutrients lead to a range of undesirable
effects, including impairment of human health (algal toxins), re-
duced biodiversity of aquatic species, reduction in amenity value,
and increased costs of treatment for drinking water (Withers
et al., 2009). Therefore, nutrient inputs must be reduced and mea-
sures for effective reduction of nutrient concentrations in rural
Abbreviations: VF, vermiltration; PCR-DGGE, polymerase chain reaction-dena-
turing gradient gel electrophoresis; COD, chemical oxygen demand; NH3-N,
ammonia nitrogen; BOD5, 5 day BOD; HLs, hydraulic loads; TN, total nitrogen; TP,
total phosphorus; DO, dissolved oxygen; PVC, polyvinyl chloride; NO3-N, nitrate
nitrogen; OM, organic matter; XRF, X-ray uorescence; S1-1, 015 cm soil in the
rst stage; S1-2, 1530 cm soil in the rst stage; S1-3, silver sand in the rst stage;
S1-4, detritus in the rst stage; S2-1, 015 cm soil in the second stage; S2-2, 15
30 cm soil in the second stage; S2-3, silver sand in the second stage; S2-4, detritus
in the second stage; S3-1, 015 cm soil in third stage; S3-2, 1530 cm soil in the
third stage; S3-3, silver sand in the third stage; S3-4, detritus in the third stage.
Corresponding authors. Tel./fax: +86 21 65643342 (Z. Zheng), Tel./fax: +86 21
65642948 (X. Luo).
E-mail addresses: zzhenghj@fudan.edu.cn (Z. Zheng), lxz@fudan.edu.cn (X. Luo).
eutrophication.
Collecting and treating rural domestic wastewater is a large
problem in China. Owing to the dispersed rural population and
construction costs of sewage collectors, centralized wastewater
treatment plants based on activated sludge or bacterial bed pro-
cesses that are utilized in large and small cities are not suitable
in rural areas (Ye and Li, 2009). Treatment systems that require rel-
atively low costs, energy, and maintenance are preferable for the
treatment of widely distributed rural domestic wastewaters. Sev-
eral proposed solutions for the treatment of diffuse sources of
domestic wastewater have been applied to on-site treatment in
spacious rural areas, including constructed wetlands, soil inltra-
tion trenches, vegetation-based wastewater treatment, and vermi-
ltration (VF) (Cuyk et al., 2001; Ham et al., 2007; Kaoru et al.,
2010; Sinha et al., 2008). Among these technologies, VF, a process
that separates wastewater solids by allowing wastewater to be
gravity-fed over the ltration material, is the most promising eco-
nomical method for treating point and diffuse sources of domestic
wastewater.
VF has been studied extensively due to its effectiveness for
removing pollutants in wastewater, and its positive effects on the
environment. For example, Sinha et al. (2008) found that VF with
earthworms increased the effectiveness of most wastewater con-
taminant treatments, whereas systems without worms showed
poor performance. Similarly, Li et al. (2009b) reported that waste-
water produced by more than 100 inhabitants per day and

