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Cyphastrea chalcidicum photo by  M Moradi 

| Nus Biosci | vol. 2 | no. 1 | pp. 1‐53 | March 2010 | ISSN 2087‐3948 (PRINT) | ISSN 2087‐3956 (ELECTRONIC)

| Nus Biosci | vol. 2 | no. 1 | pp. 1‐53 | March 2010 |  ISSN 2087‐3948 (PRINT) | ISSN 2087‐3956 (ELECTRONIC)  I S E A   J o u r n a l   o f   B i o l o g i c a l   S c i e n c e s  


ISSN: 2087-3948 (printed edition), 2087-3956 (electronic edition)

EDITORIAL BOARD: Abdulaziz M. Assaeed (King Saud University, Riyadh, Saudi Arabia), Alfiono (Sebelas Maret University, Surakarta), Edwi Mahajoeno (Sebelas Maret University, Surakarta), Ehsan Kamrani (Hormozgan University, IR Iran), Eko Handayanto (Brawijaya University, Malang), Endang Sutariningsih (Gadjah Mada University, Yogyakarta), Faturochman (Gadjah Mada University, Yogyakarta), Iwan Yahya (Sebelas Maret University, Surakarta), Jamaluddin (R.D. University, Jabalpur, India), Lien A. Sutasurya (Bandung Institute of Technology, Bandung), Magdy Ibrahim El-Bana (Suez Canal University, Al-Arish, Egypt), Mahendra K. Rai (Amravati University, India), Marsetyawan H.N. Ekandaru (Gadjah Mada University, Yogyakarta), Oemar Sri Hartanto (Sebelas Maret University, Surakarta), R. Wasito (Gadjah Mada University, Yogyakarta), Rugayah (Indonesian Institute of Science, Cibinong-Bogor), Sameer A. Masoud (Philadelphia University, Amman, Jordan), Supriyadi (Balitbiogen, Bogor), Sri Margana (Gadjah Mada University, Yogyakarta), Suranto (Sebelas Maret University, Surakarta), Sutarno (Sebelas Maret University, Surakarta), Sutiman B. Sumitro (Brawijaya University, Malang), Taufikurrahman (Bandung Institut of Technology, Bandung), Wayan T. Artama (Gadjah Mada University, Yogyakarta)

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ISSN: 2087-3948 (print) ISSN: 2087-3956 (electronic)

Vol. 2, No. 1, Pp. 1-6 March 2010

Ripening for improving the quality of inoculated cheese Rhizopus oryzae SOLIKAH ANA ESTIKOMAH1,♥, SUTARNO², ARTINI PANGASTUTI² ¹ Bioscience Program, School of Graduates, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia ² Department of Biology. Faculty of Mathematics and Natural Sciences, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia Manuscript received: 17 November 2010. Revision accepted: 26 January 2010.

Abstract. Estikomah SA, Sutarno, Pangastuti A 2010. Ripening for improving the quality of inoculated cheese Rhizopus oryzae. Nusantara Bioscience 2: 1-6. Cheese is dairy product resulted from fermented milk in which the fermentation process can be done by lactic acid bacteria or fungus. Rhizopus oryzae is able to produce lactic acid, protease and lipase. The ripening process changes the taste and texture. The purpose of this study is ripening to improve the quality of inoculated cheese R. oryzae. In this research the ripening was conducted the concentration variation of temperature (5oC, 10oC, 15oC), and time (7 days, 14 days). The procedure of research consisted of two steps, namely un-ripened cheese preparation followed by ripening cheese preparation. Cheese produced in this study analyzed the value of pH, fat content, protein content, amino acid levels and identification of microbe with ANOVA then followed by DMRT at 5% level of significance. Data results were analyzed with the like’s nonparametric statistical test, followed by Fridman Wilcoxon Signed Rank Test (WSRT) at 5% level significance. The results showed that the preferred ripened cheese panelist was at a temperature of 15oC for 14 days. Ripening conditions affect pH, fat content, protein content and do not affect the levels of amino acids that formed ripened cheese. The best quality ripened cheese i.e. at a temperature of 15°C for 14 days, had a pH value of 4.40, the highest protein content of 9.78%, and fat content of 35.02%. The results of identified microbe in un-ripened cheese and ripened cheese include Enterococcus hirae (Enterococcus faecalis), Bacillus subtilis, and Aspergillus sp. Key words: cheese, fermentation, Rhizopus oryzae, ripening, temperature.

Abstrak. Estikomah SA, Sutarno, Pangastuti A. 2010. Pemeraman untuk meningkatkan kualitas keju yang diinokulasi Rhizopus oryzae. Nusantara Bioscience 2: 1-6. Keju merupakan makanan hasil fermentasi dari susu yang proses fermentasinya dilakukan oleh bakteri asam laktat maupun jamur. Rhizopus oryzae diketahui mampu menghasilkan asam laktat, protease, dan lipase. Perubahan cita rasa dan tekstur keju terjadi selama pemeraman keju. Tujuan penelitian ini adalah untuk meningkatkan kualitas keju yang diinokulasi R. oryzae melalui pemeraman. Pemeraman dilakukan dengan variasi waktu (7, 14 hari) dan suhu (5ºC, 10ºC, 15ºC). Penelitian ini terdiri dua tahap, yaitu pembuatan keju mentah diikuti pemeraman keju mentah tersebut. Keju penelitian dianalisis nilai pH, kadar lemak, kadar protein, kadar asam amino dan diidentifikasi mikrobanya. Data hasil penelitian dianalisis dengan uji sidik ragam (ANAVA), kemudian dilanjutkan dengan uji berjarak ganda Duncan (DMRT) pada taraf signifikansi 5%. Data hasil tingkat kesukaan dianalisis dengan statistik nonparametrik uji Fridman yang dilanjutkan dengan Wilcoxon Signed Rank Test (WSRT) pada taraf sigifikansi 5%. Hasil penelitian menunjukkan bahwa keju peram yang disukai panelis adalah keju peram pada suhu 15ºC selama 14 hari. Kondisi pemeraman berpengaruh terhadap nilai pH, kadar lemak, kadar protein dan tidak berpengaruh pada kadar asam amino. Kualitas keju peram terbaik terdapat pada kondisi suhu 15°C selama 14 hari, memiliki nilai pH 4,40, kadar protein tertinggi yaitu sebesar 9,78%, dan kadar lemak sebesar 35,02%. Hasil identifikasi mikroba pada keju mentah dan keju peram meliputi Enterococcus hirae (Enterococcus faecalis), Bacillus subtilis, dan Aspergillus sp. Kata kunci: keju, fermentasi, Rhizopus oryzae, pemeraman, suhu.

INTRODUCTION Milk is a food that consists of various nutrients in balanced proportions. Its main constituent is water, protein, fat, lactose, minerals, and vitamins. Milk is yielded from livestock such as cattle, buffalo, and goats. Milk production from dairy farmers is distributed to the milk factories and processed by them into a liquid ready to drink milk. Milk produced by breeders can only be sold to a cooperative economic enterprise or factory and processed into a ready to drink milk. There are some basic problems bear down upon dairy farmers, they are low resistance on the milk or

easily damaged, the bargaining position of farmers against low milk prices and lack of absorptive capacity of milk production by the manufacturer/cooperatives as well as poor knowledge of dairy farmers. On the other hand breeders always wanted the milk that is produced can be used completely without any damage or wasted, so we need some milk processing which is aimed to preserve milk for much longer when stored. Cheese is a dairy product (Daulay 1991). The fungus Rhizopus oryzae is able to produce lactic acid (Purwoko and Pamudyanti 2004). R. oryzae also has protease enzyme which has similar characteristics as rennet


2 (1): 1-6, March 2010

(Hadiwiyoto 1983). Lactic acid will help preserve the milk, while the protease functions to wad milk casein. Besides lactic acid and protease, R. oryzae is capable to produce lipase that functions as a solver of fat that will enhance the taste of cheese. In a manufacturing of cheese, ripening is one of the important stages. Cheese product which has a ripening can change a young cheese slowly into a mature cheese. The ripening process changes the taste and texture. The changes are caused by protein breakdown into simpler peptides and amino acids, fats into fatty acid solution and volatile acids such as acetic and propionic acid, lactose fermentation, citrate, and other organic materials into acids, esters, alcohol, taste, and diacetyl other components. The process making a ripened cheese involves acidification and ripening. Acidification of milk is done by adding acid or inoculation of microbes. Direct acidification of milk by adding acid is less suitable for ripened cheese making process because during the ripening process there is no real change of proteins (proteolysis), fat (lipolysis), and lactose, whereas acidification using inoculums can cause biochemical changes, including proteolysis, lipolysis and lactose fermentation. Biochemical changes can affect flavor and texture (Septiana 1994). Ripening of cheese is done by storing the cheese for some time and at a certain temperature. The longer the ripening, the stronger the flavor of cheese is formed. In cheese ripening, maturing temperature affects the speed of proteolytic activity and acid production. High temperatures will accelerate the process, but not profitable. At high temperatures, acid production is quicker, causing a strong sour taste and accelerating evaporation so that more water loss and decay more quickly. At low temperatures a proper balance of acid production and proteolytic activity occur and water evaporation is inhibited (Daulay 1991). This research is about the cheese which was inoculated by R. oryzae by using variations of temperature and time of ripening. Cheeses are brooded in this study analyzed the value of pH, fat content, protein and amino acid contents, microbial identification and preference test.

MATERIALS AND METHODS Materials research The main material used is cow's milk from dairy cows in Boyolali District, Central Java, and Rhizopus oryzae propagated by the Faculty of Agriculture Sebelas Maret University, Surakarta. Procedures Preparation of culture media Media manufacturing process begins with mixing the ingredients PDA (Potato Dextrose Agar), which is a medium for the growth of R. oryzae. Distilled water are then inserted into the Erlenmeyer flask, then heated on hot plate and homogenized with a magnetic stirrer. Once the mixture boils, PDA media was poured into a test tube and proceed with the process of sterilization using the autoclave

at a temperature 121oC at 1 atm pressure for 30 minutes, then test tube placed in a tilted position in order to form slanted media. The working culture of R. oryzae are ready to be used for the manufacture of starter. Work culture is obtained with culture rejuvenate R. oryzae by inoculating a pure culture into the PDA side, then incubating at 37ТАC for 34 days, while the rest is stored at a temperature 4ТАC as a stock culture and rejuvenated every 6 months (Wijaya 2002; Suharyanto et al. 2006). Starter made by inoculating 250 mL fresh milk (skim milk liquid) with R. oryzae from PDA at the age of 3-4 days, propagation of R. oryzae taken as many as 50 cells/mL (3 ose) and incubated at 37 ° C for one day (Nurhidayati 2003). Making cheese Fresh cow's milk, as much as 3600 mL pasteurized up to 70°C for 30 seconds. Once pasteurized, the milk was cooled until the temperature reaches 37°C and then inserted into the 8 piece glass beaker with a volume of 200 mL. Bottles filled with milk that has been inoculated are then incubated in an incubator at a 37°C for 9 hours. During the incubation bottles were covered with aluminum foil. The part that was clotted called curd while the liquid is called whey (Ward 1996). After that the milk was heated for 30 minutes at a 40°C, then cooled for 1 hour, stirred every 5 minutes (Hadiwiyoto 1983), and then filtered with clean gauze. Filtering is done to separate curd and whey. Formed curd is taken while the whey is removed (Legowo 2003). Curd wrapped in clean gauze continued pressing to give compactness and shape of the cheese, and remove the remains of whole whey (Hadiwiyoto 1983). Formed curd was salted as much as 4%. Salt is sprinkled in the form of fine crystal, then stir until completely blended. The salted curd is then wrapped with aluminum foil and matured for 0 day (without ripening), 7 days, and 14 days, with temperature of curing 5°C, 10°C, and 15°C. Microbiological test Microbiological test involved the calculation of total microbes and microbial identification. Calculation of total microbial cheese made by weighing 25 g and then homogenized with 225 mL of distilled water (Rosa et al. 2003: Ceylan et al. 2003; Mennane et al. 2007). Calculation of total microorganisms was done on the basis of the Standard Plate Count. Fertilization is done with medium Plate Count Agar (PCA) by dropping 1 mL of inoculation into sterile petri PCA and the subsequent media that has been cold poured into sterile petri saucer as much as 12-15 mL, the mixture is homogenized with a petri saucer by moving it to form a figure eight direction. Having to harden, Petri saucer was incubated upside down at 37°C for 24-48 hours. Then the colonies formed are counted. Identification was done by isolating colonies of microbes then growing it on PDA media for mold and on MRSA media for bacteria. Identification of mold was based on its morphologic characteristics. Identification of bacteria was using the BD Phoenix TM.

ESTIKOMAH et al. – Cheese ripening by Rhizopus oryzae

Lipid analysis Soxhlet fat analysis method is as follows: Samples of 3 g was taken and then inserted into the timbel. Put the flask which has already been cleaned into the oven, then add the boiling stone and weighed as empty weight. Timbel is inserted into the soxhlet, then connected with soxhlet fat flask, and then add a liquid fat solvent of 150 mL of ether through the soxhlet. Flask fat and soxhlet are connected with bath extracted for 6 hours. After the extract is complete, flask fat is evaporated to remove solvent. Flask fat is put into the oven at 105°C for 1 hour. After it is cold, it is weighed as final weight (weight and fat flask). The sample calculation formula is: Lipid content = c-bx100% a a = weight of sample b = weight of fat and boiling flask c = weight of fat flasks, stone boiling and fat Protein content Protein content is analyzed by Lowry-Folin method by spectrophotometer (Sudarmadji et al. 1984). Measurement begins with the manufacture of standard solution of BSA (Bovine Serum Albumin). Dilution series was made from standard solutions with respective concentrations of 0.00, 0.06, 0.18, 0.24, and 0, 30 (mg/mL H2O) and inserted into each test tube. 1 ml solution D is added into the test tube and then is whirled for 5 minutes. After that, the addition of reagents E of 3 mL and then allowed to stand for 10 minutes. OD measurements performed at a wavelength of 560 nm using spectrophotometer. The next steps was taking 1g of cheese sample and dissolve it in 100 mL of distilled water and then stirring with a magnetic stirrer, the solution was filtered and added 100 mL of distilled water. 1 mL sample solution was taken and then inserted into a test tube and then added by 1 mL of Lowry reagent D, whirled with vortex for 5 minutes. Next reagent Lowry E as much as 3 mL added into test tubes and whirled with vortex and then incubated at room temperature for 45 minutes. OD measurement at a wavelength of 590 nm was using a spectrophotometer. Sample calculation formula is: % protein = axbx100% c a = concentration b = dilution factor c = a lot of sample (g)

Amino acid content of cheese Amino acid content of cheese was analyzed by HPLC (High Performance Liquid Chromatography). Cheese samples in which amino acid content will be analyzed was prepared in advance, by taking 5 g of cheese samples that has been ground smoothly into the Erlenmeyer covered with grindstones, homogenized using a magnetic stirrer and hydrolyzed at a temperature of 110 ° C for 12 hours,


filtered using Whatman filter paper 41, and the pH was adjusted to normal (pH 7). 100 ml of distilled water is added, take 3 mL of that solution and filter with millex 0, 45 μm. For injection into the HPLC, take 10 μl of milex solution + 990 mL of OPA and whirled in a vortex. Put it into reaction for 3 minutes, and the inject it into the HPLC. Preparation of standard solution. Standard stock consists of L-threonin = 1050 ppm; L-methionine = 1000 ppm; L-valine = 1010 ppm; L-thriptophan = 1010, LPhenilalanine = 1000 ppm; L-isoleusine = 1060, L-Leucine = 1010 ppm ; L-lycine = 1000 ppm, each drawn by comparison 1:1:1:1:1:1:1:1 into 10 mL + 990 mL OPA diijeksi to HPLC. Amino acid cheese detected by HPLC with a set of HPLC equipment. The prepared sample was taken as much as 20 mL using the injector. Amino acids was detected by a set of tools Eurospher 100-5 C18 HPLC column, 250x4.5 mm with pre-column P/N: l115Y535. Eluent: A = 0:01 M acetate buffer pH 5.9, B = (MeOH: 0:01 M acetate buffer pH 5.9). Organoleptic test Organoleptic test which is conducted is a test of preference. This preference test assesses the level of color, flavor, aroma, and texture of cheese. The assessment was conducted by 20 untrained panelists. This test refers to Zulaekah and Widiyaningsih (2005). A five-level scale was stated (level 1-5), start from 1 (strongly dislike), 2 (not like), 3 (somewhat like), 4 (like), and 5 (very like). Data analysis Data obtained from analysis which consists of the pH value, fat content, and protein content and total microbial was analyzed by analysis of variance (Anova) to determine whether there is any treatment effect followed by a test of Duncan's Multiple Range Test (DMRT) at the significance level of 5% to know the real difference among the treatments. Data favorite level test results were analyzed descriptively with Friedman nonparametric statistical tests (Friedman test) followed by Wilcoxon Signed Rank Test (WSRT) at 5% significance level.

RESULTS AND DISCUSSION The degree of acidity (pH) The pH is a measure of the value of dissociated hydrogen ions in solution, thus aiming to find out the pH measurements of cheese acidity caused by the presence of hydrogen ions. PH value of ripened cheese which was inoculated with R. oryzae can be seen in Table 1. According to De Souza et al. (2003), pH levels decreased during the ripening process. Decrease in pH of cheese is influenced by the amount of lactic acid produced by microorganisms, the higher the lactic acid then the pH was lower. The decrease in pH value is caused by the activity of bacteria in this cheese. BAL is in the cheese (Basillus subtilis and Enterococcus hirae) are able to produce lactic acid from sugar that will be needed in forming taste, preventing the growth of pathogenic bacteria, and the safety of the final product (Kayagil 2006). Lactic acid is


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the result of glucose metabolism. Increased lactic acid is characterized by a decrease in pH. Increased lactic acid caused by H + ions that occur because of decomposition of lactose produces acids that are easily evaporated, and the outbreak of organic phosphate contained in casein, resulting in acid (Mc Kay et al. 1971). Table 1. The pH, fat content (%), protein content (%), ripened cheese inoculated with R. oryzae Ripening period


Temperature 10°C


pH 7 days 14 days

5,44 bc 5,14 bc

5,09 b 4,88 ab

4,87 ab 4,40 a

Fat content (%) 7 days 14 days

34,56 ab 32,43a

34,48ab 33,31ab

35,30ab 35,02ab

Protein content (%) 7 days 6.28 d 7.56 c 8,34 b c c 14 days 7.22 7.60 9,78 a Note: Figures with different letters in the same column indicate significant differences (P <0.05) in Duncan's multiple range test.

Lipid content Fats are a source of some components giving flavor, aroma, and texture of cheese. Fusion of fat in the cheese occurs due to trapped fat globules at the time of the protein wadding progress (Daulay 1991). Data results of calculating the value of fat content in cheese inoculated with R. oryzae can be seen in Table 1. The results of this study indicate that the lipid content will be decreased in a longer ripening process. The results are consistent with the results of research conducted by Kayagil (2006) decreasing levels of fat in the cheese due to ripening process occurs because the degradation of fat with the help of lipase. In the process of fat degradation, fatty acid is formed. There are the volatile fatty acids and non-volatile fatty acid. According Prawisuma (2007) during the ripening process, fat is hydrolyzed into various volatile fatty acids. Volatile fatty acids are fatty acids which are easily evaporated. These easily evaporated fatty acids causes levels of fat in cheese reduced. Reduced levels of fat besides caused by the formation of volatile acid, it is also caused by the using of some fats as a source of energy in metabolism activity. Fat is used as an energy source through the renovation process initiated by the hydrolysis of triglycerides into glycerol and fatty acids with the assistance of lipase. Protein content Proteins in milk are composed by whey and casein, whereas the remaining proteins in the cheese is casein because the formed whey has been released in the process of cheese formation (Murwaningsih 2003). The value protein content in ripened cheese inoculated with R. oryzae by the treatment of ripening duration and temperature variations can be seen in Table 1. High protein levels of ripened cheese at a temperature of 15°C for 14 days are in

accordance with the results of research by Licitra et al. (2000) which showed an increase in protein content during ripening of 0 to 12 months. At 0 months of ripening protein content is 25.30% and then increased at 29.24% after 12 months of ripening. Compared with controls that have a protein content of 2.23%, the ripened cheese in this study had higher protein content. Increased levels of protein in cheese is due to the opportunity given for microbes (E. hirae, Bacillus subtilis, Aspergillus sp.), and the enzymes in the cheese curd to hydrolyze proteins during the ripening process. Protein breakdown during ripening will result in a high protein, more flexible and soft cheese structure, and aromatic taste, because the rigid proteins and insoluble nitrogen is converted into soluble form (Daulay 1991). Essential amino acid content of ripened cheese Amino acids are homologous series of compounds containing two functional groups i.e. amino groups and carboxylate groups which are attached to the same carbon atom. Essential amino acid analysis results that were inoculated with starter R. oryzae can be seen in Table 2. During ripening process the highest levels of amino acids is in the treatment of 7 days. High level of amino acid in the 7-days treatment of ripened cheese is caused by proteolysis occurs on the cheese. Meanwhile, on 14-day treatment of ripened cheese, the content of amino acid decline due to the occurrence of amino acid catabolism. Amino acids are precursory to the various flavor components in cheese (Urbach 1997; Engles et al. 1997). Catabolism of amino acids produces a number of aroma components found in the cheese. Mechanism of amino acid catabolism includes oxidation deamination, decarboxylation, transminase, and reduction reactions that would form the aldehyde, alcohol, indole, acid, phenolic and sulfur (Hansen et al. 2001; Williams et al. 2001). Table 2. Amino acid levels in cheese inoculated with R. oryzae. Essential amino acid compound L-Threonine

Essential amino acid contents (%) 0 day 7 day 1.15 1.68

Ripened cheese 14 day 1.58





L-Valine + L-Thriptophan
























Test of cheese preferences This test is conducted to know consumer preference level of cheese produced include predilection of texture, flavor, color, and flavor. The results of statistical analysis are shown in Table 3. From Table 3, it is known that the taste of ripened cheese on 15°C for 14 days has the most preferred taste over the other. Cheese in the ripening process of 5°C for 7 days has a taste of the least preferred.

ESTIKOMAH et al. – Cheese ripening by Rhizopus oryzae


Table 3. Test scores upon predilection of flavor, aroma, color and texture in ripened cheese.

Table 4. Microorganisms found during the ripening time of 7 and 14 days at 15oC on MRSA and PDA media.

Ripening duration

Type of media (cfu/mL) PDA

Flavor a

Aroma a

Color a

Texture a

5°C during 7 days 3.40 3.47 4.70 4.25 5°C during 14 days 3.75 a 4.43a 3.40 a 3.75 a 3.95a 4.22 a 4.60 a 10°C during 7 days 4.45 a a a a 10°C during 14 days 4.03 4.45 4.00 4.13 a a a a 3.83 3.85 3.17 a 15°C during 7 days 4.22 15°C during 14 days 4.50 a 4.42a 3.45 a 4.20 a a a a 3.65 4.38 3.90 a Controls 3.92 Note: The larger the value, then the ripened cheese is increasingly preferred. Same superscript indicates no significant difference in fridman test 5%. 1 = extremely dislike, 2 = dislike, 3 = somewhat like; 4 = like, 5 = very like it.

The aroma of cheese appears mainly due to the volatile formed during ripening. Results of non-parametric analysis shows the most preferred aroma on ripening of 10oC for 14 days, while the least preferred flavor is in 5oC for 7 days which is ripened at the lowest temperatures, according to Daulay (1991) Low temperature inhibits the biochemical processes that lead to process inhibited the formation of aroma. When compared with control ripened cheese preferably, in this study the control used is the cheese without ripening (unripened), which is a type of fresh cheese where the aroma has not been formed and is still dominated by the aroma of milk (Murwaningsih 2003). A non-parametric analysis result indicates a preferred color in ripened cheese 5oC for 7 days because this cheese has a more yellow color than others. According to Buckle (1987) cheese made from cow's milk without the dye will produce a yellow-white cheese. The color of cheese is influenced by fat content in cheese. Fat in cheese obtained with the help of the enzyme lipase, which can hydrolyze triglycerides into glycerol and fatty acids. The yellow color comes from carotene pigments which are fat soluble, so that more levels of fat in the cheese, the cheese color becomes more yellow, because the more soluble pigment carotene. Non-parametric analysis results show that the assessment score given by panelists on the texture of the most preferred, namely 10oC treatment for 7 days while the texture was the least preferred on 15oC treatment for 7 days. Microbiology identification Results of identification of microorganisms found in the control cheese (without ripening cheese), ripened cheese for 7 days, and ripened cheese for 14 days are shown in Table 4. In the control, 7 days ripening time and 14 days ripening time, there are 3 types of microbes which are the same, namely E. hirae, B. subtilis, Aspergillus sp. The number of mold progressively increased, while the number of bacteria become more and more declined (Table 4). This is because B. subtilis and E. hirae is thermoduric bacteria that have optimal temperature at 30-45oC while Aspergillus grows at an optimal temperature at 29-32oC so that ripening treatment at a temperature of 15oC causes Aspergillus is more able to survive than in B. subtilis and E. hirae.


Control Aspergillus sp. (1.2x104) E. hirae and B. subtilis (3.8x104)

7 days Aspergillus sp. (1.1x104) E. hirae and B. subtilis (3.3x104)

14 days Aspergillus sp. (2.8x104) E. hirae and B. subtilis (3.2x104)

The total number of microbial colonies of ripened cheese inoculated with R. oryzae The calculation of the total number of microbes in this study was conducted using SPC (Standard Plate Count) on PCA medium (Plate Count Agar) performed by dilution. The total numbers of microbes that participate in cheese ripening are shown in Table 5. The longer ripening time caused the number of microorganisms that grow less (Table 5). According to Amos (2007) microbes in cheese will grow rapidly in milk and curd during cheese making, then declines during ripening, due to a decline in pH during ripening, reduced lactose and high salt concentration. Table 5. The number of microbes (x 104) on media Total Plate Count (TPC) Ripening time 7 days 14 days

5°C 10,99x104 ab 10,12x104 a

Temperature 10°C 11, 95x104 ab 11,14x104 ab

15°C 12,99x104 b 11,30x104 ab

Note: Figures with different letters in the same column indicate significant differences (P <0.05) in Duncan's multiple range test.

The pH value of cheese in this study is ranged from 4 to 5.44 (Table 1). From Table 1, it is noted that the longer ripening time led to a lower pH value. Low acidity levels causing microbes within the cheese die due to not acid resistant (Daulay 1991). Compared with controls, which is without ripening cheese that has a large number of microbes, ripened cheese has a little amount of microbes, caused by the pH in the ripened cheese is lower (4 to 5.44) than the pH of unripened cheese (5.5) which resulted in microbes in the cheese die due to not acid resistant.

CONCLUSIONS AND SUGGESTIONS The use of long ripening variation affects the amount of microbes, pH value, fat content and protein content. The quality of the best cheese at a temperature of 15°C at 14 days ripening time, has a pH value of 4.40, the highest protein content of 9.78%, fat content of 35.02% and produces a sense of well-liked by the panelists. Identification of Bacillus subtilis using BD PhoenixTM has only 90% confidence level so it is expected that the next study will use molecular analysis to get more exact



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results. The further research is expected to have more additional secondary starter of ripened cheese making. Further research is expected to use the ripening temperature of 15°C with a time between 7 to 14 days.

REFERENCES Amos LM. 2007. Enzimes from yeast adjuncts in proteolysis during Cheddar cheese ripening. [Dissertation]. University of the Free State, Bloemfontein, South Africa. Buckle KA. 1987. Food science. UI Press. Jakarta. [Indonesia] Ceylan Z, Turgoklu H, Dayisoylu KS. 2003. The microbiological and chemical quality of sikma cheese produced in Turkey. Pakistan J Nutr 2 (2): 95-97. Daulay D. 1991. Fermentation of cheese. Bogor Agricultural University. Bogor. [Indonesia] De Souza CFV, Rosa TD, Ayub MAZ. 2003. Change in the microbiological and physicochemical of Serrano chese during manufacture and ripening. Braz J Microbiol 34 (3): 260-266. Engels WJM, Dekker R, de Jong C, Visser S. 1997. A comparative study of volatile compounds in the water-soluble fraction of various types of ripened cheese. Intl Dairy J 7: 255-263. Hadiwiyoto S. 1983. Dairy products, fish, meat, and eggs. Liberty. Yogyakarta. [Indonesia] Hansen TK, Tempel TVD, Cantor MD, Jacobsen M. 2001. Saccharomyces cerevisiae as starter culture in Mycella. Int J Food Microbiol 69: 101-111. Kayagil F. 2006. Effect of traditional starter cultures on quality of cheese. [Tesis]. Department of Biotechnology, Middle East Technical University. Ankara. Legowo A, Nurwantoro, Albaari AN. 2003. Levels of protein, fat, pH value and hedonic quality of cottage cheese with raw material of goat milk and skim milk. National Seminar on Animal Husbandry and Veterinary Technology. RD Center for Animal Husbandry, Bogor. 29-30 September 2003. [Indonesia]

Mc Kayr LL, Sandine WE, Elliker PR. 1971. Lactose utilization by lactic acid and bacteria. J Dairy Sci 37: 493. Mennaner Z, Faid M, Lagzouli M. 2007. Physico-chemical, microbial and sensory characterisation of Moroccan klila. Middle-East J Sci Res 2 (3-4): 93-97. Murwaningsih J. 2003. Chemical quality of Frisian Holstein (FH) dairy cows and cottage cheese produced in different genotypes of kappa casein. [S1 Thesis]. Bogor Agricultural University. Bogor. [Indonesia] Nurhidayati T. 2003. Effect of papain enzyme concentration and temperature of fermentation on the quality of Cottage cheese. Kappa 4 (1): 13-17. Prawisuma A. 2007. Profile of short-chain fatty acids, total solid material, and the smell of cheese from goat's milk with different ripening time. [S1 Thesis]. Bogor Agricultural University. Bogor [Indonesia] Purwoko T, Pramudyanti IR. 2004. Effect of CaCO3 on the fermentation of lactic acid by Rhizopus oryzae. J Mikrobiol Indon 9: 19-22. [Indonesia] Septiana AT. 1994. Study of the inoculated cheese ripening Lactobacillus bulgaris and Streptococcus thermophilus. [Thesis]. Gadjah Mada University. Yogyakarta. [Indonesia] Sudarmadji S, Haryono, B Suhadi. 1984. Analysis of food and agriculture. 2 nd ed. Penerbit Alumni. Bandung. [Indonesia] Urbach G. 1997. The flavour of milk and dairy product. II. cheese: contribution of volatile compound. Intl J Dairy Technol 50:79-89. Wardhani B. 1996. Studying the use of some types of rennet in the manufacture of Cottage cheese. [S1 Thesis]. Bogor Agricultural University. Bogor. [Indonesia] Wijaya S. 2002. Isolation of chitinase from Scleroderma columnare and Trichoderma harzianum. J Ilmu Dasar 3 (1): 30-35. [Indonesia] Williams AG, Noble J, Banks JM. 2001. Catabolism of amino acids by lactic acid bacteria isolated from cheddar cheese. Intl Dairy J 11:203215. Zulaekah S, Widiyaningsih EN. 2005. Effect of tea leaf extract concentration on the manufacture of hard-boiled eggs to the number of bacteria and the receipt. Penelitian Sains & Teknologi 6 (1): 1-13. [Indonesia]

ISSN: 2087-3948 (print) ISSN: 2087-3956 (electronic)

Vol. 2, No. 1, Pp. 7-13 March 2010

Comparasion of iles-iles and cassava tubers as a Saccharomyces cerevisiae substrate fermentation for bioethanol production KUSMIYATI♥ Study Center for Alternative Energy, Department of Chemical Engineering, Faculty of Engineering, University of Muhammadiyah Surakarta. Jl. Ahmad Yani Tromol Pos 1 Pabelan, Kartosuro, Sukoharjo 57102, Central Java, Indonesia. Tel./Fa: 271-717417 ext 442/ 271-715448. ♥email: Manuscript received: 8 February 2010. Revision accepted: 16 March 2010.

