Fatty acid characterization of indigenous cyanobacterial strains isolated from five hot springs in indonesia

. Cyanobacteria have been known to produce lipids that are potential for biodiesel. Cyanobacteria isolated from Indonesia are called Indonesia indigenous cyanobacteria. This study was conducted to determine the characterization of fatty acids contained in cyanobacteria originating from Indonesia which were isolated from 5 hot springs in Indonesia. For some 29 strains of cyanobacteria consisting of 8 genera have performed the analysis of fatty acids (FA) by extraction method via protocol in SHERLOCK Microbial Identification (Midi) System version 4.0, 2001 MIDI, Inc. The resulting data is as follows. All strains of 8 genera ( Synechococcus , Merismopedia , Thermosynechococcus , Stanieria , Leptolyngbia , Westiellopsis , Mastigocladus , and Nostoc ) have saturated fatty acids (SFA) and unsaturated fatty acids (MUFA and PUFA). The content of saturated fatty acids ranged from 27.77 to 50.56%, while the content of unsaturated fatty acids ranged from 7.58 to 63.31%. All strains have SFA Palmitic acid (16:00) which ranges from 23.23 to 42.64%. Meanwhile, unsaturated fatty acids Palmitoleic acid (16:1 wc7) are owned by almost all strains except Westiellopsis which range from 1.75 to 51.78%. Content of unsaturated fatty acids Oleic acid (18: w9c) ranges from 1.43 to 35.78% mainly in Leptolyngbia , Westiellopsis , and Mastigocladus . All strains have MUFA ranging from 7.58 to 63.31%, whereas PUFA is only owned by filamentous strains ( Leptolyngbia , Westiellposis , Mastigocladus , and Nostoc ). From the results of the research can be seen that 29 strains of cyanobacteria of 8 genera have potential fatty acids as raw materials of biodiesel under certain conditions.


Introduction
Concerns about petroleum supplies, high energy prices, and energy-related environmental security drew much attention to finding renewable biofuels. One renewable biofuel is biodiesel. Biodiesel is a type of liquid fuel in the form of Fatty Acid Methyl Ester (FAME) or methyl ester compound with long fatty acid chain [1]. One of the benefits of biodiesel is environmentally friendly, because it comes from renewable biological raw materials and does not increase CO2 emissions into the atmosphere due to the mechanism of carbon recirculation.
Biodiesel is generally derived from various types of vegetable oils. One of the biodiesel materials comes from microalgae. Microalgae are a group of autotrophic microorganisms that have no organs with apparent functional differences. Microalgae can be an appropriate alternative feedstock for next generation biofuels because certain species are known to contain lipids including high fatty acids in their biomass. Microalgae biomass can be extracted, processed, and refined into transportation fuels using the technology currently available. In addition, the microalgae can reduce the amount of carbon in the air simultaneously, have fast growth rates, allow unused cultivation and unreachable water, their production is not seasonal and can be harvested daily.
Microalgae are categorized into eukaryotic and prokaryotic microalgae. One of the prokaryotic microalgae is cyanobacteria. Cyanobacteria also commonly referred to as Cyanophyta, blue green algae, or Myxophyta [2,3] that have variations of form, i.e. unicellular, filament, or colony. Cyanobacteria as well as other microalgae are also known to produce lipids which can then be extracted and utilized. Some cyanobacteria have been known to produce lipids that are potential for biodiesel, i.e. Spirulina, etc.
In Cyanobacteria, the fatty acid properties of cyanobacteria cells are important for comparing the biochemical diversity of cyanobacteria. So far, the analysis shows members of a unique and varied cyanobacteria group regarding fatty acid composition. As with bacteria, fatty acid composition in cyanobacteria can be used as a marker in taxonomy [4,5,6]. For example, there is a significant correlation between the complexity of the species morphology and its fatty acid composition [7].
Fatty acid on the entire cell composed of saturated fatty acids (SFA), unsaturated fatty acids (MUFA/monounsaturated FA and PUFA/polyunsaturated FA), branched fatty acids and hydroxy-substituted fatty acids. Saturated fatty acids that are common to the entire cyanobacteria including bacteria is palmitic acid (16:0). Some cyanobacteria have a fatty acid composition as species of bacteria, some of the types of chloroplasts, and some of the type is not yet clear whether in bacteria or in chloroplasts. The fatty acid composition of bacteria is relatively common among unicellular cyanobacteria, whereas polyenoic fatty acid is most common filamentous cyanobacteria [8].
Cyanobacteria are microorganisms that can live anywhere or commonly called cosmopolitan microorganisms. Indonesia as one tropical country that always has a warm climate all year long cause often experiences blooming Cyanobacteria in freshwater. The study of microalgae community structures in some lakes and rivers in Indonesia indicates that many strains of Cyanobacteria appear to dominate certain waters. Some research has shown that Cyanobacteria is easily found in UI waters [9,10,11] or some waters of lakes and rivers in the Jakarta area [12,11,13], and at sources hot water in Indonesia [14,15,16]. Cyanobacteria found and isolated from Indonesia are called Indonesian indigenous cyanobacteria.
For the purposes of the use of cyanobacteria for biodiesel, cyanobacteria which may be used should be cultured cyanobacteria and can be grown in large quantities. In addition, strain characteristics also determine successful utilization. Optimal and stable strains can be used in observing genetic diversity, toxicity, and other benefits and applications.
Before being used as a biodiesel agent, the fatty acid content of cyanobacteria strains needs to be analyzed. Screening of fatty acids is needed to know which type of fatty acids are contained in these strains. This study was conducted to determine the characterization of fatty acids contained in cyanobacteria originating from Indonesia. The study was conducted on cyanobacteria isolated from hot springs in Indonesia.