0960-8524/$ - see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2011.07.085
 Author's personal copy
L. Wang et al. / Bioresource Technology 102 (2011) 94629470
continuously treated by VF showed efcient reduction of chemical
oxygen demand (COD), ammonia nitrogen (NH3-N), and 5-day BOD
(BOD5). Fang et al. (2010) investigated the effect of hydraulic loads
(HLs) on pollutant removal from synthetic domestic wastewater by
an earthworm ecolter. HLs exhibited varying inuences on nutri-
ent removals. Recently, the integration of VF with traditional sew-
age treatment technologies for the removal of excess nutrients
from wastewater has appeared in the literature, including a labora-
tory-scale ceramsite-vermilter and vermilter enhancement
using a converter slagcoal cinder lter for domestic wastewater
treatment (Liu et al., 2009a; Wang et al., 2010). In addition to its
usage in urban or rural domestic wastewater treatment, the design
parameters and factors inuencing earthworms have also been
investigated (Hughes et al., 2009).
Previous studies have primarily focused on the use of VF or its
combined processes in the treatment of different types of waste-
water, and the related factors contributing to its efciency in
removing pollutants. However, the efuent quality concerning
nitrogen (N) and phosphorous (P) do not always meet the I-class
A criteria specied in the Discharge Standard of Pollutants for Muni-
cipal Wastewater Treatment Plants (GB18918-2002) in China. To en-
hance the nutrients removal, a three-stage microbial-earthworm
ecolter was designed with a continuous ow conguration. This
system provided excellent aerobic conditions for nitrication,
and increased the removal rates for COD, NH3-N and P. Therefore,
to obtain a more detailed understanding of the nutrient degrada-
tion processes in the tower VF, the present study was conducted
with the following objectives: (1) evaluate the performance of
the three-stage tower earthworm ecolter for the treatment of rur-
al domestic wastewater, mainly in terms of COD, total nitrogen
(TN), total phosphorus (TP), and NH3-N removal at each VF phase
under a certain hydraulic load; (2) assess the nutrient removal
with respect to dissolved oxygen (DO) in each efuent and the
change in chemical components before and after the operation
periods; and (3) investigate the microbial community diversity
and compositions in media at different sampling times by poly-
merase chain reaction-denaturing gradient gel electrophoresis
(PCR-DGGE).
2. Methods
2.1. Experimental system
Fig. 1 presents the schematic diagram of the tower VF system,
which was constructed at a village in Changzhou, Jiangsu Province,
China. The system consists of three cubic stages and a wastewater
containment tank, all of which were made of polyvinyl chloride
(PVC) and xed by an iron base. The tank and pump were installed
to collect and transfer the inuent from rural domestic wastewater
to the apparatus. The rst and second stages were 50 cm long and
50 cm wide, with a depth of 60 cm and packed padding of 55 cm.
Their lter beds both consisted of four layers from top to bottom
to a height of 30 cm with soil (diameter 400800 lm), 10 cm with
silver sand (diameter 100800 lm), 10 cm with ne detritus
(diameter 34 mm), and 5 cm with cobblestones (diameter 4
5 cm), which were used as the supporting layer. The third stage
was lled with the same substrates with dimensions of 110 cm
in length, 65 cm in width, and 120 cm in depth. The upper, middle,
and third layers had equal depths of 30 cm and were lled with
soil, silver sand, and detritus, respectively. The lowest layer of cob-
blestones had a depth of 20 cm. All padding came from Pukou in
the suburb of Nanjing, Jiangsu Province, China. The characteristics
of the padding used in this study can be found in Fang et al. (2010).
To achieve homogeneous water distribution, the raw wastewater
was introduced to the rst stage through a rotating PVC pipe with
9463
holes, and the distribution of wastewater from the up-level to the
down-level stage was achieved using perforated PVC pipes that ran
along the entire length in the second and third phases. The vertical
distances among stages were 40 cm. All PVC pipes, which were
drilled with holes 1.5 mm in diameter evenly distributed on the
lateral surface, were kept 15 cm above each stage surface to ensure
the creation of drop-overow and thereby increase aerobic
conditions.
Eisenia fetida (Savigny), a common earthworm, was selected as
the vermilter for wastewater treatment. The vermilter had a
density of 12.5 g/L in soil for every step, and its activity ensured
that the microbial-earthworm ecolter was not in a state of inun-
dation. The average soil C to N ratio was 18.6. In March 2010, all
surfaces of the stages were planted with fully matured Penstemon
campanulatus (transplanted from the suburb of Yixing, Jiangsu
Province, China) at a density of approximately 20 plants per m2.
No mortality occurred during the running periods. Introduction
of P. campanulatus into the system improved the plant uptake of
nutrients and aesthetics.
The start-up of the tower VF process was initiated by seeding
domestic wastewater in batch mode for acclimatization of earth-
worms and plants, colonization, and accumulation of microorgan-
isms in the medium. From March 27 to July 31, the process was
operated in continuous inow mode using the 1 m3/m2 d hydraulic
load. The raw wastewater was controlled using a liquidometer, and
the treated sewage passed through the stages in sequence by grav-
ity ow. During the operation period, the average extreme air tem-
perature was 19.6 C (ranged = 7.832.9 C).
2.2. Analytical procedure
2.2.1. Water quality analysis
Wastewater samples were collected weekly from the inlet of
the rst stage and outlet of the rst to third stage. COD, NH3-N,
TN, TP, and nitrate nitrogen (NO3-N) were analyzed according to
the standard methods (APHA, 1998). COD was measured using
