JRSSEM 2022, Vol. 01, No. 11, 2038 – 2049
E-ISSN: 2807 - 6311, P-ISSN: 2807 - 6494
DOI : 10.36418/jrssem.v1i11.232 https://jrssem.publikasiindonesia.id/index.php/jrssem/index
INTERACTION OF CULTURAL MEDIA COMPOSITION
WITH EM4 DOSAGE ON POPULATION GROWTH,
BIOMASS AND NUTRITIONAL CONTENT SILK WORM
(TUBIFEX SP)
Murni
1*
Herliwati
2
Noor Arida Fauzana
1,2,3
Universitas Lambung Mangkurat, Indonesia
e-mail: murnicastle@gmail.com
1
, herli.wat[email protected].id
2
,
noor.afauzana@ulm.ac.id
3
*Correspondence: murnicastle@gmail.com
Submitted: 10 June 2022, Revised: 15 June 2022, Accepted: 20 June 2022
Abstract. Culture media as a place for the maintenance of silk worms greatly affects the growth
and nutritional content of silk worms. The purpose of this study was to analyze the interaction
effect of the composition of culture media with a dose of EM4 on population growth, biomass
and nutritional content of silk worms (Tubifex sp). This study used a completely randomized
factorial design with 9 treatments and 3 replications each. Treatment D1M1, treatment D1M2,
treatment D1M3, treatment D2M1, treatment D2M2, treatment D2M3, treatment D3M1,
treatment D3M2 and treatment D3M3. Each treatment was kept in 27 trays. The resu its showed
that the composition of the media material and the dose of EM4 had a very significant effect (P<
0.05) on population growth and biomass and nutritional content of silk worms. The highest
average population and biomass growth was obtained in the D2M2 treatment, which was 27482.9
ind/m2 and 8.52 grams, and the best nutrient content was in the D3M3 treatment with a protein
content of 50.88. Based on the results of the study, it can be concluded that the combination of
media materials with a percentage of 50% azolla pinnata and 50% chicken manure can increase
the population, biomass and nutritional content of silk worms.
Keywords: tubifex SP; chicken manure; azolla pinnata; rejected bread; EM4 dose.
Murni, Herliwati, Noor Arida Fauzana
| 2039
INTRODUCTION
Cultivation activities, especially in hatchery
activities, require natural feed which is an
important supporting factor in the successful
growth of fish larvae and fry (Silva-Aciares et al.,
2013). Natural food is vegetable and animal
plankton that is in accordance with the
development of the digestive organs of larvae
and fish seeds, one of which is silk worms
(Tubifex sp) which are always moving and
colored like blood red which will attract larvae
and fish seeds to eat them. According to (T.-H.
Liu et al., 2022), silk worms have an important
role because they can stimulate fish growth
faster than other natural foods such as water
fleas (Dapnia sp. and Moina sp).
Culture media as a place for the
maintenance of silk worms greatly affects the
growth and nutritional content of silk worms.
The growth of silk worms is influenced by
bacteria and organic particles resulting from
bacterial overhaul as food for silk worms (Chen
et al., 2020). Bacteria need organic C and N
content in the culture media for maintenance
because by utilizing high-carbon and low-
nitrogen materials, they can produce cell
proteins so that silkworms use them in their
growth (Fujita et al., 2020). One method that is
often used is the fermentation process. The use
of probiotic activator EM4 can be used in the
fermentation process. (X. Liu et al., 2020) on the
implementation method of making silkworm
cultivation media using a dose of 1 ml EM4 with
200-250 ml of water and 25 ml of molasses
drops for 1 kg of raw materials. The purpose of
this study was to analyze the effect of the
interaction of the composition of culture media
with a dose of EM4 on population growth
(Pujiastuti et al., 2018), biomass and nutritional
content of silkworms
METHODS
This study used a Completely Randomized
Factorial Design (RALF) consisting of 9
treatments and 3 replications. In this study, the
treatment is the composition of the media
material with the use of EM4 doses, namely:
The first factor is the difference in the
composition of the material consisting of 3
levels of treatment, namely as follows:
Treatment M1 = Chicken manure 25% + Azolla
pinata 25% + Bread rejected 50%. Treatment M2
= Chicken manure 25% + Azolla pinata 50% +
Bread rejected 25%. Treatment M3 = Chicken
manure 50% + Azolla pinata 25% + Bread
rejected 25%.