ABSTRACT. Kusmiyati (2010) Comparasion of iles-iles and cassava tubers as a Saccharomyces cerevisiae substrate fermentation for bioethanol production. Nusantara Bioscience 2: 7-13. The production of bioethanol increase rapidly because it is renewable energy that can be used to solve energy crisis caused by the depleting of fossil oil. The large scale production bioethanol in industry generally use feedstock such as sugarcane, corn, and cassava that are also required as food resouces. Therefore, many studies on the bioethanol process concerned with the use raw materials that were not competing with food supply. One of the alternative feedstock able to utilize for bioethanol production is the starchy material that available locally namely iles-iles (Amorphophallus mueller Blum). The contain of carbohydrate in the iles-iles tubers is around 71.12 % which is slightly lower as compared to cassava tuber (83,47%). The effect of various starting material, starch concentration, pH, fermentation time were studied. The conversion of starchy material to ethanol have three steps, liquefaction and saccharification were conducted using α-amylase and amyloglucosidase then fermentation by yeast S.cerevisiaie. The highest bioethanol was obtained at following variables starch:water ratio=1:4 ;liquefaction with 0.40 mL α-amylase (4h); saccharification with 0.40 mL amyloglucosidase (40h); fermentation with 10 mL S.cerevisiae (72h) producing bioethanol 69,81 g/L from cassava while 53,49 g/L from iles-iles tuber. At the optimum condition, total sugar produced was 33,431 g/L from cassava while 16,175 g/L from iles-iles tuber. The effect of pH revealed that the best ethanol produced was obtained at pH 5.5 during fermentation occurred for both cassava and iles-iles tubers. From the results studied shows that iles-iles tuber is promising feedstock because it is producing bioethanol almost similarly compared to cassava. Key words: alternative energy, cassava, iles-iles, bioethanol.

ABSTRAK. Kusmiyati (2010) Perbandingan umbi iles-iles dan singkong sebagai substrat fermentasi Saccharomyces cerevisiae dalam produksi bioetanol. Nusantara Bioscience 2: 7-13. Produksi bioetanol meningkat dengan cepat karena merupakan energi terbarukan untuk mengatasi krisis energi yang disebabkan oleh habisnya minyak fosil. Produksi bioetanol skala besar di industri umumnya menggunakan bahan baku seperti tebu, jagung, dan ubi kayu yang juga diperlukan sebagai sumber makanan. Oleh karena itu, banyak studi pada proses bioetanol terkait dengan penggunaan bahan baku yang tidak bersaing dengan pasokan makanan. Salah satu alternatif bahan baku dapat dimanfaatkan untuk produksi bioetanol adalah bahan berpati yang tersedia secara lokal yaitu iles-iles (Amorphophallus mueller Blum). Kandungan karbohidrat umbi iles-iles sekitar 71,12% yang sedikit lebih rendah dibandingkan dengan umbi singkong (83,47%). Pengaruh berbagai bahan awal, konsentrasi pati, pH, waktu fermentasi dipelajari. Konversi dari bahan berpati menjadi etanol memiliki tiga langkah, pencairan dan sakarifikasi dilakukan dengan α-amilase dan amyloglucosidase kemudian difermentasi dengan ragi S.cerevisiaie. Bioetanol tertinggi diperoleh pada variabel berikut rasio pati: air = 1:4; likuifaksi dengan 0,40 mL α-amilase (4h); sakarifikasi dengan amyloglucosidase 0,40 mL (40h); fermentasi dengan 10 mL S.cerevisiae (72h) memproduksi bioetanol 69,81 g/L dari singkong sementara 53,49 g/L dari umbi iles-iles. Pada kondisi optimum, gula total dihasilkan 33.431 g/L dari ubi kayu sementara 16.175 g/L dari umbi iles-iles. Pengaruh pH menunjukkan bahwa etanol yang dihasilkan terbaik diperoleh pada pH fermentasi 5,5 baik untuk ubi kayu maupun umbi iles-iles. Hasil studi menunjukkan bahwa umbi iles-iles menjanjikan sebagai bahan baku bioetanol karena menghasilkan bioetanol hampir sama dengan ubi kayu. Key words: singkong, iles-iles, etanol, energi alternatif.

INTRODUCTION Ethanol is an alternative fuel that is important in reducing the negative impacts of fossil fuel consumption (Cardona and Sanchez 2007). Fossil fuel consumption in the world reached 80%(Gozan et al. 2007). Fuel demand in Indonesia reached 5.6% per year, this has caused Indonesia to be the only OPEC member country that has to import crude oil by 487 thousand barrels/day since the end of

2004. Therefore, the use of biofuel such as bioethanol is one of the alternatives to overcome fuel crisis. Bioethanol is colorless liquid and environmentally friendly which results in the form of combustion gases, and air pollutants such as CO Nx are very small. Many researchers concluded that ethanol does not cause the greenhouse effect of fossil fuels because hazardous gases such as CO2 which is reduced by 22% (Milan 2005). Bioethanol can be used to substitute premium and kerosene. According to Kusmiyati


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processed into cassava flour, tapai, tiwul and others, while the cassava starch is used as raw materials of crackers, meatballs and pempek, and this shows that cassava has an economic value. The use of cassava for bioethanol production could affect food supply, therefore it is necessary to find the diversification of raw materials such as tubers iles-iles (Amorphophallus muelleri Blume). Iles-iles grows wild in Sumatra, Java, Flores and Timor (Jansen et al. 1996), as well as Bali and Lombok (Kurniawan et al. 2010, in press). Iles-iles belongs to the Araceae monocot plant family with compound flower of “cob" type which is covered by its leaves (spatha) (Jansen et al. 1996), it has dark brown tubers with rough rash that contain relatively high carbohydrate i.e. 7085% (Department of Agriculture 2009; Kusmiyati 2009). Iles-iles has the highest glucomannan among any other types of Amorphophallus in Indonesia, which ranged between 4446% (Sumarwoto 2004). Glucomannan is a polysaccharide Figure 1. a. 4-month-old cassava plants, b. 5 months old iles-iles plants, c. Cassava consisting of monomer β-1, 4 αtubers after harvest, d. Iles-iles tuber that have been harvested mannose and α-glucose (Widyotomo et al. 2000). The availability iles-iles in (2007), the use of bioethanol in Batik Industry has a higher Central Java is relatively abundant where forest land which efficiency than kerosene because the flame is stable, not is used reaches 640,000 hectares, while the productivity too high and not easily to handle and use. It shows that the level is 30-40 tons/ha (Department of Agriculture 2009). 60% bioethanol has better characterization and better However, this abundant amount of iles-iles has not been efficiency compared to other ethanol in the concentration highly used; therefore iles-iles does not have high below 40% and above 90%). economic value. Because the nature of the sap causes itch, Bioethanol can be produced from raw materials iles-iles tubers are not used as a food ingredient. According containing sugar, starch or cellulose. One type of natural to Imelda et al. (2008) iles-iles is easy to be cultivated, resource which has potential to make bioethanol is the either generative by using its seed or vegetative by using its tubers. Tubers are agricultural products which contain bulbs, bulbils and leaf cuttings. Iles-iles naturally grows as carbohydrates or starch, and it is known that bioethanol can secondary vegetation, on the outskirts of teak forests at an be made from raw materials containing carbohydrates. One altitude of 700-900m above sea level, with rainfall level at kind of bulbs which is often used to make bioethanol is 1000-1500 mm (Sumarwoto and Widodo 2008) cassava. Cassava is an agricultural commodity grown in Iles-iles tuber is less fully utilized by society; it will be Indonesia and is the second highest source of carbohydrates very beneficial when it is used as raw material for after rice, the carbohydrate of which is 98.4% (Osunsami et bioethanol production. This study aimed to compare the al. 1988). Cassava can grow at of 2,000 m above sea level cassava tubers and iles-iles tubers as raw material for or in sub-tropical temperature of 16 º C. These plants bioethanol production and to study the effect of variable flowers will bloom and produce bulbs properly when concentrations of substrate and pH on levels of ethanol growing at altitude of 800 m above sea level, while at produced. altitude of 300 m above sea level cassava can not bloom the flower, but they can only produce tubers. In 2005 cassava crop reached 19.5 tons with total area of 1.24 MATERIALS AND METHODS million hectares (Prihandana et al. 2007). Cassava can be harvested at the age of 9-12 months when the lower leaves growth begins to decrease. The color of the leaves begin to Materials and equipment This research materials are the cassava tubers found in turn yellow and fall off a lot ( traditional markets, while iles-iles tubers obtained from the 2000). The product of cassava tubers are used to be

KUSMIYATI – Comparison of iles-iles and cassava tubers as raw material for bioethanol

garden as a secondary plant in Wonogiri. The enzyme αamylase, β-glukoamylase obtained from Daniso (Genericor, USA). Saccharomyces obtained from the Biology laboratory. Sample preparation Iles-iles tubers are peeled, washed, and cut into small pieces and dried in the sun up to 3 days so that maximum water content is 10. After that the tubers is mashed and sifted (approximately 40 meshes) so that the obtained particle size more uniform. The iles-iles flour are stored in a dry place and used in a long time. The same process is also carried on the manufacture of cassava tuber flour. The process of iles iles tubers flour making can be seen in Figure 2. Stock culture of S. cerevisiae Pure culture of S. cerevisiae is breed on for oblique (PGY medium) which has been sterilized before at a temperature of 1210C and at pressure of 1 atm for 15 minutes. PGY medium (Peptone Yeast glucose) made by mixing 0.3 g of yeast extract, 0.3 g pantone, 0.4 g of malt and 20 g of agar agar which is dissolved in 300 auades. Stock cultures were incubated for 2-3 days at 28° C. The process of enzyme production Stock cultures of S. cerevisiae 200 is inoculated into liquid medium containing 5 g (NH) 2HPO4, 5 g KH2PO4, 1 g MgSO4.7H2O, 1 g of yeast extract. After that it is incubated with a rotary shaker at 150 rpm speed and at 300C for 24 hours. The same process for breeding preculture is done to breed the main culture; only with more liquid medium as much as 500 m. Enzyme that is formed by this process is used in the fermentation process. The process of bioethanol production The initial stage is liquification. First, dissolve 1 kg of cassava flour in to water with a ratio (1:3,5, 1:4, 1:4,5, 1:5), and then add α-amylase enzyme of 0:48 mL/kg . This process is carried out by stirring the bulb flour at the speed of 250 rpm for 4 hours until it becomes mush at a temperature of 100 ° C. The process is followed by the hydrolysis process by using the enzyme β-glukoamylase with 0:48 concentration/kg, pH 4 for 40 hours. Glucose that is produced in the process of hydrolysis is analyzed with Nelson-Somogy method (Sudarmaji et al. 1984). The same process applies for manufacturing bioethanol from iles-iles tubers. Glucose that has been generated from the


saccharification then is fermented by using the yeast S with a concentration of 10% (v/v) then DAP, urea and NaOH are added to get the pH value at 6. This process lasts for 72 hours, levels of ethanol that is produced can be known from the GC where the sample can be taken at hour 2, 8, 12, 24 and 72. Determining the water content Petri dish was dried in an oven (1050C) for ± 1 hour, then cooled down in a desiccator and weighed (A). Tubers samples (iles-iles and cassava) were weighed as much as 3 g (B). After that, the dish containing the sample was dried in an oven at a temperature of 1050C for 2 hours, then cooled in a desiccator and weighed to obtain permanent weight (C). The water content can be calculated by formula (AOAC 1984). Waterconte nt =

( A + B) − C x 100 % C

Determining the starch content Dissolve 5 g iles-iles tubers in 50, add HCl in to it, close it, heat it above the heater water until boiling for 2.5 hours. When it is cool, neutralize it with NaOH solution and dilute it until 500. The sample is titrated with Fehling solution (Sudarmaji et al. 1984). Cassava tuber starch content is measured by the same method. Crude fiber analysis Mash and then sift dry bulbs of iles-iles and cassava. Weigh as much as 2 g and then extract the fat from it by using soxhlet. Move all materials into 600 mL of Erlenmeyer and add 3 drops of anti-foaming agent. After that add 200 mL of boiling solution of H2SO4 (1.25 g concentrated H2SO4) and cover it with coolant behind. Boil it for 30 minutes and shake it a few moments. Filter the suspension with filter paper, and then wash the filter paper with boiling distilled water until no longer acidic (acidity can be tested with litmus paper). Move the residue in the filter paper into Erlenmeyer by using a spatula and then wash the rest with 200 mL of boiling NaOH (1.25 g NaOH/100 mL = 0.313 N NaOH), until all the residue gets into the Erlenmeyer. Then boil it with cooler behind while shake it for 30 minutes. Next filter it using filter paper of known weight, while wash it with a solution of K2SO4 10%. Wash the residue again with boiling distilled water and then with 15 mL alcohol 95% (Sudarmaji 1984).

a b c d e Figure 2. The process of making flour iles-iles tuber, a. Tuber crops, b. Peeled tuber, c. Tuber is cut, d. Bulbs that have been dried in the sun, e. Tuber flour iles-iles.


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Analysis sugar reduction Sugar reduction was done by using the Nelson-Somogy method. First make the standard solution of 0.1 M natrium thiosulfat was prepared by dissolving Na2S2O3 into and simmer for 5 minutes. Preparation of a solution of copper reagent was made by mixing some of Na2SO4 and KI solution, a solution of Na2CO3, KNaC4H4O6.4H2O solution, NaOH solution, a solution CuSO4.5H2O and KIO3 solution, then this copper reagent was stored in dark bottles. The Standardization of the copper reagent with the main liquor 0.005 M sodium thiosulfat was carried out with the main liquor thiosulfate. To analyze the levels of glucose in the sample, it was added with 1 sample in to the reagent solution of copper and then it was simmer at a temperature of 95°C for 30 minutes. Then add H2SO4. Next, it was titrated the sample by using a solution of Na2S2O3 with starch indicator, and TAT point is reached when the blue color turns clear. Each glucose levels is done in duplicate samples (Sudarmaji 1984). Determining ethanol by using GC To analyze the levels of ethanol, centrifuge the fluid of fermentation sample with speed 6000 rpm for 30 minutes to separate supernatant and pellet. Take as much as 1μL supernatant samples and inject it into the chromatography gas column (6890 N, Agilent Technologies Inc., USA), then equip it with a column HP-Innowax. Set the column temperature at 200°C and set carrier gas using N2 (40/min). Set the speed of gas flow rate for H2 at 40/min and for O2 at 500/min. Each sample is analyzed in duplicate.

RESULTS AND DISCUSSION The content of raw materials The material content percentage of iles-iles tubers is different from cassava. From the analysis we find that total sugar content of iles-iles tubers is 73.43% and cassava is 86.42%. Comparison of the content of other materials contained in cassava and iles-iles can be seen in Table 1. Table 1. Comparison of content of material found in the cassava and iles-iles. Percentage (%) Iles-iles Cassava Wet Dry Cellulose 1.67* 8.54* Hemicelluloses 10.5* 43.3* Lignin 0.597* 5.85* Sucrose 1.35* Water 82.82* 62.50 Total sugar 73.43 86.42 Starch 71.25 83.47 * The Center Laboratory of Food and Nutrition Studies, Gadjah Mada University. Ingredients

Table 1 shows that dry iles iles contains cellulose, hemicellulose, and lignin respectively, are 8.54%, 43.3% and 5.85%. However, in wet conditions, the contents of

cellulose, hemicellulose and lignin on iles-iles becomes lower, that is 1.67%, 10.5% and 0.597%. The content of starch in the iles-iles is 71.25%, while cassava is 83.47%. Starch is a polysaccharide compound which consists of amylose and amylopectin (Campbell et al. 2000). Starch in the iles-iles is so high that the bulb can be converted into ethanol by using the enzyme amylase that will break the monosaccharide monomers on starch into glucose. Besides we can also use yeast S. cerevisiae to break down glucose into ethanol. Glucose levels during the process of hydrolysis In general, bioethanol production from biomass consists of two main processes, i.e. hydrolysis and fermentation. Hydrolysis is a chemical process that uses H2O as a breaker of a compound (Kuswurj 2008). The reaction between water and starch goes so slowly that it needs assistance to increase the reactivity of water catalyst. Acid solution is often used in the process to accelerate the process, but in this experiment we use biological agent by using enzymes. According to Kolusheva and Marinova (2007) enzyme hydrolysis has more advantage compared to chemical hydrolysis. Chemical hydrolysis requires high temperatures (150-230° C), acid pH (1-2) and high pressure (1-4). This is different from the enzyme hydrolysis because it does not require a high temperature, medium pH of 6-8 and normal pressure. Enzymes are proteins that are catalysts, so often called biocatalysts. Enzymes have the ability to activate other specific compounds and to increase the accelerate of chemical reactions that will last longer if not using enzymes (Sun and Cheng 2002). Enzymes that is used in this study is the enzyme α-amylase and β-glucoamylase. Αamylase enzyme plays important role in hydrolyzing α-1 ,4-glucoside 19 specifically. This enzyme works at pH 5.7 and temperature 95°C. Enzyme amylase can not break down starch bond perfectly so that the process will produce dextrin with 6-10 chains units long (Schoonees 2004). The results of liquification process is then forwarded by the βglucoamylase which can hydrolyze the bond of α-1,4glucoside and α-1 0.6-glucoside with a temperature of 60°C and pH 4.2. Addition of β-glucoamylase in this experiment is aimed at producing more glucose because βglucoamylase on starch can cut the starch bond that has not been cut by the addition of α-amylase, by producing glucose which has β-configuration in contrast to the results of hydrolysis by α-amylase, so that glucose generated will multiply or abundant. According to Kholuseva (2007) hydrolysis that uses enzyme will produce higher reduction sugar if compared with the acid hydrolysis. Reduction sugar concentration during the hydrolysis process is calculated using the Nelson-Somogy method. The process lasts for 40 hours with a temperature of 60°C. The comparison of reduction sugar in cassava and in iles-iles can be seen in Figure 3. Result shows that cassava has higher glucose content than iles-iles. Measurement of glucose is conducted by using Nelson-Somogy method. Iles-iles and cassava hydrolysis that has a ratio of tub: water 1:4 shows that glucose levels are influenced by the length of time. The largest concentration of glucose is formed at the time hydrolysis for 40 hours, which are 33.431 g/L for cassava

KUSMIYATI – Comparison of iles-iles and cassava tubers as raw material for bioethanol


Figure 3. Sugar content in iles-iles and cassava.

Figure 4. Ethanol content in the tuber iles-iles and cassava tubers.

Figure 5. Effect of iles-iles substrate concentration and water concentrations toward ethanol, with yeast concentration of 10% (v/v) pH 5.5.

Figure 8. Effect of pH on ethanol production.

Figure 6. GC chromatogram resulted from fermented iles-iles for 60 hours at pH 4.5.

Figure 7. GC chromatogram of fermented cassava tuber for 60 hours pH 4.5.

and 16.175 g/L for iles-iles. This happens because the starch content in cassava is higher than in iles-iles, which is 83.47%, so that there is more glucose which can be converted. It is expected that the greater the hydrolysis of starch into glucose, the greater the ethanol produced in the fermentation process. Temperature is a factor that affects the α-amylase hydrolysis process; hence in this study we include the temperature variation in the process of α-amylase hydrolysis. Effect of temperature on variation of glucose levels can be seen in Table 2. From table we can conclude that the optimum temperature for the two tubers is 95°C, where the reduction sugar obtained from cassava and ilesiles respectively are 16. 176 g/L and 33. 431 g/L. This is the same stated by Kolusheva and Marinova (2007) where the research was performed under various temperatures (30, 60, 90 and 100ºC) and found that the optimum temperature was 90 and 100ºC where the process of hydrolysis runs faster so that result of reduction sugar is high.

Table 2. Effect of temperature variation on the hydrolysis process of the glucose levels, with concentrations of α-amylase 0:48/kg, time 40 hours, tubers and water concentration ratio (1:4).

Raw materials

Iles-iles tubers Cassava tubers

Glucose level (g/L) Temperature (°C) 90 95 100 105 14.57 16.176 16.041 15.221 29.145 33.431 30.451 28.451

110 13.245 27.219

Ethanol content during the fermentation process After hydrolysis process, the glucose which has been obtained will be converted into ethanol through a fermentation process. The basic principle is to activate the activity of microbial fermentation with the aim of changing the nature of raw materials to yield a product. The fermentation process of this study uses S because these organisms can ferment glucose, mannose, fructose and galactose in anaerobe and low pH conditions. Besides that S is resistant to high alcohol content and high sugar levels (Kartika et al. 1992; Shen et al. 2008). The process of



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fermentation by S. cerevisiae is done under anaerobic conditions, and if during the process the air enters then the ethanol formation process will be hampered. Therefore we must put small hose on the jar that serves to release CO2 gas. The purpose is to prevent a temperature raise inside the tube because S. cerevisiae is active at a temperature 432oC (Chin et al. 2010). Ethanol is fermented substrate due to the activity of S. cerevisiae. Comparison of ethanol content of fermented iles-iles and cassava are shown in Figure 4. Result analysis indicates that cassava yield higher ethanol than iles-iles. A 72-hour fermentation using the S. cerevisiae will produce highest ethanol either from iles-iles and cassava respectively 53.49 g/L and 69.81 g/L. The formation of ethanol is influenced by time, where the longer the time of fermentation the higher the level of ethanol will be. On the 24th hour ethanol content of each tuber tends to be small i.e. 30.12 g/L for cassava and 28.51 g/L for iles-iles. But the longer the fermentation time, the production of ethanol increases because the time that is used for converting glucose by S. cerevisiae is longer, resulting in higher ethanol. This is seen on the fermentation time 54 hours, where ethanol that comes from cassava and iles-iles are consecutively 43.21 g/L and 41.48 g/L. Effect of substrate concentration and pH on ethanol production Substrate concentration affect very much the production of ethanol, so to determine the effect of iles-iles substrate concentration toward the ethanol production we must perform variations of the adding water (1:3,5, 1:4, 1:4,5, 1:5 .) The level of ethanol content is then analyzed by using chromatography gas. The effect of water variation and ilesiels raw materials toward the ethanol is presented in Figure 5. From the result it is known that the highest ethanol content obtained from the ratio of raw materials: water 1:4 with ethanol content of 59.36 g/L. The proper combination of raw materials and water will make the hydrolysis reaction run fast, because if the water is too little then the course of the reaction. According to Nowak (2000), if the substrate concentration is too high (a little water), the amount of oxygen will be too small; in fact the oxygen is needed by S. cerevisiae to maintain life during the fermentation process. Levels of ethanol fermentation results were analyzed by using Chromatography Gas. Figure 6 shows the chromatogram profile of iles-iles ethanol which was fermented for 60 hours. Result of the analysis shows that iles-iles with GC will produce ethanol at retention time of 6.929 minutes, whereas cassava at retention time of 6.940 minutes. Chromatogram from GC analysis on cassava is shown in Figure 7. From the analysis using the GC we can see that the ethanol content of iles-iles tuber with early fermentation glucose concentration of 10% ethanol is formed by 44.4 g/L while the cassava 49.3 g/L. The degree of ethanol is determined by the activity of yeast with the sugar substrate which is fermented. According to Fessenden and Fessenden (1997), one molecule of glucose will form two molecules of ethanol and carbon dioxide. Too high concentration of

glucose will obstruct yeast growth, which makes the ethanol content low. Ethanol is formed by the activities of microorganisms in the substrate complex changes. Yeast growth was greatly influenced by pH, because if the pH is not appropriate yeast can not grow to a maximally, causing the death which lowers the ethanol. Effect of pH on ethanol is shown in Figure 8. Bioethanol production is influenced by acid-base conditions. According to Liu and Shen (2008) an optimum conditions of acid base can improve bioethanol production in the fermentation process because the acid-base conditions is closely related to the interaction of enzymes and raw materials. The degree of acid will affect the speed of fermentation. This is consistent with the results of research in which the process of cassava fermentation at pH of 5.5 produces highest amount of bioethanol, which is 60.85 g/L. While pH 4 produces the lowest ethanol concentration of 39.57g/L. Meanwhile iles-iles can produce maximum ethanol at pH 5.5 where the ethanol content obtained is 52.61 g/L. The results obtained prove that the acid conditions increase enzyme S. cerevisiae work. This is consistent with the study presented by Wilkins et al. (2007) where the results of ethanol will increase at pH 5 and 5.5 and will decline at pH 4, and 4.5. The optimum conditions are from pH 5 to 5.2. This result is consistent with Liu and Shen study (2008), that acid pH is an important parameter that can increase production of ethanol in fermentation processes with enzyme S. cerevisiae Test the efficiency of bioethanol fuel Bioethanol which is obtained, is then used as fuel for batik stoves. Batik stove is made by the researchers from stainless steel with tank capacity 500 mL. This experiment aims to determine the efficiency of bioethanol in melting the wax. In this experiment the researcher used some variation of ethanol degree as much as 100 mL to melt 20 g of wax. Table 3. Variations of bioethanol degree toward its use in melting wax. Parameter Time to boil (min) The time required to evaporate out (hours) Fire conditions when evaporation The condition of wax the container

Concentration of bioethanol fuel (%) 40 50 60 70 80 90 22 22 19 18 17 11 2,7 2,5 2,4 1,65 1,6 1,4 Red

Red Blue Blue Blue

Soot Soot




Blue -

From table it can be concluded 90% bioethanol grade takes the shortest time in boiling wax, with evaporation time of 1.4 hours for each 100mL. This is because the high levels of bioethanol (fuel: water = 90:10) is easier to vaporize. This is different for 40% and 50% bioethanol where the flame is red and soot appeared, but the time needed for ethanol to evaporate is longer than other levels. This is due to the water content that is high enough so that it took longer time to evaporate. However, high water

KUSMIYATI – Comparison of iles-iles and cassava tubers as raw material for bioethanol

content also produces carbon and soot during combustion. The greater the water content in ethanol is the longer it takes for the flame to run out. From the efficiency 90% bioethanol have a very high volatile nature that makes it less appropriate for use in combustion, because it tends to be wasteful and quickly burnt. While 40% and 50% bioethanol burns longer but it produces red flame and soot. This results in less energy than blue flame. Blue fire condition has more powerful energy for melting candle burning instead of changing the ethanol into carbon/soot. Based on efficiency 60% bioethanol then is more superior to other grades of ethanol. Water content that is not too high causes combustion produce blue and clean flame and clean of soot, and in turn it will burn longer than any other levels of ethanol.

CONCLUSION For 40 hours iles-iles tuber hydrolysis (Amorphophallus muelleri) has glucose content of 16.175 g/L and cassava 33.431 g/L. Degree of ethanol from iles-iles fermentation for 60 hours is 44.4 g/L, while cassava is 49.3 g/L. Temperatures difference will affect the speed of α-amylase hydrolysis in converting glucose. The result showed that the optimum temperature for hydrolysis is 95°C where the concentration of glucose obtained on cassava and cassava iles-iles are respectively 16 176 g/L and 33 431 g/L. The substrate concentration and acidity will affect the speed of fermentation. In this study the optimum ethanol degree was found in the ratio 1:4 and pH 5.5. The result shows that iles-iles tubers have the potential to be developed as a raw material for bioethanol production.

ACKNOWLEDGEMENTS The author would like to thank DP2M Higher Education Research Grant Fund for the assistance of the fiscal year 2010 (Contract No. 089/SP2H/PP/DP2M/III/2010, dated March 1, 2010). The author would like to thank Agnes H, Agus Nur Arifin, Hesthi Chandra P, Diani Mentari, and Ina Istiqomah in conducting this research.

DAFTAR PUSTAKA Assosiation of Oficial Analysis Chemis [AOAC]. 1984. Official methods of analysis of the AOAC. AOAC International. Gaithersburg, United States. Campbell NA, Reece LG, Mitchell G. 2000. Biology. 5th ed. Erlangga. Jakarta. [Indonesia] Cardona A, Sanchez OJ. 2007. Fuel ethanol production: process design trends and integration opportunities. Biores Technol 98 (12): 2415-57 Chin KL, H’ng PS, Wong LJ, Tey BT, Paridah MT. 2010. Optimization study of ethanolic fermentation from oil palm trunk, rubberwood and mixed hardwood hydrolysates using Saccharomyces cerevisiae. Biores Technol 101: 3287-3291. Department of Agriculture. 2009. Dombek KM, Ingram LO. 1987. Ethanol production during batch fermentation with Saccharomyces cerevisiae: changes in lycolytic enzyme and internal pH. Appl Environ Microbiol 53 (6):1286-1291.


Fessenden RJ, Fessenden JS. 1997. Dasar-dasar kimia organik. Binarupa Aksara. Jakarta. Gozan M, Samsuri M, Fani SH, Bambang P, Nasikin M. 2007. Sakarifikasi dan fermentasi bagas menjadi ethanol menggunakan enzim selulase dan enzim sellobiase. Jurnal Teknologi 3: 1-6. Imelda M, Wulansari A, Poerba YS. 2008. Cultivation of iles-iles (Amorphophallus muelleri Blume). Biodiversitas 9 (3): 173-176. [Indonesia] Jansen PCM, van der Wilk C, Hetterscheid WLA. 1996. Amorphophallus Blume ex Decaisne. In: Flach M, Rumawas F (eds) Plant Resources of South-East Asia No. 9. Plants yielding non-seed carbohydrate. Prosea. Bogor. Kartika B, Guritno AD, Purwadi D, Ismoyowati D. 1992. Methods and analysis of agriculture products. Inter University Center for Food and Nutrition, Gadjah Mada University. Yogyakarta. [Indonesia] Kolusheva T, Marinova A. 2007. A study of the optimal conditions forstarch hydrolysis through thermostable α-amilase, J Univ Chem Technol Metalurgy 42 (1): 93-96. Kurniawan A, Wibawa IPAH, Adjie B. 2010. Species diversity of Amorphophallus (Araceae) in Bali and Lombok with attention to genetic study in A. paeoniifolius (Dennst.) Nicolson. Biodiversitas 12 (1): 7-11. Kusmiyati. 2009. Utilization of iles-iles as a feedstock for bioethanol production used for kerosene substitute [Research report]. Institute for Research and Community Service, University of Muhammadiyah Surakarta. Surakarta.[Indonesia] Kusmiyati, Haryoto 2007. Utilization of bioethanol stove to reduce the dependence of kerosene for energy resources in the Batik home industry. [Research report]. Directorate of Research and Community Service, Higher Education, Ministry of National Education. Jakarta. [Indonesia] Kuswurj R. 2008. Sugar technology and research; proses hidrolisis dan aplikasinya di industri. [April, 2010] Liu R, Shen F. 2007. Impacts of main factors on bioethanol fermentation from stalk juice of sweet sorghum by immobilizes Saccharomyces cerevisiae (CICC 1308). Biores Technol 99: 847-854. Nowak J. 2000. Ethanol yield and productivity of Zymomonas mobilis in various fermentation methods. Elect J Polish Agric Univ Ser Food Sci Technol 3 (2): Milan JM. 2005. Bioethanol production status and prospects. J Sci Food Agric 10: 42-56. Osunsami AT, Akingbala JO, Oguntimein GB. 1988. Effect of storage on starch content and modification of cassava starch. Faculty of Technology, University of Ibadan, Department of Food Technology. Ibadan, Nigeria. Prihandana R, Noerwijari K, Adinurani G. P, Setiadi S dan Hendroko R. 2007. Bioetanol ubi kayu bahan bakar masa depan. AgroMedia Pustaka. Jakarta. Schoonees BM. 2004. Starch hydrolysis using (α-amylase: a laboratory evaluation using response surface methodology. Proceeding of 79th Annual Congress of the South African Sugar Technologists' Association. Sugar Milling Research Institute, University of KwaZulu-Natal, Durban, 4041, South Africa. Shen Y, Zhang Y, Ma T, Bao X, Du F, Zhuang G, Qu Y. 2008. Simultaneous saccharification and fermentation of acid-pretreatment corncorb with recombinant Saccharomyces cerevisiae expressing bglukosidase. Biores Technol 99: 5099-5103. Sudarmaji S, Haryono B, Suhardi. 1984. The procedures for agricultural foodstuffs. 3th ed. Liberty. Yogyakarta. [Indonesia] Sumarwoto, Widodo W. 2008. Growth and yield of elephant food yam (Amorphophallus muelleri Blume) in the first growing period with various dosages of N and K fertilizers. Agrivita 30 (1): 67-74. [Indonesia] Sumarwoto. 2005. Iles-iles (Amorphophallus muelleri Blume); description and other properties. Biodiversitas 6 (3): 185-190. [Indonesia] Sun Y, Cheng J. 2002. Hydrolysis of lignocellulosic materials for ethanol production a review. Biores Technol 83 (1): 1-11. Widyotomo S. Purwadaria HK. Syarief AM. Mulato S. 2000. Changes in particle size distribution of iles-iles flour result of processing with graded milling method. Agritech 24 (2): 82-91. [Indonesia] Wilkins MR, Widmer W, Grohmann K. 2007. Simultaneous saccharification and fermentation of citrus peel waste by Saccharomyces cerevisiae to produce ethanol. Process Biochem 42: 1614-1619.

