Jurnal Ilmu dan Teknologi Kelautan Tropis, Vol. 5, No. 1, Hlm. 162-169, Juni 2013

TRIGLYCERIDE COMPOSITION OF SIXTEEN STRAINS OF MARINE

Lily M.G. Panggabean1, Abdullah Rasyid1, Zarrah Duniani2, Yana Meliana2, dan Indah Kurniasih2 1Bidang Sumberdaya Hayati Laut, Pusat Penelitian Oseanografi-LIPI, Ancol-Jakarta Email: [email protected] 2R&D PERTAMINA, Pulo Gadung-Jakarta

ABSTRACTS Trigliceride or triacylglicerol (TAG) composition in crude oil of sixteen strain of marine diatom has been detected by spectra analyses on an Electrospray - Ion Trap – Mass Spectrometry (ESI- IT-MS) HCT Bruker-Daltonic GmbH instrument with AgNO3 used as coordination ionization agent. Biomass samples of each microalga strain were taken from early and late stationary cultures in f/2 enriched seawater and algal oils were extracted according to Bligh and Dyer. Results from spectra analysis showed that P-Pt-P (C16:0-C16:1-C16:0) were distinguished in TAG from diatom strains sp.1, Chaetoceros sp.2, Thalasiossira sp.1, Thalasiossira sp.2, Thalasiossira sp.3, Navicula sp. 1, Navicula sp. 2, Navicula sp. 3, Navicula sp. 4, Nitzschia sp. 2 and Amphora sp. In contrast, TAGs in Melosira sp. included P-P-P (C16:0-C16:0-C16:0) and P-P-O (C16:0-C16:0-C18:1) were identified. TAGs from Chaetoceros sp. were the most varies among samples, i.e. P-Pt-P (C16:0-C16:1-C16:0), A-P-M (C20:4-C16:0-C14:0), P-Pt-Lt (C16:0-C16:1-C18:3), P-Pt- A (C16:0-C16:1-C20:4), D-P-P (C22:6-C16:0-C16:0), A-Ln-P (C20:4-C18:2-C16:0). Various TAGs were also detected in Nitzschia sp.2, i.e. P-Pt-M (C16:0-C16:1-C14:0), P-Pt-P (C16:0-C16:1-C16:0), P-Pt-S (C16:0-C16:1-C18:0), P-Pt-A (C16:0-C16:1-C20:4). TAGs composition in Skeletonema strains that similar to those in Nitzschia sp.1 has longer carbon, i.e. P-P-O (C16:0-C16:0-C18:1), P-O-O (C16:0- C18:1-C18:1) and O-O-O (C18:1-C18:1-C18:1). TAGs with longer carbon chain and more double bond including highly unsaturated fatty acid C20:4 were increased with culture age in Chaetoceros sp.1, Chaetoceros sp.2, Thalasiossira sp.2, Navicula sp.1 and Nitzschia sp. 2.

Keywords: diatom, TAG, ESI-IT-MS, f/2, early and late stationary

I. INTRODUCTION reported by DOE as promising strains for development of biofuel. Panggabean et al. Nowadays development of (2009) and Bayu et al. (2009) also as a source of fuel to substitute reported some native Indonesian diatoms future fossil fuel crisis have attracted to be potential renewable feedstocks. attention worldwide (Benneman, 2008; Those findings were based on their Patil et al., 2008; Chisti, 2007; Guschina capabilities for oil production. and Harwood, 2006; NREL, 2005). The Diatoms (Bacillariophyta) are interest in microalgae, for alternative greatly diverse and abundant as the green second generation biofuel sources, is less , including 285 genera (Round et al., competitive with food and feed 1990). Diatoms belong to Ochrophytes production. Aquatic Species Program based on the presence of golden brown supported by the U.S. Department of (ocher) fucoxanthin besides chlorophyll a Energy (DOE) has collected over 3000 and c, chrysolaminarin and the silicious algal strains and screened for their oils as cell coverage –the - of various renewable source of biodiesel. Biodiesel ornamentation which reflecting taxonomic from algae contains no sulfur, nontoxic diversity. Their existance is very and highly biodegradable. There were 300 important to contribute global primary species of green and diatoms algae productivity in oceans, lake and various

©Ikatan Sarjana Oseanologi Indonesia dan 162 Departemen Ilmu dan Teknologi Kelautan, FPIK-IPB Panggabean et al.

