01 Rakhmayanti.cdr Vol.12, No.3, September 2018, p 69-73 DOI: 10.5454/mi.12.3.1 o The Growth of Leptolyngbya HS-16 and HS-36 on 35 C at Different Acidity * NURUL RAKHMAYANTI, NINING BETAWATI PRIHANTINI Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Indonesia, Kampus UI Depok 16424, Indonesia Cyanobacteria are prokaryotic organisms belonging to the kingdom Eubacteria. Cyanobacteria can be found in hot spring. Leptolyngbya is one genus of cyanobacteria that can be found in hot spring. The observation of o Leptolyngbya growth on temperature of 35 C with initial pH variation had been done. The study was experimental trial. The study aimed to determine the best initial growth pH for Leptolyngbya HS (Hot Spring)-16 and HS-36. Leptolyngbya HS-16 was isolated from Pancar Mountain hot spring, while Leptolyngbya HS-36 was isolated from Maribaya hot spring. The acidity (pH) of Pancar Mountain and Maribaya hot spring was 7. Each strain was grown o in Blue Green medium number 11 with variation of initial pH (6, 7, 8 and 9) and incubated at 35 C. Parameters was wet biomass weight of Leptolyngbya in each strain. The results of 15 days observation showed that the best initial pH for growing Leptolyngbya HS-16 is 7, while Leptolyngbya HS-36 is 9. From this study it could be seen that Leptolyngbya HS-16 and HS-36 could be cultured with alkaline condition. Key words: hot spring, Leptolyngbya, pH Cyanobacteria merupakan organisme prokariotik yang berasal dari kingdom Eubacteria. Cyanobacteria dapat ditemukan pada sumber air panas. Salah satu genus dari cyanobacteria yang ditemukan dalam sumber air panas, o yaitu Leptolyngbya. Pengamatan pertumbuhan Leptolyngbya pada suhu 35 C dengan variasi pH awal telah dilakukan. Penelitian ini merupakan penelitian eksperimental. Penelitian ini bertujuan untuk mengetahui pH pertumbuhan awal terbaik untuk Leptolyngbya HS-16 dan HS-36. Leptolyngbya HS-16 diisolasi dari sumber air panas di gunung Pancar, sedangkan Leptolyngbya HS-36 diisolasi dari sumber air panas di Maribaya. Derajat keasaman (pH) air dari sumber air panas di gunung Pancar dan Maribaya adalah 7. Masing-masing strain ditumbuhkan pada medium Blue Green nomor 11 dengan variasi pH awal (6, 7, 8 dan 9) dan diinkubasi pada suhu o 35 C. Parameter yang diteliti adalah berat basah biomassa Leptolyngbya pada masing-masing strain. Pengamatan dilakukan selama 15 hari dengan 11 sampling. Hasil pengamatan 15 hari menunjukkan bahwa pH awal terbaik untuk pertumbuhan Leptolyngbya HS-16 adalah 7, sedangkan Leptolyngbya HS-36 adalah 9. Berdasarkan penelitian dapat diketahui bahwa Leptolyngbya HS-16 dan HS-36 dapat dibiakkan dengan kondisi alkalin. Kata kunci: Leptolyngbya, pH, sumber air panas MICROBIOLOGY INDONESIA Available online at http://jurnal.permi.or.id/index.php/mionline ISSN 1978-3477, eISSN 2087-8575 *Corresponding author: Phone: +62-81297776638; Fax: +62-21-7270012; Email: nining@ui.ac.id environmental condition and their population increased twice of their first population. The population of microorganisms increased because they could used source of their new environmental. Stationary phase occurred after exponential phase. Stasioner phase is the phase that microorganisms could not do doubling cell. This phase occurred because the nutrients present in the growth medium are insufficient for doubling cell and accumulation of the metabolic waste of the microorganisms. Death phase will occured after stationer phase (Madigan et al. 2015). Cyanobacteria can be found in soil, rocks and waters (Bold et al. 1978). Another source where cyanobacteria can be found is hot spring. Maribaya hot spring and Pancar mountain hot spring are the example of source whereas cyanobacteria can be found. Maribaya hot spring