Waste Management 62 (2017) 33–42

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Waste Management
journal homepage: www.elsevier.com/locate/wasman

Stabilization of different starting materials through vermicomposting in

a continuous-feeding system: Changes in chemical and biological
parameters
Mercedes García-Sánchez ⇑, Hana Taušnerová, Aleš Hanč, Pavel Tlustoš
Department of Agro-Environmental Chemistry and Plant Nutrition, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences, Kamýcká 129, Prague
6-Suchdol, Czech Republic

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

Article history: In this study the feasibility of Eisenia andrei to digest great amount of wastes including horse manure
Received 23 September 2016 (HM), apple pomace (AP), grape pomace (GP), and digestate (DG) was monitored through a
Revised 30 January 2017 continuous-feeding system. New layers of fresh material were gradually added to form an aged-profile
Accepted 6 February 2017
of layers in order to understand the interaction between earthworms and microorganisms during vermi-
Available online 16 February 2017
composting. Thus, changes in chemical and biological parameters were evaluated for 240 days. The earth-
worm population reached maximum values in 120 d-old-layer, which was related to an increase in
Keywords:
overall microbial biomass, assayed as dehydrogenase activity, in all of the processed materials. The pH
Continuous-feeding system
Eisenia andrei
was generally alkaline or neutral in all of the materials. The electrical conductivity did not modify signif-
Enzymatic activities icantly during vermicomposting, except in the case of the processed GP, and DG. The stabilization, in all of
Microbial biomass the processed materials, was detected after 240 d of vermicomposting, as indicated the decline in the
Phytotoxicity test content of dissolved organic carbon (DOC). The N-NO3 content exhibited an enhanced in the processed
Organic materials HM and AP, while a generalized decreased was found in the GP, and DG materials in 240 d-old-layer.
The decline in microbial biomass activity, in all processed substrates, was related to a decrease in the
earthworm activity after 240 d of vermicomposting, indicating a high degree of stabilization. However,
the b-glucosidase, phosphatase, protease, and o-diphenol oxidase activities were different according to
the age of layers and type of processed material. The phytotoxicity test indicated that the end products
of the processed AP and DG were chemically stable and enriched with nutrients in comparison with the
HM and GP vermicompost. This fact indicates to stabilization (maturation) in the end product, which is
important for its safe disposal as an organic nutrient-rich product.
Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction use of livestock materials, such as manure. For instance, horse

The production of solid waste is approximately estimated in 1.3
billion metric tons, and this amount is predicted to double by 2025
(Hoornweg and Bhada-Tata, 2012). Among the total solid waste
produced, organic waste is the largest proportion which includes
food scraps, yard waste, municipal activities or sewage sludge,
agricultural, and industrial waste (Hoornweg et al., 2013). The
increase in production and concentration of intense livestock oper-
ations along with the increased urbanization of rural regions have
resulted in a greater concern for the safe disposal, treatment and
⇑ Corresponding author at: Department of Agro-Environmental Chemistry and
Plant Nutrition, Faculty of Agrobiology, Food and Natural Resources, Czech
University of Life Sciences, Kamýcká, 129 00, Prague 6-Suchdol, Czech Republic.
E-mail addresses: garcia.sanchez.mercedes@gmail.com, garcia_sanchez@af.czu.
cz (M. García-Sánchez).
manure (HM) has largely been used as a suitable organic amend-
ment due to its high amount in plant-available macro and micro-
nutrients. However, the overproduction of this material has led
to inappropriate management practices which cause detrimental
environmental problems including an excessive input of harmful
trace elements, salts and pathogens, increased nutrient loss from
soils and emission of toxic gasses (Hutchison et al., 2005). Other
industries activities, which are those derived from the juice indus-
try, generate a large amount of solid material commonly known as
apple pomace (AP). This material has extensively been used in the
food industry as a component in fruit tea (Mamma et al., 2009).
However, this material has become in a waste as the result of the
higher demand from purchasers for components with better aroma
and taste than AP. Grape pomace (GP) is a lignocellulosic material
remaining after the grape crushing and pressing in wine produc-

