Feeding Biology of Earthworms (Perionyx Excavatus and Eudrilus Eugeniae) and Bacteria Associated with their Guts and Vermicompost

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Feeding Biology of Earthworms (Perionyx Excavatus and Eudrilus Eugeniae) and Bacteria Associated
with their Guts and Vermicompost
J Microbiol Biotechnol
Feeding Biology of Earthworms (Perionyx Excavatus and
Eudrilus Eugeniae) and Bacteria Associated with their Guts and
Vermicompost
Singh NI* and Devi KB
Centre of Advanced Study in Life Sciences, Manipur (Central) University, Imphal, India
*Corresponding author: Prof. N Irabanta Singh, Nibiaa Devices, 4th Floor, L.I. Shopping
Complex, Singjamei Bazar Imphal- 795008, Imphal, India, Email: irabanta.singh@gmail.com
Research Article
Volume 9 Issue 3
Received Date: August 05, 2024
Published Date: August 29, 2024
DOI: 10.23880/oajmb-16000304
Abstract
The feeding behaviours of Perionyx excavatus and Eudrilus eugeniae were carried out. P. excavatus and E. eugeniae took
an average of 3h. and 3h. 30 mins respectively to fill their guts. So, in P. excavatus 8(eight) gut fillings could occur in a day
whereas in E. eugeniae 7(seven) gut fillings could occur in a day. Analysis of gut contents and vermicompost bacteria revealed
the presence of Bacillus sp. (A), Bacillus sp. (B) and Klebsiella sp. Micrococcus sp. was isolated only from the gut of Perionyx
excavatus. However, it was absent in the vermicompost. Further, Proteus sp. was isolated only from the gut of Eudrilus eugeniae
but it was absent in the vermicompost. Further, Proteus sp. was isolated only from the gut of Eudrilus eugeniae but it was
absent in the vermicompost. The study emphasizes importance of the rate of organic waste materials movement through the
intestine of the worm and time taken during passing through the intestine and microbial changes in the worm casts ageing in
the plastic tub.
Keywords: Feeding Biology; Perionyx excavatus; Eudrilus Eugeniae; Gut Contents; Bacteria; Vermicomposts
Introduction
An understanding of the feeding biology and digestive
capability of earthworms is essential in their role in the
complex decomposition process. It has also been known
that environmental factors greatly influence the various life
activities of many earthworms [1,2]. The epigeic (Surface-
dwelling) species Eisenia foetida, Eudrilus eugeniae and
Perionyx excavatus had gained popularity throughout the
world for their potentials as decomposer subsystem and
because of their importance various studies have been made
on their different biological aspects.
The growth and reproduction of Eisenia foetida and
Eudrilus eugeniae are greatly influenced by seasonal
environmental factors [3-9]. Further, the growth and cocoon
production pattern of Perionyx excavatus and Eudrilus
eugeniae have also been studied [10,11]. The temperature
tolerance of Eisenia foetida, Eudrilus eugeniae and Perionyx
excavatus has been investigated and found that Eudrilus
eugeniae and Perionyx excavatus could be utilized in
vermiculture [12]. The life cycles of Eudrilus eugeniae and
Perionyx excavatus have been well documented and both
the species were proved to be potential vermicomposting
agents [12-16]. Further, the growth and reproduction of

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Singh NI and Devi KB. Feeding Biology of Earthworms (Perionyx Excavatus and Eudrilus Eugeniae) and
Bacteria Associated with their Guts and Vermicompost. J Microbiol Biotechnol 2024, 9(3): 000304.
Copyright© Singh NI and Devi KB.
Eisenia foetida is also influenced by temperature [17,18].
The digestive system of earthworms consists of a tubular
alimentary canal extending from mouth to anus. The system
can be differentiated as buccal chamber/cavity, pharynx,
oesophagus, crop, gizzard, intestine and rectum. Feeding
process of earthworms is subjected to the enzymatic actions
within the gut. The capacity of mixing and turning over of
substrate by earthworms varies with species to species. Data
on substrate turnover for Eisenia foetida and Drawida calebi
are available [19,20].
Earthwormsgrazeoverawidevarietyoforganicmatters.
Influence of feeding pattern on growth and reproduction of
Eisenia foetida is known [21,22]. Preference for certain food
materials by earthworms have also been reported [23-28].
