Nus Biosci | vol. 2 | no. 1 | pp. 1-53 | March 2010

Cyphastrea chalcidicum photo by M Moradi | Nus Biosci | vol. 2 | no. 1 | pp. 1‐53 | March 2010 |
ISSN 2087‐3948 (PRINT) | ISSN 2087‐3956 (ELECTRONIC)
| Nus Biosci | vol. 2 | no. 1 | pp. 1‐53 | March 2010 | ISSN 2087‐3948 (PRINT) | ISSN 2087‐3956 (ELECTRONIC) I S E A J o u r n a l o f B i o l o g i c a l S c i e n c e s FIRST PUBLISHED:
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ISSN: 2087-3948 (print)
ISSN: 2087-3956 (electronic)
Vol. 2, No. 1, Pp. 1-6
March 2010
Ripening for improving the quality of inoculated cheese
Rhizopus oryzae
SOLIKAH ANA ESTIKOMAH1,♥, SUTARNO², ARTINI PANGASTUTI²
¹ Bioscience Program, School of Graduates, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia
² Department of Biology. Faculty of Mathematics and Natural Sciences, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia
Manuscript received: 17 November 2010. Revision accepted: 26 January 2010.
Abstract. Estikomah SA, Sutarno, Pangastuti A 2010. Ripening for improving the quality of inoculated cheese Rhizopus oryzae.
Nusantara Bioscience 2: 1-6. Cheese is dairy product resulted from fermented milk in which the fermentation process can be done by
lactic acid bacteria or fungus. Rhizopus oryzae is able to produce lactic acid, protease and lipase. The ripening process changes the taste
and texture. The purpose of this study is ripening to improve the quality of inoculated cheese R. oryzae. In this research the ripening was
conducted the concentration variation of temperature (5oC, 10oC, 15oC), and time (7 days, 14 days). The procedure of research consisted
of two steps, namely un-ripened cheese preparation followed by ripening cheese preparation. Cheese produced in this study analyzed the
value of pH, fat content, protein content, amino acid levels and identification of microbe with ANOVA then followed by DMRT at 5%
level of significance. Data results were analyzed with the like’s nonparametric statistical test, followed by Fridman Wilcoxon Signed
Rank Test (WSRT) at 5% level significance. The results showed that the preferred ripened cheese panelist was at a temperature of 15oC
for 14 days. Ripening conditions affect pH, fat content, protein content and do not affect the levels of amino acids that formed ripened
cheese. The best quality ripened cheese i.e. at a temperature of 15°C for 14 days, had a pH value of 4.40, the highest protein content of
9.78%, and fat content of 35.02%. The results of identified microbe in un-ripened cheese and ripened cheese include Enterococcus hirae
(Enterococcus faecalis), Bacillus subtilis, and Aspergillus sp.
Key words: cheese, fermentation, Rhizopus oryzae, ripening, temperature.
Abstrak. Estikomah SA, Sutarno, Pangastuti A. 2010. Pemeraman untuk meningkatkan kualitas keju yang diinokulasi Rhizopus oryzae.
Nusantara Bioscience 2: 1-6. Keju merupakan makanan hasil fermentasi dari susu yang proses fermentasinya dilakukan oleh bakteri
asam laktat maupun jamur. Rhizopus oryzae diketahui mampu menghasilkan asam laktat, protease, dan lipase. Perubahan cita rasa dan
tekstur keju terjadi selama pemeraman keju. Tujuan penelitian ini adalah untuk meningkatkan kualitas keju yang diinokulasi R. oryzae
melalui pemeraman. Pemeraman dilakukan dengan variasi waktu (7, 14 hari) dan suhu (5ºC, 10ºC, 15ºC). Penelitian ini terdiri dua
tahap, yaitu pembuatan keju mentah diikuti pemeraman keju mentah tersebut. Keju penelitian dianalisis nilai pH, kadar lemak, kadar
protein, kadar asam amino dan diidentifikasi mikrobanya. Data hasil penelitian dianalisis dengan uji sidik ragam (ANAVA), kemudian
dilanjutkan dengan uji berjarak ganda Duncan (DMRT) pada taraf signifikansi 5%. Data hasil tingkat kesukaan dianalisis dengan
statistik nonparametrik uji Fridman yang dilanjutkan dengan Wilcoxon Signed Rank Test (WSRT) pada taraf sigifikansi 5%. Hasil
penelitian menunjukkan bahwa keju peram yang disukai panelis adalah keju peram pada suhu 15ºC selama 14 hari. Kondisi pemeraman
berpengaruh terhadap nilai pH, kadar lemak, kadar protein dan tidak berpengaruh pada kadar asam amino. Kualitas keju peram terbaik
terdapat pada kondisi suhu 15°C selama 14 hari, memiliki nilai pH 4,40, kadar protein tertinggi yaitu sebesar 9,78%, dan kadar lemak
sebesar 35,02%. Hasil identifikasi mikroba pada keju mentah dan keju peram meliputi Enterococcus hirae (Enterococcus faecalis),
Bacillus subtilis, dan Aspergillus sp.
Kata kunci: keju, fermentasi, Rhizopus oryzae, pemeraman, suhu.
INTRODUCTION
Milk is a food that consists of various nutrients in
balanced proportions. Its main constituent is water, protein,
fat, lactose, minerals, and vitamins. Milk is yielded from
livestock such as cattle, buffalo, and goats. Milk production
from dairy farmers is distributed to the milk factories and
processed by them into a liquid ready to drink milk. Milk
produced by breeders can only be sold to a cooperative
economic enterprise or factory and processed into a ready
to drink milk. There are some basic problems bear down
upon dairy farmers, they are low resistance on the milk or
easily damaged, the bargaining position of farmers against
low milk prices and lack of absorptive capacity of milk
production by the manufacturer/cooperatives as well as
poor knowledge of dairy farmers. On the other hand
breeders always wanted the milk that is produced can be
used completely without any damage or wasted, so we need
some milk processing which is aimed to preserve milk for
much longer when stored. Cheese is a dairy product
(Daulay 1991).
The fungus Rhizopus oryzae is able to produce lactic
acid (Purwoko and Pamudyanti 2004). R. oryzae also has
protease enzyme which has similar characteristics as rennet
2
2 (1): 1-6, March 2010
(Hadiwiyoto 1983). Lactic acid will help preserve the milk,
while the protease functions to wad milk casein. Besides
lactic acid and protease, R. oryzae is capable to produce
lipase that functions as a solver of fat that will enhance the
taste of cheese.
In a manufacturing of cheese, ripening is one of the
important stages. Cheese product which has a ripening can
change a young cheese slowly into a mature cheese. The
ripening process changes the taste and texture. The changes
are caused by protein breakdown into simpler peptides and
amino acids, fats into fatty acid solution and volatile acids
such as acetic and propionic acid, lactose fermentation,
citrate, and other organic materials into acids, esters,
alcohol, taste, and diacetyl other components.
The process making a ripened cheese involves
acidification and ripening. Acidification of milk is done by
adding acid or inoculation of microbes. Direct acidification
of milk by adding acid is less suitable for ripened cheese
making process because during the ripening process there
is no real change of proteins (proteolysis), fat (lipolysis),
and lactose, whereas acidification using inoculums can
cause biochemical changes, including proteolysis, lipolysis
and lactose fermentation. Biochemical changes can affect
flavor and texture (Septiana 1994).
Ripening of cheese is done by storing the cheese for
some time and at a certain temperature. The longer the
ripening, the stronger the flavor of cheese is formed. In
cheese ripening, maturing temperature affects the speed of
proteolytic activity and acid production. High temperatures
will accelerate the process, but not profitable. At high
temperatures, acid production is quicker, causing a strong
sour taste and accelerating evaporation so that more water
loss and decay more quickly. At low temperatures a proper
balance of acid production and proteolytic activity occur
and water evaporation is inhibited (Daulay 1991).
This research is about the cheese which was inoculated
by R. oryzae by using variations of temperature and time of
ripening. Cheeses are brooded in this study analyzed the
value of pH, fat content, protein and amino acid contents,
microbial identification and preference test.
MATERIALS AND METHODS
Materials research
The main material used is cow's milk from dairy cows
in Boyolali District, Central Java, and Rhizopus oryzae
propagated by the Faculty of Agriculture Sebelas Maret
University, Surakarta.
Procedures
Preparation of culture media
Media manufacturing process begins with mixing the
ingredients PDA (Potato Dextrose Agar), which is a
medium for the growth of R. oryzae. Distilled water are
then inserted into the Erlenmeyer flask, then heated on hot
plate and homogenized with a magnetic stirrer. Once the
mixture boils, PDA media was poured into a test tube and
proceed with the process of sterilization using the autoclave
at a temperature 121oC at 1 atm pressure for 30 minutes,
then test tube placed in a tilted position in order to form
slanted media.
The working culture of R. oryzae are ready to be used
for the manufacture of starter. Work culture is obtained
with culture rejuvenate R. oryzae by inoculating a pure
culture into the PDA side, then incubating at 37ТАC for 34 days, while the rest is stored at a temperature 4ТАC as a
stock culture and rejuvenated every 6 months (Wijaya
2002; Suharyanto et al. 2006). Starter made by inoculating
250 mL fresh milk (skim milk liquid) with R. oryzae from
PDA at the age of 3-4 days, propagation of R. oryzae taken
as many as 50 cells/mL (3 ose) and incubated at 37 ° C for
one day (Nurhidayati 2003).
Making cheese
Fresh cow's milk, as much as 3600 mL pasteurized up
to 70°C for 30 seconds. Once pasteurized, the milk was
cooled until the temperature reaches 37°C and then inserted
into the 8 piece glass beaker with a volume of 200 mL.
Bottles filled with milk that has been inoculated are then
incubated in an incubator at a 37°C for 9 hours. During the
incubation bottles were covered with aluminum foil. The
part that was clotted called curd while the liquid is called
whey (Ward 1996). After that the milk was heated for 30
minutes at a 40°C, then cooled for 1 hour, stirred every 5
minutes (Hadiwiyoto 1983), and then filtered with clean
gauze. Filtering is done to separate curd and whey. Formed
curd is taken while the whey is removed (Legowo 2003).
Curd wrapped in clean gauze continued pressing to give
compactness and shape of the cheese, and remove the
remains of whole whey (Hadiwiyoto 1983). Formed curd
was salted as much as 4%. Salt is sprinkled in the form of
fine crystal, then stir until completely blended. The salted
curd is then wrapped with aluminum foil and matured for 0
day (without ripening), 7 days, and 14 days, with
temperature of curing 5°C, 10°C, and 15°C.
Microbiological test
Microbiological test involved the calculation of total
microbes and microbial identification. Calculation of total
microbial cheese made by weighing 25 g and then
homogenized with 225 mL of distilled water (Rosa et al.
2003: Ceylan et al. 2003; Mennane et al. 2007). Calculation
of total microorganisms was done on the basis of the
Standard Plate Count. Fertilization is done with medium
Plate Count Agar (PCA) by dropping 1 mL of inoculation
into sterile petri PCA and the subsequent media that has
been cold poured into sterile petri saucer as much as 12-15
mL, the mixture is homogenized with a petri saucer by
moving it to form a figure eight direction. Having to
harden, Petri saucer was incubated upside down at 37°C for
24-48 hours. Then the colonies formed are counted.
Identification was done by isolating colonies of microbes
then growing it on PDA media for mold and on MRSA
media for bacteria. Identification of mold was based on its
morphologic characteristics. Identification of bacteria was
using the BD Phoenix TM.
ESTIKOMAH et al. – Cheese ripening by Rhizopus oryzae
Lipid analysis
Soxhlet fat analysis method is as follows: Samples of 3
g was taken and then inserted into the timbel. Put the flask
which has already been cleaned into the oven, then add the
boiling stone and weighed as empty weight. Timbel is
inserted into the soxhlet, then connected with soxhlet fat
flask, and then add a liquid fat solvent of 150 mL of ether
through the soxhlet. Flask fat and soxhlet are connected
with bath extracted for 6 hours. After the extract is
complete, flask fat is evaporated to remove solvent. Flask
fat is put into the oven at 105°C for 1 hour. After it is cold,
it is weighed as final weight (weight and fat flask). The
sample calculation formula is:
Lipid content = c-bx100%
a
a = weight of sample
b = weight of fat and boiling flask
c = weight of fat flasks, stone boiling and fat
Protein content
Protein content is analyzed by Lowry-Folin method by
spectrophotometer (Sudarmadji et al. 1984). Measurement
begins with the manufacture of standard solution of BSA
(Bovine Serum Albumin). Dilution series was made from
standard solutions with respective concentrations of 0.00,
0.06, 0.18, 0.24, and 0, 30 (mg/mL H2O) and inserted into
each test tube. 1 ml solution D is added into the test tube
and then is whirled for 5 minutes. After that, the addition of
reagents E of 3 mL and then allowed to stand for 10
minutes. OD measurements performed at a wavelength of
560 nm using spectrophotometer. The next steps was
taking 1g of cheese sample and dissolve it in 100 mL of
distilled water and then stirring with a magnetic stirrer, the
solution was filtered and added 100 mL of distilled water. 1
mL sample solution was taken and then inserted into a test
tube and then added by 1 mL of Lowry reagent D, whirled
with vortex for 5 minutes. Next reagent Lowry E as much
as 3 mL added into test tubes and whirled with vortex and
then incubated at room temperature for 45 minutes. OD
measurement at a wavelength of 590 nm was using a
spectrophotometer. Sample calculation formula is:
% protein = axbx100%
c
a = concentration
b = dilution factor
c = a lot of sample (g)
Amino acid content of cheese
Amino acid content of cheese was analyzed by HPLC
(High Performance Liquid Chromatography). Cheese
samples in which amino acid content will be analyzed was
prepared in advance, by taking 5 g of cheese samples that
has been ground smoothly into the Erlenmeyer covered
with grindstones, homogenized using a magnetic stirrer and
hydrolyzed at a temperature of 110 ° C for 12 hours,
3
filtered using Whatman filter paper 41, and the pH was
adjusted to normal (pH 7). 100 ml of distilled water is
added, take 3 mL of that solution and filter with millex 0,
45 μm. For injection into the HPLC, take 10 μl of milex
solution + 990 mL of OPA and whirled in a vortex. Put it
into reaction for 3 minutes, and the inject it into the HPLC.
Preparation of standard solution. Standard stock
consists of L-threonin = 1050 ppm; L-methionine = 1000
ppm; L-valine = 1010 ppm; L-thriptophan = 1010, LPhenilalanine = 1000 ppm; L-isoleusine = 1060, L-Leucine
= 1010 ppm ; L-lycine = 1000 ppm, each drawn by
comparison 1:1:1:1:1:1:1:1 into 10 mL + 990 mL OPA
diijeksi to HPLC. Amino acid cheese detected by HPLC
with a set of HPLC equipment. The prepared sample was
taken as much as 20 mL using the injector. Amino acids
was detected by a set of tools Eurospher 100-5 C18 HPLC
column, 250x4.5 mm with pre-column P/N: l115Y535.
Eluent: A = 0:01 M acetate buffer pH 5.9, B = (MeOH:
0:01 M acetate buffer pH 5.9).
Organoleptic test
Organoleptic test which is conducted is a test of
preference. This preference test assesses the level of color,
flavor, aroma, and texture of cheese. The assessment was
conducted by 20 untrained panelists. This test refers to
Zulaekah and Widiyaningsih (2005). A five-level scale was
stated (level 1-5), start from 1 (strongly dislike), 2 (not
like), 3 (somewhat like), 4 (like), and 5 (very like).
Data analysis
Data obtained from analysis which consists of the pH
value, fat content, and protein content and total microbial
was analyzed by analysis of variance (Anova) to determine
whether there is any treatment effect followed by a test of
Duncan's Multiple Range Test (DMRT) at the significance
level of 5% to know the real difference among the
treatments. Data favorite level test results were analyzed
descriptively with Friedman nonparametric statistical tests
(Friedman test) followed by Wilcoxon Signed Rank Test
(WSRT) at 5% significance level.
RESULTS AND DISCUSSION
The degree of acidity (pH)
The pH is a measure of the value of dissociated
hydrogen ions in solution, thus aiming to find out the pH
measurements of cheese acidity caused by the presence of
hydrogen ions. PH value of ripened cheese which was
inoculated with R. oryzae can be seen in Table 1.
According to De Souza et al. (2003), pH levels decreased
during the ripening process. Decrease in pH of cheese is
influenced by the amount of lactic acid produced by
microorganisms, the higher the lactic acid then the pH was
lower. The decrease in pH value is caused by the activity of
bacteria in this cheese. BAL is in the cheese (Basillus
subtilis and Enterococcus hirae) are able to produce lactic
acid from sugar that will be needed in forming taste,
preventing the growth of pathogenic bacteria, and the
safety of the final product (Kayagil 2006). Lactic acid is
4
2 (1): 1-6, March 2010
the result of glucose metabolism. Increased lactic acid is
characterized by a decrease in pH. Increased lactic acid
caused by H + ions that occur because of decomposition of
lactose produces acids that are easily evaporated, and the
outbreak of organic phosphate contained in casein,
resulting in acid (Mc Kay et al. 1971).
Table 1. The pH, fat content (%), protein content (%), ripened
cheese inoculated with R. oryzae
Ripening period
5°C
Temperature
10°C
15°C
pH
7 days
14 days
5,44 bc
5,14 bc
5,09 b
4,88 ab
4,87 ab
4,40 a
Fat content (%)
7 days
14 days
34,56 ab
32,43a
34,48ab
33,31ab
35,30ab
35,02ab
Protein content (%)
7 days
6.28 d
7.56 c
8,34 b
c
c
14 days
7.22
7.60
9,78 a
Note: Figures with different letters in the same column indicate
significant differences (P <0.05) in Duncan's multiple range test.
Lipid content
Fats are a source of some components giving flavor,
aroma, and texture of cheese. Fusion of fat in the cheese
occurs due to trapped fat globules at the time of the protein
wadding progress (Daulay 1991). Data results of
calculating the value of fat content in cheese inoculated
with R. oryzae can be seen in Table 1. The results of this
study indicate that the lipid content will be decreased in a
longer ripening process. The results are consistent with the
results of research conducted by Kayagil (2006) decreasing
levels of fat in the cheese due to ripening process occurs
because the degradation of fat with the help of lipase. In the
process of fat degradation, fatty acid is formed. There are
the volatile fatty acids and non-volatile fatty acid.
According Prawisuma (2007) during the ripening process,
fat is hydrolyzed into various volatile fatty acids. Volatile
fatty acids are fatty acids which are easily evaporated.
These easily evaporated fatty acids causes levels of fat in
cheese reduced. Reduced levels of fat besides caused by the
formation of volatile acid, it is also caused by the using of
some fats as a source of energy in metabolism activity. Fat
is used as an energy source through the renovation process
initiated by the hydrolysis of triglycerides into glycerol and
fatty acids with the assistance of lipase.
Protein content
Proteins in milk are composed by whey and casein,
whereas the remaining proteins in the cheese is casein
because the formed whey has been released in the process
of cheese formation (Murwaningsih 2003). The value
protein content in ripened cheese inoculated with R. oryzae
by the treatment of ripening duration and temperature
variations can be seen in Table 1. High protein levels of
ripened cheese at a temperature of 15°C for 14 days are in
accordance with the results of research by Licitra et al.
(2000) which showed an increase in protein content during
ripening of 0 to 12 months. At 0 months of ripening protein
content is 25.30% and then increased at 29.24% after 12
months of ripening. Compared with controls that have a
protein content of 2.23%, the ripened cheese in this study
had higher protein content. Increased levels of protein in
cheese is due to the opportunity given for microbes (E.
hirae, Bacillus subtilis, Aspergillus sp.), and the enzymes
in the cheese curd to hydrolyze proteins during the ripening
process. Protein breakdown during ripening will result in a
high protein, more flexible and soft cheese structure, and
aromatic taste, because the rigid proteins and insoluble
nitrogen is converted into soluble form (Daulay 1991).
Essential amino acid content of ripened cheese
Amino acids are homologous series of compounds
containing two functional groups i.e. amino groups and
carboxylate groups which are attached to the same carbon
atom. Essential amino acid analysis results that were
inoculated with starter R. oryzae can be seen in Table 2.
During ripening process the highest levels of amino acids is
in the treatment of 7 days. High level of amino acid in the
7-days treatment of ripened cheese is caused by proteolysis
occurs on the cheese. Meanwhile, on 14-day treatment of
ripened cheese, the content of amino acid decline due to the
occurrence of amino acid catabolism. Amino acids are
precursory to the various flavor components in cheese
(Urbach 1997; Engles et al. 1997). Catabolism of amino
acids produces a number of aroma components found in the
cheese. Mechanism of amino acid catabolism includes
oxidation deamination, decarboxylation, transminase, and
reduction reactions that would form the aldehyde, alcohol,
indole, acid, phenolic and sulfur (Hansen et al. 2001;
Williams et al. 2001).
Table 2. Amino acid levels in cheese inoculated with R. oryzae.
Essential amino acid
compound
L-Threonine
Essential amino
acid contents (%)
0 day
7 day
1.15
1.68
Ripened
cheese
14 day
1.58
L-Methionine
0.47
0.62
0.58
L-Valine + L-Thriptophan
0.70
1.78
1.65
L-Phenylalanine
0.66
1.12
1.00
L-Isoleucine
0.48
0.99
0.84
L-Leucine
1.28
2.30
1.96
L-Lycine
1.64
2.42
2.44
Total
6.38
10.91
10.05
Test of cheese preferences
This test is conducted to know consumer preference
level of cheese produced include predilection of texture,
flavor, color, and flavor. The results of statistical analysis
are shown in Table 3. From Table 3, it is known that the
taste of ripened cheese on 15°C for 14 days has the most
preferred taste over the other. Cheese in the ripening
process of 5°C for 7 days has a taste of the least preferred.
ESTIKOMAH et al. – Cheese ripening by Rhizopus oryzae
5
Table 3. Test scores upon predilection of flavor, aroma, color and
texture in ripened cheese.
Table 4. Microorganisms found during the ripening time of 7 and
14 days at 15oC on MRSA and PDA media.
Ripening duration
Type of
media
(cfu/mL)
PDA
Flavor
a
Aroma
a
Color
a
Texture
a
5°C during 7 days
3.40
3.47
4.70
4.25
5°C during 14 days
3.75 a
4.43a
3.40 a
3.75 a
3.95a
4.22 a
4.60 a
10°C during 7 days
4.45 a
a
a
a
10°C during 14 days
4.03
4.45
4.00
4.13 a
a
a
a
3.83
3.85
3.17 a
15°C during 7 days
4.22
15°C during 14 days
4.50 a
4.42a
3.45 a
4.20 a
a
a
a
3.65
4.38
3.90 a
Controls
3.92
Note: The larger the value, then the ripened cheese is increasingly
preferred. Same superscript indicates no significant difference in
fridman test 5%. 1 = extremely dislike, 2 = dislike, 3 = somewhat
like; 4 = like, 5 = very like it.
The aroma of cheese appears mainly due to the volatile
formed during ripening. Results of non-parametric analysis
shows the most preferred aroma on ripening of 10oC for 14
days, while the least preferred flavor is in 5oC for 7 days
which is ripened at the lowest temperatures, according to
Daulay (1991) Low temperature inhibits the biochemical
processes that lead to process inhibited the formation of
aroma. When compared with control ripened cheese
preferably, in this study the control used is the cheese
without ripening (unripened), which is a type of fresh
cheese where the aroma has not been formed and is still
dominated by the aroma of milk (Murwaningsih 2003).
A non-parametric analysis result indicates a preferred
color in ripened cheese 5oC for 7 days because this cheese
has a more yellow color than others. According to Buckle
(1987) cheese made from cow's milk without the dye will
produce a yellow-white cheese. The color of cheese is
influenced by fat content in cheese. Fat in cheese obtained
with the help of the enzyme lipase, which can hydrolyze
triglycerides into glycerol and fatty acids. The yellow color
comes from carotene pigments which are fat soluble, so
that more levels of fat in the cheese, the cheese color
becomes more yellow, because the more soluble pigment
carotene. Non-parametric analysis results show that the
assessment score given by panelists on the texture of the
most preferred, namely 10oC treatment for 7 days while the
texture was the least preferred on 15oC treatment for 7
days.
Microbiology identification
Results of identification of microorganisms found in the
control cheese (without ripening cheese), ripened cheese
for 7 days, and ripened cheese for 14 days are shown in
Table 4. In the control, 7 days ripening time and 14 days
ripening time, there are 3 types of microbes which are the
same, namely E. hirae, B. subtilis, Aspergillus sp. The
number of mold progressively increased, while the number
of bacteria become more and more declined (Table 4). This
is because B. subtilis and E. hirae is thermoduric bacteria
that have optimal temperature at 30-45oC while Aspergillus
grows at an optimal temperature at 29-32oC so that
ripening treatment at a temperature of 15oC causes
Aspergillus is more able to survive than in B. subtilis and
E. hirae.
MRSA
Control
Aspergillus sp.
(1.2x104)
E. hirae and B.
subtilis
(3.8x104)
7 days
Aspergillus sp.
(1.1x104)
E. hirae and B.
subtilis
(3.3x104)
14 days
Aspergillus sp.
(2.8x104)
E. hirae and B.
subtilis
(3.2x104)
The total number of microbial colonies of ripened
cheese inoculated with R. oryzae
The calculation of the total number of microbes in this
study was conducted using SPC (Standard Plate Count) on
PCA medium (Plate Count Agar) performed by dilution.
The total numbers of microbes that participate in cheese
ripening are shown in Table 5. The longer ripening time
caused the number of microorganisms that grow less (Table
5). According to Amos (2007) microbes in cheese will
grow rapidly in milk and curd during cheese making, then
declines during ripening, due to a decline in pH during
ripening, reduced lactose and high salt concentration.
Table 5. The number of microbes (x 104) on media Total Plate
Count (TPC)
Ripening time
7 days
14 days
5°C
10,99x104 ab
10,12x104 a
Temperature
10°C
11, 95x104 ab
11,14x104 ab
15°C
12,99x104 b
11,30x104 ab
Note: Figures with different letters in the same column indicate
significant differences (P <0.05) in Duncan's multiple range test.
The pH value of cheese in this study is ranged from 4 to
5.44 (Table 1). From Table 1, it is noted that the longer
ripening time led to a lower pH value. Low acidity levels
causing microbes within the cheese die due to not acid
resistant (Daulay 1991). Compared with controls, which is
without ripening cheese that has a large number of
microbes, ripened cheese has a little amount of microbes,
caused by the pH in the ripened cheese is lower (4 to 5.44)
than the pH of unripened cheese (5.5) which resulted in
microbes in the cheese die due to not acid resistant.
CONCLUSIONS AND SUGGESTIONS
The use of long ripening variation affects the amount of
microbes, pH value, fat content and protein content. The
quality of the best cheese at a temperature of 15°C at 14
days ripening time, has a pH value of 4.40, the highest
protein content of 9.78%, fat content of 35.02% and
produces a sense of well-liked by the panelists.
Identification of Bacillus subtilis using BD PhoenixTM
has only 90% confidence level so it is expected that the
next study will use molecular analysis to get more exact
6
2 (1): 1-6, March 2010
results. The further research is expected to have more
additional secondary starter of ripened cheese making.
Further research is expected to use the ripening temperature
of 15°C with a time between 7 to 14 days.
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Williams AG, Noble J, Banks JM. 2001. Catabolism of amino acids by
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[Indonesia]
ISSN: 2087-3948 (print)
ISSN: 2087-3956 (electronic)
Vol. 2, No. 1, Pp. 7-13
March 2010
Comparasion of iles-iles and cassava tubers as a Saccharomyces
cerevisiae substrate fermentation for bioethanol production
KUSMIYATI♥
Study Center for Alternative Energy, Department of Chemical Engineering, Faculty of Engineering, University of Muhammadiyah Surakarta. Jl. Ahmad
Yani Tromol Pos 1 Pabelan, Kartosuro, Sukoharjo 57102, Central Java, Indonesia. Tel./Fa: 271-717417 ext 442/ 271-715448. ♥email:
[email protected]
Manuscript received: 8 February 2010. Revision accepted: 16 March 2010.