has pH 6 to 7, while Pancar Mountain hot spring has pH 7. Leptolyngbya is one genus of cyanobacteria that can be found in Maribaya and Pancar Mountain hot spring (Prihantini 2015). Cyanobacteria are prokaryotic organisms belonging to the kingdom Eubacteria (Van den hoek 2002). These organisms has photosynthetic apparatus that plays role in producing energy (Scholnick et al. 2006). Cyanobacteria has photosynthetic pigment named phycobilin. These organisms stores glycogen as its food storage (Markou et al. 2014). The growth phases of cyanobacteria are same with other microorganisms. The growth curve of population in microorganisms known as exponential growth. Exponential growth in microorganisms consist of lag phase, exponential phase, stationary phase and death phase. Lag phase occur when microorganisms has been innocculated into a new medium and adaptation with new environmental with different condition of their habitat. Exponential phase happened when microorganism could adapted with their new Leptolyngbya have filamentous as the form of the colony. Characteristic of these organisms have a thin filament with 0.5 to 3.5 mm wide with simple trichome, and some species have sheath (Komarek 2007). Leptolyngbya can be found in environmental condition with pH 7 to 8.5 (Olsson-francis et al. 2012) Leptolyngbya have many benefit for our life. It could be seen Leptolyngbya could produce lipid as biofuel feedstock, produce secondary metabolites as antibiotic, and could react as bioremediator in dairy waste (Abazari et al 2012; Beetul, 2014; Khemka et al 2015). In order to beneficial of Leptolyngbya, it is important to cultivated Leptolyngbya. The acidity (pH) in the environment can affect the growth rate of cyanobacteria especially Leptolyngbya. Cyanobacteria can also live in environmental conditions with a wide range of pH, but some species are sensitive to acidic conditions (Gerloff-Elias et al, 2005). Cyanobacteria could barely find in freshwater with range of pH 4 to 5 (Bold et al. 1978). Variation of initial pH in growth medium will affect the growth of cyanobacteria. The Leptolyngbya HS-16 and HS-36's best initial growth pH have not known yet. The aim of this study is to determine the best initial growth pH for Leptolyngbya HS-16 and HS-36 in Blue Green number 11 medium (BG-11). MATERIALS AND METHODS Microorganisms and Growth Medium. The microorganisms used in this study were cyanobacteria genus Leptolyngbya strain HS-16 and HS-36. Leptolyngbya HS-16 was isolated from Pancar Mountain hot spring, while Leptolyngbya HS-36 was isolated from Maribaya hot spring. Those strains were grown in Blue Green number 11 medium/ BG-11 (NIES 2007) with variations of pH value 6, 7, 8 and 9. The BG-11 medium were made as reported by Prihantini (2015). Cyanobacteria Cultivation in BG-11 Medium. The first step of cyanobacteria cultivation was inoculated 30 mg biomass of each strain into 100 mL growth medium in 250 mL Erlenmeyer flask. Before inoculation of cyanobacteria into medium, the medium had been adjusted the variation of pH value into 6, 7, 8 and 9. The treatment of variation pH value was repeated twice in each strain. Those strain were o incubated at temperature 35 C. Measurement the weight of wet biomass Leptolyngbya HS-16 and HS-36. Measurement of Leptolyngbya HS-16 and HS-36 biomass were done in 15 days with 11 times of sampling. Sixteen of sterile eppendorf tube 2 mL was measured at analytical measurement tool. Biomass of those strain were taken aseptically with sterile micropipet amount of 2 mL. Eppendorf tube with biomass of those strain inside were centrifuged with Biofuge Primo R machine in room temperature for 10 minute (in 10.000 rpm). The supernatant of those strain were taken out