See

discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/262939403

Earthworm eco-physiological characteristics

and quantification of earthworm feeding in...

Article in Journal of Hazardous Materials May 2014

DOI: 10.1016/j.jhazmat.2014.05.042 Source: PubMed

CITATIONS READS

8 61

4 authors, including:

Xiaowei Li Xing Meiyan

Shanghai University Tongji University
26 PUBLICATIONS 309 CITATIONS 48 PUBLICATIONS 671 CITATIONS

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

sewage sludge treatment and utilization View project

All content following this page was uploaded by Xiaowei Li on 28 November 2014.

The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the original document
and are linked to publications on ResearchGate, letting you access and read them immediately.
 Journal of Hazardous Materials 276 (2014) 353361

Contents lists available at ScienceDirect

Journal of Hazardous Materials

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

Earthworm eco-physiological characteristics and quantication of

earthworm feeding in vermiltration system for sewage sludge
stabilization using stable isotopic natural abundance
Xiaowei Li, Meiyan Xing , Jian Yang, Xiaohu Dai
State Key Laboratory of Pollution Control and Resources Reuse, The National Engineering Research Center for Urban Pollution Control,
College of Environmental Science and Engineering, Tongji University, Shanghai 200092, PR China

h i g h l i g h t s

Earthworm growth biomass and activity decreased with the VF depth.

Earthworm gut microbial communities were dominated by Gammaproteobacteria.
15 N and 13 C in earthworms decreased with time, and increased with the VF depth.
Effect of earthworm feeding in enhanced VSS reduction was analyzed quantitatively.
Earthworm feeding had low contribution to the enhanced VSS reduction.

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

Article history: Previous studies showed that the presence of earthworm improves treatment performance of vermilter
Received 10 February 2014 (VF) for sewage sludge stabilization, but earthworm eco-physiological characteristics and effects in VF
Received in revised form 15 April 2014 were not fully investigated. In this study, earthworm population, enzymatic activity, gut microbial com-
Accepted 14 May 2014
munity and stable isotopic abundance were investigated in the VF. Results showed that biomass, average
Available online 22 May 2014
weight, number and alkaline phosphatase activity of the earthworms tended to decrease, while protein
content and activities of peroxidase and catalase had an increasing tendency as the VF depth. Earthworm
Keywords:
gut microbial communities were dominated by Gammaproteobacteria, and the percentages arrived to
Sewage sludge
Vermiltration
7692% of the microbial species detected. 15 N and 13 C natural abundance of the earthworms decreased
Stable isotope with operation time, and increased as the VF depth. Quantitative analysis using 15 N showed that earth-
Earthworm feeding worm feeding and earthwormmicroorganism interaction were responsible for approximately 21% and
Earthwormmicroorganism interaction 79%, respectively, of the enhanced volatile suspended solid reduction due to the presence of earthworm.
The nding provides a quantitative insight into how earthworms inuence on sewage sludge stabilization
in vermiltration system.
2014 Elsevier B.V. All rights reserved.

1. Introduction ltration system is found to improve its treatment performance

Sewage sludge treatment and disposal represent a rising chal-
lenge for wastewater treatment plants (WWTPs) worldwide, due
to environmental, economic, social and legal factors [1]. Compared
with other technologies of sewage sludge stabilization, vermil-
tration is a low-cost and ecologically sound technique, and more
suitable for sewage sludge treatment of small or developing-
country WWTPs [2], which are widely used to treat wastewater,
sewage sludge, etc. [15]. The introduction of earthworm to
Corresponding author. Tel.: +86 13524248335; +86 021 65984275.
E-mail addresses: lixiaowei419@163.com, xingmeiyan@tongji.edu.cn (M. Xing).
for sewage sludge stabilization (e.g. an increase in volatile sus-
pended solids (VSS) by 16.6% [1]), and to promote the degradation
of organic matter, such as carbohydrates and proteinaceous mate-
rials [6], and to enhance the microbial activity in the biolm [2,6],
and to boost the quality of sewage sludge as soil organic fertil-
izer [7]. So far, the capacity of vermiltration system per volume
for sewage sludge treatment is relatively low (usually less than
1.5 kg VSS m3 d1 [2,6,7]), and needed to be further improved.
Since the earthworm is considered as a key component in ver-
miltration system, earthworm eco-physiological changes and
effects during sewage sludge stabilization is highly desirable for
the improvement of vermiltration process, but the information is
little.

