Biology: February 12-13 2014

February 12-13rd 2014
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The 4 Annual Basic Science International Conference
BIOLOGY
Batu, East Java, Indonesia | 1
2 | Batu, East Java, Indonesia
th
th
Resistance Evaluation of Large Seeded Soybean

Lines to Rust Disease
Sumartini 1 and Heru Kuswantoro 1*)
Indonesian Legume and Tuber Crops Research Institute
Indonesian Agency for Agricultural Research and Development
1)
*)
Corresponding author: heru@litbang.deptan.go.id
Abstract Rust disease is a major disease on soybean,

wide spreading, existing in almost all soybean producer
countries, and causing yield losses up to 80%. The objective was to evaluate the resistance of soybean lines to rust
disease. The research was conducted in the greenhouse of
Indonesian Legume and Tuber Crops Research Institute,
from March to June 2013. Design was randomized completely block design with three replications. Materials were
10 soybean lines and five check varieties. Inoculating rust
spores was carried out at 3 weeks after planting by using
spore suspension (104/ml density) derived from infected
soybean leaf. Inoculation was performed by spraying the
spore suspension on the first leaves at 16.00-18.00. Observations rust disease resistance based on the system of International Working Group on Soybean Rust. Results
showed that there was no soybean line identified as resistant line. Of a total ten soybean lines, seven soybean lines
(Tgm/Anj-908, Tgm/Anj-909, Tgm/Anj-910, Tgm/Anj-919,
Tgm/Anj-932, Tgm/Anj-957, and Tgm/Anj-995 were identified as moderately resistant, two soybean lines (Tgm/Anj933, and Tgm/Anj-991) were susceptible, and one soybean
line (Tgm/Anj-931) was susceptible. The three check varieties (Wilis, Tanggamus and Anjasmoro) were moderately
resistant, while two check varieties (Grobogan and Argomulyo) were moderately susceptible.
Keywords soybean lines, resistance, rust disease.
I. INTRODUCTION
ASED on the seed size, soybean seeds are classified
as small seed, medium seed and large seed. Usually,
medium and small seeds are used for soy sprouts,
soy sauce, tofu and soy milk ingredients, while large
seeds is used for tempeh ingredient. However, the main
usage is for tempeh ingredient as well as tofu. Therefore, one of the Iletris soybean breeding programs is
directed to develop large seeded soybean variety.
In developing soybean variety, the resistance of the
new variety to rust disease should be tested to provide
supporting data for release variety; because rust disease
is one of the major soybean disease in Indonesia. Rust
disease also known as the "Asian soybean rust", caused
by the fungus Phakopsora pachyrhizi. Rust disease is
widespread in the area of soybean production centers in
the world and causing significant yield loss. Distribution
of rust disease started from Japan and East Asia in 1902,
entered into Southeast Asia (Indonesia) and Australia in
1914, while in 1950 has reached India, and in 1994 en-
tered Hawaii. Then it entered South Africa in 1920 and

has reached Uganda in 1996. In the years 2001 - 2002
rust disease infestation appeared in South America, and
in 2004 had spread out to the north reaching the United
States [1]. Yield losses can reach more than 85% if suitable environment for disease development [2]. Furthermore, it is stated that there are four types of genes that
responsible for controlling rust disease resistance in
soybean, i.e. Rpp1 , Rpp2 , Rpp3, and Rpp4 [2].
One of the techniques for controlling soybean rust
disease is to grow resistant varieties. There are several
soybean varieties having rust disease resistance that
have been released. The resistance to rust disease is not
durable and someday the resistance will be broken because the fungus P. pachyrhizi can mutate to be new
races. Therefore, developing new of superior that resistant to rust disease is still needed. The purpose of the
study is to evaluate the resistance of large seeded soybean lines to rust diseases.
II. MATERIALS AND METHOD
The study was conducted in Indonesian Legume and
Tuber Crops Institute, in the dry season (March-June
2013). The design was randomized completely block,
with three replications. The materials research were 10
soybean lines (Tgm/Anj 908, Tgm/Anj-909, Tgm/Anj910, Tgm/Anj-919, Tgm/Anj-931, Tgm/Anj-932
Tgm/Anj-933, TGM / Anj-957, Tgm/Anj-991,
Tgm/Anj-995) and four check varieties (Wilis,
Tanggamus, Anjasmoro, Grobogan, Argomulyo).
Soybean seeds were grown in plastic polybag ( =
15cm), two plants per polybag. At one month old, plants
were inoculated with rust disease by spraying spore suspension (104/ml) to soybean leaves. Spore suspension
was originated of rust-infected leaves from inoculum
plantation sources. The day before inoculation, infected
leaves were taken to be incubated at 100% humidity
conditions in the laboratory. After 24 hours, the spores
were taken by using a brush, and then the spores were
diluted. The spore suspension was homogenized by using Tween 20, two drops per liter. To avoid pest attacking, spraying insecticides (carbofuran, sipemetrin, cyhalothrin) was carried out several times alternately.
Intensity of rust disease was observed in all tested
plants at 7 and 9 weeks after planting by IWGSR (International Working Group on Soybean Rust) method [3]
(Table 1).
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TABLE I
DETERMINATION OF RESISTANCE CRITERIA TO RUST DISEASE
Position of the disease on the plant
1
: 1/3 of lower part of the plant
2
2/3 of middle part of the plant
1/3 of upper part of the plant
Disease intensity
1
no pustule
mild ( 1 8 pustul/cm2 )
medium ( 9 16 pustul/cm2 )
high ( > 16 pustul/cm2 )
Resistance criteria
Imun
Score 111
Resistant
Score 122, 123, 132, 133, 222. 223
Moderately resistant
Moderately susceptible
Score 142, 143, 232, 233, 242, 243,

322, 323
Score 332, 333
Susceptible
Score 343
In addition to the intensity of rust disease, observation

was also carried out on yield component, such as plant
height, number of branches per plant, shoot fresh
weight, shoot dry weight, number of filled and unfilled
pods per plant, and seed weight.
III. RESULTS AND DISCUSSION
The results showed that until week 9th, position of
contained, there was no resistant genotype, seven

genotypes were moderately resistant (MR), two
genotypes were moderately susceptible, and one
genotype was susceptible (Table 2). In this experiment,
the three checks varieties (Wilis, Tanggamus and Anjasmoro) were classified as moderately resistance. This
results were similar to the varieties description [4].
However, Argomulyo was moderately susceptible, different to the description that Argomulyo is tolerant to
rust disease. Similar results also reported by
investigators from Nigeria using simpler methods based
solely on the intensity of rust disease on a scale 1-5.
With this method they can classify resistance of soybean
genotypes to rust disease. Of the 28 tested genotypes,
most of the genotypes were susceptible to moderatly
resistance. However, they found seven resistant
genotypes [5]. Beside, they found dominant genes
controlling rust disease found resistance in three
different loci. Investigators from the United States
classified resistance to soybean rust disease into four
types of Phakopsora pachyrhizi fungi isolates and the
symptoms by two types of symptoms i.e. reddish-brown
(RB) and TAN. Of 34 tested genotypes, 28 genotypes
were included as TAN type with many sporulating and
six genotypes were included as RB (reddish-brown) [6].
Plant height is an important character because it has a
positive correlation to grain yield [7]. Beside, plant
height had indirect effect on grain yield through number
of branches per plant, number of pods per plant, number
of grain per plant and 100 grains weight [8]. Plant height
of the tested genotypes were classified as normal.
However, three genotypes had abnormal plant height,
i.e. Tgm/Anj-995, Tgm/Anj-919 and Tgm/Anj-931
TABLE II
SCORES AND RESISTANCE CRITERIA OF LARGE SEEDED SOYBEAN GENOTYPES TO RUST DISEASE
Genotypes
Pustul position
Number of
pustul/cm2
Disease
intensitas
Spore existence
Score
Resistance criteria
Tgm/Anj-908
Tgm/Anj-909
Tgm/Anj-910
Tgm/Anj-919
Tgm/Anj-931
Tgm/Anj-932
2
2
2
2
3
2
14
11
9
13
18
11
3
3
3
3
4
3
3
3
3
3
3
3
233
233
233
233
343
233
Moderately resistance
Tgm/Anj-933
Tgm/Anj-957
3
2
11
13
3
3
Tgm/Anj-991
Tgm/Anj-995
Wilis
Tanggamus
Anjasmoro
3
2
2
2
2
10
11
12
10
10
3
3
3
3
3
Grobogan
3
16
3
plant disease was in the middle to upper canopy, or 2 to
3 score. The intensity of rust disease scores ranged 3-4,
or the number of pustules from 8 to more than 16
pustules per cm2. It means that the intensity was mild to
high, and many pustules had spores. Of the 10 genotypes
Susceptible
Moderately suscepti3
333
ble
2
232
333
ble
3
233
3
233
2
232
3
233
333
ble
Moderately
suscepti(Table 3). The three genotypes had different
response
to
rust disease; where Tgm/Anj-995 and Tgm/Anj-919
were moderately resistant and Tgm/Anj-931 was
susceptible. In this case, the rust disease did not cause
plant height character. Probably, other unknown
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TABLE III
PLANT HEIGHT, SHOOT FRESH WEIGHT AND SHOOT DRY WEIGHT OF LARGE
SEEDS SOYBEAN. ILETRI GREENHOUSE, DRY SEASON 2013
Genotypes
Plant height
(cm)
Tgm/Anj-908
Tgm/Anj-909
Tgm/Anj-910
Tgm/Anj-919
68.17
63.83
52.11
43.94
a
ab
bc
c
Tgm/Anj-931
Tgm/Anj-932
Tgm/Anj-933
Tgm/Anj-957
Tgm/Anj-991
Tgm/Anj-995
44.62
56.61
67.17
67.89
62.06
18.44
c
abc
a
a
ab
d
Wilis
Tanggamus
Anjasmoro
Grobogan
Argomulyo
61.56
56.44
62.39
52.11
65.39
ab
abc
ab
bc
a
Shoot fresh
weight (g)
Shoot dry
weight (g)
15.34 abcd
8.567 abc
17.76ab
18.75ab
10.22 ab
9.753 ab
9.40 cde
8.66 de
13.98 bcd
18.78 ab
19.54 ab
21.14 a
6.653
5.400
8.453
9.180
9.610
11.01
c
c
abc
abc
ab
a
3.03 f
16.79 ab
16.59 ab
19.14 ab
15.95 abc
14.25 bcd
1.117
8.787
8.770
9.733
9.150
7.957
d
abc
abc
ab
abc
bc
environmental factors affected the plant height

performance in this study.
The performance of fresh weight and dry weight of
plants were similar to plant height performance. Plant
with having higher fresh weight and dry weight also had
higher plant height, and vice versa. The three shortest
lines (Tgm/Anj-995, Tgm/Anj-919 and Tgm/Anj-931)
also had the lowest fresh weight and dry weight of plants
(Table 3). The low value of fresh weight and dry weight
may be caused by inability of the short plant to develop
larger or more organs than high/normal plants. Plant dry
weight described photosynthate which produced by the
plant. The photosynthate is partitioned into plant organs,
and it leads the magnitude of grain yield.
determined by the number of seeds [9], and the number

of seeds is determined by the number of pods. The
number of pods per plant varied among tested
genotypes, where Tgm/Anj-995 showed the lowest
number of filled pods and Tgm/Anj-909 showed the
highest number of filled pods. Similar to fresh weight
and dry weight of plants, soybean lines with lower plant
height also showed lower number of pods (Table 4).
The number of pods per plant and the number of
reproductive nodes have a positive correlation [10]. It is
because the formed organs (pods) are larger and more
on normally growing plants. In contrast to the number of
pods, number of unfilled pods did not seem related to
plant height (Table 4). Unfilled pod is a pod that cannot
be formed completely, because the lack of
photosynthate for pod developing in the end of seed
filling period. In addition, the number of unfilled pods is
determined by genetic factors, where the broad sense
heritability of this character was high (93.1%) [11].
The most number of branches per plant was indicated
by the check variety of Anjasmoro, while the lowest by
Tgm/Anj-995. Soybean lines with low plant height were
still able to produce number of branches as much as
normally growing genotypes (Table 4). Branch is a large
plant organ where the development of this organ
requires extremely high energy. Therefore the number of
branches was relatively similar between the tested
genotypes.
Grain yield is not an independent character [7], but a
complex character where the expression is determined
by genetic and environmental factors [12]. The highest
grain yield was showed by Tgm/Anj-991 while the
lowest by Tgm/Anj-995 (Table 4). Both of these lines
had different responses to rust disease, where Tgm/Anj991 was moderately susceptible and Tgm/Anj-995 was
TABLE IV
NUMBER OF FILLED AND UNFILLED PODS, NUMBER OF BRANCHES, AND GRAIN YIELD PER PLANT OF LARGE SOYBEAN SEEDS. ILETRI GREENHOUSE, DRY
SEASON 2013
Genotypes
Number of filled pods per

plant
Number of unfilled pods

per plant
Number of branches per

plant
Grain yield per plant (g)
Tgm/Anj-908
30.11 bcd
1.05 cd
2.17 cd
3.96 ab
Tgm/Anj-909
40.89 a
1.00 cd
2.55 bc
3.84 ab
Tgm/Anj-910
31.06 bcd
1.33 bcd
2.17 cd
3.98 ab
Tgm/Anj-919
14.05 e
2.28 ab
2.22 cd
2.67 c
Tgm/Anj-931
14.95 e
0.83 cd
2.06 cd
2.56 c
Tgm/Anj-932
27.44 cd
1.11 cd
2.61 bc
3.59 abc
Tgm/Anj-933
29.39 bcd
0.83 cd
1.94 cd
3.35 abc
Tgm/Anj-957
32.22 bc
1.06 cd
2.06 cd
3.30 abc
Tgm/Anj-991
29.22 bcd
0.95 cd
2.16 cd
4.28 a
Tgm/Anj-995
0.167 f
0.33 d
0.22 e
0.01 d
Wilis
33.56 abc
1.17 cd
3.28 ab
3.08 bc
Tanggamus
Anjasmoro
Grobogan
Argomulyo
28.33 cd
1.44 abc
1.22 cd
2.39 a
1.28 bcd
2.28 cd
3.56 a
2.00 cd
1.61 d
3.34 abc
36.61 ab
27.95 cd
23.94 d
The number of pods determine grain yield because

grain yield is the total photosynthate partitioned into
seeds, and the magnitude of grain yield was also
3.85 ab
2.93 bc
3.39 abc
moderately resistant. In this study, grain yield was not

affected by the response to rust disease. Grain yield is a
plant characters that affected by other yield components,
th
especially the number of pods and seed size. There is a

positive correlation between grain yield and 100 seeds
weight [13], [14]. Contribution of yield components to
grain yield may a direct effect or an indirect effect, and
varies depending on the environmental conditions [15],
[16].
IV. CONCLUSION
Of the 10 soybean genotypes, there was no resistant
genotype, seven genotype were moderately resistant
(Tgm/Anj-908, Tgm/Anj-909, Tgm/Anj-910, Tgm/Anj919, Tgm/Anj-932, Tgm/Anj -957, and Tgm/Anj-995),
two genotypes were moderately susceptible (Tgm/Anj933, and Tgm/Anj-991) and one genotype was
susceptible (Tgm/Anj-931). Rust disease infestation did
not affect yield and yield components.
[6]
[7]
[8]
[9]
[10]
[11]
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Shanmugasundaram. 1977. The International working group
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G. A. Iwo, M. A. Ittah, and E. O. Osai. 2012. Source of
genetics of resistance to soybean rust Phakopsora pachyrhizi
(H. Sydow & Sydow) in Nigeria. Journal of Agricultural
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C. Paul, C. B. Hill, and G. L. Hartman. 2011. Comparisons of

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2011. Investigation and comparison of some morphological
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M. El. M . El-Badawy,. and S. A. S. Mehasen, 2012. Correlation
and Path Coefficient Analysis for Yield and Yield Components
of Soybean Genotypes Under Different Planting Density. Asian
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Harmida. 2010. Respons pertumbuhan galur harapan kedelai
(Glycine max (L.) Merril) pada lahan masam. Jurnal Penelitian
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Leaf Characteristics of Some Trees Species in

Accumulating SO2 and NO2 Pollutant in Park
Area of Padjadjaran University Jatinangor
Fauziah Hafidha *), Mohamad Nurzaman,
Nurullia Fitriani, and Teguh Husodo
Department of Biology, Padjadjaran University, Sumedang, Indonesia
*)
Corresponding author: hafidhafauziah@gmail.com
Abstract Research about the relation of leaf characteristics in some trees species to the accumulation of sulfate
and nitrate has been done. The purpose of this research is
to find out the leaf characteristics that related to the sulfate
and nitrate accumulation. This research is using survey
method and correlation analysis. The plants that used in
this research were dadap merah (Erythrina crista-galli),
suren (Toona sureni), bungur (Lagerstroemia speciosa), ki
acret (Spathodea campanulata), kayu afrika (Maesopsis
eminii) dan mahagoni (Swietenia mahagoni). The results
show that bungur has the largest leaf surface and dadap
merah has the thickest leaf. Mahagoni has the highest value
of stomatal density and kayu afrika has the largest size of
stomata. Dadap merah has the highest value of total chlorophyll content. The highest value of sulfate accumulation
was in ki acret and the highest value of nitrate accumulation was in bungur. The leaf width has the positive correlation with sulfate accumulation. The leaf width, leaf thickness, stomatal size and chlorophyll content have the positive correlation with nitrate accumulation.
Keywords tree, leaf characteristics, sulfate, nitrate,
pollutant accumulation
I. INTRODUCTION
IR pollution is the introduction of pollutants into
the atmosphere that can cause disruption and discomfort to living organisms and also cause environmental damages[1]. Air pollution emissions are released naturally from smokes of forest or volcanic eruption but mostly cause due to anthropogenic sources[2,3]
such as human activities, economic development, increasing population, use of transport, industrial development and higher level of energy consumption[1,4].
Sulfur oxides (SOx) are one of the gaseous pollutants.
SOx pollution is mainly caused by sulfur dioxide (SO2)
and sulfur trioxide (SO3). Most of SO2 gases in the atmosphere are the result of human activities that comes
from burning fuel, such as coal, charcoal, wood and the
results of industrial processes[5]. SOx pollution can cause
human respiratory disease[6] and can cause tissue damage (leaf necrosis), even on the higher expose of SO2
can cause cholorosis to the plant[29].
Nitrogen oxides (NOx) can be found as nitrogen monoxides (NO) and nitrogen dioxides (NO2), but NO2 are
the most common gases that found as air pollutant. The
primary sources of NOx pollutants are motor vehicle

emissions, electric tools and human activities such as
industrial, trading and the result of the household burning[7]. Chronic exposure of NOx can cause immune system decrease and toxicity of NO2 can cause health problems, especially lungs problem to human[8]. NO absorption by plant can cause leaf flecks and higher concentration of NO2 exposures can cause leaf necrosis[9].
Plants are one of the living organisms that receive a
lot exposure from air pollution[4]. Most of pollutants
enter the plant trough leaf and can cause some damages,
although the plant still has a defense mechanism to minimize the damages. The mechanism is conducted
through the movement of opening and closing the stomata and also detoxification process[10].
Plants like trees have the potency to reduce pollutants
by providing large leaf area to accumulate pollutants[7,11]. Trees provide a leaf surface onto which particles are deposited and gases like SO2 and NO2 are removed[12]. Plant ability in absorbing gaseous pollutants
was determined by some factors such as morphology,
anatomy and physiology of the leaf. The absorption of
gaseous pollutants process is mainly occurs through
stomata[7]. The stomatal density affects the potential of
leaf to absorb pollutants[3]. Moreover, morphological
characters such as leaf surface and leaf thickness affect
the absorption of pollutant too[13]. Chlorophyll content
of the leaf may change due to air pollution[11].
Park is a green area which has a function to reduce air
pollutant concentration. The vegetation at the park is
effective to adsorb and absorb pollutants that come from
motor vehicles emissions[14]. Front park area of Padjadjaran University is areas that close to Jatinangor Sumedang highway road. The relation between plant leaf
characteristic with the absorption of SO2 and NO2 can
be discovered by calculate the accumulation of sulfate
and nitrate concentration then analyzed with correlation
analysis. The plants selected for this research are the
trees that exist in the front park area and are tolerant
species, especially SO2 and NO2 pollutant, based on the
study literature.
II. MATERIAL AND METHODS
Study area : The area of this study was in front park
th
area of Padjadjaran University near the Jatinangor

Sumedang highway road and located on 065555.7S
and 1074621.7E. This park location is near Jatinangor Sumedang highway road and Arboretum Unpad..
The trees that are used for this study is the trees that
exist on the park, near the road and have the height
around 1 3 m and also based on literature have the
ability to absorb SO2 and NO2 pollutant.
Collected sample : Leaf were collected from the study
area in the morning around 08.30 WIB. The taken
leaves were mature leaves and relative have the same
size. Temperature and light intensity were also measured when the leaves were taken.
Leaf characteristics : Leaf areas were measured using
leaf area meter and the leaf thicknesses were measured
by micrometer screw. Stomata were observed under
light microscope with 400x magnificent. Total chlorophyll content were measured using chlorophyll meter.
Sulfate and nitrate accumulation : Sulfate accumulation were measured by forming BaSO4 and then calculating SO4 concentration. Nitrate accumulation were
measured by calculating total N of leaf and then converted to NO3.
SO2 and NO2 concentration measurement : SO2 concentration were measured using the absorption method
with absorption solution of TCM. NO2 concentration
were measured using the absorption method with absorption solution of Saltzman-Griess.
Analysis of data : The data were analyzed using correlation analysis on Microsoft Excel.
III. RESULT AND DISCUSSION
Study area : The average of light intensity in study area
were
805,7 x 100 lux and the average temperature
on site were 27,2 28,5C. Average pH soils in the
location were 5.4 to 6.8.
Leaf characteristic : The data of leaf characteristic can
be seen on Table 1. All the leaves had a dark green color
on the upper side and lighter green on the bottom side.
Bungur has the largest leaf surface with 130,20 cm2 and
suren has the smallest leaf surface with 34,31 cm2. Dadap merah has the thickest leaf with 0,23 mm and suren
has the thinnest leaf with 0,07 mm. Leaf thickness is one
of internal factors that affect the transpiration process to
lowering plant temperature[16].
Stomatal type on each plant was anomocytic type, except dadap merah which has parasitic type. Stomata of
all studied species were only found on the bottom side
of leaves. Stomata were found more numerous on the
bottom side although can be found on both sides of
leaves[17]. This is an adaptation to reduce rate of transpiration by plant, because 90% of the transpiration occurs
trough stomata. Beside play a role in transpiration
process, stomata were also having a function for gases
exchange, like CO2, in the physiological process that
related to photosynthesis[18].
TABLE 1
LEAF CHARACTERISTIC
Characteristic
Leaf surface
(cm2)
Leaf thickness (mm)
Stomatal
type
Stomatal
density
2
(/mm )
Stomatal
size (m)
Total
chlorophyll
content (cci)
Suren
Bungur
Species
Ki acret
Dadap
merah
99,59
34,31
130,20
94,11
Kayu
afrika
36,32
73,09
Mahagoni
0,23
0,07
0,21
0,12
0,13
0,19
Parasi-tic
105,3
Anomocy-tic
288,95
Anomocy-tic
210,2
Anomocy-tic
122,2
Anomocy-tic
142,3
Anomocy-tic
289,9
21,5
19,2
26
21,8
29
12,1
41,5
13,5
32,8
27,7
39,4
28,6
Mahagoni has the highest value of stomatal density

with 289,9/mm2 and dadap merah has the lowest value
of stomatal density with 105,3/mm2. Kayu afrika has the
highest value of stomatal size with 29m and mahagoni
has the lowest value of stomatal size with 12,1m. Stomatal density of each plant can be different according to
the type of the plant[17]. The result shows that all the
plants have low stomatal density. Stomatal density categorized as low if the density is 200/mm2 or less[3]. This
low value may be related to plant adaptation toward
dryness. Some plants reduce the size and number of
stomata to adapt a dry environment[18].
Total chlorophyll content was measured using chlorophyll meter. Leaf plant which has the highest chlorophyll content is dadap merah with 41,5 cci and suren
leaf has the lowest with 13,5 cci. Dadap merah leaf has
darker green color, have a fairly broad leaf surface and
thicker than suren leaf which has thinner leaf, lighter
color and smaller leaf surface. Chlorophyll is one of
substantial pigment that used by plant to absorbs light
for photosynthesis process. Leaf color is closely related
to the chlorophyll content on leaf plant. Leaf plant with
normal green color relatively has higher chlorophyll
than the leaf with yellow or light green color[19].
Sulfate and nitrate accumulation : Sulfate and nitrate
accumulation can be seen in Table 2. The highest value
of sulfate accumulation was found in ki acret leaf with
15,0834 mg and the lowest value was found in kayu
afrika leaf with sulfate content around 0,0467 mg. Sulfur
(S) is secondary nutrients and generally required for
optimum growth around 0,1% - 0,5% of the dry weight
of plants. The structures of proteins in plants are largely
determined by the S group, such as methionine and
cysteine amino acids. S is also known as an essential
nutrient for the production of chlorophyll which is closely related to photosynthesis process[20]. Sulfur is absorbed by plants in the form of SO4 and stored in the
cytosol[21]. Varying value of sulfate accumulation in leaf
can be caused by genetic factors and environmental factors around plants[22].
The highest value of nitrate accumulation was found
in bungur leaf with 87,844 mg and the lowest value was
found in mahagoni with 2,982 mg. Around 5 6% nitrogen needs for the plants are come from NO2 absorption from the atmosphere. Nitrogen especially needed
for supporting the vegetative growth, such as leaf
growth rate[22]. Most of absorbed NO2 from the atmosphere remain in the leaves and only small portion were
th
distributed to stems and roots[7].NO2 gas which entering

the leaf through stomata will react with H2O and form
nitric acid (H2NO3)[22]. Nitric acid will ionized to H+ and
nitrate, so nitrate will be assimilated and turn into amino
acid which is needed by plant[8].
for sulfate uptake and transport in plants.
TABLE 2
SULFATE AND NITRATE ACCUMULATION
Species
Dadap merah
Suren
Bungur
Ki acret
Kayu afrika
Mahagoni
Leaf
weight
(g)
2,58
Sulfate
(%)
Sulfate
(mg)
Nitrate
(%)
0,0914
2,3581
2,91
75,08
0,58
3,16
1,67
0,5868
0,0993
0,9032
5,75
2,78
2,45
33,35
87,844
40,915
0,57
1,42
0,0082
0,2242
3,4034
3,1379
15,083
4
0,0467
3,1863
4,29
0,21
24,453
2,982
Nitrate
(mg)
Correlation between accumulation of sulfate and

nitrate with leaf characteristic : The result of this
study showed that sulfate accumulation on leaf is related
to some leaf characters such as leaf area, leaf thickness
and stomatal density. There was a positive correlation
between sulfate accumulations with leaf surface with a
correlation coefficient 0,2935 (Fig 1). Sulfate accumulation has negative correlation with leaf thickness with a
correlation coefficient -0,2734. Leaf thickness related to
tissue thickness and gas become relatively difficult to
enter the tissue leaf, so it causes the absorption of the
gas is relatively small[8].
The correlation between sulfate accumulation with
stomatal density and chlorophyll content showed a negative correlation with a correlation coefficient -0,2707
and -0,2789. Accumulation of air pollutant in leaf can
affect the chlorophyll content[11]. This showed that
greater accumulation sulfate can cause decreasing of leaf
chlorophyll content.
Fig. 1. Relation between sulfate accumulation with leaf area (r =

0,2935).
Ki acret leaf show greater sulfate uptake and accumulation can be caused by a relatively large leaf surface
with low leaf thickness and the value of stomatal density
which is not too high, compared to the others studied
species. Suren leaf which has the thinnest leaf among all
studied species cant accumulate sulfate as much as ki
acret because suren has relatively small leaf area that is
34,31 cm2 and also the higher value of stomatal density
which is around 288,95/mm2.
Sulfate absorption by leaf, beside affected by plant
characteristic such as leaf area, leaf thickness and stomatal density, can also influenced by genetic trait of
each plant. Each plant has a different transporter gene
Fig. 2. Relation between nitrate accumulation with leaf area (r =

0,7303) and between nitrate accumulation with leaf thickness (r =
0,4909).
The result showed that nitrate accumulation was related to some leaf characteristics such as leaf area, leaf
thickness, stomatal density and size. Figure 2 shows that
nitrate accumulation has a positive correlation with leaf
area and leaf thickness. The correlation coefficient between nitrate accumulation with leaf thickness is 0,4909.
This positive correlation is contrary to Patra[8] and Nugrahani et al.[7] which were reported that nitrate accumulation has negative correlation with leaf thickness. This
difference may be caused by the absorption of NO2
which not only depend on leaf thickness but also depend
on other factors such as leaf area and stomatal size. The
research of Patra[8] showed that there was a plant which
has relatively thick leaf were also accumulate high nitrate.
The total chlorophyll content has positive correlation
with nitrate accumulation with correlation coefficient
around 0,334 (Fig 3). Chlorophyll content will be influenced by the pollutant accumulation on leaf[11]. Chlorophyll synthesis is also influenced by several factors,
such as light, carbohydrate, water, temperature, genetic
factors and some elements such as nitrogen, magnesium,
iron, manganese, sulfur, iron and oxygen[24]. Nitrogen
(N) is closely related to chlorophyll synthesis[17], also for
synthesis protein and enzymes such as rubisco[25]. Hendriyani and Setiari[25] reported that N is main factors to
forming chlorophyll.
The result showed that nitrate accumulation has a
negative correlation with stomatal density. Patra[8] reported that the high value of stomatal density will cause
a high concentration of nitrate accumulation, but Nugrahani et al.[7] reported that there was no correlation between stomatal density and nitrate accumulation. This
difference suggests that stomatal density is not the only
one factor that affected nitrate accumulation on leaf.
showed that there is a positive correlation between nitrate accumulations with the size of stomata (r =
0,5331). This suggests that the greater the value of stomatal size tend to cause greater accumulation of nitrate
on leaf.
SO2 and NO2 concentration : SO2 gas concentration in
the air are one of factors that directly affect sulfate accumulation in leaf[21]. The results of ambient air quality
measurements showed that the concentration of SO2 gas
at the study site was not detected, so it can be categorized as an uncontaminated area. The very small concentration of sulfate accumulation on leaf may be due to the
good quality of ambient air for SO2 in the study area.
th
The concentration of SO2 gas was not detected in the

study area can be caused due to the distance of SO2
main pollutant sources, such as coal industry, not close
to the area. Concentration of SO2 is mainly influenced
by coal combustion emissions[21], coal industry, oil and
biomass burning[2]. Yanismai[26] reported that great reactions of SO2 can also cause the concentration of SO2 in
the atmosphere become not detected.
The humidity at the location when conducted air sampling was around 35 41,35%. Istantinova et al.[27] reported that the higher humidity will cause the decreasing
concentration of SO2 in the atmosphere. Temperature
measurement was 28.8 to 30C and categorized as relatively normal. Air temperature has the positive correlation with the concentration of gaseous pollutants in the
atmosphere. The increasing of air temperature will cause
to the increasing of gaseous pollutants too [28,27].
IV. CONCLUSION
Fig. 3. Relation between nitrate accumulation with the chlorophyll

content (r = 0,3340).
The results of ambient air quality measurements

showed that the concentrations of NO2 gas at the study
site was 49.64 g/Nm3/hour and it categorized as good
because the concentration of NO2 were lower than the
standard quality which is 400 g/Nm3/hour. High concentration of NO2 is generally found in areas with the
high transport activity[22]. Yanismai[26] reported that
there is tendency of increasing concentrations of NO2
along with the increasing number of vehicles.
The number of vehicles that pass through the campus
road and in front of Jatinangor Sumedang highway can
be seen in Table 3. The differences between NO2 gas
concentration in campus road and highway road were in
line with the research done by Yanismai[26] which reported that there is an increase in NO2 concentrations
with increasing number of vehicles.
TABLE 3
THE NUMBER OF VEHICLES
Location
Campus road
Highway road
Numbers of vehicles (/hour)

671
2828
NO2 gas concentration (g/Nm3/hour)

24,41
74,67
The wind speed, air temperature and humidity are

some of the meteorological factors that could affect the
concentration of gaseous pollutant in the atmosphere[27].
According to Subaid[28], wind speed can affect spreading
and mixing process of air pollutants. Higher wind speed
can cause the increasing of spreading process of air pollutants from the sources.
Istantinova et al.[27] reported that the wind speed has a
negative correlation to the concentration of gaseous pollutant in the atmosphere. According to Yanismai[26],
high wind speed can cause levels of gaseous pollutant
like NO2 are distributed to other locations, so the concentration of NO2 in the location that close to the source
is reduced. The measurement of wind speed at the study
site ranged from 0.53 to 3.02 m/sec and it categorized as
normal. The wind speed was relatively normal, but the
low concentration of SO2 and NO2 can cause gases with
a low concentration were dispersed to other locations by
wind.
1) The results are bungur (Lagerstoemia speciosa) has

the largest leaf surface with 130,20 cm2 and dadap
merah (Erythrina crista-galli) has the thickest leaf
with 0,23 mm. Mahagoni (Swietenia mahagoni)
has the highest value of stomatal density with
289,9/mm2 and kayu afrika (Maesopsis eminii) has
the largest size of stomata with 29m. Dadap merah (Erythrina crista-galli) has the highest value of
total chlorophyll content with 41,5 cci.
2) The highest value of sulfate accumulation was in ki
acret (Spathodea campanulata) with 15,0834 mg
and the highest value of nitrate accumulation was
in bungur (Lagerstoemia speciosa) with 87,848
mg.
3) The leaf width has the positive correlation with sulfate accumulation. The leaf width, leaf thickness,
stomatal size and chlorophyll content has the positive correlation with nitrate accumulation.
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th
Biological Control of Cabbage Head Caterpillar

Crocidolomia binotalis, Zeller by Using Fusants
Bacillus thuringiensis var. kurstaki and
Bacillus thuringiensis var. Israelensis Cultured
in Whole Coconut Fruits
Siti Sumarmi1*), Retno Peni Sancayaningsih2), Sebastian Margino3) and RC. Hidayat Soesilohadi4)
1),2),4)
Faculty of Biology UGM
3)
Faculty of Agriculture UGM
*)Correspondent author : siti-sumarmi@ugm.ac.id
AbstractPathogenicity of fusants Bacillus thuringiensis
isolates against cabbage head caterpillar larvae, Crocidolomia binotalis Z.was examined. Of the three Bt. fusants
(F28, F31 and F33) were tested against C. binotalis, the
second and third instar larvae. Pathogenicity of each isolate Bt. fusants varied for the second and third instar larvae. Among the 3 tested Bt. fusants. F28, F31 and F33 isolates showed that the Bt. F28 was the most pathogenic to
the second instar larvae. The mortality of the second instar
larvae were from 3,33 to 29.66 per cent recorded at 24 h
after the treatment. While the Bt. fusant F31 recorded 4,17
per cent and the Bt. fusant F33 registered 2,50 per cent
mortality of the second instar larvae. The Bt. fusant F 28
was the most pathogenic to the second instar larvae with
the value LC50 and LC90 were 2,74 x1011 and 6,10 x 1013
respectively. However the LC50 and LC90 valued of Bt.
fusant F31 and F33 were 1,03 x 10 17 and 2,14 x 10 23, and
2.33 x 1018 and 1,47x10 2 respectively. Therefore, the Bt.
fusant F33 was not pathogenic to the second instar larvae.
At 72 h of the treated time, the Bt. fusant F 28 was the
most pathogenic to the third instar larvae. The percentage
of mortality of the larvae caused by Bt. fusant F28 was
29,17 per cent, while the Bt. fusant F31 registered 13,33,
per cent mortality whereas Bt. fusant F33 showed 10 per
cent mortality. The value LC50 and LC90 of Bt. fusant F28
to the third instar larvae were 7,06 x108 and 4,98 x 109,
whereas Bt. fusant F31 , and F33 respectively were 3,12 x
10 29 and 6,4 x 10 46, whereas Bt. fusant F33 was 8,74 x
1024 and 1,20 x10 42. In conclusion, all Bt. fusant strains of
F28, F31, and F33 were pathogenic to C. Binotalis larvae.
The third instar larvae was more susceptible than the
second instar larva
KeywordsCrocidolomia binotalis, Bacillus thuringiensis, fusants, Biological control
I. INTRODUCTION
HE Cabagge head caterpillar Crocidolomia binotalis, Zeller (Lepidoptera: Pyralidae), is a serious
insect pest especially of cruciferous vegetables is
widely distributed in tropical and sub tropical region as
South and Southeast Asia, Australia, South Africa, Tanzania and the Pacific Islands [1]. In Indonesia, C. binotalis and Plutella xylostella together may caused the
yield loss up to 100 per cent, if suitable control for the

insects is not undertaken, especially in the dry season,
[2] The life history parameters and its biology has reported by [3]. Chemicals are the frontline defense for
control of insect pests. The sole reliance on pesticides is
however cause many problems. Resurgence of insect
pests, development of resistance of residue of toxic
chemicals in food stuffs are the major problems [4].
Biological control approach appears to be the best alternative to chemical control which are practical, effective,
economical and safe. Bacillus thuringiensis (Berliner), a
rod shaped gram positive entomopathogenic bacterium
is abundant in soil [5]. B. thuringiensis is the most successful commercial biocontrol agent against insect pests
more than 50th years [6]-[7]. B. huringiensis is an aerobic spore former is well known for its ability to produce
crystal proteins during sporulation [8]-[9]-[10]. The
crystal protein designated as delta endotoxin is toxic to
many insect larvae, such as lepidopterans, coleopterans
and dipteran larvae [10]-[11]. Since 2005, more than
200 crystal protein gen (cry gens) have been identified
and classified to 44 different families [12].
II. MATERIALS AND METHODS
The research was carried out in 2013. Cabbage head
caterpillar collected from Cabagge crops in Kopeng
Magelang, Central Java, Indonesia. The insect was mass
reared in the insectary of Laboratory of Entomology,
Faculty of Biology, University of Gadjah Mada, Indonesia.The larvae collected from the infested fields of cabbage were reared separately on cabbage leaves raised in
green house under insecticide free condition. Pupae thus
obtained were kept in a sterilized Petri dish and placed
in the wooden cage of 60x30 cm. for adult emergence.
When the moth started emerging, 25 30 days old small
cabbage heads were provided for oviposition. Fifth teen
per cent honey solution was provided as food for adults
in sterilized vial with cotton plug. The moth laid eggs
both on ventral and dorsal surface of cabbages leaves.
Leaves with eggs were transferred to the cage for mass
rearing of larvae. The second and the third instar
F1generation larvae were used for bioassay (Figure 3).
Of three Fusants B. thuringiensis (F28, F31,and F33
th
isolates collected from the previous research maintained

at Laboratory of Entomology, Faculty of Biology, University of Gadjah Mada, were used for bioassay to assess their pathogenicity against tested insect. [13]-[14].
To multiply the isolates they were streaked on plain
Brain Hearth Infusion Agar (BHIA) plates and incubated for 24 h which was later inoculated in whole coconut [15]-[16]-17]; and was kept for growth under
shaking condition at room temperature (28 0C) and incubated for 72 h. Then, the culture was examined under
phase-contras microscope (magnification of 1200x).
haversted in 10 ml of sterile water for taking colony
count. A concentration of B. thuringiensis (1.2X107cfu/ml) to assess its toxicity against the test insects
(Dip Leaf bioassay, described by [18] Tabashnik and
Crushing (1987) was adopted with modification. Cabbage leaves were cut square of 6 cm These leaves were
dipped in aqueous solution of the test isolates for 10
minutes. Excess fluid was drained off and the cutting
cabbage leaves were dried under shade for 10 min. before transferring to glass jars (5 cm height and 3 cm diameter) covered with absorbent gauze. Cutting Leaves
were placed slantingly so that larvae can move and feed
on either side.
Bioassays were done with three replications per
treatment and ten larvae of test insect were released on
each the container was covered with absorbent gauze.
The cabbage leaves dipped in distilled water alone
served as control. Mortality was observed at 24 h, 48 h,
and 72 h after treatment and data were subjected to
analysis of variance after suitable transformation (arcsine) and the means were separated by Duncans Multiple Range Test (DMRT)[19] and the LC50 and LC 96
were determine by Probit analysis [20]
Among three strains of fusants Bt. F28, F31 and F33
have variations result, the Bt. Fusant F28 was the most
pathogenic to the C. binotalis larvae than the others.
According to Knowles [21] explored that the variations
in efficacy against different lepidopterans may be due to
varying number of cry genes and the absence of specific
binding sites. The mortality of the second instar larvae
of C. binotaliscaused by the Bt. fusants F28 was from
3,33 to 29.66 per cent recorded at 24 h of the treatment. The Bt. fusants F31 recorded 4,17 per cent
while the Bt. fusants F33 registered 2,50 per cent mortality of the second instar larvae (Table 1). Fusant F 28
was the most pathogen to the larvae with the value LC50
and LC90 was 2,74 x1011 and 6,10 x 1013 respectively
than the fusant F31 and F33 with LC50 and LC90 valued
were 1,03 x 10 17 and 2,14 x 10 23, and 2.33 x 1018 and
1,47x10 2 respectively. But the F33 was not pathogenic to the second instar larvae.
TABEL 1.
PATHOGENICITY TEST OF Bt. FUSANTS TO THE SECOND
INSTAR LARVAE OF C. binotalis.
Mortality of the Second Instar
Fusant
Larvae of Insect at :
Total
Bt.strains
24 h
48 h
72 h
F28
A
16
12
1
29
B
C
D
Per cent
LC50
LC90
F31
A
B
C
D
Per cent
LC50
LC90
F33
A
B
C
D
Per cent
LC50
LC90
Control
1
2
1
16,67
3,05x109
1,75x1011
1
1
0
11,67
3,8x108
4,9x109
0
0
0
0,83
3
0
1
1
4,17
2,57x1017
2,61x1024
7
3
1
0
9,17
2,12x1010
8,37x1012
5
15
8
11
0
2
0
1
10,83
24,17
1,25x109
1,06x1011
1
1
1
0
2,50
2,33x1018
1,47x1025
0
0
1
0
0
0,83
1,03x1017
2,14x1023
0
0
1
0
2
0
1
0
0
0,00
3,33
1,03x1017
2,14x1023
0
0
2
3
1
29,17
3,47x108
3,89x109
Note : Concentration of the treatments

A : 1,75 x 109,; B: 1,75 x 108, C: 1,75 x 107: D: 1,75 x 106
The mortality of the second instar larvae at 48 h after

treatments can be seen at Table 1. The Fusant F28 recorded 11,67 per cent mortality to the second instar larvae while Bt. fusant F 31 was 9.17 per cent and Bt.
fusant F33 was 0,83 per cent mortality of the second
larvae. After 72 h of the treated time F28, F31, and F33
recorded 29.66 per cent, 24,83 per cent, and 3.33 per
cent mortality to the insect larvae respectively . Fusant F
28 was the most pathogenic to the second instar larvae
with the value LC50 and LC90 was 2,74 x1011 and 6,10 x
1013 respectively, whereas F31, and F33 were 12,57 x
10 17 and 2,61 x 10 24, however F33 was 2.33 x 1018
and 1,47x10 25.
The mortality of the third insect larvae can be seen at
Table 2. The Bt. fusants F28, F31, and F33 recorded
6,67 per cent while isolate F31 registered 3,33, per cent
mortality whereas F33 showed 2,50 per cent mortality.
The values LC50 and LC90 of F28 . were 2,74 x1011 and
6,10 x 1013 the value LC50 and LC90 F 31 were 1,03 x
10 17 and 2,14 x 10 23, However, F33 was not pathogenic to the third instar larvae. At 48 h of the treatments,
fusant F28 showed 20, per cent mortality of the third
instar larvae whereas F31 was 3,33 per cent mortality ,
while F33 showed 5,00 per cent mortality.
The values LC50 and LC90 of F28 . were 8,89 x108 and
4,51 x 109 whereas F 33 were 3,12 x 10 29 and 6,4 x 10
48,
respectively. However, F31 was not pathogenic to the
third instar larvae.
At 72 of the treatments, of Bt. fusant F 28 was the
most pathogenic to the third instar showed 29,17 per
cent mortality, Isolate F31 registered 13,33, per cent
mortality whereas F33 showed 10 per cent mortality. The
value LC50 and LC90 of F28 was 7,06 x108 and 4,98 x
109, whereas F31 , and F33 were 3,12 x 10 29 and 6,4 x
10 46, whereas F33 was 8,74 x 1024 and 1,20 x10 42.
The F33 was not pathogenic to the third instar larvae
th

[2]
TABEL 2.
PATHOGENICITY TEST OF Bt. FUSANTS TO THE THIRD
INSTAR LARVAE OF C. binotalis.
Fusant Bt.strains
Mortality of the third instar

larvae of insect at :
24 h.
48 h.
72 h.
Total
F28
A
B
C
D
Per cent
LC50
LC90
4
3
1
0
6,67
2,74x1011
6,1x1013
19
2
1
2
20,00
8,98x108
4,51x109
2
25
0
5
1
3
0
2
2,50
29,17
7,06x108
4,98x109
A
B
C
D
Per cent
LC50
LC90
1
2
1
0
3,33
1,03x1017
2,14x1023
0
1
3
1
3,33
0
0
3
4
3
6
1
5
0
1
5,83
13,33
3,42x1014
1,71x1022
A
B
C
D
Per cent
LC50
LC90
Control
0
1
1
1
2,50
0
0
1
Per cent
4
0
0
2
5,00
3,12x1029
6,4x1048
1
0
4
2
3
1
2
0
3
2,50
10,00
8,74x1024
1,20x1042
0
2
6,67
F31
F33
Note : : Concentration of treatments

A : 1,75 x 109,; B: 1,75 x 108, C: 1,75 x 107: D: 1,75 x 106
IV. CONCLUSION
All fusants Bt strains ; F28, F31, and F33 were
pathogenic to C. Binotalis. The third instar larvae was
more susceptible than the second. Fusant Bt F28 and F31
will be assessed their pathogenicity to the instar larvae at
small scale field.
ACKNOWLEDGEMENT
The investigators wish to thank to DP2M DIKTI RI for
the financial support of the research. 2. LPPM UGM
Indonesia for the research coordination and the Dean of
Faculty of Biology UGM, Indonesia for give me opportunity to do this research and also Special thanks to I
Nyoman Sumerta, SPd. for his sincere assistance in carrying out the experiments, and Suparmin for administration tasks.
REFERENCES
[1] Kalshoven, L.G.E. 1981. The Pests of Crops in Indonesia. Pt
Ichtiar Baru-Van Hoeve, Jakarta. P. 341-344.
Sudarwohadi S. 1975. Correlation between planting time of

cabbage and population dynamics of Plutella maculipennis
Curt. and Crocidolomia binotalis Zell. Bull. Penel. Hort. ,3, 314 (in Indonesian with English Summary)
[4] Rao, V. P., M. A. Ghani, T. Sankaran & K. C. Mathur. 1971. A
review of the biological control of insects and other pests in the
south-east Asia and the Pacific region. Commonwealth Inst. Biol. Contr. Tech. Comm. No. 6: 149 p.
[5] Bora, R.S., Murthy, M.G., Shenbagarathai, R. and Sekar, V.,
1993, Introduction of a lepidopteran specific crystal protein
gene of Bacillus thuringiensis sub sp. kurstaki by conjugal
transfer into a Bacillus megaterium strain that persist in the cotton phyllosphere. App. Envoy. Mic., 60: 214-22.
[6] Federici, B.A. (1999). Bacillus thuringiensis in Biological Control. . In: Handbook of Biological Control. T. Fisher
(Ed.)Academic Press (Ed.) 575-593, ISBN 10: 0-12-257305-6
[7]
Navon, A. (2000). Bacillus thuringiensis insecticides in crop
protection-reality and prospects. Crop Protection 19(8-10): 669
676.
[8] Krieg, A., 1961, Bacillus thuringiensis, Berliner. Mitt. Boil. Bundesantatt land- Forstwirtsch, Berlin- Dahlem, 103: 3-79.
[9] Heimpel, A.M., 1963, The status of Bacillus thuringiensis. Bull.
Am.Chem. Soc., 41: 64-74.
[10] Heimpel, A.M. and Angus, T.A., 1959, Diseases caused by
certain spore forming bacteria, In: Insect Pathology: An advanced Treatise 2, Academic Press New York, pp. 68.
[11] Feitelson, 1. S., Payne, 1. & Kim, L. (1992). Bacillus tburingiensis:insects and beyond. Biol Technology 10, 271-275.
[12] Crickmore N, Zeigler DR, Feitelson J, Schnepf E, van Rie J.
Lereclus D, BaumJ, Dean DH (1998) Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins.
Microbiol Mol Biol Rev 62:807813.
[13] Sumarmi, S., S. Margino , S. Yuwono 2006. Efikasi Fusan
Bacillus thuringiensis kurstaki dan Bt. Israelensis terhadap
larva Aedes aegypti dan Plutella xyllostela. ( laporan penelitian
Hibah bersaing 2006)
[14] Sumarmi, S., S. Margino, D.T. Buwono, dan RC. Hidayat.
2010. Pengendalian Nyamuk Vektor Malaria Anopheles aconitus
dan Ulat Jagung Helicoverpha armigera (Hubner) Hardwick Secara
Hayati dengan Fusan Bacillus thuringiensis var kurstaki dan Bt.
var israelensis) ( laporan penelitian Stranas 2010).
[15] Prabakaran, G.; Hoti, S. L.; Manonmani, A. M.; Balaraman, K.
(2008), Coconut water as a cheap source for the production of
endotoxin of Bacillus thuringiensis var israelensis - a mosquito
control agent. Acta Tropica, 105, 3538
[16] Poopathi and Kumar, 2003 S. Poopathi and K.A. Kumar, Novel
fermentation media for production of Bacillus thuringiensis
subsp. israelensis, J. Econ. Entomol. 96 (2003), pp. 10391044.
[17] Poopathi et al., 2002. S. Poopathi, K. Anup Kumar, L. Kabilan
and S. Vaithilingam, Development of low cost media for the culture of mosquito larvicides, Bacillus sphaericus and Bacillus
thuringiensis serovar. israelensis, World J. Microbiol. Biotechnol. 18 (2002), pp. 209216.
[18] Tabashnik, B.E. and Cushing, N.L., 1987, Leaf residue Vs topical bioassay for assessing insecticide resistance in the Diamond
back Moth, Plutella xylostella L. FAO Pl. Prot. Bull., 35: 11-14.
[19] Duncan, D.B., 1955, Multiple range and multiple F tests. Biometrics,11: 1-42.
[20] Finney, 1971 D.J. Finney, Probit Analysis (3rd ed.), S. Chand
and Co. Ltd., New Delhi (1971) pp. 5080.
[21] Knowles, B.H., 1994, Mode of action of Bacillus thuringiensis
upon feeding on insects. Adv. Insect Physiol., 24: 275-308.
th
The Effect of Trehalose Levels on Post-Thaw

Sperm Membrane Integrity of Boer Goat Semen
Nurul Isnaini
Faculty of Animal Husbandry, Brawijaya University, Malang, Indonesia
*)
Corresponding author: nurulisna@ub.ac.id
Abstract This study was conducted to evaluate the effect of trehalose levels on post-thaw sperm membrane
integrity at different level of trehalose in tris based diluter
of Boer Goat semen. Fresh semen was collected from six
aged male Boer goats. Immediately after collection using
artificial vagina, the semen was evaluated for quality and
diluted with tris aminomethane-base extender in 10 folds (1
semen: 9 extender). The effect of different levels of trehalose (1.5%; 2.5% and 3.5%) in the diluter on the sperm
membrane integrity was evaluated in this study. The diluted semen was freezed with standard method. The result
showed that fresh semen collected from Boer bucks in this
study indicated a normal quality and therefore, could be
used for further treatment. According to the varian analysis it was shown that 2.5% trehalose resulted higher sperm
quality than those the other levels on post-thaw of Boer
Goat semen. It was concluded that the addition 2.5% trehalose in tris-based medium resulting optimal sperm
membrane integrity of Boer goat semen post thawing. It
was suggested, that for resulting optimal sperm membrane
integrity post thawing of Boer goat semen in tris-based
medium should be supplemented with 2.5% trehalose.
Keywords Boer goat, trehalose, cryopreservation,
membrane integrity
I. INTRODUCTION
uring dilution, cooling and freezing, the sperm
quality reduce corresponding to appropriate
processing technique and cryoprotectant used. The
addition of cryoprotectant in the medium or semen extender can retard the reduce of semen quality in those
process. Disaccharides have a stabilizing effect on biological membrane. Trehalose is found in animals capable of enduring cold temperatures, whereas sucrose is
found in plants [1].
Cryopreservation are known to damage sperm
membranes [2]. This damage includes swelling and disruption of plasma and outer acrosome membranes [3],
changes in membrane fluidity [4], disregulation of intracellular Ca2+ influx [5] and changes in enzyme activity
[6].
Membrane integrity is not only important for sperm
metabolism, but also a correct change in the properties
of the membrane is required for successful union of the
male and female gametes, i.e. for sperm capacitation, the
acrosome reaction and the binding of the spermatozoa to
the egg surface. The integrity and functional activity of
the sperm membrane is of fundamental importance in
the fertilization process and assessment of membrane

function may be useful indicator of the fertilizing ability
of spermatozoa.
The objective of the present study was conducted to evaluate the effect of trehalose levels on postthaw sperm membrane integrity at different level of trehalose in tris based diluter of Boer Goat semen.
II. MATERIAL AND METHOD
a. Animal and Sperm Preparation
Ejaculated were obtained from 6 male Boer goats (originated from Australia Breeding Herd) aged of 2.0 2.5
years with about 100 kgs in weight, using artificial vagina. The bucks were maintained at Field Laboratory of
the Faculty of Animal Husbandry, University of Brawijaya Malang. After collection, the semen was evaluated
macroscopic and microscopically. Sperm motility was
evaluated by placing a drop of well mixed semen on a
prewarmed glass slide under a coverslip and examining
it at x100 and x400 magnification by phase contrast
microscopy. Motility was assessed subjectively on the
basis of spermatozoa that were moving either progressively or non progressively or those that were nonmotile.
Viability and abnormality were evaluated by eosinenegrosine staining. One hundred sperm cells were
scored per slide. Sperm concentration was measured
with a Thoma hemocytometer. Only semen with individual motility of sperm of more than 70% and mass
motility of 2+ and 3+ was used for research material.
Semen collection was regularly conducted twice a week
per individu of animal.
The selected semen was diluted with tris-base diluent containing 1.5%; 2.5% or 3.5% trehalose and equilibrated at 5C for 2 h. Before loading into 0.25 ml straw,
semen was evaluated for the quality and then straw containing diluted semen was horizontally placed on liquid
nitrogen vapour (-140C) for 9 min for pre-freezing.
Immediately thereafter, the straw was plunged into liquid nitrogen for at least 24 h. Thawing was conducted
by transfer the frozen straw into the warm water (37C)
for 30 sec. For each treatment (1.5%; 2.5% and 3.5%
trehalose) was taken for evaluation of membrane integrity by hypoosmotic swelling test (HOS test).
2. Hypoosmotic Swelling (HOS) test
The HOS test solution contained 0.49 g Na-sitrate x
2H2 O and 0.9 g fructose in 100 ml aquadest. The os-
th
motic swelling technique consisted of mixing 0.1 ml

semen with 1.0 ml hypo-osmotic solution and allowing
the mixture to incubate at 37 C for at least 30 min. The
mixture was allowed to stand for at least 1 min before
observations were made by phase-contrast microscopy
at x400 ; 100 spermatozoa were observed. The percentage spermatozoa that showed typical tail abnormalities
indicative of swelling was calculated.
III. RESULT
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selected.
a. Characteristics of fresh semen
Table 1 shows that the characteristics of Boer goat fresh
semen used in this study was normal.
TABLE 1.
CHARACTERISTICS OF FRESH SEMEN
Parameter
Mean + SD
Color
Creamy
Consistency
Less opaque
pH
7.00 + 0.0
Volume (ml)
0.64 + 0.27
Concentration (106 / ml)
3238.00 + 187.78
Mass motility
2+ - 3+
Individual motility (%)
74.50 4.38
Life sperm (%)
86.88 + 2.31
Abnormal sperm (%)
8.63 + 1.97
Membrane integrity (%)
72.59 + 6.21
b. Sperm membran integrity post thawing

Sperm membrane integrity post thawing diluted with
tris-base diluent containing different levels of trehalose
is shown in Table 2.
TABLE 2.
MEAN (+ SD) OF MEMBRANE INTEGRITY
SPERM
FOLLOWING TREATMENT WITH DIFFERENT TREHALOSE
LEVEL POST THAWING
Trehalose levels
Membrane integrity
1.5%
38.34 + 5.69b
2.5%
42.12 + 2.66 b
3.5%
29.17 + 6.93a
a.b.c within column significant difference (P<0.05)
Table 2. shows that the membrane integrity of sperm

was significantly different (P<0.05) post thawing between trehalose treatment groups. In general, it was
shown that 2.5% trehalose in tris-based diluent showed
an optimal concentration for maintaining the sperm
membrane integrity post thawing compared to the level
of 1.5% and 3.5% trehalose. The level of 1.5% trehalose
influenced suboptimal effect on the sperm membrane
integrity. Migh be due to the low concentration for
cryoprotective function in the diluter during freezing
process. Vice verse was for the level of 3.5% trehalose.
IV. DISCUSSION
The integrity and functional activity of the sperm
membrane is of fundamental importance in the fertilization process, and assessment of membrane function may
be a useful indicator of the fertilizing ability of spermatozoa. A property of the cell membrane is its ability to
permit the transport of molecules selectively. When exposed to hypo-osmotic conditions, water will enter the
spermatozoa in an attempt to reach osmotic equilibrium.
This inflow of water will increase sperm volume and the
plasma membrane will bulge (balloon), giving minimum
surface to volume ratio. The sperm tail appears to be
particularty susceptible to such hypo-osmotic conditions. These induced alteration in sperm morphology are
visible with the phase contrast microscope. The ability
of the sperm tail to swell in the presence of hypoosmotic solution is a sign that transport of water across
the membrane occurs normally, i.e. is a sign of membrane integrity and normal functional activity [7].
The role of trehalose in the protection of sperm during freezing acted by maintaining the membrane stability
of sperm (lipid bilayer) by formation of hydrogen
bounds at O2, O3 and O4 of trehalose structure with
phosphate-and carbonyl groups of lipid [8]. The former
hydrogen bounds either from phosphate- or carbonyl
groups of lipid the sperm membrane stability could
maintained and the sperm damage originated from diluent and temperature shock could be minimized. In semen freezing process, trehalose was reported used in
combining with gliserol [9] showed that 7.5% trehalose
combined with 6% glycerol resulted higher sperm quality after freezing compared to the used of Tris(hydroxymethyl-aminomethane)-citric-acid-glucose
(TGC) [10]. There is evidence that trehalose increased
the stability of sperm membrane to the physical and
morphological changes during semen dilution and freezing.
V. CONCLUSION
Based on the study, it was concluded that the

addition 2.5% trehalose in tris-based medium resulting optimal sperm membrane integrity of Boer
goat semen post thawing. It was suggested, that for
resulting optimal sperm membrane integrity post
thawing of Boer goat semen in tris-based medium
should be supplemented with 2.5% trehalose.
ACKNOWLEDGMENTS
The author thank the Dean of Animal Husbandry Faculty Brawijaya University Malang for boer goat facility
and the laboratory field staff Sumber Sekar for assistance of semen collection and evaluation.
REFERENCES
[1] K.S. Amadeu, R. Faller, and J.J. de Pablo, Molecular Simulation
Study of Phospholipid Bilayers and Insights with Disaccharides.
J Biophys, vol. 85 (5),
2003, pp.2830-2844.
[2] R.H. Hammerstedt, J.K. Graham, and J.P. Nolan, Cryopreservation of mammalian sperm: what we ask them to survive. J Androl, vol. 11, 1990 pp.73-88.
th
[3] R.C. Jones and D.L. Stewart, The effects of cooling to 5C and
freezing and thawing on the ultrastructure of bull spermatozoa. J
Reprod Fertil, vol 56, 1979 pp. 233-238.\
[4] W.V. Holt and R.D. North. Thermotropic phase transitions in the
plasma membrane of ram spermatozoa. J Reprod Fertil, vol 78,
1986 pp. 447-457.
[5] L. Robertson, J.L. Bailey, and M.M. Buhr. 1990. Effects of cold
shock and phospholipase A2 on intact boar spermatozoa and
sperm head plasma membranes. Mol Reprod Dev, vol 26, 1990
pp. 143-149.
[6] P.F. Watson, The effects of cold shock on sperm cell membranes
In: Morris GJ, Clarke A (eds), The effects of low temperatures
on biological membranes. London: Academic Press, 1981 pp.
189-218.
[7] R.S. Jeyendran, H.H. Van der Ven, M. Perez-Pelaez , B.G. Crabo,
and L.J.D. Zaneveld, Development of an assay to assess the
functional integrity of the human sperm membrane ang its relationship to other semen characteristics. J reprod Fert, vol. 70,
1989 pp. 219-228.
[8] A.K. Sum, R. Faller, and J. de Pablo, Molecular simulation
study of phospholipids bilayer and insights of the interactions
with disaccharides, Biophysical Journal, vol. 85, 2003 pp. 28302844.
[9] B.T. Storey, E.E. Noiles, and K.A. Thompson, 1998. Comparison of Glycerol, Other Polyols, Trehalose and Raffinose to Provide a Defined Cryoprotectant Medium for Mouse Sperm Cryopreservation. J.Criobiology, vol. 37, 1998 pp. 46-58.
[10] E.M.E. Aboagla and T. Terada, Trehalose-enhanced fluidity
of the goat sperm membrane and its protection during freezing.
J. Biol. Reprod, 2003.
th
Antimutagenic Effect from Brassicaceae Extracts on Swiss-Webster Mice Induced by Lead