http://dx.doi.org/10.1016/j.wasman.2017.02.008
0956-053X/Ó 2017 Elsevier Ltd. All rights reserved.
34 M. García-Sánchez et al. / Waste Management 62 (2017) 33–42
tion which consists of the stalks, skin, pulp and seeds (Flavel et al.,
2005). This waste, due to its high amount in macro and micro-
nutrients, is considered as a valuable source as a soil fertilizer
(Bertran et al., 2004). However, the inappropriate disposal of GP
into agricultural soils can produce serious environmental prob-
lems, including the excessive release of tannins and phenols in
soils, which could inhibit the root growth (Inbar et al., 1991). On
the other hand, the anaerobic digestion of materials, such as farm,
agro-industrial and organic residues, has become increasingly pop-
ular as an alternative for recycling wastes to generate biogas,
resulting in the production of a residual material known as diges-
tate (DG) (Insam et al., 2015). The use of DG as an organic amend-
ment in agriculture can therefore represent an important source of
plant-available nutrients, enhancing the soil microbial functional-
ity and biomass (García-Sánchez et al., 2015). However, biogas pro-
duction has been also accompanied by a depletion of organic
carbon (C), which may affect the turnover of nitrogen (N). In spite
of this fact, digestate appears not to negatively affect the soil
organic C status; a post-treatment of digestate has been suggested
by several authors as a mean of improving digestate quality as a
soil amendment (Insam et al., 2015; Rehl and Müller, 2011).
The environmental problems associated with the management
of these wastes, including HM, AP, GP, and DG, could be signifi-
cantly reduced by stabilizing them before their use and/or disposal.
An environmentally management option could be the vermicom-
posting which has been reported to be a low-cost technique for
the stabilization of hazardous and worthless organic materials of
different natures, converting them into safe and valuable end prod-
ucts (Domínguez and Edwards, 2011a). Vermicomposting system
is a biological process in which detritivore earthworms interact
with microorganisms, accelerating the stabilization of the organic
matter and greatly modifying its physical and biochemical proper-
ties (Domínguez et al., 2010; Sampedro and Domínguez, 2008).
This process consists of two different periods related to the earth-
worm activity: (i) a vermicomposting period (active phase) in
which the earthworm greatly modifies the physico-chemicals
characteristics of the material as well as its microbial composition
(Lores et al., 2006); (ii) a maturation period, following the earth-
worm displacement toward new layers of fresh substrate, during
which microorganisms decompose the processed material by
earthworm (Aira et al., 2007a; Gómez-Brandón and Domínguez,
2014). The duration of both, active and maturation phases of ver-
micomposting, is not fixed and depends on composition of the ini-
tial substrates, earthworm species and the rates at which they
ingest and process the material (Aira and Domínguez, 2008;
Domínguez et al., 2010). Several studies have evaluated the poten-
tial use of vermicomposting for the stabilization of different sub-
strates such as manures, AP, GP, and DG (Gómez-Brandón et al.,
2011; Hanč and Chadimova, 2014; Hanč and Vasak, 2015;
Lazcano et al., 2008; Nogales et al., 2005). Thus, Gómez-Brandón
et al. (2013) has investigated the activity of Eisenia fetida on the
microbial functionality and structure during the vermicomposting
of rabbit manure, using a continuous-feeding system. Overall, the
stabilization of the organic material was reached within 200–250
days of vermicomposting, as revealed by the lower values of micro-
bial activities compared to the fresh manure. Besides, it was
reported a great loss of total carbon in the presence of E. fetida,
indicating the importance of this epigeic earthworm in the decom-
position of organic matter because of their interactions with
microorganisms during the process. Hanč and Chadimova (2014)
and Hanč and Vasak (2015) have reported the ability of earth-
worms belong to genus Eisenia to transform substrates such as
AP and DG into an organic nutrient-rich product, but in a non-
continuous system, as indicate the optimal range found in pH,
EC, nutrient content and C:N ratio. To this date, less is reported
on microbial activities and biochemical changes that occur during
the vermicomposting process of these materials in a continuous-
feeding system. On the other hand, studies conducted by Gómez-
Brandón et al. (2011) and Nogales et al. (2005) have observed that
the combined action of microorganisms and E. andrei enhanced the
stabilization of materials such as GP using a non-continuous sys-
tem. This result was evidence by the lower values of labile C pool
and overall microbial biomass, measured as the basal respiration
and dehydrogenase activity. However, these studies were per-
formed to monitor the effectiveness of the active phase during ver-
micomposting of GP, but in lab-scale systems. Therefore, there is a
need for further studies to evaluate the efficiency of a continuous-
feeding system vermicomposting to transform a wider range of dif-
ferent starting materials, and to analyse the stability of the end
products as fertilizers. Moreover, it is essential to establish to what
extent the relationships between earthworms and microorganisms
are crucial in the decomposition of the organic matter and there-
fore, in the stabilization of organic substrates. To do this, the objec-
tive of this study was aimed in investigating the efficiency of E.
andrei to convert different types of organic materials, including
HM, AP, GP, and DG in a continuous-feeding system. With this sys-
tem, we were able to establish a profile of layers to observe the dif-
ferent phases of interaction between earthworms and
microorganisms during the decomposition of the organic matter
in the different materials. The evolution of microbial biomass and
important enzymatic activities were monitored as well as chemical
parameters through an increased aged-profile of layers with differ-
ent degree of maturation (stabilization), from the top to the bottom
of the system. Furthermore, the stabilization of the vermicompost
was evaluated during the germination of Lepidium sativum, L. seeds
to assess the feasibility of using these materials as an agricultural
resource. Information regarding this subject will contribute in bet-
ter understanding the interaction between earthworm activity and
microorganisms during the biological transformation and may
have important contributions in the large-scale valorisation of
organic materials through vermicomposting system.
2. Materials and methods
2.1. Organic materials
HM was collected from a horse farm near Prague (Kladno, Czech
Republic), which contained a mixture of solid and liquid horse fae-
ces. AP was provided by the Severofrukt company (Terezin, Czech
Republic) and was mainly obtained from apple juice production.
GP was composed mainly of pulp and stones of grapes remaining
after wine production and was provided by a winery company