ABSTRACT. Kusmiyati (2010) Comparasion of iles-iles and cassava tubers as a Saccharomyces cerevisiae substrate fermentation for
bioethanol production. Nusantara Bioscience 2: 7-13. The production of bioethanol increase rapidly because it is renewable energy that
can be used to solve energy crisis caused by the depleting of fossil oil. The large scale production bioethanol in industry generally use
feedstock such as sugarcane, corn, and cassava that are also required as food resouces. Therefore, many studies on the bioethanol
process concerned with the use raw materials that were not competing with food supply. One of the alternative feedstock able to utilize
for bioethanol production is the starchy material that available locally namely iles-iles (Amorphophallus mueller Blum). The contain of
carbohydrate in the iles-iles tubers is around 71.12 % which is slightly lower as compared to cassava tuber (83,47%). The effect of
various starting material, starch concentration, pH, fermentation time were studied. The conversion of starchy material to ethanol have
three steps, liquefaction and saccharification were conducted using α-amylase and amyloglucosidase then fermentation by yeast
S.cerevisiaie. The highest bioethanol was obtained at following variables starch:water ratio=1:4 ;liquefaction with 0.40 mL α-amylase
(4h); saccharification with 0.40 mL amyloglucosidase (40h); fermentation with 10 mL S.cerevisiae (72h) producing bioethanol 69,81
g/L from cassava while 53,49 g/L from iles-iles tuber. At the optimum condition, total sugar produced was 33,431 g/L from cassava
while 16,175 g/L from iles-iles tuber. The effect of pH revealed that the best ethanol produced was obtained at pH 5.5 during
fermentation occurred for both cassava and iles-iles tubers. From the results studied shows that iles-iles tuber is promising feedstock
because it is producing bioethanol almost similarly compared to cassava.
Key words: alternative energy, cassava, iles-iles, bioethanol.
ABSTRAK. Kusmiyati (2010) Perbandingan umbi iles-iles dan singkong sebagai substrat fermentasi Saccharomyces cerevisiae dalam
produksi bioetanol. Nusantara Bioscience 2: 7-13. Produksi bioetanol meningkat dengan cepat karena merupakan energi terbarukan
untuk mengatasi krisis energi yang disebabkan oleh habisnya minyak fosil. Produksi bioetanol skala besar di industri umumnya
menggunakan bahan baku seperti tebu, jagung, dan ubi kayu yang juga diperlukan sebagai sumber makanan. Oleh karena itu, banyak
studi pada proses bioetanol terkait dengan penggunaan bahan baku yang tidak bersaing dengan pasokan makanan. Salah satu alternatif
bahan baku dapat dimanfaatkan untuk produksi bioetanol adalah bahan berpati yang tersedia secara lokal yaitu iles-iles
(Amorphophallus mueller Blum). Kandungan karbohidrat umbi iles-iles sekitar 71,12% yang sedikit lebih rendah dibandingkan dengan
umbi singkong (83,47%). Pengaruh berbagai bahan awal, konsentrasi pati, pH, waktu fermentasi dipelajari. Konversi dari bahan berpati
menjadi etanol memiliki tiga langkah, pencairan dan sakarifikasi dilakukan dengan α-amilase dan amyloglucosidase kemudian
difermentasi dengan ragi S.cerevisiaie. Bioetanol tertinggi diperoleh pada variabel berikut rasio pati: air = 1:4; likuifaksi dengan 0,40
mL α-amilase (4h); sakarifikasi dengan amyloglucosidase 0,40 mL (40h); fermentasi dengan 10 mL S.cerevisiae (72h) memproduksi
bioetanol 69,81 g/L dari singkong sementara 53,49 g/L dari umbi iles-iles. Pada kondisi optimum, gula total dihasilkan 33.431 g/L dari
ubi kayu sementara 16.175 g/L dari umbi iles-iles. Pengaruh pH menunjukkan bahwa etanol yang dihasilkan terbaik diperoleh pada pH
fermentasi 5,5 baik untuk ubi kayu maupun umbi iles-iles. Hasil studi menunjukkan bahwa umbi iles-iles menjanjikan sebagai bahan
baku bioetanol karena menghasilkan bioetanol hampir sama dengan ubi kayu.
Key words: singkong, iles-iles, etanol, energi alternatif.
INTRODUCTION
Ethanol is an alternative fuel that is important in
reducing the negative impacts of fossil fuel consumption
(Cardona and Sanchez 2007). Fossil fuel consumption in
the world reached 80%(Gozan et al. 2007). Fuel demand in
Indonesia reached 5.6% per year, this has caused Indonesia
to be the only OPEC member country that has to import
crude oil by 487 thousand barrels/day since the end of
2004. Therefore, the use of biofuel such as bioethanol is
one of the alternatives to overcome fuel crisis. Bioethanol
is colorless liquid and environmentally friendly which
results in the form of combustion gases, and air pollutants
such as CO Nx are very small. Many researchers concluded
that ethanol does not cause the greenhouse effect of fossil
fuels because hazardous gases such as CO2 which is
reduced by 22% (Milan 2005). Bioethanol can be used to
substitute premium and kerosene. According to Kusmiyati
8
2 (1): 7-13, March 2010
processed into cassava flour, tapai,
tiwul and others, while the cassava
starch is used as raw materials of
crackers, meatballs and pempek, and
this shows that cassava has an
economic value. The use of cassava
for bioethanol production could affect
food supply, therefore it is necessary
to find the diversification of raw
materials such as tubers iles-iles
(Amorphophallus muelleri Blume).
Iles-iles grows wild in Sumatra,
Java, Flores and Timor (Jansen et al.
1996), as well as Bali and Lombok
(Kurniawan et al. 2010, in press).
Iles-iles belongs to the Araceae
monocot plant family with compound
flower of “cob" type which is
covered by its leaves (spatha) (Jansen
et al. 1996), it has dark brown tubers
with rough rash that contain
relatively high carbohydrate i.e. 7085% (Department of Agriculture
2009; Kusmiyati 2009). Iles-iles has
the highest glucomannan among any
other types of Amorphophallus in
Indonesia, which ranged between 4446%
(Sumarwoto
2004).
Glucomannan is a polysaccharide
Figure 1. a. 4-month-old cassava plants, b. 5 months old iles-iles plants, c. Cassava
consisting of monomer β-1, 4 αtubers after harvest, d. Iles-iles tuber that have been harvested
mannose and α-glucose (Widyotomo
et al. 2000).
The availability iles-iles in
(2007), the use of bioethanol in Batik Industry has a higher
Central Java is relatively abundant where forest land which
efficiency than kerosene because the flame is stable, not
is used reaches 640,000 hectares, while the productivity
too high and not easily to handle and use. It shows that the level is 30-40 tons/ha (Department of Agriculture 2009).
60% bioethanol has better characterization and better
However, this abundant amount of iles-iles has not been
efficiency compared to other ethanol in the concentration
highly used; therefore iles-iles does not have high
below 40% and above 90%).
economic value. Because the nature of the sap causes itch,
Bioethanol can be produced from raw materials
iles-iles tubers are not used as a food ingredient. According
containing sugar, starch or cellulose. One type of natural
to Imelda et al. (2008) iles-iles is easy to be cultivated,
resource which has potential to make bioethanol is the either generative by using its seed or vegetative by using its
tubers. Tubers are agricultural products which contain
bulbs, bulbils and leaf cuttings. Iles-iles naturally grows as
carbohydrates or starch, and it is known that bioethanol can secondary vegetation, on the outskirts of teak forests at an
be made from raw materials containing carbohydrates. One
altitude of 700-900m above sea level, with rainfall level at
kind of bulbs which is often used to make bioethanol is
1000-1500 mm (Sumarwoto and Widodo 2008)
cassava. Cassava is an agricultural commodity grown in
Iles-iles tuber is less fully utilized by society; it will be
Indonesia and is the second highest source of carbohydrates
very beneficial when it is used as raw material for
after rice, the carbohydrate of which is 98.4% (Osunsami et
bioethanol production. This study aimed to compare the
al. 1988). Cassava can grow at of 2,000 m above sea level cassava tubers and iles-iles tubers as raw material for
or in sub-tropical temperature of 16 º C. These plants
bioethanol production and to study the effect of variable
flowers will bloom and produce bulbs properly when
concentrations of substrate and pH on levels of ethanol
growing at altitude of 800 m above sea level, while at produced.
altitude of 300 m above sea level cassava can not bloom
the flower, but they can only produce tubers. In 2005
cassava crop reached 19.5 tons with total area of 1.24
MATERIALS AND METHODS
million hectares (Prihandana et al. 2007). Cassava can be
harvested at the age of 9-12 months when the lower leaves
growth begins to decrease. The color of the leaves begin to Materials and equipment
This research materials are the cassava tubers found in
turn yellow and fall off a lot (www.warintek.ristek.go.id
traditional
markets, while iles-iles tubers obtained from the
2000). The product of cassava tubers are used to be
KUSMIYATI – Comparison of iles-iles and cassava tubers as raw material for bioethanol
garden as a secondary plant in Wonogiri. The enzyme αamylase, β-glukoamylase obtained from Daniso
(Genericor, USA). Saccharomyces obtained from the
Biology laboratory.
Sample preparation
Iles-iles tubers are peeled, washed, and cut into small
pieces and dried in the sun up to 3 days so that maximum
water content is 10. After that the tubers is mashed and
sifted (approximately 40 meshes) so that the obtained
particle size more uniform. The iles-iles flour are stored in
a dry place and used in a long time. The same process is
also carried on the manufacture of cassava tuber flour. The
process of iles iles tubers flour making can be seen in
Figure 2.
Stock culture of S. cerevisiae
Pure culture of S. cerevisiae is breed on for oblique
(PGY medium) which has been sterilized before at a
temperature of 1210C and at pressure of 1 atm for 15
minutes. PGY medium (Peptone Yeast glucose) made by
mixing 0.3 g of yeast extract, 0.3 g pantone, 0.4 g of malt
and 20 g of agar agar which is dissolved in 300 auades.
Stock cultures were incubated for 2-3 days at 28° C.
The process of enzyme production
Stock cultures of S. cerevisiae 200 is inoculated into
liquid medium containing 5 g (NH) 2HPO4, 5 g KH2PO4,
1 g MgSO4.7H2O, 1 g of yeast extract. After that it is
incubated with a rotary shaker at 150 rpm speed and at
300C for 24 hours. The same process for breeding preculture is done to breed the main culture; only with more
liquid medium as much as 500 m. Enzyme that is formed
by this process is used in the fermentation process.
The process of bioethanol production
The initial stage is liquification. First, dissolve 1 kg of
cassava flour in to water with a ratio (1:3,5, 1:4, 1:4,5, 1:5),
and then add α-amylase enzyme of 0:48 mL/kg . This
process is carried out by stirring the bulb flour at the speed
of 250 rpm for 4 hours until it becomes mush at a
temperature of 100 ° C. The process is followed by the
hydrolysis process by using the enzyme β-glukoamylase
with 0:48 concentration/kg, pH 4 for 40 hours. Glucose that
is produced in the process of hydrolysis is analyzed with
Nelson-Somogy method (Sudarmaji et al. 1984). The same
process applies for manufacturing bioethanol from iles-iles
tubers. Glucose that has been generated from the
9
saccharification then is fermented by using the yeast S with
a concentration of 10% (v/v) then DAP, urea and NaOH
are added to get the pH value at 6. This process lasts for 72
hours, levels of ethanol that is produced can be known
from the GC where the sample can be taken at hour 2, 8,
12, 24 and 72.
Determining the water content
Petri dish was dried in an oven (1050C) for ± 1 hour,
then cooled down in a desiccator and weighed (A). Tubers
samples (iles-iles and cassava) were weighed as much as 3
g (B). After that, the dish containing the sample was dried
in an oven at a temperature of 1050C for 2 hours, then
cooled in a desiccator and weighed to obtain permanent
weight (C). The water content can be calculated by formula
(AOAC 1984).
Waterconte nt =
( A + B)
− C x 100 %
C
Determining the starch content
Dissolve 5 g iles-iles tubers in 50, add HCl in to it,
close it, heat it above the heater water until boiling for 2.5
hours. When it is cool, neutralize it with NaOH solution
and dilute it until 500. The sample is titrated with Fehling
solution (Sudarmaji et al. 1984). Cassava tuber starch
content is measured by the same method.
Crude fiber analysis
Mash and then sift dry bulbs of iles-iles and cassava.
Weigh as much as 2 g and then extract the fat from it by
using soxhlet. Move all materials into 600 mL of
Erlenmeyer and add 3 drops of anti-foaming agent. After
that add 200 mL of boiling solution of H2SO4 (1.25 g
concentrated H2SO4) and cover it with coolant behind. Boil
it for 30 minutes and shake it a few moments.
Filter the suspension with filter paper, and then wash the
filter paper with boiling distilled water until no longer
acidic (acidity can be tested with litmus paper). Move the
residue in the filter paper into Erlenmeyer by using a
spatula and then wash the rest with 200 mL of boiling
NaOH (1.25 g NaOH/100 mL = 0.313 N NaOH), until all
the residue gets into the Erlenmeyer. Then boil it with
cooler behind while shake it for 30 minutes. Next filter it
using filter paper of known weight, while wash it with a
solution of K2SO4 10%. Wash the residue again with
boiling distilled water and then with 15 mL alcohol 95%
(Sudarmaji 1984).
a
b
c
d
e
Figure 2. The process of making flour iles-iles tuber, a. Tuber crops, b. Peeled tuber, c. Tuber is cut, d. Bulbs that have been dried in the
sun, e. Tuber flour iles-iles.
10
2 (1): 7-13, March 2010
Analysis sugar reduction
Sugar reduction was done by using the Nelson-Somogy
method. First make the standard solution of 0.1 M natrium
thiosulfat was prepared by dissolving Na2S2O3 into and
simmer for 5 minutes. Preparation of a solution of copper
reagent was made by mixing some of Na2SO4 and KI
solution, a solution of Na2CO3, KNaC4H4O6.4H2O
solution, NaOH solution, a solution CuSO4.5H2O and KIO3
solution, then this copper reagent was stored in dark
bottles. The Standardization of the copper reagent with the
main liquor 0.005 M sodium thiosulfat was carried out with
the main liquor thiosulfate. To analyze the levels of glucose
in the sample, it was added with 1 sample in to the reagent
solution of copper and then it was simmer at a temperature
of 95°C for 30 minutes. Then add H2SO4. Next, it was
titrated the sample by using a solution of Na2S2O3 with
starch indicator, and TAT point is reached when the blue
color turns clear. Each glucose levels is done in duplicate
samples (Sudarmaji 1984).
Determining ethanol by using GC
To analyze the levels of ethanol, centrifuge the fluid of
fermentation sample with speed 6000 rpm for 30 minutes
to separate supernatant and pellet. Take as much as 1μL
supernatant samples and inject it into the chromatography
gas column (6890 N, Agilent Technologies Inc., USA),
then equip it with a column HP-Innowax. Set the column
temperature at 200°C and set carrier gas using N2 (40/min).
Set the speed of gas flow rate for H2 at 40/min and for O2 at
500/min. Each sample is analyzed in duplicate.
RESULTS AND DISCUSSION
The content of raw materials
The material content percentage of iles-iles tubers is
different from cassava. From the analysis we find that total
sugar content of iles-iles tubers is 73.43% and cassava is
86.42%. Comparison of the content of other materials
contained in cassava and iles-iles can be seen in Table 1.
Table 1. Comparison of content of material found in the cassava
and iles-iles.
Percentage (%)
Iles-iles
Cassava
Wet
Dry
Cellulose
1.67*
8.54*
Hemicelluloses
10.5*
43.3*
Lignin
0.597*
5.85*
Sucrose
1.35*
Water
82.82*
62.50
Total sugar
73.43
86.42
Starch
71.25
83.47
* The Center Laboratory of Food and Nutrition Studies, Gadjah
Mada University.
Ingredients
Table 1 shows that dry iles iles contains cellulose,
hemicellulose, and lignin respectively, are 8.54%, 43.3%
and 5.85%. However, in wet conditions, the contents of
cellulose, hemicellulose and lignin on iles-iles becomes
lower, that is 1.67%, 10.5% and 0.597%. The content of
starch in the iles-iles is 71.25%, while cassava is 83.47%.
Starch is a polysaccharide compound which consists of
amylose and amylopectin (Campbell et al. 2000). Starch in
the iles-iles is so high that the bulb can be converted into
ethanol by using the enzyme amylase that will break the
monosaccharide monomers on starch into glucose. Besides
we can also use yeast S. cerevisiae to break down glucose
into ethanol.
Glucose levels during the process of hydrolysis
In general, bioethanol production from biomass consists of
two main processes, i.e. hydrolysis and fermentation.
Hydrolysis is a chemical process that uses H2O as a breaker
of a compound (Kuswurj 2008). The reaction between
water and starch goes so slowly that it needs assistance to
increase the reactivity of water catalyst. Acid solution is
often used in the process to accelerate the process, but in
this experiment we use biological agent by using enzymes.
According to Kolusheva and Marinova (2007) enzyme
hydrolysis has more advantage compared to chemical
hydrolysis. Chemical hydrolysis requires high temperatures
(150-230° C), acid pH (1-2) and high pressure (1-4). This
is different from the enzyme hydrolysis because it does not
require a high temperature, medium pH of 6-8 and normal
pressure. Enzymes are proteins that are catalysts, so often
called biocatalysts. Enzymes have the ability to activate
other specific compounds and to increase the accelerate of
chemical reactions that will last longer if not using
enzymes (Sun and Cheng 2002). Enzymes that is used in
this study is the enzyme α-amylase and β-glucoamylase. Αamylase enzyme plays important role in hydrolyzing α-1
,4-glucoside 19 specifically. This enzyme works at pH 5.7
and temperature 95°C. Enzyme amylase can not break
down starch bond perfectly so that the process will produce
dextrin with 6-10 chains units long (Schoonees 2004). The
results of liquification process is then forwarded by the βglucoamylase which can hydrolyze the bond of α-1,4glucoside and α-1 0.6-glucoside with a temperature of 60°C
and pH 4.2. Addition of β-glucoamylase in this experiment
is aimed at producing more glucose because βglucoamylase on starch can cut the starch bond that has not
been cut by the addition of α-amylase, by producing
glucose which has β-configuration in contrast to the results
of hydrolysis by α-amylase, so that glucose generated will
multiply or abundant. According to Kholuseva (2007)
hydrolysis that uses enzyme will produce higher reduction
sugar if compared with the acid hydrolysis. Reduction
sugar concentration during the hydrolysis process is
calculated using the Nelson-Somogy method. The process
lasts for 40 hours with a temperature of 60°C. The
comparison of reduction sugar in cassava and in iles-iles
can be seen in Figure 3.
Result shows that cassava has higher glucose content
than iles-iles. Measurement of glucose is conducted by
using Nelson-Somogy method. Iles-iles and cassava
hydrolysis that has a ratio of tub: water 1:4 shows that
glucose levels are influenced by the length of time. The
largest concentration of glucose is formed at the time
hydrolysis for 40 hours, which are 33.431 g/L for cassava
KUSMIYATI – Comparison of iles-iles and cassava tubers as raw material for bioethanol
11
Figure 3. Sugar content in iles-iles and cassava.
Figure 4. Ethanol content in the tuber iles-iles and cassava tubers.
Figure 5. Effect of iles-iles substrate concentration and water
concentrations toward ethanol, with yeast concentration of 10%
(v/v) pH 5.5.
Figure 8. Effect of pH on ethanol production.
Figure 6. GC chromatogram resulted from fermented iles-iles for
60 hours at pH 4.5.
Figure 7. GC chromatogram of fermented cassava tuber for 60
hours pH 4.5.
and 16.175 g/L for iles-iles. This happens because the
starch content in cassava is higher than in iles-iles, which is
83.47%, so that there is more glucose which can be
converted. It is expected that the greater the hydrolysis of
starch into glucose, the greater the ethanol produced in the
fermentation process.
Temperature is a factor that affects the α-amylase
hydrolysis process; hence in this study we include the
temperature variation in the process of α-amylase
hydrolysis. Effect of temperature on variation of glucose
levels can be seen in Table 2. From table we can conclude
that the optimum temperature for the two tubers is 95°C,
where the reduction sugar obtained from cassava and ilesiles respectively are 16. 176 g/L and 33. 431 g/L. This is
the same stated by Kolusheva and Marinova (2007) where
the research was performed under various temperatures
(30, 60, 90 and 100ºC) and found that the optimum
temperature was 90 and 100ºC where the process of
hydrolysis runs faster so that result of reduction sugar is
high.
Table 2. Effect of temperature variation on the hydrolysis process
of the glucose levels, with concentrations of α-amylase 0:48/kg,
time 40 hours, tubers and water concentration ratio (1:4).
Raw materials
Iles-iles tubers
Cassava tubers
Glucose level (g/L)
Temperature (°C)
90
95
100
105
14.57 16.176 16.041 15.221
29.145 33.431 30.451 28.451
110
13.245
27.219
Ethanol content during the fermentation process
After hydrolysis process, the glucose which has been
obtained will be converted into ethanol through a
fermentation process. The basic principle is to activate the
activity of microbial fermentation with the aim of changing
the nature of raw materials to yield a product. The
fermentation process of this study uses S because these
organisms can ferment glucose, mannose, fructose and
galactose in anaerobe and low pH conditions. Besides that
S is resistant to high alcohol content and high sugar levels
(Kartika et al. 1992; Shen et al. 2008). The process of
12
2 (1): 7-13, March 2010
fermentation by S. cerevisiae is done under anaerobic
conditions, and if during the process the air enters then the
ethanol formation process will be hampered. Therefore we
must put small hose on the jar that serves to release CO2
gas. The purpose is to prevent a temperature raise inside
the tube because S. cerevisiae is active at a temperature 432oC (Chin et al. 2010). Ethanol is fermented substrate due
to the activity of S. cerevisiae. Comparison of ethanol
content of fermented iles-iles and cassava are shown in
Figure 4.
Result analysis indicates that cassava yield higher
ethanol than iles-iles. A 72-hour fermentation using the S.
cerevisiae will produce highest ethanol either from iles-iles
and cassava respectively 53.49 g/L and 69.81 g/L. The
formation of ethanol is influenced by time, where the
longer the time of fermentation the higher the level of
ethanol will be. On the 24th hour ethanol content of each
tuber tends to be small i.e. 30.12 g/L for cassava and 28.51
g/L for iles-iles. But the longer the fermentation time, the
production of ethanol increases because the time that is
used for converting glucose by S. cerevisiae is longer,
resulting in higher ethanol. This is seen on the fermentation
time 54 hours, where ethanol that comes from cassava and
iles-iles are consecutively 43.21 g/L and 41.48 g/L.
Effect of substrate concentration and pH on ethanol
production
Substrate concentration affect very much the production
of ethanol, so to determine the effect of iles-iles substrate
concentration toward the ethanol production we must
perform variations of the adding water (1:3,5, 1:4, 1:4,5,
1:5 .) The level of ethanol content is then analyzed by using
chromatography gas. The effect of water variation and ilesiels raw materials toward the ethanol is presented in Figure
5. From the result it is known that the highest ethanol
content obtained from the ratio of raw materials: water 1:4
with ethanol content of 59.36 g/L. The proper combination
of raw materials and water will make the hydrolysis
reaction run fast, because if the water is too little then the
course of the reaction. According to Nowak (2000), if the
substrate concentration is too high (a little water), the
amount of oxygen will be too small; in fact the oxygen is
needed by S. cerevisiae to maintain life during the
fermentation process.
Levels of ethanol fermentation results were analyzed by
using Chromatography Gas. Figure 6 shows the
chromatogram profile of iles-iles ethanol which was
fermented for 60 hours. Result of the analysis shows that
iles-iles with GC will produce ethanol at retention time of
6.929 minutes, whereas cassava at retention time of 6.940
minutes. Chromatogram from GC analysis on cassava is
shown in Figure 7.
From the analysis using the GC we can see that the
ethanol content of iles-iles tuber with early fermentation
glucose concentration of 10% ethanol is formed by 44.4
g/L while the cassava 49.3 g/L. The degree of ethanol is
determined by the activity of yeast with the sugar substrate
which is fermented. According to Fessenden and Fessenden
(1997), one molecule of glucose will form two molecules
of ethanol and carbon dioxide. Too high concentration of
glucose will obstruct yeast growth, which makes the
ethanol content low. Ethanol is formed by the activities of
microorganisms in the substrate complex changes. Yeast
growth was greatly influenced by pH, because if the pH is
not appropriate yeast can not grow to a maximally, causing
the death which lowers the ethanol. Effect of pH on ethanol
is shown in Figure 8.
Bioethanol production is influenced by acid-base
conditions. According to Liu and Shen (2008) an optimum
conditions of acid base can improve bioethanol production
in the fermentation process because the acid-base
conditions is closely related to the interaction of enzymes
and raw materials. The degree of acid will affect the speed
of fermentation. This is consistent with the results of
research in which the process of cassava fermentation at
pH of 5.5 produces highest amount of bioethanol, which is
60.85 g/L. While pH 4 produces the lowest ethanol
concentration of 39.57g/L. Meanwhile iles-iles can produce
maximum ethanol at pH 5.5 where the ethanol content
obtained is 52.61 g/L. The results obtained prove that the
acid conditions increase enzyme S. cerevisiae work. This is
consistent with the study presented by Wilkins et al. (2007)
where the results of ethanol will increase at pH 5 and 5.5
and will decline at pH 4, and 4.5. The optimum conditions
are from pH 5 to 5.2. This result is consistent with Liu and
Shen study (2008), that acid pH is an important parameter
that can increase production of ethanol in fermentation
processes with enzyme S. cerevisiae
Test the efficiency of bioethanol fuel
Bioethanol which is obtained, is then used as fuel for
batik stoves. Batik stove is made by the researchers from
stainless steel with tank capacity 500 mL. This experiment
aims to determine the efficiency of bioethanol in melting
the wax. In this experiment the researcher used some
variation of ethanol degree as much as 100 mL to melt 20 g
of wax.
Table 3. Variations of bioethanol degree toward its use in melting
wax.
Parameter
Time to boil (min)
The time required to
evaporate out (hours)
Fire conditions when
evaporation
The condition of wax
the container
Concentration of bioethanol fuel (%)
40
50
60
70
80
90
22
22
19
18
17
11
2,7
2,5 2,4 1,65 1,6
1,4
Red
Red Blue Blue Blue
Soot Soot
-
-
-
Blue
-
From table it can be concluded 90% bioethanol grade
takes the shortest time in boiling wax, with evaporation
time of 1.4 hours for each 100mL. This is because the high
levels of bioethanol (fuel: water = 90:10) is easier to
vaporize. This is different for 40% and 50% bioethanol
where the flame is red and soot appeared, but the time
needed for ethanol to evaporate is longer than other levels.
This is due to the water content that is high enough so that
it took longer time to evaporate. However, high water
KUSMIYATI – Comparison of iles-iles and cassava tubers as raw material for bioethanol
content also produces carbon and soot during combustion.
The greater the water content in ethanol is the longer it
takes for the flame to run out. From the efficiency 90%
bioethanol have a very high volatile nature that makes it
less appropriate for use in combustion, because it tends to
be wasteful and quickly burnt. While 40% and 50%
bioethanol burns longer but it produces red flame and soot.
This results in less energy than blue flame. Blue fire
condition has more powerful energy for melting candle
burning instead of changing the ethanol into carbon/soot.
Based on efficiency 60% bioethanol then is more superior
to other grades of ethanol. Water content that is not too
high causes combustion produce blue and clean flame and
clean of soot, and in turn it will burn longer than any other
levels of ethanol.
CONCLUSION
For 40 hours iles-iles tuber hydrolysis (Amorphophallus
muelleri) has glucose content of 16.175 g/L and cassava
33.431 g/L. Degree of ethanol from iles-iles fermentation
for 60 hours is 44.4 g/L, while cassava is 49.3 g/L.
Temperatures difference will affect the speed of α-amylase
hydrolysis in converting glucose. The result showed that
the optimum temperature for hydrolysis is 95°C where the
concentration of glucose obtained on cassava and cassava
iles-iles are respectively 16 176 g/L and 33 431 g/L. The
substrate concentration and acidity will affect the speed of
fermentation. In this study the optimum ethanol degree was
found in the ratio 1:4 and pH 5.5. The result shows that
iles-iles tubers have the potential to be developed as a raw
material for bioethanol production.
ACKNOWLEDGEMENTS
The author would like to thank DP2M Higher Education
Research Grant Fund for the assistance of the fiscal year
2010 (Contract No. 089/SP2H/PP/DP2M/III/2010, dated
March 1, 2010). The author would like to thank Agnes H,
Agus Nur Arifin, Hesthi Chandra P, Diani Mentari, and Ina
Istiqomah in conducting this research.