http://dx.doi.org/10.1016/j.jhazmat.2014.05.042
0304-3894/ 2014 Elsevier B.V. All rights reserved.
354 X. Li et al. / Journal of Hazardous Materials 276 (2014) 353361
Earthworm growth characteristics could be indicated by a range
of parameters, such as biomass, average weight, individual num-
ber, and activities of alkaline phosphatase (AKP), peroxidase (POD)
and catalase (CAT). Biomass, average weight and individual number
represent directly changes in earthworm growth. AKP activity has
an important relationship with P cycle in the body of earthworm
[8], while POD and CAT are antioxidant enzymes, which have been
used as biomarkers of environmental stress on earthworm growth
[9].
The microorganisms in earthworm gut play an important role
in the digestion of organic food. Xing et al. [1] found that passage
of intestinal tract of earthworm had a qualitatively and quanti-
tatively inuence on microbial community of the vermilter (VF)
biolm. However, the knowledge about microbial community com-
position in earthworm gut is limited and controversial. Egert et al.
[10] suggested that an abundant indigenous earthworm microbial
community was unlikely to exist, at least in the midgut region of
Lumbricus terrestris by T-RFLP analysis of 16S rRNA gene fragments,
but Sampedro et al. [11] proposed the existence of a specic micro-
bial community in the earthworm gut that provides fatty acids to
the earthworm by analysis of fatty acid proles. In the present
study, PCR-based 16S rDNA gene clone library was used to analyze
the microbial community composition in earthworm gut, which
could provide a more comprehensive analysis of microbial compo-
sitions [6,12].
Stable isotope ratios of nitrogen and carbon can be used to
determine the trophic position of an organism and to analyze
the ultimate source of energy for an organism [13], including
earthworm feeding ecology [14]. Xing et al. [1] found that the
earthworms (adults, hatchlings and cocoons) lay in the top trophic
level of food web in the vermiltration system based on 15 N
and 13 C natural ratios. Sampedro and Domnguez [15] got insight
about relative trophic position of earthworms and other soil fauna
in vermicomposting system using stable isotope natural signa-
tures of nitrogen and carbon. Eisenia fetida is widely used to
treat sewage sludge and other organic waste in vermicomposting
and vermiltration [13,6,7,16], and thus it is needful to inves-
tigate the 15 N and 13 C natural abundances of E. fetida and their
changes with operation time and the VF depth during vermil-
tration process, in order to study the feeding characteristics of
E. fetida.
Earthworm feeding is an important strategy for the earthworm
improving sewage sludge stabilization (e.g. the increase of VSS
reduction) in vermiltration system [2]. Quantitative analysis of
earthworm feeding in enhanced VSS reduction is very signicant
to reveal the mechanism of earthworm improving VF treatment
performance. A feasible method to quantitatively analyze the strat-
egy is rstly to measure the amount of earthworm cast and the
digestion ratio of earthworms for the sewage sludge, and then to
calculate digestion amount of the earthworms [2]. The amount
of earthworm cast is determined through 15 N and 13 C natural
abundances of the inuent sludge, biolm and earthworm cast,
and the amount of the efuent sludge [17]. A Visual Basic pro-
gram called ISOSOURCE is available to perform these calculations
for the data. The digestion ratio of earthworm is measured based
on the VSS/SS ratios of the ingested and digested sludge, and
the assumption that inorganic matter is not or little digested by
earthworms [2].
The objectives of the present study were: (1) to explore
depth distribution of earthworm population and enzymatic
activities in the VF, and (2) to analyze microbial commu-
nity composition in earthworm gut by 16S rDNA clone library,
and (3) to investigate the changes in earthworm 15 N and 13 C
natural abundances as the time and depth, and (4) to quanti-
tatively reveal earthworm feeding in the improvement of VSS
reduction.
2. Materials and methods
2.1. Vermiltration system
A cylindrical VF consisting of four superimposed and separated
perspex tubing (20 cm in diameter and 30 cm in depth) was set up
with a 0.5-in. polypropylene pipe with holes to ensure uniform dis-
tribution of the inuent, and assembled as previously description
[1,6]. The tubing contained a 100 cm lter bed of ceramic pellets
(1020 mm in diameter). A layer of plastic ber was placed on top of
the lter bed to avoid direct hydraulic inuence on the earthworms
and ensure even inuent distribution.
The VF was inoculated with E. fetida at an initial earthworm
density of approximately 40 g L1 . A conventional biolter (BF)
was set up without earthworms as the control. The hydraulic
loadings of the lters were kept at 4 m d1 during the experimen-
tal period. The inuent sludge was obtained from the aeration
tank of a domestic WWTP (Quyang WWTP, Shanghai) and diluted
to a constant organic load of approximately 1.12 kg VSS m3 d1
using tap water. After passing through the lter bed, the treated
sludge entered into a sedimentation tank below the VF, and the
supernatant in the sedimentation tank was recycled. The VSS
to suspended solid (SS), sludge volume index and pH value in
the inuent sludge were 704.0 33 g kg1 SS, 115 34 ml g1 and
7.5 0.4, respectively (Table S1 of the Supplementary Data, SD).
The initial earthworms were randomly picked from a Donghai farm,
Shanghai City, China, and cultured with cow dung as substrate.
SS contents of the inuent and efuent samples were measured
by drying the samples at 105 C for 24 h. VSS contents were deter-
mined by heating the samples at 600 C for 1 h in a mufe furnace.
2.2. Earthworm population analysis
Forty healthy earthworms were taken randomly from the four
tubing of VF, and kept in a culture dishes in the dark for 24 h to
empty their guts. Their excrement (cast) in the culture dishes and
adhering to the earthworms was collected for the VSS/SS ratio,
yield rate of earthworm cast and stable isotopic analysis. There-
after, the sampled earthworms were washed and dried with paper
towels and then weighed. The mean value of earthworm individual
weights was considered to be average weight of the earthworms.
Total number of earthworms in the VF was determined by hand
sorting and enumeration. Earthworm biomass was estimated by
the multiplication of the average weight and total number of earth-
worms.
2.3. Earthworm enzymatic activity
After the operation of 6 months, the earthworm samples in the
upper and under layers of VF were collected for the analysis of enzy-
matic activities. Protein content was determined according to the
method of Bradford [18]. AKP activity was measured as described
by Tabatabai and Bremner [19]. CAT activity was assayed according
to the method of Saint-Denis [20]. POD activity were determined
using kit obtained from the Nanjing Jiancheng Bioengineering Insti-
tute [21].
2.4. Earthworm gut microbial community analysis
The initial and 6th month samples of earthworm gut in the
VF were gained through dissecting earthworms in sterile condi-
tion after emptying their gut content. Bacterial genomic DNA was
extracted from gut samples using a Fast DNA Spin Kit for soil (QBIO-
gene, Carlsbad, CA, USA). PCR of bacteria 16S rDNA genes (using
primers 27F and 1492R), DNA purication, ligation and transfor-
mation, construction of clone libraries, and phylogenetic analysis
 X. Li et al. / Journal of Hazardous Materials 276 (2014) 353361
were carried out according to the method of Feng et al. [22]. The
nucleotide sequences reported in this paper have been deposited
in the GenBank, EMBL, and DDBL nucleotide database under the
accession numbers JF276457JF276652.
The Shannon index (H) was used to reect the diversity (richness
and evenness) of the gut microbial community as follows [23],