the potassium dichromate method. NH3-N, TN, TP, and NO3-N were
determined using the Nesslers reagent spectrophotometric meth-
od, potassium persulphate oxidation-ultraviolet spectrophotome-
try, molybdenum-antimony anti-spectrophotometric method, and
ultraviolet spectrophotometric screening method, respectively.
The DO, pH, and temperature of the efuent of each stage were
measured in situ using a DO meter, including temperature mea-
surement (YSI Model No. 550A, USA) and a pH meter (Shanghai
Kangyi Instrument Co. Ltd., PHS-2C, China). All analyses, including
the control, were conducted in triplicate.
2.2.2. Matrix chemical composition analysis
At the end of the experiment (July 31, 2010), different types of
lter materials in the packed tower were collected at different
depths. In each stage, ve padding sampling holes 50 mm in diam-
eter were distributed on the lateral surface. Soil samples, namely,
topsoil (010 cm), middle-level soil (1020 cm), subsoil (20
30 cm), silver sand, and detritus including the initial medium were
air-shipped to the laboratory. The earthworms and trash were re-
moved from the plant roots on the day of collection. All samples
were freeze-dried, sieved (<2 mm) (except detritus), and stored
at 20 C for analysis. Organic matter (OM) was determined by
the loss of ignition after treatment at 550 C for 2 h (Liu et al.,
2009b). Chemical compositions were analyzed using an ARL-9800
XRF spectrometer (X-ray uorescence spectroscopy-XRF,
Switzerland).
2.2.3. Microbial analysis
Filters from 015 cm soil (S1/2/3-1), 1530 cm soil (S1/2/3-2),
silver sand (S1/2/3-3), and detritus (S1/2/3-4) collected in every
 Author's personal copy
9464 L. Wang et al. / Bioresource Technology 102 (2011) 94629470
Fig. 1. Diagram of the three-stage vermiltration for rural domestic wastewater treatment used in this study (unit: cm).
stage (1/2/3 indicates rst/second/third stage) were obtained. The
sampling was conducted twice on May 26, 2010 and July 31, 2010.
The pretreatment process was complied with the matrix chemical
composition analysis. DNA was extracted from the samples (0.5 g)
using an Ultra Clean Soil DNA Isolation Kit (MO BIO Laboratories,
CA, USA) according to the manufacturers instructions. The ex-
tracted DNAs were used as the template for PCR.
Three replicates of each sample were mixed to include as much
of the entire spectrum of microbes as possible. PCR was conducted
using an EDC-810 Thermal Cycler (Eastwing, Beijing, China) and
sample mixtures with a nal volume of 50 lL, containing 5 lL
10Ex Taq Buffer, 4 lL MgCl2 (25 mM), 4 lL dNTP mixture
(2.5 mM each), 1 lL primer (20 lM each), 0.25 lL DNA polymerase
(5 U/lL) (TaKaRa, Ex Taq, Japan), and 0.52 ng of DNA. Two rounds
of nested PCR were used to amplify the 16S rDNA gene fragments
from the V3 region. The rst-round of PCR was conducted using the
general bacterial primers 63fGC and 1387r (Ovreas et al., 1997).
The PCR amplicons were then used as the template in the sec-
ond-round of PCR, which was conducted using the specic primer
pair P338fGC/P518r (Muyzer et al., 1993). Both reactions consisted
of 30 cycles of 1 min at 94 C, 1 min at 55 C, and 2 min at 72 C.
This process was followed by a nal extension step at 72 C for
10 min. The PCR products were puried using the AP-PCR-50
Clean-Up Kit (Axygen, USA).
Several molecular approaches for characterization of microbes
have been investigated (Downing and Nerenberg, 2008; Gao
et al., 2010). PCR-DGGE, which permits assessment of the complex-
ity of microbial communities with respect to the most dominant
microbial populations (>1% of the total community) (Muyzer
et al., 1993), has been widely used owing to its rapid, reliable, sen-
sitive, and convenient function. Cloning and sequencing are com-
monly subsequently applied to identify the phylogeny of
microbes in environmental samples. In this study, DGGE was con-
ducted using the Bio-Rad D gene system (Bio-Rad, USA). The poly-
acrylamide gels were made with denaturing gradients ranging
from 40% to 70% (100% denaturant is 7 M urea plus 40% formam-
ide) to separate bacteria based on their V3 region. Electrophoresis
was conducted as described by Xia et al. (2005). After electropho-
resis, the gels were soaked for 15 min in ethidium bromide and
then rinsed for 20 min with Milli-Q water. Subsequently, gel
images were captured using the Gel Doc 2000 System (Bio-Rad,
USA).
2.2.4. Sequencing
The dominant bands of interest in the DGGE gel were excised
and then placed into a micro-centrifuge tube containing 25 lL TE
buffer for elution of the DNA at 4 C for 24 h. Under the same con-
ditions as described above, 1 lL of the extracted DNA was used as
the template in a PCR process with the primers P338f without a
GC-clamp and P518r. The puried PCR products were sequenced
directly by GenScript Inc. (Nanjing, China).
2.2.5. Database analysis
The nal removal efciency was calculated as the percent re-
moval (R) for each parameter, which was determined using the fol-
lowing equation: R = (1  Ce/Ci)  100, where Ci and Ce are the
inuent and efuent 3 concentrations in mg/L, respectively. The
mean inuent and efuent 3 values of every batch sampled during
a week were used to calculate removal rates for each parameter.
All statistical analyses were conducted using the Origin 8.0
software.
The Shannon index was calculated to reect the structural
diversity (richness and evenness) of the microbial community:
X
s X
s
H pi log pi  ni =N logni =N
i1 i1
where H is the Shannon index, ni is the height of the peak, and N is
the sum of all peak heights in the curves (Zhao et al., 2010b). The
nucleotide sequences were compared with those deposited in Gen-
Bank (NCBI) using the Basic Local Alignment Search Tool (BLAST),
after which the sequences determined in this study and obtained
from the DNA database were aligned using CLUSTAL W. Distance
matrix analyses were conducted with the p-distance, and the neigh-
 Author's personal copy