The second factor is the difference in the
dose of probiotic EM4 in the composition of the
ingredients, as follows: Treatment D1 = Dose 1
ml/kg Treatment D2 = Dose 2 ml/kg Treatment
D3 = Dose 3 ml/kg Working Procedures.
Preparation Containers
Rearing silk worms using plastic trays of size
(25×20×10) cm with a multilevel shelf model.
The water channel uses a recirculation system
with a shelter using a tarpaulin which is made to
form a rectangle with a size of (100×50×25) cm.
Media Preparation
Mud material taken from the rice fields of
the Ulin River area was taken and sieved using a
strainer/sieve with a mesh size of 1 mm. Chicken
manure that will be used as a media material
comes from chicken farms (Islam et al., 2021).
Azolla pinnata which will be used as a media
material comes from aquatic plant cultivators.
The rejected bread that will be used as a media
2040 | Interaction of Cultural Media Composition with EM4 Dosage on Population Growth, Biomass and
Nutritional Content Silk Worm (Tubifex SP)
material comes from the seller of animal feed
ingredients (Kuts et al., 2021). The three
materials were dried in the sun to dry, then
crushed or kneaded by hand and filtered using
a 1 mm sieve to obtain a medium material in the
form of a fine powder.
Preparation of Fermentation Materials
Media materials Chicken manure, azolla
pinnata and rejected bread to be used were
weighed according to each treatment with the
total material for each treatment was 1 kg and
mixed with fine mud. The mixed media material
is given EM4 according to the dose that will be
used for the fermentation process (Li et al.,
2018). The use of EM4 as a fermentator is done
by mixing EM4 with 250 ml of water according
to the dose in the treatment, then mix it into the
media material to be used. Fermentation media
material with a predetermined dose is stored for
7 days in a closed container in the form of a jar.
Distribution of Silk
Worms Silkworms were first drained for
about 1 minute and weighed on an analytical
balance as much as 20 grams for each treatment
as the initial weight of silkworm biomass. The
maintenance of silk worms was carried out for
20 days. Re-fertilization was carried out after
each sampling with the fermentation of the
material used as much as 50 grams in each
treatment.
Sampling of silkworms was carried out once
every day. Silkworm sampling was carried out at
4 points in each treatment container. The
sampling process uses a inch pipe with a
diameter of 3 cm inserted into the substrate,
then removed while closing the top hole with
the palm of the hand. The taken substrate is
accommodated in a fine mesh scoop then the
substrate is washed in running water, then
spread over a transparent plastic cover. Then
the silk worms were separated using a pipette
and counted one by one. The formula for
population growth is based on (Ansyari and
Rifa'i, 2005):
Total
=
× RJIS
Description
LPP : Area of sample plots (m
2
) LLP :
Area of pipe circle (m
2
)
RJIS : Average number of individual
samples (Individual/m
2
)
Biomass weight of silkworms
Observation of biomass weight gain was
carried out by sampling method done once
every day. Measurement of the weight gain of
biomass by weighing using a digital scale from
the sampling results of each treatment.
Daily Length Growth of Silk Worms
Sampling was carried out once every day by
taking samples of 10 silkworms from the
observation of biomass weight and measured
using a digital ruler. The formula for finding
length growth according to Effendie (2002) is:
P = Pt – Po
Description:
P = Length Growth
Pt = Final Length Growth (mm) P0 = Initial
Length Growth (mm) Nutrient Content
Qualitative analysis consists of:
1. Nutrient content test Organic C, organic N
and C/N ratio of material media before and
after fermentation at the Soil Science
Laboratory, Faculty of Agriculture, ULM.
