Chemosphere 72 (2008) 1013–1019

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Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere

Comparison of the effectiveness of composting and vermicomposting

for the biological stabilization of cattle manure
Cristina Lazcano *, María Gómez-Brandón, Jorge Domínguez
Departamento de Ecoloxía e Bioloxía Animal, Universidade de Vigo, 36310 Vigo, Spain

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

Article history: Cattle manure is produced in large quantities in industrial breeding facilities and the storage and/or
Received 31 January 2008 spreading of this waste on land may cause contamination of the atmosphere, soil and water. The aim
Received in revised form 8 April 2008 of the present study was to evaluate the effectiveness of the active phases of composting, vermicompo-
Accepted 8 April 2008
sting, and also a combination of composting and vermicomposting for reducing the polluting potential
Available online 3 June 2008
and for stabilizing cattle manure in the short-term. For this, the degree of decomposition as well as
the microbial activity and microbial composition of the resulting products after the active phase of com-
Keywords:
posting and vermicomposting were analysed. None of the treatments significantly reduced the dissolved
Cattle manure
Biological stabilization
organic carbon and dissolved organic nitrogen contents relative to the control, and therefore more time
Microbial activity may be required for stabilization. Nevertheless, the lowest values of microbial biomass and activity cor-
Enzyme activities responded to the earthworm-worked substrates, in which fungal growth was also promoted; the com-
Microbial groups bined treatment (composting + vermicomposting) was the most effective in terms of stabilizing the
cattle manure. Moreover, earthworms promoted the retention of nitrogen and gradual release of P, as
well as a reduction in electrical conductivity, thereby producing improved substrates for agricultural use.
Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction depending on the characteristics of the waste, high quality

mulches can be obtained for agricultural use, and with further mat-
Cattle manure is a valuable resource as a soil fertilizer, as it pro- uration and elimination of phytotoxic compounds, high quality or-
vides high contents of macro- and micro-nutrients for crop growth ganic fertilizers.
and is a low-cost alternative to mineral fertilizers. However, over- Composting and vermicomposting are two of the best-known
production of this waste substance has led to inappropriate processes for the biological stabilization of solid organic wastes.
disposal practices such as the indiscriminate and inappropri- Composting involves the accelerated degradation of organic matter
ately-timed application to agricultural fields. Such practices can by microorganisms under controlled conditions, in which the or-
cause serious environmental problems, including an excessive ganic material undergoes a characteristic thermophilic stage that
input of potentially harmful trace metals, inorganic salts and allows sanitization of the waste by the elimination of pathogenic
pathogens, increased nutrient loss from soils through leaching, microorganisms (Lung et al., 2001). Two phases can be distin-
erosion and runoff – caused by lack of consideration of the nutrient guished in composting: (i) the thermophilic stage, where decom-
requirements of crops – and the emission of hydrogen sulphide, position takes place more intensively and which therefore
ammonia and other toxic gases (Hutchison et al., 2005). constitutes the active phase of composting; and (ii) a maturing
Animal wastes pose health and environmental risks similar to stage which is marked by the decrease of the temperature to the
those of human wastes and should be treated accordingly. Stabil- mesophilic range and where the remaining organic compounds
ization involves the decomposition of a waste substance to the ex- are degraded at a slower rate. The duration of the active phase de-
tent where the hazards are eliminated, and is normally reflected by pends on the characteristics of the waste (amount of easily decom-
decreases in microbial activity and concentrations of labile com- posable substances) and on the management of the controlling
pounds (Benito et al., 2003). Stabilization therefore reduces the parameters (aeration and watering). The extent of the maturation
environmental problems associated with the management of man- phase is also variable and it is normally marked by the disappear-
ure by transforming it into a safer and more stabilized material ance of the phytotoxical compounds. Composting is well estab-
suitable for application to soil (Carr et al., 1995). Furthermore, lished at the industrial scale for solid organic waste treatment,
although the loss of nitrogen through volatilization of NH3 during
* Corresponding author. Tel.: +34 986 812 037; fax: +34 986 812 556. the thermophilic stage of the process is one of the major draw-
E-mail address: [email protected] (C. Lazcano). backs of the process (Eghball et al., 1997).