S
H= pi ln pi
i=1
where S is the number of the species detected by 16S rDNA clone
library; Pi is the proportion of individuals belonging to the ith
species in the whole dataset.
2.5. Earthworm stable isotopic analysis
The inuent and efuent sludge, and biolms samples in the two
lters were collected after 6 months, and concentrated using cen-
trifugation at 6000 rpm for 5 min, and acidied using 1 mol L1 HCl
to remove inorganic carbon [24]. Then, the acidied samples were
washed to pH 7.0 with deionized water. The earthworm samples at
the initial period, 6th and 10th month, including adults, hatchlings
and cocoons, were randomly taken from the VF by hand sorting,
and then cleaned with distilled water. All of earthworm and sludge
samples were dried at 40 C in a drying chamber, and stored in a
desiccator containing P2 O5 at room temperature until analysis.
15 N and 13 C natural abundances of the dried samples were mea-
sured using a Thermo Finnigan Delta plus XP isotope ratio mass
spectrometer coupled to a Flash EA 1112 elemental analyzer in the
State Key Laboratory of Estuarine and Coastal Research at East China
Normal University (Shanghai, China) according to the method of
Sampedro and Domnguez [15]. Amount of the samples were pre-
viously adjusted to ensure a C:N that permit analysis in the dual
isotope mode in which C and N isotope ratios are determined simul-
taneously on the samples. Duplicate analyses were performed on
all the samples.
Stable isotope ratios of carbon and nitrogen were shown
as the ratio of heavy to light carbon and nitrogen relative to
Vienna Peedee Belemnite standard limestone (13 C/12 C) and atmo-
spheric dinitrogen standards (15 N/14 N) in parts per thousand:
X = (Rsample /Rstandard 1) 1000, where X = 13 C or 15 N and
R = 13 C/12 C or 15 N/14 N, respectively [15].
2.6. Quantitative analysis of earthworm feeding
The theoretical calculation of VSS balance in the vermiltration
system was based on the following equations.
1) VSSear.
Since the VSS content of inuent sludge in the VF is similar to
that in the BF, the enhanced VSS reduction content due to the pres-
ence of earthworm is equal to the difference in the VSS content of
efuent sludge between the VF and the BF. Thus,
VSSear. = VSSeff.B. VSSeff.V.
where VSSear. is VSS reduction content due to the presence of earth-
worm; VSSeff.V. is VSS content of efuent sludge in the VF; VSSeff.B. is
VSS content of efuent sludge in the BF.
At the other hand, the earthworms have two pathways to pro-
mote the reduction of VSS content in the VF. Thus, the VSSear. is also
written as,
VSSear. = VSSdig. + VSSint.
where VSSdig. is VSS reduction content due to the digestion by
earthworms feeding; VSSint. is VSS reduction content due to the
355
microbial-activity improvement by earthwormmicroorganism
interaction.
2) VSSdig.
VSSdig. is denoted as follows,
VSSdig. = SSfee. Rfee. SScas. Rcas. (4)
(1)
where SSfee. is SS content of earthworm feeding; Rfee. is VSS/SS ratio
of earthworm organic food (similar to the inuent sludge); SScas. is
SS content of earthworm cast; Rcas. is VSS/SS ratio of the earthworm
cast.
According to the assumption that the content of inorganic mat-
ter is constant during digestion process of the earthworms [2], there
was a following equation,
(1 Rfee. ) SSfee. = (1 Rcas. ) SScas. (5)
Thus,
Rfee. Rcas.
VSSdig. = SScas. (6)
1 Rfee.
3) SScas.
There are two methods to estimate SS content of earthworm
cast in the VF.
a) One is according to the earthworm biomass and yield rate of
earthworm cast, based on the method of Zhao et al. [2]. SScas. is
expressed as follows,
SScas. = SSyie. Wear. (7)
where SSyie. is the yield rate of earthworm cast
(g SS kg1 earthworm d1 ); Wear. is the earthworm biomass in
the whole VF.
b) The other is to base on stable isotopic ratios using ISOSOURE
software
In the BF, the efuent sludge is regarded as a mixture of the
biolm and inuent sludge. Thus,
ISOinf . Pinf ./eff.B. + ISObio.B. Pbio.B./eff.B. = ISOeff.B. (8)
Pinf ./eff.B. + Pbio.B./eff.B. = 1 (9)
where ISOinf. is isotopic ratio of the inuent sludge; ISObio.B. is iso-
topic ratio of the biolm in the BF; ISOeff.B. is isotopic ratio of the
efuent sludge in the BF; Pinf./eff.B. is contribution ratio of the inuent
sludge to the efuent sludge of the BF; Pbio.B./eff.B. is contribution
ratio of the biolm to the efuent sludge of the BF.
Similarly, the efuent sludge is regarded as a mixture of the
biolm, inuent sludge and earthworm cast in the VF. Thus,
ISOinf . Pinf ./eff.V. + ISObio.V. Pbio.V./eff.V.
(2) + ISOcas. Pcas./eff.V. = ISOeff.V. (10)
Pinf ./eff.v. + Pbio.V./eff.v. + Pcas./eff.v. = 1 (11)
where ISObio.V. is isotopic ratio of the biolm in the VF; ISOeff.V. is iso-
topic ratio of the efuent sludge in the VF; ISOcas. is isotopic ratio of
the earthworm cast; Pinf.V./eff.V. is contribution ratio of the inuent
sludge to the efuent sludge of the VF; Pbio.V./eff.V. is contribution
(3)
ratio of the biolm to the efuent sludge of the VF; Pcas./eff.V. is con-
tribution ratio of the earthworm cast in the efuent sludge of the
VF.
356 X. Li et al. / Journal of Hazardous Materials 276 (2014) 353361

Additionally, another assumption was made, which was the Earthworm biomass (g.L-1 )
presence of earthworm only changed the distribution of the iso- 0 10 20 30 40 50 60 70
topic ratio between the biolm and the cast, but did not change total
amount of the isotopic ratios. So, there was a following equation, a
ISObio.V. Pbio.V./eff.V. + ISOcas. Pcas./eff.V. 0-25

= ISObio.B. (1 Pinf ./eff.V. ) (12)

25-50

Depth (cm)
Based on Eqs. (8)(12), the Pcas./eff.V. was gained. Thus,

VSScas. = VSSeff.V. Pcas./eff.V. (13) 50-75

VSScas. VSSeff.V. Pcas./eff.V.