ISSN: 2087-3948 (print) ISSN: 2087-3956 (electronic)

Vol. 2, No. 1, Pp. 14-22 March 2010

Variation of morphological and protein pattern of cassava (Manihot esculenta) varieties of Adira1 and Cabak makao in Ngawi, East Java TRIBADI1,♥, SURANTO², SAJIDAN² ¹ SMA Negeri 1 Kendal, Ngawi. Jl. Raya Ngawi-Madiun, Kendal, Ngawi, East Java, Indonesia ² Bioscience Program, School of Graduates, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia. Manuscript received: 13 Augustus 2009. Revision accepted: 25 January 2010.

Abstract. Tribadi, Suranto, Sajidan. 2009. Variation of morphological and protein pattern of cassava (Manihot esculenta) varieties of Adira1 and Cabak makao in Ngawi, East Java. Nusantara Bioscience 2: 14-22. This research is intended to find out the morphological and anatomical variation as well as the protein band pattern of cassava (Manihot esculenta Crantz) widely spread in three different areas of height. The sample collecting is done using simple random sampling in the three different areas of height that is 50, 300, 1000 meters asl in Ngawi District, East Java while the analysis of protein band pattern is done using SDS-PAGE. The result of the reseach of morphology and anatomy is analyzed descriptively and presented in the form of tabels, histograms and figures. The analysis of protein band pattern is done using quantitative and qualitative analysis that is based on the appearance or not the gel band pattern by counting the molecular weights based on code marker S 8445 and qualitative method based on the quality of the band formed. The band pattern formed is istimated and presented in the form of zimogram. The result of the research shows that the height of the cultivating site very much influences toward variations of root, stem and leaf morphology. The longest root is at 50 meter heights asl (Cabak makao local variety, the widest stem diameter is at 50 meters asl (Cabak makao local variety) the longest leaf and branch is at 300 meters asl (Cabak makao local variety) and 1000 meters asl (Cabak makao local variety). There is no difference of anatomy in the root, stem and leaf and no difference of protein band pattern either in Adira1 or Cabak makao local variety. Key words: Manihot esculenta, morphologic variation, anatomy, protein band pattern.

Abstrak. Tribadi, Suranto, Sajidan. 2009. Variasi morfologi dan pola pita protein uni kayu (Manihot esculenta) varietas Adira1 dan Cabak makao di Ngawi, Jawa Timur. Nusantara Bioscience 2: 14-22. Penelitian ini bertujuan untuk mengetahui variasi morfologi dan anatomi serta pola pita protein ubi kayu (Manihot esculenta Crantz) yang tumbuh pada tiga daerah ketinggian berbeda. Pengambilan sampel dilakukan dengan metode sampel acak sederhana (simple random sampling) pada tiga ketinggian tempat yang berbeda yaitu 50,300,1000 m dpl di Kabupaten Ngawi, Jawa Timur serta analisis pola pita protein dilakukan dengan metode SDS-PAGE. Hasil penelitian morfologi dan anatomi diuraikan secara deskriptip dan disajikan dalam bentuk tabel, histogram dan gambar. Analisis pola pita protein dilakukan dengan menggunakan analisis kuantitatif dan kualitatif yaitu berdasarkan muncul tidaknya pola pita protein pada gel dengan menghitung berat molekul berdasarkan marker kode S 8445 dan metode kualitatif berdasarkan kualitas pita yang terbentuk.Pola pita yang terbentuk diestimasikan dan disajikan dalam bentuk zimogram. Hasil penelitian menunjukan bahwa ketinggian habitat berpengaruh terhadap variasi morfologi akar, batang, dan daun. Umbi akar terpanjang pada ketinggian 50 m dpl (Cabak makao), diameter batang terlebar pada ketinggian 50 m dpl (Cabak makao), panjang daun dan tangkai terpanjang pada ketinggian 300 m (Cabak makao) dan 1000 m dpl (Cabak makao).Tidak ada perbedaan anatomi pada akar, batang dan daun serta tidak ada perbedaan pola pita protein baik pada varietas Adira-1 maupun Cabak makao. Kata kunci: Manihot esculenta, variasi morfologi, anatomi, pola pita protein.

INTRODUCTION Cassava (Manihot esculenta Crantz) is not a plant native to Indonesia, but has become very popular in Indonesia. Cassava belongs to the family of shrubs plant that is originally from the American continents, specifically from Brazil and Central America (Mexico, Bolivia, Peru, Venezuela, Guyana, and Suriname) (Nassar 1978 1992; Olsen and Schaal 1999; Nassar et al. 1996, 2008; Allem 1994). The spread of cassava has been almost to the entire world, including Africa, Madagascar, India and China. These plants came into Indonesia in 1852. Cassava is grown in agricultural areas. Cassava plant is widespread

throughout Indonesia, which has many local names, such as katela, kentila, ubi kayee (Aceh), ubi parancik (Minangkabau), ubi singkong (Jakarta), batata kayu (Manado), bistungkel (Ambon), buari deur, vori jendral, kasapen, sampeu, ubi kayu (Sunda), balet kasame, kaspa, kaspe, ketela buding, katela jendral, katela kaspe, kaspa, kaspe, katela budin,katela mantra, katila marikan, katela menyok, katela paung, katela prasman, katela sabekan, katela sarmunah, katela tapah, katela cengkol, ubi kayu, tela pohong (Jawa), blandong, manggala menyok, pohung, pahoung, sambrang balada, same, katela balada, tengsak (Madura), kesame, ketal kayu, sabrang same (Bali), kasubi (Gorontalo), bare, padu, lame kayu (Makasar), lame ayu

TRIBADI et al. â&#x20AC;&#x201C; Diversity of cassava varieties of Adira-1 and Cabak macao in Ngawi

(Bugis Majene), and kasibi (Ternate, Tidore) (Heyne 1987; Balitkabi 2009). Cassava has been a staple food as well as comodity. It has been the major source of food in food in South Africa and certain areas in Indonesia. Cassava is a source of carbohydrates for an estimated 800 million people around the world (CIAT 1993; Nweke 1996). In Indonesia, this plant stays in the third place after rice and maize as the main source of carbohydrates. As a commodity Cassava can be processed to produce dried cassava, tapioca, ethanol, liquid sugar, sorbitol, monosodium glutamate, and modified cassava flour (mocaf) (Harnowo et al. 2006; Wargiono 2006). Cassava can be also an alternative source of energy. This is in accordance with the Presidential Regulation No. 5 of 2006 which says that the increased production of cassava can be used as bio-ethanol fuel that is mixed with 10% premium (premium mix E10). There are three subspecies of Cassava. Cultivated subspecies are all included M. esculenta subsp. esculenta, which are closely related with the wild subspecies namely M. esculenta subsp. peruviana that grows in Peru and Brazil and wild species of M. esculenta subsp. flabellifolia that grows in Brazil and Venezuela (Allem 1994, 2002). This variety cassava (M. esculenta subsp. esculenta) which consists of 7200 cultivars has been released. The varieties of superior cassava that are commonly grown today include: Adira-1, Adira 2, Adira 4, Darul Hidayah, Malang


1, Malang 2, Malang 4, Malang 6, 3 and UJ 5 (Subandi 2007). Cultivar Adira 4, Malang 6, UJ 3 and 5 has a superior characteristic in accordance with the criteria for the raw material of bioethanol (high starch content) (Balitkabi 2009). The purpose of this study are to identify the (i) morphological, (ii) anatomical, and (iii) protein banding pattern variation of cassava that exists in areas with three different level of heights (50 m, 300 m, 1000 m) above the sea level in the Ngawi District, East Java.

MATERIALS AND METHODS The experiment was conducted from June 2008 to June 2009. Research morphology and the leaves sample of M. esculenta done in several sub-centers of cultivation and production of cassava in Ngawi District, East Java, namely: (i) Northern Ngawi (+ 50 m asl), covering sub-district Karangjati, Bringin and Karanganyar; (ii) Central Ngawi (+ 300 m asl), covering sub-district Kwadungan, Paron and Mantingan; (iii) Southern Ngawi (+ 1000 m asl), covering sub-district Jogorogo, Ngrambe and Sine (Figure 1). Ngawi District represents the northern lowlands, with the height of 50 m above the sea level, so too the central part of Ngawi, with the height of about 300 m above the sea level. The area has the air temperature of 26-380ÂşC, the


6 5



9 8 4 7

Figure 1. Research sites of cassava in the Ngawi District. The north: 1. Bringin, 2. Karangjati, 3. Karanganyar; the center: 4. Kwadungan, 5. Paron, 6. Mantingan; and the south: 7. Jogorogo, 8. Ngrambe and 9. Sine.


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rainfall of 1800 mm/year, its soil type is clay that becomes hard when it is dry. The south part of Ngawi is a plateau with the average of about 1000 m above sea level, with the peak in Mount Lawu (3265 m asl). The nort part of Ngawi is dominated by several plantations, cassava, tobacco, teak, soybeans, corn and a little rice. The central part of Ngawi is dominated by plantations, rice, cassava, tobacco, soybeans, corn, sugarcane. The southern part Ngawi is dominated by plantations, rambutan, tea, coffee, cassava, soybean, corn, cocoa, and zalacca fruits (Office of Agriculture, Plantation and Horticulture, Ngawi District 2009). Material The materials used in the research is M. esculenta from three different altitudes in Ngawi District. The entire plant is used for morphological study and to test the protein banding pattern the third leaf from top of the pant is used. The pattern of protein bands revealed by SDS-PAGE method with protein dye system is Comassie blue, premises marker code S 8445 (Sigma, Germany). Samples were taken by simple random sampling method. Procedures Morphology and anatomy. Observations of morphology include cassava’s roots (skin color, tuber color and flavor), stems (distance segment and color), and leaves (shape, color and stems). Observation of cross-sectional anatomy covers the roots, stems and leaves. Protein band patterns. Protein banding pattern analysis was conducted using SDS-PAGE (Schägger et al. 1988; Artama 1991; Tarkka 2000). The third leaf from the top part of cassava plant (two varieties, three locations in Ngawi) is washed with a mortar and pestle mixed extract buffer 500 uL. Once crushed and homogenized it is put into in a tube with ependorf. Centrifugation is prepared and when it has been more or less cold (with temperature of ± 0ºC) then the tube is inserted to be centrifuged with the speed of 12,000 rpm for 5 minutes. Thus, the sample solution is divided into two parts. The top of the colored nodes (supernatant) will be used in the process of electrophoresis, which is then stored in a place with the temperature of 4ºC, while the bottom solid forms (pellets) are removed. Supernatant is boiled for two minutes with so that the protein can open. Polyacrylamide gel consists of 2 parts, ie separating gel that lies at the bottom with a concentration of 12% and stacking gel which is located at the top with a concentration of 3%. Separating gel is made by mixing ± 10 ml of stock SDS PAGE 12%, plus 7 uL Temed and 80 uL APS 10%. While the 3% stacking gel is made by mixing 5 ml of stock 3% stacking gel plus 3.5 uL Temed and 50 uL APS 10%. Polyacrylamide gel solution is mixed. After it is homogeneous the separating gel electrophoresis is put into in the glass, after somewhat thickened some saturated isobutene is added. After that the saturated isobutene is removed and the stacking gel electrophoresis is included in the glass just above the staking gel. After that the sample comb is mounted on the stacking gel and released after it gets solid, and until some holes are formed that will be filled with the supernatant. The samples of Supernatant are

loaded into the hole as much as 10 uL. Before the installation of the plate glass on the vessel electrophoresis, it must be ensured that circulator temperature is less than 4ºC. After that the clip tube clamps and shied from the glass plates are removed and then the glass plates are set face to face to each other on the vessel electrophoresis, with the notched glass plate is put inside. At the time of installation there should be no air bubbles between the glass plates, then tighten the bar holder. The running buffer solution is added into the plate glass tanks that have been installed face to face to each other so that it is just right below the notch. After that the electrode buffer is filled again until it is full and bathtub’s lid is put back again. The power supply is turned on again to run the electrophoresis process with electric current at 125 volts for 90 minutes or until the supernatant reaches the lowest limit. After the electrophoresis process is complete, the gel is taken and continued to get colored. By putting the gel on the the plastic tray, then blue comassie is poured onto it and let it there overnight. After that the gel is rinsed with the destaining until clear. When the gel is clear, then the washing is stopped by replacing the destaining with 10% glacial acetic acid. Data analysis All data are described in descriptive method. Observation of morphology, including roots (tuber), stems and leaves are tested by doing analysis of the variance followed by Duncan test to know the difference; then presented in the form of tables, images and histograms. In observation of the anatomy of the roots, stems, and leaves, the preparats are microscopically photographed, and then presented in the form of images and the results are compared descriptively based on of the heights of the area the plants are grown and the varieties. Data analysis performed by the pattern of protein bands in quantitative and qualitative method is based on gel banding pattern appears or not by calculating the molecular weight marker code based on the S 8445 (Sigma, Germany) and qualitative methods based on the quality of banding pattern formation.

RESULTS AND DISCUSSION Morphology The result of morphological study of cassava varieties with research samples of Adira-1 and Cabak macao in the areas with the height of 50 m, 300 m, 1000 m above sea level in Ngawi District showed the presence of variations. The results of morphological observation of cassava, the varieties of Adira-1 and Cabak macao are shown in Figures 2 and 3 and Tables 1 and 2. Adira-1 Adira-1 of northern Ngawi (50 m asl). The characteristics are: has roots, outer brown and yellow skin color, edible taste, stem segments with the length of 2-4 cm, an oval shape for the leaves, red color for the stalks and no flowers. Of the five samples can be gained the

TRIBADI et al. – Diversity of cassava varieties of Adira-1 and Cabak macao in Ngawi

























Figure 2. Morphology of cassava, the varieties of Adira-1 and Cabak Macao from various areas of the heights. Note: 1. Adira, 2. Cabak macao; A. 50 m asl, B. 300 m asl, C. 1000 m asl; a. tuber roots b. tuber color, c. stem, d. leaf.

following average: The length of the root is19.84 cm. The length of one segment to another is 2.32 cm. The diameter of the stem is 2.38cm. The length of the leaf is 9.72 cm. The length of the stem is 13.84 cm. The location of the study is Karangjati, Ngawi, with the average rainfall of 1800mm/year, the average temperature of 350C, 6 for the pH of the soil , with grumusol for the type of the soil.. Adira-1 of central Ngawi (300 m asl). The characteristics are: has roots, with brown skin color for the outside part and yellow for the inside, yellow for the tuber, and edible. 2-4 cm for the length of the stem, with yellow color and oval for its leaf’s shape. The color of the stem is red and the type of the flower is kind of combination of many shades of brown color. Of the five samples can be gained the following average: The length of the root is 35.28 cm. The length of one segment to another is 3.18 cm. The diameter of the stem is 2.92 cm. The length of leaf


is14.64 cm. The length of the stem is 21 , 48 cm. The research’s location in Kendal, Ngawi with the rainfall of 1885 mm rain/year, the average temperature of 250C, 6 for the soil’s PH, brown Mediterranean for the type of the soil. Adira-1 of southern Ngawi (1000 m asl). The characteristics are: has roots, with the brown skin color for the outside part and yellow for the inside, yellow for the tuber and edible. 2-4 cm for the length of the stem with yellow color and oval for its leaf’s shape. The color of the stalks is red and no flowers. Of the five samples can be gained the following average: The length of the root is 22.55 cm. The length of one segment to another is 3 cm. The diameter of the stem is 2.28 cm. The length of the leaf is 14.88 cm. The length of the stem is 23.04 cm. The research’s location is in Jamus Ngawi, with the average rainfall of 4473 mm/year, the average temperature of 100ºC, 6 for the soil’s pH, and the soil’s type is brown lithosols.

Cabak macao Cabak macao of northen Ngawi (50 m asl). The characteristics are: has roots, with the brown skin color for the outside part and red for the inside, white color for the tuber, and edible. 2-4 cm for the length of the stem, blackish green color, oval for the leaf’s shape, light green for the stalk’s color, and no flowers. Of the five samples can be gained the following average: The length of the root is 47.44 cm. The length of one segment to another is 2.96 cm. The diameter of the stem is 3.92 cm. The length of the leaf is 17.44 cm. The length of the stem is 26 , 6 cm. The research’s location is in Karangjati, Ngawi, with the average rainfall of 1800 mm/year, the average temperature of 350C, 6 for the soil’s pH, and grumusol taupe for the soil’s type. Cabak macao of Central Ngawi (300 m asl). The characteristics are: has roots, with the brown skin color for the outside part and red for the inside, white color for the


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Table 1. The morphology observation of M. esculenta, varieties Adira-1 and Cabak macao (planted in June 2008 - August 2009).

color for the tuber, and edible. 2-4 cm for the length of the stem with blackish green as its color, light Adira 1 Cabak makao green its leaf’s color and brown for Morphological Southern Central Northern Southern Central Northern its compound flowers. Of the five characteristics Ngawi Ngawi Ngawi Ngawi Ngawi Ngawi samples can be gained the following Root Outer skin (brown) √ √ √ √ √ √ average: The length of the root is Inner skin (red) √ √ √ 38.6 cm. The length of one segment Inner skin (yellow) √ √ √ to another is 3.16 cm. The diameter Tuber (yellow) √ √ √ of the stem is 1.96 cm. The length of Tuber (white) √ √ √ Taste (good) √ √ √ √ √ √ the leaf is 18.2 cm. And the length of Stem yellow √ √ √ the stem is 22.36 cm. The research’s Darken green √ √ √ location is in Jamus Ngawi with the Leaves Shape (palm) √ √ √ √ √ √ average rainfall of 4473 mm/year, Petiole (red) √ √ √ the average temperature of 10ºC, 6 Petiole (pale green) √ √ √ for the soil’s pH, and the soil’s type is brown lithosols. Morphological observations Table 2. The average morphological characteristics measurement (cm) of M. esculenta, cassava, for the varieties of Adira-1 varieties Adira 1 and Cabak macao based on altitude. and Cabak macao from three areas Length of Internode Stem Length of Length of of research with different altitudes tuber distant diameter leaves petiole Altitude of 50 m asl, 300 m above sea level, Ad Cm Ad Cm Ad Cm Ad Cm Ad Cm and 1000 m above sea level on the 50 m dpl 19.84 47.44 2.32 2.96 2.38 3.92 9.72 17.44 13.84 22.36 length of the root, the length of one 300 m dpl 35.28 41.6 3.18 3.4 2.92 3.46 14.64 25.28 21.48 27.48 segment to another , the diameter of 1000 m dpl 22.55 38.6 3 3.16 2.28 1.96 14.88 18.2 23.04 22.36 the stem, the length of the leaf and Note: ad: Adira, cm: Cabak macao. the length of the stem, indicated variations in morphological levels. This is shown by data Table 2 and Figure 3. It means that the environmental factors in this case is the altitude of the area has effects on the morphological variations, especially for the varieties of Adira-1 and Cabak macao in Ngawi. Based on data in Table 2 and Figure 3 for the varieties of Adira-1 can be gained some following data: for the measurement of the length of the root it can be concluded that there are significant differences that show that the altitude of where the cassava is planted determines the Figure 3. Comparative morphology of cassava varieties, Adira-1 and Cabak macao. Note: length of the roots. The longest root ad: Adira, cm: Cabak macao. is found in the study sample planted in the area with the height of 300 m above sea level (35.28 cm). For the tuber, and edible. 2-4 cm for the length of the stem, measurement of the length of one segment to another it is blackish green color, oval for the shape of the leaf, light also found some differences but not as significant as the green for the color of the stalks, and green for its measurement foe the length of the roots. The longest is compound flowers. Of the five samples can be gained the found in the study sample in the area with the height of 300 following average: The length of the root is 41.60 cm. The m above sea level (3.18 cm). length of one segment to another is 3.4 cm. The stem’s Altitude also affects the diameter of the trunk but not diameter is3.46 cm. The length of the leaf is 25.28 cm. And significant. For the length of the leaf it is also obtained data the length of the stem is 27.48 cm. The research’s location some differences in the results, but there are similar data in in Kendal, Ngawi with the rainfall of 1885 mm rain/year, the study sample at the height of 300 m and 1000 m above the average temperature of 250ºC, 6 for the soil’s pH, sea level that means that altitude in anyway also affects the brown Mediterranean for the type of the soil. morphology, particularly the variations for the length of the Cabak macao of southern Ngawi (1000 m asl). The leaves. Although not absolute, the altitude also affects the characteristics are: has roots white bulb with the brown length of the stalk. The data of longest stalks is obtained in skin color for the outside part and red for the inside, white

TRIBADI et al. – Diversity of cassava varieties of Adira-1 and Cabak macao in Ngawi

the study sample in the area with the height of 1000 m asl (27.48 cm). Similarly, data from Tables 2 (Figure 3) for Cabak macao can be obtained the following data: The altitude also affects the morphological variations of the length of the root. The longest root is found in the sample at a height of 50 m above sea level (47.44 cm). The altitude also affects the length of one segment to another even though not really significant. The longest is found in the altitude of 300 m above sea level (3.4 cm). The diameter of the stem is also influenced by the altitude though not significant. The altitude shows significant influence on the morphological variations particularly on the longest leaf’s length obtained in the study sample with a height of 300 m asl (25.28 cm) that is almost equal to the height of 50 m and 1000 m asl and the longest stem length data in the study sample in the area with the height of 300 m asl (27.48 cm) and almost the same with the study sample in the area with a height of 50 m and 1000 m asl. At the organism level, phenotype is something that can be seen, observed and measured. It is a natural characteristic for individuals. Phenotype is determined by some genotypes of individuals, in some cases by the environment where these individuals live, the time and in some cases also by the interaction between the genotype itself and the environment. Time is usually classified as environmental aspects (of life) this can be written as follows: P = G + E, with P means phenotypes, and E means the environment. Observation of phenotypes can be simple for instance to observe the color of flowers or the stalks or can very complicated that requires special tools and methods (Cheverud 1982). For the same type of cassava found in the three research’s locations with the height of 50 m, 300 m, and 1000 m asl showed no significant morphological variations, except for length of the root, leaf and stalk. This variation is related to the growth of each plant. The cassavas found at the altitude of 300 m asl have bigger size then the ones at the other two places with the same age. The differences that emerged are related to the physical/environmental factors where the cassava is planted. the research location with the height of 300 m asl is a good and ideal place for the growth of ideal crops. Temperatures that are too low or too high can affect the opening of stomata which in turn affects the photosynthesis process (Levitt 1980). Temperatures above 30° C tend to cause cassava stomata to open properly, so that the photosynthesis works effectively and the plants grow faster (Bueno 1986). While temperatures below 20°C tend to cause the stomata to close (Akparobi et al, 2002a, b). Low temperatures slow down the growth of cassava (ElSharkawy 2004). In addition, the response of stomata to temperatures is also strongly influenced by water content and humidity in plants (Berry and Bjorkman 1980). According to Park et al. (1997) and Sulistyono et al. (1999) anytime plants deal with environmental pressure, they always make an adaptation. They may make adjustments through changes in morphological and physiological characteristics. Suchs an adjustment is made by for


instance making the leaves wider but at the same time thinner (Taiz and Zeiger 1991). Phenotype/morphological aspects in living creatures is a combination of genotype and environmental factors (Prawoto et al. 1987). The physical environment of the northern of Ngawi is different from the one of the Central and South Ngawi (in terms of altitude, rainfall, temperature and soil type). Then the altitude for instance influences a lot towards the phenotypes that arise in the form of morphological characters in the study samples (cassava, varieties of Adira-1 and Cabak macao), except for certain characteristics such as the color of the outer and inner part of the roots, the stem’s color and the taste. This can happen because phenotypes that appear are not necessarily morphological, they can be physiological. Changes in physiological characteristics only influence the system so that the cell performance can not be detected on morphological levels. Another possibility that caused the characteristics of the study samples of the varieties of Adira-1 and Cabak macao in northern, central and the south part of Ngawi despite different environments is because genetic factors may have a stronger influence than that of the environmental factors. As stated by Suranto (2001) that the emergence of variations can be caused by two factors namely environmental factors and genetic factors. If genetic factors have a stronger influence than environmental factors, then individuals living in different environments may not show morphological variations. Anatomy Analysis is based on cross-sectional slice on the anatomical parts of cassava, for the varieties Adira-1 and Cabak macao, covers cross-sections of roots, stems, and leaves for the species planted in area with the different heights: 50 m asl, 300 m asl and 1000 m asl presented in Figure 4. Adira-1 Roots. Based on the results of the cross sectional slice with an enlargement of 4x10 mm, at an altitude of 50 m asl, 300 m asl and 1000 m asl, it can be gained that there is no difference/almost the same as between the one in the areas with the altitude of 50 m asl, 300 m asl and 1000 m asl. Stems. The result of cross-sectional slice with 2.4 magnification x10 mm. There is little difference for the density between cells shown from the plants in different altitude of 50 m asl, 300 m asl, and 1000 m asl. The altitude seems to influence the density between cells but not really significant. Leaves. parenchyma cells of the leaves are found almost the same/no significant difference. The carrier tissues (phloem and xylem) show a state that is not much different either at the altitude of 50 m asl, 300 m asl, or 1000 m asl (Figure 4). Cabak macao Roots. Analysis based on the anatomy of roots, cassava, the varieties of Cabak macao, with enlargement of 4x10 mm2. It can be found that there is no difference in density.



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Leaves. Analysis of the cross sectional slice of the leaves, focusing on the bone, with 4x10 magnification, shows similar looks in terms of structures both at the altitude of 50 m, 300 m, and 1000 m asl. Cells around the carrier 1Aa 1Ab 1Ac tissues around shows no significant difference either at the altitude of 50 m asl, 300 m asl or1000 m asl (Figure 4). Based on the above results all samples shows similar looks and characteristics, although they are planted in areas with 1Ba 1Bb 1Bc different altitudes. It can be understood that the three researchâ&#x20AC;&#x2122;s locations are still in one region that is in Ngawi, so it is possible that each sample of existing research in these three sites belongs to the same family that has no genetic difference whatsoever. 2Aa 2Ab 2Ac Genetic factors have stronger influence than that of the environmental ones, so that the plants belonging one and the same genetic characteristics show similar looks even when planted in different areas with different 2Ba 2Bb 2Bc environmental factors. This is supported by results based on morphological variation indicating that cassava with the same variety found in different locations did not show variations in morphological levels. Appearance of a phenotype 3Aa 3Ab 3Ac depends on the nature of the relationship between genotype and environment. In fact, the development of an organism is influenced by the state of the environment and gene interactions. Living organisms are always 3Ba 3Bb 3Bc responsive to the environment Figure 4. Cross sections of roots, stems, leaves of cassava varieties Adira-1 and local during its development. In a broad Cabak macao based on altitude. Note: 1. Root, 2. Stem, 3. Leaves. A: Adira-1, B: Cabak sense, environmental factors macao; a. 50 m asl, b. 300 m asl, c. 1000 m asl. including both outside and inside factors, affect how a phenotype looks. Both of these factors can The structure of the root tissues shows the similar look of provide a major influence on the phenotype (Crowder one from another. 1997). Stems. Analysis on the stems to the variety of Cabak The result of cross-sectional analysis/anatomy of roots, macao with 4x10 mm magnification. It shows that the distance or density between cells of the plants grown in the stems, leaves for the varieties of Adira-1 and Cabak macao, areas with the altitude of 50 m asl and 300 m asl appear in the areas with the altitudes of altitude of 50, 300, and smaller than at an altitude of 1000 m asl. It means that 1000 m asl in the district Ngawi can be described as altitude only has insignificant effect on the anatomy of the follows: the distance between cells of the roots cross did stem. Altitude also has little effect on the distance or not show significant differences. There was no difference in the density between cells in the stems. There was no density between cells. difference in that of the leaves, too. The final conclusion of

TRIBADI et al. â&#x20AC;&#x201C; Diversity of cassava varieties of Adira-1 and Cabak macao in Ngawi

this discussion is that the altitude where the cassavas are planted has no significant effect on the anatomy of the stems, roots and leaves. The pattern of protein bands According Suketi (1994) proteins or enzymes can be separated by using electrophoresis and the result is zimogram banding pattern. Zimogram electrophoresis results of a typical patterned so that it can be used as a characteristic phenotype to reflect genetic opener. In the electrophoresis process of gel used polyacrylamide gel was used. The percentage of polyacrylamide in the electrophoresis is 7%, usually done in a tris glycine buffer at the pH of 8.1. In certain cases the comparison between polyacrylamide and pH is various (Suranto 2001). Electrophoresis is processes in which molecules of enzymes/proteins that have electricity moves through the electric field. The speed of the molecule/protein of the enzymes depends on the amount of the electric current. The separation of one molecule/protein of enzymes from another by the electrophoresis process is influenced by two factors: the amount of the electric current and the size of the particles The results of the electrophoresis process on the cassavaâ&#x20AC;&#x2122;s leaf for the varieties of Cabak Adira-1 and Macao with code S 8445, is shown in Figure 5.

Figure 5. Protein banding pattern of cassava leaf varieties of Adira-1 (1, 3, 5) and Cabak macao (2, 4, 6). Note: 1: Adira-1 50 m asl, 2: Cabak macao 50 m asl, 3: Adira-1 300 m asl, 4: Cabak macao 300 m asl, 5: Adira-1 1000 m asl, 6: Cabak macao 1000 m asl., M = proteins marker (S 8445, Sigma).

Based on zimogram, the variety of Adira-1 (Figure 5, nos. 1, 3, 5) expresses 20 bands; nos. 1, 2 (thick) MW was not detected, no. 3 MW 158 kDa, no. 4 MW 92.6 kDa, no. 5 MW 88.2 kDa, no 6 MW 70.4 kDa, no 7 MW 66 kDa, no. 8 MW 63.8 kDa, no. 9 (thick) MW 55 kDa, no. 10 (thick) MW 45 kDa, no. 11 (thick) MW 44 kDa, no. 12 (thick) MW 42 kDa, No. 13 (thick) MW 38.3 kDa, no. 14 (thick) MW 30.4 kDa, no. 15 (thick) BM 25.8 kDa, no.