0045-6535/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.chemosphere.2008.04.016
1014 C. Lazcano et al. / Chemosphere 72 (2008) 1013–1019

Vermicomposting involves the bio-oxidation and stabilization stage in composting largely conditions microbial communities
of organic material by the joint action of earthworms and microor- (Klamer and Bååth, 1998), and so do the earthworms by ingesting
ganisms. Although it is the microorganisms that biochemically de- the waste (Lores et al., 2006). Since microorganisms are responsi-
grade the organic matter, earthworms are the crucial drivers of the ble for chemical degradation in the last term, the changes occurred
process, as they aerate, condition and fragment the substrate, on microbial communities in each process might also condition the
thereby drastically altering the microbial activity. Earthworms further decomposition of the waste, as well as the establishment
act as mechanical blenders and by comminuting the organic mat- and survival of beneficial (plant growth promoting bacteria) or del-
ter they modify its physical and chemical status by gradually eterious (fecal coliforms) microorganisms for land application. In
reducing the ratio of C:N and increasing the surface area exposed the present study we evaluated the effectiveness of the active
to microorganisms – thus making it much more favourable for phases of composting, vermicomposting, and a combination of
microbial activity and further decomposition (Domínguez et al., composting and vermicomposting, for the short-term stabilization
1997). Therefore two phases can also be distinguished here, (i) of cattle manure, by analysing the physicochemical, biochemical
an active phase where the earthworms process the waste modify- and microbiological characteristics of the final products.
ing its physical state and microbial composition (Lores et al., 2006),
and (ii) a maturation-like phase marked by the displacement of the 2. Material and methods
earthworms towards fresher layers of undigested waste, where the
microbes take over in the decomposition of the waste. Like in com- 2.1. Source materials and biostabilization processes
posting, the duration of the active phase is not fixed, and it will de-
pend on the species and density of earthworms, the main drivers of Cattle manure, consisting of a mixture of faeces, urine and straw
the process, and their ability to ingest the waste (ingestion rate). was obtained from the agricultural cattle complex ‘‘Energía Viva,
Vermicomposting is not fully adapted to the industrial scale S.A.’’ in León, Spain; the main physicochemical and microbiological
(Domínguez et al., 1997) and since the temperature is always in characteristics of the manure are summarized in Table 1.
the mesophilic range, pathogen removal is not ensured, although Composting was carried out in five trenches of 42 m long, 4.5 m
some studies have provided evidence of suppression of pathogens wide and 1.8 m deep, each of which contained approximately
(Monroy et al., 2008). In some cases, organic residues require pre- 300 m3 of material. Throughout the process, the trenches were aer-
treatment before being vermicomposted as they may contain sub- ated from the bottom with forced air (through a blower) in order to
stances that are toxic for earthworms, such as acidic compounds induce movement of air into the material and deliver oxygen to
(Nair et al., 2006). microorganisms. The functioning of the air blower varied depend-
The combination of composting and vermicomposting has re- ing on the temperature: (a) continuous aeration when the temper-
cently been considered as a way of achieving stabilized substrates ature of the composting mass exceeded 60 °C; (b) intermittent
(Tognetti et al., 2007). Composting enables sanitization of the aeration according to a preset cycle of 5 min aeration and 5 min
waste and elimination of toxic compounds, and the subsequent pause when the temperature was between 55 and 60 °C; and (c)
vermicomposting reduces particle size and increases nutrient intermittent aeration according to a preset cycle of 5 min aeration
availability; in addition, inoculation of the material resulting from followed by a pause of 10 min when the temperature was below
the thermophilic phase of composting with earthworms reduces 55 °C. In addition to the forced ventilation, the compost was turned
the expense and duration of the treatment process (Ndegwa and daily in order to homogenize the mass, and to avoid compaction of
Thompson, 2001). the substrate and subsequent low porosity and poor air distribu-
Although several studies have addressed the optimization of tion. The composting material was watered and the moisture con-
either composting, vermicomposting or composting with subse- tent was monitored daily and maintained within 55–65%. At the
quent vermicomposting (Domínguez et al., 1997; Frederickson end of the active phase (15d), ten sub-samples were randomly col-
et al., 1997; Ndegwa and Thompson, 2001; Tognetti et al., 2005; lected within each trench and composited into single samples,
Tognetti et al., 2007), there are no studies concerning the efficiency each of 10 l (Gómez-Brandón et al., 2008).
of these three processes together to stabilize a specific organic Vermicomposting was carried out in a 1 m3 vermireactor con-
waste. taining a stable and very active population of the earthworm Eise-
To obtain high quality organic fertilizers, it is necessary to nia andrei. The reactor was fed with different animal manures and
understand the changes that the material undergoes during the mixed agricultural wastes, and supported a population density of
biological stabilization process. The stability of the final products 250 g of earthworms kg1 in the top layers. The upper surface of
is essential for its successful application to crops. Thermophilic the vermireactor was divided into four independent compartments