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ISSN: 2087-3948 (print)
ISSN: 2087-3956 (electronic)
Vol. 2, No. 1, Pp. 14-22
March 2010
Variation of morphological and protein pattern of cassava (Manihot
esculenta) varieties of Adira1 and Cabak makao in Ngawi, East Java
TRIBADI1,♥, SURANTO², SAJIDAN²
¹ SMA Negeri 1 Kendal, Ngawi. Jl. Raya Ngawi-Madiun, Kendal, Ngawi, East Java, Indonesia
² Bioscience Program, School of Graduates, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia.
Manuscript received: 13 Augustus 2009. Revision accepted: 25 January 2010.
Abstract. Tribadi, Suranto, Sajidan. 2009. Variation of morphological and protein pattern of cassava (Manihot esculenta) varieties of
Adira1 and Cabak makao in Ngawi, East Java. Nusantara Bioscience 2: 14-22. This research is intended to find out the morphological
and anatomical variation as well as the protein band pattern of cassava (Manihot esculenta Crantz) widely spread in three different areas
of height. The sample collecting is done using simple random sampling in the three different areas of height that is 50, 300, 1000 meters
asl in Ngawi District, East Java while the analysis of protein band pattern is done using SDS-PAGE. The result of the reseach of
morphology and anatomy is analyzed descriptively and presented in the form of tabels, histograms and figures. The analysis of protein
band pattern is done using quantitative and qualitative analysis that is based on the appearance or not the gel band pattern by counting
the molecular weights based on code marker S 8445 and qualitative method based on the quality of the band formed. The band pattern
formed is istimated and presented in the form of zimogram. The result of the research shows that the height of the cultivating site very
much influences toward variations of root, stem and leaf morphology. The longest root is at 50 meter heights asl (Cabak makao local
variety, the widest stem diameter is at 50 meters asl (Cabak makao local variety) the longest leaf and branch is at 300 meters asl (Cabak
makao local variety) and 1000 meters asl (Cabak makao local variety). There is no difference of anatomy in the root, stem and leaf and
no difference of protein band pattern either in Adira1 or Cabak makao local variety.
Key words: Manihot esculenta, morphologic variation, anatomy, protein band pattern.
Abstrak. Tribadi, Suranto, Sajidan. 2009. Variasi morfologi dan pola pita protein uni kayu (Manihot esculenta) varietas Adira1 dan
Cabak makao di Ngawi, Jawa Timur. Nusantara Bioscience 2: 14-22. Penelitian ini bertujuan untuk mengetahui variasi morfologi dan
anatomi serta pola pita protein ubi kayu (Manihot esculenta Crantz) yang tumbuh pada tiga daerah ketinggian berbeda. Pengambilan
sampel dilakukan dengan metode sampel acak sederhana (simple random sampling) pada tiga ketinggian tempat yang berbeda yaitu
50,300,1000 m dpl di Kabupaten Ngawi, Jawa Timur serta analisis pola pita protein dilakukan dengan metode SDS-PAGE. Hasil
penelitian morfologi dan anatomi diuraikan secara deskriptip dan disajikan dalam bentuk tabel, histogram dan gambar. Analisis pola pita
protein dilakukan dengan menggunakan analisis kuantitatif dan kualitatif yaitu berdasarkan muncul tidaknya pola pita protein pada gel
dengan menghitung berat molekul berdasarkan marker kode S 8445 dan metode kualitatif berdasarkan kualitas pita yang terbentuk.Pola
pita yang terbentuk diestimasikan dan disajikan dalam bentuk zimogram. Hasil penelitian menunjukan bahwa ketinggian habitat
berpengaruh terhadap variasi morfologi akar, batang, dan daun. Umbi akar terpanjang pada ketinggian 50 m dpl (Cabak makao),
diameter batang terlebar pada ketinggian 50 m dpl (Cabak makao), panjang daun dan tangkai terpanjang pada ketinggian 300 m (Cabak
makao) dan 1000 m dpl (Cabak makao).Tidak ada perbedaan anatomi pada akar, batang dan daun serta tidak ada perbedaan pola pita
protein baik pada varietas Adira-1 maupun Cabak makao.
Kata kunci: Manihot esculenta, variasi morfologi, anatomi, pola pita protein.
INTRODUCTION
Cassava (Manihot esculenta Crantz) is not a plant
native to Indonesia, but has become very popular in
Indonesia. Cassava belongs to the family of shrubs plant
that is originally from the American continents, specifically
from Brazil and Central America (Mexico, Bolivia, Peru,
Venezuela, Guyana, and Suriname) (Nassar 1978 1992;
Olsen and Schaal 1999; Nassar et al. 1996, 2008; Allem
1994). The spread of cassava has been almost to the entire
world, including Africa, Madagascar, India and China.
These plants came into Indonesia in 1852. Cassava is
grown in agricultural areas. Cassava plant is widespread
throughout Indonesia, which has many local names, such as
katela, kentila, ubi kayee (Aceh), ubi parancik
(Minangkabau), ubi singkong (Jakarta), batata kayu
(Manado), bistungkel (Ambon), buari deur, vori jendral,
kasapen, sampeu, ubi kayu (Sunda), balet kasame, kaspa,
kaspe, ketela buding, katela jendral, katela kaspe, kaspa,
kaspe, katela budin,katela mantra, katila marikan, katela
menyok, katela paung, katela prasman, katela sabekan,
katela sarmunah, katela tapah, katela cengkol, ubi kayu,
tela pohong (Jawa), blandong, manggala menyok, pohung,
pahoung, sambrang balada, same, katela balada, tengsak
(Madura), kesame, ketal kayu, sabrang same (Bali), kasubi
(Gorontalo), bare, padu, lame kayu (Makasar), lame ayu
TRIBADI et al. – Diversity of cassava varieties of Adira-1 and Cabak macao in Ngawi
(Bugis Majene), and kasibi (Ternate, Tidore) (Heyne 1987;
Balitkabi 2009).
Cassava has been a staple food as well as comodity. It
has been the major source of food in food in South Africa
and certain areas in Indonesia. Cassava is a source of
carbohydrates for an estimated 800 million people around
the world (CIAT 1993; Nweke 1996). In Indonesia, this
plant stays in the third place after rice and maize as the
main source of carbohydrates. As a commodity Cassava
can be processed to produce dried cassava, tapioca,
ethanol, liquid sugar, sorbitol, monosodium glutamate, and
modified cassava flour (mocaf) (Harnowo et al. 2006;
Wargiono 2006). Cassava can be also an alternative source
of energy. This is in accordance with the Presidential
Regulation No. 5 of 2006 which says that the increased
production of cassava can be used as bio-ethanol fuel that
is mixed with 10% premium (premium mix E10).
There are three subspecies of Cassava. Cultivated
subspecies are all included M. esculenta subsp. esculenta,
which are closely related with the wild subspecies namely
M. esculenta subsp. peruviana that grows in Peru and
Brazil and wild species of M. esculenta subsp. flabellifolia
that grows in Brazil and Venezuela (Allem 1994, 2002).
This variety cassava (M. esculenta subsp. esculenta) which
consists of 7200 cultivars has been released. The varieties
of superior cassava that are commonly grown today
include: Adira-1, Adira 2, Adira 4, Darul Hidayah, Malang
15
1, Malang 2, Malang 4, Malang 6, 3 and UJ 5 (Subandi
2007). Cultivar Adira 4, Malang 6, UJ 3 and 5 has a
superior characteristic in accordance with the criteria for
the raw material of bioethanol (high starch content)
(Balitkabi 2009).
The purpose of this study are to identify the (i)
morphological, (ii) anatomical, and (iii) protein banding
pattern variation of cassava that exists in areas with three
different level of heights (50 m, 300 m, 1000 m) above the
sea level in the Ngawi District, East Java.
MATERIALS AND METHODS
The experiment was conducted from June 2008 to June
2009. Research morphology and the leaves sample of M.
esculenta done in several sub-centers of cultivation and
production of cassava in Ngawi District, East Java, namely:
(i) Northern Ngawi (+ 50 m asl), covering sub-district
Karangjati, Bringin and Karanganyar; (ii) Central Ngawi (+
300 m asl), covering sub-district Kwadungan, Paron and
Mantingan; (iii) Southern Ngawi (+ 1000 m asl), covering
sub-district Jogorogo, Ngrambe and Sine (Figure 1).
Ngawi District represents the northern lowlands, with
the height of 50 m above the sea level, so too the central
part of Ngawi, with the height of about 300 m above the
sea level. The area has the air temperature of 26-380ºC, the
1
6
5
3
2
9
8
4
7
Figure 1. Research sites of cassava in the Ngawi District. The north: 1. Bringin, 2. Karangjati, 3. Karanganyar; the center: 4.
Kwadungan, 5. Paron, 6. Mantingan; and the south: 7. Jogorogo, 8. Ngrambe and 9. Sine.
16
2 (1): 14-22, March 2010
rainfall of 1800 mm/year, its soil type is clay that becomes
hard when it is dry. The south part of Ngawi is a plateau
with the average of about 1000 m above sea level, with the
peak in Mount Lawu (3265 m asl). The nort part of Ngawi
is dominated by several plantations, cassava, tobacco, teak,
soybeans, corn and a little rice. The central part of Ngawi is
dominated by plantations, rice, cassava, tobacco, soybeans,
corn, sugarcane. The southern part Ngawi is dominated by
plantations, rambutan, tea, coffee, cassava, soybean, corn,
cocoa, and zalacca fruits (Office of Agriculture, Plantation
and Horticulture, Ngawi District 2009).
Material
The materials used in the research is M. esculenta from
three different altitudes in Ngawi District. The entire plant
is used for morphological study and to test the protein
banding pattern the third leaf from top of the pant is used.
The pattern of protein bands revealed by SDS-PAGE
method with protein dye system is Comassie blue, premises
marker code S 8445 (Sigma, Germany). Samples were
taken by simple random sampling method.
Procedures
Morphology and anatomy. Observations of morphology
include cassava’s roots (skin color, tuber color and flavor),
stems (distance segment and color), and leaves (shape,
color and stems). Observation of cross-sectional anatomy
covers the roots, stems and leaves.
Protein band patterns. Protein banding pattern
analysis was conducted using SDS-PAGE (Schägger et al.
1988; Artama 1991; Tarkka 2000). The third leaf from the
top part of cassava plant (two varieties, three locations in
Ngawi) is washed with a mortar and pestle mixed extract
buffer 500 uL. Once crushed and homogenized it is put into
in a tube with ependorf. Centrifugation is prepared and
when it has been more or less cold (with temperature of ±
0ºC) then the tube is inserted to be centrifuged with the
speed of 12,000 rpm for 5 minutes. Thus, the sample
solution is divided into two parts. The top of the colored
nodes (supernatant) will be used in the process of
electrophoresis, which is then stored in a place with the
temperature of 4ºC, while the bottom solid forms (pellets)
are removed. Supernatant is boiled for two minutes with so
that the protein can open.
Polyacrylamide gel consists of 2 parts, ie separating gel
that lies at the bottom with a concentration of 12% and
stacking gel which is located at the top with a concentration
of 3%. Separating gel is made by mixing ± 10 ml of stock
SDS PAGE 12%, plus 7 uL Temed and 80 uL APS 10%.
While the 3% stacking gel is made by mixing 5 ml of stock
3% stacking gel plus 3.5 uL Temed and 50 uL APS 10%.
Polyacrylamide gel solution is mixed. After it is
homogeneous the separating gel electrophoresis is put into
in the glass, after somewhat thickened some saturated
isobutene is added. After that the saturated isobutene is
removed and the stacking gel electrophoresis is included in
the glass just above the staking gel. After that the sample
comb is mounted on the stacking gel and released after it
gets solid, and until some holes are formed that will be
filled with the supernatant. The samples of Supernatant are
loaded into the hole as much as 10 uL. Before the
installation of the plate glass on the vessel electrophoresis,
it must be ensured that circulator temperature is less than
4ºC. After that the clip tube clamps and shied from the
glass plates are removed and then the glass plates are set
face to face to each other on the vessel electrophoresis,
with the notched glass plate is put inside. At the time of
installation there should be no air bubbles between the
glass plates, then tighten the bar holder. The running
buffer solution is added into the plate glass tanks that have
been installed face to face to each other so that it is just
right below the notch. After that the electrode buffer is
filled again until it is full and bathtub’s lid is put back
again. The power supply is turned on again to run the
electrophoresis process with electric current at 125 volts for
90 minutes or until the supernatant reaches the lowest limit.
After the electrophoresis process is complete, the gel is
taken and continued to get colored. By putting the gel on
the the plastic tray, then blue comassie is poured onto it and
let it there overnight. After that the gel is rinsed with the
destaining until clear. When the gel is clear, then the
washing is stopped by replacing the destaining with 10%
glacial acetic acid.
Data analysis
All data are described in descriptive method.
Observation of morphology, including roots (tuber), stems
and leaves are tested by doing analysis of the variance
followed by Duncan test to know the difference; then
presented in the form of tables, images and histograms. In
observation of the anatomy of the roots, stems, and leaves,
the preparats are microscopically photographed, and then
presented in the form of images and the results are
compared descriptively based on of the heights of the area
the plants are grown and the varieties. Data analysis
performed by the pattern of protein bands in quantitative
and qualitative method is based on gel banding pattern
appears or not by calculating the molecular weight marker
code based on the S 8445 (Sigma, Germany) and
qualitative methods based on the quality of banding pattern
formation.
RESULTS AND DISCUSSION
Morphology
The result of morphological study of cassava varieties
with research samples of Adira-1 and Cabak macao in the
areas with the height of 50 m, 300 m, 1000 m above sea
level in Ngawi District showed the presence of variations.
The results of morphological observation of cassava, the
varieties of Adira-1 and Cabak macao are shown in Figures
2 and 3 and Tables 1 and 2.
Adira-1
Adira-1 of northern Ngawi (50 m asl). The
characteristics are: has roots, outer brown and yellow skin
color, edible taste, stem segments with the length of 2-4
cm, an oval shape for the leaves, red color for the stalks
and no flowers. Of the five samples can be gained the
TRIBADI et al. – Diversity of cassava varieties of Adira-1 and Cabak macao in Ngawi
1Aa
1Ab
1Ac
1Ad
1Ba
1Bb
1Bc
1Bd
1Ca
1Cb
1Cc
1Cd
2Aa
2Ab
2Ac
2Ad
2Ba
2Bb
2Bc
2Bd
2Ca
2Cb
2Cc
2Cd
Figure 2. Morphology of cassava, the varieties of Adira-1 and Cabak Macao from various areas of
the heights. Note: 1. Adira, 2. Cabak macao; A. 50 m asl, B. 300 m asl, C. 1000 m asl; a. tuber roots
b. tuber color, c. stem, d. leaf.
following average: The length of the root is19.84 cm. The
length of one segment to another is 2.32 cm. The diameter
of the stem is 2.38cm. The length of the leaf is 9.72 cm.
The length of the stem is 13.84 cm. The location of the
study is Karangjati, Ngawi, with the average rainfall of
1800mm/year, the average temperature of 350C, 6 for the
pH of the soil , with grumusol for the type of the soil..
Adira-1 of central Ngawi (300 m asl). The
characteristics are: has roots, with brown skin color for the
outside part and yellow for the inside, yellow for the tuber,
and edible. 2-4 cm for the length of the stem, with yellow
color and oval for its leaf’s shape. The color of the stem is
red and the type of the flower is kind of combination of
many shades of brown color. Of the five samples can be
gained the following average: The length of the root is
35.28 cm. The length of one segment to another is 3.18 cm.
The diameter of the stem is 2.92 cm. The length of leaf
17
is14.64 cm. The length of
the stem is 21 , 48 cm.
The research’s location in
Kendal, Ngawi with the
rainfall of 1885 mm
rain/year, the average
temperature of 250C, 6 for
the soil’s PH, brown
Mediterranean for the type
of the soil.
Adira-1 of southern
Ngawi (1000 m asl). The
characteristics are: has
roots, with the brown skin
color for the outside part
and yellow for the inside,
yellow for the tuber and
edible. 2-4 cm for the
length of the stem with
yellow color and oval for
its leaf’s shape. The color
of the stalks is red and no
flowers. Of the five
samples can be gained the
following average: The
length of the root is 22.55
cm. The length of one
segment to another is 3
cm. The diameter of the
stem is 2.28 cm. The
length of the leaf is 14.88
cm. The length of the stem
is
23.04
cm.
The
research’s location is in
Jamus Ngawi, with the
average rainfall of 4473
mm/year, the average
temperature of 100ºC, 6
for the soil’s pH, and the
soil’s type is brown
lithosols.
Cabak macao
Cabak macao of
northen Ngawi (50 m asl). The characteristics are: has
roots, with the brown skin color for the outside part and red
for the inside, white color for the tuber, and edible. 2-4 cm
for the length of the stem, blackish green color, oval for the
leaf’s shape, light green for the stalk’s color, and no
flowers. Of the five samples can be gained the following
average: The length of the root is 47.44 cm. The length of
one segment to another is 2.96 cm. The diameter of the
stem is 3.92 cm. The length of the leaf is 17.44 cm. The
length of the stem is 26 , 6 cm. The research’s location is in
Karangjati, Ngawi, with the average rainfall of 1800
mm/year, the average temperature of 350C, 6 for the soil’s
pH, and grumusol taupe for the soil’s type.
Cabak macao of Central Ngawi (300 m asl). The
characteristics are: has roots, with the brown skin color for
the outside part and red for the inside, white color for the
18
2 (1): 14-22, March 2010
Table 1. The morphology observation of M. esculenta, varieties Adira-1 and Cabak
macao (planted in June 2008 - August 2009).
color for the tuber, and edible. 2-4
cm for the length of the stem with
blackish green as its color, light
Adira 1
Cabak makao
green its leaf’s color and brown for
Morphological
Southern Central Northern Southern Central Northern
its compound flowers. Of the five
characteristics
Ngawi Ngawi Ngawi
Ngawi Ngawi Ngawi
samples can be gained the following
Root Outer skin (brown)
√
√
√
√
√
√
average: The length of the root is
Inner skin (red)
√
√
√
38.6 cm. The length of one segment
Inner skin (yellow)
√
√
√
to another is 3.16 cm. The diameter
Tuber (yellow)
√
√
√
of the stem is 1.96 cm. The length of
Tuber (white)
√
√
√
Taste (good)
√
√
√
√
√
√
the leaf is 18.2 cm. And the length of
Stem yellow
√
√
√
the stem is 22.36 cm. The research’s
Darken green
√
√
√
location is in Jamus Ngawi with the
Leaves Shape (palm)
√
√
√
√
√
√
average rainfall of 4473 mm/year,
Petiole (red)
√
√
√
the average temperature of 10ºC, 6
Petiole (pale green)
√
√
√
for the soil’s pH, and the soil’s type
is brown lithosols.
Morphological
observations
Table 2. The average morphological characteristics measurement (cm) of M. esculenta,
cassava, for the varieties of Adira-1
varieties Adira 1 and Cabak macao based on altitude.
and Cabak macao from three areas
Length of
Internode
Stem
Length of
Length of
of research with different altitudes
tuber
distant
diameter
leaves
petiole
Altitude
of 50 m asl, 300 m above sea level,
Ad
Cm
Ad
Cm
Ad Cm Ad
Cm
Ad
Cm
and 1000 m above sea level on the
50 m dpl
19.84 47.44 2.32 2.96 2.38 3.92 9.72 17.44 13.84 22.36
length of the root, the length of one
300 m dpl
35.28 41.6 3.18 3.4
2.92 3.46 14.64 25.28 21.48 27.48
segment to another , the diameter of
1000 m dpl
22.55 38.6 3
3.16 2.28 1.96 14.88 18.2 23.04 22.36
the stem, the length of the leaf and
Note: ad: Adira, cm: Cabak macao.
the length of the stem, indicated
variations in morphological levels.
This is shown by data Table 2 and
Figure 3. It means that the
environmental factors in this case is
the altitude of the area has effects on
the
morphological
variations,
especially for the varieties of Adira-1
and Cabak macao in Ngawi.
Based on data in Table 2 and
Figure 3 for the varieties of Adira-1
can be gained some following data:
for the measurement of the length of
the root it can be concluded that
there are significant differences that
show that the altitude of where the
cassava is planted determines the
Figure 3. Comparative morphology of cassava varieties, Adira-1 and Cabak macao. Note:
length of the roots. The longest root
ad: Adira, cm: Cabak macao.
is found in the study sample planted
in the area with the height of 300 m
above sea level (35.28 cm). For the
tuber, and edible. 2-4 cm for the length of the stem,
measurement of the length of one segment to another it is
blackish green color, oval for the shape of the leaf, light
also found some differences but not as significant as the
green for the color of the stalks, and green for its
measurement foe the length of the roots. The longest is
compound flowers. Of the five samples can be gained the
found in the study sample in the area with the height of 300
following average: The length of the root is 41.60 cm. The
m above sea level (3.18 cm).
length of one segment to another is 3.4 cm. The stem’s
Altitude also affects the diameter of the trunk but not
diameter is3.46 cm. The length of the leaf is 25.28 cm. And
significant. For the length of the leaf it is also obtained data
the length of the stem is 27.48 cm. The research’s location
some differences in the results, but there are similar data in
in Kendal, Ngawi with the rainfall of 1885 mm rain/year,
the study sample at the height of 300 m and 1000 m above
the average temperature of 250ºC, 6 for the soil’s pH,
sea level that means that altitude in anyway also affects the
brown Mediterranean for the type of the soil.
morphology, particularly the variations for the length of the
Cabak macao of southern Ngawi (1000 m asl). The
leaves. Although not absolute, the altitude also affects the
characteristics are: has roots white bulb with the brown
length of the stalk. The data of longest stalks is obtained in
skin color for the outside part and red for the inside, white
TRIBADI et al. – Diversity of cassava varieties of Adira-1 and Cabak macao in Ngawi
the study sample in the area with the height of 1000 m asl
(27.48 cm).
Similarly, data from Tables 2 (Figure 3) for Cabak
macao can be obtained the following data: The altitude also
affects the morphological variations of the length of the
root. The longest root is found in the sample at a height of
50 m above sea level (47.44 cm). The altitude also affects
the length of one segment to another even though not really
significant. The longest is found in the altitude of 300 m
above sea level (3.4 cm). The diameter of the stem is also
influenced by the altitude though not significant. The
altitude shows significant influence on the morphological
variations particularly on the longest leaf’s length obtained
in the study sample with a height of 300 m asl (25.28 cm)
that is almost equal to the height of 50 m and 1000 m asl
and the longest stem length data in the study sample in the
area with the height of 300 m asl (27.48 cm) and almost the
same with the study sample in the area with a height of 50
m and 1000 m asl.
At the organism level, phenotype is something that can
be seen, observed and measured. It is a natural
characteristic for individuals. Phenotype is determined by
some genotypes of individuals, in some cases by the
environment where these individuals live, the time and in
some cases also by the interaction between the genotype
itself and the environment. Time is usually classified as
environmental aspects (of life) this can be written as
follows: P = G + E, with P means phenotypes, and E means
the environment. Observation of phenotypes can be simple
for instance to observe the color of flowers or the stalks or
can very complicated that requires special tools and
methods (Cheverud 1982).
For the same type of cassava found in the three
research’s locations with the height of 50 m, 300 m, and
1000 m asl showed no significant morphological variations,
except for length of the root, leaf and stalk. This variation
is related to the growth of each plant. The cassavas found at
the altitude of 300 m asl have bigger size then the ones at
the other two places with the same age. The differences
that emerged are related to the physical/environmental
factors where the cassava is planted. the research location
with the height of 300 m asl is a good and ideal place for
the growth of ideal crops.
Temperatures that are too low or too high can affect the
opening of stomata which in turn affects the photosynthesis
process (Levitt 1980). Temperatures above 30° C tend to
cause cassava stomata to open properly, so that the
photosynthesis works effectively and the plants grow faster
(Bueno 1986). While temperatures below 20°C tend to
cause the stomata to close (Akparobi et al, 2002a, b). Low
temperatures slow down the growth of cassava (ElSharkawy 2004). In addition, the response of stomata to
temperatures is also strongly influenced by water content
and humidity in plants (Berry and Bjorkman 1980).
According to Park et al. (1997) and Sulistyono et al.
(1999) anytime plants deal with environmental pressure,
they always make an adaptation. They may make adjustments
through changes in morphological and physiological
characteristics. Suchs an adjustment is made by for
19
instance making the leaves wider but at the same time
thinner (Taiz and Zeiger 1991).
Phenotype/morphological aspects in living creatures is
a combination of genotype and environmental factors
(Prawoto et al. 1987). The physical environment of the
northern of Ngawi is different from the one of the Central
and South Ngawi (in terms of altitude, rainfall, temperature
and soil type). Then the altitude for instance influences a
lot towards the phenotypes that arise in the form of
morphological characters in the study samples (cassava,
varieties of Adira-1 and Cabak macao), except for certain
characteristics such as the color of the outer and inner part
of the roots, the stem’s color and the taste. This can happen
because phenotypes that appear are not necessarily
morphological, they can be physiological. Changes in
physiological characteristics only influence the system so
that the cell performance can not be detected on
morphological levels.
Another possibility that caused the characteristics of
the study samples of the varieties of Adira-1 and Cabak
macao in northern, central and the south part of Ngawi
despite different environments is because genetic factors
may have a stronger influence than that of the
environmental factors. As stated by Suranto (2001) that the
emergence of variations can be caused by two factors
namely environmental factors and genetic factors. If
genetic factors have a stronger influence than
environmental factors, then individuals living in different
environments may not show morphological variations.
Anatomy
Analysis is based on cross-sectional slice on the
anatomical parts of cassava, for the varieties Adira-1 and
Cabak macao, covers cross-sections of roots, stems, and
leaves for the species planted in area with the different
heights: 50 m asl, 300 m asl and 1000 m asl presented in
Figure 4.
Adira-1
Roots. Based on the results of the cross sectional slice
with an enlargement of 4x10 mm, at an altitude of 50 m asl,
300 m asl and 1000 m asl, it can be gained that there is no
difference/almost the same as between the one in the areas
with the altitude of 50 m asl, 300 m asl and 1000 m asl.
Stems. The result of cross-sectional slice with 2.4
magnification x10 mm. There is little difference for the
density between cells shown from the plants in different
altitude of 50 m asl, 300 m asl, and 1000 m asl. The
altitude seems to influence the density between cells but
not really significant.
Leaves. parenchyma cells of the leaves are found
almost the same/no significant difference. The carrier
tissues (phloem and xylem) show a state that is not much
different either at the altitude of 50 m asl, 300 m asl, or
1000 m asl (Figure 4).
Cabak macao
Roots. Analysis based on the anatomy of roots, cassava,
the varieties of Cabak macao, with enlargement of 4x10
mm2. It can be found that there is no difference in density.
20
2 (1): 14-22, March 2010
Leaves. Analysis of the cross
sectional slice of the leaves,
focusing on the bone, with 4x10
magnification, shows similar looks
in terms of structures both at the
altitude of 50 m, 300 m, and 1000
m asl. Cells around the carrier
1Aa
1Ab
1Ac
tissues around shows no significant
difference either at the altitude of
50 m asl, 300 m asl or1000 m asl
(Figure 4). Based on the above
results all samples shows similar
looks and characteristics, although
they are planted in areas with
1Ba
1Bb
1Bc
different altitudes.
It can be understood that the
three research’s locations are still
in one region that is in Ngawi, so it
is possible that each sample of
existing research in these three sites
belongs to the same family that has
no genetic difference whatsoever.
2Aa
2Ab
2Ac
Genetic factors have stronger
influence than that of the
environmental ones, so that the
plants belonging one and the same
genetic characteristics show similar
looks even when planted in
different areas with different
2Ba
2Bb
2Bc
environmental factors. This is
supported by results based on
morphological variation indicating
that cassava with the same variety
found in different locations did not
show variations in morphological
levels.
Appearance of a phenotype
3Aa
3Ab
3Ac
depends on the nature of the
relationship between genotype and
environment.
In
fact,
the
development of an organism is
influenced by the state of the
environment and gene interactions.