SScas. = = (14)
Rcas. Rcas.
75-100
where VSScas. is VSS content of the earthworm cast.
Thus, according to Eqs. (2), (3), (6), and (14), the propor-
tion of VSS reduction by earthworm feeding in enhanced VSS
Earthworm average weight (g.individual -1 )
reduction was gained. Then, the proportion of VSS reduction by
earthwormmicroorganism interaction was also acquired based on 0.0 0.2 0.4 0.6 0.8 1.0 1.2
Eq. (3).
b
2.7. Statistical analysis 0-25

One-way ANOVA was analyzed the differences among the sam-

ples followed by Tukeys post hoc test. The probability levels used 25-50
Depth (cm)

for statistical signicance were P < 0.05.

3. Results and discussion 50-75

3.1. Earthworm population and enzymatic activity

75-100
The depth distribution of biomass, average weight and number
of earthworm individuals in the VF were shown in Fig. 1, while
the earthworm enzymatic activities in the upper and under layers
Earthworm numbers (individual)
were presented in Fig. 2. As the depth, the biomass, average weight,
number and AKP activity of the earthworms tended to decrease 0 200 400 600 800
(one-way ANOVA, P > 0.05), while the protein content and activi-
c
ties of POD and CAT had an increasing tendency (one-way ANOVA,
P > 0.05). The results implied that the earthworm growth property 0-25
in the upper layer was better than that in the under layer. The
possible reason was that organic matter content in the VF sludge
samples decreased with the depth, causing that the organic food 25-50
Depth (cm)

for earthworm growth was less in the under layer [6]. Additionally,
there were large standard deviations in mean values of earthworm
biomasses, average weights and numbers at the four depths of VF. 50-75
The reason was that the mean values of average weights and num-
bers of earthworm individuals was based on the data during the
whole experimental period at the four depths, and the data varied 75-100
considerably with the operation time, as were shown in Fig. S1 of
SD. Xing et al. [1] suggested that temperature variation might be
an important factor to cause the changes in the average weight and
number of earthworm individuals.
Fig. 1. Depth distribution of earthworm biomass (a), average weight (b) and number
(c) in the VF.
3.2. Earthworm gut microbial community

Microbial communities of earthworm gut in the initial and 6th
month samples were assayed by 16S rDNA clone library technique,
and the results were summarized in Fig. 3, and Figs. S2 and S3 of the
SD. The gut microbial communities were dominated by Gammapro-
teobacteria, and the percentages arrived to 7692% of the microbial
species detected. Vivas et al. [25] found that the most abundant
phylum in vermicomposting was Proteobacteria. Danon et al. [26]
also reported that Proteobacteria were the dominant populations
in compost. Zhao et al. [2] found that phylum Proteobacteria was
an important contributor in vermiltration or vermicomposting
processes. Li et al. [6] reported that VF biolms appeared to have
more populations of Proteobacteria than in BF biolms because
indigenous gut-associated microora from earthworms contribute
to the microbial community. Thus, the results implied that part of
Gammaproteobacteria might be the indigenous microbial species
of earthworm gut.
Most of Gammaproteobacteria in the samples belonged to
Aeromonadaceae (2978%), followed by Moraxellaceae (233%),
Enterobacteriaceae (78%), Pseudomonadaceae (12%), and 34%
of Gammaproteobacteria was not identied further. Other
 X. Li et al. / Journal of Hazardous Materials 276 (2014) 353361 357

140 35

Alkaline phosphatase (AKP) activity

30
130

(U.g protein)
Protein content
(g.kg )
-1
25
120

-1
20

110
15

100 10

40 50
Peroxidase (POD) activity

Catalase (CAT) activity

35
40

(U.g protein)
(U.g protein)

30
-1

-1
30
25

20 20
Upper layer Under layer Upper layer Under layer

Fig. 2. Protein content and activities of alkaline phosphatase, catalase and peroxidase of earthworm in the VF.

bacterial like Bacilli (313%), Betaproteobacteria (28%), Acti- sample of earthworm gut (growth substrate, sewage sludge)
nobacteria (02%), Alphaproteobacteria (02%), Clostridia (02%), (H = 1.85) seemed higher than that of the initial samples (growth
Sphingobacteria (02%), Mollicutes (02%) were also found in the substrate, cow dung) (H = 1.01), implying that the microbial diver-
gut samples. Additionally, microbial diversity of the 6th month sity of earthworm gut appeared to be enhanced using sewage

95
70 inital period
90 6th month
60

50
Percentage(%)

85
40

80 30

20
75
10
Percentage (%)

70 0
Ae En Pse Mo oth
rom ter udo r ax ers
ona oba mo ella
dac cter nad cea
eae iac ace e
eae ae
15

10

0
Ga Be Alp Ac Clo Ba Sp Mo
mm ta P hap tin stri cill hin llic
ap rot r oba dia i gob ute
rot eo ote ct act s
eob b act obac eria eri
act eri ter a
eri a ia
a