L. Wang et al. / Bioresource Technology 102 (2011) 94629470 9465

bor-joining trees were constructed by pair-wise deletion using

MEGA version 4.0 (Molecular Evolutionary Genetics Analysis). Tree
topology was evaluated by bootstrap analysis using 1000 replicates.

3. Results and discussion

3.1. Performance of wastewater treatment, organic matter (OM) and

chemical component analysis of substrates in the tower VF

The proles of the inlet concentration and removal efciency of

COD, NH3-N, TN, TP, and NO3-N distribution for rural domestic
wastewater at various stages during the 133-day operation period
are indicated in Figs. 2(ad) and 3. As shown in Fig. 2(a), the OM
measured as COD in the inuent ranged from 104.1 to 382.6 mg/
L. The removal efciency in the VF systems was more than 81.3%
for concentrations in Efuent 3 (less than 35.6 mg/L) during the
operation period. In addition, the concentration of COD sharply de-
clined while passing the rst stage, with further OM degradation
occurring while going through the second and third stage lters.
The concentration of NH3-N in the inuent ranged from 45.3 to Fig. 3. Analysis of nitrate (NO3-N) distribution in the inuent and the efuent of
89.8 mg/L. Moreover, the variations in the concentration of NH3- each stage.
N in different stage efuents resembled that of COD variation, with
a steady removal efciency reaching approximately 98%. In con- efuents. The nal outlet remained at a rather high level (mean
trast, the concentration of raw TN inow was maintained at 20.4 mg/L). Additionally, no steady-state removal efciency oc-
47.593.5 mg/L, which changed slightly from the inuent to the curred, even after treatment with the three-stage VF. Regarding

Fig. 2. Performance of the tower vermiltration system for rural domestic wastewater treatment at various stages during the 133-day operation period. (a) Changes in the
concentration of chemical oxygen demand (COD) between the inuent and efuents and its removal efciency; (b) changes in the concentration of ammonium (NH3-N)
between the inuent and efuents and its removal efciency; (c) changes in the concentration of total nitrogen (TN) between the inuent and efuents and its removal
efciency; and (d) changes in the concentration of total phosphorus (TP) between the inuent and efuents and its removal efciency.
 Author's personal copy

9466 L. Wang et al. / Bioresource Technology 102 (2011) 94629470

Detritus
the temporal changes observed in the TP concentration and re-

57.35
22.66
2.17

8.94
2.56
2.33
0.41
moval rate in the tower VF (Fig 2(d)), the efuent concentration de-
creased signicantly following a series of paths in which the rst
and third stages played a primary role. The inlet TP concentration

0.064
Silver

1.14

9.54
1.12
0.35
82.22
sand ranged from 5.04 to 9.88 mg/L, with an overall average of 98.4% in

0.4
terms of the removal rate. The concentration of NO3-N in the inu-
ent and efuents was also monitored (Fig. 3). The nitrates accumu-
2030 cm

lated in the system with an inlet concentration below 0.95 mg/L on

0.92
0.91
0.21
11.32

3.82
68.07
14.01

average, especially in the second and third phases.