Living organisms are always
3Ba
3Bb
3Bc
responsive to the environment
Figure 4. Cross sections of roots, stems, leaves of cassava varieties Adira-1 and local
during its development. In a broad
Cabak macao based on altitude. Note: 1. Root, 2. Stem, 3. Leaves. A: Adira-1, B: Cabak
sense,
environmental
factors
macao; a. 50 m asl, b. 300 m asl, c. 1000 m asl.
including both outside and inside
factors, affect how a phenotype
looks. Both of these factors can
The structure of the root tissues shows the similar look of
provide
a
major
influence
on the phenotype (Crowder
one from another.
1997).
Stems. Analysis on the stems to the variety of Cabak
The result of cross-sectional analysis/anatomy of roots,
macao with 4x10 mm magnification. It shows that the
distance or density between cells of the plants grown in the stems, leaves for the varieties of Adira-1 and Cabak macao,
areas with the altitude of 50 m asl and 300 m asl appear in the areas with the altitudes of altitude of 50, 300, and
smaller than at an altitude of 1000 m asl. It means that 1000 m asl in the district Ngawi can be described as
altitude only has insignificant effect on the anatomy of the follows: the distance between cells of the roots cross did
stem. Altitude also has little effect on the distance or not show significant differences. There was no difference
in the density between cells in the stems. There was no
density between cells.
difference in that of the leaves, too. The final conclusion of
TRIBADI et al. – Diversity of cassava varieties of Adira-1 and Cabak macao in Ngawi
this discussion is that the altitude where the cassavas are
planted has no significant effect on the anatomy of the
stems, roots and leaves.
The pattern of protein bands
According Suketi (1994) proteins or enzymes can be
separated by using electrophoresis and the result is
zimogram banding pattern. Zimogram electrophoresis
results of a typical patterned so that it can be used as a
characteristic phenotype to reflect genetic opener. In the
electrophoresis process of gel used polyacrylamide gel was
used. The percentage of polyacrylamide in the
electrophoresis is 7%, usually done in a tris glycine buffer
at the pH of 8.1. In certain cases the comparison between
polyacrylamide and pH is various (Suranto 2001).
Electrophoresis is processes in which molecules of
enzymes/proteins that have electricity moves through the
electric field. The speed of the molecule/protein of the
enzymes depends on the amount of the electric current. The
separation of one molecule/protein of enzymes from
another by the electrophoresis process is influenced by two
factors: the amount of the electric current and the size of
the particles
The results of the electrophoresis process on the
cassava’s leaf for the varieties of Cabak Adira-1 and
Macao with code S 8445, is shown in Figure 5.
Figure 5. Protein banding pattern of cassava leaf varieties of
Adira-1 (1, 3, 5) and Cabak macao (2, 4, 6). Note: 1: Adira-1 50
m asl, 2: Cabak macao 50 m asl, 3: Adira-1 300 m asl, 4: Cabak
macao 300 m asl, 5: Adira-1 1000 m asl, 6: Cabak macao 1000 m
asl., M = proteins marker (S 8445, Sigma).
Based on zimogram, the variety of Adira-1 (Figure 5,
nos. 1, 3, 5) expresses 20 bands; nos. 1, 2 (thick) MW was
not detected, no. 3 MW 158 kDa, no. 4 MW 92.6 kDa, no.
5 MW 88.2 kDa, no 6 MW 70.4 kDa, no 7 MW 66 kDa,
no. 8 MW 63.8 kDa, no. 9 (thick) MW 55 kDa, no. 10
(thick) MW 45 kDa, no. 11 (thick) MW 44 kDa, no. 12
(thick) MW 42 kDa, No. 13 (thick) MW 38.3 kDa, no. 14
(thick) MW 30.4 kDa, no. 15 (thick) BM 25.8 kDa, no.
21
MW 1623.7 kDa, no. 17 MW 20 kDa, nos. 18, 19, 20 MW
was not detected. Same banding pattern expressed both in
height (50 m, 300m 1000 m asl).
Cabak macao (Figure 5, nos. 2, 4, 6) expresses 20
bands. No.1, 2 (thick) was not detected, no 3 MW 158 kDa,
no. 4MW 92.6 kDa, no. 5 MW 88.2 kDa, no 6 MW 70.4
kDa, no. 7 MW 66 kDa, No. 8 MW 63.8 kDa, no. 9 MW
55 kDa, no. 10 MW 45 kDa, no. 11 MW 44 kDa, no. 12
MW 42 kDa, no. 13 MW 38.3 kDa, no. 14 MW 30.4 kDa,
no. 15 MW (thick) MW 25.8 kDa, no. 16 MW 23.7 kDa,
no. 17 MW 20 kDa, nos. 18, 19, 20 MW was undetected.
Band s were expressed equally well at a height of 50, 300,
1000 m asl.
Protein banding patterns for the varieties of Adira-1 and
Cabak macao in the areas with the altitude of 50 m asl (no.
1, 2) and 300 m asl (no. 3, 4) in general seem much thicker
than in those of the varieties of Adira-1 and Cabak macao
at the altitude of 1000 m asl (no 5, 6). This shows a higher
protein content that is possibly because at an altitude of 50
m and 300 m asl more sunlight is accessed that facilitates
the better photosynthesis process, including the formation
of proteins. The features of study samples of the protein
bands (Adira-1 and Cabak macao) at an altitude of 50, 300,
1000 m asl did not show any difference/variation. The
difference is only on the thickness bands due to the
differences in the number of migrated protein molecules or
the differences in the content/the quantity of protein. The
thickness of the bands does not indicate the difference of
the molecular weight, but only shows the differences in the
content/the quantity of migrated proteins (Maryati 2008).
Apparently, the limited number of samples tested may
cause the disappearance of protein polymorphism in
cassava, since several other studies have shown the
existence of polymorphism in cassava and its relatives with
the marker of proteins such as in the researches conducted
by Nassar (2003), De Souza (2002), and Nassar et al.
(2010). On the other hand, studies using isozym, which is
equivalent to the protein, to study the diversity of cassava
also show the polymorphisms in the population. Sumarani
et al. (2004) found that 37 polymorphic bands appear on
the test of 218 accessions of wild cassava with esterase
enzyme. Lefevre and Charrier (1993) found that from 365
cultivars and 109 accessions of wild cassava in Africa there
are 17 bands of polymorphism generated by 10 enzymes
dye. In Parana Brazil, Resende et al. (2000), found 28 loci
polymorphisms of local cassava samples with four enzyme
systems. Research by Montarroyos et al. (2005), on 28
accessions of cassava in Pernambuco, Brazil showed the
existence of 6 and 8 isozyme banding patterns with GOT
and peroxidase. Genetic diversity with isozyme in
populations of cassava were also found by Hussain et al.
(1987), Ramirez et al. (1987), and Sarria (1993).
CONCLUSION
The altitudes at which the plants are planted affect the
variation of morphology, the length of the root, tuber and
stalk. The longest samples are dominated by the ones from
the height of 300 m asl because of the height is a good
22
2 (1): 14-22, March 2010
habitat and an ideal place for planting cassava. Anatomical
observations indicate that the altitudes have no effect on
the anatomy of the roots, stems and leaves of the plants.
Analysis of protein band patterns showed that there were
no differences in protein band profiles of cassava samples
from different varieties (Adira-1 and Cabak macao) or
different altitudes (50 m, 300 m and 1000 m asl). The
difference is only the thickness of the bands due to the
differences in the content/quantity of migrated protein
molecules.
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Malang. [Indonesia]
ISSN: 2087-3948 (print)
ISSN: 2087-3956 (electronic)
Vol. 2, No. 1, Pp. 23-33
March 2010
Diversity analysis of mangosteen (Garcinia mangostana) irradiated by
gamma-ray based on morphological and anatomical characteristics
ALFIN WIDIASTUTI1,♥, SOBIR2,3, MUH RAHMAD SUHARTANTO2,3
¹ Center for Development of Seed Quality Testing of Food Crops and Horticulture (BBPPMBTH). Jl. Raya Tapos, P.O. Box 20, Cimanggis, Depok
16957, West Java, Indonesia. Tel.: +62-21-8755046/8754225. Fax.: 62+21 8755046/8754225
² Plant Breeding and Biotechnology Program, School of Graduates, Bogor Agricultural University, Bogor 16680, West Java, Indonesia.
3
Faculty of Agriculture, Bogor Agricultural University, Kampus IPB Darmaga, Bogor 16680, West Java, Indonesia.
Manuscript received: 13 December 2009. Revision accepted: 2 March 2010.
Abstract. Widiastuti A, Sobir, Suhartanto MR. 2010. Diversity analysis of mangosteen (Garcinia mangostana L.) irradiated by gammaray based on morphological and anatomical characteristics. Nusantara Bioscience 2: 23-33. The aim of this research was to increase
genetic variability of mangosteen (Garcinia mangostana L.) irradiated by gamma rays dosage of 0 Gy, 20 Gy, 25 Gy, 30 Gy,35 Gy and
40 Gy. Plant materials used were seeds collected from Cegal Sub-village, Karacak Village, Leuwiliang Sub-district, Bogor District,
West Java. Data was generated from morphological and anatomical characteristics. The result indicated that increasing of gamma ray
dosage had inhibited ability of seed to growth, which needed longer time and decreased seed viability. Morphologically, it also
decreased plant heigh, stem diameter, leaf seizure, and amount of leaf. Anatomically, stomatal density had positive correlation with
plant height by correlation was 90% and 74%. Gamma rays irradiation successfully increase morphological variability until 30%. Seed
creavage after irradiation increased variability and survival rate of mangosteen.
Key words: Garcinia mangostana, gamma ray, genetic variability.
Abstrak. Widiastuti A, Sobir, Suhartanto MR. 2010. Analisis keragaman manggis (Garcinia mangostana) diiradiasi dengan sinar
gamma berdasarkan karakteristik morfologi dan anatomi. Nusantara Bioscience 2: 23-33. Tujuan penelitian ini adalah meningkatkan
keragaman genetik manggis (Garcinia mangostana L.) yang diiradiasi dengan sinar gamma dosis 0 Gy, 20 Gy, 25 Gy, 30 Gy, 35 Gy dan
40 Gy. Bahan tanaman yang digunakan adalah biji yang dikumpulkan dari Kampung Cegal, Desa Karacak, Kecamatan Leuwiliang,
Kabupaten Bogor, Jawa Barat. Data dihasilkan dari karakteristik morfologi dan anatomi. Hasil penelitian menunjukkan bahwa
peningkatan dosis sinar gamma dapat menghambat pertumbuhan benih, sehingga membutuhkan waktu lebih lama untuk tumbuh dan
menurunkan viabilitas benih. Secara morfologi, hal itu juga menurunkan tinggi tanaman, diameter batang, ukuran daun, dan jumlah
daun. Secara anatomi, kepadatan stomata berkorelasi positif dengan tinggi tanaman dengan nilai korelasi adalah 90% dan 74%. Iradiasi
sinar gamma dapat meningkatkan keragaman morfologi hingga 30%. Pemotongan benih setelah iradiasi dapat meningkatkan keragaman
dan tingkat kelangsungan hidup manggis.
Kata kunci: Garcinia mangostana, sinar gamma, keragaman genetik.
INTRODUCTION
Mangosteen (Garcinia mangostana L.) is an Indonesian
original fruit commodities which have very good prospects
for further development. Mangosteen is a tropical fruit that
is very well known, and known as the Queen of Fruit
because of its delicious taste and its a lot of fans (Test
2007). In addition, the mangosteen has long been used as
medicine among them are as anti-inflammatory (Chen et al.
2008), antibacterial (Chomnawang et al. 2009), and for
treatment of infections and wounds. Improvement on
varieties of mangosteen aims to obtain high yielding
varieties that are directed to accelerate the growth of
mangosteen through improved root system, rapid
production (early maturing), high productivity and good
fruit quality. Mangosteen plant breeding to improve those
characteristics is constrained because the mangosteen plant
has a low genetic variability and no possibility of
increasing genetic variability through crossbreeding
because of the male flowers are rudimental (Morton 1987).
Mangosteen is a type of plant with very long juvenile
period, where the slow growth which is caused by poor
root system, the slow absorption of nutrients and water,
low photosynthetic rate and low cutting rate of cells in the
apical meristems (Ramlan et al. 1992; Wible et al . 1992;
Poerwanto 2000). The mangosteen seeds shape themselves
apomictically and develop from adventive’s embryos
asexually (Sobir and Poerwanto 2007). The asexual
regeneration of mangosteen leads to its low genetic
variability (Richard 1990b) and is genetically inherited
female elders’ characteristics (Koltunow et al. 1995).
According to Ramage et al. (2004), based on the study
Randomly Amplified DNA Fingerprinting (RAF) of 37
accessions of mangosteen, 70% showed no variation.
Mansyah’s reserach et al. (1999) on 30 plants of West
Sumatra’s mangosteens it can be concluded that the
24
2 (1): 23-33, March 2010
variability is narrow, although a few characters show a
wide phenotypic variability.
Efforts to improve the quality of mangosteen by
increasing genetic diversity need to be done. With the wide
variability, the selection process can be done effectively
because it will give more opportunity to gain the desired
characteristics or quality. One of the alternatives to
increase variability in apomictic plant is through artificial
mutation (Sobir and Poerwanto 2007). The use of radiation
to cause mutations or changes in genetic makeup has a lot
of positive impacts with an increase in the number of new
plant varieties. This technique contributes to the increase
genetic diversity and from the gained mutants there are
some which have superior characteristics. Fauza et al.
(2005) states that gamma ray irradiation on the mangosteen
seeds shows an increase in phenotypic variability in several
characters such as plant height, leaf number per plant, stem
diameter, and leaf width. In rice plants, radiation with
gamma rays at specific doses is known to be able induce
chlorophyll mutations and increase the genetic resistance to
blast disease (Mugiono 1996). Institute of Radiation
Breeding in Japan has been using mutation induction since
1969 to gain potential mutants. Some new varieties of
crops of apples, sugarcane, barley, and ornamental plants
have been released until 1998 (IRB 2001).
Radiation is enlightening process using radioactive rays
that can cause mutations. High energy radiation is usually
the form that release energy in large quantities and is
sometimes called ionization radiation because the ions are
generated in the material penetrated by the energy
(Crowder 1997). Mutations with radiation can increase
genetic variation. Cells that can survive well after
irradiation will undergo several changes in physiological or
genetic. These changes can produce better-looking plants
(plants superior) than before (Harahap 2005). Mutations are
resulted from all types of material changes derived. DNA,
which is a major component of genes as carriers of genetic
information from generation to generation, is the main
target of radiation delivery. DNA changes that occur as a
result of mutation, will lead to new genetic variations that
will be deployed on its derivatives. The success of mutation
can be observed through changes in morphology, anatomy,
and also at the DNA level. Mutants that show
morphological characteristics better than previous elders
and show the existence of a genetic difference is expected
to be developed into new varieties which are superior.
MATERIALS AND METHODS
Time and place
This research was conducted from January to August
2009 in the greenhouse and laboratory Research Center of
Tropical Fruits (PKBT), Bogor Agricultural University,
Laboratory of Microtechniques, Department of Biology,
Bogor Agricultural University, and Center for Research
and Development of Isotopes and Radiation Technology
(IP3TIR), National Agency of Nuclear Energy (BATAN),
Jakarta.
Plant material
Plant material used is the mangosteen seed harvested in
Kampong Cengal, Karacak Village, Leuwiliang Subdistrict,
Bogor District, West Java.
Experimental design
This study consisted of two experiments, namely: (i).
Mangosteen seeds cut after gamma ray irradiation
treatment, and (ii). Mangosteen seeds cut before gamma
irradiation treatment. Seeds were selected based on the
weight of > 1 g. The dose Gamma ray radiation used is
according to Harahap (2005) who states that 50% lethal
dose (LD 50%) derived from the mangosteen seeds were
32.09 Gy, so the doses used in this study was 0 Gy (I0) as a
control were 20 Gy (I1), 25 Gy (I2), 30 Gy (I3), 35 Gy (I4),
and 40 Gy (I5). The tool used for irradiation is a Gamma
Chamber 4000A with radiation source is a Co-60 radiation
dose rate of 96.481 krad / hr (0.96481 kGy / hr). Treatment
on cutting of the mangosteen seeds consists of three levels
i.e. : the seeds are left intact (B0), seeds are cut into two
equal parts (B1), and the seed is cut into three equal parts
(B2). Each experiment consisted of 18 experimental units
so that the total of the two experiments were 36
experimental units. Each treatment consisted of 10
replications so that the total population in this study was
360. Coefficient of variation (KK) is calculated based on
each level of treatment by using a completely randomized
design (CRD).
Implementation of experiments
Mangosteen seeds that had been extracted and cleaned
were broken to be divided into two groups. Group 1
(experiment 1) the seeds were cut after irradiation
treatment and group 2 (experiment 2) the seeds were cut
prior to irradiation treatment. Each group was divided
according to standard treatment combination of gamma
irradiation (0 Gy, 20 Gy, 25 Gy, 30 Gy, 35 Gy and 40 Gy)
and the standard of cutting seeds (whole, cut two, cut
three). Next, the seeds were inserted into a different paper
pocket and separated for each treatment combination. One
container is one experimental unit. seeds that have been
irradiated were planted in polybags in accordance with
their respective treatment.
Morphological observation
Observations were done on the seeds that formed buds
on
each
treatment
combination.
Morphological
observations were made by observing the number, the
lenghth, the width and shape of the leaves, and the diameter
and height of the stems. Observations were made since the
seeds germinated until they reached the age of 6 months.
Anatomical observations
Anatomical observations on the mangosteen’s leaves
were made on both the transferal and paradermal slices.
The paradermal slice was made using intact preparations
(whole mount) while transfersal incision was made by
following the paraffin method. The leaf was slashed using a
rotary microtome with a thickness 10 μm, and then it was
colored.
WIDIASTUTI et al. – Phenotypic variation of Garcinia mangostana after gamma ray irradiation
Data analysis
From the morphological data, the coefficient of
variation was measured using SAS ver. 9.1.3 (SAS 2004).
The data was also scored in binary measurement and was
analyzed using SAHN, after that its similar matrixs were
calculated using the program NTSYS ver. 2:01 and
displayed in a dendogram (Rohlf 2002). The grouping
showed similar relationship between each individual in the
form of morphological characteristics. Genetic distance is
the difference in value of the percentage of similarity to the
value of 100%. From the dendrogram it can be seen how
far the changes in crop radiation results when compared
with controls. The same was not reported for transverse
slices leaf anatomical parameters due to the data which was
so various, so it did not show a specific pattern.
Relationships and morphological parameters of the stomata
were gained by doing a correlation analysis using SAS
program ver. 9.1.3 (SAS 2004).
25
A
B
C
D
E
F
Figure 1. The growth of buds on the mangosteen seeds untreated
with gamma ray irradiation (A) and (B), and seeds that received
gamma irradiation treatment (C), (D), (E), and (F).
RESULTS AND DISCUSSION
At the beginning of the study, the number of seed
planted were 360 seeds and each seed was planted in a poly
bag. Not all seeds were grown to form buds, total number
of seeds capable of forming buds both in the control
treatment results and gamma irradiation were 57 seeds.
Data shown in this study is one related to the
morphological observation and the anatomy of the seed
treated by being cut and enlightened by gamma ray
irradiation. Morphological observations were carried out on
all the mangosteen seeds that formed buds, while the
anatomical observations were carried out on seedlings that
survived after 30 weeks of planting and showed normal
growth, which were not stunted.
Morphological observations
Morphological observations showed that the number
and time when the seeds to form buds, plant height, stem
diameter, leaf number, length and width of leaves of some
plants
that
underwent
irradiation,
experienced
abnormalities. Of 360 units of the experiment, only 57
seeds that formed buds, 14 of which belong control group.
This number decreased in the subsequent week of
observation, because some plants died that was preceded
by experiencing necrosis and the loss of leaves. The largest
number of buds was shown of the period 20 weeks after
planting, and in some control plants there grew more than
one sprout from one single seed. Control plants showed
normal growth in which plants did not experience any
delays in forming buds and the growth was not inhibited.
Harahap (2005) stated that the mangosteen seeds which did
not undergo gamma rays treatment, the buds generally
appear after 2 weeks of planting. Seeds which received
irradiation treatment showed a delay emergence of buds.
The formation and growth of buds on the control seeds and
the results of irradiation treatment are presented in Figure 1.
The morphological shape of mangosteen leaves both in
plants that received both treatment and controls were
generally ovate, obovate, and a small portion of them were
A
B
C
D
E
F
Figure 2. Mangosteen leaf’s morphology after 30 weeks of
planting: A. ovate, B. obovate, and C lacoleate. The color of
young leaves of mangosteen: D. control of red brick, E. brownish
green irradiated, and E. reddish brown control (f). Bar = 1 cm.
A
B
C
D
Figure 3. The color of the leaves after 30 weeks of planting: A.
control, B and C. 20 Gy irradiation treatment, D. 25 Gy
irradiation treatment (d). Bar = 1 cm.
26
2 (1): 23-33, March 2010
laceolate. From this observation, there was no specific
pattern that distinguished the shape of leaves in the control
plants from the irradiated ones. Flush that appeared on the
buds that did not receive irradiation treatment was
brownish red in color, while the flush that appeared on the
irradiated buds was usually light green in color with a very
slow growth. The morphological shape of the leaves and
the color of young leaves of mangosteen which appeared in
this study are presented in Figure 2.
Most of the young leaves of mangosteen were red brick
and reddish brown, but in some plants that received
irradiation treatment emerged young leaves that were green
with brownish color on the edges. Plant response to gamma
irradiation treatment is individual one, but there is a general
description of several variables of the outcomes. Leaves
emerged from the results of irradiation plants are generally
smaller, darker green in color, and thicker in texture.
The response of each mangosteen plant to the stress of
gamma ray irradiation was different. At the dose of 20 Gy
appeared the light green leaves and looked transparent, but
at a higher doses, ie 25 Gy and 30 Gy appeared leaves that
have a smaller size, dark green in color and thicker (Figure
3). Abnormality was a response to disruption of
physiological processes due to stress caused by gamma ray
radiation. According to Soeranto (2003), abnormalities in
the irradiated populations showed the occurrence of major
changes in the level of genomes, chromosomes and DNA,
so that physiological processes within cells that are
genetically controlled became not normal. Meanwhile,
according to Harahap (2005), changes in the leaf due to
irradiation are thought to occur because of the increased
amount of chlorophyll due to gamma ray irradiation stress.
Time and number of seeds that formed buds
Mangosteen seeds that are not treated with gamma-ray
irradiation and the average deduction showed the
emergence of buds after 3 weeks of planting (Table 1),
while seeds which received gamma irradiation treatment
alone (without cutting seed) the emergence of buds varied
between 3 and 16 weeks after planting. Buds of which
formation took the longest time was the individuals with 40
Gy irradiation treatments, but these plants grow very
slowly and not able to survive at the end of observation (25
weeks after planting). From this observation, it is seen that
the higher dose of gamma irradiation treatment is, the less
and the longer time required for the seeds for the
emergence of buds. At the doses high enough that above 30
Gy, most of seeds did not die or decay despite of being 20
weeks of sowing, but they also showed no signs of
emerging buds / sprouts. Then at the end of the study the
seeds which did not germinate eventually died and
decomposed. At a dose of 35 Gy, none of the seeds that
showed the emergence of buds, while at 40 Gy irradiation
treatment, there was one seed that emerged buds that took a
very long period which was 16 weeks after planting.
The number of plants that germinated in the control
treatment continued to increase until 15 weeks of planting
and reduced in on the 16th , 20th , and 25th week after
planting, that was from 33 plants on the 16th week getting
reduced to 30 plants on 20th week and to 25 plants on 25th
week after planting. In the treatment at 20 Gy and 25 Gy,
the number of surviving plants reduced after 20 weeks of
planting, respectively from 15 to 14 and 9 to 7 (Table 1).
Table 1. Number of mangosteen seeds that form buds.
Number of buds on the WAP
Level of
irradiation
3 4 8 9 10 11 13 14 15 16 20
( Gy)
Seeds irradiated before cut
Whole seed
0
5 7 9 8 8 8 10 10 10 10 6*
20
0 3 3 3 3 3 3 3 3 3 4*
25
0 2 2 2 2 1 3 4 4 6 5
30
0 0 0 0 0 0 0 0 0 0 0
35
0 0 0 0 0 0 0 0 0 0 0
40
0 0 0 0 0 0 0 0 0 0 0
Seed cut two
0
4 5 5 5 5 6 6 6 6 6 3*
20
0 2 2 3 3 3 3 3 3 3 3
25
0 0 0 0 0 0 0 0 0 0 0
30
0 0 0 0 0 0 0 0 0 0 0
35
0 0 0 0 0 0 0 0 0 0 0
40
0 0 0 0 0 0 0 0 0 0 0
Seed cut three
0
3 3 3 3 3 3 4 4 5 4 3*
20
0 0 0 0 1 1 1 1 1 1 1
25
0 0 0 0 1 1 2 2 2 2 1*
30
0 0 0 0 0 0 0 0 0 0 0
35
0 0 0 0 0 0 0 0 0 0 0
40
0 0 0 0 0 0 0 0 0 0 0
Seeds irradiated after cut
Whole seed
0
5 8 8 8 7 7 8 10 10 8 9
20
2 2 4 5 4 4 4 4 4 5 4*
25
0 0 0 0 0 1 1 1 1 1 1
30
0 0 0 1 1 1 1 1 1 1 1
35
0 0 0 0 0 0 0 0 0 0 0
40
0 0 0 0 0 0 0 0 0 1 1
Seed cut two
0
0 2 6 3 3 2 2 2 2 2 4
20
0 0 0 0 0 1 1 1 1 2 1
25
0 0 0 0 0 0 0 0 0 0 0
30
0 0 0 0 0 0 0 0 0 0 0
35
0 0 0 0 0 0 0 0 0 0 0
40
0 0 0 0 0 0 0 0 0 0 0
Seed cut three
0
0 0 0 0 0 0 0 0 0 0 0
20
0 0 1 1 1 1 1 1 1 1 1
25
0 0 0 0 0 0 0 0 0 0 0
30
0 0 0 0 0 0 0 0 0 0 0
35
0 0 0 0 0 0 0 0 0 0 0
40
0 0 0 0 0 0 0 0 0 0 0
Note: * Some plants were dead so that in on the 25th week the
number of plants got reduced. WAP = weeks after planting.
Treatment
This was due to some individual plants have sprouted
and emerged dying leaves, started with the drying of leaf
tips, followed by the entire leaf and eventually the fall of
the leaves. The death of those individual plants is due to the
irradiation stress. According to van Harten (1998), gamma
irradiation is destructive towards the path network it goes
through. In addition, because its penetrability is very deep,
the damage that it can cause can reach a few centimeters.
Ahnstroem (1977) and Datta (2001), state that both the
abnormality and the death of the irradiated plants are
caused by the formation of free radicals such as Ho, that
was a very unstable ion and caused a lot of collisions in
different directions that will create mutations in DNA, as
well as caused changes at the level of cellular and network.
It can even cause death in plants. In the control group, the
death of plants was suspected to be caused by
physiologically immature seeds. In the treatment of 30 Gy
WIDIASTUTI et al. – Phenotypic variation of Garcinia mangostana after gamma ray irradiation
27
only one seed that formed buds which survived until the
age of 20 weeks after planting, while at the 40 Gy
treatment, the buds that appeared was dead after 25 weeks
of planting.
Cutting the mangosteen seeds actually has effects on
the number of buds that emerged (Table 1). On the
irradiation level of 25 Gy, the truncated seeds after gamma
irradiation showed the higher number of seeds that formed
buds that was three seeds for the seeds that were cut into
two, and two seeds for the ones cut into three, compared to
seeds that were cut first before irradiation gamma rays,
which were the two seeds in the treatment group that was
two seeds for the seeds cut into two cut and no buds
appeared on the seeds cut into three after 16 weeks of
planting. Seeds treated with irradiation before being cut
showed the ability to form buds faster and higher than the
ones cut prior to irradiation. This is because that on the
seeds cut prior to irradiation; the tissues damaged due to
radiation are greater than the seeds cut after irradiation.
The decreasing number of leaves creates patterns in the
control treatment at the level of 20 Gy and 25 Gy, where
the higher the dose of the treatment using gamma ray
irradiation means the less number of the leaves. The effect
is due to physiological damage caused by gamma
irradiation. The length and width of the leaves also showed
a decrease in the size of some irradiated crops . According
to Patit (1966) and Ashri (1970), reduced size of the leaves
can occur due to irradiation and chemical mutagen treatment.
Plants height, stems diameter, and number, length and
width of leaves
The growth of the mangoosteen plants can be seen by
performing a detection on the morphological characteristics.
