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Bioresource Technology 102 (2011) 94629470

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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


eutrophication.
Non-point source pollution of enclosed watersheds (e.g., lakes, Collecting and treating rural domestic wastewater is a large
reservoirs, and parts of rivers) is a global concern. The current dete- problem in China. Owing to the dispersed rural population and
rioration of water quality in Lake Taihu in China is primarily due to construction costs of sewage collectors, centralized wastewater
pollution from domestic wastewater produced in rural areas (Li treatment plants based on activated sludge or bacterial bed pro-
et al., 2009a). The essential issue regarding the increased incidence cesses that are utilized in large and small cities are not suitable
of eutrophication is the input of nitrogen (N) and phosphorus (P). in rural areas (Ye and Li, 2009). Treatment systems that require rel-
Excessive amounts of these nutrients lead to a range of undesirable atively low costs, energy, and maintenance are preferable for the
effects, including impairment of human health (algal toxins), re- treatment of widely distributed rural domestic wastewaters. Sev-
duced biodiversity of aquatic species, reduction in amenity value, eral proposed solutions for the treatment of diffuse sources of
and increased costs of treatment for drinking water (Withers domestic wastewater have been applied to on-site treatment in
et al., 2009). Therefore, nutrient inputs must be reduced and mea- spacious rural areas, including constructed wetlands, soil inltra-
sures for effective reduction of nutrient concentrations in rural 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
Abbreviations: VF, vermiltration; PCR-DGGE, polymerase chain reaction-dena- that separates wastewater solids by allowing wastewater to be
turing gradient gel electrophoresis; COD, chemical oxygen demand; NH3-N, gravity-fed over the ltration material, is the most promising eco-
ammonia nitrogen; BOD5, 5 day BOD; HLs, hydraulic loads; TN, total nitrogen; TP, nomical method for treating point and diffuse sources of domestic
total phosphorus; DO, dissolved oxygen; PVC, polyvinyl chloride; NO3-N, nitrate
wastewater.
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; VF has been studied extensively due to its effectiveness for
S1-4, detritus in the rst stage; S2-1, 015 cm soil in the second stage; S2-2, 15 removing pollutants in wastewater, and its positive effects on the
30 cm soil in the second stage; S2-3, silver sand in the second stage; S2-4, detritus environment. For example, Sinha et al. (2008) found that VF with
in the second stage; S3-1, 015 cm soil in third stage; S3-2, 1530 cm soil in the earthworms increased the effectiveness of most wastewater con-
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 taminant treatments, whereas systems without worms showed
65642948 (X. Luo). poor performance. Similarly, Li et al. (2009b) reported that waste-
E-mail addresses: zzhenghj@fudan.edu.cn (Z. Zheng), lxz@fudan.edu.cn (X. Luo). 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 9463

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

microbial populations (>1% of the total community) (Muyzer


H pi log pi  ni =N logni =N
i1 i1
et al., 1993), has been widely used owing to its rapid, reliable, sen-
sitive, and convenient function. Cloning and sequencing are com- where H is the Shannon index, ni is the height of the peak, and N is
monly subsequently applied to identify the phylogeny of the sum of all peak heights in the curves (Zhao et al., 2010b). The
microbes in environmental samples. In this study, DGGE was con- nucleotide sequences were compared with those deposited in Gen-
ducted using the Bio-Rad D gene system (Bio-Rad, USA). The poly- Bank (NCBI) using the Basic Local Alignment Search Tool (BLAST),
acrylamide gels were made with denaturing gradients ranging after which the sequences determined in this study and obtained
from 40% to 70% (100% denaturant is 7 M urea plus 40% formam- from the DNA database were aligned using CLUSTAL W. Distance
ide) to separate bacteria based on their V3 region. Electrophoresis 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.
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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 S1-4 S2-1 S2-2 S2-3 S2-4 S3-1 S3-2 S3-3 S3-4
a
Number of bands 67 days 9 14 12 3 4 3 7 N/D 7 10 6 5
131 days 21 16 5 2 5 9 9 8 7 12 10 3
Shannon index (H) 67 days 0.94 1.11 1.08 0.48 0.60 0.44 0.81 N/Da 0.83 0.97 0.76 0.66
131 days 1.29 1.18 0.69 0.29 0.70 0.94 0.90 0.87 0.81 1.05 0.73 0.48
a
Data not detected.