Table 1
Mean values ± standard error of the physicochemical and biochemical properties in the initial raw cattle manure and the substrates produced by the different treatments:
incubation under field conditions for 15d (control); active phase of composting (composting); vermicomposting, and composting with subsequent vermicomposting
(Composting + vermicomposting)

Raw cattle manure Control Composting Vermicomposting Composting + vermicomposting

pH 7.70–8.94 8.89–8.78a 8.86–8.07a 7.73–7.51b 7.85–7.14b
EC (dS m1) 1.25 ± 0.08 1.32 ± 0.08a 2.13 ± 10b 0.78 ± 0.02c 0.72 ± 0.04c
C to N ratio 17.0 ± 0.74 15.7 ± 1.09a 17.5 ± 0.33a 11.1 ± 0.24b 11.3 ± 0.16b
Total C (g kg1 dw) 399.2 ± 2.8 395.7 ± 3.2a 384.9 ± 2.7a 314.0 ± 5.4b 309.0 ± 8.6b
Total N (g kg1 dw) 23.6 ± 0.9 25.6 ± 1.7ab 22.0 ± 0.3a 28.3 ± 0.2b 27.4 ± 0.8b
DON (mg kg1 dw) 2190 ± 380 2260 ± 244a 2571 ± 896a 3726 ± 153a 2165 ± 198a
NHþ4 —N (mg kg
1
dw) 610 ± 92 534 ± 128a 1235 ± 291b 276 ± 24a 191 ± 30a
NO3 —N (mg kg
1
dw) 19 ± 15 0 ± 0a 721 ± 184b 917±113b 829 ± 110b
1
DOC (mg kg dw) 4406 ± 704 6819 ± 772a 9338 ± 2103a 5249 ± 302a 4825 ± 387a
Available P (mg kg1 dw) 211 ± 6 175 ± 7a 342 ± 22b 111 ± 3c 109 ± 6c

Results of the Tukey HSD test for the different treatments are shown; the data corresponding to the raw manure were not included in the statistical comparisons.
dw: Dry weight.
a,b,c
Means with the same letter were not statistically different (Tukey HSD test, a = 0.05).
 C. Lazcano et al. / Chemosphere 72 (2008) 1013–1019 1015