Acetate
Supartini Syarif 1*), Melati P. Puteri 1), and Nining Ratningsih1)
1)
Jurusan Biologi, FMIPA UNPAD
Jl. Raya Bandung-Sumedang KM. 21 Jatinangor 45363
*)
Corresponding author: supartini_syarif@yahoo.co.id
AbstractThe research was undertaken to investigate

the antimutagenic effect of Brassicaceae extracts (white
headed cabbage, pak choi, and Chinese cabbage) on SwissWebster mice induced by lead acetate. The study has been
conducting using Completely Randomized Design method
consist of five treatments with five repetitions. Mice were
pretreated with white headed cabbage, pak choi, and Chinese cabbage extracts by cumulative dose 156 mg/kg bw
for 7 (1, 3, 5, 7, 9, 11 and 13) consecutive days prior to exposure of lead acetate by cumulative dose 150 mg/kg bw
(on day 5, 9 and 13). All treatment was given orally. Parameters that observed were frequency and type of chromosomal aberration. The data was analyzed statistically using
ANOVA and continued with Duncans Multiple Range
Test ( = 0.5). The highest number of chromosomal aberration (86.00%) was showed in lead acetate treatment. The
frequency of chromosomal aberration on white headed
cabbage, pak choi, and Chinese cabbage extracts treatments respectively were 41.80%, 59.80%, and 51.80%. The
result indicated that Brassicaceae extracts reduced frequency chromosomal aberration on mice causes by lead
acetate. Several types of chromosomal aberration detected
were chromosome fragment, acentric chromosome, double
point, numeric, stickiness and ring chromosome.
Keywords Antimutagenic, white headed cabbage
(Brassica oleracea var. capitata), pak choi (Brassica rapa
chinensis), Chinese cabbage (Brassica rapa pekinensis),
Lead Acetate
I. INTRODUCTION
RESENTLY, parallel to the rapid growth in industrialization environmental pollution is also increasing. Heavy metals like Pb, Cd, Cu, Fe, and Hg, contribute significantly to the growing pollution problem.
Among the heavy metals, the use of lead in industrial
caused widespread environmental contamination [1].
Internal and external factors, including environmental
pollutants induce various kind of genetic damage [2].
Lead acetate induced genotoxicity, mutagenicity and
carsinogenic Lead is genotoxic itself or enhances the
effect of other DNA-damaging agents that can trigger
cancer formation [3,4,5,6,7]. Prevention of the cancer
and other mutation related disease can be carry on both
by avoiding exposure to carcinogen and by favoring the
intake of protective factors which fortify physiological
defense mechanisms [8].
In recent years, there is increasing awareness that
certain naturally occurring substance in plants provide

protection against environmental mutagens or carcinogen. The Brassicaceae (Cruciferae) family has chemo
preventive potential. Antimutagenic and anticarcinogenic effect of these vegetables, particularly due to their
content in glucosinolate, is hydrolyzed by specific thioglucosidases called myrosinases to produces isothiocyanates, nitriles, and thiocyanates with different biological
activity [9,10,11,12,13].This study was conducted to
examine the antimutagenic potential of Brassicaceae
vegetables white headed cabbage (Brassica oleracea
var. capitata), pak choi (Brassica rapa chinensis), and
Chinese cabbage (Brassica rapa pekinensis) on mouse
chromosomal aberration induced by lead acetat.
Animal experiments were 6-8 week old Swiss Webster male mice weighing 20-25 g. Chemicals and substances, lead acetate (Merck), CMC 5% (Merck) , 70%
ethanol, colchisine (Merck), Carnoys fixative solution,
phosphate buffer saline pH 7, KCL 0.56%, Giemsa solution (Merck), extracts from Brassicaceae vegetables.
Vegetables extraction based on Harborne method [14].
a. Treatment Procedure
Twenty five 8-10 weeks old mice weighing 20-25 g
were houses allowed without food for 3-5 h before
treatment. There were five experimental groups and
each group consisted of five male mice. Group I positive
control was administered with 150 mg/kg bw lead acetate (P1) [5] . group II negative control was administered with CMC 0.5% (P2), Groups III, IV, and V were
pretreated with 156 mg/kg bw white headed cabbage
extract (P3), 156 mg/kg bw of pak choi extract (P4), and
156 mg/kg bw Chinese cabbage extract (P5) respectively for 7 (1, 3, 5, 7, 9, 11, and 13) consecutive days
(22.22 mg/kg bw/day) prior to treatment with lead acetate dose of 150 mg/kg bw on day 5, 9 and 13 (50 mg/kb
bw/day). All treatment was given orally. Chromosomal
aberration analysis was done following Bish and Devi
method with modification [15]. A hundred well spread
intact metaphase were score from each animal under
100X oil immersion.
b. Statistical analysis
Data was presented as mean SD. The significance
of difference between the data in control and in experi-
th
mental groups was analyzed with ANOVA and continued with Duncan Multiple Range test.
Pre treatment of different vegetables extracts
was given orally prior on to lead treatment in group III,
IV and V decreased frequency of chromosomal aberrations (table 1.) All types of chromosomal aberrations
induced by lead acetate including, numeric abberation,
fragment, acentric, ring, stickiness, gap, tri-radial, chromosome breaks, double point. The result showed that
lead acetate produces mutagenic effect induced chromosomal aberration. Lead compound do not damage DNA
directly, lead ion are known to participate in Fenton
reaction produces ROS which can induces oxidative
stress and free radicals. In addition, lead ions are reportedly known to inhibit the DNA polymerase , one of the
prime enzymes involved in DNA repair. There are indications for rather indirect mechanisms of genotoxicity,
which may be due to an interaction of lead and DNA
repair processes [7,16].
TABLE I
ANTIMUTAGENIC EFFECT OF BRASSICACEAE EXTRACTS
ON LEAD ACETATE INDUCED CHROMOSOMAL
ABERRATIONS IN SWISS WEBSTER MICE.
Experimental groups
Lead acetate
CMC
White headed cabbage

Pak-choi
[4]
[5]
[6]
[7]
[8]
170
34 + 3.51b
209
41.8 + 2.27b
[12]
299
59.8 + 6.12bc
[13]
259
51.8 + 3.57c
[9]
[10]
[11]
[14]
Result of one way

ANOVA
[3]
86 + 2.47a
[2]
430
REFERENCES
[1]
Average Percentage of
chromosomal
aberrations (%
+ S.D)
Chinese cabbage
The authors thanks to Rector Padjadjaran University

was supported this work by DIPA BLU Foundation
Metaphase with
chromosomal
aberrations/100
cells*
ACKNOWLEDGMENT
0.000
27.28
* One hundred cells were analyzed per animal, for a total of 500 cells
per treatment. Values, within columns, with no common superscripts are statistically different (P<0.05)
In this study revealed the antimutagenic potential of

brassicaceae extracs against chromosomal aberration
induced by lead acetate. The mechanism for protection
of brassicaceae extracts involved chemical property of
brassicaceae plants is high content of glucosinolate and
their isothiocyanate hydrolysis product as chemoprotector. Isothiocyanate inhibit phase I enzyme (cytochrome
P 450). It converts procarcinogens to highly reactive
electrophilic that can damage susceptible DNA base.
However, isothiocyanate enhanced activities family of
phase II enzyme (glutathione, glucuronic acid), these
detoxication enzyme were responsible for protective
action to xenobiotics in this case lead acetate [8,9,17] .
Isothiocyanate has a role in protection enzyme.
Overall, results from this study indicate that brassicaceae extracts inhibit mutagenic effect on Swiss Webster mice induced by lead acetate (chemopreventive).
[15]
[16]
[17]
Kumar, G; R. Tripathi. 2008. Lead Induced Cytotoxicity and

Mutagenicity in Grass Pea Turk J Biol (32): 73-78
Alekperov U. and R Guileva 1996. The Inhibition of the Genotoxic Effects of environmental Pollutans and Aging processes
by Plant mutagen. Tr J.of Biology (23):135-142.
Alghazal,M.A; N. Kovalkoviscova; J. Legath; M. Falis; J. Pistl;
R.Sobo; K.Benova;L Sabova and P.Vaczi 2008. Induction of
micronuclei in rat bone marrow after chronic exposure to lead
acetate trihydrate. Toxicology and Industrial Health 24 (9):
587-593.
Uysal, H,. 1997. Induction of Chromosomal Aberration in
Polytene Chromosomal of Drosophila melanogaster by Lead
Acetate. Cytologia.62:213-217.
Aly, F.W 2001. Potential Mutagenic Effect of Lead Acetate in
mouse Bone Marrow and culture
Mouse Spleen Cells.
http://www.jst.go.jp/article/char/en
Poma, A. Pittaluqa E; Tucci.A. 2003. Lead Aceate Genotoxiccity on Human Melanoma Cells. Melanoma Res. 13(6):
563-566
Kasuba,V, R. Rosgaj, A Fucic, V.M Varnai and M Piasek.
2004. Lead Acetate genotoxicity in suckling rats. Biologia,
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Talalay,P; J.W. Fahey 2001. Phytochemicals from Cruciferous
Plants Protect agains Cancer by Modulating Carcinogen Metabolism. American Society for Nutritional Sciences.
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th
Yield and Yield Components of Acid-Tolerant

Soybean Promising Lines on Ultisols
Heru Kuswantoro 1*), and Purwantoro 1)
Indonesian Legume and Tuber Crops Research Institute
Indonesian Agency for Agricultural Research and Development
1)
*)
Corresponding author: heru@litbang.deptan.go.id
Abstract Acid soil properties vary even in the relatively close location. Hence, yield and yield components of a
genotype is not similar from location to another location
depends on the location condition. The objective of the
research was to study the yield and yield components of
acid-tolerant soybean promising lines in Ultisols of Banjarnegara at rainy season 2009. The tested materials consisted
of
10
promising
lines
(SC2P2.99.5.4.5-1-6-1,
SC2P2.151.3.5.1-10, SC5P2P3.5.4.1-5, SC5P2P3.23.4.1-328-3,
SC5P2P3.23.4.1-5,
SC5P2P3.48.31.1-10,
SJ5/Msr.99.5.4.5-1-6-1, Msr/SJ-5.21.3.7-3-27-1, Msr/SJ5.23.4.1-3-28-3 and Msr/SJ-5.23.4.1-5) and three check
varieties (Tanggamus, Wilis and Grobogan). Experimental
design was randomized completely block design with three
replications. Plot size was 2,0 m x 4,5 m, plant spacing 40
cm x 15 cm, two plants per hill. The results showed that in
strong acid soil Ultisols of Banjarnegara, Tanggamus had
the highest grain yield than other promising lines and both
check varieties Wilis and Grobogan. Msr/SJ-5.23.4.1-3-283 was the most potential promising line having grain yield
higher than Wilis and seed size larger than Tanggamus.
Wilis was adaptive in slightly acid soil, but Grobogan was
not suitable for planting in acid soil. Yield per plant was
supported by plant height, filled pods and reproductive
nodes. Early maturity with larger seed size varieties, such
as Grobogan, was risky due to the relatively high unfilled
pods.
Keywords acid soil, soybean, Ultisols, yield, yield components.
I. INTRODUCTION
CID Acid soil covers 30-40% of the worlds total
land area [1] and approximately 69% of dryland in
Indonesia [2]. Acid soil limits crop growth potential [3] due to the many problems in supplying mineral
nutrients to the crops such as deficiencies of calcium,
magnesium, molybdenum, and phosphorus, as well as
toxicities of aluminum and manganese [4],[5]. However,
Al toxicity is the main problem in mineral acid soils [6].
Crop growth decreases as Al saturation in acid soils
increases [7]. High aluminum concentration also causes
the decrease in chlorophyll content, photosynthesis rate,
utilization efficiency of photosynthetically active radiation and water utilization efficiency, and increasing
transpiration rates [8].
Gene expression is subject to modification by the en-
vironment, consequently genotypic expression of the

phenotype is environmentally dependent [9]. Soil fertility, such as soil acidity, is one of the environmental factors which modify the gene expression. Some genotypes
evaluations to low pH at seedling level have been reported by [10] and at to reproductive level by [11]. The
high soybean yield might be the result of the high yield
capacity and favorable weather conditions [12]. Hence,
ecophysiological parameters should be noticed in selecting good varieties [13] as well as soil properties [14].
Yield is the main criterion in an evaluation of plants
adaptability, because adaptability is the ability of the
plant to maintain the production on the diverse environmental conditions [15]. However, grain yield is not an
independent character [16], but a character which supported by the yield components. As a complex character,
grain yield consists of components of quantitative nature, where the expression is determined by genetic and
environmental factors as well as their interactions [17].
Contribution of yield components to grain yield may a
direct effect of a yield component to grain yield or indirect effect through other yield components. Direct and
indirect effects also varies depending on the environmental conditions [18], [19].
II. MATERIALS AND METHOD
Experiment was conducted in rainy season 2009 in
Village of Pucung Beduk, Sub-district of Kali Sawah,
District of Banjarnegara, Central Java, Indonesia. The
design was a randomized completely block design with
three replications. The plants materials were 10 acidtolerant promising lines (i.e. SC2P2.99.5.4.5-1-6-1,
SC2P2.151.3.5.1-10,
SC5P2P3.5.4.1-5,
SC5P2P3.23.4.1-3-28-3,SC5P2P3.23.4.1-5,
SC5P2P3.48.31.1 -10, SJ-5/Msr.99.5.4.5-1-6-1, Msr/SJ5.21.3.7-3-27-1, Msr/SJ-5.23.4.1-3-28-3 and Msr/SJ5.23.4.1-5), and three check varieties (Tanggamus,
Wilis and Grobogan). Each genotype was grown on 2.0
m x 4.5 m, plant spacing of 40 cm x 15 cm, two plants
per hill. The crops were fertilized with 75 kg Urea, 100
kg SP36 and 100 kg KCl per ha on the soil before planting. Observations were conducted on plant height, numbers of branches, numbers of filled and unfilled pods,
and 100 seeds weight and grain yield. Soil properties of
the experiment location are presented in Table 1.
th

The grain yield of acid-tolerant soybean lines and the
check varieties was presented in Table 2. Tanggamus as
an acid-tolerant check variety yielded the highest grain
(1.48 t/ha) followed by Msr/SJ-5.23.4.1-3-28-3 (1.42
t/ha), while Wilis as wide adaptation variety yielded
1.34 t/ha higher than Grobogan (0.69 t/ha) as early maturity check variety. Compare to those varieties description [20], grain yield of Wilis and Grobogan was lower,
while Tanggamus was higher. No acid-tolerant soybean
promising lines showed higher grain yield than Tanggamus. However, all acid-tolerant soybean promising lines
showed higher grain yield than Grobogan, except
Msr/SJ-5.21.3.7-3-27-1 that showed similar grain yield
to Grobogan. Grobogan is a specific location variety,
and usually cultivated on favorable environments. Thus
Grobogan is not suitable for Ultisols of Banjarnegara. In
this research, the water was supplied from rainfall. It
lead water deficit on some growth and development
stages. Early maturity variety is also susceptible to water
deficit when the water deficit occur in vegetative and
flowering stage [21].
Wilis showed the highest plant height (63.3 cm) followed by SC5P2P3.48.31.1-10 (62.0 cm). Other lines
with plant height higher than 60 cm were Msr/SJ5.23.4.1-3-28-3 (60.7 cm) and Msr/SJ-5.23.4.1-5 (61.2
cm) and the check variety of Tanggamus (61.0 cm) (Table 2). On the other hand, Grobogan showed the lowest
plant height (40.9 cm) followed by SC5P2P3.23.4.1-328-3 (46.0 cm). Other lines showed plant height higher
than 50 cm. [10] also showed the low plant height in
acid soil with average of 38.78 cm. Liming can increase
plant height [22], [23].
The highest number of filled pods was showed by
SC5P2P3.23.4.1-3-28-3 (29.6 pods), while the fewest
was SJ-5/Msr.99.5.4.5-1-6-1 (18.3 pods). Among the
three check varieties, Tanggamus showed the highest
number of filled pods while Grobogan was the lowest
(Table 3). It indicated that the genotypes varied in pods
number trait. Similar results also reported by [10] where
genotypes affect the number of pods per plant. In this
experiment, number of pods did not perform optimally,
since Tanggamus could produce number of pods from
98.5 to 114.2 in acidic soil Manokwari [24]. Acid soil
might suppress number of filled pods by deleterious
effect of low pH [11] and low nutrient supply [25]. In
addition, water deficit due to the less rainfall might induce pod abortion during pod development [26].
Grobogan showed the highest unfilled pods, while the
fewest unfilled pods showed by two promising lines
(Msr/SJ-5.21.3.7-3-27-1 and Msr/SJ-5.23.4.1-3-28-3)
and the two check varieties (Tanggamus and Wilis) (Table 3). Variability of unfilled pods number was due to
the genetic constitution. [27] reported that broad sense
heritability of this trait was high (93.1%), suggested that
genetic factor had higher effect than environment effect.
Usually in one reproductive node consist of more than
one pod. Therefore, reproductive nodes fewer than
number of pods. The highest reproductive nodes number
was showed by SC5P2P3.23.4.1-3-28-3, Tanggamus
and Wilis, while the fewest was showed by Msr/SJ-
TABLE I
GRAIN YIELD AND 100 GRAIN WEIGHT OF ACID-TOLERANT SOYBEAN LINES
ON ULTISOLS IN BANJARNEGARA, RAINY SEASON 2009
Yield (t.ha-1)
Plant height (cm)
1.15ab
55.68abc
SC2P2.151.3.5.1-10
1.08
abc
56.22abc
SC5P2P3.23.4.1-3-28-3
1.15ab
46.01cd
1.18
ab
50.19bcd
1.38
ab
51.72abcd
1.00
bc
62.02ab
SJ-5/Msr.99.5.4.5-1-6-1
1.20
ab
53.37abc
Msr/SJ-5.21.3.7-3-27-1
0.68c
51.77abcd
Genotypes
SC2P2.99.5.4.5-1-6-1
SC5P2P3.5.4.1-5
SC5P2P3.23.4.1-5
SC5P2P3.48.31.1-10
1.42
ab
60.67ab
1.26
ab
61.22ab
60.98ab
Wilis
1.34
ab
63.28a
Grobogan
Average
0.68c
40.87d
1.16
54.92
LSD 5%
0.44
11.95
Msr/SJ-5.23.4.1-3-28-3
Msr/SJ-5.23.4.1-5
1.47
Tanggamus
5.23.4.1-5, SC5P2P3.48.31.1-10 and SJ-5/Msr.99.5.4.51-6-1 (Table 4). However, number of reproductive

nodes in this experiment was higher than those obtained
in the experiment in Manokwari [24] with the same soybean lines under pH 4.89 (personal communication),
suggested that lower pH decreased number of reproducTABLE II
NUMBER OF FILLED PODS AND UNFILLED PODS PER PLANT OF ACID-TOLERANT
SOYBEAN LINES ON ULTISOLS BANJARNEGARA, RAINY SEASON 2009
Filled pods.plant-1
Unfilled pods.plant-1
24.8abc
0.8d
bc
0.7d
SC5P2P3.23.4.1-3-28-3
29.6
1.6b
SC5P2P3.5.4.1-5
26.4ab
1.5bc
21.3
bc
0.9cd
20.9
bc
1.2bcd
1.0bcd
Msr/SJ-5.21.3.7-3-27-1
22.1
abc
0.7d
Msr/SJ-5.23.4.1-3-28-3
19.0bc
0.7d
Genotypes
SC2P2.99.5.4.5-1-6-1
SC2P2.151.3.5.1-10
20.3
SC5P2P3.23.4.1-5
SC5P2P3.48.31.1-10
SJ-5/Msr.99.5.4.5-1-6-1
Msr/SJ-5.23.4.1-5
Tanggamus
Wilis
Grobogan
18.3
bc
1.1bcd
23.9
abc
0.7d
22.2
abc
0.6d
bc
2.4a
20.2
20.1
Average
22.2
1.1
LSD 5%
8.05
0.63
tive nodes.
Grain size can be measured through 100 grains
weight. The largest seed size was showed by Grobogan
(14.13 g/100 seeds) as early maturity check variety with
large seed size, while the smallest seed size were
showed by Tanggamus and Wilis (7.73 and 8.57 g/100
seeds). Tanggamus and Wilis showed smaller seed size
than the description [20] suggested that the two checks
th
varieties showed seed decreasing. All promising lines

showed seed sizes between Wilis and Grobogan, where
there were three promising lines with seed size more
than 10 g/100 seeds (Table 4). The magnitude of seed
size depends on seeds filling rate [28].
Relationship among some agronomic characters of acid-tolerant soybean lines showed that there were significant positive correlation between grain yield and plant
100 seeds weight, between plant height and both of 100

seeds weight and unfilled pods (Table 5). Negative correlation between plant height and both of 100 seeds
weight were also reported by [31]. Usually, positive
correlation found between grain yield and 100 seeds
weight [32], [33]. However, larger seed size had risk for
having higher unfilled pods.
IV. CONCLUSION
TABLE III
PLANT HEIGHT AND NUMBER OF REPRODUCTIVE NODES PER PLANT OF
ACID-TOLERANT SOYBEAN LINES ON ULTISOLS IN BANJARNEGARA,
RAINY SEASON 2009
Genotypes
Reproductive
nodes.plant-1
100 grains weight

(g)
SC2P2.99.5.4.5-1-6-1
11.38ab
9.87bcde
abc
9.87bcde
SC2P2.151.3.5.1-10
SC5P2P3.23.4.1-3-283
10.56
11.54a
9.70bcde
SC5P2P3.5.4.1-5
11.39ab
8.90ef
SC5P2P3.23.4.1-5
9.20
bc
10.13bc
SC5P2P3.48.31.1-10
SJ-5/Msr.99.5.4.5-1-61
Msr/SJ-5.21.3.7-3-271
Msr/SJ-5.23.4.1-3-283
9.07c
9.10def
8.70c
9.23cdef
9.43abc
10.00bcd
9.58abc
10.50b
Msr/SJ-5.23.4.1-5
9.08c
9.63bcde
Tanggamus
11.52a
7.73g
Wilis
11.46a
8.57fg
abc
14.13a
9.98
Grobogan
Average
10.2
TABLE V
RELATIONSHIP AMONG SOME AGRONOMICAL CHARACTERS OF ACIDTOLERANT SOYBEAN LINES ON ULTISOLS BANJARNEGARA, RAINY SEASON
2009
100
Rep.
Filled
Unfilled
Yield
seeds
nodes
pods
pods
per plant
weight
Rep. nodes
Filled pods
Unfilled
pods
100 seeds
weight
-0.059
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th
BIOLOGICAL CONTROL AND

MANAGEMEN OF INSECT PEST ON
STROBERY COMMUNITY: 1. INSECT
DIVERSITY IN STRAWBERY COMMUNITY
RCH. Soesilohadi11), S. Sumarmi1), S. Marginio2) dan R. Susandarini3)
1) Lab. of Entomology, Faculty of Biology UGM,
2) Lab. of Microbiology, Faculty of Agriculture UGM,
3) Lab. of Plant Systematic, Faculty of Biology UGM
*) Corresponding author: hidayat@ugm.ac.id
Abstract Insect was the most important aspect in decreasing strawbery fruit in Kopeng, Jawa Tengah. There
are need effectively and safely method to control the insect.
The objective of the reserch was inventory insect in strobery community. Sampling was done every 3 day periode
since the strobery panted in the field. The Result showed
seven insect ordo visiting strabery community, namely
Ordo Neuroptera, Orthoptera, Hymenoptera, Hemiptera,
Diptera, Coleoptera, and Lepidoptera. Aphid (Hemiptera:
Aphididae), was predominan insect in strowbery community.
Keywords Spodoptera, Aphid, white fly dan strawbery
I. INTRODUCTION
The stroberi, Fragaria chiloensis L planted for commercially fruit production. It will delivery profit around
IDR. 54.007.500 ha-1/ year [1]. Strobery growing in
high land with low air temperatur. In Indonesia the
strowbery have been cultivated in Lembang, and cianjur,
West java for a long time ago. In central Java, the
strawbery cultivation sited in Kopeng, Central Java.
So many insect visited on strowbery community, and
some of them as its pest potency. The acarin and insect
kwown as pest in strowbery are 1) mite, Tetranychus sp.
dan Tarsonemus sp., 2) Aphid, Chaetosiphon fragaefolii
(Hemiptera: Aphididae), 3), Flower borer beetle, Anthonomus rubi, root borer beetle, Otiorhynchus rugosostriatus and stem borer beetle, O. sulcatus, 4) white fly,
Pseudococcus sp., and 5) leaves borer larvae of Lepodoptera, Spodoptera sp. (Diptera: Lycaenidae) [1].
Recenly, strobery production decreased because the pest
has resistant to insecticides [2]. Kopeng, Cental Java
(73957; 110,417) is an agroecosystem with 1.500
m asl, and air temperature 20 C at noon.
Strowbery was cultivated in Kopeng with organic method and It has problem with production caused by insect pest. Longterm outcomes of the research Will be
used Bacillus thuringiensis isolated from soil in the stroberry community, which it as pathogenic agent for reduction population of insect pest. Objective of reserch
was gathering information and the mapping of insect
visiting strowbery community in Kopeng, Cental java,
with emphazised on insect pest potencial.
II. MATERIAL AND METHOD
Research was done in strobery farm in Kopeng and

Laboratory of Entomologi UGM, and LIPI for specimen
confirmation. Two blocks area, 10 x 10 m2 with (organic block) and without cage screen (non organic block)
respectively. One hundred and fivety polybags, (Diameter: 30; Height: 50 cm, Figure. 1a) placed in each block.
(Figure 1b). A polybag contain four strabery plants
(Figure 1a). Observation was done during 5 months
from June to November 2013.
Insect collection was done using trap such as Yellow
trap, sweeping net and efford unit by choose 6 polybags
(6 polybag = 1m2) and decided five unit in the block
(Four unit in each side and one unit in the center of the
blocks). The spesimen identififying in Laboratory of
Entomologi UGM with refferencies, Boror [3], Kalshoven [4], Booth et al. [5], Bolton [6], Braby [7], and
Lawrence & Bolton [8]. The specimens will be confirmed in Laboratory of Entomologi LIPI.
50 cm
30 cm
(a)
(b)
th
Figure 1. (a) A polybag with four strobery plants, dan (b) the
pattern of polybags squence in the block.

A. Insect visitor on Strowbery Cummunity
There were seven insect order smpled from both two
block (organic and non organic) in strabery community.
The Ordo were Neuroptera, Orthoptera, Hymenoptera,
Hemiptera, Diptera, Coleoptera, and Lepidoptera (Figure 3).
Most of insect visitor on strabery dominated by Ordo
Lepidoptera, Coleoptera, Diptera, Homoptera and Hemiptera Almost member of Ordo Hemiptera, Homoptera
and Lepidoptera sampled from strabery community will
be pest potencial. There are two reasons, first the the
insects population were higher than those the others, and
all of the immature stage eat part of the strobery. Mostly
of Ordo Diptera, Coleoptera and Neuroptera were predator. Ordo Hymenoptera, such as honey bees visit
strowbery flower, so the insect will be candidate as polinator. Athough member of Ordo Orthoptera eat strabery leaves, they will not be pest, because the population always low (Figure 2).
Figure 2. Insects visitor on strabery community in organic

and non organic block
Aphid (Hemiptera: Apididae) was predominan in both

organic and non organic block, but aphid population in
non organic block more higher than those in organic
block (Figure3). The insect will be potencial as pest.
There were significant in increasing aphid population
during 30 days observation from 10 at begining to almost 800 in non organic block (Figure 3). In non organic block, aphid population decreased from 800/block to
less than 10/block in 30 days, because insecticides
treatment by the farmer. Meanwhile in organic block,
aphid population growth in low speed begining from
10/block to 200/block in 70 days and then after 70 days,
the population become stationer in around 200/block
(Figure 4). No insecticides in organic block.
Figure 3. Population Growth Comparision of aphid on strowbery in organik and non organic area
Figure 4. Natural enemies and aphid and its competitors

(Arachnida, Neuroptera, Homoptera dan Coleoptera) in organic and non organic area
Both in organic and non organic blocks, the fluctuation population of natural enemies and aphid competitor
following aphid population growth. (Figure 3 and 4).
There were competition beetween aphid and its competitors, and it cause reduction of aphid population. The
competitors was an alternative prey or host for aphid
natural enemies, so its will be maintenance the natural
enemies. Decreasing natural enemies and aphid competitor in line with decreasing aphid population not just only
pesticides treatment but the natural enemies and some of
competitors will be avoid insecticides treatment because
they have highly mobility. There were density dependend fluctuation beetween natural enemies and aphid
population (Figure 4 and 5).
Caging on stobery cultivation reduced aphid population (Figure 4), and reducing insecticides treatment.
Population of aphid natural enemies in cultivation
strowbery with cage was more higher than those in
strowbery cultivation without cage (Figure 5).
IV. CONCLUSION
1. There were Neuroptera, Orthoptera, Hymenoptera,
Hemiptera, Diptera, Coleoptera, and Lepidoptera visiting strobery cultivation
2. Population of Apid (Hemiptera: Aphididae) was higest than those other taxa.
3. Aphid population can be minimize in strobery cultivation by caging.
ACKNOWLEDGEMENT
We thanks to the management of PUPT UGM 2013 which financing
this research.
REFERENCES
[1]
Anonim 2008 .Perkebunan stroberi. http://www.ristek.go.id
http://www.warintek.ristek.go.id/ perkebunan/stroberi.pdf
[2]
Bradford MM (1976) A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing theprinciple
of protein-dye binding.Anal Biochem 72:248254
[3] Borror, D.J., L.A. Triplehorn, and N.F. Johnson. 1989. An Introduction to Study of Insect. 6th edition.Holt Reinhard and Winston.
New York.
[4]
Kalshoven, L.G.E. 1981. The Pests of Crops in Indonesia.Pt
Ichtiar Baru-Van Hoeve, Jakarta. P. 341-344.
[5] Booth, R.G. Cox, ML Madge, RB.1991. The Guide to the Insects
of Importance to Man; Coleoptera. The University Press Cambridge.
[6]
Bolton, B. 1994.Identification Guide to the Ants Genera of
World. The President and Fellows of Harvard College. USA.
[7] Braby. F. Michael 2004. The Complet Field Guide to Butterflies
of Australia.CSIRO Publishing Australia.
[8] Lawrence, JF. Britton.E. 1994. Australia Beetles. Melbourne
University Press.
th
Protein profile of Holothuria leucospilota

(Echinodermata: Holothuroidea) at Ceased
Traditional Gold Mining, Lampon Banyuwangi
District
Susintowati1*) and Nurchayati, N.2)
1,2
) Faculty of Teacher Training and Education, 17 Agustus 1945 University, Banyuwangi, Indonesia
*) Email: susintowati@yahoo.com
Abstract - Holothuria leucospilota was used as bioindicator of mercury bioaccumulation at ceased traditional gold mining at Lampon estuarine and stone
shore. We used the samples with same species as control samples from Stone Shore Beach of Rajegwesi
Resort in Meru Betiri National Park. The bioaccumulation of mercury in this research was detected by
SNI 06-6992.2-2004 (with modification). The result of
mercury bioaccumulation shows from 47.83 ppb to
0.03 ppb in the lowest, but it was undetected in samples from Meru Betiri National Park. The mercury
accumulation shows in high value from recommended standard value. Protein profile was analyzed using
SDS-PAGE method. There are three new bands and
one band of protein in bolder from the other bands
of the samples. It may show that anomaly form of
protein profile caused mercury pollution at this site.
Mercury is still remaining to pollute while the mining
was ceased. It can be predicted that recidence time of
mercury effect caused some anomaly structure to
aquatic biota because the protein profile was different from the normal profile.
Key words: protein profile, Holothuria leucospilota,
bioaccumulation, mercury
I. INTRODUCTION
Lampon shore was one of the areas to wasted of tailing traditional gold amalgamation in Banyuwangi District. They were used mercury for amalgamation
process. Although, the activity of tailing discharging
was stopped at least 2 years ago, but the effect of mercury polluted still be remaining to aquatic organisms
indeed. Mercury bioaccumulation in Gastropods was
proofed, they are Nerita argus (3.03 ppm) and Terebralia sulcata (3.10 ppm). Hepatopancreas of Nerita argus
in severe atropi [1]. The observation result in 2013
shows still be found mercury bioaccumulation in some
macrobenthos based on niches and feeding behavior [2],
[3].
Pathology effect of mercury pollution is approximately to be monitoring time after time. There is a lot of Holothuroidea in summer season. Holothuroidea as detrivor, they cleaned of the shore from detritus. They feeding behavior can be used to mercury bioindicator in
Lampon. Holothuria leucospilota very abundance beside another genus called Cucumaria. We used Holothu-
ria leucospilota as bioindicator for mercury effect to

them protein profile.
Mercury is toxic and carsinogenic heavy metal because mercury is non-degradable. Mercury accumulation can be caused lethal effect. Mercury in nature as
cinnabar (HgS) and we found mercury in sea about 0.15
g/l. Mercury value standard of water that recommended in Indonesia is 0.001 mg/l (Kep. MenLH No.51,
2004). If mercury concentration in environment higher
from standard value, it can be negative impact for organisms.
Toxic effect of mercury such as mithocondial
damage, nerve disfunction, endocrine disruption, cancer,
and memory lost. The risk for chronic toxicity depends
on the frequency, intensity, and duration of contact with
the contaminant along with the exposure route. Toxicity
risk also depends on the inherent toxic potential of the
metal itself. All forms of mercury are toxic to humans.
Their effects are organ specific and depend on the chemical form of the mercury and the exposure level, duration, and route. Different forms of mercury deposit preferentially in different tissue compartments, which explains their different toxic profiles. MeHg (methylmercury) is almost 100% absorbed across the intestines and
also crosses the blood-brain barrier (BBB). Inorganic
mercury is also absorbed through the intestines and
crosses the BBB but much less so than MeHg. MeHg is
considered the most toxic form of mercury and has a
half-life of 70 days in humans ([4]; [5]; [6]; [7]; [8];
[9]; [10]).
Manisseri dan Menon [11] shows, mercury can
make reticulum endoplasmic damage. Reticulum endoplasmic contain lots of Ribosomes. Ribosom is an organella to resposible to make protein cell. The change of
protein profile show the change of protein building by
ribosoms. Mercury pollution still be remaining in unpredictable time at Lampon. It is approximately to tracing
mercury effect, included protein profile of Holothuria
leucospilota.
Lampon estuaries was administratively located in
Banyuwangi, East Java. This research taken placed from
October-December 2013. The Method used in this research to collect samples was random sampling when
low water spring in estuary and rocky shore. The coordinate site is 83705.39S 1440511.46E. Control
th
samples taken from Stone Shore of Rajegwesi Resort,

Meru Betiri National Park.
To identified species of the specimen (Holothuria leucospilota) were used the data and sample picture in literatures at laboratory. The specimens were
fixation with 4% formaldehyde before preparation to
mercury analysis. Mercury concentration were analyse
based on SNI method 06-6992.2-2004 (with modification) and Mercury Analyser at LPPT Gadjah Mada University, Yogyakarta. Whereas, protein profile of Holothuria leucospilota used SDS PAGE (sodium dodecyl
sulfate polyacrylamyde gel electrophoresis) procedure at
Falitma Laboratory of Faculty of Biology, Gadjah Mada
University [11]. The overall analysis of the data of the
results based on protein bands of protein profile and
qualitative descriptive analysis.
III.RESULT AND DISCUSSION
Bioaccumulation of mercury in Holothuria leucospilota still high enough. Although, dischaging of
tailing stopped, the accumulation of mercury still remaining and detected. Bioaccumulation mercury in Holothuria leucospilota from 47.83 ppb to 0.03 ppb in the
lowest. But, the samples from Meru Betiri National Park
by mercury analyzer tools shows undetected. According
to Eisler, the standard concentration of mercury that
make lethal sensitivity on aquatic organisms is 0.1 2.0
ppb [4]. In Indonesia value standard of mercury based
SNI 7387:2009, did not clear yet. But here, if we look in
another country e.g. Japan and Canada, 0.3 ppm maximal [9].
Based on protein analysis by SDS PAGE all
protein bands of specimen from Lampon are same as the
specimen from Meru Betiri National Park. But, one of
them show different, there is boldest band and three new
bands (Figure 1). It is need another works to proof the
protein specification. Residence time of mercury in water and sea sediment is very long time. It could be the
reason that bioaccumulation of mercury in the sample of
Holothuria leucospilota still detected. There are a lot of
symptoms appear caused mercury toxicity in fact DNA
damage [10]. The DNA damage can be causing anomaly
of protein structure. The effect of profile protein change
is pathologycal symptoms of this animal.
Molecule weight of protein of Holothuria leucopsilota (Tabel. 1) was estimated by using empiric
pattern y=-1.049x+2.272 and R2=0.951 based on calculating of Marker molecular weight and it racing factors
(rf). There are three main protein bands of Holothuria
leucospilota from Lampon dan Meru Betiri National
Park based SDS PAGE procedure. But, there is one
sample that different from the other samples. It is show,
three new bands and one band boldest from the other.
B Mark L1
L2
L3
M1 M2
M3 B
Figure 1. Result of SDS PAGE of Holothuria leucospilota protein

profile. Bolder and addition of bands (arrow indeed). Lampon sample
(L) and Meru Betiri National Park sample (M) in normal sequence.
Figure 2. The Curve of Molecule Weight (MW) of Marker vs Marker

racing factor (rf)
TABLE 1.
PROTEIN MOLECULE WEIGHT OF HOLOTHURIA
LEUCOSPILOTA FROM LAMPON
Tracking distance
(cm)
rf
MW (kDa)
log MW
0.8
0.133
135.571
2.132
4.3*
0.717
33.127
1.520
4.9
0.817
26.018
1.415
5*
0.833
25.014
1.398
5.2
0.867
23.059
1.363
5.7*
0.950
18.856
1.275
Note: * new band; kDa = kilo Dalton
TABLE 2.
PROTEIN MOLECULE WEIGHT OF HOLOTHURIA
LEUCOSPILOTA FROM STONE SHORE OF MERU
BETIRI NATIONAL PARK
Tracking
Distance
BM
log
(cm)
rf
(kDa)
BM
0.8
0.133
135.571
2.132
4.9
0.817
26.018
1.415
5.2
0.867
23.059
1.363
th
The molecule weight of new protein bands are 33.127

kDa, 25.014 kDa and 18.856 kDa, they to be estimate
as CBD-BmFKBP13 and Lyzozyme. It need another
works to proof that new bands are anomaly proteins
caused mercury bioaccumulation in their body or caused
by another reasons. In another observation, we found
significantly different of spicules density of Holothuria
leucospilota from Lampon if compared with samples
from Meru Betiri National Park. Density spicules of
Lampon sample is less than the sample of Meru Betiri
[2]. Spicules Is a skeleton spicules on Holothuroidea.
Spicule formation is influenced genetic factors by DNA
code, because the spicules form a specific character type
recognition. Changes in the protein profile has been
implicated in the formation of spicules Holothuroidea
included Holothuria leucospilota.
III.CONCLUSION
Bioaccumulation of mercury in Holothuria leucospilota
in Lampon still detectable despite amalgamation activity
has stopped in long time enough. Bioaccumulation of
mercury in the body Holothuria leucospilota suspected
to cause a change in the protein profile. However,
another works needs to proof it.
ACKNOWLEDGMENT
Many thanks to the Directorate General of Indonesian
Higher Education which has provided research grants of
beginner lecturer for fiscal year 2013. Thank you also to
all the staff of LPPT and Laboratory Falitma, Faculty of
Biology Gadjah Mada University Yogyakarta for any
help given. Thank you to PPPM 17 Agustus 1945 Banyuwangi University to recommended this research. And
thank you to all those involved in sampling and data
processing this research.
REFFERENCES
[1] Susintowati and S. Hadisusanto, Mercury bioaccumulation and
Community Structure of Gastropoda at Ceased Traditional Mining, Lampon, Banyuwangi District, Thesis,
Gadjah Mada University, Yogyakarta, 2012.
[2] Susintowati and N. Nurchayati, Mercury Pathology Effect to
Spicules Density of Holothuroidea (Echinodermata) at
Ceased Lampon Traditional Gold Mining, Banyuwangi
District, (accepted for publication) in Saintek Journal,
Kopertis Wilayah VII Jawa Timur. 2013.
[3] Susintowati and S. Hadisusanto, Mercury bioaccumulation of
macrobenthos based on feeding behavior and niches at
Lampon ceased traditional gold mining, Banyuwangi
District, East Java, Paper (presented) in International
Conference of Biology Science, at Faculty of Biology,
Gadjah Mada University Yogyakarta, September 2122th, 2013, (accepted for proceeding).
[4] R. Eisler, Mercury hazards to fish, Wildlife and Invertebrates, A
synoptic review Biologycal Report, 1987, 85(1:10).
[5] B.T. Setiabudi, Penyebaran merkuri akibat usaha pertambangan
emas di daerah Sangon Kabupaten Kulon Progo DI Yogyakarta, Draft Report (unpublished)-DIM, 2005: 1-17.
[6] Darmono, Lingkungan hidup dan pencemaran, hubungannya
dengan toksikologi senyawa logam, UI-Press-Jakarta,
2008.
[7] H. Palar, Pencemaran dan toksikologi logam berat, Rineka Cipta
Publisher, Jakarta, 2008.
[8] W. Widowati, A. Sastiono, and R.R. Jusuf, Efek toksik logam.
Pencegahan dan penanggulangan pencemaran, Andi
Publisher, Yogyakarta, 2008.
[9] Anonimous, Maximum level of Heavy Metal in Food by SNI
7387:2009. ICS 67.220.20, Indonesia National Standarization, Jakarta, 2009.
[10] J. Neustadt, and S. Pieczenik, Heavy-metal Toxicity With
Emphasis on Mercury, Integrative Medicine, 2011,
Vol.10.No.5: 45-50.
[11] M.K. Manisseri and N.R. Menon, Ultrastructural aberrasion in
the hepatopancreas of Metapenaeus dobsoni (Miers)
Exposed to Mercury, J. Mar. Biol. Ass. India, 2006, 48
(1): 89-94.
[12] B.T. Kurien, and R.H. Scofield, Extraction of protein from gels:
a brief review, Methods Mol. Biol., 2012, 869:403-5.
The 4th Annual Basic Science International Conference
Screening and Identification of CelluloseDegrading Bacteria from The Mangrove Areas

of Sembilang National Park South Sumatera
1)
Hary Widjajanti 1*), Sarno1), and Novida Rosalia Sinaga 1)

Biology Department, Faculty of Mathematic Natural Science, Sriwijaya University,
Inderalaya, South Sumatera, Indonesia
*)
Corresponding author: haryunsri@yahoo.com
Abstract The aim of the present study was to isolate and

characterize the cellulose-degrading bacteria from the mangrove areas of Sembilang National Park South Sumatera.
The bacteria capable of growing in the liquid medium containing cellulose as the only source of carbon were isolated
and their cellulolytic activity on CMC-containing media was
confirmed by the congo red clearing zone assay. The isolates
were identified based on colony and cell morphological characteristics and biochemical characteristics. The results of
the present study show that 12 cellulose-degrading bacteria
isolated from the mangrove areas of Sembilang National
Park belonged to the species Micrococus sp.(S1SS1),
Acinetobacter sp.(S1SS2), Bacillus sp. (S1SS3), Bacillus sp.
(S1TS1), Clostridium sp.(S1SS4), Aerococus sp. (S1TS3) dan
Pseudomonas sp. (S1TS5), Pseudomonas sp. (S2TS2)
Clostridium sp. (S2SS3), Pseudomonas sp. (S3SS1), Clostridium
sp. (S3SS3), and Clostridium sp. (S3TS1).
Keywords : cellulose-degrading bacteria, identification,
mangrove areas
I. INTRODUCTION
Indonesian territory consists of 17,508 islands and has a
long coastline of about 81,000 km , is the country that has
the largest mangrove forest in the world . Extensive mangrove forests in Indonesia reached 4.25 million hectares is
the largest mangrove in the world beyond Brazil (1.3 million ha) , Nigeria (1.1 million ha) and Australia (0.97 ha)
[11]. One Indonesian mangrove areas which Sembilang
National Park located in South Sumatera . According Suwignyo et al.[9], various types of mangrove genera that
dominat in Sembilang National Park were Rhizophora,
Avicennia, Sonneratia and Bruguiera. According to Noor
et al.[6], the mangrove ecosystem is an interface between
terrestrial and ocean ecosystem. Biodiversity level of
mangrove ecosystem was high so that mangrove can grow
well. Mangrove forest ecosystems that are capable of producing a high organic matter, 90 % of organic particles in
the water coming from the mangrove vegetation and result
in 35-60 % of nutrients that are beneficial to the growth of
mangroves. Constituent components of the organic matter
is cellulose, hemicellulose and lignin .
The importance of microbial diversity in mangrove
ecosystems due to the presence of microbes capable of
providing nutrients in a mangrove ecosystem that can
grow well without organic fertilizer. Microbial diversity in

mangrove ecosystems have each function in degrading
organic materials for the growth of mangroves. Thus in
conserving the mangrove ecosystem, information and
benefit of microbes was required such as cellulosedegrading and lignin-degrading bacteria that play role in
litter decomposition in a mangrove ecosystem benefits [8].
The aims of the research was to isolate and characterize
cellulose-degrading bacteria in mangrove litter and soil, in
Sembilang National Park, South Sumatera.
Sample of mangrove litter and soil bacterial isolates were taken from a mangrove forest area Sembilang
National Park, South Sumatera. Soil samples as top soil
were taken from 20 cm depth at 3 stations with purposive
sampling method. In each station, samples were taken at 5
points of soil sampling to represent each study sites. Samples taken in the form of litter collected for 1 day at
amount 5 grams of finely ground leaf litter and 5 grams of
soil in each erlenmeyer then put in 45 mL distilled water
respectively. Serial dilutions to a concentration of 10-6
were performed. One mL sample was taken at the last 3
serial dilutions and grown on CMC (Caboxy Methyl Cellulose) medium for growth of cellulose-degrading bacteria
in a petri dish with pour plate method, and then incubated
at 37oC for 24-48 hours [2].
Selection is done by taking the pure bacterial isolates using a needle inserted in the center of the loop that
has solid medium in a petri dish, and then incubated at
37oC for 2 days. Bacteria were grown on selective media
cellulolytic after spilled congo red and 1M NaCl to form a
clear zone is a zone of cellulose-degrading bacteria [2].
Based on the research conducted showed cellulosedegrading bacteria isolates of mangrove litter and soil in
Sembilang National Park area as in Table 1.
Thirty pure isolates pure taken from isolation of cellulose-degrading bacteria were isolated using CMC medium
(Table 1), because CMC medium used is the best substrate to induce the synthesis of extracellular cellulolytic
enzymes. CMC is a synthetic substrates that serve as
model compounds of cellulose. CMC has many amorphous regions so soluble in water. CMC concentration used
was 1 %, according to research Narasimha et al. (2005), 1

% cellulose concentration is the optimum concentration
for the production of cellulase enzymes. The medium used
for bacterial growth substrate is a suitable medium for the
growth of bacteria [2].
Table 1. Cellulose-degrading bacteria isolates
Station
Sample
Number of cellulosedegrading bacteria isolates
I
10405413,2E
20925,5S
II
10405341,4E
0
2 543,6S
III
10405418E
0
2 947,4S
Total
Litter
Soil
Litter
Soil
Litter
Soil
4
30
Table 2.
Screening result of cellulose-degrading bacteria
CelluloseStation
Sample Screening result of
cellulosedegrading bacteria
degrading bacteria
isolates code
I
Litter
4
S1SS1, S1SS2,
0
104 5413,2E
S1SS3, S1SS4
20925,5S
Soil
3
S1TS1,S1TS3,
S1TS5
II
Litter
1
S2SS3
10405341,4E Soil
1
S2TS2
20543,6S
III
Litter
2
S3SS1, S3SS3
10405418E
Soil
1
S3TS1
20947,4S
Total
12
Screening of cellulolytic bacteria used selective

medium are characterized by the formation of clear zone
after spilled congo red dye that is used as an indicator of
cellulolytic bacteria. Formation of a clear zone is due cellulolytic bacteria capable of hydrolyzing cellulose into
simple compounds. The formation of clear zone indicates
that the cellulose contained in the media is hydrolyzed by
cellulase enzymes into simple compounds that cellobiose
is then simplified into two molecules of glucose [6].
Clear zone can be formed by washing use 1M
NaCl. Clear zone will be clearly after congo red addition.
Congo red is the sodium salt of benzidinediazo bis - 1 naphthylamine - 4-sulfonic acid (C32H22N6Na2O6S2) so
that the dye will dissolve and leached by other sodium
salts, such as NaCl . Thus , the clear zone is formed will
be clearly. Formation of clear zone indicates that the bacteria are able to degrade cellulose [8].
Based on the characteristics obtained, each bacterial isolate can be grouped into several genera were identified [1] [4]. Each cellulolytic bacteria belongs to the genera Micrococus, Ancinetobacter, Bacillus, Clostridium,
Pseudomonas, and Aerococus.
Isolates of bacteria belonging to the genus Micrococus is S1SS1. The bacterial isolates have characteristic
shape cocci cells, negative staining and aerobic endospores. The characteristics of the genus Micrococcus
spherical cell shape , nature of gram-positive, negative
indole test, catalase test positive, does not produce acid
fermentation of carbohydrates, is motile due to its flagella
as locomotor and there is also nonmotil. This genus can

be isolated from soil, water and food products. Isolates of
bacteria belonging to the genus Acinetobacter[1]. Genus
Acinetobacter discrete forms of cocci, gram-negative, non
motile, growth requires oxygen (aerobic), not produce
endospores, biochemical test results were positive in catalase test and negative on indole and H2S test, can be isolated of soil, water and litter decomposition [4].
Other genera that found Bacillus sp. ( S1SS3 ), Bacillus sp. (S1TS1 ), Bacillus sp. ( S1SL2 ) , Bacillus sp. (
S1SL4 ) and Bacillus sp. ( S2TL1 ). The fifth Bacillus
genera have characteristics as bacillus form, grampositive, catalase test positive, and form endospores. Differences in biochemical test on each isolate would classify the genus Bacillus isolates at the species level. Genus
Bacillus have characteristics such as bacillus shape,
straight, long and short, gram-positive, motile but there
are a few nonmotile, form endospores oval, spherical,
cylindrical and resistant to a wide range of conditions,
aerobic and facultative anaerobic, catalase test positive,
are pathogenic in vertebrates and invertebrates, its broad
habitat, and can be found in water and soil [1] [4].
Isolates S1SS4 , S2SS3 , S3SS3 , S3TS1 , and
S3TL1 belonging to the genus Clostridium. They have
bacillus shaped Clostridium characteristics, anaerobic,
gram positive, and form endospores. Clostridium discrete
cell shape in general, but there are a few bacilli, cocci,
gram-positive, motile with flagella peritric partially and
sometimes non-motile, anaerobic, form endospores, and
negative catalase test, found in water, soil, also in human
skin. The difference in the physiological test each isolate
in the genus Clostridium, enabling this genus can be
grouped in the species level [1] [4].
Isolates S1TS3, S1TL5 and S3SL3 belong to the
genus Aerococus. This genus generally have characteristics of gram-positive, non motile and catalase test negative. Aerococus belong to the gram- positive, non motile,
facultative anaerobic and some are anaerobic, not produce
catalase, so based on common characteristics of these
three isolates were grouped in a single genus. However,
the differences in the nature of the physiological test,
Aerococus this genus can be further grouped based on
species level [1].
Besides Aerococus also found the genus Pseudomonas isolates S1TS5, S2TS2, S3SS1, S1SL5, S2SL1,
S2SL2 and S3SL2. These isolates differed in physiological tests can be grouped by allowing the species level .
However , similarity of the characteristic of these isolates,
like flagella are motile, gram negative and positive catalase test that are grouped into a single genus. This is consistent with [1] and [4] Pseudomonas has the characteristics bacillus form single or in groups, motile with flagella
lies opposite, gram-negative, aerobic and facultative anaerobic, catalase test positive, can be found in soil, water
and sea. Genus of bacteria most commonly found is the
genus Pseudomonas as mention by Rao [8] that genus of
microorganisms that can degrade cellulose and lignin is
the genus Pseudomonas.
ACKNOWLEDGMENT
Thank you to Dikti with Hibah Fundamental that funding
this research and also to Sembilang National Park Author-
ity thank you very much for your help and togetherness
during at Sembilang National Park.
REFERENCES
[1]
[2]
[3]
[4]
[5]
Buchanan, R.E. & N.E Gibbons. 1974. Bergeys Manual of

Determinative Bacteriology 8th Edition. The Wiilliams & Wilkins
Company. USA. 1268 pages.
Cappuccino, J. G & N, Sherman. 2008. Microbiology A
Laboratory Manual. Rockland Community College Suffern. New
York. xvi + 557 pages.
Hartanti. 2010. Isolasi Seleksi Bakteri Selulolitik Termofilik dari
Kawah Air Panas Gunung Pancar, Bogor. Skripsi. Departemen
Biokimia Fakultas Matematika dan Ilmu Pengetahuan Alam. IPB.
Bogor + 29 hlm.
Holt, J.G,. R.K. Noel, H.A.S. Peter, T.S. James & T.W. Stanlay
1994. Bergeys Manual of Determinative Bacteriology 9th Edition.
The Wiilliams & Wilkins Company. USA. 1268 pages.
Noor, R.Y, M. Khazali & I N.N. Suryadiputra. 1999. Panduan
Pengenalan Mangrove di Indonesia. PHKA/WI-IPB. Bogor. Vii +
220 hlm.
[6]
Perez, J. J. Munoz-Dorado, T. de la Rubia & J. Martinez. 2002.

Biodegradation and biological treatments of cellulose,
hemicelluloses and lignin: an overview. Int. Microbiol.
[7] Rao, N. S. Subba. 1994. Mikroorganisme Tanah dan
Pertumbuhan Tanaman. Edisi kedua. Universitas Indonesia Press.
Jakarta. 353 hlm.
[8] Sahoo, K & N.K. Dhal. 2008. Potential microbial diversity in
mangrove ecosystems.
A review. Institute of mineral and
materials technology Bhubaneswar. India. 249-256.
[9] Suwignyo, R. A. Munandar, Sarno, T. Z. Ulqodry & E.S. Halimi.
2011. Pengalaman Pendampingan dalam Pengelolaan Hutan
Mangrove pada Masyarakat. Makalah. Balai Pengelolaan Hutan
Mangrove Wilayah II Direktorat Jenderal Bina Pengelolaan
Daerah Aliran Sungai dan Perhutanan Sosial, Kementerian
Kehutanan Hotel Swarna Dwipa. Palembang. 22 hlm.
[10] Yunasfi, 2006. Dekomposisi Serasah Daun Avicennia marina oleh
Bakteri dan Fungi pada Berbagai Tingkat Salinitas. Disertasi.
Program Studi Ilmu Pengetahuan Kehutanan, Institut Pertanian
Bogor. Bogor. 60 hlm.

Appendix 1

Tabel 3. The results of characterization and identification of cellulose degrading bacteria
Isolate Character
S1SS1
S1SS2
S1SS3
S1SS4
S1TS1
S1TS3
S1TS5
S2SS3
S2TS2
S3SS1
S3SS3
S3TS1
Plumose
Echinulate
Filamentous
Ramose
Convex Papilate
White
Plumose
Echinulate
Comensal
Undulate
Umbonate
Cream
Effuse
Villous
Crenate
Undulate
Convex
Papilate
Cream
Beaded
Villous
Crenate
Lobate
Raised with
Concave
Brown
Filliform
Villous
Circular
Entire
Effuse
white
Plumose
Beaded
Circular
Entire
Convex
Yellow
Filliform
Villous
Circular
Entire
Convex
Orange
Filliform
Villous
Irregular &
spreading
Crenate
Convex Rugose
White
Spreading
Echinulate
Irregular &
spreading
Crenate
Umbonate
Cream
Plumose
Beaded
Circular,
filamentous
Crenate
Raised with
Concave
Cream
Filliform
Villous
Circular
Entire
Convex
Brown
Spreading
Villous
Irregular,
spreading
Crenate
Convex
Papilate
Cream
Microscopic cell morphology
Coccus, Gram
positive, did
not produce
spore
Bacillus,
Gram positive, produce spore
Coccus,
Bacillus,
Bacillus, Gram
negative, did
not produce
spore
Bacillus,
Bacillus,
Anaerob
Fakultatif
Anaerob
Anaerob
Fakultatif
Bacillus,
Gram negative, did not
produce
spore
Anaerob
Fakultatif
Bacillus, Gram
negative, did
not produce
spore
Aerob
Bacillus,
Gram positive, did not
produce
spore
Anaerob
Fakultatif
Bacillus, Gram
positive, produce spore
O2 needed
Coccus
Gram negative, did not
produce
spore
Aerob
Anaerob
Anaerob Fakultatif
Anaerob
Anaerob
Anaerob
Motility test
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Macroscopic colony morphology
Biochemical test :
Simmons citrate test
Indole production
Starch hydrolysis
Urea hydrolysis
Methyl-red test
Voges Proskauer test
Catalase production
Glucose FermenGas
tation
Acid
Lactose FermenGas
tation
Acid
Sucrose FermenGas
tation
Acid
Gas
H2S and gas
Sugar fermentaproduction
tion
H2S
CONCLUSION
Notes: (+) : Positive
(-) : Negative
Micrococus
sp. (S1SS1)
Ancinetobacter sp.
(S1SS2)
Bacillus sp.
(S1SS3)
Clostridium
sp. (S1SS4)
+
Bacillus sp.
(S1TS1)
+
Aerococus
sp. (S1TS3)
Pseudomo
nas sp.
(S1TS5)
Clostridium
sp. (S2SS3)
Pseudomonas
sp. (S2TS2)
+
Pseudomonas
sp. (S3SS1)
Clostridium
sp. (S3SS3)
Clostridium
sp. (S3TS1)
th
Lipase/Amylase Ratio as The Indication of

Pancreatic Exocrine Inflammation and The
Correlation with Insulin Resistance in Type 2
Diabetes Mellitus
Srihardyastutie, A1,2*), Soeatmadji, D.W. 3), Fatchiyah 2), and Aulanniam2)
1)
Biology Doctoral Program, Faculty of Science, Brawijaya University
2)
Faculty of Science Brawijaya University, 3) Medical Faculty, Brawijaya University,
*)
Corresponding author: arie_s@ub.ac.id
Abstract Diabetes mellitus is a chronic metabolic disorder which associated with hyperglycemia. It caused by a
derangement in secretion or function of the endocrinal
portion of the pancreas. The insulin resistance and insulin
secretion are well known underlying pathophysiologies of
diabetes. There is a close anatomical and functional relationship between its exocrine and endocrine portions. An
increased in the serum concentration of pancreatic enzyme
is commonly an expression of inflammatory exocrine pancreatic disease. The aim of the study was to evaluate the
correlation between fasting blood glucose, insulin resistance and lipase/amylase ratio in type 2 DM. The subjects
were categorized into three groups which include: healthy
controls (n = 21), prediabetes (n = 12), and diabetes (n=34).
That clinical identification was assessed according to ADA
criteria in 2013. Plasma glucose, lipid profile testing, total
cholesterol, HDL cholesterol and triglycerides were measured using an auto analyzer. Amylase and lipase were
measured colorimetric method using Assay Kit. Insulin
concentration was measured using Elisa kit. The results
showed that there were elevated levels of blood glucose,
insulin resistance, and serum pancreatic enzymes (amylase
and lipase) activities in type 2 DM. The increased serum
pancreatic enzymes activities were positively correlated
with increased levels of blood glucose, insulin resistance,
and decreased proinsulin level. The ratio of serum lipase/amylase showed a positive correlation with duration
of diabetes, FPG levels, insulin resistance, decreased insulin sensitivity, and increased lipase activity. The ratio of
serum lipase/amylase will be able to determine the acute
phase of alcoholic and non-alcoholic pancreatitis.
Keywords T2DM, insulin resistance, exocrine inflammation
I. INTRODUCTION
DIABETES MELLITUS (DM) is a group of metabolic disorders characterized by hyperglycemia resulting from
defects in insulin secretion, insulin action, or both. The
vast majority of cases of diabetes fall into two categories
namely Type 1 diabetes (Type 1 DM) and the more prevalent Type 2 diabetes (Type 2 DM). Other categories
include specific types of diabetes relate to genetic defects, the exocrine pancreas disease, endocrinopathies,
drug induced and Gestational diabetes [1].
Type 2 DM is caused by a combination of genetic
factors related to impaired insulin secretion and insulin

resistance and environmental factors such as obesity,
overeating, lack of exercise and stress, as well as aging.
The main pathophysiological feature of type 2 DM are
impaired insulin secretion and increased insulin resistance [2].
Anatomically, the pancreas is a mixed exocrineendocrine gland. There is also morphological evidence
that indicates that the pancreatic exocrine function may
be influenced by the pancreatic endocrine hormones.
Insulin has a trophic effect on exocrine pancreas. There
are multiple defects in the insulin secretion and signaling in type 2 DM, which may affect the enzyme synthesis and release in the exocrine pancreas [3].
Recent studies have demonstrated an increased incidence of pancreatitis in patients with type 2 DM. An
elevated serum pancreatic enzyme (amylase and lipase)
supports clinical diagnosis of acute pancreatitis.
With this background, the present study was conducted in order to elucidate a possible correlation between insulin resistance and the pancreatic exocrine
inflammation in Indonesian Type 2 diabetes subject.
II. SUBJECTS AND METHODS
The study was approved by institutional Human ethics
committee (Medical Faculty, Brawijaya University) and
informed consent was obtained from all participants.
A. Subjects:
This study included 67 subjects having general
check-up in Central Laboratory of Dr. Saiful Anwar
Hospital for a period of six month. The inclusion criterion of them were males and females with type 2 diabetes mellitus with or without complications or any comorbid condition like hypertension, coronary artery disease, etc, and also non diabetic healthy individual as the
controls. All the subjects, including the controls, were
fully informed about the study and their voluntary informed consents were taken. The subjects were categorized into four groups which include: healthy controls (n
= 21), prediabetes (n = 10), diabetes (n=10), and prediabetes or diabetes with exocrine insufficiency (n=26).
th
B. Methods
Clinical identification of normal or healthy control,
prediabetes, and diabetes was done according to American Diabetes Association criteria. The diagnostic criteria of diabetes was assessed according to American Diabetes Association (ADA) i.e. subjects with a fasting
plasma glucose > 126 mg/dL and/or 2 hour plasma glucose level > 200 mg/dL and/or HbA1c > 6.5 % were
considered to have diabetes; subjects with a fasting
plasma glucose 100 to 125 mg/dL (IFG) or 2 hour plasma glucose level 140 to 199 mg/dL (IGT) or HbA1c 5.7
to 6.4% were considered to have increased risk for diabetes (prediabetes); subjects with a fasting plasma glucose < 110 mg/dL or 2 hour plasma glucose level < 140
mg/dL or HbA1c < 5.6 % were regarded as a having
normal glucose tolerance (NGT) [1]. The exocrine insufficiency was assessed according to increasing of amylase
and or lipase level. The considered reference range of
normal amylase was 30 - 110 U/L and lipase was 30 210 U/L.
Fasting venous blood was collected from all of subjects, it was centrifuged (at 1500 g for 15 minutes). The
separated plasma was used to assay the HbA1c. HbA1c
was measured with ion-exchange high-performance liquid chromatography using an automated analyzer (BioRad D10). The separated serum was divided into four
aliquot. One was designed for immediate assay of glucose and lipid profile which included Triglyceride (TG),
total cholesterol (CHOL), high density lipoprotein
(HDL), low density lipoprotein (LDL). The other aliquots were stored at -20oC for subsequent assay for amylase, lipase, insulin and proinsulin.
The assay of sample analysis was carried out by using different reagent kits as per procedure which was
defined by manufacturer. The immediate assay of sample analysis was measured on a fully automated analyzer.
The fasting plasma glucose was measured by the hexokinase method. The serum triglyceride was measured
by the enzymatic method (GPO-POD method, End
Point). For determination of total cholesterol, an enzymatic (CHOD-POD) colorimetric method was used. The
direct measurement for HDL and LDL were done by
using enzymatic methods.
Insulin concentration was measured by Sandwich
enzyme immunoassay method. Insulin concentration was
measured using kit from Ucsn, China. Homeostasis
model assessment of insulin resistance (HOMA-IR) was
used for the direct measurement of insulin resistance and
was calculated as follows:
HOMA-IR = [fasting insulin (U/mL)
(mg/dL)]/405 [4]
fasting glucose
The cut-off point to define insulin resistance corresponds to HOMA-IR 3.8 [4][5]
The quantitative insulin sensitivity check index
(QUICKI) was calculated from fasting plasma glucose
(mg/dL) and insulin (IU/mL) concentrations, as follows:
QUICKI = 1/(log Io + log Go )
[6]
Amylase and lipase activity were measured by photometric enzymatic method. The amylase activity was
assayed using BioAssay Systems QuantiChromTM Amylase Assay Kit (DAMY-100). Lipase activity was
assayed using BioAssay Systems QuantiChromTM Lipase Assay Kit (DLPS-100).

