4. I Made (Lipid) revisi.cdr BIOTROPIA Vol. 21 No. 2, 2014: 100 - 110 LIPID ACCUMULATION BY ATH USING PALM OIL MILL EFFLUENT AS SUBSTRATE Flavodon flavus I MADE SUDIANA , ATIT KANTI , HELBERT , SENLIE OCTAVIANA and SUPRAPEDI Received 3 April 2014/Accepted 6 October 2014 Large amount of palm oil mill effluent (POME) is generated annually. The waste would be potential for production of single cell oils (SCOs). The objective of this study was to evaluate lipid accumulation by fungi using POME as substrate. Seven filamentous fungi were initially isolated from various biomes. The study results showed that out of these 7 fungi, five of them produced endoglucanase and accumulated lipid about 34.3-87.5% of their dry cell mass using POME as substrate. The five fungi were identified as ATH, sp., sp., sp. 1 T30, and sp.2 T50. The highest lipid accumulation was obtained by ATH. The profile of trans-esterified SCOs revealed high content of saturated and mono-unsaturated fatty acids i.e., palmitic (C16:0), stearic (C18:0) and oleic (C18:1) acids similar to conventional vegetable oils used for biodiesel production. The strain that was able to use organic substrates in POME indicated that they are promising strain for biofuel feedstock as well as for fulfilling effluent quality for wastewater discharge. Fatty acid methyl ester, , Oleaginous fungi, Palm oil mill effluent (POME), Single cell oil *1 1 1 1 2 1 2 Research Center for Biology, Indonesian Institute of Sciences, Cibinong 16911, Indonesia Research Center for Physic, Indonesian Institute of Sciences, Komplek PUSPIPTEK Tangerang Selatan, Banten, Indonesia Flavodon flavus Aspergillus Trichoderma Fusarium Fusarium Flavodon flavus Flavodon flavus ABSTRACT INTRODUCTION Keywords: As awareness on the limited stock of fossil fuel and concern on environmental hazard of petroleum fuel is growing, research on sustainable biofuel is gaining its popularity (Richana 2010; Fujita 2004; Wraight & Ramos 2005; Jing 2010; Mohammadi 2011). Microbial based biofuel (micro-diesel) has become more attractive due to its environmental incentive and renewable properties (Zheng et al. et al. et al. et al. * Corresponding author : imadesudianalipi@gmail.com DOI: 10.11598/btb.2014.21.2.4 100 2012). The merit of micro-diesel over petroleum fuel is clear, but our understanding on microbial diversity and physiology of oleaginous microbes is still incomplete to produce economically viable micro-diesel (Zhao . 2012). Current practice of biodiesel production is through trans-esterification of vegetable oils or animal fats with short chain alcohols. Feedstock acquires more than 70% of total biofuel production cost, and this limit further expansion of biodiesel (Wei . 2013). Exploiting lipid accumulating microorganism would offer solution for feedstock generation (Wang . 2011). Some lipid accumulating fungi i.e. , sp., var. , sp., sp., sp. and sp. accumulate large amounts of lipids greater than 40% per dry cell weight (Wei 2013; Wynn . 2001) under N-limited cultivation conditions. Oils derived from microbes have many advantages over plant oil due to having short life cycle, less labor required, less affection by venue, season and climate, and easier to scale up (Wang . 2013). These oleaginous characters would place microbial oils as potential feedstocks for biodiesel production in the future (Liu . 2012). Lignocellulose waste are abundant in tropical region. Several microbes use lignocelluloses hydrolysate for biofuel production (Tsigie . 2011). POME contains high strength organic substances with total chemical oxygen demand (COD) about 30,000-40,000 ppm and 3% lipid (Wu . 2010). Utilizing these wastes for biofuel production offer manifold benefits through reducing total organic in effluent, and generate biofuel feedstock via bioconversion palm oil waste into triacyl glycerol rich microbial cell (Coleman 2004). Fungi would be good candidate for biofuel feedstock, since some fungi produce extracellular cellulase which break down complex polysaccharide into fermentable sugar (Goyal . 