and 15 kg of cattle manure were placed in three successive layers digested with 2 ml of methanol and 0.5 ml of 2 M NaOH in a scien-
(5 kg each) added to each compartment as the waste was pro- tific microwave oven (CEM Corporation MDS-2000), processed at
cessed by the earthworms. The moisture content of the cattle man- 2450 MHz and 630 W maximum output and irradiated at medium
ure in the vermireactor was maintained at 75–80% and the samples power (60% of maximum power output, manufacturer’s setting) for
were collected from the last layer (40d of earthworm processing) 20 s three times, with 1 min of cooling between each time. The
of the reactor once the manure was processed by the earthworms. contents were extracted with pentane (3  ca. 2 ml), the pentane
Composting plus subsequent vermicomposting was carried out extracts were then evaporated to dryness under a stream of N2
by first composting the manure for 15d, as described above, and gas and then redissolved with 1 ml of methanol and filtered
then vermicomposting in the 1 m3 vermireactor for 40d with the through a 0.2 lm syringe filter (MFS) prior to HPLC analysis
earthworm E. andrei, as described for the vermicomposting treat- (Young, 1995). Dehydrogenase enzyme activity was measured by
ment. Samples were collected from the vermireactor 40d after estimation of the rate of reduction of triphenyltetrazolium chloride
the addition of the third layer of composted manure. (TTC) (1.5%) to triphenylformazan (TPF), after incubation at 30 °C
As controls (no treatment), five manure heaps (15 kg each) were for 24 h, in a microplate reader (Bio-Rad Model 550), at 545 nm
maintained under field conditions and moistened twice a week for (Casida et al., 1964). Protease activity was measured by determina-
15d. All samples were placed in sealed plastic containers and tion of the amino acids released, after incubation of the samples
stored at 5 °C until analysis. (1 g fresh weight) with sodium caseinate (2%) for 2 h at 50 °C
The aim of this study was to compare stabilization treatments and with Folin–Ciocalteu reagent, in a microplate reader (Bio-Rad
and the duration of the treatments differed as it depended on the Model 550), at 700 nm (Ladd and Butler, 1972). b-Glucosidase
time necessary for the completion of the active phase in each pro- activity was assessed by determination of the p-nitrophenol
cess: 15d for the active or thermophilic phase of composting, 40d (PNP) released, after incubation of the samples (1 g fresh weight)
for processing of the manure by the earthworms and 55d for the with b-D-glucopyranoside (0.025 M) for 1 h at 37 °C, in a micro-
combined treatment. The duration of these processes could not plate reader (Bio-Rad Model 550), at 400 nm (Eivazi and Tabatabai,
be modified without altering the processes themselves. 1988). Alkaline phosphomonoesterase activity was estimated by
determinatio of the PNP released, after incubation of the samples
2.2. Chemical analyses (1 g fresh weight) with p-nitrophenyl phosphate (0.025 M) for
1 h at 37 °C, with a microplate reader (Bio-Rad Model 550), at
The moisture and organic matter contents of the samples were 400 nm (Eivazi and Tabatabai, 1972).
determined after drying at 105 °C for 24 h and ashing at 550 °C for For quantification of actinomycetes, samples were homoge-
4 h, respectively. The pH and electrical conductivity were deter- nized and added to a tris-buffered saline solution, and then serially
mined in water extracts (1:20, w/v). Total C and N were measured diluted and incubated at room temperature for 2 h. Dilutions were
in oven-dried (60 oC) and ball-milled sub-samples, with a Carlo plated on actinomycetes agar and incubated at 30 ° C. The number

Erba NA 1500 C/N analyzer. Inorganic nitrogen (NHþ 4 and NO3 ) of colony forming units (CFU) was counted after 72 h.
was determined in 0.5 M K2SO4 extracts (1:10 w/v) by the modified
indophenol blue technique (Sims et al., 1995), with a microplate 2.4. Statistical analyses
reader (Bio-Rad Model 550). Total extractable N was determined
after oxidation with K2S2O8 as described by Cabrera and Beare Results are means of either five replicates, for the composting
(1993) and the dissolved organic nitrogen (DON) content was cal- and control treatments, or of four replicates, for the vermicompo-

culated as (total extractable N)–(NHþ 4 –N +NO3 –N). Dissolved or- sting and composting plus vermicomposting treatments. One-way
ganic carbon (DOC) in the cattle manure and the final products analysis of variance and comparison of means based on the Tukey
were determined colorimetrically at 590 nm after moist digestion honestly significance difference test (HSD, P < 0.05) were used to
(K2Cr2O7 and H2SO4) of aliquots of 0.5 M K2SO4 extracts (1:10 w/ determine significant differences between treatments. Differences
v) of the samples. between the raw manure and the substrates after the treatments
Available P was analyzed in ammonium bicarbonate–diethyl- were analysed by t-test. All statistical tests were evaluated at the
ene triaminepentaacetic acid extracts of oven-dried and ball- 95% confidence level. The relationships between variables were
milled samples (1:6, w/v) by induced coupled plasma optical emis- defined by regression analysis. Statistical analyses were carried
sion spectrometry (Soltanpour and Schwab, 1977). out with SPSS 11.0 for Windows.