The effects of different organic wastes on vermiculture and
vermicomposting has also been known [29-31]. Earthworms
affect soil microfloral and faunal population directly or
indirectly like the joint action of earthworms and microflora
in forest soils [32]. The effects of earthworms on soil
microflora leading to changes in soil properties, processes,
micro floral and faunal community structure and plant and
soil health have been known [33]. The earthworms also graze
over soil microflora and that they have requisite enzymes to
degrade complex plant substances [24,34]. Selective grazing
by earthworms may reduce the number of some organisms
and increase others that could be beneficial [35].
The role of earthworms in the dispersal of soil
organisms had been known [36,37]. The bacterial feeding
by earthworms [38-40]. The gut of earthworm constitutes
a mobile anoxic microzone to which the microorganisms
of aerated soils are subjected [41]. It provides an optimum
incubation temperature, near neutral pH and abundant
soluble organic carbon creating an ideal condition for high
microbial activity. The cast in turn contains high assimilable
carbon, favourable water regime and higher concentrations
of many nutrients [42] providing an excellent condition
for microfloral and faunal proliferation. The intestine of
earthworms is rich in microorganisms [43].
Our previous study revealed 4 bacteria types 2 Actino-
mycetes types, 15 fungal types, 1-protozoa group, and 2
Nematodes types. For these 2 (two) types of worms, the
richest microorganism were fungal types. 15 fungal types
were isolated from the previous study [44]. The changes
in the microbial status of earthworm cast varied based on
their age [45]. The microbial composition of vermicompost
are derived from various organic sources [46,47]. A detailed
microbe of the gut of Eudrilus eugeniae and Perionyx
excavatus have also been made [48,49]. The effects of gut
passage, the age of cast material and the type of ingested
substrate on microorganisms in Lumbricus terrestris faces
are known [50]. The rate that material moves through the
intestine depends on whether the worm is feeding, food
takes about 20 hours to pass, but when burrows are being
formed materials passes in about 12 hours [43]. Further,
changes in the microbial status of the casts are noticed as
they age under field condition [45]. The present study deals
with organic wastes feeding behaviours of Perionyx excavatus
and Eudrilus eugeniae in regards to their ingestion, gut load,
transit time and egestion. And to examine further bacteria
isolated from the gut content and vermicompost (Vermicast)
produced by the worms.
Materials and Methods
Study on the Feeding Biology of Earthworms
(Perionyx Excavatus and Eudrilus Eugeniae)
Experimental rearing of two earthworms (Perionyx
excavatus and Eudrilus eugeniae) (Figures 1A & 1B) were
conducted in the Life Sciences Department, Manipur
University for a period of three years.
Figure 1A: A view of Perionyx excavatus. Figure 1B: A view of Eudrilus Eugeniae.

3
The organic wastes like cowdung, Kitchen wastes, Litter,
and agricultural wastes were mixed prior to vermicosting
(Figure 2A). For studies on the feeding biology of Perionyx
excavatus and Eudrilus eugeniae both species were reared
separately in plastic tubs (30 cm diameter and 8 cm height)
in vermicomposting shed under natural conditions (Figure
2B). Observations on the feeding behaviours like ingestion,
gut load, transit time and egestion were made (Figure 2C).
Figure 2A: Organic wastes prior to vermicomposting.
Figure 2B: Culture tubs containing cowdung and different
organic wastes.
Figure 2C: A view of harvested vermicompost.
Rate of Gut Loading and Transit Time
Five (5) worms each of Perionyx excavatus and Eudrilus
eugeniae were randomly collected from the respective culture
tubs and brought to be laboratory. The gut of each individual
worm was cleared by keeping it in half covered Petri dishes
containing distilled water. To study the rate of gut loading and
transittimeofthefeedmaterials,onegutclearedwormofeach
species were introduced to each culture tub containing 1kg of
air dried, powdered and sieved (pore size: 2mm) cowdung.
Two sets of experiments (one with Perionyx excavatus and
the other with Eudrilus eugeniae) each with three replications
were maintained. Worms were taken out at hourly intervals
from the three replications of a set and their guts were cleared.
The dry mass of the gut content was measured. The time
periods at which the worms started casting were also noted.