located in South Moravia (Czech Republic). DG was obtained from
the biogas plant of an agricultural cooperative placed in Krasna
Hora nad Vltavou (Czech Republic). The composition of the DG
was approximately 50% manure slurry, 40% corn silage, and 10%
haylage. In order to reduce the worm toxicity factors such as high
content of ammonia (HM and DG) and low pH level (AP and GP),
the starting organic materials were pre-composted in 70 L capacity
laboratory reactors following the same procedure described else-
where (Hanč et al., 2012). The physico-chemical composition of
each organic waste is shown in Table 1.
2.2. Vermicomposting process
The vermicomposting process was carried out in vertical con-
tinuous feeding vermireactors (VermiHut Worm Bin). Twelve ver-
mireactors were set up, three per each organic material tested
(HM, AP, GP, and D) and were composed of trapezoidal trays
(40  40  18 cm) with a perforated bottom to allow the move-
ment of earthworms from one tray to another. 1 L of cow manure
 M. García-Sánchez et al. / Waste Management 62 (2017) 33–42
Table 1
The physico-chemical composition of fresh materials (HM, AP, GP, and DG) used for
the experiment.
Initial material pH EC (mS cm 1
) TOC (%)
HM 7.8 1.4 40
AP 7.9 1.3 40
GP 7.7 1.2 42
DG 8.5 3.5 36
vermicompost containing 200 earthworms with an average weight
of 7.1 ± 4.1 g, fresh weight (E. andrei) was placed on the first bot-
tom tray to provide an initial habitat for the earthworms and
reduce their initial shock due to the environmental changes as pre-
viously observed by Hanč and Chadimova (2014). Cow manure ver-
micompost and E. andrei were provided by the Jakub Filip, Luzice u
Hodonina company (Czech Republic). This earthworm specie was
chosen due to its high tolerance in relation to environmental vari-
ables and its high rate of organic matter processing (Domínguez,
2004). The second tray of the reactor was fed with 5 L of the pre-
viously mentioned individual organic material. New layers with
the same amount of fresh material were added and placed gradu-
ally into the third, fourth and fifth reactor trays every sixty days
(when the earthworm feeding activity required according to pre-
liminary study). A detail graphic explaining the procedure of ver-
micomposting process is given in Fig. 1. The vermicomposting
experiment was conducted in a specially adapted laboratory with
especial conditions (temperature 22 °C, relative humidity 80%, ven-
tilation for 15 min every 12 h), as previously published elsewhere
(Hanč and Chadimova, 2014), and the process was maintained
until 240 days to enable the maturation of the vermicomposts. At
the end of the vermicomposting, the vermireactors comprised 4
trays with an increasing gradient of age from upper to lower layers
as follow: 60, 120, 180, and 240 days. Earthworm biomass and
number were handling in each tray. Five samples of material per
tray were taken at random, gently mixed and stored at 4 °C for
enzyme activity analyses, while the other was air dried and finely
ground for chemical analysis.
2.3. Chemical analysis
The pH and electrical conductivity (EC) were measured in an
aqueous extract 1:10 (sample:water v/w) after shaking for 1 h
using a CRISON MicropH and CRISON conductometer, respectively.
Dissolved organic carbon (DOC) was extracted for 1 h with distilled
water (1:10 sample:water, w/v) and then determined with potas-
sium dichromate and sulphuric acid. The mixture was incubated
for 30 min at 160 °C and the absorbance was measured at
Fig. 1. Description of the procedure used during the vermicomposting process.
35
590 nm (Mingorance et al., 2007). Inorganic N forms (N-NO3 and
N-NH+4) were extracted with 10 mL of CaCl2 (0.01 M) using a 1:10
(sample:water, v/w) ratio after shaking for 120 min at room tem-
TN (%) perature. N-NO3 and N-NH+4 contents were quantified using an
2.7 automated SKALAR SANPLUS SYSTEM continuous-flow segmented
2.5 analyser (Skalar, Netherlands).
2.1
2.2
2.4. Microbial biomass and activities
The microbial biomass was assayed by the quantification of
dehydrogenase (EC 1.1) as previously described by Camiña et al.
(1998). Sample (1 g fresh weight) was incubated with 2 mL of
iodonitrotetrazolium violet (0.5%) (INT). The reaction was incu-
bated for 1 h at 40 °C. The iodonitrotetrazolium formazan (INTF),
produced by the reduction of INT, was extracted with 10 mL of
ethanol:dimethylformamide (1:1, v/v) and the absorbance was
measured spectrophotometrically at 490 nm. Microbial activities
such as: b-glucosidase (EC 3.2.1.21) and phosphatase (EC 3.1.3.1)
activities were determined using the methods described elsewhere
(Eivazi and Tabatabai, 1988, 1977). Briefly, sample (0.5 g fresh
weight) was mixed with 2 mL of buffered substrate solution
(0.1 M modified universal buffer (MUB), pH 6.0) and incubated
for 2 h at 37 °C. The following substrate concentrations were used:
b-glucosidase, p-nitrophenyl-D-glucopyranoside (0.05 M); phos-
phatase, p-nitrophenyl-phosphate (0.115 M). The concentrations
of p-nitrophenol produced were determined at 400 nm after the
addition of 4 mL Tris buffer (0.1 M, pH 12) and 1 mL CaCl2 for b-
glucosidase (0.5 M); 4 mL NaOH (0.5 M) and 1 mL CaCl2 (0.5 M)
for phosphatase. Protease (EC 3.4.2.21-24) activity was measured
by colorimetric determination of the amino acids released, after
incubation of sample (1 g fresh weight) with 5 mL of Na-casein
(2%) for 2 h at 50 °C and with Folin-Cicalteu reagent. The absor-
bance was quantified at 700 nm (Ladd and Butler, 1972). The of
o-diphenol oxidase activity (EC 1.10.3.1.) was estimated by incu-
bating sample (1 g fresh weight) with 1.5 mL of proline (0.2 M)
and 1.5 mL of catechol (0.2 M) for 10 min at 30 °C, and subsequent
colorimetric measurement of proline-o-benzoquinone released at
525 nm (Perucci et al., 2000).
2.5. Phytotoxicity assay
To assess the maturity and phytotoxicity levels of the vermi-
composts at the end of the vermicomposting process, the germina-
tion index (GI) was calculated according to the technique described
by Zucconi et al. (1981). Seeds of L. sativum were germinated using
an aqueous extract 1:10 (sample:water, w/v) at 25 °C for 24 h in
darkness.
36 M. García-Sánchez et al. / Waste Management 62 (2017) 33–42
2.6. Statistical analysis
The data analysis was performed using the SPSSÒ software Win-
dows Version 17.0 (Chicago, Illinois, USA). The earthworm activity
(biomass and number) and the chemical parameters tested during
vermicomposting were statistically analysed using one-way analy-
sis of variance (ANOVA). The microbial activities were analysed by
two-way ANOVA to determine the differences among the type of
material used and depth of sampling (i.e. age of layers). For post
hoc comparison Tukey’s HSD test was used (p  0.05). The differ-
ences between the parameters measured in the starting material
and end-product (vermicompost) for each organic material were
analysed by a paired-sample t test. All data presented are the
means of three replicates with standard error (mean ± SE). Princi-
pal component analysis (PCA) was also carried out using PAST