and wet biomass weight were measured with analytical measurement tool. The growth curves were made by comparrasion between wet biomass weight as the ordinate axis Y with observed time as absisca X. The growth curves were made by Microsoft Excel. RESULTS The study of growth Leptolyngbya HS-16 and HS- 36 had been done. It took 15 days with 11 times of sampling. The result produced growth curve of Leptolyngbya HS-16 and HS-36. The growth curve showed the growth of both strains in the adaptation stage to the pH condition of the medium. It could be seen at the growth curve of Leptolyngbya HS-16 and HS-36, which each strain produce the growth curve unstable. The age of inoculum that used in this study were 5 months old. Macroscopic observation of Leptolyngbya were observed. Color appereance of Leptolyngbya based on Faber Castle standard color. Color appereance of Leptolyngbya HS-16 was emerald green, while Leptolyngbya HS-36 was brown ochre at day 0 (t0). At day-15 (t15), the color appereance of Leptolyngbya HS-16 in pH 9 was changed from emerald green into apple green, while Leptolyngbya HS-36 in pH 6, 7 & 8 were changed from brown ochre into apple green. The average of wet weight biomass of Leptolyngbya HS-16 and HS-36 are shown on Table 1. The growth curve of Leptolyngbya HS-16 shown on Figure 3, while the growth curve of Leptolyngbya HS- 36 shown on Figure 4. The growth curve of Leptolyngbya HS-16 and HS-36 were made based on their wet weight of biomass. After 15th day of incubation, both Leptolyngbya were able to grow on initial pH 6 medium, with wet -1 -1 weight 0.0295 g L for HS-16 and 0.02905 g L for HS- 36. The growth curve of those strain in initial pH 6 th th were slightly rise at 2 until 10 day, and drasticaly th th th increased at 13 until 14 day, but decreased at 15 day. On the other medium with initial pH 7, Leptolyngbya HS-16 were able to grow and produced maximum amount of wet weight than other initial pH, but 70 RAKHMAYANTI ET AL. Microbiol Indones Volume 12, 2018 Microbiol Indones 71 Fig 1 The color appereance of Leptolyngbya HS-16 at day-15 (t15). Fig 2 The color appereance of Leptolyngbya HS-36 at day-15 (t15). Fig 3 The growth curve of Leptolyngbya HS-16. Fig 4 The growth curve of Leptolyngbya HS-36. Leptolyngbya HS-36 produced minimum amount of wet weight than other initial pH. The wet weight of - 1 Leptolyngbya HS-36 was 0.0404 g L and -1 Leptolyngbya HS-36 was 0.01995 g L . The growth curve of Leptolyngbya HS-16 in initial pH 7 was th th th decreased at 2 until 4 day then increased at 3 until th th th 13 day and decreased at 14 until 15 day, while th Leptolyngbya HS-36 was increased constantly at 1 th th until 14 day and decreased at 15 day. Wet weight of Leptolyngbya HS-16 and HS-36 in growth medium -1 -1 with initial pH 8 were 0.03725 g L and 0.05345 g L . Both of those strain well adapted in medium with initial growth pH 8, especially Leptolyngbya HS-36. th th Leptolyngbya HS-16 was increased at 1 until 13 day th th then slightly decreased at 14 until 15 day, while th th Leptolyngbya HS-36 was increased at 1 until 15 day. Wet weight of Leptolyngbya HS-16 and HS-36 in -1 growth medium with initial pH 9 were 0,02735 g L -1 and 0,05995 g L . Leptolyngbya HS-36 produced maximum amount of wet weight than other initial pH. th th Leptolyngbya HS-16 was decreased at 1 until 3 day th th th then increased at 6 until 13 day and decreased at 14 th until 15 day, while Leptolyngbya HS-36 was th th th decreased at 1 until 3 day then increased at 6 day, but th th slightly decreased again at 7 until 8 day and increased th again until 15 day. DISCUSSION Based on the result of this experiment, the color of Leptolyngbya HS-16 changed on growth medium with initial pH 9, and