MW 1623.7 kDa, no. 17 MW 20 kDa, nos. 18, 19, 20 MW was not detected. Same banding pattern expressed both in height (50 m, 300m 1000 m asl). Cabak macao (Figure 5, nos. 2, 4, 6) expresses 20 bands. No.1, 2 (thick) was not detected, no 3 MW 158 kDa, no. 4MW 92.6 kDa, no. 5 MW 88.2 kDa, no 6 MW 70.4 kDa, no. 7 MW 66 kDa, No. 8 MW 63.8 kDa, no. 9 MW 55 kDa, no. 10 MW 45 kDa, no. 11 MW 44 kDa, no. 12 MW 42 kDa, no. 13 MW 38.3 kDa, no. 14 MW 30.4 kDa, no. 15 MW (thick) MW 25.8 kDa, no. 16 MW 23.7 kDa, no. 17 MW 20 kDa, nos. 18, 19, 20 MW was undetected. Band s were expressed equally well at a height of 50, 300, 1000 m asl. Protein banding patterns for the varieties of Adira-1 and Cabak macao in the areas with the altitude of 50 m asl (no. 1, 2) and 300 m asl (no. 3, 4) in general seem much thicker than in those of the varieties of Adira-1 and Cabak macao at the altitude of 1000 m asl (no 5, 6). This shows a higher protein content that is possibly because at an altitude of 50 m and 300 m asl more sunlight is accessed that facilitates the better photosynthesis process, including the formation of proteins. The features of study samples of the protein bands (Adira-1 and Cabak macao) at an altitude of 50, 300, 1000 m asl did not show any difference/variation. The difference is only on the thickness bands due to the differences in the number of migrated protein molecules or the differences in the content/the quantity of protein. The thickness of the bands does not indicate the difference of the molecular weight, but only shows the differences in the content/the quantity of migrated proteins (Maryati 2008). Apparently, the limited number of samples tested may cause the disappearance of protein polymorphism in cassava, since several other studies have shown the existence of polymorphism in cassava and its relatives with the marker of proteins such as in the researches conducted by Nassar (2003), De Souza (2002), and Nassar et al. (2010). On the other hand, studies using isozym, which is equivalent to the protein, to study the diversity of cassava also show the polymorphisms in the population. Sumarani et al. (2004) found that 37 polymorphic bands appear on the test of 218 accessions of wild cassava with esterase enzyme. Lefevre and Charrier (1993) found that from 365 cultivars and 109 accessions of wild cassava in Africa there are 17 bands of polymorphism generated by 10 enzymes dye. In Parana Brazil, Resende et al. (2000), found 28 loci polymorphisms of local cassava samples with four enzyme systems. Research by Montarroyos et al. (2005), on 28 accessions of cassava in Pernambuco, Brazil showed the existence of 6 and 8 isozyme banding patterns with GOT and peroxidase. Genetic diversity with isozyme in populations of cassava were also found by Hussain et al. (1987), Ramirez et al. (1987), and Sarria (1993).

CONCLUSION The altitudes at which the plants are planted affect the variation of morphology, the length of the root, tuber and stalk. The longest samples are dominated by the ones from the height of 300 m asl because of the height is a good


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habitat and an ideal place for planting cassava. Anatomical observations indicate that the altitudes have no effect on the anatomy of the roots, stems and leaves of the plants. Analysis of protein band patterns showed that there were no differences in protein band profiles of cassava samples from different varieties (Adira-1 and Cabak macao) or different altitudes (50 m, 300 m and 1000 m asl). The difference is only the thickness of the bands due to the differences in the content/quantity of migrated protein molecules.

REFERENCES Akparobi SO, Ekanayake IJ, Togun AO. 2002. Genotypic variability for cassava tuberous root development in two low altitude and mid altitude savanna sites of Nigeria. African J Root Tuber Crops 5 (1): 24-28. Akparobi SO, Togun AO, Ekanayake IJ, Idris R. 2002. Effect of low temperatures on dry matter partitioning and yield of cassava clones. Trop Sci 42: 22-29. Allem AC (2002) The origin and taxonomy of cassava. In: Hillocks RJ, Thresh JM, Bellotti AC (eds). Cassava: biology, production and utilization. CABI. New York. Allem AC. 1994. The origin of Manihot esculenta Crantz (Euphorbiaceae). Genet Resour Crop Evol 41: 133-150. Artama WT. 1991. Rekayasa genetika. PAU – Bioteknologi. Universitas Gajah Mada. Yogyakarta. [Indonesia] Balitkabi. 2009. Cultivation technique of cassava. Crops Research Institute for Legumes and Tubers. Malang. [Indonesia] Berry JA, Bjorkaman O. 1980. Photosynthetic response and adaptation to temperature in higher plants. Annu. Rev. Plant Physiol. 29: 345-378 Bueno A. 1986. Adequate number of environments to evaluate cassava cultivars. Rev. Bras. Mandioca 5: 83-93. Cheverud JM. 1982. Phenotypic, genetic, and environmental morphological integration in the cranium. Evolution 36 (3): 499-516. CIAT (Centro Internacional de Agricultura Tropical) (1993). Cassava: The latest facts about an ancient crop. CIAT. Cali, Colombia. Cock JH. 1985. Cassava: New potential for a neglected crop. Westview Press. Boulder, Colorado. Crowder. LV. 1997. Plant genetics. Gadjah Mada University Press. Yogyakarta. [Indonesia] De Souza CRB, Carvalho LJCB, De Almeida ERP, Gander ES. 2002. Identification of cassava root protein genes. Plant Foods Human Nutr 57 (3-4): 353-363. El-Sharkawy MA. 2004. Cassava biology and physiology. Plant Mol Biol 56:481-501. Harnowo D, Subandi, Saleh N. 2006. The prospect of strategy and technology development of cassava. Agribusiness and food security. Food Crops Research and Development. Bogor. [Indonesia] Heyne K. 1987. Tumbuhan Berguna Indonesia. Jilid I-IV. Koperasi Karyawan Departemen Kehutanan. Jakarta. [Indonesia] Lefèvre E, Charrier A. 1993. Heredity of seventeen isozyme loci in cassava (Manihot esculenta Crantz). Euphytica 66: 171-178. Levitt J. 1980. Responses of plants to environmental stresses. Academic Press. New York. Maryati KT. 2008. Characterization of white grub (Melolonthidae: Coleoptera) on salak cultivation based on the characteristic morphology and protein banding patterns. [Thesis]. School of Geaduates, Sebelas Maret University. Surakarta. [Indonesia] Montarroyos AVV, de Lima MAG, dos Santos EO, de Franca JGE. 2003 Isozyme analysis of an active cassava germplasm bank collection Euphytica 130 (1): 101-106. Nassar NM, Hashimoto DY, Fernandes SD. 2008. Wild Manihot species: botanical aspects, geographic distribution and economic value. Genet Mol Res 7 (1): 16-28.

Nassar NMA, Bomfim N, Chaib A, Abreu LFA, Gomes PTC. 2010. Compatibility of interspecific Manihot crosses presaged by protein electrophoresis. Genet Mol Res 9 (1): 107-112. Nassar NMA, Carvalho CG, Vieira C. 1996. Overoming crossing barrers between cassava, Manihot esculenta Crantz and a wild relative, M. pohlii Warma. Braz J Genet 19 (4): 617-620. Nassar NMA. 1978. Wild Manihot species of Central Brazil for cassava breeding. Canadian J Plant Sci 58: 257-261. Nassar NMA. 1992. Cassava, Manihot esculenta Crantz, genetic resources: origin of the crop, its evolution and relationships with wild relatives. Geneti Mol Res 1 (4): 298-305 Nassar NMA. 2003. Gene flow between cassava, Manihot esculenta Crantz, and wild relatives. Genet Mol Res 2 (4): 334-347. Nweke FI. 1996. Cassava processing in sub- Saharan Africa: Implications for expanding cassava production. IITA Res 12: 7-14. Office of Agriculture, Plantation and Horticulture, Ngawi District. 2009 Growing of cassava (Manihot esculenta Crantz) Office of Agricultural, Plantation and Horticulture, Ngawi District. Ngawi. [Indonesia] Olsen KM, Schaal BA. 1999. Evidence on the origin of cassava: Phylogeographyof Manihot esculenta. Proc Natl Acad Sci USA 96: 10 5586-105591. Park YL, Chow WS, Anderson JM. 1997. Antenna size dependency of photoinactivation of photosystem II in light-acclimated pea leaves. Plant Physiol 115: 151-157. Prawoto, Sujoko, Maryam S. 1987. Evolution. Open University. Jakarta. [Indonesia] Presidential Regulation (Perpres) 2006. Regulation of the President of the Republic of Indonesia No. 5 of 2006 about the National Energy Policy. [Indonesia] Ramirez H, Hussain H, Roca W, Bushuk W. 1987. Isozyme electrophoregrams of sixteen enzymes in five tissues of cassava (Manihot esculenta Crantz) varieties. Euphytica 36: 39-48. Resende AG, Filho PSV, Machado MDFPS. 2000. Isozyme Diversity in Cassava Cultivars (Manihot esculenta Crantz). Biochem Genet 38 (78): 203-216. Sarria R, Ocampo C, Ramirez H, Roca WM. 1993. Genetic of esterase and glutamate oxaloacetatae transaminase isozymes in cassava (Manihot esculenta Crants). In: Roca WM, Thro AM (eds). Proc 1st Intl Sci Meeting of the Cassava Biotechnology Network. CIAT, Cartagena, Columbia, 25-28 August 1992. Schägger H, Aquila H, Von Jagow G. 1988. Coomassie blue-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for direct visualization of polypeptides during electrophoresis. Analytic Biochem 173 (1): 201-205. Subandi. 2007. Superior variety of nuts and tubers. Crops Research Institute for Legumes and Tubers. Malang. [Indonesia] Suketi K. 1994. Characterization studies of durian clonal seedling based on leaf morphology and isozyme banding pattern. [Thesis]. School of Graduates, Bogor Agricultural University. [Indonesia] Sulistyono E, Sopandie D, Chozin MA, Suwarno. 1999. Adaptation to shade of upland rice: morphological and physiological approach. Komunikasi Pertanian 4 (2): 62-68. [Indonesia] Sumarani GO, Pillai SV, Harisankar P, Sundaresan S. 2004. Isozyme analysis of indigenous cassava germplasm for identification of duplicates. Genet Resour Crop Evol 5 (2): 205-209. Suranto. 2001. Isozymes studies on the morphological variation of Rannunculus nanus population. Agrivita 3 (2): 139-146 Taiz L, Zeiger E. 1991. Plant physiology. Benyamin/Cumming. Tokyo Tarkka, M. T., Vasara, R., Gorfer, M., and Raudaskoski, M. 2000. Molecular characterization of actin genes from homobasidiomycetes: two different actin genes from Schizophyllum commune and Suillus bovinus. Gene 251:27-35. Veltkamp HJ, Bruijn GH. 1996. Manihot esculenta Crantz. In: Flach M, Rumawas F (eds). Plant Resources of South-East Asia 9: Plants yielding non-seed carbohydrates. Pudoc. Wageningen. Wargiono J, Hasanudin A, Suyamto. 2006. Technology support for cassava bio-ethanol industry. Center for Food Crops Research and Development, Crops Research Institute for Legumes and Tubers. Malang. [Indonesia]

ISSN: 2087-3948 (print) ISSN: 2087-3956 (electronic)

Vol. 2, No. 1, Pp. 23-33 March 2010

Diversity analysis of mangosteen (Garcinia mangostana) irradiated by gamma-ray based on morphological and anatomical characteristics ALFIN WIDIASTUTI1,♥, SOBIR2,3, MUH RAHMAD SUHARTANTO2,3 ¹ Center for Development of Seed Quality Testing of Food Crops and Horticulture (BBPPMBTH). Jl. Raya Tapos, P.O. Box 20, Cimanggis, Depok 16957, West Java, Indonesia. Tel.: +62-21-8755046/8754225. Fax.: 62+21 8755046/8754225 ² Plant Breeding and Biotechnology Program, School of Graduates, Bogor Agricultural University, Bogor 16680, West Java, Indonesia. 3 Faculty of Agriculture, Bogor Agricultural University, Kampus IPB Darmaga, Bogor 16680, West Java, Indonesia. Manuscript received: 13 December 2009. Revision accepted: 2 March 2010.

Abstract. Widiastuti A, Sobir, Suhartanto MR. 2010. Diversity analysis of mangosteen (Garcinia mangostana L.) irradiated by gammaray based on morphological and anatomical characteristics. Nusantara Bioscience 2: 23-33. The aim of this research was to increase genetic variability of mangosteen (Garcinia mangostana L.) irradiated by gamma rays dosage of 0 Gy, 20 Gy, 25 Gy, 30 Gy,35 Gy and 40 Gy. Plant materials used were seeds collected from Cegal Sub-village, Karacak Village, Leuwiliang Sub-district, Bogor District, West Java. Data was generated from morphological and anatomical characteristics. The result indicated that increasing of gamma ray dosage had inhibited ability of seed to growth, which needed longer time and decreased seed viability. Morphologically, it also decreased plant heigh, stem diameter, leaf seizure, and amount of leaf. Anatomically, stomatal density had positive correlation with plant height by correlation was 90% and 74%. Gamma rays irradiation successfully increase morphological variability until 30%. Seed creavage after irradiation increased variability and survival rate of mangosteen. Key words: Garcinia mangostana, gamma ray, genetic variability.

Abstrak. Widiastuti A, Sobir, Suhartanto MR. 2010. Analisis keragaman manggis (Garcinia mangostana) diiradiasi dengan sinar gamma berdasarkan karakteristik morfologi dan anatomi. Nusantara Bioscience 2: 23-33. Tujuan penelitian ini adalah meningkatkan keragaman genetik manggis (Garcinia mangostana L.) yang diiradiasi dengan sinar gamma dosis 0 Gy, 20 Gy, 25 Gy, 30 Gy, 35 Gy dan 40 Gy. Bahan tanaman yang digunakan adalah biji yang dikumpulkan dari Kampung Cegal, Desa Karacak, Kecamatan Leuwiliang, Kabupaten Bogor, Jawa Barat. Data dihasilkan dari karakteristik morfologi dan anatomi. Hasil penelitian menunjukkan bahwa peningkatan dosis sinar gamma dapat menghambat pertumbuhan benih, sehingga membutuhkan waktu lebih lama untuk tumbuh dan menurunkan viabilitas benih. Secara morfologi, hal itu juga menurunkan tinggi tanaman, diameter batang, ukuran daun, dan jumlah daun. Secara anatomi, kepadatan stomata berkorelasi positif dengan tinggi tanaman dengan nilai korelasi adalah 90% dan 74%. Iradiasi sinar gamma dapat meningkatkan keragaman morfologi hingga 30%. Pemotongan benih setelah iradiasi dapat meningkatkan keragaman dan tingkat kelangsungan hidup manggis. Kata kunci: Garcinia mangostana, sinar gamma, keragaman genetik.

INTRODUCTION Mangosteen (Garcinia mangostana L.) is an Indonesian original fruit commodities which have very good prospects for further development. Mangosteen is a tropical fruit that is very well known, and known as the Queen of Fruit because of its delicious taste and its a lot of fans (Test 2007). In addition, the mangosteen has long been used as medicine among them are as anti-inflammatory (Chen et al. 2008), antibacterial (Chomnawang et al. 2009), and for treatment of infections and wounds. Improvement on varieties of mangosteen aims to obtain high yielding varieties that are directed to accelerate the growth of mangosteen through improved root system, rapid production (early maturing), high productivity and good fruit quality. Mangosteen plant breeding to improve those characteristics is constrained because the mangosteen plant has a low genetic variability and no possibility of

increasing genetic variability through crossbreeding because of the male flowers are rudimental (Morton 1987). Mangosteen is a type of plant with very long juvenile period, where the slow growth which is caused by poor root system, the slow absorption of nutrients and water, low photosynthetic rate and low cutting rate of cells in the apical meristems (Ramlan et al. 1992; Wible et al . 1992; Poerwanto 2000). The mangosteen seeds shape themselves apomictically and develop from adventive’s embryos asexually (Sobir and Poerwanto 2007). The asexual regeneration of mangosteen leads to its low genetic variability (Richard 1990b) and is genetically inherited female elders’ characteristics (Koltunow et al. 1995). According to Ramage et al. (2004), based on the study Randomly Amplified DNA Fingerprinting (RAF) of 37 accessions of mangosteen, 70% showed no variation. Mansyah’s reserach et al. (1999) on 30 plants of West Sumatra’s mangosteens it can be concluded that the


2 (1): 23-33, March 2010

variability is narrow, although a few characters show a wide phenotypic variability. Efforts to improve the quality of mangosteen by increasing genetic diversity need to be done. With the wide variability, the selection process can be done effectively because it will give more opportunity to gain the desired characteristics or quality. One of the alternatives to increase variability in apomictic plant is through artificial mutation (Sobir and Poerwanto 2007). The use of radiation to cause mutations or changes in genetic makeup has a lot of positive impacts with an increase in the number of new plant varieties. This technique contributes to the increase genetic diversity and from the gained mutants there are some which have superior characteristics. Fauza et al. (2005) states that gamma ray irradiation on the mangosteen seeds shows an increase in phenotypic variability in several characters such as plant height, leaf number per plant, stem diameter, and leaf width. In rice plants, radiation with gamma rays at specific doses is known to be able induce chlorophyll mutations and increase the genetic resistance to blast disease (Mugiono 1996). Institute of Radiation Breeding in Japan has been using mutation induction since 1969 to gain potential mutants. Some new varieties of crops of apples, sugarcane, barley, and ornamental plants have been released until 1998 (IRB 2001). Radiation is enlightening process using radioactive rays that can cause mutations. High energy radiation is usually the form that release energy in large quantities and is sometimes called ionization radiation because the ions are generated in the material penetrated by the energy (Crowder 1997). Mutations with radiation can increase genetic variation. Cells that can survive well after irradiation will undergo several changes in physiological or genetic. These changes can produce better-looking plants (plants superior) than before (Harahap 2005). Mutations are resulted from all types of material changes derived. DNA, which is a major component of genes as carriers of genetic information from generation to generation, is the main target of radiation delivery. DNA changes that occur as a result of mutation, will lead to new genetic variations that will be deployed on its derivatives. The success of mutation can be observed through changes in morphology, anatomy, and also at the DNA level. Mutants that show morphological characteristics better than previous elders and show the existence of a genetic difference is expected to be developed into new varieties which are superior.

MATERIALS AND METHODS Time and place This research was conducted from January to August 2009 in the greenhouse and laboratory Research Center of Tropical Fruits (PKBT), Bogor Agricultural University, Laboratory of Microtechniques, Department of Biology, Bogor Agricultural University, and Center for Research and Development of Isotopes and Radiation Technology (IP3TIR), National Agency of Nuclear Energy (BATAN), Jakarta.

Plant material Plant material used is the mangosteen seed harvested in Kampong Cengal, Karacak Village, Leuwiliang Subdistrict, Bogor District, West Java. Experimental design This study consisted of two experiments, namely: (i). Mangosteen seeds cut after gamma ray irradiation treatment, and (ii). Mangosteen seeds cut before gamma irradiation treatment. Seeds were selected based on the weight of > 1 g. The dose Gamma ray radiation used is according to Harahap (2005) who states that 50% lethal dose (LD 50%) derived from the mangosteen seeds were 32.09 Gy, so the doses used in this study was 0 Gy (I0) as a control were 20 Gy (I1), 25 Gy (I2), 30 Gy (I3), 35 Gy (I4), and 40 Gy (I5). The tool used for irradiation is a Gamma Chamber 4000A with radiation source is a Co-60 radiation dose rate of 96.481 krad / hr (0.96481 kGy / hr). Treatment on cutting of the mangosteen seeds consists of three levels i.e. : the seeds are left intact (B0), seeds are cut into two equal parts (B1), and the seed is cut into three equal parts (B2). Each experiment consisted of 18 experimental units so that the total of the two experiments were 36 experimental units. Each treatment consisted of 10 replications so that the total population in this study was 360. Coefficient of variation (KK) is calculated based on each level of treatment by using a completely randomized design (CRD). Implementation of experiments Mangosteen seeds that had been extracted and cleaned were broken to be divided into two groups. Group 1 (experiment 1) the seeds were cut after irradiation treatment and group 2 (experiment 2) the seeds were cut prior to irradiation treatment. Each group was divided according to standard treatment combination of gamma irradiation (0 Gy, 20 Gy, 25 Gy, 30 Gy, 35 Gy and 40 Gy) and the standard of cutting seeds (whole, cut two, cut three). Next, the seeds were inserted into a different paper pocket and separated for each treatment combination. One container is one experimental unit. seeds that have been irradiated were planted in polybags in accordance with their respective treatment. Morphological observation Observations were done on the seeds that formed buds on each treatment combination. Morphological observations were made by observing the number, the lenghth, the width and shape of the leaves, and the diameter and height of the stems. Observations were made since the seeds germinated until they reached the age of 6 months. Anatomical observations Anatomical observations on the mangosteen’s leaves were made on both the transferal and paradermal slices. The paradermal slice was made using intact preparations (whole mount) while transfersal incision was made by following the paraffin method. The leaf was slashed using a rotary microtome with a thickness 10 μm, and then it was colored.

WIDIASTUTI et al. â&#x20AC;&#x201C; Phenotypic variation of Garcinia mangostana after gamma ray irradiation

Data analysis From the morphological data, the coefficient of variation was measured using SAS ver. 9.1.3 (SAS 2004). The data was also scored in binary measurement and was analyzed using SAHN, after that its similar matrixs were calculated using the program NTSYS ver. 2:01 and displayed in a dendogram (Rohlf 2002). The grouping showed similar relationship between each individual in the form of morphological characteristics. Genetic distance is the difference in value of the percentage of similarity to the value of 100%. From the dendrogram it can be seen how far the changes in crop radiation results when compared with controls. The same was not reported for transverse slices leaf anatomical parameters due to the data which was so various, so it did not show a specific pattern. Relationships and morphological parameters of the stomata were gained by doing a correlation analysis using SAS program ver. 9.1.3 (SAS 2004).








Figure 1. The growth of buds on the mangosteen seeds untreated with gamma ray irradiation (A) and (B), and seeds that received gamma irradiation treatment (C), (D), (E), and (F).

RESULTS AND DISCUSSION At the beginning of the study, the number of seed planted were 360 seeds and each seed was planted in a poly bag. Not all seeds were grown to form buds, total number of seeds capable of forming buds both in the control treatment results and gamma irradiation were 57 seeds. Data shown in this study is one related to the morphological observation and the anatomy of the seed treated by being cut and enlightened by gamma ray irradiation. Morphological observations were carried out on all the mangosteen seeds that formed buds, while the anatomical observations were carried out on seedlings that survived after 30 weeks of planting and showed normal growth, which were not stunted. Morphological observations Morphological observations showed that the number and time when the seeds to form buds, plant height, stem diameter, leaf number, length and width of leaves of some plants that underwent irradiation, experienced abnormalities. Of 360 units of the experiment, only 57 seeds that formed buds, 14 of which belong control group. This number decreased in the subsequent week of observation, because some plants died that was preceded by experiencing necrosis and the loss of leaves. The largest number of buds was shown of the period 20 weeks after planting, and in some control plants there grew more than one sprout from one single seed. Control plants showed normal growth in which plants did not experience any delays in forming buds and the growth was not inhibited. Harahap (2005) stated that the mangosteen seeds which did not undergo gamma rays treatment, the buds generally appear after 2 weeks of planting. Seeds which received irradiation treatment showed a delay emergence of buds. The formation and growth of buds on the control seeds and the results of irradiation treatment are presented in Figure 1. The morphological shape of mangosteen leaves both in plants that received both treatment and controls were generally ovate, obovate, and a small portion of them were







Figure 2. Mangosteen leafâ&#x20AC;&#x2122;s morphology after 30 weeks of planting: A. ovate, B. obovate, and C lacoleate. The color of young leaves of mangosteen: D. control of red brick, E. brownish green irradiated, and E. reddish brown control (f). Bar = 1 cm.





Figure 3. The color of the leaves after 30 weeks of planting: A. control, B and C. 20 Gy irradiation treatment, D. 25 Gy irradiation treatment (d). Bar = 1 cm.



2 (1): 23-33, March 2010

laceolate. From this observation, there was no specific pattern that distinguished the shape of leaves in the control plants from the irradiated ones. Flush that appeared on the buds that did not receive irradiation treatment was brownish red in color, while the flush that appeared on the irradiated buds was usually light green in color with a very slow growth. The morphological shape of the leaves and the color of young leaves of mangosteen which appeared in this study are presented in Figure 2. Most of the young leaves of mangosteen were red brick and reddish brown, but in some plants that received irradiation treatment emerged young leaves that were green with brownish color on the edges. Plant response to gamma irradiation treatment is individual one, but there is a general description of several variables of the outcomes. Leaves emerged from the results of irradiation plants are generally smaller, darker green in color, and thicker in texture. The response of each mangosteen plant to the stress of gamma ray irradiation was different. At the dose of 20 Gy appeared the light green leaves and looked transparent, but at a higher doses, ie 25 Gy and 30 Gy appeared leaves that have a smaller size, dark green in color and thicker (Figure 3). Abnormality was a response to disruption of physiological processes due to stress caused by gamma ray radiation. According to Soeranto (2003), abnormalities in the irradiated populations showed the occurrence of major changes in the level of genomes, chromosomes and DNA, so that physiological processes within cells that are genetically controlled became not normal. Meanwhile, according to Harahap (2005), changes in the leaf due to irradiation are thought to occur because of the increased amount of chlorophyll due to gamma ray irradiation stress. Time and number of seeds that formed buds Mangosteen seeds that are not treated with gamma-ray irradiation and the average deduction showed the emergence of buds after 3 weeks of planting (Table 1), while seeds which received gamma irradiation treatment alone (without cutting seed) the emergence of buds varied between 3 and 16 weeks after planting. Buds of which formation took the longest time was the individuals with 40 Gy irradiation treatments, but these plants grow very slowly and not able to survive at the end of observation (25 weeks after planting). From this observation, it is seen that the higher dose of gamma irradiation treatment is, the less and the longer time required for the seeds for the emergence of buds. At the doses high enough that above 30 Gy, most of seeds did not die or decay despite of being 20 weeks of sowing, but they also showed no signs of emerging buds / sprouts. Then at the end of the study the seeds which did not germinate eventually died and decomposed. At a dose of 35 Gy, none of the seeds that showed the emergence of buds, while at 40 Gy irradiation treatment, there was one seed that emerged buds that took a very long period which was 16 weeks after planting. The number of plants that germinated in the control treatment continued to increase until 15 weeks of planting and reduced in on the 16th , 20th , and 25th week after planting, that was from 33 plants on the 16th week getting reduced to 30 plants on 20th week and to 25 plants on 25th

week after planting. In the treatment at 20 Gy and 25 Gy, the number of surviving plants reduced after 20 weeks of planting, respectively from 15 to 14 and 9 to 7 (Table 1). Table 1. Number of mangosteen seeds that form buds. Number of buds on the WAP Level of irradiation 3 4 8 9 10 11 13 14 15 16 20 ( Gy) Seeds irradiated before cut Whole seed 0 5 7 9 8 8 8 10 10 10 10 6* 20 0 3 3 3 3 3 3 3 3 3 4* 25 0 2 2 2 2 1 3 4 4 6 5 30 0 0 0 0 0 0 0 0 0 0 0 35 0 0 0 0 0 0 0 0 0 0 0 40 0 0 0 0 0 0 0 0 0 0 0 Seed cut two 0 4 5 5 5 5 6 6 6 6 6 3* 20 0 2 2 3 3 3 3 3 3 3 3 25 0 0 0 0 0 0 0 0 0 0 0 30 0 0 0 0 0 0 0 0 0 0 0 35 0 0 0 0 0 0 0 0 0 0 0 40 0 0 0 0 0 0 0 0 0 0 0 Seed cut three 0 3 3 3 3 3 3 4 4 5 4 3* 20 0 0 0 0 1 1 1 1 1 1 1 25 0 0 0 0 1 1 2 2 2 2 1* 30 0 0 0 0 0 0 0 0 0 0 0 35 0 0 0 0 0 0 0 0 0 0 0 40 0 0 0 0 0 0 0 0 0 0 0 Seeds irradiated after cut Whole seed 0 5 8 8 8 7 7 8 10 10 8 9 20 2 2 4 5 4 4 4 4 4 5 4* 25 0 0 0 0 0 1 1 1 1 1 1 30 0 0 0 1 1 1 1 1 1 1 1 35 0 0 0 0 0 0 0 0 0 0 0 40 0 0 0 0 0 0 0 0 0 1 1 Seed cut two 0 0 2 6 3 3 2 2 2 2 2 4 20 0 0 0 0 0 1 1 1 1 2 1 25 0 0 0 0 0 0 0 0 0 0 0 30 0 0 0 0 0 0 0 0 0 0 0 35 0 0 0 0 0 0 0 0 0 0 0 40 0 0 0 0 0 0 0 0 0 0 0 Seed cut three 0 0 0 0 0 0 0 0 0 0 0 0 20 0 0 1 1 1 1 1 1 1 1 1 25 0 0 0 0 0 0 0 0 0 0 0 30 0 0 0 0 0 0 0 0 0 0 0 35 0 0 0 0 0 0 0 0 0 0 0 40 0 0 0 0 0 0 0 0 0 0 0 Note: * Some plants were dead so that in on the 25th week the number of plants got reduced. WAP = weeks after planting. Treatment

This was due to some individual plants have sprouted and emerged dying leaves, started with the drying of leaf tips, followed by the entire leaf and eventually the fall of the leaves. The death of those individual plants is due to the irradiation stress. According to van Harten (1998), gamma irradiation is destructive towards the path network it goes through. In addition, because its penetrability is very deep, the damage that it can cause can reach a few centimeters. Ahnstroem (1977) and Datta (2001), state that both the abnormality and the death of the irradiated plants are caused by the formation of free radicals such as Ho, that was a very unstable ion and caused a lot of collisions in different directions that will create mutations in DNA, as well as caused changes at the level of cellular and network. It can even cause death in plants. In the control group, the death of plants was suspected to be caused by physiologically immature seeds. In the treatment of 30 Gy

WIDIASTUTI et al. – Phenotypic variation of Garcinia mangostana after gamma ray irradiation


only one seed that formed buds which survived until the age of 20 weeks after planting, while at the 40 Gy treatment, the buds that appeared was dead after 25 weeks of planting. Cutting the mangosteen seeds actually has effects on the number of buds that emerged (Table 1). On the irradiation level of 25 Gy, the truncated seeds after gamma irradiation showed the higher number of seeds that formed buds that was three seeds for the seeds that were cut into two, and two seeds for the ones cut into three, compared to seeds that were cut first before irradiation gamma rays, which were the two seeds in the treatment group that was two seeds for the seeds cut into two cut and no buds appeared on the seeds cut into three after 16 weeks of planting. Seeds treated with irradiation before being cut showed the ability to form buds faster and higher than the ones cut prior to irradiation. This is because that on the seeds cut prior to irradiation; the tissues damaged due to radiation are greater than the seeds cut after irradiation.

The decreasing number of leaves creates patterns in the control treatment at the level of 20 Gy and 25 Gy, where the higher the dose of the treatment using gamma ray irradiation means the less number of the leaves. The effect is due to physiological damage caused by gamma irradiation. The length and width of the leaves also showed a decrease in the size of some irradiated crops . According to Patit (1966) and Ashri (1970), reduced size of the leaves can occur due to irradiation and chemical mutagen treatment.

Plants height, stems diameter, and number, length and width of leaves The growth of the mangoosteen plants can be seen by performing a detection on the morphological characteristics. In this study, the measurement of morphological characters was conducted on the height of the plants, the diameter of stems, the number, the length and width of the leaves. The height of the plants was measured from the neck of the root up to the point where the plants grow, while the trunk diameter was measured at a height of 1 cm above the root’s neck. The length and width of the leaves were measured on the leaves that emerged secondly. The height of the plants, the stems’ diameter, the number, length and width of the leaves gets decreased as the doses of gamma irradiation increases (Table 2). This happens because cellular damage happens to the plant’s meristem. According Handayati et al. (2001), the damage leads to the degradation of indole acetic acid (IAA) because indole acetaldehyde dehydrogenase enzyme is inhibited (Moore 1979).

