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International Journal of Applied Agricultural Research
ISSN 0973-2683 Volume 4 Number 1 (2009) pp. 75–85
© Research India Publications
http://www.ripublication.com/ijaar.htm
Comparative Studies on Physico-Chemical,
Microbiological and Enzymatic Activities of
Vermicasts of the Earthworms, Eudrilus Eugeniae,
Lampito Mauritii and Perionyx Ceylanensis Cultured
in Press mud
M. Jayakumar1, V. Karthikeyan2 and N. Karmegam3*
1
Department of Biotechnology & Chemical Engineering,
Arulmigu Meenakshi Amman College of Engineering,
Vadamavandal-604 410, Tamil Nadu, India
2
Department of Biotechnology,
Karpaga Vinayaka College of Engineering and Technology,
Chinnakolambakkam, Madurantakam-603 308,
Tamil Nadu, India.
3
Department of Biotechnology,
V.M.K.V. Engineering College,Vinayaka Missions University,
Periya Seeragapadi, Salem– 636 308,
Tamil Nadu, India.
*Corresponding Author;
E-mail: [email protected]
Abstract
The works on the efficiency of Eudrilus eugeniae and Lampito mauritii in
vermiconversion of different organic materials are largely available. The
culturing of Perionyx ceylanensis in press mud and characterization of press
mud-vermicasts of P. ceylanensis are not reported hitherto. Hence the present
study has been carried out to analyse the physico-chemical, microbiological
and enzyme activities in the vermicasts of three different species of
earthworms, E. eugeniae, L. mauritii and P. ceylanensis cultured in press mud
(or filter cake). The results of the present study showed that the vermicasts of
E. eugeniae, L. mauritii and P. ceylanensis recorded higher values for E.C.,
total nitrogen, phosphorus and potassium than worm-unworked press mud
(WUP). The increased NPK values in vermicasts were found to be
significantly different than WUP. The organic carbon, C/N ratio and C/P ratio
in vermicasts were lower than in WUP where the values for vermicasts were
76
M. Jayakumar et al
significant. Among the three species of earthworms, E. eugeniae recorded
maximum percentage decrease of C/N, (144.73) followed by P. ceylanensis
and L .mauritii respectively. The total microbial population, viz., bacteria,
fungi and actinomycetes were found to be many-fold higher than in the initial
vermibed substrate and WUP. The activity of the enzymes, amylase, cellulase,
invertase, phosphatase and protease in vermicasts were higher than that of
WUP. The study clearly shows that P. ceylanensis is greater in activity than L.
mauritii.
Keywords: C/N ratio, Earthworms, Press mud, Vermicasts,
Vermicomposting.
Introduction
Increasing demand for safe disposal of solid wastes synchronized the use of
earthworms and microorganisms. The conversion of various wastes (leaf litters,
domestic, industrial and municipal) into compost for application to soil as plant
nutrients and organic farming serves as an effective eco-friendly, low-cost methods of
disposal of wastes [1]. These wastes are highly organic in nature, so vermicomposting
has become an appropriate alternative for the safe, hygienic and cost effective
disposal of it. Earthworms feed on the organics and convert materials into casting
(ejected matter) rich in plant nutrients [2]. The action of earthworms in the process of
vermicomposting of waste is physical and biochemical. The physical process
includes substrate aeration, mixing as well as actual grinding while the biochemical
process is influenced by microbial decomposition of substrate in the vermibed and in
the intestine of earworms. Various studies have shown that vermicomposting of
organic waste accelerates organic matter stabilization [3, 4, 5] and gives chelating and
phytohormonal elements [6] which have a higher content of microbial matter and
stabilized humic substances.
In India, the earthworm species like Eisenia fetida, Eudrilus eugeniae, Lampito
mauritii, Perionyx excavatus, P. sansibaricus and P. ceylanensis are being used for
vermicomposting [3, 4, 5, 7]. Vermicompost has been shown to influence the growth and
productivity of a variety of plants, cereals and legumes, vegetables, ornamental,
flowering plants and field crops [8, 9, 10, 11]. Studies on the efficiency of E. eugeniae
and L. mauritii in vermicomposting different organic substrates including press mud
are available. But the culturing of the earthworm species, P. ceylanensis in press mud
and the characteristics of press mud-vermicasts is not available. Hence the present
study has been carried out to compare the efficiency of P. ceylanensis with that of L.
mauritii and E. eugeniae in vermicomposting of press mud, physico-chemical
characteristics, microbial and enzymatic activities in press mud-vermicasts.