Leptolyngbya HS-36 changed on growth medium with initial pH 6, 7, and 8. It hapened because of their physiology adaptation mechanism in new environmental. The physiological adaptation caused the alteration phycobilin content (Muster et al. 1983). As we can see on the growth curve, both strains still in lag phase on growth medium with initial pH 6, 7, 8, and 9. It proven that the curve still in unstable stage. Lag phase sometimes could be the longest phase for some microorganisms because in this phase, microorganisms must adapt with new environmental conditions like new source of nutrient, pH and temperature (Hogg 2005). Both of these strains were able to grow in initial growth pH 6 medium, but not as good as in alkaline condition. Leptolyngbya were often be found in neutral to alkaline condition (Madigan et al. 2015). Leptolyngbya HS-16 had the most average in growth medium with pH 7 than other pH. It could be Leptolyngbya HS-16 was already adapted in growth medium with pH 7, because Leptolyngbya HS-16 was isolated from Pancar mountain, which has pH 7 (Prihantini 2015). Leptolyngbya HS-16 was well adapted in medium growth with pH 7, which coud be grouped into neutrophile organisms (Madigan et al. 2015). Leptolyngbya HS-36 were well adapted in growth medium with initial pH 8 and 9, especially Leptolyngbya HS-36 had the most average in growth medium with initial pH 9. It happened because Leptolyngbya were often found in environmental with pH 8 (Olsson-francis et al. 2012). It could be also that 72 RAKHMAYANTI ET AL. Microbiol Indones -1 Table 1 The average of wet weight Leptolyngbya HS-16 and HS-36 (g L ) Wet weight of biomass (g L-1) T HS-16 HS-36 6 7 8 9 6 7 8 9 0 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 1 2 3 6 7 8 10 13 14 15 0.01375 0.01345 0.00445 0.01305 0.01535 0.0107 0.0132 0.02525 0.0348 0.0295 0.00685 0.01075 0.00655 0.0151 0.01555 0.0179 0.02695 0.0681 0.06415 0.0404 0.00755 0.00755 0.00835 0.01245 0.0126 0.01465 0.033 0.05635 0.0362 0.03725 0.00645 0.00705 0.0061 0.01515 0.0188 0.02485 0.0285 0.05005 0.031 0.02735 0.00615 0.0101 0.00555 0.0129 0.0077 0.00755 0.0086 0.02285 0.0325 0.02905 0.0069 0.00545 0.0058 0.01 0.0108 0.0127 0.01675 0.01765 0.02345 0.01995 0.0046 0.0052 0.0064 0.01075 0.01205 0.01525 0.02685 0.0284 0.04965 0.05345 0.00925 0.00745 0.00645 0.0166 0.01705 0.0163 0.03555 0.0548 0.0596 0.05995 Leptolyngbya was alkalophile, which microorganisms that could live with pH 8 to 10 (Madigan et al. 2015). th Those strains were decreased at 15 day of observation. It could be happened because the nutrients in the growth medium had been reduced. The nutrients in the growth medium used those strain for metabolisms to their growth (Madigan et al. 2015). Based on the th observation of the 15 day and the discuccion that had been done, the best growth of Leptolyngbya HS-16 was on growth medium with initial pH 7, while the best growth of Leptolyngbya HS-36 was on growth medium with initial pH 9. ACKNOWLEDGMENT This work was fully funded by Hibah Publikasi Internasional Terindeks untuk Tugas Akhir Mahasiswa (PITTA) 2017 to Nining Betawati Prihantini, grant no. 667/UN2.R3.1/HKP.05.00/2017. REFERENCES Abazari M, Gholamreza Z, Iraj R. 2013. Antimicrobial potentials of Leptolyngbya sp. and its synergistic effects with antibiotics. Jundishapur J Microbiol. 6:1-6. Bold, H.C., Wynne M.J. 1985. Introduction to the algae structure and reproduction. 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Balancing benefits and risks to the photosynthetic apparatus. Plant Physiol. 141:805-810. Van Den Hoek C, Mann DG, Jahns HM. 2002. Algae: An introduction to phycology, USA: Cambridge university press. Volume 12, 2018 Microbiol Indones 73 Page 1 Page 2 Page 3 Page 4 Page 5