Ingestion, Egestion and Assimilation
Five (5) earthworms of each species were introduced
into plastic tubs (30x8cm) separately containing 1kg of air
dried, powdered and sieved cowdung. Additional powdered
cowdung was also spread thinly and uniformly in the tub.
The surface of the cowdung was carefully smoothened so
that the freshly laid cast was easily distinguished. The Cast
were collected at 24hrs interval for a week and the dry
mass of each sample was determined. Further, percentage
egestion and assimilation of the ingested materials were also
calculated on weekly basis. The intestinal loading rate and
transit time of cowdung manure of two worms were applied
following [51,52].
Isolation and Identification of Bacteria
associated with the Gut of Earthworms and
Vermicompost
Earthworm Gut Bacteria: Mature worms of Perionyx
excavatus and Eudrilus eugeniae were collected separately
in sterile containers from the vermicomposting plastic tubs
and brought to the laboratory for investigation of the gut
microflora. Five (5) adult worms each of Perionyx excavatus
and Eudrilus eugeniae were collected separately in sterile
containers at random from the culture tubs. Representative
worms were fixed in 70% alcohol for 10 mins and then
washed repeatedly with sterilized distilled water. Each worm
was cut into 3 pieces: anterior, middle and posterior with
sterilized scissors. The gut contents of the different regions
(anterior, middle and posterior) were collected in sterilized
petri dishes, containing 2 ml of sterilized water. Gut contents
of each region of individual worm were inoculated into petri
dishes containing nutrient agar for bacteria in an inverted
position at 30±1o
C. Colonies so developed were counted for
every 24 hrs interval. Morphologically dissimilar colonies
were separated and subcultured pure were obtained [44].

4
Vermicompost Bacteria
Vermicompost obtained from feed mixture of cowdung
+ kitchen wastes + litters + paddy waste in 1:1:1:1 ratio was
collected in sterilized Petri dishes using sterilized spatula
and brought to the laboratory for investigations of bacterial
(Figure 2B). Five (5) samples each of l gram vermicompost
was randomly collected from the vermicomposting tubs.
Each sample was mixed with 5ml of sterilised distilled water
separately and later subjected to serial dilution up to 10-6
dilutions. For the isolation of bacterial components, 1 ml each
of the suspension was inoculated in nutrient agar plates (in
triplicate) and incubated in an inverted position at 30±1°C.
Bacterial colonies so developed were counted for every 24h
interval. Suitable controls were maintained. Morphologically
dissimilar colonies were separated and transferred as
subcultures. Pure cultures were obtained following [53].
Results and Discussion
Different regions and number of segments found in two
species of earthworms are provided (Table 1A).
Regions and Segments Eudrilus Eugeniae (cm) Perionyx Excavatus (cm)
Contracted size 7 9
Length of anterior region 2 2.5
Length of middle region 2 2.5
Length of posterior region 3 4
No. of segments in anterior region 3 40
No. of segments in middle region 30 40
No. of segments in Posterior region 40 50
Table 1A: Size and Length (in cm) of different regions and number of segments found in two species of earthworms.
A comparative account of various biological aspect of Perionyx excavatus and Eudrilus eugeniae are provided (Table 1B).
Biological Characteristics Eudrilus Eugeniae Perionyx Excavatus
Duration of life cycle (days) ± 60 ± 46
Growth rate (mg worm-1
day-1
) 12 3.5
Max body mass (mg) 4294 600
Maturation obtained at age (days) ± 40 ± 21
Start of cocoon production (days) ± 46 ± 24
Coccon production (worm-1
day-1
) 1.3 1.1
Incubation period (days) ± 16.6 ± 18.7
No. of hatchings from one worm 1-5 1-3
Table 1B: A Comparative account of various biological aspects of Perionyx excavatus and Eudrilus eugeniae
Source: Gunathilagraj, K. and Sahaya Alfred, cited by Devi KB [49].
Gut Load and Transit Time
Transit time and rate of gut loading of cow dung and
other organic wastes by the two earthworms varied with
each other. Perionyx excavatus and Eudrilus eugeniae could
consume 9.7 mg (dry mass) and 20.96 mg (dry mass) of
cowdung respectively in one hour and 22.46 mg (dry mass)
and 47.32 mg (dry mass) respectively in two hours. In three
hours, Perionyx excavatus showed casting activity while
Eudrilus eugeniae continued gut loading. By around three
and half hours Eudrilus eugeniae started casting (Table 2A).