package software to asses overall differences in the chemical and
microbial activities of the processed HM, AP, GP, and DG through
the profile of the vermireactors layers.
3. Results and discussion
3.1. Evaluation of earthworm activity during the vermicomposting
The maximum biomass of E. andrei earthworms was reached in
120 d-old-layer of the vermireactors in all of the organic materials
(HM, AP, GP, and DG) (F = 37.16, p  0.000; F = 8.16, p  0.019;
F = 86.93, p  0.000; F = 21.04, p  0.002, respectively) (Fig. 1a).
However, a higher enhancement in the earthworm biomass was
found especially in the case of processed HM (55 ± 2.88 g kg 1 sub-
strate) and AP (50.76 ± 5.91 g kg 1 substrate) compared with other
materials. Likewise, the highest earthworm numbers were strongly
achieved in 120 d-old-layer of all the processed substrates (HM, AP,
GP, and DG) (F = 74.43, p  0.000; F = 13.08, p  0.006; F = 7.21,
p  0.025; F = 28.09, p  0.001) (Fig. 2b). Domínguez and
Edwards (2011a, 2011b) reported that the epigeic earthworms
are able to process wastes much more efficiently within a rela-
tively low range of optimal conditions, such as moisture and tem-
perature. However, this points to the biochemical qualities of the
materials, which could be one of the most important factors for
the development of E. andrei. This finding is consistent with the
general hypothesis that proposes the biochemical quality of the
substrates as the main attribute in determining the highest rates
of earthworm development and reproduction (Garg and Gupta,
2011). Conversely, a significant decline in the earthworm biomass
and number was detected with depth of layers (180 d-old-layer) of
processed HM (37.3 ± 1.8 g kg 1 substrate; 38 ± 1.1 worms kg 1
substrate, respectively) and AP (33.8 ± 1.8 g kg 1 substrate;
178.8 ± 1.8 worms kg 1 substrate, respectively) (Fig. 2a and b). In
spite of the fact that E. andrei did not significantly modify its bio-
mass with depth of layers of processed GP and DG, a decline in
the earthworm numbers was also detected in 180 d-old-layer
(16.33 ± 0.9 worms kg 1 substrate; 7.33 ± 0.9 worms kg 1 sub-
strate, respectively) (Fig. 2b). This fact points to an increase in
the ammonium content as a consequence of the mineralization
of proteins, anaerobic conditions and/or an alkaline pH due to
the decomposition of organic anions. No earthworms were found
in the remaining layer (240 d-old-layer) in all of the processed
HM, AP, GP, and DG. These findings may suggest that the earth-
worm activity during the transformation of the materials was
markedly separated into two stages. The first stage, characterized
by the presence of E. andrei, could be associated with to active
phase of vermicomposting, corresponding to the 60–120 d-old-
layers. Meanwhile, the second stage, corresponding to the 180–
240 d-old-layers, probably occurs with the maturation phase of
vermicomposting which is characterized by the earthworm
displacement to new layers of fresh materials. These results are
in concordance with the findings previously described by Aira
et al. (2007a, b) and Gómez-Brandón et al. (2013) during the
process of manure in a continuous-feeding system.
3.2. Evaluation of the chemical changes during the vermicomposting
The pH values were significantly increased with depth of layers
of processed AP, GP and DG (8 ± 0.01; 9.2 ± 0.03; 8.1 ± 0.03, respec-
tively) (Table 2). These higher pH values could be explained by the
disappearance of organic acids with the level of processed AP and
GP, and/or due to the mineralization of the organic nitrogen not
required by microorganism releasing ammonia, as suggested
Nayak et al. (2013) and Pramanik et al. (2007). This result is also
consistent with previous findings in which the pH value increased
after the vermicomposting of vegetable greenhouse wastes
(Fernández-Gómez et al., 2010a). Conversely, the reduction in pH
found in 240 d-old-layer of processed HM (7.1 ± 0.01) may be asso-
ciated with the mineralization of N and P compounds and the sub-
sequent release of CO2 and organic acids from microbial
metabolism, and the production of humic and fulvic acids, as pre-
viously reported by Gómez-Brandón et al. (2013) and Lazcano et al.
(2008). The organic salt content is also very crucial for the survival
of earthworms (Domínguez and Edwards, 2011a and Edwards,
1988). In our study, no significant changes in the EC values were
observed with the level of processed HM, and AP. Similarly, a study
performed by Gómez-Brandón et al. (2013) reported no differences
in the salt content through the profile layers during the vermicom-
posting of rabbit manure. Conversely, an increase in the EC values
was found in 240 d-old-layer of processed GP. This result is in line
with the previous finding reported by Fernández-Gómez et al.
(2010a), indicating a probably release of salts due to high rate of
mineralization of organic compounds. Meanwhile, the EC content
was significantly declined with the level of processed DG, from
upper to lower layers, reaching reduced values than those
observed in the initial material (1.6 ± 0.03) (Table 1).
Earthworms are known to accelerate the rate of organic matter
transformation during vermicomposting (Domínguez et al., 2010),
thus leading to important losses of total carbon throughout this
process of biotransformation (Aira et al., 2007a; Domínguez
et al., 2010; Garg et al., 2006). In our study, the values for the
DOC content were increased with depth of layers from 60 d to
180 d-old-layers, whereas a decline was found after 240 d of ver-
micomposting in all of the processed materials (Table 2). The
DOC represents a fraction that contains organic materials which
can be used as an energy source by microbes producing other dif-
ferent molecules such as proteins, enzymes, and polysaccharides.
For this reason, the DOC content has been suggested an important
parameter in determining the degree of stabilization of the organic
matter during vermicomposting (Campitelli and Ceppi, 2008).
However, there is still no threshold level of DOC at which vermi-
compost is considered stable. In our study, the DOC content
reached values up to 3.3 ± 0.07; 2.6 ± 0.02; 3.6 ± 0.08; 2.5 ± 0.04
in 240 d-old-layer of processed HM, AP, GP, and DG, respectively.
A study conducted by Aira et al. (2007a) has reported levels of
DOC below 1.5 g kg 1 in 252-old-layer of processed pig manure,
whereas Gómez-Brandón et al. (2013) found values close to
6 g kg 1 in 200 d-old-layer of processed rabbit manure. These dif-
ferences may be related to differences in the composition of parent
substrates and/or to the experimental set-up (i.e. age of layers).
The stability of vermicompost can be also defined in terms of
nitrification. The available fractions of dissolved organic nitrogen,
N-NH+4 forms, microbial biomass, and their C and N requirements
regulate the rate of N mineralization (Atiyeh et al., 2000; Yadav
and Garg, 2009). Vermicompost commonly exhibits an enhance-
ment in the N-NO3 content with respect to N-NH+4, as consequence
 M. García-Sánchez et al. / Waste Management 62 (2017) 33–42 37