Fig. 3. Microbial community composition in earthworm gut by PCR-based 16S rDNA clone library technique.
358 X. Li et al. / Journal of Hazardous Materials 276 (2014) 353361
sludge as growth substrate for the earthworms. The above results
showed that microbial community composition of earthworm gut
changed with the growth substrate, implying that the earthworm
gut microbial community had an interaction with that of the
sewage sludge. Meanwhile, the changes in gut microbial commu-
nity might effect the digestion of earthworm to sewage sludge fed,
and then caused an inuence on the earthworm growth.
3.3. Earthworm N and C isotopic characteristics
The life cycle of the earthworm includes three stages: hatching,
growing and mating. The N and C isotopic ratios of earthworm in the
three stages of the life cycle, including earthworm cast and growth
substrate, were measured and shown in Figs. 4 and 5.
Adults had the biggest values of 15 N, followed by hatchlings,
cocoons, cast and growth substrate, while 13 C in the biological
samples (adults, hatchlings and cocoons) were higher than that
in the non-biological ones (cast and growth substrate) (one-way
ANOVA, P < 0.001) (Fig. 4). The results indicated that there were
consistent enrichments of 15 N and 13 C in cocoons, hatchlings
and adults relative to the growth substrate, corresponding to those
of the previous studies [2729].
3.3.1. Temporal changes
The 15 N and 13 C values of earthworms in the 6th month were
signicantly lower than that in the initial period (one-way ANOVA,
P < 0.001) (Fig. 4), probably attributed to the changes in the organic
food of earthworms. After the earthworms were introduced to the
vermiltration system, their food was acclimated to convert from
the cow dung to the sewage sludge. Many researches showed that
the change in the diet with different stable isotopic abundances,
e.g. 15 N and 13 C, cause the changes in the stable isotopic ratio
of the feeder (e.g. [30,31]). Neilson et al. [28] suggested that an
organism that feeds on another near the based of a food chain is
isotopically less 13 C- and 15 N-enriched than an organism that feed
on another higher up a food chain. Sampedro and Domnguez [15]
found that there are a different variation in 15 N and 13 C of earth-
worm in different growth substrates. In the present study, 15 N and
13 C of the growth substrate for earthworm in the initial samples
(cow dung) were 5.585 and 22.714, respectively, and signi-
cantly higher than that in the 6th month samples (sewage sludge)
(one-way ANOVA, P < 0.001) (Fig. 4). Thus, the decrease in 15 N and
13 C values of the growth substrates were likely an important fac-
tor to cause the drop in 15 N and 13 C natural abundances of the
earthworms.
The 15 N and 13 C values of the earthworms in the 10th month
were lower compared with that in the 6th month (Fig. 4). Pon-
sard and Arditi [32] considered that seasonal differences in isotopic
ratios of animal samples were not large. Accordingly, we specu-
lated that the decrease in 15 N and 13 C of the earthworms from
6th month to 10th month was likely resulted from the accumula-
tion of earthworm feeding sewage sludge. Average weight of the
earthworm samples in the 10th month was 2.042.43 times that
in the 6th-month samples (Fig. S1 of the SD), showing that the
weight of the earthworm individuals rose greatly, implying that
more organic matter from sewage sludge with low 15 N and 13 C
values was assimilated to synthesis the earthworm body, and thus
15 N and 13 C natural abundances of the earthworm body decreased
further.
3.3.2. Depth distribution
15 N and 13 C natural abundances in the biolm and earthworm
cast, cocoons and adults had an upward trend as the depth of VF
(Fig. 5). 15 N and 13 C enrichment of soil organic matter with depth
was commonly observed in the forest, grassland and agricultural
ecosystems [15,3235]. C isotope values increase with soil depth is
Table 1
Trophic levels of earthworms in VF on the basis of shift in 15 N and 13 C relative to
sewage sludge.
Earthworm VF
15 Na 13 Cb
Adults 23 25
Hatchlings 3 4
Cocoons 23 25
a
On the basis of shift in 15 N relative to sewage sludge.
b
On the basis of shift in 13 C relative to sewage sludge.
perhaps resulted from decomposition processes, while the increase
of 15 N values with depth is usually related to a more intense micro-
bial activity (e.g. [34,36]). However, Scheu and Falca [33] reported
that 15 N/14 N ratios of earthworms vary little with soil depth in for-
est, due to the movement of earthworms. In the present study, the
VF was composed by four superimposed and separated perspex
tubings, causing the earthworms not to move among the tubings.
Thus, 15 N- and 13 C-enrichment of biolms with depth implied the
increase in the natural abundances of the organic food for earth-
worm, and led to 15 N- and 13 C-enrichment of the earthworms as
the depth. These ndings conrmed and complemented the nd-
ings that the N and C isotopic characteristics of the earthworms
were inuenced by that of growth substrates (sewage sludge).
3.4. Quantication of earthworm feeding based on 15 N ratio
In vermiltration system, the earthworms promote organic
matter degradation of sewage sludge (e.g. the increase in VSS reduc-
tion) mostly through two strategies, which are earthworm feeding
and earthwormmicroorganism interaction [2]. Earthworm feed-
ing improves the VF treatment performance through the digestion
action of earthworm, while earthwormmicroorganism interac-
tion promotes the organic matter degradation through improving
the microbial activity and composition. In the present study, sta-
ble isotopic abundance was rstly used to estimate the amount of
earthworm cast, and then to determine the contribution of earth-
worm feeding to the enhanced VSS reduction.
3.4.1. Why using 15 N ratio
According to the previous study, average trophic shifts in 15 N
and 13 C are 3.4 and 1, respectively in aquatic and terres-
trial ecosystems [37,38], and thus trophic levels of aquatic fauna
could be estimated on the basis of shift in 15 N and 13 C relative to
the growth substrate (sewage sludge). The trophic levels of earth-
worms (adults, hatchlings and cocoons) relative to sewage sludge
based on 15 N and 13 C were shown in Table 1. Trophic levels of the
earthworms (adults, hatchlings and cocoons) varied in the range
of 2 and 3 on the basis of the shift in 15 N, while trophic levels of
the earthworms changed in the range of 2 and 5 on the basis of
13 C. The results showed that the range of the variation based on
15 N was less than that based on 13 C, implying that 15 N natural
abundance was a more accurate choice for estimating the amount
of earthworm cast in the whole VF than that of 13 C.
3.4.2. Quantication of earthworm feeding
The data for analyzing quantitatively the contributions
of the two earthworm effects, earthworm feeding and
earthwormmicroorganism interaction, to the enhanced VSS
reduction were outlined in Table 2, and the results were shown
in Table 3. The results based on 15 N showed that the earthworm
feeding and earthwormmicroorganism interaction were respon-
sible for approximately 21% and 79%, respectively, of the enhanced
VSS reduction. However, the results according to the earthworm
biomass and yield rate of earthworm cast were distinct, and
 X. Li et al. / Journal of Hazardous Materials 276 (2014) 353361 359