2.3. Microbiological and biochemical analyses

3. Results and discussion
Microbial activity and biomass were assessed by measurement
of the rate of CO2 evolution from the samples during 6 and 12-h Although several physical, chemical, and biological parameters
incubation, for basal and substrate-induced respiration (SIR), have been suggested as indicators of compost stability, and some
respectively. Prior to incubation, 0.75 ml of glucose solution of them, such as respiration rates, constitute widely-used, rapid
(equivalent to 100 mg glucose g1 dw sample) was added to sam- and reliable measurements, it is not easy to establish the stability
ples for the SIR assay. The evolved CO2 was trapped in 0.02 and of an organic amendment based on just one parameter, and the
0.04 M NaOH (basal and SIR, respectively) and then measured by threshold values may not be applicable to all composts, given the
titration with HCl to a phenolphthalein endpoint, after addition variety of parent wastes and feedstock as well as the composting
of excess BaCl2 (Anderson, 1982). Incubation times, NaOH and glu- processes (more or less controlled) from which they are originated.
cose concentrations were adjusted in order to obtain the most In this sense, an integrated approach is recommended for a more
accurate response for this type of organic samples as shown in Aira accurate determination.
et al. (2006, 2007a).
Fungal biomass was determined by quantification of ergosterol, 3.1. Evaluation of the chemical changes
a membrane-bound molecule commonly used as a fungal bio-
marker. The ergosterol content of the samples was extracted by The main chemical properties of the cattle manure processed by
the microwave assisted extraction method and determined by composting, vermicomposting, composting plus vermicomposting
HPLC analysis. Briefly, samples (500 mg fresh weight) were and the control treatment are summarized in Table 1. The control
1016 C. Lazcano et al. / Chemosphere 72 (2008) 1013–1019

treatment and the active phase of the composting process did not tern was not observed in the present study, in which the active
significantly change the pH of the initial raw waste (t-test: phase of composting increased the DOC relative to that in the
P = 0.262, P = 0.859, respectively), whereas vermicomposting and raw manure. The increase was minor following the vermicompo-
composting plus vermicomposting significantly decreased the pH sting and the combined treatments, but nevertheless shows an
(t-test: P = 0.009, P = 0.017, respectively). Other authors have found incomplete and active degradation that mobilises the insoluble
similar results in vermicomposting experiments, and have sug- carbon from the organic matter to the soluble phase. We expect
gested that the mineralization of N and P compounds, the release that if the processing time was longer, the DOC would decrease
of CO2 and organic acids from microbial metabolism, and the pro- as shown by, for example, Gómez-Brandón et al. (2008) for compo-
duction of humic and fulvic acids, as possible causes of the de- sting, and Aira et al. (2007a) for vermicomposting.
crease in pH during vermicomposting (Ndegwa and Thompson,
2001; Kaushik and Garg, 2004). 3.2. Evaluation of the microbiological and biochemical changes
The electrical conductivity (EC) reflects the salinity of an organ-
ic amendment. High salt concentration may cause phytotoxicity From the stabilization treatments assayed, only vermicompo-
problems and therefore EC is a good indicator of the suitability sting and composting with subsequent vermicomposting reduced
and safety of a compost or vermicompost for agricultural purposes. significantly the microbial biomass of the raw manure (t-test,
EC was affected in different ways by the application of the different P = 0.029, P = 0.007 respectively). These treatments were also sig-
treatments. The value of this parameter increased after the active nificantly different from the control (Fig. 1a). On the contrary,
phase of composting, relative to the control, whereas it decreased the active phase of composting did not reduce the microbial bio-
after vermicomposting and the combined treatment of composting mass of the manure and was not significantly different from the
and vermicomposting (Table 1). A sharp increase due to the release control (Fig. 1a). Similar results were reported by Tognetti et al.
of soluble salts like ammonium and phosphate after the degrada- (2005) for comparison between composting and vermicomposting.
tion of the most labile compounds in the thermophilic stage of It is known from incubation experiments that the passage of
composting has also been reported by several authors (Villar microorganisms through the earthworm gut has a negative effect
et al., 1993). During vermicomposting the minor production of sol- and leads to a short-term decrease in microbial biomass through
uble metabolites such as ammonium (NHþ 4 ), as well as precipita- feeding and digestion (Aira et al., 2006).
tion of the dissolved salts may lead to lower EC values (Mitchell,
1997). The EC of the cattle manure after the different treatments
did not exceed the threshold value of 3 dS m1 that indicates a
material that can be safely applied to soil (Soumaré et al., 2002).
The C to N ratio indicates the degree of decomposition of a a 7·10 4
waste, as carbon is lost as CO2 during biooxidation, whereas N is
a
lost at a lower rate, and therefore the more decomposed a waste, 6·10 4

the lower the C to N ratio. In the present study, total C was signif-
SIR (mg CO 2 kg -1 OM)