environments. They live as planktonic, 1L seawater with salinity of 27ppt benthic, periphytic (on plant leave or enriched with f/2 media (Guillard, 1975). surfaces), epizoic (on The cultures were aerated and incubated crustaceans, whales etc.), endozoic in air condition room (23-25 oC) under (within foraminifera), moist teresstrial 3000 lux illumination for 12 hrs/day. At habitats, polar ice etc. Several diatom early and late stationary phases, 50mL strains of Indonesian waters are cultures were filtered through GF/F maintained in the culture collection of membrane for biomass measurement and Research Centre for Oceanography, 300 mL of culture stock for algal oils Indonesian Institute of Science. extraction according to method of Bligh Diatoms store carbon in the form and Dyer (1959) using chloroform : of natural oils or as polymer of methanol Proportion. The TAG carbohydrates known as chrysolaminarin. components of the algae oil were analyzed The natural oils from diatoms and algae by electron spray ionization-ion trap-mass are in the form of triacylglycerols (TAGs). spectrometry (ESI-IT-MS) on HCT TAGs consist of three long chains of fatty Brucker-Daltonic GmBH instruments with acids attached to a glycerol backbone. AgNO3 used as coordination ionization TAGs with methanol through agent (Sandra et al., 2002) transesterification reaction a chemical compound known as an alkyl ester or III. RESULT AND DISCUSSION generically known as biodiesel. Biodiesel is an alternative to petroleum diesel fuel. 3.1. Biomass and Lipid Production The fatty acids components of different Biomass obtained from early microalgal strains determine the fuel stationary phase were varied from 142 produced, because individual fatty acid mg/L for Amphora sp. to 698 mg/L for chains is species or strain specific (Pernet Skeletonema sp. 1. Biomass were mostly et al., 2003; Hu et al., 2008). TAG content increased from early to late stationary and variation could be change as they phase up to 880 mg/L in Skeletonema sp. grow or at different physiological 3 (Figure 1) and the increment was condition. Therefore variations in fatty greatest for Amphora sp. (> 200%). acids component in algal lipid would be However lipid content which expresses as necessary to take into consideration. The dry weight/ L (Figure 1) or percentage of objective of this present study is to biomass dry weight did not follow determine fatty acids components of TAG biomass production rate (Figure 2). The of typical diatom strains from two stages strain Skeletonema sp.1, Skeletonema sp. of culture. 2, Nitzschia sp. 1, Thalassiosira sp. 1 and Thalassiosira sp. 3 have high biomass II. METHOD production rates, but low lipid content. On the other hand those algae with lower Sixteen strains of marine diatoms biomass dry weight (Chaetoceros, (Table 1) originally taken from micro- Melosira, Navicula and Amphora) algal culture collection of Research Centre produced higher lipid content. Lipid for Oceanography, LIPI, were used in the content varied from 1.39% in experiment. Several group of algal strains Thalassiosira sp. 3 to 26% in Amphora were cultured during February through sp. after reaching early stationary phase March 2012 and others were cultured and from 0.76% in Skeletonema sp. 1 to during May though June 2012 in our 20% in Chaetoceros sp. 1 after late laboratory. Each strain were grown in two stationary phase. Lipid accumulation

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during stationary phase was observed in only 15% of its high biomass yield. most of centric diatoms (Chaetoceros, Moderate lipid contents includes Melosira Melosira sp., Skeletonema sp. 3 and sp. (18%) from the centrics and Navicula Thalassiosira) (Figure 2) which also sp. (17-22%) from the naviculoids were reported in several literatures (Bayu et al., derived from 49 mg/L and 35-43 mg/L 2010; Nurachman et al., 2012 b, lipid respectively. Earlier lipid Panggabean et al., 2009; Pratiwi et al., accumulation also observed in the 2009). Highest lipid accumulation was Navicula sp. of Nurachman et al. (2012b). observed in Melosira sp., that was up to Thus the use of the naviculoids may be 4.5 fold from the early stationary harvest. more economical in terms shorter culture It was in contrary with the naviculoids or biomass production. Achievable yield of pennate diatoms (Navicula, Nitzschia and about 142-880 mg/L biomass with 1-26% Amphora), further culturing to late oil content (as triglycerides) in this study stationary phase did not increase but was lower than our former results (Bayu decreased their lipid content. Top lipid et al., 2010; Nurachman et al., 2012 a and producer are Chaetoceros sp. 1 (20%) and b, Panggabean et al., 2012). The low TAG Amphora sp. (26%) that represented the production was due to different culture centrics and naviculoids repectively conditions, i.e. lower temperature and among our samples examined. Those light intensity. Low light intensity favours percentage were derived from 38.2 and the formation of the membrane polar lipid 30.8 mg/L lipid product from Chaetoceros associated with chloroplast (Brown et al., sp.1 and Amphora sp respectively. Higher 1996, Orcutt and Patterson, 1974, Sukenik lipid product was resulted from et al., 1989). Thalassiosira sp. 2 (65.78 mg/L) which