1991), consume it for lipid synthesis and finally accumulate it into lipid bodies which primarily consist of triacylglicerol (Rossi . 2010). This study aims to evaluate the lipid accumulation by fungi using POME as substrate. This study initially isolated 7 filamentous fungi from various biomes as listed in Table 1. et al et al et al Cunninghamella echinulata Microsphaeropsis Mortierella isabellina, M. ramanniana angulispora Mucor circinelloides, Phomopsis Cephalosporium Sclerocystis Nigrospora et al. et al et al et al et al et al et al et al MATERIALS AND METHODS Fungi Species Used Table 1. Fungi used in this study Name of species Fungi sources sp. Soil of Cibinong West Java ATH Decaying wood of South East Sulawesi Comp Insect frass sp1 T30 Sludge of wastewater treatment in West Java sp2 T50 Sludge of wastewater treatment in West Java sp Soil of Cibinong West Java sp. Soil of Cibinong West Java Aspergillus Flavodon flavus Paecilomyces lilacinus. Fusarium Fusarium Trichoderma Mucor 101 Lipid accumulation by ATH using palm oil mill effluent I Made SudianaFlavodon flavus – et al. Culture Conditions Determination of Cellulolytic Fungi Endoglucanase Assays Biomass The fungal inoculants of the 7 filamentous fungi was prepared following Mulder (1989) and Kimura . (2004). Briefly, fungal spores that had been incubated at 30 °C for 5 days on Potato Dextrose A L ×10 spores/mL was inoculated into 150 mL of the seed culture medium containing (g/L): glucose, 30; yeast extract, 5; KNO , 1; KH PO , 2.5; ZnSO ·7H O, 0.01; CuSO ·5H O, 0.002; MnSO , 0.01; MgSO ·7H O, 0.5; FeSO ·7H O, 0.02; and CaCl , 0.1. The initial pH of the medium was adjusted to 5.5 by adding 10 M NaOH in a 500 mL flask and incubated at 30 °C with shaking at 125 rpm for 24 hours. Then, 10 mL of the seed culture was transferred into 250 mL of growth medium containing POME obtained from PT Perkebunan Nusantara, Malimping, West Java. Prepared medium was autoclaved for 20 minutes before use. The culture was incubated at 30 °C with shaking at 125 rpm for 24 hours. The ability of fungi to hydrolyze cellulose was evaluated using CMC containing media following Zhou . (2004). To evaluate the effect of temperature on the endoglucanase activity, the cultures were separately incubated at 30 °C and 50 °C with shaking at 125 rpm for 5 days. Enzymatic activity was assayed following Zhou . (2004). Briefly, the culture were placed in 50 mL centrifuge tubes, then centrifuged at 2,500 g at 4 ºC for 30 minutes activity measurements, 2% carboxymethylcellulose (CMC, Sigma) was L L L s were incubated at 50 ºC for 30 minutes. Reducing sugars were determined using the 3,5-dinitrosalycilic acid (DNS) assay according to Dinis , (2009). L utes (after adding the supernatant to the reaction mixture) up to 45 minutes L utes and immediately cooled on ice for 5 minutes. Finally, L was measured at 540 nm in a spectrophotometer (UV Mini 1240 Shimadzu). One enzyme unit is defined as mMol glucose produced by 1 mL enzyme per hour. Biomass fungal was determined according to Ogbo (2010). Briefly, biomass was harvested by filtration, and fungal pellet were washed with 50 mL deionized water for removal of residual nutrients, and lyophilized at -50 °C in a vacuum of 1 mbar for 12 hours and weighed. Fungal biomass was expressed as grams of dry weight per liter of culture medium. et al. et al et al et al et al. gar plates (Difco Laboratories, Detroit, MI, USA) were harvested and suspended in seed culture medium. A spore suspension (500 μ ) containing approximately 4 . Supernatants were clarified by filtration through 0.45 μm nitrocellulose filters (Pall). For enzymatic dissolved in 50 mM citrate buffer pH 5. Enzymatic reactions contained 200 μ of supernatant, 300 μ of 50 mM citrate buffer pH 5, plus 500 μ of each substrate solution. The reaction mixture Briefly, 50 μ aliquots were taken every 5 min , then mixed with 50 μ of a DNS solution, boiled for 5 min 500 μ of water were added and absorbance 6 3 2 4 4 2 4 2 4 4 2 4 2 2 BIOTROPIA Vol. 21 No. 2, 2014 102 Lipid