Microorganisms and Growth Medium
Cyanobacteria strains were isolated from hot springs and grown on a medium suitable for the growth of each strain of cyanobacteria. The Cyanobacteria strain used in this study were cyanobacteria strains derived from 5 hot springs in West Java, Indonesia. Fatty Acid characterization performed on 29 strains of Indonesian hot springs cyanobacteria. Hot springs in Indonesia, namely Ciseeng, Mount Pancar, Rawa Danau Banten, Ciater, and Maribaya. Meanwhile, the growth medium used were CT (Cyanobacteria TAPs), MA (Microcystis Aeruginosa Medium), BG-11 (Blue Green no. 11) [17], and BBM (Bold Basal Medium) [18].

Research places
Researches were conducted at the Laboratory of Plant Taxonomy, Department of Biology, FMIPA, UI, and Laboratory Center of Excellence Indigenous Biological Resources Genome Studies (CoE IBR -GS) FMIPA UI. Maintenance of hot spring cyanobacteria strains was carried out in Algae Culture Room Laboratory of Plant Taxonomy, Department of Biology, FMIPA UI.

Culturing, preparing of starter cultures, and harvesting of cultures
Propagation of culture was begun with the inoculation of 15 mL of culture from the culture of work into Erlenmeyer flask containing 150 mL of medium. Work was done in aseptic technique. Further cultures were incubated in the rack culture. The intensity of light given was 3000-5000 lux, with photoperiodicity 12L/12D regulated by a timer. Incubation temperature which used were 20 ± 5 o C; 30 ± 5 o C; and 50 ± 5 o C. Cyanobacteria cultures in test tubes and Erlenmeyer flasks were shown at Fig.1.

Extraction of Fatty Acid
There were five (5) steps for extracting the samples. The steps were harvesting, saponification, methylation, extraction, and washing. Besides harvesting procedure, other steps procedure was done by protocols in SHERLOCK Microbial Identification (Midi) System version 4.0, 2001 MIDI, Inc. The extract preparation procedures were shown at Fig.2.
Harvesting was done when the cyanobacteria cultures reached 20 days old or already exist in the early stationary phase. Culture was put into centrifuge tubes, and then centrifuged at 4500 rpm for 20 min (modified [19]). Centrifugation process produced pellets (wet biomass) and supernatant. Wet biomass and the supernatant obtained was frozen in a freezer at a temperature of 4 o C.