The results were analyzed statistically using SPSS
version 16.0 statistical software. The results were expressed as mean SD if the variables were continuous,
and as percentage, if categorical. Multivariate analysis of
variance was used for differences in continuous variables. Multiple regression was applied for correlation
studies. All statistical tests were two-side and a P<0.05
was considered to be significant.
III. RESULT
The clinical characteristic of the study subjects
have been shown in Table 1. The serum amylase and
lipase activity in Group IV was found significantly higher than other groups. Normally, range serum amylase
activity was 30 110 U/L and lipase was 30 210 U/L.
Table I.
Clinical characteristics of the study subjects
Characteristics of the subjects
Age (years)
Fasting Blood Glucose (mg/dL)
HbA1c (%)
Cholesterol (mg/dL)
HDL (mg/dL)
LDL (mg/dL)
TG (mg/dL)
Insulin (ng/mL)
Proinsulin (ng/mL)
Amilase (U/L)
Lipase (U/L)
Group I (n = 21)
45.86 8.04
74.81 7.07
4.58 0.41
195.24 39.32
54.43 15.99
132.14 38.93
120.86 71.65
409.06 19.04
42.79 11.78
46.63 17.10
120.47 45.59
Group 2 (n = 10)
65.70 11.41
109.30 6.07
6.02 0.41
197.60 57.05
44.50 11.65
131.00 49.80
120.00 47.57
343.18 17.52
96.21 36.52
54.96 18.64
100.12 58.07
Group 3 (n = 10)
61.60 6.22
181.50 53.97
9.18 1.89
238.00 56.01
50.90 13.67
169.00 51.04
172.80 50.10
313.53 27.89
114.86 28.46
56.91 18.39
92.95 50.24
Group 4 (n = 26)
55.77 10.52
189.81 92.87
9.49 3.16
193.15 53.44
44.58 13.61
128.88 45.96
186.23 113.81
322.93 32.46
99.24 39.33
601.08 901.68
1423.10 1075.70
The difference fasting blood glucose, HbA1c levels and Insulin Resistance of the group diabetic with
and without exocrine insufficiency were not significant,
but it were different significantly with normal and prediabetes groups (Fig.1). The increasing levels of blood
glucose, HbA1c and insulin resistance were characteristic feature of the most patients with type 2 diabetes mellitus. One of other features that followed of this condition was dyslipidemia (high triglyceride and low HDL
cholesterol), as shown in Fig.1 [7], [8].
Insulin resistance is defined as reduced sensitivity
of target organs to the biological effects of insulin [9].
The quantification of insulin resistance condition can be
performed by evaluating the peripheral insulin sensitivity using mathematical formula, such as Homeostasis
model assessment (HOMA), QUICKI (Quantitative Insulin sensitivity check index), etc [7]. The HOMA index of -cell function and QUICKI index of insulin sensitivity related with decreasing of insulin and increasing
of proinsulin production by -cell, as shown in Fig 2.
The serum amylase and or lipase activity were
found to be significantly higher in case group IV as
compared to the control (group I), prediabetes (group II)
and diabetes subject (group IV). There was significant
positive correlation between lipase/amylase ratio and
duration of disease (diabetes), FPG, serum lipase activity and HOMA index of -cell. However, the correlation
of lipase/amylase ratio with HbA1c, lipid profile (TG,
Cholesterol, HDL cholesterol and LDL cholesterol),
insulin, proinsulin concentration, and amylase activity
were not found to be significant (Table 2).
p value
0.062
< 0.001
< 0.001
0.756
0.449
0.889
0.026
0.014
0.001
< 0.001
< 0.001
th
500.00
300.00
P < 0.001
b
250.00
FPG (mg/dL)
L) 450.00
m
400.00
g/
(n 350.00
n
o
tia 300.00
rt 250.00
n
e
c 200.00
n
o
c 150.00
in
l 100.00
u
s
In 50.00
200.00
150.00
a
100.00
0.00
50.00
0.00
Group I
Group II
Group III
)L
450.00
/m
g
n
(
n 400.00
io
ta
tr 350.00
n
e
c
n 300.00
o
c
n
li
su 250.00
In
b
b
HbA1c (%)
8.00
4.00
200.00
2.00
0.00
0.00
Group I
Group II
Group III
2000.00
4000.00
6000.00
8000.00
10000.00
Insulin Resistance Index
Group IV
(B)
(B)
8000
7000
200.00
(A)
10.00
x
e
d
n
I
ce
an
ts
is
e
R
n
li
u
s
n
I
150.00
500.00
P < 0.001
12.00
6.00
100.00
Group IV
(A)
14.00
50.00
Proinsulin concentration (ng/mL)
0.00
500.00
P < 0.001
L)
450.00
m
g/
(n
n 400.00
o
tia
rt 350.00
n
e
c
n 300.00
o
c
in
l
su 250.00
In
6000
5000
4000
3000
2000
1000
200.00
0
Group I
Group II
Group III
0.150
Group IV
0.155
0.160
0.165
0.170
0.175
Insulin Sensitivity Index
(C)
(C)
Fig. 1. Increasing (A) FPG levels, (B) HbA1c levels, and (C) Insulin
Resistance in control/normal (Group I), prediabetes (Group
II), type 2 DM (Group III), prediabetes and diabetes with exocrine insufficiency groups (Group IV).
Table 2.
Correlation of lipase/amylase ratio with duration of disease, blood
glucose, lipid profile (Cholesterol, TG, HDL, LDL), insulin, proinsulin, amylase, lipase and insulin resistance of the subjects.
Correlation of lipase/amylase Pearson's correlation

ratio with:
co-efficient ( r )
Duration
0.298*
FPG
0.245*
HbA1c
0.224
Cholesterol
-0.12
TG
0.082
HDL
-0.105
LDL
-0.107
Pro Insulin
0.047
Insulin
-0.059
Amilase
-0.189
Lipase
0.869**
Insulin Resistance Indeks
0.282*
Insulin Sensitivity Index
-0.333**
NS = not significant
0.175
x
e
d
in
yt
iv
tii
s
n
e
s
n
li
u
s
In
0.170
0.165
0.160
0.155
0.150
0.00
50.00
100.00
150.00
200.00
p-value
0.014
0.046
NS
NS
NS
NS
NS
NS
NS
NS
< 0.001
0.021
0.006
( D)
10000.00
9000.00
x
e
d
n
I
ce
an
t
iss
e
R
n
il
u
s
n
I
8000.00
7000.00
6000.00
5000.00
4000.00
3000.00
2000.00
1000.00
0.00
0.00
50.00
100.00
150.00
200.00
(E)
Fig. 2. Decreasing insulin concentration correlate with increasing
proinsulin concentration (A), insulin resistance index (B), decreasing insulin sensitivity index (C); Increasing proinsulin
concentration correlate with decreasing insulin sensitivity index (D) and increasing insulin resistance index (E)
IV. DISCUSSION
Diabetes is a global problem and would be the leading
cause of morbidity and mortality in the future. Diabetes
is invariably associated with derangement in secretion or
function of the endocrinal portion of the pancreas.
th
The pancreas is dual organ with dual function in

our body, as a digestive organ and as an endocrine organ. The exocrine gland produces enzymes important to
digestion. The endocrine component of the pancreas
consists of islet cells that create and release important
hormone directly into the bloodstream which act to regulate blood sugar. The human exocrine pancreatic secretions are affected in diseases which affect the pancreas. In our study, we found a significantly high amylase and or lipase activity in diabetic and prediabetic
patients in group IV. The high exocrine pancreatic enzymes may reflect the impaired exocrine-endocrine interactions of the pancreas.
There is evidence that insulin influence the enzyme
synthesis and release in the exocrine pancreas. Insulin is
a trophic hormone for the exocrine pancreas, increasing
pancreatic enzyme synthesis and cell division on acinar
tissue [10]. Our results suggest that the high serum amylase and or lipase activity in diabetes and prediabetes
with exocrine insufficiency (group IV) are associated
with an impaired insulin action due to insulin resistance
and inadequate insulin secretion, as was indicated by the
increasing level of proinsulin and proinsulin/insulin ratio.
Since diabetes is a multifactorial disorder, hypercholesterolaemia and hypertriglyceridaemia are mostly
observed. Dyslipidaemia, which characterized by raised
triglycerides, low HDL and high small dense LDL particles, is very frequently seen in diabetes and the determination of serum lipid profile in the diabetic patients is
now considered as a standard of the diabetic care [1],
[2], [3].
Pancreatitis is defined as inflammation swelling of
the pancreas. Acute pancreatitis is most commonly
caused by gallstone or excessive of alcohol use. Chronic
pancreatitis occurs over long period of time and result
when digestive enzymes destroy the pancreas and nearby tissue. To diagnose the pancreatitis, physicians will
often order blood tests to determine if the levels of pancreatic enzymes (i.e. amylase and lipase) have markedly
increased. Amylase is an enzyme made primarily in the
pancreas and released into the digestive tract to digest
starch and glycogen. Amylase levels rise at the beginning of a pancreatic attack and tapper off after 2 days.
The normal or reference range for serum amylase varies
due to patient factors and the assay used and is typically
20 300 units/L. Amylase activity can be 5 10 times
higher than normal during pancreatitis. Lipase is an enzyme made in pancreas too and release into digestive
tract to digest fats. Like amylase, reference range of
lipase is typically < 200 units/L. In acute pancreatitis,
lipase level can be 2 5 times higher than normal and
remain elevated for 4 7 days. Amylase and lipase levels often rise in parallel and are often ordered together
to diagnose acute pancreatitis, as well as monitor chronic pancreatitis [11].
In our study, we found that the lipase/amylase ratio
correlate with duration of diabetes, fasting glucose concentration, lipase activity, insulin resistance index and
insulin sensitivity index. But, the index had not significant correlation with HbA1c, lipid profile, insulin,
proinsulin and amylase activity. The serum lipase/amylase ratio had been proposed to distinguish the
etiology of pancreatitis. The serum lipase/amylase ratio

could differentiate acute episodes of pancreatitis
[12][13]. The serum lipase/amylase ratio would be able
to put an insight into possible endocrine-exocrine relationship of the pancreas in the type 2 diabetes patients.
V. CONCLUSION
Our study demonstrated that the high lipase/amylase ratio correlate with type 2 diabetes, thus
suggesting a possible exocrine-endocrine relationship in
the disease. However the cut-off value of the marker
remains to be elucidate in various other clinical condition of the pancreas, especially in Indonesian subjects.
ACKNOWLEDGMENT
Author thanks to Directorate General of Higher Education through Penelitian Unggulan Perguruan Tinggi
(Pemula) second batch for 2013 in sponsor and financial
support.
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T. Cederholm, P. Stenvinkel, B. Lindholm, U. Rise, and J.

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th
Diversity of Ammonia Oxidizers in Agricultural

Soil of Gintungan, Central Java
Rully A. Nugroho, Ph.D.,Gabriel B. Kennardi, S.Si.,andDr. Vincentia I. Meitiniarti
Faculty of Biology, Satya Wacana Christian University, Salatiga, Indonesia
AbstractDiversity of autotrophic ammonia oxidizers in
agricultural soils, which are responsible for the rate limited
step of nitrification in most soils, was investigated. DNA
samples were extracted from eight different soils collected
from Gintungan agricultural land, Central Java. DNA
samples were amplified using three different primer sets
targeting 16S rRNA genes of general bacteria, 16S rRNA
genes of ammonia-oxidizing bacteria, and amoA genes of
ammonia-oxidizing archaea, and were profiled using denaturant gradient gel electrophoresis (DGGE). Microbial
diversity was calculated using Shannon-Weiner index. The
DGGE analysis revealed that ammonia-oxidizing archaea
and ammonia-oxidizing bacteria communities were diverse
and variability in banding patterns was affected by plant
species. Our results suggest that in agricultural soil of Gintungan, archaea may be the primary ammonia oxidizers.
Keywordsagricultural
soils,
ammonia-oxidizing
archaea, ammonia-oxidizing bacteria, general bacteria,
plant species.
I. INTRODUCTION
communities play a critical role in ecosystem processes, such as nitrogen cycling, nutrient
turnover, or the production of trace gases. Soil microbial activities, populations and communities are governed by environmental variables and agricultural system, as conventional and organic system [1], [2].
In soil environments ammonia oxidizers mediate the
first, rate-limiting step of autotrophic nitrification, which
is considered to be a key control point in the nitrogen
cycle resulting in increased N mobility and loss of oxidized N forms through leaching and denitrification [3],
[4]. The ammonia oxidizers catalyze the conversion of
ammonia (NH3) to nitrite (NO2 ), which is then
converted to nitrate (NO3 ) by nitrite-oxidizing bacteria.

Ammonia-oxidizing bacteria (AOB) are affiliated with
-Proteobacteria and -Proteobacteria. In terrestrial
soil ecosystems, oxidation of ammonia is believed to be
mainly carried out by AOB within the -Proteobacteria
[5], [6]. Ammonia oxidizers are generally slow growing,
difficult to isolate and have therefore have been primarily investigated using molecular techniques based on
amplification of genes encoding either the 16S ribosomal RNA or ammonia monooxygenase (amo) [5] [9].
OILmicrobial
Recent studies have demonstrated the existence of

ammonia-oxidizing archaea (AOA) within the
Crenarchaeota in soil and marine environments using
metagenomic and cultivation approaches [10] [14].
Other studies based on the crenarchaeotal amoA gene
have revealed the numerical dominance of AOA over
AOB in natural ecosystems, such as terrestrial soil,

freshwater and marine sediments [15] [24]. However,
to date little work has been conducted to investigate the
diversity of ammonia oxidizers in Indonesian
agricultural soils.The objective of this study was to
investigate the diversity of general bacteria and
ammonia oxidizers in horticultural soils of Gintungan,
Central Java.
In Gintungan, agricultural area is planted with
flowers, fruits and vegetables. A previous study
conducted on mineral soil samples from this area has
shown that nitrification rate was low(<3g (NO3 N +
1
1
NO2 N) g d ) in these soils [25]. Environmental
factors, such as ammonia availability, organic matter
content, and/or soil pH may influence the presence of
specific types of ammonia oxidizers and nitrification
rates in soils [6], [26].
Microbial communities in the environment have traditionally been studied by conventional methods based on
cultivation of populations, by measurement of their metabolic activities or direct observation using microscopic
methods [27]. However, analysis of microbial is a
complex task that cannot be achieved by traditional
microbial culturing methods. Ward et al. [28] stated that
more than 90% of microorganisms existing in nature are
not amenable to currently available cultural methods.
Here, we examined the diversity of ammonia oxidizers
with
denaturing
gradient
gel
electrophoresis
(DGGE).DGGE has been used extensively for diversity
analysis in microbial ecology [29]. Coupled with polymerase chain reaction (PCR) and primers that target
conserved, taxonomically significant genes, it is a technique that allows the comparison and analysis of molecular fingerprints of diversity.
A. Sample collection
Soil samples from the eight sites were collected on
September 25, 2012. Table 1 describes the types of
vegetation present in each sampling plots. The location
were randomly selected and covering all the vegetation
types. The locations of sample collections were
confirmed by the use of Global Positioning System unit
(GPS). At each location, soil samples were collected
from litter layer. Samples were placed in plastic bags
and transported to the laboratory where they stored at
20 C until further processing.
B. DNA Extraction
Extraction of DNA was completed directly after
collection, using a commercial kit (PowerSoil DNA
extraction kit; MO BIO Laboratories Inc., Carlsbad, CA,
th
USA). A 0.7 g subsample from each litter soil sample

was processed for DNA extraction according to the
manufacturers instructions, except that mechanical
TABLE I
VEGETATION TYPE IN THE SAMPLING LOCATIONS SELECTED IN THE
STUDY
Plant
Capsicum annuum
Capsicum frutescens
Brassica oleracea
Allium sp.
Brassica chinensis
Solanum lycopersicum
Daucus carota
Cucumis sativus
Coordinate
Elevation
(m asl a)
71218S; 1102127E
71228S; 1102126E
71228S; 1102126E
71216S; 1102126E
71228S; 1102126E
71223S; 1102127E
71228S; 1102126E
71228S; 1102126E
1,286
1,248
1,248
1,287
1,248
1,283
1,248
1,248
disruption of cells was carried out in a mini-beadbeater1 (BioSpec). Extracted DNA samples were stored at 20
C.
C. DGGE Analysis of 16SrRNA and amoA Genes
The diversity of ammonia oxidizers community was
investigated by denaturing gradient gel electrophoresis
(PCR-DGGE) fingerprinting of ammonia oxidizers
16SrRNA and amoA genes. DGGE was performed with
a Dcode Universal Mutation Detection System (BioRad) as described previously [30]. 16S rRNA genes of
AOBwere amplified using primers CTO189f-GC and
CTO654r [31], archaeal amoA genes were amplified
using primers Arch-amoAf and Arch-amoAr-GC [32],
and 16S rRNA genes of general bacteria were amplified
using primers 357f-GC and 518r [33]. Amplification
was performed in a 25 l reaction volume containing
200 nM of each primer, 0.1 mMdNTPs, 5 g BSA, Taq
DNA polymerase (2.5 unit, Promega), the buffer conditions recommended by the manufacturer, and 1 l DNA
template. 16SrRNA genes of AOB were amplified at 95
C for 3 min; followed by 30 cycles of 95 C for 1 min,
55 C for 1 min, and 72 C for 1 min; followed by 72 C
at 10 min [34].Archaeal amoA genes were amplified at
95 C for 5 min; followed by 30 cycles of 94 C for 45 s,
53 C for 1 min, and 72 C for 1 min; followed by 72 C
at 15 min [32]. 16SrRNA genes of general bacteria were
amplified at 94 C for 4 min; followed by 35 cycles of
94 C for 1 min, 54 C for 1 min, and 72 C for 1 min;
followed by 72 C at 5 min [35].
A small portion (5 l) of each PCR product was run
on a 1% agarose gel to confirm successful amplification
of a DNA fragment of the expected length. The
remaining PCR products were subjected to DGGE on a
Dcode Universal Mutation Detection System (Bio-Rad)
with gels containing a linear formamide/urea gradient
of30% to 60%,20% to 45%,or 30% to 55% linear
gradient of denaturant for 16S rRNA genes of AOB
[36], amoA genes of AOA [23], and 16S rRNA genes of
general bacteria[33] assays, respectively. After
electrophoresis, the gel was stained with ethidium
bromide for 30 min and then visualized using a GelDoc.
D. Data Analysis
For the DGGE analysis, each band was designated as
an operational taxonomic unit (OTU) [37]. The band
richness of the samples was estimated based on the
number of bands per lane. The band diversity was

calculated using Shannon-Weiner index.All gels were
aligned using the three markers. The DNA bands that
migrated to the same position within each gel were ascribed a number. Band strengths were estimated visually; weak bands were assigned avalue of 1, intermediate
bands a value of 2, and strong bands a value of 3 [38].
Our analysis revealed that soil microorganisms in
mineral soil (0-10 cm depth) of agricultural soil of Gintunganwere in very low abundance. DNA extraction
using PowerSoil DNA extraction kit did not generate
enough amounts DNA for further molecular analysis
although a nested PCR was carried out. PCR failure was
not due to the presence of PCR inhibitors, such as humic
substances [39], since PCR products were resulted when
known DNA was added to the PCR reaction (result not
shown). For that reason, we used DNA extracted from
leaf litter for further molecular analysis.
This study found that general bacterial communities
from litter samples showed almost similar DGGE band
patterns as presented in Fig. 1.This may indicate that the
communities are almost similar or related to a degree
and share common bacterial members.DGGE also revealed high general bacteria diversity (diversity index
value was more than 2.0). Each sample produced a
complex fingerprint composed of a number of bands
(average 12 bands). Muyzer et al. [33] showed that all
the bands appearing in a DGGE profile represent differ-
Fig. 1.Image of DGGE gel for general bacteria 16S rRNA genes. M =
Marker, CF = Capsicum frutescens, BC = Brassica chinensis, DC =
Daucus carota, CS = Cucumis sativus, CA = Capsicum annuum, BO =
Brassica oleracea, AS = Allium sp., SL = Solanum lycopersicum.
ent species present in the microbial population.

However, certain bands (bands A1 A6) present in the
samples indicating that certain bacterial species may
adapted to the litter characteristics.
We also observed that the recovered DGGE
fingerprints for each population were found to be
influenced by the primer set used for the analysis. All
agricultural litters did not yield detectable PCR products
of AOBamoA genes although a nested PCR was carried
out, but PCR products were generated using CTO
primers targeting 16S rRNA of AOB. The detection of
th
non-AOB-like using CTO primers was not surprising

because these primers have a relatively low specificity
[5], [34], [40]. The PCR amplification of the AOB 16S
rRNA gene generated DGGE patterns as shown in Fig.
2with diversity index value around 2.0 3.5.Some
bands (bands B1 B7) were unique to some samples
tions which were less prevalent seemed to be equally

abundant.Further studies to sequence 16S rRNA and
amoA genes will be essential in order to reveal the presence and identity of ammonia oxidizers responsible for
nitrification in agricultural soils.
Interpretation of DGGE patterns needs to be done
cautiously as discussed in several reviews [41]. Amplified 16S rRNA fragments of different but phylogenetically related species might have the same electrophoretic mobility because they share the identical or similar
sequence in the stretch analyzed [42]. Despite several
pitfalls of PCR-DGGE based analysis, profiling of ammonia oxidizer communities by denaturing gradient gels
proved to be a powerful method allowing a cultivationindependent analysis of large number of soil and litter
samples.
IV. CONCLUSION
We can conclude that the diversity of ammonia

oxidizers in agricultural soil of Gintungan was high as
previously observed in agricultural soils. Although
DGGE could reflect the profile of ammonia oxidizer
Fig. 2.Image of DGGE gel for ammonia-oxidizing bacteria 16S rRNA communities in agricultural soils, sequencing is required
genes. For details of legends, see Fig. 1.
to reveal the presence and identity of ammonia
oxidizers.
(Fig. 2).
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th
Distribution and Accumulation of Mercury in

Kerang Bulu (Anadara sp.) at Sekotong Region, West Lombok, Nusa Tenggara Barat.
Haikal Prima Fadholi 1*), Rindra Aryandari 1), Rahadyan Aulia 1) Andhika Puspito Nugroho 1)
and Mulyati Sarto 1)
1)
Faculty of Biology, Gadjah Mada University, Yogyakarta, Indonesia
*)
Corresponding author : haikalprima@gmail.com
Abstract -- Sekotong, West Lombok, Nusa Tenggara Barat is place where people traditionally gold mining. Mercury (Hg) is used by traditional gold miners. Hg waste flow
towards and can pollute to the ocean through river. Kerang bulu (Anadara sp.) is one of the major fisheries commodity in Lombok. This organism is a filter feeder and
sessile animal, they can accumulate metals in their bodies.
This research aims to study the distribution and accumulation of Hg in Anadara sp. at the Sekotong, West Lombok,
Nusa Tenggara Barat and environmental factors that can
influence it. Research sites include Medang, Permulae and
Gili genting village. The study was conducted by collecting
same size sample with free sampling method. Environmental parameters measured include Hg levels in water and
sediment, water and air temperature , pH and salinity. The
mussel organ analyzed were gills, mantle, visceral mass and
shells. Hg level were analyzed using Mercury Analyzer.
Data were analyzed using LSD Variance Analysis and
Pearson Correlation. The results show that from the highest level of Hg is gills > mantle > visceral mass > shells. The
highest Hg levels found in gills was 0.52 0.56 mol kg-1.
From the result showed that the highest Hg level were in
Medang which has the Hg content in the water at 10.04 mg
L-1 . The content of Hg in the organs was positively correlated with Hg levels in the water.
Keywords -- Sekotong. Mercury, Bioaccumulation, Anadara sp.
I. INTRODUCTION
ndonesia has about 713 gold mining areas scattered in
Sumatra, Java, Kalimantan, Sulawesi and Nusa Tenggara Barat[1]. Sekotong, located at West Lombok regency, Nusa Tenggara Barat, it is one of a traditional
gold mining in indonesia.
Mercury (Hg) is a chemical used by traditional gold
miners to coat gold particles formed Hg-Au, after heated
by high temperature then Hg will dissolved and leave a
lump of pure gold[2]. The Hg waste then passes the
ponds and left without further treatment. Water that has
been contaminated Hg can flow out of the ponds, and
flowing toward the river and pollute the ocean.
In the aquatic environment, Hg form is organic. Hg
form in the water is Hg0 and methyl mercury ion
(CH3Hg+) both are volatile [3][4]. Hg has the ability to be
bioaccumulation and finally undergo biomagnification
in the environment when it is in organic form. Hg can be
found in the body tissues of marine biota such as clams
and others. Mercury can enter to the organism through
the gills or digestive mechanism from plankton as a

source of food [5].
Kerang Bulu (Anadara sp.) is kind of mollusk it is
class bivalves. Kerang bulu can easily found in coastal
intertidal zone. It is lived hiding in the sand and mud.
Kerang bulu is one of the major fisheries commodity,
and its become the most favored food by the people in
Sekotong. Kerang bulu (Anadara sp.) is a good bioindicator for the environment, because their characteristic as
a filter feeder and a sessile animal[6]. They feed itself by
screening food from the water, allow them to accumulate Hg from the environment, whereas a sessile animal
which mean they have slow movement allow them as an
overview what actually happened in the environment
they are lived[7].
Mercury in the water can absorbed by the mussel directly. It is through gill membranes or through food,
then transported in the blood and can be accumulated in
the mussel organs[8]. Furthermore, Hg binds to a protein
called metalothionein[9]. Metalothionein is a protein that
is highly sensitive and can cause toxic and can be used
as an indicator of pollution. This is based on the presence of metalothionein in the mussels tissue can make a
bond with Hg. If the mussel are resistant to high concentrations of Hg, then Hg can be accumulated in the tissues/organs, especially the liver, gills, kidney or muscles[10].
Bioaccumulation is also influenced by environmental
factors such as temperature, pH, salinity and substrate
conditions. Bioaccumulation can be found in large numbers, from the mussels which had lived long enough in
the substrate which containing Hg[11].
Hg accumulation also can occur in humans through
the process called biomagnification[9]. Consumed the
mussels which is contaminated Hg, bioaccumulation
process will occur in the liver and kidneys will then lead
to impaired function of organs such as neural degeneration, blindness, mental disorders, and chromosome damage[12]. This research aims to study the distribution and
accumulation of Hg in Kerang bulu (Anadara sp.) at
Sekotong, West Lombok, Nusa Tenggara Barat and environmental factors that can influence it.
II. RESEARCH METHODS
A. Research Site
Sekotong, West Lombok is located about 28.7 km
th
southwest of the Mataram city, the capital of the province Nusa Tenggara Barat. About 2.000 households
spreaded in 8 villages. Gold mining and processing area
spread over 3 villages in the sekotong it was Buwun
Mas, Kerato, and Pelangan. Kerang bulu was obtained at
3 different points in Medang, Permulae, and Gili Genting. Almost all households have gelondong unit to gather gold from stone or mud (Fig. 1) it is operated
throughout the day. And the wastes was sent into the
rivers and sewers that close to the sea. Temperature, pH
of sea water, salinity, and the content of Hg in water and
sediment is measured as environmental factors.
ences. And also showed that the Hg concentration in

different organs in the same place were significantly
different. Mercury accumulation in water and sediment
at 3 locations showed varying results. The result also
showed the correlation of Hg content in organs with Hg
content in waters, as shown in Figure. 2
Table I. Hg Accumulation in Organs of Kerang Bulu (mol kg-1)
Organs
Location
Visceral
Shells
Gills
Mantles
Mass
0,39Medang
0,13-0,37
0,52-0,56
0,36-0,46
0,57
0,220,02-0,37
0,14-0,24
0,17-0,29
Permulae
0,33
0,00020,11Gili Gent0,10-0,24
0,12-0,23
0,03
0,14
ing
Table II. Hg Accumulation in Waters and Sediments
Location
Waters (g L-1)
Sediments (mol kg-1)
Medang
Permulae
Gili Genting
10,04 10.05
0.95 0,96
0.75 0,76
0,18 0,26
0,03 0,68
0,002 0,03
Tabel III. Environmental Parameters

Parameters
Fig. 1. Gelondong unit.
B. Materials
This study need Mercury Analyzer (MA) as a main
equipment to measured the Hg levels. The chemicals
used in this study is HNO3 solution, HCl solution,
HClO4 solution for sample destruction process. Hydroxylaminehydrochloride solution, KMnO4 solution, and
SnCl2.2H2O solution is used in MA.
Location
Air Temperatures (oC)
Water Temperatures (oC)
Salinity
()
Medang
31
33.5
40
Permulae
25
29.5
39
Gili Genting
29.75
31
40
pH
6
5.1
5
6.0
9
C. Sample Preparation
The sample target is Kerang bulu which has size 3.5 5.5 cm (Fig. 2), 3 individuals from each location by free
sampling method. Kerang bulu was separated into shells,
gills, mantle and visceral mass in the laboratory. The
organs was stored in oven at 60C to obtain a constant
dry weight. Destruction sample organ by acid destruction, using hot plate. Organ and water samples was destructed by using HNO3:HCl (4:1).
D. Sample Analysis
Destructed sample then inserted into 10 mL volumetric flask and aquades was added. Then reacted with 0.1
ml KMnO4, 0.1 ml Hydroxylaminehydrochloride and
0.5 ml SnCl2.2H2O. Hg content was analyzed by using
MA.
III. RESULT
The Hg accumulation in organs of sea mussels Anadara sp. at 3 location was shown in Table I. The data of
Hg accumulation in waters and sediment was shown in
Table II, and environmental parameters was shown in
Table III.
The concentration of Hg that accumulated in organs
of Anadara sp. in 3 locations showed varying results
(Table I). ANOVA showed that the levels of Hg in the
same organ in different places showed significant differ-
Fig. 2. The correlation of Hg levels in organs with Hg levels in waters.
Correlation analysis showed that the accumulation

of Hg in water had strong positive correlation with Hg
levels in the shells (r = 0.96, p < 0.05) and mantle (r =
0.97, p < 0.05), but also had weak positive correlation to
Hg levels in the gills (r = 0.99, p > 0.05) and visceral
mass (r = 0.99, p > 0.05).
IV. DISCUSSION
In this study, the accumulation of Hg in each organ
has a varies value, it was shown that environmental
parameters in 3 locations especially the Hg
concentrations in the water and sediment were different
th
(Table II). The results show that from the highest level
of Hg is gills > mantle > visceral mass > shells.
Gills accumulated Hg from 0.10 to 0.56 mol kg-1 .
The concentration of Hg in gills was the highest.
Generally, the absorpsion of Hg in water is methyl
mercury ion form (CH3Hg+) and Hg0 and absorbed by
shellfish directly through the water that passes the gill
membranes[8]. Gills can accumulate more Hg because
Hg enter to the muscle is through the gills first. In the
gill membranes, absorbed Hg will bind to a protein
called metalothionein, then it was distributed to various
organs like mantle and visceral mass via blood vessels in
gills[9]. Mantle accumulating Hg from 0.11 to 0.57 mol
kg-1 it was the second highest. Water that enters through
the gills will be distributed into mantle cavity first,
which has located between the body and the shells, also
because of metalothionein distribution also widespread
on mantle[13]. The visceral mass accumulated Hg from
0.12 to 0.46 mol kg-1. Its lower than mantle.
Detoxification mechanisms and secretion of Hg in
visceral mass is very active. Hg which is distributed to
the visceral mass also binds to ,metalothionein and then
widespreadly distributed in the digestive gland. Hg
which absorbed in the gastro-intestinal tract will be
actively or passively diffuses and transported, then
excreted through the kidneys and intestines. Excretion
through the kidneys can can occur only when pH and the
amount of amino acids that can bind Hg are in balance
[13]
. The intestine can excrete Hg from mucus
membranes actively[13]. Whereas, shell was the lowest of
Hg accumulation in the range of 0.0002 to 0.37 mol kg1
. Shells cant select the metal which entered its hard
tissue. Although there is no metalotionein, shells still can
accumulate Hg in a few number[14].
Mercury levels in organs had positive correlation
with the Hg levels in water, but it had varied
correlatation with the Hg levels in sediment. As filter
feeder, the mussels filtered anything that present in
water. Mercury dissolve in methyl mercury (CH3Hg+)
and Hg0 in the water, the increase of Hg levels in water
also increasing Hg levels that accumulated in the body of
marine biota[15]. This is due to the accumulation of Hg
transported by metalothionein and distributed throughout
the body. Hg levels in water is different with sediments.
The sea water is so dynamic. The Hg levels in water can
fluctuate everytime[16]. Increasing content of organic
matter in a body of water and sediment also cause heavy
metal content in the sediment increased[15]. Based on
Table II., ANOVA results showed that the levels of Hg
in water at three locations were significantly different.
The highest Hg levels was found in Medang. Which has
Hg concentration in the water reached at 10.04 g L-1 or
0.1004 mg L-1. about 0.002 to 0.68 mol kg-1 on
sediment. Based on the Decree of the Minister of Health
No. 907/MENKES/SK/VII/2002 consumption water
limit is set at 0.001 mg L-1 and the maximum Hg levels
for water is 0.005 mg L-1. Hg levels in water on Medang
is above the limit of Hg levels in water. It cause the
accumulation of Hg in these area is very high. Medang
located nearest to the river that directly connect to tailing
sources that is vat to sludge processing of gold mining.
Permulae and Gili Genting, Hg concentration still below
on limit. Although on Permulae, the Hg levels in water

was very close with the limit of water consumption with
Hg levels at 0.00096 ppm. It because the location where
close to the conventional gold processing industry, so
that waste disposal activities less active than Medang.
Gili Genting had the lowest Hg levels because the gold
processing activities around the site is low, only a small
river which connects the source of waste to the sea.
The measurements of environmental parameters was
shown in Table III. Some of the parameters that may
affect the levels of Hg in sea water, besides due to increasing gold mining activity around the area, can be
caused pH and salinity become low. The results showed
pH of water was about 5.15 to 6. Methyl mercury
(CH3Hg+) can be easily formed at pH 6 or smaller, and
salinity may affect the bioavailability of Hg, methyl
mercury at high salinity will undergo demethylation
procces[17]. The temperature of the water and the air
were high. It was about 25 30oC. Increasing the water
temperature tends to increase the accumulation and toxicity of heavy metals, due to the increasing metabolic
rate of aquatic organisms[18].
V. CONCLUSION
The results show that from the highest level of Hg is
gills > mantle > visceral mass > shells. Gills accumulate
Hg in the highest level with 0.52-0.56 mol kg-1 at Medang. Medang has the highest Hg levels with 10.04 g
L-1 in water. Mercury levels in each organ is positively
correlated with Hg levels in water. Factors affecting
bioaccumulation and distribution of Hg are the human
activities near the sampling location, Hg levels in water,
Hg levels in sediment, pH, salinity, and temperature of
water and air.
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Padjadjaran.
th
The Effect of Salinity on Growth, Dry Weight

and Lipid Content of the Mixed Microalgae
Culture Isolated from Glagah
as Biodiesel Substrate
Eko Agus Suyono1*), Winarto Haryadi2), Muhammad Zusron1), Matin Nuhamunada1), Sri Rahayu1),
and Andhika Puspito Nugroho1)
1)
Faculty of Biology, University of Gadjah Mada, Indonesia
2)
Department of Chemistry, Faculty of Mathematics and Natural Sciences,
University of Gadjah Mada, Indonesia
*)Corresponding author:eko_suyono@ugm.ac.id
AbstractMicroalgae use photosynthesis to convert solar energy into chemical energy, such as lipid and they can
be a replacement for oil-based fuels. They are among the
fastest growing plants in the world, and about 50% of their
weight is oil. This lipid oil can be used to make biodiesel.
Unfortunately, there are only some of potential strains
isolated from Indonesia and most of the biodiesel productions are usually using a single strain. Then, although they
are rich of oils, their biomass productivity is still low. Salinity treatment can be used to increase their biomass as
well as their lipid content. Therefore, the research aim was
to study the effect of salinity on the growth, dry weight and
lipid content of mixed microalgae isolated from Glagah,
Yogyakarta. The mixed microalgae were cultured in
3NBBM medium with different salinities or types of water
(sea water, brackish water, and fresh water). The cultures
were incubated at light intensity 3000 lux under dark:light
exposure of 12:12 hours for 7 days. The number of cells
was counted every 24 hours with a Haemocytometer, and
the biomass was calculated based on the dry weight. The
lipid content was measured on days 0, 3, and 7 using Nile
Red (NR) staining, and then the amount of lipid was analyzed using a fluorescence microscope and measured with
CellProfiler 2.0 software. The highest dry weight and lipid
content were found in seawater medium, they accounted
for 3.42 mg/ml and 13,58% at day 7, respectively. Whereas, the highest number of cells was found in freshwater
medium, this was 9.8 x 106 cells/ml.
Keywordssalinity, growth, dry weight, lipid content
I. INTRODUCTION
HE worldwide increase of human population and
transportation has generated greater energy consumption of petroleum fossil fuels that lead energy
crisis because of depleting fossil fuel reserves [1].
Therefore, for solving the issue above, a renewable
energy asan alternative resource should be developed.
Microalgae-based biodiesel production could be a potential source for the future renewable energy [2]. Microalgae as a potential candidate for biodiesel production has generated significant interest [3][4], because the
organismis the most efficient biological producer of oil
and biomass source due to use photosynthesis to convert

solar energy and combine water with fixing CO2 into
chemical energy, such as lipid and it can be a replacement for oil-based fuel [5][6].
Currently, biodiesel productionwas dominated by
commercial single strains microalgae [3]. Most of the
commercial single cell cultivations of microalgae have
low biomass production and lipid content due to various
problems such as culture condition and susceptibility to
contaminant [7]. Those problems can be overcome
through the search and selection of the local microalgae
strains for biodiesel production which have highest
growth rate, biomass productivity, and lipid content
[8][9].Furthermore, the use of mixed culture of the selected local strainsshow that cells grow faster and high
biomass yield because the cells are capable for utilizing
organic carbon sourcesoptimally [10].
Even so, optimization technology is commonly done
by regulating environmental conditions and culture medium [11]. By adjusting the salinity was reported able to
increase biomass production, such asin Scenedesmus
almeriensis culture [12] and Scenedesmus sp. Culture
[13]. [14] also reported that Botryococcus braunii
adapted to grow in low salinity was able to increase the
biomass production, hydrocarbon, fat, carbohydrate, and
carotenoids. Microalgae have a response against increase of salinity and osmotic stress on the environment
by accumulating small molecules components for osmoregulation [6]. [11] and [15] describe that the increasing
of salinity leads to slight increase in the total lipid content of algae, but excessive salinity gives an negative
effect on growthdue to salt stress causes microalgae tend
colonies-form in the growth phase, inhibit the photosynthesis and decrease the growth rate
Therefore, it is important to study the effect of different salinities in culture medium of the mixed microalgae
culture isolated from Glagah cell on growth, dry weight,
and lipid content for biodiesel substrate.
th

A. Algal cultures
The mixed microalgae culture isolate Glagah were
obtained fromGlagah beach in the coastal area of South
Yogyakarta-Indonesia. Microalgae samples were isolated in Laboratory of Biotechnology, Faculty of Biology, UGM by using the microcapillary pipette method
and serial dilution method [16]. The isolated microalgae
was not identified yet, however, according to morphological characters, they consisted of three genus, there
were Chlorella, Scenedesmus [17], and Nannochloropsis [18].
The strains were cultivated in modified sea water medium of f/2 for selection. Then, survived strains were
cultivated in modified 3NBBM medium + vitamins
medium [19] They were grown in 500 mL glass bottles
and incubated at light intensity of 3000 lux under
dark:light exposure of 12:12 hours for 7 days at 1825oC.
B. Cultivation condition with different salinity
For all experiments, the microalgae were grown in
modified 3NBBM medium + vitamin in 500 mL glass
bottle under condition described above. The effect of
salinity was investigated at 3NBBM + vitamins diluted
in fresh water, sea water, and brackish water (mixture of
fresh water:sea water (1:1)) with three replications. The
cultures were inoculated into glass bottles of different
salinity medium with ratio 50 mL stock culture and 200
mL the medium.
C. Determination of cells growth
To compare cell growth in different salinity, cells
were counted using a light microscope and Haemocytometer Neubauer 1 mm every 24 hour. Sample was shaken to homogenize, then 900L of sample put into microtube 2 mL mixed with 100 L of alcohol 70% for
fixing. Number of cells was calculated as follows:
D. Determination of algal biomass
The biomass production was measured based on dry
weight of the culture for seven days. Sample culture (2
mL) was transferred into microtube 2 mL. Sample was
centrifuged at 3300 rpm for 10 minutes. Supernatant
was discarded, then washed using distillate water. Cell
suspensions on bottom microtube were dried in the incubator oven at 34oC to constant weight.
E. Determination of lipid content by Nile Red staining
Lipid content was measured by using Nile Red (9(Diethylamino)-5Hbenzophenoxazin-5-one) staining
(Qin, 2005). Nile red staining was conducted to detect
intracellular lipid droplets [20]. The cultured cells (1
mL) were collected into microtube and 0.01 mg/mL then
was added with Nile Redfor staining. Stained microalgaecells were observed by Fluorescent Microscope to
get the lipid fluorescence. The lipid fluorescence from
neutral lipid
B
A will be smeared Byellow-orange [21]. Then,
lipid fluorescence was quantified using image analysis
software CellProfiler 2.0 as a intracellular lipid droplet
[22].

The comparison of, cell growth, dry weight, cell
quota andlipidcontent in mixed cultures of microalgae
isolated from Glagah, Yogyakarta using various
salinities of medium in bacth cultures were investigated.
Those data are presented in Fig. 1A, Fig. 1B, Fig. 1C
and Fig. 1D.
As can be seen from Fig 1A., the microalgae were
growth better in fresh water medium as similar to their
habitat. The highest number of cells was found in
freshwater medium reached 9.8 x 106 cells/mLat day 7,
followed by brackish water and sea water treatments.
However, the highest total dry weight was found in sea
water treatment accounted for 3.42 mg/mL, followed by
brackish water and fresh water. Both sea water and
brackish water treatments reached a pick at day 5, but
fresh water treatment was at a pick at day 4. (Fig. 1B).It
can be assumed that microalgae growth in fresh water
medium were stimulated to proliferate their cells rather
than construct their cell walls. The fresh water
nutrientwas absorbed by microalgae cells did not used
much to construct of the cell wall, but to stimulate cell
division. Then, their cells became smaller and their cell
walls were relatively thinner and lighter. On the other
hand, sea water nutrient provided more and suitable
compounds for producing carbohydrate stored in the cell
walls as sellulose and hemiselluse, so that the total dry
weight was higher.
C
D
Figure 1. Growth (A), dry eight (B), cell quota (C) and lipid content
(D) of the mixed microalgae cultures isolated from Glagah were
cultured in fresh water ( ), brackish water ( ), and sea water ( ).
The trend of cellquota (dry weight per cells) of each

treatment (Fig. 1C) was different from total dry weight
(Fig. 3). The highest cell quota wasin the brackish water
treatment at day 5 with reached 3.79 ng/cell, followed
by sea water treatment and fresh water treatment, they
accounted for 1.52 ng/cell and 0.17 ng/cell, respectively
(Fig. 1C). Therefore, the total dry weight in sea water
was the highest, but the dry weight per cells in brackish
water
C was the highest. This was happen because the cell
C very slow (Fig.
division in brackish water was growth
1A).
th
Lipid contents of the microalgae in all treatments tend

to increase during their growth. The highest lipid
content of the microalgae was found in seawater
treatment at day 7 accounted for 13.58%, followed by
brackish water treatment (5.86%), and the lowest was
found in fresh water treatment (0.88%) (Fig. 1D).
Increasing of lipid content was caused by the
accumulation of lipid under stress conditions. These
results were similar to the research done by Takagi et al.
(2006) in Dunaliella cells. This was also confirmed by
[23] that the increase of salinity could accelerate the
total lipid content in microalgae cells.Under high
salinity condition, the microalgae were stimulated to
enhance their lipid production as osmoprotectant which
necessary to protect them from salt stress [24].High salinity water might effect on intracellular osmolarity due
to hypertonic medium condition over the microalgae
cells. Then, it caused release of water inside of the microalgae cells into the environment. The mechanisms for
encountering salinity stress were regulated by producing
lipid in large quantities to prevent the escape of water
from microalgae cells. So, the high amounts of lipid lead
to enhance the microalgae dry weight. Therefore, the
high salinity treatment could be used to as method to
increase the biomass productivity and stimulate lipid
production of microalgae for biodiesel substrates.
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
IV. CONCLUSION
Different salinity have potential for increase the number of cells, dry weight and lipid content on the mixed
microalgae cultures isolates from Glagah. The highest
dry weight and lipid content were found in seawater
medium, they accounted for 3.42 mg/mL and 13,58% at
day 7, respectively. Whereas, the highest number of
cells was found in freshwater medium, this was 9.8 x
106 cells/mL.
ACKNOWLEDGMENT
All authors would like to sincerely acknowledge to
Directorate General of Higher Education, Ministry of
Education and Culture, Indonesia for funding this research.
[16]
[17]
[18]
[19]
[20]
[21]
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th
Soil Mesofauna Diversity in Two Different Ages

of Cocoa (Theobroma cacao L.) Plantation
1)
Ida Kinasih 1*), Tri Cahyanto 1), Ina Andriana 1) and Ramadhani Eka Putra 2)
Department of Biology, Faculty of Science and Technology, UIN Sunan Gunung Djati Bandung,
Indonesia
2)
School of Life Sciences and Technology ITB, Indonesia
*)
Corresponding author: idakinasih@uinsgd.ac.id
AbstractIt is well established knowledge that soil animals and their interactions with microorganisms play a
primary role in the mineralization of nutrients, and hence
nutrient acquisition and the growth of plants. Soil microarthropods have a strong influence on vital ecosystem
processes, such as the decomposition of organic matter and
nutrient mineralization. The mesofauna (microarthropods
and enchytraeids) also affect soil structural processes
through their production of faecal pellets and biopores.
This has strong effect on structural stability and waterholding capacity of the soil. Human activities, such as agriculture activities, influenced abundance and diversity of
soil mesofauna due to changes on structure and composition of vegetations. This study was carried out at cocoa
plantation with different planted periods, 34-year-old and
24-year-olds. Method used for sampling was pitfall trap
with 12 sampling time during 3 months sampling periods.
In total, 22 species collected during sampling, which were
Lasius fuliginosus, Cardiocondyla sp, Netelia sp, Forficula
auricularia, Telostylinus sp, Megaselia scalaris, Drosophila
sp, Monoclona sp, Diptera larvae, Onthophagus sp, Lebia
moesta, Haptoncus luteolus, Quedius sp, Phylus coryli, Mycalesis sp, Melanoplus sp, Orocharis sp, Euryopis sp, Linyphiidae sp, Labidostomma sp, Ceratophysella pratorum,
Lumbricus terestris. This study showed higher number of
individuals collected at older plantation, 858 individuals,
than younger one, 441 individuals. On the other hand, diversity of soil fauna at older plantation were slightly higher
than younger one, 1.49 and 1.44 respectively. Based on this
result, it can be concluded that time of land use changes
caused more effect of abundance than diversity for soil
fauna community.
Keywords soil fauna, diversity, cocoa plantation, abundance, land use change.
I. INTRODUCTION
NDONESIAN cocoa plantations had rapid development since the early 1980s. In 2002 the area of Indonesian cocoa plantation recorded of 914,051 hectares,
of which most (87.4%) run by the people and the rest
6.0% of the country's farming and 6.7% of large private
farming. Indonesia is currently listed as the third largest
cocoa producer in the world with a total area of
1,563,423 hectares and a production of 795,581 tones
[1]. Cocoa plantations in PTP Nusantara VIII Cikumpay, Raamandala, West Bandung has large land area
594.70 hectares, with land-clearing system that is not
concurrent. Opening the earliest land is around in
1977 and most recently in 1991. Based on opening land,
it could be said that the age of the cocoa crop is not uniform.
The macrofauna are the most conspicuous fauna of

soil, and also the most documented in term of their biology and impact on soil fertility. Inside soil, those fauna
create various highly organized, both structural and
function, microcommunities. These communities highly
affected by the changes in soil environment both by natural [2] and human activities [2], [3]. This condition
made soil fauna as a good environmental indicator [4],
[5]. Many soil arthropods, including Collembola, Oribatida, live a sedentary life in soil in close relationship
with the external conditions of their ecological niches.
As a consequence, the structure of the microarthropod
community closely reflects the environmental factors
affecting the soil, including the impact of human activities, and could be considered a bioindicator of soil
health [6]. The macrofauna have the ability to create
their own spaces, through their burrowing activities, and
can have large influences on gross soil structure [7].
Within a heterogeneous land-use system, like cocoa
plantation in Cikumpay, Rajamandala, West Java, the
different plant species and soil management may lead to
different living condition to litter and soil fauna. Agroforestry systems are generally considered to have a positive effect on the conservation of biodiversity by minimum tillage, quantity, and quality of litter, diversification, and especially incorporation of tress (of several
species), shade, deep and perennial root systems that
create a more suitable environment for soil faunal communities [8], [9]. Soil management practice can have
dramatic effects upon soil invertebrate communities
[10], [11] and may lead to indicator change in soil functioning.
This study determined the abundance and diversity of
soil meso and macrofauna in two different planted periods cacao agroecosystem in PTP Nusantara VIII Cikumpay, Rajamandala, West Bandung.
The study was conducted on the PTP Nusantara VIII
Cikumpay, Rajamandala, located West Bandung, Indonesia (594,70 ha, 220-375 m altitude). The average values of annual temperature was 27.5C with range from
23 to 32C and annual rainfall 2453.05 mm. The topography of this area has partially flat, sloping, and hilly
with steep slopes. The soil had varied types, namely
Latosol, Regosol, Grumosol, Podsolic and Alluvial with
acidity ranged from 3.9-7. Samples collected during
December 2011, January 2012, and February 2012.
Soil fauna samples were taken from two cocoa planta-
th
tions with different planted periods, older plantation

(34-years-old) and younger plantation (24-year-olds).
There were 12 species of weeds in younger plantation,
meanwhile only 9 species of weeds in older plantation
(Table 1). Litter in older plantation was more numerous
than younger plantation. Furthermore, older plantation
was more shaded than younger plantation.
To collect ground invertebrates, each site was divided
into two plots. Six pitfall traps (plastic containers of 8
cm in diameter and 11 cm in height) were set in 5 m
distance between the traps in each plot. The traps were
their density and richness. Furthermore, those taxa were

identified into species level. Invertebrates was divided
into different functional groups: herbivores, microbial
grazers, predators, saprophagous, social, and other insect (that represent more than one feeding habit) [12].
The diversity was measured with the Shannon index.
Redundancy analysis (RDA) using CANOCO version
4.5 [13] was carried out to analyze the variation in the
faunal communities data with respect to environmental
variables.
III. RESULTS
21 species, an unidentified taxon and 1299 invertebrates were collected across two different planted periods in this study. Insecta (Hymenoptera, Diptera, Coleoptera, Lepidoptera, Hemiptera, Orthoptera) were
dominated in all sites, followed by Collembola (Poduromorpha), Dermaptera, Arachnida, Actinenida and
TABLE 2
COMPARISON OF INVERTEBRATE FAUNAL ABUNDANCE AND RICHNESS
BETWEEN TWO DIFFERENT PLANTED PERIODS
Fig. 1. Comparison of functional group of soil fauna between two

different planted periods
TABLE I
COMPARISON OF INVERTEBRATE FAUNAL ABUNDANCE AND RICHNESS
BETWEEN TWO DIFFERENT PLANTED PERIODS
+: present
-: absent
partially filled with 10% detergent and left for 48 hours.

The lids of the containers were set above the traps to
prevent the traps from flooding.
Before the sampling, the environmental variables (soil
temperature, soil moisture and soil acidity) were recorded at each point in each sampling. The average of the
values were used.
Fauna was collected in 70% alcohol and counted under a binocular microscope. The invertebrates were the
classified into higher taxonomic levels and counted for
Lumbricina (Table 2). The total number of species was

slightly higher in the site with older plantation than
younger one. Two times more invertebrate specimens
were collected in the older plantation (858) compared
with younger plantation (441). On other hand, diversity
of invertebrates at older plantation were slightly higher
than younger one, 1.49 and 1.44 respectively.
Lasius fuliginosus was dominated the invertebrate
sample both in young and old plantation (Table 2), followed by Ceratophysella pratorum, and Cardiocondyla
sp. Some species responded differently to different
planted periods sites i.e Megaselia scalaris, Forficula
auricularia, and Labidostomma sp, showed high preference to site with older plantation.
The fauna collected allocated to six functional groups.
Formicidae dominated the social insect group both in
younger plantation and older plantation and represented
48.3% and 52.6% of animal recovered in this study.
Poduromorpha (Collembola) was the only taxa which
occured as microbial grazers and represented 22.4% of
total fauna both in younger plantation and older plantation (Tables 1, Fig. 1.). The rest were listed as follows in
th
the order of abundance: herbivore (14.7% and 12%),

predator (7.9% and 4.4%), other insect (5.9% and
4.8%), and saprophagous (0.7% and 3.8%).
Generally, RDA ordination revealed a clear separation of faunal communities, except at one site in older
plantation (O9) (Fig. 2.) The first and second axes explained 17.7% and 26.6 of variation, respectively. The
species-environment correlation coefficients for the first
and second RDA axes were 0.68 and 0.83, respectively,
suggesting a strong relation between species and environment factors for the second RDA axis. The first axis
seems to represent the soil acidity (pH), and the second
axis is closely related to the soil humidity (RH) and soil
temperature (T).
IV. DISCUSSION
As a whole, soil fauna living in above ground were affected by the management practices, planted period and
characteristic of that area. Planted period can affected
density and richness of total fauna, social insects, predators, saprophagous, herbivores, and other insects. There
is not an apparent the differences for diversity of total
fauna between younger plantation and older plantation.
This probably because those sites have similar structures
and maintain steady conditions. However, RDA ordination showed clearly separated between younger plantation and older plantation.
Furthermore, the thickness of litter in older plantation
could be explain of the highest density of saprophagous
in older plantation. The number of weeds species in
agroforestry provide a diversity of microhabitas, contribute to a larger density and soil biological diversity [8],
[14] and the spatial heterogeneity of the litter layer in
mixed tree plantation lead to small scale differences in
the composition of faunal communities [15]. This litter
on the soil surface serves as a source of energy and nu-
Fig. 2. RDA ordination, showing the distribution of invertebrates in

younger plantation site (Y, open triangle) and older plantation site (O,
closed triangle). RH: soil humidity; T: soil temperature; pH: soil acidity
trients (large amount of labile C and N) that improves

faunal habitat and protects them from predators [16].
Cacao agroforestry systems provided a useful refuge
for microbial grazers (Collembola) and social insect
(majority Formicidae) [17]. Formicidae was the most
abundance individu both in younger and older plantation. Shaded cacao systems protect a large number of ant
species from several different components of the ant
community by forest-like structure of this agroecosystems [18].
Poduromorpha (Collembola) are among the second
abundant arthropod groups both in young and older

plantation, furthermore older plantation had more higher
abundant of Poduromorpha. This finding suggest that
litter thickness could affect the population of this Collembola. Collembola playing an important role in microfragmentation of litter and fungi colonie and the major
component of soil ecosystems. It is known that most of
them live in the litter and feed on fungi or decaying materials [7].
V. CONCLUSION
Soil fauna in cacao agroforestry were affected by the
management practices, plantation age and characteristic
of that area. It shown by the diversity of soil fauna at
older plantation were slightly higher than younger plantation, 1.49 and 1.44, respectively. Social insects (Formicidae) and microbial grazers had the highest abundance both in younger and older plantation.
REFERENCES
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Saragih, R. (2013, May). Indonesia's Cacao: Optimistic number

one
in
the
world
[Online].
Available:
http://ditjenbun.deptan.go.id/bbpptpmedan/berita-179-kakaoindonesia--optimis-nomor-satu-didunia.html. In Indonesian.
[2] Dindal, Daniel L. Soil Biology Guide. John Wiley & Sons, Inc.
1990.
[3] Eggleton, P., A.J. Vanbergen. D. T. Jones, M.C. Lambert, C.
Rockett, P.M. Hammond, J. Beccaloni, D. Marriot, E. Ross, and
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land-use intensification gradient: influences of habitat quality,
heterogeneity and area. Journal of Applied Ecology. 2005.
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[5] Frouz, J. Use of soil dwelling Diptera (Insecta, Diptera) as bioindicators : a review of ecological requirements and response to
disturbance. Agric. Ecosys. Environ. 1999. 74, 167-186.
[6] Knoeppa, J.D., Coleman, D.C., Crossley Jr., D.A., and Clark,
J.S. Biological indices of soil quality: an ecosystem case study
of their use. For. Ecol. Manage. 2000. 138. 357368.
[7] Coleman, David C., Crossley Jr., D.A., Hendrix, Paul F. Fundamentals of soil ecology. Elsevier Academic Press, USA.
2004.
[8] Brown GG, Rombke J, Hofer H, Verhaagh M, Sautter KD,
Santana DLQ. Biodiversity and function of soil animals in
Brazilian agroforestry systems. In: Sistemas Agroflorestais:
Bases Cientficas para o desenvolvimento sustentavel. UENF,
Campos dos Goytacazes, Gama-Rodrigues AC, Barros NF,
Gama-Rodrigues EF, Freitas MSM, Viana AP, Jasmin JM,
Marciano CR, Carneiro JGA, Eds., 2006. pp 217242.
[9] Lopez-Hernandez D, Araujo Y, Lopez A, Hernandez-
Valencial I, Hernandez C. Changes in soil properties and

earthworm populations induced by long-term organic
fertilization of the sandy soil in the Venezuelan Amazonian.
Soil Sci. 2004. 169:188194.
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Fragoso C, Lavelle P. Soil macrofaunal communities in
permanent pastures derived from tropical forest or savanna.
Agric Ecosyst Environ. 2004. 103:301312.
[11] Rossi, J. P and Blanchart, E. Seasonal and land-use induced
variations of soil macrofauna composition in the Western Ghats,
shouthern India. Soil Biology & Biochemistry. 2005. 37. 10931104.
[12] Moco MKS, Gama-Rodrigues EF, Gama-Rodrigues AC,

Correia MEF. Caracterizacao da fauna edafica em
diferentes coberturas vegetais na regiao norte fluminense.
Rev Bras Cienc Solo .2005. 29:555564.
[13] ter Braak, C.J.F. & Smilauer, P. CANOCO Reference Manual
and CanoDraw for Windows Users Guide. Software for
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Computer Power, Ithaca, New York. 2002.
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Martins Metal. Effects of plant diversity on plant biomass
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Pedobiologia (Jena). 2008. 51:397407.
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Publishing, Wallingford, 2003. pp 303324.
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Lavelle P. Development of the soil macrofauna community
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[17] Moco, MKS., Gama-Rodrigues, E.F., Gama-Rodrigues, AC.,

Nachado, RCR. Soil and litter fauna of cacao agroforestry systems in Bahia, Brazil. Agroforest Syst. 2009. 76:127-138.
[18] Delabie JHC, Jahyny B, Nascimento IC, Mariano SF, Lacau
S, Campiolo S et al. Contribution of cocoa plantations to the

conservation of native ants (Insecta: Hymenoptera:
Formicidae) with a special emphasis on the Atlantic forest
fauna of southern Bahia, Brazil. Biodivers Conserv. 2007.
16:23592384.
th
Study on Bioconvertion of Cassava Tuber Skin

and Dry Rice Stalk By Black Soldier Flies
(Hermetia illucens)
Ateng Supriatna, Ramadhani Eka Putra, Robert Manurung and Rizkita R. Esyanthi
School of Life Sciences and Technology, Institut Teknologi Bandung, Bandung Indonesia
*)
Corresponding author : ateng.supriatna@yahoo.co.id
Abstract Recent report showed increasing production

of rice and cassava due to demand for staple food and raw
material of bioethanol. This condition increases post harvest waste in which 100 and 20.8 million tons originated
from rice production and cassava production, respectively.
Commonly, farmer burn this waste or use it as compost
and ruminary fed which provide small amount of profit.
Recently, another approach for post harvest waste management by bioconversion which believed could provide
better profit to farmer has been proposed. Bioconversion
defined as conversion of biomass of macromolecule into
smaller molecule by biological processes. In this study,
larvae of black soldier flies (Hermetia illucens) were used
as bioconversion agent due to their simple biological system
and short life cycle. Previous studies also confirmed their
efficiency as bioconversion agents of various organic
wastes but agricultural wastes.
Conversion rate of waste for dried rice stalk and cassava
tuber skin was 25% and 45% after 15 days (black soldier
flies life cycle), respectively. Residue produced from bioconversion process maintained, on average, more than 50%
of chemical composition of wastes with elevated water content. It can be concluded that H. illucens showed high potency as bioconversion agent of organic wastes with high
lignin and cellulose content.
Keywords agriculture wastes, bioconversion, black
soldier flies, cassava, dried rice stalk
I. INTRODUCTION
is one of human activities that produced significant amount of waste and increasing
significantly due to increasing demand on agricultural products. In Indonesia, two agricultural products
showed increasing pattern on production are rice and
cassava. Production of dry rice stalk, as post harvest
waste of rice, estimated 100 million ton [1] while cassava tuber skin, as waste of cassava, estimated 20.8 million ton [2], annually. All of these wastes have great
potency as raw material of organic based product due to
high content of cellulose, hemicellulose, lignin [3]-[6],
small amount of protein [7][8], and significant amount
of carbon [9]-[10].
Commonly treatment of dry rice stalk is pilling and
burning while application as livestock feed not recommended due to low energy and protein content also low
digestibility [11](Sitorus, 2002). On the other hand, casGRICULTURE
sava tuber skin usually applied as livestock feed [10].