Materials and Methods
Vermicomposting studies were carried out with press mud substrate using the
earthworms, Eudrilus eugeniae, Lampito mauritii and Perionyx ceylanensis. The
Comparative Studies on Physico-Chemical
77
vermicomposting trials were performed in controlled environmental condition where
the relative humidity and the temperature were 75-85 percent and 27±1°C
respectively. The vermibeds were prepared in plastic containers with six replicates
for each experiment. Each container was fed with 250 g of press mud and 10
earthworms. For each experiment, a control set was maintained without earthworms.
After 30 days of the start of the experiment, the vermicasts from each bed was
carefully brushed aside and collected in separate containers. The vermicasts of three
different earthworm species and the worm-unworked substrates (WUP, control set)
were analysed for various physico-chemical parameters such as pH, electrical
conductivity (E.C.), organic carbon (O.C.) and total NPK, using standard procedures
[12, 13, 14]
. The C/N and C/P ratios were also found out for the worm-worked and for the
worm-unworked vermibed substrates. The percentage increase/decrease of various
physico-chemical parameters over the worm-unworked substrates was also calculated.
The percentage of increase / decrease of physico-chemical parameters in the wormworked over the worm-unworked substrates were subjected to student ‘t’ test using
the Computer Software (Microcal Origin, Version 3.1).
The total colony forming units (CFU) of bacteria, fungi and actinomycetes in the
vermibed materials at the beginning of the experiment (initial) and at the end of the
experiment (vermicasts and worm-unworked substrate i.e., control) were enumerated
using standard plate count method [15]. One gram of each sample was taken in a sterile
conical flask containing nine ml of distilled water and shaken in a vortex mixer for 30
minutes. From this stock, various dilutions were prepared from 10-1 to 10-7 with
sterile distilled water. One ml of the diluted sample was poured into petriplates
containing nutrient agar, Martin’s Rose Bengal agar and Kenknight’s media for
bacteria, fungi and actinomycetes respectively. Three replicates were maintained for
each observation. The initial microbial CFU and the final microbial CFU (wormworked and worm-unworked) in the vermibed substrates were subjected to one-way
ANOVA using the Computer Software, Microcal Origin (Version 3.1). Standard
methods were adopted for measuring the activity of the enzymes, amylase, cellulase,
invertase, protease and phosphates [16, 17, 18].
Results and Discussion
The physico-chemical characteristics of vermicasts produced by E. eugeniae showed
an increase in electrical conductivity, total nitrogen, total phosphorus and total
potassium where as organic carbon, C/N and C/P ratio showed reduction. The
electrical conductivity in WUP was 1.11±0.10 dS/m and the vermicasts produced
from the same substrate worked by E. eugeniae (VCE) showed 2.23±0.12 dS/m which
was 50.22% increase over WUP. C/N ratio was reduced up to 16.01 in VCE (93.18%
decrease). E.C., total nitrogen, phosphorus and potassium showed significantly
higher percentage increase over WUP (Table 1).
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M. Jayakumar et al
Table 1: Physico-chemical characteristics of the worm un-worked press mud (WUP)
and vermicasts of E. eugeniae (VCE).
Sl. No.
Parameters
1.
2.
3.
4.
5.
6.
7.
8.
pH
E.C. (dS/m)
O.C. (%)
N (%)
P (%)
K (%)
C/N
C/P
Press mud
WUP
VCE
7.13±0.03
6.90±0.03
1.11±0.10
2.23±0.12
46.45±0.40
32.19±0.52
1.19±0.10
2.01±0.07
1.86±0.07
2.49±0.04
0.66±0.02
0.95±0.02
39.03±1.05
16.01±0.44
24.97±1.28
12.93±0.77
% increase /
decrease(−)
-3.33NS
50.22***
-44.30**
40.80**
25.30*
30.53**
-143.73***
-93.18***
*, **, *** and NS indicates statistically significant difference at p<0.05, p<0.01,
p<0.001 and not significant by ‘t’ test.
Table 2: Physico-chemical characteristics of the worm un-worked press mud (WUP)
and vermicasts of L. mauritii (VCL).
Sl. No.
Parameters
1.
2.
3.
4.
5.
6.
7.