14 -16
initial period
6th month
12 10th month
-18

10
-20

8
N ()

C ()
-22
6
15

13
-24
4

-26
2

0 -28
gro e e e e gro e e e e
wth arthw arthw arthw arthw wth arthw arthw arthw arthw
sub orm orm orm orm sub orm orm orm orm
stra cas coc h a stra cas coc h a
te t oon atchi dults te t oon atchi dults
ng ng

Fig. 4. Temporal changes in 15 N and 13 C of the earthworms, cast and growth substrate during experimental period.

showed the two effects were responsible for approximately 40%
and 60%, respectively. In the previous study by Zhao et al. [2], the
method to estimate the amount of earthworm cast was to select
some earthworms randomly, and to culture the earthworm in a
dish in the dark for 24 h, and then to determine the excrement in
the culture dish, and nally to estimate the yield rate of earthworm
cast (g SS kg1 earthworm d1 ), combining with the earthworm
biomass in the whole VF. However, the results according to
the method might be the presence of large deviation as the
considerable difference in the environments between the dish
and VF system might lead to a signicant variation in the yield
rate of earthworm cast. Additionally, it was difcult to estimate
exactly the earthworm biomass in the whole VF, especially in the
full-scale system. Thus, the methods according to the earthworm
biomass and the yield rate of earthworm cast were less feasible
and accurate than that based on 15 N.
The results based on 15 N implied that earthworm feeding
made a less contribution to the enhanced VSS reduction than

N()
15

5 6 7 8 9 10 11

0-25

25-50
Depth(cm)

50-75

75-100

C()
13

-25.5 -25.0 -24.5 -24.0 -23.5 -23.0 -22.5 -22.0 -21.5

0-25

25-50
Depth(cm)

50-75 biofilm
earthworm cast
75-100 earthworm cocoon
earthworm adult

Fig. 5. Depth changes in 15 N and 13 C of the earthworms, cast and biolm during experimental period.
360 X. Li et al. / Journal of Hazardous Materials 276 (2014) 353361

Table 2 The results from the earthworm population and enzymatic

The data for analyzing quantitatively the contribution of earthworm feeding to
activities implied that the earthworm population growth and activ-
enhanced VSS reduction.
ity decreased with the depth of VF. Thus, in order to improve
Parameter Average the earthworm property in the under layer, a possible approach
BF VF was to input the sewage sludge stratiedly. The approach would
1
make the earthworms in the under layer gain the available
VSS content (g d )
Inuent sludge 33.16 7.38 33.16 7.38 organic food. However, the proportion of inuent sludge inputted
Efuent sludge 22.86 4.47 17.23 3.37 from the different depths needed to be improved, as different
N ()
15
proportion distributions might inuence on the treatment per-
Inuent sludge 4.354 0.932 4.354 0.932 formance of VF. Additionally, the optimization of lter media,
Efuent sludge 4.790 0.976 5.022 1.076
Biolm 6.244 0.743 5.825 1.089
including rational gradation and particle size, was another pos-
Earthworm cast a 6.732 0.589 sible approach to increase the organic loading of VF, and then to
VSS/SS ratio (%) meet more earthworm biomass for organic food demand, and in
Inuent sludge 70.40 3.3 70.40 3.3 turn to enhance the VF treatment performance for sewage sludge
Earthworm cast 54.21 2.10
stabilization.
Others
Earthworm biomass 36.63 13.94 Hartenstein observed that 1 g worm could convert approx-
(g L1 ) imately 4 g of activated sludge in 5 days in their lab-scale
Yield rate of 3.60 0.16 (10 cm-diameter petri-dishes) experiments on vermicomposting
earthworm cast of activated sludge [40]. According to the data, the earth-
(g SS kg1 earthworm d1 )
worm biomass of VF was 20.562.5 g L1 in the present study,
Earthworm digestion 36.74
rate (%) and thus the amount of sewage sludge needed might be
a
6401970 g SS (wet weight) d1 . However, the organic loading of
, not detected.
the VF was 1.12 kg VSS (dry weight) m3 d1 , and thus the inuent
sludge amount in the VF was 249 g SS (wet weight) d1 in consid-
earthwormmicroorganism interaction, and the latter strategy eration of the water content and the VSS/SS ratio of sewage sludge.
was 3.81 times of the former (Table 3). The data supplied an The data showed that the organic food from the inuent sludge
complementary explanation why the previous studies about vermi- seemed not enough to meet all the earthworm growth in the VF.
compost or vermilter highlighted the improvement of earthworm The reduction of earthworm individual numbers as the operation
to microbial activity, but paid little attention to the contribution of time (Fig. S1 of the SD) complemented and conrmed the conclu-
earthworm feeding to organic matter degradation. The previous sion. Further, quantitatively analysis of earthworm effects showed
study reported that the microbes are responsible for the biochem- that earthworm feeding played a less important role in enhanced
ical degradation of organic matter, whereas earthworms drive this VSS reduction than earthwormmicroorganism interaction in the
process by conditioning the substrate and altering its biological vermiltration system. Thus, the above results implied that the
activity in vermicomposting process [39]. strategy to improve VF treatment performance should focus on
earthwormmicroorganism interaction, but not earthworm feed-
3.5. Possible approaches to optimize earthworm growth and ing. Thus, another possible approach was to control the earthworm
activity biomass in the vermiltration system through the harvest of the
earthworms. The earthworm biomass decreased, the competition
The presence of earthworm is a key factor for improving among earthworm individuals and the demand of organic food
treatment performance of vermiltration system treating sewage reduced, and then the activity of earthworm individuals and the
sludge, compared with conventional bioltration system. Accord- interaction of earthwormmicroorganism might be improved and
ingly, a feasible strategy to enhance the treatment capacity of VF is strengthened.
to further optimize earthworm growth and activity in vermiltra-
tion system. 4. Conclusion