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Duration
Earthworm Species
Perionyx Excavatus Eudrilus Eugeniae
1h 9.7 ± 0.36 20.96 ± 0.52
2h 22.46 ± 0.48 47.32 ± 0.64
2h and 30 min Gut loading continued Gut loading continued
3h Casting Gut loading continued
3h and 30 min – Casting
Table 2A: Transit time and rate of Gut loading of cowdung (in mg dry mass) for two earthworm species.
Hence, in one day 8(eight) gut fillings could take place in Perionyx excavatus while 7(seven) gut fillings could take place in
Eudrilus eugeniae.
Ingestion, Egestion and Assimilation
In the experimental tubs with Perionyx excavatus, the
worms consumed about 98% of the cowdung and other
organic wastes in a week but 69.44% of the ingested material
was excreted while in the tubs with Eudrilus eugeniae, 95%
was ingested in a week and 60.58% of the ingested material
was excreted. Percentage assimilation of ingested material
per week was higher in Eudrilus eugeniae than Perionyx
excavatus by recording a value of 39.42% and 30.56%
respectively. The ingestion, egestion and assimilation rate is
provided (Table 2B).
Biological Activities
Earthworm Species
Perionyx Excavatus Eudrilus Eugeniae
Food ingested % week-1
98 95
Food egestion % ingested week-1
69.44 ± 0.86 60.58 ± 0.85
Assimilation % ingested week-1
30.56 ± 0.86 39.42 ± 0.85
Table 2B: Ingestion, Egestion and Metabolic rate of Perionyx excavatus and Eudrilus eugeniae in cowdung.
Bacteria associated with the Gut of Earthworms
and Vermicompost
The investigations showed a number of bacterial
colonies being developed on nutrient agar plates (Figure
3A). The bacterial colonies so developed were isolated and
identified. The results of the various tests conducted were
recorded and compared with the key to taxonomic bacteria
from standard literatures [54,55]. The bacterial types
isolated from the gut of Perionyx excavatus and Eudrilus
eugeniae and vermicompost (Figure 2C) are also presented
(Table 3). Altogether four genera of bacteria were isolated
and identified. Two species of Bacillus (Figures 3B & 3C) and
one species of Klebsiella were isolated from both Perionyx
excavatus and Eudrilus eugeniae. However, Micrococcus sp.
was isolated from Perionyx excavatus alone and Proteus sp.
was also isolated from Eudrilus eugeniae alone.
1. Bacillus sp. [A]: Rod shaped, single, short/long chain,
Gram +ve, Acid Fast – Not tested, spore +ve, Capsule/
Cyst – Not tested, Flagella – Not tested, and Motality +ve.
(Figure 3B)
2. Bacillus sp. [B]: Rod, single or chain (2-3μmL), spore
cylindrical, Gram +ve, Acid Fast – Not tested, spore +ve,
Capsule/Cyst – Not tested, Flagella – Not tested, and
Motality +ve. (Figure 3C)
3. Klebseilla sp.: Straight rod, upto 5.0μmL, singly, rarely
chain, Gram -ve, Acid Fast -ve, spore -ve, Capsule /Cyst
+ve, Flagella -ve, and Motality -ve.
4. Micrococcus sp.: Spherical, single, pair & in clusters,
Gram +ve, Acid Fast -ve, spore -ve, Capsule/Cyst -ve,
Flagella = Not tested, and Motality -ve.
5. Proteus sp.: Straight, rod about 3μmL pair or chain, Gram
-ve, Acid Fast -ve, spore -ve, Capsule/Cyst -ve, Flagella
+ve, and Motality +ve.

6
Figure 3A: Bacterial colonies developed on Nutrients agar
medium.
Figure 3B: Bacillus sp.
Figure 3C: Bacillus sp.
Different region and number of segments found in two species of earthworm is provided (Table 3).
Types of Becteria
Gut Content Vermicompost
Eudrilus Eugeniae Perionyx Excavatus Eudrilus Eugeniae Perionyx Excavatus
Bacillus sp. A Isolated Isolated Isolated Isolated
Bacillus sp. B Isolated Isolated Isolated Isolated
Klebsiella sp. Isolated Isolated Isolated Isolated
Micrococcus sp. Not Isolated Isolated Not Isolated Not Isolated
Proteus sp. Isolated Not Isolated Not Isolated Not Isolated
Table 3: Bacteria isolated from the gut content and vermicompost of Eudrilus eugeniae and Perionyx excavatus.