4
5·10
icantly reduced after the vermicomposting and combined treat-
ment, whereas it remained high after the active phase of
4·10 4
composting. In contrast, total N content was higher after vermi- ab
composting and the combined treatment than after the active 3·10
4
b
phase of composting. Consequently, the C to N ratio was
significantly lower in the treatments involving vermicomposting, 2·10
4
b
which indicates that they underwent more intense decomposition
(Table 1). 1·10
4

The concentration of DON was still high after stabilization, with

0
no differences between the treatments, indicating that in this re-
spect, stabilization was not sufficient, as established by Hue and b 6·10
3

a
Liu (1995). The concentration of mineral N (NHþ 4 and especially
NO 3 ) was significantly higher following the three treatments than
5·10 3
Basal respiration (mg CO2 kg-1OM)

ab
in the raw cattle manure, indicating an important degree of miner-
alization. The concentration of NHþ 4 only increased after the active 4·10 3
phase of the composting process, with significantly higher values ab
than in the other treatments (Table 1). 3
3·10
There were no significant differences in the DOC contents corre- b
sponding to the different treatments. The content of labile carbon
was higher following the active phase of composting, vermicompo- 2·10 3

sting and the control treatment, than in the initial manure. An or-
ganic amendment with a high DOC can cause serious damage to 1·10 3
crops, since it will continue to degrade in the soil consuming oxy-
gen, hampering root respiration and leading to the production of Composting +
Raw manure Control Composting Vermicomposting
phytotoxical compounds such as SH2 (Mathur et al, 1993). vermicomposting

Although DOC consistently decreases during a complete compo-

Fig. 1. Microbial biomass (a) and microbial activity (b) in the raw cattle manure
sting process, the initial degradation of solid polymeric material and the substrates produced by the different treatments: incubation under field
after the thermophilic stage in the composting substrate may lead conditions for 15d (control); active phase of composting (composting); vermicom-
to the formation of soluble organic matter, which would increase posting, and composting until the end of the active phase plus subsequent vermi-
the DOC concentration. Gómez-Brandón et al. (2008) observed that composting (composting + vermicomposting). Values are means ± standard error
(control and composting: n = 5; vermicomposting and composting plus vermicom-
the DOC content decreased sharply within the first two weeks of posting: n = 4). Results of the Tukey HSD test for the different treatments are sho-
composting, to values of less than 4000 mg kg1 dw by the end wn; the data corresponding to the raw manure were not included in the statistical
of the process, which would guarantee safe plant growth. This pat- comparisons. Different letters indicate significant differences at P < 0.05.
 C. Lazcano et al. / Chemosphere 72 (2008) 1013–1019 1017