Another common practice for organic wastes is composting which applicable for large amount of waste.
Generally common practice of composting is application
of bacteria and earthworm as composting agent [12] in
which final product of this process is limited to fertilizer.
Recent developments showed development of composting into bioconversion. Bioconversion is a process of
conversion of large molecules into smaller molecules by
living organism. One of bioconversion agent is insect
larvae such as Black Soldier Fly (BSF) (Hermetia illucens). Study on bioconversion by BSF showed the ability of BSF to convert organic wastes into protein and
fatty acid in form of body mass [13]-[16] that could be
used as replacement fishmeal which value of USD 1883
per metric tonne in February 2013 [17]. Even though
BSF known for their ability as bioconverter, their application for converting agriculture with high cellulose
content hardly known. Based on this information, purpose of this study is study the ability of BSF to converting dry rice stalk and cassava tuber skin.
II. METHODS
A. Hermetia illucens egg collecting
Eggs of H. illucens were collected by ovitraps filled with
bait made of combination of shrewd coconut and manure
(1:1). Ovitraps were kept in various places in Bandung.
Collected eggs were reared at Laboratory of Toxicology,
School of Life Sciences and Technology, Institut Teknologi Bandung at room temperature (24-28oC), humidity
70-80%, and photoperiod 12:12.
B. Dry rice stalk and cassava tuber skin
Dried rice stalk abd cassava tuber skin originated from
traditional farming of Soreang, Bandung Timur. Prior
application, all material was crushed into powder by
commercial food processor.
C. Research methods
Ten third instar larvae were kept inside plastic container filled with 30 g dry rice stalk and 10 g cassava
tuber skin. As control group, dry rice stalk and cassava
tuber skin was kept in similar container at same room
without any larvae. Each treatment was replicated 20
th
times and carried out for 15 days. Mortality of larvae

and conversion rate of each material by BSF were measured every 3 days. Conversion rate was defined based on
formulae
Conversion rate =
sava tuber skin by BSF. Along with reduction of fiber,

we also recorded reduction on other material (Table 1).
TABLE 1
COMPOSITION OF RESEARCH MATERIAL AFTER
CONVERSION
Composition
D. Data Analysis
Mean difference of conversion rate between treatment
and control group was analyzed by Students T-test. All
statistical analysis was carried out with Statsoft Statistica ver. 8.
III. RESULTS
A. Conversion rate of dry rice stalk by BSF
Conversion dry rice stalk by BSF showed in Figure 1.
This result showed that in average biomass of dry rice
stalk was reducing higher when treated with BSF. By the
end of experiment, biomass of dry rice stalk was reduced by 25% when treated with BSF while only 21%
reduction when BST was omitted.
Dry Rice Stalk

Before
After
Cassava Tuber Skin

Before
After
Fiber (%)
19,76
17.84
17.95
7.98
Protein (%)
Water content (%)
Ash (%)
5,35
2.96
6.87
4.05
9,61
39.80
10.63
23.84
26,69
20.69
4.68
7.72
37,89
22.82
47.40
39.77
0,86
0.67
1.05
0.72
44,18
34.05
45
55.23
0,18
0.19
0.18
0.22
2.50
2.11
0.95
2.90
Carbon (%)
Nitrogen
(%)
C/N ratio
Phosphor
(%)
Potassium
(%)
D. Composition of BSF at the end of study

Analysis on content of protein and lipid of BSF at the
end of study showed that on BSF reared on cassava tuber skin produced higher content of protein and lipid
(Table 2).
TABLE 2
PROTEIN AND LIPID CONTENT OF BSF AFTER
TREATMENT
Fig. 1. Conversion rate of dry rice stalk for 15 days by
BSF (closed bar) and without BSF (open bar). (*) indicated significant mean differences.
B. Conversion rate of dry rice stalk by BSF

Conversion cassava tuber skin by BSF showed in
Figure 2. This result showed that in average biomass of
cassava tuber reduced less when treated with BSF. By
the end of experiment, biomass of dry rice stalk was
reduced by 45% when treated with BSF while it reached
61% reduction when BST was omitted.
Fig. 2. Conversion rate of cassava tuber skin for 15

days by BSF (closed bar) and without BSF (open bar).
(*) indicated significant mean differences.
C. Composition of organic waste material after conversion by BSF

Analysis on research material showed 1.92% reduction of fiber of dry rice stalk and 9.97% of fiber of cas-
Composition
Percentage
Dry rice
Cassava
stalk
tuber skin
Protein (%)
11.66
13.14
Lipid (%)
2.03
7.25
IV. DISCUSSION
A. Conversion rate of dry rice stalk by BSF
Reduction of dry rice stalk biomass by BSF probably
due to activity of some digestive enzyme inside salivary
gland and digestive tract such as leucine arylamidase, galactosidase, -galactosidase, -mannosidase, and fucosidase. -galactosidase [18]. However, conversion
rate is relatively low which is probably caused by high
content of lignin inside stalk structure. Further study is
conducting to improve the conversion rate by application of addition decomposing agents.
B. Conversion rate of dry rice stalk by BSF
Result showed that BSF relatively less efficient in reducing biomass of cassava tuber skin. We believed this
condition caused by fermentation of cassava tuber skin.
Unlike rice starch, this material contain less lignin and
much easier to undergo fermentation.
th
C. Composition of organic waste material after conversion by BSF

Reduction of fiber and other material showed the ability of BSF to digest both dry rice stalk and cassava tuber
skin. This ability may possible due to availability of
some microbe on their digestive system. Previous study
had found Bacillus substilis inside digestive system of
BSF [19]. This bacteria produced protease [20], lipase
[21][22], and celulase [23]-[26].
D. Composition of BSF at the end of study
Compare with study by Rachmawati et al. (2010),
protein content of BSF fed with dry rice stalk and cassava tuber skin lower than BSF fed with fermented starch
and house flies artificial feed (14.6% and 15.3%, respectively). However lipid content of BSF fed on cassava tuber skin much higher than fermented starch and
house flies artificial feed (0.03% and 3.8%, respectively). We suspect this related with original content of protein and lipid of feed and fermentation process.
V. CONCLUSION
This study is preliminary study on bioconversion of
agricultural wastes by BSF. The result showed the ability of BSF to digest to material contains high cellulose
and lignin also the potency as alternative protein and
lipid source. Further study is conducting to improving
the process of bioconversion.
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
ACKNOWLEDGMENT
We thanks Rizal Jamjam and Suyitno for his valuable
help in collecting samples and maintain insect collection.
[19]
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Pollination Agents of Coffee at Small Urban

Plantation in Sumedang, West Java, Indonesia
Ramadhani Eka Putra1), Agus Dana Permana1), NdenRissa Hadikusumah1), and Ida Kinasih2)
1)
School of Life Sciences and Technology, InstitutTeknologi Bandung, Indonesia
2)
Department of Biology, Universitas Islam Negeri Bandung
AbstractCoffee (Coffeaarabica and Coffeacanephora)
are considered as important cash crop major plant for export in Indonesia. The price of product basically depends
on quality of fruit produced. One of key process that highly
influenced quality of fruit is pollination. Research showed
that high yield usually was obtained from plantation close
by forest due to benefit of pollinating insects inhabited the
forest. However, due to land use change for housing, many
plantation areas in West Java changed from classical plantation into urban plantation. We examined the possible
insects that provide pollination service to coffee planted at
urban area. This study focused on the diversity of flower
visiting insects, behavior (flower handling time and foraging rate) and pollination efficiency (determined by fruit
set) of each species. In total, 11 insects species visiting coffee flowers dominated by Hymenoptera in term of numbers
and diversity. Local honey bee, Apiscerana, exhibited longest flower handling time (4.38seconds/flower) while Amegillasp. foraged more flowers each visit that other species
(26.57 flowers/visit). Foraging rate and time of insects
highly correlated with average temperature, humidity, and
light intensity. Insect visitation significantly increased fruit
set (ANOVA P<0.05) and among all visiting insects, Amegilla sp. has highest pollination efficiency, with 80.83%.
During this study we also found the importance of wild and
garden flowers to maintain insect when coffee flowers were
not available.
Keywordscoffee, fruit set, pollination, urban plantation
I. INTRODUCTION
is human activities designed to fulfill
energy demand of human. In classical farming system, agriculture is a complex landscape mosaics
consisted of noncrop and crop habitat [1]-[3]. Increasing
human population led to development of more intensive
agricultural system that increasing isolation from natural
habitats. This condition negatively affect community
structure [4][5]and important ecological services provided by it [6][7]. Ecological service is defined as as a
wide range of conditions and processes within natural
ecosystems, and the species that are partof them that
help to sustain and fulfil human life [8] for example pollination [8]-[10].
Indonesia also experienced similar problem. Increasing population made most of agriculture area became
urban agriculture as most of surrounding area transformed into human dwelling. During this study, we examined the effect of this changing to pollination service
provided by nature. As subject of study we used coffee
as this plant considered as important cash crop and depend predominantly on wind for pollination [11] [12],
GRICULTURE
and insect pollination has been assumedto make only a

small contribution to total pollen transfer [13].
II. METHODS
A. Research Area
Research was conducted at coffee plantation of SekolahPertanian Pembangunan (SPP-SPMA) BT andUniversitasWinayaMukti (UNWIM). Both plantation located between 800-900 m above sea level and surrounded by housing of DesaGunungmanik, KecamatanTanjungsari, SumedangJawa Barat. The plantation itself
is mixed plantation whereas highland coffee (C. arabica) and lowland coffee (C. canephora) planted among
cocoa, durian, guava, and longan trees.
B. Pollination experiments
We carried out three pollination experiments on open
and bagged mature flower buds to examine the benefit
of local insects population for fruit set of coffee. We
selected three different branches on each five different
coffee shrubs and replicated this in three plantations
resulting in altogether 45 branches. During this study we
did not separate between c. arabicaand C. canephora.
The three pollination experiments were as follows: (1)
open pollination, in which flowers had access to insect
and wind pollination; (2) wind pollination, in which
insect pollination excluded by coarse mesh gauze; (3)
self-pollination (within plant pollination), hand pollination using pollen of same flowers of same plant. In
hand-pollination experiments, pollen was transferred to
stigmas with fine brush then pollinated flowers bagged
with bag made of coarse mesh gauze. Seven weeks after
the end of the flowering period, bags were removed and
total numbers of green fruit per branch were counted.
C. Flower-visiting insects
Abundance and species richness of flower visitor
were observed during flowering period. Observations
were carried out on sunny days between 06.30 to 17.00.
for 30 min. After each 30-min observation period, insects were caught by sweep netting for 5 min.
During this observation, flower handling time, foraging rate, and pollination efficiency also measured for
each insect species along with environmental factors
such as average temperature, humidity, and light intensity. Pollination efficiency was measured based on method
developed by Olsen (1997).
D. Data Analysis
Differences among fruit set produced by different pol-
th
lination procedure were analyzed using ANOVA with

Tukey as post hoc test. Foraging rates, flower handling
time, and pollination efficiency analyzed using Student
T-test.
Pearson correlation test was used to find correlation
between environmental factors and abundance of flower
visiting insects also between flower handling time and
pollination efficiency. All statistical analysis was carried
out with IBM SPSS ver. 19.
high preference to forage during midday which explained peak activity during midday (Putra, unpublished
data).
III. RESULTS
A. Fruit set of different pollination systems
Fruit set of coffee was 79.02% in open pollination
and 66.53% in wind pollination. Fruit set of open pollination was significantly higher than self pollination (Table 1).
TABLE 1
FRUIT SET AFTER DIFFERENT POLLINATION
TREATMENT
No.
Experiment
% Fruit set
Fig. 1.Daily changes of total number of individual and species visiting

coffee flowers at study site
TABLE 2
CORRELATION BETWEEN FORAGING
ACTIVITY AND SELECTED
ENVIRONMENTAL FACTORS
Open pollination
79.02 a
Temperature
Humidity
Light Intensity
Wind pollination
66.53ab
Self pollination
56.49b
(C)
- 0.842
(%rH)
0.843
(lux)
- 0.622
Different letters show significant differences

between experiments
B. Flower visiting insects

We found 11 species (4 orders) and 138 individual of
insects visiting coffee flowers within 30 hours (1800
minutes) observation. Flower mostly visited higher by
Hymenoptera than Diptera, Lepidoptera, and Coleoptera
(Fig. 1).
On average, Hymenoptera frequently visited coffee

flowers than other orders. Among all identified insects,
Amegillasp. (Hymenoptera) had highestforaging rate,
followed by Euremasp. (Lepidoptera), Xylocopasp.
(Hymenoptera), and Halictidae sp. (Hymenoptera) (Table 3).
TABLE 3
FORAGING RATE OF FLOWER VISITING INSECTS
COLLECTED AT STUDY AREA
Spesies
Fig. 1. Proportion of insect visiting coffee flowers at

study site
C. Foraging activity
Daily foraging activity of flower visiting insects determined by total number of individuals. Observation
showed three peak periods of activities in early morning,
midday, and afternoon for abundance. On the other
hand, there was no clear peak for number of species
visited coffee flowers.
There was negative correlation between temperature
and light intensity to foraging activity while humidity
showed positive correlation (Table 2). The result indicated the preference of most insects to forage during low
temperature and high humidity period while they preferred to shade area than open area which explained two
peak period foraging activities at Fig.2. On the other
hand, some species (mostly order) Lepidoptera showed
Xylocopasp
Apiscerana
Amegillasp
Halictidaesp
Rygchiumsp
Mycalesissp
Eurema sp.
Hesperidaesp
Graphiumdoson
Scatophagidaesp
Cetonidaesp
Order
Foraging Rate
(Flowers.min-1)
Hymenoptera
Hymenoptera
Hymenoptera
Hymenoptera
Hymenoptera
Lepidoptera
Lepidoptera
Lepidoptera
Lepidoptera
Diptera
Coleoptera
15
47
31
3
12
8
4
1
1
15
1
20.45
13.69
26.57
20.00
16.36
3.12
21.82
6.00
10.00
4.79
12.00
D. Flower handling time and pollination efficiency

Amegilla sp. was the most efficient insect in term of
coffee pollination at our study area even though had
short flower handling time (Table 4). Further analysis
did not show significant correlation between pollination
efficiencytoflower handling time(R2 = -0.1220, p >
0.05) and foraging rate (R2 = 0.2570, p > 0.05).
th
IV. DISCUSSION
A. Fruit set of different pollination systems
This study also showed that coffee could maintain
their fruit set even without pollination service from insects. However, cross pollination accommodated by
insect improve fruit set about 12% and 22%, of wind
and self pollination respectively(Tabel 1). Result agrees
with previous studies on improvement of fruit set due to
insect pollination [10], [15]-[17]. Fruit set recorded during this study similar to study conducted on large agroforestry coffe fields by Klein [15] which concluded that
smaller plantation may produced high fruit set as long
healthy pollination service is available.
TABLE 4
FLOWER HANDLING TIME AND POLLINATION EFFICIENCY
OF FLOWER VISITING INSECTS COLLECTED AT STUDY
AREA
Spesies
Flower Handling Time (s)
Xylocopasp
Apiscerana
Amegillasp
Halictidaesp
Rygchiumsp
Mycalesissp
Eurema sp.
Hesperidaesp
Graphiumdoson
Scatophagidaesp
Cetonidaesp
15
47
31
3
12
8
4
1
1
15
1
2.93
4.38
2.26
3.00
3.67
19.25
2.75
10.00
6.00
12.53
5.00
Pollination Efficiency
(%)
77.10
80.30
80.83
0
60.79
27.78
0
0
0
62.50
0
In this study we also found that wild bees were more

efficient pollinator than tropical honey bees (Apiscerana) which considered as best pollinator by many farmers. This could be explained by (1) honeybees spent
more time in branches with dense flowers which increase the possibility of visiting more flowers in same
plant [32]. In advance this condition could increase the
possibility of self pollination in which reduce fruit set
[15], (2) honeybees prefer to collect nectar and contact
stigma less frequent [33] compare with solitary wild
bees that more efficient in depositing pollen on stigmas
[34].
V. CONCLUSION
Wild flower visiting insects could improve productionof urban coffee plantation through improvement of
fruit set as result of perfect pollination.These wild insects had difference in activity and pollination efficiency, in which diversity of these pollination agents may
crucial to maintain production rate of urban coffee plantation.On the other hand, isolation of urban plantation
from forest threatened continuous supply of pollinating
insects to plantation. Thus it is necessary to maintain
local population of wild bees or application of domesticated pollinationagents to ensure adequate pollination of
coffee flowers.
ACKNOWLEDGMENT
We thanks Suyitno for his valuable help in preparing
field equipment and maintain insect collection.
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We only found 11 species (4 orders) within 30 hours
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developing fruit aborted because competition of neighbouring fruits [28][29] (2) Flowers probably require a
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th
Diversity and Abundance of Zooplankton in

Rawa Jombor, Klaten, Central Java
Khairunnisa Arumsari 1*), Suwarno Hadisusanto 1) Rindra Aryandari 1), and Haikal P. Fadholi 1)
1)
Faculty of Biology, Gadjah Mada University, Yogyakarta, Indonesia
*)
Corresponding author : khairunnisa@ugm.ac.id
Abstract Rawa jombor is a lenthic water ecosystem

that have similar characteristic with a reservoir or swamp,
located at Krakitan village, Klaten, Central Java. The water used for food culinary by the villagers, and also there
are a lot aquaculture can be found. Zooplankton is one an
importat biotic component in aquatic ecosystem as a secondary chain which is connect to the next trophic level.
Zooplankton can be used as controller of phytoplankton
abundance and as bio-indicators of water quality and a
productivity of fisheries in Rawa Jombor. The study aimed
to determine the diversity and abundance of zooplankton in
Rawa Jombor swamp. Data was taken at two zone: aquaculture zone and free zone. On each zone there are 3 sampling sites. Twenty litres water sample has taken used van
Dorn water sampler 5 litres on each sampling site. The
sample was filtered by Wisconsin Plankton Net and filtrate
was fixed by 4% formalin. Environmental parameters that
was measured include water temperature, air temperature,
water depth, light transparency, DO, CO2, alkalinity, pH,
nitrate and phosphate content. Zooplankton samples were
observed with a total strip counting method using SRCC
(Sedgwick Rafter Counting Cell). Results show there were
5 functional group in both locations, which consists of 30
species in the Free Zone and 21 species in Aquaculture
Zone. Functional groups found include Copepods, Cladocera, Rotifers, Rhizopods and Ostracods. Species that have
highest important value are Nauplius sp. and Cyclops sternus, belong to Class Copepoda.
Keywords Zooplankton abundance, Rawa Jombor,
Copepods
I. INTRODUCTION
Generally, zooplanktons are microscopic
aquatic animals that movement is influenced by winds,
currents, and tides. Zooplankton is an important biotic
component in the chain. In the swamp ecosystem,
zooplankton became a secondary chain linking the
primary chain (phytoplankton) with a chain on the next
level (shrimp and fish), so that the diversity and
abundance of zooplankton organisms can control
phytoplankton's abundance and become bio-indicators of
water quality and fisheries productivity in the swamp
[2]-[4],[6]. Zooplankton community are very sensitive to
environmental variations, factors that influence its
existence in swamp ecosystem is the level of dissolved
organic and inorganic compounds, the availability of
food (phytoplankton), competition, and predation.
Zooplankton diversity and abundance of information can
provide an important indication of the waters, or
disruption in the aquatic environment [12],[13] .
Zooplankton is an important component of the
food web in aquatic environments, especially fresh
water. Loss of Cladocera species members in large

quantities can reduce the predation on lower trophic
levels (phytoplankton), so that phytoplankton abundance
and disrupt ecosystem functioning, and lower trophic
levels above survival, such as planktoniovore fish [5].
Based on the function of providing food in the fishery,
zooplankton can be distinguished into two groups. There
are the zooplankton groups that eaten by fish and shrimp,
for example Copepoda and Cladocera, and the adverse
zooplankton groups (noxious) is a protozoan. Adverse
zooplankton may overflow if there is a decrease in
physico-chemical quality of the water [10].
Research on the diversity and abundance of
zooplankton was done at the turn of the rainy season to
the dry season, in the Rawa Jombor swamp. This
ecosystem is divided into four zones, namely floating
restaurant zone, aquaculture zone, water hyacinth zone,
and free zone. Water circulation in the ecosystem Rawa
Jombor can be said consequently less water will stagnate
so long on the ecosystem accumulation of organic matter
such as water hyacinth, floating stalls leftovers, and
giving pellets in ponds and other activities will affect the
diversity and abundance of zooplankton in the next
Rawa Jombor swamp ecosystem will determine the
quality and productivity of these waters.
On the diversity and abundance of zooplankton
research, the use of two zones, they are the free zone and
aquaculture zone. Both of zone (a) Aquaculture zone is
an barriers area and used for fish farming, on this waters
there is also additional of pellets provision, (b) Free
Zone is an area of open water that could be used by
peoples for fishing, animal livestock drinking, and other
activities.
This research aims to study the diversity and
abundance of zooplankton in Rawa Jombor swamp. In
focus to study about the species composer of the
community of zooplankton in the Rawa Jombor swamp.
Moreover also study about oxygen conditions, CO2,
alkalinity, pH, water temperature, air temperature, light
penetration, nutrient levels such as phosphate and nitrate
in the Rawa Jombor swamp.
II. METHODS
A. Study site
Diversity and abundance of zooplankton research was conducted in the Rawa Jombor swamp, Krakitan Village, District Bayat, Klaten, Central Java, on
the afternoon of March 16, 2013.
th

In this study, zooplankton species were
grouped into five functional groups, they were: (1)
copepods, (2) Cladocera, (3) Rotifers, (4) Ostracods,
and (5) Rhizopods. Functional group of Copepods and
Cladocera were the most abundant group in the study
locations (Figure 2), while the group that had the highest
diversity is Rotifer, reaching 10 species in free zone
(Figure 2.). The following table also presented diversity
of zooplankton in the free zone and aquaculture zone at
Rawa Jombor swamp (Table 1.).
Fig. 1. Rawa Jombor swamp. Location and sampling site (taken

from Google earth June 6, 2013)
B. Methods
Plankton sample in this study were taken from
two locations, they are free zone and aquaculture zone,
every zone taken from 3 sampling site (Fig. 1.), every
sampling site taken 2 bottle sample as replicates and 1
bottle extra samples. Every sampling sites, samples of
plankton sampled by water sampler modification of Van
Dorn water sampler 5 liters. Water samples were collected in a 10 liter bucket. In this study, water samples
were taken are composite sample, was sampled 4 times,
so that the total volume taken is 20 L. Then sample was
filtered with a Wisconsin plankton net. Then the filtrate
was collected in a flacon 10cc bottle properly labeled,
then fixed with 4% formalin. Then the flacons wrapped
in plastic and put in a box samples [11].In addition to
several physical and chemical variables, such as the light
transparency, DO, CO2, alkalinity, pH, water temperature, air temperature and dissolved nutrient levels include phosphate (PO43-), and nitrate (NO3) were measured. Then sample was identified at laboratory with total
strip counting method [13], use sedgewick rafter counting cell (SRCC), and light microscope and also plankton identify books [3,11].
Based on the results, 30 species of zooplankton
were distributed randomly in the free zone and aquaculture zone (Figure 2.). Most number of species were
found in the free zone consist of 30 species of zooplankton, while in aquaculture zone observed 21 species of
zooplankton (Figure 2.). From the results indicate that
the diversity of zooplankton in the free zone was more
diverse than aquaculture zone.
TABLE 1.
ZOOPLANKTON DIVERSITY IN THE FREE ZONE AND
AQUACULTURE ZONE
Agriculture
No
Species Name
Free Zone
Zone
COPEPODA
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Nauplius
Ergacillus versicolor
Cyclops sternus
Calanus minor
Spesies A
Spesies B
Eodiaptomus japonicas
Diaptomus glaciaris
CLADOCERA
Cheriodaphnia lacustris
Cheriodaphnia reticulate
Diaphanosoma branchyurum
Daphnia pulex
Moina macrocopa
ROTIFERA
Brachionus pala
Lepadella acuminata
Brachionus forficula
Brachionus falcatus
Epiphanes clavilata
Euclanis dilatsis
Keratella valga
Keratella serrulata
Filinia terminalis
Tricocera sp.
OSTRACODA
Cypria apthalmica
Notodromus monocha
Candona hyalina
Spesies C
RHIZOPODA
Centropyxis aculeta
Centropyxia ecornis
Euglypha cristata
Based on the results, it was known that the

most abundance zooplankton in the both of zone were
copepods, the second group were Cladocera and the
third were Rotifer. It is same with Trykov results of the
study in 2011 in Reservoir Kardzhaly also found that the
most abundant zooplankton were copepods, Cladocera
and the third level was Rotifera. They were three zooplankton groups that can be eaten by the fish. The three
groups of zooplankton abundance was an indicator that
the water quality of Rawa Jombor swamp was good, it
mean lush and sufficient to provide nutrients for the
predators (fish). Besides that, in this study noxius zooplankton group such as Euglypha cristata also rarely
found (Figure 3.).
Fig. 2. Number of species in the Free Zone and Agriculture Zone.
th

.
Figure 4. physico-chemical quality (1) free zone and (2) aquaculture

zone (2) include Dept, Transpiration, DO, CO2, Alkalinity , pH, Water temperature, Air Temperature.
Fig. 3. : Number of Individuals per liter in the Free Zone (blue

histogram with black value) and Agriculture Zones (red histogram
with red value).
Although in both zones, the total of functional groups

were same, but the abundance values was different, can
be seen in (Figure 3.), overall, zooplankton in the free
zone was more abundance than in aquaculture zone.
Copepod such as Cyclop sternus and Nauplius were the
most abundance species in both places, and the
abundance value of Cyclop sternus in free zone reached
232,750 individuals per liter, while in aquaculture zone
111,000 individuals per liter and the abaundance value
of Nauplius in free zone reached 229,833 individuals per
liter, while in aquaculture zone
was 138, 417
individuals per liter.
It was related to the physico-chemical quality of water
that supports the zooplankton growth. Although the free
zone and aquaculture zone in one swamp but there were
some different physico-chemical quality between these
zones, such as DO, light transparency, water
temperature, and phosphate (Table 1.).
The water temperature in the aquaculture zone was
higher than the water temperature in the free zone,
which was 29.6C in aquaculture zone and 28.5C in the
free zone (Fig. 4.). According research [4] the high
temperatures can be zooplankton lethal, its affect lower
abundance of zooplankton.
Light transparency in the free zone was higher than
the aquaculture zone, which respectively were 0.42 m
and 0.36 m (Fig. 4.). Light transparency affects the
distribution and abundance of phytoplankton, which
affects zooplankton abundance as primary consumers.
High phosphate content stimulus the abundance of

zooplankton population, according [1] phosphate was a
primary component of ATP was used in DNA replication, so the higher phosphate in free zone than aquaculture zone was the ones of determining factor in the abundance of zooplankton. Besides that, there were fish
culture in aquaculture zone. So it was possible that
zooplankton in the aquaculture zone had been eaten by
fish culture.
IV. CONCLUSION
From this study it can be concluded that the 5
functional groups of zooplankton found in both places,
which consists of 30 species in the free zone and 21
species in aquaculture zone. Species that has the highest
abundance was Nauplius then Cyclops sternus, derived
from Class Copepoda. Physicochemical parameters that
regulate the diversity and abundance of zooplankton in
the Rawa Jombor swamp are DO, water temperature,
transparency, light and phosphate levels.
ACKNOWLEDGMENT
I would like to express my very great appreciation to
my friend, Riana Nindita, for her valuable support on
this project. NungkeDiah for her supporting.I would also
like to my thanks to Abid Zulfikar, Mr. Bambang, Miss
Shinta for their contribution in collecting my data. Laboratory assistant is also gave useful contribution on
providing of equipment.

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th
Novel Cellulolytic Yeast Isolated From South

East Sulawesi, Indonesia
Atit Kanti1,2*), Nampiah Sukarno3), Latifah K Darusman4), Endang Sukara5), I Made Sudiana2),
Helbert6), and Kyria Boundy-Mills7)
1)
Microbiology Study Program. Graduate School of Bogor Agricultural University, Indonesia

Research Centre for Biology, Indonesian Institute of Sciences (LIPI), Cibinong Science Centre,
3)
Department of Biology, Faculty of Mathematics and Natural Sciences, Bogor Agricultural University, Darmaga Campus, Bogor 16680
4)
Department of Chemistry, Faculty of Mathematics and Natural Sciences, Bogor Agricultural University, Darmaga Campus, Bogor 16680
5)
Research Centre for Biotechnology, Indonesian Institute of Sciences (LIPI), Cibinong Science Centre
6)
R&D Units For Biomaterials, Indonesian Institute of Sciences (LIPI), Cibinong Science Centre, Indonesia
7)
Phaff Yeast Culture Collection, Department of Food Science and Technology, College of Agricultural and Environmental Science, University of California, One Shield Avenue, Davis, CA 95616,
USA
2)
*)
Corresponding author: atityeast@gmail.com
AbstractThe objective of study was to isolate, identify

and characterize the cellulolytic yeast isolated from secondary forest in South East Sulawesi, Indonesia. We isolated 142 strains and obtain 43 strains (32.28%) were cellulolytic yeasts consist of 26 species residing within 10 genera. Candida was the most diverse genus consisting of 15
species. It is important to note that several strains residing
within this genus could be candidate for new taxa, among
others Candida aff. Cylindracea PL2W1, Candida aff. Insectorum PL3W6, Candida afffriedrichiiMKL7W3, Candida afflessepsii, Candida aff. tenuis. Five new candidates for
novel species of cellulolytic yeast close to Yamadazymamexicana: were Yamadazymaaff. mexicana(5 strains). Pichia,
Pseudozyma, Sporodiobolus, and Sporobolomyceswere other cellulolytic yeasts found in South East Sulawesi. It is
obvious, that leaf litter was a good source of cellulolytic
yeasts. This cellulase producing yeasts dominate this biome, and production of extracellular cellulase is critical
strategy for such yeast to survive in cellulose rich ecosystem such as leaf-litter. This finding would suggest that
yeasts play key role on hydrolyzes of cellulose and important resources for sustainable energy research.
Keywords Cellulolytic yeasts, secondary forest, South
East Sulawesi, leaf-litter.
I. INTRODUCTION
p to date ecological studies of yeast from natural
habitats have been conducted extensively mostly in
temperate regions [1],[2]. Taxonomy and ecology
data indicate a need for additional studies in tropical
ecosystems, particularly in Asia [3],[4]. Indonesia is a
tropical nation comprised of over 17,000 islands is rich
in biodiversity, having unique flora and fauna [5],and

presumably microbes as well. Rifai[6] estimated Indonesia has more than 200,000 species of fungi. However,
little information on species diversity of Indonesia indigenous yeast and yeast-like fungi has been generated.
Studies of Indonesian yeasts primarily related to their
role in fermented foods [7],[8]. Early studies of yeast
from natural environment in Indonesia include Deinema
in 1961, who found Candida bogoriensis from the surface of leaves of the flowering shrubs Randiamelleifera
(Rubiaceae) in Bogor. In recent years, more studies have
been performed to explore yeast diversity in Indonesia
[9]-[10]. While microorganism are very important
sources for bioindustry however few study conducted to
assess the important of forest as genetic resources for
many interest. Cellulase are primarily obtained from
cellulolytic fungi: Trichodermareseei, T. viride, T. lignorum[11], T. koningi, Chrysosporiumlignorum, C.
pruinosum, Fusariumsolani, Neusrosporacrassa, Aspergillus[12],
Penicelllium,
Acremoniumstrictum[13],[14], including that from several group of cellulolytic bacteria: Cellulomonas, Cellulosimicrobiumcellulans [15] Clostridium clariflavum, Bacillus group,
Flavobacterium, and Paenibacillus[16], and Fermicutes
and Actinomycetes group ([17];[18]). On the hand the
role of yeast on biodegration of cellulose is few explored, particularly using yeast from tropical forest
([19];[20]).
Sulawesi has high biodiversity, and reported to have
high biodiversity of flora,[21] and fauna [22];[23]. At
the microbial diversity level, further study is needed to
verify the species richness of this area, particularly cellulolytic yeast. We evaluated two areas in South East
th
Sulawesi that have different covering vegetation. Leaf

litter, soil and leaf surfaces are common habitats for
yeast [24]-[26]. This paper is concerned with the isolation of cellulolytic yeast from natural habitats in Southeast Sulawesi, Indonesia and their phylogenetic affiliation based on partial 26S ribosomal RNA and ITS sequences.
A. Sampling sites and Samples collection
Leaf and leaf litter samples were aseptically collected
from two sites that differ in elevation, forest type, and
land use type. PapaliaProtected Forest is a tropical lowland monsoon wet forest dominated by evergreen tree,
with a high humidity and high rain fall. Papalia, located
in South Konawe, GPS S 04 13 526, E 122 44 301,
having altitude <1000 m asl. Whereas MekonggaProtected Forest covers lowland and upland rain forest sites
included hill forest, montane forest with elevation gradient ranges from 100 m to 2500 m asl. Land use type is
dominated by cocoa plantations. Mekongga rain forest is
located near Tinukari, North Kolaka, GPS S 03 38
085, E 121 04 311. Leaf and leaf litter sampling were
conducted in 2009 and 2010. Six leaves and 6 leaf litter
samples were collected in Papalia, and six leaves and 10
leaf litter were collected from Mekongga Protected Forest.
B. Isolation of Yeasts
Yeasts were isolated from leaf, leaf litter, and
soil using published methods [27]. One g of soil or leaf
litter was added to 25 mL of saline/Tween (0.85% NaCl,
0.01% Tween 80, v/v) buffer in a 7 oz Whirl-Pak filter
bag (Cat.# B01385WA, Nasco, Salida, CA, USA) and
shaken to suspend the microbes. The bag had two separated compartments which allowed separation of suspension from debris. Two hundreds L of suspension
were plated on Rose Bengal Chlorampenicol Agar
(RBCA) plates supplemented how much chloramphenicol. Leaves were plated using two methods, washing and
direct plating. For washing, leaves were added to 10 mL
of saline/Tween buffer in a 7 oz. Whirl-Pak filter bag
and processed as detailed previously. Aliquots of 200
L and 50 L of these samples were plated on RBCA,
which prevents growth of bacteria and slows down the
spread of molds. For direct plating, the leaf and leaf
litter were weighed and cut into small pieces of about
2cm2. The leaf and leaf litter were washed with 30 ml of
sterile distilled water and vortexed for 5 min. Washed
materials were placed directly onto RBCA plates.
Ballistospore-producing yeasts were isolated from
leaves using the ballistospore-fall method. Briefly, aseptically collected segments of leaves were attached to the
underside of a Petri dish lid using Vaseline, and the
plate was incubated lid-side up. Ballistospores ejected
onto the surface of the agar germinated, and yeasts were
cultivated. Incubation of the plates was done for 5 days
at room temperature. Strain purification was done at
least twice by selecting the different type of yeast colony
and plated on potato dextrose agar (PDA, Cat.#
CM0139, OXOID,city, country) at room temperature.
C. rDNA sequence determination

Yeast DNA template was prepared from freshlygrown cells on the PDA plate and used for colony PCR.
Five uL of lysed yeast cell suspension was used for PCR
amplification of the partial 26S rDNAsubunitwith primers NL1 and NL4 [28], using GoTaq master mix
(Promega, M7122). PCR products were visualized on
2% agarose and sequenced with both primers using Big
Dye terminator v3.1. Cycle Sequencing Ready Reaction
Kit (Applied Biosystems) following the manufacturers
instructions. The partial 26S sequences determined in
this study were compared to those in the
EMBL/GenBank/DDBJ databases using the nucleotide
Basic Local Alignment Search Tool (BLASTn) [40].
The ITS1/5.8S/ITS2 region of selected strains was also
amplified with primers ITS1 and ITS4 [29] when species identifications were ambiguous.
D. Phylogenetic Analysis
Sequence were aligned using CLUSTALX [30]. The
distance matrix for the aligned sequence was calculated
using the two-parameter methods of [31]. The neighborjoining (NJ) method [32] was used to construct all phylogenetic trees.
E. Screening of CMC activities
Cellulolytic yeast is defined as yeast grow on CMC as
the sole carbon sources, and produce CMC-ase. To obtain the cellulolytic each yeast was grown on CMC agar
containing 3 g L-1 yeast extract, 5 g L-1 peptone ,10 g L-1
CMC, K2HPO4 5 g L-1,(NH4)2SO40,5 g
L1
-1
,MgSO4.H2O0,2 L , FeCl3.6H2O 0,01 g L-1,
MnSO4.H2O 0.001 gL-1. ,20 g L-1 agar (pH 6.2 0.2)
[33]and incubated for 5 days at room temperature. Cellulolytic yeast was determined by pouring 2 mL congo
red 1 M solution into grown colonies, and kept for 10
minutes. Observing clear zone was done by pouring the
congo red solution and replaced with NaCl 0.1N. Cellulolytic yeast was indicated by formation of clear zone
surrounding colonies and the ratio between clear zone
divided by colonys size indicating cellulolytic capacity.
F. Production of CMCase
Cellulolytic yeast was grown in CMC broth medium
(3 g L-1 yeast extract, 5 g L-1 peptone ,10 g L-1 CMC,
K2HPO4 5 g L-1,(NH4)2SO40,5 g L-1,MgSO4.H2O0,2 L-1,
FeCl3.6H2O 0,01 g L-1, MnSO4.H2O 0.001 gL-1. (pH 6.2
0.2)pH 6.2 0.2) at 25C for 48 h as a pre-culture.
The culture medium was inoculated with 2% of the preculture and incubated at 25C with shaking at 120 rpm
for 72 h. To assess the ability of culture to hydrolyze
cellulose from agriculture waste, 2 % grinded printed
paper, straw and bamboo leaf were used separately as
carbon sources to medium containing 3 g L-1 Yeast extract, 5 g L-1 peptone, pH 6.2 0.2 and grown at rotary
shaker (125 rpm) at 25 C.
G. Preservation of Yeast Cultures
Yeast isolates were preserved by two methods, in 20
% glycerol solution at -80C [46], and by lyophilization[34]. Yeasts were deposited in the Indonesian Culture Collection (InaCC, www.biologi.lipi.go.id) at the
Indonesian Institute of Sciences, Research Center for
th
Biology; the Forest Microbes Collection (FORDACC,

http://forda-mof.org/) at the Forestry Research and Development Agency, Indonesian Ministry of Forestry; and
the Phaff Yeast Culture Collection, Department of Food
Science and Technology, University of California Davis
(UCDFST, phaffcollection.ucdavis.edu).
A. Diversity of cellulolytic yeast
Numerous of yeasts were isolated from two sites
(Mekongga and Papalia) in South East Sulawesi. The
cellulolytic yeast was defined as yeast grow on CMC
used as the sole carbon sources and produce CMC-ase
(Endoglucanases EG1, EC. 3.2.1.4 ) hydrolyze soluble,
substituted celluloses such as Carboxymethyl Cellulose
(CMC) by attacking the carbohydrate chain (1-4, glucosidic bond) internally and randomly. This can be visualized by culturing yeast both on liquid and solid media
contain CMC. Formation of clearing zone on CMCmedia poured with 1 % congored and production of
CMC-ase were used to screen cellulolytic yeast. We
obtained numerous species of yeasts (Fig. 1). Of 142
strains tested 43 strains were cellulolytic yeasts consist
of 10 genera and 26 species, of whose Candida was the
most diverse genus consisting of 15 species (Figure 2),
and more divers was the non-cellulolytic yeast (Fig.1)-.
Novel taxa of cellulolytic yeasts
Comparison of the D1/D2 region of LSU rDNA data
showed 14 strains belonging to 11 different species had
homology values less than 99%, indicating that they may
be novel species. The results of ITS sequence analysis
of these isolates confirmed that they are likely novel
species. Our molecular analysis revealed that these yeast
isolates are phylogenetically diverse and distributed
within the phyla Ascomycota (genera Candida and Yamadazyma).
The novel species candidates were mostly residing
within genus Candida (13 species: Candida aff. cylindraceaPL2W1, Candida affinsectorumPL3W6, Candida
afffriedrichiiMKL7W3,
Candida
afflessepsiiPLE3W1, MKL7W4, Candida aff. tenuisPL3DP3,
MKL6W4. We isolated 5 strains of cellulolytic yeast
close to Yamadazyma Mexicana: Yamadazymaaff. mexicanaMKL6DP1, MKL6DP2, MKL8W2, MKL6W2,
MKL6W1), and our study reveal detection of many undescribed yeast from Indonesia. Further study is needed
to describe the novel taxa found in this study.
B. Phylogeography of cellulolytic
Sample sources were collected from secondary forest
in Mekongga and Papalia, South East Sulawesi. Both
places harbor numerous species of Cellulolytic (Fig. 4
and 5).There were 15 species including 6 candidates for
novel species (3 strains of Yamadazymaaff. scolyti, Y.
aff. Mexicana ,Candida aff. tenuis, C. aff. lessepsii, C.
aff. friedrichii, and C. aff. endomychidarum ) Fig.
4).Eleven species including 3 candidates for novel species of cellulolytic yeast (Candida aff. tenuis, C. afff.
insectorium, C. aff. cylindracea) were found in Papalia
(Fig. 5). Yamadazyma were only isolated from Mekongga, andAsterotremella and Sporodiobolus were
only cultivated from Papalia.
Number of strains
Fig. 1. Diversity of non-cellulolytic yeast isolated
from South East Sulawesi
th
Fig. 4. Cellulolytic yeast isolated from Mekongga.
Fig. 2. Diversity of cellulolytic yeast from South East Sulawesi t
Fig. 5. Cellulolytic yeast isolated from Papalia
C. Litter cellulolytic yeast

Litter are god sources for cellulolytic yeast, as indicated by numerous cellulolytic yeasts obtained from liter
collected both from Mekongga (Fig. 5) and Papalia (
Fig. 6).
Fig. 3. Candidate for novel species of cellulolytic yeast
D. Cellulolytic yeast on leaf

Seven species of cellulolytic yeasts were isolated
from Papalia leaf, and none was from Mekongga. Sporodiobolus were common genera, and Sporodiobolusruineneniaeand Candidaintermediawere dominant in
leaf (Fig 8).
th
Fig. 6. Mekongga cellulolytic yeast from litter
Fig.7. Papalia cellulolytic yeast from litter
and habitat range of these known species.

A variety of ascomycetous and basidiomycetous
yeasts were cultivated. Basidiomycetous yeasts are more
likely to utilize a broader range of carbon sources than
ascomycetes, and have been cultivated more frequently
from low-nutrient habitats such as leaf surfaces [39].The
most frequently cellulolytic yeast isolated from South
East Sulawesi were Yamadazyma, Pseudozyma and
Candida. Candida was isolated from both locations and
ubiquitous on litter. Candida is a polyphyletic genus,
with species placed in 14 families within the class Saccharomycotina. In fact, over 400 of the 1600 known
species of yeasts have been placed in the genus Candida
[40]. Due to its taxonomic diversity, it is not surprising
that Candida is ecologically diverse also, occupying
niches including human infections, soil [54], insect
frass, fruit[55]. Important applications using Candida
species include agent for bioremediation, Candida catenulata[56], and biofertilizer, Candida tropicalis HY
[54].
Yamadazyma, Pseudozymaand Candida, isolated repeatedly in this study, is a well known species having
wide distribution and having high xylose transport capacity [57].
This study supports other studies that concluded leaf
litter and plant leaf surfaces are good sources of a diversity of yeast cultures for research [1],[30],[58]. We
found leaf litter are good sources for cellulolytic yeast.
The strains isolated in this study were deposited in three
public culture collections (InaCC, FORDACC and
UCDFST).
Isolation of numerous species of yeasts include novel
taxa reaffirm that South East Sulawesi are rich in biodiversity of flora, fauna and microorganism, and potential
genetic resources for sustainable development.
ACKNOWLEDGMENT
Fig. 8. Cellulolytic yeasts obtained from Papalia leaf
Little information was previously available about

yeasts on the island of Sulawesi, Indonesia, one of the
five largest islands that makes up this richly biodiversity
and biogeographically significant region [9],[35],[36].
We found a broad taxonomic diversity of cellulolytic
yeast species from this exploratory survey. Because
plant surfaces and leaf litter have been sampled extensively [1]; [37]; [38], novel taxa were not expected.
However, numerous potentially novel species of cellulolytic yeast were obtained. Novel strains of known species were obtained, expanding the known geographic
Yeasts used in this study were isolated and identified

as part of a collaborative project between the University
of California Davis and the Government of the Republic
of Indonesia, funded by Grant Number U01TW008160
from the US National Institutes of Health Fogarty International Center, the NIH Office of Dietary Supplements,
the National Science Foundation and the Department of
Energy. This project was supported by the USDA Agricultural Food Research Initiative of the National Food
and Agriculture, USDA, Grant #35621-04750. The content is solely the responsibility of the authors and does
not necessarily represent the official views of the Fogarty International Center or the National Institutes of
Health, the Office of Dietary Supplements, the National
Science Foundation, the Department of Energy, or the
Department of Agriculture.
This research was a part of the Ph.D. thesis dissertation of AtitKanti, submitted to Bogor Agricultural University as fulfillment to obtain a doctorate degree in microbiology. We would like to thank AnisMutirani, and
YeniYuliani of LIPI; Sarah A. Faulina, Aryanto and
SiraSilaban of FORDA for their assistance.
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10.1007/s10482-013-0098-8
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CHEMISTRY
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EFFECT OF AGRICULTURAL WASTE ON

THEGROWTHAND MANGANESE
PEROXIDASE PRODUCTION OF
Phanerochaete chrysosporium
1
Evi Susanti1), Aulaniam2), Suhardjono3)and Tri Ardiyati3)

Doctoral Program of Biology Department, Brawijaya University, Malang, Indonesia
and Department of Chemistry, The State University of Malang, Indonesia
2 Department of Chemistry, Brawijaya University, Malang, Indonesia
3 Department of Biology, Brawijaya University, Malang, Indonesia
*)
Corresponding author:esusanti.kim@gmail.com
Abstract Types of carbohydrate affect the growth and

manganese peroxide production of Phanerochaete chrysosporium. Agricultural waste is a carbohydrate composed of
cellulose, hemicelluloses and lignin. The purpose of this
study was to determine the effect of agricultural wastes on
growth of P. Chrysosporium and itsmanganese peroxides
enzyme activity (MnP). The N-limited media were used as
the growth media. Agricultural wastes that tested are bagasse, rice husks, corn cob, straw and sawdust. Glucose
was used as positive control while medium without carbon
source as negative control. The results showed that the
formation and an increase in the number pellet cell of P.
chrysosporiumwas clearly visible in media containing rice
husk and bagasse, while corncob, sawdust and straw was
not seem to clear the formation of the cell pellet. Media
contaning of straw, corn cob, bagasse and rice husk produces MnP with the highest activity as much as 0,090,
0,033, 0,014, 0,011 U/mL respectively while the sawdust
was not able to produce MnP.
of lignin degradation [3]. This enzyme is known to be

applied to the bioremediation process various xenobiotic
compounds such as phenols, polycyclic aromatic
hydrocarbons, trinitrotoluen and textile dyes [4,5,6]
Growth of P. chrysosporium and production the
MnP enzyme is strongly influenced by the carbon
source. Previous research claimed that the MnP
produced by P. chrysosporium in response to the limited
amounts of nitrogen, carbon and sulfur in the growth
medium [7], but the other research showed that the
highest LiP and MnP activity produced on the amount of
excess glucose in the medium [8]. The difference of
extracellular proteins profil was produced by P.
chrysosporium grown on different carbon sources [2].
Uncertainly, how the influence of agricultural waste to
the growth and activity of manganese peroxidase
produced by P. chrysosporium is still unknown.
Keywordsagriculture,
waste,
Phanerochaete and mangan peroxide
carbon
source,
I. INTRODUCTION
enomics and proteomics studies showed that Phanerochaete chrysosporium can be a source of enzymes that play a role in the metabolism of lignin.
Genomic studies indicate that P. chrysosporium has
hundreds predictable sequence encodes an enzyme lignin peroxidase (LiP), manganese peroxidase (MnP),
copper radical oxides, cytochrome, flavin and multicopper oxides [1]. Proteomics studies show that there are
117 enzymes that are composed of cellulose-degrading
enzymes i.e. endoglucanase, beta-glucosidase and exogluconase,hemicelluloses decomposition enzymes i.e.
xylanase, acetilxylanase, esterase, mannosidase and
mannanase, pectin decomposer enzymes i.e. poligalakturonase, rhamnogalakturonase and arabinose, as well as
lignin-degrading enzyme consists of a group oxidoreductases[2].
Manganese peroxidase (MnP) is one of the
extracellular heme proteins involved in the metabolism
A. Microorganism
Phanerochaete chrysosporium cultures were obtained
from the collection of the Laboratory of MicrobiologyITB, were sub cultured in PDA agar slant at 30oC. Nlimited mediacontaining carbon source were used as
growth medium. The compositionof N-limited media per
liter consists of: 0.1 g NaCl, 1.2 g of K3PO4 (Na2HPO4),
1.0 g of NH4Cl, 0.2 g KCl, 1.2 g MgSO4.7H2O, 0.1 g of
CaCl2 and 10 g of carbon source.The glucosewas used
as a positive control and N-limited media without a carbon source as a negative control. Agricultural
wasteswere used as carbon sources are bagasse, straw,
rice husks, sawdust and corn cob
B. Preparation of agricultural waste as carbon
source
Bagasse and sawdust can be directly washed several
times with aquades to remove impurities, dried at 100
C until constant weight, and then sieved with a sieve
flour ( 30 mess). The part that is not filtered mashed in
a blender and sieve again.
th
Straw, rice husks and corn cob washed, cut into

small pieces, and then dried under the sun until
completely dry before it wasblended and sieved with
flour sieve.
C. Growth and isolation manganese peroxidase ofP.
chrysosporium
Growth and isolation of the manganese peroxidase
done by adding suspension sporesof P. chrysosporiuminto 250 mlErlenmeyer flask containing 100 ml of
growth medium. The number of spores around 1,1.106
spores/ml for each Erlenmeyer. The inoculum were incubated at 30 C and 100 rpm.
The suspension spores obtained by adding four milliliters of 0.02 % Tween 80 into subculturedof P. chrysosporium that had been grown for 14 days. The spores
were extracted with the help of a sterile needle ose, left
it to stand for 5 minutes, and poured it in a sterile container. The suspension spores were vortexing for 10
minutes and left to stand for 30 minutes before were
used.
Observations texture residue, residue dry weight and
enzyme activities carried out since the 4th to 9th. The
experiments were performed two times repetition. The
residue obtained by the growth medium was poured past
the filter paper. Filter paper containing the residue was
dried at 105 C for 2 hours .
D. Determine of Manganese peroxidase activity
Determination of MnP activity refers Paszczynski
based on the ability of MnP oxidizes Mn (II) to Mn
(III). Mn (II) do not absorb at 238 nm, whereas Mn (III)
absorbs strongly with ekstinsik molar coefficient of
6500 M-1 cm-1 [9].
III.
RESULT AND DISCUSSTION
A. Effect of agricultural waste to the growth of the P.

chrysosporium
The residual dry weight data were obtained (Fig. 1)
can not directly reflect the growth of P. chrysosporium
except in the control treatment. The negative control
does not contain a carbon source and a positive control
containing glucose. Glucose was soluble of carbon
source, while agricultural wastes werw highly insoluble.
It makes that the increase ofresidual dry weight each day
in the control just derived from the growth of P. chrysosporium, whereas the residual dry weight on growth
medium containing agricultural wastes suspected influenced of two events i.e. the degradation of agricultural
waste and growth of P. chrysosporium.
Assuming weight of cells at zero day are none, and it
was happen in all treatment, then the increase in the
number of cells at negative control was very low compared to the positive control. Residual dry weight of the
positive control in the fourth and fifth day almost ten
times higher than the negative control. This reinforces
the notion that P. chrysosporium requires a carbon
source for growth.
Fig. 1. Residue dry weight of each growth medium containing various

carbonsources for the growth time of 4 to 9 days
In general, the decreased of residues dry weight from

growth medium containing agricultural waste was done
between fourthuntil sixth days incubating, while the increased between sixth until eighth days. The decreased
presumably due to the rate of degradation of agricultural
wastes by P. chrysosporiumwasgreater than the growth
rate. Furthermore, the increase in residue dry weight
presumably due to a faster growth rate of P.
chrysosporium.
At the fourth day seemed to be two groups of agricultural waste that is difficult to degrade group of agricultural wastes and easily degraded. Agricultural waste
that is difficult to degrade were corn cob, sawdust and
straw. The one that are easily degraded were bagasse
and corncob . Theoretically agricultural waste easily
degradable carbon source will supply much faster than a
difficult to degraded agricultural waste. The faster the
carbon source available then the cell growth was also
faster. This opinion was in line with observations on the
shape of the resulting residue (Fig. 2). Formation and an
increase in the number of cell pellet P. chrysosporium in
the group that is difficult to degrade agricultural waste
couldnot be observed. The shape of cell pellet was
unclear form because almost all of which seemed to be
an agricultural waste that has not been degraded. In contrast, the agricultural waste that easily degraded result
cell pelet that had observed as at the positive control.
Based on these results can be concluded that bagasse
and rice husk carbon source can be a potential alternative for the growth of P. chrysosporium than corn cob,
straw and rice husk.
(a)
(b)
th

IV. CONCLUTION
Bagasse and rice husk influence the formation of pellet cells of P. chrysosporium whereas corncobs, sawdust
and straw are not known yet. Straw and corn cob produce manganese peroxidase by high activity that is equal
to 0.090 and 0.033 U/ mL, bagasse and rice husk produce low activity manganese peroxidase that is equal to
0.014 and 0.11 U/mL whereas sawdust completely unable to produce manganese peroxidase.
(c)
(d)
REFERENCES
[1]
[2]
[3]
(e)
(f)
(g)
Fig. 2. Residuesof P. chrysosporium in growth medium with

different carbon sources.(a) negative control,(b) positive control, (c)
rice husk, (d) bagasse, (e)corncob, (f). straw and (g) sawdust.
B. Effect of agricultural waste in the activity of manganese peroxidase (MnP) produced by P. chrysosporium
Effect of agricultural waste to the activity which
produced manganese peroxide showed no clear pattern
(Fig. 3). Agricultural wastes are easily degraded
produced low MnP activity, while the agricultural waste
that is difficult to degrade produce higher MnP activity
whereas sawdust did not produce MnP.The highest MnP
was produced at different day. Growth medium containing straw on 5thday, corncobs on 8th day, baggase on
9th day and rice husk on 6th daywith MnP activity values respectively 0.090, 0.033, 0.014 and 0, 11 U / mL,
Based on this, the straw and corn stalks can be used as
an alternative carbon source to produce MnP from P.
chrysosporium.
[4]
[5]
[6]
[7]
[8]
[9]
Kersten, P. and Cullen, D., 2007, Review of Extracellular Oxidative System of the Lignin-Degrading Basidiomycetes Phanerochaete chrysosporium, Fungal Genetics and Biology, No. 44,
pp. 77-87
Manavalan, A., S.S. Adav & S.K.Sze. 2011. iTRAQ-based
quantitative secretome analysis of Phanerochatea chrysosporium. Journal of Proteomics. No. 75, pp. 642-654.
Dashtban, M., Heidi Schraft., T.A. Syed & W. Qin. 2010. Fungal biodegradation and enzymatic modification of lignin. International Journal Biochemistry Molecular Biology.Vol 1, No.1,
pp. 36-50
Abo-state, M. A. M., B. Reyad., M. Ali., O. Gomaa & E.A.
Youssif. 2011. Comparing decolorization of dye by white rot
fungi, free enzyme and immobilized enzyme, World Applied
Science Journal. Vol 14, No.10, pp. 1469-1486
Ruggaber, T. P. & J. W. Talley. 2006, Enhancing Bioremediation With Enzymatic Processes: A Review. Practice periodical
of Hazardous, Toxic and Radioactive Waste Managemen
Wesenberg, D. Irine K. & Spiros N. A. 2003. White-rot fungi
and their enzyme for treatment of industrial dye effluents, Biotechnology Advances, No 22. Pp. 161-187.
Li, D., M. Alic., J.A. Brown & M.H. Gold. 1995. Regulation of
manganese peroxidase gene transcription by hydrogen peroxide,
chemical stress and molecular oxygen, Applied and Environmetal Microbiology,Vol 61, No. 1, pp.341-345
Wang, P., X. Hu, S. Cook, M.Begonia, Lee, S.Ken & H.Hwang.
2008. Effect of culture condition on the production of ligninolytic enzymes by white rot fungi Phanerochaete chrysosporium
(ATCC 20696) and separation of its lignin peroxidase. World
Journal Microbiology Biotechnology. No. 24, pp. 2205-2212
Zahmatkesh, M., Tabandeh F., & Ebrahimi, S. 2010. Biodegradation of Reactive Orange 16 by Phnerochaete chrysosporium
Fungus: Application in a Fluidized Bed Reactor. Iran Journal
Environmental Health Science Engineering, Vol .7, No. 5, pp.
385-390
Fig. 3. Activity of manganese peroxidase (MnP) of P. chrysosporium

in various carbon sources during the growth period of 4 to 9 days
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THE STUDY OF ADSORPTION ON CR(VI)

IN NATURAL CLAY SURFACE MODIFIED
WITH SURFACTANT CTAB
(CETYLTRIMETHYLAMMONIUM
BROMIDE)
Maksum1*), Susi Nurul Khalifah, 1), and Anton Prasetyo1)
1)Departement of Chemistry, Science and Technology Faculty,
State Islamic University of Maulana Malik Ibrahim Malang
*)Correspondence author: susikhalifah@gmail.com
AbstractClay is a mineral particles composed of silicaalumina base frame, has a layered structure and a hollowed space causing surface becomes very widespread and
effec-tive as an adsorbent. Adsorbent of clay is very low to
ad-sorb anion, such as Cr(VI) formed in HCrO4- at pH 2.
This research has been conducted in the clay of activation
pro-cess chemically with the variation of H2SO4 0.5, 1.0,
1.5 and 2.0 M and physically with the variation in the temperature of 200, 300, 400 C as well as modifying the surface with the variation of CTAB surfactant 25, 50, 75, and
100 mM to enhance the adsorption of Cr(VI). The results
of study showed that the treatment on the activation and
modifica-tion of natural clay can increase the adsorption
capacity of Cr(VI) is greater. Adsorption capacity (Qe)
before activat-ing the natural clay is 0.0971 mg/g, while the
Na-clay in-creased adsorption of Cr(VI) at 7.85 % as indicated by Qe = 0.1756 mg/g of natural clay. The use of 0.5
M H2SO4 acti-vation of adsorption increased by 21.44 %
to the value of Qe = 0.3115 mg/g. The treatment of physically activation after activating the best chemical showed at
a temperature of 200 C with an increase of 29.82 % adsorption of value Qe = 0.3953 mg/g. While modification to
the clay treatment results in the best physical activation get
CTAB 25 mM concentration with increased adsorption of
94.54 % with a value of Qe = 1.0425 mg/g.
Keywordsnatural clay, activation, modification and adsorption of Cr(VI)
I. INTRODUCTION
Indonesia has abundant clay as one of its natural materials, one of them is placed in Gandusari Area Trenggalek Regency that have been used by people around as
tile-making material. Besides being used as the tilemaking material, clay also can be used as an economical
adsorbent which has large adsorption capacity. This
happens because clay has high surface area, chemically
stable, varying surface structure, high capacity of ion
exchange and the presence of Bronsted and Lewis acids
[3].
The application of clay as an adsorbent material is
widely used as an alternative material to overcome waste
problems, especially the waste of heavy metal like
chromium. Chromium usually comes from industrial
metal plating, metal corrosion inhibitors, tanneries,
paint, textile and wood preservatives [9]. Chromium
which stable in water can be found as Cr (III) in the
form of cationic compound (Cr3+) and Cr (VI) in the

form of anionic compounds such as HCrO4-,CrO42- and
Cr2O72- [4]. Cr(VI) Metal is more toxic, has a higher
solubility, more corrosive and more carcinogenic than
Cr(III) [9].
The ability of clay to adsorb anion like Cr(VI) is very
low because clay has negatively charged surface. Several methods have known to increase the adsorption capacity of clay by chemical and physical activation and
modifying the clay surface.
In this research, chemical activation has done by using a variation of H2SO4 and physical activation by
heating at high temperature and modification on the surface also has carried out with variation of CTAB to enhance the adsorption ability of clay to Cr(VI).
The addition of CTAB concentration has done above
CMC (critical micelle concentration) in order to change
the clay surface in which negatively charged becomes
positively charged, thereby the ability of clay to adsorb
Cr(VI) in anionic form will be greater. Furthermore, a
comparison of the adsorption ability of each natural clay
is done, Na-clay, Na-clay which is chemically and physically activated and the surface of Na-clay modified by
CTAB.
This study consists of several stages, namely beneficiation, sample preparation, Na-clay preparation, Naclay activation, modification of clay surface activation
result with CTAB surfactant and determination of
Cr(VI) by using UV-Vis Spectroscopy method and characterization with FTIR.
A. Beneficiation
Natural clay dried at room temperature, then crushed
and dissolved by aquades. The mixture is stirred until
the clay is soluble and then left a few days until the clay
forms into three parts. The top part is an organic compound and salt which dissolved in water. The middle
part is clay, while the bottom is gravel, sand and other
impurities which have a high specific gravity. The result
of clay which has obtained is repeated ones more to obtain perfect separation. After that, the result of beneficiation dried under the sun [1].
th
B. Sample Preparation
Clay fraction which has been dried is pulverized to a
powder, and then sieved by a sieve of 200 meshes. Furthermore, natural clay is characterized with FTIR.
C. Preparation of Na-Clay
A total of 100 g of clay incorporated into 1000 mL of
NaCl 1 M and stirred for 24 hours at a temperature of
70-80 0C. Residues incorporated into 1000 mL of NaCL
6 M while stirring for 24 hours. The residue washed
with aquades to remove residue of chloride ions. The
filtrate is tested with a solution of AgNO3 1 M until
unformed white precipitate of AgCl. Then, Clay that has
been free of chloride ions is dried in an oven at 100 0C
for 24 hours (Wijaya, et al., 2005 in [9]).
D. Na-Clay Activation
Na-clay is chemically activated by means each of
them is weighed for 5 g and put into 4 Erlenmeyer 250
mL. Then added 100 mL of H2SO4 with various concen-tration of 0.5, 1.0, 1.5, and 2.0 M [12], while stirring for 5 hours [2]. The mixture is filtered and washed
with hot water (60-70 0C) until free of sulfate ions
which can be characterized by the formation of white
precipitate of BaSO4 (Negative test to BaCl2) [12]. After that, do the adsorption to Cr(VI).
The result of chemically clay activation continued to
physical activation by drying sample in a furnace at
temperature of 200; 300; and 400 0C for 6 hours [2],
and then stored in a desiccators. After that, do the adsorption to Cr(VI).
E. Modification of clay surface activation result with
CTAB surfactant
A total of 2 g of sample result of the best physical activation added 100 mL of CTAB with concentration of
25, 50, 75 and 100 mM and stirred in a shaker at room
temperature for 4 hours at speed of 200 rpm. The suspension which obtained is filtered and washed with 100
mL of aquades in 2 times. The result of clay which has
modified is dried at room temperature [10]. After that,
do the adsorption to Cr(VI).
F. Determination of Cr(VI) with Spectrophotometry Method
Cr(VI) Solution of 20 ppm 50 mL conditioned at pH
2 by using H2SO4 0.1 M/NaOH 0.1 M, and then added
3 mL of buffer solution pH 2. Then as much as 0.5 g of
natural clay put into Erlenmeyer 250 mL, then added
Cr(VI) solution of 25 mL. The mixture is shaked for 30
minutes at speed of 200 rpm at room temperature of 25
0
C.
The mixture which is obtained filtered with a filter
paper. The filtrate which is obtained pipetted in 5 mL
and placed in a glass baker which is conditioned at pH 2
by using H2SO4 0.1 M/NaOH 0.1 M. Then added 3 mL
of buffer solution pH 2 and 2 mL of diphenylcarbazide
(0.25 %). After that, put in a volumetric flask 25 mL and
diluted with aquades up to mark boundaries. The solution is allowed to stand 5-10 minutes. Then absorbance
measured by UV-Vis Spectroscopy at a wavelength of
540 nm.
Procedure above is repeated for the adsorption of

Cr(VI) on Na-clay, Na-clay which chemically activated
with concentration of 0.5, 1.0, 1.5, and 2.0 M, Na-clay
which physically activated with concentration of 200;
300; and 400 0C, and the modification of clay surface
activation proceed with CTAB concentration of 25; 50;
75 and 100 mM.
Metal ion concentration calculated by using a standard curve. The difference in initial and final concentration of the Cr(VI) solution is the amount of Cr(VI)
which can be adsorbed by clay. Furthermore, the adsorption capacity can be calculated by equation [2]:
Qe =
(Co Ce) V
m
In which Qe is the adsorption capacity per weight of

clay (mg/g), V is the solution volume (L), C0 is the initial concentration of solution (ppm), Ce is the final concen-tration of solution (ppm), m is the clay mass (g).
G. Characterization of FTIR (Fourier Transform Infrared)
FTIR is used to identify the presence of key functional groups in the compound structure like clay. In this
study, natural clay, Na-clay, Na-clay which activated by
the best chemistry, Na-clay which activated by the best
physics, Na-clay which modified CTAB and CTAB are
characterized by using infrared light. Infrared spectra
are recorded using a Fourier Transform Infrared Spectrophotometer (FTIR) with KBr pellet method in the
region of 4000-400 cm-1 [5].
Natural clay which used in this study come from
Gandusari area Trenggalek regency which has analyzed
XRD by [7] and the result shows that natural clay is
composed of many components such as chloritoid, feldspar, kyanite, potassium magnesium, iron, alumunium,
titanium, phylloalumosilicate, cronstadite, and fayalite.
The XRD data also shows that the clay which used has
low crystallinity, it is characterized by diffractogram
peaks which are not sharp as in figure 1.
Figure 1. The result of XRD clay in Gandusari area [7].
A. Natural Clay Preparation with Beneficiation Method