Earthworm growth biomass and activity decreased with the VF

Table 3
Calculated results from quantitative analysis about the contribution of earthworm
feeding to enhanced VSS reduction.
Parameters
VSS content from earthworm cast
(g VSS d1 )
VSS content by earthworm feeding
(g VSS d1 )
VSS reduction by earthworm
feeding (g VSS d1 )
VSS reduction by
earthwormmicroorganism
interaction (g VSS d1 )
The contribution of earthworm
feeding to enhanced VSS
reduction (%)
The contribution of
earthwormmicroorganism
interaction to enhanced VSS
reduction (%)
depth. 16S rDNA clone library indicated that part of Gammapro-
teobacteria was indigenous microbial species of earthworm gut,
and gut microbial composition had an interaction with that of
Value
sewage sludge. 15 N and 13 C in the earthworms (cocoons, hatch-
Based on yield rate Based on 15 N lings and adults) enriched consistently relative to sewage sludge,
of earthworm cast
and meanwhile were inuenced by that of sewage sludge. Quanti-
2.24 2.81 tative analysis showed earthworm feeding had less effect in the
improvement of VSS reduction than earthwormmicroorganism
4.51 4.00
interaction. Possible approaches to further optimize earthworm
2.26 1.17 property of VF included stratied input of sewage sludge and con-
trol of earthworm biomass.
3.36 4.45
Supplementary data
40 21
One table showing the characteristics of the inuent sewage
60 79
sludge; one gure presenting changes in earthworm average
weight and number in the VF; two gures showing phyloge-
netic tree based on neighbor-joining analysis of 16S rDNA gene
sequences from earthworm gut microorganism.
 X. Li et al. / Journal of Hazardous Materials 276 (2014) 353361
Acknowledgements
The research was funded by the China Postdoctoral Science
Foundation (2013M541545), National Science Foundation of China
(51109161), National Water Pollution Control and Management
Technology Major Projects (2011ZX07303004) and National Tech-
nology Research and Development Program of China (863 Program)
(2012AA063502).
Appendix A. Supplementary data
Supplementary material related to this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.jhazmat.
2014.05.042.
References
[1] M. Xing, X. Li, J. Yang, B. Lv, Y. Lu, Performance and mechanism of vermiltration
system for liquid-state sewage sludge treatment using molecular and stable
isotopic techniques, Chem. Eng. J. 197 (2012) 143150.
[2] L. Zhao, Y. Wang, J. Yang, M. Xing, X. Li, D. Yi, D. Deng,
Earthwormmicroorganism interactions: a strategy to stabilize domestic
wastewater sludge, Water Res. 44 (2010) 25722582.
[3] M. Xing, X. Li, J. Yang, Z. Huang, Y. Lu, Changes in the chemical characteristics of
water-extracted organic matter from vermicomposting of sewage sludge and
cow dung, J. Hazard. Mater. 205206 (2011) 2431.
[4] L. Wang, Z. Zheng, X. Luo, J. Zhang, Performance and mechanisms of a microbial-
earthworm ecolter for removing organic matter and nitrogen from synthetic
domestic wastewater, J. Hazard. Mater. 195 (2011) 245253.
[5] R.K. Sinha, G. Bharambe, U. Chaudhari, Sewage treatment by vermiltration
with synchronous treatment of sludge by earthworms: a low-cost sustainable
technology over conventional systems with potential for decentralization, The
Environmentalist 28 (2008) 409420.
[6] X. Li, M. Xing, J. Yang, Y. Lu, Properties of biolm in a vermiltration sys-
tem for domestic wastewater sludge stabilization, Chem. Eng. J. 223 (2013)
932943.
[7] X. Li, M. Xing, J. Yang, L. Zhao, X. Dai, Organic matter humication in vermil-
tration process for domestic sewage sludge treatment by excitationemission
matrix uorescence and Fourier transform infrared spectroscopy, J. Hazard.
Mater. 261 (2013) 491499.
[8] M. Aira, F. Monroy, J. Domnguez, Earthworms strongly modify microbial
biomass and activity triggering enzymatic activities during vermicomposting
independently of the application rates of pig slurry, Sci. Total Environ. 385
(2007) 252261.
[9] Z.Z. Zhou, Z.F. Liu, L.D. Guo, Chemical evolution of Macondo crude oil during lab-
oratory degradation as characterized by uorescence EEMs and hydrocarbon
composition, Mar. Pollut. Bull. 66 (2013) 164175.
[10] M. Egert, S. Marhan, B. Wagner, S. Scheu, M.W. Friedrich, Molecular proling of
16S rRNA genes reveals diet-related differences of microbial communities in
soil, gut, and casts of Lumbricus terrestris L. (Oligochaeta: Lumbricidae), FEMS
Microbiol. Ecol. 48 (2004) 187197.
[11] L. Sampedro, R. Jeannotte, J.K. Whalen, Trophic transfer of fatty acids from
gut microbiota to the earthworm Lumbricus terrestris L., Soil Biol. Biochem. 38
(2006) 21882198.
[12] K.-L. Ho, Y.-C. Chung, C.-P. Tseng, Continuous deodorization and bacterial com-
munity analysis of a biolter treating nitrogen-containing gases from swine
waste storage pits, Bioresour. Technol. 99 (2008) 27572765.
[13] D.M. Post, M.L. Pace, N.G. Hairston, Ecosystem size determines food-chain
length in lakes, Nature 405 (2000) 10471049.
[14] J.P. Curry, O. Schmidt, The feeding ecology of earthworms a review, Pedobio-