8.
pH
E.C. (dS/m)
O.C. (%)
N2 (%)
P (%)
K (%)
C/N
C/P
Press mud
WUP
VCL
7.13±0.03
7.06±0.10
1.11±0.10
1.43±0.08
46.45±0.40
36.29±0.70
1.19±0.10
1.87±0.14
1.86±0.07
2.20±0.02
0.66±0.02
0.82±0.02
33.03±1.05
19.41±1.78
24.97±1.28
16.50±0.87
% increase /
decrease(−)
-0.99NS
22.38*
-28.00*
36.36**
15.45*
19.51*
-101.14***
-51.39***
*, **, *** and NS indicates statistically significant difference at p<0.05, p<0.01,
p<0.001 and not significant by ‘t’ test.
Table 3: Physico-chemical characteristics of the worm un-worked press mud (WUP)
and vermicasts of P. ceylanensis (VCP).
Sl. No.
Parameters
1.
2.
3.
4.
5.
6.
7.
8.
pH
E.C. (dS/m)
O.C. (%)
N2 (%)
P (%)
K (%)
C/N
C/P
Press mud
WUP
VCP
7.13±0.03
7.02±0.02
1.11±0.10
1.96±0.02
46.45±0.40
35.17±0.40
1.19±0.10
1.95±0.04
1.86±0.07
2.24±0.13
0.66±0.02
1.15±0.10
39.03±1.05
18.04±0.04
24.97±1.28
15.70±1.16
% increase /
decrease(−)
-1.57NS
43.37**
-32.07*
38.97**
16.96*
42.61**
-116.42***
-59.06**
*, **, *** and NS indicates statistically significant difference at p<0.05, p<0.01,
p<0.001 and not significant by ‘t’ test.
Comparative Studies on Physico-Chemical
79
50
45
2.5
40
35
30
2.0
1.5
25
20
15
10
1.0
0.5
O.C.
5
0
Total Nitrogen (%)
Organic Carbon (%)
The same trend has been observed in the vermicasts of L. mauritii (VCL) and P.
ceylanensis (VCP) (Tables 2 and 3). C/N ratio is a good index of the maturity of the
organic substrates and it also indicates the quality of the organic manure. Among the
three species of earthworms, E. eugeniae recorded maximum percentage decrease of
C/N, (144.73) followed by P. ceylanensis and L .mauritii respectively.
The changes in total organic carbon and total nitrogen was found to be inversely
proportional where total nitrogen showed increase and organic carbon showed
decrease (Figure 1). Next to C/N ratio, C/P ratio showed highest percentage increase
in the vermicasts of E. eugeniae, L. mauritii and P. ceylanensis (Figure 2). The
percentage increase/decrease of physico-chemical characteristics of vermicasts over
WUP are represented in Figures 3, 4 and 5.
The total microbial populations in the vermicasts were higher than the WUP and
initial levels (Table 4). An initial population of 108 CFU x 107 g-1 was observed in
press mud where it showed an increased level of 123.42 CFU x 107 g-1 and of 202.10
CFU x 107 g-1 in WUP and in VCE respectively. Initial bacterial population level did
not differ significantly with WUP at 5% level where as the increase in bacterial
population differed (p<0.05) significantly from WUP and initial levels in the
vermicasts of E. eugeniae, L. mauritii and P. ceylanensis. The fungal population in
the vermicasts of E. eugeniae, L. mauritii and P. ceylanensis significantly differed
from initial levels and from WUP except the casts of E. eugeniae. The actinomycetes
population showed the similar trend as that of bacterial population.
Nitrogen
0.0
WUP
VCE
VCL
VCP
Figure 1: Total organic carbon and total nitrogen in vermicasts of earthworms, E.
eugeniae (VCE), L. mauritii (VCL) and P. ceylanensis (VCP) and in worm-unworked
press mud (WUP).
45
C/N
40
C/P
35
Ratio
30
25
20
15
10
5
0
WUP
VCE
VCL
VCP
Figure 2: C/N ratio and C/P ratio in the vermicasts of earthworms, E. eugeniae
(VCE), L. mauritii (VCL) and P. ceylanensis (VCP) and in worm-unworked press
mud (WUP).
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M. Jayakumar et al
60
Percentage increase/decrease(-)
40
20
0
-20
-40
-60
-80
-100
-120
-140
-160
pH
E.C. O.C. (%)
(dS/m)
N2 (%)
P (%)
K (%)
C/N
C/P
Physico-chemical parameters
Figure 3: Percentage increase/decrease of physico-chemical parameters in the
vermicasts of earthworm, E. eugeniae (VCE) over worm-unworked press mud
(WUP).