Natural clay beneficiation process uses aquades as the
solvent for few days. Natural clay which diluted with
aquades will separate into 3 parts. Organic compounds
th
and dissolving salt are in the upper part, middle part of

clay, sand and other impurities with a higher specific
gravity than the clay which is at the bottom.
Clay beneficiation result sieved by using a sieve of
200 meshes to get smaller particles size and equal so it
will be obtained a larger clay surface area. The result of
the sieve which passes 200 meshes is used as a sample
test and is characterized with FTIR.
Adsorption method of Cr(VI) carried out by Batch
method in which Cr(VI) solution is contacted with natural clay, Na-clay, Na-clay which chemically activated,
Na-clay which physically activated, and Na-clay which
modified CTAB surfactant. Determination Cr(VI) uses
UV-Vis Spectroscopy method which reacted Cr(VI) solution with 1,5-difenilkarbazida until produces complex
compound that has purple color.
B. Adsorption of Cr(VI) in Natural Clays
The calculation result of adsorption capacity shows that
natural clay has Qe of 0.0971 mg/g. The adsorption result in natural clay is relatively small because there are a
lot of impurities so that its adsorption capability is low.
C. Na-clay Preparation and adsorption of Cr(VI) in Naclay
Natural clay which obtained still has a lot of other cations such as K+, Mg2+, and Ca2+ and also has unequal
cation size so that it should be done a uniformity in the
cation size which existing in interlayer clay area. This
uniformity is intended to produce the same layer distance and improve its adsorption ability.
The first stage uses a solution of NaCl 1 M which
done at 70 0C for 24 hours, it has an aim to initiate the
reaction of cations exchange besides Na+ so that it can
open and activate the space inter layers of clay. The
cations exchange expects that mostly cations other than
Na+ can be replaced by Na+. Heating process which
done has an aim to increase the likelihood of collisions
between Na+ ions with negative charge clay [15].
The second stage uses a solution of NaCl 6 M which
done at room temperature for 24 hours, it has an aim to
replace cations which have greater valence than Na+ can
be replaced by Na+.
The adsorption process of Na-clay with Cr(VI) obtains adsorption capacity of (Qe) 0,1756 mg/g with an
increase of adsorption capacity of 7,85 % from natural
clay. The result of Na-clay adsorption is higher than the
natural clay. This happens because Na-clay surface
which has negatively charge to cation Na+ can improve
the adsorption of Cr(VI).
D. Adsorption of Cr(VI) on Na-clay which chemically
activated
Chemically Clay activation has an aim to increase the
clay activation as an adsorbent. According to [5], they
stated that the using of H2SO4 can eliminate other impu-rities from lattice structure so that the clay physically
becomes active.
Chemical activation process of Na-clay done by soaking the variation of H2SO4 concentration of 0.5; 1.0;
1.5 and 2.0 M for 5 hours. The aim of treatment with
various concentrations to determine the effect of the
active property of resulting clay to the H2SO4 concentration in adsorbing Cr(VI).
The result of Cr(VI) adsorption on Na-clay which
chemically activated obtains a relation between Na-clay
which activated by various concentration of H2SO4 and
the amount of Cr(VI) which adsorbed per unit weight of
adsorbent (Qe) as Table 1 and figure 2.
Based on table 1 and figure 2, it can be observed that
with the addition of H2CO4 on the surface of Na-clay
can provide the result of adsorption of Cr(VI) which is
greater than the natural clay and Na-clay. This happens
because the activation of acid can increase the acidity
quite highly and can eliminate other impurities from the
lattice structure so that clay surface area becomes high
in which it can adsorb Cr(VI) greater [2].
Variations of H2CO4 concentration which used in
chemical activation affect the adsorption capacity of
Cr(VI). H2CO4 concentration which highly can adsorb
Cr(VI) anions is 0.5 M that indicated by Qe value of
0.3115 mg/g with an increase of adsorption capacity
21.44 %. This provides that acid activation process can
neutralize negative charge on the clay surface so that on
the side of active clay can be positive charge.
TABLE 1
THE RELATION Of ADSORPTION CAPACITY (Qe) Cr(VI) ON NATURAL CLAY, Na-CLAY AND Na-CLAY WHICH CHEMICALLY
ACTIVATED WITH VARIOUS CONCENTRATION OF H2SO4
Sample
Natural Clay
Na-Clay
Qe (U1)
(mg/g)
0.0963
Qe (U2)
(mg/g)
0.0851
Qe (U3)
(mg/g)
0.1098
0.1847
0.1664
0.1756
Na-Clay+H2SO4 0.5 M
0.3159
0.3099
0.3087
Na-Clay +H2SO4 1.0 M
0.2832
0.2748
0.2735
Na- Clay +H2SO4 1.5 M
0.2403
0.2429
0.2429
Na- Clay +H2SO4 2.0 M
0.2158
0.2089
0.2130
The higher of H2SO4 concentration of 1.0, 1.5, and

2.0 M decreased adsorption of Cr(VI) than with H2SO4
0.5 M. This is due to the possibility of damage and unstable clay structure which caused by the highest acid
concen-tration.
E. Adsorption of Cr(VI) on Na-clay which physically

activated
Clay structure with three-dimension cause clay has cavities which filled by water molecules. This research has
warmed clay which has chemically activated by using an
electric furnace at temperature of 200; 300 and 400 0C
th
for 6 hours. The heating process on clay has an aim to

evaporate the water molecules that cover clay surface so
that its specific surface area can increase [7].
Figure 2 The relation of adsorption capacity (Qe) Cr(VI) on natural

clay, Na-clay and Na-clay which chemically activated with various
concentration of H2SO4
The result of clay physical activation temperature variation followed by the adsorption process on Cr(VI)
which obtains a relation between clay physical activa-
tion temperature variation and the amount of Cr(VI)

which adsorbed by per unit weight of adsorbent (Qe) as
shown in Table 2 and figure 3. Determination of the
adsorption best temperature on Cr(VI) has an aim to
know the temperature of physical activation in which
Cr(VI) solution can be maximally adsorbed by adsorbent.
Based on the Table 2 and Figure 3 it can be seen that
when it heated, Na-clay which activated by the best
chemistry can provide greater Cr(VI) adsorption result.
This is due to the physical activation can vaporize water
molecules that cover clay surface so that the specific
surface area increases.
In this research, the temperature of physical activation
which is capable to adsorb Cr(VI) greater is temperature
of 200 0C that indicated by Qe value = 0.3953 mg/g
with an increase of adsorption capacity of 29.82 %. It
can be stated that at this temperature occurs a water release which placed in interlayer clay and H+ ions cannot
be separated [14].
TABLE 2
THE RELATION OF ADSORPTION CAPACITY (Qe) Cr(VI) ON NATURAL CLAY, Na-CLAY, Na-CLAY WHICH ACTIVATED BY THE
BEST CHEMISTRY, AND Na-CLAY ACTIVATED BY PHYSICS WITH VARIOUS TEMPERATURE
Qe (U1)
Qe (U2)
Qe (U3)
Sample
(mg/g)
(mg/g)
(mg/g)
Natural Clay
0.0963
0.0851
0.1098
Na-Clay
0.1847
0.1664
0.1756
Na-Clay+H2SO4 0,5 M
0.3159
0.3099
0.3087
Na-Clay+H2SO4 0,5 M+ 200 C
0.3965
0.3958
0.3937
0.3053
0.2970
0.3008
02757
0.2711
0.2731
creasing calcinations temperature (100-200 0C) so that

it can absorb dyes and decreased surface area on calcinations temperature (300-400 0C) [9].
Figure 3 The relation of adsorption capacity (Qe) Cr(VI) on natural

clay, Na-clay, Na-clay which activated by the best chemistry , and
Na-clay activated by physics with various temperature
At a temperature of 300-400 0C the adsorption capacity of clay to adsorb Cr(VI) decreased as indicated by
the value of Qe 0.3010 mg/g and 0.2733 mg/g. This is
might be due to the higher heating causes the higher
density of crystal structure and more regularly, so that
the clay is less reactive as adsorbent. According to [15],
heating at 300 0C to Na-bentonite was able to cause
damage to the 001 field. Clay will experience structural
and inter-layer damage at temperature above 250 0C
[12]. The surface area of the alunite increased with in-
F. Adsorption of Cr(VI) on Na-Clay Modified CTAB

Increasing adsorption of Cr(VI) can be done by modify-ing clay surface with CTAB surfactant as has reported by [2] that the result of modified clay with
HDTMA surfactant is the most effective process to increase the adsorption ca-pacity of Cr(VI). Adsorption of
Cr(VI) by natural clay modified CTAB is based on its
CMC concentration (critical micelle concentration) of 1
mM. This study has used the CTAB concentration exceed its CMC, they are 25, 50, 75 and 100 mM. It is
intended to form a bilayer or multilayer coating in which
positive charge groups are placed on the clay surface so
that by the form of bilayer or multilayer coating, it
makes the surface property of the clay will change to be
positive charge, so the ability of clay to adsorb Cr(VI) in
the form of anions will be more maximal [11].
The result of clay modification which activated by the
best physics with CTAB followed by adsorption of
Cr(VI) process. The result of adsorption can be known
that there is a relation between Na-clay modified with
various concen-tration of CTAB surfactant and the
amount of Cr(VI) which adsorbed per unit weight of
adsorbent (Qe) as in the Table 3 and Figure 4.
th
Based on the Table 3 and Figure 4, it can be seen that

with the CTAB modified on the clay surface which activat-ed by the best chemistry and physics can provide the
best adsorption result of Cr(VI) which is greater than
natural clay without modified with CTAB. This is due to
the pres-ence of CTAB surfactant which places on the
clay surface adsorb Cr(VI) greater.
The best concentration of CTAB modified on the clay
surface is highly able to adsorb Cr(VI), that is concentration of CTAB 25 mM of Qe 1.0425 mg/g with an
increase of adsorption capacity 94.54%. It can be stated
that at this concentration the positive side of CTAB is
on the clay sur-face, so that the clay surface is filled by
positive sides which neutralized by B- ion. In which this
Br- ion potentially as anion exchange with HCrO4anion so that the clay modified by CTAB is able to adsorb Cr(VI) maximally. At the concentration of CTAB
50; 75 and 100 mM decreased the adsorption of Cr(VI)
than with 25 mM CTAB which does not have any significant difference. It is assumed that the external surface
of the clay is not all filled by the posi-tive side of
CTAB.
TABLE 3
THE RELATION OF ADSORPTION CAPACITY (Qe) Cr(VI) ON
NATURAL CLAY, Na-CLAY, Na-CLAY ACTIVATED BY THE
BEST CHEMISTRY, Na-CLAY ACTIVATED BY PHYSICS AND
Na-CLAY MODIFIED WITH CTAB VARIATION
Sampel
Natural Clay
Na-Clay
Na-Clay+H2SO4
0,5 M
Na-Clay+H2SO4
0,5 M+200C
Na-Clay+H2SO4 0,5
M+200C+25
mM
Na-Clay+H2SO4
0,5
M+200C+50
mM
Na-Clay+H2SO4
0,5
M+200C+75
mM
Na-Clay+H2SO4
0,5
M+200C+100
mM
Qe (U1)
(mg/g)
0.0963
0.1847
0.3159
Qe (U2)
(mg/g)
0.0851
0.1664
0.3099
Qe (U3)
(mg/g)
0.1098
0.1756
0.3087
0.3965
0.3958
0.3937
1.0400
1.0454
1.0421
1.0333
1.0318
1.0336
1.0278
1.0279
1.0281
1.0271
1.0256
1.0259
Figure 4 The relation of adsorption capacity (Qe) Cr(VI) on Natural

clay, Na-clay, Na-clay activated by the best chemistry, Na-clay activated by physics and Na-clay modified with CTAB variation
Figure 5 Illustration of adsorption Cr(VI) in the form of HCrO4- on

the clay modified CTAB over its CMC.
The mechanism of adsorption Cr(VI) on natural claymodified by CTAB over its CMC as follows [14]:
Clay-(CTA)2+-Br- + HCrO4-
Clay-(CTAB)2HCrO4 + BrThe process of adsorption Cr(VI) on the clay which
modi-fied CTAB surfactant exceeds its CMC involves
anion ex-change between bromide ion of CTAB with
ionic species Cr(VI), so that there are more Cr(VI)
which adsorbed [14].
G. FTIR characterization
FTIR characterization has an aim to know the main
functional groups of clay. Spectra clay for infrared area
divided into two frequency groups, namely area between
4000 and 3000 cm-1 is the stretching vibration of the
adsorbed water or -OH octahedral groups and area in
1400 to 800 cm-1 which caused by a vibration of Al-OH
or Si-O.
Figure 6. IR spectra of natural clays, clay-Na, Na-activated clays

best chemistry, Na- The best physics of activated clay and Na-clay
modified CTAB best
Based on Figure 6, it can be stated that during the

treat-ment of chemical activation, physics and its surface
modification with CTAB surfactant do not
change/damage the clay structure. On the clay surface
modification treat-ments are new spectra at wavelengths
of 2851 and 2920 cm-1 indicating the presence of CH
stretching [11].
IV. CONCLUSION
The treatment of activation and modification on natural
clay can highly improve the adsorption capacity of
Cr(VI) as indicated by the value of Qe (adsorption capacity). Ad-sorption capacity of natural clay has Qe =
0.0971 mg/g and Na-clay with Qe = 0.1756 mg/g with
an increase 7.85 % of natural clay. Clay which activated
by H2SO4 0.5 M has an adsorption increase of 21.44 %
with Qe = 0.3115 mg/g. Natural clay physically activated after activated by the best chemistry has obtained
at temperature of 200 0C with Qe = 0.3953 mg/g which
indicates an adsorption increase of 29.82 %. While the
best concentration of CTAB surfactant on modified natural clay has obtained at CTAB concentra-tion 25 mM
with the largest value of Qe 1.0425 mg/g which indicated by an adsorption increase of 94.54 %.
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Selective Hydrogenation of FurfuralUsing

Ni/-Al2O3 Catalyst
SitiMariyahUlfa*and ElvinaDhiaulIftitah
Faculty of Science, Brawijaya University, Malang, Indonesia
*
Corresponding author:ulfa.ms@ub.ac.id
AbstractLiquid phase selective hydrogenation of furfural to furfuryl alcohol on Ni/-Al2O3catalyst was studied.
The influence of different amount of impregnated nickel
salt in aluminum oxide and reaction time on catalytic activity and selectivity were examined. Among 5%, 10%, and
15% Ni/-Al2O3, the last catalysts provide the best result.It
has been found that15% Ni/-Al2O3gave the conversion of
furfural up to 15% and selectivity for furfuryl alcohol as
high as 100%. This result is obtained under 150oCafter 150
minutes of reaction time.
Keywordshydrogenation, furfural, furfuryl alcohol,
nickel
ported as a furfural hydrogenation catalyst [6], but the

utilized of such precious metal in this system is economically high cost. Thus, different catalysts, such as copper
supported on activated carbon, Raney Ni, and amorphous Ni alloys dispersed on different support have been
used in the hydrogenation of furfural, not only in liquid
phase but also in vapor phasereaction [7].
In the present work, the hydrogenation of furfural on
a series of 5%, 10%, and 15% Ni supported on -Al2O3
have been studied. The effect reaction time on the activity and conversion of furfural (FFald) to furfuryl alcohol
(FFalc) is investigated.
I. INTRODUCTION
II. EXPERIMENTAL
The chemistry of furfural is well developed and reported

as promising chemicals since its derivatives gives variety applications in the chemical industry. Furfural is
mainly used as intermediate chemical in the manufacturing of solvent, such astetrahydrofuran (THF), methyl
tetrahydrofuran (MeTHF), furfuryl alcohol, plastics, and
agrochemicals [1]. One important chemical coming from
furfural is furfuryl alcohol. This compound is mainly
used in the manufacture of resins, as a starting material
for the synthesis of tetrahydrofurfuryl alcohol (THFalc),
important intermediate for the manufacture of fragrance,
vitamin C, and lysine [2].
Furfuryl alcohol (FFalc) is prepared industrially by
catalytic hydrogenation of furfural.The two ways to produceFFalc is liquid and vapor hydrogenation. In liquid
phase, high-pressure and temperature is required. In a
vapor phase, it is depend on the type catalyst used in the
reaction [3]. For over five decades, copper cromite has
been the most powerful catalyst for furfural hydrogenation in liquid and vapor phase with selectivity up to 98%
[4]. The severe drawback from Cu-Cr based catalyst is
their toxicity, which causes experimental problem. Many
attempts have been made to develop new catalysts that
are environmentally friendly.
Furfural is suitable compound to test the selectivity of
catalysts because the presence of both C=O and unsaturated C=C bonds in the structure. It is well known that
group 9 and 10 metal, such as Rh, Ir, Ni, Pd, and Pt,
generally an active catalysts to hydrogenate C=C rather
than C=O in ,-unsaturated system [5]. Then, the necessary modification is required to improve selective
hydrogenation of C=O group. Platinum has been re-
A. Chemicals
Furfural, with a purity of >98% was obtained fromMerck Germany used without distillation.High-purity
hydrogen gas (>99.99%) from local vendor was used
without further purification. Nickel nitrate hexahydrate
(Ni(NO3)2.6H2O)and aluminum oxide (-Al2O3) were
supplied by Merck Germany.
B. Preparation of catalysts
The catalysts were prepared according to the method
given by Miloneet al. [8]. Aluminum oxide (-Al2O3)
having surface area 150-400 m2/g, pore size 0.5-1 cm3/g
with radius 3-12 nm was slowly added to methanol solution contain5%, 10%, and 15% ofNi salt and stirred for
24 h at ambient temperature. The solvent were slowly
removed by rotary evaporator at 35 oC for 1 hour. All
the catalysts were dried at 120 oC for 2 hours and calcined at 450 oC for 3 hours. Before hydrogenation reaction, all the catalysts were reduced at 450 oC for 5 hours.
C. Catalysts characterization
The characteristic of Ni/-Al2O3 catalyst were investigated by X-ray diffraction (XRD, Philips Xpert with
Ni-filtered Cu K radiation) operated at 40 kV and 30
mA. The surface morphology and the particle size was
determinedby Scanning Electronic Microscopy (SEM)
TM3000. The BET surface area was evaluated from
nitrogen adsorption isotherms at 77 K performed in
Quantochrome NovaWin2.
th
D. Liquid phase hydrogenation of furfural

Catalytic hydrogenation of furfural was performed in
modified two-neckedglass reactor fitted with sampling
and hydrogen valve. Before used, the Ni/-Al2O3catalyst
(0.5 g) wasreduced in glass reactor with hydrogen
flowed for 30 minute at 100 oC(95 mmHg). The activation of catalysts was repeated for three times to completely replace air in the reactor. Then 5 ml of furfural
were added to the reactor and stirred. After the necessary connection between reactor and the hydrogen gas
cylinder was duly made, H2 passed into the reactor and
reaction temperature gradually increased up to 150 oC.
The reaction time was counted after the setting temperature obtained. The progress of the hydrogenation reaction maintain by sampling a sufficient number of microsamplein 30, 60, 90, 120, 150, and 180 minutes. After
the reaction complete, all the products analyzed by
means of a Shimadzu QCMS-QP2010S gas chromatograph spectrometer massa with RastekstabilwakR-DA
column and FID detector.
The XRD patterns of the 5Ni/Al and 10Ni/Al catalysts compared to -Al2O3 after reduced at 450oC under
hydrogen were depicted in Figure 2. The sharp diffraction at 2 = 44 and 66o correspond to the -Al2O3(440).

A. Catalysts characterization
Figure 1 compile the profile of surface morphology
(SEM) of 5% Ni/-Al2O3(5Ni/Al), 10% Ni/-Al2O3
(10Ni/Al), and 15% Ni/-Al2O3 (15Ni/Al). Due to the
different concentration of nickel deposited on Al2O3, the
distinct differences of the morphology and particle size
were observed. The particle size of catalyst (A) 5Ni/Al
is in the range 50-100 m. The sample (B) 10Ni/Al andsample (C) 15Ni/Al is in the similar particle size range
30-60 m. The smaller particle size implies a higher
surface energy of the particle [9].
Figure 2. XRD spectra of the -Al2O3, 5Ni/Al, and 10Ni/Al reduced at 450oC
The new peaks appear at the 2 = 44.5, 52, and 76o in

agreement with Ni(111), Ni(200), and Ni(222) species,
respectively [5]. The specific diffraction of NiO at 2 =
43 and 63 didnt observed which indicate that Ni2+
transformed to Ni0 after H2 treatment at 450oC. Another
peak detected were at 2 = 37 and 66o correspond with
NiAl2O4 from the reaction of Ni2+ with -Al2O3.
TABLE 1.
BET SURFACE AREA AND POROSITY OF THE CATALYSTS
Catalyst
BET surface
area (m2/g)
Pore volume
(cm2/g)
Average pore
diameter ()
5Ni/Al
10Ni/Al
15Ni/Al
123.919
108.270
101.905
0.252
40.8268
0.221
41.0688
40.7094
0.206
Figure 1. SEM of (A) 5Ni/Al; (B) 10Ni/Al; (C) 15Ni/Al calcined

at 450oC
The BET surface area and the pore volume of the synthesized Ni/-Al2O3 catalysts are shown in Table 1. The
BET surface and the pore volume of 5Ni/Al is the largest compared with 10Ni/Al, and 15Ni/Al. No significant
change in the pore size distribution from 5Ni/Al,
10Ni/Al, and 15Ni/Al was observed. The increasing of
the salt loading decreases the surface area as well as the
pore volume of -Al2O3. The decreasing of surface area
th
was attributed to nickel which fills up the pores of the

support [8].
B. Influence of reaction time on activity and selectivity
The hydrogenation of furfural (FFald) was studied at
150 oC (95 mmHg) in the modified glass reactor.
Scheme 1 showed the hydrogenation of furfural on
5~15% Ni/-Al2O3. The result showed a continuous decrease in the furfural (FFald) concentration and the formation of furfuryl alcohol (FFalc) as a majorreaction
product. Unreacted 5-methylfurfural (MF) was also observed in the final reaction product. It is suggested that
the catalyst is highly selective to reduce C=O bond from
furfural but unreactive for any starting material.According to the detection of MF, lies in fact that all
raw materials used for the manufacture of furfural contain some methyl pentosanwhich might hydrolyzed to
MF [1]. The direct catalytic reaction of alkylmethylfurfuralwithin the starting materialalso reported to give MF
[10].
Scheme 1. Hydrogenation product of furfural on 5~15% Ni/-Al2O3
The conversion of FFald and the selectivity of FFalcon 5Ni/Al, 10Ni/Al, and 15Ni/Al are shown in Figure
3. Product conversion by the activity of15Ni/Al gave the
highest result compared to 5Ni/Aland 10Ni/Al. This
catalyst converted FFald to FFalc up to 15% within 150
minutes of reaction time. The 5Ni/Al catalyst is considerably as good catalyst by 2.1% conversion of starting
material after 120 minutes. Theactivity of 10Ni/Al
showed 2.0% conversion ofFFald after 180 minutes. It is
noteworthy that the higher the salt loading gives the increasing activity of the catalysts. However, conversion
of FFald to FFalc should be optimizedto increase the
product formation. Mki-Arvela reported that temperature of the reductionand time were influence the activity
of the catalysts. Catalytic activity and selectivity of
Au/TiO2 catalyst in the hydrogenation of crotonaldehyde
exhibit a maximum after increasing catalysts-reduction
temperature [11].
The selectivity formation of FFalcbyhydrogenation
reaction of FFaldon Ni/-Al2O3 is depicted in Figure 3.
It observed that within 60 minute of reaction time, the
selectivity of 5Ni/Al increased sharply from 27.9% to
85.7% then prolonged the reaction to 120 minutes completely reduce FFald to FFalc up to 100% conversion.
Similarly, 10Ni/Al and 15Ni/Al also exhibit a similar
selectivity, raised up from 63.4% and 77.5% to 100%,
after 60 minute of reaction time. However, prolonged
the reaction time until 180 minutes reduce the selectivity
of 5Ni/Al catalyst to 46.9%, but 10Ni/Al and 15Ni/Al
remain unchanged until 180 minutes of reaction time.
Figure 3. Conversion of furfural and selectivity for furfuryl alcohol as a function of reaction time. Reaction conditions: T = 150 oC
(90 mmHg); furfural = 3 ml; catalyst = 0.5 g;
and
for
5Ni/Al;
and
for 10Ni/Al;
and
for
15Ni/Al.
It is likely that within the range of the reaction time, all

the catalysts showed the higher selectivity to the formation of FFald. However, the decreasing selectivity of
5Ni/Al after 180 minutes is considered bythe formation
of side product.Structure determination of side product
is under consideration.Baijunet al.reported the formation of furfuryl alcohol (FFald) and tetrahydrofurfuryl
alcohol (THFald) are parallel reaction, whereas THFald
is the final product from further hydrogenation of FFald
[12].
IV. CONCLUSION
A series of 5~15% Ni/-Al2O3 catalysts were prepared
by wet impregnation method. The characterization of
catalysts conducted by SEM, XRD and BET surface
area. The activity of these catalysts utilized for hydrogenation reaction of furfural to furfuryl alcohol at 150 oC
for 30 to 180 minutes of reaction time. On the basis of
the result presented herein, 15% Ni/-Al2O3showed the
higher activities among others by 15% conversion of
furfural to furfuryl alcohol with the selectivity up to
100% after 150 minutes reaction.
ACKNOWLEDGMENT
This work was supported by DPP/SPP research grant
through DIPA Faculty of Science, University of Brawijaya No. 16/UN10.9/PG/2013and Student Creativity
Program (PKM-P) research grant from The Directorate
General of Higher Education, Indonesian Ministry of
Education.
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A. Corma, S. Iborra, and A. Velty, Chemical routes for the
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Selective one step synthesis of ()menthol from (+)citronellal
on Ru supported on modified SiO2, Appl. Catal. A: Gen., vol.
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NiP, NiB, and NiPB ultrafine materials, Ind. Eng. Chem.
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[12] L. Baijun, L. Lianhai, W. Bingchun, C. Tianxi, and K. Iwatani,
Liquid phase selective hydrogenation of furfural on Raney
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Appl. Catal. A: Gen., vol. 171, pp. 117122, 1998.
th
A New Oxalate Ion Sensors Based on Chitosan

Membrane
Atikah1*), R. Retnowati2), H. Sulistyarti3), B.Siswojo4), Z. Rismiati5)
Department of Chemistry, Faculty of Mathematics and Natural Sciences,
University of Brawijaya
4)
Department of Electrical Engineering, Faculty of Engineering, University of Brawijaya
1,2,3,5)
*)
Corresponding author: atikah_chem@ub.ac.id
AbstractThe aim of this research is to prepare

potentiometric sensor prototype as a coated wire oxalate
ion selective electrodes (CWE) for urinary oxalate
determination by potentiometric method. The sensor is
composed of a platinum (Pt) wire that is coated directly on
the membrane surface.The membranes sensor consist of a
mixture an active material of Chitosan was protonation
using acetic acid 3%v/v in order to have anion exchange
properties and Aliquat-336-oxalate as additive material,
polyvinylchloride (PVC) as supporting material,
dibuthylphtalate (DBP) as plasticizer = 4:1:33.5:61.5 (%
w/w) dissolved in tetrahydrofuran (THF) solvent (1:3
w/v).The characterization of the basic properties of sensor
included : sensitivity and linearity of response (detection
limit), response time, influence of pH and temperature,
soaking time, selectivity against foreign ions and also life
time. The sensor shows a good Nernstian slope of 29.9
0.1mV/ decade in wide linear range concentration from 1.0
10 -5 to 1.0 10 -1 M .The detection limit of 2,56x10-6M
(0.22 ppm),respond time fast (20 seconds) and was found
usable in pH range of 3.0 7.0 and temperature of 2050oC. Selectivity was obtained over HPO42-,SO42-, PO43-, Cl,H2PO4-,I-,SCN- ,creatinine and also urea thats contained
in the urine. The electrode is reproducible and stable for
nearly 2 months. This kind of CWE was successfully applied in determination of oxalate anion in urine samples at
concentrations corresponding levels of oxalate kidney
stones light and medium provide average accuracy of
98.72% and an average precision of 99.81%.
KeywordsCoated Wire Ion Selective Electrode (CWE),
potentiometric sensor ,Chitosan, oxalate,membrane.
I. INTRODUCTION
ROLITHIASIS is a symptom that is mostly caused
by a multifactorial metabolic disorder. Originators
in part because a diet rich in fat and protein,
lacking fiber intake combined with inactivity, resembling the so-called modern industrialised life style and
genetic predisposition enhance developing urolithiasis
[4] Typical symptoms of an acute stone colic are, inter
alia, agony, sickness and hematuria. Urinary calculus
formation is caused by disturbed urinary compositions
with altered urinary pH, increased concentrations of
lithogenic components as, e.g. calcium, oxalate, phosphate and a lack in inhibitoric substances as, e.g. citrate
and magnesium. Calcium oxalate represents the most
frequent mineral phase found in uroliths with a frequency of approximately 7075% [1]
Oxalate is one of the important nutrients in the human

diet found principally in spinach, beet leaves, etc. Oxalate is primary chelator of calcium ion, so it forms chelates with calcium. Oxalate ion also inhibits the calcium
adsorption in the body and if it is not sufficiently degraded, it may accumulate in the body. It plays a crucial
role in the formation of most renal stones. In the body
oxalates can be found as two forms in vivo: oxalate ions
and calcium oxalate monohydrate (COM) crystals that
readily form in the presence of calcium[ 2]. Owing to
the high recurrence rate of calcium oxalate stone formation in case of inadequate treatment, evaluation of the
individual causes for calcium oxalate urolithiasis is of
utmost clinical importance [1]. Urinary stone formation
has been evolved to a widespread disease during the last
years. The reasons for the formation of urinary stones are
little crystals, mostly composed of calcium oxalate,
which are formed in human kidneys. The early diagnosis
of the risk for urinary stone formation of patients can be
determined by the Bonn-Risk-Index method based on
the potentiometric detection of the Ca2+-ion or oxalate
ion concentration and an optical determination of the
triggered crystallisation of calcium oxalate in unprocessed urine [1].
Most of the analytical methods like ion chromatography, neutron activation analysis, atomic absorption spectrophotometry and mass spectrometry etc. have been
reported for the determination of oxalate ions at a very
low concentration levels, but these methods require expensive instrumentation, delicate and expert handling of
the sample and instrument. Hence potentiometric determination based on ion selective electrodes offer several
advantages such as ease of preparation, low cost, simple
procedure and easy instrumentation. It gives relatively
fast response, wide linear range, good selectivity, high
detection limit and online applications as compared to
other analytical methods[1,3].
During the last decade, a number of studies have focused on properties of functionalized chitosan, a relatively new class of an adsorbent, chelating agent and ion
exchanger. Chitosan, a poly-[1-4]-D-glucosamine, is a
derivative of chitin, a naturally occurring polysaccharide
found in insects, arthropods and crustaceans. Chitosan is
hydrophobic, bio-degradable, bio-compatible and low
toxicity so widely used for various applications including pharmaceutical, biotechnology and wastewater
treatment. Chitosan is well known for complexing transition metal ions, through chelation at its amino
group[1]. It was also shown that chitosan can be used as
th
potent ionophores for the preparation of ion selectivesensors[4,5]. The potentiometric selective coefficients of
ISEs sensitive to organic anion included oxalate ion
have been reviewed in detail. The application of function so far, several experimental studies have demonstrated that the generation of a membrane potential of
those type of ISEs could be attributed to permselective
ion transport across the liquid membrane/solution interface, i.e., charge separation through a preferential uptake
of a primary ion by a sensing element in the liquid
membrane, leaving its hydrophilic counter ion in an
aqueous sample solution and usually exhibit the Hofmeister pattern with the largest selectivity to lipophilic
cations [6,7].
These can also be used in complex and coloured media. Therefore, there has been progressive growth in the
development and application of potentiometric sensors
based on polymeric membrane ion selective electrodes
incorporating ionophores for the detection of different
cations and anions and other biologically important
compounds[3]. Recent studies in different laboratories
showed incorporation of different novel materials as
ionophores for the ion selective electrodes[4]. A strong
interaction of the anions and the ionophore as well as the
steric effect associated with the structure of the ligand
gives rise to selectivity sequence. Thus the research on
sensing materials for anion as well as developments including new synthetic ionophores, miniaturization of the
detecting device like coated wire selective ion electrode
(CWE) etc. makes it an ever-expanding culture for research in chemical sensors [2,8].
In this paper, we wish to introduce a highly oxalate
ion selective potentiometric sensor based on a heterogeneous membrane of chitosan as ion carrier membranes
and Aliquat-336-oxalae as additive material supported
by polymeric polyvinyl chloride (PVC) of high molecular weight and plasticizer dibutyl phthalate (DBP) then
its application for the determination of urinary oxalae
ion as early diagnosis of the risk for urinary stone formation of patients can be determined by the Bonn-RiskIndex method based on the potentiometric detection of
the oxalae ion concentration combine with Ca2+ determination by atomic absorption spectrophotometric (AAS)
and their result compared by an optical determination of
the triggered crystallisation of calcium oxalate in unprocessed urine.
II. METHODOLOGY
A. Apparatus and emf measurements
All potential measurements were performed using the
following assembly: Hg, Hg2Cl2 (Satd)//sample solution/PVC membrane/Pt-wire electrode. A pH-meter
(Fisher E 520) was used for potential measurements at
26C 0.5oC. The activities of ioxalae ion (C2O42-) ions
in the urine were calculated according to the Debye
Hckel approximation.
B.Reagent and solution
Chitosan powder isolation results from the shell of
jerbung shrimp (Penaeus merguinensis) with a degree of
deacetylation 68% (w/w) is use as ionophore was
protonated using Acetic Acid (3%), Aliquat-336 oxalae
as additive material,polyvinyl chloride (PVC) of high

molecular weight , dibutylphtalate (DBP) as a plsticizer
were purchased from sigma, tetrahydrofuran is products
from E.Merck. Platinum wire (99,9% ; 0.5 mm) is
products from Aldrich and RG-58 Coaxial cable as
connector ISE to mV potentiometer. All other reagent
used were of analytical reagent grade, and doubly distilled water was used throughout. Ca oxalate, Acetic
Acid (3%), Aliquat 336-S,NaOH, Na3PO4, CaCl2,
creatinine, uric acid.
C. Construction and calibration of the electrodes
The membranes electrode was prepared by mixing
thoroughly by dissolving protonated chitosan, Aliquat336-Oxalat, PVC, DBP plasticizer in THF solvent (1:2
v/w). This solution was deposited directly onto a platinum wire approximately 0.5 mm in diameter and 10 cm
in length whose tip had been melted in flame to form a
spherical button was soldered to a length of RG-58
coaxial cable, and the solvent was evaporated for approximately 30 minutes and then allowed to stand overnight in the oven at 50oC. A membrane was formed on
the platinum surface and the electrode was allowed to
stabilize overnight. Prior to use the electrode was initially conditioned by soaking it overnight in a 0.1M solution of Naoxalate (Na2C2O4) to be measured. When not
use, the electrode was store in air between use and reconditioning immediately before using by soaking for at
least 1 hour in a 0.1M solution of Na2C2O4. The utility,
composition of polymer membrane, respond characteristic, and selectivity coated wire oxalae ion selective electrode (CWE) were investigated. The electrode potential
measurement was made under constant conditions by
taking 25 mL of solution for each measurement in a cell
thermostated at 26 0.5 oC , immersing the electrode to
a constant depth in the solution, and stirring at a constant rate by means of a magnetic stirring bar. In all experiments the electrode potential measurement was carried out from low concentration to high concentration.
The electrode tip was rinsed with deionized water and
then immersed in one of the standard solution.
D. determination of oxalate ion in urine
Urine samples were taken from the Central
Laboratory of Clinical Pathology Hospital laboratory
which have completed used for clinical pathology
examination (samples are no longer used or discarded)
of patients with kidney stones (calcium oxalate crystals
containing microscopic examination based on positive
optical crystal with no indication of the patients showed
symptomatic urolithiasis) taken from 50 patients with the
risk of kidney stones and high light each 25 samples and
10 urine samples taken from normal patients as a whole
amounted to 92 samples. Each urine sampel put in a
polyethylene tubes. The samples were immediately centrifuged and stored at 4oC. A 1.0 mL aliquot of the sample was transferred into a 10-mL measuring flash and
diluted with distilled water. For each analysis, the oxalae
sensor and double junction Ag/AgCl reference electrode were immersed in the same solution, and the potential reading were recorded. A typical potentiometric
calibration plot was made by plotting the potential
th
change against the logarithm [C2O42-] concentration. The

obtain calibration curve was used for subsequent determination of C2O42- in unknown samples. The results of
determination of C2O42- on both the optical microscopy
method and the potentiometric method using oxalate
ion sensors tested for their accuracy, precision and also
for early diagnosis of the risk for urinary stone formation
of patients can be determined by the Bonn-Risk-Index
(BRI) method based on the ratio of potentiometric detection of the urinary oxalae ion and Ca2+-ion concentration
by AAS and their result compare to an optical microscopy determination of the triggered crystallisation of calcium oxalate in unprocessed urine.
III.
RESULT AND DISCUSSION
A. Influence of membrane composition

The different aspect of membrane preparation based
on protonated chitosan as ionophore and Aliquat 336oxalate as additive material containing different
PVC/plasticizer ratios were mix in THF solvent (1:2
ratio v/w) were studied and the results revealed that the
amount of ionophore, the nature of solvent mediator, the
plasticizer/PVC ratio significantly influence the sensitivity of ion selective electrodes. Membrane with a
composition ratio of wt% PVC: DBP: the active
ingredient Chitosan-oxalate; additives Aliquat 336oxalate = 4: 1: 33: 62 in THF 1: 3 volume gives
Nernstian properties with prices Nernst factor of 29.9
mV / decade concentration, means that the optimum
composition meets the theoretical Nernst factor for
monovalent anion, because the active ingredient
membrane forming a homogeneous phase with
membranes visible supporter of the smallest dm price
for the active ingredient chitosan [4,5]. Non Nernstian
response of the oxalate ion CWE, most probably due to
saturation or non-uniformity of the membrane. Use of
the DBP plasticizer as a solvent mediator for preparing
a coated wire oxalate ion-selective electrode(oxalae ion
CWE) need to fulfill four principal criteria: high lipophilicity, solubility in the polymeric membrane (no crystallization) as well as no exudation (one phase system) and
good selectivity behavior of the resulting membrane. It
should be noted that the nature of plasticizer influences
both the dielectric constant of the membrane and the
mobility of ionophore and its complexed associatiated
with oxalae ion [9,10]. Thus, based on the result obtained on the optimazation of the membrane composition, the membrane 4 with the optimized composition of
percent ratio (w/w) of the active ingredient protonated
chitosan -oxalate; Aliquat 336-oxalate additives::
PVC:DBP = 4: 1: 4: 1 in THF 1: 3 volume was selected
for preparation the polymeric membrane electrode for Iion. Nernstian responses obtained on the composition
ratio of PVC / plasticizer 1.2 as obtained by other
researchers [3].
The specifications of oxalate ion CWE base on
chitosan carrier are as follows: Sensitivity (Nernst factor) of 29,9 0,252 mV / concentration decade of oxalate concentration over the range 1.10-5-1.10-1 M, with
the detection limit of 42.56x10-6 (0.22 ppm), They have
relatively fast response (20 seconds), satisfactory
reproducibility, and life times more than two months
and was found to be very selective toward oxalate ions

with the selectivity sequence against foreign ions in
order: oksalat2-> HPO42->SO42-> PO43->Cl-H2PO4-ISCN-> kreatinin> urea. The observed selectivity pattern for proposed sensor significantly same from the
Hofmeister selectivity sequence (i.e. selectivity based on
lipophilicity and charge density of anions), usable in
wide pH range of 3-7and temperature of 20-50oC, need
soaking time of 75 minutes in 0.1M in oxalate solution.
This result states that oxalate ion CWE has a optimal
character for the potentiometric measurement of oxalate
analysis. However, the lipophilicity of the anion still
plays an important role, and only the simultaneous consideration of both the lipophilicity and interaction of the
anion with zeolite allows one to explain the selectivity
patterns. Therefore, ion exchange selectivity is mainly
determined by two factors:i.e the charge of an ion and
its solvation, since the interaction between anions and
ion exchange groups on chitosan is electrostatic [8].
B. Application
The new coated wire oxalae ion selective electrode
was satisfactorily applied to the determination of oxalae
ion cover from 9 urine samples of kidney stone patients
were examined in the Clinical Pathology Laboratory and
measurements performed 20 times at room temperature
27 1oC.The analysis were performed by direct potentiometry using the standard curve technique. Good recoveries in all matrices were obtained. From this results
we can conclude that the proposed sensor was successfully applied to determining the oxalae content in biological samples .The results obtained that coated wire
oxalate ion selective electrode can be used as sensors for
the determination of oxalate in urine to detect renal
stone disease before clinical symptoms arise with giving
rat average accuracy of 98.72% and an average
precision of 99, 81% which shows measurement
accuracy and good precision.
Distribution potentiometric measurements oxalate
concentration in urine and measurement of calcium ions
in the urine by AAS method and also early detection of
urolithiasis risk categories based on price of BRI and
optical microscopy base on the statistical test Chi
Square. Percentage error determination urolithiasis risk
category according the Bonn-Risk-Index (BRI)
method (The BRI method is based on the potentiometric
detection of the free Ca2+-ion concentration(activity) by
means of an ion-selective electrode (ISE) together with
an optical determination of the induced crystallization of
calcium oxalate in native urine. The BRI is determined
as ratio: BRI = [Ca2+]/(Ox2-) [1] and optical microscopy
said that Count X2 = 72.35 while the table on the
confidence limits P X2 = 95% and degrees of freedom
(DB) = (3-1) (3-1) = 4, then X2 (0.95) (4) = 9.49.
Arithmetic mean X2> X2 table, which means there is a
very real relationship between the % difference BRI
results using oxalate sensor with each category on the
risk of urolitiasis. It means the measurement results
urinary oxalate causing urolithiasis in all population
categories are independent. The validation results of
potentiometric method according to the calculation
th
results indicated by the percentage of correspondence

between the two methods, i.e., 69.6% or 30.5% gives an
error, that is 12% sensor method smaller than the
microscope method and 18.5% error sensor method
greater than microscope metod.To test whether %
differences in BRI outcomes toward the optical
microscope with a population of BRI category is
independent or not chi squared test needs to be done and
the result test state that BRI method are sensitive and
specific for high-risk category of patients suffering from
urolithiasis because of the formation of calcium oxalate
crystals. For patients with a high risk of urolithiasis
giving a sensitivity of 75% and specificity of 75%.
Moderate risk for urolithiasis patients, giving a
sensitivity of 95% and specificity was also 60%, whereas
the risk for urolithiasis patients with mild risk gave a
sensitivity of 86.6% and a specificity of 72.1%. Thus
potentiometric method can be used as an alternative
method besides optical microscopy method
IV. CONCLUSION
The membrane composition influence the Nernstian
character of oxalae sensor. The membrane with the
composition of ratio of wt% the active ingredient
Chitosan-oxalate; additives Aliquat 336-oxalate: PVC:
DBP= 4: 1: 33: 62 in THF 1: 3 dissolved in THF solvent
(1:2 w/v) was selected for preparation the polymeric
membrane electrode for oxalae ion and can be use as
chemical sensor for oxalae ion in the construction of
coated wire oxalae ion selective electrode which has
optimum characteristics for oxalate ion analysis. Method
validation results showed that oxalate ion CWE
produced have optimum characteristics for sensor
oxalate ions suitable for urinary oxalate analysis provides of an accuracy of 98.72% and precision of
99.81%beside to the soptical microscopy.
This kind of CWE was successfully applied to detect
high-risk patients with urolithiasis (sensitivity 75% and
specificity 75%), moderate risk of urolithiasis
(sensitivity 95% and specificity 60%) as well as mild
risk of urolithiasis (sensitivity 86.8% and specificity of
72.1% ), have compatibility with the optical microscope
method of error of 69.6% or 30.5% error, that is 12%
smaller than of optical microscope and 18.5% larger

than of optical microscopy method
ACKNOWLEDGMENT
The study was funded by Competitive Research Grant,
the Directorate General of Higher Education, Indonesia
Ministry of National Education with the contract number: 366/SK/2012 To the Ministry of National Education
and University of Brawijaya are gratefully Acknowledged
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[6] Pretsch,E. (2007). The New Wave of Potentiometric Ion Sensors,Trends in Analytical Chemistry., 26(1): 46-51 from
http://www.elsevier.com/locate/trac
[7] Okada,M.H, and T.Ohki, (2009), Hydration of Ions in Confines
Spaces and IonRecognition Selectivity, Analytical Science., vol.
25,pp 167-175,
[8]
Gustavo , A.D., A.Z-Guilln, G. A. Crespo, S. Macho and J. R.

F. X. Rius.(2011). Nanostructured Materials in Potentiometry,
Anal
Bioanal
Chem,
399:171181
from
http://www.elsevier.com/
[9] Sulekh C, S. Raizada and S. Sharma (2012) Highly selective
oxalate
membrane electrode based on [CuL, IOSR Journal
of Applied Chemistry (IOSRJAC) ISSN : 2278-5736 Volume 1,
Issue 5 (July-Aug 2012), PP 39-48 www.iosrjournals.org
[10] Stefan R. I. Draghici, G. E. Baiulescu (2000), Sensors and
Actuators
B, 65, 250252
th
PHYSICS
th
Relocation of Hypocentrum Earthquake in

Mentawai Island Region as Data Support
Determination of Earthquake Early Warning
1)
Ahmad Marzuki S 1*), Munawarah1), and Iven Ganesja 1)

Department of Physics, University of Indonesia, Depok, Indonesia
*)
Corresponding author : ahmad.marzuki@ui.ac.id
AbstractIndonesia is one of the most country in the

world which has the high tectonic activity. It caused by
ring of fire passed Indonesia Region. Ring of fire zone is
the subduction zone or convergent plate motion. It cause
earthquake often occured on Indonesia region. Mentawai
Island region is one of the common area of earthquake in
Indonesia region. This area located at west of Sumatera
which subduction zone between Eurasia plate tectonic
(continental crust) and Indo-Australia plate tectonic
(oceanic crust). Relocation of hypocentrum earthquake on
this area is important because it as early warning
determination. The relocation hypocentrum earthquake on
this
area
using
Modified
Joint
Hypocentrum
Determination (MJHD) method.The earthquake data on
this area is taken from 2009-2012. Moreover our research
create seismicity mapping in the Mentawai Island region
based on result of relocation hypocentrum earthquake
using MJHD method. The results used to early warning
determination on Mentawai island as to minimize seismic
hazard
II. RESEARCH METHOD

The method on this research is using Modified Joint
Hypocentrum Determination (MJHD). This method is
created by [1], Japan seismologist (1990-1992), This
method is used to determination relocation of
earthquake. The following inversion steps of this method
is :
Start
Reformat data to
mjhd format
Input data for mjhd07.f

: mjhd07.inp
No
mjhd07.f
KeywordsEarthquake, MJHD, Subduction zone, Plate

tectonic
I. INTRODUCTION
NDONESIA is one of the most country in the world
which has the high tectonic activity. It caused by
indonesia has three plate tectonic motion, they are
Eurasia, Indo-Australia, and Pasifik. This subduction
zone is called ring of fire zone. It starts from west of
Indonesia region continuous to east of Indonesia region.
That region to common area of earthquake on Indonesia.
One of our focus is Mentawai island, it located on the
west of Sumatera. This zone belonging subduction zone
(Eurasia plate tectonic and Indo-Australia plate
tectonic), and the common area of earthquake on the
west of Sumatera Island. Moreover, the geological
setting at this area is dominated sedimentary and mixing
from continental and oceanic. Relocation of
hypocentrum earthquake on this area is important
because this relocation of hypocentrum to identification
tectonic condition on this area including the knowing the
seismic gap which may be a big source of earthquake in
the future. The results is used to early warning
determination as to minimisize seismic hazard especially
for occupant on this area and the surrounding.
Station selection
process : station.f
The desired residual
mjhdoutselect.f
mjhd.outp
mjhd.out
mjhd.print
Yes
STOP
STOP
The earthquake parameters is lattitude and longitude

of earthquake, time of common earthquake, the change
of that parameters influence the change significant the
hyprocentrum of earthquake depth. The excellent of this
method calculate many hypocentrum of earthquake
simultaneously and correction about lateral heterogenity
in subsurface. Lateral heterogenity in subsurface
attenuate the p-wave propagation, the effect is
mislocation the hypocentrum of earthquake. The input
data is taken from 2009-2012 years and recorded by
th
BMKG (Badan Meteorologi dan Geofisika). The data

has magnitude > 5 SR, including time of common
earthquake, the recorded station, arrival time p-wave,
longitude and lattitude coordinate, and the depth of
hypocentrum.
The start of inversion using MJHD step of determine
the value of Minimum Number of Earthquake at Each
Station (MNEQ), and Minimum Number of station at
Each Earthquake (MNST) This value as input data of
station program, the function to determine the number of
station that match the requirments of the value MNEQ
and MNST. The others parameters is lattitude and
longitude the earthquake as initial data, the depth of
margin earthquake in km, maximum residu of travel
time (RESS), the number of maximum iteration (ITRT)
is 5, standar deviation, the number of station and the
number of earthquake which not participate in
calculation, reading accurate (RANKAB), minimum
magnitude is > 5 SR, the value of slope, the
hypocentrum corrected using MJHD is plotting in
General Mapping Tool (GMT), the function of GMT
showing the hypocentrum corrrected distribution from
the surface and their cross section.
Picture 2. Hypocentrum of Earthquake in Mentawai Island before

relocation correction 2009-2012 data with all magnitude > 5 SR
III. RESULTS, ANALYSIS, AND DISCUSSION

At this part we show the inversion results using
MJHD method, compare the results before relocation
correction (picture 1), and after relocation correction
(picture2),then interpretation the results. At the picture 1
(before relocation correction), the distribution of
hypocentrum on Mentawai island dominated at shallow
depth (0-20 km), and but at picture 2 (after relocation
correction), the distribution of hypocentrum depth on
Mentawai island dominated at 25-50 km, and the
distribution on seismicity mapping show dominated at
shallow-intermediet depth earthquake near the Mentawai
island, and deeper far from Mentawai island. In
geological setting the enough great angle of subduction
zone between Eurasia plate motion and Indo-Australia
plate motion at west of Sumatera island. It caused by as
oceanic crust ages and cools, so it thickens. Thick crust
(far the spreading zone) tends to subduct at a greater
angle than thin crust (near the spreading zone). The
more depth of hypocentrum, indicating that the great
angle of convergent plate motion. The plate has point of
rupture, where if the force between the plate (ocean
crust and continental crust) at subduction zone more
great than rupture point of the plate then the plate will be
rupture. This rupture create a propogate wave on the
subsurface medium. Its traveling on the subsurface
medium get attenuate the energy when this wave
propagate on the weathering zone. So, the observer at the
surface calculated the time traveling of wave from
source to surface. In reality, time of calculation and time
of observe get shiftting because weathering zone at
subsurface. So, time of calculated and time of of
observed must be correction. Its match with seismicity
mapping.
Based on seismicity mapping after relocation
correction, the Mentawai island dominated by shallow to
intermediet depth earthquake, this earthquake dangerous
for Mentawai occupant and its surrounding.
Picture 3. Hypocentrum of Earthquake in Mentawai Island after

relocation correction 2009-2012 data with all magnitude > 5 SR
Picture 3. The Seismicity Mapping After Relocation Correction using

MJHD
On this seismicity mapping we can look that the red

circle indicate that the earthquake at the shallow to
intermediet depth, and the yellow circle indicate that the
earthquake at the intermediet to deeper. The red circle
dominated at the subduction zone with dominated
th
intensity is 3-7 SR (Richter Scale), and the yellow circle

dominated at the far of subduction zone with dominated
intensity is 3-5 SR ( Richter Scale ). It indicated that the
collison between Eurasia plate and Indo-Australia plate
at subduction on the flattening angle (0-80 degree).
Based on seismicity mapping the Mentawai island
dominated with variate earthquake intensity with the
focal depth near the Mentawai island. Based on
geological setting, lithology of Mentawai island
dominated from continental and oceanic rock ( Melange
formation ) with active fault such as reversed fault or
thrust fault. This zone called fore arc zone. Focal depth
shallow to intermediet because Mentawai island near the
subduction zone, it very dangerous for local citizens
because source of earthquake near the surface. The
effect of this earthquake very destruct. We can not to
predict when the earthquake create, but we can predict
that the earthquake at the Mentawai island variate
intensity, because the lithology at the Mentawai island is
dominated clastic sediment. In general the earthquake at
the Mentawai island created by collision between
Eurasia plate and Indo-Australia plate at the subduction
zone with focal depth shallow to intermediet.
IV. CONCLUSION
The depth of hypocentrum on the Mentawai island is
shallow to intermediet (25-50 km), and their distribution
on the west of Mentawai island indicating that
subduction zone between Eurasia plate motion and IndoAustralia plate motion. The convergent plate motion
between Eurasia and Indo-australia has the enough great
angle.
ACKNOWLEDGEMENT
The Author Thank to BMKG for their cooperation in
providing the earthquake data on the Mentawai island
from 2009-2012 year used in this study and Mr. DR.
ENG. Supriyanto as geophysics lecture on Department
of Physics University of Indonesia for his constructive
suggestion and transfer his knowledge to interpretation
the result.
REFERENCES
[1]
N. Hurukawa and Imoto. 1992. Modified Joint Hypocentrum

Determination. Japan.
[2]
Thompson &Turk. 2000. Introduction to Physical Geologi.

USA.
th
Reducing Deforestation and GHG Emission

with UB Biomass Stove and Fuel as Alternative
Energy for Community
Muhammad Nurhuda 1*), Setyowati Rahayu 2), and Dhoni Saputra 2)
1)
2)
Yayasan Inovasi Indonesia (INOTEK), Jl. Jenggala 2 No 9, Kebayoran Baru, Jakarta
*)
Corresponding author: mnurhuda@ub.ac.id
nesias consumption on wood-fuel was among the highest

Abstract A pilot project for implementing the UB bio- in the world i.e. 70.72 million m3 in 2006 and 65.03 milmass stoves and measuring their impacts over 100 households lion m3 in 2008 [4].
in Palangka Raya rural area has been performed. The reciCentral Kalimantan is one of province in Indonesia
pients were located in two villages, one village (Petuk Bukit) which has been experiencing highest rate of deforestation.
whose inhabitants was predominated by Dayak Native and
Deforestation is mostly attributed to logging for the conthe other (Habaring Hurung) by transmigrants from Java.
version of forests to plantations for palm oil and to supply
The pilot project was supported by Energy and Environment
the pulp and paper industry. In addition, it also has major
Partnership (EEP) Indonesia for one year.
It was found that after intensive coaching and monitoring, energy crisis whereas supply of fossil fuel is limited. Due
beneficiaries increase the usage of biomass stoves and adapt to poor availability of kerosene and gas fuel, households in
new habits in cooking preparation. By the end of project, 100 Central Kalimantan commonly use firewood for cooking
% of households in Habaring Hurung used the UB biomass harvesting it from forests surrounding their villages.
stoves for their daily cooking, while in Petuk Bukit the numAs an agricultural-based nation, Indonesia possesses an
ber was 78%. It was also found that 60% households in Ha- abundant source of biomass from residues, e.g. rice, subaring Hurung relied on UB biomass stove only, while the
garcane, palm oil, logging, sawn timber, coconut, and othrest 40% were in combination with traditional woodstove and
er agricultural wastes. These residues and wastes are ackerosene stoves. In practical use, the firewood reduction accounted for 59% less than that of traditional wood stove and tually sources of energy that can provide fuel for rural
the stoves produce much less smoke than the traditional households. It was with this justification that INOTEK
Foundation had been partnering with Muhammad Nurhuda
woodstoves.
in developing and disseminating an innovative UB BioKeywordsUB stove, biomass, deforestation.
mass Stove and a range of biomass fuel. Nurhuda has
received mentoring facilitation in RAMP Indonesia proI. INTRODUCTION
gram that is implemented by INOTEK. Technologically,
VER 40% of the world's population still burns vari- the UB biomass Stove is designed with innovative preous forms of biomass, such as wood, dung, charcoal, heating, gasification, counter flow and turbulence mechanor crop residues or coal as a cooking fuel, see e.g [1,2]. ism [5,6]. The external laboratory tests has shown that the
They cook over open fires or on rudimentary cookstoves. stoves can reach thermal efficiency as high as 49%, which
This way of cooking is not only inefficient, but also emits is comparable to the fossil-based cooking stove [7]. With
a harmful smoke that causes range of deadly chronic and such high efficiency, it is expected that the use of UB bioacute health effects such as child pneumonia, lung cancer, mass stove can reduce the amount of firewood needed for
chronic obstructive pulmonary disease, heart disease, and daily cooking as well as reduce the green house gas (GHG)
produced from combustion process
low birth-weight [3].
Though currently the Indonesias government has
II. METHOD
launched kerosene to LPG national conversion program,
Through
the
project,
100 energy efficient UB biomass
the use of biomass as daily cooking fuels is still predominate in most rural area. Moreover, inadequate supply of stoves were taken into use in the villages of Habaring Huhydrocarbon fuel at an affordable price and unsafe lique- rung and Petuk Bukit, which are rural areas in Palangka
fied petroleum gas stoves for low income communities Raya, Central Kalimantan. Petuk Bukit village is predomihave led to the growing use of firewood as fuel for daily nated by Dayak Native whereas Habaring Hurung is by
activities, including by cutting down trees in nearby fo- transmigrants from Java. The choice of two villages sociorests. The combination of deforestation, inefficient fuel logically were based on different characters of inhabitants.
combustion and high consumption has significantly created Each village received 50 stoves, and the stoves were then
vast environmental degradation and global warming. Indo- distributed among the households readily for usage in their
th
daily cooking activities. The project was funded by the

Energy and Environment Partnership (EEP) Indonesia
(www.eepindonesia.org) for one year.
The data collection were made by interviewing the beneficiaries of the stoves in every three months. Socialization, coaching and mentoring were carried out by involving Yayasan Mitra Insani as a local partner of the project.
The project started after the signing of the contract between INOTEK and EEP-Indonesia in May 24, 2012 and
were accomplished in the period of June 2012 up to June
2013. The project was designed from the beginning to involve active participation of local people with the goal to
raise awareness among the people on the significance of
health and nature conservation.
In the beginning of the project, there were some constraints, both technically and non-technical. The non technical problem was due to refusal from the local leaders,
such as installment scheme, the selection criteria of beneficiaries, and also collecting data base and conducting interview. All barriers could be solved with intensive approach
and explanation on the purpose of the project, and by identifying formal and informal leaders and opinion leaders to
get support from them. The technical problem was due to
fuel requirement, which, if not carefully prepared, could
lead into improper usage of the stove such that the combustion emits a lot of smokes in their kitchen. However,
after extensive coaching and monitoring, the beneficiaries
started to accept the new habits in cooking using the biomass stove.
To introduce the UB biomass stoves, knowledge transfer
was achieved by giving training, and one to one coaching.
The training did not relate the technical aspects only, such
as operating biomass stove, preparing woods or biomass
fuel, maintenance of the stoves, but also income generating
activities and simple accounting. To involve active participation among beneficiaries and raising awareness and
ownership, the selected beneficiaries are requested to pay
IDR 200,000,- . The payment was made using an installment scheme. The installment payment was collected by
representative of beneficiaries and the money will be used
as revolving fund. Dissemination of project were achieved
through media and by distributing promotion materials.
In the beginning of the project, very rare beneficiaries
used the stoves for daily cooking. It was found that most
beneficiaries put their stoves in rack after non-successful
first usage. The reason was the beneficiaries considering
the combustion in the stoves as it in their traditional clay
stove, though each stove was accompanied with manual
use procedure. However, after 6 month the project was
running, the monitoring track showed some good trends
and encouraging results. The beneficiaries were getting
used to biomass stoves. It was found that 99% of beneficiaries continue to use biomass stoves every day or in
combination with other stoves. It was only one beneficiary
in Petuk Bukit which returned the stove to the project implementers, and claimed to be reluctant to cutting woods
into pieces.
Fig. 1. Usage of biomass stove in kitchen.