Figure 1. Biomass and lipid (mg/L dry weight) yield of sixteen diatom strains from early (Be and Le) and late (Bl and Ll) stationary phase.

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Figure 2. Lipid content (% biomass) of sixteen diatom strains from early and late stationary phase.

3.2. Fatty Acids Composition examining individual lipid classes. The Fatty acids are building blocks for results showed that TAGs in most diatom the formation of various types of lipids, strains (Bacillariophyta) consisted of long- which may include neutral lipids, polar chain C16 fatty acids (Table 1). The C16 lipids, wax esters, sterols, hydrocarbons component in Chaetoceros sp.1, and derivates prenyl e.g. carotenoids and Chaetoceros sp.2, Thalasiossira sp.1, phytylated pyrrole chlorophyls (Hu et al., Thalasiossira sp.2, Thalasiossira sp.3, 2008). Lipid from growing algae are Navicula sp. 1, Navicula sp. 2, Navicula mostly membrane polar glycerol-based sp. 3, Navicula sp. 4, Nitzschia sp. 2 and lipids, which constitute about 5-20% of Amphora sp. were predominated by dry weight. Under stress condition, they saturated and mono-unsaturated P-Pt-P shift their membrane lipids into storage (C16:0-C16:1-C16:0), but in Melosira sp. was neutral lipids in the form of triacyl predominated by saturated P-P-P (C16:0- glycerol (TAG) (Brown et al., 1993; C16:0-C16:0). Other TAG species from Grimma et al., 1992; Parish and Melosira sp., also contained longer C18 Wangersky, 1987, 1990; Lombardi and mono-saturated fatty acid P-P-O (C16:0- Wangersky, 1991,1995; Leonardos and C16:0-C18:1) also occured. TAGs species Geider, 2004; McGinnis et al., 1997). from Skeletonema and Nitzschia sp.1 are Component of fatty acids are mostly contained C18 carbon chains, i.e. P- either saturated or unsaturated with double P-O (C16:0-C16:0-C18:1), P-O-O (C16:0-C18:1- bonds on the carbon chain backbone. C18:1) and O-O-O (C18:1-C18:1-C18:1). On They are classified into medium chain the other hand, the chain length in C10-C14; long chain C16-C18 and very Nitzschia sp.2 varied from C14-C20, i.e. P- long >C20 species fatty acids and Pt-M (C16:0-C16:1-C14:0), P-Pt-P (C16:0- derivatives (Hu et al., 2008). Fatty acid C16:1-C16:0), P-Pt-S (C16:0-C16:1-C18:0), P- composition of lipid alga is species or Pt-A (C16:0-C16:1-C20:4). strain specific and varies with different Variation in chain length and culture conditions (Guschina and degree of saturation in Chaetoceros sp.1 Harwood, 2006; Chisti, 2009). oils are much greater than those in the The analysis of fatty acid other diatoms, which were particularly composition in this study was conducting detected from the early stationary phase using the total lipid extracts rather than specimens, namely from medium-chain

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C14 saturated to very-long-chain C20-22 stationary phase, which also observed in poly-unsaturated fatty acids (PUFA) or other diatoms (Hatate et al., 1998; highly-unsaturated fatty acids (HUFA). Chrismada et al., 1993; Bayu et al., 2009; TAGs detected in Chaetoceros sp.1. are Tonon, 2002). The oils of high PUFA P-Pt-P (C16:0-C16:1-C16:0), A-P-M (C20:4- contents are not stable and more C16:0-C14:0), P-Pt-Lt (C16:0-C16:1-C18:3), P- susceptible to oxidation during storage Pt-A (C16:0-C16:1-C20:4), D-P-P (C22:6- which not suitable for diesel fuel, C16:0-C16:0), A-Ln-P (C20:4-C18:2-C16:0) according to EU standard requirement EN The very long PUFA above C18 is typical 14214 and EN 14213 (Chisti, 2007). With marine algae which can not be synthesized the current technology, i.e. partial by higher plants (Hu et al., 2008). catalytic hydrogenations of the oil, the TAGs with very-long-chain HUFA unsaturation of more than four double C20:4 in Chaetoceros sp.1, Chaetoceros bonds can be reduced (Jay et al. 2005; sp.2, Thalasiossira sp.2, Navicula sp.1, Dijkstra 2006 cited by Chisti, 2007). and Nitzschia sp. 2 increased during late