Percentage increase/decrease(-)
40
20
0
-20
-40
-60
-80
-100
-120
pH
E.C. O.C. (%) N2 (%)
(dS/m)
P (%)
K (%)
C/N
C/P
Physico-chemical parameters
Figure 4: Percentage increase/decrease of physico-chemical parameters in the
vermicasts of earthworm, L. mauritii (VCL) over worm-unworked press mud (WUP).
Percentage increase/decrease(-)
60
40
20
0
-20
-40
-60
-80
-100
-120
pH
E.C. O.C. (%) N2 (%)
(dS/m)
P (%)
K (%)
C/N
C/P
Physico-chemical parameters
Figure 5: Percentage increase/decrease of physico-chemical parameters in the
vermicasts of earthworm, P. ceylanensis (VCP) over worm-unworked press mud
(WUP).
Comparative Studies on Physico-Chemical
81
Table 4: Colony forming units (CFU) of bacteria, fungi and actinomycetes in the
initial vermibed substrate (INI), worm un-worked (WUP) and vermicasts of E.
eugeniae (VCE), L. mauritii (VCL) and P. ceylanensis (VCP).
Vermicasts of
earthworm species
analysed
E. eugeniae (VCE)
L. mauritii (VCL)
P. ceylanensis (VCP)
Bacteria x 107 g-1
INI
WUP
a
108.30
108.30 a
108.30 a
a, b
123.42
123.42 a, b
123.42 a, b
Fungi x 10 3 g-1
Vermicast
c
202.10
238.40 c
268.62 c
INI
WUP
a
98.25
98.25 a
98.25 a
Vermicast
b
159.64
159.64 b
159.64 b
182.23 b
196.27 c
223.39 c
Actinomycetes x 104 g-1
INI
WUP
Vermicast
64.27 a
86.90 a, b
128.77 c
a
a, b
64.27
86.90
135.64 c
a
a, b
86.90
141.09 c
64.27
The difference in mean values between the columns followed by the same letter
are not significant from each other at 5% level (ANOVA, p<0.05).
The enzymes, amylase, cellulase, invertase, phosphatase and protease in VCE,
VCL and VCP showed higher activity than WUP (Table 5). The vermicasts of E.
eugeniae showed maximum activity of all the enzymes analysed in their casts than
WUP where it recorded statistically (ANOVA) significant increase in the enzyme
activities over WUP. VCL also showed increased enzyme activity in their casts, but
they did not show significant difference (p<0.05), except protease (VCP). Excluding
amylase and protease activities, VCP showed significant increase in enzyme
activities.
Table 5: Enzyme activities in the vermicasts of the earthworms, E. eugeniae, L.
mauritii and P. ceylanensis (WUP-Worm Unworked Pressmud; VC-Vermicast).
Earthworm
species
E. eugeniae
L. mauritii
P. ceylanensis
Amylase
WUP
VC
a
2.96
6.11 b
a
2.96
4.80 a
a
2.96
4.01 a
Cellulase
WUP
VC
a
2.23
7.68 b
a
2.23
3.57 a
a
2.23
6.92 b
Invertase
WUP
VC
a
3.46
10.28 b
a
3.46
6.13 a
a
3.46
8.25 b
Phosphatase
WUP
VC
a
1.21
6.16 b
a
1.21
3.32 a
a
1.21
5.33 b
Protease
WUP
VC
a
4.32
12.80 b
a
4.32
8.36 b
a
4.32
7.25 a
(Activities of amylase, cellulose and invertase are expressed as mg of glucose/g of
oven dry samples for 24 hr of incubation; Protease activity: mg of glutamic acid/g of
oven dry substrates for 24 hr incubation; Phosphatase activity: mg/phenol/g of oven
dry substrates for 24 hr of incubation; Mean values followed by same letters are not
significantly different from each other at 5% level (ANOVA, p<0.05)
Vermicasts are highly valuable organic manure hold micro and macronutrients,
microorganisms and a variety of enzymes. In the present study vermicasts collected
from three different earthworm species, E. eugeniae, L. mauritii and P. ceylanensis
showed increased nutrients, microbial population and enzyme activities. The compost
characteristics of vermicasts, such as pH, electrical conductivity, organic carbon,
NPK, C/N and C/P ratios showed significant variation with WUP. The physico-
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M. Jayakumar et al
chemical characteristics of vermicasts and worm-unworked composts showed notable
changes in their composition. The pH values showed a slight reduction in the wormworked vermicompost when compared to the worm-unworked compost but the
difference in pH values between WUM and vermicasts was not significant. The
reduction in pH in the final products could also have been due to the production of
CO2 and organic acids by microbial activity during the process of bioconversion of
the different substrates in the beds [19]. A decrease in pH is an important factor in
nitrogen retention, as this element is lost as volatile ammonia at high pH values [20].