TABEL 1
PERCENTAGE OF USAGE OF UB BIOMASS STOVE IN
COMBINATION WITH OTHER COOKSTOVES:
Type of stoves
UB stove only
UB + kerosene
UB + clay
Clay+UB+
Kerosene
Clay+UB+LPG+
Kerosene
NA
Total
Habaring
Hurung
60%
10%
16%
14%
Pethok Bukit
16%
76%
0%
4%
0%
2%
0%
100%
2%
100%
Referring to table 1, it is shown that among 100 households, 38% have used the UB stove exclusively. Further
data analysis has shown that the stoves were used to boil
water (78%), side dish (81%), and cooking rice (20%).
The lower percentage in using the stove for cooking rice
were due the fact that the size of the stoves were too small
for cooking rice for large family members, since it is normal in the rural area of Palangka Raya that one house is
occupied with family member that is larger than 10. The
controllable flame intensity of UB biomass stove may be
the reason why the stoves are mostly used for preparing the
side dish.
Finally, in Tab. 2 we show the saving potential of both
firewood and kerosene. The data was collected at the time
the project was approaching to end. On average, the use of
UB biomass stove could reduce the real consumption of
woods up to 59% compared to using traditional clay
stoves, and reduce the use of kerosene up to 50%. The
comparisons were made based on the monthly needs, before and after implementing the UB biomass stove. The
saving in using kerosene was found to be depend on the
kerosene supply and whether the biomass stoves were used
exclusively or in combination with other stoves.
From Tab. 1 and Tab 2 we can immediately see that the
households in Habaring Hurung, which are mostly transmigrant from Java, could be better accepting the cooking
new habits compared to households in Petuk Bukit, which
are predominated by Dayak native. The reason are proba-
th
bly the Java transmigrant could better adapt the new habits, that by the condition, they are far away separated
from their families in Java and thus must hardly struggle
for surviving in the new land.
TABLE. 2:
THE COMPARISON OF MONTHLY NEEDS OF FUELS, BEFORE
AND AFTER IMPLEMENTING THE UB STOVE.
Project
tion
Loca-
Habaring Hurung
Petuk Bukit
Type of
fuel
Monthly amount of fuel

Before
After
Woods
Kerosene
Wood
Kerosene
102 kg
11.8 L
86.9 kg
15.1 L
41.3kg
5.8 L
40.8 kg
7.7 L
Saving
(%)
59%
51%
53%
49%
which represent two different cultures. It is found that the

potential reduction for fire woods accounts for 60% compared to the traditional clay stove. Furthermore, adaptation
of new cooking habits can only be realized after intensive
coaching and monitoring.
ACKNOWLEDGMENT
The authors and involved project members greatly appreciate the Energy and Environment Partnership with
Indonesia (EEP Indonesia) for their financial supports. The
project is of coded under project ID
2055060401741509211.
REFERENCES
Despite the difference level of results in habaring Hurung and Petuk Bukit, it is clear for Tab. 2 that the beneficiaries required less wood for their daily cooking. Less
fuel means also less smoke in the kitchen, since the emission is always proportional the amount of combusted fuel.
Thus, in regards to the deforestation, the use of UB biomass stove could help preventing the people from cutting
the woods in forest and thus reducing the potential of deforestation.
IV. CONCLUSION
As conclusion, a UB biomass stove pilot project to
measure the real reduction of biomass usage for cooking
has been performed in rural area of Palangka Raya district,
Central Kalimantan for one year. The sample areas were
chosen to be the Habaring Hurung and Petuk Bukit village,
[13] Global
Alliances
for
Clean
Cookstoves:
http://www.cleancookstoves.org/
[14] Hedon Household Energy Network: http://www.hedon.info/tikiindex.php
[15] Carlos Torres-Duque, Daro Maldonado, Rogelio Prez-Padilla,
Majid Ezzati, and Giovanni Viegi "Biomass Fuels and Respiratory
Diseases", Proceedings of the American Thoracic Society, Vol. 5,
No. 5 (2008), pp. 577-590.
[16] http://www.fao.org/docrep/013/i2000e/i2000e00.htm.
[17] M. Nurhuda, Kompor Biomass Dengan Gasifikasi Terpanaskan
Dan Pembakaran Turbulen, paten register number P00201000217,
2010.
[18] M, Nurhuda, Kompor Briket Dengan Pre-Heating dan
Pembakaran Secara Counter Flow, patent register number
P00201100059, 2011.
[19] David Beritault and Veronique Lim, Stove Performance Report,
UB03, Envirofit G3300 and M5000, Ezystove, Geres, Phnom Penh,
Cambodia, 2013.
[20] Anonymous, INOTEK END-PROJECT COMPLETION REPORT FOR EEP
INDONESIA, 2013.
th
Measurement of Bioefficacy and Its Effects on

One Push Aerosol Insecticide by Using Glass
Chamber
Firdy Yuana1), Chomsin S. Widodo2), and Sukainah Quraisyiyah3)
1)
2)
*)
Corresponding author: fyuana@yahoo.com
Abstract Research has been conducted to examine the

bioefficacy of one push aerosol that is currently on the market. This study aims to determine bioefficacy (KT) of one
push aerosols on insects and determine whether there is a
residue generated by these insecticides within 1 hour of observation by using a glass chamber size of 70x70x70 cm and
particle counters Ptrak 8525 models. The average percentage
mortality of Aedes aegypti mosquito is entirely equal 100 %.
The fastest KT 50 and KT 90 on a product A (transfluthrin
21.3 %) is 573 s and 1462 s , followed by product B ( metofluthrin 3.5 % ) which has a value of 792 s and 1879 s . Product C (transfluthrin 25 %) has KT 50 and KT in 1277 s and
2867 s . Within 60 minutes of observation was found residue
on each product , product A have particle concentration 897
pt / cc , product B 1047 pt/cc , and the product C 493 pt/cc
.Product A is the the most effective to kill mosquitoes because
it has a greater concentration of particles is 17703 pt / cc rather than product C and also for product) as the active ingredient contained in a product that is transfluthrin 21.3 % even
though the product B has an average concentration greater
than the average concentration of product A is 18350 pt /cc .
Keywords bioefficacy, one push aerosol, insecticide
I. INTRODUCTION
HE use of insecticides is one of the effective way to
control mosquitoes, cockroaches, ants or other insects
that are common in the home. Insecticides are sold
widely in various forms both fuels and aerosols. For aerosol itself there are various brands available in the market
such as Baygon, vape, Mortein and others.
Bioefficacy emerging insecticides that used today is one
push aerosols. Aerosol is a term used for the preparation of
thin mist spray with high-pressure system. Aerosol types
can also be distinguished by size. Aerosol particle size is
usually expressed in particle radius assuming a sphereshaped particles. According to the version of the Aitken
particle size divided into three categories, namely:
Aitken particles (nucleation mode) with a size range between 0.001-0.1 m;
large particles (accumulation mode) measuring between
0.1-1 m, and
giant particles (particle coarsa mode) which size > 1 m
radius. [2].
The use of one push aerosol somewhat more efisient because just a single tap is enough to free us from mosqui-
toes for a 10 hours . At the time inhaled, aerosol particles

can get rid of the respiratory system's natural defenses and
lodge deep in the lungs. Aerosol very dangerous for people
with diseases such as asthma, bronchitis, and emphysema
(swelling in the lungs because blood vessels intruding air),
as dangerous for people with liver disease. High levels of
these objects in the air can trigger asthma attacks, lung
damage, and supports carcinogenesis, and premature
death.
In contrast to the usual aerosol spray which should in
some places into the room to obtain the same results. Given these differences, there are needs to determine the bioefficacy (knock down time) of one push aerosols on insects
which is Aedes aegypti and determine whether there is a
residue generated by these insecticides.
Stages of the study are as follows :
1 . Testing Using Glass Chamber
Glass Chamber is a box made of glass measuring
70x70x70cm with one wall open the door. Glass Chamber
must be ensured in a contaminated state. Insecticides are
used in this case is one push Vape ( Transflutrin 21.3 % ),
Force Magic Microns ( 3.5 % Metoflurin ) and Hit (Transflutrin 25 %). Gauging levels of mosquito repellent spray
is done in the following way : one pushaerosol mosquito
to be tested , weighed , and then sprayed for one second
outdoors . Then after a severe insect repellent sprayed
weighed again and the difference in weight is recorded (in
grams). Gauging levels of spray made with three replications.
A total of 20 Aedes aegypti (age 7-8 days) is released
into the Glass Chamber and wait one minute. One pushaerosol insect repellent sprayed 1 time press into the Glass
Chamber. Observations were made for 60 minutes. The
number of mosquitoes fainting calculated at any specified
time interval , which are : 0:50 ; 1:25 ; 2:00 ; 2:50 ; 3:00 ;
3:50 ; 5:00 ; 15:00 ; 30.00 and 60.00 minutes . Then all the
mosquitoes moved into the plastic tube , given the wet
cotton sugar solution and stored ( holding ) for 24 hours at
room temperature 27 C. To determine the time fall / lame
( Knock-down Time ) 50 % ( KT-50 ) used probit analysis.
KT-50 is the time required for the drop / knock out 50 %
of the population of mosquitoes in certain doses. As for
knowing the difference bioefficacy between the treatment
th
and control groups performed the t-test as a condition of

ANOVA test. Bioefficacy insecticides have a sense of the
effectiveness of the insecticide itself. Percentage collapsed
and died in the experiment was 100 % after bioefficacy
calculated using the formula :
{ ( P + Q ) : R } X 100 %
(1)
Description : P : Number of mosquitoes fainting
Q : The number of dead mosquitoes
R : Number of mosquitoes tested
2 . Preparation of test animals
Test animals used is dengue fever mosquito (Aedes aegypti) were sterile dengue virus androgynous females ,
aged 7-8 days as many as 400 individuals .
3 . Particle measurements using P-trak 8525 models
The next stage is to measure the concentration of particles contained in a spray product using the particle counter is the P-Trak 8525 Model. The first step is to measure
the temperature in the chamber by using a thermometer
and the pressure using a barometer. Then, the P-trak hose
connected to a glass chamber as well as the hose connects
to the glass chamber with air pump. After that, one push
aerosol insect repellent manually sprayed into the glass
chamber for a second . Then spray the glass allowed to
stand in the chamber during a predetermined time is: 0:50 ;
1:25 ; 2:00 ; 2:50 ; 3:00 ; 3:50 ; 5:00 ; 15:00 ; 30.00 and
60.00 minutes . After that, the concentration of particles is
measured using a P-Trak 8525 Model and be repeated
three times for each time. Automatically particle measurement data will be stored in the P-Trak then the data will be
processed by using the 0rigin 8.1 software. The concentration of particles obtained from the difference between the
minimum and maximum values contained in the P-Trak
program. Shape measurement circuit is shown in Figure 1.
minutes and 2:50 minutes. This can be happen because

most of the particles of the active materials undergo deposition (particles to the surface of the glass forming chamber, causing droplets with larger size).
In the next minute is minute 3:00, the particle concentration increased on average significantly due to the 3:00
minute droplets stick to the glass chamber will change the
shape of solids into gases that can be caught by the P-Trak.
And at minute of 3:50, the particle concentration decreased
again from the previous minutes. The decrease is largely
due to the concentration of the particles that have accumulated particles into the air to experience deposition on the
surface of the glass chamber. In the next minute, the concentration of particles continued to decline slowly, until
the last minute which are 60.00 , a product of particle concentration 897 pt/cc , product B 1047 pt/cc , and the product C 493 pt/cc. This indicates that after 60 minutes the
particle is not evaporated entirely or the particles accumulate in the air is not all evaporated.
Fig. 1. Total concentration of particles.
Figure 1. Shape measurement circuit is shown
To calculate the total concentration of particles of insecticide used the following equation
(2)
Information:
III. RESULT
From the measurement, the total concentration of aerosol particles in one push can be seen in Figure 1. The concentration of particles in the early minutes to 0.50 minutes
product A the average particle concentration reached
17703 pt/cc , product B 18350 pt / cc, and for products C
10037 pt /cc. And then decreased at minute 1:25, 2:00
By using ANOVA analysis shows that the three products had average concentrations were significantly different. From the ANOVA analysis was obtained that Product
B has an average particle concentrations are higher than
most other products, whis are 7427.7 pt/cc . The average
particle concentration of the second highest is a product
that is 4478.9 pt / cc and the lowest concentration is the
product C is 3340.9 pt / cc.
Can be seen in Figure 1 that at minute 3:00, the particle
concentration decreased , suggesting that at minute 1:25 ;
2:00 and 2:50 of the particles undergo deposition around
the surface of the glass and experience the deposition
chamber where the material will undergo a process of
change in the form of a gas or liquid that is small be solid
also called desublimasi so that the particles can not be exploited by the P - trak .
At minute 3:00, the active ingredient volatile at temperatures above the room temperature, causing the particles are
shaped colorless crystals undergo evaporation. When it is
evaporated, the solid particles are transformed into a gas
that can be sucked back by the P-Trak. This is why at the
minute 3:00, particle concentration values increase. The
workings of the active ingredients contained in insect repellent one push aerosol is is pyrethroid which attack the
nervous system of the mosquito Aedes aegypti that can
th
cause paralysis. The active ingredients are micro-sized so

it also attacks the circulatory system, the hormonal system,
and respiratory system and can settle in the lungs and led
to the death of the Aedes aegypti mosquitoes.
Henceforth , the testing of bioefficacy KT 50 and KT 90
by using three types of drugs push one of the aerosol mosquito Aedes aegypti mosquitoes were placed in a glass
chamber. Test results bioefficacy KT 50 and KT 90 can be
seen in table 1.
TABLE 1.
The relationship between the concentration of particles produced per
insect repellent spray for each product with the duration of paralysis
Test bioefficacy one push repellent aerosol showed that

mosquito mortality on all products are 100 % . KT 50 and
KT 90 of the most effective is a product containing 21.3 %
with variation transfluthrin paralysis time mosquitoes. After that followed by B product with the active ingredient
metofluthrin 3.5 %. KT 50 and KT 90 a C product with the
active ingredient transfluthrin 25 % has the lowest
amount. Differences KT 50 and KT 90 at any one push
repellent products due to differences in aerosol particle
number concentrations in each product has a different
amount.
It is seen that the product A , which contains 21.3 %
transfluthrin , faster crippling mosquito Aedes aegypti is
11 seconds ( KT 50 minutes to 0.5 ) than the product C is
33 seconds ( KT 50 minutes to 0.5 ) which contains transfluthrin 25 % , even though the active ingredient product C
higher than product A. This is because the value of the
average concentration of product A is higher than product
C. Types of active ingredients listed on the packaging of
the product also affect the timing of mosquitoes death. It is
shown in the tables above indicate that the product kills
mosquito A faster than product B even though the products
have an average concentration value higher than a product
, for example in 0.5 minutes , the product A has a value of
17703 particle concentration pt / cc whereas product B has
a particle concentration of 1830 pt / cc
IV. CONCLUSION
From the results of the research can be concluded that

Product B has the largest concentration followed by product A and product C. Bioefficacy test bioefikasi of three
different insect repellent products have a value of KT 50
and KT 90 distinct and not depending on the magnitude of
the concentration of the active substance and the number
of particles generated by one push-aerosol insect repellent.
Product A has the most rapid efficacy followed by product
B and C. There is still a residue of insecticide after 60 mi-
nutes of observation, this is indicated by the presence of a

concentration of particles in the observation area
REFERENCES
[1] Ardley, Neil. 1998. Percobaan Ilmu Pengetahuan. Semarang. PT Mandiri Jaya.
[2] Biswas, P. 2009. Measurement and Capture of Fine and
Ultrafine Particles from a Pilot-Scale Pulverized Coal
Combuster with an Electrostatic Precipitator. J.
Air&Waste Manage. Assoc.
[3] Departemen Pendidikan Nasional. 2004. Ensoklopedi Sains
dan Kehidupan. Jakarta. CV Tarity Samudra Berlian.
[4] Djojosumarto, Panut. 2012. Available :
http://www.google.co.id/Panduan%20Lengkap%20Pestisida
%20&Aplikasinya.html.
[5] Ganiswara, S.G., Setiabudi, R., Suyatna, F.D., Purwantyastuti, Nafrialdi. 1995. Farmakologi dan Terapi (Edisi 4). Bagian Farmakologi FK UI: Jakarta
[6] Gillett, J. D. 1972. The Mosquito: Its life, Activities and
Impact on Human Affairs. Doubleday, Garden City. New
York
[7] Hartanto, L.N. 2004. Biologi Dasar. Yogyakarta: Penebar
Surabaya.
[8] Heller, J.L. 2010. Insectisida Poisoning. Medline plus.
English.
[9] Kurnianti,
Novik.
2013.
Available:
www.google.co.id/Petunjuk Aplikasi Pestisida.html
[10] Lucas, JR; Shono, Y; Iwasaki, T; Ishiwatari, T; Spero, N;
Benzon, G. 2007. Journal of the American Mosquito Control Association. Laboratory and field trials of metofluthrin
(SumiOne) emanators for reducing mosqito biting outdoors.
US. 23(1): 47-54
[11] M, Sugono. 2005. The biological activity of a novel pyrethroid
:Metofluthrin. Sumitomo
Chemical Co., Ltd., Hyogo. Japan
[12] Sigit SH, Koesharto FX, Hadi UK, Gunandini DJ, Soviana
S, Wirawan IA, Chalidaputra M, Rivai M, Priyambodo, Yusuf S dan Utomo S. 2006. Hama Pemukiman Indonesia.
Pengenalan, Biologi, dan Pengendalian. Bogor. Penerbit
Unit Kajian Pengendalian Hama Pemukiman. Fakultas Kedokteran Hewan IPB.
[13] Staf pengajar Farmokologi. 1995. Absorbsi dan Ekskresi.
Bagian Farmakologi FK UNLAM: Banjarbaru.
[14] T, Nazimek. et al. Content of Transfluthin. Departement of
Toxicology, Institute of Rural health. Lublin. Poland
[15] Widiarti, dkk. 1997. Uji Bioefikasi Beberapa Insektisida
Rumah Tangga terhadap Nyamuk Vektor Demam Berdarah. Stasiun Penelitian Vektor Penyakit, Pusat Penelitian
Ekologi Kesehatan Badan Penelitian dan Pengembangan
Kesehatan. Salatiga.
[16] Womack, M. 1993. The yellow fever mosqito, Aedes
aegypti. Wing Beats. Florida
[17] Zoolner, G; Orshan, L. 2011. Journal of the Society for
vector Ecology. Evaluation of metofluthrin fan vaporizer
device against phlebotomine sand flies (Diptera: Psychodidae) in a cutaneous leishmaniasis focus in the judean
Desert. Israel. 36 Suppl: S157-65
th
The Effect of Probe Pulse in a Double Pulses

Experiment
Abdurrouf
Faculty of Mathematics and Natural Sciences, Brawijaya University, Malang, Indonesia
Corresponding author: a.abdurrouf@gmail.com
AbstractThis paper investigates numerically the probe the double pulses experiment can be arranged in several
pulse effect on the dynamic alignment, generated by pump different ways.
pulse, in a double pulses experiment. To this end, we compare
the dynamic alignment by pump pulse only, with that obtained by using both pump and probe pulses. The numerical
calculation demonstrates that the probe pulse slightly raises
the degree of alignment without changing its general characteristics, both in time and frequency domain
Keywordsdouble pulses experiment, probe, alignment,
time domain, frequency domain
I. INTRODUCTION
he double pulses experiment is one of the interesting

research topics in atomic and molecular physics, no
less because of its potential applications as a source of
coherent ultraviolet light and/or generation of ultrashort
attosecond laser pulses [1], for molecular imaging [2], for
controlling molecule-laser interaction [3], etc.
In double pulses experiments, an ensemble of gas is
first subject to a femtosecond pump pulse to controls their
wave-function to set them in rotational motions and wait
for the molecules to be aligned at a later time (a few picoseconds) when they could undergo a rotational revival
[3]. The second more intense femtosecond probe pulse,
was delayed with respect to the first by successively increasing the time intervals td, is given to generate the observed signals [4], either in ionization [5], dissociation,
optical Kerr effect (OKE) [6], photoelectron angular distribution (PAD) [7], above threshold ionization (ATI) [8],
or high harmonic generation (HHG) schema [4]. By plotting the observed signal as a function of delay time between two pulses, one obtains the dynamic signal describing the molecular behavior due to the interaction.
The typical pump-probe experiment set up by using
HHG as a probe, is depicted in Fig. 1. A laser beam is split
into two parts by a beam splitter (BS) to separate the pump
and probe pulses, with a specific ratio of intensity between
them. The probe pulse is then delayed with a controllable
delay line system D and, if needed, can be also rotated
with a polarizer P. The two pulses is then mixed again with
a beam mixer (BM) where the probe pulse is delayed by
from the pump pulse and its polarization is rotated
with respect to that of the pump pulse by an angle , if
desired. The pump-probe pulse sequence is focused by a
system of lenses (F) and is then subjected to a molecular
gas jet: the pump pulse dynamically aligns the molecules
whereas the probe pulse generates HHG signal from the
aligned molecules. The HHG signal is then recorded by a
detector system. Depending on the experimental purpose,
Fig. 1. Schema of pump-probe experiment. The explanation is given in

text
So far it is assumed that the alignment is due to the

pump pulse only, while the second one only gives the observed signal, without rotating it during the short duration
of the probe pulse. However, it is worthwhile to observe,
whether the probe pulse affects on the dynamic signal, or
not. The question has been raised in Ref. [9]. Here, the
discussion is extended, including the eighth revival and its
Fourier spectrum.
II. COMPUTATIONAL DETAILS
To overcome the question, I directly compare here the
alignment by the pump pulse only, with that obtained by
using both the pump and the probe pulses.
For the second schema, we allow the possibility that the
probe pulse also may rotate the molecule before generating
the observed signal. The total field is assumed to consist of
the sum of the two fields:
In the above indices 1 and 2 stand for pump and probe

pulse, respectively. td is the delay time between the two
pulses. In above, 10 and 10 are peak field whereas
and
are time profile of the field. The
effective pulse then reads
For two pulses, dynamic alignment is characterized by

pump and probe peak intensity, pump and probe time profiles, delay time between them, and interaction time (i.e.
delay time between entering probe pulse and observing
th
signal).
The schema for alignment by the pump and the probe
pulses is the following. The probe pulse is given
after
the pump pulse. We let the probe pulse to interact with the
molecule at a given time and then take the some expectation values
and plot as function of the delay
is the quantum measurement of dytime .
namical alignment of a rotating molecule, given by
, which is the expectation value of the
with is the angle between the molecular axis and the pump/probe polarization direction; the
double angular brackets stand for the expectation value
with respect to the wave packet states (inner brackets) and
the statistical average with respect to the Boltzmann distribution (outer brackets) of the initially occupied rotational
states.
In this paper, I calculate two expectation values of a diatomic linear molecule
, those are the first alignment
degree
, which is the first term in ionization
signal [5], and the second alignment degree
, which is the first term in high harmonic generation signal [4]. The properties of
are
listed in Tab. I.
TABLE I
PROPERTIES OF O2
Symbol
Quantity
Value
Rotational constant
parallel polarizability
perpendicular polarizability
number of even-j wave function
number of odd-j wave function
1.4297 cm-1
2.35 (A3)
1.21 (A3)
0
1
Fig. 2. Dynamics alignment of

(upper panel) and
(lower panel) of
at 300 K
subject to laser pulses of
and
with FWHM 40 fs. We
In experiment, I use here the pump and probe pulses keep the second pulse to interact for 40 fs. Solid lines for
with peak intensity of
and double pulse and dashed lines for one pulse.
. Both pulses have Gaussian
profile with FWHM (full width at half maximum) 40 fs.
In general, the probe pulse does not change the dynamThe gas of
is kept at room temperature 300 K. I choose ics of
, except in three aspects. First, the
the pulse duration, 40 fs, as the delay time between enter- probe pulse intensity adds up the intensity of the pump
ing probe pulse and observing signal
pulse and enhances the alignment process prepared and
The results are shown in Fig. 2, for
(up- generated by the pump pulse, so that the expectation value
per panel) and
(lower panel). From of
with two pulses is higher than the one
the upper figure, it is shown that there is no difference be- with pump pulse only. Second, the alignment enhancement
tween the signal due to the double pulses (black, solid by the probe pulse depends on the delay time between the
lines) compare to that of single pulse (blue, dashed lines). two pulses and reaches a maximum when the slope of
Both signal revive with a period of
, which is given alignment degree is positive. This means that the degree of
by
alignment is enhanced before its maximum and as a consequence reaches the maximum earlier than the alignment
with one pulse only. Third, there are a small revival structure at
in signal due to two pulses (as shown in Fig.
3), where they are absent in the case of single pulse signal.
For O2,
. The signals also show a half and
The similar situation occurs in
(Fig
quarter revival, which is a character of
.
2. lower panel). Both signal has a period of 11.6 ps and
show a half, quarter, and eighth revivals, as a characteristics of
. In general, there is no significant difference between the signal due to single and double
pulses.
th
because of its small depth modulation, which is about one

by hundred compare to that of half and fourth revival.
Fig. 4. The Fourier spectrum of

of
for single
pulse (upper panel) and double pulses (lower panel). The pulses parameters and temperature are similar to those in Fig. 2.
Fig.
shows
the
Fourier
spectrum
of
. The dynamic of
is associated with phase differences
. While
the
transition
with
is
related
to
series
of
Fig. 3. The revival structure at
(panel a),
(panel
(10,18,26,34,.)Bc, the transition with
is assob),
(panel c), and
(panel d) of
of ciated with phase differences with
for double pulses. The pulses parameters and temperature are similar to those in Fig. 2.
,
(5)
To see more clearly, we Fourier transform the dynamic
signal and get the spectrum in frequency domain, as shown
in Fig. 4 for
and Fig. 5 for
.
From Fig. 4, one can see that there is no difference between spectrum of single and double pulses. Both spectrum consist of peak series located at (10,18,26,34,.)Bc
or
. As we know, the
generates a transition with
The transition with
J = 0 creates a peak at frequency zero, whereas the transition with
is associated with phase differences
with
(4)
with c in cm/s. The absence of series (4Jeven+6)Bc indicating that O2 has Jodd only. The series is peaked at 50 Bc
corresponding to
. I note here, that the revival
structure at
does not contribute in Fourier spectrum
and related to series of (28, 44, 60, 76, 92, 108, 124,
140,.) Bc. According to Eqs. (4) and (5), the Fourier
spectrum of
of O2 consists of series of
and
. It was shown in Fig. 5,
that both Fourier spectrums show series of (10, 18, 26,
34,.) Bc as representation of
and series
of (28, 44, 60, 76, 92.)Bc due to
transition. The series
is peaked at 50 Bc corresponding to
, similar to those of
. On the other hand, the series
is peaked at 140 Bc corresponding to
. The detail of Fourier spectrum and its role in
dynamic alignment can be found in Ref. [10].
I also note that the Fourier spectrum of
of
double pulses has similar intensity compare to that of single pulse. It is related to the depth modulation of two dynamic signals. On the other hand, the intensity of the
Fourier spectrum of
is smaller than that
of single pulse. It can be understood, because the depth

modulation of
similar to that of single pulse.
for
th
double pulse is not in the observed signal in a double pulses experiment.

REFERENCES
Fig. 5. The Fourier spectrum of

of
for
single pulse (upper panel) and double pulses (lower panel). The pulses
parameters and temperature are similar to those in Fig. 2.
IV. CONCLUSION
In summary, it has been demonstrated that the probe
pulse does not change the signal, in both time and frequency domain. Then, we can neglect the effect of probe pulse
1) A.H. Zewail, Femtochemistry: Atomic-scale dynamics of the

chemical bond, J. Phys. Chem. A, vol. 104, no. 24, pp. 5660
5694, June 2000.
2) J. Itatani, J. Levesque, D. Zeidler, H. Niikura, H. Pepin, J.C. Kieffer,
P.B. Corkum, and D.M. Villeneuve, Tomographic imaging of
molecular orbitals, Nature, Vol 432, no 7019, pp. 867 871, Dec.
2004.
3) J. Itatani, D. Zeidler, J. Levesque, M. Spanner, D.M. Villaneuve, and
P.B. Corkum, Controlling high harmonics generation with
molecular wave packets, Phys. Rev. Lett., vol. 94, no. 12, pp.
123902, March 2005.
4) K. Miyazaki, M. Kaku, G. Miyaji, A. Abdurrouf, and F.H.M. Faisal,
Field-Free Alignment of Molecules Observed with High-Order
Harmonic Generation, Phys. Rev. Lett., vol. 95, no. 24, pp.
243903 (1-4), Dec. 2005.
5) P. W. Dooley, I. V. Litvinyuk, K. F. Lee, D. M. Rayner, M. Spanner,
D. M. Villeneuve, and P. B. Corkum, Direct imaging of rotational
wave-packet dynamics of diatomic molecules,. Phys. Rev. A, vol.
68, no. 2, pp 023406, August 2003.
6) B.J. Sussman, J.G. Underwood, R. Lausten, M.Y. Ivanov, and A.
Stolllow, \Quantum control via the dynamic Strak effect:
Application to switch rotational wave packets and molecular axis
alignment, Phys. Rev. A, vol. 73, no. 5, pp. 053403, May 2006.
7) M. Tsubouchi and T. Suzuki, Photoionization of homonuclear
diatomic molecules aligned by an intense femtosecond laser pulse,
Phys. Rev. A, vol. 72, no. 2, pp. 022512, 2005.
8) T. K. Kjeldsen and L. B. Madse, Alignment-dependent abovethreshold ionization of molecules, J. Phys. B: At. Mol. Opt. Phys.,
vol. 40, no. 1, pp. 237 245, Jan. 2007
9) A. Abdurrouf and F. H. M. Faisal, Theory of intense-field dynamic
alignment and high-order harmonic generation from coherently
rotating molecules and interpretation of intense-field ultrafast
pump-probe experiments, Phys Rev. A, vol. 79, no 2, pp. 023405,
Feb. 2009
10) F. H. M. Faisal, A. Abdurrouf, K. Miyazaki, and G. Miyaji, Origin
of Anomalous Spectra of Dynamic Alignments Observed in N2 and
O2, Phys. Rev. Lett., vol 98, no, 14 pp. 143001, April 2007.
th
Gravity Data Analysis of Teluk Mandar,

Makassar Strait
Supriyanto 1*), T. Noor 1), Y. Mark 1), and Y. Sofyan 2) 1)
Department of Physics, Faculty of Mathematics and Natural Science, University of Indonesia, Kampus UI
Depok, Indonesia, 16424
2)
Aso Volcanological Laboratory, Graduate School of Science, Kyoto University, Minami Aso, Aso, Kumamoto
869-1404, Japan
*)
Corresponding author: supriyanto@sci.ui.ac.id

II. GRAVITY METHOD
Abstract The Teluk Mandar is located around Makassar

strait, and is situated within a complex tectonic region at the
edge of Eurasian plate. Gravity data analysis have been
performed to identify the subsurface structure Complete
Bouguer gravity anomalies derived from satellites altimeter
measurements were used in this analysis. We separate the
Bouguer gravity into two parts, the regional and the residual
gravity, by using Power Spectrum based filtering. The
residual gravity data were then analyzed using integrated
gradient interpretation techniques, such as the Horizontal
Gradient (HG), and Second Vertical Derivative (SVD). These
techniques detected many faults and several sedimentary
basin that characterized by negative Bouguer gravity
anomalies. The results of present study will lead to an
improved understanding of the geological structure in Parepare region, especialy inside Sengkang sedimentary basin
area.
In principal, the goal of gravity surveying is to locate

and describe subsurface structures from the gravity effects
caused by their anomalous densities. Gravity studies in
regioanal basin area, in particular, can provide unique insights into shallow sub-surface density variations associated with the structural and magmatic activity of basinal
system [2].
KeywordsGravity, Teluk Mandar, Horizaontal Gradient,

Second Vertical Derivative
I. INTRODUCTION
HE Teluk Mandar is located around Makassar strait
between 3.556 oS and 4.9853 oS latitudes; and
119.4083 oE -- 120.3083 oE longitudes, is situated
within a complex tectonic region at the edge of Eurasian
plate, covers an area of about 12.869 km2(Figure 1). It is
well-known that Sengkang sedimentary basin is located in
the onshore of South Sulawesi province.
Gravity data from Geosat and ERS-1 satelites are
analyzed using advanced gravity data processing to
determine structures that affected the basin and to estimate
the sedimentary thickness. It is known that gravity
interpretation suffers from non-uniqueness. Different
subsurface features produce same gravity field. Care was
taken to constrain the analysis results using integrated
gradient method [4].
1)
Department of Physics, Faculty of Mathematics and Natural Science,
University of Indonesia, Kampus UI Depok, Indonesia, 16424
2)
Aso Volcanological Laboratory, Graduate School of Science, Kyoto
University, Minami Aso, Aso, Kumamoto 869-1404, Japan
*)
Corresponding author: supriyanto@sci.ui.ac.id
Fig 1. Teluk Mandar region, South Sulawesi Province
Nowadays, gravity data could be acquared from

satellites. Satellite altimetry has provided the most
comprehensive images of gravity field at ocean basins with
accuracies and resolution approaching typical shipboard
gravity data.
The analysis of gravity data uses three approaches to
reduce the error in satellite-derived gravity anomalies to 2
3 mGal from 5 to 7 mGal. First, the raw waveforms were
retracked from 11 months of ERS-1 satellite data [4] and
18 months of Geosat/GM satellite data resulting in
improvements in range precision of 40% and 27%,
respectively. Second, the recently published EGM2008
global gravity model at 5 min resolution [1] were used in
the remove/restore method to provide 5-min resolution
gravity over the land and 1-min resolution (8 km 1/2
wavelength) over the ocean with a seamless land to ocean
transition. Third, a biharmonic spline interpolation method
including tension [6] were used to construct residual
th
vertical deflection grids from seven types of inconsistent

along-track slope measurements.
Comparisons between shipboard gravity and the global
gravity grid show errors ranging from 2.0 mGal in the Gulf
of Mexico to 4.0 mGal in areas with rugged seafloor
topography. The largest errors of up to 20 mGal occur on
the crests of large seamounts.
III. GRAVITY MEASUREMENT AND DATA PROCESSING
Gravity data retracked from Geosat and ERS-1 altimetry
satelites have been provided by The Satellite Geodesy
research group at Scripps Institution of Oceanography,
University of California San Diego. A total of 3904 point
of gravity measurement were distributed at Teluk Mandar
region at the total area of about 12.869 km2 ; with point
interval of about 1.8 km (1 minute). The gravity data set
consists of longitude and latitude coordinates; and
complete bouguer anomaly (CBA).
All data processing steps are shown in Figure 2. In this
study, we assume that the final output of several steps of
processing of satellite-derived gravity anomaly explained
above is a Complete Bouguer Anomaly (CBA) as shown in
3. Complete Bouguer Anomaly (CBA) map of Teluk Mandar
Figure 3. The CBA data ranges between -21 and 114 Fig.
region, South of Sulawesi. Coast line and Sengkang Basin boundary are
mGal. Negative anomalies were observed mainly in the shown in blue line and black line respectiveley.
offshore of Teluk Mandar region. Meanwhile, highest
positive gravity values were detected on the onshore of
Teluk Mandar region and, it can be interpreted by high
density of volcanic rock instrusion.
Fig 2. Flow processing of gravity data
There is a general assumption that the negative value of

the CBA could be interpreted as a sedimentary basin.
However, the CBA value come from all bodies anomaly at
various depth. Moreover, in contrast, the Sengkang
sedimentary basin is covered by relatively high of CBA,
not negative value. Therefore, to be more confident for
determining the location of sedimentary basin, we need to
extract residual gravity anomaly from the CBA.
We have separated the CBA into two parts, the regional
and the residual gravity anomalies, by using power
spectrum based filtering. The regional gravity reflects
source of gravity anomaly from deeper part which is more
than 10 km. We have decided to analize residual gravity,
rather than regional gravity. In term of gravity data, we
have expected that sedimentary basin indicated by low
residual gravity anomaly inside Sengkang sedimentary
basin area (Figure 4).
Fig 4. Residual gravity anomaly of Teluk mandar region
The residual gravity resulted from filtering were then

analyzed using two integrated gradient methods, such as
the Horizontal Gradient (HG) and Second Vertical
Derivative (SVD) methods. These techniques detected
many faults as the boundary of the sedimentary basin.
Moreover, the depth structure of formation inside the
Sengkang sedimentary basin have been delineated.
IV. RESULT AND DISCUSSION
A. Horizontal Gradient
The horizontal gradient method was used extensively to
locate the boundaries of regions of contrasting density
from gravity data [3]. The greatest advantage of the
th
horizontal gradient method is that it is least susceptible to

noise in the data because it requires the calculation of only
two first-order, horizontal derivatives of the field [4].
H H
H ( x, y ) =
+
x y
2
(1)
The map of horizontal gradient from Teluk Mandar is
shown in Figure 5. It shows that the boundaries/faults are
located at the maxima of the horizontal gradient. Some
new faults were detected as well. These faults are located
at, or near the volcanic area. Moreover, some geological
faults are not corroborated by the horizontal gradient
technique. This discrepancy may be the fact that the
horizontal gradient detects only faults that displaced
formations vertically causing density contrasts.
Fig 6. Second Vertical Derivative map of Teluk Mandar region
Fig 5. Result of Horizontal Gradient analysis
B. Second Vertical Derivative

In order to identify certain type of fault, the Second
Vertical Derivative (SVD) analysis has been aplied to the
CBA free noise. The SVD shows and isolates structural
features that are identical and complimentary to those
already identified with the horizontal gradient maxima.
The Euler solution superimposed on the SVD clearly
outlines the various contacts and structures of the faults
and basin boundaries.
To determine fault type, we first make a line that
perpendicular to the certain fault line interpreted by Euler
calculation. Second, evaluate the maxima and minima of
the SVD value along the line. If the absolute of maxima
number is greater than absolute minima number, the fault
type is identified as a normal fault. Otherwise, the fault
type is reverse fault.
Fig 7. SVD value variation along AB line. The fault type is normal fault
because absolute maxima number is greater than absolute minima
number.
V. CONCLUSION
Integrated gradient interpretation techniques, Horizontal
Gradient (HG) and Second Vertical Derivative (SVD).
Have ssuccessfully detected many faults and several
sedimentary basin that are characterized by negative
Bouguer gravity anomalies. The structural high interpreted
from SVD analysis high has covered the area around of
250 km2.
ACKNOWLEDGMENT
We would like to acknowledge the support of the
Directorate of Research and Public Service, University of
Indonesia, who gave us financial support under the Hibah
Penelitian Unggulan Perguruan Tinggi (PUPT) BOPTN
2013
scheme
with
the
contract
number:
1350/H2.PPK/HKP.05.00/2013.
th
REFERENCES
[1]
[2]
Mikhailov, V., Galdeano, A., Diament, M., Gvishiani, A., Agayan,

S.,
Bogoutdinov, S., Graeva, E., Sailhac, P., (2003). Application of
artifcial intelligence for Euler solutions clustering. Geophysics 68,
168e180. Mikhailov et al., 2003
Pavlis, , N. K., S. A. Holmes, S. C. Kenyon, and J. K. Factor
(2008), An Earth Gravitational model to degree 2160, paper
presented at General Assembly, Eur. Geosci. Union, Vienna.
[3]
[4]
[5]
[6]
Reid, A.B., Allsop, J.M., Granser, H., Millett, A.J., Somerton, I.W.,
(1990). Magnetic interpretation in three dimensions using Euler
deconvolution. Geophysics 55, 80e91
Sandwell, D. T., and W. H. F. Smith (2005), Retracking ERS-1
altimeter waveforms for optimal gravity field recovery, Geophys. J.
Int., 163, 7989, doi:10.1111/j.1365-246X.2005.02724.x.
Thompson, D.T., (1982). EULDPHda new technique for making
computer-assisted depth estimates from magnetic data. Geophysics
47, 31e37.
Wessel, P., and D. Bercovici (1998), Interpolation with splines in
tension: A Greens function approach, Math. Geol., 30(1), 77 93,
doi:10.1023/A:1021713421882.
th
Modeling of Relative Dating Using

Spectroscopy Analysis of Tambans Subfossil
T.B. Susilo*#, U. B. L. Utami*, R. Nurmasari*, R. Mujianti*, M. Habibi***, S. Mawadah***, A. Pradana***, S. Hayati***, Y. Seftiawan***, O. Soesanto**, B. I. Prayogo**** and Satrio *****
*
Prodi Kimia FMIPA UNLAM, Jl. A. Yani Km 35,5 Banjarbaru, Kal Sel; ***Mahasiswa Kimia FMIPA
UNLAM, Jl. A. Yani Km 35,5 Banjarbaru, Kal Sel; **Prodi Matematika FMIPA UNLAM, Jl. A. Yani
Km 35,5 Banjarbaru, Kal Sel; ****Museum Lambung Mangkurat, Jl. A Yani Km 35, Banjarbaru,
*****
Pusat Aplikasi Teknologi Isotop dan Radioaktif-Badan Teknologi Atom Nasional (PATIRBATAN), Jl. Lebak Bulus Raya No. 49, Pasar Jumat, Jakarta,
*#
Corresponding author: tbi.susilo@yahoo.co.id
AbstractArchaeological fragments of bone that are exposed to a wetland environment take up fluorine from the
surrounding soil. The fluorine ion exchanged the hydroxyl
group in the hydroxyapatite (Ca10(PO4)6(OH)2)of the bone,
forming
chemically
more
stable
fluorapatite(Ca10(PO4)6(F)2). Based on our data 14C radiocarbon,
the age of two Tambans subfossil are 485- 5 and 5684
16year ago, respectively. The IR spectrum is sharp band
3500-4000 cm-1 in the hydroxyapatite. Tambans subfossil
and Tahuras bone that spectrum is assigned to the OH stretching mode and considering the fossilization have been a conservations in wetland environment. In the region 800-900 cm1, the subfossil and bone implies that carbonate and silicon
substitution dont induce vacancies at the OH- site. In here,
we report that modeling Ca2+replaces Cu2+, Cd2+ and
Zn2+ions, which can be described by a diffusion model, contain information on the exposure duration of the Tambans
subfossil object, several attempts to use metal profiling as a
relative dating method.
Keywords14C, FTIR and relative dating method.
I. INTRODUCTION
HE chemical composition of the mineral and the
organic part of bones has been used palaeodiet and
palaeoclimate reconstruction. However, the burial
period, bones have been in contact with sediments, soil
and water. Partial of complete dissolution, erosion, and
precipitation, recrystallization, ion uptake by sorption and
diffusion, hydrolysis, and polymerization may lead to
changes in the chemical composition and structures. The
state of preservation is very variable and depends mainly
on direct environmental conditionsuch as groundwater and
sediment temperature, soil hydrology, and pH, reductionoxidation (redox) potential and temperature, mechanical
pressure, biological factors and particle transport. They are
of great importance to understand the alteration process in
soils and the impact of environments conditions on
bone/fossil conservation[1]. Very few studies have addressed multi-element ionic exchanges between soil solution and bones. Ionic interactions with soil solution would
involve competition between a wide range of ions for the

various lattice position in bone mineral (apatites) [2]. In
this context, the preservation of the geochemical signal in
biological apatites that are relevant for studies related to
between 14C dating, rate diffusion of ion metal (Cu2+,
Cd2+ and Zn2+), and shift of FTIR spectra.
In the Borneo, elephants have a very limited distribution, being restricted to approximately 5% in northeast.
Fossil evidence for the prehistoric presence of elephants
on Borneo is limited to a single specimen of tooth from a
cave in Brunei. Two history popular, considering the geographic proximity to Borneo, the elephant trade that flourished in Sumatra, Java and Paninsular Malaya during 16th18th centuries may have been the source. Alternatively,
Borneos elephant presented to the Sultan of Sulu in 1750
to Borneo northeast by East India Trading Company.
These animals presumably originated in India [3].
Conversely, if elephant occurred naturally on Borneo,
they would have colonized the island during Pleistocene
glaciations, when much of the Sunda shelf was exposed
and the western Indo-Malayan archipelago formed a single
landmass designated Sundaland. Thus, the isolation of
Borneos elephant from other conspecific population
would minimally date from the last glacial maximum
18,000 year ago, when land bridges last linked the Sunda
islands and the mainland [4]. If Borneos elephants are
indigenous origin, this would push the natural range of
Asian elephant 1300 Km to east, and as a unique population at an extreme of the species range, Borneo elephants
in situ conservation would be apriority and ex situ crossbreeding with other population would be contraindicated
[3].
Initially, Borneo elephant were classified as a unique
subspecies (Elephas maximus borneoensis) based on morphological differences other population [5]. Subsequently,
they were subsumed under the Indian Elephas maximus
indicus[6] or the Sumatran Elephas maximus sumatrensis[7] subspecies, based on assumption of their introduction to the region or on the reasoning that morphological
divergence was insufficient to warrant separated status.
According to Fernando (2003), HVS mtDNA analysis
showed that Borneos elephant are genetically distinct with
th
molecular divergence indicative of a Pleistocene coloniza- conclusions. The sharp band 3500-4000 cm-1 in the HAp,
tion of Borneo and subsequent isolation.
Tambans subfossil and Tahuras bone spectrum is assigned to the OH stretching mode; presence this mode
from the spectra of Tahuras bone and Tambans subfossil
The Fourier Transform Infrared (FTIR) spectra were implies the carbonate content and silicon substitutions did
recorded on Bruker Optic IFS66s/S interferometer not induce vacancies at the OH sites. The increase in inequipped with an attenuated total reflectance (ATR) unit. tensity with decrease in carbonate content and the presence
The range frequencies was 650-4000 cm-1 and the typical of structurally bound OH in the little carbonated Tahuras
experimental condition utilized a resolution of 4cm-1, a bone and Tambans subfossil has been reported in a very
velocity of 6-10 KHz, a gain of 16x, an apodization Black recent work.
Table 1, shows the 650-900cm-1 region of the IR specHarris 3-term, a Mertz phase correction and zero filling 2,
on a double sided, forward-backward acquisition mode. A tra of all the samples. In the region, all substituted samples
KBr beam splitter was used for the M-IR source. Subse- display very weak bands that can be assigned to the vibraquently, aliquots of approximately 2 mg subfossil Tam- tion4CO32- (4 CO32-)and 2 CO32- modes at energies
bans elephant were ground and pressed into a KBr pellet similar to the previously reported exceptfor the Tahuras
and the infrared spectra were measured on a Perkin Elmer bone and Tambans subfossil [8]. The absence 2CO32-,
the subfossil and bone implies that carbonate and silicon
Spectrum One instrument [8].
For isotope 14C dating, carbonate in calcined subfossil substitution dont induce vacancies at the OH- site, probaTambans elephant obtained is the most reliable source of bly considering the fossilization have been a conservation
inorganic carbon. The subfossil was demineralized in a 1% in wetland environment. The weak intensity of the absorphydrochloric acid (HCl) solution several days. The ex- tion band near 1640cm-1 corresponding to CNH of the
tracted gelatin-like collagen was thoroughly washed with amide group [1]. According to Abeyratne et al (1997) [14],
distilled water. In order to remove the humic acid, the col- FTIR trace for bone and fossil, phosphate is indicated by
lagen was treated with an 0.1 N sodium hydroxide (NaOH) double troughs around 600cm-1 and width trough at 1036
for several days. The remaining collagen was again washed cm-1. Carbonate is shown bay the narrow dip at about 875
with distilled water, dried and carbonized by heating at cm-1 and the wider one at 1425cm-1. The Tambans sub800oC in an oxygen-free environment. The phosphorous fossil and Tahuras bone was measured with FTIR by excompounds were removed by treating the collagen with amining the splitting factor of PO4 anti-asymmetric bendaqua regia, a mixture of nitric acid (HNO3) and hy- ing mode peak at wave number 563cm-1.
The FTIR spectra of both bone and subfossil are characdrochloric acid (HCl). The cleaned collagen was then
terized
by intense band between 1300-2000cm-1 and
washed with distilled water, dried, and used for carbon
2300-3000
cm-1 indicating an abundant contribution of
dioxide gas (CO2) preparation [9].For metal Cu, Cd and
Zn carried out by atomic absorption spectroscopy (AAS) alkyl chains. Strong aliphatic absorptions centered at
[10]. Modeling dating relative referenced to Lagrange me- around 2860-2930cm-1 are assigned to asymmetric stretching vibrations from CH2 and symmetric stretching vibrathod of interpolation [11], [12] and [13].
tions from CH2 methylene group, respectively. The absorption of symmetric bending of CH3 with possible conIII. RESULTS AND DISCUSSION
tribution from (CH)n bending. The presence a long polymethelynic chains (n 4) is indicated by the absorption at
A. Examination of sample preservation by FTIR
around 720cm-1. The absorption at 1710cm-1 shows the
The major peculiarities for a diagenetically altered bone
presence of carboxyl group. The weak absorption signal
are an increase in crystal size and a decrease in protein
attributed to aromatics C=C ring stretching vibration peaks
content. Thus information on the state of degradation can
at round 1620cm-1.
be obtained from FTIR spectroscopy (Fourier Transform
Infrared Spectroscopy) by observing the characteristic
splitting of the double peaks at 563-604 cm-1 correspondB. Radiocarbon Dating and Originality of Boring to phosphate vibration (PO4)3- indicating mineral
neosElephant
phase modification, e. g changes in crystallinity and ion
In the Indian subcontinent, the dispersal elephants carexchange. A low value for the splitting factor (SF) indi- ried out by human intervention through trade and warfare.
cates a high amount of amorphous material in the mineral Human activity has changed the natural distribution pattern
phase and obtained as described in Reiche et al. (2003). of elephants. Special originality Borneos elephants still
The intensity of the organic CO-signal at 1650 cm- discussion. There are two popular hypotheses, first, refer
1(compared to the signal of inorganic CO32- at 1450 to the documents that the sultan of Sulu has received a gift
cm-1 provides an indication of the degree of collagen de- elephant from the Indian (Elephas maximus indicus) in
gradation and expressed as C=O and C-C bonding, where a 1750 and domesticated in North Borneo[6]. According to
high value represents a high degradation of collagen in the Medway (1977)[7], elephant Borneo derived from Sumasample [8].
tran elephant or Elephas maximus sumatrensis. The next
The IR spectra of all specimens (Tahuras bone , Tam- hypothesis is indigenous or not introducer. Fernando
bans subfossil and Antonakoss data) show broad bands in (2003) [3] stated that based on the analysis of D-loop
the high energy region that are propably water related band mtDNA fragment, population showed evidence of indi(table 1). They show peak positions without leading to any genous and not introducer. Mac Kinnon (1996) [4] also
th
stated that the Borneo elephants have colonized the Pleistocene about 18,000 years ago when the bridge was
formed Sundaland shift. Our 14C dating data also show that
the findings of the elephant subfosil from Tamban village,
district Batola shows the age range 4855 and 568416
years ago. This information supported the second hypothesis.
TABLE 1.
ROOM TEMPERATURE OBSERVED IR MODES AND THEIR
ASSIGNMENT
HAp
574s
856
wbr
CHAp
15M
577s
CHAp
25M
584s
SiHAp
CFAp 530
CFAp 840
592s
590s
602s
674
vm
noisy
[759vw
805vw
844m]
872s 880m
893w
954sh 961s
668 m
[713vm
760vw
813vw
847sh]
873s
880m
933sh
961s
680vw
spectrum
668 vw 690w
720w
668w
754vm
865s 877s
864m 876m
2 CO3
956sh
992sh
1 PO4
1029vs
1060sh
1094s
1029vs1061sh1093vs
961s
noisy
873w 882w
936sh 962s
964s
Tambans
Subfossil
563s
727w
871vw
vm
Tahuras
Bone
563s
760
871vw
963s
1015s
1029vs
1060sh
1029vs
3400br
3567m
1029vs
1045vs
1060sh
1091s
1174m
1223m
1409m
1427m
1444m
1468sh
3700br
4073br
1410s
1450s
1470s
1498sh
1568sh
3553br
3460br
1030vs1060sh
1093vs 1160m
1427s
1456s
1468s
[1482sh
1506vm
1518vm
1538vm
1558vm]
3400br
1025vs1045sh
1093s
1146m
1162w 1424m
1452m
[1470sh
1506vm
1518vm
1538vm
1558vm]
3750br
1033vs
1334vm
1411m
1550vm
1658m
1982vm
2075vm
2252vm
2337m
2939m
2970m
3425br
3927m
1041vs
Assignment
4 CO3and
1 PO4
4 CO3
3 PO4 and
1 CO3
3 CO3 and
SiO4
1411m
1627m
2291vm
2368m
3410br
3749m
OH ion or
moisture
Code: [HAp: (Ca10(PO4)6(OH)2)]; [CHAp 15M: (Ca10(PO4)6(OH)2) 15M];

[CHAp25M: (Ca10(PO4)6(OH)2) 25M]; [SiHAp : (Ca10(PO4)6-x(SiO4)x(OH)2)];
[CFAp 530: (Ca10(PO4)6(F)2) heat treat at 530 oC]
[CFAp 840: (Ca10(PO4)6(F)2) heat treat at 840 oC, 4,8 % loss CO2]
C. Modeling Relative Datingand Cation Exchange Cu2+,

Cd2+ and Zn2+
The use of entropy of hydration (Hh) in addition to
crystallographic ionic radii improve predictions concerning the abilities of various cationic to substitute in to Ca2+
lattice position. The heat of hydration provides a measure
of the strength of ion water molecule bond. Strong bonds
are indicated by highly negative Hh values. In the context
fossilization,
the
Cu2+(-2100
kalJ/mol),
Zn2+(2+
2044kalJ/mol), Cd (-1806 kalJ/mol) cationic substitutein
to Ca2+(-1592 kalJ/mol) lattice hydroxyapatite. Larger cationic tend to associate less strongly with water due to their
increased radii () and reduced surfaced area[2].For radii
Cu2+, Zn2+, Cd2+and Ca2+cations are 0.73; 0.74, 0.95,
and 1.00, respectively.The degree of hydration increases
with increasing ionic charge. In the case of this experimental, the third cationicchargeare the same so that is 2+. According Scimiklas (2003) [15] and Smiliklas (2007) [16]
and Chen (1997) [17], the exchange Cu2+, Zn2+, and
Cd2+cationicin to Ca2+ lattice hydroxyapatite can be expressed as follows.
substitution of solution ions for normal hydroxyapatite

lattice position [2]. In our data, Cu2+, Cd2+ and
Zn2+cationic replace lattice Ca2+, after elephant subfossil
from Tamban (Batola-Kalsel) and elephant bone (Tahura
park) have been buried for 1, 2, 4, and 6 month, respectively. Carbon dating showed 485- 5 and 5684 16 BP
(Before Present) (table 2). Modeling dating relative have
been construct based on radiocarbon dating versus spectroscopy data (table 2) using multi-interpolation method
(table 3 and figure 1) [12].e. g. relative dating Y(Zn) = 1.4333e-007t2 +0.00063794t + 0.14854 and rate diffusion
Y(Zn)/dt = -2.8666e-007t + 0.00063794 (table 3 and figure
1).
TABLE 2.
PERCENT OF MATERIAL SUBFOSSIL VERSUS FOR BURIED
(YEAR)
Year
485.08
3
485.16
7
485.25
485.5
7.083
7.167
7.250
7.500
485
Sample
FG
Ash%
Organic%
Cu%
Cd%
76.667
21.260
0.011
0.008
61.290
38.710
0.003
0.005
60.000
61.290
70.968
73.333
67.857
63.333
23
40.000
34.630
18.832
14.907
18.143
24.907
70
Zn%
0.488
0.004
0.009
0.007
0.253
0.005
0.007
0.002
0.008
0.008
0.4
0.009
0.009
0.499
RG
0.013
0.007
0.406
0.006
0.008
0.32
0.118
0.017
0.692
0.0008 0.0003 0.0180
4148
59.70
32.29
3
1
FG: Elephant subfossil buried 485 and 4148 year (BP) from Tamban
village; RG: Elephant bone buried 7 year from Tahura park
TABLE 3.
MODELING DATING RELATIVE BASED ON MATERIAL VS TIME
(YEAR).
Materit2 (year)
t (year)
C
(conal
stant)
Ash
1.2178e-005
-0.049814
64.138
Organic -1.163e-005
0.045528
33.06
Cu
-3.2659e-008
0.00013609
0.004815
Cd
-3.1.3912e1.3912e0.0053648
005
005
Zn
-1.4333e-007
0.00063794
0.14854
[Ca10(PO4)6(OH)2] + n X2+ [CA10-nXn(PO4)6(OH)2] + n

Ca2+
X2+ : Cu2+, Zn2+, or Cd2+cationic
n : number of moles
A
B
Figure 1. Equation shown that type related between ash and organic
material (%) vs t (time-year) (A), equation Cu, Cd and Zn content(%)
versus t (time-year) (B).
Ionic exchange between soil solution and bone should

IV. CONCLUSION
be a dominant process in apatite mineral diagenesis. ExpeFigure FTIR analysisand14Cdatingsuggest thatthere is
rimental evidence indicates that Ba2+, Mg2+, Pb2+, Sr2+,
in the Borneo.
Na+, CO32- an F- enter the crystal surface from the bound Elephas maximusborneensisandindigenous
A
Combinationanalysis
ofCu,
CdandZncontent
of elephant
hydration
layer
of
mineral
hydroxyapatite
14
subfossiland
Cradiocarbondatingshownnon-linear
equa(Ca10(PO4)6(OH)2). Ion exchange involves the isomorphic
tions.
th

[7]
Medway L., (1977), Mammals of Borneo, Monogr Malay Br, R
Asian Soc, 7: 1-72.

ACKNOWLEDGEMENT
[8] Antonakos, A., Liarokapis, E, and Leventaouri, T., (2007), MicroHigest thank to head of Lambung MangkuratMuseuRaman and FTIR studies of synthetic and natural apamandheadof community forestpark(Tahura-Banjarbaru) for
tites,Biomaterilas, 28: 3043-3054.
[9] Keates, S. G., (2010), The Chronology of Pleistocene Modern
gave elephant bone and subfossil sample.
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Fernando, P., Vidya, T. N. C., Payne, J., Stuewe, M., Davison, G.,
Alfred, R. J., Andau, Patrick, Edwin, B., Kilbaourn, K., Melnick,
D. J., (2003), DNA Analysis Indicates That Asians Elephant Are
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Mac Kinnon, K., Hatta, G., Halim, H., Manggalik, A., (1996), The
Ecology of Kalimantan, Hong Kong, Periplus Edition, Ltd. 802 p.
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Shoshani, J., dan Eisenberg, J. F., (1982), Elephas Maximus,
Mamm sp., 182: 1-8.
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[10] Walsh, A., (1955), The application of atomic absorption spectra to
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Ahmadi, M. B., (2009), Lagrange two-dimentional interpolation
method for modeling nanoparticle formation during RESS process,
Int. J. Industrial Mathematics vol. 1, No. 2, 175-181.
[13] Gasca,M., and Sauer, T., (2001), Polynomial interpolation in variables, Advances in Computational Mathematics,
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Multidating studies of Batadomba Cave, Sri Langka, Quaternary
Science Reviews, vol.16, pp. 243-255.
[15] Scimiklas I., D., (2003), Cadmium immobilization by Hydroxyapatite, Chem, Ind, 57(3), 101-106.
[16] Scimiklas, I., Onjia, A., Raicevic, S., Janakovic, D., and Mitric,
M., (2007), Factor influencing the removal of divalent cations by
hydroxyapatite, Journal Hazardous Material, 152, 876-884.
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(1997), Effect of pH on Heavy Metal Sorption on Mineral Apatite,
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th
Particle Motion of Seismic Waves Recorded

from Hydrothermal Area
at Cangar, East Java, Indonesia
1
Wasis1*), Sukir Maryanto1, Dahlia Kurniawati1

Geophysics Lab., Dept. of Physics, Brawijaya Univ., Jl. Veteran, Malang 65145, Indonesia
*)email : wasis_55@yahoo.co.id
AbstractThe particle motion analysis of seismic waves

around Cangar hydrothermal area has been investigated to
estimate the epicenter and hypocenter. The determination of
epicenter and hypocenter are based on the direction of the
particle motion, by using single-h methods. On each station CGR01 and CGR02 three events are chosen. From the
particle motion analysis, five epicenters obtained, based on
the intersection of particle motion direction on both stations. They located at (112322,04 E ; 74432,208 S),
(112322,04 E; 74432,1 S), (112320,96 E ;
74431,49 S), (112323,57 E ; 74432,58 S), and
(112323,44 E; 7,67 S). Whereas the hypocenter of earthquake ranging from 3060 meters depth. The epicenter and
hypocenter related to hydrothermal activities in subsurface.
Keywordssparticle motion analysis, seismic, hydrothermal,

Cangar.
I. INTRODUCTION
EOTHERMAL is one of the natural energy
sources originated from the rocks interaction and
heat flow in the earth. Indonesia has 40% of the world
geothermal sources, from Sumatera, Java, Nusa Tenggara
to Sulawesi. One of them is Cangar hotspring in East Java.
Some researchs have been conducted to find geothermal
potential using some geophysics methods such as:
geoelectric, geomagnetic and gravity. Research results
used geoelectric method showed the existence of the
geothermal potential south to the hotspring in the depth of
24,7 meter [1]. Research used geomagnetic method
showed the existence of the geothermal sources in the
north and west direction from the hotsprings[2]. Meanwhile,
researchs used gravity method predicting that there is
geothermal potential as much as 2.064.640 m3 in the
coordinates of 7.7406 S and 112.5339E [3]. Nevertheless,
research using seismic method based on the microseimic
analysis to find area having geothermal potential has never
been conducted yet. Geothermal energy can be defined as
energy that naturally produced by the earth. Earthquake in
the geothermal area connected to sesar movement along
the geothermal fluids flow [4]. Earthquake with magnitude
less than 3, known as microearthquake. According to Holland (2002) [5], by studying microeartquake on the
geothermal location, inrerrelationship of the crack sytems
that control fluids migration on the geothermal area can be

determined.
According to Utama et al (2013) [6], microseismic

or microtremor is one of the passive seismic
methods for recording the vibration of the earth
caused by vulcanic activity, waves, meteorology
regional
condition,
human
activities,
etc.
Microseismic method usually used for exploration,
mining, as well as geothermal.
One of the methods to investigate the crack existence in
the geothermal field is the microseimic particle motion.
Horizontal and vertical components of the particle motion
used for determining epicenter and hypocenter of the microseismic.
II. RESEARCH PROCEDURE
This research used microseismic data in Cangar area,
East Java, with two recording stations CGR01 and CGR02.
Research flow as seen in figure 1. Data recorded by TDS
have 3 components which are North-South (NS), EastWest (EW) and Up-Down (UD). These three components
will be used for analysing microseismic particle motion.
Data in the time domain will be transformed to the
frequency domain using FFT (Fast Fourier Transform) so
that frequency spectrum for each component obtained.
Spectogram analysis is needed to know variation of the
harmonic signal frequency to time. This process aims to
determine frequency limit that will be used in filtering
process. Filtering used Butterworth band-pass filter,
because filter of this type has an advantage in band-pass
filtering.
Particle motion plotting in horizontal and vertical
components was used for determining epicenter and hypocenter of a microseismic. Microseismic epicenter predicted
by examining the particle motion direction, and then
calculated roughly. The same process was applied for
determining hypocenter distance of a microseismic in a
grothermal field. Interpretation on the existence of the
geothermal potential, need to be correlated to the results of
the prior researchs.
th

350
Start
300
Amplitudo
250
Secondary
Data
200
150
100
50
0
0
10
15
20
25
30
35
40
45
50
Frekuensi
Data Selection
Figure 2. Frequency Spectrum of Microseismic
Data Conversion
FFT
Spectrogram
Frequency
Spectrum
Figure 3. Microseismic Spectrogram
Filtering
Particle Motion
Interpretation
Frequency limit determined by the frequency spectrum

analysis and spectogram will be applied in filtering
process. Filtered signal will be sampled every 1 second in
order to know particle motion direction. Based on the
particle motion plotting for horizontal and vertical
components, position of the epicenter point can be
determined.
N
70
60
50
50
40
40
30
30
20
20
10
10
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-2 5 0 -2 0 0 -1 5 0 -1 0 0
Epicenter; Hypocenter
0
-5 0
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E
0
50
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250
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0
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250
-10
-2 0
-20
-3 0
-30
-4 0
-40
-5 0
-50
-6 0
-7 0
.
Finish
Figure 1. Research Flow Chart
Frequency spectrum analyzed using FFT, where data

recorded in time domain will be transformed to frequency
domain. According to Ihsan (2011) [7], frequency limit
determination based on dominant frequency, showed that
there was a signal in that frequency. Microseismic data in
Cangar area has a dominant frequency more than 15 Hz
(Figure 2). This high dominant frequency could becaused
by hydrothermal activities influence. The same result
yielded by the spectogram that applied the STFT (Short
Time Fourier Transform) principles that is frequency
variation to time.
Figure 4. Particle Motion CGR01: a) horizontal component (b) vertical

component
Based on the particle motion analysis, seismic activities in

Cangar area, East Java, there are 5 epicenter points with
subsurface hydrothermal activities. This is supported by
geothermal manifestation distribution around that area.
The dominant rocks in Cangar, according to geoelectric,
geomagnetic, and gravity surveys are basalt and tuff. Tuff
rocks contain many cracks from where fluids (water) flow
(Rakhmanto, 2011). Cracks happened because of volcanic
activities or tectonic in Mount Arjuno-Welirang area.
Fluids under the surface will be heated by hot rocks, so
that the hot fluids activities increased and earthquake took
place. Figure 5 shows three epicenter points of the
microseismic that could be represent unrevealed
hydrothermal sources
T
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Keterangan:
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30 Meters
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Figure 5. Geothermal Distribution Map in Cangar Area, East Java
III. CONCLUSION
Particle motion analysis on the horizontal and
vertical components can be used for predicting the
location of the epicenter and hypocenter of the
microeartquake. Based on the research, the center of
the earthquake is in depth of 16 to 60 meter.
Epicenter distribution are on the 5 points which are:
(112322,04 E; 74432,208 S), (112322,04 E;
74432,1 S), (112320,96 E; 74431,49 S),
(112323,57 E;74432,58 S), and (112323,44
E; 74432,67 S).
1. Epicenter and hypocenter determination related
to the hydrothermal activities in the subsurface. A
high dominant frequency spectrum showed that there
are fluid activities heated by the hot rocks around
them.
REFERENCES
[1] Rakhmanto, F., 2011. Tomografi Geolistrik Daerah Panasbumi Welirang-Arjuno (Studi Sumber Air Panas Cangar Batu). Tesis S2. Universitas Brawijaya Malang.
[2] Afandi, Akhmad. 2011. Studi Potensi Panas Bumi Di Daerah Cangar
Kota Batu Jawa Timur
[3] Zaman, Muhammad Badaruz. 2011. Studi Potensi Panas Bumi Di
Pemandian Air Panas Cangar, Kota Batu, Jawa Timur Dengan
Menggunakan Metode Gayaberat. Skripsi S1. Universitas Brawijaya
Malang.
[4] Holland, Austin Adams. 2002. Microearthquake Study Of The Salton
Sea Geothermal Field, California: Evidence Of Stress Triggering.
The University Of Texas. El Paso.
[5] Utama, W., Tri Martha Kp, Dwa Desa W., And Makky S. Jaya. 2013.
Application Of Ensemble Empirical Mode Decomposition (Eemd)
For Identification Of Hydrothermal Dynamics In The Subsurface,
Case Study Mt. Lamongan, East Java. Proceeding Itb Geothermal
Workshop. Bandung.
[6] Ihsan, Agung Budi. 2011. Karakterisasi Mikrotremor Di Daerah
Sekitar Sungai Porong Desa Kebonagung Sidoarjo. Skripsi S1. Universitas Brawijaya Malang.
th
MATHEMATIC
AND
STATISTIC
th
Model Components Selection in Bayesian

Model Averaging Using Occam's Window for
Microarray Data
1)
Ani Budi Astuti1*), Nur Iriawan2), Irhamah3) and Heri Kuswanto4)

PhD. Student at Statistics Department of Mathematics and Natural Sciences,
Institut Teknologi Sepuluh Nopember (ITS), Surabaya, Indonesia
1)
Mathematics Department of Mathematics and Natural Sciences,

Brawijaya University, Malang, Indonesia
2), 3), 4)
Statistics Department of Mathematics and Natural Sciences,

Institut Teknologi Sepuluh Nopember (ITS), Surabaya, Indonesia
*)
e-mail: ani_budi@ub.ac.id
AbstractMicroarray is an analysis for monitoring gene expression activity simultaneously. Microarray data is data generated from microarray experiments having characteristics of
very few numbers of samples where the shape of distribution is
very complex and the number of measured variables is very
large. Due to this specific characteristic, it requires special method to overcome this. Bayesian Model Averaging (BMA) is a
Bayesian solution method that is capable to handle microarray
data with a best single model constructed by combining all possible models in which the posterior distribution of all the best
models will be averaged. There are several method that can be
used to select the model components in Bayesian Model Averaging (BMA). One of the methods that can be used is the Occam's
Window method. The purpose of this study is to measure the
performance of Occam's Window method in the selection of the
best model components in the Bayesian Model Averaging
(BMA). The data used in this study are some of the gene expression data as a result microarray experiments used in the study
of Sebastiani, Xie and Ramoni in 2006. The results showed that
the Occam's Window method can reduce a number of models
that may be formed as much as 65.7% so that the formation of a
single model with BMA only involves the model of 34.3%.
KeywordsBayesian Model Averaging, Microarray Data,
Model Components Selection, Occams Window Method.
I. INTRODUCTION
Microarray data is the data obtained from a microarray
experiment that is an experiment with a particular analysis
technique to monitor the activity of thousands genes expression simultaneously [1]. Microarray data have several characteristics i.e. -limited availability of the number of samples
because of limited budget, resources and time. Though the
availability of the number of samples is limited, the measurable characteristic variables can be hundreds or even thousands of gene expression. By these special characteristics, it
is possible that the nature of the distribution of gene expression data will be very complex in which the distribution of
the data is probably not a normal distribution [2]. Due to
these specific characteristics, it requires special method to
overcome this.
Bayesian is a statistical analysis method that does not
consider the number of samples (especially for very small
sample size) and to any form of distribution. Moreover,
Bayesian method is based on information from the original
data (driven data) to obtain the posterior probability distribution which is a product of the prior distribution and the likelihood function [3]. Model Parameter in Bayesian method is
viewed as a random variable in the space of model parameter
and allows for the formal combination of different from the
prior distribution and facilitates the iterative updating of new
information which thus overcome the problem of uncertainty
and complexity of the model in the data [4] .
Bayesian Model Averaging (BMA) is a Bayesian solution to model uncertainty in which the completion of the
model by averaging the posterior distribution of all the best
models. The basic principles of the BMA is form the best
single model by considering all possible models that could
be formed so that the purpose of the BMA is models incorporate uncertainty and obtain the best model [5]. There are
several method that can be used for the model components
selection in the BMA of which Occam 's Window method of
[5]. This method is quite simple and widely used in research
related the BMA in which obtained quite good results in the
model components selection in the modeling of the BMA [5]
and [6].
Various studies have been done related to the Bayesian
Model Averaging (BMA), among others [6], [7], [8], [9],
[10] and [11]. In this study will be used Occam's Window
method of [5] to select the component model in the modeling
of the BMA for microarray data.
II. MICROARRAY, BAYESIAN MODEL AVERAGING

AND OCCAMS WINDOW METHOD
A. Microarray Techniques and Microarray Data.
According to [1], microarray technique is a technique
of data collection by using the platform (reference) which is
a duplicate of the original object identifier. The measurement
data of a microarray technique called Microarray Data [12].
There are a variety of different technologies have been developed for microarray techniques, among which is a Synthetic Oligonucleotide Microarray Technology [13]. Gene
expression data is the measurement data from Microarray
techniques so that the gene expression data has the characteristics of microarray data. According to [2], the data obtained
from experiments with microarray technique has the following characteristics:
1. The number of samples that can be observed very limited
(slightly) because of limited budget, resources and time.
Though the availability of the number of samples is
limited, the measurable characteristic variables can be
hundreds or even thousands of gene expression.
2. The nature of the distribution of data will be very complex
in which the distribution of the data is probably not a
normal distribution.
By looking at the characteristics possessed by the microarray data then to analyze of the microarray data requires
special handling because it is generally the basis of parametric statistical method, especially for the comparative analysis requires a large number of samples. If the basis of this
statistical method is not fulfilled then the conclusion of the
analysis can not be accounted for [9].
Bayesian Method.
Bayesian is a statistical method based on the combination of two information that is the past of data information as
the prior information and the observations data as a constituent likelihood function to update the prior information in the
form of posterior probability distribution model. Bayesian
method is based on information from the original data (driven data) to obtain the posterior probability distribution and
it is does not consider the number of samples (especially for
very small sample size) and to any form of distribution.
Bayesian method allow for the formal combination of different from the prior distribution and facilitates the iterative
updating of new information which thus overcome the problem of uncertainty and complexity of the model in the data.
The Rational of Bayesian method derived from Bayes Theorem thinking concept invented by Thomas Bayes in 17021761[3], [4], [14] and [15].
In Bayesian method, the parameters of the model is
seen as a random variable in the parameter space . Suppose there are x observational data with likelihood function f ( x | ) then the known information about the parameters
before
th
the observations were made is referred to as
namely p( ) . Posterior probability distribution of

, namely p( | x) determined by the rules of probability
prior
in Bayes theorem [3] as follows:
f ( x | ) p ( )
f (x)
p ( | x ) =
( 2 . 1)
where
f ( x) = E[ f ( x | )] =
f ( x | ) f ( ) d
if
x R
continous and
f ( x) = E[ f ( x | )] = f ( x | ) p( ) if discrete.
xB
f (x) is a constant called the normalized constant [4]. So

that the equation (2.1) can be written as:
p( | x) f ( x | ) p( )
Posterior Likelihood Function x Prior
(2.2)
Equation (2.2) shows that the posterior probability is proportional to the product of the likelihood function and the prior
probability of the model parameters. This means that the
update's information prior to use information of samples in
the data likelihood to obtain the posterior information that
will be used for decision making [16].
B.1.1. Markov Chain Monte Carlo (MCMC) Algorithms with
Gibbs Sampler Approach.
According to [17], [18] and [19], MCMC algorithms
with Gibbs sampler approach can be described as:
Step 1. Set initial values for
(0)
(0)
1
,...,
(0)
r
(k )
at
k = 0 so that
Step 2. Sampling process to obtain the value of
j from the
conditional distribution by the sampling for

r steps as follows:
2.1. Sampling
1( k +1)
from
p 1 | x, 2( k ) ,..., r( k )
2( k +1)
2.2. Sampling
( k +1) in
from
from
p 2 | x, 1( k ) , 3( k ) ,..., r( k )
.
.
r( k +1)
2.r. Sampling
p r | x,
(k )
1
(k )
2
,...,
(k )
r 1
Step 3. Doing iteration in step 2 as M times with
C. Bayesian Model Averaging (BMA).