Table 1. Triacyl glycerol from sixteen strains of diatoms.

No. Strain Name Specimens from early stationary Specimens from late stationary phase phase 1 Chaetoceros sp.1 P-Pt-P (C16:0-C16:1-C16:0) P-Pt-P (C16:0-C16:1-C16:0) A-P-M (C20:4-C16:0-C14:0) P-Pt-A (C16:0-C16:1-C20:4) P-Pt-Lt (C16:0-C16:1-C18:3) P-Ln-P (C16:0-C18:2-C16:0) P-Pt-A (C16:0-C16:1-C20:4) D-P-P (C22:6-C16:0-C16:0) A-Ln-P (C20:4-C18:2-C16:0) 2 Chaetoceros Sp. 2. P-Pt-P (C16:0-C16:1-C16:0) P-A-P (C16:0-C20:4-C16:0) S-Pt-P (C18:0-C16:1-C16:0) 3 Melosira sp. P-P-P (C16:0-C16:0-C16:0) nd P-P-O (C16:0-C16:0-C18:1) 4 Skeletonema sp. 1. nd P-P-O (C16:0-C16:0-C18:1) P-O-O (C16:0-C18:1-C18:1) S-Ln-S (C18:0-C18:2-C18:0) 5 Skeletonema sp. 2. P-P-O (C16:0-C16:0-C18:1) nd P-O-O (C16:0-C18:1-C18:1) 6 Skeletonema sp. 3. P-O-O (C16:0-C18:1-C18:1) nd O-O-O (C18:1-C18:1-C18:1) 7 Thalasiossira sp.1. P-Pt-P (C16:0-C16:1-C16:0) nd 8 Thalasiossira sp.2. P-Pt-P (C16:0-C16:1-C16:0) P-Pt-P (C16:0-C16:1-C16:0) P-Pt-A (C16:0-C16:1-C20:4) A-P-M (C20:4-C16:0-C14:0) P-Pt-Lt (C16:0-C16:1-C18:3) P-Pt-A (C16:0-C16:1-C20:4) 9 Thalasiossira sp.3. P-Pt-P (C16:0-C16:1-C16:0) P-Pt-P (C16:0-C16:1-C16:0) P-Pt-O (C16:0-C16:1-C18:1) 10 Navicula sp. 1. P-Pt-P (C16:0-C16:1-C16:0) P-Pt-P (C16:0-C16:1-C16:0) P-Pt-A (C16:0-C16:1-C20:4) 11 Navicula sp. 2. nd P-Pt-P (C16:0-C16:1-C16:0) 12 Navicula sp. 3. P-Pt-P (C16:0-C16:1-C16:0) P-Pt-P (C16:0-C16:1-C16:0) S-Pt-P (C18:0-C16:1-C16:0) P-Pt-S (C16:0-C16:1-C18:0) 13 Navicula sp .4. nd P-Pt-P (C16:0-C16:1-C16:0) 14 Nitzschia sp.1. P-P-O (C16:0-C16:0-C18:1) P-O-O (C16:0-C18:1-C18:1) O-S-O (C18:1-C18:0-C18:1) O-O-O (C18:1-C18:1-C18:1) 15 Nitzschia sp. 2. P-Pt-M (C16:0-C16:1-C14:0) P-Pt-A (C16:0-C16:1-C20:4) P-Pt-P (C16:0-C16:1-C16:0) P-Pt-S (C16:0-C16:1-C18:0) P-Pt-A (C16:0-C16:1-C20:4) 16 Amphora sp. P-Pt-P (C16:0-C16:1-C16:0) nd Ln-P-P (C18:2-C16:0-C16:0)

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Accepted : March 7, 2013 Revised : 21 Mei 2013 Approved : 20 Juni 2013

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