2. Proximate test to determine the
composition of protein, carbohydrates, fat
Murni, Herliwati, Noor Arida Fauzana
| 2041
and water content contained in silk worms
(Tubifex sp) after being cultured at the
Nutrition and Animal Feed Laboratory,
Faculty of Agriculture, ULM.
3. Test of Amino Acid Content of Silkworm
(Tubifex sp) after being cultured at SIG
Laboratory PT. Saraswanti Indo Genetech,
Bogor.
Water Quality
Physical and chemical parameters of water
measured include temperature, pH, dissolved
oxygen, and ammonia. The research data were
statistically analyzed using analysis of variance
to determine the effect of the treatment given
on the observed changes and if F count > F
table 5% or 1% then the calculation will
continue with further analysis with the
provisions of the Small KK BNJ test (KK < 5% ),
medium KK BNT test (5% < HH < 10%) and large
KK Duncan test (> 10%) (Hanafiah, 2013).
Meanwhile, for water quality data, noanalysis of
variance was carried out, but quantitative tests
were carried out and will be explained
descriptively.
RESULTS AND DISCUSSION
Nutrient Content of Silk Worm Material Media
The nutrient content of N, P, K and C/N ratio
in silk worm material media by conducting
laboratory tests on the media material. The
results of the laboratory test of the material
media can be seen in Table 1.
Table 1. Elemental Content of N, P, K and C/N Ratio
Treatment
Nutrient content
N
C
P
K
Ratio C/N
M1
1.23
9.18
0.67
0.74
7.46
M2
1.32
9.03
1.22
1.13
6.84
M3
1.28
8.14
1.46
0.92
6.35
D1M1
1.30
10.7
4
0.55
0.39
8.26
D1M2
0.83
4.45
0.34
0.45
5.36
D1M3
1.25
8.01
1.12
1, 09
6.40
D2M1
0.97
8.17
0.45
0.24
8.42
D2M2
1.10
5.41
0.36
0.33
4.91
D2M3
1.04
7.82
1.07
1.02
7 ,51
D3M1
1.08
8.25
0.59
0.36
7.63
D3M2
1.00
4.79
0.39
0.39
4.79
D3M3
1.23
9.03
1.55
1.23
7.34
2042 | Interaction of Cultural Media Composition with EM4 Dosage on Population Growth, Biomass and
Nutritional Content Silk Worm (Tubifex SP)
The C/N ratio in this study is the result of
analysis before and after the fermentation
process. The principle of fermentation for media
materials is to reduce the C/N ratio of organic
matter (Hastuti et al., 2022). This can be seen in
Table 1. which shows that before and after the
fermentation process the organic material has
completely decomposed or degraded so that
the material media can be used in the
maintenance of silk worms. During the
fermentation process, microorganisms utilize
only a small amount of phosphate and
potassium elements for their metabolic
activities. The increase in the growth of silk
worms in the culture material media plays a very
important role in the success of silk worm
cultivation. Likewise, the nutritional quality of
silk worms from cultivation is largely
determined by the material media that the silk
worms feed on to live during the maintenance
period (Sudarman & Velina, 2021).
The increase in the average growth of
biomass in the D2M2 treatment was 8.52 gr
which can be seen in Table 4.8. assumed to be
due to the presence of a higher number of
bacteria and organic particles, which became
food for silk worms. According to Anggraini
(2017), fermentation of different media
materials can affect the increase in organic
matter in the media, so that it can increase the
amount of food ingredients and subsequently
have an impact on the population and biomass
of silk worms.
In the D2M1 treatment where the growth in
length, population and biomass were found to
be low, it showed that the use of 25% chicken
manure + 25% pinnata azola + 50% rejected
bread caused the growth of weight and biomass
to be less than optimal. Allegedly this is because
the combination of media materials is quite
difficult to digest, besides the D2M1 treatment
contains a higher C/N ratio than other
treatments, so it takes longer time for silkworms
to digest.