Gamma ray irradiation has effects the on morphological characters of mangosteen. These changes appear to be individual, although they were irradiated at the same dose. The comparison between control plants and plants produced with gamma ray irradiation is presented in Figure 4. Gamma ray irradiation can affect the growth and morphology of mangosteen. In this research it is gained that at higher doses than 25 Gy, the mangosteen seeds require just 9 weeks to form buds, while seeds without irradiation only takes 3 weeks. The height of the plants, the number, length and width of the leaves also shows the response to irradiation dose of gamma rays, where the higher dose of irradiation, the less the characteristics in value. This suggests that high doses cause stunted growth and even cause the seeds not able to grow. Barriers to growth are in the form of physiological damage due to gamma ray irradiation. The length and width of the leaves also showed a decrease in the size of some irradiated crops. Qosim (2006) stated that nodular callus of mangosteen with



Table 2. Average high of mangosteen, leaf number, stem diameter, leaf length and width on the 25th week after planting. Characteristics Plant height Stem diameter Leaf number Leaf length Leaf width


0 Gy 5.85 ± 2.58 0.19 ± 0.09 1.65 ± 0.47 3.79 ± 1.67 2.32 ± 0.68

20 Gy 5.00 ± 1.77 0.15 ± 0.04 1.33 ± 0.47 3.36 ± 1.40 2.23 ± 0.48

25 Gy 4.40 ± 2.74 0.15 ± 0.06 1.33 ± 0.47 2.7 ± 1.30 1.45 ± 0.91


Figure 4. Comparison of control plants and the results of irradiation on the 25th week: A. Results of irradiation at the level of 25 Gy and control. B. Results of irradiation at the level 25 Gy and 20 Gy, the combined seed is cut into two equal after irradiation. C. Control and irradiated 25 Gy combinations of seeds cut into three equal after irradiation. D. Results of irradiation 25 Gy combined seeds cut into three equal after irradiation.


2 (1): 23-33, March 2010

Table 3. The coefficient of variation (%) of each morphological character in (i) the extent of irradiation control (I0), 20 Gy (I1), and 25 Gy (I2), (ii) cutting seed: whole, cut in half, and cut into three (iii) treatment of seeds irradiated before being cut, and seeds irradiated after the cut.

Morphological character Number of buds

16 WAP 20 WAP 25 WAP Plant height (cm) 16 WAP 20 WAP 25 WAP Number of leaves 16 WAP 20 WAP 25 WAP Diametar stem (cm) 16 WAP 20 WAP 25 WAP Leaf length (cm) 16 WAP 20 WAP 25 WAP Leaf width (cm) 16 WAP 20 WAP 25 WAP Note: WAP = weeks after planting.

level of gamma irradiation 0 Gy

20 Gy

25 Gy

67.16 78.53 84.219 35.023 37.26 36.190 34.10 27.08 27.91 30.82 25.49 35.79 35.51 38.41 43.15 32.59 26.85 21.15

188.91 170.44 170.44 36.780 31.28 39.6 27.3 35.18 39.95 24.84 19.84 23.47 45.38 40.85 43.94 49.17 42.23 23.81

267.46 222.51 257.64 80.07 80.07 76.08 35 33.126 41.92 25.064 27.59 34.65 52.906 54.16 55.50 31.4 45.08 72.53

gamma ray irradiation at the level above 25 Gy took 129 weeks to form buds. Irradiation gamma rays can also cause changes in the anatomy of mangosteen’s leaves. The number of seeds capable of forming buds also decreases as the dose of irradiation increases. At the doses greater than 25 Gy, only one seed that is able to form buds, i.e. at a dose of 30 Gy on the 10th week of planting, and one seed at 40 Gy on the 16th week of planting. Decreased ability to form buds or seed germination occurring with increasing dose of irradiation was also observed in wheat (Gou et al. 2007), peanut (Baddiganavar and Murty 2007), and soyseed (Manjaya and Nandawar 2007). Diversity test towards the mangosteen phenotypic Effect of gamma irradiation According Baihaki (1999), to determine the variation of a population, the following rules of measurement and analysis that are in accordance with statistical way need to be done. The various populations will have specific characteristics that can be seen from the coefficient value describing diversity in a single treatment. Gamma ray irradiation is a physical mutagen which can cause an increase in the diversity of initial population. In this study, the dose of gamma irradiation which provides the highest variability based on coefficient of variation is 25 Gy, while the dose that gives the lowest diversity is 0 Gy (control) (Table 3). Almost all the characters showed increased coefficients of variability as the levels of irradiation increased, except for the stem’s diameter, which is at the dose of 20 Gy the coefficients of variability on the 16th, 20th, and 25 week of planting, are lower than those in the control group. At the dose of 25 Gy, the coefficient of variation for the stem’s diameter that is greater than that of the control group is only found in the one on the 20th week of planting (Table 3).

Coefficient of variance (%) Cutting seed Whole 137.82 131.76 162.01 42.93 43.65 41.99 35.49 30.37 31.38 25.47 24.60 27.37 39.20 41.06 42.99 36.38 34.10 31.04

Cut two 243.01 236.79 218.60 40.78 38.50 44.79 0 30.73 43.47 33.14 20.39 26.51 45.53 46.81 64.29 37.99 31.56 23.78

Seed cutting time After Before Cut tree irradiation irradiation 306.81 112.38 161.34 342.26 114.28 178.37 367.15 122.90 143.11 30.53 29.47 41.13 48.01 34.26 34.22 17.06 42.94 22.26 37.50 36.36 17.42 0 41.14 14.67 31.25 52.01 22.98 102 29.33 32.97 91.20 36.52 30.39 101.03 0 0 39.21 35.13 34.50 16.15 0 34.77 11.78 0 33.94 19.10 0 31.95 8.97 0 23.94 23.10 0 24.77

Increasing dose for irradiation led to the less ability of seeds to form buds, which is marked by the declining number of seeds that form the buds compared with the control (Table 1). At doses where the seeds are still able to form buds and grown into plants, it is known that 25 Gy is the dose that most suppressing dose towards the growth of mangosteen, which is characterized by small average value for each observation of morphological characters compared to those belonging to control group and at lower doses (Table 2). Effect of seed cutting level Mangosteen seeds are poliembrionyc ones which can grow more than one bud. Coefficient of variation on a level of cutting seed treatment with the highest number of buds is obtained from the seed treatment which is cut crosswise into three equal sizes. For the height of the plant, the highest coefficient of variation on 16th and 20th week of planting is found in the treatment of intact seeds, while on the 25th week of planting the highest coefficient diversity is found from the seed treatment which is cut into two equal sizes. The highest coefficient of the number of leaves from the ones on the16th week of planting is found in the seed treatment which is cut into three equal sizes, while from the ones on the 20th and 25th week of planting is found in the treatment of seed which is cut into two equal sizes. For the stem’s diameter, the highest coefficient of diversity is found on the seed treatment which is cut into three equal sizes, while for the length of leaves the highest coefficient is obtained in the treatment of the seed which is into two equal sizes. The highest coefficient of diversity for the width of the leaves found in the plants on 16th and 20th week of planting is obtained on the seed treatment which is cut into two equal sizes, meanwhile the highest coefficient of

WIDIASTUTI et al. – Phenotypic variation of Garcinia mangostana after gamma ray irradiation

diversity for the plants on the 25th week of planting is found on the seed treatment which is left intact (Table 3). Coefficient of variation which is higher on the morphological characters derived from the cutting of mangosteen seeds is related to the nature of poliembrionyc of the mangosteen seeds. A single of mangosteen seed can grow more than one bud, where each bud emerges from different sections, and allegedly carries different genetic constitutions as well. Mansyah et al. (2008) stated that of the nine seeds of poliembrionyc mangosteen it can be seen the differences on the DNA bands on the buds that grow from the seeds of the same mangosteen. Effect of seed cutting time The time for cutting seeds can be divided into after and before irradiation. The treatment for the seeds cut before irradiation has a higher coefficient of variation than the ones cut after irradiation (Table 3). This is because in the treatment for the seeds cut before irradiation, the exposure of irradiation directly hits the surface of the seed that experiences injury from the cutting so that the effect becomes larger, while in the seeds that are cut after irradiation, the exposure to gamma rays only hit the surface of the seeds so that the radiation effect is smaller. Increasing of morphological diversity due to gamma ray irradiation Mangosteen is included in obligate apomixes plants, whose seeds are not derived from the results of fertilization but developed from adventive’s embryos asexually (Sobir and Poerwanto 2007), thus might have low genetic diversity (Richards 1990b; Varheij 1992; Cox 1996). Apomixes on mangosteen plants cause the same genetic trait in the progeny the same as with that in the parents (Koltunow et al. 1995). Induction of irradiation with gamma rays is one alternative to increase genetic diversity in plants in which the occurrence of cross-fertilization is not possible. In this study, the dendogram drawn from the morphological observation of mangosteen plants showed that gamma irradiation treatments can increase the diversity compared to ones belonging to control group. The similarity on values in the plants that do not have gammaray irradiation treatment ranged from 13-83% (Figure 5A), while the ones the plants are treated with gamma-ray irradiation ranged from 0-100% (Figure 5B). Diversity increased by 30% after the induction of gamma ray irradiation. Gamma rays include mutagens that produce ions and free radicals in the form of hydroxyl (OH-). If hydroxyl radicals are attached to the chain of nucleotides in DNA, the single strand of DNA will be broken and undergo some genomic changes (Mohr and Schopfer 1995). Visually the diversity in maize growth due to the influence of gamma irradiation becomes larger (Herison et al. 2008). In the mangosteen plants that do not get gamma-ray irradiation treatments, the greatest similarity is found in plants without the cutting seed treatment which are I0B0 and B0I0, respectively by 83%. Broadly speaking, the research results are divided into two that are plants derived


from intact seeds and plants whose seeds are cut into two or three equal size. MXComp cophenetic value generated from the control plants is (r = 0.967) with very appropriate goodness fit, while the value of the irradiated ones is (r = 0.956). Clustering analysis on the results of gamma irradiation plants do not provide specific grouping between control plants and plants produced with gamma ray irradiation. This happens because the nature of mutations caused by irradiation of gamma rays is random. One mutant plant derived from irradiation of 25 Gy (B0I2) is in the group of plants without irradiation treatment that is on the similarity of 100%. This shows that at the morphological level, these plants do not differ from the ones untreated with gamma ray irradiation. Increasing morphological diversity by cutting seed process The time of cutting the mangosteen seeds, whether before or after irradiation, makes difference in improving the diversity based on morphological observations. Mangosteen seeds irradiated prior to getting cut gives a smaller similarity of 43-88% (Figure 5C), while the mangosteen seeds receiving irradiation after the cut has a similarity of 0-83% (Figure 5D). The pattern in cutting the seeds is also seen to lead to diversity, both in the irradiation treatment before and after cutting. In Figure 5C, based on the cutting pattern of mangosteen seeds, the dendogram is divided into two groups on the similarity of 43%, i.e. the first group only consisted of plants from the intact seeds only(I0B0) and group two consisted of plants from the seeds of the mangosteen which are cut into two equal (I0B1) or three equal (I0B2)sizes. At intervals of 60% similarity, the individual plants I0B2 and I0B1 are in different groups, indicating the existence of diversity between both of them. Mangosteen seeds are poliembrionyc ones, meaning that one seed can grow more than one bud. Each bud has a different genetic constitution because they come from different embryos. Mansyah et al. (2008) stated that four out of nine seeds of poliembrionyc mangosteen seeds it can be seen the different DNA bands in the buds that grow from the seeds of the same mangosteen. Anatomical observations Transfersal section The structure of mangosteen leaves on the transferal slice consists of the layers of cuticle, upper epidermis, palisade parenchyma, spongy parenchyma and lower epidermis. The epidermal tissue is covered by cuticles which are spread throughout the upper and lower leaf surfaces. The structure of mangosteen’s leaves belong to the type of dorsiventral as a palisade parenchyma tissue is between the upper epidermis and spongy tissue. The results showed that the cuticle is on the upper and lower surfaces. The palisade parenchyma of Mangosteen’s leaf consists of two layers which are under the upper epidermis, while the sponge layer is under the parenchyma palisade (Figure 7). The observation on the mangosteen leaves transversally sliced was conducted towards 16 plants that visually show a good growth.


2 (1): 23-33, March 2010

I0B0 B0I0 I0B1 I0B2 B1I0 0.13




Koefisien kemiripan





I0B0 B0I2 B0I0 I1B0 I2B2 B0I3 I0B1 B0I1 I1B1 I2B0 B1I1 I0B2 B1I0 B0I5 0.75




Koefisien kemiripan

I0B0 I1B0 I2B2 I0B1 I2B0 I1B1 I0B2 0.43






Koefisien kemiripan

B0I0 B0I2 B0I1 B0I3 B1I1 B1I0 B0I5 0.00






Koefisien kemiripan

Based on Table 4 it can be gained that the range of values in a thick cuticle, upper epidermis, lower epidermis, palisade parenchyma, spongy parenchyma and the thickness of the mangosteen’s leaves vary greatly. There is no particular pattern between the thickness of cuticle of the individual belonging to control plants and that of the irradiated ones. Table 4 shows that the thickness of upper epidermis and lower epidermis for most crops is almost the same. Epidermal tissue is the tissue that serves to protect the underlying tissue and serves as a coating for gas exchange to and from outside the body through the hole plant stomata. Changes in the thickness of the epidermis can be caused by the ionizing nature of gamma rays which can penetrate the epidermal layer and cause the changes. Other factors affecting the changes in leaf anatomical characters beside the regenerant are the environment factors such as the availability of water, light intensity, the concentration of CO2, and the temperature which can affect the density of stomata (Willmer 1983). Some plants which are the result of irradiation treatment showed substantial palisade thickness values, namely B0I3, I1B01, and I0B02. The thickness of sponges and the highest thickness of leaves are obtained from the control plants (B0I03), while the lowest obtained from the plants produced at the irradiation level of 25 Gy. The thickness of the sponge tissue is associated with the thickness of space between cells, where the thicker the sponge tissue, the greater the spaces between cells that are useful for storing water and CO2. According to Fahn (1991) the important factors that can increase the efficiency of photosynthesis is the space between cells which is very well located in the mesophyll, thus facilitating gas exchange quickly.

Figure 5. Mangosteen dendogram based on morphology: A. Control. B. Gamma ray irradiation treatment. C. Cutting the seed after irradiation. D. Cutting the seed prior to irradiation.

Table 4. Observations of leaf transversal section in some individuals of mangosteen (µm). Individual plants


Average thickness (µm) Upper epidermis Under epidermis Palisade

Sponges Leaf Seeds irradiated before cut I0B01 3.88 ± 1.24 10.00 ± 2.04 7.75 ± 1.53 42.50 ± 6.87 184.51 ± 25.02 64.37 ± 7.85 I0B02a 2.88 ± 0.60 14 .00± 1.29 10.25 ± 1.42 48.00 ± 6.21 180.75 ± 5.01 75.38 ± 5.76 I0B03 4.00 ± 1.29 7.75 ± 1.84 9.25 ± 2.64 44.5 1± 11.59 202.25 ± 31.27 65.77 ± 13.81 I1B02b 2.50 ± 0 10.13 ± 2.53 11 ± 3.37 46.5 3± 5.29 176.51 ± 6.89 70.37 ± 7.56 I1B01 3.75 ± 1.17 12.00 ± 2.58 9.25 ± 1.20 51.52 ± 8.26 178.75 ± 24.24 76.75 ± 6.61 I1B1 2.63 ± 0.39 10.52 ± 2.29 9 ± 1.29 42.52 ± 6.97 188.25 ± 13.64 64.88 ± 7.45 I2B0 3.38 ± 1.19 7.13 ± 1.87 6.25 ± 1.77 42.03 ± 8.32 151.03 ± 8.99 58.96 ± 10.08 I2B2 3.13 ± 1.06 9.13 ± 2.04 7.5 ± 1.67 43.25 ± 6.13 140.75 ± 20.01 63.20 ± 6.06 Seeds irradiated after cut B0I02 3.52 ± 1.49 10.25 ± 1.84 9.25 ± 2.059 48.25 ± 6.46 195.25 ± 27.11 71.51 ± 7.06 B0I03 3.75 ± 1.18 10.50 ± 3.07 10.00 ± 2.63 55.00 ± 8.97 203.25 ± 19.04 79.53 ± 9.95 B0I04 4.54 ± 0.87 9.75 ± 2.75 9.75 ± 2.19 55 .00± 10.99 167.50 ± 19.01 79.24 ± 12.52 B0I01 4.50 ± 1.05 9.87 ± 2.8 8.50 ± 1.74 53.50 ± 5.02 168.50 ± 12.08 76.62 ± 5.08 B0I2 4 .01± 1.15 9.25 ± 2.37 8.88 ± 1.71 51.25 ± 7.38 185.5 0± 11.71 73.63 ± 6.16 B0I3 5.37 ± 2.13 13.12 ± 3.19 8.63 ± 2.91 57.25 ± 11.27 176.5 1± 24.58 84.63 ± 13.59 B1I02 3.13 ± 0.88 10.52 ± 1.97 8.5 0± 2.11 52.75 ± 9.75 173.01 ± 10.85 75.12 ± 8.77 Note: I0 = 0 Gy irradiation, I1 = 20 Gy irradiation, I2 = 25 Gy irradiation, I3 = 30 Gy irradiation, B0 = seeds intact, B1 = cut two seeds, B2 = cut three seeds

WIDIASTUTI et al. – Phenotypic variation of Garcinia mangostana after gamma ray irradiation



a c h

d g

i e f

A B C D E E Figure 6. The comparison of the anatomical structure of mangosteen leaf transversely sliced (magnification 400x). A. Control, B. Irradiation of 20 Gy the whole seeds, C. Irradiation of 25 Gy seeds cut in half, D. Irradiation of 30 Gy whole seeds, E. Irradiation of 20 Gy seeds cut in half, F. Seeds cut into two without irradiation. Description: Cuticle of (a), upper epidermis (b), palisade parenchyma (c), sponges (d), below the epidermis (e), lower cuticle (f), beam vessel (g), idioblas (h), gland secretion (i).


b c


A B C D E Figure 7. Comparison of leaf anatomical structure paradermal slices. (100x). Description: A. Control, B. Seed is cut in two, C. 20 Gy, seed cut in two, D. 25 Gy, seed cut three, E. Irradiation of 25 Gy whole seeds. Description: opening of stomata (a), guard cells (b), the neighboring cell (c), and epidermal cells (d).

Almost all plants produced by gamma ray irradiation show a thick palisade and leaves except for I2B2 plants that instead showed the smallest number in thickness. According to Dickison (2000), the plants’ response to gamma-ray radiation that has the nature of ionization can cause changes in the leaf’s anatomy. Leaves may experience changes such as tissue necrosis, distortion of leaves’ bones, changes in the composition and size of palisade tissue and the enlargement of spongy tissue. The differences of the sectional of paradermal slices on the mangosteen’s leaves both the control plants and the ones treated with gamma irradiation are presented in Figure 6. Paradermal section This observation show the stomata of mangosteen leaf is found on the upper part of the leaf. Paradermal slices show that in the epidermal layer of mangosteens leaves there are stomata, guard cells, neighboring cells, and epidermis cells. Observations on the paradermal slice on the mangosteen’s leaves show the characteristics of the observed variations. The highest number of stomata on the plants is found in the plants without irradiation treatment and the plants irradiated with 25 Gy combined with the cutting of the seeds into three equal sizes, while the lowest is obtained in the plants irradiated with gamma ray irradiation at the level of 25 Gy, with an intact form of the seeds. The highest number for epidermis is obtained in the plants with 25 Gy irradiation with seeds cut into 3 (I2B2), while the lowest number is obtained from the plants without irradiation treatment (I0B03). The index for the

stomata ranges from 5.223 to 9.78 (Table 5). The highest index and density of stomata is obtained in the plants without the treatment of gamma irradiation (I0B03), while the lowest ones is obtained from the plants with irradiation of 25 Gy (Figure 7). The mangosteens which are the results of gamma irradiation which survive experience anatomical changes on the leaves. The same thing is also reported by Harahap (2005) and Qosim (2006) of mangosteen leaves that is grown in vitro. Dickison (2000) states that gamma-ray radiation that has ionization in nature can cause changes in the leaf’s anatomical structure. Gamma ray irradiation is known to increase the thickness of the cuticle, epidermis, palisade and leaves in some individuals which are the result of gamma irradiation, although the range of increase varied and showed no pattern of increasing doses of irradiation. According Qosim (2006), the plants that have thick cuticle is more likely to have properties more tolerant to drought because a thicker cuticle can reduce the rate of transpiration of water and can reflect sunlight. Cuticle also serves to protect plants from pests and diseases. Stomatal index observations on paradermal slices shows that in the gamma-ray irradiation treatment, the stomata index has a smaller number than the plants without irradiation (control), the smallest density of stomata is also obtained in the plants irradiated with the level of 25 Gy, whose seed is cut into two equal size. Mangosteen which has stomata with a high density allows a high gas exchange or absorption of CO2 so that the photosynthetic rate becomes higher. With a higher rate, fotosintat, which is the result of

2 (1): 23-33, March 2010

Leaf length -0.16 0.02 -0.07 1

Leaf width 0.13 0.17 0.07 0.74* 1

Results of correlation analysis between variables of the cuticle’s thickness, upper epidermis, lower epidermis, palisade and spongy to the morphologic variables of the mangosteen plants showed that plant’s height, leaf’s width and length are not significantly affected by the thick of the cuticle, upper and lower epidermis, the thickness of palisade parenchyma and the sponges of mangosteen’s leaf (Table 7). The height of the was significantly affected by the width of the leaf with a correlation value of 76%.

Cuticle Upper epidermis Under epidermis Palisade Sponges Leaf length Leaf width Plant height

1 -0.17 0.04 0.01 1 0.08 -0.04 1 0.17 1

0.08 0.17 0.1 0.15 1

Plant height


Leaf width

Table 7. The value of correlation between the thickness of cuticle, upper epidermis, lower epidermis, palisade, and sponges and the length, the width of the leaves and the height of the plant.

Leaf length

Correlation between morphological and anatomical characters Plants growth and development is influenced by factors that are interrelated, both internal and external. The correlation test between the morphological characters namely the height of the plants, the length and width of the leaves, with the stomatal index character and the density of stomata, indicates a positive correlation between the height of the plants with the density of stomata, and the length of the leaves with the their width. The existence of a correlation between the height of the plants and the density of stomata indicates that characteristic is influenced by the density of stomata. The level of correlation between the height of the plants and the density of stomata was 90% (Table 6) which means that the density of stomata has 90% role in determining the height of the plants, while the correlation between width and the length of leaves was 74% (Table 6). High density of stomata allows easier process of photosynthesis so that the fotosintat that can be generated will be greater in results, and then the growth and development of the plant are more supported. Positive correlation between the height of the plant and the density of stomata enable the stomata density parameter to become

Anatomical Stomatal Stomatal Plant characteristics index density height Stomatal index 1 0.02 0.01 Stomatal density 1 0.90* Plant height 1 Leaf length Leaf width


Average Individual Number of Number of Stomatal Stomatal plants stomata epidermis index density b/(mm2) Seeds irradiated before cut B0I01a 13.00 ± 1.22 205.00 ± 27.07 6.06 ± 1.19 184.03 ± 17.33 B0I03b 14.2 0± 1.48 176.20± 17.16 7.49 ± 0.93 200.99 ± 20.99 B0I02 13.20 ± 4.38 205.80± 16.02 6.09 ± 2.23 186.84 ± 62.02 B0I2 10.00 ± 2.01 182.41± 15.46 5.22 ± 1.16 141.54 ± 28.30 B1I01b 14.02 ± 0.71 207.02 ± 26.90 6.38 ± 0.61 198.04 ± 10.01 Seeds irradiated after cut I0B01b 11.61 ± 1.14 194.81 ± 12.29 5.62 ± 0.38 164.18 ± 16.14 I0B02b 14.22 ± 1.64 204.42 ± 22.81 6.56 ± 1.09 200.99 ± 23.25 I0B03 18.81 ± 2.59 174.60 ± 14.18 9.78 ± 1.62 266.10 ± 36.63 I1B1 13.82 ± 2.38 195.00 ± 28.46 6.67 ± 1.25 195.33 ± 33.79 I2B2 18.63 ± 3.13 213.20 ± 39.51 8.23 ± 2.46 263.27 ± 44.30 Note: I0 = 0 Gy irradiation, I1 = 20 Gy irradiation, I2 = 25 Gy irradiation, I3 = 30 Gy irradiation, B0 = seeds intact, B1 = cut two seeds, B2 = cut three seeds.

Table 6. The value of correlation between stomatal index, stomatal density, plant height, leaf length and width.


Table 5. The average number of stomata, number of epidermis, stomatal index, and stomatal density of mangosteen leaf.

a criterion to measure the growth of mangosteen.

Under epidermis

photosynthesis process, the plant’s growth is more supported. Qosim (2006), states the regeneran mangosteen which has a high density of stomata, palisade parenchyma and a high number of file vessels can be used as indirect selection criteria for efficiency. Harahap (2005) states that the study of anatomical structure of the mutant is very useful to explain the changes in the genetic control of certain processes. Fahn (1991) found a recessive mutant of maize which has survived is found to have changed its anatomy in the form of the obstruction towards the differentiation process on the stem’s vessels. Cutter (1969) states that the cells that can grow after irradiation are expected to experience physiological or genetic changes. Irradiated plants that will survive are expected to add diversity to increase the effectiveness of selection.

Upper epidermis



0.02 0.12 0.18 0.25 -0.12 1

-0.1 0.12 0.12 0.01 -0.08 0.24 1

-0.16 0.35 0.29 0.06 -0.07 0.3 0.76* 1

CONCLUSIONS AND RECOMENDATIONS Gamma ray irradiations with the doses of 0, 20 Gy, 25 Gy, 30 Gy, 35 Gy and 40 Gy increase the diversity of morphology of mangosteen by 30%. The highest increase of diversity in the mangosteen obtained in the plants with: (i) the dose of 25 Gy irradiation, (ii) with the seed cut into two equal size, and (iii) the cutting of the seeds done after gamma ray irradiation. The biggest increase of the diversity of the mangosteen is obtained by the method of irradiation on the seeds with the dose of 25 Gy and then arecut across seed into two equal sizes. The density of stomata has a positive correlation with the height of the plants by 90%. The density of stomata can be used as a criteria to estimate the growth of mangosteen. To get a mangosteen with greater diversity, it is advisable to perform irradiation on the mangosteen with the dose of 25 Gy with a more number of the research materials.

WIDIASTUTI et al. – Phenotypic variation of Garcinia mangostana after gamma ray irradiation

REFERENCES Ahnstroem G. 1977. Radiobiology. In: Manual on mutation breeding. 2nd ed. Tech. Report Series No. 119. Joint FAO/IAEA. Division of Atomic Energy in Food and Agriculture. Vienna. Ashri A. 1970. Inheritance of small leaflets in a wide cross in peanuts, Aracis hypogaea. Oleagineux 35: 153-154. Baddiganavar AM, Murty GSS. 2007. Genetic enhancement of groundnut throught gamma ray induced mutagenesis. Plant Mutation Rep 1 (3): 16-21. Baihaki A. 1999. College textbooks engineering design and analysis of breeding research. Faculty of Agriculture, Padjadjaran University. Jatinangor. [Indonesia] Chen LG, Yang LL, Wang CC. 2008. Anti-Inflamantry activity of mangosteen from Garcinia mangostana. Food Chem Toxicol 46: 688693. Chomnawang MT, Surassmo S, Wongsariya K, Bunyapraphatsara N. 2009. Antibacterial activity of Thai medicinal plants against methicillin-resistant Staphylococcus aureus. Fitoterapia 80 (2): 102104. Cox JEK. 1988. Garcinia mangostana, mangosteen In: Gardner RJ, Chaudari SA (eds). The propagation of tropical fruits trees. Anthony Rowe Ltd. Chippenham, Wiltshire, England. Crowder LV. 1997. Plants genetics. Gadjah Mada University Press. Yogyakarta. [Indonesia] Cutter EG. 1969. Plant anatomy: experiment and interpretation. part 1. pell and tissues. William Clowers and Sons. London. Datta SK. 2001. Mutation studies on garden Chrysanthemum. A Review. Sci. Hort. 7 : 159-199. Dickison WC. 2000. Intregative plant anatomy. Academic Press. Tokyo. Fahn A. 1991. Anatomi tumbuhan. Gajah Mada University Press. Yogyakarta. Fauza H, Karmana MH, Rostini N, Mariska I. 2005. Growth and phenotypic variability of gamma ray irradiated mangosteens. Zuriat 16 (2): 133-144. [Indonesia] Gou HJ, Liu LX, Han WB, Zhao SR, Zhao LS, Sui L, Zhao K, Kong FQ, Wang J. 2007. Biological effect of high energy Li ion beams implantation in wheat. Plant Mutation Rep 1 (3): 31-35. Handayati W, Darliah, Mariska I, Purnamaningsih R. 2001. Increasing genetic diversity of the mini roses through in-vitro culture and gamma irradiation. Berita Biol 5 (4): 365-371. [Indonesia] Harahap F. 2005. Induction of genetic variation of mangosteen (Garcinia mangostana) with gamma radiation. [Dissertation]. School of Graduates, Bogor Agricultural University. Bogor. [Indonesia] IRB [Institut of Radiation Breeding]. 2001. National Institute of Agrobiological Resurces, MAFF. PO Box 3, Ohmiya Macni, Nacagun, Ibaraki, 312-22, Japan. Koltunow AM, Grossniklaus U. 2003. Apomixis: a developmental prespective. Ann Rev Plant Biol 54: 547-574.


Manjaya JG, Nandanwar RS. 2007. Genetic improvement of soybean variety JS 80-21 trough induced mutation. Plant Mutation Rep 1 (3): 36-40. Mansyah E, Anwarudinsyah MJ, Sadwiyanti L, Susilohadi A. 1999. Genetic variability of mangosteen through isozyme analysis and its relation to phenotypic variability. Zuriat 10 (1): 1-10. [Indonesia] Mansyah E, Santoso PJ, Muas I, Sobir. 2008. Evaluation of genetic diversity among and within mangosteen (Garcinia mangostana L.) trees. 4th International Symposium on Tropical and Subtropical Fruits. Bogor, West Java. Indonesia. November 3-7, 2008. Mohr H, Schopfer P. 1995. Plant physiology. Springer. Berlin. Moore TC. 1979. Biocemistry and physiology of plant hormones. Springer. New York. Morton J. 1987. Breadfruit. In: Fruits of warm climates. Miami, FL. Mugiono. 1996. Effect of gamma irradiation on chlorophyll mutation and genetic variation of blast disease resistance in upland rice. Zuriat 7 (1): 15-21. [Indonesia] Poerwanto R. 2000. Mangosteen cultivation technology. National Discussion of Business and Technology Mangosteen. Bogor, 15-16th November 2000. [Indonesia] Qosim WA. 2006. Studies of gamma irradiation on the culture of nodular callus of mangosteen to enhance genetic diversity and morphology regenerant. [Dissertation]. School of Graduates, Bogor Agricultural University. Bogor. [Indonesia] Ramage CM, Sando L, Peace CP, Carroll BJ, Drew RA. 2004. Genetic diversity revealed in the apomictic fruit species Garcinia mangostana L. (mangosteen). Euphytica 136: 1-10. Richards AJ. 1990. Studies in Garcinia, dioecious tropical fruit trees: agamospermy. Bot J Linn Soc. 103: 233-250. Rohlf FJ. 2002. NTSYS-PC numerical taxonomy and multivariate analysis system, Version 2.01. Owner's manual. Exeter Publication. Setauket, NY, USA. SAS [SAS Institute Inc]. 2004. What’s new in SAS® 9.0, 9.1,9.1.2, and 9.1.3. SAS Institute Inc. Cary, NC. Sobir, Poerwanto R. 2007. Mangosteen genetic and improvement. Intl J Plant Breed 1(2): 105-111. Soeranto H. 2003. The role of nuclear science and technology in plant breeding to support the agricultural industry. Center for Research and Development of Isotopes and Radiation Technology. National Agency of Nuclear Energy (BATAN) Jakarta. [Indonesia] Uji T. 2007. Diversity, distribution, and the potential of Garcinia species in Indonesia. Berk Penel Hayati 12: 129-135. [Indonesia] Van Harten AM. 1998. Mutation breeding. Theory and Practical Aplication. Press Syndicate. University of Cambridge, UK. Verheij EWM, Coronel RE. 1991. Plants Resources of South East Asia No 2. Edible fruits and nuts. Pudoc. Wageningen. Wible J, Chack EK, Downtown WJS. 1992. Mangosteen (Garcinia mangostana L.) a potential crop for tropical Northern Australia. Acta Hor 321: 132-137. Willmer CM. 1983. Stomata. Longman. London.