Fig. 2. The fatty acid extract preparation procedures
After harvesting, the cells were saponified. There were 3 kinds of chemical compounds for saponify the cells, i.e. sodium hydroxide (certified ACS), methanol (HPLC grade), and deionized distilled water. Those compounds were mixed (150 mL deionized distilled water, 150 mL methanol, 45 gram sodium hydroxide/NaOH), and namely reagent 1 (the methalonic base). A strong methanolic base combined with heat kills and lysis the cells. Fatty acids were hydrolyzed form the cell lipids and were converted to their sodium salts (Midi: 18).
Samples (5 ± 10 mg cells) were transferred into a clean, dry 13 mm x 100 mm screw cap culture tube. Reagent 1 (1.0 ± 0.1 mL) was pipetted in to each of the culture tubes in the batch, and each tube was sealed tightly with a clean Teflon-lined screw cap. The tubes were vortexed for 5-10 seconds. The batched samples tubes rack was placed into a boiling or circulating water bath at 95 0 C -100 0 C for 5 minutes. After five (5) minutes, the tubes were removed from the boiling water, and cooling the tubes slightly. Then again, the tubes were vortexed for 5-10 seconds and then returned to the water bath for additional 25 minutes.
Next step was methylation. There were 2 kinds of chemical compounds for methylation, i.e. 6.00N hydrochloric Acid (HCl) and methanol (HPLC grade). The mixing of two chemicals was called reagents 2 or the methylation reagent (325 mL HCl and 275 mL methanol). Methylation converts the fatty acids (as sodium salts) to fatty acid methyl esters, which increases the volatility of the fatty acids for the Gas Chromatography (GC) analysis. The cap of tube from saponification steps was opened in the batch, the tubes then were added with 2.0 ± 0.1 mL of reagent 2 to each tube. Each tube was sealed tightly with a clean Teflon-lined screw cap, and the tubes were vortexed for 5-10 seconds. Because of an excess of reagents, a granular precipitate (salt) may form, and proceed with the following. The tubes were heated in an 80 ± 1 0 C water bath for 10 ± 1 minute. Then, the tubes were removed and quickly cool to room temperature by placing tubes in a tray of cold tap water. Ice-cold water was not necessary. The tubes were shaken to speed the cooling process.
Extraction was done using extraction solvent that comprises 2 kinds of chemical compounds, i.e. hexane (HPLC grade) and methyl tert-butyl ether/MTBE (HPLC grade). MTBE was added to the hexane and stirred well. Fatty acid methyl esters were removed from acidic aqueous phase and transferred to an organic phase with a liquid-liquid extraction procedure. The cap of tube from methylation steps was opened. Each tube was then added with 2.0 ± 0.1 mL of reagent 3. Each tube was sealed tightly with a clean Teflon-lined screw cap. Batch of tubes in a laboratory rotator was placed and gently mix end-over-end for 10 minutes. The cap of tube was opened again. The aqueous (lower) phase was removed and discarded using a clean Pasteur pipette for each sample.
Final step was called Base Wash. Base wash was done using reagent 4 which were included sodium hydroxide/NaOH (certified ACS) and deionized distilled water. Sodium hydroxide (NaOH) was added to stirring water until the pellets were dissolved. A dilute base solution was added to the sample preparation tubes to remove free fatty acids and residual reagents from the organic extract. Residual reagents will damage the chromatographic system, resulting in tailing and loss of the hydroxy fatty acid methyl esters.
Each tube then was added with 3.0 ± 0.1 mL of reagent 4. Each tube was sealed tightly with a clean Teflon-lined screw cap. The tubes were rotated gently end-over-end for 5 minutes. Brief centrifugation (3 minutes at 2000 rpm) was recommended to clarify the interface between the phases when an emulsion was present.

Fatty Acid Analysis
Furthermore, the upper solvent phase was removed and placed in a sample vial suitable for the automatic sampler mounted on the gas chromatograph. The extract was transferred to sample Vial and analyzing of samples according to the procedure of SHERLOCK MIS (Microbial Identification System) version 4, MIDI, Inc.

The growth of cyanobacteria strains
Twenty-nine (29) strains were characterized as cellular fatty acids. The process of analysis of fatty acids (FAs) was very dependent on the ability to grow and the speed of growth of cyanobacteria strains.
Strains were analyzed and grouped into three (3) groups. The first group was strains that grew fast enough or normal, which entered a period of peak growth of about 20 days (20 days of age culture). The second group were strains that had slow growth (30 to 40 days), while the third group were the strains that had very slow growth (more than 40 days). The first group (fast enough/normal growth) consists of strains of Synechococcus (HS-1
On the other hand, strains of hot spring filamentous cyanobacteria did not show specific groupings as well as the grouping system of Kenyon and Stanier 1972. This was likely due to the data of hot spring strains that exists only in di-unsaturated fatty acid (especially 18:2), while the Kenyon and Stanier system (1972) using Triunsaturated fatty acids (especially 18:3) and Tetraunsaturated fatty acids (especially 18:4) as distinguishing characteristics among filamentous cyanobacteria [21].
In this study, it could only differentiate between unicellular coccoid cyanobacteria (genus Synechococcus, Merismopedia, Thermosynechococcus, and Stanieria) with filamentous cyanobacteria (genus Leptolyngbia, Westiellopsis, Mastigocladus, and Nostoc) based on total polyunsaturated. In the Table 2, it shows that the total PUPA in the group of unicellular cyanobacteria strains coccoid lower than in the group of strains of filamentous cyanobacteria. Table 2 showed the total of each type of fatty acid.

Conclusions
The research showed that fatty acids were analyzed from 29 strains of Indonesian indigenous cyanobacteria are vary widely and independent of the genus cyanobacteria. The same genus may not necessarily have the same fatty acids. All strains have saturated fatty acids of 16