soil

The chemical composition of the lters used as the VF substrate

for domestic wastewater treatment in each stage at different
1020 cm

depths (including OM content) is listed in Table 1. The OM percent-

11.47
62.56
14.78
3.45
1.19
3.19
0.27
soil

age of all samples was more or less enhanced by the end of the
operation period, and no signicant differences were detected
010 cm

among stages. When compared to the initial soils, the average

Stage 3

0.24
12.38
60.21

3.96

3.26
15.06

OM content of the nal soils increased by 3.23% in each stage;

1.2
soil

however, when the silver sand and detritus before and after treat-
ment were compared, the mean quality only increased by 0.16%
Detritus

2.34

8.37
2.79
3.46
0.48
56.23
22.76

and 1.41%, respectively. Moreover, the OM content of soil did not

vary greatly between the upper and bottom layers in each level.
XRF analysis revealed that SiO2, Al2O3, and Fe2O3 were the main
0.072

chemical components of the initial samples. Specically, these

Silver

81.36
9.33
0.87

0.31
0.41
1.02
sand

compounds were present in ratios of 68.07/14.01/3.82%, 82.22/

9.54/1.12%, and 57.35/22.66/8.94% (SiO2/Al2O3/Fe2O3) in the soil,
2030 cm

silver sand and detritus substrates, respectively. A low abundance

1.21
3.45
0.27
11.24
62.14
14.57
4.06

of MgO was also observed in all samples (Table 1). Before and after
soil

a long-term run, the contents of SiO2, Al2O3, Fe2O3 and MgO in the
tower VF did not change signicantly with depth during any
1020 cm

stages. However, the concentration of CaO and P2O5 was higher

0.24
61.24
13.16
10.67

3.27
1.19
3.33

in the nal soils than in the initial soil, as indicated by increases

soil

of 2.312.79 and 2.685.32 times, respectively. No similar in-

creases were observed in the rest of the lters (Table 1).
010 cm
Stage 2

11.47
63.52
14.26
3.14
1.17
3.18
0.35

VF is an efcient OM removal system due to its various physical,

soil
Organic matter and chemical components quality in media of different stages before and after a long-term run.

chemical, and biological functions. Specically, VF promotes the

adsorption of small-particle organisms, colloid organisms, mole-
Detritus

cules and ions, oxidationreduction of OM, and activity of earth-

54.47
22.13
3.75

8.36

3.34
0.37
3.09

worms and their synergistic effects with microorganisms (Wang

et al., 2010). In response to the high COD removal efciency during
0.051

wastewater treatment, the OM content in the substrate indicated

Silver

0.84

0.99
0.32
0.33
84.55
8.11
sand

that soil was more benecial for the removal of organic contami-
nants by precipitation and adsorption than silver sand and detritus.
2030 cm

In the present study, aside from the aforementioned positive ef-

fects of VF, plenty of biolms in the silver sand and detritus also
59.26
15.22

1.23
3.29
0.36
12.90

4.01
soil

contributed to the removal of OM. As the dominant type of N in

domestic wastewater, NH3-N was removed from the inuent
1020 cm

through rapid adsorption by the padding media and biomass. Sub-

12.29

14.85
60.39

3.95
1.24

0.34

sequently, the adsorbed NH3-N was converted to NO3-N through

3.2
soil

biological nitrication (Kadam et al., 2009). Nitrication coupled

with denitrication appears to be the major N removal process in-
010 cm
Stage 1

volved in many VFs, while insufcient available organic C is con-

12.64
59.94
14.65

1.22
3.27
0.32
4.06
soil

sidered to be responsible for the inhibition of denitrication

(Sinha et al., 2008; Wang et al., 2010). In the present study, all sam-
Detritus

ples were rich in SiO2, which primarily has a negative charge (Yu
0.51
57.35
22.66
1.34

8.94
2.56
2.33

et al., 1996). The matrix consisted of anions similar to NO3-N,

and a small amount of NO3-N was adsorbed by padding. Hence,
the concentration of NO3-N increased in the efuents because of
0.068
Silver

9.54
1.12
0.79

0.35
82.22
sand

0.4
Initial sample

the TN concentration. From the above discussion, no steady, rela-

tively low TN removal and NO3-N accumulation with OM degrada-
0.057

tion occurred with depth. The remarkable phosphorus reduction of

8.59

3.82
0.92
0.91
68.07
14.01
Soil

the VF was attributed to its biological effects, which included direct

absorption of phosphorus by suspended growing cells and plants,
Parameters

enhanced storage capacity of phosphorus as polyphosphate by

Fe2O3 (%)
Al2O3 (%)