In this study, the measurement of morphological characters
was conducted on the height of the plants, the diameter of
stems, the number, the length and width of the leaves. The
height of the plants was measured from the neck of the root
up to the point where the plants grow, while the trunk
diameter was measured at a height of 1 cm above the root’s
neck. The length and width of the leaves were measured on
the leaves that emerged secondly.
The height of the plants, the stems’ diameter, the
number, length and width of the leaves gets decreased as
the doses of gamma irradiation increases (Table 2). This
happens because cellular damage happens to the plant’s
meristem. According Handayati et al. (2001), the damage
leads to the degradation of indole acetic acid (IAA)
because indole acetaldehyde dehydrogenase enzyme is
inhibited (Moore 1979).
Gamma ray irradiation has effects the on morphological
characters of mangosteen. These changes appear to be
individual, although they were irradiated at the same dose.
The comparison between control plants and plants
produced with gamma ray irradiation is presented in Figure
4. Gamma ray irradiation can affect the growth and
morphology of mangosteen. In this research it is gained
that at higher doses than 25 Gy, the mangosteen seeds
require just 9 weeks to form buds, while seeds without
irradiation only takes 3 weeks. The height of the plants, the
number, length and width of the leaves also shows the
response to irradiation dose of gamma rays, where the
higher dose of irradiation, the less the characteristics in
value. This suggests that high doses cause stunted growth
and even cause the seeds not able to grow. Barriers to
growth are in the form of physiological damage due to
gamma ray irradiation. The length and width of the leaves
also showed a decrease in the size of some irradiated crops.
Qosim (2006) stated that nodular callus of mangosteen with
A
B
Table 2. Average high of mangosteen, leaf number, stem
diameter, leaf length and width on the 25th week after planting.
Characteristics
Plant height
Stem diameter
Leaf number
Leaf length
Leaf width
C
0 Gy
5.85 ± 2.58
0.19 ± 0.09
1.65 ± 0.47
3.79 ± 1.67
2.32 ± 0.68
20 Gy
5.00 ± 1.77
0.15 ± 0.04
1.33 ± 0.47
3.36 ± 1.40
2.23 ± 0.48
25 Gy
4.40 ± 2.74
0.15 ± 0.06
1.33 ± 0.47
2.7 ± 1.30
1.45 ± 0.91
D
Figure 4. Comparison of control plants and the results of irradiation on the 25th week: A. Results of irradiation at the level of 25 Gy and
control. B. Results of irradiation at the level 25 Gy and 20 Gy, the combined seed is cut into two equal after irradiation. C. Control and
irradiated 25 Gy combinations of seeds cut into three equal after irradiation. D. Results of irradiation 25 Gy combined seeds cut into
three equal after irradiation.
28
2 (1): 23-33, March 2010
Table 3. The coefficient of variation (%) of each morphological character in (i) the extent of irradiation control (I0), 20 Gy (I1), and 25
Gy (I2), (ii) cutting seed: whole, cut in half, and cut into three (iii) treatment of seeds irradiated before being cut, and seeds irradiated
after the cut.
Morphological character
Number of buds
16 WAP
20 WAP
25 WAP
Plant height (cm)
16 WAP
20 WAP
25 WAP
Number of leaves
16 WAP
20 WAP
25 WAP
Diametar stem (cm)
16 WAP
20 WAP
25 WAP
Leaf length (cm)
16 WAP
20 WAP
25 WAP
Leaf width (cm)
16 WAP
20 WAP
25 WAP
Note: WAP = weeks after planting.
level of gamma irradiation
0 Gy
20 Gy
25 Gy
67.16
78.53
84.219
35.023
37.26
36.190
34.10
27.08
27.91
30.82
25.49
35.79
35.51
38.41
43.15
32.59
26.85
21.15
188.91
170.44
170.44
36.780
31.28
39.6
27.3
35.18
39.95
24.84
19.84
23.47
45.38
40.85
43.94
49.17
42.23
23.81
267.46
222.51
257.64
80.07
80.07
76.08
35
33.126
41.92
25.064
27.59
34.65
52.906
54.16
55.50
31.4
45.08
72.53
gamma ray irradiation at the level above 25 Gy took 129
weeks to form buds. Irradiation gamma rays can also cause
changes in the anatomy of mangosteen’s leaves. The
number of seeds capable of forming buds also decreases as
the dose of irradiation increases. At the doses greater than
25 Gy, only one seed that is able to form buds, i.e. at a dose
of 30 Gy on the 10th week of planting, and one seed at 40
Gy on the 16th week of planting. Decreased ability to form
buds or seed germination occurring with increasing dose of
irradiation was also observed in wheat (Gou et al. 2007),
peanut (Baddiganavar and Murty 2007), and soyseed
(Manjaya and Nandawar 2007).
Diversity test towards the mangosteen phenotypic
Effect of gamma irradiation
According Baihaki (1999), to determine the variation of
a population, the following rules of measurement and
analysis that are in accordance with statistical way need to
be done. The various populations will have specific
characteristics that can be seen from the coefficient value
describing diversity in a single treatment. Gamma ray
irradiation is a physical mutagen which can cause an
increase in the diversity of initial population.
In this study, the dose of gamma irradiation which
provides the highest variability based on coefficient of
variation is 25 Gy, while the dose that gives the lowest
diversity is 0 Gy (control) (Table 3). Almost all the
characters showed increased coefficients of variability as
the levels of irradiation increased, except for the stem’s
diameter, which is at the dose of 20 Gy the coefficients of
variability on the 16th, 20th, and 25 week of planting, are
lower than those in the control group. At the dose of 25 Gy,
the coefficient of variation for the stem’s diameter that is
greater than that of the control group is only found in the
one on the 20th week of planting (Table 3).
Coefficient of variance (%)
Cutting seed
Whole
137.82
131.76
162.01
42.93
43.65
41.99
35.49
30.37
31.38
25.47
24.60
27.37
39.20
41.06
42.99
36.38
34.10
31.04
Cut two
243.01
236.79
218.60
40.78
38.50
44.79
0
30.73
43.47
33.14
20.39
26.51
45.53
46.81
64.29
37.99
31.56
23.78
Seed cutting time
After
Before
Cut tree
irradiation irradiation
306.81
112.38
161.34
342.26
114.28
178.37
367.15
122.90
143.11
30.53
29.47
41.13
48.01
34.26
34.22
17.06
42.94
22.26
37.50
36.36
17.42
0
41.14
14.67
31.25
52.01
22.98
102
29.33
32.97
91.20
36.52
30.39
101.03
0
0
39.21
35.13
34.50
16.15
0
34.77
11.78
0
33.94
19.10
0
31.95
8.97
0
23.94
23.10
0
24.77
Increasing dose for irradiation led to the less ability of
seeds to form buds, which is marked by the declining
number of seeds that form the buds compared with the
control (Table 1). At doses where the seeds are still able to
form buds and grown into plants, it is known that 25 Gy is
the dose that most suppressing dose towards the growth of
mangosteen, which is characterized by small average value
for each observation of morphological characters compared
to those belonging to control group and at lower doses
(Table 2).
Effect of seed cutting level
Mangosteen seeds are poliembrionyc ones which can
grow more than one bud. Coefficient of variation on a level
of cutting seed treatment with the highest number of buds
is obtained from the seed treatment which is cut crosswise
into three equal sizes. For the height of the plant, the
highest coefficient of variation on 16th and 20th week of
planting is found in the treatment of intact seeds, while on
the 25th week of planting the highest coefficient diversity is
found from the seed treatment which is cut into two equal
sizes. The highest coefficient of the number of leaves from
the ones on the16th week of planting is found in the seed
treatment which is cut into three equal sizes, while from the
ones on the 20th and 25th week of planting is found in the
treatment of seed which is cut into two equal sizes. For the
stem’s diameter, the highest coefficient of diversity is
found on the seed treatment which is cut into three equal
sizes, while for the length of leaves the highest coefficient
is obtained in the treatment of the seed which is into two
equal sizes. The highest coefficient of diversity for the
width of the leaves found in the plants on 16th and 20th week
of planting is obtained on the seed treatment which is cut
into two equal sizes, meanwhile the highest coefficient of
WIDIASTUTI et al. – Phenotypic variation of Garcinia mangostana after gamma ray irradiation
diversity for the plants on the 25th week of planting is
found on the seed treatment which is left intact (Table 3).
Coefficient of variation which is higher on the
morphological characters derived from the cutting of
mangosteen seeds is related to the nature of poliembrionyc
of the mangosteen seeds. A single of mangosteen seed can
grow more than one bud, where each bud emerges from
different sections, and allegedly carries different genetic
constitutions as well. Mansyah et al. (2008) stated that of
the nine seeds of poliembrionyc mangosteen it can be seen
the differences on the DNA bands on the buds that grow
from the seeds of the same mangosteen.
Effect of seed cutting time
The time for cutting seeds can be divided into after and
before irradiation. The treatment for the seeds cut before
irradiation has a higher coefficient of variation than the
ones cut after irradiation (Table 3). This is because in the
treatment for the seeds cut before irradiation, the exposure
of irradiation directly hits the surface of the seed that
experiences injury from the cutting so that the effect
becomes larger, while in the seeds that are cut after
irradiation, the exposure to gamma rays only hit the surface
of the seeds so that the radiation effect is smaller.
Increasing of morphological diversity due to gamma
ray irradiation
Mangosteen is included in obligate apomixes plants,
whose seeds are not derived from the results of fertilization
but developed from adventive’s embryos asexually (Sobir
and Poerwanto 2007), thus might have low genetic
diversity (Richards 1990b; Varheij 1992; Cox 1996).
Apomixes on mangosteen plants cause the same genetic
trait in the progeny the same as with that in the parents
(Koltunow et al. 1995). Induction of irradiation with
gamma rays is one alternative to increase genetic diversity
in plants in which the occurrence of cross-fertilization is
not possible.
In this study, the dendogram drawn from the
morphological observation of mangosteen plants showed
that gamma irradiation treatments can increase the diversity
compared to ones belonging to control group. The
similarity on values in the plants that do not have gammaray irradiation treatment ranged from 13-83% (Figure 5A),
while the ones the plants are treated with gamma-ray
irradiation ranged from 0-100% (Figure 5B). Diversity
increased by 30% after the induction of gamma ray
irradiation. Gamma rays include mutagens that produce
ions and free radicals in the form of hydroxyl (OH-). If
hydroxyl radicals are attached to the chain of nucleotides in
DNA, the single strand of DNA will be broken and
undergo some genomic changes (Mohr and Schopfer
1995). Visually the diversity in maize growth due to the
influence of gamma irradiation becomes larger (Herison et
al. 2008).
In the mangosteen plants that do not get gamma-ray
irradiation treatments, the greatest similarity is found in
plants without the cutting seed treatment which are I0B0
and B0I0, respectively by 83%. Broadly speaking, the
research results are divided into two that are plants derived
29
from intact seeds and plants whose seeds are cut into two
or three equal size. MXComp cophenetic value generated
from the control plants is (r = 0.967) with very appropriate
goodness fit, while the value of the irradiated ones is (r =
0.956). Clustering analysis on the results of gamma
irradiation plants do not provide specific grouping between
control plants and plants produced with gamma ray
irradiation. This happens because the nature of mutations
caused by irradiation of gamma rays is random. One
mutant plant derived from irradiation of 25 Gy (B0I2) is in
the group of plants without irradiation treatment that is on
the similarity of 100%. This shows that at the
morphological level, these plants do not differ from the
ones untreated with gamma ray irradiation.
Increasing morphological diversity by cutting seed process
The time of cutting the mangosteen seeds, whether
before or after irradiation, makes difference in improving
the diversity based on morphological observations.
Mangosteen seeds irradiated prior to getting cut gives a
smaller similarity of 43-88% (Figure 5C), while the
mangosteen seeds receiving irradiation after the cut has a
similarity of 0-83% (Figure 5D). The pattern in cutting the
seeds is also seen to lead to diversity, both in the irradiation
treatment before and after cutting. In Figure 5C, based on
the cutting pattern of mangosteen seeds, the dendogram is
divided into two groups on the similarity of 43%, i.e. the
first group only consisted of plants from the intact seeds
only(I0B0) and group two consisted of plants from the
seeds of the mangosteen which are cut into two equal
(I0B1) or three equal (I0B2)sizes. At intervals of 60%
similarity, the individual plants I0B2 and I0B1 are in
different groups, indicating the existence of diversity
between both of them.
Mangosteen seeds are poliembrionyc ones, meaning
that one seed can grow more than one bud. Each bud has a
different genetic constitution because they come from
different embryos. Mansyah et al. (2008) stated that four
out of nine seeds of poliembrionyc mangosteen seeds it can
be seen the different DNA bands in the buds that grow
from the seeds of the same mangosteen.
Anatomical observations
Transfersal section
The structure of mangosteen leaves on the transferal
slice consists of the layers of cuticle, upper epidermis,
palisade parenchyma, spongy parenchyma and lower
epidermis. The epidermal tissue is covered by cuticles
which are spread throughout the upper and lower leaf
surfaces. The structure of mangosteen’s leaves belong to
the type of dorsiventral as a palisade parenchyma tissue is
between the upper epidermis and spongy tissue. The results
showed that the cuticle is on the upper and lower surfaces.
The palisade parenchyma of Mangosteen’s leaf consists of
two layers which are under the upper epidermis, while the
sponge layer is under the parenchyma palisade (Figure 7).
The observation on the mangosteen leaves transversally
sliced was conducted towards 16 plants that visually show
a good growth.
30
2 (1): 23-33, March 2010
I0B0
B0I0
I0B1
I0B2
B1I0
0.13
0.30
0.48
0.66
Koefisien kemiripan
0.00
0.25
0.50
0.83
I0B0
B0I2
B0I0
I1B0
I2B2
B0I3
I0B1
B0I1
I1B1
I2B0
B1I1
I0B2
B1I0
B0I5
0.75
1.00
A
B
Koefisien kemiripan
I0B0
I1B0
I2B2
I0B1
I2B0
I1B1
I0B2
0.43
0.54
0.65
0.76
0.88
C
Koefisien kemiripan
B0I0
B0I2
B0I1
B0I3
B1I1
B1I0
B0I5
0.00
0.21
0.42
0.62
0.83
D
Koefisien kemiripan
Based on Table 4 it can be gained that the range of
values in a thick cuticle, upper epidermis, lower epidermis,
palisade parenchyma, spongy parenchyma and the
thickness of the mangosteen’s leaves vary greatly. There is
no particular pattern between the thickness of cuticle of the
individual belonging to control plants and that of the
irradiated ones.
Table 4 shows that the thickness of upper epidermis and
lower epidermis for most crops is almost the same.
Epidermal tissue is the tissue that serves to protect the
underlying tissue and serves as a coating for gas exchange
to and from outside the body through the hole plant
stomata. Changes in the thickness of the epidermis can be
caused by the ionizing nature of gamma rays which can
penetrate the epidermal layer and cause the changes. Other
factors affecting the changes in leaf anatomical characters
beside the regenerant are the environment factors such as
the availability of water, light intensity, the concentration
of CO2, and the temperature which can affect the density of
stomata (Willmer 1983).
Some plants which are the result of irradiation
treatment showed substantial palisade thickness values,
namely B0I3, I1B01, and I0B02. The thickness of sponges
and the highest thickness of leaves are obtained from the
control plants (B0I03), while the lowest obtained from the
plants produced at the irradiation level of 25 Gy. The
thickness of the sponge tissue is associated with the
thickness of space between cells, where the thicker the
sponge tissue, the greater the spaces between cells that are
useful for storing water and CO2. According to Fahn (1991)
the important factors that can increase the efficiency of
photosynthesis is the space between cells which is very
well located in the mesophyll, thus facilitating gas
exchange quickly.
Figure 5. Mangosteen dendogram based on morphology: A.
Control. B. Gamma ray irradiation treatment. C. Cutting the seed
after irradiation. D. Cutting the seed prior to irradiation.
Table 4. Observations of leaf transversal section in some individuals of mangosteen (µm).
Individual plants
Cuticle
Average thickness (µm)
Upper epidermis Under epidermis
Palisade
Sponges
Leaf
Seeds irradiated before cut
I0B01
3.88 ± 1.24
10.00 ± 2.04
7.75 ± 1.53
42.50 ± 6.87
184.51 ± 25.02
64.37 ± 7.85
I0B02a
2.88 ± 0.60
14 .00± 1.29
10.25 ± 1.42
48.00 ± 6.21
180.75 ± 5.01
75.38 ± 5.76
I0B03
4.00 ± 1.29
7.75 ± 1.84
9.25 ± 2.64
44.5 1± 11.59
202.25 ± 31.27
65.77 ± 13.81
I1B02b
2.50 ± 0
10.13 ± 2.53
11 ± 3.37
46.5 3± 5.29
176.51 ± 6.89
70.37 ± 7.56
I1B01
3.75 ± 1.17
12.00 ± 2.58
9.25 ± 1.20
51.52 ± 8.26
178.75 ± 24.24
76.75 ± 6.61
I1B1
2.63 ± 0.39
10.52 ± 2.29
9 ± 1.29
42.52 ± 6.97
188.25 ± 13.64
64.88 ± 7.45
I2B0
3.38 ± 1.19
7.13 ± 1.87
6.25 ± 1.77
42.03 ± 8.32
151.03 ± 8.99
58.96 ± 10.08
I2B2
3.13 ± 1.06
9.13 ± 2.04
7.5 ± 1.67
43.25 ± 6.13
140.75 ± 20.01
63.20 ± 6.06
Seeds irradiated after cut
B0I02
3.52 ± 1.49
10.25 ± 1.84
9.25 ± 2.059
48.25 ± 6.46
195.25 ± 27.11
71.51 ± 7.06
B0I03
3.75 ± 1.18
10.50 ± 3.07
10.00 ± 2.63
55.00 ± 8.97
203.25 ± 19.04
79.53 ± 9.95
B0I04
4.54 ± 0.87
9.75 ± 2.75
9.75 ± 2.19
55 .00± 10.99
167.50 ± 19.01
79.24 ± 12.52
B0I01
4.50 ± 1.05
9.87 ± 2.8
8.50 ± 1.74
53.50 ± 5.02
168.50 ± 12.08
76.62 ± 5.08
B0I2
4 .01± 1.15
9.25 ± 2.37
8.88 ± 1.71
51.25 ± 7.38
185.5 0± 11.71
73.63 ± 6.16
B0I3
5.37 ± 2.13
13.12 ± 3.19
8.63 ± 2.91
57.25 ± 11.27
176.5 1± 24.58
84.63 ± 13.59
B1I02
3.13 ± 0.88
10.52 ± 1.97
8.5 0± 2.11
52.75 ± 9.75
173.01 ± 10.85
75.12 ± 8.77
Note: I0 = 0 Gy irradiation, I1 = 20 Gy irradiation, I2 = 25 Gy irradiation, I3 = 30 Gy irradiation, B0 = seeds intact, B1 = cut two seeds,
B2 = cut three seeds
WIDIASTUTI et al. – Phenotypic variation of Garcinia mangostana after gamma ray irradiation
b
31
a
c
h
d
g
i
e f
A
B
C
D
E
E
Figure 6. The comparison of the anatomical structure of mangosteen leaf transversely sliced (magnification 400x). A. Control, B.
Irradiation of 20 Gy the whole seeds, C. Irradiation of 25 Gy seeds cut in half, D. Irradiation of 30 Gy whole seeds, E. Irradiation of 20
Gy seeds cut in half, F. Seeds cut into two without irradiation. Description: Cuticle of (a), upper epidermis (b), palisade parenchyma (c),
sponges (d), below the epidermis (e), lower cuticle (f), beam vessel (g), idioblas (h), gland secretion (i).
a
b
c
d
A
B
C
D
E
Figure 7. Comparison of leaf anatomical structure paradermal slices. (100x). Description: A. Control, B. Seed is cut in two, C. 20 Gy,
seed cut in two, D. 25 Gy, seed cut three, E. Irradiation of 25 Gy whole seeds. Description: opening of stomata (a), guard cells (b), the
neighboring cell (c), and epidermal cells (d).
Almost all plants produced by gamma ray irradiation
show a thick palisade and leaves except for I2B2 plants that
instead showed the smallest number in thickness.
According to Dickison (2000), the plants’ response to
gamma-ray radiation that has the nature of ionization can
cause changes in the leaf’s anatomy. Leaves may
experience changes such as tissue necrosis, distortion of
leaves’ bones, changes in the composition and size of
palisade tissue and the enlargement of spongy tissue. The
differences of the sectional of paradermal slices on the
mangosteen’s leaves both the control plants and the ones
treated with gamma irradiation are presented in Figure 6.
Paradermal section
This observation show the stomata of mangosteen leaf
is found on the upper part of the leaf. Paradermal slices
show that in the epidermal layer of mangosteens leaves
there are stomata, guard cells, neighboring cells, and
epidermis cells. Observations on the paradermal slice on
the mangosteen’s leaves show the characteristics of the
observed variations. The highest number of stomata on the
plants is found in the plants without irradiation treatment
and the plants irradiated with 25 Gy combined with the
cutting of the seeds into three equal sizes, while the lowest
is obtained in the plants irradiated with gamma ray
irradiation at the level of 25 Gy, with an intact form of the
seeds. The highest number for epidermis is obtained in the
plants with 25 Gy irradiation with seeds cut into 3 (I2B2),
while the lowest number is obtained from the plants
without irradiation treatment (I0B03). The index for the
stomata ranges from 5.223 to 9.78 (Table 5). The highest
index and density of stomata is obtained in the plants
without the treatment of gamma irradiation (I0B03), while
the lowest ones is obtained from the plants with irradiation
of 25 Gy (Figure 7).
The mangosteens which are the results of gamma
irradiation which survive experience anatomical changes
on the leaves. The same thing is also reported by Harahap
(2005) and Qosim (2006) of mangosteen leaves that is
grown in vitro. Dickison (2000) states that gamma-ray
radiation that has ionization in nature can cause changes in
the leaf’s anatomical structure. Gamma ray irradiation is
known to increase the thickness of the cuticle, epidermis,
palisade and leaves in some individuals which are the result
of gamma irradiation, although the range of increase varied
and showed no pattern of increasing doses of irradiation.
According Qosim (2006), the plants that have thick cuticle
is more likely to have properties more tolerant to drought
because a thicker cuticle can reduce the rate of
transpiration of water and can reflect sunlight. Cuticle also
serves to protect plants from pests and diseases. Stomatal
index observations on paradermal slices shows that in the
gamma-ray irradiation treatment, the stomata index has a
smaller number than the plants without irradiation
(control), the smallest density of stomata is also obtained
in the plants irradiated with the level of 25 Gy, whose seed
is cut into two equal size. Mangosteen which has stomata
with a high density allows a high gas exchange or
absorption of CO2 so that the photosynthetic rate becomes
higher. With a higher rate, fotosintat, which is the result of
2 (1): 23-33, March 2010
Leaf
length
-0.16
0.02
-0.07
1
Leaf
width
0.13
0.17
0.07
0.74*
1
Results of correlation analysis between variables of the
cuticle’s thickness, upper epidermis, lower epidermis,
palisade and spongy to the morphologic variables of the
mangosteen plants showed that plant’s height, leaf’s width
and length are not significantly affected by the thick of the
cuticle, upper and lower epidermis, the thickness of
palisade parenchyma and the sponges of mangosteen’s leaf
(Table 7). The height of the was significantly affected by
the width of the leaf with a correlation value of 76%.
Cuticle
Upper epidermis
Under epidermis
Palisade
Sponges
Leaf length
Leaf width
Plant height
1 -0.17 0.04 0.01
1 0.08 -0.04
1 0.17
1
0.08
0.17
0.1
0.15
1
Plant height
Karakter
Leaf width
Table 7. The value of correlation between the thickness of cuticle,
upper epidermis, lower epidermis, palisade, and sponges and the
length, the width of the leaves and the height of the plant.
Leaf length
Correlation between morphological and anatomical
characters
Plants growth and development is influenced by factors
that are interrelated, both internal and external. The
correlation test between the morphological characters
namely the height of the plants, the length and width of the
leaves, with the stomatal index character and the density of
stomata, indicates a positive correlation between the height
of the plants with the density of stomata, and the length of
the leaves with the their width. The existence of a
correlation between the height of the plants and the density
of stomata indicates that characteristic is influenced by the
density of stomata. The level of correlation between the
height of the plants and the density of stomata was 90%
(Table 6) which means that the density of stomata has 90%
role in determining the height of the plants, while the
correlation between width and the length of leaves was
74% (Table 6). High density of stomata allows easier
process of photosynthesis so that the fotosintat that can be
generated will be greater in results, and then the growth
and development of the plant are more supported. Positive
correlation between the height of the plant and the density
of stomata enable the stomata density parameter to become
Anatomical
Stomatal Stomatal
Plant
characteristics
index
density
height
Stomatal index 1
0.02
0.01
Stomatal density
1
0.90*
Plant height
1
Leaf length
Leaf width
Sponges
Average
Individual
Number of Number of
Stomatal
Stomatal
plants
stomata
epidermis
index density b/(mm2)
Seeds irradiated before cut
B0I01a
13.00 ± 1.22 205.00 ± 27.07 6.06 ± 1.19 184.03 ± 17.33
B0I03b
14.2 0± 1.48 176.20± 17.16 7.49 ± 0.93 200.99 ± 20.99
B0I02
13.20 ± 4.38 205.80± 16.02 6.09 ± 2.23 186.84 ± 62.02
B0I2
10.00 ± 2.01 182.41± 15.46 5.22 ± 1.16 141.54 ± 28.30
B1I01b
14.02 ± 0.71 207.02 ± 26.90 6.38 ± 0.61 198.04 ± 10.01
Seeds irradiated after cut
I0B01b
11.61 ± 1.14 194.81 ± 12.29 5.62 ± 0.38 164.18 ± 16.14
I0B02b
14.22 ± 1.64 204.42 ± 22.81 6.56 ± 1.09 200.99 ± 23.25
I0B03
18.81 ± 2.59 174.60 ± 14.18 9.78 ± 1.62 266.10 ± 36.63
I1B1
13.82 ± 2.38 195.00 ± 28.46 6.67 ± 1.25 195.33 ± 33.79
I2B2
18.63 ± 3.13 213.20 ± 39.51 8.23 ± 2.46 263.27 ± 44.30
Note: I0 = 0 Gy irradiation, I1 = 20 Gy irradiation, I2 = 25 Gy
irradiation, I3 = 30 Gy irradiation, B0 = seeds intact, B1 = cut two
seeds, B2 = cut three seeds.
Table 6. The value of correlation between stomatal index,
stomatal density, plant height, leaf length and width.
Palisade
Table 5. The average number of stomata, number of epidermis,
stomatal index, and stomatal density of mangosteen leaf.
a criterion to measure the growth of mangosteen.
Under epidermis
photosynthesis process, the plant’s growth is more
supported. Qosim (2006), states the regeneran mangosteen
which has a high density of stomata, palisade parenchyma
and a high number of file vessels can be used as indirect
selection criteria for efficiency. Harahap (2005) states that
the study of anatomical structure of the mutant is very
useful to explain the changes in the genetic control of
certain processes. Fahn (1991) found a recessive mutant of
maize which has survived is found to have changed its
anatomy in the form of the obstruction towards the
differentiation process on the stem’s vessels. Cutter (1969)
states that the cells that can grow after irradiation are
expected to experience physiological or genetic changes.
Irradiated plants that will survive are expected to add
diversity to increase the effectiveness of selection.
Upper epidermis
Cuticle
32
0.02
0.12
0.18
0.25
-0.12
1
-0.1
0.12
0.12
0.01
-0.08
0.24
1
-0.16
0.35
0.29
0.06
-0.07
0.3
0.76*
1
CONCLUSIONS AND RECOMENDATIONS
Gamma ray irradiations with the doses of 0, 20 Gy, 25
Gy, 30 Gy, 35 Gy and 40 Gy increase the diversity of
morphology of mangosteen by 30%. The highest increase
of diversity in the mangosteen obtained in the plants with:
(i) the dose of 25 Gy irradiation, (ii) with the seed cut into
two equal size, and (iii) the cutting of the seeds done after
gamma ray irradiation. The biggest increase of the diversity
of the mangosteen is obtained by the method of irradiation
on the seeds with the dose of 25 Gy and then arecut across
seed into two equal sizes. The density of stomata has a
positive correlation with the height of the plants by 90%.