logia 50 (2007) 463477.
[15] L. Sampedro, J. Domnguez, Stable isotope natural abundances (13 C and
15 N) of the earthworm Eisenia fetida and other soil fauna living in
two different vermicomposting environments, Appl. Soil Ecol. 38 (2008)
9199.
View publication stats
361
[16] X. Li, M. Xing, J. Yang, Z. Huang, Compositional and functional features of humic
acid-like fractions from vermicomposting of sewage sludge and cow dung, J.
Hazard. Mater. 185 (2011) 740748.
[17] D.L. Phillips, J.W. Gregg, Source partitioning using stable isotopes: coping with
too many sources, Oecologia 136 (2003) 261269.
[18] M.M. Bradford, A rapid and sensitive method for the quantitation of micro-
gram quantities of protein utilizing the principle of protein-dye binding, Anal.
Biochem. 72 (1976) 248254.
[19] M. Tabatabai, J. Bremner, Use of p-nitrophenyl phosphate for assay of soil phos-
phatase activity, Soil Biol. Biochem. 1 (1969) 301307.
[20] M. Saint-Denis, F. Labrot, J. Narbonne, D. Ribera, Glutathione, glutathione-
related enzymes, and catalase activities in the earthworm Eisenia fetida andrei,
Arch. Environ. Contam. Toxicol. 35 (1998) 602614.
[21] D. Xu, C. Li, Y. Wen, W. Liu, Antioxidant defense system responses and DNA
damage of earthworms exposed to Peruorooctane sulfonate (PFOS), Environ.
Pollut. 174 (2013) 121127.
[22] L. Feng, Y. Chen, X. Zheng, Enhancement of waste activated sludge protein con-
version and volatile fatty acids accumulation during waste activated sludge
anaerobic fermentation by carbohydrate substrate addition: the effect of pH,
Environ. Sci. Technol. 43 (2009) 43734380.
[23] C.E. Shannon, A mathematical theory of communication, ACM SIGMOBILE Mob.
Comput. Commun. Rev. 5 (2001) 355.
[24] S. Carabel, E. Godnez-Domnguez, P. Versimo, L. Fernndez, J. Freire, An assess-
ment of sample processing methods for stable isotope analyses of marine food
webs, J. Exp. Mar. Biol. Ecol. 336 (2006) 254261.
[25] A. Vivas, B. Moreno, S. Garcia-Rodriguez, E. Bentez, Assessing the impact of
composting and vermicomposting on bacterial community size and structure,
and microbial functional diversity of an olive-mill waste, Bioresour. Technol.
100 (2009) 13191326.
[26] M. Danon, I.H. Franke-Whittle, H. Insam, Y. Chen, Y. Hadar, Molecular analysis
of bacterial community succession during prolonged compost curing, FEMS
Microbiol. Ecol. 65 (2008) 133144.
[27] P.F. Hendrix, S.L. Lachnicht, M.A. Callaham, X. Zou, Stable isotopic studies of
earthworm feeding ecology in tropical ecosystems of Puerto Rico, Rapid Com-
mun. Mass Spectrom. 13 (1999) 12951299.
[28] R. Neilson, B. Boag, M. Smith, Earthworm 13 C and 15 N analyses suggest that
putative functional classications of earthworms are site-specic and may also
indicate habitat diversity, Soil Biol. Biochem. 32 (2000) 10531061.
[29] T. Uchida, N. Kaneko, M. Ito, K. Futagami, T. Sasaki, A. Sugimoto, Analysis of
the feeding ecology of earthworms (Megascolecidae) in Japanese forests using
gut content fractionation and 15 N and 13 C stable isotope natural abundances,
Appl. Soil Ecol. 27 (2004) 153163.
[30] A. Tiunov, Stable isotopes of carbon and nitrogen in soil ecological studies, Biol.
Bull. 34 (2007) 395407.
[31] A. El-Wakeil, K. Fouad, Trophic structure of macro- and meso-invertebrates
in Japanese coniferous forest: carbon and nitrogen stable isotopes analyses,
Biochem. Syst. Ecol. 37 (2009) 317324.
[32] S. Ponsard, R. Arditi, What can stable isotopes (15 N and 13 C) tell about the
food web of soil macro-invertebrates, Ecology 81 (2000) 852864.
[33] S. Scheu, M. Falca, The soil food web of two beech forests (Fagus sylvatica) of
contrasting humus type: stable isotope analysis of a macro-and a mesofauna-
dominated community, Oecologia 123 (2000) 285296.
[34] M.J.I. Briones, R. Bol, Natural abundance of 13 C and 15 N in earthworms from
different cropping treatments, Pedobiologia 47 (2003) 560567.
[35] M. Briones, O. Schmidt, S. Pandalai, Stable isotope techniques in studies of the
ecological diversity and functions of earthworm communities in agricultural
soils, Recent Res. Dev. Crop Sci. 1 (2004) 1126.
[36] M. Briones, R. Bol, D. Sleep, D. Allen, L. Sampedro, Spatio-temporal variation
of stable isotope ratios in earthworms under grassland and maize cropping
systems, Soil Biol. Biochem. 33 (2001) 16731682.
[37] R. France, R. Peters, Ecosystem differences in the trophic enrichment of 13 C in
aquatic food webs, Can. J. Fish. Aquat. Sci. 54 (1997) 12551258.
[38] M. Minagawa, E. Wada, Stepwise enrichment of 15 N along food chains: further
evidence and the relation between 15 N and animal age, Geochim. Cosmochim.
Acta 48 (1984) 11351140.
[39] J. Domnguez, C.A. Edwards, Vermicomposting organic wastes, in: S.H. Shakir
Hanna, W.Z.A. Mikhal (Eds.), Soil Zoology for Sustainable Development in the
21st Century, Self-Publisher, Cairo, 2004, pp. 369395.
[40] P. Ndegwa, S. Thompson, K. Das, Effects of stocking density and feeding rate on
vermicomposting of biosolids, Bioresour. Technol. 71 (2000) 512.