C.1. Basic Concepts of Bayesian Model Averaging (BMA)
The basic concept of Bayesian Model Averaging
(BMA) is the best single model formed by considering all
th
possible models that could be formed. BMA is a Bayesian

solution for model uncertainty in which the completion of the
model uncertainty by averaging the posterior distribution of
all the best models. The purpose of the BMA is to combine
models of uncertainty in order to obtain the best model [5]
and [6].
According to [20], the prediction parameters using the
BMA approach uses data derived from a combination of
hierarchical models. If known {M 1 , M 2 ,..., M q } is the set
of models which may be formed from M and is the value
to be predicted, then the BMA prediction begins with determining the prior probability distribution of all the parameters
of the model and the model M k . Posterior distribution of
if x is known to the data as follows:

q
P( | x) = P( | M k , x) P( M k | x)
( 2.3)
k =1
where q is the sum of all the models that may have formed.
Posterior distribution of if known the data x is the average of the posterior distribution if known models weighted
by posterior probability models. While the posterior probability of the model M k is:
P( M k | x) =
P(Y | M k ) P( M k )
q
P(Y | M
l =1
Mk .
is
p ( k | M k ) and
and
p ( M k ) is the prior probability of M k if model M k is true.
Implicitly, all probabilities depend on the model M so that
the expected value of the coefficient of obtained by averaging the model of M , that is:
q
( 2 .6 )
k =1
E ( | x) in the equation (2.6) shows the

weighted expected value of in every model possible comThe value of
bination (weights determined by the prior and the model).

While the variance of ( | x) is:
Var( | x) = (var( | x, M k ) + [E( | M k , x]2 )P(M k | x) E( | x)2 ) (2.7)
k =1
where A is the posterior odds to the model- k and c values
highest posterior probability score and P ( M k
k if known model M k
p( k | M k ) is the likelihood
( 2.8)
( 2.5)
Equation (2.5) is the marginal likelihood of the model
E ( | x) = P( M k | x) E ( | M k , x)
max l ( P ( M l | x ))
c}
P( M k | x)
(2.4)
where
Prior probability of
A= {M k :
of 20 is equivalent to = 5% if using the test criteria with

p-value [21]. If a model has a value of A is greater than 20,
then the model is not included in the modeling of the BMA
and must be removed from the equation (2.3) and otherwise
if a model has a value of A is less than or equal to 20, then
the model will be included in the modeling of the BMA and
should be included in the calculation of equation (2.3). In the
equation (2.8), max l ( P ( M l | x )) is the model with the
) P(M l )
P ( x | M k ) = P ( x | k , M k ) P ( k | M k ) d k
C.2. Model Components Selection in Bayesian Model Averaging (BMA).

Based on the basic concept of Bayesian Model Averaging (BMA), the components of the model will be selected to
be included in the equation (2.3) of q number of models
that may be formed. There are several method for selecting
the components model that will be included in the equation
(2.3) based on its posterior probabilities, which are Occam's
Window method [5]. Occam's Window method is quite simple and widely used in research related to the BMA and give
good results in the selection of components model in the
BMA [5] and [6]. According to [5], the rationale of Occam's
Window method in selecting the component model in the
BMA modeling based on the posterior probability of the
model. The model that will be accepted by this method (the
model can fit in modeling BMA) must satisfy the following
equation:
| x) is the
value of the posterior probability of the model-k. In the various applications of Occam's Window method is generally
able to reduce the large number of components model so that
it becomes less than 100 models of even less than 10 models.
Reduction of component model that only one or two models
are very rare but may occur [5].
III. MATERIAL AND METHODS
The data used in this study are some of the data used in
the study [22]. Selection of component models in the BMA
modeling begins with determining the most appropriate form
of distribution to the data and parameter estimator and then
based on the distribution model raised several distribution
models by MCMC method with the Gibbs sampler approach
to obtain some models that might be formed. Selection of
component in the BMA modeling using Occam's window
method [5] with the following formula:
A= {M k :
maxl ( P(M l | x))

c}. The BMA Modeling in
P(M k | x)
the equation (2.3) is based on the result of model components

selection from Occam's Window method.
th
IV. RESULTS AND DISCUSSION
DISTRIBUTION SHAPE DAN ESTIMATOR PARAMETER

FOR GENE EXPRESSION DATA WITH POLY DETECTOR METHOD
A. Description of Gene Expression Data on Diseased and

Health Conditions with Poly Detector and mRNA Method.
Results of Descriptive statistics for gene expression data on the deseased and healthy condition can be seen in the
following figure:
TABLE 4.2
DISTRIBUTION SHAPE DAN ESTIMATOR PARAMETER
FOR GENE EXPRESSION DATA WITH MRNA METHOD
Figure 4.1. Mean Value of Gene Expression with Poly Detector Method
Figure 4.2. Mean Value of Gene Expression with mRNA Method
Based on Figure 4.1 and Figure 4.2 for the 10 ID genes were
observed known that there are differences in gene expression
for diseased and healthy conditions in which there are several ID genes showed that in healthy condition is more expressive than the deseased condition that is H55933, R39465-2,
U14973, R02593, T51496, H80240 and T55131 for Poly
Detector method and U14973 for mRNA method and otherwise there are several ID genes showed that in deseased condition is more expressive than the healthy condition that is
R39465-1, R85482 and T65938 for Poly Detector method
and H55933, R39465-1, R39465-2, R85482, R02593,
T51496, H80240, T65938 and T55131 for the mRNA method.
B. Identification of Distribution and Parameter Estimator for
the Data
The results of the identification to distribution and parameter estimator for gene expression data can be seen in
Table 4.1 and Table 4.2 below:
TABLE 4.1
Based on Table 4.1 and Table 4.2, it can be seen that there
are some differences in the distribution of ID genes in diseased and healthy conditions that is 6 ID genes with Poly
Detector method and 5 on the mRNA method and some other ID genes that have the same distribution that is 4 ID genes
in Poly Detector method and 5 on the mRNA method. In
addition, most of the data have non normal distributions that
is lognormal distribution and there are some others have 2parameter exponential distibution.
C. Model Components Selection in BMA with Occam's
Window method.
The results of the identification to model components
selection in BMA with Occams Window method can be
seen in Table 4.3 and Table 4.4 below:
TABLE 4.3
th
PERCENTAGE OF COMPONENT MODELS INCLUDED IN THE BMA

MODELING WITH OCCAM'S WINDOW
FOR POLY DETECTOR METHOD.
eral genes ID that have the value of the expression in healthy

condition is stronger than diseased condition. The average
percentage of the component model that can be included in
the BMA modeling with Occam's Window method as much
as 34.3%. This means that the Occam 's Window method can
reduce the component model may be formed as much as
65.7% so that in the form of the BMA modeling involve only
34.3% where it would further simplify the model without
reducing the validity of the model is formed.
ACKNOWLEDGMENT
TABLE 4.4
PERCENTAGE OF COMPONENT MODELS INCLUDED IN THE BMA
MODELING WITH OCCAM'S WINDOW
FOR MRNA METHOD.
This research is part of the doctoral research at the Statistics Department of Institut Teknologi Sepuluh Nopember
(ITS), Surabaya, Indonesia. We would like to thank the
group of researchers Sebastiani, P., Xie H. and Ramoni,
M.F. to the use of data, Head of the Mathematics Department and Dean of FMIPA UB Malang.
REFERENCES
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Based on Table 4.3 and Table 4.4, it can be seen that the
total of overall mean to percentage of the component models
included in the BMA modeling at 34.3% that is derived from
this calculations (32.54+63.64+4.17+36.88)/4). This means
that in a study with Occam's Window method can reduce the
component models in the BMA modeling was 65.7% so that
in the formation of the BMA modeling involves only 34.3%
of the overall model may be formed.
V. CONCLUSION
Based on the results of research conducted, it can be
concluded that most of the gene expression data as a result of
microarray experiments have nonnormal distributions both in
healthy and diseased conditions. In addition, there are different type of data distribution in healthy and diseased conditions and there is also the same type of data distribution in
healthy and diseased conditions . There are several genes ID
that have the value of the expression in diseased condition is
stronger than healthy condition and otherwise there are sev-
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[3] Gosh, J. K., Delampady, M. and Samanta, T. (2006). An Introduction to
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London.
th
Monitoring of High-Yielding Varieties of Rice

Plants Using Multi Temporal Landsat-8 Data
Candra Dewi 1)
1)
Program of Information Technology and Computer Science, Brawijaya University
AbstractMonitoring spectral characteristic of rice plant is

important to obtain information about the age of rice during it
growth. This study examines multi temporal spectral
characteristics of three varieties of high yielding rice plant in
Malang using Landsat 8 image. The varieties consist of IR64,
Ciherang and Memberamo. Normalized Difference Vegetation
Index (NDVI) is used to detect the condition and the age of rice
plants. The comparison of their vegetation indices shows that all
these three varieties have different growth patterns, where the
most distinct pattern found in IR64.
KeywordsHigh Yielding Rice Plant, Landsat-8, Monitoring,
Spectral Characteristic
I. INTRODUCTION
ICE is a staple food source in almost all regions of
Indonesia. Therefore, the increasing of population
resulted the increasing of the demand for rice. However,
the extent of agricultural area more and more reduced and
turned into residential area and other uses. Thus, continuos
monitoring and identification of the rice plant are needed to
determine the availability of rice.
Satellite image is one of the method that can be used to
monitor rice plant during its growth. This process can be
done by using the data of spectral characteristics of the plant
during its growth phase. Some research on monitoring crop
growth have been carried out. Most of these research utilizes
a medium resolution imagery such as NOAA-AVHRR and
MODIS [1]-[5] and RADARSAT [6][7]. In Indonesia, the
prediction of the greenery rate of agricultural crops,
especially rice has been conducted continuously by Space
Agency (LAPAN) using NOAA and MODIS satellite
imagery. But, for a small scale of agricultural land parcell,
this image could not be used because in one pixel contains a
variety information of land uses. This will reduce the
accuracy of the identification process [8]. Occording to this
limitation of small resolution imagery, some research were
conducted by using medium resolution imagery such as
Landsat ETM + [9][10]. This image with a resolution of 30
m could be used for small scale agricultural land parcel.
ETM+ imagery also has a revisit time every 16 days, so that
appropriate if it is used for monitoring the growth of rice that
has a growth cicle between 110 to 125 days .
In the study was conducted by Nuarsa et al, the spectral
identification process was carried out on Ciherang varieties.

As in Indonesia, the type of rice planted is vary widely. In
East Java, especially in Malang district, a type of rice that
are usually planted is high yielding varieties of rice. Each
type of high yielding varieties has difference characteristics
and difference growth cycle until harvest time. In addition,
each type also has different yields product. According to the
Center for Rice Research Bereau [11] , rice IR64 ages
ranged from 110 to 120 days with average yield is 5 tons/ ha
and can reach a maximum of 6. For Ciherang variety has a
life cycle of 116 to 125 days with an average yield about 6
tons/ha and can reach a maximum of around 8 tons. While
Membramo variety has a lifespan of 115 to 120 days with the
average yield is 6.5 tons/ha.
As already known, that the Landsat 7 satellite was
damaged since May 2003 and resulted the captured images
that contain stripping data. This image causes the
identification process does not produce optimum accuracy.
To replace this satellite, in February 2013, NASA launched
Landsat 8 with characteristics similar to the Landsat ETM+
in contextual of resolution, correction methods, and the
characteristics of the sensor (http://landsat.usgs.gov).
However, Landsat 8 has added characteristic as perfecting of
Landsat ETM+ such as the number of bands, the lower range
of the electromagnetic waves spectrum that can be captured
by the sensor and 16 bit value range of each pixel. The
increasing of quantification for each pixel will improve the
ability to distinguish each interpreted object.
To determine the sufficiency of rice in more detail, needs
a research to monitor the growth of each rice plant varieties.
Therefore, this study is aimed to monitor several varieties of
rice plant during their growth using Landsat 8 image data.
A. Study Area
Location of study is Malang regency. Administratively,
the area is part of East Java province, Indonesia. Geographically, the study area is located between 7o50 ' 8o09 ' South
Latitude and 112o33 ' 112o44 ' East Longitude.
Rice plant phase consists of three phases namely vegetative (early growth until panicle formation/primordial), reproductive (primordial until flowering) and maturation (flowering to mature grain). In general, most tropical rice varieties
th
have reproductive phase of approximately 35 days and a

maturation phase of approximately 30 days. According to the
Center for Agricultural Research and Development of Agriculture Ministry (BPPT), the rice planting follows certain
patterns season called dasarian. The dasarian is calendar
system for rice cultivating established by the Ministry of
Agriculture. However, based on the data obtained from the
field survey, the majority of agricultural areas have cultivating time that does not comply with this calendar system
(TABLE 1). The field survey found that the average rice cultivating season is twice a year with each planting cycle was
about 4 months (120 days).
TABLE I
RICE CULTIVATING SEASON AT SOME SUB DISTRIC IN MALANG
(SOURCE: [12], [13] AND FIELD SURVEY)
No
Sub District
Dasarian (BPPT)
*MT I/
*MT III /
MH
MK II
Field Survey
Blimbing
Oct II - III
Jun II III
Jul, Sep
Kedungkandang
Nov I - II
Jul I II
Jul, Aug, Sep,

Oct
Lowokwaru
Oct II - III
Jun II - III
Aug, Sep, Oct
Sukun
Nov I - II
Jul I II
Aug, Sep., Oct
Karangploso
Oct II - III
Jun II - III
Kepanjen
Nov I - II
Jul I II
Lawang
Oct II - III
Jun II - III
Pakis
Nov I - II
Jul I II
Sep, Oct, Nov
Pakisaji
Oct II - III
Jun II - III
Aug, Sep, Oct
10
Singosari
Oct II - III
Jun II - III
Aug, Sep, Oct
Sep, Oct
Aug, Sep, Oct,
Nov
Sep, Oct
*MT (Cultivating Season), MT I (Wet Season/MH), MT III (Dry Season/MK).
B. Landsat-8 Data
Landsat 8 satellite provide data in the form of digital values with a spatial resolution (pixel) 30m to the visible
region, near infrared and middle infrared. The characteristics
of Landsat 8 are recognized using Operational Land Manager sensors (OLI). Landsat 8 has shorter bands interval than
Landsat ETM+ intervals and with the addition of two bands.
Landsat-8 allegedly had better geodetic and geometric accuracy.
Data collected to LDCM Mission by the Operational Land Imager (OLI) instrument will improve the measurement capability in the future. With the "Ultra-Blue" band
(Band 1) which is used for coastal and aerosols study, as
well as Band 9 is useful for detecting cirrus clouds and two
thermal bands provide more accurate surface temperature
(TIRS 1 and TIRS 2). The spectral characteristics of Landsat-8 are shown in TABLE 2.
TABLE 2
SPECTRAL CHARACTERISTICS OF LANDSAT-8
Band
Spectral Range
(
m)
Band Division
Spatial Resolution (m)
1
2
3
4
5
6
7
8
9
10
11
0,43 0,45
0,45 0,51
0,53 0,59
0,64 0,67
0,85 0,88
1,57 1,65
2,11 2,29
0,50 0,68
1,36 1,38
10,6 11,19
11,5 12,51
Ultra Blue
Blue
Green
Red
NIR
SWIR1
SWIR2
PAN
Cirrus
TIRS 1
TIRS2
30
30
30
30
30
30
30
15
30
100
100
Overall Malang is located in Path 118 Row 066 on Landsat 8 image. Based on the field survey, the monitoring is examined for three varieties namely Ciherang, IR64 and Membramo. For this study, the sample data of rice field is planted
on early August. To monitor the characteristics for one rice
growth cycle is used as much as 6 time series images with
different acquisition date (August 13, 2013; August 29,
2013; September 14, 2013; September 30, 2013; October 16,
2013 and November 1, 2013). Some of the images that are
acquired on November and December can not be used due to
the existing of the clouds with a fairly high percentage.
C. Methods
The step of monitoring spectral characteristic for this
study consists of two parts. The first is calculating the vegetation index value to determine differences in the pattern of
the three varieties in one rice growth cycle. The second is
determines the relationship between the vegetation index and
rice age.
This study uses 135 pixels that representing the pixels for
IR64, Ciherang and Membramo for the analysis. These pixels are taken from 5 different agricultural areas in each acquisition date of Landsat image. Then, the average value was
used to represent the value of each variety. The vegetation
indices analysis in this study used normalized difference
vegetation index (NDVI). The calculation of NDVI uses
formula as in (1).
NDVI =
NIR R
NIR + R
(1)
Where NIR and R are Near Infra Red and Red bands

Spectral reflectance patterns of some plants will be different depending on the color of the leaves (chlorophyll), leaf
structure and water content in the leaves. This spectral will
of course influence the vegetation indices pattern of three
rice varieties (Figure 1).
The picture shows that the differences of vegetation indices patterns for the three varieties are clearly seen by using
NDVI. Of the three varieties, IR64 has the most different
pattern than the other two varieties. The peak reflectance of
th
IR64 occurs at the age of 33 days and 68 days. For Ciherang,

the peaks reflectance can be found on days 17 and 68. While
peak reflectance of Membramo found at days 68. Of the
three varieties, it can be identified that in average the peak
reflectance is at the age of 68 days. At this age, the three
varieties are in the reproductive phase in which the leaves of
paddy is lush and entered a flowering period.
Ciherang
100
Rice Age
80
NDVI
y = -2106,1x 2 + 1483,1x - 203,37

R2 = 0,1034
60
40
20
0,5
NDVI
0,4
0,05
0,1
0,15
0,2
0,25
0,3
0,35
NDVI
0,3
0,2
0,1
Fig. 3. Relationship between NDVI and rice age of Ciherang
0
17
33
49
65
81
Memberam o
Age (Day)
IR64
Ciherang
100
Memberamo
Rice Age
80
Fig. 1. NDVI graph of IR64, Ciherang and Memberamo.
Due to the study was conducted by Nuarsa et all, using

multiple bands give better relationship than using single
band. Therefore, this study uses NDVI to utilize relationship between rice age and spectral value of Landsat-8 data. Furthermore, the relationship between rice age and
NDVI was evaluated using determination coefficient (R2).
Base on the experiment, the best equation that can be
used to show the relationship between rice age and NDVI
was polynomial for Ciherang and Memberamo, while for
IR64 was power. Figure 2 to Figure 4 show this relationship for three varieties of rice that are evaluated in this
study. The higher of R2 value shows the stronger relationship between rice age and NDVI. The figure shows that
the strong relationship of these varieties can be seen on
Memberamo variety with the value of R2 is 0.9175. While
the other shows the weak relationship with the values of
R2 are about 0.2928 and 0.1034 respectively for IR64 and
Ciherang.
y = -4290,7x 2 + 2739,4x - 361,5

R2 = 0,9175
60
40
20
0
0
0,1
0,2
0,3
0,4
NDVI
Fig.3.Relationship between NDVI and rice age of Memberamo
IV. CONCLUSION
This study shows that all three varieties have different pattern of growth since it is evaluated using NDVI. Based on the
calculation of vegetation indices can be seen that the three
varieties have a different growth pattern, where IR64 variety
has a growth pattern that is most easily recognized than
Membramo and Ciherang varieties. Otherwise, the stronger
relationship between rice age and NDVI can be found in
Memberamo variety.
IR64
100
REFERENCES
Rice Age
80
[1]
y = 228,64x 1,5556
R2 = 0,2928
60
40
[2]
20
0
0
0,1
0,2
0,3
0,4
0,5
[3]
NDVI
Fig. 2. Relationship between NDVI and rice age of IR64
[4]
S. Panigrahy, J. S. Parihar, and N. K. Patel, Rice Estimation in

Orissa Using NOAA-AVHRR Data, Journal of the Indian Society, of
Remote Sensing, 20, 35-42, 1992.
X. Xiao, S. Boles, J. Liu, D. Zhuang, S. Frolking, C. Li, W. Salas, and
B. Moore, Mapping Paddy Rice Agriculture in South and Southeast
Asia Using Multi-Temporal MODIS Image, Remote Sensing of
Environment, 100, 95-113, 2005.
T. Wataru, O. Taikan, and Y. Yoshifumi, Investigating an Integrated
Approach on Rice Paddy Monitoring Over Asia with MODIS and
AMSR-E, Proceedings of The Conference of The Remote Sensing
Society in Japan, 40, 173-174, 2006.
H. Sun, J. Huang, A. R. Huete, D. Peng, F. Zhang, Mapping Paddy
Rice with Multi-Date Moderate-Resolution Imaging Spectroradiome-
[5]
[6]
[7]
[8]
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ter (MODIS) Data in China, Journal of Zhejiang University

SCIENCE A, Vol. 10, Issue 10, 1509-1522, 2009.
H. O. Kim, and J. M. Yeom, Multi-Temporal Spectral Analysis of
Rice Fields in South Korea Using MODIS and RapidEye Satellite Imagery, Journal of Astronomy and Space Sciences, 29(4), 407-411,
2012, http://dx.doi.org/10.5140/JASS.2012.29.4.407.
F. Ribbes, T. le Toan, Rice Field Mapping and Monitoring with
RADARSAT Data, International Journal Remote Sensing, 20(4):
745-765, 1999.
Y. Shao, X. Fan, H. Liu, J. Xiao, S. Ross, B. Brisco, R. Brown, and
G. Staples, Rice Monitoring and Production Estimation Using
Multitemporal RADARSAT, Journal of Remote Sensing for Environment, 76, 310-325, 2001.
A. H. Strahler, L. Boschetti, G. M. Foody, M. A Friedl, M. C.
Hansen, M. Herold, P. Mayaux, J. T. Morisette, S. V. Stehman, and
C. E. Woodcock, Global Land Cover Validation : Recommendation
for Evaluation and Accuracy Assessment of Global Land Cover
Maps, Office for Official Publication of European Communities,
Available:http://wgcv.ceos.org/docs/wgcv26/GloballandCoverValidati
on_JefMorisette.pdf, 2006.
[9]
[10]
[11]
[12]
[13]
S. Uchida, Monitoring of Planting Paddy Rice With Complex

Cropping Pattern int The Tropical Humid Climate Region Using
Landsat and MODIS Data A Case of West Java, Indonesia,
International Archieves of The Photogrammetry, Remote Sensing and
Spatial Information Science, Volume XXXVIII, Paet 8, Kyoto, Japan,
477- 481, 2010.
I. W. Nuarsa, F. Nishio, and C. Hongo, Spectral Characteristics and
Mapping of Rice Plants Using Multi-Temporal Landsat Data, Journal of Agricultural Science Vol. 3, No. 1: 54-67, 2011.
BPPT, Description the Variety of Paddy, Centre of Research of
Paddy Plant (BPPT), Agriculture Department, 2009.
BPPT, Integrated Planting Calendar on Cultivating Season (MT) I
2013/2014 Malang City, East Java Province, The Center for
Agricultural Research and Development (BPPT), Ministry of
Agriculture, 2013.
BPPT, Integrated Planting Calendar on Cultivating Season (MT) I
2013/2014 Malang Regency, East Java Province, The Center for
Agricultural Research and Development (BPPT), Ministry of
Agriculture, 2013.
th
The CourantFriedrichsLewy Number

Influences the Accuracy of Finite
Volume Methods
Sudi Mungkasi1,*) and Noor Hidayat2,3)
Department of Mathematics, Faculty of Science and Technology, Sanata Dharma University, Mrican, Tromol Pos 29, Yogyakarta 55002, Indonesia
2)
Doctoral Program, Faculty of Science and Technology, Airlangga University, Surabaya, Indonesia
3)
Department of Mathematics, Brawijaya University, Malang, Indonesia
1)
*)
Corresponding author: sudi@usd.ac.id
Abstract The shallow water (wave) equations govern shallow

water flows. We solve the shallow water equations using a finite
volume method. A necessary condition for a consistent finite volume method to be stable (hence, convergent) is that the method
satisfies the CourantFriedrichsLewy (CFL) condition. Numbers representing this condition are called CFL numbers. In this
paper, the effects of CFL numbers to the convergence rate of the
finite volume method are investigated. Setting a CFL number to
the method gives varying time steps in the numerical evolution.
We compare results between those produced by imposing a CFL
number and imposing a fixed time step to the numerical method.
We shall show which strategy is more efficient and produces more
accurate solutions in solving the shallow water equations.
Keywordsconvergence rate, CourantFriedrichsLewy, finite
volume method, shallow water equations.
I. INTRODUCTION
HE system of shallow water equations is a well-known
mathematical model that describes shallow water waves
and flows. We are interested in solving these equations as
the solutions are useful in the simulations of real world problems such as dam break floods and tsunamis. In this paper we
implement a finite volume method to solve the shallow water
equations. The method is chosen due to its robustness in dealing with smooth and non-smooth solutions [9, 10].
In finite volume methods, a necessary condition for convergence is that the CourantFriedrichsLewy (CFL) condition be
satisfied [3, 9, 10]. This condition is related to the time stepping in the integration of the shallow water equations with
respect to time after the equations are discretized with respect
to space. This means that we can use either a fixed time step as
long as the CFL condition is satisfied at every time step or a
varying time step based on a fixed CFL number. Here a CFL
number represents a positive number such that the CFL condition is satisfied.
This paper investigates the influence of CFL number to the
accuracy of numerical solutions produced by the finite volume
method. The accuracy of the finite volume method, of course,
depends on the accuracy of the integration technique implemented to the space and time. To focus on our investigation,
we use a single integration technique for the space variable,
that is, we use a second order method for the space integration.
Then we compare the performance of a second order method
for the time integration by presenting the errors between implementing a fixed time step and a fixed CFL number.
This paper is organized as follows. In Section II we recall
the shallow water equations in one dimension. The finite volume method is presented in Section III. Numerical results are
given in Section IV. Finally some concluding remarks are
drawn in Section V.
II. GOVERNING EQUATIONS
The shallow water equations are
ht + (hu ) x = 0 ,
(hu ) t +
1
2
gh + hu
2
(1)
= ghBx .
(2)
where t denotes the time variable, x denotes the space variable, h( x, t ) is water height or depth, u ( x, t ) is velocity, B ( x)
represents the bottom elevation or topography, and g is the
acceleration due to gravity. The absolute water level (stage) is
defined as
(3)
w( x, t ) := h( x, t ) + B ( x) .
A number of authors have proposed numerical techniques to
solve these shallow water equations (1) and (2). Some of them
are [1, 2, 5-8, 11, 12, 15, 16].
III. NUMERICAL METHOD
As we mentioned, we use a finite volume numerical method
to solve the shallow water equations. In a semi-discrete form,
the finite volume method is
d
1
Q j (t ) =
F 1 (t ) F j 1 (t ) + S nj
(4)
2
dt
x j + 2
where Q is an approximation of the conserved quantity, F is
an approximation of the analytical flux and S is a discretization of the analytical source term. See the References [1, 6, 15]
th
for more details of this type of scheme. This scheme is called

semi-discrete because we have discretize the shallow water
equations with respect to space, but the time variable is still
continuous [3, 9, 10].
To get a second order method in space, we use a linear reconstruction for quantities stage, height, bed, velocity and
momentum. Then in order to suppress artificial oscillation due
to the space reconstruction, we implement the minmod limiter.
This limiter gives a limitation to the values of the gradients in
the linear reconstruction of the aforementioned quantities. After that, numerical fluxes are computed based on these reconstructions. We use the Lax-Friedrichs numerical flux function.
We refer to [9, 10] for the formulation of this flux function.
The next step is to integrate the semi-discrete form (4) with
respect to time. We actually can use any standard method of
Ordinary Differential Equations (ODEs) solver. However, because we have used a second order method in space, it is better
to use either a first or second order method in time. This is
because we will never get a finite volume method of order
higher than two, even if we use higher order method in time. In
this paper we implement the second order Runge-Kutta method to integrate the semi-discrete form (4) with respect to
time.
IV. NUMERICAL RESULTS
This section provides numerical results regarding two different strategies for the numerical evolution. The first strategy
is imposing a fixed time step in the second order Runge-Kutta
integration. The second strategy is imposing a fixed CFL number where in our simulations we use CFL number to be 1.0 in
one case and 0.01 in another case. Details about CFL conditions and CFL numbers can be found in the References [9, 10,
17].
Numerical settings are as follow. We use SI units for measured quantities, so we omit the writing of units. Errors are
quantified using absolute L1 formula as used in [13, 14]. In
this paper we consider one test case. Standard test cases are
available in the References [4, 18].
As a test case we consider the dam break problem. We assume that the topography is given by a flat horizontal bottom
B( x) = 0 , where 1 x 1 . Therefore we have that stage
equals to water height. The water height is initially given by
10 , x < 0 ,
h( x,0) =
(5)
4 , x > 0.
The analytical solution of this problem has been found by
Stoker [18] and an extension to the debris avalanche problem
has been solved by Mungkasi and Roberts [13, 14].
TABLE I
COMPARISON OF STAGE ERRORS BETWEEN IMPOSING A FIXED TIME STEP AND
IMPOSING FIXED CFL NUMBERS. THE FIXED TIME STEP IS 0.05 TIMES THE CELLWIDTH, WHEREAS FIXED CFL NUMBER ARE 1.0 AND 0.01.
Cell
number
100
Fixed time step

Error
0.058
9
RC
CFL=1.0
Error
0.058
2
RC
CFL=0.01
Error
0.056
9
RC
200
0.030
8
400
0.014
4
800
0.007
2
1600
0.003
6
Average rate
of convergence
0.934
3
1.094
0
1.001
4
0.992
5
1.005
5
0.030
4
0.014
3
0.007
1
0.003
6
0.935
9
1.091
7
1.001
4
0.990
0
1.004
7
0.029
6
0.014
0
0.007
0
0.003
5
0.940
5
1.082
3
0.999
4
0.986
0
1.002
0
TABLE II
COMPARISON OF DISCHARGE ERRORS BETWEEN IMPOSING A FIXED TIME STEP
AND IMPOSING FIXED CFL NUMBERS. THE FIXED TIME STEP IS 0.05 TIMES THE
CELL-WIDTH, WHEREAS FIXED CFL NUMBER ARE 1.0 AND 0.01.
Cell
number
Fixed time step

Error
0.471
4
200
0.244
8
400
0.117
7
800
0.058
9
1600
0.029
9
Average rate
of convergence
RC
100
0.945
5
1.056
2
0.998
4
0.979
1
0.994
8
CFL=1.0
Error
0.465
2
0.241
6
0.116
4
0.058
3
0.029
6
RC
0.945
3
1.053
3
0.996
4
0.977
8
0.993
2
CFL=0.01
Error
0.452
0
0.234
1
0.113
8
0.057
1
0.029
1
RC
0.948
8
1.041
4
0.993
8
0.970
7
0.988
7
TABLE III
COMPARISON OF VELOCITY ERRORS BETWEEN IMPOSING A FIXED TIME STEP
AND IMPOSING FIXED CFL NUMBERS. THE FIXED TIME STEP IS 0.05 TIMES THE
CELL-WIDTH, WHEREAS FIXED CFL NUMBER ARE 1.0 AND 0.01.
Cell
number
Fixed time step

Error
0.073
2
200
0.038
8
400
0.017
8
800
0.008
8
1600
0.004
4
Average rate
of convergence
RC
100
0.915
7
1.127
6
1.009
0
1.003
2
1.013
9
CFL=1.0
Error
0.072
4
0.038
4
0.017
6
0.008
7
0.004
4
RC
0.915
6
1.126
8
1.008
8
1.003
5
1.013
7
CFL=0.01
Error
0.070
5
0.037
3
0.017
2
0.008
5
0.004
3
RC
0.916
6
1.120
9
1.006
7
0.996
6
1.010
2
Our simulation results are summarized in Tables 1-3. Table 1 shows error comparison for stage (water surface) between three scenarios of simulations. Errors for discharge
(momentum) and velocity are summarized in Table 1 and Table 2 respectively. From these three tables, the highest convergence rate is achieved by setting a fixed time step, rather than
imposing a fixed CFL number. We should note that the average rate of convergence for imposing CFL number 1.0 produces a very close average rate of convergence to the fixed time
step setting. Furthermore imposing CFL number to be 1.0
gives the most efficient computation as it takes the shortest
running time. Setting CFL to be too small such as 0.01 gives a
low rate of convergence. Of course setting CFL number too
th
small makes the computation be expensive, so the running

computation is long. Here the fixed time step scenario used is
t = 0.05 x , with the time step t and the cell width x .
true only when the solution of the shallow water equations is

smooth [9, 10]. As our solution in this paper is nonsmooth due
to discontinuities, it is not surprising that we obtain that the
rate of convergence is less than two, that is, about one.
Even though we have a fixed formal order, the numerical
order or rate of convergence is obviously dependent on the
numerical strategy that we use. This has been shown in this
paper. Taking a fixed time step in the finite volume method
gives different convergence rate from taking a varying time
step with imposing a CFL number. In addition, imposing a
specific CFL number gives different convergence rate from
imposing another CFL number.
V. CONCLUSION
Fig. 1. The initial condition of the dam break problem (at time t = 0 using 100 cells. Solid line represents the exact solution. Dotted line represents
the numerical solution.
We have investigated the CFL effects on the convergence

rate of finite volume methods used to solve the shallow water
equations. Our simulations indicate that the use of CFL number 1.0 for solving the dam break problem gives the best combination between efficiency and accuracy. Note that setting the
CFL number greater than 1.0 may make the numerical method
unstable when we solve the shallow water equations in general.
REFERENCES
[1]
[2]
[3]
[4]
Fig. 2. Solution to the dam break problem at time t = 0.05 using 100
cells with the fixed time step. Solid line represents the exact solution. Dotted
line represents the numerical solution.
Figure 1 shows the initial condition for the test case. The
first subfigure is the stage or water level (free surface). The
second and third subfigures are the momentum and velocity
respectively. It is clear that initially we have only discontinuity
in the stage, while the momentum and velocity are continuous.
Figure 2 shows the stage, discharge and velocity of water after 0.05 seconds of dam break using the fixed time step. The
numerical solutions approximate the analytical solution well
based on this Figure 2. Here we see discontinuities in the
stage, momentum and velocity.
The convergence rate in our simulation is about 1.0 even
though we have implemented a second order finite volume
method, that is, second order in space and second order in
time. This is because the discontinuities of the solution occur.
The discontinuity appears in the measured quantities as well as
the derivative of the quantities. Again, see Figure 2 for these
discontinuities.
It is worth to note that the formal convergence rate of our
numerical method is two, because we use a second order method in space as well as in time. However this formal order is
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th
Bayesian Migration Schedule Model:

An Aplication to Migration in East Java
1)
Preatin1), Iriawan, N. 1), Zain, I.1), and Hartanto, W. 2)

Statistics Departement , Sepuluh Nopember Institute of Technology, Indonesia
2)
National Family Planning Coordination Board (BKKBN), Indonesia
Abstract From several migration models for individual data,

the schedule model has advantages over logistic models and event
histories analysis. In terms of data, the schedules model is more
simple because does not involve non-migrants such as the logistic
model. While the event history analysis requires special survey
because it need detailed information. Schedule model show regular features as a peak in young, declining migration in old age,
and may be elevated migration in retirement age. These features
can characterize migration flows that associated with labour migration, return migration or familial affiliations. This paper using
Bayesian approach to apply schedule model to see the pattern of
in-migration to East Java by age so that it can be used for development planning.
Migration
Keywords Migration, Schedule Model, Bayesian, East Java
I. INTRODUCTION
ANY discipline of sciences interested in developing
migration model. It is because migrations are a complex
phenomenon that involves many dimensions. It requires
a comprehensive understanding which is not limited to particular disciplines. Multidisciplinary modeling approach couple
with the right chosen variables would be more beneficial than
just using any particular theory approach [2]. There are two
aspects that follow the process, those are individuals and regions. The individual data or the micro data requires specific
modeling to the individual characteristic related to the decision
to migrate. While the region data or macro data requires different modeling to characterize the region, as the origin and
the destination of migration. Figure 1 shows separation some
models that are used to elaborate migration viewed from the
availability of data.
This paper using individual data from populations census
in 2010 by BPS. For individual data there are 3 options,
namely logistic model, event history analysis, and schedule
models. Each model has its advantages and weakness when
applied to migration data in Indonesia. Based on the data used
in logistic model, it would be very hard in preparation for
analysis. It is due to the difficulty to have the entire population
data. Using logistic models on individual data migration, on
the other hand, will involve migrants and non-migrants.
Indonesia which still relies on census data for the analysis of
migrations, therefore, need the use of computational intensive
approaches due to the involving large data.
Figure 1. Separation of Selected Migration Models

Event history analysis requires a special survey to see the
migration history of each individual during every individual
lifetime, and Indonesia not already have migration surveys. So,
the using of schedule model is the most probable to applied.
II. BAYESIAN MIGRATION SCHEDULE MODEL
Schedule model show regular features as a peak in young,
declining migration in old age, and may be elevated migration
in retirement age. These features can characterize migration
flows that associated with labour migration, return migration
or familial affiliations [6].This paper using Bayesian approach
to apply schedule model with purely parametric model. Let Yx
is migration flows at individual years of ages (x=1,2,3,), Nx
is mid year populations, and assume Yx ~ Poi(Nxmx) where mx
are migrations rates.
th
mx = a1 exp(1 x)
+ a2 exp{ 2 ( x 2 ) exp[2 ( x 2 )]}
+ a3 exp{ 3 ( x 3 ) exp[3 ( x 3 )]}
+ a4 exp(4 x) + c
The component with parameters 1 and 1 represent child migration, the component with parameters (2, 2, 2, 2)
represent young migration which in mainly labour migration,
the retirement age represented by the shifted exponential term
with parameters (3, 3, 3, 3), and post retirement age
represented by parameters 4 and 4.
Figure 3. Type of Migration Schedules Model
Where is an additional positive parameter. The mean mx and
the variance mx2/ of mx represent that mx declines as increases.
III. MIGRATION FLOWS IN EAST JAVA
1
2
2
3
3
c
=
=
=
=
=
=
rate of descent of pre-labor force component

rate of ascent of labor force component
rate of descent of labor force component
rate of ascent of post-labor force component
rate of descent of post-labor force component
Constant
xj
xh
xr
X
A
B
=
=
=
=
=
=
low peak
high peak
retirement peak
labor force shift
parental shift
Jump
East Java province including the migrants sender to other

provinces in Indonesia and abroad. In the province itself there
is mobility between district / city or even in-migration from
outside. In-migration data in 2010 was 1.5% of the total
population.
Figure 2 Migration Schedule Model

Figure 2 shows migration patterns according to age. Its
graduation was changed by a scheduled model, which is defined as a sum of four components:
1. Pre-labor force, a single negative exponential curve with its
rate of decent 1.
2. Labor-force, a left skewed unimodal curve with mean age
2, rate of ascent 2, and rate of decent 2.
3. Post-labor force, an almost bell shaped curve, with mean age
3, rate of ascent 3, and rate of decent 3.
4. Post-retirement peak, exponential curve with rate of ascent
4.
5. Constant c.
Combination from above components form the 4 types of
schedule models adjusted of data conditions, as shown in
figure 3.
Substantively there is some conditions likely to be over dispersion that excess heterogeneity, this lead to allow hierarchical model. The conjugate option for mx as gamma mixing.
Yx ~ Poi(Nxmx)
mx ~ Ga(, /mx)
mx = a0 + a1 exp(1 x)
Figure 4. In-migration Rates by Age and Gender

Figure 4 shows that the in-migration balanced between male
and female, where higher female in-migration in young ages
and vice versa in old age. From the image identification shows
that schedule models is appropriate to type 2 and type 4, where
there
is
a
post-retirement
peak.
Table 1 and Table 2 contains the estimated parameters and
summary measure of fit. Schedule models for male and female
have the same conclusion, that fit with type 2 if seen from DIC
values, but fit with type 4 if seen from MSE values.
+ a2 exp{ 2 ( x 2 ) exp[2 ( x 2 )]}

+ a3 exp{ 3 ( x 3 ) exp[3 ( x 3 )]}
+ a4 exp(4 x)
TABLE I
MALE SCHEDULE MODEL PROPERTIES
Parameters
1 a0
Type 1
Type 2
Type 3
Type 4
0,003712
0,000128
0,003916
0,000004

2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
a1
a2
a3
a4
alpha
alpha1
alpha2
alpha3
lambda2
lambda3
lambda4
mu2
mu3
deviance
DIC
mse
0,027810
0,104500
0,008710
0,069230
6,15
0,230700
0,137100
0,000000
23,97
0,026480
0,093340
0,326000
0,646100
15,81
0,207100
13,33
903,79
994,12
0,000021
875,10
957,22
0,000009
th

0,077260
0,087320
0,022260
4,86
0,459800
0,181800
0,351200
0,534000
0,448100
13,84
29,29
894,40
985,20
0,000015
TABLE 2
FEMALE SCHEDULE MODEL PROPERTIES
Parameters
Type 1
Type 2
Type 3
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
a0
a1
a2
a3
a4
alpha
alpha1
alpha2
alpha3
lambda2
lambda3
lambda4
mu2
mu3
deviance
DIC
mse
0,003026
0,030100
0,097130
0,000267
0,008568
0,068920
4,50
0,223700
0,151800
0,000000
20,34
0,031550
0,116800
0,511800
0,900600
14,20
0,196700
12,73
882,30
874,02
0,000022
858,30
942,76
0,000010
0,003106
0,069240
0,103500
0,014190
4,01
0,366500
0,192300
0,366800
0,635400
0,517600
13,42
29,95
878,10
969,89
0,000023
0,004615
0,082050
0,007638
0,000000
154,10
0,056460
0,121400
0,058040
0,509000
0,024270
0,186900
13,97
59,45
897,60
964,48
0,000006
[4]
[5]
[6]
[7]
[8]
[9]
[10]
Type 4
0,000022
0,008067
0,133600
0,002120
0,000001
141,60
0,024090
0,329600
0,280700
0,419700
0,089270
0,185300
15,43
55,24
900,70
969,74
0,000004
IV. CONCLUSION
Modeling migration must be adapted to the purpose of
research and the availability of data. For in-migration in East
Java having limited data requires the selection of an
appropriate model. For individual data, schedule models is
more probable because does not involve non-migrants such as
the logistic model and special surveis as event history analysis.
Bayesian approach was recommended, because it would be
more flexible as data driven approaches, but it requires
computational intensive capabilities. Pattern of in-migrations
to East Java by ages still characterize as young migration at
labor force migrants. Post retirement peak shows return
migrations is significant but further research is needed.
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th
A Totally Irregular Total Labeling Disjoint

Union of Wheel Graph
Diana Kurnia Sari Sudirman1*), Rismawati Ramdani 1), and Siti Julaeha2)
Department of Mathematics, SunanGunungDjati State Islamic University Bandung, Indonesia
2)
Department of Mathematics, SunanGunungDjati State Islamic University Bandung, Indonesia
1)
*)
Diana Kurnia Sari Sudirman:dianakurniasarisudirman@yahoo.com
AbstractA total labeling

is called
totally irregular total -labeling of
if every twodistinct
vertices and in
satisfies
, and every two
distinct
edges
and
in
satisfies
,
where
and
. The minimum for which
a graph has totally irregular total k-labeling is called the total
irregularity strength of , denoted by
. In this paper
determined
for disjoint union from
copies of wheel
denoted by
.
Keywordsthe total edge irregularity strength, the total vertex
irregularity strength, total irregularity strength, totally irregular
total k-labeling, wheel.
in a paper entitled On the total Irregularity strength on cycles

and paths [3].
Suppose is
a
graph.
Total
ling
is calleda total k - labeling irregular total of G if any two points and are different in
and any two sides
and
satisfies
different in
satisfies
, where
and
. The minimum value of
k so that G has a total k - labeling irregular total called total
value of the total (total Irregularity strength) of G and is denoted by
[3]. In this paper, we determined the total irregular total labeling disjoint union of wheel graph.
II. THEORY
I. INTRODUCTION
athematics is a branch of science known as the Queen of
Science. Evident from many other disciplines that employ methods contained in mathematics. One area in
mathematics that great attention is graf.Teori graph theory is
part of mathematics that is widely used as a tool to describe or
represent a problem so that it is easier to understand, be understood and resolved. Many issues will be clearer to explain
if it can be formed into a graph [5].
Until now the use of graph theory is perceived role in various sectors of other sciences. One of the uses of science graph
in other disciplines, namely in the fields of chemistry, including hydrocarbon compounds that can be formed into a tree
graph.
Over time, the growing study of graph theory. One of the
topics in graph theory is graph labeling. Labeling graphs was
first introduced by Rosa in 1967 [2]. Labeling on the graph is
the mapping that carries graph elements to the values [1].
Based on the domain, labeling is divided into three, namely the
point of labeling, the labeling, and the labeling of the total.
Labeling is the point of labeling with domain the set point, the
labeling is labeling with domain the set of sides, and the total
labeling is labeling combined with domain the set of points
with the set side. One topic of total labeling of a graph is irregular total labeling introduced by Marzuki, Salman, and Miller
Irregular total labeling was introduced by Baca, et al in

2007 in a paper entitled On irregular total labelings. In this
paper, Baca, et al introduce two types of irregular total labeling, ie irregular total labeling and labeling the total irregular
point. Definitions and results-labeling studies of labeling is
given below.
A. An Edge IrregularTotal Labeling
is called an edge irA total labeling
regular total -labelingin if every two different edges
and
in
satisfies
,
where
.
The smallestvalue ofksuch that agraph has anedge irregular total -labeling is called the total edgeIrregularitystrengthof
agraph isdenoted by
[1].
In the paper [1] Ba a, et al provides a lower limit of
the
andthe total edgeIrregularitystrength for some
graph, including path and circle graph. The results of these
studies are given in the following theorems.
Theorem 2.1 [1]
Let
is a graph with a nonempty and , then
Theorem 2.2 [1]

Let is a path, with
, then
th
Let n be a positive integer number and

n vertex, then
Theorem 2.3 [1]

Let is a circle graph, with
, then
B. A Vertex Irregular Total Labeling

is called a vertex irA total labeling
regular total -labelingin if every two different vertex and
in
satisfies
,
where
.
The smallestvalue ofksuch that agraph has a vertex irregular total -labeling is called the total vertex Irregularity
strength of a graph is denoted by
[1].
In the paper [1] Ba a, et al provides a lower limit of
the
and the total vertex Irregularity strength for some
graph, ie star graph. The results of these studies are given in
the following theorems.
Theorem 2.4 [1]
Let
is a graph with
and
, minimum
degree , and maksimum degree , then
Theorem 2.5 [1]

Let
is a star graph with
then
C. Totally Irregular Total Labeling

Marzuki, et al. [3]combine the idea of an edge Irregular total
labeling and a vertex irregular total labeling into a new labeling called a totally irregular total labeling.
A total labeling
is called a totally irregular total -labelingin if every two different vertex and
in
satisfies
and every two different
edges
and
in
satisfies
where
and
.
The smallest value of k such that a graph has a totally irregular total -labeling is called the total Irregularity strength of
a graph is denoted by
[3].
In the paper [3], Marzuki, et al provides a lower limit of
. In addition, the same paper has determined the total
irregularity strength of path and circle graphs are summarized
in the following theorems.
Theorem 2.6 [3]
Let is a graph. Then
Theorem 2.7 [3]
Let
be a positive integer number and
is a circle
graph with n edge, then
Theorem 2.8 [5]
is a path graph with
Research on the total irregular total labeling was also performed by Ramdani and Salmanin a paperen titled on the total
Irregularity strength of some Cartesian product graphs[6].
Inthe paper, given the total irregulariy strength of some Cartesian
product
graphs,
ie
, where
is
a path graph with n order,
is a circle graph with n order and
is a star graph with
order. The results are summarized in the following theorems.
Theorem 2.9 [6]
For
Theorem 2.10 [6]
For
Theorem 2.11 [6]
For
Theorem 2.12 [6]
For
D. Disjoint Union
Definition 2.13 [2]
Two graphs
and
said disjoint if
.
Definition 2.14 [2]
Let
and
are two disjoint graph.
Disjoint union from and , denoted
are graphwith
the set of vertex
and the set of edges
.
Example 2.15
Definition 2.16 [2]
Figure 2.1 Disjoint Union form
and
III. RESULT
A. Wheel Graph
Definition 3.1 [2]
th
Wheel graph with

vertices denotedby
formed from the cycle
by adding a vertex
ing each point in
to the vertex .
Example 3.2
is a graph
and connect-
Moreover, based on theorem 2.4
Thus
Moreover, based on theorem 2.6
Thus,
(3.1)
2. Will prof that

Total labeling given the graph
is as follows:
untuk
b. Labeling on
is as follows:
Figure3.1Wheel Graph
B. A Totally Irregular Total Labeling Disjoint Union of
Wheel Graph
Definition 3.2 [2]
graphis a disjoint union from copies of wheel graph
Figure 3.2
graph has a set of vertex
, where:
as follows:
a. Labeling on
Graph
and a set of edges
and
Theorem 3.3
Let
graph is a disjoint union from
graph
, then
copies of wheel
Proof.
There are two steps to proof theorem 3.3, ieby determining the
lower limit and the upper limit from
, as can be seen
in the following description
1. Will proof that

graph has
vertices and
edges. The
smallest degree from
graph is
andthe greatest degree from
is
.
Based on the theorem 2.1,
To show that is totally irregular total

labeling, then it will be shown that by
labeling , the weights of all vertices
on
and weights on all edges
are
different.The weight of a vertices and an
edges obtained by labeling is as follows:
(i)
The weight of the edge
for
(ii)
for
is
Thus
is
(iii)
th
The weight of edge
for
is
(ix)
for
is
.
(iv)
The weight of edge

is
for
(x)
for
is
(v)
(vi)
The weight of edge
for
is
(xi)
for
(xii)
for
is
for
is
is
(vii)
The weight of theedge
, for
(xiii)
The weight of the vertices
for
(xiv)
The weight of the vertices , for
is
is
(viii)

is
for
is
th
(xv)
for
(xvi)
, for
is
(xvii)

is
, for
(xviii)
, for
is
Based on the formula weigh to the vertices and edges above,

it can be seen that the weight of all edges and the weight of all
vertices are different.
Thus, the total labeling satisfy a totally irregular total labeling with the biggest labels
. Therefore,
(3.2)
Based on theequation(3.1) and(3.2), it can be concludedthat
Example 3.4
As an illustration, the following will be given a totally irregular total labeling disjoint union of
based formula totally
irregular total labeling on theorem 3.3
is
Figure 3.3
Graph
Labeling the edges and vertices of
graph as follows
(xix)
, for
(xx)
, for
is
Figure 3.4 A totally irregular labeling of
graph
Sothere is noequal weight toeach vertices and no equal
weight on each edge.
th

[6]
[7]
[8]
Figure 3.5Weight on all edges
Figure 3.6 Weight on all vertices
R. Ramdani dan A. N. M. Salman. On the total irregularity

strength of some cartesian product graphs,AKCE Int. J. Graphs
Comb., 10, No 2 (2013), pp. 199-209.
S. Slamet.PengantarTeori Graf, Universitas Indonesia, Jakarta
(1998).
R. J. Wilson. dan J. J. Watkins. Graph:An Introductory Approach,
Simultaneously, Canada (1990).
graph
graph
IV. CONCLUSION
In this paper determined
for disjoint union from
copies of wheel denoted by
obtained from theorem 3.3
that
.The discussion of thetotalirregulartotallabelingis
stillopenfor other researcherstoconductsimilarstudieswithdifferenttypes ofgraphs, includinggraph
with
.
REFERENCES
[1]
[2]
[3]
[4]
[5]
M. Ba a, S. Jendrol. M. Miller. Dan J. Ryan. On irregular total

labellings.Discrete Mathematics 307(2007) 1378-1388.
J.A.Bondydan U.S.R. Murty, Graf Theory with Application, The
Macmillan Press Ltd, New York (1976).
J. A. Galian. A Dynamic Survey of Graph Labeling. The
Electronic journal of combinatorics18 (2011).
V. E. Levit danE.Mandrescu.The Independence Polynomial of a
Graph--A Survey.Dalam proses untuk the 1st International Conference on Algebraic Informatics(2005).
C. C. Marzuki, A. N. M. Salman, dan M. Miller. On the total
irregularity strength on cycles and paths. Diterima untuk
dipublikasikan di Far East Journal of Mathematical Sciences.
th
The Implementation of The Meshless Local PetrovGalerkin on Calculating Volume of River

Sedimentation in The Confluence of Two Rivers
Inu Laksito Wibowo1, Suhariningsih2 and Basuki Widodo3
PhD Student in FST UNAIR/Lecturer of Math. Dept. of ITS,
2
Professor in Physics FST UNAIR
3
Professor in Applied Mathematics Department Math of ITS
1
Abstract---The occurrence of sedimentation in a confluence two

rivers can be formulated into mathematical model and simulated
numerically, so the morphological changes due to the sedimentation of the river can be unpredictable. Mathematical modeling
and numerical simulation results of solutions can be used as a
material consideration in the adoption of a policy, so the impact
will be caused by the sedimentation of the river can be prevented
as early as possible or at least be reduced. In this paper, we
consider about a model of sedimentation in the river which is
formulated by using control volume and be solved using the method of Meshless Local Petrov- Galerkin (MLPG). Themain purpose of meshless method is to get rid of the grid or to reduce the
difficulty in making a grid with points. We obtain that the
sedimentation distribution in the confluences of two rivers is
influenced by the shape of river morphology. The higher of the
river velocity the higher the erotion in the river.
Keywords: Sedimentation, Confluence two rivers, MLPG
method is predicted to replace the FEM method in the future

(Atlury and Lin, 2001).
In this paper, we consider, the method Meshless Local PetrovGalerkin (MLPG) which is used to solve the model which has
been obtained from the Finite Volume Method approach on
the sedimentation at the confluence of two rivers. Furthermore,
by using the approach of Moving Least Square (MLS) as a
function of the shape and Heavyside function as a function of
test completion the solution sought. Settlement obtained is
then made using the computer program MATLAB programming language to be solved numerically using a computer, and
by varying the input variables and parameters subsequently
simulated to obtain the characteristics of the variables and parameters of the system being modeled. The results of the simulation is then visualized in the form of pictures of the calculation results of the numerical simulations and compared with
the results of visualization using the software.
I. INTRODUCTION
ne of the benefits of river is very important is to store

water during the rainy season. Siltation of the river due to
sediment deposition causes water or undrained cannot be
accommodated to the maximum, it cause flooding.
The process of sedimentation in the river can be constructed
into a mathematical model and numerically simulated, so that
the process of morphological changes due to sedimentation of
the river can be predicted. Mathematical modeling and numerical simulation results that the solution can be used as one consideration in making a policy, so the impact will be caused by
the presence of river sedimentation can be prevented as early
as possible or at least be reduced.
River sedimentation model is built using the approach volume method and it is solved by using the meshless local Petrov-Galerkin (MLPG). This method is relatively new, and
still being developed in the fluid dynamics problems. Meshless
method which is developed in this study is used to resolve
those problems. The main purpose meshless method is to eliminate or to reduce the difficulty in making the grid by using the
points (nodes) as his successor. This method is very flexible,
accurate and not at all in the use of grid application , either for
interpolation purposes or for purposes of calculating the
integral . Complexity thing, the MLPG is good when compared with the method that uses mesh (Widodo, 2009).This
II. SEDIMENTATION FORMATION PROCESS

The main function of the river is flowing rain water so that it is
possible silting. This is due to deposition of sediment in certain places at the bottom of the river. Sedimentation occurs
because of the presence of solid particles (sediment) that is
carried on by the flow of water. Sediment transport mechanism
is categorized into two, namely bed load and suspended load.
Bed load sediment movement is sediment moves on the river
bottom by rolling, sliding and jumping around. While the
suspended load, consisting of fine granules suspended in water
(Widodo 2012).
2.1 Sedimentation Calculation Basic Equation
Bed load is grains / particles / sediment material that generally occurs in the watershed. There are several kinds of mathematical formulas that can be used to calculate the amount
of sediment in this type of sediment transport. One of the formulas / mathematical formula which is popular is formula
Meyer - Peter & Mller (Yang, 1996).
In the study Yang (1996), changes in river morphology is
assumed to occur only at the bottom of the river and caused by
the presence of scour and deposition processes. Changes in the
river bed can be calculated using the equation of conservation
th
of mass equation for sediment transport. Yang (1996), Widodo

(2012), namely :
y
1 qb
+
=0
t (1 p ) x
where: y = height of the river bed,
p = porosity,
qb= bed load
2.2 Confluence of Watershed
Confluence of two rivers is an interesting phenomenon and
very complex. Soburo Komura (Widodo, 2009) conducted a
study of the phenomena occurring at the confluence of two
streams using the balance equation, which is obtained by using
the equations of motion, continuity equation for sediment
transport, and the continuity equation for the shear velocity of
water flow. The equation obtained is less reflect the real situation on the ground. So that this equation can be used or can be
applied to things that are ideal. It is therefore necessary to find
or develop a new mathematical model to another or explain the
phenomenon approaching real state (Widodo,2009).
2.3 Two forms of Morphology Meeting Watershed
Morphology shape of confluence of two rivers is a natural
phenomenon that is very interesting, because we will see the
confluence of two rivers form the model that various kinds.
Some of these models have been widely studied as a model
and a model developed Shazy Shabayek, ie numeca.
In models shaped river Main stream and lateral stream has
been shown to result in sedimentation in the riverbed on the
research results (Widodo, 2009). In the study described also
that sedimentation is not only dependent on the flow of the
river upstream but also the morphology of the river, the river
mouth will be eroded due to the presence of scours caused as a
result of back water gate block of sedimentation (Widodo
2009).
Model confluence of two rivers that form a quarter-circle is
arc numeca as depicted at the Figure (2.1).
Figure 2.1 Watershed Model Numeca Bow Quarter Circle.
In the quarter-circle model of domain Numeca river is divided into 2 parts by volume control. The main river (main
stream) and two tributaries (lateral stream) on the flow curve is
expressed as a volume control 1 while at the confluence
straight expressed as volume control 2. For the forces acting
on the second volume of this control include hydrostatic force,
the frictional force on the bottom of the river, the frictional

forces that occur at the boundary between the two volume control, gravitydue to the influence of Earth's gravity and friction
forces
on
the
surface
of
the
river
water.
Some of the characteristics of the main river will change with
the influx of tributary streams. Such changes include the
change in mass of the depth, direction , and flow rate , as well
as other changes .
Markup (2001) have mathematically derive the equation of
conservation of mass and momentum for flow in tributaries
entering from the side of the main stream. Mass.and momentum equations are:
z Q
+
=q
t x
Q Q 2
z gQ | Q |
+
= vq cos
+ gA +
t x A
x AC 2 R
With:
A = cross sectional area of the flow
B = width of the surface flow
Q = flow rate
z = height of surface flow
v = velocity of lateral flow
= angle between the main flow and lateral flow
q = lateral flow width unity
C = Chezy coefficient
g = gravity
2.4 Method of Meshless Local Petrov - Galerkin (MLPG)
The main purpose of the meshless method is to avoid the
use mesh / grid. This method is very useful in problems with
the domain boundary that is not continuous or moving, or other difficulties may be found in the use of the finite element
method (Atlury and Lin, 2001).
Meshless method is known to be very effective implemented
in the field of computational science and engineering, but in
terms of speed and reliability still needs to be developed. Integration numerically to determine the convergence of this method numerical solution generated. Nodal shape functions of
the Moving Least Square (MLS) is used in this method is very
complex, so as to obtain accurate numerical Integration results
in weak form is very difficult to do , especially for a method
that is included in this type meshlessGalerkin (Ottevanger,
2005). MLPG predicted could replace the finite element method (FEM) in the future (Widodo, 2009).
2.5 Numerical Methods Basic Search Volume
Search volume can be approximated by the approach area
of the base multiplied by the height, so that the search is necessary to find the approach area of the base and height function numerically.
III.
FRAMEWORK CONCEPT
Numerical methods, in addition to methods meshless local