Reduction in pH towards neutrality is an important factor in retaining nitrogen, for it
seems to promote the nutrient availability for plants [21]. The vermicomposting
studies carried out with various organic materials using E. eugeniae and E. fetida also
gave similar results [22, 23, 24].
The electrical conductivity has shown significant increase in the vermicasts over
the worm un-worked composts. This shows that during vermicomposting process, the
soluble salt level increases due to the mineralization activity of earthworms and
microorganisms in the organic substance and as well in the gut of earthworms. The
vermicasts of earthworms, E. eugeniae, L. mauritii and P. ceylanensis showed highly
significant reduction of organic carbon, C/N and C/P ratio than in the worm unworked composts. Because of the combined action of microorganisms and the
earthworms, a large fraction of the organic matter in the initial substrates was lost as
The lowering of C/N ratio during
CO2 by the end of the vermicomposting period.
vermicomposting is achieved by the combustion of carbon substrates during
respiration. The nutrient level of vermicompost depends on the nature of the organic
waste used as food source for earthworms [25].
It has been reported that mineral aggregates are more stable in the presence of
organic particles [26]. Deficiency in organic carbon reduces the storage capacity of
soil for nitrogen, sulphur and phosphorus and leads to atmospheric activity and
reduction in soil fertility. The C/N and C/P ratio is of importance since plants cannot
assimilate mineral nitrogen and phosphorus unless the ratio is between 20:1 and 15:1
or lower [27]. Increased level of P during vermicomposting is due to earthworm gut
derived phosphatase activity and also increased microbial activity in the cast. The rise
in the level of P content during vermicomposting is probably due to mineralization
and mobilization of P due to bacterial and faecal phosphatase activity of earthworms
[28]
. The increased level of macro and micronutrients in the vermicomposts were in
conformity with the results of earlier works [3, 4, 5, 23, 24].
Studies on the microbial colony forming units have established the superiority of
the vermicast over the worm-unworked compost by their presence in higher counts
indicating their symbiotic association with the earthworms, which is essential for the
biodegradation of organic wastes. The inter-relationship of microorganisms with
macroorganisms by their presence inside or outside the body of macroorganism and in
their environment has been well established [29, 30].
In the present study, amylase, cellulase, invertase, phosphatase and protease in
VCE, VCL and VCP showed enhanced activities of all the enzymes studied with
respect to the action of individual earthworm species. Earthworms are important in
turnover of soil, contribute most to soil invertebrate biomass, contain the highest
Comparative Studies on Physico-Chemical
83
international units of cellulase and are believed to be capable of producing cellulases
and chitinases other than through intestinal microflora. Amylase activities may also
be increased by the addition of organic materials. The enhanced level of amylase
activity upon addition to the organic amendments promoted the recycling of nutrients
in the soil ecosystem [31]. Because of the feeding activity of the earthworms on press
mud, there occurred variation in the changes and activities of enzymes. Especially in
case of E. eugeniae, its voracious feeding habit and gut transit time, it showed
maximum activity of the enzymes investigated in the present study.
In conclusion, press mud, a wealthy organic resource may serve as good feeding
material for the earthworms, E. eugeniae, L. mauritii and P. ceylanensis. Comparing
the physico-chemical, microbiological and enzymatic activities in the vermicasts of
three species of earthworms studied, E. eugeniae showed very good results followed
by P. ceylanensis and L. mauritii. As observed from the results of the present study,
by feeding the earthworms with press mud, nutritionally and microbiologically rich
vermicasts with good enzymatic activities can be produced which can supplement the
nutrients to the soil and help maintaining fertility status.
Acknowledgements
The authors sincerely acknowledge Dr. J. M. Julka, Solan, Himachal Pradesh for the
identification of the earthworm, Perionyx ceylanensis.
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