Daily Length of Silk Worm
9.40
9.20
9.00
8.80
8.60
8.40
8.20
8.00
7.80
7.60
D1M1 D1M2 D1M3 D2M1 D2M2 D2M3 D3M1 D3M2 D3M3
Treatment
Figure 1. Graph of Average Daily Length of Silkworms
Length (mm)
Murni, Herliwati, Noor Arida Fauzana
| 2043
DOI : 10.36418/jrssem.v1i11.232 https://jrssem.publikasiindonesia.id/index.php/jrssem/index
The figure above shows the daily
length growth of different silkworms
during 20 days of rearing. The highest
daily length growth was obtained in the
combination treatment of 25% chicken
manure + azolla pinnata + 25% rejected
bread with a dose of EM4 of 2 ml (D2M2)
with an average length growth of 9.27
mm. While the lowest length growth
results were achieved in the combination
treatment of 25% chicken manure
+ azolla pinnata + 50% rejected bread
with a dose of EM4 of 2 ml (D2M1) with an
average length growth of 8.25 mm.
The results of analysis of variance in
daily length of silk worms for 20 days of
maintenance showed that the
combination of media material and dose
of EM4 obtained (P < 0.05) so the
treatment had a very significant effect. In
figure 4.1. It can be seen that the daily
length of silk worms ranged from 8.25-
9.27 mm, where observations on days 6-
10 the average length of silk worms
decreased because on that day silk worms
had already laid eggs and silkworms were
seen inhabiting colored substrates.
smaller redness than other silkworms.
Silkworm Population
30000
25000
20000
15000
10000
5000
0
D1M1 D1M2 D1M3 D2M1 D2M2 D2M3 D3M1 D3M2 D3M3
Treatment
Figure 2. The Average Silkworm Population
Graph Figure 2. The above shows the
total population of silk worms for all
treatments with a combination of different
media materials and EM4 doses used
during the study. The highest population
was achieved in the combination treatment
of 25% chicken manure + azolla pinnata +
25% rejected bread with a dose of EM4 as
much as 2 ml (D2M2), obtaining a
population average of 27428.9 ind/m
2
.
While the lowest population results were
achieved in the treatment of a combination
of 25% chicken manure + 25% azolla
pinnata + 50% rejected bread with a dose
of EM4 as much as 2 ml (D2M1), obtaining
a population average of 19863.1 ind/m
2
.
The results of the analysis of variance of the
silkworm population during 20 days of
rearing showed that the combination of
media material and EM4 dose obtained (P
< 0.05), the treatment had a very significant
effect.
Populasi
(ind/m2)
Murni, Herliwati, Noor Arida Fauzana
| 2044
DOI : 10.36418/jrssem.v1i11.232 https://jrssem.publikasiindonesia.id/index.php/jrssem/index
Silkworm biomass
9.00
8.00
7.00
6.00
5.00
4.00
3.00
2.00
1.00
0.00
D1M1 D1M2 D1M3 D2M1 D2M2 D2M3 D3M1 D3M2 D3M3
Treatment
Figure 3. Graph of Average Silkworm Biomass
Figure 3. The above shows that the
silkworm biomass reared for 20 days with a
combination of different media materials
and EM4 doses increased until the last day
of rearing. The highest biomass growth was
in the treatment of a combination of 25%
chicken manure + azolla pinnata + 25%
rejected bread (D2M2) with a dose of EM4
of 2 ml to obtain 8.52 grams of biomass.
This is because the nutrient content
in the media material can increase
organic matter as a food source for silk
worms. Provision of 50% azolla pinnata as
the media is suspected as the largest
supplier of N elements because it has a
high protein content, so it can add food to
the media. According to Lestari et al. (2020),
Azolla pinnata is a plant that has the
privilege of being able to bind free N from
the air due to its symbiosis with blue algae
(Anabaena azollae) so that it contains high
protein. According to Alalade and lyayi
(2006), the crude protein content of Azolla
pinnata is 21.4%, this can be seen in Table
4.1. The amount of N content in the
combination media with a dose of EM4
amounted to 1.10. The results of the
analysis of silkworm biomass variance for
20 days of rearing showed that the
combination of media material and EM4
dose obtained (P < 0.05) so the treatment
had a very significant effect.