and 2 than in the inuent and Efuent 3. This occurred as a result bial biomass to stabilize during the operation period of a con-
of passive oxygenation of the dropping overow between the rst structed wetland (Ragusa et al., 2004). Similarly, the present
and second stages of VF, as well as between the second and third study found that VF did not tend to mature due to variation in
stages. The mean DO increased from 0.87 0.42 mg/L in the inu- the community ngerprints in some lter materials at different
ent to 2.12 0.67 mg/L in the nal efuent of the third stage. More- sampling times within a short period. Based on the comparison,
over, the wastewater temperature between the inuent and no obvious changes in dominant banding distribution occurred in
efuent did not vary greatly (data not shown). Throughout the partial samples with variations in time. These results are similar
study period, the average temperature was 18.6 C (ran- to the results of a study conducted by Tao et al. (2006, 2007),
ged = 10.532.1 C). and may be due to the mixed population at different temperatures,
The present study obtained higher COD, NH3-N, and TP removal as well as stable and optimal pH for heterotrophic bacterial pro-
efciency than Wang et al. (2010). Specically, Wang et al. ob- duction at near-neutral values of treated wastewater. This stable
tained removal of 78% COD, 90.3% NH3-N, and 62.4% TP using a microbial community composition was benecial for OM and
VF with converter slagcoal cinder to treat rural domestic waste- nutrient removal in wastewater treatment of the VF at all times.
water. With the tower VF system, higher average DO concentra- As shown in Fig. 4 and Table 3, the banding patterns and inten-
tions were obtained in Efuents 1 and 2, which most likely sities in the tower VF for lter samples had distinct differences in
resulted from the formation of cascades in the three-stage VF con- distribution with depth. Higher bacterial diversity and richness
guration. Moreover, the large amount of earthworms in the mul- were observed in the middle layers of the lter bed in every stage
tilevel VF played an important role in adjusting the redox on Day 67, as well as in the second and third stages on Day 133.
environment (Lin et al., 2006). These results indicate that the DO The number of bands and diversity decreased gradually as the
concentration and earthworm activity support nitrication and depth increased in the rst stage on Day 133. No data were ob-
COD removal. Because the tower VF alternately produced anaero- tained for the bottom of the second stage on Day 67. The DGGE
bic and aerobic circumstances, the corresponding P showed exces- banding pattern also showed that some dominant bands at S1-2
sive release and excessive accumulation when compared with the (band 9), S1-1 (band 17), and S1-1/S1-2 (band 20) were in the soil
single stage, which facilitated the high removal rate of P. The roots samples, but were not present in the other matrices. Moreover, a
of plants worked as a giant biological lter that removed all types maximum of 21 and 16 dominant bands were observed at S1-1
of OM, including microorganisms residing in submerged roots in and S1-2, respectively, in the rst stage on Day 133. Therefore,
the wastewater and OM that were later absorbed by the plants the activity of the earthworms resulted in a more dominant micro-
(de-Bashan and Bashan, 2004). Furthermore, the COD, NH3-N and bial community.
TP removal efciencies were not signicantly inuenced by the The differences in banding patterns and intensities obtained
variations in water temperature, possibly due to the presence of after analysis of the samples from various depths of the lter beds,
the growing plants. These ndings were in agreement with the re- as well as the presence of some unique bands that were only found
sults of other studies (Ye and Li, 2009; Zhao et al., 2010a). 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
3.2. Analysis of and successive changes in the denaturing gradient gel
oxygen and improved aerobic conditions for burrowing action of
electrophoresis (DGGE) proles
the earthworms favor the microenvironment for aerobic microor-
ganisms. In addition, the mucus and casts produced by the earth-
PCR-DGGE was used to compare the 16S rDNA gene fragment
worms are conducive to the development of a diverse microbial
proles of the V3 region of bacteria in samples collected during dif-
community (Vivas et al., 2009). Both earthworm functions may ex-
ferent operational periods and at each stage of the tower VF waste-
plain the higher community complexity observed at the middle-
water treatment process. Fig. 4 shows the bacterial community of
lower part of the soil layer. Additionally, the bacterial diversity
the media samples in the three-stage VF process at two operating
and richness were higher in the soil and silver sand than in the
times (67 and 133 days) in Lanes S1-1S1-4, S2-1S2-4, and S3-1
detritus. The small particles in soil and silver sand have a greater
S3-4. Both Lanes S1-1S3-4 in Fig. 4(a and b) show community n-
capacity to protect microbial biomass by providing a sorptive, high
gerprints somewhat similar to each other, except for Lane S2-4 in
surface area environment for closer microorganism attachment
Fig. 4(a). The complexity of the DGGE bands increased with operat-
(Ladd et al., 2004). Finally, decreased substrate and nutrient avail-
ing time in Lanes S1-1, S1-2, S2-2, S2-4, and S3-3, which indicates
ability was observed in the lower part of VF due to the removal of
that some media of the tower VF on Day 133 had a higher bacterial
organic pollutants in the upper layer, which was caused by the late
diversity than on Day 67. Similarly, evaluation of the bacterial rich-
growth of microbes in these areas.
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 3.3. Sequence diversity of bacteria
Lane S1-3 decreased as time progressed. Moreover, no signicant
change in dominant banding distribution was observed in Lanes Dominant bands recovered from the tower VF were individually
S1-4, S2-1, S2-3, S3-1, S3-2, and S3-4 at different sampling periods. identied as different members based on comparison with those in
Previously conducted laboratory experiments in batch reactors the GenBank database, and their phylogenetic positions are illus-
have shown that it would take more than 100 days for the micro- 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 9469