The density of stomata can be used as a criteria to estimate
the growth of mangosteen. To get a mangosteen with
greater diversity, it is advisable to perform irradiation on
the mangosteen with the dose of 25 Gy with a more
number of the research materials.
WIDIASTUTI et al. – Phenotypic variation of Garcinia mangostana after gamma ray irradiation
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Handayati W, Darliah, Mariska I, Purnamaningsih R. 2001. Increasing
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Manjaya JG, Nandanwar RS. 2007. Genetic improvement of soybean
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ISSN: 2087-3948 (print)
ISSN: 2087-3956 (electronic)
Vol. 2, No. 1, Pp. 34-37
March 2010
First record of two hard coral species (Faviidae and Siderastreidae)
from Qeshm Island (Persian Gulf, Iran)
MAHDI MORADI1,♥, EHSAN KAMRANI1, MOHAMMAD R. SHOKRI2, MOHAMMAD SHARIF RANJBAR1,
MAJID ASKARI HESNI3
1
Department of Marine Biology, School of Basic Sciences, University of Hormozgan, P.O. Box 3995, Bandar Abbas, Iran. Tel: +98-914-9818304; Fax:
+98-761-766 0012. ♥email: [email protected]
2
Department of Marine Biology, Faculty of Biological Sciences, Shahid Beheshti University, G.C., Evin, Tehran 1983963113, Iran
3
Department of Biology, Faculty of Science, Shahid Bahonar University of Kerman, Kerman, Iran.
Manuscript received: 30 December 2009. Revision accepted: 13 February 2010.
ABSTRACT
Abstract. Moradi M, Kamrani E, Shokri MR, Ranjbar MS, Hesni MA (2009) First record of two hard coral species (Faviidae and
Siderastreidae) from Qeshm Island (Persian Gulf, Iran). Nusantara Bioscience 2: 34-37. Two species of hard corals including
Cyphastrea chalcidicum (Forskal 1775) (Faviidae) and Coscinaraea monile (Forskal 1775) (Siderastreidae) were collected from the
south of Qeshm Island (Persian Gulf, Iran) in the late of 2008. These species were previously reported from southern Persian Gulf, Gulf
of Aden, Southeast Africa and Indo-Pacific. The literature review on the distribution of these two species revealed that these species
were firstly recorded from the Persian Gulf. These findings further emphasize the high diversity of coral fauna in the Iranian waters of
the northern Persian Gulf.
Key word: first record, Coscinaraea monile, Cyphastrea chalcidicum, Qeshm Island, Persian Gulf.
Abstrak. Moradi M, Kamrani E, Shokri MR, Ranjbar MS, Hesni MA (2009) Rekaman pertama dua spesies karang keras (Faviidae dan
Siderastreidae) dari Pulau Qeshm (Teluk Persia, Iran). Nusantara Bioscience 2: 34-37. Dua jenis karang keras termasuk Cyphastrea
chalcidicum (Forskal 1775) (Faviidae) dan Coscinaraea monile (Forskal 1775) (Siderastreidae) dikumpulkan dari selatan Pulau Qeshm
(Teluk Persia, Iran) pada akhir tahun 2008. Spesies ini sebelumnya dilaporkan terdapat di Teluk Persia selatan, Teluk Aden, Afrika
Tenggara dan Indo-Pasifik. Tinjauan literatur pada distribusi kedua jenis mengungkapkan bahwa spesies ini pertama kali tercatat dari
Teluk Persia. Temuan ini semakin menunjukkan tingginya keragaman fauna karang di perairan Iran di bagian utara Teluk Persia.
Kata kunci: catatan pertama, Coscinaraea monile, Cyphastrea chalcidicum, Qeshm island, Persian gulf.
INTRODUCTION
The Persian Gulf has a complex and unique tropical
marine ecosystem, especially coral reefs, with relatively
low biological diversity and many endemic species (Price
1993) In this area, the coral reef communities are occurred
in the form of non-reef setting (Riegl 1999) and surrounded
by some of the driest landmasses in the world, such that
continental influences are limited (Price 1993). While large
parts of the region are still in a pristine condition, several
anthropogenic threats notably habitat destruction, overexploitation and pollution are ever-increasingly disturbing
the coral reef communities, . The coral reef communities in
the Persian Gulf are less diverse than that of Indian Ocean
(Price 1993). This is due to the high salinity; high daily
amplitude of temperature (Coles and Fadlallah 1991) and
occasional extreme low tides (Reynolds 1993) that make
the environmental condition is unfavorable to coral reef
communities.
The largest island in Persian Gulf is Qeshm Island (ca.
122 km long, 18 km wide on average, 1,445 sq km). This
island is located a few kilometers off the southern coast of
Iran (Persian Gulf), about 22 km south of Bandar Abbâs
and not far from Bandar Khamir (DHI 1976).
A study was conducted to explore the species diversity
of hard corals in Qeshm Island, in order to bridge the gap
of knowledge on species inventory of hard corals in this
area. The results of the study have been presented in detail
elsewhere (Moradi et al., in prep.) and this paper presents
only the new recordings of two hard coral species from
Qeshm Island (Persian Gulf, Iran).
MATERIAL AND METHODS
The coral survey was conducted in August 2008. Hard
coral specimens were collected by SCUBA diving from
two sites (Naz and Zeitoon) within shallow non-reef
MORADI et al. – First record of two hard coral species from Qeshm Island
settings (6 to 10 m deep) with hard ground substrate in the
south of Qeshm Island, (Iran, Northern Persian Gulf). The
geographical positions of the sampling sites were N 26º
49' 19.4" and E 56º 07' 23.1" for Naz station, and N 26º
55' 40.15" and E 56º 15' 54.82" for Zeitoon Station (Figure
1). The coral specimens were bleached using hydro
peroxide and photographed showing the whole specimen
and the corallite structures. Identifications were performed
using available references, especially Veron (2000), and
through communication with Prof. Charles Sheppard at the
Dept. of Biological Sciences, Warwick University for
further checking. The materials are deposited in at the
Faculty of Marine Biology, University of Hormozgan, Iran.
RESULTS AND DISCUSSION
Results
Twenty one species of hard corals belonging to 8
families were identified and with resulting Poritidae and
Faviidae as the dominant families. Two species,
Coscinaraea
monile,
(Forskal
1775)
(Family:
Siderastreidae) and Cyphastrea chalcidicum (Forskal 1775)
(Family: Faviidae) were new records from northern Persian
Gulf.
Coscinaraea monile (Forskal 1775)
Kingdom Animalia
Phylum Coelenterata Frey and Leuckart 1847
Subphylum Cnidaria Hatschek 1888
Class Anthozoa Ehrenberg 1831
Subclass Zoantharia de Blainville 1830
Order Scleractinia Bourne 1900
Family Siderastreidae (Vaughan and Wells, 1943)
Genus Coscinaraea (Forskal 1775)
Coscinaraea monile (Forskal 1775) (Figure 2)
Taxonomic references: Scheer and Pillai (1983)
Material examined: Qeshm Island, Naz Island, depth 811m, collector M. Moradi, 24 August 2008.
Diagnosis characters: Colonies are encrusting or
massive, 10-30 cm in diameter sometimes larger. Corallites
is are 2.5 to 3.5 millimeters in diameter and form a liner
series in meandroid valleys. In some cases, there is no
demarcation between adjacent corallites and others. Tthere
is an irregular, low, thin wall marking the boundary. In
massive colonies, calices are 2-4 mm in diameter; in
explanate corolla, calices are 3-6 mm in diameter. Up to 30
septa occur at the wall, but only 8-9 reach the columella
due to fusion of adjacent septa;, septa and septocostae are
lightly granulated and the marigine (?) are divided into
sharp dentations.
Color: Light brown
Habitat: Abundant in 8-10 meter depths
Distribution: This species is confined to the Indian
Ocean and is are mostly common along the shores of the
southern Persian Gulf, Oman Sea Red Sea, Gulf of Aden
(Veron 2000) and Southeast Africa, (Riegl 1996).
Cyphastrea chalcidicum (Forskal 1775)
Family Faviidae (Gregory, 1900)
Genus Cyphastrea (Forskal 1775)
35
Cyphastrea chalcidicum (Forskal 1775) (Figure 3)
Taxonomic references: Veron, Pichon and WijsmanBest (1977), Wijsman-Best (1980).
Material examined: Qeshm Island, Zeyton Park, depth
2-5 m, collector M. Moradi, 19 June 2008.
Diagnosis characters: Colonies are encrusting to
massive, usually about 20-35 cm in diameter Corallites are
round, variably exsert, usually about 1.5-2.5 mm diameter,
budding is extratentacular. There are 20-26 septa arranged
in 2 orders, inner septal margins of primaries and
secondaries carry rounded dentations and descend into the
calices at about 45 degree angle. All primaries reach the
columella, some secondaries do not. Septa are sparsely
granulated and septa are not continuous with those of
adjacent corallites. Septocostae are sub-equally exsert
about 0.5 mm above the wall. Costae are equally exsert, the
columella is small less than 0.4 mm in diameter composed
of tangled by synapticular ring. The coenosteum is covered
with short tapering spines.
Color: Usually uniform brown, green or cream with
corallite walls and calices of contrasting colors.
Habitat: Abundant in 3 meter depths.
Distribution: This species is confined to the Indian
Ocean and are mostly common along the shores of Red
Sea, Gulf of Aden (Veron 2000) and Southeast Africa
(Riegl 1996).
Discussion
Harger (1984) reported 19 species of corals at Hormuz
Island in the east of Qeshm Island, Persian Gulf. Staghorn
corals (Acropora sp.) were found to be the dominant
species around the islands in the Persian Gulf (Sheppard
and Sheppard 1991), whereas the massive corals (Poritidae,
Favidae) are dominant corals at present. Staghorn corals
are defined as disturbance-adapted types for their rapid
growth rate and fragility (Done, 1982; Karlson and Hurd
1993). Massive and submassive corals being defined as
stress-tolerators (Veron, 1986; Rogers 1990) are shown to
tolerate to the high sedimentation and/or eutrophication.
Presence of massive corals in Qeshm Island suggests that
the reef corals reefs in this island are likely subjected to
high sedimentation and/or eutrophication. The species
found in the present study are massive types that are
confined to the Indian Ocean, mostly common along the
shores of Persian Gulf, Oman Sea, Red Sea and Gulf of
Aden (Veron 2000). Cyphastrea chalcidicum (Forskal
1775) was reported from Southeast Africa (Riegl 1996),
Gulf of Aden, Indo-Pacific and Indiana Ocean (Veron
2000) and Coscinaraea monile (Forskal 1775) was reported
from Northern Red Sea (Riegl & Velimirov 1994),
Southeast Africa (Riegl 1996), Dubai, (Riegl 1999), Oman
sea (Coles 1996), Gulf of Aden, Indo-Pacific and Indiana
Ocean (Veron 2000).
The shift in coral diversity from disturbance-adapted
types (Acropora branching corals) in the past to stresstolerators (Favia and Porites massive and submassive
corals) at present indicates that coral species composition
in Qeshm Island have been altered over three decades
(1984 to present). The corals in Persian Gulf have recently
experienced multiple bleaching events (1996 1998 2002)
36
2 (1): 34-37, March 2010
Naz Island
1
2
Figure 1. Study area and location of sampling sites, Qeshm Island (Persian Gulf, Iran). 1. Zeyton Park, 2. Naz Island (above insert).
A
B
Figure 2. Concinarae monile, A: Colony, B: Corallites. Bar = 2 cm
A
Figure 3. Cyphastrea chalcidicum, A. Colony, B. Corallites. . Bar = 2 cm
B
MORADI et al. – First record of two hard coral species from Qeshm Island
(Pilcher, et al. 2000; Wilkinson 2000; Wilson et al. 2002;
Rezaei et al. 2004) causing mass mortality of Acropora
corals in the entire region. The climatic change revealed in
multiple bleaching events associated with high
sedimentation and/or eutrophication in this area may be
possible factors altering the coral species diversity in the
study area. Further studies are required in the Persian Gulf
to reveal the possible effects of climate change on reef
corals.
CONCLUSION
Two species of hard corals including Cyphastrea
chalcidicum (Forskal 1775) (Faviidae) and Coscinaraea
monile (Forskal 1775) (Siderastreidae) were firstly
recorded from the south of Qeshm Island (Persian Gulf,
Iran). These species were previously reported from
southern Persian Gulf, Gulf of Aden, Southeast Africa and
Indo-Pacific. These findings further emphasize the high
diversity of coral fauna in the in Iranian waters of the
northern Persian Gulf.
ACKNOWLEDGEMENT
We would like to express our appreciation to Mrs. Lale
Daraei management of GEF/SGP in Iran, Abdullah Salehi
and Mohammad Dakhte management of Geopark Institute
of Qeshm Island who assisted us in filed sampling. We are
also greatly honored and thankful to Prof. Charles
Sheppard at the Department of Biological Sciences,
Warwick University, U.K. for assistance in identifications
of the specimens.
37
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ISSN: 2087-3948 (print)
ISSN: 2087-3956 (electronic)
Vol. 2, No. 1, Pp. 38-42
March 2010
Isolation and identification of lactic acid bacteria from abalone (Haliotis
asinina) as a potential candidate of probiotic
1
SARKONO1,♥, FATURRAHMAN1, YAYAN SOFYAN2
Faculty of Mathematics and Natural Sciences, Mataram University, Jl. Majapahit 62 Mataram 83125, West Nusa Tenggara, Indonesia. Tel./Fax.: +62370-646506 ♥email: [email protected]:
2
Institute for Marine Aquaculture, Grupuk, Sengkol, Pujut, Central Lombok 83511, West Nusa Tenggara, Indonesia.
Manuscript received: 13 December 2009. Revision accepted: 15 March 2010.
Abstract. Sarkono, Faturrahman, Sofyan Y. 2010. Isolation and identification of lactic acid bacteria from abalone (Haliotis asinina) as
a potential candidate of probiotic. Nusantara Bioscience 2: 38-42. The purpose of this study was to isolate, select and characterize lactic
acid bacteria (LAB) from abalone as a potential candidate probiotic in abalone cultivation system. Selective isolation of LAB performed
using de Man Rogosa Sharpe medium. LAB isolate that potential as probiotics was screened. Selection was based on its ability to
suppress the growth of pathogenic bacteria, bacterial resistance to acidic conditions and bacterial resistance to bile salts (bile). Further
characterization and identification conducted to determine the species. The results showed that two of the ten isolates potential to be
developed as probiotic bacteria that have the ability to inhibit several pathogenic bacteria such as Eschericia coli, Bacillus cereus dan
Staphylococus aureus, able to grow at acidic condition and bile tolerance during the incubation for 24 hour. Based on the API test kit,
the both of isolate identified as members of the species Lactobacillus paracasei ssp. paracasei.
Key word: lactic acid bacteria, isolation, identification, Lactobacillus paracasei ssp. paracasei.
Abstrak. Sarkono, Faturrahman, Sofyan Y. 2010. Isolasi dan identifikasi bakteri asam laktat dari induk abalon (Haliotis asinina) yang
berpotensi sebagai kandidat probiotik. Nusantara Bioscience 2: 38-42. Tujuan penelitian ini adalah untuk mengisolasi, menyeleksi dan
mengkarakterisasi Bakteri Asam Laktat (BAL) dari induk abalon yang berpotensi sebagai kandidat probiotik pada sistem budidaya
abalon. Isolasi selektif BAL dilakukan menggunakan media de Man Rogosa Sharpe Agar. Isolat BAL yang berpotensi sebagai probiotik
diskrining. Pemilihan ini didasarkan atas kemampuannya dalam menekan pertumbuhan bakteri patogen, resistensi terhadap kondisi
asam, resistensi terhadap bile salt (empedu). Selanjutnya dilakukan karakterisasi dan identifikasi untuk mengetahui spesiesnya. Hasil
penelitian menunjukkan bahwa 2 di antara 10 isolat yang berhasil diisolasi dari abalon berpotensi untuk dikembangkan menjadi bakteri
probiotik karena mempunyai kemampuan menghambat beberapa bakteri patogen yaitu Eschericia coli, Bacillus cereus dan
Staphylococus aureus, mampu tumbuh pada kondisi asam dan toleran terhadap cairan empedu selama inkubasi 24 jam. Berdasarkan uji
API Kit, kedua isolat teridentifikasi sebagai anggota spesies Lactobacillus paracasei ssp. paracasei.
Kata kunci: bakteri asam laktat, isolasi, identifikasi, Lactobacillus paracasei ssp. paracasei.
INTRODUCTION
One type of shellfish that has the potential and
economic value is the seven eyes shelfish. Seven eyes
shelfish (Haliotis asinina) is also called abalone, awabi,
mutton fish, sea ear and in local language (Sasak, Lombok)
it is called medau. Abalone is included in univalve shellfish
species (Cholik et al. 2005) which the meat has high
nutritional value with protein content of 71.99% and 3.20%
fat. Its shell also has an aesthetic value that can be used for
jewelry, the manufacture of buttons and various other
forms
of
handicraft
goods
(Imai
1997).
Abalone is mostly found in Eastern Indonesia (Bali,
Lombok, Sumbawa, Sulawesi, Maluku and Papua). On the
island of Lombok abalone is often found in southern coast
of central Lombok called Kute Beach and surrounding
areas. During this time abalone have been exploited by local
residents without any proper selection, resulting reduction
in the catch and in the long term may threaten its sustainability.
The effort of Abalone cultivation technology ranging
from domestication, a trial of gonal maturation in a
controlled basin, spawning, larval rearing, and larval food
preparation have been done (Sofyan et al. 2005), but these
activities do not give a satisfactory result. Survival rate of
abalone larvae in larval rearing tanks until this time is still
very low at around 1.0%. Mortality was happening a lot on
planktonic stage until attachment to the substrate (first
weeks). Low larval survival rate is among others due to
water filtration systems that is poor which resulted in the
emergence of protozoa, worms and various types of
pathogenic microorganisms that can cause death of larvae.
One effort to prevent the occurrence of population
shifts (split population) and also suppress the growth of
pathogenic microorganisms is to maintain the natural
balance of microorganisms in the larval rearing system
(Haryanti et al. 1997) through the addition of probiotic
microorganisms (Fuller 1989). Prevention of disease was
taking place by controlling the growth of potentially
SARKONO et al. – Probiotic candidate of lactic acid bacteria from abalone
pathogenic microbes in the gastrointestinal tract
(Strompfova et al. 2005; Iñiguez-Palomares et al. 2007)
and a number of positive effects of probiotic bacteria
including immunomodulastion (Wallace et al. 2003).
Development of probiotics for the cultivation of abalone
would be better if the probiotic microbes are indigenous
abalone itself, so as to avoid the problem of microbial
adaptation on larval rearing tanks and seven channels of the
body of this seven eyes shellfish when applied. It is
therefore important to do research on indigenous bacteria
isolation and identification of potentially probiotic abalone.
MATERIALS AND METHODS
Isolation of LAB strains from abalone
A total of 20 examples of healthy male and female
seven eyes shellfishes are obtained from the Institute for
Marine Aquaculture Lombok. Then the fluid from the
digestive tract is taken in a sterile way as much as 1-10 g.
Selective isolation of Lactic Acid Bacteria (LAB) is
performed with Spread plate method developed by
Brashear et al. (2003) and Ray et al. (1997). A total of 1 g
sample added into 10 mL of fever Rogosa Sharpe (MRS)
broth sterile and mixed until homogeneous. The suspension
is then spread on MRS medium pH 5.5 plus 0.1% Naazide, each in-trade with calcium carbonate 1%.
Furthermore, the petri plates were incubated at 37oC for 48
hours in an incubator in a microaerophilic atmosphere.
Single colonies that grew were taken from each plate and
transferred into test tubes containing 10 mL MRS broth.
Then they were incubated at 37oC for 18-72 hours to obtain
maximum growth cultures. Culture isolates were streaked
again on MRS media for the petri plates and incubated at
37oC for 48 hours to obtain a single colony/pure culture. In
pure culture Gram stain is done for initial identification.
Lactic acid bacteria culture obtained is stored by freezing
the temperatures. Stock to be used was prepared by
growing the isolated bacteria in MRS liquid medium and
incubated at 37oC for 24-48 hours (Rahayu et al. 2004).
Test of LAB strains antibacterial power
LAB isolates were tested their ability to inhibit the
growth of pathogenic bacteria namely Eschericia coli,
Bacillus cereus and Staphylococcus aureus by using well
diffusion assay. Each isolate was treated in the form of
fermentation result supernatant containing the extracellular
metabolites, which are obtained by inoculating liquid
culture of isolate lactic acid bacteria as much as 2% into
the liquid media fever Rogosa Sharpe (pH 6.5) and then
incubated at 37°C for 96 hours (Bar et al. 1987). After
incubation pH measurements were taken, subsequently
centrifugation was done upon liquid culture using a
centrifuge with a speed of 3500 rpm for 20 minutes.
Supernatant obtained was sterilized with bacterial filter
(porous diameter of 0.2 μm, Whatman) in order to obtain
sterile extracellular metabolites.
Antibacterial test was conducted using well diffusion
assay developed by Djafaar et al. (1996) and modified by
Sarkono et al (1996), by plating test bacterium E. coli, B.
39
cereus and S. aureus in petri disk with Nutrient Agar solid
medium, then added by Nutrien Agar soft medium on it.
After being cooled for 1 hour in a refrigerator room, a well
was made with a diameter of 0.7 mm and then isolates of
bacterial supernatant was inserted and incubated at 37oC
for 24-48 hours. The diameter of each isolate contained
clear zone is measured.
Test of tolerance towards acid and bile
The tolerance Isolates LAB which inhibits the growth
of pathogenic bacteria extensively was screened towards
acids and bile. Tolerance test towards the acid uses the
method of Brashear et al. (2003). LAB fresh culture
harvested from MRS broth by centrifugation and the pellet
obtained was washed and suspended with sterile phosphate
buffer saline (PBS). Each strain was added by 4 mL of
sterile PBS and pH was adjusted to pH 2, 4, 5 and 7
(control) and incubated for 2, 4 and 24 hours in a water
bath at a temperature of 37°C. After each incubation
period, the growth of strains can be identified by measuring
the absorbance at 620 nm. Bile tolerance test was using the
method of Gilliland et al. (1984). Fresh cultures of selected
LAB isolates were inoculated into tubes containing 10 mL
MRS broth with levels 0 (control), 0:05, 0:15 and 0.3%
oxgall. Inoculated tubes were incubated at 37°C in a water
bath. Growth of isolates was observed at 2, 4, 6, and 24
hours by measuring absorbance at 600 nm.
Early identification of isolates with the API
Initial identification made to isolated LAB with
inhibitory activity on the growth of E. coli, B. cereus and S.
aureus and their tolerance to acid and bile. LAB isolates
were identified through fermentation patterns with index of
profile analysis standard test with 50CHL API Kit
(Biomerieux 2009).
RESULTS AND DISCUSSION
Selective isolation of Lactic Acid Bacteria from abalone
Isolation process yields 10 colonies which were
suspected as isolates Lactic Acid Bacteria (LAB) because
they produces a clear zone in isolation medium (Figure 1),
then a strengthened test was conducted by growing on solid
MRS medium plus CaCO3 1%. From this confirmation test
by re-growing process showed that all 10 isolates LAB
could grow well and produce clear zones around colonies.
The characterization results further prove that the 10
isolates allegedly a member of the lactic acid bacteria
(Table 1).
Results of identification at the genus level confirm that
the four isolates that were characterized are members of the
genus Lactobacillus. These isolates have a phenotypic
characters among others the forms of stem cell are long, the
structure resembles a fence and row of cells singly
scattered, gram positive reaction, not motile and do not
form endospores (Sneath et al. 1986). Images of each
isolate cell can be seen in Figure 2.
40
2 (1): 38-42, March 2010
Table 1. Test results that characterized the feature of Lactic Acid Bacteria
isolated from abalone
Isolates
OPA1
OPA2
OPA3
OPA4
OPA5
OPA6
OPA7
AL1
RL1
KA1
Resources
Digestive organs
Digestive organs
Digestive organs
Digestive organs
Digestive organs
Digestive organs
Digestive organs
Sea water
Seaweed
Abalone feces
Feature that characterized Lactic Acid Bacteria
Cell
Gram
EndoKataase Motility
shape reaction
spora
Stem
+
Stem
+
Stem
+
Stem
+
Stem
+
Stem
+
Stem
+
Stem
+
Stem
+
Stem
+
-
Test of antibacterial power LAB strains
against pathogenic bacteria E. coli, B.
cereus and S. aureus
The result of bacterial growth inhibition
test with the indicator diffusion method
showed that seven among ten isolates
showed the ability to inhibit the growth of
bacteria, characterized by the formation of
clear zones around the wells with varied
sizes. Three isolates had the ability to
inhibit the three bacterial indicators as well
as the isolates OPA3, OPA4 and AL1.
Three isolates could inhibit the growth of
two indicator bacteria namely OPA5, OPA6
and OPA7.
Holozone (mm)
Figure 1. Colonies are indicated as LAB with
clear zones around colonies
E. coli
S. aureus
B. cereus
Type of isolates
Figure 3. Antibacterial test isolates LAB supernatant against indicator bacteria
Eschericia coli, Staphylococcus aureus and Bacillus cereus with well diffusion
method
OPA1
OPA2
OPA3
OPA4
OPA5
OPA6
OPA7
AL1
RL1
KA1
Figure 2. Gram reaction and cell shape of LAB isolates which were isolated by seven eye mussel (abalone) and their habitats
SARKONO et al. – Probiotic candidate of lactic acid bacteria from abalone
Meanwhile, only one isolate which is only able to inhibit
the growth of one indicator bacteria namely OPA1 isolates,
whereas three other isolates namely OPA1, RL1 and KA1
did not have the ability to inhibit any bacterial indicator
(Figure 3).
Based on the character of inhibition zone, ten isolates
tested showed different characters of inhibitions, but in
general some of them showed inhibition zone with blurred
edges (not firm) and others showed inhibition zone with
firm edges. Blurred edges zone indicates that the active
metabolite found in the supernatant is bacteriostatic, which
only inhibit cell growth of indicator bacteria but not kill the
cell. According Rahayu (2004), inhibition with vague zone
might be the action of acid and other antibacterial
components which are only bacteriostatic, since most
bacterial test (indicator) remains alive in the clear zone,
although with very slow growth. Meanwhile inhibition
zone with a firm edge indicates that isolates have the ability
to produce metabolites which are bactericidal, where
metabolites can kill bacterial cells indicator. This is one of
the expected ability of probiotic bacteria so it can control
the growth of pathogenic bacteria in their applications.
Test of tolerance to acid and bile
Based on the results of testing the ability of inhibition
on the growth of pathogenic bacteria in a previous study
phase and then continued by selecting two isolates that had
the best inhibitory then proceed with the test of isolates
growth in an atmosphere of acid and bile. Data obtained
from this test form absorbance data using a
spectrophotometer. The addition of absorbance values in
line with the addition of incubation time showed the
growth of LAB isolates tested (Figure 4).
Figure 4 show that both of tested Lactic Acid Bacteria
isolates showed the ability to grow in acidic environment
which is relatively similar. Isolate OPA4 and AL1 have
excellent adaptability to acid atmosphere, because an
increase in growth at 3 pH levels in 24-hour period. At pH
2 the two isolates did not grow, because the pH of 2 is a
very extreme pH for growth of microorganisms, including
lactic acid bacteria which are generally well adapted to
living in habitats with a relatively low pH environment. At
pH 4, 5 and 7 both isolates are able to grow well, the
exponential increase in growth occurred in the observation
at 24th hour because of the incubation period is long enough
from the 4th hour up to the 24th hour resulting in significant
cell division. Isolate OPA4 achieve the best growth at pH 7
whereas at pH 5 isolates AL1. Lactic acid bacteria
41
generally prefer the atmosphere of a pH slightly below a
neutral pH for best growth (Axellson 1998). The result of
the endurance test isolates of bile showed that the four
isolates had a very, very good ability, because of an
increase in growth in the overall level of concentration of
bile (0.05%, 0.15%, and 0.30%) in 24-hour period (Figure
4).
The ability to grow of the two isolates namely AL1
OPA4 in the bile can not be distinguished from each other.
This is predicted caused by the very low concentration of
bile that is used. The tests for resistance toward bile liquid
used method that was developed by Gilliand et al. (1984)
which uses bile concentration of 0.05%, 0.15%, and 0.30%.