Fig. 2. Evolution of E. andrei biomass (a) and number (b) in 60 d, 120 d, 180 d, and 240 d-old-layers of processed HM, AP, GP and DG throughout the process of
vermicomposting. Each symbol indicates the mean ± standard error (n = 3).

Table 2
Changes in the chemical properties (pH, EC, DOC, N-NO3 , and N-NH+4) in 60 d, 120 d, 180 d, and 240 d-old-layers of processed HM, AP, GP, and DG throughout the process of
vermicomposting. The values represent the mean (±SE) of three replicates (n = 3). The different letters represent significant differences between layers in each processed material
according to the HSD Tukey test (P  0.05).

Time (days) Processed materials

HM AP GP DG
a
pH 60 7.3 ± 0.04 7 ± 0.04 b 7.9 ± 0.02 c
7 ± 0.04 b
a
120 7.4 ± 0.04 8 ± 0.05 a 8.3 ± 0.03 b
6.5 ± 0.02 c

c b b c
180 6.9 ± 0.02 7.2 ± 0.04 8.1 ± 0.02 6.4 ± 0.02
b
240 7.1 ± 0.01 8 ± 0.01 a 9.2 ± 0.03 a
8.1 ± 0.03 a

1 b b b a
EC (mS cm ) 60 1 ± 0.05 0.4 ± 0.1 0.7 ± 0.07 2 ± 0.03
120 1.2 ± 0 a 0.9 ± 0.01 a
0.7 ± 0.03 b
2 ± 0.03 a
180 1 ± 0.01 b 0.4 ± 0.01 b
0.7 ± 0.01 b
1.8 ± 0.01 b

b b a b
240 1.1 ± 0.02 0.4 ± 0.01 1.0 ± 0.03 1.6 ± 0.03
1 c d d d
DOC (g kg ) 60 2.4 ± 0.03 1.8 ± 0.04 1 ± 0.01 2.1 ± 0.04
120 3.1 ± 0.1 b 3.7 ± 0.08 b
1.9 ± 0.02 c
3.2 ± 0.04 b

180 4.7 ± 0.04 a 5.2 ± 0.06 a

2.6 ± 0.02 b
5.4 ± 0.05 a

240 3.3 ± 0.07 b 2.6 ± 0.02 c

3.6 ± 0.08 a
2.5 ± 0.04 c

1 c b a
N-NO3 (mg kg ) 60 583.07 ± 17 36 ± 4.4 138.6 ± 23.6 1568.6 ± 116 a
120 1053.4 ± 36 a 36.5 ± 3.7 b
54 ± 10.8 b 1056 ± 41.21 b
180 974.2 ± 8.6 a 50.9 ± 1.4 a
55 ± 14.6 b 1488.8 ± 57.4 a
240 735 ± 60.5 b 56.2 ± 4 a 36.6 ± 1.2 b 1170.5 ± 26.1 b
N-NH+4 (mg kg 1
) 60 125.7 ± 3.5 b 257 ± 28.5 a 331.5 ± 6.1 a 108.6 ± 1.3 b
120 184 ± 13.2 b 103.4 ± 19.4 b 113 ± 27.1 b 80.3 ± 9.3 b
180 243.8 ± 10.5 a 146.7 ± 9.2 b 343.3 ± 19.4 a 145.5 ± 10.6 a
240 215.6 ± 11.1 a 163.7 ± 1.5 b 162.3 ± 8.4 b 159.6 ± 4.9 a

EC, electrical conductivity; DOC, dissolved organic carbon.