The content of the proximate analysis
of silk worms
The results of the proximate analysis of
silkworms reared for 20 days on a
combination of media materials with
different doses of EM4 can be seen in Table
3. following.
Biomassa
(gram)
Murni, Herliwati, Noor Arida Fauzana
|
2045
Table 3. Analysis Content of Silk Worms Reared on Different Media Materials and
Doses of EM4
Proximate content (%)
Treatmen
t
cont
ent
Ash
conte
nt
Protei
n
Fat
Crude
content
fiber
content
Carbohydrate
D1M1
12.6
3
46.19
16.06
2.43
38.75
D1M2
11 ,4
8
42.45
15.33
2.31
43.76
D1M3
10.1
9
42.67
16.42
2.28
48.98
D2M1
11.0
9
42.07
17.94
1.62
45.21
D2M2
13.5
5
43.47
17.34
1.38
41.60
D2M3
12.8
2
45.33
17.11
1.63
40.21
D3M1
13.8
5
49.11
24.03
1.94
35.09
D3M2
14.9
9
47, 17
20.08
1.81
36.03
D3M3
15.0
5
50.88
20.66
1.56
32.51
Amino acid content of silk worms Amino
acid
content analysis was carried out to
determine the amino acid composition of
silk worms (Tubifex sp) reared with a
combination of media materials with a dose
of EM4. The amino acid content of silk
worms reared in a combination of media
materials with a dose of EM4 can be seen
in Table 5.
Table 5. Amino Acid Content of Silk Worms Maintained on Different Media Materials
Aad EM4 Doses
Test Parameters
(%)
D1M
1
DIM
2
D1M
3
D2M
1
D2M
2
D2M
3
D3M
1
D3M
2
D3M3
L- serine
0.31
0.22
0.18
0.35
0.45
0.25
0.25
0.28
0.30
Acid
0.60
0.39
0.39
0.71
Gluta
mic
0.60
0.57
0.57
0.64
2046 | Interaction of Cultural Media Composition with EM4 Dosage on Population Growth,
Biomass and Nutritional Content Silk Worm (Tubifex SP)
L- Phenylalanine
0.38
0.23
0.18
0.45
0.48
0.26
0.24
0.27
0.30
L- Isoleucine
0.19
0.17
0.30
0.37
0.24
0.23
0.24
0.27
L-
Valine
0.33
0.23
0.21
0.38
0.46
0.30
0.29
0.30
0.33
L- Alanine
0.34
0.24
0.23
0.46
0.43
0.27
0.23
0.34
0.36
L-Arginine
0.41
0.19
0.16
0.49
0.63
0.28
0.28
0.33
0.35
Glycine
0.35
0.26
0.21
0.42
0.52
0.31
0.29
0.31
0.34
L-Lysine
0.32
0.21
0.21
0.31
0.36
0.24
0.29
0.28
0.26
L-Aspartic Acid
0.41
0.28
0.27
0.34
0.54
0.34
L
0.34
0.34
L-Leucine
0.46
0.32
0.29
0.51
0.63
0.41
0.39
0.41
0.46
L-Tyrosine
0.27
0.15
0.12
0.30
0.32
0.18
0.16
0.18
0.21
L-Proline
0.22
0.15
0.14
0.26
0.30
0.23
0.19
0.21
0.23
L-Threonine
0.33
0.23
0.19
0.39
0.47
0.26
0.26
0.28
0.31
Hisidine
0.19
0.12
0.09
0.20
0.20
0.11
0.1 1
0.12
0.13
Based on the amino acid content test
of silkworms reared for 20 days in a
combination of media materials with a
dose of EM4 showed that each treatment
had different amino acid content. The
amino acid content in the D2M2
treatment looked better than the other
treatments. The different amino acid
content of silkworms is thought to be due
to differences in the nutrient content of
nitrogen, carbon, phosphorus and
potassium which can be seen in Table 4.1.