As a comparison, other researcher (Ljungh et al. 2002)
tested the resistance of isolates Lactobacillus paracasei
subsp. paracasei F19 in 20% bile and continues to show
growth on incubation time of 2 hours.
Early identification of isolates with the API
API biochemical test kits are used to determine the
biochemical characteristics of LAB isolates that are tested
so that it can be used for identification purposes. Because
the two LAB isolates tested are members of the genus
Lactobacillus, so we only use the API Kit 50CHL content
of which is 49 kinds of sugar and its derivatives, plus one
negative control so in overall there are 50 types of test
(Biomerieux 2009). Visually Kit API 50CHL represented
by Figure 5.
A
B
Figure 5. Visualization of the results of sugar fermentation test
with API Kit 50CHL (a) isolates AL1 48 hours and (b) isolates
OPA4 48 hours
A
B
C
D
Figure 4. Selected isolate growth test during 24 hours incubation time. A. Isolates OPA4 in bile, B. Isolates AL1 in bile, C. Isolates
OPA4 in in the acid atmosphere, D. Isolates AL1 in the acid atmosphere
42
2 (1): 38-42, March 2010
The test of sugar fermentation is a very important
characterization process in the genus Lactobacillus to know
the character to the identification of species diversity (Holt
et al. 1994). The result of sugar test with the API kit toward
10 isolates of the isolated form of positive character (+) and
negative (-) which in total amounted to 50 characters, then
analyzed by a computer program ApiwebTM Version 1.2.1
to identify the species name.
The test results showed that after 48 hours incubation
AL1 OPA4 isolates gave the same results, that are positive
reactions on sugar numbers 5, 10, 11, 12, 13, 14, 16, 18,
19, 21, 22, 23, 24, 25, 26, 27, 28, 31, 32, 34, 39.40, 41, 42
and 47, the rest react negatively. This shows the level of
characters with very high similarity between the two
isolates, so it is possible that they are from the same strain,
at least a member of the same species. The test results
which is in the form of sugar fermentation profile was
analyzed with the program ApiwebTM version 1.2.1, the
result is that the two isolates tested is a member of the same
species of Lactobacillus paracasei ssp. paracasei. This
species has a very close relationship, and even considered
as neotype strain of Lactobacillus lactic species (Dellaglio
et al. 2002). According Vlieger et al. (2009) members of
this species have been applied as probiotic bacteria in
infant milk together with Bifidobacterium.
CONCLUSIONS AND SUGGESTIONS
A total of ten isolates of LAB can be isolated from
gastrointestinal tract of abalone and their habitats. After the
selection there are two isolates obtained potentially to be
the candidates for probiotic that is OPA4 and AL1. Both
isolates have the ability to inhibit the growth of
enteropathogenic bacteria namely Eschericia coli, Bacillus
cereus and Staphylococcus aureus with inhibition zone
varied widely, and able to grow in acidic conditions and
tolerant of bile during 24 hours incubation. Based on the
API test kit and analyzed with software 50CHL ApiwebTM
Version 1.2.1, the both isolates are identified as members
of the species Lactobacillus paracasei ssp. paracasei.
Isolates of this research which have the potential to be a
candidate of probiotics in abalone larval rearing system of
(Haliotis asinina) are expected to be studied further in order
to know its potential in improving the survival ability of
abalone larvae in vitro and in vivo, so it can be
recommended as probiotic bacteria, especially in the
abalone farming systems in the future.
ACKNOWLEDGEMENTS
The authors thanks the Directorate General of Higher
Education, Ministry of National Education which has
funded this research through research project Fiscal Year
2009 Competitive Grant Contract Number: 0234.0/02304.2/XXI/2009, 31December 2008.
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bacteria from meat and processed products as a producer of
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ISSN: 2087-3948 (print)
ISSN: 2087-3956 (electronic)
Vol. 2, No. 1, Pp. 43-47
March 2010
Productivity of sugarcane plants of ratooning with fertilizing treatment
A. SUTOWO LATIEF1,♥, RIZAL SYARIEF2, BAMBANG PRAMUDYA2, MUHADIONO3
¹ Semarang Polytechnic State (Polines). Jl. Prof. Sudharto, SH, Tembalang, Semarang, Central Java, Indonesia; ♥e-mail: [email protected]
2
Faculty of Agriculture Technology, Bogor Agricultural University (IPB), Darmaga Bogor 16680, West Java, Indonesia
3
Faculty of Mathematic and Natural Science, Bogor Agricultural University (IPB), Darmaga Bogor 16680, West Java, Indonesia
Manuscript received: 30 September 2009. Revision accepted: 26 January 2010.
Abstract. Latief AS, Syarief R, Pramudya B, Muhadiono. 2010. Productivity of sugarcane plants of ratooning with various fertilizing
treatments. Nusantara Bioscience 2: 43-47. This research aims to determine the sugarcane plants of ratooning productivity with low
external input of fertilization treatment towards farmers can increase profits. The method used is the Completely Randomized Block
Design (CRBD) with four treatments and three repetitions (4x3). Sugarcane varieties R 579 planted in each patch experiment 5x5 m2.
Dosage of fertilizer: P0 = 3.6 kg/year plot experiment was 100% dosage usage of chemical fertilizers used by farmers. Further dosages
were P1 (75%) = 2.7 kg/plot, P2 (50%) = 1.8 kg/plot and P3 (0.25%) = 0.9 kg/plot, each supplemented with fertilizer 5 mL of liquid
organic/patch a year. Sugarcane crops with a variety of treatment showed no significant difference. The highest productivity was
achieved at dosages of P2 (50% chemical fertilizers plus organic fertilizer) is 21.67 kg per square meter. Chemical fertilizers can be
saved 7 quintals per hectare a year or Rp 997,500 per year. Additional costs of liquid organic fertilizer Rp. 100,000 per hectare year and
labor Rp 100,000 per hectare, so the additional advantage of saving farmers fertilizer Rp. 797,500 per year.
Key words: sugarcane plant, ratooning, fertilizing, profits.
Abstrak. Latief AS, Syarief R, Pramudya B, Muhadiono. 2010. Productivity of sugarcane plants of ratooning with fertilizing treatment.
Nusantara Bioscience 2: 43-47. Penelitian ini bertujuan untuk menentukan produktivitas tebu keprasan dengan perlakuan pemupukan
input eksternal rendah, sehingga petani dapat meningkatkan keuntungan. Metode yang digunakan adalah Blok Rancangan Acak
Lengkap dengan empat perlakuan dan tiga ulangan (4x3). Tebu varietas R 579 ditanam pada masing-masing plot percobaan seluas 5x5
meter2. Dosis pupuk: P0 = 3,6 kg/plot yaitu 100% dosis penggunaan pupuk kimia yang digunakan oleh petani. Selanjutnya dosis: P1
(75%) = 2,7 kg/plot, P2 (50%) = 1,8 kg/plot dan P3 (0,25%) = 0,9 kg/plot, masing-masing dilengkapi dengan 5 mL pupuk organik cair
plot/tahun. Tanaman tebu dengan berbagai perlakuan tidak menunjukkan perbedaan yang signifikan. Produktivitas tertinggi dicapai pada
dosis P2 (pupuk kimia 50% plus pupuk organik) adalah 21,67 kg/m2. Pupuk kimia dapat dihemat 700 kg/ha/tahun atau Rp 997.500 per
tahun. Tambahan biaya pupuk cair organik Rp 100.000 per tahun hektar dan tenaga kerja Rp 100.000 per hektar, sehingga keuntungan
tambahan petani dari tabungan pupuk Rp. 797.500 per tahun.
Kata kunci: tanaman tebu, keprasan, pemupukan, keuntungan.
INTRODUCTION
In this time government is inciting sugarcane planting
of superior variety to overcome the low sugar production in
Indonesia. To be in the triumph time as sugar exporter in
the year of 1930 is done by increasing sugarcane product
either through quantity and quality with paying attention to
the environment preservation. Indonesia sugar productivity
has declined, not only because of less field, irrigation and
the increasing dry field or dry farming that planted
sugarcane, but also that sugarcane variety doesn't support
productivity and the ratooning is done more than 10 times.
Therefore the company of Plantation Nusantara XI in East
Java does penetration to develop new variety of arcane
plants namely R-579 (MoA 2002). This new variety can
produce average sugar of 10, 07 ton of /ha, while the
average national productivity is 4 ton /ha (Anon 2002).
Development of sugarcane is quite reasonable where it
is produced more than half of the world’s sugar production
from sugarcane (Mubyarto and Daryanti 1994). The
productivity of sugarcane crop in Indonesia that has been
achieved is 4.924 tons/ha (Anon 1996), but in the last 5
years it has increased from 5.7 tons/ha in 2004 to 6.8
tons/ha in 2009 (Lestari 2009); while in Papua New Guinea
to reach 5.5 tons/ha (Hartemink 1996), and South Africa
11.0 tons/ha (McGlinchey and Inman-Bamber 1996).
The administrator of Sugar Factory of Rendeng, Kudus,
said, most of 5,679 hectare sugarcane plants were
cultivated by farmers farmer with ratooning system, with
the average 10 times. Sugarcane productivity moment
harvests the highest products of 70 ton/ha, and yield only
5,76%. Begin in the year 2003, farmers plant a kind of
superior varieties namely PS 851 (MoA 2004) and R 579
(BR 579) in the area of 728 hectare. The superior variety R
579 has been experimented at some amount in the Sugar
Factory in East Java and has produced the minimum crops
of 150 ton/ha 8% (Krismanu 2003).
The ratooning system is growing return sugarcane that
felled. Anon (2005), ratooning sugarcane management has
been intensively done since the issue of the President
44
2 (1): 43-47, March 2010
Instruction number 9 in the 1975 about intensification.
Since 1990, the trend of the use of ratooning sytem of
sugarcane has continued to increase, that is around 60%
from total square existing sugarcane.
Since Green Revolution was proclaimed in the 1970’s
farmers’ dependence in inorganic fertilizer use has been
there. Inorganic fertilizer used that is over dosage or more
causes the depletion of the soil quality, and it leads to the
decrease of sugarcane’s productivity. Aryantha said that
(2002) this condition causes inhibited of root absorption
process towards water and nutrient that was dissolved so
that the existence of nutrient in total low is not taken by the
roots in maximally. Thereby certain dosage of fertilizer is
needed to make the roots able to absorb the nutrient in
enough number from the nutrients available in the soil.
Suprapta (2005) said that chemical fertilizer causes bad
impacts as we have witnessed. He added that we should
organic fertilizer and at the same time also slowly reduces
the use of chemical fertilizer. While According to
Darutama (2008), organic fertilizer the use organic
fertilizer for sugarcane plants obviously shows good
significance in comparison with the use of the chemical
fertilizer such as urea or NPK.
The success sugarcane farming means giving the profits
to the farmers and being able to keep the environment
healthy. Therefore it is necessary to conduct a research
aimed at decreasing the use chemical/inorganic fertilizer
and encouraging the use of organic fertilizer to do the
rationing system for sugarcane farming to make the
productivity stable.
MATERIALS AND METHOD
Location and time of research
The research location based on fertilizing variation
treatment effort plan towards ratooning sugarcane plants is
chosen to be conducted at Jurang Village, Gebog
Subdistrict, Kudus District, Central Java. The place that is
used to do the analysis towards the chemical element of the
soil nutrient, good macro and micro element is in the
Laboratory of Department of Soil Science and Land
Resources, Faculty of Agriculture, Bogor Agricultural
University (IPB), Bogor. Research time is carried out to
begin in July 2008 and end in June 2009, during one
sugarcane harvest season.
Materials and tools
Principal material is a variety of sugarcane plants
namely R 579. Other materials are fertilizers namely: (i)
inorganic fertilizer ZA (ammonium sulphate), and NPK
(Phonska), (ii) liquid organic fertilizer.
Method
The design of the research was Completely
Randomized Block Design with 4 (four) treatments and for
each treatment there are 3 (three) repetitions. Fertilizing
treatment is done towards ratooning sugarcane plants.
Ratooning sugarcane plants that is analyzed is the variety
of sugarcane namely R 579 that can undergo the ratooning
process three times (can be four in the future) in the area in
Jurang village, district Gebog, Kudus regency. The size of
trial compartment each 5x5 square meters = 25 m2 (poled
to be clear the limit).
The fertilizing treatment that is: (i) P0 = the use
chemical fertilizer (inorganic fertilizer/factory fertilizer)
done by the farmers up to that time (100% inorganic
fertilizer), without organic fertilizer. (ii) P1 = chemistry
fertilizer use is reduced by 25% from the usual use (75%)
then replaced by the organic fertilizer. (iii) P2 = chemistry
fertilizer use is reduced by 50% from the usual use (50%)
and replaced by the organic fertilizer. (iv) P3 = chemistry
fertilizer use is reduced by 75% from the usual use (25%)
then replaced by the organic fertilizer.
The addition of organic fertilizer is done towards P1,
P2, and P3 with the same dosage, that is 2 L every hectare
a year, while P0 as a group control doesn't uses organic
fertilizer. Organic fertilizer kind use result of Fadiluddin
(personal communication, 2009).
The use dose 2 L/ha of land, atomized twice (each time
spraying 1 L/ha), before atomized in soil surround plants,
liquid organic fertilizer is thinned with water first of all
with comparison 100 mL to 1 (one) tank sprayer (15 L
water) or 15 mL (size bottle plug) to 2 L water.
Liquid organic fertilizer use to each size compartment
25 m2: 25/10,000x2 liters = 5 mL. Overall use from 9 trial
compartments (P1, P2, and P3 with repetition 3 times) a
year need: 5x9 = 45 mL then thinned with 6 clean water
liters. Fertilizing with liquid organic fertilizer was done by
spraying, one year done 2 times, as according to inorganic
fertilizing, not concurrent but done 3-5 days before or after
fertilizing with inorganic fertilizer.
Inorganic fertilizer use usually is done by farmer
towards sugarcane plants each time fertilizing is 100
kilogram/sector of rice field is do twice a year (200
kilogram/year sector of rice field) consist of 50% fertilizer
ZA (ammonium sulfate): nitrogen (N) = 21% and sulfur (S)
= 24% and 50% fertilizer NPK (Phonska: N = 15%; P2O5 =
15%; K2O = 15%; S = 10%)
One hectare there is 7 sectors of rice field, every sector
of rice field approximately 1400 m2. Inorganic fertilizer use
for size of trial compartment 25 m2 a yearlong is need: P0 =
25/1400x200 = 3.6 kg, P1 = 0.75x3.6 kg = 2.7 kg, P2 =
0.50x3.6 = 1.8 kg, and P3 = 0.25x3.6 = 0.9 kg.
Soil is taken as the sample to analyze as many as three
times during research, that is: (i) before fertilizing, (ii) after
fertilizing and (iii) approach harvest. Soil analysis is done
in laboratory to detect element of nutrition completely.
Sugarcane plants observation is done according to in a
flash with take when soil samples taking. The finals
research is sugarcane harvest result ready mill from each
trial compartment. Sugarcane observation is done towards:
(i) amount of sugarcane plants every square meters or
every meter makes, (ii) tall/long sugarcane stick ready mill
and (iii) sugarcane stick diameter (measured 15 cm from
base). Sample taking at random every square meters (meter
makes from each trial compartment). Heaviness each
weighed and analyzed to detect treatment difference with
statistical methods that are Analysis of Variance (ANOVA).
LATIEF et al. – Sugarcane plant ratooning
45
fertilizer has begun to react towards soil so that root
absorption towards water and nutrition is better.
RESULT AND DISCUSSION
Soil evaluation criteria
Soil sample taking is done 3 times, that is: (i) before
fertilizing in 9 Novembers 2008, (ii) after fertilizing in 22
February 2009 and (iii) approach harvest in 21 May 2009.
Based on soil analysis result from Department of Soil
Science and Land Resource, Faculty Agriculture, Bogor
Agricultural University (IPB) Bogor, follow Hardjowigeno
(2007) determinable the criteria as be showed in Table 1,
Table 2 and Table 3.
Criteria of nutrition N before fertilizing, after fertilizing
and approach harvest shows low, while P in the form of
P2O5 there are increase a little, but K does not change.
Another macro element that is: Ca, Mg and Na are fair.
Table 1. Soil chemistry properties evaluation criteria before
fertilizing
Soil properties
C (%)
N (%)
C/N
P2O5 HCl (mg/100 g)
P2O5 Bray 1 (ppm)
KTK (me/100 g)
K (me/100 g)
Na (me/100 g)
Mg (me/100 g)
Ca (me/100 g)
Saturation of basic (%)
pH H2O
pH KCl
Sugarcane productivity
Based on observation towards sugarcane plant when
taking second soil sample 22 February 2009 known that for
treatment P0, green appear sugarcane leaf, while for
treatment P1, P2, and P3 appear sugarcane leaf more
becomes yellow. But when taking third soil sample 21 May
2009 that is approach sugarcane leaf color harvest visible
hasn't showed difference. This matter caused by organic
Treatment:
P0 = P1 = P2 = P3
1.2
0.13
9.23
23.6
2.2
14.82
0.44
0.34
1.67
5.34
52.56
4.5
3.6
C-org (%)
N-total (%)
C/N
P2O5 HCl (mg/100 g)
P2O5 Bray 1 (ppm)
KTK (me/100 g)
K (me/100 g)
Na (me/100 g)
Mg (me/100 g)
Ca (me/100 g)
Saturation of basic (%)
pH H2O
pH KCl
P0
0.96
0.12
8
25.86
53.1
15.35
0.28
0.24
1.48
6.77
57.13
4.00
3.3
Treatment
P1
P2
1.36
1.2
0.13
0.11
10.46
10.90
30.43
49.76
32.5
60.0
14.96
14.56
0.28
0.58
0.23
0.30
1.67
2.43
6.95
5.65
61.03
61.54
4.30
4.40
3.5
3.5
P3
0.96
0.09
10.66
48.91
52.4
15.55
0.28
0.22
2.57
7.87
70.35
4.40
3.7
Criteria
P0 very low; P1 low; P2 low; P3 very low
P0 low; P1 low; P2 low; P3 very low
P0 low; P1 fair; P2 fair; P3 fair
P0 fair; P1 fair; P2 high; P3 high
P0 very high; P1 high; P2 very high; P3 very high
P0 low; P1 low; P2 low; P3 low
P0 fair; P1 fair; P2 high; P3 high
P0 low; P1 low; P2 low; P3 low
P0 low; P1 fair; P2 high; P3 high
P0 fair; P1 fair; P2 fair; P3 fair
P0 high; P1 high; P2 high; P3 very high
P0 very acid; P1 very acid; P2 very acid; P3 very acid
P0 very acid; P1 very acid; P2 very acid; P3 very acid
Table 3. Soil chemistry properties evaluation criteria approach harvest
Soil properties
C-org (%)
N-total (%)
C/N
P2O5 HCl (mg/100 g)
P2O5 Bray 1 (ppm)
KTK (me/100 g)
K (me/100 g)
Na (me/100 g)
Mg (me/100 g)
Ca (me/100 g)
Saturation of basic (%)
pH H2O
pH KCl
P0
1.43
0.13
11
34.01
49.0
18.62
0.35
0.40
2.70
8.63
64.88
5.40
4.50
Treatment
P1
P2
1.27
0.95
0.11
0.10
11.5
9.5
33.16
36.21
47.3
49.3
27.75
20.35
0.08
0.10
0.19
0.21
0.20
0.18
4.3
2.6
32.4
36.9
5.50
5.20
4.70
4.00
P3
0.71
0.09
7.9
43.99
56.2
22.2
0.29
O.90
0.31
3.5
83.3
5.30
4.10
low
low
low
fair
very low
low
fair
fair
fair
fair
high
acid
very acid
Sugarcane harvest is done at dry season because
moment that is has high yield, after cutting down sugarcane
soon be processed to be sugar. The cutting down of
Table 2. Soil chemistry properties evaluation criteria after fertilizing
Soil properties
Criteria
Criteria
P0 low; P1 low; P2 very low; P3 very low
P0 low; P1 low; P2 low; P3 very low
P0 fair; P1 fair; P2 low; P3 low
P0 fair; P1 fair; P2 fair; P3 high
P0 very high; P1 very high; P2 very high; P3 very high
P0 fair; P1 high; P2 fair; P3 fair
P0 fair; P1 very low; P2 low; P3 low
P0 fair; P1 low; P2 low; P3 high
P0 high; P1 very low; P2 very low; P3 very low
P0 fair; P1 low; P2 low; P3 low
P0 high; P1 low; P2 fair; P3 very high
P0 acid; P1 acid; P2 acid; P3 acid
P0 acid; P1 acid; P2 acid; P3 acid
46
2 (1): 43-47, March 2010
sugarcane in this research is done after age approximately
one year, that is on 16 June 2009.
Amount of sugarcane plant/stick every square meters
based on observation in the harvest in the range from 16 up
to 24 stick of sugarcanes. Long sugarcane stick ready mill
also vary that is between 1.5 meters up to 3.5 meters.
Sugarcane stick diameter ranges from 2.5 cm up to 4.5 cm.
The average of amount stick, length stick, and sugarcane
stick diameter is presented in Figure 1.
Figure 1. Amount average of stem, length and diameter of
sugarcane plant every square meters in experimental land.
The model of relation between fertilizing treatment with
sugarcane productivity is shown in Figure 2.
Produktivitas Tebu (kg/m2)
Series1
Series2
Poly. (Series2)
25
20
3
2
y = -2.6667x + 20.33x - 44.983x + 45.65
15
2
R =1
10
5
0
P0
P1
P2
P3
Perlakuan Pemupukan
Figure 2. Relation between fertilizing treatment and sugarcane
productivity.
Based on the Analysis of Varian with signification
standard 1%, sugarcane productivity with variation
fertilizing treatment, it doesn't show real difference.
Highest productivity is achieved in treatment (P2) that is
fertilizing combination with reduction 50% chemistry
fertilizer from the usual one done by farmers, added with
organic fertilizer. Thereby it can be saves the chemistry
fertilizer purchasing cost-saving as big as 50%, although
the liquid organic fertilizer purchasing cost and labor cost
for fertilizer spraying increase.
Farm operation analysis of sugarcane and cost-saving
Farm operation analysis of sugarcane is done to
determine profit and business feasibility based on income
ratio criteria towards net (B/C). Farm operation of
sugarcane is said feasible when value B/C bigger than one
Based on primary data that is got and cultivated with
one hectare land square production cost: C = Rp
12,000,000. Land lease were Rp 5,000,000 per year. Labor,
cultivation, fertilizer and pesticide were Rp 7,000,000 per
year. Sugarcane sales revenue: Rp 160,000 per ton,
sugarcane harvest result 150 ton/ha, so that Benefit total: B
= Rp. 24,000,000.
Farm operation profit of sugarcane: B-C = Rp
24,000,000-Rp 12,000,000 = Rp 12,000,000 per year.
Benefit per Cost Ratio: Net B/C = Rp 24,000,000/Rp
12,000,000 = 2.0.
Based on analysis result above (B/C = 2.0 > 1), it can
be known that the farming operation of sugarcane is
feasible.
Cost-saving analysis is based on fertilizer chemistry
(inorganic fertilizer) use reduction 50% from habit that is
as much as 7 quintal (700 kg) fertilizer that can be saved
without decreasing of productivity. Chemistry fertilizer
dosage that used farmers usually is 1.4 ton/ha. Despite of
organic fertilizer use cost and labor increasing, but still
more beneficial because liquid organic fertilizer use lower
than chemistry fertilizer, beside that is also cheaper the
price.
The price of kind inorganic/chemical fertilizer ZA: Rp
110,000 per quintal, kind fertilizer Phonska: Rp 175,000
per quintal. Fertilizer use ZA and Phonska proportional,
which is each 50%. Cost addition for liquid organic
fertilizer: Rp. 50,000 per liter, as much as 2 L/ha and labor
wage: Rp 25,000 per day as much as 4 persons.
Based on this research result when applied manifestly
with chemistry fertilizer reduction 50% is 7 quintal/year is
land square base one hectare, so cost-saving can be done by
farmer:
Chemistry fertilizer cost-saving-(organic fertilizer cost
+ worker wage) = 7x(110,000 + 175,000/2-(2x50,000 +
100,000) = Rp. 797,500/hectare.
Cost-saving a kind of this be concept LEISA (Low
External Input Sustainable Agriculture), that is a concept
that promoting system and that agriculture manners by
using a little chemical addition. Principle applications
LEISA make possible Good Agriculture Practices (GAP)
where productivity and economy profit is increased in the
way of that pay attention ecological aspect. For example,
livestock animal maintenance to make use in stable
fertilizer maker with agriculture rubbishes utilization like
foliage to be used as supplement plants.
CONCLUSIONS AND RECOMENDATIONS
The productivity of sugarcane with fertilization
treatment variations P0, P1, P2 and P3, showed no
significant difference. The highest results achieved by
treatment of P2, which is 21.67 kg/m² of land area.
Reduction of chemical fertilizers without the addition of
organic fertilizer is not done because the experience of
farmers who have tried to reduce the dosage of chemical
fertilizers without the addition of organic fertilizers, the
productivity of sugarcane declined. Thus the combination
of reduction in the use of chemical fertilizers and organic
LATIEF et al. – Sugarcane plant ratooning
fertilizers can stabilize the productivity of sugarcane and
input cost savings. Input cost savings made by farmers is
an advantage, is Rp. 797,500/hectare during the season
(year)
This study should be followed up at various locations
mainly on dry land, and the land with more extensive
experiments, and the use of chemical fertilizers ZA and
Phonska
varied
to
obtain
optimal
savings.
Future research needs to be done reducing the use of
chemical fertilizers or without the use of chemical
fertilizers at all. The use of organic fertilizer without
chemical fertilizers is conducting agricultural/organic
sugarcane plantations, so that farming guidelines and good
agricultural products (Good Agriculture Practices/GAP).
REFERENCES
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[Indonesia]
Anon. 2002. Increasing sugar production by finding new varieties
of sugarcane. Situs Hijau Media Pertanian Online.
www.situshijau.co.id [27 Mei 2009]. [Indonesia]
Anon. 2005. Management of ratooning sugarcane. Sugarcane
Development Project, Plantation Office, East Java..
www.ratoonjatim.co.cc/tebu_keprasan/pengelolaan_tebu_kepra
san.htm [27 Mei 2009] [Indonesia]
Aryantha IP. 2002. Development of sustainable agricultural
systems. One Day Discussion on the Minimization of
Fertilizer Usage. Menristek-BPPT, Jakarta, 6 May 2002.
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Darutama BE. 2008. Pupuk organik tingkatkan rendemen tebu.
www.beritacerbon.com/berita/2008-09/Pupuk-OrganikTingkatkan-Rendemen-Tebu.html [10 October 2008].
[Indonesia]
Fadiluddin M. 2009. The effectiveness of biological fertilizer
formula in promoting nutrient uptake, production, and quality
of maize and upland rice in the field [Thesis]. School of
Graduates. Bagor Agricultural University. Bogor. [Indonesia]
Hardjowigeno HS. 2007. Soil science. Akademika Pressindo.
Jakarta.
Hartemink A. Kuniata. 1996. Some factors influencing the trend
of sugarcane yield in Papua New Guinea. Outlook Agric
25(4): 227-234.
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Indonesia 07-12-2001. [Indonesia]
McGlinchey MG, Inman-Bamber Ng. 1996. Effect of irrigation
scheduling on water use efficiency and yield. Proc S Afr Sug
Technol Ass 70: 55-56.
Minister of Agriculture of the Republic of Indonesia. 2002.
Decree of the Minister of Agriculture Number:
372/TU.210/A/XI/2002 on release of sugarcane variety R 579
as a superior variety. [Indonesia]
Minister of Agriculture of the Republic of Indonesia. 2004.
Decree of the Minister of Agriculture number:
55/Kpts/Sr.120/1/2004 on release of sugarcane variety PS 851
as a superior variety. [Indonesia]
Mubyarto, Daryanti. 1994. Sugar, a national study of economics.
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ISSN: 2087-3940 (print)
ISSN: 2087-3956 (electronic)
Vol. 2, No. 1, Pp. 48-53
March 2010
Exposure copper heavy metal (Cu) on freshwater mussel (Anodonta
woodiana) and its relation to Cu and protein content in the body shell
AHMAD INTAN KURNIA1,♥, EDI PURWANTO², EDWI MAHAJOENO²
¹Sekolah Tinggi Ilmu Kesehatan (STIKES) Karya Husada Pare Kediri. Jl. Soekarno Hatta Po Box 153, Kediri 64225, Jawa Timur, Indonesia. Tel.: +92354-393888, Fax: +92-354-393888, ♥email: [email protected]
² Bioscience Program, School of Graduates, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia
Manuscript received: 8 October 2009. Revision accepted: 19 February 2010.