of the accelerated mineralization of organic matter (Edwards,
2011). However, in our study, it was found a generalized increased
in the N-NH+4 content with depth of layers of processed HM and DG
(215.6 ± 11.1 mg kg 1 dw and 159.6 ± 4.9 mg kg 1 dw, respec-
tively) after vermicomposting for 240 d (Table 2). It has been
reported that an increase in the concentration of N-NH+4 could also
be associated with the excretion products of E. andrei (Gómez-
Brandón et al., 2011), which could explain the results found in both
materials, HM and DG. These findings are also in line with the
enhancement in the N-NH+4 content observed during the vermi-
composting of tomato-fruit waste in a continuous-feeding system
(Fernández-Gómez et al., 2010a). Conversely, the N-NO3 values
increased significantly in 240 d-old-layer of processed AP
(56.2 ± 4 mg kg 1dw), whereas the N-NH+4 content decreased dras-
tically (163.7 ± 1.5 mg kg 1dw) (Table 2). This fact suggests that
the earthworm decomposition provoked N mineralization after
vermicomposting for 240 d, probably favouring the stabilization
of the AP material. On the other hand, inorganic N-forms were sig-
nificantly declined with depth of layers of processed GP, from
upper layers (60 d; N-NO3 : 138.6 ± 23.6 mg kg 1dw; N-NH+4:
331.5 ± 6.1 mg kg 1 dw, respectively) to lower layers (240 d; N-
NO3 : 36.6 ± 1.2 mg kg 1 dw; N-NH+4: 162.3 ± 8.4 mg kg 1 dw)
(Table 2). The transformation in the inorganic N-forms is associ-
ated with the nitrification and denitrification (ammonification)
and/or the immobilization of N in a microbial biomass (Atiyeh
et al., 2000). However, these values could also be associated with
38 M. García-Sánchez et al. / Waste Management 62 (2017) 33–42
the process of volatilization of ammonium, as a consequence of the
alkaline pH and/or denitrification of N-NH+4 and N-NO3 as sources
to produce NO2 which is a typical process of the anaerobic earth-
worm gut (Horn et al., 2003).
3.3. Evaluation of microbial biomass and activities changes during the
vermicomposting
The microbiological properties are considered reliable indica-
tors of vermicompost stability due to the key role of microorgan-
ism during the process. It has been reported that the digestion of
the organic material by earthworms can have a negative effect
on microbial biomass by depletion of the resources of the microbes
(Domínguez, 2004). In this study, the overall microbial biomass
was measured as dehydrogenase activity because it occurs intra-
cellularly in all living microbial cells, and it is involved in microbial
respiratory processes (Masciandaro et al., 2000). The highest dehy-
drogenase activity was reached in 120 d-old-layer in all of the pro-
cessed materials (HM, AP, GP, and DG) (F = 151.42; P  0.000),
which can suggest the release of available substances for microor-
ganism as the result of the earthworm activity as suggested by
Benitez et al. (2005). Parhasarathi and Ranganathan (1999)
reported that the fresh casts of earthworms released into the ver-
micomposting material have higher microbial activities and cell
numbers, which could also explain the maximum earthworm bio-
mass and dehydrogenase activity found after vermicomposting for
120 d. The type of material can markedly influence the organic
matter decomposition and thus, the performance of earthworms.
In fact, in our study differences in the dehydrogenase activity were
found according to the type of processed material (F = 62.08;
P  0.000). Thus, the AP vermicomposting showed the highest
dehydrogenase activity when compared with other materials
(P  0.000) (Fig. 3a). This fact points to the biochemical character-
istics found in processed AP which influenced the transformation
of the organic matter, through the E. andrei gut, by significantly
increasing the activity of dehydrogenase with respect to other
materials. However, the dehydrogenase activity recorded in 180
d-old-layer decline significantly (P  0.000) reaching values up to
0.3–8 mg INTF g 1 h 1 (P  0.000) after 240 d of vermicomposting
in all of the processed materials (Fig. 3a). Our findings are in con-
cordance with the previous results reported by Fernández-Gómez
et al. (2010a), indicating that during a continuous feeding-system
a strong increase in the dehydrogenase activity along with the
maximum earthworm biomass would allow the end of the active
phase to be distinguished from the start of the maturation phase.
Therefore, these results point to the dehydrogenase activity as a
reliable and good indicator of microbial biomass for monitoring
vermicomposting in a continuous feeding-system.
The analysis of microbial activities has been reported as prof-
itable tools for characterizing the evolution of the organic matter
decomposition during vermicomposting, as they are involved in
many biochemical processes leading to the transformation of
organic materials into stabilized end products (Aira et al., 2007b;
Benitez et al., 1999, 2005). However, it is not easy to establish gen-
eral threshold values to apply enzymes activities as stability
indexes due to the huge variety of organic materials involved in
the process. Thus, in this study the trend followed by the b-
glucosidase activity was statistically different according to the type
of materials (F = 174.63; P  0.000) and depth of sampling (i.e. age
of layers) (F = 7.63; P  0.001). In fact, the vermicomposting of sub-
strates such as HM provoked a decline in the b-glucosidase activity
in 120-d-old layer (P  0.000) (Fig. 3b). The decrease in this activity
could be associated with the highest earthworm biomass detected
after vermicomposting for 120 d, which may indicate that E. andrei
was strongly involved in the stabilization of organic matter during
the process of this material. Earthworms belong to genus Eisenia
are classified as epigeic (litter feeding), and thereby they possess
a complex enzymatic system (Lavelle, 2010). No changes in the
b-glucosidase activity were found with the level of processed AP
and DG (Fig. 3b). Moreover, these two substrates showed lower
values with respect to this activity compared with the HM and
GP materials (P  0.000). The lack of effects in this activity may
indicate that the passage through the gut of E. andrei could signif-
icantly modify the cellulolytic compounds and/or other organic C
compounds in these both materials. Conversely, a significant
enhancement in the b-glucosidase activity was found to the 60–
120 d-old-layers, and then a subsequent decline was recorded to
the 180–240 d-old-layers of processed GP (P  0.000) (Fig. 3b).
GP is suggested to be a material rich in carbohydrates and sugars,
and therefore it could provide an available substrate for this
enzyme. Moreover, the earthworm activity could greatly increase
the b-glucosidase through the casting released into the substrate
which has been suggested to have a high content of cellulose activ-
ity (Parhasarathi and Ranganathan, 1999). However, the sharp
decline in this activity with depth of layers may suggest that the
stabilization was achieved after the active phase of vermicompost-
ing, as previously suggested Gómez-Brandón et al. (2011). On the
other hand, the phosphatase activity reached higher values in the
case of processed HM and AP compared with other materials
(F = 30.54; P  0.000) (Fig. 3c). However, the most significant
changes in this activity were achieved in 240 d-old-layer of pro-
cessed HM, GP, and DG (F = 18.98; P  0.000). For instance, the
HM and DG materials increased significantly the phosphatase
activity (P  0.000), whereas it was declined in the case of pro-
cessed GP (P  0.000) (Fig. 3c). This result could suggest the pres-
ence of residual amounts of organic-phosphate compounds in
castings from E. andrei, which may act as an inducer of phos-
phatase activity in these substrates. Conversely, a high content in
inorganic P-compounds as consequence of microbial activity and
higher pH values have been shown to inhibit the phosphatase
activity (Alef and Nannipieri, 1995; Fernández-Gómez et al.,
2010a). The phosphatase activity was not significantly modified
after vermicomposting for 240 d of processed AP, probably as the
result of the higher degradation of organic P-compounds during
their passage through the earthworm gut, as previously reported
by Garg et al. (2012). The AP and GP materials seemed to be the
most significant materials in which the highest protease activity
was achieved (F = 73.57; P  0.000) (Fig. 3c). The depth of sampling
(i.e. age of layers) determined differences in the protease activity,
showing different patterns depending on the type of material
(F = 34.15; P  0.000). Thus, an increase in the protease activity
was recorded in 240 d-old-layer of processed HM (Fig. 3d). This
fact points to the hydrolysis of organic N-compounds into available
organic N, which can then be converted into ammonium, and
therefore could explain the enhancement in the N-NH+4 content
found after vermicomposting for 240 days. The protease activity
patterns are highly influenced by the microbial biomass as the high
correlation between the overall microbial biomass and the activity
of this enzyme indicated (Aira et al., 2007a). This fact could explain
the higher activity in protease found in 120 d-old-layer of pro-
cessed GP (Fig. 3d). The protease activity has been suggested as
reliable indicator of the degree of substrate decomposition due to
it is strongly dependent on substrate availability (Lazcano et al.,
2008). This finding is in concordance with our results which
showed a decline in the oldest layers (240 d) of processed AP,
GP, and DG, suggesting a high degree of stability as consequence
of the transformation of available N-containing compounds in
the microbial biomass. The o-diphenol oxidase is related to the
capacity of microorganism to break down recalcitrant compounds
such as aromatic compounds and therefore, this microbial activity
is involved in the stabilization of the organic matter (Benitez et al.,
2004; Perucci et al., 2000). In our study, the type of material used
 M. García-Sánchez et al. / Waste Management 62 (2017) 33–42 39