The stabilization treatments resulted in substrates with similar was higher in the earthworm gut and vermicompost than in fresh
(t-test, control and combined treatments, P = 0.614, P = 0.322, substrate in vermicomposting facilities with the earthworm E.
respectively) or even higher microbial activity (t-test, active andrei.
phase of composting, vermicomposting, P = 0.018, P = 0.010, The number of actinomycetes was also significantly affected by
respectively) than in the raw manure (Fig. 1b); after the stabiliza- the treatment applied (Fig. 3); it was very high in the raw manure
tion treatments none of the substrates differed significantly from and the active phase of composting increased the abundance
the control, which indicates that decomposition was incomplete slightly. The most remarkable result was that both vermicompo-
and total stabilization was not achieved. The high activity regis- sting treatments showed abundances of these microorganisms
tered after the composting treatment may be due to the short 100 times lower than the active phase of composting. Actinomy-
duration of the process. Gómez-Brandón et al. (2008) reported cetes compete rather ineffectively when nutrient levels are high,
high metabolic rates within the first weeks in composting of cat- develop far more slowly than most bacteria and fungi, and are typ-
tle manure, which then tended to decrease during the maturation ical during the final stages of decomposition (Alexander, 1980).
phase. Despite the above-mentioned reduction in microbial bio- Thus their abundance in the fresh waste and in the control treat-
mass by the earthworms, microbial activity was not affected, ment is difficult to explain. Actinomycetes are tolerant to high
and it is possible that the passage through the earthworms’ gut temperatures, some of them being facultative thermophilic micro-
favoured the appearance of a reduced but more catabolically ac- organisms, and thus they may be expected to survive the thermo-
tive microflora (Aira et al., 2007a). Nevertheless, this pattern philic stage of the composting treatment. Nakasaki et al. (1985)
was not observed after the earthworms had digested the compost studied the changes in the different microbial groups in compo-
from the active phase, when a significant reduction in microbial sting of sewage sludge and reported that the increase in actinomy-
activity was produced. cetes was typical of the final stage of the thermophilic phase. The
Fungal biomass was not affected by the active phase of compo- significantly lower numbers of these microorganisms in both ver-
sting in comparison with the control (Fig. 2) and the ergosterol micompost treatments showed that the earthworms may have
content remained at approximately the same levels as in the raw some effect. Earthworms have been found to cause changes in
manure (t-test, P = 0.652). This was not true for vermicomposting the microbial communities in several organic wastes during vermi-
and the combined treatment, as these had a marked effect on fun- composting (Lores et al., 2006), moreover, these microbial commu-
gal growth, and the ergosterol content of the raw manure was in- nities have been found to be more efficient metabolically (Aira
creased by 28 and 41 times respectively following these et al., 2007a). Development of actinomycetes was probably not fa-
treatments (t-test, P < 0.001 and P = 0.001 respectively); this indi- voured during vermicomposting of the cattle manure because their
cates significantly higher fungal contents than both the control ineffective competition with the more active microbial communi-
and the compost. Enhanced fungal growth in degrading organic ties promoted by the earthworms.
matter has been attributed to the depletion of easily degradable or- The changes in dehydrogenase, protease, glucosidase, and phos-
ganic compounds and the subsequent decrease in bacteria. Never- phatase (key enzymes involved in aerobic metabolism, as well as
theless, this was not the cause of the increase in fungal abundance degradation of polypeptides, polysaccharides and phosphate es-
in both vermicomposts since DOC and DON contents were still ters, respectively) are summarized in Fig. 4. Dehydrogenase activ-
high in these substrates. The higher ergosterol content in both kind ity was significantly lower after both vermicomposting treatments
of vermicompost shows that earthworms enhanced fungal growth than after the active phase of composting (Fig. 4a). However, the
in the short-term, either indirectly through the modification of the activity was not significantly lower in any of the substrates than
substrate, or directly through their feeding activity. Enhanced fun- in the control probably due to the high DOC content which corre-
gal biomass after gut transit was also found by Aira et al. (2006) lated positively with this parameter (R2 = 0.235; P = 0.019). This
after vermicomposting of pig slurry with Eisenia fetida, and Pizl indicates that the substrates were not sufficiently stabilized.
and Nováková, 2004, who found that the density of microfungi Dehydrogenase activity is used as a measure of overall microbial

20
200·10 6

b a
15
150·10 6
Actinomycetes (CFU g-1 )
Ergosterol (mg·g-1 OM)

10 b 6 ab
100·10

5
6
50·10

a
a
b b
0
0
Composting + Composting +
Raw manure Control Composting Vermicomposting Raw manure Control Composting Vermicomposting
vermicomposting vermicomposting

Fig. 2. Ergosterol content of the raw cattle manure and the substrates produced by Fig. 3. Abundance of actinomycetes in the raw cattle manure and the substrates
the different treatments: incubation under field conditions for 15d (control); active produced by the different treatments: incubation under field conditions for 15d
phase of composting (composting); vermicomposting, and composting until the (control); active phase of composting (composting); vermicomposting, and com-
active phase plus subsequent vermicomposting (composting + vermicomposting). posting until the end of the active phase plus subsequent vermicomposting (com-
Values are means ± standard error (control and composting: n = 5; vermicompo- posting + vermicomposting). Values are means ± standard error (control and
sting and composting plus vermicomposting: n = 4). Results of the Tukey HSD test composting: n = 5; vermicomposting and composting plus vermicomposting: n =
for the different treatments are shown; the data corresponding to the raw manure 4). Results of the Tukey HSD test for the different treatments are shown; the data
were not included in the statistical comparisons. Different letters indicate signif- corresponding to the raw manure were not included in the statistical comparisons.
icant differences at P < 0.05. Different letters indicate significant differences at P < 0.05.
1018 C. Lazcano et al. / Chemosphere 72 (2008) 1013–1019

a a
b b
400 25000

β - Glucosidase activity (µg PNP g-1 dw h-1)