Concentration Lipid Composition Analysis The lipid concentration of fungi broth was determined from a standard curve obtained by plotting absorbance against the corresponding lipid concentration determined by the conventional method of acid hydrolysis followed by solvent extraction and gravimetric estimation. Forty milligrams samples were extracted with 3 mL of chloroform/methanol (1/2, v/v) by vortexing (1 minute) and centrifugating at 2,500 g for 15 minutes at room temperature. The supernatants were collected and residues were re-extracted twice with 2 mL of chloroform/methanol (1/1, v/v) by centrifugation as stated above. All the supernatants were pooled together, filtered with Whatman filter No. 1 (Whatman, USA), and washed with 2 mL of Milli-Q water, followed by centrifugation at 2,500 g for 5 minutes. The lower organic phases were collected and evaporated to dryness under nitrogen and total lipid contents were determined gravimetrically (Sitepu . 2013). The total lipid concentration was determined by gas chromatographic analysis of the total fatty acids directly trans-methylesterified from dried cell (Liu 2008 ; Kosa & Ragauskas 2011). One milliliter of 10% methanolic-HCl and 0.5 mL methylene chloride were added to the dried biomass and placed at 60 °C for 3 hours for direct methyl-esterification. The reaction was stopped by the addition of 2 mL saturated NaCl solution and 1 mL hexane. The resultant methyl esters recovered in the hexane layer were then applied to a gas chromatograph (GCMS-QP 2010-Ultra; Shimadzu, Kyoto, Japan) equipped with a FAMEWAX capillary column (30 m× 0.25 mm i.d., GL Science, Tokyo, Japan) under temperature programming (150-250 °C at 5 °C/minute increments). Peanut oil (Nacalai Tesque, Kyoto, Japan) was trans-methylesterified and used as the reference material. Overall, all fungi studied showed CMC-ase activity (Fig.1), but the strains of sp., and Comp grew slowly. Therefore, we omitted these strains for further study. ATH produced the highest CMC-ase at 96 hours incubation. Other strains having slightly lower activity were sp. and Comp. Maximum activity attained at 96 hours, and decreased at 144 hours. The ability of cultures to produce CMC-ase indicated these fungi produced endoglucanases, EC 3.2.1.4, and played important role in cellulose hydrolyses (Hasper 2002). Kitcha and Cheirsilp (2014) observed newly isolated fungi as being able to hydrolyze cellulose of palm by products. To gain cellulolytic character of fungi in this study, we further evaluated their ability to hydrolyze POME at 30 °C and 50 °C. et al et al. Mucor Paecilomyces lilacinus Flavodon flavus Aspergillus Paecilomyces lilacinus et al. Aspergillus tubingensis RESULTS AND DISCUSSION Lipid accumulation by ATH using palm oil mill effluent I Made SudianaFlavodon flavus – et al. 103 Endoglucanase Activity at 30 °C Overall, endoglucanase activity of fungi grown in POME is summarized in Figure 2. The highest endoglucanase activity was obtained by ATH and sp. grown at 30 °C after 96 hours cultivation. Slightly lower activity was observed on sp.2 St50. Generally lower CMC-ase was observed at 144 hours fermentation. Flavodon flavus Aspergillus Fusarium Figure 1. CMC-ase (endoglucanase) activity of fungi grown on CMC-medium Figure 2. Endoglucanase activity of selected isolates grown on POME at 30 °C Aspergillus sp. Flavodon flavus ATH Paecilomyces sp. Comp Fusarium sp1 T30 Fusarium sp2 T50 Trichoderma sp. Mucor sp. Aspergillus sp. Flavodon flavus Fusarium sp1 T30 Fusarium sp2 T50 Trichoderma sp. 104 BIOTROPIA Vol. 21 No. 2, 2014 Endoglucanase Activity at 50 °C Effect of Growth Temperature on Endoglucanase Activity Endoglucanase activity of selected fungi grown on POME at 50 °C varies among fungal species depending on incubation period (Fig.3). Maximum activity was showed by ATH at 96 hours incubation. Lower activity was obtained after 144 hours cultivation. In general, CMC-ase activity was higher in cultures incubated at higher temperature (Fig.4). Almost 50% increase of CMC-ase activity was