MgO (%)

P2O5 (%)
SiO2 (%)

CaO (%)
OM (%)

the microbial biomass in padding, and improved nutrient transfor-

Table 1

mation and absorption capacity for phosphate from the wastewa-

ter in response to earthworm activity (de-Bashan and Bashan,
2004; Wen et al., 2006). The precipitation of P retained in the sub-
 Author's personal copy

L. Wang et al. / Bioresource Technology 102 (2011) 94629470 9467

Table 2 Moreover, the enhancement of the percentage of Ca listed in Ta-

The values of pH and DO changes in the inuent and efuents of every stage. ble 1 facilitates P retention via precipitation, and formation of
Parameters Inuenta Efuent 1a Efuent 2a Efuent 3a CaP deposition on the solids surfaces (Yang et al., 2009). In addi-
pH 7.15 0.16 7.24 0.12 7.18 0.19 7.07 0.18 tion, the rst and third stages likely took the leading role in TP re-
DO (mg/L) 0.87 0.42 3.89 1.10 4.08 1.24 2.12 0.67 moval because the upper stage received higher P loading than the
a
lower one (Pant et al., 2001), while the third-stage provided more
Data represent the mean standard deviation of 20 samplings.
lter materials that were favorable for TP removal after precipita-
tion and adsorption.
stratum also played an important role in decreasing the TP concen- The changes in the physicochemical parameters of the inuent
tration in the efuents since an increase in P2O5 contents in the - and efuent in all stages during the operation periods are pre-
nal soils was observed. This chemical P precipitation could sented in Table 2. The pH of the efuent increased throughout
partially be ascribed to the presence of Al and Fe, which formed the rst and second stages, then decreased slightly during the third
oxides that reacted with phosphate ions from the inuent under- period, reaching approximately 7.07, which was near the original
going ligand exchange, imparting the ability to retain P in the soils. value of the inuent. The DO was relatively higher in Efuents 1