Petrov - Galerkin (MLPG), there have been widely applied in
th
solving fluid dynamics problems. One fundamental thing in

common and that became the basis of the above methods is the
use grid or cells in its application. The use of the grid determines the level of accuracy of these methods. The smaller the
grid is created, or in other words the more the number of the
grid , the more accurate the output (output) produced , but the
more expensive the cost of computation to be done (Atlury and
Lin, 2001). Even the grid itself to the manufacture of complex
domains is very hard to do. Then Meshless Local PetrovGalerkin (MLPG) was introduced and applied to the problem known
as the ideal fluid Navier Stokes equations (Atlury and Lin
2001) which followed (Atlury and Shen 2002).
Meshless method (without mesh / grid) that developed in
subsequent studies used to address the problem of sedimentation. The main purpose meshless method (without mesh / grid)
grid is to eliminate or to reduce the difficulty in making the
grid by using the points (nodes) as successor (Widodo 2011).
This method is very flexible, accurate and not at all in the use
of grid application, either for interpolation purposes or for
purposes of calculating the integral. This method is known as
the MLPG (Meshless Local PetrovGalerkin) method is applied
to obtain the distribution pattern of sedimentation with a case
study times SurabayaWidodo 2011), and then proceed to the
case of the application of MLPG shaped river Numeca. This
study followed a combination of curved and straight rivers
with MLPG and refined with the application of the Moving
Least Square (MLS) (Wibowo 2012) which is then used as an
initial study of this dissertation.
Sedimentation volume produced will be very instrumental in
determining the gate that creates large blocks scour the river
mouth. This phenomenon makes it clear that the large volume
of sedimentation factor is an important factor to be addressed
in managing a river in relation to keeping the river mouth from
fell out.
Sedimentation volume will be constructed from the results
of the determination of the location of sedimentation to be
made sedimentation area L (A) multiply by a function of position in the sediment height F (P) and summedto determine the
volume of sedimentation can be done with a numerical approach. Shape 3 dimensions are sought and can use some help
in getting the visualization software.
IV. BUILDING A MODEL SEDIMENTATION
As has been described in the literature review , that the sedimentation process can be divided into two parts , namely :
the hydrodynamic flow of the river and river morphology .
lume control to be modeled is described as follows:
Figure 4.1 (a) Model Numeca Bow River Quarter Circle, and (b) Volume
Control
4.2 Morphology River

Changes in river morphology is assumed to occur only in
the river bed due to scour and deposition processes. Changes
in the river bed can be calculated using the equation of conservation of mass transport of sediment. As for the formula used
to calculate the amount of sediment Meyer - Peter & Muller.
In the Application, the second equation is expanded into a
two-dimensional equation for. So the formula used to calculate
the change of the river bed due to sediment transport and sediment transport to calculate the amount is as follows :
Sediment mass conservation :
Lateral Stream (Meandering Flow):
Main Stream :
Sediment transport :
With,
Number of bed load sediment
4.1 Hydrodynamic Watershed

Profile river models numeca quarter-circle arc and the vo-
: 8.0
s=density of the sediment and = density of water,
g = acceleration due to gravity ,
d50 = median diameter of sediment,
= 1.0,
c = 0.047,
th
In Figure 4.10 that seemingly stream with initial condition

velocity = 0.9 at all positions (x) increased approximately
0.068586 .
,
U : velocity of the river flow,
h :depth
4.3 Simulation Calculation of Volume Sedimentation
Sedimentation volume will be constructed from the results
of the determination of the location of sedimentation to be
made sedimentation area L(A) multiply by a function of position in the sediment height F(P). This volume is calculated
from the two creeks to flow Cornering and meet at the creek
with a straight flow.
4.3.1 Simulation Flow Creeks Cornering
This volume is calculated from the two creeks to flow Cornering obtained by simulating it as follows :
Simulation I
Initial depth h, = 0.3
Initial velocity v, = 0.9
Initial height of the sediment, =0.3
Time t, = 20
Delta t, = 5
Figure 4.9 Plot Depth River in Simulation I
simulation I, it appears that the flow with initial conditions at

depth = 0.3 speed = 0.9 and after the time of the decline in
river depth of approximately 0.015832.
Figure 4.10 Plot of Speed on the Flow Simulation I
Figure 4.11 Plot Sediment Elevation in Simulation I
In Figure 4.11 shows that the flow with initial conditions

sediment height = 0.3 in all positions (x) and after the time of
the change of height of the sediment that is down about
0.166570.
From the results shown above plot (Figure 4.6 - 4:11) shows
that the depth h, velocity v, and the sediment height zb undergo different changes at the position (x) after a certain time.
When the river flow rate increased from 0.1 into 0.9 visible
increase in the depth of the river, a decrease in flow velocity,
and rise sediments occur also increases.
4.3.2 Simulation Flow Straight Creeks
Search volumes were then computed sediment yield of peertemuan two creeks with Cornering the flow then continues on
sediments from tributary streams straight.
simulation II
Velocity v, = 0.2
Time t, = 5
=1
Delta t,
The angle of the river 1, =
The angle of the river 2, =
The river1 discharge,
=0.5
The river 2 discharge,
=0.5
Figure 4.12 Plot of the depth of the river simulation II
In the third simulation, shows that the flow with the initial
condition and depth = 0.3 after which time an increase in depth
up approximately 2.792678.
th
Two river discharge,

= 0.9
The resulting volume is = 1.5414e +007 = 1.5414 x 107 m3
Sediment volume charts
Figure 4.13 Plot of flow velocity in simulation II
In Figure 4.13 that seemingly stream with initial conditions

a = streamflowtwo = 0.5 and after the time of the change is the
speed drops 5.172373.
Figure 4.18 The graph plots the volume of sediment
V. CONCLUSIONS
In this chapter provides the conclusions of the analysis and
discussion that has been done. Moreover, given also the advice
to do as a continuation or development of this research.
Figure 4.14 Plot Sediment height on Simulation II
In Figure 4.14shows that the flow with initial conditions sediment height = 0.3 in all positions (x) and after the time of the
change in height of the sediment that is down approximately
2.792678.
4.4 Calculation Of Total Volume Sedimentation
Sedimentation Volume Calculation constructed from the results of the determination of the location of sedimentation to
be made sedimentation area L(A) multiply by a function of
position in the sediment height F (P) and summed according to
the grid (interval) is the sum Reimann as follows :
Limit
L(A) F(P) ap = l(a) f(p) dp da

p 0
a0
and used GAUSS Quadrature order to determine the sedimentation volume , this technique can produce a numerical 3D
shapes using software assistance in getting visualization.
For the data:
Velocity v, = 0.2
Time t, = 5
Delta t, = 1
The angle of the river, =
The angle of the river, =
The river discharge,
=0.3
5.1 Conclusion
From the analysis and discussion that has been done, it
is concludedthat:
Sediment distribution patterns along the flow is influenced by the shape of its morphology. Streams quarter-circle arc -shaped curve or a straight stream to experience the difference at each change of position of
the point, either change the depth, speed, and changes
in sediment height after a certain time interval.
River velocity will increase the speed of the bow section of the river that can allow scouring the bow section of the river. At confluence, the vector velocity
will increase and form a vortex as a result of the convergence of two different direction vectors river.
Sedimentation volume can be constructed from the results of the determination of the location of sedimentation to be made sedimentation area L(A) multiply
by a function of position in the sediment height F (P)
and summed according to the grid (interval) is the
sum numerical approaches can be searched and visualized in the form of 3 dimensions .
REFERENCES
1. Apsley, D. 2005. Computational Fluid Dynamic, Springer. New
York.
2. Asahi. 2003. Estimation of Sediment Discharge into Account Tributaries to the Ishikari River, Journal of Natural Disaster
Science. Vol 25 No 1 pp. 17-22.
3. Atlury and Lin. 2000.The meshless local Petrov-Galerkin (MLPG)
method for convection-diffusion problems, CMES. Vol. 1, No. 2,
pp. 42-60.
4. Atlury and Lin. 2001.The meshless local Petrov-Galerkin (MLPG)
method for solving incompressible Navier-Stokes Equations,
CMES. Vol. 2, No. 2, pp. 117-142.
5. Atlury and Shen. 2002. The Meshless Local Petrov-Galerkin
(MLPG) Method: A Simple & Less-costly Alternative to the Finite
Element and Boundary Element Methods, CMES, vol.3, no.1,
pp.11-51.
th
6. Atlury and Zhu. 1998. A New Meshless Local Patrov-Galerkin, In

Computational mechanics. New York.
7. Koolahdoozan 2003. Three-Dimensional Geo-Morphological Modeling of Astuarine Waters,International Journal of Sediment Research. Vol 18, No.1, pp. 1-16.
8. Ottevanger, W. 2005.Discontinuous Finite Element Modeling of
River Hydraulics and Morphology with Application to the Parana
River, Master Tesis.Department of Applied Mathematics.University of Twente.
9. Shabayek, S., dkk. 2002. Dynamic model for sub critical combining flows in channel junction, Journal of Hydraulic Engineering, ASCE, pp. 821-828
10. Wang. 2004. River Sedimentation and Morphology Modeling-The

State of The Art and Future Development, Proceedings of the
Ninth Symposium on River Sedimentation, Yichang-China.
11. Wibowo, I L and Widodo B, 2013, Numerical Simulation on Calculating Volume Sedimentation On Two Rivers Confluences Far
East Journal of Mathemathical Sciences (FJMS) Vol 76, ISSN
0972-0871, PUSPHA Publishing House India.
12. Yang, C. T. 1996.Sediment Transport, Theory and Practice,
McGraw Hill. New York.
On The Total Irregularity Strength Offriendship

Rismawati Ramdani1), A.N.M. Salman2), and Hilda Assiyatun2)
1)
Faculty of Sciences and Technologies Universitas Islam Negeri Sunan Gunung Djati Bandung
2) 3)
Combinatorial Mathematics Research Group Faculty of Mathematics and Natural Sciences,
Institut Teknologi Bandung
*)
Corresponding author : rismawatiramdani@gmail.com
AbstractLet
be a graph and k be a positive integer.
Total
k-labeling
of
G
is
a
mapping
. A total k-labeling of Gis called totally
irregular total k-labeling of G if every two distinct vertices x
and y inV satisfies
and every two distinct
edges
and
in Esatisfies
,
and
. The minimum k for
which a graph G has a totally irregulartotal k-labeling is
called the total irregularity strength of G, denoted by ts(G).
Thefriendship
is a graph obtained from wheel
by
missing every alternate rimedge. In this paper, we consider
the total irregularity strength of friendship.
where
Keywords friendship, the edge irregularity strength, the

total irregularity strength, the vertex irregularity strength,
totally irregular total k-labeling.
I. INTRODUCTION
et
be a graph. A labeling of a graph is a
mapping that carries graphelements to the numbers
(usually to the positive or non-negative integers). A
labeling f is called edge labeling if the domain of f is E, a
labeling f is called vertex labeling if the domain of f is V ,
, then the labeling
and if the domain of a labeling f is
fis called total labeling. Graph labeling was introduced in
1963 by Sadlacek. There aremany kinds of graph labeling,
such as graceful labeling, harmonious labeling, magic
labeling, and anti magic labeling.
In 2007, Ba a, Jendro , Miller, and Ryan [1] introduced irregular total k-labeling.They studied two kinds of
irregular total labeling, namely edge irregular total labelingand vertex irregular total labeling. Let
be a
graph.
For
an
integer
k,
atotal
labeling
is called an edge irregular total klabeling ofG if every two distinct edges
and
in
E satisfy
, where
=
and
. The minimum k
for which a graph G has anedge irregular total k-labeling,
denoted by
, is called the total edge irregularitystrength of G. Some results about the edge irregular total
klabelingweregiven by Nurdin, Salman, and Baskoro in
[7] and Jendro , Mi kuf, and Sot k in [2].For an integer k,

a total labeling
is called avertex
irregular total k-labeling of G if every two distinct vertices
,
where
x
and
y
in
Vsatisfy
. The minimum k for
which a graphG has a vertex irregular total k-labeling,
denoted by
, is called the total vertexirregularity
strength of G.Some results about the vertex irregular total
k-labeling were given by Nurdin, Baskoro, Salman, and
Gaos in [4]-[5]-[8].
Combining both of these notions, Marzuki, Salman,
and Miller [3] introduced anew irregular total k-labeling of
a graph G called 'totally irregular total k-labeling'.A totally
irregular total k-labeling of G is a mapping
such
that
is
distinct
for
every
and
is distinct for every
. The minimum k for which a graph
Ghas a totally irregular total k-labelng, denoted by
,
is called the total irregularity strength of G. Marzuki, Salman, and Miller [3] provided an upper bound and a lower. Besides that, they determined the total
bound on
irregularity strength of cyclesand paths.
In [1], Ba a, Jendro , Miller, and Ryanderive a lower and
an upper bounds on the total edge irregularitystrength of
any graph
as follows.
In the same paper, Ba a, Jendro , Miller, and Ryan[1]

also derive a lower and an upper bounds of the total vertex
irregularity strength of any graph
with minimum degree and maximum degree , thefollowing
bounds hold.
Marzuki, Salman, and Miller [3] given a lower bound

of
as follows.
Some results about the totally irregular total k-labeling
were given by Ramdaniand Salman in [9]. In the paper,
they have given the total irregularity strength ofsome Cartesian product graphs.
II. MAIN RESULTS

Friendship
The weights of all vertices and the weights of all edges

under the totally irregulartotal 6-labeling are given in Figure 3.
is a graph with the vertex set
and the edge set

For ilustration, friendship are given in Figure 1.
Figure 3.The weights of vertices and edges under the labeling of Figure 2
Figure 1. Friendship
III. CONCLUSION
, then
.
Theorem 2.1.Let
Proof. has
vertices and
edges. The minimum
degree of is
and the maximum degree of is
.
From
(1)
and
(2),
weget
and
. There-
Friendship is a graph which has the total irregularity

strength which is equal to its lower bound, so that it completes other graph other graphs classes on paper [9].
fore, from (3), we get
[1] M. Ba a, S. Jendro , M. Miller, and J. Ryan, On irregular total labelings,Discrete Mathematics, vol. 307, 1378-1388, 2007.
[2] S. Jendro , J. Mi kuf, and R. Sot k, Total edge irregularity strength
of completegraphs and complete bipartite graphs, Discrete Mathematicsvol. 310, 400-407, 2010.
[3] C. C. Marzuki, A. N. M. Salman, and M. Miller, On the total irregularitystrength on cycles and paths, Far East Journal of Mathematical Sciences, to be published.
[4] Nurdin, E. T. Baskoro, A. N. M. Salman, and N. N. Gaos, On the
total vertexirregularity strength of trees, Discrete Mathematics, vol.
310, 3043-3048, 2010.
[5] Nurdin, E. T. Baskoro, A. N. M. Salman, and N. N. Gaos, On the
total vertexirregularlabelings for several types of trees, UtilitasMathematica, vol. 83, 277-290, 2010.
[6] Nurdin, E. T. Baskoro, and A. N. M. Salman, The total edge irregular strengthof the union of
,JurnalMatematikadanSains
FMIPA-ITB, vol. 11,105-109, 2006.
[7] Nurdin, A. N. M. Salman, and E. T. Baskoro, The total edgeirregular strengthof the corona product of paths with some graphs,
Journal of CombinatorialMathematics and Combinatorial Computing,vol. 65, 163-175, 2008.
[8] Nurdin, A. N. M. Salman, N. N. Gaos, and E. T. Baskoro, On the
total vertex-irregular strength of a disjoint union of t copies of a
path, Journal of Combinatorial Mathematics and Combinatorial
Computing, vol. 71, 227-233, 2009.
[9] R. Ramdani, A.N.M. Salman, On the total irregularity strength of
some Cartesian product graphs, AKCE International. Journal of
Graphs and Combinatorics, vol. 10, No.2, pp. 199-209, 2013.
Next, we will show that

Define a total labeling of
.
.
as follows:
We can see that f is a labeling from

. Next,we can check that:
into
Hence, there are no two vertices of the same weight and

there are no two edgesof the same weight. So, f is a totally
irregular total
-labeling. We conclude that
REFERENCES
For ilustration, we give a totally irregular total 6-labeling

for in Figure 2.
Figure 2. A totally irregular total 6-labeling for
EXISTENCE AND UNIQUENESS

SOLUTION
OF EULER-LAGRANGE EQUATION
IN Rn
Ratna Dwi Christyanti 1*), Ratno Bagus Edy Wibowo 2), Abdul Rouf Alghofari 2)
Student of Magister Mathematics, Faculty of Mathematics and Natural Sciences, Brawijaya
University, Malang
2)
Department of Mathematics, Faculty of Mathematics and Natural Sciences, Brawijaya University,
Malang
1)
Keywords Euler-Lagrange equation, Existence, Uniqueness
Fu ( u%, u%, x )
where Fu =
ALCULUS of variations is a branch of mathematics

that deals with optimization problem to find the extremum for a functional. Functionals is function of the
functions. It can be expressed as integrals and derivatives
of the function.
Some methods that used to solve the problem of the
calculus of variations are classical and direct methods. In
the classical method, the most important tool is the EulerLagrange equation, see [2]. The existence and uniqueness
of the solution of Euler-Lagrange equation in the finite
dimensional has been discussed in [5]-[7]. Unlike in the
finite dimensional, the classical method in the infinite
dimensional can not be used directly since u% in
(C
or C
) is difficult to be proved, see [2].
call such equation as Euler-Lagrange equation in
Rn.
In order to prove existence and uniqueness of minimizers

for a functional in Sobolev spaces, we need some theorem
as in the following
Theorem 1 (Holder inequality)
Let
R n be open and 1 p . If u Lp ( )
and
v Lp ( ) where
and moreover
uv
where
u : R, F C1 ( RRn ) , F = F( u,u, x) and
u =
u
, i = 1, 2, L , n .
xi
Moreover, we find the minimizer u% for a functional (P)

which satisfy the equation
L1
Lp
Lp
'
Theorem 2 (Poincare inequality)
Let
R n be a bounded open set and 1 p .

u
Lp
= ( , p ) > 0
Lp
W 1, p
so that
, u W01, p ( ) ,
or equivalently
u
(P)
1 1
+ = 1, then uv L1 ( )
p p'
'
Then there exists
In this paper, we prove the existence and uniqueness

solution u% of Euler-Lagrange equation in R n for a
functional
I ( u ) = F ( u, u, x ) dx,
II. PRELIMINARIES
F
F
and Fu x =
, i = 1, 2,L , n. We
i
u
u xi
I. INTRODUCTION
Fux ( u%, u%, x ) = 0,

i
i =1 xi
n
Abstract This paper discusses the existence and uniqueness of minimizers of a functional in Sobolev spaces with
Direct method, and by introducing of Euler-Lagrange equation with the Classical method. Finally, we prove the existence solution of Euler-Lagrange equation in R n .
Lp
, u W01, p ( ) .
Theorem 3 Let R be convex. The function

f : R said to be convex if for every x, y
n
and every
[ 0,1] ,
the following inequality holds
f ( x + (1 ) y ) f ( x ) + (1 ) f ( y ) .
Theorem 4 Let
f : R n R and f C1 ( R ) , the
function f is convex if only if
f ( x ) f ( y ) + f ( y ) ; x y , x, y R n
f ( y ) f ( y )
f ( y )
,
,L ,
f ( y ) =
and
x2
xn
x1
.;. denotes the scalar product in n .
where
Direct method.
Step 1: (Compactness)
I ( u0 ) < and from (A4), we get
Since
< m I ( u0 ) < .
Theorem 5 (Fundamental Lemma of the Calculus of variations)
Let
Let R be an open set and
(1), i.e.
u L1loc ( ) so that
u ( x ) ( x ) dx = 0,
uv u0 + W01, p ( ) be a minimizing sequence of
I ( uv ) inf { I ( u )} = m, as v .
From (A4) that for v large enough
C0 ( )
m + 1 I ( u v ) 1 u
then u = 0, almost everywhere in .
and hence there exists

III. RESULTS
Let R
boundary.
Let F C
be a bounded open set with Lipschitz
( R R n ) , F = F ( u, u, x ) , satisfy
u F ( u , u , x )
(A1)
is
convex
for
Applying Theorem 2 (Poincare inequality), we can find

constants 5 , 6 > 0 so that
4 u L 5 uv
and we can find
uv
1 > 0, 2 , 3 R
such that
use
the
fact
W 1, p
Step 2: (lower semicontinuity)
inf I ( u) = F ( u, u, x) dx : u u0 +W01, p ( ) = m (1)
1, p
where u0 W ( ) with I ( u0 ) < . Then there
We now show that
u% u0 + W
()
Proof: We will assume that F C
( u, u ) F ( u, u, x )
(A3)
x .
(A4) there exist p > 1 and
(R R
) and
is convex for every
1 > 0, 3 R
I ( u% ) weakly lower semicontinuity,
uv u% in W 1, p ( ) lim inf I ( uv ) I ( u% ) .
v
( uv , uv ) F ( uv , uv , x )
x , then from Theorem 4 we get

F ( uv , uv , x) F ( u%, u%, x) + Fu ( u%, u%, x)( uv u% )
+ Fu ( u%, u%, x) ;uv u% .
'
( u , u , x ) R R n
p 1
p 1
Fu ( u , u , x ) (1 + u + u )
Fu ( u, u , x ) 1 + u
where
and
Fu =
p 1
+ u
Fu = Fux , Fux ,L, Fux , Fux =

1
p 1
(2)
Fu ( u%, u%, x ) Lp ( ) and
F ( u, u, x ) 1 u + 3 , ( u, u, x ) R R .
'
Fu ( u%, u%, x ) Lp ( ; R n ) .
(A5) there exist a constant 0 so that for every
and
is convex for every
Furthermore, we need to show that
such that
exists
this mean that
Since
a minimizer of (1).
p > 1, there
that
Let
exists
7.
uv u% di W 1, p ( ) as v .
F ( u, u, x) 1 u +2 u +3, ( u, u, x) R R .
1, p
0
6 ,
u% u0 + W01, p ( ) and subsequence (still denoted uv )

so that
W 1, p
7 > 0 so that
every
We
4.
Lp
( u , x ) R ,
(A2) there exist p > q 1 and
4 > 0 so that
u
Using Direct method we have

Theorem 6 (Existence)
+ 3 ,
p
Lp
F
, i =1,2,L, n
uxi
F
.
u
From
(A5)
and
u% W1, p ( )
where
1 1
p
+ ' = 1 p' =
,
p p
p 1
we have
Fu ( u% , u%, x )
p'
F ( u%, u%, x )
p'
1 1 + u%
W 1, p
W 1, p
)<
and
u
1 1 + u%
) < ,
The proof of Theorem 6 are devided into theree steps by
y% u0 +W01, p ( ) .
1 being a constant.
We get
Since
Fu ( u% , u% , x ) Lp ( ) and
'
Fu ( u%, u%, x ) L ( ; R ) .
p'
Using Theorem 1 (Holder inequality) we find that for
uv W 1, p ( )
( uv u% ) Fu ( u%, u%, x ) L1 ( )
and
( u,u) F( u,u, x) is
convex
for
every
x , then
1
1
1 1 1 1
%,wx
%, ) F v%+ w
%, v%+ wx
%, =F( y%,y%, x)
F( v%,v%, x) + F( w
2
2
2 2 2 2
We get,
m=
Fu ( u%, u%, x ) ; uv u% L1 ( )
1
1
I ( v% ) + I ( w% ) I ( y% ) m.
2
2
We therefore get,
We next integrate (2), to get
1 1
%, ) F v% + w
%, v% + wx
%, dx =0.
2F( v%,v%, x) +2F( w%,wx
2
2 2 2
I ( uv ) I ( u%) +Fu ( u%,u%, x)( uv u%) dx+ Fu ( u%,u%, x) ;uv u% dx.
Since
uv u% 0 in W
1, p
()
(i.e.
uv u% 0 di L
uv u% 0 di L ), then we get
p
and
F , positive
% and v% = w% we deduce that v% = w% alunless v% = w
most everywhere in .
Since the integrand is, by strict convexity
Theorem 8 (Euler-Lagrange equation)
1, p
lim Fu ( u%, u%, x )( uv u% ) dx = 0,Fu ( u%, u%, x ) Lp ( ) Let u% u0 + W0 ( ) be a minimizer of
v
inf I ( u) = F ( u, u, x) dx : u u0 +W01, p ( ) = m, (3)

and
1, p
'
lim F u%,u%, x ;u u% dx = 0, F u%,u%, x Lp . where u0 W ( ) , then u% satisfies the weak form of
'
( )
We have
Step
( F (u%,u%, x) + F (u%,u%, x) ;) dx = 0,W ( ) .
3:
I ( uv ) inf { I ( u)} =m
Since
liminf I ( uv ) I ( u% )
Moreover,
and
I ( u% ) = m, or in other words u% is a mi-
( u, u ) F ( u, u, x ) is strictly con-
vex for every x then the minimizers is unique.
W01, p ( )
u% + u0 + W
().
Since
DI ( u% )( ) =
and let
then
that
x .
DI ( u% )( ) = ( Fu ( u%, u%, x ) + Fu ( u%, u%, x ) ; ) dx.
1, p
0
I ( u ) = F ( u, u, x ) dx,
PrProof: The proof of Theorem 8 are devided into two

steps by Classical method.
Step 1: By using Gateaux derivative, we prove that
Let R ,
v%, w% u0 + W01, p ( ) be two solutions of
inf I ( u ) = F ( u, u, x ) dx = m.
uA
%.
We prove v% = w
1 1
y% = v% + w% and observe
Denote
by
2 2
Fux ( u%, u%, x ) = 0,

i
i =1 xi
n
nimizers of (1).
Theorem 7 (uniqueness)
Proof: Let
F C 2 ( R R n ) and u% C 2 ( ) , then u%
Fu ( u%, u%, x )
If the function
If
satisfies the Euler-Lagrange equation
m = lim inf I ( uv ) I ( u% ) .
We deduce that
(4)
then
1, p
0
lim inf I ( uv ) I ( u% ) .
v
the Euler-Lagrange equation
d
I ( u% + ) ,
d
=0
and
DI ( u% )( ) =
d
F ( u% + , u% + , x ) dx
d
=0
F( u,u, x) F( u%,u%, x) +Fu ( u%,u%, x)( uu%) +Fu ( u%,u%, x) ;( uu%)

Integrating the above inequality.
Since
= Fu ( u%, u%, x ) + Fu ( u%, u%, x ) ; dx.
Fu (u%,u%, x) .( u u%) dx = (u u%) (.Fu (u%, u%, x)) dx,

Step 2: Indeed since u% is a minimizers of (3), then
I ( u% + ) I ( u% ) , W01, p ( )
then
and thus
I ( u ) I ( u% ) + ( Fu ( u%, u%, x) .Fu ( u%, u%, x) ) ( u u% ) dx
DI ( u% )( ) = 0.
Furthermore, we get
DI ( u%)() = Fu ( u%,u%, x) + Fu ( u%,u%, x) ; dx = 0. (5) Using

Since
F ( u%, u%, x ) ; dx = ( .F ( u%, u%, x ) ) dx,

u
i =1
F ( u%, u%, x) ( .F ( u%, u%, x) )dx. and

u
if
every
From (5), we have that
1, p
0
( ) satisfies
Fux ( u%, u%, x) = 0,

i
i =1 xi
and if
( u, u ) F ( u, u, x ) is
x .
(6)
x , then u% is a minimizer of
From this paper we can conclude that by Direct

method, optimization problems in the infinite dimensional
space have solution if
u F( u,u, x) is convex and
1 > 0, 2 , 3 R such
there exist p > q 1 and

that
F ( u, u, x) 1 u +1 u +3, ( u, u, x) R Rn .
p
It is shown that if function
( u , u ) F ( u , u , x )
is
R n have
solution if function ( u , u ) F ( u , u , x ) is convex.
Moreover, Euler-Lagrange equation in
Furthermore, we shown that Euler-Lagrange equation in

(7)
u% be a solution of (4).
Since ( u , u ) F ( u , u , x ) is convex for every
Proof: Let
x , then from Theorem 4 we deduce that for every

u u0 + W01, p ( ) the following holds
(9)
strictly convex, then the minimizer of (P) is unique.
convex for every
I ( u ) = F ( u , u, x ) dx.
x , then u% is a unique minimizer of
IV. CONCLUSIONS
Theorem 9 (Existence solution of Euler-Lagrange

equation)
If u% satisfies
n
Fu ( u%,u%, x) Fux ( u%,u%, x) = 0,x

i
i=1 xi
stricly convex for
Proof: The proof of Corollary 10 is analog with Theorem

9, and by Theorem 7 we deduce u% is a unique minimizer
of (9).
or in other words
n
(8)
Fu ( u%, u%, x ) ( .Fu ( u%, u%, x ) ) = 0,

Fu ( u%, u%, x)
( u, u ) F ( u, u, x ) is
I ( u ) = F ( u , u, x ) dx.
F (u%,u%, x) (.F ( u%, u%, x))dx = 0, W ( ) .

From Theorem 5, u% C
Fu ( u%,u%, x) Fux ( u%,u%, x) = 0,x

i
i=1 xi
I ( u ) I ( u% ) . We deduce u% that is
we get indeed that
F ( u%, u%, x) + F ( u%, u%, x) ; dx =

u
Fux ( u% , u%, x ) = 0,
i
xi
a minimizer of (7).
Corollary 10 (uniqueness)
If u% satisfies
then
u
Fu ( u%, u%, x )
R n have a unique solution if function

( u, u ) F ( u, u, x ) is stricly convex.
REFERENCES
[1]
[2]
Adams R.A., Sobolev spaces, Academic Press, New York, 1975.

Dacorogna B., Introduction to the Calculus of Variations, Imperial
College Press, French, 1992.

[3]
[4]
[5]
Sun W. and Yuan Y., Optimization theory and methods, Springer,

New York, 2006.
Clarke F.H., The Euler-Lagrange Differential Inclusion, J. Of
Diferential Equations. 19 (1975), 80-90.
Bohner M. and Guseinov G.Sh., Double integral calculus of variations on time scales, J. of Computers and Mathematics with Applications. 54 (2007), 45-57.
[6]
[7]
Christyanti R.D, Euler-Lagrange Equation, Research report, 2011.

Orpel A., The existence of minimizers of the action functional
without convexity assumption, J. of the Juliusz Schauder Center.
20 (2002), 179-193
APPLICATION BARRO MODEL ON

ECONOMIC GROWTH VIA HEALTH IN
CENTRAL JAVA
Caroline
Universitas Sultan Fatah Demak, Demak, Indonesia
*)
Corresponding author: Carolinehamboro@yahoo.com
AbstractCentral Java Through Health Spending, Education, health and income are the three pillars that are important in the economic development of a region (World Bank,
1993). By considering the importance of health for the improvement of the health of a region need to get the government's attention. Barro model offers economic growth
through health channels. With the healthy person's productivity will increase, so that the output will be generated will
increase the economic growth of a region. Central Java is
one of the provinces in Indonesia, which has a human development index (HDI) which is lower than the HDI and the
Indonesian island of Java. With the improvement of health in
Central Java, is expected to boost economic growth in Central Java, so that with the economic growth of Central Java
which will increase Indonesia's economic growth.
Keywords Economic growth, health
I. INTRODUCTION
evelopment is a tool used to achieve the goals of ecoDnomic growth of the nation and is one of the indicators
to assess the success of a country's development. Development paradigm that is being developed at this time is
economic growth measured by the human development
that seen with the level of quality of human life in each
country. One of the benchmarks used in looking at the
quality of human life is the Human Development Index
(HDI) which is measured by the quality of education,
health and economic (purchasing power). Through the
third increase this indicator is expected to increase the
quality of human life.
To see the extent of development and human wellbeing, UNDP has issued an indicator of the Human Development Index (HDI) to measure the success of a country's development and prosperity. Human Development
Index (HDI) is a benchmark figure of a region or state
welfare is seen by three dimensions: life expectancy at
birth, literacy rates and average length of the school, and
purchasing power. Life expectancy is an indicator to
measure
the
health,
indicator of the adult population literacy rate and the average length of the school to measure education and the
last indicator measures the purchasing power of the standard of living. (United Nations Development Programme,
UNDP, 1990).
The rate of economic growth in Central Java Province from 2005 to 2012 has increased. This suggests that
the economic development in Central Java Province has
increased. It will boost economic development and human
development. Regional economic growth positively and

significantly influenced by human development.
TABLE I
GROSS REGIONAL DOMESTIC PRODUCT AT CONSTAN
2000 MARKET PRICE 2005-2012
Year
Gross Regional Domestic Product at Constan

2000 Market Price
2005
5.35
2006
5.33
2007
5.59
2008
5.61
2009
5.14
2010
5.84
2011
6.03
2012
6.34
Source : BPS-Statistics of Central Java Province
HDI achievement targets in Central Java in 2013 is

expected to experience a significant increase in the
amount of 74.3% with a life expectancy indicators (life
expectancy) of 73.8 years, the average length of 7.0 years
of school, literacy rates for 97.3%, and the per capita expenditure of IDR. 626,200. It became a Central Java in
order to make the target unable to compete with other
regions, especially in Java and outside Java in general,
which is expected to improve the competitiveness in terms
of quality of human resources.
The elements of human development underlines
explicitly targets to be achieved, namely a healthy life and
longevity, educated and can enjoy a decent living. This
means that human development aims to improve the welfare of the community with regard to the quality of human
and society. Therefore, human is central to the development process.
Health, education and income has been regarded as
the three pillars of human development in the Human Development Index (HDI) (UNDP, 1990). Health is an important form of human capital. This can increase worker
productivity by improving their physical capacity, such as
strength and durability, as well as their mental capacity,
such as cognitive functioning and reasoning abilities.
Health factors closely related to the quality of human resources (quality of human resources) itself. High
and low quality of human resources (HR) will be determined by health status, education and income levels per
capita (Ananta and Hatmadji, 1985). In economic activities, the three indicators of the quality of human resources
will also indirectly impact on the level of productivity of
human resources, in this case, especially labor productivity.
Year
s
TABLE II
HUMAN DEVELOPMENT INDEX IN CENTRAL JAVA
PROVINCE 2005-2013
Life
The averReal exLiteracy
Expecage Old
penditure /
rates
HDI
tation
School
per capita
(%)
(Year)
(Years)
(Rp.000)
2005
70.60
6.60
87.40
621.40
69.8
2006
70.80
6.80
88.20
621.70
70.25
2007
70.90
6.80
88.62
628.53
70.92
2008
71.10
6.86
89.24
633.58
71.60
2009
71.25
7.07
95.60
624.20
72.10
2010
71.40
7.24
96.10
624.60
72.49
2011
73.20
6.90
96.60
625.30
72.94
2012
73.50
7.00
97.00
625.80
71.72
97.30
626.20
71.68
2013
73.80
7.00
Source : BPS-Statistics of
Central Java Tengah Province
The quality of labor, in the form of human capital,

contributing significantly to economic growth. Workers
are physically fit and mentally more energetic and robust.
They are more productive so that it will get higher wages.
They also tend to be absent from work due to illness (or
disease in their family). Disease and disability reduce
hourly wages substantially.
Todaro and Smith (2006) that health is at the core
of the welfare and education is essential to achieve a satisfying and worthwhile life. Education plays a major role in
shaping the ability of developing countries to absorb
modern technology and to develop the capacity to create
sustainable growth and development. Health is a prerequisite for improved productivity, while educational success
also relies on good health. Its dual role as both input and
output cause health and education is very important in
economic development and economic growth. So research
on the application dieprlukan barro models in promoting
economic growth through health channels in Central Java.
II. THEORY
A. Economic Growh Teory
The theory of economic growth has a long history
dating back to the late 18 century when the analysis of
economic growth was at the center of attention of classical
economists such as Smith (1776), Malthus (1798) and
Ricardo (1817). These studies identified important causes
and mechanisms that affect economic growth. The most
important result from them is that the accumulation and
investment of the production output is the main driving
force behind economic growth.
The much later works of Ramsey (1928), Young
(1928), Schumpeter (1934) and Knight (1944), which
emphasize the elements of competition, equilibrium dynamics, diminishing returns, the accumulation of physical
and human capital and the monopoly power gained from
technology advances, formed a good basis for the neoclassical growth theories and the endogenous growth theories
developed after the middle 20 century.
The models of Solow (1956) and Swan (1956) use
a production function approach where there are constant
returns to scale but diminishing return to each input. The
equilibrium will exist if certain conditions are satisfied.
The growth rate of the economy is determined exclusively
by the exogenous technology. In other words, there will
be long-term economic growth only if there are continuous new technologies available. One important finding
of the neoclassical model is neoclassical models explain
everything except long-term growth. To overcome this
modeling deficiency, researches on endogenous growth
such as Romer (1986), Lucas (1988) and Romer (1990),
which emphasize the roles technology changes and human
capital accumulation in the form of education play, help to
generate some important results confirming the important
roles of technology changes and education in promoting
long-term growth. The theory of "conditional convergence" which shows that the growth rate of the economy
will be faster the further this economy is below its own
equilibrium level. The historical facts show that the positive rate of economic growth persists over a century and
there is no trend of decline. The property of diminishing
return of the inputs determines that the neoclassical models explain everything but long-term growth. To overcome
this modeling deficiency, researches on endogenous
growth such as Romer (1986), Lucas (1988) and Romer
(1990), which emphasize the roles technology changes
and human capital accumulation in the form of education
play, help to generate some important results confirming
the important roles of technology changes and education
in promoting long-term growth.
A. Economi Growth Via Health with Barro Model
Fogel (1991, 1997, and 2000) have used historical facts to demonstrate that health is a powerful engine of
economic growth. Barro (1991), used a-cross sectional
framework; that is, the growth and the explanatory were
observed only once per country. The main reason extend
to a panel set up is to expand the sample information. Although the main evidence turn out to ceme from the crosssectional (between-country) variation, the time-series
(within-country) dimension provides some additional information. Barro Model with initial level of GDP,initial
level of schooling and initial health status.
Initial Level of GDP
For given of the explanatory variables, the neoclassical model predicts a negatif coefficient on initial GDP,
which enters in the system in logarithmic form. The coefficient on log of initial GDP has the interpretation of a
conditional rate of convergence.
Initial Level of Schooling
Education appears in two in system : average years
of attaiment for male age 25 and over in secondary and
higher schools at the start of each period and an interaction between the log of initial GDP and theyears of male
secondary and higher schooling. Female schooling is importanat for other indicators of economic development,
such as fertility, infant morality and political freedom.
Specifically, female primary education has a strong negative relation with the fertility rate (see Schultz [1989],
Behrman [1990], and Barro [1994]).
Initial Health Status

The populations overall health status is measured
here by the log of life expectancy at birth at the start of
each period. The results are, however, similar with some
alternative aggregate indicators of health, such at the infant mortality rate, the mortality rate up to age five, or
expectancy at age five.
Fertility Rate
If the population is growing, then a portion of the
economys investment is used to provide capital for new
workers, rather than to raisecapital per worker. For this
reason, a higher rate of population groeth has a negative
effect on y*, the steady-state level of output per effective
workers in the neoclassical growth model. Another, reinforcing, effect is that a higher fertility rate means that increased must be devoted to childerearing, rather than to
production of goods (see Beckers and Barro [1988]). Fertility decision are surely endogenous; previous research
has shown that fertility rypically declines with measures
of prosperity, especiall female primary education and
health status (see Schultz [1989], Berhman [1990], and
Barro and Lee [1994]).
Based on the Barro (1996) framework, inspired

by the argument made by Grossman (1972) that health
depreciation rate should not be constant, we endogenize
the health depreciation rate by considering the following
two cases: (1) health depreciation is determined exclusively by health; (2) health depreciation rate is jointly determined by health and education.
In these two cases, the negative effect of health
on economic growth is reflected explicitly by the endogenous health depreciation rate which is a positive function of health. The optimization results show that when
the endogenous health depreciation rate is determined
only by health, the negative effect of health on economic
growth would be too strong to generate endogenous
growth in the long-term. In contrast, if we consider the
effect of education on lowering the health depreciation
rate simultaneously with the positive effect of health on
health.
In the Barro (1996b) model, health affects economic growth by entering the production function directly, which corresponds to part I of Figure 1.
Health affect economic growth through labor
productivity. Improvement in health would allow the
worker to work more efficiently, increase the amount of
effective working hours and lower the probability of being
absent from work either by the worker or his/her family
members. Better health status would also increase the life
expectancy and thus prolong the working ages which
would encourage investment in education because the
return on education investment is higher with longer effective working time. All these channels would lead to improvement in labor productivity which results in economic
growth.
Our idea of endogenous health depreciation rate is supported by Grossman (1972). In the Grossman (1972) paper, health has been identified as another important form
of human capital, which provides a good starting point for
researchers to analyze the relationship between health and
economic growth. However, as accepted by Grossman,
health depreciation rate should vary over time. To understand why the health depreciation rate should not be constant, we should first understand the definition of health
depreciation rate, which is the cost of maintaining the
current level of health.
There are many examples to show why the health
depreciation rate should not be a
constant. For example, before a major competition like the
Olympic Games, an athlete needs to spend time on training, to eat following the instruction of dietitian and to
check his/her body fitness regularly. In order to keep the
match fitness, the investment is huge. However, after the
competition, he/she no longer needs to keep that high level of match fitness and the
expenditure to keep his/her non-match fitness level of
health would be lower. Another example is that one of the
significant indicators of better health is life expectancy.
C. The Barro model of health and economic growth
Barro (1996b) proposed a one-sector model which
extended the neoclassical model to incorporate the impact
of health on economic growth. In his model, health affects
economic growth both directly and indirectly. First, health
directly enters production function indicating a direct impact of health on productivity. In other words, if other
determinants of the production function, such as physical
capital, labor and schooling, are all constant, an improvement in an individual's health would increase the productivity. Second, health also determines the depreciation rate
of both health and education. Therefore, health contributes to economic growth indirectly through its effect on
education.
D. The Barro growth model revisited
In the Barro model, health is a private good that is
financed totally by the individuals themselves. Investments in health include activities such as the purchase of
nutrition products, the leisure time spent on sports, the
money paid on doctors and medicines, a regular body
check, etc.
The economy is a one sector economy. First, total
output Y is determined in a Cobb-Douglas function by
physical capital inputs, K,individual's schooling and other
education related factors, S, the health capital of individual, H, and the amount of labor provided, L:
Y = AK S H (L)1---
where A is the knowledge stock parameter, which
represents the exogenously determined technology level.
The model assumes that > 0, > 0 , > 0 and + +
< 1.
B. The health depreciation rate

As health depreciation is one component in the
health accumulation function, we are interested in endogenizing the health depreciation rate in order to reflect the
negative effect of health in promoting economic growth.
th
Higher labor
productivity
Less illness
Increased
effective
labor
Longe working
hours
More Work
Energy
Higher Cognitive
Ability
Increasing
Return on
Education
Longer years
of working
Health
Higher life
expectancy
Population
structure
change
Lower
fertility
Lower
morbidity
Goods production
function:
Y=AF (K, H)
Fig. 1 The interaction network between health and growth at Barro Model
That is, in this production function, Barro assumes constant

returns to scale with respect to the four inputs (physical capital, education, health and labor) but diminishing returns with
respect to each of the inputs respectively. This is a key assumption to derive the results of the Barro model. One thing to
pay attention to these assumptions is that constant returns to
scale with respect to the four inputs imply diminishing returns
to scale with respect to the inputs of physical capital, education
and health together.
IV. ESTIMATION AND RESULTS

From the SPSS 16 output display, Model summary
magnitude of R2 is 0.746, meaning 74.6 GDP variation is explained by the variation of the four independent variables
POP, LABORFORCE, LABOR and LIFE EXPECTANCY.
While the remaining (100% - 74.6% = 25.4%) is explained by
other causes outside the model.
TABEL III
DESCRIPTIVE STATISTICS
Mean
III.DATA
PDRB
We construct a panel of Central Java over 1988-2012.

Output data Gross Regional Domestic Product Constant 2000
from BPS-Statisctics of Jawa Tengah Province.
We measure a provinces labor supply by the size of its
economically active population using data from BPSStatisctics of Jawa Tengah Province. Life expectancy date
from BPS-Statisctics of Jawa Tengah Province.
Std. Deviation
8.49E7
2.935E11
19
Pop
3.1136E7
1.00281E10
19
Laborforce
1.7185E7
3.73047E10
19
Labor
1.4683E7
5.92126E9
19
69.7355
4335.65823
19
Lifeexpectation
a. Weighted Least Squares Regression - Weighted by Education
Standard error of estimate (SEE) is 1.478 Milyar . The smaller the value of SEE will make more precise regression models
in predicting the dependent variable.
TABEL IV
Model Summaryb,c
Change Statistics
Model
1
R
.896a
R Square
.803
Adjusted R
Square
.746
Std. Error of the

Estimate
1.478E11
a. Predictors: (Constant), Lifeexpectation, Laborforce, Labor, Pop

b. Dependent Variable: PDRB
c. Weighted Least Squares Regression - Weighted by Education
R Square
Change
.803
F Change
14.242
df1
Sig. F
Change
df2
4
14
.000
DurbinWatson
2.065
th
From the ANOVA F test obtained or calculated value F calculated at 14.242 with probability 0.000. Because the probability
is much smaller than 0.05 then the regression model can be
used to predict the GDP or POP, LABORFORCE, LABOR

and
Life
Expectancy
jointly
affect
the
GDP.
TABLE V
ANOVAb,c
Model
Sum of Squares
df
Mean Square
Regression
1.244E24
3.111E23
Residual
3.058E23
14
2.184E22
Total
1.550E24
18
Sig.
14.242
.000a
a. Predictors: (Constant), Lifeexpectation, Laborforce, Labor, Pop

b. Dependent Variable: PDRB
c. Weighted Least Squares Regression - Weighted by Education
TABLE VI
Coefficientsa,b
Unstandardized
Coefficients
Model
1
(Constant)
Std.
Error
Standardized Coefficients
Beta
-4.290E9 7.221E8
95% Confidence
Interval for B
Lower
Bound
Upper
Bound
Sig.
-5.941
.000
-5.839E9 -2.741E9
Collinearity
Statistics
Correlations
Zeroorder
Partial
Part
Tolerance
VIF
Pop
-3.948
5.198
-.135
-.759
.460
-15.096
7.201
.576
-.199
-.090
.447
2.239
Laborforce
-.837
1.027
-.106
-.816
.428
-3.040
1.365
-.339
-.213
-.097
.827
1.209
Labor
-1.250
6.512
-.025
-.192
.851
-15.217
12.718
.327
-.051
-.023
.816
1.225
3.867E7
9.126E7
Lifeexpectation 6.496E7 1.226E7
.960 5.299
.000
.888
.817
.629
.429 2.329
a. Dependent Variable: PDRB

b. Weighted Least Squares Regression - Weighted by
Education
The coefficient of the independent variable (independent) can use unstandarized coefficients and standarized coefficients.
Unstandarized beta coefficients :
The four independent variables included in the model
OLS, Life expectancy variables significant at 0.05, while the
other three variables were not significant (because of the above
0:05).
Mathematical equation:
PDRB = -4.290E9 -3.948 Pop 0,837 Laborforce 1.250
Labor + 6.496E7 Life expectancy.
REFERENCES
[1] Baldacci, E. Hillman, A. and Kojo, N. 2004. Growth governance, and
fiscal policy transmission channels in low-income countries. European
Journal of Political Economy, 20 (3), 517-549.
[2] BPS-Statistics of Jawa Tengah Province
[3] Barro, Robert J. 1990. Government spending in a simple model of endogenous growth. Journal of Political Economy, 98, October, part II,103125.
[4] Barro, Robert J. 1991. Economic growth in a cross section of countries.
Quarterly Journal of Economics, 106, May, 407-443.
[5] Barro, Robert J. and Xavier Sala-I-Martin. 1991. Convergence across
states and regions. Brookings Papers on Economic Activity, 1, 107-158.
[6] Barro, Robert J. and Xavier Sala-i-Martin. 1992. Convergence. Journalof
Political Economy, 100, 223-251.
[7] Barro, Robert J. and Lee, J. 1993. International comparisons of educational attainment. Journal of Monetary Economics, 32 (3),363-394.
[8] Barro, Robert J. 1996a. Determinants of economic growth: A crosscountry empirical study. NEBR Working Paper No.5968. Cambridge,
MA: National Bureau of Economic Research
[9] Barro, Robert J. 1996b. Health, human capital and economic growth,
Paper for the program on Public Policy and Health, Pan American
Health Organization and World Health Organization. Washington, DC:
Pan American Health Organization
[10] Barro, Robert. J. 1997. Determinants of economic growth: a cross country empirical study, MIT Press
[11] Barro, Robert. J. and Lee, J. 2000. International data on education attainment: Updates and implications. Center for International Development Working Paper No 42. Cambridge, MA: Harvard University.
[12] Barro, Robert J. and Xavier Sala-i-Martin. 2005. Economic
Growth.New York: McGraw-Hill, Inc.
[13] Bassanini, A., and Scarpetta, S. 2001. Does human capital matter for
growth in OECD countries? Evidence from pooled mean-group estimates, Economics Department Working Paper No. 282, Paris, France:
OECD.
[14] Becker, G.S. 1962. Investment in human capital: a theoretical analysis.
Journal of Political Economy, 70, 9-49.
[15] Becker, G.S. and Barro, Robert J. 1989. Fertility choice in a model of
economic growth. Econometrica. 16, 481-501.
[16] Bloom David E. and David Canning. 2000. Demographic change and
economic growth: The role of cumulative causality, in Nancy Birdsall,
Allen C. Kelley, and Stephen Sinding, eds., Population Does Matter:
Demography, Growth, and Poverty in the Developing World. New
York: Oxford University Press
[17] Bloom, David E., Canning, D., and Sevilla, J. 2001. The effect of health
on economic growth: theory and evidence. NBER Working Papers
8587,National Bureau of Economic Research, Inc.
[18] Bloom, David E. and Canning, D. 2003. The health and poverty of
nations: From theory to practice. Journal of Human Development, 4(1),
47-71.
th
[19] Bloom, David E. and Canning, D., and Sevilla, D. 2004. The effect of
health on economic growth: A production function approach. World
Development, 32 (1), 1-13
[20] Chakraborty, S., and Das, M. 2005. Mortality, human capital and persistent inequality. Journal of Economic Growth, 10, 159-192.
[21] Durlauf, S. 1996. A theory of persistent income inequality. Journal of
Economic Growth, 1, 75-94.
[22] Fogel, Robert W. 1991. New sources and new techniques for the study
of secular trends in nutritional status, health, mortality, and the process
of aging, National Bureau of Economic Research Working Paper Series
on Historical Factors and Long Run Growth: 26, May.
[23] Fogel, Robert W. and Wimmer, L. T. 1992. Early indicators of later
work levels, disease, and death, Bureau of Economic Research Working
Paper Series on Historical Factors and Long Run Growth: 38, June.
[24] Grossman, M. 1972. The Demand for Health: A Theoretical and Empirical Investigation. NBER, Occasional Paper 119, Columbia University
Press.
[25] Gupta, S., Verhoeven, M., and Tiongson, E. 2003. Public spending on
health care and the poor. Health Economics, 12(8), 685-696.
[26] Islam, N. 1995. Growth empirics: A panel data approach. Quarterly
Journal of Economics, 110, 1127-1170
[27] Musgrove, P. 1996. Public and private roles in health: Theory and financing patterns, World Bank Discussion Paper No. 339, Washington
DC: World Bank.
[28] Mushkin, S.J. 1962. Health as an investment. Journal of Political
Economy, 70, 129-157.
[29] Romer, P. M. 1986. Increasing returns and long-run growth. Journal of
Political Economy, 94 (5), 1002-37
[30] Schultz, T.P. 1961. Investment in human capital. American Economic
Review, 51, 1-17.
[31] Solow, R.M. 1956. A contribution to the theory of economic growth
Quarterly Journal of Economics, 70, 65-94.
th
The Development of Gui Simulation-Based

Media for Geometry Transformation
Lilik Hidayati1*)
1)
SMKN 2 Lingsar West Lombok, West Nusa Tenggara, Indonesia
*)
Corresponding author: liliknyagatan@gmail.com
Abstract This study aimed to develop Graphical User

Interface (GUI) simulation-based media that involved material concepts and problem-solving of mathematical processes, particularly pertaining to the transformation of geometry material. The rationale for this is that the language
of mathematics that employs symbols and abstract meanings are hardly understood by students, and therefore it is
regarded as a difficult subject to study. To deal with this
condition, it is necessary to develop teaching media that
can transform the abstract mathematical concepts into
more concrete ones. Generally, the mathematics software
for learning simulation does not show the process of solving
mathematical problems. The software only generates the
final result without helping students to do the thinking
process. With this in mind, it is necessary to develop a
mathematical learning simulation that contains mathematical processes pertaining to the mathematical problemsolving. To conduct the current research, the researcher
used a flow chart design.
Keywords Abstract, Math Problems, GUI-Based Media, Symbols, Software
I. INTRODUCTION
HE specific objective of mathematics teaching at
school is to train students with logical, critical,
precise, and applicale thinking as well as providing
students with ability to study sciences and technology for
further education. The teaching of mathematics has
experienced paradigm changes of learning technique.
This can be seen from the development of the
cooperative models in mathematics teachings which
focus on the students as the learning indivduals.
[Herman]1
On the other hand, according to Rastaman &
Rastaman (1997), laboratorium is a supporting media in
the process of teaching and learning. Optimum result
will be achieved when students are involved both
physically and mentally in the teaching process. Through
the simulation media being developed, it is expected that
teachers can easily explain the abstract mathematical
concepts becoming more concrete ones.
The problem to solve is how the Graphic User
interface (GUI-based) simulation media in the teaching
of mathematics can help convey materials and concepts
of mathematics problems solving. This study aims at
developing a GUI-based simulation media for
mathematics teaching that contain maerials and concepts
of mathematics problems solving.
II. LITERATURE RIVIEW
A. The Mathematics Learning Theory of Dienes

According to Dienes, mathematical games are really
important because the math operation in the game illustrates concrete rules that guide and sharpen the mathematic understanding of the learners. Thus, the concrete
objects in the form of game play an important role in
mathematics teaching when manipulated well. The more
varied the the concepts introduced, the clearer the understanding of the students is.
B. Graphical User Interface (GUI)
So far, the teaching of mathematics is dominantly performed in the traditional way in which everything is
written on the board. Nowadays, the teaching process
has has progressed where Matlab is used as computation
device that help teachers in teaching mathematics. Matlab is a programming language with high performance
in computation that is highly qualified for technical
computation. It is also an interactive system for numeric
computation and data visuaization.
C. Simulation Method
This simulation method develops learners skills, both
mental and pysical/technical skills. The method transfers
a real-life situation into a learning activity or learning
room as there is difficulty to do a practice in the real
situation. For instance, an aviation school student does a
flying operation simulaton. The situation faced in the
simulation must be created in such that it appears like
the real true one (reality replication).
III. RESEARCH METHOD
Research design is the procedure concept to guide the

research implementation so that it runs correctly to
achieve the goal. This is a developing type of research
with the following design:
th
Start
Designing GUI
Model
IV. UNITS
Scripting
Implementation
No
Successful
Yes
Project Application
%
TRANSLASI, by itself, creates a new
TRANSLASI or raises the existing
%
singleton*.
%
%
H = TRANSLASI returns the handle to a
new TRANSLASI or the handle to
%
the existing singleton*.
%
%
TRANSLASI('CALLBACK',hObject,eventData,handles,
...) calls the local
%
function named CALLBACK in TRANSLASI.M
with the given input arguments.
%
%
TRANSLASI('Property','Value',...)
creates a new TRANSLASI or raises the
%
existing singleton*. Starting from the
left, property value pairs are
%
applied to the GUI before
translasi_OpeningFcn gets called. An
%
unrecognized property name or invalid
value makes property application
%
stop. All inputs are passed to
translasi_OpeningFcn via varargin.
%
%
*See GUI Options on GUIDE's Tools menu.
Choose "GUI allows only one
%
instance to run (singleton)".
%
% See also: GUIDE, GUIDATA, GUIHANDLES
% Edit the above text to modify the response to
help translasi
% Last Modified by GUIDE v2.5 23-Nov-2013
11:58:56
% Begin initialization code - DO NOT EDIT
End
Chart 1. Diagram of the Developing of GUI-Application for Maths
Teaching Simulation
IV. RESULT AND DISCUSSION
........
function edit7_CreateFcn(hObject, eventdata,
handles)
% hObject
handle to edit7 (see GCBO)
% eventdata reserved - to be defined in a
future version of MATLAB
% handles
empty - handles not created until
after all CreateFcns called
A. Design of the GUI Model

% Hint: edit controls usually have a white
background on Windows.
%
See ISPC and COMPUTER.
if ispc &&
isequal(get(hObject,'BackgroundColor'),
get(0,'defaultUicontrolBackgroundColor'))
set(hObject,'BackgroundColor','white');
end
Figure 1.This is GUI Geometry Transformation concept

Note the Figure is designed for GUI model. The program is used for
calculating geometric transfomation. By input the data into the boxes
(A,B,and C). The data is operated by pressing the button (translasi,
refleksi, rotasi, dilatasi) and the result will appear in the screen.
B. Scripting
After designing the GUI model, the computation code
is added to the m-file. This scripting process can be seen
in details in the appendix. Some parts of the stages are
as the following:
function varargout = translasi(varargin)
% TRANSLASI MATLAB code for translasi.fig
C. Implementation and Discussion

The result of the developing of the simulation media
is to be applied for problem solving in transformational
geometry. The samples of problems are chosen on the
basis of their representativeness covering items such as
translation, reflection, rotation, and dilation.
The project application from the development of of
the GUI-based simulation media is in the form of software compilation in CD application. The GUI - Model
is equipped with scripting (computation language). After
verification and application test in which the media is
considered successful, it is then applied into teaching
practice. The data of the implementation/application
stage is analyzed to see the relation among variables and
also to identify the pattern of the data.
The advantage for teacher: The first, process of
teaching leaarning is effective and effesient, the second
the target of learning achieved or sucessfull, for
students: The first, the whole process appear in this

program, the second the program appearing the
animation process make the process of teaching reality
without leaving the mathematic terms, third constructed
the students knowledge systematicly.
V.
CONCLUSION
Based on the implementation result of the GUIModel simulation media and the study on the learning
theory of Dienes, the teaching media is found to be potential to contribute much in increasing the students
understanding towards the concept of transformational
geometry.
REFERENCES
[1]
[2]
[3]
Bloom, Benyamin S., et. all (1971), Handbook on Formative

and Summative Evaluation of Student Learning, McGraw-Hill
Book Company, New York.
Djamarah, Syaiful Bahri, Drs. Strategi Belajar Mengajar. Jakarta: Rineka Cipta. 2002.
Gronlund, Norman E. (1985) Measurement and Evaluation in
Teaching, Fifth Edition, Macmillan Publishing Company New
York.
th

[4]
Guilford (1973). Fundamental Statistic in Psychology and

Education, Tokyo: Mc Graw-Hill Kogakusha.
[5] Herman Hudoyo. 1998. Belajar Mengajar Matematika. Jakarta:
Depdikbud P2LPTK.
[6] Hidayati, L., (2012), Aplikasi Berbasis Gui (Grafik User Interfaces) Untuk Simulasi Limit Fungsi, Jurnal, AVISENA,
Vol.4/No.2/ISSN2086-8960/Desember/2012 UNIZAR Mataram.
[7] Hidayati, L., (2013), Pengembangan Media Pembelajaran
Matematika Berbasis Gui (Grafik User Interfaces), Makalah
Seminar Nasional MIPA dan Pendidikan Matematika, UNESA,
2013.
[8] Ibrahim, (2012), Teori Dienis, Makalah tugas mata kuliah Psikologi Belajar Matematika, Jurusan Matematika UIN Sunan
Kalijaga Yogyakarta.
[9] Muslihati, (2012), Teori Belajar Permainan Dienes Dalam Pembelajaran matematika, Jurnal, STKIP PGRI Metro.
[10] Purwanto, Drs. Strategi Pembelajaran Matematika. Surakarta:
Sebelas Maret University Press. 2003.
[11] Sardiman, A.M. Interaksi dan Motivasi Belajar Mengajar. Cet.
IV; Jakarta: Rajawali Pers. 1992.
[12] Sri Wulandari Danoebroto. 2008. Meningkatkan Kemampuan
Pemecahan Masalah Melalui Pendekatan PMRI dan Pelatihan
Metakognitif. Jurnal Penelitian dan Evaluasi Pendidikan. Nomor 1 Tahun XI. 69 81.

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February 12-13rd 2014

The 4 Annual Basic Science International Conference

Batu, East Java, Indonesia | 1

February 12-13rd 2014

2 | Batu, East Java, Indonesia

The 4 Annual Basic Science International Conference

February 12-13rd 2014

The 4 Annual Basic Science International Conference

Resistance Evaluation of Large Seeded Soybean

Corresponding author: heru@litbang.deptan.go.id

Abstract Rust disease is a major disease on soybean,

tered Hawaii. Then it entered South Africa in 1920 and

Batu, East Java, Indonesia | 3

February 12-13rd 2014

The 4 Annual Basic Science International Conference

2/3 of middle part of the plant

1/3 of upper part of the plant

high ( > 16 pustul/cm2 )

Score 122, 123, 132, 133, 222. 223

Score 142, 143, 232, 233, 242, 243,

In addition to the intensity of rust disease, observation

contained, there was no resistant genotype, seven

4 | Batu, East Java, Indonesia

February 12-13rd 2014

The 4 Annual Basic Science International Conference

environmental factors affected the plant height

determined by the number of seeds [9], and the number

Number of filled pods per

Number of unfilled pods

Number of branches per

Grain yield per plant (g)

The number of pods determine grain yield because

moderately resistant. In this study, grain yield was not

Batu, East Java, Indonesia | 5

February 12-13rd 2014

The 4 Annual Basic Science International Conference

especially the number of pods and seed size. There is a

R. M. Miles, R. D. Frederick, and G. L. Hartman. 2003.

6 | Batu, East Java, Indonesia

C. Paul, C. B. Hill, and G. L. Hartman. 2011. Comparisons of

February 12-13rd 2014

The 4 Annual Basic Science International Conference

Leaf Characteristics of Some Trees Species in

Corresponding author: hafidhafauziah@gmail.com

primary sources of NOx pollutants are motor vehicle

Batu, East Java, Indonesia | 7

February 12-13rd 2014

The 4 Annual Basic Science International Conference

area of Padjadjaran University near the Jatinangor

8 | Batu, East Java, Indonesia

Mahagoni has the highest value of stomatal density

February 12-13rd 2014

The 4 Annual Basic Science International Conference

distributed to stems and roots[7].NO2 gas which entering

for sulfate uptake and transport in plants.

Correlation between accumulation of sulfate and

Fig. 1. Relation between sulfate accumulation with leaf area (r =

Fig. 2. Relation between nitrate accumulation with leaf area (r =

Batu, East Java, Indonesia | 9

February 12-13rd 2014

The 4 Annual Basic Science International Conference

The concentration of SO2 gas was not detected in the

Fig. 3. Relation between nitrate accumulation with the chlorophyll