achieved by ATH. Fungi have been intensively explored for hydrolyses of lignocellulose materials. ATH is wood decayed fungi isolated from South East Sulawesi, Indonesia. appears to be widely distributed lignocellulolytic fungi isolated from decaying sea grass from a coral lagoon off the west coast of India (Raghukumar 1999; Mtui & Nakamura 2008) and produces extracellular lignin-modifying enzymes (LMEs): manganese-dependent peroxidase (MNP), lignin peroxidase (LIP), and laccase when grown in N-limited media (Mtui & Nakamura 2008). These enzyme characters would be advantageous for producing fermentable substances for triacylglicerol synthesis using lignin containing waste such as POME (Duarte 2013; Lam & Lee 2011). We also noticed that a culture of ATH having high CMC-ase or endoglucanase activity indicated that hydrolyses and product hydrolyses consumption affected enzyme synthesis (Vlasenko 2010). Flavodon flavus Flavodon flavus Flavodon flavus Flavodon flavus et al. Flavodon flavus et al. Figure 3. Endoglucanase activity of selected isolates grown on POME at 50 °C Aspergillus sp.Flavodon flavus Fusarium sp1 T30 Fusarium sp2 T50 Trichoderma sp. Lipid accumulation by ATH using palm oil mill effluent I Made SudianaFlavodon flavus – et al. 105 Lipid Accumulation Fatty Acid Profiles of Fungal SCOs POME is good substrate for growing lipid accumulating fungi (Fig.5), as shown by the lipid production (75% lipid per cell dry-weight). The highest lipid accumulation was attained by ATH when grown at 30 °C, but less when the culture was grown at 50 °C. Incubation temperature affected lipogenesis of fungi (Fig.5). The suitability of SCOs from oleaginous fungi was evaluated and observed contained 92% (w/w) neutral lipids in its SCO (Wu 2011). Other lipid producer, , contained lower neutral lipid fractions (18.5%, w/w), higher amount of polar lipids (35%, w/w ), and free fatty acids (32% w/w) in its SCO (Wynn . 2001). Fatty acid profiles compositions of selected fungi were obtained through trans- esterification of triacyl glycerol with methanol under alkaline condition. Fatty Acid Methyl Esters (FAME) was mainly composed of Methyl palmitate ( C H O ), Methyl cis-10-heptadecenoate (C H O ) and Methyl oleate (C H O ) (Table 2), indicating that the FAME is closely related to palm and soybean fatty acids (Ratledge & Wynn, 2002). There was slight differences in concentration of lipid species among fungi evaluated (Wynn 2001; Khot 2012; Chan 2010). The variability in fatty acid composition of oleaginous organism could be due to culture technique (Chi 2011) and species dependence (Gasmi . 2011). Flavodon flavus Cunninghamella echinulata et al. Mucor circinelloides et al et al. et al. et al. et al. et al 17 34 2 18 34 2 19 36 2 Figure 4. Comparison of endoglucanase activity of selected isolates grown on POME at 30 °C and 50 °C Aspergillus sp. Flavodon flavus Fusarium sp1 Fusarium sp2 Trichoderma sp. Aspergillus sp. Flavodon flavus Fusarium sp1 Fusarium sp2 Trichoderma sp. 106 BIOTROPIA Vol. 21 No. 2, 2014 Figure 5. Lipid accumulation by fungi grown on POME at 30 °C and 50 °C after 6-day fermentation Table 2. Lipid composition of fungi grown on POME cultured at 30 °C after 6 days fermentation Fatty Acid Methyl Esters (FAME) Trichoderma sp. Flavodon flavus ATH sp. Fusarium sp1 T30 Fusarium sp2 T50 Methyl palmitate(C17H34O2) 31.39 27.05 23.62 21.36 20.21 Methyl palmitoleate(C17H32O2) 29.65 26.28 21.08 21.08 20.68 Methyl cis-10- heptadecenoate (C18H34O2) 3.1 5.32 4.36 5.32 4.32 Methyl stearate (C19H38O2 ) 4.14 4.28 5.23 4.23 3.69 Methyl linoleate (C19H34O2) 5.41 6.21 5.21 4.65 6.1 Methyl butanoate 6.72 11.2 7.06 6.1 9.2 Methyl oleate (C19H36O2) 6.06 6.21 7.32 7.36 6.35 Methyl linolenate (C19H32O2) 4.11 3.11 9.81 9.98 8.21 Methyl cis- vaccenate(C19H32O2) 2.38 2.32 6.8 8.69 6.98 Methyl cis-12- Octadecenoate 2.71 4.82 5.91 4.91 8.6 Methyl myristate(C15H30O2) 4.33 3.2 3.6 6.32 5.66 Aspergillus sp.Flavodon flavus Fusarium sp1 T30 Fusarium sp2 T50 Trichoderma sp. 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