7
From the vermicompost (Figure 2C) produced by using
Perionyx excavatus and Eudrilus eugeniae, one species each
of Bacillus and Klebsiella could be isolated as the common
bacterial flora. However, Bacillus sp. (B), Micrococcus Sp. and
Proteus sp. could not be isolated from the vermicompost
produced by using both the earthworms. In the present
study, both Perionyx excavatus and Eudrilus eugeniae grow
well and actively performed their various feeding and casting
activities in cowdung and other organic wastes. In fact,
cowdung can be considered as the most suitable medium for
rearing of these earthworm species. These findings agreed
with the previous findings [4] for Eudrilus eugeniae and
[56] for Perionyx excavatus. Transit time and the rate of gut
loading of Perionyx excavatus showed similar results with
Octochaetona surensis [57] and that of Eudrilus eugeniae with
Allobophora rosea [58] and L. mauritti [57]. The results of the
ingestion, egestion and assimilation rates agreed previous
workers [56]. In nature, it is observed that organisms
ingesting smaller amount of cellulose and other plant
materials exhibit higher assimilation efficiency, whereas
organisms which have a lower assimilation capability always
ingest larger amounts of food materials to meet their need.
Hence, the lower ingestion and higher assimilation efficiency
in Eudrilus eugeniae and higher ingestion and lower
assimilation efficiency in Perionyx excavatus confirmed the
above hypothesis.
Vermicomposting is one of the most efficient methods
for converting organic wastes into valuable plant nutrients
[59]. Eudrilus eugeniae and Perionyx excavatus are nowadays
widely used in vermicomposting as they are prolific
breeders and can adapt easily to various organic waste
materials [56,57]. Production of vermicompost depends on
the consumability of the worms and also on the nutritional
status and texture of organic wastes. The rate of breakdown
of waste depends on the types of litter used [27]. In the
present study, Perionyx excavatus showed better production
of vermicompost than Eudrilus eugeniae. These results
agreed with the previous findings [7,60].
Earlier study revealed influence of environmental
factors on survival and growth of Earthworms. P. excavatus
does not grow much at low temperatures although it can
survive them 4°C (39.2°F) but it is less susceptible to high
temperature over 30°C (86°F) than E. eugeniae. Even in
tropical areas, P. excavatus does not grow during low-
winter temperatures but can survive the high-summer
temperatures, whereas E. eugeniae has a much narrower
tolerance range for temperature and cannot survive either
the extreme low winter or the high summer temperatures
[61]. In the present study too, earthworms move to more
suitable areas in the culture tub if the environmental limits
are greatly exceeded. Bacteria in the gut flora increased
greatly in number [43] but the result suggest that changes
in the microbial population during passage through the
worm gut tended to be logarithmic, indicating that increases
were by bacteria growth and not by the worm selecting food
material with high bacteria count. The results of the present
investigation revealed the presence of a number of bacteria
in the gut of the earthworms. However, in the vermicompost,
some of the microbes present in the gut were not isolated.
Among the bacterial flora, one species of Bacillus and one
species of Klebsiella were isolated from the vermicompost.
The outcome of the present study shows the presence of
bacteria like Bacillus and Klebsielal in the vermicompost
indicating that they were not digested. These may be due to
the production of antibiotic and/or presence of strong outer
coat and/or production of phytotoxic metabolites. Both
Perionyx excavatus and Eudrilus eugeniae could thrive well
in organic waste materials and they can be used as potential
vermicomposting agents. Through their decomposing
activities, they help in the recycling of organic wastes.
Conflict of Interest
The authors have no financial conflict of interest to
declare.
Acknowledgement
TheworkwassupportedbyDepartmentofBiotechnology,
MinistryofScience&Technology,GovernmentofIndia.(Grant
No. BT/PR 4078/SPD/09/313/2003 Dt. 23/03/2004). The
funders had no role in study, design, data collection and
analysis, decision to publish. Thanks, are also due to Prof. P.K.
Bandyopadhyay of Zoology Department, Kalyani University,
Kalyani, (W.B.), India for confirming the Identification of the
Earthworms.
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