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 tes, Zhao et al. (2010b) analyzed the bacterial communities in the
close relationship was observed among different undetermined VF for the treatment of domestic wastewater sludge and found that
bands, with the phylogenetic relationship between bands 11, 14, species belonging to Proteobacteria were the dominant organisms
15 and 16 being closest. With regard to the identied bands, all in the VF biolm. Vivas et al. (2009) found that Proteobacteria
of them showed between 97% and 100% similarity with previously was the most abundant phylum in vermicompost made from a
identied gene sequences. Among these, half of the bands (9, 17, 20 toxic olive-mill waste. In addition, Fracchia et al. (2006) found that
and 22) were placed within Class Bacilli of Firmicutes. Specically, the most abundant bacteria in their vermicompost were related to
bands 9, 17 and 20 belonged to Trichococcus and band 22 belonged Acidobacteria and Bacteroidetes. This apparently distinct microbial
to Bacillus. Among the bands that were identied as Proteobacteria, distribution in different VFs could arise for two reasons. (1) If there
band 4 belonged to Class Alphaproteobacteria, and exhibited was an insufcient quantity of bacterial template DNA for PCR
homology to Rhodobacter sp., while band 13 was clustered with amplication for rarer groups among the bacteria, the most abun-
Pseudomonas sp. in the Class Gammaproteobacteria. The presence dant group could appear to be the only one present. (2) The system
of Flavobacterium sp. was observed in the sequences of band 8 may be relatively immature and thus the different populations
and 21; hence, they were ascribed to Class Flavobacteria of would not have had time to reach a long-term equilibrium.
Bacteroidetes.
In the present study, the microbial community of the three-
stage VF microcosm sediment was dominated by unclassied bac- 4. Conclusions
terium, followed by Bacilli of Firmicutes. Less abundant bacteria
were observed and classied according to phylogenetic clusters re- The present study showed that the tower VF efciently re-
lated to Proteobacteria and Flavobacteria of Bacteroidetes. Similar moved water pollutants. Compared with traditional VF, the tower
ndings concerning Firmicutes have been reported in other studies. VF had higher COD, NH3-N, and TP removal efciencies. This may
For example, Lu et al. (2008) found that Bacillus spp. (belonging to have occurred as a result of the adsorption and precipitation for l-
Firmicutes) comprised the dominant population of a VF. Bacillus ter materials during the longer running distance, increasing nitri-
spp. are antagonistic and functional bacteria that are crucial for cation ability between each stage, and the functioning of Al-, Fe-
the treatment efcacy of VF. However, Vivas et al. (2009) reported and Ca-oxides. The changes in microbial community diversity in
that Firmicutes was not the primary phylum in the vermicompost different paddings and stages likely contributed to the introduction
they used. In contrast, in the case of Proteobacteria and Bacteroide- of earthworms, media properties, and nutrient variations. The
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9470 L. Wang et al. / Bioresource Technology 102 (2011) 94629470

dominant populations were distinct and the microbe compositions International Conference on Energy and Environment Technology. Guilin, China,
pp. 472475.
were not similar to previous reports.
Liu, J.Y., Wang, H., Yang, H.J., Ma, Y.J., Cai, O.C., 2009b. Detection of phosphorus
species in sediments of articial landscape lakes in China by fractionation and
Acknowledgements phosphorus-31 nuclear magnetic resonance spectroscopy. Environ. Pollut. 157,
4956.
Lu, Z.B., Xing, M.Y., Yang, J., Liu, J., 2008. Analysis of microbial community in
The authors thank the National Water Pollution Project for Tai- vermiltration by PCR-SSCP method. Huanjing Kexue (Chinese) 29, 19921997.
hu Lake Pollution Control of China (2008ZX07101-004, Muyzer, G., Ellen, C.D.W., Andre, G.U., 1993. Proling of complex microbial
2008ZX07101-005) and Shanghai Natural Science Foundation populations by denaturing gradient gel electrophoresis analysis of polymerase
chain reaction-amplied genes coding for 16S rRNA. Appl. Environ. Microbiol.
(11ZR1402900) for their nancial support. The nancial assistance 59, 695700.
provided by the National Natural Science Foundation of China Ovreas, L., Forney, L., Daae, F.L., Torsvik, V., 1997. Distribution of bacterioplankton in
(40803025, 11075039) is also acknowledged. meromictic Lake Saelenvannet, as determined by denaturing gradient gel
electrophoresis of PCR-amplied gene fragments coding for 16S rRNA. Appl.
Environ. Microbiol. 63, 33673373.
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