ISSN: 2087-3948 (print) ISSN: 2087-3956 (electronic)

Vol. 2, No. 1, Pp. 34-37 March 2010

First record of two hard coral species (Faviidae and Siderastreidae) from Qeshm Island (Persian Gulf, Iran) MAHDI MORADI1,♥, EHSAN KAMRANI1, MOHAMMAD R. SHOKRI2, MOHAMMAD SHARIF RANJBAR1, MAJID ASKARI HESNI3 1

Department of Marine Biology, School of Basic Sciences, University of Hormozgan, P.O. Box 3995, Bandar Abbas, Iran. Tel: +98-914-9818304; Fax: +98-761-766 0012. ♥email: 2 Department of Marine Biology, Faculty of Biological Sciences, Shahid Beheshti University, G.C., Evin, Tehran 1983963113, Iran 3 Department of Biology, Faculty of Science, Shahid Bahonar University of Kerman, Kerman, Iran. Manuscript received: 30 December 2009. Revision accepted: 13 February 2010.

ABSTRACT Abstract. Moradi M, Kamrani E, Shokri MR, Ranjbar MS, Hesni MA (2009) First record of two hard coral species (Faviidae and Siderastreidae) from Qeshm Island (Persian Gulf, Iran). Nusantara Bioscience 2: 34-37. Two species of hard corals including Cyphastrea chalcidicum (Forskal 1775) (Faviidae) and Coscinaraea monile (Forskal 1775) (Siderastreidae) were collected from the south of Qeshm Island (Persian Gulf, Iran) in the late of 2008. These species were previously reported from southern Persian Gulf, Gulf of Aden, Southeast Africa and Indo-Pacific. The literature review on the distribution of these two species revealed that these species were firstly recorded from the Persian Gulf. These findings further emphasize the high diversity of coral fauna in the Iranian waters of the northern Persian Gulf. Key word: first record, Coscinaraea monile, Cyphastrea chalcidicum, Qeshm Island, Persian Gulf.

Abstrak. Moradi M, Kamrani E, Shokri MR, Ranjbar MS, Hesni MA (2009) Rekaman pertama dua spesies karang keras (Faviidae dan Siderastreidae) dari Pulau Qeshm (Teluk Persia, Iran). Nusantara Bioscience 2: 34-37. Dua jenis karang keras termasuk Cyphastrea chalcidicum (Forskal 1775) (Faviidae) dan Coscinaraea monile (Forskal 1775) (Siderastreidae) dikumpulkan dari selatan Pulau Qeshm (Teluk Persia, Iran) pada akhir tahun 2008. Spesies ini sebelumnya dilaporkan terdapat di Teluk Persia selatan, Teluk Aden, Afrika Tenggara dan Indo-Pasifik. Tinjauan literatur pada distribusi kedua jenis mengungkapkan bahwa spesies ini pertama kali tercatat dari Teluk Persia. Temuan ini semakin menunjukkan tingginya keragaman fauna karang di perairan Iran di bagian utara Teluk Persia.

Kata kunci: catatan pertama, Coscinaraea monile, Cyphastrea chalcidicum, Qeshm island, Persian gulf.

INTRODUCTION The Persian Gulf has a complex and unique tropical marine ecosystem, especially coral reefs, with relatively low biological diversity and many endemic species (Price 1993) In this area, the coral reef communities are occurred in the form of non-reef setting (Riegl 1999) and surrounded by some of the driest landmasses in the world, such that continental influences are limited (Price 1993). While large parts of the region are still in a pristine condition, several anthropogenic threats notably habitat destruction, overexploitation and pollution are ever-increasingly disturbing the coral reef communities, . The coral reef communities in the Persian Gulf are less diverse than that of Indian Ocean (Price 1993). This is due to the high salinity; high daily amplitude of temperature (Coles and Fadlallah 1991) and occasional extreme low tides (Reynolds 1993) that make the environmental condition is unfavorable to coral reef communities.

The largest island in Persian Gulf is Qeshm Island (ca. 122 km long, 18 km wide on average, 1,445 sq km). This island is located a few kilometers off the southern coast of Iran (Persian Gulf), about 22 km south of Bandar Abbâs and not far from Bandar Khamir (DHI 1976). A study was conducted to explore the species diversity of hard corals in Qeshm Island, in order to bridge the gap of knowledge on species inventory of hard corals in this area. The results of the study have been presented in detail elsewhere (Moradi et al., in prep.) and this paper presents only the new recordings of two hard coral species from Qeshm Island (Persian Gulf, Iran).

MATERIAL AND METHODS The coral survey was conducted in August 2008. Hard coral specimens were collected by SCUBA diving from two sites (Naz and Zeitoon) within shallow non-reef

MORADI et al. – First record of two hard coral species from Qeshm Island

settings (6 to 10 m deep) with hard ground substrate in the south of Qeshm Island, (Iran, Northern Persian Gulf). The geographical positions of the sampling sites were N 26º 49' 19.4" and E 56º 07' 23.1" for Naz station, and N 26º 55' 40.15" and E 56º 15' 54.82" for Zeitoon Station (Figure 1). The coral specimens were bleached using hydro peroxide and photographed showing the whole specimen and the corallite structures. Identifications were performed using available references, especially Veron (2000), and through communication with Prof. Charles Sheppard at the Dept. of Biological Sciences, Warwick University for further checking. The materials are deposited in at the Faculty of Marine Biology, University of Hormozgan, Iran.

RESULTS AND DISCUSSION Results Twenty one species of hard corals belonging to 8 families were identified and with resulting Poritidae and Faviidae as the dominant families. Two species, Coscinaraea monile, (Forskal 1775) (Family: Siderastreidae) and Cyphastrea chalcidicum (Forskal 1775) (Family: Faviidae) were new records from northern Persian Gulf. Coscinaraea monile (Forskal 1775) Kingdom Animalia Phylum Coelenterata Frey and Leuckart 1847 Subphylum Cnidaria Hatschek 1888 Class Anthozoa Ehrenberg 1831 Subclass Zoantharia de Blainville 1830 Order Scleractinia Bourne 1900 Family Siderastreidae (Vaughan and Wells, 1943) Genus Coscinaraea (Forskal 1775) Coscinaraea monile (Forskal 1775) (Figure 2) Taxonomic references: Scheer and Pillai (1983) Material examined: Qeshm Island, Naz Island, depth 811m, collector M. Moradi, 24 August 2008. Diagnosis characters: Colonies are encrusting or massive, 10-30 cm in diameter sometimes larger. Corallites is are 2.5 to 3.5 millimeters in diameter and form a liner series in meandroid valleys. In some cases, there is no demarcation between adjacent corallites and others. Tthere is an irregular, low, thin wall marking the boundary. In massive colonies, calices are 2-4 mm in diameter; in explanate corolla, calices are 3-6 mm in diameter. Up to 30 septa occur at the wall, but only 8-9 reach the columella due to fusion of adjacent septa;, septa and septocostae are lightly granulated and the marigine (?) are divided into sharp dentations. Color: Light brown Habitat: Abundant in 8-10 meter depths Distribution: This species is confined to the Indian Ocean and is are mostly common along the shores of the southern Persian Gulf, Oman Sea Red Sea, Gulf of Aden (Veron 2000) and Southeast Africa, (Riegl 1996). Cyphastrea chalcidicum (Forskal 1775) Family Faviidae (Gregory, 1900) Genus Cyphastrea (Forskal 1775)


Cyphastrea chalcidicum (Forskal 1775) (Figure 3) Taxonomic references: Veron, Pichon and WijsmanBest (1977), Wijsman-Best (1980). Material examined: Qeshm Island, Zeyton Park, depth 2-5 m, collector M. Moradi, 19 June 2008. Diagnosis characters: Colonies are encrusting to massive, usually about 20-35 cm in diameter Corallites are round, variably exsert, usually about 1.5-2.5 mm diameter, budding is extratentacular. There are 20-26 septa arranged in 2 orders, inner septal margins of primaries and secondaries carry rounded dentations and descend into the calices at about 45 degree angle. All primaries reach the columella, some secondaries do not. Septa are sparsely granulated and septa are not continuous with those of adjacent corallites. Septocostae are sub-equally exsert about 0.5 mm above the wall. Costae are equally exsert, the columella is small less than 0.4 mm in diameter composed of tangled by synapticular ring. The coenosteum is covered with short tapering spines. Color: Usually uniform brown, green or cream with corallite walls and calices of contrasting colors. Habitat: Abundant in 3 meter depths. Distribution: This species is confined to the Indian Ocean and are mostly common along the shores of Red Sea, Gulf of Aden (Veron 2000) and Southeast Africa (Riegl 1996). Discussion Harger (1984) reported 19 species of corals at Hormuz Island in the east of Qeshm Island, Persian Gulf. Staghorn corals (Acropora sp.) were found to be the dominant species around the islands in the Persian Gulf (Sheppard and Sheppard 1991), whereas the massive corals (Poritidae, Favidae) are dominant corals at present. Staghorn corals are defined as disturbance-adapted types for their rapid growth rate and fragility (Done, 1982; Karlson and Hurd 1993). Massive and submassive corals being defined as stress-tolerators (Veron, 1986; Rogers 1990) are shown to tolerate to the high sedimentation and/or eutrophication. Presence of massive corals in Qeshm Island suggests that the reef corals reefs in this island are likely subjected to high sedimentation and/or eutrophication. The species found in the present study are massive types that are confined to the Indian Ocean, mostly common along the shores of Persian Gulf, Oman Sea, Red Sea and Gulf of Aden (Veron 2000). Cyphastrea chalcidicum (Forskal 1775) was reported from Southeast Africa (Riegl 1996), Gulf of Aden, Indo-Pacific and Indiana Ocean (Veron 2000) and Coscinaraea monile (Forskal 1775) was reported from Northern Red Sea (Riegl & Velimirov 1994), Southeast Africa (Riegl 1996), Dubai, (Riegl 1999), Oman sea (Coles 1996), Gulf of Aden, Indo-Pacific and Indiana Ocean (Veron 2000). The shift in coral diversity from disturbance-adapted types (Acropora branching corals) in the past to stresstolerators (Favia and Porites massive and submassive corals) at present indicates that coral species composition in Qeshm Island have been altered over three decades (1984 to present). The corals in Persian Gulf have recently experienced multiple bleaching events (1996 1998 2002)



2 (1): 34-37, March 2010

Naz Island



Figure 1. Study area and location of sampling sites, Qeshm Island (Persian Gulf, Iran). 1. Zeyton Park, 2. Naz Island (above insert).



Figure 2. Concinarae monile, A: Colony, B: Corallites. Bar = 2 cm


Figure 3. Cyphastrea chalcidicum, A. Colony, B. Corallites. . Bar = 2 cm


MORADI et al. â&#x20AC;&#x201C; First record of two hard coral species from Qeshm Island

(Pilcher, et al. 2000; Wilkinson 2000; Wilson et al. 2002; Rezaei et al. 2004) causing mass mortality of Acropora corals in the entire region. The climatic change revealed in multiple bleaching events associated with high sedimentation and/or eutrophication in this area may be possible factors altering the coral species diversity in the study area. Further studies are required in the Persian Gulf to reveal the possible effects of climate change on reef corals.


Two species of hard corals including Cyphastrea chalcidicum (Forskal 1775) (Faviidae) and Coscinaraea monile (Forskal 1775) (Siderastreidae) were firstly recorded from the south of Qeshm Island (Persian Gulf, Iran). These species were previously reported from southern Persian Gulf, Gulf of Aden, Southeast Africa and Indo-Pacific. These findings further emphasize the high diversity of coral fauna in the in Iranian waters of the northern Persian Gulf.


We would like to express our appreciation to Mrs. Lale Daraei management of GEF/SGP in Iran, Abdullah Salehi and Mohammad Dakhte management of Geopark Institute of Qeshm Island who assisted us in filed sampling. We are also greatly honored and thankful to Prof. Charles Sheppard at the Department of Biological Sciences, Warwick University, U.K. for assistance in identifications of the specimens.


REFERENCES Coles SL, Fadlallah YH (1991) Reef coral survival and mortality at low temperatures in the Arabian Gulf: New species-specific lower temperature limits. Coral Reefs 9:231-237. Coles SL (1996) Corals of Oman. R. Keech Publ., Thorns, Hawes, UK. Deutsches Hydrographisches Institut [DHI] (1976) Handbuch des Persischen Golfs. 5th ed. Deutsches Hydrographisches Institut, Hamburg. Done TJ (1982) Patterns in the distribution of coral communities across the central Great Barrier Reef. Coral Reefs 1:95-107. Harger JRE (1984) Rapid survey techniques to determine distribution and structure of coral communities. In: Comparing coral reef survey methods. UNEP-UNESCO Workshop. [Thailand] Karlson RH, Hurd LE (1993) Disturbance, coral reef communities, and changing ecological paradigms. Coral Reefs 12: 117-125. Pilcher NJ, Wilson S, Alhazeem SH, Shokri MR (2000). Status of coral reefs in the Persian Gulf and Arabian Sea Region (Middle East). In: Wilkinson C (ed) Status of coral reefs of the world 2000. AIMS Press, Australia. Price ARG (1993) The Gulf: Human impact and management initiatives. Mar Poll Bull 27: 17-27. Rezaei H, Wilson S, Claereboudt M, Riegl B (2004) Coral reef status in the ROPME Sea Area: Arabian/Persian Gulf, Gulf of Oman and Arabian Sea. In: Wilkinson C (ed) Status of coral reefs of the world 2004. AIMS Press, Australia. Riegl B, Velimirov B (1994). The structure of coral communities at Hurghada in the northern Red Sea. P.S.Z.N. I: Mar Ecol 15: 213-231. Riegl B (1996) Hermatypic coral fauna of subtropical southeast Africa: A checklist. Pacific Science 50: 404-414. Riegl B (1999) Corals in a non-reef setting in the southern Arabian Gulf (Dubai, UAE): fauna and community structure in response to recurring mass mortality. Coral Reefs 18: 63-73. Reynolds RW, Marisco DC (1993) An improved real-time global sea surface temperature analysis. J Climate 6: 114-119. Rogers CS (1990) Responses of coral reefs and reef organisms to sedimentation. Mar Ecol Prog Ser 62: 185-202. Sheppard CRS, Sheppard ALS (1991) Corals and coral communities of Arabia. Fauna of Saudi Arabia 12: 3-170. Veron JEN (1986) Corals of Australia and the Indo-Pacific. Angus and Robertson publishers, Australia. Veron J (2000) Corals of the world. Australian Institute of Marine Science, Australia. Wilkinson C (2004) Status of coral reefs of the world. AIMS Press, Townsville, Australia. Wilson S, Fatemi SMR, Shokri MR, Claerebout M (2002) Status of coral reefs of the Persian Gulf and Arabian Sea Region. In: Wilkinson C, (ed) Status of coral reefs of the world 2002. AIMS Press, Australia.


ISSN: 2087-3948 (print) ISSN: 2087-3956 (electronic)

Vol. 2, No. 1, Pp. 38-42 March 2010

Isolation and identification of lactic acid bacteria from abalone (Haliotis asinina) as a potential candidate of probiotic 1

SARKONO1,â&#x2122;Ľ, FATURRAHMAN1, YAYAN SOFYAN2 Faculty of Mathematics and Natural Sciences, Mataram University, Jl. Majapahit 62 Mataram 83125, West Nusa Tenggara, Indonesia. Tel./Fax.: +62370-646506 â&#x2122;Ľemail: 2 Institute for Marine Aquaculture, Grupuk, Sengkol, Pujut, Central Lombok 83511, West Nusa Tenggara, Indonesia. Manuscript received: 13 December 2009. Revision accepted: 15 March 2010.

Abstract. Sarkono, Faturrahman, Sofyan Y. 2010. Isolation and identification of lactic acid bacteria from abalone (Haliotis asinina) as a potential candidate of probiotic. Nusantara Bioscience 2: 38-42. The purpose of this study was to isolate, select and characterize lactic acid bacteria (LAB) from abalone as a potential candidate probiotic in abalone cultivation system. Selective isolation of LAB performed using de Man Rogosa Sharpe medium. LAB isolate that potential as probiotics was screened. Selection was based on its ability to suppress the growth of pathogenic bacteria, bacterial resistance to acidic conditions and bacterial resistance to bile salts (bile). Further characterization and identification conducted to determine the species. The results showed that two of the ten isolates potential to be developed as probiotic bacteria that have the ability to inhibit several pathogenic bacteria such as Eschericia coli, Bacillus cereus dan Staphylococus aureus, able to grow at acidic condition and bile tolerance during the incubation for 24 hour. Based on the API test kit, the both of isolate identified as members of the species Lactobacillus paracasei ssp. paracasei. Key word: lactic acid bacteria, isolation, identification, Lactobacillus paracasei ssp. paracasei.

Abstrak. Sarkono, Faturrahman, Sofyan Y. 2010. Isolasi dan identifikasi bakteri asam laktat dari induk abalon (Haliotis asinina) yang berpotensi sebagai kandidat probiotik. Nusantara Bioscience 2: 38-42. Tujuan penelitian ini adalah untuk mengisolasi, menyeleksi dan mengkarakterisasi Bakteri Asam Laktat (BAL) dari induk abalon yang berpotensi sebagai kandidat probiotik pada sistem budidaya abalon. Isolasi selektif BAL dilakukan menggunakan media de Man Rogosa Sharpe Agar. Isolat BAL yang berpotensi sebagai probiotik diskrining. Pemilihan ini didasarkan atas kemampuannya dalam menekan pertumbuhan bakteri patogen, resistensi terhadap kondisi asam, resistensi terhadap bile salt (empedu). Selanjutnya dilakukan karakterisasi dan identifikasi untuk mengetahui spesiesnya. Hasil penelitian menunjukkan bahwa 2 di antara 10 isolat yang berhasil diisolasi dari abalon berpotensi untuk dikembangkan menjadi bakteri probiotik karena mempunyai kemampuan menghambat beberapa bakteri patogen yaitu Eschericia coli, Bacillus cereus dan Staphylococus aureus, mampu tumbuh pada kondisi asam dan toleran terhadap cairan empedu selama inkubasi 24 jam. Berdasarkan uji API Kit, kedua isolat teridentifikasi sebagai anggota spesies Lactobacillus paracasei ssp. paracasei. Kata kunci: bakteri asam laktat, isolasi, identifikasi, Lactobacillus paracasei ssp. paracasei.

INTRODUCTION One type of shellfish that has the potential and economic value is the seven eyes shelfish. Seven eyes shelfish (Haliotis asinina) is also called abalone, awabi, mutton fish, sea ear and in local language (Sasak, Lombok) it is called medau. Abalone is included in univalve shellfish species (Cholik et al. 2005) which the meat has high nutritional value with protein content of 71.99% and 3.20% fat. Its shell also has an aesthetic value that can be used for jewelry, the manufacture of buttons and various other forms of handicraft goods (Imai 1997). Abalone is mostly found in Eastern Indonesia (Bali, Lombok, Sumbawa, Sulawesi, Maluku and Papua). On the island of Lombok abalone is often found in southern coast of central Lombok called Kute Beach and surrounding areas. During this time abalone have been exploited by local residents without any proper selection, resulting reduction in the catch and in the long term may threaten its sustainability.

The effort of Abalone cultivation technology ranging from domestication, a trial of gonal maturation in a controlled basin, spawning, larval rearing, and larval food preparation have been done (Sofyan et al. 2005), but these activities do not give a satisfactory result. Survival rate of abalone larvae in larval rearing tanks until this time is still very low at around 1.0%. Mortality was happening a lot on planktonic stage until attachment to the substrate (first weeks). Low larval survival rate is among others due to water filtration systems that is poor which resulted in the emergence of protozoa, worms and various types of pathogenic microorganisms that can cause death of larvae. One effort to prevent the occurrence of population shifts (split population) and also suppress the growth of pathogenic microorganisms is to maintain the natural balance of microorganisms in the larval rearing system (Haryanti et al. 1997) through the addition of probiotic microorganisms (Fuller 1989). Prevention of disease was taking place by controlling the growth of potentially

SARKONO et al. – Probiotic candidate of lactic acid bacteria from abalone

pathogenic microbes in the gastrointestinal tract (Strompfova et al. 2005; Iñiguez-Palomares et al. 2007) and a number of positive effects of probiotic bacteria including immunomodulastion (Wallace et al. 2003). Development of probiotics for the cultivation of abalone would be better if the probiotic microbes are indigenous abalone itself, so as to avoid the problem of microbial adaptation on larval rearing tanks and seven channels of the body of this seven eyes shellfish when applied. It is therefore important to do research on indigenous bacteria isolation and identification of potentially probiotic abalone.

MATERIALS AND METHODS Isolation of LAB strains from abalone A total of 20 examples of healthy male and female seven eyes shellfishes are obtained from the Institute for Marine Aquaculture Lombok. Then the fluid from the digestive tract is taken in a sterile way as much as 1-10 g. Selective isolation of Lactic Acid Bacteria (LAB) is performed with Spread plate method developed by Brashear et al. (2003) and Ray et al. (1997). A total of 1 g sample added into 10 mL of fever Rogosa Sharpe (MRS) broth sterile and mixed until homogeneous. The suspension is then spread on MRS medium pH 5.5 plus 0.1% Naazide, each in-trade with calcium carbonate 1%. Furthermore, the petri plates were incubated at 37oC for 48 hours in an incubator in a microaerophilic atmosphere. Single colonies that grew were taken from each plate and transferred into test tubes containing 10 mL MRS broth. Then they were incubated at 37oC for 18-72 hours to obtain maximum growth cultures. Culture isolates were streaked again on MRS media for the petri plates and incubated at 37oC for 48 hours to obtain a single colony/pure culture. In pure culture Gram stain is done for initial identification. Lactic acid bacteria culture obtained is stored by freezing the temperatures. Stock to be used was prepared by growing the isolated bacteria in MRS liquid medium and incubated at 37oC for 24-48 hours (Rahayu et al. 2004). Test of LAB strains antibacterial power LAB isolates were tested their ability to inhibit the growth of pathogenic bacteria namely Eschericia coli, Bacillus cereus and Staphylococcus aureus by using well diffusion assay. Each isolate was treated in the form of fermentation result supernatant containing the extracellular metabolites, which are obtained by inoculating liquid culture of isolate lactic acid bacteria as much as 2% into the liquid media fever Rogosa Sharpe (pH 6.5) and then incubated at 37°C for 96 hours (Bar et al. 1987). After incubation pH measurements were taken, subsequently centrifugation was done upon liquid culture using a centrifuge with a speed of 3500 rpm for 20 minutes. Supernatant obtained was sterilized with bacterial filter (porous diameter of 0.2 μm, Whatman) in order to obtain sterile extracellular metabolites. Antibacterial test was conducted using well diffusion assay developed by Djafaar et al. (1996) and modified by Sarkono et al (1996), by plating test bacterium E. coli, B.


cereus and S. aureus in petri disk with Nutrient Agar solid medium, then added by Nutrien Agar soft medium on it. After being cooled for 1 hour in a refrigerator room, a well was made with a diameter of 0.7 mm and then isolates of bacterial supernatant was inserted and incubated at 37oC for 24-48 hours. The diameter of each isolate contained clear zone is measured. Test of tolerance towards acid and bile The tolerance Isolates LAB which inhibits the growth of pathogenic bacteria extensively was screened towards acids and bile. Tolerance test towards the acid uses the method of Brashear et al. (2003). LAB fresh culture harvested from MRS broth by centrifugation and the pellet obtained was washed and suspended with sterile phosphate buffer saline (PBS). Each strain was added by 4 mL of sterile PBS and pH was adjusted to pH 2, 4, 5 and 7 (control) and incubated for 2, 4 and 24 hours in a water bath at a temperature of 37°C. After each incubation period, the growth of strains can be identified by measuring the absorbance at 620 nm. Bile tolerance test was using the method of Gilliland et al. (1984). Fresh cultures of selected LAB isolates were inoculated into tubes containing 10 mL MRS broth with levels 0 (control), 0:05, 0:15 and 0.3% oxgall. Inoculated tubes were incubated at 37°C in a water bath. Growth of isolates was observed at 2, 4, 6, and 24 hours by measuring absorbance at 600 nm. Early identification of isolates with the API Initial identification made to isolated LAB with inhibitory activity on the growth of E. coli, B. cereus and S. aureus and their tolerance to acid and bile. LAB isolates were identified through fermentation patterns with index of profile analysis standard test with 50CHL API Kit (Biomerieux 2009).

RESULTS AND DISCUSSION Selective isolation of Lactic Acid Bacteria from abalone Isolation process yields 10 colonies which were suspected as isolates Lactic Acid Bacteria (LAB) because they produces a clear zone in isolation medium (Figure 1), then a strengthened test was conducted by growing on solid MRS medium plus CaCO3 1%. From this confirmation test by re-growing process showed that all 10 isolates LAB could grow well and produce clear zones around colonies. The characterization results further prove that the 10 isolates allegedly a member of the lactic acid bacteria (Table 1). Results of identification at the genus level confirm that the four isolates that were characterized are members of the genus Lactobacillus. These isolates have a phenotypic characters among others the forms of stem cell are long, the structure resembles a fence and row of cells singly scattered, gram positive reaction, not motile and do not form endospores (Sneath et al. 1986). Images of each isolate cell can be seen in Figure 2.



2 (1): 38-42, March 2010 Table 1. Test results that characterized the feature of Lactic Acid Bacteria isolated from abalone


Resources Digestive organs Digestive organs Digestive organs Digestive organs Digestive organs Digestive organs Digestive organs Sea water Seaweed Abalone feces

Feature that characterized Lactic Acid Bacteria Cell Gram EndoKataase Motility shape reaction spora Stem + Stem + Stem + Stem + Stem + Stem + Stem + Stem + Stem + Stem + -

Test of antibacterial power LAB strains against pathogenic bacteria E. coli, B. cereus and S. aureus The result of bacterial growth inhibition test with the indicator diffusion method showed that seven among ten isolates showed the ability to inhibit the growth of bacteria, characterized by the formation of clear zones around the wells with varied sizes. Three isolates had the ability to inhibit the three bacterial indicators as well as the isolates OPA3, OPA4 and AL1. Three isolates could inhibit the growth of two indicator bacteria namely OPA5, OPA6 and OPA7.

Holozone (mm)

Figure 1. Colonies are indicated as LAB with clear zones around colonies

E. coli S. aureus B. cereus

Type of isolates Figure 3. Antibacterial test isolates LAB supernatant against indicator bacteria Eschericia coli, Staphylococcus aureus and Bacillus cereus with well diffusion method











Figure 2. Gram reaction and cell shape of LAB isolates which were isolated by seven eye mussel (abalone) and their habitats

SARKONO et al. â&#x20AC;&#x201C; Probiotic candidate of lactic acid bacteria from abalone

Meanwhile, only one isolate which is only able to inhibit the growth of one indicator bacteria namely OPA1 isolates, whereas three other isolates namely OPA1, RL1 and KA1 did not have the ability to inhibit any bacterial indicator (Figure 3). Based on the character of inhibition zone, ten isolates tested showed different characters of inhibitions, but in general some of them showed inhibition zone with blurred edges (not firm) and others showed inhibition zone with firm edges. Blurred edges zone indicates that the active metabolite found in the supernatant is bacteriostatic, which only inhibit cell growth of indicator bacteria but not kill the cell. According Rahayu (2004), inhibition with vague zone might be the action of acid and other antibacterial components which are only bacteriostatic, since most bacterial test (indicator) remains alive in the clear zone, although with very slow growth. Meanwhile inhibition zone with a firm edge indicates that isolates have the ability to produce metabolites which are bactericidal, where metabolites can kill bacterial cells indicator. This is one of the expected ability of probiotic bacteria so it can control the growth of pathogenic bacteria in their applications. Test of tolerance to acid and bile Based on the results of testing the ability of inhibition on the growth of pathogenic bacteria in a previous study phase and then continued by selecting two isolates that had the best inhibitory then proceed with the test of isolates growth in an atmosphere of acid and bile. Data obtained from this test form absorbance data using a spectrophotometer. The addition of absorbance values in line with the addition of incubation time showed the growth of LAB isolates tested (Figure 4). Figure 4 show that both of tested Lactic Acid Bacteria isolates showed the ability to grow in acidic environment which is relatively similar. Isolate OPA4 and AL1 have excellent adaptability to acid atmosphere, because an increase in growth at 3 pH levels in 24-hour period. At pH 2 the two isolates did not grow, because the pH of 2 is a very extreme pH for growth of microorganisms, including lactic acid bacteria which are generally well adapted to living in habitats with a relatively low pH environment. At pH 4, 5 and 7 both isolates are able to grow well, the exponential increase in growth occurred in the observation at 24th hour because of the incubation period is long enough from the 4th hour up to the 24th hour resulting in significant cell division. Isolate OPA4 achieve the best growth at pH 7 whereas at pH 5 isolates AL1. Lactic acid bacteria


generally prefer the atmosphere of a pH slightly below a neutral pH for best growth (Axellson 1998). The result of the endurance test isolates of bile showed that the four isolates had a very, very good ability, because of an increase in growth in the overall level of concentration of bile (0.05%, 0.15%, and 0.30%) in 24-hour period (Figure 4). The ability to grow of the two isolates namely AL1 OPA4 in the bile can not be distinguished from each other. This is predicted caused by the very low concentration of bile that is used. The tests for resistance toward bile liquid used method that was developed by Gilliand et al. (1984) which uses bile concentration of 0.05%, 0.15%, and 0.30%. As a comparison, other researcher (Ljungh et al. 2002) tested the resistance of isolates Lactobacillus paracasei subsp. paracasei F19 in 20% bile and continues to show growth on incubation time of 2 hours. Early identification of isolates with the API API biochemical test kits are used to determine the biochemical characteristics of LAB isolates that are tested so that it can be used for identification purposes. Because the two LAB isolates tested are members of the genus Lactobacillus, so we only use the API Kit 50CHL content of which is 49 kinds of sugar and its derivatives, plus one negative control so in overall there are 50 types of test (Biomerieux 2009). Visually Kit API 50CHL represented by Figure 5.



Figure 5. Visualization of the results of sugar fermentation test with API Kit 50CHL (a) isolates AL1 48 hours and (b) isolates OPA4 48 hours

A B C D Figure 4. Selected isolate growth test during 24 hours incubation time. A. Isolates OPA4 in bile, B. Isolates AL1 in bile, C. Isolates OPA4 in in the acid atmosphere, D. Isolates AL1 in the acid atmosphere


2 (1): 38-42, March 2010

The test of sugar fermentation is a very important characterization process in the genus Lactobacillus to know the character to the identification of species diversity (Holt et al. 1994). The result of sugar test with the API kit toward 10 isolates of the isolated form of positive character (+) and negative (-) which in total amounted to 50 characters, then analyzed by a computer program ApiwebTM Version 1.2.1 to identify the species name. The test results showed that after 48 hours incubation AL1 OPA4 isolates gave the same results, that are positive reactions on sugar numbers 5, 10, 11, 12, 13, 14, 16, 18, 19, 21, 22, 23, 24, 25, 26, 27, 28, 31, 32, 34, 39.40, 41, 42 and 47, the rest react negatively. This shows the level of characters with very high similarity between the two isolates, so it is possible that they are from the same strain, at least a member of the same species. The test results which is in the form of sugar fermentation profile was analyzed with the program ApiwebTM version 1.2.1, the result is that the two isolates tested is a member of the same species of Lactobacillus paracasei ssp. paracasei. This species has a very close relationship, and even considered as neotype strain of Lactobacillus lactic species (Dellaglio et al. 2002). According Vlieger et al. (2009) members of this species have been applied as probiotic bacteria in infant milk together with Bifidobacterium.