Fig. 3. Dehydrogenase activity (a), b-glucosidase activity (b), phosphatase activity (c), protease activity (d), and o-diphenol oxidase activity (e) in 60 d, 120 d, 180 d, and 240
d-old-layers of processed HM, AP, GP and DG throughout the process of vermicomposting. Each symbol indicates the mean ± standard error (n = 3).

such as GP, and DG significantly influenced the activity of o-
diphenol oxidase (F = 34.15; P  0.000) (Fig. 3e), and it was also
affected by the age of layers in all of the processes materials
(F = 24.51; P  0.000). Thus, the o-diphenol oxidase activity was
increased in 120 d-old-layer of processed HM and AP (Fig. 3e). This
fact points to an intimate mixing during the transit of organic com-
pounds through the gut of earthworm, which encourages the sta-
bilization of phenolic compounds by the stimulation of microbial
activities in the castings, as previously found by Castillo et al.
(2013). However, a decline in this activity was found after vermi-
composting for 240 d of processed HM and AP. The lower microbial
biomass as the result of the earthworm activity could be responsi-
ble for the reduction found in the activity of this enzyme. However,
this fact could also indicate a possible implication of this microbial
activity in the stabilization of raw materials such as HM, and AP,
and therefore o-diphenol oxidase appears to be useful to monitor
processes of vermicomposting in a continuous-feeding system.
Conversely, an enhancement in the activity of the o-diphenol oxi-
dase was found in 240 d-old-layer of processed GP and DG (Fig. 3e).
3.4. Integrated multivariate analysis
In order to evaluate further insights into similarities or differ-
ences among treatments (HM, AP, GP, and DG) according to vari-
ables (pH, EC, DOC, N-NH+4, N-NO-3, dehydrogenase, b-glucosidase,
phosphatase, protease, and o-diphenol oxidase) tested in this
study, a multivariate approach based on the principal components
analysis was used (Fig. 4). The PCA study indicated that nearly the
80% of the variability of the data was explained by the first two
principal components (PC1 and PC2; accounting for 58.5%, and
21.6% of the variance, respectively) (Fig. 4). The processed DG
was clearly separated by the first grouping factor suggesting that
PC1 in this study was the type of material used. Thus, 60-d, 120-
d, 180-d, and 240-d-old-layers of processed DG were located in
the right-upper quadrant (Fig. 4a) and the most influential vari-
ables were N-NO3 content and o-diphenol oxidase as ranked by
the respective vector lengths (Fig. 4b). The pH was not significantly
separated by any component. Conversely, processed HM, AP, and
GP were strongly differentiated by the PC2 and clustered in two
40 M. García-Sánchez et al. / Waste Management 62 (2017) 33–42