Fig. 4. Denaturing gradient gel electrophoresis (DGGE) analysis of the V3 region bacteria fragments retrieved from lter materials in the tower vermiltration system on Days
67 (a) and 131 (b). The dominant bands were numbered 122. S1/2/3-1, S1/2/3-2, S1/2/3-3, and S1/2/3-4 represent the 015 cm soil, 1530 cm soil, silver sand, and detritus,
respectively. 1/2/3 denote the rst, second, and third stage, respectively.
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9468 L. Wang et al. / Bioresource Technology 102 (2011) 94629470
Table 3
Contrast of Shannon-wiener diversity index in different paddings of the tower VF at two sampling times.
S1-1 S1-2 S1-3
Number of bands 67 days 9 14 12
131 days 21 16 5 2
Shannon index (H) 67 days 0.94 1.11 1.08
131 days 1.29 1.18 0.69
a
Data not detected.
and 2 than in the inuent and Efuent 3. This occurred as a result
of passive oxygenation of the dropping overow between the rst
and second stages of VF, as well as between the second and third
stages. The mean DO increased from 0.87 0.42 mg/L in the inu-
ent to 2.12 0.67 mg/L in the nal efuent of the third stage. More-
over, the wastewater temperature between the inuent and
efuent did not vary greatly (data not shown). Throughout the
study period, the average temperature was 18.6 C (ran-
ged = 10.532.1 C).
The present study obtained higher COD, NH3-N, and TP removal
efciency than Wang et al. (2010). Specically, Wang et al. ob-
tained removal of 78% COD, 90.3% NH3-N, and 62.4% TP using a
VF with converter slagcoal cinder to treat rural domestic waste-
water. With the tower VF system, higher average DO concentra-
tions were obtained in Efuents 1 and 2, which most likely
resulted from the formation of cascades in the three-stage VF con-
guration. Moreover, the large amount of earthworms in the mul-
tilevel VF played an important role in adjusting the redox
environment (Lin et al., 2006). These results indicate that the DO
concentration and earthworm activity support nitrication and
COD removal. Because the tower VF alternately produced anaero-
bic and aerobic circumstances, the corresponding P showed exces-
sive release and excessive accumulation when compared with the
single stage, which facilitated the high removal rate of P. The roots
of plants worked as a giant biological lter that removed all types
of OM, including microorganisms residing in submerged roots in
the wastewater and OM that were later absorbed by the plants
(de-Bashan and Bashan, 2004). Furthermore, the COD, NH3-N and
TP removal efciencies were not signicantly inuenced by the
variations in water temperature, possibly due to the presence of
the growing plants. These ndings were in agreement with the re-
sults of other studies (Ye and Li, 2009; Zhao et al., 2010a).
3.2. Analysis of and successive changes in the denaturing gradient gel
electrophoresis (DGGE) proles
PCR-DGGE was used to compare the 16S rDNA gene fragment
proles of the V3 region of bacteria in samples collected during dif-
ferent operational periods and at each stage of the tower VF waste-
water treatment process. Fig. 4 shows the bacterial community of
the media samples in the three-stage VF process at two operating
times (67 and 133 days) in Lanes S1-1S1-4, S2-1S2-4, and S3-1
S3-4. Both Lanes S1-1S3-4 in Fig. 4(a and b) show community n-
gerprints somewhat similar to each other, except for Lane S2-4 in
Fig. 4(a). The complexity of the DGGE bands increased with operat-
ing time in Lanes S1-1, S1-2, S2-2, S2-4, and S3-3, which indicates
that some media of the tower VF on Day 133 had a higher bacterial
diversity than on Day 67. Similarly, evaluation of the bacterial rich-
ness based on the Shannon index revealed that the bacterial rich-
ness in the above lters was higher on Day 133 than on Day 67
(Table 3). In contrast, the banding pattern and the complexity of
Lane S1-3 decreased as time progressed. Moreover, no signicant
change in dominant banding distribution was observed in Lanes
S1-4, S2-1, S2-3, S3-1, S3-2, and S3-4 at different sampling periods.
Previously conducted laboratory experiments in batch reactors
have shown that it would take more than 100 days for the micro-
S1-4 S2-1 S2-2 S2-3 S2-4 S3-1 S3-2 S3-3 S3-4
a
3 4 3 7 N/D 7 10 6 5
5 9 9 8 7 12 10 3
0.48 0.60 0.44 0.81 N/Da 0.83 0.97 0.76 0.66
0.29 0.70 0.94 0.90 0.87 0.81 1.05 0.73 0.48
bial biomass to stabilize during the operation period of a con-
structed wetland (Ragusa et al., 2004). Similarly, the present
study found that VF did not tend to mature due to variation in
the community ngerprints in some lter materials at different
sampling times within a short period. Based on the comparison,
no obvious changes in dominant banding distribution occurred in
partial samples with variations in time. These results are similar
to the results of a study conducted by Tao et al. (2006, 2007),
and may be due to the mixed population at different temperatures,
as well as stable and optimal pH for heterotrophic bacterial pro-
duction at near-neutral values of treated wastewater. This stable
microbial community composition was benecial for OM and
nutrient removal in wastewater treatment of the VF at all times.
As shown in Fig. 4 and Table 3, the banding patterns and inten-
sities in the tower VF for lter samples had distinct differences in
distribution with depth. Higher bacterial diversity and richness
were observed in the middle layers of the lter bed in every stage
on Day 67, as well as in the second and third stages on Day 133.
The number of bands and diversity decreased gradually as the
depth increased in the rst stage on Day 133. No data were ob-
tained for the bottom of the second stage on Day 67. The DGGE
banding pattern also showed that some dominant bands at S1-2
(band 9), S1-1 (band 17), and S1-1/S1-2 (band 20) were in the soil
samples, but were not present in the other matrices. Moreover, a
maximum of 21 and 16 dominant bands were observed at S1-1