Abstract. Kurnia AI, Purwanto E, Mahajoeno E. 2010. Exposure copper heavy metal (Cu) on freshwater mussel (Anodonta woodiana)
and its relation to Cu and protein content in the body shell. Nusantara Bioscience 2: 48-53. To determine the relationship of Cu
exposure in water to the freshwater mussel exposure experiment is conducted with water containing Cu. Which measured the influence
of Cu and protein content in the body shell. This study used the freshwater mussel species, Anodonta woodiana. Oysters were exposed
for four weeks in the water with Cu concentration of 0.02 ppm, 0.04 ppm, 0.06 ppm and 0.00 ppm control. Cu content and protein
content in the body shells are checked every week. Cu analysis was done by AAS method and the protein content using Kjeldahl
method. Cu analysis showed elevated levels of Cu in mussel body after exposure. The pattern of increase in Cu content was not the
same, where the pattern of the largest increases occurred after the fourth week. The statistical test showed no significant effect between
the treatment with Cu accumulation in the body shell. Protein analysis showed an increase of protein content after exposure of the
second week and decreased after the third and fourth weeks. The pattern of changes in protein content varied among the various
treatments. The statistical test showed no significant effect between treatment with the protein changes in the body shell. Correlation test
of the relationship between concentration of Cu in mussel body protein level showed a positive correlation between them with a fairly
good level of relationship (correlation coefficient r = 0.836).
Key words: Anodonta woodiana, exposure, Cu, protein.
Abstrak. Kurnia AI, Purwanto E, Mahajoeno E. 2010. Paparan logam berat tembaga (Cu) pada kerang air tawar (Anodonta
woodiana) dan hubungannya dengan kandungan Cu dan protein dalam tubuh kerang. Nusantara Bioscience 2: 48-53. Untuk
mengetahui hubungan pemaparan logam Cu dalam air terhadap kerang air tawar maka dilakukan percobaan pemaparan dengan air yang
mengandung Cu. Pengaruh yang diukur adalah kadar Cu dan kadar protein dalam tubuh kerang. Penelitian ini menggunakan kerang air
tawar Anodonta woodiana. Kerang dipaparkan selama empat minggu dalam air dengan konsentrasi Cu 0,02 ppm, 0,04 ppm, 0,06 ppm
dan kontrol 0,00 ppm. Kadar Cu dan kadar protein dalam tubuh kerang diperiksa setiap minggu. Analisis Cu dilakukan dengan metode
AAS dan kadar protein menggunakan metode Kjeldahl. Analisis Cu menunjukkan peningkatan kadar Cu dalam tubuh kerang setelah
pemaparan. Pola kenaikan kadar Cu tidak sama, dimana pola kenaikan terbesar terjadi setelah minggu keempat. Uji statistik
menunjukkan tidak adanya pengaruh signifikan antara perlakuan dengan akumulasi Cu dalam tubuh kerang. Analisis protein
menunjukkan kenaikan kadar protein setelah pemaparan minggu kedua dan menurun setelah minggu ketiga dan keempat. Pola
perubahan kadar protein bervariasi antar berbagai perlakuan. Uji statistik menunjukkan tidak adanya pengaruh signifikan antara
perlakuan dengan perubahan protein dalam tubuh kerang. Uji korelasi hubungan antara kadar Cu dalam tubuh kerang dengan kadar
protein menunjukkan adanya korelasi positif antara keduanya dengan tingkat hubungan yang cukup baik (koefisien korelasi r = 0,836).
Kata kunci: Anodonta woodiana, pemaparan, Cu, protein.
INTRODUCTION
The idea of environmental pollution in Indonesian Act
No. 23 of 1997 on Environmental Management is the
inclusion of living things, matter, energy, and/or other
components into the environment by human activities so
that its quality decreases to a certain level that causes
environment can not function as intended. One of the
pollutants that are often found as contaminants in the
aquatic environment that is detected in the organism body
is heavy metals, copper (Ward 2001). Copper (Cu) is a
metal that widely used in chemical industry, metallurgy,
textiles and anti-rust paint (Effendi, 2003). Like other
heavy metals, Cu, is difficult to be soluted in the
environment and it can enter the food chain through
organisms that exist in the water. Aquatic organisms that
are known to accumulate heavy metals are phytoplankton,
zooplankton, class of fish, crustaceans (crustaceans) and
mollusks or shellfish species (Ward 2001).
Freshwater mussels (Anodonta woodiana) are bivalves
included in phylum mollusks that have two symmetrical
shells. They live in the riverbed, channels, ponds and lakes.
They can have rough or smooth shell depending on the
habitat where they live. Freshwater mussels are
microscopic plant-eating animals and perform it by sucking
water through a siphon and removing the particles. As a
KURNIA et al. – Cu exposure on freshwater mussels (Anodonta woodiana)
filter organism, freshwater mussels can serve to clean water
and reduce algae, particles, toxic materials and some diseases.
Freshwater mussels is an organism that can be used as
biological indicators or bioindicator (EPA 2009).
Biondicators are organisms or biological responses that
indicate the entry of certain substances in the environment.
Species or species group for bioindicator are selected based
on several factors which is; they are easily measured and
give response observed in ecosystems, have specific
response that is able to predict on how a species or
ecosystem will respond to a certain pressure, measure the
response with accuracy and precision that can be accepted
based on knowledge about contaminants and characteristics
(Mulgrew et al. 2006).
Umar et al. (2001) which researched on the relationship
between Cu in the aquatic environment with the content Cu
in the body of sea shells Marcia sp. concluded that the
higher the Cu in water and sediment, the higher the metal
content of Cu accumulated by shellfish that live in the area.
According to Sunarto (2007) there is a relationship between
structure of microanatomy, the efficiency of gill function,
morphology and condition of freshwater mussels Anodonta
woodiana with Cd concentrations in water. So from that
relationship and the condition of the shell morphology can
be used as a bioindicator macroscopic beginning at A.
woodiana due to exposure to heavy metals Cd. Stolyar et
al. (2005) says that there are significant effects caused by
heavy metals Cu to the protein of freshwater mussels. In
the laboratory experiments on a freshwater mussel
Anodonta cygnea shows that at the time of Cu exposure
concentrations in water increased by 10 micrograms/liter,
the protein content of Cu in Methallotionin will experience
increased 100.7% compared to control groups of organisms
(without any treatment of heavy metal exposure Cu).
To find out how the relationship of heavy metal Cu
exposure concentration to A. woodiana a laboratory study
is conductedf with observations of variable concentrations
of Cu and protein content in the body of a freshwater
bivalve A. woodiana. This study aims to: (i) know the
relationship between exposure concentration of Cu in water
with Cu concentration in the body of A. woodiana, (ii) to
study the relationship between the concentration of Cu in
water with the protein of A. woodiana; (iii) know the
relationship between the concentration of Cu in the body A.
woodiana with protein A. woodiana.
MATERIALS AND METHODS
Place and time of study
The study was conducted in three places.
Acclimatization activities, maintenance and treatment of
Anodonta woodiana was conducted at the Laboratory of
Environment STIKES Karya Husada Kediri, East Java.
Analysis of heavy metal Cu content in the body A.
woodiana was conducted in Chemical sub-Laboratory,
Center Laboratory of MIPA for UNS Surakarta. Analysis
of protein content in the body of A. woodiana was
conducted at the Laboratory of Agriculture, Faculty of
Agricultural Technology, Sebelas Maret University, Surakarta
49
Material
Materials research is a freshwater bivalve species A.
woodiana. Samples were taken from Fresh Water Fish
Seed Board (BBI) Janti, Klaten, Central Java. Shells used
are wit the following specifications size is 7-9 cm long with
a total weight of 30-45 grams.
Research design
The design of the research done on these activity was
completely randomized design (CRD), with 3 (three)
concentrations of Cu exposure treatment plus 1 (one)
controls. Type of concentration are:
(i) Group 1: 0.00 ppm Cu concentration (C0)
(ii) Group 2: Cu concentration of 0.02 ppm (C1)
(iii) Group 3: Cu concentration of 0.04 ppm (C2)
(iv) Group 4: Cu concentration of 0.06 ppm (C3)
Selection of exposure concentration of Cu is based on
PAN Pesticide Database (2009) which states that the lethal
concentration (LC50) for copper (CuSO (iv) toward fresh
water mussel A. woodiana is of 0.1 ppm.
A four time examination will be conducted to the four
groups based on the length of exposure time, namely:
(i) Examination 1: end of week-1 (T1)
(ii) Examination 2: the end of week-2 (T2)
(iii) The examination 3: end of week 3 (T3)
(iv) The examination 4: end of week 4 (T4)
From this experiment 16 units of the experiment will be
obtained (4 x 4 examination treatment group). In each
sample the variables observed are: concentration of Cu in
the shells A. woodiana and protein levels in the shells A.
woodiana.
Procedures
Experimental research
The animals tested were taken from fish ponds in Janti,
Klaten, Central Java then brought to the Laboratory of
Chemical STIKES Husada Kediri, East Java. The first step
is acclimatization. The shells were looked after in an
aquarium filled with clean water as much as 15 liters. Each
aquarium filled with 15 shells. The water in the aquarium
was changed every 3 to 4 days. Mussels were fed every
other day. The food provided is brand Takari manufactured
by PT. Protein Prima Tbk. Feeding is done by making food
that is still a solid into a liquid form and then dissolved into
the water in the aquarium. During the acclimatization if
there are dead shells then they will be replaced with new
ones. Acclimatization process was conducted for 2 weeks.
And then, experiments on heavy metals Cu exposure was
conducted. The standard solution containing heavy metals
such as Cu in certain concentrations was put into the
aquarium as planned. Each type of concentration was
carried out in three aquariums.
Aquarium grouping is as follow: aquarium 1, 2, and 3
for the concentration of Cu 0 ppm (control), aquarium 4, 5,
6 are for 0.02 ppm Cu concentration, aquarium 7, 8, and 9
for 0.04 ppm and 10, 11 , 12 for Cu concentrations of 0.06
ppm. Standard solution is inserted into the tank together
with replacing the water. So during the exposure time, the
shells will continuously be in the water containing copper
50
2 (1): 48-53, March 2010
in that amount. Every water replacement will be
accompanied by water temperature and pH measure.
Furthermore, mussels/shells are taken for analysis of Cu
content and protein content at the end of week 1, the end of
week 2, 3, and 4.
body A. woodiana, the relationship between protein content
with the content of Cu in the body A. woodiana.
Analysis of Cu content
Shells samples were taken, opened, and its feces was
dumped. And then the samples were weighed in analytical
scale and the weights were recorded. Samples were placed
in a glass beaker. Pour 5 mL of HClO4 into the sample and
let stand for 3-5 minutes. Add 50 mL of distilled water.
The sample was heated to form a homogeneous solution
and leaving a volume of 20 mL. Remove the sample from
the heater. Then 5 mL HNO3 was added. It was again
heated for 10-15 minutes. Add 50 mL distilled water. Filter
with filter paper and insert it into the sample bottle. And
then the sample was injected into the Flammable Atomic
Adsorption Spectrophotometer (FAAS). From the
injection, the data on levels of Cu would be obtained (in
units of ppm).
Relations between Cu concentration in water with the
levels of Cu in the body
To describe the magnitude of changes in Cu levels in
the body of the shells in each treatment the results are
expressed as a histogram. Image histogram shown in
Figure 1 which shows the movement or changes in Cu
levels in the body shells at each treatment concentration.
Analysis of protein content
Shells samples were taken, opened, and its feces was
dumped. Samples were crushed with a blender until
smooth, then given 50 mL of distilled water and stirred
until homogeneous. As much as 10 mL of sample solution
were taken and put into 100 mL of glass, diluted until it
reached the mark. From this solution (point 1) it was taken
as much as 10 mL and put into 500 mL Kjeldahl flask and
add 10 mL of H2SO4. Added 5 g mixture of Na2SO4-HgO
(20:1) for the catalyst. The solution was boiled until clear
and continued boiling for 30 minutes more. Once cool,
wash the Kjeldahl flask walls with water and simmer again
for 30 minutes, then cooled. When it was cool it was then
added with 140 mL distilled water and add 35 mL of
NaOH-Na2SO3 and some granules of Zink. Then do the
distillation. Distillate was gathered as much as 100 mL in
elmeyer tube containing 25 mL of saturated boric acid and
a few drops of red indicator. Titrate the solution obtained
with 0.02 HCl. Number of total N (% protein) was
calculated with the formula as follows:
The number N total = mL HCl x N HCl x 14,008 x f
mL sample solution
f = dilution factor
Data analysis
Analysis of variance (ANOVA) is used to see if there is
variation among the treatments. Rank data analysis with
analysis variant will be conducted on data obtained from
the examination of Cu content in the network and data
examination of protein levels in the body A. woodiana.
Correlation analysis is used to determine the relationship
between variables observed in the study. Data to be tested
with correlation analysis is the relationship between the
concentration of Cu in water with high levels of Cu in the
body A. woodiana, the relationship between the
concentration of Cu in water with protein content in the
RESULTS AND DISCUSSION
Figure 1. Cu levels in the body of shells A. woodiana
The ANOVA test on these calculations using degrees of
freedom of 3 and with a confidence level of 95% (ρ =
0.05). From the table ANOVA F test result greater than the
F table. The results of this test gives the sense that there is
no significant difference in Cu concentrations in water
treated with Cu levels in the body shells, or the variation of
treatment to be given in the form of concentration variation
of Cu in the water did not give significant effect on levels
of Cu in the body shells. Based on ANOVA statistical test
the results stating that there is no significant difference in
the concentration of Cu treatment. It means that the results
of statistical tests can be analyzed from several points of
the view.
According Sǎrkǎny-Kiss et al. (2000), the ability of
mussels organisms to accumulate heavy metals in the body
is influenced by two factors, namely exterior complex and
inner complex. Exterior complex is the condition of water
environment in which these organisms live. While the inner
complex factors are matters related to the metabolic
capabilities of organisms with the presence of heavy metal
components in the body. Referring to the opinion, the
phenomenon in this study can be viewed from two factors,
namely the live shells is based on water conditions as the
life media, and based on the characters of organisms A.
woodiana.
In the factor of environmental, this experiment has been
made causes the environment in which mussels live are
relatively similar and homogeneous. All the variables and
parameters associated with mussels habitat in this
experiment have been controlled and made uniform. Only
KURNIA et al. – Cu exposure on freshwater mussels (Anodonta woodiana)
the concentration of Cu which is made vary according to
the research design.
The first is about water. In this experiment the water
used to perform the experiment originated from the same
water source. For each type of treatment, water used is
handled in the same way and taken in the same time.
Physical factors of the water as place for living such as
temperature and pH was monitoring in every water change.
Water temperature during the experiment ranged in around
27oC. The food given were also made similar for each
treatment. In this experiment, mussels were fed every other
day. Food provided is Takari fish feed manufactured by
PT. Protein Prima Tbk. Each concentration treatment was
the same food and made at the same time.
Based on existing levels of Cu in the body of samples
mussels, it appears that among the individual samples there
are significant differences in the ability to absorb copper, to
process of metabolism and to accumulate copper particles
that are soluble in water. Then this different ability results
in different Cu accumulation in the body. This fact can be
seen on the results of analysis of Cu for each sample
(Figure 1). From this figure, it appears that the same type
of treatment concentration and exposure at the same time
still shows that there are a few samples that gave results
that differ greatly. The difference of the Cu accumulation
got here almost hit 100% difference. For example in the
treatment concentration of 0.02 ppm Cu (week 2), Cu 0.04
(week-1 and 2) and Cu 0.06 at week 1. The same table also
shows that from the same type of treatment and longer
exposure time resulted in the lower accumulation of
copper, while the other treatments showed an increase.
This is visible on the treatment concentration of 0.02
ppm, where in the first week its average Cu accumulation
was 0.0107, but in the next week the number decreased as
much as 24.80% to 0.0086 ppm. Difference like that is
what the statistics would cause the effect of exposure
concentration of Cu in the water has no significant effect
on Cu content in the body A. woodiana. A pattern of Cu
accumulation in the mussels body that vary at the same
time shows that the system of mussels biology and
metabolism is not a simple system which has a linear
mechanism, where the same input (treatment) would results
that were very different.
According to Krolak et al. (2001) the fact that the Cu
content varying in individual of A. woodiana living with
the same treatment is the effect of a selective retrieval
capabilities of various components in the surrounding
environment and is influenced also by the ability of
different parts of each organ in the body of the mussels to
accumulate Cu.
From these quotations steps of analysis can be taken
that the Cu content accumulated in A. woodiana after
exposure to the metal Cu is influenced by two things.
These two things are how the Cu ions get inside of the
organism and how the Cu ions are accumulated in the
organism. It can be said that the difference in levels of Cu
accumulated in this experiment was caused by the
differences in both processes.
51
The process of inclusion of Cu ions into the mussels body.
In Soto et al. (2008) it is explained that metal enters the
oysters in two ways. First is through the gills and second
through the organs of the body which is in direct contact
with the water containing the heavy metals. From these
quotations, we can say that the quantity of Cu goes into the
shells is influenced by two things. The first is related to the
gills and the second is related to the contact or exposure of
organs with heavy metals.
If Cu enter through the gills: in bivalves, including
Anodonta wodiana, metal ions in water will get into the
gills by diffusion, which is a more passive process (Soto et
al. 2008). Because this process is passive, the decisive part
the diffusion process is the condition of the gills, i.e. the
width of the philamen surface. If the greater the surface of
gill filaments, then the diffusion will occur the more and
more, thus more Cu ions enters the body of the mussels.
Besides the diffusion process, the process of Cu enters the
gill also involves active transport mechanism, i.e. when the
gills must work against the pressure difference between the
water pressure and the pressure in the gills. Gill’s ability to
perform the active transport depends on the availability of
ATP (Soto et al. 2008). From the description of this process
shows that the extent of Cu entering through an active
transport process depends on each individual. Where the
influential part is the availability of energy in the form of
ATP.
Cu influx through the organ of the mussels which is in
direct contact with the water. In A. woodiana, organ which
is in direct contact with water is the organs which
anatomically Fox calls(2005) the external anatomy, that is
the shell, muscle cells (mantle), gill, the body (visceral
mass), foot, and labial palps. In the closed shell condition,
the organs which will be in contact with water are the two
pieces of shells, while the organs that are inside it are not in
contact with water. In the condition where the shell is open,
all the organs of external anatomy will be in contact with
water.
At the time of contact with the media/water, the Cu ions
dissolved in water will penetrate the surface of the organ
and enter into the cell. The process when Cu ions enter into
the cell occurs in two ways, namely by passive diffusion
and active transport. In the process of passive diffusion, the
parts that play important role are the area in contact with
water. While on active transport processes the most
influential thing is the availability of energy (Soto et al.
2008).
From these conditions, the process of Cu entering
through the contact is determined by the surface area of the
water contacts. The greater the surface area that contacts
the more and more the Cu that can enter the cell. Similarly,
the greater the available energy of ATP, the more active
transport process and Cu will be more included in the cell.
The process of Cu ion accumulation in the body of the
mussels
After the copper enters the body, on a scale of cellular
the metals will experience stabilization process, from the
original shape that is still in the form of free ions then form
a ligand binding with other components. Cu bond
52
2 (1): 48-53, March 2010
stabilization process in the body bivalves takes place in two
processes. The first is in the process of synthesizing metalbinding proteins, where metal ions play role as promoter
and will be bound in a protein that is formed. The second
process is the formation of granulation of mineral grains or
mineralized granules, in which metal ions would be bound
in mineral granules that are not dissolved (Soto et al. 2008).
According to Stanley (2003) the process of stabilization
and detoxification of heavy metals in the body are made in
three types of processes, namely: (i) through the formation
of soluble compounds that bind metals, in this case it is
methallotionin protein synthesis, (ii) through the storage
process (compartmentalization) of metal in one of the cell
organelles cell which is in lysosomes, (iii) through the
formation of the precipitate that was dissolved in the form
of granulation of mineral grains that can bind metals.
When it is known that the metal accumulation process
is done in two or three streets, as mentioned above, when
the results of this study found no significant relationship
between metal concentration exposure to metal
accumulation in A. woodiana, meaning three Cu
accumulation process occurring in the body are varied and
not uniform. This variation that ultimately gives the
resultant in the form of accumulated Cu levels varying
between individual samples of shellfish. The existence of
these differences were in line with Viarengo et al. (1993)
who said that the detoxification process that involves the
stabilization and the storage of metals in the body of
bivalves have a level of effectiveness that varies among
species, in the same species between different individuals
and on the same individual between different organs. These
differences, according to Soto et al. (2008) defined by two
biotic factors, namely the age and the weight of individual.
Age influences the level of sensitivity of a shellfish
organisms to absorb metal ions. The young individuals are
more sensitive and able to absorb heavy metals more than
the older individual. While the weight ratio between weight
and volume of organs which can be exposed by metal.
Relations between Cu concentration in water with levels
of protein in the body of the mussels
Results of analysis of protein levels in the body is the
result of the examination of laboratory analysis of total
protein content in samples of shellfish A. woodiana.
presented in Figure 2, which shows the changing levels of
protein in the body shells in each - each concentration
treatment.
Figure 2. Protein levels in the body of A. woodiana.
From the table ANOVA, F test result is greater than the
F table. The results of this test gives the sense that there is
no significant difference in differences of treatment Cu
concentration in water with levels of protein in the body of
the mussels. The variation of treatment provided in the
form of various concentration of Cu in the water does not
have a significant impact on levels of Cu in the body of the
mussels.
In the study of the relationship between the effects of
heavy metals with the change in levels of protein in
organisms, the review is on protein Methallotionin.
According to Soto et al. (2007) Methallotionin protein (MT
protein) is a Cytosol protein with low molecular weight,
soluble, resistant to high temperatures (thermophilic
proteins), rich in sulfur elements (more than 30%) and has
a strong affinity with metal ties. In aquatic organisms, MT
proteins responsible for maintaining the metal
concentration remain at low levels. Protein synthesis was
induced by the presence of metal in the cell. MT protein
specifically binds with metal Cd, Cu, Hg, and Zn ions.
Increase in levels of MT proteins associated with an
increase in the capacity of cells to bind heavy metal ions
which increases the protection against toxicity of heavy
metals.
In this study, based on statistical tests it is concluded
that there is no significant impact from the variation of Cu
in water treatment to changes in protein levels in mussels.
In other words that the difference of treatment and duration
gave no significant impact on the changing levels of
protein in the body of the mussels.
The result of these statistical tests can actually be read
visually from the pattern of change protein content. In the
Figure 2. above it appears that there is an increase of
protein levels in the second week of exposure. After that in
week three the protein levels decreased and in the next
week there was also a decline, except in the 0.04 ppm
treatment the protein content increased in the fourth week.
The existence of patterns of change that are not regular
showed a wide variation that occurred in this experiment.
The results of this study more or less is in line with Stolyar
et al. (2005) on Cu exposure toward Anodonta cygnea. In
the study it is illustrated that at low concentrations of Cu
exposure, increase in levels of protein will be the same as
the control organisms or, no change at all. While the
highest concentration of Cu exposure (0.2 ppm) did not
provide a significant impact on changes in protein levels in
the body of the mussels that were tested.
The reasons of why there is no relationship between
levels of Cu in the water with the change of protein in the
body of the mussels can be seen in two views. The first
refers to the opinions of Stanley (2003). In this study it is
explained that the heavy metals are stored in the body of
freshwater mussels in three forms, namely as a metalbound in the precipitate minerals, metals stored in cell
organelles as lysosomes and metal that is bound by the
metal-binding-protein binds, or Methallotionin. Soto et al.
(2008) adds that in the body of freshwater mussels there is
also the ability to excrete heavy metals that enter directly,
besides the three processes mentioned above.
KURNIA et al. – Cu exposure on freshwater mussels (Anodonta woodiana)
From the statement above, the presence of protein
synthesis formed in response to the entry of metal into the
cell is one of four other responsive processes. In other
words the existing copper metal in water will not fully
trigger a protein synthesis in cells. That is because there are
other mechanisms of storage in the lysosomes, the process
of granulation with a mineral and excretion of Cu ions
directly. In addition, up to now it has not been known for
certain about which one is the most dominant process in
response to the entry of copper ions into the body of the
mussels.
53
conducted by Stolyar et al. (2004) which illustrates that
copper metal ions in water will increase the metal content
in the cells of the body. And it will lead to increased levels
of MT proteins in animals Anodonta cygnea. Soto et al.
(2007) explains the elevated levels of Cu linkages with
elevated levels of this protein. Increased levels of this
protein are a response to elevated levels of Cu, where MT
protein will bind Cu ions to prevent the toxicity of these
metals.
CONCLUSION
Relations between Cu concentration in the body with
levels of protein in the body of the mussels
The relationship between Cu levels in shellfish with
high levels of protein in the shells is presented in Figure 3
below:
Provision of Cu concentration in water did not affect
significantly to the amount of Cu content accumulated in
the body of A. woodiana. Provision of Cu concentration in
water did not give significant effect on changes in body
protein content of A. woodiana. Cu levels in the body have
a positive correlation with levels of protein in the body of
A. woodiana.
REFERENCES
Figure 3. The relationship between Cu content with protein
content in the body of A. woodiana
Figure 3 shows that there are variations in the pattern of
relationship between Cu with proteins in the body shells.
At 0.0035 ppm Cu values up to 0.0107 ppm, protein
content has a pattern of relationships that fluctuate up and
down. Meanwhile, in the range 0.0287 ppm Cu values up
to 0.1375 ppm, which was conceived by the protein content
of oysters tend to have patterns of increase with increasing
Cu content. To determine the relationship between Cu
content with protein content in the body shell, then the data
was analyzed by correlation method of Pearson Product
Moment (Nazir 2005).
The computation with the working table, obtained that
the correlation coefficient amounted to 0.848. This value
illustrates the positive correlation between two variables
measured, namely Cu levels in the body shells with protein
levels in the body shells. Where the degree of relationship
is quite good. In other words the change of Cu content in
the body shells have a fairly strong correlation with
changes in protein levels in the body shells.
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Chapter in book:
Webb CO, Cannon CH, Davies SJ. 2008. Ecological organization, biogeography,
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| Nus Biosci | vol. 2 | no. 1 | pp. 1‐53 | March 2010 | ISSN 2087‐3948 (PRINT) | ISSN 2087‐3956 (ELECTRONIC) I S E A J o u r n a l o f B i o l o g i c a l S c i e n c e s Ripening for improving the quality of inoculated cheese Rhizopus oryzae SOLIKAH ANA ESTIKOMAH, SUTARNO, ARTINI PANGASTUTI 1‐6 Comparasion of iles‐iles and cassava tubers as a Saccharomyces cerevisiae substrate fermentation for bioethanol production KUSMIYATI 7‐13 Variation of morphological and protein pattern of cassava (Manihot esculenta) varieties of Adira1 and Cabak makao in Ngawi, East Java TRIBADI, SURANTO, SAJIDAN 14‐22 Diversity analysis of mangosteen (Garcinia mangostana) irradiated by gamma‐ray based on morphological and anatomical characteristics ALFIN WIDIASTUTI, SOBIR, MUH RAHMAD SUHARTANTO 23‐33 First record of two hard coral species (Faviidae and Siderastreidae) from Qeshm Island (Persian Gulf, Iran) MAHDI MORADI, EHSAN KAMRANI, MOHAMMAD R. SHOKRI, MOHAMMAD SHARIF RANJBAR, MAJID ASKARI HESNI 34‐37 Isolation and identification of lactic acid bacteria from abalone (Haliotis asinina) as a potential candidate of probiotic SARKONO, FATURRAHMAN, YAYAN SOFYAN 38‐42 Productivity of sugarcane plants of ratooning with fertilizing treatment A SUTOWO LATIEF, RIZAL SYARIEF , BAMBANG PRAMUDYA, MUHADIONO 43‐47 Exposure copper heavy metal (Cu) on freshwater mussel (Anodonta woodiana) and its relation to Cu and protein content in the body shell AHMAD INTAN KURNIA, EDI PURWANTO, EDWI MAHAJOENO 48‐53 Published three times in one year PRINTED IN INDONESIA ISSN 2087‐3948 (print) ISSN 2087‐3956 (electronic)