main groups, indicating the age of layers of processed material as
the second grouping factor (PC2). Thus, the upper layers (60 and
120 d-old) of processed materials (HM, AP, and GP), which had
been shown to achieve higher values of dehydrogenase activity
after vermicomposting for 120 d, were clearly located in the left-
upper quadrant and were strongly correlated to this microbial
activity. Meanwhile, the lower layers (180 and 240 d-old) of pro-
cessed HM, AP, and GP were placed in the left-lower quadrant
(Fig. 4a). The DOC content, b-glucosidase, and phosphatase were
negatively correlated to PC2, which indicates that these variables
decreased with depth of layers (Fig. 4b). Overall, this analysis sug-
gests that the particular characteristic found in each type of mate-
rial may had influenced the earthworm digestion, and therefore
could be responsible for the way the process evolved in the DG
respect to HM, AP, and GP materials. Besides, in this study the
age of layers indicated that the activity of E. andrei during the
transformation of the HM, AP, and GP materials was clearly sepa-
rated in two stages which reinforces the finding previously
above-mentioned. Thereby, this PCA study indicates that in the
first stage, corresponding to the 60–120 d-old-layers, in which E.
andrei was still present, the most significant changes with regards
to chemical variables, and microbial biomass were recorded.
Meanwhile, the second stage, corresponding to the 180 d–240
d-old-layers, characterized by the lack of earthworms (totally
absence at 240 d) and the stabilization of the organic matter,
showed an intense decline in DOC and microbial activities
(b-glucosidase, and phosphatse).
3.5. Germination index
The germination index (GI) values were relatively low in the
starting material, but this parameter was significantly increased
after vermicomposting for 240 d of processed AP and DG when
compared with the HM and GP material (F = 5.33; P  0.026)

Fig. 4. (a) Scores from the different samples and (b) loadings of the chemical variables and microbial activities analysed in 60 d, 120 d, 180 d, and 240 d-old-layers of
processed HM, AP, GP and DG throughout the process of vermicomposting. The variables analysed were: pH; electrical conductivity (EC); dissolved organic carbon (DOC); N-
NO3 ; N-NH+4; dehydrogenase (DH), b-glucosidase (G), phosphatase (P), protease (Pr) and o-diphenol oxidase (o-dPO). Percentage of variability explained by each principal
component is shown between round brackets after each axis legend.
 M. García-Sánchez et al. / Waste Management 62 (2017) 33–42
Fig. 5. Germination index (%) in the initial materials and the final vermicomposts (HM, AP, GP, and DG). Values represent the mean (±SE) of three replicates (n = 3). Lowercase
and uppercase letters represent significant differences among the starting materials and final vermicomposts, respectively, according to Tukey test (p < 0.05). Asterisks
indicate a significant difference between the initial material and the final vermicompost from each organic waste according to a paired-sample t-test (p < 0.01).
(Fig. 5). This result is in concordance with the previous findings
reported by Iannotti et al. (1994) which suggested that the high
salinity levels in compost could inhibit the cress seed germination.
However, the EC values found in each initial material can be con-
sidered lower than the optimal range, which suggests that the low-
est GI values (particularly in HM material) may be associated with
the presence of some highly phytotoxic water soluble substance.
After vermicomposting, the GI values were strongly enhanced in
all of the vermicomposts with respect to the starting materials
(t = 21; P  0.000) (Fig. 5). It has been reported that vermicomposts
which determinates a GI value higher than 50% can be considered
as stabilized end products, and thus they can be stated as organic
fertilizers (Fernández-Gómez et al., 2010b). Vermicomposts con-
taining AP and DG showed greater abilities to enhance the GI when
compared with HM and GP (F = 28.30; P  0.000). This result may
indicate the presence of humic substances as the result of the ver-
micomposting of materials such as AP and DG which may be influ-
ence the root length as suggested Canellas et al. (2012). However,
this hypothesis requires of further research.
4. Conclusions
This present demonstrated the potential of E. andrei to process
HM, AP, GP, and DG through an aged-profile layer. Clearly, the nat-
ure of the starting material, such as DG, resulted in differences in
the evolution of the vermicomposting compared with HM, AP,
and GP, as the PCA analysis indicated. The earthworm activity dur-
ing the decomposition of organic materials was markedly sepa-
rated in two stages: (i) an active phase characterized by the
presence of E. andrei and an increase in the overall microbial bio-
mass; (ii) maturation phase where earthworms were absent. The
age of the layers indicated that the role of earthworms modifying
the decomposition of the organic matter was different in relation
to type of substrates, and thereby leading to different trends in
the enzymatic activities of the processed materials. The DOC con-
tent, dehydrogenase, protease and o-diphenol oxidase activities
resulted in good indicators in terms of stabilization of the pro-
cessed substrates. Meanwhile, phosphatase activity was shown to
be highly sensitive to alkaline pH. Moreover, the phytotoxicity test
indicated that besides the amount of organic C compounds, the
presence of biologically active plant-growth in the processed AP
and DG, may be responsible of the increased values in the germina-
tion index (%). These results point out that the vermicomposting in
41
a continuous-feeding system can be considered as an environmen-
tally management technique to process a large amount of materi-
als including HM, AP, GP, and DG with further agronomical
application.
Acknowledgements
The authors are grateful for the financial support of the ESF/
MŠMT project (No. CZ.1.07/2.3.00/30.0040), and the NAZV project
(No. QJ1530034). The authors also acknowledge to reviewers for
their valuable comments and suggestions.
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