The difference in potassium content in the
combination of material media affects the
amino acid content of silk worms where
the nitrogen and potassium content of the
D2M1 treatment is lower than that of the
D3M3 treatment. It is presumed that in
the D2M1 study with the lowest
population and biomass, silk worms fully
utilized the nitrogen content for growth,
so that the amino acid content obtained
was higher than in the D3M3 treatment,
but because the potassium content in the
D2M1 treatment medium was lower than
D3M3 and D2M2 then the formation
protein N levels will be disrupted and can
produce high ammonia content and cause
death in silkworms.
In the feed laboratory, protein is
separated from carbohydrates and lipids
due to the nitrogen content of the protein.
According to Syam et al. (2011),
sufficient N content will increase the
activity of microorganisms so that the
decomposition process can take place
effectively. Organic C is the main
constituent of carbohydrates and fats
which are an energy source for the
metabolism of silk worms, so that a
balanced content of organic N and C can
affect the high population growth and
biomass of silk worms (Anggaraini, 2017).
The important role of nutrient K in
determining products with chemical
composition and physical appearance. For
example, in plants that lack K, protein
formation will be disrupted so that protein
Murni, Herliwati, Noor Arida Fauzana
| 2047
N levels decrease and non-protein N
levels increase (Wiraatmaja, 2017). If the K
deficiency has reached a serious level,
plant tissue contains a lot of nitrate and
free ammonium, amides and organic acids
which will reduce the quality of the
product.
Table 4. Water Quality Parameter
Treatme
nt
Parameter
Te
mp
era
tur
e
p
H
D
O
Ammo
nia
D1M1
27.
65
6.
52
4.
43
1.6
D1M2
27.
79
6.
59
4.
21
1.8
D1M3
27.
59
6.
46
4.
55
1.6
D2M1
27 ,
71
6.
42
4.
21
2.4
D2M2
27.
81
6.
48
4.
45
2.4
D2M3
27.
85
6.
44
4.
71
2.3
D3M1
27.
65
6.
62
4.
56
2.4
D3M2
27.87
6.3
9
4.
47
2.
4
D3M3
27.67
6.4
5
4.
73
2.
4
The
Results of the water temperature
measurement in Table 4.13 above are
suspected to be the optimum temperature
for good metabolism and nutrient
absorption in organic matter media.
maximum. Lestari et al (2020), said the
optimum range of temperature in the
growth of silkworms was 25–28
0
C. The
optimum range of dissolved oxygen could
meet the needs for the growth of silkworms
range. Dissolved oxygen for silkworm
growth is 2.5-7 mg/L (Effendi, 2013). It is
also stated by Lestari et al (2020) that the
dissolved oxygen requirement for the
growth of silk worms is in the range of 2.24-
6.48 mg/L. the pH value during the research
2048 | Interaction of Cultural Media Composition with EM4 Dosage on Population Growth,
Biomass and Nutritional Content Silk Worm (Tubifex SP)
on the maintenance media was still in the
optimal value, namely 6.39-6.59. Safrudin et
al (2005) stated that at neutral pH, bacteria
can break down organic matter normally
into simpler ones that are ready to be used
by silkworms as food, ranging from 6.02-
7.76. The ammonia content was still at the
optimum limit for the growth of silk worms.
Ammonia content during the study ranged
from 1.6 to 2.4 mg/L. This is in accordance
with Anggraini's (2017) statement that the
optimum range for the growth of silk
worms is <3.6 mg/L.
CONCLUSIONS
The interaction of the composition of
the culture media with the dose of EM4 on
population growth and biomass of
silkworms (Tubifex sp) had a very
significant effect. The highest daily length,
population and biomass growth was
obtained in the combination treatment of
25% chicken manure + azolla pinnata +
25% rejected bread with a dose of EM4 of
2 ml (D2M2) on day 20 with an average
length growth of 9.27 mm. , the average
population was 27482.9 ind/m2
and
the
average biomass was 8.52 grams, while
the nutrient content of silk worms (Tubifex
sp) reared with different compositions of
culture media and EM4 doses obtained
results. D3M3 treatment obtained the
highest protein content. ie 50.88, then
D2M2 obtained a protein content of
43.47.
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