CONCLUSIONS AND SUGGESTIONS A total of ten isolates of LAB can be isolated from gastrointestinal tract of abalone and their habitats. After the selection there are two isolates obtained potentially to be the candidates for probiotic that is OPA4 and AL1. Both isolates have the ability to inhibit the growth of enteropathogenic bacteria namely Eschericia coli, Bacillus cereus and Staphylococcus aureus with inhibition zone varied widely, and able to grow in acidic conditions and tolerant of bile during 24 hours incubation. Based on the API test kit and analyzed with software 50CHL ApiwebTM Version 1.2.1, the both isolates are identified as members of the species Lactobacillus paracasei ssp. paracasei. Isolates of this research which have the potential to be a candidate of probiotics in abalone larval rearing system of (Haliotis asinina) are expected to be studied further in order to know its potential in improving the survival ability of abalone larvae in vitro and in vivo, so it can be recommended as probiotic bacteria, especially in the abalone farming systems in the future.

ACKNOWLEDGEMENTS The authors thanks the Directorate General of Higher Education, Ministry of National Education which has funded this research through research project Fiscal Year 2009 Competitive Grant Contract Number: 0234.0/02304.2/XXI/2009, 31December 2008.

REFERENCES Axelsson L. 1998. Lactid acid bacteria: clasification and physiology. In: Salminen S,Wright AV (eds). Lactid acid bacteria: microbiology and functional aspects. Marcel Dekker, New York. Bar NA, Harns ND, Hill RI. 1987. Purification and properties of an antimicrobial substance produced by Lactobacillus. J Food Sci 52: 411-415. Biomerieux. 2009. API 50CHL medium for invitro diagnostic use. Brashears MM, Jaroni D, Trimble J. 2003. Isolation, selection and characterization of lactic acid bacteria for a competitive exclusion product to reduce shedding of Eschericia coli 0157:H7 in cattle. J Food Protect 66 (3): 355-363. Cholik F, Ateng G, Jagatraya, Poernomo RP, Ahmad A. 2005. Aquaculture hopes the future of the nation. Kerjasama Masyarakat Perikanan Nusantara dan Taman Akuarium Air Tawar, Taman Mini Indonesia Indah. Jakarta.[Indonesia] Dellaglio F, Felis GE, Torriani G. 2002. The status of the species Lactobacillus casei (Orla-Jensen 1916) Hansen and Lessel 1971 and Lactobacillus paracasei Collins et al. 1989 request for an opinion. Intl J Syst Evol Microbiol 52: 285-287. Djaafar TF, Rahayu ES, Wibowo D, Sudarmadji S. 1996. Antimicrobial substance produced by Lactobacillus sp. TGR-2 isolated from growol. Food and Nutrition Development and Research Center, Gadjah Mada University. Yogyakarta. Fuller R. 1989. Probiotic in man and animal. J Applied Bacteriol 66: 365. Gilliland SE, Stanley TE, Bush LJ. 1984. Importance of bile tolerance of Lactobacillus acidophilus used as a dietary adjunct. J Dairy Sci 67: 3045-3051. Haryanti, Samuel, Tsumura S. 1997. Preliminary study of the use of bacteria Flamimonas By-9 as probiotics in larval rearing of shrimp. Research and Development Center for Fisheries. Jakarta. [Indonesia] Holt G, Kreig NR, Sneath PHA, Stanley JT, Williams ST. 1994. Bergeys manual of determinative bacteriology. 9th ed. William and Wilkins, Baltimore. Imai T. 1997. Aquacultur in shallows seas; progress in shallow sea culture. Oxford and IBH. New Delhi. Iñiguez-Palomares C, Pérez-Morales R, Acedo-Félix E. 2007. Evaluation of probiotic properties in Lactobacillus isolated from small intestine of piglets. Rev Latinoam Microbiol 49 (3-4): 46-54. Ljung A, Lan J, Yanagisawa N. 2002. Isolation, selection and characteristics of Lactobacillus paracasei subsp. paracasei F19. Microb Ecol Health Disease 14 (3) Suppl. 3: 4-6. Rahayu ES, Wardani AK, Margino S. 2004. Screening of lactic acid bacteria from meat and processed products as a producer of bacteriocin. Agritech. Yogyakarta. [Indonesia] Ray B, Rahayu ES, Margino S. 1997. LAB: isolation and identification. IUC for Food and Nutrition, Gadjah Mada Yogyakarta. [Indonesia] Sarkono, Sembiring L, Rahayu ES. 2006. Isolation, selection, characterization and identification of lactic acid bacteria (LAB) producing bacteriocin from a variety of ripe fruit. Sains dan Sibernatika 19 (2): 1-5. [Indonesia] Sneath PHA, Mair NS, Sharpe ME, Holt JG (eds). 1986. Bergey’s manual of systematic bacteriology. William and Wilkins. Baltimore. Sofyan Y, Sukriadi, Ade Y, Bagja I, Dadan K. 2005. Abalone hatchery (Haliotis asinina) in Lombok Marine Aquaculture Station. Directorate General of Aquaculture, Ministry of Marine Affairs and Fisheries. Mataram. [Indonesia] Sofyan Y, Sukriadi, Ade Y. 2005. Fry production engineering abalone (H. asinina) in Lombok Marine Aquaculture Station. Directorate General of Aquaculture, Ministry of Marine Affairs and Fisheries. Mataram. [Indonesia] Strompfova V, Marcinakova M, Gancarcikova S, Jonecova Z, Scirankova L, Guba P, Boldizarova K, Laukova A. 2005. New probiotic strain Lactobacillus fermentum AD1 and its effect in Japanese quail. Vet Med Czech 50 (9): 415-420. Vlieger AM, Robroch A, Buuren SV, Kiers J, Rijkers G, Benninga MA, Biesebeke R. 2009. Tolerance and safety of Lactobacillus paracasei ssp. paracasei in combination with Bifidobacterium animalis ssp. lactis in a prebiotic-containing infant formula: a randomised controlled trial. Br J Nutr 31: 1-7. Wallace TD, Bradley S, Buckley ND, Green-Johnson JM. 2003. Interaction of lactic acid bacteria with human intestinal epithelial cells: effect on cytokine production. J Food Protect 66 (3): 466-472.

ISSN: 2087-3948 (print) ISSN: 2087-3956 (electronic)

Vol. 2, No. 1, Pp. 43-47 March 2010

Productivity of sugarcane plants of ratooning with fertilizing treatment A. SUTOWO LATIEF1,♥, RIZAL SYARIEF2, BAMBANG PRAMUDYA2, MUHADIONO3

¹ Semarang Polytechnic State (Polines). Jl. Prof. Sudharto, SH, Tembalang, Semarang, Central Java, Indonesia; ♥e-mail: 2 Faculty of Agriculture Technology, Bogor Agricultural University (IPB), Darmaga Bogor 16680, West Java, Indonesia 3 Faculty of Mathematic and Natural Science, Bogor Agricultural University (IPB), Darmaga Bogor 16680, West Java, Indonesia Manuscript received: 30 September 2009. Revision accepted: 26 January 2010.

Abstract. Latief AS, Syarief R, Pramudya B, Muhadiono. 2010. Productivity of sugarcane plants of ratooning with various fertilizing treatments. Nusantara Bioscience 2: 43-47. This research aims to determine the sugarcane plants of ratooning productivity with low external input of fertilization treatment towards farmers can increase profits. The method used is the Completely Randomized Block Design (CRBD) with four treatments and three repetitions (4x3). Sugarcane varieties R 579 planted in each patch experiment 5x5 m2. Dosage of fertilizer: P0 = 3.6 kg/year plot experiment was 100% dosage usage of chemical fertilizers used by farmers. Further dosages were P1 (75%) = 2.7 kg/plot, P2 (50%) = 1.8 kg/plot and P3 (0.25%) = 0.9 kg/plot, each supplemented with fertilizer 5 mL of liquid organic/patch a year. Sugarcane crops with a variety of treatment showed no significant difference. The highest productivity was achieved at dosages of P2 (50% chemical fertilizers plus organic fertilizer) is 21.67 kg per square meter. Chemical fertilizers can be saved 7 quintals per hectare a year or Rp 997,500 per year. Additional costs of liquid organic fertilizer Rp. 100,000 per hectare year and labor Rp 100,000 per hectare, so the additional advantage of saving farmers fertilizer Rp. 797,500 per year. Key words: sugarcane plant, ratooning, fertilizing, profits.

Abstrak. Latief AS, Syarief R, Pramudya B, Muhadiono. 2010. Productivity of sugarcane plants of ratooning with fertilizing treatment. Nusantara Bioscience 2: 43-47. Penelitian ini bertujuan untuk menentukan produktivitas tebu keprasan dengan perlakuan pemupukan input eksternal rendah, sehingga petani dapat meningkatkan keuntungan. Metode yang digunakan adalah Blok Rancangan Acak Lengkap dengan empat perlakuan dan tiga ulangan (4x3). Tebu varietas R 579 ditanam pada masing-masing plot percobaan seluas 5x5 meter2. Dosis pupuk: P0 = 3,6 kg/plot yaitu 100% dosis penggunaan pupuk kimia yang digunakan oleh petani. Selanjutnya dosis: P1 (75%) = 2,7 kg/plot, P2 (50%) = 1,8 kg/plot dan P3 (0,25%) = 0,9 kg/plot, masing-masing dilengkapi dengan 5 mL pupuk organik cair plot/tahun. Tanaman tebu dengan berbagai perlakuan tidak menunjukkan perbedaan yang signifikan. Produktivitas tertinggi dicapai pada dosis P2 (pupuk kimia 50% plus pupuk organik) adalah 21,67 kg/m2. Pupuk kimia dapat dihemat 700 kg/ha/tahun atau Rp 997.500 per tahun. Tambahan biaya pupuk cair organik Rp 100.000 per tahun hektar dan tenaga kerja Rp 100.000 per hektar, sehingga keuntungan tambahan petani dari tabungan pupuk Rp. 797.500 per tahun. Kata kunci: tanaman tebu, keprasan, pemupukan, keuntungan.

INTRODUCTION In this time government is inciting sugarcane planting of superior variety to overcome the low sugar production in Indonesia. To be in the triumph time as sugar exporter in the year of 1930 is done by increasing sugarcane product either through quantity and quality with paying attention to the environment preservation. Indonesia sugar productivity has declined, not only because of less field, irrigation and the increasing dry field or dry farming that planted sugarcane, but also that sugarcane variety doesn't support productivity and the ratooning is done more than 10 times. Therefore the company of Plantation Nusantara XI in East Java does penetration to develop new variety of arcane plants namely R-579 (MoA 2002). This new variety can produce average sugar of 10, 07 ton of /ha, while the average national productivity is 4 ton /ha (Anon 2002). Development of sugarcane is quite reasonable where it is produced more than half of the world’s sugar production from sugarcane (Mubyarto and Daryanti 1994). The

productivity of sugarcane crop in Indonesia that has been achieved is 4.924 tons/ha (Anon 1996), but in the last 5 years it has increased from 5.7 tons/ha in 2004 to 6.8 tons/ha in 2009 (Lestari 2009); while in Papua New Guinea to reach 5.5 tons/ha (Hartemink 1996), and South Africa 11.0 tons/ha (McGlinchey and Inman-Bamber 1996). The administrator of Sugar Factory of Rendeng, Kudus, said, most of 5,679 hectare sugarcane plants were cultivated by farmers farmer with ratooning system, with the average 10 times. Sugarcane productivity moment harvests the highest products of 70 ton/ha, and yield only 5,76%. Begin in the year 2003, farmers plant a kind of superior varieties namely PS 851 (MoA 2004) and R 579 (BR 579) in the area of 728 hectare. The superior variety R 579 has been experimented at some amount in the Sugar Factory in East Java and has produced the minimum crops of 150 ton/ha 8% (Krismanu 2003). The ratooning system is growing return sugarcane that felled. Anon (2005), ratooning sugarcane management has been intensively done since the issue of the President


2 (1): 43-47, March 2010

Instruction number 9 in the 1975 about intensification. Since 1990, the trend of the use of ratooning sytem of sugarcane has continued to increase, that is around 60% from total square existing sugarcane. Since Green Revolution was proclaimed in the 1970’s farmers’ dependence in inorganic fertilizer use has been there. Inorganic fertilizer used that is over dosage or more causes the depletion of the soil quality, and it leads to the decrease of sugarcane’s productivity. Aryantha said that (2002) this condition causes inhibited of root absorption process towards water and nutrient that was dissolved so that the existence of nutrient in total low is not taken by the roots in maximally. Thereby certain dosage of fertilizer is needed to make the roots able to absorb the nutrient in enough number from the nutrients available in the soil. Suprapta (2005) said that chemical fertilizer causes bad impacts as we have witnessed. He added that we should organic fertilizer and at the same time also slowly reduces the use of chemical fertilizer. While According to Darutama (2008), organic fertilizer the use organic fertilizer for sugarcane plants obviously shows good significance in comparison with the use of the chemical fertilizer such as urea or NPK. The success sugarcane farming means giving the profits to the farmers and being able to keep the environment healthy. Therefore it is necessary to conduct a research aimed at decreasing the use chemical/inorganic fertilizer and encouraging the use of organic fertilizer to do the rationing system for sugarcane farming to make the productivity stable. MATERIALS AND METHOD Location and time of research The research location based on fertilizing variation treatment effort plan towards ratooning sugarcane plants is chosen to be conducted at Jurang Village, Gebog Subdistrict, Kudus District, Central Java. The place that is used to do the analysis towards the chemical element of the soil nutrient, good macro and micro element is in the Laboratory of Department of Soil Science and Land Resources, Faculty of Agriculture, Bogor Agricultural University (IPB), Bogor. Research time is carried out to begin in July 2008 and end in June 2009, during one sugarcane harvest season. Materials and tools Principal material is a variety of sugarcane plants namely R 579. Other materials are fertilizers namely: (i) inorganic fertilizer ZA (ammonium sulphate), and NPK (Phonska), (ii) liquid organic fertilizer. Method The design of the research was Completely Randomized Block Design with 4 (four) treatments and for each treatment there are 3 (three) repetitions. Fertilizing treatment is done towards ratooning sugarcane plants. Ratooning sugarcane plants that is analyzed is the variety of sugarcane namely R 579 that can undergo the ratooning

process three times (can be four in the future) in the area in Jurang village, district Gebog, Kudus regency. The size of trial compartment each 5x5 square meters = 25 m2 (poled to be clear the limit). The fertilizing treatment that is: (i) P0 = the use chemical fertilizer (inorganic fertilizer/factory fertilizer) done by the farmers up to that time (100% inorganic fertilizer), without organic fertilizer. (ii) P1 = chemistry fertilizer use is reduced by 25% from the usual use (75%) then replaced by the organic fertilizer. (iii) P2 = chemistry fertilizer use is reduced by 50% from the usual use (50%) and replaced by the organic fertilizer. (iv) P3 = chemistry fertilizer use is reduced by 75% from the usual use (25%) then replaced by the organic fertilizer. The addition of organic fertilizer is done towards P1, P2, and P3 with the same dosage, that is 2 L every hectare a year, while P0 as a group control doesn't uses organic fertilizer. Organic fertilizer kind use result of Fadiluddin (personal communication, 2009). The use dose 2 L/ha of land, atomized twice (each time spraying 1 L/ha), before atomized in soil surround plants, liquid organic fertilizer is thinned with water first of all with comparison 100 mL to 1 (one) tank sprayer (15 L water) or 15 mL (size bottle plug) to 2 L water. Liquid organic fertilizer use to each size compartment 25 m2: 25/10,000x2 liters = 5 mL. Overall use from 9 trial compartments (P1, P2, and P3 with repetition 3 times) a year need: 5x9 = 45 mL then thinned with 6 clean water liters. Fertilizing with liquid organic fertilizer was done by spraying, one year done 2 times, as according to inorganic fertilizing, not concurrent but done 3-5 days before or after fertilizing with inorganic fertilizer. Inorganic fertilizer use usually is done by farmer towards sugarcane plants each time fertilizing is 100 kilogram/sector of rice field is do twice a year (200 kilogram/year sector of rice field) consist of 50% fertilizer ZA (ammonium sulfate): nitrogen (N) = 21% and sulfur (S) = 24% and 50% fertilizer NPK (Phonska: N = 15%; P2O5 = 15%; K2O = 15%; S = 10%) One hectare there is 7 sectors of rice field, every sector of rice field approximately 1400 m2. Inorganic fertilizer use for size of trial compartment 25 m2 a yearlong is need: P0 = 25/1400x200 = 3.6 kg, P1 = 0.75x3.6 kg = 2.7 kg, P2 = 0.50x3.6 = 1.8 kg, and P3 = 0.25x3.6 = 0.9 kg. Soil is taken as the sample to analyze as many as three times during research, that is: (i) before fertilizing, (ii) after fertilizing and (iii) approach harvest. Soil analysis is done in laboratory to detect element of nutrition completely. Sugarcane plants observation is done according to in a flash with take when soil samples taking. The finals research is sugarcane harvest result ready mill from each trial compartment. Sugarcane observation is done towards: (i) amount of sugarcane plants every square meters or every meter makes, (ii) tall/long sugarcane stick ready mill and (iii) sugarcane stick diameter (measured 15 cm from base). Sample taking at random every square meters (meter makes from each trial compartment). Heaviness each weighed and analyzed to detect treatment difference with statistical methods that are Analysis of Variance (ANOVA).

LATIEF et al. â&#x20AC;&#x201C; Sugarcane plant ratooning


fertilizer has begun to react towards soil so that root absorption towards water and nutrition is better.

RESULT AND DISCUSSION Soil evaluation criteria Soil sample taking is done 3 times, that is: (i) before fertilizing in 9 Novembers 2008, (ii) after fertilizing in 22 February 2009 and (iii) approach harvest in 21 May 2009. Based on soil analysis result from Department of Soil Science and Land Resource, Faculty Agriculture, Bogor Agricultural University (IPB) Bogor, follow Hardjowigeno (2007) determinable the criteria as be showed in Table 1, Table 2 and Table 3. Criteria of nutrition N before fertilizing, after fertilizing and approach harvest shows low, while P in the form of P2O5 there are increase a little, but K does not change. Another macro element that is: Ca, Mg and Na are fair.

Table 1. Soil chemistry properties evaluation criteria before fertilizing Soil properties C (%) N (%) C/N P2O5 HCl (mg/100 g) P2O5 Bray 1 (ppm) KTK (me/100 g) K (me/100 g) Na (me/100 g) Mg (me/100 g) Ca (me/100 g) Saturation of basic (%) pH H2O pH KCl

Sugarcane productivity Based on observation towards sugarcane plant when taking second soil sample 22 February 2009 known that for treatment P0, green appear sugarcane leaf, while for treatment P1, P2, and P3 appear sugarcane leaf more becomes yellow. But when taking third soil sample 21 May 2009 that is approach sugarcane leaf color harvest visible hasn't showed difference. This matter caused by organic

Treatment: P0 = P1 = P2 = P3 1.2 0.13 9.23 23.6 2.2 14.82 0.44 0.34 1.67 5.34 52.56 4.5 3.6

C-org (%) N-total (%) C/N P2O5 HCl (mg/100 g) P2O5 Bray 1 (ppm) KTK (me/100 g) K (me/100 g) Na (me/100 g) Mg (me/100 g) Ca (me/100 g) Saturation of basic (%) pH H2O pH KCl

P0 0.96 0.12 8 25.86 53.1 15.35 0.28 0.24 1.48 6.77 57.13 4.00 3.3

Treatment P1 P2 1.36 1.2 0.13 0.11 10.46 10.90 30.43 49.76 32.5 60.0 14.96 14.56 0.28 0.58 0.23 0.30 1.67 2.43 6.95 5.65 61.03 61.54 4.30 4.40 3.5 3.5

P3 0.96 0.09 10.66 48.91 52.4 15.55 0.28 0.22 2.57 7.87 70.35 4.40 3.7

Criteria P0 very low; P1 low; P2 low; P3 very low P0 low; P1 low; P2 low; P3 very low P0 low; P1 fair; P2 fair; P3 fair P0 fair; P1 fair; P2 high; P3 high P0 very high; P1 high; P2 very high; P3 very high P0 low; P1 low; P2 low; P3 low P0 fair; P1 fair; P2 high; P3 high P0 low; P1 low; P2 low; P3 low P0 low; P1 fair; P2 high; P3 high P0 fair; P1 fair; P2 fair; P3 fair P0 high; P1 high; P2 high; P3 very high P0 very acid; P1 very acid; P2 very acid; P3 very acid P0 very acid; P1 very acid; P2 very acid; P3 very acid

Table 3. Soil chemistry properties evaluation criteria approach harvest Soil properties C-org (%) N-total (%) C/N P2O5 HCl (mg/100 g) P2O5 Bray 1 (ppm) KTK (me/100 g) K (me/100 g) Na (me/100 g) Mg (me/100 g) Ca (me/100 g) Saturation of basic (%) pH H2O pH KCl

P0 1.43 0.13 11 34.01 49.0 18.62 0.35 0.40 2.70 8.63 64.88 5.40 4.50

Treatment P1 P2 1.27 0.95 0.11 0.10 11.5 9.5 33.16 36.21 47.3 49.3 27.75 20.35 0.08 0.10 0.19 0.21 0.20 0.18 4.3 2.6 32.4 36.9 5.50 5.20 4.70 4.00

P3 0.71 0.09 7.9 43.99 56.2 22.2 0.29 O.90 0.31 3.5 83.3 5.30 4.10

low low low fair very low low fair fair fair fair high acid very acid

Sugarcane harvest is done at dry season because moment that is has high yield, after cutting down sugarcane soon be processed to be sugar. The cutting down of

Table 2. Soil chemistry properties evaluation criteria after fertilizing Soil properties


Criteria P0 low; P1 low; P2 very low; P3 very low P0 low; P1 low; P2 low; P3 very low P0 fair; P1 fair; P2 low; P3 low P0 fair; P1 fair; P2 fair; P3 high P0 very high; P1 very high; P2 very high; P3 very high P0 fair; P1 high; P2 fair; P3 fair P0 fair; P1 very low; P2 low; P3 low P0 fair; P1 low; P2 low; P3 high P0 high; P1 very low; P2 very low; P3 very low P0 fair; P1 low; P2 low; P3 low P0 high; P1 low; P2 fair; P3 very high P0 acid; P1 acid; P2 acid; P3 acid P0 acid; P1 acid; P2 acid; P3 acid



2 (1): 43-47, March 2010

sugarcane in this research is done after age approximately one year, that is on 16 June 2009. Amount of sugarcane plant/stick every square meters based on observation in the harvest in the range from 16 up to 24 stick of sugarcanes. Long sugarcane stick ready mill also vary that is between 1.5 meters up to 3.5 meters. Sugarcane stick diameter ranges from 2.5 cm up to 4.5 cm. The average of amount stick, length stick, and sugarcane stick diameter is presented in Figure 1.

Figure 1. Amount average of stem, length and diameter of sugarcane plant every square meters in experimental land.

The model of relation between fertilizing treatment with sugarcane productivity is shown in Figure 2.

Produktivitas Tebu (kg/m2)



Poly. (Series2)

25 20



y = -2.6667x + 20.33x - 44.983x + 45.65 15


R =1

10 5 0 P0




Perlakuan Pemupukan

Figure 2. Relation between fertilizing treatment and sugarcane productivity.

Based on the Analysis of Varian with signification standard 1%, sugarcane productivity with variation fertilizing treatment, it doesn't show real difference. Highest productivity is achieved in treatment (P2) that is fertilizing combination with reduction 50% chemistry fertilizer from the usual one done by farmers, added with organic fertilizer. Thereby it can be saves the chemistry fertilizer purchasing cost-saving as big as 50%, although the liquid organic fertilizer purchasing cost and labor cost for fertilizer spraying increase. Farm operation analysis of sugarcane and cost-saving Farm operation analysis of sugarcane is done to determine profit and business feasibility based on income ratio criteria towards net (B/C). Farm operation of sugarcane is said feasible when value B/C bigger than one

Based on primary data that is got and cultivated with one hectare land square production cost: C = Rp 12,000,000. Land lease were Rp 5,000,000 per year. Labor, cultivation, fertilizer and pesticide were Rp 7,000,000 per year. Sugarcane sales revenue: Rp 160,000 per ton, sugarcane harvest result 150 ton/ha, so that Benefit total: B = Rp. 24,000,000. Farm operation profit of sugarcane: B-C = Rp 24,000,000-Rp 12,000,000 = Rp 12,000,000 per year. Benefit per Cost Ratio: Net B/C = Rp 24,000,000/Rp 12,000,000 = 2.0. Based on analysis result above (B/C = 2.0 > 1), it can be known that the farming operation of sugarcane is feasible. Cost-saving analysis is based on fertilizer chemistry (inorganic fertilizer) use reduction 50% from habit that is as much as 7 quintal (700 kg) fertilizer that can be saved without decreasing of productivity. Chemistry fertilizer dosage that used farmers usually is 1.4 ton/ha. Despite of organic fertilizer use cost and labor increasing, but still more beneficial because liquid organic fertilizer use lower than chemistry fertilizer, beside that is also cheaper the price. The price of kind inorganic/chemical fertilizer ZA: Rp 110,000 per quintal, kind fertilizer Phonska: Rp 175,000 per quintal. Fertilizer use ZA and Phonska proportional, which is each 50%. Cost addition for liquid organic fertilizer: Rp. 50,000 per liter, as much as 2 L/ha and labor wage: Rp 25,000 per day as much as 4 persons. Based on this research result when applied manifestly with chemistry fertilizer reduction 50% is 7 quintal/year is land square base one hectare, so cost-saving can be done by farmer: Chemistry fertilizer cost-saving-(organic fertilizer cost + worker wage) = 7x(110,000 + 175,000/2-(2x50,000 + 100,000) = Rp. 797,500/hectare. Cost-saving a kind of this be concept LEISA (Low External Input Sustainable Agriculture), that is a concept that promoting system and that agriculture manners by using a little chemical addition. Principle applications LEISA make possible Good Agriculture Practices (GAP) where productivity and economy profit is increased in the way of that pay attention ecological aspect. For example, livestock animal maintenance to make use in stable fertilizer maker with agriculture rubbishes utilization like foliage to be used as supplement plants. CONCLUSIONS AND RECOMENDATIONS The productivity of sugarcane with fertilization treatment variations P0, P1, P2 and P3, showed no significant difference. The highest results achieved by treatment of P2, which is 21.67 kg/m² of land area. Reduction of chemical fertilizers without the addition of organic fertilizer is not done because the experience of farmers who have tried to reduce the dosage of chemical fertilizers without the addition of organic fertilizers, the productivity of sugarcane declined. Thus the combination of reduction in the use of chemical fertilizers and organic

LATIEF et al. â&#x20AC;&#x201C; Sugarcane plant ratooning

fertilizers can stabilize the productivity of sugarcane and input cost savings. Input cost savings made by farmers is an advantage, is Rp. 797,500/hectare during the season (year) This study should be followed up at various locations mainly on dry land, and the land with more extensive experiments, and the use of chemical fertilizers ZA and Phonska varied to obtain optimal savings. Future research needs to be done reducing the use of chemical fertilizers or without the use of chemical fertilizers at all. The use of organic fertilizer without chemical fertilizers is conducting agricultural/organic sugarcane plantations, so that farming guidelines and good agricultural products (Good Agriculture Practices/GAP). REFERENCES Anon. 1996. Superior varieties of sugarcane, sugar selfsufficiency guide. Neraca (March edition). Jakarta. [Indonesia] Anon. 2002. Increasing sugar production by finding new varieties of sugarcane. Situs Hijau Media Pertanian Online. [27 Mei 2009]. [Indonesia] Anon. 2005. Management of ratooning sugarcane. Sugarcane Development Project, Plantation Office, East Java.. san.htm [27 Mei 2009] [Indonesia] Aryantha IP. 2002. Development of sustainable agricultural systems. One Day Discussion on the Minimization of Fertilizer Usage. Menristek-BPPT, Jakarta, 6 May 2002.


Darutama BE. 2008. Pupuk organik tingkatkan rendemen tebu. [10 October 2008]. [Indonesia] Fadiluddin M. 2009. The effectiveness of biological fertilizer formula in promoting nutrient uptake, production, and quality of maize and upland rice in the field [Thesis]. School of Graduates. Bagor Agricultural University. Bogor. [Indonesia] Hardjowigeno HS. 2007. Soil science. Akademika Pressindo. Jakarta. Hartemink A. Kuniata. 1996. Some factors influencing the trend of sugarcane yield in Papua New Guinea. Outlook Agric 25(4): 227-234. Krismanu H. 2003. Sugar mill are loss, but the yield improved. PTPN IX PG Rendeng, Kudus. [Indonesia] Lestari D. 2009. Sugarcane acreage will increase. Bisnis Indonesia 07-12-2001. [Indonesia] McGlinchey MG, Inman-Bamber Ng. 1996. Effect of irrigation scheduling on water use efficiency and yield. Proc S Afr Sug Technol Ass 70: 55-56. Minister of Agriculture of the Republic of Indonesia. 2002. Decree of the Minister of Agriculture Number: 372/TU.210/A/XI/2002 on release of sugarcane variety R 579 as a superior variety. [Indonesia] Minister of Agriculture of the Republic of Indonesia. 2004. Decree of the Minister of Agriculture number: 55/Kpts/Sr.120/1/2004 on release of sugarcane variety PS 851 as a superior variety. [Indonesia] Mubyarto, Daryanti. 1994. Sugar, a national study of economics. Aditya Medya. Yogyakarta. [Indonesia] Suprapta DN. 2005. It should be a national movement of organic fertilizer use. Kompas 24-02-2005. [Indonesia]

ISSN: 2087-3940 (print) ISSN: 2087-3956 (electronic)

Vol. 2, No. 1, Pp. 48-53 March 2010

Exposure copper heavy metal (Cu) on freshwater mussel (Anodonta woodiana) and its relation to Cu and protein content in the body shell AHMAD INTAN KURNIA1,♥, EDI PURWANTO², EDWI MAHAJOENO² ¹Sekolah Tinggi Ilmu Kesehatan (STIKES) Karya Husada Pare Kediri. Jl. Soekarno Hatta Po Box 153, Kediri 64225, Jawa Timur, Indonesia. Tel.: +92354-393888, Fax: +92-354-393888, ♥email: ² Bioscience Program, School of Graduates, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia Manuscript received: 8 October 2009. Revision accepted: 19 February 2010.

Abstract. Kurnia AI, Purwanto E, Mahajoeno E. 2010. Exposure copper heavy metal (Cu) on freshwater mussel (Anodonta woodiana) and its relation to Cu and protein content in the body shell. Nusantara Bioscience 2: 48-53. To determine the relationship of Cu exposure in water to the freshwater mussel exposure experiment is conducted with water containing Cu. Which measured the influence of Cu and protein content in the body shell. This study used the freshwater mussel species, Anodonta woodiana. Oysters were exposed for four weeks in the water with Cu concentration of 0.02 ppm, 0.04 ppm, 0.06 ppm and 0.00 ppm control. Cu content and protein content in the body shells are checked every week. Cu analysis was done by AAS method and the protein content using Kjeldahl method. Cu analysis showed elevated levels of Cu in mussel body after exposure. The pattern of increase in Cu content was not the same, where the pattern of the largest increases occurred after the fourth week. The statistical test showed no significant effect between the treatment with Cu accumulation in the body shell. Protein analysis showed an increase of protein content after exposure of the second week and decreased after the third and fourth weeks. The pattern of changes in protein content varied among the various treatments. The statistical test showed no significant effect between treatment with the protein changes in the body shell. Correlation test of the relationship between concentration of Cu in mussel body protein level showed a positive correlation between them with a fairly good level of relationship (correlation coefficient r = 0.836). Key words: Anodonta woodiana, exposure, Cu, protein.