Dehydrogenase activity (μg TPF g-1 dw)

350 ab
20000
300

250
15000

200 c

10000
150

100 b
b 5000
a
50 a

0 0

c 20000 d 8000
b

Phosphatase activity (µg p-nitrophenol g -1dw h )

a

-1
Protease activity (µg tyrosine g dw-1 h-1)

ab
6000
15000 ab
b

4000
10000

b 2000

b a
5000

0
Composting + Composting +
Raw manure Control Composting Vermicomposting Raw manure Control Composting Vermicomposting
vermicomposting vermicomposting

Fig. 4. Dehydrogenase (a), protease (b), b-glucosidase (c), and phosphatase (d) activities in the raw cattle manure and the substrates produced by the different treatments:
incubation under field conditions for 15d (control); active phase of composting (composting); vermicomposting, and composting until the end of the active phase plus
subsequent vermicomposting (composting + vermicomposting). Values are means ± standard error (control and composting: n = 5; vermicomposting and composting plus
vermicomposting: n = 4). Results of the Tukey HSD test for the different treatments are shown; the data corresponding to the raw manure were not included in the statistical
comparisons. Different letters indicate significant differences at P < 0.05.

activity; and the levels therefore indicated that both vermicompo- enzyme is mainly produced by fungi (Hayano and Tubaki, 1985). In
sting treatments produced more stabilized substrates than the ac- the present study, a significant correlation between the ergosterol
tive phase of composting, in accordance with the basal respiration content of the substrates and their ß-glucosidase activity
levels obtained. Nevertheless, it should be noted that this analysis (R2 = 0.406, P = 0.004) was observed, which is consistent with the
only accounts for a limited percentage of respiration since oxygen results of previous studies (Aira et al 2006). The higher activity de-
is a better electron acceptor than the TTC used in our assay (Nann- tected after both vermicomposting treatments therefore corre-
ipieri et al., 1990). The active phase of the composting process sponded to the higher fungal abundance than observed after
exhibited the highest degree of protease activity, and differed sig- composting. None of the assayed treatments showed significant
nificantly from the control; this high activity explains the high N– differences in alkaline phosphatase activity in comparison with
NHþ 4 concentration found after both treatments. On the contrary, the control; nevertheless, the substrate produced after the active
treatments with earthworms resulted in significantly lower prote- phase of composting showed significantly lower activity than both
ase activity than in the control (three times lower following vermi- kinds of vermicompost (22 and 13 times lower) (Fig. 4d). Phospha-
composting, and 4.4 times lower following the combined tase catalyzes hydrolysis of the phosphoric esters to inorganic P,
treatment: Fig. 4c). Significant reductions in the activity of this en- which inhibits the activity of the enzyme when present at high
zyme were also observed by Aira et al. (2007b) in vermicomposting quantities in the substrate. In the present study we found a signif-
experiments with pig slurry. Protease activity is highly dependent icant negative correlation between phosphatase activity and avail-
on substrate availability (Aira et al., 2007b), and therefore it is a able P (R2 = 0.532; P < 0.0001). This explains the low activity after
very good indicator of the level of decomposition of a substrate. the active phase of composting, as a higher concentration of solu-
According to this criterion, the vermicomposted materials were ble P than in the rest of substrates was detected (Table 1). The high
significantly more stabilized than the compost. b-Glucosidase activity following vermicomposting and the combined treatment
activity of the manure was significantly affected by the treatments shows that there were still sufficient amounts of phosphate esters
applied. The active phase of composting resulted in the lowest val- available and that the release of P was not sufficient to produce en-
ues, which were not significantly different from the control values, zyme inhibition. Benítez et al. (2005) observed that phosphatase
whilst both vermicomposting treatments resulted in significantly activity increased gradually throughout the vermicomposting pro-
higher activity, the highest corresponding to the vermicomposting cess, first reaching stability and then decreasing slightly. The
treatment (21 times higher than the control). b-Glucosidase cata- recovery of the phosphatase activity after vermicomposting the
lyzes the breakdown of glucosides to glucose, one of the last steps compost from the active phase was remarkable and was probably
in the degradation of cellulose, and it is assumed that in soils this due to a more gradual decomposition by the earthworms.
 C. Lazcano et al. / Chemosphere 72 (2008) 1013–1019 1019

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