and S1-2, respectively, in the rst stage on Day 133. Therefore,
the activity of the earthworms resulted in a more dominant micro-
bial community.
The differences in banding patterns and intensities obtained
after analysis of the samples from various depths of the lter beds,
as well as the presence of some unique bands that were only found
in soils of the tower VF were most likely due to earthworm activity,
lter body properties, and nutrient availability. Indeed, earth-
worms were found to dwell at a depth of 1530 cm soil. Sufcient
oxygen and improved aerobic conditions for burrowing action of
the earthworms favor the microenvironment for aerobic microor-
ganisms. In addition, the mucus and casts produced by the earth-
worms are conducive to the development of a diverse microbial
community (Vivas et al., 2009). Both earthworm functions may ex-
plain the higher community complexity observed at the middle-
lower part of the soil layer. Additionally, the bacterial diversity
and richness were higher in the soil and silver sand than in the
detritus. The small particles in soil and silver sand have a greater
capacity to protect microbial biomass by providing a sorptive, high
surface area environment for closer microorganism attachment
(Ladd et al., 2004). Finally, decreased substrate and nutrient avail-
ability was observed in the lower part of VF due to the removal of
organic pollutants in the upper layer, which was caused by the late
growth of microbes in these areas.
3.3. Sequence diversity of bacteria
Dominant bands recovered from the tower VF were individually
identied as different members based on comparison with those in
the GenBank database, and their phylogenetic positions are illus-
trated in the neighbor-joining tree shown in Fig. 5. Most of the
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L. Wang et al. / Bioresource Technology 102 (2011) 94629470
Fig. 5. Phylogenetic tree based on neighbor-joining analysis of gene sequences from the V3 region of bacteria with a length of 197 bp. Bands 122 represent the sample
sequences. Numbers at the branches represent bootstrap values, and the scale bar indicates 5 changes per 100 amino acid positions.
bands were not related to the identied bacterium group, but a
close relationship was observed among different undetermined
bands, with the phylogenetic relationship between bands 11, 14,
15 and 16 being closest. With regard to the identied bands, all
of them showed between 97% and 100% similarity with previously
identied gene sequences. Among these, half of the bands (9, 17, 20
and 22) were placed within Class Bacilli of Firmicutes. Specically,
bands 9, 17 and 20 belonged to Trichococcus and band 22 belonged
to Bacillus. Among the bands that were identied as Proteobacteria,
band 4 belonged to Class Alphaproteobacteria, and exhibited
homology to Rhodobacter sp., while band 13 was clustered with
Pseudomonas sp. in the Class Gammaproteobacteria. The presence
of Flavobacterium sp. was observed in the sequences of band 8
and 21; hence, they were ascribed to Class Flavobacteria of
Bacteroidetes.
In the present study, the microbial community of the three-
stage VF microcosm sediment was dominated by unclassied bac-
terium, followed by Bacilli of Firmicutes. Less abundant bacteria
were observed and classied according to phylogenetic clusters re-
lated to Proteobacteria and Flavobacteria of Bacteroidetes. Similar
ndings concerning Firmicutes have been reported in other studies.
For example, Lu et al. (2008) found that Bacillus spp. (belonging to
Firmicutes) comprised the dominant population of a VF. Bacillus
spp. are antagonistic and functional bacteria that are crucial for
the treatment efcacy of VF. However, Vivas et al. (2009) reported
that Firmicutes was not the primary phylum in the vermicompost
they used. In contrast, in the case of Proteobacteria and Bacteroide-
9469
tes, Zhao et al. (2010b) analyzed the bacterial communities in the
VF for the treatment of domestic wastewater sludge and found that
species belonging to Proteobacteria were the dominant organisms
in the VF biolm. Vivas et al. (2009) found that Proteobacteria
was the most abundant phylum in vermicompost made from a
toxic olive-mill waste. In addition, Fracchia et al. (2006) found that
the most abundant bacteria in their vermicompost were related to
Acidobacteria and Bacteroidetes. This apparently distinct microbial
distribution in different VFs could arise for two reasons. (1) If there
was an insufcient quantity of bacterial template DNA for PCR
amplication for rarer groups among the bacteria, the most abun-
dant group could appear to be the only one present. (2) The system
may be relatively immature and thus the different populations
would not have had time to reach a long-term equilibrium.
4. Conclusions
The present study showed that the tower VF efciently re-
moved water pollutants. Compared with traditional VF, the tower
VF had higher COD, NH3-N, and TP removal efciencies. This may
have occurred as a result of the adsorption and precipitation for l-
ter materials during the longer running distance, increasing nitri-
cation ability between each stage, and the functioning of Al-, Fe-
and Ca-oxides. The changes in microbial community diversity in
different paddings and stages likely contributed to the introduction
of earthworms, media properties, and nutrient variations. The
 Author's personal copy
9470 L. Wang et al. / Bioresource Technology 102 (2011) 94629470
dominant populations were distinct and the microbe compositions
were not similar to previous reports.
Acknowledgements
The authors thank the National Water Pollution Project for Tai-
hu Lake Pollution Control of China (2008ZX07101-004,
2008ZX07101-005) and Shanghai Natural Science Foundation
(11ZR1402900) for their nancial support. The nancial assistance
provided by the National Natural Science Foundation of China
(40803025, 11075039) is also acknowledged.
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