Abstrak. Kurnia AI, Purwanto E, Mahajoeno E. 2010. Paparan logam berat tembaga (Cu) pada kerang air tawar (Anodonta woodiana) dan hubungannya dengan kandungan Cu dan protein dalam tubuh kerang. Nusantara Bioscience 2: 48-53. Untuk mengetahui hubungan pemaparan logam Cu dalam air terhadap kerang air tawar maka dilakukan percobaan pemaparan dengan air yang mengandung Cu. Pengaruh yang diukur adalah kadar Cu dan kadar protein dalam tubuh kerang. Penelitian ini menggunakan kerang air tawar Anodonta woodiana. Kerang dipaparkan selama empat minggu dalam air dengan konsentrasi Cu 0,02 ppm, 0,04 ppm, 0,06 ppm dan kontrol 0,00 ppm. Kadar Cu dan kadar protein dalam tubuh kerang diperiksa setiap minggu. Analisis Cu dilakukan dengan metode AAS dan kadar protein menggunakan metode Kjeldahl. Analisis Cu menunjukkan peningkatan kadar Cu dalam tubuh kerang setelah pemaparan. Pola kenaikan kadar Cu tidak sama, dimana pola kenaikan terbesar terjadi setelah minggu keempat. Uji statistik menunjukkan tidak adanya pengaruh signifikan antara perlakuan dengan akumulasi Cu dalam tubuh kerang. Analisis protein menunjukkan kenaikan kadar protein setelah pemaparan minggu kedua dan menurun setelah minggu ketiga dan keempat. Pola perubahan kadar protein bervariasi antar berbagai perlakuan. Uji statistik menunjukkan tidak adanya pengaruh signifikan antara perlakuan dengan perubahan protein dalam tubuh kerang. Uji korelasi hubungan antara kadar Cu dalam tubuh kerang dengan kadar protein menunjukkan adanya korelasi positif antara keduanya dengan tingkat hubungan yang cukup baik (koefisien korelasi r = 0,836). Kata kunci: Anodonta woodiana, pemaparan, Cu, protein.

INTRODUCTION The idea of environmental pollution in Indonesian Act No. 23 of 1997 on Environmental Management is the inclusion of living things, matter, energy, and/or other components into the environment by human activities so that its quality decreases to a certain level that causes environment can not function as intended. One of the pollutants that are often found as contaminants in the aquatic environment that is detected in the organism body is heavy metals, copper (Ward 2001). Copper (Cu) is a metal that widely used in chemical industry, metallurgy, textiles and anti-rust paint (Effendi, 2003). Like other

heavy metals, Cu, is difficult to be soluted in the environment and it can enter the food chain through organisms that exist in the water. Aquatic organisms that are known to accumulate heavy metals are phytoplankton, zooplankton, class of fish, crustaceans (crustaceans) and mollusks or shellfish species (Ward 2001). Freshwater mussels (Anodonta woodiana) are bivalves included in phylum mollusks that have two symmetrical shells. They live in the riverbed, channels, ponds and lakes. They can have rough or smooth shell depending on the habitat where they live. Freshwater mussels are microscopic plant-eating animals and perform it by sucking water through a siphon and removing the particles. As a

KURNIA et al. â&#x20AC;&#x201C; Cu exposure on freshwater mussels (Anodonta woodiana)

filter organism, freshwater mussels can serve to clean water and reduce algae, particles, toxic materials and some diseases. Freshwater mussels is an organism that can be used as biological indicators or bioindicator (EPA 2009). Biondicators are organisms or biological responses that indicate the entry of certain substances in the environment. Species or species group for bioindicator are selected based on several factors which is; they are easily measured and give response observed in ecosystems, have specific response that is able to predict on how a species or ecosystem will respond to a certain pressure, measure the response with accuracy and precision that can be accepted based on knowledge about contaminants and characteristics (Mulgrew et al. 2006). Umar et al. (2001) which researched on the relationship between Cu in the aquatic environment with the content Cu in the body of sea shells Marcia sp. concluded that the higher the Cu in water and sediment, the higher the metal content of Cu accumulated by shellfish that live in the area. According to Sunarto (2007) there is a relationship between structure of microanatomy, the efficiency of gill function, morphology and condition of freshwater mussels Anodonta woodiana with Cd concentrations in water. So from that relationship and the condition of the shell morphology can be used as a bioindicator macroscopic beginning at A. woodiana due to exposure to heavy metals Cd. Stolyar et al. (2005) says that there are significant effects caused by heavy metals Cu to the protein of freshwater mussels. In the laboratory experiments on a freshwater mussel Anodonta cygnea shows that at the time of Cu exposure concentrations in water increased by 10 micrograms/liter, the protein content of Cu in Methallotionin will experience increased 100.7% compared to control groups of organisms (without any treatment of heavy metal exposure Cu). To find out how the relationship of heavy metal Cu exposure concentration to A. woodiana a laboratory study is conductedf with observations of variable concentrations of Cu and protein content in the body of a freshwater bivalve A. woodiana. This study aims to: (i) know the relationship between exposure concentration of Cu in water with Cu concentration in the body of A. woodiana, (ii) to study the relationship between the concentration of Cu in water with the protein of A. woodiana; (iii) know the relationship between the concentration of Cu in the body A. woodiana with protein A. woodiana.

MATERIALS AND METHODS Place and time of study The study was conducted in three places. Acclimatization activities, maintenance and treatment of Anodonta woodiana was conducted at the Laboratory of Environment STIKES Karya Husada Kediri, East Java. Analysis of heavy metal Cu content in the body A. woodiana was conducted in Chemical sub-Laboratory, Center Laboratory of MIPA for UNS Surakarta. Analysis of protein content in the body of A. woodiana was conducted at the Laboratory of Agriculture, Faculty of Agricultural Technology, Sebelas Maret University, Surakarta


Material Materials research is a freshwater bivalve species A. woodiana. Samples were taken from Fresh Water Fish Seed Board (BBI) Janti, Klaten, Central Java. Shells used are wit the following specifications size is 7-9 cm long with a total weight of 30-45 grams. Research design The design of the research done on these activity was completely randomized design (CRD), with 3 (three) concentrations of Cu exposure treatment plus 1 (one) controls. Type of concentration are: (i) Group 1: 0.00 ppm Cu concentration (C0) (ii) Group 2: Cu concentration of 0.02 ppm (C1) (iii) Group 3: Cu concentration of 0.04 ppm (C2) (iv) Group 4: Cu concentration of 0.06 ppm (C3) Selection of exposure concentration of Cu is based on PAN Pesticide Database (2009) which states that the lethal concentration (LC50) for copper (CuSO (iv) toward fresh water mussel A. woodiana is of 0.1 ppm. A four time examination will be conducted to the four groups based on the length of exposure time, namely: (i) Examination 1: end of week-1 (T1) (ii) Examination 2: the end of week-2 (T2) (iii) The examination 3: end of week 3 (T3) (iv) The examination 4: end of week 4 (T4) From this experiment 16 units of the experiment will be obtained (4 x 4 examination treatment group). In each sample the variables observed are: concentration of Cu in the shells A. woodiana and protein levels in the shells A. woodiana. Procedures Experimental research The animals tested were taken from fish ponds in Janti, Klaten, Central Java then brought to the Laboratory of Chemical STIKES Husada Kediri, East Java. The first step is acclimatization. The shells were looked after in an aquarium filled with clean water as much as 15 liters. Each aquarium filled with 15 shells. The water in the aquarium was changed every 3 to 4 days. Mussels were fed every other day. The food provided is brand Takari manufactured by PT. Protein Prima Tbk. Feeding is done by making food that is still a solid into a liquid form and then dissolved into the water in the aquarium. During the acclimatization if there are dead shells then they will be replaced with new ones. Acclimatization process was conducted for 2 weeks. And then, experiments on heavy metals Cu exposure was conducted. The standard solution containing heavy metals such as Cu in certain concentrations was put into the aquarium as planned. Each type of concentration was carried out in three aquariums. Aquarium grouping is as follow: aquarium 1, 2, and 3 for the concentration of Cu 0 ppm (control), aquarium 4, 5, 6 are for 0.02 ppm Cu concentration, aquarium 7, 8, and 9 for 0.04 ppm and 10, 11 , 12 for Cu concentrations of 0.06 ppm. Standard solution is inserted into the tank together with replacing the water. So during the exposure time, the shells will continuously be in the water containing copper


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in that amount. Every water replacement will be accompanied by water temperature and pH measure. Furthermore, mussels/shells are taken for analysis of Cu content and protein content at the end of week 1, the end of week 2, 3, and 4.

body A. woodiana, the relationship between protein content with the content of Cu in the body A. woodiana.

Analysis of Cu content Shells samples were taken, opened, and its feces was dumped. And then the samples were weighed in analytical scale and the weights were recorded. Samples were placed in a glass beaker. Pour 5 mL of HClO4 into the sample and let stand for 3-5 minutes. Add 50 mL of distilled water. The sample was heated to form a homogeneous solution and leaving a volume of 20 mL. Remove the sample from the heater. Then 5 mL HNO3 was added. It was again heated for 10-15 minutes. Add 50 mL distilled water. Filter with filter paper and insert it into the sample bottle. And then the sample was injected into the Flammable Atomic Adsorption Spectrophotometer (FAAS). From the injection, the data on levels of Cu would be obtained (in units of ppm).

Relations between Cu concentration in water with the levels of Cu in the body To describe the magnitude of changes in Cu levels in the body of the shells in each treatment the results are expressed as a histogram. Image histogram shown in Figure 1 which shows the movement or changes in Cu levels in the body shells at each treatment concentration.

Analysis of protein content Shells samples were taken, opened, and its feces was dumped. Samples were crushed with a blender until smooth, then given 50 mL of distilled water and stirred until homogeneous. As much as 10 mL of sample solution were taken and put into 100 mL of glass, diluted until it reached the mark. From this solution (point 1) it was taken as much as 10 mL and put into 500 mL Kjeldahl flask and add 10 mL of H2SO4. Added 5 g mixture of Na2SO4-HgO (20:1) for the catalyst. The solution was boiled until clear and continued boiling for 30 minutes more. Once cool, wash the Kjeldahl flask walls with water and simmer again for 30 minutes, then cooled. When it was cool it was then added with 140 mL distilled water and add 35 mL of NaOH-Na2SO3 and some granules of Zink. Then do the distillation. Distillate was gathered as much as 100 mL in elmeyer tube containing 25 mL of saturated boric acid and a few drops of red indicator. Titrate the solution obtained with 0.02 HCl. Number of total N (% protein) was calculated with the formula as follows: The number N total = mL HCl x N HCl x 14,008 x f mL sample solution f = dilution factor Data analysis Analysis of variance (ANOVA) is used to see if there is variation among the treatments. Rank data analysis with analysis variant will be conducted on data obtained from the examination of Cu content in the network and data examination of protein levels in the body A. woodiana. Correlation analysis is used to determine the relationship between variables observed in the study. Data to be tested with correlation analysis is the relationship between the concentration of Cu in water with high levels of Cu in the body A. woodiana, the relationship between the concentration of Cu in water with protein content in the


Figure 1. Cu levels in the body of shells A. woodiana

The ANOVA test on these calculations using degrees of freedom of 3 and with a confidence level of 95% (ρ = 0.05). From the table ANOVA F test result greater than the F table. The results of this test gives the sense that there is no significant difference in Cu concentrations in water treated with Cu levels in the body shells, or the variation of treatment to be given in the form of concentration variation of Cu in the water did not give significant effect on levels of Cu in the body shells. Based on ANOVA statistical test the results stating that there is no significant difference in the concentration of Cu treatment. It means that the results of statistical tests can be analyzed from several points of the view. According Sǎrkǎny-Kiss et al. (2000), the ability of mussels organisms to accumulate heavy metals in the body is influenced by two factors, namely exterior complex and inner complex. Exterior complex is the condition of water environment in which these organisms live. While the inner complex factors are matters related to the metabolic capabilities of organisms with the presence of heavy metal components in the body. Referring to the opinion, the phenomenon in this study can be viewed from two factors, namely the live shells is based on water conditions as the life media, and based on the characters of organisms A. woodiana. In the factor of environmental, this experiment has been made causes the environment in which mussels live are relatively similar and homogeneous. All the variables and parameters associated with mussels habitat in this experiment have been controlled and made uniform. Only

KURNIA et al. â&#x20AC;&#x201C; Cu exposure on freshwater mussels (Anodonta woodiana)

the concentration of Cu which is made vary according to the research design. The first is about water. In this experiment the water used to perform the experiment originated from the same water source. For each type of treatment, water used is handled in the same way and taken in the same time. Physical factors of the water as place for living such as temperature and pH was monitoring in every water change. Water temperature during the experiment ranged in around 27oC. The food given were also made similar for each treatment. In this experiment, mussels were fed every other day. Food provided is Takari fish feed manufactured by PT. Protein Prima Tbk. Each concentration treatment was the same food and made at the same time. Based on existing levels of Cu in the body of samples mussels, it appears that among the individual samples there are significant differences in the ability to absorb copper, to process of metabolism and to accumulate copper particles that are soluble in water. Then this different ability results in different Cu accumulation in the body. This fact can be seen on the results of analysis of Cu for each sample (Figure 1). From this figure, it appears that the same type of treatment concentration and exposure at the same time still shows that there are a few samples that gave results that differ greatly. The difference of the Cu accumulation got here almost hit 100% difference. For example in the treatment concentration of 0.02 ppm Cu (week 2), Cu 0.04 (week-1 and 2) and Cu 0.06 at week 1. The same table also shows that from the same type of treatment and longer exposure time resulted in the lower accumulation of copper, while the other treatments showed an increase. This is visible on the treatment concentration of 0.02 ppm, where in the first week its average Cu accumulation was 0.0107, but in the next week the number decreased as much as 24.80% to 0.0086 ppm. Difference like that is what the statistics would cause the effect of exposure concentration of Cu in the water has no significant effect on Cu content in the body A. woodiana. A pattern of Cu accumulation in the mussels body that vary at the same time shows that the system of mussels biology and metabolism is not a simple system which has a linear mechanism, where the same input (treatment) would results that were very different. According to Krolak et al. (2001) the fact that the Cu content varying in individual of A. woodiana living with the same treatment is the effect of a selective retrieval capabilities of various components in the surrounding environment and is influenced also by the ability of different parts of each organ in the body of the mussels to accumulate Cu. From these quotations steps of analysis can be taken that the Cu content accumulated in A. woodiana after exposure to the metal Cu is influenced by two things. These two things are how the Cu ions get inside of the organism and how the Cu ions are accumulated in the organism. It can be said that the difference in levels of Cu accumulated in this experiment was caused by the differences in both processes.


The process of inclusion of Cu ions into the mussels body. In Soto et al. (2008) it is explained that metal enters the oysters in two ways. First is through the gills and second through the organs of the body which is in direct contact with the water containing the heavy metals. From these quotations, we can say that the quantity of Cu goes into the shells is influenced by two things. The first is related to the gills and the second is related to the contact or exposure of organs with heavy metals. If Cu enter through the gills: in bivalves, including Anodonta wodiana, metal ions in water will get into the gills by diffusion, which is a more passive process (Soto et al. 2008). Because this process is passive, the decisive part the diffusion process is the condition of the gills, i.e. the width of the philamen surface. If the greater the surface of gill filaments, then the diffusion will occur the more and more, thus more Cu ions enters the body of the mussels. Besides the diffusion process, the process of Cu enters the gill also involves active transport mechanism, i.e. when the gills must work against the pressure difference between the water pressure and the pressure in the gills. Gillâ&#x20AC;&#x2122;s ability to perform the active transport depends on the availability of ATP (Soto et al. 2008). From the description of this process shows that the extent of Cu entering through an active transport process depends on each individual. Where the influential part is the availability of energy in the form of ATP. Cu influx through the organ of the mussels which is in direct contact with the water. In A. woodiana, organ which is in direct contact with water is the organs which anatomically Fox calls(2005) the external anatomy, that is the shell, muscle cells (mantle), gill, the body (visceral mass), foot, and labial palps. In the closed shell condition, the organs which will be in contact with water are the two pieces of shells, while the organs that are inside it are not in contact with water. In the condition where the shell is open, all the organs of external anatomy will be in contact with water. At the time of contact with the media/water, the Cu ions dissolved in water will penetrate the surface of the organ and enter into the cell. The process when Cu ions enter into the cell occurs in two ways, namely by passive diffusion and active transport. In the process of passive diffusion, the parts that play important role are the area in contact with water. While on active transport processes the most influential thing is the availability of energy (Soto et al. 2008). From these conditions, the process of Cu entering through the contact is determined by the surface area of the water contacts. The greater the surface area that contacts the more and more the Cu that can enter the cell. Similarly, the greater the available energy of ATP, the more active transport process and Cu will be more included in the cell. The process of Cu ion accumulation in the body of the mussels After the copper enters the body, on a scale of cellular the metals will experience stabilization process, from the original shape that is still in the form of free ions then form a ligand binding with other components. Cu bond



2 (1): 48-53, March 2010

stabilization process in the body bivalves takes place in two processes. The first is in the process of synthesizing metalbinding proteins, where metal ions play role as promoter and will be bound in a protein that is formed. The second process is the formation of granulation of mineral grains or mineralized granules, in which metal ions would be bound in mineral granules that are not dissolved (Soto et al. 2008). According to Stanley (2003) the process of stabilization and detoxification of heavy metals in the body are made in three types of processes, namely: (i) through the formation of soluble compounds that bind metals, in this case it is methallotionin protein synthesis, (ii) through the storage process (compartmentalization) of metal in one of the cell organelles cell which is in lysosomes, (iii) through the formation of the precipitate that was dissolved in the form of granulation of mineral grains that can bind metals. When it is known that the metal accumulation process is done in two or three streets, as mentioned above, when the results of this study found no significant relationship between metal concentration exposure to metal accumulation in A. woodiana, meaning three Cu accumulation process occurring in the body are varied and not uniform. This variation that ultimately gives the resultant in the form of accumulated Cu levels varying between individual samples of shellfish. The existence of these differences were in line with Viarengo et al. (1993) who said that the detoxification process that involves the stabilization and the storage of metals in the body of bivalves have a level of effectiveness that varies among species, in the same species between different individuals and on the same individual between different organs. These differences, according to Soto et al. (2008) defined by two biotic factors, namely the age and the weight of individual. Age influences the level of sensitivity of a shellfish organisms to absorb metal ions. The young individuals are more sensitive and able to absorb heavy metals more than the older individual. While the weight ratio between weight and volume of organs which can be exposed by metal. Relations between Cu concentration in water with levels of protein in the body of the mussels Results of analysis of protein levels in the body is the result of the examination of laboratory analysis of total protein content in samples of shellfish A. woodiana. presented in Figure 2, which shows the changing levels of protein in the body shells in each - each concentration treatment.

Figure 2. Protein levels in the body of A. woodiana.

From the table ANOVA, F test result is greater than the F table. The results of this test gives the sense that there is no significant difference in differences of treatment Cu concentration in water with levels of protein in the body of the mussels. The variation of treatment provided in the form of various concentration of Cu in the water does not have a significant impact on levels of Cu in the body of the mussels. In the study of the relationship between the effects of heavy metals with the change in levels of protein in organisms, the review is on protein Methallotionin. According to Soto et al. (2007) Methallotionin protein (MT protein) is a Cytosol protein with low molecular weight, soluble, resistant to high temperatures (thermophilic proteins), rich in sulfur elements (more than 30%) and has a strong affinity with metal ties. In aquatic organisms, MT proteins responsible for maintaining the metal concentration remain at low levels. Protein synthesis was induced by the presence of metal in the cell. MT protein specifically binds with metal Cd, Cu, Hg, and Zn ions. Increase in levels of MT proteins associated with an increase in the capacity of cells to bind heavy metal ions which increases the protection against toxicity of heavy metals. In this study, based on statistical tests it is concluded that there is no significant impact from the variation of Cu in water treatment to changes in protein levels in mussels. In other words that the difference of treatment and duration gave no significant impact on the changing levels of protein in the body of the mussels. The result of these statistical tests can actually be read visually from the pattern of change protein content. In the Figure 2. above it appears that there is an increase of protein levels in the second week of exposure. After that in week three the protein levels decreased and in the next week there was also a decline, except in the 0.04 ppm treatment the protein content increased in the fourth week. The existence of patterns of change that are not regular showed a wide variation that occurred in this experiment. The results of this study more or less is in line with Stolyar et al. (2005) on Cu exposure toward Anodonta cygnea. In the study it is illustrated that at low concentrations of Cu exposure, increase in levels of protein will be the same as the control organisms or, no change at all. While the highest concentration of Cu exposure (0.2 ppm) did not provide a significant impact on changes in protein levels in the body of the mussels that were tested. The reasons of why there is no relationship between levels of Cu in the water with the change of protein in the body of the mussels can be seen in two views. The first refers to the opinions of Stanley (2003). In this study it is explained that the heavy metals are stored in the body of freshwater mussels in three forms, namely as a metalbound in the precipitate minerals, metals stored in cell organelles as lysosomes and metal that is bound by the metal-binding-protein binds, or Methallotionin. Soto et al. (2008) adds that in the body of freshwater mussels there is also the ability to excrete heavy metals that enter directly, besides the three processes mentioned above.

KURNIA et al. â&#x20AC;&#x201C; Cu exposure on freshwater mussels (Anodonta woodiana)

From the statement above, the presence of protein synthesis formed in response to the entry of metal into the cell is one of four other responsive processes. In other words the existing copper metal in water will not fully trigger a protein synthesis in cells. That is because there are other mechanisms of storage in the lysosomes, the process of granulation with a mineral and excretion of Cu ions directly. In addition, up to now it has not been known for certain about which one is the most dominant process in response to the entry of copper ions into the body of the mussels.


conducted by Stolyar et al. (2004) which illustrates that copper metal ions in water will increase the metal content in the cells of the body. And it will lead to increased levels of MT proteins in animals Anodonta cygnea. Soto et al. (2007) explains the elevated levels of Cu linkages with elevated levels of this protein. Increased levels of this protein are a response to elevated levels of Cu, where MT protein will bind Cu ions to prevent the toxicity of these metals.

CONCLUSION Relations between Cu concentration in the body with levels of protein in the body of the mussels The relationship between Cu levels in shellfish with high levels of protein in the shells is presented in Figure 3 below:

Provision of Cu concentration in water did not affect significantly to the amount of Cu content accumulated in the body of A. woodiana. Provision of Cu concentration in water did not give significant effect on changes in body protein content of A. woodiana. Cu levels in the body have a positive correlation with levels of protein in the body of A. woodiana.


Figure 3. The relationship between Cu content with protein content in the body of A. woodiana

Figure 3 shows that there are variations in the pattern of relationship between Cu with proteins in the body shells. At 0.0035 ppm Cu values up to 0.0107 ppm, protein content has a pattern of relationships that fluctuate up and down. Meanwhile, in the range 0.0287 ppm Cu values up to 0.1375 ppm, which was conceived by the protein content of oysters tend to have patterns of increase with increasing Cu content. To determine the relationship between Cu content with protein content in the body shell, then the data was analyzed by correlation method of Pearson Product Moment (Nazir 2005). The computation with the working table, obtained that the correlation coefficient amounted to 0.848. This value illustrates the positive correlation between two variables measured, namely Cu levels in the body shells with protein levels in the body shells. Where the degree of relationship is quite good. In other words the change of Cu content in the body shells have a fairly strong correlation with changes in protein levels in the body shells. These results are in line with the opinion delivered by Couillard et al. (1993) who argued that in studies with bivalve species, Anodonta grandis subjects showed a strong correlation between elevated levels of Cu in the body with protein content in the cell body of organisms such shells. These results are in line with research

Couillard Y, Campbell PGC, Tessier A. 1993. Response of metallothionein concentrations in a freshwater bivalve (Anodonta grandis) along an environmental cadmium gradient. Limnol Oceanogr 38 (2): 299-313. Effendi H. 2003. Study of water quality for management of resource and water environment. Kanisius. Yogyakarta. [Indonesia] Krolak E, Zdanowski B. 2001. The bioaccumulation of heavy metals by the mussels Anodonta woodiana (LEA 1834) and Dreissena polymorpha (Pall) in the Heated Konin lakes. Polish Fish 9: 229-237. Mulgrew A, Williams P. 2000. Biomonitoring of air quality using plants. WHO Collaborating Centre for Air Quality Management and Air Pollution Control. Berlin. PAN Pesticide Database. 2009. Freshwater mussel (Anodonta sp.) toxicity studies. Pesticide Action Network, San Francisco. Republic of Indonesia Act No. 23 of 1997 on Environmental Management. [Indonesia] Sarkany-Kiss A, Fodor A, Ponta M. 1997. Bioacumulation of certain heavy metals by unionidae molluscs in Crig/Ktirisl rivers. In: Sarkany-Kiss A, Hamar J (eds). Tiscia monographseries. Szolnok, Szeged, Tg. Mureg. Cluj, Romania. Soto M, MarigĂłmez I, Cancio I. 2008. Biological aspects of metal accumulation and storage. University of the Basque Country. Bilbo, Basque Country, Spain. Stolyar OB, Myhayliv RL, Mischuk OV. 2005. The concentration-specific response of metallothioneins in copper loading freshwater bivalve Anodonta cygnea. Toxicon 48: 359-372. Sunarto. 2007. Bioindicator of heavy metal pollutant cadmium (Cd) with microanatomical structure analysis, the efficiency of gill function, morphology and shells condition of freshwater mussels Anodonta woodiana Lea. Graduate Program, University of Airlangga. Surabaya. [Indonesia] Umar MT, Winarni MM, Fachruddin L. 2001. The content of heavy metals copper (Cu) in water, sediment and shellfish Marcia sp. in Pare Pare Bay, South Sulawesi. Sci Tech 2 (2): 35-44. [Indonesia] Viarengo A, Moore MN, Mancinelli G, Mazzucotelli A, Pipe RK, Farrar SV. 1987. Metallothioneins and lysosomes in metal toxicity and accumulation in marine mussels: the effect of cadmium in the presence and absence of phenanthrene. J Mar Biol 94 (2): 251-257. Wardhana WA. 2001. Impact of environmental pollution. Penerbit Andi. Yogyakarta. [Indonesia]


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presented. Title of a picture is written down below the picture, while title of a table is written in the above the table. Colored picture and photo can be accepted if information in manuscript can lose without those images. Photos and pictures are preferably presented in a digital file. JPEG format should be sent in the final (accepted) article. Author could consign any picture or photo for front cover, although it does not print in the manuscript. There is no appendix, all data or data analysis are incorporated into Results and Discussions. For broad data, it can be displayed in website as Supplement. Citation in manuscript is written in “name and year” system; and is arranged from oldest to newest and from A to Z. The sentence sourced from many authors, should be structured based on the year of recently. In citing an article written by two authors, both of them should be mentioned, however, for three and more authors only the family (last) name of the first author is mentioned followed by et al., for example: Saharjo and Nurhayati (2006) or (Boonkerd 2003a, b, c; Sugiyarto 2004; El-Bana and Nijs 2005; Balagadde et al. 2008; Webb et al. 2008). Extent citation as shown with word “cit” should be avoided, and suggested to refer an original reference. References. APA style in double space is used in the journal reference. Only published or in-press papers and books may be cited in the reference list. Unpublished abstracts of papers presented at meetings or references to "data not shown" are not permitted. References should be cited in alphabetic order. All authors should be named in the citation (unless there are more than five). If there are more than five, list the first author's name followed by et al. Include the full title for each cited article. Authors must translate foreign language titles into English, with a notation of the original language (except for Spanish, France, and Germany). For Indonesian manuscript, translation of Indonesian title to English is not necessary. For correct abbreviations of journal titles, refer to Chemical Abstracts Service Source Index (CASSI). Provide inclusive volume, number, and page ranges for journal articles, but not for book or book chapters. Journal: Saharjo BH, Nurhayati AD. 2006. Domination and composition structure change at hemic peat natural regeneration following burning; a case study in Pelalawan, Riau Province. Biodiversitas 7: 154-158. Book: Rai MK, Carpinella C. 2006. Naturally occurring bioactive compounds. Elsevier, Amsterdam. Chapter in book: Webb CO, Cannon CH, Davies SJ. 2008. Ecological organization, biogeography, and the phylogenetic structure of rainforest tree communities. In: Carson W, Schnitzer S (eds) Tropical forest community ecology. WileyBlackwell, New York. Abstract: Assaeed AM. 2007. Seed production and dispersal of Rhazya stricta. 50th annual symposium of the International Association for Vegetation Science, Swansea, UK, 23-27 July 2007. Proceeding: Alikodra HS. 2000. Biodiversity for development of local autonomous government. In: Setyawan AD, Sutarno (eds) Toward mount Lawu national park; proceeding of national seminary and workshop on biodiversity conservation to protect and save germplasm in Java island. Sebelas Maret University, Surakarta, 17-20 July 2000. [Indonesia] Thesis, Dissertation: Sugiyarto. 2004. Soil macro-invertebrates diversity and inter-cropping plants productivity in agroforestry system based on sengon. [Dissertation]. Brawijaya University, Malang. [Indonesia] Information from internet: Balagadde FK, Song H, Ozaki J, Collins CH, Barnet M, Arnold FH, Quake SR, You L. 2008. A synthetic Escherichia coli predator-prey ecosystem. Mol Syst Biol 4: 187. Progress of manuscript. Notification of manuscript whether it is accepted or refused will be notified in about three months since the manuscript received. Manuscript is refused if the content does not in line with the journal mission, low quality, inappropriate format, complicated language style, dishonesty of research authenticity, or no answer of correspondence in a certain period. Author or first authors at a group manuscript will get one original copy of journal containing manuscript submitted not more than a month after publication. Offprint or reprint is only available with special request. NOTE: Author(s) agree to transfer copy right of published paper to NUSANTARA BIOSCIENCE, the ISEA Journal of Biological Science. Authors shall no longer be allowed to publish manuscript completely without publisher permission. Authors or others allowed multiplying article in this journal as long as not for commercial purposes. For the new invention, authors suggested to manage its patent before publishing in this journal.

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| Nus Biosci | vol. 2 | no. 1 | pp. 1‐53 | March 2010 |  ISSN 2087‐3948 (PRINT) | ISSN 2087‐3956 (ELECTRONIC)  I S E A   J o u r n a l   o f   B i o l o g i c a l   S c i e n c e s  

Ripening for improving the quality of inoculated cheese Rhizopus oryzae  SOLIKAH ANA ESTIKOMAH, SUTARNO, ARTINI PANGASTUTI 


Comparasion of iles‐iles and cassava tubers as a Saccharomyces cerevisiae substrate  fermentation for bioethanol production  KUSMIYATI 


Variation of morphological and protein pattern of cassava (Manihot esculenta) varieties of  Adira1 and Cabak makao in Ngawi, East Java  TRIBADI, SURANTO, SAJIDAN 


Diversity analysis of mangosteen (Garcinia mangostana) irradiated by gamma‐ray based  on morphological and anatomical characteristics  ALFIN WIDIASTUTI, SOBIR, MUH RAHMAD SUHARTANTO 


First record of two hard coral species (Faviidae and Siderastreidae) from Qeshm Island  (Persian Gulf, Iran)  MAHDI MORADI, EHSAN KAMRANI, MOHAMMAD R. SHOKRI, MOHAMMAD SHARIF  RANJBAR, MAJID ASKARI HESNI 


Isolation and identification of lactic acid bacteria from abalone (Haliotis asinina) as a  potential candidate of probiotic  SARKONO, FATURRAHMAN, YAYAN SOFYAN 


Productivity of sugarcane plants of ratooning with fertilizing treatment  A SUTOWO LATIEF, RIZAL SYARIEF , BAMBANG PRAMUDYA, MUHADIONO 


Exposure copper heavy metal (Cu) on freshwater mussel (Anodonta woodiana) and its  relation to Cu and protein content in the body shell  AHMAD INTAN KURNIA, EDI PURWANTO, EDWI MAHAJOENO 


        Published three times in one year  PRINTED IN INDONESIA 

ISSN 2087‐3948 (print) 

ISSN 2087‐3956 (electronic) 

Nusantara bioscience vol. 2, no. 1, March 2010  
Nusantara bioscience vol. 2, no. 1, March 2010  

Nusantara Bioscience (Nus Biosci) is an official publication of the Society for Indonesian Biodiversity (SIB). The journal encourages submis...